See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/313309046

Synthesis of new Diclofenac Derivatives

Book · January 2016

CITATIONS READS
0 2,223

2 authors:

Noor Hatef Naser Monther Faisal

University of Kufa/ college of pharmacy Ashur University College
15 PUBLICATIONS   3 CITATIONS    42 PUBLICATIONS   49 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

medicinal chemistry View project

Preparation and Comparative Evaluation of Fe+2/Fe+3 and Mg+2/Fe+3 LDHs as Promising Nanocarriers for Class II and Class IV Drugs View project

All content following this page was uploaded by Noor Hatef Naser on 03 February 2017.

The user has requested enhancement of the downloaded file.



  
 

           


  
 

      
  
  
 !"# 

    ! $%      #   &
  
              
&    
 ! $       
 
  #    #    &
        & #     
 %   '
     #          '
&          
   
     
    &
    !

 
  %(!)
!* 
 
 

&
  
&#   
   ! ) 
(+ 
,   -.!


  


 



 

 

 
 

 

 

 
 




 




 

 

 
   

 
   
 

 


 
  

 

 
 




 ! 

 

"



 
# 

 

$  !%
 !

   
& 
$

'  

 
'  

($
 

' 

# %
 %
)   %
*  %
'  
  $


'
 

 

+ 
 


" %
 
 
 

&

'  !

#  




  
$,

 

( 

$



  

 
 
-
 
 
   
. 
 
   

 
 

 
 
  

 
  
 

  

 


 ! 

"-
 
 


 


 


(

  %
 

 



  


  


 


. 



 
 %

 %

 %
 
 %




$ 

  
 

 
$



$ -




 
 

 
 -

 






  

 

 


 


-
-

/

/
 
$$$   

0 

 

&  1








  


2&
)*
3
/
+)

  
 4
56%
77888
& %
  

) -
9 

: 

*
 

&





 
 



;

"
-
  %
<-


  %
 %
5=8=

/- 
>

"   %
# 
" 

/- 
>
5=87
2&
)*
3
/
+)
"
? 
 

"
 

& 
5=87
To…

My mother and father

who taught me how to
fight for my goals..

To…

anyone who believe in

the richness of learning

1
 Acknowledgment
Praise is to almighty Allah gracious for enabling me finish what I started and for
helping me to present this work .
My gratitude and deepest appreciation is expressed to my supervisor Ass. Prof. Dr.
Monther F. Mahdi for his incessant encouragement, valuable advice and support
throughout my work. He has been available whenever I needed his guidance and
assistance. I really appreciate his positive comments for improving and bring out the
utmost optimum consequences out of my endeavors. So; without his guidance,
support and optimism, I would have never been able to organize and complete the
thesis tasks as optimal as it is and within time constraints.

My sincere thanks and deepest appreciation to Ass. Prof. Dr. Kawkab Y. Saour for
her help in carrying out CHNOS elemental microanalysis.
I am grateful to the College of Pharmacy-University of Baghdad, specially Prof. Dr.
Alaa Abdel Rasool, Dean of the college for offering me the opportunity to continue
my graduate study.
My appreciation and indebtedness to Dr. Mohamed Hassan, Head of department of
pharmaceutical chemistry for his assistance in providing some chemicals and for his
advice discussions and suggestions throughout my study.
I am heartily thankful to Dr. Ghada Mahdi, Head of department of Pharmacognosy
in Kufa University for her encouragement and kindness support throughout all my
study.
My deepest gratitude and special thanks to Ass. Prof. Dr. Naji, Head of department
of pharmaceutical chemistry in Kufa University for his guidance and support.
I would like to express my appreciation to Prof. Dr. Hussain Abed Mohammed Saleh
College of Science-Babylon University for his help and support.
My great thankful expressed to Dr. Sabah Jawad for helping me to obtain the
Diclofenac Sodium.

2
I would like to express my sincere thanks to the College of Pharmacy University of
Kufa for offering the opportunity of having M.Sc. degree. Genuine gratitude and
appreciation extended to family of uncle Esam , whom brought me throughout the
two years of M.Sc. study, and provided me their love and kindness.
I would like to extend my thanks to Mr. Amar, the director of the animal house,
College of Pharmacy-Baghdad University for his help in performing the
pharmacological study.

I wish to express grateful thanks to my colleagues the post graduate students Zainab
Abdel Hadi and Othman Makki for their help and support that make us as integral
network during working in the laboratory.

Finally, I would like to express my genuine thanks and deepest appreciation to my

brother Zaid for his generous help in presenting this thesis in its final shape.

3
 Contents Page

Dedication 1

Acknowledgment 2

List of Contents 4

List of Tables 9

List of Schemes 10

List of Figures 11

List of Abbreviations 13

Abstract 15

Chapter One : Introduction

1.1. Non-steroidal anti-inflammatory drugs 17

1.2. Current approaches to counteract NSAIDs-induced toxicity 17

1.3. Inflammation 18

1.4. Cyclooxygenase mechanism 20

1.5. Types of COX-enzymes 21

1.6 Cyclooxygenase structure 23

1.7 Pharmacological inhibition of COX-enzymes 26

1.8 Classification of non-steroidal anti-inflammatory drugs 28

1.8.1 Reversibility of cyclooxygenase inhibitors 28

1.8.1.1 Irreversible cyclooxygenase inhibitors 28

1.8.1.2 Reversible cyclooxygenase inhibitors 28

1.8.2 Basic chemical structures 28

4
1.8.2.1 Arylacetic acid analogues 28

1.8.2.2 Heteroarylacetic Acid analogues 29

1.8.2.3 Arylpropionic Acid analogues 29

1.8.2.4 Naphthalene Acetic Acid analogues 30

1.8.2.5 Salicylic acid analogues 30

1.8.2.6 Pyrazolones and Pyrazolodiones 31

1.8.2.7 Anthranilates 32

1.8.2.8 Oxicames (enolic acid) 32

1.8.2.9 Gold compounds 33

1.8.3 Selectivity of cyclooxygenase inhibitors 33

1.8.3.1 Classical NSAIDs 33

1.8.3.2 Newer NSAIDs 34

1.9 A common pharmacophoric groups of selective COX-2 inhibitors 35

Cyclooxygenase activity is important for efficient replication of
1.10 37
hepatitis virus at an early stage of infection
1.11 Significance of COX-2 activity in brain diseases 37

1.12 Cyclooxygenase inhibitors in cancer chemotherapy 38

Effect of selective COX-2 inhibitors in treatment of glucose-stimulated
1.13 41
insulin secretion
1.14 Selective COX-2 inhibitors in endometriosis 42

1.15 Aim of the work 43

1.16 Strategy of the work 44

Chapter Two: materials and methods

2.1. Chemicals and Equipments 45

2.1.1. Chemicals 45

5
2.1.2. Equipments and Instruments 46

2.2. Methods of Identification 47

2.2.1. Thin Layer Chromatography 47

2.2.2. Melting Points 47

2.2.3. Infrared Spectra 47

2.2.4. Elemental Microanalysis ''CHNOS'' 47

2.2.5 Chemical tests 48

2.2.5.1 Sodium fusion test 48

2.2.5.2. Carboxylic acid test 48

2.2.5.3 Sulfonamide test 48

2.3 Methods of synthesis 49

2.3.1 Conversion of diclofenac salt to its acidic counterpart 49

2.3.2 Protection of the carboxylic acid of the diclofenac moiety 49

2.3.3 Bromination of protected diclofenac 49

2.3.4 Amination of brominated intermediate 49

2.3.5 Deprotection of esterified carboxyl moiety 49

2.4 Chemical synthesis 49

Conversion of diclofenac salt to 2-[2-(2,6-dichloro phenyl amino )
2.4.1 49
phenyl]acetic acid (Intermediate Ia)
Synthesis of methyl 2-[2-(2,6-dichlorophenylamino)phenyl]acetate
2.4.2 50
(intermediate Ib)
Synthesis of methyl 2-bromo-2-[2-(2,6-dichlorophenylamino)phenyl]
2.4.3 50
acetate (intermediate Ic)
Synthesis of methyl2-[2-(2,6-dichlorophenylamino)phenyl]-2-(4-
2.4.4 51
sulfamoylphenylamino)acetate (1ntermediate Id)
Synthesis of methyl 2-[4-(N-acetylsulfamoyl)phenylamino]-2-[2-(2,6-
2.4.5 51
dichlorophenylamino)phenyl]acetate (intermediate IIa)
Synthesis of methyl 2-[2-(2,6-dichlorophenylamino)phenyl]-2-[4-(N-
2.4.6 52
methylsulfamoyl)phenylamino]acetate (intermediate IIIa)
Synthesis of methyl 2-[2-(2,6-dichlorophenylamino)phenyl]-2-
2.4.7 52
(pyridine-2-ylamino)acetate (intermediate IVa)

6
 Synthesis of methyl 2-[2-(2,6-dichlorophenylamino)phenyl]-2-(thiazol-
2.4.8 53
2-ylamino)acetate (intermediate Va)
Synthesis of 2-[2(2,6-dichlorophenylamino)phenyl]-2-(4-
2.4.9 53
sulfamoylphenylamino )acetic acid (compound I)
Synthesis of 2-[4-(N-acetylsulfamoyl)phenylamino]-2-[2-(2,6-
2.4.10 54
dichlorophenylamino)phenyl]acetic acid (compound II)
Synthesis of 2-[2-(2,6-dichlorophenylamino)phenyl]-2-[4-(N-
2.4.11 55
methylsulfamoyl)phenylamino]acetic acid (compound III)
Synthesis of 2-[2-(2,6-dichlorophenylamino)phenyl]-2-(pyridine-2-
2.4.12 55
ylamino)acetic acid (compound IV)
Synthesis of 2-[2-(2,6-dichlorophenylamino)phenyl]-2-(thiazol-2-
2.4.13 56
ylamino)acetic acid (compound V)
2.5 Preliminary Pharmacological Study 56

2.5.1 Methods 57

2.5.2 Dose Determination 57

2.5.3 Experimental Design 58

2.5.4 Statistical Analysis 58

Chapter Three: Results & Discussion

3.1. Chemistry 59

3.1.1. Protection of the carboxylic moiety of diclofenac acid 62

3.1.2. Bromination of diclofenac methyl ester 64

3.1.3. Amination of brominated intermediate 65

Alkaline hydrolysis of diclofenac methyl ester derivatives
3.1.4. 66
(Saponification)
3.2 Identification of the synthesized compounds and their intermediates 68

3.2.1 Melting point determination 68

3.2.2 Thin layer chromatography (TLC) 68

3.2.3 Infrared spectra 68

3.2.4 Elemental microanalysis 69

3.2.5 Chemical tests discussion 69

3.2.5.1 Sodium fusion test discussion 69

7
3.2.5.2 Sulfonamide test discussion 69

3.3 Pharmacological study 91

3.3.1 Dose determination of the tested compounds 91

3.3.2 Anti-inflammatory effect of the tested compounds 91

3.3.3 The in vivo method for the evaluation of anti-inflammatory activity 92

3.3.4 Advantages of the paw edema method 92

3.3.5 Evaluation of the anti-inflammatory activity of the tested compounds 93

Effect of the reference anti-inflammatory drug (diclofenac sodium) on
3.3.5.1 93
paw edema
3.3.5.2 Effect of compounds I, IV and V on paw edema 96

3.3.5.3 Comparative analysis 99

3.4. Conclusions 105

3.5. Recommendations 105

References

References 106

8
Table Page
Title
No. No.

1-1 Cyclooxygenase activities and their inhibitors 22

COX-2 expression in human normal tissues and malignant

1-2 39
tumors

2-1 Chemicals and their Suppliers 45

2-2 Equipments, Instruments and their suppliers 46

The characterization and physical data of the intermediate

3-1 70
compounds and final compounds

3-2 Elemental microanalysis of the final compounds 71

Characteristic IR absorptions of compounds(Diclofenac

3-3 72
sodium, Ia, Ib and Ic).

3-4 Characteristic IR absorptions of compounds(Id, I and IIa) 73

3-5 Characteristic IR absorptions of compounds (II, IIIa and III) 74

3-6 Characteristic IR absorptions of compounds (Iva, IV and Va) 75

3-7 Characteristic IR absorptions of compound V 76

Effect of diclofenac sodium (reference )and propylene glycol

3-8 94
(control) on egg-white induced paw edema in rats

Effect of compounds I, IV, V and propylene glycol on egg-

3-9 97
white induced paw edema in rats

9
 Effect of compounds I, IV, V, diclofenac sodium (reference)
3-10 and propylene glycol (control) on egg-white induced paw 100
edema in rats

Percent inhibition of diclofenac sodium, compounds I, IV and

3-11 103
V on egg-white induced paw edema in rats

No. of Scheme Title Page

3-1 The synthesis of intermediates Ia, Ib and Ic 60

The synthesis of intermediates (Id, Iia-Va) and target

3-2 61
compounds (I-V)

Mechanism of conversion of carboxylic acid to acid

3-3a 63
chloride

3-3b Mechanism of conversion of acid chloride to ester 63

3-4 Mechanism of bromination by using NBS 65

Mechanism of base-induced ester hydrolysis

3-5 67
(saponification)

10
No. of Figure Title Page

Biochemical pathway shows that the formation of

1-1 19
prostaglandins occur by both COX-1 and COX-2.

Catalytic activities of COX and peroxidase for the synthesis

1-2 21
of PGs and TX from AA.

1-3 COX-1 and COX-2 binding sites 24

Comparison of the NSAIDs binding sites of COX-1 and

1-4 27
COX-2.

1-5 Structural modification of Nimesulide 36

Design and structure-functional analysis of nimesulide

1-6 41
analogues.

3-1 FTIR spectrum of Diclofenac sodium in KBr disc 77

3-2 FTIR spectrum of intermediate Ia in KBr disc 78

3-3 FTIR spectrum of intermediate Ib in KBr disc 79

3-4 FTIR spectrum of intermediate Ic in KBr disc 80

3-5 FTIR spectrum of intermediate Id in KBr disc 81

3-6 FTIR spectrum of compound I in KBr disc 82

3-7 FTIR spectrum of intermediate Iia in KBr disc 83

11
3-8 FTIR spectrum of compound II in KBr disc 84

3-9 FTIR spectrum of intermediate IIIa in KBr disc 85

3-10 FTIR spectrum of compound III in KBr disc 86

3-11 FTIR spectrum of intermediate Iva in KBr disc 87

3-12 FTIR spectrum of compound IV in KBr disc 88

3-13 FTIR spectrum of intermediate Va in KBr disc 89

3-14 FTIR spectrum of compound V in KBr disc 90

Effect of diclofenac sodium (reference) and propylene

3-15 95
glycol (control) on egg-white induced paw edema in rats
Effect of diclofenac sodium (reference) and propylene
3-16 95
glycol (control) on egg-white induced paw edema in rats

Effect of propylene glycol (control), compounds I, IV, and V

3-17 98
on egg-white induced paw edema in rats

Effect of propylene glycol (control), compounds I, IV, and V

3-18 98
on egg-white induced paw edema in rats
Effect of diclofenac sodium, propylene glycol (control),
3-19 compounds I, IV, and V on egg-white induced paw edema in 101
rats
Effect of diclofenac sodium, propylene glycol (control),
3-20 compounds I, IV, and V on egg-white induced paw edema in 101
rats
Percent inhibition produced by diclofenac sodium
3-21 104
(reference), compounds I, IV and V on paw edema
Percent inhibition produced by diclofenac sodium
3-22 (reference), compounds I, IV and V on paw edema, at time 104
intervals 60,180, 240 and 300 min.

