Chymotrypsin inhibitors for selective dissolution of thrombus in thrombotic or thromboembolic conditions

Abstract

The present invention encompasses the use of general chymotrypsin inhibitors, and more specifically, substituted bicyclic substituted uracil of general formula (I) as described and defined herein, and 3-methylbenzo-[b] thiophene)-2-sulfonamido derivatives of general formula (II) for the manufacture of pharmaceutical compositions for the treatment or prevention of stroke, pulmonary embolism, pulmonary embolism, and the like. The traditional Chinese medicine composition can be used for treating thrombosis, deep vein or superficial vein thrombosis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy in a hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion at a vascular access part.

Description

[0001] The present invention covers the use of chymase inhibitors in general and, more specifically, substituted bicyclic substituted uracils of general formula (I) as described and defined herein, and 3-methylbenzo-[b]thiophene)-2-sulfonylamino derivatives of general formula (II) for the manufacture of pharmaceutical compositions for the treatment or prevention of stroke, pulmonary embolism, deep or superficial vein thrombosis, thrombotic microangiopathy, infection, inflammation, post-transplant hypercoagulable state thrombotic microangiopathy, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, vascular access site thrombosis or occlusion.

[0002] Hemostasis is a protective physiological mechanism that quickly and reliably covers leaky lesions in the vessel wall with the goal of avoiding excessive blood loss or keeping it to a minimum. After such a vessel wall injury, the activation of the coagulation system, the activation of platelets, adhesion and aggregation, and the formation of insoluble fibrin lead to the rapid formation of a blood clot, which seals the leak in the vessel wall. However, clot formation must maintain a balance - to prevent (severe) bleeding, but not to lead to excessive clot formation, which could occupy a large part of the vessel lumen and thus partially or completely reduce blood flow and ultimately lead to insufficient oxygen and nutrient supply to the surrounding tissues. Although there are physiological mechanisms to: a) control the activity of the coagulation system with endogenous inhibitors; b) break down the already generated fibrin clot material (via the fibrinolytic system), these mechanisms are not always sufficient. Uncontrolled, excessive activation of the coagulation system or defects in the hemostatic balance may lead to thrombosis by forming local thrombi or emboli, and the blood clots so formed break off from other points in the circulatory system and block distant vessels. Blockage of arteries in the heart, brain, and lungs, known as myocardial infarction, stroke, or pulmonary embolism, respectively, is one of the most common causes of death and morbidity worldwide. The occurrence of large numbers of microthrombi blocking blood vessels in small organs has attracted increasing attention during the COVID-19 pandemic.

[0003] The prognosis of patients with thrombotic events depends largely on the timely resupply of fresh blood to the corresponding organs. Therefore, determining a safe method to quickly reopen occluded blood vessels is an important goal of clinical research.

[0004] Although current anticoagulant drugs, vitamin K antagonists, direct factor Xa or thrombin inhibitors, or heparin as an indirect dual FXa / thrombin inhibitor, reduce the coagulability of blood and have been shown to be effective in preventing thrombotic events, their primary dissolving effect on pre-existing clots is thought to be only indirect: reduced activity of the coagulation system shifts the hemostatic balance toward the physiological fibrinolytic system, which aids in the degradation of fibrinous material. However, high doses of anticoagulants may increase the risk of bleeding, and because of the indirect effect, clot dissolution is rather slow, limiting the use of anticoagulants to indications that do not require rapid reopening of the vessel.

[0005] Current approaches to rapidly reopening a vessel in many indications are based on mechanical removal of the obstructing clot or degradation of the fibrin clot by forced fibrinolysis.

[0006] Recently, mechanical approaches, such as thrombectomy, have been increasingly used in patients with large artery stroke. However, despite technological advances, accessibility and clinical availability of thrombectomy are often limited, so that this procedure can only be applied to a minority of patients.

[0007] The major enzyme of fibrin degradation, plasmin, is activated in the presence of fibrin by the plasma protein plasminogen via plasminogen activators (tissue plasminogen activator (tPA), urokinase-type plasminogen activator (uPA)) secreted by endothelial cells. In the blood, active plasmin is rapidly inactivated by a large excess of α2-antiplasmin, but is known to remain active when bound to fibrin. Current fibrinolytic strategies use systemically or locally administered plasminogen activators to generate plasmin. Recombinant tissue plasminogen activator (rtPA, alteplase) is the most widely used fibrinolytic drug, which is FDA-approved for myocardial infarction, ischemic stroke, pulmonary embolism, or reopening of blocked intravenous catheters (Hughes RH et al., StatPearls 2020) and has become the standard of care for the treatment of patients with acute ischemic stroke in many fields, and patients can be treated within a 4.5-hour time window after the event (Powes WJ et al., Stroke 50 (2019) e344-e418). However, the administration of tPA is associated with a dose-dependent risk of bleeding, including life-threatening intracranial hemorrhage (Emberson J et al., Lancet 384 (2014) 1929), which limits the dose of tPA and thus its fibrinolytic efficacy. In addition to the risk / benefit profile of this drug, the short half-life in human plasma (major half-life <5 min; Tanswell P et al., Arzneimittelforschung 41 (1991) 1310-9), and other adverse reactions, including rethrombosis and the more rare but morbid tPA-related angioedema (Rathburn KM, Oxf. Med. Case Rep. 2019; K et al., Stroke 50 (2019) 1682), representing an overall challenging drug profile, which is responsible for poor efficacy and limited use.

[0008] Therefore, identifying rapid, reliable, and safe medical revascularization strategies remains a challenging task with a very high medical need.

[0009] To date, the role of mast cells and their contents in the pathology of thrombotic events is probably severely underappreciated and underestimated, and there are no therapeutic approaches that target mast cell granule components for thrombolysis.

[0010] Mast cells are long-lived perivascular resident hematopoietic cells that are distributed in most tissues, usually at the interface between tissues and the external environment. For example, they can be found in the mucosa or epithelial surfaces of the intestines and lungs, in the skin, and tend to gather around blood vessels, lymphatic vessels, and nerves. (Prussin C, Metcalfe DD, J.Allergy Clin. Immunol. 111 (2003) S486-94). Only a small amount of progenitor cells can be found in the blood before repositioning to their mature tissues. They are multifunctional immune cells involved in a variety of health and disease states, and the most widely known is their role in allergic and anaphylactic reactions in the lungs, intestines, or skin. However, they are also involved in other processes, including protective strategies such as host defense against pathogens, immune tolerance, wound healing, angiogenesis, and several pathophysiological processes (Miyazaki et al., Pharmacol. Ther. 112 (2006), 668-676; Shiota et al., J. Hypertens. 21 (2003) 1823-1825). Their number is increased in patients with heart failure, myocardial infarction and ischemia, human atherosclerotic plaques and abdominal aortic aneurysms (Kovanen PT et al., Circ. 92 (1995) 1084-1088; Libby P, Shi GP, Circ. 115 (2007) 2555-2558; Bankl HC et al., Circ. 91 (1995) 275-83). Recently, the role of mast cell activation in the etiology of SARS-CoV-2 infection has been discussed (Wu ML et al., Signal Transduct. Targ. Ther. 6 (2021) 428; Kempuraj D. et al., Neuroscientist 26 (2020) 402-414).

[0011] Mast cells contain many large cytoplasmic granules, which, when stimulated, release a large amount of histamine and heparin, as well as many other active ingredients, including several cytokines, proteoglycans and serine proteases, such as tryptase, chymotrypsin, chymotrypsin, cathepsin G and carboxypeptidase A (Lindstedt KA et al., J. Cell. Mol. Med. 11 (2007) 739-758). Some stored compounds, such as heparin, polyphosphates or histamine, are known to interact with the coagulation system (Guilarte M et al., Front. Immunol 2017), but the overall effect of mast cells is anticoagulation or procoagulation, and whether there is a relevant effect on clot formation, which is still controversial. So far, there has been no description of mast cells or granular components having an antifibrinolytic effect on thrombi in the vascular system.

[0012] Recently, in a mouse model of experimental venous stenosis, two types of mast cell-deficient mice were completely protected from venous thrombosis compared to untreated mice (Ponomaryov et al., Circ Res. 121 (2017) 941-950). It was concluded that a potential procoagulant effect of mast cells might be related to the release of histamine, but this could not be confirmed using histamine receptor blockers. No potential effects on the fibrinolytic system and, therefore, on thrombolysis were documented. In a mouse model of arterial thrombosis (Ponomaryov et al. (Circ Res. 121 (2017) 941-950), in which ferric chloride-induced injury to mesenteric arterioles triggered thrombosis, no statistically significant differences in occlusion time were observed between wild-type and mast cell-deficient mice. The authors concluded that mast cells might be involved only in venous thrombosis and might not be involved in arterial thrombosis.

[0013] In a review of mast cell interactions with the fibrinolytic system, Bankl and Valent (Thromb. Res. 105 (2002) 359) suggested that mast cells themselves may be profibrinolytic due to selective production of tPA rather than its endogenous inhibitor PAI-1. Their conclusion that mast cells are profibrinolytic is in contrast to the surprising findings of the present invention, which show that chymotrypsin is antifibrinolytic and that inhibitors can be used to reopen blood vessels.

[0014] Chymotrypsin is a serine protease that is stored in mast cell granules as a complex with heparin. After mast cell activation, it is released into the extracellular space, where chymotrypsin is activated, and -- like other mast cell proteases -- strictly controlled to prevent damage to host tissues (Pejler G. et al., Adv. Immunol. 95 (2007) 167-255). However, these control mechanisms have not yet been fully elucidated: in human whole blood, chymotrypsin is rapidly inhibited by endogenous circulating inhibitors, including α-1 antitrypsin, α-1 antichymotrypsin, α-2 macroglobulin (Metcalfe DD et al., World Allergy Organ. J. 11 (2016) 7), so that the activity of chymotrypsin cannot usually be measured in blood, serum or plasma. Therefore, it is expected that chymotrypsin activity cannot be measured in vascular thrombosis.

[0015] Several substrates of chymotrypsin have been documented: it is involved in the production of angiotensin II in the heart, arterial wall and lungs (Fleming I., Circ. Res. 98 (2006), 887-896) and endothelin-1 (Nakano et al., J. Immunology, 1997; 159: 1987-1992; D'Orléans-Juste P et al., Vascular Pharm. 49 (2008) 51-62). In addition, chymotrypsin leads to the degradation of extracellular matrix proteins such as fibronectin, procollagen and vitronectin, and leads to the disruption of focal adhesions (Pejler, J Innate Immun 2020; 12: 357-372). It activates and releases latent forms of TGFβ, which plays an important role in the development of cardiac hypertrophy and cardiac fibrosis (Cho et al., Am J Respir Cell Mol Biol. 2015 Jan; 52(1): 88-95; Oyamada S et al., J Pharmacol Exp Ther. 2011 Oct; 339(1): 143-51). The enzyme has atherogenic effects by degrading apolipoproteins and preventing the absorption of cholesterol by HDL (Lee et al., ATVB 2002; 22: 1475-1481). The action of chymotrypsin also leads to the release and activation of interleukin-1, a cytokine with pro-inflammatory properties (Mizutani et al., J Exp Med. 1991; 174(4): 821-825).

[0016] So far, the connection of chymotrypsin to the fibrinolytic system, its potential role and relevance are still unclear:

[0017] Tchougounova and Pejler studied the effects of peritoneal mast cells from heparin-deficient or wild-type mice on the activation and inactivation of thrombin and plasmin in in vitro experiments (Tchougounova E, Pejler G., FASEB J. 15 (2002) 2763-5). In their study, the regulatory effects on thrombin and plasmin were heparin-dependent and could be inhibited by the nonspecific endogenous protease inhibitor α1-antichymotrypsin. Therefore, they attributed these proteolytic findings to chymotrypsin or elastase. However, the validity or translatability of these findings is unclear because there are several chymotrypsin-like enzymes in murine peritoneal mast cells that are different from the relevance of translation to humans, and no specific chymotrypsin inhibitors were used for further verification. In addition, this suggests that this regulation of thrombin and plasmin may play a role outside the blood vessels, rather than inside the blood vessels. This is contrary to the results of the present invention, which show that chymotrypsin activity and its inhibition in intravascular fibrin clots play a central role.

[0018] recent, et al. reported histological experiments revealing the colocalization of chymotrypsin and fibrin in vasculitis samples. Subsequent in vitro experiments, in which chymotrypsin was added to fibrinogen and fibrin, showed that chymotrypsin potentially slowly degraded fibrinogen and fibrin, which was only observed in the absence of plasma ( T. et al., Brit. J. Dermatol 181 (2019) 296-303). It is inferred that in this case, chymotrypsin itself may have fibrinolytic properties. The hypothesis that chymotrypsin may be pro-fibrinolytic and therefore may represent a therapeutic opportunity for vasculitis is in stark contrast to the findings described in the present invention.

[0019] In summary, it is unclear from current common sense what the role of mast cells in experimental venous stenosis in mice is and whether it can be translated to arterial models and clinical situations. What role chymotrypsin may play in the context of revascularization or prevention of thrombotic events has not been investigated.

[0020] Chymotrypsin inhibitors are disclosed in WO2013167495, WO2015067650, WO2015067651 and WO0222595 and have been evaluated in several pharmaceutical companies (Ahmad S, Ferrario CM, Expert. Opin. Ther. Pat 28 (2018) 755-764). Fulacimstat (1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid) and TY-51469 ((2-(4-((5-fluoro-3-methylbenzo-[b]thiophene)-2-sulfonylamino)-3-(methylsulfonyl)phenyl)thiazole-4-carboxylic acid) are selective chymotrypsin inhibitors of different structural classes, IC 50 The values ​​were 4 nM and 20 nM, respectively. Its selectivity has been tested against various proteases. The IC values ​​obtained for plasmin, plasma kallikrein, thrombin, trypsin, tissue plasminogen activator, factor Xa and factor XIa 50The values ​​are all greater than 2 μM. Compared with chymotrypsin, TY-51469 is 60 times less potent in inhibiting the closest protease, cathepsin G, and fulacimstat is 35 times less potent in inhibiting the closest protease, cathepsin G, and is therefore considered irrelevant in vivo. These two compounds have been evaluated in many preclinical models, covering most inflammatory and fibrotic diseases in various organs including the heart, kidney, lung, intestine, and skin, as well as autoimmune diseases (Pejler G, J. Innate Immun. 12 (2020) 357-372). Fulacimstat is bioavailable after oral administration. The compound was well tolerated in clinical studies without any signs of an effect on bleeding risk and had favorable pharmacokinetic properties with once-daily dosing (Kanefendt F et al., Clin. Pharmacol. Drug Dev. 8 (2019) 467-479), Düngen HD et al., Clin. Pharmacol. Drug Dev. 8 (2019) 942-951), Düngen HD et al., Am. Heart J. 224 (2020) 129-137), Rossing P et al., Nephrol. Dial. Transplant 26 (2021) 2263-2273).

[0021] For drugs used in acute thrombotic events, rapid clot dissolution with low bleeding risk is critical.

[0022] Therefore, the object of the present invention is to provide a new thrombolytic strategy to treat thrombotic events in veins, capillaries, arteries and lymphatic vessels in humans and animals without increasing the risk of bleeding.

[0023] Surprisingly, as part of the present invention, it has now been found that plasmin is a chymotrypsin substrate and that the degradation and inactivation of plasmin can be prevented in animals and humans by the chymotrypsin inhibitors of the present invention (Experimental part, section 3; Figure 1 and 2 ), making these chymotrypsin inhibitors useful for thrombolysis, particularly in the etiologies of acute thrombotic and thromboembolic events.

[0024] Furthermore, since chymotrypsin is rapidly inactivated in plasma, it was not expected to remain active in the local microenvironment of thrombus, and the addition of the chymotrypsin inhibitor of the present invention resulted in a decrease in chymotrypsin activity and an increase in its substrate plasmin in this environment (Experimental Section, Section 9; Fig.10 , 15 ).

[0025] Surprisingly, it was shown that in samples of human venous, arterial and pulmonary embolic clots, chymotrypsin was present not only in clots from animal experiments but also in human clots and therefore plays an important role in human thrombotic events (Experimental Section, Section 8; Fig. 9 ).

[0026] Surprisingly, in the case of inferior vena cava stenosis, the administration of the chymotrypsin inhibitors of the invention leads to a reduction in thrombus weight and length - even in the setting of a clear intervention, e.g., when administered 24 h after the onset of thrombosis. These results were obtained by using two structurally different chymotrypsin inhibitors, 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (fulacimstat) and TY-51469, in an established mouse DVT model and fulacimstat in a newly developed hamster DVT model, both animal models being associated with inferior vena cava stenosis, to confirm the general nature of these findings (Experimental Section, Section 4; Figure 4).

[0027] Although it is generally believed that the main mechanism behind the rat vein stenosis experiments performed by Ponomaryov et al. is related to coagulation, in our study, the activity measurement of chymotrypsin and plasmin in thrombi removed from animals showed a strong link between chymotrypsin and the fibrinolytic system: while the inhibitors reduced chymotrypsin activity, plasmin activity was increased in thrombi of these animals (Experimental section, section 9.2; Fig.10 ).

[0028] Compared to the ferric chloride-induced injury experiments known from the prior art Ponomaryov et al. performed in mesenteric arteries, which showed that mast cells had no significant effect on thrombus growth, the ferric chloride-induced injury experiments on different blood vessels described in the present invention showed significant differences between the groups treated with the chymotrypsin inhibitors of the present invention and the vehicle group. In these acute thrombosis experiments, the compounds were administered before thrombus formation was initiated by ferric chloride, which means that even at an early stage during clot formation, chymotrypsin inhibitors affect the weight of the thrombus and thus the risk of vessel occlusion (Experimental part, section 7; Figure 7 , 13 ). This is observed not only in venous vessels, but also in arteries, such as the carotid artery.

[0029] Surprisingly, when the lungs were examined in an inferior vena cava (IVC) stenosis experiment in mice and hamsters, a reduced number of pulmonary emboli was observed in the lungs of animals with chymotrypsin inhibition compared with control animals ( Experimental Section , Section 6; Figure 5 and 6 ). This very important and relevant case of thromboembolism demonstrates that chymotrypsin not only acts at the local site of thrombus formation, but also has an antifibrinolytic effect within the embolus. Chymotrypsin inhibitors are therefore effective agents for lytic thromboembolism, including, for example, deep vein emboli causing pulmonary embolism or left atrial appendage emboli causing stroke or systemic embolism in patients with atrial fibrillation.

[0030] In the mouse tail bleeding model and the hamster femoral vein puncture model, no effect on bleeding time was observed, which is noteworthy because the experiments were conducted at doses that resulted in a robust effect in thrombolysis—making the chymotrypsin inhibition approach not only effective but also unexpectedly safe (Experimental Section, Section 10; Fig.12 ).

[0031] Thus, a particularly important and surprising finding of the present invention is that - through the discovery of the unexpected effect of chymotrypsin on plasmin and its correlation - a mechanism has been identified that leads to reduced thrombolysis under certain circumstances, including inflammation of the vessel wall and pathological blood flow reduction from mast cell degranulation, but may not interfere with hemostasis in bleeding events. Thus, the chymotrypsin inhibitors of the present invention are not non-specific fibrinolytic agents, but rather novel selective fibrinolytic modulators within thrombi.

[0032] In summary, in the various parts of the present invention, it is demonstrated for the first time that chymotrypsin inhibitors inhibit the degradation of plasmin by chymotrypsin in thrombi, which is determined for the first time in vivo as a pathophysiologically relevant method for thrombi in animals and humans, wherein the size of the thrombi is affected. The inhibition of plasmin degradation by the chymotrypsin inhibitors of the present invention accelerates the dissolution of fibrin clots in blood vessels without affecting bleeding time or hemostasis, thus representing a new method for safe revascularization of blood vessels blocked by thrombi or emboli (Experimental Section, Fig.16 ), potentially saving the lives of patients experiencing thrombotic events such as stroke, pulmonary embolism, or myocardial infarction.

[0033] The present invention relates to compounds of general formula (I) and salts, solvates and solvates of salts thereof

[0034]

[0035] in

[0036] R 1 is hydrogen, methyl or ethyl,

[0037] R2 A group of the formula

[0038]

[0039] in

[0040] * is the attachment site to the nitrogen atom of uracil,

[0041] A is -CH2-, -CH2-CH2-, -O-CH2-## or oxygen,

[0042] in

[0043] ## is the connection site with the benzene ring,

[0044] R 4A is hydrogen, fluorine, chlorine, trifluoromethyl or methyl,

[0045] R 4B is hydrogen, fluorine, chlorine, trifluoromethyl or methyl,

[0046] The condition is R 4A and R 4B At least one of the groups is not hydrogen.

[0047] R 5A For hydrogen,

[0048] R 5B For hydrogen,

[0049] R 6 For hydrogen,

[0050] R 7 For hydrogen,

[0051] R 8 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl,

[0052] R 9 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl,

[0053] R 3 A group of the formula

[0054]

[0055] in

[0056] # is the connection site with the nitrogen atom of uracil,

[0057] E 1 CR 11 or N,

[0058] in

[0059] R 11is hydrogen, (C1-C4)-alkyl, (C3-C7)-cycloalkyl or aminocarbonyl,

[0060] E 2 CR 12 or N,

[0061] in

[0062] R 12 is hydrogen, (C1-C4)-alkyl or (C3-C7)-cycloalkyl,

[0063] G 1 is C=O or SO2,

[0064] G 2 CR 16A R 16B NR 17 , O or S,

[0065] in

[0066] R 16A is hydrogen, fluorine, (C1-C4)-alkyl or hydroxy,

[0067] R 16B is hydrogen, fluorine, chlorine, (C1-C4)-alkyl or trifluoromethyl,

[0068] or

[0069] R 16A and R 16B Together with the carbon atoms to which they are bonded, they form a 3- to 6-membered carbon ring.

[0070] R 17 is hydrogen, (C1-C6)-alkyl, (C3-C7)-cycloalkyl or (C1-C4)-alkoxycarbonyl,

[0071] wherein the (C1-C6)-alkyl radical may be substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, (C3-C7)-cycloalkyl, hydroxy, trifluoromethoxy, (C1-C4)-alkoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0072] R 24 is fluorine or methyl,

[0073] n is a number 0 or 1,

[0074] R 10 is (C1-C4)-alkyl or (C3-C7)-cycloalkyl,

[0075] wherein the (C1-C4)-alkyl group may be substituted by 1 or 2 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0076] R 15 is hydrogen, (C1-C6)-alkyl or (C3-C7)-cycloalkyl,

[0077] Wherein the (C1-C6)-alkyl group may be substituted by 1 or 2 substituents independently selected from the group consisting of fluoro, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl.

[0078] The present invention relates to compounds of general formula (I) and salts, solvates and solvates of salts thereof

[0079]

[0080] in

[0081] R 1 is hydrogen, methyl or ethyl,

[0082] R 2 A group of the formula

[0083]

[0084] in

[0085] * is the attachment site to the nitrogen atom of uracil,

[0086] A is -CH2-, -CH2-CH2-, -O-CH2-## or oxygen,

[0087] in

[0088] ## is the connection site with the benzene ring,

[0089] R 4A is hydrogen, fluorine, chlorine, trifluoromethyl or methyl,

[0090] R 4B is hydrogen, fluorine, chlorine, trifluoromethyl or methyl,

[0091] The condition is R 4A and R 4B At least one of the groups is not hydrogen.

[0092] R 5A For hydrogen,

[0093] R 5B For hydrogen,

[0094] R6 For hydrogen,

[0095] R 7 For hydrogen,

[0096] R 8 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl,

[0097] R 9 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl,

[0098] R 3 A group of the formula

[0099]

[0100] in

[0101] # is the connection site with the nitrogen atom of uracil,

[0102] E 1 CR 11 or N,

[0103] in

[0104] R 11 is hydrogen, (C1-C4)-alkyl, (C3-C7)-cycloalkyl or aminocarbonyl,

[0105] E 2 CR 12 or N,

[0106] in

[0107] R 12 is hydrogen, (C1-C4)-alkyl or (C3-C7)-cycloalkyl,

[0108] G 1 is C=O or SO2,

[0109] G 2 CR 16A R 16B NR 17 , O or S,

[0110] in

[0111] R 16A is hydrogen, fluorine, (C1-C4)-alkyl or hydroxy,

[0112] R 16B is hydrogen, fluorine, chlorine, (C1-C4)-alkyl or trifluoromethyl,

[0113] or

[0114] R 16A and R16B Together with the carbon atoms to which they are bonded, they form a 3- to 6-membered carbon ring.

[0115] R 17 is hydrogen, (C1-C6)-alkyl, (C3-C7)-cycloalkyl or (C1-C4)-alkoxycarbonyl,

[0116] wherein the (C1-C6)-alkyl radical may be substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, (C3-C7)-cycloalkyl, hydroxy, trifluoromethoxy, (C1-C4)-alkoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0117] R 24 is fluorine or methyl,

[0118] n is a number 0 or 1,

[0119] R 10 is (C1-C4)-alkyl or (C3-C7)-cycloalkyl,

[0120] wherein the (C1-C4)-alkyl group may be substituted by 1 or 2 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0121] R 15 is hydrogen, (C1-C6)-alkyl or (C3-C7)-cycloalkyl,

[0122] wherein the (C1-C6)-alkyl group may be substituted by 1 or 2 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0123] It is used to treat and / or prevent stroke, pulmonary embolism, deep vein or superficial vein thrombosis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy of hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites.

