Synthesis, Characterization and Antimalarial Studies of Cd(II), Cu(I) and Ni(II) Complexes of 5-(4-chlorophenyl)-6-ethyl-2,4-Pyrimidinediamine and 4-Amino-N-(5,6-dimethoxy-4-pyrimidinyl) benzenesulfonamide Mixed Ligand

I.E. Otuokere¹, M.C. Ndukwe¹ and D. Akachukwu²

¹Department of Chemistry, Michael Okpara University of Agriculture, Umudike

²Department of Biochemistry, Michael Okpara University of Agriculture, Umudike

*Corresponding Author E-mail: tosmanbaba@yahoo.com

ABSTRACT:

Cd(II), Cu(I) and Ni(II) complexes of 5-(4-chlorophenyl)-6-ethyl-2,4-pyrimidinediamine (pyrimethamine) and 4-Amino-N-(5,6-dimethoxy-4-pyrimidinyl)benzenesulfonamide (sulfadoxine) mixed ligand have been synthesized. The melting point, solubility, yield and colour of the complexes were determined. The mixed ligand and complexes are stable, non hygroscopic, exhibited high melting points and are soluble in polar solvents. The complexes were characterized based on ultraviolet- visible and infrared spectroscopy. The electronic spectra of the ligands and complexes showed intraligand charge transfer (ILCT), ligand to metal charge transfer (LMCT) and d→d transitions. Infrared spectrum of cadmium complex suggested coordination through NH₂ groups of pyrimethamine and sulfadoxine. The infrared spectra of Cu(I) and Zn(II) suggested complexation through one NH₂ group of pyrimethamine (monodentate) and S=O group of sulfadoxine (bidentate). A trigonal geometry was suggested for the metal complexes. Antimalarial investigations showed that sulfadoxine/pyrimethamine - metal complexes are more effective than sulfadoxine/pyrimethamine alone against strains of Plasmodium berghei.

KEYWORDS: Sulfadoxine, pyrimethamine, mixed ligand, metal complexes, antimalarial. spectroscopy.

INTRODUCTION:

Malaria continues to be one of the major public health problems in Africa, Asia and Latin America. Plasmodium falciparum malaria is estimated to be the direct cause of 500 million cases and over 1 million deaths per year, mostly in women and children under the age of 5 years ^1,
2. In children, progression of disease from mild to severe is particularly rapid ³. Human malaria is caused by parasite of the genus Plasmodium. Four species are known to cause human malaria namely, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae. Nevertheless, P. falciparum has been found to be the most lethal of all human malaria parasites. This increase in malaria burden is largely accounted for by increasing antimalarial resistance.

For decades, chloroquine was the most widely used treatment of malaria, but resistance has increased to such an extent that it has become ineffective in almost all malaria endemic countries⁴.A fixed-dose combination of sulfadoxine and pyrimethamine, the usual successor to failing chloroquine, has been widely implemented in the last decade and is now one of the most widely used antimalarial treatments in the world ⁴. Sulfadoxine-pyrimethamine has the great advantage that the entire treatment can be given as a single dose. Sulfadoxine and pyrimethamine are folic acid antagonists. Sulfadoxine inhibits the activity of dihydropteroate synthase whereas pyrimethamine inhibits dihydrofolate reductase⁴. Sulfadoxine and pyrimethamine are active against the asexual erythrocytic stages of Plasmodium falciparum ⁵ The dihydrofolate reductase (DHFR) domain of Plasmodium falciparum bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is one of the few well-defined targets in malarial chemotherapy ⁵. The rapid emergence of antifolate resistant P. falciparum has unfortunately compromised the clinical utilities of the drugs, and thus highlights the urgent need to search for new effective antifolate antimalarials.

