Design and synthesis of isoxazoline derivatives as factor Xa inhibitors. 2.

Intravascular clot formation is an important factor in a number of cardiovascular diseases. Therefore, the prevention of blood coagulation has become a major target for new therapeutic agents. One attractive approach is the inhibition of factor Xa (FXa), the enzyme directly responsible for thrombin activation. Herein we report a series of isoxazoline derivatives which are potent FXa inhibitors. Optimization of the side chain at the quaternary position of the isoxazoline ring led to SK549 which showed subnanomolar FXa potency (K(i) 0.52 nM). SK549 shows good selectivity for FXa compared to thrombin and trypsin, potent antithrombotic effect in the rabbit arterio-venous thrombosis model, and improved pharmacokinetics relative to other compounds evaluated from this series.     

Non-hemostatic activity of coagulation factor Xa: potential implications for various diseases.

Because of its unique position at the convergence point of the intrinsic (contact) and extrinsic (tissue factor/factor VIIa) pathways in the coagulation system, coagulation factor Xa (FXa) has been a theoretically interesting therapeutic target for antithrombotic drugs for many years. More recently, the discovery of naturally occurring FXa inhibitors, such as tick anticoagulant peptide and antistasin, has helped substantiate FXa as a desirable target by demonstrating the efficacy and potential safety advantages of FXa inhibition over conventional antithrombotic therapy. These discoveries led to the design and development of many small-molecule inhibitors of FXa, which have provided potent and selective tools for evaluating the potential role of FXa in various diseases. In addition, these advances have been instrumental in defining the biology of FXa and have aided in the discovery of specific receptors and intracellular signaling pathways for FXa that may be important in the progression of, or the response to, various diseases.     

Synthesis, characterization, and structure-activity relationships of amidine-substituted (bis)benzylidene-cycloketone olefin isomers as potent and selective factor Xa inhibitors

Factor Xa (FXa) is a trypsin-like serine protease that plays a key role in blood coagulation linking the intrinsic and extrinsic pathways to the final common pathway of the coagulation cascade. During our initial studies, we observed facile photochemical conversion of the known FXa/tPA inhibitor, BABCH ?(E,E)-2, 7-bis(4-amidinobenzylidene)cycloheptan-1-one, 1a, to the corresponding (Z,Z) olefin isomer, 1c (FXa K(i) = 0.66 nM), which was over 25,000 times more potent than the corresponding (E,E) isomer (1a, FXa K(i) = 17 000 nM). In order to determine the scope of this observation, we expanded on our initial investigation through the preparation of the olefin isomers in a homologous series of cycloalkanone rings, 4-substituted cyclohexanone analogues, and modified amidine derivatives. In most cases the order of potency of the olefin isomers was (Z,Z) > (E,Z) > (E,E) with the cycloheptanone analogue (1c) showing the most potent factor Xa inhibitory activity. In addition, we found that selectivity versus thrombin (FIIa) can be dramatically improved by the addition of a carboxylic acid group to the cycloalkanone ring as seen with 8c (FXa K(i) = 6.9 nM, FIIa K(i) > 50,000 nM). Compounds with one or both of the amidine groups substituted with N-alkyl substituents or replaced with amide groups led to a significant loss of activity. In this report we have demonstrated the importance of the two amidine groups, the cycloheptanone ring, and the (Z,Z) olefin configuration for maximum inhibition of FXa within the BABCH template. The results from this study provided the foundation for the discovery of potent, selective, and orally active FXa inhibitors.     

