Compounds useful as factor XIa inhibitors

Abstract

The present invention relates to factor XIa inhibitors, their tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts and solvates thereof, pharmaceutical compositions containing the compounds and the use of the compounds in the treatment and / or prevention of thromboembolic disorders, inflammatory disorders and diseases or conditions in which plasma kallikrein activity is involved.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 054,826, filed on July 22, 2020, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to factor XIa inhibitors, their tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts and solvates thereof, pharmaceutical compositions containing the compounds and the use of the compounds in the treatment and / or prevention of thromboembolic disorders, inflammatory disorders and diseases or conditions in which plasma kallikrein activity is involved. Background Art

[0004] Although anticoagulants such as warfarin are available Heparin, low molecular weight heparin (LMWH) and synthetic pentasaccharides and antiplatelet agents such as aspirin and clopidogrel However, thromboembolic diseases remain the leading cause of death in developed countries. The oral anticoagulant warfarin inhibits the post-translational maturation of coagulation factors VII, IX, X and prothrombin, and has been shown to be effective in venous and arterial thrombosis. However, due to its narrow therapeutic index, slow onset of therapeutic effect, multiple diets and drug interactions, and the need for monitoring and dosage adjustment, its use is limited. Therefore, it has become increasingly important to find and develop safe and effective oral anticoagulants for the prevention and treatment of a wide range of thromboembolic disorders.

[0005] Factor XIa is a plasma serine protease involved in the regulation of blood coagulation. Although blood coagulation is a necessary and important part of regulating the homeostasis of an organism, abnormal blood coagulation may also have deleterious effects. For example, thrombosis is the formation or presence of blood clots inside a blood vessel or heart chamber. Such clots may remain in a blood vessel, blocking circulation and inducing a heart attack or stroke. Thromboembolic disorders are the largest cause of death and disability in the industrialized world.

[0006] Blood coagulation is a process that controls blood flow essential for mammalian survival. The process of coagulation and subsequent dissolution of the clot after wound healing has already occurred, begins after vascular injury and can be divided into four stages. In the first stage, vasoconstriction or vasoconstriction leads to reduced blood loss in the damaged area. In the next stage, platelets are activated by thrombin, which attaches to the site of vascular wall injury and forms platelet aggregates. In the third stage, the formation of the coagulation complex leads to the massive formation of thrombin, which converts soluble fibrinogen into fibrin by cleavage of two small peptides. In the fourth stage, after wound healing, the clot is dissolved by the action of plasmin, a key enzyme of the endogenous fibrinolytic system.

[0007] Two kinds of alternative pathways can lead to the formation of fibrin clots, i.e., intrinsic pathways and extrinsic pathways. These pathways are triggered by different mechanisms, but in the latter stage they converge to produce the common final pathway of the coagulation cascade. In this final coagulation pathway, coagulation factor X is activated. Activated factor X is responsible for forming thrombin by the inactive precursor prothrombin circulating in the blood. Forming thrombus at the bottom of the abnormal vascular wall without wound is the result of intrinsic pathway. The formation of fibrin clots as a response to tissue damage or damage is the result of extrinsic pathway. Both pathways contain relatively large amounts of proteins, which are referred to as coagulation factors. The intrinsic pathway requires coagulation factors V, VIII, IX, X, XI and XII and also requires prekallikrein, high molecular weight kininogen, calcium ions and phospholipids from platelets.

[0008] Factor XIa, a plasma serine protease involved in the regulation of blood coagulation, is triggered in vivo by the binding of tissue factor (TF) to factor VII (FVII) to generate factor VIIa (FVIIa). The resulting TF:FVIIa complex activates factor IX (FIX) and factor X (FX), leading to the production of factor Xa (FXa). The generated FXa catalyzes the conversion of prothrombin into a small amount of thrombin before the pathway is shut down by tissue factor pathway inhibitor (TFPI). The coagulation process is then further propagated via feedback activation of factors V, VIII, and XI by catalytic amounts of thrombin. (Gailani, D. et al., Arterioscler. Thromb. Vasc. Biol., 27: 2507-2513 (2007)). The resulting thrombin burst converts fibrinogen to fibrin, which polymerizes to form the structural framework of the clot and activates platelets, which are key cellular components of coagulation (Hoffman, M., Blood Reviews, 17: S1-S5 (2003)). Factor XIa therefore plays a key role in propagating this amplification loop and is therefore an attractive target for antithrombotic therapy.

[0009] In addition to stimulation via tissue factor, the coagulation system can be activated on negatively charged surfaces, which not only include the surface structures of foreign cells (e.g., bacteria), but also include artificial surfaces, such as vascular prostheses, stents, and extracorporeal circulation. On the surface, initially factor XII (FXII) is activated to factor XIIa, which subsequently activates the factor XI attached to the cell surface to factor XIa. This results in the further activation of the coagulation cascade as described above. In addition, factor XIIa also activates the combined plasma prekallikrein to plasma kallikrein (PK), which causes further factor XII activation in the enhancement loop, generally causing the amplification of the initial coagulation cascade. In addition, PK is an important bradykinin-releasing protease, which causes an increase in endothelial permeability. Other substrates described are prorenin and prourokinase, and their activation can affect the regulatory process of the renin-angiotensin system and fibrinolysis. Therefore, the activation of PK is an important link between coagulation and inflammatory processes.

[0010] Therefore, there remains a need for Factor XIa inhibitor compounds having pharmacokinetic and pharmacodynamic properties suitable for use as human pharmaceuticals for the treatment and / or prevention of thromboembolic disorders. Summary of the invention

[0011] The present invention relates to compounds of formula (I)

[0012]

[0013] It is also known as 7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0014] The present invention also relates to compounds of formula (II)

[0015]

[0016] It is also called 4-(2-(7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizin-3-yl)-1H-imidazol-5-yl)-3-fluoropicolinic acid and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0017] The present invention also relates to compounds of formula (III)

[0018]

[0019] It is also known as 4-(2-(7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizin-3-yl)-1H-imidazol-5-yl)-3-fluoro-2-(hydroxymethyl)pyridine-1-oxide and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0020] The present invention also relates to compounds of formula (IV)

[0021]

[0022] It is also called 7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindole-3-carboxylic acid and its stereoisomers, isotopomers and salts.

[0023] The present invention also relates to compounds of formula (V)

[0024]

[0025] Among them A 1 It is C 1-4 Alkyl groups, and stereoisomers, isotopes and salts thereof.

[0026] The present invention also relates to compounds of formula (VI)

[0027]

[0028] Among them A 2 C 1-4 Alkyl, and A 3 Selected from -O-SO2-CF3, -O-SO2-CF2CF2H or -O-SO2-CF2CF2CF3, and stereoisomers, isotopes and salts thereof.

[0029] The present invention also relates to a method for preparing a compound of formula (I), a compound of formula (II), a compound of formula (III), a compound of formula (IV), a compound of formula (V), or a compound of formula (VI). The present invention also relates to a compound of formula (I), a compound of formula (II), a compound of formula (III), a compound of formula (IV), a compound of formula (V), or a compound of formula (VI) prepared according to any method described herein.

[0030] An example of the invention is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of formula (I), a compound of formula (II), or a compound of formula (III) as described herein. An example of the invention is a pharmaceutical composition made by mixing a compound of formula (I), a compound of formula (II), or a compound of formula (III) as described herein with a pharmaceutically acceptable carrier. Description The invention is a method of preparing a pharmaceutical composition, the method comprising mixing a compound of formula (I), a compound of formula (II), or a compound of formula (III) as described herein with a pharmaceutically acceptable carrier.

[0031] Exemplifying the invention are methods for treating and / or preventing a thromboembolic disorder, an inflammatory disorder, or a disease or condition in which plasma kallikrein activity is implicated as described herein, comprising administering to a subject in need thereof a therapeutically effective amount of any of the compounds or pharmaceutical compositions described above.

[0032] Exemplifying the invention are methods for treating and / or preventing thromboembolic disorders, such as arterial cardiovascular thromboembolic disorders, venous cardiovascular thromboembolic disorders, arterial cerebrovascular thromboembolic disorders, and venous cerebrovascular thromboembolic disorders, comprising administering to a subject in need thereof a therapeutically effective amount of any of the compounds or pharmaceutical compositions described above. Examples of thromboembolic disorders include, but are not limited to, unstable angina, acute coronary syndrome, atrial fibrillation, first myocardial infarction, recurrent myocardial infarction, sudden ischemic death, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary embolism, cerebral artery embolism, cerebral embolism, renal embolism, pulmonary embolism, and thrombosis caused by medical implants, devices, or surgeries in which blood is exposed to artificial surfaces that promote thrombosis.

[0033] In one embodiment, the invention relates to compounds of formula (I), (II) or (III) for use as medicaments. In another embodiment, the invention relates to compounds of formula (I), (II) or (III) for use in the treatment and / or prevention of thromboembolic disorders, inflammatory disorders or diseases or conditions in which plasma kallikrein activity is implicated.

[0034] In another embodiment, the invention relates to compounds of formula (I), (II) or (III) for use in the treatment and / or prevention of thromboembolic disorders, such as arterial cardiovascular thromboembolic disorders, venous cardiovascular thromboembolic disorders, arterial cerebrovascular thromboembolic disorders and venous cerebrovascular thromboembolic disorders. In another embodiment, the invention relates to a compound of formula (I), a compound of formula (II) or a compound of formula (III) for use in the treatment and / or prevention of a thromboembolic disorder selected from the group consisting of unstable angina, acute coronary syndrome, atrial fibrillation, first myocardial infarction, recurrent myocardial infarction, ischemic sudden death, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary embolism, cerebral artery embolism, cerebral embolism, renal embolism, pulmonary embolism and thrombosis caused by medical implants, devices or surgeries in which blood is exposed to artificial surfaces that promote thrombosis. In another embodiment, the invention relates to a compound of formula (I) or a compound of formula (II) for use in the treatment and / or prevention of a thromboembolic disorder selected from the group consisting of hereditary angioedema (HAE) and diabetic macular edema (DME).

[0035] In another embodiment, the invention relates to a composition comprising a compound of formula (I), a compound of formula (II), or a compound of formula (III) for use in treating and / or preventing a disorder, disease, or condition as described herein. In another embodiment, the invention relates to a compound comprising a compound of formula (I), a compound of formula (II), or a compound of formula (III) for use in treating and / or preventing a thromboembolic disorder, an inflammatory disorder, or a disease or condition in which plasma kallikrein activity is implicated.

