Article pubs.acs.org/jmc
N‑Aryl-2-aminobenzimidazoles: Novel, Efficacious, Antimalarial Lead Compounds Sreekanth Ramachandran,† Shahul Hameed P.,†,¶ Abhishek Srivastava,§,¶ Gajanan Shanbhag,† Sapna Morayya,‡ Nikhil Rautela,‡ Disha Awasthy,‡ Stefan Kavanagh,∥ Sowmya Bharath,§ Jitendar Reddy,§ Vijender Panduga,§ K. R. Prabhakar,§ Ramanatha Saralaya,§ Robert Nanduri,§ Anandkumar Raichurkar,† Sreenivasaiah Menasinakai,† Vijayashree Achar,† María Belén Jiménez-Díaz,○ María Santos Martínez,○ Iñigo Angulo-Barturen,○ Santiago Ferrer,○ Laura María Sanz,○ Francisco Javier Gamo,○ Sandra Duffy,# Vicky M. Avery,# David Waterson,⊥ Marcus C. S. Lee,▽ Olivia Coburn-Flynn,▽ David A. Fidock,▽ Pravin S. Iyer,† Shridhar Narayanan,‡ Vinayak Hosagrahara,*,§ and Vasan K. Sambandamurthy*,‡ †
Department of Medicinal Chemistry, ‡Department of Biosciences, and §DMPK and Animal Sciences, AstraZeneca India Pvt. Ltd., Bellary Road, Hebbal, Bangalore 560024, India ∥ Safety Assessment, AstraZeneca, Alderley Park, Macclesfield, U.K. ⊥ Medicines for Malaria Venture, International Center Cointrin, Route de Pré-Bois 20, Post Office Box 1826, 1215 Geneva, Switzerland # Discovery Biology, Eskitis Institute for Drug Discovery, Griffith University, 170 Kessels Road, Nathan, Queensland 4111, Australia ○ Tres Cantos Medicines Development Campus, Diseases of the Developing World (DDW), GlaxoSmithKline, Severo Ochoa 2, Tres Cantos 28760, Madrid, Spain ▽ Department of Microbiology and Immunology and Division of Infectious Diseases, Department of Medicine, Columbia University Medical Center, Columbia University, 116th Street and Broadway, New York, New York 10027, United States S Supporting Information *
ABSTRACT: From the phenotypic screening of the AstraZeneca corporate compound collection, N-aryl-2-aminobenzimidazoles have emerged as novel hits against the asexual blood stage of Plasmodium falciparum (Pf). Medicinal chemistry optimization of the potency against Pf and ADME properties resulted in the identification of 12 as a lead molecule. Compound 12 was efficacious in the P. berghei (Pb) model of malaria. This compound displayed an excellent pharmacokinetic profile with a long half-life (19 h) in rat blood. This profile led to an extended survival of animals for over 30 days following a dose of 50 mg/kg in the Pb malaria model. Compound 12 retains its potency against a panel of Pf isolates with known mechanisms of resistance. The fast killing observed in the in vitro parasite reduction ratio (PRR) assay coupled with the extended survival highlights the promise of this novel chemical class for the treatment of malaria.
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INTRODUCTION Malaria caused by the protozoan parasite belonging to the genus Plasmodium continues to be a global public health problem with an estimated 219 million cases and 627,000 deaths in 2012.1 Malaria caused by Plasmodium falciparum (Pf) accounts for over 95% of all malaria cases occurring in Africa and South East Asia. The cerebral form of malaria caused by Pf leads to high mortality among children below 5 years of age. Malaria is transmitted to humans by the bite of infected female anopheline mosquitoes. The emergence of Pf strains resistant to the two former first-line antimalarial drugs, chloroquine (CQ) and sulphadoxine-pyrimethamine (SP), have seriously impeded efforts to effectively treat malaria.1 Even more worrisome is the most recent emergence of Pf strains resistant to artemisinin derivatives; the frontline agents in the treatment of malaria.2 The current treatment for malaria, recommended © XXXX American Chemical Society
by the WHO, is an artemisinin-based combination therapy of two or more drugs administered over a period of 3 to 7 days. The global efforts to treat and eliminate malaria would be immensely benefitted by introducing novel agents that require a single exposure radical cure and prophylaxis (SERCAP).3 Such a treatment regimen would entail a combination of antimalarial agents that kill the asexual blood stages, sexual gametocyte stages (block transmission), and hypnozoites in the liver (provide radical cure).3 It is envisaged that a single dose therapy would greatly reduce the emergence and dissemination of drug resistance and significantly improve patient compliance. Substantial progress has been made in the past decade to advance several novel antimalarials targeting Pf into clinical Received: May 8, 2014
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Table 1. Structure−Activity Relationship and Cytotoxicity Selectivity Index of Compounds 1−23a
a NDa: not determined. IC50 against NF54 and K1 strains of Pf were determined using a SYBR-Green based assay as previously reported.6 The cytotoxicity of compounds against a mammalian THP-1 cell line was performed as described (see Supporting Information).
