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Analytical Profiles

of
Volume 5
Edited b y

Klaus Florey
The Squibb Institute for Medical Research
New Brunswick, New Jersey

Contributing Editors

Norman W. Atwater Boen T. Kho


Glenn A. Brewer, Jr. Gerald J. Papariello
Lester Chafetz Bernard 2. Senkowski

Compiled under the auspices of the


Pharmaceutical Analysis and Control Section
Academy of Pharmaceutical Sciences

Academic Press New York San Francisco London 1976


A Subsidiary of Harcourt Brace Jovanovich, Publishers
EDITORIAL BOARD

Norman W. Atwater Erik H. Jensen


Jerome I. Bodin Boen T. Kho
Glenn A. Brewer, Jr. Arthur F. Michaelis
Lester Chafetz Gerald .J.Papariello
Edward M. Cohen Bruce C. Rudy
Jack P. Comer Bernard Z. Senkowski
Klaus Florey Frederick Tishler
Salvatore A. Fusari

Academic Press Rapid Manuscript Reproduction


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Library of Congress Cataloging in Publication Data

Main entry under title:

Analytical profiles of drug substances.

Includes bibliographical references.


Compiled under the auspices of the Pharmaceutical
Analysis and Control Section, Academy of Pharmaceutical
Sciences.
1. Drugs-Analysis-Collected works. 2. Chemistry,
Medical and pharmaceutical-Collected works. I. Florey,
Klaus, ed. 11. Brewer, Glenn A. 111. Academy of
Pharmaceutical Sciences. Pharmaceutical Analysis and
Control Section. [DNLM: 1. Drugs-Analysis-Yearbooks.
QV740 AA1 A551
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PRINTED IN THE UNITED STATES OF AMERICA


AFFILIATIONS OF EDITORS
AND CONTRIBUTORS

H. Y. Aboul-Enein, USV Pharmaceuticals, Tuckahoe, New York, now: Riyadh


University, Riyadh, Saudi Arabia

G. D. Anthony, Searle and Company, Chicago, Illinois

N. W.Atwuter, Searle and Company, Chicago, Illinois


J. I. Bodin, Carter-Wallace Inc., Cranbury, New Jersey

G.A. Brewer, The Squibb Institute for Medical Research, New Brunswick, New
Jersey

D. E. Cudwulluder,School of Pharmacy, University of Georgia, Athens, Georgia

L. Chufefz,Warner-Lambert Research Institute, Morris Plains, New Jersey

A. R. Chamberlin, Stanford Research Institute, Menlo Park, California

2.L. Chung,Searle and Company, Chicago, Illinois

A. P. K . Chatng, Stanford Research Institute, Menlo Park, California

E. M.Cohen, Merck, Sharp and Dohme, West Point, Pennsylvania

J. P. Comer, Eli Ully and Company, Indianapolis, Indiana

R. D. Duley, Ayerst Laboratories, Rouses Point, New York

K. Florey, The Squibb Institute for Medical Research, New Brunswick, New
Jersey

S. A. Fusuri, Parke, Davis and Company, Detroit, Michigan

vii
AFFILIATIONS OF EDITORS A N D CONTRIBUTORS

G. G. Gallo, Gruppo LePetit, Milan, Italy

R. Gomez, Hoffmann-LaRoche, Inc., Nutley, New Jersey

R. B. Hagel, Hoffmann-LaRoche, Inc., Nutley, New Jersey

D.D.Hong, The Sterling-Winthrop Research Institute, Rensselaer, New York

E. H. Jensen, The Upjohn Company, Kalamazoo, Michigan

J , H. Johnson, Hoffmann-LaRoche, Inc., Nutley, New Jersey

H. W. Jun, School of Pharmacy, University of Georgia, Athens, Georgia

B. T. Kho, Ayerst Laboratories, Rouses Point, New York

P. Lim, Stanford Research Institute, Menlo Park, California


J. P. McDonnell, Salsbury Laboratories, Charles City, Iowa

E. A. MacMuZlan, Hoffmann-LaRoche, Inc., Nutley, New Jersey

A. F. Michaelis, Sandoz Pharmaceuticals, East Hanover, New Jersey

S. M.Miller, Wyeth Laboratories, Philadelphia, Pennsylvania

N. G.Nash, Ayerst Laboratories, Rouses Point, New York

C E. Onech, Ayerst Laboratories, Rouses Point, New York


G. J. Papanello, Wyeth Laboratories, Philadelphia, Pennsylvania

A. Post, Smith, Kline and French Laboratories, Philadelphia, Pennsylvania

P. Radaelli, Gruppo LePetit, Milan, Italy

J, A. Raihle, Abbott Laboratories, North Chicago, Illinois

R, J. Rucki, Hoffmann-LaRoche, Inc., Nutley, New Jersey

B. C. Rudy, Schering-Plough,Corp., Bloomfield, New Jersey

viii
AFFILIATIONS OF EDITORS A N D CONTRIBUTORS

F. Russo-Alesi, The Squibb Institute for Medical Research, New Brunswick,


New Jersey

B. Z. Senkowski, Hoffmann-LaRoche, Inc., Nutley, New Jersey

C.M. Shearer, Wyeth Laboratories, Philadelphia, Pennsylvania


F. Tishler, Ciba-Geigy, Summit, New Jersey

R J. Warren, Smith, Kline and French Laboratories, Philadelphia, Pennsylvania


E. H. Waysek,Hoffmann-LaRoche, Inc., Nutley, New Jersey

L. L. Wearley, Searle and Company, Chicago, Illinois

ix
PREFACE
Although the official compendia list tests and limits for drug substances
related to identity, purity, and strength, they normally do not provide other
physical or chemical data, nor do they list methods of synthesis or pathways of
physical or biological degredation and metabolism. For drug substances impor-
tant enough to be accorded monographs in the official compendia such supple-
mental information should also be made readily available. To this end the Phar-
maceutical Analysis and Control Section, Academy of Pharmaceutical Sciences,
has undertaken a cooperative venture to compile and publish Analytical Profiles
of Drug Substances in a series of volumes of which this is the fifth.
The concept of Analytical Profiles is taking hold not only for cornpendial
drugs but, increasingly, in the industrial research laboratories. Analytical Profiles
are being prepared and periodically updated to provide physico-chemical and
analytical information of new drug substances during the consecutive stages of
research and development. Hopefully, then, in the not too distant future, the
publication of an Analytical Profile will require a minimum of effort whenever a
new drug substance is selected for compendial status.
The cooperative spirit of our contributors had made this venture possible.
All those who have found the profiles useful are earnestly requested to contrib-
ute a monograph of their own. The editors stand ready to receive such contribu-
tions.

Klaus Florey

xi
BENDROFLUMETHIAZIDE

Klaus FIorey and Frank M.Russo-Alesi


KLAUS FLOREY A N D FRANK M . RUSSO-ALES)

CONTENTS
1. Description
1.1 History
1.2 Name, Formula, Molecuiar Weight
1.3 Appearance, Color,Odor
2. Physical Properties
2.1 Infrared Spectrum
2.2 Nuclear Magnetic Resonance Spectrum
2.3 Ultraviolet Spectrum
2.4 Mass Spectrum
2.5 Melting Range
2.6 Differential Thermal Analysis
2.7 Solubility
2.8 Crystal Properties
2.9 pKa

3. Synthesis
4. Stability and Degradation
5. Drug Metabolic Products - Pharmacokinetics
6. Methods of Analysis
6.1 Elemental Analysis
6.2 Spectrophotometric Analysis
6.3 Colorimetric Analysis
6.4 Nonaqueous Titration
6.5 Chromatographic Analysis
6.51 Paper
6.52 Thin-Layer
7. Identification and Determination in
Biological Fluids
8. Miscellaneous
9. References

2
BENDROFLUMETH IAZIDE

1. Description
1.1 Histor
d l u m e t h i a z i d e belongs to the class
of thiazide diuretics. Its synthesis was first
reported by Holdrege, Babel and Cheneyl in
1959 and its diuretic activity was first
described in 960 almost simultaneously by Lund
and Kobing
Cunningham s.and by Kennedy, Buchanan and

1.2
Name, Formula, Molecular Weight
Bendroflumethiazide (also bendrofluazide,
benydroflumethiazide, and benzylhydroflumethiazide)
is 2H-1,2,4-Benzothiadiazine-7-sulfonamide, 3,4-
dihydro-3-(phenyl methyl)-6-trifluoromethyl-l,l-

a
dioxide or 3-Benzyl-3,4-dihydro-6-(trifluoro-
methyl)-2H-1,2,4-benzothiad~az~ne-7-sulfonam~de-
1,l dioxide [73-48-31,
0 0
H > ' ( NH@ SO'

HC-CH2
CF 3
H
Mol. Wt. 421.41
1' SH1qF 3N304 2

1.3 Appearance, Color, Odor


White or sliqhtly off-white, uniform free
flowing crystalline powder i slight floral odor.
2. Physical Properties
2.1 Infrared Spectrum
P.
The infrared spectrum f bendroflu-
methiazide is given in Figure 1

2.2
Nuclear Magnetic Resonance
The 60 MHz proton magnetic resonance
spectrum in d4-methanol containing tetramethyl-
silane as an intern93 reference (Figure 2) is
assigned as follows :

3
4

Figure 1. Infrared Spectrum of Bendroflumethiazide (batch #15); KBr, MeOH.


rl

wrn

=w=
H H

cu

h
a

rl
m
cl
al

kC\
fdk
G G

fd
al

..
Instrument: Perkin-Elmer 621.
m

Figure 2. NMR Spectrum of Bendroflumethiazide (batch 15) in deuterated


methanol (Instrument: Perkin Elmer R12B)
KLAUS FLOREY A N D FRANK M. RUSSO-ALES1

H ~1.67(s)

not assigned
Q T2.7
I n d6-DMSO ( F i g u r e 3 1 , t h e NH p r o t o n r e s o n a n c e s
were observed near ~ 1 . 7 5( s i n g l e t , superimposed
w i t h a r y l p r o t o n ) , ~2 ( b r o a d ) , 2 . 4 5 ( s i n g l e t ) and
2.57 ( s i n g l e t ) ( F i g u r e 3) i n a d d i t i o n t o t h e o t h e r
p r o t o n s a s s i g n e d from t h e dq-methanol spectrum.
The h i g h e r f i e l d s i n g l e t s a t ~ 2 . 4 5and 2.57 are
t e n t a t i v e l y a s s i g n e d t o t h e sulfonamide -NH2
p r o t o n s . N o p r e f e r e n c e of assignment i s made of
t h e o t h e r two -NH p r o t o n s . On a d d i t i o n of D 2 0 , t h e
-NH p r o t o n s a r e r a p i d l y exchanged f o r d e u t e r i u m ,
r e s u l t i n g i n t h e d i s a p p e a r a n c e of t h e NH p r o t o n s
and sharpening of t h e p r o t o n r e s o n a n c e n e a r ~5
which now a p p e a r s l i k e t h a t of t h e dq-methanol
spectrum .
2.3 U l t r a v i o l e t Spectrum
Squibb House S t a n d a r d ( b a t c h #15) i n
methanol f x h i b i t e d t h r e e maxima a t 208 mu (El 7 4 5 ) ,
273 mp (E 565) and 326 mu (Ei 9 6 ) 1 3 . 1
T h i s ag i s w i t h measurements by P i l s b u and
JacksonfF and by Kracmar and L a s t o v k o v a f i who
d i s c u s s t h e U . V . spectrophotometry of b e n z o t h i a -
d i a z i n e s and p r e s e n t s p e c t r a .
2.4 Mass Spectrum
The l o w r e s o l u t i o n mass spectrum
( F i g u r e 4 ) shows o n l y a v e r y weak m o l e c u l a r i o n
(M+) of m / e 4 2 1 and a b a s e peak o f m / e 319 t h a t
could a r i s e by a double p r o t o n r e a r r a n g e m e n t ,
which i s n o t a common f r a g m e n t a t i o n pathway. The
assignment of some of t h e fragment i o n s i s shown
below:

6
0 L I 4 c 7 r S in

Figure 3. NMR Spectrum of Bendroflumethiazide (batch 15) in deuterated DMSO


(Instrument: Perkin-Elmer R12B)
3753 SO15497 BFI#15

12-FEB-76 MR0587
I00 I

90..
I
80.-

70.-

60.-

5 0.-

40-

ie
MRSS/CHRRGE
INTENSITY SUM = 1 8 3 3 9 BRSE PERK Z = 8 . 3 2

Figure 4. Low Resolution Mass Spectrum of Bendroflumethiazide


(Instrument: AEI MS9)
BENDROFLUMETHIAZIDE

319
H
2"H2

330191 H
77
m/e 402 6 M+ m/e 4 2 1 m/e 303
-so2NH2 m/e 302 (-HI
m/e 239 \<
-
-so2
m/e 255 4 m/e 319- m/e 302 -so2NH2, m/e 2 2 2

m/e 174 m/e 158


m/e 175 (+HI m/e 159 (+HI
m/e 176 (+2H)

Although t h e s e a p p e a r t o be r e a s o n a b l e a s s i g n m e n t s ,
t h e y should be c o n s i d e r e d t e n t a t i v e s i n c e t h e
s
a s s i nments a r e n o t confirmed by h i g h - r e s o l u t i o n
d a t a 3.

2.5 Melting Range


The m e l t i n g p o i n t d o e s n o t o c c u r
s h a r p l y and depends on t h e r a t e of h e a t i n g . The
m e l t i n g p o i n t of t h e Squibb House S t a n d a r d ( b a t c h
15) w a s r e p o r t e d a t 223.20 - 226.20 C. L i t e r a t u r e
v a l u e s r a n g e from 220' - 228'.
2.6 D i f f e r e n t i a l Thermal A n a l y s i s
Melting endotherm: 2210 C. (17).
2.7 Solubilit
d i n water and c h l o r o f o r m .

9
KLAUS FLOREY AND FRANK M. RUSSO-ALES1

S o l u b l e i n a l k a l i , a c e t o n e and a l c o h o l . S o l u b l e
i n one e q u i v a l e n t of 0 . 1 N NaOH. S l i g h t l y s o l u b l e
i n e t h e r . I n s o l u b l e i n a c i d , benzene, l i g r o i n and
petroleum e t h e r l 7 . S t a b l e c o n c e n t r a t e d s o l u t i o n
i n p o l y e t h y l e n e g l y c o l , water and dimethyl-
a c e t a n i l i d e o r N-methyl-2-pyrolidinone have been
claimed (18).
2.8 Crystal Properties
The powder x-ray d i f f r a c t i o n p a t t e r n i s
p r e s e n t e d i n Table I and F i g u r e 517.
TABLE I
0
d (A) * I/I0**
17.23 0.528
13.27 0.356
11.63 0.103
8.94 0*246 *d = i n t e r p l a n a r d i s t a n c e
8.11 0.148
7.64 0.118 - 1
11 n
7.36 0.100
6.70 0.199 Z @
6.34 0.124
5.91 0.341 X = 1.539 A
5.48
5.31 0*141 Radiation: K a l and
0.194
4.82 0.162 Ka2 Copper
4.67 0.545
4.53
0.206 ** R
4.46 l'ooo elative intensity
4.31 0.175 based on h i g h e s t
4.19 0.516 i n t e n s i t y of 1 . 0 0
3.96 0.213
3.84 0.357
3.75 0.185
3.69 0.248
3.63 0.289
3.54 0.205
3.44 0.140
3.40 0.145
3.19 0.166
3.11 0.145
2.99 0.157

10
I
I

Figure 5. Powder X-Ray Diffraction Pattern of Bendroflumethiazide


(Instrument: Norelco)
KLAUS FLOREY AND FRANK M. RUSSO-ALES1

2.9 pKa
A pKa of 8.5320.05 was determined b
using the solubility variation with pH method4<
3. Synthesis
(see Figure 6)
The synthesis of bendroflumethiazide (I)
by cyclization of 4-amino-6-trifluoromethyl-m-
benzenedisulfonamide (11) with phenylacetaldehydel
(111) was e c 1 e by Holdrege, Babel and Cheney .
and others 4~'~r;'t6.Alternate approaches are
condensation of 4-amino-6-trifluoromethyl-m-
benzenedisulfonyl chloride (IV) with phenyl-
acetaldehyde (111) in the presence of ammonia8,9 ,
condensation of I11 with phenylacetimine (V)10,
reaction of 4-amino-6-trifluoromethyl-m-benzene-
disulfonamide (11) with cetoxystyrene (VI) in the
presence of acetic acid" and by hydrogenation of
3-benzyl-6-trifl~oromethyl-7-sulfamoyl-l,2~4-
benzothiadiazine 1,l-dioxide (VII) with lithium
aluminum hydridel2.
4. Stability and Degradation
Bendroflumethiazide, as a solid, appears to be
very stable. The solid, exposed to.606-C. for a
period of two weeks showed no decomposition as
measured by I.R. and modified Bratton-Marshal
reaction. In ethanol (1 mg/ml), it showed
significant changes (22% decomposition) at the
end of two weeks at 60 C. In aqueous suspension,
almost complete breakdown to the disulfonamide (11,
Figurfg6) occurred at the end of one week at
6OOC. .
In alkaline solution (pH 12) bendroflu-
methiazide undergoes complete hydro1 sis
disulfonamide (11) in one hour at 351: C. 2@2the 21
It is also unstable under certain acid conditions .
5. -
Drug Metabolic Products Pharmacokinetics
No drug metabolites have been identified so
far. The Pharmacokinetics ha55 been studied in
the rat24 and in human beings .
In human beings,
orally or intravenously administered doses of
S35-labeled bendroflumethiazide a excreted
almost quantitatively in 24 hourss5. The
stability of the trifluoromethyl group was

12
H2NSOZ
+
CF3

@ H=CHOCOCH I1 @H2-CH0

"'mC
0!
J"/' I11

H2NS0
0 0
LiAlH4
v1
'
Y NH2 - 0 0
+S4
-CH2
4
13

0
x-
V
I

C F'3 - C H 2 a 3 H

VII NaO/ I k 4 0 H
H
H
3

Q
B
c H -cH=NH SO'C1 +
v I11

NH
CF 3
IV
w
l-l
a
m
4J

S y n t h e t i c Pathways t o B e n d r o f l u m e t h i a z i d e
0
k
0

a,
c
In

Figure 6.
KLAUS FLOREY AND FRANK M. RUSSO-ALES1

studied in ratsz6. There was no detectable


fluoride uptake in the teeth of rats on a carious
diet. Bendrof umethiazide was found to be 94%
protein bound4 i! .
6. Methods of Analysis
6.1 Elemental Analysis
Found :
Calc. % (Squibb House Standard Batch 15)
C 42.75 42.63
H 3.35 3.27
F 13.53 13.83
N 9.97 9.92
0 15.21 -
S 15.22 15.60
6.2 Spectrophotometric Analysis
The ultraviolet absorption maximum at
273 mp (Ei 565) can be used for the quantitation
of bendro lumethiazide in dosage forms23I 27.
6.3 Colorimetric Analysis
Bendroflumethiazide can be quantitatively
assayed in dosage forms by alkaline hydrolysis to-
the disulfonamide (11, Figure 6) diazotization,
coupling with N-(1-naphthyl) ethylenediamine
and determ'nation of the absorption maximum at
515 nm28r3'. Other coupling agents have been
used29r31. This assay can also be used to
determine the presence2pS2&he disulfonamide (11)
in bendroflumethiazide
6.4 Non-Aqueous Titration
An assay based on titration with sodium
methoxide in dimethylformamide using p-nitro-
benzene-azo-resorcinol as indicator has been
developed32. Pyridine as solvent and azo-violet
as indicator can also be used2*.
6.5 Chromatographic Analysis
6.51 Paper Chromatographic
Bendroflumethiazide (Rf 0.76) can
be separated from the disulfonamide (11, Figure 6)
using methylisobutylketone saturated with
formamide as mobile phase and 30% formamide

14
in methanol as stationary phase. For quantitation, the spots are eluted with
methanol and the concentration is determined spectrophotometrically (see 6.2)33.
Identification and separation from other thiazide diuretics by
paper chromatography has also been reportedl5.
6.52 Thin-Layer Chromatography
Thin layer chromatographic systems are compiled in the
following table.
Absorbent Solvent System -
Ref. -
Rf
250 Silica Gel G Benzene-Ethyl Acetate (2:8) and 34 0.91
Ethyl Acetate-Methanol
Ammonium Hydroxide (85:10 :5)
G 250 Silica Gel G Propanol-2/12N Ammonia (8:2) 34 0.88
n 11
G Propanol-l/12N Ammonia (8:2) 34 0.93
11 II
" G Butanol-l/12N Ammonia (8 :2) 34 0.70
11 II
G Pentanol-l/12N Ammonia (8:2) 34 0.54
11 n
G
'I Ethyl Acetate/l2N Ammonia (8:2) 34 0.98
11 I1
" G Chloroformflethanol (8:2) 34 0.52
I1 11
" G Cyclohexane/Glacial Acetic Acid/ 34 0.98
Acetone (4:1:5)
Silica Gel Toluene/xylene/l-4-Dioxane/Isopropanol/ 35
25% Ammonia (50:10 :30 :10)
Silica Gel Ethanol/Chloroform/Heptane (1:l:l) 36 0.98
Alumina GF-254 (1st) Ethanol: (2nd) Chloroform/ 37 -
(Two Dimensional) Bu tanol
Silica Gel Ethanol containing 1.5% Water 37 0.71
Alumina G Ethanol 38 -
Absorbent Solvent System Ref.
- -
Rf
Silica Gel G Ethanol/Benzene (80:20) 38 -
Silica Gel Ethyl Acetate/Benzene (8:2) 39 0.70
It I1
Benzene/Ethyl Acetate/25% Ammonia/ 39 0.75
Methanol (20:80 :1:10)
I1 I1
Ethyl Acetate/Benzene/25% Ammonia/ 39 0.40
Methanol (60:4 0 :20)
Silica Gel Ethyl Acetate/Benzene/Ammonia/ 39 0.68
25% Methanol (50:40:2:10)
Silica Gel n-Hexane/Acetone/Ethylamine (60:30:10) 39 0.10
n-Hexane/Acetone/Diethylamine (40:40:20) 39 0.46
Silica Gel Chloroform/Methanol/Diethylamine 39 0.75
(80:15:5)
Silica Gel G Benzene/Ethyl Acetate (2:8) 40 0.82
I1
" G Ethyl Acetate/Methanol/Ammonium 40 0.82
Hydroxide (85:10:5
11
G Ethyl Acetate/Benzene (8:2) 41 0.98
Identification of oral hypoglycemic and diuretic drugs by TLC using metal ions
has been described42.
7. Identification and Determination in Biological Fluids
In plasma: Colorimetrically12
In urine: Calorimetrically 24
Radioactivity25
TLC39 r 40 t 41

8. Miscellaneous 43,44
Pharmaceutical preparation of bendroflumethiazide have been patented
BEN DROFLUMETH IAZIDE

9. References
1. C. T. Holdrege, R. B. Babel and L. C. Cheney,
J. Am. Chem. SOC. 81, 4807 (1959)
2. A. C. Kennedy, K. b. Buchanan and
C. Cunningham, Lancet 1960-1, 1267.
3. F. Lund, W. 0. Godtfredsen, Brit. Pat.
863,474 (1961); C.A. 55, 19,971d (1961) also
U . S . Pat. 3,392,168 n968).
4. J. G. Topliss, M. H. Sherlock, F. H. Clarke,
M. C. Daly, B. W. Pettersen, J. Lipski and
N. Sperber, J. Org. Chem. 26, 3842 (1961).
5. J. Klosa and H. Voigt, J. =act. Chem. 16,
264 (1962), also Ger. (East) Pat. 3 1 , 4 8 r
(1964); C.A. 63, 14,887g (1965), Brit. Pat.
1,049,322 (1960) C.A. 66, 65,5343 (1967) and
Belg. Pat. 631,232 (1963); C.A. - 60, 14,528b
(1964).
6. K. Abildgaard, Fr. Pat. 1,586,602 (1970)
C.A. 74, 100,112J (1971).
7. E. Schoenfeldt and H. Thorsteinson, Brit. Pat.
-
879,592 (1961); C.A. 57, 844f (1962).
8. L. C. Cheney and C. T. Holdrege, Fr. Pat.
1,368,708 (1964); C . A . 62, 9157c (1965).
9. J. Klosa, Brit. Pat. 1,063,102 (1967);
C.A. 67,- 11,514e (1967).
10. Brit. Pat. 898,109 (1962); C.A. - 57 12,516a
(1962).
-
11. Fr, Pat. 1,388,607 (1965); C.A. 63, 7,024b
(1965).
12. Gl Hurka, Austrian Pat. 253,513 (1967);
C.A. 67, 11,512~(1967).
13. J. D ux a m , The Squibb Institute, Personal
Communjcation.
14. J. Kracmar and M. Lastovkovi, Pharmazie -
V V
25,
464 (1970); Cesk. Farm. 20, 287 (1971);
C.A. 76, 144,8783 (1972).
15. V. B.Pilsbury and J. V. Jackson, J. Pharm.
Pharmac. 18, 713 (1966).
16. F. J. Lunrand W. Kobinger, Acta Pharmacol.
-
et Toxicol. 16, 297 (1960).
17. H. Jacobson, The Squibb Institute, Personal
Communication.
18. J. Ueda, Japan Pat. 25,692 (1963); C.A. - 60,
9,107f (1964).
19. M. Everhard, The Squibb Institute, Private
Communication.

17
KLAUS FLOREY AND FRANK M. RUSSO-ALES1

20. K. Matsushima and K. Kiyota, Iryo 23, 1561 -


(1969); C.A. 73, 112,897m (1970).
21. J. C. Turner, A. W. Nichols and J. E. Sloman,
The Pharmaceutical J. 1970, 622.
22. J. Dobrecky and B. G. Gonzalez, Rev. Farm.
-
114, 34 (1972); C.A. 78, 7,794f (1973).
23. A. I. Cohen, The S q u i s Institute, Personal
Communication.
24. J. J. Piala, J. W. Poutsiaka, C. J. Smith,
J. C. Burke and B. N. Graves, J. Pharmacol.
Expt. Therapeutics -134, 273 (1961).
25. H. R. Brettell, J. G. Smith and J, K. Aikawa,
Arch. Internal. Med. 113, 373 (1964).
26. G. Hasselmann and K. m o l d , J. Pharm.
Pharmacol. 15, 339 (1963).
27. A. M. Leal and M. B. S. Ramos Lopez, Rev.
Port. Farm. 2, 48(1963); C.A. - 59, 11,188e
(1964).
28. National Formulary XIV, p. 61ff (1975).
29. J. Bermejo, Galenica Acta - 14, 255 (1961);
C.A. 56, 10,286e (1962).
30. M. Ghxardoni and M. Fedi, Boll. Chim. Farm.
-
101, 26 (1962); C.A. 57, 9582 (1962).
31. J. F. Magalhaes and
.G.M Piros, Rev. Farm.
Bioquh. Univ. Sao Paulo - 8, 273 (1971);
C.A. 75, 121,466~ (1971).
32. H. C.Chiang, J. Pharm. Sci. 5 0 , 885 (1961).
33. H. Roberts, The Squibb Institute, Personal
Communication.
34. P. J. Smith and T. S. Herman, Anal. Biochem.
-
22, 134 (1968).
35. K. C. Guven and S. Cobanlar, Ecsacilik Bul. - 9,
98 (1967); C.A. 67, 102,839f (1967).
36. S. Gecgil, Ecsacmik Bul. - 7, 100 (1965);
C.A. 64, 14,032d (1966).
37. M. Duzene and L. Lapiere, J. Pharm. Belg.
-
20, 275 (1965); C.A. 64, 7,970d (1966).
38. R. Adam and C. L. Lapsre, J. Pharm. Belg. - 19,
79 (1964); C.A. 61, 8,134 (1964).
39. R. Maes, M. Gijbxs and L. Larvelle,
J. Chromatogr. 53, 408 (1970).
40. D. Sohn, J. Simon, H. Moheeb, G. Shali and
R. Tolba, J. Chromatogr. 87, 570 (1973).
41. B. G. Osborne, J. ChromatGr. 70, 190 (1972).
42. S. Agarwal, M. Walash, and M. I. Blake,
Indian J. Pharm. -35, 181 (1973).

18
BENDROFLUMETHIZAIDE

43. M. G o l d b e r g , U.S. Pat. 3,265,573 (1960);


C.A. 6 5 , 1 3 , 4 5 9 ~ ( 1 9 6 6 ) .
44. C . RifSirin and M. G o l d b e r g , U . S . P a t .
3 , 4 2 6 , 1 3 0 ( 1 9 6 9 ) ; C.A. 7 1 , 33,41513 ( 1 9 6 9 ) .
45. A. xgren and T. Bzck, A z a . Pharm. S u e c i n a
-
10, 2 2 3 ( 1 9 7 3 ) .

L i t e r a t u r e surveyed through June 1 9 7 4 .

19
CEPHRADINE

Klaus Florey
KLAUS FLOREY

TABLE OF CONTENTS
1. Description
1.1 Name
1.2 Definition
1.3 Formula and Molecular Weight
1.4 Appearance, Color, Odor
2. Physical Properties
2.1 Infrared Spectra
2.2 NMR Spectra
2.3 Mass Spectrum
2.4 Ultraviolet Spectrum
2.5 Optical Rotation
2.6 Melting Range
2.7 Differential Thermal Analysis
2.8 Thermogravimetric Analysis
2.9 Ionization Constant, pK
2.10 Solubility
2.11 Crystal Properties
3. Synthesis
4. Stability-Degradation
4.1 Bulk Stability
4.2 Solution Stability
5. Drug Metabolism, Pharmacokinetics
6. Methods of Analysis
6.1 Elemental Analysis
6.2 Microbiological Analysis
6.3 Iodometric Analysis
6.4 Spectrophotometric Analysis
6.5 Fluorometric Analysis
6.6 Colorimetric Analysis
6.7 Chromatographic Analysis
6.71 Paper
6.72 Thin-layer
6.73 Column
7. Determination in Body Fluids and Tissues
8. Determination in Pharmaceutical Preparations
9. References

22
CEPHRADINE

1. Description
1.1 Names
Cephradine is @,
-
7 ( - ) -2-amino- (1,4
cyclohexadien-l-yl)acetamido-3-rnethyl-8-0~0-5-
thia-l-azabicyclo-oct-2-ene-2-carboxylic acid;
also 7-p-amino-2-(1,4 cyclohexadienyl)
acetamido7-desacetyl-cephalosporanic acid.
1.2 -Definition
Cephradine, unless specified otherwise,
is defined as a hydrated form containing 3-6% of
water (for further discussion see Section 2.11).
1.3 Formula,Molecular Weiqht
/+
U C IH - 8- !jT~++-cH3
NH2
0 CO2H
C16H19N304S
Molecular Weights: 349.41 anhydrous
367.43 monohydra te
385.45 dihydrate
1.4 Appearance,Color,Odor
White crystalline powder, odorless to
slightly sulphurous.

2. Physical Properties
2.1 Infrared Spectra
Spectra of cephradine (Batch #NN005NC)
(Figure l), cephradine dihydrate(house standard
batch #NNOO5NB) (Figure 2), cephradine mono-
hydrate recrystallized from acetonitrile-water
(sample MSA 38719) (Figure 3 ) and cephradine
monohydrate recrystallized from methanol (sample
MSA 38680) (Figure 4) are presented'.

23
FREQUENCY (a')

WAVELENGTH (MICRONS)

Figure 1. Infrared Spectrum of Cephradine Batch #NN005NC,


KBr P e l l e t , Instrument, Perkin E l m e r 21.
F i g u r e 2. I n f r a r e d Spectrum of Cephradine Dihydrate(House S t a n d a r d
Batch # NN005NB) KBr P e l l e t , I n s t r u m e n t , P e r k i n E l m e r 621.
Figure 3. Infrared Spectrum of Cephradine Monohydrate Recrystallized From
Acetonitrile (Water Sample MSA 38719) KBr Pellet, Instrument,
Perkin Elmer 621.
Figure 4. Infrared Spectrum of Cephradine Monohydrate, Recrystallized
From Methanol (Sample MSA 38680) KBr Pellet, Instrument,
Perkin Elmer 621.
KLAUS FLOREY

2.2 NMR S p e c t r a
NMR s p e c t r a i n CF3COOH ( F i g u r e 5 ) and
D20 (Figure 6) a r e presented2.
I n t r i f l u o r o a c e t i c a c i d (TFA), t h e
compound e x i s t s i n p r o t o n a t e d form w i t h the NMR
s h i f t o f the NH+ a t Z 2 . 4 4 w i t h r e f e r e n c e t o i n -
t e r n a l t e t r a m e t h y l s i l a n e ( T M S ) . The i m i d e NH
p r o t o n a p p e a r i n g a s a d o u b l e t (J = 9.0 H z ) a t
Z 2 . 0 0 i s c o u p l e d t o o n e o f B-lactam r i n g p r o t o n s ,
which a p p e a r a s a q u a r t e t (J = 9 . 0 , 4 . 0 Hz) a t
C4.20. The s e c o n d @ - l a c t a m p r o t o n r e s o n a n c e i s
a d o u b l e t (J = 4 . 0 H z ) a t % = 4.74. The S-CH2
g r o u p p r o t o n s a p p e a r a s AB q u a r t e t (J = 1 8 . 0 Hz)
a t C 6 . 3 6 and 6.54 w h i l e t h e m e t h y l g r o u p a p p e a r s
a t C7.61. I n t h e dihydrophenyl r i n g , t h e o l e f i n i c
p r o t o n s a p p e a r a t f 3.62 (1 H ) and 4 . 2 1 ( 2 H ) . T h e
r e s o n a n c e a t Z 7 . 1 3 i s a s s i g n e d t o f o u r p r o t o n s of
the dihydro ring. F i n a l l y t h e m e t h i n e p r o t o n of
the CHNHf g r o u p a p p e a r s a s a m u l t i p l e t a t f 5 . 0 0 .
The s p e c t r u m o b t a i n e d i n d e u t e r i u m o x i d e
c o n t a i n i n g a d r o p of sodium d e u t e r i u m oxide was
recorded using 3-(trimethyl sily1)-1-propansul-
f o n i c a c i d sodium s a l t a s an i n t e r n a l r e f e r e n c e .
The amine a n d NH g r o u p p r o t o n s a r e exchanged w i t h
D20. I n t h e dihydrophenyl r i n g , t h e chemical
s h i f t s a r e t w o o l e f i n i c hydrogens a t C 4 . 2 5 , one
o l e f i n i c p r o t o n a t X 4 . 1 5 and t h e o t h e r f o u r hy-
drogens a t Z 7 . 3 1 . The @ - l a c t a m r i n g p r o t o n s a r e
a p a i r o f d o u b l e t s (J = 4 . 0 H z ) a t t 4 . 4 2 and 4 . 9 4
The S - m 2 g r o u p p r o t o n s a p p e a r a s a AB q u a r t e t
(J = 1 8 . 0 ) a t C 6 . 8 1 and 6 . 4 3 . The m e t h y l
r e s o n a n c e i s a d o u b l e t a t C 8 . 2 1 and may be a
r e s u l t o f p a r t i a l i s o m e r i z a t i o n of the d o u b l e bond
i n t h e b a s i c s o l u t i o n . F i n a l l y , t h e m e t h i n e (CHNHd
p r o t o n h a s a c h e m i c a l s h i f t o f 6.00C 2 .
NMR c a n a l s o be u s e d t o d e t e r m i n e t h e
amount o f r e s i d u a l c e p h a l e x i n by comparing t h e
a r e a u n d e r t h e p h e n y l r o o n s ( . C 2 . 5 0 ) and
o l e f i n i c p r o t o n s ( z 3. E4) 5.
28
F i g u r e 5. N M R S p e c t r u m of C e p h r a d i n e B a t c h NN005NC i n CF3COOH
I n s t r u m e n t : v a r i a n XL-100.
- ., , ,
n
I:

F i g u r e 6. NMR S p e c t r u m of C e p h r a d i n e B a t c h NNO05NC i n D20


I n s t r u m e n t : V a r i a n XL-100.
CEPHRADINE

2.3 Mass Spectrum


B e c a u s e o f t h e low v o l a t i l i t y of ceph-
r a d i n e , a t r i m e t h y l s i l y l d e r i v a t i v e was made
u s i n g t h e r e a g e n t BSA (N,O-Bis- ( T r i m e t h y l s i l y l )
a c e t a m i d e ) . The low r e s o l u t i o n mass s p e c t r u m
( F i g u r e 7 ) i n d i c a t e s t h a t t w o and t h r e e t r i m e t h y l
s i l y l g r o u p s were added. The compound h a v i n g t w o
t r i m e t h y l s i l y l g r o u p s was t h e p r e d o m i n a n t
p r o d u c t w i t h m o l e c u l a r i o n a t m / e 493. The loss
of CH3 from t h e m o l e c u l a r i o n ( t y p i c a l o f t r i -
methyl s i l y l g r o u p s ) y i e l d e d an i o n a t m / e 4783.
The d a t a s u p p o r t s t r u c t u r e I.

U
An i n t e n s e i o n a t m/e 180 (see s p e c t r u m )
c o r r e s p o n d s t o 11,

I1
a n o t h e r i n t e r v a l i o n a t m/e 230 c o r r e s p o n d s t o
111.

C - 0 Si(CH3I3
II
O I11
which i s a t y p i c a l f r a g m e n t o b t a i n e d from
8-lactam r i n g fragmentations3.

31
1FIB

>
t.
f.n
Z
lL
k-
96l

815

715

68
/
-
Z

Id
56l

2 9
15
$ 3 ;
j 315
K
zh:
161

MASS/CHARGE

F i g u r e 7. Low R e s o l u t i o n Mass Spectrum of T r i m e t h y l S i l y l D e r i v a t i v e of


Cephradine. I n s t r u m e n t : A E I MS-902.
2.4 U l t r a v i o l e t Spectrum
Cephradine e x h i b i t s a s i n g l e a b s o r b a n c e peak a t 262 nm
= 220 f o r b a t c h ~ N 0 0 5 N c ) ~ .
2.5 Optical Rotation
The s p e c i f i c r o t a t i o n o f t h e h o u s e s t a n d a r d ( d i h y d r a t e , b a t c h
#NN005NB) i n a pH 8 b u f f e r was found t o be + 8 8 . 3 O ( a s i s ) 5. The a v e r a g e
s p e c i f i c r o t a t i o n of s e v e n b a t c h e s of c e p h r a d i n e a t a c o n c e n t r a t i o n o f
0.5% i n 0.1M a c e t a t e b u f f e r (pH 4 . 6 ) and c a l c u l a t e d on an anhydrous b a s i s
was found t o be + 91.6O w i t h a r a n g e from + 89.22O t o 92.90°. The e f f e c t
o f pH on r o t a t i o n was found t o be s l i g h t , a s c a n be s e e n from Table 15.

Table 1
27O
L 3 -7 D conc. Approx. 1%

pH Initial 1 Hour 3 Hours 5 Hours 24 Hours


4.01 + 77.450 + 79.45O + 76.58O + 78.45O + 75.380
5.00 + 78.250 + 78.75O + 77.550 + 77.65O + 75.86O
6.03 -t 78.32O + 79.10° + 78. loo + 77.83O + 77.770
7.00 + 80.47O + 78.37O + 77.78O + 76.68O + 76.95O
8.20 + 87.24O + 86.04O + 85.05O + 85.74O + 84.32O
KLAUS FLOREY

2.6 M e l t i n q Range
Cephradine m e l t s w i t h decomposition.
The m e l t i n g range (USP) of cephradine d i h y d r a t e
house s t a n d a r d (Batch NN005NB) was 183-185O.
The m e l t i n g range of t y p i c a l b a t c h e s of cephra-
d i n e h a s v a r i e d from 175-192O.
2 . 7 D i f f e r e n t i a l Thermal A n a l y s i s
The thermogram of cephradine e x h i b i t s a
s i n g l e exotherm a t approximately 200-203O,
depending on h e a t i n g r a t e . T h i s exotherm
i n d i c a t e s o x i d a t i v e decomposition accompanied by
melting6. On t h e o t h e r hand t h e thermogram of
c e p h r a d i n e d i h y d r a t e e x h i b i t s two endothermic
peaks a t a b o u t 90° and a t a b o u t 102O which a r e
r e l a t e d t o t h e h y d r a t i o n of t h e compound. A de-
composition exotherm i s observed a t about 20OoC.
The d i f f e r e n c e i n t e m p e r a t u r e between t h e two
endothermic peaks i s a measure of t h e stress
( g r i n d i n g , h e a t i n g ) l e a d i n g t o d e h y d r a t i o n , to
which t h e sample i s s u b j e c t e d 6 .
Thermograms of c e p h r a d i n e d i h y d r a t e ,
cephradine, c e p h a l e x i n7 , and cephradine mono-
h y d r a t e ( r e c r y s t a l l i z e d from a c e t o n i t r i l e - w a t e r ) '
a r e p r e s e n t e d i n F i g u r e a6. The absence Of an
endotherm f o r cephradine s u g g e s t s t h a t cephradine
i s n o t a t r u e h y d r a t e and t h e w a t e r i s n o t
s t r u c t u r a l l y bound i n t h e c r y s t a l - l a t t i c e ( s e e
a l s o s e c t i o n 2.11). On t h e o t h e r hand t h e d i -
h y d r a t e , t h e monohydrates o b t a i n e d by
r e c r y s t a l l i z a t i o n from a c e t o n i t r i l e - w a t e r 7 ( s h a r p
endotherm a t 150-152O) and methanol8 (Endotherm
a t 152O)as w e l l a s cephalexin7 a r e t r u e h y d r a t e s ?
DTA h a s a l s o been used a s a s c r e e n i n g
technique t o s t u d y t h e i n t e r a c t i o n o f c e p h r a d i n e
with p o t e n t i a l adjuvants t o a parenteral
formula t ion9 .

34
CEPHR AD IN E

Figure 8. D i f f e r e n t i a l
The rmog rams

35
KLAUS FLOREY

2.8 Thermogravimetric A n a l y s i s
By t h e r m o g r a v i m e t r i c a n a l y s i s (TGA) o f
t y p i c a l batches o f c e p h r a d i n e from 3 t o 6%
v o l a t i l e s ( w a t e r ) , w e r e found ( t h e o r y f o r
monohydrate w a t e r 4 . 9 3 % ) . The d i h y d r a t e ( b a t c h
B49988D)by TGA g a v e 9.8% v o l a t i l e s ( t h e o r y f o r
d i h y d r a t e w a t e r 9 . 3 5 % ) . VPC was u s e d t o c o n f i r m
the v o l a t i l e compound a s w a t e r 4 8 . The 13% loss
shown b y TGA a t a b o u t 200-210°(decomposition
p o i n t ) i s assumed t o be due t o d e c a r b o x y l a t i o n 6 .
2 . 9 I o n i z a t i o n C o n s t a n t , pK
By t i t r a t i o n w i t h sodium h y d r o x i d e a
pK1 of 2 . 6 3 and pK2 o f 7 . 2 7 w a s found6.
2.10 S o l u b i l i t y
T h e r e i s no d i f f e r e n c e i n s o l u b i l i t y of
t h e v a r i o u s c r y s t a l forms. The s o l u b i l i t y o f
c e p h r a d i n e i n b u f f e r s a t d i f f e r e n t pH v a l u e s i s
reported i n Table 2.
Table 2
S o l u b i l i t y of Cephradine a t D i f f e r e n t pH Values
pH of S a t u r a t e d Solubility
pH of b u f f e r Solution mq/ml
4.00 4.02 35.8
water 4.91 21.3
60% s u c r o s e 5.0 17.1
5.00 5.04 21.1
6.03 5.90 20.5
7.20 6.12 28.2
8. 20 7.09 36.7
9. i a 7.41 49.6
Cephradine i s p r a c t i c a l l y i n s o l u b l e i n
e t h y l e t h e r , c h l o r o f o r m , b e n z e n e and h e x a n e . The
a n t i b i o t i c i s very s l i g h t l y soluble i n acetone
and a b s o l u t e e t h a n o l . I t i s f r e e l y soluble i n
propylene glycol. The s o l u b i l i t y terminology
u s e d i s t a k e n from U S P X V I I I .
The i n t r i n s i c d i s s o l u t i o n r a t e s of
c e p h r a d i n e and i t s d i h y d r a t e a s w e l l as

36
CEPHRADINE

c e p h a l e x i n were found i d e n t i c a l a t an a g i t a t i o n
i n t e n s i t y of 100 rpm 10 . The observed i n t r i n s i c
d i s s o l u t i o n r a t e s (mg/ml/min/cm2) a r e a s f o l l o w s :
Intrinsic
Compound Temperature D i s s o l u t i o n Rate
Cephradine 2 2oc 0.08
37oc 0.1
Cephradine
dihydrate 2 2oc 0.08
37OC 0.09

Cephalexin 2 2% 0.08
3 7% 0.09
2 . 1 1 Crystal Properties
There i s c o n s i d e r a b l e evidence f o r
polymorphism and f o u r polymorphs o r r a t h e r
pseudopolymorphs have been c h a r a c t e r i z e d s o f a r .
1. ) Cephradine, h y d r a t e d . Although t h e
w a t e r c o n t e n t v a r i e s from 3-6% i t i s n o t a
s t o i c h i o m e t r i c hydrate s i n c e t h e water apparently
moves f r e e l y i n t h e c r y s t a l l a t t i c e ( s e e s e c t i o n
2 . 7 ) . I t i s o b t a i n e d from aqueous s o l u t i o n .
Anhydrous cephradine h a s been p r e p a r e d and was
found t o be v e r y s t a b l e and r e s i s t a n t t o
o x i d a t i o n t o c e p h a l e x i n , when k e p t a n h y d r o u s ( s e e
s e c t i o n 4 . 1 ) . However, i t cannot be p r o p e r l y
c h a r a c t e r i z e d , s i n c e i t immediately h y d r a t e s on
exposure t o t h e atmosphere.
2 . ) Cephradine d i h y d r a t e . T h i s
compound which c r y s t a l l i z e s from aqueous s o l u t i o n
under c o n t r o l l e d c o n d i t i o n s l l , i s a t r u e dihy-
drate (see section 2 . 7 ) . I t i s v e r y s t a b l e and
r e s i s t a n t t o o x i d a t i o n t o c e p h a l e x i n . However, on
d e h y d r a t i o n ( l o s s of c r y s t a l s t r u c t u r e ) t h e
d i h y d r a t e becomes v e r y u n s t a b l e ( s e e s e c t i o n 4 . 1 ) .
The s t r u c t u r e of c e p h r a d i n e d i h y d r a t e
was determined by s i n g l e c r y s t a l x-ray d i f f r a c -
t i o n i n o r d e r t o i n v e s t i g a t e t h e p o s i t i o n of t h e
w a t e r molecules i n t h e c r y s t a l and t h e hydrogen

37
KLAUS FLOREY

bonding of t h e w a t e r t o v a r i o u s s i t e s i n t h e
cephradine molecule50. The hydrogen bonding
p a t t e r n i s i n d i c a t e d i n f i g u r e 9 . There are t w o
molecules i n t h e u n i t c e l l . The p r o j e c t i o n of
one i s shown. The second i s symmetry r e l a t e d by
180° r o t a t i o n i n t h e p l a n e of p r o j e c t i o n , and
t r a n s l a t i o n of 1 / 2 of t h e b r e p e a t , Atoms of t h e
second molecule i n v o l v e d i n hydrogen bonding a r e
i n d i c a t e d w i t h a prime n o t a t i o n .
The bonding may b e summarized a s
follows: Water oxygens a r e numbered 69 and 71.
Water oxygen #69 bonds w i t h t h e @-lactam carbonyl
of molecule 1, t h e amide n i t r o g e n 13”, of molecule
2 , and w i t h t h e second w a t e r molecule # 7 1 . Water
oxygen # 7 1 Ponds w i t h one of t h e carboxyl
oxygens, 64 , of molecule 2 , and w i t h w a t e r
molecule #69. The amino n i t r o g e n , N5’ of
molecule 2 bonds w i t h b o t h carboxyl oxygens.
3 . ) Cephradine monohydrate r e c r y s t a l l i z e d
from a c e t o n i t r i l e water8. T h i s i s a t r u e h y d r a t e
(see s e c t i o n 2 . 7 ) .
4 . ) Cephradine r e c r y s t a l l i z e d from an-
hydrous methanol8 a l s o a p p e a r s t o b e a t r u e mono-
h y d r a t e , a l t h o u g h a n o t h e r one h a l f mole of
unbound w a t e r was a l s o p r e s e n t . N o r e s i d u a l
methanol was found.
Cephradine, g e n e r a l l y , seems t o have
a tendency t o form s o l v a t e s s i n c e a c e t o n i t r i l e
and e t h y l e n e g l y c o l s o l v a t e s have been observed 1 2 .
Powder x-ray p a t t e r n s of t h e f o u r c r y s t a l forms
a r e p r e s e n t e d i n Tables 3-613.
The u n i t c e l l dimension, s p a c e group
and c e l l c o n t e n t d e t e r m i n a t i o n s of t h e f o u r
c r y s t a l forms w e r e made by s i n g l e x-ray c r y s t a l l -
ographyl and a r e p r e s e n t e d i n Table 7.
For f u r t h e r d i s c u s s i o n on t h e s e
c r y s t a l forms, s e e s e c t i o n s on I R ( 2 . 1 ) , DTA(2.7)
and Bulk S t a b i l i t y ( 4 . 1 ) .

38
Ia oc

0
W

F i g u r e 9. D i s t r i b u t i o n of water molecules i n cephradine dihydr at e .


KLAUS FLOREY

Table 3
Powder X-Ray P a t t e r n of Cephradine,Hydrated
Batch NN005NC
d 1/10 d 1/10
15.80 14.1 3.76 14.1
11.90 47.4 3.61 35.9
8.04 15.4 3.47 12.8
5.98 19.2 3.33 17.9
5.61 24.4 3.24 12.8
5.34 100.0 3.18 11.5
4.92 21.8 3.08 25.6
4.66 11.5 2.90 12.8
4.48 21.8 2.80 11.5
4.32 57.7 2.74 10.3
4.20 26.9 2.67 14.1
4.00 30.8 2.57 9.0
3.93 14.1 2.43 11.5
Table 4
Powder X-Ray P a t t e r n of Cephradine Dihydrate
Batch NN005NB (House S t a n d a r d )
d 1/10 d 1/10
11.60 35.4 3.76 22.9
10.50 15,6 3.67 26.0
8.75 10.4 3.55 100.0
7.05 19.8 3.45 43.8
6.20 37.5 3.42 46.9
6.00 17.7 3.19 28.1
5.80 24.0 3.08 30.2
5.61 56.3 2.92 54.2
5.23 20.8 2.80 10.4
5.10 37.5 2.68 15.6
4.68 7.3 2.61 15.6
4.55 9.4 2.57 13.5
4.45 26.0 2.49 14.6
4.25 17.7 2.46 17.7
3.94 9.4 2.39 7.3
3.87 12.5 2.31 12.5
3.80 28.1
CEPHRADINE

Table 5
Powder X-Ray Pattern of Cephradine Monohydrate
Recrystallized from Acetonitrile-Water
Sample #38720
d 1/10 d 1/10
9.92 100.0 3.05 17.0
8.83 94.0 3.02 18.0
6.96 82.0 2.96 25.0
6.45 24.0 2.90 14.0
5.64 80.0 2.80 17.0
5.43 35.0 2.77 35.0
4.92 15.0 2.75 14.0
4.84 22.0 2.70 13.0
4.74 91.0 2.65 18.0
4.39 45.0 2.60 26.0
4.23 100.0 2.47 11.0
4.00 45.0 2.42 26.0
3.91 35.0 2.38 23.0
3.64 21.0 2.35 31.0
3.41 32.0 2.24 25.0
3.36 29.0 2.21 17.0
3.22 24.0 2.14 25.0
3.10 61.0

41
KLAUS FLOREY

Table 6
Powder X-Ray Pattern of Cephradine Monohydrate
Recrystallized from Methanol
Sample # 38708

d 1/10 d 1/10
11.20 100.0 3.78 14.0
8.83 17.0 3.63 26.0
8.18 19.0 3.51 33.0
7.83 10.0 3.30 24.0
6.80 18.0 3.17 15.0
6.23 19.0 3.08 16.0
5.82 25.0 2.94 26.0
5.64 13.0 2.82 18.0
5.21 14.0 2.74 21.0
4.86 37.0 2.65 17.0
4.74 45.0 2.62 18.0
4.59 43.0 2.54 16.0
4.34 32.0 2.45 14.0
4.19 34.0 2.42 14.0
4.13 25.0 2.36 17.0
4.00 26.0 2.30 14.0
3.89 32.0

42
Table 7

- CELL coNsTAms-> SPACE MEASURED C E L L COJTI'ENTS

12 cephradine

P
0

4 cephradine
Acetonitrile
Monohydra t'e I
Recrystallized
from 17.58 9.4 21.6 90 3568 P212121 1.33 8 cephradine
M e th an o 1 8 water
3. Synthesis
Cephradine (111, Figure 10) is synthesized by coupling 7-aminodesace-
toxycephalosporanic acid (7-ADCA) (11) with a protected derivative of
dihydrophenylglycine (I),
Figure 10
Synthetic Pathway to Cephradine

0hz I
CH - COOH Intermediate + H2N

COOH

0:" m2
- CO - J
<m
-J

/ CH3
0
COOH
I11
such as the tert.-butoxylcarbonyl derivative which can be converted to a
mixed anhydride with ethylchloroformate and reacted with 7-ADCA14.
Cephradine can also be made by forming the methyl acetoacetic ester
enamine derivative of dihydrophenylglycine-which is converted to a mixed
CEPHRADINE

anhydride w i t h benzoyl c h l o r i d e p r i o r t o coupling


w i t h 7-ADCAI2. Cephradine i s t h e n cr s t a l l i z e d
from a b i p h a s i c MIBK-aqueous s o l u t i o nY2 .
It also
can be c r y s t a l l i z e d from w a t e r a l o n e , a s w e l l a s
from o t h e r s o l v e n t s ( s e e s e c t i o n 2 . 1 1 ) .

4. S t a b i 1i ty-Degrada t i o n
4 . 1 Bulk S t a b i l i t y
C e p h r a d i n q w h e n k e p t u n d e r d r y and c o o l
s t o r a g e c o n d i t i o n s , h a s shown r e a s o n a b l e b u l k
s t a b i l i t y 1 2 . Like o t h e r 1,4 cyclohexadienes
such a s 2,5 d i h y d r ~ p h e n y l a l a n i n e ~c~e p, h r a d i n e i s
prone t o a slow r a t e of o x i d a t i o n of t h e c y c l o -
hexadiene r i n g t o t h e benzenoid r i n g . The e x a c t
mechanism of t h i s r e a c t i o n i s n o t known, b u t i n
a d d i t i o n t o oxygen and w a t e r , t r a c e m e t a l s s u c h
a s i r o n seem t o have an a c c e l e r a t i n g e f f e c t .
The o x i d a t i o n t o c e p h a l e x i n a s w e l l a s
d e g r a d a t i o n (loss of b i o p o t e n c y ) can be p r e v e n t e d
o r s i g n i f i c a n t l y r e d u c e d b y s t o r a g e a t low
t e m p e r a t u r e and e x c l u s i o n o f oxygen, a s w e l l a s
removal of w a t e r . T h i s l a s t method, however, i s
i m p r a c t i c a l due t o t h e e x t r e m e l y h y g r o s c o p i c
n a t u r e o f anhydrous c e p h r a d i n e 1 2 .
On t h e o t h e r hand c e p h r a d i n e d i h y d r a t e 11
b e i n g a t r u e s o l v a t e (see s e c t i o n 2 , l l ) w h e r e
water c a n n o t move f r e e l y i n t h e c r y s t a l l a t t i c e
e x h i b i t s remarkable r e s i s t e n c e t o c e p h a l e x i n
f o r m a t i o n , l o s s o f b i o p o t e n c y and c o l o r d e v e l o p -
m e n t d u r i n g e x t e n d e d s t o r a g e u n d e r a i r l 2 . The
d i f f e r e n c e b e t w e e n c e p h r a d i n e and i t s d i h y d r a t e
i s i l l u s t r a t e d i n Table 812.
However upon p a r t i a l o r f u l l d e h y d r a t i o n
of c e p h r a d i n e d i h y d r a t e u n d e r a v a r i e t y of
conditions, drying a t higher temperatures o r
c e r t a i n kinds of m i l l i n g t h i s e x c e l l e n t s t a b i l i t y
i s not maintainedlz.

45
KLAUS FLOREY

Table 8
Average Bulk S t a b i l i t y Data for Three Batches of
Cephradine and f o r Three Batches of Cephradine
Dihydrate A f t e r 9 Months of S t o r a g e Under A i r

Cephradine ( " a s i s " )

Initial 5Oc RT 4OoC 5OoC


Bioassay, mcg/ml 949 947 942 924 849
Cephalexin, % 2.7 3.0 3.5 4.6 7.0
Cephradine Dihydra t e ( " a s i s " )
Bioassay, mcg/ml 909 870 870 897 906
Cepha l e x i n , % 2.3 2.3 2.3 2.2 2.4

Cephradine i s moderately l i g h t
s e n s i t i v e . When exposed t o U.V. l i g h t , t h e s o l i d
t u r n s yellow on t h s u r f a c e b u t no loss of b i o -
a c t i v i t y w a s noted f 2 . The s e n s i t i v i t y of
c e p h a l o s p o r i n C t o l i g h t h a s been p r e v i o u s l y
no t e d l 6 .
Other than c e p h a l e x i n , no d e g r a d a t i o n
p r o d u c t s of s o l i d cephradine have been i d e n t i f i e d
TLC examination of degraded (loss of biopotency)
samples r e v e a l e d a m u l t i p l i c i t y of U.V. a b s o r b i n g
and f l u o r e s c e n t s p o t s 1 7 .
4.2 S t a b i l i t y i n Solution
I n aqueous s o l u t i o n c e p h r a d i n e t e n d s t o
be q u i t e s t a b l e a t pH 4.0 and below and much l e s s
s t a b l e a t h i g h e r pH u n i t s . A p H - s t a b i l i t y p r o f i l e
i s p r e s e n t e d i n Table 96 . Cephradine was found
to be f u l l y p o t e n t for at l e a s t e i g h t h o u r s i n a
v a r i e t y of p a r e n t e r a l i n f u s i o n s o l u t i o n s 2 3 .
Although cephradine, l i k e o t h e r cephal-
o s p o r i n s i s much more r e s i s t a n t t o opening of
t h e B-lactam r i n g by a l k a l i t h a n p e n i c i l l i n s , t h e
r i n g does open up w i t h subsequent f u r t h e r
d e g r a d a t i o n , Ring opening a l s o l e a d s t h e loss
of U.V. absorbance a t 262 nm.

46
Table 9
S t a b i l i t y of C e p h r a d i n e i n Phosphate B u f f e r
a t Room Temperature

P e r c e n t of Remaining B i o a c t i v i t y
p H of S o l u t i o n
* 2 days 4 days 7 days 10 days 14 days
4.0 95.9 105.1 99.3 99.3 95.2
6.0 73.0 86.9 69.5 30.2 18. 5
8.0 67.1 43.1 24.5 13.4 10.9
10.0 64.0 41.1 25.2 13. 3 11.7
*
ph of samples a t s t a r t of s t u d y . The pH o f t h e h i g h pH samples d r i f t e d
toward lower pH v a l u e s a s t h e s t u d y p r o g r e s s e d .
5
One of t h e a l k a l i n e d e g r a d a t i o n p r o d u c t s p r e c i p i t a t e s from
t h e s o l u t i o n and h a s been i d e n t i f i e d a s 2-[6-(1,4 cyclohexadien-l-yl)-2,
5-dioxo-3-piperazinyl~-5,6-dihydro-5-methyl-2H-l,3-thiazine-4-carboxylic
a c i d , sodium s a l tI8.
KLAUS FLOREY

The B-lactam r i n g o f c e p h r a d i n e i s q u i t e re-


s i s t a n t t o p e n i c i l l i n a s e , b u t opens r e a d i l y w i t h
cephalosporinasel9.
On t h e a c i d s i d e NMR s t a b i l i t y
s t u d i e s w i t h a 2% s o l u t i o n a t pH 1 . 6 , h e l d a t
60°C, d e m o n s t r a t e d t h a t t h e r e i s no 0-lactam
r i n g opening. However p o s s i b l e s h i f t i n g o f t h e
d o u b l e bond from c a r b o n s 2-3 t o 3-4 was o b s e r v e d
b y NMR and c o n f i r m e d by loss of U.V. a b s o r b a n c y
a t 262 nm. A f t e r 93 h o u r s NMR i n d i c a t e d a 50%
d o u b l e bond i s o m e r i z a t i o n 2 . Very v i g o r o u s
treatment with strong a c i d w i l l lead a t l e a s t i n
p a r t t o a s p l i t o f t h e amide l i n k a g e t o form
7-ADCA and d i h y d r o p h e n y l g l y c i n e 1 2 . This cleavage
can a l s o be a c h i e v e d e n z y m a t i c a l l y w i t h
p e n i c i l l i n acylase19. I n a phosphate b u f f e r a t
pH 6 v i g o r o u s a e r a t i o n o r e x p o s u r e t o room l i g h t
d i d n o t a c c e l e r a t e degradation2'. However 10
h o u r s of e x p o s u r e t o a Hanovia U.V. lamp of an
aqueous s o l u t i o n (pH 5 ) of c e p h r a d i n e more t h a n
doubled t h e c e p h a l e x i n c o n t e n t w i t h l i t t l e
change i n b i o p o t e n c y 1 2 . Cephradine was found t o
be s t a b l e f o r a t l e a s t t h i r t y days i n f r o z e n
(-S0C) human s e r u m and u r i n e . No significant
l o s s was d e t e c t e d by r e p e a t e d thawing and
refreezing2'. On t h e o t h e r hand when i n c u b a t e d
w i t h s er u m a t 37OC f o r 6 h o u r s t h e r e was a 20%
loss of a c t i v i t y . I n c u b a t i o n w i t h whole b l o o d
under t h e same c o n d i t i o n s c a u s e d l i t t l e o r no
loss of b i o p o t e n c y 2 2 .
I t was found5I, t h a t t h e a c t i v i t y
of c e p h a l o s p o r i n s c o n t a i n i n g a p h e n y l g l y c i n e
m o i e t y ( c e p h a l o g l y c i n and t o a l e s s e r d e g r e e
c e p h a l e x i n and c e p h r a d i n e ) i s p r o g r e s s i v e l y 10s t
i n t h e p r e s e n c e of c u p r i c i o n . T h i s d e g r a d i n g
e f f e c t of c u p r i c i o n can be i n h i b i t e d b y
d-penicillamine.

