Factor XIa Inhibitors as New Anticoagulants - Journal of Medicinal

Perspective Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX

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Factor XIa Inhibitors as New Anticoagulants Mimi L. Quan,* Donald J. P. Pinto, Joanne M. Smallheer, William R. Ewing, Karen A. Rossi, Joseph M. Luettgen, Dietmar A. Seiffert, and Ruth R. Wexler Research and Development, Bristol-Myers Squibb Company, P.O. Box 5400, Princeton, New Jersey 08543, United States

ABSTRACT: With the introduction of thrombin and factor Xa inhibitors to the oral anticoagulant market, significant improvements in both efficacy and safety have been achieved. Early clinical and preclinical data suggest that inhibitors of factor XIa can provide a still safer alternative, with expanded efficacy for arterial indications. This Perspective provides an overview of target rationale and details of the discovery and development of inhibitors of factor XIa as next generation antithrombotic agents. patients with atrial fibrillation.4 Thus, there remains a significant unmet medical need for effective anticoagulant approaches with little or no impact on hemostasis. Blood coagulation enzymes are serine proteases. Coagulation is initiated when tissue factor (TF) at sites of vascular injury becomes exposed to factor VIIa (FVIIa) present in the circulation. The TF/FVIIa complex activates factor X (FX) in the common pathway to FXa, which subsequently generates thrombin.5 Thrombin has numerous functions, including inducing fibrin formation, platelet activation, and factor XI (FXI) activation. Other activators of FXI are factor XIa (FXIa) (autoactivation) and factor XIIa (FXIIa). Factor XII (FXII) activation can occur following autoactivation of FXII to FXIIa upon contact with collagen (plaque rupture) or with platelet polyphosphates released from platelet storage granules upon activation.6−8 Other activators of FXII include DNA and histones, with neutrophils as a potential source. FXI is centrally located at the interface of the contact activation/intrinsic pathway and the final common pathway and receives activation signals from both (Figure 1). If FXI is involved in pathological thrombosis, the prediction is that increased levels of FXI, resulting in increased FXIa activity, will be associated with increased risk for thrombotic events. A second prediction is that reduced FXI levels will provide protection from thrombosis. These predictions are confirmed by epidemiological studies as increased circulating FXI levels in humans have been associated with increased risk for venous and arterial thrombosis, including stroke.6−8 In

1. INTRODUCTION Cardiovascular disease continues to be the leading cause of death worldwide.1 Thrombus formation in arterial circulation can cause heart attacks or ischemic strokes. In venous circulation, blood clots can cause local pain and swelling. A venous blood clot can break free and travel to the lungs causing a pulmonary embolism (PE) and lead to sudden death. Atrial fibrillation is a condition where the upper chambers of the heart (atria) fail to pump blood effectively to the ventricles due to an irregular heartbeat. Inefficient blood flow can allow a blood clot to form in the atria, and if dislodged, the clot may travel to the brain causing a stroke. Together, ischemic heart disease, ischemic stroke, and venous thromboembolism (VTE) account for a large portion of deaths due to cardiovascular disease.1 The goal of anticoagulation therapy is to prevent and treat thrombotic events including heart attacks, strokes, venous thrombosis and PE, as well as systemic embolization in atrial fibrillation, with minimal effects on hemostasis. For decades, vitamin K antagonists such as warfarin, which prevent posttranslational processing of multiple coagulation factors, were the only oral anticoagulants available.2 However, drug−drug interactions, dietary interactions, and a narrow therapeutic index with respect to bleeding safety combine to make warfarin a difficult drug to manage.2 Novel oral anticoagulants (NOACs) directly targeting either thrombin or factor Xa (FXa) emerged over the past decade as alternatives to warfarin and have been approved for both venous and arterial indications.3 Marketed NOACs such as dabigatran, apixaban, betrixaban, edoxaban, and rivaroxaban are highly effective anticoagulant medications.3 However, the risk of bleeding is not completely eliminated and can be as high as 2−3% per year in © XXXX American Chemical Society

Received: February 2, 2018

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DOI: 10.1021/acs.jmedchem.8b00173 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Spontaneous bleeding is rare in homozygous hemophilia C subjects. Factor XI deficiency is often first diagnosed when a prolonged activated partial thromboplastin time (aPTT) is found serendipitously. When bleeding is observed in hemophilia C patients, it is often associated with trauma or surgery and found in tissues with high fibrinolytic activity, such as the oropharynx and urogenital tract.6−8 Thus, the bleeding risk in hemophilia C is mild compared to classic hemophilias [factors VIII (FVIII) and factor IX (FIX) deficiency]. In contrast, complete deficiencies of prothrombin and FX have not been reported in humans and are presumed to be lethal based on mouse knockout data.10 Currently available data support the notion that inhibition of FXI/FXIa is a means to achieve effective anticoagulation in humans. Bleeding differentiation from existing anticoagulants is suggested by human and mouse genetics and animal pharmacology studies using selective inhibitors.11 These findings triggered an intensive research effort in the biopharmaceutical industry to identify inhibitors of FXIa for clinical development. FXIa inhibitors from various sources have now been reported, including naturally occurring peptides, antibodies, antisense oligonucleotides (ASOs), RNA and DNA aptamers, and numerous small molecule inhibitors designed using structure-based drug design (SBDD) techniques combined with crystallographic data. This Perspective will review compounds from published journal articles and meeting abstracts describing the discovery and development of the various classes of FXIa inhibitors but does not include a survey of the patent applications covering FXIa inhibitors. A survey of FXIa inhibitors disclosed in patent applications was published in 2016.12 1.1. Molecular Features of the FXIa Active Site. Serine proteases all have a similar secondary structure, but the shape and composition of the substrate binding pockets serve as recognition sites for their specific substrates.13,14 Substrate amino acid nomenclature is Pn, ...P2, P1, P1′, P2′, ...Pn′ where P1−P1′ denotes the cleaved peptide bond. The binding pockets are named in a corresponding manner to the substrate:

Figure 1. Schematic of the coagulation system.

contrast, patients with congenital FXI deficiency (hemophilia C) are protected from ischemic stroke and venous thromboembolism.6−8 The role of FXI in myocardial infarction is less clear.8 The results of the historical hemophilia C cohort studies were recently confirmed using electronic medical records of a large Israeli health system that contain information from over 1000 patients with reduced FXI activity.9 The hazard ratios for cardiovascular events and deep vein thrombosis were significantly reduced. In the same patient group, subjects with lower FXI levels had a higher likelihood of gastrointestinal bleeding. A potential limitation of the hemophilia C cohort studies as well as the medical record study are relatively small patient numbers compared to typical cardiovascular outcome trials and the focus on a single ethnic group (in this case Ashkenazi Jews).

