Benzimidazole-based fXa inhibitors with improved thrombin and trypsin selectivity

Optimization of the benzimidazole-based fXa inhibitors for selectivity versus thrombin and trypsin was achieved by substitution on the benzimidazole ring and replacement of the naphthylamidine group. Substitution of a nitro group at the 4-position on the benzimidazole improves both potency against fXa and selectivity versus thrombin. Alternatively, replacement of the naphthylamidine with either a biphenylamidine or propenylbenzamidine not only improves fXa potency and selectivity versus thrombin, but selectivity versus trypsin as well.     

Design, structure-activity relationship, and pharmacokinetic profile of pyrazole-based indoline factor Xa inhibitors

A new series of pyrazole-based factor Xa inhibitors have been identified as part of our ongoing efforts to optimize previously reported clinical candidate razaxaban. Concern over the possible formation of primary aniline metabolites via amide hydrolysis led to the replacement of the primary amide linker between the pyrazole and phenyl moieties with secondary amides. This was accomplished by replacing the aniline with a variety of heterobicycles, of which indolines were the most potent. The indoline series demonstrated subnanomolar factor Xa binding K(i)s, modest to high selectivity versus other serine proteases, and good in vitro clotting activity. A small number of indoline fXa inhibitors were profiled in a dog pharmacokinetic model, one of which demonstrated pharmacokinetic parameters similar to that of clinical candidate razaxaban.     

Discovery of potent, efficacious, and orally bioavailable inhibitors of blood coagulation factor Xa with neutral P1 moieties

The bicyclic dihydropyrazolopyridinone scaffold allowed for incorporation of multiple P1 moieties with subnanomolar binding affinities for blood coagulation factor Xa. The compound 3-[6-(2'-dimethylaminomethyl-biphenyl-4-yl)-7-oxo-3-trifluoro-methyl-4,5,6,7-tetrahydro-pyrazolo[3,4-c]pyridine-l-yl]-benzamide 6d shows good fXa potency, selectivity, in vivo efficacy and oral bioavailability. Compound 6d was selected for further pre-clinical evaluations.     

Optimization of a coagulation factor VIIa inhibitor found in factor Xa inhibitor library

An inhibitor of the complex of factor VIIa and tissue factor (fVIIa/TF), 2-substituted-4-amidinophenylpyruvic acid 1a, was structurally modified with the aim of increasing its potency and selectivity. The lead compound 1a was originally found in our factor Xa (fXa) inhibitor library on the basis of structural similarity of the primary binding sites of fVIIa and fXa. The design was based on computational docking studies using the extracted active site of fVIIa. Compound 1j was found to inhibit factor VIIa/TF at nanomolar concentration with improved selectivity versus fXa and thrombin and it preferentially prolonged the clotting time in the TF-dependent extrinsic pathway.     

Role of P2 glycine in determining the specificity of antithrombin reaction with coagulation proteases

Structural data suggests that bulky hydrophobic residues at the S2-S4 sub-sites of factor Xa (fXa) restrict the preference of this pocket for small and non-polar residues like Gly at the P2 position of substrates and inhibitors. However, kinetic studies monitoring the cleavage specificity of 10-residue peptides by fXa have identified Phe as the most preferred P2 residue and Gln-Phe-Arg-Ser-Leu-Ser as the most preferred P3-P3′ residues for recognition by fXa. To determine whether this mechanism of specificity is also true for fXa reaction with antithrombin (AT), we prepared two AT mutants having either a Phe at the P2 or Gln-Phe-Arg-Ser-Leu-Ser at the P3-P3′ positions of the reactive center loop. Inhibition kinetic studies indicated that the reactivity of P2-Phe with fXa was significantly (∼5-fold) impaired, however, the P3-P3′ mutant exhibited 1.5-fold improved reactivity with the protease, suggesting cooperative effects between P3-P3′ residues influence the P2 specificity of AT. Substitution of Tyr-99 of fXa with a Gly dramatically impaired the reactivity of fXa with wild-type AT, but improved its reactivity with the serpin mutants in the absence, but not in the presence of pentasaccharide. AT with a P2-Phe inhibited thrombin with >150-fold impaired reactivity, however, the defect was restored by either pentasaccharide or by replacing Leu-99 of thrombin with a Gly. The P3-P3′ mutant rapidly inhibited factors VIIa and XIa independent of pentasaccharide. These results indicate that P2-Gly plays a key role in determining the S2 sub-site specificity and target protease selectivity of AT in circulation.     