40
CEPHRADINE

5. Druq Metabolism
Cephradine-3H 250 mg d r y f i l l e d c a p s u l e s w e r e
a d m i n i s t e r e d t o e i g h t normal male s u b j e c t s a s a
s i n g l e o r a l dose i n a b i o a v a i l a b i l i t y study24.
Serum and u r i n e s a m p l e s were a s s a y e d i n a d o u b l e
b l i n d f a s h i o n f o r b i o l o g i c a l a c t i v i t y and i n a n
open f a s h i o n f o r r a d i o c h e m i c a l a c t i v i t y .
The mean p e a k c e p h r a d i n e s e r u m c o n c e n t r a t i o n
(7.0 2 1 . 0 kgm/ml - SE by b i o a s s a y a n d 7 . 8 f
0 . 9 pgm/ml - SE b y r a d i o a s s a y ) o c c u r r e d 55
m i n u t e s a f t e r d o s i n g and d e c r e a s e d w i t h a b i o l o g -
i c a l h a l f - t i m e o f 40 m i n u t e s . The c u m u l a t i v e
a r e a s u n d e r t h e c u r v e s f o r c e p h r a d i n e were 1 6 1 . 5 f
29 and 1 7 7 . 3 f 34.7 min. pgm/ml f o r b i o a s s a y and
radioassay curves respectively. C e p h r a d i n e was
r a p i d l y e x c r e t e d . A p p r o x i m a t e l y 77% of
c e p h r a d i n e was e x c r e t e d w i t h i n t h e t h r e e h o u r
p e r i o d f o l l o w i n g d r u g a d m i n i s t r a t i o n . A f t e r 24
h o u r s a p p r o x i m a t e l y 87% o f t h e a d m i n i s t e r e d d o s e
of c e p h r a d i n e was r e c o v e r e d i n t h e u r i n e a s
m e a s u r e d b y both b i o and r a d i o c h e m i c a l a s s a y25
Four h e a l t h y f e m a l e s u b j e c t s r e c e i v e d a s i n g l e
.
i n t r a m u s c u l a r i n j e c t i o n of 1 gram o f
~ e p h r a d i n e - ~ H ~The ~ . mean p e a k c o n c e n t r a t i o n o f
1 0 f 1 . 7 ug/ml - SE i n plasma w a s r e a c h e d a t 2
h o u r s a f t e r a d m i n i s t r a t i o n and t h e n d e c r e a s e d
with a b i o l o g i c a l h a l f - l i f e of 2 hours. Binding
of c e p h r a d i n e t o plasma p r o t e i n was f o u n d t o be
6% o v e r t h e r a n g e of c o n c e n t r a t i o n s found i n
plasma d u r i n g t h e a b s o r p t i v e a n d e x c r e t o r y phases.
A b s o r p t i o n , b a s e d on e x c r e t i o n o f r a d i o -
a c t i v i t y i n u r i n e o v e r t h e 24 h o u r p e r i o d
-
f o l l o w i n g a d m i n i s t r a t i o n , w a s 92
+ 6.6% + SE f o r
-
t h e f o u r s u b j e c t s . A n o t h e r 1%was e x c r e t e d i n
f e c e s w i t h i n t h e 72-hr p e r i o d o f t h e e x p e r i m e n t .
T h e r e w a s no s i g n i f i c a n t d i f f e r e n c e i n e x c r e t i o n
b a s e d on t h e r e c o v e r y o f r a d i o a c t i v i t y o r
antimicrobial a c t i v i t y i n urine. E x c r e t i o n was
66 f 6.9% f SE a t t h e e n d o f 6 h r and 8 5 f 6.5%

49
5 S E a t t h e end of 1 2 hours. Recovery of antimicrobial a c t i v i t y i n u r i n e
and t h e a r e a under t h e c u r v e f o r c o n c e n t r a t i o n o f a n t i b i o t i c i n plasma
were i n e x c e l l e n t agreement w i t h t h o s e found when l a b e l l e d c e p h r a d i n e w a s
administered o r a l l y .
Cephradine a p p e a r s t o be s l o w l y r e l e a s e d from t h e s i t e o f i n j e c t i o n
t o g i v e l e v e l s of a n t i b i o t i c i n plasma which, though r e a c h i n g a maximum
a t 1/3 t h o s e found a f t e r o r a l a d m i n i s t r a t i o n , p r o v i d e t h e same amount o f
b i o a v a i l a b l e a n t i b i o t i c o v e r a l o n g e r p e r i o d of t i m e 2 4 -
N o c a n i n e o r human drug m e t a b o l i t e s w e r e found so f a r e x c e p t t r a c e
amounts of c e p h a l e x i n , a s i d e n t i f i e d by m a s s spectrometry26.
The above i n f o r m a t i o n was p u b l i s h e d ( r e f e r e n c e s 27-31).

6. Methods o f A n a l y s i s
6 . 1 Elemental A n a l y s i s % C H N S
Calc. f o r anhydrous 55.01 5.48 12.03 9.16
Calc. f o r monohydrate 52.30 5.76 11.44 8.73
Found f o r c e p h r a d i n e
b a t c h NN057NC 51.96 5.83 11.16 9.00
Calc. f o r d i h y d r a t e 49.85 6.01 10.90 8.31
Found f o r c e p h r a d i n e
d i h y d r a t e b a t c h NNOO5NB 49.77 6.06 10.95 8.39
CEPHRADINE

6.2 M i c r o b i o l o g i c a l Assay
For b u l k and f o r m u l a t e d p r o d u c t s a
t u r b i d i m e t r i c method u s i n g S t r e p t o c o c c u s f a e c a l i s
A.T. C. C. 1 0 , 5 4 1 i s c o n v e n i e n t . A l t e r n a t i v e l y
a g a r p l a t e methods u s i n g S a r c i n a l u t e a A . T . C. C.
9341, B a c i l l u s pumilus A . T . C. C. 14,884 o r
S t a p h y l o c o c c u s a u r e u s , A.T.C. C. 6538P = FDA 209P,
a r e a l s o employed. F o r b l o o d and body f l u i d
samples an a g a r p l a t e method i s u s e d employing
S a r c i n a l u t e a a s t e s t o r anism b e c a u s e of i t s
g r e a t s e n s i t i v i t y3 2 , 4 6 , 4 7 ,
The minimum i n h i b i t o r y c o n c e n t r a t i o n
(MIC) of cephradine f o r t h e f o u r t e s t c u l t u r e s
i s a s follows:
mcq/ml
Sarcina lutea 0.04
Staphylococcus aureus 0.40
Bacillus pumilus 0.40
Streptococcus f a e c a l i s 50.0

Cephradine i s s l i g h t l y more b i o a c t i v e
a g a i n s t S t r e p t o c o c c u s f a e c a l i s and S a r c i n a l u t e a
than cephalexin, while the reverse hold t r u e f o r
Staphylococcus a ~ r e u s ~ ~ .
6.3 Iodometric Analysis
Cephradine can be d e t e r m i n e d by t h e
i o d o m e t r i c a s s a y . The B-lactam r i n g i s opened
w i t h a l k a l i o r c e p h a l o s p o r i n a s e f o l l o w e d by
i o d i n a t i o n a t an a c i d pH (pH 4 . 5 p h o s p h a t e
b u f f e r ) . About 4-5 moles o f i o d i n e a r e c o n s u m e d34 ,
I t i s i n t e r e s t i n g t o n o t e t h a t p e n i c i l l i n s under
t h e same c o n d i t i o n consume 8-9 moles o f i o d i n e .
The p r e c i s i o n of t h e i o d o m e t r i c a s s a y f o r
c e p h r a d i n e i s n o t a s good a s t h a t f o r p e n i c i l l i n s
when a l k a l i i s u s e d f o r i n a c t i v a t i o n . The
p r e c i s i o n can be c o n s i d e r a b l y improved by u s i n g
cephalos o r i n a s e i n s t e a d of a l k a l i f o r r i n g
opening 16. A l s o , w i t h t h e l a t t e r method much
b e t t e r agreement was o b t a i n e d w i t h t h e m i c r o b i o -

51
logical assay (see Table 10) even with severely degraded bulk samples.
It therefore can be considered stability indicating.

Table 10
Comparison of the cephradinase-iodometric, alkali-iodometric
and bioassay methods for the determination of cephradine in bulk powders
*
Cephradine potency = mcg/mg

Sample Bioassay Cephalosporinase- Alkali-iodometric


iodometric assay assay
NN054ND 812 813 979
NNO 5 9ND 854 043 991
NN061ND 778 846 980
NN054N.D 578 578 744
NNO59ND 424 466 643
NNO61ND 405 464 639
* Resultsare the means of determinations on two consecutive days. The
powders were stored at 5OoC two years in bottles with varying volumes
of head space.
CEPHRADINE

The p r e s e n c e of 7-ADCA d o e s n o t i n t e r -
.
f e r e w i t h t h e i o d o m e t r i c a s s a y 19
I t was found t h a t when i o d i n a t i o n was
c a r r i e d o u t a t an a l k a l i n e r a t h e r t h a n a c i d pH,
1 3 e q u i v a l e n t s o f i o d i n e were consumed 34 .
6.4 Spectrophotometric Analysis
The u l t r a v i o l e t a b s o r b a n c e peak of
c e p h r a d i n e a t 262 nm ( s e e s e c t i o n 2 . 4 ) can be
u s e d a s an i d e n t i f y a n d homogeneity a s s a y i n
formulations35.
Opening of t h e B-lactam r i n g w i t h
a l k a l i o r preferably, with cephalosporinase
a b o l i s h e s t h e U.V. a b s o r b a n c e a t 260nm. This
h a s been made t h e p r i n c i p a e o f a q u a n t i t a t i v e
a s s a y which a p p e a r s t o be s t a b i l i t y i n d i c a t i n g
i n t h e " p r a c t i c a l " r a n g e ( l e s s t h a n 20% loss o f
b i o a c t i v i t y ) 19.
6.5 Fluorometric Analysis
Cephradine can be a s s a y e d q u a n t i t a t i v e -
l y i n a l k a l i n e medium b y f l u o r i m e t r y , ( e x c i t a t i o n
wave l e n g t h 350 nm, e m i s s i o n wave l e n g t h 495 nm).
T h i s method h a s been used f o r b l o o d l e v e l s t u d i e s
b u t i s n o t recommended f o r t h e d e t e r m i n a t i o n i n
u r i n e because of e r r a t i c r e s u l t s .
6.6 Colorimetric Analysis
The w e l l known hydroxylamine method
f o r p e n i c i l l i n h a s been a d a p t e d b y FDA t o
cephradine a s a batching assay. It i s not
s t a b i l i t y indicating. Strongly alkaline reaction
c o n d i t i o n s and f e r r i c n i t r a t e w e r e used 5 . When
f e r r i c ammonium s u l f a t e was u s e d a t pH 7 o n l y
15% o f t h e r e s p o n s e normal f o r p e n i c i l l i n s was
o b t a i n e d 3 7.
-
A c o l o r i m e t r i c method u s i n g 5 , 5 '
d i t h i o b i s ( 2 - n i t r o b e n z o i c a c i d ) a t pH 9.2
p r o d u c e s a y e l l o w c o l o r which can be q u a n t i t a t e d
b y measuring t h e peak a b s o r b a n c e a t 412 nm38.

53
KLAUS FLOREY

6.7 Chromatoqraphic A n a l y s i s
6 . 7 1 Paper
Cephalexin (slower moving
component) can be s e p a r a t e d from c e p h r a d i n e w i t h
a n-butanol-t-amyl alcohol-~ater(7:l:4)system
and q u a n t i t a t e d b y b i o a u t o g r a p h y u s i n g
S t r e p t o c o c c u s a u r e u s 209P a s t h e a s s a y organisn??
Dihydrophenlyglycine(faster moving component)
can be s e p a r a t e d from c e p h r a d i n e w i t h a t e r t . -
amyl a l c o h o l - s e c . - b u t a n o l - w a t e r (4:4: 1) s y s t e m

.
and q u a n t i t a t e d w i t h a n i n h y d r i n - c o p p e r
complex 40
6.72 Thin-Layer
T h e r e a r e two TLC methods b y
which c e p h a l e x i n and o t h e r i m p u r i t i e s can be
s e p a r a t e d from c e p h r a d i n e a n d b o t h c e p h a l e x i n a n d
c e p h r a d i n e can be q u a n t i t a t e d . Both methods
40
g i v e comparable r e s u l t s . I n t h e f i r s t method ,
s i l i c a g e l p l a t e s a r e impregnated w i t h s i l i c o n e
f l u i d and d e v e l o p e d f o r 2-1/2 h o u r s i n a pH 4 . 1
M c I l v a i n e b u f f e r and a c e t o n e ( 1 0 0 : l . S ) . T h e z o n e s
a r e l o c a t e d w i t h U.V. l i g h t , e l u t e d a n d q u a n t i -
t a t e d s p e c t r o p h o t o m e t r i c a l l y a t 260 nm. The
s e p a r a t i o n scheme of t h e known components is a s
follows :
A t 22OC
compound Rf Cephradine
Dihydrocephradine 0.31 0.72
Cephradine 0.43 1.00
Cephalexin 0.51 1.20
7-ADCA 0.65 1.51
7 -ACA 0.65 1.51
Dihydrophenylglycine 0.74 1.72
Phenylglycine 0.80 1.88
To d e t e r m i n e r e s i d u a l 7-ADCA
q u a n t i t a t i v e l y , i t i s a d v a n t a g e o u s t o change t o
a s o l v e n t s y s t e m o f pH 6.5 M c I l v a i n e s b u f f e r -
acetone(50:l). The 7-ADCA zone i s e l u t e d w i t h
0.24 M sodium b i c a r b o n a t e a n d t h e a b s o r b a n c e a t

54
CEPHRADINE

260 nm i s measured41.
The s e c o n d method42, a m o d i f i c a -
t i o n of t h e f i r s t i s p r e f e r r e d because of g r e a t e r
e a s e of performance. The p l a t e s a r e i m p r e g n a t e d
w i t h t e t r a d e c a n e i n s t e a d of s i l i c o n e and s i n c e
t h i s i n t e r f e r e s w i t h t h e u. v. a b s o r b a n c e ,
q u a n t i t a t i o n is c a r r i e d o u t with ninhydrin.
T h i s method can a l s o b e u s e d t o
d e t e r m i n e d i h y d r o p h e n l y g l y c i n e and 7-ADCA semi-
q u a n t i t a t i v e l y . Approximate Rf v a l u e s a r e a s
follows:
C ep h r a d i n e 0.2
Cephalexin 0.3
7 -ADCA 0.6
Dihydrophenyl- 0.7
glycine
6 . 7 3 Column Chromatoqraphy
High p r e s s u r e l i q u i d chromoto-
g r a p h y (HPLC) h a s b e e n u s e d t o q u a n t i t a t e c e p h r a -
d i n e a n d r e s i d u a l c e p h a l e x i n i n c e p h r a d i n e . The
mobile p h a s e i s a pH 4 . 3 g l a c i a l a c e t i c -
a n h y d r o u s sodium s u l f a t e s y s t e m , a Dupont s t r o n g
c a t i o n exchange r e s i n , a t ambient t e m p e r a t u r e
and a p r e s s u r e o f 1000 p s i g . Sulfamethazine is
u s e d as i n t e r n a l s t a n d a r d and t h e o r d e r of elu-
t i o n i s s u l f a m e t h a z i n e , c e p h a l e x i n and
~ e p h r a d i n e ~ A~ .m o d i f i c a t i o n , u s e s a n a c e t a t e -
0.17M sodium s u l f a t e b u f f e r pH 4 . 7 , Dupont
Zipax c a t i o n exchange r e s i n , n- (4-methoxy-methyl-
6 - m e t h y l - 2 - p y r i m i d i n y l s u l f a n i l a m i d e as i n t e r n a l
s t a n d a r d and a p r e s s u r e o f 300 p s i g . The o r d e r
of e l u t i o n is cephalexin, cephradine, i n t e r n a l
s t a n d a r d . 7-ADCA, when p r e s e n t , w i l l a p p e a r i n
t h e v o i d volume44. When a d j u s t i n g t h e p H o f
t h e mobile p h a s e t o 3.70 a n d r a i s i n g t h e
p r e s s u r e t o 1800 p s i g , t h e s y s t e m was a b l e t o
s e p a r a t e c e p h r a d i n e ( d - i s o m e r ) from t h e s y n t h e -
t i c a l l y p r e p a r e d l-isomer which p e a k s between
c e p h a l e x i n and c e p h r a d i n e . N o l-isomer was

55
KLAUS FLOREY

d e t e c t e d i n r e g u l a r c e p h r a d i n e samples4’. A
r e v e r s e phase system, u s e f u l f o r q u a n t i t a t i o n i n
formulation h a s a l s o been d e s c r i b e d 4 9 . A
1 mm x 2.1 mm i . d . column, ODS-Sil-X-I1 packing
and 7 % methanol, 93% 0.05M ammonium c a r b o n a t e a s
mobile phase were used. P r e s s u r e , 1 0 0 0 p i g ;
f l o w 0 . 6 ml/min; d e t e c t o r W (254 nm);
s e n s i t i v i t y , 0.08 AUFS.
7. Determination i n Body F l u i d s and T i s s u e s
Cephradine h a s been determined microbio-
l o g i c a l l y (see s e c t i o n 6.2) i n human serum, i n
human u r i n e , human lung t i s s u e , human eye t i s s u e
and i n s p i n a l f l u i d .
I t has been determined f l u o r o m e t r i c a l l y
(see s e c t i o n 6.5) i n dog serum.
8. Determination i n Pharmaceutical P r e p a r a t i o n
I n pharmaceutical p r e p a r a t i o n s ( c a p s u l e s ,
o r a l suspensions and i n j e c t a b l e s ) i n f r a r e d has
been used f o r i d e n t i t y t e s t s , t h e hydroxylamine
and iodometric a s s a y f o r b a t c h i n g , t h e micro-
b i o l o g i c a l and cephalosporinase-iodometric a s s a y s
f o r s t a b i l i t y and chromatography f o r d e t e c t i o n of
impurities.

56
CEPHRADI N E

9. References

1. B. T o e p l i t z , The S q u i b b I n s t i t u t e , P e r s o n a l
communi c a t i o n .
2. M. S. P u a r , The S q u i b b I n s t i t u t e , P e r s o n a l
communication.
3. P. T. Funke, The S q u i b b I n s t i t u t e , P e r s o n a l
communication.
4. J. Dunham, The S q u i b b I n s t i t u t e , P e r s o n a l
communication.
5. F. M. R u s s o - A l e s i , The S q u i b b I n s t i t u t e ,
P e r s o n a l communication.
6. H. J a c o b s o n , The S q u i b b I n s t i t u t e , P e r s o n a l
communication.
7. L. P. M a r e l l i , A n a l y t i c a l P r o f i l e s o f Drug
S u b s t a n c e s , Vol. 4 , p 21, A c a d e m i c Press,
1974.
8. M. S. A t w a l , The S q u i b b I n s t i t u t e , P e r s o n a l
communication.
9. H. J a c o b s o n and I. G i b b s , J. Pharm. S c i . 62,
1543 ( 1 9 7 3 ) .
10. D. E. A u s l a n d e r , The S q u i b b I n s t i t u t e ,
P e r s o n a l communication,
11. F. D u r s c h , The S q u i b b I n s t i t u t e , U . S .
P a t e n t 3,829,620, June 25, 1 9 7 4 .
12. F. D u r s c h , The S q u i b b I n s t i t u t e , P e r s o n a l
communication.
13. Q. Ochs, T h e S q u i b b I n s t i t u t e , P e r s o n a l
communication.
14. J. E. D o l f i n i , H. E. A p p l e g a t e , G. Bach,
H. Basch, J. B e r n s t e i n , J. S c h w a r t z a n d
F. L. Weisenborn, J. Med. Chem. 1 4 , 117
(1971) : U . S . P a t e n t 3 , 4 8 5 , 8 1 9 ( 1 9 6 9 ) .
15. M. L. Snow, C. L a u i n g e r and C. R e s s l e r ,
J. Org. Chem. 3 3 , 1 7 7 4 ( 1 9 6 8 ) .
16. L. Demain, N a t u r e 2 1 0 , 4 2 6 ( 1 9 6 6 ) .
17. H. R. Roberts, The S q u i b b I n s t i t u t e ,
P e r s o n a l communication.

57
KLAUS FLOREY

18. A. I. Cohen, P. T. Funke and M. S. Puar,


J. Pharm. S c i , 6 2 , 1559 ( 1 9 7 3 ) .
19. B. M. F r a n t z , The Squibb I n s t i t u t e , P e r s o n a l
communication; J. Pharm. S c i . , i n p r e s s ,
20. R. V a l e n t i and H. J a c o b s o n , The Squibb
I n s t i t u t e , P e r s o n a l communication.
21. M. A. L e i t z , The S q u i b b I n s t i t u t e , P e r s o n a l
commun i c a ti o n ,
22. K. J. K r i p a l a n i and A. V. Dean, The S q u i b b
I n s t i t u t e , P e r s o n a l communication.
23. D. Adam, Munch. Med. WSchr. 116, 1 9 4 5 ( 1 9 7 4 ) .
24. I. Weliky and R. Vukovich, The S q u i b b
I n s t i t u t e , P e r s o n a l communication.
25. A. V a h i d i , R. Vukovich, E. S. N e i s s and
E. S c h r e i b e r , The S q u i b b I n s t i t u t e , P e r s o n a l
communication.
26. P. T. Funke, The Squibb I n s t i t u t e , P e r s o n a l
communication.
27. Weliky, I . , and Z a k i , A . , S e l e c t e d
P r o c e e d i n g s from t h e 8 t h I n t e r n a t i o n a l
Congress o f Chemotherapy, S e p t . 8-14,1973,
A t h e n s , Greece, pp. 1-5.
28. Vukovich, R . , M a r t i n e z , M . , and N e i s s , E . S . ,
S e l e c t e d P r o c e e d i n g s from t h e 8 t h I n t e r n a -
t i o n a l Congress o f Chemotherapy,
S e p t . 8-14, 1973, A t h e n s , Greece, pp. 30-34.
29. Z a k i , A. , S c h r e i b e r , E . C . , Weliky, I . ,
K n i l l , J . R . , and Hubsher, J . A . , J. C l i n .
Pharmacol, New Drugs 14: 118-126 ( 1 9 7 4 ) .
30. I. Weliky, H. H. Gadebusch, K. K r i p i l a n i ,
P. Arnow and E. C. S c h r e i b e r , A n t i m i c r o b i a l
Agents and Chemotherapy 2, 4 9 ( 1 9 7 4 ) .
31. G. R e n z i n i , G. Ravagnan, B. O l i v a ,
E. S a l v e t t i , and R. A u r i t i , Q u a d e r n i di
A n t i b i o t i c a ; 1972, 17.
32. T. B. P l a t t , The S q u i b b I n s t i t u t e , P e r s o n a l
communication.
33. S. Wind, The Squibb I n s t i t u t e , P e r s o n a l
c o m u n i c at i o n .

58
CEPHRADINE

34. J. Alicino, The Squibb Institute, Personal


communication.
35. H. Lerner and S. Willis, The Squibb
Institute, Personal communication.
36. A. F. Heald, C. E. Ita and E. Solney, The
Squibb Institute, Personal communication.
37. C. Sherman, The Squibb Institute, Personal
communication.
38. J. Kirschbaum, J. Pharm. Sci., 63 923
(1974).
39. A. Vahidi and H. R. Roberts, The Squibb
Institute, Personal communication.
40. H. R. Roberts, The Squibb Institute,
Personal communication.
41. F. P. Targos, The Squibb Institute,Personal
communication.
42. I. R. Salmon, The Squibb Institute,Personal
communica tion.
43. A. Peterson and D. Guttman, Smith Kline &
French Laboratories, Personal communication.
44. J. Kirschbaum, The Squibb Institute,
Personal communication.
45. H. H. Pu and R. B. Poet, The Squibb
Institute, Personal communication.
46. D. M. Isaacson, The Squibb Institute,
Personal communication.
47. J. R. Ster, H. Weisblatt and J. D. Levin,
The Squibb Institute, Personal
communication.
48. A. N. Niedermayer, The Squibb Institute,
Persona1 communication.
49. E. R. White, M. A . Carroll, J.E. Zarembo
and A. D. Bender, J. Antibiotics
-
28, 205 (1975).
50. J. Z. Gougoutas and B. K. Toeplitz, Univ.of
Minnesota and Squibb Institute, Personal
communication.
51. J. V. Uri, P. Actor, L. Phillips and
J. A. Weisbach, Experientia &54(1975).

59
CHMROQUINE PHOSPHATE

Donald D,Hong
DONALD D. HONG

CONTENTS

Analytical Profile - Chloroquine Phosphate

1. Description
1.1 Name, Formula, Molecular Weight
1.2 Appearance, Color, Odor

2. Physical Properties
2.1 Ultraviolet
- Spectrum
2.2 rescence Spectrum
2.3 NA ear Magnetic Resonance Spectrum
2.4 Ma&% Spectrum
2.5 ~ p cal
t i Rotation
2.6 morphism and Melting Range
2.7 Dissociation Constant
2.8 DH
2.9 seezing Point Depression
2.10 Solubility
2.11 Differential Scanning Calorimetry

3. Synthesis

4. Drug Metabolic Products


4.1 Biotransformation Products
4.2 Distribution in Human Tissues
5. Methods of Analysis
5.1 Phase Solubility Analysis
5.2 Identification by Spot Tests
5.3 Non-Aqueous Titration
5.4 Spectrophotometric Analysis
5.5 Fluorometric Analysis
5.6 Gravimetric Analysis
5.7 Chromatographic Analysis
5.71 Paper
5.72 Thin-Layer
5.73 Gas
5.8 Miscellaneous Methods
6. Referencea

62
CHLOROQUINE PHOSPHATE

1. Description

1.1 Name, Formula, Molecular Weight

The chemical name of chloroquine phosphate i n


Chemical Abstracts is found under t h e heading Quinoline and
designated as 7-Chloro- [4- (4-diethylamino-1-methylbutyl-
amino) Iquinoline diphosphate. The hydrochloride and s u l f a t e
e a l t e are a l s o a v a i l a b l e .

2H2PO4-

C@&CN3 2H3Po4 Molecular Weight: 515.87


1 . 2 Appearance, Color, Odor

Chloroquine phosphate i s a w h i t e , o d o r l e s s ,
c r y s t a l l i n e powder having a b i t t e r taste: it d i s c o l o r e
gradually on exposure t o l i g h t .

2. Physical P r o p e r t i e s

2 . 1 U l t r a v i o l e t Spectrum

A 10 y/ml s o l u t i o n of chloroquine phosphate i n


0.01 N H C 1 when scanned between 360 and 210 nm e x h i b i t s
t h r e e maxima, three minima and s e v e r a l shoulders i n t h e
region from 270 t o 225 nm, as shown i n Figure 1. The maxima
are located a t 343 nm ( a = 36.1), 328 nm ( a = 32.6) and
222 nm ( a = 59.9). The r a t i o of /%% i s 1.11. Minima
were observed a t 335 nm, 280 nm a342h3 nm.

2.2 Fluorescence Spectrum

Figure 2 shows t h e fluorescence spectrum of


chloroquine phosphate obtained on a s o l u t i o n of 0.2 mg/ml
pH 7.9 phosphate b u f f e r using an Aminco-Bowman spectro-
photofluorometer. Excitation a t e i t h e r 320 nm o r 370 nm
produced emission s p e c t r a with a maximum a t 400 nm, t h e

63
- si
..- . . . . . . . . .
...~. . . . . . . . .
........
... . . . . .
-
... . . . . . . . .
... ......
... . . .
' . 1.! .
-
. . .
. . .. ,.
I
... j
. . .... I
- 1
,
i (
+
. . I
"I
i
, . .
!.
3- I
i
i
I
!
,
64
CHLOR OQUlNE PHOSPHATE

Fluorescence

Figure2. F luorescence Spectrum of Chloroquine Phosphate


i n pH 7.9 Phosphote Buffer (Sterling-Winthrop
House Reference Standard Lot N-087- JF)
70

WAVE L ENGTH-MILL IMICRONS

65
DONALD D. HONG

l a t t e r e x c i t a t i o n wavelength providing a higher emission


response.

2.3 Nuclear Magnetic Resonance Spectrum (NMRl


The spectrum i n Figure 3 was obtained w i t h a
Varian A60 NMR Spectrometer using a 20$ s o l u t i o n i n D20
containing TMS as an e x t e r n a l standard. The s p e c t r a l
assignments are summarized below (1).

c1
No. Protons
Derived from a
Protons Integration Chemical Shi ; Multiplic

CH3 -CH2 6 1-68-1-80 doublet


3 1.90-1.9
Cg-
CH -CH

CH2-N
CH2 4
6
2.35
3 -55-3 a 9 1
doublet
broad s i n g l e t
quintet
CH-N 1 4.38-4.65 broad s i n g l e t
NH exchanged 5 -39 sharp s i n g l e t

c 1 -
7.15-7 28 doublet
b
a
1 -
7 58-7 -75 multiplet
singlet
1 7.75
1 8-33-8.40 doublet
C
d 1 -
8 48-8-55 doublet

2.4 Mass Spectrum

The mass spectrum is shown i n Figure 4 and was


obtained using a J o e l JMS-01SC mass spectrometer with an
i o n i z i n g energy of 75 eV. The h i g h e s t mass observed a t
m/e 319 i s a thermal breakdown product where two phosphoric
acid moieties were l o s t from t h e parent compound. The base
peak a t m/e 86 i s due t o t h e N,N,N-diethYlmethylene fragment
(1).

66
A/-+-

L
I . . . . , . . . .I . , . I I 1 I I I I I . , . . I . .
, . I . ' . . I . , . I , ' ' I I 1 I I I I
U 71 00 (0 Y I 4 I 40 an 10 $8 8

Figure 3. NMR Spectrum of Chloroquine Phosphate. Instrument: Varian A60


DONALD D. HONG

Intensity

100
>
t 80
v) 319
z
W
F
5 60
W
L
+ 40
a BACKGROUND
A
W
a 20

0 . .
20 40 60 ' 80 100 I20 140160 I80 250 300
m/e
Fig.4 Mass Spectrum of Chloroquine Phosphate (Sterling-Winthrop
House Reference Standard Lot N-087-JF)

60
CHLOROQU IN E PHOSPHATE

2.5 Optical Rotation

Chloroquine phosphate e x h i b i t s e s s e n t i a l l y no
o p t i c a l a c t i v i t y , e x i s t i n g as a racemic mixture. Riegel
and Sherwood ( 2 ) have shown t h a t n e i t h e r of t h e o p t i c a l l y
a c t i v e enantiomorphs showed any s i g n i f i c a n t d i f f e r e n c e s i n
a n t i m a l a r i a l a c t i v i t y i n birds and f o r t o x i c i t y i n dogs.

2.6 Polymorphism and Melting Range

Chloroquine phosphate e x i s t s i n two polymorphic


forms giving rise t o two melting ranges. USP xn ( 3 ) r e p o r t s
one melting between 193" and 195" and t h e o t h e r between 210"
and 215". Mixtures of t h e forms melt between 193" and 215".
It i s possible t o obtain one form s e l e c t i v e l y by r e g u l a t i n g
t h e rate of c r y s t a l l i z a t i o n ( 4 ) .

2.7 Dissociation Constant

The pKa's f o r chloroquine phosphate by t h e


t i t r i m e t r i c method were found t o be 8.10 and 9.94 ( 5 ) .

2.8 @

A 18 aqueous s o l u t i o n has a pH of about 4.2.

2.9 Freezing Point Depression

Cryoscopic measurements were made on lo$ and 2o$


(w/v) solutions of t h e drug.

Chloroquine phosphate **
Freezing Point Depression

0.73 "
Calculated

Isotonic
7.oe

69
DONALD 0.HONG

The value f o r "calculated i s o t o n i c " s o l u t i o n was


obtained by graphic i n t e r p o l a t i o n t o FPD of 0.550",
representing 0 . d sodium chloride s o l u t i o n ( 5 ) .

2.10 S o l u b i l i t y

Chloroquine phosphate i s f r e e l y soluble i n water:


p r a c t i c a l l y insoluble i n alcohol, i n chloroform and i n
ether (3).

2.11 D i f f e r e n t i a l ScanninR Calorimetry (DSC)

Two polymorphic forms of chloroquine phosphate


a r e exhibited by DSC. A mixture of t h e two c r y s t a l forms
may be demonstrated a l s o by t h e t r a n s i t i o n temperatures ( 6 ) .
The DSC thermogram of a chloroquine phosphate s t a n d a r d shown
i n Figure 5 was obtained on a Perkin-Elmer DSC-lB d i f f e r e n -
t i a l scanning calorimeter a t a heating rate of 10°C per
minute under nitrogen. T h i s i s an example of t h e low melting
form. Figure 6 shows another sample of chloroquine phosphate
containing a mixture of t h e low and high melting forms.

Both t h e low and high melting polymorphs may be


obtained from t h e same aqueous s o l u t i o n of chloroquine
phosphate by s e l e c t i v e c r y s t a l l i z a t i o n . The high melting
form usually occurs as small c r y s t a l s while t h e low melting
polymorph c r y s t a l l i z e s as s i g n i f i c a n t l y l a r g e r c r y s t a l s .
The two forms e x h i b i t s l i g h t d i f f e r e n c e s i n t h e i r I R curves
from a KBr matrix.

3. Synthesis

E l d e r f i e l d (7) and Kenyon i s collaboration w i t h


Wiesner and K w a r t l e r (8) and more r e c e n t l y Basu, e t a1 ( 9 )
have summarized t h e American e f f o r t t o develop an a n t i -
malarial drug necessitated by World W a r 11. T h i s need was
compounded when Japan seized c o n t r o l of t h e East Indies,
e f f e c t i v e l y c u t t i n g o f f t h e n a t u r a l sources of quinine,
which was t h e drug of choice f o r malaria a t t h e time.
Chloroquine was one of t h e f r u i t s of t h i s concerted e f f o r t .

The synthesis of chloroquine was f i r s t reported


by t h e German chemists Andersag, Breitner and Jung (10, 11).
Since t h e p a t e n t l i t e r a t u r e lacked t h e d e t a i l information
required t o prepare t h e necessary intermediates, a research
program w a s s t a r t e d a t Winthrop Chemical Company r e s u l t i n g
i n a method o f synthesis for chloroquine by Surrey and

70
CHLOROQUINE PHOSPHATE

Figure 5. OSC Thermogram of Chloroquine Phosphate


(Sterling-Winthrop House Reference Standard
Lot N - 0 8 7 - J F ) Low Melting Form
189-202.5-207.5 OC (Corr.)
Ic -4

0 0 0
0
N
t
0
m
t
*t
0
0
0
(D
t
r-
*
0
OD
t
0
Q,
t
1 I I I I I I I
TEMPERATURE O K

71
DONALD D. HONG

Figure 6 DSC Thermogram of Chloroquine Phosphate


(Lot An-K-67) Mixture of Low and High
Melting Forms

1- 189-202-1
-207.5 OC (Corrl

215-223-
2 2 6 O C (Corr)

ENDO
t
1
EX0
0 0 0 0 0
m a 0
e
1
*
O

I
P c
Q
I
00
d
I
*
Q,

I
In
J
TEMPERATURE O K

72
CH LOROQUINE PHOSPHATE

Kammer (12).

The two key intermediates required i n t h e


synthes is a r e 4 7-d i ch l o r oquinoline and 4-d i e t h y lami no 1 --
methylbutylamine ( “novol diamine”). The s y n t h e t i c scheme
i s shown i n Figure 7.

4. Drug Metabolic Products

4.1 Biotransformation Products

Several metabolites in a d d i t i o n t o t h e unchanged


drug have been i s o l a t e d from human t i s s u e s and urine. Titus,
--
e t a1 (13) used counter-current d i s t r i b u t i o n t o i s o l a t e t h e
d e s e t h y l compound ( A ) from t h e u r i n e of human volunteers.
Kuroda (14) i s o l a t e d four metabolites of chloroquine from
human t i s s u e s using a combination of paper chromatography
and W spectroscopy. I n a d d i t i o n t o t h e desethyl compound
( A ) of T i t u s , they were t h e b i s d e s e t h y l (B), t h e c a r b i n o l
(C) and t h e ~-amino-7-chloroquinoline( D ) d e r i v a t i v e s .
Similar observations were seen from u r i n e of
human s u b j e c t s b u t no t r a c e of t h e 4-hydroxy-7-chloro-
quinoline w a s found. The unchanged drug was always found
t o be t h e major compound. McChesney, e t a1 (15, 16) using
a fluorescence technique confirmed t h e above findings and
i n a d d i t i o n found t r a c e s of t h e hl-aldehyde (E) and t h e
b’-carboxy (F) d e r i v a t i v e s . They l i s t e d t h e amount of
determinable excretory products as TO$ chloroquine, 23s
desethylchloroquine, 1-2$ bisdesethylchloroquine and t h e
o t h e r s as t r a c e degradation products. I n a d d i t i o n they
reported t h a t about one-third of t h e administered
chloroquine was unaccounted and remains obscure i n t h i s
very complex blotransformation of t h e drug.

73
DONALD D. HONG

Figure 7: Synthesis of Chloroquine Phosphate

-
m-Chloroaniline Ethyl oxalacetate Ethyl(m-chloropheny1imino)-
succinate

and isomer

(1) NaOH
heat

(2) HC1
kseparation

OH
I

c1 COOH co0c.$l5

7-Chloro-4-hydroxy- Ethyl-7-chloro-4-hydroxy-
quinoline-2-carboxylic quinoline-2-carboxylate
acid

a>
-COOH
1
CH3CHCH2CH2CH9 (CpHg )2

-. I
!
H
2
Cl c1 N “novol diamine” .1,
7-Chloro - 4- 4,7-Dichloroquinoline
hydroxyquinoline

Chloroquine
diphosphate -<H3P04
CL
Chloroquine.base

74
CHLOROQUIN E PHOSPHATE

Compound R

Chloroquine -CHCH$H2CH$I (C2H5 )2


I
CH3
A -CHCH2CH$H$IHC2Hrj
I
CH3
B -CHCH$H$H$H2
I
CH3
C -CHCH2CH2CH20H

D
4
H

E
-FHO
CH3
F
-rHC82CH2CwH
CH3
4.2 D i s t r i b u t i o n in Human T i s s u e s

Chloroquine i s predominantly l o c a l i z e d i n t h e
l i v e r and t o lesser d e g r e e s i n t h e s p l e e n , h e a r t , kidney,
lung, b r a i n , l e u c o c y t e s and s k i n . Chloroquine was o r i g i n a l l y
used t o treat malaria. Subsequently it was found t o be
e f f e c t i v e a g a i n s t o t h e r p a r a s i t i c d i s o r d e r s . According t o
Rubin, e t a1 (17) t r a c e s o f t h e d r u g and its m e t a b o l i t e s
were found i n t h e blood and u r i n e of s u b j e c t s up t o f i v e
y e a r s after d i s c o n t i n u i n g long-term t h e r a p y .

75
DONALD D. HONG

5. Methods of Analysis

5 . 1 Phase S o l u b i l i t y Analysis (PSA)


PSA i s probably n o t promising f o r c h l o r o q u i n e
phosphate because t h e m u l t i p l e pKa's would allow s u b s t a n -
t i a l d i s p r o p o r t i o n a t i o n (18).

5.2 I d e n t i f i c a t i o n of Chloroquine Phosphate by


Spot Tests

I n t h e approximate period of two decades f o l l o w i n g


World War I1 where t h e r e was i n c r e a s i n g use of c h l o r o q u i n e
phosphate f o r mass p r o p h y l a x i s of malaria it became n e c e s s a r y
t o have a r e l a t i v e l y s i m p l e method t o i n s u r e t h a t t h e d r u g
was t a k e n r e g u l a r l y . T e s t s of t h i s t y p e u s u a l l y measure
t h e d r u g i n u r i n e . Other tests u t i l i z e v a r i o u s r e a g e n t s t o
r e a c t e i t h e r w i t h t h e pure d r u g o r t h e d r u g i n dosage form.
Some of t h e s e t e s t s are summarized i n Table 1.

5.3 Non-Aqueous T i t r a t i o n
Chloroquine phosphate can be t i t r a t e d w i t h
a c e t o u s 0.1 N p e r c h l o r i c a c i d . The t i t r a t i o n may be c a r r i e d
o u t manually w i t h c r y s t a l v i o l e t as i n d i c a t o r o r determined
p o t e n t l o m e t r i c a l l y . The t i t r a t i o n is r a p i d a l t h o u g h
n o n - s e l e c t i v e . T h i s n o n - s p e c i f i c i t y i s no drawback,
however, so long as good i d e n t i f i c a t i o n tests are a l s o
adopted ( 3 0 ) . Wu, e t a1 (31) have r e p o r t e d t h e determin-
a t i o n of e l e v e n a n t i m a l a r i a l d r u g s u s i n g t h i s t i t r i m e t r i c
procedure.

Each ml of 0.1 N HClO4 i s e q u i v a l e n t t o 25.79 mg


of chloroquine phosphate.

5.4 S p e c t r o p h o t o m e t r i c Assay

The W s p e c t r a of c h l o r o q u i n e base and phosphate


s a l t are s i m i l a r i n 0.01 N HC1. Absorption maxima a r e
observed a t 343, 328, 256 and 222 nm. Measurements are
most f a v o r a b l y made a t 343 nm where a b s o r p t i o n i s most
i n t e n s e and least a f f e c t e d by i n t e r f e r i n g s u b s t a n c e s i n t h e
b i o l o g i c a l sample.

The c h l o r o q u i n e b a s e is o b t a i n e d by e t h e r o r
chloroform e x t r a c t i o n of an a l k a l i n e homogenate of t h e
b i o l o g i c a l sample. After s e p a r a t i o n of i n t e r f e r i n g

76
CH LOROQUIN E PHOSPHATE

Table 1 - Spot T e s t s

Test Form Color Sensitivity Ref.


1. Complex w i t h Copper Tablet P a l e green NA 19
2. Complex w i t h Cobalt Tablet Violet NA 1.9
3 . Dimethylaminobenz- Tablet Yellow NA 20
aldehyde ( E h r l i c h ' s
reagent )

4. Styphnic a c i d Pure drug Rosettes 0.5~ 21


of p l a t e s

5. N i t r o p r u s s i d e and Pure drug NA SOY 22


P i p e r a z i n e (Lewin's
r e a g e n t )*

6. Eosin yellowish Urine Yellow t o NA 23


( D i l l & Glazko) v i o l e t -red

7. Mercuric iodide/KI Urine ** 2.5-9- 5~ 24


( W e r -Tanre t ' s per rnl urine
reagent )

8. Methyl orange Urine Yellow 2 mg/liter 25

9. Complex w i t h Pure drug Rosettes 0.4~ 26


HClO 4/AuClj o r b i o l . and d e n d r i t e s
extract

10. Aconitic a c i d / Pure drug Red 57 27


a c e t i c anhydride/
e t h y l e n e di c h l o ri de

11. H2SO4/KClOj Biol. Red - v i o l e t 5Y 20


extract

12. H C ~ / K C ~ O ~ Biol. Yellow 10Y 28


extract

15. 25$ H2SOl+/Chlorinated Biol. Yellow 10Y 28


line extract

1 4 . BPB/boric a c i d Free b a s e Blue-violet 0.8 mg$ 29


to b l u e -green

* S p e c i f i c f o r N-ethyl group
** T u r b i d i t y i s measured

77
DONALD D. HONG

materials, t h e base i s i n t u r n extracted i n t o a s o l u t i o n of


0.1 or 0.01 N H C 1 and q u a n t i t a t i v e l y determined by measuring
i t s W absorption. A l t e r n a t i v e l y t h e acid s o l u t i o n can be
made a l k a l i n e and t h e base extracted w i t h e t h e r o r chloro-
form. The organic phase i s evaporated t o dryness and t h e
residue further examined by I R and paper o r TLC.

A b r i e f summary of t h e spectrophotometric
procedures f o r t h e q u a n t i t a t i v e determination of chloroquine
and metabolites i s summarized i n Table 2.

Table 2 - Spectrophotometric Methods

Method of Analysis I s o l a t i o n from Human Reference

uv Tissues 32
uv Blood 33
UV, I R , TLC, GLC Tissues 34
uv, I R , PC Tissues+ 14
uv Urine 35
* Includes i d e n t i f i c a t i o n of chloroquine metabolites a l s o

5.5 Fluorometric Analysis

Fluorometric procedures have been extensively


u t i l i z e d f o r t h e q u a n t i t a t i v e determination of chloroquine
i n b i o l o g i c a l materials. I n t h e e a r l y days of fluorescence
when instrumentation was n o t s u f f i c i e n t l y advanced, an
a d d i t i o n a l i r r a d i a t i o n s t e p was required t o convert t h e
chloroquine t o a more i n t e n s e fluorophore ( 3 6 ) . With t h e
advent during t h e mid 1950's of t h e highly s e n s i t i v e
spectrophotofluorometers u t i l i z i n g t h e xenon a r c source
and monochromators, it i s now possible t o measure t h e
chloroquine fluorescence d i r e c t l y (15, 16, 37, 3 8 ) .

Brodie, e t a1 (36) found t h a t t h e fluorescence


of chloroquine i n pH 9.5 b o r a t e b u f f e r after i s o l a t i o n
from b i o l o g i c a l specimen may be s t a b i l i z e d by t h e a d d i t i o n
of cysteine. The sample i s then irradiated with UV l i g h t
and t h e measurement made using a s u i t a b l e fluorometer. The
s e n s i t i v i t y of t h e procedure i s about 0.lmcg.

5.6 Gravimetric Analysis


Parikh and Mukherji ( 3 9 ) reported t h e q u a n t i t a -
t i v e formation of chloroquine-silicatungatate CSi02-

78
CH LOROQUI N E PHOSPHATE

1803 -2(C18H2@3Cl). 2H20] p r e c i p i t a t e from chloroquine


phosphate and s i l i c a t u n g s t i c a c i d . By use o f t h e appro-
p r i a t e g r a v i m e t r i c f a c t o r t h e v a r i o u s s a l t s o f chloroquine
could be determined. USP XVI (10) a l s o contained a
g r a v i m e t r i c method whereby t h e chloroquine base is measured.

5.7 Chromatographic Analysis

5.71 Paper Chromatosaphic Analysis


A number o f paper chromatographic systems for
chloroquine phosphate and i t s base are summarized i n Table 3.
Goldbaum and Kazyak ( 4 1 ) use i o d o p l a t i n a t e r e a g e n t t o
v i s u a l i z e t h e chloroquine which i n t u r n may be e l u t e d o f f
t h e paper and t h e s p o t q u a n t i t a t e d by o t h e r means.

5.72 Thin-Layer Chromatographic Analysis

The f o l l o w i n g TU: systems (Table 4 ) are u s e f u l


as an i d e n t i t y t e s t and i n t h e e v a l u a t i o n of t h e p u r i t y o f
t h e drug substance. The n a t u r e of t h e i m p u r i t i e s p r e s e n t
is a l s o h e l p f u l i n t h a t it t e l l s i n d i r e c t l y , f o r example,
s i m i l a r i t y o r d i e s i m i l a r i t y o f t h e manufacturing process.

A l l of t h e systems u t i l i z e precoatcd s i l i c a g e l
containing a fluorescent indicator.

5.73 Gas-Liquid Chromatographic Analysis (GLC)


The f o l l o w i n g procedures have been demonstrated
t o be a p p l i c a b l e t o t h e GLC examination of chloroquine base.
Vanden Heuvel, - et - a1 ( 4 6 ) Viala, e t a1 (47) and Kazyak and
Knoblock (48) have r e p o r t e d t h e d e t e c t i o n of t h e drug i n
microgram amounts from b i o l o g i c a l samples after s o l v e n t
e x t r a c t i o n . Holtzman (49) has shown t h a t a8 l i t t l e as 5
nanogram q u a n t i t i e s of chloroquine base could be d e t e c t e d
u s i n g e l e c t r o n c a p t u r e . The c o n d i t i o n s r e p o r t e d , however,
are f o r pure drug and m a y be o f p o t e n t i a l value i n t h e
a n a l y s i s o f t h e substance i n b i o l o g i c a l m a t e r i a l e .

The following c o n d i t i o n s have been used f o r t h e


CIA: d e t e r m i n a t i o n of chloroquine base (50).

Column: 3.8s S i l i c o n e Gum SE 30, 4 f t , g l a s e


Support : D i a t o p o r t S
Detection: FID

79
Table 3 - Paper Chromatographic Systems

S o l v e n t System Species Paper Detection -


Rf Reference

1. n-Butanol s a t ' d base Whatman No. 2 s a t ' d w i t h uv 0.15 41


w i t h buffer pH 3 .O M a c I l v a i n e ' s buffer

2. n-Butanol sat 'd base Whatman No. 2 sat 'd w i t h uv 0.16 41


w i t h buffer pH 5 .O MacIlvaine ' s buffer

3. n-Butanol s a t ' d base Whatman No. 2 sat 'd w i t h W 0.26 41


w i t h buffer pH 6.5 Sorensen's b u f f e r
4. n-Butanol sat 'd base Whatman No. 2 sat 'd w i t h uv 0.89 41
W
0 w i t h buffer pH 7.5 S o r e n s e n ' s buffer

5. Ethanol-Water-Conc base Whatman N o . 1 s a t ' d w i t h w 0.36 42


ammonia (35-63-2) petroleum (195-220' f r a c t i o n )

6. AS NO. 5 (45-53-2) base as above w 0.60 42

7 - AS NO. 5 (55-43-2) base as above w 0.79 42

8. AS NO. 5 (65-33-2) base as above uv 0.87 42

9. AS NO. 5 (75-23-2) base as above W 0.88 42

10. As No. 5 (85-13-2) base as above W 0.89 42

11. As No. 5 (95-3-2) base as above uv 0.89 42


CHLOROQUINE PHOSPHATE

Table 4 - TLC Systems


System S p o t t i n g Soln Rf x 100 Reference
1
Methanolwater-conc Chloroform 2% 43
a m o n i a (72:25:3)

Benzene-methanol- Chloroform' 41 43
ieopropylamine (87:10:3)

Chlorof orm-aethanol- MeOH-H 0 40 43


isopropylamine (94:3 :3) (737
Chloroform-isopropylamine Chloroform2 15 43
(97:3 1
-
E t h e r -hexane i sopropyl- H20/MeOH/CHC13 / 28 43
m i n e (90:~:~) i sopropylaaine

n-Butanol-conc mmonia- Water 59 43


water (85:k:ll)
Ethylacetate-conc ammonia- Water 60 44
e b s . a l c o h o l (5:2:2)

25$ ammonia-benzene-dioxane- Water 28 44


e t h a n o l (1:10:8 :1)

Chloroform-cyclohexane- Water 40 44
diethylamine ( 5 :4:1)

25% ammonia-methanol Water 20 44


(3:200)

Acetone -water -ammonia abs. a l c o h o l S 15 45


(90 :40 :1 )

Spotting solution
1. The drug i s e x t r a c t e d i n t o chloroform a f t e r b a s i f y i n g w i t h 10% Na2C03.
2. S i m i l a r t o above b u t from human plasma.
3 . Spotted as t h e b a s e .

Detection
A - W-254
B - I o d i n e vapor/20$ H2S04
C - Dragendorff's r e a g e n t
D - w-360
E - Iodoplatinate reagent

81
DONALD D. HONG

Temperature :
.
I n j port 275 "C
Column 240 O C
Detector 250 "C
Flaw Rate: 30 ml/min helium
Retention Time: 7.0 min

5.8 Miscellaneous Methods of Analysis

Roushdi and Shafik (51) reported t h e determina-


t i o n of chloroquine phosphate by t i t r a t i o n with an anionic
s u r f a c t a n t , d i o c t y l sodium sulfosuccinate (Aerosol O.T. ),
using dlmethyl yellow as i n d i c a t o r .

A complexometric method using bismuth complexonate


t o p r e c i p i t a t e t h e chloroquine base and t i t r a t i n g t h e
liberated EWIA w i t h zinc s u l f a t e was also reported by t h e s e
authors (52).

Various quinine salts and chloroquine phosphate


have been determined using ammonium reinckate (53). For
chloroquine phosphate t h e insoluble r e i n c k a t e s a l t I s formed
a t pH 1, removed, and t h e amount of excess reagent I n t h e
f i l t r a t e i s measured colorirnetrlcally. The d i f f e r e n c e i n
absorbance between t h e sample and blank and t h e standard
and blank r e p r e s e n t t h e absorbances of t h e sample and
standard s o l u t i o n s , r e s p e c t i v e l y .

82
CH LOROQU IN E PHOSPHATE

6. References

1. S. Clemans, Sterling-Winthrop Research I n s t i t u t e ,


Personal Communication.
2. B. Riegel and L. T. Sherwood , Jr. , JACS, 71, 1129
(1949)-
3- United S t a t e s Pharmacopeia X M , p. 82 (1975).
4. R. L. Kenyon, J. A. Wiesner and C. E. K w a r t l e r , Ind.
and Ehg. Chem. , 41, 659 (1949).
5. P. Dederick, Sterling-Winthrop Research I n s t i t u t e ,
Unpublished Data.
6. W. Houghtaling, Sterling-Winthrop Research I n s t i t u t e ,
Personal Communication.
7. R . C. E l d e r f i e l d , Chem. Eng. News,6,
2598 (1946).
8. R . L. Kenyon, J. A. Wiesner and C. E. K w a r t l e r , Ind.
and Eng. Chem. , 2, 654 (1949).
9. U. P. Basu, A. Raychaudhuri and R. De, Res. and Ind.,
10, 165 (1965).
10. H. Andersag, S. B r e i t n e r and II. Jung, German Patent
683,692 (1939).
-.
11. Ibid , U . S. P a t e n t 2,233,970 ( t o Winthrop Chemical
Company) (1941).
12. A. R. Surrey and H. F. Hammer, JACS, 68, 113 (1946).
13 E. 0. T i t u s , L. C. Craig, C. Golumbic, H. R. Mighton,
I. M. Wampen and R. C. E l d e r f i e l d , J. Org. Chem.,
UJ39 (194a)*
14. K. Kuroda, J. Pharmacol. Exp. Ther., u,
156 (1962).
E. W . McChesney, W. D. Conway, W. F. Banks, Jr. ,
15.
J. E. Rogers, and J. M. Shekosky, fbid., 151, 482
( 1966 .
16. E. W . McChesney, M. J. Fasco and W. F. Banks, Jr.,
- 158,
Ibid., 323-(1967).
17 * M. Rubin. H. N . Bernstein and N. J. Z v a i f l e r , Arch.
Ophthalmil. , 70, 474 (1963 ) .
18. L. T. Grady, Drug Standards Laboratory, Personal
Communication.
19 P. Cooper, Pharmaceutical J., 177, 53 (1956)-
20. -
I b i d . , p. 495.
21. E. G. C. Clarke, J. Pharm. and Pharmacol., l0, 194
(1958 *
22. E. S i l v a , Anal. Chem., Proc. I n t . Symp. i n Honor of
F r i t z F e i g l , Birmingham, Engl., A p r i l 1962.
Elsevier, 1963 , p. 78.
23 * J. L e l i j v e l d and H. Kortmann, Bull. WHO, 42, 477

24.
( 1970 -
G. Fuhrmann, w., 22, 663 (1960).
25 W. T. Haskins, Am. J. Trop. Med. Hyg., 7, 199 (1958).

83
DONALD D. HONG

26 A. F. Fartushnyi, Farmatsiya, l8, 77 (1969);


CA 71: 47882 v (1969).
27. R. S t r u f e , Clin. Chlm. Acta, 2, 753 (1960).
28. A. F. Fartusknyl, Sudebro-Med. Ekspertiza. Mln.
Zdravokhl SSR, lo, 45 (1967); CA 66:114281 k (1967).
29 0. Fuhrmann and K. Werrbach, 2. Tropenmed.
Parasitol., l ., 269 (1965): CA 65:12725 b (1966).
6
M. E. Auerbach, Sterling-Winthrop Research I n s t i t u t e ,
Unpublished Data.
T. S. Wu, T. 0 . Sun and T. H. Tang, Yao Hsueh Hsueh
Pao, 6, 253 (1958); CA 53~20691d (1959).
32. R. W. Prouty and K. Kuroda, J. Lab. Clin. Med., 2,
477 (1958)-
33 V. Waaret, Archiv. For Pharmacl. Og. Cheml., 71, 116
(1964)
34. A. E. Robinson, A. I. Coffer and F. E. Camps, J.
Pharm. and Pharmacol., 22, 700 (1970).
J. Stepan and B. Kakac, Med. Exp., ll, 352 (1964).
B. B. Brodie, S. Udenfrlend, W. D i l l and T. Chenkin,

37
J. Biol. Chem., x, 319 (1947).
E. M. Ensor, Trans. Roy. SOC. Trop. Med. Hyg., 60,
75 (1966).
38- E. W. McChesney, W. F. Banks and J. P. McAullff,
A n t i b i o t i c s and Chemotherapy, 12, 583 (1962).
39 P. M. Parlkh and S. P. Mukherji, Ind. J. Pharm.,
5, 269 (1.963).
40. United S t a t e s Pharmacopeia XVI, p. 151 (1960).
41. L. R. Goldbaum and L. Kazyak, Anal. Chem., 28, 1289
(1956).
42. J. Vecerkova, J. Solc and K. Kacl, J. Chromat., l0,
479 (l963).
43 R. Crain, Sterling-Winthrop Research I n s t i t u t e ,
Unpublished Data.
44. L. T. Grady, Drug Standards Laboratory, APhA,
Unpublished Data.
45 R. Castagnou and M. Sylvestre, Bull. SOC. Pharm.
Bordeaux, 105, 121 (1966); CA 6 7 ~ 6 2 8 2 1k.
46. W. J. A. VandenHeuvel, B. 0. A. Kaahti and E. C.
Horning, C l t n . Chem., g, 351 (1962).
47 A. Viala, J. P. C a n 0 and A. Durand, J. Chromatog.,
111, 299 (1975).
48. L. KaWsk and B. C. Knoblock, A n a l . Chem., s, 1448
(1963)
49 J. L. Holtzman, Anal. Biochem., u,
66 (1965).
50 J. Grego, Sterling-Wlnthrop Research I n s t i t u t e ,
Unpublished Data.

84
CHLOROOU IN E PHOSPHATE

51. I. M. Roushdi and R . M. Shafik, J. Pharm. Sci.


U.A.R., 2, 65 (1968).
52. -
Ibid., p . 57.
53. S-K. Sheng, C - I . Hsieh and S-H. Peng, Yao Hsueh
Hsueh Pao, l2, 662 (1965); CA 64:7967 c (1966).

Acknowledgments

The writer wishes t o thank M r . E. L. P r a t t and


D r . R . S. Browning f o r reviewing t h e m n u a c r i p t , D r . S. D.
Clemans f o r t h e NMR and M.S. data, Mr. W. W. Houghtaling
f o r t h e DSC data, Dr. L. T. Grady (USP Laboratories) f o r
t h e TLC data, Mrs. E. V. Miller f o r t h e drawings and Miss
S. F. Casey f o r typing t h e p r o f i l e .

85
DAFSONE

Chester E. Orzech, Norris G. Nash, and Raymond D. Daley


CHESTER E. ORZECH ma/.

CONTENTS

1. DESCRIPTION
1.1 Name, Formula, Molecular Weight
1.2 Appearance, Color, Odor
2. PHYSICAL PROPERTIES
2.1 Infrared Spectra
2.2 Nuclear Magnetic Resonance Spectrum
2.3 Ultraviolet Spectra
2.4 Mass Spectrum
2.5 Differential Thermal Analysis (DTA)
2.6 Differential Scanning Calorimetry (DSC)
2.7 Crystal Properties
2.8 Solubility
2.9 Fluorescence Spectra
2.10 Melting Point
3. SYNTHESIS
4. STABILITY-DEGRADATION
5. DRUG METABOLIC PRODUCTS
6. METHODS OF ANALYSIS
6.1 Identification Tests
6.2 Elemental Analysis
6.3 Colorimetric Methods
6.4 Titration Methods
6.5 Fluorometric Methods
6.6 Paper Chromatography
6.7 High Pressure Liquid Chromatography
6.8 Gas Chromatography
6.9 Thin Layer Chromatography
7. ACKNOWLEDGMENTS
8. REFERENCES

88
DAPSONE

1. DESCRIPTION

1.1 Name, Formula, Molecular Weight


Dapsone i s 4,4'-diaminodiphenyl sulfone, a l s o
known as p i p '-sulf onyldianiline and b i s (4-aminophenyl)
sulfone. Chemical Abstracts indexes dapsone a s benzen-
amine, 4,4'-sulfonylbis-, s t a r t i n g w i t h Volume 76; pre-
viously the index name was a n i l i n e , 4,4'-sulfonyldi-.
The CAS Registry Number is [80-08-0].

0
H2N 2*c
H $
0

Mol. W t . : 248.31

1.2 Appearance, Color, Odor


Dapsone i s a white or creamy white odorless crys-
t a l l i n e powder.

