Antimalarial Pyrido[1,2- a ]benzimidazoles

ARTICLE pubs.acs.org/jmc Antimalarial Pyrido[1,2-a]benzimidazoles Albert J. Ndakala,† Richard K. Gessner,† Patricia W. Gitari,† Natasha October,† Karen L. White,§ Alan Hudson,|| Foluke Fakorede,^ David M. Shackleford,§ Marcel Kaiser,# Clive Yeates,¥ Susan A. Charman,§ and Kelly Chibale*,†,‡ Department of Chemistry, and ‡Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa § Centre for Drug Candidate Optimisation, Monash University (Parkville Campus), 381 Royal Parade, Parkville, Victoria 3052, Australia Pharmacons, Kent, U.K. ^ Tropical Diseases Research, World Health Organization, 20 Avenue Appia, CH-1211, Geneva 27, Switzerland # Swiss Tropical and Public Health Institute, Postfach, Socinstrasse 57, 4002 Basel, Switzerland ¥ InPharma Consultancy, Herts, U.K. ) † bS Supporting Information ABSTRACT: A novel class of antimalarial pyrido[1,2-a]benzimidazoles were synthesized and evaluated for antiplasmodial activity and cytotoxicity following hits identified from screening commercially available compound collections. The most active of these, TDR86919 (4c), showed improved in vitro activity vs the drugresistant K1 strain of Plasmodium falciparum relative to chloroquine (IC50 = 0.047 μM v 0.17 μM); potency was retained against a range of drug-sensitive and drug-resistant strains, with negligible cytotoxicity against the mammalian (L-6) cell line (selectivity index of >600). 4c and several close analogues (as HCl or mesylate salts) showed significant efficacy in P. berghei infected mice following both intraperitoneal (ip) and oral (po) administration, with >90% inhibition of parasitemia, accompanied by an increase in the mean survival time (MSD). The pyrido[1,2-a]benzimidazoles appeared to be relatively slow acting in vivo compared to chloroquine, and metabolic stability of the alkylamino side chain was identified as a key issue in influencing in vivo activity. (1994), artemether/lumefantrine (1999), atovaquone/proguanil (1999), chlorproguanil/dapsone (2003), but all come with some issue limiting use.3 There is thus a clear need to develop new more affordable and effective antimalarials.4 As part of a TDR collaboration with the Belgian company Tibotec, in 2000 a small library of 1440 diverse nonpropriety compounds donated by SPECS were screened against a panel of protozoa in vitro. This led to the identification of a pyrido[1,2-a]benzimidazole coded TDR15087 (1, Figure 1), with moderate in vitro activity toward P. falciparum GHA and W2 strains (IC50 = 0.17
0.37 μM), significant because there is no published prior art relating to the antimalarial activity of pyrido[1,2-a]benzimidazoles, although derivatives related to 1 have previously been investigated for antifungal,5 antibacterial,6 and antitumor6
11 activity. Other pyrido[1,2-a]benzimidazoles were then sought to further explore the in vitro antimalarial structure
activity relationships (SARs). Initially this involved the selection of 535 ’ INTRODUCTION Malaria continues to take an enormous toll on human health, particularly in tropical regions. The drugs used to treat this disease are far from ideal, and many of these were introduced decades ago. The utility of these medicaments in resource-poor settings is limited by a number of factors, such as high cost, poor compliance, drug resistance, low efficacy, and poor safety.1 Malaria results from infection with four different species of the genus Plasmodium, all transmitted by mosquitoes, namely, falciparum, vivax, ovale, and malariae. The most important of these in terms of virulence and mortality is P. falciparum, although P. vivax also has a huge impact on populations with regard to morbidity.2 The enormous public health problem posed by malaria across the developing world is reflected by the grim estimates that each year the disease causes between 1.7 and 2.5 million deaths. Over the past few decades the mainstays of antimalarial chemotherapy, chloroquine and pyrimethamine/ sulfadoxine, have been significantly compromised in many regions by the spread of drug-resistant