12
AA Arachidonic acid
Arg Argenine
ASA Acetylsalicylic acid
oC Degree centigrade
COX Cyclooxygenase
Carbon, hydrogen, oxygen, nitrogen and sulfur elemental
CHONS
microanalysis
DMF Dimethylformamide
DNA Deoxyribonucleic acid
EGF Epidermal growth factors
FGF Fibroblast growth factors
Fig Figure
FTIR Fourier transform infrared spectra
g Gram
GI Gastrointestinal
His Histidine
IL Interleukin
Ile Isoleucine
IR Infrared spectroscopy
i.p Intraperitoneal
Kda Kilo Dalton
M.wt Molecular weight
N Normality
NF-NB Nuclear factor kappa B
NSAIDs Non-steroidal anti-inflammatory drugs
P Probability value
13
PG Prostaglandin
PGE2 Prostaglandin E2
PGH2 Prostaglandin H 2 (endoperoxide)
PGI2 Prostaglandin I2 (prostacyclin)
Phe Phenylalanine
Rf Retention factor
rt Room temperature
SEM Standard error of the mean
Ser Serine
SN2 Second order nucleophilic substitution reaction
TB Tuberculosis
THF Tetrahydrofuran
TNF Tumor necrosis factor
TXA2 Thromboxane A2
Tyr Tyrosine
Val Valine
VEGF Vascular endothelial growth factor

14
 ABSTRACT

Non-steroidal anti-inflammatory drugs represent one of the most

widely used classes of drugs, and are used primarily for the treatment of
osteoarthritis, rheumatoid arthritis and other inflammatory disorders; however,
the use of NSAIDs is significantly limited by their ability to induce the
formation of erosions and ulcers in the gastrointestinal (GI) tract. The analgesic
effects of NSAIDs are ascribed primarily to COX-2 inhibition, whereas several
adverse effects are believed to be mediated by COX-1 inhibition. Preferential
inhibition of COX-2 is thought to be due to the additional space in the COX-2
hydrophobic channel, as well as to the presence of a side pocket in the channel.
Therefore, a group of amine derivatives (4-aminobenzenesulfonamide
derivatives, 2-aminopyridine and 2-aminothiazole) incorporated in the Į-carbon
of diclofenac; a well-known NSAID, to increase its bulkiness were designed,
synthesized and evaluated as potential anti-inflammatory agents with expected
inhibitory selectivity toward COX-2 enzyme.

Synthetic procedures have been successfully developed for the generation of the
target compounds (I-V). The synthetic approach involved multi-steps procedures
which include:

1. Protection of carboxyl group of diclofenac.

2. Bromination of Į-carbon of the diclofenac ester.

3. Amination of the brominated intermediate by selected aromatic amino groups.

4. Alkaline hydrolysis of the methyl ester group to liberate the target compounds.

The structures of these compounds were confirmed using elemental microanalysis

(CHNOS), infrared spectroscopy (IR) and some physicochemical properties.

In vivo acute anti-inflammatory activity of the selected final compounds (I,

IV and V) was evaluated in rats using egg-white induced edema model of
15
inflammation in a dose equivalent to (3 mg/Kg) of diclofenac sodium. All
tested compounds produced a significant reduction in paw edema with
respect to the effect of propylene glycol 50% v/v (control group). Moreover,
the 4-aminobenzenesulfonamide derivative (compound I) exhibited superior
anti-inflammatory activity compared to diclofenac sodium at times 180-300
minutes with the same onset of action. The results of this study indicate
that the incorporation of the selected aromatic amino groups into diclofenac
maintain its anti-inflammatory activity and encourage further evaluation of
these compounds to demonstrate or identify their selectivity toward COX-2
iso-enzyme.

16
 CHAPTER ONE
INTRODUCTION

1.6 Non steroidal Anti-Inflammatory Drugs

The term non-steroidal anti-inflammatory drugs (NSAIDs), is a collective term for a

chemically heterogeneous group of drugs synthesized since the early 1900s that have
analgesic, anti-inflammatory, and antipyretic properties(1).

NSAIDs represent one of the most widely used classes of drugs, and are used
primarily for treatment of osteoarthritis, rheumatoid arthritis and other inflammatory
disorders; however, the use of NSAIDs is significantly limited by their ability to
induce the formation of erosions and ulcers in the gastrointestinal (GI) tract.
Approximately 50% of individuals who use NSAIDs develop gastric erosions, while
an estimated 2 to 4% of these individuals develop clinically significant GI ulcers and
bleeding, sometimes leading to death. There is therefore a need for anti-inflammatory
and analgesic drugs that will provide symptom relief without causing GI injury(2).

1.2 Current approaches to counteract NSAID-induced toxicity

Different strategies have emerged to improve the safety profile of NSAIDs:

1) The development of selective COX-2 inhibitors: selective inhibition of the COX-2

isoform was considered more therapeutically desirable – the hypothesis being that
suppression of COX-2 activity would reduce the production of PGs at sites of
inflammation while sparing COX-1-mediated PG synthesis in the stomach(3). The
cardiovascular toxicity of selective COX-2 inhibitors is possibly a consequence of the
inhibition of the synthesis of prostacyclin (PGI2), which has anti-thrombotic
properties, while sparing the synthesis of thromboxane A2 (TXA2), a pro-thrombotic
substance, PGE2 is the PG primarily associated with inflammation. Therefore,
17
selective inhibition of PGE2 synthesis could be a rational approach for reducing
inflammation without producing the cardiovascular and GI toxicity associated with
NSAIDs(4).

2) Gaseous mediator-releasing NSAIDs: The discovery that other endogenous

mediators produced many of the same effects of PGs in terms of mucosal defense has
led to the development of novel NSAIDs that slowly release 'gastro protective'
substances. Nitric oxide (NO) and hydrogen sulfide (H2S) are endogenous gaseous
mediators that exhibit many PG-like effects in the GI tract(5).

3) Prodrug synthesis: Gastric mucosal injury produced by NSAIDs is generally

aggravated by the local irritation caused by acidic group of NSAIDs. Thus temporary
masking of this group gives relief from GI irritation; hence prodrug approach is
suitable technique for this purpose(6).

1.3 Inflammation (Latin, inflammation, a setting on fire)

Is the complex biological response of vascular tissues to harmful stimuli, such as
pathogens, damaged cells, or irritants. Inflammation is a protective attempt by the
organism to remove the injurious stimuli as well as initiate the healing process for the
tissue (7).

Inflammation can be classified as either acute or chronic. Acute inflammation

describe the rapid response of innate immune components to a challenge. While
chronic inflammation may arise because of susceptibility in the individual to
perpetuate inflammatory response, failure to eradicate the agents or factors triggering
inflammation (e.g. foreign body embedded in injured tissue), persistent microbial
infection (e.g. TB, or continuing tissue damage) and pro-inflammatory stimuli, such
as those encountered in the atherosclerotic plaque(8). Almost, steroids namely :
prednisolone, dexamethasone, betamethasone, 18rostaglandin and hydrocortisone
were considered to be the drug of choice as anti-inflammatory agents. Owing to the
several adverse effects caused by either short-term or long-term steroid therapy, these
18
have been more or less replaced by much safer and better tolerated non-steroidal anti
–inflammatory drugs (NSAIDs)(9). The seriousness and enormous after effects of
steroid therapy necessitated an accelerated research towards the development of non-
steroidal anti-inflammatory drugs since the past. A good number of these agents have
been put into clinical usage widely and confidently thereby exhibiting positive
therapeutic efficacy accompanied with fewer untoward reactions(9). The synthesis of
prostaglandins is the primary target of all NSAIDs.

Prostaglandins are known to be involved in numerous physiological systems .The

regulation of vascular tone and platelet aggregation is affected by endothelial
prostacyclin and platelet-derived thromboxane. Prostaglandins of the E-series exert
protective effects on the gastric mucosa. Prostaglandins are also of major importance
in the regulation of the inflammatory cascade and they act as sensitizers of peripheral
nociceptors(10).as shown in fig .(1-1)

Fig.(1-1) Biochemical pathway shows that the formation of prostaglandins occur by

both COX-1 and COX-2 (11).

19
In addition to their involvement as mediators of inflammation, prostaglandins have
been implicated in diseases such as cancer specifically lung, breast, and colon),
Alzheimer’s disease, Parkinson’s disease, and cardiovascular diseases including
stroke, myocardial infarction, and atherosclerosis(12).

The mechanism of action principally responsible for most of the NSAIDs seems to
be inhibition of prostaglandin synthesis by causing almost complete blockade of the
activity of the precursor enzymes, cyclooxygenases(9).

These are the rate-limiting enzymes in the synthesis of the inflammatory

prostaglandins PGE2 and PGF2a, the cytoprotective prostaglandin PGE1, and the
vasoactive prostanoids thromboxane A2 (TXA2) and prostacyclin (PGI2) (13).

1.4 Cyclooxygenase mechanism

The cyclooxygenases (COXs) are responsible for the formation of important

biological mediators called prostanoids, which play a critical role in various
biological processes. Arachidonic acid (AA) is a 20 carbon unsaturated fatty acid
distributed throughout the lipid bi-layer of the cell membranes. Phospholipase-A2
enzyme cleave membrane bound arachidonate for the conversion into bioactive
precursors(14).

COX converts arachidonic acid, to prostaglandin H2 (PGH2), the precursor of the

series-2 prostanoids. The enzyme contains two active sites; a cyclooxygenase active
site, where arachidonic acid is converted into the hydroperoxy endoperoxide
prostaglandin G2 (PGG2), and heme with peroxidase activity(15), the PGG2 defuses
from the cyclooxygenase active site where it is reduced to the 20rostagl
endoperoxide, PGH2(16), as shown in fig.(1-2). This is converted by isomerase to
20rostaglandins and thromboxane, which exert numerous physiological and
pathophysiological effects(17).

20
1.5 Types of COX enzymes

Three COX isozymes are known; COX-1, COX-2,

X 2 and COX
COX-3. COX-3 is a splice
variant of COX-1,which retains intron one and has a frame shift mutation, thus some
prefer the name COX-1b or COX-1 variant(COX-1v) COX-3 could play a role in
fever and pain processes(15).

COX-1 is constitutively expressed, widely distributed and has "housekeeping"

function. It is of particular importance in maintaining gastric mucosal integrity, renal
function and homeostasis(19).In contrast,COX-2 is induced in setting of inflammation
by cytokines and inflammatory mediators or physiological stress (20,21).

In addition constitutive expression of COX-2 was discovered in the kidneys, uterus,

ovary(22), the vascular system, in wound healing, the lung, bone(23), pancreatic islets of
mice(24), heart(25), the spinal cord(26), and in the brain(27). The cyclooxygenases
activities and their inhibitors are shown in table (1-1).

The analgesic effects of NSAIDs are ascribed primarily to COX-2 inhibition, whereas
several adverse effects are believed to be mediated by COX-1 inhibition. The
inhibition of COX-1 prolongs the bleeding time due to an inhibition of TXA2

21
synthesis from platelets and may lead to the formation of gastric ulcerations due to
PGE1 inhibition. COX-1 inhibition may, under certain circumstances, decrease renal
glomerular filtration rate. COX-2 selectivity may theoretically attenuate such adverse
effects(10).

Tab.(1-1) Cyclooxygenases Activities and their Inhibitors(28)

COX Expression Function Inhibitors

organ pain, platelet

NSAIDs including
COX 1 constitutively function, stomach
aspirin
protection

induced by growth
factors, Inducible
neurotransmitters, COX2: inflammation,
NSAIDs, COX 2
inflammatory pain and fever
COX 2 inhibitors including
cytokines, oxidative Constitutive
celecoxib
stress, COX2: synaptic
injury. Constitutively plasticity
in brain and kidney

COX 3 Acetaminophen (no

pain pathways, not
constitutively, high in GI problems, great
inflammation
brain and heart fever reducer),
pathways
some NSAIDs

22
1.6 Cyclooxygenase structure

The cyclooxygenase enzyme existed as a homo dimer with 587 amino acid residues
per chain. Upon investigation of the homodimer, it was determined that within a
single monomer of this structure, three domains existed; the N-terminal epidermal
growth factor domain, the membrane binding motif and the C-terminal catalytic
domain, there are five disulfide

bonds which contribute to the interface binding of the two individual monomers to
complete the enzyme(29).

In terms of their molecular biology, COX-1 and COX-2 are of similar molecular
weight, approximately 70 and 72 kDa, respectively, with just over 600 amino acids,
of which 63% of the amino acids are in an identical sequence (30).

Various important residues that affect the catalytic activity of these enzymes, include:
Tyr 385 in its radical form is thought to be responsible for abstracting a proton from
arachidonic acid during its conversion to PGG2 (29). The role of the active site Arg 120
in anchoring arachidonic acid and NSAIDs to the active site via electrostatic forces
which explains why an acidic functional group capable of becoming anionic at
physiologic Ph is an essential NSAID structural feature(13), the important exchange of
Ile for Val at the ‘‘lip’’ of the allosteric pocket of COX-1 and COX-2, respectively,
coupled with a distinctly different orientation of Tyr 355 (31), the smaller Val residues
(particularly Val 523) open up the COX-2 allosteric pocket for therapeutic
manipulation. In contrast, the bulkier Ile residues and the uninviting conformation of
Tyr 355 in COX-1 prohibit binding (and, therefore, enzyme inhibition) of
diarylheteroaromatic NSAIDs(13), Substitution of Ile 434 for Val 434 allows the side
chain of Phe 518 to move back and make some extra space, this side pocket allows
for interactions with Arg 513. So, within the hydrophilic side pocket of COX-2 the
oxygen of the sulfonamide or sulfone group interacts with His 90, Arg 513 and forms
hydrogen bonds(32,33). As shown in fig.(1-3)

23
Fig. (1-3) COX-1 and COX-2 binding sites

24
Carboxylic acid moieties are by far the most common, although an acidic enol group
fulfills the anionic moiety requirement in oxicams (34), examples are piroxicam (1) and
meloxicam (2)(35).

A ratio of aromatic and/or lipophilic active site residues explains how two aromatic
NSAID moieties enhance anti-inflammatory potency, both by augmenting COX
receptor affinity through hydrophobic or ʌ electron requiring van der Waals
interactions, and by providing the lipophilicity that propels the drug across biological
membranes to reach those receptors. The 3-dimensional models of COX active and
allosteric site residues, document that these amino acids are out of plane with one
another, which demands a non-coplanar orientation of NSAID aromatic moieties for
optimal binding affinity(34).The 25-fold anti-inflammatory activity enhancement of
meclofenamic acid (3) over mefenamic acid (4)(13), due to a more consistently non-
coplanar orientation of aromatic rings and enhanced lipophilicity were provided by
the 2 large ortho-chlorine atoms. The non-coplanar orientation of rings is important
for optimal binding and to the ratio of aromatic/ aliphatic COX active-site
(36)
residues.

25
1.7 Pharmacological Inhibition of COX Enzymes..

The "classical" nonselective NSAIDs bind to both COX-1 and COX-2, interacting
with the hydrophobic channel of the COX iso-enzymes(37). The binding mechanism of
selective COX-2 inhibitors involve two reversible steps with both COX-1 and COX-
2, but the selectivity for COX-2 is due to another step that is slow and is only seen in
the inhibition of COX-2 but not COX-1. The slow step has been attributed to the
presence of the sulfonamide or sulfone that fits into the side pocket of COX-2. The
first step accounts for the contact of the inhibitor with the gate of the hydrophobic
channel (called the lobby region). The second step could account for the movement
of the inhibitor from the lobby region to the active site of the COX enzyme. The last
step probably represents repositioning of the inhibitor at the active site which leads to
strong interactions of the phenylsulfonamide or phenylsulfone group of the inhibitor
and the amino acids of the side pocket (38). Coxibs are selective agents because they
bind to COX-1 poorly and in a rapidly reversible manner, where as they bind to
COX-2 more tightly. Preferential inhibition of COX-2 is thought to be due to the
additional space in the COX-2 hydrophobic channel, as well as to the presence of a
side pocket in the channel. This side pocket can discriminate the coxibs from
nonselective agents based on the different overall structures of these agents(37), as
shown in fig.(1-4).

26
Fig.(1-4) Comparison of the NSAIDs binding sites of COX-1 and COX-2.(39)

27
1.8 Claasification of Non-steroidal Anti-inflammatory Drugs

NSAIDs are classified in different ways:

1.8.1 Reversibility of cyclooxygenases inhibitors:

COX-inhibitors can be claaaassified according to reversibility to;

1.8.1.1 Irreversible cyclooxygenase inhibitor: where aspirin (5) is the only

medically used agent covalently and irreversibly acetylates the enzyme COX-1, so
inactivating it, while acetylating of the enzyme COX-2 by aspirin will not
inactivating this enzyme but leading to modification of their products. The 2-(hex-1-
ynylthio) phenyl acetate (6) is a selective irreversible COX-2 inhibitor which has the
ability to inactivate this enzyme(40).

1.8.1.2. Reversible cyclooxygenase inhibitor: this group includes all NSAIDs (non
selective and selective COX inhibitors)(41).

1.8.2 Basic chemical structures

NSAIDs are classified according to their basic chemical structures into;

1.8.2.1 Arylacetic Acid Analogues

It has been observed that organic compounds which bear some sort of resemblance
either with respect to their structural features or functionally often display similar
biological actions(9,42).

A few potent analogues are belonging to this class e.g. : diclofenac (7)(43).

28
1.8.2.2 Heteroarylacetic Acid Analogues

This constitutes an important class of non-steroidal anti-inflammatory drugs which

have gained recognition in the recent past. A few classical examples of this group
are(9,42), indomethacin (8)(43) , Etodolac (9)(42) .