Crystal structures of human factor Xa complexed with potent inhibitors

Involved in the coagulation cascade, factor Xa (FXa) is a serine protease which has received great interest as a potential target for the development of new antithrombotics. Although there is a great wealth of structural data on thrombin complexes, few structures of ligand/FXa complexes have been reported, presumably because of the difficulty in growing crystals. Reproducible crystallization conditions for human des-Gla1-45 coagulation FXa have been found. This has led to an improvement in the diffraction quality of the crystals (about 2.1 A) when compared to the previously reported forms (2.3-2.8 A) thus providing a suitable platform for a structure-based drug design approach. A series of crystal structures of noncovalent inhibitors complexed with FXa have been determined, three of which are presented herein. These include compounds containing the benzamidine moiety and surrogates of the basic group. The benzamidine-containing compound binds in a canonical fashion typical of synthetic serine protease inhibitors. On the contrary, molecules that contain surrogates of the benzamidine group do not make direct hydrogen-bonding interactions with the carboxylate of Asp189 at the bottom of the S1 pocket. The structural data provide a likely explanation for the specificity of these inhibitors and a great aid in the design of bioavailable potent FXa inhibitors.     

Recent advances in serine protease inhibitors as anticoagulant agents

The drawbacks and limitations of existing anticoagulant therapy which may result in serious adverse effects and a high mortality rate, have given rise to many anticoagulant development programmes in the last decade, focusing mainly at development of thrombin and FXa low-molecular weight inhibitors. A detailed understanding of blood coagulation pathways, functioning of the serine proteases thrombin, FXa, FVIIa and FIXa and elucidation of their crystal structures resulted in many potent compounds, among which some have entered the clinical phase or have been approved for use in clinical practice. Recently, the focus of anticoagulant research turned to inhibition of the TF:FVIIa complex, with some promising clinical candidates on the horizon. This article provides an overview of the current development status of serine protease inhibitors as anticoagulants, including new trends such as dual coagulation factor inhibitors.     

Combined chemical feature-based assessment and Bayesian model studies to identify potential inhibitors for Factor Xa

In our study, we have described chemical feature-based 3D QSAR pharmacophore models with help of known inhibitors of Factor Xa (FXa). The best model, Hypo1, has validated by various techniques to prove its robustness and statistical significance. The well validated Hypo1 was used as 3D query in the virtual screening to retrieve potential leads for FXa inhibition. The hit molecules were sort out by applying drug-like filters and molecular docking. Bayesian model was developed using training set compounds which provides molecular feature that are favoring or not favoring for FXa inhibition.     

Clinical pharmacology of direct and indirect factor Xa inhibitors

The limitations of conventional anticoagulants have stimulated the development of new anticoagulants. The central position of factor Xa (FXa) at the junction of the intrinsic and extrinsic pathways in the coagulation cascade means that direct and indirect FXa inhibitors have increasingly changed antithrombotic strategies. FXa inhibitors potently and selectively inhibit thrombin formation rather than thrombin activity. Direct FXa inhibitors may directly bind to FXa, whereas indirect inhibitors are dependent on antithrombin. Direct inhibitors may bind free FXa and, in contrast to indirect inhibitors, FXa within the prothrombinase complex or within clots as well. Fondaparinux is the prototype indirect FXa inhibitor and has been extensively studied in the prevention and treatment of thromboembolic diseases, including acute coronary syndromes. Due to a favourable efficacy and safety profile and convenient once-daily dosing without the need for monitoring, fondaparinux is preferentially recommended in recent guidelines dealing with antithrombotic treatment. A number of small-molecule direct FXa inhibitors are currently at different stages of clinical development. After an extensive clinical trial programme demonstrating superior efficacy without a significant increase in major bleeds compared with enoxaparin, rivaroxaban is now available for the prevention of thromboembolic events in patients undergoing orthopaedic surgery. Rivaroxaban also offers the convenience of oral once-daily dosing without the need for monitoring. Whereas most direct FXa inhibitors are orally active, otamixaban is administered intravenously, offering rapid on-off anticoagulant activity. Other compounds under development may offer additional options for tailored antithrombotic strategies according to differing indications, clinical situations and patient variables.     

Design and structure-activity relationships of potent and selective inhibitors of blood coagulation factor Xa.

The discovery of a series of non-peptide factor Xa (FXa) inhibitors incorporating 3-(S)-amino-2-pyrrolidinone as a central template is described. After identifying compound 4, improvements in in vitro potency involved modifications of the liphophilic group and optimizing the angle of presentation of the amidine group to the S1 pocket of FXa. These studies ultimately led to compound RPR120844, a potent inhibitor of FXa (K(i) = 7 nM) which shows selectivity for FXa over trypsin, thrombin, and several fibrinolytic serine proteinases. RPR120844 is an effective anticoagulant in both the rat model of FeCl(2)-induced carotid artery thrombosis and the rabbit model of jugular vein thrombus formation.     