Figure 2. Surface of the FXIa active site is shown in tan with catalytic triad residues Asp102, His57, and Ser195 and S1 residue Asp189 shown in green. Selected residues of the ecotin M84R mutant peptide are shown in orange and highlight the recognition binding pockets (PDB code 1XX9). B



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Figure 3. (a) Surface of FXa bound to apixaban (PDB code 2P16). (b) Surface of thrombin bound to fragment of bivalirudin (PDB code 3VXE). (c) Surface of FXIa bound to 24 (PDB code 4X60). (d) Overlay of bound conformations of FXa (yellow), thrombin (green), and FXIa (purple) ligands from panels a−c to illustrate extension into different binding pockets.

Sn, ...S2, S1, S1′, S2′, ...Sn′.13 To date there are over 70 publicly available FXIa crystal structures. The majority are of the catalytic domain (or light chain); however, four structures were published containing the heavy chain apple domains as well as the light chain catalytic domain. There are four apple domains that form a planar structure that the catalytic domain sits upon, analogous to a cup and saucer arrangement. These apple domains bind to platelets, heparin, high molecular weight kininogen, and many proteins such as thrombin, factor XIIa, and glycoprotein Ibα.15 The composition of the ligands bound to the FXIa light chain crystal structures is quite variable, including peptides, covalent and noncovalent ligands, and also fragment-like molecules. The first crystal structures of FXIa bound to peptide substrates were solved in late 2004.16 Not only did these crystal structures elucidate the structure of FXIa, the bound peptides indicated where small molecule ligands may bind (Figure 2). The secondary structure of FXIa is similar to other trypsin-like serine proteases and is composed of two β-barrels, two helical regions, and several loops. The S1 pocket is the only deep, well-defined pocket in the FXIa active site. The acidic residue Asp189 is situated in the base of the pocket and often serves as an anchor for ligands containing basic P1 substituents via formation of a salt bridge. The S1 pocket is lined by a β-strand and loop that ends with a disulfide bridge between Cys191-Cys219 at the top of the pocket. The oxyanion hole completes the perimeter of the S1 pocket and is formed by the backbone nitrogen atoms of residues Glu193 and Ser195 and stabilizes the negative charge formed during proteolysis. The smaller S2 pocket of FXIa is located near His57 and is capped by the 60s loop residue Tyr58B, while the S3 and S4 pockets are shallow pockets. The S1′ pocket is the region opposite the catalytic triad of Asp102, His57, and Ser195, near another disulfide bridge formed by Cys40-Cys58. The S2′ pocket contains a β-strand that includes residues Arg39, His40, and Leu41, as well as residues Ile151 and Tyr143, where polar interactions are frequently observed.

Typically, crystal structures of ligand/protein complexes of trypsin-like serine proteases show that ligands anchor in the deep S1 pocket and extend out into different pockets depending on the serine protease. In factor Xa, the main interactions take place in the S1 and S4 pockets.17,18 In contrast, thrombin ligands tend to bind in the S1, S2, and S4 pockets.19 Like thrombin and FXa, FXIa crystal structures contain ligands that always bind in the S1 pocket; however, FXIa inhibitors bind in different pockets dependent upon chemotype, extending from the S1 pocket into several combinations of pockets including the S1′, S2, and S2′ pockets.20 The different shapes of the FXa, thrombin, and FXIa binding pockets are illustrated in Figure 3, wherein the proteins are shown in similar orientations. Information from crystal structures was used to design novel chemotypes that can bind in multiple pockets in FXIa leading to improved affinity and selectivity. The FXIa X-ray crystal structures of various ligands will be discussed in more detail below to show how structural information helped to guide the successive design of different chemotypes.

2. SMALL MOLECULE FXIa INHIBITORS Significant progress has been made in the field of small molecule FXIa inhibitors. Many potent and selective FXIa inhibitors have been identified, and moderate oral bioavailability has been achieved. It is anticipated that the remarkable enhancement of FXIa affinity that has been achieved in various chemotypes will enable further optimization of ADME properties to provide the potency and oral bioavailability needed to advance additional small molecule FXIa inhibitors into clinical trials and ultimately into clinical practice. 2.1. Covalent Inhibitors. Incorporation of a serine trap such as a boronic ester or boronic acid,21,22 aldehyde,23 or ketothiazole,24 which have the potential to form a covalent bond with the catalytic Ser195 in the active site, was successfully applied in the early discovery of thrombin and FXa inhibitors. A conserved Ser195 residue is also present in FXIa, and a similar approach was utilized to identify the first C



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Figure 4. 2-Ketothiazole FXIa inhibitors from Daiichi-Sankyo.

Figure 5. Crystal structure of FXIa complexed with inhibitor 4. The red sphere represents a water molecule, and hydrogen bonds are displayed as dashed lines (PDB code 1ZPZ).

active site inhibitors of FXIa. The first reported pharmacologic proof of concept (PoC) studies in animal models with small molecules were conducted with compounds designed to form a covalent interaction with FXIa. An example of this approach is a series of ketothiazolecontaining compounds reported by Lin et al. from DaiichiSankyo.25 Compound 1 (Figure 4) inhibited FXIa with an IC50 of 12 nM and showed >100-fold selectivity over thrombin and >1000-fold over FXa. Further optimization provided the 2-(4bromophenyl)ethylurea derivative 2 with similar FXIa affinity (IC50 = 10 nM). Single digit nanomolar affinity was achieved in this series with the incorporation of tyrosine and asparagine residues, as in analogs 3 and 4. The structure of the αketothiazole 4 bound to FXIa25 (Figure 5) illustrates that 4 is anchored in the S1 pocket and makes a covalent linkage to Ser195. It also extends into the S2, S3, and S4 pockets, yielding improved protein/ligand interactions that result in an increase in potency. The backbone nitrogen atom of the Val residue hydrogen bonds to the backbone carbonyl of Ser214, orienting the isopropyl moiety into the S2 pocket, making van der Waals contact with His57. The backbone carbonyl of the Asn residue makes a hydrogen bond to Gly216, orienting the Asn side chain toward the S3 and S4 pockets and making hydrogen-bond interactions with Glu98 directly and with Glu217 through a water molecule. The urea and Val carbonyls form hydrogen bonds with the side chain of Lys192, orienting the