[0124] The present invention relates to compounds of general formula (I) and their salts, solvates or solvates of salts

[0125]

[0126] in

[0127] R 1 is hydrogen, methyl or ethyl,

[0128] R2 A group of the formula

[0129]

[0130] in

[0131] * is the attachment site to the nitrogen atom of uracil,

[0132] A is -CH2-, -CH2-CH2-, -O-CH2-## or oxygen,

[0133] in

[0134] ## is the connection site with the benzene ring,

[0135] R 4A is hydrogen, fluorine, chlorine, trifluoromethyl or methyl,

[0136] R 4B is hydrogen, fluorine, chlorine, trifluoromethyl or methyl,

[0137] The condition is R 4A and R 4B At least one of the groups is not hydrogen.

[0138] R 5A For hydrogen,

[0139] R 5B For hydrogen,

[0140] R 6 For hydrogen,

[0141] R 7 For hydrogen,

[0142] R 8 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl,

[0143] R 9 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl,

[0144] R 3 A group of the formula

[0145]

[0146] in

[0147] # is the connection site with the nitrogen atom of uracil,

[0148] E 1 CR 11 or N,

[0149] in

[0150] R 11is hydrogen, (C1-C4)-alkyl, (C3-C7)-cycloalkyl or aminocarbonyl,

[0151] E 2 CR 12 or N,

[0152] in

[0153] R 12 is hydrogen, (C1-C4)-alkyl or (C3-C7)-cycloalkyl,

[0154] G 1 is C=O or SO2,

[0155] G 2 CR 16A R 16B NR 17 , O or S,

[0156] in

[0157] R 16A is hydrogen, fluorine, (C1-C4)-alkyl or hydroxy,

[0158] R 16B is hydrogen, fluorine, chlorine, (C1-C4)-alkyl or trifluoromethyl,

[0159] or

[0160] R 16A and R 16B Together with the carbon atoms to which they are bonded, they form a 3- to 6-membered carbon ring.

[0161] R 17 is hydrogen, (C1-C6)-alkyl, (C3-C7)-cycloalkyl or (C1-C4)-alkoxycarbonyl,

[0162] wherein the (C1-C6)-alkyl radical may be substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, (C3-C7)-cycloalkyl, hydroxy, trifluoromethoxy, (C1-C4)-alkoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0163] R 24 is fluorine or methyl,

[0164] n is a number 0 or 1,

[0165] R 10 is (C1-C4)-alkyl or (C3-C7)-cycloalkyl,

[0166] wherein the (C1-C4)-alkyl group may be substituted by 1 or 2 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0167] R 15 is hydrogen, (C1-C6)-alkyl or (C3-C7)-cycloalkyl,

[0168] wherein the (C1-C6)-alkyl group may be substituted by 1 or 2 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0169] It is used for thrombolytic methods without the risk of bleeding and is therefore used for thrombolysis in stroke, pulmonary embolism, deep or superficial vein thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in infection, inflammation, post-transplant hypercoagulable state, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombolysis in vascular access site thrombosis or occlusion.

[0170] The present invention relates to compounds of general formula (I) and salts, solvates and solvates of salts thereof

[0171]

[0172] in

[0173] R 1 is hydrogen, methyl or ethyl,

[0174] R 2 A group of the formula

[0175]

[0176] in

[0177] * is the attachment site to the nitrogen atom of uracil,

[0178] A is -CH2-, -CH2-CH2-, -O-CH2-## or oxygen,

[0179] in

[0180] ## is the connection site with the benzene ring,

[0181] R 4A is hydrogen, fluorine, chlorine, trifluoromethyl or methyl,

[0182] R 4B is hydrogen, fluorine, chlorine, trifluoromethyl or methyl,

[0183] The condition is R4A and R 4B At least one of the groups is not hydrogen.

[0184] R 5A For hydrogen,

[0185] R 5B For hydrogen,

[0186] R 6 For hydrogen,

[0187] R 7 For hydrogen,

[0188] R 8 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl,

[0189] R 9 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl,

[0190] R 3 A group of the formula

[0191]

[0192] in

[0193] # is the connection site with the nitrogen atom of uracil,

[0194] E 1 CR 11 or N,

[0195] in

[0196] R 11 is hydrogen, (C1-C4)-alkyl, (C3-C7)-cycloalkyl or aminocarbonyl,

[0197] E 2 CR 12 or N,

[0198] in

[0199] R 12 is hydrogen, (C1-C4)-alkyl or (C3-C7)-cycloalkyl,

[0200] G 1 is C=O or SO2,

[0201] G 2 CR 16A R 16B NR 17 , O or S,

[0202] in

[0203] R 16Ais hydrogen, fluorine, (C1-C4)-alkyl or hydroxy,

[0204] R 16B is hydrogen, fluorine, chlorine, (C1-C4)-alkyl or trifluoromethyl,

[0205] or

[0206] R 16A and R 16B Together with the carbon atoms to which they are bonded, they form a 3- to 6-membered carbon ring.

[0207] R 17 is hydrogen, (C1-C6)-alkyl, (C3-C7)-cycloalkyl or (C1-C4)-alkoxycarbonyl,

[0208] wherein the (C1-C6)-alkyl radical may be substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, (C3-C7)-cycloalkyl, hydroxy, trifluoromethoxy, (C1-C4)-alkoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0209] R 24 is fluorine or methyl,

[0210] n is a number 0 or 1,

[0211] R 10 is (C1-C4)-alkyl or (C3-C7)-cycloalkyl,

[0212] wherein the (C1-C4)-alkyl group may be substituted by 1 or 2 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0213] R 15 is hydrogen, (C1-C6)-alkyl or (C3-C7)-cycloalkyl,

[0214] wherein the (C1-C6)-alkyl group may be substituted by 1 or 2 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0215] It is used to treat and / or prevent stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep vein or superficial vein thrombosis, phlebitis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy of hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites.

[0216] The present invention relates to compounds of general formula (I) and salts, solvates and solvates of salts thereof

[0217]

[0218] in

[0219] R 1 is hydrogen, methyl or ethyl,

[0220] R 2 A group of the formula

[0221]

[0222] in

[0223] * is the attachment site to the nitrogen atom of uracil,

[0224] A is -CH2-, -CH2-CH2-, -O-CH2-## or oxygen,

[0225] in

[0226] ## is the connection site with the benzene ring,

[0227] R 4A is hydrogen, fluorine, chlorine, trifluoromethyl or methyl,

[0228] R 4B is hydrogen, fluorine, chlorine, trifluoromethyl or methyl,

[0229] The condition is R 4A and R 4B At least one of the groups is not hydrogen.

[0230] R 5A For hydrogen,

[0231] R 5B For hydrogen,

[0232] R 6 For hydrogen,

[0233] R 7 For hydrogen,

[0234] R 8 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl,

[0235] R 9 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl,

[0236] R 3 A group of the formula

[0237]

[0238] in

[0239] # is the connection site with the nitrogen atom of uracil,

[0240] E 1 CR 11 or N,

[0241] in

[0242] R 11 is hydrogen, (C1-C4)-alkyl, (C3-C7)-cycloalkyl or aminocarbonyl,

[0243] E 2 CR 12 or N,

[0244] in

[0245] R 12 is hydrogen, (C1-C4)-alkyl or (C3-C7)-cycloalkyl,

[0246] G 1 is C=O or SO2,

[0247] G 2 CR 16A R 16B NR 17 , O or S,

[0248] in

[0249] R 16A is hydrogen, fluorine, (C1-C4)-alkyl or hydroxy,

[0250] R 16B is hydrogen, fluorine, chlorine, (C1-C4)-alkyl or trifluoromethyl,

[0251] or

[0252] R 16A and R 16B Together with the carbon atoms to which they are bonded, they form a 3- to 6-membered carbon ring.

[0253] R 17 is hydrogen, (C1-C6)-alkyl, (C3-C7)-cycloalkyl or (C1-C4)-alkoxycarbonyl,

[0254] wherein the (C1-C6)-alkyl radical may be substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, (C3-C7)-cycloalkyl, hydroxy, trifluoromethoxy, (C1-C4)-alkoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0255] R 24is fluorine or methyl,

[0256] n is a number 0 or 1,

[0257] R 10 is (C1-C4)-alkyl or (C3-C7)-cycloalkyl,

[0258] wherein the (C1-C4)-alkyl group may be substituted by 1 or 2 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0259] R 15 is hydrogen, (C1-C6)-alkyl or (C3-C7)-cycloalkyl,

[0260] wherein the (C1-C6)-alkyl group may be substituted by 1 or 2 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0261] It is used for a method of thrombolysis without the risk of bleeding and is therefore used for thrombolysis in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep or superficial vein thrombosis, phlebitis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy in post-transplant hypercoagulable states, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombolysis in thrombosis or occlusion of vascular access sites.

[0262] The present invention relates to compounds of general formula (I) and salts, solvates and solvates of salts thereof

[0263]

[0264] in

[0265] R 1 is hydrogen, methyl or ethyl,

[0266] R 2 A group of the formula

[0267]

[0268] in

[0269] * is the attachment site to the nitrogen atom of uracil,

[0270] A is -CH2-, -CH2-CH2-, -O-CH2-## or oxygen,

[0271] in

[0272] ## is the connection site with the benzene ring,

[0273] R 4A is hydrogen, fluorine, chlorine, trifluoromethyl or methyl,

[0274] R 4B is hydrogen, fluorine, chlorine, trifluoromethyl or methyl,

[0275] The condition is R 4A and R 4B At least one of the groups is not hydrogen.

[0276] R 5A For hydrogen,

[0277] R 5B For hydrogen,

[0278] R 6 For hydrogen,

[0279] R 7 For hydrogen,

[0280] R 8 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl,

[0281] R 9 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl,

[0282] R 3 A group of the formula

[0283]

[0284] in

[0285] # is the connection site with the nitrogen atom of uracil,

[0286] E 1 CR 11 or N,

[0287] in

[0288] R 11 is hydrogen, (C1-C4)-alkyl, (C3-C7)-cycloalkyl or aminocarbonyl,

[0289] E 2 CR 12 or N,

[0290] in

[0291] R 12 is hydrogen, (C1-C4)-alkyl or (C3-C7)-cycloalkyl,

[0292] G 1 is C=O or SO2,

[0293] G 2 CR 16A R 16B NR 17 , O or S,

[0294] in

[0295] R 16A is hydrogen, fluorine, (C1-C4)-alkyl or hydroxy,

[0296] R 16B is hydrogen, fluorine, chlorine, (C1-C4)-alkyl or trifluoromethyl,

[0297] or

[0298] R 16A and R 16B Together with the carbon atoms to which they are bonded, they form a 3- to 6-membered carbon ring.

[0299] R 17 is hydrogen, (C1-C6)-alkyl, (C3-C7)-cycloalkyl or (C1-C4)-alkoxycarbonyl,

[0300] wherein the (C1-C6)-alkyl radical may be substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, (C3-C7)-cycloalkyl, hydroxy, trifluoromethoxy, (C1-C4)-alkoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0301] R 24 is fluorine or methyl,

[0302] n is a number 0 or 1,

[0303] R 10 is (C1-C4)-alkyl or (C3-C7)-cycloalkyl,

[0304] wherein the (C1-C4)-alkyl group may be substituted by 1 or 2 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0305] R 15 is hydrogen, (C1-C6)-alkyl or (C3-C7)-cycloalkyl,

[0306] wherein the (C1-C6)-alkyl group may be substituted by 1 or 2 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0307] It is used in a method for treating and / or preventing stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, phlebitis or microangiopathy.

[0308] The present invention relates to compounds of general formula (I) and salts, solvates and solvates of salts thereof

[0309]

[0310] in

[0311] R 1 is hydrogen, methyl or ethyl,

[0312] R 2 A group of the formula

[0313]

[0314] in

[0315] * is the attachment site to the nitrogen atom of uracil,

[0316] A is -CH2-, -CH2-CH2-, -O-CH2-## or oxygen,

[0317] in

[0318] ## is the connection site with the benzene ring,

[0319] R 4A is hydrogen, fluorine, chlorine, trifluoromethyl or methyl,

[0320] R 4B is hydrogen, fluorine, chlorine, trifluoromethyl or methyl,

[0321] The condition is R 4A and R 4B At least one of the groups is not hydrogen.

[0322] R 5A For hydrogen,

[0323] R 5B For hydrogen,

[0324] R 6 For hydrogen,

[0325] R 7 For hydrogen,

[0326] R 8 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl,

[0327] R 9 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl,

[0328] R 3A group of the formula

[0329]

[0330] in

[0331] # is the connection site with the nitrogen atom of uracil,

[0332] E 1 CR 11 or N,

[0333] in

[0334] R 11 is hydrogen, (C1-C4)-alkyl, (C3-C7)-cycloalkyl or aminocarbonyl,

[0335] E 2 CR 12 or N,

[0336] in

[0337] R 12 is hydrogen, (C1-C4)-alkyl or (C3-C7)-cycloalkyl,

[0338] G 1 is C=O or SO2,

[0339] G 2 CR 16A R 16B NR 17 , O or S,

[0340] in

[0341] R 16A is hydrogen, fluorine, (C1-C4)-alkyl or hydroxy,

[0342] R 16B is hydrogen, fluorine, chlorine, (C1-C4)-alkyl or trifluoromethyl,

[0343] or

[0344] R 16A and R 16B Together with the carbon atoms to which they are bonded, they form a 3- to 6-membered carbon ring.

[0345] R 17 is hydrogen, (C1-C6)-alkyl, (C3-C7)-cycloalkyl or (C1-C4)-alkoxycarbonyl,

[0346] wherein the (C1-C6)-alkyl radical may be substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, (C3-C7)-cycloalkyl, hydroxy, trifluoromethoxy, (C1-C4)-alkoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0347] R 24 is fluorine or methyl,

[0348] n is a number 0 or 1,

[0349] R 10 is (C1-C4)-alkyl or (C3-C7)-cycloalkyl,

[0350] wherein the (C1-C4)-alkyl group may be substituted by 1 or 2 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0351] R 15 is hydrogen, (C1-C6)-alkyl or (C3-C7)-cycloalkyl,

[0352] wherein the (C1-C6)-alkyl group may be substituted by 1 or 2 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0353] It is used for thrombolysis without the risk of bleeding and is therefore used for thrombolysis in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, phlebitis or microangiopathy.

[0354] In another embodiment, the present invention encompasses compounds of the above general formula (I) and salts, solvates and solvates of salts thereof, wherein

[0355] R 1 is hydrogen, methyl or ethyl,

[0356] R 2 A group of the formula

[0357]

[0358] in

[0359] * is the attachment site to the nitrogen atom of uracil,

[0360] A is -CH2-,

[0361] R 4A is chloro or trifluoromethyl,

[0362] R4B For hydrogen,

[0363] R 3 A group of the formula

[0364]

[0365] in

[0366] # is the connection site with the nitrogen atom of uracil,

[0367] E 1 CR 11 ,

[0368] in

[0369] R 11 For hydrogen,

[0370] E 2 For N,

[0371] G 1 is C=O,

[0372] G 2 CR 16A R 16B NR 17 , O or S,

[0373] in

[0374] R 16A is hydrogen, fluorine, methyl or hydroxyl,

[0375] R 16B is hydrogen, fluorine, methyl or trifluoromethyl,

[0376] or

[0377] R 16A and R 16B Together with the carbon atoms to which they are bonded, they form a cyclopropyl ring,

[0378] R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl,

[0379] wherein the (C1-C4)-alkyl group may be substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl,

[0380] Cyclobutyl, hydroxy, trifluoromethoxy, methoxy, ethoxy,

[0381] azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0382] R 24 is hydrogen or fluorine,

[0383] R 10 is (C1-C4)-alkyl,

[0384] R 15 is hydrogen, methyl or ethyl,

[0385] The methyl group and the ethyl group may be substituted by one substituent selected from the group consisting of fluorine,

[0386] trifluoromethyl and cyclopropyl,

[0387] It is used to treat and / or prevent stroke, pulmonary embolism, deep vein or superficial vein thrombosis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy of hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites.

[0388] In another embodiment, the present invention encompasses compounds of the above general formula (I) and salts, solvates and solvates of salts thereof, wherein

[0389] R 1 is hydrogen, methyl or ethyl,

[0390] R 2 A group of the formula

[0391]

[0392] in

[0393] * is the connection site with the nitrogen atom of uracil,

[0394] A is -CH2-,

[0395] R 4A is chloro or trifluoromethyl,

[0396] R 4B Hydrogen

[0397] R 3 A group of the formula

[0398]

[0399] in

[0400] # is the connection site with the nitrogen atom of uracil,

[0401] E 1 CR 11 ,

[0402] in

[0403] R 11 For hydrogen,

[0404] E 2 For N,

[0405] G 1 is C=O,

[0406] G 2 CR 16A R 16B NR 17 , O or S,

[0407] in

[0408] R 16A is hydrogen, fluorine, methyl or hydroxyl,

[0409] R 16B is hydrogen, fluorine, methyl or trifluoromethyl,

[0410] or

[0411] R 16A and R 16B Together with the carbon atoms to which they are bonded, they form a cyclopropyl ring,

[0412] R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl,

[0413] wherein the (C1-C4)-alkyl group may be substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl,

[0414] Cyclobutyl, hydroxy, trifluoromethoxy, methoxy, ethoxy,

[0415] azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0416] R 24 is hydrogen or fluorine,

[0417] R 10 is (C1-C4)-alkyl,

[0418] R 15 is hydrogen, methyl or ethyl,

[0419] The methyl group and the ethyl group may be substituted by one substituent selected from the group consisting of fluorine,

[0420] trifluoromethyl and cyclopropyl,

[0421] It is used for thrombolytic methods without the risk of bleeding and is therefore used for thrombolysis in stroke, pulmonary embolism, deep or superficial vein thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in infection, inflammation, post-transplant hypercoagulable state, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombolysis in vascular access site thrombosis or occlusion.

[0422] In another embodiment, the present invention encompasses compounds of the above general formula (I) and salts, solvates and solvates of salts thereof, wherein

[0423] R 1 is hydrogen, methyl or ethyl,

[0424] R 2 A group of the formula

[0425]

[0426] in

[0427] * is the connection site with the nitrogen atom of uracil,

[0428] A is -CH2-,

[0429] R 4A is chloro or trifluoromethyl,

[0430] R 4B For hydrogen,

[0431] R 3 A group of the formula

[0432]

[0433] in

[0434] # is the connection site with the nitrogen atom of uracil,

[0435] E 1 CR 11 ,

[0436] in

[0437] R 11 For hydrogen,

[0438] E 2 For N,

[0439] G 1 is C=O,

[0440] G 2 CR 16A R 16B NR 17 , O or S,

[0441] in

[0442] R 16A is hydrogen, fluorine, methyl or hydroxyl,

[0443] R 16B is hydrogen, fluorine, methyl or trifluoromethyl,

[0444] or

[0445] R 16A and R 16B Together with the carbon atoms to which they are bonded, they form a cyclopropyl ring,

[0446] R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl,

[0447] wherein the (C1-C4)-alkyl group may be substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxy, trifluoromethoxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0448] R 24 is hydrogen or fluorine,

[0449] R 10 is (C1-C4)-alkyl,

[0450] R 15 is hydrogen, methyl or ethyl,

[0451] wherein the methyl group and the ethyl group may be substituted by one substituent selected from the group consisting of fluorine, trifluoromethyl and cyclopropyl,

[0452] It is used to treat and / or prevent stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep vein or superficial vein thrombosis, phlebitis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy of hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites.

[0453] In another embodiment, the present invention encompasses compounds of the above general formula (I) and salts, solvates and solvates of salts thereof, wherein

[0454] R 1 is hydrogen, methyl or ethyl,

[0455] R 2 A group of the formula

[0456]

[0457] in

[0458] * is the connection site with the nitrogen atom of uracil,

[0459] A is -CH2-,

[0460] R 4A is chloro or trifluoromethyl,

[0461] R 4B Hydrogen

[0462] R 3 A group of the formula

[0463]

[0464] in

[0465] # is the connection site with the nitrogen atom of uracil,

[0466] E 1 CR 11 ,

[0467] in

[0468] R 11 For hydrogen,

[0469] E 2 For N,

[0470] G 1 is C=O,

[0471] G 2 CR 16A R 16B NR 17 , O or S,

[0472] in

[0473] R 16A is hydrogen, fluorine, methyl or hydroxyl,

[0474] R 16B is hydrogen, fluorine, methyl or trifluoromethyl,

[0475] or

[0476] R 16A and R 16B Together with the carbon atoms to which they are bonded, they form a cyclopropyl ring,

[0477] R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl,

[0478] wherein the (C1-C4)-alkyl group may be substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxy, trifluoromethoxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0479] R 24 is hydrogen or fluorine,

[0480] R 10 is (C1-C4)-alkyl,

[0481] R 15 is hydrogen, methyl or ethyl,

[0482] wherein the methyl group and the ethyl group may be substituted by one substituent selected from the group consisting of fluorine, trifluoromethyl and cyclopropyl,

[0483] It is used for a method of thrombolysis without the risk of bleeding and is therefore used for thrombolysis in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep or superficial vein thrombosis, phlebitis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy in post-transplant hypercoagulable states, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombolysis in thrombosis or occlusion of vascular access sites.

[0484] In another embodiment, the present invention encompasses compounds of the above general formula (I) and salts, solvates and solvates of salts thereof, wherein

[0485] R 1 is hydrogen, methyl or ethyl,

[0486] R 2 A group of the formula

[0487]

[0488] in

[0489] * is the connection site with the nitrogen atom of uracil,

[0490] A is -CH2-,

[0491] R 4A is chloro or trifluoromethyl,

[0492] R 4B For hydrogen,

[0493] R 3 A group of the formula

[0494]

[0495] in

[0496] # is the connection site with the nitrogen atom of uracil,

[0497] E 1 CR 11 ,

[0498] in

[0499] R 11 For hydrogen,

[0500] E 2 For N,

[0501] G 1 For C=O,

[0502] G 2 CR 16A R 16B NR 17 , O or S,

[0503] in

[0504] R 16A is hydrogen, fluorine, methyl or hydroxyl,

[0505] R 16B is hydrogen, fluorine, methyl or trifluoromethyl,

[0506] or

[0507] R 16A and R 16B Together with the carbon atoms to which they are bonded, they form a cyclopropyl ring,

[0508] R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl,

[0509] wherein the (C1-C4)-alkyl group may be substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxy, trifluoromethoxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0510] R 24 is hydrogen or fluorine,

[0511] R 10 is (C1-C4)-alkyl,

[0512] R 15 is hydrogen, methyl or ethyl,

[0513] wherein the methyl group and the ethyl group may be substituted by one substituent selected from the group consisting of fluorine, trifluoromethyl and cyclopropyl,

[0514] It is used in a method for treating and / or preventing stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep or superficial vein thrombosis, phlebitis or microangiopathy.

[0515] In another embodiment, the present invention encompasses compounds of the above general formula (I) and salts, solvates and solvates of salts thereof, wherein

[0516] R 1 is hydrogen, methyl or ethyl,

[0517] R 2 A group of the formula

[0518]

[0519] in

[0520] * is the connection site with the nitrogen atom of uracil,

[0521] A is -CH2-,

[0522] R 4A is chloro or trifluoromethyl,

[0523] R 4B For hydrogen,

[0524] R 3 A group of the formula

[0525]

[0526] in

[0527] # is the connection site with the nitrogen atom of uracil,

[0528] E 1 CR 11 ,

[0529] in

[0530] R 11 For hydrogen,

[0531] E 2 For N,

[0532] G 1 is C=O,

[0533] G 2 CR 16A R 16B NR 17 , O or S,

[0534] in

[0535] R 16A is hydrogen, fluorine, methyl or hydroxyl,

[0536] R 16B is hydrogen, fluorine, methyl or trifluoromethyl,

[0537] or

[0538] R 16A and R 16B Together with the carbon atoms to which they are bonded, they form a cyclopropyl ring,

[0539] R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl,

[0540] wherein the (C1-C4)-alkyl group may be substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxy, trifluoromethoxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0541] R 24 is hydrogen or fluorine,

[0542] R 10 is (C1-C4)-alkyl,

[0543] R 15 is hydrogen, methyl or ethyl,

[0544] wherein the methyl group and the ethyl group may be substituted by one substituent selected from the group consisting of fluorine, trifluoromethyl and cyclopropyl,

[0545] It is used for thrombolysis without the risk of bleeding and is therefore used for thrombolysis in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, phlebitis or microangiopathy.