parasites. To counter this, a range of newer drugs and combinations have gradually been introduced into use, e.g., mefloquine (1984), artemisinins r XXXX American Chemical Society Received: A February 28, 2011 dx.doi.org/10.1021/jm200227r | J. Med. Chem. XXXX, XXX, 000–000 Journal of Medicinal Chemistry ARTICLE commercially available analogues, primarily on the basis of providing diversity around the pyridobenzimidazole “core”, which were evaluated in a medium throughput screen (MTS) using a multidrug resistant strain of P. falciparum (K1), with cytotoxicity assessed against the murine L-6 cell-line; actives from the MTS were re-evaluated for IC50 against both P. falciparum and L-6. From this exercise, 49 compounds were identified with P. falciparum IC50 of <0.1 μg/mL; notable were the N-benzylpiperazinyl derivatives TDR35885 (2) and TDR44047 (3) (Figure 1), which showed good selectivity and greater activity in vitro compared to 1 (Table 2). However, 1
3 all proved to be inactive in the standard P. berghei mouse model at doses up to 4  100 mg/kg ip, most likely because of a combination of poor solubility and metabolic stability. To find compounds active in vivo in the P. berghei mouse model, we have investigated the antimalarial SAR of pyridobenzimidazoles further. Here we describe part of these investigations focused on 3-aryl derivatives, with alkylamino side chains (Figure 2). Metabolic stability emerged as a significant factor influencing activity in vivo, and in vitro microsome stability studies proved to be useful in guiding the selection of compounds for animal assessment. ’ RESULTS AND DISCUSSION Chemistry. A relatively straightforward synthetic approach (Scheme 1) was followed for the synthesis of target pyrido[1,2-a]benzimidazoles, adapting published procedures.6
12 The final stage involving nucleophilic substitution with the appropriate amine was initially carried out with external heating, requiring relatively long reaction times of up to 18 h; microwave irradiation was subsequently found to reduce the reaction time to approximately 20 min. As the pyridobenzimidazoles generally show low aqueous solubility, hydrochloride salts were made of compounds selected for in vivo evaluation to aid formulation; for a few compounds mesylate salts were also made for comparison to provide confidence that the salt form was not significantly affecting activity. Chloroquine, administered as the free base, shows significantly lower activity in the P. berghei mouse model compared to the diphosphate salt, the standard used in our studies. Hydrochloride salts were initially prepared by bubbling excess HCl(g) through a mixture of the free base in dichloromethane or more conveniently with HCl(aq) in methanol; the compounds in the free base form were generally insoluble in methanol, and solubilization occurred on addition of the aqueous acid. The latter method allowed for easy isolation of the salts by removal of the solvent followed by trituration with dichloromethane to remove any impurities. Mesylate salts were prepared by addition of methanesulfonic acid to a solution of the appropriate freebase in dichloromethane. Biology. In Vitro Activity. Initial SAR studies (Figure 2, Table 1) involved exploration around the aminoalkyl side chain and variation in substitution at position R1 (including CF3 and positions 30 and 40 substituted aromatic groups). Compounds 4a
10f were evaluated for antimalarial activity against the multidrug-resistant P. falciparum K1 strain and cytotoxicity against the mammalian L-6 cell line (Table 1). Many of the compounds showed antimalarial activity comparable to that of chloroquine (IC50 = 0.17
0.20 μM), with compound 4c being the most active (IC50 = 0.047 μM, ∼3.5 times more potent than chloroquine) and 6a the least active (IC50 = 4.48 μM, ∼22 times less potent than chloroquine and ∼95 times less potent than 4c). In general lipophilicity appears to be an important factor influencing in vitro activity and selectivity, although other structural features in the alkylamino side chain also seem to have a significant influence: 1. N-Alkylated derivatives (R3 = N(Me)2, N(Et)2, or N(H)(Et)) showed greater potency (∼2