However, the medicinal uses and applications of metals complexes are of increasing clinical and commercial importance. The essential trace metals cannot be over emphasized in a living system. Transition metal ions are responsible for the proper functioning of different enzymes ⁶. Metal ions play a vital role in all living systems and any malfunctioning of these ions can initiate a number of physiological anomalies and symptoms of clinical disorders⁷. The synthesis, characterization and antimalarial studies of sulfadoxine-pyrimethamine complexes of Fe (III), Co(II) and Ni(II) has been reported ⁸. Obaleye reported that the potency of sulfadoxine-pyrimethamine increased when coordinated to transition metal ions ⁸. The synthesis, characterization, antimicrobial and toxicology study of Co(II), Mn(II) and Ni(II) complexes of pyrimethamine mixed with sulfadoxine showed that the metal complexes are not toxic ⁹.

In our effort to search for novel antimalarial drugs, we hereby report the synthesis, characterization and antimalarial studies of Cd(II), Cu(I) and Ni(II) complexes of 5-(4-chlorophenyl)-6-ethyl-2,4-Pyrimidinediamine and 4-Amino-N-(5,6-dimethoxy-4-pyrimidinyl) benzene sulfonamide mixed ligand.

MATERIALS AND METHODS:

All chemicals and solvents used in this work were of analytical grade. 5-(4-chlorophenyl)-6-ethyl-2,4-Pyrimidinediamine (Pyrimethamine) and 4-Amino-N-(5,6-dimethoxy-4-pyrimidinyl) benzenesulfonamide (sulfadoxine) were obtained from Madreich Limited, India. The melting points were determined by capillary method. The Infrared spectra of the ligand and complexes were carried out using FT-IR spectrometer by Perkin Elmer (Model Spectrum BX) equipped with caesium widow (4000-350cm^-1) in KBr pellets. The UV- visible spectra of the complexes in solution were scanned between 200 – 800 nm on a Perkin Elmer model spectrum BX using chloroform as the solvent. The melting of the ligand and complexes were determined using capillary tube method

Synthesis of the complexes:

Equimolar ratio of Pyrimethamine/Sulphadoxine mixed ligand and CdCl₂ were accurately measured into a beaker. 50 cm³ of distilled water was used as solvent. The solution was mixed thoroughly in a round bottom flask and was refluxed for 2 hours. The solution was allowed to cool at room temperature and was then dried in a dessicator. Yield was recorded. The same procedure was performed using CuCl₂ and ZnCl₂.

Antimalarial investigations:

Plasmodium berghei (NK 65) parasitized mice were obtained from Lagos State University Teaching Hospital (LUTH). Swiss mice were obtained from College of Veterinary Medicine in Michael Okpara University of Agriculture, Umudike’s animal house.

Inoculation of parasite:

The parasite was inoculated on 4^th November 2014. NK-65 Plasmodium Berghei was obtained from the infected Albino Swiss mice using a haemotocuit capillary tube through an ocular puncture. 0.1ml of the infected blood was added to 5ml of saline water (pH 7.0). A preparation 0.110⁶cell per ml was obtained. 0.2ml was taken from the already prepared solution and inoculated into each of the experimental mice intraperitoneally (exclusion of the control).

Determination of % parasitemiea ⁹

After 5 days, the degree of % parasitaemia was determined. A thin blood smear film of the blood samples collected from the tail of each mouse was made on a clean grease free slide. The film was allowed to air dry and was stained using Lishman stain. The films were air dried after wash off the stains with water and then viewed under a binocular microscope using oil immersion objective. The percentage of the infected Red Blood Cells (RBCs) was determined by enumerating the number of infected RBCs using a haematology tally counter in relation to the number of uninfected RBCs.

Number of infected cells 100

% Parasitaemia =---------------------------------- × -------

Total number of RB cells counted 1

The parent drug and the complexes were administered to the mice orally. The inhibitory of the complexes administered on the mice was based on the standard dose per the animal body weight. The inhibitory values for the parent drugs and complexes were calculated.

RESULTS AND DISCUSSION:

The melting point, yield, colour and solubility of the mixed ligand and complexes have been summarized in Table 1. The electronic absorption spectra of the mixed ligand and the complexes have been reported in Figures 1- 4, while the infrared spectra are presented in Figures 5–8. The Inhibition of Plasmodium Berghei with standard dose of Antimalarial drugs and their complexes have been reported in Table 2.

Table 1: Melting point, yield, colour and solubility of the mixed ligand and complexes.

