Design,synthesis and biological activity of YM-60828 derivatives: potent and orally-bioavailable factor Xa inhibitors based on naphthoanilide and naphthalensulfonanilide templates

Factor Xa (FXa) is a serine protease which plays a pivotal role in the coagulation cascade. The inhibition of FXa has received great interest as a potential target for the development of new antithrombotic drug. Herein we describe a series of novel 7-amidino-2-naphthoanilide and 7-amidino-2-naphthalensulfonanilide derivatives which are potent FXa inhibitors. These scaffolds are rigid and are allowed to adopt an L-shape conformation which was estimated as the active conformation based on a docking study of YM-60828 with FXa. Optimization of the side chain at the central aniline nitrogen of 7-amidino-2-naphthoanilide has led to several potent and orally active FXa inhibitors. 5h (YM-169964), the best compound of these series, showed potent FXa inhibitory activity (IC(50)=3.9nM) and effectively prolonged prothrombin time by 9.6-fold ex vivo at an oral dose of 3mg/kg in squirrel monkeys.     

Design,synthesis and biological activity of YM-60828 derivatives. Part 2: potent and orally-bioavailable factor Xa inhibitors based on benzothiadiazine-4-one template

Compound YM-60828 was previously characterized in our laboratory as a potent, selective and orally-bioavailable Factor Xa (FXa) inhibitor. The L-shape conformation of this compound in the active site of FXa was recognized as an important factor in displaying its FXa inhibitory activity. This led to the exploration of conformationally restricted cyclic scaffolds bearing a similar active conformation. The current study investigated a novel series of benzothiadiazine-4-one based compounds as FXa inhibitors. Structure-activity relationship (SAR) investigations revealed some potent FXa inhibitors that were selected for further in vitro and ex vivo anticoagulant studies. Among them, compound 6j (YM-169920) was proved to be most effective anticoagulant in this series. The synthesis and SAR in addition to docking studies of this class of inhibitors are described.     

Arylsulfonamidopiperidone derivatives as a novel class of factor Xa inhibitors

The design, synthesis and SAR of a novel class of valerolactam-based arylsulfonamides as potent and selective FXa inhibitors is reported. The arylsulfonamide–valerolactam scaffold was derived based on the proposed bioisosterism to the arylcyanoguanidine-caprolactam core in known FXa inhibitors. The SAR study led to compound 46 as the most potent FXa inhibitor in this series, with an IC₅₀ of 7 nM and EC_2×PT of 1.7 μM. The X-ray structure of compound 40 bound to FXa shows that the sulfonamide–valerolactam scaffold anchors the aryl group in the S1 and the novel acylcytisine pharmacophore in the S4 pockets.     

The discovery of YM-60828: a potent, selective and orally-bioavailable factor Xa inhibitor

Since Factor Xa (FXa) is well known to play a central role in thrombosis and hemostasis, inhibition of FXa is an attractive target for antithrombotic strategies. As a part of our investigation of a non-peptide, orally available FXa inhibitor, we found that a series of N-[(7-amidino-2-naphthyl)methyl]aniline derivatives possessed potent and selective inhibitory activities. Structure--activity relationship (SAR) of the substituent (R(1)) on the central aniline moiety suggested that increasing lipophilicity caused a detrimental effect on anticoagulant activity (prothrombin time assay) in plasma. Several compounds bearing a hydrophilic substituent in R(1) showed not only potent FXa inhibitory activities but also high anticoagulant activities. The best compound in this series was sulfamoylacetic acid derivative (YM-60828) which was a potent, selective and orally bioavailable FXa inhibitor and was chosen for clinical development.     

Design, synthesis, and in vitro biological activity of indole-based factor Xa inhibitors

A series of indole and carbazole based inhibitors of factor Xa (FXa) has been investigated. The most potent compound inhibits FXa with a Ki of 0.2 nM and has 900- and 750-fold selectivity over thrombin and trypsin, respectively.     

Enantiopure five-membered cyclicdiamine derivatives as potent and selective inhibitors of factor Xa. Improving in vitro metabolic stability via core modifications

We previously reported a series of enantiopure cis-(1R,2S)-cyclopentyldiamine derivatives as potent and selective inhibitors of Factor Xa (FXa). Herein, we describe our approach to improve the metabolic stability of this series via core modifications. Multiple resulting series of compounds demonstrated similarly high FXa potency and improved metabolic stability in human liver microsomes compared with the cyclopentyldiamide 1. (3R,4S)-Pyrrolidinyldiamide 31 was the best overall compound with human FXa K(i) of 0.50 nM, PT EC(2x) of 2.1 microM in human plasma, bioavailability of 25% and t(1/2)of 2.7h in dogs. Further biochemical characterization of compound 31 is also presented.     