bromophenyl moiety to stack above the Lys192 side chain to make a hydrophobic interaction with the protein. The Val substituent extends into a region of FXIa that would result in a clash with Trp60D in the S2 pocket of thrombin and Tyr99 in the S4 pocket of FXa, potentially contributing to the good selectivity for FXIa that is observed. Ketothiazole 1 was evaluated in a rat vena cava model of thrombosis and provided antithrombotic efficacy at a total intravenous (iv) dose of 0.25 mg kg−1.25 It was evaluated at 4fold this efficacious dose (1 mg/kg, continuous iv infusion) in a rat mesenteric bleeding model and did not alter the bleeding time. Analogs 3 and 4 exhibited good anticoagulant activity in vitro, as measured by the concentration required for doubling of the activated partial thromboplastin time (aPTT) with an aPTT EC2× of 2.4 and 0.56 μM, respectively. In rats, compound 3 had a short half-life (t1/2 = 45 min) and moderately high clearance (Cl = 32 mL min−1 kg−1) and was shown to be efficacious in the rat venous thrombosis model.25 β-Lactam 5 (BMS-262084, FXIa IC50 = 2.8 nM, Figure 6) was reported by Bristol-Myers Squibb (BMS) scientists as a potent covalent FXIa inhibitor that was used as a tool compound for preclinical PoC studies in rats and rabbits.26,27 Compound 5 was >70-fold more selective for FXIa compared to other relevant serine proteases (i.e., thrombin, FXa, FIXa, FXIIa, TF/VIIa, tissue plasminogen activator, urokinase, and plasmin), with the exception of tryptase (IC50 = 5 nM) and D



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Figure 6. Covalent β-lactam inhibitor from BMS.

trypsin (IC50 = 50 nM). Although no X-ray structure was reported, a covalent interaction between the β-lactam carbonyl and Ser195 was proposed. Antithrombotic efficacy of 5 was observed in the rat vena cava and carotid artery FeCl2-induced thrombosis models. Bleeding time was unchanged in cuticle, mesenteric, or renal bleeding time models compared to vehicle controls.26 Compound 5 was further evaluated in the arteriovenous-shunt thrombosis (A-V Shunt), venous thrombosis (VT), and electrolytic-mediated carotid arterial thrombosis (ECAT) models in rabbits.27 The bleeding time was measured using a cuticle bleeding time (CBT) model. Compound 5 produced dose-dependent antithrombotic effect in rabbits, with ED50 values of 0.4, 0.7, and 1.5 mg kg−1 h−1 iv in the A-V shunt, VT, and ECAT models, respectively. Minimal effects on bleeding time in rabbits were observed at doses up to 10 mg kg−1 h−1. Lazarova et al. at Daiichi-Sankyo reported the functionalized boronates shown in Figure 7 as covalent FXIa inhibitors with

Figure 8. Crystal structure of glycerol boronate ester of compound 7 covalently bound to FXIa. The red sphere represents a water molecule, and hydrogen bonds are displayed as dashed lines (PDB code 1ZLR).

nitrogen of Lys192, as well as van der Waals contact with the side chain of Leu146, are also observed. Clavatadine A (8, Figure 9), a natural product isolated from a marine sponge, was reported by Buchanan et al. as a selective,

Figure 9. Other covalent inhibitors of FXIa.

covalent FXIa inhibitor. 29 A FXIa-bound structure of clavatadine A shows that the P1 guanidine unit interacts with Asp189 in the S1 pocket. The carboxylic acid group of 8 is within H-bonding distance of Arg37D and Lys192 and facilitates a covalent interaction between Ser195 and the carbamate carbonyl of 8. Coumarin derivative 9 (Figure 9, FXIa IC50 = 0.77 μM) was recently reported by Obaidullah and AlHorani.30 The coumarin carbonyl of 9 was shown to interact with Tyr228, placing the furanyl ester within covalent bonding distance of Ser195. 2.2. Allosteric Factor XIa Inhibitors. Al-Horani et al. have published a series of sulfated pentagalloylglucopyranosides as allosteric inhibitors of FXIa.31,32 Allosteric inhibitors do not bind to the FXIa active site, where features are similar across serine proteases, and may achieve selectivity by targeting unique features of FXIa at other sites. Sulfated pentagalloylglucopyranoside 10 (β-SPGG-1, Figure 10) was reported to have a Ki of 521 nM, with 200-fold selectivity over other relevant enzymes.31 A competitive study between 10 and heparin indicated that 10 bound to the heparin binding site on the catalytic domain of FXIa and confirmed it was an allosteric inhibitor.33 Allosteric monosulfated benzofuran dimer and trimer FXIa inhibitors were reported by Argade et al.34 Compound 11 (Figure 10) was the most potent inhibitor with an IC50 of 820 nM and was proposed to bind at the apple 3 domain of FXIa based upon molecular modeling.34 For comparison, heparin also binds to the apple 3 domain of FXIa with Kd ≈ 0.7 nM.35 To date, the allosteric inhibitors of FXIa

Figure 7. Boronic ester FXIa inhibitors from Daiichi-Sankyo.

low micromolar affinity.28 Starting with commercially available (4-(aminomethyl)phenyl)boronic acid, the Daiichi-Sankyo group incorporated a highly basic guanidine moiety and extended the linker to provide a relatively weak phenethyl guanidine inhibitor (6). The potency was further improved by incorporation of the pyridyl ester moiety in 7. These inhibitors are reminiscent of early thrombin inhibitors22 that contain a covalent linkage between the boronic acid and Ser195. An Xray crystal structure of 7 (Figure 8), which crystallized as the glycerol boronic ester analog, confirmed a covalent interaction between the boronate moiety of the (S)-isomer of 7 and Ser195. One oxygen of the boronate ester makes a hydrogen bond to the side chain of His57, and the other oxygen makes interactions with the oxyanion hole. The guanidine P1 substituents make a bidentate interaction with Asp189 at the base of the S1 pocket, as well as a hydrogen bond with the backbone carbonyl of Gly218. The ligands extend above Ser195, making a water-mediated hydrogen bond between the alcohol and the backbone carbonyl of His57. Additional interactions between the ester carbonyl and the backbone E



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Figure 10. Allosteric FXIa inhibitors.

that have been disclosed do not show the level of affinity obtainable with active site inhibitors. However, allosteric inhibition provides an alternative approach to FXIa inhibition, and further research could lead to more potent molecules. 2.3. Acyclic Reversible FXIa Inhibitors. The first PoC study with a reversible small molecule FXIa inhibitor was reported by Quan et al. from BMS.36 Tetrahydroquinoline (THQ) 12 (Figure 11) is a potent FXIa inhibitor with a FXIa

