Factor XIIa inhibitor

Abstract

The present invention relates to compounds of formula (I) and methods of treatment using the compounds. The present invention also relates to processes and methods for producing the compounds of the present invention. The compounds of the present invention are modulators of factor XII (e.g., factor XIIa). In particular, the compounds are inhibitors of factor XIIa and can be useful as anticoagulants. [Formula 1]

Description

[Technical Field]

[0001] The present invention relates to compounds and methods for treatment (or prevention) using the compounds. The present invention also relates to processes and methods for producing the compounds of the present invention. The compounds of the present invention are modulators of factor XII (e.g., factor XIIa). In particular, the compounds are inhibitors of factor XIIa and may be useful as anticoagulants. [Background technology]

[0002] Cardiovascular disease is a leading cause of death in developed countries, affecting millions of people worldwide each year. This disease is generally caused by atherosclerosis of the arterial walls, which progresses over many years, and is characterized by endothelial inflammation, subendothelial lipid deposition, macrophage infiltration, and plaque formation. In the acute phase of the disease, atherosclerotic plaques become unstable, rupture, and cause thrombosis. The formation of a blood clot (thrombus) that blocks a blood vessel, resulting in oxygen deprivation of tissue, constitutes a sudden major event leading to morbidity and death. Clot formation is initiated by the activation and aggregation of platelets. Platelet thrombi solidify through the activation of coagulation and the formation of a fibrin network. Arterial occlusion by thrombus leads to tissue death downstream and, depending on where it occurs, is associated with the development of myocardial infarction, stroke, or claudication.

[0003] Venous thrombosis has a different etiology, as it is not dependent on atherosclerosis and is caused by circulatory stagnation resulting from fixation, and is often associated with spontaneously occurring deficiencies of blood clot inhibitors (e.g., antithrombin, protein C and S) and surgical procedures. Venous thrombosis usually occurs in the legs or arms (deep vein thrombosis, DVT) and especially in the lungs (pulmonary embolism, PE), which can lead to embolus (thrombus fragments) that block smaller blood vessels downstream. Other triggers for DVT include cancer, nephrotic syndrome, antiphospholipid antibody syndrome, and heart failure.

[0004] Thrombosis is a very serious condition, and in the UK alone, it is associated with up to 25,000 deaths per year and 200,000 deaths per year from venous and arterial thrombosis, respectively. In January 2010, the UK National Institute for Health and Clinical Excellence (NICE) published new guidelines to increase screening for early signs of thrombosis in hospitalized patients.

[0005] Current drug therapies for treating or preventing thrombosis target either platelets or coagulation. Generally, antiplatelet agonists are used to prevent arterial disease, while anticoagulants are used to prevent stroke in patients with atrial fibrillation, deep vein thrombosis (DVT), and pulmonary embolism (PE). The biggest clinical problem associated with current anticoagulant use is the risk of bleeding. Depending on the patient group and the choice of anticoagulant, as many as 1-3% of patients experience major bleeding during anticoagulation treatment, or 15-18% experience minor bleeding.

[0006] Warfarin and heparin (including all its derivatives) are the most commonly used anticoagulants. Warfarin, the first approved long-term oral anticoagulant, requires regular monitoring via prothrombin time (PT) coagulation assays to determine the optimal dosage, which places a significant burden on healthcare systems and patients' quality of life. Warfarin is nonspecific and targets several coagulation enzymes, while heparin is administered subcutaneously or intravenously and targets activated factor X (FXa) and / or thrombin depending on its molecular weight. Furthermore, new oral anticoagulants (NOACs) currently on the market or under development that target thrombin or FXa also carry a significant risk of bleeding comparable to that of heparin and warfarin, with the exception of intracranial hemorrhage, where NOACs have a better outcome than warfarin. However, gastrointestinal bleeding is increased by NOACs compared to vitamin K antagonists, including low molecular weight heparin and warfarin [New Oral Anticoagulants Increase Risk for Gastrointestinal Bleeding: A Systematic Review and Meta-analysis Holster IL, Valkhoff VE, Kuipers EJ, Tjwa ET Gastroenterology. 2013 Jul;145(1):105-112].

[0007] Therefore, there is a great unmet clinical need for novel anticoagulants that do not cause bleeding. This goal has been desired in this field for over 60 years. However, since the mechanisms involved in thrombosis are thought to be the same as those involved in hemostasis, it has always been assumed that anticoagulants inevitably carry a risk of bleeding.

[0008] Factor XII (FXII) was identified 50 years ago as a coagulation protein in the intrinsic pathway of blood coagulation because patients with FXII deficiency exhibited a marked prolongation of in vitro surface-activated coagulation time. However, a series of studies have convincingly shown that FXII has no role in normal hemostasis. Evidence from the past decade has identified FXII as essential for thrombus formation in vivo (Renne T, Pozgajova M, Gruner S, Schuh K, Pauer HU, Burfeind P, Gailani D, Nieswandt B. Defective thrombus formation in mice lacking coagulation factor XII. J Exp Med 2005;202:271-281; ​​Kleinschnitz C, Stoll G, Bendszus M, Schuh K, Pauer HU, Burfeind P, Renne C, Gailani D, Nieswandt B, Renne T. Targeting coagulation factor XII provides protection from pathological thrombosis in cerebral ischemia without interfering with hemostasis. J Exp Med 2006;203:513-518; Renne T, Nieswandt B, Gailani D. The intrinsic Pathway of coagulation is essential for thrombus stability in mice. Blood Cells Mol Dis 2006;36:148-151, Hagedorn I, Schmidbauer S, Pleines I, Kleinschnitz C, Kronthaler U, Stoll G,Dickneite G, Nieswandt B.Factor XIIa inhibitor recombinant human albumin Infestin-4 abolishes occlusive arterial thrombus formation without affecting bleeding. Circulation 2010;121:1510-1517; and Matafonov A, Leung PY, Gailani AE, Grach SL, Puy C, Cheng Q, Sun MF, McCarty OJ, Tucker EI, Kataoka H, ​​Renne T, Morrissey JH, Gruber A, Gailani D. Factor XII inhibition reduces thrombus formation in a primate thrombosis model. Blood. 2014;13;123(11):1739-46). A unique characteristic of FXII is that, unlike deficiencies of all other coagulation factors, its deficiency does not lead to bleeding. Therefore, FXIIa is a very attractive target for the discovery of anticoagulants with the potential for a significantly improved safety profile.

[0009] Recent studies are challenging dogmas in the field of hemostasis and thrombosis by demonstrating novel mechanisms of thrombosis involving FXII. These studies provide clear evidence that FXII is essential for thrombosis but plays no role in hemostasis. FXII-deficient mice, when sensitized with collagen and epinephrine injections, were significantly protected against thrombosis while not showing prolonged bleeding time during surgery or tail clipping. Similar protection against thrombosis was observed in mesenteric arterioles exposed to FeCl3 and in the aorta after physical injury. Injection of human FXII in these models reversed thrombosis. The novel nature of these findings is illustrated by the debate, which until recently dominated the field, regarding the role of FXII function and the contact coagulation pathway activated by FXIIa. This debate was stimulated by the fact that FXII deficiency does not lead to bleeding, while deficiencies in all other coagulation proteases do, leading to the idea that FXII is not required for physiological coagulation and that FXII activation is an in vitro phenomenon.

[0010] However, recent studies have shown that FXII is activated by negatively charged surfaces and the surface of activated platelets (Zakharova et al, PLoS One. 2015 Feb 17;10(2):e0116665). These in vivo and in vitro studies demonstrate that FXII plays a previously unrecognized role in thrombosis. The production of FXIIa stabilizes thrombi by enhancing thrombin generation, fibrin deposition, and direct thrombus formation-promoting effects on fibrin structure. Since FXII deficiency does not show a phenotype in humans and mice, this mechanism does not appear to play a role in normal hemostasis, making FXII an ideal target for the development of novel anticoagulants to treat thrombosis.

[0011] The effectiveness of FXII deficiency in reducing thrombosis has been demonstrated in several different in vivo thrombosis models. In addition to the models mentioned above, the role of FXII in thrombosis has been demonstrated in a mouse model of carotid artery ligation-induced thrombosis and a mouse model of cerebral microvascular thrombosis secondary to transient occlusion of the middle cerebral artery. Stroke size was significantly reduced in FXII-deficient mice and returned to large infarcts upon injection of human FXII. FXII inhibition has also been shown to reduce the risk of venous thrombosis. One study demonstrated that a contact-activated Kunitz inhibitor isolated from tick salivary gland (Ir-CPI) effectively reduces thrombosis in mouse and rat models of vascular ligation-induced venous thrombosis. This inhibitory protein was also effective in reducing PE in a mouse model induced by collagen and epinephrine injection and in a mouse model of cutaneous arteriovenous thrombosis. As expected, there was no effect on bleeding time in animals treated with Ir-CPI. Inhibition of FXIIa with HD-Pro-Phe-Arg-chloromethyl ketone (PCK) has also been shown to provide protection against thrombosis. These studies provide preclinical evidence for the concept that inhibition of FXIIa is effective in the treatment of thrombosis.

[0012] More recently, Magnus Larsson et al., “A Factor XIIa Inhibitory Antibody Provides Thromboprotection in Extracorporeal Circulation Without Increasing Bleeding Risk” Sci Transl Med 6, 222ra17 (2014), demonstrated that recombinant fully human antibody 3F7 binds to the FXIIa enzyme pocket. 3F7 inhibited FXIIa-mediated coagulation in mice and rabbits, eliminating thrombus formation in flow and blocking experimental thrombosis. In rabbits, 3F7 provided thromboprotection as effectively as heparin, but unlike heparin, 3F7 treatment did not impair hemostatic ability or increase bleeding from wounds. Larsson et al. concluded that targeting FXIIa is a safe mode of thromboprotection in bypass systems and provides a clinically valid anticoagulant strategy without excessive bleeding.

[0013] Dabigatran, apixaban, rivaroxaban, edoxaban, and betrixaban are each approved as oral FXa / thrombin inhibitors for short-term use. Dabigatran is 3-({2-[(4-carbamimidoylphenylamino)-methyl]-1-methyl-1H-benzimidazole-5-carbonyl}pyridine-2-yl-amino)-propionic acid.

[0014] Dabigatran is also approved for the long-term prevention of stroke in patients with atrial fibrillation (AF), as described in U.S. Patent No. 6,087,380.

[0015] [ka]

[0016] Rivaroxaban is (S)-5-chloro-N-{[2-oxo-3-[4-(3-oxomorpholin-4-yl)phenyl]oxazolidine-5-yl]methyl}thiophene-2-carboxamide.

[0017] [ka]

[0018] Rivaroxaban is also approved for reducing the risk of stroke in patients with non-valvular atrial fibrillation (AF). Rivaroxaban has been shown to be superior to warfarin in protecting AF patients from stroke and non-CNS systemic embolism when administered once daily. Rivaroxaban has also demonstrated clinically relevant major and non-major bleeding rates comparable to warfarin, as well as significantly lower rates of intracranial hemorrhage than warfarin. Rivaroxaban is listed in U.S. Patent No. 7,157,456.

[0019] Apixaban is also a factor Xa inhibitor approved for use in the prevention of stroke and systemic embolism in patients with non-valvular atrial fibrillation.

[0020] [ka]

[0021] Apixaban is 1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxopiperidine-1-yl)phenyl]-4,5-dihydropyrazolo[5,4-c]pyridine-3-carboxamide.

[0022] Apixaban is listed in U.S. Patent No. 6,413,980.

[0023] Edoxaban is N'-(5-chloropyridine-2-yl)-N 2 It is -((1S,2R,4S)-4-[(dimethylamino)carbonyl]-2-{[(5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine-2-yl)carbonyl]amino}cyclohexyl)ethanediamide.

[0024] [ka]

[0025] Edoxaban is another factor Xa inhibitor approved for use in the prevention of stroke and systemic embolism in patients with non-valvular atrial fibrillation, as well as for the treatment of deep vein thrombosis. Edoxaban is described in U.S. Patent No. 7,365,205.

[0026] Betrixaban is N-(5-chloropyridine-2-yl)-2-[4-(N,N-dimethylcarbamimidoyl)benzamide]-5-methoxybenzamide.

[0027] [ka]

[0028] Betrixaban is a factor Xa inhibitor approved for use in the prevention of venous thromboembolism in patients with moderate to severe behavioral disorders. Betrixaban is listed in U.S. Patent No. 6,376,515.

[0029] Recent surveys of major pharmaceutical companies' cardiovascular pipelines have not identified any oral inhibitors of FXIIa currently under investigation. Infestin-4 is a biologic manufactured by CSL Behring that targets FXIIa and has demonstrated efficacy in FeCl3 induction models of thrombosis in mice and rabbits. Other antibody approaches targeting FXII(a) have also shown in vivo efficacy. However, if infestin-4 or antibody approaches are successful, they require intravenous administration, and therefore are not well-suited for long-term anticoagulant use.

[0030] In humans, FXII deficiency causes bleeding, and unlike other coagulation factor deficiencies where FXII activity deficiency or inhibition exhibits anticoagulant effects, it is asymptomatic. Therefore, selective FXIIa inhibitors may have the potential to reduce the risk of bleeding associated with currently available anticoagulant therapies.

[0031] European Patent Application No. 0672658 (Eli Lilly) describes a phenylalanine proline derivative useful as a thrombin inhibitor.

[0032] International Publication No. 2002 / 064559 (Merck) also describes a phenylalanine proline derivative useful as a thrombin inhibitor. This compound is a selective inhibitor of cyclooxygenase-2 compared to cyclooxygenase-1.

[0033] International Publication No. 02 / 50056 (Merck) describes benzylamine and cyclohexylamine derivatives that are useful as thrombin inhibitors.

[0034] An object of one aspect of the present invention is to alleviate, at least partially, the problems associated with the prior art.

[0035] An object of a particular embodiment of the present invention is to provide a compound that inhibits FXII activity, particularly FXIIa activity, for example, the serine protease activity of FXIIa.

[0036] An objective of certain embodiments of the present invention is to provide compounds having physicochemical and pharmacokinetic properties consistent with the potential for oral bioavailability.

[0037] An objective of certain embodiments of the present invention is to provide compounds that exhibit reduced cytotoxicity or increased solubility compared to conventional compounds and existing treatments.

[0038] Another objective of certain embodiments of the present invention is to provide a compound having a convenient pharmacokinetic profile and a suitable duration of action after dosing. A further objective of certain embodiments of the present invention is to provide a compound in which one or more metabolites of the drug after absorption are GRAS (Generally Recognized as Safe).

[0039] An objective of certain embodiments of the present invention is to provide a modulator of a target, and this embodiment selectively modulates this target compared to other targets. An objective of certain embodiments of the present invention is to provide a compound that is a selective FXIIa inhibitor. In particular, an objective of certain embodiments of the present invention is to provide a compound that selectively inhibits FXIIa compared to thrombin and FXa.

[0040] Certain embodiments of the present invention satisfy some or all of the above objectives. Summary of the Invention

[0041] According to the present invention, a compound according to formula (I) and a pharmaceutically acceptable salt thereof: [[ID=QQ]] [[ID=QQ]]

Chemical formula

[0042] According to the present invention, a compound according to formula (I) and a pharmaceutically acceptable salt thereof:

Chemical formula

[0043] R 3In embodiments where is option (a) or option (b), if X is a combination, then m is not 0. 3 In the embodiment where option (c) is the case, if X is a combination, then m can be 0.

[0044] The compounds according to formula (I) are the compounds of formula (Ia) and their pharmaceutically acceptable salts: [ka] (In the formula, Y is [ka] Selected from, R 1a and R 1b They combine to form a substituted or unsubstituted 5 or 6-membered heteroaromatic ring or phenyl ring; R 1a and R 1b If the ring formed from is replaced, it is 1, 2, or 3 R z It is substituted with the base, where R z In each occurrence, independently, =O, CN, -OH, or -OC 1~6 alkyl, halo, and C 1~6 Selected from alkyl groups; R 3a is H, or C 1~6 It is alkyl; (m is selected from 1, 2, or 3) It is possible.

[0045] In one embodiment, R 1a and R 1b It is substituted by an adjacent atom. Therefore, Y is [ka] It can be selected from the following.

[0046] In this embodiment, Y is [ka] Selected from.

[0047] In one embodiment, R 1a and R 1b is 1, 2, or 3 R z It forms a phenyl ring substituted with a group.

[0048] In this embodiment, Y is [ka] (In the formula, R z is =O, CN, -OH, or -OC 1~6 alkyl, halo, C 1~6 Alkyl, and C 1~6 (Selected from haloalkyls) Selected from.

[0049] In the embodiment, m is 2 or 3, or m is 1 or 2.

[0050] In an embodiment, X is either a bond, -C(O)NH-, or -C(O)-.

[0051] In one embodiment, the compound of formula (I) is a compound according to formula (IIa), (IIb), (IIc), or (IId): [ka] That is the case.

[0052] In the embodiment of formula (IIa), m is 2 or 3. In the embodiment of formula (IIa), X is a bond. Therefore, in the embodiment, the compound of formula (I) is the compound of formula (IIIa) or (IIIb): [ka] That is the case.

[0053] In embodiments of formula (IIb), m is 1 or 2. In embodiments of formula (IIb), X is a bond, -C(O)NH-, or -C(O)-, preferably X is -C(O)-.

[0054] In one embodiment, R z is selected from H, OH, Cl, or OMe. Therefore, in the embodiment, the compound of formula (I) is the compound according to formula (IV): [ka] It is possible.

[0055] In the embodiment, o is 1. In the embodiment, R e and R f H is H.

[0056] In a preferred embodiment, o is 1, and R e and R f is H. Therefore, in one embodiment, the compound of formula (I) is the compound of formula (V) or (Va): [ka] That is the case.

[0057] In one embodiment, R 2 H is H. In this embodiment, R 3 H is H. In this embodiment, R 5 H is H. In this embodiment, R 2 , R 3 and R 5 Each of these is H. In this embodiment, R a and R b Each of these is H. In this embodiment, R 2 , R 3 , R 5 , R a and R b Each of these is H.

[0058] In embodiments, the compound of formula (I) is a compound of formula (VIa), (VIb), (VIc), or (VId): [ka] It is possible.

[0059] In embodiments of the compound of formula (VIa) or (VIb), R 2 , R 3 and R 5 Each of these is H. In embodiments of the compound of formula (VIc) or (VId), R 2 and R 5 Each of these is H. In embodiments of the compound of formula (VIa) or (VIb), R a and R b Each of these is H. In embodiments of the compound of formula (VIa) or (VIb), R 2 , R 3 , R 5 , R a and R b Each of these is H.

[0060] In embodiments, the compound of formula (I) is a compound according to formula (VIIa), (VIIb), (VIIc), or (VIId): [ka] It is possible.

[0061] In embodiments of the compound of formula (VIIa) or (VIIb), R 2 , R 3 and R 5 Each of these is H. In embodiments of the compound of formula (VIIc) or (VIId), R 2 and R 5 Each of these is H. In embodiments of the compound of formula (VIIa) or (VIIb), R a and R b Each of these is H. In embodiments of the compound of formula (VIIa) or (VIIb), R 2 , R 3 , R5 , R a and R b Each of these is H.

[0062] In one embodiment, R 4a This can be selected from H, OH, or F. Preferably, R 4a H is H.

[0063] In the embodiment, the compound of formula (I) is a compound according to formula (VIIIa), (VIIIb), (VIIIc), or (VIIId): [ka] It is possible.

[0064] In embodiments, Ar is selected from phenyl, a 6-membered heteroaryl, or a 9-10 membered bicyclic heteroaromatic ring system (preferably 9 members), where Ar is unsubstituted or C 1~6 Alkyl, -OR g , -NR g R h , or -NR g R h C replaced by 1~4 It is substituted with alkyl. In some cases, Ar is unsubstituted or substituted with methyl, chloro, -OMe, -NH2, or -CH2NH2.

[0065] In embodiments, Ar is selected from a 9-10 membered bicyclic heteroaromatic ring system (preferably 9 members), where Ar is unsubstituted or C 1~6 Alkyl, -OR g , -NR g R h , or -NR g R h C replaced by 1~4 It is substituted with alkyl. In some cases, Ar is unsubstituted or substituted with methyl, chloro, -OMe, -NH2, or -CH2NH2.

[0066] R g and Rh In each occurrence, it can be independently selected from H and methyl.

[0067] In embodiments, Ar is selected from phenyl, pyridyl, benzotriazole, imidazopyridine, pyridofuran, azaindole, benzopyrazole, pyridozathiophene, benzoxazole, quinoline, thiophenyl, and isoquinoline, where Ar is unsubstituted or substituted with methyl, chloro, -OMe, -NH2, or -CH2NH2.

[0068] In embodiments, Ar is selected from benzotriazole, imidazopyridine, pyridofuran, azaindole, benzopyrazole, pyridozathiophene, benzoxazole, quinoline, and isoquinoline, where Ar is unsubstituted or substituted with methyl, chloro, -OMe, -NH2, or -CH2NH2.

[0069] In embodiments, Ar is selected from phenyl, pyridyl, benzotriazole, imidazopyridine, pyridofuran, azaindole, benzopyrazole, pyridozathiophene, benzoxazole, and thiophenyl, where Ar is unsubstituted or substituted with methyl, chloro, -OMe, -NH2, or -CH2NH2.

[0070] In embodiments, Ar is selected from benzotriazole, imidazopyridine, pyridofuran, azaindole, benzopyrazole, pyridozathiophene, and benzoxazole, where Ar is unsubstituted or substituted with methyl, chloro, -OMe, -NH2, or -CH2NH2.

[0071] In one embodiment, Ar is [ka] Selected from.

[0072] In one embodiment, Ar is [Chemical formula] is selected from.

[0073] In an embodiment, Ar is not a substituted or unsubstituted phenyl.

[0074] In a preferred embodiment, Ar is an azaindole, benzotriazole, or N-methylbenzotriazole.

[0075] In a preferred embodiment, Ar is [Chemical formula] is.

[0076] In an embodiment, the compound of formula (I) is a compound according to formula (IXa), (IXb), (IXc) or (IXd): [Chemical formula] is.

[0077] In an embodiment of the compound of formula (IXa) or (IXb), R 2 , R 3 and R 5 are each H. In an embodiment of the compound of formula (IXa) or (IXb), R a and R b are each H. In an embodiment of the compound of formula (IXc) or (IXd), R 2 and R 5 are each H. In an embodiment of the compound of formula (IXa) or (IXb), R 2 , R 3 , R 5 , R a and R b are each H.

[0078] In an embodiment, L is selected from a bond, -NR 6 -, and -NR 7 C(O)-.

[0079] R 6 R can be H, Me, or -C(O)Me. 7 This can be H. In this embodiment, R 6 H is H.

[0080] In embodiments, L is selected from bond, -NH-, -NMe-, -N(C(O)Me)-, and -NHC(O)-.

[0081] In the embodiment, n is 0, 1, 2, or 3. In the embodiment, n is 0 or 1.

[0082] R c and R d In each occurrence, is independently selected from H and methyl. Preferably, R c and R d H is H.

[0083] In an embodiment, -L-(CR c R d ) n - is selected from bond, CH2, -NH-, -NHCH2-, -NH(CH2)2-, -NH(CH2)3-, -N(Me)-, -N(C(O)Me)CH2-, -NHC(O)-, -NHC(O)CH2-, -NHC(O)(CH2)2-, or NHC(O)(CH2)3-.

[0084] In an embodiment, -L-(CR c R d ) n - is selected from a bond or CH2.

[0085] R 4 is =CH2, -CN, halo, C 1~4 Alkyl, C 1~4 Haloalkyl, -OR 10 , -NR 10 R 11 , 6-10 member aryl, C 3~8The following can be selected from cycloalkyl, 3-6 member heterocycloalkyl, and 5-10 member heteroaryl, where C 3~8 Cycloalkyl, 3-6 membered heterocycloalkyl, 6-10 membered aryl or heteroaryl groups are unsubstituted or have 1, 2, or 3 R groups. 12 It has been replaced with.

[0086] R 4 is =CH2, -NR 10 R 11 The group may be selected from 6-10 membered aryl groups, 3-6 membered heterocycloalkyl groups, or 5-10 membered heteroaryl groups, where the 3-6 membered heterocycloalkyl group, 6-10 membered aryl group, or heteroaryl group may be unsubstituted or have 1, 2, or 3 R groups. 12 It has been replaced with.

[0087] R 4 The 6-10 membered aryl can be selected from phenyl or naphthalenyl. 4 C 3~8 The cycloalkyl group can be selected from cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. 4 The 3-6 member heterocycloalkyl can be selected from tetrahydropyranil, tetrahydrofuranil, piperidinil, piperazinil, morpholinil, pyrrolidinil, pyrazolidinil, or imidazolidinil (preferably tetrahydropyranil or piperazinil). 4 The 5-10 member heteroaryls may be selected from pyridinyl, pyrazinyl, pyrazolyl, imidazolyl, dihydrobenzofuran, benzodioxolanil, or isoindolinyl (depending on the case, pyridinyl, pyrazinyl, pyrazolyl, imidazolyl, dihydrobenzofuran, benzodioxolanil, or isoindolinyl). 3~6 Any of the cycloalkyl, 3-6 membered heterocycloalkyl, 6-10 membered aryl, or heteroaryl groups may be unsubstituted or have 1, 2, or 3 R groups. 12 It may be replaced with .

[0088] R4 The 6-10 membered aryl can be selected from phenyl or naphthalenyl. 4 The 3-6 member heterocycloalkyl can be selected from tetrahydropyranil, tetrahydrofuranil, piperidinil, piperazinil, morpholinil, pyrrolidinil, pyrazolidinil, or imidazolidinil (preferably tetrahydropyranil or piperazinil). 4 The 5-10 membered heteroaryl group may be selected from pyridinyl, pyrazinyl, pyrazolyl, imidazolyl, dihydrobenzofuran, benzodioxolanil, or isoindlinyl (or, depending on the case, pyridinyl, pyrazinyl, pyrazolyl, imidazolyl, dihydrobenzofuran, benzodioxolanil, or isoindlinyl). Any of the 3-6 membered heterocycloalkyl, 6-10 membered aryl, or heteroaryl group may be unsubstituted or have 1, 2, or 3 R groups. 12 It may be replaced with .

[0089] In one embodiment, R 4 is =CH2, -CN, halo, C 1~4 Alkyl, C 1~4 Haloalkyl, -OR 4b , -NR 4b R 4c Selected from phenyl or naphthalenyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, tetrahydropyranyl, tetrahydrofuranyl, piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, pyridinyl, pyrazinyl, pyrazolyl, imidazolyl, dihydrobenzofuran, benzodioxolanil, or isoindolinyl, where any cyclic group is unsubstituted or has 1, 2, or 3 R groups. 12 It has been replaced with.

[0090] In one embodiment, R 4 is =CH2, -NR 4b R 4c, selected from phenyl or naphthalenyl, tetrahydropyranil, tetrahydrofuranil, piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, pyridinyl, pyrazinyl, pyrazolyl, imidazolyl, dihydrobenzofuran, benzodioxolanil, or isoindolinyl, where any cyclic group is unsubstituted or has 1, 2, or 3 R groups 12 It has been replaced with.

[0091] In one embodiment, R 12 They are independent, Hello, C 1~4 Alkyl, C 1~4 Haloalkyl, -OR 13 -CN, -C(O)R 10 ,=O,SO2R 10 Selected from benzyl, phenyl, unsubstituted 5- or 6-membered heteroaryls, or methyl-substituted 5- or 6-membered heteroaryls. In some cases, R 12 The following are independently selected from Cl, Br, F, CF3, OMe, OEt, OPh, CN, SO2Me, methyl, pyridinyl, or methylpyrazole.

[0092] In one embodiment, R 4a is H, OH, or F (preferably H), and -L-(CR c R d ) n -R 4is selected from -CF3, -OH, -NH2, =CH2, -CN, -NHC(O)Me, -NHC(O)Ph, -NHC(O)Bn, -NHC(O)CH2CH2Ph, -NHC(O)(CH2)3Ph, -NHC(O)OMe, -NHC(O)NHMe, -N(C(O)Me)benzyl, -N(C(O)Me)CH2pyridinyl, -N(Me)cyclohexyl, phenyl, isoindoline, piperazine, benzyl, -CH2phenyl, -CH2pyridinyl, -CH2cyclopentyl, -CH2tetrahydropyranyl, -CH2pyrazolyl, -CH2dihydrobenzofuran, -CH2imidazolyl, -CH2benzodioxolanyl, -NHcyclohexane, -NHpyrazinyl, -NHCH2Ph, -NHCH2cyclohexane, -NHCH2CH2Ph, and -NHCH2CH2CH2Ph, wherein any of the above cyclic groups is unsubstituted or substituted with 1, 2 or 3 groups selected from Cl, Br, F, CF3, OMe, OEt, -O-phenyl, -O-benzyl, CN, SO2Me, methyl, pyridinyl, or methylpyrazole.

[0093] In an embodiment, R 4a is H, OH, or F (preferably H), and -L-(CR c R d ) n -R 4 is selected from =CH2, -NHC(O)Ph, -NHC(O)Bn, -NHC(O)CH2CH2Ph, -NHC(O)(CH2)3Ph, -NHC(O)NHMe, -N(C(O)Me)benzyl, -N(C(O)Me)CH2pyridinyl, phenyl, isoindoline, piperazine, benzyl, -CH2phenyl, -CH2pyridinyl, -CH2tetrahydropyranyl, -CH2pyrazolyl, -CH2dihydrobenzofuran, -CH~2imidazolyl, -CH2benzodioxolanyl, -NHpyrazinyl, -NHCH2Ph, -NHCH2CH2Ph, and -NHCH2CH2CH2Ph, Here, any of the above cyclic groups is either unsubstituted or substituted with one, two, or three groups selected from Cl, Br, F, CF3, OMe, OEt, -O-phenyl, -O-benzyl, CN, SO2Me, methyl, pyridinyl, or methylpyrazole.

[0094] In one embodiment, R 4a H, and -L-(CR c R d ) n -R 4 teeth, [ka] That is the case.

[0095] In the embodiment, the compound of formula (I) is the compound of formula (Xa) or (Xb): [ka] That is the case.

[0096] In the embodiment, the compound of formula (I) is the compound according to formula (XIa) or (XIb): [ka] That is the case.