2. PHYSICAL PROPERTIES

2.1 Infrared Spectra


Infrared s p e c t r a of two c r y s t a l forms of dapsone
(designated Form 1-and Form 11) are shown i n Figures 1
and 2. The samples were prepared a s mineral o i l m u l l s
between potassium bromide p l a t e s . A Beckman Model I R - 1 2
spectrophotometer was used. The spectrum of Form I i s
similar t o those published by Pouchert (1) and by Hayden
e t a 1 (2). The spectrum of a hydrate of dapsone has a l s o
been observed; s i n c e i t i s d i f f e r e n t from the s p e c t r a of
the anhydrous forms, samples should be dried a t 105OC
before obtaining spectra. Some of the absorption bands
may be assigned a s follaws (3):

89
(D
0

Figure 1. Infrared Spectrum of Dapsone (Form I ) , Mineral Oil Mull


Figure 2. Infrared Spectrum of Dapsone (Form II), Mineral O i l Mull
CHESTER E. ORZECH H a l .

Band frequency, cm -’ Assignment

3200-3500 N-H S t r e t c h

3000-3 100 Aromatic C-H S t r e t c h

2800-3000 Mineral O i l

1635 N-H Deformation

1590, 1500, 1440 Aromatic C=C S t r e t c h

1460, 1380 Mineral O i l

1300 Region Asymmetric -SO2- S t r e t c h

1150 Synunetric -SO2- S t r e t c h

830-840 2 Adjacent H on aromatic


ring

550 -SOz- Scissoring

2.2 Nuclear Magnetic Resonance Spectrum


The NMR spectrum shown i n Figure 3 was obtained
by dissolving USP reference standard dapsone i n acetone-
d containing tetramethylsilane as i n t e r n a l reference.
Tke spectrum was produced using a Varian EM-360 NMR
spectrometer. The s e r i e s of peaks centered a t 7.2 ppm is
an A2Bp p a t t e r n t h a t is t y p i c a l of para s u b s t i t u t i o n and
the broad s i n g l e t a t 5.4 ppm is due t o the amine protons.
The peaks a t 2 ppm and 3 ppm a r e due t o the solvent.
These values c o r r e l a t e well with those previously reported
(4)

2.3 U l t r a v i o l e t Spectra
Figure 4 i s t h e u l t r a v i o l e t absorption spectrum
of dapsone i n methanol s o l u t i o n , run on a Cary Model 14
spectrophotometer. The s o l u t i o n contained 8.0 mg of
dapsone per l i t e r of methanol, and was r u n a g a i n s t
methanol ( 1 cm c e l l s ) . The spectrum e x h i b i t s peaks a t
295 nm and 260 nmwith a b s o r p t i v i t i e s of 30,100 and
18,300 l./mole c m respectively. The u l t r a v i o l e t i d e n t i t y
t e s t of the B r i t i s h Pharmacopoeia (5) s p e c i f i e s peaks a t

92
.

I---

I I I I I I I I I I I 1
10 9 8 7 6 5 4 3 2 I 0
6
Figure 3. Nuclear Magnetic Resonance Spectrum of Dapsone
.
.
.
w
v
z
94

(D
P
2
a
%
m
a

I L

m
In
0
210 250 300 350
0
I
W
z
I-

z
-

WAVELENGTH IN NANOMETERS

Figure 4 . Ultraviolet Spectrum of Dapsone, 8 mg/l., in Methanol, 1 cm Cells


CHESTER E. ORZECH eta/.

295 and 260 nm with a b s o r p t i v i t i e s of about 30,000 and


18,100 l./mole cm respectively.
A s i m i l a r spectrum i s obtained i n 95% ethanol
s o l u t i o n , except t h a t the a b s o r p t i v i t i e s a r e apparently
lower. Baliah and Ramakrishnan ( 6 ) r e p o r t peaks a t 295
and 261 nm w i t h a b s o r p t i v i t i e s of 27,000 and 17,800 1./
mole cm. Hayden e t a 1 (2) r e p o r t peaks a t 295 and 260 nm.
Maschka e t a 1 (7) reported u l t r a v i o l e t s p e c t r a of
dapsone a t various hydrogen ion concentrations; the d a t a
a r e a s follows:

Wave length Molar Absorptivity,


Condition of Maxima, nm l./mole cm

1. pH 11.0 291.0 19,820


258.5 12,190

2. pH 6.8 291.0 17,950


259.0 10,810

3. pH 1.8 289.5 13,490

4. 22 HCl 273.5 1,850


264.5 2,000
234.5 10,790

2.4 Mass Spectrum


Figure 5 shows the low r e s o l u t i o n mass spectrum
of dapsone. The d a t a w a s obtained w i t h an LKB 9000s mass
spectrometer, with an i o n i z a t i o n voltage of 70 eV, source
temperature 250'C. Some of the peaks may be assigned a s
follows (8):

-
m/e Assignment

248 M+

140

108

92

95
96

50
MASS/ CHARGE R AT10
Figure 5. Mass Spectrum of Dapsone
v1
a
0)
DAPSONE

2.5 D i f f e r e n t i a l Thermal Analysis (DTA)


The DTA curve i n Figure 6 was obtained with a
DuPont Model 900 instrument. The curve shows a sharp endo-
thermic s o l i d - s o l i d phase t r a n s i t i o n a t 84OC and a melting
endotherm a t 178OC.

2.6 D i f f e r e n t i a l Scanning Calorimetry (DSC)


The DSC melting thermogram of dapsone c r y s t a l l i z e d
from methanol and water i s shown i n Figure 7 , 'The thermo-
gram was obtained with a Perkin-Elmer DSC-1B d i f f e r e n t i a l
scanning calorimeter a t a heating r a t e of 1.25°C/minute i n
a nitrogen atmosphere. The p u r i t y of samples of t h i s com-
pound can be determined by a n a l y s i s of the melting thermo-
gram (9).

2.7 C r y s t a l Properties
B u t t (10) reported t h a t dapsone can be obtained i n
a t l e a s t two c r y s t a l forms, with d i f f e r e n t melting points.
Infrared s p e c t r a (see Section 2.1) and x-ray powder d i f -
f r a c t i o n p a t t e r n s a l s o i n d i c a t e t h a t dapsone occurs i n a t
l e a s t two c r y s t a l forms, end t h a t it forms a c r y s t a l l i n e
hydrate. The powder d i f f r a c t i o n p a t t e r n s a r e given i n
Table 1. These p a t t e r n s were obtained with a Norelco d i f -
fractometer, using n i c k e l - f i l t e r e d copper Kcr r a d i a t i o n .
The c r y s t a l and molecular s t r u c t u r e of dapsone has
been determined by Alleaume and Decap (ll), and by
Dickinson e t a 1 (12, 13). Both groups r e p o r t 4 molecules
i n an orthorhombic u n i t c e l l , symmetry P212121, with
s i m i l a r dimensions (I):

a=25.57 -+ 0.01, b=8.07 +- 0.01, c=5.77 5 0.01 (11)


a=8.065 -+ 0.005, b=25.57 + 0.02, c=5.760 2 0.001 ( 1 2 )
c

97
Figure 6. Differential Thermal Analysis Curve of Dapsone
to
6

20
21
OAPSONE
hHF ~4.90

a
Tf
a,
0
i

00
Irz
25
30
x KCAL.

II
6 PURITY = 99.99%
99

(0
(0

I-I
0

Figure 7 . Differential Scanning Calorimeter Curve of Dapsone


CHESTER E. ORZECH e t a / .

TABLE 1
DAPSONE X-RAY POWDER DIFFRACTION PATTERNS

F o r m I1 Hydrate
d
c -
1/10 -d -
1/10 -d -
1/10

12.79 4 13.07 5 12.55 1


7.74 8 10.78 2 12.25 4
6.86 32 9.21 1 11.13 3
6.42 53 7.41 7 8.54 16
5.88 4 6.85 8 7.73 29
5.66 9 6.66 89 7.62 13
5.28 60 6.57 18 7.54 47
5.03 34 6.40 18 7.45 28
4.71 17 5.80 3 6 -82 1
4.64 100 5.68 3 6.39 7
4.42 49 5.45 27 6.25 7
4.30 62 5.35 54 5.98 4
4.12 1 5.01 23 5.74 28
4.00 48 4.66 97 5.68 35
3.84 22 4.57 20 5.62 19
3.78 44 4.37 96 5.53 17
3.65 2 4.27 9 5.45 11
3.46 1 3.99 8 5.32 4
3.42 10 3.86 21 5.12 3
3.32 2 3.82 100 5.00 21
3.21 10 3.52 23 4.93 58
3.16 4 3.50 25 4.86 9
3.09 46 3.42 8 4.79 2
2.98 2 3.37 9 4.68 100
2.94 9 3.33 17 4.59 39
2.88 4 3.28 2 4.55 75
2.80 6 3.20 8 4.45 6
2.78 4 3.13 22 4.39 27
2.74 4 3.09 14 4.31 13
2.64 7 3.02 26 4.27 54
2.56 3 2.89 9 4.22 31
2.51 2 2.86 17 4.18 12
2.44 5 2.73 3 4.09 87
2.40 5 2.68 3 4.05 19
2.35 3 2.52 6 4.01 13
2.31 2 2.43 2 3.95 4
2.25 5 2.40 2 3.86 15
2.20 3 2.34 5 3.83 19
2.17 4 2.28 2 3.77 4
2.13 8 2.21 2 3.72 3

100
DAPSONE

2.08 5 2.17 3 3.65 1


2.06 1 2.10 7 3.58 14
1.96 2 3.46 11
3.42 9
3.37 13
3.35 6
3.30 15
3.28 8
3.23 30
3.20 10
3.14 21
3.09 14
3.02 8
2.96 6
2.87 5
2.84 3
2.82 3
2.79 6
2.72 6
2.69 5
2.65 7
2.62 7
2.58 1
2.50 3
2.45 3
2 -42 2
2.27 6
2.15 5
2.11 2
2.10 2
2.07 3
1.96 3

2.8 S o l u b i l i t y
The s o l u b i l i t y a t room temperature is a s f o l l o w s :

Solvent Approximate S o l u b i l i t y , mg/ml

Methanol 52
Ethanol (95%) 28
2-Propanol 6
Ethyl Acetate 34
Ethyl Ether 0.9
Chloroform 3
Benzene 0.5
Water 0.2 (14)
2,2,4-trimethylpentane c0.2

101
CHESTER E. ORZECH eta/.

Dapsone is very soluble i n acetone and a c e t o n i t r i l e ; one


gram of dapsone dissolves i n 1.8 m l of acetone or 5 m l of
a c e t o n i t r i l e . It is a l s o soluble i n d i l u t e mineral a c i d s ;
one gram dissolves i n about 10 m l of 1E hydrochloric acid.

2.9 Fluorescence Spectra


Peters e t a 1 (15) found the e x c i t a t i o n maximum a t
285 nm and the fluorescence maximum a t 350 nm, i n ethylene
dichloride s o l u t i o n , Ellard and Gammon (16) reported an
e x c i t a t i o n maximum a t 298 nm and the fluorescence maximum
a t 345 nm, i n e t h y l a c e t a t e . Glazko e t a 1 (73) and Cucinell
e t a 1 (17) reported the e x c i t a t i o n maximum a t 297 nm and
the fluorescence maximum a t 340 nm, i n e t h y l a c e t a t e ,

2.10 Melting Point


A range of melting points has been reported for
dapsone :

172'C
172-173OC
-
172 174'C
174OC
174- 176'C
175OC
175-176'C
176OC
176.3-177.5OC
178-179OC
1 7 9- 1 8 O o C

Bu t (10) reported t h a t dapsone can -e obtained ,n two


forms, one melting a t about 178.5OC, the other a t about
180.5'C.

3. SYNTHESIS

Dapsone has been prepared by various procedures, s t a r t -


ing with 4,4'-dinitrodiphenyl s u l f i d e ( 2 1 , 2 2 , 31, 32) pre-
pared from p-chloronitrobenzene (33), or by oxidation of
4,4'-diacetylaminodiphenyl s u l f i d e (19, 23, 25) prepared
from 4-nitro-4'-aminodiphenyl s u l f i d e (19, 23, 24) or 4,4'-
diaminodiphenyl s u l f i d e (25). It has a l s o been made from a
s u l f i n a t e and a halonitrobenzene (24, 26, 2 9 , 3 5 ) ; from 4-
acetylaminobenzenesulfonyl chloride and a c e t a n i l i d e (20,
36, 3 7 , 38); from a c e t a n i l i d e and thionyl chloride ( 2 7 , 39,
40); from 4,4'-dichlorodiphenyl sulfone (18, 28, 30, 41,
42, 43); from the diphthaloyl derivative of 4,4'-diaminodi-

102
0
"
fn
cu
0
-
rn 9)
G
0
i ? dma
F
D U
rnx
r A

I4
0
cw
0
0
"
0
"
cn
0
0
U
m
2
cn
x
d
4
U
.
co
x 9 aJ
I4
z
V=O
a
U ii
'4
2
" a
$1
.r
0
Xm
z
rnx "
8
X"
I
4
U
X xm
0 0
"
rn rfl
103
CHESTER E. ORZECH era/.

phenyl sulfide (44, 45); and from 4,4'-dimethyldiphenyl


sulfone by oxidation to the dicarboxylic acid and Hofmann
degradation of the diamide (46). The 4,4'-diacetylaminodi-
phenyl sulfone obtained by any of these methods can easily
be hydrolyzed to the diamino compound (23). Sanghavi (47)
has reviewed the various methods of synthesis. A few of
these procedures are outlined in Figure 8.

4. STAB1LITY-DEGRADATION
No reports of stability studies or degradation of dap-
sone were found.
5, DRUG METABOLIC PRODUCTS

Dapsone metabolic products have been reported as


follows:
Me tabo1i te Species References
Dapsone glucuronide Man (48,49,50)
Monkey (51,521
Rabbit (48,51,53,
54,55)
Dapsone sulfamate Man (48,501
Rat (51,521
Dapsone monohydroxylamine Man (56,57,58)
Dog (56,581
Monoace tyl dapsone Man (15,51,57,
59,60,61)
Monkey (51,52,62)
Rabbit (51,61,62)
Dapsone monohydroxylamine Man (58)
su 1f amate

Dapsone monohydroxylamine Man (581


glucuronide
Monoace tyl dapsone sulfamate Man (50)
Monkey (51,52)

104
0
x x
0
ru

n
Dapsone sulfamate (DDS-S) Dapsone glucuronide (DDS-G)

rl
5

Y
cn

cd

al
1

u
a
a
..
ocv)+o
8
(I
X
z,

X
hl
Dap s one (DDS ) Dapsone monohydroxylamine Az ox y -d aps one

2-
82
105

(DDS-NOH) (AZmy-DDS)
o=u0

ot;;:
V
X

z
X

p:
z
II
Monoace t y l dapsone Monoace t y l dapsone hydroxylamine Diace t y l azoxy-dapsone

rl
x
(MADDS (MADDS-NOH)

o=u
V
5:

Xm
Figure 9. Some Dapsone Metabolites
CHESTER E. ORZECH era/.

Monoacetyl dapsone glucuronide Man (50,511


Monkey (52)
Rabbit (51)
Rat (51)

Monoacet y l dapsone hydroxylamine Man (57)

Az my-da p s one Man (571


Rat (57)

Diace t y l azoxy-dapsone Man (57)

The s t r u c t u r e s of some of these metabolites, a r e shown i n


Figure 9.

6. METHODS OF ANALYSIS

6.1 I d e n t i f i c a t i o n Tests
Dapsone is e a s i l y i d e n t i f i e d by the physical prop-
e r t i e s described i n s e c t i o n 2 above. Where i d e n t i f i c a t i o n
of dapsone i n formulations i s necessary, i t can be e x t r a c t e d
by various organic solvents such a s methanol, chloroform,
ethylene d i c h l o r i d e , e t c . I f the i d e n t i f i c a t i o n of very
small amounts of dapsone i s necessary, the colorimetric
method of P e t e r s e t a1 (63), the c o l o r i m e t r i c t e s t s des-
cribed i n s e c t i o n 6.3, or the fluorometric t e s t s described
i n s e c t i o n 6.5 may be useful.

6.2 Elemental Analysis


The elemental composition of dapsone i s as follows:

Element %Theory

Carbon 58.04
Hydrogen 4.87
Nitrogen 11.28
Su If u r 12.91
Oxygen 12.89

6.3 Colorimetric Methods


There a r e two b a s i c colorimetric methods f o r dap-
sone. The f i r s t and most widely u s e d is the method of
Bratton and Marshall (64). Dapsone is diazotized, then
coupled with N-(1-naphthy1)-ethylenediamine t o form a n 820
dye. Other i n v e s t i g a t o r s have used t h i s b a s i c method but
have used d i f f e r e n t coupling reagents: N i t t i e t a 1 (651,
dimethyl- 0 -naphthylamine; Rose and Bevan (66), N-@-sulfa-

106
DAPSONE

toethyl-m-toluidine; Schoog (67), l-sulfomethylaminonaph-


thalene-8-sulfonic a c i d ; Merland (68), N-naphthyl-N' ,N'-di-
e thy 1pro py lene d iamine .
The second type of method, reported by Levy and
Higgins (69), i s based on the formation of a Schiff base
between dapsone and 4-dimethylaminobenzaldehyde. This
method is reported t o be 2.4 times more s e n s i t i v e than the
Bratton-Marshall technique and, i n a d d i t i o n , i t i s reported
t o be s p e c i f i c f o r dapsone i n the presence of i t s metabo-
li tes.

6.4 T i t r a t i o n Methods
Tomicek (70) t i t r a t e d dapsone potentiometrically
with perchloric acid i n g l a c i a l a c e t i c acid. Wojahn bro-
minated dapsone with bromide-bromate and b a c k - t i t r a t e d the
excess bromate with sodium t h i o s u l f a t e s o l u t i o n ( 7 1 ) . The
USP method of assay i s t i t r a t i o n of the cooled, s t r o n g l y
acid sample s o l u t i o n with sodium n i t r i t e s o l u t i o n t o a n
electrometric endpoint (72).

6.5 Fluorometric Methods


Glazko (73) reported a fluorometric method f o r
dapsone i n plasma and u r i n e samples. Complete d e t a i l s of
sample preparation a r e given, The fluorescence of the
e t h y l a c e t a t e e x t r a c t is determined by a c t i v a t i n g a t 297 nm
and measuring the emission a t 340 nm. Ellard and Gammon
(16) developed an e x t r a c t i o n scheme t o determine dapsone
and two possible metabolites, MADDS and DADDS, fluoro-
m e t r i c a l l y i n plasma and urine samples. Peters e t a1 (15)
modified and expanded the above procedure. They extracted
with ethylene dichloride instead of e t h y l a c e t a t e and thus
eliminated the problem of quenching caused by small quan-
t i t i e s of water i n the e t h y l a c e t a t e e x t r a c t .

6.6 Paper Chromatography


Longenecker (74) reported procedures f o r p a r t i t i o n
chromatography of dapsone by both ascending and descending
chromatography on paper and s t r i n g . Bushby and Woiwood
(54, 55) u s e d paper chromatography t o i s o l a t e and i d e n t i f y
dapsone and some d i a z o t i z a b l e metabolites by paper chroma-
tography and electrophoresis. Wadia e t a 1 (75) reported
Rf values f o r dapsone and twenty f i v e other diaminodiphenyl
s u l f i d e s , sulfoxides, and sulfones using four d i f f e r e n t
solvent systems. J a r d i n and S t o l l (76), Khosla e t a 1 (77),
and T s u t s u m i (48) a l l used paper chromatography i n t h e i r
s t u d i e s on the metabolism of dapsone.

107
CHESTER E. ORZECH eta/.

6.7 High Pressure Liquid Chromatography


Gordon and Peters (78) separated dapsone, mono-
a c e t y l dapsone (MADDS), and d i a c e t y l dapsone (DADDS) a t the
1 t o 20 ,,g l e v e l on a s i l i c a g e l column with e t h y l a c e t a t e
as solvent. The e f f l u e n t was monitored a t 280 nm. Murray
e t a1 (79) used a fluorometric d e t e c t o r and increased the
s e n s i t i v i t y of the determination t o the 10 ng level. This
procedure was modified by Murray e t a 1 (80) t o increase the
s e n s i t i v i t y t o 0.1 ng i n 0.5 m l . Ribi e t a 1 (81) used
pressure accelerated chromatography through microparticulate
s i l i c a g e l columns, packed by c e n t r i f u g a t i o n , t o separate
dapsone, MADDS, and DADDS, with chloroform-methanol (97:3)
as solvent and u l t r a v i o l e t absorption a t 254 nm f o r detec-
tion.
Gordon e t a1 (82) used a s i l i c a g e l column with
chloroform-carbon t e t r a c h l o r i d e (7:3) solvent t o examine
dapsone f o r impurities. Column e f f l u e n t was monitored by
the absorption a t 280 nm. Fractions were c o l l e c t e d and
evaporated, and the residues were dissolved i n ethylene d i -
chloride. The fluorescence of the re-dissolved f r a c t i o n s
was measured. Impurities determined were 2,4'-diaminodi-
phenyl sulfone and 4-aminodiphenyl sulfone .
Krol and Mannan (83) developed a method f o r the
a n a l y s i s af dapsone using a commercially a v a i l a b l e s i l i c a
g e l column 6- P o r a s i P ) , a solvent containing isopropyl a l -
cohol, a c e t o n i t r i l e , e t h y l a c e t a t e , and pentane (1:1:1:7),
and d e t e c t i o n a t 254 m o A 30 cm by 4 mm I.D. column was
s u i t a b l e e i t h e r f o r assay f o r dapsone i n t a b l e t s or f o r
analyzing f o r impurities i n dapsone. The following r e l a t e d
compounds can be separated from dapsone: (a) 2,4'-diamino-
diphenyl sulfone; (b) 4-aminodiphenyl sulfone; (c) 4-amino-
4'-chlorod iphenyl s u l f one ; (d ) 4,4'-d iace tamidod iphenyl
sulfone (DADDS); (e) 4-amino-4I-ace tamidodiphenyl s u l f one
(MADDS); ( f ) 4-amino-4'-hydroxydiphenyl sulfone; ( g ) 4,4'-
diace tamidodiphenyl sulfoxide; 4,4'-diaminodiphenyl s u l f i d e
(90).

6.8 Gas Chromatography


Burchfield e t a 1 (84, 85) reported t h a t dapsone can
be chromatographed d i r e c t l y . Because t h e i r l a t e r work r e -
quired g r e a t e r s e n s i t i v i t y , and, i n a d d i t i o n , the determi-
nation of MADDS and DADDS, the iodo d e r i v a t i v e s were pre-
pared by d i a z o t i z a t i o n and an e l e c t r o n capture d e t e c t o r w a s
used. Using a 4 f o o t by 0.25 inch O.D. g l a s s U-tube packed
with 3% Poly-A-103 on 100-120 mesh Gas Chrom Q a t 285'C
with nitrogen, 280 pg of iodo d e r i v a t i v e was e a s i l y d e t e c t -
able.

108
DAPSONE

Chang e t a 1 (52) investigated the metabolism of


dapsone by chromatographing t r i m e t h y l s i l y l d e r i v a t i v e s of
the metabolic products on 3% OV-17 (Gas Chrom Q) a t 275OC.

6.9 Thin Layer Chromatography


Thin layer chromatography systems have been com-
piled i n Table 2 .

7. ACKNOWLEDGMENTS

The w r i t e r s wish t o thank D r . B. T. Kho f o r h i s review


of the manuscript, D r . G. S c h i l l i n g of Ayerst Research
Laboratories f o r h i s mass s p e c t r a l data and i n t e r p r e t a t i o n ,
the l i b r a r y s t a f f f o r t h e i r l i t e r a t u r e search, and Mrs. B.
Juneau f o r typing the p r o f i l e .

109
TABLE 2

Adsorbent SoLvent Sys tem !% -


Ref.

Silica gel Chloroform-Butanol-Pe troleum Ether (1:1:1) 0.62 86


Silica gel DF-5 Toluene-Ethyl acetate (1:1) 0.30 87
Silica gel G Chlorofom-Hep tane-E thanol (1: 1: 1) 0.60 88
11
Ace tone-Chloroform (1:9) 0.20 89
11
Chloroform-Ethyl Ether (85: 15) 0.11 11

11
Toluene-Ethyl acetate (1:l) 0.27 11

11
Chloroform-Methanol (95:5) 0.47 11

11
Chloroform-Methanol (9: 1) 0.48 11

11
Chloroform-Ethanol (9:l) 0.65 11

I1
Chlorof orm-Ace tone-Die thanolamine (5 :4:1) 0.69 11

I1
Me thano1-Ace tone-Die thanolamine (50:50 :1.5 ) 0.78 11

11
Ace tone -Chlorofo m (9:1 ) 0.84 11

11
Isopropyl alcohol-cyclohexane-259. Axunonia 0.85 11

(65:25:10)
11
Dime thy1 formamide-Die thylamine-Ethanol- 0.88 11

Ethyl acetate (1:1:6:12)


DAPSONE

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Ca.

L i t e r a t u r e surveyed through June, 1975.

114
FLUCYTOSINE

Edward H. Waysek and James H. Johnson


EDWARD H. WAYSEK AND JAMES H. JOHNSON

INDEX

Analytical P r o f i l e - Flucytosine

1. Description

1.1 Name, Formula, M o l e c u l a r Weight


1.2 Appearance, Color, Odor

2. Physical Properties

2.1 I n f r a r e d Spectrum
2.2 N u c l e a r M a g n e t i c Resonance Spectrum
2.3 U 1 t r a v i o l e t Spectrum
2.4 F l u o r e s c e n c e Spectrum
2.5 Mass Spectrum
2.6 Opt i ca 1 Rota t i o n
2.7 M e l t i n g Range
2.8 D i f f e r e n t i a l Scanni ng C a l o r i m e t r y
2.9 Thermogravirnetric Analysis
2.10 Solubility
2.11 X-Ray C r y s t a l P r o p e r t i e s
2.12 D i s s o c i a t i on Constant

3. Synthesis

4. Stability

5. Drug M e t a b o l i c P r o d u c t s

5.1 Toxicology

6. Methods o f A n a l y s i s

6.1 Elemental A n a l y s i s
6.2 Phase S o l u b i l i t y A n a l y s i s
6.3 Th i n-Layer Chromatograph i c Ana 1 ys i s
6.4 Non-Aqueous T i t r a t i o n
6.5 Fluorometric Analysis
6.6 D i r e c t Spect rophotornetr i c A n a l y s i s
6.7 Fluorine Analysis

6.71 Free F l u o r i d e Analysis


6.72 O r g a n i c a l l y Bound F l u o r i n e A n a l y s i s

116
FLUCYTOSIN E

6.8 Biological Assays

7. Acknowledgements

8. References

117
EDWARD H. WAYSEK AND JAMES H. JOHNSON

1. Description

1.1 Name, Formula, Molecular Weight

F 1 ucytos ine i s 4-ami no-5-f 1uoro-2 ( 1 H) -pyr i m i done.

C4H4FN30 Molecular Weight: 129.1

1.2 Appearance, Color, Odor

F l u c y t o s i n e i s a white, odorless, c r y s t a l l i n e
powder.

2. Physical P r o p e r t i e s

2.1 I n f r a r e d Spectrum ( I R )

The i n f r a r e d spectrum o f f l u c y t o s i n e i s presen-


t e d i n F i g u r e 1. The spectrum was recorded
w i t h a Perkin-Elmer Model 621 G r a t i n g I n f r a r e d
Spectrophotometer as a Fluorolube suspension
from 4000 t o 1350 cm’l and as a m i n e r a l o i l
suspension from 1350 t o 400 cm”. The assign-
ments f o r t h e c h a r a c t e r i s t i c bands i n t h e I R
spectrum a r e l i s t e d i n Table 1 ( I ) .

118
FIGURE 1
I n f r a r e d Spectrum o f Flucytosine

W AVEL ENGTH MICROMETERS


2.5 3 4 5 6 7 8 9 10 12 14 1822 35 50
100 100

80
8 80
W

260 60
I-

540 40
a
CtT
t-
20 20

0 0
4000 3500 3000 2500 2000 1700 1400 1100 800 500 200
FREQUENCY (CM-')
EDWARD H. WAYSEK AND JAMES H. JOHNSON

Table I

I n f r a r e d Ass ignmen t s For F I ucytos ine

Frequency (cm-I) Characteristic O f

3374, 3138 Bonded NH


2800-2200 (broad) Enol i c OH (associated)
1684, 1672 C=O s t r e t c h
1647 N-C=H (amidine)
1554 C=C (extended conjugation)

2.2 Nuclear Magnetic Resonance Spectrum (NMR)

The NMR spectrum o f f l u c y t o s i n e i n F i g u r e 2 was


determined on a JEOL C-60 HL Spectrometer a t
ambient temperature (ca. 25"). The sample was
d i s s o l v e d i n 0.1N deuterium c h l o r i d e c o n t a i n i n g
sodium 2,2-dimethyl-2-silapentane s u l f o n a t e as
an i n t e r n a l reference (MeSi = 0 ppm). The C-6
p r o t o n e x h i b i t e d a doublet ( J = 5.5 Hz) a t
h- f
8.03 ppm.

2.3 U l t r a v i o l e t Spectrum

The u l t r a v i o l e t spectrum o f f l u c y t o s i n e i n 0.1N


h y d r o c h l o r i c a c i d i n the r e g i o n 340 t o 210 nm
e x h i b i t s a maximum a t 283-287 nm (E = 9.2 x 103 )
and a minimum a t 243-247 nm. The spectrum p r e -
sented i n F i g u r e 3 was obtained from a reference
standard s o l u t i o n o f f l u c y t o s i n e a t a concen-
t r a t i o n o f 0.85 mg per 100 m l o f 0.1N H C I ( 2 ) .

2.4 Fluorescence Spectrum

F i g u r e 4 shows the e x c i t a t i o n and emission


spectra o f a s o l u t i o n o f reference standard
f l u c y t o s i n e from 250 t o 650 nm. The s p e c t r a
measured i n a methanol s o l u t i o n o f f l u c y t o s i n e
(0.025 mg/ml u s i n g a Farrand MK-1 spectro-
f luorometer, showed one e x c i t a t i o n maximum a t
290 nm and one emission maximum a t 366 nm.

120
FLUCYTOSINE

FI GURE 2
NMR Spectrum of Flucytosine

121
EDWARD H. WAYSEK AND JAMES H. JOHNSON

FIGURE 3
Ultraviolet Spectrum of Flucytosine

.7

.6 -

.5 -

w.4
0
-
f8
v)

9.3
-

*‘t -

L
.I

n
210 -
250 I _ I-
_ 300
NANOMETERS
35

122
FIGURE 4: Fluorescence Spectra of Flucytosine

I I

Excitation
Spectrum

\ Spectrum

250 300 350 400 450 500 550 600 650


NANOMETERS
EDWARD H. WAYSEK AND JAMES H. JOHNSON

2.5 Mass Spectrum

The mass spectrum o f f l u c y t o s i n e was o b t a i n e d


u s i n g a CEC 21-110 mass spectrometer w i t h an
i o n i z i n g energy o f 70 eV. The o u t p u t from the
mass spectrometer was analyzed and presented i n
t h e form o f the bar graph, shown i n F i g u r e 5,
by a Varian 100 MS dedicated computer system
(3)
The spectrum shows a s t r o n g molecular ion peak
a t m/e 129. The peak a t m/e 101 i s due t o t h e
loss o f CO from the p a r e n t mass and t h e f r a g -
ment o f m/e 86 i s due t o t h e loss o f HNCO, a l s o
from t h e molecular i o n .

2.6 Optical Rotation

F l u c y t o s i n e e x h i b i t s no o p t i c a l a c t i v i t y .

2.7 M e l t i n q Range

A sharp m e l t i n g p o i n t i s n o t observed w i t h f l u -
c y t o s i n e . The m e l t i n g range depends on t h e
r a t e o f h e a t i n g . When the USP Class l a pro-
cedure (4) i s used, the m e l t i n g p o i n t l i e s
between 292" and 298°C. M e l t i n g i s accompanied
by m e l t decomposition.

2.8 D i f f e r e n t i a l Scanning C a l o r i m e t r y (DSC)

When scanned a t a programmed r a t e o f 10°C per


minute using n i t r o g e n as the i n e r t gas, decom-
p o s i t i o n occurs b e f o r e any observed t r a n s i t i o n ,
t h e r e f o r e , DSC i s n o t u s e f u l f o r c h a r a c t e r i z a -
t i o n o f t h i s compound ( 5 ) .

2.9 Thermogravimetric A n a l y s i s (TGA)

No weight loss was observed by TGA between


ambient temperature and 210°C a t a h e a t i n g r a t e
o f 10°C per minute. A weight loss began a t
about 210°C and amounted t o about 64% o f t h e
sample weight a t 5OO0C ( 6 ) .

124
5: Mass Spectrum of F l u c y t o s i n e

v\
FIGURE

..
125
EDWARD H. WAYSEK AND JAMES H. JOHNSON

2.10 Solubi 1 i t y

The s o l u b i l i t y data f o r f l u c y t o s i n e obtained a t


25'C i s g i v e n i n Table I1 ( 7 ) . The s o l u b i l i t i e s
were measured a f t e r an e q u i l i b r a t i o n p e r i o d of
t h r e e hours.

Table I 1

Flucytosine S o l u b i l i t y

So 1 ven t Solubi 1 i t y (mg/ml)

3A a l c o h o l 1.3
Absolute ethanol 0.5
Ace tone <o. 1
Benzene <o. 1
Chloroform <o. 1
Diethyl ether <o. 1
Ethyl acetate <o. 1
Hexane <o. 1
-
2 p ropa no 1 1.1
Met hano 1 5.0
USP a l c o h o l 1.7
Water 14.2

2.11 X-Ray C r y s t a l P r o p e r t i e s

The x-ray powder d i f f r a c t i o n p a t t e r n o f f l u c y -


t o s i n e i s presented i n Table I l l ( 8 ) . The
instrumental c o n d i t i o n s are g i v e n below,

Instrumental Conditions

General E l e c t r i c Model XRD-6 Spectrogoniometer

Genera t o r 50 KV, 12.5 mA


Tube Target Copper 0

Radiation Cu (Cu Ka = 1.5418 A)


Optics 0.1' Detector s l i t
M.R. S o l l e r s l i t
3" Beam s l i t
0.0007" N i f i l t e r
4' take o f f angle

126
FLUCYTOSINE

Goniometer Scan a t 0.2"/minute 20


A m p l i f i e r g a i n - 16 coarse
8.17 f i n e
Sealed p r o p o r t i o n a l c o u n t e r tube
and D.C. v o l t a g e a t p l a t e a u ,
p u l s e h e i g h t s e l e c t o r EL; 8.0
v o l t s , Eu; o u t
Rate meter T.C. 4, 2000 c/s f u l l
scale
Recorder Chart speed 1" p e r 5 min.
Sample Prepared by g r i n d i n g a t room
temperature

Table I l l

X-Ray Powder D i f f r a c t i o n P a t t e r n o f F l u c y t o s i n e

-
20 d(i)
13.74 6.45 0.4
14.76 6 .OO * 39
15.14 5.85 .76
17.26 5.14 .09
18.76 4.73 .22
20.18 4.40 .81
21.81 4.08 .98
22.75 3.91 .16
23.94 3.72 1 .oo
25.94 3.44 .29
26.44 3.37 .67
29.24 3.06 .72
29.94 2.99 .14
30.65;: 2.92 .15
32.01* 2.80 .55
33.48 2.68 .22
37.57 2.39 .22
39.98 2.26 .07
40.70 2.22 .09
42.74 2.12 .15
51.75 1.77 .15
*Broad Peaks

127
EDWARD H. WAYSEK AND JAMES H. JOHNSON

(1) d - ( i n t e r p l a n a r d i s t a n c e ) nA
2 sin 0
(2) [ / I 1= r e l a t i v e i n t e n s i t y

2.12 D i s s o c i a t i o n Constant

The apparent pKa values f o r f l u c y t o s i n e have


been determined s p e c t r o p h o t o m e t r i c a l l y t o be
2.90 2 0.05 and 10.71 2 0.05 ( 9 ) . The apparent
d i s s o c i a t i o n constants were c a l c u l a t e d from t h e
UV s p e c t r a l data i n water a t v a r i o u s pH values.
pKal (2.90) represents the p r o t o n a t i o n o f N-3
and pKa (10.71) represents d e p r o t o n a t i o n a t
the ami8e moiety. These values a r e i n good
agreement w i t h t h e pKa's reported by Ueda and
Fox f o r 3-methylcytosine (10).

3. Synthesis

F l u c y t o s i n e may be prepared by the r e a c t i o n scheme


shown i n F i g u r e 6. 5 - F l u o r o u r a c i l i s r e f l u x e d w i t h
d i m e t h y l a n l l i n e and phosphorous o x y c h l o r i d e . The
2,4-di ch l o r o - 5 - f 1 uoro-pyr i m i d i n e formed i s reacted
w i t h ammonia and ethanol t o g i v e 2-chloro-4-amino-
5-f l u o r o p y r i m i d i n e which y i e l d s f l u c y t o s i n e hydro-
c h l o r i d e upon being t r e a t e d w i t h concentrated hydro-
c h l o r i c acid. F l u c y t o s i n e h y d r o c h l o r i d e i s neutra-
l i z e d w i t h ammonium hydroxide forming 5 - f l u o r o c y t o s i n e
(11).

4. Stability

F l u c y t o s i n e s t o r e d a t room temperature f o r f i v e years


was s t a b l e when t e s t e d by u l t r a v i o l e t a b s o r p t i o n and
t h i n - l a y e r chromatography u s i n g e t h y l a c e t a t e :
methano1:conc. NHhOH (50:50:2) as t h e s o l v e n t system
(12). The s t a b i l i t y o f 0.14; b u f f e r e d s o l u t i o n s i s
shown i n Table I V (13).

128
FIGURE 6: Synthesis of Flucytosine

0 CI

LL
=
z
-4

A
o
H
5-Fluorouracil 2.4-Dichloro- 2-Chloro-4-amino-
5-f luoropyrimidine 5-f luoropyrimidine
129

y 2

fi

Lt
NH40H

0
I
HCI

H H
Flucytosine Flucytosine
hydrochloride
EDWARD H. WAYSEK AND JAMES H. JOHNSON

Table I V

Stabi 1 i t y o f 0.1% B u f f e r e d S o l u t i o n

1
~~

I HBefore n i ng
z THeat g A f t e r 1 Hour a t 100°C

APHA APHA
pH Color Clarity pH Color Clarity % Recover:

3.10 0-10 Clear 3.15 0-10 Clear 92.9


5.50 0-10 I1
5.60 0-10 I1
95.9
6.90 0-10 I1 6.90 0-10 I1
99.0
9.40 0-10 I1 9.40 0-10 I1
98.5

Method Used: U l t r a v i o l e t a b s o r p t i o n and t h i n - l a y e r


chromatography.

The s t a b i l i t y o f f l u c y t o s i n e has a l s o been s t u d i e d i n


i t s dosage forms (14). I n ampuls f l u c y t o s i n e was
s t a b l e t o the e x t e n t o f 2% o r less breakdown a t room
temperature f o r 36 months. No degradation products,
urea, u r a c i 1, and 5 - f l u o r o u r a c i 1 , were detected i n
capsules a f t e r s i x months a t 37°C and twelve months
a t 25°C.

5. Drug M e t a b o l i c Products

I t has been r e p o r t e d t h a t f l u c y t o s i n e d i d n o t under-


go s i g n i f i c a n t b i o t r a n s f o r m a t i o n i n humans a f t e r o r a l
dosage and 90% o f the f l u c y t o s i n e administered was
e x c r e t e d unchanged i n t h e u r i n e (15). Koechl i n ,
e t a l . have s t u d i e d t h e metabolism o f f l u c y t o s i n e
i n t h e r a t and i n man u s i n g drug l a b e l e d i n the 2-
p o s i t i o n w i t h carbon-14. Rats given a p a r e n t e r a l
dosage d i d n o t metabolize f l u c y t o s i n e and the dosage
was recovered q u a n t i t a t i v e l y , mostly i n t h e u r i n e .
F l u c y t o s i n e was deaminated t o 5 - f l u o r o u r a c i l t o t h e
e x t e n t o f 20-30% a f t e r o r a l a d m i n i s t r a t i o n t o r a t s ,
which was i n t u r n metabolized f u r t h e r t o a - f l u o r o -
6 - u r e i d o - p r o p i o n i c a c i d , urea, and carbon d i o x i d e .

No m e t a b o l i c degradation o f f l u c y t o s i n e i n humans was

130
F LUCY TOSl N E

found a f t e r a s i n g l e o r a l 2 g dose (16). Polak and


Scholer (17, 18) using r a d i o l a b e l e d f l u c y t o s i n e i n
Candida a l b i c a n s , determined t h a t f l u c y t o s i n e e n t e r s
the c e l l by means o f the enzyme c y t o s i n e permease and
i s deaminated t o 5 - f l u o r o u r a c i 1 by c y t o s i n e deaminase.
The 5 - f l u o r o u r a c i 1 thus formed i s i n c o r p o r a t e d i n t o
t h e RNA i n place o f u r a c i l by means o f t h e f o l l o w i n g
pathway ( 1 9 ) .

5- f 1 uorocy tos in e 5 - f 1 uorourac i l+5- f 1 uorour id i n e


monophosphat e
-+5-f1uorouridine diphosphate+5-fluorouridine
triphosphate
+RNA (up t o 50% o f u r a c i 1 replaced by
5-f luorouraci 1)

5.1 Toxicology

The f o l l o w i n g e x c e r p t on t h e t o x i c o l o g y o f f l u -
c y t o s i n e was taken from Drug E v a l u a t i o n Data
(15):

"The LD f o r f l u c y t o s i n e has been s t u d i e d i n


r a t s a d o m i c e . The LD i n r a t s i s r e p o r t e d as
8 g/kg o r a l l y and 100 $&kg i n t r a v e n o u s l y . The
LD f o r mice i s r e p o r t e d t o be 2 g/kg o r a l l y
anaOsubcutaneously, 1.2 g/kg i n t r a p e r i toneal ly
and 500 mg/kg in t ravenous 1 y I ' .
6. Methods o f A n a l y s i s

6.1 Elemental A n a l y s i s

The r e s u l t s from t h e elemental a n a l y s i s a r e


l i s t e d i n Table V (20).

131
EDWARD H. WAYSEK A N D JAMES H. JOHNSON

Table V

Elemental A n a l y s i s o f F l u c y t o s i n e

E 1emen t % Theory % Found

C 37.22 37.24
H 3.12 3.13
N 32.55 32.40
F 14.72 14.60

6.2 Phase S o l u b i l i t y

Phase s o l u b i l i t y a n a l y s i s o f f l u c y t o s i n e may be
c a r r i e d o u t u s i n g methanol as t h e s o l v e n t .
F i g u r e 7 shows a t y p i c a l example and l i s t s t h e
c o n d i t i o n s o f t h e a n a l y s i s (21).

6.3 Thin-Layer Chromatographic A n a l y s i s (TLC)

The t h i n - l a y e r chromatography o f f l u c y t o s i n e
and o t h e r f l u o r o p y r i m i d i n e s has been e x t e n s i v e l y
s t u d i e d by Hawrylyshyn, Senkowski, and W o l l i s h
(22). The R f values presented i n Table V I were
o b t a i n e d u s i n g v a r i o u s s o l v e n t systems w i t h
10 c1g o f sample s p o t t e d on a s i l i c a g e l p l a t e
and developed f o r a t l e a s t 10 cm. The p l a t e s
were a i r d r i e d and viewed under shortwave u l t r a -
v i o l e t radiation.

One and two-dimensional TLC systems a r e g i v e n


by Koechlin, e t a l . (16) f o r the s e p a r a t i o n o f
f l u c y t o s i n e , a-fluoro-8-ureido-propionic a c i d ,
5 - f l u o r o u r a c i l , and urea i n u r i n e . The s o l v e n t
systems used were e t h y l a c e t a t e : f o r m i c a c i d :
water (60:5:35), 2-propanol :ammonia (20: l ) ,
and methanol :water (85: 15).

The f o l l o w i n g TLC system i s used f o r the detec-


t i o n o f f l u o r o u r a c i l i n f l u c y t o s i n e drug sub-
stance (23). Twenty p1 o f a 25 mg/ml s o l u t i o n
o f f ucytosine i n a mixture o f g l a c i a l acetic
a c i d and w a t e r (8:2) i s s p o t t e d o n a s i 1 i c a gel
GF P a t e and developed by ascending chromato-

132
FIGURE 7

PHASE SOLUBILITY ANALYSIS


SAMPLE 8 FLUCYTOSINE
SOLVENT METHANOL
SLOPE: O.57t0.2%
EQUILIBRATION: 20 HRS at 25.
EXTRAPOLATED SOLUBILITY’ 7.01t0.1 mq wr q of SOLVENT

I
I

25 50 75
SYSTEM COMPOSITION rng of SAMPLE per g of SOLVENT
EDWARD H. WAYSEK AND JAMES H. JOHNSON

graphy i n a m i x t u r e o f c h 1 o r o f o r m : g l a c i a l
a c e t i c a c i d (65:35). A f t e r development for a t
l e a s t 14 cm, t h e a i r - d r i e d p l a t e i s viewed u n d e r
shortwave u l t r a v i o l e t r a d i a t i o n . Flucytosine
has an a p p r o x i m a t e R v a l u e o f 0.3 w i t h t h i s
f
system and f l u o r o u r a c i I 0.45 (24).

Table V 1

R f Values f o r F l u c y t o s i n e i n V a r i o u s S o l v e n t Systems (22)


R Value
S o l v e n t System f

1. Ethy 1 a c e t a t e : acetone: w a t e r
(70:40: 10) 0.1
2. E t h y l acetate:methanol:conc.
ammonium h y d r o x i d e (75:25: 1 ) 0.3
3. Acetone 0.02
4. Ether 0.0
5. Ethyl acetate 0.0
6. Methanol 0.6
7. 2-propanol 0.1
8. E t h y 1 a c e t a t e :methano 1 (80 :20) 0.2
9. E t h y l a c e t a t e : m e t h a n o l (75:25) 0.2
10. E t h y l a c e t a t e : w a t e r (100: I ) 0.0
11. E t h y l a c e t a t e : w a t e r (100:3) 0.0
12. E t h y 1 acetate:me t h a n o l :a c e t i c
a c i d (75:25: 1) 0.4

6.4 Non-Aqueous T i t r a t i o n

F l u c y t o s i n e may be t i t r a t e d i n a m i x t u r e of
g l a c i a l a c e t i c a c i d : a c e t i c a n h y d r i d e (2: 1)
using acetous p e r c h l o r i c a c i d w i t h a glass/
calomel e l e c t r o d e system ( 2 3 ) .

6.5 F l u o r m e t r i c Analysis

A s p e c t r o f l u o r o m e t r i c method o f a s s a y i n g f l u c y -
t o s i n e i n b i o l o g i c a l f l u i d s has been r e p o r t e d
b y Wade and Sudlow ( 2 5 ) . Flucytosine i s iso-
l a t e d from p r o t e i n - f r e e b i o o g i c a l f l u i d s by
TLC and e l u t e d f r o m t h e s i l ca g e l w i t h w a t e r .
A f t e r making t h e s o l u t i o n a k a l i n e , t h e

134
FLUCYTOSINE

fluorescence i s measured. E x c i t a t i o n was c a r -


r i e d o u t a t a wavelength o f 300 nm and t h e
emission was measured a t 365 nm.

6.6 D i r e c t Spect rophotomet r i c Ana l y s i s

D i r e c t spectrophotometric a n a l y s i s can be used


t o o b t a i n a q u a n t i t a t i v e assay o f f l u c y t o s i n e
i n capsules (23). I n d i l u t e hydrochloric acid
( 1 i n 100) the r e p o r t e d maximum i s 283-287 w i t h
= 9.2 x 103.

6.7 Fluorine Analysis

6.71 Free F l u o r i d e A n a l y s i s

The d e t e r m i n a t i o n o f f r e e f l u o r i d e i n
f l u c y t o s i n e drug substance can be c a r r i e d
o u t by d i r e c t measurement w i t h a f l u o r i d e -
s p e c i f i c i o n e l e c t r o d e (23). Electrode
p o t e n t i a l measurements a r e made i n a
c i t r a t e containing acetate b u f f e r solution
(pH between 5.0 and 5 . 5 ) . The p o t e n t i a l
measurements a r e converted t o f r e e f l u -
o r i d e c o n c e n t r a t i o n by r e f e r e n c e t o a
c a l i b r a t i o n curve.

6.72 O r g a n i c a l l y Bound F l u o r i n e A n a l y s i s

Bound f l u o r i n e may be determined by


SchHniger combustion f o l l o w e d by measure-
ment w i t h a f l u o r i d e - s p e c i f i c i o n e l e c -
t r o d e (26).

6.8 B i o l o q i c a l Assays

Shadomy, e t a l . (27) i n d i c a t e d t h a t t h e a c t i v i t y
o f f l u c y t o s i n e should be s t u d i e d i n a completely
s y n t h e t i c medium. I n l a t e r s t u d i e s (28) Shadomy
described a c y l i n d e r p l a t e bioassay f o r t h e
d e t e r m i n a t i o n o f f l u c y t o s i n e i n sera, u r i n e s ,
cerebrosp i na f l u i d s , and t ssue homogena tes .
The i n d i c a t o organ ism used was & c e r e v i s i a e
ATCC 9763 on yeas t n i t rogen base agar supple-

135-
EDWARD H. WAYSEK AND JAMES H. JOHNSON

mented w i t h L-asparagine and d e x t r o s e . For s e r a


and o t h e r b i o l o g i c a l f l u i d s t h e lower s e n s i t i v i t y
1 i m i t was 0.4 t o 0 . 5 pg f l u c y t o s i n e / m l . Block
and Bennett (29) r e p o r t e d a c y l i n d e r p l a t e b i o -
assay t h a t p e r m i t t e d t h e d e t e r m i n a t i o n o f f l u -
c y t o s i n e i n b i o l o g i c a l f l u i d s i n t h e presence
o f a m p h o t e r i c i n B which o t h e r w i s e would i n t e r -
f e r e . B l a k e r and D o u t t (30) d e s c r i b e d a d i s c
d i f f u s i o n method f o r a s s a y i n g f l u c y t o s i n e i n
serum and o t h e r body f l u i d s u s i n g a s t r a i n o f
Saccharomyces c e r e v i s i a e as t h e t e s t organism
on y e a s t n i t r a t e base agar. The method was use-
f u l i n t h e c o n c e n t r a t i o n range 2 t o 6 pg/ml.
Holt and Newman (31) a l s o r e p o r t e d a method f o r
t h e r o u t i n e assay o f f l u c y t o s i n e i n b i o l o g i c a l
f l u i d s u s i n g C . a l b i c a n s ( C a r s h a l t o n 2606) as
t h e i n d i c a t o r o r g a n i s m . The method r e a d i l y
d e t e c t e d 0.1 pg f l u c y t o s i n e / m l . Ma ks and
E i c k h o f f (32) s t u d i e d t h e a n t i f u n g a a c t i v i t y
o f f l u c y t o s i n e using b r o t h - d i l u t i o n micro-
t i t e r d i l u t i o n , a g a r - d i l u t i o n , and d i s c
s u s c e p t i b i l i t y tests. Their invest gation
indicated t h a t the disc s u s c e p t i b i l t y t e s t
may be a p p l i c a b l e i n d i a g n o s t i c m i c r o b i o l o g y
l a b o r a t o r i e s f o r y e a s t s such as Candida and
T o r u lops i s .

7. Acknow 1edgemen t s

The a u t h o r s w i s h t o acknowledge M.V. Go,


D r . K. B l e s s e l , E . Kohler, and t h e S c i e n t i f i c
L i t e r a t u r e Department o f Hoffmann-La Roche I n c . for
t h e i r assistance i n the preparation o f t h i s a n a l y t i c a l
p r o f i le.

136
FLUCYTOSINE

8. References

1. S . Traiman, Hoffmann-La Roche Inc., Personal


Communication.
2. R. Gomez, Hoffmann-La Roche Inc., Personal
Communication.
3. W. Benz, Hoffmann-La Roche Inc., Personal
Comnun i c a t ion.
4. Pharmacopeia of t h e h i t e d S t a t e s X I X , 652
(1975).
5. R.J. Rucki, Hoffmann-La Roche Inc., Personal
Communication.
6. R.J. Rucki, Hoffmann-La Roche Inc., Personal
Commun i c a t ion.
7. E . Bass, Hoffmann-La Roche Inc., Personal
Communi c a t i o n .
8. A.M. Chiu and W. McSharry, Hoffmann-La Roche
I n c . , Personal Communication.
9. V . Toome, Hoffmann-La Roche l n c . , Unpublished
Data.
10. T. Ueda and J.J. Fox, J . Am. Chem. Soc., 85,
4024 (1963).
11. L.A. Dolan, Hoffmann-La Roche Inc., Unpublished
Data .
12. R. Gomez, Hoffmann-La Roche Inc., Unpublished
Data.
13. J. Guastella, Hoffmann-La Roche Inc., Unpub-
l i s h e d Data.
14. J.B. Johnson, Hoffrnann-La Roche Inc., Unpub-
l i s h e d Data.
15. Drug I n t e l l i g e n c e and ClinicaZ Pharmacy, 1,75
(1973).
16. B. Koechlin, F. Rubio, S. Palmer, T. G a b r i e l ,
and R. Duschinsky, Biochemical PhamcoZogy,
-
15, 435 (1966).
17. A . Polak and H. Scholer, Path. Microbiol., 2,
148-159 (1973).
18. A. Polak and H. Scholer, Path. MicrobioZ., 39,
334-347 ( 1973) .
19. A . Polak and H. Scholer, Hoffmann-La Roche Inc.,
Basle, Switzerland, Unpublished Data.
20. F. S c h e i d l , Hoffmann-La Roche I n c . , Unpublished
Data .

137
EDWARD H. WAYSEK AND JAMES H. JOHNSON

21. E. Bass, Hoffmann-La Roche Inc., Personal


Commun i c a t i o n .
22. M. Hawrylyshyn, B.Z. Senkowski, and E.G. W o l l i s h
MicrochemicaZ Journal, 8, 15-22 (1 964) .
23. Pharmcopeia of the UniTed States, X I X , 199
(1975).
24. B.C. Rudy and B.Z. Senkowski, AnaZyticaZ
Profiles of Drug Substances, Vol. 2, 1973,
Academic Press, p . 239.
25. D. Wade and H . Sudlow, J . Pharm. Sci., 62, 828
(1973).
26. B . C . Jones, J.E. Heveran, and B.Z. Senkowski,
J . Pharm. S c i . , &, I036 (1971).
27. S. Shadomy, H.J. Shadomy, J.A. McCay, and
..
J P U t z , Antimicrobial Agents and Chemotherapy,
452 (1968).
28. .
S Shadomy, AppZied Microbiology, 17, (6) ,
871-877 . . (1969).
. . ..

29. E.R. B l o c k and J.E. Bennett, AntimkrobiaZ


Agents and Chemotherapy, 1, (61, 476-482
.
( 1 972)
30. R.G. B l a k e r and B.J. D o u t t , AntimicrobiaZ
Agents and Chemotherapy, 2, (6), 502-503
(1972).
31. R.J. H o l t and R.L. Newman, J . Clin. Path.,
-
26, 167-174 (1973).
32. M . I . Marks and T.C. E i c k h o f f , AntimicrobiaZ
Agents and Chemotherapy, 49 1 (1970) .

138
GLUTETHIMIDE

Hassan Y. Aboul-Enein
HASSAN Y. ABOUL-ENEIN

CONTENTS
Analytical Profile - Glutethimide
1. Description
1.1 Nomenc lature
1.11 Chemical Names
1.12 Generic Name
1.13 Trade Names
1.2 Formulae
1.21 Empirical
1.22 Structural
1.3 Molecular Weight
1.4 Elemental Composition
1.5 Appearance, Color, Odor
2. Physical Properties
2.1 Crystal Properties
2.11 Crystallinity
2.12 X-Ray Diffraction
2.13 Melting Range
2.14 Differential Scanning Calorimetry
2.2 Solubility
2.3 Optical Activity
2.4 Molecular Orbital Calculations & Dipole
Moment
2.5 Acid Dissociation Constant
2.6 Identification
2.7 Spectral Properties
2.71 Ultraviolet
2.72 Infrared
2.73 Nuclear Magnetic Resonance
2.74 Mass Spectrum
3. Synthesis
4. Stability and Decomposition Products
5. Metabolism
6. Method of Analysis
6.1 Titrimetric Methods
6.11 Aqueous
6.12 Non-Aqueous
6.2 Colorimetric
6.21 Qualitative
6.22 Quantitative
6.3 Polarographic Analysis
6.4 Ultraviolet Spectrophotometric
6.5 Fluorometric Analysis

140
GLUTETHIMIDE

CONTENTS (cont'd)

6.6 Chromatographic Analysis


6.61 Paper
6.62 Thin Layer
6.63 Gas Chromatography
6.7 High Voltage Electrophoresis
6.8 Biological Assay
6.9 Nuclear Magnetic Resonance

141
HASSAN Y, ABOUL-ENEIN

1. Description
1.1 Nomenclature
1.11 Chemical Names:
a. (+)
- 2-Ethyl-2-phenylglutari-
mide . b. O(-ethyl-O(-phenylglutarimide.
c. 3-ethyl-3-phenyl-2,6-piperi-
dinedione.
d. 3-ethyl-3-phenyl-2,6-dioxo-
piperidine.
e. 3-ethyl-3-phenyl-2,6-diketo-
piperidine.
1.12 Generic Name:
Glutethimide
1.13 Trade Names:
Doriden, Noxyron, Elrodorm.

1.2 Formulae:
1.21 Empirical :
C13H15N02
1.22 Structural :

1.3 Molecular Weight:


217.26
1.4 Elemental Composition:
C, 71.86; H, 6.96; N, 6.45; 0, 14.73

142
GLUTETH lMl DE

1.5 Appearance, Color, Odor:


Glutethimide is a colorless crystalline
white solid, odorless, has a bitter
taste.
2. Physical Properties:
2.1 Crystal Properties:
2.11 Crystallinity
Glutethimide is a crystalline
solid. A typical photomicrograph of glutethi-
mide obtained by sublimation, recrystallization
from 50% aqueous ethanol and recrystallization
from concentrated ammonia is shown in Fig. (1).
These crystalline structures can be used as a
microscopic means of identification of glutethi-
mide (1). The behavior of glutethimide crystals
extracted from the tablet formulations was dis-
cussed by Penprose and Biles (1).
2.12 X-Ray Diffraction:
Analytical x-ray diffraction data
for glutethimide, its anhydrous, hydrous forms
and optical active antipodes were studied in
detail by Bonamico et al. (2) along with other
analogs. The elementarcrystal structure for
these forms are shown in Table I.
Further information regarding
conformation of the chemical structure of glu-
tethimide through analysis of x-ray diffraction
patterns are given by Bonamico e t &. (2).
The optical crystallographic
properties for glutethimide crystals are as
follows ( 3 ) :
q1.572; p 1.585, x 1 . 5 9 0
Optic Sign: Negative 2V. large
Extinction: parallel and inclined
Elongation: positive
System: 6-sided rods and plates

143
f

FA. -
1 - Photomicrographs showing variation in crystal structure of
glutethimide (1). A-sublimation, B-from 50% EtOH, C-from
conc. NH40H.
Table I
X-Ray D i f f r a c t i o n Data f o r G l u t e t h i m i d e
0 0 0

Form a,A b,A C ,A


_ . -z v -B Special Groz

G l u t e t h i m i d e hydrous 7.5320.02 30.50+0.09 10.83+0.03 8 2487E3 D;5 - ?bca


A

8
rhombic prism
G l u t e t h i m i d e anhydrous 20.40+0.06 11.3920.03 2 0 . 6 0 2 0 . 0 6 16 4791A3
0 0.
92 40 220
- P - c&
m o n o c l i n i c prisms 2’COr
Pc - c;-
0
G l u t e t h i m i d e (+)or (-1 6.8720.02 25.7550.07 6.9020.02 4 1221A3
antipode
rhombic p r i s m s
HASSAN Y. ABOUL-ENEIN

2.12 X-ray D i f f r a c t i o n ( c o n t i n u e d )
The c h a r a c t e r i s t i c d i f f r a c t i o n
p a t t e r n s were o b t a i n e d by s u b j e c t i n g g l u t e t h i -
mide powder t o C u K ) I - r a d i a t i o n from x-ray spec-
trometer and r e c o r d i n g t h e d i f f r a c t e d r a d i a t i o n
on a c h a r t , u s i n g a modified GM-tube w i t h a re-
c o r d i n g p o t e n t i o m e t e r (1). The D-distances were
c a l c u l a t e d as shown i n T a b l e 11.
Table I1
10.3
7.23
6.22a
6.01
5. lla
4.59
3.78a
3.52
3.27
3.20

a - Strong
2.13 M e l t i n g Range
The N a t i o n a l Formulary X I V ( 4 )
s p e c i f i e s a m e l t i n g r a n g e f o r g l u t e t h i m i d e be-
tween 8 6 0 and 89O a s a c r i t e r i a of a c c e p t a b i l i t y .
Furthermore, it w a s r e p o r t e d t h a t g l u t e t h i m i d e
showed a m.p. range of 85O-880 w i t h r e s i d u a l
c r y s t a l s growing s l u g g i s h l y o n l y below 80° i n t o
g r a i n s and prisms ( 5 ) . The m e l t s o l i d i f i e s t o
a g l a s s . The g l a s s y powder showed nD 1.5403.
The e u t e c t i c t e m p e r a t u r e of g l u -
t e t h i m i d e w i t h azobenzene and w i t h b e n z i l w a s
r e p o r t e d t o be 53O and 61°, r e s p e c t i v e l y .

Table I11 shows t h e m e l t i n g range


of g l u t e t h i m i d e r e p o r t e d i n t h e l i t e r a t u r e .

