Introduction

Leishmaniasis is a parasitic disease caused by 17 species of the protozoan parasite Leishmania (Chandra et al., 2005) and transmitted by the bite of a sand-fly genus Phlebotomus. Three different forms of the diseases: visceral leishmaniasis, cutaneous leishmaniasis, and mucocutaneous leishmaniasis are present in the world (Bouhlel et al., 2010). This disease is mostly endemic in 88 countries in the world, and visceral form of leishmaniasis is the most severe type among all types of leishmaniasis, known as kala-azar, which is caused by Leishmania donovani and is nearly always fatal, if untreated (Sunduru et al., 2009). More than 12 million people were infected by this disease around the world (Bhandari et al., 2010). Five lakh new cases of visceral leishmaniasis or kala-azar were found every year (Perez-Victoria et al., 2006). Currently available drugs are ineffective due to emergence of resistance among parasites; also, chemotherapy of leishmaniasis is quite difficult due to significant toxicity, variable efficacy, lack of oral bioavailability, and high cost of the therapeutic agents. So novel antileishmanial agents are urgently needed (Srinivas et al., 2009).

Mostly the nitrogen heterocycles such as quinolines, acridines, phenothiazines, pyrimidines, purines, bis-benzamidines, pyrazolo[3,4b]pyridine, benzothiazoles, and imidazolidine were reported as antileishmanial agents (Agarwal et al., 2009).

Most of the clinically used DHFR inhibitor shows less selectivity for leishmanial enzymes because the gene for pteridine reductase (PTR1) is amplified in some leishmanial mutants. Antifolate drugs simultaneously target both DHFR and PTR1 to be successful antileishmanials because PTR1 can reduce pterins and folates, and therefore, act as a bypass for DHFR inhibition. A number of compounds having pyrimidine moiety is reported to be potent inhibitor of PTR1 in Leishmania (Sunduru et al., 2006).

Based on the above observations, we have synthesized hybrid derivatives of pyrimidine containing imidazole or benzimidazole. The synthesized compounds act as an inhibitor of PTR1 as well as an inhibitor of DHFR and thus act as potential antileishmanial agents.

Chemistry

N-arylation of imidazole or benzimidazole was carried out with para-fluorobenzaldehyde using hexadecyltrimethylammonium bromide as a catalyst to yield compounds 4-(1H-imidazol-1-yl) benzaldehyde (4) or 4-(1H-benzo[d]imidazol-1-yl)benzaldehyde (5). Compounds 4 and 5 were reacted with substituted acetophenones in the presence of base (40 % NaOH in methanol) to afford substituted chalcones by Claisen Schmidt condensation (6a–d, Scheme 1I, IIa and 7a–f, 1I, IIb), (Hussain et al., 2009).

Scheme 1
scheme 1

Synthesis of 2,4,6-trisubstituted pyrimidine derivatives. Reagent and conditions: (I) K2CO3/C16H33(CH3)3N+Br−, 100 °C, stirrer, 28 h; (II) MeOH/NaOH/25 °C, stirrer, 50–60 min; (IIIa & IIIb) EtOH/NaOH/90 °C, reflux, 16 h

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The formation of pyrimidine ring from chalcone proceeds via Michael reaction followed by cyclization (Micky et al., 2006). Different substituted chalcones were cyclized by guanidine hydrochloride in the presence of base using ethanol as a solvent to yield pyrimidine derivative (8a–j, Scheme 1I, III); (Trivedi et al., 2008; Rashinkar et al., 2009).

Result and discussion

Chemistry

Absorbtion band at 3,323–3,487 cm−1 which indicates the presence of aromatic primary amino group (-NH2). IR data confirms the presence of specific functional groups present in all the synthesized compounds. Absorbtion at 813–740 cm−1; 542–594 cm−1; 1,053–1,230 cm−1, and 1,232–1,255 cm−1 indicates the presence of C–Cl, C–Br, C–F, and C–OCH3 substitution groups in synthesized compound, respectively.

The presence of 7.26–7.15 δ ppm (1H, s, pyrimidine) indicates the formation of pyrimidine ring at synthesized compound. The presence of 5.89–6.45 δ ppm (2H, s, -NH2) indicates the amino group (-NH2) at synthesized compound. 3.84 δ ppm values indicate the presence of methoxy group in synthesized compound.