[0097] In one embodiment, R 1 -NR is either substituted or non-substituted. 8 R 9 Selected from 5-10 membered carbon ring systems or 5-6 membered heterocyclic ring systems; If it is substituted, R 1 is =O, CN, -OH, or -OC 1~6 alkyl, halo, C 1~6 Alkyl, and C 1~6 It is substituted with one, two, or three groups selected from haloalkyl groups.

[0098] In one embodiment, R 1 -NR is either substituted or non-substituted. 8 R9 If substituted, R is selected from 5-10 member cycloalkyl groups or 5-6 member heterocycloalkyl groups. 1 is =O, CN, -OH, or -OC 1~6 alkyl, halo, C 1~6 Alkyl, and C 1~6 It is substituted with one, two, or three groups selected from haloalkyl groups.

[0099] In one embodiment, R 1 -NR is either substituted or non-substituted. 8 R 9 If R is substituted with a 5- or 6-membered carboncyclic group or a 5- or 6-membered heterocyclic group, 1 is =O, CN, -OH, or -OC 1~6 alkyl, halo, C 1~6 Alkyl, and C 1~6 It is substituted with one, two, or three groups selected from haloalkyl groups.

[0100] In one embodiment, R 1 -NR is either substituted or non-substituted. 8 R 9 If R is substituted, selected from a 6 or 10-membered aryl, a 5, 6 or 9-membered heteroaryl, or a 3- to 7-membered heterocycloalkyl, 1 is =O, CN, -OH, or -OC 1~6 alkyl, halo, and C 1~6 It is substituted with one, two, or three groups selected from alkyl groups.

[0101] In one embodiment, R 1 -NR is either substituted or non-substituted. 8 R 9 If R is substituted, selected from 6 or 10-membered aryls, 5 or 6-membered heteroaryls, or 3-7 membered heterocycloalkyls, 1 is =O, CN, -OH, or -OC 1~6 alkyl, halo, and C 1~6 It is substituted with one, two, or three groups selected from alkyl groups.

[0102] In one embodiment, R 1 is neither a 10-membered aryl group nor an 8, 9, 10, or 13-membered heteroaryl group. In the embodiment, R 1 It is not a 10-membered aryl group. In the embodiment, R 1 It is not a 9-membered heteroaryl group. In the embodiment, R 1 It is not an 8, 10, or 13-membered heteroaryl group.

[0103] In one embodiment, R 1 It is not indole. In the embodiment, R 1 It is not a pyrazole.

[0104] R 1 -NMe2, -N(Me)i-Pr, -NH-cyclopropyl, cyclopropyl, phenyl, pyridinyl, pyridinonyl, pyrimidinyl, imidazolyl, oxazolyl, pyrrolidinyl, methylpyrrolidinyl, fluoropyrrolidinyl, azetidinyl, piperidinyl, piperazinyl, azepanil, indoline, tetrahydronaphthalenyl, or [ka] It can be selected from the following.

[0105] R 1 It may be substituted with a group selected from F, CN, =O, -OH, -OCF3, -OMe, Me, i-Pr, or -CF3.

[0106] Preferably, R 1 R may be selected from phenyl, pyridinyl, piperidinyl, pyrrolidinyl, or methylpyrrolidinyl, where R 1 It is either unsubstituted or substituted with a group selected from F, CN, -OH, -OCF3, -OMe, Me, i-Pr, or -CF3.

[0107] In this embodiment, the compound of formula (I) is [ka] JPEG0007901865000029.jpg232169 JPEG0007901865000030.jpg76169 JPEG0007901865000031.jpg251169 JPEG0007901865000032.jpg232169 JPEG0007901865000033.jpg207169 JPEG0007901865000034.jpg226169 JPEG0007901865000035.jpg219169 JPEG0007901865000036.jpg219169 JPEG0007901865000037.jpg219169 JPEG0007901865000038.jpg219169 JPEG0007901865000039.jpg213169 JPEG0007901865000040.jpg219169 JPEG0007901865000041.jpg219169 JPEG0007901865000042.jpg207169 JPEG0007901865000043.jpg226169 JPEG0007901865000044.jpg219169 JPEG0007901865000045.jpg226169 JPEG0007901865000046.jpg226169 JPEG0007901865000047.jpg238169 JPEG0007901865000048.jpg238169 JPEG0007901865000049.jpg213169 JPEG0007901865000050.jpg232169 JPEG0007901865000051.jpg238169 JPEG0007901865000052.jpg232169 JPEG0007901865000053.jpg232169 JPEG0007901865000054.jpg238169 Selected from JPEG0007901865000055.jpg219169.

[0108] As will be apparent to those skilled in the art, the compounds of the present invention contain several stereocenters. The present invention encompasses all possible stereoisomers of the present invention, whether in a single stereoisomer or a mixture thereof. Preferred stereoisomers are S enantiomers at the 2-position of the pyrrolidine ring. For example: [ka]

[0109] -NR 2 R 3 The preferred stereochemistry of the group is R. For example: [ka]

[0110] Therefore, in one embodiment, the compound of the present invention has an S configuration and -NR at the 2 position of pyrrolidine. 2 R 3 It is a diastereomer having an R configuration in the group. Therefore, the compound of formula (I) is [ka] It is possible.

[0111] In one embodiment, pyrrolidine -L-(CR c R d ) n -R 4 The base has a transform relationship with the base at position 2 if position 2 is in an S configuration. For example: [ka]

[0112] In one embodiment, the compound of the present invention has the stereochemistry shown below.

[0113] [ka]

[0114] In one embodiment, -(CR) is substituted on the pyrrolidine of a compound such as formula (IIa). a R b )-XR 1 The group has a cis relationship with the -C(=O)- group at position 2. For example: [ka]

[0115] In one embodiment, R 1 This is not indolin. In the embodiment, R 1 It is not a 9-membered bicyclic heteroaromatic group.

[0116] In one embodiment of the present invention, a compound of the present invention is provided for use as a pharmaceutical.

[0117] In another embodiment, the present invention provides a pharmaceutical formulation comprising the compound of the present invention and a pharmaceutically acceptable excipient.

[0118] In one embodiment, the pharmaceutical composition may be a combination product containing additional pharmaceutically active agents.

[0119] In a preferred embodiment of the present invention, the compound is a selective FXIIa inhibitor. The term "selective FXIIa inhibitor" means a compound that selectively inhibits FXIIa compared to thrombin and FXa. Generally, the compounds of the present invention may have at least 10 times, preferably at least 100 times, selectivity for FXIIa compared to thrombin.

[0120] According to another aspect of the present invention, compounds of the present invention are provided for use in the prevention or treatment of symptoms modulated by factor XIIa. Symptoms preventable or treatable by modulation of factor XIIa are generally considered to be symptoms preventable or treatable by inhibition of factor XIIa. Therefore, compounds of the present invention may be used for use in the prevention or treatment of symptoms preventable or treatable by inhibition of factor XIIa.

[0121] The compounds of this invention are used for thrombosis, deep vein thrombosis, complex left-sided ablation (pulmonary vein isolation; VT ablation), reperfusion injury also known as ischemic reperfusion injury, transcatheter aortic valve replacement (TAVR) also known as transcatheter aortic valve implantation (TAVI), spinal or epidural anesthesia, lumbar puncture diagnosis, thoracic surgery, abdominal surgery, major orthopedic surgery, liver biopsy, transurethral resection of the prostate, kidney biopsy, renal insufficiency, liver disease, endoscopic biopsy, prostate or bladder biopsy, and electrical stimulation for supraventricular tachycardia. Physiological studies or radiofrequency catheter ablation (including left-sided ablation via a single transseptal puncture), angiography, pacemaker or implantable cardioverter-defibrillator (ICD) implantation (unless it is a complex anatomical situation, e.g., congenital heart disease), mechanical valve implantation, prosthetic valve implantation, myocardial infarction, angina pectoris (including unstable angina), reocclusion and restenosis after angioplasty or coronary artery bypass, stroke, patients with atrial fibrillation to reduce the risk of stroke, atrial fibrillation It may be used in the treatment or prevention of, or in combination therapy for, conditions in which inhibition of FXIIa may be beneficial, such as Alzheimer's disease, vascular dementia, macular degeneration, diabetic retinopathy, diabetic macular edema, cerebral edema in stroke, edema of other causes, hereditary angioedema or acquired angioedema, or in combination therapy for, or in combination therapy for, conditions in which inhibition of FXIIa may be beneficial, such as, Alzheimer's disease, vascular dementia, macular degeneration, diabetic retinopathy, diabetic macular edema, cerebral edema in stroke, edema of other causes, hereditary angioedema or acquired angioedema.

[0122] Symptoms that can be prevented or treated by inhibiting factor XIIa may be those related to blood thickening, blood clotting, or blood clot formation, for example, the symptom may be thrombosis.

[0123] Embodiments of the present invention provide compounds for use in the prevention or treatment of high-risk bleeding, low-risk bleeding, or symptoms associated with thromboembolic disorders, or for use as combination therapies for these purposes.

[0124] Embodiments of the present invention provide compounds for use in the prevention or treatment of high-risk bleeding and associated conditions, or as adjunctive therapies for the same purposes.

[0125] Embodiments of the present invention provide compounds for use in the prevention or treatment of low-risk bleeding and associated conditions, or as adjunctive therapies for the same purposes.

[0126] In embodiments of the present invention, compounds are provided for use in the prevention or treatment of symptoms associated with thromboembolic disorders, or as combination therapies for the same purposes.

[0127] In embodiments of the present invention, the compounds of the present invention are for use as part of the prevention or treatment of high-risk bleeding and associated conditions, wherein the treatment is selected from left-sided complex ablation (pulmonary vein isolation; VT ablation), spinal or epidural anesthesia, lumbar puncture diagnosis, thoracic surgery, abdominal surgery, major orthopedic surgery, liver biopsy, transurethral resection of the prostate, renal biopsy, liver disease, or renal insufficiency.

[0128] In embodiments of the present invention, the compounds of the present invention are intended for use as part of the prevention or treatment of low-risk bleeding and associated conditions, wherein the treatment is selected from endoscopic biopsy, prostate or bladder biopsy, electrophysiological study or radiofrequency catheter ablation for supraventricular tachycardia (including left-sided ablation via a single transseptal puncture), angiography, pacemaker or implantable cardioverter-defibrillator (ICD) implantation (unless there is a complex anatomical situation, e.g., congenital heart disease), mechanical valve implantation, or prosthetic valve implantation.

[0129] In one embodiment, the compounds of the present invention are intended for use to avoid or mitigate contraindications of existing anticoagulant therapies, such as dabigatran, rivaroxaban, apixaban, warfarin, edoxaban, and betrixaban.

[0130] In one embodiment of the present invention, the use of the compounds of the present invention is provided to avoid or mitigate contraindications of existing anticoagulant therapies, such as dabigatran, rivaroxaban, apixaban, warfarin, edoxaban, and betrixaban.

[0131] In one embodiment, the compound of the present invention is intended for use in reducing contraindications of rivaroxaban therapy, where the contraindications include 15 mL / min / 1.73 m 2 This includes individuals with an estimated glomerular filtration rate (eGFR) of less than 50%, active bleeding, current or recent gastrointestinal ulcers, esophageal varices, recent brain or spinal injury, recent brain, spine or ophthalmic surgery, recent intracranial hemorrhage, malignant neoplasms, vascular aneurysms, artificial heart valves, significant risk of major bleeding from liver disease associated with coagulation disorders, and clinically relevant bleeding risks, as well as individuals with Child-Pugh B and C cirrhosis, or those taking any other anticoagulant unless on or switching from warfarin treatment, and those taking potent inhibitors of cytochrome P3A4 enzymes and P-glycoproteins such as ketoconazole, or HIV protease inhibitors such as ritonavir.

[0132] In one embodiment, the compounds of the present invention are intended for use in mitigating contraindications of apixaban therapy, where contraindications include a creatinine clearance (CrCl) of less than 15 mL / min or 15 mL / min / 1.73 m 2This includes individuals with an eGFR of less than 50%, active bleeding, current or recent gastrointestinal ulcers, esophageal varices, recent brain or spinal injury, recent brain, spine or ophthalmic surgery, recent intracranial hemorrhage, malignant neoplasms, vascular aneurysms, liver disease associated with coagulation disorders, and clinically relevant bleeding risks, significant risk of major bleeding such as artificial heart valves, taking any other anticoagulant unless on or switching from warfarin treatment, or taking potent inhibitors of cytochrome P3A4 enzymes and P-glycoproteins such as ketoconazole, or HIV protease inhibitors such as ritonavir.

[0133] In one embodiment, the compounds of the present invention are intended for use in reducing contraindications to the use of edoxaban in treatment, where contraindications include edoxaban not being used in NVAF patients with CrCl greater than 95 mL / min due to an increased risk of ischemic stroke compared to warfarin.

[0134] In one embodiment, the compounds of the present invention are intended for use in reducing contraindications to therapies using dabigatran, where contraindications include: prevention of stroke due to atrial fibrillation (prevention of stroke and systemic embolism associated with non-valvular atrial fibrillation), renal impairment (CrCl < 15 mL / min or dialysis), treatment of DVT or PE (indicated for the treatment of deep vein thrombosis (DVT) and pulmonary embolism (PE) in patients treated with parenteral anticoagulants for 5 to 10 days), CrCl This includes use at a flow rate of 30 mL / min or less or during dialysis, and for the prevention of DVT or PE (indicated for the prevention of deep vein thrombosis (DVT) and pulmonary embolism (PE) after hip replacement surgery). Dabigatran is contraindicated with defibrotide, mifepristone, and human prothrombin complex concentrate. Dabigatran should not be used with antithrombin alfa, antithrombin iii, apixaban, carbamazepine, dalteparin, dexamethasone, doxorubicin, doxorubicin liposome, doronedarone, edoxaban, enoxaparin, fondaparinux, fosphenytoin, heparin, ketoconazole, repiridine, nefazodone, phenobarbital, phenytoin, primidone, rifampin, St. John's wort, tenofovir df, tipranavir, vinblastine, and warfarin.

[0135] In one embodiment, the compounds of the present invention are intended for use in mitigating contraindications to treatment with dabigatran, where contraindications include renal impairment (CrCl < 15 mL / min), hemodialysis, hypersensitivity, active pathological bleeding, impaired hemostasis, mechanical or artificial heart valves, thromboembolic events (e.g., valve thrombosis, stroke, TIA, MI), excessive major bleeding (mainly postoperative pericardial effusion requiring hemodynamic compromise intervention), increased bleeding risk during childbirth and delivery, anticoagulants for active bleeding, elective surgery or invasive procedures, patients at increased risk of stroke, antiplatelet agents, warfarin, heparin, fibrinolytic therapy, and long-term NSAIDs or aspirin. This includes the additional risk of bleeding when administered concurrently with other drugs, congenital or acquired coagulation disorders, ulcerative GI disease and other gastritis-like symptoms, recent bleeding, recent brain, spine, or ophthalmic surgery, patients undergoing spinal anesthesia (spinal / epidural anesthesia), patients undergoing spinal puncture with a risk of developing epidural or spinal hematoma, which may result in prolonged or permanent paralysis, and concurrent administration with P-gp inducers and inhibitors, P-gp inducers (e.g., rifampin), or any combination thereof.

[0136] In one embodiment, the compounds of the present invention are intended for use in reducing contraindications to treatment with betrixaban, which include patients taking P-gp inhibitors, patients with severe renal impairment, patients with hepatic impairment, patients with intrinsic coagulation disorders, or patients with artificial heart valves, co-administration with drugs that affect hemostasis (and thereby increase the risk of bleeding), co-administration with aspirin, co-administration with other antiplatelet agents, co-administration with other anticoagulants, co-administration with heparin, co-administration with thrombolytic agents, co-administration with selective serotonin reuptake inhibitors (SSRIs), co-administration with serotonin-norepinephrine reuptake inhibitors (SNRIs), and co-administration with nonsteroidal anti-inflammatory drugs (NSAIDs).

[0137] In one embodiment, the compounds of the present invention may be used as anticoagulants for the prevention and / or treatment of thromboembolic disorders, where the disorder is one of the following: myocardial infarction, angina pectoris (including unstable angina), reocclusion and restenosis after angioplasty or coronary artery bypass, stroke, patients with atrial fibrillation to reduce the risk of stroke, patients with atrial fibrillation and chronic kidney disease, transient ischemic attack, peripheral artery occlusion disorder, reperfusion injury also known as ischemic reperfusion injury, transcatheter aortic valve replacement (TAVR) also known as transcatheter aortic valve implantation (TAVI), pulmonary embolism, deep vein microangiopathy, or patients requiring extracorporeal membrane oxygenation (ECMO).

[0138] In one embodiment, the compounds according to the present invention may be suitable for preventing and / or treating disseminated intravascular coagulation (DIC).

[0139] In one embodiment, the compounds of the present invention are also suitable for the prevention and / or treatment of atherosclerosis and arthritis, and furthermore, for the prevention and / or treatment of thrombosis in patients with cancer.

[0140] In one embodiment, the compounds of the present invention are intended for use in methods for preventing and / or treating thrombosis.

[0141] In one embodiment of the present invention, the compounds disclosed herein may be used as anticoagulants.

[0142] In one embodiment of the present invention, prevention of thrombosis or deep vein thrombosis, thrombosis, complex left-sided ablation (pulmonary vein isolation; VT ablation), spinal or epidural anesthesia, lumbar puncture diagnosis, thoracic surgery, abdominal surgery, major orthopedic surgery, liver biopsy, transurethral resection of the prostate, kidney biopsy, renal insufficiency, liver disease, endoscopic biopsy, prostate or bladder biopsy, electrophysiological studies or radiofrequency catheter ablation for supraventricular tachycardia (including left-sided ablation via a single transseptal puncture), angiography, pacemaker or implantable cardioverter-defibrillator (ICD) implantation (unless in a complex anatomical situation, e.g., congenital heart disease), mechanical valve implantation, prosthetic valve implantation, myocardial infarction, angina pectoris (including unstable angina), reocclusion and restenosis after angioplasty or coronary artery bypass, stroke, patients with atrial fibrillation to reduce the risk of stroke, atrium A method is provided for the prevention and / or treatment of a patient with fibrillation and chronic kidney disease, transient ischemic attack, peripheral artery occlusion, pulmonary embolism, deep vein microangiopathy, a patient requiring extracorporeal membrane oxygenation (ECMO), a patient requiring extracorporeal circulation such as coronary artery bypass grafting (CABG), disseminated intravascular coagulation (DIC), atherosclerosis, arthritis, thrombosis in patients with cancer, asymptomatic cerebral ischemia, stroke, neurotraumatic injury, neuroinflammatory injury, medical procedures including contact with artificial surfaces including renal dialysis, and other conditions for which inhibition of FXIIa may be beneficial, such as Alzheimer's disease, vascular dementia, macular degeneration, diabetic retinopathy, diabetic macular edema, cerebral edema in stroke, edema of other causes, hereditary angioedema or acquired angioedema, comprising administering a therapeutically effective amount of the compound of the present invention, or administering both therapeutically effective amounts of the compound of the present invention in combination.

[0143] In one embodiment of the present invention, a method for preventing coagulation is provided, comprising administering a therapeutically effective amount of the compound of the present invention.

[0144] In one embodiment of the present invention, a method for preventing and / or treating thrombosis is provided, comprising administering a therapeutically effective amount of the compound of the present invention.

[0145] In one embodiment of the present invention, the use of the compounds of the present invention in the manufacture of a pharmaceutical product for use in the prevention and / or treatment of a condition that is preventable and / or treatable by inhibition of factor XII (and optionally factor XIIa), for example, the condition being thrombosis.

[0146] In another embodiment of the present invention, a pharmaceutical composition comprising the compound of the present invention and a pharmaceutically acceptable excipient is provided.

[0147] In one embodiment, the pharmaceutical composition may be a combination product containing additional pharmaceutically active agents, which may be disclosed elsewhere herein.

[0148] The compounds of the present invention may be used for the prevention and / or treatment of any of the conditions disclosed above. Alternatively, the compounds of the present invention may be used as adjunctive therapy in the prevention and / or treatment of the conditions disclosed above. When the compounds of the present invention are used as adjunctive therapy for a particular condition, this means that the compounds of the present invention may be used in combination with other treatments known in the art for that condition. For example, FXII(a) inhibitor may be used in combination with antiplatelet therapy to enhance the antithrombotic effect without increasing the risk of bleeding compared to antiplatelet therapy alone. Furthermore, FXII(a) inhibitor may be used in combination with other treatments. [Brief explanation of the drawing]

[0149] [Figure 1] This figure shows the anticoagulant effect of compound M05272 administered intravenously at a standard dose in a thrombosis model induced by ferric chloride into the femoral blood vessels. The percentage inhibition of blood clot formation was calculated at 60 minutes compared to mice administered only the vehicle. A minimum of four mice were used in each group. [Modes for carrying out the invention]

[0150] The following are definitions of terms used in this application. Terms not defined herein have the ordinary meanings that a person skilled in the art would expect to understand.

[0151] The term "halo" refers to one of the halogens in Group 17 of the periodic table. In particular, the term refers to fluorine, chlorine, bromine, and iodine. Preferably, the term refers to bromine and iodine.

[0152] The term "alkyl" refers to a linear or branched hydrocarbon chain. For example, the term "C 1~6 "Alkyl" refers to linear or branched hydrocarbon chains containing 1, 2, 3, 4, 5, or 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, and n-hexyl. Alkylene groups can similarly be linear or branched and may have two bonds to the rest of the molecule. Furthermore, an alkylene group may correspond, for example, to one of the alkyl groups listed in this paragraph. Alkyl and alkylene groups may be unsubstituted or substituted with one or more substituents. Possible substituents are listed below. Substituents for alkyl groups include halogens, such as fluorine, chlorine, bromine, and iodine, OH, C 1~6 It could be an alkoxy.

[0153] The term "alkoxy" refers to an alkyl group that is bonded to a molecule via oxygen. For example, the term "C 1~6"Alkoxy" refers to a group in which the alkyl portion may be linear or branched and may contain 1, 2, 3, 4, 5, or 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, and n-hexyl. Therefore, alkoxy groups can be methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-hexoxy. The alkyl portion of the alkoxy group may be unsubstituted or substituted with one or more substituents. Possible substituents are listed below. Substituents for alkyl groups include halogens, such as fluorine, chlorine, bromine, and iodine, OH, C 1~6 It could be an alkoxy.

[0154] The term "haloalkyl" refers to a hydrocarbon chain in which, in each instance, is independently substituted with at least one halogen atom selected from, for example, fluorine, chlorine, bromine, and iodine. For example, the term "C 1~6 "Haloalkyl" refers to a linear or branched hydrocarbon chain containing 1, 2, 3, 4, 5, or 6 carbon atoms, substituted with at least one halogen. The halogen atom may be located at any position in the hydrocarbon chain. For example, C 1~6 Haloalkyl can refer to chloromethyl, fluoromethyl, trifluoromethyl, chloroethyl, e.g., 1-chloromethyl and 2-chloroethyl; trichloroethyl, e.g., 1,2,2-trichloroethyl and 2,2,2-trichloroethyl; fluoroethyl, e.g., 1-fluoromethyl and 2-fluoroethyl; trifluoroethyl, e.g., 1,2,2-trifluoroethyl and 2,2,2-trifluoroethyl; chloropropyl, trichloropropyl, fluoropropyl, and trifluoropropyl.

[0155] The term "alkenyl" refers to a branched or linear hydrocarbon chain containing at least one double bond. For example, the term "C 2~6An "alkenyl" refers to a branched or linear hydrocarbon chain containing at least one double bond and having 2, 3, 4, 5, or 6 carbon atoms. The double bond may exist as an E or Z isomer. The double bond can be at any possible position in the hydrocarbon chain. For example, "C 2~6 "Alkenyl" can be etenyl, propenyl, butenyl, butadienyl, pentenyl, pentadienyl, hexenyl, and hexadienyl.

[0156] The term "alkynyl" refers to a branched or linear hydrocarbon chain containing at least one triple bond. For example, the term "C 2~6 "Alkynnyl" refers to a branched or linear hydrocarbon chain having 2, 3, 4, 5, or 6 carbon atoms and containing at least one triple bond. The triple bond can be at any possible position in the hydrocarbon chain. For example, "C 2~6 "Alkinyl" can be ethinyl, propynyl, butynyl, pentynyl, and hexynyl.

[0157] The term "heteroalkyl" refers to a branched or linear hydrocarbon chain containing at least one heteroatom selected from N, O, and S located between any carbon atoms in the chain or at the end of the chain. For example, the term "C 1~6 A "heteroalkyl" refers to a branched or linear hydrocarbon chain containing 1, 2, 3, 4, 5, or 6 carbon atoms, plus at least one heteroatom selected from N, O, and S located any between carbon atoms in the chain or at the ends of the chain. For example, a hydrocarbon chain may contain 1 or 2 heteroatoms. 1~6 Heteroalkyls can bond to the rest of the molecule through carbon or heteroatoms. For example, "C 1~6 "Heteroalkyl" is C 1~6 N-alkyl, C 1~6 N,N-alkyl, or C 1~6 It can be O-alkyl.

[0158] The term "carbocyclic" refers to a saturated or unsaturated carbon-containing cyclic system. A "carbocyclic" system can be a monocyclic or a fused polycyclic system, such as a bicyclic or tricyclic system. The "carbocyclic" portion may contain 3 to 14 carbon atoms, for example, 3 to 8 carbon atoms in a monocyclic system and 7 to 14 carbon atoms in a polycyclic system. "Carbocyclic" systems include fused cyclic systems containing cycloalkyl, cycloalkenyl, aryl, and aromatic moies.

[0159] The term "heterocyclic" refers to a saturated or unsaturated cyclic system containing at least one heteroatom selected from N, O, or S. A "heterocyclic" system may contain one, two, three, or four heteroatoms, for example, one or two. A "heterocyclic" system may be monocyclic or fused polycyclic, for example, bicyclic or tricyclic. A "heterocyclic" moiety may contain 3 to 14 carbon atoms, for example, 3 to 8 carbon atoms in a monocyclic system and 7 to 14 carbon atoms in a polycyclic system. "Heterocyclic" includes heterocycloalkyl moies, heterocycloalkenyl moies, and heteroaromatic moies. For example, heterocyclic groups can be oxirane, aziridine, azetidine, oxetane, tetrahydrofuran, pyrrolidine, imidazolidine, succinimide, pyrazolidine, oxazolidine, isoxazolidine, thiazolidin, isothiazolidine, piperidine, morpholine, thiomorpholine, piperazine, and tetrahydropyran.

[0160] The term cycloalkyl refers to a saturated hydrocarbon ring system. For example, "C 3~8 A "cycloalkyl" refers to a ring system containing 3, 4, 5, 6, 7, or 8 carbon atoms. For example, "C 3~8 "Cycloalkyl" can be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0161] The term “C 3~8 A "cycloalkenyl" refers to a non-aromatic unsaturated hydrocarbon ring system containing 3, 4, 5, 6, 7, or 8 carbon atoms. The ring may contain two or more double bonds, however the ring system is not aromatic. For example, "C3~8 "Cycloalkyl" can be cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, cycloheptadiene, cyclooctenyl, and cyclooctadienyl.

[0162] The term "heterocycloalkyl" refers to a saturated hydrocarbon ring system containing carbon atoms and at least one heteroatom in the ring selected from N, O, and S. For example, there may be one, two, or three heteroatoms, and possibly one or two. A "heterocycloalkyl" can be bonded to the rest of the molecule through any carbon or heteroatom. A "heterocycloalkyl" may have one or more bonds to the rest of the molecule, for example, one or two bonds, and these bonds may be through any of the atoms of the ring. For example, a "heterocycloalkyl" is a "C 3~8 It could be a heterocycloalkyl compound. 3~8 A "heterocycloalkyl" refers to a saturated hydrocarbon ring system containing 3, 4, 5, 6, 7, or 8 atoms, where at least one of the atoms is a heteroatom in the ring selected from N, O, and S. "Heterocycloalkyl" can be oxiran, aziridine, azetidine, oxetane, tetrahydrofuran, pyrrolidine, imidazolidine, succinimide, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, piperidine, morpholine, thiomorpholine, piperazine, and tetrahydropyran.

[0163] The term "heterocycloalkenyl" refers to a non-aromatic unsaturated hydrocarbon ring system containing a carbon atom and at least one heteroatom selected from N, O, and S within the ring. For example, there may be one, two, or three heteroatoms, and possibly one or two. A "heterocycloalkenyl" can be bonded to the rest of the molecule through any carbon atom or heteroatom. A "heterocycloalkenyl" may have one or more bonds to the rest of the molecule, for example, one or two bonds, and these bonds may be through any of the atoms of the ring. For example, a "heterocycloalkenyl" may be "C 3~8 It could be a heterocycloalkenyl. Term "C 3~8 A "heterocycloalkenyl" refers to a saturated hydrocarbon ring system containing 3, 4, 5, 6, 7, or 8 atoms, where at least one of the atoms is a heteroatom selected from N, O, and S. "Heterocycloalkenyls" can be tetrahydropyridines, dihydropyrans, dihydrofurans, or pyrrolines.

[0164] The term "aromatic," when applied to the entire substituent, refers to a single ring or polycyclic ring system in which the conjugate π system within the ring or ring system has 4n+2 electrons and all atoms contributing to the conjugate π system are in the same plane.

[0165] The term "aryl" refers to an aromatic hydrocarbon ring system. The ring system has 4n+2 electrons in the conjugate π system within the ring, and all atoms contributing to the conjugate π system are in the same plane. For example, "aryl" can be phenyl and naphthyl. The aryl system itself may be substituted with other groups. The term "aryl" also includes bicyclic or tricyclic ring systems that are not fully aromatic but contain an aromatic ring within the ring system, such as indanes or tetralines.

[0166] The term "heteroaryl" refers to an aromatic hydrocarbon ring system having at least one heteroatom selected from O, N, and S, either within a single ring or a fused ring system. The ring or ring system has 4n+2 electrons in the conjugate π system, and all atoms contributing to the conjugate π system are in the same plane. For example, "heteroaryls" may include imidazole, thien, furan, thiantrene, pyrrole, benzimidazole, pyrazole, pyrazine, pyridine, pyrimidine, and indole. The term "heteroaryl" also includes bicyclic or tricyclic ring systems that are not fully aromatic but contain an aromatic ring. The heteroatom can be present in the ring system, either in the aromatic or non-aromatic ring. For example, heteroaryls also include chromene, chroman, indoline, and tetrahydroquinoline.