1.8.2.3 Arylpropionic acid analogues

Like the aryl acetic acids the aryl propionic acid analogues also exhibit potent anti-
inflammatory properties besides usual antipyretic and analgesic characteristics (9,42). A
few examples of this category of compounds are presented here, ibuprofen (10),
fenoprofen (11)(35), flurbiprofen (12), ketoprofen (13)(44).

29
1.8.2.4 Naphthalene Acetic Acid Analogues

The recent intensive quest for non-steroidal anti-inflammatory drugs and aryl acetic
acids in particular offer a brighter scope that the naphthalene acetic acid analogues
might turn out to be the leading compounds of an extensive series of promising
clinical agents(9). Example : Naproxen (14)(35,43).

1.8.2.5 Salicylic Acid Analogues

The salicylate are derivatives of 2-hydroxybenzoic acid (salicylic acid). Salicylic acid
was used medicinally as the sodium salt but replaced therapeutically in the late 1800s
by the acetylated derivative, acetylsalicylic acid (ASA) or aspirin(42).

The derivatives of salicylic acid are of two types:

30
Type I represents those that are formed by modifying the carboxyl group (e.g., salts,
esters, or amides). Type II , represents those that are derived by substitution on the
hydroxyl group of salicylic acid(35).

Therapeutic utility is enhanced by esterification of the phenolic hydroxyl group as in

aspirin, and by substitution of a hydrophobic group at C-5 as in diflunisal (15)(42).

A good number of salicylic acid analogues have also been found to possess anti-
inflammatory actions, e.g. aspirin (5)(35), salsalate (16), sodium salicylate,
salicylamide (17), benorilate (18), choline salicylate, flufenisal(9).

1.8.2.6 Pyrazolones and Pyrazolodiones

This class of agents is characterized by the 1-aryl-3,5-pyrazolidinedione structure.

The presence of a proton which is situated to two electron withdrawing carbonyl
groups renders these compounds acidic. The pKa for phenylbutazone (19)(44) is 4.5 .
Oxyphenbutazone (20) is a hydroxylated metabolite of phenylbutazone, which are
used primarily in the treatment of rheumatoid arthritis and osteoarthritis (42).

Drugs like phenazone, aminophenazone (aminopyrine), dipyrone, phenylbutazone,

oxyphenbutazone, sulfinpyrazone, belonging to this category, besides their
antipyretic-analgesic action, have also been reported to exhibit anti-inflammatory
properties(9).
31
1.8.2.7 Anthranilates

These agents are considered to be N-aryl substituted derivatives of anthranilic acid

(21) which is itself a bio-isostere of salicylic acid. These include meclofenamic acid
(3) and mefenamic acid (4). The most active fenamates have small alkyl or halogen
substituent at the 2',3' and/or 6' position of the N-aryl moiety. Among the
disubstituted N-aryl fenamates the 2',3'-derivatives are most active suggesting that the
substituent at the 2',3'-positions serve to force the N-aryl ring out of co-planarity with
the anthranilic acid. Hence this steric effect is proposed to be important in the
effective interaction of the fenamates at their inhibitory site on the
(42)
cyclooxygenase .

1.8.2.8 Oxicams (Enolic Acids)

Oxicams, Piroxicam (1) and Meloxicam (2) are characterized by the 4-

hydroxybenzothiazine heterocycle. The acidity of the oxicams is attributed to the 4-
32
OH with the enolate anion being stabilized by intramolecular H-bonding to the amide
N-H group. Also, the presence of the carboxamide substituent at the 3-position of the
benzothiazine ring contributes toward acidity by stabilizing the negative charge
formed during ionization (resonance stabilization). Although these compounds are
acidic (pKa = 6.3), they are somewhat less acidic than carboxylic acid NSAIDs. Yet
the oxicams are primarily ionized at physiologic pH and acidity is required for COX
inhibitory activity(42).

1.8.2.9 Gold Compounds

In general, gold compounds either suppress or prevent, but do not cure arthritis and
synovitis. The use of organic gold derivatives for the treatment of rheumatoid arthritis
was first reported. However, (the mono valent ) bring symptomatic relief to
rheumatoid arthritis in patients. A few classical examples of this class of
(9)
compounds, Example : auranfin (22) .

(22)

1.8.3 selectivity of cyclooxygenase inhibitors:

NSAIDs are classified according to their selectivity to inhibit COX enzymes into;

1.8.3.1 Classical NSAIDs

The classical COX inhibitors are not selective and inhibiting all types of COX, and
cause peptic ulceration and dyspepsia. It is believed that such lack of selectivity
33
(inhibition of prostaglandin synthesis by COX-1), and direct irritation of the gastric
mucosa (many NSAIDs are acids), are caused the ''dual-insult'' of NSAIDs.
Prostaglandins have a protective role in the gastrointestinal tract, preventing acid-
insult to the mucosa(45).

1.8.3.2 Newer NSAIDs

The discovery of a second cyclooxygenase isoform (COX-2) in the early 1990

created a new scenario where selective COX-2 inhibitors(46) represented a second
generation of non-steroidal anti-inflammatory drugs (NSAIDs), without undesirable
side effects of gastrointestinal (GI) damage (47).

Because COX-2 is usually specific to inflamed tissue, there is much less gastric
irritation associated with COX-2 inhibitors, with a decreased risk of peptic
ulceration(48). Selective COX-2 inhibitors differ from traditional NSAIDs in two
major ways, Coxibs are less likely to result in NSAID-induced gastropathy, and they
do not inhibit platelet function(49). As a result, the major benefit of coxibs are the
reduction in gastric ulcer formation and bleeding from those ulcers (50). Another
benefit of the platelet sparing coxibs is their use as analgesic and anti-inflammatory
agents in situations in which bleeding may limit the use of the traditional NSAIDs,
such as in trauma and surgical procedures(51-53).

However, it is now clear that both COX-1 and COX-2 contribute to mucosal
defense(54). Selective COX-2 inhibitors elicit less clinically significant GI damage and
bleeding than conventional NSAIDs(55,56). In addition, several other clinical uses of
selective COX-2 inhibitors have been investigated(57), some of these indications
including the treatment of the neurodegenerative disease like Alzheimer (58) and
Parkinson disease(59) are now under clinical trials to validate the therapeutic
possibilities of COX-2 inhibitors(60). Moreover studies show that the COX-2 enzyme
would be an interesting target in the treatment of some cancers(61).

34
1.9 A common Pharmacophoric Groups of Selective COX-2 Inhibitors

Enormous effort was expended in the development of NSAIDs between the 1960s
and 1980s so there were numerous pharmacophores to test when COX-2 was
discovered. The first breakthrough came with reports that the diarylheterocycle Du-
P697 (23) and the acidic sulfonamide NS-398 (24) were anti-inflammatory but non-
ulcerogenic(62).

So, selective inhibition of COX-2 can be achieved with the compounds of the general
form of arylmethylsulfonyl and arylmethyl sulfonamides as shown in the above
mentioned compounds respectively(63).

Structure-activity studies indicate that a cis-stilbene moiety containing 4-

methylsulfonyl or sulfonamide substituent in one of the phenyl rings is required for
COX-2 specificity. The oxidation state of the sulfur in the methylsulfon moiety is
required for selective COX-2 inhibition because the sulfoxide or sulfide are inactive
or non-selective. The ring system that is fused to the stilbene framework has been
extensively manipulated to include heterocyclic ring(62), example celecoxib (25)(60)
and valdecoxib (26)(64).

35
In place of the carboxyl group of the non-steroidal anti-inflammatory acids, the
structure of celecoxib contains a sulfonamide group, while DuP-697 contains
methylsulfone group, the sulfur contains phenyl ring of these drugs binds into the
side pocket of the cyclooxygenase catalytic channel of COX-2 but interact weakly
with the active site of COX-1(65). The SO2NHCOCH3 moiety is a novel COX-2
pharmacophore that also has the potential to serve as a prodrug moiety to the
respective SO2NH2 COX- 2 pharmacophore, as in parecoxib, which is a prodrug for
valdecoxib, is 105 - 106 more reactive acetylating agent of enzyme serine hydroxyl
groups than simple amides (66).
(44)
Nimesulide (27) [N-(4-nitro-2-phenoxyphenyl)methane sulfonamide] is a
selective COX-2 inhibitor, investigated the physiochemical properties and structural
analysis of nimesulide-based analogs for COX inhibitory activity and found that N-
methylation on the sulfonamide group of nimesulide, compound (28) Fig.(1-5)
caused complete loss of COX-2 inhibitory activity due to deprotonation of N atom.
Thus, the N-H bond seemed to be critical for maintaining COX-2 inhibitory activity,
while, replacement of nitrophenyl methyl sulfonamide group with trifluoromethyl
pyridyl group (29) enhanced the COX-2 inhibitory activity(18).

(28) (27) (29)

Fig.(1-5) Structural modification of Nimesulide

36
1.10 Cyclooxygenase Activity is Important for Efficient Replication of
. Hepatitis Virus at An Early Stage of Infection

virus infections often cause acute inflammatory responses, which are mediated by
several cellular effectors and soluble factors. Although these responses have an
important protective role, they may also have deleterious effects on the host. PGs are
important regulators of this inflammatory reaction. PGs from the E series, such as
PGE2, also exhibit immunomodulatory activities, preventing hyper activation of the
innate cellular immunity. Furthermore, they can inhibit the secretion of gamma
interferon, a cytokine with antiviral activity(67). A direct role for COXs and PGs in
controlling viral replication has been described for a wide range of virus infections,
but their actions appear to be dependent on both the virus and cell type (68). For
instance, COXs and/or PGs are required for efficient replication of herpes viruses (69-
73)
, bovine leukemia virus(74), and rotavirus(75). On the other hand, COX/PGs
negatively affect adenovirus replication(76).

1.11 Significance of COX-2 Activity in Brain Diseases

COX- 2 expression in brain has been associated with pro-inflammatory activities,

thought to be instrumental in neurodegenerative processes of several acute and
chronic diseases. However, two major aspects should be borne in mind when
considering the significance of COX-2 activity in brain diseases. First, COX-2 is
expressed under normal conditions and contributes to fundamental brain functions
such as synaptic activity, memory consolidation, and functional hyperemia. Second,
‘‘neuro inflammation’’ is a much more controlled reaction than inflammation in
peripheral tissues, and in many cases is triggered and sustained by activation of
resident cells, particularly microglia. In degenerative diseases, it mainly occurs in the
absence of blood-borne infiltrating cells and is sustained by activated glial cells,
particularly microglia(77). Neurons are particularly susceptible to damage caused by
free radicals generated through COX-2 peroxidase activity, whereas glial cells are
more resistant. There are several evidence of a direct role of COX-2 in
37
neurodegenerative events, major human neurological diseases, multiple sclerosis(78),
amyotrophic lateral sclerosis(79), Parkinson disease(59,80), Creutzfeldt-Jakob disease(81),
and Alzheimer disease(58).

1.12 Cyclooxygenase Inhibitors in Cancer Chemotherapy

The COX-2 enzyme is responsible for the production of various PGs in pathological
states including inflammation and cancer. Increasing number of evidences suggest
that COX-2 expression is associated with aggressive tumor growth. Significant
studies have shown that COX-2 selective inhibitors exert anticancer effect through
prevention of angiogenesis, induction of apoptosis and modulation of immune
response, although COX-2 -independent pathways are involved. Thus, COX-2 has
become an important molecular target and the accumulation of data suggests that
COX-2 inhibition is an exciting anticancer strategy(18).

The involvement of COX in cancer development was first evidenced by

pharmacological analysis of PGs in different human cancers(82). Prostaglandin E2
(PGE2) exert especially carcinogenic effects in the human body. It was found that
premalignant lesions and established cancers produce excessive quantities of PGE2
and this enhances tumor cell growth and increases tumor invasiveness(83). A
significant relation between over expression of COX-2 and survival of patients with
breast, colon, gastric, and lung cancers has been reported(84).

A multitude of evidences supports the COX-2 contribution to cancer by increase in

the production of PGs, carcinogenesis, inhibition of apoptosis, angiogenesis and
invasion of cancer cells(85). The published data on the expression of COX-2 in
different human tumors and normal tissues are summarized in Table (1-2).

38
 Cancer site/organ COX-2 expression in COX-2 expression in

normal tissues (%) malignant lesion (%)

Head and neck Absent 100

Breast (ductal carcinoma) 23 40

Pancreatic Absent 47

Colorectal Absent 85–90

Bladder Absent 75–85

Skin/Melanoma Absent 83

Table (1- 2) COX-2 expression in human normal tissues and malignant

tumors(18).
COX-2 catalyze the conversion of procarcinogens to carcinogens to initiate tumor
formation. The peroxidase part of the COX oxidizes aromatic and heterocyclic
amines to dihydrodiol derivatives and it can be induced by procarcinogens (18).
Moreover, by-products of AA metabolism, like malondialdehyde are chemically
highly reactive and form adducts with DNA(86,87). Furthermore, COX-2 not only
regulate the production of PGs but also regulates other angiogenic promoters such as
(EGF), (VEGF), (FGF) and cytokines (TNF-Į), (IL)-1ȕ and IL-2(88). COX-2
expression also favors adhesion of cancer cells to endothelial cells and selective
COX-2 inhibitors were found to inhibit metastasis(89).

NS-398 (24) is a selective COX-2 inhibitor and has no activity on COX-1 activity,
investigation of the effect of NS-398 on invasion of colon cancer cell show that the
invasion of these cells was significantly suppressed 72 hr after NS-398 treatment at
the concentrations of 0.1, 1.0, and 10—M with 22.74,42.35,and 58.61% inhibitions,
respectively.

39
However, NS-398 (24) had no significant effect on cell viability at the experimental
concentrations, indicating that anti-invasive effect of NS-398 does not result from its
cytotoxicity(90,91).

SC-236,(30),(4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-
yl]benzenesulfonamide) is a selective COX-2 inhibitor that is highly selective and
potent for COX-2 with antitumor properties(92).

Nimesulide (27) can induce apoptosis in liver and lung cancer cells; it also
suppressed mammary gland carcinogenesis in rats. In all the structures used for the
library synthesis, sulfonamide or carboxamide functionality ortho to aryloxy group
was the basic motif for the anticancer activity, Fig.(1- 6). These compounds showed
anticancer effects on various breast cancer cell lines via COX-2-dependent and COX-
2-independent pathways(18).

40
 Fig. (1-6)

It is found that methanesulfonyl or aminosulfonyl moieties are important for their

optimal COX-2 inhibitory and anti-proliferative activities. Replacement of sulfonyl
moieties with other substituent such as methyl or chloro has reduced COX-2 as well
as anti-proliferative activities Thus, both aminosulfonyl and methanesulfonyl
moieties are important for optimal COX-2 inhibition(93).

1.13 Effect of Selective Cyclooxygenase-2 Inhibitors in Treatment of

. Glucose-Stimulated Insulin Secretion

Both COX-1 and COX-2 were localized in the insulin-producing ȕ cells. PGE2 is the
major prostanoid derived from COX-1 and COX-2. PGE2 inhibits glucose-stimulated
insulin secretion .

Therefore, reducing PGE2 production in pancreatic islets would be predicted to

enhance glucose-stimulated insulin secretion. Although both COX-1 and COX-2 are
expressed in normal murine islets, only COX-2 inhibition contributes to basal PGE2
41
production in isolated islets, it is shown that selective COX-2 inhibitors induce a 4-
fold increase in insulin secretion by the islet in the presence of 20 mM glucose, On
the other hand, selective COX-1 inhibitor did not improve insulin secretion by the
islets isolated from B6 mice. This finding could explain why selective inhibition of
COX-2, but not COX-1, enhanced glucose-stimulated insulin secretion in the present
in vivo and in vitro examinations using B6 mice. Therefore, a reduction in PGE2
production in pancreatic islets by selective COX-2 inhibition may contribute to an
amelioration of pancreatic ȕ-cell dysfunction such as poor glucose-stimulated insulin
secretion observed in B6 mice (94).