Compound	Melting point	% Yield	Colour	Solubility in different solvents
Compound	Melting point	% Yield	Colour	Ethanol	Methanol	Acetone	H₂O
SP	190 - 194		white	VS	VS	VS	VS
Cd(II)SP	198 - 200	53.6	Milky	VS	VS	VS	VS
Cu(I)SP	202 - 205	89.0	Green	VS	S	Sp.S	VS
Zn(II)SP	220 - 222	22.9	Milky	VS	VS	VS	VS

SP = sulfadoxine-pyrimethamine, VS = very soluble, S = soluble and Sp.S = sparingly soluble

Table 2: Inhibition of Plasmodium berghei with standard dose of Antimalarial drugs and their complexes.

Treatment Group/ Conc./ 25.37g Animal weight	% Parasitaemia	% inhibition
Sulfadoxine-pyrimethamine 0.10g/ml	0.40	96.30
Cd(II)SP 0.10g/ml	0.20	98.20
Cu(I)SP 0.10g/ml	0.00	100.00
Zn(II)SP 0.10g/ml	0.00	100.00
Control nil	1.10

SP = sulfadoxine-pyrimethamine

The mixed ligand and complexes are stable, non hygroscopic with high melting points and are soluble in polar solvents. The electronic spectrum of the sulfudoxine / pyrimethamine mixed ligand (Figure 1) appeared in the ultraviolet region only. The absorption band at 192.63 nm which is in the vacuum ultraviolent region has been assigned n-σ* transition. The absorption bands 222.38, 281.02, 318.65 nm have been assigned π-π* transition in the ligand. The chromophores that might exhibit this type of transitions are S=O, C=C and C=N respectively. These transitions are also called Intra-ligand charge transfer (ILCT). In the electronic spectrum of Cd(II)SP complex (Figure 2), the absorption band at 197.00 nm which is in the vacuum ultraviolet region have been assigned n- transition. The absorption band 227.63 and 274.89nm have been assigned π-π* transition. The absorption band at 282.77 nm have also been assigned ligand to metal charge transfer (LMCT). The absorption bands of the Cu(I)SP complex ( Figure 3) appeared in the ultraviolet and visible region. In the electronic spectrum of the Cu(I)SP complex, the absorption band at 196.13 nm which is in the vacuum ultraviolet region has been assigned n-σ* transition. The absorption 320.40, 328.28 and 382.54nm, have also been assigned π-π* transition. The chromophores that might exhibit this type of transitions are S=O, C=C and C=N. The absorption band at 430.67nm indicated ligand to metal charge transfer (LMCT). The bands at 781.62 and 790.37 nm have been assigned to d→d transition (E_gT_2g). Lastly in the Zn(II)SP complex spectrum, (Figure 4) the absorption bands 218.01, 239.01, and 268.77 nm, have been assigned π-π*, while the peaks at 295.02 and 316.03 nm were attributed to ligand to metal charge transfer (LMCT)⁸.

The IR spectra of the complexes were compared with those of the free ligand in order to determine the involvement of coordination sites in chelation. Characteristic peaks in the spectra of the ligand and complexes were considered and compared. The infrared spectra of the mixed ligand, Cd(II), Cu(I) and Zn(II) complexes have been reported in Figures 5, 6, 7 and 8 respectively. In the Infrared spectrum of the mixed ligand, the vibrational frequency 1155.24 and 1313.12 cm^-1 have been assigned ν(S=O). In the metal complexes ν(S=O) functional group have been shifted to 1146.82cm^-1 and1295.10cm-1(Zn(II)SP complex),1141.25 and 1297.90 cm^-1 (Cu(I)SP complex), these blue shifts indicated the involvement of S=O in complexation. The coordination of Zn and Cu to S=O will cause a weakening of the S=O bond since the electron density was increased and consequently the decrease in vibration frequency to a lower wave number. There was no shift in the S=O vibrational frequency for the Cd(II)SP complex, implying that cadmium did not coordinate with S=O. In the spectrum of the mixed ligand, the vibrational frequency 3456.58 and 3381.78 cm^-1have been assigned to ν(N-H) stretching vibration. In the spectrum of Cd(II) SP complex, the ν(N-H) stretching vibration shifted to 3373.54, 3462.1 and 3240.18 cm^-1.These shifts indicated the involvement of three –NH groups in coordination. The shift from 3381 cm^-1 in the mixed ligand to 3412.00 cm^-1 in the Cu(I)SP complex suggested the involvement of –NH group in complexation. In the Zn(II) complex spectrum the ν(N-H) stretching vibration shifted to 3535.01 cm^-1 .This shift also suggested coordination through –NH group. In the low frequency region, vibration frequency 413.98 and 687.5cm^-1 for Cu(I) SP complex and 481.11cm^-1 , 662.93cm^-1 for Zn(II)SP complex, have been attributed to metal-oxygen (M-O) bond and metal-nitrogen (M-N) bond respectively. The frequency 543.37cm^-1 in Cd(II) complex also suggested metal- nitrogen bond (M-N) bond ¹⁰.