20 times) than their N-dealkylated (R3 = NH2) counterparts and also had more favorable selectivity to L-6. 2. Phenyl or monosubstituted aryl groups at R1 in place of a CF3 group showed superior potency of up to 10-fold in all cases, and activity was generally further enhanced when the R1 aryl group was substituted (CF3, Cl, or F) at the 40 position. Figure 1. Pyrido[1,2-a]benzimidazole screening hits with potent activity toward P. falciparum. Figure 2. Target pyrido[1,2-a]benzimidazoles for SAR exploration. Scheme 1. General Synthetic Approach to 4-Cyanopyrido[1,2-a]benzimidazolesa a Reagents and conditions: (i) NH4OAc, 140
150 C, 30
60 min; (ii) POCl3, reflux, 2 h; (iii) DMF or THF, Et3N, 80
90 C, 18 h or microwave (150 W), 20 min; R1, R2, R3, n as defined in Table 1. B dx.doi.org/10.1021/jm200227r |J. Med. Chem. XXXX, XXX, 000–000 Journal of Medicinal Chemistry ARTICLE Table 1. In Vitro Antimalarial Activity Evaluation of Pyrido[1,2a]benzimidazole Derivatives IC50 (μM)a R1 compd R2 R3 log DpH7.4 b n P. fal (K1) chloroquine 0.17
0.20 artemisinin 0.006
0.011 podophyllotoxin cytotox (L6) SI 0.009
0.014 4a 4-CF3Ph H NH2 1 1.80 1.49 5.11 3.4 5a 3-CF3Ph H NH2 1 1.80 1.00 11.7 11.7 6a Ph H NH2 1 0.83 4.48 18.9 4.2 7a CF3 H NH2 1 1.37 3.09 40.1 13 4b 4-CF3Ph H N(Me)2 1 2.45 0.13 >210 >1600 5b 3-CF3Ph H N(Me)2 1 2.45 0.29 7.14 24.6 6b Ph H N(Me)2 1 1.48 1.49 >80 >50 7b CF3 H N(Me)2 1 2.01 3.43 44.7 13 8b 4-FPh H N(Me)2 1 1.41 0.28 19.6 70 9b 3-FPh H N(Me)2 1 1.50 0.84 5.88 7 10b 4-ClPh H N(Me)2 1 2.05 0.31 19.5 62.9 4c 4-CF3Ph H N(Et)2 1 2.94 0.047 28.8 612 5c 3-CF3Ph H N(Et)2 1 2.94 0.42 102 243 6c Ph H N(Et)2 1 1.89 1.11 120 108 7c CF3 H N(Et)2 1 2.51 1.90 121 63.7 8c 4-FPh H N(Et)2 1 1.90 0.25 56.5 226 9c 3-FPh H N(Et)2 1 1.99 0.55 124 225 10c 4-ClPh H N(Et)2 1 2.54 0.06 41 683 4d 4-CF3Ph H NH2 2 1.46 0.92 6.26 6.8 5d 3-CF3Ph H NH2 2 1.46 0.63 4.33 6.9 6d Ph H NH2 2 0.49 2.27 11.9 5.2 4e 4-CF3Ph H N(Me)2 2 2.49 0.44 >200 455 5e 3-CF3Ph H N(Me)2 2 2.49 0.38 180 474 6e Ph H N(Me)2 2 1.52 1.52 108 71 7e CF3 H N(Me)2 2 2.06 3.16 166 52.5 8e 4-FPh H N(Me)2 2 1.45 0.27 11.6 43 9e 3-FPh H N(Me)2 2 1.54 0.53 15.4 29 10e 4-ClPh H N(Me)2 2 2.09 0.21 >210 >1000 4f 4-CF3Ph H N(Et)2 2 2.97 0.30 >190 >633 5f 3-CF3Ph H N(Et)2 2 2.97 0.19 24.3 128 6f Ph H N(Et)2 2 2.00 1.53 >230 >150 7f CF3 H N(Et)2 2 2.54 2.08 87.8 42.2 8f 4-FPh H N(Et)2 2 1.93 0.71 86.6 122 9f 3-FPh H N(Et)2 2 2.02 0.46 55.7 121 10f 4-ClPh H N(Et)2 2 2.57 0.17 48.8 287 4g 4-CF3Ph Et N(H)(Et) 1 3.52 0.94 21.5 22.9 4h 4-CF3Ph H N(H)(Et) 1 2.01 0.12 2.46 20.5 4i 4-CF3Ph H piperidine 1 3.78 0.054 193 3581 4j 4-CF3Ph H pyrrolidine 1 2.58 0.053 146 2751 4k 4-CF3Ph H morpholine 1 3.72 0.49 33.3 68 4l 4-CF3Ph H morpholine 2 3.79 1.22 98.3 80.6 a Mean from n g 2 independent experiments. Individual values varied by less than a factor of 2. b The log DpH7.4 values were calculated using the ACD LogD Suite of software (version 9.0, Advanced Chemistry Development Inc., Toronto, Canada). C dx.doi.org/10.1021/jm200227r |J. Med. Chem. XXXX, XXX, 000–000 Journal of Medicinal Chemistry ARTICLE Table 2. In Vitro Activity against Drug-Sensitive and Drug-Resistant Strains of P. falciparum P. falciparum IC50 (μM) W2 mef HB3 NF54 FC27 FCR3 MAD20 K1 chloroquine 0.118 0.041 0.006 0.004 0.085 0.010 0.151
0.173 artemesinin 0.008 0.003 0.005 0.008 0.005 0.008 0.005 mefloquine 0.011 0.012 0.013 <0.040 0.005 0.013 0.008 pyrimethamine 29.5 1.146 0.019 <0.060 0.012 <0.060 9.9 1 ND ND ND ND ND ND 0.17
0.36 2 ND ND 0.052 ND ND ND 0.05
0.078 3 ND ND 0.053 ND ND ND 0.023
0.095 4c 4h 0.084 0.123 0.060 0.090 0.11 0.11 0.175 0.11 0.137 0.154 0.104 0.106 0.047
0.058 0.116 Table 3. In Vivo Antimalarial Activity of Pyrido[1,2a]benzimidazole Aminoalkyl Derivatives and Corresponding Salts P. berghei in vivo % reduction parasitemia (MSD)a IC50 (μM) compd chloroquine X R P. fal (K1) cytotox (L6) b ip 99.6% (20) control po 99.9% (17) (5
7) 4b 40 -CF3