Discovery of the novel antithrombotic agent 5-chloro-N-({(5S)-2-oxo-3- [4-(3-oxomorpholin-4-yl)phenyl]-1,3-oxazolidin-5-yl}methyl)thiophene- 2-carboxamide (BAY 59-7939): an oral, direct factor Xa inhibitor

Despite recent progress in antithrombotic therapy, there is still an unmet medical need for safe and orally available anticoagulants. The coagulation enzyme Factor Xa (FXa) is a particularly promising target, and recent efforts in this field have focused on the identification of small-molecule inhibitors with good oral bioavailability. We identified oxazolidinone derivatives as a new class of potent FXa inhibitors. Lead optimization led to the discovery of BAY 59-7939 (5), a highly potent and selective, direct FXa inhibitor with excellent in vivo antithrombotic activity. The X-ray crystal structure of 5 in complex with human FXa clarified the binding mode and the stringent requirements for high affinity. The interaction of the neutral ligand chlorothiophene in the S1 subsite allows for the combination of good oral bioavailability and high potency for nonbasic 5. Compound 5 is currently under clinical development for the prevention and treatment of thromboembolic diseases.     

Contemporary medicinal-chemistry strategies for discovery of blood coagulation factor Xa inhibitors

ABSTRACT

Introduction: Thrombosis is a common causal pathology for stroke, acute coronary syndrome and venous thromboembolism disorders, which are the leading cause of death worldwide. Anticoagulants have exhibited a crucial role in the prevention and treatment of thrombotic diseases. Factor Xa (FXa) is a serine protease with a central role in activating the complex blood coagulation cascade, and it is therefore regarded as an attractive target for antithrombotic agents.

Areas covered: The authors review the current status of medicinal chemistry strategies for the discovery of novel FXa inhibitors and provide their expert perspectives on their future development.

Expert opinion: Even if only a number of small-molecule FXa inhibitors have been reported to date, all currently available FXa inhibitors are associated with significant risk of bleeding, which may become life-threatening. There is, therefore, an urgent and unmet demand for potent novel FXa inhibitors that are potent treatments for thrombotic disorders, but which have a reduced risk of bleeding if their use is to be increasingly favored.     

The therapeutic potential of novel anticoagulants

Conventional anticoagulant therapy has been based on indirect inhibition of coagulation factors with heparin and warfarin. These agents display liabilities prompting the development of new anticoagulants over the last two decades. The first to be developed was a series of low molecular weight heparins(LMWHs). Their favourable pharmacokinetic profiles and risk/benefit ratios led to widespread use in Europe and, more recently, approval for their use in the USA. Paralleling the development of LMWHs has been the pursuit of a different strategy focused on direct rather than indirect inhibition of enzymes in the coagulation cascade. In contrast to heparin, LMWHs, or other glycosaminoglycans, direct inhibitors exert their effects independent of either antithrombin III (ATIII) or heparin cofactor II (HCII) and more effectively inhibit clot-bound thrombin or FXa. Highly potent, selective (versus other serine proteases)direct thrombin and FXa inhibitors have been identified and isolated from natural sources, such as leeches, ticks and hookworms. The recombinant forms and analogues of the senatural proteins have been produced using molecular biology techniques, i.e., rHirudin, Hirulogs, recombinant tick anticoagulant peptide (rTAP), recombinant antistasin (rATS) and recombinant nematode anticoagulant peptide-5 (rNAP-5). The design of novel structures or the modification of existing chemicals has led to the synthesis of many non-peptide, low molecular weight inhibitors of thrombin and FXa. Some of them are orally active and may be suitable for long-term clinical use. In addition, considerable progress has been made in developing specific TF/VIIa complex inhibitors. The anticoagulation properties of the new agents are being characterised in experimental studies. Some of them have been advanced to large scale clinical trials and their effectiveness, and sometimes relative ineffectiveness,in arterial and venous thromboembolic disorders has been demonstrated. They are being tested for their potential as new antithrombotic agents that act via direct enzyme inhibition. Thus,the clinician should in future be able to target different thrombotic conditions with proven, specific anticoagulant interventions.     