[0546] The compounds of the invention are compounds of formula (I) and their salts, solvates and solvates of salts, compounds covered by formula (I) as specified below and their salts, solvates and solvates of salts, and compounds covered by formula (I) and specified below as working examples and their salts, solvates and solvates of salts, as long as the compounds covered by formula (I) and specified below are not already salts, solvates and solvates of salts.

[0547] In the context of the present invention, preferred salts are physiologically acceptable salts of the compounds according to the invention. Also included are salts which are not suitable for pharmaceutical applications per se but which can be used, for example, for isolating, purifying or storing the compounds according to the invention.

[0548] Physiologically acceptable salts of the compounds of the invention include acid addition salts of inorganic acids, carboxylic acids and sulfonic acids, for example, salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, naphthalene disulfonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid and benzoic acid.

[0549] Physiologically acceptable salts of the compounds according to the invention also include salts with customary bases, such as and preferably alkali metal salts (e.g. sodium and potassium salts), alkaline earth metal salts (e.g. calcium and magnesium salts) and ammonium salts derived from ammonia or organic amines having 1 to 16 carbon atoms, such as and preferably ethylamine, diethylamine, triethylamine, N,N-ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dimethylaminoethanol, diethylaminoethanol, procaine, dicyclohexylamine, dibenzylamine, N-methylpiperidine, N-methylmorpholine, arginine, lysine, choline and 1,2-ethylenediamine.

[0550] In the context of the present invention, solvates are those forms of the compounds according to the invention which form complexes in the solid or liquid state by coordination with solvent molecules. Hydrates are a special form of solvates in which the coordination takes place with water. In the context of the present invention, preferred solvates are hydrates.

[0551] Depending on the structure, the compounds according to the invention may exist in different stereoisomeric forms, i.e. in the form of configurational isomers or, where appropriate, also as conformational isomers (enantiomers and / or diastereomers, including those in the case of atropisomers). The present invention therefore includes the enantiomers or diastereomers and their respective mixtures. Stereoisomerically identical components can be separated in a known manner from mixtures of enantiomers and / or diastereomers; for this purpose, preferably chromatography is used, in particular HPLC chromatography on an achiral or chiral phase.

[0552] Where the compounds of the present invention can occur in tautomeric forms, the present invention includes all tautomeric forms.

[0553] The present invention also includes all suitable isotopic variants of the compounds of the present invention. Isotopic variants of the compounds of the present invention are understood herein to mean such a compound: wherein at least one atom in the compounds of the present invention is exchanged for another atom with the same atomic number but with an atomic mass that is usually or mainly present in nature. Examples of isotopes that can be incorporated into the compounds of the present invention are isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine and iodine, such as 2H (deuterium), 3H (tritium), 13C, 14C, 15N, 17O, 18O, 32P, 33P, 33S, 34S, 35S, 36S, 18F, 36Cl, 82Br, 123I, 124I, 129I and 131I. Specific isotopic variants of the compounds of the present invention, especially those that have been incorporated with one or more radioactive isotopes, may be useful, for example, to examine the mechanism of action or the distribution of the active ingredient in the body; due to the relatively easy preparation and detectability, compounds labeled with 3H or 14C isotopes are particularly suitable for this purpose. In addition, since the incorporation of isotopes (e.g., deuterium) makes the compound metabolically more stable, it can bring specific therapeutic benefits, such as extending the half-life in the body or reducing the required active dose; therefore, such modifications of the compounds of the present invention may also constitute preferred embodiments of the present invention in some cases. Isotopic variants of the compounds of the present invention can be prepared by methods known to those skilled in the art, for example, by the following methods and the description reproduced in the working examples, by using the corresponding isotopic modifications of the specific reagents and / or starting compounds therein.

[0554] In addition, the present invention also includes prodrugs of the compounds of the present invention. The term "prodrug" herein refers to a compound that may itself be biologically active or inactive but is converted into a compound of the present invention when present in the body, for example by metabolic or hydrolytic pathways.

[0555] In the context of the present invention, unless otherwise stated, substituents are each defined as follows:

[0556] Alkyl in the context of the present invention is a straight-chain or branched alkyl radical having the number of carbon atoms specified in each case. Preferred examples include: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, 1-methylpropyl, tert-butyl, n-pentyl, isopentyl, 1-ethylpropyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,4-dimethylpentyl, 4,4-dimethylpentyl and 1,4,4-trimethylpentyl.

[0557] Cycloalkyl in the context of the present invention is a monocyclic saturated alkyl group having 3 to 7 carbon atoms. Preferred examples include: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

[0558] Alkylcarbonyl in the context of the present invention is a straight-chain or branched alkyl group having 1 to 4 carbon atoms and having a carbonyl group attached at position 1. Preferred examples include: methylcarbonyl, ethylcarbonyl, n-propylcarbonyl, isopropylcarbonyl, n-butylcarbonyl, isobutylcarbonyl and tert-butylcarbonyl.

[0559] Alkoxy in the context of the present invention is a straight-chain or branched alkoxy group having 1 to 4 carbon atoms. Preferred examples include: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy and tert-butoxy.

[0560] Alkoxycarbonyl in the context of the present invention is a straight-chain or branched alkoxy group having 1 to 4 carbon atoms in which the carbonyl group is attached to oxygen. Preferred examples include: methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl and tert-butoxycarbonyl.

[0561] Alkylthio in the context of the present invention is a straight or branched alkyl group having 1 to 4 carbon atoms and bonded via a sulfur atom. Preferred examples include: methylthio, ethylthio, n-propylthio, isopropylthio, 1-methylpropylthio, n-butylthio, isobutylthio and tert-butylthio.

[0562] Alkylsulfonyl in the context of the present invention is a straight-chain or branched alkyl group having 1 to 4 carbon atoms and bonded via a sulfonyl group. Preferred examples include: methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, isopropylsulfonyl, n-butylsulfonyl and tert-butylsulfonyl.

[0563] 4- to 7-membered heterocyclic radicals in the context of the present invention are monocyclic saturated heterocyclic rings having 4 to 7 ring atoms, comprising 1 or 2 ring heteroatoms selected from N, O, S, SO and / or SO2, and connected via ring carbon atoms or, if appropriate, via ring nitrogen atoms. Examples include: azetidinyl, oxetanyl, pyrrolidinyl, pyrazolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl. Preferably: azetidinyl, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl and morpholinyl.

[0564] A 5- to 7-membered heterocyclyl group in the context of the present invention is a partially unsaturated heterocycle having a total of 5 to 7 ring atoms, including 1 to 3 ring heteroatoms selected from N, O, S and / or SO2, and having R 3 Examples include: dihydropyrrolyl, dihydroimidazolyl, dihydrothiazolidine dioxide, dihydrooxazolyl, dihydropyridinyl, tetrahydropyrazinyl and dihydrooxazinyl.

[0565] Heteroaryl in the context of the present invention is a monocyclic aromatic heterocycle (heteroaromatic) having a total of 5 or 6 ring atoms, including up to 3 identical or different ring heteroatoms selected from N, O and / or S, and having R 3 The benzene ring in is fused. Examples include furanyl, pyrrolyl, thienyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl and triazinyl. Pyrazolyl, imidazolyl, thiazolyl and triazolyl are preferred.

[0566] Halogen in the context of the present invention includes fluorine, chlorine, bromine and iodine. Chlorine or fluorine is preferred.

[0567] An oxo group in the context of the present invention is an oxygen atom which is bonded to a carbon atom or a sulfur atom via a double bond.

[0568] In R 2 and R 3 In the formulae of the groups which may be represented, the end points of the lines marked with the symbols * or # or ## do not represent carbon atoms or CH2 groups, but are connected to R 2 and R 3 The portion of the bond to which each atom is bonded.

[0569] If a radical in a compound according to the invention is substituted, the radical may be monosubstituted or polysubstituted, unless otherwise indicated. In the context of the present invention, all radicals which occur more than once are defined independently of one another. Preference is given to substitution with one or two identical or different substituents. Very particular preference is given to substitution with one substituent.

[0570] In the context of the present invention, the term "treatment" or "treating" includes inhibiting, delaying, checking, alleviating, attenuating, limiting, reducing, suppressing, resisting or curing a disease, condition, disorder, injury or health problem, or the development, course or progression of these states and / or symptoms of these states. The term "therapy" is understood herein to be synonymous with the term "treat".

[0571] The terms "prevention," "prophylaxis," or "preclusion" are used synonymously in the context of the present invention and refer to avoiding or reducing the risk of contracting, experiencing, afflicting or having a disease, condition, disorder, injury or health problem, or the development or progression of these states and / or symptoms of these states.

[0572] Treatment or prevention of a disease, condition, disorder, injury or health problem may be partial or complete.

[0573] In another embodiment, the present invention encompasses compounds of the above general formula (I) and salts, solvates and solvates of salts thereof, wherein

[0574] R 1 For hydrogen,

[0575] R 2 A group of the formula

[0576]

[0577] in

[0578] * is the connection site with the nitrogen atom of uracil,

[0579] R 5A For hydrogen,

[0580] R 5B For hydrogen,

[0581] R 6 For hydrogen,

[0582] R 7 For hydrogen,

[0583] R 8 is fluorine, chlorine or trifluoromethyl,

[0584] R 9 is fluorine, chlorine, trifluoromethyl or methyl,

[0585] R 3 A group of the formula

[0586]

[0587] in

[0588] # is the connection site with the nitrogen atom of uracil,

[0589] E 1 CR 11 ,

[0590] in

[0591] R 11 For hydrogen,

[0592] E 2 For N,

[0593] G 1 is C=O,

[0594] G 2 CR 16A R 16B NR 17 , O or S,

[0595] in

[0596] R 16A is hydrogen, fluorine, methyl or hydroxyl,

[0597] R 16B is hydrogen, fluorine, methyl or trifluoromethyl,

[0598] or

[0599] R 16A and R 16B Together with the carbon atoms to which they are bonded, they form a cyclopropyl ring,

[0600] R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl,

[0601] wherein the (C1-C4)-alkyl group may be substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxy, trifluoromethoxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0602] R 24 is hydrogen or fluorine,

[0603] R 10 is (C1-C4)-alkyl,

[0604] R 15 is hydrogen, methyl or ethyl,

[0605] The methyl group and the ethyl group may be substituted by one substituent selected from the group consisting of fluorine,

[0606] trifluoromethyl and cyclopropyl,

[0607] It is used to treat and / or prevent stroke, pulmonary embolism, deep vein or superficial vein thrombosis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy of hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites.

[0608] In another embodiment, the present invention encompasses compounds of the above general formula (I) and salts, solvates and solvates of salts thereof, wherein

[0609] R 1 For hydrogen,

[0610] R 2 A group of the formula

[0611]

[0612] in

[0613] * is the connection site with the nitrogen atom of uracil,

[0614] R 5A For hydrogen,

[0615] R 5B For hydrogen,

[0616] R 6 For hydrogen,

[0617] R 7 For hydrogen,

[0618] R 8 is fluorine, chlorine or trifluoromethyl,

[0619] R 9 is fluorine, chlorine, trifluoromethyl or methyl,

[0620] R 3 A group of the formula

[0621]

[0622] in

[0623] # is the connection site with the nitrogen atom of uracil,

[0624] E 1 CR 11 ,

[0625] in

[0626] R 11 For hydrogen,

[0627] E 2 For N,

[0628] G 1 is C=O,

[0629] G 2 CR 16A R 16B NR 17 , O or S,

[0630] in

[0631] R 16A is hydrogen, fluorine, methyl or hydroxyl,

[0632] R 16B is hydrogen, fluorine, methyl or trifluoromethyl,

[0633] or

[0634] R 16A and R 16BTogether with the carbon atoms to which they are bonded, they form a cyclopropyl ring,

[0635] R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl,

[0636] wherein the (C1-C4)-alkyl group may be substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxy, trifluoromethoxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0637] R 24 is hydrogen or fluorine,

[0638] R 10 is (C1-C4)-alkyl,

[0639] R 15 is hydrogen, methyl or ethyl,

[0640] wherein the methyl group and the ethyl group may be substituted by one substituent selected from the group consisting of fluorine, trifluoromethyl and cyclopropyl,

[0641] It is used for thrombolytic methods without the risk of bleeding and is therefore used for thrombolysis in stroke, pulmonary embolism, deep or superficial vein thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in infection, inflammation, post-transplant hypercoagulable state, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombolysis in vascular access site thrombosis or occlusion.

[0642] In another embodiment, the present invention encompasses compounds of the above general formula (I) and salts, solvates and solvates of salts thereof, wherein

[0643] R 1 For hydrogen,

[0644] R 2 A group of the formula

[0645]

[0646] in

[0647] * is the connection site with the nitrogen atom of uracil,

[0648] R 5A For hydrogen,

[0649] R 5B For hydrogen,

[0650] R 6 For hydrogen,

[0651] R 7 For hydrogen,

[0652] R 8 is fluorine, chlorine or trifluoromethyl,

[0653] R 9 is fluorine, chlorine, trifluoromethyl or methyl,

[0654] R 3 A group of the formula

[0655]

[0656] in

[0657] # is the connection site with the nitrogen atom of uracil,

[0658] E 1 CR 11 ,

[0659] in

[0660] R 11 For hydrogen,

[0661] E 2 For N,

[0662] G 1 is C=O,

[0663] G 2 CR 16A R 16B NR 17 , O or S,

[0664] in

[0665] R 16A is hydrogen, fluorine, methyl or hydroxyl,

[0666] R 16B is hydrogen, fluorine, methyl or trifluoromethyl,

[0667] or

[0668] R 16A and R 16B Together with the carbon atoms to which they are bonded, they form a cyclopropyl ring,

[0669] R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl,

[0670] wherein the (C1-C4)-alkyl group may be substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxy, trifluoromethoxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0671] R 24 is hydrogen or fluorine,

[0672] R 10 is (C1-C4)-alkyl,

[0673] R 15 is hydrogen, methyl or ethyl,

[0674] wherein the methyl group and the ethyl group may be substituted by one substituent selected from the group consisting of fluorine, trifluoromethyl and cyclopropyl,

[0675] It is used to treat and / or prevent stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep vein or superficial vein thrombosis, phlebitis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy of hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites.

[0676] In another embodiment, the present invention encompasses compounds of the above general formula (I) and salts, solvates and solvates of salts thereof, wherein

[0677] R 1 For hydrogen,

[0678] R 2 A group of the formula

[0679]

[0680] in

[0681] * is the connection site with the nitrogen atom of uracil,

[0682] R 5A For hydrogen,

[0683] R 5B For hydrogen,

[0684] R 6 For hydrogen,

[0685] R 7 For hydrogen,

[0686] R 8 is fluorine, chlorine or trifluoromethyl,

[0687] R 9 is fluorine, chlorine, trifluoromethyl or methyl,

[0688] R 3 A group of the formula

[0689]

[0690] in

[0691] # is the connection site with the nitrogen atom of uracil,

[0692] E 1 CR 11 ,

[0693] in

[0694] R 11 For hydrogen,

[0695] E 2 For N,

[0696] G 1 is C=O,

[0697] G 2 CR 16A R 16B NR 17 , O or S,

[0698] in

[0699] R 16A is hydrogen, fluorine, methyl or hydroxyl,

[0700] R 16B is hydrogen, fluorine, methyl or trifluoromethyl,

[0701] or

[0702] R 16A and R 16B Together with the carbon atoms to which they are bonded, they form a cyclopropyl ring,

[0703] R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl,

[0704] wherein the (C1-C4)-alkyl group may be substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxy, trifluoromethoxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0705] R 24 is hydrogen or fluorine,

[0706] R 10 is (C1-C4)-alkyl,

[0707] R 15 is hydrogen, methyl or ethyl,

[0708] wherein the methyl group and the ethyl group may be substituted by one substituent selected from the group consisting of fluorine, trifluoromethyl and cyclopropyl,

[0709] It is used for a method of thrombolysis without the risk of bleeding and is therefore used for thrombolysis in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep or superficial vein thrombosis, phlebitis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy in post-transplant hypercoagulable states, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombolysis in thrombosis or occlusion of vascular access sites.

[0710] In another embodiment, the present invention encompasses compounds of the above general formula (I) and salts, solvates and solvates of salts thereof, wherein

[0711] R 1 For hydrogen,

[0712] R 2 A group of the formula

[0713]

[0714] in

[0715] * is the connection site with the nitrogen atom of uracil,

[0716] R 5A For hydrogen,

[0717] R 5B For hydrogen,

[0718] R 6 For hydrogen,

[0719] R 7 For hydrogen,

[0720] R 8 is fluorine, chlorine or trifluoromethyl,

[0721] R 9 is fluorine, chlorine, trifluoromethyl or methyl,

[0722] R 3 A group of the formula

[0723]

[0724] in

[0725] # is the connection site with the nitrogen atom of uracil,

[0726] E 1 CR 11 ,

[0727] in

[0728] R 11 For hydrogen,

[0729] E 2 For N,

[0730] G 1 For C=O,

[0731] G 2 CR 16A R 16B NR 17 , O or S,

[0732] in

[0733] R 16A is hydrogen, fluorine, methyl or hydroxyl,

[0734] R 16B is hydrogen, fluorine, methyl or trifluoromethyl,

[0735] or

[0736] R 16A and R 16B Together with the carbon atoms to which they are bonded, they form a cyclopropyl ring,

[0737] R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl,

[0738] wherein the (C1-C4)-alkyl group may be substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxy, trifluoromethoxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0739] R 24 is hydrogen or fluorine,

[0740] R 10 is (C1-C4)-alkyl,

[0741] R 15 is hydrogen, methyl or ethyl,

[0742] wherein the methyl group and the ethyl group may be substituted by one substituent selected from the group consisting of fluorine, trifluoromethyl and cyclopropyl,

[0743] It is used in a method for treating and / or preventing stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep or superficial vein thrombosis, phlebitis or microangiopathy.

[0744] In another embodiment, the present invention encompasses compounds of the above general formula (I) and salts, solvates and solvates of salts thereof, wherein

[0745] R 1 For hydrogen,

[0746] R 2 A group of the formula

[0747]

[0748] in

[0749] * is the connection site with the nitrogen atom of uracil,

[0750] R 5A For hydrogen,

[0751] R 5B For hydrogen,

[0752] R 6 For hydrogen,

[0753] R 7 For hydrogen,

[0754] R 8 is fluorine, chlorine or trifluoromethyl,

[0755] R 9 is fluorine, chlorine, trifluoromethyl or methyl,

[0756] R 3 A group of the formula

[0757]

[0758] in

[0759] # is the connection site with the nitrogen atom of uracil,

[0760] E 1 CR 11 ,

[0761] in

[0762] R 11 For hydrogen,

[0763] E 2 For N,

[0764] G 1 is C=O,

[0765] G 2 CR 16A R 16B NR 17 , O or S,

[0766] in

[0767] R 16A is hydrogen, fluorine, methyl or hydroxyl,

[0768] R 16B is hydrogen, fluorine, methyl or trifluoromethyl,

[0769] or

[0770] R 16A and R 16B Together with the carbon atoms to which they are bonded, they form a cyclopropyl ring,

[0771] R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl,

[0772] wherein the (C1-C4)-alkyl group may be substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxy, trifluoromethoxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl,

[0773] R 24 is hydrogen or fluorine,

[0774] R 10 is (C1-C4)-alkyl,

[0775] R 15 is hydrogen, methyl or ethyl,

[0776] wherein the methyl group and the ethyl group may be substituted by one substituent selected from the group consisting of fluorine, trifluoromethyl and cyclopropyl,

[0777] It is used for thrombolysis without the risk of bleeding and is therefore used for thrombolysis in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, phlebitis or microangiopathy.

[0778] In the context of the present invention, preference is given to compounds selected from the group consisting of 1-(1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(6-fluoro-1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), pyrimidine-5-carboxylic acid (R enantiomer), 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1-(1,3,3-trimethyl-2-oxo-2,3-dihydro-1H-indol-5-yl)-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid ( R enantiomer), 1-(1'-methyl-2'-oxo-1',2'-dihydrospiro[cyclopropane-1,3'-indol]-5'-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzothiazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid ( R enantiomer) and 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid ethyl ester (R enantiomer), which are used for treating and / or preventing stroke, pulmonary embolism, deep vein or superficial vein thrombosis, thrombotic microangiopathy, infection, inflammation, post-transplant hypercoagulable state thrombotic microangiopathy, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, vascular access site thrombosis or occlusion.

[0779] In the context of the present invention, preference is given to compounds selected from the group consisting of the following compounds and their salts, solvates and solvates of salts: 1-(1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(6-fluoro-1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1 ,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1-(1,3,3-trimethyl-2-oxo-2,3-dihydro-1H-indol-5-yl)-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(1'-methyl-2'-oxo-1',2'-dihydrospiro[cyclopropane-1,3'-indol]-5'-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzothiazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) and ethyl 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylate (R enantiomer), which are used in a thrombolytic method without bleeding risk and are therefore used in stroke, pulmonary embolism, deep or superficial vein thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in infection, inflammation, post-transplant hypercoagulable state, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombolysis in vascular access site thrombosis or occlusion.

[0780] In the context of the present invention, preference is given to compounds selected from the group consisting of 1-(1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(6-fluoro-1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 5-carboxylic acid (R enantiomer), 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1-(1,3,3-trimethyl-2-oxo-2,3-dihydro-1H-indol-5-yl)-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(1'-methyl-2'-oxo-1',2'-dihydrospiro[cyclopropane-1,3'-indol]-5'-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzothiazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) and 1-(3 -methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid ethyl ester (R enantiomer), which is used for treating and / or preventing stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep vein or superficial vein thrombosis, phlebitis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy in hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access site.

[0781] In the context of the present invention, preference is given to compounds selected from the group consisting of the following compounds and their salts, solvates and solvates of salts: 1-(1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(6-fluoro-1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3 ,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1-(1,3,3-trimethyl-2-oxo-2,3-dihydro-1H-indol-5-yl)-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) , 1-(1'-methyl-2'-oxo-1',2'-dihydrospiro[cyclopropane-1,3'-indol]-5'-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzothiazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) and 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzothiazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer). The invention relates to ethyl 2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylate (R enantiomer), which is used for a thrombolytic method without bleeding risk and is therefore used for stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep or superficial vein thrombosis, phlebitis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy in hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombolysis in thrombosis or occlusion of vascular access sites.

[0782] In the context of the present invention, preference is given to compounds selected from the group consisting of 1-(1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzoimidazol-5-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(6-fluoro-1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzoimidazol-5-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1-(1,3,3-trimethyl-2-oxo-2,3-dihydro-1H-inden-1-yl)- 1-(1'-methyl-2'-oxo-1',2'-dihydrospiro[cyclopropane-1,3'-indol]-5'-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzothiazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), )-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) and 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid ethyl ester (R enantiomer) for use in a method for treating and / or preventing stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep vein or superficial vein thrombosis, phlebitis or microangiopathy.

[0783] In the context of the present invention, preference is given to compounds selected from the group consisting of the following compounds and their salts, solvates and solvates of salts: 1-(1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(6-fluoro-1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3 -dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1-(1,3,3-trimethyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl] dihydro-1H-indol-5-yl)-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(1'-methyl-2'-oxo-1',2'-dihydrospiro[cyclopropane-1,3'-indol]-5'-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzothiazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), The invention relates to ethyl 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylate (R enantiomer) and ethyl 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylate (R enantiomer) for use in a thrombolytic method without the risk of bleeding and therefore for use in thrombolysis in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, phlebitis or microangiopathy.

[0784] In the context of the present invention, 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) is preferred for use in a method for treating and / or preventing stroke, pulmonary embolism, deep or superficial vein thrombosis, thrombotic microangiopathy, thrombotic microangiopathy of infection, inflammation, hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites.

[0785] In the context of the present invention, 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) is preferred for use in a method for treating and / or preventing stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep or superficial vein thrombosis, phlebitis, thrombotic microangiopathy, thrombotic microangiopathy of infection, inflammation, hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites.

[0786] In the context of the present invention, preference is given to 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) for use in a thrombolytic method without the risk of bleeding and therefore for use in stroke, pulmonary embolism, deep or superficial vein thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in infection, inflammation, post-transplant hypercoagulable state, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombolysis in vascular access site thrombosis or occlusion.

[0787] In the context of the present invention, preference is given to 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) for use in a thrombolytic method without the risk of bleeding and therefore for use in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep or superficial vein thrombosis, phlebitis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy in hypercoagulable states after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombolysis in vascular access site thrombosis or occlusion.

[0788] In the context of the present invention, preference is given to 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) for use in a method for the treatment and / or prevention of stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, microangiopathy or phlebitis.

[0789] In the context of the present invention, preference is given to 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) for use in a thrombolytic method without bleeding risk and therefore for use in thrombolysis in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, microangiopathy or phlebitis.

[0790] In the context of the present invention, preference is given to 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) for use in a method for the treatment and / or prevention of stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease or phlebitis.

[0791] In the context of the present invention, preference is given to 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) for use in a thrombolytic method without bleeding risk and therefore for use in thrombolysis in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease or phlebitis.