NH(CH2)2N(Me)2 0.13 >210 4ba 40 -CF3 2HCl salt of 4b 0.1 147.2 0%c 4bb 40 -CF3 mesylate salt of 4b 0.14 >175 94.1% (11) 96.8% (13.7) 4c 40 -CF3
NH(CH2)2N(Et)2 0.05 28.8 4ca 40 -CF3 2HCl salt of 4c 0.08 89.6 91.9% (10) 96.2% (16) 4cb 4e 40 -CF3 40 -CF3 mesylate salt of 4c
NH(CH2)3N(Me)2 0.09 0.44 123.6 >200 95.2% (11) 97.4% (11) 4ea 40 -CF3 2HCl salt of 4e 0.26 21.1 30.7%c 5e 30 -CF3
NH(CH2)3N(Me)2 0.38 180 5ea 30 -CF3 2HCl salt of 5e 0.49 19.2 >190 4f 4 -CF3
NH(CH2)3N(Et)2 0.30 4fa 40 -CF3 2HCl salt of 4f 0.16 2.23 5f 30 -CF3
NH(CH2)3N(Et)2 0.19 24.3 5fa 30 -CF3 2HCl salt of 5f 0.25 11.8 0 0%c 72.6% (9) 93.4% (11) 0%c a MSD = mean survival time (in days). 50 (mg/kg)/day  4 ip or 100 (mg/kg)/day  4 po (formulated in 10% aq DMSO). b Chloroquine diphosphate at 10 (mg/kg)/day  4 ip or po. c Mice euthanized on day 4, 24 h after last treatment, because of inactivity. 3. Variation in the alkyl chain length had practically no significant effect on the in vitro antiplasmodial activity, although this was only varied by one methylene unit. 4. The morpholino derivative 4k was ∼10 less active than other cycloalkylamino derivatives 4i and 4j. 5. In the one example in which there was additional alkyl substitution on the N attached to the ring (4g), activity was reduced compared to the des-ethyl derivative (4h). 6. 4h, the N-des-ethyl metabolite of compound 4c, retained its activity to an extent, albeit with reduced selectivity vs L-6.The activity of compounds 4c and 4h was found to be more or less invariant across a broad range of drug sensitive and drug resistant strains of P. falciparum (Table 2). In Vivo Studies. Six compounds with the most favorable activity and selectivity from the in vitro screen (4b, 4c, 4e, 5e, 4f, and 5f) were chosen for preliminary in vivo evaluation in P. berghei infected mice. The corresponding hydrochloride salts (4ba
5fa), as well as two mesylate salts (4bb and 4cb), were used for the in vivo studies. At a repeat dose of 50 (mg/kg)/day ip for 4 days four of these (4bb, 4ca, 4cb, and 4fa) showed significant efficacy. Subsequently they were also found to be active following oral (po) administration with 100 (mg/kg)/day  4, giving >90% D dx.doi.org/10.1021/jm200227r |J. Med. Chem. XXXX, XXX, 000–000 Journal of Medicinal Chemistry ARTICLE Table 4. Oral Efficacy of Selected Compounds (Bis-hydrochloride Salts) in the P. berghei Mouse Modela a Test compounds were formulated in hydroxypropyl methylcellulose (HPMC) and administered po (3 mice per group). inhibition of P. berghei parasitemia and a significant increase in the mean survival time (MSD) of the mice from 10 to 16 days, relative to controls (5
7 days, Table 3). The lack of in vivo activity with the hydrochloride salt of 4b (4ba) compared to the mesylate salt (4bb) when given ip may be due to a poorer solubility or dissolution rate adversely affecting distribution. In single dose studies in the P. berghei mouse model (Table 4), the hydrochloride salts of four of the most active compounds 4ca, 4ha, 4ja, 4ia showed significant activity at 25 mg/kg, which was not significantly improved on going to a higher dose of 50 mg/kg, presumably because of saturation of systemic exposure; 4ia was ineffectual at 50 mg/kg, possibly because of a problem with formulation arising from poor solubility (cloudiness of the solution was observed during administration of 4ia at both doses). In any case the rate of reduction in parasitemia after treatment with the pyridobenzimidazoles was significantly slower than with chloroquine, with maximum reduction generally observed on day 5 compared to day 2 with chloroquine. In Vitro Metabolic Stability. Studies were conducted using human, mouse, and rat liver microsomes (Table 5). Compounds 4ca and 4ja
4ka exhibited moderate to high rates of metabolic degradation, while degradation of 4ha was in the low to moderate range. Generally for the alkylated derivatives 4ca and 4ha, N-dealkylated metabolites were observed, and for the cycloalkylated derivatives (4ja
