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Synthesis, Characterization and antimicrobial activity of mixed Amodiaquine

and sulphadoxine drug – metal complexes

Conference Paper · September 2013

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 Synthesis, Characterization and antimicrobial
activity of mixed Amodiaquine and sulphadoxine
drug – metal complexes
1Obaleye, J.A., 1Lawal, A., 1Rajee A.O.*, 1Babamale H.F., 1Afolayan S.S.,
and 2Lawal M.
1Department of Chemistry, Faculty of Science, University of Ilorin, Ilorin, Nigeria.
2Department of pure and Applied Chemistry, Faculty of Science, Kebbi state University
of Science and Technology, Aliero, Kebbi
E-mail: olarajee@gmail.com*

ABSTRACT

Four mixed ligands metal complexes of [Co(AMD)(SUL)Cl2], [Cu(AMD)(SUL)Cl2],

[Ni(AMD)(SUL)Cl2] and [Zn(AMD)(SUL)Cl2] were synthesized. The complexes formed
were characterized by solubility, melting point, conductivity and infrared
spectroscopy, ultraviolet spectroscopy, NMR spectroscopy and elemental analysis. The
chemical shifts observed on the proton-NMR also revealed the formation of new
complexes. The ligands used were found to acts as monodentates. In sulphadoxine,
coordination occurs through the N atom of the NH2 group in the complexes while in
Amodiaquine, coordination occurs also through the nitrogen of the quinoline as shown
on the Infrared spectra data. Hence, tetrahedral geometry was assigned for all the
synthesized complexes. Investigation of the antibacterial study was also carried out
which indicated that the synthesized complexes exhibits higher activities than their
parent ligands.

Keywords: Sulphadoxine, Ammodiaquine, Metal Complex, Antibacterial studies

INTRODUCTION
Multi-drug resistance has been developed in most parts of the world, and world
health organization recommends that combination treatment rather than
monotherapy should be used in areas where multi-drug resistance to Plasmodium

1
falciparum is a problem. This ongoing battle against malaria is far from over. In the
1950s and 1960s, there was a massive drive to try and eradicate malaria worldwide
following the successful eradication of the disease in the United States. The
population at risk from malaria was reduced to 10%. However, the banning of
dicophane (DDT) and the concurrent emergence of chloroquine resistance led to the
collapse of this campaign. A quarter of a century later, over 300 million clinical cases
of malaria occur annually and over 40% of the world’s population is at risk of
contracting the disease. Of these cases, over one million will prove fatal [1]. The
greatest tragedy of malaria is that 90% of fatalities occur in sub-Saharan Africa, and
the overwhelming majority of those fatalities are children under the age of 5.

Malaria is still essentially a tropical disease, but continues to claim the title of one of
the leading killers among infectious diseases. The importance of developing new
antiplasmodial drugs cannot be overemphasised. The Roll Back Malaria campaign
which began in 1998 has yet to show a decrease in malaria mortality rates [2]. Over
the last decade, there has been an increasing interest in metal-containing
antiplasmodials. This trend was in part initiated by the successes of metal-
containing antitumour drugs such as cisplatin. In the 1980's and 1990’s, interest in
coordination complexes of known chemotherapeutic agents began to emerge [3, 4].
It was not long before coordination complexes of chloroquine were synthesised and
evaluated for efficacy against both chloroquine-sensitive and chloroquine-resistant
strains of P. falciparum [5–7]. It is not surprising that antiplasmodial efficacy proved
to be somewhat dependent on both metal and ligand. [8]

EXPERIMENTAL

Materials and Instrumentation: All reagents and chemicals were of analytical

grade and used as obtained from Aldrich. Amodiaquine and Sulphadoxine were
obtained as gift from Emzor Pharmaceuticals Company Limited, Isolo, Lagos,
Nigeria. Metal salts used include Copper chloride dehydrate [CuCl2.2H2O], Cobalt

2
chloride hexahydrate [CoCl2.6H2O], Nickel (II) chloride [NiCl2.6H2O] and Zinc (II)
chloride [ZnCl2.H2O]were sourced from Chemistry Department, University of Ilorin.
The IR spectra of the samples in KBr pellets were obtained in the ranges of 4000-
400 cm-1 using an IR-435 Shimadzu spectrometer. Metal Analyses were determined
by atomic absorption spectroscopy with Perkin-Elmer Spectrometer, model 3110.
UV-Vis spectra were obtained on Aquamate v4.60 spectrophotometer.
Microanalyses for C, H, O and N were performed on Perkin Elmer 204C micro-
analyser, while NMR measurements were also carried out on a Bruker AV 400 NMR
Spectrophotometer.