Figure 12. Crystal structure of FXIa complexed with 12. The red sphere represents a water molecule. Hydrogen bonds are displayed as dashed lines (PDB code 4NA7). Figure 11. Reversible FXIa inhibitor used for early PoC studies at BMS.

side chain OH of Ser195, and the phenyl substituent at the 4position on the THQ ring provides the most significant nonpolar interaction with a hydrophobic region above the S1 pocket near the Cys191-Cys219 disulfide. The phenyl ring at the 2-position on the THQ orients its 3- and 5-substituents to the S2 and S2′ pockets, with the acid group at the ortho position of the pendent phenyl binding in the oxyanion hole. A new S2′ interaction shows two key hydrogen bonds between the terminal carboxamide nitrogen and the backbone carbonyls of His40 and Leu41. Small molecule compounds binding in the S2′ pocket had not been previously observed, and efforts to understand these interactions stimulated the discovery of new P2′ substituents in this and subsequent chemotypes. Hangeland et al. described a series of phenylimidazole derivatives with a 4-aminomethyl-trans-cyclohexyl P1 moiety replacing the guanidine and amidine P1 moieties of previously reported inhibitors (Figure 13).38 Phenylimidazole 13 (FXIa Ki = 120 nM) was identified through screening of a targeted set of compounds. A comparison of a model of 13 with the FXIa Xray cocrystal structure of 12 prompted the introduction of the carboxamide P2′ substituent in 14 resulting in a 4-fold improvement in FXIa affinity (FXIa Ki = 30 nM). Addition of a chlorine on the imidazole core further improved FXIa affinity in this series (15, FXIa Ki = 4 nM).

Ki of 0.20 nM and >1000-fold selectivity over thrombin, FXa, FIXa, FVIIa, trypsin, plasmin, tPA, urokinase, and chymotrypsin, 23-fold selectivity over plasma kallikrein, and 365-fold selectivity over activated protein C. Compound 12 had similar potency in prolonging the aPTT in both human and rabbit plasma (EC2× of 2.2 and 2.4 μM, respectively). In the rabbit ECAT model, it produced a dose-dependent antithrombotic effect with an EC50 of ∼300 nM, with a minimal increase in bleeding time (1.3-fold) in the rabbit CBT model at the dose that produced an 80% inhibition of thrombosis.37 The pharmacological profile of 12 prompted BMS researchers to focus on reversible small molecule FXIa inhibitors. The crystal structure of 12 bound to FXIa shows binding interactions in the S1, S2, and S2′ pockets, making several polar interactions with the protein (Figure 12).36 Like arginine P1 substituents, ligands containing amidine P1 groups are shown to be extremely potent due to charge-reinforced hydrogen bonds between the basic amidine and the acidic Asp189 side chain, as well as hydrogen bonds with the Gly218 backbone and to the backbone carbonyl of Val227 via a water molecule. The nitrogen of the THQ makes a weak hydrogen bond to the F



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Figure 13. Basic P1 imidazole core FXIa inhibitors from BMS.

A FXIa X-ray cocrystal structure with phenylimidazole 1438 indicates that this ligand also occupies the S1 and S2′ pockets of FXIa, as shown in Figure 14. However, unlike 12, 14 does

of His40, the inhibitor P2′-carboxamide and the hydroxyl group of Tyr143. It was hypothesized that replacing the P2′ phenylcarboxamide with a bicyclic heterocycle capable of forming a hydrogen bond with the hydroxyl group of Tyr143 would improve affinity. This resulted in aminoindazole 16 (Figure 13), a selective inhibitor of FXIa with subnanomolar affinity and excellent in vitro anticoagulant activity (aPTT EC2× = 1 μM). Compound 16 was evaluated in the rabbit A-V shunt thrombosis model and showed robust antithrombotic efficacy (ID50 = 0.6 mg kg−1 iv bolus followed by 1 mg kg−1 h−1 iv infusion). Other P2′ moieties identified through this effort include 4-hydroxyquinolinone40 and 4-methyl phenylcarbamate.20,41 These groups are all able to hydrogen-bond directly to the backbone carbonyl of His40, form a second interaction with the backbone NH of His40 through a water molecule and a third hydrogen bond either through a water molecule or directly to Tyr143 OH. The indazole and phenylcarbamate each make a fourth hydrogen bond through a conserved water molecule to Ile151 (Figure 15).

Figure 14. Crystal structure of FXIa complexed with 14. The red spheres represent water molecules; hydrogen bonds are displayed as dashed lines. The gray molecule is ethylenediol, which is part of the crystallization conditions and mimics water in the structure (PDB code 4TY6).

not make any interactions in the S2 pocket. The benzyl substituent of 14 binds in the S1′ pocket near the Cys42-Cys58 disulfide bridge. The S2′ interactions of 14 also differ slightly from those of 12 since the ligand is further shifted into the S2′ pocket, positioning the nitrogen of the primary carboxamide to form a hydrogen bond with the backbone carbonyl of His40. There are numerous water molecules in this structure that mediate key hydrogen bonds between the protein and the ligand. The nitrogen of the P2′ carboxamide of 14 participates in a second hydrogen bond through two water molecules to the backbone NH of His40, and the carboxamide carbonyl makes a hydrogen bond through a water molecule to the backbone NH of Ile151 and interacts with the phenol of Tyr143 via another water molecule. This network of hydrogen bonds through water molecules is notable in that most of the key polar interactions to the protein are mediated by water molecules. Understanding the effects that water molecules have on protein/ligand complexes can therefore be important in predicting binding affinity.39 Information from the FXIa-bound structure of 14 was utilized to target additional P2′ modifications, and several different substituents were incorporated to mimic the same interactions with the protein. A key observation from this structure was the coplanar orientation of the backbone carbonyl

Figure 15. Crystal structures of the different P2′ substituents show similar hydrogen-bonding patterns. The ligands, corresponding crystallographic water molecules, and hydrogen bonds are colored as follows: aminoindazole = orange, methyl phenylcarbamate = cyan, hydroxyquinolinone = pink.