[0792] In the context of the present invention, preference is given to 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) for use in a method for the treatment and / or prevention of stroke, pulmonary embolism, microangiopathy or phlebitis.

[0793] In the context of the present invention, preference is given to 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) for use in a thrombolytic method without bleeding risk and therefore for use in thrombolysis in stroke, pulmonary embolism, microangiopathy or phlebitis.

[0794] In the context of the present invention, preference is given to 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) for use in a method for the treatment and / or prevention of stroke.

[0795] In the context of the present invention, preference is given to 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) for use in a thrombolytic method without bleeding risk and therefore for use in thrombolysis in stroke.

[0796] In the context of the present invention, 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) is preferred for use in a method for the treatment and / or prevention of pulmonary embolism.

[0797] In the context of the present invention, preference is given to 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) for use in a thrombolytic method without the risk of bleeding and therefore for use in thrombolysis in pulmonary embolism.

[0798] In the context of the present invention, preference is given to 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) for use in a method for the treatment and / or prevention of myocardial infarction.

[0799] In the context of the present invention, preference is given to 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) for use in a thrombolytic method without bleeding risk and therefore for use in thrombolysis in myocardial infarction.

[0800] In the context of the present invention, preference is given to 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) for use in a method for the treatment and / or prevention of peripheral arterial disease.

[0801] In the context of the present invention, preference is given to 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) for use in a thrombolytic method without the risk of bleeding and therefore for use in thrombolysis in peripheral arterial disease.

[0802] In the context of the present invention, preference is given to 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) for use in a method for the treatment and / or prevention of microangiopathy.

[0803] In the context of the present invention, preference is given to 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) for use in a thrombolytic method without bleeding risk and therefore for use in thrombolysis in microangiopathy.

[0804] In the context of the present invention, preference is given to 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) for use in a method for the treatment and / or prevention of phlebitis.

[0805] In the context of the present invention, preference is given to 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) for use in a thrombolytic method without the risk of bleeding and therefore for use in thrombolysis in phlebitis.

[0806] In the context of the present invention, preferred are compounds of formula (I) wherein

[0807] R 1 For hydrogen,

[0808] and salts, solvates and solvates of salts thereof.

[0809] In the context of the present invention, preference is given to compounds of formula (I) and their salts, solvates and solvates of salts, wherein

[0810] R 1 For hydrogen,

[0811] R 2 A group of the formula

[0812]

[0813] in

[0814] * is the connection site with the nitrogen atom of uracil,

[0815] A is -CH2-,

[0816] R 4A is chloro or trifluoromethyl,

[0817] R 4B For hydrogen.

[0818] In the context of the present invention, preference is given to compounds of formula (I) and their salts, solvates and solvates of salts, wherein

[0819] R 2 A group of the formula

[0820]

[0821] in

[0822] * is the connection site with the nitrogen atom of uracil,

[0823] A is -CH2-,

[0824] R 4A is chloro or trifluoromethyl,

[0825] R 4B For hydrogen.

[0826] In another embodiment, the present invention encompasses compounds of formula (II),

[0827]

[0828] in

[0829] R 1 represents a hydrogen atom, a halogen atom or a lower alkyl group,

[0830] R 2 represents a lower alkyl group,

[0831] R 3 and R 4 Each may be the same or different and represents a hydrogen atom, a lower alkoxycarbonyl group, a lower alkylsulfonyl group, a benzoyl group, an acyl group having 1 to 4 carbon atoms, a lower alkoxy group, a lower alkoxycarbonylmethylthioacetyl group, a nitro group, -CONHR 6 , where R 6 represents a hydrogen atom, a lower alkoxycarbonylmethyl group, a carboxymethyl group or -CH(CH2OH)COOR 7 , where R 7 represents a hydrogen atom or a lower alkyl group,

[0832] The group represented by the following formula

[0833]

[0834] Where R 7 The same meaning as above, the group represented by the following formula

[0835]

[0836] Where R 8 and R 9 Each may be the same or different and represents a hydrogen atom, a lower alkyl group, a lower alkylthio group, a lower alkylsulfinyl group, a lower alkylsulfonyl group or a lower alkoxycarbonyl group, a hydroxy lower alkyl group, a cyano group or a monocyclic heterocyclic group represented by the following formula:

[0837]

[0838] wherein A represents an oxygen atom, a sulfur atom or NH, and the dotted part represents a single bond or a double bond, provided that the hydrogen atoms on the ring can be replaced by a lower alkyl group, a lower alkoxy group, a hydroxy-lower alkyl group, a lower alkoxycarbonyl group or a carboxyl group which can be replaced by a halogen atom, and provided that R 3 or R 4 At least one of

[0839]

[0840] and

[0841] R 5 represents a hydrogen atom, a lower alkoxy group or a lower alkyl group, except for the compounds represented by the following formula:

[0842]

[0843] It is used to treat and / or prevent stroke, pulmonary embolism, deep vein or superficial vein thrombosis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy of hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites.

[0844] In another embodiment, the present invention encompasses compounds of formula (II),

[0845]

[0846] in

[0847] R 1 represents a hydrogen atom, a halogen atom or a lower alkyl group,

[0848] R 2 represents a lower alkyl group,

[0849] R 3 and R 4 Each may be the same or different and represents a hydrogen atom, a lower alkoxycarbonyl group, a lower alkylsulfonyl group, a benzoyl group, an acyl group having 1 to 4 carbon atoms, a lower alkoxy group, a lower alkoxycarbonylmethylthioacetyl group, a nitro group, -CONHR 6 , where R 6 represents a hydrogen atom, a lower alkoxycarbonylmethyl group, a carboxymethyl group or -CH(CH2OH)COOR 7 , where R 7 represents a hydrogen atom or a lower alkyl group,

[0850] The group represented by the following formula

[0851]

[0852] Where R 7 The same meaning as above, the group represented by the following formula

[0853]

[0854] Where R 8 and R 9Each may be the same or different and represents a hydrogen atom, a lower alkyl group, a lower alkylthio group, a lower alkylsulfinyl group, a lower alkylsulfonyl group or a lower alkoxycarbonyl group, a hydroxy lower alkyl group, a cyano group

[0855] Or a monocyclic heterocyclic group represented by the following formula:

[0856]

[0857] wherein A represents an oxygen atom, a sulfur atom or NH, and the dotted part represents a single bond or a double bond, provided that the hydrogen atoms on the ring can be replaced by a lower alkyl group, a lower alkoxy group, a hydroxy-lower alkyl group, a lower alkoxycarbonyl group or a carboxyl group which can be replaced by a halogen atom, and provided that R 3 or R 4 At least one of

[0858]

[0859] and

[0860] R 5 represents a hydrogen atom, a lower alkoxy group or a lower alkyl group, except for the compounds represented by the following formula:

[0861]

[0862]

[0863] It is used for stroke, pulmonary embolism, deep or superficial vein thrombosis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy in post-transplant hypercoagulable state, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombolytic methods in thrombosis or occlusion of vascular access sites. 1 Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, and a fluorine atom or a chlorine atom is particularly preferred.

[0864] R 1 , R 2 , R 5 , R 7 , R 8 and R 9 Examples of the lower alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group or a tert-butyl group, and a methyl group or an ethyl group is particularly preferred.

[0865] R 3 , R 4 , R 8 and R 9Examples of the lower alkoxycarbonyl group include methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, sec-butoxycarbonyl or tert-butoxycarbonyl, and particularly preferably methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl or tert-butoxycarbonyl.

[0866] R 3 , R 4 , R 8 and R 9 Examples of the lower alkylsulfonyl group include methylsulfonyl, ethylsulfonyl, propylsulfonyl, isopropylsulfonyl, butylsulfonyl, isobutylsulfonyl, sec-butylsulfonyl or tert-butylsulfonyl, and particularly preferably methylsulfonyl or ethylsulfonyl.

[0867] R 3 and R 4 Examples of the acyl group having 1 to 4 carbon atoms include a formyl group, an acetyl group, a propionyl group, a butyryl group or an isobutyryl group, and an acetyl group is particularly preferred.

[0868] R 3 , R 4 and R 5 Examples of the lower alkoxy group include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy or tert-butoxy, and particularly preferably methoxy or ethoxy.

[0869] R 3 and R 4 Examples of the lower alkoxycarbonylmethylthioacetyl group include methoxycarbonylmethylthioacetyl, ethoxycarbonylmethylthioacetyl, propoxycarbonylmethylthioacetyl, isopropoxycarbonylmethylthioacetyl, butoxycarbonylmethylthioacetyl, isobutoxycarbonylmethylthioacetyl, sec-butoxycarbonylmethylthioacetyl or tert-butoxycarbonylmethylthioacetyl, and particularly preferably methoxycarbonylmethylthioacetyl or ethoxycarbonylmethylthioacetyl.

[0870] In R 3 and R 4 Each is -CONHR 6 In the case of R 6 Examples of the lower alkoxycarbonylmethyl group include methoxycarbonylmethyl, ethoxycarbonylmethyl, propoxycarbonylmethyl, isopropoxycarbonylmethyl, butoxycarbonylmethyl, isobutoxycarbonylmethyl, sec-butoxycarbonylmethyl or tert-butoxycarbonylmethyl, and particularly preferably methoxycarbonylmethyl or ethoxycarbonylmethyl, or isopropoxycarbonylmethyl.

[0871] R 3 and R 4Examples of the hydroxy lower alkyl group include a linear or branched hydroxy lower alkyl group having 1 to 4 carbon atoms, such as a hydroxymethyl group, a hydroxyethyl group, a hydroxypropyl group or a hydroxybutyl group, and particularly preferably a hydroxymethyl group or a hydroxyethyl group.

[0872] R 8 and R 9 Examples of the lower alkylthio group include straight-chain or branched lower alkylthio groups having 1 to 4 carbon atoms, such as methylthio, ethylthio, propylthio or butylthio, particularly methylthio or ethylthio.

[0873] R 8 and R 9 Examples of the lower alkylsulfinyl group include a linear or branched lower alkylsulfinyl group having 1 to 4 carbon atoms such as methylsulfinyl, ethylsulfinyl, propylsulfinyl or butylsulfinyl, and particularly preferably methylsulfinyl or ethylsulfinyl.

[0874] As a group represented by the following formula:

[0875]

[0876] For example, vinyl, methylthiovinyl, methylsulfinylvinyl or 2-methylsulfinyl-2-methyl-thiovinyl is preferred.

[0877] The following formula

[0878]

[0879] The meaning of the lower alkoxy, hydroxy lower alkyl and lower alkoxycarbonyl groups which the hydrogen atom on the represented ring can be substituted by is as described above. In addition, the lower alkyl group which can be substituted by a halogen atom means a lower alkyl group which is substituted by a fluorine atom, a chlorine atom, a bromine atom or an iodine atom in addition to the above-mentioned lower alkyl group. Examples thereof include chloromethyl, bromomethyl, dichloromethyl or 1-chloroethyl.

[0880] One or two lower alkyl groups, lower alkoxy groups, hydroxy-lower alkyl groups, lower alkoxycarbonyl groups or carboxyl groups which may be substituted with a halogen atom may be present as substituents on each heterocyclic ring, and these substituents may be the same or different.

[0881] Examples of monocyclic heterocyclic groups are represented by the formula:

[0882]

[0883] Wherein A represents an oxygen atom, a sulfur atom or NH, and the dotted part represents a single bond or a double bond, including those represented by the following formula:

[0884]

[0885] Specific examples of the monocyclic heterocyclic group are represented by the following formula:

[0886]

[0887] Wherein A represents an oxygen atom, a sulfur atom or NH, the dotted line portion represents a single bond or a double bond, and the hydrogen atoms on the ring may be replaced by a lower alkyl group, a lower alkoxy group, a hydroxy-lower alkyl group, a lower alkoxycarbonyl group or a carboxyl group which may be substituted by a halogen atom, preferably including those represented by the following formula:

[0888]

[0889] These substituents are preferably 4 In this case, it is further preferred that R 3 is methylsulfonyl and R 5 A hydrogen atom.

[0890] In the context of the present invention, preference is given to compounds selected from the group consisting of 2-[4-(5-chloro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methylsulfonylphenyl]oxazole-4-carboxylic acid, 2-[4-(5-fluoro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methylsulfonylphenyl]oxazole-4-carboxylic acid, 2-[4-(5-chloro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methylsulfonylphenyl]oxazole-4-carboxylic acid, )-3-methylsulfonylphenyl]oxazole-4-carboxylic acid disodium, 2-[4-(5-fluoro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methylsulfonylphenyl]oxazole-4-carboxylic acid disodium, 2-[4-(5-fluoro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methylsulfonylphenyl]thiazole-4-carboxylic acid, 5-fluoro-N-[4-(4-hydroxymethylthiazol-2-yl)-2 -methylsulfonylphenyl]-3-methylbenzo[b]thiophene-2-sulfonamide, 5-fluoro-N-[2-methylsulfonyl-4-(5-methoxy-4-methyloxazol-2-yl)phenyl]-3-methylbenzo[b]thiophene-2-sulfonamide, ((2-(4-((5-fluoro-3-methylbenzo[b]thiophene)-2-sulfonylamino)-3-(methylsulfonyl)-phenyl)thiazole-4-carboxylic acid and 5-fluoro-N-[2 -methanesulfonyl-4-(5-methyloxazol-2-yl)phenyl]-3-methylbenzo[b]thiophene-2-sulfonamide, which is used to treat and / or prevent stroke, pulmonary embolism, deep vein or superficial vein thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in infection, inflammation, hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites.

[0891] In the context of the present invention, preference is given to compounds selected from the group consisting of 2-[4-(5-chloro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methylsulfonylphenyl]oxazole-4-carboxylic acid, 2-[4-(5-fluoro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methylsulfonylphenyl]oxazole-4-carboxylic acid, 2-[4-(5-chloro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methylsulfonylphenyl]oxazole-4-carboxylic acid, )-3-methylsulfonylphenyl]oxazole-4-carboxylic acid disodium, 2-[4-(5-fluoro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methylsulfonylphenyl]oxazole-4-carboxylic acid disodium, 2-[4-(5-fluoro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methylsulfonylphenyl]thiazole-4-carboxylic acid, 5-fluoro-N-[4-(4-hydroxymethylthiazol-2-yl)- 2-methylsulfonylphenyl]-3-methylbenzo[b]thiophene-2-sulfonamide, 5-fluoro-N-[2-methylsulfonyl-4-(5-methoxy-4-methyloxazol-2-yl)phenyl]-3-methylbenzo[b]thiophene-2-sulfonamide, ((2-(4-((5-fluoro-3-methylbenzo[b]thiophene)-2-sulfonylamino)-3-(methylsulfonyl)-phenyl)thiazole-4-carboxylic acid and 5-fluoro-N- [2-Methanesulfonyl-4-(5-methyloxazol-2-yl)phenyl]-3-methylbenzo[b]thiophene-2-sulfonamide, which is used for stroke, pulmonary embolism, deep vein or superficial vein thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in infection, inflammation, and post-transplant hypercoagulable state, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombolytic methods in thrombosis or occlusion of vascular access sites.

[0892] In the context of the present invention, (2-(4-((5-fluoro-3-methylbenzo[b]thiophene)-2-sulfonylamino)-3-(methylsulfonyl)phenyl)thiazole-4-carboxylic acid is preferred for use in the treatment and / or prevention of stroke, pulmonary embolism, deep vein or superficial vein thrombosis, thrombotic microangiopathy, infection, inflammation, post-transplant hypercoagulable state thrombotic microangiopathy, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites.

[0893] In the context of the present invention, (2-(4-((5-fluoro-3-methylbenzo[b]thiophene)-2-sulfonylamino)-3-(methylsulfonyl)phenyl)thiazole-4-carboxylic acid is preferred for use in stroke, pulmonary embolism, deep or superficial vein thrombosis, thrombotic microangiopathy, infection, inflammation, post-transplant hypercoagulable state thrombotic microangiopathy, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombolysis methods in thrombosis or occlusion of vascular access sites. The compounds according to the invention are chymotrypsin inhibitors, in particular compounds of formula (I), as well as salts, solvates and solvates of salts thereof, and have an unexpectedly useful pharmacological activity spectrum. These compounds influence the proteolytic activity of the serine protease chymotrypsin. The compounds according to the invention inhibit the enzymatic cleavage of chymotrypsin substrates.

[0894] The compounds of formula (I), their preparation methods and their use as selective inhibitors of chymotrypsin for the treatment and / or prevention of heart failure, pulmonary hypertension, chronic obstructive pulmonary disease, asthma, renal failure, kidney disease, fibrotic disorders of internal organs and fibrotic skin diseases are generally disclosed in WO 2013167495A1, in particular the specifically disclosed compounds are an explicitly described part of the present invention and are hereby incorporated by reference.

[0895] US 7,071,220 B2 generally discloses compounds of formula (II), their preparation methods and their use as chymotrypsin inhibitors in the treatment of hypertension, cardiac hypertrophy, heart failure, myocardial infarction, arteriosclerosis, diabetic or non-diabetic nephropathy, diabetic retinopathy, ischemic reperfusion disorders, restenosis after percutaneous coronary angioplasty, intimal thickening after bypass grafting, chronic rheumatic diseases, keloids, psoriasis, allergies, inflammation, asthma, atopic dermatitis, solid tumors and pulmonary hypertension, and in particular the specifically disclosed compounds are explicitly described as part of the present invention and are hereby incorporated by reference.

[0896] In the context of the present invention, the term "treatment" or "treating" includes inhibiting, delaying, checking, alleviating, attenuating, limiting, reducing, suppressing, resisting or curing a disease, condition, disorder, injury or health problem, or the development, course or progression of these states and / or symptoms of these states. The term "therapy" is understood herein to be synonymous with the term "treat".

[0897] The terms "prevent," "preventing," or "inhibiting" are used synonymously in the context of the present invention and refer to avoiding or reducing the risk of contracting, experiencing, afflicting, or having a disease, condition, disorder, injury, or health problem, or the development or progression of these states and / or symptoms of these states.

[0898] Treatment or prevention of a disease, condition, disorder, injury or health problem may be partial or complete.

[0899] For the purposes of the present invention, "lysis of thrombi during thrombotic or thromboembolic events" includes disorders and complications occurring in the arterial, venous vasculature, microcirculation and lymphatic system, which can be treated with the compounds according to the invention.

[0900] Compound according to the present invention is applicable to the treatment and prevention of the venous vascular system disorders causing venous vascular occlusion and thus causing venous thrombosis. It includes the thrombotic events of the venous blood vessels of the lower and upper extremities, such as the lower deep vein thrombosis of the calf vein, popliteal vein, femoral vein, common femoral vein and iliac vein (containing or not containing the inferior vena cava), the upper deep vein thrombosis of the jugular vein, brachiocephalic vein, subclavian vein and axillary vein, and the thrombosis of the more distant brachial vein, ulnar vein and radial vein, or the superficial vein thrombosis of the small saphenous vein, great saphenous vein, cephalic vein and basilic vein, the event of the organ vein blood vessels, including retinal vein occlusion, cerebral vein and skull venous sinus thrombosis, renal vein thrombosis, Budd-Chiari syndrome and portal vein thrombosis, mesenteric vein thrombosis, ovarian vein thrombosis and cavernous part thrombosis.

[0901] The compounds according to the invention are suitable for preventing complications following venous thrombosis, including, for example, post-thrombotic syndrome, pulmonary hypertension or pulmonary embolism, cryptogenic stroke associated with paradoxical thromboembolism with patent foramen ovale, and deep vein thrombosis of the extremities.

[0902] Compounds according to the present invention are suitable for treating and preventing arterial vascular system disorders that cause arterial vascular occlusion and thus cause arterial thrombosis. It particularly includes cerebrovascular arterial disorders, such as transient ischemic attack (TIA), ischemic stroke, including cardioembolic stroke, such as a stroke caused by atrial fibrillation, non-cardioembolic stroke, such as lacunar stroke, stroke caused by large artery or small artery disease, or stroke caused by unknown causes, cryptogenic stroke, embolic stroke, embolic stroke of unknown origin, or thrombotic and / or thromboembolic events leading to stroke or TIA, coronary artery disorders, such as acute coronary syndrome (ACS), ST-segment elevation heart disease. myocardial infarction (STEMI) and non-ST-segment elevation myocardial infarction (non-STEMI), stable angina, unstable angina, reocclusion and restenosis after coronary artery interventions such as angioplasty, stenting or major coronary artery bypass grafting, peripheral arterial disorders, which lead to peripheral arterial disease, including peripheral artery occlusion, acute limb ischemia, amputation, reocclusion and restenosis after interventions such as angioplasty, stenting or surgery and bypass grafting, and / or stent thrombosis, renal, hepatic or mesenteric artery thrombosis, retinal artery thrombosis.

[0903] The compounds according to the invention are suitable for the treatment and prevention of local or systemic microcirculatory disorders caused by small vessel thrombosis, including primary thrombotic microangiopathy (TMAs), such as thrombotic thrombocytopenic purpura (TTP) or hemolytic uremic syndrome (HUS), and secondary thrombotic microangiopathy. It includes microthrombosis caused by the hypercoagulable state of infectious diseases caused by bacteria, viruses or fungi, such as the hypercoagulable state of viral infections caused by coronaviruses (such as SARS-Cov1, MERS and SARS-Cov2 infections), sepsis, microthrombosis caused by drugs or vaccination (such as vaccination-induced thrombotic thrombocytopenia (VITT), which is a very rare adverse event after COVID vaccination), disseminated intravascular coagulation (DIC), pulmonary thrombosis with or without ARDS, autoimmune diseases (such as systemic lupus erythematosus), antiphospholipid syndrome, malignant hypertension, transplantation (such as solid organs, bone marrow), pregnancy-related thrombotic microangiopathy (such as eclampsia, HELLP syndrome), placental microthrombosis, hepatic veno-occlusive disease, diabetic microangiopathy (such as diabetic retinopathy, glomerulopathy, gangrene), and small vessel inflammation of the capillary system (such as cerebral vasculitis).

[0904] The compounds according to the invention are suitable for the treatment and prevention of venous thromboembolism (VT of arterial vessels) in relevant diseases with unmet clinical needs including acute ischemic stroke, peripheral arterial occlusive disease and myocardial infarction. artery Atherothrombotic occlusion , treatment and / or prevention of cerebrovascular arterial disorders such as transient ischemic attack (TIA), ischemic stroke, including cardioembolic stroke, such as stroke caused by atrial fibrillation, non-cardioembolic stroke, such as lacunar stroke, stroke caused by large artery or small artery disease, or stroke of unknown cause, cryptogenic stroke, embolic stroke, embolic stroke of unknown source, or thrombotic and / or thromboembolic events leading to stroke or TIA, and / or peripheral arterial disorders, which lead to peripheral arterial disease, including peripheral arterial occlusion, acute limb ischemia, amputation, reocclusion and restenosis after interventions such as angioplasty, stenting or surgery and bypass, and / or stent thrombosis.

[0905] The compounds according to the invention are suitable for the treatment and prevention of diseases related to pulmonary embolism, cardioembolic stroke and myocardial infarction. Thrombotic embolus Caused by arterial occlusion Ischemic events .

[0906] The compounds according to the invention are suitable for the treatment and prevention of venous thrombosis, including thrombosis in the deep and superficial veins of the limbs and in the cerebral veins.

[0907] The compounds according to the invention are suitable for the treatment and prevention of Thrombotic microangiopathy Diseases or conditions with thrombotic complications in the microcirculation, including infectious diseases caused by bacteria, viruses or fungi, sepsis, isolated pulmonary embolism / thrombosis, autoimmune diseases.

[0908] The compounds of the present invention are useful for treating and preventing thrombosis or occlusion at vascular access sites, including arteriovenous fistulas and central venous ports.

[0909] The compounds of the invention are useful in the treatment and prevention of extravascular fibrin deposition, for example in the lung, placenta, brain.

[0910] This includes the dissolution of venous thromboembolism (VTE) in cancer patients receiving hormone therapy, chemotherapy or radiotherapy, which does not meet clinical needs. These patients are often prone to visceral local or catheter-induced thrombosis, they are anticoagulant and therefore have bleeding disorders associated with the latter class of drugs (Giustozi et al., Trends Cardiovasc Med, 2022; Received). In addition, mast cell activation is increasingly reported to play a role in cancer development (Noto et al., Front cell Dev Biol. October 12, 2021; 9: 752350; Segura-Villalobos et al., Cells. January 20, 2022; 11 (3): 349).

[0911] This includes conditions resulting from exposure of the biomaterial or artificial surface to blood and / or the vessel wall, including procedures to reopen vascular access sites, including arteriovenous fistulas, central venous ports for central line therapy, or dialysis procedures, procedures for revascularization or clot degradation in stents, grafts, cardiovascular assist devices, artificial hearts, mechanical or bioprosthetic heart valves, or during transplantation.

[0912] This includes conditions associated with extravascular fibrin deposition, including fibrin deposition in the interstitial spaces of the lung.

[0913] This includes the use of chymotrypsin inhibition in diagnostic methods for disease and / or therapeutic management.