[0167] The term "halogen" in this specification includes references to F, Cl, Br, and I. A halogen may be Br. A halogen may be I.

[0168] The end is [ka] A bond that is " " indicates that the bond is connected to another atom not shown in the structure. A bond whose ends are inside the cyclic structure and whose ends are not atoms of the cyclic structure indicates that the bond may connect to any of the atoms in the cyclic structure, provided that the valence allows it.

[0169] Solid and dashed lines represent bonds that, if chemically possible, can be either single or double bonds. For example, the bonds shown below can be either single or double bonds.

[0170] [ka]

[0171] If a portion is substituted, it may be substituted at any position of the portion, provided that it is chemically possible and matches the valence conditions of the atom. The portion may be substituted with one or more substituents, e.g., 1, 2, 3, or 4 substituents, and the group may have 1 or 2 substituents. If there are two or more substituents, the substituents may be the same or different. Substituents include OH, NHR, amidino, guanidino, hydroxyguanidino, formamidino, isothioureido, ureido, mercapto, C(O)H, acyl, acyloxy, carboxy, sulfo, sulfamoyl, carbamoyl, cyano, azo, nitro, halo, and C. 1~6 Alkyl, C 1~6 Alkoxy, C 1~6 Haloalkyl, C 3~8 Cycloalkyl, C 2~6 Alkenil, C 2~6 The substituents can be selected from alkynyl, aryl, heteroaryl, or alkaryl. If the substituted group is an alkyl group, the substituent can be =O. R is H, C 1~6 Alkyl, C 3~8 The group can be selected from cycloalkyl, phenyl, benzyl, or phenethyl groups, for example, R is H, or C 1~3 It is alkyl. If the part is substituted with two or more substituents, and two of the substituents are adjacent, the adjacent substituents are C together with the atoms of the substituted part. 4~8 A ring may be formed, where C 4~8 The ring is a saturated or unsaturated hydrocarbon ring containing 4, 5, 6, 7, or 8 carbon atoms, or a saturated or unsaturated hydrocarbon ring containing 4, 5, 6, 7, or 8 carbon atoms and 1, 2, or 3 heteroatoms.

[0172] Substituents exist only at positions where they are chemically possible, and a person skilled in the art can determine which substituents are chemically possible and which are not without unnecessary effort (either experimentally or theoretically).

[0173] Ortho, meta, and para substitutions are well understood in the art. To avoid any doubt, an "ortho" substitution is when the adjacent carbon is a single group, for example, a fluoro group in the following example, or [ka] As indicated by the bonded end of the molecule, this is a substitution pattern with substituents regardless of the rest of the molecule.

[0174] [ka]

[0175] A "meta" substitution is a substitution pattern in which two substituents lie on carbon atoms from which one carbon atom has been removed; in other words, there is a single carbon atom between the substituted carbon atoms. In other words, the substituent lies on a second atom separate from the atom with the other substituent. For example, the following groups are meta-substituted.

[0176] [ka]

[0177] A "para" substitution is a substitution pattern in which two substituents lie on carbon atoms from which two carbon atoms have been removed from each other; in other words, there are two carbon atoms between the substituted carbon atoms. In other words, the substituents are located on a third atom separate from the atom with the other substituent. For example, the following groups are para-substituted.

[0178] [ka]

[0179] "Acyl" refers to an organic radical derived from an organic acid by, for example, the removal of a hydroxyl group, such as the formula RC(O)-(wherein R is H, C). 1~6 Alkyl, C 3~8The group can be selected from cycloalkyl, phenyl, benzyl, or phenethyl groups, for example, R is H or C 1~3 This refers to a radical having an alkyl group. In one embodiment, the acyl is an alkyl-carbonyl group. Examples of acyl groups include, but are not limited to, formyl, acetyl, propionyl, and butyryl. A specific acyl group is acetyl.

[0180] Throughout this description, the present disclosure of compounds also includes pharmaceutically acceptable salts, solvates, and stereoisomers thereof. Where a compound has a stereocenter, both (R) and (S) stereoisomers are contemplated by the present invention, and similarly, mixtures or racemic mixtures of stereoisomers are completed by this application. Where a compound of the present invention has two or more stereocenters, any combination of (R) and (S) stereoisomers is contemplated. Combinations of (R) and (S) stereoisomers may result in a mixture of diastereomers or a single diastereoisomer. The compounds of the present invention may exist as a single stereoisomer, or as a mixture of stereoisomers, e.g., a racemic mixture and a mixture of other enantiomers, as well as a mixture of diastereomers. Where the mixture is a mixture of enantiomers, the enantiomer excess may be any of those disclosed above. Where the compound is a single stereoisomer, the compound may still contain other diastereomers or enantiomers as impurities. Therefore, a single stereoisomer does not necessarily have a 100% enantiomer excess (ee) or diastereomer excess (de), but may have at least about 85%, at least about 60%, or less than that ee or de. For example, the ee or de could be 90% or more, 90% or more, 80% or more, 70% or more, 60% or more, 50% or more, 40% or more, 30% or more, 20% or more, or 10% or more.

[0181] The present invention aims to provide pharmaceutically acceptable salts of the compounds of the present invention. These may include acid adducts and base salts of the compounds. These may be acid adducts and base salts of the compounds. Furthermore, the present invention aims to provide solvates of the compounds. These may be hydrates or other solvated forms of the compounds.

[0182] Suitable acid addition salts are formed from acids that form non-toxic salts. Examples include acetates, aspartates, benzoates, besilates, bicarbonates / carbonates, bisulfates / sulfates, borates, cansilates, citrates, edisylates, esylates, formates, fumarates, gluceptates, glucons, glucurons, hexafluorophosphates, hibenzates, hydrochlorides / chlorides, hydrobroms / bromids, hydroiodides / iodides, isethionates, lactates, malates, maleates, malons, mesilates, methyl sulfates, naphthylates, 1,5-naphthalenedisulfonates, 2-napsylates, nicotinates, nitrates, orotates, oxalates, palmitates, pamoates, phosphates / hydrogen phosphates / dihydrogen phosphates, sugarates, stearates, succinates, tartrates, tosylates, and trifluoroacetates.

[0183] Suitable base salts are formed from bases that form non-toxic salts. Examples include aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc salts. Hemi salts of acids and bases, such as hemisulfates and hemicalcium salts, may also be formed. For an overview of suitable salts, see "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

[0184] The pharmaceutically acceptable salts of the compound of formula (I) are: (i) by reacting the compound of the present invention with a desired acid or base; (ii) By removing an acid or base unstable protecting group from a suitable precursor of the compound of the present invention, or by opening the ring of a suitable cyclic precursor, such as a lactone or lactam, using a desired acid or base; or (iii) By converting one salt of the compound of the present invention to another salt by reaction with a suitable acid or base, or by a suitable ion exchange column. It can be prepared by one or more of the following three methods.

[0185] All three reactions are typically carried out in solution. The resulting salts can precipitate and be collected by filtration, or recovered by evaporation of the solvent. The degree of ionization of the resulting salts can vary from complete ionization to nearly non-ionization.

[0186] The compounds of the present invention may exist in both non-solvated and solvated forms. The term "solvate" is used herein to describe a molecular complex comprising the compounds of the present invention and one or more pharmaceutically acceptable solvent molecules, such as ethanol, in stoichiometric amounts. The term "hydrate" is used when the solvent is water.

[0187] Complexes such as clathrates and drug-host inclusion complexes are included within the scope of the present invention, where, in contrast to the solvates described above, the drug and host are present in stoichiometric or non-stoichiometric quantities. Drug complexes containing two or more organic and / or inorganic components are also included, which may be in stoichiometric or non-stoichiometric quantities. The resulting complexes may be ionized, partially ionized, or non-ionized. For an overview of such complexes, see Haleblian (August 1975) in J Pharm Sci, 64 (8), 1269-1288.

[0188] Hereafter, all references to any compound in any given formula include references to its salts, solvates, and complexes, as well as references to the solvates and complexes of its salts.

[0189] The compounds of the present invention include all of their polymorphs and crystal habits, their prodrugs and isomers (including optical, geometric and tautomers) as defined below, and compounds of several formulas as defined herein, including the isotope-labeled compounds of the present invention.

[0190] The present invention also includes all pharmaceutically acceptable isotope-labeled compounds of the present invention, in which one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature.

[0191] Examples of isotopes suitable for inclusion in the compound of the present invention include: 2 H and 3 Hydrogen such as H, 11 C, 13 C and 14 Carbon such as C, 36 Chlorine such as Cl 18 Fluorine such as F 123 I and 125 Iodine such as I 13 N and 15 Nitrogen such as N, 15 O, 17 O and 18 Oxygen such as O, 32 Phosphorus such as P, and 35 It contains sulfur, such as S.

[0192] Certain isotope-labeled compounds, for example, those incorporating radioactive isotopes, are useful in drug and / or substrate tissue distribution studies. One such radioactive isotope is tritium, i.e. 3 H, and carbon-14, i.e. 14 C is particularly useful for this purpose due to its ease of integration and the availability of off-the-shelf detection methods.

[0193] Deuterium, that is 2Substitution with heavier isotopes, such as 1H, may yield certain therapeutic benefits resulting from greater metabolic stability, such as an increased in vivo half-life or reduced dosage requirements, and may therefore be preferable in some situations.

[0194] Before purification, the compounds of the present invention may exist as a mixture of enantiomers depending on the synthetic procedure used. These enantiomers can be separated by prior art known in the art. Therefore, the present invention encompasses not only individual enantiomers but also mixtures thereof.

[0195] For some process steps in the preparation of the compounds of the present invention, it may be necessary to protect potential reactive functional groups that are undesirable to react, and consequently cleave the protecting groups. In such cases, any suitable protective radical can be used. Specific methods of protection and deprotection can be used, such as those described by TW GREENE (Protective Groups in Organic Synthesis, A. Wiley International Science Publication, 1981) or PJ Kocienski (Protecting groups, Georg Thieme Verlag, 1994). All preparations of the novel starting materials used in the above reactions and preceding methods are conventional and appropriate reagents and reaction conditions for their performance or preparation, and the procedures for isolating the desired product are well known to those skilled in the art by the conventions of the literature, as well as by reference to its examples and preparations.

[0196] Furthermore, the compounds of the present invention and the intermediates for their preparation can be purified by various well-known methods, such as crystallization or chromatography.

[0197] One or more compounds of the present invention may be combined with one or more pharmaceuticals, such as anti-inflammatory agents, anti-fibrotic agents, chemotherapy, anticancer agents, immunosuppressants, antitumor vaccines, cytokine therapy, or tyrosine kinase inhibitors, for the treatment of symptoms mediated by inhibition of ROCK, such as fibrotic diseases, autoimmune inflammatory fibrosis, inflammatory symptoms, central nervous system disorders, or cancer.

[0198] Such combination treatments can be achieved by simultaneous, sequential, or separate administration of the individual components of the treatment. Such combination products utilize the compounds of the present invention within the therapeutically effective dosage range already described, and other pharmaceutically active agents within the approved dosage range.

[0199] The compounds of the present invention can be administered in vivo either alone or in combination with other pharmaceutically active agents, for example, agents particularly effective in treating and / or preventing the aforementioned diseases. Preferred combinations consist of the compounds of the present invention and one or more active substances, the active substances of which, for example, preferably, are lipid-lowering agents, particularly HMG-CoA-(3-hydroxy-3-methylglutaryl-coenzyme A)-reductase inhibitors; coronary artery treatment / vasodilators, particularly ACE (angiotensin-converting enzyme) inhibitors; AII (angiotensin II) receptor antagonists; β-adrenergic receptor antagonists; alpha-1-adrenergic receptor antagonists; urination promoters; calcium channel blockers; cyclic guanosine monophosphate (cOMP). Substances that increase thrombolytic activity, such as soluble guanylate cyclase stimulants; plasminogen activators (thrombolytic agents / fibrinolytic agents) and thrombolytic / fibrinolytic-increasing compounds, such as inhibitors of plasminogen activator inhibitors (PAI inhibitors) or inhibitors of thrombin-activated fibrinolytic inhibitors (TAFI); substances having anticoagulant activity (anticoagulants); substances that inhibit platelet aggregation (platelet aggregation inhibitors, thrombus aggregation inhibitors); and fibrinogen receptor antagonists (glycoprotein IIb / IIIa antagonists).

[0200] Since cancer patients may have thrombosis-promoting symptoms and require anticoagulants, the compounds of the present invention may be advantageous for cancer treatment. This usually needs to be balanced with the risk of bleeding, and therefore, the compounds described herein provide safer anticoagulants in cancer patients due to the reduced risk of bleeding. For cancer treatment, the compounds of the present invention may be administered in combination with known cancer treatments.

[0201] The compounds of the present invention may exist in a single crystalline form or as a mixture of crystalline forms, or they may be amorphous. Therefore, the compounds of the present invention intended for pharmaceutical use may be administered as crystalline or amorphous products. They can be obtained, for example, as solid plugs, powders, or films by methods such as precipitation, crystallization, freeze-drying, spray-drying, or evaporative drying. Microwave or radio frequency drying may also be used for this purpose.

[0202] With respect to the compounds of the present invention listed above, the dosage administered will naturally vary depending on the compound used, the method of administration, the desired treatment, and the disorder being treated. A person skilled in the art of drug dosage would readily be able to identify a suitable dosage, for example, the dosage could be a standard dosage.

[0203] The compounds of the present invention, or pharmaceutically acceptable salts thereof, can be used on their own, but are generally administered in the form of pharmaceutical compositions in which the compounds of the present invention, or pharmaceutically acceptable salts thereof, are associated with pharmaceutically acceptable adjuvants, diluents, or carriers. Conventional procedures for selecting and preparing suitable pharmaceutical compositions are described, for example, in "Pharmaceuticals - The Science of Dosage Form Designs", ME Aulton, Churchill Livingstone, 1988.

[0204] Depending on the method of administering the compound of the present invention, the pharmaceutical composition used to administer the compound of the present invention preferably contains 0.05 to 99% w (weight percent) of the compound of the present invention, more preferably 0.05 to 80% w of the compound of the present invention, even more preferably 0.10 to 70% w of the compound of the present invention, and even more preferably 0.10 to 50% w of the compound of the present invention, all weight percentages being based on the total composition.

[0205] Pharmaceutical compositions may be administered topically (e.g., on the skin) in the form of, for example, creams, gels, lotions, solutions, or suspensions; systemically, for example, by oral administration, in the form of tablets, capsules, syrups, powders, or granules; parenterally (including intravenous, subcutaneous, intramuscular, intravascular, or injectable) in the form of sterile solutions, suspensions, or emulsions for injection; rectally in the form of suppositories; or by inhalation in the form of aerosols.

[0206] For oral administration, the compounds of the present invention may be mixed with an adjuvant or carrier, such as lactose, sucrose, sorbitol, or mannitol; starch, such as potato starch, corn starch, or amylopectin; a cellulose derivative; a binder, such as gelatin or polyvinylpyrrolidone; and / or a lubricant, such as magnesium stearate, calcium stearate, polyethylene glycol, wax, or paraffin, and then compressed into tablets. If coated tablets are required, the cores prepared as described above may be coated with a concentrated sugar solution, for example, one containing gum arabic, gelatin, talcum, and titanium dioxide. Alternatively, the tablets may be coated with a suitable polymer dissolved in an easily evaporable organic solvent.

[0207] For the preparation of soft gelatin capsules, the compounds of the present invention may be mixed with, for example, vegetable oil or polyethylene glycol. Hard gelatin capsules may contain granules of the compounds using any of the tablet excipients described above. Liquid or semi-solid formulations of the compounds of the present invention may also be filled into hard gelatin capsules. Liquid preparations for oral administration may be in the form of a syrup or suspension, for example, a solution comprising the compounds of the present invention, with the remainder being sugar, as well as a mixture of ethanol, water, glycerol, and propylene glycol. Optionally, such liquid preparations may contain carboxymethylcellulose or other excipients known to those skilled in the art as colorants, flavorings, sweeteners (e.g., sucrose), preservatives, and / or thickeners.

[0208] For intravenous (parenteral) administration, the compounds of the present invention may be administered as a sterile aqueous solution or an oily solution.

[0209] The therapeutic dose size of the compounds of the present invention varies naturally according to well-known medical principles, depending on the nature and severity of the symptoms, the age and sex of the animal or patient, and the route of administration.

[0210] The dosage level, frequency of administration, and duration of treatment of the compounds of the present invention are expected to vary depending on the formulation and clinical indication, the patient's age, and any co-existing medical conditions. The standard duration of treatment with the compounds of the present invention may be any length of time. For example, the treatment period may be in days, weeks, months, or years. Treatment may be indefinite. For most clinical indications, treatment is thought to be 1 to 7 months. In cases of recurrent infection or infection related to tissues or implant materials, including bone / joint, respiratory, endocardium, and dental tissue, where blood supply is insufficient, the treatment period may need to be extended beyond 7 days. [Examples]

[0211] Examples and Synthesis ¹H-NMR: Spectra are obtained using a Bruker DRX 400 MHz or Jeol ECS 400 MHz spectrometer. Spectra are measured at 294 K (unless otherwise specified), and chemical shifts (δ values) are reported in parts per million (ppm) with reference to one of the following: TMS (0.0 ppm), DMSO-d6 (2.50 ppm), or CDCl3 (7.26 ppm). Coupling constants (J) are reported in Hertz (Hz), and spectral resolving patterns are shown as singlelines (s), doublelines (d), triplelines (t), quadruplelines (q), multi-line or more overlap signals (m), and broadband signals (br). The solvent is indicated in parentheses.

[0212] abbreviation The following abbreviations are used in the examples and other parts of this description.

[0213] ABCN: Azobiscyclohexanecarbonyl; Boc: tert-butoxycarbonyl; Cbz: Carbobenzyloxy; DavePhos: 2-Dichlorohexylphosphino-2'-(N,N-dimethylamino)biphenyl; dba: Tris(dibenzylideneacetone); DBU: 1,8-Diazabicyclo[5.4.0]undeca-7-ene; DCE: 1,2-Dichloroethane; DCM: Dichloromethane; DIAD: Diisopropylazodicarboxylate; Dioxane: 1,4-Dioxane; DIPEA: Diisopropylethylamine; DMA: Dimethylacetamide; DMAP: 4-(Dimethylamino)pyridine; DMF: N,N-Dimethylformamide; DMS: Dimethyl sulfide; DMSO: Dimethyl sulfoxide; Dppf: 1,1'-Bis(diphenylphosphino)ferrocene; dtbpf: ([1,1'-Bis(di-tert-butylphosphino)ferrocene]; Â: Ethyl acetate; Fmoc: 9-Fluorenyl methoxycal Bonyl; h: hour; HATU: 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HBTU: (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HPLC: high-performance liquid chromatography; MIDA: N-methyliminodiacetic acid; min: minute; LCMS: liquid chromatography-mass spectrometry; MS: mass spectrometry; Ms: mesyl; Pet-ether: Petroleum ether (boiling point 60-80°C); quant.: quantitative (conversion); Rt: retention time; RT: room temperature; SCX: strong cation exchange; TEA: triethylamine; TFA: trifluoroacetic acid; THF: tetrahydrofuran; TsCl: p-toluenesulfonyl chloride; XPhos: 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl; XantPhos: 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene.

[0214] Analysis method The products and intermediates were analyzed using reversed-phase HPLC-MS with the parameters shown below.

[0215] HPLC analytical method: AnalpH2_MeOH_4min:Phenomenex Luna C18(2)3μm、50×4.6mm;A=water+0.1% acetic acid;B=MeOH+0.1% acetic acid;45℃;%B:0.0 min 5%,1.0 min 37.5%,3.0 min 95%,3.5 min 95%,3.51 min 5%,4.0 min 5%;2.25mL / min.

[0216] AnalpH2_50-95MeOH_4min:Phenomenex Luna C18(2) 3μm, 50×4.6mm; A=water+0.1% acid; B=MeOH+0.1% acid; 45℃; %B: 0.0 minutes 50%, 1.5 minutes 95%, 3.5 minutes 95%, 3.51 minutes 5%, 4.0 minutes 5%; 2.25mL / minute.

[0217] AnalpH9_MeOH_4min:Phenomenex Luna C18(2)3μm, 50×4.6mm; A=water pH9 (hydrogen carbonate 10mM); B=MeOH+0.1% acid; 45℃; %B: 0.0 points 5%, 1.0 points 37.5%, 3.0 points 95%, 3.5 points 95%, 3.51 5%, 4.0 minutes 5%; 2.25mL / minute.

[0218] AnalpH2_MeOH_QC_V1: Phenomenex Gemini NX C18 5μm, 150×4.6mm; A = Water + 0.1% ethyl acetate; B = MeOH + 0.1% ethyl acetate; 40℃; %B: 0.0 min 5%, 0.5 min 5%, 7.5 min 95%, 10.0 min 95%, 10.1 min 5%, 13.0 min 5%; 1.5mL / min.

[0219] AnalpH9_MeOH_QC_V1:Phenomenex Gemini NX C18 5μm, 150×4.6mm; A=water+pH9 (hydrogen carbonate 10mM); B=MeOH; 40℃; %B: 0.0min 5%, 0.50min 5%, 7.5min 95%, 10.0min 95%, 10.1min 5%, 13.0min 5%; 1.5mL / min.

[0220] Agilent MeCN HPLC 3min: Phenomenex Luna C18, 50×2mm: A=Water + 0.1% Formic Acid; B=MeCN + 0.1% Formic Acid; 5~95% B 0~3 min; 1 mL / min

[0221] UPLC analysis method AnalpH2_MeCN_UPLC_3.8min: Acquity UPLC BEH C-18 1.7um, 2.1×50mm, A=Water + 0.05% Formic Acid; B:Acetonitrile + 0.05% Formic Acid; 35℃; %B: 0.0 min 10%, 0.5 min 10%, 1 min 35%, 1.5 min 45%, 2.3 min 90%, 3.2 min 90%, 3.8 min 10%; 0.55mL / min

[0222] AnalpH2_MeCN_UPLC_4.0min:Acquity UPLC BEH C-18 1.7um, 2.1×50mm, A=Water + 0.05% Formic Acid; B:Acetonitrile + 0.05% Formic Acid; 35℃; %B: 0.0 min 10%, 0.5 min 10%, 1 min 35%, 1.5 min 45%, 2.3 min 90%, 3.2 min 90%, 3.6 min 10%, 4.0 min 10%; 0.55mL / min

[0223] AnalpH2_MeCN_UPLC_4.2min:Acquity UPLC BEH C-18 1.7um, 2.1×50mm, A=Water + 0.05% Formic Acid; B:Acetonitrile + 0.05% Formic Acid; 40℃; %A: 0.0 min 95%, 0.3 min 95%, 2 min 5%, 3.5 min 5%, 3.6 min 95%, 4.2 min 95%; 0.6mL / min

[0224] AnalpH2_MeCN_UPLC_5.0min: Acquity UPLC BEH C-18 1.7um, 2.1×50mm, A=Water + 0.05% Formic Acid; B:Acetonitrile + 0.05% Formic Acid; 40℃; %A: 50% at 0.0 min, 90% at 3.0 min, 90% at 5.0 min, 50% at 5.1 min; 0.4mL / min

[0225] AnalpH2_MeCN_UPLC_6.1min: Acquity UPLC BEH C-18 1.7um, 2.1×100mm, A=Water + 0.05% Formic Acid; B:Acetonitrile + 0.05% Formic Acid; 40℃; %A: 0.0 min 60%, 2.0 min 90%, 6.0 min 90%, 6.1 min 60%; 0.3mL / min

[0226] AnalpH9_MeCN_UPLC_10min: Acquity UPLC BEH C-18 1.7um, 2.1×50mm, A=5mM ammonium acetate in water; B: acetonitrile; 40℃; %B: 0.0 min 3%, 1.0 min 3%, 7.0 min 100%, 7.5 min 100%, 9.0 min 3%, 10 min 3%; 0.5mL / min

[0227] Thermometer MeOH UHPLC 1.2 min: Phenomenex Kinetex, 2.6 μM, 50 × 2.1 mm, A = water + 0.1% formic acid; B = MeOH + 0.1% formic acid; 2-95% B; 0-1.0 min; 1.3 mL / min

[0228] General method General method 1 (GM1): Amide coupling A mixture of a carboxylic acid (1.0 eq), an amine (1.0-1.5 eq), N,N-diisopropylethylamine or triethylamine (1.5-3.0 eq), and a coupling agent (1.0-1.5 eq) such as HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate), or HCTU (O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) was stirred at room temperature for 1-72 hours in an anhydrous solvent such as DMF or DCM. The product was isolated and purified using one of the following methods.

[0229] The reaction mixture was diluted with a mixture of water and saturated NaCl aqueous solution and extracted with SiO2. The organic phase was dried over Na2SO4 or MgSO4, filtered, and concentrated under vacuum to obtain the crude material, which was used without further purification or purified by column chromatography.

[0230] The solvent was removed under vacuum, the residue was dissolved in siRNA, and the organic phase was washed with NaHCO3(aq) solution, H2O, and then brine. The organic phase was dried over Na2SO4 or MgSO4, filtered, and the filtrate was concentrated under vacuum to obtain the crude material, which was used without further purification or purified by column chromatography.

[0231] The reaction was diluted with water or saturated NaCl aqueous solution and extracted with DCM. The organic phase was dried over MgSO4, filtered, or passed through hydrophobic frit and concentrated under vacuum. The crude material was used without further purification or purified by column chromatography.

[0232] The reaction was cooled in an ice bath, diluted with water, and extracted with RINKAN. The organic phase was sequentially washed with NaHCO33(aq) solution, NH4Cl(aq), and brine, then dried over Na2SO4, filtered, and the filtrate was concentrated under vacuum to obtain the crude material, which was purified by column chromatography.

[0233] General method 2 (GM2): Boc deprotection Boc deprotection method 2A: The Boc-protecting amine was stirred for 1 to 18 hours in a mixture of DCM:TFA (in a ratio of 10:1 to 1:1).

[0234] Boc deprotection method 2B: The Boc-protecting amine was dissolved in ethyl acetate or dioxane, and either 4M HCl in dioxane, 2M HCl in Et2O, or 1M HCl in Et2O was added. The reaction mixture was stirred at room temperature for 0.25 to 18 hours.

[0235] Boc deprotection method 2C: Boc-protected amines prepared in medium-column iontophoresis (DCM) were passed through an MP-TsOH cartridge, washed with MeOH (maximum 5 column volumes), and eluted with 2M NH3-MeOH.

[0236] The reaction mixture (or product-containing fraction: Method 2C) was concentrated in vacuum to obtain the crude material, which was either used as is or purified by one of the following methods.

[0237] Preparative HPLC after SCX-2.

[0238] The solution was basicized by adding 1M NH3 in MeOH, concentrated under vacuum, and purified by preparative HPLC.

[0239] 0.5N HCl (aq) The aqueous phase was diluted with ethyl acetate, and the layers were separated. The aqueous phase may be washed with ethyl acetate, then basicized (pH approximately 10), and extracted with ethyl acetate. The combined organic extracts were dried (MgSO4), filtered, and the solvent was evaporated under vacuum.

[0240] Reverse-phase chromatography Water and saturated aqueous NaHCO3 were added. The product was extracted in ethyl acetate, the organic extract was dried (MgSO4), and the solvent was removed under vacuum.

[0241] After preparative HPLC, SCX-2 may be used if necessary.

[0242] After SCX-2, 4M HCl was added to the dioxane, and the solvent was removed to obtain the HCl salt.

[0243] General method 3 (GM3): Hydrogenation General method 3A (Hydrogenation by H2 balloon): Alkenes or Cbz / benzyl protected species (1 eq) were dissolved in EtOH or methanol and placed under an N2 atmosphere. Pd / C or Pd(OH)2 / C (10 wt%) was added. An H2 atmosphere was introduced, and the reaction mixture was stirred at room temperature for 1 to 72 hours. The mixture was filtered through Celite, and the filtrate was concentrated to obtain the crude product, which was used without further purification.

[0244] If necessary, additional aliquots of Pd / C may be added during the course of the reaction.

[0245] General method 3B (Hydrogenation with ammonium formate): Alkene (1 eq) was dissolved in EtOH and Pd / C (0.5 eq), and NH4HCO2 (10 eq) was added. The mixture was stirred under reflux for 1 to 72 hours. The solution was cooled to room temperature, filtered through Celite, and washed with MeOH or HCl. The solvent was evaporated under vacuum, and the residue was separated between HCl and saturated aqueous NaHCO3. The organic phase was dried (MgSO4), filtered, and the solvent was removed to obtain the crude product, which was used without further purification.

[0246] If necessary, additional aliquots of Pd / C and / or NH4HCO2 may be added during the course of the reaction.

[0247] General method 3C (Hydrogenation by H-Cube): Alkenes were dissolved in a protic solvent and passed through an H-cube reactor (Pd / C cartridge) under typical conditions: 30°C, 20 bar, 1 mL / min. The solvent was evaporated under vacuum, and the material was used without further purification.

[0248] General method 4 (GM4): Ester hydrolysis Ester hydrolysis method 4A: Ester (1.0 eq) was dissolved in MeOH or 1,4-dioxane, 1 M LiOH (1-2 eq) was added, and the mixture was stirred at room temperature for 1-64 hours.

[0249] If necessary, an additional equivalent amount of 1 M LiOH(aq) may be added during the reaction.

[0250] Ester hydrolysis method 4B: Ester (1.0 eq) was dissolved in a 1:1:1 solution of 1 M NaOH / MeOH / THF and stirred at room temperature for 1 to 18 hours.

[0251] Ester hydrolysis method 4C: Ester (1.0 eq) was dissolved in 10M NaOH (5 equivalents) in MeOH and stirred at 60°C for 1 to 18 hours.

[0252] The solvent was removed under vacuum, and the product was isolated using one of the following methods.

[0253] The crude product was used without further purification.

[0254] The extract was diluted with water, acidified to pH 1-4, and then extracted with toluene or DCM. The organic extract was dried over MgSO4, filtered to remove the solvent, and the product was used without further purification.

[0255] The crude product was dissolved in water, and the aqueous layer was washed with toluene or dimethylcellulose. The aqueous layer was acidified with 1 M HCl, and the product was extracted in toluene or dimethylcellulose. The combined organic extract was dried (MgSO4), filtered, and the solvent was removed to obtain the product, which was used without further purification.