[0036] In another embodiment, the invention relates to a composition comprising a compound of formula (I), a compound of formula (II) or a compound of formula (III) for use in the treatment and / or prevention of thromboembolic disorders, such as arterial cardiovascular thromboembolic disorders, venous cardiovascular thromboembolic disorders, arterial cerebrovascular thromboembolic disorders and venous cerebrovascular thromboembolic disorders. In another embodiment, the present invention relates to a composition comprising a compound of formula (I), a compound of formula (II) or a compound of formula (III) for use in the treatment and / or prevention of thromboembolic disorders selected from the group consisting of unstable angina, acute coronary syndrome, atrial fibrillation, first myocardial infarction, recurrent myocardial infarction, ischemic sudden death, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary artery embolism, cerebral artery embolism, cerebral embolism, renal embolism, pulmonary embolism and thrombosis caused by medical implants, devices or surgeries in which blood is exposed to artificial surfaces that promote thrombosis. In another embodiment, the present invention relates to a composition comprising a compound of formula (I) or a compound of formula (II) for use in the treatment and / or prevention of thromboembolic disorders such as hereditary angioedema (HAE) and diabetic macular edema (DME).

[0037] Another example of the invention is the use of any of the compounds described herein in the preparation of a medicament for treating and / or preventing a disorder, disease or condition as described herein. Another example of the invention is the use of any of the compounds described herein in the preparation of a medicament for treating and / or preventing a thromboembolic disorder, an inflammatory disorder, or a disorder or condition in which plasma kallikrein activity is implicated.

[0038] Another illustration of the invention is the use of any of the compounds described herein in the preparation of a medicament for the treatment and / or prevention of a thromboembolic disorder selected from the group consisting of: (a) an arterial cardiovascular thromboembolic disorder, (b) a venous cardiovascular thromboembolic disorder, (c) an arterial cerebrovascular thromboembolic disorder, and (d) a venous cerebrovascular thromboembolic disorder. Another example of the invention is the use of any of the compounds described herein in the preparation of a medicament for the treatment and / or prevention of (a) unstable angina, (b) acute coronary syndrome, (c) atrial fibrillation, (d) first myocardial infarction, (e) recurrent myocardial infarction, (f) ischemic sudden death, (g) transient ischemic attack, (h) stroke, (i) atherosclerosis, (j) peripheral occlusive arterial disease, (k) venous thrombosis, (l) deep vein thrombosis, (m) thrombophlebitis, (n) arterial embolism, (o) coronary embolism, (p) cerebral artery embolism, (q) cerebral embolism, (r) renal embolism, (s) pulmonary embolism, or (t) thrombosis caused by a medical implant, device, or surgery in which blood is exposed to an artificial surface that promotes thrombosis. Another illustration of the invention is the use of any of the compounds described herein in the preparation of a medicament for the treatment and / or prevention of: (a) hereditary angioedema (HAE) or (b) diabetic macular edema (DME).

[0039] Another illustration of the invention is the use of any of the compounds described herein in a method for treating a thromboembolic, inflammatory, or disease or condition in which plasma kallikrein activity is implicated as described herein in a subject in need thereof.

[0040] Another exemplary of the invention is the use of any of the compounds described herein in a method for treating and / or preventing (a) an arterial cardiovascular thromboembolic disorder, (b) a venous cardiovascular thromboembolic disorder, (c) an arterial cerebrovascular thromboembolic disorder, or (d) a venous cerebrovascular thromboembolic disorder in a subject in need thereof. Another exemplary of the invention is the use of any of the compounds described herein in a method for treating and / or preventing the following diseases in a subject in need thereof: (a) unstable angina, (b) acute coronary syndrome, (c) atrial fibrillation, (d) first myocardial infarction, (e) recurrent myocardial infarction, (f) ischemic sudden death, (g) transient ischemic attack, (h) stroke, (i) atherosclerosis, (j) peripheral occlusive arterial disease, (k) venous thrombosis, (l) deep vein thrombosis, (m) thrombophlebitis, (n) arterial embolism, (o) coronary artery embolism, (p) cerebral artery embolism, (q) cerebral embolism, (r) renal embolism, (s) pulmonary embolism, or (t) thrombosis caused by a medical implant, device, or surgery in which blood is exposed to an artificial surface that promotes thrombosis. Another illustration of the invention is the use of any of the compounds described herein in a method for treating and / or preventing (a) hereditary angioedema (HAE) or (b) diabetic macular edema (DME) in a subject in need thereof.

[0041] In another example, the invention relates to a compound as described herein for use in a method of treating and / or preventing a disorder, disease or condition as described herein in a subject in need thereof. In another example, the invention relates to a compound as described herein for use in a method of treating and / or preventing a thromboembolic disorder, an inflammatory disorder, or a disease or condition in which plasma kallikrein activity is implicated as described herein in a subject in need thereof.

[0042] In another example, the invention relates to a compound as described herein for use in a method of treating and / or preventing a thromboembolic disorder in a subject in need thereof, such as an arterial cardiovascular thromboembolic disorder, a venous cardiovascular thromboembolic disorder, an arterial cerebrovascular thromboembolic disorder, and a venous cerebrovascular thromboembolic disorder. In another example, the invention relates to a compound as described herein for use in a method of treating and / or preventing the following diseases in a subject in need thereof: unstable angina, acute coronary syndrome, atrial fibrillation, first myocardial infarction, recurrent myocardial infarction, ischemic sudden death, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary embolism, cerebral artery embolism, cerebral embolism, renal embolism, pulmonary embolism, and thrombosis caused by medical implants, devices, or surgeries in which blood is exposed to artificial surfaces that promote thrombosis. In another example, the invention relates to a compound as described herein for use in a method of treating and / or preventing hereditary angioedema (HAE) or diabetic macular edema (DME) in a subject in need thereof.

[0043] The present invention also relates to compounds, compositions (eg, pharmaceutical compositions), methods of treatment, methods of preparation or uses as described herein. DETAILED DESCRIPTION

[0044] The present invention relates to compounds of formula (I)

[0045]

[0046] and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates. The present invention also relates to a compound of formula (II):

[0047]

[0048] and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates. The present invention also relates to a compound of formula (III):

[0049]

[0050] and their tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0051] The compounds of formula (I), (II) and (III) of the present invention are useful for the treatment and / or prevention of thromboembolic disorders, inflammatory disorders and diseases or conditions in which plasma kallikrein activity is implicated.

[0052] In certain embodiments, the present invention relates to compounds of formula (IA)

[0053]

[0054] Also known as (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizine-5(1H)-one and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0055] In certain embodiments, the present invention relates to compounds of formula (IB)

[0056]

[0057] Also known as (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl-4-d)-2,3,8,8a-tetrahydroindolizine-5(1H)-one and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0058] In certain embodiments, the present invention relates to compounds of formula (IC)

[0059]

[0060] Also known as (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(4-(3-fluoro-2-(hydroxymethyl-d2)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizine-5(1H)-one and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0061] In certain embodiments, the present invention relates to compounds of formula (ID)

[0062]

[0063] Also known as (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizine-5(1H)-one-8a-d and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0064] In certain embodiments, the present invention relates to compounds of formula (IE)

[0065]

[0066] Also known as (3S*,8aS)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizine-5(1H)-one-8a-d and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0067] In certain embodiments, the present invention relates to compounds of formula (IF)

[0068]

[0069] Also known as (3S,8aR*)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl-d2)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizine-5(1H)-one-8a-d and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0070] In certain embodiments, the present invention relates to compounds of formula (IG)

[0071]

[0072] Also known as (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizine-5(1H)-one-1,1,8,8,8a-d5 and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0073] In certain embodiments, the present invention relates to compounds of formula (IH)

[0074]

[0075] Also known as (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl-d2)pyridin-4-yl)-1H-imidazol-2-yl-4-d)-2,3,8,8a-tetrahydroindolizine-5(1H)-one and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0076] In certain embodiments, the present invention relates to compounds of formula (IJ)

[0077]

[0078] Also known as (3R,8aS)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl-d)phenyl)-3-(4-(3-fluoro-2-(hydroxymethyl-d2)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizine-5(1H)-one and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0079] In certain embodiments, the present invention relates to compounds of formula (IK)

[0080]

[0081] Also known as (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizine-5(1H)-one-8,8,8a-d3 and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0082] In certain embodiments, the present invention relates to compounds of formula (IL)

[0083]

[0084] Also known as (3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizine-5(1H)-one and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0085] In certain embodiments, the present invention relates to compounds of formula (II-A)

[0086]

[0087] It is also called 4-(2-((3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizin-3-yl)-1H-imidazol-5-yl)-3-fluoropicolinic acid and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0088] In certain embodiments, the present invention relates to compounds of formula (II-B)

[0089]

[0090] Also known as 4-(2-((3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizin-3-yl)-1H-imidazol-5-yl-4-d)-3-fluoropicolinic acid and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0091] In certain embodiments, the present invention relates to compounds of formula (II-D)

[0092]

[0093] It is also called 4-(2-((3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizin-3-yl-8a-d)-1H-imidazol-5-yl)-3-fluoropicolinic acid and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0094] In certain embodiments, the present invention relates to compounds of formula (II-E)

[0095]

[0096] Also known as 4-(2-((3S,8aS)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizin-3-yl-8a-d)-1H-imidazol-5-yl)-3-fluoropicolinic acid and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0097] In certain embodiments, the present invention relates to compounds of formula (II-F)

[0098]

[0099] Also known as 4-(2-((3S,8aR*)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizin-3-yl-8a-d)-1H-imidazol-5-yl)-3-fluoropicolinic acid and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0100] In certain embodiments, the present invention relates to compounds of formula (II-G)

[0101]

[0102] Also known as 4-(2-((3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizin-3-yl-1,1,8,8,8a-d5)-1H-imidazol-5-yl)-3-fluoropicolinic acid and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0103] In certain embodiments, the present invention relates to compounds of formula (II-J)

[0104]

[0105] Also known as 4-(2-((3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl-d)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizin-3-yl)-1H-imidazol-5-yl)-3-fluoropicolinic acid and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0106] In certain embodiments, the present invention relates to compounds of formula (II-K)

[0107]

[0108] Also known as 4-(2-((3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-yl-8,8,8a-d3)-1H-imidazol-5-yl)-3-fluoropicolinic acid and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0109] In certain embodiments, the present invention relates to compounds of formula (II-L)

