Inhibitors of chymase for use in the selective lysis of thrombi in thrombotic or thromboembolic disorders - Patent Application 20070233334

Abstract

The present invention encompasses the use of chymase inhibitors in general, more particularly substituted bicyclic substituted uracils of general formula (I) and 3-methylbenzo-[b]thiophene)-2-sulfonamide derivatives of general formula (II) as defined and described herein, for the manufacture of a pharmaceutical composition for the treatment or prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states following inflammation, transplantation, infection, disseminated intravascular coagulation, vaccine-induced immune thrombocytopenia, thrombosis or occlusion of vascular access sites.

Description

[Technical field]

[0001] The present invention encompasses the use of chymase inhibitors, generally and more specifically of the substituted bicyclic substituted uracils of general formula (I) and 3-methylbenzo-[b]thiophene)-2-sulfonamide derivatives of general formula (II), as described and defined herein, for the manufacture of a pharmaceutical composition for the treatment or prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states following inflammation, transplantation, infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access sites. [Background technology]

[0002] Hemostasis is a protective physiological mechanism that rapidly and reliably covers leakage damage in the vessel wall with the aim of avoiding or minimizing excessive blood loss. Following such damage to the vessel wall, activation, adhesion and aggregation of platelets after activation of the coagulation system, as well as the formation of insoluble fibrin, lead to the rapid formation of a clot that closes the leakage in the vessel wall. However, clot formation must be balanced to protect against (severe) bleeding, but not to excessive clot formation that would fill most of the vessel lumen, thereby partially or completely reducing blood flow and, ultimately, causing a lack of oxygen and nutrient supply in the surrounding tissues. Although physiological mechanisms exist that a) keep the activity of the coagulation system under control by endogenous inhibitors and b) dissolve already generated fibrin clot material (by the fibrinolytic system), they are not always sufficient. Uncontrolled excessive activation of the coagulation system or defects in hemostatic balance can lead to thrombosis, either by the formation of a local thrombus or embolus, or by a clot that breaks off from another site in the circulatory system and occludes a distal vessel. Blockages in arteries of the heart, brain, and lungs, known as myocardial infarction, stroke, or pulmonary embolism, respectively, are among the most common causes of mortality and morbidity worldwide. The occurrence of numerous microthrombi that block smaller organ blood vessels has recently attracted increased attention during the COVID-19 pandemic.

[0003] The prognosis of patients with thrombotic events strongly depends on the timely re-supply of fresh blood to the respective organs, therefore identifying safe methods for rapid recanalization of occluded vessels is an important goal in clinical research.

[0004] Current anticoagulants, vitamin k antagonists, direct factor Xa or thrombin inhibitors, or heparin as an indirect dual FXa / thrombin inhibitor, have been shown to be effective in reducing blood coagulability and preventing thrombotic events, but their main effect on the dissolution of existing clots is thought to be only indirect: by reducing the activity of the coagulation system, the hemostatic balance is shifted towards the physiological fibrinolytic system, thereby promoting the degradation of fibrin material. However, higher doses of anticoagulants may increase the risk of bleeding, and the indirect effect slows down thrombolysis considerably, limiting their use to indications that do not require rapid recanalization.

[0005] Current approaches for rapid vascular recanalization, which is required in many indications, are based on mechanical removal of the occluding clot or on degradation of the fibrin clot by forced fibrinolysis.

[0006] In recent years, mechanical approaches such as thrombectomy have become increasingly applicable to patients with aortic stroke, but despite all technical improvements, accessibility and clinical availability of the thrombus are often limited, so that the procedure is applicable to only a minority of patients.

[0007] Plasmin, the main enzyme for fibrin degradation, is activated from the plasma protein plasminogen in the presence of fibrin by plasminogen activators (tissue plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA)) secreted by endothelial cells. It is known that in blood, active plasmin is rapidly inactivated by a large excess of α2-antiplasmin, but remains active while bound to fibrin. Current fibrinolysis strategies use plasminogen activating enzymes, administered systemically or locally, to generate plasmin. Recombinant tissue-type plasminogen activator (rtPA, alteplase) is the most widely used fibrinolytic drug, FDA approved for myocardial infarction, ischemic stroke, pulmonary embolism, or re-establishment of patency in occluded intravenous catheters (Hughes RH et al., StatPearls 2020), and in many areas has become the standard of care for the treatment of acute ischemic stroke in patients who can be treated within 4.5 hours after the event (Powes WJ et al., Stroke 50 (2019) e344-e418). However, administration of tPA carries a dose-dependent risk of bleeding, including life-threatening intracranial hemorrhage (Emberson J et al., Lancet 384 (2014) 1929), which limits the administration of tPA and thereby its fibrinolytic efficacy. In addition to the risk / benefit profile of this agent, its short half-life in human plasma (predominant half-life <5 min; Tanswell P et al., Arzneimittelforschung41(1991)1310-9), and further adverse effects including re-thrombosis and more rare but severe tPA-associated angioedema (Rathburn KM, Oxf. Med. Case Rep. 2019; Froehlich K et al., Stroke50(2019)1682) represent an overall challenging drug profile, which is often the cause of suboptimal efficacy and limited use in many cases.

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

[0009] So far, the role of mast cells and their content in the pathology of thrombotic events has been greatly underestimated and no therapeutic approaches exist that target mast cell granule components for thrombolysis.

[0010] Mast cells are long-lived perivascular resident cells of hematopoietic origin that are distributed in most tissues, often located at the interface between tissues and the external environment. For example, they can be found in the skin, in the mucosal or epithelial surfaces of the intestine and lungs, and tend to cluster around blood and lymphatic vessels and nerves (Prussin C, Metcalfe DD, J. Allergy Clin. Immunol. 111 (2003) S486-94). Only a small number of progenitor cells can be found in the blood before relocalizing to tissues where their maturation is completed. They are multifunctional immune cells involved in a variety of health and disease conditions, and are best known for their role in allergy and anaphylaxis in the lungs, intestine or skin. However, they are involved in additional processes, including host defense against pathogens, immune tolerance, wound healing, protective strategies such as angiogenesis, as well as several pathophysiological processes (Miyazaki et al., Pharmacol. Ther. 112 (2006), 668-676; Shiota et al., J. Hypertens. 21 (2003) 1823-1825). Their increased numbers have been observed in patients with heart failure, myocardial infarction and ischemia, in 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 pathogenesis 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 numerous large cytoplasmic granules and upon stimulation release large amounts of histamine and heparin as well as many other active components, including several cytokines, proteoglycans and serine proteases, such as tryptase, chymase, chymotrypsin, cathepsin G and carboxypeptidase A (Lindstedt KA et al., J. Cell. Mol. Med. 11 (2007) 739-758). Although some of the stored compounds, such as heparin, polyphosphate or histamine, are known to interact with the coagulation system (Guilarte M et al., Front. Immunol 2017), it is still a matter of debate whether the overall effect of mast cells can be anticoagulant or procoagulant and whether it has an impact on clot formation. An antifibrinolytic effect on thrombi in the vasculature has not been described so far for mast cells or granule components.

[0012] Recently, in an experimental venous stenosis model in mice, two strains of mast cell-deficient mice were completely protected against venous thrombosis, in contrast to untreated mice (Ponomaryov et al., CircRes.121(2017)941-950). It was concluded that the potential procoagulant effect of mast cells could be due to the release of histamine, but could not be verified with histamine receptor blockers. The potential effect on the fibrinolytic system and therefore on thrombolysis was not described. In a mouse model of arterial thrombosis, in which thrombus formation was induced by ferric chloride-induced injury of mesenteric arterioles (Ponomaryov et al., CircRes.121(2017)941-950), no statistically significant differences in occlusion times between wild-type and mast cell-deficient mice were observed. The authors concluded that mast cells may be relevant only for venous thrombosis, but not for arterial thrombosis.

[0013] In a review of mast cell interactions with the fibrinolytic system, Bankl and Valent (Thromb. Res. 105 (2002) 359) state that mast cells themselves may be profibrinolytic due to their selective production of tPA, but not its endogenous inhibitor PAI-1. Their conclusion that mast cells are profibrinolytic is in contrast to the results associated with the surprising findings of the present invention, which show that chymase has antifibrinolytic properties and that inhibitors can be used for vascular recanalization.

[0014] Chymase is a serine protease that is stored as a complex with heparin in mast cell granules. Upon mast cell activation, it is released into the extracellular space where chymase is activated and, like other mast cell proteases, is tightly 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, chymase is rapidly inhibited by endogenous circulating inhibitors, including α-1 antitrypsin, α-1 antichymotrypsin, and α-2 macroglobulin (Metcalfe DD et al., World Allergy Organ J. 11 (2016) 7), so chymase activity cannot usually be measured in blood, serum or plasma. Therefore, it was not expected that chymase activity could be measured in intravascular thrombi.

[0015] Several substrates of chymase have been described: 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 in the production of endothelin-1 (Nakano et al., J. Immunology, 1997; 159: 1987-1992; D'Orleans-Juste P et al., Vascular Pharm. 49 (2008) 51-62). In addition, chymase leads to the degradation of extracellular matrix proteins such as fibronectin, procollagen and vitronectin, as well as the disruption of focal adhesions (Pejler, J Innate Immun 2020; 12: 357-372). This leads to the activation and release of TGFβ from its latent form, which plays a key role in the development of cardiac hypertrophy and cardiac fibrosis (Cho et al., Am J Respir Cell Mol Biol. 2015Jan;52(1):88-95; Oyamada S. et al., J Pharmacol Exp Ther. 2011Oct;339(1):143-51). The enzyme has an atherogenic effect by degrading apolipoproteins and preventing cholesterol absorption by HDL (Lee et al., ATVB 2002;22:1475-1481). The action of chymase also leads to the release and activation of the cytokine interleukin-1, which has its proinflammatory properties (Mizutani et al., J Exp Med. 1991;174(4):821-825).

[0016] The relationship of chymase to the fibrinolytic system, its potential role and relevance were previously unknown: Tchougounova and Pejler investigated the effect 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 effect on the regulation of thrombin and plasmin was heparin-dependent and inhibited by the nonspecific endogenous protease inhibitor α1-antichymotrypsin. Thus, they attributed these proteolytic findings to chymase or elastase. However, since several chymase-like enzymes with different relevance for translation to humans are present in mouse peritoneal mast cells and further validation with specific chymase inhibitors was not performed, the validity or translatability of these findings is unclear. Furthermore, it is suggested that this regulation of thrombin and plasmin may play a role extravascularly rather than intravascularly. This is in contrast to the results of the present invention, where chymase activity and its inhibition in intravascular fibrin clots play a central role.

[0017] Recently, Lipitsae et al. reported histological experiments revealing the colocalization of chymase and fibrin in vasculitis specimens. Subsequent in vitro experiments with chymase added to fibrinogen and fibrin revealed a potential slow degradation of fibrinogen and fibrin by chymase, which could only be found in the absence of plasma (Lipitsae T. et al., Brit. J. Dermatol 181 (2019) 296-303). Under these circumstances, it was concluded that chymase itself may have fibrinolytic properties. The hypothesis that chymase is profibrinolytic and thus may represent a therapeutic opportunity for vasculitis is in clear contrast to the findings described in this invention.

[0018] In conclusion, according to current knowledge, it is very unclear what is the cause of the mast cell effect in experimental venous stenosis in mice and whether it is translatable to arterial models and clinical situations. What role chymase may play in the context of revascularization or in the prevention of thrombotic events has not been investigated at all.

[0019] Chymase inhibitors have been disclosed in WO2013167495, WO2015067650, WO2015067651, and WO0222595 and are being evaluated by several pharmaceutical companies (Ahmad S, Ferrario CM, Expert. Opin. Ther. Pat28(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-sulfonamido)-3-(methylsulfonyl)phenyl)thiazole-4-carboxylic acid) were each IC 50 They are selective chymase inhibitors of different structural classes with IC values ​​of 4 nM and 20 nM. Their selectivity has been tested against various proteases. They have IC values ​​of more than 2 μM for plasmin, plasma kallikrein, thrombin, trypsin, tissue plasminogen activator, factor Xa and factor XIa. 50values ​​were obtained. The closest protease, cathepsin G, is inhibited 60-fold less potently by TY-51469 than chymase and 35-fold less potently by furasymstat than chymase and is therefore considered to be of no relevance in vivo. Both compounds have been evaluated in many preclinical models, encompassing mainly inflammatory and fibrotic diseases as well as autoimmune diseases in various organs, including the heart, kidney, lung, intestine and skin (Pejler G, J. Innate Immun. 12 (2020) 357-372). Furasymstat shows bioavailability after oral ingestion. The compound showed no effect on bleeding risk, was well tolerated in clinical trials, and has a favorable pharmacokinetic profile with a once-daily dosing regimen (Kanefendt F et al., Clin. Pharmacol. Drug Dev. 8 (2019) 467-479; Duengen HD et al., Clin. Pharmacol. Drug Dev. 8 (2019) 942-951; Duengen HD et al., Am. Heart J. 224 (2020) 129-137); Rossing P et al., Nephrol. Dial. Transplant 26 (2021) 2263-2273).

[0020] For agents applied in the setting of an acute thrombotic event, rapid lysis of the clot with low risk of bleeding is of paramount importance. [Prior art documents] [Patent documents]

[0021] [Patent Document 1] International Publication No. 2013167495 [Patent Document 2] International Publication No. 2015067650 [Patent Document 3] International Publication No. 2015067651 [Patent Document 4] WO 0222595 [Patent Document 5] U.S. Patent No. 7,071,220 [Non-patent literature]

[0022] [Non-licensed document 1] Hughes RHら、StatPearls2020 [Non-licensed document 2] Powes WJら、Stroke50(2019)e344-e418 [Non-licensed document 3] Emberson Jら, Lancet 384(2014)1929

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[0023] It is therefore an object of the present invention to provide a novel strategy for thrombolysis to treat thrombotic events in veins, capillaries, arteries and lymphatics in humans and animals without increasing the bleeding risk. [Means for solving the problem]

[0024] Surprisingly, as part of the present invention, it was found that plasmin is a chymase substrate, the degradation and inactivation of which can be prevented by the chymase inhibitors of the present invention in animals and humans (Experimental Section, Section 3; Figures 1 and 2), and that these chymase inhibitors are suitable for thrombolysis, particularly in the cause of acute thrombotic and thromboembolic events.

[0025] Furthermore, due to its rapid inactivation in plasma, it was not expected that chymase would remain active in the local microenvironment of the thrombus and that addition of the chymase inhibitor of the present invention would result in a decrease in chymase activity and an increase in its substrate plasmin in this environment (Experimental Section, section 9; Figures 10, 15).

[0026] Surprisingly, in specimens from human venous thrombi, arterial thrombi, and pulmonary embolic thrombi, chymase was not only present in thrombi from animal studies, but was also found in human thrombi as well, thereby indicating that it plays an important role in human thrombotic events (Experimental Section, Section 8; Figure 9).

[0027] Surprisingly, under conditions of inferior vena cava stenosis, administration of the chymase inhibitors of the present invention leads to a reduction in the weight and length of the thrombus, even in a well-defined intervention setting, when administered, for example, 24 hours after the onset of thrombus formation. These results were obtained by using two structurally different chymase 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 (furasymstat) and TY-51469, in an established mouse DVT model, as well as furasymstat in a newly developed hamster DVT model (both animal models involve inferior vena cava stenosis) to confirm the general nature of these findings (Experimental section, section 4, Figure 4).

[0028] Although it was generally believed that the main mechanism in the mouse venous stenosis experiments by Ponomaryov et al. was related to coagulation, in our study, activity measurements of chymase and plasmin activity in thrombi taken from animals indicate a strong association between chymase and the fibrinolytic system. Meanwhile, chymase activity was decreased by inhibitors, whereas plasmin activity was increased in thrombi from these animals (Experimental section, section 9.2; Figure 10).

[0029] Whereas the ferric chloride-induced injury experiments in mesenteric arterioles known in the state of the art by Ponomaryov et al. showed that mast cells had no significant effect on thrombus growth, the ferric chloride-induced injury experiments in different vessels described in this invention demonstrate a significant difference between the group treated with the chymase inhibitor of the invention and the vehicle group. In these acute thrombosis experiments, the compound was given before the initiation of thrombus formation by ferric chloride, which means that even at the early stage of ongoing thrombus formation, the chymase inhibitor affects thrombus weight and thus the risk of vascular occlusion (Experimental section, section 7; Figures 7, 13). This was observed not only in venous vessels, but also in arteries, such as the carotid artery.

[0030] Surprisingly, when the lungs were examined in inferior vena cava (IVC) stenosis experiments in mice and hamsters, a reduction in the number of pulmonary emboli in the lungs of animals that received chymase inhibition was observed compared to control animals (Experimental section, section 6; Figures 5 and 6). This very important and relevant case of thromboembolism implies that chymase not only has a role at the local site of thrombus formation, but also has an antifibrinolytic effect in emboli. Thus, chymase inhibitors are effective agents in the resolution of thromboembolism, including, for example, the dissolution of emboli from deep veins causing pulmonary embolism or from the left atrial appendix in patients with atrial fibrillation causing stroke or systemic embolism.

[0031] No effect on bleeding times was observed in the mouse tail bleed model and the hamster femoral vein puncture model, which is noteworthy because the experiments were performed at doses that have a strong effect on thrombolysis, meaning that the chymase inhibition approach is not only effective but also unexpectedly safe (Experimental Section, section 10; Figure 12).

[0032] Therefore, the special importance and surprising discovery of the present invention is that by discovering the unexpected effect of chymase on plasmin and its relevance, a mechanism that may reduce thrombolysis in certain situations, including mast cell degranulation and inflammation of the vessel wall with pathologically reduced blood flow, but not prevent hemostasis in hemorrhagic events, is identified.Thus, the chymase inhibitor of the present invention is not a non-specific fibrinolytic agent, but rather a novel selective fibrinolysis modulator within the thrombus.

[0033] In summary, in various parts of the present invention, it has been shown for the first time that chymase inhibitors inhibit the degradation of plasmin by chymase in thrombi, which has been identified for the first time in in vivo experiments as a pathophysiologically relevant process in animal and human thrombi that affects thrombus size. Inhibiting the degradation of plasmin via the chymase inhibitors of the present invention represents a novel approach for the safe revascularization of blood vessels occluded by thrombi or emboli, which has the potential to accelerate the dissolution of fibrin clots in blood vessels without affecting bleeding time or hemostasis, thereby saving the lives of patients experiencing thrombotic events such as stroke, pulmonary embolism or myocardial infarction (Experimental section, Figure 16). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The present invention relates to a compound represented by the general formula (I) [ka] [During the ceremony, R 1 is hydrogen, methyl or ethyl, R 2is the formula [ka] Based on where * is the site of attachment to the nitrogen atom of uracil, A is -CH2-, -CH2-CH2-, -O-CH2-## or oxygen; where ## is the site of attachment to the phenyl ring, R 4A is hydrogen, fluorine, chlorine, trifluoromethyl or methyl, R 4B is hydrogen, fluorine, chlorine, trifluoromethyl or methyl, However, R 4A and R 4B At least one of the groups is not hydrogen; R 5A is hydrogen, R 5B is hydrogen, R 6 is hydrogen, R 7 is hydrogen, R 8 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl, R 9 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl, R 3 is the formula [ka] Based on where # is the attachment site for the nitrogen atom of uracil, E 1 CR 11 or N, Here, R 11 is hydrogen, (C1-C4)-alkyl, (C3-C7)-cycloalkyl or aminocarbonyl, E 2 CR 12 or N, Here, R12 is hydrogen, (C1-C4)-alkyl or (C3-C7)-cycloalkyl, G 1 is C=O or SO2, G 2 CR 16A R 16B , N.R. 17 , O or S; Here, 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 atom to which they are attached form a 3- to 6-membered carbocyclic ring, R 17 is hydrogen, (C1-C6)-alkyl, (C3-C7)-cycloalkyl or (C1-C4)-alkoxycarbonyl, wherein (C1-C6)-alkyl is optionally substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, (C3-C7)-cycloalkyl, hydroxyl, 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 (C1-C4)-alkyl is optionally substituted by one or two substituents independently selected from the group: fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxyl, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl, R 15 is hydrogen, (C1-C6)-alkyl or (C3-C7)-cycloalkyl, wherein (C1-C6)-alkyl may be substituted with 1 or 2 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxyl, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl. and salts, solvates and solvates of the salts thereof.

[0035] The present invention relates to a compound of general formula (I) for use in a method for the treatment and / or prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states following inflammation, transplantation, infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion at vascular access sites [ka] [During the ceremony, R 1 is hydrogen, methyl or ethyl, R 2 is the formula [ka] Based on where * is the site of attachment to the nitrogen atom of uracil, A is -CH2-, -CH2-CH2-, -O-CH2-## or oxygen; where ## is the site of attachment to the phenyl ring, R 4A is hydrogen, fluorine, chlorine, trifluoromethyl or methyl, R 4B is hydrogen, fluorine, chlorine, trifluoromethyl or methyl, However, R 4A and R 4B At least one of the groups is not hydrogen; R 5A is hydrogen, R 5B is hydrogen, R 6 is hydrogen, R 7 is hydrogen, R 8 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl, R 9 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl, R 3 is the formula [ka] Based on where # is the attachment site for the nitrogen atom of uracil, E 1 CR 11 or N, Here, R 11 is hydrogen, (C1-C4)-alkyl, (C3-C7)-cycloalkyl or aminocarbonyl, E 2 CR 12 or N, Here, R 12 is hydrogen, (C1-C4)-alkyl or (C3-C7)-cycloalkyl, G 1 is C=O or SO2, G 2 CR 16A R 16B , N.R. 17 , O or S; Here, 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 atom to which they are attached form a 3- to 6-membered carbocyclic ring, R 17 is hydrogen, (C1-C6)-alkyl, (C3-C7)-cycloalkyl or (C1-C4)-alkoxycarbonyl, wherein (C1-C6)-alkyl is optionally substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, (C3-C7)-cycloalkyl, hydroxyl, 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 (C1-C4)-alkyl is optionally substituted by one or two substituents independently selected from the group: fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxyl, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl, R 15 is hydrogen, (C1-C6)-alkyl or (C3-C7)-cycloalkyl, wherein (C1-C6)-alkyl may be substituted with 1 or 2 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxyl, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl. and salts, solvates and solvates of the salts thereof.