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development.4 However, there is a dearth of leads with long half-life in humans that could be used in combination with suitable partner drugs to prevent the emergence of drugresistant forms of the parasite. The present study describes the discovery and optimization of an antimalarial series with the potential for long half-life in humans and activity against a panel of clinical strains harboring resistance to known antimalarial agents.
reference drugs in all of the experiments. Chloroquine exhibited an IC50 of 17.1 ± 5 nM (NF54 strain) and 347.3 ± 82 nM (K1 strain) and artemisinin an IC50 of 7.1 ± 4 nM (NF54 strain) and 6.5 ± 2 nM (K1 strain) in this assay. Structure−Activity Relationship (SAR). Initial efforts to explore the SAR requirements to improve potency focused on the optimization of the site 1 ring substitution of the benzimidazole scaffold (Table 1). The introduction of 2-pyridyl (compound 2) instead of a phenyl group at site 1 resulted in a 4-fold improvement in Pf potency (NF54 IC50, 53 nM; and K1 IC50, 80 nM). The R3 position at site 1 turned out to be critical for the observed antimalarial activity, which was evident from the activity of compounds 3−5, in which lipophilic electron withdrawing groups displayed better antiplasmodial activity. The importance of the pyridine-2-ylamino moiety for potency was further substantiated by compound 6, wherein the 3pyridyl derivative was found to be inactive. On the basis of the encouraging activity observed for the pyridine-2-ylamino moiety at site 1, this motif was retained during further medicinal chemistry optimization. The focus was then shifted to the core benzimidazole; the introduction of additional nitrogen at the C-5 position of the benzimidazole core retained the antiplasmodial activity (7) (match pair; compound 5 versus 7, Table 1). Additionally, chloro substitution at the C-4 position of benzimidazole was tolerated for potency (compound 2 versus 8, Table 1). However, converting the benzimidazole core to 4-aza benzimidazole with additional fluoro substitution completely abolished the potency (compound 2 versus 9, Table 1). This may be due to the disburbance of electron density of the benzimidazole core. Efforts were made to identify the best basic side chain at the C6 position of benzimidazoles (Table 1). Compounds with basic side chains having calculated pKa ≥ 7 (10, 12−15, and 17−18) retained their antiplasmodial activity, whereas compounds 11 and 16 with weak basic/neutral side chains were found to be inactive. Having observed a substitution effect on the antiplasmodial activity at site 1 for these inhibitors, we shifted our attention to further understand the multisubstitution pattern on the 2amino pyridines (Table 1). Toward this objective, compounds
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RESULTS AND DISCUSSION Antiplasmodial Activity and Structure−Activity Relationship (SAR). A whole cell phenotypic screen of 500,000 compounds from the AstraZeneca corporate library, assayed against the asexual blood stage of Pf, was carried out using a high content image-based approach previously reported.5 This screen led to the identification of N-aryl-2-aminobenzimidazoles 1 as a hit compound suitable for further medicinal chemistry optimization. Compound 1 displayed moderate potency against both sensitive (NF54) and resistant (K1) strains of Pf and a good cytotoxicity index (>1190-fold) against a mammalian THP-1 cell line. These findings encouraged further exploration of structure−activity relationship (Table 1) of this chemical class. The hit compound 1 consists of a core benzimidazole ring attached to 2-aminophenyl (site 1) and a basic piperazine ring at the C-6 position (site 2) (Figure 1).
Figure 1. Generic structure of the N-aryl-2-amino benzimidazole scaffold.
