A series of aryl carboxamide and benzylamino dispiro 1,2,4,5-tetraoxane analogues have been designed and synthesized in a short synthetic sequence from readily available starting materials. From this series of endoperoxides, molecules with in vitro IC50s versus Plasmodium falciparum (3D7) as low as 0.84 nM were identified. Based on an assessment of blood stability and in vitro microsomal stability, N205 (10a) was selected for rodent pharmacokinetic and in vivo antimalarial efficacy studies in the mouse Plasmodium berghei and Plasmodium falciparum Pf3D70087/N9 severe combined immunodeficiency (SCID) mouse models. The results indicate that the 4-benzylamino derivatives have excellent profiles with a representative of this series, N205, an excellent starting point for further lead optimization studies.

New antimicrobials are needed for the treatment of extensively drug-resistant Acinetobacter baumannii. The de novo pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) is a validated drug target for malaria and human autoimmune diseases. We provide genetic evidence that A. baumannii DHODH (AbDHODH) is essential for bacterial survival in rodent infection models. We chemically validate the target by repurposing a unique library of ~450 triazolopyrimidine/imidazopyrimidine analogs developed for our malaria DHODH program to identify 21 compounds with submicromolar activity on AbDHODH. The most potent (DSM186, DHODH IC50 28 nM) had a minimal inhibitory concentration of ≤1 µg/ml against geographically diverse A. baumannii strains, including meropenem-resistant isolates. A structurally related analog (DSM161) with a long in vivo half-life conferred significant protection in the neutropenic mouse thigh infection model. Encouragingly, the development of resistance to these compounds was not identified in vitro or in vivo. Lastly, the X-ray structure of AbDHODH bound to DSM186 was solved to 1.4 Å resolution. These data support the potential of AbDHODH as a drug target for the development of antimicrobials for the treatment of A. baumannii and potentially other high-risk bacterial infections.

Targeting cell division by chemotherapy is a highly effective strategy to treat a wide range of cancers. However, there are limitations of many standard-of-care chemotherapies: undesirable drug toxicity, side-effects, resistance and high cost. New small molecules which kill a wide range of cancer subtypes, with good therapeutic window in vivo, have the potential to complement the current arsenal of anti-cancer agents and deliver improved safety profiles for cancer patients. We describe results with a new anti-cancer small molecule, WEHI-7326, which causes cell cycle arrest in G2/M, cell death in vitro, and displays efficacious anti-tumor activity in vivo. WEHI-7326 induces cell death in a broad range of cancer cell lines, including taxane-resistant cells, and inhibits growth of human colon, brain, lung, prostate and breast tumors in mice xenografts. Importantly, the compound elicits tumor responses as a single agent in patient-derived xenografts of clinically aggressive, treatment-refractory neuroblastoma, breast, lung and ovarian cancer. In combination with standard-of-care, WEHI-7326 induces a remarkable complete response in a mouse model of high-risk neuroblastoma. WEHI-7326 is mechanistically distinct from known microtubule-targeting agents and blocks cells early in mitosis to inhibit cell division, ultimately leading to apoptotic cell death. The compound is simple to produce and possesses favorable pharmacokinetic and toxicity profiles in rodents. It represents a novel class of anti-cancer therapeutics with excellent potential for further development due to the ease of synthesis, simple formulation, moderate side effects and potent in vivo activity. WEHI-7326 has the potential to complement current frontline anti-cancer drugs and to overcome drug resistance in a wide range of cancers.

The ongoing global malaria eradication campaign requires development of potent, safe, and cost-effective drugs lacking cross-resistance with existing chemotherapies. One critical step in drug development is selecting a suitable clinical candidate from late leads. The process used to select the clinical candidate SJ733 from two potent dihydroisoquinolone (DHIQ) late leads, SJ733 and SJ311, based on their physicochemical, pharmacokinetic (PK), and toxicity profiles is described.