4la), there was evidence for deamination, N-dealkylation, or ring cleavage. In Vivo Pharmacokinetic Studies. The in vivo pharmacokinetic properties of both 4c and 4h were assessed following administration of the bis-hydrochloride salts (i.e., 4ca and 4ha) at dose levels of 5 mg/kg intravenously and 20 mg/kg orally to male Sprague
Dawley rats (Table 6). For compound 4c, the apparent half-life ranged between 6 and 8 h; volume of distribution was high, and plasma clearance was moderate. The rate of absorption was relatively slow after oral administration, and the apparent oral bioavailability was ∼22%. The des-ethyl derivative 4h was identified as a metabolite of 4c in plasma samples following both routes of administration, in agreement with the in vitro microsome studies. The in vivo conversion of 4c to 4h was estimated to be approximately 70%, suggesting that N-dealkylation is likely to be a major in vivo clearance pathway for 4c. Direct urinary excretion of 4c was negligible following both intravenous and oral administration. When 4h was administered intravenously as the bis-hydrochloride salt (4ha) at a dose of 5 mg/kg, it exhibited a long apparent half-life, high volume of distribution, and low plasma clearance; direct urinary excretion of 4h was negligible. Compound 4h exhibited high protein binding in human and mouse plasma with fraction bound values being in the ranges 98.3
99.9% and 95.3
96.9%, respectively. In comparison 4c had 96.7
99.2% (human) and 97.9% (mouse) protein binding. On the basis of the high conversion of 4c to 4h observed in vivo, the long half-life, and low blood clearance of 4h and its intrinsic activity, it is likely that the N-dealkylation product 4h contributes significantly to the efficacy of 4c. Single-dose and multidose oral efficacy studies of 4ha against P. berghei ANKA GFP in mice indicated that the activity plateaus (reaches a limit) with dose concentrations higher than 25 mg/kg E dx.doi.org/10.1021/jm200227r |J. Med. Chem. XXXX, XXX, 000–000 Journal of Medicinal Chemistry ARTICLE Table 5. In Vitro Microsomal Stability a EH (hepatic extraction ratio) = fraction of dose entering liver, which is metabolized during one pass through the liver. b P-28: monodeethylation. P-56: bis-deethylation. P + 14: carbonyl addition. P-53: deamination of the pyrrolidine ring. P-97, P-68, P-127, P-113: N-dealkylation of the respective R side chain. P + 16: oxygenation. P-70: morpholine dealkylation. P-26: morpholine ring cleavage. Table 6. Pharmacokinetic Parameters for 4c and 4h after iv and Oral Administration of Their Bis-hydrochloride Salts (4ca and 4ha) to Male Rats 4h (after administration of 4ca) 4c parameter iva orala iva orala iva 2.6 19.1 cncd cncd ∼16.3 cncd nominal dose (mg/kg)b 4.3 19.0 apparent t1/2 (h) 7.3 6.4 plasma CLtotal 30 VZ (L/kg) 18.9 % dose in urinec Cmax (μM) 0.39 0.05 0.38 248 640 1200 304 290 666 1032 Tmax (min) AUC0
tlast (μM 3 min) bioavailability (%) 4h orala 5.4 7.5 0.08 0.54 0.05 0.92 1.13 0.21 0.95 1000 2076 960 >28e >22 a b Compounds were formulated as suspensions in 0.5% hydroxypropyl methylcellulose. Values are the mean from two animals. As the bishydrochloride salt. c % of dose present in pooled urine (collected over 0
24 or 0
48 h). d Given the flat nature of the profile, the terminal elimination half-life could not be determined. e The terminal elimination half-life and oral bioavailability could not be accurately determined. On the basis of the dose-normalized AUC0
tlast, the exposure after oral administration was approximately 25
30% of that after iv administration; however, this will likely be an underestimation of the actual oral bioavailability. in vivo (Tables 7 and 8). A similar plateau in efficacy was also observed following subcutaneous administration. As a preliminary indication of systemic exposure, in vivo studies in mice following oral and subcutaneous administration of 4ha at F dx.doi.org/10.1021/jm200227r |J. Med. Chem. XXXX, XXX, 000–000 Journal of Medicinal Chemistry ARTICLE escalating dose levels (6