All compounds were screened for their antiplasmodial activity against a fully sensitive (NF54) and multidrug resistant (K1) strain of Pf using a SYBR-Green based readout.6 Chloroquine, pyrimethamine, and artesunate were included as
Scheme 1. General Synthetic Scheme for Compounds 1−11, 14, 19−20, and 22−23
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Table 2. In Vitro DMPK Profile and hERG Activity for Representative Compoundsa compd
aqueous solubility (μM)
log D at pH 7.4
human plasma protein binding (% free)
human microsomal clearance (μL/min/mg)
rat hepatocyte clearance (μL/min/106 cells)
hERG IC50 (μM)
2 4 8 12 17 22
60 1170 >1000 121 >1000 5.6
2.7 2.2 3 3 3.2 3.9
11.5 18.3 30.1 13.4 27.5 5.1
4.1 4.8 95% as determined using the following conditions: a Shimadzu HPLC instrument with a Hamilton reverse phase column (HxSil, C18, 3 μm, 2.1 mm × 50 mm (H2). Eluent A, 5% CH3CN in H2O; eluent B, 90% CH3CN in H2O. A flow rate of 0.2 mL/min was used with UV detection at 254 and 214 nm. The structures of the intermediates and end products were confirmed by 1H NMR and mass spectroscopy. Chemical shifts are reported in ppm (δ) relative to the residual solvent peak in the corresponding spectra; chloroform δ 7.26, methanol δ 3.31, DMSO-d6 δ 3.33, and coupling constants (J) are reported in hertz (Hz) (where s = singlet, bs = broad singlet, d = doublet, dd = double doublet, bd = broad doublet, ddd = double doublet of doublet, t = triplet, tt−triple triplet, q = quartet, and m = multiplet) and analyzed using ACD NMR data processing software. Mass spectra values are reported as m/z (HRMS). All reactions were conducted under nitrogen and monitored using LCMS unless otherwise noted. Solvents were removed in vacuo on a rotary evaporator. General Synthetic Procedures. N-(4-Bromophenyl)-6-(4-methylpiperazin-1-yl)-1H-benzo[d]imidazol-2-amine (1). To a solution of 4-(4-methylpiperazin-1-yl)benzene-1,2-diamine (1b, 0.4 g, 1.93 mmol) in THF (10 mL) were added 1-bromo-4-isothiocyanatobenze (1e, 0.415 g, 2.41 mmol) and EDC·HCl (0.743 g, 3.87 mmol) at room temperature. The resulting mixture was subjected to microwave irradiation in a sealed tube at 100 °C for 2 h. Product formation was confirmed by LCMS. The solvent was evaporated to dryness. The crude obtained was purified by column chromatography using 1% MeOH in DCM saturated with NH3 to get N-(4-bromophenyl)-6-(4methylpiperazin-1-yl)-1H-benzo[d]imidazol-2-amine as a white solid. Yield: 0.075 g (8.57%). HRMS calculated for [C18H20BrN5]+386.09993 [M + H]+; found, 386.09754. 1H NMR 400 MHz, DMSO-d6: δ 10.72 (s, 1H), 9.48 (d, J = 20.1 Hz, 1H), 7.73 (t, J = 2.7 Hz, 2H), 7.45 (t, J = 1.6 Hz, 2H), 7.21 (d, J = 8.5 Hz, 1H), 7.13 (d, J = 8.4 Hz, 1H), 6.95 (s, 1H), 6.86 (d, J = 1.6 Hz, 1H), 6.71 (q, J = 6.9 Hz, 1H), 3.05 (t, J = 4.9 Hz, 4H), 2.49 (t, J = 8.2 Hz, 4H), and 2.23 (s, 3H). HPLC 99.78%. 5-(4-Methylpiperazin-1-yl)-2-nitroaniline (1b). To the solution of 2-chloro-5-nitropyridin-4-amine (1a10.0 g, 57.9 mmol) in anhydrous DMF (100 mL) were added 1-methyl piperazine (12.85 mL, 11.0 mmol) and potassium carbonate (24.03 g, 17.3 mmol). The resulting mixture was heated at 130 °C for 16 h. The reaction mixture was poured into cold water. The precipitate formed was filtered and dried H