From the results of the activities of these compounds against malaria parasites (Table 2), it was evident that the addition of the metal to the mixed ligand did not impede/hinder the therapeutic value of the mixed ligand. Thus, it can be deduced that sulfadoxine/pyrimethamine-metal complexes were more effective than sulfadoxine /pyrimethamine alone against strains of Plasmodium Berghei. Percentage parasitaemia for sulfadoxine /pyrimethamine, Cd(II), Cu(I) and Zn(II) complexes were 0.40, 0.20, 0.00, and 0.00 respectively. The percentage inhibitions were 96.30, 98.20, 100, and 100. The results showed that Zn(II)SP and Cu(I)SP complexes were more potent than Cd(II)SP complex. However, the Cd(II)SP complex still had an inhibitory value higher than that of the parent drug.

Based on spectroscopic investigations, the following structures (Figures 9, 10 and 11) have been proposed for the metal complexes.

REFERENCES:

1 Guerra CA, et al. The limits and intensity of Plasmodium falciparum transmission: implications for malaria control and elimination worldwide. PLoS Med. 5; 2008: E38.

2 Lewison G and Srivastava D. Malaria research, 1980–2004, and the burden of disease. Acta Trop. 106; 2008: 96–103

3 Greenwood BM et al. Mortality and morbidity of malaria among children in a rural area of The Gambia, West Africa. Trans. R. Soc. Trop. Med. Hyg. 81; 1987: 478 – 486.

4 Venkata RBV et al. HPLC Method Development and Validation for Simultaneous Estimation of Sulfadoxine and Pyrimethamine in Tablet Dosage Form. International Journal of Pharma Sciences, 3(4); 2013: 295-298

5 Karen IB et al. Sulfadoxine-pyrimethamine pharmacokinetics in malaria: Pediatric dosing implications, Cli. Pharmacol.&Therapeu. 80; 2006: 582–596.

6 Roat-Malone RM. A short course, Bioinorganic Chemistry, 2^nd edition, John Wiley & Sons, Inc., 2007: 3-9.

7 Wattoo MHS. Bioanalytical investigation of bottled mineral water and soft drinks. M. Phil Thesis, University of Sindh, Jamshoro, Pakistan.

8 Obaleye JA et al. Some metal-antimalarial drug complexes: synthesis characterization and their effect against malaria parasites, Proceedings of the Second Conference on Science and National development, 2006: 114 – 119.

9 Ogunniran KO Synthesis, antibacterial, and toxicology study of Mn(II), Co(II) and Ni(II) metal complexes of Sulfadoxine mixed with pyrimethamine, Canadian Journal of Pure and Applied Sciences, 7(3);2013: 2619 – 2627.

9 Gracia LS. Diagnostic Medical Parasitology, 4^thedition ASM press Washington D.C., 2001

10 Nakamoto K. Infrared and Raman spectra of inorganic, coordination compounds, 5^th edition, John Wiley and Sons, Part A and B, New York, 1998.

Received on 12.12.2014 Accepted on 19.12.2014

Asian J. Pharm. Tech. 2014; Vol. 4: Issue 4, Oct.-Dec., Pg 211-217