[0256] 2N HCl was added to adjust the pH to 7. The solvent was evaporated under vacuum, and the residue was dissolved in DCM. The solution was filtered, the filtrate was concentrated under reduced pressure, and the resulting solid residue was dried under vacuum.

[0257] The crude mixture was diluted with water and extracted with Et2O. The organic phase was extracted with water. The combined aqueous phase was concentrated under vacuum, and the crude product was dissolved in toluene and concentrated under vacuum to obtain the carboxylic acid as a lithium salt.

[0258] General method 5 (GM5): Fmoc deprotection The Fmoc-protected amine was stirred at room temperature for 1 to 18 hours in a 10:1 mixture of piperidine and either DCM or DMF. The solvent was removed under reduced pressure, and the residue was used without further purification or purified by column chromatography or preparative HPLC.

[0259] General method 6 (GM6): Nitrile reduction by NaBH4 / NiCl2.6H2O NaBH4 / NiCl2.6H2O Method 6A: Benzonitrile (1 eq) in MeOH was added with NiCl2.6H2O (0.1 eq) at 0°C, followed by the gradual addition of NaBH4 (10 eq). The reaction temperature was maintained below 5°C. The mixture was stirred for 1-3 hours.

[0260] NaBH4 / NiCl2.6H2O in situ Boc protection method 6B: Benzonitrile (1 eq) in MeOH was added with NiCl2.6H2O (0.1 eq) and Boc2O (2 eq) at 0°C, followed by the gradual addition of NaBH4 (10 eq). The reaction temperature was maintained below 5°C. The mixture was stirred for 1-2 hours.

[0261] The reaction mixture was concentrated in a vacuum, and then saturated with NaHCO3. 3(aq) The solution and suspension in water were extracted with ELISA. The organic phase was dried over Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by column chromatography.

[0262] General method 7 (GM7): Alkylation of methyl N-(diphenylmethylene)glycinate To a solution of methyl N-(diphenylmethylene)glycinate (1 eq) in anhydrous DMF and toluene (1:1), potassium tert-butoxide (1 eq) was added under N2, and the reaction mixture was stirred at RT. After 20 minutes, appropriate phenethyl bromide (1 eq) in anhydrous DMF was added dropwise, and the reaction was stirred at RT (18-24 hours). The reaction mixture was then diluted with water and extracted with SiO2. The combined organic phases were washed with brine, dried over Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by column chromatography.

[0263] General method 8 (GM8): Benzophenone deprotection and in situ Boc protection To a solution of methyl N-(diphenylmethylene)glycinate derivative (1.0 eq) in DCM, 2M HCl (approximately 20 eq) in diethyl ether was added, and the reaction was stirred at RT (24-72 hours). The reaction mixture was concentrated under vacuum. To the solution of the crude mixture in DCM, Boc2O (1.2 eq) and DIPEA (3.0 eq) were added. Once the crude intermediate was completely consumed (1-23 hours), the reaction mixture was diluted in DCM with water and saturated NH4Cl. (aq) The mixture was then washed with brine. The organic phase was dried over Na2SO4, filtered, and concentrated under vacuum. The crude product was purified using column chromatography.

[0264] General method 9 (GM9): Aryl iodine-alkyl iodine coupling Zinc powder (6.0 eq), anhydrous DMF, and 1,2-dibromoethane (0.3 eq) were sequentially added to an oven-dried, N2-purged flask. The reaction mixture was heated to 60°C for 30 minutes and then cooled to RT. Trimethylsilyl chloride (0.06 eq) was added, and the reaction was vigorously stirred for 30 minutes. Benzyl (2R)-2-{[(tert-butoxy)carbonyl]amino}-4-iodobutanoate (1.0 eq) was added to anhydrous DMF, and the reaction was heated to 35°C for 30 minutes and then cooled to RT. Pd2dba3 (0.02 eq), P(o-tol)3 (0.08 eq), and a suitable aryl iodine species (0.75 eq) were sequentially added, and the reaction was stirred for 18-48 hours. The reaction was then diluted with ELISA and washed with brine. Next, the organic solution was dried with Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by column chromatography.

[0265] General method 10 (GM10): Iodine substitution with amine A solution of iodine species (1.0 eq) in anhydrous DMF was mixed with amine (2.5 eq), and the reaction was heated to 50°C. After 1 hour, the reaction mixture was cooled to RT and diluted with water. The aqueous solution was extracted with ethyl acetate, and the combined organic phase was dissolved in LiCl (aq) The solution was sequentially washed with (10% w / v) and brine, dried over Na2SO4, and concentrated under vacuum. If necessary, the crude product was purified by column chromatography.

[0266] General method 11 (GM11): Mesylation and substitution To a solution of alcohol (1.0 eq) in anhydrous DCM, Et3N (1.2 eq) was added under nitrogen. The reaction mixture was cooled to 0°C, and methanesulfonyl chloride (1.2 eq) was added dropwise. The temperature was maintained at 0°C during the complete conversion to the mesylated species (1–17 hours). The reaction mixture was then diluted with DCM, sequentially washed with 1 M HCl, saturated NaHCO3 solution, and brine, dried over Na2SO4, and concentrated under vacuum. It was immediately used in one of the following reactions without further purification.

[0267] 11A: Substitution with cyclic amine: 1.0 eq of mesylate in anhydrous DMF was mixed with 10 eq of cyclic amine, and the reaction was heated to 90°C for 17-24 hours. The reaction was cooled to RT and diluted with water. The aqueous solution was extracted with SiO(3x), the combined organic phase was washed with brine(1x), dried over Na2SO4, and concentrated under vacuum. Residual starting material was removed using an SCX column, using only the fraction eluted with ammonium methane (2M). The crude product was purified by reversed-phase column chromatography.

[0268] Substitution with cyclic amide 11B: To a solution of cyclic amide (10 eq) in anhydrous DMF, NaH (60% in mineral oil) (10 eq) was carefully added under N2. After 30 minutes, a solution of mesylate (1.0 eq) in anhydrous DMF was added to the flask, and the reaction was heated to 90°C. Once the mesylate was completely consumed (19 hours), the reaction was cooled to RT and diluted with water. The aqueous solution was extracted with ELISA, the combined organic phase was washed with brine, dried over Na2SO4, and concentrated under vacuum. If necessary, the crude product was purified by column chromatography.

[0269] General method 12 (GM12): Amide coupling with T3P To a stirred suspension of acid (1.0–1.1 eq depending on whether the acid or amine is the restriction reagent), amine (1.0–1.1 eq depending on whether the acid or amine is the restriction reagent), and Et3N (4.0–5.0 eq), T3P (50% in HCl) (2.0 eq) was added dropwise at 0°C. The suspension was heated to RT and stirred until the starting materials were completely consumed, then cooled to 0°C, diluted with water, and stirred for a further 30 minutes. The aqueous solution was extracted with HCl (×3), the combined organic phase was washed with water (×1) and brine (×1), dried over Na2SO4, and concentrated under vacuum. If necessary, the crude product was purified by column chromatography.

[0270] Synthesis of RgA Synthesis of (4-aminomethyl-benzyl)-carbamate 9H-fluoren-9-ylmethyl ester: [ka]

[0271] Step 1: 1-(N-boc-aminomethyl)-4-(aminomethyl)benzene (3.80 g, 16.1 mmol) was dissolved in DCM (100 mL) and DIPEA (5.0 mL, 29 mmol), followed by the addition of FmocCl (5.0 g, 19 mmol). The reaction mixture was stirred at room temperature for 1 hour, after which a precipitate formed. Water (100 mL) was added, the precipitate was filtered, and dried to obtain [4-(tert-butoxycarbonylaminomethyl)-benzyl]-carbamate 9H-fluoren-9-yl methyl ester (6.32 g, 86%) as a white solid. AnalpH2_MeOH_4MIN:Rt:3.52 min, m / z 481.3[M+H] +

[0272] Step 2: [4-(tert-butoxycarbonylaminomethyl)-benzyl]-carbamate 9H-fluoren-9-yl methyl ester (6.32 g, 13.2 mmol) was suspended in dioxane (50 mL), and HCl solution (4 M in dioxane, 20 mL) was slowly added. The reaction mixture was stirred overnight at room temperature, then an additional aliquot of 4 M HCl in dioxane (10 mL) was added, and the mixture was stirred at room temperature for a further 2 hours. The reaction mixture was diluted with hexane (150 mL), the product was collected by filtration, and dried under vacuum to obtain (4-aminomethyl-benzyl)-carbamate 9H-fluoren-9-yl methyl ester (4.0 g, 73%) as an off-white solid. AnalpH2_MeOH_4MIN:Rt:2.32 min, m / z 359.3[M+H] +

[0273] 5-aminomethyl-3-methoxypyridine-2-carbonitrile (regarding GS7) [ka]

[0274] (5-Methoxy-1-oxypyridine-3-ylmethyl)-carbamate tert-butyl ester [ka] 1 eq of tert-butyl(5-methoxypyridine-3-yl)methylcarbamate was dissolved in 5 mL of chloroform. 1.9 eq of meta-chloroperoxybenzoic acid was added, and the reaction mixture was stirred overnight under RT. The reaction mixture was diluted with dichloromethane and washed with a 2 M aqueous solution of sodium hydroxide. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum to obtain tert-butyl ester (5-methoxy-1-oxypyridine-3-ylmethyl)-carbamate (208 mg, 92%) as a white solid. AnalpH2_MeOH_4MIN:Rt:2.26 min, m / z 255.3[M+H] +

[0275] (6-cyano-5-methoxypyridine-3-ylmethyl)-carbamate tert-butyl ester [ka] Acetonitrile (2 mL), followed by dimethylcarbamyl chloride (1.56 eq), was added to (5-methoxy-1-oxypyridine-3-ylmethyl)-carbamate tert-butyl ester (1 eq). The reaction mixture was heated at 40°C for 4 hours. The reaction mixture was then cooled to 0°C, and a solution of sodium cyanide (3.3 eq) in water (2 mL) was added. The reaction mixture was stirred overnight in RT. A 2 M aqueous solution of sodium hydroxide (5 mL) was added, and the reaction mixture was stirred in RT for 3 hours. A further 2 M aqueous sodium hydroxide (10 mL) was added, and the mixture was extracted with ethyl acetate (×3). The organic layers were combined, dried over MgSO4, filtered, and concentrated under vacuum. The crude material was purified by preparative HPLC to obtain (6-cyano-5-methoxypyridine-3-ylmethyl)-carbamate tert-butyl ester (86 mg, 40%) as a white solid. AnalpH2_MeOH_4MIN:Rt:2.60 min, m / z 264.3[M+H] +

[0276] 5-aminomethyl-3-methoxypyridine-2-carbonitrile [ka] (6-cyano-5-methoxypyridine-3-ylmethyl)-carbamate tert-butyl ester (1 eq) was treated with a 1:1 mixture of TFA:DCM. The reaction mixture was monitored by LC-MS. Once the starting material was consumed, the reaction mixture was concentrated under vacuum. The crude material was purified by SCX-2 ion exchange column to obtain 5-aminomethyl-3-methoxypyridine-2-carbonitrile (55 mg, quantitative) as an off-white solid. AnalpH9_MeOH_4MIN:Rt:1.50 min, m / z 164.3[M+H] +

[0277] Scheme: Synthesis of 1-amino-isoquinoline-6-carbonitride: [ka] 1-amino-6-bromoisoquinoline (500 mg, 2.24 mmol) was dissolved in DMF (5 mL), Pd(PPh3)4 (260 mg, 0.22 mmol) was added, and N2 was passed through the solution for 10 minutes. Zn(CN)2 (157 mg, 1.34 mmol) was added, and the reaction mixture was heated in a microwave at 120°C for 1 hour. Water and ethyl acetate were added, the layers were separated, and the aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with brine, passed through a phase separator, and the solvent was removed under vacuum. The crude product was purified by column chromatography (biotage, 25 g SNAP cartridge, 0-100% ethyl acetate in hexane, followed by 0-20% MeOH in ethyl acetate). After removing the solvent, 1-amino-isoquinoline-6-carbonitrile (293 mg, 77%) was obtained as a yellow solid. AnalpH9_MeOH_4MIN:Rt:2.12 min, m / z 170.1[M+H] +

[0278] The following compounds were prepared using the same method.

[0279] [Table 1]

[0280] 3-Chloro-1-methyl-1H-indazole-6-carbonitrile [ka] To a stirred solution of 1-methyl-1H-indazole-6-carbonitrile (20.0 g, 105 mmol) in 250 ml of dry acetonitrile, N-chlorosuccinimide (18.0 g, 150 mmol) was added at room temperature. After the reaction was complete (monitored by thin-layer chromatography), the reaction mixture was concentrated and purified by silica gel flash column chromatography using ethyl acetate and petroleum ether (10-30%) solutions to obtain 3-chloro-1-methyl-1H-indazole-6-carbonitrile (13.0 g, 59%) as a white solid. AnalpH2_MeCN_UPLC_4.2min:Rt:1.96min, m / z 192.1[M+H] +

[0281] C-(1-methyl-1H-benzotriazol-5-yl)-methylamine [ka]

[0282] Step 1: tert-butyl(1-methyl-1H-benzo[d][1,2,3]triazole-5-yl)methylcarbamate To a stirred solution of 1-methyl-1H-benzotriazole-5-carbonitride (5.0 g, 32 mmol) in dry methanol (200 mL), Boc₂O (13.8 g, 63.0 mmol) and NiCl₂·6H₂O (0.41 g, 3.2 mmol) were added at 0°C. Then, NaBH₄ (8.4 g, 220 mmol) was gradually added over 20 minutes, and the reaction mixture was heated to room temperature overnight. Diethylenetriamine (3.4 mL, 32 mmol) was added at room temperature, and the mixture was stirred for a further 30 minutes. siRNA and saturated aqueous NaHCO₃ (300 mL) were added, and the layers were separated. The aqueous layer was extracted with siRNA, and the combined organic extract was washed with saturated aqueous NaHCO₃ and brine, dried, and concentrated under reduced pressure. The crude product was purified by column chromatography (biotage, 100 g, 0-100% siRNA / hexane) to obtain (1-methyl-1H-benzotriazole-5-ylmethyl)-carbamate tert-butyl ester (5.05 g, 61%) as a brown solid.

[0283] Step 2: (1-methyl-1H-benzo[d][1,2,3]triazole-5-yl)methanamine hydrochloride (1-methyl-1H-benzotriazole-5-ylmethyl)-carbamate tert-butyl ester (5.05 g, 19.2 mmol) was dissolved in 4 M HCl in dioxane (100 mL) and stirred at room temperature for 45 minutes. The reaction mixture was filtered and dried under vacuum to obtain (1-methyl-1H-benzo[d][1,2,3]triazole-5-yl)methanamine hydrochloride (2.67 g, 53%) as a white solid. AnalpH2_MeOH_4MIN:Rt:0.38 min, m / z 163.2[M+H] +

[0284] The following compounds were prepared using the same method.

[0285] [Table 2]

[0286] Synthesis of (5-aminomethyl-thiazole-2-ylmethyl)-carbamate tert-butyl ester [ka]

[0287] Step 1: Di-tert-butyl dicarbonate (746 mg, 3.42 mmol) was added to 2-aminomethyl-thiazole-5-carbonitrile (5.0 g, 2.9 mmol) in MeCN (10 mL). DMAP (355 mg, 0.29 mmol), followed by Et3N (0.4 mL, 2.8 mmol), was added, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with ethyl acetate and washed with saturated aqueous NaHCO3 solution. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum to obtain (5-cyanothiazole-2-ylmethyl)-carbamate tert-butyl ester, which was used without further purification.

[0288] Step 2: (5-cyano-thiazole-2-ylmethyl)-carbamate tert-butyl ester was dissolved in MeOH (20 mL) and cooled to 0°C. NiCl2.6H2O (135 mg, 0.57 mmol), followed by NaBH4 (1.08 g, 28.5 mmol) was added little by little. The reaction mixture was heated to room temperature for 2 hours, then diluted with siRNA and washed with saturated NaHCO3 aqueous solution. The organic layer was dried over MgSO4, filtered through Celite, and purified by SCX-2 (washed with MeOH and eluted with 0.5 M NH3 in MeOH). After removing the solvent, the crude product was purified by preparative HPLC to obtain (5-aminomethyl-thiazole-2-ylmethyl)-carbamate tert-butyl ester (33.3 mg, 5%) as a yellow oil. AnalpH2_MeOH_4MIN:Rt:0.82~1.34 min, m / z 244.4[M+H] +

[0289] Synthesis of c-Flo[2,3-c]pyridine-2-ylmethylamine [ka]

[0290] Step 1: 7-Chloroflou[2,3-c]pyridine (433 mg, 2.82 mmol) was dissolved in acetic acid (6 mL), and zinc powder (1.03 g, 15.8 mmol) was added. The reaction mixture was heated under reflux for 2 hours and then cooled to room temperature. The mixture was then filtered, and the filtrate was concentrated under vacuum. The resulting solid was dissolved in water, basicized with 2 M NaOH, and extracted using DCM (×3). The combined organic matter was dried over MgSO4 and concentrated under vacuum to produce flou[2,3-c]pyridine (206 mg, 62%) as an orange oil. ANALPH2_MEOH_4min, Rt:0.36min, m / z 120.2[M+H]+

[0291] Step 2: Dissolve flo[2,3-c]pyridine (206 mg, 1.72 mmol) in anhydrous THF (8 mL) and cool to -78°C. Then add n-butyllithium (0.87 mL, 2.15 mmol). Stir the reaction mixture for 30 minutes, then add DMF (0.27 mL, 3.44 mmol). Warm the reaction mixture to room temperature and stir for 18 hours. Quench the reaction with 1 M HCl, neutralize with NaHCO3, and extract with DCM (×3). Dry the combined organic matter with MgSO4 and concentrate under vacuum to produce flo[2,3-c]pyridine-2-carbaldehyde (165 mg, 65%) as an orange solid. ANALPH2_MEOH_4min, Rt:0.36min, m / z 148.1[M+H]+

[0292] Step 3: Flo[2,3-c]pyridine-2-carbaldehyde (165 mg, 1.12 mmol) was dissolved in ethanol (2.5 mL) while heating. Hydroxyamine hydrochloride (164 mg, 2.36 mmol) in water (0.33 mL) and potassium carbonate (164 mg, 1.19 mmol) in water (0.83 mL) were added, and the reaction mixture was cooled to room temperature and stirred for 18 hours. The mixture was filtered, and the residue was dried under reduced pressure to obtain flo[2,3-c]pyridine-2-carbaldehyde oxime (130 mg, 72%) as an off-white solid. ANALPH2_MEOH_4min, Rt:0.37min, m / z 163.1[M+H]+

[0293] Step 4: Dissolve flu[2,3-c]pyridine-2-carbaldehyde oxime (130 mg, 0.80 mmol) in acetic acid (2 mL) and add zinc powder (293 mg, 4.48 mmol). Stir the reaction mixture at room temperature for 30 minutes. The mixture was then filtered, and the filtrate was concentrated under vacuum to produce c-flu[2,3-c]pyridine-2-ylmethylamine (123 mg, quantitatively) as an orange oil, which was used "as is" in the subsequent reaction. ANALPH2_MEOH_4min, Rt:0.27min, m / z 149.1[M+H]+

[0294] RgB synthesis General scheme for RgB cores: Scheme: Synthesis of 4-(bromomethyl)-2,3-dihydrobenzofuran (2,3-dihydro-benzofuran-4-yl)methanol [ka] 2,3-Dihydrobenzofuran-4-carbaldehyde (2.00 g, 13.5 mmol) was dissolved in dry MeOH (30 mL), and the solution was cooled to 0°C. NaBH4 (510 mg, 13.5 mmol) was added, and the solution was stirred at room temperature for 1 hour. Ice water was added, and the layers were separated. The organic phase was evaporated under vacuum to obtain the title compound as a colorless crystalline solid (1.90 g, 94%). AnalpH2_MeOH_4MIN:Rt:2.00 min, m / z 151.3[M+H]+

[0295] 4-bromomethyl-2,3-dihydrobenzofuran [ka] 2,3-Dihydrobenzofuran-4-ylmethanol (1.90 g, 12.6 mmol) was dissolved in dry DCM (30 mL), and the mixture was cooled to 0°C. PBr3 (2.40 mL, 25.4 mmol) was added dropwise over 10 minutes, and the mixture was stirred at room temperature for 1 hour. The mixture was cooled to 0°C, and saturated NaHCO3 (aq) (50 mL) was added little by little. The mixture was stirred at 0°C for 10 minutes, and then diluted with DCM (30 mL). The layers were separated, and the organic phase was washed with saturated NaHCO3 (aq) (50 mL), and then with saturated brine solution (50 mL). The organic phase was dried (MgSO4), filtered, and the solvent was evaporated under vacuum to obtain the title compound (2.35 g, 87%) as a pale yellow oil. AnalpH2_MeOH_4MIN:Rt:2.99 min, m / z did not show any identifiable ionization.

[0296] Scheme 2A (Acid) [ka] Route 1: Synthesis of (2S)-4-(1,3-benzodioxol-5-ylmethyl)-1-tert-butoxycarbonyl-pyrrolidine-2-carboxylic acid (S)-4-benzo[1,3]dioxol-5-ylmethyl-5-oxopyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester [ka]

[0297] Methyl Boc-L-pyroglutamate (1.05 g, 4.32 mmol) was dissolved in dry THF (20 mL). The mixture was cooled to -78°C. LiHMDS (1 M, 4.40 mL, 4.40 mmol in THF) was added dropwise over 10 minutes at -78°C. The solution was stirred at -78°C for 1 hour. Next, a solution of 5-(bromomethyl)-1,3-benzodioxole (0.975 g, 4.53 mmol) in dry THF (5 mL) was added dropwise over 30 minutes at -78°C. The solution was stirred at -78°C for 2 hours. Saturated NaHCO3 (aq) (40 mL) was added dropwise while continuously stirring until the solution reached room temperature. The mixture was diluted with ELISA (50 mL) and the layers were separated. The organic phase was washed with saturated brine (aq) (40 mL), then dried (MgSO4), filtered, and the solvent was evaporated under vacuum. The crude product was purified by flash chromatography (Biotage Isolera; 50 g SNAP cartridge) using isohexane followed by elution with 40% alkylammonium ammonium ammonium ammonium eluate to obtain the title compound (1.08 g, 66%) as a white solid. AnalpH2_MeOH_4MIN:Rt:3.03 min, m / z 378.3[M+H]+

[0298] If necessary, the reaction may be stirred at room temperature for up to 18 hours after adding the required amount of benzyl bromide.

[0299] If necessary, after the reaction is complete, the mixture may be warmed to room temperature and quenched with saturated NH4Cl (aq) or saturated brine (aq).

[0300] (2S,4R)-4-benzo[1,3]dioxol-5-ylmethylpyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester [ka]

[0301] (S)-4-benzo[1,3]dioxol-5-ylmethyl-5-oxopyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (550 mg, 1.46 mmol) was dissolved in dry DCM (15 mL). The mixture was cooled to 0°C, and 1 M BH3-THF (1.80 mL, 1.80 mmol) was added dropwise over 5 minutes under N2, followed by the dropwise addition of BF3-Et2O (200 μL, 1.62 mmol) under N2 at 0°C for 5 minutes. Subsequently, 1 M BH3-THF (1.80 mL, 1.80 mmol) and BF3-Et2O (200 μL, 1.62 mmol) were added sequentially dropwise over 5 minutes at 0°C. The mixture was stirred at 0°C for 1 hour, and then stirred at room temperature for 1 hour. Saturated NH4Cl(aq) (30 mL) and HCl (40 mL) were added at 0°C, and the mixture was vigorously stirred for 15 minutes. The layers were separated, the organic phase was washed with saturated brine (aq) (40 mL), then dried (MgSO4), filtered, and the solvent was evaporated under vacuum. The crude product was purified by flash chromatography (Biotage Isolera; 25 g SNAP cartridge) eluting with isohexane → 40% HCl-isohexane to obtain the title compound (352 mg, 66%) as a pale yellow oil. AnalpH2_MeOH_4MIN:Rt:3.26 min, m / z 364.3[M+H]+

[0302] In some cases, 1M BH3-THF was added all at once, followed by BF3-Et2O, under N2 conditions at 0°C. Immediately afterward, the solution was heated to room temperature and stirred for a maximum of 3 hours.

[0303] If the reaction is incomplete, additional aliquots of 1M BH3-THF (10 mol%) and / or BF3.Et2O (20 mol%) may be added, and the mixture may be stirred at room temperature for a further 16 hours. Then, if the reaction is incomplete, BH3-THF and BF3.Et2O may be added as needed, and the mixture may be stirred for up to 24 hours.

[0304] In some cases, the product may be purified by reverse-phase HPLC.

[0305] (2S,4R)-4-benzo[1,3]dioxol-5-ylmethylpyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester [ka] (2S,4R)-4-benzo[1,3]dioxol-5-ylmethyl-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (352 mg, 0.969 mmol) was dissolved in MeOH (7.5 mL). 1 M LiOH (H2O, 2.50 mL, 2.50 mmol) was added, and the mixture was stirred at room temperature for 16 hours. The solvent was evaporated under vacuum, and the residue was divided between ELISA (50 mL) and 0.25 M HCl (aq) (40 mL). The aqueous phase was extracted with ELISA (40 mL), the combined organic phase was dried (MgSO4), filtered, and the solvent was evaporated under vacuum to obtain (2S,4R)-4-benzo[1,3]dioxol-5-ylmethyl-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester (262 mg, 78%) as a colorless rubbery substance. AnalpH2_MeOH_4MIN:Rt:3.10 min, m / z 350.3[M+H]+

[0306] The following compounds were prepared using the same method.

[0307] [Table 3-1] [Table 3-2] [Table 3-3]

[0308] Scheme 2B (Carboxylic Acid): [ka] Route 2: Synthesis of (S)-4-(2-trifluoromethylpyridine-4-ylmethyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester (S)-5-oxo-4-[1-(2-trifluoromethylpyridine-4-yl)-methylidene]-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester [ka] Methyl Boc-L-pyroglutamate (1.00 g, 4.11 mmol) was dissolved in dry THF (20 mL). The mixture was cooled to -78°C, and LiHMDS (1 M in THF, 4.30 mL, 4.30 mmol) was added dropwise over 10 minutes. The solution was stirred at -78°C for 1 hour. Next, a solution of 2-(trifluoromethyl)isonicotinaldehyde (0.720 g, 4.11 mmol) in dry THF (5 mL) was added dropwise over 30 minutes at -78°C. The resulting solution was then stirred at -78°C for 2.5 hours. H2O (10 mL) was added dropwise while continuously stirring at approximately -40°C, and then siRNA (50 mL) was added. The solution was warmed to room temperature while continuously stirring. The layers were separated, the organic phase was washed with saturated brine (aq) (30 mL), dried (MgSO4), filtered, and the solvent was evaporated under reduced pressure. The crude product was obtained as a yellow rubbery substance (918 mg), which was dissolved in dried DCM (20 mL). TEA (750 μL, 5.38 mmol), followed by MsCl (200 μL, 2.58 mmol), was added, and the mixture was stirred at room temperature for 3 hours. The mixture was diluted with DCM (30 mL), the solution was washed with saturated NaHCO3 (aq), followed by saturated aqueous brine (30 mL), dried (MgSO4), filtered, and the solvent was evaporated under vacuum. The title compound was obtained as a pale yellow rubbery substance (0.800 g, 48%). AnalpH2_MeOH_4MIN:Rt:2.92 min, m / z 401.2[M+H]+

[0309] In some cases, TsCl may be used instead of MsCl to achieve dehydration.

[0310] (S)-5-oxo-4-(2-trifluoromethylpyridine-4-ylmethyl)pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester [ka] (S)-5-oxo-4-[1-(2-trifluoromethylpyridine-4-yl)-methylidene]-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (0.800 g, 2.00 mmol) and NH4OAc (1.26 g, 20.0 mmol) were dissolved in EtOH (20 mL). Pd / C (10% wt., 64 mg, 0.60 mmol) was added, and the reaction mixture was stirred under reflux for 1 hour. The reaction mixture was filtered through Celite, and the residue was washed with ELISA (50 mL). The filtrate was evaporated under vacuum, and the resulting residue was dissolved in ELISA (60 mL). The organic phase was washed with saturated NaHCO3 (aq), followed by saturated brine (aq) (40 mL), then dried (MgSO4), filtered, and the solvent was evaporated under vacuum. The crude product was purified by flash chromatography (Biotage; 25g SNAP cartridge) using elution with 8:2 isohexane-siRNA followed by 2:8 isohexane-siRNA to obtain the title compound as a pale yellow, rubbery substance (0.328g, 41%). AnalpH2_MeOH_4MIN:Rt:2.82 min, m / z 403.2[M+H]+

[0311] (S)-2-hydroxymethyl-4-(2-trifluoromethylpyridine-4-ylmethyl)pyrrolidine-1-carboxylate tert-butyl ester [ka] (S)-5-oxo-4-(2-trifluoromethylpyridine-4-ylmethyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (270 mg, 0.671 mmol) was dissolved in dry DCM (15 mL). 1 M BH3-THF (3.40 mL, 3.40 mmol) and BF3.Et2O (1.25 mL, 1.01 mmol) were added, and the mixture was stirred in a microwave at 80°C for 30 minutes. The mixture was diluted with DCM (30 mL), the solution was washed with saturated NaHCO3 (aq), then dried (MgSO4), filtered, and the solvent was evaporated under vacuum. The title compound was obtained as a yellow solid (217 mg, 90%). AnalpH2_MeOH_4MIN:Rt:3.00 min, m / z 361.2[M+H]+

[0312] Depending on the circumstances, BF3.Et2O may be added up to 1 hour after the addition of BH3-THF.

[0313] (S)-4-(2-trifluoromethylpyridine-4-ylmethyl)pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester [ka] ((S)-2-hydroxymethyl-4-(2-trifluoromethylpyridine-4-ylmethyl)-pyrrolidine-1-carboxylic acid tert-butyl ester (217 mg, 0.602 mmol), TEMPO (28 mg, 0.179 mmol), and PhI(OAc)2 (427 mg, 1.33 mmol) were dissolved in 1:1 MeCN-H2O (8 mL), and the reaction mixture was stirred at room temperature for 6 hours. The mixture was diluted with H2O (20 mL), and the solution was extracted with ELISA (3 × 30 mL). The combined organic matter was washed with saturated brine (aq) (30 mL), then dried (MgSO4), filtered, and the solvent was evaporated under vacuum to obtain the title compound as an off-white solid (50 mg, 22%). AnalpH2_MeOH_4MIN:Rt:3.17 min, m / z 375.4[M+H]+

[0314] The following compounds were prepared using the same method.