[0110]

[0111] It is also called 4-(2-((3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizin-3-yl)-1H-imidazol-5-yl)-3-fluoropicolinic acid and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0112] In certain embodiments, the present invention relates to compounds of formula (III-A)

[0113]

[0114] Also known as (4-(2-((3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizin-3-yl)-1H-imidazol-5-yl)-3-fluoro-2-(hydroxymethyl)pyridine-1-oxide and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0115] In certain embodiments, the present invention relates to compounds of formula (III-B)

[0116]

[0117] Also known as 4-(2-((3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizin-3-yl)-1H-imidazol-5-yl-4-d)-3-fluoro-2-(hydroxymethyl)pyridine-1-oxide, its stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0118] In certain embodiments, the present invention relates to compounds of formula (III-C)

[0119]

[0120] Also known as 4-(2-((3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-yl)-1H-imidazol-5-yl)-3-fluoro-2-(hydroxymethyl-d2)pyridine-1-oxide and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0121] In certain embodiments, the present invention relates to compounds of formula (III-D)

[0122]

[0123] Also known as 4-(2-((3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizin-3-yl-8a-d)-1H-imidazol-5-yl)-3-fluoro-2-(hydroxymethyl)pyridine-1-oxide and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0124] In certain embodiments, the present invention relates to compounds of formula (III-E)

[0125]

[0126] Also known as 4-(2-((3S,8aS)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizin-3-yl-8a-d)-1H-imidazol-5-yl)-3-fluoro-2-(hydroxymethyl)pyridine-1-oxide and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0127] In certain embodiments, the present invention relates to compounds of formula (III-F)

[0128]

[0129] Also known as 4-(2-((3S,8aR*)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-yl-8a-d)-1H-imidazol-5-yl)-3-fluoro-2-(hydroxymethyl-d2)pyridine-1-oxide and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0130] In certain embodiments, the present invention relates to compounds of formula (III-G)

[0131]

[0132] Also known as 4-(2-((3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizin-3-yl-1,1,8,8,8a-d5)-1H-imidazol-5-yl)-3-fluoro-2-(hydroxymethyl)pyridine-1-oxide and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0133] In certain embodiments, the present invention relates to compounds of formula (III-H)

[0134]

[0135] Also known as 4-(2-((3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-yl)-1H-imidazol-5-yl-4-d)-3-fluoro-2-(hydroxymethyl-d2)pyridine-1-oxide and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0136] In certain embodiments, the present invention relates to compounds of formula (III-J)

[0137]

[0138] Also known as 4-(2-((3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl-d)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-yl)-1H-imidazol-5-yl)-3-fluoro-2-(hydroxymethyl-d2)pyridine-1-oxide and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0139] In certain embodiments, the present invention relates to compounds of formula (III-K)

[0140]

[0141] Also known as 4-(2-((3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizin-3-yl-8,8,8a-d3)-1H-imidazol-5-yl)-3-fluoro-2-(hydroxymethyl)pyridine-1-oxide and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0142] In certain embodiments, the present invention relates to compounds of formula (III-L)

[0143]

[0144] Also known as 4-(2-((3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizin-3-yl)-1H-imidazol-5-yl)-3-fluoro-2-(hydroxymethyl)pyridine-1-oxide and its tautomers, stereoisomers, isotopomers and pharmaceutically acceptable salts or solvates.

[0145] The present invention also relates to compounds of formula (IV)

[0146]

[0147] Also known as 7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindole-3-carboxylic acid and its stereoisomers, isotopomers and salts. The present invention also relates to compounds of formula (V):

[0148]

[0149] and its stereoisomers, isotopes and salts. The present invention also relates to a compound of formula (VI):

[0150]

[0151] Among them A 2 C 1-4 Alkyl, and A 3 Selected from -O-SO2-CF3, -O-SO2-CF2CF2H or -O-SO2-CF2CF2CF3, and stereoisomers, isotopes and salts thereof.

[0152] The compound of formula (IV), the compound of formula (V) and the compound of formula (VI) can be used as intermediates in the synthesis of the compound of formula (I), the compound of formula (II) and / or the compound of formula (III).

[0153] In certain embodiments, the present invention also relates to compounds of formula (IV-A)

[0154]

[0155] It is also called (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindole-3-carboxylic acid and its stereoisomers, isotopomers and salts.

[0156] In certain embodiments of the present invention, A 1 It is C 2-4 In certain embodiments of the present invention, A 1 is selected from the group consisting of methyl, ethyl, isopropyl and tert-butyl. 1 is selected from the group consisting of methyl, ethyl and tert-butyl. 1 In certain embodiments of the present invention, A 1 is methyl or tert-butyl. In certain embodiments of the present invention, A 1 In certain embodiments of the present invention, A 1 is ethyl or tert-butyl. In certain embodiments of the present invention, A 1 It is ethyl.

[0157] In certain embodiments, the present invention relates to compounds of formula (VS)

[0158]

[0159] Also known as 5,7-dioxooctahydroindoleazine-3-carboxylic acid methyl ester and its stereoisomers, isotopologues and salts. In certain embodiments, the present invention also relates to compounds of formula (VA)

[0160]

[0161] It is also called (3S)-5,7-dioxooctahydroindoleazine-3-carboxylic acid methyl ester and its stereoisomers, isotopomers and salts.

[0162] In certain embodiments of the present invention, A 2 is selected from the group consisting of methyl, ethyl, isopropyl and tert-butyl. 2 is selected from the group consisting of methyl, ethyl and tert-butyl. 2 In certain embodiments of the present invention, A 2 is methyl or tert-butyl. In certain embodiments of the present invention, A 2 It's methyl.

[0163] In certain embodiments of the present invention, A 3 In certain embodiments of the present invention, A 3 In certain embodiments of the present invention, A 3 In certain embodiments of the present invention, A 3 In certain embodiments of the present invention, A 3 It is -O-SO2-CF2CF2CF3.

[0164] The present invention relates to compounds of formula (VI-S)

[0165]

[0166] Also known as 5-oxo-7-(((trifluoromethyl)sulfonyl)oxy)-1,2,3,5,8,8a-hexahydroindole-3-carboxylic acid methyl ester and its stereoisomers, isotopomers and salts. In certain embodiments, the present invention relates to a compound of formula (VI-A)

[0167]

[0168] It is also called (3S,8aR)-5-oxo-7-(((trifluoromethyl)sulfonyl)oxy)-1,2,3,5,8,8a-hexahydroindoleazine-3-carboxylic acid methyl ester and its stereoisomers, isotopomers and salts.

[0169] In certain embodiments of the invention, the compound of formula (IV), the compound of formula (V) and / or the compound of formula (VI) is present in excess of the corresponding 8S-enantiomer.

[0170] In certain embodiments, the present invention relates to a method for treating and / or preventing thromboembolic disorders, which method comprises administering to a patient in need of such treatment and / or prevention a therapeutically effective amount of a compound of the present invention or at least one of its stereoisomers, isotopomers, or pharmaceutically acceptable salts or solvates.

[0171] As used herein, the term "thromboembolic disorder" includes arterial cardiovascular thromboembolic disorders, venous cardiovascular or cerebrovascular thromboembolic disorders, and thromboembolic disorders in the cardiac chambers or in the peripheral circulation. As used herein, the term "thromboembolic disorder" also includes specific disorders selected from, but not limited to, unstable angina or other acute coronary syndromes, atrial fibrillation, first or recurrent myocardial infarction, ischemic sudden death, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary embolism, cerebral artery embolism, cerebral embolism, renal embolism, pulmonary embolism, and thrombosis caused by medical implants, devices, or surgeries in which blood is exposed to artificial surfaces that promote thrombosis. Medical implants or devices include, but are not limited to, prosthetic valves, artificial valves, indwelling catheters, stents, blood oxygenators, shunts, vascular access ports, ventricular assist devices, and artificial hearts or heart chambers and vascular grafts. Surgeries include, but are not limited to, cardiopulmonary bypass, percutaneous coronary intervention, and hemodialysis. In certain embodiments, the term "thromboembolic disorder" includes acute coronary syndrome, stroke, deep vein thrombosis, and pulmonary embolism. In certain embodiments, "thromboembolic disorder" includes hereditary angioedema (HAE) and diabetic macular edema (DME).

[0172] In certain embodiments, the present invention relates to a method for treating and / or preventing an inflammatory disorder, the method comprising: administering to a patient in need of such treatment and / or prevention a therapeutically effective amount of a compound of the present invention or at least one of its stereoisomers, isotopomers, or pharmaceutically acceptable salts or solvates. Examples of inflammatory disorders include, but are not limited to, sepsis, acute respiratory distress syndrome, and systemic inflammatory response syndrome.

[0173] In certain embodiments, the present invention relates to a method for treating and / or preventing a disease or condition in which plasma kallikrein activity is involved, the method comprising administering to a patient in need of such treatment and / or prevention a therapeutically effective amount of a compound of the present invention or at least one of its stereoisomers, isotopomers, or pharmaceutically acceptable salts or solvates. Diseases or conditions in which plasma kallikrein activity is involved include, but are not limited to, impaired visual acuity, diabetic retinopathy, diabetic macular edema, hereditary angioedema, diabetes, pancreatitis, nephropathy, cardiomyopathy, neuropathy, inflammatory bowel disease, arthritis, inflammation, septic shock, hypotension, cancer, adult respiratory distress syndrome, disseminated intravascular coagulation, and cardiopulmonary bypass surgery.

[0174] In certain embodiments, the present invention provides methods for the primary prevention of thromboembolic disorders. In certain embodiments, the present invention provides methods for the primary prevention of thromboembolic disorders, wherein the thromboembolic disorder is selected from unstable angina, acute coronary syndrome, atrial fibrillation, myocardial infarction, ischemic sudden death, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary artery embolism, cerebral artery embolism, cerebral embolism, renal embolism, pulmonary embolism, and thrombosis caused by medical implants, devices, or surgeries in which blood is exposed to artificial surfaces that promote thrombosis. In another embodiment, the present invention provides methods for the primary prevention of thromboembolic disorders, wherein the thromboembolic disorder is selected from acute coronary syndrome, stroke, venous thrombosis, and thrombosis caused by medical implants and devices.

