Single-Dose Pharmacokinetics, Excretion, and Metabolism of Zoliflodacin, a Novel Spiropyrimidinetrione Antibiotic, in Healthy Volunteers.

Zoliflodacin is a novel spiropyrimidinetrione with activity against bacterial type II topoisomerases that inhibits DNA biosynthesis and results in accumulation of double-strand cleavages in bacteria. We report results from two phase 1 studies that investigated the safety, tolerability, and pharmacokinetics (PK) of zoliflodacin and absorption, distribution, metabolism, and excretion (ADME) after single doses in healthy volunteers. In the single ascending dose study, zoliflodacin was rapidly absorbed, with a time to maximum concentration of drug in serum (Tmax) between 1.5 and 2.3 h. Exposure increased dose proportionally up to 800 mg and less than dose proportionally between 800 and 4,000 mg. Urinary excretion of unchanged zoliflodacin was <5.0% of the total dose. In the fed state, absorption was delayed (Tmax, 4 h), accompanied by an increase in the area under the concentration-time curve (AUC) at 1,500- and 3,000-mg doses. In the ADME study (3,000 mg orally), the PK profile of zoliflodacin had exposure (AUC and maximum concentration of drug in serum [Cmax]) similar to that of the ascending dose study and a median Tmax of 2.5 h. A total of 97.8% of the administered radioactivity was recovered in excreta, with urine and fecal elimination accounting for approximately 18.2% and 79.6% of the dose, respectively. The major clearance pathway was via metabolism and elimination in feces with low urinary recovery of unchanged drug (approximately 2.5%) and metabolites accounting for 56% of the dose excreted in the feces. Zoliflodacin represented 72.3% and metabolite M3 accounted for 16.4% of total circulating radioactivity in human plasma. Along with the results from these studies and based upon safety, PK, and PK/pharmacodynamics targets, a dosage regimen was selected for evaluation in a phase 2 study in urogenital gonorrhea. (The studies discussed in this paper have been registered at ClinicalTrials.gov under identifiers NCT01929629 and NCT02298920.).

Evaluation of the metabolism, bioactivation and pharmacokinetics of triaminopyrimidine analogs toward selection of a potential candidate for antimalarial therapy.

1. During the course of metabolic profiling of lead Compound 1, glutathione (GSH) conjugates were detected in rat bile, suggesting the formation of reactive intermediate precursor(s). This was confirmed by the identification of GSH and N-acetylcysteine (NAC) conjugates in microsomal incubations. 2. It was proposed that bioactivation of Compound 1 occurs via the formation of a di-iminoquinone reactive intermediate through the involvement of the C-2 and C-5 nitrogens of the pyrimidine core. 3. To further investigate this hypothesis, structural analogs with modifications at the C-5 nitrogen were studied for metabolic activation in human liver microsomes supplemented with GSH/NAC. 4. Compounds 1 and 2, which bear secondary nitrogens at the C-5 of the pyrimidine core, were observed to form significant amounts of GSH/NAC-conjugates in vitro, whereas compounds with tertiary nitrogens at C-5 (Compound 3 and 4) formed no such conjugates. 5. These observations provide evidence that electron/hydrogen abstraction is required for the bioactivation of the triaminopyrimidines, potentially via a di-iminoquinone intermediate. The lack of a hydrogen and/or steric hindrance rendered Compound 3 and 4 incapable of forming thiol conjugates. 6. This finding enabled advancement of compound 4, with a desirable potency, safety and PK profile, as a lead candidate for further development in the treatment of malaria.

Lipid Peroxide-Mediated Oxidative Rearrangement of the Pyrazinone Carboxamide Core of Neutrophil Elastase Inhibitor AZD9819 in Blood Plasma Samples.

This study focused on the mechanistic interpretation of ex vivo oxidation of a candidate drug in blood plasma samples. An unexpected lipid peroxide-mediated epoxidation followed by a dramatic rearrangement led to production of a five-membered oxazole derivative from the original six-membered pyrazinone-carboxamide core of a human neutrophil elastase inhibitor, 6-(1-(4-cyanophenyl)-1H-pyrazol-5-yl)-N-ethyl-5-methyl-3-oxo-4-(3-(trifluoromethyl)phenyl)-3,4-dihydropyrazine-2-carboxamide (AZD9819). The rearranged oxidation product 2-(1-(4-cyanophenyl)-1H-pyrazol-5-yl)-5-(N-ethylacetamido)-N-(3-(trifluoromethyl)phenyl)oxazole-4-carboxamide was characterized by accurate-mass tandem mass spectrometry fragmentations, by two-dimensional NMR and X-ray crystallography of an authentic standard, and by incorporation of an (18)O atom from molecular (18)O2 to the location predicted by our proposed mechanism. The lipid peroxide-mediated oxidation was demonstrated by using human low-density lipoprotein (LDL) in pH 7.4 phosphate buffer and by inhibiting the oxidation with ascorbic acid or l-glutathione, two antioxidants effective in both plasma and the LDL incubation. A nucleophilic mechanism for the epoxidation of AZD9819 by lipid hydroperoxides explains the prevention of its ex vivo oxidation by acidification of the plasma samples. The discovery of the lipid peroxide-dependent oxidation of an analyte and the means of prevention could provide valuable information for biotransformation and bioanalysis.