100 mg/kg) were conducted. Exposure was found to become saturated at dose levels above 25 mg/kg (orally) or 50 mg/kg (subcutaneously), consistent with the observed plateau in efficacy (Figure 3). It is likely that solubility or dissolution-limited absorption is a contributory factor in limiting systemic exposure and efficacy with increasing dose. Table 7. Single-Dose Oral Efficacy of 4ha against P. berghei ANKA GFP in Mice % reduction parasitemia compd a a dose (mg/kg) day 2 day 3 day 4 day 5 MSD (day) 4ha 1  25 23.51 55.45 62.25 68.92 13.3 4ha 1  50 30.78 50.24 71.22 79.79 12.7 chloroquine control 1  10 99.9 ’ CONCLUSIONS A novel series of pyrido[1,2-a]benzimidazole derivatives have been identified that combine good in vitro activity against P. falciparum with oral efficacy in a P. berghei mouse model. The pyridobenzimidazoles appear to be slower acting in vivo relative to chloroquine, pointing to a different mode of action, which has not been established. The most significant feature of the series is that the pharmacokinetic profile of the lead compounds needs considerable improvement if a pyridobenzimidazole is to be identified as worthy of further progression. Although 4h shows good stability in rat and mouse microsomes and has a long halflife in rats, pharmacokinetic studies indicate oral absorption becomes saturated at relatively low doses, most likely because of poor dissolution or solubility. Further work is needed to identify compounds that have the potential for improved pharmacokinetics, most likely achievable through a combination of improved solubility and metabolic stability. 9 5 Compounds were formulated in HPMC. Table 8. Multidose Oral Efficacy of 4ha against P. berghei ANKA GFP in Mice % reduction 4ha (mg/kg)a cured/infected parasitemia at day 4 MSD (day) 43 0/3 0 4b 46 0/3 38.35 7 4  12.5 0/3 80.73 14 4  25 0/3 95.72 14.3 4  50 0/3 95.56 control 14 6.5 a Compounds were formulated in HPMC. b Mice euthanized on day 4, 24 h after last treatment, because of inactivity. ’ EXPERIMENTAL SECTION Chemistry. All commercially available chemicals were purchased from either Sigma-Aldrich or Merck. All solvents were dried by appropriate techniques. Unless otherwise stated, all solvents used were anhydrous. Reactions were monitored by TLC using Merck silica gel plates (60 F-254), and were visualized by ultraviolet light. Silica gel chromatography was performed using Merck Kieselgel 60: 70
230 mesh for gravity columns. Melting points were determined on a Reichert-Jung Thermovar hotstage microscope and are uncorrected. Infrared spectra were recorded on a Thermo Nicolete FTIR instrument in the 4000
500 cm
1 range using KBr disks. Microanalyses were determined using a Fisons EA 1108 CHNO-S instrument. Mass spectra were recorded at the School of Chemistry, University of the Witwatersrand, South Africa. NMR spectra were recorded on either a Varian Mercury 300 (1H, 300.13 MHz; 13C, 75.5 MHz) or a Varian Unity 400 (1H, 400.13 MHz; 13C, 100.6 MHz) spectrometer. Chemical shifts (δ) are given in ppm downfield from TMS as the internal standard. Coupling constants, J, are recorded in hertz (Hz). LC purity traces were obtained using the Kinetex C18 (2.1 mm  150 mm, 2.6 mm fused-core particles) column, 1 mL injection volume, flow of 0.4 mL/min, gradient 0
100% B in 9 min (hold 3 min) (mobile phase A of 10 mM ammonium formate, pH 3, in 10% MeCN and mobile phase B of 10 mM ammonium formate, pH 3, in 90% MeCN) with a diode array detector operating at a wavelength range from 190 to 400 nm. Purity was determined by combustion analysis and/or HPLC, and all compounds were confirmed to have >95% purity. General Procedure for the Synthesis of 1-Oxo-3-alkyl/ aryl-5H-pyrido[1,2-a]benzimidazole-4-carbonitriles (A). A mixture of 2-benzimidazole acetonitrile (1.0 g, 6.36 mmol), NH4OAc (0.98 g, 12.72 mmol), and ethyl (4-alkanoyl/aroyl)acetate (7.63 mmol) was heated to reflux at 150 C for 1 h and allowed to cool to 100 C. MeCN (10 mL) was added. The mixture was stirred for 15 min, allowed to cool to room temperature, and then cooled on ice. The cold mixture was filtered and the residue washed with cold MeCN (4  10 mL), dried in vacuo, and used without further purification. 1-Oxo-3-[4-(trifluoromethyl)phenyl]-5H-pyrido[1,2-a]benzimidazole-4-carbonitrile. Silvery tan powder, mp 341