Biological activity

Biological evaluation was performed against L. donovani strain, and percentage lysis inhibition was calculated by maglumine antimoniate as positive control (Table 1). 4-Cl substitution on aromatic phenyl ring increases the biological activity and 2,4 dichloro substitution on aromatic phenyl ring further increases the biological activity. A 4-OCH3 substituted benzimidazole derivative was found to be more potent than 4-OCH3 substituted imidazole derivatives. 8h and 8j were the most potent compounds of the series and shows more than 50 % inhibition against promastigotes.

Table 1 General structures of synthesized compounds and anti-leishmanial activity of with respect to control
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Conclusion

2,4,6-Trisubstituted pyrimidine derivatives were synthesized and confirmed by IR, 1H NMR and Mass spectral data and screened for antileishmanial activity. Compounds 8h and 8j exhibited 56.42 and 51.18 % inhibition against promastigotes, respectively, thus providing lead structure for antileishmanials.

Experimental

Melting points of synthesized compounds were identified by open capillary method and were uncorrected. Infrared (IR) spectra were recorded using Shimadzu FTIR (SGSITS Indore). Nuclear magnetic resonance (1H NMR) spectra were recorded on Bruker Avance II, 400 MHz Spectrophotometer (Panjab University) using DMSO as a solvent and TMS as internal standard (Chemical shift in δ ppm). Spin multiplates are given as s (singlet), d (doublet), t (triplet), and m (multiplet). Mass spectra were recorded on GCMS solution\system\tune1\Auto Tuning-EI (IICT Hyderabad). Biological activity of synthesized compounds was performed from Pharmbio Research Center Pvt. Ltd., Indore.

Synthesis of 4-(1H-imidazol-1-yl)benzaldehyde (4; Scheme 1I, Ia)

A mixture of imidazole 2 (6.8 g, 100 mmol), anhydrous potassium carbonate (13.80 g, 100 mmol), 4-fluorobenzaldehyde 1 (12.40 g, 100 mmol), hexadecyltrimethylammonium bromide (20 mg), and DMF (50 ml) was stirred for a period of 28 h at 100 °C, and after cooling to room temperature, was poured on to crushed ice (200 ml). Pale yellow crystals obtained were filtered, dried, and recrystallized from methanol (Hussain et al., 2009).

Synthesis of 4-(1H-benzo[d]imidazol-1-yl)benzaldehyde (5; Scheme 1I, Ib)

A mixture of benzimidazole 3 (11.8 g, 100 mmol), anhydrous potassium carbonate (13.80 g, 100 mmol), 4-fluorobenzaldehyde 1 (12.40 g, 100 mmol), hexadecyltrimethylammonium bromide (20 mg), and DMF (50 ml) was stirred for a period of 28 h at 100 °C, and after cooling to room temperature, was poured on to crushed ice (200 ml). Pale yellow crystals obtained were filtered, dried, and recrystallized from methanol.

Synthesis of 3-(4-(1H-imidazol-1-yl)phenyl)-1-phenylprop-2-en-1-one (6a–d; Scheme 1I, IIa)

A methanolic sodium hydroxide solution (40 %, 10 mmol) was added dropwise to a mixture of 4-(1H-imidazol-1-yl)benzaldehyde (10 mmol,), acetophenone (10 mmol), and methanol over a period of 50–60 min with continuous stirring till completion of reaction (as indicated by TLC). Precipitates obtained were filtered and washed with cold methanol–water mixture (1:10). Finally, the product was recrystallized from methanol (Hussain et al., 2009).

Synthesis of 3-(4-(1H-benzo[d]imidazol-1-yl)phenyl)-1-phenylprop-2-en-1-one (7a–f; Scheme 1I, IIb)

A methanolic sodium hydroxide solution (40 %, 10 mmol) was added dropwise to a mixture of 4-(1H-benzo[d]imidazol-1-yl)benzaldehyde (10 mmol), acetophenone (10 mmol), and methanol over a period of 50–60 min with continuous stirring till completion of reaction (as indicated by TLC). Precipitates obtained were filtered and washed with cold methanol–water mixture (1:10). Finally, the product was recrystallized from methanol (Hussain et al., 2009).

Synthesis of 4-(4-(1H-imidazol-1-yl)phenyl)-6-phenylpyridine-2-amine (8a–d; Scheme 1I, IIIa)

Guanidine hydrochloride (3 mmol) was dissolved in 12 ml 5 % ethanolic solution of NaOH. Synthesized chalcones (6a–d) was added to it and refluxed for 16 h. Solution was cooled at room temperature and poured into the crushed ice. Precipitate was filtered and washed with cold water. The crude product was dried and recrystalised from ethanol than methanol (Rashinkar et al., 2009).