[0036] The present invention relates to General formula (I) [ka] [During the ceremony, R 1 is hydrogen, methyl or ethyl, R 2 is the formula [ka] Based on where * is the site of attachment to the nitrogen atom of uracil, A is -CH2-, -CH2-CH2-, -O-CH2-## or oxygen; where ## is the site of attachment to the phenyl ring, R 4A is hydrogen, fluorine, chlorine, trifluoromethyl or methyl, R 4B is hydrogen, fluorine, chlorine, trifluoromethyl or methyl, However, R 4A and R 4B At least one of the groups is not hydrogen; R 5A is hydrogen, R 5B is hydrogen, R 6 is hydrogen, R 7 is hydrogen, R 8 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl, R 9 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl, R 3 is the formula [ka] Based on where # is the attachment site for the nitrogen atom of uracil, E 1 CR 11 or N, Here, R 11 is hydrogen, (C1-C4)-alkyl, (C3-C7)-cycloalkyl or aminocarbonyl, E 2 CR 12 or N, Here, R 12 is hydrogen, (C1-C4)-alkyl or (C3-C7)-cycloalkyl, G 1 is C=O or SO2, G 2 CR 16A R 16B , N.R. 17 , O or S; Here, 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 atom to which they are attached form a 3- to 6-membered carbocyclic ring, R 17 is hydrogen, (C1-C6)-alkyl, (C3-C7)-cycloalkyl or (C1-C4)-alkoxycarbonyl, wherein (C1-C6)-alkyl is optionally substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, (C3-C7)-cycloalkyl, hydroxyl, 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 (C1-C6)-alkyl is optionally substituted by 1 or 2 substituents independently selected from the group: fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxyl, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl, R 15 is hydrogen, (C1-C6)-alkyl or (C3-C7)-cycloalkyl, wherein (C1-C6)-alkyl may be substituted with 1 or 2 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxyl, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl. and salts, solvates and solvates of the salts thereof.

[0037] The present invention relates to a compound of general formula (I) for use in a method for the treatment and / or prevention of stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep or superficial venous thrombosis, phlebitis, thrombotic microangiopathy, inflammation, transplantation, thrombotic microangiopathy in hypercoagulable states following infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access sites. [ka] [During the ceremony, R 1 is hydrogen, methyl or ethyl, R 2 is the formula [ka] Based on where * is the site of attachment to the nitrogen atom of uracil, A is -CH2-, -CH2-CH2-, -O-CH2-## or oxygen; where ## is the site of attachment to the phenyl ring, R 4A is hydrogen, fluorine, chlorine, trifluoromethyl or methyl, R 4B is hydrogen, fluorine, chlorine, trifluoromethyl or methyl, However, R 4A and R 4B At least one of the groups is not hydrogen; R 5A is hydrogen, R 5B is hydrogen, R 6is hydrogen, R 7 is hydrogen, R 8 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl, R 9 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl, R 3 is the formula [ka] Based on where # is the attachment site for the nitrogen atom of uracil, E 1 CR 11 or N, Here, R 11 is hydrogen, (C1-C4)-alkyl, (C3-C7)-cycloalkyl or aminocarbonyl, E 2 CR 12 or N, Here, R 12 is hydrogen, (C1-C4)-alkyl or (C3-C7)-cycloalkyl, G 1 is C=O or SO2, G 2 CR 16A R 16B , N.R. 17 , O or S; Here, 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 atom to which they are attached form a 3- to 6-membered carbocyclic ring, R 17 is hydrogen, (C1-C6)-alkyl, (C3-C7)-cycloalkyl or (C1-C4)-alkoxycarbonyl, wherein (C1-C6)-alkyl is optionally substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, (C3-C7)-cycloalkyl, hydroxyl, 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 (C1-C4)-alkyl is optionally substituted by one or two substituents independently selected from the group: fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxyl, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl, R 15 is hydrogen, (C1-C6)-alkyl or (C3-C7)-cycloalkyl, wherein (C1-C6)-alkyl may be substituted with 1 or 2 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxyl, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl. and salts, solvates and solvates of the salts thereof.

[0038] The present invention relates to a compound of general formula (I) for use in a method for thrombolysis without bleeding risk and thereby in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep or superficial venous thrombosis, phlebitis, thrombotic microangiopathy, inflammation, transplantation, thrombotic microangiopathy in hypercoagulable states following infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access sites. [ka] [During the ceremony, R1 is hydrogen, methyl or ethyl, R 2 is the formula [ka] Based on where * is the site of attachment to the nitrogen atom of uracil, A is -CH2-, -CH2-CH2-, -O-CH2-## or oxygen; where ## is the site of attachment to the phenyl ring, R 4A is hydrogen, fluorine, chlorine, trifluoromethyl or methyl, R 4B is hydrogen, fluorine, chlorine, trifluoromethyl or methyl, However, R 4A and R 4B At least one of the groups is not hydrogen; R 5A is hydrogen, R 5B is hydrogen, R 6 is hydrogen, R 7 is hydrogen, R 8 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl, R 9 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl, R 3 is the formula [ka] Based on where # is the attachment site for the nitrogen atom of uracil, E 1 CR 11 or N, Here, R 11 is hydrogen, (C1-C4)-alkyl, (C3-C7)-cycloalkyl or aminocarbonyl, E2 CR 12 or N, Here, R 12 is hydrogen, (C1-C4)-alkyl or (C3-C7)-cycloalkyl, G 1 is C=O or SO2, G 2 CR 16A R 16B , N.R. 17 , O or S; Here, R 16A is hydrogen, fluorine, (C1-C4)-alkyl or hydroxyl, R 16B is hydrogen, fluorine, chlorine, (C1-C4)-alkyl or trifluoromethyl, or R 16A and R 16B together with the carbon atom to which they are attached form a 3- to 6-membered carbocyclic ring, R 17 is hydrogen, (C1-C6)-alkyl, (C3-C7)-cycloalkyl or (C1-C4)-alkoxycarbonyl, wherein (C1-C6)-alkyl is optionally substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, (C3-C7)-cycloalkyl, hydroxyl, 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 (C1-C4)-alkyl is optionally substituted by one or two substituents independently selected from the group: fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxyl, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl, R 15is hydrogen, (C1-C6)-alkyl or (C3-C7)-cycloalkyl, wherein (C1-C6)-alkyl may be substituted with 1 or 2 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxyl, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl. and salts, solvates and solvates of the salts thereof.

[0039] The present invention relates to a compound of general formula (I) [ka] [During the ceremony, R 1 is hydrogen, methyl or ethyl, R 2 is the formula [ka] Based on where * is the site of attachment to the nitrogen atom of uracil, A is -CH2-, -CH2-CH2-, -O-CH2-## or oxygen; where ## is the site of attachment to the phenyl ring, R 4A is hydrogen, fluorine, chlorine, trifluoromethyl or methyl, R 4B is hydrogen, fluorine, chlorine, trifluoromethyl or methyl, However, R 4A and R 4B At least one of the groups is not hydrogen; R 5A is hydrogen, R 5B is hydrogen, R 6 is hydrogen, R 7is hydrogen, R 8 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl, R 9 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl, R 3 is the formula [ka] Based on where # is the attachment site for the nitrogen atom of uracil, E 1 CR 11 or N, Here, R 11 is hydrogen, (C1-C4)-alkyl, (C3-C7)-cycloalkyl or aminocarbonyl, E 2 CR 12 or N, Here, R 12 is hydrogen, (C1-C4)-alkyl or (C3-C7)-cycloalkyl, G 1 is C=O or SO2, G 2 CR 16A R 16B , N.R. 17 , O or S; Here, R 16A is hydrogen, fluorine, (C1-C4)-alkyl or hydroxyl, R 16B is hydrogen, fluorine, chlorine, (C1-C4)-alkyl or trifluoromethyl, or R 16A and R 16B together with the carbon atom to which they are attached form a 3- to 6-membered carbocyclic ring, R 17 is hydrogen, (C1-C6)-alkyl, (C3-C7)-cycloalkyl or (C1-C4)-alkoxycarbonyl, wherein (C1-C6)-alkyl is optionally substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, (C3-C7)-cycloalkyl, hydroxyl, 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 (C1-C4)-alkyl is optionally substituted by one or two substituents independently selected from the group: fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxyl, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl, R 15 is hydrogen, (C1-C6)-alkyl or (C3-C7)-cycloalkyl, wherein (C1-C6)-alkyl may be substituted with 1 or 2 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxyl, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl. and salts, solvates and solvates of the salts thereof.

[0040] The present invention relates to a method for thrombolysis without bleeding risk and thereby for thrombolysis in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, phlebitis or microangiopathy, comprising the compound of general formula (I) [ka] [During the ceremony, R 1 is hydrogen, methyl or ethyl, R 2 is the formula [ka] Based on where * is the site of attachment to the nitrogen atom of uracil, A is -CH2-, -CH2-CH2-, -O-CH2-## or oxygen; where ## is the site of attachment to the phenyl ring, R 4A is hydrogen, fluorine, chlorine, trifluoromethyl or methyl, R 4B is hydrogen, fluorine, chlorine, trifluoromethyl or methyl, However, R 4A and R 4B At least one of the groups is not hydrogen; R 5A is hydrogen, R 5B is hydrogen, R 6 is hydrogen, R 7 is hydrogen, R 8 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl, R 9 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl, R 3 is the formula [ka] Based on where # is the attachment site for the nitrogen atom of uracil, E 1 CR 11 or N, Here, R 11 is hydrogen, (C1-C4)-alkyl, (C3-C7)-cycloalkyl or aminocarbonyl, E 2 CR 12 or N, Here, R 12 is hydrogen, (C1-C4)-alkyl or (C3-C7)-cycloalkyl, G 1 is C=O or SO2, G 2 CR 16A R 16B , N.R. 17 , O or S; Here, R 16A is hydrogen, fluorine, (C1-C4)-alkyl or hydroxyl, R 16B is hydrogen, fluorine, chlorine, (C1-C4)-alkyl or trifluoromethyl, or R 16A and R 16B together with the carbon atom to which they are attached form a 3- to 6-membered carbocyclic ring, R 17 is hydrogen, (C1-C6)-alkyl, (C3-C7)-cycloalkyl or (C1-C4)-alkoxycarbonyl, wherein (C1-C6)-alkyl is optionally substituted by 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, (C3-C7)-cycloalkyl, hydroxyl, 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 (C1-C4)-alkyl is optionally substituted by 1 or 2 substituents independently selected from the group: fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxyl, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl, R 15 is hydrogen, (C1-C6)-alkyl or (C3-C7)-cycloalkyl, wherein (C1-C6)-alkyl may be substituted by 1 or 2 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxyl, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl. and salts, solvates and solvates of the salts thereof.

[0041] In a further embodiment, the present invention relates to a compound of general formula (I) above for use in a method for the treatment and / or prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states following inflammation, transplantation, infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access sites. [During the ceremony, R 1 is hydrogen, methyl or ethyl, R 2 is the formula [ka] Based on where * is the site of attachment to the nitrogen atom of uracil, A is -CH2-, R 4A is chlorine or trifluoromethyl, R 4B is hydrogen, R 3 is the formula [ka] Based on where # is the attachment site for the nitrogen atom of uracil, E 1 CR 11 and Here, R 11 is hydrogen, E 2 is N, G 1 is C=O, G 2 CR 16A R 16B , N.R. 17 , O or S; Here, R 16A is hydrogen, fluorine, methyl or hydroxyl, and R 16B is hydrogen, fluorine, methyl or trifluoromethyl, or R 16A and R 16B together with the carbon atom to which they are attached form a cyclopropyl ring, R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl, wherein (C1-C4)-alkyl is optionally substituted with 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxyl, 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, wherein methyl and ethyl may be substituted with one substituent selected from the group consisting of fluorine, trifluoromethyl and cyclopropyl. and salts, solvates and solvates of the salts thereof.

[0042] In a further embodiment, the present invention relates to a compound of general formula (I) above for use in a method for thrombolysis without bleeding risk and thereby in stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states following inflammation, transplantation, infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access sites. [During the ceremony, R 1 is hydrogen, methyl or ethyl, R 2 is the formula [ka] Based on where * is the site of attachment to the uracil nitrogen atom, A is -CH2-, R 4A is chlorine or trifluoromethyl, R 4B is hydrogen, R 3 is the formula [ka] Based on where # is the attachment site for the nitrogen atom of uracil, E 1 CR 11 and Here, R 11 is hydrogen, E 2 is N, G 1 is C=O, G 2 CR 16A R 16B , N.R. 17 , O or S; Here, R 16A is hydrogen, fluorine, methyl or hydroxyl, and R 16B is hydrogen, fluorine, methyl or trifluoromethyl, or R 16A and R 16B together with the carbon atom to which they are attached form a cyclopropyl ring, R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl, wherein (C1-C4)-alkyl is optionally substituted with 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxyl, 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, wherein methyl and ethyl may be substituted with one substituent selected from the group consisting of fluorine, trifluoromethyl and cyclopropyl. and salts, solvates and solvates of the salts thereof.

[0043] In a further embodiment, the present invention relates to a compound of general formula (I) above for use in a method for the treatment and / or prevention of stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep or superficial venous thrombosis, phlebitis, thrombotic microangiopathy, inflammation, transplantation, thrombotic microangiopathy in hypercoagulable states following infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access sites. [During the ceremony, R 1 is hydrogen, methyl or ethyl, R 2 is the formula [ka] Based on where * is the site of attachment to the uracil nitrogen atom, A is -CH2-, R 4A is chlorine or trifluoromethyl, R 4B is hydrogen, R 3 is the formula [ka] Based on where # is the attachment site for the nitrogen atom of uracil, E 1 CR 11 and Here, R 11 is hydrogen, E 2 is N, G 1 is C=O, G 2 CR 16A R 16B , N.R. 17 , O or S; Here, R 16A is hydrogen, fluorine, methyl or hydroxyl, and R 16B is hydrogen, fluorine, methyl or trifluoromethyl, or R 16A and R 16B together with the carbon atom to which they are attached form a cyclopropyl ring, R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl, wherein (C1-C4)-alkyl is optionally substituted with 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxyl, 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, wherein methyl and ethyl may be substituted with one substituent selected from the group consisting of fluorine, trifluoromethyl and cyclopropyl. and salts, solvates and solvates of the salts thereof.

[0044] In a further embodiment, the present invention relates to a compound of general formula (I) above for use in a method for thrombolysis without bleeding risk and thereby in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep or superficial venous thrombosis, phlebitis, thrombotic microangiopathy, inflammation, transplantation, thrombotic microangiopathy in hypercoagulable states after infection, disseminated intravascular coagulation, vaccine induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access sites. [During the ceremony, R 1 is hydrogen, methyl or ethyl, R 2 is the formula [ka] Based on where * is the site of attachment to the nitrogen atom of uracil, A is -CH2-, R 4A is chlorine or trifluoromethyl, R 4B is hydrogen, R 3 is the formula [ka] Based on where # is the attachment site for the nitrogen atom of uracil, E 1 CR 11 and Here, R 11 is hydrogen, E 2 is N, G 1 is C=O, G 2 CR 16A R 16B , N.R. 17 , O or S; Here, R 16A is hydrogen, fluorine, methyl or hydroxyl, and R 16Bis hydrogen, fluorine, methyl or trifluoromethyl, or R 16A and R 16B together with the carbon atom to which they are attached form a cyclopropyl ring, R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl, wherein (C1-C4)-alkyl is optionally substituted with 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxyl, 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, wherein methyl and ethyl may be substituted with one substituent selected from the group consisting of fluorine, trifluoromethyl and cyclopropyl. and salts, solvates and solvates of the salts thereof.

[0045] In a further embodiment, the present invention relates to a compound of general formula (I) above for use in a method for the treatment and / or prevention of stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep or superficial venous thrombosis, phlebitis or microangiopathy. [During the ceremony, R 1 is hydrogen, methyl or ethyl, R 2 is the formula [ka] Based on where * is the site of attachment to the nitrogen atom of uracil, A is -CH2-, R 4Ais chlorine or trifluoromethyl, R 4B is hydrogen, R 3 is the formula [ka] Based on where # is the attachment site for the nitrogen atom of uracil, E 1 CR 11 and Here, R 11 is hydrogen, E 2 is N, G 1 is C=O, G 2 CR 16A R 16B , N.R. 17 , O or S; Here, R 16A is hydrogen, fluorine, methyl or hydroxyl, and R 16B is hydrogen, fluorine, methyl or trifluoromethyl, or R 16A and R 16B together with the carbon atom to which they are attached form a cyclopropyl ring, R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl, wherein (C1-C4)-alkyl is optionally substituted with 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxyl, 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, wherein methyl and ethyl may be substituted with one substituent selected from the group consisting of fluorine, trifluoromethyl and cyclopropyl. and salts, solvates and solvates of the salts thereof.

[0046] In a further embodiment, the present invention relates to a compound of general formula (I) above for use in a method for thrombolysis without bleeding risk and thereby thrombolysis in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, phlebitis or microangiopathy. [During the ceremony, R 1 is hydrogen, methyl or ethyl, R 2 is the formula [ka] Based on where * is the site of attachment to the nitrogen atom of uracil, A is -CH2-, R 4A is chlorine or trifluoromethyl, R 4B is hydrogen, R 3 is the formula [ka] Based on where # is the attachment site for the nitrogen atom of uracil, E 1 CR 11 and Here, R 11 is hydrogen, E 2 is N, G 1 is C=O, G 2 CR 16A R 16B , N.R. 17 , O or S; Here, R 16Ais hydrogen, fluorine, methyl or hydroxyl, and R 16B is hydrogen, fluorine, methyl or trifluoromethyl, or R 16A and R 16B together with the carbon atom to which they are attached form a cyclopropyl ring, R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl, wherein (C1-C4)-alkyl is optionally substituted with 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxyl, 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, wherein methyl and ethyl may be substituted with one substituent selected from the group consisting of fluorine, trifluoromethyl and cyclopropyl. and salts, solvates and solvates of the salts thereof.

[0047] The compounds of the present invention are compounds of formula (I) and salts, solvates and solvates of salts thereof, compounds encompassed by formula (I) specified below and salts, solvates and solvates of salts thereof, and compounds encompassed by formula (I) and specified below as examples and salts, solvates and solvates thereof, to the extent that the compounds encompassed by formula (I) and specified below are not already salts, solvates and solvates of salts.

[0048] In the context of the present invention, preferred salts are physiologically acceptable salts of the compounds of the invention. Also included are salts which are not themselves suitable for pharmaceutical applications but which can be used, for example, for the isolation, purification or storage of the compounds of the invention.

[0049] Physiologically acceptable salts of the compounds of the invention include acid addition salts of mineral acids, carboxylic acids and sulfonic acids, such as the salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid and benzoic acid.

[0050] Physiologically acceptable salts of the compounds of the invention also include salts of conventional bases, by way of example and preferably the alkali metal salts (e.g., sodium and potassium salts), alkaline earth metal salts (e.g., calcium and magnesium salts), as well as ammonium salts derived from ammonia or organic amines having 1 to 16 carbon atoms, by way of example 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.

[0051] In the context of the present invention, solvates refer to those forms of the compounds of the present invention that form a complex in solid or liquid state by coordination with solvent molecules. Hydrates are a specific form of solvates in which the coordination is with water. The preferred solvates in the context of the present invention are hydrates.

[0052] Depending on their structure, the compounds of the present 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 the case of atropisomers).The present invention therefore encompasses enantiomers or diastereomers and their respective mixtures.Stereoisomeric homogeneous components can be isolated from such mixtures of enantiomers and / or diastereomers in known manner, and chromatographic processes, in particular HPLC chromatography on achiral or chiral phases, are preferably used for this purpose.

[0053] Where compounds of the invention can exist in tautomeric forms, the present invention encompasses all tautomeric forms.

[0054] The present invention also encompasses 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 compounds in which at least one atom in the compounds of the present invention is replaced with another atom that has the same atomic number but has a different atomic mass than the atomic mass that usually or mainly occurs in nature.The examples of isotopes that can be incorporated into the compounds of the present invention are the 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. Certain isotopic variants of the compounds of the present invention, especially those incorporating one or more radioisotopes, may be useful, for example, for studying the mechanism of action or the distribution of active ingredients in the body; due to their relatively easy preparation and detectability, in particular compounds labeled with 3H or 14C isotopes are suitable for this purpose. Furthermore, the incorporation of an isotope, for example the incorporation of deuterium, may result in certain therapeutic benefits, such as an increase in the half-life in the body or a reduction in the required active dose, as a result of the greater metabolic stability of the compound; therefore, such modifications of the compounds of the present invention may in some cases also constitute preferred embodiments of the present invention. Isotopic variants of the compounds of the present invention may be prepared by methods known to those skilled in the art, for example by the methods described below and the instructions reproduced in the examples, by using the corresponding isotopic modifications of certain reagents and / or starting compounds therein.