Recent advances in clinical trials of the direct and indirect selective Factor Xa inhibitors

Over the years, pharmacological intervention to prevent undesired intravascular coagulation and the associated detrimental effects has been a clinical challenge. The first generation of anticoagulant agents, warfarin and unfractionated heparin (UFH), involve indirect mechanisms of inhibiting the coagulation cascade. Fractionated, or low-molecular weight, heparins (LMWHs) are more selective for coagulation Factor Xa (FXa) over thrombin (FIIa). LMWHs also utilise an indirect mechanism of inhibition and have improved pharmacokinetic, pharmacodynamic and therapeutic profiles over UFH. The success of LMWHs, along with the pivotal location of FXa in the coagulation cascade, has prompted interest in the discovery and development of selective FXa inhibitors. There are two general classes of FXa inhibitors in development, of which SR90107A/ORG31540, an antithrombin-III-dependent pentasaccharide and DX-9065a, a small molecule direct FXa inhibitor, have published clinical data. SR90107A/ORG31540 and DX-9065a offer safe, predictable pharmacokinetic and pharmacodynamic profiles when administered subcutaneously and intravenously, respectively, to healthy volunteers and appear to be progressing through clinical development. The purpose of this review is to compile and summarise the published Phase I and II clinical data for SR90107A/ORG31540 and DX-9065a.     

Discovery of N-[2-hydroxy-6-(4-methoxybenzamido)phenyl]-4- (4-methyl-1,4-diazepan-1-yl)benzamide (darexaban, YM150) as a potent and orally available factor Xa inhibitor

Inhibitors of factor Xa (FXa), a crucial serine protease in the coagulation cascade, have attracted a great deal of attention as a target for developing antithrombotic agents. We previously reported findings from our optimization study of a high-throughput screening (HTS) derived lead compound 1a that resulted in the discovery of potent amidine-containing FXa inhibitors represented by compound 2. We also conducted an alternative optimization study of 1a without incorporating a strong basic amidine group, which generally has an adverse effect on the pharmacokinetic profile after oral administration. Replacement of 4-methoxybenzene with a 1,4-benzodiazepine structure and introduction of a hydroxy group at the central benzene led to the discovery of the potent and orally effective factor Xa inhibitor 14i (darexaban, YM150). Subsequent extensive study revealed a unique aspect to the pharmacokinetic profile of this compound, wherein the hydroxy moiety of 14i is rapidly transformed into its glucuronide conjugate 16 (YM-222714) as an active metabolite after oral administration and it plays a major role in expression of potent anticoagulant activity in plasma. The distinctive, potent activity of inhibitor 14i after oral dosing was explained by this unique pharmacokinetic profile and its favorable membrane permeability. Compound 14i is currently undergoing clinical development for prevention and treatment of thromboembolic diseases.     

Heterogeneity of synthetic factor Xa inhibitors

Heparins and vitamin K antagonists are the landmarks of antithrombotic treatment. Both of them were discovered by serendipity; they are multi-targeted drugs and share several limitations. New molecules have been designed in order to be both more selective concerning their biological target and more homogeneous in their biochemical structure aiming at an improved benefit/risk ratio in the treatment of thrombotic disease. In this article, we will review the pharmacological characteristics of the new synthetic direct or antithrombin dependent inhibitors of FXa in the light of the modern concept of blood coagulation process. We will also present the most recent data from the clinical trials with synthetic inhibitors of FXa. Among them, the synthetic pentasaccharide fondaparinux is the first synthetic and specific FXa inhibitor, which has been approved by health authorities in Europe and in the USA for the prophylaxis of venous thromboembolism in major orthopaedic surgery and is being approved for the treatment of pulmonary embolism and DVT as a single daily subcutaneous injection. The phase II dose-finding trial of the "meta-pentasaccharide" idraparinux administered subcutaneously once weekly in the secondary prevention of VTE has been completed. DX-9065a is the first direct synthetic inhibitor which has been studied in patients with coronary disease. Razaxaban, BAY59-7939, ZK-807834 and JTV-803 are orally active direct FXa inhibitors, which have been studied in phase II trials. Several other synthetic direct inhibitors of FXa (such as FXV673, YM60828, KFA-1411) are in a pre-clinical stage of research. From a clinical point of view, the results of recent trials with the synthetic specific FXa inhibitors clearly show that the inhibition of FXa is a critical point in the antithrombotic strategy.     