[0175] In certain embodiments, the present invention provides methods for secondary prevention of thromboembolic disorders. In certain embodiments, the present invention provides methods for secondary prevention of thromboembolic disorders, wherein the thromboembolic disorder is selected from unstable angina, acute coronary syndrome, atrial fibrillation, recurrent myocardial infarction, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary embolism, cerebral artery embolism, cerebral embolism, renal embolism, pulmonary embolism and thrombosis caused by medical implants, devices or surgeries in which blood is exposed to artificial surfaces that promote thrombosis. In another embodiment, the present invention provides methods for secondary prevention of thromboembolic disorders, wherein the thromboembolic disorder is selected from acute coronary syndrome, stroke, atrial fibrillation and venous thrombosis.

[0176] In certain embodiments of the invention, the compound of formula (I), the compound of formula (II), or the compound of formula (III) may be administered in combination with one or more anticoagulants, antithrombin agents, antiplatelet agents, fibrinolytic agents, hypolipidemic agents, antihypertensive agents, and / or anti-ischemic agents. Suitable examples include, but are not limited to, warfarin, heparin, aprotinin, synthetic pentasaccharides, boroarginine derivatives, boropeptides, heparin, hirudin, argatroban, thromboxane-A2-receptor antagonists, thromboxane-A2-synthetase inhibitors, PDE-III inhibitors, PDE V inhibitors, ADP receptor antagonists, purinergic receptor P2Y1 antagonists, purinergic receptor P2Y12 antagonists, tissue plasminogen activator and its modified forms, anistreplase, urokinase, streptokinase, tenecteplase, lanoteplase, PAI-I inhibitors, alpha-2-antiplasmin inhibitors, anisoylated plasminogen streptokinase activator complex, HMG-CoA reductase inhibitors, squalene synthetase inhibitors, fibrate e), bile acid sequestrants, ACAT inhibitors, MTP inhibitors, lipoxygenase inhibitors, cholesterol absorption inhibitors, cholesterol ester transfer protein inhibitors, alpha adrenergic blockers, beta adrenergic blockers, calcium channel blockers, diuretics, renin inhibitors, angiotensin converting enzyme inhibitors, angiotensin-II-receptor antagonists, ET receptor antagonists, dual ET / A11 antagonists, neutral endopeptidase inhibitors, vasopeptidase inhibitors, class I agents, class II agents, class III agents, class IV agents, IAch inhibitors, IKur inhibitors and cardiac glycosides.

[0177] As used herein, the symbol "*" shall indicate the presence of a stereocenter.

[0178] If the compounds according to the invention have at least one chiral center, they may exist in enantiomeric form accordingly. If the compounds have two or more chiral centers, they may exist in diastereomeric form in addition. It should be understood that all such isomers and mixtures thereof are encompassed within the scope of the present invention.

[0179] The skilled artisan will recognize that in naming the compounds of the present invention, the S* (or *S) and R* (or *R) designations indicate that while the compound is prepared and / or present in excess of the corresponding stereoisomer, the exact stereoconfiguration is undetermined. The S and R designations indicate that the compound is prepared and / or present in excess of the corresponding S or R stereoisomer, the exact stereoconfiguration is as indicated.

[0180] Preferably, if the compound is present in enantiomeric form, the enantiomer is present in an enantiomeric excess of greater than or equal to about 80%, more preferably greater than or equal to about 90%, still more preferably greater than or equal to about 95%, still more preferably greater than or equal to about 98%, and most preferably greater than or equal to about 99%. Similarly, if the compound is present in diastereomeric form, the diastereomeric excess is greater than or equal to about 80%, more preferably greater than or equal to about 90%, still more preferably greater than or equal to about 95%, still more preferably greater than or equal to about 98%, and most preferably greater than or equal to about 99%.

[0181] In certain embodiments, the invention relates to a compound of formula (I), a compound of formula (II), or a compound of formula (III) that is an enantiomeric excess of one of the R-enantiomer or the S-enantiomer (at any stereocenter marked with a "*"). In certain embodiments of the invention, the compound of formula (I), a compound of formula (II), or a compound of formula (III) is present in an enantiomeric excess of one of the R-enantiomer or the S-enantiomer (at any stereocenter marked with a "*"). Preferably, the compound of formula (I), the compound of formula (II) or the compound of formula (III) is present in an enantiomeric excess of greater than or equal to about 80%, preferably greater than or equal to about 90%, more preferably greater than or equal to about 93%, more preferably greater than or equal to about 95%, more preferably greater than or equal to about 97%, more preferably greater than or equal to about 98%, more preferably greater than or equal to about 99% of one of the R-enantiomer or the S-enantiomer (at any stereocenter marked with a "*").

[0182] In certain embodiments, the invention relates to a compound of formula (I), a compound of formula (II), or a compound of formula (III) that is a diastereomeric or stereoisomeric excess of one of the possible diastereomers or stereoisomers. In certain embodiments of the invention, a compound of formula (I), a compound of formula (II), or a compound of formula (III) is present in a diastereomeric or stereoisomeric excess of one of the possible diastereomers or stereoisomers of about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%. Preferably, the compound of formula (I), the compound of formula (II) or the compound of formula (III) is present in a diastereomeric or stereoisomeric excess of one of the possible diastereomers or stereoisomers of greater than or equal to about 80%, preferably greater than or equal to about 90%, more preferably greater than or equal to about 93%, more preferably greater than or equal to about 95%, more preferably greater than or equal to about 97%, more preferably greater than or equal to about 98%, more preferably greater than or equal to about 99%.

[0183] In certain embodiments, the present invention relates to a compound of formula (I), a compound of formula (II), or a compound of formula (III), wherein the stereogenic center at the 3-position of hexahydroindole-5(1H)-one is present in stereoisomeric excess of the corresponding S stereoisomer or R stereoisomer.

[0184] In certain embodiments, the present invention relates to a compound of formula (I), a compound of formula (II), or a compound of formula (III), wherein the stereogenic center at the 8-position of hexahydroindole-5(1H)-one is present in stereoisomeric excess of the corresponding S stereoisomer or R stereoisomer.

[0185] In certain embodiments, the present invention relates to a compound of formula (I), a compound of formula (II), or a compound of formula (III), wherein the stereocenter at the 3-position of hexahydroindole-5(1H)-one is present in stereoisomeric excess of the corresponding S stereoisomer, and wherein the stereocenter at the 8-position of hexahydroindole-5(1H)-one is present in stereoisomeric excess of the corresponding R stereoisomer.

[0186] In addition, some crystalline forms of the compounds of the present invention can exist in polymorphic form, and are equally intended to be included in the present invention. In addition, some compounds of the present invention can form solvates (i.e. hydrates) with water or form solvates with common organic solvents, and such solvates are also intended to be included in the scope of the present invention.

[0187] Unless otherwise specified, as used herein, the term "isotopologue" shall mean molecules that differ only in their isotopic composition. More specifically, an isotopologue of a molecule differs from the parent molecule in that it contains at least one atom that is an isotope (i.e., has a different number of neutrons than its parent atom).

[0188] For example, isotopologues of water include, but are not limited to, "light water" (HOH or H2O), "semi-heavy water" (HDO or H2O) having an equal ratio of deuterium isotopes to protium. 1 H 2 HO), "heavy water" (D2O or 2 H2O), in which the hydrogen atoms are replaced by tritium ( 3 H) isotope-substituted "super heavy water" or tritiated water (T2O or 3 H2O), two heavy-oxygen water isotopes (H2 18 O and H2 17 O) and isotopologues in which hydrogen atoms and oxygen atoms can be independently replaced by isotopes, such as doubly labeled water isotopologue D2 18 O. In certain embodiments, an isotopologue may be an "isotopomer". Isotopologues include structural isomers and stereoisomers based solely on the position of the isotopologue. For example, CH3CHDCH3 and CH3CH2CH2D are a pair of structural isotopologues of n-propane; and (R)-CH3CHDOH and (S)-CH3CHDOH or (Z)-CH3CH=CHD and (E)-CH3CH=CHD are examples of isotopologue stereoisomers of ethanol and n-propylene, respectively.

[0189] Within the scope of the present invention, any one or more elements (especially when referred to in relation to a compound of formula (I), a compound of formula (II) or a compound of formula (III)) are intended to include all isotopologues or isotopic mixtures of said element (naturally occurring or synthetically prepared) in their natural abundance or in their isotopically enriched form. For example, a reference to hydrogen includes within its scope 1 H. 2 H(D) and 3 H(T). Similarly, references to carbon and oxygen include within their scope 12 C. 13 C and 14 C and 16 O and 18 O. Isotopes may be radioactive or non-radioactive. The radiolabeled compound of the compound of formula (I) or the compound of formula (II) may contain one or more radioactive isotopes selected from 3 H. 11 C.18 F. 122 I. 123 I. 125 I. 131 I. 75 Br, 76 Br, 77 Br and 82 Preferably, the radioisotope is selected from the group consisting of 3 H. 11 C and 18 F's group.

[0190] In certain embodiments of the invention, the compound of formula (I), the compound of formula (II) or the compound of formula (III) contains one or more, preferably one to eight, more preferably one to six, more preferably one to five, more preferably one to three, more preferably one to two deuterium atoms bound to ring or chain carbon atoms. In certain embodiments of the invention, the compound of formula (I), the compound of formula (II) or the compound of formula (III) contains one or more, preferably one to three, more preferably one to two deuterium atoms bound to ring or chain carbon atoms.

[0191] Those skilled in the art will further recognize that compounds of formula (I), compounds of formula (II) and compounds of formula (III) can exist as tautomers (i.e., structural isomers that can easily interconvert), more particularly as tautomers of the imidazolyl moiety of the compound. Those skilled in the art will further recognize that compounds of formula (I), compounds of formula (II) and compounds of formula (III) can exist as any tautomeric form or as any mixture of their tautomeric forms.

[0192] It is also intended that the present invention includes the compounds described herein, including all isomers thereof (including but not limited to stereoisomers, enantiomers, diastereomers, tautomers, isotopomers, atropisomers, and the like).

[0193] Under standard nomenclature used throughout this disclosure, the terminal portion of the designated side chain is described first, followed by the adjacent functionality toward the point of attachment. Thus, for example, "phenyl C1-C6 alkylaminocarbonyl C1-C6 alkyl" refers to a radical of the formula:

[0194]

[0195] The abbreviations used in this specification, especially in the schemes and examples are listed in Table A below:

[0196] Table A: Abbreviations

[0197]

[0198]

[0199]

[0200] As used herein, unless otherwise indicated, the term "isolated form" shall mean that the compound exists in a form separated from any solid mixture with another compound, from a solvent system, or from a biological environment. In one embodiment of the invention, the compound of formula (I) exists in an isolated form. In one embodiment of the invention, the compound of formula (II) exists in an isolated form. In one embodiment of the invention, the compound of formula (III) exists in an isolated form.