[0055] Additionally, the present invention also encompasses prodrugs of the compounds of the present invention. The term "prodrug" as used herein refers to a compound which may itself be biologically active or inactive, but which is converted to a compound of the present invention while present in the body, for example, by a metabolic or hydrolytic pathway.

[0056] For purposes of this invention, unless otherwise indicated, the substituents are each defined as follows: In the context of the present invention, alkyl 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-trimethylphenyl.

[0057] Cycloalkyl in the context of the present invention is a monocyclic saturated alkyl radical having 3 to 7 carbon atoms. Preferred examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

[0058] Alkylcarbonyl in the context of the present invention is a straight or branched alkyl group having 1 to 4 carbon atoms and a carbonyl group attached in position 1. Preferred examples include methylcarbonyl, ethylcarbonyl, n-propylcarbonyl, isopropylcarbonyl, n-butylcarbonyl, isobutylcarbonyl and tert-butylcarbonyl.

[0059] Alkoxy in the context of the present invention is a straight or branched alkoxy group having 1 to 4 carbon atoms. Preferred examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy and tert-butoxy.

[0060] In the context of the present invention, alkoxycarbonyl is a straight or branched alkoxy group having 1 to 4 carbon atoms and a carbonyl group bonded to oxygen. Preferred examples include methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl and tert-butoxycarbonyl.

[0061] In the context of the present invention, alkylthio 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, iso-propylthio, 1-methylpropylthio, n-butylthio, iso-butylthio and tert-butylthio.

[0062] Alkylsulfonyl in the context of the present invention is a straight 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.

[0063] In the context of the present invention, a 4-7 membered heterocyclyl is a monocyclic saturated heterocycle having a total of 4-7 ring atoms, containing 1 or 2 ring heteroatoms from the group N, O, S, SO and / or SO2, bonded via a ring carbon atom or, where appropriate, via a ring nitrogen atom. Examples include azetidinyl, oxetanyl, pyrrolidinyl, pyrazolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl. Azetidinyl, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl and morpholinyl are preferred.

[0064] A 5-7 membered heterocyclyl in the context of the present invention is a partially unsaturated heterocycle having a total of 5-7 ring atoms, containing 1-3 ring heteroatoms from the group N, O, S and / or SO2 and fused to a phenyl ring in R3. Examples include dihydropyrrolyl, dihydroimidazolyl, dihydrothiazole dioxide, dihydrooxazolyl, dihydropyridyl, tetrahydropyrazinyl and dihydrooxazinyl.

[0065] In the context of the present invention, heteroaryl is a monocyclic aromatic heterocycle (heteroaromatic) with a total of 5 or 6 ring atoms, containing up to 3 identical or different ring heteroatoms from the group N, O and / or S, fused to a phenyl ring in R3. Examples include furyl, pyrrolyl, thienyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl and triazinyl. Pyrazolyl, imidazolyl, thiazolyl and triazolyl are preferred.

[0066] Halogen in the context of the present invention includes fluorine, chlorine, bromine and iodine, with chlorine or fluorine being preferred.

[0067] An oxo group in the context of the present invention is an oxygen atom attached via a double bond to a carbon or sulfur atom.

[0068] R 2 and R 3 In the formula of the radical which may be represented by R, the end of the line marked by the symbols * or # or ## does not represent a carbon atom or a CH2 group, 2 and R 3 is part of the bond to each atom it is bonded to.

[0069] When radicals in the compounds according to the invention are substituted, they may be mono- or polysubstituted, unless otherwise specified.In the context of the present invention, all radicals that occur more than once are defined independently of each other.Preferably, they are substituted by one or two identical or different substituents.Very particularly preferred is the substitution by one substituent.

[0070] In the context of the present invention, the term "treatment" or "treating" includes inhibiting, delaying, checking, mitigating, attenuating, limiting, reducing, suppressing, warding off or curing a disease, condition, disorder, injury or health problem, or the onset, course, progression of such a condition and / or symptoms of such a condition. The term "therapy" is understood herein to be synonymous with the term "treatment."

[0071] The terms "prevention", "prophylaxis" or "prevention" are used interchangeably in relation to the present invention and refer to the avoidance or reduction of the risk of developing, experiencing, suffering from or having a disease, condition, disorder, injury or health problem, or the development or progression of such a condition and / or symptoms of such a condition.

[0072] The treatment or prevention of a disease, condition, disorder, injury or health problem may be partial or complete.

[0073] In a further embodiment, the present invention relates to a compound of general formula (I) above for use in a method for the treatment and / or prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states following inflammation, transplantation, infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access sites. [During the ceremony, R 1 is hydrogen, R 2 is the formula [ka] Based on where * is the site of attachment to the nitrogen atom of uracil, R 5A is hydrogen, R 5B is hydrogen, R 6 is hydrogen, R 7 is hydrogen, R 8 is fluorine, chlorine or trifluoromethyl, R 9 is fluorine, chlorine, trifluoromethyl or methyl, R 3 is the formula [ka] Based on where # is the attachment site for the nitrogen atom of uracil, E 1 CR 11 and Here, R 11 is hydrogen, E 2 is N, G 1 is C=O, G 2 CR 16A R 16B , N.R. 17 , O or S; Here, R 16A is hydrogen, fluorine, methyl or hydroxyl, and R 16B is hydrogen, fluorine, methyl or trifluoromethyl, or R 16A and R 16B together with the carbon atom to which they are attached form a cyclopropyl ring, R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl, wherein (C1-C4)-alkyl is optionally substituted with 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxyl, 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, wherein methyl and ethyl may be substituted with one substituent selected from the group consisting of fluorine, trifluoromethyl and cyclopropyl, and salts, solvates and solvates of the salts thereof.

[0074] In a further embodiment, the present invention relates to a compound of general formula (I) above for use in a method for thrombolysis without bleeding risk and thereby in stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states following inflammation, transplantation, infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access sites. [During the ceremony, R 1 is hydrogen, R 2 is the formula [ka] Based on where * is the site of attachment to the nitrogen atom of uracil, R 5A is hydrogen, R 5B is hydrogen, R 6 is hydrogen, R 7 is hydrogen, R 8 is fluorine, chlorine or trifluoromethyl, R 9 is fluorine, chlorine, trifluoromethyl or methyl, R 3 is the formula [ka] Based on where # is the attachment site for the nitrogen atom of uracil, E 1 CR 11and Here, R 11 is hydrogen, E 2 is N, G 1 is C=O, G 2 CR 16A R 16B , N.R. 17 , O or S; Here, R 16A is hydrogen, fluorine, methyl or hydroxyl, and R 16B is hydrogen, fluorine, methyl or trifluoromethyl, or R 16A and R 16B together with the carbon atom to which they are attached form a cyclopropyl ring, R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl, wherein (C1-C4)-alkyl is optionally substituted with 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxyl, 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, wherein methyl and ethyl may be substituted with one substituent selected from the group consisting of fluorine, trifluoromethyl and cyclopropyl. and salts, solvates and solvates of the salts thereof.

[0075] In a further embodiment, the present invention relates to a compound of general formula (I) above for use in a method for thrombolysis without bleeding risk and thereby in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep or superficial venous thrombosis, phlebitis, thrombotic microangiopathy, inflammation, transplantation, thrombotic microangiopathy in hypercoagulable states after infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access sites. [During the ceremony, R 1 is hydrogen, R 2 is the formula [ka] Based on where * is the site of attachment to the nitrogen atom of uracil, R 5A is hydrogen, R 5B is hydrogen, R 6 is hydrogen, R 7 is hydrogen, R 8 is fluorine, chlorine or trifluoromethyl, R 9 is fluorine, chlorine, trifluoromethyl or methyl, R 3 is the formula [ka] Based on where # is the attachment site for the nitrogen atom of uracil, E 1 CR 11 and Here, R 11 is hydrogen, E 2 is N, G 1 is C=O, G 2 CR 16A R 16B , N.R.17 , O or S; Here, R 16A is hydrogen, fluorine, methyl or hydroxyl, and R 16B is hydrogen, fluorine, methyl or trifluoromethyl, or R 16A and R 16B together with the carbon atom to which they are attached form a cyclopropyl ring, R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl, wherein (C1-C4)-alkyl is optionally substituted with 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxyl, 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, wherein methyl and ethyl may be substituted with one substituent selected from the group consisting of fluorine, trifluoromethyl and cyclopropyl. and salts, solvates and solvates of the salts thereof.

[0076] In a further embodiment, the present invention relates to a compound of general formula (I) above for use in a method for thrombolysis without bleeding risk and thereby in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep or superficial venous thrombosis, phlebitis, thrombotic microangiopathy, inflammation, transplantation, thrombotic microangiopathy in hypercoagulable states after infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access sites. [During the ceremony, R 1 is hydrogen, R2 is the formula [ka] Based on where * is the site of attachment to the nitrogen atom of uracil, R 5A is hydrogen, R 5B is hydrogen, R 6 is hydrogen, R 7 is hydrogen, R 8 is fluorine, chlorine or trifluoromethyl, R 9 is fluorine, chlorine, trifluoromethyl or methyl, R 3 is the formula [ka] Based on where # is the attachment site for the nitrogen atom of uracil, E 1 CR 11 and Here, R 11 is hydrogen, E 2 is N, G 1 is C=O, G 2 CR 16A R 16B , N.R. 17 , O or S; Here, R 16A is hydrogen, fluorine, methyl or hydroxyl, and R 16B is hydrogen, fluorine, methyl or trifluoromethyl, or R 16A and R 16B together with the carbon atom to which they are attached form a cyclopropyl ring, R 17is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl, wherein (C1-C4)-alkyl is optionally substituted with 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxyl, 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, wherein methyl and ethyl may be substituted with one substituent selected from the group consisting of fluorine, trifluoromethyl and cyclopropyl. and salts, solvates and solvates of the salts thereof.

[0077] In a further embodiment, the present invention relates to a compound of general formula (I) above for use in a method for the treatment and / or prevention of stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep or superficial venous thrombosis, phlebitis or microangiopathy. [During the ceremony, R 1 is hydrogen, R 2 is the formula [ka] Based on where * is the site of attachment to the nitrogen atom of uracil, R 5A is hydrogen, R 5B is hydrogen, R 6 is hydrogen, R 7 is hydrogen, R 8 is fluorine, chlorine or trifluoromethyl, R 9is fluorine, chlorine, trifluoromethyl or methyl, R 3 is the formula [ka] Based on where # is the attachment site for the nitrogen atom of uracil, E 1 CR 11 and Here, R 11 is hydrogen, E 2 is N, G 1 is C=O, G 2 CR 16A R 16B , N.R. 17 , O or S; Here, R 16A is hydrogen, fluorine, methyl or hydroxyl, and R 16B is hydrogen, fluorine, methyl or trifluoromethyl, or R 16A and R 16B together with the carbon atom to which they are attached form a cyclopropyl ring, R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl, wherein (C1-C4)-alkyl is optionally substituted with 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxyl, 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, wherein methyl and ethyl may be substituted with one substituent selected from the group consisting of fluorine, trifluoromethyl and cyclopropyl. and salts, solvates and solvates of the salts thereof.

[0078] In a further embodiment, the present invention relates to a compound of general formula (I) above for use in a method for thrombolysis without bleeding risk and thereby thrombolysis in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, phlebitis or microangiopathy. [During the ceremony, R 1 is hydrogen, R 2 is the formula [ka] Based on where * is the site of attachment to the nitrogen atom of uracil, R 5A is hydrogen, R 5B is hydrogen, R 6 is hydrogen, R 7 is hydrogen, R 8 is fluorine, chlorine or trifluoromethyl, R 9 is fluorine, chlorine, trifluoromethyl or methyl, R 3 is the formula [ka] Based on where # is the attachment site for the nitrogen atom of uracil, E 1 CR 11 and Here, R 11 is hydrogen, E 2 is N, G 1 is C=O, G 2 CR 16A R 16B , N.R. 17 , O or S; Here, R 16A is hydrogen, fluorine, methyl or hydroxyl, and R 16B is hydrogen, fluorine, methyl or trifluoromethyl, or R 16A and R 16B together with the carbon atom to which they are attached form a cyclopropyl ring, R 17 is hydrogen, (C1-C4)-alkyl or (C3-C5)-cycloalkyl, wherein (C1-C4)-alkyl is optionally substituted with 1 to 3 substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxyl, 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, wherein methyl and ethyl may be substituted with one substituent selected from the group consisting of fluorine, trifluoromethyl and cyclopropyl. and salts, solvates and solvates of the salts thereof.

[0079] 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-tetrahydropiperidin, for use in a method for the treatment and / or prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, inflammatory, transplantation, thrombotic microangiopathy in hypercoagulable states following infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion at a vascular access site. 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-dihy- 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,Compounds selected from the group consisting of 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) are preferred in the context of the present invention.

[0080] 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,Compounds selected from the group of 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) and the salts, solvates and solvates of the salts thereof are preferred in the context of the present invention.

[0081] 1-(1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-benzoimidazol-5-yl] ... H-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,Compounds selected from the group 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) are preferred in the context of the present invention.

[0082] 1-(1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,4-dioxo-3-(trifluoromethyl)-2,5-dioxo-4-(trifluoromethyl)-2,6-dioxo-1,7-dimethyl-2,7-dimethyl-2,8-dimethyl-2,9-dimethyl-2,10-trifluoromethyl-2,11-dimethyl-2,20-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dioxo-1,7-dimethyl ...3-dioxo-1,7-dimethyl-2,3-dioxo-1,7-dimethyl-2,3-dioxo-1,7-dimethyl-2,3-dioxo-1,7-dimethyl-2,3-dioxo-1,7-dimethyl-2,3-dioxo-1,7-dimethyl-2,3-dioxo-1,7-dimethyl-2,3 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-benzoxazo 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-2,3-dihydro-1H-indol-5-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), 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,Compounds selected from the group of 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) and the salts, solvates and solvates of the salts thereof are preferred in the context of the present invention.

[0083] 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-benzimid ... 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), 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 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-(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,In the context of the present invention, compounds selected from the group consisting of 3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylate (R enantiomer) and salts, solvates and solvates of the salts thereof are preferred.

[0084] 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-inden-1-yl)-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), for use in a method for thrombolysis without bleeding risk and therefore for thrombolysis in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, phlebitis or microangiopathy. H-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,In the context of the present invention, compounds selected from the group consisting of 3-dihydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylate (R enantiomer) and salts, solvates and solvates of the salts thereof are preferred.

[0085] Preferred in the context of the present invention 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) for use in a method for the treatment and / or prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in inflammation, transplantation, post-infectious hypercoagulable states, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access sites.

[0086] Preferred in the context of the present invention 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) for use in a method for the treatment and / or prevention of stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep or superficial venous thrombosis, phlebitis, thrombotic microangiopathy, inflammation, transplantation, thrombotic microangiopathy in hypercoagulable states after infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access sites.

[0087] Preferred in the context of the present invention 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) for use in a method for thrombolysis without bleeding risk and thereby in stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states after inflammation, transplantation, infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access sites.

[0088] Preferred in the context of the present invention 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) for use in a method for thrombolysis without bleeding risk and thereby in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep or superficial venous thrombosis, phlebitis, thrombotic microangiopathy, inflammation, transplantation, thrombotic microangiopathy in hypercoagulable states after infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access sites.

[0089] Preferred in the context of the present invention 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) for use in a method for the treatment and / or prevention of stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, microangiopathy or phlebitis.

[0090] Preferred in the context of the present invention 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) for use in methods for thrombolysis without bleeding risk and thereby thrombolysis in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, thrombotic microangiopathy or phlebitis.

[0091] Preferred in the context of the present invention 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) for use in a method for the treatment and / or prophylaxis of stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease or phlebitis.

[0092] Preferred in the context of the present invention 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) for use in methods for thrombolysis without bleeding risk and thereby thrombolysis in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease or phlebitis.

[0093] Preferred in the context of the present invention 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) for use in a method for the treatment and / or prevention of stroke, pulmonary embolism, microangiopathy or phlebitis.

[0094] Preferred in the context of the present invention 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) for use in methods for thrombolysis without bleeding risk and thereby thrombolysis in stroke, pulmonary embolism, myocardial infarction, microangiopathy or phlebitis.

[0095] Preferred in the context of the present invention 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) for use in a method for the treatment and / or prevention of stroke.

[0096] Preferred in the context of the present invention 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) for use in a method for thrombolysis without bleeding risk and thereby for thrombolysis in stroke.

[0097] Preferred in the context of the present invention 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) for use in a method for the treatment and / or prophylaxis of pulmonary embolism.

[0098] Preferred in the context of the present invention 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) for use in a method for thrombolysis without bleeding risk and thereby for thrombolysis in pulmonary embolism.

[0099] For use in a method for the treatment and / or prevention of myocardial infarction, 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 in the context of the present invention.

[0100] Preferred in the context of the present invention 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) for use in a method for thrombolysis without bleeding risk and thereby for thrombolysis in myocardial infarction.

[0101] Preferred in the context of the present invention 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) for use in a method for the treatment and / or prevention of peripheral arterial disease.

[0102] Preferred in the context of the present invention 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) for use in methods for thrombolysis without bleeding risks and thereby for thrombolysis in peripheral arterial diseases.

[0103] Preferred in the context of the present invention 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) for use in a method for the treatment and / or prevention of microangiopathy.

[0104] Preferred in the context of the present invention 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) for use in a method for thrombolysis without bleeding risk and thereby thrombolysis in microangiopathy.

[0105] Preferred in the context of the present invention 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) for use in a method for the treatment and / or prophylaxis of phlebitis.

[0106] Preferred in the context of the present invention 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) for use in a method for thrombolysis without bleeding risk and therefore thrombolysis in phlebitis.

[0107] In the context of the present invention, a compound of formula (I) 1 is hydrogen. and salts, solvates and solvates of the salts thereof are preferred.

[0108] In the context of the present invention, the compound of formula (I) [During the ceremony, R 1 is hydrogen, R 2 is the formula [ka] Based on where * is the site of attachment to the nitrogen atom of uracil, A is -CH2-, R 4A is chlorine or trifluoromethyl, R 4B is hydrogen. and salts, solvates and solvates of the salts thereof are preferred.

[0109] In the context of the present invention, the compound of formula (I) [During the ceremony, R 2 is the formula [ka] Based on where * is the site of attachment to the nitrogen atom of uracil, A is -CH2-, R 4A is chlorine or trifluoromethyl, R 4B is hydrogen. and salts, solvates and solvates of the salts thereof are preferred.

[0110] In a further embodiment the present invention relates to a compound of general formula (II) [ka] [During the ceremony, 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 may be the same or different, and each may be 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 is a hydrogen atom, a lower alkoxycarbonylmethyl group, a carboxymethyl group, or -CH(CHOH)COOR 7 (where R 7 represents a hydrogen atom or a lower alkyl group, formula: [ka] A group represented by In the formula, R 7 has the same meaning as above, formula: [ka] A group represented by Here, R 8 and R9 may be the same or different and each represents a hydrogen atom, a lower alkyl group, a lower alkylsulfanyl group, a lower alkylsulfinyl group, a lower alkylsulfonyl group or a lower alkoxycarbonyl group, a hydroxy lower alkyl group, or a cyano group; Or, the expression: [ka] represents a monocyclic heterocyclic group represented by the formula: In the formula, A represents an oxygen atom, a sulfur atom or NH, the dotted line represents a single bond or a double bond, and a hydrogen atom on the ring may be replaced by a halogen atom, a lower alkoxy group, a hydroxy-lower alkyl group, a lower alkoxycarbonyl group or a lower alkyl group which may be substituted by a carboxyl group; However, R 3 or R 4 At least one of [ka] and and R 5 represents a hydrogen atom, a lower alkoxy group, or a lower alkyl group, excluding compounds represented by the following formula: [ka]

[0111] In a further embodiment the present invention relates to a compound of general formula (II) [ka] [During the ceremony, R 1represents a hydrogen atom, a halogen atom or a lower alkyl group; R 2 represents a lower alkyl group; R 3 and R 4 may be the same or different, and each may be 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 is a hydrogen atom, a lower alkoxycarbonylmethyl group, a carboxymethyl group, or -CH(CHOH)COOR 7 (where R 7 represents a hydrogen atom or a lower alkyl group); formula [ka] (wherein R 7 has the same meaning as above), formula [ka] Here, R 8 and R 9 may be the same or different and each represent a hydrogen atom, a lower alkyl group, a lower alkylsulfanyl group, a lower alkylsulfinyl group, a lower alkylsulfonyl group or a lower alkoxycarbonyl group, a hydroxy-lower alkyl group, or a cyano group; or the expression: [ka] represents a monocyclic heterocyclic group represented by the formula: In the formula, A represents an oxygen atom, a sulfur atom or NH, the dotted line represents a single bond or a double bond, and a hydrogen atom on the ring may be replaced by a halogen atom, a lower alkoxy group, a hydroxy-lower alkyl group, a lower alkoxycarbonyl group or a lower alkyl group which may be substituted by a carboxyl group; However, R 3or R 4 At least one of [ka] and and R 5 represents a hydrogen atom, a lower alkoxy group, or a lower alkyl group. but excluding compounds represented by the formula: [ka]

[0112] R 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.

[0113] 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, with a methyl group or an ethyl group being particularly preferred.

[0114] R 3 , R 4 , R 8 and R 9 Examples of the lower alkoxycarbonyl group include a methoxycarbonyl group, an ethoxycarbonyl group, a propoxycarbonyl group, an isopropoxycarbonyl group, a butoxycarbonyl group, an isobutoxycarbonyl group, a sec-butoxycarbonyl group, and a tert-butoxycarbonyl group, and particularly a methoxycarbonyl group, an ethoxycarbonyl group, an isopropoxycarbonyl group, and a tert-butoxycarbonyl group are preferred.