Recent advances in clinical trials of the direct and indirect selective Factor Xa inhibitors

Over the years, pharmacological intervention to prevent undesired intravascular coagulation and the associated detrimental effects has been a clinical challenge. The first generation of anticoagulant agents, warfarin and unfractionated heparin (UFH), involve indirect mechanisms of inhibiting the coagulation cascade. Fractionated, or low-molecular weight, heparins (LMWHs) are more selective for coagulation Factor Xa (FXa) over thrombin (FIIa). LMWHs also utilise an indirect mechanism of inhibition and have improved pharmacokinetic, pharmacodynamic and therapeutic profiles over UFH. The success of LMWHs, along with the pivotal location of FXa in the coagulation cascade, has prompted interest in the discovery and development of selective FXa inhibitors. There are two general classes of FXa inhibitors in development, of which SR90107A/ORG31540, an antithrombin-III-dependent pentasaccharide and DX-9065a, a small molecule direct FXa inhibitor, have published clinical data. SR90107A/ORG31540 and DX-9065a offer safe, predictable pharmacokinetic and pharmacodynamic profiles when administered subcutaneously and intravenously, respectively, to healthy volunteers and appear to be progressing through clinical development. The purpose of this review is to compile and summarise the published Phase I and II clinical data for SR90107A/ORG31540 and DX-9065a.     

Design, structure-activity relationships, X-ray crystal structure, and energetic contributions of a critical P1 pharmacophore: 3-chloroindole-7-yl-based factor Xa inhibitors

An indole-based P1 moiety was incorporated into a previously established factor Xa inhibitor series. The indole group was designed to hydrogen-bond with the carbonyl of Gly218, while its 3-methyl or 3-chloro substituent was intended to interact with Tyr228. These interactions were subsequently observed in the X-ray crystal structure of compound 18. SAR studies led to the identification of compound 20 as the most potent FXa inhibitor in this series (IC(50) = 2.4 nM, EC(2xPT) = 1.2 microM). An in-depth energetic analysis suggests that the increased binding energy of 3-chloroindole-versus 3-methylindole-containing compounds in this series is due primarily to (a) the more hydrophobic nature of chloro- versus methyl-containing compounds and (b) an increased interaction of 3-chloroindole versus 3-methylindole with Gly218 backbone. The stronger hydrophobicity of chloro- versus methyl-substituted aromatics may partly explain the general preference for chloro- versus methyl-substituted P1 groups in FXa, which extends beyond the current series.     

Inhibitors of Factor Xa

The development of directly acting, orally-active anticoagulants is an important drug discovery target. Many companies are involved in this area and the most popular target is Factor Xa (FXa). Inhibitors of FXa prevent the generation of thrombin which is the active enzyme in clot formation as well as platelet activation. Patents for FXa inhibitors from Aventis and AstraZeneca are reviewed here. The AstraZeneca patent is a continuation of a previous patent and contains virtually no biological data. The Aventis patent covers a large number of compounds. The compounds described have two amidino groups, which are important in many different protease inhibitors. However, this can lead to problems including lack of oral activity as well as lack of specificity. The only biological data presented are the K_ivalues for a number of compounds in an in vitro FXa assay.     

