Discovery of Milvexian, a High-Affinity, Orally Bioavailable Inhibitor of Factor XIa in Clinical Studies for Antithrombotic Therapy.

Factor XIa (FXIa) is an enzyme in the coagulation cascade thought to amplify thrombin generation but has a limited role in hemostasis. From preclinical models and human genetics, an inhibitor of FXIa has the potential to be an antithrombotic agent with superior efficacy and safety. Reversible and irreversible inhibitors of FXIa have demonstrated excellent antithrombotic efficacy without increased bleeding time in animal models (Weitz, J. I., Chan, N. C. Arterioscler. Thromb. Vasc. Biol. 2019, 39 (1), 7-12). Herein, we report the discovery of a novel series of macrocyclic FXIa inhibitors containing a pyrazole P2' moiety. Optimization of the series for (pharmacokinetic) PK properties, free fraction, and solubility resulted in the identification of milvexian (BMS-986177/JNJ-70033093, 17, FXIa Ki = 0.11 nM) as a clinical candidate for the prevention and treatment of thromboembolic disorders, suitable for oral administration.

Discovery of a High Affinity, Orally Bioavailable Macrocyclic FXIa Inhibitor with Antithrombotic Activity in Preclinical Species.

Oral factor XIa (FXIa) inhibitors may provide a promising new antithrombotic therapy with an improved benefit to bleeding risk profile over existing antithrombotic agents. Herein, we report application of a previously disclosed cyclic carbamate P1 linker which provided improved oral bioavailability in the imidazole-based 13-membered macrocycle to the 12-membered macrocycle. This resulted in identification of compound 4 with desired FXIa inhibitory potency and good oral bioavailability but high in vivo clearance. Further structure-activity relationship (SAR) studies of heterocyclic core modifications to replace the imidazole core as well as various linkers to the P1 group led to the discovery of compound 6f, a potent FXIa inhibitor with selectivity against most of the relevant serine proteases. Compound 6f also demonstrated excellent pharmacokinetics (PK) profile (high oral bioavailability and low clearance) in multiple preclinical species. Compound 6f achieved robust antithrombotic efficacy in a rabbit efficacy model at doses which preserved hemostasis.

Orally bioavailable amine-linked macrocyclic inhibitors of factor XIa.

The discovery of orally bioavailable FXIa inhibitors has been a challenge. Herein, we describe our efforts to address this challenge by optimization of our imidazole-based macrocyclic series. Our optimization strategy focused on modifications to the P2 prime, macrocyclic amide linker, and the imidazole scaffold. Replacing the amide of the macrocyclic linker with amide isosteres led to the discovery of substituted amine linkers which not only maintained FXIa binding affinity but also improved oral exposure in rats. Combining the optimized macrocyclic amine linker with a pyridine scaffold afforded compounds 23 and 24 that were orally bioavailable, single-digit nanomolar FXIa inhibitors with excellent selectivity against relevant blood coagulation enzymes.

Potent, Orally Bioavailable, and Efficacious Macrocyclic Inhibitors of Factor XIa. Discovery of Pyridine-Based Macrocycles Possessing Phenylazole Carboxamide P1 Groups.

Factor XIa (FXIa) inhibitors are promising novel anticoagulants, which show excellent efficacy in preclinical thrombosis models with minimal effects on hemostasis. The discovery of potent and selective FXIa inhibitors which are also orally bioavailable has been a challenge. Here, we describe optimization of the imidazole-based macrocyclic series and our initial progress toward meeting this challenge. A two-pronged strategy, which focused on replacement of the imidazole scaffold and the design of new P1 groups, led to the discovery of potent, orally bioavailable pyridine-based macrocyclic FXIa inhibitors. Moreover, pyridine-based macrocycle 19, possessing the phenylimidazole carboxamide P1, exhibited excellent selectivity against relevant blood coagulation enzymes and displayed antithrombotic efficacy in a rabbit thrombosis model.

Structure based design of macrocyclic factor XIa inhibitors: Discovery of cyclic P1 linker moieties with improved oral bioavailability.

This manuscript describes the discovery of a series of macrocyclic inhibitors of FXIa with oral bioavailability. Assisted by structure based drug design and ligand bound X-ray crystal structures, the group linking the P1 moiety to the macrocyclic core was modified with the goal of reducing H-bond donors to improve pharmacokinetic performance versus 9. This effort resulted in the discovery of several cyclic P1 linkers, exemplified by 10, that are constrained mimics of the bioactive conformation displayed by the acrylamide linker of 9. These cyclic P1 linkers demonstrated enhanced bioavailability and improved potency.

Discovery of a Parenteral Small Molecule Coagulation Factor XIa Inhibitor Clinical Candidate (BMS-962212).

