StructureâActivity-Relationship Studies around the 2-Amino Group

Article pubs.acs.org/jmc

Structure−Activity-Relationship Studies around the 2‑Amino Group and Pyridine Core of Antimalarial 3,5-Diarylaminopyridines Lead to a Novel Series of Pyrazine Analogues with Oral in Vivo Activity Yassir Younis,† Frederic Douelle,† Diego González Cabrera,† Claire Le Manach,† Aloysius T. Nchinda,† Tanya Paquet,† Leslie J. Street,† Karen L. White,‡ K. Mohammed Zabiulla,§ Jayan T. Joseph,§ Sridevi Bashyam,§ David Waterson,∥ Michael J. Witty,∥ Sergio Wittlin,⊥,# Susan A. Charman,‡ and Kelly Chibale*,†,∇ †

Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa Centre for Drug Candidate Optimisation, Monash University (Parkville Campus), 381 Royal Parade, Parkville, Victoria 3052, Australia § Syngene International Ltd., Biocon Park, Plot No. 2 & 3. Bommasandra IV Phase, Jigani Link Road, Bangalore 560099, India ∥ Medicines for Malaria Venture, ICC Building, Route de Pré-Bois 20, P.O. Box 1826, 1215 Geneva, Switzerland ⊥ Swiss Tropical and Public Health Institute, Socinstrasse 57, 4002 Basel, Switzerland # University of Basel, Socinstrasse 57, 4002 Basel, Switzerland ∇ Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa ‡

S Supporting Information *

ABSTRACT: Replacement of the pyridine core of antimalarial 3,5-diaryl-2-aminopyridines led to the identification of a novel series of pyrazine analogues with potent oral antimalarial activity. However, other changes to the pyridine core and replacement or substitution of the 2-amino group led to loss of antimalarial activity. The 3,5-diaryl-2-aminopyrazine series showed impressive in vitro antiplasmodial activity against the K1 (multidrug resistant) and NF54 (sensitive) strains of Plasmodium falciparum in the nanomolar IC50 range of 6−94 nM while also demonstrating good in vitro metabolic stability in human liver microsomes. In the Plasmodium berghei mouse model, this series generally exhibited good efficacy at low oral doses. One of the frontrunner compounds, 4, displayed potent in vitro antiplasmodial activity with IC50 values of 8.4 and 10 nM against the K1 and NF54 strains, respectively. When evaluated in P. berghei-infected mice, compound 4 was completely curative at an oral dose of 4 × 10 mg/kg.

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INTRODUCTION

affordable drugs against multidrug-resistant Plasmodium strains are long-term and vital tasks for researchers.3−10 In our previous work, we disclosed the aminopyridine series of compounds as novel orally active antimalarial agents.11 SAR exploration around positions 3 and 5 of the 2-aminopyridine core led to the identification of 1 and 2 as frontrunner

Malaria is one of the most widespread and deadly infectious diseases in the world and is responsible for an estimated 500 000−900 000 deaths each year, especially among children and pregnant women.1 Plasmodium falciparum and Plasmodium vivax are the two most prevalent species responsible for causing disease in humans.2 Because of the continuing development of the resistance of malaria parasites to conventional antimalarial drugs, efforts to search for novel, structurally diverse, and © 2013 American Chemical Society

Received: August 19, 2013 Published: October 7, 2013 8860

dx.doi.org/10.1021/jm401278d | J. Med. Chem. 2013, 56, 8860−8871

Journal of Medicinal Chemistry

Article

candidates (Figure 1).11,12 Inspired by this initial success and in our quest to identify more potent analogs with improved