Converting blood coagulation factor IXa into factor Xa: dramatic increase in amidolytic activity identifies important active site determinants.

The coagulation factors IXa (fIXa) and Xa (fXa) share extensive structural and functional homology; both cleave natural substrates effectively only with a cofactor at a phospholipid surface. However, the amidolytic activity of fIXa is 10(4)-fold lower than that of fXa. To identify determinants of this poor reactivity, we expressed variants of truncated fIXa (rf9a) and fXa (rf10a) in Escherichia coli. The crystal structures of fIXa and fXa revealed four characteristic active site components which were subsequently exchanged between rf9a and rf10a. Exchanging Glu219 by Gly or exchanging the 148 loop did not increase activity of rf9a, whereas corresponding mutations abolished reactivity of rf10a. Exchanging Ile213 by Val only moderately increased reactivity of rf9a. Exchanging the 99 loop, however, dramatically increased reactivity. Furthermore, combining all four mutations essentially introduced fXa properties into rf9a: the amidolytic activity was increased 130-fold with fXa substrate selectivity. The results suggest a 2-fold origin of fIXa's poor reactivity. A narrowed S3/S4 subsite disfavours interaction with substrate P3/P4 residues, while a distorted S1 subsite disfavours effective cleavage of the scissile bond. Both defects could be repaired by introducing fXa residues. Such engineered coagulation enzymes will be useful in diagnostics and in the development of therapeutics.     

1-(2-Naphthyl)-1H-pyrazole-5-carboxylamides as potent factor Xa inhibitors. Part 2: A survey of P4 motifs

A variety of P4 motifs have been examined to increase the binding affinity and in vitro anticoagulant potency of our biphenyl 1-(2-naphthyl)-1H-pyrazole-5-carboxylamide-based fXa inhibitors. Highly potent 2-naphthyl-P1 fXa inhibitors (K(i)< or =2 nM) with improved in vitro anticoagulant activity (2xTG< or =1 microM) and respectable pharmacokinetic properties have been discovered.     

Aminobenzisoxazoles with biaryl P4 moieties as potent, selective, and orally bioavailable factor Xa inhibitors

We have previously reported on a series of aminobenzisoxazoles as potent, selective, and orally bioavailable factor Xa inhibitors, which culminated in the discovery of razaxaban. Herein, we describe another approach to improve factor Xa inhibitory potency and pharmacokinetic profile by incorporating basic and water soluble functionalities on the terminal ring of the P4 biaryl group found in our earlier Xa inhibitors. This approach resulted in a series of potent, selective, and orally bioavailable factor Xa inhibitors.     

Synthesis and biological activity of novel 1,2-disubstituted benzene derivatives as factor Xa inhibitors

Factor Xa (fXa) is a serine protease that plays a pivotal role in the coagulation cascade. High-throughput screening of the Yamanouchi compound library yielded lead compound 1 with the ability to inhibit fXa at micromolar concentrations. To improve its fXa inhibitory activity and its oral anticoagulant activity, the linker between benzamidine and the central benzene ring was modified and a carboxyl group was introduced at the central benzene ring. The resulting compounds 40b (YM-203552), 41a (YM-202054), and 41c (YM-203558) exhibited potent fXa inhibitory activity and oral anticoagulant activity. In particular, YM-203558 exhibited the most potent oral anticoagulant activity, prolonging PT more than 3-fold at 0.5 and 2.0 h. Additionally, these compounds showed a high degree of selectivity for other serine proteases.     