Factor XIa (FXIa) is a blood coagulation enzyme that is involved in the amplification of thrombin generation. Mounting evidence suggests that direct inhibition of FXIa can block pathologic thrombus formation while preserving normal hemostasis. Preclinical studies using a variety of approaches to reduce FXIa activity, including direct inhibitors of FXIa, have demonstrated good antithrombotic efficacy without increasing bleeding. On the basis of this potential, we targeted our efforts at identifying potent inhibitors of FXIa with a focus on discovering an acute antithrombotic agent for use in a hospital setting. Herein we describe the discovery of a potent FXIa clinical candidate, 55 (FXIa Ki = 0.7 nM), with excellent preclinical efficacy in thrombosis models and aqueous solubility suitable for intravenous administration. BMS-962212 is a reversible, direct, and highly selective small molecule inhibitor of FXIa.

Macrocyclic inhibitors of Factor XIa: Discovery of alkyl-substituted macrocyclic amide linkers with improved potency.

Optimization of macrocyclic inhibitors of FXIa is described which focused on modifications to both the macrocyclic linker and the P1 group. Increases in potency were discovered through interactions with a key hydrophobic region near the S1 prime pocket by substitution of the macrocyclic linker with small alkyl groups. Both the position of substitution and the absolute stereochemistry of the alkyl groups on the macrocyclic linker which led to improved potency varied depending on the ring size of the macrocycle. Replacement of the chlorophenyltetrazole cinnamide P1 in these optimized macrocycles reduced the polar surface area and improved the oral bioavailability for the series, albeit at the cost of a decrease in potency.

Structure-Based Design of Macrocyclic Factor XIa Inhibitors: Discovery of the Macrocyclic Amide Linker.

A novel series of macrocyclic FXIa inhibitors was designed based on our lead acyclic phenyl imidazole chemotype. Our initial macrocycles, which were double-digit nanomolar FXIa inhibitors, were further optimized with assistance from utilization of structure-based drug design and ligand bound X-ray crystal structures. This effort resulted in the discovery of a macrocyclic amide linker which was found to form a key hydrogen bond with the carbonyl of Leu41 in the FXIa active site, resulting in potent FXIa inhibitors. The macrocyclic FXIa series, exemplified by compound 16, had a FXIa Ki = 0.16 nM with potent anticoagulant activity in an in vitro clotting assay (aPTT EC1.5x = 0.27 µM) and excellent selectivity against the relevant blood coagulation enzymes.

The utility of stable isotope labeled (SIL) analogues in the bioanalysis of endogenous compounds by LC-MS applied to the study of bile acids in a metabolomics assay.

The growing field of biomarker bioanalysis by liquid chromatography mass spectrometry (LC-MS) is challenged with the selection of suitable matrices to construct relevant and valid calibration curves resulting in not only precise but also accurate data. Because surrogate matrices are often employed with the associated concerns about the accuracy of the obtained data, here we present an assay using surrogate analytes in naive biological matrices. This approach is illustrated with the analysis of endogenous bile acids (e-BAs) in serum and plasma using stable isotope-labeled (SIL) analogues as calibration standards to address the matrix concerns. Several deuterated BAs (d-BAs) were used as standards representing respectively grouped e-BAs with structural similarity allowing for the simultaneous bioanalysis of 16 e-BA. The utility of this LC-MS assay employing d-BAs is demonstrated with the analysis of samples resultant of a controlled metabolomics study where a cohort of rats was fed/fasted to investigate the change of e-BAs dependent on food consumption and fasting time.

Orally bioavailable pyridine and pyrimidine-based Factor XIa inhibitors: Discovery of the methyl N-phenyl carbamate P2 prime group.

Pyridine-based Factor XIa (FXIa) inhibitor (S)-2 was optimized by modifying the P2 prime, P1, and scaffold regions. This work resulted in the discovery of the methyl N-phenyl carbamate P2 prime group which maintained FXIa activity, reduced the number of H-bond donors, and improved the physicochemical properties compared to the amino indazole P2 prime moiety. Compound (S)-17 was identified as a potent and selective FXIa inhibitor that was orally bioavailable. Replacement of the basic cyclohexyl methyl amine P1 in (S)-17 with the neutral p-chlorophenyltetrazole P1 resulted in the discovery of (S)-24 which showed a significant improvement in oral bioavailability compared to the previously reported imidazole (S)-23. Additional improvements in FXIa binding affinity, while maintaining oral bioavailability, was achieved by replacing the pyridine scaffold with either a regioisomeric pyridine or pyrimidine ring system.

Novel phenylalanine derived diamides as Factor XIa inhibitors.

