RESUMO
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.
Assuntos
Trombose das Artérias Carótidas , Fator XIa , Fibrinolíticos , Pirimidinas , Triazóis , Animais , Camundongos , Coelhos , Administração Oral , Trombose das Artérias Carótidas/tratamento farmacológico , Fator XIa/antagonistas & inibidores , Fibrinolíticos/administração & dosagem , Fibrinolíticos/síntese química , Fibrinolíticos/farmacocinética , Fibrinolíticos/uso terapêutico , Macaca fascicularis , Estrutura Molecular , Pirazóis/administração & dosagem , Pirazóis/síntese química , Pirazóis/farmacocinética , Pirazóis/uso terapêutico , Pirimidinas/administração & dosagem , Pirimidinas/síntese química , Pirimidinas/farmacocinética , Pirimidinas/uso terapêutico , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Triazóis/administração & dosagem , Triazóis/síntese química , Triazóis/farmacocinética , Triazóis/uso terapêuticoRESUMO
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.
Assuntos
Fator XIa/antagonistas & inibidores , Fibrinolíticos/administração & dosagem , Fibrinolíticos/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , Cristalografia por Raios X , Cães , Avaliação Pré-Clínica de Medicamentos , Fator XIa/química , Fator XIa/metabolismo , Fibrinolíticos/química , Fibrinolíticos/farmacocinética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Compostos Macrocíclicos/administração & dosagem , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacocinética , Compostos Macrocíclicos/farmacologia , Modelos Moleculares , Coelhos , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Aminas/química , Fator XIa/antagonistas & inibidores , Compostos Macrocíclicos/química , Inibidores de Serina Proteinase/síntese química , Administração Oral , Animais , Sítios de Ligação , Desenho de Fármacos , Fator XIa/metabolismo , Meia-Vida , Compostos Macrocíclicos/metabolismo , Compostos Macrocíclicos/farmacocinética , Simulação de Dinâmica Molecular , Estrutura Terciária de Proteína , Piridinas/química , Ratos , Inibidores de Serina Proteinase/metabolismo , Inibidores de Serina Proteinase/farmacocinética , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Fator XIa/antagonistas & inibidores , Fibrinolíticos/síntese química , Fibrinolíticos/farmacologia , Piridinas/síntese química , Piridinas/farmacologia , Animais , Disponibilidade Biológica , Coagulação Sanguínea/efeitos dos fármacos , Cristalografia por Raios X , Desenho de Fármacos , Descoberta de Drogas , Fibrinolíticos/farmacocinética , Humanos , Imidazóis/síntese química , Imidazóis/farmacologia , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/farmacologia , Modelos Moleculares , Tempo de Tromboplastina Parcial , Coelhos , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/farmacologia , Relação Estrutura-Atividade , Trombose/tratamento farmacológicoRESUMO
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.
Assuntos
Anticoagulantes/química , Anticoagulantes/uso terapêutico , Fator XIa/antagonistas & inibidores , Isoquinolinas/química , Isoquinolinas/uso terapêutico , Trombose/tratamento farmacológico , para-Aminobenzoatos/química , para-Aminobenzoatos/uso terapêutico , Animais , Anticoagulantes/farmacocinética , Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Cristalografia por Raios X , Cães , Descoberta de Drogas , Fator XIa/química , Fator XIa/metabolismo , Humanos , Isoquinolinas/farmacocinética , Isoquinolinas/farmacologia , Masculino , Simulação de Acoplamento Molecular , Coelhos , Ratos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacocinética , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Trombose/sangue , para-Aminobenzoatos/farmacocinética , para-Aminobenzoatos/farmacologiaRESUMO
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.
Assuntos
Amidas/farmacologia , Descoberta de Drogas , Fator XIa/antagonistas & inibidores , Compostos Macrocíclicos/farmacologia , Inibidores de Serina Proteinase/farmacologia , Amidas/síntese química , Amidas/química , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Fator XIa/metabolismo , Humanos , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/química , Modelos Moleculares , Estrutura Molecular , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/química , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Amidas/química , Fator XIa/antagonistas & inibidores , Compostos Macrocíclicos/farmacologia , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Descoberta de Drogas , Ligação de Hidrogênio , Ligantes , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacocinética , Estrutura Molecular , Inibidores de Serina Proteinase/farmacocinéticaRESUMO
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.
Assuntos
Anticoagulantes/química , Anticoagulantes/farmacologia , Fator XIa/antagonistas & inibidores , Piridinas/química , Piridinas/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Administração Oral , Animais , Anticoagulantes/administração & dosagem , Anticoagulantes/farmacocinética , Coagulação Sanguínea/efeitos dos fármacos , Cristalografia por Raios X , Cães , Fator XIa/metabolismo , Humanos , Modelos Moleculares , Fenilcarbamatos/administração & dosagem , Fenilcarbamatos/química , Fenilcarbamatos/farmacocinética , Fenilcarbamatos/farmacologia , Piridinas/administração & dosagem , Piridinas/farmacocinética , Pirimidinas/administração & dosagem , Pirimidinas/farmacocinéticaRESUMO
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.
Assuntos
Ácidos e Sais Biliares/sangue , Ácidos e Sais Biliares/metabolismo , Marcação por Isótopo , Metabolômica , Animais , Ácidos e Sais Biliares/química , Cromatografia Líquida , Humanos , Espectrometria de Massas , Estrutura Molecular , RatosRESUMO
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.
RESUMO
Twenty-seven highly diversified pharmaceutical compounds were used as a test set to evaluate matrix effects from microsomal media on LC/MS analyses. The individual effects of Tris buffer, NADPH and microsomes on ESI response were investigated. Direct flow injection MS/MS analysis, using no sample preparation or HPLC separation, gave an average of 2.2-5-fold matrix suppression in MS response from Tris buffer and NADPH. More polar analytes were affected the greatest. To reduce the loss in response, an automated solid phase extraction (SPE) procedure was developed. A much smaller average matrix effect was observed when samples were prepared using a Waters Oasis HLB 96-well SPE. As little as 1 ml of methanol (MeOH) was sufficient to elute most compounds with more than 80% recovery. Comparable results were obtained by directly injecting a protein-precipitated incubation onto a fast gradient LC separation prior to MS/MS detection. No advantage was seen by using both SPE and a fast LC separation prior to MS/MS analysis.