5-bromo-3-iodopyrazin-2-amine 24, which underwent Suzuki cross-coupling reactions using 6-(trifluoromethyl)pyridin-3ylboronic acid or 4-(trifluoromethyl)phenylboronic acid resulting in intermediates 25 and 25a (Scheme 5). The aryl groups were introduced at position 5 of the 2-aminopyrazine core via Suzuki cross-coupling reactions with appropriate boronic acids to deliver the desired target compounds 4−10 and 26−36.11−13All compounds were purified using column chromatography and were fully characterized by a range of analytical and spectroscopic techniques. In Vitro Antiplasmodial Activity. Initial studies involved exploration around the 2-amino group and pyridyl core to establish SAR, as shown in Tables 1 and 2, respectively. The in vitro antiplasmodial activities of all synthesized compounds were determined against a sensitive (NF54) and multidrug resistant (K1) strain of P. falciparum.6 Chloroquine and artesunate were used as the reference drugs in all of the experiments. Among the 7 compounds evaluated with varying 2-amino group substituents, the unsubstituted 2-amino group retained the best activity (1; IC50 = 51 nM (K1) and (NF54)). Complete removal of the 2-amino substituent (13; IC50 = 538 nM (K1) and 421 nM (NF54)) led to a decrease in antiplasmodial potency. Introducing electron-withdrawing or -donating substituents in place of the 2-amino group resulted in a significant loss in antiplasmodial activity as exemplified by 17 (IC50 = >24 505 nM (K1) and (NF54)) and 15 (IC50 > 28 083 nM (K1) and (NF54)). In general, the unsubstituted 2-amino group of the pyridyl core is critical to potent in vitro antiplasmodial activity, suggesting that this group is essential for specific binding to an as yet unknown target. The next phase of SAR exploration was directed at optimizing the pyridyl core while fixing the optimal unsubstituted 2-amino group. Compounds 18−21 (Table 2) were synthesized for this purpose. It was observed that the 2aminopyridine core remained critical for antiplasmodial activity (1; IC50 = 51 nM (K1) and (NF54)), whereas its replacement with either phenyl, pyridazine, and/or thienopyridine groups was detrimental to activity as was the case for 19 (IC50 >2821 nM (K1) and (NF54)), 20 (IC50 > 281 nM (K1) and (NF54)), and 21 (IC50 = 1173 nM (K1) and >2433 nM (NF54)). Moving the position of the pyridine nitrogen, as exemplified by compound 18 (IC50 >28 136 nM (K1) and (NF54)), led to a significant loss in antiplasmodial potency. However, introducing the pyrazine core (3; IC50 = 45 nM (K1) and 48 nM (NF54)) resulted in significant potency, which was comparable

Figure 1. Structures of 2-aminopyridine frontrunners compounds, 1 and 2.9,10

ADME properties, our attention was diverted to the investigation of SAR around the 2-amino group and pyridine core. In this article, we report the synthesis and SAR exploration around the 2-amino group and the pyridine core of 3,5diarylaminopyridines, leading to the identification of the corresponding 2-aminopyrazines with potent antimalarial activity (Figure 2).

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RESULTS AND DISCUSSION Chemistry. Target compounds 1, 11, 13, and 18−20 were synthesized using a previously reported general route, which involved iodination followed by two Suzuki cross-coupling reactions, as depicted in Scheme 1.11−13 Compounds 14−16 were obtained from compound 1 by converting the 2-amino group of the pyridyl core to the corresponding 2-iodo-, 2ethoxy-, and 2-hydroxyl-substituted pyridines using previously described procedures to give the desired products, as depicted in Scheme 2.14−16 The 2-trifluoromethyl analogue 17 was prepared from 2-iodo analogue 14 using methyl (fluorosulfonyl)difluoroacetate and copper iodide in DMF, as shown in Scheme 2.17 Compounds 12 and 21 were accessed as outlined in Schemes 3 and 4, respectively. The aminopyrazine analogues 4−10 and 26−36 shown in Table 3 were synthesized from 2-aminopyrazine 22, which was brominated at position 5 using NBS in DCM at 25 °C for 4 h to afford intermediate 5bromopyrazin-2-amine 23. This intermediate was iodinated at position 3 using NIS in DCM to give the second intermediate

Figure 2. SAR around 2-aminopyrdines led to 2-aminopyrazine series. 8861



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Scheme 1. General Synthetic Approach for Synthesized Compounds 1, 3, 11, 13, and 18−20a