1.14 Selective Cox-2 Inhibitors in Endometriosis

Endometriosis, a common gynecologic disorder, accounts for infertility and pelvic

pain in 10%–15% of women of reproductive age. Recently, it was detected that
significantly higher expression of COX-2 in glandular epithelial cells from
endometriotic lesions than in cells from matched ectopic endometrium, regardless of
menstrual cycle stage. There are now numerous reports that COX-2 is involved in
development and progression of a variety of neoplasm and that treatment with COX-
2 inhibitor can lead to a marked reduction in tumor growth. Although endometriosis
is not a neoplasm, they hypothesized that over expression of COX-2 may be involved
in the patho-physiology of endometriosis and that a COX-2 selective inhibitor could
be effective in its prevention or treatment. Recently, Williams et al. demonstrated that
COX-2 may regulate intratumoral vascular density, and one mechanism for the
antineoplastic effects of COX-2 inhibitor use may be through inhibited production of
proangiogenic factors by tumor and stromal cells. One potential mechanism may be
that COX-2 inhibitors have antiangiogenic effects on ectopic implants. Aberrant
aromatase expression may be one of the most important molecular mechanisms in the
pathogenesis of endometriosis. COX-2 inhibitors may inhibit the production of
aromatase through decreased production of PGE2(95).

42
1.15. Aim of the Work:

The aim of this work is to design, synthesize and preliminary pharmacological

evaluation of new non-steroidal anti-inflammatory agents (compounds I-V) with
expected selectivity against COX-2 enzyme.

The structures and numbers of these compounds are:-

compound I

compound II

compound III compound IV

Compound V

43
1.16. Strategy of the Work:

The direction of the present work is to design and synthesize potential non-steroidal
anti-inflammatory agents that are derivatives of diclofenac, which is a well-known
non-steroidal anti-inflammatory drug , it is available with low cost and the chemical
structure of it has no additional functional groups that may undergo conversion to
another intermediates throughout the overall reaction, so it will undergo straight line
reaction. These newly synthesized compounds may represent potent anti-
inflammatory agents and exhibit expected selectivity towards COX-2 enzyme due to
the properties of the selected groups that linked to the Į-carbon of the diclofenac as
followings:

1. In accordance with the current data on selective COX-2 inhibitors, one aromatic
ring must be substituted with a methyl sulfonyl or sulfonamide moiety in the para-
position for good COX-2 selectivity. These structural pre-requisites for COX-2
selectivity can mainly attributed to the occupancy of the side pocket, and
hydrogen bonding of the sulfone part with Arg 513 in COX-2 enzyme and
methylsulfonamide group present in many selective COX-2 inhibitors(63). These
characters shown in compounds I and III.
2. N-acyl derivatives are more potent as acylating agents for serine hydroxyl moiety
in the COX enzymes than the unsubstituted amide and also more selective (66), as
shown in compound II.
3. The heterocyclic ring 2-aminopyridine selected as it is present in a well-known
NSAID piroxicam.
4. The meloxicam which is a selective NSAID has 2-amino-4-methylthiazole
heterocyclic ring , so accordingly the 2-aminothiazole was selected and
incorporated in diclofenac to give compound V.

44
 CHAPTER TWO

Materials and methods

2.1: Chemicals and Equipments:

2.1.1: Chemicals:
The specific chemicals used in this work are listed in table (2-1) with their
suppliers.

Table (2-1): Chemicals and their suppliers.

Materials Company Country

Acetic acid BDH England

2-Aminopyridine Fluka Switzerland
The general chemical&
2-Aminothiazole England
pharmaceutical CO.LTD
Anhydrous magnesium
Merck Germany
sulfate
South
Cyclohexane Chem-supply
Asturalia
Dichloromethane GCC U.K
Diclofenac Sodium Cox. Tx. LT Chem China
Dimethylformamide Biosolve Netherlands
Ethylacetate Riedel-Dehaen Germany
Glacial acetic acid BDH England
N-bromosuccinamide Aldrich Germany
N-hexane Riedel-Dehaen Germany
Nickel chloride Fluka Switzerland
Nitric acid BDH England

45
 Petrolium ether BDH England
Hopkin and Wiliams
Propane diol England
Searle
Silver nitrate Riedel-Dehaen Germany
Sulfacetamide BDH England
Sulfanilamide Fluka Switzerland
Tetrahydrofuran Fluka Switzerland
Thionyl chloride Merck Germany

All the solvents and materials used were of Analar type and used without further
purification.

2.1.2: Equipments and Instruments:

The epuipments and their suppliers that are used in this work are listed in table (2-2).

Table (2-2): Equipments and instruments and their suppliers.

Equipment Company Country

Chiller Julabo VC (F30) GMBH Germany

Precisa instrument-
Electrical balance Switzerland
AG
Electrical melting point
Electrothermal 9300 USA
apparatus

Elemental analyzer 1106 Carlo Erba Italy

FTIR-spectrophotometer Shimadzu Japan

Hot plate stirrer Daihan lab. Tech. Korea

Oven Astell Hearso H2272 England

46
2.2. Methods of Identification:

The general methods used for the identification of the synthesized compounds
includes:

2.2.1. Thin Layer Chromatography:

Ascending thin layer chromatography was run on DC-Kartan SI Alumina gel 0.2 mm
to check the purity and progress of reactions. The identification of compounds was
done using iodine vapor and the chromatograms were eluted by the following solvent
systems:

A/ n-hexane Ethyl acetate Acetic acid (7: 2.5: 0.5) (96)

B/ THF ether cyclohexane (4: 4: 2) (97)

2.2.2. Melting Points:

Electro-thermal melting point apparatus was used to determine the melting points
reported in this work, and were uncorrected.

2.2.3. Infrared Spectra:

Determination of infrared spectra by using FTIR-spectrophotometer, were done at the

College of Science-Kufa University, and the determination of the spectra taken were
performed as KBr discs.

2.2.4. Elemental Microanalysis (CHONS):

The CHNOS microanalysis of the synthesized final products were done in Cleveland
clinical foundation learner research institute-France, by using Carlo Erba elemental
analyzer.

47
2.2.5.Chemical tests: Different chemical tests were performed on the target
compounds and their intermediates:

2.2.5.1 Sodium fusion test;

This test used to indicate the presence of bromine in intermediate Ic, and it was
carried out as follow; Intermediate Ic (10 mg) and a freshly cut, pea-size (about 50
mg) piece of sodium metal, were placed in a small test tube, with precaution. The
test tube was heated until the bottom is a glowing red. Allowing the glowing and
charred to cool to room temperature, then a few drop of ethanol was added, with
stirring, to ensure decomposition of all remaining elemental sodium, repeating until
no further bubbles of hydrogen gas are evolved. Water (2ml) was added to the
solution, then heating and filtering the solution. In the hood acidify the fusion filtrate
(2ml) with 5% nitric acid in a small test tube, boiling gently for a few minutes.
Cooling the solution and add then three drops of 0.1M silver nitrate solution to the
liquid. The formation of a pale yellow solid indicate the presence of bromine in the
compound(98).

2.2.5.2 Carboxylic acid test; Few milligrams of the tested compound was dissolved in
methanol, then added to a test tube containing saturated solution of sodium
bicarbonate (1ml). The evolution of bubbles indicate the presence of carboxyl group
which liberate the CO2 gas(99).

2.2.5.3 Sulfonamides test; Compound I (0.0625g) was fused with powdered sodium
hydroxide (0.375g) by heating with bunsen burner. Pink litmus paper was placed in
the top of the test tube to test the liberation of ammonia. The test tube was cooled and
enough distilled water was added to dissolve the sample, then acidify the solution
with 2M HCl. Suspended a filter paper that has been covered with a paste of nickel
hydroxide (that prepared from nickel chloride) over the test tube. Gently warm the
test tube to speed up the production of SO2 gas. The formation of gray-black paste
give evidence about the presence of sulfonamide group (99).

48
2.3. Methods of Synthesis

2.3.1.Conversion of diclofenac salt to its acid (100).

Diclofenac acid was prepared by using an equivalent of 2N hydrochloric acid

solution.

2.3.2.Protection of the carboxyl moiety of diclofenac acid (101).

Methyl ester of the diclofenac acid in this work was prepared according to a method
of Schwarz et.al.

2.3.3.Bromination of the protected diclofenac acid (64).

N-bromosuccinamide was used for bromination of the Į-carbon of diclofenac methyl

ester.

2.3.4.Amination of brominated intermediate (102).

Nucleophilic substitution reaction, was performed by using sulfonamide derivatives

and different heterocyclic rings.

2.3.5.Deprotection of the esterified carboxyl moiety(100).

The removal of the methyl ester moiety was performed using 1.2 equivalent of 2N
sodium hydroxide solution.

2.4 Chemical Synthesis:

2.4.1. Conversion of diclofenac salt to 2-[2-(2,6-dichlorophenylamino)

phenyl]acetic acid (Intermediate Ia):

49
Diclofenac sodium (2g, 6.28mmol), was dissolved in a minimum volume of ethanol
99 %: THF (3:1) mixture. The solution was cooled to 18o C, while stirring for 10
minutes, then HCl (2N, 3.1ml, 6.28mmol) was added, followed by addition of excess
cold water, the acid was precipitated then filtered and dried to give intermediate Ia
which was used in the next step without further purification. The percent yield,
physical data, and Rf values were given in table (3-1). IR spectrum for this
compound was shown in figure (3-2).

2.4.2 Synthesis of methyl 2-[2-(2,6-dichlorophenylamino)phenyl] acetate

(Intermediate Ib):

A suspension of intermediate Ia (1g, 3.37mmol) in absolute methanol (50ml), was

cooled down to -15o C, then thionyl chloride (0.25ml, 3.37mmol) was added drop
wise, (the temperature should be kept below -10o C). Then the reaction mixture was
kept at 40 o C for three hours, followed by refluxing for three hours, and left at room
temperature overnight. The solvent was evaporated to dryness, re-dissolved in
methanol and evaporated. The process was repeated several times to ensure complete
removal of thionyl chloride. The residue was collected and re-crystallized from
methanol-ether. The percent yield, physical data and Rf values were given in table (3-
1). IR spectrum for this compound was shown in figure (3-3).

2.4.3 Synthesis of methyl 2-bromo-2-[2-(2,6-dichlorophenylamino)

phenyl]acetate (Intermediate Ic):

50
Intermediate Ib (1g, 3.23mmol) was dissolved in methylene chloride (15ml), then
NBS (0.57g, 3.23mmol) was added gradually with continuous stirring. The reaction
was allowed to proceed at room temperature with stirring for three hours, then the
solvent was evaporated. Ether was added to the residue, and then filtered, the filtrate
is dried to give intermediate Ic. The percent yield, physical data and Rf values were
given in table (3-1). IR spectrum for this compound was shown in figure (3-4).

2.4.4. Synthesis of methyl 2-[2-(2,6-dichlorophenylamino)phenyl]-2- (4-

sulfamoylphenylamino)acetate (Intermediate Id):

A mixture of intermediate Ic (1g, 2.57mmol), and sulfanilamide (0.44g, 2.57mmol)

was placed in round flask, then dissolved with ethanol 99%:DMF (50:50) mixture
(30ml). The reaction mixture was refluxed gently for three hours. The solvent was
evaporated, the residue was dissolved in ethyl acetate, washed with NaOH (5%, 3X),
filtered over anhydrous magnesium sulfate, the filtrate was evaporated to give
intermediate Id. The percent yield, physical data and Rf values were given in table (3-
1). IR spectrum for this compound was shown in figure (3-5).

2.4.5. Synthesis of methyl 2-[4-(N-acetylsulfamoyl)phenylamino]-2[2-

(2,6-dichlorophenylamino)phenyl]acetate (Intermediate IIa):

51
A mixture of intermediate Ic (1g, 2.57mmol), and sulfacetamide (0.55g, 2.57mmol)
was placed in round flask, then dissolved with ethanol 99%: DMF (50:50) mixture
(40ml). The reaction mixture was refluxed gently for three hours, then it was worked
up as described in section 2.4.4, to liberate intermediate IIa. The percent yield,
physical data and Rf values were given in table (3-1). IR spectrum for this compound
was shown in figure (3-6).

2.4.6. Synthesis of methyl 2-[2-(2,6-dichlorophenylamino)phenyl]-2 [4-(N-

methylsulfamoyl)phenylamino]acetate ( Intermediate IIIa):

A mixture of intermediate Ic (1g, 2.57mmol), and 4-amino-N-methylbenzene

sulfonamide* (0.47g, 2.57mmol) was placed in round flask, then dissolved with
ethanol 99%:DMF (50:50) mixture (40ml). The reaction mixture was refluxed gently
for three hours, then it was worked up as described in section 2.4.4, to liberate
intermediate IIIa. The percent yield, physical data and Rf values were given in table
(3-1). IR spectrum for this compound was shown in figure (3-7).

2.4.7. Synthesis of methyl 2-[2-(2,6-dichlorophenylamino)phenyl]-2-

(pyridine-2-ylamino)acetate (Intermediate IVa):

52
A mixture of intermediate Ic (0.5g, 1.28mmol), and 2-aminopyridine (0.12g,
1.28mmol) was placed in round flask, then dissolved with ethanol: DMF (50:50)
mixture (40ml). The reaction mixture was refluxed gently for three hours, then it was
worked up as described in section 2.4.4, to liberate intermediate IVa. The percent
yield, physical data and Rf values were given in table (3-1). IR spectrum for this
compound was shown in figure (3-8).

2.4.8. Synthesis of methyl 2-[2-(2,6-dichlorophenylamino)phenyl]-2-

(thiazol-2-ylamino)acetate (Intermediate Va):

A mixture of intermediate Ic (0.5g, 1.28mmol), and 2-aminothiazol (0.128g,

1.28mmol) was placed in round flask, then dissolved with ethanol 99%:DMF (50:50)
mixture (20ml). The reaction mixture was refluxed gently for three hours, then it was
worked up as described in section 2.4.4, to liberate intermediate Va. The percent
yield, physical data and Rf values were given in table (3-1). IR spectrum for this
compound was shown in figure (3-9).

2.4.9. Synthesis of 2-[2-(2,6-dichlorophenylamino)phenyl]-2-(4-

sulfamoylphenylamino)acetic acid (Compound I):

53
Intermediate Id (0.3g, 0.62mmol) was dissolved in minimum volume of ethanol
99%:THF (7:1) mixture. The solution was cooled down 18 o C, and then sodium
hydroxide (2N, 0.37ml, 0.75mmol ) was added drop wise, with continuous stirring
over a period of 30 minutes. Stirring was continued at 18 oC for additional three
hours. The reaction mixture was acidified with HCl (2N, 0.37ml, 0.75mmol), then
excess of cold water was added and the acidic compound was precipitated, and
filtered thendried to give compound I. The percent yield, physical data and Rf values
were given in table (3-1). IR spectrum for this compound was shown in figure (3-10).
Elemental microanalysis for this compound was given in table (3-2).

2.4.10. Synthesis of 2-[4-(N-acetylsulfamoyl)phenylamino]-2-[2-(2,6-

dichlorophenylamino)phenyl]acetic acid (Compound II):

Intermediate IIa (0.25g, 0.47mmol) was dissolved in minimum volume of ethanol

99%:THF (20:1) mixture. The solution was cooled down 18 o C, and then sodium
hydroxide (2N, 0.28ml, 0.56mmol) was added drop wise, with continuous stirring
over a period of 30 minutes. Then the reaction mixture was worked up as described in
section 2.4.9, to give compound II. The percent yield, physical data and Rf values
were given in table (3-1). IR spectrum for this compound was shown in figure (3-11).
Elemental microanalysis for this compound was given in table (3-2).

54
2.4.11. Synthesis of 2-[2-(2,6-dichlorophenylamino)phenyl]-2-[4-(N
methylsulfamoyl)phenylamino]acetic acid (Compound III):

Intermediate IIIa (0.26g, 0.52mmol) was dissolved in minimum volume of ethanol

99%:THF (20:1) mixture. The solution was cooled down 18 o C, and then sodium
hydroxide (2N, 0.31ml, 0.63mmol) was added drop wise, with continuous stirring
over a period of 30 minutes. Then the reaction mixture was worked up as described in
section 2.4.9, to liberate compound III. The percent yield, physical data and Rf
values were given in table (3-1). IR spectrum for this compound was shown in figure
(3-12). Elemental microanalysis for this compound was given in table (3-2).

2.4.12. Synthesis of 2-[2-(2,6-dichlorophenylamino)phenyl]-2-(pyridine-2-

ylamino)acetic acid (Compound IV):

Intermediate IVa (0.5g, 1.24mmol) was dissolved in minimum volume of ethanol

99%:THF (20:1) mixture. The solution was cooled down 18 o C, and then sodium
hydroxide (2N, 0.74ml, 1.49mmol) was added drop wise, with continuous stirring
over a period of 30 minutes. Then the reaction mixture was worked up as described
in section 2.4.9, to liberate compound IV. The percent yield, physical data and Rf

55
values were given in table (3-1). IR spectrum for this compound was shown in figure
(3-13). Elemental microanalysis for this compound was given in table (3-2).