[0201] As used herein, unless otherwise indicated, the term "substantially pure form" shall mean that the mole percent of impurities in the isolated compound is less than about 5 mole percent, preferably less than about 2 mole percent, more preferably less than about 0.5 mole percent, and most preferably less than about 0.1 mole percent. In one embodiment of the invention, the compound of formula (I) exists as a substantially pure form. In one embodiment of the invention, the compound of formula (II) exists as a substantially pure form. In one embodiment of the invention, the compound of formula (III) exists as a substantially pure form.

[0202] Unless otherwise indicated, the term "substantially free of corresponding salt forms" as used herein, when used to describe a compound of formula (I), a compound of formula (II) or a compound of formula (III), should mean that the molar percentage of the corresponding salt forms in the separated compound of formula (I), the separated compound of formula (II) or the separated compound of formula (III) is less than about 5 mole%, preferably less than about 2 mole%, more preferably less than about 0.5 mole, and most preferably less than about 0.1 mole%. In one embodiment of the invention, the compound of formula (I) exists in the form of substantially free of corresponding salt forms. In one embodiment of the invention, the compound of formula (II) exists in the form of substantially free of corresponding salt forms. In one embodiment of the invention, the compound of formula (III) exists in the form of substantially free of corresponding salt forms.

[0203] As used herein, unless otherwise indicated, the term "treatment" and the like shall include the management and care of a subject or patient (preferably a mammal, more preferably a human) for the purpose of combating a disease, condition or disorder, and includes the administration of the compounds of the present invention to prevent the onset of symptoms or complications, alleviate symptoms or complications, slow the progression of the disease or disorder, or eliminate the disease, condition or disorder. The term "treatment" also includes: (a) inhibiting the disease state, i.e., preventing its development; and / or (b) alleviating the disease state, i.e., causing regression of the disease state.

[0204] As used herein, "prevention" is protective treatment of a disease state by administering to a patient a therapeutically effective amount of a compound of the invention or at least one of its stereoisomers, isotopomers, pharmaceutically acceptable salts or solvates to reduce and / or minimize the risk of the disease state and / or reduce the risk of recurrence of the disease state. Patients for preventive therapy can be selected based on factors known to increase the risk of developing a clinical disease state compared to the general population. For preventive treatment, symptoms of a clinical disease state may or may not be present. "Preventive" treatment can be divided into (a) primary prevention and (b) secondary prevention. Primary prevention is defined as treatment that reduces or minimizes the risk of a disease state in a patient who has not yet presented with a clinical disease state, while secondary prevention is defined as minimizing or reducing the risk of recurrence or a second occurrence of the same or similar clinical disease state.

[0205] As used herein, "prevention" encompasses preventive treatment of subclinical disease states in mammals, particularly humans, with the goal of reducing the likelihood of clinical disease states occurring. Patients for preventive therapy are selected based on factors known to increase the risk of developing clinical disease states compared to the general population.

[0206] As used herein, "risk reduction" encompasses therapies that reduce the incidence of developing a clinical disease state. Thus, primary and secondary preventive therapies are examples of risk reduction.

[0207] Those skilled in the art will recognize that, in the case of the present invention involving a method of prevention, a subject in need thereof (i.e., a subject in need of prevention) shall include any subject or patient (preferably a mammal, more preferably a human) who has experienced or exhibited at least one symptom of the disorder, disease, or condition to be prevented. In addition, a subject in need thereof may also be a subject (preferably a mammal, more preferably a human) who does not exhibit any symptoms of the disorder, disease, or condition to be prevented, but is considered by a physician, clinician, or other medical professional to be at risk of developing the disorder, disease, or condition. For example, due to the subject's medical history, including but not limited to family history, a predisposing constitution, coexisting (comorbid) disorders or conditions, genetic testing, etc., the subject may be considered to be at risk of developing a disorder, disease, or condition (and therefore in need of prevention or preventive treatment).

[0208] As used herein, the term "subject" refers to an animal, preferably a mammal, and most preferably a human, that has been the subject of treatment, observation or experiment. Preferably, the subject has experienced and / or exhibited at least one symptom of the disease or disorder to be treated and / or prevented.

[0209] As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

[0210] As used herein, the term "therapeutically effective amount" means that amount of active compound or pharmaceutical agent that will elicit the biological or pharmaceutical response being sought by the researcher, veterinarian, medical doctor or other clinician in a tissue system, animal or human, including alleviation of symptoms of the disease or condition being treated.

[0211] The compounds of the present invention are preferably administered to mammals alone in a therapeutically effective amount. However, the compounds of the present invention may also be administered to mammals in combination with another therapeutic agent as defined below in a therapeutically effective amount. When administered in combination, the combination of compounds is preferably but not necessarily a synergistic combination. For example, when the effect of the compound (in this case, the inhibitory effect on the desired target) is greater than the additive effect of the compound when administered alone as a single agent, synergy may occur. In general, synergy is most clearly demonstrated at suboptimal concentrations of the compound. Compared to individual components, synergy can be in terms of the lower cytotoxicity of the combination, the increased anticoagulant effect, or some other beneficial effects.

[0212] "Combination administration" or "combination therapy" means that the compounds of the invention and one or more additional therapeutic agents are administered to the mammal being treated simultaneously or sequentially. When administered in combination, each component may be administered at the same time or sequentially at different time points in any order. Thus, each component may be administered separately but sufficiently close in time to provide the desired therapeutic effect.

[0213] One or more other pharmacologically active agents can be used in combination with the compounds of the present invention. Other active agents (or multiple other active agents) are intended to mean pharmaceutically active agents (or multiple pharmaceutically active agents) that are active in vivo, including prodrugs that are converted into pharmaceutically active forms of compounds different from the compounds of formula (I) or formula (II) after administration, and also include free acids, free bases and pharmaceutically acceptable salts (when such forms are sold commercially or chemically possible in addition) of the other active agents. Generally, any suitable one or more other active agents, including but not limited to antihypertensive agents, other diuretics, antiatherosclerotic agents (such as lipid-modified compounds), antidiabetic agents and / or antiobesity agents can be used with compounds of formula (I) or formula (II) in any combination with single dose formulations (fixed dose drug combinations), or can be applied to patients with one or more separate dosage formulations, and the separate dosage formulations allow the simultaneous or sequential application of active agents (co-administered separate active agents).

[0214] Examples of additional active agents that may be employed include, but are not limited to, angiotensin converting enzyme inhibitors (e.g., alacepril, benazepril, captopril, ceronapril, cilazapril, delapril, enalapril, enalaprilat, fosinopril, imidapril, lisinopril, moveltipril, perindopril, quinapril, ramipril, spirapril, temocapril, or trandolapril); angiotensin II receptor antagonists, also known as angiotensin receptor blockers (ARBs) (e.g., losartan (i.e., ), valsartan, candesartan, olmesartan, telmesartan, eprosartan, irbesartan, and any of these drugs used in combination with hydrochlorothiazide, such as (losartan and hydrochlorothiazide combination)); diuretics, such as hydrochlorothiazide (HCTZ); potassium-sparing diuretics, such as amiloride hydrochloride ( HCl), spironolactone, epleranone, triamterene, each with or without HCTZ; neutral endopeptidase inhibitors (e.g., thiorphan and phosphoramidon); aldosterone antagonists; aldosterone synthase inhibitors; renin inhibitors (e.g., urea derivatives of dipeptides and tripeptides (see U.S. Pat. No. 5,116,835), amino acids and derivatives (U.S. Pat. Nos. 5,095,119 and 5,104,869), amino acid chains linked by non-peptide bonds (U.S. Pat. No. 5,114,937), dipeptide and tripeptide derivatives (U.S. Pat. No. 5,106,835), peptidylaminodiols (U.S. Pat. Nos. 5,063,208 and 4,845,079), and peptidyl β-amino acylaminodiol carbamates (U.S. Pat. No. 5,089,471); in addition, various other peptide analogs disclosed in the following U.S. Pat. Nos. 5,071,837; 5,064,965; 5,063,207; 5,036,054; 5,036,053; 5,034,512 and 4,894,437, and small molecule renin inhibitors (including diol sulfonamides and sulfinyl groups (U.S. Pat. No. 5,098,924), N-morpholino derivatives (U.S. Pat. No. 5,055,466), N-heterocyclic alcohols (U.S. Pat. No. 4,885,292) and pyrrolidinones (U.S. Pat. No. 5,075,451); in addition, pepstatin derivatives (U.S. Pat. No. 4,980,283) and fluorine and chlorine derivatives of sterone-containing peptides (U.S. Pat. No. 5,066,643); enalkrein; RO 42-5892 (CAS Reg. No. 126222-34-2, also known as remikiren); A 65317 (CAS Reg. No. 119625-78-4); CP 80794 (CAS Reg. No. 119625-78-4, also known as terlakiren); ES 1005 (CAS Reg. No. 115404-79-0); ES 8891 (CAS Reg. No. 129445-88-1); SQ 34017 (CAS Reg. No. 695226-77-8); aliskiren (2(S),4(S),5(S),7(S)-N-(2-carbamoyl-2-methylpropyl)-5-amino-4-hydroxy-2,7-diisopropyl-8-[4-methoxy-3-(3-methoxypropoxy)-phenyl]-octanamide hemifumarate); endothelin receptor antagonist; vasodilator (e.g., nitroprusside salt);Calcium channel blockers (e.g., amlodipine, nifedipine, verapamil, diltiazem, felodipine, gallopamil, niludipine, nimodipine, nicardipine); potassium channel activators (e.g., nicorandil, pinacidil, cromakalim, minoxidil, aprilkalim, loprazolam); sympathomimetics; beta-adrenergic blocking drugs (e.g., acebutolol, atenolol, betaxolol, bisoprolol, carvedilol, metoprolol, metoprolol tartrate, tartate, nadolol, propranolol, sotalol, timolol); alpha adrenergic blocking drugs (e.g., doxazocin, prazocin or alpha methyldopa); central alpha adrenergic agonists; peripheral vasodilators (e.g., hydralazine); lipid-lowering agents, for example, HMG-CoA reductase inhibitors such as simvastatin and lovastatin, which act as; and The pharmaceutically acceptable salts of dihydroxy-open-acid HMG-CoA reductase inhibitors, such as atorvastatin (particularly as calcium salts sold), rosuvastatin (particularly calcium salts sold), pravastatin (especially sodium salt) and fluvastatin (particularly as Cholesterol absorption inhibitors such as ezetimibe and ezetimibe with any other lipid-lowering agent such as the above-mentioned HMG-CoA reductase inhibitors, especially with simvastatin or in combination with atorvastatin calcium; immediate-release or controlled-release forms of niacin, particularly niacin with a DP antagonist such as laropiprant and / or in combination with HMG-CoA reductase inhibitors; immediate-release or controlled-release forms of niacin, particularly niacin with a DP antagonist such as laropipran and / or in combination with HMG-CoA reductase inhibitors; nicotinic acid receptor agonists, such as acipimox and acifuran, and nicotinic acid receptor partial agonists; metabolic altering agents, including insulin sensitizers and related compounds used to treat diabetes, such as biguanides (e.g., metformin), meglitinides (e.g., repaglinide, nateglinide), sulfonylureas (e.g., chlorpropamide, glimepiride, glimepiride), and phentermine. imepiride, glipizide, glyburide, tolazamide, tolbutamide); thiazolidinediones, also known as glitazones (e.g., pioglitazone, rosiglitazone), alpha-glucosidase inhibitors (e.g., acarbose, miglitol), dipeptidyl peptidase inhibitors (e.g., sitagliptin), alogliptin, vildagliptin, saxagliptin, linagliptin, dutogliptin, gemigliptin), ergot alkaloids (e.g., bromocriptine), combination drugs such as (sitagliptin and metformin) and injectable diabetes drugs such as exenatide and pramlintide acetate; or used together with other drugs that are beneficial to the prevention or treatment of the above diseases, including but not limited to diazoxide; and including free acid, free base and pharmaceutically acceptable salt forms of the above active agents where chemically possible. Compounds that can be administered in combination with the compounds of the present invention alternatively or additionally include but are not limited to anticoagulants, antithrombin agents, antiplatelet agents, fibrinolytic agents, hypolipidemic agents, antihypertensive agents and anti-ischemic agents.