146
Table I
X-Ray D i f f r a c t i o n Data f o r G l u t e t h i m i d e

0 0 0
*F -
a,A b,A -
C ,A -2 !! -
B S p e c i a l Group
0
Glutethirnide h y d r o u s 7.5320.02 30.50fp.09 10.83+0.03 8 2 4 8 7 ~ ~ D215 - ’bca
rhombic prism
Glutethimide a n h y d r o u s 20.40+_0.06 11.39+_0.03 20.60+0.06 16
0 o* -
4 7 9 1 ~ ~9 2 4 0 5 2 0 P - Cjh
m o n o c l i n i c prisms 2’COr
Pc - c;

Glutethimide (+)or ( - ) 6.8720.02 25.7550.07 6.9020.02 4 1 2 d 3 P212121


antipode
rhombic prisms
HASSAN Y . ABOUL-ENEIN

Table I11
m.p., C O Reference
91-92 0 1
85-87, bo 3168 6
82-83 ( is6propanal) 7
86-88 anhydrous glutethimide 8
80-85 (EtOAc-pet. ether) 9
83-85 10
68.5.70.5 (ail EtOH) 11
2.14 Differential Scanning Calorimetry
Reubke and Mollica (12) reported
the application of the differential scanning
calorimetry in the quantitative estimation of
purity of glutethimide and the detection of
polymorphism. They reportedAH value for glu-
- cal/gm. at 86.80 - 87O.
tethimide to be 28.7+1
2.2 Solubilit :
d i n water (1.0 mg/ml) , soluble
1 in 5 of ethanol, 1 in 12 of ether and 1 in less
than 1 of chloroform, dichloromethane. Soluble
in acetone, ethyl acetate. A saturated solution
in water is acidic to litmus.
Recently, it was reported that the
tetramethyl-substituted amides of pimelamide,
suberamide, azelamide and sebacamide markedly
enhance the solubility of glutethimide in aqueous
solution (13). The solubility of glutethimide
was increased significantly above the critical
concentrations and from the nature of the
solubility curves, a micellar type of solubili-
zation appears to be dominant.
2.3 Optical Activity
Glutethimide is marketed as the racemic
mixture of 2-ethyl-2-phenylglutarimide. Keberle
et al. (14) describes a procedure for resolution
the racemic mixture to the pure optical anti-
podes as shown in Scheme 1. The sedative-
hypnotic activity of the (+) isomer is 2-3 times
more potent than the ( - 1 isomer (15).

148
GLUTETH IMI DE

H
+,

Pi""
(

It
c 6H 5 -:
-co2 H
(CH2)2C02H

( 2 )-2 -e thy l-2 -pheny lg lu t a r i c acid

I r e s o l v e d w i t h (+) and
(-1 d -phenethylamine

Jy3

H
(-)-isomer
Scheme 1
phn
0

H
(+)-isomer

149
\D
rl
m
rl
*
rl
W
rl
I-
rl
W
rl
*
0
rl

*
0
rl
I
Pl
0

Pl
0
rl
N
0

I
ui
*
0
rl
-
0
rl

*
0
rl
m
0
I

Pl
0
rl
I
N
0
rl rl rl 4
-
X
0
-
X
0
- h : 2
X 8 X
s
w
v

+,
w
rl
s
W
Y -
rl
II
V
I1 N N N
V
Y
+I +I +I
W
I-
W
W
*
W
rl
m
rl rl rl 4
-k I I I
-
ui
NO
U
II
0
ND
T
U

ui
N O
T
u

m
N O
n
8
0
N O
T
u
Ll
Ll a,
a,
8 d
.r(
I
+
h
Y
150
GLUTETHIMIDE

2.3 Optical Activity (continued)


The specific rotations, melting points for
the (+) and (-1 isomers are given in Table IV
as recorded'in the literature.
2.4 Molecular Orbital Calculations and Dipole
Moment
The molecular orbital calculations of
glutethimide was discussed by Andrews (19) using
two methods, extended Huckel theory (EHT) and
complete neglect of differential overlap CND0/2.
Calculated dipole moment (D) of glutethimide was
found to be 8.67 using EHT, while the experi-
mental dipole moment (D) was reported by Lee and
Kumler (20) to be 2.83. Andrews showed that
CNDO/2 method is more suitable for assessing net
atomic charges than the EHT.

H
2.83D

Furthermore, Lee and Kumler (20) con-


cluded from their study that of the dipole
moments of several six-membered cyclic imides
the imide groups in these compounds are in a
cis-cis conformation.
2.5 Acid Dissociation Constant
Doornbos and De-Zeeuw (21) measured the
"proton lost" dissociation constant K H and the
"proton gained" K3H dissociation conszant for
glutethimide by a previously described potentio-
metric titration method at 20°. K2H and K
for glutethimide at ionic strength of p=O.?O was
found to be 4.518.

151
HASSAN Y . ABOUL-ENEIN

2.6 Identification
The f o l l o w i n a i d e n t i f i c a t i o n t e s t s a r e
p u b l i s h e d i n B . P . 1973 ( 2 2 ) a s a p a r t of t h e
i d e n t i f i c a t i o n of g l u t e t h i m i d e .

1. The l i g h t a b s o r p t i o n , i n t h e r a n g e
230 t o 350 nm, o f a 2-cm l a y e r o f a 0.03 p e r c e n t
w/v s o l u t i o n i n d e h y d r a t e d a l c o h o l e x h i b i t s t h r e e
maxima, a t 252 nm, 258 nm, and 2 6 4 nm; e x t i n c t i o n
a t 252 nm, a b o u t 1.0, a t 258 nm, a b o u t 1.1, a n d
a t 2 6 4 nm, a b o u t 0 . 8 6 .
2. Heat 1 g w i t h 5 ml of sodium hydrox-
i d e s o l u t i o n and 1 5 m l o f water on a w a t e r - b a t h
f o r t h i r t y m i n u t e s , cool and a c i d i f y t o l i t m u s
paper w i t h d i l u t e h y d r o c h l o r i c acid; f i l t e r ;
m e l t i n g p o i n t of t h e p r e c i p i t a t e , a f t e r washing
w i t h water and d r y i n g a t l o o o , a b o u t 159'.

F u r t h e r m o r e , Imaoka and Ogura ( 2 3 ) pub-


l i s h e d a p r o c e d u r e f o r i d e n t i f i c a t i o n of g l u -
t e t h i m i d e i n t a b l e t s by s p o t t e s t . A powdered
sample (2-3 mg) on Whatman's T e s t P a p e r i s w e t t e d
w i t h 2 d r o p s a c e t o n e , 1 d r o p 1%c o p p e r acetate o r
1%c o b a l t acetate s o l u t i o n , k e p t f o r 30 s e c o n d s
then 1 drop 1 0 % isopropylamine s o l u t i o n i n
a c e t o n e w a s a d d e d , g l u t e t h i m i d e g i v e s a clear
v i o l e t colored spot.

Most b u l k i n g a g e n t s , e . g . l a c t o s e , s t a r c h ,
s u c r o s e , gun a r a b i c , g l u c o s e o r t a l c w i t h t h e
e x c e p t i o n of a l g i n i c a c i d d o e s n o t i n t e r f e r e w i t h
t h e test.

2.7 Spectral Properties

2.71 Ultraviolet
Glutethimide i n s o l u t i o n absorbs
u l t r a v i o l e t r a d i a t i o n over a b r o a d range t o pro-
d u c e a s p e c t r u m w i t h maximum a t 257 251, 263 nm
( t y p i c a l spectrum, F i g . 2 1 , w i t h A
t h e wave l e n g t h 257 nm ( 2 4 - 2 6 ) .
!&,
= 19 for

152
g0.40
OL

I
0

U
0 20

000 I I
L I
220 260 300 340
WAVE L€ NGT t4, rni i1i m ic rons

Fig. 2-

U l t r a v i o l e t spectrum of g l u t e t h i m i d e i n methanol ( 2 4 ) .

153
HASSAN Y . AEOUL-ENEIN

2. 72 I n f r a r e d Spectrum
The i n f r a r e d s p e c t r u m of g l u t e t h i -
mide i s shown i n F i g . 3. The s p e c t r u m w a s ob-
t a i n e d on a P e r k i n - E l m e r 621 s p e c t r o p h o t o m e t e r
from a K B r p e l l e t .

The s t r u c t u r a l a s s i g n m e n t s h a v e
b e e n c o r r e l a t e d w i t h t h e f o l l o w i n g band f r e -
quencies:

Frequency ( c m - l ) Assignment
319 0-3100 NH s t r e t c h i n g i m i d e
1710 C=O i m i d e c a r b o n y l
1680 C=O i m i d e c a r b o n y l n e x t
t o t h e d -p h en y l group
1200 C-0 stretching
760-700 Monosubstituted phenyl

Further information with regard t o


t h e i n f r a r e d s p e c t r a of g l u t e t h i m i d e i s g i v e n b y
s e v e r a l authors (27-29).

2.73 N u c l e a r M a g n e t i c Resonance S p e c t r u m
A t y p i c a l NMR s p e c t r u m of g l u t e t h i -
mide i s shown i n F i g . ( 4 ) . The sample w a s
d i s s o l v e d i n c a r b o n t e t r a c h l o r i d e . The s p e c t r u m
w a s d e t e r m i n e d on a V a r i a n T-60 NMR S p e c t r o m e t e r
w i t h TMS as the i n t e r n a l s t a n d a r d .

The f o l l o w i n g s t r u c t u r a l a s s i g n -
ments h a v e b e e n made f o r F i g . ( 4 ) .

C h e m i c a l S h i f t (dl Assignment
T r i o l e t 0.80 -CH 9 -CH
--J
7

M u l k p l e t 1 . 8 t o 1.97 -CH2-CH3 a n d t w o
-C>- of glutarimide
ring
S i n g l e t 6.9 7 A r o m a t i c phenyl protons
Broad s i n g l e t 8.27 NH i m i d e , e x c h a n g e a b l e
with D20.

Further information concernins t h e


i n t e r p r e t a t i o n of t h e NMR s p e c t r u m of g l u t e t h i -
m i d e and r e l a t e d c y c l i c imides c a n b e o b t a i n e d

154
FA.
3 -
- I n f r a r e d spectrum of g l u t e t h i m i d e , K B r p e l l e t .
Glutithimide
(1) C

-i g-. 4
F - NMR spectrum of g l u t e t h i m i d e i n CC14 c o n t a i n i n g TMS a s i n t e r n a l s t a n d a r d .
GLUTETH IMlDE

from C a s i n i and S a l v i ' s ( 2 9 ) s t u d y of t h e NMR o f


a s e r i e s of c y c l i c i m i d e s and a l s o by Defay and
Dorlet (30).

2.74 Mass S p e c t r a
The mass s p e c t r u m of g l u t e t h i m i d e
o b t a i n e d by c o n v e n t i o n a l e l e c t r o n impact i o n i -
z a t i o n shows a m o l e c u l a r i o n M+ a t m/e 2 1 7 . The
M+ i o n p e a k i s o n l y 5 % ( F i g . 5 ) . The b a s e p e a k
i s a t m / e 1 1 7 . The mass s p e c t r a l f r a g m e n t a t i o n
mechanism w a s d i s c u s s e d by Rucker ( 3 1 ) a s shown
i n Scheme 2 . Mass s p e c t r o m e t r y w a s a l s o u s e d as
an analytical tool f o r rapid, accurate identi-
f i c a t i o n o f and d i a g n o s i s o f g l u t e t h i m i d e o v e r -
d o s e s and p o i s o n i n g cases ( 3 2 - 3 3 ) .

The c h e m i c a l i o n i z a t i o n ( C I ) mass
s p e c t r o m e t r y o f g l u t e t h i m i d e w a s r e c e n t l y re-
p o r t e d by S a f e r s t e i n and Chao ( 3 4 ) . The i o n i -
z a t i o n w a s a c c o m p l i s h e d by t h e r e a c t i o n of t h e
d r u g w i t h e i t h e r i o n i z e d g a s e o u s methane or i s o -
b u t a n e . The r e s u l t i n g s p e c t r u m w a s l e s s complex
t h a n t h a t p r o d u c e d by c o n v e n t i o n a l E I i o n i z a t i o n
and u s u a l l y r e s u l t s i n t h e f o r m a t i o n of a
molecular i o n p l u s o n e (M+ + 1) i . e . , 218 f o r
glutethimide.
3. Synthesis
X ( C H 2 ) 2-C-CN

@ C2H5C:
CH2CN N aNH2

basic
catalyst

X=-CN
or
roac
H2S04

E t

Scheme 3

157
SRMPLE NUMBER 73Z
G L uT ET H1 M ID E

'001
hi CDI
G I T I 2ED 3
YH3d 35HB m
117
'0R
'09 '0h

132
I
XI 3EltllN3Xf3d

i 115
158

1 1 GO

i I
'032
'0

5.m. I am. I !in.


M/E

- -
Fig. 5 - Mass Spectrum of Glutethimide (EI).
w
(I)

c,
5
a,

0
0
k
a
GLUTETHIMIDE

\ rnlr = 189 m/c .


146

m/e = 137 m/e = 117

Scheme -
2 - Mass spectral fragmentation mechanism
of glutethimide (31).

159
HASSAN Y . ABOUL-ENEIN

Glutethimide is synthesized as
shown in Scheme 3. Starting with benzylcyanide
which is treated with ethyl chloride or bromide
in the presence of sodium amide to yieldd-ethyl
benzylcyanide. This is then allowed to react
with methyl acrylate or acrylonitrile (Michael
condensation) under the catalytic influence of
piperidine or another suitable basic catalyst,
thus, forming methyl 4-cyano-4-phenylhexanoate
or its corresponding dinitrile derivative,
respectively. The latter intermediate was
purified by distillation and cyclized in the
presence of acetic acid and 9 0 % sulfuric acid
to yield glutethimide (6, 35-37).
Another synthetic route for glu-
tethimide is described by Iwase -
et -
al. ( 9 )
shown in Scheme 4. A mixture of l-phenyl-l-
ethylpropane-1,3-dicarboxylic acid and p-nitro-
soaniline was treated at 180-185O under nitrogen
atmosphere for 5 hrs., refluxed with 2% sodium
hydroxide for 3 hrs., acidified, washed with
ether, neutralized with sodium carbonate, ex-
tracted with chloroform to yield glutethimide.

NO Et

1
H
NH2

Scheme 4

160
GLUTETHIMIDE 13.

O t h e r r o u t e (38) f o r g l u t e t h i -
mide s y n t h e s i s i s shown i n Scheme 5.

HC 1
CH 2 =CHCN 9- C1CH2CH2C02CH (CH3)
isopropanol

i s o p r o p y l /3-chlarnproDionate

dimethyl p i p e r i d i n e
sulfate

1
‘gH5
C gHg$HCN
C2H5

Scheme 5

161
HASSAN Y. ABOUL-ENEIN

4. Stability & Decomposition Products


Several reports showed that glutethimide is
hydrolyzed to h-ethyl-4-phenylglutaramic acid in
alkaline solution (36, 39). Yamaha and Mitzukami
(40) reported that this decomposition occurs in
sodium hydroxide and sodium carbonate solutions
by second order reaction but not in sodium bicar-
bonate solution. The degradation rate constants
at 20°, 30° and 400 in 0.01N sodium hydroxide
were 7.66 x 1.67 x 10-1 and 3.56 x 10-1
a?d,
those in 0.01M sodium carbonate were 3.56 x 10-
respectively. The activation energies in 0.01N
sodium hydroxide and 0.01M sodium carbonate were
14.3 and 14.6 K cal., respectively. Furthermore,
Wesolowski et al. (41) studied the kinetics of
degradationoflutethimide in buffered aqueous
solutions in the pH range 1.5-8.0. They sup-
ported the previous reports that the degradation
of glutethimide is a base-catalyzed reaction
since the contribution of ionized species to the
reaction rate in the pH range studied is very
small. The rate is first order with respect to
the concentration of glutethimide. The apparent
energy of activation Ea, for the degradation of
glutethimide at pH8 is found to be in the order
of 25.86 K cal./mole. However, the energy of
activation corrected for heat of ionization of
water at SOo, 60°, and 650 was found to be
13.47 K cal., 13.92 K cal., and 14.15 K cal./mole,
respectively.
The mechanism proposed for the glutethimide
degradation is shown in Fig. 6 and involves
direct attack by a hydroxyl ion on the unhindered
carbonyl of the glutarimide ring followed by the
cleavage of the ring to 4-ethyl-4-phenyl-glu-
taramic acid.

162
GLUTETHIMIDE

r 1
8
Ph OH,
7
0 0 Slow
HO c =o
N
H H

Fig. 6
The Mechanism of G l u t e t h i m i d e
Degradation ( 4 1 ) .

A p l o t of l o g t+ v e r s u s pH ( F i g . 7) shows t h a t
below pH5, g l u t e t h i m i d e i s v e r y s t a b l e s i n c e a t
pH 1.0 and 5.0 t h e r a t e i s independent of hydro-
gen i o n c o n c e n t r a t i o n .

163
24
2.2
t-
20 -
1.8 -
1.6 -
CI
v)
5C
r”
I4

1.2
k\
i?
-J
Q8C

0.6 -
04-

0.2 -
0.0 0

40 5.0 6.O 7.0 t3c 9


PH

- - 7
Fig. -
Log t4, the half-life of the reaction in months
against pH at 25’ (41).

164
GLUTETH l MI DE

5. Metabolism
The distribution and metabolism of glutethi-
mide in the dog and rat were extensively studied
and summarized by Keberle - et al.
- (42). The
metabolic fate in man was studied by B’itikofer
et al.
- - (43). Both of these studies indicated
that the (+)-isomers are metabolized via two
different Eiochemical routes. The ( r
e
m
o
s
i
-
)
+ was
hydroxylated on the glutarimide ring at the 4-
position to yield 4-hydroxy-2-ethyl-2-phenyl-
glutarimide, a small percent of which undergoes
dehydration to give 2-ethyl-2-phenylglutaconimide.
The (-)-isomer predominantly hydroxylated on the
ethyl side chain to form 2-(l-hydroxyethyl)-2-
phenylglutarimide, of which a small percent lose
a moleculae of acetaldehyde to form 2-phenyl--.
glutarimide, Fig. ( 8 ) . In 1969, Post and Schutz
(44) identified a phenolic metabolite which they
assumed to be 2-(4-hydroxyphenyl) glutarimide.
Recently Stillwell -
et -
al. (45) identified
more polar metabolites present in the urine of
rats and guinea pigs, (Fig. 9). They char-
acterized these metabolites by combined GC/MS
as follows:
a. 4-hydroxy-2-ethyl-2-phenylglutarimide
b. 3-hydroxy-2-ethyl-2-phenylglutarimide
C. 2-(l-hydroxyethyl)-2-phenylglutarimide
d. 2-ethyl-2-(4-hydroxyphenyl)glutarimide
e. 2-ethyl-2-(3,4-dihydroxyphenyl)glutarimide
f. 2-ethyl-2-(3,4-dihydroxy-l,5-~yclohexa-
dien- l-yl)glutarimide
g. 2 - e t h y l - 2 - p h e n y l g l u t a c o n i m i d e
h. 2-phenylglutarimide
They demonstrated that the hydroxylation of
the aromatic ring in the rat and guinea pig occurs
V J an epoxide pathway. These authors also
supported the fact that the rat and guinea pig,
like the dog and man, metabolize the (-)-isomer
of glutethimide by hydroxylation of the ethyl
side chain to form 2- (1-hydroxyethyl)-2-phenyl-
glutarimide. However, the metabolism of the ( + I -

165
(+)-isomer
Dextrorotatory

T i 0

H
.

glucuronidation dog
man
rat
O

guinea pig
C

2%
~

0
H S G 1 u - o ~ o C 6 H 5

0
H
do9
man
rat
45%

guinea pig

Glu = glucuronic acid

H
(+) Glutethimide

(-)-isomer
Levorotatory

0:
0
0 -CH3CHO,

0
C6H5

0 0 0
-N-
h- n
L 1 H
45%
4%
man do9
rat man
r a t and guinea pig
guinea pig and dog

Fig. 8 Metabolism of Glutethimide


OH

H H H
man rat Man (minor)
guinea p i g

OH

0 N
N
u
n
H H
rat rat rat
guinea p i g guinea p i g (minor) guinea p i g

F i g . 9 P o l a r M e t a b o l i t e s of G l u t e t h i m i d e
HASSAN Y . ABOUL-ENEIN

isomer seems to be species dependent. In con-


trast to man and dog, the rat apparently metabo-
lized the (+)-isomer of glutethimide by hydroxy-
lating the aromatic as opposed to the hetero-
cyclic ring to form 2-ethyl-2-(4-hydroxyphenyl)-
glutarimide, however, some hydroxylation of the
heterocyclic ring occurs. The guinea pig hy-
droxylates both the aromatic and the hetero-
cyclic rings and these metabolites are presumably
formed from the (+)-isomer. Further studies on
the metabolism of the ( - ) and (+) isomers by the
rat and guinea pig are needed to validate these
conclusions.
Ambre and Fischer (46) showed that monohy-
droxylated metabolite of glutethimide accumulated
in the plasma of humans intoxicated with glutethi-
mide overdose. This metabolite was subsequently
isolated from urine of dogs given large doses of
glutethimide and was chemically identified as
4-hydroxyglutethimide (47). This metabolite was
found to possess twice the sedative-hypnotic
activity of glutethimide (47).
Recently, it was shown that the concentration
of 4-hydroxyglutethimide is highest when the
respiratory status of the patient was near its
lowest (48, 49). This led to the conclusion that
4-hydroxyglutethimide accumulation plays an
important role in acute glutethimide poisoning.
4-Hydroxyglutethimide and other potential glu-
tethimide metabolites were chemically synthesized
and pharmacologically tested by Aboul-Enein -
et -al.
(50).

Among other metabolites previously identified


in animals and man, Andreson _ al. (51) re-
et -
cently reported the identification and chemical
characterization of 2-ethyl-2-(4-hydroxyphenyl)
glutarimide in human urine from patients over-
dosed with glutethimide as a major metabolite.
They also characterized 2-ethyl-2-(3-methoxy-4-
hydroxypheny1)-glutarimide as a new metabolite of
glutethimide.

168
GLUTETHIMIDE

The toxic effects possibly caused by the


metabolites of glutethimide has interested many
research laboratories to continue investigating
this problem.
6. Methods of Analysis
6.1 Titrimetric Methods
6.11 Aqueous
A titrimetric method was developed
for analysis of glutethimide. The procedure in-
volves the alkaline hydrolysis of glutethimide
with standard alcoholic potassium hydroxide and
subsequent back titratation of the unconsumed
alkali with standard hydrochloric acid using
phenolphthalein as indicator (521, a method
adapted by the British Pharmacopeia (22).
6.12 Non-Aqueous
Ellert et
-- al. (53)
. . described a
method for determination of glutethimide by non-
aqueous titration with 0.1N sodium methoxide in
methanol-benzene in a solution of ethylenedi-
amines (against 0-nitroaniline) or in pyridine
(against Azo Violet). Glutethimide can also be
quantitated in tablet formulation by non-aqueous
titration in dimethyl formamide and titrating
with propanol-benzene 0.1N potassium hydroxide
(using metanil yellow as indicator) (54).
6.2 Colorimetric
6.21 Qualitative
The following color reaction is
published in the B.P. 1973 (22) as part of an
identification scheme.
Shake 10 mg with 2 ml of water,
0.1 g of hydroxylammonium chloride and 1 ml of
sodium hydroxide solution, allow to stand for
ten minutes and add 2 ml of dilute hydrochloric
acid and 1 ml of ferric chloride test-solution;
a deep brownish-red color is produced.

169
HASSAN Y . ABOUL-ENEIN

6.22 Quantitative
Sheppard ( 3 9 ) and a s s o c i a t e s
u t i l i z e d a method based on t h e f o r m a t i o n of t h e
c o l o r e d complex between f e r r i c i o n and t h e
hydroxamate r e s u l t i n g from t h e r e a c t i o n of g l u -
t e t h i m i d e w i t h a l k a l i n e hydroxylamine f o r d e t e r -
m i n a t i o n of t h e drug i n u r i n e . The c o l o r w a s
r e a d w i t h i n f i v e minutes a t 5 1 0 nm. The
p r e s e n c e of l a c t o s e and a n i o n s which complex w i t h
f e r r i c i o n o r s a l t s which form p r e c i p i t a t e s w i t h
f e r r i c i o n i n t e r f e r e w i t h t h i s method. Phang
-
e t - a l . ( 5 5 ) i n t r o d u c e d a m o d i f i c a t i o n t o Shep-
p a r d ' s procedure so t h e f a t t y i m p u r i t i e s do n o t
i n t e r f e r e w i t h t h e q u a n t i t a t i o n of g l u t e t h i m i d e
i n vomitus m a t e r i a l .
Belova and Zinakova (56) re-
p o r t e d a s i m i l a r c o l o r i m e t r i c method i n which
t h e c o l o r developed was measured a t 4 9 0 nm w i t h
a no. 5 l i g h t f i l t e r and claimed t h a t t h e
p r e s e n c e of l a c t o s e does n o t i n t e r f e r e w i t h t h e
r e s u l t of g l u t e t h i m i d e d e t e r m i n a t i o n .
T h i s p r o c e d u r e , however, i s n o t
v e r y s e n s i t i v e o r a p p l i c a b l e t o blood specimens
and i s r e l a t i v e l y n o n - s p e c i f i c ( 5 7 ) .

6.3 Polarographic Analysis


G l u t e t h i m i d e f a i l e d t o have p o l a r o -
g r a p h i c wave on a . c . and d . c . polarograms u s i n g
0.1M l i t h i u m c h l o r i d e , o r tetramethylammonium
c h l o r i d e as s u p p o r t i n g e l e c t r o l y t e (58).
R e c e n t l y , a d e t a i l e d s t u d y of a n i n -
d i r e c t p o l a r o g r a p h i c d e t e r m i n a t i o n of g l u t e t h i -
mide a f t e r n i t r a t i o n was r e p o r t e d by Lauermann
( 5 9 ) . The method c a n be used t o q u a n t i t a t e
g l u t e t h i m i d e i n post-mortum t i s s u e s and b i o -
l o g i c a l f l u i d s , w i t h s e n s i t i v i t y of 0 . 8 mg/100g
of m a t e r i a l . The c o n v e r s i o n of t h e phenyl group
t o n i t r o p h e n y l group a f t e r n i t r a t i o n makes it
possible t o u t i l i z e t h e polarographic technique
i n a n a l y s i s of g l u t e t h i m i d e .

170
GLUTETHlMl DE

6.4 Ultraviolet Spectrophotometric


The ultraviolet absorption of glutethi-
mide in methanol at 257 nm (60) is-used as
a sensitive criteria for its analysis. This
method is sensitive to a concentration range of
50-500 pg/ml (61).
Ultraviolet spectroscopy has been exten-
sively applied to glutethimide determination in
biological fluids such as (62-64) serum and urine
and pharmaceutical preparations (54, 65) with
high degree of sensitivity.
6.5 Fluorometric Analysis
A fluorometric procedure was described
for the quantitative determination of glutethi-
mide in table formulations (66). The method
is based on the fluorogen resulting from the
reaction of glutethimide with conc. sulfuric
acid containing formaldehyde. The fluorescent
intensity is a straight line function of con-
centration over a wide range. The excitation
maxima were observed at 280 and 365 nm and the
corresponding maximum fluorescent emission
occurred at 450 nm. The procedure is applicable
for the determination of glutethimide in the
presence of its degradation product 4-ethyl-4-
phenylglutaramic acid, since the latter yields
only 1/10 the fluorescence of glutethimide. The
fluorescent reaction does not occur with com-
pounds lacking a phenyl group or containing a
substituted phenyl ring and there is no inter-
ference with the tablet excipients.
6.6 Chromatographic Analysis
6.61 Paper
The chromatographic behavior of
qlutethimide and related analogs was established
6y Davies and Nicholls (67) without interference
from the chemically related barbiturates by
ascending paper chromatography. Whatman No. 1
paper was used and some paper was impregnated
with liquid paraffin ( 4 % in hexane), olive oil

171
HASSAN Y. ABOUL-ENEIN

(20% in acetone) or tributyrin (10% in acetone).


All compounds were applied in amounts of 100 )Ig
by the window technique.
Several reagents were used to detect glu-
tethimide after chromatography. These include
the hypochlorite reagent by Creig and Leaback
(681, alkaline, hydroxylamine spray of Sheppard
-
et -
al. (39), and 1% HgN03 solution (69). Some of
the most useful solvent systems are shown in
Table V.
Table V
Solvent System Reference
PhMe-HOAC-H 0 10:5:4 67
CC1 HOACmH20 1:2:1 67
aq. 4iaC1 1 0 % w/v 67
0.066M Na3P04(pH 7.3 on tri- 67
butyrin paper
A method for rapid detection of
glutethimide and other hypnotic was described
(70) using pigment impregnated paper, and a
combination of 2 dimensional chromatographs.
Solvent system used in this technique were
piperidine-petroleum ether (1:9) followed by
cyclohexane, CHC13 (1:3) or solvent system
piperidine-petroleum ether (1:9) followed by
benzene: CHC13 (1: 5 ) .
The applicability of pigment
impregnated paper for the resolution of hypnotics
in cadaveric material was also discussed.
Kloecking (69) described the use
of wedge-strip procedure to achieve better
separation of glutethimide and other barbiturates
in chemical-toxicological analysis than the usual
ascending descending chromatograms. A good
separation was obtained by using ascending
chromatograms with dioxane-xylene-toulene-iso-
propanol-258 NH (1:2:1:4:2) upper phase on
Schleicher and Zchnell 2045 Bgl paper for 24

172
GLUTETHlMl DE

h o u r s a t 20°. Other p e r t i n e n t
i n f o r m a t i o n i n t h i s r e g a r d i s t o b e found i n
references (71-74).

6.62 Thin Layer


S e v e r a l t h i n l a y e r chromato-
g r a p h i c s y s t e m s have been d e v e l o p e d t o s t u d y
v a r i o u s a s p e c t s of g l u t e t h i m i d e . S e v e r a l p r o -
cedures w e r e described f o r its detection,
identification i n biological fluids, forensic
and t o x i c o l o g i c a l a n a l y s i s , (82-90). S e v e r a l
s o l v e n t s y s t e m s u s e d f o r i t s i d e n t i f i c a t i o n and
s e m i q u a n t i t a t i o n of g l u t e t h i m i d e a r e shown i n
Table V I .

6.63 G a s Chromatography
Many i n v e s t i g a t o r s h a v e u s e d
g a s chromatography t o a n a l y z e g l u t e t h i m i d e . A l -
t h o u g h most of t h e p u b l i s h e d w o r k i s c o n c e r n e d
almost e n t i r e l y w i t h a n a l y s i s of g l u t e t h i m i d e
and i t s m e t a b o l i t e s i n b i o l o g i c a l f l u i d s and
t i s s u e s , many of t h e methods c o u l d b e m o d i f i e d
e a s i l y f o r a n a l y s i s of g l u t e t h i m i d e i n pharma-
c e u t i c a l dosage forms.

A number o f t h e s y s t e m s u s e d f o r
GC are l i s t e d i n T a b l e VII. Berry ( 9 1 ) conducted
i n - d e p t h s t u d y f o r t h e g a s c h r o m a t o g r a p h i c be-
h a v i o r of g l u t e t h i m i d e i n p r e s e n c e of s e v e r a l
b a r b i t u r a t e s on 1 2 GC columns. OV-225 and CDMS
were t h e most u s e f u l of t h e columns t e s t e d f o r
a n a l y s i s of t h e b a r b i t u r a t e s , g l u t e t h i m i d e and
i n t e r f e r i n g d r u g s a t t h e r a p e u t i c and o v e r d o s e
l e v e l s i n p l a s m a . The method w a s s e n s i t i v e ,
r a p i d a n d s u i t a b l e f o r emergency t o x i c o l o g i c a l
cases.
Bloomer e - a l . (92) d e v e l o p e d a
t -
r a p i d method f o r d i a g n o s i s of s e v e r a l a c i d i c and
n e u t r a l s e d a t i v e - h y p n o t i c s u s i n g GC i n i n t o x i -
c a t e d p a t i e n t s . The p r o c e d u r e p e r m i t s s i m u l -
t a n e o u s i d e n t i f i c a t i o n and measurements of a

173
Table VI
Solvent Svstem Developer Rf Reference
CHC13:Et20 85:15 KI-benzidine 0.66 75
CHC13 :EtOH 90:10 KI-o-tolidine HC1 0.96 75
(after treatment
with gaseous C12)
EtOAcaeOH:NH385:5:2.5 0.1% KMnO4 and 1% 0.89 76
Ag NO3
CHCI3:Me2CO 9: 1 1% HgS04 Rf were reptd. 77
on various
commerc. avail. 77
2
-4
P
EtOAc:MeOH:NH40H 85:lO :5
silica gel tlc,
Cyc10hexane:CgHg :Et2NH 15 :3:2 plates, sheets, 77
films.
CHC13 :Me2C0 9 :1 0.2% aq. solution 78
CuSO with pyridine
(50:4,-grey color
developed.
IsoPrOH :CHCl :2 5%NH4OH 0.84 79
45: 45:lO
Table VI (cont'd)

Solvent System
Et0Ac:cyclohexane-dioxane:
Developer 3 Reference
79
MeOH:H20:NH40H 50:50:10:10:
1.5:0.5
50:50:10:10:0.5:1.5
EtOAc-cyclohexane:MeOH :NH40H 0.1% diphenyl carba- 0.89 79
56:40:0.8:0.4 zone (orange pink-
70:15:10:5 spot):1% AgOAc (blue-
purple spot: HgS04
(purple fades away).
EtOAc:cyclohexane:NH~0H 79
50: 40 :O .1

EtOAc :cyclohexane:NH40H : 79
MeOH:H20 70:15;2:8:0.5
CHC13:BuOH:HC02H 70:40:3.5 1% diphenyl carba-
(chamber saturated with zone;Hg (NO3) 80
NH40H)
EtOAc (redisti1led) W at 254 nm C12 0.68 81
Dioxane: CH2C1 :H20 l:2:1 and starch KI 0.96
1
CHC13 :Me2C0 9 : solution 0.55
Table VII
Gas Chromatographic Systems Used for Glutethimide Analysis
All systems listed used flame ionization detectors.
Column Carrier Gas Column Temp, Co Ref.
3% OV-17 on Supelcoport 55 ml/min He 160-2200 at 7"/min 95
(100-120 mesh) (derivatized
with dimethylformanide
dimethyl acetal) .
3% OV-1 on 100-120 mesh Gas 30 ml/min He 120-2200 at 1o0/min 96
Chrom Q
A
4 % OV-17 on 80-100 mesh 15 183 97
o, Chromosorb WHPAW-DMSC psi N2
3.8% SE-30 on 80-100 mesh 25 183 97
Chromosorb WHPAW-DMSC psi N2
10% Dexsil 300 on 800-100 N2 220 98
mesh Chromosorb WHP
17% Dexsil 300 on 80-100 220 98
N2
mesh
Chromosorb WHP
1% Carbowax 20M* 50-60 ml/min N2 205 91
* on 80-100 mesh Chromosorb W, HP.
T a b l e V I I (cont'd)

Column Carrier Gas Column T e m p , Co Ref.


3% P o l y A - 1 0 3 * 50-60 m l / m i n N2 2 15 91

3% NGA+0.07% t r i m e r acid* 50-60 m l / m i n N2 230 91

1%SP-1000* 50-60 m l / m i n N2 230 91

3% PPE-20* 50-60 m l / m i n N2 200 91

1 0 % A p i e z o n L* 50-60 m l / m i n N2 1 90 91

2
4 % CDMS* 50-60 m l / m i n N2 220-240 91
-I
-J
3 % OV-l* 50-60 m l / m i n N2 155 91

5% OV-17* 50-60 ml/min N2 200 91

3% OV-25* 50-60 m l / m i n N2 1 65 91
4% ov-210* 50-60 ml/min N2 160 91

4 % OV-225* 50-60 m l / m i n N2 205 91

3% OV-225 o n 8 0 - 1 0 0 mesh 4 0 m l / m i n N2 200 99


Chromosorb W
Table VII (cont'd)
Column Carrier G a s Column Temp, Co Ref.
8% X F 1112 on 60-80 mesh 25 ml/min N.,4 19 5 100
Chromosorb WHMDS

7% DC-200 on G a s Chrom Q 50 ml/min He 19 0 10 1


(80-100 mesh) derivatized
as N methyl derivative
5% SE-30 on 70-80 mesh AW 60 ml/min N2 19 5 10 2
Chromosorb W

4
-l
Q)
* on 80-100 mesh chromosorb, W, HP.
GLUTETHIMIDE

variety of sedative agents. By virtue of acid-


base extraction with chloroform, the acidic
and neutral agents are separated. The chloro-
form extracts were dried, evaporated and the
residue dissolved in isopropanol. Column used
was 3% SE-30 on 80-100 mesh Chromosorb W.
Gel filteration using Sephadex
LH-20 was used to remove impurities from chloro-
form extracts of acidified blood, urine and
homogenized tissue sample. Quantitative deter-
mination was made by GC using 15% SE-30 in
Chromosorb W at column temperature of 15Oo-25O0
(93).

Fischer and Ambre ( 9 4 ) showed


that analysis of urine from patients intoxi-
cated with glutethimide on columns containing
SE-30, OV-1 and OV-17 lead to an overestimation
of the unchanged drug in urine. These columns
were considered non-selective. However, 3% OV-
225 on 80-100 mesh Supelcoport and 2% Carbowax
20M on 100-120 mesh Supelcoport were selective
to separate the drug from its potentially
interfering metabolites and thus, eliminates the
possible overestimation of glutethimide in
biological samples.
Several gas chromatographic
methods for measurement of glutethimide in
biological fluids are reported employing a
variety of extraction procedures and gas chro-
matographic conditions (103-123).
6.7 High Voltage Electrophoresis
Hoffman -
et -
al. (124) described a method
for the separation, detection and semiquanti-
tation of glutethimide and other barbiturates
from urine and other biological fluids using
high voltage electrophoresis. It was reported
that glutethimide migrates at the cathode side
at a distance of 0.22 relative to crotylbarbital.

179
HASSAN Y . ABOUL-ENEIN

6.8 B i o l o g i c a l Assay
G l u t e t h i m i d e c a n b e d e t e c t e d by hemag-
g l u t i n a t i o n i n h i b i t i o n method d e s c r i b e d by
Valentour e t a l . (125). A n t i s e r a from r a b b i t s
i n j e c t e d w i t h g l u t e t h i m i d e - b o v i n e serum a l b u m i n
( 1 0 mg/ml) w e r e s e n s i t i v e enough t o d e t e c t 50 n g
g l u t e t h i m i d e /0.1 m l plasma o r u r i n e .

The method w a s u s e d t o a n a l y z e u r i n e
and plasma p a t i e n t s s u s p e c t e d of g l u t e t h i m i d e
intoxication.

6.9 N u c l e a r M a g n e t i c Resonance
Aboul-Enein (126) h a s r e p o r t e d a method
f o r q u a n t i t a t i v e d e t e r m i n a t i o n of- g l u t e t h i m i d e by
NMR b o t h i n powder and t a b l e t f o r m u l a t i o n s . The
method o f f e r s a r a p i d , a c c u r a t e p r o c e d u r e f o r
a n a l y s i s of t h e d r u g . F u r t h e r m o r e , i t p r o v i d e s
a confirmatory i d e n t i f i c a t i o n f o r glutethimide.

180
GLUTETHIMIDE

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184
GLUTETHIMI DE

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186
GLUTETHIMIOE

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Acknowledgment
The author expresses appreciation to
Miss Marie Belmonte for typing the manuscript and
Mr. S. Kobrin for his technical assistance in
photographing the figures in this profile.

187
LEVODOPA

Ralph Gomez, Robert B. Hagei, and Edward A . MacMulian


RALPH GOMEZ eta/.

INDEX

Analytical P r o f i l e - Levodopa

1. Description
1.1 Name, Formula, Mol ecul a r Weight
1.2 Appearance, Color, Odor

2. Physical P r o p e r t i e s
2.1 I n f r a r e d Spectrum
2.2 Nuclear Magnetic Resonance Spectrum
2.3 U l t r a v i o l e t Spectrum
2.4 Fluorescence Spectrum
2.5 Mass Spectrum
2.6 Optical Rotation
2.7 M e l t i n g Range
2.8 D i f f e r e n t i a l Scanning C a l o r i m e t r y
2.9 Thermogravi m e t r i c A n a l y s i s
2.10 S o l u b i l i t y
2.11 C r y s t a l P r o p e r t i e s
2.111 X-Ray D i f f r a c t i o n
2.1 12 C r y s t a l S t r u c t u r e
2.12 D i s s o c i a t i o n Constant

3. Synthesis

4. Separation o f Racemates

5. Stabi 1 it y and Degradation

6. Drug M e t a b o l i c Products
7. Methods o f A n a l y s i s
7.1 E 1ernent a 1- An a 1y s is
7.2 Phase S o l u b i l i t y A n a l y s i s
7.3 Chromatographic Methods
7.31 Thin-Layer Chromatography
7.32 Paper Chromatography
7.33 Gas-Liquid Chromatography
7.34 Column Chromatography
7.4 D i r e c t Spectrophotometric Analysis
7.5 Col o r i met ri c Ana 1y s is
7.6 Non-Aqueous T i t r a t i on
7.7 Determination o f D-Dopa

190
LEVODOPA

8. Acknow 1 edgements
9. References

191
RALPH GOMEZ et al.

1. Descri p t i on
1.1 Name, Formula, Molecular Wei q h t
Levodopa i s (-)-3-(3,4-Dihydroxyphenyl)-L-
alanine.

HO b C H 2 C H C OI O H

NH2

1.2 Appearance, Color, Odor


Levodopa i s an odorless w h i t e t o o f f - w h i t e
c r y s t a l 1ine powder.

2. Physical P r o p e r t i e s
2.1 I n f r a r e d Spectrum
The i n f r a r e d spectrum o f levodopa is shown i n
Figure 1 ( 1 ) .
The spectrum was recorded on a Perkin-
Elmer Model 621 G r a t i n g I n f r a r e d Spectrophotometer and was
measured i n a K B r p e l l e t which contained 1 mg o f levodopa
i n 300 mg o f KBr.

The f o l 1owi ng a b s o r p t i ons have been assigned


f o r Figure 1 :
a. OH s t r e t c h i n g (bonded): 3375 cm'l, 3210 cm-l
b. NH +: 3070 cm-1, 2700-2300 cm-1 (broad)
c. CO&: 1656 cm-1, 1569 cm-1
d. Aromatic CH o u t o f plane bending o f two a d j a c e n t
free H's: 821 cm-1, 816 cm-1

2.2 Nuclear Magnetic Resonance Spectrum (NMR)


The NMR spectrum o f levodopa, recorded on a JEOL
C60HL spectrometer, i s shown i n F i g u r e 2 ( 2 ) . The spectrum
was recorded u s i n g a s o l u t i o n o f 60 mg o f levodopa/0.4 m l
D20 t 0.1 m l DCL. The s p e c t r a l assignments a r e l i s t e d i n
Table I .

192
Id
P
0
-0
0
>
W
-1
cc
0
5
L
c,
V
Q)
P
Ln
-0
W
L
Id
L
+
ET
U
t-
lo-
rD \, I
0
1 I
“8 0
co
0
(0 d- 8
3 3 N V l l l W S N V H l Yo
193
FIGURE 2
NMR Spectrum o f Levodopa
LEVODOPA

TABLE I

NMR Spectral Assignments f o r Levodopa

Proton Chemical S h i f t (6) Spectral S t r u c t u r e (J i n H z l


3.22 ppm Mu1 t i p l e t (7.5)
4.44 ppm T r i p le t
6.80 ppm Doublet (2 sets; 2.0, 8.0)
6.92 ppm Doublet
7.05 ppm Doublet

The remaining protons exchange i n t h e deuterated solvent


used.

2.3 U l t r a v i o l e t Spectrum
The u l t r a v i o l e t spectrum o f levodopa ( 4 mg o f
levodopa/100 m l o f 0.1N h y d r o c h l o r i c a c i d ) i n t h e r e g i o n
o f 230 t o 370 nm e x h i b i t s one maximum a t 280 nm ( E =
2.8 x 103) and one minimum a t 250 nm. The spectrum i s
shown i n Figure 3 ( 3 ) .
2.4 F1uorescence Spectrum
The e x c i t a t i o n and emission spectra o f levodopa
are presented i n Figure 4 ( 4 ) . The sample was dissolved
i n methanol a t a concentration o f 1.43 mg o f levodopa/ml
and t h e spectra were recorded using a Farrand MK-1
recording spectrofluorometer. Levodopa e x h i b i t s e x c i t a t i o n
maxima a t 236 nm and 286 nm and emission peaks a t 320 nm
and 620 nm.
2.5 Mass Spectrum
The low r e s o l u t i o n mass spectrum o f levodopa i s
shown i n Figure 5 ( 5 ) . The spectrum was obtained using a

195
RALPH GOMEZ etal.

FIGURE 3
U1t r a v i ol e t Spectrum o f Levodopa

0.6

0.f

0.4

w
u
O.!
2a
8
m
0.2

0.I

I I I
250 300 350
NANOMETERS

196
- 8W
0
a
-8
P
0
-0
0 - 8In
>
a
-I
cc 0
0
-8, K
5L I-
U
c, O U
V
a
P
-s., z
Ln a
a 0 2
V
c -$
a
V
v)
a
L
0
- 8m
3
F
LL
- 8m
-s:
(u
197
FIGURE 5
Mass Spectrum o f Levodopa

100

90

80
A l I S N 3 1 N I 3hllWl3tl

70
5
3
I-
60
198

5 50
w
2
I-
40
a
-I
W
30
a
20

10

0
50 100 150 200
m/e
LEVOOOPA

Varian MAT spectrometer w i t h an ionizing voltage of 70 eV,


which was interfaced w i t h a Varian data system 6201. The
data system accepts the output of the spectrometer,
calculates the masses, compares the i n t e n s i t i e s t o the base
peak and plots this information as a s e r i e s of l i n e s whose
heights are proportional t o the i n t e n s i t i e s .
The molecular ion f o r levodopa was measured a t
m/e = 197. Other c h a r a c t e r i s t i c masses were observed a t
m/e = 179 corresponding t o the loss of H20 from the
molecular ion, m/e = 152 which corresponds t o the loss of
COOH from the parent mass, and m/e = 123, the base peak,
which i s the 3,4 dihydrovphenyl moiety. A h i g h
resolution scan confirmed the r e s u l t s o f the low
resolution spectrum. Table I1 l i s t s the elemental
compositions f o r the ions as determined by h i g h
resolution mass spectrometry.

TABLE I 1
High Resolution Mass Spectrum of Levodopa
Found Mass Calcd. Mass C
- H
- N
- 0
-
197.0678 197.0689 9 11 1 4
179.0540 1 79.0583 9 9 1 3
177.01 37 177.01 89 9 5 0 4
175.9964 1 75.9984 8 2 1
165.0149 165.01 88 8 5 0
161.0472 161.0477 9 7 1
152.0722 152.071 3 8 10 1
151.0633 151.0634 8 9 1
139.0207 139.0270 6 5 1
136.0537 136.0525 8 8 0
134.0612 134.0580 5 10 0
132.0446 132.0423 5 a 0
1 30.0032 1 30.0055 8 2 0
127.01 62 1 27.01 84 9 3 0
123.0415 123.0447 7 7 0 2

2.6 Optical Rotation


The s p e c i f i c rotation of an aluminum complex
of levodopa i n an acetate buffer a t 25OC i s plotted versus

199
RALPH GOMEZ et a/.

wavelength i n Figure 6 ( 6 ) . The s p e c i f i c rotation


observed a t 589 nm was approximately -41" while t h e
rotation a t 365 nm was approximately -122". In 0.1N hydro-
c h l o r i c acid the s p e c i f i c rotation of levodopa a t 589 nm
has been reported t o be -11" ( 7 ) .
Chafetz and Chen (8) have reported the
s p e c i f i c rotation of an acidified solution of levodopa
containing methenamine t o be approximately -165" a t
589 nm.
2.7 Melting Range
Levodopa me1 t s w i t h decomposition above 270°C
(9).
2.8 Differential Scanning Calorimetry
An endotherm was obtained i n the 290"-300°C
region where me1 ti ng , accompanied by sample decomposi t i on,
occurred. The temperatures observed f o r the decomposition
t r a n s i t i o n s a r e dependent on instrumental conditions as
well as sample size and cannot be considered c h a r a c t e r i s t i c
f o r the compound (10).
2.9 Thermogravimetric Analysis (TGA)
A TGA scan showed no loss o f weight as the
temperature increased from 30" t o 270°C a t a r a t e of 1O"C/
minute (10).
2.10Solubility
The approximate s o l u b i l i t y data obtained f o r a
sample of levodopa a t 25°C i s l i s t e d i n Table I11 (11).
The equilibration time was 20 hours f o r each system.

TABLE I11
Sol ubi 1i t y Data f o r Levodopa
Sol vent Sol ubi 1i t y (mg/ml )
Water 3.0
95% Ethanol 0.3
3A Alcohol 0.15

200
FIGURE 6
Specific Rotati on Versus Wavelength for Levodopa

-
-
-50" -
z -
NOtlVlOtl 3M133dS

2 -
I-
2 -
201

0
a
0 -
LL
2 -100" -
a
v) -
-
-
-
-150"
300 400 500 600
WAVELENGTH IN NANOMETERS
RALPH GOMEZ eta/.

TABLE I11 (cont.)


Sol ubi 1i t y Data f o r Levodopa
So 1 vent Sol ubi 1 i t y (mg/ml )

Me t h a no1 0.1
2-Propanol <0.01
Chloroform 0.1
D i ethyl Ether <o. 01
Petroleum Ether (3O"-6O0C) <o * 01
Benzene <o. 01
Dimethylacetami de <0.01
Propylene Glycol 0.2
Benzyl A1 coho1 <0.01
Acetone 0.03
Acetoni tri 1 e 0.07

2.11 Crystal Properties


2.111 X-Ray Diffraction
The X-ray powder d i f f r a c t i o n data f o r
levodopa a r e presented i n Table IV (12); instrumental
conditions are gi ven be1 ow.
Instrument and Operating Conditions
I n s t ru me n t GE Model XRD-6 Spectrogoniometer
Generator 50 KV, 12.5 mA 0

Tube Target Copper (Cu Ka = 1.5418 A)


Optics 0.1" Detector s l i t
M.R. S o l l e r s l i t
3" Beam s l i t
0.0007" Ni F i l t e r
4" Take o f f angle
Goni ometer Scan a t 0.2" 20 per minute
Detector Amplifier gain-16 coarse; 8.7 f i n e .
Sealed proportional counter t u b e
and DC voltage a t plateau.
Pulse height selection El 5 v o l t s ,
Eu out.
Rate meter T.C. 4, 2000 CIS
full scale.

202
LEVODOPA

Recorder Chart speed 1 "/5 minutes.


Samples Prepared by g r i n d i n g a t room
temperature.

TABLE I V

Levodopa Powder D i f f r a c t i o n Data

-
20 d (!)* I/Io**
6.54 13.52 4
13.08 6.77 2
14.75 6.01 4
15.31 5.79 2
16.85 5.26 20
17.91 4.95 4
18.45 4.81 53
19.75 4.50 14
21.21 4.19 100
22.38 3.97 21
22.73 3.91 42
23.05 3.86 38
23.85 3.73 3
24.91 3.58 60
25.86 3.45 55
26.35 3.38 6
26.92 3.31 28
28.57 3.13 10
29.61 3.02 9
31.25 2.86 21
33.19 2.70 8
33.64 2.66 12
34.31 2.61 6
35.43 2.53 5
36.15 2.49 6
36.28 2.48 21
37.25 2.41 9
37.74 2.38 25
38.34 2.35 8
39.35 2.29 5
40.04 2.25 10

203
RALPH GOMEZ er a/.

TABLE IV (cont.)

Levodopa Powder Diffraction Data


0
-
20 d (A)* I/Io**
40.80 2.21 6
41.24 2.19 8
41.82 2.16 8

- n h
*d rn (interplanar distance)
**I/Io = percent r e l a t i v e intensity (based on maximum
intensity o f 1 .OO)

112 Crystal Structure


Levodopa has been observed t o e x i s t as
needles and plates. Rapid recrystallization from water a t
low tempera ures w i t h o u t agitation produced the needle-
1i ke crysta habit. The needles were found t o exist i n two
crystalline forms (13).
Mostad, e t a l . (14) have determined the
structures of two monoclinic crystal forms of levodopa. A
s t a b l e form was produced by the slow diffusion of absolute
alcohol i n t o a half-saturated solution of levodopa in fgrmic
acid, The c e l l dimensi ns are as follows: a = 13.629 A;
1
b = 5.308 8; c = 6.049 ; and B = 97.53'.

A l e s s s t a b l e form of levodopa was


obtained as t h i n needles by using ethyl ether as a
precipitating agent. This form i s n o t s t a b l e when separated
from the p t h e r liquo
a = 16.9 A; b = 5.88 8;.I t s cell dimensions are:
c = 9.0 8; and B = 99'.
Additional data on crystal structures
have been reported (15, 16).
2.12Dissociation Constants
The dissociation constants f o r levodopa have been
determined t i trimetri cal ly and are reported as fol lows (1 7) :

204
LEVODOPA

iH2 NHZ

3. Synthesis
Yamada, e t a l . (18) report the synthesis of levodopa
starting with three different compounds: 3-(3,4-methylene-
dioxypheny1)-L-alanine ( I ) ; N-acetyl-3-(3,4-methylenedioxy-
pheny1)-L-alanine ( 1 1 ) ; or N-acetyl-3-(3,4-methylenedioxy-
pheny1)-L-alanine Z-menthyl ester (111). The s t a r t i n g
materi a1 , red phosphorous and a mixture of HI and acetic
anhydride are refluxed for several hours t o give (-)-3-(3,4
di hydroxyphenyl )-L-a1 ani ne. The reaction scheme i s shown
in Figure 7.
4. Separation of Racemates
Methods described in the l i t e r a t u r e for the separation
of DL-3-( 3,4-dihydroxyphenyl )alanine into i t s opti cal
isomers general ly i nvol ve the resol uti on of an i ntermedi a t e
in the synthesis. The optically pure intermediate i s then
reacted further t o give pure levodopa or D-Dopa (19-24).
A procedure has been described which was used t o resolve
DL-3-(3,4-dihydroxyphenyl)alanine into i t s optically active
antipodes ( 2 5 ) . A supersaturated solution of the racemic
mixture was seeded with crystals of one pure enantiomorph
which crystallized pure enantiomorph. The mother liquor
was transferred t o a separatory vessel, heated and treated
with fresh finely ground racemic mixture; the amount added
was approximately twice the amount of pure enantiomorph
obtained in the f i r s t recrystallized stage. A preferential
solution of the deficient enantiomorph occurred which l e f t
the antipode behind as a crystalline product. The method

205
FIGURE 7
Synthesis o f Levodopa

G !
CH2- CH-COOH

?HZ
I
t
CH2 -CH-COOH
I
Iu
0 NH- COCH3
UJ

II LEVODOPA

d n CH2 - CH -COO
I

0
m
LEVODOPA

i s applicable only when the optically active antipodes form


a racemic mixture and. not a compound.
5. Stabi 1 i t y and Degradati on
Levodopa, i n the presence of moisture, i s rapidly
oxidized by atmospheric oxygen and turns green (9). The
oxidation of levodopa i n basic solution r e s u l t s i n the
formati on of me1 ani n and re1 ated intermediates (26). The
s t a b i l i t y of levodopa i n the bulk form was studied under
conditions of elevated temperature by storing samples a t
105°C f o r varying periods of time. The samples were
examined f o r evidence of degradation by measuring the color
o f a 10%w/v solution i n 10% hydrochloric acid and by t h i n -
layer chromatography. The color of the solutions showed
some darkening a f t e r 24 hours, and increased w i t h increasing
heating time. However, no degradation was detected i n
these solutions by thin-layer chromatography (27).
6. Drug Metabolic Products
The major metabolites of levodopa i n humans have been
reported t o be 3-methoyy-4-hydroxyphenylacetic acid, 3,4-
d i hydroxyphenylacetic acid, dopamine, and 3-methoxytyrosine
(28-33). Barbeau (34) has reported t h a t patients given
levodopa show an increase i n the excretion of methylated
derivatives such as o-methyldopa, 3-methoxytyramine and 5-
hydroxyi ndol eaceti c acid. Wada and Fel lman (35) have
presented evidence t h a t 2,4,5-trihydroxyphenylacetic acid i s
a metabol i c product of 3,4-di hydroxyphenylpyruvate, a
levodopa metabolite. Gjessing and Borud (36) and O'Gorman,
e t a l . (30) have also studied the metabolic f a t e o f levodopa
i n humans, Figure 8 shows the metabolism of levodopa as
described by them. The chemical names o f the compounds i n
Figure 8 are l i s t e d i n Table V.
TABLE V
Metabolites of Levodopa Referred t o i n Figure 8
I, 3,4-dihydroxyphenylalanine
11. 3,4-dihydroxyphenylethylarnine
111. 3,4-dihydroxyphenylpyruvic acid
IV. 3,4-di hydroyyphenyll a c t i c acid
V. 3,4-dihydroxyphenylacetic acid

207
RALPH GOMEZ e t a / .

FIGURE 8
Metabolism o f Levodopa

208
LEVODOPA

TABLE V (cont.)
M e t a b o l i t e s o f Levodopa R e f e r r e d t o i n F i g u r e 8

VI. 3,4-dihydroxyphenylethanol
VI I. 3,4-di hydroyyphenyl a c e t a l dehyde
VI I I . 3-methoxy-4-hydroxyphenyl a1 a n i ne
IX. 3-methoxy-4-hydroxyphenyl p y r u v i c a c i d
X. 3-methoxy-4-hydroxyphenyll a c t i c a c i d
XI. 3-methoxy-4-hydroxyphenyl a c e t i c a c i d
XI I. 3-methoxy-4-hydroxyphenylethanol
XIII. 3-methoxy-4-hydroxyphenylacetaldehyde
XI V . 3-methoxy- 4- hyd roxy pheny 1e t hy 1ami ne

7. Methods o f A n a l y s i s
7.1 Elemental A n a l y s i s
A t y p i c a l elemental a n a l y s i s o f a sample o f
levodopa i s presented i n Table V I (37).

TABLE VI
Elemental A n a l y s i s o f Levodopa

Element % Theory % Found


C 54.82 54.82
H 5.62 5.74
N 7.10 7.10
0 32.46 32.34 (by d i f f e r e n c e )

7.2Phase S o l u b i l i t y A n a l y s i s
Phase s o l u b i 1it y a n a l y s i s has been c a r r i e d o u t
f o r levodopa u s i n g 1 :1 methanol :water as t h e s o l v e n t . An
example i s presented i n F i g u r e 9 along w i t h t h e c o n d i t i o n s
under which t h e a n a l y s i s was performed ( 3 8 ) .

7.3 Chromatographic Methods


7.31 Thin-Layer Chromatography (TLC)
The f o l l o w i n g TLC procedure appears i n
USP X I X and i s u s e f u l f o r s e p a r a t i n g levodopa from 3-
methoxytyrosine and 3,4,6-trihydroxyphenylalanine ( 6 ) . An
A v i c e l p l a t e , p r e v i o u s l y predeveloped i n t h e d e v e l o p i n g

209
2.5 -

2.0-
Y
0

1.5:
-- -- -- -- -
- - -

LEVOWPA PHASE SOLUBILITYANALYSIS

3
LEVODOPA

solvent, is spotted w i t h 0.1 mg of levodopa from 9:l


acetone:4% hydrochloric acid. The p l a t e i s subjected t o
ascending chromatography i n n-butanol :gl aci a1 a c e t i c acid:
double d i s t i l l e d water:methanol (1 50: 75:75:15). After
development of a t l e a s t 15 cm, t h e p l a t e i s a i r dried and
sprayed w i t h 2:l 10%w/v f e r r i c chloride:5% w/v potassium
ferricyanide. The approximate Rf values a r e summarized i n
Table VII.
TABLE VII
Summary of TLC Data
Compound Approximate Rf -
3,4,6 Trihydroyyphenylalanine 0.25
Levodopa 0.4
3-Methoxytyrosi ne 0.5
Additional TLC separations of levodopa
from related compounds o r metabolites have been reported
(39-42). A summary of these methods i s found i n Table VIII.

TABLE VIII
Thin-Layer Chromatographic Systems f o r Levodopa
Rf Value
Adsorbent Solvent of Levodopa Reference
Cellulose MN 300 Awl Alcoho1:Formic 0.50 39
Acid: Water (40 :40 :
20 1
Cellulose MN 300 n-Propano :Water 0.39 39
(65:25)
Cellulose MN 300 n-Heptane Carbon 0.02 39
Tetrachloride:
Methanol ( 70 :40 :30)
Polyami de Isobutanol :G1 aci a1 0.18 40
Acetic Acid:Cyclo-
hexane (80:7:10)

211
RALPH GOMEZ et a/.

TABLE V I I I ( c o n t . )

Thin-Layer Chromatographi C Systems f o r Levodopa

Rf Value
Adsorbent Sol vent of Levodopa Reference

Cellulose n-Butanol :61 a c i a1 0.28 41


A c e t i c A c i d :Water
( 5 : l :3)

C e l l u l ose E t h y l Acetate: 0.23 41


G1a c i a1 A c e t i c
A c i d :Water
(5:1.5:3)

Cellulose E t h y l Acetate:n- 0.33 41


Butanol :G1 a c i a1
A c e t i c Acid:
Water (3:2:1:3)

Cell ulose Methyl E t h y l 0.16 42


Ketone :Acetone:
2.5N G l a c i a l A c e t i c
A c i d (40:20:20)

7.32 Paper Chromatography


PaDer chromatowaohi c s e o a r a t i ons o f
levodopa have been used f o r i d e n t i f i c a t i o n as w e l l as f o r
i t s s e p a r a t i o n from r e l a t e d compounds (41, 43, 44, 4 5 ) .
Table I X i s a summary o f some o f t h e paper chromatographic
methods employed f o r levodopa. Whatman number 1 paper was
used i n each case.