[0115] R 3 , R 4 , R 8 and R9 Examples of the lower alkylsulfonyl group include a methanesulfonyl group, an ethanesulfonyl group, a propanesulfonyl group, an isopropanesulfonyl group, a butanesulfonyl group, an isobutanesulfonyl group, a sec-butanesulfonyl group, or a tert-butanesulfonyl group, and a methanesulfonyl group or an ethanesulfonyl group is particularly preferred.

[0116] 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, and an isobutyryl group, with an acetyl group being particularly preferred.

[0117] R 3 , R 4 and R 5 Examples of lower alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy and tert-butoxy groups, with methoxy and ethoxy groups being particularly preferred.

[0118] R 3 and R 4 Examples of the lower alkoxycarbonylmethylthioacetyl group include a methoxycarbonylmethylthio-acetyl group, an ethoxycarbonylmethylthioacetyl group, a propoxycarbonylmethylthioacetyl group, an isopropoxycarbonylmethylthioacetyl group, a butoxycarbonylmethylthio-acetyl group, an isobutoxycarbonylmethylthioacetyl group, a sec-butoxycarbonylmethylthioacetyl group, and a tert-butoxycarbonylmethylthioacetyl group, and a methoxycarbonylmethylthioacetyl group and an ethoxycarbonylmethylthioacetyl group are particularly preferred.

[0119] R 3 and R 4 are -CONHR 6 When R 6Examples of the lower alkoxycarbonylmethyl group include a methoxycarbonylmethyl group, an ethoxycarbonylmethyl group, a propoxycarbonylmethyl group, an isopropoxycarbonylmethyl group, a butoxycarbonylmethyl group, an isobutoxycarbonylmethyl group, a sec-butoxycarbonylmethyl group or a tert-butoxycarbonylmethyl group, and particularly preferred are a methoxycarbonylmethyl group, an ethoxycarbonylmethyl group or an isopropoxycarbonylmethyl group.

[0120] R 3 and R 4 Examples of the hydroxy lower alkyl group include linear or branched hydroxy lower alkyl groups having 1 to 4 carbon atoms, such as a hydroxymethyl group, a hydroxyethyl group, a hydroxypropyl group, and a hydroxybutyl group, with a hydroxymethyl group or a hydroxyethyl group being particularly preferred.

[0121] R 8 and R 9 Examples of the lower alkylsulfanyl group include linear or branched lower alkylsulfanyl groups having 1 to 4 carbon atoms, such as methylsulfanyl, ethylsulfanyl, propylsulfanyl, and butylsulfanyl groups, particularly methylsulfanyl or ethylsulfanyl groups.

[0122] R 8 and R 9 Examples of the lower alkylsulfinyl group include linear or branched lower alkylsulfinyl groups having 1 to 4 carbon atoms, such as a methanesulfinyl group, an ethanesulfinyl group, a propanesulfinyl group, or a butanesulfinyl group, with a methanesulfinyl group or an ethanesulfinyl group being particularly preferred.

[0123] formula: [ka] The group represented by the formula (I) is preferably, for example, a vinyl group, a methylsulfanylvinyl group, a methanesulfinylvinyl group or a 2-methanesulfinyl-2-methyl-sulfanylvinyl group.

[0124] formula [ka] The lower alkoxy group, hydroxy lower alkyl group and lower alkoxycarbonyl group, which may replace the hydrogen atom on the ring represented by the formula (I), are as defined above. The lower alkyl group which may be substituted with a halogen atom means a lower alkyl group substituted with a fluorine atom, a chlorine atom, a bromine atom or an iodine atom in addition to the above lower alkyl groups. Examples of such groups include a chloromethyl group, a bromoethyl group, a dichloromethyl group and a 1-chloroethyl group.

[0125] One or two of a halogen atom, a lower alkoxy group, a hydroxy-lower alkyl group, a lower alkoxycarbonyl group or a lower alkyl group optionally substituted with a carboxyl group may be present as substituents on each heterocycle, and the substituents may be the same or different.

[0126] formula: [ka] Examples of monocyclic heterocyclic groups represented by the formula (wherein A represents an oxygen atom, a sulfur atom, or NH, and the dotted line portion represents a single bond or a double bond) include those represented by the formula: [ka] Examples of the compound include compounds represented by the following formula:

[0127] formula: [ka] As a specific example of the monocyclic heterocyclic group represented by the formula: (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, [ka] These substituents are represented by R 4 In this case, R 3 is a methanesulfonyl group, and R 5 It is more preferable that is a hydrogen atom.

[0128] 2-[4-(5-chloro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methane-sulfonylphenyl]oxazole-4-carboxylic acid, ... for use in a method for the treatment and / or prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, inflammatory, transplant, thrombotic microangiopathy in hypercoagulable states following infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion at a vascular access site. (5-fluoro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methanesulfonylphenyl]oxazole-4-carboxylic acid, 2-[4-(5-chloro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methanesulfonylphenyl]oxazole-4-carboxylic acid disodium, 2-[4-(5-fluoro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methanesulfonylphenyl]oxazole-4-capric acid disodium disodium carboxylate, 2-[4-(5-fluoro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methane-sulfonylphenyl]thiazole-4-carboxylic acid, 5-fluoro-N-[4-(4-hydroxymethylthiazol-2-yl)-2-methane-sulfonylphenyl]-3-methylbenzo[b]thiophene-2-sulfonamide, 5-fluoro-N-[2-methane-sulfonyl-4-(5-methoxy-4-methyloxazol-2-yl) Preference is given in the context of the present invention to compounds selected from the group ((2-(4-((5-fluoro-3-methylbenzo-[b]thiophene)-2-sulfonamide)-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.

[0129] 2-[4-(5-chloro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methane-sulfonylphenyl]oxazole-4-carboxylic acid, ... for use in a method for the treatment and / or prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, inflammatory, transplant, thrombotic microangiopathy in hypercoagulable states following infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion at a vascular access site. (5-fluoro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methanesulfonylphenyl]oxazole-4-carboxylic acid, 2-[4-(5-chloro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methanesulfonylphenyl]oxazole-4-carboxylic acid disodium, 2-[4-(5-fluoro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methanesulfonylphenyl]oxazole-4-capric acid disodium disodium carboxylate, 2-[4-(5-fluoro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methane-sulfonylphenyl]thiazole-4-carboxylic acid, 5-fluoro-N-[4-(4-hydroxymethylthiazol-2-yl)-2-methane-sulfonylphenyl]-3-methylbenzo[b]thiophene-2-sulfonamide, 5-fluoro-N-[2-methane-sulfonyl-4-(5-methoxy-4-methyloxazol-2-yl) Preference is given in the context of the present invention to compounds selected from the group ((2-(4-((5-fluoro-3-methylbenzo-[b]thiophene)-2-sulfonamide)-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.

[0130] Preferred in the context of the present invention is 2-(4-((5-fluoro-3-methylbenzo-[b]thiophene)-2-sulfonamido)-3-(methylsulfonyl)phenyl)thiazole-4-carboxylic acid for use in a method for the treatment and / or prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states following inflammation, transplantation, infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion at vascular access sites.

[0131] Preferred in the context of the present invention is 2-(4-((5-fluoro-3-methylbenzo-[b]thiophene)-2-sulfonamido)-3-(methylsulfonyl)phenyl)thiazole-4-carboxylic acid for use in a method for the treatment and / or prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states following inflammation, transplantation, infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access sites. The compounds according to the present invention are chymase inhibitors, in particular the compounds of formula (I), as well as salts, solvates and solvates of the salts thereof, and have an unpredictable and useful spectrum of pharmacological activity. These compounds affect the proteolytic activity of the serine protease chymase. The compounds according to the present invention inhibit the enzymatic cleavage of the chymase substrate.

[0132] The compounds of formula (I), their preparation and their action as selective inhibitors of chymase for the treatment and / or prevention of heart failure, pulmonary hypertension, chronic obstructive pulmonary disease, asthma, renal failure, nephropathy, visceral fibrotic disorders and skin fibrosis are generally disclosed in WO2013167495A1, in particular the specifically disclosed compounds are an express part of the description of the present invention and are incorporated herein by reference.

[0133] Compounds of formula (II), their preparation and action as inhibitors of chymase for the treatment of hypertension, cardiac hypertrophy, heart failure, cardiac infarction, arteriosclerosis, diabetic or non-diabetic kidney disease, diabetic retinopathy, ischemic reperfusion injury, restenosis after percutaneous transluminal coronary angioplasty, intimal thickening after bypass grafts, chronic rheumatism, keloids, psoriasis, allergy, inflammation, asthma, atopic dermatitis, solid tumors, and pulmonary hypertension are generally disclosed in US 7,071,220 B2, and in particular the specifically disclosed compounds are an express part of the description of the present invention and are incorporated herein by reference.

[0134] In the context of the present invention, the term "treatment" or "treating" includes inhibiting, delaying, checking, mitigating, attenuating, limiting, reducing, suppressing, warding off or curing a disease, condition, disorder, injury or health problem, or the onset, course, progression of such a condition and / or symptoms of such a condition. The term "therapy" is understood herein to be synonymous with the term "treatment."

[0135] The terms "prevention", "prophylaxis" or "prevention" are used interchangeably in relation to the present invention and refer to the avoidance or reduction of the risk of developing, experiencing, suffering from or having a disease, condition, disorder, injury or health problem, or the development or progression of such a condition and / or symptoms of such a condition.

[0136] The treatment or prevention of a disease, condition, disorder, injury or health problem may be partial or complete.

[0137] For the purposes of the present invention, "thrombolysis during a thrombotic or thromboembolic event" includes disorders and complications occurring in the arterial and venous vasculature, microcirculation and lymphatic systems, which can be treated with the compounds according to the invention.

[0138] The compounds according to the invention are suitable for the treatment and prevention of disorders of the venous vasculature that lead to venous occlusion and thus to venous thrombosis.These include thrombotic events in the venous vessels of the lower and upper limbs, such as the lower deep vein thrombosis of the calf vein, popliteal vein, femoral vein, common femoral vein, iliac vein with or without the inferior vena cava, the upper deep vein thrombosis of the jugular vein, brachiocephalic vein, subclavian vein, axillary vein, and more distal brachial vein, ulnar vein, and radial vein, or the superficial vein thrombosis of the small saphenous vein, the large saphenous vein, the cephalic vein, and the basilic vein, events of the venous vessels of the organs, including retinal vein occlusion, thrombosis of the cerebral vein and the cranial sinus, thrombosis of the renal vein, Budd-Chiari syndrome and portal vein thrombosis, mesenteric vein thrombosis, ovarian vein thrombosis, and partial thrombosis of the corpus cavernosum.

[0139] The compounds according to the invention are suitable for example for the prevention of latent strokes associated with post-thrombotic syndrome, complications after venous thrombosis, including pulmonary hypertension or pulmonary embolism, and paradoxical thromboembolism with patent foramen ovale due to deep vein thrombosis of the limbs.

[0140] The compounds according to the invention are suitable for the treatment and prevention of disorders of the arterial vasculature which lead to the occlusion of the arterial vessels and thus to arterial thrombosis. This includes, in particular, disorders of the cerebrovascular system, such as transient ischemic attacks (TIA), cardiogenic cerebral embolism, such as stroke due to atrial fibrillation, non-cardiogenic cerebral embolism, such as lacunar infarction, ischemic stroke, including stroke due to diseases of the aorta or small arteries, or cryptogenic stroke, ... strokes, embolic stroke, embolic stroke of unknown etiology, or thrombotic and / or thromboembolic events leading to stroke or TIA, disorders of the coronary arteries of the heart, e.g. acute coronary syndromes (ACS), myocardial infarction with ST elevation (STEMI) and without ST elevation (non-STEMI, stable angina, unstable angina, re-occlusion and restenosis after coronary interventions such as angioplasty, stent grafting or aortocoronary bypass, and peripheral artery disease, including peripheral arterial occlusion, acute limb ischemia, amputation, re-occlusion and restenosis after interventions such as angioplasty, stent grafting or surgery and bypass, leading to peripheral artery disease, and / or stent thrombosis, renal artery, hepatic artery or mesenteric artery thrombosis, retinal artery thrombosis.

[0141] The compounds according to the invention are suitable for the treatment and prevention of local or systemic disorders in the microcirculation caused by small vessel thrombosis, including primary thrombotic microangiopathy (TMA), such as thrombotic thrombocytopenic purpura (TTP) or hemolytic uraemic syndrome (HUS), and secondary thrombotic microangiopathy. These include hypercoagulable states caused by bacteria, viruses or fungi, hypercoagulable states resulting from viral infections, e.g., SARS-Cov1, MERS, and SARS-Cov2 infections, sepsis, microthrombosis as a result of drugs or vaccinations (e.g., vaccine-induced thrombotic thrombocytopenia (VITT), which occurred as a very rare adverse event after COVID vaccination), disseminated intravascular coagulation (DIC), pulmonary thrombosis with and without ARDS, autoimmune diseases (such as systemic lupus erythematosus), antiphospholipid syndrome, malignant hypertension, transplants (e.g., solid organs, bone marrow), pregnancy-associated thrombotic microangiopathies (e.g., preeclampsia, HELLP syndrome), placental microthrombosis, veno-occlusive disease of the liver, diabetic microangiopathies (e.g., diabetic retinopathy, glomerulopathy, gangrene), small vasculitis of the capillary system (e.g., cerebral vasculitis).

[0142] The compounds according to the invention are useful in preventing and treating venous thromboembolism (VT) in acute ischemic stroke, peripheral arterial occlusion and related diseases including myocardial infarction. Atherothrombotic The present invention is suitable for the treatment and prevention of cerebrovascular disorders, e.g., cerebrovascular diseases, such as cerebrovascular accidents ...

[0143] The compounds according to the present invention have the following effects in pulmonary embolism, cardiogenic cerebral embolism, myocardial infarction and other related diseases: Thromboembolic Caused by arterial blockage Ischemic events The compounds are suitable for the treatment and prevention of

[0144] The compounds according to the invention are effective in preventing thrombosis in deep and superficial veins of the extremities, as well as in cerebral veins. Venous thrombosis The compounds are suitable for the treatment and prevention of

[0145] The compound according to the invention is thrombotic microangiopathy It is suitable for the treatment and prevention of diseases with thrombotic complications in the microcirculation, including infectious diseases caused by bacteria, viruses or fungi, sepsis, isolated pulmonary embolism / thrombosis, autoimmune diseases.

[0146] The compounds according to the invention are suitable for the treatment and prevention of thrombosis or occlusion of vascular access sites, including arteriovenous fistulas, central venous ports.

[0147] The compounds according to the invention are suitable for the treatment and prevention of extravascular fibrin deposits, for example in the lungs, placenta and brain.

[0148] This includes unmet clinical needs in solving venous thromboembolism (VTE) in cancer patients undergoing hormonal, chemotherapy or radiotherapy. These patients are often prone to visceral localized or catheter-induced thrombosis and are anticoagulated, thus suffering from bleeding disorders associated with the latter class of drug therapy (Giustozi et al., Trends Cardiovasc Med, 2022; In press). Moreover, mast cell activation has increasingly been reported to play a role in cancer development (Noto et al., Front Cell Dev Biol. 2021 Oct 12;9:752350; Segura-Villalobos et al., Cells. 2022 Jan 20;11(3):349).

[0149] This includes damage caused by exposure of biomaterials or artificial surfaces to blood and / or vessel walls, including procedures for recanalization of vascular access sites, arteriovenous fistulas, central venous ports for central line therapy or dialysis procedures, stents, grafts, cardiovascular assist devices, artificial hearts, mechanical or bioprosthetic heart valves, or procedures for revascularization or thrombus dissolution during transplantation.

[0150] This includes disorders associated with extravascular fibrin deposition, including fibrin deposition in the interstitial spaces of the lung.

[0151] This includes the use of chymase inhibition in diagnostic approaches for disease and / or therapeutic control.

[0152] Depending on their structure, the compounds of the present invention may exist in different stereoisomeric forms, i.e. in the form of configurational isomers or, if appropriate, in the form of conformational isomers (including in the case of enantiomers and / or diastereomers, rotamers and atropisomers).The present invention therefore encompasses enantiomers and diastereomers, as well as their respective mixtures.Stereoisomerically homogeneous components can be isolated from such mixtures of enantiomers and / or diastereomers in known manner, and chromatographic processes, in particular HPLC chromatography on achiral or chiral phases, are preferably used for this purpose.

[0153] Preferred in relation to the present invention salt are physiologically acceptable salts of the compounds according to the invention. However, the invention also encompasses salts which are not suitable per se for pharmaceutical applications but which can be used, for example, for the isolation or purification of the compounds according to the invention.

[0154] Physiologically acceptable salts of the compounds according to the invention include acid addition salts of mineral acids, carboxylic acids and sulfonic acids, such as the salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid and benzoic acid.

[0155] Physiologically acceptable salts of the compounds according to the invention also include the salts of conventional bases, for example and preferably the alkali metal salts (e.g. sodium and potassium salts), the 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, for example and preferably ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, N-methylmorpholine, arginine, lysine, ethylenediamine, N-methylpiperidine and choline.

[0156] solvate are described in the context of the present invention as forms of the compounds of the invention which form complexes in the solid or liquid state by coordination with solvent molecules. Hydrates are a specific form of solvates in which the coordination is with water.

[0157] The present invention further includes prodrugs of the compounds of the present invention. The term "prodrug" includes compounds that may be biologically active or inactive for their part, but are converted (e.g., by metabolism or hydrolysis) into a compound according to the present invention during their residence in the body.

[0158] The treatment or prevention of a disease, condition, disorder, injury or health problem may be partial or complete.

[0159] Chymase inhibitors such as those 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) furasimstat and TY-51469, are therefore suitable for use as medicines for the treatment and / or prevention of diseases in humans and animals.

[0160] Chymase inhibitors such as those 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) furasimstat, are therefore suitable for use as medicines for the treatment and / or prevention of diseases in humans and animals.

[0161] The present invention further provides the use of the compounds according to the invention for the treatment and / or prevention of disorders, in particular vascular disorders, preferably thrombotic or thromboembolic disorders and / or thrombotic or thromboembolic complications.

[0162] The compounds according to the invention are therefore suitable for the treatment and / or prevention of disorders or complications which may result from the formation of blood clots.

[0163] The present invention further provides the use of the chymase inhibitors of the present invention for the treatment and prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states following inflammation, transplantation, infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion at vascular access sites.

[0164] The present invention further provides the use of a chymase inhibitor of the present invention for thrombolysis in stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states following inflammation, transplantation, infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access sites.

[0165] The present invention further provides the use of the chymase inhibitors of the present invention for the treatment and prevention of stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states following inflammation, transplantation, infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion at vascular access sites.

[0166] The present invention further provides the use of a chymase inhibitor of the present invention for thrombolysis in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states following inflammation, transplantation, infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion at vascular access sites.

[0167] The present invention further provides the use of the chymase inhibitors of the present invention for the treatment and prevention of stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, phlebitis, and microangiopathy.

[0168] The present invention further provides the use of the chymase inhibitors of the present invention for thrombolysis in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, phlebitis, and microangiopathy.

[0169] The present invention further provides the use of chymase inhibitors of general formula I for the treatment and prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states following inflammation, transplantation, infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion at vascular access sites.

[0170] The present invention further provides the use of a chymase inhibitor of general formula I for the treatment and prevention of stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep or superficial venous thrombosis, phlebitis, thrombotic microangiopathy, inflammation, transplantation, thrombotic microangiopathy in hypercoagulable states following infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion at vascular access sites.

[0171] The present invention further provides the use of a chymase inhibitor of general formula I for thrombolysis in stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states following inflammation, transplantation, infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion at vascular access sites.

[0172] The present invention further provides the use of a chymase inhibitor of general formula I for thrombolysis in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, deep or superficial venous thrombosis, phlebitis, thrombotic microangiopathy, inflammation, transplantation, thrombotic microangiopathy in hypercoagulable states after infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access sites.

[0173] The present invention further provides the use of a chymase inhibitor of general formula I for the treatment and prevention of stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, phlebitis or microangiopathy.

[0174] The present invention further provides the use of a chymase inhibitor of general formula I for thrombolysis in stroke, pulmonary embolism, myocardial infarction, peripheral arterial disease, phlebitis or microangiopathy.

[0175] The present invention further provides the use of a chymase inhibitor of general formula I for the treatment and prevention of stroke, pulmonary embolism, phlebitis or microangiopathy.

[0176] The present invention further provides the use of a chymase inhibitor of general formula I for thrombolysis in stroke, pulmonary embolism, phlebitis or microangiopathy.

[0177] The present invention further provides the use of chymase inhibitors of general formula II for the treatment and prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states following inflammation, transplantation, infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion at vascular access sites.

[0178] The present invention further provides the use of a chymase inhibitor of general formula II for thrombolysis in stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states following inflammation, transplantation, infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion at vascular access sites.

[0179] The present invention further provides the use of a chymase inhibitor of general formula II for thrombolysis in stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states following inflammation, transplantation, infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion at vascular access sites.

[0180] The present invention further provides the use of a chymase inhibitor of general formula II for the treatment and prevention of stroke, pulmonary embolism, phlebitis or microangiopathy.

[0181] The present invention further provides the use of a chymase inhibitor of general formula II for thrombolysis in stroke, pulmonary embolism, phlebitis or microangiopathy.

[0182] For 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 and which can be treated with the compounds according to the invention.

[0183] The present invention further provides the use of the compounds according to the invention for the treatment and / or prevention of disorders, in particular the disorders mentioned above.