4-(4-(1H-imidazol-1-yl)phenyl)-6-(4 chlorophenyl)pyrimidin-2-amine (8a)

% Yield: 42. MP (°C):234–236. IR (KBr, cm−1): 3109, 3454, 1520, 1051, 813. 1H NMR (DMSO, 400 MHz) δ: 8.33(2H, d, Ar–H), 8.20(3H, t, Ar–H), 8.05(1H, s, imidazole), 7.71–7.64(3H, m, Ar–H), 7.51(2H, d, imidazole), 7.15(1H, s, pyrimidine), 6.45(2H, s, -NH2). Mass (m/z): 347.

4-(4-(1H-imidazol-1-yl)phenyl)-6-phenylpyrimidin-2-amine (8b)

% Yield: 40. MP (°C): 136–138. IR (KBr, cm−1): 3128, 3371, 1543, 1053. 1H NMR (DMSO, 400 MHz) δ: 8.36(2H, d, Ar–H), 8.28(3H, t, Ar–H), 8.23(1H, s, imidazole), 7.72(2H, d, imidazole), 7.66–7.61(4H, m, Ar–H), 7.26(1H, s, pyrimidine), 5.89(2H, s, -NH2). Mass (m/z): 313.

4-(4-(1H-imidazol-1-yl)phenyl)-6-(4-bromophenyl)pyrimidin-2-amine (8c)

% Yield: 46. MP (°C): 230–233. IR (KBr, cm−1): 3113, 3458, 1519, 1051, 586. 1H NMR (DMSO, 400 MHz) δ: 8.31(2H, d, Ar–H), 8.10(3H, t, Ar–H), 7.94(1H, s, imidazole), 7.67–7.59(5H, m, Ar–H), 7.16(1H, s, pyrimidine), 6.31(2H, s, -NH2). Mass (m/z): 391.

4-(4-(1H-imidazol-1-yl)phenyl)-6-(4-methoxyphenyl)pyrimidin-2-amine (8d)

% Yield: 40. MP (°C): 205–208. IR (KBr, cm−1): 3107, 3454, 1514, 1051, 1255. 1H NMR (DMSO, 400 MHz) δ: 8.33(3H, t, Ar–H), 8.18(2H, d, imidazole), 7.76(3H, d, Ar–H), 7.63(1H, s, imidazole), 7.14(1H, s, pyrimidine), 7.02(2H, d, Ar–H), 6.51(2H, s, -NH2), 3.85(3H, s, -OCH3). Mass (m/z): 343.

Synthesis of 4-(4-(1H-benzo[d]imidazol-1-yl)phenyl)-6-phenylpyridine-2 amine (8e–j; Scheme 1I, IIIb)

Guanidine hydrochloride (3 mmol) was dissolved in 12 ml 5% ethanolic solution of NaOH. Synthesized chalcones (7a–f) were added to it and refluxed for 16 h. Solution was cooled at room temperature and poured into the crushed ice. Precipitate was filtered and washed with cold water. The crude product was dried and recrystallized from ethanol then methanol (Rashinkar et al., 2009).

4-(4-(1H-benzo[d]imidazol-1-yl)phenyl)-6-(4 fluorophenyl) pyrimidin-2-amine (8e)

% Yield: 42. MP (°C): 182–184. IR (KBr, cm−1):3086, 3487, 1510, 1014, 1230. 1H NMR (DMSO, 400 MHz) δ: 8.38(2H, d, Ar–H), 8.33(1H, s, imidazole), 8.28(2H, d, Ar–H), 8.20–8.16(2H, m, Ar–H), 7.84(1H, d, imidazole), 7.75(3H, t, Ar–H), 7.38–7.35(2H, m, Ar–H), 7.26(1H, s, pyrimidine), 6.04(2H, s, -NH2). Mass (m/z): 381.

4-(4-(1H-benzo[d]imidazol-1-yl) phenyl)-6-(4-bromophenyl) pyrimidin-2-amine (8f)

% Yield: 45. MP (°C): 138–140. IR (KBr, cm−1): 3086, 3410, 1521, 1234, 594. 1H NMR (DMSO, 400 MHz) δ: 8.41(3H, t, Ar–H), 8.12(2H, d, imidazole), 7.81–7.73(3H, m, Ar–H), 7.67–7.63(4H, m, Ar–H), 7.38–7.31(2H, m, Ar–H), 6.38(2H, s, -NH2). Mass (m/z): 442.