2.4.13. Synthesis of 2-[2-(2,6-dichlorophenylamino)phenyl]-2-(thiazol-2-

ylamino)acetic acid (Compound V):

Intermediate Va (0.17g, 0.42mmol) was dissolved in minimum volume of ethanol

99%:THF (15:1) mixture. The solution was cooled down 18 o C, and then sodium
hydroxide (2N, 0.25ml,0.5mmol) was added drop wise, with continuous stirring over
a period of 30 minutes. Then the reaction mixture was worked up as described in
section 2.4.9, to give compound V. The percent yield, physical data and Rf values
were given in table (3-1). IR spectrum for this compound was shown in figure (3-
14). Elemental microanalysis for this compound was given in table (3-2).

2.5. Preliminary Pharmacological Study: (104)

In vivo acute anti-inflammatory effects of the chemically synthesized compounds (I,

IV and V) were evaluated in egg-white induced paw edema, to compare the anti-
inflammatory activity of sulphonamide derivatives with the selected heterocyclic
rings.

The decrease of paw thickness is the basis of screening of the newly synthesized
compounds for their anti-inflammatory activity.

56
2.5.1. Methods:

Albino rats of either sex weighing (220 ± 10 g) were supplied by the animal house of
the College of Pharmacy, University of Baghdad. and were housed in the same
location under standardized conditions. Animals were fed commercial chaw and had
free access to water ad libitum. Animals were divided into five groups (each group
consist of 6 rats) as follow:

Group A: six rats served as control; and treated with the vehicle (propylene glycol
50% v/v).

Group B: six rats treated with diclofenac sodium as reference substance in a dose of
3mg/ kg(105,106).suspended in propylene glycol 50% (v/v).

Group C-E: six rats/group treated with the tested compounds (I, IV and V) in doses
that determined below. (Suspended in propylene glycol 50% v/v).

2.5.2.Dose Determination:

All the synthesized compounds are derivatives of diclofenac sodium which is given
in a dose of 3mg/kg, so; the doses of synthesized compounds are calculated as
bellow:

Calculations:

For compound I

M.wt of diclofenac sodium=318

M.wt of compound 1=465

3mg / kg dose
318 465

Dose=4.38mg/kg of compound I (i.e. dose of 3mg/kg diclofenac sodium is equivalent to 4.38mg/kg of

compound I).

57
For compound IV

M.wt of diclofenac sodium=318

Mw.wt of compound IV=387.12

3mg / kg dose
318 387.12

Dose=3.65mg/kg of compound IV (i.e. dose of 3mg/kg diclofenac sodium is equivalent to 3.65mg/kg of

compound IV).

For compound V

M.wt of diclofenac sodium=318

M.wt of compound V=393.14

3mg / kg dose
318 393.14

Dose=3.7mg/kg of compound V (i.e. dose of 3mg/kg of diclofenac sodium is equivalent to 3.7mg/kg of

compound V).

2.5.3.Experimental Design:

The anti-inflammatory activity of the tested compounds was studied using egg-white
induced edema model(104). Acute inflammation was produced by a subcutaneous
injection of 0.05ml of undiluted egg-white into the planter side of the hind paw of the
rats.; 30 minutes after i.p. administration of the drugs or their vehicle. The paw
thickness was measured by vernea at seven time intervals (0, 30, 60, 120, 180, 240,
and 300 minutes) after drug administration.

2.5.4. Statistical Analysis

The data are expressed as the mean ± SEM and results were analyzed for statistical
significance using student t-test (Two Sample Assuming Equal Variances) for
comparison between mean values. While comparisons between different groups were
made using ANOVA: Two factors without Replication. Probability (P) value of less
than 0.05 was considered significant.

58
 CHAPTER THREE

Results & Discussion

In this chapter the synthesis of the target compounds (I-V) through their intermediate
will be discussed as well as the results of their identification and their evaluation as
anti-inflammatory agents through the preliminary pharmacological study.

3.1: Chemistry:

The synthetic procedures that actually resulted in the generation of intermediates (Ia-
Ic) were represented in scheme (3-1). Then generation of intermediates (Id, IIa-Va)
by amination, followed by conversion to the target compounds (I-V) were
represented in scheme (3-2).

The characterization and purity of these compounds (percent yields, melting points
and Rf values) were given in table (3-1).

The structures of the proposed compounds were confirmed by using the IR

spectroscopy, as shown in figures (3-1 to 3-14), while their elemental microanalysis
was presented in table (3-2).

59
60
Scheme (3-2): The synthesis of intermediates (Id, IIa-Va) and target compounds (I-V).

61
3.1.1 protection of the carboxyl moiety of diclofenac acid

Carboxyl group is normally protected as an ester(107), carboxylic acids are converted

firstly into acid halide, because the direct nucleophilic acyl substitution of carboxylic
acid is difficult in the laboratory because –OH group is a poor leaving group. Thus, it
is necessary to enhance the reactivity of the acid, either by using strong acid catalyst
to protonate the carboxylic group and make it a better acceptor or by converting the –
OH group into a better leaving group (108).

1. conversion of the carboxylic acid into acid chloride

Carboxylic acids are converted into acid chlorides by treatment with thionyl chloride
in a cold alcoholic solution (109).

General reaction

Mechanism: -The reaction occurs by a nucleophilic acyl substitution pathway in

which the carboxylic acid is first converted into a chlorosulfite intermediate, thereby
replacing the –OH group of the acid with a much better leaving group. The
chlorosulfite then reacts with a nucleophilic chloride ion to give acid chloride(108), as
shown in scheme (3-3a).

62
 Scheme (3-3a): The mechanism of conversion of the carboxylic acid to acid chloride.

2. Conversion of acid chloride into ester (Alcoholysis):

Acid chloride reacts with alcohol to yield ester.

Mechanism: - This reaction is acid catalyzed ester formation and involve a

tetrahedral intermediate formation. Acids will catalyze the nucleophilic substitution
by protonation of the carbonyl oxygen and this will increase the electrophilicity of
the carbonyl group(110), as shown in scheme (3-3b).

Scheme (3-3b): The mechanism of conversion of the acid chloride to an ester.

63
the reaction of an alcohol with an acid chloride is strongly affected by steric
hindrance. Bulky group of either partner will slow down the reaction considerably,
resulting in a reactivity order among alcohol of primary>secondary>tertiary(108).

3.1.2 Bromination of diclofenac methyl ester

N-Bromosuccinimide (NBS) is a chemical reagent which is used in radical

substitution and electrophilic addition reaction, NBS can be considered as a
convenient source of cationic bromine. NBS can Į-brominates the carbonyl
derivatives via either a radical pathway, or via acid-catalysis(111).

The mechanism of the NBS halogen substitution reaction has been the subject of a
number of investigations. However, the mechanism involves either succinimidyl
radical or the bromine atom, as the chain carrying species (112). When NBS is used to
brominates non-alkenyl substrates, the mechanism involves the abstraction of the
hydrogen from the substrate by the succinimidyl radical (113), as shown in scheme (3-
4). This mechanism is facilitated by solvents as CH 2Cl2 and CCL4 in which the NBS
is more soluble. Due to the toxicity of CCL4, so;CH2Cl2 was considered as the best
solvent for this reaction(111). At the end of the reaction, ether solvent was added in
order to isolate the succinimide by-product, as it is insoluble in ether, so; it was
separated by filtration(114).

64
 Scheme (3-4): Mechanism of bromination by using NBS .

3.1.3 Amination of the brominated compound

The amination of brominated compound occur through nucleophilic substitution

reaction (SN2).

65
Nucleophilic substitution is the reaction of an electron pair donor (the nucleophile,
Nu) with an electron pair acceptor (the electrophile). An sp 3-hybridized electrophile
must have a leaving group (X) in order for the reaction to take place.

Mechanism: The term SN2 means that two molecules are involved in the actual
transition state of the reaction:

The departure of the leaving group occurs simultaneously with the backside attack by
the nucleophile. The SN2 reaction thus leads to a predictable configuration of the
stereo-center, it proceeds with inversion of the configuration.

The rate of an SN2 reaction follows second order kinetics, as the rate limiting step
depend on the nucleophile concentration, (Nu) as well as the concentration of the
substrate, (RX)(115).

Rate =k [RX] [Nu]

This mechanism depends on solvent, temperature, and concentration of the

nucleophile or that of the leaving group. It is generally favored in primary or
secondary alkyl halides with an aprotic solvent as (DMF)(116). The reaction of
sulfanilamide is regioselective(117), where the basicity of the amine has a considerable
effect on the rate and extent of the reaction, sulfonamides are of low basicity, being
fairly strong acids, in addition to the sulfonamide group the molecule also possess a
free amino group, so, generally the reaction will take place on the free amino
group(118).

3.1.4 Alkaline Hydrolysis of diclofenac methyl ester derivatives

(Saponification):

Ester hydrolysis in basic solution is called saponification. Ester hydrolysis occurs

through a typical nucleophilic acyl substitution pathway in which hydroxide ion is the
66
nucleophile that adds to the ester carbonyl group to give a tetrahedral intermediate.
Loss of alkoxide anion to give a carboxylic acid, which is deprotonated to give the
carboxylate ion. The addition of aqueous HCl in a separate step after the
saponification is completed to protonate the carboxylate anion and give the
carboxylic acid(108), as shown in scheme (3-5).This reaction is preferably to be done
below room temperature usually about 18oC in order to prevent racemization at
elevated temperature(119).

Scheme (3-5): The mechanism of base-induced ester hydrolysis (saponification).

67
3.2 Identification of the synthesized compounds and their

intermediates:

3.2.1 Melting point determination

Uncorrected melting points of the synthesized final compounds (I-V) and their
intermediates were listed in the table (3-1) alongside with their physical appearance
and yield. Sharp melting points were obtained which reveal the purity of these
compounds.

3.2.2 Thin layer chromatography (TLC)

TLC has been performed using two different solvent systems, in order to follow up
the reaction pattern and reveal purity of the synthesized compounds and their
intermediates. Rf values of the synthesized compounds and their intermediates were
listed in the table (3-1), and it is calculated according to the equation (120).

3.2.3 Infrared spectra

The Fourier transform infrared (FT-IR) spectra of the final synthesized compounds
and their intermediates showed the characteristic absorption bands by which their
functional groups were identified. The values of the interesting bands of these
spectra have been discussed according to the Silverstein(121), Shriner(122)and Mcc
Murry(123) , and are presented in tables (3-3) to (3-7).

68
3.2.4 Elemental microanalysis

Elemental microanalysis were performed for the target compounds (I-V) to confirm
their basic chemical structures. The results were presented in the table (3-2) and
revealed a reasonable good agreement with calculated percentages, the percent
deviation of the observed/calculated values was found to comply with accepted
accurate analysis.

3.2.5 Chemical tests discussions:

3.2.5.1 sodium fusion test discussion (98): After the addition of the diluted nitric acid it
must be boiled for few minutes to expel any hydrogen cyanide that present due to
nitrogen in the original compound, as shown in the equation bellow;

Cooling the solution and adding 3 drops of 0.1M silver nitrate. The immediate
formation of a pale yellow solid, indicate the presence of bromine in the compound,
as illustrated in the follow equation

3.2.5.2 Sulfonamide test discussion (99): The oxidation of green nickel (II) hydroxide
to gray-black nickel (IV) oxyhydrate, give evidence about the liberation of SO 2 gas
from sulfonamide group, as shown in the following equations

69
Table (3-1): The characterization and physical data of the intermediate compounds
and final compounds.
Compounds
Molecular % Melting
and Empirical formula Description Rf value
weight yield point oC
intermediates
A=0.85
Ia C14H11Cl2NO2 296 White powder 98.98 157-158
B=0.65

A=0.88
Ib C15H13Cl2NO2 310 White crystals 94.9 72-75
B=0.80

Faint pink A=0.93

Ic C15H12BrCl2NO2 389 75 86-88
crystals
B=0.82

Yellowish A=0.83
Id C21H19Cl2N3O4S 480 75.9 95-97
powder
B=0.86

Faint orange A=0.92

IIa C23H21Cl2N3O5S 522 74.5 166-168
powder B=0.89

Faint yellow A=0.93

IIIa C22H21Cl2N3O4S 494 74.4 71-73
powder
B=0.85

A=0.76
IVa C20H17Cl2N3O2 402 White powder 68.9 95-98
B=0.88

A=0.92
Va C18H15Cl2N3O2S 408 Black powder 50.6 51-53
B=0.8

A=0.77
I C20H17Cl2N3O4S 466 Yellow powder 25.7 100-101d
B=0.74

Faint yellow A=0.87

II C22H19Cl2N3O5S 508 24.6 182-184
powder B=0.82

A=0.9
III C21H19Cl2N3O4S 480 white powder 40.2 168-170
B=0.78

A=0.65
IV C19H15Cl2N3O2 388 Gray powder 23 172-173d
B=0.71

A=0.87
V C17H13Cl2N3O2S 394 Black powder 31.9 161-163d
B=0.67

d: decomposition.

70
Table (3-2): Elemental microanalysis of the final compounds.

Molecular Empirical Elemental microanalysis %

Compound
weight formula
Element Calculated Observed

C 51.51 51.85

H 3.67 3.70

I 466 C20H17Cl2N3O4S N 9.01 9.36

O 13.72 13.39

S 6.88 7.34

C 51.98 52.21

H 3.77 3.85

II 508 C22H19Cl2N3O5S N 8.27 8.57

O 15.74 16.01

S 6.31 6.54

C 52.51 53.01

H 3.99 4.12

III 480 C21H19Cl2N3O4S N 8.75 9.07

O 13.32 13.56

S 6.68 6.62

C 58.78 59.21

H 3.89 4.01
IV 388 C19H15Cl2N3O2
N 10.82 11.28

O 8.24 8.66

C 51.79 52.23
V 394 C17H13Cl2N3O2S H 3.32 3.49

N 10.66 11.20

O 8.12 8.56

S 8.13 8.79

71
Table (3-3): Characteristic IR absorptions of compounds (diclofenac sodium, Ia, Ib
and Ic ).

Compound Band (cm-1) Interpretation

3261 N-H stretching vibration of secondary amine

3030 C-H stretching vibration of aromatic

Diclofenac
1570 C=O strong asymmetrical stretching vibration of carboxylate
sodium*
1558, 1500, 1454 C=C stretching vibration of aromatic (skeletal vibration)

1398 COO weak asymmetrical stretching vibration

3323 N-H stretching vibration of secondary amine

3200-2500 O-H broad stretching vibration of carboxylic acid

Ia** 2891 C-H stretching vibration of alkane

1710 C=O stretching vibration of carboxylic acid

1508, 1450 C=C stretching vibration of aromatic (skeletal vibration)

1303 C-O stretching vibration

3352 N-H stretching vibration of secondary amine

2952 C-H stretching vibration of alkane

Ib***
1745 C=O stretching vibration of ester

1587, 1506 and 1450 C=C stretching vibration of aromatic

3319 N-H stretching vibration of secondary amine

3080 C-H stretching vibration of aromatic

Ic****
2987 C-H stretching vibration of CH3

1740 C=O stretching vibration of ester

1579, 1508 and 1450 C=C stretching vibration of aromatic

* see figure (3-1). ** see figure (3-2)

*** see figure (3-3). ****see figure (3-4).

72
Table (3-4): Characteristic IR absorptions of compounds (Id, I and IIa).

Compound Band (cm-1) Interpretation

3461 and 3373 N-H stretching vibration of primary sulfonamide

3245 N-H stretching vibration of secondary amine

3089 C-H stretching vibration of aromatic

Id*
2950 C-H stretching vibration of alkane

1740 C=O stretching vibration of ester

1311 and 1150 S=O stretching vibration of primary sulfonamide

Broad, moderately absorption region result from overlapping

3200-3600 of O-H stretching vibration of carboxylic acid and N-H
stretching vibration of secondary amine

1726 C=O stretching vibration of carboxylic acid

I** 1500 and 1454 C=C stretching vibration of aromatic

1313 and 1164 S=O stretching vibration of primary sulfonamide

1500 and 1454 C=C stretching vibration of aromatic

1438 O-H bending vibration

3352 N-H stretching vibration of secondary amine

3249 N-H stretching vibration of secondary sulfonamide

3033 C-H stretching vibration of aromatic

IIa*** 2842 C-H stretching vibration of alkane

1738 C=O stretching vibration of ester

1650 C=O stretching vibration of amide (Amide I)

1326 and 1153 S=O stretching vibration of secondary sulfonamide

* see figure (3-5).