[0315] [Table 4]

[0316] Scheme 2C (Ester) [ka]

[0317] The following compounds were synthesized using a similar method to the sequence shown in Schemes 2A and 2B. The difference lies in the final deprotection step, which is switched from saponification to Boc deprotection. The general BOC deprotection methodology is described in General Method 2. Examples of the synthetic procedures for the preceding steps are also discussed in the previous section (Schemes 2A and 2B).

[0318] [Table 5-1] [Table 5-2]

[0319] Scheme: Synthesis of (2S,4R)-4-pyridine-2-ylmethyl-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester: [ka]

[0320] Step 1: To a solution of methyl Boc-L-pyroglutamate (10.0 g, 41.2 mmol) in dry THF (200 mL), LiHMDS (1 M in THF, 45.2 ml, 45.2 mmol, 1.1 eq) was added dropwise under nitrogen at -78°C. The reaction mixture was then stirred at -78°C for 2 hours. 2-pyridinecarboxaldehyde (4.8 g, 45.2 mmol) was added to the reaction mixture at -78°C, and the mixture was stirred for a further 4 hours at -78°C. The reaction was quenched with saturated ammonium chloride (100 ml). The crude product was extracted with RINKAN (3 × 200 ml), and the combined extracts were dried over anhydrous magnesium sulfate and concentrated under vacuum. The crude product was purified by flash column (15% ethyl acetate / hexane) chromatography to obtain (S)-4-(hydroxypyridine-2-yl-methyl)-5-oxo-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (6.5 g, 45%) as an off-white solid. AnalpH2_MeCN_UPLC_4.0min:Rt:1.38min, m / z 351.5[M+H]+

[0321] Step 2: To a solution of (S)-4-(hydroxypyridine-2-yl-methyl)-5-oxo-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl (6.50 g, 18.6 mmol) in DCM (65 ml), TEA (25.8 ml, 186 mmol) and methanesulfonyl chloride (2.3 g, 20.4 mmol) were added at 0°C. The reaction mixture was stirred at RT for 3 days. The reaction mixture was poured into ice-cold water and extracted with ethyl acetate (2 × 500 mL). The combined organic extract was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to obtain (S)-5-oxo-4-[1-pyridine-2-yl-methylidene]-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl (3.5 g, 58%) as a brown oil. AnalpH2_MeCN_UPLC_4.0min:Rt:1.94min, m / z 333.4[M+H]+

[0322] Step 3: To a solution of (S)-5-oxo-4-[1-pyridine-2-ylmethylmethyl-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl (3.5 g, 10.5 mmol) in methanol (35 ml), 10% palladium carbon (1.75 g, 16.8 mmol) was added under a nitrogen atmosphere. The mixture was hydrogenated at 60 psi for 3 hours. The reaction mixture was filtered through Celite, washed with methanol, and concentrated under reduced pressure to obtain crude (S)-5-oxo-4-pyridine-2-ylmethyl-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl as an off-white solid (3.5 g, 100%). LC-MS showed the compound as a mixture of diastereomers. AnalpH2_MeCN_UPLC_4.0min:Rt:1.23min, m / z 335.5[M+H]+

[0323] Step 4: To a solution of (S)-5-oxo-4-pyridine-2-ylmethyl-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (2.8 g, 8.38 mmol) in THF (28 ml), lithium triethylborohydride (1.1 ml, 10 mmol) was added at -78 °C, and the reaction mixture was stirred for 1 hour. This was quenched with saturated sodium bicarbonate and warmed to 0 °C. 35% H2O2 was added at 0 °C, and the mixture was stirred at RT for a further 20 minutes. The reaction mixture was concentrated under reduced pressure, extracted with DCM, and dried. The residue was dissolved in DCM, and triethylsilane (2.6 ml, 16.8 mmol) and BF3.EtO2 (2.1 ml, 16.8 mmol) were added at RT. The mixture was stirred for 30 minutes and quenched with saturated sodium bicarbonate solution. The aqueous layer was extracted with DCM and concentrated to obtain the crude product, which was purified by flash column (35% siRNA / isohexane) chromatography using silica gel to obtain (S)-4-pyridine-2-ylmethyl-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (950 mg, 36.5%) as a white solid. AnalpH2_MeCN_UPLC_4.0min:Rt:1.28min, m / z 321.5[M+H]+

[0324] Step 5: To a solution of (S)-4-pyridine-2-ylmethyl-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (650 mg, 2.05 mmol) in MeOH (10 ml) and THF (10 ml), 10 ml of 1 N sodium hydroxide solution was added at 0°C. The reaction mixture was stirred at RT for 1 hour. The reaction mixture was concentrated under reduced pressure and acidified with 1 N HCl to adjust the pH to approximately 4-5. The aqueous layer was extracted with ethyl acetate, and the combined organic extract was concentrated under reduced pressure to obtain the crude product, which was purified by SFC to obtain (2S,4R)-4-pyridine-2-ylmethyl-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester (276 mg, 44%) as a white solid. AnalpH2_MeOH_4MIN:Rt:1.78 min, m / z 307.3[M+H]+

[0325] Scheme: Synthesis of (2S,4R)-4-cyclopentylmethylpyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester. [ka]

[0326] Step 1: To a solution of cyclopenta-1-enecarboxylate ethyl ester (9.5 g, 75.3 mmol) in hexane (100 ml), DIBAL (1 M in THF, 82 mL, 83 mmol) was added dropwise at -78°C under a nitrogen atmosphere. The reaction mixture was stirred at -78°C for 3 hours. The reaction mixture was quenched with saturated ammonium chloride (100 mL). The crude product was extracted with diethyl ether (3 × 200 mL), and the combined organic extract was dried over anhydrous magnesium sulfate and concentrated under vacuum to obtain cyclopenta-1-enyl-methanol (5.5 g, 75%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 5.62 (1H, br s), 4.20 (2H, s), 2.37 (4H, m), 1.92 (2H, J = 7.6 Hz, q), 1.36 (1H, br s)

[0327] Step 2: To a stirred solution of cyclopenta-1-enyl-methanol (5.5 g, 56 mmol) in diethyl ether (230 ml), PBr3 (2.3 mL, 28 mmol) was added at -78 °C. The reaction mixture was stirred under N2 at -78 °C for 2 hours, then warmed to room temperature and stirred for 10 hours. The reaction was quenched with saturated sodium bicarbonate solution and extracted with diethyl ether (2 × 100 ml). The combined organic layers were washed with water (2 × 50 ml), followed by brine (100 ml), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (100-200 mesh) using 5-10% EA / Pet ether as the eluent to obtain 1-bromomethyl-cyclopentene (6.0 g, 66%) as a yellow liquid. 1 H NMR (400 MHz, CDCl3) δ 5.80 (1H, br s), 4.09 (2H, s), 2.39 (4H, m), 1.95 (2H, J = 7.2 Hz, q)

[0328] Step 3: (S)-5-oxo-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (4.0 g, 16.4 mmol) was dissolved in dry THF (40 ml). The mixture was cooled to -78°C and LiHMDS (1 M in THF, 18 mL, 18 mmol) was added dropwise under a nitrogen atmosphere. The reaction mixture was stirred for 2 hours and a solution of 1-bromomethyl-cyclopentene (2.91 g, 18.1 mmol) was added dropwise. The mixture was stirred further at -78°C for 3 hours. The reaction was quenched with saturated ammonium chloride (100 ml). The crude product was extracted with SiO2 (3 × 200 ml), and the combined extracts were dried over anhydrous magnesium sulfate and concentrated under vacuum. The crude product was purified by flash column (0-5% siRNA / ihexane) chromatography to obtain (S)-4-cyclopenta-1-enylmethyl-5-oxo-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (mixture of diastereomers) (2.8 g, 53%) as a white solid. 1 H NMR (400 MHz, CDCl3) δ 5.39 (1H, br s), 4.65 (1H, J = 9.6, 1.2 Hz, dd), 3.78 (3H, s), 2.81-2.70 (2H, m), 2.33-2.11 (7H, m), 1.97-1.82 (3H, m), 1.50 (9H, s)

[0329] Step 4: To a solution of (S)-4-cyclopenta-1-enylmethyl-5-oxo-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl (3.0, 9.3 mmol) in methanol, 10% palladium-carbon (1.48 g, 13.9 mmol) was added under a nitrogen atmosphere. The reaction mixture was hydrogenated under balloon pressure for 3 hours. The reaction mixture was filtered through Celite, washed with hot methanol, and concentrated under reduced pressure to obtain crude (S)-4-cyclopentylmethyl-5-oxo-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl (2.7 g, 96%) as a yellow liquid, which was used crudely in subsequent reactions.

[0330] Step 5: To a stirred solution of (S)-4-cyclopentylmethyl-5-oxo-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (2.7 g, 8.3 mmol) in THF (30 ml), BH3.DMS (946 mg, 12.5 mmol) was added at 0°C under argon. The mixture was heated at 90°C for 12 hours. The reaction was quenched with methanol (10 mL) and extracted with ethyl acetate (50 ml). The organic layer was washed with brine, dried over MgSO4, and concentrated to obtain the crude product as an oil. The crude product was purified by column chromatography on silica gel (100-200 mesh) using 30% EA / Pet ether as the eluent to obtain (S)-4-cyclopentylmethyl-2-hydroxymethyl-pyrrolidine-1-carboxylic acid tert-butyl ester (1.1 g, 46.8%) as a green liquid. AnalpH2_MeCN_UPLC_4.0min:Rt:2.49&2.54min, m / z 284.4[M+H]+

[0331] Step 6: To a solution of (S)-4-cyclopentylmethyl-2-hydroxymethyl-pyrrolidine-1-carboxylic acid tert-butyl ester (1.1 g, 3.88 mmol) in acetonitrile (10 ml) and water (10 ml), TEMPO (182 mg, 1.16 mmol) and iodobenzene diacetate (2.75 g, 8.54 mmol) were added at 0°C. The mixture was stirred at RT for 16 hours and then extracted with ethyl acetate (2 × 100 mL). The combined organic layer was washed with brine (100 mL), dried over Na₂SO₄, and concentrated under reduced pressure to obtain the crude product, which was purified by column chromatography on silica gel (100-200 mesh) using 40% EA / Pet ether as the eluent to obtain (S)-4-cyclopentylmethyl-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester (1.0 g, 86.9%) as an off-white solid. AnalpH2_MeCN_UPLC_6.1min:Rt:2.04min, mass not reported

[0332] Step 7: To a stirred solution of (S)-4-cyclopentylmethylpyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester (780 mg, 2.62 mmol) in DMF (15 ml), cesium carbonate (1.28 g, 3.93 mmol) and benzyl bromide (0.3 ml, 2.88 mmol) were added at 0°C. The mixture was stirred under N2 and RT for 2 hours, then diluted with water (50 ml) and extracted with ethyl acetate (2 × 100 ml). The combined organic layers were washed with water and brine (50 ml), dried over anhydrous Na₂SO₄, and concentrated under reduced pressure to obtain the crude product, which was purified by column chromatography on silica gel (100-200 mesh) using 5% EA / Pet ether as the eluent to obtain the title compound as a mixture of diastereomers (1.0 g). The product was further purified by SFC to separate the diastereomer, yielding (2S,4R)-4-cyclopentylmethylpyrrolidine-1,2-dicarboxylic acid 2-benzyl ester 1-tert-butyl ester (820 mg, 79%) as a white solid. AnalpH2_MeCN_UPLC_5.0min:Rt:2.99min, m / z 388.5[M+H]+

[0333] Step 8: In a Parr shaker, a solution of (2S,4R)-4-cyclopentylmethylpyrrolidine-1,2-dicarboxylic acid 2-benzyl ester 1-tert-butyl (820 mg, 2.11 mmol) in methanol was mixed with 10% palladium carbon (811 mg) under a nitrogen atmosphere. The reaction mixture was hydrogenated at 60 psi for 3 hours. The mixture was filtered through Celite, the residue was washed with hot methanol, and the filtrate was concentrated under reduced pressure to obtain (2S,4R)-4-cyclopentylmethylpyrrolidine-1,2-dicarboxylic acid 1-tert-butyl (480 mg, 75.5%) as an off-white solid. AnalpH2_MeCN_UPLC_5.0min:Rt:1.48min, m / z 242.4[M+H]+

[0334] Scheme: Synthesis of (2S,4S)-4-(tetrahydropyran-4-ylmethyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester [ka]

[0335] Steps 1 & 2: To a stirred mixture of tetrahydropyran-4-one (25.0 g, 250 mmol) and water (250 ml), sodium cyanide (12.2 g, 250 mmol), followed by sodium disulfate, was added at 0°C until the pH reached 4.3–5. The reaction mixture was stirred at 10°C for 1 hour. The organic layer was separated, and the aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with a small amount of water (50 ml) and then concentrated under reduced pressure. The resulting product was redissolved in toluene (100 ml), heated to 65°C, and pyridine (50.0 ml, 614 mmol) was added, followed by the slow addition of POCl3 (27.0 ml, 288 mmol). The reaction mixture was stirred at 65°C for 2 hours, then cooled to 35°C, followed by the slow addition of water (200 ml). The layers were separated, and the aqueous layer was extracted with toluene (200 ml). The combined organic layers were washed with water (300 ml) and concentrated under reduced pressure to obtain 3,6-dihydro-2H-pyran-4-carbonitrile (28 g crude) as a white solid, which was used directly in the next step without purification.

[0336] Step 3: In 40 ml of THF cooled to -5°C under a nitrogen atmosphere, a solution of crude 3,6-dihydro-2H-pyran-4-carbonitrile (28.0 g, 256 mmol) was added dropwise to a solution of DIBAL (1 M in THF, 256 ml, 256 mmol). The reaction mixture was stirred for 3 hours, quenched with saturated aqueous ammonium chloride (200 mL), and extracted with diethyl ether (3 × 300 mL). The combined extracts were dried over MgSO4 and concentrated under vacuum to obtain a yellow oil. The crude product was purified by column chromatography using silica gel (100-200 mesh) with 15% EA / Pet ether as the eluent to obtain 3,6-dihydro-2H-pyran-4-carbaldehyde (3 g, 11%) as a yellow oil. AnalpH2_MeCN_UPLC_3.8min:Rt:1.28min, m / z 113.1[M+H]+

[0337] Step 4: A stirred solution of (S)-5-oxo-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (12.0 g, 49.3 mmol) in dry THF (120 mL) was cooled to -78°C under a nitrogen atmosphere. LiHMDS (1 M, 54 mL, 54 mmol in THF) was added dropwise to the reaction mixture. The mixture was stirred at -78°C for 2 hours, and then a solution of 3,6-dihydro-2H-pyran-4-carbaldehyde (6.0 g, 54.3 mmol) was slowly added. The mixture was stirred further at -78°C for 2 hours, and then the reaction was quenched with saturated ammonium chloride (200 mL) solution. The crude product was extracted with RINKAN (3 × 300 mL), and the combined extracts were dried over anhydrous magnesium sulfate and concentrated under vacuum. The crude material was purified by flash column chromatography (0-5% ethyl acetate / hexane) to obtain (S)-4-[(3,6-dihydro-2H-pyran-4-yl)-hydroxymethyl]-5-oxo-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (7.0 g, 40%) as a white solid. AnalpH2_MeCN_UPLC_3.8min:Rt:1.45&1.50 min, ion mass not observed

[0338] Step 5: To a solution of (S)-4-[(3,6-dihydro-2H-pyran-4-yl)-hydroxymethyl]-5-oxo-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (7.00 g, 19.6 mmol) in DCM (70 ml), TEA (27.4 ml, 196 mmol) and MsCl (4.5 ml, 58.7 mmol) were added at 0°C. The reaction mixture was stirred at RT for 4 hours and then concentrated under reduced pressure. The crude product was dissolved in water and extracted with ethyl acetate (2 × 300 mL). The combined organic extracts were dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain (S)-4-(3,6-dihydro-2H-pyran-4-ylmethyl)-5-oxo-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (1.7 g, 26%) as a yellow liquid. AnalpH2_MeCN_UPLC_5.0min:Rt:2.39min, m / z 338.4[M+Na]+

[0339] Step 6: A solution of (S)-4-(3,6-dihydro-2H-pyran-4-ylmethyl)-5-oxo-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl (1.7 g, 5.0 mmol) and 10% palladium carbon (644 mg, 6.05 mmol) were added to methanol (50 ml) under a nitrogen atmosphere. The mixture was hydrogenated at 60 psi for 2 hours. The reaction mixture was filtered through Celite, the residue was washed with hot methanol, and the combined filtrate and washings were concentrated under reduced pressure to obtain crude (S)-5-oxo-4-(tetrahydro-pyran-4-ylmethyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl (1.4 g, 81%) as an off-white solid, which was used in subsequent reactions without further purification.

[0340] Step 7: To a stirred solution of (S)-5-oxo-4-(tetrahydropyran-4-ylmethyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (1.4 g, 4.1 mmol) in THF (60 ml), a solution of BH3.DMS (1.2 ml, 12 mmol) was added to the reaction mixture at 0°C under argon. The mixture was stirred at 90°C for 12 hours, then quenched with methanol, and the reaction mixture was extracted with ethyl acetate (50 ml). The combined organic fraction was washed with brine, dried over MgSO4, and concentrated under reduced pressure. The crude product was purified by column chromatography using silica gel (100-200 mesh) with 30% EA / Pet ether as the eluent to obtain (S)-2-hydroxymethyl-4-(tetrahydropyran-4-ylmethyl)-pyrrolidine-1-carboxylic acid tert-butyl ester (400 mg, 33%) as a yellow liquid. AnalpH2_MeCN_UPLC_3.8min:Rt:1.86min, m / z 300.6[M+H]+

[0341] Step 8: To a solution of (S)-2-hydroxymethyl-4-(tetrahydropyran-4-ylmethyl)-pyrrolidine-1-carboxylic acid tert-butyl ester (400 mg, 1.33 mmol) in acetonitrile (10 ml) and water (10 ml), TEMPO (250 mg, 1.59 mmol) and iodobenzene diacetate (945 mg, 2.93 mmol) were added at 0°C. The mixture was stirred at room temperature for 16 hours. The reaction mixture was extracted with ethyl acetate (2 × 100 mL), the combined organic layer was washed with brine (100 mL), dried over Na₂SO₄, and concentrated under reduced pressure to obtain the crude product. This was purified by column chromatography using silica gel (100-200 mesh) with 40% EA / Pet ether as the eluent to obtain (S)-4-(tetrahydropyran-4-ylmethyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester (150 mg, 36%) as an off-white solid. AnalpH2_MeCN_UPLC_4.0min:Rt:2.24min, m / z 314.4[M+H]+

[0342] Step 9: To a solution of (S)-4-(tetrahydropyran-4-ylmethyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester (150 mg, 0.479 mmol) in DMF (15 ml), cesium carbonate (234 mg, 0.718 mmol) and benzyl bromide (0.060 ml, 0.53 mmol) were added at 0°C. The mixture was stirred at RT for 2 hours, then diluted with water (10 ml) and extracted with ethyl acetate (2 × 25 ml). The combined organic layers were washed with water and brine (10 ml), dried over anhydrous sodium 2SO4, and concentrated under reduced pressure. The crude product was purified by column chromatography using silica gel (100-200 mesh) with 15% EA / Pet ether as the eluent, followed by SFC purification to obtain (2S,4S)-4-(tetrahydropyran-4-ylmethyl)-pyrrolidine-1,2-dicarboxylic acid 2-benzyl ester 1-tert-butyl ester (40 mg, 79%) as a white solid. AnalpH2_MeCN_UPLC_4.0min:Rt:2.39min, m / z 404.5[M+H]+

[0343] Step 10: In a Parr shaker, a solution of (2S,4S)-4-(tetrahydropyran-4-ylmethyl)-pyrrolidine-1,2-dicarboxylic acid 2-benzyl ester 1-tert-butyl ester (40 mg, 0.10 mmol) in methanol was mixed with 10% palladium carbon (40 mg) under a nitrogen atmosphere. The reaction mixture was hydrogenated at 60 psi for 2 hours. The mixture was filtered through Celite, the residue was washed with methanol, and the mixture was concentrated under reduced pressure to obtain (2S,4S)-4-(tetrahydropyran-4-ylmethyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester (28 mg, 90%) as an off-white semi-solid. AnalpH2_MeCN_UPLC_3.8min:Rt:1.66min, m / z 312.3[MH]- [ka]

[0344] Synthesis of (2S,4R)-4-(3,4-difluorophenyl)-4-hydroxypyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester (M05128-int) [ka] N-boc-4-oxo-L-proline-methyl ester (300 mg, 1.23 mmol) was dissolved in THF and cooled to 0°C. 3,4-difluorophenylmagnesium bromide (0.5 M in THF, 4.93 mL, 2.47 mmol) was slowly added, and the mixture was stirred at 0°C for 1 hour. Water and ethyl acetate were added, and the reaction was filtered through Celite to separate the layers. The aqueous layer was extracted with ethyl acetate, the combined organic matter was dried (MgSO4), and the solvent was removed to obtain a crude sample of (2S,4R)-4-(3,4-difluorophenyl)-4-hydroxypyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester as an orange oily substance, which was used directly in the subsequent reaction.

[0345] (2S,4R)-4-(3,4-difluorophenyl)-4-hydroxy-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl was hydrolyzed with 6 mL of 1 M NaOH / MeOH / THF according to general method 4B and stirred at rt for 1 hour. The solvent was removed and the residue was acidified to pH 5 with 1 M HCl. The aqueous layer was extracted with ethyl acetate, and the combined organic layers were dried over (MgSO4) and the solvent was removed to obtain (2S,4R)-4-(3,4-difluorophenyl)-4-hydroxy-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester (310 mg, 73% in 2 steps) as an orange solid. AnalpH2_MeOH_4MIN:Rt:3.07 min, m / z 366.3[M+Na]+

[0346] The following compounds were prepared using the same method.

[0347] [Table 6]

[0348] [ka] Scheme: Synthesis of (2S,4R)-4-(3,4-difluorophenyl)-4-hydroxypyrrolidine-2-carboxylate methyl ester (M05128-int) [ka] N-boc-4-oxo-L-proline-methyl ester (300 g, 1.23 mmol) was dissolved in THF and cooled to 0°C. 3,4-difluorophenylmagnesium bromide (0.5 M in THF, 4.93 mL, 2.47 mmol) was slowly added, and the reaction mixture was stirred at 0°C for 1 hour. Water and ethyl acetate were added, and the reaction mixture was filtered through Celite to separate the layers. The aqueous layer was extracted with ethyl acetate, the combined organic matter was dried (MgSO4), and the solvent was removed to obtain (2S,4R)-4-(3,4-difluorophenyl)-4-hydroxy-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester as an orange oil, which was used directly in the subsequent reaction.

[0349] (2S,4R)-4-(3,4-difluorophenyl)-4-hydroxy-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl was deprotected by Boc according to general method 2a and stirred at rt for 1 hour. The solvent was removed, and the residue was azeotropically removed with toluene to obtain (2S,4R)-4-(3,4-difluorophenyl)-4-hydroxy-pyrrolidine-2-carboxylic acid methyl, which was used crudely in the next step.

[0350] Scheme: Synthesis of (2S,4S)-4-(3-fluorobenzyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester. [ka]

[0351] Step 1: To a stirred solution of (3-fluorobenzyl)(triphenyl)phosphonium chloride (250 mg, 1.03 mmol) in DCM (5 mL), KOtBu (1 M in THF, 1.54 mL, 1.54 mmol) was added. After 45 minutes, N-boc-4-oxo-L-proline methyl ester (250 mg, 1.03 mmol) was added, and the reaction mixture was stirred at room temperature for 1 hour. Water (20 mL) was added, and the mixture was extracted with DCM (2 × 20 mL). The combined organic extract was dried (MgSO4), the solvent was removed, and the residue was purified by column chromatography (Biotage, 25 g SNAP, 0-50% siRNA / hexane) to obtain (S)-4-[1-(3-fluorophenyl)-meth-ylden]-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (206 mg, 60%) as a yellow oil. AnalpH2_MeOH_4MIN:Rt:3.40 min, m / z 358.3[M+Na]+

[0352] Step 2: Using general method 3A, (S)-4-[1-(3-fluorophenyl)-meth-(E)-ylden]-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (206 mg, 0.61 mmol) was hydrogenated for 2 hours to obtain (2S,4S)-4-(3-fluorobenzyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (180 mg, 87%) as a colorless oil. AnalpH2_MeOH_4MIN:Rt:3.29 min, m / z 360.3[M+Na]+

[0353] Step 3: (2S,4S)-4-(3-fluorobenzyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester was hydrolyzed for 3 hours according to general method 4B, and isolated by purification method b to obtain (2S,4S)-4-(3-fluorobenzyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester (175 mg, 100%) as a colorless oil. AnalpH2_MeOH_4MIN:Rt:3.23 min, m / z 324.2[M+H]+

[0354] Scheme: Synthesis of (2S,4S)-4-(pyrazine-2-ylamino)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester M05243-int [ka] cis-4-amino-B-boc-L-proline methyl hydrochloride (250 mg, 0.89 mmol), DavePhos (35 mg, 0.089 mmol), Pd2dba3 (41 mg, 0.044 mmol), 2-chloropyrazine (80 μL, 0.89 mmol), and NaOtBu (215 mg, 2.23 mmol) were suspended in dioxane (5 mL), and the reaction mixture was degassed for 10 minutes. The reaction mixture was heated in a microwave at 120°C for 30 minutes, filtered through Celite, and the filtrate was extracted with a basic aqueous solution. The basic aqueous extract was acidified and extracted with DCM (3 × 30 mL). The organic extract was dried (MgSO4), and the solvent was removed to obtain (2S,4S)-4-(pyrazine-2-ylamino)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester as a brown oil (59 mg, 21%). AnalpH2_MeOH_4MIN:Rt:2.56 min, m / z 309.3[M+H]+

[0355] Scheme: [ka] Synthesis of (2S,4R)-4-(3-bromo-benzyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester (M05366) 3-Bromobenzyl-L-proline (500 mg, 1.56 mmol) was dissolved in 1,4-dioxane (15 mL) and aqueous NaOH (2 M, 1.6 mL, 3.12 mmol), and a solution of Boc2O (511 mg, 2.34 mmol) in dioxane was added dropwise. The reaction mixture was stirred overnight at room temperature, then diluted with DCM and water, and the layers were separated. The organic layer was washed with water and brine, then passed through a phase separator to remove the solvent, yielding (2S,4R)-4-(3-bromo-benzyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester (313 mg, 52%) as a colorless oil, which was allowed to stand and solidify. AnalpH2_MeOH_4MIN:Rt:3.28 min, m / z 384.3 / 386.2[M+H]+

[0356] The following compounds were prepared using the same method.

[0357] [Table 7]

[0358] Scheme: Synthesis of (2S,4R)-4-(1,3-dihydro-isoindole-2-yl)-pyrrolidine-2-carboxylate methyl ester (M05181-int) [ka]

[0359] Step 1: Boc-4-oxo-proline methyl ester (100 mg, 0.41 mmol) and isoindoline (51 μL, 0.43 mmol) were dissolved in DCE (1.5 mL), and acetic acid (25 μL, 0.41 mmol) was added. After 1 hour, NaBH(OAc)3 (261 mg, 1.23 mmol) was added, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with saturated aqueous NaHCO3 and extracted with DCM. The combined organic matter was passed through a phase separator to remove the solvent and obtain the crude product, which was purified by column chromatography (Biotage, 10 g SNAP, 0-80% ELISA / i-hexane) to obtain (2S,4R)-4-(1,3-dihydro-isoindole-2-yl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (0.13 g, 92%) as a brown oil. AnalpH2_MeOH_4MIN:Rt:1.99 min, m / z 347.3[M+H]+

[0360] Step 2: (2S,4R)-4-(1,3-dihydro-isoindole-2-yl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (110 mg, 0.32 mmol) was subjected to 1 hour of BOC deprotection according to general method 2A. The solvent was removed under vacuum to obtain (2S,4R)-4-(1,3-dihydro-isoindole-2-yl)-pyrrolidine-2-carboxylic acid methyl ester, which was used crudely in subsequent reactions. AnalpH9_MeOH_4MIN:Rt:2.41 min, m / z 247.4[M+H]+

[0361] Synthesis of RgD Scheme: Synthesis of (R)-2-dimethylamino-5-oxo-5-pyrrolidine-1-ylpentanoic acid [ka]

[0362] Step 1: Amide coupling of Boc-D-Glu-OBzl (2.0 g, 5.9 mmol) and pyrrolidine (0.6 mL, 7.1 mmol) using HATU and DIPEA in DCM according to general method 1c. The product was purified by column chromatography (Biotage, 25 g SNAP, 20-80% siRNA / hexane) to obtain (R)-2-tert-butoxycarbonylamino-5-oxo-5-pyrrolidine-1-ylpentanoate benzyl ester as a colorless oil, which was used directly in the subsequent reaction. ANALPH2_MEOH_4min, Rt:3.04min, m / z 391.5[M+H]+

[0363] Step 2: Using general method 2A, (R)-2-tert-butoxycarbonylamino-5-oxo-5-pyrrolidine-1-ylpentanoate benzyl ester was deprotected by Boc for 1.5 hours, followed by purification by SCX-2, and then dried under vacuum to obtain (R)-2-amino-5-oxo-5-pyrrolidine-1-ylpentanoate benzyl ester (1.4 g, 82% in 2 steps), which was used crudely in subsequent reactions. ANALPH2_MEOH_4min, Rt:1.68min, m / z 291.3[M+H]+

[0364] Step 3: Formaldehyde (37% in water, 1 mL), acetic acid (0.5 mL), and NaBH3CN (0.6 g, 9.4 mmol) were added to a solution of (R)-2-amino-5-oxo-5-pyrrolidine-1-ylpentanoate benzyl ester (1.37 g, 4.7 mmol) in methanol (30 mL). The reaction mixture was stirred at room temperature for 2 hours, and then the solvent was removed under vacuum. The residue was separated between DCM and 10% K2CO3 (aq), the aqueous layer was extracted with DCM, the combined organic layers were dried (MgSO4), and the solvent was removed under vacuum to obtain (R)-2-dimethylamino-5-oxo-5-pyrrolidine-1-ylpentanoate benzyl ester (1.7 g, quantitatively) as an opaque oil. ANALPH2_MEOH_4min, Rt:2.99min, m / z 319.4[M+H]+

[0365] Step 4: Hydrogenation of (R)-2-dimethylamino-5-oxo-5-pyrrolidine-1-ylpentanoate benzyl ester using general method 3A for 36 hours. The product was dried in vacuum to obtain (R)-2-dimethylamino-5-oxo-5-pyrrolidine-1-ylpentanoic acid (1.1 g, 100%) as a white solid. ANALPH2_MEOH_4min, Rt:0.75min, m / z 229.3[M+H]+

[0366] Scheme: Synthesis of (2R,4R)-4-(pyrrolidine-1-carbonyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester. [ka]

[0367] Step 1: Following general method 1c, (2R,4R)-pyrrolidine-1,2,4-tricarboxylic acid 1-tert-butyl ester 2-methyl ester (172 mg, 0.63 mmol) was amide-coupled with pyrrolidine (52 μL, 0.63 mmol) using HBTU (239 mg, 0.63 mmol) and DIPEA (329 μL, 1.89 mmol) in DCM (5 mL) to obtain (2R,4R)-4-(pyrrolidine-1-carbonyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (380 mg, quantitative), which was used crudely in the next step. ANALPH2_MEOH_4min, Rt:2.75min, m / z 327.4[M+H]+

[0368] Step 2: Using general method 4B (and isolation method c), (2R,4R)-4-(pyrrolidine-1-carbonyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester was hydrolyzed to obtain (2R,4R)-4-(pyrrolidine-1-carbonyl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester (159 mg, 81% in 2 steps) as a colorless oil. ANALPH2_MEOH_4min, Rt:2.57min, m / z 313.3[M+H]+

[0369] The following analogues were prepared using the same method.

