Cell-Based Medicinal Chemistry Optimization of High Throughput

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Cell-Based Medicinal Chemistry Optimization of High Throughput Screening Hits for Orally Active Antimalarials. Part 2: Hits from SoftFocus Kinase and other Libraries Miniperspectives Series on Phenotypic Screening for Antiinfective Targets Yassir Younis,† Leslie J. Street,† David Waterson,‡ Michael J. Witty,‡ and Kelly Chibale*,†,§ †

Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa Medicines for Malaria Venture, ICC, Route de Pré-Bois 20, PO Box 1826, 1215 Geneva, Switzerland § Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa ‡

ABSTRACT: In the second part of this Miniperspectives series, we highlight our medicinal chemistry efforts involving progression of hits from whole cell highthroughput screening (HTS) of a SoftFocus kinase library against the malaria parasite Plasmodium falciparum. Successful SAR exploration in Hit-to-Lead and Lead Optimization efforts leading to the selection of a preclinical development candidate are demonstrated. Related efforts by researchers from Broad/ Genzyme, Anacor, and GSK are briefly covered.

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Harvard Medical School. The hit compound 18 from this series showed moderate activity across the strains (3D7/Dd2; IC50 = 216/507 nM) and a reasonable pharmacokinetic profile, resulting in demonstrable in vivo efficacy in Plasmodium berghei mouse model with an ED50 ip of 15 mg/kg (Figure 1). The

INTRODUCTION Malaria continues to be a major public health problem that predominantly affects developing countries and is responsible for an estimated 544,700−904,000 deaths each year, especially among children and pregnant women.1 The most important causative parasite, Plasmodium falciparum, is responsible for a majority of the serious morbidity and mortality of the disease.2 While there are a number of effective therapeutics available, resistance to even the newest medications is appearing and is a significant concern.3 Therefore, there is an urgent need to discover new medications that counter resistance and that are safe and easy for use in the most vulnerable populations. In recent years, phenotypic whole cell high-throughput screening (HTS) against the malaria parasite Plasmodium falciparum has made a major contribution toward the search for new antimalarial medicines as new chemotypes have emerged from these efforts. In a significant move displaying exceptional leadership, GlaxoSmithKline (GSK), Tres Cantos, Spain,4 St. Jude Children’s Research Hospital, Memphis, TN,5 and Novartis6 made available their confirmed compound hits from a combined malaria whole cell screening effort of around 4.5 million compounds. Following on from Miniperspective Part 1, authored by Chatterjee,7 we introduce Miniperspective Part 2 on our medicinal chemistry efforts involving progression of HTS hits from screening of a SoftFocus kinase library. We commence Miniperspective Part 2 by briefly covering related efforts by researchers from Broad/Genzyme, Anacor, and GSK. Researchers at Genzyme identified a novel 2-amino-3hydroxy-indole series of antimalarial compounds from screening of the compound library at the Institute of Chemical and Cell Biology-Longwood (ICCB-L), of the Broad Institute at © XXXX American Chemical Society

Figure 1. Lead optimization for aminoindole series.8

medicinal chemistry optimization of the molecule focused on the phenyl substituent, resulting in compound 28 (Genz668764) (chirality not disclosed) with good in vitro activity (3D7/Dd2; IC50 = 28/65 nM) and improved efficacy in the Pf SCID mouse assay (ED50 = 40 mg/kg po). In the four-day P. berghei in vivo mouse model, when compound 2 was dosed at 100 mg/kg po, no parasites were detected on day 4 post Special Issue: Miniperspectives Series on Phenotypic Screening for Antiinfective Targets Received: February 22, 2013