Synthesis and biological evaluation of novel pyrrolidine acid analogs as potent dual PPARα/γ agonists.

The design, synthesis and structure-activity relationships of a novel series of 3,4-disubstituted pyrrolidine acid analogs as PPAR ligands is outlined. In both the 1,3- and 1,4-oxybenzyl pyrrolidine acid series, the preferred stereochemistry was shown to be the cis-3R,4S isomer, as exemplified by the potent dual PPARα/γ agonists 3k and 4i. The N-4-trifluoromethyl-pyrimidinyl pyrrolidine acid analog 4i was efficacious in lowering fasting glucose and triglyceride levels in diabetic db/db mice.

Identification and mitigation of a reactive metabolite liability associated with aminoimidazoles.

Reactive metabolites (RMs) have been implicated as causal factors in many drug-associated idiosyncratic toxicities. This study aims at identification and mitigation of an RM liability associated with aminoimidazole and amino(aza)benzimidazole structural motifs from an antimalarial project. Nineteen compounds with different structural modifications were studied in rat and human liver microsomes using glutathione (GSH) and N-acetyl cysteine (NAC) as trapping agents for RM. Metabolite profiling of aminoimidazole compounds in initial studies revealed the presence of dihydrodiol metabolites suggestive of reactive epoxide precursors, confirmed by the identification of a dihydrohydroxy GSH conjugate in GSH supplemented incubations. Substitution of methyl group at a potential site of metabolism blocked the epoxidation; however, formation of an imine-methide RM was suspected. Masking the site of metabolism via benzimidazole and 4/7-azabenzimidazole resulted in the possible formation of quinone-imine intermediates as a product of bioactivation. Further, substitutions with electron withdrawing groups and steric crowding did not address this liability. Mitigation of bioactivation was achieved with 5/6-azabenzimidazole and with CF3 substitution at the 6-position of the 7-azabenzimidazole ring. Moreover, compounds devoid of imidazole -NH2 do not undergo bioactivation. This study, therefore, establishes aminoimidazole and amino(aza)benzimidazoles as potential toxicophores and describes ways to mitigate this bioactivation liability by chemical modification.

Discovery of a Potent and Selective Tyrosine Kinase 2 Inhibitor: TAK-279.

TYK2 is a key mediator of IL12, IL23, and type I interferon signaling, and these cytokines have been implicated in the pathogenesis of multiple inflammatory and autoimmune diseases such as psoriasis, rheumatoid arthritis, lupus, and inflammatory bowel diseases. Supported by compelling data from human genome-wide association studies and clinical results, TYK2 inhibition through small molecules is an attractive therapeutic strategy to treat these diseases. Herein, we report the discovery of a series of highly selective pseudokinase (Janus homology 2, JH2) domain inhibitors of TYK2 enzymatic activity. A computationally enabled design strategy, including the use of FEP+, was instrumental in identifying a pyrazolo-pyrimidine core. We highlight the utility of computational physics-based predictions used to optimize this series of molecules to identify the development candidate 30, a potent, exquisitely selective cellular TYK2 inhibitor that is currently in Phase 2 clinical trials for the treatment of psoriasis and psoriatic arthritis.

3,5-diarylazoles as novel and selective inhibitors of protein kinase D.

The synthesis and preliminary studies of the SAR of novel 3,5-diarylazole inhibitors of Protein Kinase D (PKD) are reported. Notably, optimized compounds in this class have been found to be active in cellular assays of phosphorylation-dependant HDAC5 nuclear export, orally bioavailable, and highly selective versus a panel of additional putative histone deacetylase (HDAC) kinases. Therefore these compounds could provide attractive tools for the further study of PKD/HDAC5 signaling.

Identification of a novel N-carbamoyl glucuronide: in vitro, in vivo, and mechanistic studies.