Amino(methyl) pyrrolidines as novel scaffolds for factor Xa inhibitors

The design and synthesis of a novel class of amino(methyl) pyrrolidine-based sulfonamides as potent and selective FXa inhibitors is reported. The amino(methyl) pyrrolidine scaffolds were designed based on the proposed bioisosterism to the piperazine core in known FXa inhibitors. The SAR study led to compound 15 as the most potent FXa inhibitor in this series, with an IC(50) of 5.5 nM and PT EC(2x) of 1.7 microM. The proposed binding models show that the pyrrolidine cores are in van der Waals contact with the enzyme surface, and the flexibility of amino(methyl) pyrrolidines allows the two nitrogen atoms to anchor both the P1 and P4 groups to fit similarly in the S1 and S4 pockets.     

Novel, potent and selective chimeric FXa inhibitors featuring hydrophobic P1-ketoamide moieties

Judicious combination of P-region sequences of highly potent anticoagulant proteins including NAP5, NAP6, Ecotin, and Antistasin with SAR from small molecule FXa inhibitors led to a series of chimeric inhibitors of formula 1a-j. We report herein the design, synthesis, and biological activity of this novel family of FXa inhibitors that express both high in vitro potency and superb selectivity against related serine proteases.     

The discovery of glycine and related amino acid-based factor Xa inhibitors

Herein, we report on the identification of three potent glycine and related amino acid-based series of FXa inhibitors containing a neutral P1 chlorophenyl pharmacophore. A X-ray crystal structure has shown that constrained glycine derivatives with optimized N-substitution can greatly increase hydrophobic interactions in the FXa active site. Also, the substitution of a pyridone ring for a phenylsulfone ring in the P4 sidechain resulted in an inhibitor with enhanced oral bioavailability.     

Exploratory solid-phase synthesis of factor Xa inhibitors: discovery and application of p3-heterocyclic amides as novel types of non-basic arginine surrogates

A series of novel FXa inhibitors 2a-m and 3a-f was discovered that feature heterocyclic carboxamides tethered to a d-diaminobutyric acid sidechain. These neutral amide derivatives serve as novel P₃ d-arginine mimics. Pyrazine carboxamide scaffolds afforded the most potent FXa inhibitors (e.g., 2b IC₅₀ = 4.6 nM). The synthesis and biological activity of two focused libraries are reported.     

Design,synthesis, and SAR of monobenzamidines and aminoisoquinolines as factor Xa inhibitors

Monoamidine FXa inhibitors 3 were designed and synthesized. SAR studies and molecular modeling led to the design of conformationally constrained diaryl ethers 4 and 5, as well as benzopyrrolidinone 7 as potent FXa inhibitors. The monoamidines show high efficacy in a DVT model, but lack desirable oral bioavailability. The benzopyrrolidinone-based aminoisoquinolines 8 do not show significant improvement in oral bioavailability.     

Synthesis and biological evaluation of 4'-(6,7-disubstituted-2,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-biphenyl-4-ol as potent Chk1 inhibitors

A new series of potent tricyclic pyrazole-based Chk1 inhibitors are described. Analogues disubstituted on the 6- and 7-positions show improved Chk1 inhibition potency compared with analogues with a single substituent on either the 6- or 7-position. Based on the lead compound 4'-(6,7-dimethoxy-2,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-biphenyl-4-ol (2), detailed SAR studies on the 6- and 7-positions were performed. 3'-morpholin-4'-yl-propoxy, pyridin-4'-ylmethoxy, pyridin-3'-ylmethoxy, 2'-(5'-ethyl-pyridin-2'-yl)-ethoxy, pyridin-2'-ylethoxy, (6'-methyl-pyridin-2'-yl)-propoxyethoxy, 2',3'-dihydroxyl-1'-yl-propoxy, and tetrahydro-furan-3'-yloxy have been identified as the best groups on the 6-position when the 7-position is substituted with methoxyl group. Pyridin-2'-ylmethoxy and pyridin-3'-ylmethoxy have been identified as the best substituents at the 7-position while the 6-position bearing methoxyl group. These compounds significantly potentiate the cytotoxicity of DNA-damaging antitumor agents in a cell-based assay and efficiently abrogate the doxorubicin-induced G2/M and the camptothecin-induced S checkpoints, suggesting that their potent biological activities are mechanism-based through Chk1 inhibition.     

Discovery and SAR of novel pyrazole-based thioethers as cathepsin S inhibitors: Part 1

A series of pyrazole-based thioethers were prepared and found to be potent cathepsin S inhibitors. A crystal structure of 13 suggests that the thioether moiety may bind to the S3 pocket of the enzyme. Additional optimization led to the discovery of aminoethylthioethers with improved enzymatic activity and submicromolar cellular potency.     