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dx.doi.org/10.1021/jm400279y | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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particular protein target or protein family in mind because they offered several advantages over more standard empirical screening libraries. These advantages include access to several novel pure drug-like molecule libraries with tractable chemistry and built-in SAR as well as facilitating target identification studies with a focus of initial efforts on specific gene families. Thus image-based screening of 35,000 compounds, spanning more than 200 scaffolds contained in BioFocus libraries, identified 222 hits against P. falciparum sensitive (3D7) and resistant (Dd2) strains from which we focused on hits demonstrating a greater than 80% reduction in parasites at a primary and retest concentration of 1.82 μM that additionally showed no cytotoxicity in HEK293 cells.11,12 From this effort, a series of ureas (6),11 aminomethyl thiazole amides (7),11 and 3,5-diaryl-2-aminopyridines (8)12 were identified as promising selective in vitro antiplasmodial hits (Figure 4).11,12 Representative active compounds from the two

infection; however, parasites recrudesced by day 9. Dosing at 200 mg/kg/day twice a day resulted in cures of 3/5 animals and 2 was selected as a candidate for preclinical development.8 It is noteworthy that the stereochemistry of compound 2 is not indicated in the published work.8 From screening the Anacor boron-containing compound collection against the malaria parasite P. falciparum, Plattner and his group identified an oxaborole series in which the lead compound 39 (AN3661) was shown to have good in vitro activity (3D7; IC50 = 44 nM), an apparent effect on all blood stages and impressive in vivo activity despite the short half-life, with an ED90 (four-day Peters test in NOD SCID mice with injected erythrocytes) of 0.6 mg/kg as shown in Figure 2. The

Figure 2. Oxaborole compound 3.9 Figure 4. Ureas (6) and aminomethyl thiazole amides (7) and 3,5diaryl-2-aminopyridines (8).11,12

medicinal chemistry optimization focused on side-chain variation and substituent modification on the benzene ring to deliver compounds with good efficacy and acceptable safety.9 From the GSK group, cyclopropyl carboxamides were selected from the Tres Cantos Anti-Malaria Set (TCAMS). The hit compound 410 showed good attractive properties from a medicinal chemistry viewpoint (Figure 3). SAR plans focused

series were selected for resynthesis, repurification, and confirmatory testing for potency and cytotoxicity. Representatives were then profiled in physical property and in vitro metabolism screens, and all data suggested both series had good properties. Thus they represented a good starting point for medicinal chemistry progression. Analysis of HTS SAR led to the first set of compounds with medicinal chemistry efforts focused in two areas. The first was rapid exploitation of data to deliver optimized molecules. The second was making drastic changes to probe fundamental understanding of SAR. These efforts led to the identification of aminomethylthiazole pyrazole carboxamide 911 (NF54/K1; IC50 = 0.08 μM) and 1012 (NF54/K1; IC50 = 0.05 μM) as selective promising structures upon which further SAR investigations were based (Figure 5). The requirement for the aminomethyl group as well as the thiazole and pyrazole moieties for 9 were consequently established. On the other hand, drastic changes to aminopyridines confirmed essentiality of 2-NH2 pyridine and preferred 3,5-diaryl substitution in 10.12 The aforementioned moieties were then exploited for further SAR studies. From SAR studies, it emerged that the thiazole series generally showed good antiplasmodial activity and good metabolic stability. The main challenge in lead optimization was to improve the in vivo efficacy and pharmacokinetic properties. The very limited range of structural modifications that could be tolerated resulted in flat SAR and was a bottleneck in further lead optimization efforts. The lead compound 9 exhibited in vivo activity in the P. berghei mouse model at 4 × 50 mg/kg dose administration via the oral route, showing 99.5% activity and 9 days survival (Figure 5). Furthermore, determination of the effective doses where 50% and 90% reduction in parasitemia was observed (ED50/ED90) indicated that compound 9 is more than 10-fold less potent compared to chloroquine, with ED50/ED90 values of 37 and 86 mg/kg versus 1.9 and 4.2 mg/kg (single oral dosing). No significant increase in survival could be achieved with a 4 × 50