The therapeutic potential of novel anticoagulants

For several decades, conventional anticoagulant therapy has been based on indirect inhibition of coagulation factors with heparin and warfarin (coumadin). Although used widely and effectively, heparin and warfarin display liabilities which have prompted the development of new anticoagulants over the last two decades. The first to be developed was a series of low molecular weight heparins (LMWHs). Their favourable pharmacokinetic profiles and risk/benefit ratios led to widespread use in Europe and more recently, approval for their use in the USA. Paralleling the development of LMWHs, but lagging behind in terms of clinical development, has been the pursuit of a different strategy focused on direct rather than indirect inhibition of enzymes in the coagulation cascade. In contrast to heparin, LMWHs, or other glycosaminoglycans, direct inhibitors, exert their effects independent of either antithrombin III (ATIII) or heparin co-factor II (HCII) and more effectively inhibit clot-bound thrombin or FXa. Highly potent, selective (versus other serine proteases), direct thrombin and FXa inhibitors have been identified and isolated from natural sources, such as leeches, ticks and hookworms. The recombinant forms and analogues of these natural proteins have been produced using molecular biology techniques, i.e., rHirudin, Hirulogs, recombinant tick anticoagulant peptide (rTAP), recombinant antistasin (rATS) and recombinant nematode anticoagulant factor (rNAP-5). The design of novel structures or the modification of existing chemicals has led to the synthesis of many non-peptide, low molecular weight inhibitors of thrombin and FXa. Some of them are orally active and may be suitable for long-term clinical use. In addition, considerable progress has been made in developing specific TF/VIIa complex inhibitors. The anticoagulation properties of the new agents have been and are being characterised in experimental studies. Some of them have been advanced to large scale clinical trials and their effectiveness (and sometimes relative ineffectiveness compared with conventional therapy) in arterial and venous thromboembolic disorders has been demonstrated. These novel anticoagulents are curently being tested or will for their validity and potential as new antithrombotic agents acting via direct enzyme inhibition. In doing so, it is hoped that the clinician will in future be able to turn to proven specific anticoagulant interventions, targeted at each respective thrombotic state.     

3,6-disubstituted coumarins as mechanism-based inhibitors of thrombin and factor Xa

In this work, coumarins were screened on thrombin (THR) and factor Xa (FXa), two of the most promising targets for the development of anticoagulant drugs. This allowed us to highlight compound 30, characterized by a 2,5-dichlorophenyl ester in the 3-position and a chloromethyl moiety in the 6-position, as a very potent THR inhibitor (ki/KI= 37,000 M(-1) s(-1)). Moreover, this compound exhibits good selectivity over FXa (168-fold) and trypsin (54-fold). The mechanism of inactivation was investigated in this series and significantly differs from that previously observed with alpha-chymotrypsin. Indeed, the addition of hydrazine on the THR-inhibitor complex promotes a partial induced THR reactivation. This reactivation, confirmed by LC/MS, showed the resurgence of the native THR and a new dihydrazide complex. Docking experiments were then efficiently used to explain the trends observed in the enzymatic assays as well as to corroborate the postulated inhibition mechanism. Finally, the cell permeability of our derivatives was estimated using a computational approach.     

AcAP5蛋白N端片段的表达、纯化与活性评价(英文)

犬钩虫抗凝肽5(Ancylostoma anticoagulant peptide 5,AcAP5)N端40个氨基酸片段(N40)含有与FXa结合的结构域。为研究N40的FXa抑制作用,我们克隆、表达和纯化了N40,并检测其生物活性。用PCR扩增N40基因;将PCR产物克隆至原核表达载体pET-30a中,构建质粒pET30a-N40;将其转化入大肠杆菌E.coli.BL21(DE3)中,IPTG诱导目的蛋白表达,SDS-PAGE电泳鉴定,经过镍柱亲和层析纯化,用BCA法进行蛋白定量,测定该蛋白抑制FXa的活性。限制性酶切鉴定和基因测序结果显示重组质粒构建成功;SDS-PAGE结果显示目的蛋白在E.coli.BL21(DE3)中为可溶性表达,亲和层析纯化后获得了纯度约90%的蛋白,经活性鉴定该蛋白确有抑制FXa的活性。