The synthesis, structural activity relationships (SAR), and selectivity profile of a potent series of phenylalanine diamide FXIa inhibitors will be discussed. Exploration of P1 prime and P2 prime groups led to the discovery of compounds with high FXIa affinity, good potency in our clotting assay (aPPT), and high selectivity against a panel of relevant serine proteases as exemplified by compound 21. Compound 21 demonstrated good in vivo efficacy (EC50=2.8µM) in the rabbit electrically induced carotid arterial thrombosis model (ECAT).

Discovery of a Potent Parenterally Administered Factor XIa Inhibitor with Hydroxyquinolin-2(1H)-one as the P2' Moiety.

Structure-activity relationship optimization of phenylalanine P1' and P2' regions with a phenylimidazole core resulted in a series of potent FXIa inhibitors. Introducing 4-hydroxyquinolin-2-one as the P2' group enhanced FXIa affinity and metabolic stability. Incorporation of an N-methyl piperazine amide group to replace the phenylalanine improved both FXIa potency and aqueous solubility. Combination of the optimization led to the discovery of FXIa inhibitor 13 with a FXIa K i of 0.04 nM and an aPTT EC2x of 1.0 µM. Dose-dependent efficacy (EC50 of 0.53 µM) was achieved in the rabbit ECAT model with minimal bleeding time prolongation.

P2Y6 receptor potentiates pro-inflammatory responses in macrophages and exhibits differential roles in atherosclerotic lesion development.

BACKGROUND: P2Y(6), a purinergic receptor for UDP, is enriched in atherosclerotic lesions and is implicated in pro-inflammatory responses of key vascular cell types and macrophages. Evidence for its involvement in atherogenesis, however, has been lacking. Here we use cell-based studies and three murine models of atherogenesis to evaluate the impact of P2Y(6) deficiency on atherosclerosis. METHODOLOGY/PRINCIPAL FINDINGS: Cell-based studies in 1321N1 astrocytoma cells, which lack functional P2Y(6) receptors, showed that exogenous expression of P2Y(6) induces a robust, receptor- and agonist-dependent secretion of inflammatory mediators IL-8, IL-6, MCP-1 and GRO1. P2Y(6)-mediated inflammatory responses were also observed, albeit to a lesser extent, in macrophages endogenously expressing P2Y(6) and in acute peritonitis models of inflammation. To evaluate the role of P2Y(6) in atherosclerotic lesion development, we used P2Y(6)-deficient mice in three mouse models of atherosclerosis. A 43% reduction in aortic arch plaque was observed in high fat-fed LDLR knockout mice lacking P2Y(6) receptors in bone marrow-derived cells. In contrast, no effect on lesion development was observed in fat-fed whole body P2Y(6)xLDLR double knockout mice. Interestingly, in a model of enhanced vascular inflammation using angiotensin II, P2Y(6) deficiency enhanced formation of aneurysms and exhibited a trend towards increased atherosclerosis in the aorta of LDLR knockout mice. CONCLUSIONS: P2Y(6) receptor augments pro-inflammatory responses in macrophages and exhibits a pro-atherogenic role in hematopoietic cells. However, the overall impact of whole body P2Y(6) deficiency on atherosclerosis appears to be modest and could reflect additional roles of P2Y(6) in vascular disease pathophysiologies, such as aneurysm formation.

Application and challenges in using LC-MS assays for absolute quantitative analysis of therapeutic proteins in drug discovery.

As more protein therapeutics enter the drug-discovery pipeline, the traditional ligand-binding assay (LBA) faces additional challenges to meet the rapid and diverse bioanalytical needs in the early drug-discovery stage. The high specificity and sensitivity afforded by LC-MS, along with its rapid method development, is proving invaluable for the analysis of protein therapeutics in support of drug discovery. LC-MS not only serves as a quantitative tool to complement LBA in drug discovery, it also provides structural details at a molecular level, which are used to address issues that cannot be resolved using LBA alone. This review will describe the key benefits and applications, as well as the techniques and challenges for applying LC-MS to support protein quantification in drug discovery.

Potent P2Y1 urea antagonists bearing various cyclic amine scaffolds.

A number of new amine scaffolds with good inhibitory activity in the ADP-induced platelet aggregation assay have been found to be potent antagonists of the P2Y1 receptor. SAR optimization led to the identification of isoindoline 3c and piperidine 4a which showed good in vitro binding and functional activities, as well as improved aqueous solubility. Among them, the piperidine 4a showed the best overall profile with favorable PK parameters.

Conformationally constrained ortho-anilino diaryl ureas: discovery of 1-(2-(1'-neopentylspiro[indoline-3,4'-piperidine]-1-yl)phenyl)-3-(4-(trifluoromethoxy)phenyl)urea, a potent, selective, and bioavailable P2Y1 antagonist.