Trypsin selectivity and oral bioavailability were the two key issues with the early BMS FXIa inhibitors such as 16. One approach to achieve selectivity over trypsin was to take advantage of the size difference in the S1 pocket. Like FXa and thrombin, the FXIa S1 pocket contains Ala190 flanking the Asp residue at the base of the pocket instead of Ser190 as in trypsin. Larger P1 substituents can bind in the S1 pocket of FXIa but not in trypsin due to a steric clash with Ser190. This approach is exemplified by 17 (Figure 16),20 wherein the aminomethylcyclohexyl P1 was replaced with the 1-amino5,6,7,8-tetrahydroisoquinolinyl group. FXIa affinity was mainG



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less basic and neutral replacements for the basic P1 group of 16 (Figure 16). 20 Although 1-amino-5,6,7,8-tetrahydroisoquinolinyl 17 had a lower pKa (calculated pKa 7.8 for 17 vs 10.2 for 16), oral bioavailability was not improved. Compounds with a neutral P1, such as the trans-4methylcyclohexyl (18) and 2,6-difluoro-4-methoxyphenyl (19) analogs, showed moderate oral bioavailability in dogs (26% and 49%, respectively) but lost some FXIa affinity relative to 16 and 17. The basic P1 of 16 was also replaced with a urealinked 3-chloro-2,6-difluorophenyl P1 to afford 20. The FXIa affinity was further improved in this series by incorporation of the benzylamine-containing P1 moiety in 21 (Ki = 5.8 nM). Subsequent replacement of the benzylamine substituent with a 1-tetrazolyl group provided 22 which retained FXIa affinity (Ki = 1.5 nM) and potency in the aPTT assay (EC1.5× = 1.8 μM). Replacement of the urea with an acrylamide linker provided 23. Substitution of the indazole P2′ substituent of 23 with the methyl phenylcarbamate P2′ group resulted in 24, which maintained similar FXIa affinity (Figure 16). Despite all of the changes that led to 24, membrane permeability remained an issue, and 24 had low oral exposure in dogs (F ≈ 3%). The crystal structure of 2220 (Figure 17) shows a typical chlorophenyltetrazole-serine protease binding mode,42 where the chlorine displaces an unstable, yet conserved water molecule above Tyr228 at the base of the S1 pocket. The tetrazole heterocycle increases potency by making hydrogen bonds to the backbone NH of Lys192 and through water molecules to the backbone carbonyl of Gly216. The polarized C−H interacts directly with the same carbonyl, and the ring makes van der Waals contact with Cys219. The linkers, however, bind in rarely observed and quite unexpected conformations. The bound crystallographic conformation of the urea of compound 22 is in a cis/trans orientation, whereas other known crystal structures of acyclic aliphatic substituted ureas strongly favor a trans/trans orientation.43 The flip in conformation of the urea allows its carbonyl to make a significantly better interaction with the oxyanion hole than the

Figure 16. Evolution of potent neutral P1 imidazole based FXIa inhibitors at BMS.

tained for 17 (FXIa Ki = 2.6 nM), and trypsin selectivity was improved to >1000-fold (trypsin Ki = 2800 nM vs 23 nM for 16). In an attempt to improve oral bioavailability in the phenylimidazole series, Pinto and Smallheer et al. explored

Figure 17. Crystal structure of FXIa complexed with 22 shows the cis/trans orientation of urea. The red spheres represent water molecules; hydrogen bonds are displayed as dashed lines. The gray molecule is ethylenediol which is part of the crystallization conditions and mimics water molecules in the structure (PDB code 4X6N). H



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Figure 18. Pyridine, pyrimidine, and pyridone core compounds from BMS.

Figure 19. FXIa inhibitors with polar P1′ and P2′ groups from BMS.

trans/trans conformation. Additionally, the P1 urea NH makes a hydrogen bond to the backbone carbonyl of Cys191. An Xray crystal structure of 24 bound to FXIa shows the acrylamide linker in a similar binding mode to the urea linker of 22. The methyl carbamate moiety of 24 interacts with the S2′ enzyme residues His40 and Ile151, and with Tyr143, either directly or through a water network. To address the issue of low oral bioavailability, Corte et al. explored imidazole core replacements, such as the pyridyl-, pyrimidinyl-, phenyl-, and 2-hydroxypyridine-containing compounds 25−27 shown in Figure 18.44 These compounds mostly maintained FXIa affinity, and in combination with P2′ modifications, moderate oral bioavailability was achieved.41 Incorporation of a methylcarbamate P2′ led to compounds 28− 30 with single digit nanomolar FXIa affinity. Compound 28 (aPTT EC1.5× = 2.6 μM) showed high clearance (Cl = 54 mL min−1 kg−1) and a high volume of distribution (Vdss = 10 L kg−1) but surprisingly good oral bioavailability (F = 63%) in dogs. The corresponding pyrimidinyl compound 30 (aPTT EC1.5× = 1.2 μM) also showed high oral bioavailability in dogs (F = 66%). These were the first reported compounds containing the P1 trans-aminomethylcyclohexyl amide that

showed good oral exposure. Further optimization with the pyridine, pyrimidine, and pyridone cores by incorporating the chlorophenyltetrazole acrylamide P1 resulted in 31−34. These compounds maintain modest FXIa activity (Figure 18). Compound 31 was also orally bioavailable (F = 53%) in dogs at a dose of 1 mg kg−1.41 A parallel effort at BMS targeted compounds that could be used as parenteral agents in an acute setting. One approach taken was the replacement of the benzyl P1′ group in 24 with aspartate-derived amides, as described by Hu et al.40 This approach proved successful and led to the potent FXIa inhibitors 35 and 36 which incorporate N-methylmorpholino and N-methylpiperazino amide P1′ moieties (Figure 19). Hydrophilic P2′ groups such as 4-hydroxyquinolinone were also explored in combination with the chlorophenyltetrazole P1 and resulted in a significant boost in FXIa affinity for 37 (FXIa Ki of 0.18 nM, Figure 19).40 Incorporation of this P2′ group in the aspartate series provided compound 38, which demonstrated picomolar affinity for FXIa (Ki = 0.04 nM), excellent anticoagulant activity with an aPTT EC2× value of 1.0 μM, and solubility suitable for parenteral administration. In the rabbit ECAT model, 38 provided dose-dependent efficacy with an I



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Figure 20. Crystal structure of FXIa complexed with 35. The red spheres represent water molecules; hydrogen bonds are displayed as dashed lines (PDB code 4Y8Y).

Figure 21. 6-Chloroquinolinone P1 compounds from AstraZeneca.

