INTRODUCTION:

Malaria is a mosquito-borne infectious disease of humans and other animals caused by parasitic protozoans (a type of single cell microorganism) of the Plasmodium type.^[1] Malaria causes symptoms that typically include fever, fatigue, vomiting and headaches. In severe cases it can cause yellow skin, seizures, coma or death. ^[2] These symptoms usually begin ten to fifteen days after being bitten. In those who have not been appropriately treated disease may recur months later. ^[1
]

In those who have recently survived an infection, re-infection typically causes milder symptoms. This partial resistance disappears over months to years if there is no ongoing exposure to malaria.^[3] The disease is widespread in tropical and subtropical regions that are present in a broad band around the equator.^[2] This includes much of Sub-Saharan Africa, Asia, and Latin America. The World Health Organization estimates that in 2012, there were 207 million cases of malaria. That year, the disease is estimated to have killed between 473,000 and 789,000 people, many of whom were children in Africa.^[4] Malaria is commonly associated with poverty and has a major negative effect on economic development.^{[5, 6 ]} In Africa it is estimated to result in losses of $12 billion USD a year due to increased healthcare costs, lost ability to work and effects on tourism.^[7] Malaria is treated with antimalarial medications; the ones used depends on the type and severity of the disease. While medications against fever are commonly used, their effects on outcomes are not clear.^[8] Uncomplicated malaria may be treated with oral medications. The most effective treatment for P. falciparum infection is the use of artemisinins in combination with other antimalarials (known as artemisinin-combination therapy, or ACT), which decreases resistance to any single drug component.^[9] These additional antimalarials include: amodiaquine, lumefantrine, mefloquine or sulfadoxine/pyrimethamine.^[10] Another recommended combination is dihydroartemisinin and piperaquine.^[11,12] Antimalarial drugs using synthetic metal-based complexes are attracting research interest.^{[13, 14]}The syntheses, in vitro and in vivo studies of Rhodium and Ruthenium chloroquine complexes have been reported. ^[15] The Ruthenium complex was five times more active than chloroquine in vitro while rhonium and ruthenium complexes reduces parasitaemia by 73% and 94% in vivo without any sign of acute toxicity being observed for up to 30 days of treatment. ^[15] The synthesis, in vitro and in vivo studies of gold-chloroquine complexes which were considerably more active than chloroquinediphosphate against chloroquine-sensitive and chloroquine resistant strains of P. falciparum have also been reported. ^[16] 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) benzenesulfonamide mixed ligand have been studied ^[17]. From the results of the activities of the compounds against malaria parasites, 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 was deduced that sulfadoxine/ pyrimethamine metal complexes were more effective than sulfadoxine /pyrimethamine alone against strains of Plasmodium Berghei.Based on the attracting research interest in antimalaria metal –based drugs, we hereby report the synthesis, characterization, in vivo antimalarial studies and geometry optimization of selected metal-based Lumefantrine/artemether complexes.

MATERIALS AND METHODS:

All chemicals and solvents used in this work were of analytical grade. Artemether and Lumefantrine were obtained from Novartis Pharmaceutical company. FeSO₄. 7H₂O, ZnSO₄, CuSO₄, CoSO₄, NiSO₄, CdSO₄were purchased from Sigma-Aldrich Chemical Company. 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 metal complexes

Equimolar ratio of artemether and Lumefantrine was dissolved in 30ml methanol. Corresponding methanolic metal salt solution was added to the reaction mixture. The reaction mixture was stirred at 40^oC for 1 hour. After 1 hour, the solution was allowed to cool for 24 hours for the reaction to go to completion. The product obtained was filtered off, washed with methanol and dried in vacuum. The yield was recorded.

Antimalarial investigations:

Plasmodium berghei (NK 65) parasitized mice were obtained from Lagos State University Teaching Hospital

(LUTH) Nigeria. 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 4th 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 0.1x 10⁶cell 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

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.

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.

Geometry optimization

Geometry optimization study was performed on a window based computer using Argus lab and ACD Lab Chem Sketch software’s. Argus Lab is the electronic structure program that is based on the quantum mechanics, it predicts the potential energies, molecular structures; geometry optimization of structure, vibration frequencies of coordinates of atoms, bond length, bond angle and reactions pathway. ^[18]

RESULTS:

The melting point, yield, colour and solubility of the antimalarial drug and metal complexes re reported in Table 1. The electronic spectra, Infrared vibrational spectra frequency, % Parasitaemia and % inhibition of Plasmodium berghei with standard dose of antimalarial drug and complexes have been presented in Tables 2, 3 and 4 respectively. Suggested structures of the metal complexes are found in Figures 1–6. 3D molecular modeling and geometrical energy are presented in Figures 7 - 12

Table 1: Melting point, yield, colour and solubility of the antimalarial drug and metal complexes