[0914] The compounds according to the invention may exist in different stereoisomeric forms, i.e. in the form of configurational isomers or, where appropriate, in the form of conformers (enantiomers and / or diastereomers, including those in the case of rotational isomers and atropisomers), depending on their structure. The present invention therefore includes enantiomers and diastereomers and their respective mixtures. Stereoisomerically identical components can be separated in a known manner from mixtures of enantiomers and / or diastereomers; for this purpose, preferably chromatography is used, in particular HPLC chromatography on an achiral or chiral phase.

[0915] Preferred salts in the context of the present invention are physiologically acceptable salts of the compounds according to the invention. However, the invention also comprises salts which are not themselves suitable for pharmaceutical applications but which can be used, for example, for isolating or purifying the compounds according to the invention.

[0916] Physiologically acceptable salts of the compounds of the invention include acid addition salts of inorganic acids, carboxylic acids and sulfonic acids, for example, salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, naphthalene disulfonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid and benzoic acid.

[0917] Physiologically acceptable salts of the compounds according to the invention also include salts with customary bases, such as and preferably alkali metal salts (e.g. sodium and potassium salts), alkaline earth metal salts (e.g. calcium and magnesium salts) and ammonium salts derived from ammonia or organic amines having 1 to 16 carbon atoms, such as and preferably ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, N-methylmorpholine, arginine, lysine, ethylenediamine, N-methylpiperidine and choline.

[0918] In the context of the present invention, Solvate Described as those forms of the compounds of the invention which form complexes in the solid or liquid state by coordination with solvent molecules. Hydrates are a special form of solvates, in which the coordination takes place with water.

[0919] Furthermore, the present invention also includes prodrugs of the compounds of the present invention. The term "prodrug" includes compounds which may themselves be biologically active or inactive but which are converted into compounds of the present invention during their residence time in the body (eg by metabolism or hydrolysis).

[0920] Treatment or prevention of a disease, condition, disorder, injury or health problem may be partial or complete.

[0921] Chymotrypsin inhibitors, such as the inhibitors encompassed by the present invention, in particular 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) fulacimstat and TY-51469 are therefore suitable for use as drugs for the treatment and / or prevention of diseases in humans and animals.

[0922] Chymotrypsin inhibitors, such as the inhibitors encompassed by the present invention, in particular 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) fulacimstat are therefore suitable for use as drugs for the treatment and / or prevention of diseases in humans and animals.

[0923] The present invention further provides the use of the compounds according to the invention for the treatment and / or prophylaxis of disorders, in particular vascular disorders, preferably thrombotic or thromboembolic disorders and / or thrombotic or thromboembolic complications.

[0924] The compounds according to the invention are therefore suitable for the treatment and / or prevention of disorders or complications which may be caused by clot formation.

[0925] The present invention further provides the use of the chymotrypsin inhibitor of the present invention for treating and preventing stroke, pulmonary embolism, deep vein or superficial vein thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in infection, inflammation, and hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites.

[0926] The present invention further provides the use of the chymotrypsin inhibitor of the present invention for stroke, pulmonary embolism, deep vein or superficial vein thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in infection, inflammation, hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombolysis in thrombosis or occlusion of vascular access sites.

[0927] The present invention further provides the use of the chymotrypsin inhibitor of the present invention for treating and preventing stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep vein or superficial vein thrombosis, thrombotic microangiopathy, thrombotic microangiopathy caused by infection, inflammation, and hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites.

[0928] The present invention further provides the use of the chymotrypsin inhibitor of the present invention for stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep vein or superficial vein thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in infection, inflammation, and hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombolysis in thrombosis or occlusion of vascular access sites.

[0929] The present invention further provides the use of the chymotrypsin inhibitor of the present invention for treating and preventing stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, phlebitis, and microangiopathy.

[0930] The present invention further provides the use of the chymotrypsin inhibitor of the present invention for thrombolysis in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, phlebitis, and microangiopathy.

[0931] The present invention further provides the use of a chymotrypsin inhibitor of general formula I for treating and preventing stroke, pulmonary embolism, deep vein or superficial vein thrombosis, thrombotic microangiopathy, thrombotic microangiopathy caused by infection, inflammation, and hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites.

[0932] The present invention further provides a use of a chymotrypsin inhibitor of general formula I for treating and preventing stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep vein or superficial vein thrombosis, phlebitis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy in a hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites.

[0933] The present invention further provides the use of the chymotrypsin inhibitor of general formula I for stroke, pulmonary embolism, deep vein or superficial vein thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in infection, inflammation, and hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombolysis in thrombosis or occlusion of vascular access sites.

[0934] The present invention further provides a chymotrypsin inhibitor of general formula I for use in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep vein or superficial vein thrombosis, phlebitis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy in a hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombolysis in thrombosis or occlusion of vascular access sites.

[0935] The present invention further provides use of the chymotrypsin inhibitor of general formula I for treating and preventing stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, phlebitis or microangiopathy.

[0936] The present invention further provides the use of the chymotrypsin inhibitor of general formula I for thrombolysis in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, phlebitis or microangiopathy.

[0937] The present invention further provides use of the chymotrypsin inhibitor of general formula I for treating and preventing stroke, pulmonary embolism, phlebitis or microangiopathy.

[0938] The present invention further provides the use of the chymotrypsin inhibitor of general formula I for thrombolysis in stroke, pulmonary embolism, phlebitis or microangiopathy.

[0939] The present invention further provides the use of a chymotrypsin inhibitor of general formula II for treating and preventing stroke, pulmonary embolism, deep vein or superficial vein thrombosis, thrombotic microangiopathy, thrombotic microangiopathy caused by infection, inflammation, and hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites.

[0940] The present invention further provides the use of the chymotrypsin inhibitor of general formula II for stroke, pulmonary embolism, deep vein or superficial vein thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in infection, inflammation, and hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombolysis in thrombosis or occlusion of vascular access sites.

[0941] The present invention further provides the use of the chymotrypsin inhibitor of general formula II for stroke, pulmonary embolism, deep vein or superficial vein thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in infection, inflammation, and hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombolysis in thrombosis or occlusion of vascular access sites.

[0942] The present invention further provides use of the chymotrypsin inhibitor of general formula II for treating and preventing stroke, pulmonary embolism, phlebitis or microangiopathy.

[0943] The present invention further provides the use of the chymotrypsin inhibitor of general formula II for thrombolysis in stroke, pulmonary embolism, phlebitis or microangiopathy.

[0944] For the purposes of the present invention, "thrombotic or thromboembolic disorders and / or thrombotic or thromboembolic complications" include disorders and complications occurring in the arterial, venous vascular system and lymphatic system, which can be treated with the compounds of the present invention.

[0945] The present invention further provides the use of the compounds according to the invention for the treatment and / or prophylaxis of disorders, in particular the disorders mentioned above.

[0946] The present invention further provides the use of the compounds according to the invention for the preparation of a medicament for the treatment and / or prevention of disorders, in particular the aforementioned disorders.

[0947] The present invention further provides the use of the compounds according to the invention for the preparation of a medicament for thrombolysis in disorders, in particular in the aforementioned disorders.

[0948] The present invention further provides methods for the treatment and / or prevention of disorders, in particular the aforementioned disorders, using a therapeutically effective amount of a compound according to the invention.

[0949] The present invention further provides a method for thrombolysis in disorders, especially in the aforementioned disorders, using a therapeutically effective amount of a compound according to the invention.

[0950] The present invention further provides the compounds according to the invention for use in a method for the treatment and / or prophylaxis of disorders, in particular the aforementioned disorders, using a therapeutically effective amount of the compounds according to the invention.

[0951] The present invention further provides compounds of the invention for use in a method for thrombolysis in disorders, especially in the aforementioned disorders, using a therapeutically effective amount of a compound of the invention.

[0952] The invention further provides medicaments comprising a compound according to the invention and one or more further active compounds.

[0953] The present invention further provides medicaments comprising the compounds according to the invention and one or more further active compounds, in particular for use in thrombolysis in the aforementioned disorders.

[0954] The present invention further provides medicaments comprising the compounds according to the invention and one or more further active compounds, in particular for the treatment and / or prophylaxis of the aforementioned disorders.

[0955] Preferred examples of active compounds suitable for combination include:

[0956] Plasminogen activators (thrombolytics / fibrinolytics), such as but not limited to tissue plasminogen activator (t-PA, e.g. ), streptokinase, tenecteplase, reteplase and urokinase, compounds that promote fibrinolysis, such as inhibitors of plasminogen activator inhibitors (PAI inhibitors), inhibitors of thrombin-activated fibrinolysis inhibitor (TAFI inhibitors) or α2 antiplasmin inhibitors, or plasminogen modulating substances that cause increased plasmin formation, such as SMTP-7;

[0957] Lipid-lowering substances, especially HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase inhibitors, such as lovastatin (Mevacor), simvastatin (Zocor), pravastatin (Pravachol), fluvastatin (Lescol), and atorvastatin (Lipitor); PCSK9 inhibitors (e.g. evolocumab, alirocumab)

[0958] Coronary artery therapy / vasodilators, especially ACE (angiotensin converting enzyme) inhibitors such as captopril, lisinopril, enalapril, ramipril, cilazapril, benazepril, fosinopril, quinapril and perindopril, or AII (angiotensin II) receptor antagonists such as embusartan, losartan, valsartan valsartan, irbesartan, candesartan, eprosartan, and temisartan, or beta-adrenergic receptor antagonists such as carvedilol, alprenolol, bisoprolol, acebutolol, atenolol, betaxolol, carteolol, metoprolol, nadolol, and tadalafil. l), penbutolol, pindolol, propanolol and timolol, or alpha-1-adrenoceptor antagonists such as prazosine, bunazosine, doxazosine and terazosine, or diuretics such as hydrochlorothiazide, furosemide, bumetanide, piretanide, torsemide, asemide), amiloride and dihydralazine, or calcium channel blockers, such as verapamil and diltiazem, or dihydropyridine derivatives, such as nifedipin (Adalat) and nitrendipine (Bayotensin), or nitro preparations, such as isosorbide 5-mononitrate, isosorbide dinitrate and glyceryl trinitrate, or substances that lead to an increase in cyclic guanosine monophosphate (cGMP), such as stimulators of soluble guanylate cyclase, such as riociguat;

[0959] Anticoagulants, such as:

[0960] indirect anticoagulants, such as proteoglycans, for example heparin (UFH), low molecular weight heparin (LMW), such as tinzaparin, certoparin, parnaparin, nadroparin, ardeparin, enoxaparin, reviparin, dalteparin, danaparoid, semuloparin (AVE 5026), adomiparin (M118);

[0961] Direct thrombin inhibitors (DTIs), such as dabigatran, atecegatran, argatroban, bivalirudin, tanogitran, and hirudin;

[0962] Direct factor Xa inhibitors, such as rivaroxaban, apixaban, edoxaban, betrixaban, darexaban, otamixaban, and fondaparinux

[0963] Direct inhibitors of more than one coagulation factor, such as dual FXa / IIa inhibitors, such as SATI (Lopez M. et al., Thromb Res. 193 (2020) 15-21)

[0964] Inhibitors of FXI or FXIa, FXII or FXIIa, or inhibitors of FXI or FXII synthesis, such as asundexian, milvxian, osocimab, abelacicumab, fesomersen

[0965] Antiplatelet drugs, such as:

[0966] Substances that inhibit platelet aggregation (platelet aggregation inhibitors, thrombocyte aggregation inhibitors), for example acetylsalicylic acid, P2Y12 antagonists such as ticlopidine (Ticlid), clopidogrel (Plavix), prasugrel, ticagrelor, cangrelor, elinogrel, PAR-1 antagonists such as vorapaxar, PAR-4 antagonists, EP3 antagonists such as DG041;

[0967] • Platelet adhesion inhibitors, such as GPVI and / or GPIb antagonists, such as, for example, revacept, glenzocimab or caplacizumab;

[0968] Fibrinogen receptor antagonists (glycoprotein-IIb / IIIa antagonists), such as abciximab, eptifibatide, tirofiban, lamifiban, lefradafiban, fradafiban, and zalunfiban;

[0969] Recombinant human activated protein C, such as Xigris, recombinant thrombomodulin, such as ART-123, or recombinant antithrombin-III;

[0970] Neuroprotective approaches, such as NMDA receptor antagonists, PSD-95 inhibitors, tissue kallikrein preparations, endothelin-B agonists, PARP inhibitors,

[0971] Anti-inflammatory compounds, such as glucocorticoids (e.g., prednison, prednisolone, methylprednisolone, triamcinolone, dexamethasone, beclometason, betamethasone, flunisolid, budesonide, or fluticasone), nonsteroidal anti-inflammatory agents (NSAIDs) (e.g., acetylsalicylic acid (aspirin), ibuprofen, or naproxen) Naproxen), 5-aminosalicylic acid derivatives, leukotriene receptor antagonists (e.g., montelukast), TNF-α inhibitors (e.g., etanercept, infliximab, adalimumab, golimumab, certolizumab) and / or chemokine receptor antagonists (e.g., CCR1, 2, and / or 5 antagonists), IRAK4 inhibitors, cannabinoid receptor agonists, interleukin-1β antibodies (e.g., canakinumab)

[0972] Anti-infective measures, such as and preferably antibacterial substances (e.g. penicillins, vancomycin, ciprofloxacin, mupirocin, azithromycin and metronidazole), antifungal substances (e.g. naftifin, nystatin) and / or antiviral and / or antiparasitic substances

[0973] Additional intensive care treatments, such as fluid therapy (eg, crystalloid or colloid fluids); vasopressors (eg, norepinephrine, dopamine, or vasopressin); inotropic therapy (eg, dobutamine); blood products (eg, red blood cell concentrates, platelet concentrates, erythropoietin, or fresh frozen plasma); assisted ventilation for sepsis-induced acute lung injury (ALI) or acute respiratory distress syndrome (ARDS; eg, permissive hypercapnia, low tidal volumes); sedation (eg, diazepam, lorazepam, midazolam, or propofol); glucose control (eg, insulin, dextrose); renal replacement therapy (eg, continuous venovenous hemofiltration or intermittent hemodialysis)

[0974] - Mast cell stabilizing compounds (nedocromil, cromolyn sodium), trypsin inhibitors, cathepsin G inhibitors or elastase inhibitors.

[0975] Antihistamines, such as cetirizine, loratadine, and astemizole

[0976] For the purposes of the present invention, a "combination" according to the invention is understood not only to be a dosage form containing all components (so-called fixed combinations) and a combination package containing components separated from one another, but also to be a combination of components which are administered simultaneously or sequentially, provided that they are used for the prevention and / or treatment of the same disease. It is likewise possible to combine two or more active ingredients with one another, which means that they are therefore both two-component or multi-component combinations.

[0977] The compounds of the invention can act systemically and / or locally. For this purpose, they can be administered in a suitable manner, for example by oral, parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal, dermal, transdermal, conjunctival or otic routes, or as implants or stents.

[0978] The compounds according to the invention can be administered in administration forms suitable for these administration routes.

[0979] Suitable administration forms for oral administration are those which act according to the prior art and deliver the compounds of the invention quickly and / or in a slowed manner and which contain the compounds of the invention in crystalline form and / or in amorphous form and / or dissolved form, for example tablets (uncoated or coated tablets, for example with an enteric coating or an insoluble or delayed dissolving coating which controls the release of the compounds of the invention), tablets which disintegrate rapidly in the mouth, or films / wafers, films / lyophilisates, capsules (for example hard or soft gelatin capsules), sugar-coated tablets, granules, pills, powders, emulsions, suspensions, aerosols or solutions.

[0980] Parenteral administration can be accomplished by avoiding an absorption step (e.g., by intravenous, intraarterial, intracardiac, intraspinal, or intralumbar routes) or by including absorption (e.g., by intramuscular, subcutaneous, intradermal, transdermal, or intraperitoneal routes). Administration forms suitable for parenteral administration include formulations for injection and infusion in the form of solutions, suspensions, emulsions, lyophilizates, or sterile powders. For topical parenteral administration, catheter-based intra-clot administration methods can be used.

[0981] Oral administration is preferred.

[0982] Suitable administration forms for other administration routes are, for example, pharmaceutical forms for inhalation (including powder inhalers, sprays), nasal drops, nasal solutions or nasal sprays; tablets, films / wafers or capsules for translingual, sublingual or buccal administration, suppositories, preparations for the ears or eyes, vaginal capsules, aqueous suspensions (lotions, shaking mixtures), lipophilic suspensions, ointments, creams, transdermal therapeutic systems (e.g. patches), emulsions (milk), pastes, foams, sprinkles, implants or stents.

[0983] The compounds of the invention can be converted into the above-mentioned administration forms. This can be done in a manner known per se by mixing with inert, non-toxic, pharmaceutically suitable excipients. These excipients include carriers (e.g. microcrystalline cellulose, lactose, mannitol), solvents (e.g. liquid polyethylene glycol), emulsifiers and dispersants or wetting agents (e.g. sodium lauryl sulfate, polyoxysorbitan oleate), binders (e.g. polyvinyl pyrrolidone), synthetic and natural polymers (e.g. albumin), stabilizers (e.g. antioxidants, e.g. ascorbic acid), colorants (e.g. inorganic pigments, e.g. iron oxides) and flavorings and / or odor correctors.

[0984] The invention further provides medicaments comprising at least one compound according to the invention, preferably together with one or more inert, nontoxic, pharmaceutically suitable excipients, and their use for the purposes mentioned above.

[0985] Generally, it has been found that in the case of parenteral administration, an amount of about 0.001 to 1 mg / kg body weight, preferably about 0.01 to 0.5 mg / kg body weight is beneficial to achieve an effective effect. In the case of oral administration, the dosage is about 0.01 to 100 mg / kg body weight. Preferably, the oral daily dose is about 1 mg to 100 mg, most preferably 2 mg to 50 mg. Nevertheless, if necessary, it may be necessary to deviate from the prescribed amount, depending on body weight, route of administration, personal performance of the active ingredient, type of formulation, and time or interval of administration.

[0986] Unless otherwise stated, the percentages in the following tests and examples are percentages by weight; parts are parts by weight. The concentration data for solvent ratios, dilution ratios and liquid / liquid solutions are based on volume in each case. "w / v" means "weight / volume". For example, "10% w / v" means: 100 mL of solution or suspension contains 10 g of substance.

[0987] Experimental methods

[0988] 1. Abbreviations

[0989] AA—Abdominal Aorta LC—Liquid Chromatography

[0990] ACN—Acetonitrile LFQ—Label-free Quantitation

[0991] ACS—acute coronary syndrome LMW—low molecular weight heparin

[0992] AMC—aminomethylcoumarin MCP-4—mast cell protease 4

[0993] ANOVA—Analysis of variance MES—2-(N-morpholino)ethanesulfonic acid

[0994] ARDS—Acute Respiratory Distress Syndrome MS—Mass Spectrometry

[0995] BSA—bovine serum albumin NEM—N-ethylmaleimide

[0996] C57BL / 6—mouse strain C57 Black 6 non-STEMI—non-ST-segment elevation myocardial infarction

[0997] cGMP—cyclic guanosine monophosphate

[0998] CMA1—chymotrypsin 1 PAI—plasminogen activator inhibitor

[0999] CNS—Central Nervous System PBS—Phosphate Buffered Saline

[1000] Ctr—control PEG—polyethylene glycol

[1001] DAB—3,3'-diaminobenzidine PSM—peptide matching spectrum

[1002] DIC - Disseminated Intravascular Coagulation PVP - Polyvinylpyrrolidone

[1003] DMSO—dimethyl sulfoxide rCMA-1—recombinant human chymotrypsin 1

[1004] DTI—direct thrombin inhibitor rmMCP-4—recombinant mouse mast cell protease 4

[1005] DTT—dithiothreitol

[1006] DVT—deep vein thrombosis rtPA—recombinant human tissue plasminogen activator

[1007] EDTA—ethylenediaminetetraacetic acid

[1008] EtOH—ethanol SEM—standard error of the mean

[1009] FA—formic acid SLPI—secretory leukocyte protease inhibitor

[1010] FDR—False Discovery Rate

[1011] HEPES—(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) STEMI—ST-segment elevation myocardial infarction

[1012] TAFI - thrombin-activatable fibrinolysis inhibitor

[1013] HPLC—High Performance Liquid Chromatography

[1014] im—intramuscular TCA—trichloroacetic acid

[1015] iv—intravenous TFA—trifluoroacetic acid

[1016] IVC - inferior vena cava TIA - transient ischemic attack

[1017] KO—knockout 60tPA—tissue plasminogen activator

[1018] UFH—unfractionated heparin uPA—urokinase

[1019] 2. Evaluation of in vitro potency and selectivity of chymotrypsin inhibitors

[1020] The suitability of the compounds according to the invention for the treatment of thromboembolic disorders by inhibiting chymotrypsin can be demonstrated in the following assay system:

[1021] 2.1 Chymotrypsin activity assay

[1022] Recombinant human chymotrypsin protein and recombinant hamster chymotrypsin (Protein Technologies, Bayer Healthcare) were used. The substrate Abz-HPFHL-Lys (Dnp) -NH2 (10 μM) was monitored in a fluorescence reader (excitation 340 nM, emission 465 nM) in an assay buffer containing 3 nM of recombinant human chymotrypsin or 1.2 nM of recombinant hamster chymotrypsin, Tris 50 mM (pH 7.5), NaCl 150 mM, BSA 0.10%, Chaps 0.10%, glutathione 1 mM, EDTA 1 mM for cleavage. The data were normalized (enzyme reaction without inhibitor = 0% inhibition, all other assay components but no enzyme = 100% inhibition). Typically test compounds were tested at 9 different concentrations ranging from 10 μM to 1 nM on the same microtiter plate (10 μM, 3.1 μM, 1.0 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM, 0.003 μM, 0.001 μM). IC was calculated using in-house software. 50 value.

[1023] result

[1024] The compounds in the context of the present invention effectively inhibit the activity of chymotrypsin, IC 50 See Table 1 for values.

[1025] Table 1 Efficacy of chymotrypsin inhibitors

[1026] Species Example <![CDATA[IC 50 [nM]]]> Example <![CDATA[IC 50 [nM]]]> people fulacimstat 4 TY-51469 20 Hamster fulacimstat 3 TY-51469 66

[1027] 2.2 Determination of selectivity

[1028] The selectivity of the substances with regard to the inhibition of cathepsin G and other proteases (eg in the area of ​​coagulation and fibrinolysis) was demonstrated.

[1029] Recombinant human cathepsin G (CathG) protein (Protein Technologies, Bayer AG) was used. The cleavage of the substrate DABCYL-Ser-Thr_Leu-Ser-Glu-Lys_Ala_Lys_Pro_Ala-Glu (EDANS) (3 μM) in an assay buffer containing recombinant CathG (2.5 nM) Hepes 100 mM (pH 7.0), NaCl 50 mM, BSA 0.01%, Tween 20 0.001%, glutathione 0.3 mM was monitored in a fluorescence reader (excitation 334 nM, emission 536 nM). The data were normalized (enzyme reaction without inhibitor = 0% inhibition, all other assay components but no enzyme = 100% inhibition). Typically test compounds were tested at 9 different concentrations ranging from 10 μM to 1 nM on the same microtiter plate (10 μM, 3.1 μM, 1.0 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM, 0.003 μM, 0.001 μM). IC was calculated using in-house software. 50 value.

[1030] In a biochemical assay, the potency of the test compounds was analyzed in buffer against several other human serine proteases, including coagulation proteases, based on the fluorescent detection of aminomethylcoumarin (AMC) cleaved from a specific synthetic peptide substrate by the corresponding protease.

[1031] The serine protease assay consists of the following enzyme and substrate (final assay concentrations are given). Active proteases and corresponding substrates are generally commercially available purified from human plasma.

[1032] Thrombin (Kordia; 0.02 nM), Boc-Asp(OBzl)-Pro-Arg-AMC (Bachem I-1560; 5 μM).

[1033] · Factor Xa (Kordia; 1.3 nM), Boc-Ile-Glu-Gly-Arg-AMC (Bachem I-1100; 5 μM).

[1034] · Factor XIa (Kordia; 0.15 nM), Boc-Glu(OBzl)-Ala-Arg-AMC (Bachem I-1575; 5 μM).

[1035] Plasmin (Kordia; 0.1 μg / ml, 1.2 nM), MeOSuc-Ala-Phe-Lys-AMC (Bachem I-1275; 50 μM).

[1036] · Tissue plasminogen activator (tPA; Loxo; 1 nM), CH3SO2-D-Phe-Gly-Arg-AMC (Pentapharm091-06; 5 μM).

[1037] Plasma kallikrein (Kordia; 0.2 nM), H-Pro-Phe-Arg-AMC (Bachem I-1295; 5 μM).

[1038] Trypsin (Sigma; 0.042 U / ml), Boc-Ile-Glu-Gly-Arg-AMC (Bachem I-1100; 5 μM).

