A 900 compound nitroimidazole-based library derived from our pretomanid backup program with TB Alliance was screened for utility against human African trypanosomiasis (HAT) by the Drugs for Neglected Diseases initiative. Potent hits included 2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]thiazine 8-oxides, which surprisingly displayed good metabolic stability and excellent cell permeability. Following comprehensive mouse pharmacokinetic assessments on four hits and determination of the most active chiral form, a thiazine oxide counterpart of pretomanid (24) was identified as the best lead. With once daily oral dosing, this compound delivered complete cures in an acute infection mouse model of HAT and increased survival times in a stage 2 model, implying the need for more prolonged CNS exposure. In preliminary SAR findings, antitrypanosomal activity was reduced by removal of the benzylic methylene but enhanced through a phenylpyridine-based side chain, providing important direction for future studies.

Journal of Eukaryotic MicrobiologyVolume 64, Issue 4 p. 558-559 In Memoriam In Memoriam: Michael Levandowsky (1935–2016) Cyrus J. Bacchi, Cyrus J. Bacchi Haskins Laboratories, Pace University, New York, 10038 New YorkSearch for more papers by this authorThomas Gorrell, Thomas Gorrell Haskins Laboratories, Pace University, New York, 10038 New York School of Visual Arts, New York, 10010 New YorkSearch for more papers by this authorGeorge McManus, George McManus Marine Sciences, University of Connecticut, Groton, 06340 ConnecticutSearch for more papers by this authorNigel Yarlett, Corresponding Author Nigel Yarlett nyarlett@pace.edu orcid.org/0000-0002-7493-4346 Haskins Laboratories, Pace University, New York, 10038 New York Department of Chemistry and Physical Sciences, Pace University, New York, 10038 New York Correspondence N. Yarlett, Haskins Laboratories, Pace University, New York, NY 10038, USA Telephone number: +1 212 346-1853; FAX number: +1 212 346-1586; e-mail: nyarlett@pace.eduSearch for more papers by this author Cyrus J. Bacchi, Cyrus J. Bacchi Haskins Laboratories, Pace University, New York, 10038 New YorkSearch for more papers by this authorThomas Gorrell, Thomas Gorrell Haskins Laboratories, Pace University, New York, 10038 New York School of Visual Arts, New York, 10010 New YorkSearch for more papers by this authorGeorge McManus, George McManus Marine Sciences, University of Connecticut, Groton, 06340 ConnecticutSearch for more papers by this authorNigel Yarlett, Corresponding Author Nigel Yarlett nyarlett@pace.edu orcid.org/0000-0002-7493-4346 Haskins Laboratories, Pace University, New York, 10038 New York Department of Chemistry and Physical Sciences, Pace University, New York, 10038 New York Correspondence N. Yarlett, Haskins Laboratories, Pace University, New York, NY 10038, USA Telephone number: +1 212 346-1853; FAX number: +1 212 346-1586; e-mail: nyarlett@pace.eduSearch for more papers by this author First published: 11 May 2017 https://doi.org/10.1111/jeu.12416Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume64, Issue4July/August 2017Pages 558-559 RelatedInformation

A series of 15 alkanediamide-linked bisbenzamidines and related analogs was synthesized and tested in vitro against two Trypanosoma brucei (T.b.) subspecies: T.b. brucei and T.b. rhodesiense, Trypanosoma cruzi, Leishmania donovani and two Plasmodium falciparum subspecies: a chloroquine-sensitive strain (NF54) and a chloroquine-resistant strain (K1). The in vitro cytotoxicity was determined against rat myoblast cells (L6). Seven compounds (5, 6, 10, 11, 12, 14, 15) showed high potency against both strains of T. brucei and P. falciparum with the inhibitory concentrations for 50% (IC50) in the nanomolar range (IC50 = 1–96 nM). None of the tested derivatives was significantly active against T. cruzi or L. donovani. Three of the more potent compounds (5, 6, 11) were evaluated in vivo in mice infected with the drug-sensitive (Lab 110 EATRO and KETRI 2002) or drug-resistant (KETRI 2538 and KETRI 1992) clinical isolates of T. brucei. Compounds 5 and 6 were highly effective in curing mice infected with the drug-sensitive strains, including a drug-resistant strain KETRI 2538, but were ineffective against KETRI 1992. Thermal melting of DNA and molecular modeling studies indicate AT-rich DNA sequences as possible binding sites for these compounds. Several of the tested compounds are suitable leads for the development of improved antiparasitic agents.