Antimicrobial resistance (AMR) is widely acknowledged as one of the most serious public health threats facing the world, yet the private sector finds it challenging to generate much-needed medicines. As an alternative discovery approach, a small array of diarylimidazoles was screened against the ESKAPE pathogens, and the results were made publicly available through the Open Source Antibiotics (OSA) consortium (https://github.com/opensourceantibiotics). Of the 18 compounds tested (at 32 μg/mL), 15 showed >90% growth inhibition activity against methicillin-resistant Staphylococcus aureus (MRSA) alone. In the subsequent hit-to-lead optimization of this chemotype, 147 new heterocyclic compounds containing the diarylimidazole and other core motifs were synthesized and tested against MRSA, and their structure-activity relationships were identified. While potent, these compounds have moderate to high intrinsic clearance and some associated toxicity. The best overall balance of parameters was found with OSA_975, a compound with good potency, good solubility, and reduced intrinsic clearance in rat hepatocytes. We have progressed toward the knowledge of the molecular target of these phenotypically active compounds, with proteomic techniques suggesting TGFBR1 is potentially involved in the mechanism of action. Further development of these compounds toward antimicrobial medicines is available to anyone under the licensing terms of the project.

Malaria is one of the most serious global infectious diseases. The pyrimidine biosynthetic enzyme Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) is an important target for antimalarial chemotherapy. We describe a detailed analysis of protein-ligand interactions between DHODH and a triazolopyrimidine-based inhibitor series to explore the effects of fluorine on affinity and species selectivity. We show that increasing fluorination dramatically increases binding to mammalian DHODHs, leading to a loss of species selectivity. Triazolopyrimidines bind Plasmodium and mammalian DHODHs in overlapping but distinct binding sites. Key hydrogen-bond and stacking interactions underlying strong binding to PfDHODH are absent in the mammalian enzymes. Increasing fluorine substitution leads to an increase in the entropic contribution to binding, suggesting that strong binding to mammalian DHODH is a consequence of an enhanced hydrophobic effect upon binding to an apolar pocket. We conclude that hydrophobic interactions between fluorine and hydrocarbons provide significant binding energy to protein-ligand interactions. Our studies define the requirements for species-selective binding to PfDHODH and show that the triazolopyrimidine scaffold can alternatively be tuned to inhibit human DHODH, an important target for autoimmune diseases.

The historical antimalarial compound endochin served as a structural lead for optimization. Endochin-like quinolones (ELQ) were prepared by a novel chemical route and assessed for in vitro activity against multidrug resistant strains of Plasmodium falciparum and against malaria infections in mice. Here we describe the pathway to discovery of a potent class of orally active antimalarial 4(1H)-quinolone-3-diarylethers. The initial prototype, ELQ-233, exhibited low nanomolar IC50 values against all tested strains including clinical isolates harboring resistance to atovaquone. ELQ-271 represented the next critical step in the iterative optimization process, as it was stable to metabolism and highly effective in vivo. Continued analoging revealed that the substitution pattern on the benzenoid ring of the quinolone core significantly influenced reactivity with the host enzyme. This finding led to the rational design of highly selective ELQs with outstanding oral efficacy against murine malaria that is superior to established antimalarials chloroquine and atovaquone.

Tuberculosis (TB) is responsible for enormous global morbidity and mortality, and current treatment regimens rely on the use of drugs that have been in use for more than 40 years. Owing to widespread resistance to these therapies, new drugs are desperately needed to control the TB disease burden. Herein, we describe the rapid synthesis of analogues of the sansanmycin uridylpeptide natural products that represent promising new TB drug leads. The compounds exhibit potent and selective inhibition of Mycobacterium tuberculosis, the etiological agent of TB, both in vitro and intracellularly. The natural product analogues are nanomolar inhibitors of Mtb phospho-MurNAc-pentapeptide translocase, the enzyme responsible for the synthesis of lipid I in mycobacteria. This work lays the foundation for the development of uridylpeptide natural product analogues as new TB drug candidates that operate through the inhibition of peptidoglycan biosynthesis.

The aryl hydantoin 1 (Ro 13-3978) was identified in the early 1980s as a promising antischistosomal lead compound. However, this series of aryl hydantoins produced antiandrogenic side effects in the host, a not unexpected outcome given their close structural similarity to the antiandrogenic drug nilutamide. Building on the known SAR of this compound series, we now describe a number of analogs of 1 designed to maximize structural diversity guided by incorporation of substructures and functional groups known to diminish ligand-androgen receptor interactions. These analogs had calculated polar surface area (PSA), measured LogD7.4, aqueous kinetic solubility, and estimated plasma protein binding values in ranges predictive of good ADME profiles. The principal SAR insight was that the hydantoin core of 1 is required for high antischistosomal activity. We identified several compounds with high antischistosomal efficacy that were less antiandrogenic than 1. These data provide direction for the ongoing optimization of antischistosomal hydantoins.