1-[4-Aminomethyl-4-(3-chlorophenyl)-cyclohexyl]-tetrahydro-pyrimidin- 2-one, 1, was developed as an inhibitor of dipeptidyl peptidase-4 enzyme. Biotransformation studies with 1 revealed the presence of an N-carbamoyl glucuronide metabolite (M1) in rat bile and urine. N-Carbamoyl glucuronides are rarely observed, and little is understood regarding the mechanism of N-carbamoyl glucuronidation. The objectives of the current investigation were to elucidate the structure of the novel N-carbamoyl glucuronide, to investigate the mechanism of N-carbamoyl glucuronide formation in vitro using stable labeled CO(2), UDP glucuronosyltransferase (UGT) reaction phenotyping, and to assess whether M1 was formed to the same extent in vitro across species-mouse, rat, hamster, dog, monkey, and human. Structure elucidation was performed on a mass spectrometer with accurate mass measurement and MS(n) capabilities. (13)C-labeled carbon dioxide was used for identification of the mechanism of N-carbamoyl glucuronidation. Mechanistic studies with (13)C-labeled CO(2) in rat liver microsomes revealed that CO(2) from the bicarbonate buffer (in equilibrium with exogenous CO(2)) may be responsible for the formation of M1. M1 was formed in vitro in liver microsomes from multiple species, mainly rat and hamster, followed by similar formation in dog, monkey, mouse, and human. M1 could be detected in UGT1A1, UGT1A3, and UGT2B7 Supersomes in a CO(2)-rich environment. In conclusion, our study demonstrates that formation of M1 was observed in microsomal incubations across various species and strongly suggests incorporation of CO(2) from the bicarbonate buffer, in equilibrium with exogenous CO(2), into the carbamoyl moiety of the formed N-carbamoyl glucuronide.

Synthesis and structure-activity relationships of 2-aryl-4-oxazolylmethoxy benzylglycines and 2-aryl-4-thiazolylmethoxy benzylglycines as novel, potent PPARalpha selective activators- PPARalpha and PPARgamma selectivity modulation.

The synthesis and follow-up SAR studies of our development candidate 1 by incorporating 2-aryl-4-oxazolylmethoxy and 2-aryl-4-thiazolylmethoxy moieties into the oxybenzylglycine framework of the PPARalpha/gamma dual agonist muraglitazar is described. SAR studies indicate that different substituents on the aryloxazole/thiazole moieties as well as the choice of carbamate substituent on the glycine moiety can significantly modulate the selectivity of PPARalpha versus PPARgamma. Potent, highly selective PPARalpha activators 2a and 2l, as well as PPARalpha activators with significant PPARgamma activity, such as 2s, were identified. The in vivo pharmacology of these compounds in preclinical animal models as well as their ADME profiles are discussed.

Preclinical investigations and a first-in-human phase I trial of M4112, the first dual inhibitor of indoleamine 2,3-dioxygenase 1 and tryptophan 2,3-dioxygenase 2, in patients with advanced solid tumors.

BACKGROUND: M4112 is an oral, potent, and selective indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2) dual inhibitor. Here, we report preclinical data and first-in-human phase I data, including safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary efficacy, of M4112 monotherapy in patients with advanced solid tumors. METHODS: In preclinical studies, M4112 was administered to mice with IDO1-expressing tumors to determine tumor IDO1 and liver TDO2 inhibition. In the phase I trial, patients received doses of M4112 two times per day in 28-day cycles until progression, toxicity, or withdrawal of consent. The primary objective was to determine the maximum tolerated dose (MTD) and recommended phase II dose (RP2D). The primary endpoint was the incidence of dose-limiting toxicities (DLTs), treatment-emergent adverse events (TEAEs), and treatment-emergent changes in safety parameters. Other endpoints included pharmacokinetics, pharmacodynamics, and antitumor effects. RESULTS: In mice, M4112 significantly decreased the kynurenine:tryptophan ratio in the liver and tumor. Fifteen patients received M4112 at five distinct dose levels (three patients per cohort: 100, 200, 400, 600, and 800 mg two times per day orally). Initially, all doses inhibited IDO1 ex vivo, but plasma kynurenine levels returned to or exceeded baseline levels after day 15. Despite initial changes in kynurenine, there was no significant reduction of plasma kynurenine at steady state. There was one DLT (grade 3 allergic dermatitis; 800 mg two times per day) and one grade 2 QT prolongation (800 mg two times per day), resulting in dose reduction (not a DLT). M4112 was well tolerated, and neither the MTD nor the RP2D was established. TEAEs included fatigue, nausea, and vomiting. The best overall response was stable disease (n=9, 60%). CONCLUSIONS: There were no serious safety concerns at any dose. Although M4112 inhibited IDO1 activity ex vivo, plasma kynurenine levels were not reduced despite achieving target exposure.Trial registration number NCT03306420.