Figure 3. Lead optimization for cyclopropyl carboxamide series.10

on the triazole ring, the CF3 substituent on ring B, and the cyclopropyl group adjacent to the amide carbonyl, which resulted in no improvement in the overall properties of the series. A variety of substituents were explored in optimization of the substitution on the C ring and a meta-CF3 proved highly beneficial with a 30-fold increase in activity against Pf (3D7; IC50 = 3 nM). The lead compound 510 inhibited P. falciparum growth in vivo, with ED90 and ED50 values of 20 and 12 mg/kg, respectively. The ED50 of 5 was 2−3 times higher than that of chloroquine, and lead optimization is ongoing to assess the real potential of the series.10 In recent work by our group at the University of Cape Town (UCT) in collaboration with Medicines for Malaria Venture (MMV), Eskitis Institute at Griffith University, Swiss Tropical and Public Health Institute, Monash University, and Syngene, we have undertaken a HTS campaign of BioFocus DPI SoftFocus libraries and subsequent medicinal chemistry progression of hits from this campaign. We were attracted to the BioFocus SoftFocus libraries, which were designed with a B



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displayed varying degrees of activity, the most notable improvement in the hERG profile was when more polar substituents were introduced in the 5-aryl group, as exemplified in methyl piperazine 12 (Figure 6),13 (IC50 = 20.5 μM).

Figure 6. Aminopyridines 12 and 13 with an improved hERG profile.12,13

Improvements in the hERG profile were also observed by the introduction of 6-trifluoromethylpyridin-3-yl groups at the 3aryl position as exemplified in morpholino compound 13 (Figure 6)13 (IC50 = 20.2 μM). Unfortunately, improvements of in vitro antiplasmodial and hERG activity were not accompanied by exceptional curative oral efficacy in the P. berghei mouse model, which was observed in frontrunner compounds 10 and 11 due to the less prolonged drug exposure achieved with these more polar compounds.13 Thus further optimization of these leads is required.

Figure 5. Frontrunner compound 9 from aminomethylthiazole series and aminopyridine series lead compound 10 and selected candidate 11.12

mg/kg dosing regimen.11 With respect to the aminopyridines series, the medicinal chemistry effort around the 3- and 5substituents of the 2-aminopyridine ring led to identification of the initial lead 10. This compound demonstrated good efficacy in vivo (Pf SCID Peters test) ED90 3.6 mg/kg with good pharmacokinetics in rats (F% 83% at 5 mg/kg; t1/2 8.7 h). The major flag at this stage of the process was hERG (IC50 = 3.5 μM in the Ionworks patch clamp electrophysiology assay). In spite of this, the compound was profiled extensively to establish preclinical candidate potential. However, additional major drawbacks included a low predicted human t1/2 of 12 h and high predicted human dose. Thus the lead optimization campaign focused on improving potency, decreasing clearance, and improving hERG. Modification of −OCH3 to −CF3 led to optimal balance as demonstrated by compound 11,12 which was more potent against multiple asexual strains (IC50: 18−30 nM) that were stable and had less hERG activity. This compound completely cured mice (99.3%) at a single oral dose of 30 mg/ kg (>30 MSD) in the P. berghei mouse model. This result is noteworthy in view of the fact that clinically used drugs such as CQ, mefloquine, and the artemisinins do not achieve a single oral dose cure in the P. berghei in vivo model. Furthermore, dose−response studies generated ED50 and ED90 values of 0.83 and 1.74 mg/kg for 11 in the standard four-dose Peters test. The good in vivo efficacy of this series correlated well with plasma exposure following oral dosing of these compounds in mice.12 Coupled with a good exploratory rat safety profile, good developability, and human pharmacokinetic prediction, this compound was approved as a preclinical drug development candidate. Although optimized lead 11 showed reduced hERG activity, we deemed it essential to embark on a campaign aimed at identifying compounds that would either further minimize or eliminate the hERG liability while retaining high in vitro potency along with good ADME properties and in vivo efficacy. Optimization of aryl substituents at positions 3 and 5 of the 2aminopyridine core led to several analogues being synthesized and tested for inhibition of the hERG channel. While analogues