[1039] The test compound was diluted in dimethyl sulfoxide (DMSO) to a final assay concentration of 0-50 μM. The corresponding human serine protease and the corresponding substrate were diluted in a buffer solution (pH 7.4, consisting of 50 mM Tris / HCl, 100 mM NaCl, 5 mM CaCl2, and 0.1% bovine serum albumin) to the final concentrations shown below. The diluted test compound or DMSO (1 μL) in the assay buffer (20 μL), the diluted serine protease solution (20 μL) and the substrate solution (20 μL) were added to a 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany). The fluorescence (excitation 360 nm, emission 460 nm) was measured at room temperature using a microtiter plate fluorescence reader (Safire II, Tecan, Maennedorf, Switzerland) for 30 minutes. The production of 50% serine protease activity inhibition (IC) was determined by a nonlinear logistic regression model. 50 ) of the test compound.

[1040] result

[1041] Compounds in the context of the present invention were selected along with several other enzymes. 50 The values ​​are shown in Table 2. Importantly, none of the proteases of the coagulation or fibrinolytic systems tested were inhibited by 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (fulacimstat) or TY-51469.

[1042] Table 2 Selectivity of chymotrypsin inhibitors

[1043]

[1044] 3. In in vitro assays, plasmin is a substrate for chymotrypsin and its degradation by chymotrypsin can be inhibited by chymotrypsin inhibitors

[1045] To evaluate the effect of chymotrypsin on plasmin, the temporal changes in plasmin activity in the presence of chymotrypsin were determined in mouse and human in vitro systems. In addition, the appearance of potential plasmin degradation products of mouse and human plasmin in the presence of chymotrypsin in buffer systems was investigated.

[1046] 3.1 Activity of plasmin in the presence of mouse and human chymotrypsin and the effect of chymotrypsin inhibitors

[1047] As previously described, recombinant mouse mMCP-4 (rmMCP-4) or recombinant human (rCMA-1) was produced in precursor form and activated with recombinant mouse cathepsin C (R&D Systems, Minneapolis, MN, USA) to have enzymatic activity (Semaan W et al., Biochem. Pharmacol. 94 (2015): 91-100). rmMCP-4 or rCMA-1 was thawed and diluted to a concentration of 20 μg / mL in maturation buffer (50 mM MES, 0.1% (w / v) BSA, pH 5.5). Active mouse cathepsin C was diluted to 20 μg / mL in cathepsin C buffer (50 mM MES, 50 mM NaCl, 5 mM DTT, pH 5.5). Activation was performed by adding equal volumes of recombinant chymotrypsin and cathepsin C, adding 50 μg / mL heparin and incubating at room temperature for 1 h. Chymotrypsin activation was stopped with 3 mM N-ethylmaleimide (NEM), followed by dilution with assay buffer (20 mM Tris, 2 M KCl, 0.02% (v / v) Triton X-100, pH 9.0) to a recombinant chymotrypsin concentration of 2 μg / mL; 5 min was sufficient to completely stop the cathepsin C-dependent reaction.

[1048] 37 nM rmMCP-4 or rCMA-1 was preincubated with vehicle or TY-51469 (final concentration of 0.1 or 1 μM) or 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (fulacimstat) (concentration range of 0.1 to 2.5 μM) at 37°C for 20 min and then incubated with 0.12 nM mouse plasmin or 0.24 nM human plasmin (Innovative Research, Inc., Novi, MI, USA) in 100 μL at 37°C for 24 h. Plasmin activity was determined by the hydrolysis rate of 50 μM substrate D-Ala-Leu-Lys-7-amino-4-methylcoumarin (Sigma Aldrich, Saint-Louis, MO, USA) at 37°C for 1 h in a white 96-well microtiter plate. Switzerland) in ex =370nm and λ em The released fluorescence was measured at 460 nm. A standard curve of 7-amino-4-methylcoumarin (AMC) was also obtained to estimate the concentration (nM) of the cleaved fluorescent substrate.

[1049] Figure 1 — Kinetics of chymotrypsin-dependent inactivation of human (A) and mouse (B) plasmin and its prevention by 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (fulacimstat) or TY-51469.

[1050] Figure 2 —Chymotrypsin-dependent inactivation of human (A) and mouse (B) plasmin was prevented by 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (fulacimstat) or TY-51469.

[1051] result

[1052] Figure 1The inactivation of human purified plasmin by recombinant human chymotrypsin (rCMA-1) in the absence or presence of increasing concentrations of 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (fulacimstat) (Figure i) or TY-51469 (Figure ii) is shown. Figure 1 A) and recombinant mouse mMCP-4-induced inactivation of mouse purified plasmin ( Figure 1 B) Kinetics. Each point represents mean ± SEM (n = 6-15). Statistical significance was determined using one-way ANOVA with Dunnett's multiple comparison test, where *P < 0.05, **P < 0.01, ***P < 0.001.

[1053] While when plasmin alone was exposed to the fluorescent substrate it resulted in a strong continuous increase in fluorescence over time as a measure of enzyme activity, the presence of chymotrypsin resulted in a strong reduction in fluorescence production which was increased again concentration-dependently by the chymotrypsin inhibitors fulacimstat and TY-51469, respectively. Figure 2 The maximum fluorescence obtained for each group at the end of the experiment (60 min) is shown.

[1054] These results surprisingly demonstrate that plasmin is a substrate for chymotrypsin in mice and humans and that its degradation and inactivation can be prevented by chymotrypsin inhibitors in animals and humans, making chymotrypsin inhibitors suitable for thrombolysis, especially in the etiology of acute thrombotic and thromboembolic events.

[1055] 3.2 Degradation of plasmin by chymotrypsin, which can be inhibited by chymotrypsin inhibitors

[1056] Mouse chymotrypsin (rmMCP-4) or human chymotrypsin (rCMA-1) was activated as described above. 74 nM rmMCP-4 or rCMA-1 was pre-incubated with vehicle or chymotrypsin inhibitor TY-51469 (10 or 300 μM) at 37°C for 20 min, and then incubated with 235 nM mouse plasmin (Innovative Research Inc., Novi, MI, USA) for rmMCP-4 or 235 nM human plasmin (Innovative Research Inc., Novi, MI, USA) for rCMA-1 for 30 min at 37°C. The reaction was stopped with an equal volume of formic acid (FA) (final concentration 4% (v / v) in water) and the samples were kept on ice until protein precipitation.

[1057] In-solution trypsin digestion, peptide purification and desalting on a C18 column

[1058] Proteins from in vitro cleavage assays were first precipitated by adding trichloroacetic acid (TCA) to a final concentration of 10% (v / v) and incubated at -20°C for 30 min. Proteins were centrifuged at 10,000 x g for 15 min. Protein pellets were washed with cold 100% acetone, air-dried, and resuspended in 50 μL of a solution containing 8 M urea, 10 mM HEPES-KOH (pH 7.4). The protein reduction step was performed by adding DTT (Thermo Fisher Scientific, Waltham, MA, USA) to a final concentration of 5 mM, boiling at 95°C for 2 min, and then incubating at room temperature for 30 min. Alkylation of proteins was performed by adding iodoacetamide (Sigma-Aldrich, Saint-Louis, MO, USA) to a final concentration of 7.5 mM, and then incubating at room temperature away from light for 20 min. By adding 150 μ L of 50 mM ammonium bicarbonate (NH4HCO3), the urea concentration was reduced to 2 M. The protein was digested by adding 1 μ g Pierce MS grade trypsin (Thermo Fisher Scientific, Waltham, MA, USA) and incubated overnight at 30 ° C. Digestion was stopped by adding trifluoroacetic acid (TFA) to a final concentration of 0.2% (v / v). The peptide was purified using Pierce C18 100 μ L tip column (Tip column) (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, the C18 tip column was first wetted by sucking 100 μ L of 100% acetonitrile (ACN) 3 times, and then balanced by sucking 100 μ L of 0.1% (v / v) TFA buffer 3 times. Each peptide sample was transferred to the balanced C18 tip column by sucking 100 μ L of sample up and down for 10 consecutive times. This step was performed twice to apply all samples to the column. The C18 tip column was then washed 3 times with 100 μL 0.1% (v / v) TFA buffer. The peptide was eluted by pumping 100 μL of 50% (v / v) ACN and 1% (v / v) FA buffer up and down in a new low binding microtube. This step was performed 3 times to obtain a final volume of 300 μL. The peptide was then concentrated at 65°C with a centrifugal evaporator until completely dry (about 60 min) and then resuspended in 25 μL 1% (v / v) FA buffer. The peptide was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and the absorbance at 205 nm was read. The peptide was then transferred to a glass vial (Thermo Fisher Scientific, Waltham, MA, USA) and stored at -20°C until mass spectrometry analysis was performed.

[1059] LC-MS / MS analysis

[1060] Tryptic peptides were separated using a Dionex Ultimate 3000 nanoHPLC system. 10 μL of sample (2 μg total) in 1% (v / v) FA was loaded onto an Acclaim PepMap100 C18 column (0.3 mm id×5 mm, Dionex Corporation) at a constant flow rate of 4 μL / min. After trap enrichment, the peptides were eluted onto an EasySpray PepMap C18 nanocolumn (75 μm×50 cm, Dionex Corporation) with a linear gradient of 5-35% solvent B (90% ACN with 0.1% FA) over 240 min at a constant flow rate of 200 nL / min. The HPLC system was coupled to an Orbitrap QExactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) via an EasySpray source. The spray voltage was set to 2.0 kV and the column temperature was set to 40°C. Full scan MS spectra (m / z 350-1600) in profile mode were acquired in the Orbitrap at a resolution of 70,000 after accumulating 1,000,000 ions. The 10 most intense peptide ions from the preview scan of the Orbitrap were fragmented by collision-induced dissociation (normalized collision energy 35% and resolution 17,500) after accumulating 50,000 ions. The maximal filling time was 250 ms for full scans and 60 ms for MS / MS scans. Precursor ion charge state screening was enabled and all unassigned charge states as well as single 7- and 8-charged species were excluded. The dynamic exclusion list was limited to a maximum of 500 entries, with a maximum retention time of 40 s and a relative mass window of 10 ppm. The lock mass option was enabled for full spectrum scans to improve mass accuracy. Data were acquired using Xcalibur software (ThermoFisher Scientific, Waltham, MA, USA).

[1061] Protein identification by MaxQuant analysis

[1062] The raw files were analyzed using MaxQuant software (version 1.6.17.0) and the Uniprot mouse proteome database (March 7, 2021, 55 366 entries) or the Uniprot human proteome database (March 21, 2020, 75 776 entries), which contains the mouse plasminogen database (UniProtKB-P20918 (PLMN_MOUSE)) and the human plasminogen database (UniProtKB-P00747 (PLMN_HUMAN)). The settings used for MaxQuant analysis were: 4 cleavage errors were allowed; the minimum peptide length was 6, the fixed modification was carbamidomethylation on cysteine; the enzymes were trypsin (K / R) and CMA1 (F / Y); the variable modifications included in the analysis were methionine oxidation, protein N-terminal acetylation, and protein carbamylation (K, N-terminal). For precursor ions, the mass tolerance used was 10 ppm, and for fragment ions, the tolerance used was 20 ppm. The identification values ​​"PSMFDR", "Protein FDR" and "Site decoy fraction" were set to 0.05. The minimum peptide count was set to 1. Label-free quantification (LFQ) was also selected with a LFQ minimum ratio count of 1. The "Second Peptide" option was also allowed. After the analysis, the results were classified according to several parameters. Proteins that were positive for at least one of the categories "Reverse", "Only.identified.by.site" or "Potential.contaminant" were excluded. The numbering of plasmin / plasminogen amino acids is based on the methionine at position 1.

[1063] Figure 3 —Human (A) and mouse (B) plasmin is a substrate for chymotrypsin, and its degradation by the latter protease is prevented by 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (fulacimstat) or TY-51469.

[1064] result:

[1065] Identification of the cleavage products by LC-MS / MS showed that there were several chymotrypsin-induced cleavage sites in the plasmin sequence, and their intensities are shown in Figure 3 A: human plasmin, and Figure 3 B: mouse plasmin (arbitrary units, each bar graph represents mean ± SEM; n = 4).

[1066] The homologous cleavage sites between human and mouse plasmin are Y283 / 283, F602 / 603, F711 / 713 and F734 / 735 (human / mouse; Y=tyrosine, F=phenylalanine). Black arrows indicate that these cleavages are absent in plasmin alone, while grey arrows indicate that these cleavages are absent when chymotrypsin inhibitors are added to plasmin and chymotrypsin, indicating that the identified cleavage sites are caused by chymotrypsin.

[1067] 4. Inhibition of chymotrypsin activity in vivo by gene knockout or pharmacological chymotrypsin inhibition effectively and dose-dependently reduced thrombus weight in the stenosis model and improved blood flow

[1068] Since chymotrypsin is normally rapidly inactivated in plasma, it is unclear whether the plasmin-degrading properties of chymotrypsin might be pathophysiologically relevant and lead to reduced fibrin degradation in vivo. We therefore explored the question in vivo whether chymotrypsin inhibition affects clot lysis and, therefore, vascular patency by keeping plasmin activity intact. Venous stenosis experiments were performed in mMCP4 knockout mice or mice and hamsters treated with the chymotrypsin inhibitors 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (fulacimstat) or TY-51469 and compared with untreated or vehicle-treated animals.

[1069] We investigated the impact of thromboprophylaxis and thrombotherapy settings by varying the time point of chymotrypsin inhibitor administration relative to stenosis onset.

[1070] 4.1 Thrombus measurement in the mouse venous stenosis model

[1071] Inferior vena cava (IVC) stenosis was performed in anesthetized mice (a mixture of 2% isoflurane and 2.5% oxygen) as previously described by Payne & Brill and Canobbio et al. (Payne H., Brill A., J. Visc. Exp. (2017) doi: 10.3791 / 56697; Canobbio I et al., Blood 130 (2017) 527-536). A laparotomy was performed, and the viscera were gently removed from the abdominal cavity without injury and stored in a 0.9% NaCl solution (37°C). The IVC was exposed by dividing the vessels between the renal vein and the iliac lumbar vein. For partial ligation, a 30G needle was used as a spacer. Neither the IVC lateral branches nor the posterior branches were ligated. After ligation with polypropylene 7.0 sutures (FST, Foster City, CA) around the IVC and the spacer, the spacer was gently removed. By doing this, it was ensured that the endothelium was not stripped and that 10% of residual blood flow was still retained in the IVC. The viscera were then moved back to their original position in the abdomen, and the abdominal cavity was closed layer by layer with braided 5.0 absorbable sutures. Buprenorphine (0.1 mg / kg subcutaneously every 6-9 hours) was used to maintain analgesia for 24 hours after surgery. After euthanasia, the IVC was incised, the thrombus was separated, measured, weighed, and finally stored at -80°C.

[1072] Under these conditions, thrombi weighing ~10 mg and measuring ~4-5 mm in length were observed in wild-type males.

[1073] The results of these "control animals" were compared to animals in which chymotrypsin enzyme activity was altered, genetically suppressed (i.e., mMCP-4KO- / -), or pharmacologically inhibited:

[1074] i) Knockout mice

[1075] mMCP-4KO mice have been backcrossed with C57BL / 6 for more than 10 generations and are highly homologous to the latter strain (Tchougounova E. et al., J. Experiment. Med. 198 (2003) 423-431). The genotype of mMCP-4KO mice used in this study was confirmed by polymerase chain reaction.

[1076] ii) 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo

[1077] -3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine

[1078] -5-Carboxylic acid (fulacimstat) dosing regimen

[1079] Mice were treated with a single oral dose of vehicle or 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (fulacimstat) (0.1, 1, or 3 mg / kg) 1 or 24 hours after ligation as described previously. Stenosis was assessed 24 or 48 hours after ligation. The IVC was opened, thrombi were isolated, measured, and weighed.

[1080] iii) TY-51469 Dosing Regimen

[1081] TY-51469 (0.1, 1 or 10 mg / kg) was administered intraperitoneally 1 or 24 hours after ligation of the inferior vena cava (IVC). Stenosis was assessed 24 or 48 hours after ligation. The IVC was opened, the thrombus was isolated, measured and weighed.

[1082] FIG4 - Fulacimstat or TY-51469 prevents (i) or resolves (ii) DVT in mouse (A) and hamster models (B) of IVC ligation.

[1083] result

[1084] The results of thrombus weight and length measurements are as follows Figure 4A As shown and summarized in Table 3 (results using 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (fulacimstat)) and Table 4 (results using TY-51469), it shows that inhibition of chymotrypsin activity in knockout animals and animals treated with chymotrypsin inhibitors resulted in a dose-dependent reduction in thrombi compared with control animals (n=6-13; each point represents data from one animal; bar graphs represent mean ± standard error of the mean; one-way ANOVA followed by Bonferroni multiple comparison test or Mann-Whitney T test, *P<0.05, **P<0.01, ***P<0.001).

[1085] Figure 4Ai) shows the thrombus weight measured 24 h after the onset of stenosis in mMCP4 knockout mice and groups of animals treated with different doses of 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (fulacimstat) (oral administration) or TY-514691 (intraperitoneal administration) 1 h after the onset of inferior vena cava (IVC) stenosis. In the early post-stenosis setting (administration 1 h after ligation), both compounds were observed to have a statistically significant effect on thrombus weight in a dose-dependent manner. Compounds were administered 24 h after the onset of stenosis, and thrombi were collected 48 h later ( Figure 4A ii) also showed potent efficacy, resulting in a near complete reduction of thrombi.

[1086] Surprisingly, in the setting of inferior vena cava stenosis, a single administration of a chymotrypsin inhibitor reduced clot weight and length not only in the prevention setting, i.e., administration of the compound before the onset of clot-induced blood flow reduction, but also in the clearance intervention setting, i.e., 24 h after the onset of thrombosis. These results were obtained by using two structurally distinct chymotrypsin inhibitors, 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (fulacimstat) and TY-51469, in an established mouse DVT model, as well as in a mouse model of DVT. The results obtained using only 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (fulacimstat) in a newly developed hamster DVT model (both animal models have inferior vena cava stenosis) confirm the general nature of these findings. These effects were achieved with two unrelated chymotrypsin inhibitors from two different structural classes, indicating that this is not a compound-specific finding but is related to general chymotrypsin inhibition.

[1087] Table 3 Effects of fulacimstat on thrombus weight and length in the mouse venous stenosis model

[1088]

[1089] One-way ANOVA followed by Bonferroni multiple comparison test or Mann-Whitney T-test. (*) symbol indicates statistical significance between vehicle and Fula-treatment. *P<0.05, **P<0.01, ***P<0.001. Error type: standard error of the mean (±SEM).

[1090] Table 4 Effects of mMCP-4 knockout and TY-51469 on thrombus weight and length in the mouse venous stenosis model

[1091]

[1092] One-way ANOVA followed by Bonferroni multiple comparison test or Mann-Whitney T-test. (*) symbol indicates statistical significance between control and TY-treatment. *P<0.05,

[1093] **P<0.01, ***P<0.001. Error type: standard error of the mean (±SEM).

[1094] 4.2 Thrombus Measurement in Hamster Venous Stenosis Model

[1095] To ensure that the surprising effects in the mouse venous stenosis model were not species-specific, additional experiments were performed in the novel hamster venous stenosis model described below.

[1096] Inferior vena cava (IVC) stenosis was achieved by anesthetizing 108-120 g hamsters with a mixture of isoflurane and oxygen (2%; 2.5%, respectively). Laparotomy was performed, and the viscera were gently removed from the abdominal cavity without injury and stored in 0.9% NaCl solution (37°C). The IVC was exposed by dividing the vessels between the renal vein and the iliac lumbar vein. A 27G (circumferential 0.4 mm) needle was used as a spacer. Neither the IVC lateral branches nor the posterior branches were ligated. After ligation with polypropylene 7.0 sutures (FST, Foster City, CA) around the IVC and the spacer, the spacer was gently removed. The viscera were then moved back to their original position in the abdomen, and the abdominal cavity was closed layer by layer with braided 5.0 absorbable sutures. Buprenorphine (0.1 mg / kg subcutaneously every 6-9 h) was used to maintain analgesia for 24 h after surgery. Thrombosis was evaluated 24 or 48 h after stenosis: the IVC was opened, the thrombus was isolated, measured, and weighed.

[1097] Hamsters were treated 1 or 24 h after stenosis onset with a single oral dose of vehicle (10% EtOH, 40% solutol, 50% sterile water) or fulacimstat (0.01, 0.1 and 1 mg / kg at 1 h and 1 mg / kg at 24 h after stenosis onset) as described previously.

[1098] result:

[1099] The results of thrombus weight and length measurements are as follows Figure 4B As shown in Figure 5 and summarized in Table 5, it is shown that inhibition of chymotrypsin activity in hamsters treated with the chymotrypsin inhibitor 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (fulacimstat) resulted in a dose-dependent reduction in thrombi compared to control animals, with a statistically significant reduction in thrombi at the high dose in the early post-stenotic setting (administered 1 h after stenosis onset, collected 24 h later; Figure 4B i)). 1 mg / kg fulacimstat was given 24 hours after the onset of IVC stenosis, and thrombi were collected 48 hours later ( Figure 4B ii)) also showed strong efficacy, resulting in complete resolution of the thrombus.

[1100] The results of the hamster venous stenosis model were very similar when compared with the mouse venous stenosis model, demonstrating that the reduction of thrombi by chymotrypsin inhibition is not a species-specific phenomenon.

[1101] It is noteworthy that in this case, the size of thrombi collected at 24 h in the vehicle group was very similar in weight and length to those collected at 48 h in the vehicle group, indicating that after 24 h, when fulacimstat was administered, the size of thrombi had reached a plateau (n=6-8; mean ± standard error of the mean; one-way ANOVA followed by Bonferroni multiple comparison test or Mann-Whitney T test, *P<0.05, **P<0.01, ***P<0.001).

[1102] Table 5 Effect of fulacimstat on thrombus weight and length in the hamster venous stenosis model

[1103]

[1104] One-way ANOVA followed by Bonferroni multiple comparison test or Mann-Whitney T-test. *P<0.05, **P<0.01, ***P<0.001. Error type: standard error of the mean (±SEM).

[1105] 5. Direct chymotrypsin inhibition improves blood flow in a mouse venous stenosis model

[1106] The main purpose of thrombosis is to ensure adequate blood flow to properly maintain the tissue around the blood vessels. In order to evaluate whether the reduction of thrombus by chymotrypsin inhibition described in 4.1 and 4.2 is associated with increased patency of the inferior vena cava (IVC) compared with the control, a high-frequency ultrasound imaging system (Vevo3100; Fujifilm VisualSonics Inc.) equipped with an MX400 linear array transducer (40MHz) was used to visualize the blood flow in the inferior vena cava (IVC) and abdominal aorta (AA) at different time points in the venous stenosis model as described in 4.1 by Doppler color flow mapping. During this procedure, mice were anesthetized (2% isoflurane; 4% oxygen), their abdomens were shaved and covered with ultrasound gel. In control mice and mice given 10 mg / kg TY-51469 intraperitoneally 1 h after the onset of stenosis, blood flow in the vessels was measured 12 h before ligation and 6 h and 24 h after ligation.

[1107] result

[1108] The results of blood flow velocity analysis in the inferior vena cava (IVC) and abdominal aorta (AA) measured before, 6h and 24h after the onset of stenosis in control animals and animals treated with TY-51469 1h after the onset of stenosis are summarized in Table 6.

[1109] These results indicate that, while a complete loss of blood flow was observed in control IVC-ligated animals, blood flow in the vena cava was restored 6 h after the onset of stenosis in animals treated with chymotrypsin inhibitors. This result suggests that chymotrypsin inhibition not only leads to a reduction in thrombus formation but also has a significant effect on blood flow in the injured vessel.

[1110] Table 6 Blood flow velocity in rat IVC and AA 1 h after stenosis onset + / - TY-51469

[1111]

[1112] One-way ANOVA followed by Bonferroni multiple comparison test. *P<0.05, ***P<0.001. Error type: standard error of the mean (±SEM).

[1113] 6. In mice and hamster stenosis models, reducing chymotrypsin activity in vivo by direct chymotrypsin inhibitors eliminates pulmonary embolism

[1114] A very important question for any thrombolytic therapy is whether the disintegration of the clot will result in the generation of emboli that may be transported to the lungs and cause pulmonary embolism. To address the question of whether chymotrypsin inhibition has an effect on embolism, the lungs of mice and hamsters treated or not with fulacimstat in a venous stenosis experiment were isolated. The right lung lobe was used to visually quantify the number of lungs with embolism.

[1115] Figure 5 -- Mice treated with fulacimstat had a reduced number of pulmonary emboli in their lungs

[1116] Figure 6 -- Hamsters treated with fulacimstat had a reduced number of pulmonary emboli in their lungs

[1117] result

[1118] Visual lung analysis results Figure 5 (mouse) and Figure 6 (hamster) and are summarized in Table 7.