Trypanothione reductase (TR) is an enzyme critical to the maintenance of the thiol redox balance in trypanosomatids, including the genera Trypanosoma and Leishmania that are parasites responsible for several serious diseases. Analogs of clomipramine were prepared since clomipramine is reported to inhibit TR and cure mice infected with trypanosomes, however its psychotropic activity precludes its use as an anti-trypanosomal therapeutic. The clomipramine analogs contained a tricyclic dibenzosuberyl moiety. Additionally a series of polyamines with N-dibenzosuberyl substituents were prepared. All compounds studied were competitive inhibitors of TR and showed trypanocidal activities against Trypanosoma brucei in vitro. The analogs of clomipramine were poor inhibitors of TR, whereas the polyamine derivatives were effective TR inhibitors with the most potent compound, N(4),N(8)-bis(dibenzosuberyl)spermine (7), having a Ki value of 0.26μM. However, compound (7) did not prolong the lives of mice infected with trypanosomes. Analysis of docking studies indicated: the tricyclic groups of inhibitors bind at four distinct hydrophobic regions in the active site of TR; the importance of the chlorine substituent of clomipramine in binding to TR; and binding of the dibenzosuberyl groups of (7) occur at separate and distinct hydrophobic regions within the active site of TR.

A series of 15 alkanediamide-linked bisbenzamidines and related analogs were synthesized and tested in vitro against two Trypanosoma brucei (Tb) strains: T. b. brucei (Tbb) and T. b. rhodesiense (Tbr), two Plasmodium falciparum (Pf) strains: a chloroquine-sensitive strain (NF54) and a chloroquine-resistant strain (K1), Trypanosoma cruzi (Tc), and Leishmania donovani (Ld). The in vitro cytotoxicity was determined against rat myoblast cells (L6). Seven compounds (5, 6, 10, 11, 12, 14, 15) showed high potency toward both strains of Tb and Pf with the inhibitory concentrations for 50% (IC50) in the nanomolar range (IC50 = 1-96 nM). None of the tested derivatives was significantly active against Tc or Ld. Three of the more potent compounds (5, 6, 11) were evaluated in vivo in mice infected with the drug-sensitive (Lab 110 EATRO and KETRI 2002) or drug-resistant (KETRI 2538 and KETRI 1992) clinical isolates of T. brucei. Compounds 5 and 6 were highly effective in curing 100% mice infected with the drug-sensitive strains, including a drug-resistant strain KETRI 2538, but were ineffective against KETRI 1992. Thermal melting of DNA and molecular modeling studies indicate AT-rich DNA sequences in the minor grove as possible binding sites for these compounds. Several of the tested compounds are excellent leads for the development of improved antiparasitic agents.

SUMMARY This review presents a progression strategy for the discovery of new anti-parasitic drugs that uses in vitro susceptibility, time-kill and reversibility measures to define the therapeutically relevant exposure required in target tissues of animal infection models. The strategy is exemplified by the discovery of SCYX-7158 as a potential oral treatment for stage 2 (CNS) Human African Trypanosomiasis (HAT). A critique of current treatments for stage 2 HAT is included to provide context for the challenges of achieving target tissue disposition and the need for establishing pharmacokinetic–pharmacodynamic (PK–PD) measures early in the discovery paradigm. The strategy comprises 3 stages. Initially, compounds demonstrating promising in vitro activity and selectivity for the target organism over mammalian cells are advanced to in vitro metabolic stability, barrier permeability and tissue binding assays to establish that they will likely achieve and maintain therapeutic concentrations during in-life efficacy studies. Secondly, in vitro time-kill and reversibility kinetics are employed to correlate exposure (based on unbound concentrations) with in vitro activity, and to identify pharmacodynamic measures that would best predict efficacy. Lastly, this information is used to design dosing regimens for pivotal pharmacokinetic–pharmacodyamic studies in animal infection models.