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CONCLUSION The use of a phenotypic whole cell screening approach and successful SAR exploration in Hit-to-Lead and Lead Optimization efforts leading to the selection of a preclinical development candidate by our team has been ably demonstrated, particularly in the case of 3,5-diaryl-2-aminopyridines.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +27-21-6502553. Fax: +27-21-6505195. E-mail: Kelly. [email protected]. Notes

The authors declare no competing financial interest. Biographies Yassir Younis received his Ph.D. in synthetic organic chemistry from the University of Cape Town (UCT) in June 2009 under supervision of Prof. Roger Hunter in collaboration with Professor Karen Anderson at Yale University in the USA. His work focused on the design and synthesis of potential HIV double-drugs (NRTI/NNRTI) against HIV reverse-transcriptase (RT). Following this, he embarked on a one year postdoctoral stint, again in the Hunter group, working on new synthetic strategies for making thymidine on small and large scales. As of 2010, Dr. Younis joined Prof. Chibale’s Lab in the Hit to Lead (H2L) and Lead Optimization (LO) programs. Dr. Younis has made significant contributions in the delivery of a preclinical development candidate arising from the UCT project. Leslie J. Street has over 25 years’ of drug discovery experience. During this time, he identified clinical candidates for various neurological and psychiatric disorders at drug companies in both the U.K. and U.S. At Merck in the U.K., Dr. Street led groups on targeting muscarinic agonists, 5-HT1D agonists, and GABAAα2/α3 agonists that led to C



