Protein Databank Accession Figures are as follows: 4CRG, 4CR5, 4CR9, 4CRA, 4CRB, 4CRC, 4CRD, 4CRE, 4CRF.. fragments towards FXIa prime part binding sites was aided by solving the X-ray constructions of reported FXIa inhibitors that we found to bind in the S1-S1-S2 FXIa binding pouches. Combining the X-ray structure information from Pristinamycin your recognized S1 binding 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment and the S1-S1-S2 binding research compounds enabled structure guided linking and growth work to accomplish probably one of the most potent and selective FXIa inhibitors reported to day, compound 13, having a FXIa IC50 of 1 1.0 nM. The hydrophilicity and large polar surface area of the potent S1-S1-S2 binding FXIa inhibitors jeopardized permeability. Initial work to increase the 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment towards prime part to yield molecules with less hydrophilicity shows promise to afford potent, selective and orally bioavailable compounds. Intro A well balanced haemostasis system is definitely important to both minimize blood loss and disturbances of blood flow. Upon injury of the vessel wall, blood is exposed to cells element which via a cascade reaction prospects to thrombin generation and a fibrin cross-linked clot to mend the injury and stop bleeding. Element XI (FXI) has an important part in thrombin generation in the amplification phase of the coagulation process. However, over-production of thrombin may lead to excessive clots resulting in thrombosis. Also, high levels of thrombin cause activation of thrombin triggered fibrinolysis inhibitor which hinders fibrinolysis. Consequently, decreased levels of thrombin will indirectly increase the rate of fibrinolysis. Inhibition of triggered FXI (FXIa) should decrease thrombin generation in the amplification phase, but not in the initiation phase, and thus yield an antithrombotic and profibrinolytic effect with minimal risk of bleeding (observe reviews [1C3]). Bleeding is definitely a serious concern with current antithrombotic medicines and FXIa inhibitors could address this problem. The part of FXIa in haemostasis and thrombosis in human being has been extensively analyzed. Human being haemophilia C individuals who are seriously deficient in FXI display reduced incidence of ischemic stroke [4]. Unlike haemophilia A and B individuals, who are deficient in FVIII and FIX, respectively, haemophilia C individuals seldom encounter spontaneous bleeding [5]. The bleeding associated with FXI deficiency usually happens after stress or surgery in the cells with high fibrinolytic activity [6,7]. An increased level of factor XI has been reported as a risk factor for deep venous thrombosis [8,9], myocardial infarction [10] and ischemic stroke [11,12]. There is also much research around the role of FXI in animals. Several studies have exhibited that FXI-null mice are guarded against venous and arterial thrombosis without an adverse effect on bleeding time [13C18]. Recent reports present similar effects in mice [19] and primates [20] using antisense oligonucleotides to inhibit FXI production [19]. Antibodies against FXI/FXIa have been Rabbit Polyclonal to KITH_HHV11 shown in one study to reduce thrombus growth in the rabbit iliac artery in the presence of repeated balloon injury [21], and in another study to increase endogenous thrombolysis in rabbit about two-fold in comparison to control antibodies [22]. Also, an anti-human antibody, aXIMab, prevented vascular graft occlusion in baboons [23]. In summary, there is ample evidence in support of FXIa as a stylish antithrombotic and profibrinolytic target. Pristinamycin FXIa small molecule inhibitors have not reached the same level of maturity as thrombin and activated factor X (FXa) inhibitors. The thrombin inhibitor dabigatran [24] and the FXa inhibitor rivaroxaban and apixaban [25] are approved anticoagulant drugs in several markets, but adverse bleeding remains an area where improvement is usually requested. In contrast, inhibitors of FXIa are still in preclinical development. Daiichi Sankyo Co has reported on potent and selective peptidomimetic alpha-ketothiazole arginine based covalent FXIa inhibitors [26,27], and one compound was shown to display similar antithrombotic efficacy as heparin in a rat venous thrombosis model [26]. Similarly, Bristol Myers Squibb (BMS) exhibited antithrombotic efficacy in rat models with BMS-262084, a potent and selective beta-lactam arginine that irreversibly inhibits FXIa with an IC50 of 2.8 nM [28]. Recently, BMS also showed antithrombotic efficacy without increased bleeding in a rabbit model with a reversible selective small molecule FXIa inhibitor [29]. Patent applications from BMS display lists of selective FXIa inhibitors,.This protein was used for crystallisation, NMR and SPR measurements. NMR screening NMR samples were prepared in aqueous buffer containing 50 mM deuterated TRIS, pH 7.4, 3 mM