342 C Figure 3. Systemic exposure of 4h following single dose oral and subcutaneous administration of the bis-hydrochloride salt (4ha) to mice. G dx.doi.org/10.1021/jm200227r |J. Med. Chem. XXXX, XXX, 000–000 Journal of Medicinal Chemistry ARTICLE General Procedure for the Synthesis of 1-(Alkylamino/ piperido/morpholino/pyrrolidino)ethyl/propylamino)-3-alkyl/arylpyrido[1,2-a]benzimidazolecarbonitrile Bis-hydrochloride Salts. HCl in methanol (1.25 M, 0.57 mL, 1.32 mmol) was (ethanol); IR (KBr) 3250
2500 bm, 2203 m (CN), 1664 s (CO), 1548 s, 1509 s, 1458 m, 1324 s, 1169 m, 1107 s, 1075 m, 1064 s cm
1; 1H NMR (400 MHz, DMF-d7) δ 8.64 (1H, d, J = 7.3 Hz, ArH), 7.93 (4H, s, ArH), 7.64 (1H, d, J = 7.3 Hz, ArH), 7.57 (1H, dd, J = 7.3, 7.3 Hz, ArH), 7.42 (1H, dd, J = 8.8, 7.3 Hz, ArH), 6.11 (1H, s, dCH
); 13C NMR (100 MHz, DMF-d7) δ 157.9, 150.9, 147.2, 140.9, 131.6, 130.0, 129.7, 128.4, 127.6, 126.4, 125.1, 122.5, 122.3, 115.9, 115.7, 111.0, 104.6, 67.9; LRMS (EI) m/z 354.2 (M + H). added to a stirred mixture of the pyrido[1,2-a]benzimidazole-carbonitrile derivative (0.354 mmol) in methanol (20 mL). After the mixture was stirred at room temperature for 2.5 h, the solvent was removed in vacuo and the residue washed with minimum amounts of ice-cold methanol followed by DCM (4  3 mL), dried in vacuo, and used without further purification. General Procedure for the Synthesis of 1-Chloro-3-alkyl/ arylpyrido[1,2-a]benzimidazole-4-carbonitriles (B). A mixture 1-(2-Diethylaminoethylamino)-3-[4-(trifluoromethyl)phenyl]pyrido[1,2-a]benzimidazole-4-carbonitrile Bis-hydrochloride Salt (4ca). Pale yellow, mp 161
162 C; 1H NMR (300 MHz, of 1-oxo-3-alkyl/aryl-5H-pyrido[1,2-a]benzimidazole-4-carbonitrile (2.83 mmol) and POCl3 (8.69 g, 5.28 mL, 56.68 mmol) was heated to reflux at 130 C for 2 h. Excess POCl3 was removed under reduced pressure and ice-cold water (20 mL) added to the residue, stirring to yield a precipitate. The mixture was neutralized with saturated NaHCO3 and filtered. The resultant solid was washed with ice-cold water (4  15 mL), dried in vacuo, and used without further purification. DMSO) δ 11.07 (1H, broad s, NH), 8.88 (1H, d, J = 8.4 Hz, ArH), 8.00 (4H, q, J = 8.4 Hz, ArH), 7.87 (1H, d, J = 7.5 Hz, ArH), 7.68 (1H, d, J = 7.7 Hz, ArH), 7.48 (1H, ddd, J = 1.2, 7.6, 8.4, ArH), 6.74 (1H, s, dCH
), 4.14 (2H, t, J = 6.6 Hz), 3.47 (2H, td, J = 5.1, 6.0 Hz), 3.23 (2H, quintet, J = 4.8, 7.2 Hz), 1.25 (6H, t, J = 7.2 Hz). 13C NMR (100 MHz, DMSO): δ 151.2, 148.4, 147.6, 130.0, 127.1, 125.5, 121.7, 116.2, 93.4, 48.9, 46.4, 8.1 (CH3). LRMS (APCI): m/z 452 (M+ + 1
HCl). 1-Chloro-3-[4-(trifluoromethyl)phenyl]pyrido[1,2-a]benzimidazole-4-carbonitrile. Yellow solid, mp 247
248 C (ethanol); IR (KBr) 3094 w, 3065 w, 2217 m (CN), 1617 w, 1591 m, 1487 s, 1444 s, 1339 s, 1172 s, 1129 s, 1067 s cm
1; 1H NMR (300 MHz, CDCl3) δ 8.59 (1H, d, J = 8.8 Hz, ArH), 8.10 (1H, d, J = 7.8 Hz, ArH), 7.85 (4H, s, ArH), 7.67 (1H, dd, J = 8.8, 6.8 Hz, ArH), 7.49 (1H, dd, J = 8.8, 5.9 Hz, ArH), 7.05 (1H, s, dCH
); 13C NMR (100 MHz, CDCl3) δ 147.2, 145.3, 138.6, 134.5, 132.7, 132.3, 132.0, 129.7, 129.1, 127.3, 126.2, 125.0, 123.0, 122.3, 120.8, 115.4, 114.2, 111.8, 98.4; LRMS (EI) m/z 372.1 (M + H), 374.0 (M + 2 + H); 1-(2-Ethylaminoethylamino)-3-[4-(trifluoromethyl)phenyl]pyrido[1,2-a]benzimidazole-4-carbonitrile Bis-hydrochloride Salt (4ha). Pale yellow solid, mp 181
183 C; purity 97.8% by LC (tR = 5.94 min); 1H NMR (300 MHz, DMSO) δ 9.36 (2H, broad s), 8.85 (1H, d, J = 8.4 Hz, ArH), 8.00 (5H, m, ArH), 7.88 (1H, d, J = 8.1 Hz, ArH), 7.68 (1H, t, J = 7.5 Hz, ArH), 7.48 (1H, t, J = 7.5 Hz, ArH), 6.70 (1H, broad s, dCH
), 4.08 (2H, m,
NHCH2C
), 3.31 (2H, m,
NHCH2CH3), 3.02 (2H, m,
NHCH2C
), 1.25 (3H, m,
CH3); 13 C NMR (100 MHz, DMSO) δ 151.0, 148.5, 147.6, 140.3, 129.7, 127.1, 126.8, 125.4, 121.4, 116.5, 116.0, 92.8, 44.3, 42.0, 10.7. General Procedure for the Synthesis of 1-[(Alkylamino/ piperido/morpholino/pyrrolidino)ethyl/propylamino]-3-alkyl/ arylpyrido[1,2-a]benzimidazolecarbonitriles (C). Method 1. The appropriate amine (2.69 mmol) was added to a stirred mixture of the 1-chloro-3-alkyl/arylpyrido[1,2-a]benzimidazole-4-carbonitrile (1.345 mmol) and triethylamine (0.27 g, 0.37 mL, 2.69 mmol) in THF or DMF (10 mL). The mixture was heated at 80