** see figure (3-6).

*** see figure (3-7).

73
Table (3-5): Characteristic IR absorptions of compounds (II,IIIa, and III).

Compound Band (cm-1) Interpretation

Broad absorption band result from overlapping of O-H

3000-3600 stretching vibration of carboxylic acid and N-H stretching
vibration of amine

3080 C-H stretching vibration of aromatic

II* 2923 C-H stretching vibration of alkane

1720 C=O stretching vibration of carboxylic acid

1610 C=O stretching vibration of amid (Amide I)

1301 and 1163 S=O stretching vibration of sulfonamide

3319 N-H stretching vibration of secondary amine

3217 N-H stretching vibration of secondary sulfonamide

3080 C-H stretching vibration of aromatic

IIIa**
2952 C-H stretching vibration of alkane

1745 C=O stretching vibration of ester

1301 and 1150 S=O stretching vibration of sulfonamide

Broad absorption band result from overlapping of O-H

2800-3600 stretching vibration of carboxylic acid and N-H stretching
vibration of amine and N-H stretching of sulfonamide

1720 C=O stretching vibration of carboxylic acid

III***
1566 and 1490 C=C stretching vibration of aromatic

1353 and 1163 S=O stretching vibration of sulfonamide

O-H bending and C-O stretching vibration of carboxylic acid

1438 and 1313
respectively

* see figure (3-8).

** see figure (3-9).

*** see figure (3-10).

74
Table (3-6): Characteristic IR absorptions of compounds (IVa, IV and Va)

Compound Band (cm-1) Interpretation

3260 and 3161 N-H stretching vibration of secondary amine

3082 C-H stretching vibration of aromatic

2952 C-H stretching vibration of alkane

IVa*
1743 C=O stretching vibration of ester

1579 and 1508 C=C stretching vibration of aromatic

1460 C=N stretching vibration of pyridine

Broad absorption band result from overlapping of O-H

2985-3627 stretching vibration of carboxylic acid and N-H stretching
vibration of secondary amine.

3078 C-H stretching vibration of aromatic

IV**
2906 C-H stretching vibration of alkane

1739 C=O stretching vibration of carboxylic acid

1454 C=N stretching vibration of pyridine

3315 N-H stretching vibration of secondary amine

3078 C-H stretching vibration of aromatic

1745 C=O stretching vibration of ester

Va***
1504 C=N stretching vibration of thiazole ring

1191 C-O stretching vibration

638 C-S stretching vibration of thiazole ring

* see figure (3-11).

** see figure (3-12).

*** see figure (3-13).

75
Table (3-7): Characteristic IR absorptions of compound V.

Compound Band (cm-1) Interpretation

Broad absorption band result from overlapping of O-H

2543-3200 stretching vibration of carboxylic acid and N-H stretching
vibration of secondary amine

3020 C-H stretching vibration of aromatic

V* 2922 C-H stretching vibration of alkane

1719 C=O stretching vibration of acid

1509 C=N stretching vibration of thiazole ring

668 C-S stretching vibration of thiazole ring

* see figure (3-14).

76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
3.3. Pharmacological Study:

The following sections are concerned with the results of preliminary pharmacological
evaluation of tested compounds as anti-inflammatory agents using paw-edema
method following intra-plantar injection of egg-white into rat hind paw.

3.3.1. Dose Determination of the Tested Compounds:

The determination of the dose of the newly synthesized compounds was done
depending on the dose of the diclofenac sodium (reference compound) in which the
tested compounds are derived from it.

Then according to its molecular weight, the dose of the tested compound was
calculated using the following equation:

Dose of reference compound Dose of tested compound

reference compound molecular weight tested compound molecular weight

3.3.2. Anti-inflammatory Effect of Tested Compounds:

Tables (3-9) and (3-10) show the effect of tested compounds on egg-white induced
edema as an indicator for their anti-inflammatory activity. The intra-plantar injection
of egg-white into rat hind paw induce a progressive edema, which was reached
maximum (measured by millimeter) after 1 hour of injection.

In this study, the intra-peritoneal injection of tested compounds produced varying

degree of anti-inflammatory effect. Compounds IV and V exhibited comparable
effect to that of diclofenac (3mg/kg, i.p.). While compound I exhibited superior anti-
inflammatory effect when compared to diclofenac. The tested compounds and the
reference drug produced significant reduction of paw edema with respect to the effect
of propylene glycol 50%v/v (control group).

91
3.3.3. In Vivo Method for Evaluation of Anti-inflammatory Activity:

The most widely used primary test to screen new anti-inflammatory agents measures
the ability of the compound to reduce local edema induced in the rat paw by injection
of an irritant agent(124). Many irritant agents have been used in the paw-edema method
like dextran, egg-white and carrageenan solution. Subcutaneous injection of
carrageenan into the rat paw produces inflammation resulting from plasma
extravasations, increased tissue water and plasma protein exudation along with
neutrophil extravasations, all due to the metabolism of arachidonic acid(125).
Carrageenan induced edema has been commonly used as an experimental animal
model for acute inflammation and is believed to be biphasic. The early phase (1 – 2
h) of the carrageenan model is mainly mediated by histamine, serotonin and increased
synthesis of prostaglandins in the damaged tissue surroundings. The late phase is
sustained by prostaglandin release and mediated by bradykinin, leukotrienes,
polymorph nuclear cells and prostaglandins produced by tissue macrophages(124).
Many methods have been described how to measure the paw volume, while in our
work, vernea has been used for the measuring of paw-volume which is a simple and
more practically valid tool than others in which the change in volume need a very
sensitive capillary tube or microcomputer which may not be an available(126).

3.3.4. Advantages of Paw Edema Method:

This in vivo method of evaluation of anti-inflammatory agents have many advantages

over the other methods including:

1. Rapid evaluation in which the inflammation is measured immediately and during

short time course, i.e. no need for overnight or waiting for several days.
2. The paw is very sensitive for inflammatory substances.

92
3. Low cost method because it involves no anesthetic procedures or expensive
chemical agents or dyes and the rat will be conscious and alive after the end of the
experiment.
3.3.5. Evaluation of the Anti-inflammatory Activity of the Tested Compounds:

The anti-inflammatory activity of the tested compounds have been evaluated in

comparison with their vehicle (control group) and reference drugs and presented
below.

3.3.5.1. Effect of Reference Anti-inflammatory drug (diclofenac sodium) on Paw

Edema:

To assess the validity of the method (paw edema) used for the evaluation of newly
synthesized anti-inflammatory compounds, diclofenac sodium was used as a
reference compound of known profile of anti-inflammatory activity. Table (3-8)
shows that the intra-plantar injection of 0.05mL egg-white into the right hind paw
produced a significant increase in paw thickness in all animals designed as control
(5.83 ±0.12mm) and diclofenac sodium (5.78 ±0.11 mm) groups with respect to their
baseline reading (zero time) (P<0.05); furthermore, no significant difference in
induced paw edema was observed among these two groups.

In control group, paw edema was shown to be continually elevated reaching

maximum (5.85 ±0.06) after 0.5 hour of induction (1 hour after i.p. injection of
vehicle or reference drug). For this reason, this time interval is used for the
comparative analysis of the anti-inflammatory effect of the reference drug and of the
tested compounds. However; paw thickness was reduced back to lower value (4.91±
0.02 mm) after 5 hours (the end of experiment) as shown in figure (3-15). While paw
edema in animals treated with diclofenac sodium (3mg/kg, i.p.) reached (5.42 ±0.12
mm) after 0.5 hour of induction which is significantly lower when compared to that
in control group (P<0.05), and reduced back to (4.54±0.13 mm) after 5 hours, a
value found to be significantly lower in comparison with that of the control group
(P<0.05) as shown in figures (3-15 and 3-16).

93
The differences in paw thickness readings among control and diclofenac sodium
groups indicates that the method used in this study (paw edema) is a valid method
and can effectively be used for the assessment of the anti-inflammatory effect of the
newly synthesized compounds.

Table (3-8): Effect of diclofenac sodium (reference) and propylene glycol (control)
on egg-white induced paw edema in rats.

Diclofenac
Time Control
sodium
(min) (n=6)
(n=6)

0 4.07±0.05 3.98±0.06

30 5.83±0.12 5.78±0.11

60 5.85±0.06 5.42±0.12*
Paw
thickness 120 5.5±0.11 5.11±0.04*
(mm)
180 5.25±0.03 4.95±0.1*

240 5.03±0.1 4.69±0.12*

300 4.91±0.02 4.54±0.13*

Data are expressed in mm paw thickness as mean ± SEM.

n= number of animals.

Time (0) is the time of i.p. injection of diclofenac sodium (reference) and propylene glycol (control).

Time (30) is the time of injection of egg-white (induction of paw edema).

* significantly different compared to control (p<0.05).

94
 6

5.5
paw thickness (mm)

5
Diclofenac
control
4.5

3.5
0 30 60 120 180 240 300
duration (min)

Fig. (3-15): Effect of diclofenac sodium (reference), and propylene glycol (control) on egg-white
induced paw edema in rats. Results are expressed as mean ± SEM (n=6 for each group). Time (30) is
the time of egg-white injection.

6
5.5
paw thickness (mm)

5
4.5
Diclofenac
4
control
3.5
3
2.5
2
0 30 60 120 180 240 300
duration (min)

Fig. (3-16): Effect of diclofenac sodium (reference), and propylene glycol (control) on egg-white
induced paw edema in rats. Time (30) is the time of egg-white injection.

95
3.3.5.2. Effect of Compounds I, IV and V on Paw Edema:

Table (3-9) showed the effect of compounds I, IV and V on paw thickness after intra-
plantar injection of 0.05mL egg-white. Paw edema in animals treated with compound
I (4.38mg/kg, i.p.) reached (5.26 ±0.13 mm) after 0.5 hour of induction which is
significantly lower in comparison to that in control group (P<0.05) and reduced back
to (4.15 ± 0.1 mm) after 5 hours, which is significantly lower with respect to that in
control group (P<0.05) as shown in figures (3-17 and 3-18). On the other hand,
animals treated with compound IV (3.65mg/kg, i.p.) exhibited (5.23± 0.12 mm)
elevation in paw thickness after 0.5 hour of induction, a value that is significantly
lower than that in control group (P<0.05). Paw thickness was decreased to (4.49
±0.02 mm) at the end of experiment, a value found significantly lower than that in
control group (P<0.05) as shown in figures (3-17 and 3-18). On the other hand,
animals treated with compound V (3.7mg/kg, i.p.) exhibited (5.8± 0.11 mm)
elevation in paw thickness after 0.5 hour of induction, a value that is not significantly
lower than in control group (P<0.05). Paw thickness was decreased to (4.56 ±0.06
mm) at the end of experiment, a value found significantly lower than that in control
group (P<0.05) as shown in figures (3-17 and 3-18).

96
Table (3-9): Effect of compounds I, IV, V and propylene glycol on egg-white induced
paw edema in rats.

Treatment groups

Control Compound I Compound IV Compound V

Time (min)
(n=6) (n=6) (n=6) (n=6)

0 4.07±0.05 4.06±0.04 4.09±0.1 4.10±0.04

30 5.83±0.12 5.78±0.12 5.85±0.06 5.87±0.12

60 5.85±0.06 5.26±0.13*a 5.23±0.12*a 5.80±0.11b

Paw
thickness 120 5.5±0.11 4.94±0.13* 4.97±0.05* 5.13±0.03*
(mm)
180 5.25±0.03 4.59±0.05*b 4.83±0.05*a 4.96±0.05*a

240 5.03±0.1 4.28±0.04*b 4.6±0.11*a 4.70±0.02*a

300 4.91±0.02 4.15±0.01*b 4.49±0.02*a 4.56±0.06*a

Data are expressed in mm paw thickness as mean ± SEM.

n= number of animals.

Time (0) is the time of i.p. injection of tested compounds and propylene glycol.

Time (30) is the time of injection of egg-white (induction of paw edema).

* significantly different compared to control (p<0.05).

Non-identical superscripts (a and b) among different groups are considered significantly different (p<0.05).

97
 6

5.5
paw thickness (mm)

4.5 co.V
co.IV
4
co.I
3.5 control
3

2.5
2
0 30 60 120 180 240 300
duration (min)

Fig. (3-17): Effect of propylene glycol (control), compounds I, IV, and V on egg-white induced paw
edema in rats. Results are expressed as mean ± SEM (n=6 for each group). Time (30) is the time of
egg-white injection.

5.5
paw thickness (mm)

4.5 co.V
co.IV
4
co.I
3.5 control
3

2.5

2
0 30 60 120 180 240 300
duration (min)

Fig. (3-18): Effect of propylene glycol (control), compounds I, IV, and V on egg-white induced paw
edema in rats. Results are expressed as mean ± SEM (n=6 for each group). Time (30) is the time of
egg-white injection.

98
3.3.5.3. Comparative Analysis:

Multi-way comparison between reference drug and tested compounds revealed the
following:

1. All tested compounds were effectively limited the increase in paw edema.
2. The effect of compounds I and IV started 1 hour after injection of drug, while that
for compound V the effect started 2 hours after injection of it, and continued till
the end of the experiment, this indicate late onset of action of compound V, as
shown in figures (3-19 and 3-20 ).
3. Compound I expressed a comparable effect to that of diclofenac sodium at 60-120
min. of injection of drug and significantly more effective at time 180-300 min. as
shown in figures (3-19 and 3-20).
4. Compound IV showed a comparable effect to that of diclofenac sodium at all the
time of experiment.
5. The effect of compound I was significantly higher than that of diclofenac sodium,
compounds IV and V from the time 180-300 minutes of the experiment, as shown
in figures (3-19 and 3-20).
6. No significant difference among reference, compound IV and V at the time 180-
300 minutes of experiment, as shown in figures (3-19 and 3-20).

99
Table (3-10): Effect of compounds I, IV, V, diclofenac (reference) and propylene
glycol (control) on egg-white induced paw edema in rats.

Treatment groups

Compound Compound Compound

Control Diclofenac
Time I IV V
(min)
(n=6) (n=6)
(n=6) (n=6) (n=6)

0 4.07±0.05 3.98±0.06 4.06±0.04 4.09±0.1 4.10±0.04

30 5.83±0.12 5.78±0.11 5.78±0.12 5.85±0.06 5.87±0.12

60 5.85±0.06 5.42±0.12* 5.26±0.13*a 5.23±0.12*a 5.80±0.11b

Paw thickness
120 5.5±0.11 5.11±0.04* 4.94±0.13* 4.97±0.05* 5.13±0.03*
(mm)

180 5.25±0.03 4.95±0.1*a 4.59±0.05*b 4.83±0.05*a 4.96±0.05*a

240 5.03±0.1 4.69±0.12*a 4.28±0.04*b 4.6±0.11*a 4.70±0.02*a

300 4.91±0.02 4.54±0.13*a 4.15±0.1*b 4.49±0.02*a 4.56±0.06*a

Data are expressed in mm paw thickness as mean ± SEM.

n= number of animals.

Time (0) is the time of i.p. injection of tested compounds and propylene glycol.

Time (30) is the time of injection of egg-white (induction of paw edema).

* significantly different compared to control (p<0.05).

Non-identical superscripts (a and b) among different groups are considered significantly different (p<0.05).

100
 6

5.5

5
paw thickness (mm)

4.5 co.V
co.IV
4 co.I
Diclofenac
3.5 control

2.5

2
0 30 60 120 180 240 300
duration (m in)

Fig. (3-19): Effect of diclofenac, control, compound I, IV and V on egg-white induced paw edema in
rats. Results are expressed as mean ± SEM (n=6 for each group). Time (30) is the time of egg-white
injection.

6
5.5
paw thickness (mm)

5 co.V
4.5 co.IV
4 co.I
3.5 Diclofenac

3 control

2.5
2
0 30 60 120 180 240 300
duration (min)

Fig. (3-20): Effect of diclofenac, control, compound I, IV and V on egg-white induced paw edema in
rats. Results are expressed as mean ± SEM (n=6 for each group). Time (30) is the time of egg-white
injection.

101
Table (3-11) showed the percent inhibition in the paw edema that produced with the
treatment with diclofenac sodium, compounds I, IV and V. At the first hour,
diclofenac sodium, compounds I and IV produced significant inhibition in edema
thickness (19%, 31.8% and 35%) respectively. While compound V did not produce
significant inhibition in edema thickness at the first hour, but it started from the
second hour to produce significant inhibition in paw thickness. The percent inhibition
noticed for compound I is significantly higher than that of diclofenac sodium,
compound IV and V at the last three hours of the experiment, this indicate that the
effect of compound I is more potent in decreasing the paw edema. At the other hand
compounds IV and V produced comparable inhibition in the paw edema with that of
diclofenac sodium at the last three hours, as shown in figures (3-21 and 3-22).