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(2) Arora, N.; Banerjee, K. A. New Targets, New Hope: Novel Drug Targets for Curbing Malaria. Mini-Rev. Med. Chem. 2012, 12, 210− 226. (3) Burrows, N. J.; Chibale, K.; Wells, T. N. C. The state of the art in anti-malarial drug discovery and development. Curr. Top. Med. Chem. 2011, 11, 1226−1254. (4) Gamo, F.-J.; Sanz, L. M. S.; Vidal, J.; de Cozar, C.; Alvarez, E.; Jose-Luis Lavandera, J.-L.; Vanderwall, D. E.; Green, D. V. S.; Kumar, V.; Hasan, S.; Brown, J. R.; Peishoff, C. E.; Cardon, L. R.; GarciaBustos, J. F. Thousands of chemical starting points for antimalarial lead identification. Nature 2010, 465, 305−310. (5) Guiguemde, W. A.; Shelat, A. A.; Bouck, D.; Duffy, S.; Crowther, G. J.; Davis, P. H.; Smithson, D. C.; Connelly, M.; Clark, J.; Zhu, F.; Jimenez-Dıaz, M. B.; Martinez, M. S.; Wilson, E. B.; Tripathi, A. K.; Gut, J.; Sharlow, E. R.; Bathurst, I.; El Mazouni, F.; Fowble, J. W.; Forquer, I.; McGinley, P. L.; Castro, S.; Angulo-Barturen, I.; Ferrer, S.; Rosenthal, P. J.; DeRisi, J. L.; Sullivan, D. J.; Lazo, J. S.; Roos, D. S.; Riscoe, M. K.; Phillips, M. A.; Rathod, P. K.; Van Voorhis, W. C.; Avery, V. M.; Guy, R. K. Chemical genetics of Plasmodium falciparum. Nature 2010, 465, 311−315. (6) Plouffe, D.; Brinke, A.; McNamara, C.; Henson, K.; Kato, N.; Kuhen, K.; Nagle, A.; Adrián, F.; Matzen, J.; Anderson, P.; Nam, T-G; Gray, N.; Chatterjee, A.; Jeff Janes, J.; Frank Yan, S.; Trager, R.; Caldwell, J.; Schultz, P.; Zhou, Y.; Winzeler, E. In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen. Proc. Natl Acad. Sci. U. S. A. 2008, 105, 9059−9064. (7) ChatterjeeA. Cell-based medicinal chemistry optimization of high-througput screening (HTS) hits for orally active antimalarials. Part 1: challenges in potency and absorption, distribution, metabolism, excretion/pharmacokinetics (ADME/PK). J. Med. Chem. 2013, DOI 10.1021/jm400314m. (8) Barker, R.; Urgaonkar, S.; Mazitschek, R.; Celatka, C.; Skerlj, R.; Cortese, J.; Tyndall, E.; Hanlan Liu, H.; Cromwell, M.; Bir Sidhu, A.; Guerrero-Bravo, J.; Crespo-Llado, K.; Serrano, A.; Lin, J.-W.; Janse, C.; Khan, S.; Duraisingh, M.; Coleman, B.; Angulo-Barturen, I.; Belén Jiménez-Díaz, M.; Magán, N.; Gomez, V.; Ferrer, S.; Santos Martínez, M.; Wittlin, S.; Papastogiannidis, P.; O’Shea, T.; Klinger, J.; Bree, M.; Lee, E.; Levine, M.; Wiegand, R.; Munoz, B.; Wirth, D.; Clardy, J.; Bathurst, I.; Sybertz, E. Aminoindoles, a Novel Scaffold with Potent Activity against Plasmodium falciparum. Antimicrob. Agents Chemother. 2011, 55, 2612−2622. (9) Zhang, Y.-K.; Plattner, J.; Freund, Y.; Easom, E.; Zhou, Y.; Ye, L.; Zhou, H.; Waterson, D.; Gamo, F.-J.; Sanz, L.; Ge, M.; Li, Z.; Li, L.; Wange, H.; Cui, H. Benzoxaborole antimalarial agents. Part 2: Discovery of fluoro-substituted 7-(2-carboxyethyl)-1,3-dihydro-1hydroxy-2,1-benzoxaboroles. Bioorg. Med. Chem. Lett. 2012, 22, 1299−1307. (10) Rueda, L.; Castellote, I.; Castro-Pichel, J.; Chaparro, M.; la Rosa, J. C.; Garcia-Perez, A.; Gordo, M.; Jimenez-Diaz, M. B.; Kessler, A.; Macdonald, S.; Martinez, M. S.; Sanz, L.; Gamo, F. J.; Fernandez, E. Cyclopropyl Carboxamides: A New Oral Antimalarial Series Derived from the Tres Cantos Anti-Malarial Set (TCAMS). ACS Med. Chem. Lett. 2011, 2, 840−844. (11) Cabrera, D. G.; F. Douelle, F.; Feng, T.-S.; Nchinda, A. T.; Younis, Y.; White, K. L.; Wu, Q.; Q. Ryan, E.; Burrows, J. N.; Waterson, D.; Witty, M. J.; Wittlin, S.; Charman, S. A.; Chibale, K. Novel Orally Active Antimalarial Thiazoles. J. Med. Chem. 2011, 54, 7713−7719. (12) Younis, Y.; Douelle, F.; Feng, T.-S.; González Cabrera, D.; Le Manach, C.; Nchinda, T. A.; Duffy, S.; White, K. L.; Shackleford, D. M.; Morizzi, J.; Mannila, J.; Katneni, K.; Bhamidipati, R.; Zabiulla, K. M.; Joseph, J. T.; Bashyam, S.; Waterson, D.; Witty, M. J.; Hardick, D.; Wittlin, S.; Avery, V.; Charman, S. A.; Chibale, K. 3,5-Diaryl-2aminopyridines as a Novel Class of Orally Active Antimalarials Demonstrating Single Dose Cure in Mice and Clinical Candidate Potential. J. Med. Chem. 2012, 55, 3479−3487. (13) González Cabrera, D.; Douelle, F.; Younis, Y.; Feng, T.-S.; Le Manach, C.; Nchinda, A. T; Street, L.; Scheurer, C.; Kamba, J.; White,