Compound	Melting point ^oC	Yield %	Colour	Solubility in different solvents
Compound	Melting point ^oC	Yield %	Colour	Ethanol	Chloroform	Methanol
AL	205 - 208	---	Yellow	Soluble	Soluble	Soluble
FeAL	214 - 220	86.2	Light brown	Soluble	Soluble	Insoluble
ZnAL	210 - 218	98.8	Milky	Soluble	Soluble	Insoluble
CuAL	210 - 216	91.8	Pale blue	Soluble	Soluble	Insoluble
CdAL	215 - 230	66.7	Milky	Soluble	Soluble	Insoluble
NiAL	218 - 228	95.6	Light green	Soluble	Soluble	Insoluble
CoAL	216 - 226	92.8	Milky	Soluble	Soluble	Insoluble

A = Artemether, L = Lumefantrine

Table 2: Electronic specta of antimalarial drug and metal complexes

Compound	Wavelength (nm)	Assignment
AL	205.42, 229.55, 253.60, 261.71, 268.44, 300.89	n → π, π → π (ILCT)
FeAL	204.38, 234.07, 265.26, 300.83 334.95	n → π, π → π (ILCT) LMCT
ZnAL	194.45, 234.45, 266.38, 302.15 338.50	n → π, π → π (ILCT) LMCT
CuAL	199.34 211.07, 236.09, 267.26, 302.30 338.13	n → σ* (ILCT) n → π, π → π (ILCT) LMCT
CdAL	234.79, 204.79, 265.48, 300.34 334.61	n → π, π → π (ILCT) LMCT
NiAlL	193.71, 211.51, 236.40, 266.79, 302.84 388.42	n → σ* (ILCT) n → π, π → π (ILCT) LMCT
CoAL	203.03, 235.46, 267.10, 302.96 338.37	n → π, π → π (ILCT) LMCT

A = Artemether, L = Lumefantrine, ILCT = Intraligand Charge Transfer, LMCT = Ligand to Metal Charge Transfer

Table 3: Selected Infrared vibrational spectra frequency of antimalarial drug and metal complexes (cm^-1)

Compound	(O-H)	(C-H) Alkanes	(C=C) Alkenes	(C-O-CH₃) Stretch	(C-N) Stretch of R₂NH	(C-Cl)	S=O
AL	3402.00	2934.24	1638.00	1255.94	1024.21	825.17	1397.22
FeAL	3423.00	2916.00	1636.62	Absent	1033.46	Absent	1398.60
ZnAL	3456.58	2941.17	1643.00	Absent	1064.00	Absent	1426.57
CuAL	3440.00	2932.70	1637.61	Absent	1043.66	Absent	1409.00
CdAL	3445.37	2933.00	1644.75	Absent	1058.00	Absent	1448.95
NiAL	3399.00	2931.00	1632.00	Absent	1043.18	Absent	1405.88
CoAL	3413.00	2929.90	1631.80	Absent	1040.29	Absent	1407.00

A = Artemether, L = Lumefantrine

Table 4: % Parasitaemia and % inhibition of Plasmodium berghei with standard dose of antimalarial drug and complexes

Treatment Group/ Conc/ 26.85g Animal weight	% Parasitaemia	% Inhibition
Artemether/Lumefantrine 0.1g/ml	0.80	91.10
Ni- Artemether/ Lumefantrine 0.05g/ml	9.00	67.80
Ni- Artemether/Lumefantrine 0.1g/ml	0.00	100.00
Cu- Artemether/ Lumefantrine 0.05g/ml	3.00	80.00
Cu- Artemether/Lumefantrine 0.1g/ml	0.00	100.00
Control (Nil)	1.10	0.00

Table 5: Geometry optimization of the metal complexes

Complex	Final geometrical energy (Kcal/mol)
Fe-Artemether/Lumefantrine	607.7777
Zn-Artemether/Lumefantrine	727.0168
Cu-Artermether/Lumefantrine	697.6756
Cd-Artemether/Lumefantrine	680.1726
Ni-Artemether/Lumefantrine	649.5898
Co-Artermether/Lumefantrine	591.5027

DISCUSSION:

The mixed ligand and complexes are stable, non hygroscopic with high melting points. The melting point range of the complexes were higher than that of the antimalarial drug (Table 1). The difference in melting point suggested that the metal ions complexed with the drug. The change in colour from yellow (drug) to light brown, milky, pale blue, light green in the complexes also suggested the formation of coordination compounds. All the complexes were soluble in ethanol and chloroform but insoluble in methanol. The solubilities suggested that the complexes were mildly polar.