[0215] Anticoagulants (or coagulation inhibitors) that can be used in combination with the compounds of the present invention include warfarin, heparin (unfractionated heparin or any commercially available low molecular weight heparin, such as enoxaparin and dalteparin), aprotinin, synthetic pentasaccharide inhibitors of factor Xa (such as fondaparinux and idraparinux), direct factor Xa inhibitors (such as rivaroxaban, apixaban, betrixaban, edoxaban, otamixaban), direct acting coagulation inhibitors (such as styrax, ... Enzyme inhibitors (including hirudin, dabigatran, argatroban, ximelagatran, melagatran, lepirudin, desirudin and bivalirudin), as well as other factor VIla inhibitors, VIlla inhibitors, DCa inhibitors, Xa inhibitors, XIa inhibitors, fibrinogen receptor antagonists (including abciximab, eptifibatide and tirofiban), TAFI inhibitors and other substances known in the art. Factor DCa inhibitors include synthetic active site blocking competitive inhibitors, oral inhibitors and RNA aptamers. These are described in the previously cited reference by Howard et al. (Howard, EL, Becker KC, Rusconi, CP, Becker RC. Factor IXa Inhibitors as Novel Anticoagulants. Arterioscler Thromb Vase Biol. 2007; 27: 722-727.).

[0216] As used herein, the term antiplatelet agent (or platelet inhibitor) refers to an agent that inhibits platelet function, for example, by inhibiting platelet aggregation, adhesion, or granule secretion. Such agents include, but are not limited to, various known nonsteroidal anti-inflammatory drugs (NSAIDS), such as aspirin, ibuprofen, naproxen, sulindac, indomethacin, mefenamic acid, chrysene sulfonate, phenoxyethanol, chloramphenicol ... The present invention relates to NSAIDS, including droxicam, diclofenac, sulfinpyrazone and piroxicam, including pharmaceutically acceptable salts or prodrugs thereof. In NSAIDS, aspirin (acetylsalicylic acid or ASA) and piroxicam are preferred. Other suitable platelet inhibitors include Ilb / IIIa antagonists (e.g., tirofiban, eptifibatide and abciximab), thromboxane-A2-receptor antagonists (e.g., ifetroban), thromboxane-A2-synthetase inhibitors, phosphodiesterase-III (PDE-III) inhibitors (e.g., dipyridamole, cilostazol) and PDE V inhibitors (such as sildenafil), and pharmaceutically acceptable salts or prodrugs thereof.

[0217] As used herein, the term antiplatelet agent (or platelet inhibitor) is also intended to include ADP (adenosine diphosphate) receptor antagonists, preferably antagonists of the purinergic receptors P2Y1 and P2Y12, and even more preferably P2Y12. Preferred P2Y12 receptor antagonists include ticlopidine, prasugrel, clopidogrel, elinogrel, ticagrelor and cangrelor, including pharmaceutically acceptable salts or prodrugs thereof. Clopidogrel is an even more preferred agent. Ticlopidine and clopidogrel are also preferred compounds because they are known to be mild to the gastrointestinal tract in use. The compounds of the present invention can also be administered in combination with aprotinin.

[0218] As used herein, the term thrombin inhibitor (or antithrombin agent) represents an inhibitor of the serine protease thrombin. By inhibiting thrombin, various thrombin-mediated processes are destroyed, such as thrombin-mediated platelet activation (i.e., for example, platelet aggregation, and / or plasminogen activator inhibitor-I and / or serotonin granule secretion), endothelial cell activation, inflammatory response, and / or fibrin formation. Many thrombin inhibitors are known to those skilled in the art, and these inhibitors are expected to be used in combination with the compounds of this invention. Such inhibitors include, but are not limited to, boroarginine derivatives, boro peptides, heparin, hirudin, dabigatran and argatroban, including pharmaceutically acceptable salts and prodrugs thereof. Boroarginine derivatives and boro peptides include N-acetyl and peptide derivatives of boric acid, such as lysine, ornithine, arginine, homoarginine and its corresponding isothiourea. C-terminal α-aminoboronic acid derivatives of the analogs. As used herein, the term hirudin includes suitable derivatives or analogs of hirudin, referred to herein as divalent hirudin, such as disulfato hirudin.

[0219] The term "thrombin receptor antagonist", also known as a protease activated receptor (PAR) antagonist or PAR-1 antagonist, can be used to treat thrombotic, inflammatory, atherosclerotic and fibroproliferative disorders, as well as other disorders in which thrombin and its receptor play a pathological role. Thrombin receptor antagonist peptides have been identified based on structure-activity studies involving amino acid substitutions on thrombin receptors. In Bernatowicz et al., J Med. Chem., Vol. 39, pp. 4879-4887 (1996), tetrapeptides and pentapeptides such as N-trans-cinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-NH2 and N-trans-cinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Arg-NH2 are disclosed as effective thrombin receptor antagonists. Peptide thrombin receptor antagonists are also disclosed in WO 94 / 03479. Substituted tricyclic thrombin receptor antagonists are disclosed in US Pat. Nos. 6,063,847, 6,326,380 and WO 01 / 96330 and US 7,037,920. Other thrombin receptor antagonists include those disclosed in US Pat. Nos. 7,304,078, 7,235,567, 7,037,920, 6,645,987 and European Patents EP1495018 and EP1294714.

[0220] As used herein, the term thrombolytic (or fibrinolytic) (or thrombolytic or fibrinolytic) refers to an agent that dissolves a blood clot (thrombus). Such agents include tissue plasminogen activator (TPA, natural or recombinant) and modified forms thereof, anistreplase, urokinase, streptokinase, tenecteplase (TNK), lanoteplasmase (nPA), factor VIla inhibitors, PAI-I inhibitors (i.e., inactivators of tissue plasminogen activator inhibitors), alpha-2-antiplasmin inhibitors, and anisoylated plasminogen streptokinase activator complexes, including pharmaceutically acceptable salts or prodrugs thereof. As used herein, the term anistreplase refers to anisoylated plasminogen streptokinase activator complexes, as described, for example, in European Patent 0028489. As used herein, the term urokinase is intended to refer to double-chain and single-chain urokinase, the latter also referred to herein as pro-urokinase. Examples of suitable antiarrhythmic agents for use in combination with the compounds of the invention include: Class I agents (such as propafenone); Class II agents (such as carvedilol and propranolol); Class III agents (such as sotalol, dofetilide, amiodarone, azimilide and ibutilide); Class IV agents (such as ditiazem and verapamil); IAch inhibitors and IKur inhibitors (e.g., compounds such as those disclosed in WO01 / 40231).

[0221] As provided more generally in this written specification, as used herein, terms such as "reacting" and "reacting" are used to refer to chemical entities in either: (a) the actual enumerated form of such chemical entity, and (b) any form of such chemical entity in the medium in which the compound is contemplated at the time of naming.

[0222] Those skilled in the art will recognize that, without further indication, the reaction steps are carried out under suitable conditions according to known methods to provide the desired product. Those skilled in the art will also recognize that in the description and claims given herein, if a reagent or reagent category / type (e.g., alkali, solvent, etc.) is mentioned in more than one step of the method, each reagent is independently selected for each reaction step and each reagent may be the same or different from each other. For example, if two steps of the method list an organic base or an inorganic base as a reagent, the organic base or inorganic base selected for the first step may be the same or different from the organic base or inorganic base of the second step. In addition, those skilled in the art will recognize that if the reaction steps of the present invention can be carried out in a variety of solvents or solvent systems, the reaction steps may also be carried out in a mixture of suitable solvents or solvent systems.

[0223] One skilled in the art will recognize that if a reaction step of the present invention can be carried out in a variety of solvents or solvent systems, the reaction step can also be carried out in a mixture of suitable solvents or solvent systems.

[0224] Those skilled in the art will further recognize that a reaction or process step as described herein is allowed to proceed for a sufficient period of time until the reaction is complete, as determined by any method known to those skilled in the art, such as chromatography (e.g., HPLC). As used herein, a "completed reaction or process step" shall mean that the reaction mixture contains significantly reduced amounts of starting materials / reagents and significantly increased amounts of the desired product compared to the amounts each present at the start of the reaction.