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synthetic route similar to that described earlier. HRMS calculated for [C18H19F3N6]+ 377.17016 [M + H]+; found, 377.16921. 1H NMR 400 MHz, DMSO-d6: δ 11.63 (s, 1H), 11.19 (s, 1H), 8.58 (s, 1H), 8.04 (q, J = 2.1 Hz, 1H), 7.45 (d, J = 8.6 Hz, 1H), 7.28 (d, J = 8.6 Hz, 1H), 6.98 (s, 1H), 6.80 (q, J = 2.0 Hz, 1H), 3.07 (t, J = 4.7 Hz, 4H), 2.51 (t, J = 1.6 Hz, 4H), and 2.25 (s, 3H). HPLC 99.07%. 6-(4-Methylpiperazin-1-yl)-N-(6-(trifluoromethyl) pyridin-3-yl)-1H-benzo[d]-imidazol-2-amine (6). Compound 6 was prepared by using a synthetic route similar to that described earlier. HRMS calculated for [C18 H19 F3N 6 ] + 377.17016 [M + H]+ ; found, 377.169170. 1H NMR: 400 MHz, DMSO-d6: δ 8.83 (d, J = 2.4 Hz, 1H), 8.32−8.35 (m, 1H), 7.75 (m, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.10 (d, J = 2.0 Hz, 1H), 6.92−6.95 (m, 1H), 3.31−3.32 (m, 8H), and 2.95 (s, 3H). HPLC 99.27%. 6-(4-Methylpiperazin-1-yl)-N-(5-(trifluoromethyl)pyridin-2-yl)-1Himidazo[4,5-c]pyridin-2-amine (7). Compound 7 was prepared by using a synthetic route similar to that described earlier. HRMS calculated for [C17H18F3N7]+ 378.16541 [M + H]+; found, 378.16239. 1 H NMR 400 MHz, DMSO-d6: δ 11.64 (s, 1H), 11.37 (s, 1H), 8.60 (s, 1H), 8.28 (s, 1H), 8.10 (dd, J = 2.2, 8.8 Hz, 1H), 7.50 (s, 1H), 6.77 (s, 1H), 3.36 (dd, J = 2.6, 13.1 Hz, 4H), 2.45 (s, 4H), and 2.23 (s, 3H). HPLC 99.0%. N-(5-Bromopyridin-2-yl)-4-chloro-6-(4-methylpiperazin-1-yl)-1Hbenzo[d]imidazol-2-amine (8). Compound 8 was prepared by using a synthetic route similar to that described earlier. HRMS calculated for [C17H18BrClN6]+421.05431 [M + H]+; found, 421.05341.1H NMR 400 MHz, DMSO-d6: δ 11.85 (s, 1H), 10.94 (s, 1H), 8.34 (s, 1H), 7.92−7.94 (m, 1H), 7.18−7.20 (m, 1H), 7.00 (s, 1H), 6.83 (d, J = 2.0 Hz, 1H), 3.08 (m, 4H), 2.50 (m, 4H), and 2.23 (s, 3H). HPLC purity 98%. N-(5-Bromopyridin-2-yl)-6-fluoro-5-(4-methylpiperazin-1-yl)-3Himidazo[4,5-b]pyridin-2-amine (9). Compound 9 was prepared by using a synthetic route similar to that described earlier. HRMS calculated for [C16H17BrFN7]+ 406.07911 [M + H]+; found, 406.07849.1H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 8.51 (d, J = 2.1 Hz, 1H), 8.12 (dd, J = 2.4, 8.8 Hz, 1H), 7.87 (d, J = 11.6 Hz, 1H), 7.24 (d, J = 8.7 Hz, 1H), 3.95−3.98 (m, 2H), 3.55−3.57 (m, 2H), 3.22−3.24 (m, 4H), and 2.88 (s, 3H). HPLC purity 99.51%. N-(5-Bromopyridin-2-yl)-6-(piperazin-1-yl)-1H-benzo[d]imidazol2-amine Hydrochloride (10). Compound 10 was prepared by using a synthetic route similar to that described earlier. HRMS calculated for [C16H17BrN6]+ 373.07764 [M + H]+; found, 373.07743. 1H NMR (400 MHz, DMSO-d6) δ 13.21 (bs, 1H), 13.04 (bs, 1H), 9.36 (bs, 2H), 8.52 (d, J = 2.4 Hz, 1H), 8.14 (dd, J = 2.4, 8.7 Hz, 1H), 7.55 (d, J = 8.8 Hz, 1H), 7.38 (d, J = 8.7 Hz, 1H), 7.21 (d, J = 1.9 Hz, 1H), 7.11 (dd, J = 2.1, 8.8 Hz, 1H), 3.37−3.39 (m, 4H), and 3.25−3.27 (m, 4H). HPLC 96.81%. 6-Morpholino-N-(5-(trifluoromethyl)pyridin-2-yl)-1H-benzo[d]imidazol-2-amine (11). Compound 11 was prepared by using a synthetic route similar to that described earlier. HRMS calculated for [C16H16BrN5O]+ 374.06165 [M + H]+; found, 374.06114. 