Design, synthesis and structure-activity relationships of azole acids as novel, potent dual PPAR alpha/gamma agonists.

The design, synthesis and structure-activity relationships of a novel series of N-phenyl-substituted pyrrole, 1,2-pyrazole and 1,2,3-triazole acid analogs as PPAR ligands are outlined. The triazole acid analogs 3f and 4f were identified as potent dual PPARalpha/gamma agonists both in binding and functional assays in vitro. The 3-oxybenzyl triazole acetic acid analog 3f showed excellent glucose and triglyceride lowering in diabetic db/db mice.

Discovery of azetidinone acids as conformationally-constrained dual PPARalpha/gamma agonists.

A novel class of azetidinone acid-derived dual PPARalpha/gamma agonists has been synthesized for the treatment of diabetes and dyslipidemia. The preferred stereochemistry in this series for binding and functional agonist activity against both PPARalpha and PPARgamma receptors was shown to be 3S,4S. Synthesis, in vitro and in vivo activities of compounds in this series are described. A high-yielding method for N-arylation of azetidinone esters is also described.

The Discovery of N-(1-Methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)-5-((6- ((methylamino)methyl)pyrimidin-4-yl)oxy)-1H-indole-1-carboxamide (Acrizanib), a VEGFR-2 Inhibitor Specifically Designed for Topical Ocular Delivery, as a Therapy for Neovascular Age-Related Macular Degeneration.

A noninvasive topical ocular therapy for the treatment of neovascular or "wet" age-related macular degeneration would provide a patient administered alternative to the current standard of care, which requires physician administered intravitreal injections. This manuscript describes a novel strategy for the use of in vivo models of choroidal neovascularization (CNV) as the primary means of developing SAR related to efficacy from topical administration. Ultimately, this effort led to the discovery of acrizanib (LHA510), a small-molecule VEGFR-2 inhibitor with potency and efficacy in rodent CNV models, limited systemic exposure after topical ocular administration, multiple formulation options, and an acceptable rabbit ocular PK profile.

Discovery and Preclinical Evaluation of BMS-711939, an Oxybenzylglycine Based PPARα Selective Agonist.

BMS-711939 (3) is a potent and selective peroxisome proliferator-activated receptor (PPAR) α agonist, with an EC50 of 4 nM for human PPARα and >1000-fold selectivity vs human PPARγ (EC50 = 4.5 µM) and PPARδ (EC50 > 100 µM) in PPAR-GAL4 transactivation assays. Compound 3 also demonstrated excellent in vivo efficacy and safety profiles in preclinical studies and thus was chosen for further preclinical evaluation. The synthesis, structure-activity relationship (SAR) studies, and in vivo pharmacology of 3 in preclinical animal models as well as its ADME profile are described.

Discovery of Oral VEGFR-2 Inhibitors with Prolonged Ocular Retention That Are Efficacious in Models of Wet Age-Related Macular Degeneration.

The benefit of intravitreal anti-VEGF therapy in treating wet age-related macular degeneration (AMD) is well established. Identification of VEGFR-2 inhibitors with optimal ADME properties for an ocular indication provides opportunities for dosing routes beyond intravitreal injection. We employed a high-throughput in vivo screening strategy with rodent models of choroidal neovascularization and iterative compound design to identify VEGFR-2 inhibitors with potential to benefit wet AMD patients. These compounds demonstrate preferential ocular tissue distribution and efficacy after oral administration while minimizing systemic exposure.

Triaminopyrimidine is a fast-killing and long-acting antimalarial clinical candidate.

The widespread emergence of Plasmodium falciparum (Pf) strains resistant to frontline agents has fuelled the search for fast-acting agents with novel mechanism of action. Here, we report the discovery and optimization of novel antimalarial compounds, the triaminopyrimidines (TAPs), which emerged from a phenotypic screen against the blood stages of Pf. The clinical candidate (compound 12) is efficacious in a mouse model of Pf malaria with an ED99 <30 mg kg(-1) and displays good in vivo safety margins in guinea pigs and rats. With a predicted half-life of 36 h in humans, a single dose of 260 mg might be sufficient to maintain therapeutic blood concentration for 4-5 days. Whole-genome sequencing of resistant mutants implicates the vacuolar ATP synthase as a genetic determinant of resistance to TAPs. Our studies highlight the potential of TAPs for single-dose treatment of Pf malaria in combination with other agents in clinical development.