[0184] The present invention further provides the use of the compounds according to the invention for the manufacture of a medicament for the treatment and / or prevention of disorders, in particular the disorders mentioned above.

[0185] The present invention further provides the use of the compounds according to the invention for the manufacture of a medicament for thrombolysis in disorders, in particular in the disorders mentioned above.

[0186] The present invention further provides a method for the treatment and / or prevention of disorders, in particular the aforementioned disorders, using a therapeutically effective amount of the compounds according to the invention.

[0187] The present invention further provides a method for thrombolysis in disorders, especially the aforementioned disorders, using a therapeutically effective amount of a compound according to the invention.

[0188] The present invention further provides the compounds of 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 a compound of the invention.

[0189] The present invention further provides a compound of the invention for use in a method for thrombolysis in disorders, especially the disorders mentioned above, using a therapeutically effective amount of a compound of the invention.

[0190] The present invention further provides a medicament comprising a compound according to the invention and one or more further active compounds.

[0191] The present invention further provides medicaments for thrombolysis, in particular in the aforementioned disorders, which comprise a compound according to the invention and one or more further active compounds.

[0192] The present invention further provides medicaments comprising a compound according to the invention and one or more further active compounds, in particular for the treatment and / or prevention of the aforementioned disorders.

[0193] Preferred examples of active compounds suitable for combination include: Plasminogen activators (thrombolytic / fibrinolytic agents), such as, but not limited to, tissue plasminogen activator (t-PA, e.g. Actilyse®), streptokinase, tenecteplase, reteplase and urokinase, compounds that promote fibrinolysis, such as inhibitors of plasminogen activator inhibitors (PAI inhibitors), inhibitors of thrombin-activated fibrinolysis inhibitors (TAFI inhibitors) or inhibitors of α2-antiplasmin, or plasminogen regulators that cause an increase in the formation of plasmin, such as SMTP-7; 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) coronary artery therapy / vasodilator drugs, in particular 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, irbesartan, candesartan, eprosartan and telmisartan, or beta-adrenergic receptor antagonists, such as carvedilol, alprenolol, bisoprolol, acebutolol, atenolol, betaxolol, carteolol, metoprolol, nadolol, penbutolol, pindolol, propanolol and timolol, or alpha-1 -adrenergic receptor antagonists, such as prazosin, bunazosin, doxazosin and terazosin, or diuretics, such as hydrochlorothiazide, furosemide, bumetanide, piretanide, torasemide, amiloride and dihydralazine, or calcium channel blockers, such as verapamil and diltiazem, or dihydropyridine derivatives, such as nifedipine (Adalat) and nitrendipine (Bayotensin), or nitropreparations, such as isosorbide 5-mononitrate, isosorbide dinitrate and glycerol trinitrate, or substances that cause an increase in cyclic guanosine monophosphate (cGMP), such as stimulators of soluble guanylate cyclase, such as riociguat; Anticoagulants, e.g.: · Indirect anticoagulants, such as proteoglycans, e.g. heparin (UFH), low molecular weight heparins (LMW), e.g. tinzaparin, certoparin, parnaparin, nadroparin, ardeparin, enoxaparin, reviparin, dalteparin, danaparoid, semuloparin (AVE 5026), admiparin (M118); Direct thrombin inhibitors (DTIs), such as dabigatran, atesegatran, argatroban, bivalirudin, tanogitran and hirudin. Direct factor Xa inhibitors, such as rivaroxaban, apixaban, edoxaban, betrixaban, darexaban, otamixaban and fondaparinux Direct inhibitors of multiple coagulation factors, e.g. dual FXa / IIa inhibitors, e.g. SATI (Lopez M et al., Thromb Res. 193(2020)15-21) Inhibitors of FXI or FXIa, FXII or FXIIa, or inhibitors of FXI or FXII synthesis, such as asandexan, milbekian, osocimab, avelacimab, fesomersen Antiplatelet drugs, such as: Substances which inhibit platelet aggregation (platelet aggregation inhibitors, thrombocyte aggregation inhibitors), e.g. acetylsalicylic acid, P2Y12 antagonists, e.g. ticlopidine (Ticlid), clopidogrel (Plavix), prasugrel, ticagrelor, cangrelor, elinogrel, PAR-1 antagonists, e.g. vorapaxal, PAR-4 antagonists, EP3 antagonists, e.g. DG041; Platelet adhesion inhibitors, such as GPVI and / or GPIb antagonists, such as revacept, glinzocimab or caplacizumab; Fibrinogen receptor antagonists (glycoprotein-IIb / IIIa antagonists), such as abciximab, eptifibatide, tirofiban, lamifiban, lefladafiban, furadafiban, zarunfiban; · recombinant human activated protein C, e.g., Xigris, recombinant thrombomodulin, e.g., ART-123, or recombinant antithrombin-III; Neuroprotective approaches, e.g. NMDA receptor antagonists, PSD-95 inhibitors, tissue kallikrein preparations, endothelin-B agonists, PARP inhibitors, Anti-inflammatory compounds, such as glucocorticoids (e.g. prednisolone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, beclomethasone, betamethasone, flunisolide, budesonide or fluticasone), non-steroidal anti-inflammatory drugs (NSAIDs) (e.g. acetylsalicylic acid (aspirin), ibuprofen or 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). Anti-infective approaches, for example preferably antibacterial drugs (e.g. penicillin, vancomycin, ciprofloxacin, mupirocin, azithromycin and metronidazole), antifungal drugs (e.g. naftifine, nystatin) and / or antiviral drugs and / or antiparasitic drugs. Additional intensive care, e.g. fluid therapy (e.g. crystalloid or colloid fluids); vasopressors (e.g. norepinephrine, dopamine or vasopressin); inotropic therapy (e.g. dobutamine); blood products (e.g. packed red blood cells, platelet concentrates, erythropoietin or fresh frozen plasma); assisted ventilation in sepsis-induced acute lung injury (ALI) or acute respiratory distress syndrome (ARDS; e.g. permissive hypercapnia, hypovolemia); sedatives (e.g. diazepam, lorazepam, midazolam or propofol); glycemic control, e.g. insulin, glucose, renal replacement therapy (e.g. continuous venovenous hemofiltration or intermittent hemodialysis). Mast cell stabilizers (nedocromil, cromolyn sodium), tryptase inhibitors, cathepsin G inhibitors or elastase inhibitors Antihistamines, such as cetirizine, loratadine, and astemizole.

[0194] "Combination" for the purposes of the present invention means not only dosage forms containing all components (so-called fixed combinations) and combination packs containing components separately from each other, but also components that 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 each other, thus meaning that they are binary or multi-component combinations, respectively.

[0195] The compounds of the present invention can act systemically and / or locally.To this end, they can be administered in an appropriate manner, for example by the oral, parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal, cutaneous, transdermal, conjunctival or auricular route, or as an implant or stent.

[0196] The compounds of the present invention can be administered in dosage forms suitable for these administration routes.

[0197] Dosage forms suitable for oral administration are those which function according to the prior art and deliver the compound of the invention rapidly and / or in a modified manner and contain the compound of the invention in crystalline and / or amorphous and / or dissolved form, such as tablets (uncoated or coated, e.g. with an enteric coating or an insoluble or slow-dissolving coating to control the release of the compound of the invention), tablets which disintegrate rapidly in the mouth, or films / wafers, films / lyophilisates, capsules (e.g. hard or soft gelatin capsules), dragees, granules, pellets, powders, emulsions, suspensions, aerosols or solutions.

[0198] Parenteral administration can be achieved by avoiding the absorption step (e.g., by intravenous, intraarterial, intracardiac, intraspinal or intralumbar routes) or by including the absorption step (e.g., by intramuscular, subcutaneous, intradermal, transdermal or intraperitoneal routes). Suitable dosage forms for parenteral administration include preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophilisates or sterile powders. For local parenteral administration, a catheter-based approach for intraclotal administration can be used.

[0199] Oral administration is preferred.

[0200] Dosage forms suitable for other administration routes are, for example, pharmaceutical forms for inhalation (including powder inhalers, nebulizers), nasal drops, solutions or sprays; tablets, films / wafers or capsules for lingual, sublingual or buccal administration, suppositories, ear or eye preparations, vaginal capsules, aqueous suspensions (lotions, shaking mixtures), lipophilic suspensions, ointments, creams, transdermal therapeutic systems (e.g. patches), milks, pastes, foams, powders, implants or stents.

[0201] The compound of the present invention can be converted into the above-mentioned dosage form.This can be achieved by mixing with inert, non-toxic, pharma- ceutically suitable excipients in a manner known per se.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 dodecyl sulfate, polyoxysorbitan oleate), binders (e.g., polyvinylpyrrolidone), synthetic and natural polymers (e.g., albumin), stabilizers (e.g., antioxidants, e.g., ascorbic acid), colorants (e.g., inorganic pigments, e.g., iron oxide), and flavor and / or odor correctors.

[0202] The present invention further provides medicaments comprising at least one compound of the invention, preferably together with one or more inert, non-toxic pharma- ceutically suitable excipients, and the use thereof for the above-mentioned purposes.

[0203] In general, in the case of parenteral administration, it has been found advantageous to administer an amount of about 0.001 to 1 mg per kg of body weight, preferably about 0.01 to 0.5 mg per kg of body weight, to achieve effective results. In the case of oral administration, the dosage is about 0.01 to 100 mg per kg of body weight. Preferably, the daily oral dosage is about 1 mg to 100 mg, most preferably 2 mg to 50 mg. Notwithstanding this, it may be necessary to deviate from the amounts specified, in appropriate cases, in particular depending on the body weight, the route of administration, the individual behavior towards the active ingredient, the type of formulation, and the time or interval of administration.

[0204] Unless otherwise stated, the percentages in the following tests and examples are percentages by weight and the parts are parts by weight. Solvent ratios, dilution ratios and concentration data for liquid / liquid solutions are in each case based on volume. "w / v" means "weight / volume". For example, "10% w / v" means that 100 ml of solution or suspension contains 10 g of substance. EXAMPLES

[0205] Experimental Method 1. Abbreviation AA - abdominal aorta ACN - Acetonitrile ACS - Acute Coronary Syndrome AMC - Aminomethylcoumarin ANOVA - analysis of variance ARDS - Acute Respiratory Distress Syndrome BSA - Bovine Serum Albumin C57BL / 6 - Mouse strain C57 Black 6 cGMP - cyclic guanosine monophosphate CMA1 - Chymase 1 CNS - Central Nervous System Ctr - Control DAB - 3,3'-diaminobenzidine DIC - Disseminated intravascular coagulation DMSO - Dimethyl Sulfoxide DTI - Direct Thrombin Inhibitors DTT - Dithiothreitol DVT - Deep Vein Thrombosis EDTA - Ethylenediaminetetraacetic acid EtOH - Ethanol FA - Formic Acid FDR - False Discovery Rate HEPES - (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) HPLC - High Performance Liquid Chromatography im - intramuscular iv. - Intravenous IVC - inferior vena cava KO - Knockout LC - Liquid Chromatography LFQ - Label-Free Quantitation LMW - Low Molecular Weight Heparin MCP-4 - Mast cell protease 4 MES - 2-(N-Morpholino)ethanesulfonic acid MS - mass spectrometry NEM-N - Ethylmaleimide non-STEMI - myocardial infarction without ST-segment elevation PAI - Plasminogen Activator Inhibitor PBS - Phosphate Buffered Saline PEG - Polyethylene glycol PSM - Peptide Spectral Match PVP - Polyvinylpyrrolidone rCMA-1 - recombinant human chymase 1 rmMCP-4 - recombinant mouse mast cell protease 4 rtPA - recombinant human tissue plasminogen activator SEM - Standard Error of the Mean SLPI - Secretory Leukocyte Protease Inhibitor STEMI - ST segment elevation myocardial infarction TAFI - Thrombin Activatable Fibrinolysis Inhibitor TCA - Trichloroacetic acid TFA - Trifluoroacetic acid TIA - Transient Ischemic Attack tPA - tissue plasminogen activator UFH - Unfractionated Heparin uPA- urokinase

[0206] 2. In vitro potency and selectivity evaluation of chymase inhibitors The suitability of the compounds according to the invention for treating thromboembolic disorders by inhibition of chymase can be demonstrated in the following assay system:

[0207] 2.1 Enzyme chymase activity assay Recombinant human chymase protein and recombinant hamster chymase (Protein Technologies, Bayer Healthcare) were used. Cleavage of the substrate Abz-HPFHL-Lys(Dnp)-NH2 (10 μM) by either 3 nM recombinant human chymase or 1.2 nM recombinant hamster chymase in assay buffer Tris 50 mM (pH 7.5), NaCl 150 mM, BSA 0,10%, Chaps 0,10%, glutathione 1 mM, EDTA 1 mM was monitored in a fluorescence reader (excitation 340 nm, emission 465 nm). Data were normalized (enzyme reaction without inhibitor = 0% inhibition, all other assay components without enzyme = 100% inhibition). Typically, test compounds were tested on the same microtiter plate at nine different concentrations ranging from 10 μM to 1 nM (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). 50 was calculated using in-house software.

[0208] result Compounds related to the present invention are those shown in Table 1 50 It potently inhibited chymase activity.

[0209] Table 1: Chymase inhibitor potency [Table 1]

[0210] 2.2 Determining selectivity The selectivity of the substances was demonstrated with respect to inhibition of cathepsin G and other proteases (eg in the coagulation and fibrinolysis fields).

[0211] Recombinant human cathepsin G (CathG) protein (Protein Technologies, Bayer AG) was used. Cleavage of the substrate DABCYL-Ser-Thr_Leu-Ser-Glu-Lys_Ala_Lys_Pro_Ala-Glu (EDANS) (3 μM) in assay buffer Hepes 100 mM (pH 7.0), NaCl 50 mM, BSA 0,01%, Tween 20 0,001%, glutathione 0.3 mM and recombinant CathG (2.5 nM) was monitored in a fluorescence reader (excitation 334 nm, emission 536 nm). Data were normalized (enzyme reaction without inhibitor = 0% inhibition, all other assay components without enzyme = 100% inhibition). Typically, test compounds were tested on the same microtiter plate at nine different concentrations ranging from 10 μM to 1 nM (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). IC50 values ​​were calculated using in-house software.

[0212] The potency of test compounds against several other human serine proteases, including coagulation proteases, in buffer was analyzed in a biochemical assay based on fluorometric detection of aminomethylcoumarin (AMC) cleaved from specific synthetic peptide substrates by the respective proteases.

[0213] The serine protease assay consisted of the following enzymes and substrates (final assay concentrations indicated): Active proteases, typically purified from human plasma, and the corresponding substrates are commercially available.

[0214] Thrombin (Kordia; 0.02 nM), Boc-Asp(OBzl)-Pro-Arg-AMC (Bachem I-1560; 5 μM).

[0215] · Factor Xa (Kordia; 1.3nM), Boc-Ile-Glu-Gly-Arg-AMC (Bachem I-1100; 5μM).

[0216] · Factor XIa (Kordia; 0.15nM), Boc-Glu(OBzl)-Ala-Arg-AMC (Bachem I-1575; 5μM) · Plasmin (Kordia; 0.1 μg / ml, 1.2 nM), MeOSuc-Ala-Phe-Lys-AMC (Bachem I-1275; 50 μM).

[0217] Tissue plasminogen activator (tPA; Loxo; 1 nM), CH3SO2-D-Phe-Gly-Arg-AMC (Pentapharm 091-06; 5 μM).

[0218] Plasma kallikrein (Kordia; 0.2 nM), H-Pro-Phe-Arg-AMC (Bachem I-1295; 5 μM).

[0219] · Trypsin (Sigma; 0.042 U / ml), Boc-Ile-Glu-Gly-Arg-AMC (Bachem I-1100; 5 μM).

[0220] Test compounds were diluted in dimethyl sulfoxide (DMSO) to final assay concentrations of 0-50 μM. Each human serine protease and each substrate were diluted to the final concentrations indicated below in a buffer consisting of 50 mM Tris / HCl, 100 mM NaCl, 5 mM CaCl2, and 0.1% bovine serum albumin (pH 7.4). The diluted test compound or DMSO (1 μL) in 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). Fluorescence (excitation 360 nm, emission 460 nm) was measured for 30 min at room temperature using a microtiter plate fluorescence reader (Safire II, Tecan, Maennedorf, Switzerland). The 50% inhibition of serine protease activity (IC 50 ) was determined via a nonlinear logistic regression model.

[0221] result Compounds related to the present invention were selected against several other enzymes. IC 50 are shown in Table 2. Importantly, none of the coagulation or fibrinolytic system proteases 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 (Furasymstat) or TY-51469.

[0222] Table 2: Selectivity of chymase inhibitors [Table 2]

[0223] 3. Plasmin is a substrate for chymase in in vitro assays, and its degradation by chymase can be inhibited by chymase inhibitors To evaluate the effect of chymase on plasmin, plasmin activity in the presence of chymase was determined over time in mouse and human in vitro systems. Furthermore, the occurrence of potential plasmin degradation products was investigated for mouse and human plasmin in the presence of chymase in buffer systems.

[0224] 3.1. Plasmin activity in the presence of mouse and human chymase and the effect of chymase inhibitors Recombinant mouse mMCP-4 (rmMCP-4) or recombinant human (rCMA-1) were produced as proforms and activated with recombinant mouse cathepsin C (R&D Systems, Minneapolis, MN, USA) to make them enzymatically active as previously described (Semaan W et al., Biochem. Pharmacol., 94(2015):91-100). rmMCP-4 or rCMA-1 were 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 chymase and cathepsin C, adding 50 μg / mL heparin, and incubating at room temperature for 1 h. Chymase activation was stopped with 3 mM N-ethylmaleimide (NEM) followed by dilution in assay buffer (20 mM Tris, 2 M KCl, 0.02% (v / v) Triton X-100, pH 9.0) to a recombinant chymase concentration of 2 μg / mL; 5 min was sufficient to completely stop the cathepsin C-dependent reaction.

[0225] 37 nM rmMCP-4 or rCMA-1 were preincubated with either vehicle or TY-51469 (final concentration 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 (Furasymstat) (concentrations 0.1–2.5 μM) for 20 min at 37°C, 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 rate of hydrolysis of 50 μM of the substrate, D-Ala-Leu-Lys-7-amido-4-methylcoumarin (Sigma Aldrich, Saint-Louis, MO, USA), combined in a white 96-well microtiter plate for 1 h at 37° C. The fluorescence emitted was expressed as λ ex = 370 nm and λ em = 460 nm on an Infinite M1000 spectrophotometer (Tecan Group Ltd., Maennerdorf, Switzerland). A standard curve of 7-amino-4-methyl-coumarin (AMC) was also obtained to estimate the concentration of fluorescent substrate cleavage (nM).

[0226] Figure 1. Kinetics of chymase-dependent inactivation of human (A) and mouse (B) plasmin and its 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 (furasymstat) or TY-51469.

[0227] Figure 2 Chymase-dependent inactivation of human (A) and mouse (B) plasmin is 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 (furasymstat) or TY-51469.

[0228] result Figure 1 shows the kinetics of recombinant human purified chymase (rCMA-1)-induced inactivation of human purified plasmin (Figure 1A) and recombinant mouse mMCP-4-induced inactivation of mouse purified plasmin (Figure 1B) 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 (furasymstat) (graph i) or TY-51469 (graph ii). Each point represents the mean ± SEM (n = 6-15). One-way analysis of variance with Dunnett's multiple comparison test was used to determine statistical significance: *p<0.05, **p<0.01, ***p<0.001.

[0229] Whereas plasmin, when exposed to the fluorogenic substrate alone, led to a strong and continuous increase in fluorescence over time, as a measure of enzyme activity, the presence of chymase led to a strong decrease in fluorescence generation, which was then increased again in a concentration-dependent manner by both chymase inhibitors, furasymstat and TY-51469, respectively. Figure 2 shows the maximum fluorescence obtained in each group at the end of the experiment (60 min).

[0230] These results surprisingly reveal that plasmin is a substrate for chymase in mice and humans and that its degradation and inactivation can be suppressed by chymase inhibitors in animals and humans, making chymase inhibitors suitable for thrombolysis, especially in the context of acute thrombotic and thromboembolic events.

[0231] 3.2 Plasmin is degraded by chymase, which can be inhibited by chymase inhibitors Mouse chymase (rmMCP-4) or human chymase (rCMA-1) was activated as described above. 74 nM rmMCP-4 or rCMA-1 was preincubated with either vehicle or chymase inhibitor TY-51469 (10 or 300 μM) for 20 min at 37° C., 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 of 4% (v / v) in water) and samples were kept on ice until protein precipitation.

[0232] In-solution tryptic digestion, purification and desalting of peptides on a C18 column Proteins from the in vitro cleavage assay 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 × 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. Protein alkylation was performed by adding iodoacetamide (Sigma-Aldrich, Saint-Louis, MO, USA) to a final concentration of 7.5 mM, followed by incubation at room temperature for 20 min away from light. The urea concentration was reduced to 2 M by adding 150 μL of 50 mM ammonium bicarbonate (NH4HCO3). Proteins were digested by adding 1 μg of 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). Peptides were purified on a Pierce C18 100 μL tip column (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, the C18 tip column was first wetted by aspirating 100 μL of 100% acetonitrile (ACN) three times, then equilibrated by aspirating 100 μL of 0.1% (v / v) TFA buffer three times. Each peptide sample was passed through the equilibrated C18 tip column by pumping 100 μL of the sample up and down ten consecutive times. This step was performed twice to load the entire sample onto the column. The C18 tip column was then washed three times with 100 μL of 0.1% (v / v) TFA buffer. Elution of peptides was performed in a new low-binding microtube by successively pumping up and down 10 times with 100 μL volumes of 50% (v / v) ACN and 1% (v / v) FA buffer.This step was performed three times to obtain a final volume of 300 μL. The peptides were then concentrated by centrifugal evaporator at 65° C. until completely dry (approximately 60 min) and then resuspended in 25 μL of 1% (v / v) FA buffer. The peptides were assayed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and read at an absorbance of 205 nm. The peptides were then transferred to glass vials (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −20° C. until analysis by mass spectrometry.