1H NMR 400 MHz, DMSO-d6: δ 11.56 (s, 1H), 10.60 (s, 1H), 8.32 (d, J = 2.4 Hz, 1H), 7.90 (q, J = 2.4 Hz, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.26 (d, J = 8.4 Hz, 1H), 6.97 (s, 1H), 6.76 (dd, J = 2.0, 8.4 Hz, 1H), 3.75 (t, J = 4.8 Hz, 4H), and 3.03 (t, J = 4.8 Hz, 4H). HPLC 99.07%. Compounds 12−18 and 21 were synthesized by alternate routes. The detailed synthetic scheme for each compound is provided in the Supporting Information. 6-(1-Methylpiperidin-4-yl)-N-(5-(trifluoromethyl)pyridin-2-yl)-1Hbenzo[d]imidazol-2-amine (12). HRMS for C19H20F3N5 [M + H]+: 376.17666. 1H NMR: 400 MHz. MeOD: δ 8.67 (s, 1H), 7.95−7.98 (m, 1H), 7.37−7.41 (m, 2H), 7.18 (dd, J = 2.1, 8.0 Hz, 1H), 7.06− 7.09 (m, 1H), 3.15−3.18 (m, 2H), 2.68−2.76 (m, 4H), 2.49 (s, 3H), 2.41−2.46 (m, 1H), and 1.93−1.99 (m, 4H). HPLC purity: 98%. 6-(1-Methylpyrrolidin-3-yl)-N-(5-(trifluoromethyl)pyridin-2-yl)1H-benzo[d]imidazol-2-amine (13). HRMS calculated for [C18H18F3N5]+ 362.15926 [M + H]+; found, 362.15897. 1H NMR (400 MHz), MeOD: δ 8.66 (s, 1H), 7.94−7.97 (m, 1H), 7.40 (d, J = 8.0 Hz, 2H), 7.16 (m, 1H), 7.10 (t, J = 1.6 Hz, 1H), 3.67 (s, 1H), 3.55 (t, J = 8.8 Hz, 1H), 3.20 (t, J = 8.4 Hz, 1H), 2.97−2.99 (m, 1H), 2.78−
2.81 (m, 1H), 2.50 (s, 3H), 2.40−2.42 (m, 1H), and 2.02−2.05 (m, 2H). HPLC purity: 97.87%. 6-(Hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl)-N-(5(trifluoromethyl)pyridin-2-yl)-1H-benzo-[d]imidazol-2-amine (14). Compound 14 was prepared by using a synthetic route similar to that described for compound 1. HRMS calculated for [C20H21F3N6]+ 403.18581 [M + H]+; found, 403.18236. 1H NMR 400 MHz, DMSOd6: δ 11.70 (s, 1H), 10.92 (s, 1H), 8.58 (s, 1H), 8.05 (t, J = 6.9 Hz, 1H), 7.45 (d, J = 7.4 Hz, 1H), 7.28 (d, J = −8.4 Hz, 1H), 7.00 (s, 1H), 6.82 (d, J = 8.6 Hz, 1H), 3.62 (s, 1H), 3.55 (d, J = 7.2 Hz, 1H), 3.00− 3.06 (m, 3H), 2.77 (s, 1H), 2.14−2.25 (m, 2H), 1.75−1.86 (m, 3H), and 1.42 (d, J = 10.8 Hz, 1H). HPLC 98.84%. 6-(8-Methyl-8-azabicyclo[3.2.1]octan-3-yl)-N-(5(trifluoromethyl)pyridin-2-yl)-1H-benzo[d]imidazol-2-amine (15). HRMS calculated for [C21H22F3N5]+ 402.19056 [M + H]+; found, 402.18973. 1H NMR 400 MHz, DMSO-d6: δ 11.42 (s, 2H), 8.59 (s, 1H), 8.06 (d, J = 8.5 Hz, 1H), 7.47 (d, J = 8.6 Hz, 1H), 7.32 (t, J = 8.4 Hz, 2H), 6.99 (d, J = 8.1 Hz, 1H), 3.20 (s, 2H), 3.04 (d, J = 8.2 Hz, 1H), 2.31−2.36 (m, 2H), 2.18 (s, 3H), 2.01 (d, J = 3.9 Hz, 2H), and 1.47 (d, J = 7.0 Hz, 3H). HPLC purity: 94.6%. 2-((5-Bromopyridin-2-yl)amino)-1H-benzo[d]imidazol-6-yl)(4methylpiperazin-1-yl)methanone (16). HRMS calculated for [C18H19BrN6O]+ 415.0882 [M + H]+; found, 415.0876. 1H NMR 400 MHz, DMSO-d6: δ 11.97 (s, 1H), 10.87 (s, 1H), 8.38 (d, J = 2.2 Hz, 1H), 7.96 (dd, J = 2.5, 8.8 Hz, 1H), 7.33−7.53 (m, 3H), 7.10 (s, 1H), 3.54 (s, 4H), 2.40 (s, 4H), and 2.25 (s, 3H). HPLC purity: 98.13%. 6-((4-Methylpiperazin-1-yl)-N-(5-trifluoromethyl) pyridine-2-yl)1H-benzo[d]imidazol-2-amine (17). HRMS calculated for [C19H21F3N6]+ 391.18581 [M + H]+; found, 391.18625. 1H NMR: 400 MHz, DMSO-d6: δ 8.70 (s, 1H), 8.22 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 7.6 Hz, 2H), 7.44 (d, J = 8.4 Hz, 1H), 7.27 (d, J = 8.0 Hz, 1H), 3.45 (s, 2H), 3.38−3.44 (m, 4H), 3.08−3.17 (m, 4H), and 2.50 (s, 3H). HPLC purity; 98.43%. 2-(4-(2-((5-(Trifluoromethyl)pyridin-2-yl)amino)-1H-benzo[d]imidazol-6-yl)piperazin-1-yl)ethan-1-ol (18). HRMS calculated for [C19H21F3N6O]+ 407.18072 [M + H]+; found, 407.18021. 