Functional analysis of human microsomal epoxide hydrolase genetic variants.

Human microsomal epoxide hydrolase (EPHX1) is active in the metabolism of many potentially carcinogenic or otherwise genotoxic epoxides, such as those derived from the oxidation of polyaromatic hydrocarbons. EPHX1 is polymorphic and encodes allelic variation at least two amino acid positions, Y113H and H139R. In a number of recent molecular epidemiological investigations, EPHX1 polymorphism has been suggested as a susceptibility factor for several human diseases. To better evaluate the functional contribution of EPHX1 genetic polymorphism, we characterized the enzymatic properties associated with each of the respective variant proteins. Enzymatic profiles were evaluated with cis-stilbene oxide (cSO) and benzo[a]pyrene-4,5-epoxide (BaPO), two prototypical substrates for the hydrolase. In one series of experiments, activities of recombinant EPHX1 proteins were analyzed subsequent to their expression using the pFastbac baculovirus vector in Spodoptera frugiperda-9 (Sf9) insect cells, and purification by column chromatography. In parallel studies, EPHX1 activities were evaluated with human liver microsomes derived from individuals of known EPHX1 genotype. Using the purified protein preparations, rates of cSO and BaPO hydrolysis for the reference protein, Y113/H139, were approximately 2-fold greater than those measured with the other EPHX1 allelic variants. However, when activities were analyzed using human liver microsomal fractions, no major differences were evident in the reaction rates generated among preparations representing the different EPHX1 alleles. Collectively, these results suggest that the structural differences encoded by the Y113H and H139R variant alleles exert only modest impact on EPHX1-specific enzymatic activities in vivo.

Discovery of pyrazolopyridones as a novel class of noncovalent DprE1 inhibitor with potent anti-mycobacterial activity.

A novel pyrazolopyridone class of inhibitors was identified from whole cell screening against Mycobacterium tuberculosis (Mtb). The series exhibits excellent bactericidality in vitro, resulting in a 4 log reduction in colony forming units following compound exposure. The significant modulation of minimum inhibitory concentration (MIC) against a Mtb strain overexpressing the Rv3790 gene suggested the target of pyrazolopyridones to be decaprenylphosphoryl-ß-D-ribose-2'-epimerase (DprE1). Genetic mapping of resistance mutation coupled with potent enzyme inhibition activity confirmed the molecular target. Detailed biochemical characterization revealed the series to be a noncovalent inhibitor of DprE1. Docking studies at the active site suggest that the series can be further diversified to improve the physicochemical properties without compromising the antimycobacterial activity. The pyrazolopyridone class of inhibitors offers an attractive non-nitro lead series targeting the essential and vulnerable DprE1 enzyme for the discovery of novel antimycobacterial agents to treat both drug susceptible and drug resistant strains of Mtb.

N-aryl-2-aminobenzimidazoles: novel, efficacious, antimalarial lead compounds.

From the phenotypic screening of the AstraZeneca corporate compound collection, N-aryl-2-aminobenzimidazoles have emerged as novel hits against the asexual blood stage of Plasmodium falciparum (Pf). Medicinal chemistry optimization of the potency against Pf and ADME properties resulted in the identification of 12 as a lead molecule. Compound 12 was efficacious in the P. berghei (Pb) model of malaria. This compound displayed an excellent pharmacokinetic profile with a long half-life (19 h) in rat blood. This profile led to an extended survival of animals for over 30 days following a dose of 50 mg/kg in the Pb malaria model. Compound 12 retains its potency against a panel of Pf isolates with known mechanisms of resistance. The fast killing observed in the in vitro parasite reduction ratio (PRR) assay coupled with the extended survival highlights the promise of this novel chemical class for the treatment of malaria.

Aminoazabenzimidazoles, a novel class of orally active antimalarial agents.

Whole-cell high-throughput screening of the AstraZeneca compound library against the asexual blood stage of Plasmodium falciparum (Pf) led to the identification of amino imidazoles, a robust starting point for initiating a hit-to-lead medicinal chemistry effort. Structure-activity relationship studies followed by pharmacokinetics optimization resulted in the identification of 23 as an attractive lead with good oral bioavailability. Compound 23 was found to be efficacious (ED90 of 28.6 mg·kg(-1)) in the humanized P. falciparum mouse model of malaria (Pf/SCID model). Representative compounds displayed a moderate to fast killing profile that is comparable to that of chloroquine. This series demonstrates no cross-resistance against a panel of Pf strains with mutations to known antimalarial drugs, thereby suggesting a novel mechanism of action for this chemical class.