Conditions: (a) I2, DMSO, 100 °C, 4 h, (75%); (b) 1,4-dioxane, K2CO3 (aq, 1 M), Pd(PPh3)2Cl2 (5 mol %), 110 °C, 16 h, (55%); (c) 1,4-dioxane, K2CO3 (aq, 1 M), Pd(PPh3)2Cl2 (7 mol %), 110 °C, 16 h, (40−60%).

a

Scheme 2. General Synthetic Approach for Synthesized Compounds 14−17a

Conditions: (a) CH2I2, t-BuONO, I2, 25 °C, 24 h, (49%); (b) DMF, CuI, FSO2CF2CO2Me, microW, 125 °C, 30 min (45%); (c) NaNO2, TFA, °C, 15 min, rt, 2 h, (88%); (d) CH3CH2I, K2CO3, THF/DMSO, 50 °C, 16 h, (52%).

a

Aminopyrazines and their derivatives have been reported to possess a variety of pharmacological activities, including antioxidant and kinase inhibition activities.18−20 Prior to our work, some examples of this class were reported as having in vitro antimalarial activity among the many thousands of compounds in the published GlaxoSmithKline (GSK) Tres Cantos Antimalarial Set (TCAMS) and Novartis (GNF) Malaria Screening.21−23 However, this activity has not been followed up on in the literature. As demonstrated in previous studies for the 5-aryl position,12 introducing polar, electronwithdrawing substituents such as morpholinylcarbonyl, isopropylsufonyl, and methylsulfonamido resulted in superior antiplasmodial activity, as displayed by compound 6 (IC50 = 10 nM (K1) and 9.0 nM (NF54)), 7 (IC50 = 7.0 nM (K1) and 6.0 nM (NF54)), 8 (IC50 = 17 nM (K1) and 20 nM (NF54)), 9 (IC50 = 11 nM (K1) and 12 nM (NF54)), and 10 (IC50 = 21 nM (K1) and 23 nM (NF54)). Adding cyclopropylmethylsul-

to that of the corresponding 2-aminopyridine compound 1 (IC50 = 51 nM (K1) and (NF54)). 3 was profiled for its in vitro ADME properties and found to have moderate metabolic stability in human liver microsomes, with a predicted human hepatic extraction ratio (EH) of 0.39. On the basis of our initial SAR knowledge of the metabolic vulnerability of the methoxy group of the 3-pyridyl substituent, a trifluoromethyl group was introduced, leading to the identification of compound 4 with impressive in vitro antiplasmodial activity (IC50 = 8.4 nM (K1) and 10 nM (NF54)) and significantly improved metabolic stability in human and rat liver microsomes (EH < 0.28 and < 0.19, respectively) (Figure 2). In addition, no cytotoxicity was observed for compound 4 when tested against a Chinese hamster ovary (CHO) cell line (IC50 >254 μM; selectivity index, SI, >20 000). The 4-trifluoromethylphenyl substituent at position 3 also led to impressive antiplasmodial activity in 5 (IC50 = 11 nM (K1) and (NF54)). 8862



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Scheme 3. General Synthetic Approach for Synthesized Compound 12a

Conditions: (a) (i) 1,4-diaminobutane, 115 °C, 16 h; (ii) (Boc)2O, DCM/MeOH, Et3N, rt, 16 h; (b) NBS, PEG, rt, 30 min; (c) 1,4-dioxane, K2CO3 (aq, 1 M), Pd(PPh3)2Cl2 (5 mol %), 110 °C, 16 h, (55%); (d) NBS, PEG, rt, 30 min; (e) 1,4-dioxane, K2CO3 (aq, 1 M), Pd(PPh3)2Cl2 (7 mol %), 110 °C, 16 h, (60%); (f) (i) TFA, DCM, rt, 3h, (ii) Amberlyst, DCM/MeOH, rt, 3 min (89.8%). a

Scheme 4. General Synthetic Approach for Synthesized Compounds 21a

a Conditions: (a) 1,4-dioxane, K2CO3 (aq, 1 M), Pd(PPh3)2Cl2 (5 mol %), 110 °C, 16 h, (55%); (b) NBS, DMF, rt, 15 min, (50%); (c) 1,4-dioxane, K2CO3 (aq, 1 M), Pd(PPh3)2Cl2 (7 mol %), 110 °C, 16 h, (42%).