Synthesis of the complexes

a. Synthesis of [Cu(AMD)(SUL)Cl2] complex: 2mmol of each of Amodiaquine

and Sulphadoxine were dissolved in 10ml of each of methanol and acetone in a
conical flask and 1mmol of [CuCl2.2H2O] in 10ml of ethanol in another flask. The
bluish coloured solution observed after refluxing for 4 hours was concentrated at
50oC. Precipitate formed was allowed to cool, was filtered, washed and dried. The
product was labelled for analysis/characterization.

methanol/acetone
CuCl2 .2H2O + AMD + SUL [Cu(AMD)(SUL)Cl2]
reflux/4 hrs

b. Synthesis of [Co(AMD)(SUL)Cl2] complex: 2mmol of each of Amodiaquine

and Sulphadoxine were dissolved in 10ml of each of methanol and acetone in a
conical flask and 1mmol of [CoCl2.2H2O] in 10ml of ethanol in another flask. The
brown coloured solution observed after refluxing for 4 hours was concentrated at
50oC and left to stand for 4 days for precipitate formation. Precipitate formed was
filtered, washed and dried. The product was labelled for analysis/characterization.

methanol/acetone
CoCl2 .2H2O + AMD + SUL [Co(AMD)(SUL)Cl2]
reflux/4 hrs

3
c. Synthesis of [Zn(AMD)(SUL)Cl2] complex: 2mmol of each of Amodiaquine
and Sulphadoxine were dissolved in 10ml of each of methanol and acetone in a
conical flask and 1mmol of [ZnCl2.H2O] in 10ml of ethanol in another flask. The
cream coloured solution observed after refluxing for 4 hours was concentrated at
50oC and left to stand for 48hrs for precipitate formation. The cream coloured
precipitate formed was filtered, washed and dried. The product was labelled for
analysis/characterization.

methanol/acetone
ZnCl2 .2H2O + AMD + SUL [Zn(AMD)(SUL)Cl2]
reflux/4 hrs

d. Synthesis of [Ni(AMD)(SUL)Cl2] complex: 2mmol of each of Amodiaquine

and Sulphadoxine were dissolved in 10ml of each of methanol and acetone in a
conical flask and 1mmol of [NiCl2.6H2O] in 10ml of ethanol in another flask. The
green coloured solution observed after refluxing for 4 hours was concentrated at
50oC and left to stand for 48hrs for precipitate formation. The army green
precipitate formed was filtered, washed and dried. The product was labelled for
analysis/characterization.

methanol/acetone
NiCl2.6H2O + AMD + SUL [Ni(AMD)(SUL)Cl2]
reflux/4 hrs

Antimicrobial Test: The inhibitory action of the ligands and complexes were
screened against three human pathogenic bacteria viz; Bacillus substilis, Escherichia
coli, and klebsiella pneumonia.

The filter paper disc agar diffusion method was used. Pure Amodiaquine and
Sulphadoxine were used separately as standard for antibacterial activities test.
Nutrient agar (NA) was used as basal medium for the cultured bacteria. 0.1cm3 of
each of the compounds was applied to the agar media on which 1.0 cm diameter
wells were punched and incubated at 37oC for one to three days. 1.0% w/v of the
sterile filtered solutions of the ligands and the metal complexes were made using

4
methanol. Discs with only methanol were used as control. Inhibitory activities were
measured (mm) as the diameter of the observed inhibition zone formed around the
wall of the seeded agar plates. The antibacterial activities were based on percentage
inhibition calculated by using the average diameter of bacterial colony on the
growth medium compared with their respective control.