Discovery of sulfonylalkylamides: A new class of orally active factor Xa inhibitors

Factor Xa (FXa) is a trypsin-like serine protease involved in the coagulation cascade and has received great interest as a potential target for the development of new antithrombotic agents. Most of amidine-type FXa inhibitors reported have been found to show extremely poor oral bioavailability. Compound 1 is one of the first reported non-amidine type FXa inhibitors. To discover novel and orally active FXa inhibitors, we investigated flexible linear linkers between the 6-chloronaphthalene ring and the 1-(pyridin-4-yl)piperidine moiety of 1 and found the orally active sulfonylalkylamide 2f with an FXa IC(50) of 0.05 microM, comparable with that of 1. Further modification to reduce the CYP3A4 inhibitory activity of 2f resulted in the potent, selective, and orally active 2-methylpyridine analogue 2s (FXa IC(50) of 0.061 microM), for which the liability of CYP3A4 inhibition was significantly weakened compared to 2f. Compound 2s also showed long lasting anticoagulant activity in cynomolgus monkeys.     

Synthesis and evaluation of acylguanidine FXa inhibitors

A series of acylguanidine derivatives were prepared and investigated as inhibitors of Factor Xa (FXa). These compounds were made by guanidine acylation with carboxylic acids using carbonyl diimidazole (CDI) as the coupling reagent. Conditions for the rapid synthesis and purification of these compounds are described along with their ability to inhibit FXa. The best FXa inhibitor is 1 with a FXa IC(50) of 6 nM.     

Structure-based optimization and biological evaluation of trisubstituted pyrazole as a core structure of potent ROS1 kinase inhibitors

Recently inhibition of ROS1 kinase has proven to be a promising strategy for several indications such as glioblastoma, non-small cell lung cancer (NSCLC), and cholangiocarcinoma. Our team reported trisubstituted pyrazole-based ROS1 inhibitors by which two inhibitors showed good IC₅₀ values in enzyme-based screening. To develop more advanced ROS1 inhibitors through SAR this trisubstituted pyrazole-based scaffold has been built. Consequently, 16 compounds have been designed, synthesized and shown potent IC₅₀ values in the enzymatic assay, which are from 13.6 to 283 nM. Molecular modeling studies explain how these ROS1 kinase inhibitors revealed effectively the key interactions with ROS1 ATP binding site. Among these compounds, compound 9a (IC₅₀ = 13.6 nM) has exerted 5 fold potency than crizotinib and exhibited high degree of selectivity (selectivity score value = 0.028) representing the number of non-mutant kinases with biological activity over 90% at 10 μM.     

Structure-activity relationships of potent and selective factor Xa inhibitors: benzimidazole derivatives with the side chain oriented to the prime site of factor Xa

A series of benzimidazole derivatives with the side chain on the nitrogen atom oriented to the prime site of factor Xa (FXa) were designed and synthesized. Compounds with substituted aminocarbonylmethyl groups as the side chain showed potent FXa inhibitory activity. Compounds 1 and 2 exhibited most potent inhibitory activity and were effective as anticoagulants in a DIC model.     

Discovery of 4'-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-benzonitriles and 4'-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-pyridine-2'-carbonitriles as potent checkpoint kinase 1 (Chk1) inhibitors

An extensive structure-activity relationship study of the 3-position of a series of tricyclic pyrazole-based Chk1 inhibitors is described. As a result, 4'-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-benzonitriles (4) and 4'-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-pyridine-2'-carbonitriles (29) emerged as new lead series. Compared with the original lead compound 2, these new leads fully retain the biological activity in both enzymatic inhibition and cell-based assays. More importantly, the new leads 4 and 29 exhibit favorable physicochemical properties such as lower molecular weight, lower Clog P, and the absence of a hydroxyl group. Furthermore, structure-activity relationship studies were performed at the 6- and 7-positions of 4, which led to the identification of ideal Chk1 inhibitors 49, 50, 51, and 55. These compounds not only potently inhibit Chk1 in an enzymatic assay but also significantly potentiate the cytotoxicity of DNA-damaging agents in cell-based assays while they show little single agent activity. A cell cycle analysis by FACS confirmed that these Chk1 inhibitors efficiently abrogate the G2/M and S checkpoints induced by DNA-damaging agent. The current work paved the way to the identification of several potent Chk1 inhibitors with good pharmacokinetics that are suitable for in vivo study with oral dosing.     

Synthesis and activity of new aryl- and heteroaryl-substituted 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole inhibitors of the transforming growth factor-beta type I receptor kinase domain

We have expanded our previously reported series of pyrazole-based inhibitors of the TGF-beta type I receptor kinase domain (TbetaR-I) to now include new 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole analogues. Limited examination of the SAR of this new series in both enzyme and cell based in vitro assays has revealed selectivity differences with respect to p38 MAP kinase (p38 MAPK) depending on the nature of the 'warhead' group on the dihydropyrrolopyrazole ring. As with our original pyrazole series, phenyl substituents tended to show greater selectivity against p38 MAPK than those comprised of the quinoline-4-yl moiety. We have also achieved co-crystallization and X-ray analysis of compounds 3 and 15, two potent examples of this new series, with the TbetaR-I receptor kinase domain.     

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.