EC50 of 0.53 μM. A minimal increase in bleeding time was observed in the rabbit CBT model at exposures more than 10 times the EC50. The FXIa-bound X-ray crystal structure of 35 (Figure 20) shows a change in orientation of the morpholine amide compared to the phenylalanine P1′ of 22 (Figure 17). The aspartate carbonyl of 35 makes a previously unseen hydrogen bond through a water molecule in the S1′ pocket to the backbone carbonyl of Cys42. The position of the water molecule in this structure is located where the phenylalanine of 22 binds. The morpholine ring does not extend into the S1′ pocket but instead orients toward the S2 pocket near His57 and Try58B. The projection of the morpholine ring is similar to that seen with THQ compound 12, where the isobutyl group of 12 extends toward the S2 pocket. Fjellström et al.45 at AstraZeneca used a fragment-based approach to identify a 6-chloroquinolin-2(1H)-one fragment 39 with weak FXIa affinity (FXIa IC50 = 140 μM) and phenylalanine fragment 40 (FXIa KD = 1400 μM). One of the goals of fragment-based drug design is to identify a fragment and either optimize it via small, iterative modifications or splice it into other known chemotypes, ideally without changing the binding pose of the fragment once it is elaborated. Grafting the two fragments together afforded 41 (Figure 21) with a significant boost in FXIa affinity (FXIa IC50 = 1.0 nM), thus confirming that the latter goal was achieved. Figure 22 shows the overlay of crystal structures of fragment 39 and the

Figure 22. Overlay of crystal structures of 39 (burgundy) and 41 (tan) shows that the fragment and the full molecule bind nearly identically (PDB codes 4CR5, 4CRF).

elaborated diamide 41; the fragment and the full ligand bind almost identically. The binding mode of the 6-chlorophenyl portion of the molecule is very similar to other known chlorophenyl P1 substituents. However, the nitrogen atom of the quinolone makes a hydrogen bond to the backbone NH of Gly218, as is often observed in more polar systems such as J



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Figure 23. Substituted phenylalanine FXIa inhibitors from BMS and Bayer.

Figure 24. Overlay of crystal structures of imidazole compound 22 (magenta) and diamide compound 42 (blue) show that the chemotypes have similar S1 and S1′ interactions; however, the pendent phenyl ring in the S2′ subsite rotates, resulting in divergent SAR (PDB codes 4X6N, 5E2O).

position of the P1′ benzyl group provided a significant improvement in both binding and anticoagulant activity, as exemplified by urea 43 (FXIa Ki = 2.0 nM, aPTT EC1.5× = 0.5 μM). These compounds also showed good efficacy in rabbit models of thrombosis. An extended P1′ phenylalanine diamide 44, which incorporates a 4-aminomethylcyclohexyl P1′ group and a novel triazolyltetrafluoropropanoic acid P2′ group, was reported by Finkelmann et al. at Bayer.47 However, FXIa inhibitory data were not reported in this reference. Alternative diamide scaffolds were designed by Pinto et al. at BMS, wherein the amide nitrogen atom in the P1 linker was cyclized with the P1′ substituent. This resulted in the discovery of the tetrahydroisoquinoline (THIQ) scaffold shown in 45 and

benzamidine P1 substituents. The carbonyl of the fragment structure makes a hydrogen bond through a water molecule to the backbone NH of Gly216. The quinolone ring is within van der Waals contact with the disulfide Cys191-Cys219 above the S1 pocket. Phenylalanine-derived diamides were also explored by Smith et al.46 at BMS in combination with a p-aminobenzoic acid P2′, as shown in 42 (Figure 23). The overlay of the X-ray structures of imidazole 22 and diamide 42 (Figure 24) indicates that the binding modes in the S1 and S1′ pockets are almost identical. The second amide overlays well with the imidazole but the P2′ phenyl ring is oriented in a different way, resulting in divergent SAR between the two series. Additional substitution at the 4K



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Figure 25. THIQ FXIa inhibitors from BMS.

Figure 26. Overlay of crystal structures 42 (dark blue) and 45 (tan) shows that the diamide and THIQ have very similar binding poses (PDB codes 5E2O, 5QCL).

forms an edge-to-face interaction with His57.48 This finding prompted an exploration of the SAR at the 5-position of the THIQ phenyl ring, and a wide variety of substituents were tolerated at this position. For example, compounds 47 and 48 both showed the same FXIa affinity (Ki = 6 nM, Figure 25).

46 and provided good FXIa binding affinity (Figure 25).48 An X-ray cocrystal structure of 45 bound to FXIa shows a similar binding mode to the linear diamide 42 (Figure 26). Additionally, it indicates the phenyl ring of the THIQ is in close proximity to the P1′ Cys42 - Cys58 disulfide bond and L



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position of the indole P1 with a five-carbon alkyl linker led to macrocycle 52, a weak inhibitor of FXIa with a Kic of 88 μM, where Kic is the Ki assuming competitive inhibition with the substrate. However, no further work has been reported with this chemotype. Building on their success in identifying potent acyclic FXIa inhibitors, BMS scientists sought to preorganize the bioactive conformation of 24 via a macrocyclization strategy. On the basis of FXIa enzyme/ligand crystal structures, the observation was made that the P1′ phenyl ring and the phenyl ring containing the P2′ carbamate of linear inhibitors such as 24 were in close enough proximity to form a macrocycle. Two macrocyclization strategies were pursued, as shown in Figure 28.54,55 One strategy (approach A) reported by Corte et al.54 involved removing the P1′ phenyl ring and connecting P1′ to the ortho-position of the P2′ phenyl ring via a 6−7 atom linker, resulting in 12- and 13-membered macrocycles. Alternatively, as described by Wang et al.,55 the P1′ phenyl ring was retained, and the P1′ and P2′ phenyl rings were joined by a linker L (approach B). In the survey of 12-membered macrocycles, the alkyl-linked compound 53 had moderate affinity for FXIa (Ki = 87 nM, Figure 29).54 Similar to the acyclic series, the affinity of 53 was improved by the addition of a chloro substituent at the C5position of the imidazole, as shown in 54 (FXIa Ki = 42 nM). The alkyl-linked macrocycle was about 6-fold more potent than the corresponding ether-linked analog 55. In the 13-membered macrocycle series, the alkyl-linked 56 was much less potent, whereas chloroimidazole analog 57 and the ether-linked analog 58 regained ∼10-fold improvement in FXIa affinity.54 In general, it was observed that FXIa affinity enhancements for chloroimidazole macrocycles ranged from 2- to 10-fold, dependent on the specific ring system. Compound 53 was found to have modest oral bioavailability in dogs (F = 12%), with low clearance and a low volume of distribution (Cl = 2.6 mL min−1 kg−1; Vdss = 0.7 L kg−1). To further enhance the affinity of these first-generation macrocycles, modifications were sought to capture a hydrogenbonding interaction with FXIa from the macrocycle linker. Xray cocrystal structures of macrocyclic inhibitors bound to FXIa suggested that the methylene of the macrocycle adjacent to the P2′ phenyl ring was proximal to the S2′ residue Leu41. To explore this possibility, an amide bond was introduced into the