RESULTS AND DISCUSSION

The Cu(II), Co(II), Zn(II) and Ni(II) complexes of Amodiaquine- Sulphadoxine

ligands were synthesized by reaction of the metal chlorides with mixed
Amodiaquine and Sulphadoxine. The complexes were characterized by AAS,
Conductivity, TLC, Infrared, UV-Visible and NMR spectroscopy and antimicrobial
studies was investigated. The complexes exhibit varying solubility in the solvents.
The complexes are non-hygroscopic solids with melting points higher than the
parent ligands. The physical properties of the various complexes are collected in
Table I and Table II. The molar conductance values measured in DMSO solution (1 -3
M) for these complexes are 4.41 x 10-6, 3.54 x 10-6, 5.31 x 10-6 and 3.45 x 10-6 for the
Cu, Co, Zn and Ni complexes respectively (Table II). Similarly, from the results, the
complexes are non-electrolytes. The proposed structure of the complexes is shown
in Figures below.

Table I. Result of Solubility of the ligands and drug-metal complexes

Distilled H2O Methanol Acetone Ethanol DMSO

COMPOUND
Cold Hot Cold Hot Cold Hot Cold Hot Cold Hot
Amodiaquine NS NS NS NS NS S NS NS SS S
Sulphadoxine NS NS NS NS NS NS NS NS SS S
[Cu(AMD)(SUL)Cl2] NS NS NS NS NS NS NS NS S S
[Co(AMD)(SUL)Cl2] NS NS NS S NS S NS NS S S
[Zn(AMD)(SUL)Cl2] NS NS NS SS NS S NS NS SS S

[Ni(AMD)(SUL)Cl2] NS NS NS S NS S NS NS S S

5
Table II. Results of Physical Properties, Melting Point, Conductivity and % Metal

COMPOUND Melting Conductivity

Colour % Yield TLC Rf
point (oC) Ω-1mol-1cm2

Amodiaquine Yellow 210-212 6.10 - 0.34

Sulphadoxine White 237-238 4.83 - 0.47

[Cu(AMD)(SUL)Cl2] Blue 267-269 3.46 68 0.35

[Co(AMD)(SUL)Cl2] Brown 270-271 4.10 61 0.34

[Zn(AMD)(SUL)Cl2] Cream 251-253 4.31 77 0.36

[Ni(AMD)(SUL)Cl2] Green 310-312 3.58 64 0.48

 UV-Visible Spectroscopy Results

Table III: UV-Visible spectra of the ligands and drug-metal complexes

COMPOUND WAVELENGTH (nm) ENERGIES (cm-1) ASSIGNMENT

205 48780   *
Amodiaquine 325 33223 n  *
364 27473 n  *

235 42553   *
Sulphadoxine
304 32895 n  *
325 30769 n  *
[Cu(AMD)(SUL)Cl2]
820 12195 2Eg 2T2g

499 20040 4T1g  4A2g

[Co(AMD)(SUL)Cl2] 4T1g  4T2g
679 14738

256 39063   *
[Zn(AMD)(SUL)Cl2] 2T2g  2Eg
430 23256

562 17800 3T1(F) 3T1(P)

[Ni(AMD)(SUL)Cl2] 3T1(F) 3T2(F)
602 16600

6
The UV-Vis. Spectra of amodiaquine in methanol present three absorption bands
maxima at 205, 325 and 364 which were assigned to  *, n * and n *
transitions respectively. The UV-Vis spectra of sulphadoxine in acetone present two
absorption band maxima at 235 and 304 which were also assigned to  * and
n * transitions respectively. Copper(II) complex exhibits two absorption bands
at 325nm and 820nm which may be tentatively assigned to n * and 2Eg  2T2g
transitions respectively. The cobalt (II) complex also exhibits two bands at 499 nm
and 679 nm. These bands are assigned to 4T1g  4A2g and 4T1g  4T2g transitions
respectively. Zinc(II) exhibits two absorption bands centered at 256nm and 430nm
which may also be assigned to n  * and 2T2g  2Eg transitions respectively. All
these characteristic bands observed in the UV-Vis. spectra confirm tetrahedral
configuration for Cu(II), Co(II), Zn(II) and Ni(II) complexes respectively.