[0233] LC-MS / MS analysis Tryptic digested peptides were separated using a Dionex Ultimate 3000 nano HPLC system. Samples (2 μg total) in 10 μL of 1% (v / v) FA were loaded onto an Acclaim PepMap100 C18 column (0.3 mm id x 5 mm, Dionex Corporation) at a constant flow rate of 4 μL / min. After trap concentration, peptides were eluted on an EasySpray PepMap C18 nano column (75 μm x 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 survey spectra (m / z 350-1600) in profile mode were acquired in an Orbitrap at a resolution of 70,000 after accumulation of 1,000,000 ions. The 10 most intense peptide ions from the preview scan in the Orbitrap were fragmented by collision-induced dissociation (normalized collision energy 35% and resolution 17,500) after accumulation of 50,000 ions. Maximum fill times were 250 ms for full scans and 60 ms for ms / ms scans. Precursor ion charge state screening was enabled, rejecting all unassigned charge states as well as singly, seventh, and eighth charged species. The dynamic exclusion list was limited to a maximum of 500 entries with a maximum hold time of 40 s and a relative mass window of 10 ppm. The lock mass option was enabled for the survey scan to improve mass accuracy. Data were acquired using Xcalibur software (Thermo Fisher Scientific, Waltham, MA, USA).

[0234] Protein identification by MaxQuant analysis Raw files were analyzed using MaxQuant software (version 1.6.17.0) and the Uniprot mouse proteome database (07 / Mar / 2021, 55 366 entries) or the Uniprot human proteome database (21 / Mar / 2020, 75 776 entries), which includes the mouse plasminogen database (UniProtKB-P20918(PLMN_MOUSE)) and the human plasminogen database (UniProtKB-P00747(PLMN_HUMAN)). The settings used for MaxQuant analysis were as follows: 4 miscleavages allowed; minimum peptide length was 6, fixed modification was carbamidomethylation on cysteines; enzymes were trypsin (K / R) and CMA1 (F / Y); variable modifications included in the analysis were methionine oxidation, protein N-terminal acetylation and protein carbamylation (K, N-terminus). A mass tolerance of 10 ppm was used for precursor ions and 20 ppm for fragment ions. Identification values ​​"PSM FDR", "Protein FDR" and "Site decoy fraction" were set to 0.05. The minimum number of peptides was set to 1. Label-free quantification (LFQ) was also selected with an LFQ minimum ratio count of 1. A "second peptide" option was also allowed. Following the analysis, the results were sorted according to several parameters. Proteins positive for at least one of the "Reverse", "Only.identified.by.site" or "Potential.contaminant" categories were excluded. Plasmin / plasminogen amino acid numbering was according to methionine at position 1.

[0235] Figure 3. Human (A) and mouse (B) plasmin are substrates for chymase, and degradation of the latter by the protease is 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 (Furasymstat) or TY-51469.

[0236] result: Cleavage product identification by LC-MS / MS revealed several cleavage sites in the plasmin sequence caused by chymase, which are shown with intensities in Figure 3A for human plasmin and in Figure 3B for mouse plasmin (arbitrary units, each bar represents the mean ± SEM; n = 4).

[0237] 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 the absence of these cleavages by plasmin alone, while grey arrows indicate their absence when a chymase inhibitor is added to plasmin and chymase, thereby demonstrating that the identified cleavage sites are caused by chymase.

[0238] 4. Inhibition of chymase activity via gene knockout or pharmacological chymase inhibition in vivo effectively and dose-dependently reduces thrombus weight and improves blood flow in stenosis models As chymase is generally rapidly inactivated in plasma, it was unclear whether the plasminolytic properties of chymase are pathophysiologically relevant and result in reduced fibrin degradation in vivo. We therefore addressed the question of whether chymase inhibition affects clot lysis and thereby vascular patency by keeping plasmin activity intact in vivo. Venous stenosis experiments were performed in mMCP4 knockout mice or mice and hamsters treated with the chymase 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 (furasymstat) or TY-51469 and compared with untreated or vehicle-treated animals.

[0239] By varying the time point of chymase inhibitor administration in relation to the onset of stenosis, we investigated its impact in thromboprophylaxis and thrombotherapy settings.

[0240] 4.1 Thrombus measurement in a mouse venous stenosis model Constriction of the inferior vena cava (IVC) 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). Laparotomy was performed and the intestine was gently displaced without damage from the abdominal cavity and stored in 0.9% NaCl solution (37 °C). The IVC was exposed by separation of the vessels between the renal vein and the iliolumbar vein. For partial ligation, a 30 G needle was used as a spacer. Neither the side nor the posterior branch of the IVC was ligated. After ligation with polypropylene 7.0 sutures (FST, Foster City, CA) around both the IVC and the spacer, the latter was gently removed. By doing this, it ensures that the endothelium is not denuded and that a residual flow of 10% in the IVC still remains. Afterwards, the intestines were returned to their original position in the abdomen and the abdominal cavity was closed in layers with braided 5.0 absorbable sutures. Analgesia protocol was maintained for 24 h after surgery with buprenorphine (0.1 mg / kg, subcutaneously every 6-9 h). The IVC was dissected after euthanasia and the thrombus was isolated, measured, weighed, and finally stored at -80 °C.

[0241] Under these conditions, thrombi weighing approximately 10 mg and measuring approximately 4-5 mm in length were observed in wild-type male animals.

[0242] The results in these "control animals" were compared to animals in which chymase activity was altered, either genetically (i.e., mMCP-4 KO - / -) or pharmacologically suppressed:

[0243] i) Knockout mice The mMCP-4 KO mice have been backcrossed with their C57BL / 6 homologues for over 10 generations and are highly homozygous for the latter strain (Tchougounova E. et al., J. Experiment. Med. 198 (2003) 423-431). The genotype of the mMCP-4 KO mice used in this study was confirmed by polymerase chain reaction.

[0244] ii) 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 (Furasimstat) dosing regime Mice were treated 1 or 24 h after ligation 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 (Furasimstat) (0.1, 1, or 3 mg / kg) as previously described. Stenosis was assessed 24 or 48 h after ligation. The IVC was dissected and the thrombus was isolated, measured, and weighed.

[0245] iii) TY-51469 administration regime 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 evaluated 24 or 48 hours after ligation. The IVC was dissected and the thrombus was isolated, measured and weighed.

[0246] Figure 4. Flasymustat or TY-51469 (i) inhibit or (ii) resolve DVT in IVC-ligated mouse (A) and hamster models (B).

[0247] result The results of thrombus weight and length measurements are shown in Figure 4A 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 (Furasymstat)) and Table 4 (results using TY-51469). These results show that inhibition of chymase activity in quorum animals and in animals treated with chymase inhibitors resulted in a dose-dependent reduction in thrombus compared with control animals (n = 6–13; each point represents data from one animal; columns represent mean ± standard error of the mean; one-way ANOVA followed by Bonferroni's multiple comparison test or Mann-Whitney T test, *P < 0.05, **P < 0.01, ***P < 0.001).

[0248] Graph 4Ai) shows thrombus weight measured 24 hours after the start of stenosis in mMCP4 knockout mice and animal groups treated with different doses of either 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 (Furasimstat) (administered orally) or TY-51469 (administered intraperitoneally) 1 hour after the start of the inferior vena cava (IVC) stenosis. Statistically significant effects on thrombus weight in the early period after stenosis (administered 1 hour after ligation) were observed with both compounds in a dose-dependent manner. Administration of the compound 24 hours after the initiation of stenosis with thrombus harvest 48 hours later (Graph 4Aii) showed a similarly strong effect, resulting in almost complete thrombus reduction.

[0249] Surprisingly, under conditions of stenosis of the inferior vena cava, a single administration of a chymase inhibitor leads to a reduction in thrombus weight and length not only in a prophylactic setting, i.e., administration of the compound before the onset of clot-induced blood flow reduction, but also in a clear intervention setting, i.e., 24 hours after the onset of thrombus formation. These results confirm the general character of these findings by comparing two structurally different chymase 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 (furasymstat) and TY-51469, with well-established markers. These results were obtained using only 1-(3-methyl-2-oxo-3,3-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 (furasimstat) in a newly developed mouse DVT model (both animal models are constrictions of the inferior vena cava). Achieving these effects with two unrelated chymase inhibitors from different structural classes demonstrates that this is not a compound-specific finding, but is related to chymase inhibition in general.

[0250] Table 3: Effect of furasymstat on thrombus weight and length in a mouse venous stenosis model. [Table 3] One-way ANOVA followed by Bonferroni's multiple comparison test or Mann-Whitney T-test. (*) symbol represents statistical significance between vehicle and Fula treatment. *P<0.05, **P<0.01, ***P<0.001. Error bars: standard error of the mean (±SEM).

[0251] Table 4: Effects of mMCP-4 knockout and TY-51469 on thrombus weight and length in a venous stenosis model in mice. [Table 4] One-way ANOVA followed by Bonferroni's multiple comparison test or Mann-Whitney T-test. (*) symbols represent statistical significance between control and TY treatment. *P<0.05, **P<0.01, ***P<0.001. Error bars: standard error of the mean (±SEM).

[0252] 4.2 Thrombus measurement in a hamster venous stenosis model To ensure that the surprising effects in the mouse venous stenosis model were not species specific, additional experiments were performed in a novel hamster venous stenosis model described below.

[0253] Constriction of the inferior vena cava (IVC) was achieved by anesthetizing hamsters weighing 108–120 g with a mixture of isoflurane and oxygen (2%; 2.5%, respectively). A laparotomy was performed, and the intestine was gently displaced without damage from the abdominal cavity and stored in 0.9% NaCl solution (37 °C). The IVC was exposed by separation of the vessels between the renal vein and the iliolumbar vein. An A27G (circumference 0.4 mm) needle was used as a spacer. Neither the side nor the posterior branch of the IVC was ligated. After ligation with polypropylene 7.0 sutures (FST, Foster City, CA) around both the IVC and the spacer, the latter was gently removed. The intestine was then returned to its original position in the abdomen, and the abdominal cavity was closed in layers with braided 5.0 absorbable sutures. An analgesic protocol with buprenorphine (0.1 mg / kg, subcutaneously every 6–9 h) was maintained for 24 h after surgery. The thrombus was evaluated 24 or 48 hours after stenosis: the IVC was dissected and the thrombus was isolated, measured, and weighed.

[0254] Hamsters were treated 1 or 24 h after the initiation of stenosis with a single oral dose of vehicle (10% EtOH, 40% solute, 50% sterile water) or furasymstat (0.01, 0.1, and 1 mg / kg given 1 h after the initiation of stenosis and 1 mg / kg given 24 h after the initiation of stenosis) as described above.

[0255] result: The results of thrombus weight and length measurements are shown in FIG. 4B and summarized in Table 5, which show that inhibition of chymase activity in hamsters treated with the chymase 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 (furasymstat) resulted in a dose-dependent reduction in thrombus at higher doses in the early post-stenosis setting (administered 1 hour after the onset of stenosis, harvested 24 hours later; graph 4Bi) compared to control animals. Administration of 1 mg / kg furasymstat 24 hours after the onset of IVC stenosis, with thrombus harvested 48 hours later (graph 4Bii), showed a similarly robust effect, resulting in complete removal of the thrombus.

[0256] The venous stenosis model in hamsters compared to the mouse venous stenosis model yielded very similar results, thus demonstrating that thrombosis reduction by chymase inhibition is not a species-specific phenomenon.

[0257] Of note, in this case, thrombus size in the vehicle group harvested at 24 h was very similar in weight and length to the vehicle group with thrombus harvested at 48 h, thus demonstrating that 24 h after furasymstat was administered, thrombi had already reached a certain size (n=6-8; mean ± standard error of the mean; one-way ANOVA followed by Bonferroni's multiple comparison test or Mann-Whitney T-test, *P<0.05, **P<0.01, ***P<0.001).

[0258] Table 5. Effect of furasymstat on thrombus weight and length in a hamster venous stenosis model [Table 5] One-way ANOVA followed by Bonferroni's multiple comparison test or Mann-Whitney T-test. *P<0.05, **P<0.01, ***P<0.001. Error bars: standard error of the mean (±SEM).

[0259] 5. Direct chymase inhibition improves blood flow in a mouse venous stenosis model The main goal in thrombosis is to ensure sufficient blood flow for proper maintenance of perivascular tissue. To evaluate whether the thrombus reduction by chymase inhibition described in 4.1 and 4.2 is associated with increased patency of the inferior vena cava (IVC) compared to controls, blood flow in the inferior vena cava (IVC) and abdominal aorta (AA) was visualized by Doppler color blood flow mapping at different time points in the venous stenosis model, as described in 4.1, using a high-frequency ultrasound imaging system (Vevo 3100; Fujifilm VisualSonics Inc.) equipped with an MX400 linear array transducer (40 MHz). During the procedure, mice were anesthetized (2% isoflurane; 4% oxygen), their abdomens were shaved and covered with ultrasound gel. Measurements of blood flow in the vessels were performed 12 h before ligation and 6 and 24 h after ligation, 1 h after the onset of stenosis in control mice and mice administered 10 mg / kg TY-51469 intraperitoneally.

[0260] result The results of blood flow velocity analysis in the inferior vena cava (IVC) and abdominal aorta (AA) measured before, 6 hours after, and 24 hours after the initiation of stenosis in control animals and animals treated with TY-51469 1 hour after the initiation of stenosis are summarized in Table 6.

[0261] These results show that while a complete loss of blood flow was observed in control IVC-ligated animals, blood flow in the vena cava was restored in chymase inhibitor-treated animals already 6 hours after the onset of stenosis. The results demonstrate that chymase inhibition not only results in a reduction of thrombus but also has a clear effect on blood flow in the injured vessels.

[0262] Table 6. Blood flow velocity 1 hour after the start of stenosis in mouse IVC and AA+ / -TY-51469 [Table 6] One-way ANOVA with Bonferroni's multiple comparison test. *P<0.05, ***P<0.001. Error bars: standard error of the mean (±SEM).

[0263] 6. Reduction of chymase activity in vivo via a direct chymase inhibitor eliminates pulmonary embolism formation in mouse and hamster stenosis models A very important question for any thrombolytic therapy is whether the disruption of thrombi leads to the generation of emboli that can be transported to the lungs and cause pulmonary embolism. To address the question of whether chymase inhibition affects embolization, lungs were isolated from mice and hamsters with or without administration of fluroxime and from venous stenosis experiments. The right lung lobe was used to visually quantify the number of embolic lungs.

[0264] Figure 5 - Reduction in the number of pulmonary emboli in the lungs of mice treated with Flasymstat Figure 6 - Reduction in the number of pulmonary emboli in the lungs of hamsters treated with Flasymstat

[0265] result: The results of the macroscopic analysis of the lungs are shown in FIG. 5 (mice) and FIG. 6 (hamsters) and summarized in Table 7.

[0266] Figures 5A and 6A show randomly selected macroscopic images of right lung lobes extracted from five vehicle- or five 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 (furasymstat)-treated mice or hamsters (average 6–10 mice / group). Pulmonary thromboemboli are indicated by black arrows and were observed in vehicle-treated mice or hamsters in the top panels of Figures 5A and 6A, respectively. Right lung lobes from mice and hamsters orally administered 3 mg / kg (mice) and 1 mg / kg (hamsters) of furasymstat 24 h after venous stenosis are shown in the bottom panels of Figures 5A and 6A. They demonstrated normal pulmonary anatomy and no signs of pulmonary embolism 24 hours after induction of stenosis.

[0267] Part B of both figures summarizes in pie charts the incidence of pulmonary embolism in vehicle-treated mice (Figure 5) and hamsters (Figure 6) in the stenosis model 24 or 48 h after the initiation of stenosis. Embolism was observed in 30-50% of vehicle animals.

[0268] Mice treated with 3 mg / kg furasymstat (FIG. 5) and hamsters treated with 1 mg / kg furasymstat (FIG. 6) showed no signs of embolism at any time point, either when dosed 1 hour after the start of stenosis and read at 24 hours, or when dosed 24 hours after the start of stenosis and read at 48 hours.

[0269] Thus, the profibrinolytic approach by chymase inhibition not only did not show an increase in emboli compared to control animals, but surprisingly also led to a strong reduction in pulmonary emboli.

[0270] The vehicle data after 24 hours may suggest that emboli may already be present in the animals 24 hours after the onset of stenosis, prior to treatment with Flasymstat. The finding in both species that no emboli were observed in treated mice and hamsters after 48 hours suggests, to our surprise, that chymase inhibition may not only reduce thrombi in the local environment of their formation, as shown for thrombi in the vena cava, but may also reduce emboli.

[0271] Surprisingly, when examining the lung in vein (IVC) stenosis experiments in mice and hamsters, a reduction in the number of pulmonary emboli in the lungs of animals under chymase inhibition was observed compared to control animals. This very important and relevant case of thromboembolism implies that chymase not only has a role at the local site of thrombus formation, but also has an antifibrinolytic effect in emboli. Therefore, chymase inhibitors are effective drugs for the treatment of embolic diseases, including, for example, dissolution of emboli from deep veins causing pulmonary embolism and from the left atrial appendix in patients with atrial fibrillation causing stroke or systemic embolism.

[0272] Table 7. Effect of furasimstat on the incidence of pulmonary embolism in a venous stenosis model [Table 7] One-way ANOVA followed by Bonferroni's multiple comparison test. *P<0.05, error bars: standard error of the mean (±SEM).

[0273] 7. Chymase inhibitors inhibit thrombosis models of vessel wall injury (FeCl 3 Effectively and dose-dependently reduces thrombus weight in a thrombus-induced injury model To exclude that the effect of chymase inhibition is limited to the stenosis model, we addressed the question of whether chymase inhibition could prevent thrombus formation induced by vessel wall injury in a short-term ferric chloride-induced injury model in mice and hamsters.

[0274] 7.1 Mouse Inferior vena cava Ferric chloride-induced damage in 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 either vehicle, 10 mg / kg furasymstat or 10 mg / kg TY-51469 1 h prior to anesthesia of the animals, whereas mMCP-4 knockout animals were not treated prior to anesthesia. The endothelial injury model of the inferior vena cava (IVC) was achieved by anesthetizing mice with ketamine / xylazine (87 / 13 mg / kg IM). Laparotomy was performed and the intestine was gently displaced without damage from the abdominal cavity and stored in 0.9% NaCl solution (37 °C). The IVC was exposed by separation of the vessels between the renal and iliolumbar veins. A 2 × 2 mm double-layered absorbent gauze saturated with 0.37 M (10% w / v in sterile water) ferric chloride hydrate (FeCl3) (Sigma-Aldrich, St-Louis, MO, USA) was applied directly onto the IVC for 3 min and then removed. The intestine was then returned to its original position in the abdomen. Mice were sacrificed 30 min later to access the thrombus area of ​​the IVC. The IVC was dissected and the thrombus was isolated and weighed on an analytical balance (0.1 mg precision, Entris II, Sartorius).

[0275] Figure 7 Chymase inhibition reduces thrombus weight in FeCl3-induced injury models in the inferior vena cava (IVC) in mice (A) or hamsters (B) and in the femoral vein (hamster, C).

[0276] result The results of the thrombus measurements are shown in Figure 7A and summarized in Table 8. They show 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 (Furasimstat) and intraperitoneal administration of 10 mg / kg TY-51469 prior to injury, thus in a prophylactic setting, or mMCP-4 KO, resulted in a statistically significant reduction in thrombus compared to control animals.

[0277] Table 8. Effect of chymase inhibition on thrombus weight in FeCl3-induced injury thrombosis model in mouse inferior vena cava 1 [Table 8] One-way ANOVA followed by Bonferroni's multiple comparison test or Mann-Whitney T test. (*) symbol indicates statistical significance between vehicle and flasymstat administration. & ) Symbols represent statistical significance between control, TY-51469-treated and mMCP-4 KO mice. *P<0.05, **P<0.01, ***P<0.001. Error bars: standard error of the mean (±SEM).

[0278] 7.2 Ferric chloride-induced injury in the vena cava or femoral vein of hamsters The FeCl3-induced injury model in hamsters was heavily inspired by the methodology used in mice (section 7.1). Golden Syrian hamsters were orally treated with either 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 (Furasimstat) 1 h prior to anesthetization of the animals.

[0279] Ferric chloride-induced injury in the vena cava Endothelial injury of the inferior vena cava (IVC) was achieved by anesthetizing hamsters, weighing 115–130 g, with ketamine / xylazine (87–200 / 13 mg / kg IM). The animals were kept at a body temperature of 37 °C using a heating pad during the experiment. A laparotomy was performed and the digestive tract was gently removed without damage from the abdominal cavity and preserved in 0.9% NaCl solution (37 °C). The IVC was then exposed by separation of the vessels between the renal and iliolumbar veins. A 2 × 2 mm bilayer absorbent gauze saturated with 0.37 M (10% w / v in sterile water) ferric chloride hydrate (FeCl3) (Sigma-Aldrich, St-Louis, MO, USA) was applied directly onto the IVC for 3 min and then removed. The intestine was then returned to its original position in the abdomen. The hamsters were sacrificed 30 min later to localize the thrombus area within the injured IVC.