Malaria kills nearly 0.5 million people yearly and impacts the lives of those living in over 90 countries where it is endemic. The current treatment programs are threatened by increasing drug resistance. Dihydroorotate dehydrogenase (DHODH) is now clinically validated as a target for antimalarial drug discovery as a triazolopyrimidine class inhibitor (DSM265) is currently undergoing clinical development. We discovered a related isoxazolopyrimidine series in a phenotypic screen, later determining that it targeted DHODH. To determine if the isoxazolopyrimidines could yield a drug candidate, we initiated hit-to-lead medicinal chemistry. Several potent analogues were identified, including a compound that showed in vivo antimalarial activity. The isoxazolopyrimidines were more rapidly metabolized than their triazolopyrimidine counterparts, and the pharmacokinetic data were not consistent with the goal of a single-dose treatment for malaria.

Modelling and simulation are being increasingly utilized to support the discovery and development of new anti-malarial drugs. These approaches require reliable in vitro data for physicochemical properties, permeability, binding, intrinsic clearance and cytochrome P450 inhibition. This work was conducted to generate an in vitro data toolbox using standardized methods for a set of 45 anti-malarial drugs and to assess changes in physicochemical properties in relation to changing target product and candidate profiles.

Perforin is a key effector of lymphocyte-mediated cell death pathways and contributes to transplant rejection of immunologically mismatched grafts. We have developed a novel series of benzenesulfonamide (BZS) inhibitors of perforin that can mitigate graft rejection during allogeneic bone marrow/stem cell transplantation. Eight such perforin inhibitors were tested for their murine pharmacokinetics, plasma protein binding, and their ability to block perforin-mediated lysis in vitro and to block the rejection of major histocompatibility complex (MHC)-mismatched mouse bone marrow cells. All compounds showed >99% binding to plasma proteins and demonstrated perforin inhibitory activity in vitro and in vivo. A lead compound, compound 1, that showed significant increases in allogeneic bone marrow preservation was evaluated for its plasma pharmacokinetics and in vivo efficacy at multiple dosing regimens to establish a pharmacokinetic/pharmacodynamic (PK/PD) relationship. The strongest PK/PD correlation was observed between perforin inhibition in vivo and time that total plasma concentrations remained above 900 μM, which correlates to unbound concentrations similar to 3× the unbound in vitro IC90 of compound 1. This PK/PD relationship will inform future dosing strategies of BZS perforin inhibitors to maintain concentrations above 3× the unbound IC90 for as long as possible to maximize efficacy and enhance progression toward clinical evaluation.

The quest for new antimalarial drugs, especially those with novel modes of action, is essential in the face of emerging drug-resistant parasites. Here we describe a new chemical class of molecules, pyrazoleamides, with potent activity against human malaria parasites and showing remarkably rapid parasite clearance in an in vivo model. Investigations involving pyrazoleamide-resistant parasites, whole-genome sequencing and gene transfers reveal that mutations in two proteins, a calcium-dependent protein kinase (PfCDPK5) and a P-type cation-ATPase (PfATP4), are necessary to impart full resistance to these compounds. A pyrazoleamide compound causes a rapid disruption of Na(+) regulation in blood-stage Plasmodium falciparum parasites. Similar effect on Na(+) homeostasis was recently reported for spiroindolones, which are antimalarials of a chemical class quite distinct from pyrazoleamides. Our results reveal that disruption of Na(+) homeostasis in malaria parasites is a promising mode of antimalarial action mediated by at least two distinct chemical classes.

Malaria persists as one of the most devastating global infectious diseases. The pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) has been identified as a new malaria drug target, and a triazolopyrimidine-based DHODH inhibitor 1 (DSM265) is in clinical development. We sought to identify compounds with higher potency against Plasmodium DHODH while showing greater selectivity toward animal DHODHs. Herein we describe a series of novel triazolopyrimidines wherein the p-SF5-aniline was replaced with substituted 1,2,3,4-tetrahydro-2-naphthyl or 2-indanyl amines. These compounds showed strong species selectivity, and several highly potent tetrahydro-2-naphthyl derivatives were identified. Compounds with halogen substitutions displayed sustained plasma levels after oral dosing in rodents leading to efficacy in the P. falciparum SCID mouse malaria model. These data suggest that tetrahydro-2-naphthyl derivatives have the potential to be efficacious for the treatment of malaria, but due to higher metabolic clearance than 1, they most likely would need to be part of a multidose regimen.