[0370] [Table 8]

[0371] scheme Synthesis of 2-amino-4-methyl-4-phenylpentanoic acid [ka]

[0372] Step 1: 3-methyl-3-phenylbutanal (500 mg, 3.08 mmol) was dissolved in EtOH (10 mL) and H2O (10 mL), and KCN (400 mg, 6.16 mmol) and NH4Cl (489 mg, 9.24 mmol) were added. The reaction mixture was heated overnight at 60 °C. After cooling to room temperature, water (20 mL) was added, and the mixture was extracted with ELISA (2 × 30 mL). The combined organic layers were extracted with 1 M HCl (3 × 20 mL), the combined aqueous layer was basicized to pH 9, and then the product was extracted with ELISA (3 × 20 mL). These combined organic extracts were dried (MgSO4), the solvent was removed, and 2-amino-4-methyl-4-phenyl-pentanenitrile (125 mg, 22%) was obtained as an orange oil, which was used directly in the subsequent reaction. ANALPH2_MEOH_4min, Rt:1.92min, m / z 189.3[M+H]+

[0373] Step 2: 125 mg of 2-amino-4-methyl-4-phenylpentanenitrile was dissolved in 5 mL of 6 M HCl and heated under reflux overnight. The reaction mixture was cooled to room temperature, the solvent was removed under reduced pressure, and the residue was dissolved in 5 mL of H₂O / DCM. 158 mg of Boc₂O and 0.73 mmol of Et₃N were added, and the reaction mixture was stirred at room temperature for 4 days. The layers were separated, the aqueous layer was acidified to pH 4, and extracted with 2 × 20 mL of DCM. The combined organic extracts were passed through a hydrophobic frit, and the solvent was removed to obtain 2-amino-4-methyl-4-phenylpentanoic acid (210 mg, quantitatively) as a colorless oil. ANALPH2_MEOH_4min, Rt:3.31min, m / z 308.3[M+H]+

[0374] Synthesis of (R)-2-tert-butoxycarbonylamino-4-oxo-4-phenylbutyric acid [ka]

[0375] Step 1: Z-AspOMe (500 mg, 1.77 mmol) was dissolved in THF (5 mL), and SOCl2 (200 μL) was added. The reaction mixture was heated at 70°C for 1 hour, then cooled to room temperature and the solvent was removed. The residue was dissolved in the minimum volume of DCM, cooled to 0°C, and isohexane was added until a precipitate formed. The product was collected by filtration and dried to obtain (R)-2-benzyloxycarbonylamino-3-chlorocarbonylpropionate methyl ester (370 mg, 70%) as a white solid.

[0376] Step 2: (R)-2-benzyloxycarbonylamino-3-chlorocarbonylpropionate methyl ester (370 mg, 1.23 mmol) and CuI (22.8 mg, 0.12 mmol) were dissolved in THF (5 mL) and cooled to -78°C. Phenylmagnesium bromide (1 M solution in THF, 1.2 mL, 1.2 mmol) was added, and the reaction mixture was heated to room temperature overnight. The mixture was divided between NH4Cl (aq.) and Â, the organic layer was dried, and the solvent was removed. The residue was purified by column chromatography (Biotage, 10 g, Â / hexane) to obtain (R)-2-benzyloxycarbonylamino-4-oxo-4-phenylbutyrate methyl ester (50 mg, 0.15 mmol) as a yellow oil. ANALPH2_MEOH_4min, Rt:2.98min, m / z 342.3[M+H]+

[0377] Step 3: (R)-2-benzyloxycarbonylamino-4-oxo-4-phenylbutyrate methyl ester (50 mg, 0.15 mmol) was hydrolyzed according to general method 4A to obtain (R)-2-benzyloxycarbonylamino-4-oxo-4-phenylbutyrate, which was used crudely in the subsequent reaction. ANALPH2_MEOH_4min, Rt:2.88min, m / z 328.3[M+H]+

[0378] Synthesis of (R)-2-tert-butoxycarbonylamino-3-(1,2,3,4-tetrahydronaphthalene-1-yl)-propionic acid [ka]

[0379] Step 1: Dissolve (R)-2-amino-3-(1,2,3,4-tetrahydro-naphthalene-1-yl)-propionate methyl ester (100 mg, 0.46 mmol) in DCM (5 mL), add Et3N (0.10 mL, 0.69 mmol) and Boc2O (110 mg, 0.51 mmol), and stir the reaction mixture overnight at room temperature. Remove the solvent, dilute the residue with water, and extract with DCM. Dry the combined organic extract, remove the solvent, and obtain (R)-2-tert-butoxycarbonylamino-3-(1,2,3,4-tetrahydro-naphthalene-1-yl)-propionate methyl ester (94 mg, 61%) as a colorless oil. ANALPH2_MEOH_4min, Rt:3.51min, m / z 334.3[M+H]+

[0380] Step 2: Methyl (R)-2-tert-butoxycarbonylamino-3-(1,2,3,4-tetrahydro-naphthalen-1-yl)-propionate (94 mg, 0.28 mmol) was dissolved in MeOH, 2 M NaOH was added (0.28 mL, 0.56 mmol), and the mixture was stirred at room temperature for 2 hours. 2 M HCl(aq) (0.28 mL, 0.56 mmol) was added, then the solvent was removed, and the residue was divided between water and ethyl acetate. The aqueous layer was extracted with ethyl acetate, then the combined organic layers were washed with brine, dried, and the solvent was removed to obtain (R)-2-tert-butoxycarbonylamino-3-(1,2,3,4-tetrahydro-naphthalen-1-yl)-propionic acid (60 mg, 41%) as a white solid. ANALPH2_MEOH_4min, Rt:3.42min, m / z 320.3[M+H]+

[0381] Scheme 3: Constrained RgD & RgB Intermediate [ka]

[0382] Procedure Example X: Synthesis of (R)-4-(1-methyl-1H-pyrazole-4-yl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester [ka]

[0383] Step 1: Triflate formation (R)-4-trifluoromethanesulfonyloxy-2,5-dihydropyrrole-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester [ka] Lithium bis(trimethylsilyl)amide (1M, 44.0 mmol, 44.0 mL in THF) was added dropwise to a stirred solution of (R)-4-oxo-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (40.0 mmol, 9.72 g) in anhydrous THF (50 mL) under an N2 atmosphere at -78°C for 20 minutes. The resulting solution was stirred at -78°C for 1 hour, and then a solution of N-phenyl-bis(trifluoromethanesulfonamide) (44.0 mmol, 15.7 g) in anhydrous THF (40 mL) was added dropwise over 20 minutes. The resulting solution was stirred at -78°C for 2 hours, and then warmed to room temperature over 2 hours. Saturated aqueous ammonium chloride (100 mL), followed by DCM (100 mL) and water (100 mL) were added to the reaction mixture. The layers were separated, and the aqueous layer was extracted with DCM (3 × 50 mL). The combined organic extracts were washed with water (100 mL) and brine (100 mL), passed through hydrophobic frit, and concentrated under vacuum. The crude material was partially purified by column chromatography (Biotage Isolera, SNAP KP-Sil 340 g, dry-packed in Celite, gradient i-hexane / siRNA from 99:1 to 90:10) to obtain a sample of (R)-4-trifluoromethanesulfonyloxy-2,5-dihydropyrrole-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (8.22 g, 30%) as a yellow oil, which was used "as is" in the subsequent reaction.

[0384] Step 2: Pd-mediated coupling Example of Pd-mediated coupling using Suzuki conditions: (R)-4-(1-methyl-1H-pyrazole-4-yl)-2,5-dihydropyrrole-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester [ka] (R)-4-trifluoromethanesulfonyloxy-2,5-dihydropyrrole-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (2.67 mmol, 1.00 g); 1-methyl-pyrazole-4-boronic acid, pinacol ester (1.78 mmol, 1 equivalent, 0.370 g); tetrakis(triphenylphosphine)palladium (0) (0.178 mmol, 0.206 g); and potassium carbonate (3.56 mmol, 0.492 g) were suspended in 1,4-dioxane / water (1:1 v / v, 16 mL) and stirred with nitrogen injection for 10 minutes. The resulting solution was heated in a microwave at 100°C for 10 minutes. The reaction mixture was then diluted with DCM (20 mL) and water (20 mL). The organic phase was washed with water (2 × 20 mL) and brine (20 mL), then passed through hydrophobic frit and concentrated under vacuum. The crude product was purified by flash column chromatography (Biotage Isolera, SNAP KP-Sil 50 g, dry-packed in Celite, gradient i-hexane / siRNA from 70:30 to 50:50) to obtain (R)-4-(1-methyl-1H-pyrazole-4-yl)-2,5-dihydropyrrole-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (0.425 g, 78%) as a colorless oil. AnalpH2_MeOH_4MIN:Rt:2.89 min, m / z 308.2[M+H]+

[0385] Example of Pd-mediated coupling using Stille conditions: (R)-4-pyrimidine-4-yl-2,5-dihydropyrrole-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester [ka] (R)-4-trifluoromethanesulfonyloxy-2,5-dihydropyrrole-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (0.799 mmol, 1.5 equivalents, 0.300 g), 4-(tributylstannyl)pyrimidine (0.532 mmol, 1.0 equivalent, 0.196 g), tetrakis(triphenylphosphine)palladium(0) (0.053 mmol, 0.1 equivalent, 0.061 g), and copper(I) iodide (0.053 mmol, 0.1 equivalent, 0.010 g) were suspended in 1,4-dioxane and stirred with nitrogen injection for 10 minutes. The resulting suspension was heated in a microwave at 100°C for 5 minutes. The crude product was concentrated in vacuum and then purified by flash column chromatography (Biotage Isolera, SNAP KP-Sil 25g, dry-packed in Celite, gradient i-hexane / siRNA 70:60 to 50:50) to obtain (R)-4-pyrimidine-4-yl-2,5-dihydropyrrole-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (0.157g, 0.515 mmol, 97%) as a yellow oil, which was used without further purification. AnalpH2_MeOH_4MIN:Rt:2.96 min, m / z 306.3[M+H]+

[0386] Step 3: Hydrogenation (R)-4-(1-methyl-1H-pyrazole-4-yl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (530-150-2-1) [ka] (R)-4-(1-methyl-1H-pyrazole-4-yl)-2,5-dihydropyrrole-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl (1.38 mmol, 0.425 g), 10% palladium carbon, and hydrogen gas were reacted according to general procedure 3A to obtain (R)-4-(1-methyl-1H-pyrazole-4-yl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl as a colorless oil (0.351 g, 1.13 mmol, 82%), which was used without further purification. AnalpH2_MeOH_4MIN:Rt:2.80 min, m / z 310.2[M+H]+

[0387] Step 4: Ester hydrolysis (R)-4-(1-methyl-1H-pyrazole-4-yl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester [ka] (R)-4-(1-methyl-1H-pyrazole-4-yl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (1.13 mmol, 1.0 equivalent, 0.351 g) and LiOH (1 M in H2O, 4.52 mmol, 4.0 equivalent, 0.452 mL) were reacted according to general procedure 4A and purified to obtain (R)-4-(1-methyl-1H-pyrazole-4-yl)-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester (0.333 g, 1.13 mmol, 100%) as a colorless rubbery substance, which was used without further purification. AnalpH2_MeOH_4MIN:Rt:2.48 min, m / z 296.3[M+H]+

[0388] The following compounds were synthesized by the same method.

[0389] [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4]

[0390] Scheme 4 Carboxamide Library [ka] Synthesis of (R)-2-tert-butoxycarbonylamino-5-oxo-5-pyrrolidine-1-ylpentanoic acid [ka]

[0391] Step 1: Boc-D-Glu-OBzl (400 mg, 1.19 mmol) was dissolved in DCM (20 mL), and HATU (498 mg, 1.31 mmol) and DIPEA (0.62 mL, 3.57 mmol) were added. 2-Methylpyrrolidine (0.15 mL, 1.42 mmol) was added, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was washed with water (3 × 40 mL), the organic layer was dried (MgSO4), and the solvent was removed under vacuum. The residue was purified by column chromatography to obtain (R)-2-tert-butoxycarbonylamino-5-(2-methylpyrrolidine-1-yl)-5-oxopentanoate benzyl ester (0.37 g, 77%) as a colorless oil.

[0392] Step 2: (R)-2-tert-butoxycarbonylamino-5-(2-methyl-pyrrolidine-1-yl)-5-oxopentanoic acid benzyl ester (0.37 g, 0.91 mmol) was dissolved in EtOH (10 mL) and placed under an N2 atmosphere, and Pd / C (37 mg) was added. An H2 atmosphere was introduced, and the reaction mixture was stirred at room temperature for 72 hours. The reaction mixture was filtered through Celite, and the solvent was removed under vacuum to obtain (R)-2-tert-butoxycarbonylamino-5-(2-methyl-pyrrolidine-1-yl)-5-oxopentanoic acid (273 mg, 95%) as a colorless oil. AnalpH2_MeOH_4MIN:Rt:2.75 min, m / z 315.2[M+H]+

[0393] The following compounds were synthesized by the same method.

[0394] [Table 10]

[0395] Scheme 5: Synthesis of (2R)-2-{[(tert-butoxy)carbonyl]amino}-4-(4-fluorophenyl)butanoic acid [ka]

[0396] Step i: To (R)-N-boc-2,2-dimethyl-4-vinyloxazolidine (95 mg, 0.43 mmol) in anhydrous THF (1.5 mL), 9-BBN (0.5 M in THF, 1.7 mL, 0.84 mmol) was added dropwise at 0°C under N2. The flask was wrapped in foil and stirred at RT for 2 hours. Then, 3M K3PO4 was added. 4(aq)(0.28 mL, 0.84 mmol) was added dropwise, followed by 4-fluoroiodobenzene (0.5 mL, 0.46 mmol) in anhydrous degassed DMF (1.5 mL) and PdCl2 (dppf).DCM (17 mg, 0.02 mmol). The reaction was then stirred for 19 hours, after which the reaction mixture was concentrated under vacuum. The crude mixture was suspended in Et2O (30 mL) and saturated NaHCO3 3(aq) The aqueous phase was washed with solution (40 mL). The aqueous phase was further washed with Et2O (30 mL). The combined organic phase was washed with brine (30 mL), dried over Na2SO4, and concentrated under vacuum. Tert-butyl(4R)-4-[2-(4-fluorophenyl)ethyl]-2,2-dimethyl-1,3-oxazolidine-3-carboxylate was isolated as a colorless oil (55 mg, 0.17 mmol, 40%) by column chromatography (0-20% ethyl t = 46.3 seconds m / z 224.36[M-Boc+H] +

[0397] Step ii: To a solution of tert-butyl(4R)-4-[2-(4-fluorophenyl)ethyl]-2,2-dimethyl-1,3-oxazolidine-3-carboxylate (22 mg, 0.069 mmol) in methanol (2 mL), p-toluenesulfonic acid monohydrate (1 mg, 0.007 mmol) was added at 0°C. The reaction was stirred at 0°C for 1 hour, then heated to RT and stirred for a further 20 hours. The reaction mixture was concentrated under vacuum and then divided between DCM (20 mL) and saturated NaHCO3 solution (20 mL). The aqueous phase was further extracted with DCM (2 × 20 mL). The combined organic phase was dried over Na2SO4 and concentrated under vacuum. tert-butyl N-[(2R)-4-(4-fluorophenyl)-1-hydroxybutan-2-yl]carbamate was isolated as a colorless, rubbery substance by column chromatography (20-60% ethyl acetate in petroleum). This was allowed to stand and solidify into a white solid (10 mg, 0.035 mmol, 51%). LCMS:R t =0.6 min m / z 589.57[2M+Na] +

[0398] Step III: To a solution of tert-butyl N-[(2R)-4-(4-fluorophenyl)-1-hydroxybutan-2-yl]carbamate (36 mg, 0.13 mmol) in acetone (0.6 mL), 5% NaHCO3 solution (0.3 mL) was added, and the reaction mixture was cooled to 0°C. Potassium bromide (2 mg, 0.013 mmol) and TEMPO (24 mg, 0.15 mmol) were added, followed by dropwise addition of 5% sodium hypochlorite solution (0.32 mL, 0.24 mmol). The reaction temperature was maintained below 5°C for 1 hour. The reaction mixture was then diluted with water (3 mL) and extracted with RINKAN (2 × 7 mL). The combined organic phase was washed with brine (10 mL), dried over Na2SO4, and concentrated under vacuum. (2R)-2-{[(tert-butoxy)carbonyl]amino}-4-(4-fluorophenyl)butanoic acid was used without further purification (8 mg, 0.027 mmol, 21%). LCMS:R t =0.6 min m / z 617.51[2M+Na] +

[0399] Synthesis of a homophenylalanine library (A) [ka]

[0400] The following intermediates were prepared using this general methodology.

[0401] [Table 11]

[0402] Synthesis of a homophenylalanine library (B) [ka]

[0403] [Table 12-1] [Table 12-2]

[0404] Synthesis of 1-tert-butyl 2-lithino(2R,4R)-4-(piperidine-1-yl)pyrrolidine-1,2-dicarboxylate [ka]

[0405] Step 1: To a solution of (2R,4S)-1-tert-butyl-2-methyl-4-hydroxypyrrolidine-1,2-dicarboxylate (267 mg, 1.09 mmol) and Et3N (0.18 mL, 1.31 mmol) in anhydrous DCM (5 mL), mesyl chloride (0.10 mL, 1.31 mmol) was added under N2 at 0°C. The temperature was maintained below 5°C for 1 hour, and then the reaction was diluted with DCM (20 mL), 1 M HCl (30 mL), and saturated NaHCO3. 3(aq) The mixture was then sequentially washed with brine (30 mL), dried over Na2CO3, and concentrated under vacuum. Next, the crude mesylate was dissolved in THF (1 mL) and piperidine (1 mL) and heated at 120°C by microwave heating. After 110 minutes, the reaction was diluted with water (30 mL) and extracted with ELISA (3 × 30 mL). The combined organic phase was washed with brine (40 L), dried over Na2CO3, and concentrated under vacuum. The crude product was purified using SCX-2 to obtain 1-tert-butyl 2-methyl(2R,4R)-4-(piperidine-1-yl)pyrrolidine-1,2-dicarboxylate (137 mg, 40%). Thermo_MeOH_UHPLC_1.2min LCMS:Rt=0.6min m / z 313.44[M+H] +

[0406] Step 2: To a solution of 1-tert-butyl 2-methyl(2R,4R)-4-(piperidine-1-yl)pyrrolidine-1,2-dicarboxylate (137 mg, 0.44 mmol) in MeOH (5 mL), LiOH.H2O (18 mg, 0.44 mmol) was added. The reaction was stirred at RT for 48 hours, and then LiOH.H2O (18 mg, 0.44 mmol) was added again. After stirring at RT for a further 2.5 hours, the reaction was heated to 60°C for 3 hours. After adding LiOH.H2O (18 mg, 0.44 mmol) and heating the reaction to 60°C for a further 1 hour, complete consumption of the starting material was shown by LC-MS. After cooling the reaction mixture to RT, it was concentrated under vacuum, and 1-tert-butyl2-lithio(2R,4R)-4-(piperidine-1-yl)pyrrolidine-1,2-dicarboxylate (148 mg, 95%) was used without further purification. Thermo_MeOH_UHPLC_1.2min LCMS:Rt=27.8 seconds m / z 299.40[M+H] +

[0407] General Scheme 1 [ka] (2S,4S)-1-((R)-2-amino-4-phenyl-butyryl)-4-phenyl-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide M05119 [ka]

[0408] Step 1: (2S,4S)-1-boc-4-phenylpyrrolidine-2-carboxylic acid (90 mg, 0.31 mmol), 5-(aminomethyl)-1-methyl-1H-benzotriazole (50 mg, 0.31 mmol), and HATU (117 mg, 0.31 mmol) were dissolved in DMF (2 mL), and Et3N (43 μL, 0.31 mmol) was added. The reaction mixture was stirred overnight at room temperature and then diluted with ELISA (20 mL). The organic layer was washed with water (3 × 10 mL) and brine (10 mL), dried over Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by column chromatography (biotage, 10 g, 0-100% ethyl acetate / hexane) to obtain (2S,4S)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-4-phenyl-pyrrolidine-1-carboxylic acid tert-butyl ester (100 mg, 82%) as a colorless oil. ANALPH2_MEOH_4min, Rt:3.03min, m / z 436[M+H]+

[0409] Step 2: (2S,4S)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-4-phenyl-pyrrolidine-1-carboxylic acid tert-butyl ester (110 mg, 0.25 mmol) was dissolved in DCM (2 mL), TFA (1 mL) was added, and the mixture was stirred at room temperature for 1 hour. The solution was concentrated under vacuum to obtain (2S,4S)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-4-phenyl-pyrrolidine as a clear oil, which was used in subsequent reactions without further purification. ANALPH2_MEOH_4min, Rt:1.71min, m / z 336[M+H]+

[0410] Step 3: (2S,4S)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-4-phenyl-pyrrolidine (85 mg, 0.25 mmol), Boc-D-homophenylalanine (71 mg, 0.25 mmol), and HATU (96 mg, 0.25 mmol) were combined in DMF (2 mL), then Et3N (60 μL, 0.25 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction mixture was diluted with Â. The organic layer was washed with water (2 × 10 mL), saturated NH4Cl aqueous solution (2 × 10 mL), saturated NaHCO3 aqueous solution (2 × 10 mL), and brine, dried over Na2SO4, filtered, and concentrated under vacuum to obtain ((R)-1-{(2S,4S)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-4-phenyl-pyrrolidine-1-carbonyl}-3-phenyl-propyl)-carbamate tert-butyl ester (110 mg, 74%) as a colorless oil, which was used in subsequent reactions without further purification. ANALPH2_MEOH_4min, Rt:3.40min, m / z 597.4[M+H]+

[0411] Step 4: ((R)-1-{(2S,4S)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-4-phenyl-pyrrolidine-1-carbonyl}-3-phenyl-propyl)-carbamate tert-butyl ester (110 mg, 0.19 mmol) was dissolved in DCM (2 mL), TFA (1 mL) was added, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was washed with MeOH and purified with SCX-2 (2 g) eluted with NH3 / MeOH to obtain a yellow oily substance, which was freeze-dried from MeCN / H2O to obtain (2S,4S)-1-((R)-2-amino-4-phenyl-butyryl)-4-phenyl-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)-amide (65 mg, 71%) as a white solid. ANALPH9_MEOH_QC_v1, Rt:7.58 min, m / z 497.33[M+H]+ ANALPH2_MEOH_QC_v1, Retention time: 5.70 minutes, m / z 497.26 [M+H]+ 1H NMR (400 MHz, DMSO-D6) δ 8.86 (t, J = 5.9 Hz, 0.2H), 8.51 (t, J = 6.0 Hz, 0.8H), 7.90 (s, 0.2H), 7.89 (s, 0.8H), 7.80 (d, J = 8.6 Hz, 0.8H), 7.74 (d, J = 8.5 Hz, 0.2H), 7.48 (dd, J = 8.6, 1.5 Hz, 1H), 7.39 - 7.09 (m, 9.5H), 6.97 - 6.91 (m, 0.5H), 4.55 - 4.37 (m, 3H), 4.29 (s, 3H), 4.22 (s, 1H), 3.95 (dd, J = 9.5, 7.6 Hz, 1H), 3.50 (d, J = 9.6 Hz, 1H), 3.22 (t, J = 9.8 Hz, 1H), 2.88 - 2.56 (m, 2H), 2.46 - 2.13 (m, 2H), 2.08 (s, 2H), 1.86 - 1.71 (m, 1H), 1.63 - 1.43 (m, 1H).

[0412] The following compounds were prepared by the same method.

[0413] [Table 13] JPEG0007901865000141.jpg232169 JPEG0007901865000142.jpg172169 JPEG0007901865000143.jpg169169 JPEG0007901865000144.jpg251169 JPEG0007901865000145.jpg169169 JPEG0007901865000146.jpg188169 JPEG0007901865000147.jpg188169 JPEG0007901865000148.jpg201169 JPEG0007901865000149.jpg207169 JPEG0007901865000150.jpg194169 JPEG0007901865000151.jpg194169 JPEG0007901865000152.jpg219169 JPEG0007901865000153.jpg238169 JPEG0007901865000154.jpg213169 JPEG0007901865000155.jpg238169 JPEG0007901865000156.jpg213169 JPEG0007901865000157.jpg219169 JPEG0007901865000158.jpg232169 JPEG0007901865000159.jpg226169 JPEG0007901865000160.jpg244169 JPEG0007901865000161.jpg213169 JPEG0007901865000162.jpg213169 JPEG0007901865000163.jpg194169 JPEG0007901865000164.jpg232169 JPEG0007901865000165.jpg219169 JPEG0007901865000166.jpg213169 JPEG0007901865000167.jpg201169 JPEG0007901865000168.jpg226169 JPEG0007901865000169.jpg226169 JPEG0007901865000170.jpg226169 JPEG0007901865000171.jpg226169 JPEG0007901865000172.jpg232169 JPEG0007901865000173.jpg238169 JPEG0007901865000174.jpg244169 JPEG0007901865000175.jpg244169 JPEG0007901865000176.jpg226169 JPEG0007901865000177.jpg238169 JPEG0007901865000178.jpg238169 JPEG0007901865000179.jpg251169 JPEG0007901865000180.jpg244169 JPEG0007901865000181.jpg244169 JPEG0007901865000182.jpg188169 JPEG0007901865000183.jpg169169 JPEG0007901865000184.jpg176169 JPEG0007901865000185.jpg176169 JPEG0007901865000186.jpg188169 JPEG0007901865000187.jpg188169 JPEG0007901865000188.jpg182169 JPEG0007901865000189.jpg188169 JPEG0007901865000190.jpg182169 JPEG0007901865000191.jpg169169 JPEG0007901865000192.jpg188169 JPEG0007901865000193.jpg188169 JPEG0007901865000194.jpg182169 JPEG0007901865000195.jpg188169 JPEG0007901865000196.jpg169169 JPEG0007901865000197.jpg232169 JPEG0007901865000198.jpg188169 JPEG0007901865000199.jpg182169 JPEG0007901865000200.jpg176169 JPEG0007901865000201.jpg176169 JPEG0007901865000202.jpg169169 JPEG0007901865000203.jpg176169 JPEG0007901865000204.jpg176169 JPEG0007901865000205.jpg182169 JPEG0007901865000206.jpg176169 JPEG0007901865000207.jpg194169

[0414] The following compounds were prepared by the same method (general scheme 1), omitting step 4.