90 C for 18 h, filtered hot, and allowed to cool. The solvent was removed in vacuo, and the residue was washed with minimum amounts of ice-cold ethanol. The resulting solid was recrystallized from acetone or ethanol. Method 2. Microwave irradiation (150 W) was substituted for external heating, reducing the reaction time to approximately 20 min. Workup followed the same protocol as method 1. ’ ASSOCIATED CONTENT bS Supporting Information. Additional details of the characterization of selected compounds and the procedures used for the in vitro and in vivo antimalarial studies and cytotoxicity assays. This material is available free of charge via the Internet at http://pubs.acs.org. ’ AUTHOR INFORMATION 1-(2-Diethylaminoethylamino)-3-[4-(trifluoromethyl)phenyl]pyrido[1,2-a]benzimidazole-4-carbonitrile (4c). Yellow pow- Corresponding Author der, mp 219
220 C (ethanol); purity 98% by LC (tR = 5.82 min); IR (KBr) 3333 bm, 2971 m, 2841 w, 2210 s (CN), 1624 s, 1595 s, 1552 s, 1458 m, 1371 m, 1371 m, 1324 s, 1165 m, 1129 s, 1067 s, 1014 m cm
1; 1 H NMR (300 MHz, CDCl3) δ 8.13 (1H, d, J = 7.8 Hz, ArH), 8.05 (1H, d, J = 7.8 Hz, ArH), 7.83 (2H, d, J = 7.8 Hz, ArH), 7.77 (2H, d, J = 8.8 Hz, ArH), 7.59 (1H, t, J = 7.8 Hz, ArH), 7.44 (1H, br s, NH), 7.36 (1H, dd, J = 7.8, 6.8 Hz, ArH), 5.90 (1H, s, dCH
), 3.49 (2H, t, J = 5.9 Hz, CH2CH2N(Et)2), 2.97 (2H, t, J = 5.9 Hz, CH2CH2N(Et)2), 2.73 (4H, q, J = 7.8 Hz, N(CH2CH3)2), 1.16 (6H, t, J = 7.8 Hz, N(CH2CH3)2); 13C NMR (75 MHz, CDCl3) δ 150.5, 149.0, 147.9, 145.7, 141.0, 129.0, 128.0, 126.0, 125.7, 121.0, 120.1, 116.7, 112.8, 89.3, 50.1, 46.0, 39.9, 11.7; LRMS (EI) m/z 452.2 (M + H). *Phone: +27 21 650 2553. Fax: +27 21 650 5195. E-mail: [email protected]. ’ ACKNOWLEDGMENT We thank the WHO Special Programme for Research and Training in Tropical Diseases for financial support for this research (Project A50868). The University of Cape Town, South African Medical Research Council (MRC), and South African Research Chairs Initiative (SARChI) of the Department of Science and Technology (DST) administered through the South African National Research Foundation are gratefully acknowledged for support (K.C.). We also acknowledge the generous gifts of the early samples of TDR15087 and related analogues from SPECS, with the valuable roles of Herman Verheij in the initial selection of the 1440 compound library, Louis Maes in the screening of this library at Tibotec, and Reto Brun in overseeing further screening at Swiss TPH. 1-(2-Ethylaminoethylamino)-3-[4-(trifluoromethyl)phenyl]pyrido[1,2-a]benzimidazole-4-carbonitrile (4h). Yellow fluffy powder, mp 222
224 C (dec). Purity >99% by LC (tR = 5.94 min). 1H NMR (400 MHz, CDCl3) δ: 8.57 (1H, d, J = 8.4 Hz, ArH), 7.94 (4H, m, ArH), 7.79 (1H, d, J = 8.0 Hz, ArH), 7.52 (1H, m, ArH), 7.33 (1H, m, ArH), 6.18 (1H, s, =CH-), 3.11 (2H, t, J = 6.2 Hz, -NHCH2C
), 2.83 (2H, q, J = 7.2 Hz, -NHCH2CH3), 2.48 (2H, m, -NHCH2C
), 1.14 (3H, t, J = 7.2 Hz,
CH3). 13C NMR (100 MHz, CDCl3) δ: 150.5, 149.1, 147.9, 145.8, 141.1, 129.0, 128.1, 126.1, 125.8, 121.0, 120.3, 116.7, 112.9, 89.3, 50.1, 46.1, 40.0, 11.8. m/z (EI, positive ion) 423.5 (M+), 417, 352, 333, 274, 237, 210, 162, 88. ’ ABBREVIATIONS USED ip, intraperitoneal; po, oral administration; HPLC, high pressure liquid chromatography; HPMC, hydroxypropyl methylcellulose; H dx.doi.org/10.1021/jm200227r |J. Med. Chem. XXXX, XXX, 000–000 Journal of Medicinal Chemistry ARTICLE MSD, mean survival time; MTS, medium throughput screen; SAR, structure
activity relationship; TDR, tropical diseases research; TLC, thin layer chromatography; TMS, tetramethylsilane; WHO, World Health Organization ’ REFERENCES (1) Nwaka, S.; Ridley, R. G. Virtual drug discovery and development for neglected diseases through public
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