Acute and Subchronic Toxicity?of Teucrium polium Total Extract in Rats

Copyright ? 2009 by School of Pharmacy
Shaheed Beheshti University of Medical Sciences and Health Services

Original Article

Synthesis and In Vitro Leishmanicidal Effects of
Conformationally Restricted Analogues of Pentamidine

Farzin Hadizadeha,b*, Mahmoud Reza Jaafaria,b, Afshin Samieib,
Azam Mostafavirada and Ebrahim Mohammadianb

aDepartment of Medicinal Chemistry, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. bBiotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.

Four conformationally restricted analogues of pentamidine were prepared. Then, different concentrations (0.039, 0.078, 0.156, 0.312 and 0.625 mg/mL) of each compound and two positive controls (amphotericin B and pentamidine, 0.625 mg/mL), one negative control (culture medium) and one solvent control (DMSO) were prepared and placed in 24-well plates containing 50000 parasite per well. Promastigotes of Leishmania major were incubated over a period of 2 days at 25?C; subsequently, percent of viable parasite in each well determined spectrophotometrically using MTT assay. The average EC50 for compounds 4a,b and 8a,b in DMSO was 0.098, 0.410, 0.150, 0.720 mg/mL, respectively. The average EC50 for positive controls pentamidine and amphotericin B was found to be 0.062 and 0.026 mg/mL. The control solvent had no significant effect on L. major promastigotes. All compounds had significant effect compared to DMSO and were less potent than positive controls.

Keywords: Pentamidine; Conformationally restricted; Analogues; Leishmaniasis.

Introduction

Protozoal parasitic diseases continue to pose serious public health in the world. Among them, Leishmania spp. are causative agents of mortality and morbidity in the human known as leishmaniasis, and estimated to affect 12 million people, with approximately 350 million individuals at the risk, worldwide. During the last 10 years, there has been the global burden and extensive epidemics form of the disease due to human migration, particularly its coincidence with HIV and the capacity of Leishmania to infect specialized immune cells and then to inhibit induction and activation of the immune system (1, 2). Leishmaniases are transmitted by the bite of the infected female phlebotomine sandfly and manifest with visceral, cutaneous, and mucocutaneous forms (3).

They are obligatory intracellular parasites that proliferate and develop to virulence metacyclic stage in an invertebrate vector then can infect diverse vertebrate hosts. The two distinct developmental stages of Leishmania are recognized as promastigotes and amastigotes, the first form which is found within midgut of the sandfly has an elongated shape and long flagellum. However, in the mammalian hosts upon the bite of an infected sandfly, it differentiates to amastigote stage, which lack flagella and is an obligatory intracellular pathogen infecting hematopoietic cells of the monocyte/macrophage lineage as professional phagocytic cells (4, 5).

Chemotherapy for these parasitic diseases is generally ineffective mainly due to the emergence of drug-resistant strains and toxicity of the therapeutic agents (6). The pentavalent antimonials are widely used as primary therapy whereas alternative drugs include amphotericin B, pentamidine, paromomycin, and azoles (7, 8). However, resistance to antimonials is common (9) and treatments with amphotericin B and pentamidine are plagued by severe toxic side effects (10, 11).

It has been hypothesized that multiple pharmacological actions of the pentamidine (12) might be due to its conformational flexibility resulting in indiscriminate binding to both target and non-target macromolecules. Hence, as a part of our research program on the development of novel antiparasitic drug candidates, we decided to synthesize series of conformationally restricted analogues of pentamidine and investigated their biological effects against the Leishmania parasites.

Experimental

Synthesis of compounds

The synthesis of compounds 4a, b and 8a, b was accomplished according to the procedure shown in scheme 1. Pyridine-3,5-dicarboxylic acid (1) was heated with SOCl2 to give the dichloride (2), which was then reacted with 4-cyanoaniline (3a) and 4-aminobenzamidine (3b) to give pyridine -3,5-dinitrile (4a) and diamidine (4b).

Similar procedures was used to synthesize pyrrazole-3,5-dinitrile (8a) and diamidine (8b) (scheme 2).

Characterization of compounds

Melting points were determined on Electrothermal Capillary apparatus and are uncorrected. The IR spectra were obtained using a Perkin-Elmer Model 1000. 1H NMR were obtained on Bruker Ac-80 spectrophotometer and chemical shifts (δ) are in ppm relative to internal tetramethylsilane. Errors of elemental analyses were within ?0.4% of theoretical values.

General procedure for the synthesis of dinitriles (4a, 8a)

Following a known procedure (12), a solution of the appropriate acid dichloride in THF was added to a stirred solution of 4-aminobenzonitrile (2 mmol) and triethylamine (2 mmol) in THF at 0?C. After 30 min another portion of triethylamine (2 mmol) was added and the mixture stirred overnight. The solvent was removed under reduced pressure. Chromatography of the residue with appropriate solvent gave the corresponding dinitriles.

N, N?-bis(4-cyanophenyl)pyridine-3,5-dicarboxamide (4a)

A mixture of 1 (0.5 g, 3 mmol) and SOCl2 (20 mL) was refluxed for 6 h. Removal of the excess SOCl2 gave 0.6 g of 2 which was used without further purification. This compound was immediately used in the next reaction because of its instability.