[0415] [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4] [Table 14-5]

[0416] General Scheme 2 [ka] Synthesis of (S)-1-((R)-2-amino-5-oxo-5-pyrrolidine-1-ylpentanoyl)-4-(4-trifluoromethoxybenzyl)-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide (M05306) [ka]

[0417] Step 1: (S)-4-(4-trifluoromethoxybenzyl)-pyrrolidine-2-carboxylate methyl ester (100 mg, 0.33 mmol), (R)-2-tert-butoxycarbonylamino-5-oxo-5-pyrrolidine-1-ylpentanoic acid (109 mg, 0.36 mmol), HATU (125 mg, 0.33 mmol), and DIPEA (172 μL, 0.99 mmol) were dissolved in DMF (2 mL) and stirred overnight at room temperature. The reaction mixture was diluted with DCM, washed with water (20 mL) and brine (20 mL), then passed through a phase separator and the solvent was removed under vacuum. The residue was purified by column chromatography (biotage, 0-100% ethyl hexane) to obtain (S)-1-((R)-2-tert-butoxycarbonylamino-5-oxo-5-pyrrolidine-1-ylpentanoyl)-4-(4-trifluoromethoxybenzyl)-pyrrolidine-2-carboxylate methyl ester as a yellow oil (79 mg, 41%). ANALPH2_MEOH_4min, Rt:3.35min, m / z 586[M+H]+

[0418] Step 2: (S)-1-((R)-2-tert-butoxycarbonylamino-5-oxo-5-pyrrolidine-1-ylpentanoyl)-4-(4-trifluoromethoxybenzyl)-pyrrolidine-2-carboxylic acid methyl ester (79 mg, 0.14 mmol) was dissolved in MeOH (1 mL) and aqueous LiOH (1 M solution, 1 mL) and stirred at room temperature for 2 hours. The solvent was removed under reduced pressure to obtain (S)-1-((R)-2-tert-butoxycarbonylamino-5-oxo-5-pyrrolidine-1-ylpentanoyl)-4-(4-trifluoromethoxybenzyl)-pyrrolidine-2-carboxylic acid (77 mg, 40%), which was used crudely in the next step. ANALPH2_MEOH_4min, Rt:3.32min, m / z 572.2[M+H]+

[0419] Step 3: Dissolve (S)-1-((R)-2-tert-butoxycarbonylamino-5-oxo-5-pyrrolidine-1-ylpentanoyl)-4-(4-trifluoromethoxybenzyl)-pyrrolidine-2-carboxylic acid (77 mg, 0.14 mmol), C-(1-methyl-1H-benzotriazole-5-yl)-methylamine (29 mg, 0.15 mmol), HATU (57 mg, 0.14 mmol), and DIPEA (94 μL, 0.54 mmol) in DMF (2 mL) and stir overnight at room temperature. Add an additional DIPEA (24 μL, 0.14 mmol), HATU (57 mg, 0.14 mmol), and C-(1-methyl-1H-benzotriazole-5-yl)-methylamine (29 mg, 0.15 mmol), and stir the reaction mixture at room temperature for 3 days. The reaction mixture was diluted with DCM and washed with water (2 × 20 mL), then with brine (20 mL). The organic matter was passed through a phase separator, and the solvent was removed under vacuum to obtain {(R)-1-[(S)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-4-(4-trifluoromethoxybenzyl)-pyrrolidine-1-carbonyl]-4-oxo-4-pyrrolidine-1-yl-butyl}carbamate tert-butyl ester, which was used crudely in the next step. ANALPH2_MEOH_4min, Rt:3.26min, m / z 716[M+H]+

[0420] Step 4: {(R)-1-[(S)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-4-(4-trifluoromethoxy-benzyl)-pyrrolidine-1-carbonyl]-4-oxo-4-pyrrolidine-1-yl-butyl}carbamate tert-butyl ester was dissolved in 10% TFA / DCM and stirred overnight at room temperature. The reaction mixture was passed through an SCX-2 cartridge eluted with 1M NH3 / MeOH to remove the solvent. The residue was purified by preparative HPLC to obtain (S)-1-((R)-2-amino-5-oxo-5-pyrrolidine-1-ylpentanoyl)-4-(4-trifluoromethoxy-benzyl)-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide as a white solid (4 mg, 5%). ANALPH9_MEOH_QC_v1, Rt:7.74 min, m / z 616.3[M+H]+ ANALPH2_MEOH_QC_v1, Rt:6.27 min, m / z 616.3[M+H]+

[0421] The following compounds were prepared in the same manner as those listed above, except that modifications were used, and in that case, the properties of the modified protocol are described in detail.

[0422] [Table 15-1] [Table 15-2] [Table 15-3] [Table 15-4] [Table 15-5] [Table 15-6] [Table 15-7]

[0423] General Scheme 3 (Amino RgB) [ka] Synthesis of (2S,4S)-4-acetylamino-1-((R)-2-amino-4-phenyl-butyryl)-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide (M05144) [ka]

[0424] Step 1: Piperidine (1 mL) was slowly added to a solution of {(3S,5S)-1-((R)-2-amino-4-phenyl-butyryl)-5-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-3-yl}carbamate 9H-fluoren-9-yl methyl ester (1.42 g, 1.87 mmol) in DCM (10 mL), and the reaction mixture was stirred at room temperature for 4 hours. The solvent was removed under vacuum, and the product was purified by column chromatography (Biotage, 100 g SNAP, in DCM (0-(1% Et3N and 10% MeOH)) to obtain ((R)-1-{(S)-4-amino-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-carbonyl}-3-phenyl-propyl)-carbamate tert-butyl ester (878 mg, 88%) as an off-white solid. ANALPH2_MEOH_4min, Rt:2.28min, m / z 536.4[M+H]+

[0425] Step 2: Amine Functionalization Method 1 Example of amine functionalization using alkylation: ((R)-1-{(S)-4-amino-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-carbonyl}-3-phenyl-propyl)-carbamate tert-butyl ester (50 mg, 0.093 mmol) was dissolved in DMF (3 mL), and DIPEA (49 μL, 0.14 mmol, 1.5 eq) was added, followed by acetyl chloride (20 μL, 0.28 mmol, 3.0 eq). The reaction mixture was stirred at room temperature for 1.5 hours, then brine was added, and the mixture was extracted with ELISA. The combined organic compounds were washed with brine, dried (MgSO4), and the solvent was removed under vacuum to obtain crude ((R)-1-{(S)-4-acetylamino-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-carbonyl}-3-phenyl-propyl)-carbamate tert-butyl ester (51 mg, 95%), which was used directly in subsequent reactions without further purification. ANALPH2_MEOH_4min, Rt:3.05min, m / z 600.4[M+Na]+

[0426] Method 2: An example of amine functionalization using reductive amination: Synthesis of ((R)-1-{(2S,4S)-4-benzylamino-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-carbonyl}-3-phenyl-propyl)-carbamate tert-butyl ester [ka] ((R)-1-{(S)-4-amino-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-carbonyl}-3-phenyl-propyl)-carbamate tert-butyl ester (100 mg, 0.19 mmol) was dissolved in DCE, and AcOH (22 μL, 0.38 mmol), followed by benzoaldehyde (21 μL, 0.21 mmol) and NaBH(OAc)3 (61 mg, 0.29 mmol) were added. The reaction mixture was stirred overnight at room temperature, followed by the addition of NaBH(OAc)3 (41 mg, 0.19 mol) and AcOH (11 μL, 0.19 mmol), and the mixture was stirred for 1.5 hours. Na2CO3 solution (saturated aqueous solution) was added, and the mixture was passed through a phase separator, after which the aqueous layer was further extracted with DCM. The combined organic extracts were purified using SCX-2 elution with 0.5 M NH3 / MeOH. The solvent was removed under reduced pressure to obtain ((R)-1-{(2S,4S)-4-benzylamino-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-carbonyl}-3-phenyl-propyl)-carbamate tert-butyl ester (96 mg, 81%), which was used in subsequent reactions without further purification. ANALPH2_MEOH_4min, Rt:3.36min, m / z 626.3[M+H]+

[0427] Method 3: An example of amine functionalization using urea formation: Synthesis of {(R)-1-[(2S,4S)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-4-(3-methylureido)-pyrrolidine-1-carbonyl]-3-phenyl-propyl}-carbamate tert-butyl ester [ka] ((R)-1-{(S)-4-amino-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-carbonyl}-3-phenyl-propyl)-carbamate tert-butyl ester (75 mg, 0.14 mmol) was dissolved in DCM, and Et3N (29 μL, 0.21 mmol) and CDI (24 mg, 0.15 mmol) were added. The reaction mixture was stirred at room temperature for 1.75 hours. Methylamine (2 M in THF, 0.35 mL, 0.7 mmol) was added, and the reaction mixture was stirred for 30 minutes. Further aliquots of methylamine (2 M in THF, 0.21 mL, 0.42 mmol) were added, and the mixture was stirred at room temperature for 3 days. The reaction mixture was diluted with brine and DCM, the aqueous layer was then extracted with DCM, and the combined organic layer was concentrated to obtain {(R)-1-[(2S,4S)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-4-(3-methyl-ureido)-pyrrolidine-1-carbonyl]-3-phenyl-propyl}carbamate tert-butyl ester (70 mg, 84%) as a colorless oil, which was used directly in subsequent reactions without further purification. ANALPH2_MEOH_4min, Rt:2.99min, m / z 593[M+H]+

[0428] Step 3: Final Deprotection Acetylamino-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-carbonyl}-3-phenyl-propyl)-carbamate tert-butyl ester (50 mg, 0.090 mmol) was dissolved in DCM (4 mL), and TFA (2 mL) was added. The reaction mixture was stirred at room temperature for 2 hours, then the solvent was removed under reduced pressure, and the crude material was purified by preparative HPLC to obtain (S)-4-acetylamino-1-((R)-2-amino-4-phenyl-butyryl)-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)-amide (22.1 mg, 51%) as a white solid. ANALPH2_MEOH_QC_v1, Rt:4.21 min, m / z 478.3[M+H]+ ANALPH9_MEOH_QC_v1, Rt:6.18 min, m / z 478.4[M+H]+

[0429] The following examples were prepared using the same method.

[0430] [Table 16-1] [Table 16-2] [Table 16-3] [Table 16-4] [Table 16-5]

[0431] General Scheme 4 [ka]

[0432] (Method 1): Synthesis of (2S,4R)-1-((R)-2-benzylamino-5-oxo-5-pyrrolidine-1-ylpentanoyl)-4-pyridine-4-ylmethylpyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide (M05261) [ka] Benzoaldehyde (2.0 mg, 0.020 mmol, 1.05 equivalents), sodium borohydride cyanohydride (11 mg, 0.180 mmol, 10 equivalents), and acetic acid (0.1 mL) were added to a solution of (2S,4R)-1-((R)-2-amino-5-oxo-5-pyrrolidine-1-ylpentanoyl)-4-pyridine-4-ylmethylpyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide (10 mg, 0.019 mmol, 1.0 equivalent) in methanol (1 mL). The resulting solution was stirred at room temperature for 2 hours and then concentrated under reduced pressure. Next, the obtained product was purified by reverse-phase preparative LC-MS to obtain (2S,4R)-1-((R)-2-benzylamino-5-oxo-5-pyrrolidine-1-ylpentanoyl)-4-pyridine-4-ylmethylpyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide (6 mg, 53%) as a white solid. ANALPH2_MEOH_QC_v1, Rt:3.91 min, m / z 623.3[M+H]+ ANALPH9_MEOH_QC_v1, Rt:6.95 min, m / z 623.3[M+H]+

[0433] (Method 2) Synthesis of (2S,4R)-1-((R)-5-oxo-2-phenylacetylamino-5-pyrrolidine-1-ylpentanoyl)-4-pyridine-4-ylmethylpyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide (M05267) [ka] N,N-diisopropylethylamine (15.0 mg, 0.113 mmol, 1.2 equivalents) and phenylacetyl chloride (17 mg, 0.113 mmol, 1.2 equivalents) were added to a solution of (2S,4R)-1-((R)-2-amino-5-oxo-5-pyrrolidine-1-ylpentanoyl)-4-pyridine-4-ylmethylpyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide (10 mg, 0.019 mmol, 1.0 equivalent) in dichloromethane (1 mL). The resulting solution was stirred at room temperature for 1 hour and then concentrated under reduced pressure. The obtained product was purified by reverse-phase preparative LC-MS to obtain (2S,4R)-1-((R)-5-oxo-2-phenylacetylamino-5-pyrrolidine-1-ylpentanoyl)-4-pyridine-4-ylmethylpyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide (12 mg, 95%) as a yellow oily substance. ANALPH2_MEOH_QC_v1, Rt:5.19 min, m / z 651.3[M+H]+ ANALPH9_MEOH_QC_v1, Rt:6.90 min, m / z 651.3[M+H]+ 1H NMR (400 MHz, DMSO-d6) δ 8.79 (d, J = 6.0 Hz, 0.3H), 8.55 (dd, J = 12.4, 7.1 Hz, 1H), 8.46 (td, J = 3.9, 1.5 Hz, 2H), 8.16 (t, J = 6.1 Hz, 0.7H), 7.84 - 7.69 (m, 2H), 7.48 - 7.34 (m, 1H), 7.32 - 7.21 (m, 3H), 7.21 - 7.12 (m, 4H), 5.01 - 4.90 (m, 0.5H), 4.48 - 4.34 (m, 2H), 4.34 - 4.23 (m, 5H), 4.12 (q, J = 7.7 Hz, 0.5H), 3.80 (dd, J = 10.1, 7.5 Hz, 1H), 3.54 - 3.37 (m, 2H), 3.30 - 3.08 (m, 5H), 3.00 (t, J = 6.6 Hz, 1H), 2.75 - 2.59 (m, 2H), 2.47 - 2.20 (m, 2H), 1.98 - 1.64 (m, 7H).

[0434] The following examples were prepared using the same method.

[0435] [Table 17-1] [Table 17-2]

[0436] General Scheme 5: Final Step Suzuki [ka] Synthesis of (2S,4R)-1-((R)-2-amino-5-oxo-5-pyrrolidine-1-ylpentanoyl)-4-[3-(2-methyl-2H-pyrazole-3-yl)-benzyl]-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide (M05350) [ka] (2S,4R)-1-((R)-2-amino-5-oxo-5-pyrrolidine-1-ylpentanoyl)-4-(4-bromo-benzyl)-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide (37 mg, 0.061 mmol), 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole (15 mg, 0.072 mmol), Pd(dppf)2Cl2.DCM (2.5 mg, 0.0031 mmol), and K2CO3 (16 mg, 0.12 mmol) were dissolved in 1,4-dioxane (1.8 mL) and H2O (0.2 mL). The reaction mixture was degassed with N2 for 5 minutes. The stirred mixture was then heated in a microwave at 90°C for 45 minutes. The mixture was filtered through Celite, and the residue was washed with DCM. The filtrate was washed twice with H2O and then evaporated under vacuum. The crude product was purified by reverse-phase HPLC, and the product-containing fraction was passed through a catch-release cartridge (Biotage SCX-2; 5g) eluted with NH3-MeOH to obtain the title compound (14 mg, 38%) as a white solid. ANALPH2_MEOH_QC_v1, Rt:5.49 min, m / z 612.5[M+H]+ ANALPH9_MEOH_QC_v1, Rt:6.98 min, m / z 612.5[M+H]+

[0437] Depending on the circumstances, the finishing procedure may also include further washing with saturated brine (aq) and catch-release purification by elution with 0.5 M NH3-MeOH (Biotage SCX-2).

[0438] The following examples were synthesized using the same method.

[0439] [Table 18-1] [Table 18-2]

[0440] General Scheme 6: Late-stage carboxamide synthesis [ka]

[0441] Synthesis of (2S,4R)-1-[(R)-2-amino-5-((S)-2-methylpiperazine-1-yl)-5-oxopentanoyl]-4-(4-methoxybenzyl)-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide (M05417) [ka]

[0442] Step 1: (R)-4-tert-butoxycarbonylamino-5-{(2S,4R)-4-(4-methoxybenzyl)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-yl}-5-oxopentanoate benzyl ester (GS6-int1) (918 mg, 1.32 mmol) was dissolved in ethanol (10 mL) and hydrogenated by circulating it through an H-cube at 60°C for 5 hours using a 10% Pd / C CatCart. The reaction mixture was dried under vacuum to obtain (R)-4-tert-butoxycarbonylamino-5-{(2S,4R)-4-(4-methoxybenzyl)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-yl}-5-oxopentanoic acid (775 mg, 96%) as a colorless oil. ANALPH2_MEOH_4min, Rt:3.03min, m / z 609.8[M+H]+

[0443] Step 2: Amide coupling of (R)-4-tert-butoxycarbonylamino-5-{(2S,4R)-4-(4-methoxybenzyl)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-yl}-5-oxopentanoic acid with (3S)-3-methylpiperazine-1-carboxylic acid tert-butyl ester, isolated by HATU, DIPEA in DCM and General Method 1. The procedure was carried out using method c for 1 hour to obtain (S)-4-((R)-4-tert-butoxycarbonylamino-5-{(2S,4R)-4-(4-methoxybenzyl)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-yl}-5-oxopentanoyl)-3-methyl-piperazine-1-carboxylic acid tert-butyl ester (117 mg, 94%) as a colorless oil. ANALPH2_MEOH_4min, Rt:3.40min, m / z 791.8[M+H]+

[0444] Step 3: Boc deprotection of (S)-4-((R)-4-tert-butoxycarbonylamino-5-{(2S,4R)-4-(4-methoxybenzyl)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-yl}-5-oxopentanoyl)-3-methyl-piperazine-1-carboxylic acid tert-butyl ester was carried out for 1.5 hours using TFA in DCM and the general method 2A. The reaction mixture was concentrated under vacuum, and the residue was purified using an SCX-2 cartridge (5 g) and eluted with 4 M ammonia in methanol. The product-containing fraction was dried under vacuum and purified using preparative HPLC to obtain (2S,4R)-1-[(R)-2-amino-5-((S)-2-methyl-piperazin-1-yl)-5-oxo-pentanoyl]-4-(4-methoxy-benzyl)-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide (15 mg, 16%) as a white solid. ANALPH2_MEOH_QC_v1, RT: 4.33 min, m / z 591.4[M+H]+ ANALPH9_MEOH_QC_v1, RT: 6.63 min, m / z 591.4[M+H]+

[0445] The following examples were synthesized using the same method.

[0446] [Table 19-1] [Table 19-2]

[0447] General Scheme 7 [ka] Synthesis of (2S,4R)-1-((R)-2-amino-4-phenyl-butyryl)-4-cyclohexyl-pyrrolidine-2-carboxylic acid 4-aminomethyl-benzylamide (M05076) [ka]

[0448] {(R)-1-[(2S,4R)-2-(4-cyano-benzylcarbamoyl)-4-cyclohexyl-pyrrolidine-1-carbonyl]-3-phenyl-propyl}-carbamate tert-butyl ester (GS7-int1, 140 mg, 0.24 mmol) was dissolved in AcOH (15 mL) and 10% Pd / C (50 mg) was added. The mixture was stirred at 55°C for 4 hours under an H2 atmosphere. The reaction mixture was filtered through Celite and then purified by SCX-2 elution with 2 M NH3 / MeOH. The solvent was removed and the residue was dissolved in TFA / DCM and stirred at room temperature for 1 hour. After removing the solvent, the residue was purified using an SCX-2 cartridge, followed by preparative HPLC, to obtain (2S,4R)-1-((R)-2-amino-4-phenyl-butyryl)-4-cyclohexyl-pyrrolidine-2-carboxylic acid 4-aminomethyl-benzylamide (60 mg, 53%) as a white solid. ANALPH2_MEOH_QC_v1, Rt: 5.25 minutes, m / z 477.3 [M+H]+ ANALPH9_MEOH_QC_v1, Rt: 8.21 minutes, m / z 477.3 [M+H]+ 1H NMR (400 MHz, CDCl3) δ 7.36 - 7.26 (m, 2H), 7.30 - 7.11 (m, 7H), 6.91 (t, J = 5.8 Hz, 1H), 4.49 (dd, J = 14.9, 6.0 Hz, 1H), 4.42 - 4.29 (m, 2H), 3.86 (s, 2H), 3.43 - 3.27 (m, 2H), 2.89 (t, J = 10.7 Hz, 1H), 2.80 (m, 2H), 2.26 (dt, J = 12.7, 7.6 Hz, 1H), 1.93 (ddd, J = 12.8, 11.4, 8.6 Hz, 1H), 1.87 - 1.64 (m, 7H), 1.45 (d, J = 12.3 Hz, 1H), 1.30 - 1.11 (m, 4H), 0.99 - 0.81 (m, 2H).

[0449] The following examples were synthesized by the same method.

[0450] [Table 20-1] [Table 20-2] [Table 20-3]

[0451] [Table 21]

[0452] General Scheme 8 (Fmoc deprotection, followed by boc deprotection) [Chemical formula]

[0453] Synthesis of (2S,4R)-1-((R)-2-amino-4-phenyl-butyryl)-4-cyano-pyrrolidine-2-carboxylic acid 4-aminomethyl-benzylamide (M05091) [ka]

[0454] Step 1: [(R)-1-((2S,4R)-4-cyano-2-{4-[(9H-fluoren-9-ylmethoxycarbonylamino)-methyl]-benzylcarbamoyl}-pyrrolidine-1-carbonyl)-3-phenyl-propyl]-carbamate tert-butyl ester (GS8-int1, 207 mg, 0.28 mmol) was dissolved in DCM (2 mL), and piperidine (0.2 mL) was added. The reaction mixture was stirred at room temperature for 2 hours, then the solvent was removed, and the residue was purified by preparative LC-MS to obtain {(R)-1-[(2S,4R)-2-(4-aminomethyl-benzylcarbamoyl)-4-cyano-pyrrolidine-1-carbonyl]-3-phenyl-propyl}-carbamate tert-butyl ester (133 mg, 93%) as a clear oil. AnalpH2_MeOH_4MIN:Rt:2.27min, m / z 520.4[M+H] +

[0455] Step 2: {(R)-1-[(2S,4R)-2-(4-aminomethyl-benzylcarbamoyl)-4-cyano-pyrrolidine-1-carbonyl]-3-phenyl-propyl}carbamate tert-butyl ester (133 mg, 0.226 mmol) was subjected to BOC deprotection using general method 2A and purified by preparative LC-MS to obtain (2S,4R)-1-((R)-2-amino-4-phenyl-butyryl)-4-cyano-pyrrolidine-2-carboxylic acid 4-aminomethyl-benzylamide (81.4 mg, 62%) as a white solid. ANALPH9_MEOH_QC_v1, Rt:5.95 min, m / z 420.4[M+H]+ ANALPH2_MEOH_QC_v1, Rt:2.76 min, m / z 420.3[M+H]+ 1 H NMR (400 MHz, DMSO-d6) δ 8.66 (t, J = 6.0 Hz, 1H), 7.42 - 7.36 (m, 2H), 7.36 - 7.27 (m, 5H), 7.28 - 7.17 (m, 3H), 4.45 (dd, J = 8.6, 3.7 Hz, 1H), 4.30 (d, J = 5.9 Hz, 2H), 4.21 (t, J = 6.0 Hz, 1H), 4.02 (s, 2H), 3.99 - 3.88 (m, 1H), 3.76 (dd, J = 10.3, 7.2 Hz, 1H), 3.63 - 3.42 (m, 1H), 2.77 - 2.59 (m, 2H), 2.49 - 2.41 (m, 1H), 2.23 (ddd, J = 12.7, 6.7, 3.8 Hz, 1H), 2.02 (m, 2H).

[0456] The following examples were synthesized using the same method.

[0457] [Table 22-1] [Table 22-2]

[0458] General Scheme 9: [ka] Synthesis of (2S,4S)-1-((R)-2-amino-4-phenyl-butyryl)-4-(4-benzyl-piperazin-1-yl)-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide M05200

[0459] Step 1: ((R)-1-{(S)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-4-oxo-pyrrolidine-1-carbonyl}-3-phenyl-propyl)-carbamate tert-butyl ester (70 mg, 0.13 mmol), 1-benzylpiperazine (25 μL, 0.13 mmol), and AcOH (7.0 μL, 0.13 mmol) were stirred in DCE (1 mL) for 1 hour. NaBH(OAc)3 (83 mg, 0.39 mmol) was added, and the reaction was stirred overnight at room temperature. The reaction was monitored by LC-MS. Further aliquots of amine, AcOH, and NaBH(OAc)3 (0.13 mmol each) were added, followed by stirring for 1 hour. Further aliquots of amine and AcOH (0.13 mmol each) were added, and the reaction mixture was stirred overnight. The reaction was washed with saturated aqueous solution NaHCO3, dried over MgSO4, and the solvent was removed. The residue was purified using an SCX-2 cartridge (2g) packed with MeOH and eluted with 0.5M NH3 / MeOH. The product-containing fraction was concentrated to obtain ((R)-1-{(2S,4S)-4-(4-benzylpiperazin-1-yl)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-carbonyl}-3-phenyl-propyl)-carbamate tert-butyl ester (61 mg, 68%) as a brown oil. AnalpH2_MeOH_4MIN:Rt:3.44 min, m / z 695.5[M+H]+

[0460] Step 2: ((R)-1-{(2S,4S)-4-(4-benzylpiperazine-1-yl)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-carbonyl}-3-phenyl-propyl)-carbamate tert-butyl ester (61 mg, 0.090 mmol) was dissolved in DCM (3 mL) and TFA (3 mL) was added. The mixture was stirred at room temperature for 1 hour, then the solvent was removed and the residue was purified by preparative LC-MS to obtain (2S,4S)-1-((R)-2-amino-4-phenyl-butyryl)-4-(4-benzylpiperazine-1-yl)-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)-amide (24.7 mg, 47%) as a white solid. ANALPH9_MEOH_QC_v1, Rt:7.65 min, m / z 595.5[M+H]+ ANALPH2_MEOH_QC_v1, Rt: 3.7 min, m / z 595.5[M+H]+

[0461] The following examples were synthesized using the same method.

[0462] [Table 23]

[0463] General Scheme 10: Synthesis of (S)-1-((R)-2-methylamino-4-phenyl-butyryl)-pyrrolidine-2-carboxylic acid 4-aminomethyl-benzylamide [ka]

[0464] Step 1: Amide coupling of (S)-pyrrolidine-2-carboxylate methyl hydrochloride (3.00 g, 18.1 mmol) and Boc-D-homophenylalanine (5.00 g, 17.9 mmol) was carried out according to General Method 1 using HATU (7.0 g, 18.4 mmol) and Et3N (6 mL, 26.7 mmol) in DMF (100 mL). Purification by column chromatography (Biotage, 0-50% siRNA / hexane) yielded (S)-1-((R)-2-tert-butoxycarbonylamino-4-phenyl-butyryl)-pyrrolidine-2-carboxylate methyl hydrochloride (6.1 g, 87%) as a clear oil. AnalpH2_MeOH_4MIN:Rt:3.21 min, m / z 291.3[M+H-boc] +

[0465] Step 2: (S)-1-((R)-2-tert-butoxycarbonylamino-4-phenyl-butyryl)-pyrrolidine-2-carboxylate methyl ester (500 mg, 1.3 mmol) was dissolved in DMF (20 mL), and NaH (60% suspension in mineral oil, 76 mg, 1.9 mmol) was added. The reaction mixture was stirred for 10 minutes, then methyl iodide (0.12 mL, 1.9 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was cooled to 0°C, quenched with H2O, and separated between ethyl acetate and H2O. The organic matter was washed with brine, and the solvent was removed. The crude product was purified by column chromatography (Biotage, 0-50% ethyl acetate / hexane) to obtain (S)-1-[(R)-2-(tert-butoxycarbonylmethylamino)-4-phenyl-butyryl]-pyrrolidine-2-carboxylate methyl ester (690 mg, 70%) as a colorless oil. AnalpH2_MeOH_4MIN:Rt:3.30 min, m / z 405[M+H] +

[0466] Step 3: (S)-1-[(R)-2-(tert-butoxycarbonylmethylamino)-4-phenyl-butyryl]-pyrrolidine-2-carboxylic acid methyl ester (690 mg, 1.7 mmol) was dissolved in aqueous KOH (5 M, 6 mL, 12 mmol), MeOH (20 mL), and THF (20 mL), and heated at 50°C for 1 hour. The reaction mixture was cooled in an ice bath, water was added, and the pH was adjusted to pH 2 by adding 2 M aqueous HCl. The mixture was extracted with ELISA, the organic matter was washed with brine, dried (MgSO4), and the solvent was removed to obtain (S)-1-[(R)-2-(tert-butoxycarbonylmethylamino)-4-phenyl-butyryl]-pyrrolidine-2-carboxylic acid (450 mg, 68%) as a colorless oil, which was used crudely in the next step. AnalpH2_MeOH_4MIN:Rt:3.27 min, m / z 391.4[M+H] +

[0467] Step 4: The amide coupling of (S)-1-[(R)-2-(tert-butoxycarbonylmethylamino)-4-phenyl-butyryl]-pyrrolidine-2-carboxylic acid (280 mg, 0.72 mmol) and 1-(N-boc-aminomethyl)-4-(aminomethyl)benzene (170 mg, 0.72 mmol) was carried out according to General Method 1 using HATU (330 mg, 0.86 mmol) and DIPEA (0.30 mL, 1.7 mmol) in DCM (50 mL). Purification by column chromatography (biotage, 0-80% ethyl acetate / petroleum) yielded ((R)-1-{(S)-2-[4-(tert-butoxycarbonylaminomethyl)-benzylcarbamoyl]-pyrrolidine-1-carbonyl}-3-phenyl-propyl)-methylcarbamate tert-butyl ester (230 mg, 51%) as a colorless oil. AnalpH2_MeOH_4MIN:Rt:3.51 min, m / z 609[M+H] +

[0468] Step 5: ((R)-1-{(S)-2-[4-(tert-butoxycarbonylamino-methyl)-benzylcarbamoyl]-pyrrolidine-1-carbonyl}-3-phenyl-propyl)-methylcarbamate tert-butyl ester (230 mg, 0.34 mmol) was deprotected using 5:1 DCM:TFA (12 mL) according to general method 2A, stirred at room temperature for 1 hour, and then purified to obtain (S)-1-((R)-2-methylamino-4-phenyl-butyryl)-pyrrolidine-2-carboxylic acid 4-aminomethyl-benzylamide (59 mg, 38%) as a white foam. ANALPH9_MEOH_QC_v1, Rt:6.61 min, m / z 409.3[M+H]+ ANALPH2_MEOH_QC_v1, Rt:2.81 min, m / z 409.3[M+H]+

[0469] Scheme 12 (Methylation of the final compound) [ka] Synthesis of (2S,4R)-4-(4-methoxybenzyl)-1-[(2R,4S)-1-methyl-4-(1-methyl-1H-pyrazole-4-yl)-pyrrolidine-2-carbonyl]-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide (M05474) [ka] Formaldehyde (37% wt in water, 22 mg, 0.27 mmol, 3.0 equivalents) and sodium triacetoxyborohydride (57 mg, 0.27 mmol, 3.0 equivalents), followed by acetic acid (5.0 mg, 0.081 mmol, 0.9 equivalents), were added to a solution of ((2S,4R)-4-(4-methoxybenzyl)-1-[(2R,4S)-4-(1-methyl-1H-pyrazole-4-yl)-pyrrolidine-2-carbonyl]-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide (50 mg, 0.090 mmol, 1.0 equivalent) in DCM (10 mL). The resulting solution was heated in a room. The mixture was stirred overnight at warm temperature. The reaction mixture was diluted with saturated aqueous NaHCO3 (10 mL) and then extracted with dichloromethane (3 × 10 mL). The combined organic phase was washed with brine, passed through a hydrophobic membrane, and concentrated under vacuum. The crude product was purified with SCX-2 (2.5 g) to obtain (2S,4R)-4-(4-methoxybenzyl)-1-[(2R,4S)-1-methyl-4-(1-methyl-1H-pyrazole-4-yl)-pyrrolidine-2-carbonyl]-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide (0.045 g, 0.078 mmol, 87%) as a white solid. ANALPH2_MEOH_QC_v1, Rt:5.28 min, m / z 571.4[M+H]+ ANALPH9_MEOH_QC_v1, Rt:7.32 min, m / z 571.4[M+H]+ 1H-NMR (400 MHz, DMSO-d6) δ 8.64 (t, J = 6.2 Hz, 0.3H), 8.37 (t, J = 6.2 Hz, 0.7H), 7.81 (s, 1H), 7.72 (t, J = 9.2 Hz, 1H), 7.47 (s, 1H), 7.39 (dd, J = 8.0, 4.8 Hz, 1H), 7.33 (s, 0.3H), 7.22 (d, J = 8.2 Hz, 0.7H), 7.13-7.02 (m, 2H), 6.92-6.77 (2H), 4.60-4.27 (m, 3H), 4.27-4.18 (3H), 3.77-3.64 (m, 7H), 3.52-3.40 (0.3H), 3.22 (dd, J = 18.3, 7.8 Hz, 1H), 3.12-3.00 (m, 0.7H), 2.91-2.67 (m, 1H), 2.65-2.49 (m, 4H), 2.39-2.26 (m, 1H), 2.27-2.09 (m, 3H), 2.05-1.85 (1H), 1.84-1.70 (m, 2H), 1.69-1.49 (1H)

[0470] The following examples were prepared using the same method.