Preclinical antithrombotic efficacy and bleeding models have demonstrated that P2Y1 antagonists are efficacious as antiplatelet agents and may offer a safety advantage over P2Y12 antagonists in terms of reduced bleeding liabilities. In this article, we describe the structural modification of the tert-butyl phenoxy portion of lead compound 1 and the subsequent discovery of a novel series of conformationally constrained ortho-anilino diaryl ureas. In particular, spiropiperidine indoline-substituted diaryl ureas are described as potent, orally bioavailable small-molecule P2Y1 antagonists with improved activity in functional assays and improved oral bioavailability in rats. Homology modeling and rat PK/PD studies on benchmark compound 3l will also be presented. Compound 3l was our first P2Y1 antagonist to demonstrate a robust oral antithrombotic effect with mild bleeding liability in the rat thrombosis and hemostasis models.

2-Aminothiazole based P2Y(1) antagonists as novel antiplatelet agents.

ADP receptors, P2Y1 and P2Y12 have been recognized as potential targets for antithrombotic drugs. A series of P2Y1 antagonists that contain 2-aminothiazoles as urea surrogates were discovered. Extensive SAR of the thiazole ring is described. The most potent compound 7j showed good P2Y1 binding (Ki=12nM), moderate antagonism of platelet aggregation (PA IC50=5.2µM) and acceptable PK in rats.

Discovery of diarylurea P2Y(1) antagonists with improved aqueous solubility.

Preclinical data suggests that P2Y1 antagonists, such as diarylurea compound 1, may provide antithrombotic efficacy similar to P2Y12 antagonists and may have the potential of providing reduced bleeding liabilities. This manuscript describes a series of diarylureas bearing solublizing amine side chains as potent P2Y1 antagonists. Among them, compounds 2l and 3h had improved aqueous solubility and maintained antiplatelet activity compared with compound 1. Compound 2l was moderately efficacious in both rat and rabbit thrombosis models and had a moderate prolongation of bleeding time in rats similar to that of compound 1.

In silico prediction of biliary excretion of drugs in rats based on physicochemical properties.

Evaluating biliary excretion, a major elimination pathway for many compounds, is important in drug discovery. The bile duct-cannulated (BDC) rat model is commonly used to determine the percentage of dose excreted as intact parent into bile. However, a study using BDC rats is time-consuming and cost-ineffective. The present report describes a computational model that has been established to predict biliary excretion of intact parent in rats as a percentage of dose. The model was based on biliary excretion data of 50 Bristol-Myers Squibb Co. compounds with diverse chemical structures. The compounds were given intravenously at <10 mg/kg to BDC rats, and bile was collected for at least 8 h after dosing. Recoveries of intact parents in bile were determined by liquid chromatography with tandem mass spectrometry. Biliary excretion was found to have a fairly good correlation with polar surface area (r = 0.76) and with free energy of aqueous solvation (DeltaG(solv aq)) (r = -0.67). In addition, biliary excretion was also highly corrected with the presence of a carboxylic acid moiety in the test compounds (r = 0.87). An equation to calculate biliary excretion in rats was then established based on physiochemical properties via a multiple linear regression. This model successfully predicted rat biliary excretion for 50 BMS compounds (r = 0.94) and for 25 previously reported compounds (r = 0.86) whose structures are markedly different from those of the 50 BMS compounds. Additional calculations were conducted to verify the reliability of this computation model.

Bioactivation of coumarin in rat olfactory mucosal microsomes: Detection of protein covalent binding and identification of reactive intermediates through analysis of glutathione adducts.

The presence of high levels, as well as tissue-specific forms, of cytochrome P450 enzymes in mammalian olfactory mucosa (OM) has important implications in the bioactivation and toxicity of xenobiotics entering the tissue. Previous studies have shown that coumarin, a known olfactory toxicant in rats, is bioactivated by OM microsomal P450s to a number of products, presumably via coumarin-3,4-epoxide and other epoxide intermediates. The aim of the current study was to obtain direct evidence for the formation of such reactive intermediates in rat OM through the detection of protein covalent binding and glutathione (GSH) adduct formation. Protein covalent binding experiments with [(14)C]coumarin (10microM) displayed a 7-9-fold higher NADPH-dependent radioactivity binding in rat OM microsomes (2.5nmol/mg/30min) compared to those in rat and human liver microsomes; the binding value in rat OM microsomes was substantially but not completely reduced by the addition of GSH (5mM). LC/MS analyses detected a number of GSH adducts in GSH-supplemented coumarin metabolism reaction in rat OM microsomes; 3-glutathionyl coumarin was found to be the major one, indicating 3,4-epoxidation as the main bioactivation pathway. Additional GSH adducts were identified, presumably forming via the same pathway or epoxidation on the benzene moiety. Our findings provide direct evidence for the formation of multiple coumarin reactive intermediates in rat OM, leading to protein covalent binding and GSH conjugation.