The electronic spectrum of the antimalarial drug (Table 2) showed absorption at 205.42, 229.55, 253.60, 261.71, 268.44, 300.89 nm. This absorption bands have been assigned n → π* and π → π* transitions also known as intraligand charge transfer ( ILCT). These transitions were due to C=C chromophoric system in Lumefantrine. The absorption bands in the electronic spectrum of Fe-artemether/ lumefantrine complex appeared at 204.38, 234.07, 265.26, 300.83 and 334.95 nm. The bands 204.38, 234.07 and 265.26 nm were assigned intraligand charge transfer ( ILCT) transitions from n → π* and π → π* . These transitions were due to the C=C chromophores in Lumefantrine. 334.95 nm absorption band was attributed to ligand metal charge transfer (LMCT). Zn-artemether/lumefantrine complex electronic absorption at 194.45, 234.45, 266.38 and 302.15 nm were attributed to n → π* and π → π* (ILCT) transitions in C=C lumefantrine chromophoric system while 338.50 nm was assigned ligand metal charge transfer (LMCT). In Cu-artemether/ lumefantrine complex, the absorption band at 199.34 nm indicated n → σ* transition because it occurred in the vacuum ultraviolent region. The bands at 211.07, 236.09, 267.26 and 302.30 nm indicated intaligand charge transfer transition (ILCT) because of the n → π* and π → π* transition in C=C chromophore. 338.13 nm was assigned LMCT. Cd-artemether/lumefantrine complex absorption bands 234.79, 204.79, 265.48 and 300.34 nm suggested ILCT transitions while 334.61nm absorptions was assigned to LMCT. In the electronic spectrum of Ni-artemether/ lumefantrine complex, 193.71 nm was attributed to n → σ* transition because it occurred in the vacuum ultraviolent region, < 200 nm. The ILCT bands were 211.51, 236.40, 266.79 and 302.84 nm while 388.42 nm was attributed to LMCT transition. The absorption bands 203.03, 235.46, 267.10 and 302.96 nm were assigned ILCT while 338.37 nm were atributed to LMCT transitions.

The infrared spectrum of the antimalarial drug (Table 3) showed vibrational frequency at 3402.00, 2934.24, 1638.00, 1255.94, 1024.21, 825.17 and 1397.22 cm-¹. These vibrational frequency have been assigned (O-H), (C-H) of alkanes, (C=C) Alkenes, (C-O) Stretch of Ethers, (C-N) Stretch of R₂NH, (C-Cl) and S=O functional groups. The spectra of the metal complexes also showed similar absorption for (O-H), (C-H) of alkanes and (C=C) Alkenes. The vibrational frequency for (C-O-CH₃) stretch in ethers and (C-Cl) were absent in the complexes. Elimination of CH₃ group of (C-O-CH₃) suggested the formation of metal –oxygen bond. These absence of C-Cl) functional groups suggested their elimination during complex formation. The vibration frequency for (C-N) Stretch was shifted in the spectra of the complexes. These shifts also suggested the involvement of lone pair electrons of nitrogen in complexation. S=O functional group appeared in the antimalarial drug at 1397.22 cm^-1. This band shifted 1426.57, 1409.00, 1448.95, 1405.88 and 1407.00 cm^-1in the spectra of the metal complexes. These shift indicated complexation through Sulphur atom of S=O group. There was no shift in the S=O vibrational frequency of Fe-artemether/lumefantrine complex.

From the results of the activities of these compounds against malaria parasites (Table 4), 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 was deduced that Ni-artermether- lumefantrine and Cu-artermether- lumefantrine complexes were more effective than artermether- lumefantrine alone against strains of Plasmodium Berghei. Percentage parasitaemia (0.1g/ml dosage) for artemether-/lumefantrine, Ni-artemether/ lumefantrine and Cu-artermether/ lumefantrine were 0.80, 0.00, and 0.00 respectively. The percentage inhibitions were 91.10, 100, and 100. The results showed that Ni-artemether/ lumefantrine and Cu-artemether/ lumefantrine complexes were more potent than artemether/ lumefantrine.

Geometry optimization of artemether/lumefantrine complexes were performed using ArgusLab 4.0.1 software. The minimum potential energy was calculated by geometry convergence function using ArgusLab software. The most feasible position for the complexes to inhibit angiogenesis and modulate host immune function was found to be in the range 591.5027-727.0168 Kcal/mol. Co-artemether/ lumefantrine exhibited the lowest geometrical energy. Based on geometry optimization, Co-artemether/ lumefantrine would be the most effective complex in the treatment of Plasmodium Berghei.

Based on the electronic and Infrared spectra characterization, the following structures (Figures 1-6) have been proposed for the metal complexes.

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Received on 17.01.2015 Modified on 22.01.2015

Res. J. Pharm. Dosage Form. & Tech. 7(1): Jan.-Mar. 2015; Page 59-68

DOI: 10.5958/0975-4377.2015.00009.9