 Infrared Spectroscopy Results

Table IV: Infrared spectra data for the ligands and drug-metal complexes

COMPOUND ν(N-H) ν(S=O) ν(C=O) ν(C-O) ν(C-N) ν(O-H) ν(C-S)

3356.02 1262.31 1270.10 1220.98 3426.04

Amodiaquine - -
s s s,b b s,b
3625.80
1195.76 1396.79 1218.19 1173.60 3484.29
Sulphadoxine 3582.42 -
w,b s s,b s s,b
m,b
3538.68 1184.82 1268.55 1126.52 1108.47 3429.94 697.52
[Cu(AMD)(SUL)Cl2]
s s,b s m,b m s,b m,b
3413.14 1205.93 1356.61 1127.76 3452.17 687.45
[Co(AMD)(SUL)Cl2] -
s w,b s m w,b M
3403.53 1196.55 1357.77 1157.71 1127.56 3441.04 689.10
[Zn(AMD)(SUL)Cl2]
s w,b s s w s M
3465.73 1326.32 1157.77 1108.15 3425.03 691.34
[Ni(AMD)(SUL)Cl2] 1212.45
s s w w s,b M

The coordination of Amodiaquine occurs through the nitrogen of the secondary

amine group while in Sulphadoxine, coordination occurs also through nitrogen of

7
the primary amine group as shown on the Infrared spectra. The IR frequency values
of both ligands and their complexes have been assigned mainly for those specific
frequencies directly involved in complex formation. The infrared spectra of the
ligands were compared with those of metal complexes (Table IV). The spectra data
of the ligands and their metal complexes are in agreement with the expected range.
The strong band in the range of 3413 to 3625cm-1 was attributed to (N-H) stretching
vibration8,9. The same band was observed in the metal complexes spectra at lower
wavelength [Cu(AMD)(SUL)Cl2] (3538.7cm-1), in [Co(AMD)(SUL)Cl2] (3413.1cm-1),
[Zn(AMD)(SUL)Cl2] (3403.5cm-1) and in [Ni(AMD)(SUL)Cl2] (3465.7cm-1). The
shifting of this group to lower frequency when compared with the Amodiaquine and
Sulphadoxine free ligands suggesting a coordination of Cu(II), Co(II) Zn(II) and
Ni(II) ions, respectively through nitrogen atom of the respective amine group 10,11.
However, this observation was confirmed by C-N bending vibration which appeared
as medium band at 1120.9cm-1 in Amodiaquine and 1173.6 cm-1 in Sulphadoxine.
The band was observed to have shifted to lower frequencies in the metal complexes
coupled with reduction in intensity13.

The appearance of C-N bending at this position further supports the involvement of
nitrogen atoms in complexation with metal ions under investigation 8. Also, the
infrared spectra display strong absorption band at 697.5cm-1 [Cu(AMD)(SUL)Cl2],
687.5cm-1 [Co(AMD)(SUL)Cl2], 689.1cm-1 [Zn(AMD)(SUL)Cl2] and at 691.3 cm-1
[Ni(AMD)(SUL)Cl2] attributed to M-N vibration12. The band was conspicuously
absent in the spectra of the ligands. The appearance of M-N vibration further
supports the involvement of nitrogen in the complexation. Thus, indicating that
these amine groups are involved in coordination of all complexes. Other bands
observed in the spectra of the ligands were also observed in the metal complexes
with shifting in their position due to the effect of complexation.