4-(4-(1H-benzo[d]imidazol-1-yl) phenyl)-6-phenylpyrimidin-2-amine (8g)

% Yield: 45. MP (°C): 185–187. IR (KBr, cm−1): 3086, 3323, 1544, 1224. 1H NMR (DMSO, 400 MHz) δ: 8.30(2H, d, Ar–H), 8.19(1H, s, imidazole), 8.10–8.07(2H, m, Ar–H), 7.92–7.8(1H, m, Ar–H), 7.67(2H,d, imidazole), 7.53–7.39(4H, m, Ar–H), 7.39–7.35(2H, m, Ar–H), 7.26(1H, s, pyrimidine), 5.28(2H, s, -NH2). Mass (m/z): 363.

4-(4-(1H-benzo[d]imidazol-1-yl) phenyl)-6-(4-chlorophenyl) pyrimidin-2-amine (8h)

% Yield: 46. MP (oC): 161–164. IR (KBr, cm−1): 3101, 3433, 1514, 1010, 742. 1H NMR (DMSO, 400 MHz) δ:8.40(3H, t, Ar–H), 8.18(2H, d, Ar–H), 7.93(1H, s, imidazole), 7.79(2H, d, imidazole), 7.65 (2H, t, Ar–H), 7.50(2H,d, Ar–H), 7.36–7.33(2H, m, Ar–H), 6.33(2H,s, -NH2). Mass (m/z): 397.

4-(4-(1H-benzo[d]imidazol-1-yl) phenyl)-6-(4-methoxyphenyl) pyrimidin-2-amine (8i)

% Yield: 48. MP (°C): 186–189. IR (KBr, cm−1):3070, 3477, 1512, 1031, 1232. 1H NMR (DMSO, 400 MHz) δ: 8.80(3H, t, Ar–H), 8.14(2H, d, Ar–H), 7.79(1H, d), 7.7(2H, d, imidazole), 7.76(1H, d, Ar–H),7.58 (1H, s, pyrimidine), 7.38–7.30(2H, m, Ar–H),7.02(2H, d, Ar–H), 6.29(2H, s, -NH2), 3.86(3H, s, -OCH3). Mass (m/z): 393.

4-(4-(1H-benzo[d]imidazol-1-yl) phenyl)-6-(2,4-dichlorophenyl) pyrimidin-2-amine (8j)

% Yield: 50. MP (°C):128–130. IR (KBr, cm−1): 3070, 3485, 1521, 1105, 736. 1H NMR (DMSO, 400 MHz) δ:8.49(1H, s, imidazole), 8.23(2H, d, imidazole), 7.78(3H, t, Ar–H), 7.76(2H, t, Ar–H), 7.59 (1H, s, pyrimidine), 7.47(1H, d, Ar–H), 7.37–7.29(3H, m, Ar–H), 6.73(2H, s,-NH2). Mass (m/z): 431.

Biological activity

The splenic culture L. donovani was made in Medium-199 (l-glutamine with Hepes buffer without NaHCO3) supplemented with 10% fetal bovine serum (pH 7.2). The logarithm phases of promastigotes (2 × 106 cells/ml) were incubated with or without the isolates along with Medium-199 at 22 °C. The samples were dissolved in 0.2% DMSO and then added to the culture in doses of 10 μg/ml. After 2 h of treatment, all tubes were centrifuged at 8,000×g for 10 min, the supernatant was decanted and the pellets were washed with 20 mM phosphate buffer saline (PBS). Each pellet was dissolved in 100 μl (2 mg/ml) of MTT in phosphate buffer saline.

All the tubes were incubated at 22 °C for 4 h and then centrifuged at 8,000×g for 10 min. All the pellets were dissolved in 500 μl DMSO and assessed by UV spectrophotometry (UV 2060 plus—Analytical India) at 570 nm. Percent of lysis of promastigotes by the compounds was calculated using the standard formula of Tim Mosmann. Chloroform (0.1% CHCl3) treatment served as control.

$$ {\text{Lysis }}\left( \% \right) = 100-\left( {T \, - \, PC} \right)/\left( {C \, - \, PC} \right) \times 100 $$
(1)

T is the test isolate, PC is the positive control—meglumine antimoliate, C is the control.