Calculation of the percent inhibition in edema thickness

The percent inhibition of edema thickness at each time interval was calculated from
the mean effect in control and treated animals according to the equation:

Percent inhibition in edema thickness= [(Vc-Vt) /Vc]×100.

Where Vc and Vt are the mean paw thickness of the control group and tested group
(at time t-time zero) respectively(127,128).

The comparison among the diclofenac sodium, compounds I, IV and V was confirm
in the following results of the comparative analysis and shown in table (3-11) and
figures (3-21) and (3-22).

102
Table (3-11): Percent inhibition of diclofenac, compounds I, IV, and V

on egg-white induced paw edema in rats.

Treatment groups

Diclofenac Compound Compound

Compound I
Time (min) sodium IV V
(n=6)
(n=6) (n=6) (n=6)

60 19% 31.8% 35% 3.4%

120 20.9% 38.4% 38.4% 27.9%

Percent
180 17.7% 55% 37% 27%
inhibition
240 26% 77% 46.8% 37.5%

300 33% 89% 52% 45.2%

103
 80.5
70.5
% of inhibition

60.5 co.V
50.5 co.IV
40.5 co.I
30.5 diclofenac
20.5
10.5
0.5
60 120 180 240 300
Time of inhibition (min)

Fig. (3-21): Percent inhibition produced by diclofenac sodium (reference), compounds I, IV and V on
paw edema.

100
90
80
% of inhibition

70
co.V
60
co.IV
50
co.I
40
diclofenac
30
20
10
0
60 180 240 300
Time of inhiobition (min)

Fig. (3-22): Percent inhibition produced by diclofenac sodium, compounds I, IV and V on paw edema,
at time intervals 60, 180, 240 and 300 min.

104
3.4. Conclusions:

1. The synthesis of the designed compounds has been successfully achieved.

2. Purity and structural formulas of the synthesized compounds were confirmed by
melting points determination, Rf values, FT-IR spectroscopy and elemental
microanalysis.
3. In vivo anti-inflammatory study for the diclofenac derivative (compounds I)
showed that the incorporation of 4- aminobenzenesulfonamide onto Į-carbon of
diclofenac a well-known non-steroidal anti-inflammatory drug, increases its anti-
inflammatory activity.
4. The incorporation of 2-aminopyridine and 2-aminothiazole maintain its anti-
inflammatory activity, as shown in compounds IV and V, as confirmed by the in
vivo study.
5. The superior anti-inflammatory activity produced by compound I, attributed to the
sulfonamide group that participated in enzyme inhibition by tight bonds.

3.5. Recommendations:

1. Incorporation of another amino groups as C 6H5-NH2 and R-C6H4-NH2 into Į-

carbon of diclofenac and compare their anti-inflammatory activity. (R= halogen,
alkyl, aromatic,….etc.)
2. Incorporation of the pharmacophoric group (NH2-C6H4-SO2NH2) into different
well known NSAIDs (e.g. indomethacin) to improve their potency as anti-
inflammatory activity.
3. Study the sub-acute and chronic anti-inflammatory effect of the tested compounds
on formalin induced paw edema and cotton pellet-induced granuloma, glass rod
granuloma in rats.
4. Study the acute anti-inflammatory activity of compounds II and III.
5. Determination of COX-2 selectivity of the tested compounds by assessing COX-
2:COX-1 inhibitory ratio using human whole blood assay.

105
 References

1) Raymond S. Sinatra, Oscar A, de Leon-Casasola, Brian Ginsberg, and Eugene

R Viscusi (Eds): Acute Pain Management (1st ed). Cambridge University
Press, New York, 2009; pp.53.
2) John L Wallace & Linda Vong: NSAID-induced gastrointestinal damage and
the design of GI-sparing NSAIDs. Current Opinion in Investigational Drugs
2008; 9(11):1151-1156.
3) Ma L, del Soldato P, Wallace JL: Divergent effects of new cyclooxygenase
inhibitors on gastric ulcer healing: Shifting the angiogenic balance. Proc Natl
Acad Sci USA 2002; 99(20):13243-13247.
4) Samuelsson B, Morgenstern R, Jakobsson PJ: Membrane prostaglandin E
synthase-1: A novel therapeutic target. Pharmacol Rev. 2007; 59(3):207-224.
5) Wallace JL: ''Building a better aspirin: Gaseous solutions to a century-old
problem''. Br J Pharmacol . 2007; 152(4):421-428.
6) Dinesh T. Makhija and Rakesh R. Somani: Improvement of GI tolerance of
NSAIDs using oral prodrug approach. Der Pharmacia Lettre 2010; 2(2): 300-
309.
7) Ferrero-Miliani L, Nielsen OH, Andersen PS, Girardin SE: ''Chronic
inflammation: importance of NOD2 and NALP3 in interleukin-1beta
generation''. Clin. Exp. Immunol. 2007; 147 (2): 227-235.
8) P. N. Bennett, M. J. Brown (Eds.): Clinical Pharmacology (10 th ed).Churchill
Livingstone, Spain, 2008; pp.250.
9) Ashutosh Kar (Ed.): Medicinal Chemistry (4th ed). New Age International
Publishers, New Delhi, 2007; pp.522-535.
10) Ole Naesh: ''Back to the future: postoperative pain management beyond COX-
2 inhibitors''. Jornal of the New Zealand Medical Association 2006; 119(1242):
1-9.
11) Steven D. Stovitz, Robert J. Johnson: NSAIDs and Musculoskeletal
Treatment. The physician and sportsmedicine. 2003; 31(1).
106
12) Chubb AJ, Fitzgerald DJ, Nolan KB, Moman E: ''The productive conformation
of prostaglandin G2 at peroxidase site of prostaglandin endoperoide H
synthase: docking, molecular dynamics, and site directed mutagenesis studies''.
Biochemistry 2006;45(3):811-820.
13) V. F. Roche: A Receptor-Grounded Approach to Teaching Non-steroidal Anti-
inflammatory Drug Chemistry and Structure-Activity Relationships. American
Journal of Pharmaceutical Education 2009; 73 (8) Article 143.
14) Smith WL, Langenbach R: Why there are two cyclooxygenase isozymes. J
Clin Invest. 2001;107:1491-1495.
15) Chandrasekharan NV, Dai H, Roos KL, Evanson NK, Tomsik J, Elton TS,
Simmons DL: COX-3, a cyclooxygenase-1variant inhibited by acetaminophen
and other analgesic/antipyretic drugs: Cloning, structure, and expression. Proc.
Natl. Acad. Sci. USA 2002; 99(21):13926-13931.
16) Rowlinson SW, Kiefer JR, Prusakiewicz JJ. et al: A novel mechanism of
cyclooxygenase-2 inhibition involving interactions with Ser-530 and Tyr-385.
J Biol Chem. 2003; 278:45763-45769.
17) L. J. Marnett: Cyclooxygenase Mechanisms. Current Opinion in Chemical
Biology 2000; 4:545-552.
18) Mohammed Naser A. Kban and Yong Sup Lee: "Cyclooxygenase inhibitors:
Scope of their use and development in cancer chemotherapy". Medicinal
research reviews. 2009 Wily periodicals, Inc. DOI 10.1002/med.20182.
19) Katzung, B.G. (Ed.): Basic and clinical pharmacology, (9th Ed.).
McGraw-Hill, New York, 2004; pp. 298.
20) Hardman, J.G; Limbird, L.E. and Molinoff, P.B.(Eds.): Goodman and
Gilman’s The Pharmacological Basis of Therapeutics (10th ed.), McGraw-Hill,
New York, 2001; pp. 689.
21) Lipsky, P.E.; Abramson, S.B.; Breedveld, F.C. et al. : Analysis of the effect of
COX-2 specific inhibitors and recommendations for their user in clinical
practice. J. Rheumatol. 2000; 27:1338-1340.

107
22) Thotakura Balaji, Manickam Ramanathan and Marimuthu Srinivasan:
Distribution of cyclooxygenase-1 and cyclooxygenase-2 in the mouse seminal
vesicle. J. of Biomedicine. 2008; 6:97-104.
23) G. Dannhardt and S. Laufer: ''structural approaches to explain the selectivity of
COX-2 inhibitors: Is their a common pharmacophore?''. Current medicinal
chemistry. 2000; 7: 1101-1112.
24) F. Burdan, J. Szumito, J. Dudka and M. Szumito: Association of maternal
pancreatic function and foetal growth in rats treated with DFU, a selective
cyclooxygenase-2 inhibitor. Folia Morphol. 2007; 66 (3): 172-180.
25) Nina Zidar, Katarina Odar, Damjan Glavac and Maja Jerse: Cyclooxygenase in
normal human tissue-is cox-1 really a constitutive isoform, and cox-2 an
inducible isoform? J. of cellular and molecular medicine. 2009; 13: 3753-
3763.
26) Peter Curtis-Prior: The Eicosanoid (1st ed). John Wiley and Sons, England,
2004; pp. 449.
27) Parente L. et al.: ''Advances in the pathophysiology of constitutive and
inducible cyclooxygenases: two enzymes in the spotlight''. Biochem.
Pharmacol. 2003; 65: 153-159.
28) Kiefer, et al.:Dioxygenase. Nature 2000; 405: 97-101.
29) Kristen Huber: ''My Favorite Protein: Cyclooxygenase. CH 791A-
Biomolecular Structures''. 2005; 1-9.
30) Vane JR, Bakhle YS, Botting RM.: Cyclooxygenases 1 and 2. Annu Rev
Pharmacol Toxicol. 1998; 38: 97-120.
31) Anana R, Rap PNP, Chen Q-H, Knaus EE: Synthesis and biological evaluation
of linear phenyethylbenzenesulfonamide regioisomers as cyclooxygenase-1/-2
(COX-1/-2) inhibitors. Bioorganic Med Chem. 2006;14: 5259-5265.
32) Michaux, C. and C. Charlier: Structural Approach for COX-2 Inhibition. Mini
Reviews in Medicinal Chemistry 2004; 4(6): 603-615..

108
33) Ermondi, G., G. Caron, et al. : Docking studies on NSAID/COX-2 isozyme
complexes using Contact Statistics analysis. Journal of Computer-Aided
Molecular Design 2004; 18(11): 683-696.
34) Harman CA, Turman MV, Kozak KR, et al. : Structural basis of
enantioselective inhibition of cyclooxygenase-1 by S-alphasubstituted
indomethacin ethanolamides. J Biol Chem. 2007; 282: pp.28096-28105.
35) John H. Block, and John. M. Beale: Wilson and Gisvold's Textbook of Organic
Medicinal and Pharmaceutical Chemistry, (11th ed), Lippincott Williams and
Wilkins, Philadelphia, 2004; pp.753-761.
36) Borne R, Levi M, Wilson N. Nonsteroidal anti-inflammatory drugs. In:
Williams DA, Lemke TL, eds. Foye’s Principles of Medicinal Chemistry, (6th
ed). Baltimore, MD: Lippincott Williams & Wilkins; 2008: pp. 970.
37) Cronstein BN. : ''Cyclooxygenase-2-selective inhibitors: translating
pharmacology into clinical utility''. Cleve Clin J Med. 2002;69:13-19.
38) Walker, M. C., R. G. Kurumbail, et al. : A three-step kinetic mechanism for
selective inhibition of cyclo-oxygenase-2 by diarylheterocyclic inhibitors.
Biochem. J. 2001; 357(3): 709-718.
39) Rod J. Flower: The development of COX-2 inhibitors. Nature Reviews Drug
Discovery 2003; 2: 179-191.
40) D. J. Abraham (Ed.): Burger's Medicinal Chemistry and Drug Discovery, (6 th
ed), Volume 4, John Wiley and Sons, Inc., Publisher, 2003; pp. 234-244.
41) Derek G. Waller, Andrew G. Renwick, Keith Hillier: Medical Pharmacology
and Therapeutics (1st ed). Harcourt publisher limited, Spain, 2001; pp.309.
42) Jack DeRuiter: Non-Steroidal Anti-inflammatory Drugs, Principles of drug
action 2, Fall 2002.
43) Radovan Toth, Marco Ferrone and Stanislav Miertus: ''Structure and
Energetics of Biocompatible Polymer Nanocomposite Systems: A Molecular
Dynamics Study''. J Biomacromolecules 2006; 7: 1714-1719.
44) Maria Victoria Gomez-Gaviro, Isidoro Gonzalez-Alvaro and Carmen
Dominguez-Jimenez: Structure-Function Relationship and Role of Tumor
109
 Necrosis Factor-Į-converting Enzyme in the Down-regulation of L-selectin by
Non-steroidal Anti-inflammatory Drugs. J Biol. Chem. 2002 ; 277: 38212-
38221.
45) S. K. Kulkarni, N. K. Jain :''Coxibs: The new super aspirins or unsafe pain
killers?''. 2005;37(2): 86-89.
46) Rodrigues, C. R.; Veloso, M. P., Verli, H.; Fraga, C. A. M.; Miranda, A. L. P.;
Barreiro, E. J. Medicinal Chemistry Reviews on line 2004; 1:pp. 73.
47) Hawkey, C. J.; Skelly, M. M. Curr. Pharm. Design 2002; 8: 1077.
48) John L. Wallace: Commonality of Defensive Roles of COX-2 in the Lung and
Gut. American Journal of Pathology 2006;168(4):1060-1063.
49) Hawkey C. J. :COX-2 inhibitors. Lancet 1999; 353: pp.307 -314.
50) Bombardier C, Laine L, Reicin A, Shapiro D, Burgos-Vargas R, Davis B, et al.
: Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in
patients with rheumatoid arthritis. N Engl. J Med. 2000; 343:1520 -1528.
51) Raymond M. Pertusi: Selective Cyclooxygenase Inhibition in Pain
Management. JAOA. 2004;104(11): 19-24.
52) Lees PT, Recker DP, Kent JD: ''The COX-2 selective inhibitor, valdecoxib,
does not impair platelet function in the elderly: results of a randomized
controlled trial''. J Clin Pharmacol. 2003; 43:504 -513.
53) Silverman DG, Halaszynski T, Sinatra R, Luther M, Rinder CS.: Rofecoxib
does not compromise platelet aggregation during anesthesia and surgery. Can J
Anaesth.2003; 50:1004 -1008.
54) Wallace JL, Devchand PR.: Emerging roles for cyclooxygenase-2 in
gastrointestinal mucosal defence. Br J Pharmacol. 2005;145: pp.275-282.
55) Bombardier C, Laine L, Reicin A, Shapiro D, Burgos-Vargas R, Davis B, Day
R, Ferraz MB, Hawkey CJ, Hochberg MC, Kvien TK, Schnitzer TJ.:
Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in
patient with rheumatoid arthritis. New Engl. J Med 2002;343:1520-1528.
56) Schnitzer TJ, Burmester GR, Mysler E, Hochberg MC, Doherty M, Ehrsam E,
Gitton X, Krammer G, Mellein B, Matchaba P, Gimona A, Haekey CJ.:
110
 ''Comparison of lumiracoxib with naproxen and ibuprofen in the Therapeutic
Arthritis Research and Gastrointestinal Event Trial (TARGET), reduction in
ulcer complications: randomized controlled trial''. Lancet 2004; 364: 665-674.
57) Michael Spink, D.D.S.,M.D., Saul Bahn, D.M.D., M.S.D., and Robert
Glickman, D.M.D: Clinical implication of cyclooxygenase-2 inhibitors for
acute dental pain management. J. of the American dental association. 2005;
136(10): 1439-1448.
58) Xiang Z et al.: Cyclooxygenase (COX-2) and cell cycle activity in a transgenic
mouse model of Alzheimer's disease neuropathology. Neurobiol. Aging 2002;
23: 327-334.
59) Teismann P, Tieu K, Choi DK, et al.: Cyclooxygenase-2 is instrumental in
Parkinson’s disease neurodegeneration. Proc Natl Acad Sci USA 2003;100:
5473–5478.
60) L. S. Varandas, C. A. M. Fraga, A. L. P. Miranda and E. J. Barreiro: Design,
Synthesis and Pharmacological Evaluation of New Nonsteroidal Anti-
inflammatory 1,3,4-Thiadiazole Derivatives. Letters in Drug Design and
Discovery 2005; 2: 62-67.
61) Nataraj Sekhar P, Kavi Kishor PB, Sunita MSL, Premkumar E and Ananda
Reddy L.: Computational designing of new inhibitors against COX-2 involved
in human diseases based on binding energy calculations. IJIB. 2008; 2(2): 182-
189.
62) Lawrence J. Marnett and Amit S. Kalgutkar: ''Cyclooxygenase-2 inhibitors:
discovery, selectivity and the future''. Rev. TiPS 1999; 20: pp.465-469.
63) Robert A., Williams, Jean M., Giannaras, John, Nurnberg, Sherrill and
Covington: Mechanism of selective inhibition of the inducible isoform of
prostaglandin G/H synthase. Proceeding of the National Academy of Sciences
of the United States of America. 1994; 91(23): pp.11202-11206.
64) Hae-Sun Prk, Hee-Jeon Choi, Hea-Soon Shin, Sang Kook Lee and Myung-
Sook Park: "Synthesis and Characterization of Novel Hydantoins as Potential