[1119] exist Figure 5 A and Figure 6 In A, randomly selected visual images of the right lung lobes extracted from 5 vehicle or 5 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (fulacimstat)-treated mice or hamsters (average 6-10 animals / group) are shown. Pulmonary thrombi are indicated by black arrows and are shown in Figure 5 A and Figure 6 In the upper panel of A, this was observed in vehicle-treated mice or hamsters. Figure 5 A and Figure 6 The right lung lobes of mice and hamsters treated with 3 mg / kg (mice) and 1 mg / kg (hamsters) of fulacimstat orally 24 hours after venous stenosis are shown in the lower panel of A. It shows normal lung anatomy and no signs of pulmonary embolism 24 hours after stenosis induction.

[1120] Part B of both figures summarizes the results of vehicle-treated mice in the stenosis model in the form of pie charts ( Figure 5 ) and hamsters ( Figure 6 ) Incidence of pulmonary embolism 24h or 48h after the onset of stenosis. Emboli were observed in 30-50% of vehicle-treated animals.

[1121] Mice treated with 3 mg / kg fulacimstat ( Figure 5 ) and hamsters treated with 1 mg / kg fulacimstat ( Figure 6 ) showed no signs of embolism at any time point (i.e., administration 1 h after stenosis onset and reading 24 h, or administration 24 h after stenosis onset and reading 48 h).

[1122] Thus, the profibrinolytic approach by chymotrypsin inhibition not only showed no increase in emboli compared to control animals, but surprisingly, it led to a strong reduction in pulmonary emboli.

[1123] The dissolution data after 24 h may suggest that emboli may have been present in the animals 24 h after the onset of stenosis, prior to treatment with fulacimstat. The finding in both species, that after 48 h, no emboli were observed in treated mice and hamsters, surprisingly suggests that chymotrypsin inhibition can reduce thrombi not only in the local environment of thrombus generation, as shown for thrombi in the vena cava, but also in the presence of emboli.

[1124] Surprisingly, when the lungs were examined in an IVC stenosis experiment in mice and hamsters, it was observed that the number of pulmonary emboli in the lungs of animals under chymotrypsin inhibition was reduced compared to control animals. This very important and relevant case of thromboembolism shows that chymotrypsin not only acts at the local site of thrombus formation, but also has an antifibrinolytic effect in emboli. Chymotrypsin inhibitors are therefore effective agents in embolic disease, including, for example, dissolving deep vein emboli causing pulmonary embolism or left atrial appendage emboli causing stroke or systemic embolism in patients with atrial fibrillation.

[1125] Table 7 Effect of fulacimstat on the incidence of pulmonary embolism in the venous stenosis model

[1126]

[1127] One-way ANOVA followed by Bonferroni multiple comparison test. *P<0.05. Error type: standard error of the mean (±SEM).

[1128] 7. In the thrombosis model of vascular wall injury (FeCl3-induced injury model), chymotrypsin inhibitors effectively and dose-dependently reduce thrombus weight

[1129] To exclude that the effect of chymotrypsin inhibition was limited to the stenosis model, we investigated whether chymotrypsin inhibition could prevent thrombosis triggered by vessel wall injury in a short-term ferric chloride-induced injury model in mice and hamsters.

[1130] 7.1 In mice Inferior vena cava Ferric chloride-induced injury

[1131] The FeCl3-induced injury model was performed as previously described (Aghourian MN et al., J. Thromb. Hemost. 10 (2012) 447-452; Li W et al., J. Vis. Exp. 54479 (2016) doi: 10.3791 / 54479; Wang X et al., J. Thromb. Hemost. 4 (2006) 403-416). Wild-type male mice were treated with vehicle, 10 mg / kg fulacimstat or 10 mg / kg TY-51469 1 h before animals were anesthetized, while mMCP-4 knockout animals were not treated before anesthesia. The endothelial injury model of the inferior vena cava (IVC) was obtained by anesthetizing mice with ketamine / xylazine (87 / 13 mg / kg IM). Laparotomy was performed, and the viscera were gently removed from the abdominal cavity without damage and stored in 0.9% NaCl solution (37°C). The IVC was exposed by separating the blood vessels between the renal vein and the iliac lumbar vein. A 2×2mm double-layer absorbent gauze soaked in 0.37M (10% w / v, in sterile water) hydrated ferric chloride (FeCl3) (Sigma-Aldrich, St-Louis, MO, USA) was applied directly to the IVC for 3 minutes and then removed. The viscera were then moved back to their original position in the abdomen. The mice were killed 30 minutes later to obtain the thrombotic area in the IVC. The IVC was cut open, the thrombus was separated and weighed with an analytical balance (accuracy 0.1 mg, EntrisII, Sartorius).

[1132] Figure 7 In the FeCl3-induced injury model, chymotrypsin inhibition reduces thrombus weight in the inferior vena cava (IVC) and femoral vein (hamster, C) of mice (A) or hamsters (B).

[1133] result

[1134] The results of thrombus measurement are as follows Figure 7 A and summarized in Table 8. It shows that oral administration of 10 mg / kg 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (fulacimstat) and intraperitoneal administration of 10 mg / kg TY-51469 before injury - and therefore in the prevention setting - or mMCP-4KO resulted in a statistically significant reduction in thrombi compared to control animals.

[1135] Table 8 Effect of chymotrypsin inhibition on thrombus weight in the FeCl3-induced injury thrombosis model in the inferior vena cava of mice

[1136]

[1137] One-way ANOVA followed by Bonferroni multiple comparison test or Mann-Whitney T-test. (*) symbol indicates statistical significance between vehicle and fulacimstat treatment. & ) Symbols indicate statistical significance between control, TY-51469-treated and mMCP-4KO mice. *P<0.05, **P<0.01, ***P<0.001. Error type: standard error of the mean (±SEM).

[1138] 7.2 Ferric chloride-induced injury in the vena cava or femoral vein of hamsters

[1139] The FeCl3-induced injury model in hamsters was inspired by the methods used in mice (Section 7.1). Golden Syrian hamsters were treated orally with vehicle, 3 or 10 mg / kg 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (fulacimstat) 1 h before animals were anesthetized.

[1140] Ferric chloride-induced injury in the vena cava

[1141] Endothelial injury of the inferior vena cava (IVC) was achieved by anesthetizing hamsters weighing between 115-130 g with ketamine / xylazine (87-200 / 13 mg / kg IM). The body temperature of the animals was maintained at 37°C with a heating pad throughout the experiment. A laparotomy was performed and the viscera were gently removed from the abdominal cavity without injury and stored in a 0.9% NaCl solution (37°C). The IVC was then exposed by dividing the vessels between the renal vein and the iliac lumbar vein. A 2×2 mm double layer of absorbent gauze soaked in 0.37 M (10% w / v in sterile water) hydrated ferric chloride (FeCl3) (Sigma-Aldrich, St-Louis, MO, USA) was applied directly to the IVC for 3 min and then removed. The viscera were then moved back to their original position in the abdomen. The hamsters were sacrificed 30 min later to locate the thrombotic area within the injured IVC.

[1142] Ferric chloride-induced injury in the femoral vein

[1143] The femoral vein (FV) endothelial injury model was obtained by anesthetizing 115-130 g hamsters with ketamine / xylazine (87-200 / 13 mg / kg IM). The body temperature of the animals was maintained at 37°C with a heating pad throughout the experiment. The left femoral vein was exposed after incision of the skin and separation from the femoral artery. A 2×8 mm double layer of degreased gauze soaked with 3 μL of 5% hydrated ferric chloride (Sigma-Aldrich, St-Louis, MO, USA) (5% w / v in sterile water) was applied directly to the femoral vein for 5 minutes and then removed. The hamsters were sacrificed 30 minutes later and the femoral vein was incised.

[1144] Collection and characterization of thrombus

[1145] The relevant pieces of the inferior vena cava and femoral vein were dissected, and the thrombi were isolated and weighed using an analytical balance (Sartorius).

[1146] result

[1147] The results of thrombus measurement were as follows: Figure 7 B and Figure 7 C, and summarized in Table 9, which shows that the reduction of chymotrypsin activity with 3 and 10 mg / kg 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (fulacimstat) resulted in a statistically significant reduction in thrombus weight compared to control animals. This was demonstrated not only in the inferior vena cava, but also in the much smaller femoral vein, indicating that this effect of chymotrypsin inhibition is not limited to large vessels.

[1148] Table 9 Effect of fulacimstat inhibition of chymotrypsin on thrombus weight in the FeCl3-induced injury thrombosis model in the inferior vena cava and femoral vein

[1149]

[1150] One-way ANOVA followed by Bonferroni multiple comparison test or Mann-Whitney T-test. *P<0.05, **P<0.01, ***P<0.001. Error type: standard error of the mean (±SEM).

[1151] 7.3 Mice and hamsters Ferric chloride induced Carotid artery damage

[1152] To exclude that the effect of chymotrypsin inhibition was limited to the venous thrombosis model, we investigated whether chymotrypsin inhibition could prevent thrombosis triggered by arterial wall injury in a short-term ferric chloride-induced carotid artery injury model in mice and hamsters.

[1153] Fig.13 -- Fulacimstat reduces thrombus-induced occlusion in the FeCl3-induced injury model of mouse or hamster common carotid artery (CCA). Each series of images is representative of 6 independent experiments.

[1154] Ferric chloride-induced carotid artery injury in mice

[1155] The FeCl3-induced injury model was performed as previously described (Bonnard T et al., J. Vis. Exp. 2015; doi: 10.3791 / 52838). Wild-type male mice were treated with vehicle, 10 or 20 mg / kg fulacimstat 1 h before anesthesia.

[1156] Endothelial injury model of common carotid artery (CCA) was established by anesthetizing mice with ketamine / xylazine (87 / 13 mg / kg IM). A 1 cm vertical skin incision was made on the neck to expose and isolate the right CCA and vagus nerve. A 2×2 mm double layer of degreased gauze soaked in 0.28 M (7.5% w / v, in sterile water) ferric chloride (FeCl3) (Sigma-Aldrich, St-Louis, MO, USA) was applied directly to the exposed CCA for 1 min and then removed. The CCA was then moved back to its original position near the trachea. Mice were killed 30 min later. The chemically injured and intact contralateral CCA were cut off and stored in PFA (4% w / v) overnight and then stored in EtOH (70%) at 4°C for subsequent histological analysis.

[1157] result

[1158] Fig.13 CCA histology and quantification of thrombus area after FeCl3 administration (Image J) are shown in A and B, as well as identification of the reference section for thrombus area measurement (the first section shows FeCl3-induced vascular injury). Each series of images represents 6 independent experiments. It shows that oral administration of 10 or 20 mg / kg fulacimstat before injury resulted in a statistically significant reduction in thrombus size in mice in a dose-dependent manner compared to control animals ( Fig.13C; One-way ANOVA followed by Bonferroni multiple comparison test or Mann-Whitney T-test. *P<0.05, **P<0.01, ***P<0.001. Error type: standard error of the mean (±SEM).

[1159] Ferric chloride-induced carotid artery injury in hamsters

[1160] The FeCl3-induced injury model in hamsters was highly inspired by the methods used in mice (Section 7.3). Golden Syrian hamsters were treated orally with vehicle or 10 mg / kg 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (fulacimstat) 1 h before animals were anesthetized.

[1161] Endothelial injury of the CCA was achieved by anesthetizing hamsters weighing between 115-130 g with ketamine / xylazine (87-200 / 13 mg / kg IM). The body temperature of the animals was maintained at 37°C with a heating pad throughout the experiment. A 2 cm vertical skin incision was made in the neck, and the right CCA and vagus nerve were exposed by separation. A 2×2 mm double-layer absorbent gauze soaked in 1.11 M (30% w / v, in sterile water) hydrated ferric chloride (FeCl3) (Sigma-Aldrich, St-Louis, MO, USA) was applied directly to the exposed CCA for 3 min and then removed (Kuo et al., J. Biomed. Sci, 2009). The CCA was then moved back to its original position near the trachea. The hamsters were killed 30 min later and the thrombotic area in the CCA was obtained. The chemically injured and intact contralateral CCA were excised, and the thrombi were isolated and weighed using an analytical balance (precision 0.1 mg, Entris II, Sartorius).

[1162] result

[1163] exist Fig.13 The results of thrombus measurements are quantified in D. They show that oral administration of 10 mg / kg fulacimstat prior to injury resulted in a statistically significant reduction in thrombi in hamsters compared to control animals (one-way ANOVA followed by Bonferroni's multiple comparisons test T-test. **P<0.0. Error type: standard error of the mean (±SEM)).

[1164] 7.4 Overall interpretation of the results of the FeCl3-induced damage model

[1165] In these models of venous and arterial vessel wall injury, mice and hamsters in which chymotrypsin activity was reduced by chymotrypsin inhibitors or mMCP-4 knockout had reduced thrombus weight compared with vehicle control animals. This is surprising because, in contrast to the stenosis model, thrombi in this model arise rapidly after vessel wall injury and are already detached after 30 min. It can therefore be considered a model of thrombogenesis rather than thrombolysis. However, even under these conditions, the profibrinolytic effects of the prophylactically administered compounds appear to be strong enough to balance the procoagulant effects of the growing thrombus.

[1166] Surprisingly, in contrast to the ferric chloride-induced injury experiments performed by Ponomaryov et al. in mesenteric arterioles, the ferric chloride-induced injury experiments described in the present invention at different venous and arterial vessels showed significant differences between the groups treated with chymotrypsin inhibitors and the vehicle group. In these short-term thrombosis experiments, the compounds were given before ferric chloride-induced thrombosis, which means that even at an early stage during clot formation, chymotrypsin inhibitors can have an effect on thrombus weight and thus on the risk of vascular occlusion.

[1167] 8. Chymotrypsin is present in thrombi generated in vivo in mice and in human thrombus samples

[1168] Immunohistochemistry experiments were performed to investigate the presence of chymotrypsin in thrombi from animal studies and in human thrombi, and to assess the feasibility of transfer to the clinical setting.

[1169] Due to the lack of antibodies that bind to hamster chymotrypsin, experiments focused on studying thrombi in mice.

[1170] 8.1 Immunohistochemical analysis of mouse chymotrypsin and perivascular mast cells in mouse thrombi

[1171] Immunochemical analysis was performed on healthy and ligated sections of the inferior vena cava of wild-type and mMCP-4KO mice. The samples were fixed with 10% buffered formalin solution, processed, embedded in paraffin, and finally sectioned (4 μm sections) on the histopathology platform of RI-MUHC (Montreal, Québec, Canada). Mast cells were stained with toluidine blue (TB), and mMCP-4 was detected using goat polyclonal anti-MCPT4 antibody (USBiological life science, cat. #M2414-20A, 1×400 dilution) and biotinylated horse anti-goat IgG (Vector Laboratories, cat. #BA9500, 1×200 dilution). Chymotrypsin was detected using goat polyclonal IgG against human MCPT4 (Abcam, cat. #ab11239, 1×200 dilution).

[1172] Figure 8 -Deep vein thrombosis showing mast cells containing immunoreactive chymotrypsin in wild-type mice with IVC ligation. Each image represents 3 different tissue samples.

[1173] result

[1174] Figure 8 Representative images of immunochemical detection of chymotrypsin in thrombi from experiments are shown (scale bar: 50 μm; black arrows in the left image: mast cells; black arrows in the right image: mMCP-4 immunoreactivity). These images are representative of 3 independent experiments.

[1175] 24 h after the onset of stenosis, mMCP-4-specific immunoreactivity was localized around and within the thrombus in the ligated vena cava of wild-type animals ( Figure 8 D), but in thrombus-free ligated veins isolated from mMCP-4KO mice ( Figure 8 F) and in unligated, non-thrombus-free vessels from wild-type animals ( Figure 8 In the latter samples, most mMCP-4-specific immunoreactivity was detected in perivascular mast cells. On the other hand, toluidine blue-stained mast cells were identified in the adventitia of vena cava samples from both WT and mMCP-4KO mice ( Figure 8 A, E).

[1176] 8.2 Immunohistochemical analysis of human chymotrypsin and mast cells in human samples of deep vein thrombosis, pulmonary embolism, and popliteal artery occlusion

[1177] To demonstrate that chymotrypsin is not only present in thrombi from animal experiments but also plays a role in the degradation of plasmin in human thrombi, chymotrypsin immunoreactivity was assessed in samples of human thrombotic events.

[1178] Preformed sections of human venous thrombi were obtained from Tissue for Research Ltd (Ellingham, Suffolk, UK), human arterial thrombi were obtained from Discovery Life Science (Powell, OH, USA), and pulmonary embolism samples were obtained from the Dept of Pathology, McGill University.

[1179] For identification and quantification of mast cells, toluidine blue staining of basophilic granules was used. This is an established method, which metastains red-purple against a blue background. The staining solution was from Newcomers Supply.

[1180] To identify chymotrypsin in human samples, an immunochemical analysis was performed. Dewaxed and rehydrated tissue slides (4 microns) were placed on a Leica Bond Max automated staining system using the Bond Polymer RefineDetection kit (DS9800). HIER (heat-induced antigen retrieval) was performed at 95°C for 20 minutes using ER2 solution (AR9640). Peroxidase blocking was then performed for 5 minutes. The primary antibody (mouse anti-human mast cell chymotrypsin antibody [CC1] (Abeam: ab2377)) was then added and incubated for 15 minutes. Rabbit anti-mouse secondary antibody solution was added for 8 minutes. Anti-rabbit poly-HRP reagent from the Leica kit was added for 15 minutes. DAB (3,3'-diaminobenzidine) solution was added for 5 minutes. Counterstaining: Hematoxylin was added for 2 minutes. The slides were rehydrated with gradient ethanol (70%, 95%, and 100%), washed by three changes of xylene, then air-dried and mounted. Human tonsil tissue was used as a quality control.

[1181] Fig. 9 - Human venous thrombosis, pulmonary embolism and arterial thrombosis showing mast cells containing immunoreactive chymotrypsin. Each image represents 3 different tissue samples.

[1182] result

[1183] Fig. 9 Representative images of chymotrypsin-specific immunostaining in human samples are shown (dilution of human chymotrypsin antibody: 1 / 200; black arrows indicate mMCP-4 immunoreactivity). All images are representative of 3 independent experiments.

[1184] Chymotrypsin is located in Fig. 9 A human deep vein thrombosis (from left iliac, right femoral and left inguinal varicose veins), as shown in Example Fig. 9 The interior of the pulmonary embolus shown in B and the popliteal artery thrombus ( Fig. 9 C) All samples showed chymotrypsin immunoreactivity as well as chymotrypsin-positive mast cells within the thrombus (black arrows).

[1185] These results demonstrate for the first time that chymotrypsin is present in human thrombi and therefore plays a role in the degradation of plasmin, thus having an antifibrinolytic effect in human pathology. Chymotrypsin inhibitors exhibit profibrinolytic effects by maintaining the integrity of plasmin and leading to rapid lysis of thrombi - as has been demonstrated in animal models.

[1186] 9. Chymotrypsin activity is present in thrombi and can be inhibited by chymotrypsin inhibitors, which leads to increased plasmin activity in thrombi

[1187] Although the presence of chymotrypsin in thrombi could be verified, its role in thrombi was unclear. Therefore, experiments were performed to measure the activities of chymotrypsin and plasmin in thrombus homogenates from control animals and animals treated with a chymotrypsin inhibitor.

[1188] 9.1 In thrombi treated with chymotrypsin inhibitors, ex vivo chymotrypsin activity is reduced, while plasmin activity is increased

[1189] To demonstrate that the reduction in thrombi in animals treated with chymotrypsin inhibitors was associated with a decrease in chymotrypsin activity and an increase in plasmin activity in thrombi, in the experiments described subsequently, chymotrypsin and plasmin were extracted from mouse and hamster thrombi and the enzyme activities were measured in mice and hamsters.

[1190] Extraction of chymotrypsin from mouse and hamster thrombi in vitro

[1191] The thrombus was homogenized in 10 volumes (w / v) of 20 mM PBS (pH 7.4) (supplemented with 0.5 mg / mL BSA) using a glass-Teflon homogenizer (Kakizoe E. et al., J. Invest. Dermatol. 116 (2001) 118-123). The homogenate was centrifuged at 18,000 x g for 30 min at 4°C and the supernatant was discarded. This last step was repeated twice more, but the supernatant was retained during the last centrifugation. The supernatant was stored at 4°C until the enzymatic activity of chymotrypsin was tested on the same day.

[1192] Extraction of plasmin from mouse and hamster thrombi in vitro

[1193] The thrombus was homogenized in 10 volumes (w / v) of 20 mM PBS (pH 7.4) (supplemented with 1.5 mg / mL BSA) using a glass-Teflon homogenizer. The homogenate was transferred to a 1 mL ultracentrifuge tube and incubated at 4 °C. 48 The supernatant was stored at 4°C until the enzyme activity of plasmin was tested on the same day.

[1194] Measurement of plasmin or chymotrypsin activity in thrombus

[1195] 30 μL of thrombus extract supernatant was used and placed directly in a white 96-well plate. The Bradford protein assay did not find changes in protein concentration per unit volume of thrombus (results not shown). Some homogenates were treated with 10 μM TY-51469 or fulacimstat (volume 5 μL) before fluorescence reading. 65-70 μL of 20 mM PBS (pH 7.4) supplemented with 1.5 mg / mL BSA (for plasmin) or 0.5 mg / mL BSA (for chymotrypsin) was added to each well to reach a final volume of 100 μL. Plasmin activity was determined by the hydrolysis rate of 50 μM substrate D-Ala-Leu-Lys-7-amino-4-methylcoumarin (SigmaAldrich, Saint-Louis, MO, USA) at 37°C for 1 h. Chymotrypsin activity was determined by the hydrolysis rate of 10 μM substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Peptide Institute Inc., Osaka, Japan) at 37°C for 1 h. The chromatograms were taken using an Infinite M1000 spectrophotometer (Tecan Group Ltd., Switzerland) measures the released fluorescence, where λ ex =370nm and λ em = 460 nm. A standard curve of AMC was also obtained to estimate the concentration (nM) of fluorescent substrate cleavage.

[1196] Fig.10 Chymotrypsin inhibitors increase plasmin activity in thrombi of mice (A) or hamsters (B)

[1197] result

[1198] The results of intrathrombotic enzyme activity of chymotrypsin or plasmin are as follows Fig.10As shown, and summarized in Table 10. Plasmin and chymotrypsin activities were measured for 60 min using AMC-specific cleavage (nM) of the fluorescent substrate D-Ala-Leu-Lys-AMC (for plasmin) or Suc-Leu-Leu-Val-Tyr-AMC (for chymotrypsin). Each bar graph represents the mean ± SEM of at least 6 experiments. **P<0.01, ***P<0.001.

[1199] Chymotrypsin activity was observed in mouse and hamster thrombi ( Fig.10 Ai and Fig.10 Bi). To our knowledge, this is the first time that chymotrypsin activity has been shown in vitro in thrombotic material. These experiments show that - although chymotrypsin is known to be very rapidly inactivated in whole blood or plasma - chymotrypsin activity is surprisingly intact in the local microenvironment between the vessel wall and the thrombus. Chymotrypsin activity was almost completely reduced in thrombi treated with the chymotrypsin inhibitors 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (fulacimstat) or TY-51469 (respectively). Fig.10 Ai and Fig.10 Measurement of plasmin activity in the same thrombi showed that the addition of chymotrypsin inhibitors strongly increased plasmin activity in both mouse and hamster thrombi ( Fig.10 Aii, Fig.10 Bii), which confirms the proposed mechanism of action.

[1200] Although chymotrypsin is rapidly inactivated in plasma, surprisingly it remains active in the local microenvironment of thrombus, and the addition of chymotrypsin inhibitors leads to a decrease in chymotrypsin activity and an increase in its substrate plasmin in this environment.

[1201] Table 10 Intrathrombotic enzyme activities of chymotrypsin and plasmin in thrombi from venous stenosis experiments in mice and hamsters

[1202]

[1203] One-way ANOVA followed by Bonferroni multiple comparison test or Mann-Whitney T-test. *P<0.05, **P<0.01, ***P<0.001. Error type: standard error of the mean (±SEM).

[1204] Surprisingly, although it is generally believed that the main mechanism of the rat vein stenosis experiment by Ponomaryov et al. (Ponomaryov et al., CircRes.121 (2017) 941-950) is related to coagulation, the activity measurement of chymotrypsin and plasmin activity in thrombi removed from animals in our study revealed a strong association between chymotrypsin and the fibrinolytic system. The present invention demonstrates that in thrombi of animals treated with chymotrypsin inhibitors, chymotrypsin activity is reduced, while plasmin activity is increased.

[1205] 9.2 Effect of chymotrypsin inhibitors on human clots in vitro (method “Halo assay”)

[1206] To demonstrate that human chymotrypsin reduces plasmin-dependent fibrinolysis of human thrombi, the effect of this mast cell-derived serine protease was tested in halo-type human blood concentric clot formation.