[0225] In order to provide a more concise description, some quantitative expressions given herein are not modified by the term "about". It should be understood that, whether or not the term "about" is explicitly used, each quantity given herein is intended to refer to the actual given value, and is also intended to refer to the approximate values ​​of these given values ​​that can be reasonably inferred by ordinary technicians in the field, including the approximate values ​​of these given values ​​caused by experimental and / or measurement conditions.

[0226] To provide a more concise description, some quantitative expressions herein are described as a range of about X amount to about Y amount. It should be understood that when describing a range, the range is not limited to the upper and lower limits described, but should include the entire range of about X amount to about Y amount or any amount or range therein.

[0227] Examples of suitable solvents, bases, reaction temperatures and other reaction parameters and components are given in the detailed description that follows below. Those skilled in the art will recognize that listing such examples is not intended and should not be construed as limiting in any way the invention as set forth in the claims appended hereto.

[0228] Unless otherwise indicated, as used herein, the term "leaving group" shall mean a charged or uncharged atom or group that leaves during a substitution or displacement reaction. Suitable examples include, but are not limited to, Br, Cl, I, mesylate, tosylate, and the like.

[0229] In any method for preparing the compounds of the invention, it may be necessary and / or desirable to protect sensitive or reactive groups in any of the molecules involved. This may be achieved by conventional protecting groups, such as those described in Protective Groups in Organic Chemistry, edited by JFW McOmie, Plenum Press, 1973; and TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991. Protecting groups may be removed at a convenient subsequent stage using methods known in the art.

[0230] Unless otherwise indicated, as used herein, the term "nitrogen protecting group" shall mean a group that can be attached to a nitrogen atom to protect the nitrogen atom from participating in the reaction and can be easily removed after the reaction. Suitable nitrogen protecting groups include, but are not limited to, carbamates, i.e., groups of formula -C(O)OR, wherein R is, for example, methyl, ethyl, tert-butyl, benzyl, phenethyl, CH2=CH-CH2-, etc.; amides, i.e., groups represented by formula -C(O)-R', wherein R' is (for example) methyl, phenyl, trifluoromethyl, etc.; N-sulfonyl derivatives, i.e., groups of formula -SO2-R", wherein R" is, for example, tolyl, phenyl, trifluoromethyl, 2,2,5,7,8-pentamethylchroman-6-yl-, 2,3,6-trimethyl-4-methoxybenzene, etc. Other suitable nitrogen protecting groups can be determined in texts such as TWGreene and PGM Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991.

[0231] Unless otherwise indicated, as used herein, the term "oxygen protecting group" shall mean a group that can be connected to an oxygen atom to protect the oxygen atom from participating in the reaction and can be easily removed after the reaction. Suitable oxygen protecting groups include, but are not limited to, acetyl, benzoyl, tert-butyl-dimethylsilyl, trimethylsilyl (TMS), MOM, THP, etc. Other suitable oxygen protecting groups can be determined in texts such as TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991.

[0232] If the method for preparing the compound according to the present invention produces a mixture of stereoisomers, these isomers can be separated by conventional techniques such as preparative chromatography. The compound can be prepared in racemic form, or can be synthesized by enantiomer specificity or prepared by splitting a single enantiomer. For example, by standard techniques, such as by forming a salt with an optically active acid (such as (-)-di-p-toluoyl-D-tartaric acid and / or (+)-di-p-toluoyl-L-tartaric acid) to form a diastereomeric pair, then fractional crystallization and regeneration of free alkali, the compound is split into their component enantiomers. These compounds can also be split by forming diastereomeric esters or amides, followed by chromatographic separation and removal of chiral auxiliary agents. Alternatively, the compound can be split using a chiral HPLC column.

[0233] Alternatively, chiral HPLC can be used to determine the enantiomeric excess (%ee) by comparison with standards. Enantiomeric excess can be calculated as follows:

[0234] [(R mole - S mole) / (R mole + S mole)]×100%

[0235] Wherein R mol and S mol are the mole fractions of R and S in the mixture, such that R mol + S mol = 1. Alternatively, the enantiomeric excess can also be calculated from the specific rotation of the desired enantiomer and the prepared mixture as follows:

[0236] ee=([α-obs] / [α-max])×100.

[0237] The present invention includes within its scope prodrugs of the compounds of the present invention. Generally speaking, such prodrugs will be functional derivatives of the compounds, which can be easily converted into the desired compounds in vivo. Therefore, in the therapeutic methods of the present invention, the term "administering" should cover the use of specifically disclosed compounds or undisclosed compounds to treat the various conditions described, and the undisclosed compounds can be converted into specified compounds in vivo after being administered to the patient. For example, conventional procedures for selecting and preparing suitable prodrug derivatives are described in "Design of Prodrugs", edited by H. Bundgaard, Elsevier, 1985.

[0238] For use in medicine, salts of the compounds of the present invention refer to non-toxic "pharmaceutically acceptable salts". However, other salts may also be used to prepare compounds according to the present invention or their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compounds include acid addition salts, which can be formed, for example, by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. In addition, if the compounds of the present invention have an acidic portion, suitable pharmaceutically acceptable salts thereof may include alkali metal salts, such as sodium or potassium salts; alkaline earth metal salts (such as calcium or magnesium salts); and salts formed with suitable organic ligands (such as quaternary ammonium salts). Thus, representative pharmaceutically acceptable salts include, but are not limited to, the following: acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camphorsulfonate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, etopate, esylate, fumarate, glucoheptonate, gluconate, glutamate, para-glycolylaminophenylarsonic acid salt, hexylbenzene diphenolate, hydrazine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide iodide, isothiosulfate, lactate, lactobionate, laurate, malate, maleate, mandelate, methanesulfonate, methylbromide, methylnitrate, methylsulfate, mucate, naphthenate, nitrate, N-methylglucamine ammonium salt, oleate, pamoate (enbolate), palmitate, pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, theoclate, toluenesulfonate, triethyl iodide, and valerate.

[0239] Representative acids that can be used to prepare pharmaceutically acceptable salts include, but are not limited to, the following: acids including acetic acid, 2,2-dichloroacetic acid, acylated amino acids, adipic acid, alginic acid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, D-glucuronic acid, L-Glutamic acid, α-oxo-glutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, (+)-L-lactic acid, (±)-DL-lactic acid, lactobionic acid, maleic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, and undecylenic acid.

[0240] Representative bases that can be used to prepare pharmaceutically acceptable salts include, but are not limited to, the following: bases including ammonia, L-arginine, phenethoxybenzylamine, benzathine, calcium hydroxide, choline, decanol, diethanolamine, diethylamine, 2-(diethylamino)-ethanol, ethanolamine, ethylenediamine, N-methyl-glucamine, hydrabamine, 1H-imidazole, L-lysine, magnesium hydroxide, 4-(2-hydroxyethyl)-morpholine, piperazine, potassium hydroxide, 1-(2-hydroxyethyl)-pyrrolidine, sodium hydroxide, triethanolamine, tromethamine, and zinc hydroxide.

[0241] synthesis

[0242] The compound of formula (I), the compound of formula (II), the compound of formula (III), the compound of formula (IV), the compound of formula (V) and the compound of formula (VI) of the present invention can be prepared as described in the synthesis examples below. The preparation of the starting materials used in the synthesis examples below is completely within the technical scope of those skilled in the art.

[0243] Pharmaceutical composition

[0244] The present invention also includes a pharmaceutical composition comprising a compound of formula (I) and / or a compound of formula (II) and / or a compound of formula (III) and a pharmaceutically acceptable carrier. A pharmaceutical composition containing the compound as an active ingredient can be prepared by intimately mixing one or more of the compounds of the present invention described herein with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. Depending on the desired route of administration (such as oral administration, parenteral administration), the carrier can take a variety of forms. Therefore, for liquid oral preparations such as suspensions, elixirs and solutions, suitable carriers and additives include water, glycols, oils, alcohols, flavoring agents, preservatives, stabilizers, colorants, etc.; for solid oral preparations such as powders, capsules and tablets, suitable carriers and additives include starch, sugar, diluents, granulating agents, lubricants, binders, disintegrants, etc. Solid oral preparations may also be coated with substances such as sugar or coated with enteric coatings to adjust the main absorption site. For parenteral administration, the carrier will generally consist of sterile water, and other ingredients may be added to increase solubility or preservatives. Injectable suspensions or solutions can also be prepared using water-based vehicles together with suitable additives.

[0245] In order to prepare the pharmaceutical composition of the present invention, one or more compounds of the present invention as active ingredients are intimately mixed with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques, and the carrier may take a variety of forms depending on the desired form of preparation for administration (e.g. oral administration or parenteral administration such as intramuscular administration). When preparing a composition in oral dosage form, any available pharmaceutical medium may be used. Thus, for liquid oral preparations such as suspensions, elixirs and solutions, suitable carriers and additives include water, glycols, oils, alcohols, flavoring agents, preservatives, colorants, etc.; for solid oral preparations (such as, for example, powders, capsules, caplets, capsule lozenges and tablets), suitable carriers and additives include starch, sugar, diluents, granulating agents, lubricants, binders, disintegrants, etc. Due to their convenience in administration, tablets and capsules represent the most advantageous oral unit dosage forms, in which case solid pharmaceutically acceptable carriers are obviously used. If desired, tablets can be coated with sugar or enteric coating by standard techniques. For parenteral administration, the carrier will generally include sterile water, but may also include other ingredients, for example, for purposes such as aiding dissolution or preservation. Injectable suspensions may also be prepared, in which case suitable liquid carriers, suspending agents, etc. may be used. The pharmaceutical compositions of the present invention will contain an amount of the active ingredient necessary to deliver the above-mentioned effective dose per dosage unit (e.g., per tablet, per capsule, per powder, per injection, per teaspoon, etc.). The pharmaceutical compositions herein will contain about 0.01 mg to about 1000 mg or any amount or range thereof per unit dosage unit (e.g., per tablet, per capsule, per powder, per injection, per suppository, per teaspoon, etc.), and may be administered at a dosage of about 0.05 mg / day to about 1000 mg / day or any amount or range thereof, about 0.1 mg / day to about 500 mg / day or any amount or range thereof, preferably about 1 mg / day to about 300 mg / day or any amount or range thereof.

[0246] However, dosages may vary depending on the needs of the patient, the severity of the condition being treated and the compound being employed. Daily dosing or post-cyclic dosing may be employed.