111
 COX-2 Inhibitors:1,5-Diarylhydantoins". Bull. Korean Chem. Soc. 2007;
28(5): 751-757.
65) R. M. Botting: "Inhibitore of Cyclooxygenases: Mechanisms, Selectivity and
Uses", Journal of physiology and pharmacology 2006; 57:113-124.
66) Afshin Zarghi, P. N. Praveen Rao and Edward E. Knaus: "Design and
Synthesis of new rofecoxib analogues as selective Cyclooxygenase-2 (COX-2)
inhibitors: Replacement of the methanesulfonyl pharmacophore by an N-
acetylsulfonamido bioisostere". J Pharm pharmaceutic Sci. 2007:10(2): 159-
167.
67) Matthijs Raaben, Alexandra WC Einerhand, Lucas JA Taminiau, Michel van
Houdt and Janneke Bouma: Cyclooxygenase activity is important for efficient
replication of mouse hepatitis virus at an early stage of infection. Virology
Journal 2007; 4:pp.55.
68) Steer SA, Corbett JA: The role and regulation of COX-2 during viral infection.
Viral Immunol 2003; 16(4): 447-460.
69) Janelle ME, Gravel A, Gosselin J, Tremblay MJ, Flamand L: Activation of
monocyte cyclooxygenase-2 gene expression by human herpesvirus 6. Role for
cyclic AMP-responsive element-binding protein and activator protein-1. J Biol
Chem 2002; 277(34): pp.30665-30674.
70) Symensma TL, Martinez-Guzman D, Jia Q, Bortz E, Wu TT, Rudra-Ganguly
N, Cole S, Herschman H, Sun R: COX-2 induction during murine
gammaherpesvirus 68 infection leads to enhancement of viral gene expression.
J Virol. 2003; 77(23):12753-12763.
71) Zhu H, Cong JP, Yu D, Bresnahan WA, Shenk TE: Inhibition of
cyclooxygenase 2 blocks human cytomegalovirus replication. Proc Natl Acad
Sci SA 2002; 99(6): 3932-3937.
72) Ray N, Bisher ME, Enquist LW: Cyclooxygenase-1 and -2 are required for
production of infectious pseudorabies virus. J Virol 2004; 78(23): 12964-
12974.

112
73) Rott D, Zhu J, Burnett MS, Zhou YF, Zalles-Ganley A, Ogunmakinwa J,
Epstein SE: Effects of MF-tricyclic, a selective cyclooxygenase-2 inhibitor, on
atherosclerosis progression and susceptibility to cytomegalovirus replication in
apolipoprotein-E knockout mice. J Am Coll Cardiol 2003; 41(10):1812-1819.
74) Pyeon D, Diaz FJ, Splitter GA: Prostaglandin E(2) increases bovine leukemia
virus tax and pol mRNA levels via cyclooxygenase 2: regulation by
interleukin-2, interleukin-10, and bovine leukemia virus. J Virol. 2000;
74(12): 5740-5745.
75) Rossen JW, Bouma J, Raatgeep RH, Buller HA, Einerhand AW: Inhibition of
cyclooxygenase activity reduces rotavirus infection at a postbinding step. J
Virol. 2004; 78(18): 9721-9730.
76) Hayes MM, Lane BR, King SR, Markovitz DM, Coffey MJ: Prostaglandin
E(2) inhibits replication of HIV-1 in macrophages through activation of protein
kinase A. Cell Immunol. 2002; 215(1): pp.61-71.
77) Luisa Minghetti: Cyclooxygenase-2 (COX-2) in inflammatory and
Degenerative Brain Disease. J of neuropathology and experimental neurology
2004; 63(9): 901-910.
78) Martino G, Adorini L, Rieckmann P, et al. :''Inflammation in multiple
sclerosis: The good, the bad, and the complex''. Lancet Neurol 2002;1: pp.499–
509.
79) Maihofner C, Probst-Cousin S, Bergmann M, Neuhuber W, NeundorferB,
Heuss D.: Expression and localization of cyclooxygenase-1 and -2 in human
sporadic amyotrophic lateral sclerosis. Eur J Neuro. sci 2003;18:1527–34.
80) Gao HM, Liu B, Zhang W, Hong JS.: Novel anti-inflammatory therapy for
Parkinson’s disease. Trends Pharmacol Sci 2003; 24: pp.395–401.
81) Perry VH, Newman TA, Cunningham C.: The impact of systemic infection on
the progression of neurodegenerative disease. Nat Rev Neurosci 2003; 4: 103–
112.
82) Botha JH, Robinson KM, Ramchurren N, Reddi K, Norman RJ.: ''Human
esophageal carcinoma cell lines: Prostaglandin production, biological
113
 properties, and behavior in nude mice''. J Natl Cancer Inst. 1986; 76: 1053–
1056.
83) Morgan GD.: Deleterious effects of prostaglandin E2 in oesophageal
carcinogenesis. Med Hypotheses 1997; 48: 177–181.
84) Koki AT, Masferrer JL.: ''Celecoxib: A specific COX-2 inhibitor with
anticancer properties''. Cancer Control 2002; 9: 28–35.
85) Liao Z, Mason KA, Milas L.: ''Cyclo-oxygenase-2 and its inhibition in cancer:
Is there a role? Drugs''. 2007; 67: 821–845.
86) Wiese FW, Thompson PA, Kadlubar FF.: Carcinogen substrate specificity of
human COX-1 and COX-2. Carcinogenesis 2001; 22: pp.5–10.
87) Rajinder Singh, Chiara Leuratti, Shylaja Josyula and Marek A. Sipowicz:
Lobe-spesific increase in malondialdehyde DNA adduct formation in the liver
of mice following infection with Helicobacter hepaticus. Oxford Journals Life
Sciences and Medicine Carcinogenesis. 2001; 22(8): 1281-1287.
88) Chen CC, Sun YT, Chen JJ, Chang, YJ.: Tumor necrosis factor-alpha-induced
cyclooxygenase-2 expression via sequential activation of ceramide-dependent
mitogen-activated protein kinases, and IkappaB kinase 1/2 in human alveolar
epithelial cells. Mol Pharmacol 2001; 59: 493–500.
89) Kakiuchi Y, Tsuji S, Tsujii M, Murata H, Kawai N, Yasumaru M, Kimura A,
Komori M, Irie T, Miyoshi E, Sasaki Y, Hayashi N, Kawano S, Hori M.:
Cyclooxygenase-2 activity altered the cell surface carbohydrate antigens on
colon cancer cells and enhanced liver metastasis. Cancer Res. 2002; 62: 1567–
1572.
90) Han JH, Roh MS, Park CH, Park KC, Cho KH, Kim KH, Eun HC, Chung JH.:
Selective COX-2 inhibitor, NS-398, inhibits the replicative senescence of
cultured dermal fibroblasts. Mech Ageing Dev 2004; 125: 359–366.
91) Jia XQ, Zhong N, Han LH, Wang JH, Yan M, Meng FL, Zhang SZ.: Effect of
NS-398 on colon cancer cells. World J Gastroenterol 2005; 11: 353–356.
92) Kim SJ, Jeong HJ, Choi IY, Lee KM, Park RK, Hong SH, Kim HM.:
Cyclooxygenase-2 inhibitor SC-236 [4-[5-(4-chlorophenyl)-3
114
 (trifluoromethyl)-1-pyrazol-1-l] benzenesulfonamide] suppresses nuclear
factor-{kappa}B activation and phosphorylation of p38 mitogen-activated
protein kinase, extracellular signal-regulated kinase, and c-Jun N-terminal
kinase in human mast cell line cells. J Pharmacol Exp Ther. 2005; 314: 27- 34.
93) Charlier C, Michaux C.: Dual inhibiton of cyclooxygenase-2 (COX-2) and 5-
lipoxygenase (5-LOX) as a new strategy to provide safer non-steroidal anti-
inflammatory drugs. Eur J Med Chem 2003; 38: pp.645–659.
94) Hiroki Fujita, Masafumi Kakei , Hiromi Fujishima , Tsukasa Morii,Yuichiro
Yamada , Zhonghua Qi and Matthew D. Breyer: Effect of selective
cyclooxygenase-2 (COX-2) inhibitor treatment on glucose-stimulated insulin
secretion in C57BL/6 mice. Biochemical and Biophysical Research
Communications 2007; 363: 37–43.
95) Sachiko Matsuzaki, M.D., Michel Canis, M.D., Claude Darcha, M.D.,
Radhouane Dallel, D.D., Kunihiro Okamura, M.D., and Gerard Mage, M.D.:
Cyclooxygenase-2 selective inhibitor prevents implantation of eutopic
endometrium to ectopic sites in rats. Fertility and Sterility 2004; 82(6): 1609-
1615.
96) Ali Mohammad, Sudhanshu Sharma and Showkat Ahmad Bhawni : Reversed
Phase thin layer chromatography of five co-administerea drugs with surfactant
modified solvent systems. Indian Journal of chemical Technology 2009; 16:
344-350.
97) AL-Mikhlafi Sadik, A.S.: Synthesis and evaluation of novel nonsteroidal anti-
inflammatory agents. Ph.D. Thesis, College of Pharmacy, Baghdad University,
Baghdad, 2004.
98) Ralph L. Shriner, Christine K. F. Hermann, Terence C. Morrill, David Y.
Curtin, Reynold C. Fuson: The systemic identification of organic compounds
(8th ed). John Wiley and Sons, Inc. United States of America, 2004; Chapter
3,pp. 58.
99) Ralph L. Shriner, Christine K. F. Hermann, Terence C. Morrill, David Y.
Curtin, Reynold C. Fuson: The systemic identification of organic compounds
115
 (8th ed). John Wiley and Sons, Inc. United States of America, 2004; Chapter 9,
314-348.
100) Bodansky, M.; Klausner, Y.S. and Ondetti, M.A. (Eds): Peptide synthesis (2 nd
ed.). John Wiley and Sons, New York, 1976.
101) H. Schwarz, and F. M. Bumpus, J.Am. Chem. Soc.,1959; 81: 890-897.
102) Park, M. S.; Park, H.S. Yakhak Hoeji 2003; 47: pp.276.
103) Monther F. Mahdi: Synthesis and Preliminary Pharmacological Evaluation of
New Non-steroidal Anti-inflammatory Agents. Ph.D. Thesis, College of
Pharmacy, Baghdad University, Baghdad, 2006.
104) Vogel, H.G. and Goethe, J.H.(Eds.) : Drug discover and evaluation.
pharmacological assays (2nd ed). Springer-Verlag. Berlin Heidelbers., 2002;
pp.751.
105) Assessment and alleviation of post-operative pain. Animal Welfare
Information Center Newsletter ,1998; 8: pp.3-4.
106) Chandrashekhar, S.P.; Naveen, K.J.; Amarjit, S. and Shinivas, K.K.:
Modulatory effect of COX inhibitors on sildenafil-induced antinociception.
Pharmacology 2003; 69: 183-189.
107) Francis A. Carey: Organic Chemistry (3rd ed). McGraw–Hill, New York,1996;
pp. 1092 –1124.
108) John Mc Murry: Organic Chemistry (7th ed). Thomson Learning. Inc., U. S.
A., 2008; Chapter 21, pp.794-811.
109) M. Brenner, and W.Huber, Helv. Chem. Acta., 1953; 36: pp. 1109-1115.
110) M. B. Smith, and J. March: March Advanced Organic Chemistry (6th ed). John
Wiley and Sons, Inc., New Jersey, U.S.A.,2007; pp.1411.
111) Rajesh Thapa: Rigioselectivity in free radical bromination of unsymmetrical
dimethylated pyridines. Thesis for Master of Science, Miamy University,
Oxford, Ohio, 2009.
112) Dennis D. Tanner, Tomoki C.S. Ruo, Hideki Takiguchi, Andre Guillaume,
Darwin W. Reed, B.P. Setiloane, Seet L. Tan, Christian P. Meintzer: ''On the
mechanism of N-bromosuccinimide bromination. Bromination of cyclohexane

116
 and cyclopentane with N-bromosuccinimide. J. Org. Chem. 1983; 48(16):
2743-2747.
113) M. B. Smith, and J. March: March Advanced Organic Chemistry (6 th ed). John
Wiley and Sons, Inc., New Jersey, U.S.A.,2007, pp.963.
114) Susan Budavari (Ed): The Merck Index, An Encyclopedia of chemicals, drugs
and biologicals (12th ed). Merck Research Laboratories. Division of Merck and
co., Inc. Rahway, New Jersey. U. S. A. 1996. PP. 1467.
115) Francis A. Carey, Richard J. Sundberg: ''Advance Organic Chemistry:
Structures and Mechanisms'' (5th ed). Springer Science and Business Media,
LLC, Spring Sheet, New York, U.S.A., 2007; pp. 393.
116) Marye Anne Fox, James K. Whitesell: Organic Chemistry (3 rd ed). Jones and
Barlett publisher Inc., Canada, 2004; pp.376.
117) Anand, N.: Sulfonamides and Sulfones. In Burger‫ތ‬s Medicinal Chemistry and
Drug Discovery. Wiley, New York, 1996;Vol.2 pp. 527-544.
118) N. A. Kravchenya: Selective N-acylation of 4-aminobenzenesulfonamide.
Phar. Chem. J. 1989; 32(4): 454-456.
119) C. K. Ingold: Structures and Mechanisms in Organic Chemistry (2 nd ed).
Corenell University Press, U.S.A., 1969; pp.457-463.
120) R. M. Smith: Thin-Layer Chromatography. A modern Practical Approach.
Royal Society of chemistry, U.K., 2005; pp.70.
121) Robert M. Silverstein, Francis X. Webster and David J. Kiemle: Spectrometric
Identification of Organic Compounds (7th ed). John Wiley and Sons, Inc.
U.S.A., 2005; PP. 72-108.
122) Ralph L. Shriner, Christine K. F. Hermann, Terence C. Morrill, David Y.
Curtin, Reynold C. Fuson: The systemic identification of organic compounds
(8th ed). John Wiley and Sons, Inc. United States of America, 2004; Chapter 7,
pp.194-213.
123) John Mc Murry: Organic Chemistry (7th ed). Thomson Learning. Inc., U. S. A.,
2008; Chapter 12, pp.422-429.

117
124) Ravi V, Saleem TSM, Patel SS, Raamamurthy J and Gauthaman K: Anti-
inflammatory Effect of Methanolic Extract of Solanum nigrum Linn Berries,
2009; 2(2): 33-36.

125) Anupama A. Suralkar: In Vivo Animal Models for Evaluation of Anti-

inflammatory activity. Latest Reviews. 2008; 6(2): pp. 3991.
126) Webb, E.F. and Griswold, D.E.: Microprocessor-assisted plethysmograph for
the measurement of mouse paw volume. J. Pharmacol. Meth. 1984; 12: 149-
153.
127) Shekhar Verma, Anurekha Jain and V. B. Gupta: Synergestive and Sustained
Anti-inflammatory activity of Guguul with Ibuprofen: A preliminary study.
International Journal of Pharma and Bio Science. 2010; 1(2): pp. 1-7.
128) Ahmed Z. Abdel-Azeem, Atef A. Abdel Hafez, Gamal S. El-kara Mang.
Hassan H. Farag: Chlorozoxazone esters of some Non-steroidal anti-
inflammatory carboxylic acids as mutual prodrugs. Bioorganic and Medicinal
Chemistry.2009; 17: 3665-3670.

118
Buy your books fast and straightforward online - at one of the world’s
fastest growing online book stores! Environmentally sound due to
Print-on-Demand technologies.

Buy your books online at

www.get-morebooks.com
Kaufen Sie Ihre Bücher schnell und unkompliziert online – auf einer der am
schnellsten wachsenden Buchhandelsplattformen weltweit!
Dank Print-On-Demand umwelt- und ressourcenschonend produziert.

Bücher schneller online kaufen

www.morebooks.de
OmniScriptum Marketing DEU GmbH

#BIOIPGTUS

D - 6611 Saarbrücken info@omniscriptum.com
Telefax: +49 681 93 81 567-9 www.omniscriptum.com
View publication stats