[1207] Formation of halo-shaped blood clots containing human chymotrypsin

[1208] Clot formation according to the method of Bonnard et al. (Sci Rep. 2017 May 24;7(1):2346) was induced by adding 31.2 mM calcium chloride (CaCl2) to blood samples collected from healthy donors. 1 μL of human chymotrypsin, at a final concentration of 0.5 μM or 1 μM, was deposited at the bottom edge of the wells of a flat-bottomed transparent 96-well plate in the presence or absence of 10 μM fulacimstat (only for the latter concentration of rCMA-1). 20 μL of blood supplemented with CaCl2 was then added in a circular motion around the edge of each well, thereby forming a halo of blood clots (fluid cohesion effect) leaving the center of the well empty. The plate was then sealed and incubated at 37°C for 30 min to allow the blood halo to coagulate.

[1209] Research on thrombolysis with rtPA

[1210] Recombinant human tissue plasminogen activator (rtPA, Genentech Inc., SF, USA) induced clot fibrinolysis by activating endogenous plasmin. rtPA was diluted to a concentration of 1.5 nM in 80 μL of PBS, pH 7.4, and added to each well. The 96-well plate was then incubated at 37°C for 30 min. Negative control (A total ) was obtained by adding 80 μL of PBS without fibrinolytic agent, and the positive control (A zero ) corresponds to the wells containing 20 μL fresh blood (not supplemented with CaCl2) and 80 μL PBS. The plate was read using a plate reader (Infinite M1000 spectrophotometer (Tecan Group Ltd., Switzerland)) to measure the fibrinolysis rate. The degradation of the corona was measured in real time as the coverage of each experimental well with thrombolytic blood increased the absorbance at 510 nm. After 5 seconds of orbital shaking at 195.3 rpm with an amplitude of 3.5 mm, the change in absorbance was measured every minute for 2 h at 37 ° C. Each absorbance measurement was converted to a degradation percentage according to the following formula:

[1211] Dx(t)=100(A x (t)-A zero (t)) / (A total (t)-A zero (t)).

[1212] Fibrinolysis kinetics analysis

[1213] In addition to real-time monitoring and plotting of blood degradation kinetics, other parameters can be derived from the same curves:

[1214] i) Activation time required to induce fibrinolysis (A) t ), which is defined as D x The first t value for which (t) / dt>1.

[1215] ii) The time required to achieve 50% dissolution (T0.5), which is defined as D x The first t value when (t) = 50%.

[1216] iii) Maximum clot lysis rate (CLR) max ) corresponds to D x Maximum value of (t) / dt.

[1217] Fig.11 Human chymotrypsin (CMA-1) reduces plasmin-dependent fibrinolysis of human clots in a fulacimstat-sensitive manner

[1218] result

[1219] like Fig.11 As shown, human chymotrypsin added before CaCl2-induced clotting significantly reduced tPA-induced fibrinolytic activity in human blood clots in a fulacimstat-sensitive manner. Blood clots pre-incubated with human chymotrypsin (0.5 μM and 1 μM) for 30 minutes showed a concentration-dependent reduction in rtPA-dependent fibrinolysis, which was reduced by fulacimstat ( Fig.11 A) B) At: fibrinolytic activation time; C) T0.5: time required to achieve 50% lysis; D) CLR max: Maximum clot lysis rate. Each point and bar corresponds to the mean ± SEM (n = 5-7). (*P < 0.05; **P < 0.01; ***P < 0.001).

[1220] Normally, human chymotrypsin is rapidly inactivated in whole blood. However, when added directly to whole blood before initiation of coagulation by recalcification, human chymotrypsin is active in thrombi and causes a concentration-dependent reduction in thrombus degradation ( Fig.11 A). By adding fulacimstat, the action of chymotrypsin was reversed, thereby accelerating clot lysis. These observations complement the fulacimstat-dependent enhancement of plasmin activity found in mouse and hamster thrombus homogenates as previously described in Section 9.1.

[1221] Fig.14 A to C show that human blood halo clots require activation of endogenous plasmin to be degraded. In additional control experiments, rivaroxaban ( Fig.14 D) or plasminogen / plasmin inhibitor 10-chloro-4-(piperidin-4-yl)pyrimido[1,2-b]indazol-2(1H)-one hydrochloride (Example 3 in WO2015067549; Fig.14 B, C, and E) Concentration response experiments were performed to investigate the role of endogenous factor Xa and plasmin in inducing or dissolving halo-shaped human blood clots in the absence of rCMA-1. Each point represents the mean + SEM of at least 6 experiments. (*P < 0.05, **P < 0.01, ***P < 0.001.)

[1222] A halo-shaped blood clot was formed, and then human chymotrypsin was added

[1223] Clot formation according to the method of Bonnard et al. (Sci Rep. 2017 May 24;7(1):2346) was induced by adding 31.2 mM calcium chloride (CaCl2) to blood samples collected from healthy donors. 20 μL of blood supplemented with CaCl2 was deposited in the wells of a flat-bottomed transparent 96-well plate in a circular motion around the edge of each well, thereby forming a halo-shaped blood clot (fluid cohesion effect), leaving the center of the well empty. The plate was then sealed and incubated at 37°C for 30 min to allow the blood halo to coagulate. Alteplase (1.5 nM) and recombinant human chymotrypsin (final concentration of 0.5 μM or 1 μM) were added simultaneously in the presence or absence of 10 μM fulacimstat (only for the latter concentration of rCMA-1) ( Fig.15A). In another series of experiments, 10-chloro-4-(piperidin-4-yl)pyrimido[1,2-b]indazol-2(1H)-one hydrochloride (Example 3 in WO2015067549) (0.1 μM or 1 μM) was co-administered with tPA in the presence of a preformed halo. The plate was incubated at 37°C for 2 h with horizontal mixing (2 rpm) and then the absorbance at 510 nm was measured.

[1224] result

[1225] Normally, human chymotrypsin is rapidly inactivated in whole blood. Fig.15 As shown in B, the addition of human chymotrypsin after CaCl2-induced clot formation significantly reduced tPA-induced fibrinolytic activity in human halo clots in a fulacimstat-sensitive manner. Each point and bar graph corresponds to the mean ± SEM of 6 to 13 experiments (***P < 0.001). By adding fulacimstat, the effect of chymotrypsin was reversed, thereby accelerating clot lysis.

[1226] In a final series of control experiments, the plasminogen / plasmin inhibitor 10-chloro-4-(piperidin-4-yl)pyrimido[1,2-b]indazol-2(1H)-one hydrochloride (Example 3 in WO2015067549) very significantly reduced tPA-induced fibrinolysis in human blood ( Fig.15 B).

[1227] 10. Reduction of chymotrypsin activity in vivo by gene knockout or direct chymotrypsin inhibition has no effect on bleeding time in animal models

[1228] A very important disadvantage of current thrombolytic strategies is the increased risk of bleeding, which limits the dose of, for example, rtPA that can be used and further limits the number of patients that can be treated with this fibrinolytic approach. Therefore, a new thrombolytic strategy that does not affect hemostasis is clearly needed. To explore the question of whether chymotrypsin inhibition has an effect on hemostasis, bleeding time experiments with and without chymotrypsin inhibitors were performed in mice and hamsters.

[1229] 10.1 Effect of chymotrypsin inhibitor on tail bleeding time in mice

[1230] Mice were anesthetized with a mixture of ketamine / xylazine (87 / 13 mg / kg) intramuscularly (IM). 300 U / kg (via jugular vein) of unfractionated heparin (as per the manufacturer's instructions) or 10 mg / kg of TY-51469 (IP) or 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (fulacimstat) (PO) were injected 1 hour before testing. DVT mice were evaluated 24 hours after ligation with or without a single dose of TY-51469 (10 mg / kg) given 1 hour after ligation. They were then fixed in a vertical position with the tail in a 2 mL tube filled with 1.5 mL of Drabkin reagent (Sigma Aldrich Canada Co., Oakville, ON, Canada) (Saito MS et al., Int. J. Exp. Pathol. 97 (2016) 285-292). The tail was cut off 3 mm from the end. The time for bleeding to stop was recorded. After 30 min, all animals were euthanized.

[1231] result

[1232] The results of the bleeding time experiment are as follows Fig.12 As shown in Figures A and summarized in Table 11, it is shown that chymotrypsin inhibition by 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (fulacimstat) or TY-51469 has no effect on bleeding time at doses that are very effective for thrombolysis. Even in mMCP-4KO animals that completely lack chymotrypsin, no prolongation of bleeding time was observed. In contrast, heparin, a typical control compound in bleeding models, led to a strong increase in bleeding time. Each bar graph represents the mean ± SEM of 6 mice. *P<0.05, **P<0.01, ***P<0.001 compared to control-treated animals.

[1233] Table 11 Effect of chymotrypsin inhibition on bleeding time in the mouse tail bleeding model

[1234]

[1235] One-way ANOVA followed by Bonferroni multiple comparison test. ***P<0.001. Error type: standard error of the mean (±SEM).

[1236] Fig.12 -- Fulacimstat or TY-51469 does not increase bleeding time in mice (A) or hamsters (B)

[1237] 10.2 Effect of chymotrypsin inhibitors on bleeding time in the hamster femoral vein puncture model

[1238] 300 U / kg of unfractionated heparin (as per the manufacturer's instructions) or 10 mg / kg of fulacimstat were injected intrajugally or orally, respectively, 1 hour before testing. Hamsters were anesthetized with a mixture of intramuscular ketamine / xylazine (87-200 / 13 mg / kg). The femoral vein of the right hind limb was exposed. A small incision was made in the middle of the femoral vein using micro scissors and excess blood was absorbed with filter paper without touching the incision site. The time until bleeding completely stopped was recorded as the bleeding time. 43 .

[1239] result

[1240] The results of bleeding time measurements in hamsters are as follows Fig.12 B and summarized in Table 12, which shows that while heparin statistically significantly prolonged bleeding time, no effect on bleeding time was observed in hamsters treated with 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (fulacimstat) compared to control animals.

[1241] Table 12 Effect of chymotrypsin inhibitor fulacimstat on bleeding time in hamster femoral vein puncture model

[1242]

[1243] One-way ANOVA followed by Bonferroni multiple comparison test. ***P<0.001. Error type: standard error of the mean (±SEM).

[1244] Comprehensive analysis of mouse and hamster bleeding time results combined with thrombolysis results

[1245] In both mouse and hamster bleeding time models, no prolongation of bleeding time was observed at doses of chymotrypsin inhibitor that resulted in a robust thrombolytic effect.

[1246] Therefore, it was surprising that with this chymotrypsin inhibition approach, rapid lysis of the clot could be achieved without any signs of bleeding - making the chymotrypsin inhibition approach not only effective but also unexpectedly safe.

[1247] The proposed mechanism of action is Fig.16 shown.

[1248] Fig.16 ——A new method to safely dissolve blood clots and reopen blood vessels by targeting chymotrypsin in blood clots

[1249] In summary, it has been shown for the first time in various parts of the present invention that chymotrypsin inhibitors inhibit the degradation of plasmin by chymotrypsin, which has been identified for the first time in vivo as a pathophysiologically relevant process in animal and human thrombosis, with an effect on thrombus size. The inhibition of plasmin degradation by chymotrypsin inhibitors accelerates the dissolution of fibrin clots in blood vessels without affecting bleeding time or hemostasis, thus representing a new method for extremely safe revascularization of blood vessels blocked by thrombi or emboli (Experimental Section, Fig.16 ).

[1250] Therefore, the chymotrypsin inhibitor of the present invention is an effective agent for treating and preventing stroke, pulmonary embolism, deep vein or superficial vein thrombosis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy of hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites.

[1251] 11. Working Examples of Pharmaceutical Compositions

[1252] The substances of the present invention can be converted into the following pharmaceutical preparations:

[1253] tablet:

[1254] composition:

[1255] 100 mg compound, 50 mg lactose (monohydrate), 50 mg corn starch, 10 mg polyvinylpyrrolidone (PVP25) (BASF, Germany) and 2 mg magnesium stearate.

[1256] Tablet weight: 212 mg. Diameter: 8 mm, radius of curvature: 12 mm.

[1257] preparation:

[1258] A mixture of the compound of Example 1, lactose and starch is granulated with a 5% solution of PVP in water (m / m). After drying, the granules are mixed with magnesium stearate for 5 min. The mixture is compressed in a conventional tablet press (see above for tablet format).

[1259] Oral suspension:

[1260] composition:

[1261] 1000 mg of the compound of Example 1, 1000 mg of ethanol (96%), 400 mg of Rhodigel (xanthan gum) (FMC, USA) and 99 g of water.

[1262] 10 mL of the oral suspension corresponds to a single dose of 100 mg of the compound according to the invention.

[1263] preparation:

[1264] Rhodigel was suspended in ethanol, and the compound of Example 1 was added to the suspension. Water was added under stirring. The mixture was stirred for about 6 h until the Rhodigel was completely swollen.

[1265] Solution for oral administration:

[1266] composition:

[1267] 500 mg of the compound according to the invention, 2.5 g of polysorbate and 97 g of polyethylene glycol 400. A single dose of 100 mg of the compound according to the invention corresponds to 20 g of oral solution.

[1268] preparation:

[1269] Under stirring, the compound of the present invention is suspended in the mixture of polyethylene glycol and polysorbate. The stirring operation is continued for 15 minutes until the compound of the present invention is completely dissolved.

[1270] IV solution

[1271] The compound of the invention is dissolved in a physiologically acceptable solvent (eg, isotonic sodium chloride solution, glucose solution 5% and / or PEG 400 solution 30%) at a concentration below saturation solubility. The solution is sterile filtered and dispensed into sterile and pyrogen-free injection containers.

Claims

1. Chymotrypsin inhibitors are used to treat and prevent stroke, pulmonary embolism, deep vein or superficial vein thrombosis, thrombotic microangiopathy, thrombotic microangiopathy caused by infection, inflammation, and hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites.

2. A chymotrypsin inhibitor for the treatment and prevention of stroke, pulmonary embolism, deep or superficial vein thrombosis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy in hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access sites, wherein the chymotrypsin inhibitor is a compound of formula (I) and its salt, solvate and solvate of the salt: in R 1 is hydrogen, methyl or ethyl, R 2 A group of the formula in * is the connection site with the nitrogen atom of uracil, A is -CH2-, -CH2-CH2-, -O-CH2-## or oxygen, in ## is the connection site with the benzene ring, R 4A is hydrogen, fluorine, chlorine, trifluoromethyl or methyl, R 4B is hydrogen, fluorine, chlorine, trifluoromethyl or methyl, The condition is R 4A and R 4B At least one of the groups is not hydrogen. R 5A For hydrogen, R 5B For hydrogen, R 6 For hydrogen, R 7 For hydrogen, R 8 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl, R 9 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl, R 3 A group of the formula in # is the connection site with the nitrogen atom of uracil, E 1 CR 11 or N, in R 11 is hydrogen, (C1-C4)-alkyl, (C3-C7)-cycloalkyl or aminocarbonyl, E 2 CR 12 or N, in R 12 is hydrogen, (C1-C4)-alkyl or (C3-C7)-cycloalkyl, G 1 is C=O or SO2, G 2 CR 16A R 16B NR 17 , O or S, in R 16A is hydrogen, fluorine, (C1-C4)-alkyl or hydroxy, R 16B is hydrogen, fluorine, chlorine, (C1-C4)-alkyl or trifluoromethyl, or R 16A and R 16B Together with the carbon atoms to which they are bonded, they form a 3- to 6-membered carbon ring. R 17 is hydrogen, (C1-C6)-alkyl, (C3-C7)-cycloalkyl or (C1-C4)-alkoxycarbonyl, wherein the (C1-C6)-alkyl radical may be substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, (C3-C7)-cycloalkyl, hydroxy, trifluoromethoxy, (C1-C4)-alkoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl, R 24 is fluorine or methyl, n is a number 0 or 1, R 10 is (C1-C4)-alkyl or (C3-C7)-cycloalkyl, wherein the (C1-C4)-alkyl group may be substituted by 1 or 2 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl, R 15 is hydrogen, (C1-C6)-alkyl or (C3-C7)-cycloalkyl, Wherein the (C1-C6)-alkyl group may be substituted by 1 or 2 substituents independently selected from the group consisting of fluoro, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl.

3. The chymotrypsin inhibitor according to claim 2, which is used to treat and prevent stroke, pulmonary embolism, deep vein or superficial vein thrombosis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy in hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access site, wherein the chymotrypsin inhibitor is a compound of formula (I) and its salt, solvate and solvate of salt, in R 1 is hydrogen, methyl or ethyl, R 2 A group of the formula in * is the connection site with the nitrogen atom of uracil, A is -CH2-, R 4A is chloro or trifluoromethyl, R 4B Hydrogen R 3 A group of the formula in # is the connection site with the nitrogen atom of uracil, E 1 CR 11 , in R 11 For hydrogen, E 2 For N, G 1 For C=O, G 2 CR 16A R 16B NR 17 , O or S, in R 16A is hydrogen, fluorine, methyl or hydroxyl, R 16B is hydrogen, fluorine, methyl or trifluoromethyl, or R 16A and R 16B Together with the carbon atoms to which they are bonded, they form a cyclopropyl ring, R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl, wherein the (C1-C4)-alkyl group may be substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, Cyclobutyl, hydroxy, trifluoromethoxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl, R 24 is hydrogen or fluorine, R 10 is (C1-C4)-alkyl, R 15 is hydrogen, methyl or ethyl, The methyl group and the ethyl group may be substituted by one substituent selected from the group consisting of fluorine, trifluoromethyl and cyclopropyl.

4. The chymotrypsin inhibitor according to claim 2, which is used to treat and prevent stroke, pulmonary embolism, deep vein or superficial vein thrombosis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy in hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access site, wherein the chymotrypsin inhibitor is a compound of formula (I) and its salt, solvate and solvate of salt, in R 1 For hydrogen, R 2 A group of the formula in * is the connection site with the nitrogen atom of uracil, R 5A For hydrogen, R 5B For hydrogen, R 6 For hydrogen, R 7 For hydrogen, R 8 is fluorine, chlorine or trifluoromethyl, R 9 is fluorine, chlorine, trifluoromethyl or methyl, R 3 A group of the formula in # is the connection site with the nitrogen atom of uracil, E 1 CR 11 , in R 11 For hydrogen, E 2 For N, G 1 For C=O, G 2 CR 16A R 16B NR 17 , O or S, in R 16A is hydrogen, fluorine, methyl or hydroxyl, R 16B is hydrogen, fluorine, methyl or trifluoromethyl, or R 16A and R 16B Together with the carbon atoms to which they are bonded, they form a cyclopropyl ring, R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl, wherein the (C1-C4)-alkyl group may be substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, Cyclobutyl, hydroxy, trifluoromethoxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl, R 24 is hydrogen or fluorine, R 10 is (C1-C4)-alkyl, R 15 is hydrogen, methyl or ethyl, The methyl group and the ethyl group may be substituted by one substituent selected from the group consisting of fluorine, trifluoromethyl and cyclopropyl.

5. The chymotrypsin inhibitor according to claim 2 or 3, which is used to treat and prevent stroke, pulmonary embolism, deep vein or superficial vein thrombosis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy in hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, vascular access site thrombosis or occlusion, wherein the chymotrypsin inhibitor is a compound of formula (I), which is selected from: 1-(1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-indene-1-yl ]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(6-fluoro-1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzoimidazol-5-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), Tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1-(1,3,3-trimethyl-2-oxo-2,3-dihydro-1H-indol-5-yl)-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(1'-methyl-2'-oxo-1',2'-dihydrospiro[cyclopropane-1,3'-indol]-5'-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine -5-carboxylic acid (R enantiomer), 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzothiazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), and 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid ethyl ester (R enantiomer).

6. The chymotrypsin inhibitor according to claim 2, 3 or 5, which is used for treating and preventing stroke, pulmonary embolism, deep vein or superficial vein thrombosis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy of hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, vascular access site thrombosis or occlusion, wherein the chymotrypsin inhibitor of formula (I) is 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer) of the following formula 7. A chymotrypsin inhibitor for the treatment and prevention of stroke, pulmonary embolism, deep or superficial vein thrombosis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy in post-transplant hypercoagulable state, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access sites, wherein the chymotrypsin inhibitor is a compound of formula (II): in R 1 represents a hydrogen atom, a halogen atom or a lower alkyl group, R 2 represents a lower alkyl group, R 3 and R 4 each may be the same or different and represents a hydrogen atom, a lower alkoxycarbonyl group, a lower alkylsulfonyl group, a benzoyl group, an acyl group having 1 to 4 carbon atoms, a lower alkoxy group, a lower alkoxycarbonylmethylthioacetyl group, a nitro group, -CONHR 6 , where R 6 represents a hydrogen atom, a lower alkoxycarbonylmethyl group, a carboxymethyl group or -CH(CH2OH)COOR 7 , where R 7 represents a hydrogen atom or a lower alkyl group, The group represented by the following formula Where R 7 The same meaning as above, the group represented by the following formula Where R 8 and R 9 Each may be the same or different and represents a hydrogen atom, a lower alkyl group, a lower alkylthio group, a lower alkylsulfinyl group, a lower alkylsulfonyl group or a lower alkoxycarbonyl group, a hydroxy lower alkyl group, a cyano group Or a monocyclic heterocyclic group represented by the formula: wherein A represents an oxygen atom, a sulfur atom or NH, and the dotted part represents a single bond or a double bond, provided that the hydrogen atoms on the ring can be replaced by a lower alkyl group, a lower alkoxy group, a hydroxy lower alkyl group, a lower alkoxycarbonyl group or a carboxyl group which can be replaced by a halogen atom, and provided that R 3 or R 4 At least one and R 5 represents a hydrogen atom, a lower alkoxy group or a lower alkyl group, except for the compounds represented by the following formula:

8. The chymotrypsin inhibitor according to claim 7, which is used for treating and preventing stroke, pulmonary embolism, deep vein or superficial vein thrombosis, thrombotic microangiopathy, infection, inflammation, thrombotic microangiopathy of hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access site, wherein the compound is selected from: 2-[4-(5-chloro-3-methylbenzo[b]thiol] 2-[4-(5-fluoro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methylsulfonylphenyl]oxazole-4-carboxylic acid, 2-[4-(5-chloro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methylsulfonylphenyl]oxazole-4-carboxylic acid disodium salt, 2-[4-(5-fluoro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methylsulfonylphenyl]oxazole-4-carboxylic acid thiophene-2-sulfonylamino)-3-methylsulfonylphenyl] oxazole-4-carboxylic acid disodium, 2-[4-(5-fluoro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methylsulfonylphenyl] thiazole-4-carboxylic acid, 5-fluoro-N-[4-(4-hydroxymethylthiazol-2-yl)-2-methylsulfonylphenyl]-3-methylbenzo[b]thiophene-2-sulfonamide, 5-fluoro-N-[2-methyl-sulfonyl -4-(5-methoxy-4-methyloxazol-2-yl)phenyl]-3-methylbenzo[b]thiophene-2-sulfonamide, ((2-(4-((5-fluoro-3-methylbenzo-[b]thiophene)-2-sulfonylamino)-3-(methylsulfonyl)-phenyl)thiazole-4-carboxylic acid and 5-fluoro-N-[2-methylsulfonyl-4-(5-methyloxazol-2-yl)phenyl]-3-methylbenzo[b]thiophene-2-sulfonamide.

9. A chymotrypsin inhibitor according to claim 7 or 8, which is used to treat and prevent stroke, pulmonary embolism, deep vein or superficial vein thrombosis, thrombotic microangiopathy, thrombotic microangiopathy caused by infection, inflammation, and hypercoagulable state after transplantation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites, wherein the compound is (2-(4-((5-fluoro-3-methylbenzo-[b]thiophene)-2-sulfonylamino)-3-(methylsulfonyl)phenyl)thiazole-4-carboxylic acid.

10. A combination of one or more compounds of the formula (I) with one or more other active compounds for use according to any one of claims 2 to 6.

11. A combination of one or more compounds of the formula (II) with one or more other active compounds for use according to any one of claims 7 to 9.

12. A pharmaceutical composition comprising at least one compound of formula (I) according to any one of claims 2 to 6 and one or more inert, non-toxic, pharmaceutically suitable excipients for use according to any one of claims 2 to 6.

13. A pharmaceutical composition comprising at least one compound of formula (II) according to any one of claims 7 to 9 and one or more inert, non-toxic, pharmaceutically suitable excipients for use according to any one of claims 7 to 9.

14. A method for treating and / or preventing stroke, pulmonary embolism, deep or superficial vein thrombosis, thrombotic microangiopathy, infection, inflammation, post-transplant hypercoagulable state thrombotic microangiopathy, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, vascular access site thrombosis or occlusion, by systemically and / or locally administering a therapeutically effective amount of at least one compound according to any one of claims 1, claims 2 to 6, or claims 7 to 9, or a medicament comprising at least one compound according to any one of claims 1 to 9 and an inert, non-toxic, pharmaceutically acceptable additive.

15. The method according to claim 14, wherein the medicament further comprises at least one other active compound selected from anticoagulants and / or antiplatelet drugs.

16. A medicament comprising a compound of formula (I) as defined in any one of claims 2 to 6 and one or more other active ingredients selected from anticoagulants and / or antiplatelet agents.

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