Human African trypanosomiasis (African sleeping sickness) is a disease caused by various subspecies of the parasite Trypanosoma brucei and spread through an insect vector, the tsetse fly, in sub-Saharan Africa. Although only relatively small numbers of patients (estimated by the World Health Organization to be approximately 10 000 per annum) are diagnosed each year, all victims of this disease will progress to a second-stage central nervous system disease that is 100% fatal if untreated. Current treatment options for HAT are limited to old, toxic, and ineffective drugs that are difficult to administer in the disease endemic area, particularly for the second-stage disease. Consequently, there is an urgent need for the discovery and development of new agents to treat Stage 2 HAT. This chapter reviews some recent efforts in this area, in particular the discovery and development of a new class of novel boron-containing compounds, the benzoxaboroles.

A series of 2,4-diaminopyrimidines was investigated and compounds were found to have in vivo efficacy against Trypanosoma brucei in an acute mouse model. However, in vitro permeability data suggested the 2,4-diaminopyrimidenes would have poor permeability through the blood brain barrier. Consequently a series of 4-desamino analogs were synthesized and found to have improved in vitro permeability.

Human African trypanosomiasis, caused by the kinetoplastid parasite Trypanosoma brucei, affects thousands of people across sub-Saharan Africa, and is fatal if left untreated. Treatment options for this disease, particularly stage 2 disease, which occurs after parasites have infected brain tissue, are limited due to inadequate efficacy, toxicity and the complexity of treatment regimens. We have discovered and optimized a series of benzoxaborole-6-carboxamides to provide trypanocidal compounds that are orally active in murine models of human African trypanosomiasis. A key feature of this series is the presence of a boron atom in the heterocyclic core structure, which is essential to the observed trypanocidal activity. We also report the in vivo pharmacokinetic properties of lead compounds from the series and selection of SCYX-7158 as a preclinical candidate.

Background Human African trypanosomiasis (HAT) is an important public health problem in sub-Saharan Africa, affecting hundreds of thousands of individuals. An urgent need exists for the discovery and development of new, safe, and effective drugs to treat HAT, as existing therapies suffer from poor safety profiles, difficult treatment regimens, limited effectiveness, and a high cost of goods. We have discovered and optimized a novel class of small-molecule boron-containing compounds, benzoxaboroles, to identify SCYX-7158 as an effective, safe and orally active treatment for HAT. Methodology/Principal Findings A drug discovery project employing integrated biological screening, medicinal chemistry and pharmacokinetic characterization identified SCYX-7158 as an optimized analog, as it is active in vitro against relevant strains of Trypanosoma brucei, including T. b. rhodesiense and T. b. gambiense, is efficacious in both stage 1 and stage 2 murine HAT models and has physicochemical and in vitro absorption, distribution, metabolism, elimination and toxicology (ADMET) properties consistent with the compound being orally available, metabolically stable and CNS permeable. In a murine stage 2 study, SCYX-7158 is effective orally at doses as low as 12.5 mg/kg (QD×7 days). In vivo pharmacokinetic characterization of SCYX-7158 demonstrates that the compound is highly bioavailable in rodents and non-human primates, has low intravenous plasma clearance and has a 24-h elimination half-life and a volume of distribution that indicate good tissue distribution. Most importantly, in rodents brain exposure of SCYX-7158 is high, with Cmax >10 µg/mL and AUC0–24 hr >100 µg*h/mL following a 25 mg/kg oral dose. Furthermore, SCYX-7158 readily distributes into cerebrospinal fluid to achieve therapeutically relevant concentrations in this compartment. Conclusions/Significance The biological and pharmacokinetic properties of SCYX-7158 suggest that this compound will be efficacious and safe to treat stage 2 HAT. SCYX-7158 has been selected to enter preclinical studies, with expected progression to phase 1 clinical trials in 2011.

Background: There is an urgent need to develop new, safe and effective treatments for human African trypanosomiasis (HAT) because current drugs have extremely poor safety profiles and are difficult to administer. Here we report the discovery of 2,4-diaminopyrimidines, exemplified by 4-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid phenylamide (SCYX-5070), as potent inhibitors of Trypanosoma brucei and the related trypanosomatid protozoans Leishmania spp.Methodology/Principal Findings: In this work we show that loss of T. brucei viability following SCYX-5070 exposure was dependent on compound concentration and incubation time. Pulse incubation of T. brucei with SCYX-5070 demonstrates that a short period of exposure (10-12 hrs) is required to produce irreversible effects on survival or commit the parasites to death. SCYX-5070 cured an acute trypanosomiasis infection in mice without exhibiting signs of compound related acute or chronic toxicity. To identify the molecular target(s) responsible for the mechanism of action of 2,4-diaminopyrimidines against trypanosomatid protozoa, a representative analogue was immobilized on a solid matrix (sepharose) and used to isolate target proteins from parasite extracts. Mitogen-activated protein kinases (MAPKs) and cdc2-related kinases (CRKs) were identified as the major proteins specifically bound to the immobilized compound, suggesting their participation in the pharmacological effects of 2,4-diaminopyrimidines against trypanosomatid protozoan parasites.Conclusions/Significance: Results show that 2,4-diaminopyrimidines have a good in vitro and in vivo pharmacological profile against trypanosomatid protozoans and that MAPKs and CRKs are potential molecular targets of these compounds. The 2,4-diminipyrimidines may serve as suitable leads for the development of novel treatments for HAT.