NaN3, and 10% D2O. inhibitors that we found to bind in the S1-S1-S2 FXIa binding pockets. Combining the X-ray structure information from the identified S1 binding 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment and the S1-S1-S2 binding reference compounds enabled structure guided linking and growth work to achieve one of the most potent and selective FXIa inhibitors reported to date, compound 13, with a FXIa IC50 of 1 1.0 nM. The hydrophilicity and large polar surface area of the potent S1-S1-S2 binding FXIa inhibitors compromised permeability. Initial work to expand the 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment towards prime side to yield molecules with less hydrophilicity shows promise to afford potent, selective and orally bioavailable compounds. Introduction A well balanced haemostasis system is usually important to both minimize blood loss and disturbances of blood flow. Upon injury of the vessel wall, blood is exposed to tissue factor which via a cascade reaction leads to thrombin generation and a fibrin cross-linked clot to mend the injury and stop bleeding. Factor XI (FXI) has an important role in thrombin generation in the amplification phase of the coagulation process. However, over-production of thrombin may lead to excessive clots resulting in thrombosis. Also, high levels of thrombin cause activation of thrombin activated fibrinolysis inhibitor which hinders fibrinolysis. Therefore, decreased levels of thrombin will indirectly increase the rate of fibrinolysis. Inhibition of activated FXI (FXIa) should decrease thrombin generation in the amplification phase, but not in the initiation phase, and thus yield an antithrombotic and profibrinolytic effect with minimal risk of bleeding (see reviews [1C3]). Bleeding is usually a serious concern with current antithrombotic drugs and FXIa inhibitors could address this issue. The role of FXIa in haemostasis and thrombosis in human has been extensively studied. Human haemophilia C patients who are severely deficient in FXI display reduced incidence of ischemic stroke [4]. Unlike haemophilia A and B patients, who are deficient in FVIII and FIX, respectively, haemophilia C patients seldom experience spontaneous bleeding [5]. The bleeding associated with FXI deficiency usually occurs after trauma or surgery in the tissues with high fibrinolytic activity [6,7]. An increased level of factor XI has been reported as a risk factor for deep venous thrombosis [8,9], myocardial infarction [10] and ischemic stroke [11,12]. There is also much research around the role of FXI in animals. Several studies have exhibited that FXI-null mice are guarded against venous and arterial thrombosis without an adverse effect on bleeding time [13C18]. Recent reports present similar effects in mice [19] and primates [20] using antisense oligonucleotides to inhibit FXI production [19]. Antibodies against FXI/FXIa have been shown in one study to reduce thrombus growth in the rabbit iliac artery in the presence of repeated balloon injury [21], and in another study to increase Pristinamycin endogenous thrombolysis in rabbit Pristinamycin about two-fold in comparison to control antibodies [22]. Also, an anti-human antibody, aXIMab, prevented vascular graft occlusion in baboons [23]. In summary, there is ample evidence in support of FXIa as a stylish antithrombotic and profibrinolytic target. FXIa small molecule inhibitors have not reached the same level of maturity as thrombin and activated factor X (FXa) inhibitors. The thrombin inhibitor dabigatran [24] and the FXa inhibitor rivaroxaban and apixaban [25] are approved anticoagulant drugs in several markets, but adverse bleeding remains an area where improvement is usually requested. In contrast, inhibitors of FXIa are still in preclinical development. Daiichi Sankyo Co has reported on potent and selective peptidomimetic alpha-ketothiazole arginine based covalent FXIa inhibitors [26,27], and one compound was shown to display similar antithrombotic efficacy as heparin in a rat venous thrombosis model [26]. Similarly, Bristol Myers Squibb (BMS) exhibited antithrombotic efficacy in rat models with BMS-262084, a potent and selective beta-lactam arginine that irreversibly inhibits FXIa with an IC50 of 2.8 nM [28]. Recently, BMS also showed antithrombotic efficacy without increased bleeding in a rabbit model with a reversible selective small molecule FXIa inhibitor [29]. Patent applications from BMS display lists of selective FXIa inhibitors, or dual FXIa and plasma kallikrein inhibitors, with IC50 values in the low nM range [30C32]. These examples encourage further work with the aim of reaching the clinical setting for small molecule FXIa inhibitors. In-house high throughput screening (HTS) attempts had previously failed to identify viable leads. Therefore, structure aided fragment based lead generation (FBLG) was chosen as a rescue strategy to create new FXIa inhibitor leads. The choice.