TABLE I X

Paper Chromatographic Systems f o r Levodopa


Rf Value
Sol vent Development o f Levodopa Reference
n-Butanol :Gl a c i a1 Descending 0.27 41
A c e t i c Acid:Water
(5:1:3)

212
LEVODOPA

TABLE IX (cont.)
Paper Chromatographic Systems f o r Levodopa

Rf Value
Sol vent Development o f Levodopa Reference
Ethyl Acetate: Des cendi n g 0.21 41
Glacial Acetic
Acid: Water
(5:1.5:3)
Methanol :Water: Des cendi n g 0.42 43
Quinoline (160:
40 :8)
n-Butanol :Pyridine: Descending 0.47 44
0.2N Sodium Acetate
(1:l:l)
n-Butanol :Pyridine: Descending 0.68 44
1M Sodium Acetate
(1 :1:2)
n-Butanol :Pyridine Descending 0.40 44
(1 :1 ) saturated w i t h
1M Sodium Acetate
n-Butanol :Pyridine: Descending 0.54 44
Water (1 :1:1)
Benzene :Methanol : Descending 0.36 44
n-Butanol :Pyridine:
Water (1 :2: 1 :1:1)
Methanol :Water: Descend i n g 0.46 44
Pyridine (20:5:1)
Methanol :Water: Ascending 0.37 44
Pyri d i ne (20:5: 1 )

213
RALPH GOMEZ etal.

TABLE IX (cont.)
Paper Chromatographic Systems f o r Levodopa
Rf Value
Sol vent Development o f Levodopa Reference
To1uene:Ethyl Descending 0.59 44
Acetate :Py ri d i ne :
Water :Methanol
(1:l:l:l:l)
To1uene:Ethyl Descending 0.62 44
Acetate:Methanol :
Water (1 :1:1 : l ) -
Aqueous Phase
Water saturated Descending 0.05 44
w i t h methyl ethyl
ketone
Water saturated Ascending 0.02 44
w i t h methyl ethyl
ketone
n-Butanol :Ethanol : Descend 0.23 44
Water ( 2 : l : l )
Methanol :n-Butanol : Descend 0.3 44
Benzene: Water ( 2 :1 :
1:1)
Methanol :n-Butanol : Descending 0.2 44
Benzene:Water (4:3:
2:l)
Methanol :n-Butanol : Ascending 0.20 44
Benzene:Water (4: 3:
2:l)
To1 uene: Ethyl Descending 0.75 44
Acetate: Methanol :
0.1N HC1 ( 1 : l : l : l )

214
LEVODOPA

TABLE IX (cont.)
Paper Chromatographic Systems for Levodopa
Rf Value
Sol vent Development of Levodopa Reference
Methanol :Awl Descending 0.51 44
A1 coho1 :Benzene :
2N HC1 (37:17.5:
35: 12.5)
n-Butanol saturated Descending 0.19 44
w i t h 1N HC1
n-Butanol saturated Ascending 0.18 44
w i t h 1N HC1
$ e r t . -Butanol : Descending 0.08 44
Acetone: Formic Acid:
Water (160 :160: 1 :39)
t e r t . -8utanol: Ascending 0.06 44
Acetone: Formic Acid:
Water (160:160:1:39)
Ch1oroform:Glacial Descending 0.80 44
Acet i c Acid: Water
(2:l:l)
n-Butanol :G1 aci a1 Descending 0.21 44
Acetic Acid:Water
(4:l:l)
t e r t . -Butanol : Descending 0.06 44
Acetone:Propionic
Acid:Water (160:
160: 1 :39)
Benzene: Propionic Descending 0.83 44
Acid:Water ( 2 : l : l )

215
RALPH GOMEZ et al.

TABLE I X ( c o n t . )

Paper Chromatographic Systems f o r Levodopa

Rf Value
Sol vent Development o f Levodopa Reference

t e r t . -Butanol : Ascending 0.42 45


Methyl E t h y l
Ketone :Formi c
Aci d :Water
(40:30: 15: 15)
n-Butanol : Ascending 0.44 45
G1a c i a1 A c e t i c
Acid: Water (50:
25 :25)

Phenol-Water Ascending 0.38 45


(Lower Phase)
Water:sec. - Ascending 0.33 45
Butanol :t e r t . -
Butanol (48.4:43:
-
8.6 ) Upper Phase

2-propanol :Water: Ascending 0.40 45


Concentrated HC1
(65:18.4: 16.6)

see. -Butanol : Ascending 0.17 45


Water-Upper Phase
Ethyl Acetate: Ascending 0.30 45
Formi c Acid :Water
( 70 :20 :10)

E t h y l Acetate: Ascending 0.00 45


Water:Formic Acid
(60: 35 :5)-Upper
Phase

216
LEVODOPA

TABLE IX (cont.)
Paper Chromatographic Systems f o r Levodopa
Rf Value
Sol vent Development of Levodopa Reference
t e r t . -Butanol : As cendi n g 0.10 45
Methyl Ethyl
Ketone: Water:
Formic Acid:
(44: 44: 11 :
0.26)

7.33 -G s-Liquid Chrom tography


A gas-1 i q u i d chromatographic procedure
f o r the determination of levodopa purity and detection of
possible impurities has been developed (46).
Gas-1 i q u i d chromatography permits the
q u a l i t a t i v e identification of the impurities by r e l a t i n g
the retention times r e l a t i v e t o an added internal standard.
Deri vati z a t i on was carried out by the reaction o f 1evodopa
and/or proposed impurities w i t h bis-trimethylsilyl-
acetamide reagent (BSA). Because levodopa is v i r t u a l l y
insoluble i n most non-acidic o r non-aqueous solvents, the
conversion t o the trimethylsi lyl (TMS) derivative was
accomplished without the use o f a solvent. Direct
addition of the BSA reagent t o the compound followed by
moderate heating was s u f f i c i e n t for complete derivative
formati on.
The TMS deri vati ves were successful ly
chromatographed on a column packed w i t h 20% SE-30 on Gas
Chrom Q under isothermal conditions and detected using a
thermal conductivity detector. In order t o compensate for
column c h a r a c t e r i s t i c s , instrumental variati ons , and
sample introduction technique, an internal standard,
docosane, was employed for r e l a t i v e retention time data and
response data. Assay values calculated by area normaliza-
tion yielded a precision o f f 0.5% f o r f i v e degrees of
freedom.

217
RALPH GOMEZ et al.

An absolute determination u s i n g an
internal standard yielded a precision of 2 1.2% f o r f i v e
degrees of freedom.
Gehrke and S t a l l i n g (47) reported the
quantitative gas-liquid chromatographic separation of the
N-trifluoroacetyl-n-butyl e s t e r of levodopa from 14 other
.
non-protei n amino acids The temperature programmed
chromatography was carried out on columns of 60/80 mesh
acid-washed Chromosorb W coated w i t h 5% w/w DC-550.
Yields of 98 -+ 3% were obtained.
Atkinson, Brown and Gelby (48) separated
the trimethylsilyl derivatives of levodopa, tyrosine,
phenylalanine, N-acetyl tyramine, tyramine, N-acetyldopamine
and dopamine isothermally on various columns. By using the
column i n conjunction w i t h a flame ionization detector,
levodopa was detected a t levels as low as 1-2 pg.
7.34 Col u r n Chromatography
As part of a study of the biosynthesis and
metabolism of catecholamines, Masuoka, e t a l . (49)
developed a separation of the constituents of tissue
extracts by column chromatography. The e x t r a c t s were
separated i n t o three fractions on an alumina column by
eluting successively w i t h 0.5M (pH 6.1) ammonium acetate
buffer, 0.01M (pH 4.0) ammonium acetate buffer and 2N
a c e t i c acid. The t h i r d fraction was then passed through a
Dowex 50 column which separated levodopa from the other
constituents. The fractions were monitored by measuring
the absorbance a t 279 nm.
Spiegel and Tonchen (50) described a
method f o r the separation of levodopa from catecholamines
found i n plasma. The sample was adsorbed on alumina,
eluted w i t h 0.1N hydrochloric a c i d , then adsorbed on and
eluted from an AG50W-X4 cation-exchange column.
A method f o r the separation of catechol
derivatives, including levodopa, from guinea p i g brains by
column chromatography wi t h Duo1 i t e C-25 was described by
Nakajima (51).

218
LEVODOPA

Rolland, Lasry and Lissitzky (52)


separated levodopa from L-tyrosine and other amino acids
contained in protein or natural extracts on Dowex 50. The
limit of detection of levodopa was reported t o be 50-200~.
7.4 Direct Spectrophotometric Analysis
Levodopa, i n tablets or capsules, can be
assayed directly by an ultraviolet absorption procedure,
The tablets are finely ground or the contents of the
capsules are mixed. A portion of the powder i s weighed and
quantitatively diluted w i t h 0.4N hydrochloric acid, The
contents are mixed, f i l t e r e d and the absorbance of an
appropriately diluted solution i s measured a t 280 nm. The
amount of levodopa i n tablets or capsules i s determined by
comparing the absorptivity of the sample a t 280 nm with the
absorptivity of a solution of levodopa reference sample
simi larly prepared and measured ( 6 ) .
7.5 Colorimetric Analysis
An automated method utilizing the Doty reaction
has been successfully applied t o the uantitative
determination o f levodopa in tablets 753). The method i s
specific f o r the catechol confi gurati on and wi 11 indicate
any decomposition due t o the oxidation of t h i s moiety.
The tablets are dissolved i n 1N sulfuric acid, homogenized
w i t h d i s t i l l e d water, and quickly combined w i t h sodium
b i s u l f i t e solution t o prevent oxidation. A ferrous
c i t r a t e solution i s introduced into the system followed by
a strongly basic buffer which produces a stable purple
color measureable a t 545 nm. The amount of levodopa i s
calculated by comparison with a cal ibration curve prepared
from pure levodopa, similarly treated (54).
7.6 Non-Aqueous Titration
A potenti ometri c t i t r a t i on w i t h perch1 oric acid
i n glacial acetic acid is the method o f choice t o assay
levodopa. The sample i s dissolved in formic acid, glacial
acetic acid i s added, and the t i t r a t i o n i s carried o u t with
0.1N perchloric acid i n glacial acetic acid. Each ml of
0.100N perchloric acid i s equivalent t o 19.72 mg of
levodopa ( 6 ) .

219
RALPH GOMEZ et a/.

7.7 Determi n a t i on o f G-Dopa


Coppi, V i d i and Bonardi (55) described a
method f o r t h e determination o f D-Dopa i n levodopa. The
method i s based on t h e q u a n t i t a t i v e r e a c t i o n o f a levodopa
decarboxyl ase, present i n a Streptococcus frecaZis
suspension, which converts 1evodopa t o dopami ne w i t h o u t
a f f e c t i n g D-Dopa. D-Dopa was separated from dopamine by
e l u t i n g a s o l u t i o n o f t h e m i x t u r e w i t h 0.05M pH 6.0
phosphate b u f f e r on an Amber1 it e I RC50 ion-exchange c o l urn.
The e l u a t e was assayed f l u o r i m e t r i c a l l y f o r D-Dopa
according t o Anton and Sayre (56).

8. Acknowledgements
The authors wish t o acknowledge t h e assistance o f
t h e S c i e n t i f i c L i t e r a t u r e Department and t h e Research
Records O f f i c e o f Hoffmann-La Roche Inc. f o r t h e i r
1it e r a t u r e searches.

220
LEVODOPA

9. References

1. Waysek, E., Hoffmann-La Roche I n c . , Personal


Communication.
2. Johnson, J., Hoffmann-La Roche I n c . , Personal
Communi c a t i o n .
3. Corrade, C., Hoffmann-La Roche, Inc., Personal
Communi c a t i o n .
4. Boatman, J., Hoffmann-La Roche, Inc., Personal
Communication.
5. Benz, W., Hoffmann-La Roche Inc., Personal
Communication.
6. "The U n i t e d States Pharmacopei a X I X " , pp. 280,
281 (1975).
7. Osborn, S., Hoffmann-La Roche Inc., Personal
Communic a t ion.
8. Chafetz, L. and Chen, T.M., J . Pharm. S c i . ,
63, 807 (1974).
9. T e r c k Index", E i g h t h Ed., p. 397.
10. Rucki, R., Hoffmann-La Roche Inc., Personal
Communication.
11. MacMull an, E. , Hoffmann-La Roche I n c . , Personal
Communication.
12. Chiu, A. M., Hoffmann-La Roche I n c . , Personal
Communication.
13. . .
Sci are1 10 , J and Sheridan, J , Hoffmann-La
Roche I n c . , Personal Communi c a t i o n .
14. Mostad, A,, Ottersen, T. and Romming, Chr.,
Acta Chem. Scand. , 24, 1864 (1970).
15. Jandacek, R. J . and E a r l e , K. M., Acta C r y s t . ,
-
B27, 841 (1971).
16. Becker, J. W., Thathachari , Y. T. and
Simpson, P. G., Biochem. Biophys. Res. C o m n . ,
-
41, 444 (1970).
17. Gorton, J. E. and Jameson, R. F., J . Chem. SOC.
( A ) , 2615 (1968).
18. Yamada, S., F u j i i , T. and S h i o i r i , T., Gem.
Pharm. BuZZ., 10, 693 (1962).
19. H a r r i n g t o n , C . T . and Randall, S. S., Biochem. J . ,
-
25, 1028 (1931).
20. Vogler, K. and Baumgartner, H., HeZv. a i m . Acta,
35, 1776 (1952).
21. Emada, S., F u j i i , T. and S h i o r i , T., Chern. Pham.
BUZZ., 10, 680 (1962).

221
RALPH GOMEZ er a/.

22 * Berenyi, E., Budar, Z . , Pallos, L.,


Magdanyi, L. and Benko, P . , Hung. Teljes 858,
28 Aug. 1970, Appl. 28 Oct. 1969; CA:74:64389u.
23. Berenyi, E. Budai, Z. Pallos, L . , Benko P. and
Magdanyi, L . , Hung. Teljes 859, 28 Aug. 1970,
24.
. .
Appl 28 Oct 1969; CA: 64390n.
Hever, I. and Villanyi, E., Hung. Teljes 383,
6 June 1970, Appl. 16 Nov. 1968; CA: 64391~.
25. Merck and Co., Inc., Neth. Appl. 6,514, 950,
May 18, 1966.
26. Lerner, A. 6. and Fitzpatrick, T. B . , Physical
Rev,, 30, 91 (1950).
27. Burke, C. M . , Sokoloff, H . K. and Heveran, J . E . ,
Hoffmann-La Roche Inc., Personal Communication.
28. Abrams, W. B . , Spiegel, H . E., Leon, A. S . ,
Pocelinko, R. M., and Solomon, H. M., Am. Med.
Assoc. 119th Ann. Conv., Chicago, I l l i n o i s ,
June 21-25, 1970.
29. Bronaugh, R. L . , McMurtry, R. J . , Hoehn, M. M.,
-
and Rutledge, C. O . , Pharmacologist, 15, 247
(1973).
30. O'Gorman, L. P . , Borud, 0. Khan, I. A. and
Gjessing, L. R., CZin. Chim. Acta, 29, 111
(1 970) .
31. Routh, J . I . , Bannow, R. E . , Fincham, R. W . ,
and S t o l l , J . L . , CZin. Chim., 1, 867 (1971).
32. S i i r t o l a , T. and Hirviaho, L . , Scand. J. CZin.
Lab. Invest., 3, ( S u p p l . 130), 14 (1973).
33. Tyce, G. M., Muentner, M. D. and Owen, C. A., J r . ,
Mayo CZin. Proc., 45, 645 (1970).
34. Barbeau, A , , and Mzowell, F. H . , "L-Dopa and
Parkinsonism," F. A. Davis Co., P h i l a . , Pa.,
pp. 360-362, 1970.
35. Wada, G. H. and Fel lman, J . H . , Biochemistry,
-
12, 5212 (1973).
36. Gjessing, L. R. and Borud, O . , S c m d . J . CZin.
Lab. Invest., 17,80 (1965).
37. Scheidl, F . , Hoffmann-La Roche Inc., Personal
Communication.
38. Bass, E . , Hoffmann-La Roche Inc., Personal
Communication.
39 O i ttmann J., J . Chromatogr., 32, 764 (1968).
40. .
Sapi r a , J D. , J , hromatogr. ,TL,134 ( 1969).

222
LEVODOPA

41. Vahidi, A. and Siva Sankar, D. V.,


J . Chromatogr., 43, 135 (1969).
42. Baumann, P. , Scherer, B. , Kramer, W . and
Matussek, N . , J . Chromatogr., 59, 463 (1971).
43. Mattok, G. L. , J . Chromatogr.,T~, 254 (1964).
44 * McGeer, E. G. and Clark, W. H. , J . Chromatogr.,
14, 107 (1964).
--r
45. F i n k , K . , Cline, R. E . and F i n k , R. M.,
Anal. Cham., 35, 389 (1963).
46. Mahn, F. P . , a n i u s , G . , Venturella, V. S .
and Senkowski, B. Z . , Hoffmann-La Roche Inc.,
Personal Communication.
47. Gehrke, C. W . and S t a l l i n g , D. L . , S e p a r . Sci.,
-2, 101 (1967).
48. Atkinson, P. W . , Brown, W . V. and Gilby, A. R . ,
AnaZ. Biochem., 40, 236 (1971).
49. Matsuoka, D. T . , Drell, W . , Schatt, H. F. ,
Alcaraz, A. F. and James, E . C . , AnaZ. Biochem.,
-5, 426 (1963).
50. Spiegel, H . E . and Tonchen, A. E . , CZin. Chem.,
16, 763 (1970).
51. Nakajima, K . , Osaka f/iagaku Igaku Zasshi, 72,
897 (1960).
52. Rolland, M. Lasry, S . and Lissitzky, S . , BUZZ.
SOC. Chim. BioZ., 42, 1065 (1960).
53. Doty, J . R., Anal. &em., 20, 1166 (1948).
54. Billmeyer, A . , Geller, M., Venturella, V. and
Senkowski , B. , Hoffmann-La Roche Inc. , Personal
Communication.
55. Coppi, G., Vidi, A. and Bonardi, G . , J . Pharm.
S c i . , 61, 1460 (1972).
56. Anton,T. H. and Sayre, D. F. , J . PharmacoZ.
Exp. Ther., 145,326 (1964).

223
SODIUM LEVOTHYROXINE

Alex Post and Richard J. Warren


ALEX POST AND RICHARD J. WARREN

CONTENTS

I. Description

1.1 Nomenclature

1.11 Chemical Names

1.12 G e n e r i c Names

I . 13 Trade Names

I .2 Formula, M o l e c u l a r Weight, S t r u c t u r e

I .21 E m p i r i c a l Formula, M o l e c u l a r Weight

1.22 Structure

I .3 Appearance, C o l o r , Odor

2. Physical Properties

2. I Spectra I P r o p e r t i e s

2.11 U l t r a v i o l e t A b s o r p t i o n Spectra

2.12 I n f r a r e d Spectrum

2.13 N u c l e a r Magnetic Resonance S p e c t r a

2.131 P r o t o n NMR Spectrum

2. I 3 2 Carbon- I 3 NMR Spectrum

2.14 Mass Spectrum

2. I5 Specific Optical Rotation

2.2 Solubility

2.3 Crystal Properties

2.31 Crystallinity

2.32 X-Ray D i f f r a c t i o n

226
SODIUM LEVOTHY ROX IN E

CONTENTS (continued)
2.4 pKa, Ionization Constant
2.5 Melting Range
2.6 Differential Thermal Analysis
2.7 Thermogravimetric Analysis
3. Synthesis
3.1 Chemical Synthesis
3.11 L-Thyroxine, Monosodium Salt
3.12 D-Thyroxine, Monosodium Salt
3.2 Nonenzymic Synthesis of L-Thyroxine
3.3 Synthesis of Radiolabeled L-Thyroxine
4. Stability
5. Drug Metabol ism
5.1 Metabolic Products
5.2 Biological Half-Life
6. Elemental Analysis

6.1 Determination of Organically Bound Iodine


6.11 Oxygen Flask Combustion t lodometric Titration
6.12 Ashing t N-Bromosuccinimide Titration
6.13 Oxygen Flask Combustion + Coulometric Titration

6.14 Specific Ion Electrode

6.2 Determination o f Water


6.21 USP X I X

227
ALEX POST AND RICHARD J. WARREN

CONTENTS ( c o n t i n u e d )

6.22 Thermogravimetric Analysis

6.3 Chromatographic A n a l y s i s

6.3 I Paper Chromatography

6.32 T h i n Layer Chromatography

6.33 Column Chromatography

6.331 Ion Exchange Chromatography

6.332 Gel F i l t r a t i o n Chromatography

6.333 Gas L i q u i d Chromatography

6.334 H i g h Performance L i q u i d Ckomatography

6.4 Neutron A c t i v a t i o n Analysis

6.5 Po I arograph i c Ana I y s i s

6.6 K i n e t i c Methods o f A n a l y s i s

6.7 Double-Isotope D i l u t i o n A n a l y s i s

6.8 Determination of Stereoisomeric P u r i t y

6.9 Equilibrium Dialysis

7. Methods o f A n a l y s i s - A Compilation

8. References

228
SODIUM LEVOTHYROXINE

I. Description

Sodium l e v o t h y r o x i n e i s a p h y s i o l o g i c a l l y a c t i v e m a t e r l a l
b e i n g t h e levo-isomer o f t h y r o x i n e . The d a t a p r e s e n t e d i n
t h l s a n a l y t i c a l p r o f i l e , unless otherwise stated, w i l l r e f e r
t o t h e levo-isomer. References t o d a t a o b t a i n e d f o r t h e
dextro-isomer, sodium d e x t r o t h y r o x i n e , t h e DL-form, o r f o r
t h e f r e e amino a c i d of e i t h e r s t e r e o i s o m e r w i l l be so
des i gnated.

I. I Nomenclature

1.11 Chemical Names

Several chemical names have been used t o


d e s c r i b e sodium l e v o t h y r o x i n e and sodium d e x t r o t h y r o x i n e :

( a ) Sodium d e r i v a t i v e o f 3-[4-(4-hydroxy-3,5-
d i iodophenoxy)-3,5-di iodophenyll-L-a l a n i n e l

( b 1 L-3,3', 5,5'-Tetra i o d o t h y r o n 1 ne, sod i um


sa I t, p e n t a h y d r a t e 2

( c ) !3-[(3,5-D i iodo-4-hydroxyphenoxy -3,5-


d i i o d o p h e n y l l - a l a n i n e , sodium s a l t , p e n t a h y d r a t e z

( d ) D-Tyrosine, 0-(4-hydroxy-3,5-di odophenyll-


3,5-d i odo-, monosod i um sa I t, h y d r a t e d

( e l L-Tyrosine, 0-(4-hydroxy-3,5-di iodophenyll-


3,5-d iodo-, monosodium s a l t , h y d r a t e 4

I . 12 G e n e r i c Names

Sodium d e x t r o t h y r o x i n e (D-T4, Na); sodium


l e v o t h y r o x i n e (L-T4, Na); and L - t h y r o x i n e , sodium (L-T4, Na).

I . 13 Trade Names

C h o l o x i n , S y n t h r o i d , L e t t e r , C y t o l e n , Levoid,
E l t r o x i n , Laevoxin, Levaxin, Oroxine, and S y n t h r o i d Sodium.

1.2 Formula, M o l e c u l a r Weight, S t r u c t u r e

229
ALEX POST AND RICHARD J. WARREN

I .21 E m p i r i c a l Formula, M o l e c u l a r Weight

(a) Sodium S a l t , P e n t a h y d r a t e :
C15H1014NNa04 5H20 888.96

(b) Anhydrous Sodium S a l t :


cl
S H l 01qNNa04 798.86

(c) Free A c i d :
*
15 11 4 776.93

I .22 Structure

I I

Na' -0 O--@-CH 2- YH-COOH 5H20

NH2
I' I
I.3 Appearance, C o l o r , Odor

t o p a l e b u f f powder o r c r y s t a l l i n e powder s.
The sodium s a l t , pentahydrate, i an o d o r l e s s , w h i t e
The anhydrous
powder I s l i g h t ye1 low t o b u f f c o l o r e d and hygroscopic3.

2. Physical Properties

2. I S p e c t r a I P r o p e r t i es

2.11 U l t r a v i o l e t Absorption Spectra

Gemml I I s r e p o r t e d X max 325 ( E = 6207) I n 0.4 N


KOH and A max 295 ( E = 4160) i n 0.4 N HCI. Evidence has been-
p r e s e n t e d t h a t t h e s h l f t I n t h e maxima I s due t o t h e d i s s o c i -
a t i o n o f t h e p h e n o l i c h y d r o x y l group. Edelhoch6 a l s o r e p o r t e d
X max 325 ( E = 6180) i n 0.1 - N NaOH.

The u l t r a v i o l e t spectrum o f t h y r o x i n e i n a c i d i -
f i e d e t h a n o l (pH 2.017 i s shown i n F i g u r e I . A maximum a t
300 nm ( E = 4600) and a s h o u l d e r a t 290 nm a r e bands c h a r a c t e r -
i s t i c o f t h e c o n j u g a t e d a r o m a t i c system.

F i u r e 2 i s t h e u l t r a v i o l e t spectrum i n a l k a l i n e
9
e t h a n o l (pH 13.0) which produces t h e sodium s a l t o f t h e

230
:$
0.8

0.7

0.6

0.5 -
-
CONC: 2.004 x W5Y
SOLVENT: Acidifird Ethrnd (pH 2.0)
CELL: 5 an

ru
Y 0.4 -

0.3 -
0.2 -

0.1 -
?

I I I I
260 300 350 400
Figure 1
13.0)

0
400

- 0
d
0
350

-u)
m
0

300
- 0
0
0

260
-to
(v
N

Figure 2
Q)
232
SOD I UM LEVOTHY ROXl N E

p h e n o l i c h y d r o x y l on t h e a r o m a t i c system. The s p e c t r u m
shows t h e expected s h i f t i n maximum t o 328 nm ( E = 6580)
w l t h corresoonding increase i n i n t e n s i t y .
2.12 I n f r a r e d SDectrum

F i g u r e 3 i s t h e i n f r a r e d spectrum o f t h y r o x i n e ,
USP, t a k e n as a m i n e r a l o i l d i s p e r s i o n from 4000-625 c m - l
on a Perkin-Elmer Model 457 i n f r a r e d s p e c t r o p h o t m e t e r . The
f o l l o w i n g a b s o r p t i o n bands a r e a ~ s i g n e d : ~
Table I
I n f r a r e d S p e c t r a l Assignments
Wavelength (cm-l) Assignment

3600 OH
3500-3200 broad, NH2 + H20
1610
1415) coo-
I245 R-0-R

The i n f r a r e d spectrum o f L - t h y r o x i ne has been r e p o r t e d by


Hagen, e t a1.8

2.13 N u c l e a r Magnetic Resonance S p e c t r a

2.131 P r o t o n NMR Spectrum

The p r o t o n NMR spectrum ( F i g u r e 4 ) was


o b t a i n e d I n a DMs0-d~ s o l u t i o n which c o n t a i n e d a b o u t 100 mg
t h y r o x i n e / m l and t e t r a m e t h y l s i l a n e as t h e i n t e r n a l r e f e r e n c e .
The spectrum was o b t a i n e d on a P e r k i n - E l m e r Model R32 90 MHz
s p e c t r o m e t e r . The assignments a r e as f o l l o w s :

Table 2
P r o t o n NMR S p e c t r a l Assignments

233
Figure 3
MATERIAL:
INSTRUMENT: P r L R E l m r R32.90 MHz
RUN IN: DMSO-dg

I
235

k
L I I I I I I I 1 I
m 9 B 1 6 6 4 3 2 1 0

Figure 4
ALEX POST AND RICHARD J. WARREN

Table 2 (continued)

Proton P o s i t i o n S i g n a l Appearance Chemical S h i f t (ppm)

a broad m u l t i p l e t 3.00
b broad m u l t i p l e t 3.51
c broad, NH: t H20 5.30
d singlet 7.09
e singlet 7.83

2.132 Carbon-13 NMR Spectrum


The carbon-13 NMR spectrum o f t h y r o x i n e
was t a k e n i n DMSO-d, s o l u t i o n on a V a r i a n CFT-20 s p e c t r o m e t e r .
The spectrum i s shown i n F i g u r e 5. The assignments a r e a s
f o l lows:?

Table 3

Carbon-13 NMR S p e c t r a l Assignments

I
COOH
I

Carbon-13 P o s i t i o n Chemical S h i f t (ppm)

a 169.30
b 151.27
c 149.80
d 140.90
e 139.06
f and k 125.00
9 91.50
h 87.59
i 54.78
j o v e r l a p p e d by s o l v e n t

2.14 Mass Spectrum

The f i e l d d e s o r p t i o n mass spectrum o f

236
MATERIAL Thyroxine
INSTRUMENT: Varian CFT 20
RUN IN: DMSOd6

N
Y

4000 3000 2000 1000 0

Figure 5
ALEX POST AND RICHARD J. WARREN

9
L-thyroxine was o b t a i n e d on a Varian MAT CH-5 spectrometer.
The r e s u l t s a r e presented as a bar graph I n F i g u r e 6. The
presence o f a t r a c e o f t r l iodothyronlne i s evidenced by f r a g -
ments a t m/e 577, 607 and 651. Mass s p e c t r o m e t r i c s t u d i e s o f
t h y r o x i n e t r l m e t h y l s i l y l d e r i v a t i v e s have been reported.10

2.15 Specific Optical Rotation

As b o t h t h e lev0 and d e x t r o isomers a r e


pharmacologically important substances, t h e i r r e s p e c t i v e
s p e c i f i c r o t a t i o n values have been l i s t e d i n several compen-
d i a as c r i t e r i a o f a c c e p t a b i l i t y . The f o l l o w i n g a r e
severa I r e p o r t e d va I ues, t o g e t h e r w It h references ( R I ) :

I somer Concentration Solvent - X max


*C - - [a]

6.07 g 0.5 N NaOH


Lev& 0.66 g + 13.03 g CZH50H 21 546 -3.2 I 1
6.07 g 0.5 N NaOH
Lev4 0.66 g + 13.03 g CTH50H 25 546 -3.2 2
0.13 N NaOH i n 2o
Lev& 3% D -4.4 1 2
70% CFH5OH
I N NaOH : C2H50H
Levoa 2.2% 2o D -5.7 1 2
(1-72)
I N NaOH : C2H5OH
Levoa 3.28% 2, D -5.4 1 3
( 172)
24 g 0.5 N NaOH
Levoa 3.25% t 56 g C2A5OH
17,5 D -4.6 1 3

Levoa I N HCI : C2H50H


5% 25 D +I5 14
(172)
Lev& 0.2 N NaOH I n 20 D
3% -4.5 14
70% & H ~ O H
Lev& I N HCIC: C ~ H S O H 20 D +I6 t o
2%
( 1-74] +20
Lev& I N NaOH : C2HsOH -5 t o
3% 20 D 4
( 1-72) -6
Levoa 2% I N HCI : C2H50H 2o D t i 9 . 1 25
( 174)
I N HCI : C2H50H 2o
Dextro' 2% D -19.2 15
( 1741

Dextrob I N NaOH i n 25 D t5 t o 3
3%
C2HsOH +6
aAnhydrous f r e e amino ac i d bAnhydrous sod iurn s a l t

238
Matsrial: Thyroxine
EmitbrWireCumnt: 23mA
win dippad m DMSO sdution

A
InmUm*n: Vrirn MAT CH-5 O f

Q
Soura T a n ~ e m re:
u 50°C
P.akr>m

El 'r
HI
+-
a
I
+'
+-
-
E
239

L
t
:il
10

El
3

0
0

Figure 6
ALEX POST AND RICHARD J. WARREN

S p e c i f i c r o t a t i o n s o f L - t h y r o x i n e (C = 29, 0.2 N H C I - e t h a n o l )
a t s e v e r a l wave1 engths and t e m p e r a t u r e s have been r e p o r t e d : 1 5

Temp Cal
(OC)
- 589 nm 578 nm 546 nm 436 nm 364 nm

10 +19.5 t20.3 +23.3 +42.2 +7 I .O


20 t19. I +20. I t23.2 +42.6 +70. I
30 +18.9 +19.7 +22.8 +42.0 +64.5
40 +18.4 +19.3 +22. I +41.5 +62.7

Equivalent s p e c i f i c r o t a t i o n values, b u t o f opposite sign,


were a I so r e p o r t e d f o r D - t h y r o x i ne.I5

U s i n g t h e above data, F e l d e r , e t a l l 5 o b t a i n e d t h e o p t i c a l
r o t a t o r y d i s p e r s i o n c u r v e s and e s t a b l ished t h e L- c o n f i g u r a -
t i o n o f t h e samp l e o f L - t h y r o x i n e .

2.2 Sol u b i I i t v

Table 4

L-Thyroxine Solubi I i t y
Ref
So I v e n t g/lOO ml
#
-
H20 0. 14 3
95% e t h a n o l 0.3, 0.4 1
a1 k a l i h y d r o x i d e s so I ub 1 e 1
chI orofom almost l n s o l u b l e 1
ethyl ether almost i n s o l u b l e 1
pH 7.4 b u f f e r 0.022-0.044 16

E v e r t 1 6 used a phosphate b u f f e r a t pH 7.2-7.8 a t 38OC


and a range o f i o n i c s t r e n g t h from 0.032-0.162. He p o s t u l a t e d
t h a t t h e i n c r e a s e i n i o n i c s t r e n g t h by t h e a d d i t i o n o f sodium
c h l o r i d e produced a change i n t h e i o n i c atmosphere o f t h e
c e n t r a l i o n r e s u l t i n g i n a ' s a l t i n g - i n and s a l t i n g - o u t '
e f f e c t . U l t r a v i o l e t a b s o r p t i o n a n a l y s i s was used t o e s t a b l i s h
t h e s o l u b i l i t i e s . E v e r t has p r e s e n t e d s o l u b i l i t y c u r v e s which
show t h e e f f e c t o f t e m p e r a t u r e (25' and 38OC) and o f i o n i c
s t r e n g t h s on t h e s o l u b i l i t y o f L - t h y r o x i n e sodium. S o l u b i l i t y
i s s i g n i f i c a n t l y increased above pH 7.4.

240
SODIUM LEVOTHYROXINE

2.3 Crysta I Properties

2.31 Crystallinity

C r y s t a l d a t a o n L - t h y r o x i n e , sodium s a l t ,
p e n t a h y d r a t e , have been r e p o r t e d by Cody, e t a l .I7

2.32 X-Ray D i f f r a c t i o n

The c r y s t a l and m o l e c u l a r s t r u c t u r e s o f
L - t h y r o x i n e h y d r o c h l o r i d e , monohydrate have been determined
b y X-ray c r y s t a l lography and r e p o r t e d i n t h e I i t e r a t u r e . 2 8
The c r y s t a l system i s m o n o c l i n i c , and t h e sample c r y s t a l l i z e d
i n space group C 2 w i t h a = 17.23, b = 5.14, C = 25. 15 R;
$ = 90.47'; Z = 4.

2.4 pKa, I o n i z a t i o n Constant

The a p p a r e n t pKa o f t h e phenol i c h y d r o x y l , c a r b o x y l


and amino f u n c t i o n s has been r e p o r t e d :
Ref Ref
Funct i o n pKa #
ca r b o x y I 2.2 19 3.832 20
phenolic hydroxyl 6.7 5 8.085 20
amino 10. I 19 9.141 20

a l n 75% d l m e t h y l s u l f o x i d e - w a t e r and 0.1 M KNO3


T i t r a n t : p o t e n t i o m e t r i c w i t h sodium h y d r o x i d e

2.5 M e l t i n g Range

The fol l o w i n g m e l t i n g ranges have been r e p o r t e d f o r


thyroxine:

I somer M e l t i n g Range (OC)


Reference # -
L-T4 233-235 ( d e c m p 1 12
L-T4 235-236 (decomp 1 2
D-T4 237 ( d e c m p ) 2
L-T4 236 ( c o r r ) 8

241
ALEX POST AND RICHARD J. WARREN

The m e l t i n g r a n g e o f L - t h y r o x l ne (SKBF r e f e r e n c e 88-2746-227)


employing t h e USP X I X , C l a s s I procedure, was 235-236OC
(decomp 1. 21

2.6 D i f f e r e n t i a l Thermal A n a l y s i s

A d i f f e r e n t i a l thermal a n a l y s i s curve o f L-thyroxine


(SK&F r e f e r e n c e 88-2746-2271, o b t a i n e d on a DuPont Model 900
D i f f e r e n t i a l Thermal A n a l y z e r from r o o m t e m p e r a t u r e t o 25OOC
a t a h e a t i n g r a t e o f 10°C p e r m i n u t e under n i t r o g e n sweep, i s
shown i n F i g u r e 7. A sharp e x o t h e r m i c change o c c u r r e d from
230-235OC, i n d l c a t l n decomposition w i t h no o b s e r v a b l e p r i o r
m e l t i n g (endotherm). Be

2.7 Thermogravlmetrlc Analysis

The t h e r m o g r a v l m e t r l c a n a l y s i s o f L - t h y r o x i n e sodium,
pentahydrate, was o b t a i n e d on a DuPont T h e r m o g r a v l m e t r i c
A n a l y z e r ( s e e F i g u r e 8).z1 The compound was heated a t a r a t e
o f 2OoC p e r m i n u t e under n i t r o g e n sweep t o 475OC. A w e i g h t
loss of ~ 9 was % observed. As expected, i n c e p t i o n o f r a p i d
decomposition appeared t o o c c u r a t a p p r o x i m a t e l y 20OoC. The
r e s u l t o b t a i n e d by t h e loss on d r y i n g p r o c e d u r e o f t h e USP
X I X 4 was 8.75%.

3. Synthesis

3.1 Chemical S y n t h e s i s

3. I I L-Thyroxl ne, Monosodi um Sa I t

The s y n t h e t i c r o u t e t o L - t h y r o x i n e , and subse-


q u e n t l y t o I t s monosodium s a l t , pentahydrate, was d e s c r i b e d
by Chalmers, e t a l l 2 and i s p r e s e n t e d i n F i g u r e 9. Evidence
was a l s o p r e s e n t e d showing t h e s t e r e o s p e c i f i c i t y o f t h i s
s y n t h e s i s . The o v e r a l l y i e l d was 26%.

To a c o o l e d suspension o f L - t y r o s i n e ( 1 ) i n
s u l f u r l c a c i d was added n i t r i c a c i d which on n e u t r a l i z a t i o n
yielded 3,5-dinitro-L-tyrosine ( I I ) . An a l k a l i n e s o l u t i o n o f
( I I ) a c e t y l a t e d w i t h a c e t i c a n h y d r i d e y i e l d e d t h e amide ( I l l ) .
E s t e r i f i c a t i o n o f ( I l l ) t o ( I V ) was e f f e c t e d u s i n g e t h y l
a l c o h o l and p - t o l u e n e s u l f o n i c a c i d and t h e n a z e o t r o p i n g t h e
w a t e r w i t h c h l o r o f o r m . The d l p h e n y l e t h e r ( V ) was p r e p a r e d
from ( I V ) by t r e a t m e n t w i t h p - t o l u e n e s u l f o n y l c h l o r i d e and

242
h)
P
0

0 50 100 150 200 250


T, %(CORRECTED FOR CHROME1 ALUMEL THERMOCOUPLES)

Figure 7
N
P
P

1 1 I I 1 I I I I I I
0 50 100 150 200 250 300 350 400 450 500

T. 'C(CORRECTE0 FOR CHROM€l AlUMEl THERMOCOUPLES)

Figure 8
t
N r n
I I
0 0
81
0v=o
I
0
0
1
I I
;
0 0--z
1 1
I N IN
0-2 I
I 0
N
I
0
I
I
."
0
I
I
\m
m
I
0 I 0
z 0
I 0 1
0 v=o
I
1 1
1
0--z
I
8
0
I N
f
1 1
V -2
I
C
0
Y-
O
I
245
m
I
o
c v m
I Z
0 0
t
I
O I 0
8 o=o 0
I I 0,
I 1
0-2 6
\
I,
I
W
I
u m
I
o
N m
I 1
0 0
Q, 81
o o=o
I I
1 1
0 --z
IN
I
0,
I
o
Y a,
.-UL
l;9
\'
0
0,
I
0
246
c
h
0
0
u I
r x
N I N I
0--z I 1 cu
0--z I
I I 0
iu I
I cu
I
OH
0
Jp q\-
H H Y
H 0
I
0
I
H
H H
0 0
+0
(0
I I z
247
ALEX POST AND RICHARD J. WARREN

p-methoxyphenol. Reduction o f ( V ) t o t h e d i a m i n e ( V I ) was


r e a d i l y o b t a i n e d by t r e a t i n g an a l c o h o l s o l u t i o n o f ( V ) w i t h
Raney N i .

Replacement o f t h e amino groups was b r o u g h t


about v i a t e t r a z o t i s a t i o n and Sandmeyer procedures. T e t r a -
z o t i s a t i o n was c a r r i e d o u t by t h e slow a d d i t i o n of a s o l u t i o n
o f ( V I ) i n a m i x t u r e o f a c e t i c and s u l f u r i c a c i d s t o one o f
sodium n i t r i t e i n t h e m i x t u r e o f t h e same a c i d s . The d l a z o -
nium groups were removed by t h e a d d i t i o n o f a s o l u t i o n o f
i o d i n e i n aqueous sodium i o d i d e .

A l I t h e p r o t e c t i v e groups p r e s e n t i n ( V I I )
were removed by t r e a t m e n t w i t h a m i x t u r e o f h y d r o i o d i c a c i d
and g l a c i a l a c e t i c a c i d . lodination of (VIII) wlth a
s o l u t i o n o f Iodine i n ethylamlne yielded L-thyroxine ( I X ) .
The a d d i t i o n o f L - t h y r o x i n e t o a b o i l i n g 2 2
sodium c a r b o n a t e
s o l u t i o n produced t h e d e s i r e d p r o d u c t ( X I .

3.12 D-Thyroxine, Monosodium S a l t

E l k s and Wal l e r Z 3 prepared t h e D-isomer by


f i r s t inverting the 3,5-dinitro-L-tyrosine ( I I-from Figure 9 )
t o t h e D-form ( X I I - F i g u r e 10) by t r e a t m e n t o f ( I 1 ) w l t h
n i t r o s y l b r o m l d e which, a f t e r ammonolysis o f ( X I - F i g u r e l o ) ,
yielded t h e 3,5-dinitro-D-tyrosine (XI I - F i g u r e 10). The
D - t h y r o x i n e was t h e n p r e p a r e d I n a s e r i e s of r e a c t i o n s
e s s e n t i a l l y e q u i v a l e n t t o t h a t used b y Chalmers, e t a l l 2
[see ( I I I ) - ( X I i n F i g u r e 91.

3.2 -
Nonenzymic S y n t h e s i s of L-Thyroxine

I n an a t t e m p t t o e x p l a i n t h e b i o s y n t h e s i s o f
t h y r o x i n e from 3,5-di i o d o - L - t y r o s l n e , Shi ba, e t a I z 4 pro-
posed a nonenzymic model f o r t h i s pathway ( F i g u r e I I ) . A t
n e u t r a l pH and a t room t e m p e r a t u r e and i n t h e presence o f
sodium g l y o x y l a t e ( I l l , c u p r i c a c e t a t e , and oxygen, t h e
r e a c t i o n proceeded r a p i d l y t h r o u g h a t r a n s a m i n a t i o n v i a a
m e t a l c h e l a t e o f t h e S c h i f f base o f d l i o d o t y r o s i n e and g l y -
o x y l i c a c i d ( I I I ) . O x i d a t i v e coup1 i n g o f 4-hydroxy-3,5-
d i l o d o p h e n y l p y r u v i c a c i d ( I V ) w i t h t y r o s i n e y i e l d e d t h e L-
thyroxine (V). The a n a l y t i c a l d a t a p r e s e n t e d e s t a b l i s h e d
t h e s t e r e o s p e c i f i c i t y of t h i s s e r i e s o f r e a c f i o n s . The
o v e r a l I y i e l d was 8%.

240
NO2

KBr, NaN02
H O V CH2 -CH - COOH
\
L-HO-I=)..-CH-COOH / I
NH2 >- H2S04 BI r

NO2 n02
(I1 - from F i g u r e 9) (XI 1

-
aqueous NH3
H O O C H 2 - CH-COOH
\ I
1
NH2
NO2

(XI I )

F i g u r e 10
N
3
+
3 z
0 -0--0 N
I
/ + I +
"Y
' "Z," -O
I 2
+ Y 4 ?
Y
%Y&
0-0
P

I
+
+
N
r:
250
SODIUM LEVOTHYROXINE

3.3 Synthesis o f Radiolabeled L-Thyroxine

Weeke and O r s k o ~have ~ ~ developed a r a t h e r f a c i l e


rocedure f o r t h e s y n t h e s i s and p u r i f i c a t i o n o f monolabeled
P251-LT4 w i t h a v e r y h i g h s p e c i f i c a c t i v i t y f o r use i n
radiolmnune assay.

F i v e mCI o f 1251 ( s p e c i f i c a c t i v i t y > 14 mCi/ug)


were added t o 50 p1 o f 50 mmol/l phosphate b u f f e r , pH 7 . 5 ,
f o r b u f f e r i n g t h e a l k a l i n e 1251 s o l u t i o n . A d d i t i o n o f 2 pg
(20 pI 1 o f 3 , 5 , 3 ' - t r l i o d o - L - t h y r o n l n e and 90 pg (25 1.11) o f
chloramine-T, m i x i n g f o r 15 seconds and h a l t i n g t h e r e a c t i o n
w i t h 240 pg ( I 0 0 p l ) o f sodium m e t a b i s u l f l t e , y i e l d e d a
1251-LT4 o f very h i g h s p e c i f i c a c t i v i t y (~3000mCl/mg T4).
The 1 2 5 1 l a b e l i s presumably i n t h e 5' p o s i t i o n .

A more general and simple procedure f o r t h e synthe-


s i s o f r a d i o a c t i v e L-thyroxine, c a r r y i n g t h e label I3lI or
14C e i t h e r i n t h e phenol i c r i n g o r i n t h e non-phenol i c r i n g
and t h e s i d e chain, was r e p o r t e d b y Shiba and Cahnmann.26
I t i s based on t h e c o u p l i n g o f 4-hydroxy-3,5-diiodophenyI-
p y r u v i c a c i d and d i i o d o t y r o s i n e . Labeled 4-hydroxy-3,S-
d l l o d o p h e n y l p y r u v i c a c i d was prepared by t h e condensation of
i o d i n a t e d p-hydroxybenzaldehyde w i t h a c e t y l g l y c i n e and t h e
h y d r o l y s i s o f t h e azlactone. Depending on t h e s p e c i f i c
labeled compound prepared, y i e l d s ranged from 12-209.

4. Stability

The USP,4 NF,3 and B P I s t a t e t h a t levothyrox ne and


d e x t r o t h y r o x i n e sodium a r e t o be p r o t e c t e d from i g h t . On
exposure to l i g h t these compounds may assume a p nk c o l o r .
I n a d d i t i o n , t h e USP4 s t a t e s t h a t I e v o t h y r o x i n e s stable in
dry a i r .

Several i n v e s t i g a t o r s have s t u d i e d t h e s t a b i l i t y o f
t h y r o x i n e as a f u n c t i o n o f t h e d e l o d i n a t i o n process which
y i e l d s f r e e i o d i n e and t r i iodothyronine. StanburyaP has
reviewed t h e pathway and mechanism o f t h i s process by b o t h
i n v i t r o and I n v i v o experimentation. He i n d i c a t e d t h a t t h e
major f a c t o r I n t h e d e i o d i n a t i o n process i s i n v o l v e d w i t h
t h e f a c t l e i o n l z a b l I i t y o f t h e hydroxyl group. Because o f
t h i s , one can expect t h a t t h e lodlnes i n t h e 3' and 5'
p o s i t i o n s a r e more l a b i l e than those i n t h e 3 and 5 p o s i t i o n s

251
ALEX POST AND RICHARD J. WARREN

( r e f e r t o § I ,221. The r e l e a s e d i o d i d e i s t h e n o x i d i z e d t o
f r e e i o d i n e . T h a t t h i s appears t o be t h e i n v i v o mechanism
has subsequently been borne o u t by o t h e r i n v e s t i g a t o r s .

High energy g a m a r a d i a t i o n has been shown t o cause


r a p i d d e i o d l n a t i o n and t h e f o r r n a t l o n o f o t h e r i o d l n a t e d
o r g a n i c molecu 1 es .28

The r a t i o n a l e and e x p l a n a t i o n f o r t h e spontaneous


d e i o d i n a t i o n o f 1311 l a b e l e d t h y r o x i n e Is s t i I I o f concern.
TaurogBg has shown t h a t t h e s p o t t i n g o f d i l u t e aqueous
b u f f e r s o l u t i o n s on f i l t e r p a p e r and d r y i n g f o r 10-20
minutes l e d t o a s i g n i f i c a n t and v a r i a b l e loss o f 1311 from
t h e 1 3 1 1 - t h y r o x i n e . T h i s e f f e c t was a l s o observed when g l a s s
paper and t h i n l a y e r s i I i c a g e l were used. R e d u c t i o n i n t h e
amount o f d e i o d i n a t i o n o c c u r r e d when e t h a n o l or p r o p y l e n e
g l y c o l was added t o t h e sample. Shortwave u l t r a v i o l e t l i g h t
g r e a t l y enhanced t h e r a t e o f d e i o d i n a t i o n .

These spontaneous d e i o d i n a t i o n e f f e c t s were a l s o s t u d i e d


by J o l i n , e t a l . 3 0 They i n d i c a t e d t h a t t h i s e f f e c t a r i s e s
when one o f t h e components o f t h e s o l u t i o n under s t u d y be-
comes an a c t i v e d e i o d i n a t i o n agent d u r i n g t h e a n a l y t i c a l
procedure. Thus, a s t r i c t adherence t o p r o c e d u r e must b e
f o l lowed.

Reviczky and Nagy3l found t h a t under u l t r a v i o l e t r a d i -


a t i o n d e i o d i n a t i o n o c c u r r e d t o a g r e a t e r e x t e n t and more
r a p i d l y i n aqueous s o l u t i o n s t h a n i n b u t a n o l , t h e r e l e a s e o f
I o d i d e i s p r o p o r t i o n a l t o t h e decrease i n pH, and chromatog-
raphy showed t h a t i n a d d i t i o n t o t h e presence o f f r e e i o d i d e
t r i i o d o t h y r o n i n e was t h e p r i m e decomposition p r o d u c t .

5. Drug Metabolism

5. I Biological Half-Life

I n a s t u d y s i m i l a r t o t h a t of S t e r l i n g , e t a1,32
Demeester-Mi r k i ne showed t h a t t h e ha I f - I i f e o f 1 3 1 1 - t h y r o x i ne
i s 7 days i n serum and 0.4 days i n t i s s u e s . 3 3

5.2 Metabol i c P r o d u c t s

T h y r o x i n e i s c o n v e r t e d t o t r i i o d o t h y r o n i n e i n hypo-
t h y r o i d human s u b j e c t s m a i n t a i n e d on s y n t h e t i c sodium L-

252
SODIUM LEVOTHYROXINE

t h y r o x i n e a d m i n i s t e r e d o r a l ly.34 T h i s was c o n f Inned by


S t e r l i n g , e t al,32 who i n j e c t e d p u r i f i e d t h y r o x i n e l a b e l e d
w i t h 14C i n r i n g A and i n t h e a l a n i n e s i d e c h a i n . Methods
f o r d e t e r m i n i n g t h i s c o n v e r s i o n have been reviewed by Surks
and Oppenheimer. 35

6. Elemental A n a l v s i s

The elemental c o m p o s i t i o n o f L - t h y r o x i n e as t h e mono-


sodium s a l t p e n t a h y d r a t e , t h e anhydrous monosodium s a l t , and
t h e anhydrous f r e e a c i d i s as f o l l o w s :

% (Theory)

Monosodium Monosodium Amino A c i d


(Pentahydra t e ) (Anhydrous) (An hy d r o u s 1

Carbon 20.27 22.55 23.19


Hyd r o g en 2.27 I .26 I .43
N it rogen I .58 1.75 I .80
Oxygen 16.20 8.01 8.24
Sod i urn 2.59 2.88 -
Iodine 57.10 63.54 65.34

The t h e o r e t i c a l percentage o f w a t e r i n t h e monosodium penta-


h y d r a t e form i s 10.13%.

6.1 D e t e r m i n a t i o n of O r g a n i c a l l y Bound l o d l n e

As t h e c r i t e r i o n o f a c c e p t a b i I i t y i n b o t h t h e USPd
and t h e NF3 i s t h e assay v a l u e f o r i o d i n e , t h e methods o f
a n a l y s i s employed t o d e t e r m i n e t h i s element have been c a r e -
f u l l y investigated.

6. I I Oxygen F l a s k Combustion36 + lodometric


T i t r a t ion37

This method i s d e s c r i b e d i n b o t h t h e USP X I X


and NF X I V monographs f o r sodium l e v o t h y r o x i n e and sodium
d e x t r o t h y r o x i ne.

Apparatus - The a p p a r a t u s c o n s i s t s o f a heavy-


w a l l e d c o n i c a l , d e e p l y l i p p e d o r cupped 500-ml f l a s k ( u n l e s s
a l a r g e r f l a s k Is s p e c i f i e d ) , f i t t e d w i t h a ground-glass
s t o p p e r t o which i s fused a sample c a r r i e r c o n s i s t i n g o f

253
ALEX POST AND RICHARD J. WARREN

heavy-gauge p l a t i n u m w i r e and a p i e c e o f welded p l a t i n u m


gauze measuring about 1.5 x 2 cm.

Procedure - Weigh a c c u r a t e l y about 25 mg o f


samp e on a p i e c e o f h a l i d e - f r e e f i l t e r paper measuring about
4 cm square, and f o l d t h e paper t o e n c l o s e it. P l a c e t h e
samp e, t o g e t h e r w i t h a f i l t e r paper f u s e - s t r i p , i n t h e
p l a t num gauze sample h o l d e r . P l a c e t h e a b s o r b i n g l i q u i d ,
cons s t i n q of a m i x t u r e of 10 m i of sodium h y d r o x i d e
s o l u i o n 71 i n 1001, i n t h e f l a s k and m o i s t e n t h e j o i n t o f
t h e stoDoer w i t h w a t e r .
I ,
F l u s h t h e a i r from t h e f l a s k w i t h a
stream o f r a p l d l y f l o w i n g oxygen, s w i r l i n g t h e I i q u i d t o
f a v o r i t s t a k i n g up oxygen. [NOTE--saturation o f t h e l i q u i d
w i t h oxygen i s e s s e n t i a l f o r t h e s u c c e s s f u l performance o f
t h e combustion procedure.] I g n i t e t h e f u s e - s t r i p by s u i t a b l e
means. I f the s t r i p i s ignited outside the flask, invert the
f l a s k so t h a t t h e a b s o r p t l o n s o l u t i o n makes a seal around t h e
stopper, and h o l d t h e s t o p p e r f i r m l y i n p l a c e . When t h e
combustion i s complete, p l a c e a few m l o f w a t e r around t h e
s t o p p e r and a l l o w t h e f l a s k t o s t a n d f o r a b o u t 15 minutes.
Loosen t h e s t o p p e r , and r i n s e t h e s t o p p e r , sample h o l d e r , and
s i d e s o f t h e f l a s k w i t h about 20 m l o f water, added i n s m a l l
p o r t i o n s . Add I m l o f an o x i d i z i n g , s o l u t i o n prepared by
a d d i n g 5 m l of bromide t o 100 ml of a I i n 10 s o l u t i o n o f
sodium a c e t a t e i n g l a c i a l a c e t i c a c i d . I n s e r t t h e stopper I n
t h e f l a s k , and shake v i g o r o u s l y f o r I minute. Add 0.5 m l o f
f o r m i c a c i d , r e p l a c e t h e s t o p p e r , and shake v i g o r o u s l y f o r I
m i n u t e . Remove t h e s t o p p e r , and r i n s e t h e s t o p p e r , t h e
sample h o l d e r , and t h e s i d e s o f t h e f l a s k w i t h s e v e r a l small
p o r t i o n s o f water. Bubble n i t r o g e n t h r o u g h t h e f l a s k t o
remove t h e oxygen and excess bromine, add 500 mg o f p o t a s s i u m
i o d i d e , s w i r l t o d i s s o l v e , add 3 ml o f d i l u t e d s u l f u r i c a c i d ,
s w i r l t o mix, and a l l o w t o s t a n d f o r 2 minutes. T i t r a t e w i t h
0.02 N sodium t h i o s u l f a t e , adding 3 m l o f s t a r c h TS as t h e
endpoTnt i s approached. Each ml o f 0.02 - N sodium t h i o s u l f a t e
i s e q u i v a l e n t t o 0.1057 mg o f i o d i n e .

6.12 Ash i ng t N-Bromosucc i n i m i de T i t r a t i o n

Bakarat, e t al,38 ashed t h e sample i n t h e


presence o f potassium c a r b o n a t e and, a f t e r d i l u t i o n w i t h
water, t i t r a t e d t h e s o l u t i o n w i t h 0.02 N N-bromosuccinimide.
Accuracy o f g r e a t e r t h a n 99% was o b t a i n z d f o r samples con-
t a i n i n g 5-10 mg o f t h y r o x i n e .

254
SODIUM LEVOTHYROXINE

6.13 Oxygen F l a s k Combustion + Coulometric


T i t r a t i on

A modif i c a t i o n d g o f t h e oxygen f l a s k
combustion method has been used p r i o r t o t h e c o u l o m e t r i c
t i t r a t i o n 4 0 o f t h e iodide:

To a 500-ml oxygen f l a s k f i l l e d w i t h oxygen


I s added 10.00 m i o f 0.4 N potassium h y d r o x i d e and s e v e r a l
m i l l i g r a m s o f h y d r a z i n e s u l f a t e . The f i l t e r paper t a b i s
i g n i t e d , and a f t e r combustion i s c o m p l e t e t h e f l a s k i s
c o o l e d f o r a few seconds i n a stream o f c o l d w a t e r and s e t
a s i d e f o r a t l e a s t 30 minutes f o r complete a b s o r p t i o n o f t h e
combustion p r o d u c t s . The a b s o r b i n g s o l u t i o n i s a c i d i f i e d
w i t h 10.00 m l o f a s o l u t i o n o f 0.6 N n i t r i c a c i d i n 20%
g l a c i a l a c e t i c a c i d . The f l a s k i s s w i r l e d t o remove some o f
t h e carbon d i o x i d e evolved, stoppered and v i g o r o u s l y shaken.
An a l i q u o t o f 4.00 m l i s t i t r a t e d c o u l o m e t r i c a l l y on a s u i t -
a b l e c o u l o m e t r i c t i t r a t o r , such as t h e Aminco-Cotlove Auto-
mat i c T i t r a t o r . 4 2 j 42

6. I4 S p e c i f i c Ion E l e c t r o d e

The s p e c i f i c i o n e l e c t r o d e s e n s i t i v e t o i o d i d e
has been used by P a l e t t a and Pantenbeck43 f o r samples of L-T4.
The i o d i n e i s s t r i p p e d from t h e compound w i t h a c t i v a t e d a l u -
minum f o i I a t pH I I a t 6OoC i n 10 minutes. A f t e r n e u t r a l l z -
a t i o n w i t h d i l u t e h y d r o c h l o r i c a c i d , t h e p o t e n t i a l is meas-
u r e d u s i n g t h e s p e c i f i c i o n e l e c t r o d e 4 4 versus t h e calomel
r e f e r e n c e e l e c t r o d e . The amount o f i o d i d e p r e s e n t i s d e t e r -
mined by comparing t h e p o t e n t i a l r e a d i n g w i t h t h o s e o b t a i n e d
f o r a s e r i e s o f standard s o l u t i o n s c o n t a i n i n g t o lo-’
grams o f i o d i d e p e r m l . The assay r e s u l t s compared f a v o r a b l y
w i t h t h o s e o b t a i n e d by t h e c o n v e n t i o n a l t i t r i m e t r i c p r o c e d u r e
u s i n g a sodium t h i o s u l f a t e t i t r a n t .

6.2 D e t e r m i n a t i o n o f Water

L - t h y r o x i n e monosodium i s g e n e r a l l y p r e p a r e d as a
h y d r a t e . Thus, i n o r d e r t o compare samples on an anhydrous
b a s i s , t h e m o i s t u r e c o n t e n t i s determined.

6.21 G r a v i m e t r i c Method (USP X I X , Method I l l , p.668)

From t h e monograph f o r l e v o t h y r o x i n e sodium

255
ALEX POST AND RICHARD J. WARREN

t h e d e t e r m i n a t i o n i s as f o l l o w s :

' D r y about 500 mg, a c c u r a t e l y weighed, o v e r phosporous


p e n t o x i d e a t 6OoC and a t p r e s s u r e n o t exceeding 10 mm
o f mercury f o r 4 hours.

6.22 Thermogravi m e t r i c Ana I y s i s (TGA)

The procedure d e s c r i b e d i n 2.7 has been found


t o y i e l d e q u i v a l e n t r e s u l t s t o t h e USP X I X procedure. A
sample ( B a t c h #27) analyzed by t h e USP X I X p r o c e d u r e showed
t h e presence y f 8.75% m o i s t u r e , and by thermogravimetry I t
showed 8.85%.

6.3 Chromatographic A n a l y s i s

As t h y r o x i n e i s o f t e n contaminated w i t h t r i i o d o -
t h y r o n l n e , and i n t h r y o i d powders w i t h a s e r i e s o f i o d i n a t e d
t h y r o n i n e s a n d / o r t y r o s i n e s , and i n s e r a w i t h s i m i l a r
compounds, it i s i m p o r t a n t t h a t t h e chromatographic system
employed be c a p a b l e o f s e p a r a t i n g t h e s e r e l a t e d compounds.
Thus, i n t h e s e v e r a l chromatographic t e c h n i q u e s used t o
e v a l u a t e t h r y o x i n e and t h r y o x i n e - c o n t a i n i n g m a t e r i a l s , t h e
R f , RT, e t c . o f t h e s e r e l a t e d compounds a r e a l s o r e p o r t e d
when a v a i l a b l e .