1H NMR 400 MHz, DMSO-d6: δ 11.59 (s, 1H), 10.80 (s, 1H), 8.58 (s, 1H), 8.04 (t, J = 1.9 Hz, 1H), 7.45 (d, J = 8.1 Hz, 1H), 7.27 (d, J = 2.0 Hz, 1H), 6.98 (s, 1H), 6.79 (t, J = 2.0 Hz, 1H), 4.48 (d, J = 7.1 Hz, 1H), 3.55 (d, J = 4.8 Hz, 2H), 3.08 (s, 4H), and 2.62 (s, 4H). HPLC purity: 95.94%. N-(5-Fluoro-6-methylpyridin-2-yl)-6-(4-methylpiperazin-1-yl)-1Hbenzo[d]imidazol-2-amine (19). Compound 19 was prepared by using a synthetic route similar to that described for compound 1. HRMS calculated for [C18H21FN6]+ 341.18900 [M + H]+; found, 341.18815. 1H NMR 400 MHz, DMSO-d6: δ 11.33 (s, 1H), 10.33 (s, 1H), 7.59 (t, J = 9.0 Hz, 1H), 7.30 (s, 2H), 6.91 (d, J = 2.2 Hz, 1H), 6.74 (d, J = 6.1 Hz, 1H), 3.06 (s, 4H), 2.51 (s, 4H), and 2.23 (s, 3H). HPLC purity: 99.63%. N-(5-Chloro-6-methylpyridin-2-yl)-6-(4-methylpiperazin-1-yl)-1Himidazo[4,5-c]pyridin-2-amine (20). Compound 20 was prepared by using a synthetic route similar to that described for compound 1. HRMS calculated for [C17H20ClN7]+ 358.15470 [M + H]+; found, 358.15022. 1H NMR (300 MHz, DMSO-d6) δ ppm 2.23 (s, 3 H) 2.44 (br. s., 4 H) 2.61 (br. s., 3 H) 6.55−6.98 (m, 1 H) 7.07−7.49 (m, 1 H) 7.66−7.90 (m, 1 H) 8.08−8.42 (m, 1 H) 10.47−11.05 (m, 1 H) 11.32−11.70 (m, 1 H). HPLC purity: 98%. N-(3-Chloro-5-(trifluoromethyl)pyridin-2-yl)-6-(1-methylpiperidin-4-yl)-1H-benzo[d]imidazol-2-amine (21). HRMS calculated for [C19H19ClF3N5]+ 410.13594 [M + H]+; found, 410.13504. 1H NMR (300 MHz, DMSO-d6) δ ppm 1.55−1.87 (m, 4 H) 1.91−2.08 (m, 2 H) 2.14−2.26 (m, 3 H) 2.82−2.95 (m, 2 H) 7.01−7.07 (m, 1 H) 7.15−7.20 (m,1 H) 7.22−7.29 (m, 1 H) 7.92−8.12 (m, 1 H) 8.33− 8.53 (m, 1 H) 12.08−12.47 (m, 2 H). HPLC purity: 98%. N-(4-Cyclopropyl-5-fluoro-6-methylpyridin-2-yl)-6-(4-methylpiperazin-1-yl)-1H-imidazo[4,5-c]pyridin-2-amine (22). Compound 22 was prepared by using a synthetic route similar to that described for compound 1. HRMS calculated for [C20H24FN7]+ 382.21555 [M + H]+; found, 382.21254. 1H NMR (300 MHz, DMSO-d6) δ ppm 0.69− 0.83 (m, 2 H) 1.06−1.17 (m, 2 H) 2.01−2.14 (m, 1 H) 2.17−2.26 (m, I
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3 H) 2.43 (br. s., 4 H) 6.56−6.73 (m, 1H) 6.73−6.95 (m, 1 H) 8.10− 8.35 (m, 1 H) 10.16−10.74 (m, 1 H) 11.40−11.70 (m, 1 H). HPLC purity: 99%. N-(4-Cyclopropyl-5-fluoro-6-methylpyridin-2-yl)-6-(1-methylpiperidin-4-yl)-1H-benzo[d]imidazol-2-amine (23). Compound 23 was prepared by using a synthetic route similar to that described for compound 1. HRMS calculated for [C22H26FN5]+ 380.22505 [M + H]+; found, 380.22443. 1H NMR (300 MHz, DMSO-d6) δ ppm 0.72− 0.80 (m, 2 H) 1.04−1.15 (m, 2 H) 1.61−1.80 (m, 4 H) 1.91−2.02 (m, 2 H) 2.02−2.10 (m, 1 H) 2.15−2.25 (m,3 H) 2.81−2.95 (m, 2 H) 6.66−6.80 (m, 1 H) 6.82−6.98 (m, 1 H) 7.06−7.46 (m, 2 H) 10.00− 10.34 (m, 1 H) 11.37−11.66 (m, 1 H). HPLC purity: 97%. Bioactivation Analysis. Chemicals. HLMs were obtained from BD Biosciences (Singapore). Reduced L-glutathione (GSH), βnicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt (β-NADPH), formic acid (ultra pure), N-acetyl cysteine (NAC), dimethyl sulfoxide, and 1-aminobenzotriazole (ABT) were purchased from Sigma-Aldrich (Bangalore, India). All compounds used in this article were synthesized internally. Methanol, water, and acetonitrile were HPLC grade from Thermo Fisher Scientific India (Mumbai, India). All the other solvents were HPLC grade, and unless otherwise specified, all of the other reagents were purchased from Sigma-Aldrich. In Vitro