1-[3-Aminobenzisoxazol-5'-yl]-3-trifluoromethyl-6-[2'-(3-(R)-hydroxy-N-pyrrolidinyl)methyl-[1,1']-biphen-4-yl]-1,4,5,6-tetrahydropyrazolo-[3,4-c]-pyridin-7-one (BMS-740808) a highly potent, selective, efficacious, and orally bioavailable inhibitor of blood coagulation factor Xa

Attempts to further optimize the pyrazole factor Xa inhibitors centered on masking the aryl aniline P4 moiety. Scaffold optimization resulted in the identification of a novel bicyclic pyrazolo-pyridinone scaffold which retained fXa potency. The novel bicyclic scaffold preserved all binding interactions observed with the monocyclic counterpart and importantly the carboxamido moiety was integrated within the scaffold making it less susceptible to hydrolysis. These efforts led to the identification of 1-[3-aminobenzisoxazol-5'-yl]-3-trifluoromethyl-6-[2'-(3-(R)-hydroxy-N-pyrrolidinyl)methyl-[1,1']-biphen-4-yl]-1,4,5,6-tetrahydropyrazolo-[3,4-c]-pyridin-7-one 6f (BMS-740808), a highly potent (fXa Ki=30 pM) with a rapid onset of inhibition (2.7x10(7) M-1 s-1) in vitro, selective (>1000-fold over other proteases), efficacious in the AVShunt thrombosis model, and orally bioavailable inhibitor of blood coagulation factor Xa.     

Design and synthesis of aminophenol-based factor Xa inhibitors

A novel potent and selective aminophenol scaffold for fXa inhibitors was developed from a previously reported benzimidazole-based naphthylamidine template. The aminophenol template is more synthetically accessible than the benzimidazole template, which simplified the introduction of carboxylic acid groups. Substitution of a propenyl-para-hydroxy-benzamidine group on the aminophenol template produced selective, sub-nanomolar fXa inhibitors. The potency of the inhibitors is partially explained with the aid of a trypsin complex crystal structure.     

Prodrug-based design, synthesis, and biological evaluation of N-benzenesulfonylpiperidine derivatives as novel, orally active factor Xa inhibitors

We describe here our investigation of a new series of orally active fXa inhibitors based on a prodrug strategy. Solid-phase parallel synthesis identified a unique series of fXa inhibitors with a substituted benzenesulfonyl group as a novel S4 binding element. This series resulted in compound 39, which exhibited potent inhibitory activity against fXa (IC50 = 13 nM) and excellent selectivity over thrombin (>7000-fold). The masking of its highly hydrophilic groups led to the creation of related prodrug 28, which demonstrated an anticoagulant effect after oral dosing.     

alpha-Amino-beta-sulphone hydroxamates as potent MMP-13 inhibitors that spare MMP-1

A series of alpha-amino-beta-sulphone hydroxamates was prepared and evaluated for potency versus MMP-13 and selectivity versus MMP-1. Various substituents were employed on the alpha-amino group (P(1) position), as well as different groups attached to the sulphone group extending into P(1)'. Low nanomolar potency was obtained for MMP-13 with selectivity versus MMP-1 of >1000x for a number of analogues.     

Novel bicyclic lactam inhibitors of thrombin: highly potent and selective inhibitors

The potency and selectivity of a previous series of low molecular weight thrombin inhibitors were improved through modifications of the P1 and P3 residues. Introduction of diphenyl substituted sulfonamides in the P3 moiety led to highly efficacious compounds. By correctly selecting the combination of P1 and P3 residues, high levels of potency, selectivity and in vivo efficacy were obtained.     

Structure- and property-based design of factor Xa inhibitors: pyrrolidin-2-ones with acyclic alanyl amides as P4 motifs

Structure-based drug design was exploited in the synthesis of 3-(6-chloronaphth-2-ylsulfonyl)aminopyrrolidin-2-one-based factor Xa (fXa) inhibitors, incorporating an alanylamide P4 group with acyclic tertiary amide termini. Optimized hydrophobic contacts of one amide substituent in P4 were complemented by hydrophobicity-modulating features in the second, producing potent fXa inhibitors including examples with excellent anticoagulant properties.     

The de novo design and synthesis of cyclic urea inhibitors of factor Xa: optimization of the S4 ligand

In this report refinements to the S4 ligand group leads to compound 19, an inhibitor of fXa with good potency in vitro and an improved pharmacokinetic profile in rabbit. The X-ray crystallographic study of a representative analogue confirms our binding model for this series.     