The f o l l o w i n g a b b r e v i a t i o n s o f t h r y o x i n e and
r e l a t e d compounds w i l l be used:

T4 thyroxine
T3 3,3', 5 - t r i io d o t h y r o n i ne
T2 3,5-d i i o d o t h y r o n i ne
TI 3-iodothyron i n e
T t h y r o n i ne
I- i norgan ic iod i de
TY t y r o s i ne
MIT 3-iodotyrosine
DIT 3,5-diiodotyrosine

6.31 Paper Chromatography (PC)

A s i g n i f i c a n t amount o f l i t e r a t u r e i s a v a i l a b l e
concern i ng t h e app I i c a t i o n o f paper chromatography t o t h e
s e p a r a t i o n o f t h e iodoamino-acids. A few o f t h e r e l e v a n t
pub1 i c a t i o n s have been c i t e d . 4 5 - 5 3

256
I&Lci
R f Values o f Thyroxine, Analogs, and R e l a t e d Compounds (Paper Chromatography)
M o b i l e Phase
2 3 4 5 6 7 8 9 10 II 12
ComDound I Rf
T4 0.89 0.47 0.45 0.45 0.51 0.43 0.86 0.70 0.28 0.43 0.22 0.07
T3 0.91 0.70 0.63 0.65 0.63 0.58 0.86 0.85 0.36 0.78 0.33 0.25
T2 0.69 0.06 0.11 0.11 0.68 0.62 0.82 0.85 0.47 0.42
TI 0.87 0.62 0.35
T 0.26
'I 0.26 0.40 0.90 0.89
T 0.46 0.26 0.25 0.17 0.17
MIT 0.58 0.39 0.18 0.28 0.72
DIT 0.67 0.59
Mobile Phase: Reference #
I. n-Butanol:2N acetic a c i d ( I : I ) 46
2. n-Butanol :&hano1 :0.5N NH40H ( 5 : 1 :2) 47
3. n-Butano I :d I oxane :2& m 4 0 H ( 4 : I :5 1 48
4. C o l l i d i n e (conc. NHt,OH vapor) 46
5. n-5utanol :ethanol :2N NH40H (5:I :2) 49
6. n-Butanol :2N NH40H T5:2) 49
7. n-Butanol :Zv f o r m i c a c i d 50
8. n-Butanol :6F NH40H 50
9. lsoamyl alcohol : t e r t - a m y l a l c o h o l :6N NH40H ( 1 : I :2-upper phase) 45
10. t e r t - a m y l a l c o h o l :2N NH40H:hexane ( 5 : 6 : I ) 53
I I. I, I-dimethy I propanol- I s a t u r a t e d w i t h 6N - NH40H 52
12. 3% sodium c h l o r i d e 53
ALEX POST AND RICHARD J. WARREN

Table 6
D e t e c t 1on Met hods
Used i n Paper Chromatography o f Thyroxine

A comprehensive d e s c r i p t i o n o f t h e f o l l o w i n g d e t e c t i o n
methods i s l i s t e d i n Reference 46 - they a r e o n l y b r i e f l y
descr i bed here :

'I. C e r i c - a r s e n i t e reagent:

T h i s method o f d e t e c t i o n depends upon t h e l i b e r a t i o n


o f i o d i n e from t h e compounds by o x i d a t i o n w i t h c e r l c
s u l f a t e . The i o d i n e - c o n t a i n i n g compounds appear as
w h i t e spots on a y e l l o w background. As l i t t l e as 0.1
pg o f L-T4 and L-T3 and 0.01 pg o f I- c o u l d be
detected. A d e t a i l e d account of t h e a p p l i c a t i o n
o f t h i s method can be found i n Reference 139.

2. F e r r i c h l o r 1 d e - f e r r i c y a n ide-arsenious a c i d reagent:

&el I n and Virtanen"' demonstrated t h a t t h e c a t a l y t i c


a c c e l e r a t i o n by 'I o f t h e simultaneous r e d u c t i o n o f
f e r r l c h l o r l d e and f e r r i c y a n t d e t o y i e l d a m i x t u r e of
T u r n b u l l ' s b l u e and Prusslan b l u e was a s e n s i t i v e
d e t e c t i o n method f o r iodothyronines and i o d o t y r o s i n e s
(0.002 p g f o r each o f t h e amino a c i d s and 0.001 pg
f o r I-.)

3. Starch:

Datta, e t a I .49 showed t h e use o f a s t a r c h spray,


an overspray w i t h KI03, and exposure t o UV l i g h t
t o be a s e n s l t i v e d e t e c t i o n reagent. A t r a n s i e n t
b l u e c o l o r from as l i t t l e as 0.05 pg o f an iodoamino-
a c i d can be detected.

Less s e n s i t i v e d e t e c t i o n reagents a r e :

4. D l a t o t i z e d s u l f a n i l l c acid:

10-20 pg o f t h y r o x i n e can be detected. A range o f


c o l o r s from r e d d i s h p u r p l e (L-T4) t o orange ( L - T I )
i s obtained.

258
SOD1 UM LEVOTHY ROX I N E

Table 6 (continued)

5. D i a z o t i zed N,N-di e t h y I su I f a n i lami de:

T h i s r e a g e n t i s o n l y s l i g h t l y more s e n s i t i v e t h a n t h e
above, and t h e c o l o r s a r e i n t h e p u r p l e range.

6. N i n h y d r in :

A l e s s s p e c i f i c reagent, s i n c e it r e a c t s w i t h amino
a c i d s . L-T4 appears as a p u r p l i s h brown c o l o r
r a t h e r t h a n t h e t y p i c a l p u r p l e . The l i m i t o f
d e t e c t i o n o f L-T4, L-T3, and L-T2 i s I ~ 9 . ~ ~

7. Ultraviolet light:

These compounds absorb s t r o n g l y i n UV l i g h t and can


be d e t e c t e d i f 20-30 pg a r e p r e s e n t .

A u n i ue r e a g e n t (Emerson's)'41 was used by E i s d o r f e r and


2
P o s t 4 t o qua1 i t a t i v e l y and q u a n t i t a t i v e l y d e t e r m i n e t h e
presence and amount o f L-T2 and L-T3 i n L-T4. The r e a g e n t
i s p r e p a r e d by d i s s o l v i n g 4 - a m i n o a n t i p y r i n e h y d r o c h l o r i d e i n
aqueous sodium c a r b o n a t e s o l u t i o n . The r e a g e n t i s s e n s i t i v e
i n d e t e c t i n g I Pg o f each o f t h e iodoamino a c i d s f o r m l n g a
range o f c o l o r s : L-T2 (orange), L-T3 ( r e d ) , and L-T4 ( p u r p l e ) .

D e t e c t i o n o f l a b e l e d iodoarnino a c i d s can b e accomplished by:

I. Neutron a c t i v a t i o n a n a l y s i s

2. Radioautography (X-ray f i Im)

3. Automatic chromatographfc scanners f i t t e d w i t h a Geiger-


Mu I I e r d e t e c t o r

259
Table 7

R f Values o f T h y r o x i n e , Analogs, and R e l a t e d Compounds


( T h i n Layer Chromatography)

(see notes)
M o b i l e Phase
I 2 3 4 5 6 7 8 9 10

Rf
0.55 0.25 0.38 0.48 1.28 0.89 0.14 0.18 0.24 0.58
0.73 0.36 0.46 0.59 1.76 1.13 0.34 0.50 0.30 0.49
0.50 0.66 0.48 0.58 0.27 0.38
0.27
1- 0.87 0.75 0.70 0.53 0.90 0.62 0.31
0.12
MIT 0.18 0.29 0.23 0.67 0.39 0.83 0.07 0.14 0.18
DIT 0.73 0.24 0.19 0.15 0.17 0.72 0.04 0.09 0.30

NOTES

Mob; I e Phase Adsorbent Detect ion Ref #

I. n-Butanol : c h l o r o f o r m (4:7) s a t u r a t e d Kieselguhr G Rad ioscan 55


w i t h NH40H atmosphere

2. 30% NHhOH (w/v):methanol : c h l o r o f o r m S i l i c a Gel H Ceric sulfate: 56


(0. I :2:2, v / v ) sodium a r s e n i t e :
methy I ene b I ue 57
h
c

+
.-
Table 7 ( c o n t i n u e d )

V
0
c

+
.-
Ref #
.--
n

a
D

n
+I

t
r
Mob i l e Phase Adsorbent Detect ion

ul
L

a,
a,
V

0
a,

m
ul

C
a,
58

.-

a
0
-N
-V.->

Z
I

-acn,
-a,
W

-0
L

0
3. Tert-amy alcohol:acetone:NH40H Kieselgel G N i n h y d r i n , PdCI2

s
a,

a,
ul
?!
nJ\

u
a,

..
t
( 2 5 : 8 : 7 , v/v)

>

.-

.-L
n
0
-N
t

-
-V
-..

cn
L

z
L
-0

D
- -f
t-

t W
4. Tert-but-nol:tert-amyl alcohol: Eastman Sheets N i n h y d r i n , PdC12 59

0 0

m
E 0

Q)

c
L >

E t

ul 0

m
C

a,

ul

C
a,L

ma,
2-0
1 Y

4
* Lo

c3
NH40H :met hy I e t hy I ketone :H20 #6060 ( S i l i c a Gel)

a,
C
a,

N
(Rf &ven as r e Z a t i v e Rf of I-, Rf of I- not r e p o r t e d )

!-I
5.

.-
._.-
n ulna

t c

D V
Tert-amyl alcoho1:dioxane:iN- NH40H 20% S i I i c a Gel G+ Diazotized 60

(0--0
N + -
o m w
N -
a, .-

.-
uz

D
-
(2:2: I ) 80% C e l l u l o s e s u l f a n i l i c acid,

a,

0
ul
a,

3 D

m
V
0
0
z
M
MN 300 PdC I 2
(Rf given as reZative Rf of I-, Rf of I- not r e p o r t e d )
L

G O -

k
%W

I
H
6.
>-
h)
rub
- NH40H

v,

Ln
Same as 5
' F -V

9 Ethano I :methy I e t h y I ketone:ZN Same as 5 60


x

m
E
a,
m
ul
( I :4: I )
( I:5)
-..u-3

u
-a,

.-
V

-
u .. .-
Cel I u l o s e Powder Ferric chloride:
LL

7. Formic acid:H20 61
0

a,
0
3
a,
I

O D
L O U

a,
v

I:

..
.-
f e r r i cyan i de:

u
arsenious a c i d

(0
- NH40H:ch l o r o f o r m

Cn

P
Z
2I
I

'

8. Tert-butanol :2N Same as 7 Same as 7 61


P 8I

5
a,
m
ul
0

0
E
..
I

(376:70:60)
I
..

- NH40H
U

9.

.-0

c3
L3
-a,
S i l i c a Gel G
-

Ceric sulfate:
-

Ethy1acetate:methanoI:ZN 62
2

m
2

1
..
I
:
-

(100:40:60, V / V ) sod i um a r s e n i t e
.
..

(not r e p o r t e d )
+

.-V
.-V

-
c3
t h

-..

T;I

.-V
c3
S i l i c a Gel G
Y-

10. Ch1oroform:methanol : f o r m i c a c i d 66
E0 In
E \>

(0

0

(0

(0
e -..

a, >

a,
0
c
M
0

( 7 0 : 15: 15, v / v )
..
ALEX POST AND RICHARD J. WARREN

T a b l e 5 l i s t s t h e R f o f s e v e r a l o f t h e iodo-
aminoacids i n d i f f e r e n t systems. D e t e c t i o n methods a r e
l i s t e d i n T a b l e 6. Several Whatman papers have been used:
Whatman I , 3, 4, 3MM b e i n g t h e most p o p u l a r . I n addition,
t h e procedure has been c a r r i e d o u t i n t h e ascending,
descending and c i r c u l a r modes. I n each case, i t has been
t h e e x p e r i e n c e o f t h e a u t h o r s t h a t , under t h e p r o p e r c o n d i -
t i o n s o f temperature, equi I i b r a t i o n time, and l e n g t h o f run,
t h e Rfs o b t a i n e d w i t h any o f t h e s e modes gave e s s e n t i a l l y
e q u i v a l e n t r e s o l u t i o n from day t o day and l a b o r a t o r y t o
Iaboratory . Dei od I n a t I on e f f e c t s d u r i ng paper chromatog r a p hy
have been e v a l u a t e d and described I n Section 4.

Paper chromatography, u s i n g f o u r d i f f e r e n t
s o l v e n t systems on Whatman # I paper, was used t o e s t a b l i s h
t h e chromatographic p u r i t y o f t h e L - t h y r o x i n e sodium r e f e r e n c e
substance p r i o r t o i t s a d d i t i o n t o t h e B r i t i s h .Pharmacopoeia.54
e f h y r o x i n e v a l u e s a r e r e p o r t e d f o r t h e compounds I i s t e d i n
T a b l e 7.

6.32 T h i n Layer Chromatography (TLC)

T h i n l a y e r chromatography o f f e r s some
advantages t o paper chromatography i n t h a t b e t t e r s e p a r a t i o n s
a r e general l y o b t a i n e d w i t h h i g h e r l o a d i n g s . However,
r e p r o d u c i b i l i t y o f R f values i s oftentimes d i f f i c u l t t o
o b t a i n because o f t h e b a t c h t o b a t c h d i f f e r e n c e s i n adsorbents,
t e m p e r a t u r e v a r i a t i o n s w i t h i n a l a b o r a t o r y , and e q u i l i b r a t i o n
t i m e s used by d i f f e r e n t i n v e s t i g a t o r s . Thus, t h e d a t a
p r e s e n t e d i n T a b l e 7 s h o u l d be c o n s i d e r e d i n l i g h t o f t h e s e
v a r i a b l e s ; and t h e a n a l y s t should be expected t o a l t e r t h e
m o b i l e phase, a l t h o u g h s l i g h t l y , t o e f f e c t a s u i t a b l e
separation.

The r e f e r e n c e s c i t e d 5 5 - 6 2 i n d i c a t e t h e v a r i e t y
o f systems a v a i l a b l e f o r t h e s e p a r a t i o n o f t h e s e compounds.
Chapters from s e v e r a l t e x t ~ 4 6 , ~ 3 > @c o n t a i n a d d i t i o n a l
i nformation.

A c r i t i c a l t h i n l a y e r chromatographic a n a l y s i s
o f L - t h y r o x i n e sodium was made by a j o i n t committee o f t h e
Pharmaceutical S o c i e t y o f G r e a t B r i t a i n and t h e B r i t i s h
Pharmacopoeia p r i o r t o e s t a b l i s h i n g t h e sample as a r e f e r e n c e
s ~ b s t a n c e . 5 ~F i v e d i f f e r e n t m o b i l e phases on f i v e d i f f e r e n t
adsorbants were used t o e s t a b l i s h i t s p u r i t y .

262
SODIUM LEVOTHYAOXINE

Schorn and W i n k l e r S 5 s y s t e m a t i c a l y investi-


g a t e d more t h a n two dozen s o l v e n t systems on S i I ca g e l G
p l a t e s i n t h e s e p a r a t i o n o f L-T4, L-T3, L-T2, L- I and I-.
The r e s u l t s c e a r l y showed t h e v i a b i l i t y o f t h i s technicrue
t o t h e separa i o n o f t h e iodoaminoacids.

6.33 Col umn Chromatography

As column chromatography i s a r a t h e r non-


s p e c i f i c t e r m t o d e s c r i b e a s e D a r a t i o n orocedure. we have
d e l i neated t h i s t e c h n i q u e i n t o ' f o u r s p e c i f i c t y p e s : Ion
exchange, g e l f i l t r a t i o n , gas l i q u i d , and h i g h performance
I i q u i d chromatography. T h e i r i n d i v i d u a l a p p l i c a t i o n s a r e
d e s c r i b e d i n f o l lowing s e c t i o n s .

6.331 I o n Exchange Chromatography ( I E C )

Resin column chromatography has been


e v a l u a t e d and employed by many i n v e s t i g a t o r s i n t h e separa-
t i o n and q u a n t i t a i t o n o f iodoamino a c i d s and i o d o t h y r o n i n e s
i s o l a t e d from b i o l o g i c a I mater i a I ~ . 6 ~ - 7 8A I though t h e s e
procedures a r e amenable t o a s s a y i n g t h y r o x i n e and t h y r o i d
powders, t h e new s e p a r a t i o n t e c h n i q u e s d e s c r i b e d i n S e c t i o n s
6.332, 6.333, and 6.334, have, i n g e n e r a l , s u p p l a n t e d i o n
exchange chromatographic s e p a r a t i o n s .

A n i o n i c r e s i n s , Dowex I-X2 and 50-X4


(Dow Chemical Co., Midland, Mich.), u s i n g ammonium a c e t a t e ,
sodium a c e t a t e or ammonium formate b u f f e r s a t pH rnages from
3.2-5.6, w i t h o u t o r c o n t a i n i n g up t o 30% e t h a n o l , have been
used t o s e p a r a t e s e v e r a l o f t h e i o d o t h y r o n i n e s . Automated
procedures f o r d e t e c t i n g t h e components i n e f f l u e n t s have
a l s o been reported.68J73J74J77J7&? The c o n v e n t i o n a l c e r i c -
a r s e n i t e r e a c t i o n d e t e r m i n a t i o n f o r i o d i n e i s t h e most
f r e q u e n t l y used d e t e c t i o n method.

The a p p l i c a t i o n o f c a t i o n exchange
r e s i n s t o s e p a r a t e t h e i o d o t h y r o n i n e s has been r e p o r t e d . 71J
7 7 J 7 8 The c i t e d r e f e r e n c e s deal a l m o s t e x c l u s i v e l y w i t h two
iodoaminoacids and two i o d o t h y r o n i n e s , MIT, DIT, T3 and T4,
r e s p e c t i v e l y . R e c e n t l y , Sorimachi and U i 7 9 r e p o r t e d t h e
s e p a r a t i o n o f e i g h t d i f f e r e n t i o d o t h y r o n i n e s on (1.0 x 15 cm)
c a t i o n exchange r e s i n , AG 50W-X4 (30-35 pm), e q u i l i b r a t e d
w i t h 0.04 M ammonium a c e t a t e b u f f e r , pH 4.7, c o n t a i n i n g 30%
( v / v ) e t h a n o l a t 5OoC and a g r a d i e n t o f I n c r e a s i n g pH. The

263
ALEX POST AND RICHARD J. WARREN

g r a d i e n t c o n s i s t e d o f s t a r t i n g b u f f e r and 0.65 N NaOH.


D e t e c t i o n was by t h e i o d i n e c a t a l y z e d c e r i c - a r s s n l t e r e a c t i o n .
T a b l e 8 l i s t s t h e e l u t i o n volumes o f a s e r i e s o f lodoamino-
a c i d s and i o d i d e o b t a i n e d by t h i s procedure.

Table 8

E l u t i o n Volumes o f lodoam noac ds


Approximate E l u t on
Com pou nd
Volumes ( m l 1

Iodide 6
MIT 37
DIT 50
TI 80
T2 80
T3 I06
T4 91
3'-T I 96
3,3'-T2 I13
' '
3 , 5 -T2 88
'
3,3 ,5' -T3 96

6.332 Gel F i l t r a t i o n Chromatography (GFC)

The appl i c a t i o n o f g e l f i l t r a t i o n
s e p a r a t i o n o f t h e i o d o t h y r o n i n e s has been p r i m a r i l y used t o
d e t e r m i n e t h e i r i n d i v i d u a l c o n t e n t s I n b i o l o g i c a l samples.
The i n f o r m a t i o n p r o v i d e d i n T a b l e 9 i n d i c a t e s t h e chromato-
g r a p h i c systems used t o s e p a r a t e t h e iodoaminoacids i s o l a t e d
from t h e s e samples. Each o f t h e c i t e d r e f e r e n c e s d e s c r i b e s
t h e i m p o r t a n t s t e p s r e q u i r e d t o p r e p a r e t h e s e columns (e.g.,
t h e i r dimensions, mesh s i z e o f t h e g e l p a r t i c l e s , e t c . ) , t h e
d e t e c t i o n systems used ( g e n e r a l l y t h e c e r i c - a r s e n i t e r e a c t i o n ,
u l t r a v i o l e t absorption, l i q u i d s c i n t i l l a t i o n counting o f
tagged i s o l a t e s , e t c . ) , and t h e p r e c i s i o n and accuracy o f t h e
p a r t i c u I a r v a r i a t i o n employed by t h e r e s p e c t i v e i n v e s i t g a t o r .

B l a s i and DeMasiB0 have l i s t e d t h e


p a r t i t i o n c o e f f i c i e n t s , Kd, o f s e v e r a l t y r o s i n e s and t h y r o -
n i n e s as o b t a i n e d from t h e Sephadex G-25 column and t h e i r
e l u t i o n conditions. From t h e s e data, r e l a t i o n s h i p s between
t h e s t r u c t u r e o f t h e compound and t h e e l u t i o n volume can be
e s t a b l i s h e d . The Kd v a l u e s a r e l i s t e d I n T a b l e 10.

264
S O D I UM LEVOTHY ROX INE

Table 9
Gel F i l t r a t i o n Chromatographic S e p a r a t i o n Systems
Co I umn Compounds Ref
E1uent
Mater i a I Separated -
#
(a )
Sephadex G-25 0.01 ,N NaOH DIT, T3, T4 81
Sephadex G-25 tert-amyl alcohol T3, T4 82
saturated w i t h
2 -
N NH40H
Sephadex G-25 0.02 -
N NaOH Ty, MIT, DIT, T, 80
T I , ( b ) T2, T3, T4
Sephadex LH-20(a) ethy I acetate: MIT, DIT, T3, T4 83
methanol : 2 N NH40H
( l00:25: 10, T/v)

Sephadex G-25 0.1 N NaOH I-, T3, T4 84


0.007 -
N NaCl
Sephadex G- I 5
fa) 0.02 -
N NaOH T3, T4 85

fa)Pharmac i a F i ne Chemi ca I s, I nc. , P i scataway , NJ


fb)3- i o d o t h y r o n i ne. I n a d d i t i o n 3 ' ,5'-d i i o d o t h y r o n i ne and
3,3' ,5'- t r i io d o t h y r o n i ne were a I so separated.

T a b l e 10
Kd Values of l o d o t h y r o n i n e s and R e l a t e d Compounds
(a ) Kd
Comoound
TY 0.32
MIT 0.36
DIT 0.52
T 0.52
3- l o d o t h y r o n i ne 0.93
T2 1.13
3 , 5 '-d i io d o t h y r o n i ne I .95
T3 2.35
3,3',5'-tri iodothyron ine 4.40
T4 5.20

(a)l.5 mg i n 0.5 ml 0.02 -


N NaOH

265
Table I I
Gas L i q u i d Chromatographic S e p a r a t i o n Systems

Compound Co I umn Amount Ref


Derivative Co I umn Detector
Separated Temperature I nj e c t e d #
-
T4, T3 N,O-d p i v a l y l 0.5% SE-30 Program FID(~) ug 66
MIT, DIT, T met hy e s t e r on Gas Chrom Q I30-305OC
IOo/mi n
T4, T3, T2 N,O-b s t r i f I u o r o 3.8% SE-30 25OoC FID 0.01 umol 86
MIT, D I T a c e t y methy I on D i a t o p o r t S
ester
, T4, T3, N,O-d p i v a l y I 1 % polysulfone 232OC ECD f b ) ng 87
8 MIT, DIT met hy e s t e r on Gas Chrom Q
T4, T3, N, 0-d p i v a l y l 3% OV-17 on 282OC ECD P9 87
MIT, DIT methy e s t e r Gas Chrom P
T4, T3, TMS (CJ 3% O V - l on 285OC ECD 50-150 ng 88
MIT, DIT Gas Chrom Q
T4, T3, T2, TMS 0.5% SE-30 o n 75-250°C FIC 20 ng 89
MIT, DIT, T DMSC-treated f d ) 8 4.6'/rnin
Chromosorb G
T4, T3, T2 N,O-dipivalyl 5% OV-17 on 225-235OC FID < I ug 90
methyl e s t e r Gas Chrom Q @ 5O/min
T4, T3, T2, N,O-dipivalyl 5% OV-17 on 285OC ECD 3 ng 90
MIT, DIT, T methyl e s t e r Gas Chrom Q
Table I I (continued)

Compound Amount Ref


Der i v a t iv e Co I umn Co I umn Detector
Separated TemDerature Injected #--

T4, T3, T2 TMS 2% SE-33 on I 50-280'C FID 50 ng 91


DIT, Ty Gas Chrom Q @ 1o0/min
T4, T3, T TMS 3% OV-17 o n 165'C @ 3 m i n FID 3-15 u g 92
DIT, MIT, Ty Gas Chrom Q t o 265' @
10 m i n
T4, T3, T2, T, TMS 1 % OV-1 on 135-255'C FID 'L4 llg 93
M l T DIT, Ty C h romosor b W HP @ 5'/min
T4, T3, T2, T, TMS I % OV- I on 180-225OC ECD 0.3-1.5 ng 93
ru
3 MIT, DIT, Ty Chromosorb WHP 8 25'/min
T4, T3, T2 TMS 3% O V - 1 7 o n (el FID 5-20 ng 94
D i a t o m i t e CQ
T4, (f)T3, N,O-dimethyl 3% OV-1 on 25OoC FID
T2, DIT methyl e s t e r Gas C h r m Q
T4, T3, T2 TFAA(9) 2.3% O V - l on fe) ECD 1.4-2.5 ng 96
methyl e s t e r s Gas Chrom Q
T4, T3, T2 N,O-d i p i v a l y I 2.3% OV-1 on 29OoC ECD 6-8 ng 96
methy I e s t e r s Gas Chrom Q
T4, T3, TMS 1 % OV-l on 165-285'C FID 4-16 97
Gas Chrom Q 8 1o0/min
Tab I e I I (continued 1

Compou nd Co I umn Amount Ref


Separated
Der i v a t i ve Col umn Temper a t ure Detector Injected -
#

T4, T3 N,O-dipivalyl 3% DEXS 1 L 300 GC 305’C ECD 0.25-3 ng 98


methyl e s t e r on Chromosorb WHP

fa’FID = Flame I o n i z a t i o n D e t e c t o r

ECD = E l e c t r o n Capture Detector

fC’Trimethylsl l y l d e r i v a t i v e


OD ‘d’Dimethylchlorosi lane

fe’Variable, depending on which compounds a r e t o be separated

ff’As t h e sod i um sa I t , h y d r a t e
SODIUM LEVOTHYROXINE

6.333 Gas L i q u i d Chromatography (GLC)

S i n c e t h e iodoaminoacids a r e n o t
v o l a t i l e and t h u s n o t amenable t o a gas c h r o m a t o g r a p h i c
analysis, the preparation o f suitable stable v o l a t i l e
derivatives p r i o r t o analysis i s a prerequisite.

The f i r s t s u c c e s s f u l gas chromato-


g r a p h i c s e p a r a t i o n of T4, T3, DIT, MIT, and Ty, a s t h e i r
N , O - d i p i v a l y l m e t h y l e s t e r d e r i v a t i v e s , was r e p o r t e d by
Stouffer, e t S i n c e t h e n t h i s t e c h n i q u e s has been
c r i t i c a l l y e v a l u a t e d because o f i t s i n h e r e n t s e n s i t i v i t y ,
speed o f a n a l y s i s , and a p p l i c a b i l i t y t o t h e q u a n t i t a t i o n o f
t h e s e compounds i n b i o l o g i c a l p r e p a r a t i o n s . As i t i s
beyond t h e scope o f t h i s monograph t o p r o v i d e p r e c i s e
d e t a i l s o f t h e gas chromatographic methods, a t a b u l a t i o n o f
the various r e p o r t s i s l i s t e d i n Table 1 1 . I t i s recommended
t h a t t h e c i t e d r e f e r e n c e s be r e f e r r e d t o f o r t h e p r e p a r a t i o n
o f t h e v o l a t i l e d e r i v a t i v e s , t h e use o f i n t e r n a l s t a n d a r d s
f o r q u a n t i t a t i v e a n a l y s i s , and f o r t h e p r e p a r a t i o n o f t h e
column s u b s t r a t e s .

From t h e i n f o r m a t i o n I n T a b l e I 1 i t i s
apparent h a t t h e two f a v o r e d d e r i v a t i v e s a r e t h e N,O-
d i p i va I y I methyl e s t e r and t h e TMS. The advantage o f t h e
former i s t h a t t h e e s t e r s have g r e a t e r s t a b i l i t y . However,
they requ r e a two-step s y n t h e s i s , whereas t h e l a t t e r can be
prepared n a s i n g l e step but a r e s e n s i t i v e t o moisture.

6.334 H i g h Performance L i q u i d Chromatography

High performance I i q u i d chromatography


(HPLC) has been used by Karger, e t a l . 9 9 t o s e p a r a t e T4, T3
and T2 i n about two m i n u t e s i n a m o b i l e phase o f b u t a n o l and
m e t h y l e n e c h l o r i d e on a s i l i c a g e l column c o a t e d w i t h a
m i x t u r e o f p e r c h l o r l c a c i d and sodium p e r c h l o r a t e . On a
s i m i l a r system T4, MIT and DIT s e p a r a t e d i n l e s s t h a n e i g h t
m i n u t e s . DuPont I n s t r u m e n t s l o o r e p o r t e d t h e s e p a r a t i o n o f
T4 and T3 on a s t r o n g c a t i o n exchange (SCX) column. Both of
t h e s e methods showed e x c e l l e n t s e n s i t i v i t y , i n t h a t nanogram
q u a n t i t i e s c o u l d be r e a d i l y d e t e c t e d u s i n g h i g h l y s e n s i t i v e
UV d e t e c t o r s a t 254 nm.

T h y r o x i n e and t r i i o d o t h y r o n i n e have
a l s o been s e p a r a t e d i n l e s s t h a n 12 m i n u t e s on a M i c r o p a k

269
ALEX POST AND RICHARD J. WARREN

C-18 ( r e v e r s e phase) column u s l n g a methanol-ammonium c l t r a t e


mobi l e phase.101 Waters Associates102 r e p o r t e d a s l m i l a r
s e p a r a t i o n on a C I & o r a s i I column u s i n g a mob i l e phase con-
s i s t i n g o f a c e t o n i t r i le-n-butanol :0.005 M sodium p e r c h l o r a t e .
A c l e a r s e p a r a t i o n was e f f e c t e d i n l e s s t h a n 20 minutes.

6.4 Neutron Act i v a t i on Ana I ys i s

Neutron a c t Iv a t i o n ana I ys 1 s was used by A I soszo3 t o


d e t e r m i n e t h e L - t h y r o x i n e sodium c o n t e n t i n t a b l e t s . In this
procedure, t h e t a b l e t was i r r a d i a t e d f o r 5 m i n u t e s I n a
n e u t r o n f l u x of = 2.5 x 1012n/cm2/sec and q u i c k l y t r a n s f e r r e d
t o a c o u n t i n g t u b e f o r measurement o f 1281. Comparison w i t h
standards o f potassium i o d i d e I r r a d i a t e d f o r t h e same t i m e
y i e l d e d t h e c o n t e n t o f L - t h y r o x i n e sodium. Interfering
n u c l e a r r e a c t i o n s were n e g l l g l b l e , and t h e e r r o r was l e s s
t h a n 2%.

Neutron a c t i v a t i on ana I ys Is was a I so used by G I obe I,


e t a I . 104 t o determl ne t h e r e 1 a t i v e amounts o f L - t h y r o x i ne
( T 4 ) and 3 , 5 , 3 ' - t r I i o d o t h y r o n i n e (T3) i n serum. These
hormones were removed from t h e p r o t e l ns by pass i ng 20 m I o f
serum ( a t pH I I ) t h r o u g h a Dowex IX-2 i o n exchange column i n
t h e a c e t a t e form. The e l u a t e was c o n c e n t r a t e d and chromato-
graphed on a c e l l u l o s e t h i n l a y e r p l a t e t o s e p a r a t e t h e T4
and T3 from t h e i n o r g a n i c i o d i d e . The i s o l a t e d T4 and T3
f r a c t i o n s were i r r a d i a t e d I n a t h e r m a l n e u t r o n f l u x o f 5 x
1O2n/cm2/sec u s i n g a T r i g a Mark I r e a c t o r , f o l l o w e d by
i d e n t i f i c a t i o n and measurement of induced 1281 a c t i v i t y w i t h
a germanium ( L i ) sol i d s t a t e d e t e c t o r . The I i m i t s o f
d e t e c t i o n were 5 ng.

Schmoelzer and Muel Ier1O5 used a s l m l l a r approach


b u t separated t h e T4 and T3 o n a QAE Sephadex A-25 column
p r i o r t o activation analysis.

6.5 Po I a r o g r a p h i c Ana I y s i s

P o l a r o raphy was employed by Wacholz and P f e i f e r t o


assay I h y r o x i n e ~ O 6and t o d e t e r m i n e t h e t h y r o x i n e c o n t e n t o f
t h y r o l d powders and t a b I e t s .

I n t h e assay o f t h y r o x i n e , 5-50 pg of sample i n I m i


o f 2 N n i t r i c a c i d i s heated a t 6OoC f o r I hour. A f t e r c o o l -
i n g aKd t h e a d d i t i o n o f 5 m l o f 0.06 - N sodium h y d r o x i d e , t h e

270
SOD1 U M LEVOTHY R OX INE

s o l u t i o n I s deoxygenated w i t h n i t r o g e n . The t h y r o x i n e
c o n t e n t I s determined by comparison o f wave h e i g h t of t h e
sample a t E& -0.6 t o -0.7V w i t h t h a t o f standards. The
method i s s u f f l c l e n t l y s e n s i t i v e t o determine t h e t h y r o x i n e
c o n t e n t o f spots i s o l a t e d by t h i n l a y e r chromatography.

I n t h e assay f o r t h y r o x i n e I n t a b l e t s and powders,


p r i o r e x t r a c t i o n procedures a r e r e q u i r e d t o remove t h e
t h y r o x i n e and o t h e r i o d l n a t e d amino acids. A f t e r s e p a r a t i o n
by t h i n l a y e r chromatography, e l u t i o n o f t h e t h r y o x i n e spot,
c o n c e n t r a t i o n and n l t r a t i o n , 1 0 6 t h e wave h e i g h t a t t h e
p r e v i o u s l y s p e c i f i e d E+ i s obtained.

6.6 K i n e t i c Methods o f A n a l y s i s

The a p p l i c a t i o n of k i n e t i c measurements of t h e
i o d i n e c a t a l y z e d c e r i c arseni t e reaction108,109 has been
u t i I lzed f o r t h e d e t e r m i n a t i o n o f t h y r o x i n e i o d i n e i n
chromatograph I c e l u a t e s . 76~1*0,111 The r e p o r t e d methods a r e
rapid, have h i g h p r e c i s i o n and a r e s e n s i t i v e , g e n e r a l l y
d e t e c t i n g less than I ng o f T4.

6.7 Double-losotope D i l u t i o n A n a l y s i s

A double-isotope d e r i v a t i v e assay f o r serum iodo-


t h y r o n In e s l l 2 (L-T4 and L-T3) has been mod i f l e d and improved
upon by Hagen, e t a1.8 I n t h i s procedure, t h e unknown
t h y r o x i n e i s labeled by formation o f an a c e t y l d e r i v a t l v e l l 3
u s i n g t r i t i u m - l a b e l e d a c e t i c anhydride. As t h e s p e c i f i c
a c t i v i t y o f t h e t r i t i a t e d d e r i v a t i v e i s known, t h e t h y r o x i n e
c o n t e n t of t h e sample can be c a l c u l a t e d . Losses I n t h e
complex p u r i f i c a t i o n steps a r e accounted f o r by t h e a d d i t i o n
of a h i g h s p e c i f i c a c t i v i t y 1311-labeled t h y r o x i n e .

6.8 Determination o f Stereoisomeric P u r i t y

I n f o r m a t i o n presented by t h e J o i n t Committee of t h e
Pharmaceutical S o c i e t y of Great B r i t a i n and t h e B r i t i s h
Pharmaceutical Committee54 i n d i c a t e s t h a t a l I D-thyroxine
c o n t a i n s t r a c e amounts o f t h e L-isomer. A method f o r d e t e r -
mining t h e amount o f L-T3, an i n t e r m e d i a t e i n t h e s y n t h e s i s
o f D-T4, has been reported.45 An a d a p t a t i o n o f t h i s method
has been appl l e d t o D-T4 c o n t a i n i n g less than I % o f t h e L-
i somer. 114

271
ALEX POST AND RICHARD J. WARREN

6.9 Equi I i b r i u m D i a l y s i s

Several i n v e s t r g a t o r s 115-117 have used equ i I ib r i urn


d i a l y s i s t o d e t e r m i n e t h e f r e e t h r y o x i n e i n serum. A c r i t i -
c a l s t u d y o t h i s p r o c e d u r e was made b y Lee and P i leggT.115
The e f f e c t s of pH, i n c u b a t i o n t i m e and temperature, b u f f e r
c o m p o s i t i o n and c o n c e n t r a t i o n , p r o t e i n c o n c e n t r a t i o n , and
specimen d l u t i o n were s t u d i e d . U s i n g 1 3 1 1 - L - t h y r o x i n e and
a r e u s a b l e p l a s t l c d i a l y s i s c e l I , r e c o v e r i e s of 92-96% were
o b t a i n e d when t h e d i a l y s i s was r u n a g a i n s t 0.05 M phosphate
pH 7.6 b u f f e r a t 37OC f o r 18 hours. W i t h i n - r u n and between-
r u n p r e c i s i o n s were 10.6% and 14.2%, r e s p e c t i v e l y .

Fang and Selenkow,'" using t h e conventional d i a l y s i s


bag and 1251-L-thyroxine, determined t h e f r e e t h y r o x i n e con-
t e n t a f t e r d i a l y s i s a t 4 O C , a g a l n s t pH 7.4 phosphate b u f f e r
f o r 18-24 hours.

B i r d and A b i o d u n l Z 7 employed e q u i l i b r i u m d i a l y s i s
and i o n exchange chromatography t o d e t e r m i n e free t h y r o x i n e .
I o n exchange chromatography was used t o s e p a r a t e t h e 1251
i o d i d e from t h e 1251-L-thyroxine p r i o r t o d e t e r m i n i n g t h e
f r e e thyroxine content.

The l i t e r a t u r e i s r e p l e t e w i t h a s i g n i f i c a n t number
of p u b l i c a t i o n s d e s c r i b i n g m o d i f i c a t i o n s o f e q u i l i b r i u m
d y a l y s i s o r a c o m b i n a t i o n of t h i s p r o c e d u r e w i t h o t h e r
s e p a r a t i o n t e c h n l q u e s . l 1 8 - Z 2 2 The c i t e d papers o f f e r a good
b a s i c background t o t h e a p p l i c a t i o n of t h i s method t o t h e
s t u d y o f t h e t h r y o x i n e - p r o t e i n b i n d i n g phenomenon.

7. Methods of A n a l y s i s - A Compilation

The f o l l o w i n g t a b l e s (12, 13, 14 and 15) i n c l u d e t h e


r e f e r e n c e s t o a n a l y t i c a l procedures f o r t h e a n a l y s i s of
chemicals, t a b l e t s , powders, and serum and t i s s u e . S e v e r a l
a d d i t i o n a l r e f e r e n c e s which were n o t c i t e d i n t h e s p e c i f i c
sections a r e included i n t h e fol lowing compi'lations Two
r e v i e w a r t i c l e s , by CahnmannlZ3 and by Ral I , e t a l .i24, a r e
a l s o noted.

272
SODIUM LEVOTHYROXINE

Table 12

Analysis o f Thyroxine Chemicals

Ana l y s i s : See Reference # :

I dent I f i c a t ion 3, 3, 4, 54, 125, 1 2 6


T I t r I metry :
I odornet r ic 1, 3, 37, 107
N-Bromosuccinimide 38
Coulometric 39, 4 0
S p e c i f i c Ion E l e c t r o d e 43
Cer i rnetry 129
Chromatography:
PC 45, 54, 130, 131, 132, 1 3 3
T LC 54, 55, 58, 59, 61, 62, 107,
130, 131, 134
I EC 68, 69, 77, 79, 1 3 6
G FC 120, 127, 137
GLC 86, 87, 89, 90, 91, 92, 93, 94,
97, 98, 138
HP LC 101, 142, 143
Neutron A c t i v a t i o n 103
Po Ia rog r a p hy 97, 106, 107
Kinetic 76, 78, 111
Spect rophotornet r y 5, 6, 7, 107, 144
Automation 68, 77, 78, 110, 136, 145, 146,
147
Electrophoresis 54
Phase S o l u b i l i t y 54

Table 13

A n a l y s l s o f Thyroxine T a b l e t s

Ana I ys i s See Reference #

Titrimetry:
I odometr i c 1, 3, 4

273
ALEX POST AND RICHARD J. WARREN

Table 13 (continued

Chromatog rap hy :
PC 148
TLC 107, 144, 149
Pol arography 107
Spectrop hotometry 107, 144, 148, 149, 150, 151,
152, 153

Table 14

A n a l y s i s o f Thyroxine Powders

Ana I ys i s See Reference #

Cer i metry 61
Chromat o g rap hy
PC 154
T LC 59, 61, 149, 155, 156
I EC 154
GLC 94, 97, 157
S pectrophotometry 149, 150, 156
Titrlmetry 1

Table 15

Analysis o f Sera and/or Tissues f o r Thyroxine and Analogs

Anal y s 1 s See Reference #

Cer i met r y 68, 69, 71, 73, 74, 75, 76, 77,
78, 79, 120, 133, 145, 146,
158, 159, 160, 161
Chromatography :
I EC 67, 69, 70, 72, 72, 73, 74, 76,
77, 120, 117, 136, 145, 146,
GFC 147, 159, 163, 164
G FC 85, 110, 137, 165, 166, 167
GLC 87, 90, 98, 138
Neutron Act i v a t ion 104, 105

274
SODIUM LEVOTHYROXINE

T a b l e 15 ( c o n t i n u e d )

Kinetic 211
D i a iy s i s 215, 116, 117, 118, 719, 168,
169, 170, 171, 172
Spectrometry 163, 169, 173
Au toma t ion 73, 74, 78, 136, 145, 146, 147,
159, 170
Radloimmunoassay 174, 175, 176
Competitive P r o t e i n Binding 105, 128, 170, 171, 176, 177,
178, 179, 180, 181, 182, 183,
184, 185, 186; 187-
Nephelometry 188
E I e c t r o p hores i s 70, 135, 172
F I uoromet r y 162

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SODIUM LEVOTHYROXINE

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78, 32 (1975).
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39, 327 (1972) *
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S. J . P a t t e r s o n and R. L. Clements, A n a l y s t , 89, 328
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279
ALEX POST AND RICHARD J. WARREN

131. J . M. Ensor and P. Kendal I - T a y l o r , E n d o c r i n o l ., 46,


527 (1970).
132. P. R. Larsen, Metabolism, 20, 609 (1971).
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W i lmington, DE.
144. E. Wachholz and S . P f e i e r , Pharma i e , - 24, 459 (1969).
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146. G. K e s s l e r and V. J . P i eggi, C I i n . Chem., - 16, 382
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147. H. Y . Yee, e t a l . , C I i n Chem., 17, 622 ( 1971 1.
148. R. Lemieux and J. M. Ta mage, J . Pharm. Pharmacol
94 (1966).
149. J . E. Moody, J r . , e t a l , J . Pharm. Sci., 57, 634 ( I968
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252. I. C o v e l l i , e t a t . , Ana y t . B i o s e m . , 42, 82 (197 1.
152. .
J . H. Graham, e t a l , J Pharm. S c i . , 63, 763 ( 1 9 4 ) .
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J. H. Graham. J . Pharm. Sci , 64, 1 3 9 3 7 1 975).
154. S . k b l o g l u , e t a l . , E n d o c r i n o l . , 78, 231 (1966).
155. H. Frey, Scand. J. C I i n . Lab. I n v e s t . , 16, 470 (1964).
156. J o i n t Committee o f t h e Pharmaceutical S o c i e t y and t h e
S o c i e t y f o r A n a l y t i c a l Chemistry on Recommended
Methods f o r t h e E v a l u a t l o n o f Drugs, A n a l y s t , Lond.,
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92, 328 (1967).
157.
158. E. Makowetz, e t a l . , Mlcrochem. J.,
-
R. B l l o u s , e t a l . , J . Pharm. S c i . , 60, 1270 (1971).
10, 194 (19661.

280
SODIUM LEVOTHYROXINE

159. K. Horn, e t a t . , Z . K I i n . Chem. u k i n . B ochem., 2,


99 (1972).
160. N. E. K o n t a x i s and D. E. P i c k e r i n c.,I
l . J. CI n. E n d o c r i n o l .
Metab., 18, 774 (1958).
161. J . D. A c E n d , Biochem., g , 177 (1957).
162. E. E. G u s s a k o v s k i i , e t a l . , Mol. B i o l . , 7, 598 (1974
163. J . A. Hathaway, e t a t . , Am. J . CI i n . Path., - 53, 635
(1970).
164. V. V. Row, e t a l . , C I i n . Chim. Acta, 31, 473 (1971).
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I , 401 (1974).
166. R; Liewendahl, e t a l . , Acta E n d o c r i n o l . , 67, 793 ( 1 9 7 1 ) .
167. P. J . D a v i s and R. I . Gregerman, J. CI i n . E n d o c r i n o l .
Metab., 30, 236 (1970).
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126 (1975).
169. R. A. Pages, e t a l . , Biochem., 12, 2773 (1973).
170. W. C. G r i f f i t h s , e t a l . , C I i n . Biochem., 5, 13 (1972
171. N . M. Alexander and J . F. Jenni ngs, CI in.-Chem., -
20,
553 ( 1974).
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( I 970).
173. I . Posner, J . Lab. C I i n . Med., 57, 314 (1961).
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175. M. I . Surks, e t a l . , J. C l i n T l n v e s t . , 52, 805 ( 1 9 7 3 ) .
176. I . J . Chopra, e t a l . , J . C l i n . E n d o c r i n o l . Metab., - 36,
31 I ( 1 9 7 3 ) .
177. F. W. S p i e r t o , e t a t . , C l i n . Chim. Acta, 5 , 281 ( 1 9 7 4 ) .
178. B. Murphy and C. Patee, J . C I i n . E n d o c r i n o * ’ 26, 247
(1966) *
-
179. €3. Murphy and C. Patee, J . C I i n . E n d o c r i n o .’ -
24, 187
( I 964 1 *
180. R. Ekins, C I i n . Chim. Acta, 5, 453 ( 1 9 6 0 ) -
181. R. Ekins. e t a l . , C I l n . BiocFem., 2, 253 ( 969) *
182. F. J . L. Crombag, e t I., C l i n . Ch%. Acta - 46, 345
( 1 973).
183. D. E. D a l r y m p l e and R D. U t i g e r , J . Lab. C l i n . Ped.,

184.
-
75, 325 ( 1 9 7 0 ) .
S . Barbadoro, Can. J . Med. Technol., 35, 5 ( 1 9 7 3 ) .
185. H. Sel igson and D. Se igson, C I i n . C h x . Acta, - 38, 199
(1972) *
186. S. C. Thorson, e t a l . , Acta E n d o c r i n o l . , 64, 630 ( 1 9 7 0 ) .
287. S. Nobel and F. B a r n h a r t , C I i n . Chem., 15, 509 ( 1 9 6 9 ) .
188. G. Hocman and L. Hegedus, E n d o k r i n o l o g i e , - 55, 194
(1969).

261
METHOTREXATE

Arthur R. Chamberlin, Andrew P.K. Cheung, and Peter Lim


ARTHUR R . CHAMBERLIN etal.

Contents
--
1. Description
1.1 Name, Formulae, Molecular Weight
1.2 Isomeric Forms
1.3 Appearance, Color, Odor
2. Physical P r o p e r t i e s
2.1 Infrared Spectrum
2.2 Proton Magnetic SpeCtrUm
2.3 Carbon-13 Magnetic Spectrum
2.4 U l t r a v i o l e t spectrum
2 . 5 Mass Spectrum
2.6 Optical Rotation
2.7 D i s s o c i a t i o n Constants
2.8 S o l u b i l i t y

3. Synthesis

4. Stability
4.1 Bulk
4.2 S o l u t i o n

5. M e t a bol i s m
6. Methods of A n a l y s i s
6.1 Elemental Analysis
6.2 Equivalent Weight Determination
6.21 Nonaqueous t i t r a t ion
6.22 Complex-formation t i t r a t i o n
6.3 Biological Assay
6.31 Microbiological Assay
6.32 Enzymic Assay
6.4 Polarographic Assay
6.5 Spectrophotometric Analysis
6.51 Fluoromet r i c
6.52 U l t r a v i o l e t / v i s i b l e
6.6 Chromatography
6.61 Paper
6.62 Thin-Layer
6.63 column
6.64 High Speed Liquid
6.7 Proton Magnetic Resonance

284
METHOTREXATE

7. Acknowledgment

8. References

285
ARTHUR R . CHAMBERLIN eta/.

1 . Description

1.1 Name, S t r u c t u r a l and Empirical Formulae, Molecular


Weight
Methotrexate is N-[4-{[(2,4-diamino-6-pteridinyl)-
met hy 1]met hy 1amine } benz oy1] g l u t ami c a c i d . ,
Frequent 1y t h e
name i s abbreviated t o MTX. Methotrexate a l s o i s known as
~-amino-l0-methylfolic a c i d and amethopterin and i s
i d e n t i f i e d by t h e National Cancer I n s t i t u t e code number
NSC-740.

' 6'
7 0 Y FooH

COOH

CZ0Hz2N8O5 Mol. w t . 454.46


1.2 Isomeric Forms
The presence of an asymmetric carbon i n t h e
glutamic a c i d moiety provides f o r o p t i c a l isomerism. Unless
sp2cif ied, commercially a v a i l a b l e methotrexate i s prepared
from L-glutamic a c i d , Recently, L e e and co-workers'
prepared methotrexate s t a r t i n g with D-glutamic a c i d . The
D-enantioxer of methotrexate was a c t i v e a g a i n s t L-1210 i n
t h e mouse and had less t o x i c e f f e c t s t h a n methotrexate
i t s e l f , t h e L-enant iomer.
1.3 Appearance, Color, Odor
Methotrexate i s a bright yellow-orange, o d o r l e s s
powder. It g e n e r a l l y is hydrated t o t h e e x t e n t of 8 t o lo$
w a t e r . I t a l s o has been prepared a s t h e hydrochloride
( 1:0.3) and hydrate (1:2.5)*.
li. P r i v a t e communication from D r . H.B. Wood, J r . , of t h e
National Cancer I n s t i t u t e and M r . D.F. Worth of Parke,
Davis and Co.

286
METHOTREXATE

2. P h y s i c a l Properties

2.1 --
I n f r a r e d Spectrum
--
Recorded as a s u s p e n s i o n i n m i n e r a l o i l , t h e
spectrum i n F i g u r e 1 shows r e l a t i v e l y broad s t r u c t u r e s ,
i n d i c a t i n g t h e complexity of t h e molecule and s u g g e s t i n g
t h e l a c k of c r y s t a l l i n i t y of t h e sample. T a b l e I g i v e s
t h e assignments t o t h e major bands.

TABLE 1

I n f r a r e d Assignments f o r Methotrexate

I R Absorption B a n d b )
-I-----
Interpretation
---
2.90-3.10 H20, -W2)
3 .O0-4.03 -
-COOH

5.90-6.10
9
-C-(
R B
COOH, -C-N-)

6.20, 6.50-6.60 Aryl systems

6.50-6.60 Amide I1

11.9 H

2.2
ElH
H
P r o t o n Magnetic Resonance Spzctrum (pmr)
H

The pmr spectrum i n F i g u r e 2 w a s r e c o r d e d on a


Varian A&-A s p e c t r o m e t e r w i t h t h e sample a s a s o l u t i o n i n
DMSO-de. The chemical s h i f t s are i n ppm r e l a t i v e t o TMS
d e s i g n a t e d a s 0.00.

Table I1 g i v e s t h e s t r u c t u r a l assignments t o t h e
resonances i n F i g u r e 2 .

287
-I

O
0.10 r
w
V
z 0.20
2K 0.30
N
a, 0
a, 0.40
Q

-
2 3 4 5 6 7 8 9 10 11 12 13 14 15
WAVELENGTH - microns

FIGURE 1 INFRARED SPECTRUM OF METHOTREXATE


0
w
L-
h
m X
w
a
L
d U
0
f
N
Lu
289
TABLE I1

pnr Assignments for Methotrexate

Assignments
___-I_
s i c a l Shifts (ppm) Multiplicity J(Hz)

H2N-2 4 7.42 s( broad) --


H-7 8.64 S --
H-9 4.81 s( broad) --
HSC-11 3.21 S --
~ - 2 ~ , 6 ~ ,5'
,3' 6.81,7.78 d 8.2
H-8' 8.21 d 8 .O
H u 4.43 q 7 .o
H-B9y 1.70-2.60 m --
H(COOH, HOH) 6.21 s( broad) --
The values agree reasonable w e l l w i t h those reported by Pastore2 who
studied the p m spectra of methotrexate i n solutions a t pH 7.5.
METHOTREXATE

2.3 -
Carbon-13 magnetic spectrum (cmr)
The "cmr spectrum i n Figure 3 w a s recorded on a
Varian XL-100 spectrometer with t h e sample as a s o l u t i o n i n
IMSO. The assignments given i n Table 111 f o r Figure 3 are
i n general agreement with those reported by Ewers and
co-workers,' but minor d i f f e r e n c e s e x i s t .

TABLE I11

13C Assignment For Methotrexate (TMS 0 .OO ppm)

Chemical S h i f t ( ppm) Assignment


162.67 3 c-2
160.01 c-4
148.31 C-6
148.96 1 c-7
150.94 c-8a
121.66 C-4a
rv 40 (amid DMSO) c-9
51.96 c-11
121.24 c-1
128.94 ~ - 2 1 , C-6'
111.12 c-31, c-5'
150.85 c-4 '
166.41 c-7'
54.82 ca
26.17 c-B
39.55 CY
173.96 3 U-CoOH
174 .ll Y -COOH

2.1; ---
Ultraviolet
Figure =the
spectru~
UV spectrum of methotrexate i n 0.1 N
NaOH. The longest wavelength maximum appears a t 372 nm and
i s a s c r i b a b l e t o t h e diaminopteridine moiety. The i n t e r -
mediate wavelength maximum occurs a t 303 nm and i s due
primarily t o t h e aminobenzoyl group. The s h o r t wavelength
maximum is a t 258 nm and is a t t r i b u t e d t o both chromophores.
In 0.1 N HC1 methotrexate expsriences a hypsochromic
s h i f t r e s u l t i n g i n a UV spzctrum (Figure 5 ) t h a t e x h i b i t s

* UV s p e c t r a were recorded on a Cary Model. 14 and t h e


molar absorpt i v i t i e s a r e based on anhydrous methotrexate .
291
i
W
I-
a
X
W
a
I-
0
r
L
z
LL
0
H
9
a
I-
0
W
n
v)
E'
(?
F
u
m
W
a
3
(3
U
3
292
0
I 1 I 1 I I I 1 I In
d
I
P
0
0
d
0
I-
z
w
2
2
w
I-
a
X
w
&I a
m a I-
W 0
I
t; I-
w
5 z
0
z LL
a
Z
0
H
3
0 a
0 I-
0 u
w
L
v)
t;
J
0
>
a
E
0
In 3
3
hl
t
w
a
3
12
LL
293
ARTHUR R. CHAMBERLIN eta:.

250 300 350 400


NANOMETERS

FIGURE 5

294
METHOTREXATE

maxima a t 307 and 243 nm. The W d a t a are summarized i n


T a b l e I V and i n g e n e r a l are i n agreement w i t h t h o s e
r e p o r t e d by S e e g e r and co-workers4,

TABLE I V

A b s o r p t i o n S p a c t r a of M e t h o t r e x a t e

-
Solvent
I

0.1 N HC1
0.1 M ~ ~ 6 T. r 7i s buffer
0.1 N NaOH

2.5 Mass Spectrum


M e t h o t r e x a t e it self d o e s not y i e l d a s a t i s f a c t o r y
mass spectrum because of non-volat i l i t y .
HoNever, t r e a t m e n t
of m e t h o t r e x a t e w i t h a TMS r e a g e n t a f f o r d s a m i x t u r e of tri-,
tetra-, and psnta-TMS d e r i v a t i v e s t h a t d o e s g i v e u s e f u l mass
spzctral d a t a . That s e v e r a l TMS-containing d e r i v a t i v e s
are formed i s n o t s u r p r i s i n g , c o n s i d e r i n g t h e number and
v a r i e t y of f u n c t i o n a l g r o u p s i n v o l v e d .

The mass s p s c t r a l f r a g m e n t a t i o n p a t t e r n o b t a i n e d
f o r t h e tri-, tetra-, and psnta-TMS d e r i v a t i v e s is
summarized as follows:
I
RZ-NH
c

R1=R,=TMS: m/e 319 I


I
'1 COOTMS
Rl,R2=1TMS, 1H; m/e 247
I Rl=R2=TMS m / e 452
Rl,R,=lTMS, 1 H m / e 380
I
M1bR1=R2=R3=TMS m / e 814
M-, R1=R2=TMS, R,=H m/e 742
Mf-R1,R2=1TMS, l H , R3=H m/e 670
M-CH, m / e 727
M-HOTMS m/e 652
M-HOTMS-CH, m/e 637

295
ARTHUR R . CHAMBERLIN et al.

2.6 Optical Rotation


46

21
589
= 20.4 2 0.6' (c 1, N/10 NaOH)
546 = 26.9 0.8'

2.7 ~
D i s s o c i-
a t i o- --
n Constants
Neither t h e a c i d i c nor t h e b a s i c pKa f o r methotrex-
ate has been r e p o r t e d . However, Albert and co-workers5
reported t h a t t h e basic pKa values f o r 2 , b d i a m i n o p t e r i d i n e
w e r e < 0.5 and 5.32. Kallen and Jencks' r e p o r t e d t h e a c i d i c
pKa values f o r p-aminobenzoylglutamic a c i d a s 4.83 and 3.76.
By spectrometry, w e found a pKa of 5.60 2 0.03, which i s
a s s i g n a b l e t o t h e diaminopteridinyl moiety.

2.8 ___--
Solubility7
Methotrexate is p r a c t i c a l l y i n s o l u b l e i n water,
alcohol, chloroform, and e t h e r . I t i s f r e e l y s o l u b l e i n
d i l u t e s o l u t i o n s of a l k a l i n e and carbonates; it i s
s l i g h t l y s o l u b l e i n d i l u t e hydrochloric acid (1 i n 2 ) .

3. Synthesis
The f i r s t reported s y n t h e s i s of methotrexate, t h a t by
Seeger and co-workers' is a s follows:

CH2Br
I
+ CHBr +H-N C-NH-
I
CHO

COOH

-------- ----
+C These s p e c i f i c r o t a t i o n values a r e based on anhydrous
methotrexate.

296
METHOTREXATE

T h i s r e a c t i o n o f t e n i s r e f e r r e d t o as t h e Waller r e a c t i o n ,
because Waller i n i t i a l l y p r e p a r e d p t e r o y l g l u t a m i c a c i d by
an analogous method.

T h e o r e t i c a l l y , the s u b s t i t u t i o n on t h e p y r a z i n e r i n g
c a n be at t h e C-6 o r t h e C-7 p o s i t i o n . Proof of s t r u c t u r e
is based on t h e p t e r i n e c a r b o x y l i c a c i d o b t a i n e d from a n
a l k a l i n e p3rmanganat.e o x i d a t i o n of m e t h o t r e x a t e . Of t h e
two a c i d s p o s s i b l e , o n l y t h e C-6 h a s been found. D i s -
t i n c t i o n between t h e two p o s s i b l e a c i d s h a s been based on
comparative paper chromatography, a l t h o u g h pmr or c m r a l s o
c o u l d be u s e d .

Because t h e Waller r e a c t i o n g e n e r a l l y y i e l d s impure


p r o d u c t s t h a t are v e r y d i f f i c u l t t o p i r i f y, many r e s e a r c h e r s
have a t t e m p t e d t o improve t h e s y n t h e s i s of m e t h o t r e x a t e .
Among t h e more s u c c e s s f u l a t t e m p t s are t h o s e by Taylor’
and by P i p 2 r and Montgomery”. The r e s p e c t i v e methods
are o n t l i n e d as f o l l o w s :
Taylor

NC N CHZC1 0

0 0 I DMF

R=N-( g l u t a m y l )

297
ARTHUR R . CHAMEERLIN eta/.

Piper and Montgomery

NH2 NH2

PMABG = N-( p-methylaminobenzoy1)glutamic acid


DMAC = N,N-dimethylacetamide

These improved procedures not only y i e l d products


t h a t are easier t o purify, but t h e s u b s t i t u t i o n on t h e
pyrazine is unambiguous.

4. ----
Stability

4.1 Bulk
When stored i n a cappsd, brown b o t t l e a t room
temperature, samplings removed over a twelve-month period
yielded i n d i s t i n g u i s h a b l e UV and papsr chromatographic
d a t a , These r e s u l t s i n d i c a t e t h a t methotrexate is stable
f o r a t least one y e a r under these conditions.

4.2 Solution
As d i l u t e s o l u t i o n s ( 0 . 5 mg and 0.05 mg/ml) i n pH
7 .O aqueous sodium bicarbonate maintained a t room tempera-
t u r e i n darkness and under laboratory illumination, a l i q u o t s
removed over a 24-hour period y i e l d e d i d e n t i c a l papergrams,
within experimental e r r o r s . On t h i s basis, these s o l u t i o n s
were considered stable under t h e s e conditions.

298
METHOTREXATE

Under s t r o n g l y a c i d i c aqueous conditions, t h e amide i s


s u b j e c t t o hydrolysis, y i e l d i n g N10-methyl-4-amino-4-
deoxypteroic a c i d and glutarnic a c i d . Under highly a l k a l i n e
aqueous conditions, e s p x i a l l y a t e l e v a t e d temperatures,
t h e p r i n c i p a l decomposition products a r e N1'-methylfolic
acid, N1'-methylpteroic acid, and glutamic a c i d - - a l l as
t h e carboxylate i o n s .

Photodecompwition of c e r t a i n p t e r i n e s is well
documented .I1 However, no s p e c i f i c r e p o r t on t h e
photochemistry of methotrexate has been found i n t h e
literature.