R. T. Jacobs1, B. Nare1, S. A. Wring1, C. J. Bacchi2, R. Brun3, J. J. Plattner4, B. Beaudet1, T. S. Bowling1, D. Chen1, Y. Freund4, E. Gaukel1, M. Jenks1, M. Kaiser3, L. T. Mercer1, A. Noe1, M. D. Orr1, R. Parham1, R. Randolph1, C. Rewerts1, J. M. Sligar1, N. Yarlett2, R. Don5 1SCYNEXIS, Inc., Research Triangle Park, North Carolina, United States 2Haskins Laboratory, Pace University, New York, New York, United States 3Swiss Tropical Institute, Basel, Switzerland 4Anacor Pharmaceuticals, Inc., Palo Alto, California, United States 5Drugs for Neglected Diseases initiative, Geneva, Switzerland

We report the discovery of benzoxaborole antitrypanosomal agents and their structure-activity relationships on central linkage groups and different substitution patterns in the sulfur-linked series. The compounds showed in vitro growth inhibition IC50 values as low as 0.02 μg/mL and in vivo efficacy in acute murine infection models against Tryapnosoma brucei.

ABSTRACT We report the discovery of novel boron-containing molecules, exemplified by N -(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)-2-trifluoromethylbenzamide (AN3520) and 4-fluoro- N -(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)-2-trifluoromethylbenzamide (SCYX-6759), as potent compounds against Trypanosoma brucei in vitro , including the two subspecies responsible for human disease T. b. rhodesiense and T. b. gambiense. These oxaborole carboxamides cured stage 1 (hemolymphatic) trypanosomiasis infection in mice when administered orally at 2.5 to 10 mg/kg of body weight for 4 consecutive days. In stage 2 disease (central nervous system [CNS] involvement), mice infected with T. b. brucei were cured when AN3520 or SCYX-6759 were administered intraperitoneally or orally (50 mg/kg) twice daily for 7 days. Oxaborole-treated animals did not exhibit gross signs of compound-related acute or subchronic toxicity. Metabolism and pharmacokinetic studies in several species, including nonhuman primates, demonstrate that both SCYX-6759 and AN3520 are low-clearance compounds. Both compounds were well absorbed following oral dosing in multiple species and also demonstrated the ability to cross the blood-brain barrier with no evidence of interaction with the P-glycoprotein transporter. Overall, SCYX-6759 demonstrated superior pharmacokinetics, and this was reflected in better efficacy against stage 2 disease in the mouse model. On the whole, oxaboroles demonstrate potent activity against all T. brucei subspecies, excellent physicochemical profiles, in vitro metabolic stability, a low potential for CYP450 inhibition, a lack of active efflux by the P-glycoprotein transporter, and high permeability. These properties strongly suggest that these novel chemical entities are suitable leads for the development of new and effective orally administered treatments for human African trypanosomiasis.

Human Africa trypanosomiasis is a centuries-old disease which has disrupted sub-Saharan Africa in both physical suffering and economic loss. This article presents an update of classic chemotherapeutic agents, in use for >50 years and the recent development of promising non-toxic combination chemotherapy suitable for use in rural clinics.