Incubations for the Reactive Metabolite Screen. Each compound was incubated at a concentration of 10 μM with HLM/ RLM (1 mg/mL protein) in 100 mM phosphate buffer (pH 7.4) in the presence of NADPH (2 mM). In order to assess the ability of the microsomes to bioactivate the compounds by trapping the reactive intermediates, some incubations also contained either GSH or NAC (2 mM). Incubations were performed for 60 min (t60) in a shaking incubator at 37 °C. The microsomal reaction was terminated by the addition of an equal volume of ice-cold acetonitrile, vortexed, and centrifuged at 13000 rpm for 10 min. The supernatant was transferred for HPLC-UV/MS analysis. Control incubation samples (500 μL) were quenched at time = 0 (t0) (prior to the addition of NADPH) with an equal volume of ice-cold acetonitrile. LC-MS/MS Analysis. The LC was an Agilent 1100 series system consisting of an autosampler, vacuum degasser, binary pump system, column oven, and PDA detector. The metabolites were resolved with a Phenomenex Gemini C18 column (25 × 0.46 cm, 5-μm particle size). The mobile phase consisted of a combination of acetonitrile containing formic acid (0.05%, v/v) and 10 mM ammonium formate buffer. A sample aliquot of 40 μL was injected onto the column and eluted at 1 mL/min with a gradient starting at 5% ACN, followed by 5 to 45% ACN from 5 to 30 min and maintaining 45% ACN for 35 min, and finally returning to 5% ACN in next 5 min. The UV data was collected for a scan range of 220−320 nm using an in-line Agilent PDA detector. The HPLC eluent was directly coupled to an Agilent 6520 Q-TOF mass spectrometer with 500 μL/min eluate split-flow to the LC-MS interface. The instrument was operated in electrospray ionization (ESI) mode with positive polarity. Nitrogen was used as collision gas. Capillary voltage was +4000. Drying gas was set at 350 °C with a flow rate of 13 L/min. Mass analysis was performed in autoMSMS mode with mass range (m/z) 100−800 in full scan and mass range (m/z) 40−800 in MSMS mode. In Vitro ADME and Pharmacokinetic Studies. All studies were performed as described earlier.7 In Vivo P. berghei Efficacy Model. Efficacy studies in the surrogate P. berghei model was performed as described earlier.13 P. falciparum Humanized Mouse Efficacy Model. The therapeutic efficacy of 2 against P. falciparum Pf 3D70087/N9 was studied using a “4day test” protocol as described earlier.14 Pharmacokinetic Analysis. The levels of 2 were evaluated in whole blood using a protocol described earlier.14 In Vitro Parasite Reduction Ratio (PRR) Assay. This kill rate of compound 2 was assessed using a protocol described earlier.17 Assessment of Cytotoxicity against a THP-1 Cell Line. The THP-1 cytotoxicity screen is based upon the determination of the fluorescent signal generated by the reduction of nonfluorescent resazurin (7hydroxy-3H-phenoxazin-3-one 10-oxide) to the fluorescent resorufin (Alamar blue assay). Cellular reduction of resazurin is dependent on a