5. Metabolism
I n man, a very l a r g e p o r t i o n of t h e methotrexate
administered is excreted unchanged, 'la i n d i c a t i n g t h a t
l i t t l e metabolism of t h i s drug occurs.

Johns and Loo" reported t h a t methotrexate is


metabolized r a p i d l y by r a b b i t l i v e r aldehyde oxidase,
and t h e metabolite w a s i d e n t i f i e d as 4-amino-k-deoxy-7-
hydroxy-N1O-methylpteroylglut amic acid (7-hydroxy-MTX) .
Johns and Valerino13 have observed t h a t , when
methotrexate i s incubated with cecal c o n t e n t s from t h e
mouse i n v i t r o , ~-amino-~-deoxy-N1O-methylpteroica c i d i s
produced. Levy and Goldman'* have demonstrated t h a t t h i s
metabolite can be produced from methotrexate by a c t i o n
of carboxypiptidases from s t r a i n s of Psudoxona_t.

6. Methods
--- -
of Analysis

6.1 ----
Elemental Analysis
Table I V p r e s e n t s t h e r e s u l t s from a r e p r e s e n t a t i v e
elemental a n a l y s i s of methotrexate ( r e f e r e n c e standard) .
TABLE V
Elemental Analysis of Methotrexate
9
Element -Theory -- $ Found
C 52.85 52.72
H 4.88 4.85
N 24.66 24.51
* Adjusted f o r t h e found water

299
ARTHUR R . CHAMEERLIN e t a / .

6.2 Equivalent Weight Determinations


_ _ l _ l
--I_---_

6.21 Nonaqueous T i t r a t i o n
D e Carnevale e t a1.l’ d e s c r i b e d a n
e q u i v a l e n t weight d e t e r m i n a t i o n based on a sodium methoxide
t i t r a t i o n i n p y r i d i n e t o an a z o - v i o l e t end p o i n t . Because
t h e b a s i s of t h e t i t r a t i o n i s a n a c i d l b a s e n e u t r a l i z a t i o n ,
it l a c k s s p e c i f i c i t y . Consequently, t h e method has not
been employed widely as a n a s s a y f o r m e t h o t r e x a t e .

6.22 -
Complex-Formation -----
Titration
G u e r e l l o h a s described’’ a second t i t r a t i o n
by which e q u i v a l e n t w e i g h t s of m e t h o t r e x a t e samples can be
obtain??. The aethod i s basad on t h e complexation between
the Ca and t h e glutamyl moiety and t h e r e b y a f f o r d s
higher s p e c i f i c i t y than an acidlbase neutralization.
Because g l u t a m i c a c i d c o n t a i n i n g i m p u r i t i e s a r e commonly
found i n m e t h o t r e x a t e samples, r e s u l t s from t h i s t i t r a t i o n
must be i n t e r p r e t e d c a r e f u l l y .

6.3 ---------
B i o l o g i c a l Assay

6.31 M i c r o b i o l o g i c a l Assay
S e v e r a l m i c r o b i o l o g i c a l a s s a y s are d e s c r i b e d
i n the 1 i t e r a t ~ r e . l ~
A l l are r e l a t i v e l y n o n s p e c i f i c and
very time consuming and t h u s have l i t t l e u s e f u l n e s s f o r
routine analyses.

6.32 -Enzymic
- - - Assay
~ -
Werkheiser and co-workers” have developed
an enzymatic a s s a y f o r m e t h o t r e x a t e u s i n g f o l i c acid
reductase . M e t h o t r e x a t e b i n d s v e r y t e n a c i o u s l y and
s t o i c h i o n e t r i c a l l y t o t h i s enzyme and i s determined
c o l o r o m e t r i c a l l y by t i t r a t i o n of t h e drug w i t h t h e
enzyme.

6.4 P o l a r o g r a p h i c Assay
Asahi” has r e p o r t e d p o l a r o g r a p h i c r e d u c t i o n of
m e t h o t r e x a t e . H e a t t r i b J t e s t h e f i r s t wave as being t h e
r e d u c t i o n t o dihydromethotrexate, t h e second wave a s being
t h e r e d u c t i v e c l e a v a g e of t h e CH2-N bond, and t h e t h i r d
wave a s being t h e r e d u c t i o n t o 2,4,-diamino-6-methyl-5,6,7,
8-tetrahydropteridine ,

300
METHOTREXATE

6.5 S p e c t r o p h-------
otometric Analysis

6.51 ---
Fluorometric Analysis
F l u o r o m e t r i c methods2' have been used
w i d e l y i n t h e a n a l y s i s of m e t h o t r e x a t e . The methods a r e
based on a n o x i d a t i v e t r a n s f o r m a t i o n of m e t h o t r e x a t e t o
the presumed p t e r i n e c a r b o x y l i c a c i d which f l u o r e s c e s
i n t e n s e l y . Because many of t h e i m p u r i t i e s commonly found
i n m e t h o t r e x a t e a l s o undergo t h e same r e a c t i o n , t h e s e
methods l a c k s p c i f i c i t y u n l e s s t h e o x i d a t i o n i s preceded
by a s e p a r a t i o n scheme i n which m e t h o t r e x a t e i s i s o l a t e d .

6.52 --
Ultraviolet/Visible Analysis
Because commercial m e t h o t r e x a t e s a n p l e s a r e
impure, and because t h e o r g a n i c i m p u - i t i e s have W absorp-
t i o n c h a r a c t e r i s t i c s t h a t a r e similar t o t h o s e of
methotrexate, d i r e c t u v / v i s s p s c t r o p h o t o n e t r i c a n a l y s i s i s
e n t i r e l y t o o n o n s p e c i f i c t o be of any v a l u e i n t h e
q u a n t i t a t i v e a n a l y s i s of m e t h o t r e x a t e . P r a c t i c a l l y a l l
t h e contaminants l i k e l y t o be p r e s e n t i n a sample of
met h o t r e x a t e- -N1' -me t h y 1p t e r o i c a c i d , and so f o r t h - -
w m l d i n t e r f e r e w i t h a direct W o r v i s i b l e method.

6.61 --
Paper
Nichol and co-workers21 have r e p o r t e d t h e
s e p a r a t i o n s of m e t h o t r e x a t e from related compounds on
Whatman No. 1 w i t h pH 7.0, 0.1 M sodium phosphate and w i t h
pH 5.0, 0.1 M sodium acetate. I n our l a b o r a t o r y , w e have
used 0.5% NaHC03 and 0.5% Na2C03 w i t h t h e sane papzr.

Balazs and co-workers22 r e p o r t e d a r a p i d


a s s a y f o r methotrexate based on ps.per chromatographic
s e p a r a t i o n followed by W measurement of t h e i s o l a t e d
m e t h o t r e x a t e component. The l i m i t a t i o n s of t h i s a s s a y a r e
t h e accuracy of t h e r e f e r e n c e UV v a l u e and t h e r e c o v e r y
of m e t h o t r e x a t e from t h e papergram. The molar a b s o r p t i v i t y
a t 303 nm f o r m e t h o t r e x a t e i n N / 1 0 NaOH r e p o r t e d by
B a l a z s and co-workers i s i n e r r o r ; it should be 24,830.

The a s s a y procedure f o r m e t h o t r e x a t e c i t e d
i n USP XVIII is s i m i l a r t o t h e a s s a y r e p o r t e d by Balazs et
a 1 e x c e p t t h a t t h e r e f e r e n c e s t a n d a r d i s USP m e t h o t r e x a t e

301
ARTHUR R . CHAMEERLIN etal.

which i t s e l f i s impure.

6.62 Thin-layer
Copenhaver and O'Brienz3 have used ion-
exchange thin-layer chromatography t o s e p a r a t e a number of
f o l i c a c i d analogs including methotrexate .
The cation-
exchange r e s i n was AG50W-X4, and the developing solvent
was 154 Na2HF04'12 H20, p H 8.5 b u f f e r containing 0.1 M
mercaptoethanol .
6.63 Column
Heinrich and c o - ~ o r k e r sreported ~~ the
use of Dowex 1-chloride w i t h very d i l u t e HC1 or N@ to
separate f o l i c acid and r e l a t e d compounds. Noble
described a p u r i f i c a t i o n procedure based on the use of a
Dowex 1-acetate w i t h pH 3.2, M a c e t a t e b u f f e r . Oliverio2'
cited t h e use of DEAE c e l l u l o s e and pH 8 phosphate b u f f e r
gradient t o s e p a r a t e f o l i c acid analogs. G a l l e l l i and
YokoyamaZ7 developed a methotrexate assay procedure
e n t a i l i n g a DEAE c e l l u l o s e s e p a r a t i o n followed by a
spectrophotometric measurement of t h e i s o l a t e d component.

The use of DEAE cellulose t o separate f o l i c


a c i d analogs is w e l l e s t a b l i s h e d . The p r i n c i p a l
l i m i t a t i o n s of t h i s method are t h e t i m e required t o pack
t h e column and t h e t i m e needed t o develop t h e chromatogram.

6.64 High -
Speed Liquid
In our work w i t h other f o l i c a c i d antagon-
ists, cat ion exchange high speed l i q u i d chromatography
(HSLC) has been very e f f e c t i v e i n separating c l o s e l y
r e l a t e d p t e r i d i n e s . Taking advantage of t h i s s e l e c t i v i t y ,
we have developed a HSLC method of a n a l y s i s f o r methotrexate
that is specific, f a s t , and s e n s i t i v e . W e use it t o assay
b u l k and formulated methotrexate. The procedure r e q u i r e s
a reference methotrexate sample of known purity, which i s
used a s an e x t e r n a l reference o r i n conjunction w i t h a n
i n t e r n a l reference that, i n our laboratory, has been
2-amino-bmethylpyridine . The column, l m x 3mm 0 .D.
Vydac Cation Exchange Packing, i s held a t 55' during t h e
developnent w i t h pH 4.30, 0 . 1 M KH2P04 a t a f l o w rate of
1.0 ml/min. The e f f l u e n t is monitored by a UV-detector set
a t 254 nm. With t h e d e t e c t o r s e t a t i t s highest s e n s i t i v i t y ,
s o l u t i o n s a s d i l u t e d a s 1 pg methotrexate/ml can be i n j e c t e d

302
METHOTREXATE

d i r e c t l y and d e t e c t e d .

A second procedure employing a l m x 3mm O.D.


column packed with reverse-phase phenyl and a mobile phase
of 5% MeOH i n 0.05 M KH2P04, pH 7.0, b u f f e r a l s o has been
developed and found u s e f u l , The reverse-phase system is
less s e n s i t i v e t o minor pH v a r i a t i o n s r e s u l t i n g from sample
i m p u r i t i e s . For t h i s reason, t h i s column produces high
p r e c i s i o n more r e a d i l y .

6.7 Proton Magnetic Resonance Assay


K t h o t r e x a t e has been assayed by q u a n t i t a t i v e
pmr on a Varian A-&A spectrometer. Accurately weighed
portions of methotrexate and of an i n t e r n a l standard
(2,4-dimethoxy-5-methylpyrimidine) were dissolved i n DMSO,
and t h e spectrum was recorded. The psrcentage of anhydrous
methotrexate i n t h e sample then was c a l c u l a t e d according
t o t h e formula

$ MTX = Wr x A s x454.4 x
- - 1 P
Ws Ar 54.2
where W r = weight of i n t e r n a l standard used

W s = weight of methotrexate sample

A r = i n t e g r a t e d a r e a of i n t e r n a l
standard He s i n g l e t (8.02 6 )

As = i n t e g r a t e d a r e a of methotrexate
H7 proton (8.64 6)

154.2 = molecular weight of i n t e r n a l standard

454.4 = molecular weight of anhydrous MTX

P = p u r i t y of t h e i n t e r n a l standard

Although t h e pmr method may lack s e n s i t i v i t y and possibly


s p e c i f i c i t y i n complex mixtures, it i s one method of assay
t h a t does not r e q u i r e a methotrexate reference sample of
known p u r i t y . Thus, it may be u s e f u l i n cases i n which no
reference material is a v a i l a b l e .

303
ARTHUR R. CHAMBERLIN etal.

7 , Acknowledgments
----
Supported by C o n t r a c t N01-CM-33723 from t h e d i v i s i o n of
Cancer Treatment, N a t i o n a l Cancer I n s t i t u t e , N a t i o n a l
I n s t i t u t e s of Health, Department of Health, Education, and
Welfare. The o p i n i o n s e x p r e s s e d are t h o s e of t h e a u t h o r s
and not n e c e s s a r i l y t h o s e of t h e N a t i o n a l Cancer I n s t i t u t e .

The a u t h o r s wish t o acknowledge t h e t e c h n i c a l


a s s i s t a n c e r e n d e r e d by Ms. Leslie Pont, M s . Barbara Senuta,
Ms. F l o r e n c e Yoshikawa, M r . Martin S t r u b l e and I r John .
Jee of S t a n f o r d Research I n s t i t u t e .

304
METHOTREXATE

8. Reference?

1. W.W. Lee, A.P. Martinez, and L . Goodman, J . Med.


Chem. lJ, 326 (1974) .
2. E . J . Pastore, Ann. N.Y. Acad. S c i . -9185 43 (1971).
3. U . Ewers, H. Gunther, and L . Jaenicke, Chem. B e r .
-
106, 3351 (1973) *
4. D.R. Seeger, D.B. Coslllich, J . M . Smith, and M.E.
Hultquist, J . Am. Chein. SOC. 2, 1753 (1943).
5. A . A l b e r t , D.J. Brawn, and G . Chessman, J . Chem.
SOC., 4219 (1952).
6. R.G. Kallen and W.P. Jencks, J . B i o l . Chem. 24,
531.~5(1956) . I

7. The United S t a t e s Pharlnacopzia, The XVIII


Revision (1970), p. 418.
8. D . R . Seeger, J.M. Smith, and M.E. Hulquist, J . Am.
Chem. S O C . 69, 2567 (1947) .
9 . E .C. Taylor,'Chernistry and Biology of P t e r i d i n e s ,
Proceedings of the Fourth I n t e r n a t i o n a l SympDsium
on P t e r i d i n e s , Toba, 19s9, I n t e r n a t i o n a l Academic
P r i n t i n g Co., Ltd., Tokyo, 1970, p. 79.
10* J . R . Pipzr and J . A . Montgoaery, J . H e t . Chem. 11,
273 (1974) *
11. R.L. B l a k e l y , The Biochemistry of F o l i c Acid and
Related P t e r i d i n e s , Nort h-Holland Pub1i s h i n g
Compnny, London, 1959, p. 77.
l l a . I b i d p.495.
12. D.G. Johns and T.L. Loo, J . Pharm. S c i . - 56, 356
(1957) *
13. D.G. Johns and D.M. Valerino, Ann. N.Y. Acad. S c i .
-
186, 384 (1971).
14. C.C. Levy and P. Goldman, J . Biol. Chem. --
242, 2933
(1967) *
15. R.C.D. D e Carnevale, J . Dobrecky, and L.O. Guerello,
Rev. F a m . (Buenos Aires) -
113, 15 (1971).
16. L.O. Guerello, Rev. Asoc. Bioquim. Argent. - 34, 33
(1969) *
17. J.H. Burchenal, G.B. Waring, R.R. E l l i s o n , and
H.D. R e i l l y , Proc. SOC. Exp. B i o l . N.Y. i '8, 603
.
( 1951) ; A . Z Smolyanskaya and N.N. Agadzhanova,
.
vop. Onkol 13, 58 (1957) Chem. Abst , .,46, 26036;
D.E. Hunt a n r R . F . P i t t i l l o , Cancer R e s . -28, 1095
(1958) .

305
ARTHUR R . CHAMBERLIN etal.

18. W.C. Werkheiser, S . F . Zakrzewski, and C.A. Nichol,


.
J . Plannacol Exp. Therap. m,
162 (19.52) .
19. Y, Asahi, Yakugaku Zasshi E,
1570 (1959), Chea.
.
Abst 54, 10593d.
20. S.G. CcGkrabarti and I . Bernstein, C l i n . Chem. 9,
1157 (1959); S . F . Zakrzewski and C.A. Nichol, J .
Biol Chem. - -- 364 (1953); M.V. Freeman, J .
205,
Pharmacol. E X ~ .Therap. - -- 1, (1957) and 121,
120,
154 (1958) *
21. C.A. Nichol, S . F . Zakrewski, and A.D. Welch, Proc.
So. Exp. Biol. Med. 5, 272 (1953); S.F. Zakrrewski
and C . A . Nichol, J . Biol. Chea. 205, 352 (1953).
M.K. Balazs, C.A. Anderson, and K-Lim, J . Pharm.
22.
sci. z, 2002 (1968).
J.H. Copmhaver and K.L. O'Brien, Anal. Biochem.
23.

24.
s, 454 (1969) '
M.K. Heinrich, V.C. Dewey, and G.W. Kidder, J .
Chromatog. - 2, 296 (1959).
25. E.P. Noble, Biochem. Prep. 8, 20 (1961).
26. V.T. Oliverio, Anal. Chem. 3, 263, (1951).
27. J . F . G a l l e l l i and G. Yokoyama, J . Pharm. S c i . 55,
357 (1957)

306
METHYCLOTHIAZLDE

James A . Raihle
JAMES A. RAIHLE

Contents

1. Description
1.1 Nomenclature
1.11 Chemical Names
1.12 Generic Name
1.13 Trade Names
1.2 Formulae
1.21 Empirical
1.22 Structural
1.3 Molecular Weight
1.4 Elemental Composition
1.5 General

2. Physical Properties
2.1 Infrared Spectrum
2.2 Raman Spectrum
2.3 Nuclear Magnetic Resonance Spectrum
2.4 Ultraviolet Spectrum
2.5 Mass Spectrum
2.6 Melting Range
2.7 Differential Thermal Analysis
2.8 Dissociation Constant
2.9 Solubility
2.10 Crystal Properties

3. Synthesis

4. Stability-Degradation

5. Drug Metabolic Products

6. Methods of Analysis
6.1 Titrimetric Methods
6.11 Argentimetric Titration
6.12 Potentiometric Titration
6.2 Chromatographic Methods
6.21 Column Chromatography
6.22 Paper and Thin-Layer Chromatography
6.3 Spectrophotometric Methods
6.4 Polarographic Method

7. References

308
METHYCLOTH lAZl DE

Methyclothiazide

1. Description

1.1 Nomenclature
1.11 Chemical Name

Methyclothiazide is 6-chloro-3-(chloromethyl)
-3,4-dihydro-2-methy1-2H-1,2,4-benzothiadiazine-7-su1fon-
amide 1,l-dioxide. (1) It is also known as 6-chloro-3-
ch1oromethy1-3,4-dihydro-2-methy1-7-su1famoy1-1,2,4-benzo-
thiadiazine lyl-dioxide; 6-chloro-3-chloromethyl-2-methyl-
7-sulfamyl-3,4-dihydro-1,2,4-benzothiadiazine 1,l-dioxide
(2) and by many slight variations of the particular nomen-
clature. The GAS Registry No. is [135-07-91.

1.12 Generic Name

Methyclothiazide

1.13 Trade Names

Enduro@ and Aquatensen@

1.2 Formulae
1.21 Empirical

‘gHl 1C12N304S 2
1.22 Structural

309
JAMES A. RAIHLE

1.3 Molecular Weight

360.23

1.4 Elemental Composition

C -
30.00; H - 3.08; C1 - 19.68; N - 11.66;
0 - -
17.77; S 17.80.

1.5 General

Methyclothiazide occurs as a white to practically


white crystalline powder which is principally odorless.

2. Physi'cal Properties

2.1 Infrared Spectrum (IR)

The infrared spectrum of methyclothiazide (NF Ref-


erence Standard, Lot No. 69196) is presented in Figure 1.
The spectrum of a KBr pellet is taken on a Perkin-Elmer
Model 521 Spectrophotometer. The assignments for the char-
acteristic bands in the IR spectrum are listed in Table I.
(3)
Table I

Characteristic Bands in the IR Spectrum


of Methyclothiazide

Wavelength (em-l) Characteristic of

3270 and 3360 NH stretching vibration


of sulfonamide group

1595 and 1502 C = C stretch of aromatics


1155 and 1330 S = 0 stretching vibra-
(doublet) tions of sulfonamide
groups
This spectrum is consistent with that published by Fazzari
and co-workers. (4)

310
WAVELENGTH (MICRONS)
2.5 3 4 5 6 7 8 9 10 12 15

FIGURE 1 - INFRARED SPECTRUM


0F METHY CLOTHIAZIDE
JAMES A. RAIHLE

2.2 Raman Spectrum

The raman spectrum of the methyclothiazide refer-


ence standard was determined in the solid phase on a Cary
Model 83 Spectrophotometer. The assignments of the char-
acteristic bands as shown in Figure 2 are listed in Table
11. (3)

Table I1

Characteristic Bands in the Raman Spectrum


of Methyclothiazide

Wavelength (cm-l) Characteristic of

3270 and 3363 NH stretching vibration


of sulfonamide group
1600 C = C stretch of aromatics

1160 s = 0 stretching vibrations


of sulfonamide groups

2.3 Nuclear Magnetic Resonance Spectrum ("El)

The 60 MHz NMR spectrum of methyclothiazide is


presented in Figure 3. The spectrum was determined in
deuterated acetone (d6) on a Varian T-60 Spectrometer.
Spectral assignments are given in Table 111. (5)

312
- 0
AlISN31NI
0 0
0 0 0 0 0
Qo 9 v el
313
FIGURE 3 - NUCLEAR MAGNETIC RESONANCE
SPECTRUM OF METHYCLOTHIAZIDE
r

d
8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.o
PPM (6)
METHYCLOTH IAZIDE

Table 111

NMR Assignments for Methyclothiazide

Number of Chemical
Assignment Protons Shift (ppm) Multiplicity

Aromatic proton 1 8.23 S


at carbon 8

Aromatic proton 1 7.22 S


at carbon 5

Exchangeable 3 6.87 M(b)


protons: NH, NH2

Methyne proton 1 5.53 T


at carbon 3

Methylene protons 2 4.07 D


of chloromethyl
group at carbon 3

Methyl protons 3 2.77 S


of 2-methyl group

2.4 Ultraviolet Spectrum (W)


The UV spectrum of methyclothiazide prepared as a
1 in 100,000 solution in methanol is shown in Figure 4 .
The spectrum exhibits three maxima and two minima charac-
teristic of substituted benzothiadiazines. The maxima are
at 226 nm (Em = 39,300), 267 nm (Em = 21,250) and ca 311 nm
(Em = 3,300). Minima were observed at 240 nm and 290 nm.
The spectrum is consistent with previously published re-
ports by Furman ( 6 ) and Fazzari, et. al. (4)

2.5 Mass Spectrum

The mass spectrum shown in Figure 5 was obtained


using an Associated Electrical Industries Model 902 Mass
Spectrometer with an ionizing energy of 50 eV and a temper-
ature of 150°C. Methyclothiazide yields a spectrum with
a base peak at m/e 359. Subsequent fragments, Table IV,

315
JAMES A. RAIHLE

FIGURE 4 - ULTRAVIOLET SPECTRUM


OF METHYCLOTHIAZIDE

Ly
v
2
a
m
=
0
VI
m
a

200 250 300 350


WAVE LENGTH (nm)

316
FIGURE 5, MASS 5PEClRUM OF MElHYCLOlHIAZIDE

I ; I,, ,
317

I,, .I, I .#I,II


I ' I ' L ' I ' I
250 300 350
JAMES A. RAIHLE

reflect the loss of the chloromethyl and sulfonamide groups


as well as the fragmentation of the ring system. (7)
Table IV

High Resolution Mass Spectrum of Methyclothiazide

Composition
Mass Relative Error
Found Intensity (mu) -c L! ! 0 s c135
358.9565 3.06 -0.32 9 1 1 3 4 2 2

309.9711 100.00 -1.21 8 9 3 4 2 1

291.9629 0.56 1.18 8 7 3 3 2 1

229.9914 6.02 -0.26 8 7 2 2 1 1

166.0280 3.52 -1.78 8 7 2 0 0 1

131.0615 5.42 0.57 8 7 2 0 0 0

123.9952 7.89 -0.22 6 3 1 0 0 1

96.9847 5.95 0.16 5 2 0 0 0 1

90.0113 6.77 0.25 3 5 1 0 0 1

42.0345 63.94 0.13 2 4 1 0 0 0

2.6 Melting Range

Methyclothiazide melts with rapid decomposition


between 225°C and 227°C. Slight discoloration of the solid
may be observed at about 215°C.

2.7 Differential Thermal Analysis

The thermogram depicted in Figure 6 shows a large


endothermic melt between 224°C and 231°C. The thermogram
shows a subsequent exothermic response confirming the
visual observation of rapid decomposition.

318
FIGURE 6 - DIFFERENTIAL THERMAL ANALYSIS CURVE OF METHYCLOTHIAZIDE

0 20 40 40 80 100 no 140 160 in0 200 220 240


1, OC (CORRECTED FOR CHROME1 ALVMEL THERMOCOUPLES)
JAMES A. RAIHLE

2.8 Dissociation Constant

The pKa of methyclothiazide was determined by the


titrimetric method by extrapolation of data from acetone-
water mixed solvents to 100% water. The pKa is 9.4 (proton
lost).

2.9 Solubility

The following solubilities have been determined


for methyclothiazide at room temperature.

National Formulary
Solvent Solubility Descriptive Term( 1)

Water 0.06 mg/ml Very Slightly Soluble


Chloroform 0.03 mg/ml Very Slightly Soluble
Benzene < 1 mg/ml Very Slightly Soluble
n-Butanol 1 mg/ml ----
PEG-600 1 mg/ml ----
Ethano1 11 mg/ml Slightly Soluble
Acetonitrile 5 mg/ml ----
Methanol > 10 mg/ml Sparingly Soluble
Acetone - 200 mg/ml Freely Soluble
Pyridine -
N

- 200 mg/ml Freely Soluble

2.10 Crystal Properties

The X-ray powder diffraction pattern of methyclo-


thiazide was determined by visual observation of a film
obtained with a 143.2 mm Debye-Scherrer Powder Camera. An
Enraf-Nonius Diffractis 601 Generator; 38 KV and 18 MA
with nicker filtered copper radiation; ), = 1.5418 was
employed. A listing of d-spacings and intensities is pre-
sented in Table V. (8)

320
METHYCLOTH IAZIDE

Table V

X-Ray Powder Diffraction Pattern


d-Spacings and Intensities

-
dA -111
1 -
dA I/I1
9.8 5 2.95 5
7.75 50 2.90 LO
7.5 30 2.81 60
7.2 5 2.72 3
6.25 20 2.68 5
5.75 50 2.62 15
5.3 5 2.51 10
5.1 10 2.42 3
4.85 100 2.27 15
4.56 80 2.24 10
4.42 20 2.15 3
4.3 15 2.11 5
4.11 20 1.99 8
4.00 2 1.88 3
3.90 2 1.85 5
3.75B 20 1.81 8
3.6 3 1.77 2
3.52 20 1.75 3
3.44 5 1.73 3
3.32 5 1.69 2
3.30 8 1.64 4
3.12 5 1.5 B 4
3.07 8 1.35B 3
3.00 5 1.3 B 3

3. Synthesis

Methyclothiazide is synthesized by the reaction


sequence shown in Figure 7. Sprague (9) described the
reaction of 4-amino-6-chloro-1,3-benzenedisulfonamide with
urea to form the 3-keto derivative. Close, et. al. (10)
have preferentially alkylated the more acidic cyclic sul-
fonamide with methyl iodide to form 6-chloro-2-methyl-3-
oxo-7-sulfamyl-3,4-dihydro-l,2,4-benzothiadiazine 1,l-
dioxide. This intermediate is readily ring opened by
alkaline hydrolysis and then re-cyclized with chloro-
acetaldehyde to form the desired product.

321
Figure 7

Synthesis of Methyclothiazide

0=0
0

0
II
+ CH31

z
z
I
+HpNCNHz

N
N

z
I
N
322

*
-
6
I
N
t
0
I

%
0
2

I
z
I

I
z
I
0
6

6
N

0
METHYCLOTH IAZIDE

4. Stability-Degradation

Methyclothiazide is stable in the solid state and under


ordinary ambient conditions. It is rapidly decomposed in
boiling acidic solutions to 2-methylsulfamyl-4-sulfarnyl-5-
chloroaniline. In alkaline solution it rapidly loses one
chlorine atom to form the 3-hydroxymethyl analog. This
product has been shown to degrade further under severe con-
ditions, however none of the alkaline degradation products
contain primary aromatic amine centers. Solutions buffered
at pH 4.0 show 22% hydrolysis after 7 days at 60"C, 10%
after 28 days at 40"C, but only 2% after 28 days at 25°C.
Solutions buffered at pH 6.0 gave 1.6% hydrolysis after 7
days at 6OoC, and less than 1% after 28 days at 40°C or
25°C.

5. Drug Metabolic Products

No report of metabolic products related to methyclo-


thiazide has been published.

6. Methods of Analysis
6.1 Titrimetric Methods

6.11 Argentimetric Titration

The compendia1 procedure for the purity de-


termination of the drug substance is based upon the argen-
timetric titration of the chloride liberated after reflux
in methanolic potassium hydroxide. (1) The equivalent
weight is 360.23 since only the chlorine from the 3-chloro-
methyl group is liberated during reflux. The method is
specific since this group is added during the final step
of the synthesis and a limit of 0.02% free chloride is
imposed on the drug substance.

6.12 Potentiometric Titration


Methyclothiazide can be titrated as an acid
using either tetra-butylamonium hydroxide in chlorobenzene
(11) or potassium hydroxide in isopropanol (12) as the
titrant. The solvents are pyridine and acetone, respective-
ly. Methyclothiazide consumes two equivalents of base per

323
JAMES A. RAIHLE

mole of drug substance. The first equivalent is from the


neutralization of the free sulfamyl group. The exact lo-
cation for the reaction of the second equivalent has not
been determined, however, it may result from rapid hydroly-
sis of the 3-chloromethyl function.

6.2 Chromatographic Methods

6.21 Column Chromatography

Fazzari (13) has published a collaborative


study on a column chromatographic method for the analysis
of methyclothiazide from tablets. The drug is eluted from
a 0.1 NaHC03-Celite column with chloroform and measured
directly at 267 nm by W spectrophotometry. The precision
of the method was 99.8 4 1.64% on a commercial preparation
of 2.5 mg tablets.

6.22 Paper and Thin-Layer Chromatography

Paper chromatography has been applied by


Pilsbury and Jackson (14) for the rapid detection and iden-
tification of thiazide diuretics in tablets, gastric fluid,
and urine. Identification is accomplished by ascending
reverse phase chromatography using tributyn treated paper
and developing for 20 minutes at 90°C with a phosphate buf-
fer (pH 7.4). The thiazides are located by ultraviolet
light (254 nm) and confirmed by the red color given by
alkaline sodium 1,2-naphthaquinone-4-sulfonate spray rea-
gent. Paper chromatography can also monitor the extent of
manufacturing by-products. Ascending chromatography using
butanol saturated with 3% ammonium hydroxide and descending
chromatography using butano1:acetic acid:water (50:15:60)
have been employed. Visualization is by ultraviolet light.

Thin-layer chromatography using the system


ch1oroform:methanol:ammonium hydroxide (170:30:2) on 0.25
mm silica gel GF254 plates and ultraviolet detection rapid-
ly isolates and identifies the common manufacturing inter-
mediate s ,

324
METHYCLOTH IAZIDE

6.3 Spectrophotometric Assays

Methyclothiazide can be determined after acid


hydrolysis to 2-methylsulfamyl-4-sulfa~l-5-chloroaniline
by a modified Bratton-Marshall procedure. This procedure
without prior acid hydrolysis also monitors diazotizable
substances in the drug substance. (1) Ultraviolet absorp-
tion at 267 nm is seldom directly employed since the inter-
mediates and degradation products have similar absorption
spectra. The ultraviolet procedure has been utilized after
prior separation by chromatography (13) or for non-specific
content uniformity measurements. (1)

6.4 Polarographic Method

The current compendia1 assay for methyclothiazide


tablets is a polarographic assay in an aqueous system con-
taining 6% v/v dimethylformamide and 0 . 1 _M tetra-n-butyl-
ammonium chloride as the supporting electrolyte. A mercury
anode is used in conjunction with the DME since the half-
wave potential of methyclothiazide occurs in the region
where potassium ions from classical agar electrodes would
interfere.

7. References

1. The National Formulary, 14th Ed., Mack Publishing


G o . , Easton, PA (1975).

2. The Merck Index, 8th Ed. , Merck and Go. , Inc.,


Rahway, NJ (1968).

3. Washburn, W., Abbott Laboratories, Personal


Comunication.

4. Fazzari, F. R., Sharkey, M. F. , Yaciw, C. A. and


Brannon, W. L., J. Ass. Offic. Anal. Chem., 2,
1154, (1968).

5. Egan, R. S., Abbott Laboratories, Personal


Communication.

325
JAMES A. RAIHLE

6. Furman, W. B., J . Ass. Offic. Anal. Chem., 51,


1111, (1968).

7. Mueller, S . , Abbott Laboratories, Personal


Communication.

8. Quick, J. E. , Abbott Laboratories, Personal


Communication.

9. Sprague, J. M., Ann. N. Y. Acad. Sci. , 71, 328,


(1958).

10. Close, W. J., Swett, L. R., Brady, L. E., Short,


J. H. , and Versten, M. , J. Am. Chem. SOC. , 82,
1132 (1960).

11. Luebke, D. 'R., Abbott Laboratories, Personal


Communication.

12. Chang, S . L., Acta. Phann Sinica, 7, (1959).


(Anal. Abstr., 1, 1909, (1960)).

13. Fazzari, R. , J. Ass. Offic. Anal. Chem., 56, 677,


(1973).

14. Pilsbury, V. B., and Jackson, J. V., J. Pharm.


Pharmacol., J-8,713, (1966).

326
M ETRONIDAZOLE

Lorraine L. Wearley and Gaylord D. Anthony


LORRAINE L. WEARLEY AND GAYLORD D. ANTHONY

Cantents

1. Description
1.1 Name, Formula, Molecular Weight
1.2 Appearance, Color, Odor
2. Physical Properties
2.1 Infrared Spectrum
2.2 Nuclear Magnetic Resonance Spectrum
2.3 Ultraviolet Spectrum
2.4 Mass Spectrum
2.5 Optical Rotation
2.6 Melting Range
2.7 Differential Scanning Calorimetry
2.8 Thermogravimetric Analysis
2.9 Solubility
3. Metabolism
4. Pharmacokinetics
5. Methods of Analysis
5.1 Titrimetric Analysis
5.2 Spectrophotometric Analysis
5.3 Colorimetric Analysis
5.4 Chromatographic Analysis
5.41 Thin Layer Chromatography
5.42 Gas Chromatography
5.5 Polarographic Analysis
6. Synthesis
7. References
8. Acknowledgments

328
METRON IDAZOLE

1. Description
1.1 Name, Formula, Molecular Weight
Metronidazole is 1-(2-hydroxyethyl) -2-methyl- 5-
n i troimidazole

CH2 CHZOH

N02a l4olecular Weight: 171.16


'SHSN303

1.2 ADDearance. Color. Odor


Metronidazole is a white t o pale yellow, odorless
c r y s t a l l i n e powder.
2. Physical Properties
2.1 Infrared S p e c t m
The infrared absorption spectrum of a metronidazole
reference standard compressed i n a KBr p e l l e t is
shown i n Figure 1. The following assignmenfs have
been made f o r absorption bands i n Figure 1.

Band (an-1) Assignment


32 30 OH s t r e t c h

3105 C=CH; C-H s t r e t c h


1538 6 1375 NO2; N-0 s t r e t c h
1078 C-OH; C-0 s t r e t c h

830 C-N02; C-N s t r e t c h

329
W

Figure 1
Infrared Spectrum of Metronidazole
METRON I DAZO LE

2.2 Nuclear Magnetic Resonance Spectrum


The M I spectrum of metronidazole in deuterated
acetic acid is shown in Figure 2. Below are the
assignments of the major signals. Positions of
absorption bands are reported as shifts downfield
from the signal of the protons in te ramethylsilane
which was used as internal standard. I
u \\
Assignment
155 singlet ,C - CH3
-
241 (triplet) CH2 --ez- OH
274 (triplet) $2 -cFr2-oH
481 (singlet)
a -
,C - H
2 . 3 Ultraviolet Spectrum
Metronidazole exhibits an absorption maxima at
about 274 run. using 0.1 N sulfuric acid in
methanol as solvent. The molar absorptivity in
this solvent is 6333. The ult2aviolet absorption
spectrum is shown in Figure 3.
2.4 Mass Spectrum
The low resolution mass spectrum of metronidazole
is shown h3Figure 4. Structure assignments are
as follows:
!YE Assignment % Relative Intensity
171 M**(molecular ion) 12.5
154 M-OH 4.0
125 M - NO2 22.1
125 M-NOZ-H 25.7
41 M - CH3CN 100

331
. . . . 5.0 . ..m
( T ). .6.0
, .. . . . . . . . . .8.0. . . . . . . .91) . . . . , . . . . ? . . .
. . . . . 7.0
l ' " - I ' ' ~ ' . '

00 PD tm m
tm
o m
**,

0
0
1u

I I . . . . I
I . . . 1 1 . . r....l...., . . . II . . .. . , . . . . l I . .
.. I
I
..I . . . . ,
I
. I
I . . . . ' . .. 1 .
....LA
- 1 .
..
ko 74 4.a
Figure 2
Nuclear Magnetic Resonance Spectrum of Metronidazole
I

Figure 3
u1 traviolet Spectrum of Metronidazole in 0.1 N Sulfuric Acid in Methanol
E

1
J
NBMINFIL- MRSSi-
Figure 4
Mass Spectnnn of Metronidazole
METRON IDAZOLE

2.5 Optical Rotation


Metronidszole exhibits no optical activity.
2.6 Melting Range
Theomelting range given in the USP XIX is 159' to
163 C.
2.7 Differential Scanning Calorimetry
The DSC thermogramoof metronidazole obtained at a
heating rate of 20 C/minute is shown in Figuse 5,
The endothermic change observed at about 163 C
corresponds to the melting of the compound.
2.8 Thermogravimetric Analysis
The TGAof metronidazole obtained a t a heatinq

r a t e o f 10°C/minute i s shown i n f i g u r e 6. 4

2.9 Solubility
Solubilities in various solveps at 2S0C are
given in the following table.
Solvent Solubility, mg/ml
Water 10.5
Methanol 32.5
Ethanol 15.4
Chlorofo m 3.8
Heptane co.01
3. Metabolism
Stambaugh et a1.6 found that the major urinary excretioA
products of metronidazole in man and 0 - 1 mice were
unchanged metronidazole, 1-(2-hyroxyethyl)-2-hydroxy-
methyl-5-nitroimidazole and their ether glucuronides .
335
-
OaN3
OX3
336

Figure 5
DSC Thennogram of Metronidazole
2-

.c-+m
E W E R A T W E OC
------
Figure 6
TGA of Metronidazole
LORRAINE L. WEARLEY AND GAYLORD D. ANTHONY

These products represented 70-90% of the total urinary


nitro fraction. Minor metabolites were reported to be
1-(2 -hydroxyethyl)- 2 -carboxylic acid-5-nitraimidazole
and 1-acetic acid-2-methyl-5-nitroimidazole(see Fig. 7).
Oxidation of the methyl group of metronidazole appears
to occur more facilely than the hydroxyethyl group.
Nitro reduction products have not been found in animal
or human urine.
The major vaginal products in females given oral doses
of drug were found by Manthei et a1 to be unchanged
drug, and 1-(2 -hydroxyethyl) - 2 -hydroxymethyl- 5-nitro-
imidazole. These products were also present in the
urine. In addition a fluorescent lipophilic product
thought to be a cyclized lactone was found.
4. Phannacokinetics
In a study on healthy females receiving single and
multiple doses of 200 mg metronidazole tablets, Welling
and Monro reported the biological half-life to be
6.2 hr. Serum concentration data fit a one compart-
ment open model. Steady state serum concentrations
of metronidazole on a regimen of 200 mg twice daily
averaged 7.07 ug/ml maximum and 2.47 ug/ml m i n h .
Ings et al,’ studied the distribution of p4C]jmetroni-
dazole in rats. They found that oral doses were
rapidly absorbed from the gastrointestinal tract;
and rapidly equilibrated between blood and most
tissues. Radioactivity was found to concentrate
in the liver, kidney, gastro-intestinal tract and
vaginal secretions. The half life of clearance was
longest for the gastro-intestinal tract. I.V. doses
showed similar distribution.
5. Methods of Analysis
5.1 Titrimetric Analysis
The titration with perchloric acid is the method of
choice to assay metronidazole. The sample is
dissolved in acetic anhydride, and warmed slightly
to effect solution. After cooling, one drop of
mlachite green T.S. is added, and the titration
with 0.1 N perchloric acid to a yellow-green end-
point is carried out. A blank determination is

338
METRON IDAZOLE

Figure 7: Pathways proposed by Stambaugh et al. for the


metabolism of metronidazole in man.
(1) metronidazole (2) the corresponding ether
glucuronide (R = glucuronide) (3) 1 - (2-hydroxyethy1)-
2-hydroxymethyl-5-nitroimidazole (4) its corresponding
glucuronide (5) 1-acetic acid-2-methyl- S-nitroimidazole
.
(6) 1-(2- hydroxyethyl) - 2-carboxylic acid-5-nitroimidazole

339
L O R R A I N E L. WEARLEY A N D G A Y L O R D D. ANTHONY

performed and any necessary correction is


One equivalent of the compound is t i t r a t e d .
?ae *

5.2 Spectrophotometric Analysis

Spectrophotometric analysis of metronidazole m y


be carried out using 0 . 1 N sulfuric acid in methanol
a s the solvent. The ultraviolet absorption maxima
is a t about 274 nm.
5.3 Colorimetric Analysis
5.31 Metronidazole can be analyzed colorimetrically
by reducing the n i t r o group t o t h e correspond-
ing m i n e , which is subsequently determined
by diazotization and coupling w i t h N- (l-naph-
thyl) - ethylenediamine dihydrochlor ide (Brat ton-
Marshall Reagent) .11
5.32 A variation of the above method involves
the alkaline hydrolysis of the n i t r o group
of metronidazole. The nitrous acid produced
diazotizes sulfanilamide i n acidic m e d i u m
t o form a diazonium s a l t . After coupling
with Bratton-Marshall reagent the concentra-
t i o n is d e t e n i n e d spectrophotmetrically
by canparison t o n i t r i t e standards which have
been arried through the colorimetric proced-
ure 15
5.4 Chromatographic Analysis
5.41 Thin Layer Chromatography - Several TLC
systems and corre onding Rf values are
smnarized below. Pi

340
METRONIDAZOLE

Solvent System Absorbent Detection E€


Acetone Silica Gel 1 0.65

Ch1orofonn:Methanol: Silica Gel 1 0.76


Water:Acetic Acid
74:20:4:2

Benzene:methanol: Silica Gel 1 0.36


ammonium hydroxide
79:20:1

Ch1orofonn:methanol: Silica Gel 1, 2 0.66


water:acetic acid
70:24 :4: 2
1. Spray Vith 1% aqueous titanium trichloride; heat
at 130 C for 3 minutes. Spray with 1% Dimethyl-
aminobenzaldehyde in 2 N HC1.
2. Saturate plate with t-butyl hypochlorite vapors.
Spray with aqueous 1% starch/l% potassium iodide
solution. (This spray has been found to be much
more sensitive than #1.)
5.42 Gas-Liquid Chromatography - Metronidazole
can be chranatographed as the trimethyl silyl
derivative. The silyl derivative is prepared
by dissolving metronidazole in a 1:l mixture
of dimethylfomide and bis (trimethylsilyl)-
trifluoroacetamide. The silylation reaction
is compete in 30 minutes at room tempera-
ture.1
Instrumental Conditions
Column: 6 ft. glass column packed with 3%
OV-1 on Gas Chrom Q
Column Temp.: 160' C
Carrier: Nitrogen at 70 ml/min
Detector: Hydrogen Flame Ionization
Retention Time: 4 . 1 minutes

341
LORRAINE L. WEARLEY AND GAYLORD D. ANTHONY

-0.6 V

Figure 8
Polarograph of Metronidazole,
2% Solution in pH 3.8f 0.2 Buffer

342
METRONIDAZOLE

5.5 Polarographic Analysis


A suitable polarographic analysis of metronidazole
may be carried out at a concentration of approxi-
mately 5 ug/ml in a 2% solution of pH 3 . 8 + 0.2
buffer. The scan shown in figure 8 was ohained
on such a solution, using differential pulse
mode, 3 electrode system. For less concentrated
solutions addition of a maxima suppressant may
be necessary. Peak maximuni value is approximately
-0.23 volts vs saturated calomel ele~trode.~
6.0 Synthesis
2-methyl-imidazole (I) is nitrated by reacting
with nitric acid in the presence of sulfuric acid
catalyst. The resulting 5-nitro product (11) can
then be reacted with either chloroethanol or
ethylene oxide to produce 1-(2-hydroxyethy1)-2-
methyl-5-nitroimidazole (111). See figure 9.

CICH2CH20H
or H 2 C ~ F H 2
0
’ NwH3 CH2CH20H

m
Figure 9
Synthesis o f Metronidazole

343
LORRAINE L. WEARLEY AND GAYLORD D. ANTHONY

7. References
1. Damascus, J., Searle Laboratories, personal
conmumication.
2. Aranda, E., Searle Laboratories, personal
cammicat ion.
3. Hribar, J., Searle Laboratories, personal
conmumication.
4. Marshall, S., Searle Laboratories, personal
cammication.
5. Aranda, E., Searle Laboratories, personal
camnrnicat ion.
6. Stambaugh, J., Feo, L., Manthei, R., J, Phaxm.
and Fxp. Therapeutics, Vol. 161, No. 2,
1968.
7. Manthei, R., Feo, L., Stambaugh, J., Wiadmosci
Parazytologiczne T. XV, Nr. 3-4, 1969.
8. Welling, P., Monro, A., Arzneim - Forsch
(drug Research) 22, Nr. 1 2 (1972).
9. Ings, R., McFadzian J., Onnerod, W., Xenobio-
tica, 1975, Vol. 5, No. 4, 223-235.
10. USP XIX, p. 327.
11. Bratton, A., Marshall, E., Babbitt, D., Herdrick-
son, A., J. Biol. Chm., 128, 537, (1939).
12. Lau, E., Yao, C., Lewis, M., Senkwski, B.,
J. Phaxm. Sci., 58, No. 1, 55, (1969).
13. Quirk, P., Chow,T., Searle Laboratories,
personal cammication.
14. Wood, N., Chow, R., Searle Laboratories,
personal commmication.
8. Acknowledgments
The authors wish t o express special thanks t o
Dr. J. W i t t for the information on synthesis; t o
Dr. J. Opperman for his assistance i n preparing
the sections on metabolism and pharmacokinetics;
to Mr. S. Marshall and Mrs. M. Gerry for generating
and interpreting the data on TGA, DSC and Polaro-
graphy; to Mr, J. Damascus and Dr. J. Hribar for
the information on IR, W, and Mass Spectra; and
t o Ms. J. Janik for her secretarial assistance i n
preparing t h i s manuscript.

344
NITROFURANTOIN

Donald E. Cadwallader and Hung Won Jun


DONALD E. CADWALLADER AND HUNG WON JUN

T a b l e of Contents

1. Description
1. 1 N a m e , F o r m u l a , and Molecular Weight
1. 2 Appearance, C o l o r , Odor and T a s t e

2. P h y s i c a l P r o p e r t i e s
2.1 Ultraviolet S p e c t r a
2.2 Infrared Spectrum
2 . 3 N u c l e a r Magnetic Resonance S p e c t r u m
2.4 Dissociation Constant
2.5 Melting Range
2.6 Crystal Properties
2.61 C r y s t a l Shape
2 . 6 2 C r y s t a l Size
2.63 Hydrates
2.7 Salts
2.8 Solubility
2.81 Solubility in Aqueous Media
2.82 Solubility in Organic Solvents

3 . Synthesis

4. Stability
4.1 Stability to Light and M e t a l
4.2 Shelf-Life and S t o r a g e Conditions

5 . Metabolism

6 . Methods of Analysis
6.1 Identification
6.2 Color Reaction T e s t
6. 3 E l e m e n t a l Analysis
6.4 Chromatographic Systems
6.41 Thin L a y e r Chromatography
6.42 P a p e r Chromatography
6 . 4 3 Column Chromatography

346
NITROFURANTOIN

T a b l e of Contents, continued.

6.5 Quantitative Analysis


6.51 A s s a y of Dosage F o r m s
6 . 5 2 Quantitative D e t e r m i n a t i o n in
Biological S a m p l e s

7. B i o p h a r m a c e u t i c s and P h a r m a c o k i n e t i c s
7.1 Absorption
7.2 Distribution
7. 3 Elimination
7.4 Bioavailability
7.5 Pharmacokinetics

8. R e f e r e n c e s

347
DONALD E. CADWALLADER AND HUNG WON JUN

I. Description

1. 1 N a m e , F o r m u l a , and M o l e c u l a r Weight

Nitrofurantoin is N - ( 5 -Nitro-2-furfurylidene) -1-

hydantoin. F u r a d a n t i n 8
aminohydantoin; I - ( 5- N i ro-2-furfuryliden
and Macrodantin a r e the
m o s t commonly u s e d t r a d e m a r k s ; 11 additional a r e
smino)
listed in the M e r c k Index (1).

I
CH2- ,C
/
‘ 0

‘gHgN4O5 Mol. w t . : 238.16

1.2 A p p e a r a n c e , C o l o r , Odor and T a s t e

Nitrofurantoin i s d e s c r i b e d as lemon-yellow,
o d o r l e s s c r y s t a l s o r fine powder, having a b i t t e r
t a s t e (1,2).

2. P h y s i c a l P r o p e r t i e s

2.1 U l t r a v i o l e t S p e c t r a

Values of E (170, l c m ) in w a t e r a t 367.5 and 265


n m a r e found to be 760 and 540, r e s p e c t i v e l y (3).
T h e m o l a r extinction coefficients, e
at 367 and 265
n m a r e 13,100 and 17, 300, r e s p e c t i v e l y ( 4 ) .

340
NITROFURANTOIN

When t h e U V s p e c t r u m of nitrofurantoin i n 270


d i m e t h y l f o r m a m i d e ( D M F ) w a s s c a n n e d f r o m 260 to
400 n m , t w o maxima o c c u r r e d a t 265 and 367 n m .
T h e s p e c t r u m shown in F i g u r e 1 w a s obtained f r o m a
solution of 10.00 m g of n i t r o f u r a n t o i n / l i t e r of 270
D M F in w a t e r ( 3 ) .

F i g u r e 2 shows the effect of pH on m a x i m a l w a v e -


length and E (170,I c m ) v a l u e s of n i t r o f u r a n t o i n ( 3 ) .
K H 2 P 0 4 - NaOH and b o r i c acid - NaOH buffer s y s t e m s
w e r e employed. T h e s p e c t r a l shift w a s n o t due to
hydantoin r i n g - opening in the alkaline solution s i n c e
the shift w a s r e v e r s i b l e upon reacidification ( 3 ) .

2.2 Infrared Spectrum

T h e i n f r a r e d s p e c t r u m of nitrofurantoin (Norwich
P h a r m a c a l Reference Standard Purity) a s a m i n e r a l
oil m u l l is shown in F i g u r e 3. I n t e r p r e t a t i o n of the
s p e c t r u m w a s m a d e by M i c h e l s ( 3 ) using C r o s s ,
Stevens and Watts ( 5 ) a s a r e f e r e n c e .

Table I

IR Band A s s i g n m e n t s f o r Nitrofurantoin

Wavelength ( m i c r o n s ) Assignment

3.05 NH
5.6-5.75 hydantoin C = 0
6.6-7.45 d, - n i t r o f u r a n
10.4 2,5-disubstituted furan
8.05 asymmetrical C-0-C
9. a symmetrical C-0-C

349
DONALD E. CADWALLADER AND HUNG WON JUN

1.c

.e

W 6
0
z
Q
m
K
0
m
m
a 4

NANOMETERS

F i g u r e 1. U l t r a v i o l e t S p e c t r u m of Nitrofurantoin
(Coleman 124)

350
NITROFURANTOIN

390

380
r
-

a 370 -
z
I I I
360
5 6 7 8 9 10

F i g u r e 2. U l t r a v i o l e t A b s o r p t i o n of N i t r o f u r a n t o i n
as a F u n c t i o n of pH

351
4000 3000 2000 1500 1000 900 800 700
100

h 80
FR
v

Lu
60
2
k
v) 40
Z
2
fi
20

rcc
m
a
.rl
cn

Figure 3 . Infrared Spectrum of Nitrofurantoin (Perkin Elmer 467)


M

k
k

5
(d

k
E5
0

0
M

k
k

a
V
k
5

a,
a,
NITROFURANTOIN

2. 3 N u c l e a r Magnetic R e s o n a n c e S p e c t r u m

T h e n u c l e a r magnetic r e s o n a n c e s p e c t r u m f o r
nit r ofurantoin ( N o rwic h P h a r m a c a l R e f e r e n c e
S t a n d a r d P u r i t y ) in DMSO - d6 containing a t e t r a -
m e t h y l s i l a n e a s the i n t e r n a l r e f e r e n c e i s shown in
F i g u r e 4 ( 3 ) . T h e s p e c t r a l a s s i g n m e n t s a r e presented
in T a b l e I1 ( 3 ) .

T a b l e I1

NMR S p e c t r a l A s s i g n m e n t s f r Nitr furantoin

Band ( p p m , d ) No. of P r o t o n s Assignment

Singlet 4.40 2 hydantoin CH2


Doublet 7. 15 ( J = 4 ) 1 f u r a n 3H
Doublet 7 . 6 2 ( J = 4 ) 1 f u r a n 4H
Singlet 7 . 8 3 1 -CH=N-
B r o a d Singlet 1 1 . 4 1 hydantoin NH
(exchangeable)

2.5 - solvent

2 . 4 Dissociation Constant

M i c h e l s ( 3 ) d e t e r m i n e d the pKa of the f r e e a c i d to


b e 7 . 0 using t h e method d e s c r i b e d by Stockton and
Johnson ( 6 ) . A pKa of 7 . 2 i s a l s o r e p o r t e d f o r n i t r o -
furantoin ( 1).

2 . 5 Melting Range

Nitrofurantoin m e l t s a t 270-272' C. (1,7).

353
2.0 3.0 4.0 ' !
5.0 . PPM(TJ
' ' ' . 6.0
! ' 7.0
! ',' ' 8.0
! '.' I 9.0
I ' i Io ' , '
I "
I , ' ' : ' , ' ;' ! '
'.' ' , ' ', ' ' , ' ' ' ' . ' ' ' ' , ' ' ' ' ' ' ' ' ' '; ' , ' ' ' ' ' . ' ',' '

a0 ,m m lm o cn
++

I
1 . . . . I . . . . I . . . . I . . . . 1 . . . . 1 . . . . 1 . . . . 1 , . . . I
I . . _ . , . . . . I . . . . , . . . _ I . _ . . , . . . . I . _ . . , _ . . _ I . . . . I . . . . I . _ . . I . . . . I _ . . . I . . . . I . . . . , . . . . l . . .
a.0 7.0 6.0 5.0 PPM( 6 ) 4.0 3.0 2.0 1.o 0

Figure 4. NMR Spectrum of Nitrofurantoin (Varian A60A)


NITROFURANTOIN

2.6 Crystal Properties

2.61 C r y s t a l Shape

C r y s t a l l i z a t i o n f r o m diluted a c e t i c a c i d
yields needle-like c r y s t a l s (1).

2.62 C r y s t a l S i z e

T h e c r y s t a l s i z e of n i t r o f u r a n t o i n h a s been
found to affect the d e g r e e of emesis a n d the r a t e s
of g a s t r o i n t e s t i n a l a b s o r p t i o n and u r i n a r y excre-
tion following o r a l a d m i n i s t r a t i o n (8). T h e d i f -
f e r e n t c r y s t a l s i z e s of n i t r o f u r a n t o i n w e r e p r e -
p a r e d by recrystallization f r o m nitromethane.

2.63 Hydrates

It h a s b e e n found that n i t r o f u r a n t o i n c a n
e x i s t i n anhydrous and monohydrate f o r m ( 9 ) .
Anhydrous n i t r o f u r a n t o i n and p r e v i o u s l y dried
n i t r ofurantoin monohydrate b e c o m e h y d r a t e d only
a t v e r y high humidity (>92% R . H. ). N i t r o f u r a n -
toin monohydrate d o e s not l o s e o r gain m o i s t u r e
upon s t o r a g e a t v a r i o u s r e l a t i v e humidities i n the
r a n g e of 31-9270 R.H. Monohydrate f o r m i s v e r y
s t a b l e i n terms of retaining w a t e r a t 5OoC.

2. 7 S a l t s

T h e s o d i u m s a l t of nitrofurantoin i s available and


is u s e d t o p r e p a r e p a r e n t e r a l f o r m u l a t i o n s ( 1 0 ) .
Aqueous solutions of the s a l t a r e v e r y u n s t a b l e .

355
DONALD E. CADWALLADER AND HUNG WON JUN

2 . 8 Solubility

2 . 8 1 Solubility in Aqueous Media

Solubilities of nitrofurantoin in aqueous


m e d i a have been r e p o r t e d f o r v a r i o u s t e m p e r a -
t u r e and pH conditions. T h e s e solubility values
a r e p r e s e n t e d in T a b l e 111.

Table I11

Solubilities of Nitrofurantoin i n Aqueous Media

T e m p e r a t u r e , OC @ Solubility ( m g / l ) References.

24 5 76. 3 11
24 7 131.1 11
24 d. H 2 0 79.5 11
25 7 190 1,12
30 d. H 2 0 113.4 11
37 1.12 154 13
37 4.8 125 14
37 5 167.8 11
37 d. H 2 0 174. 1 11
37 7 312. 1 11
7.2 374 13
45 d. H 2 0 251.2 11

2 . 8 2 Solubility in Organic Solvents

The solubilities of nitrofurantoin in v a r i o u s


organic solvents a r e p r e s e n t e d in T a b l e IV.

356
NlTR 0 FUR A N T 0 I N

T a b l e IV

Solubilities of Nitrofurantoin in Organic Solvents:%

Solvent Solubility ( m g / l )

Acetone 5,100

Dime thy lfo r t n a m i d e 80 ,000

Ethanol 47.570 189

70 % 712

9 570 5 10

Glycerin 600

Peanut Oil 20.7

Polyethylene glycol 300 15,100

Propylene glycol 2070 1,560

;::The solubility values w e r e r e p o r t e d i n R e f e r e n c e s


( 1 ) and (15). T h e t e m p e r a t u r e w a s not indicated.

357
DONALD E. CADWALLADER AND HUNG WON JUN

3. Synthesis

Nitrofurantoin c a n b e p r e p a r e d by the r e a c t i o n of 1-
aminohydantoin a s the sulfate (16) and as the hydrochlo-
r i d e (17) with 5 - n i t r o f u r f u r a l diacetate in isopropyl-
alcohol-water media. Details of the production of n i t r o -
furantoin w e r e d e s c r i b e d by S a n d e r s -- e t at. ( 1 8 ) . T h e
r e a c t i o n s c h e m e is shown in F i g u r e 5. Nitrofurantoin
w a s a l s o p r e p a r e d by t h e condensation of 1-aminohydantoin
with 5-nitro-2-furaldehyde (19). Another method f o r the
s y n t h e s i s of nitrofurantoin w a s d e s c r i b e d by J a c k (20).

4. Stability

4. 1 Stability to Light and M e t a l

Nitrofurantoin c r y s t a l s and its solutions a r e d i s -


colored by alkali and by e x p o s u r e to light, and a r e
decomposed upon contact with m e t a l s o t h e r than
s t a i n l e s s s t e e l and aluminum ( 2 ) . Since n i t r o f u r a n -
toin solutions a r e photosensitive, all analytical o p e r a -
tions m u s t b e conducted under subdued light. Nitro-
furantoin solutions a r e a l s o e x t r e m e l y s e n s i t i v e to
alkali; t h e r e f o r e , a l l g l a s s w a r e m u s t b e analytically
clean and d r y f o r a s s a y p r o c e d u r e s .

4 . 2 Shelf-Life and S t o r a g e Conditions

S t o r a g e conditions of nitrofurantoin and o r a l


suspensions a r e r e c o m m e n d e d t o be in tight, light-
r e s i s t a n t c o n t a i n e r s ( 2 ) . Recommended shelf-life of
tablets and suspensions i s f i v e y e a r s when s t o r e d a t
r o o m t e m p e r a t u r e and in r e g u l a r g l a s s c o n t a i n e r s (21).

358
z-
I I
% .c_
z
I = 0
t
C
0
i
II
..
3
Y-
I-u .-
t
I 2
\
a,
c
Z
0 (v
t
a,
o
t:. C U I
i I
0 T
L
0 2 0
.-
L
z
t \\/
0
I
zI-
I

\4
0
I m
.r(
m
Q
I z = 8
In cu
I ul
m
.
Q
$
M
.r(
Fr
I
0
359
DONALD E. CADWALLADER AND HUNG WON JUN

When the products a r e packaged in l i g h t - r e s i s t a n t


c o n t a i n e r s and s t o r e d a t r o o m t e m p e r a t u r e , the d r u g
showed negligible l o s s of potency o v e r a f i v e - y e a r
period of t i m e . T h e nitrofurantoin products should
not be s t o r e d w h e r e t e m p e r a t u r e s a r e expected to
exceed 86'F.

5. Metabolism

Reckendorf -- e t a l . ( 2 2 ) r e p o r t e d that l a r g e amounts of


nitrofurantoin (30 -50%) of a n o r a l l y and intravenously a d -
m i n i s t e r e d d o s e w e r e r e c o v e r e d intact in the u r i n e of r a t ,
dog and m a n . Beutner _ et -at. ( 2 3 ) a l s o found a s i m i l a r r e -
covery in urine after o r a l administration. T h e s e studies
suggest that nitrofurantoin undergoes metabolic t r a n s f o r -
mation in t h e body to a significant extent. T h e possible
metabolic pathways of nitrofurantoin a r e not completely
elucidated i n t h e l i t e r a t u r e . However, nitrofurantoin
would follow somewhat s i m i l a r pathways in m e t a b o l i s m to
that f o r n i t r o f u r a z o n e which undergoes reduction in the
n i t r o g r o u p and hydrolysis in the aaomethane linkage.