[0471] [Table 24]

[0472] General Scheme 13: Synthesis of (2S,4R)-1-{(R)-2-amino-3-[pyrrolidine-1-carbonyl)-amino]-propionyl}-4-(4-methoxy-benzyl)-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide (M05414) [ka]

[0473] Step 1: Fmoc deprotection of (2S,4R)-4-(4-methoxybenzyl)-1-((R)-2-methylbutyryl)-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide (204 mg, 0.26 mmol) (GS13-int1) using piperidine (2 mL) in DMF (8 mL) was carried out according to general method 5. The product was purified using column chromatography (Biotage, 25 g SNAP, 0-15% DCM / methanol) to obtain (2S,4R)-1-((R)-3-amino-2-methylpropionyl)-4-(4-methoxybenzyl)-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide (73 mg, 56%) as a white solid. ANALPH2_MEOH_4min, Rt:2.42min, m / z 566.5[M+H]+

[0474] Step 2: (2S,4R)-1-((R)-3-amino-2-methyl-propionyl)-4-(4-methoxy-benzyl)-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide (73 mg, 0.13 mmol) was dissolved in DCM (2 mL), and CDI (31 mg, 0.19 mmol) and DMAP (17 mg, 0.14 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour, after which pyrrolidine (16 μL, 0.19 mmol) was added. The reaction mixture was stirred for a further 24 hours, and then quenched with water. The layers were separated, and the aqueous layer was extracted with DCM (×3). The combined organic layers were washed with brine, dried (MgSO4), and the solvent was removed under vacuum to obtain ((R)-2-{(2S,4R)-4-(4-methoxybenzyl)-2-[1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-yl}-2-oxo-1-{[pyrrolidine-1-carbonyl)-amino]-methyl}-ethyl)-carbamate tert-butyl ester (57 mg, 66%) as a colorless oil. ANALPH2_MEOH_4min, Rt:3.16min, m / z 663.4[M+H]+

[0475] Step 3: Boc deprotection of ((R)-2-{(2S,4R)-4-(4-methoxybenzyl)-2-[1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-yl}-2-oxo-1-{[pyrrolidine-1-carbonyl)-amino]-methyl}-ethyl)-carbamate tert-butyl ester (57 mg, 0.086 mmol) was carried out for 1 hour using TFA in DCM according to general method 2A. The solvent was removed under vacuum, and the residue was purified using SCX-2 and eluted with 4 M NH3 in methanol. The product-containing fraction was concentrated under vacuum and purified using preparative HPLC to obtain (2S,4R)-1-{(R)-2-amino-3-[pyrrolidine-1-carbonyl)-amino]-propionyl}-4-(4-methoxy-benzyl)-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide (13 mg, 27%) as a white solid. ANALPH2_MEOH_QC_v1, RT: 5.49 min, m / z 563.4[M+H]+ ANALPH9_MEOH_QC_v1, RT: 7.05 min, m / z 563.4[M+H]+ 1 H NMR (400 MHz, DMSO-d6) δ 8.41 (s, 1H), 7.79 (d, J = 7.2 Hz, 1H), 7.71 (d, J = 8.6 Hz, 1H), 7.38 (dd, J = 8.7, 1.4 Hz, 1H), 7.09 - 7.01 (m, 2H), 6.83 - 6.77 (m, 2H), 6.32 (s, 1H), 4.44 - 4.27 (m, 3H), 4.24 (s, 3H), 3.84 (s, 1H), 3.75 (s, 1H), 3.67 (s, 3H), 3.29 - 3.09 (m, 6H), 2.62 - 2.46 (m, 2H), 1.79-1.69 (m, J = 6.2 Hz, 8H).

[0476] General Scheme 14 Synthesis of cis-benzyl RgB compounds (M05210 and M05211) [ka]

[0477] Step 1: (3-Chlorobenzyl)(triphenyl)phosphonium bromide (400 mg, 0.86 mmol) was dissolved in DCM (10 mL), and KOtBu (1 M in THF, 0.86 mL, 0.86 mmol) was added. After 45 minutes, N-boc-4-oxo-L-proline methyl ester (180 mg, 0.74 mmol) was added, and the reaction mixture was stirred at room temperature for 1 hour. Water (20 mL) was added, and the mixture was extracted with DCM (2 × 20 mL). The combined organic layers were dried (MgSO4), the solvent was removed, and the residue was purified by column chromatography (Biotage, 25g SNAP, 0-50% SiO2 / ihexane) to obtain (S)-4-[1-(3-chlorophenyl)-meth-(E)-ylidene]-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (148 mg, 57%) as a colorless oil. ANALPH2_MEOH_4min, Rt:3.44min, m / z 352[M+H]+

[0478] Step 2: (S)-4-[1-(3-chlorophenyl)-meth-(E)-ylidene]-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (150 mg, 0.42 mmol) was dissolved in THF (1 mL), and MeOH (1 mL) and NaOH (1 M aq, 1 mL) were added. The reaction mixture was stirred at room temperature for 1 hour. The solvent was removed, and saturated aqueous solution NH4Cl (5 mL) and 1 M aqueous solution HCl were added to acidify the mixture to pH 5. The aqueous layer was extracted with ELISA, and the combined organic extracts were dried over MgSO4. The solvent was removed to obtain (S)-4-[1-(3-chlorophenyl)-meth-(E)-ylidene]-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester (120 mg) as a pale yellow oil, which was used directly in the subsequent reaction without further purification. ANALPH2_MEOH_4min, Rt:3.43min, m / z 338[M+H]+

[0479] Step 3: (S)-4-[1-(3-chlorophenyl)-meth-(E)-ylidene]-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester (120 mg, 0.36 mmol) was dissolved in DCM (5 mL) and HBTU (159 mg, 0.42 mmol), and C-(1-methyl-1H-benzotriazole-5-yl)-methylamine (68 mg, 0.42 mmol) and DIPEA (217 μL, 1.26 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour, then water (20 mL) was added, and the aqueous layer was extracted with DCM. The combined organic extract was dried (MgSO4) to remove the solvent. The residue was purified by column chromatography (Biotage, 10g SNAP, 0-100% ethyl acetate / hexane) to obtain (S)-4-[1-(3-chlorophenyl)-meth-(E)-ylidene]-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-carboxylate tert-butyl ester as a colorless oil (174 mg, 86% in 2 steps). ANALPH2_MEOH_4min, Rt:3.19min, m / z 482[M+H]+

[0480] Step 4: (S)-4-[1-(3-chlorophenyl)-meth-(E)-ylidene]-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-carboxylate tert-butyl ester (174 mg, 0.36 mmol) was dissolved in EtOH (5 mL) under N2, and Pd / C (20 mg) was added. An H2 atmosphere was introduced, and the reaction mixture was stirred at room temperature for 7 hours. An additional Pd / C (20 mg) was added, and the reaction mixture was stirred overnight, then heated to 45°C for 2 hours. The mixture was cooled to room temperature, then filtered through Celite, the residue was washed with EtOH, and the combined filtrate and washing solution were concentrated to obtain a combination of (2S,4S)-4-(3-chlorobenzyl)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-carboxylate tert-butyl ester and (2S,4S)-4-benzyl-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-carboxylate tert-butyl ester (140 mg, 80%) as a colorless oil. ANALPH2_MEOH_4min, Rt: 3.23 min, m / z 484[M+H]+(Cl); Rt: 3.09 min, m / z 450[M+H]+(H)

[0481] Step 5: Dissolve the combination of (2S,4S)-4-(3-chlorobenzyl)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-carboxylate tert-butyl ester and (2S,4S)-4-benzyl-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-carboxylate tert-butyl ester (140 mg, 0.29 mmol) in ELISA (3 mL), add HCl (1 M in Et2O, 3 mL), and stir at room temperature for 1 hour. After removing the solvent, the combination of (2S,4S)-4-(3-chlorobenzyl)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]pyrrolidine and (2S,4S)-4-benzyl-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]pyrrolidine was obtained as a white solid, which was used crudely in the next step.

[0482] Step 6: A crude mixture of (2S,4S)-4-(3-chlorobenzyl)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]pyrrolidine and (2S,4S)-4-benzyl-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]pyrrolidine was dissolved in DCM (5 mL), and HBTU (110 mg, 0.29 mmol), (R)-2-tert-butoxycarbonylamino-5-oxo-5-pyrrolidine-1-ylpentanoic acid (87 mg, 0.29 mmol) and DIPEA (300 μL, 1.74 mmol) were added. The mixture was stirred overnight at room temperature, then water (20 mL) was added, and the aqueous layer was extracted with DCM. The combined organic layer was dried (MgSO4) to remove the solvent. The residue was purified by column chromatography (Biotage, 10 g SNAP, 0-100% siRNA / hexane followed by 0-20% MeOH / siRNA) to obtain a mixture of ((R)-1-{(2S,4S)-4-(3-chlorobenzyl)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-carbonyl}-4-oxo-4-pyrrolidine-1-yl-butyl)-carbamate tert-butyl ester and ((R)-1-{(2S,4S)-4-benzyl-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-carbonyl}-4-oxo-4-pyrrolidine-1-yl-butyl)-carbamate tert-butyl ester as a colorless oil (119 mg, 62% in 2 steps). ANALPH2_MEOH_4min, Rt: 3.29 min, m / z 666[M+H]+(Cl); Rt: 3.17 min, m / z 632[M+H]+(H)

[0483] Step 7: Dissolve ((R)-1-{(2S,4S)-4-(3-chlorobenzyl)-2-[(1-methyl-1H-benzotriazole-5-ylmethyl)-carbamoyl]-pyrrolidine-1-carbonyl}-4-oxo-4-pyrrolidine-1-yl-butyl)-carbamate tert-butyl ester (119 mg, 0.18 mmol) in ELISA (5 mL), and add HCl (1 M in Et2O, 3 mL). Stir the reaction mixture at room temperature for 2 hours. The solvent was removed, 1 M NH3 in MeOH (10 mL) was added, the solvent was removed, and the residue was purified by preparative HPLC to obtain (2S,4S)-1-((R)-2-amino-5-oxo-5-pyrrolidine-1-ylpentanoyl)-4-(3-chloro-benzyl)-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide and (2S,4S)-1-((R)-2-amino-5-oxo-5-pyrrolidine-1-ylpentanoyl)-4-benzyl-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide as white solids.

[0484] M05210(2S,4S)-1-((R)-2-amino-5-oxo-5-pyrrolidine-1-ylpentanoyl)-4-(3-chlorobenzyl)-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide (9mg, 9%) ANALPH9_MEOH_QC_v1, Rt:7.34 min, m / z 566.3[M+H]+ ANALPH2_MEOH_QC_v1, Rt:5.57 min, m / z 566.2[M+H]+

[0485] M05211(2S,4S)-1-((R)-2-amino-5-oxo-5-pyrrolidine-1-ylpentanoyl)-4-benzyl-pyrrolidine-2-carboxylic acid (1-methyl-1H-benzotriazole-5-ylmethyl)amide (33 mg, 33%) ANALPH9_MEOH_QC_v1, Rt:6.97 min, m / z 532.3[M+H]+ ANALPH2_MEOH_QC_v1, Rt:5.2 min, m / z 532.2[M+H]+

[0486] General scheme 15. Late N-alkylation: [ka]

[0487] The following compounds were synthesized using general methods.

[0488] [Table 25]

[0489] General Scheme 16. Synthesis of RgD Glutamate Derivatives [ka]

[0490] The following compounds were synthesized using general methods.

[0491] [Table 26]

[0492] General Scheme 17: Synthesis of Late-Functionalized RgD Carboxamide [ka]

[0493] The following compounds were synthesized using general methods.

[0494] [Table 27-1] [Table 27-2]

[0495] General examination methods The activity of the compounds of the present invention was determined in vitro using the following assay protocols for screening the activity of FXIIa and other proteases. Each of these assays was performed in a purification system using a microplate well chromogenic assay. The chromogenic peptide substrate, which mimics a native protein substrate, is bound to the chromogenic group via an amide bond. Para-nitroaniline (pNA) is released from the peptide after catalysis by a protease, increasing its absorbance and allowing monitoring at 405 nm.

[0496] All compounds were dissolved in 100% (v / v) DMSO to a stock concentration of 10 mM. The maximum concentration of each compound used in each assay was 500 μM. The final concentration of DMSO was 5% (v / v) in 50 mM Tris 137 mM NaCl pH 7.4. When no test compound was added, the final concentration of 5% DMSO was used.

[0497] Determination of factor XIIa inhibition Factor XIIa activity was measured using the chromogenic substrate S-2302 (Chromogenix). Various concentrations of the compound were incubated with 10 nM FXIIa, incubated in 50 mM Tris, 137 mM NaCl, pH 7.4 at 37°C for 10 minutes, and then a final concentration of 450 μM S-2302 chromogenic substrate was added. Dynamic readings at 405 nm were monitored every 12 seconds over a total duration of 3 hours at 37°C. The initial velocity gradient was determined, and IC was calculated. 50 It was used to calculate the value. IC 50 The value of, expression: K i =IC 50 / (1+[substrate] / Km) The Ki value was converted based on this.

[0498] K obtained by the above method i The data is shown in Table 1 below. The activity of the compound of the present invention is K i Classification is based on values, and the classification is " * " **" and " *** It was ". Classification " * " is K exceeding 2 μM i This refers to compounds that possess a value. Classification: ** " is 0.2 μM to 2 μM K i This refers to compounds that possess a value. Classification: *** " is K less than 0.2 μM i This refers to compounds that possess a value.

[0499] Determining Selectivity To determine the selectivity of the test compounds, these compounds were assayed for inhibitory activity against FXa and other serine proteases, including thrombin. Essentially, compounds at increasing concentrations were incubated with FXa (5 nM) and thrombin (5 nM) enzymes at 37°C for 10 minutes, followed by incubation with appropriate chromogenic substrates S2765 (350 μM) and GPR (250 μM), respectively, in 50 mM Tris, 137 mM NaCl, pH 7.4. Chromogenic substrate S2765 was from Chromogenix, and GPR was from Bachem. Dynamic readings at 405 nm were monitored every 12 seconds over a total duration of 3 hours at 37°C. The initial velocity gradient was determined, and IC was calculated. 50 It was used to calculate the value. IC 50 The value of, expression: K i =IC 50 / (1+[substrate] / Km) The Ki value was converted based on this.

[0500] Here, [substrate] indicates the concentration of the substrate used in the assay, and Km is the determinant value for each enzyme by its own substrate. Compounds in this chemical series exhibit competitive inhibition.

[0501] The selectivity ratios for thrombin and FXa are also shown in Table 1 below. The selectivity ratio indicates the preferred inhibition of FXIIa compared to FXa and thrombin. For the compounds of the present invention, the selectivity ratios for FXIIa compared to thrombin are classified based on the selectivity ratio value, and the classifications are "+", "++", and "+++". Classification "+" refers to selectivity ratio values ​​less than 10. Classification "++" refers to selectivity ratio values ​​between 10 and 100. Classification "+++" refers to selectivity ratio values ​​greater than 100.

[0502] The selection ratio of the compounds of the present invention compared to FXa with respect to FXIIa is classified based on the selection ratio value, and the classifications are "o", "oo", and "ooo". Classification "o" refers to a selection ratio value of less than 10. Classification "oo" refers to a selection ratio value of 10 to 100. Classification "ooo" refers to a selection ratio value greater than 100.

[0503] Determination of anticoagulant efficacy in vivo reagent I purchased AlexaFluor488 conjugate fibrinogen from Invitrogen (Paisley, UK).

[0504] animal All experiments used C57BL / 6 male mice weighing 20-30g. All procedures were approved by the University of Sheffield Bioethics Committee and carried out in accordance with the UK Home Office Animals (Scientific Procedures) Act 1985.

[0505] In vivo microscopy for real-time evaluation of fibrin formation Microscopic observation of thrombus formation after ferric chloride (FeCl3)-induced injury in vivo was performed using a bright-field upright microscope (Nikon eclipse E600-FN, Nikon UK, Kingston upon Thames, UK) and a fluorescence microscope, with a water immersion objective lens (40 / 0.80W).

[0506] Mice were anesthetized by intravenous injection of 125 mg / kg ketamine hydrochloride (Ketaset; Willows Francis Veterinary, Crawley, UK), 12.5 mg / kg xylazine hydrochloride (Bayer Suffolk, UK), and 0.025 mg / kg atropine sulfate (phoenix Pharmaceuticals Ltd, UK). Cannulas were inserted into the trachea (to assist respiration) and carotid artery (for anesthesia maintenance and substance delivery) to expose the femoral vein. Five minutes after administration of 100 μl of AlexaFluor488 conjugate fibrinogen (2 mg / ml) and 100 μl of the compound (diluted in 10% DMSO and 90% saline in 100 μl) or vehicle (10% DMSO in 100 μl saline) via the carotid artery, a 3 mm × 2 mm filter paper saturated with 10% (v / v) FeCl3 was applied directly to the femoral vein for 3 minutes.

[0507] Real-time Alexa 488nm (green channel) images were acquired using Slidebook imaging software (Version 5.0; Intelligent Imaging Innovations, 3i, Denver, USA) to monitor in vivo thrombus formation at regular 1-hour intervals. After exposure to FeCl3 and throughout the experiment, the area was flushed with warm PBS.

[0508] Data analysis for determining fibrin clot formation in real time (Slidebook) Real-time images of thrombus formation were analyzed using Slidebook image analysis software by setting a background region outside the thrombus area and measuring the Alexa 680nm signal intensity on the background across the entire area of ​​injury. By setting individual background intensities for the green channel in this way, it is possible to select pixels that show only background signals for both probes at each time frame. Then, the signal intensity of pixels for FITC 488nm is determined (including signal intensity and area) in the resulting selected pixels or "masked" region (defined as the region used for data analysis). Slidebook software enables automated background calculation for each image file representing different time points, thus enabling background removal at each time point. The thrombus area is determined by quantifying the pixel intensity on the background (at each time point) in the FITC 488nm channel, and masked pixels are represented as the total pixel area. When establishing a background region, all time frames within the background are run as video to ensure that the region selected as background does not develop clot growth over the duration of the experiment. The background signal before ferric chloride injury is determined and subtracted from the reading after ferric chloride injury. This is important for analysis using Slidebook because the same area of ​​background is used for signal determination in each time frame. The generated data reflects the area intensity of each pixel, and when background removal is performed on the same image / time frame, this data provides an accurate assessment of the FITC area by intensity. The data is plotted as relative fluorescence units (RFU) over time.

[0509] The percentage inhibition of blood clot formation was calculated at 60 minutes compared to mice administered only the vehicle. The results are shown in Figure 1.

[0510] [Table 28-1] [Table 28-2] [Table 28-3]

[0511] Throughout this specification and the claims, the words “contains” and “includes,” and their variations, mean “contains, but not limited to,” and they are not intended (and do not exclude) other parts, additives, components, ingredients, or steps. Throughout this specification and the claims, the singular forms include the plural unless the context requires otherwise. In particular, where the indefinite article is used, this specification is understood to intend both the plural and the singular unless the context requires otherwise.

[0512] Any features, integers, properties, compounds, chemical parts, or groups described in connection with a particular aspect, embodiment, or example of the present invention will be understood to be applicable to any other aspect, embodiment, or example described herein, unless incompatible therewith. All features disclosed herein (including the appended claims, abstract, and drawings) and / or all steps of any method or process so herein may be combined in any combination, except for any combination in which at least some of such features and / or steps are mutually exclusive. The present invention is not limited to any of the details of any of the embodiments described herein. The present invention extends to any novel features or any novel combination of features disclosed herein (including the appended claims, abstract, and drawings) and any novel steps or any novel combination of any method or process so herein may be disclosed.

[0513] The reader's attention is directed to all papers and documents filed concurrently with or prior to this specification in connection with this application and published together with this specification, the contents of all such papers and documents by reference forming part of this specification.

Claims

1. Compounds of formula (Ia) and pharmaceutically acceptable salts thereof: 【Chemistry 1】 (In the formula, Y is, 【Chemistry 2】 (In the formula, R ｚ (This is selected from -OH, Cl, and OMe.) Selected from; X is either a bond, -C(O)NH-, or -C(O)-; -L-(CR ｃ R ｄ ) ｎ - is selected from bonding, -CH ２ -, -NH-, -NHCH ２ (CH ２ ) ２ -, -NH(CH ２ ) ３ -, -N(Me)-, -N(C(O)Me)CH ２ -, -NHC(O)-, -NHC(O)CH ２ -, -NHC(O)(CH ２ ) ２ -, or NHC(O)(CH ２ ) ３ -; Ar is a substituted or unsubstituted 9-10 membered bicyclic heteroaromatic ring, wherein the 9-10 membered bicyclic heteroaromatic ring is a heteroaromatic ring having at least one heteroatom selected from O, N, and S within the ring, or a bicyclic ring containing an aromatic ring that is not fully aromatic, and the at least one heteroatom is present in the aromatic or non-aromatic ring and is substituted, then the 9-10 membered bicyclic heteroaromatic ring is a halo, C １～６ Alkyl, -OR ｇ , -NR ｇ R ｈ , or -NR ｇ R ｈ C replaced by １～４ Substituting with one, two, or three substituents selected from alkyl groups; m is selected from 1 or 2; o is selected from 1 or 2; R １ -NR is either substituted or unsubstituted. ８ R ９ Selected from 6- or 10-membered aryls, 5- or 6-membered heteroaryls, and 3- to 7-membered heterocycloalkyls; If substituted, R １ is =O, CN, -OH, -OC １～６ Alkyl, Halo, C １～６ Alkyl and C １～６ Substituted with one, two, or three groups selected from haloalkyl groups; R ２ H, C １～６ Alkyl, C １～６ Haloalkyl, benzyl, and -C(O)R ２ａ Selected from; R ２ａ C １～６ Selected from alkyl, phenyl, and benzyl; R ３ａ is H, or C １～６ It is alkyl; R ４ is -CN, Halo, C １～４ Alkyl, C １～４ Haloalkyl, -OR １０ , -NR １０ R １１ , 6-10 member aryl, C ３～８ Selected from cycloalkyl, 3-6 member heterocycloalkyl, and 5-10 member heteroaryl, where the C ３～８ Cycloalkyls, 3-6 member heterocycloalkyls, 6-10 member aryls, or 5-10 member heteroaryls are unsubstituted or have 1, 2, or 3 R groups. １２ It has been replaced with; R ４ａ H is; R ５ is H, or C １～６ It is alkyl; R ８ and R ９ In each occurrence, H and C appear independently. １～６ Alkyl, C ３～６ Cycloalkyl, phenyl, -OR ｉ C replaced by １～４ C substituted with alkyl or phenyl １～４ Selected from alkyl groups, or R ８ and R ９ They, together with the atoms they are bonded to, are either unsubstituted or CN, halo, C １～６ Alkyl or -OR ｉ Forms a 3- to 8-membered heterocycloalkyl ring substituted with; R １２ In each occurrence, independently, Halo, C １～４ Alkyl, C １～４ Haloalkyl, -OR １３ , -CN, -C(O)R １０ , = O, SO ２ R １０ Selected from benzyl, phenyl, unsubstituted 5 or 6-membered heteroaryl, or methyl-substituted 5 or 6-membered heteroaryl; R １０ and R １１ In each occurrence, H and C appear independently. １～４ Selected from alkyl groups; R １３ H, C １～４ Alkyl, C １～４ Selected from haloalkyl, phenyl, or benzyl; R ａ and R ｂ In each occurrence, H and C appear independently. １～４ Alkyl and -OR ｊ Selected from; R ｅ , R ｆ , R ｇ , R ｈ , R ｉ , and R ｊ In each occurrence, H and C appear independently. １～４ (Selected from alkyl groups).

2. o is 1, and / or R ｅ and R ｆ The compound according to claim 1, wherein is H.

3. R ２ The compound according to claim 1 or 2, wherein is H.

4. R ５ The compound according to any one of claims 1 to 3, wherein is H.

5. R ａ and R ｂ is each H, the compound according to any one of claims 1 to 4.

6. Ar is either unsubstituted or methyl, chloro, -OMe, -NH ２ or -CH ２ NH ２ A compound according to any one of claims 1 to 5, which is substituted with [the compound].

7. R ｇ and R ｈ The compound according to any one of claims 1 to 6, wherein each appearance is independently selected from H and methyl.

8. Ar is selected from benzotriazole, imidazopyridine, pyridofuran, azaindole, benzopyrazole, pyridoazathiophene, benzoxazole, quinoline, and isoquinoline, where Ar is unsubstituted or substituted with methyl, chloro, -OMe, -NH ２ or -CH ２ NH ２ The compound according to any one of claims 1 to 7, which is substituted with.

9. Ar is, 【Transformation 3】 A compound selected from any one of claims 1 to 8.

10. The compound according to claim 8, wherein Ar is azaindole, benzotriazole, or N-methylbenzotriazole.

11. -L-(CR ｃ R ｄ ) ｎ -R ４ However, -CF ３ -OH, -NH ２ , -CN, -NHC(O)Me, -NHC(O)Ph, -NHC(O)Bn, -NHC(O)CH ２ CH ２ Ph, -NHC(O)(CH ２ ) ３ Ph, -NHC(O)OMe, -NHC(O)NHMe, -N(C(O)Me)benzyl, -N(C(O)Me)CH ２ Pyridinyl, -N(Me)cyclohexyl, phenyl, isoindoline, piperazine, benzyl, -CH ２ Pyridinyl, -CH ２ Cyclopentyl, -CH ２ Tetrahydropyranyl, -CH ２ Pyrazolyl, -CH ２ Dihydrobenzofuran, -CH ２ Imidazolyl, -CH ２ Benzodioxolanil, -NHcyclohexane, -NHpyrazinil, -NHCH ２ Ph, -NHCH ２ Cyclohexane, -NHCH ２ CH ２ Ph, and -NHCH ２ CH ２ CH ２ Selected from Ph, Here, any of the above cyclic groups is either unsubstituted or Cl, Br, F, CF ３ , OMe, OEt, -O-phenyl, -O-benzyl, CN, SO ２ The compound according to any one of claims 1 to 10, which is substituted with one, two, or three groups selected from Me, methyl, pyridinyl, or methylpyrazole.

12. R １ However, -NMe ２ -N(Me)-isopropyl, -NH-cyclopropyl, phenyl, pyridinyl, pyridinonyl, pyrimidinyl, imidazolyl, oxazolyl, pyrrolidinyl, methylpyrrolidinyl, fluoropyrrolidinyl, azetidinyl, piperidinyl, piperazinyl, azepanil, tetrahydronaphthalenyl, or 【Chemistry 4】 A compound according to any one of claims 1 to 11, which may be selected from among them.

13. R １ is F, CN, =O, -OH, -OCF ３ -OMe, Me, isopropyl, or -CF ３ The compound according to any one of claims 1 to 12, which may be substituted with a group selected from the above. 【Request Item 14】 【Chemistry 5】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 A compound according to any one of claims 1 to 12, selected from the above.

15. A compound according to any one of claims 1 to 14, for use as a pharmaceutical.

16. A pharmaceutical formulation comprising a compound according to any one of claims 1 to 14 and a pharmaceutically acceptable excipient.

17. The pharmaceutical formulation according to claim 16, which is a combination product comprising an additional pharmaceutically active agent selected from an anti-inflammatory agent, an anti-fibrotic agent, a chemotherapeutic agent, an anticancer agent, an immunosuppressant, an antitumor vaccine, a cytokine therapeutic agent, or a tyrosine kinase inhibitor.

18. For use in preventing thrombosis in medical procedures including pulmonary vein isolation, VT ablation, transcatheter aortic valve implantation (TAVI), transcatheter aortic valve replacement (TAVR), spinal or epidural anesthesia, lumbar puncture diagnosis, thoracic surgery, abdominal surgery, major orthopedic surgery, liver biopsy, transurethral resection of the prostate, kidney biopsy, biopsy-based endoscopy, prostate or bladder biopsy, electrophysiological studies or radiofrequency catheter ablation for supraventricular tachycardia (including left-sided ablation via a single transseptal puncture), angiography, pacemaker or implantable cardioverter-defibrillator (ICD) implantation, mechanical valve implantation, prosthetic valve implantation, angioplasty or reocclusion and restenosis after coronary artery bypass, patients requiring extracorporeal membrane oxygenation (ECMO), patients requiring coronary artery bypass grafting (CABG), or renal dialysis; or For use in the prevention of thrombosis in a condition selected from thrombosis, deep vein thrombosis, reperfusion injury also known as ischemic reperfusion injury, renal insufficiency, liver disease, myocardial infarction, angina pectoris (including unstable angina), stroke, patients with atrial fibrillation that reduces the risk of stroke, patients with atrial fibrillation and chronic kidney disease, transient ischemic attack, peripheral artery occlusion, pulmonary embolism, deep vein microangiopathy, disseminated intravascular coagulation (DIC), atherosclerosis, arthritis, thrombosis in patients with cancer, asymptomatic cerebral ischemia, stroke, neurotraumatic injury, neuroinflammatory disorder, Alzheimer's disease, vascular dementia, macular degeneration, diabetic retinopathy, diabetic macular edema, cerebral edema in stroke, edema of other causes, hereditary angioedema or acquired angioedema, or for use in the prevention and / or treatment of such condition, or for use as an adjunctive therapy in the treatment or prevention of such condition. The compound according to any one of claims 1 to 14.

19. A compound according to any one of claims 1 to 14, for use in the prevention or treatment of symptoms related to blood thickening, blood coagulation, or blood clot formation.

20. A compound according to any one of claims 1 to 14, for use as an anticoagulant.

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