Following the general procedure described above, 4a was prepared from 2 with 63% yield after chromatography (ethyl acetate). This compound was found to have following characteristics: melting point (M.P.) >300?C; 1H-NMR (DMSO-d6): δ 10.66 (s, 2H, NH), 9.23 (s, 2H, H-pyridine), 9.00 (s, 1H, H-pyridine), 7.92 (d, 4H, arom, J=10 Hz), 7.5 ppm (d, 4H, arom, J=10 Hz); IR (KBr): 3500 (NH), 2230 cm-1 (CN), 1690 (CO); elemental analysis, C21H13N5O2 (367.36) with calculated results of C, 68.66; H, 3.57; N, 19.06, and found results of C, 68.61; H, 3.43; N, 19.15.

N, N?-bis(4-cyanophenyl)pyrrazol -3,5-dicarboxamide (8a)

Following the general procedure described for 4a, it was prepared from 6 in 58% yield after chromatography (ethyl acetate). This compound was found to have following characteristics: M.P.>300?C; 1H-NMR (DMSO-d6): 8.2-7.6 (m, 10H, arom); IR (KBr): 3500 (NH), 2230 cm-1 (CN), 1690 (CO); elemental analysis, C19H12N6O2 (356.34) with calculated results of C, 64.04; H, 3.39; N, 23.58, and found results of C, 64.10; H, 3.28; N, 23.71.

General procedure for the synthesis of diamidines (4b, 8b)

A solution of the appropriate acid dichloride (2.3 mmol) in DMA (5 mL) was added stepwise to a stirring solution of arylamine (4.6 mmol) in DMA (25 mL) at 0?C. After 24 h, the mixture was treated with concentrated ammonium hydroxide. The product was collected by filtration.

N, N?-bis(4-amidinophenyl)pyridine-3,5-dicarboxamide (4b)

Following the general procedure described above, 4a was prepared from 2 in 87% yield. This compound was found to have following characteristics: M.P. 219-225?C; 1H-NMR (CD3OD): δ 9.26 (s, 2H, H-pyridine), 9.00 (s, 1H, H-pyridine), 7.86 (d, 4H, arom, J=10 Hz), 6.97 ppm (d, 4H, arom, J=10 Hz); IR (KBr): 3328 (NH), 1670 cm-1 (CO); elemental analysis, C21H19N7O2 (401.42) with calculated results of C, 62.83; H, 4.77; N, 24.42, and found results of C, 62.94; H, 4.897; N, 24.31.

N, N?-bis(4-amidinophenyl)pyrrazol -3,5-dicarboxamide (8a)

Following the general procedure described for 4b, it was prepared from 6 in 83% yield. This compound was found to have following characteristics: M.P. >300?C; 1H-NMR (DMSO-d6): 8.2-7.4 (m, 8H, arom), 7.1 ppm (s, 1H, H-pyrrazole); IR (KBr): 3300 (NH), 1680 (CO); elemental analysis, C19H18N8O2 (390.40) with calculated results of C, 58.45; H, 4.65; N, 28.70, and found results of C, 58.25; H, 4.81; N, 28.63.

Leishmania parasites

Leishmania major strain MRHO/IR75/ER was maintained with passage in BALB/c female mice. The amastigotes were isolated from lesions of infected BALB/c mice and transformed to promastigotes on NNN medium then subcultured in RPMI 1640 (Sigma) containing 10% v/v heat inactivated FCS, 2 Mm glutamine, 100 U/mL of penicillin and 100 mg/mL of streptomycin sulfate (RPMI-FCS) at 25?C. Leishmanicidal assays were conducted using stationary-phase promastigotes.

Assay for leishmanicidal activity

The antileishmanial activity against promastigotes was determined as described elsewhere (13, 14). Briefly, L. major promastigotes in stationary phase were seeded at 50.000 parasites/200?L/well in 24-well plate in RPMI-FCS. Samples 4a,b and 8a,b were dissolved in DMSO and added further 200 ?L/well to give final concentrations of 1 mg/mL and serial two fold dilutions thereof. Promastigotes were incubated over a period of 2 days at 25?C and after that percent of viable parasite in each well determined spectrophotometrically using MTT assay (15). Amphotericin B and pentamidine was used as positive control, culture media was used as negative control and DMSO alone was used as solvent control.

Statistical analysis

Statistical analysis was carried out using one-way ANOVA and multiple comparison Tukey-Kramer test was used to compare the means of different treatment groups. The EC50 was determined by Litchfield and Wilcoxon method.

Results and Discussion

Amphotericin B (0.625 mg/mL) and pentamidine (0.625 mg/mL) in DMSO killed all of the L. major promastigotes (Figures 1-4). All synthesized compounds 4a,b and 8a,b in DMSO killed L. major promastigotes dose-dependently (Figures 1-4). Dinitriles 4a, 8a showed higher activity (EC50 0.098 and 0.150 mg/mL, respectively) in comparison to diamidines 4b, 8b (EC50 0.410 and 0.720 mg/mL, respectively) (see Table 1 for EC50 values). The control solvent had no significant effect on L. major promastigotes. All compounds were weaker in comparison to positive controls pentamidine and amphotericin B (EC50 0.062 and 0.026 mg/mL, respectively).

Acknowledgement

This work was supported by grant number 85026 from Vice Chancellor of Research, Mashhad University of Medical Sciences.

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