6. Methods of Analysis

6. 1 Identification

Nitrofurantoin may b e identified by i t s melting


point (270-272OC) and by m e a n s of i t s c h a r a c t e r i s t i c
infrared spectra (see Figure 3 ) .

6. 2 Color Reaction T e s t

A n u m b e r of c o l o r r e a c t i o n t e s t s f o r the identi-
fication of nitrofurantoin has b e e n p r e s e n t e d in a
publication ( 2 4 ) .

360
NITROFURANTOIN

6. 3 Elemental A n a l y s i s

T h e r e s u l t s of a n e l e m e n t a l a n a l y s i s of nitrofuran-
toin (Norwich P h a r m a c a l C o . , Lot. No. E3769) a r e
presented in Table V (25).

Table V

E l e m e n t a l Analysis of Nitrofurantoin

Element yo T h e o r y yo Found

C 40. 34 40.22

H 2.54 2.57

N 23.53 23.53

0 33.59

6.4 Chromatographic S y s t e m s

6.41 Thin Layer Chromatography

T h e following T L C s y s t e m s have b e e n found


suitable f o r detection of p o s s i b l e postulated im-
purities (26).

Solvent S y s t e m I

Acetone: Glacial Acetic Acid: Methanol


90 5 5 (v/v)

361
DONALD E. CADWALLADER AND HUNG WON JUN

Rf = 0 . 8 1 on B r i n k m a n Silica Gel plate with-


out f l u o r e s c e n t indicator as visualized by s h o r t -
wave u l t r a v i o l e t light. It w a s found that the
l i n e a r dynamic range w a s 0.25-1.10 p g / s p o t .

Solvent S y s t e m I1

Acetone: Benzene: Glacial a c e t i c a c i d


80 20 1 (v/v)

Rf = 0 . 9 5 on Brinkman Silica Gel plate


without f l u o r e s c e n t indicator as visualized by
s h o r t - w a v e ultraviolet light and H2SO4 c h a r r i n g .

Other T L C p r o c e d u r e s f o r the identification


and s e p a r a t i o n of nitrofurantoin have been d e s -
cribed (27).

6.42 P a p e r Chromatography

T h e u s e of paper chromatography i n the


a n a l y s i s of nitrofurantoin and o t h e r n i t r o f u r a n
compounds h a s been r e p o r t e d by B r e i n l i c h ( 2 8 ) ,
who examined s e v e r a l solvent s y s t e m s and
methods f o r identification of the s p o t s .

6.43 Column Chromatography

Bender -- e t al. (29) u s e d column c h r o m a t o -


g r a p h i c p r o c e d u r e s to s e p a r a t e nitrofurantoin
f r o m o t h e r components of u r i n e s a m p l e s and s u b -
sequently d e t e r m i n e d the d r u g concentrations by
s p e c t r o p h o t o m e t r i c a s say.

362
NITROFURANTOIN

6 . 5 Quantitative Analysis

6.51 Assay of Dosage F o r m s

The methods of analysis that a r e used € o r


the determination of nitrofurantoin depend on i t s
ultraviolet absorption c h a r a c t e r i s t i c s . The
U. S. P. XIX ( 3 0 ) d e s c r i b e s a spectrophotometric
determination of nitrofurantoin in an acidic 27'0
dimethylformamide -wate r solution. This g e n e r a l
method i s applicable to nitrofurantoin in tablet,
capsule and suspension dosage f o r m s w h e r e the
excipients a r e U V non-absorbing. Under condi-
tions w h e r e separations a r e not made, any drug
degradation products a r e reasonably expected to
be reflected in the value of the U V ratio; con-
v e r s e l y , constancy of the U V r a t i o a s compared
to the initial formulation value can be taken a s
evidence f o r stability (31). A polarographic ( 32)
and gravimetric ( 3 3 ) determination of nitrofuran-
toin in tablets have been described.

6 . 5 2 Quantitative Determination in Biological


Samples

Paul -- et al. (34) utilized the ultraviolet ab-


sorption of nitrofurantoin to determine drug con-
centrations in r a t urine followed by extraction
with e t h e r , but the absorption maximum was pH
dependent a s indicated by Stoll and c o - w o r k e r s
(35).

363
DONALD E. CADWALLADER AND HUNG WON JUN

In 1965 Conklin and Hollifield (36) i n t r o -


duced the nitromethane-Hyamine p r o c e d u r e f o r
the d e t e r m i n a t i o n of nitrofurantoin i n u r i n e , and
this method h a s been established a s the p r e -
f e r r e d a s s a y f o r nitrofurantoin in biological
s a m p l e s . T h e original p r o c e d u r e h a s been
modified to a s s a y the d r u g in whole blood o r
p l a s m a (37). T h i s p r o c e d u r e h a s a sensitivity of
2 r g / m l and i s specific f o r nitrofurantoin.
However, this method w a s inadequate f o r quantita-
ting nitrofurantoin blood levels in s u b j e c t s who r e -
ceived n o r m a l therapeutic d o s e s . Mattock,
McGilveray and C h a r e t t e (38) improved the n i t r o -
methane-Hyamine method f o r the determination
of nitrofurantoin in blood s a m p l e s s o that s m a l l e r
volumes a r e r e q u i r e d ( 0 . 8 ml) and the sensitivity
i s g r e a t e r ( 0 . 2 u g / m l ) . A modified nitromethane-
Hyamine method w a s d e s c r i b e d by Hollifield and
Conklin (39) f o r the a s s a y of nitrofurantoin in
u r i n e i n the p r e s e n c e of phenazopyridine and i t s
metabolites .
Buzard --e t at. ( 4 0 ) developed a c o l o r i m e t r i c
method f o r the a n a l y s i s of nitrofurantoin in plasma
o r s e r u m of the r a t a f t e r v a r i o u s o r a l d o s e s .

J o n e s e t at. (41) d e s c r i b e d a polarographic


method f o r the d e t e r m i n a t i o n of nitrofurantoin i n
u r i n e . T h e sensitivity l i m i t of a s s a y i s l p g / m l .
S e v e r a l a u t h o r s (42, 43) have r e p o r t e d polaro-
g r a p h i c p r o c e d u r e s f o r the a s s a y of the drug.

364
NITROFURANTOIN

A microbiological p r o c e d u r e f o r the a s s a y
of nitrofurantoin in u r i n e w a s a l s o d e s c r i b e d by
--
J o n e s e t a l . ( 4 1 ) . Gang and Shaikh (44) u s e d a n
indicator o r g a n i s m i n a t u r b i d i m e t r i c method f o r
the a s s a y of nitrofurantoin and o t h e r n i t r o f u r a n
d e r i v a t i v e s in s e r u m and u r i n e s a m p l e s .

A column c h r o m a t o g r a p h i c method f o r the


d e t e r m i n a t i o n of nitrofurantoin i n u r i n e w a s r e -
ported by Bender e t a l . ( 2 9 ) .

Stone (45) and P u g l i s i (46) have m e a s u r e d a


t r a c e of nitrofurantoin i n m i l k by the c o l o r i m e t r i c
and s p e c t r o pho t o m e t r i c methods , r e s p e c tive 1y .
Both p r o c e d u r e s a r e b a s e d on t h e conversion of
nitrofurantoin to 5 -nitrofurfuraldehyde phenylhy-
d r a z o n e and a r e followed by the e x t r a c t i o n and
concentration on a c h r o m a t o g r a p h i c column.
F i n a l d e t e r m i n a t i o n s depend on the development
of a blue c o l o r by the addition of Hyamine base.

7. Biopharmaceutics and P h a r m a c o k i n e t i c s

7 . 1 Absorption

Nitrofurantoin i s efficiently and rapidly a b s o r b e d


a f t e r o r a l a d m i n i s t r a t i o n ( 4 7 ) . Limited d r u g a b s o r p -
tion o c c u r s when nitrofurantoin is a d m i n i s t e r e d
r e c t a l l y (48).

T h e f i r s t evidence that a b s o r p t i o n and e x c r e t i o n


w e r e affected by d i f f e r e n c e s in p a r t i c l e s i z e of the
d r u g w a s published in 1967 ( 8 ) . T h i s paper r e p o r t e d
on the r e l a t i o n s h i p of p a r t i c l e s i z e of nitrofurantoin
to emesis in dogs and to absorption and u r i n a r y

365
DONALD E. CADWALLADER AND HUNG WON JUN

excretion in man and r a t s . It was found that l a r g e r


c r y s t a l s of the drug caused l e s s e m e s i s in dogs and
slower absorption in man and r a t s . Thus, i t was
concluded that the u s e of l a r g e c r y s t a l s of nitrofuran-
toin ( M a c r o d a n t i n e could minimize a d v e r s e effects
of this drug such a s nausea and vomiting by slowing the
r a t e of absorption in the gastrointestinal tract.

Nitrofurantoin occasionally causes nausea,


vomiting, drowsiness, headache and skin r a s h e s (49).
Incidence of these reactions may be reduced to some
degree by administration of the drug with food (50,51).

Bates and co-workers (52) studied the effect of


food on the absorption of nitrofurantoin f r o m c o m m e r -
cial dosage f o r m s . They found considerably i n -
c r e a s e d absorption in nonfasting a s compared to
fasting subjects.

7 . 2 Distribution

After absorption into the blood circulation, n i t r o -


furantoin is rapidly distributed into most body fluids
(53 ) . During n o r m a l o r a l therapeutic regimen, blood
o r plasma levels of the drug a r e usually v e r y low, in
the neighborhood of 1 p g / m l . However, nitrofuran-
toin levels about 3-5 t i m e s g r e a t e r than this a r e
usually found in these fluids when drug i s adminis-
t e r ed intravenous Iy o r intramuscularly. Detailed
studies of the absorption and distribution of nitro-
furantoin have been c a r r i e d out by Buzard e t al. (54)
in small animals.

366
NITROFURANTOIN

7. 3 Elimination

The biological half-life of nitrofurantoin in man


a p p e a r s to b e 30 min. o r l e s s (55, 56, 57, 22). In
clinical studies in human subjects, u r i n a r y drug con-
centration of 200 to 400 m g / l i t e r have been reported
(50, 58). Renal excretion involves glomerular f i l t r a -
tion and active tubular secretion. S c h i r m e i s t e r --
et a t .
(59)found that the clearance of nitrofurantoin in
human subjects was lower in acid urine than in alka-
line urine. During renal impairment, nitrofurantoin
u r i n a r y excretion was significantly reduced (60).

Conklin and Wagner (61)and Conklin -- e t al. (62)


reported that a s much a s 20% of the intravenous dose
of nitrofurantoin sodium was excreted in hepatic bile
in the dog.

7.4 Bioavailability

Bioavailability of nitrofurantoin has received a


considerable attention in recent y e a r s . Cadwallader
(63)presented a monograph on the bioavailability of
nitrofurantoin in the special APhA Bioavailability
pilot project. The general c h a r a c t e r i s t i c s and
experimental c r i t e r i a f o r bioavailability testing of
nitrofurantoin w e r e discussed. More recently, Meyer
and co-workers (64)evaluated the bioavailabilities
of 14 commercially available nitrofurantoin p r e p a r a -
tions by in vivo and -- in vitro procedures. They found
that some products that m e t the official Compendia1
requirement, w e r e l e s s bioavailable than other p r o -
ducts tested. E a r l i e r studies by various authors

367
DONALD E. CADWALLADER AND HUNG WON JUN

(65, 66, 67) a l s o r e p o r t e d bioavailability p r o b l e m s


a s s o c i a t e d with the u s e of c o m m e r c i a l n i t r o f u r a n t o i n
tablets. An updated monograph on the bioavailability
of nitrofurantoin w a s r e c e n t l y p r e s e n t e d by
Cadwallader (68).

7. 5 P h a r m a c o k i n e t i c s

In m a n , p l a s m a levels of nitrofurantoin a p p e a r t o
decline exponentially with a half-life v a l u e of about 20
to 30 m i n u t e s (56, 57, 22). U r i n a r y e x c r e t i o n and
b i o t r a n s f o r m a t i o n a p p e a r to b e mainly a n d equally
r e s p o n s i b l e f o r the elimination of nitrofurantoin. The
o n e - c o m p a r t m e n t m o d e l a p p e a r s to be adequate f o r
d e s c r i b i n g the k i n e t i c s involved in nitrofurantoin
absorption and elimination.

8. R e f e r e n c e s

1. T h e M e r c k Index, 8 t h e d . , M e r c k and C o . , I n c . ,
Rahway, N . J . , 1968, p. 738.
2. T h e United S t a t e s P h a r m a c o p e i a , 19th e d . , M a c k
Publishing C o . , E a s t o n , Pennsylvania 1975, p. 341.
3. J. G. M i c h e l s , Norwich P h a r m a c a l C o . ,
P e r s o n a l Communication.
4. V. E g e r t s , J. S t r a d i n s a n d M. S h i m a n s k a ,
"Analysis of 5 - n i t r o f u r a n D e r i v a t i v e s , t r a n s -
l a t e d by J. S c h m a r a k , Ann A r b o r S c i e n c e
P u b l i s h e r s , Ann A r b o r , Michigan, 1970, p. 83.
5. A. H. J. C r o s s , S. G. E. S t e v e n s , a n d T . H. E.
W a t t s , J . Appl. C h e m . , London, 1,562 (1957).
6. J. Stockton and C. R. Johnson, J. A m , P h a r m .
A s s o c . , Sci. E d . , 33, 383 (1944).
7. T h e N i t r o f u r a n s , Vol. 1, "Introduction to the
N i t r o f u r a n s , Eaton L a b o r a t o r i e s , Norwich, N. Y.
1958, p. 16.
8 . H. E. P a u l , K . J . H a y e s , M . F. P a u l and A . R.
Borgmann, J. P h a r m . S c i . , 56, 882 (1967).

368
NITROFURANTOIN

9. B. G. P a t e l , Norwich P h a r m a c a l C o . , P e r s o n a l
Communication.
10. F u r a d a n t i n a Sodium P a r e n t e r a l , Eaton L a b o r a -
t o r i e s , Norwich, N. Y.
1 1. L. K. Chen, M.S. T h e s i s , U n i v e r s i t y of G e o r g i a ,
1975.
12. H. E. P a u l and M. F. P a u l i n E x p e r i m e n t a l
C h e m o t h e r a p y , vol. 11. R. J. S c h n i t z e r and F.
Hawking, e d s . , A c a d e m i c P r e s s , New Y o r k , N .
Y . , 1964, p. 334.
13. T . R. B a t e s , J . M. Young, C . M. Wu a n d H. A.
R o s e n b e r g , J. P h a r m . S c i . , 63, 643 (1974); T . R.
B a t e s , SUNY a t Buffalo, P e r s o n a l Communication.
14. M. F. P a u l , R . C. B e n d e r , and E. G. Nohle,
A m e r . J . P h y s i o l . , 197,580 (1959).
15. T h e N i t r o f u r a n s , vol. 1, "Introduction to the
N i t r o f u r a n s , I ' Eaton L a b o r a t o r i e s , Norwich,
N. Y . , 1958, pp. 14-15.
16. A . S w i r s k a , J . L a n g e , and Z . Buczkowski,
P r z e m y s l . C h e m . , 11 ( 3 4 ) , 306-308 (1965).
Through C. A . , 52, 14079 b (1958).
17. C. J. O'Keefe, Norwich P h a r m a c a l G o . ,
P e r s o n a 1 C ommunication ,
18. H. J. S a n d e r s , R. T . E d m u n d s , and W. B.
Stillman, Ind. Enp. C h e m . , 47, 358 (1955).
19. K. H a y e s , U. S. P a t e n t , 2,610, 181 (1952).
20. D. J a c k , J. P h a r m . P h a r m a c o l . , 11,Suppl.,
108 T (1959).
21. J. F. S t a r k , Norwich P h a r m a c a l C o . , P e r s o n a l
C ommuni ca ti on.
22. H. K. Reckendorf, R . C a s t r i n g i u s , a n d H .
Spingler, Med. Welt, 15, 816 (1963).
23. E. H. B e u t n e r , J. J . P e t r o n i o , H. E. Lind, H.
M. T r a f t o n , and M. C o r r e i a - B r a n c o , Antibiotics
Ann., 1954-1955, 988 (1955).

369
DONALD E. CADWALLADER AND HUNG WON JUN

24. V. E g e r t s , J. S t r a d i n s , and M. Shimanska,


I'Analysis of 5-Nitrofuran D e r i v a t i v e s , I ' t r a n s -
lated by J. S c h m a r a k , Ann A r b o r Science
P u b l i s h e r s , Ann A r b o r , Michigan, 1970, p. 18.
25. Atlantic M i c r o l a b , Inc. , Atlanta, Georgia.
26. M. J. Cardone, Norwich P h a r m a c a l Co.,
P e r s o n a l Communication.
27. V. E g e r t s , J. S t r a d i n s , and M. Shimanska,
"Analysis of 5-Nitrofuran D e r i v a t i v e s , t r a n s -
lated by J . S c h m a r a k , Ann A r b o r Science
P u b l i s h e r s , Ann A r b o r , Michigan, 1970, pp. 126-
127.
28. J. Breinlich, Dtsch. Apotheker. Z t g . , 104, 535(1964).
29. R . C . B e n d e r , E . G. Nohle, and M. F . P a u l ,
Clin. C h e m . , 2, 420 (1956).
30. T h e United S t a t e s P h a r m a c o p e i a , 19th e d . , Mack
Publishing Co. , Easton, Pennsylvania, 1975,
p. 342.
31. R. A. Boice, Norwich P h a r m a c a l C o . ,
P e r s o n a l Communication.
32. V. E g e r t s , J. S t r a d i n s , and M. Shimanska,
"Analysis of 5-Nitrofuran D e r i v a t i v e s , ' I t r a n s -
lated by J. S c h m a r a k , Ann A r b o r Science
P u b l i s h e r s , Ann A r b o r , Michigan, 1970, pp. 73-
74.
33. Ibid. , pp. 39-40.
34. H. E. P a u l , F. L. Austin, M. F. P a u l and V. R .
E l l s , J. Biol. C h e m . , 180,345 (1949).
35. R . G. Stoll, T. R . B a t e s and J. S w a r b r i c k ,
J. P h a r m . S c i . , 62, 65 (1973).
36. J . D. Conklin and R . D. Hollifield, Clin. C h e m . ,
-
11, 925 (1965).

370
NITROFURANTOIN

37. J . D. Conklin and R. D. Hollifield, w.,3,


690 (1966).
38. G. L. Mattock, I. J . McGilveray, and C.
Charette, ibid., 16,
820 (1970).
39. R. D. Hollifield and J. D. Conklin, ibid., 16,
335 (1970).
40. J. A . B u z a r d , D. M. V r a b l i c , and M. F. P a u l ,
Antibiotics and Chemotherapy, 6, 702 (1958).
41. B. M. J o n e s , R. J . M. Ratcliffe and S. G. E.
S t e v e n s , J. P h a r m . P h a r m a c o l . , 17,525 (1965).
42. T . S a s a k i , P h a r m . Bull. (Tokyo), 2, 104 (1954).
43. H. P. M o o r e and C . R. G u e r t a l , J. A s s o c . Offic.
Agr. C h e m i s t s . , 43, 308 (1960).
44. D. M. Gang and K. Z. Shaikh, J. P h a r m . S c i . ,
-
61, 462 (1972).
45. L. R. Stone, J. A s s o c . Offic. A g r . C h e m i s t s , -
44,
2 (1961).
46. E. P u g l i s i , J. A s s o c . Offic. A g r . C h e m i s t s , 44,
30 (1961).
47. J. D. Conklin, R. J. S o b e r s , and D. L. Wagner,
1. P h a r m . S c i . , 58, 1365 (1969).
48. J. D. Conklin, P h a r m a c o l o g y , S, 178 (1972).
49. G. C a r r o l l and R . V. Brennan, J. U r o l . , 71,650
( 1954).
50. P. F. MacLeod, G. S. R o g e r s , and B. R .
Anylowar, Intern. R e c o r d Med. and Gen. P r a c t .
C l i n . , 169, 5 6 1 (1956).
--
51. A. G. S m i t h , J . A m . Med. A S S O C . , 195,1061
(1966).
52. T . R. B a t e s , J. A. S e q u e i r a , and A. V. T e m b o ,
Clin. P h a r m a c o l . T h e r a p . , 16,63 (1974).

371
DONALD E. CADWALLADER AND HUNG WON JUN

5 3. M. F. P a u l , H. E . P a u l , R . C. B e n d e r , F.
Kopko, C. M. H a r r i n g t o n , V. R . E l l s , and J. A.
B u z a r d , Antibiotics and C h e m o t h e r a p y , 10,287
(1960).
54. J . A. B u z a r d , J . D . Conklin, E. O'Keefe and
M. F. P a u l , J . P h a r m a c o l . Exptl. T h e r a p . -
131,
38 (1961).
55. W. M. Bennett, I. S i n g e r , and C. H. Coggins,
J. Am. Med. A s s o c . , 214, 1468 (1970).
56. V . D. Kobvletzki.
, - -
Med. W e l t . .. 19. 2010 (19681.
. .
67,
- I . I I

57. C. M. Kunin, Ann. I n t e r n a l Med. , 1 5 1 (1967).


58. W. A . R i c h a r d , E. R i s s , E. H. K a s s a n d M .
Finland, A.M.A. Arch. Internal Med., 96,
4 3 7 (1955).
59. J . S c h i r m e i s t e r , F. Stefani, H. Willmann, and
W. H a l l o u e r , Antimicrobiol. Agents and C h e m o -
t h e r a p y , 1965, p. 223.
60. J. S a c h s , T . G e e r , P. Noell, and C. M. Kunin,
New Engl. J. M e d . , 9 ,1032 (1968).
61. J . D. Conklin and D. L. Wagner, J. P h a r m a c o l . ,
-
43, 140 (1971).
62. J . D. Conklin and R . J. S o b e r s and D . L.
W a g n e r , B r . J. P h a r m a c o l o g y , 48, 2 7 3 (1973).
63. D. E. C a d w a l l a d e r , "Nitrofurantoin, I f T h e APhA
Bioavailability P i l o t P r o j e c t , A m e r i c a n P h a r m a -
c e u t i c a l Association, Washington, D. C. J u l y 1973
64. M. C. M e y e r , G. W . A. Slywka, R. E. Dann and
P. L . Whyatt, J . P h a r m . S c i . , 63, 1693 (1974).
65. I. J. M c G i l v e r a y , G. L. Mattok, a n d R . D.
H o s s i e , J. P h a r m . P h a r m a c o l . , 23, 246 S ( 1 9 7 1 ) .

372
NITROFUR ANT0 IN

66. G. L. Mattok, R . D. H o s s i e , and I. J . McGilveray


Can. J. P h a r m . S c i . , 1,8 4 (1972).
67. I. J. McGilveray, G. L. Mattok, and R . D. Hossie
Rev. Can. Biol. 32, 99 (1973).
68. D. E. Cadwallader, J. Am. P h a r m . A S S O C . ,
NS15, 413 (1975).

T h e a u t h o r s w i s h to thank D r . John S t a r k and


o t h e r p e r s o n n e l at the Norwich P h a r m a c a l Company f o r
t h e i r valuable a s s i s t a n c e and support. T h e excellent
secretarial support of M r s . Kay W. Oliver i s
acknowledged.

373
PIPERAZINE ESTRONE SULFATE

Zui L. Chang
ZUI L. CHANG

Contents

Analytical Profile - Piperazine Estrone Sulfate

1. Description

1.1 Name, Formula, Molecular Weight


1.2 Appearance, color, Odor
1.3 Elemental Composition

2. Physical Properties

2.1 Infrared Spectrum


2.2 Nuclear Magnetic Resonance Spectrum
2.3 Ultraviolet Spectrum
2.4 Mass Spectrum
2.5 Raman Spectrum
2.6 Optical Rotation
2.7 Melting Range
2.8 Differential Thermal Analysis
2.9 Solubility
2.10 Crystal Properties
2.11 Dissociation Constant
2.12 Fluorescence
2.13 Hygroscopic Behavior
2.14 Sublimation

3. Synthesis

4. Stability - Degradation

5. Drug Metabolic Products and Pharmacokinetics

6. Method of Analysis

6.1 Identification
6.2 Chromatographic Analysis
6.21 Thin-Layer Chromatography
6.22 Gas-Liquid Chromatography
6.3 Spectrophotometric Analysis
6.4 Colorimetric Analysis
6.5 Nitrogen Analysis
6.6 Titration

376
PlPERAZlNE ESTRONE SULFATE

7. Acknowledgements

8. References

377
ZUI L. CHANG

1. Description

1.1 Name, Formula, Molecular Weight


Piperazine estrone sulfate is estra-1,3,5 (10)-
trien-l7-one, 3-(su1fooxy)-, compound with piperazine
(1:1). y 2

HO-SO'
II
0

Piperazine Estrone Sulfate

c18H2205 s' C4H10N2 Molecular Weight 436.56

1.2 Appearance, Color, Odor


Piperazine estrone sulfate is a white to yellow-
ish white, fine crystalline powder. It is odorless.

1.3 Elemental Composition


C-60.53; H-7.39; N-6.42; 0-18.32; S-7.34.

2. Physical Properties

2.1 Infrared Spectrum


The infrared spectrum of piperazine estrone sul-
fate is presented in Figure 1. The spectrum was measured
in the solid state as a potassium bromide dispersion. The
following bands (cm-1) have been assigned for Figure 1.3

a. 3400-2300 cm-l broad complex of bands due mainly to


N-H stretching vibrations of the amine
salt.

378
FIGURE 1 - INFRARED S P E C T R U M O F PIPERAZINE E S T R O N E SULFATE
WAVELENGTH (MICRONS)
2.5 3 4 5 6 7 8 9 10 12 15

0
-4
(D

4000 3500 3000 2 500 2000 1500 1000 700


FREQUENCY (CM-1)
ZUI L. CHANG

b. 1730 cm-l characteristic C=O stretching vibra-


tion of the 17-keto group.

c. 1600 and 1490 cm-I characteristic skeletal stretching


vibrations of the aromatic ring.
d. 1040 cm-l due to S-0 linkage

2.2 Nuclear Magnetic Resonance Spectrum (NMR)


The nuclear magnetic resonance spectrum of
piperazine estrone sulfate as shown in Figure 2 was
obtained on a Varian HA-100 NMR Spectrometer in deuterated
dimethylsulfoxide (d6) containing tetramethylsilane as the
internal standard. The spectral peak assignments4 are
presented in Table I.

2.3 Ultraviolet Spectrum (W)


When the W spectrum of 0.1% solution of pipera-
zine estrone sulfate in 0.04% sodium hydroxide solution
was scanned from 400 to 210 nm, two maxima and two minima
were observed (Figure 3).l The maxima are at 275 nm
( c = 838) and 268 nm ( c = 851). The minima occur at 272
nm and 239 nm. The spectrum was obtained with a Cary
Model 14 Recording Spectrophotometer.

2.4 Mass Spectrum


The mass spectrum shown in Figure 4 was obtained
using an Associated Electrical Industries Model MS-902
Mass Spectrometer with an ionizing energy of 70 ‘eV. The
mass spectrum of piperazine estrone sulfate indicates the
presence of estrone, piperazine, and a sulfur-oxygen
constituent, but it does not yield a molecular ion f o r the
complete chemical entity. This is attributed to the follow-
ing behavior:

(a) Dissociation of the amine salt into the free


amine (piperazine) and the free acid (estrone hydrogen
sulfate).

(b) Possible thermal decomposition of estrone hydro-


gen sulfate to estrone, S02, OH’, etc.

380
FIGURE 2 - NUCLEAR MAGNETIC RESONANCE SPECTRUM OF PIPERAZINE ESTRONE SULFATE
ZUI L. CHANG

Table I

NMR Spectral Assignments for Piperazine


Estrone Sulfate

Proton Chemical
Assignment Shift (ppm) Mu 1 tip 1i c i ty
H

doublet

fl 0
7.17 J=9. 5Hz

QIH 0 6.92 Mu1 t ipl e t

5.5 Broad

2.89 Singlet

382
PIPERAZINE ESTRONE SULFATE

Table I Cont.

Proton Chemi c a 1
Assignment Shift (ppm) Mu1 tip 1ic i ty

2 - 3.0 Comp 1ex

C -CH, 0.84 Singlet

383
ZUI L. CHANG

I
0.8

0.7 -
FIGURE 3 - ULTRAVIOLET SPECT
SPECTRUM
RUM
OF PIPERAZINE ESTRONE SULFATE
SULFATE
0.6 =

\
0.5

0.4

0.3

0.2

0.1

2 00 250 300 350


WAVELENGTH (nm)

384
FIGURE 4 - MASS SPECTRUM OF
PIPERAZINE ESTRONE SULFATE

I'
ZUI L. CHANG

The mass spectrum assignments of the prominent


ions and subsequent fragments are shown in Table I1 and
Figure 5.5

2.5 Raman Spectrum


The Raman spectrum of piperazine estrone sulfate
a s shown in Figure 6; was obtained in the solid state on
a Cary Model 83 Spectrometer. The following bands (cm-l)
have been assigned for Figure 6.3

a. 1733 ane1 due to the C=O stretching of the 17-keto


group.

b. 1608 cm-l due to skeletal stretching mode of the


aromatic ring.

c. 1050 cm-l due to s-0 linkage

2.6 Optical Rotation


A 1% solution of piperazine estrone sulfate in
0.4% sodium hydroxide solution exhibited a rotation of
[cu]~~ + 87.8" when determined on a Perkin-Elmer Model 141
Po1ar imeter . 6

2.7 Melting Range


Piperazine estrone sulfate melts at about 190°C
to a light brown, viscous liquid which re-solidifies, on
further heatin and finally melts at about 245°C with
decomposition. f3
2.8 Differential Thermal Analysis (DTA)
The DTA curve obtained on a Dupont Model 900
Analyzer as shown in Figure 7 confirms the observed
melting characteristics described in section 2.7.

2.9 Solubility
Approximate solubility data obtained at room
temperature are given in the following table:'s8

386
PIPERAZINE ESTRONE SULFATE

Table I1

High Resolution Mass Spectrum of Piperazine


Estrone Sulfate

Found Mass Calculated Mass -


C -
H -
N -
O -
5

270.1620 270.1620 18 22 0 2 0

213.1287 213.1279 15 17 0 1 0

185.0960 185.0966 13 13 0 1 0

172.0887 172.0888 12 12 0 1 0

146.0725 146.0732 10 LO 0 1 0

86.0843 86.0844 4 10 2 0 0

85.0771 85.0766 4 9 2 0 0
63.9618 63.9619 0 0 0 2 1

47.9669 47.9670 0 0 0 1 1

387
ZUI L. CHANG

FIGURE 5 - FRAGMENTATION PATHWAYS OF PIPERAZINE ESTRONE SULFATE

H
I
y '&
H
I
II
HO-S-0
O

II
0

HO-S-0
0
II
W

m / a 350
bN>1+* not observed

...I- -- [ H O

I
W 1 m/a 270
L J
m/a 64

[ so]+'

[..m]+' m/a 172

I
[ H o r n ] " m/a 146

388
FIGURE 6 - RAMAN SPECTRUM OF PIPERAZINE ESTRONE SULFATE
2000 1800 1600 1400 1200 loo0 800 600 400 200 0
I I I I I I I I I

80 - - 80

60 - - 60

- 40

- 20

0 - - 0
I 1 I 1 I I I 1 I
2000 1800 1600 1400 1200 lo00 800 600 400 200 0
RAMAN SHIFT ACM-’
FIGURE 7 - DIFFERENTIAL THERMAL ANALYSIS CURVE OF PIPERAZINE ESTRONE SULFATE

0
X
Ly

A -

-
s

w I-
(D
0 d '

v -
0
n
t
1 I I I I I I I I I
0 50 100 150 200 250 300 3 50 400 450

T " C (CORRECTED FOR CHROME1 ALUMEL THERMOCOUPLES)


PlPERAZlNE ESTRONE SULFATE

Solvent Solubility (mg/ml)


Water 8

95% Ethanol 7.4

Chloroform 1

Ether Practically insoluble

Acetone 0.2

Benzene Practically insoluble

Methylene dichloride 1.6

Isopropanol Practically insoluble

0.1 Sodium hydroxide 57

Propylene glycol 35

Mineral oil 2

Sesame oil 2

2.10 Crystal Properties


The X-ray powder diffraction pattern of pipera-
zine estrone sulfate was determined by visual observation
of a film obtained with a 1 4 3 . 2 mm Debye-Scherrer Powder
Camera (Table 111). An Enraf-Nonius Diffractis 601
Generator; 38 KV and 18 MA with nickel filtered copper
radiation; ), = 1 . 5 4 1 8 , were employed.9

2.11 Dissociation Constant


The apparent pKa value of the unprotonated
piperazine nitrogen (proton gained) was found to be 3 . 6 ,
by titration in acetonitrile-water ( 8 0 1 2 0 , v/v) with
aqueous sodium hydroxide. Attempts to find systems to
extrapolate the pKa to 100% water were unsuccessful.

The pKa value of the protonated piperazine


nitrogen (proton lost) was found to be 9 . 7 by titration
in pyridine-water mixtures with methanolic KOH, and
extrapolation to 100% water. 8

391
ZUI L. CHANG

Table 111

X-Ray Powder Diffraction Pattern


d-Spacings and Intensities

dA I/I -
dA if1
__.

16.5 10 2.98 2
7.7 10 2.92 2
7.4 40 2.86 5
6.6 40 2.73 10
6.0 20 2.53 5
5.67 30 2.46 8
5.23 40 2.37 1
4.55B 20 2.32 5
4.38 80 2. 28 5
4.22 60 2.21 1
4.05 5 2.16B 5
3.86 30 2.11 1
3.77 100 2.07B 8
3.61 5 1.95 2
3.54 2 1.89 2
3.4013 20 1.85B 3
3.22 5 1.75B 5
3.05 1 1.70 3

392
PlPERAZlNE ESTRONE SULFATE

2.12 Fluorescence
Piperazine estrone sulfate does not exhibit
fluorescent properties in either methanol or in an
alkaline aqueous solution. However, it does exhibit
fluorescence at 488 nmwhen excited at 465 nm in 65%
sulfuric acid solution.6 The strong sulfuric acid con-
verts the piperazine estrone sulfate to estrone which
reacts with sulfuric acid to yield the fluorescent
species.

2.13 Hygroscopic Behavior


Piperazine estrone sulfate was not hygroscopic
when e posed to a relative humidity of 40-50% for three
weeks.3

Piperazine estrone sulfate does not absorb


moisture at 79% relative humidity, and is only very
slight1y hygroscopic (0.89%) at 100% relative humidity. 7

2.14 Sublimation
Piperazine estrone sulfate did not sublime when
it was stored at 105'C for one month.8 No evidence of
sublimation was noted when it was heated with a hot stage
to 204°c.7

3. Synthesis

Piperazipg estrone sulfate was first prepared by Hasbrouck


in 1951.

The compound can also be prepared from estrone by a fast,


complete conversion reaction using a dimethylformamide/
sulfur troxide complex as the sulfating reactant. Excess
dimethylformamide is the solvent. Th reaction is com-
pleted by the addition of piperazine.'11

4. Stability-Degradation
Piperazine estrone sulfate was found to be stable
when refluxed in water for 3 hours. But it degrades
completely after 3 hours in refluxing 1 3 hydrochloric
acid to yield estrone and piperazine sulfate. It degrades
slightly in refluxing 1 sodium hydroxide after 3 hours
to yield less than 10% free estrone.12

393
ZUI L. CHANG

Piperazine estrone sulfate yields about 10% free


estrone when heated at 105OC for one month.

The rate of hydrolysis of piperazine estrone sulfate


to estrone at 90°C was studied over the pH range of
2.5-9.1. The extent of degradation was determined by a
spectrophotometric measurement in 0.1 1 sodium hydroxide
solution.13

The hydrolysis of piperazine estrone sulfate to


estrone follows first order kinetics with respect to the
piperazine estrone sulfate concentration remaining.
Futh rmore, the degradation is first order with respect
to t' 2 hydrogen ion concentration, resulting in a 10-fold
rate increase with each pH unit decrease.13 Some rate
cons ants at different pH and temperature values are shown
in I? oles IV and V. The activation energy of the degra-
datit n reaction, Ea (obtained from the slope of the plot
of 11 K, as a function of 1/T where T is the absolute
temp rature), for three pH levels is shown in Table V.

5. Drug Metabolic Products and Pharmacokinetics


The drug substance is hydrolyzed to estrone in acidic
media. The known metabolic intercon ersions of the
estrone are summarized in Figure 8.1Z

Purdy's15 work suggests that estrone, as the sulfate


conjugate, is an important transport form of estrone in
human plasma.

Urinary excretion studies of sodium estrone sulfate


have been performed by Twombly and Levitz16, and Brown. 17

Biliary excretion was also studied by Twombly and


Levitz.16

A very exhaustive study of urinary and biliary meta-


bolites was described by Jirku and Levitz. 18

Quantitative determination of plasma estrogens by


radioimmmunoassay has been developed by Vega. l9

394
PlPERAZlNE ESTRONE S U L F A T E

Table IV

First Order Rate Constants of Piperazine Estrone


Sulfate as a Function of pH at 90°C

Initial kl
- 95% Conf. Lim.
Concentration No. of
-1
pH mg. /ml. Samp1e s X lo4, hour

2.50 0.3 8 - 250


1,830 +
3.03 0.3 8 480 +
- 25
3.07 1.5 12 396 +
- 16
3.40 1.5 13 201 +- 14

3.57 0.3 7 130 +


- 16

3.88 1.5 15 59.l 2.9

4.26 1.5 15 22.4 +


- 1.5
4.86 1.5 15 7.51 +
- 1.03

5.16 1.5 15 3.63 +


- .68

5.98 1.5 13 1.85 +


- .77

6.11 1.5 14 +
1.12 - .44

6.48 1.5 15 .68 +


- .30
6.88 1.5 14 .72 +
- .17
7.50 1.5 13 .42 +
- .12
7.90 1.5 15 + .11
.14 -

8.43 1.5 13 + .08


22 -

9.10 1.5 12 .18+


- .11

395
ZUI L. CHANG

Table V

F i r s t Order Rate C o n s t a n t s and A c t i v a t i o n E n e r g i e s


of P i p e r a z i n e E s t r o n e S u l f a t e a t Three pH l e v e l s

pH 2.50 pH 3.03 pH 3.57

kl x lo4, hr.-’
+ 480 +_ 25 +
90°C.
8OOC.
1,830
695 z+ 250
31 150 + 4.6
52.2 7
130
44.8
f
16
4.0
7OOC. 262 - 15 - 2.2 14.6 2.3

Arrhenius R e l a t i o n s h i p

Linear C o r r e l a t i o n Coeff. 1.000 .999 1.000


A c t i v a t i o n Energy, Ea 24,100 27,500 27,100

396
-

Y
2!
0
z
Ly

v)
3
4
4
&
4
c
-3
A

z
Ly

Ly
v)
c

c
Ly
FIGURE 8 METABOLIC PATHWAYS OF PIPERAZINE ESTERONE SULFATE

HO

HO \
2 -h yd rox yertrone HO
178 -errradio1

OH

0
I
397

W
(0
-J
7

HO
2-methoxyestrone estriol
f
F(

- U
2
E
Q
Ef
0

P)
X
L
P

HO
M a -h y drox y estrone HO
15a-hydroxyestradiol 16-epiestriol
(also 168-)
ZUI L. CHANG

6. Methods of Analysis
6.1 Identification
The presence of piperazine may be identified with
quinone T. S. or thin-layer chromatography (Section
6.21).
The presence of estrone hydrogen sulfate may be
identified by thin-layer chromatography (Section 6.21).

The presence of free estrone may be ident


with a 2.5% solution of @-naphthol in sulfuric acid Idied
or
thin-layer chromatography (Section 6.21).

6.2 Chromatographic Analysis

6.21 Thin-Layer Chromatography


A number of thin-layer chromatographic
systems on silica gel have been described for the separa-
tion of hydrolysis products of i erazine estrone sulfate
from the parent substance. 1 9 6 2f 9 $2 piper azine estrone
9

sulfate chromatographs as the piperazine and estrone


sulfate moieties. Various systems,methods of detection
and Rf valves are summarized in Table VI.

6.22 Gas-Liquid Chromatography


Piperazine estrone sulfate can not be di-
rectly chromatographed. However, the principal degrada-
tion product, estrone, may be silylated with BSA and
chromatographed using 3% QF-1 on Gas Chrom Qf2, or it may
be silylated with a silylating mixture containing
N-trimethylsilylimidazole, BSA and trimethylchorosilane in
the ratio 1:30:bf, and chromatographed using 10% SE-30 on
-
Chrom0 sorb W AW .

6.3 Spectrophotometric Analysis


Direct spectrophotometric analysis of piperazine
estrone sulfate is applicable provided significant quan-
tities of interferring contaminents are not present. The
drug substance may be examined directly in a methanol
solution at 269 nm ( 6 = 860) or in 0.1 -
N sodium hydroxide
solution.6

398
Table VI

TLC Systems f o r P i p e r a z i n e E s t r o n e S u l f a t e

Rf Rf Rf
Solvent Estrone Refe-
System Detection Piperazine Sulfate Estrone Pence

Chloroform: A r senomolybda t e Origin 0.64 0.82 6


Methanol (5:4) spray

n-Propanol: S h o r t wave W 0.42 0.78 0.92 21


Ethanol : o r 10% S u l f u r i c
Conc. Ammonia Acid i n Ethanol
0
(D (2:1:2)
(0

Chlorof o m : S h o r t wave W 0.03 0.35 0.72 21


Methanol (3:1 ) o r 10% S u l f u r i c
Acid i n Ethanol

n-Butanol: s h o r t wave W
Water: Conc. Ammonia o r 10% S u l f u r i c
(1: 1: 1) Acid i n Ethanol

Chloroform: Methanol: I o d i n e Vapor


S t r o n g e r Ammonia TS
(85:15: 1 )
ZUI L. CHANG

The estrone content of piperazine estrone sulfate


can be determined with a suitable spectrophotometer at
238 nm after the conversion of piperazine estrone sulfate
to estrone with the use of hydrochloric acid.l

The degradation product, estrone,may be quantitated


in the drug substance by a liquid-liquid extraction into
chloroform and comparison to an estrone reference standard
at 280 nm. Alternately, the chloroform extract may be
evaporated and the residue dissolved in 65% suifuric acid
solution. The estrone is dehydrated to a species which
fluorescence at 488 nm with excitation at 465 nm.6

6.4 Colorimetric Analysis


Piperazine estrone sulfate may be determined as
dehydrated estrone using a phenol-sulfuric acid mixture as
the col r development reagent, The chromophore absorbs at
522 nm.40

Piperazine estrone sulfate may also be determined


using 65% sulfuric acid as the reagent. The reaction pro-6
duct with sulfuric acid has a yellow chromophore at 4 5 0 nm.

These colorimetric methods may be used for the


analysis of piperazine estrone sulfate in tablets or creams.
The primary degradation product, estrone, can be removed
by prior chloroform extraction.

6.5 Nitrogen Analysis


The amount of nitrogen present in piperazine
estrone sulfate may be determined by converting the
nitroqgn to ammonia and titrating with 0.05 sulfuric
acid.

6.6 Titration
Piperazine estrone sulfate may be potentiometri-
cally titrated in pyridine-water mixtures using methanolic
potassium hydroxide and glass-calomel electrodes.8

7. Acknowledgements

The author wishes to thank Mr. V. E. Papendick and


Mr. J. A. Raihle for their review of the manuscript, and
Miss Sandra Hudson for typing and drawing of the manu-
script.

400
ZUI L. CHANG

8. References

1. The National Formulary, 14th Revision, Mack


Publishing Co., Easton, PA (1974).

2. Chemical Abstracts Service Registry No. 7280-37-7.

3. Washburn, W., Abbott Laboratories, Personal


Communication.

4. Cirovic, M., Abbott Laboratories, Personal


Comunicat ion.

5. Mueller, S., Abbott Laboratories, Personal


Communication.

6. Chang, 2. L., Abbott Laboratories, unpublished work.

7. Yunker, M., Abbott Laboratories, Personal


Communication.

8. Wimer, D. C., Abbott Laboratories, Personal


Communicat ion.

9. Quick, J., Abbott Laboratories, Personal


Communication.

10. Hasbrouck, R. B., Assignor to Abbott Laboratories,


U. S. Patent 2,642,427.

11. Lex, C. G., Assignor to Abbott Laboratories, U. S.


Patent 3,525,738.

12. Williamson, D. E., Abbott Laboratories, Personal


communication.

13. Borodkin, S., Abbott Laboratories, Personal


Communication.

14. Kohn, F. E . , Abbott Laboratories, Personal


Communication.

15. Purdy, R. H., Engel, L. L., and Oncley, J. L.,


J. Biol. Chem., 236, 1043, (1961).

401
ZUI L. CHANG

16. Twombly, G H., and Levitz, M., Am. J. Obstet.


and Gynec., 80, 889, (1960).
17. Brown, J. B., J. Obstet, and Gynec. Brit. Emp.,
-
66, 795, (1969).
18. Jirku, H. and Levitz, M., J. Clin. Endocr., 2,
615, (1969).

19. Vega., S. M., Abbott Laboratories, Personal


Communication.

20. Abbott Laboratories, J. Am. Med. Assoc., 149 (5),


443, (1952).

21. Birch, R. A., Dale, B. J. and Earl, J. M., Abbott


Laboratories, Personal Communication.

22. Luebke, D. R., Abbott Laboratories, Personal


Communication.

402
PROCARBAZINE HYDROCHLORIDE

Richard J. Rucki
RICHARD J. RUCK1

INDEX

Analytical Prof il e - Procarbazine H y d r o c h l o r i d e

1. Description
1.1 Name, Formula, Molecular Weight
1.2 Appearance, Color, Odor

2. Phys i ca 1 Proper t ies


2.1 I n f r a r e d Spectrum
2.2 Nuclear Magnetic Resonance Spectrum
2.3 U l t r a v i o l e t Spectrum
2.4 F1uorescence Spectrum
2.5 Mass Spectrum
2.6 O p t i c a l R o t a t i o n
2.7 M e l t i n g Range
2.8 D i f f e r e n t i a1 Scanning C a l o r i m e t r y
2.9 Thermogravi met r ic Anal ys i s
2.10 Solubi 1 i t y
2.11 C r y s t a l P r o p e r t i e s
2.12 D i s s o c i a t i o n Constant

3. Synthesis

4. S t a b i 1 i t y and Degradation

5. Drug Metabolic Products

6. Toxi c i t y

7. Methods o f A n a l y s i s
7.1 Elemental A n a l y s i s
7.2 Thin-Layer Chromatographic A n a l y s i s
7.3 D i r e c t Spectrophotometric A n a l y s i s
7.4 Coulometric A n a l y s i s
7.5 Pol arog raph ic Ana 1ys i s
7.6 T i t r i r n e t r i c A n a l y s i s

a. Acknowledgements

9. References

404
PROCARBAZINE HYDROCHLORIDE

1. Description

1.1 Name, Formula, Molecular Weight

P roca r baz i ne hyd roch 1o r i de i s N- i sop ropy 1-a-


(2-methylhydrazino)-~-toluamid~h y d r o c h l o r i d e .

Procarbazi ne H y d r o c h l o r i d e

C12H19N30-HCl Molecular Weight: 257.76

1.2 Appearance, Color, Odor

White t o p a l e ye1 low c r y s t a l 1 i n e powder w i t h


a s l i g h t c h a r a c t e r i s t i c odor.

2. Phys ic a l P r o p e r t i e s

2.1 I n f r a r e d Spectrum ( 1 R )

The i n f r a r e d spectrum o f procarbazine hydro-


c h l o r i d e i s presented i n F i g u r e 1 ( 1 ) . The
instrument used was a Perkin-Elmer Model 621
G r a t i n g Spectrophotometer. The sample was
dispersed i n FluorolubeR t o r e c o r d t h e spec-
trum i n t h e r e g i o n o f 4000-1340 cm’l and i n
m i n e r a l o i l f o r the r e g i o n o f 1340-400 cm-l.
The f o l l o w i n g assignments have been made f o r
the bands i n F i g u r e 1 ( 1 ) .

Band (cm-’) Assignment


3277 and 3200 NH s t r e t c h
3035 Aromatic CH s t r e t c h
2961 and 2853 A 1 i p h a t i c CH s t r e t c h
2760-2300, main
NH:
band a t 2725

405
FIGURE 1
I n f r a r e d Spectrum of Procarbazine Hydrochloride

WAVELENGTH (MICRONS)
2.5 3 4 5 6 7 8 9 10 12 14 18 22 3550
33NWlllWSNVW %
406

4000 3500 3000 2500 2000 1700 1400 I loo 800 500 200
FREQUENCY (CM-’1
PROCARBAZINE HYDROCHLORIDE

1660 and 1636 C=O stretch (Amide I )


1556 Amide I t
1299 Amide I l l
1361 and 1351

857 Aromatic CH out-of-


p 1 ane bend i ng

2.2 Nuclear Magnetic Resonance Spectrum (NMR)

The N M R spectrum shown in Figure 2 was


obtained by dissolving 50.4 mg o f procar-
bazine hydrochloride in 0.5 ml of DMSO-d6
containing tetramethylsilane as internal
reference. The spectral assignments are
shown in Table 1 (2).
a

Table 1
Chemi ca 1 Coup 1 i ng
Shift Constant ,
Protons 6 (ppm) Mu1 tip1 ici ty J (in Hz)

1.20 Doub 1 et 6.0


2.72 Singlet --
4.18 Mu1 ti p let 6.0
4.23 Sing 1 et --
7.55 Doub 1 e t 8.5
7.99 Doublet 8.5
8.36 Doublet 7.0
6.33- Singlet --
10.33 (broad)

407
FIGURE 2
N M R Spectrum o f Procarbazine Hydrochloride

P
408

0
0)

1 1 1 1 1 1
6 5 4 3 2 1 0
PPM (8)
PROCARBAZINE HYDROCHLORIDE

2.3 U I t r a v i o l e t Spectrum (UV)

The u l t r a v i o l e t spectrum o f procarbazine hydro-


c h l o r i d e i n the r e g i o n o f 350 t o 200 nm i s
shown i n F i g u r e 3. ( 3 ) . The s p e c t r u q e x h i b i t s
one maximum a t 232 nm ( E = 1.3 x 10 ) and a m i n i -
mum a t 213 nm. The s o l u t i o n c o n c e n t r a t i o n was
0.01 mg/ml i n 0 . 1 N h y d r o c h l o r i c a c i d and t h e
q u a r t z c e l l width-was 1 cm.

2.4 FI uorescence Spectrum


E x c i t a t i o n and emission scans were c a r r i e d
o u t f o r procarbazine h y d r o c h l o r i d e i n s o l u t i o n s
o f 0 . 1 N h y d r o c h l o r i c a c i d , 0.1N sodium hydrox-
i d e a n 7 water. No fluorescence, however, was
observed under these c o n d i t i o n s (2).

2.5 Mass Spectrum

The low r e s o l u t i o n mass spectrum shown i n


F i g u r e 4 was o b t a i n e d u s i n g a Varian MAT CH5
mass spectrometer, i n t e r f a c e d w i t h a V a r i a n
d a t a system, w i t h an i o n i z i n g energy o f
70 eV ( 4 ) . The d a t a system accepted t h e o u t -
p u t o f t h e spectrometer, c a l c u l a t e d t h e masses,
compared t h e i r i n t e n s i t i e s t o the base peak
and p l o t t e d t h i s i n f o r m a t i o n as a s e r i e s o f
l i n e s whose h e i g h t s were p r o p o r t i o n a l t o t h e
intensities.

The molecular i o n o f t h e f r e e base was observed


a t m/e 221. The H C l moiety was observed a t
m/e 36. The ions a t m/e 191, 177 and 163 c o r -
respond t o a loss from t h e f r e e base o f NHCH
NHNHCH , and NHCH(CH ) r e s p e c t i v e l y . The
3’
ions a ? m/e 149 and 3j$’correspond t o t h e loss
o f C H by M c L a f f e r t y rearrangement from m/e
I91 an9 177, r e s p e c t i v e l y . The base peak a t
m/e 118 r e s u l t s from t h e l o s s o f NHNHCH f r o m
3
m/e 163.
A h i g h r e s o l u t i o n scan confirmed t h e r e s u l t s of
t h e low r e s o l u t i o n spectrum. Table I I l i s t s
t h e elemental compositions f o r t h e i o n s as

409
RICHARD J. RUCK1

FIGURE 3
U l t r a v i o l e t Spectrum o f
Procarbazi ne Hydrochlor i de

0.5-

0.4 -

w
V
z
a
8 0.3-
$
m
a

1 I I I
200 250 300 35c
NANOMETERS

410
FIGURE 4
Mass Spectrum o f Procarbazine Hydrochloride

100
L
90
80
A l l S N 31N I 3A I1 V 138

70
60
411

50
40
30
20
10

0 1 1 I I I I I I I I I I I I I I I I I I I
50 100 Iio 200 2 0
m/e
RICHARD J. RUCK1

determined by h i g h r e s o l u t i o n mass spectroscopy (4).

Table I I

High R e s o l u t i o n Mass Spectrum o f


Procarbazine H y d r o c h l o r i d e

Found Mass Calcd. Mass -


C H -N
118.0428 118.0419 8 6 0
135.0698 135.0684 8 9 1
149.0753 149.07 1 5 8 9 2
163.0870 163.0872 9 11 2
177.1170 177.11 54 11 15 1
191.1288 191. I 3 1 1 12 17 1
221.1533 221.1529 12 19 3
2.6 Optical Rotation

Procarbazine h y d r o c h l o r i d e e x h i b i t s no
o p t i c a l a c t i v i t y (3).

2.7 Me1 t i ng Range

According t o USP X I X , procarbazine hydro-


c h l o r i d e m e l t s a t about 223"C, w i t h decorn-
p o s i t i o n (5).

2.8 D i f f e r e n t i a l Scanning C a l o r i m e t r y (DSC)

DSC s p e c t r a f o r procarbazine h y d r o c h l o r i d e
a t a scan r a t e o f 10°C/min. e x h i b i t e d an
endotherm a t about 23OoC, where m e l t i n g was
accompanied by sample decomposition. The endo-
therm was f o l l o w e d immediately by a slow
exotherm (con t inu i n g decompos it ion). The
observed temperature o f t h e melting/decomposi-
t i o n endotherm i s dependent upon i n s t r u m e n t a l
c o n d i t i o n s and, t h e r e f o r e , i s n o t c h a r a c t e r i s t i c
o f t h e compound.

2.9 Thermogravimetric A n a l y s i s (TGA)

A thermogravimetric a n a l y s i s performed on

412
PROCARBAZINE HYDROCHLORIDE

procarbazine h y d r o c h l o r i d e e x h i b i t e d n o l o s s
o f w e i g h t from 30-150"C. A t about 150"C,
decomposition weight losses began and con-
t i n u e d t o 500°C (upper l i m i t o f i n s t r u m e n t ) .

2.10 Solubi 1 i t y

Approximate s o l u b i l i t y data o b t a i n e d a f t e r
3 hours a t 25°C a r e g i v e n i n Table I l l (6,7).

Table I l l

S o l u b i l i t y o f Procarbazine H y d r o c h l o r i d e

Solvent Solubi 1 i t y (mg/ml)

Water 200
95% Ethanol 27
Absolute Ethanol 8
Methanol 59
Ace tone 4.5
Diethyl Ether <o. 1
Petroleum E t h e r (30-60") <0.1
C h 1o r o f o r m 2
Benzene <0.5
3A A l c o h o l 12
l s o p r o p y l Alcohol 1
Cyclohexane <0.5
Ethyl Acetate <O. 5
D ioxane 0.7
Acetonitri l e <O. 5
Carbon Tet rach l o r i d e <0.5
Methylene C h l o r i d e <O. 5

2.11 Crystal Properties

The x-ray powder d i f f r a c t i o n p a t t e r n of p r o -


carbazine h y d r o c h l o r i d e i s presented i n
Table I V (8).

Instrument C o n d i t i o n s

Instrument GE Mode XRD-6 Spect rogon i o -


meter
Genera t o r 50 K V , 2.5 m~

413
RICHARD J. RUCK1

Tube T a r g e t Copper (Cu Ka = 1.5418 A)


Optics 0.1" D e t e c t o r s l i t
M.R. S o l l e r s l i t
3" Beam s l i t
0.0007'' N i F i l t e r
4" Take o f f a n g l e
Goniometer Scan a t 0.2" 2 W m i n u t e
Detector A m p l i f i e r gain-16 coarse
8.7 f i n e
Sealed p r o p o r t i o n a l c o u n t e r
t u b e and DC v o l t a g e a t .
plateau.
P u l s e h e i g h t s e l e c t i o n El 5
v o l t s , Eu o u t .
Rate meter T.C. 4, 2000 c / s
f u l l scale.
Recorder Chart speed 1"/5 minutes
Samp 1 es Prepared by g r i nd i ng a t room
temperature.

Table I V

Procarbazine Hydrochloride

-
20 d (A)+:
0

-''
/ Oft
11.240 7.8719 0.24
12.400 7.1380 0.46
18.020 4.9225 0.48
18.640 4.7601 0.23
19.800 4.4838 0.17
20.370 4.3596 0.40
21.550 4.1235 0.61
22.550 3.9428 0.35
23.930 3.7185 0.30
25.190 3.5352 1 .oo
25.800 3.4530 0.10
28.800 3.2090 0.14
28.410 3.1415 0.04
29.550 3.0228 0.22
29.650 3.0128 0.23
33.740 2.6564 0.07
;fd ( i n t e r p l a n a r d i s t a n c e ) = nX/2 s i n 0 1
**l/lo = r e l a t i v e i n t e n s i t y ( h i g h e s t i n t e n s i t y = 1.00)

414
PROCARBAZINE HYDROCHLORIDE

2.12 D i s s o c i a t i o n Constant

The d i s s o c i a t i o n c o n s t a n t f o r p r o c a r b a z i n e
h y d r o c h l o r i d e was determined t i t r i m e t r i c a l l y
u s i n g 0.1N sodium hydroxide and a s o l u t i o n
i o n i c s t r e n g t h o f 0.1. The v a l u e f o r t h e pKa
determined by t h i s method was 6.8 ( 9 ) .

3. Synthesis

Procarbazine h y d r o c h l o r i d e may be prepared by t h e


r e a c t i o n scheme shown i n F i g u r e 5. 4-Formylben-
z o i c a c i d isopropylamide ( I ) i s combined w i t h m e t h y l -
h y d r a z i n e i n dimethylformamide t o prepare t h e c o r r e s -
ponding methylhydrazone ( I I ) , which i s then reduced,
u s i n g p a l l a d i u m charcoal c a t a l y s t , t o N - i s o p r o p y l -
a-(Z-methylhydrazino) -p-toluamide ( I IT). Hydrogen
c h l o r i d e i s added t o t h e r e a c t i o n m i x t u r e o f I l l con-
v e r t i n g i t t o the h y d r o c h l o r i d e o f N-isopropyl-a-
(2-methyl h y d r a z i no) -e-toluami de ( I VT.

4. S t a b i 1 i t y and Degradation

Procarbazine h y d r o c h l o r i d e , i n t h e presence o f
m o i s t u r e o r i n aqueous s o l u t i o n , undergoes o x i d a t i o n
by atmospheric oxygen. T h i s a u t o x i d a t i o n has beeg
2
r e p o r t $ $ t o be c a t a l y z e d by metal ions such as Mn
and Cu (10,ll). The major products o f t h i s o x i -
d a t i o n a r e N- i sopropyl -a- (2-methylazo) -p- to1 uami de
( I I ) and N-Tsopropyl-a-(2-methyl hydrazof;o)-p-tolua-
mide ( I 1 IT. To a much l e s s e r e x t e n t , t o l u i c a c i d
isopropylamide ( I V ) and 4-formylbenzoic a c i d i s o -
propylamide (V) can a l s o be formed (12,13). The
o x i d a t i v e degradation scheme i s shown i n F i g u r e 6.
I n a d d i t i o n , procarbazine h y d r o c h l o r i d e i s v e r y
s e n s i t i v e t o u l t r a v i o l e t l i g h t (14), n e c e s s i t a t i n g
t h e use o f l o w - a c t i n i c glassware d u r i n g analyses.
I n closed, amber b o t t l e s a t room temperature, degra-
d a t i o n o f procarbazine h y d r o c h l o r i d e i n t h e s o l i d
s t a t e i s slow, and t h e compound remains s u i t a b l y
s t a b l e f o r a t l e a s t t h r e e years (15). A n i t r o g e n
atmosphere has been found t o r e t a r d degradation.

U t i l i z i n g spectrophotometric (16) and p o l a r o g r a p h i c


time s t u d i e s , i t was determined t h a t p r o c a r b a z i n e

415
FIGURE 5
Synthesis o f Procarbazine Hydrochloride

CH3 H 0 H CH3 H 0
I
HC-N-CI IIo A = O + C H ~HIN - N H Z ---+ H
H;-i-!!OC=N-NCH3 I
H
I
I I
CH3 CH3
(I)

CH3 H 0 H H CH3 H 0 H H
H h - l ! l - ! o C H 2 - N - N - - C H 3I I +-HCI I
HC-l!l-: O C H z - N - N IC HI 3
I I
CH3 -HCI CH3
(Im (nI)
FIGURE 6
Deg rada t ion o f Procarbaz ine Hydroch l o r i de

0-0-0
I
0

0=0
y 3

c)l

6F
7
(I)

z
I
HC-NH-leCH2-NH-NH-CH3 HCI

2
I
2
r
I

0
I

Y
I
0
-

LI
IN

H
I
I
m
I

I"
CH3

A
(-2H) \2HI