Further optimization led to the 5-piperazinyl example 49, with a significant enhancement in both FXIa affinity and anticoagulant activity (aPTT EC1.5× = 0.56 μM). Ultimately, compound 50 (BMS-962212) was identified with excellent FXIa binding affinity (Ki = 0.71 nM), anticoagulant activity (aPTT EC1.5× = 0.30 μM), and selectivity over a broad panel of serine proteases (>3000-fold over FVIIa, FIXa, FXa, FXIIa, thrombin, trypsin, activated protein C, plasmin, TPA, urokinase, and chymotrypsin).48 Compound 50 met the solubility criteria for a parenteral agent at physiological pH. It was highly efficacious in the rabbit A-V shunt model of thrombosis, producing 83% inhibition of thrombus formation at a dose of 0.23 mg kg−1 iv bolus + 1.44 mg kg−1 iv infusion. In the rabbit cuticle bleeding model, no increase in bleeding time was observed at 2 times the A-V shunt dose. Bleeding times in combination with aspirin were not prolonged compared to aspirin alone. Compound 50 satisfied preclinical safety assessments and was advanced into phase I clinical trials as a first-in-class, reversible, parenteral FXIa inhibitor.49 2.4. Macrocyclic Factor XIa Inhibitors. Macrocycles have been recognized as an important structural class in drug discovery and have been shown to improve one or more of binding affinity, selectivity, metabolic stability, and pharmacokinetic properties compared to the corresponding acyclic precursors.50−52 The first disclosed macrocyclic inhibitors of FXIa were reported by Hanessian et al. (Figure 27).53 Docking

Figure 27. First reported macrocyclic inhibitor of FXIa.

experiments with linear diamide 51 in the FXIa active site revealed that there was a relatively short distance between the methylene of the sulfonamide and one of the carbon atoms of the phenylamidine. Replacing the benzamidine P1 group with 5-chloroindole and connecting the sulfonamide to the 2-

Figure 28. Macrocyclization approaches pursued at BMS. M



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Figure 29. Evolution of macrocyclic FXIa inhibitors at BMS.

Figure 30. Cocrystal structure of FXIa complexed with 59. The red spheres represent water molecules, and hydrogen bonds are displayed as dashed lines. The gray molecules are ethylenediol, which is part of the crystallization conditions and mimics water molecules in the structure (PDB code 5TKT).

macrocycles, the (R)-α-Me substituent showed FXIa affinity improvements and maintained good anticoagulant potency in compounds 62 (FXIa Ki = 0.39 nM, aPTT EC1.5× = 0.39 μM) and 63 (FXIa Ki = 0.07 nM, aPTT EC1.5× = 1.2 μM), whereas β-methyl substitution resulted in a 30-fold loss in potency. Ethyl and isopropyl groups were also accommodated in the αposition as illustrated by ethyl compound 64 (FXIa Ki = 0.18 nM, aPTT EC1.5× = 0.52 μM). In the case of the 13-membered ring macrocycles, only the (S)-β-Me compounds, 65 (FXIa Ki = 0.69 nM) and 66 (FXIa Ki = 0.06 nM, aPTT EC1.5× = 0.57 μM), showed improved FXIa affinity over the parent 59, while the corresponding (R)-β-Me analog was >20-fold less active. The cocrystal structures of 12-membered, α-ethyl substituted macrocycle 64 and 13-membered β-methyl substituted macrocycle 68 demonstrate that appropriately placed substituents are able to access this hydrophobic region despite the slightly different conformation of the two ring systems (Figure 32). Improvements in oral bioavailability in this series were achieved by replacing the chlorophenyltetrazole P1 element

linker of the 13-membered macrocycle, resulting in a dramatic improvement in affinity for compound 59 (FXIa Ki = 3.2 nM), compared to the all carbon-linked analog 56. The cocrystal structure of 59 shown in Figure 30 confirms the rationale for functionalization of the macrocycle ring, with a hydrogen bond observed between the macrocycle amide NH and the backbone carbonyl of Leu41. An unsaturated amide linker that incorporated an E-alkene into the 13-membered macrocycle provided subnanomolar FXIa affinity (60, Ki = 0.16 nM), which was further enhanced with chloroimidazole 61 (Ki = 0.03 nM). Both compounds had excellent aPTT potency (EC1.5× = 0.27 and 0.28 μM, respectively). Modeling studies based on the X-ray cocrystal structure of 59 carried out by Corte and Yang et al.56 identified a hydrophobic region in the S1′ pocket near the Cys42-Cys58 disulfide, suggesting that substitution on the macrocycle ring could provide improved potency. The S1′ pocket was targeted with small alkyl substitutions on the linker at positions α, β, or γ to the amide carbonyl (Figure 31). In the 12-membered ring N



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Figure 31. Optimization of BMS macrocycle linkers to improve oral bioavailability.

Figure 32. Overlay of crystal structures of 12-membered macrocycle 64 (cyan) and 13-membered macrocycle 68 (yellow) shows the alkyl substituents of the different ring sizes are similarly placed to interact with the disulfide (PDB codes 5Q0G, 5Q0H).

with the more lipophilic 2,6-difluoro-4-methylbenzamide. This modification resulted in 67 and 68, both of which demonstrate moderate FXIa affinity (Figure 31). Compound 61 with the chlorophenyltetrazole P1 was a highly potent FXIa inhibitor but had no measurable rat oral bioavailability. This could be partially due to the high calculated polar surface area (PSA) value of 169 Å2 for 61. To test this theory, 67 (FXIa Ki = 16 nM; aPTT EC1.5× = 6.6 μM) and 68 (FXIa Ki = 12 nM; aPTT EC1.5× = 2.7 μM), with lower PSA values of 125 Å2, were made. These compounds showed improved, but still modest, oral

exposure in rats (F of 16% and 41%, respectively) but had much reduced FXIa affinity and aPTT potency compared to 61. In the case of compounds reported by Wang et al.55 that retain the P1′ phenyl substituent in the macrocyclic ring, the meta-linked, 14-membered macrocycle 69 (Figure 33) was found to have the best FXIa affinity (FXIa Ki = 17 nM). Incorporation of an amide bond into the linker and optimization via a substituent at the para position of the P1′ phenyl provided compounds 70 and 71, both of which exhibited improved FXIa affinity. Replacement of the P1′ phenyl with a 2-pyridyl ring led to the discovery of 72, which O



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when administered iv to rabbits, produced an antithrombotic effect comparable to that of the thrombin inhibitor, dabigatran, or the FXa inhibitor, rivaroxaban, but with a much lower bleeding risk.