Tick anticoagulant peptide: kinetic analysis of the recombinant inhibitor with blood coagulation factor Xa

Tick anticoagulant peptide (TAP) is a 60 amino acid protein which is a highly specific inhibitor of human blood coagulation factor Xa (fXa) isolated from the tick Ornithodoros moubata [Waxman, L., Smith, D. E., Arcuri, K. E., & Vlasuk, G. P. (1990) Science 248, 593-596]. Due to the limited quantities of native TAP, a recombinant version of TAP produced in Saccharomyces cerevisiae was used for a detailed kinetic analysis of the inhibition interaction with human fXa. rTAP was determined to be a reversible, slow, tight-binding inhibitor of fXa, displaying a competitive type of inhibition. The binding of rTAP to fXa is stoichiometric with a dissociation constant of (1.8 +/- 0.02) x 10(-10) M, a calculated association rate constant of (2.85 +/- 0.07) x 10(6) M-1 s-1, and a dissociation rate constant of (0.554 +/- 0.178) x 10(-3) s-1. Binding studies show that 35S-rTAP binds only to fXa and not to DFP-treated fXa or zymogen factor X, which suggests the active site of fXa is required for rTAP inhibition. That rTAP is a unique serine proteinase inhibitor is suggested both by its high specificity for its target enzyme, fXa, and also by its unique structure.     

Basis for the Specificity and Activation of the Serpin Protein Z-dependent Proteinase Inhibitor (ZPI) as an Inhibitor of Membrane-associated Factor Xa

The serpin ZPI is a protein Z (PZ)-dependent specific inhibitor of membrane-associated factor Xa (fXa) despite having an unfavorable P1 Tyr. PZ accelerates the inhibition reaction ∼2000-fold in the presence of phospholipid and Ca²⁺. To elucidate the role of PZ, we determined the x-ray structure of Gla-domainless PZ (PZ_ΔGD) complexed with protein Z-dependent proteinase inhibitor (ZPI). The PZ pseudocatalytic domain bound ZPI at a novel site through ionic and polar interactions. Mutation of four ZPI contact residues eliminated PZ binding and membrane-dependent PZ acceleration of fXa inhibition. Modeling of the ternary Michaelis complex implicated ZPI residues Glu-313 and Glu-383 in fXa binding. Mutagenesis established that only Glu-313 is important, contributing ∼5–10-fold to rate acceleration of fXa and fXIa inhibition. Limited conformational change in ZPI resulted from PZ binding, which contributed only ∼2-fold to rate enhancement. Instead, template bridging from membrane association, together with previously demonstrated interaction of the fXa and ZPI Gla domains, resulted in an additional ∼1000-fold rate enhancement. To understand why ZPI has P1 tyrosine, we examined a P1 Arg variant. This reacted at a diffusion-limited rate with fXa, even without PZ, and predominantly as substrate, reflecting both rapid acylation and deacylation. P1 tyrosine thus ensures that reaction with fXa or most other arginine-specific proteinases is insignificant unless PZ binds and localizes ZPI and fXa on the membrane, where the combined effects of Gla-Gla interaction, template bridging, and interaction of fXa with Glu-313 overcome the unfavorability of P1 Tyr and ensure a high rate of reaction as an inhibitor.     

Design and synthesis of orally active pyrrolidin-2-one-based factor Xa inhibitors

A series of novel, non-basic 3-(6-chloronaphth-2-ylsulfonyl)aminopyrrolidin-2-one-based factor Xa (fXa) inhibitors, incorporating an alanylamide P4 group, was designed and synthesised. Within this series, the N-2-(morpholin-4-yl)-2-oxoethyl derivative 24 was shown to be a potent, selective fXa inhibitor with good anticoagulant activity. Moreover, 24 possessed highly encouraging rat and dog pharmacokinetic profiles with excellent oral bioavailabilities in both species.     

Nonbenzamidine tetrazole derivatives as factor Xa inhibitors

Factor Xa (fXa) is an important serine protease that holds the central position linking the intrinsic and extrinsic activation mechanisms in the blood coagulation cascade. Therefore, inhibition of fXa has potential therapeutic applications in the treatments of both arterial and venous thrombosis. Herein we describe a series of tetrazole fXa inhibitors containing benzamidine mimics as the P(1) substrate, of which the aminobenzisoxazole moiety was found to be the most potent benzamidine mimic. SR374 (12) inhibits fXa with a K(i) value of 0.35 nM and is very selective for fXa over thrombin and trypsin.