Scheme 5. General Synthetic Approach for Synthesized Compounds 4−10 and 26−36a

Conditions: (a) NBS, DCM, rt, 4 h, (50.4%); (b) NIS, 1,4-dioxane, 80 °C, 4 h, (58%); (c) 1,4-dioxane, K2CO3 (aq, 1 M), Pd(PPh3)2Cl2 (5 mol %), 110 °C, 16 h, (55%); (d) 1,4-dioxane, K2CO3 (aq, 1 M), Pd(PPh3)2Cl2 (7 mol %), 110 °C, 16 h, (40−60%).

a

Interestingly, compound 28 (IC50 = 20 nM (K1) and 26 nM (NF54)) with a 4-pyridyl substituent displayed good activity, whereas 3-pyridyl-containing 29 (IC50 = 316 nM (NF54)) and pyrimidine-based 30 (IC50 = 735 nM (K1) and 1043 nM (NF54)) substituents showed significantly decreased in vitro antiplasmodial activity. Furthermore, compounds containing the 2-aminopyridyl 33 (IC50 = 28 nM (K1) and 33 nM (NF54)) and 2-methylsulfonylpyridyl (35) (IC50 = 41 nM (K1) and 48 nM (NF54)) substituents also demonstrated good

fonyl and cyclopropylsulfonyl aryl substituents at position 4 also resulted in good potency, as achieved with compounds 27 (IC50 = 13 nM (K1) and 19 nM (NF54)) and 26 (IC50 = 10 nM (K1) and 11 nM (NF54)), respectively. Introducing 4methylsulfoxide aryl substituent maintained good potency (36; IC50 = 16 nM (K1) and 21 nM (NF54)) in comparison with corresponding methylsulfonyl analogue 4. Moreover, we investigated aryl substitution at position 5 of the 2-aminopyrazine core by introducing heteroaromatic substituents. 8863



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Table 1. In Vitro Antiplasmodial Activity of Compounds 1 and 11−17

a Mean from n values of ≥2 independent experiments. bLogD pH 7.4 values were determined using a chromatographic estimation method; nd = not determined. cData from Younis et al.11

speaking, an improvement in the hERG profile was observed for some aminopyrazine analogues. In Vitro ADME Profiling. The physicochemical properties of all of the compounds were evaluated through a combination of in silico and experimental techniques,5,11,12 and the parameters were generally found to be within the ranges typically considered acceptable for “drug-like” compounds, as shown in Tables 1, 2, and 3. Generally, most of the compounds displayed poor to moderate kinetic solubility at pH 6.5 but increased solubility under acidic conditions, consistent with expected ionization behavior. Partition coefficient values of the compounds were generally moderate, with LogD values at pH 7.4 ranging from 1.7 to 3.2.

activity. However, compounds with 2-fluoropyridyl 31 (IC50 = 189 nM (K1) and 282 nM (NF54)) and 2-trifluoromethylpyridyl 32 (IC50 = 680 nM (NF54)) substituents displayed inferior activity. Generally speaking, 2-aminopyrazine analogues displayed potent antiplasmodial activity of slightly greater potency than their previously reported corresponding 2-aminopyridines. Toward hERG derisking, some analogues from the aminopyrazine series were tested for their activity against the hERG potassium channel using in vitro IonWorks patch-clamp electrophysiology. The data is shown in Table 3. Lead compound 4, displayed a hERG IC50 value of 8.1 μM. However, the high antiplasmodial (low IC50) potency of this compound should ensure a good safety margin. Generally 8864