3. NONSMALL MOLECULE APPROACHES 3.1. Factor XIa Inhibitors from Natural Sources. The pursuit of inhibitors of the coagulation cascade from leeches, ticks, snakes, bats, etc. has been documented and has led to fundamental insights into coagulation cascade pharmacology.63 Interestingly, relatively few selective inhibitors of FXIa have been identified, while several inhibitors with multiple serine protease activities have been found.64 Recombinant AduNAP4, an anticoagulant peptide isolated from the human hookworm, Ancylostoma duodenale, was found to inhibit both FXIa (Ki = 42 nM) and FXa (Ki = 7.3 nM).65 This 104-amino acid protein was the first FXIa inhibitor isolated from blood-feeding animals. Hookworms reside in the small intestine of mammalian hosts where they feed on blood. These nematodes have developed systems for overcoming the host’s hemostatic and thrombotic responses to bleeding that are referred to as nematode anticoagulant protein/peptides (NAPs). AduNAP4 was found to significantly prolong both aPTT (EC2× = 23 nM) and PT (EC2× = 155 nM) consistent with a dual FXIa/FXa mechanism. Desmolaris is an anticoagulant protein isolated from the salivary gland of the vampire bat (Desmodus rotundus).66 Structurally, it is related to tissue factor pathway inhibitor and has a mass of 21.5 kDa. Desmolaris binds to FXIa with a Kd of 0.6 nM in a slow, tight-binding manner. Demolaris inhibits peptide hydrolysis by FXIa, FXa, and plasma kallikrein in a noncompetitive manner, with Ki values of 12 nM, 18 nM, and 115 nM, respectively. Intravenous administration of desmolaris (0.1 mg kg−1) inhibited FeCl3-induced carotid arterial thrombus formation in a mouse model but did not impair hemostasis. Fasxiator is a FXIa inhibitor isolated from the venom of the banded krait snake (Bungarus fasciatus) that has a molecular mass of 7 kDa.67 The potency and selectivity of recombinant fasxiator were improved by mutagenesis to achieve a slowbinding type of competitive inhibitor with a FXIa Ki of 0.86 nM. Recombinant fasxiatorN17R,L19E doubled the aPTT at 300 nM but had no effect on PT up to 40 μM in human plasma. It also prolonged aPTT in mouse plasma but was ∼10-fold weaker. In a FeCl3-induced mouse carotid artery thrombosis model, rfasxiatorN17R,L19E at a dose of 0.3 mg/mouse prevented occlusion for at least 30 min. In comparison, six of seven control animals showed occlusion within 10 min, while the seventh mouse occluded at 30 min. 3.2. RNA and DNA Aptamers. RNA and DNA aptamers are short, single-stranded oligonucleotides (6−30 kDa) that bind specific protein targets with high selectivity.68 RNA aptamers targeting FXIa were identified using systematic evolution of ligands by exponential enrichment (SELEX).69 Aptamers were selected on the basis of inhibition of FXIamediated peptide substrate cleavage and FXIa-mediated activation of FIX. The selected aptamers, which bind either to both FXI and FXIa or only to FXIa, lack specific binding to a panel of other coagulation enzymes, including plasma kallikrein. These aptamers appear to interact with the positively charged anion-binding sites present in FXIa. Aptamer 12.7 increased the human plasma aPTT 2-fold at a concentration of 4.2 μM.69 A DNA aptamer, FELIAP, which binds with high affinity to FXIa

Figure 33. Potent macrocycles incorporating a P1′ phenyl moiety.

achieved picomolar FXIa binding affinity (FXIa Ki = 0.02 nM) and potent in vitro anticoagulant activity (aPTT EC1.5× = 0.27 μM). However, the oral bioavailability of this compound was not reported. It is anticipated that the enhanced FXIa affinity of these macrocycles could enable further optimization to provide orally bioavailable inhibitors. In a benchmarking study conducted at Merck,57 Wang et al. evaluated macrocycle 73, previously disclosed by BMS58 (Figure 34), in a rabbit FeCl3-induced injury model. In this

Figure 34. Macrocyclic FXIa inhibitor studied in vivo at Merck.

study, when given iv, 73 dose-dependently inhibited thrombus formation with ED50 = 0.003 mg kg−1 h−1. In addition, 73 inhibited cerebral microembolic signals (MES), a predictor of stroke or transient ischemic attack (TIA), with ED50 = 0.106 mg kg−1 h−1. 2.5. Compounds without Disclosed Structures. Data, but no structures, have been published for several additional FXIa inhibitors. Sakai et al. from Ono Pharmaceuticals have disclosed data for ONO-8610539,59 a selective, injectable small molecule FXIa inhibitor (Ki = 0.9 nM). ONO-8610539 was reported to inhibit thrombus formation in rabbits and monkeys, without affecting blood loss volume at a dose 33% above the maximum antithrombotic effect. Another compound described by Koyama et al., ONO-7750512,60 was reported as a potent FXIa inhibitor (Ki = 3.8 nM) with 22% oral bioavailability in rats. ONO-7750512 significantly inhibited thrombus formation at a dose of 4.0 mg kg−1 h−1 in a deep vein thrombosis model, and no increased blood loss was observed in a rabbit model of ear bleeding. Data for a second orally bioavailable small molecule, ONO-5450598, were presented by Kouyama et al.61 This compound has a FXIa Ki = 2 nM with aPTT EC2x < 1 μM, and demonstrated high oral bioavailability in multiple species (F = 81%, 88%, and 59% in monkeys, dogs, and rats, respectively). In a monkey A-V shunt model, it inhibited thrombus formation at iv doses above 0.097 mg kg−1 h−1 and did not prolong bleeding time in a monkey nail-cut model at an oral dose of 30 mg kg−1. DSR-130787,62 described by Mori et al. of Sumitomo Dainippon Pharma, is an orally active FXIa inhibitor prodrug with an IC50 value of

Factor XIa Inhibitors as New Anticoagulants - Journal of Medicinal

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