In vitro activity against Trypanosoma brucei strains Summary SCYX-7158, a 3,3-dimethyloxaborole-6-carboxamide, is distinguished from earlier trypanocidal oxaboroles by enhanced pharmacokinetic and CNS disposition properties allowing for a once per day (QD) oral dosing regimen at a markedly lower efficacious dose in a Stage 2 murine Human African Trypanosomiasis (HAT) model. The discovery of SCYX-7158 was achieved through application of integrated lead optimization strategies across medicinal chemistry, parasitology and pharmacokinetic disciplines. SCYX-7158 is active in vitro against relevant strains of Trypanosoma brucei, including T. b. rhodesiense and T. b. gambiense and is efficacious in both Stage 1 and Stage 2 murine HAT models. Physicochemical and in vitro ADME properties of SCYX-7158 are consistent with the compound being orally available, metabolically stable, readily CNS permeable and with low risk for drug-drug interactions. In an ongoing murine Stage 2 study, SCYX-7158 is effective orally at doses as low as 12.5 mg/kg (QD x 7 days). In vivo pharmacokinetic characterization of SCYX-7158 demonstrates that the compound is highly bioavailable in rodents, has low intravenous plasma clearance, a 24 hr elimination half-life and a volume of distribution that indicates good tissue distribution. Most importantly, SCYX7158 readily distributes into brain and CSF and crosses the blood-testicular barrier to achieve therapeutically-relevant concentrations in potential trypanosomal sanctuary sites. Based on these properties, which promise lower rates of recrudescence than with current standard of care, SCYX-7158 has been selected as a pre-clinical candidate for treatment of Stage 2 HAT. In vitro ADMET profile of SCYX-7158 In vivo PK of SCYX-7158 in rodents Stage 2 CNS Model (Chronic) Stage 1 non-CNS Model (Acute) Synthesis of SCYX-7158

ABSTRACTTrypanosomiasis remains a significant disease across the sub-Saharan African continent, with 50,000 to 70,000 individuals infected. The utility of current therapies is limited by issues of toxicity and the need to administer compounds intravenously. We have begun a program to pursue lead optimization around MDL 73811, an irreversible inhibitor ofS-adenosylmethionine decarboxylase (AdoMetDC). This compound is potent but in previous studies cleared rapidly from the blood of rats (T. L. Byers, T. L. Bush, P. P. McCann, and A. J. Bitonti, Biochem. J. 274:527-533). One of the analogs synthesized (Genz-644131) was shown to be highly active againstTrypanosoma brucei rhodesiensein vitro (50% inhibitory concentration, 400 pg/ml). Enzyme kinetic studies showed Genz-644131 to be approximately fivefold more potent than MDL 73811 against theT. brucei bruceiAdoMetDC-prozyme complex. This compound was stable in vitro in rat and human liver microsomal and hepatocyte assays, was stable in rat whole-blood assays, did not significantly inhibit human cytochrome P450 enzymes, had no measurable efflux in CaCo-2 cells, and was only 41% bound by serum proteins. Pharmacokinetic studies of mice following intraperitoneal dosing showed that the half-life of Genz-644131 was threefold greater than that of MDL 73811 (7.4 h versus 2.5 h). Furthermore, brain penetration of Genz-644131 was 4.3-fold higher than that of MDL 73811. Finally, in vivo efficacy studies ofT. b. bruceistrain STIB 795-infected mice showed that Genz-644131 significantly extended survival (from 6.75 days for controls to >30 days for treated animals) and cured animals infected withT. b. bruceistrain LAB 110 EATRO. Taken together, the data strengthen validation of AdoMetDC as an important parasite target, and these studies have shown that analogs of MDL 73811 can be synthesized with improved potency and brain penetration.

Human African Trypanosomiasis (HAT) represents a significant public health problem in sub-Saharan Africa affecting hundreds of thousands of individuals. An urgent need exists for the discovery and development of new, safe, and effective drugs to treat HAT, as existing therapies suffer from poor safety profiles, difficult treatment regimens, limited effectiveness, and a high cost of goods. From a collaborative effort between SCYNEXIS, Anacor Pharmaceuticals, Pace University, and DNDi, we report ongoing lead optimization efforts on a novel class of small molecule boron-containing compounds, exemplified by SCYX-6759. These compounds inhibit in vitro growth of T. brucei with IC50 ’s ~100 nM, are not cytotoxic to mammalian cells, and exhibit good physiochemical and pharmacokinetic properties. In a murine model of CNS-stage disease utilizing the TREU667 strain of T.b.brucei, treatment with 50 mg/kg of SCYX-6759 BID for 14 days has demonstrated 100% efficacy, resulting in absence of blood parasites for >180 days. Development of a structure-activity relationship (SAR) profile for this chemical series and efforts to improve biological and pharmacokinetic profiles in the CNS-disease model through chemical modifications are reported herein.