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Table 2. In Vitro Antiplasmodial Activity of Compounds 1, 3, and 18−21

a Mean from n values of ≥2 independent experiments. bLogD pH 7.4 values were determined using a chromatographic estimation method; nd = not determined. cData from Younis et al.11

disclosed aminopyridines, presumably because of superior in vitro potency, generally, there was no improvement in the ADME properties. In Vivo Efficacy. The in vivo antimalarial activity of the most promising compounds was evaluated using a P. berghei mouse model.6 Parasitemia reduction and mean survival days (MSD) for single- or multidose regimens were determined. Table 5 summarizes the in vivo efficacy test results of compounds 4−7 following oral (p.o.) administration compared with the standard antimalarial chloroquine (CQ). As shown in Table 5, compounds 6 and 7 at low daily oral doses of 4 × 10 and 4 × 3 mg/kg exhibited good in vivo activity with a >80% reduction in parasitemia. Three compounds, 8−10, were evaluated for their efficacy at a lower (4 × 3 mg/kg) oral

In vitro metabolic stability of all the compounds was evaluated using human liver microsomes as a preliminary indication of the likely in vivo metabolic clearance (Table 4). No measurable degradation was observed for many of the compounds, suggesting that they would have low hepatic clearance in vivo. Only one compound showed instability in human liver microsomes, 13 with (EH = 0.7), likely due to pyridine N-oxide formation, whereas five compounds, 16, 3, 5, 9, and 10, exhibited a moderate rate of degradation. Generally speaking, the pyrazine analogues displayed good metabolic stability in human liver microsomal preparation. Notably, compound 4 demonstrated stability in both rat and human liver microsomes. Although some compounds in the aminopyrazine series displayed superior mouse efficacy compared to the previously 8865



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Table 3. In Vitro Antiplasmodial Activity and hERG Data of Compounds 4−10 and 26−36

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Table 3. continued

Mean from n values of ≥2 independent experiments. bLogD pH 7.4 values were determined using a chromatographic estimation method; nd = not determined. cData from Younis et al.11 dAverage of two independent experiments. eNo hERG inhibition at the highest measured concentration (11 μM). a

Mouse Exposure Studies. To interpret the in vivo efficacy results obtained, five representative analogues, 4, 5, 7, 9, and 10, were selected for mouse exposure studies on the basis of their varying degrees of in vivo efficacy. The plasma concentrations in P. berghei-infected mice were measured after an oral dose of 3 mg/kg and followed for 24 h using a limited sampling schedule (i.e; sampling at 1, 4, and 24 h), as depicted in Figure 3 and Table 6.24 As shown in Table 6, compound 5 exhibited a relatively lower Cmax but a more extended duration of exposure. However, it was shown that analogues 7, 9, and 10 exhibited a generally higher Cmax but a short duration of exposure, as evidenced by plasma concentrations being undetectable at 24 h after administration. It is noteworthy that even the relatively low systemic exposure

dose. Compound 8 was less potent at this dose, with a 45% reduction in parasitemia (MSD 6 days). However, compounds 9 and 10 exhibited significant activity at 4 × 3 mg/kg with a 99.7 (MSD 8 days) and 99.6% (MSD 8 days) reduction in parasitemia respectively. In general, 4 showed potent in vivo activity at all low multiple and single doses, with a >99.0% suppression of parasitemia. Interestingly, compound 4 remained active at a single oral dose of 3 mg/kg and multiple oral doses of 4 × 1 mg/kg with 99.4 (MSD 9 days) and 99.9%, (MSD 10 days) reduction in parasitemia, respectively. No toxicity symptoms were observed during in vivo studies with these compounds. Notably, an oral dose of 4 × 10 mg/kg of 4, resulting in 99.9% reduction in parasitemia, was completely curative (MSD >30 days). 8867



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Table 4. In Vitro Microsomal Stability for Selected Analogues metabolism in human liver microsome compound

t1/2 (min)

CLint (μL/min/mg)

EHa

(1) (11) (12) (13) (14) (15) (16) (17) (18) (19) (3) (20) (21) (4) (5) (6) (7) (9) (10)

281 c.n.c. >250 41 238.4 c.n.c. 121.7 nd c.n.c. >250 154 >250 >250 >250 155 >250 >250 217 111

6.2 c.n.c.

StructureâActivity-Relationship Studies around the 2-Amino Group

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StructureâActivity-Relationship Studies around the 2-Amino Group