several compounds entering human clinical trials and a marketed drug. In the U.S., at Cortex Pharmaceuticals, Leslie was responsible for directing the chemistry research based on AMPA modulators, for neurological and psychiatric disorders, e.g., ADHD, and opiate induced respiratory depression, and from this work a compound successfully completed phase I clinical studies. Leslie is currently head of medicinal chemistry for the H3-D centre at UCT. David Waterson obtained his Ph.D. in organic chemistry at Cambridge University. He has over 25 years of experience in the global pharmaceutical sector (principally at AstraZeneca UK) as a medicinal chemist working within multiple therapeutic areas. He also spent two years at AstraZeneca’s Bangalore site, where the focus was on the identification of new treatments for tuberculosis. David is currently Director within the Discovery Team at Medicines for Malaria Venture (MMV) and is responsible for a number of Lead Generation and Lead Optimization projects within the MMV portfolio. Michael J. Witty obtained his Ph.D. in organic chemistry at Oxford University and then joined Pfizer Animal Health, working in drug and vaccine R&D for most therapeutic areas of livestock and companion animal research with a focus and expertise in parasitology. He later moved to a divisional HQ role responsible for Portfolio and Strategic Planning. Since retiring in 2008, he has been a scientific consultant supporting various, mainly not-for-profit, organizations involved in human and animal neglected disease research. He has been a member of the MMV Expert Scientific Advisory Committee since 2007 and a mentor and scientific consultant to various of their projects. He has been a member of several WHO-TDR, Wellcome Trust, B&M Gates Foundation, and GALVmed scientific advisory boards. Kelly Chibale obtained his Ph.D. in synthetic organic chemistry from the University of Cambridge in the U.K. with Stuart Warren (1989− 1992). This was followed by postdoctoral stints at the University of Liverpool in the U.K. as a British Ramsay Research Fellow with Nick Greeves (1992−1994) and at the Scripps Research Institute in the USA as a Wellcome Trust International Prize Research Fellow with K .C. Nicolaou (1994−1996). He joined the University of Cape Town (UCT) in 1996 and has so far spent sabbaticals at the University of California San Francisco (2002), University of Pennsylvania (2008), and Pfizer (2008). He is the Founder and Director of the UCT Drug Discovery and Development Centre (H3-D).

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ACKNOWLEDGMENTS We thank Medicines for Malaria Venture (MMV) for financial support for this research (project MMV09/0002). We thank Dr. Jeremy N. Burrows (MMV) for helpful discussions. The University of Cape Town, South African Medical Research Council, and South African Research Chairs initiative of the Department of Science and Technology administered through the South African National Research Foundation are gratefully acknowledged for support (K.C.).

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ABBREVIATIONS USED hERG, the human ether-à-go-go-related gene; ICCB-L, Institute of Chemical and Cell Biology, Longwood; ip, Intraperitoneal administration; NOD, non-obese diabetic; po, oral administration; MSD, mean survival time; SAR, structure− activity relationship; PK, pharmacokinetics

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REFERENCES

(1) World Malaria Report 2012; World Health Organization: Geneva, Switzerland, 2012. D



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K. L.; Montagnat, O. D.; Ryan, E.; Katneni, K.; Zabiulla, K. M.; Joseph, J. T.; Bashyam, S.; Waterson, D.; Witty, M. J.; Charman, S. A.; Wittlin, S.; Chibale, K. Synthesis and Structure−Activity Relationship Studies of Antimalarial 3,5-Substituted 2-Aminopyridines. J. Med. Chem. 2012, 55, 11022−11030.

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Cell-Based Medicinal Chemistry Optimization of High Throughput

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