 Proton NMR Spectroscopy Results

Table V: NMR spectra data of the ligands and drug-metal complexes

CH(,ppm CH(,ppm CH (, ppm) CH3(,pp NH(,ppm) OH(,

COMPOUND ) ) 4- m) Aromatic C- ppm)
I-benzene Quinoline pyrimidine Methyl NH Hydroxyl

Amodiaquine 7.26 8.00 - 1.00 4.00 5.00

Sulphadoxine 6.78 - 7.64 3.73 4.00 -

8
 7.26 8.64 1.20
[Cu(AMD)(SUL)Cl2] 7.64 4.12 5.00
6.74 8.00 3.73
7.22 8.64 1.20
[Co(AMD)(SUL)Cl2] 7.44 4.18 5.00
6.71 8.00 3.72
7.26 8.64 1.22
[Zn(AMD)(SUL)Cl2] 7.66 4.12 5.00
6.74 8.00 3.72
7.26 8.64 1.20
[Ni(AMD)(SUL)Cl2] 7.40 4.10 5.00
6.74 8.00 3.70

 Results of C,H,N & O Elemental Analysis and Metal estimation

Table VI. Elemental analysis & metal estimation of the ligands and drug-metal
complexes
Hydrogen Metal content
COMPOUND Carbon Oxygen Nitrogen
found found
found (Calc.) found (Calc.) found (Calc.)
(Calc.) (calculated)
Amodiaquine 67.01 (67.50) 6.20 (6.23) 4.32 (4.50) 11.48 (11.81) -

Sulphadoxine 48.90 (46.44) 6.43 (4.55) 21.10 (20.62) 18.62 (18.05) -

[Cu(AMD)(SUL)Cl2] 48.93 (48.13) 4.31 (4.29) 10.14 (10.02) 12.34 (12.28) 8.14 (7.96)

[Co(AMD)(SUL)Cl2] 49.20 (48.41) 4.64 (4.32) 10.08 (10.08) 12.68 (12.35) 7.69 (7.42)

[Zn(AMD)(SUL)Cl2] 48.86 (48.01) 4.30 (4.28) 10.02 (9.99) 12.38 (12.25) 8.56 (8.17)

[Ni(AMD)(SUL)Cl2] 48.61 (48.42) 4.45 (4.32) 10.14 (10.08) 12.62 (12.35) 8.56 (7.39)

The results of the elemental analysis (as shown in Table VI) of the complexes also
agreed well with 1:1:1 metal to ligands stoichiometry for all the complexes.

Table VII. Result of Antimicrobial activity of the ligands and drug-metal complexes

ʹEscherichia coli (nm) Staphylococcus (nm) Klebsiella (nm)

COMPOUND (-ve) (-ve) (-ve)

20 40 50 100 20 40 50 100 20 40 50 100

Amodiaquine
0 0 o 7.8 0 9.7 0 3.50 0 4.5 0 0
Sulphadoxine
0 3.4 0 0 2.5 0 0 0 4.5 0 0 0
[Cu(AMD)(SUL)Cl2]
26.8 22.2 13.5 37.0 0 0 0 0 15.5 17.0 7.8 17.1
[Co(AMD)(SUL)Cl2]

9
 23.4 25.6 14.4 38.5 0 0 0 0 16.5 15.7 7.2 15.3
[Zn(AMD)(SUL)Cl2]

0 0 34.9 35.6 0 0 0 3.1 15.7 16.9 8.9 16.9

[Ni(AMD)(SUL)Cl2]

From the result in Table 5 above, it is evident that the overall zone of inhibition
against bacterial species is highest in Co(II) complex, followed by Cu(II) complex
while Cd(II) complex appears to have the least zone of inhibition. It is also evident
that nearly all the metal complexes posses significant zone of inhibition acting
against bacterial species than their parent ligands thus, giving the metal-chelators
potentiality as antidotes for metal–overload.

Structure of the Complexes

The results obtained from spectroscopic analysis gave the tentative structure of the
complexes. This mode of coordination correspond to those in literatures [6,7].

Evidence of formation of complex was demonstrated by examining the IR and UV

spectra carefully. The suggested structure can be seen in the figure below.

N Cl

HO

N N

Cl
M
Cl
OCH3
O
H OCH3
NH S N
O
N N

Where M = Cu(II), Co(II), Zn(II) and Ni(II)

Figure: Proposed Structures of Amodiaquine - Sulphadoxine metal complexes

10
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