pool of reductase or diaphorase enxymes derived from the mitochondria and cytosol. Therefore, Alamar blue can be used as an oxidation−reduction indicator in cell viability assays for mammalian cells.18 THP-1 cells were seeded at 40,000/well in growth medium (95 μL) and 5 μL of compound added immediately at the indicated concentrations. Five microliters of solvent (5% (v/v) DMSO in growth medium) was used in the control wells giving a final 0.5% v/v DMSO. All stock compounds in 50 mM neat DMSO were diluted in growth medium to 10% (v/v) DMSO and 5 μL used in the assay, giving a final 0.5% v/v DMSO (final assay volume 100 μL). Menadione, the standard cytotoxic agent, was used in each experiment for quality control at a top test concentration of 125 μM. Ninety-sixwell plates containing THP-1 cells with compounds or solvent were incubated for 24 h under standard cell culture conditions. Eleven microliters of stock resazurin solution (450 μM; 11.3 mg dissolved in 100 mL of PBS) was added to all wells, mixed, and incubated under standard cell culture conditions for a further 2 h. Plates were read on an Envision reader using an excitation λ of 560 nm and emission λ of 590 nm. Data is normalized to the median of the plate controls. Concentration−effect curves are fitted to the primary (fluorescent) signal and IC50 values calculated.
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ASSOCIATED CONTENT
S Supporting Information *
Details of the synthesis of all compounds and Pf drug resistant strains. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*(V.H.) Phone: +1 781-386-7725. E-mail: vinayak.
[email protected]. *(V.K.S.) Phone: +91-9740089874. E-mail: vasan2005@gmail. com. Author Contributions ¶
S.H.P. and A.S. contributed equally to this work. S.R., S.H.P., and A.R. were responsible for medicinal chemistry design and analysis. A.S. and V.H. were responsible for the bioactivation study design and interpretation. S.R. and G.S. were responsible for synthetic chemistry. S.M. performed the preparative HPLC. V.A. was responsible for dispensing the compounds for various biochemical and DMPK assays. N.R., D.A., S.M., P.V., S.B., S.R., J.R., V.P., K.R.P., R. N., and V.K.S. were responsible for biological profiling of compounds in various assays. S.D. and V.M.A. were responsible for the high throughput screening and analysis of the AstraZeneca library. M.C. L. and D. A. F. generated the cross-resistance data with the PI4K or NITD609-resistant mutant clones. B.J. and M.S.M. were responsible for generating the efficacy data in the Pf/ SCID model and assessing the kill rates in the in vitro PRR assay. A.S., S.H.P, S.R., and V.K.S. wrote the manuscript with comments from all coauthors.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the Medicines for Malaria Venture (MMV) for their financial support of this project (MMV 09/5400). Our heartfelt thanks are given to Dr. Simon Campbell and Dr. Steve Ward for their scientific advice during the course of this project. Our special thanks go to Rajkumar Thimmaiah and Subhash Rajanna for their help with the in vivo efficacy studies. Special thanks go to Syngene International, Bangalore for their support in synthetic chemistry. J
dx.doi.org/10.1021/jm500715u | J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
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Article
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ABBREVIATIONS USED Pf , Plasmodium falciparum; SAR, structure−activity relationship; PRR, parasite reduction ratio; SCID, severe combined immunodeficiency; PK, pharmacokinetics; GSH, glutathione; NAC, N-acetyl cysteine
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