[0280] Ferric chloride-induced injury in the femoral vein The femoral vein (FV) endothelial injury model was achieved by anesthetizing 115–130 g hamsters with ketamine / xylazine (87–200 / 13 mg / kg IM). The animals were kept at a body temperature of 37 °C using a heating pad during the experiment. The left femoral vein was exposed after skin incision and separation from the femoral artery. A 2 × 8 mm double-layered absorbent gauze saturated with 3 μl of 5% ferric chloride hydrate (Sigma-Aldrich, St-Louis, MO, USA) dissolved in water (5% w / v in sterile water) was applied directly to the femoral vein for 5 min and then removed. Hamsters were sacrificed 30 min later and the femoral vein was dissected.

[0281] Clot harvesting and characterization Relevant segments of the inferior vena cava and femoral vein were dissected and the thrombus was isolated and weighed on an analytical balance (Sartorius).

[0282] result The results of the thrombus measurements are shown in Figure 7B and Figure 7C, respectively, and summarized in Table 9, which show that reduction of chymase activity by 3 and 10 mg / kg 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 (Furasimstat) 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, thereby indicating that this effect of chymase inhibition is not limited to large vessels.

[0283] Table 9. Effect of chymase inhibition by furasymstat on thrombus weight in FeCl3-induced injury thrombosis model in inferior vena cava and femoral vein. [Table 9] One-way ANOVA followed by Bonferroni's multiple comparison test or Mann-Whitney T-test. *P<0.05, **P<0.01, ***P<0.001. Error bars: standard error of the mean (±SEM).

[0284] 7.3 Mouse and Hamster of carotid artery Ferric chloride-induced damage in To exclude that the effect of chymase inhibition is limited to venous thrombosis models, we addressed the question of whether chymase inhibition could prevent thrombus formation induced by arterial vessel wall injury in a short-term ferric chloride-induced carotid artery injury model performed in mice and hamsters.

[0285] Figure 13 - Flasymstat attenuates thrombus-induced occlusion in an FeCl3-induced injury model in the common carotid artery (CCA) of mice or hamsters. Each series of images is representative of six independent experiments.

[0286] Ferric chloride-induced injury of mouse carotid arteries 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 either vehicle, 10 or 20 mg / kg furasymstat 1 h prior to anesthesia.

[0287] The endothelial injury model of the common carotid artery (CCA) was performed by anesthetizing mice with ketamine / xylazine (87 / 13 mg / kg IM). A 1 cm vertical skin incision was made in the neck, and the right CCA and vagus nerve were exposed and isolated. A 2 × 2 mm double-layered absorbent gauze saturated in 0.28 M (7.5% w / v in sterile water) ferric chloride hydrate (FeCl3) (Sigma-Aldrich, St-Louis, MO, USA) was applied directly onto the exposed CCA for 1 min and then removed. The CCA was then returned to its original position near the trachea. Mice were sacrificed 30 min later. The chemically injured CCA and the intact contralateral CCA were dissected and stored in PFA (4% w / v) overnight and then in EtOH (70%) at 4 °C for subsequent histological analysis.

[0288] result Histology of the CCA after FeCl3 administration and quantification of thrombus area (Image J) are shown in Figure 13A and B, along with identification of the reference section for thrombus area measurement (the first section showing FeCl3-induced vascular injury). Each series of images is representative of six independent experiments. They show that oral administration of 10 or 20 mg / kg of furasymstat prior to injury resulted in a dose-dependent and statistically significant reduction in thrombus in mice compared to control animals (Figure 13C; one-way ANOVA followed by Bonferroni's 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)).

[0289] Ferric chloride-induced injury to the carotid artery of hamsters. The FeCl3-induced injury model in hamsters was heavily inspired by the methodology used in mice (section 7.3). Golden Syrian hamsters were orally treated with either 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 (Furasimstat) 1 hour prior to anesthetization of the animals.

[0290] Endothelial injury of the CCA was achieved by anesthetizing hamsters, weighing 115–130 g, with ketamine / xylazine (87–200 / 13 mg / kg IM). The animals were kept at a body temperature of 37 °C using a heating pad during 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 bilayer absorbent gauze saturated with 1.11 M (30% w / v ferric chloride hydrate (FeCl3) in sterile water (Sigma-Aldrich, St-Louis, MO, USA)) was applied directly onto the exposed CCA for 3 min and then removed (Kuo et al., J. Biomed. Sci, 2009). The CCA was then returned to its original position near the trachea. The hamsters were sacrificed 30 min later to access the thrombosed area of ​​the CCA. The chemically injured CCA and the intact contralateral CCA were dissected, and the thrombus was isolated and weighed with an analytical balance (precision 0.1 mg, Entris II, Sartorius).

[0291] result The results of the thrombus measurements are quantified in FIG. 13D. They show that oral administration of 10 mg / kg furasymstat prior to injury resulted in a statistically significant reduction in thrombus in hamsters compared to control animals. (One-way ANOVA followed by Bonferroni's multiple comparisons T-test. **P<0.01. Error type: standard error of the mean (± SEM)).

[0292] 7.4 Overall interpretation of the results of the FeCl3-induced injury model Mice and hamsters with reduced chymase activity, either by chymase inhibitors or by mMCP-4 knockout, had reduced thrombus weights in these models of venous and arterial vessel wall injury compared to vehicle control animals. This is surprising because, in contrast to the stenosis model, thrombi in this model are generated quickly after injury to the vessel wall and are already isolated after 30 minutes. It can therefore be considered as a thrombus generation model rather than a thrombolytic model. However, even under these conditions, the profibrinolytic effect of prophylactically administered compounds seems strong enough to balance the procoagulant effect of the growing thrombus.

[0293] Surprisingly, in contrast to the ferric chloride-induced injury experiments in mesenteric arterioles by Ponomaryov et al., the ferric chloride-induced injury experiments in different venous and arterial vessels described in this invention demonstrate significant differences in the groups treated with chymase inhibitors compared to the vehicle group. In these short-term thrombosis experiments, the compounds were given before the initiation of thrombus formation by ferric chloride, which means that even at the early stage of ongoing clot formation, the chymase inhibitors affect thrombus weight and thereby the risk of vascular occlusion.

[0294] 8. Chymase is present in thrombi generated in mice and in vivo studies in human thrombus specimens To address the question of whether chymase is present in thrombi in animal studies and in human thrombi, and thereby evaluate aspects of translatability towards the clinical situation, immunohistochemical experiments were performed.

[0295] Due to the lack of antibodies that bind to hamster chymase, experiments focused on thrombus from in vivo studies in mice.

[0296] 8.1 Immunohistochemical analysis of mouse chymase and perivascular mast cells in mouse thrombi Immunochemistry was performed on healthy ligated sections of the inferior vena cava of wild-type and mMCP-4 KO mice. Specimens were fixed in 10% buffered formalin solution, processed, embedded in paraffin, and finally sliced ​​(4 μm slices) at the service of the histopathology platform of the RI-MUHC (Montreal, Quebec, Canada). Toluidine blue (TB) was used to stain mast cells, mMCP-4 was detected using goat polyclonal anti-MCPT4 antibody (US Biological life science, Cat. No. M2414-20A, 1x400 dilution) and biotinylated horse anti-goat IgG (Vector Laboratories, Cat. No. BA9500, 1x200 dilution), and chymase was detected using goat polyclonal IgG against human MCPT4 (Abcam, Cat. No. ab111239, 1x200 dilution).

[0297] Figure 8 - Deep vein thrombosis showing immunoreactive chymase-containing mast cells in IVC-ligated wild-type mice. Each image represents three different tissue samples. result Representative images of immunochemical detection of chymase in thrombi obtained from an experiment are shown in Figure 8 (scale bar: 50 μm; black arrows in left panel: mast cells; black arrows in right panel: mMCP-4 immunoreactivity). Images are representative of three independent experiments.

[0298] Twenty-four hours after the initiation of stenosis, mMCP-4-specific immunoreactivity was localized peripherally and within the thrombus in ligated vena cava of wild-type animals (Fig. 8D), but not in thrombus-free ligated veins isolated from mMCP-4 KO mice (Fig. 8F) and in unligated, thrombus-free vessels from wild-type animals (Fig. 8B). In late specimens, the majority of mMCP-4-specific immunoreactivity was detected in perivascular mast cells. Meanwhile, toluidine blue-stained mast cells were identified in the adventitia of vena cava specimens from both WT and mMCP-4 KO mice (Fig. 8A, E).

[0299] 8.2 Immunohistochemical analysis of human chymase and mast cells in human specimens with deep vein thrombosis, pulmonary embolism, and popliteal artery occlusion To demonstrate that chymase is not only present in thrombi in animal experiments but also plays a role in the degradation of plasmin in human thrombi, specimens from human thrombotic events were evaluated for their chymase immunoreactivity.

[0300] Pre-prepared slides 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), whereas pulmonary embolism samples were obtained from the Department of Pathology, McGill University.

[0301] Toluidine blue staining of basophilic granules was used for identification and quantification of mast cells. This is an established method that produces a metachromatic magenta stain on a blue background. Staining solution was obtained from Newcomers Supply.

[0302] Immunochemical analysis was performed for identification of chymase in human samples. Deparaffinized and rehydrated tissue slides (4 microns) were placed on a Leica Bond Max automated staining system using the Bond Polymer Refine Detection kit (DS9800). HIER (heat-induced antigen retrieval) was performed with ER2 solution (AR9640) at 95°C for 20 minutes. Peroxidase block was then performed for 5 minutes. Primary antibody (mouse anti-human mast cell chymase antibody [CC1] (Abcam: 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. Counterstain: Hematoxylin was added for 2 minutes. Slides were rehydrated in graded ethanol (70%, 95%, and 100%), washed through three changes of xylene, then air-dried and mounted. Human tonsil tissue was used as a quality control.

[0303] Figure 9 - Human venous thrombosis, pulmonary embolism and arterial thrombosis showing immunoreactive chymase-containing mast cells. Each image is representative of three different tissue samples.

[0304] result Representative images of chymase-specific immunostaining in human specimens are shown in Figure 9 (dilution of human chymase antibody: 1 / 200; black arrows indicate mMCP-4 immunoreactivity). All images are representative of three independent experiments.

[0305] Chymase is located inside human deep vein thrombi (left iliac vein, right femoral vein, left inguinal varicose vein) as shown in the examples in Figure 9 A, inside pulmonary emboli as shown in Figure 9 B, and inside popliteal artery thrombi as shown in Figure 9 C. All samples also show chymase immunoreactivity and chymase-positive mast cells within the thrombi (black arrows).

[0306] These results show for the first time that chymase is present in human thrombi and therefore plays a role in the degradation of plasmin and has an antifibrinolytic effect in human pathology.Chymase inhibitors exert a profibrinolytic effect by keeping plasmin intact, resulting in rapid thrombolysis as shown in animal models.

[0307] 9. Chymase activity is present in thrombi and can be inhibited by chymase inhibitors, which leads to increased plasmin activity in the thrombi. Although the presence of chymase in thrombi could be verified, the effect of chymase in thrombi was not clear. Therefore, experiments were performed to measure the activity of chymase and plasmin in thrombus homogenates of control animals and animals treated with chymase inhibitors.

[0308] 9.1 Chymase inhibitor-treated thrombi show decreased intrathrombotic chymase activity and increased plasmin activity as determined ex vivo To demonstrate that thrombus reduction in animals treated with chymase inhibitors is associated with reduced chymase activity and increased plasmin activity in the thrombi, chymase and plasmin were extracted from mouse and hamster thrombi from the experiments described subsequently, and enzyme activity was measured in mice and hamsters.

[0309] Ex vivo chymase extraction from mouse and hamster thrombi The clots were 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×g for 30 min at 4° C. and the supernatant was discarded. We repeated this last step two more times, except for the final centrifugation, in which the supernatant was retained. The supernatant was kept at 4° C. until it was tested for chymase enzyme activity on the same day.

[0310] Ex vivo plasmin extraction from mouse and hamster thrombi The clots were 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 centrifuged at 100,000×g for 20 min at 4° C. 48 The supernatant was kept at 4°C until it was tested for plasmin enzyme activity on the same day.

[0311] Measurement of intrathrombotic plasmin or chymase enzyme activity 30 μL of supernatant from the clot extract was used and placed directly into a white 96-well plate. No variation in protein concentration per unit volume of clot was found by Bradford protein assay (results not shown). Some homogenates were treated with either 10 μM TY-51469 or furasymstat (volume of 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 (for chymase) was added to each well to achieve a final volume of 100 μL. Plasmin activity was determined by the rate of hydrolysis of 50 μM of the substrate, D-Ala-Leu-Lys-7-amido-4-methylcoumarin (Sigma Aldrich, Saint-Louis, MO, USA) at 37 °C for 1 h. Chymase activity was determined by the rate of hydrolysis of 10 μM of the substrate, Suc-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (Peptide Institute Inc., Osaka, Japan) at 37° C. for 1 h. The fluorescence emitted was expressed as λ ex = 370 nm and λ em = 460 nm on an Infinite M1000 spectrophotometer (Tecan Group Ltd., Maennerdorf, Switzerland). A standard curve of AMC was also obtained to estimate the concentration of fluorescent substrate cleavage (nM).

[0312] Figure 10 - Chymase inhibitors increase plasmin activity in clots from mice (A) or hamsters (B).

[0313] result The results of intrathrombus enzymatic activity of chymase or plasmin are shown in Figure 10 and summarized in Table 10. Plasmin and chymase activities were measured as AMC-specific cleavage (nM) of the fluorogenic substrate D-Ala-Leu-Lys-AMC (for the former) or Suc-Leu-Leu-Val-Tyr-AMC (for the latter enzyme), respectively, for 60 min. Each bar represents the mean ± SEM of a minimum of six experiments. **P<0.01, ***P<0.001.

[0314] Chymase activity was observed in mouse and hamster thrombi (Figures 10Ai and 10Bi, respectively). To the inventors' knowledge, this is the first time that chymase activity has been shown ex vivo in thrombotic material. These experiments demonstrate that, although chymase is known to be inactivated very rapidly in whole blood or plasma, in the local microenvironment between the vessel wall and the thrombus, chymase activity is surprisingly maintained intact. Chymase activity is almost completely reduced in thrombi treated with the chymase 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 (Furasimstat) or TY-51469 (right bars in Fig. 10Ai and Fig. 10Bi, respectively). Measurement of plasmin activity in the same thrombi demonstrated that after addition of chymase inhibitors, plasmin activity was strongly increased in both mouse and hamster thrombi (Fig. 10Aii, Fig. 10Bii), confirming the proposed mechanism of action.

[0315] Although rapidly inactivated in plasma, surprisingly, chymase remains active in the local microenvironment of the thrombus, and addition of a chymase inhibitor leads to a decrease in chymase activity and an increase in its substrate plasmin in this environment.

[0316] Table 10. Intrathrombus enzyme activities of chymase and plasmin in thrombi derived from venous stenosis experiments in mice and hamsters [Table 10] One-way ANOVA followed by Bonferroni's multiple comparison test or Mann-Whitney T-test. *P<0.05, **P<0.01, ***P<0.001. Error bars: standard error of the mean (±SEM).

[0317] Surprisingly, while it was generally believed that the main mechanism behind the mouse venous stenosis experiments of Ponomaryov et al. (Ponomaryov et al., CircRes.121(2017)941-950) was related to coagulation, the activity measurements of chymase and plasmin activity in thrombi removed from animals in our study reveal a strong link between chymase and the fibrinolytic system. The present invention demonstrated that chymase activity was decreased, but plasmin activity was increased in thrombi of animals treated with chymase inhibitors.

[0318] 9.2 Effect of chymase inhibitors on human blood clots in vitro (mod. “Halo assay”) To demonstrate that plasmin-dependent fibrinolysis of human thrombi is reduced by human chymase, the effect of this mast cell-derived serine protease was examined in halo-type human blood concentric clot formation.

[0319] Formation of halo-shaped blood clots containing human chymase Clot formation, based on the method of Bonnard et al. (Sci Rep. 2017 May 24;7(1):2346), is induced by adding 31.2 mM calcium chloride (CaCl2) to blood samples taken from healthy donors. 1 μl of human chymase was deposited on the bottom edge of the wells of a flat-bottomed transparent 96-well plate to a final concentration of 0.5 μM or 1 μM, in the presence or absence of 10 μM flasimstat (only for the latter concentration of rCMA-1). Then, 20 μl of blood spiked with CaCl2 was added in a circular motion around the edge of each well, leaving the center of the well empty, forming a halo-shaped clot (fluid agglutination action). The plate was then sealed and incubated at 37 °C for 30 min to allow the solidification of the blood halo.

[0320] Thrombolysis test using rtPA Recombinant human tissue plasminogen activator (rtPA, Cathflo®, Genentech Inc, SF, USA) induces clot fibrinolysis by activating endogenous plasmin. rtPA was diluted to a concentration of 1.5 nM in 80 ul of PBS, pH 7.4, and added to each well. The 96-well plate was then incubated at 37°C for 30 min. A negative control (A total ) was obtained by adding 80 μl of PBS without fibrinolytic agent, and the positive control (A zero ) corresponds to wells containing 20 μl fresh blood and 80 μl PBS without added CaCl2. The fibrinolysis rate was measured using a plate reader (Infinite M1000 spectrophotometer (Tecan Group LtdMaennerdorf, Switzerland)). The dissolution of the halo was measured in real time, as coverage of each experimental well with thrombolyzed blood increases the absorbance at 510 nm. The change in absorbance was measured every minute for 2 h at 37°C, after orbital shaking for 5 s at 195.3 rpm with an amplitude of 3.5 mm. The respective absorbances were calculated as Dx(t)=100(A x (t)-A zero (t)) / (A total (t)-A zeroThe percentage of decomposition is converted according to the formula (t).

[0321] Analysis of fibrinolysis kinetics In addition to real-time monitoring and plotting of blood degradation kinetics, other parameters were derived from the same curves: i) Activation time required for fibrinolysis induction (A t ) is D x It is defined as the first t-value for which (t) / dt>1. ii) The time required to obtain 50% dissolution (T0.5) is D x It is defined as the first t-value for which (t)=50%. iii) Maximum clot lysis rate (CLR) max ) is D x It corresponds to the maximum value of (t) / dt.

[0322] FIG. 11 Plasmin-dependent fibrinolysis of human clots is reduced by human chymase (CMA-1) in a flasymstat-sensitive manner.

[0323] result As shown in Figure 11, human chymase incorporated prior to CaCl2-induced coagulation significantly reduces tPA-induced fibrinolytic activity in human blood halo clots in a furasymstat-sensitive manner. Blood halos preincubated for 30 min with human chymase (0.5 μM and 1 μM) show a t-PA-dependent decrease in fibrinolysis in a concentration-dependent manner, which is reduced by furasymstat (Figure 11A). B) Fibrinolytic activation time; C) T0.5: time required to obtain 50% lysis; D) CLRmax: 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).

[0324] Normally, human chymase is rapidly inactivated in whole blood. However, when added to whole blood just before the initiation of clotting by recalcification, human chymase was activated within the clot and reduced clot dissolution in a concentration-dependent manner (Figure 11A). The effect of chymase was restored by the addition of furasymstat, which accelerated clot lysis. These observations complement the furasymstat-dependent enhancement of plasmin activity seen in mouse and hamster clot homogenates, as previously described in section 9.1.

[0325] Figures 14A-C show that human blood halo clots require activation of endogenous plasmin to be broken down. In additional control experiments, the role of endogenous factor Xa and plasmin in the induction or lysis of halo-type human clots in the absence of rCMA-1 was investigated in concentration-response experiments using rivaroxaban (Figure 14D) or the plasminogen / plasmin inhibitor, 10-chloro-4-(piperidin-4-yl)pyrimido[1,2-b]indazol-2(1H)-one hydrochloride (Example 3 in WO2015067549; Figures 14B, C and E). Each point represents the mean + SEM of at least six experiments. (*P<0.05, **P<0.01, ***P<0.001.)

[0326] Subsequent addition of human chymase resulted in the formation of a halo-shaped clot. Clot formation, based on the method of Bonnard et al. (Sci Rep. 2017 May 24;7(1):2346), is induced by adding 31.2 mM calcium chloride (CaCl2) to blood samples taken from healthy donors. 20 μl of CaCl2-spiked blood is deposited in the wells of a flat-bottomed transparent 96-well plate in a circular motion around the edge of each well, allowing the formation of a halo-shaped clot (fluid clumping effect) and emptying the center of the well. The plate is then sealed and incubated at 37 °C for 30 min to allow clotting of the blood halo. Alteplase (1.5 nM) and recombinant human chymase (final concentration 0.5 μM or 1 μM) are added simultaneously in the presence or absence of 10 μM furasymstat (only for the latter concentration of rCMA-1) (Figure 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) is co-administered with tPA in the presence of a preformed halo. The plate is incubated at 37° C. with lateral mixing (2 rpm) for 2 hours, after which the absorbance at 510 nm is measured.

[0327] result Normally, human chymase is rapidly inactivated in whole blood. However, as shown in FIG. 15B, human chymase incorporated after CaCl2-induced clotting significantly reduces tPA-induced fibrinolytic activity in human blood halo clots in a furasymstat-sensitive manner. Each point and bar corresponds to the mean ± SEM of 6–13 experiments (***P<0.001). The effect of chymase was restored by the addition of furasymstat, and clot lysis was accelerated.

[0328] 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 of WO2015067549) highly significantly reduced tPA-induced fibrinolysis of human blood halos (Figure 15B).