Elevated iron in the SNpc may play a key role in Parkinson's disease (PD) neurodegeneration since drug candidates with high iron affinity rescue PD animal models, and one candidate, deferirpone, has shown efficacy recently in a phase two clinical trial. However, strong iron chelators may perturb essential iron metabolism, and it is not yet known whether the damage associated with iron is mediated by a tightly bound (eg ferritin) or lower-affinity, labile, iron pool. Here we report the preclinical characterization of PBT434, a novel quinazolinone compound bearing a moderate affinity metal-binding motif, which is in development for Parkinsonian conditions. In vitro, PBT434 was far less potent than deferiprone or deferoxamine at lowering cellular iron levels, yet was found to inhibit iron-mediated redox activity and iron-mediated aggregation of α-synuclein, a protein that aggregates in the neuropathology. In vivo, PBT434 did not deplete tissue iron stores in normal rodents, yet prevented loss of substantia nigra pars compacta neurons (SNpc), lowered nigral α-synuclein accumulation, and rescued motor performance in mice exposed to the Parkinsonian toxins 6-OHDA and MPTP, and in a transgenic animal model (hA53T α-synuclein) of PD. These improvements were associated with reduced markers of oxidative damage, and increased levels of ferroportin (an iron exporter) and DJ-1. We conclude that compounds designed to target a pool of pathological iron that is not held in high-affinity complexes in the tissue can maintain the survival of SNpc neurons and could be disease-modifying in PD.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), is an increasing threat to global health. Available medicines were introduced over 40 years ago, have undesirable side effects, and give equivocal results of cure in the chronic stage of the disease. We report the development of two compounds, 6 and (S)-7, with PCR-confirmed curative activity in a mouse model of established T. cruzi infection after once daily oral dosing for 20 days at 20 mg/kg 6 and 10 mg/kg (S)-7. Compounds 6 and (S)-7 have potent in vitro activity, are noncytotoxic, show no adverse effects in vivo following repeat dosing, are prepared by a short synthetic route, and have druglike properties suitable for preclinical development.

In the pursuit of new antimalarial leads, a phenotypic screening of various commercially sourced compound libraries was undertaken by the World Health Organisation Programme for Research and Training in Tropical Diseases (WHO-TDR). We report here the detailed characterization of one of the hits from this process, TDR32750 (8a), which showed potent activity against Plasmodium falciparum K1 (EC(50) ~ 9 nM), good selectivity (>2000-fold) compared to a mammalian cell line (L6), and significant activity against a rodent model of malaria when administered intraperitoneally. Structure-activity relationship studies have indicated ways in which the molecule could be optimized. This compound represents an exciting start point for a drug discovery program for the development of a novel antimalarial.

To examine the utility of human plasma as an assay medium in Caco-2 permeability studies to overcome poor mass balance and inadequate sink conditions frequently encountered with lipophilic compounds.

The continued proliferation of malaria throughout temperate and tropical regions of the world has promoted a push for more efficacious treatments to combat the disease. Unfortunately, more recent remedies such as artemisinin combination therapies have been rendered less effective due to developing parasite resistance, and new drugs are required that target the parasite in the liver to support the disease elimination efforts. Research was initiated to revisit antimalarials developed in the 1940s and 1960s that were deemed unsuitable for use as therapeutic agents as a result of poor understanding of both physicochemical properties and parasitology. Structure-activity and structure-property relationship studies were conducted to generate a set of compounds with the general 6-chloro-7-methoxy-2-methyl-4(1H)-quinolone scaffold which were substituted at the 3-position with a variety of phenyl moieties possessing various properties. Extensive physicochemical evaluation of the quinolone series was carried out to downselect the most promising 4(1H)-quinolones, 7, 62, 66, and 67, which possessed low-nanomolar EC50 values against W2 and TM90-C2B as well as improved microsomal stability. Additionally, in vivo Thompson test results using Plasmodium berghei in mice showed that these 4(1H)-quinolones were efficacious for the reduction of parasitemia at >99% after 6 days.

The clinical use of mefloquine (MQ) has declined due to dose-related neurological events. Next generation quinoline methanols (NGQMs) that do not accumulate in the central nervous system (CNS) to the same extent may have utility. In this study, CNS levels of NGQMs relative to MQ were measured and an early lead chemotype was identified for further optimization.