[0329] 10. Reduction of chymase activity in vivo via gene knockout or direct chymase inhibition does not affect bleeding times in animal models A very important drawback of current strategies to resolve thrombi is the increased bleeding risk, which not only limits the dose of rtPA that can be used, but also further limits the number of patients treated with such fibrinolytic approaches.Therefore, there is a clear need for novel thrombolytic strategies that do not affect hemostasis.To address the question of whether chymase inhibition affects hemostasis, bleeding time experiments with and without chymase inhibitors were performed in mice and hamsters.

[0330] 10.1 Effect of chymase inhibitors on tail bleeding time in mice Mice were anesthetized with a mixture of intramuscular (IM) ketamine / xylazine (87 / 13 mg / kg). 300 U / kg (via the jugular vein) of unfractionated heparin (as specified by the manufacturer) 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 (Furasimstat) (PO) were injected 1 hour prior to testing. DVT mice were evaluated 24 hours after ligation and with or without a single dose of TY-51469 (10 mg / kg) 1 hour after ligation. The tail was then fixed in a vertical position in a 2 mL tube filled with 1.5 mL of Drabkin's reagent (Sigma Aldrich Canada Co., Oakville, ON, Canada) (Saito MS et al., Int. J. Exp. Pathol. 97 (2016) 285-292). The tail was cut 3 mm from the tip. The time when bleeding stopped was recorded. After 30 min, all animals were euthanized.

[0331] result The results of the bleeding time experiments are shown in FIG. 12A and summarized in Table 11, which show that chymase inhibition 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 (Furasimstat) or TY-51469 did not affect bleeding time at doses that were highly effective in thrombolysis. No prolongation of bleeding time was observed even in mMCP-4 KO animals that are completely devoid of chymase. In contrast, heparin, as a typical control compound in bleeding models, led to a significant increase in bleeding time. Each bar represents the mean value ± SEM of six mice. *P<0.05, **P<0.01, ***P<0.001 compared to control-treated animals.

[0332] Table 11: Effect of chymase inhibition on bleeding time in a mouse tail bleeding model [Table 11] One-way ANOVA with Bonferroni's multiple comparison test. ***P<0.001. Error bars: standard error of the mean (±SEM).

[0333] FIG. 12 - Flasymstat or TY-51469 do not increase bleeding time in mice (A) or hamsters (B)

[0334] 10.2 Effect of chymase inhibitors on bleeding time in a femoral vein puncture model in hamsters 300 U / kg unfractionated heparin (specified by the manufacturer) or 10 mg / kg furasymstat was injected via the jugular vein or orally, respectively, 1 h before testing. Hamsters were anesthetized with a mixture of intramuscular ketamine / xylazine (87-200 / 13 mg / kg). The femoral vein was exposed in the right hind limb. A small incision was made with microscissors in the middle of the femoral vein, and excess blood was absorbed with filter paper without touching the incision. The time until bleeding completely stopped was defined as the bleeding time. 43 was recorded as.

[0335] result The results of bleeding time measurements in hamsters are shown in FIG. 12B and summarized in Table 12, which showed that heparin statistically significantly prolonged bleeding time, but no effect on bleeding time could be 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 (Furasimstat) compared to control animals.

[0336] Table 12. Effect of the chymase inhibitor furasimustat on bleeding time in a femoral vein puncture model in hamsters [Table 12] One-way ANOVA with Bonferroni's multiple comparison test. ***P<0.001. Error bars: standard error of the mean (±SEM).

[0337] Global analysis of the results of bleeding time experiments in mice and hamsters in relation to thrombolysis outcomes In both mouse and hamster bleeding time models, no prolongation of bleeding time was observed at doses of chymase inhibitors, which resulted in a strong thrombolytic effect.

[0338] Thus, surprisingly, this chymase inhibition approach achieved rapid clot resolution without any signs of bleeding, making the chymase inhibition approach not only effective but also unexpectedly safe.

[0339] The proposed mechanism of action is shown in FIG.

[0340] Figure 16 - A novel approach for safe thrombolysis and vascular recanalization by targeting intrathrombus chymase In summary, in the various parts of this invention, it has been shown for the first time that chymase inhibitors inhibit plasmin degradation by chymase, identifying for the first time as a pathophysiologically relevant process in animal and human thrombi, affecting thrombus size in in vivo experiments. Inhibiting plasmin degradation via chymase inhibitors accelerates the dissolution of fibrin clots in blood vessels without affecting bleeding time or hemostasis, thereby representing a novel approach for extremely safe revascularization of blood vessels occluded by thrombi or emboli (Experimental part, Figure 16).

[0341] Therefore, the chymase inhibitors of the present invention are effective agents for the treatment and prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states following inflammation, transplantation, and infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion at vascular access sites.

[0342] 11. Pharmaceutical Composition Examples The substances according to the invention can be converted into pharmaceutical preparations as follows: tablet: composition: 100 mg of the compound, 50 mg of lactose (monohydrate), 50 mg of corn starch, 10 mg of polyvinylpyrrolidone (PVP25) (from BASF, Germany) and 2 mg of magnesium stearate.

[0343] Tablet weight 212mg. Diameter 8mm, radius of curvature 12mm.

[0344] Manufacturing: The mixture of the compound of Example 1, lactose and starch is granulated with a 5% strength solution (m / m) of PVP in water.After drying, the granules are mixed with magnesium stearate for 5 minutes.This mixture is compressed in a conventional tablet press (tablet format see above).

[0345] Oral suspension: composition: 1000 mg of the compound of Example 1, 1000 mg of ethanol (96%), 400 mg of Rhodigel (Xanthan gum) (from FMC, USA) and 99 g of water.

[0346] 10 ml of oral suspension corresponds to a single dose of 100 mg of a compound of the invention.

[0347] Manufacturing: Rhodigel is suspended in ethanol and the compound of Example 1 is added to the suspension. Water is added with stirring. The mixture is stirred for approximately 6 hours until swelling of the Rhodigel is complete.

[0348] Solutions for oral administration: composition: 500 mg of a compound of the invention, 2.5 g of polysorbate and 97 g of polyethylene glycol 400. A single dose of 100 mg of a compound of the invention is equivalent to 20 g of oral solution.

[0349] Manufacturing: The compound of the present invention is suspended in the mixture of polyethylene glycol and polysorbate with stirring.15 Stirring is continued until dissolution of the compound of the present invention is complete.

[0350] Intravenous fluids: The compounds of the present invention are dissolved in a physiologically acceptable solvent (e.g., isotonic sodium chloride solution, 5% glucose solution and / or 30% PEG 400 solution) at a concentration less than the saturated solubility. The solution is subjected to sterile filtration and dispensed into sterile and pyrogen-free injection containers. [Brief description of the drawings]

[0351] [Figure 1]FIG. 1 depicts the kinetics of chymase-dependent inactivation of human (A) and mouse (B) plasmin and its 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 (Furasymstat) or TY-51469. [Diagram 2] FIG. 2 shows that chymase-dependent inactivation of human (A) and mouse (B) plasmin is 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 (furasymstat) or TY-51469. [Diagram 3] FIG. 3 shows that human (A) and mouse (B) plasmin are substrates for chymase and that degradation of the latter by the protease is 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 (flasymstat) or TY-51469. [Figure 4-1] FIG. 4 shows that furasymustat or TY-51469 (i) inhibit or (ii) resolve DVT in IVC-ligated mouse (A) and hamster models (B). [Figure 4-2] FIG. 4 shows that furasymustat or TY-51469 (i) inhibit or (ii) resolve DVT in IVC-ligated mouse (A) and hamster models (B). [Diagram 5] FIG. 5 depicts the reduction in the number of pulmonary emboli in the lungs of mice treated with furasimstat. [Figure 6]FIG. 6 depicts the reduction in the number of pulmonary emboli in the lungs of hamsters treated with furasimustat. [Figure 7] FIG. 7 shows that chymase inhibition reduces thrombus weight in an FeCl 3 -induced injury model in the inferior vena cava (IVC) in mice (A) or hamsters (B) and in the femoral vein (hamster, C). [Figure 8] Figure 8 shows deep vein thrombosis showing immunoreactive chymase-containing mast cells in IVC-ligated wild-type mice. Each image represents three different tissue samples. [Figure 9] Figure 9 shows that human venous thrombosis, pulmonary embolism and arterial thrombosis show immunoreactive chymase-containing mast cells. Each image is representative of three different tissue samples. [Figure 10] FIG. 10 shows that chymase inhibitors increase plasmin activity in thrombi from mice (A) or hamsters (B). [Figure 11] FIG. 11 shows that plasmin-dependent fibrinolysis of human clots is reduced by human chymase (CMA-1) in a flasymstat-sensitive manner. [Figure 12] FIG. 12 shows that furasymustat or TY-51469 do not increase bleeding time in mice (A) or hamsters (B). [Figure 13] Figure 13 shows that furasymstat reduces thrombus-induced occlusion in a FeCl3-induced injury model in the common carotid artery (CCA) of mice or hamsters. Each series of images is representative of six independent experiments. [Figure 14] Figures 14A-C show that human blood halo clots require activation of endogenous plasmin to be broken down. Figures 14D and E show the results investigated in concentration-response experiments using rivaroxaban or the plasminogen / plasmin inhibitor 10-chloro-4-(piperidin-4-yl)pyrimido[1,2-b]indazol-2(1H)-one hydrochloride (Example 3 in WO2015067549). [Figure 15]Figure 15A shows how human chymase is added to form a halo-shaped clot. Figure 15B shows that human chymase incorporated after CaCl2-induced clotting significantly reduces tPA-induced fibrinolytic activity in human blood halo clots in a flasymstat-sensitive manner. [Figure 16] FIG. 16 depicts a novel approach for safe thrombolysis and vascular recanalization by targeting intrathrombus chymase.

Claims

1. Chymase inhibitors for use in the treatment and prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, inflammation, transplantation, thrombotic microangiopathy in post-infection hypercoagulable states, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites.

2. A chymase inhibitor for use in the treatment and prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states after inflammation, transplantation, or infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites, Here, the chymase inhibitor is given by formula (I) 【Chemistry 1】 [During the ceremony, R １ is hydrogen, methyl, or ethyl, R ２ is, formula 【Chemistry 2】 It is the basis of, Here, * represents the bonding site of uracil to the nitrogen atom. A is -CH ２ -ien-CH ２ -CH ２ -, -O-CH ２ -## or oxygen, Here, ## is the binding site to the phenyl ring, R ４Ａ These are hydrogen, fluorine, chlorine, trifluoromethyl, or methyl. R ４Ｂ These are hydrogen, fluorine, chlorine, trifluoromethyl, or methyl. However, R ４Ａ and R ４Ｂ at least one of the groups is not hydrogen, R ５Ａ It is hydrogen, R ５Ｂ It is hydrogen, R ６ It is hydrogen, R ７ It is hydrogen, R ８ These are fluorine, chlorine, difluoromethyl, trifluoromethyl, or methyl. R ９ These are fluorine, chlorine, difluoromethyl, trifluoromethyl, or methyl. R ３ is, formula 【Transformation 3】 It is the basis of, Here, # represents the bonding site of uracil to the nitrogen atom. E １ CR １１ or N, Here, R １１ is hydrogen, (C １ -C ４ )-alkyl, (C ３ -C ７ ) - Cycloalkyl or aminocarbonyl, E ２ CR １２ or N, Here, R １２ is hydrogen, (C １ -C ４ )-alkyl or (C ３ -C ７ ) - Cycloalkyl, G １ C=O or SO ２ And, G ２ CR １６Ａ R １６Ｂ , NR １７ , O or S, Here, R １６Ａ is hydrogen, fluorine, (C １ -C ４ ) - Alkyl or hydroxyl, R １６Ｂ (C) １ -C ４ ) - Alkyl or trifluoromethyl, or R １６Ａ and R １６Ｂ These, together with the carbon atoms to which they are bonded, form a 3- to 6-membered carbon ring. R １７ is hydrogen, (C １ -C ６ )-alkyl, (C ３ -C ７ )-cycloalkyl or (C １ -C ４ ) - alkoxycarbonyl, Here, (C １ -C ６ )-Alkyl is fluorine, trifluoromethyl, cyano, (C ３ -C ７ )-Cycloalkyl, Hydroxyl, Trifluoromethoxy, (C １ -C ４ )- May be substituted with 1 to 3 substituents independently selected from the group consisting of alkoxy, azetidinyl, oxetanyl, tetrahydrofuranyl, and pyrrolidinyl. R ２４ is fluorine or methyl, n is a number that is either 0 or 1. R １０ is, (C １ -C ４ )-alkyl or (C ３ -C ７ ) - Cycloalkyl, Here, (C １ -C ４ )-alkyl may be substituted with one or two substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxyl, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl, and pyrrolidinyl. R １５ is hydrogen, (C １ -C ６ )-alkyl or (C ３ -C ７ ) - Cycloalkyl, Here, (C １ -C ６ )-Alkyl may be substituted with one or two substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxyl, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl, and pyrrolidinyl. A chymase inhibitor, which is a compound of the same, as well as its salts, solvates, and solvates of salts.

3. A chymase inhibitor according to claim 2 for use in the treatment and prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states after inflammation, transplantation, or infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites, Here, the chymase inhibitor is given by formula (I) [During the ceremony, R １ is hydrogen, methyl, or ethyl, R ２ is, formula 【Chemistry 4】 It is the basis of, Here, * represents the bonding site of uracil to the nitrogen atom. A is -CH ２ - and R ４Ａ is chlorine or trifluoromethyl, R ４Ｂ It is hydrogen, R ３ is, formula 【Transformation 5】 It is the basis of, Here, # represents the bonding site of uracil to the nitrogen atom. E １ CR １１ And, Here, R １１ It is hydrogen, E ２ N is, G １ C=O, G ２ CR １６Ａ R １６Ｂ , NR １７ , O or S, Here, R １６Ａ is hydrogen, fluorine, methyl or hydroxyl, and 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, (C １ -C ４ )-alkyl or (C ３ )-cycloalkyl, and ５ ​ Here, (C １ -C ４ )-alkyl may be substituted with one to three substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxyl, trifluoromethoxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl, and pyrrolidinyl. R ２４ is hydrogen or fluorine, R １０ is, (C １ -C ４ ) - Alkyl, R １５ is hydrogen, methyl, or ethyl, Here, methyl and ethyl may be substituted with one substituent selected from the group consisting of fluorine, trifluoromethyl, and cyclopropyl. A chymase inhibitor, which is a compound of the same, as well as its salts, solvates, and solvates of salts.

4. A chymase inhibitor according to claim 2 for use in the treatment and prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states after inflammation, transplantation, or infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites, Here, the chymase inhibitor is given by formula (I) [During the ceremony, R １ It is hydrogen, R ２ is, formula 【Transformation 6】 It is the basis of, Here, * represents the bonding site of uracil to the nitrogen atom. R ５Ａ It is hydrogen, R ５Ｂ It is hydrogen, R ６ It is hydrogen, R ７ It is hydrogen, R ８ These are fluorine, chlorine, or trifluoromethyl, R ９ These are fluorine, chlorine, trifluoromethyl, or methyl. R ３ is, formula 【Transformation 7】 It is the basis of, Here, # represents the bonding site of uracil to the nitrogen atom. E １ CR １１ And, Here, R １１ It is hydrogen, E ２ N is, G １ C=O, G ２ CR １６Ａ R １６Ｂ , NR １７ , O or S, Here, R １６Ａ R is hydrogen, fluorine, methyl or hydroxyl, １６Ｂ These are 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, (C １ -C ４ )-alkyl or (C ３ -C ５ ) - Cycloalkyl, Here, (C １ -C ４ )-alkyl may be substituted with one to three substituents independently selected from the group consisting of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxyl, trifluoromethoxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl, and pyrrolidinyl. R ２４ is hydrogen or fluorine, R １０ is, (C １ -C ４ ) - Alkyl, R １５ is hydrogen, methyl, or ethyl, Here, methyl and ethyl may be substituted with one substituent selected from the group consisting of fluorine, trifluoromethyl, and cyclopropyl. These are compounds, as well as their salts, solvates, and solvates of salts.

5. A chymase inhibitor according to claim 2 for use in the treatment and prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states after inflammation, transplantation, or infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites, Here, the chymase inhibitor is 1-(1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzimidazole-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-benzimidazole-5-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-di Hydro-1H-inden-1-yl]-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazole-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-indole-5-yl)-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid (R enantiomer), 1-(1'-methyl-2'-oxo-1',2'-dihydrospiro[cyclopropane-1,3'-indole]-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-benzothiazole-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-benzoxazole-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-1,2,3,A chymase inhibitor, which is a compound of formula (I) selected from the group of 4-tetrahydropyrimidine-5-carboxylate (R enantiomer).

6. A chymase inhibitor according to claim 2 for use in the treatment and prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states after inflammation, transplantation, or infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites, Here, the chymase inhibitor of formula (I) is given by formula: 【Transformation 8】 The chymase inhibitor is 1-(3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazole-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).

7. A chymase inhibitor for use in the treatment and prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states after inflammation, transplantation, or infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites, wherein the chymase inhibitor is formula (II) 【Chemistry 9】 [During the ceremony, 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 These may be the same or different, and include 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, and -CONHR. ６ (Here, R ６ This includes a hydrogen atom, a lower alkoxycarbonylmethyl group, a carboxymethyl group, or CH(CH ２ OH) COOR ７ (Here, R ７ (represents a hydrogen atom or a lower alkyl group) formula 【Chemistry 10】 A group represented by (where R ７ This is synonymous with the above.) formula 【Chemistry 11】 A base represented by Here, R ８ and R ９ These may be the same or different, and represent a hydrogen atom, a lower alkyl group, a lower alkylsulfanyl group, a lower alkylsulfinyl group, a lower alkylsulfonyl group or a lower alkoxycarbonyl group, a hydroxy lower alkyl group, or a cyano group. Or formula: 【Chemistry 12】 This represents a monocyclic heterocyclic group, Here, A represents an oxygen atom, a sulfur atom, or NH, and the dotted line portion represents a single bond or a double bond, however, the hydrogen atoms on the ring may be replaced by a halogen atom, a lower alkoxy group, a hydroxy lower alkyl group, a lower alkoxycarbonyl group, or a lower alkyl group which may be substituted with a carboxyl group, however, R ３ or R ４ At least one of them is 【Chemistry 13】 And, and R ５ [This represents a hydrogen atom, a lower alkoxy group, or a lower alkyl group.] It is a compound of the following formula: 【Chemistry 14】 Chymase inhibitors, excluding compounds represented by [the specified formula].

8. A chymase inhibitor according to claim 7 for use in the treatment and prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, inflammation, transplantation, thrombotic microangiopathy in post-infection hypercoagulation, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, and thrombosis or occlusion of vascular access sites, wherein the compound is 2-[4-(5-chloro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methane-sulfonylphenyl] Oxazole-4-carboxylic acid, 2-[4-(5-fluoro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methanesulfonylphenyl]oxazole-4-carboxylic acid, 2-[4-(5-chloro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methanesulfonylphenyl]oxazole-4-carboxylic acid disodium, 2-[4-(5-fluoro-3-methylbenzo[b]thiophene-2-sulfonylamino)-3-methanesulfonyl [Triphenyl]oxazole-4-carboxylate disodium, 2-[4-(5-fluoro-3-methylbenzo[b]thiophen-2-sulfonylamino)-3-methane-sulfonylphenyl]thiazole-4-carboxylic acid, 5-fluoro-N-[4-(4-hydroxymethylthiazole-2-yl)-2-methane-sulfonylphenyl]-3-methylbenzo[b]thiophen-2-sulfonamide, 5-fluoro-N-[2-methane-sulfonyl-4-(5-methoxy-4-methyl A chymase inhibitor selected from the group consisting of oxazole-2-yl)phenyl]-3-methylbenzo[b]thiophene-2-sulfonamide, ((2-(4-((5-fluoro-3-methylbenzo-[b]thiophene)-2-sulfonamide)-3-(methylsulfonyl)-phenyl)thiazole-4-carboxylic acid and 5-fluoro-N-[2-methanesulfonyl-4-(5-methyloxazole-2-yl)phenyl]-3-methylbenzo[b]thiophene-2-sulfonamide.

9. A chymase inhibitor according to claim 7 for use in the treatment and prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states after inflammation, transplantation, or infection, 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-sulfonamide)-3-(methylsulfonyl)phenyl)thiazole-4-carboxylic acid.

10. A combination of one or more compounds of formula (I) as described in claim 2 and one or more other active compounds.

11. A combination of one or more compounds of formula (II) as described in claim 7 and one or more other active compounds.

12. A pharmaceutical composition comprising at least one compound of formula (I) as described in claim 2, in combination with one or more inert, non-toxic, pharmaceutically suitable excipients.

13. A pharmaceutical composition comprising at least one compound of formula (II) as described in claim 7, in combination with one or more inert, non-toxic, pharmaceutically suitable excipients.

14. A pharmaceutical composition for the treatment and / or prevention of stroke, pulmonary embolism, deep or superficial venous thrombosis, thrombotic microangiopathy, thrombotic microangiopathy in hypercoagulable states following inflammation, transplantation, or infection, disseminated intravascular coagulation, vaccine-induced immune thrombotic thrombocytopenia, thrombosis or occlusion of vascular access sites, comprising a therapeutically effective amount of at least one compound according to any one of claim 1, claim 2, or claim 7 in combination with an inert, non-toxic, pharmaceutically acceptable additive, wherein the pharmaceutical composition is administered systemically and / or topically.

15. The pharmaceutical composition according to claim 14, wherein the composition further comprises at least one further active compound selected from the group consisting of anticoagulants and / or antiplatelet agents.

16. A pharmaceutical product comprising a compound of formula (I) as described in claim 2, in combination with one or more further active ingredients selected from the group consisting of anticoagulants and / or antiplatelet agents.

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