[0247] Preferably, these compositions are in unit dosage form, such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, metered aerosols or liquid sprays, drops, ampoules, automatic injection devices or suppositories; for oral, parenteral, intranasal, sublingual or rectal administration, or for administration by inhalation or insufflation. Alternatively, the composition can be provided in a manner suitable for once-weekly or once-monthly administration; for example, an insoluble salt of the active compound (such as a decanoate) can be suitable for providing a long-acting preparation for intramuscular injection. To prepare solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutically acceptable carrier (e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gum) and other pharmaceutical diluents (e.g., water) to form a solid preformulation composition containing a uniform mixture of a compound of the invention or a pharmaceutically acceptable salt thereof. When these preformulated compositions are referred to as uniform, it is meant that the active ingredient is uniformly dispersed throughout the composition so that the composition can be easily subdivided into equivalent dosage forms, such as tablets, pills, and capsules. The solid preformulation composition is then subdivided into unit dosage forms of the above-mentioned type containing about 0.01 mg to about 1,000 mg or any amount or range of the active ingredient of the present invention. The tablets or pills of the novel composition can be coated or otherwise compounded to generate a dosage form that can provide a long-acting advantage. For example, a tablet or pill can include an inner dosage form component and an outer dosage form component, the latter being in the form of a coating on the former. The two components can be separated by an enteric layer that acts to prevent disintegration in the stomach and allows the inner component to enter the duodenum intact or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, including a variety of polymeric acid materials together with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0248] Liquid preparations that can be incorporated into the novel compositions of the present invention for oral or injection administration include aqueous solutions, appropriately flavored syrups, aqueous or oily suspensions, and emulsions flavored with edible oils (cottonseed oil, sesame oil, coconut oil, or peanut oil), as well as elixirs and similar pharmaceutical vehicles. Dispersants or suspending agents suitable for aqueous suspensions include synthetic or natural gums, such as tragacanth, gum arabic, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone, or gelatin.

[0249] The method for treating and / or preventing thromboembolic disorders described in the present invention may also be performed using a pharmaceutical composition comprising any compound as defined herein and a pharmaceutically acceptable carrier. The pharmaceutical composition may contain from about 0.01 mg to about 1000 mg of the compound or any amount or range therein, preferably from about 0.05 mg to about 300 mg of the compound or any amount or range therein, more preferably from about 0.1 mg to about 100 mg of the compound or any amount or range therein, more preferably from about 0.1 mg to about 50 mg of the compound or any amount or range therein; and may be constituted in any form suitable for the selected mode of administration. The carrier includes necessary and inert pharmaceutical excipients, including but not limited to binders, suspending agents, lubricants, flavoring agents, sweeteners, preservatives, dyes and coatings. Compositions suitable for oral administration include solid forms such as pills, tablets, caplets, capsules (including immediate release, timed release and sustained release, respectively), granules and powders; and liquid forms such as solutions, syrups, elixirs, emulsions and suspensions. Useful forms for parenteral administration include sterile solutions, emulsions and suspensions.

[0250] Advantageously, the compositions of the present invention can be administered in a single daily dose, or the total daily dose can be administered in divided doses twice, three times or four times a day. In addition, the compounds of the present invention can be administered in intranasal form by topical use of a suitable intranasal vehicle, or by transdermal patches well known to those of ordinary skill in the art. To be administered in the form of a transdermal delivery system, the dosage administration will of course be continuous rather than discontinuous throughout the dosage regimen.

[0251] For example, for oral administration in the form of tablets or capsules, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier, such as ethanol, glycerol, water, etc. In addition, suitable binders, lubricants, disintegrants and colorants can also be incorporated into the mixture when desired or necessary. Suitable binders include, but are not limited to, starch, gelatin, natural sugars (e.g., glucose or β-lactose), corn sweeteners, natural gums and synthetic gums (e.g., gum arabic, tragacanth) or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, etc.

[0252] Liquid forms may include appropriately flavored suspensions or dispersions, such as synthetic and natural gums, for example, gum tragacanth, gum arabic, methylcellulose, etc. For parenteral administration, sterile suspensions and solutions are desirable. When intravenous administration is desired, isotonic preparations are used that generally contain suitable preservatives.

[0253] In order to prepare the pharmaceutical composition of the present invention, according to conventional drug compounding technology, the compound of formula (I) and / or the compound of formula (II) and / or the compound of formula (III) as active ingredient are closely mixed with a pharmaceutical carrier, and the carrier can take various forms according to the desired formulation form of administration (e.g., oral administration or parenteral administration). Suitable pharmaceutically acceptable carriers are well known in the art. Descriptions of some of these pharmaceutically acceptable carriers can be found in The Handbook of Pharmaceutical Excipients published by the American Pharmaceutical Association and the Pharmaceutical Society of Great Britain. Methods of formulating pharmaceutical compositions are described in multiple publications, such as Pharmaceutical Dosage Forms: Tablets, Second Edition, Revised and Expanded, Volumes 1-3, edited by Lieberman et al.; Pharmaceutical Dosage Forms: Parenteral Medications, Volumes 1-2, edited by Avis et al.; and Pharmaceutical Dosage Forms: Disperse Systems, Volumes 1-2, edited by Lieberman et al.; all of which are published by Marcel Dekker, Inc.

[0254] The compounds of the invention may be administered in any of the aforementioned compositions and according to dosing regimens established in the art whenever treatment or prevention of thromboembolic disorders, inflammatory disorders, and diseases or conditions in which plasma kallikrein activity is implicated is required.

[0255] The daily dose of the product can vary in a wide range of about 0.01 mg to about 1,000 mg per adult per day, or in any amount or range therein. For oral administration, the composition is preferably provided in the form of a tablet containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 150, 200, 250 and 500 milligrams of active ingredient for adjusting the dosage according to the symptoms of the patient to be treated. An effective amount of the drug can generally be provided at a dosage level of about 0.005 mg / kg body weight / day to about 10 mg / kg body weight / day or any amount or range therein. Preferably, the range is from about 0.01 mg / kg body weight / day to about 5.0 mg / kg body weight / day or any amount or range therein, more preferably, from about 0.1 mg / kg body weight / day to about 1.0 mg / kg body weight / day or any amount or range therein, more preferably, from about 0.1 mg / kg body weight / day to about 0.5 mg / kg body weight / day or any amount or range therein. The compound can be administered in a regimen of 1 to 4 times per day.

[0256] The optimal dosage to be administered can be easily determined by those skilled in the art, and the optimal dosage will vary with the specific compound used, the mode of administration, the strength of the preparation, and the advancement of the disease condition. In addition, factors related to the specific patient being treated, including patient age, weight, diet, and administration time, will result in the need to adjust the dosage.

[0257] Those skilled in the art will recognize that both in vivo and in vitro assays using appropriate, known and generally accepted cell and / or animal models are predictive of the ability of a test compound to treat or prevent a given disorder.

[0258] Those skilled in the art will also recognize that human clinical trials (including first-in-human, dose-ranging and efficacy trials) in healthy patients and / or those suffering from a given condition can be accomplished according to methods well known in the clinical and medical arts.

[0259] The following examples are presented to aid in understanding the present invention and are not intended to, and should not be construed to, limit in any way the present invention set forth in the claims that follow the examples.

[0260] In the following examples, some synthetic products that have been isolated as residues are listed. It will be appreciated by those skilled in the art that the term "residue" does not limit the physical state of the product when it is isolated, and may include, for example, solids, oils, foams, jelly-like substances, slurries, etc.

[0261] Synthesis Example

[0262] Example 1: Intermediate 1 Compound of Formula (IV-A)

[0263] (3S)-7-(6-Amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylic acid

[0264]

[0265] Step 1: Synthesis of 1-(tert-butyl)-2-methyl (2S)-5-methoxypyrrolidine-1,2-dicarboxylate

[0266] To a solution of (S)-5-oxopyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester (2.1 g, 8.63 mmol, 1.0 eq) in THF (40 mL) was added lithium triethylborohydride (13 mL, 13 mmol, 1.5 eq, 1 M in THF) dropwise at -78 °C under N2. The reaction was stirred at -78 °C for 40 min, then Na2CO3 (aq., 10 mL) was added at -78 °C, and the mixture was warmed to 0 °C. Hydrogen peroxide (1 mL, 30%) was added and the resulting mixture was stirred at room temperature for 30 min. THF was removed from the mixture under vacuum, and the resulting residue was extracted with ether, washed with brine, dried and concentrated in vacuo to give a colorless oil. The colorless oil is dissolved in methanol (40mL), and p-toluenesulfonic acid (149mg, 0.86mmol, 0.1 equivalent) is added to the mixture. The resulting solution is stirred overnight at room temperature. The reaction is quenched with water, and the resulting mixture is extracted with ether. The combined organic layer is washed with salt water, dried over Na2SO4 and concentrated to obtain (2S)-5-methoxypyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl ester) 2-methyl ester as a colorless oil.

[0267] Step 2: Synthesis of 1-(tert-butyl)-2-methyl (2S)-5-allylpyrrolidine-1,2-dicarboxylate

[0268] To a solution of (2S)-5-methoxypyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester (10 g, 38.6 mmol, 1.0 equiv) in Et2O (200 mL) was added allyltrimethylsilane (27 ml, 169.7 mmol, 4.4 equiv) and boron trifluoride etherate (6.57 g, 46.3 mmol, 1.2 equiv) at -40 °C and under N2 atmosphere. The resulting mixture was stirred at -40 °C for 30 min, warmed to room temperature, and then stirred for 40 min. The reaction was quenched with Na2CO3 (aq.) and the resulting mixture was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated. The resulting residue was purified by silica gel chromatography (0-20% EtOAc / petroleum ether) to give 1-(tert-butyl)2-methyl 5-allylpyrrolidine-1,2-dicarboxylate as a yellow oil. LC / MS: C 14 H 23 The calculated mass value of NO4 is 269.16, and the measured value is 270.20 [M+H] + .

[0269] Step 3: Synthesis of 1-(tert-butyl) 2-methyl (2S)-5-(2-oxoethyl)pyrrolidine-1,2-dicarboxylate

[0270] To a solution of (2S)-5-allylpyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester (7.3 g, 27.1 mmol, 1.0 equiv) in THF / HO (120 mL) was added OsO4 (347 mg, 1.36 mmol, 0.05 equiv). The resulting mixture was stirred at room temperature in the dark for 5 min, and then sodium periodate (14.5 g, 67.8 mmol, 2.5 equiv) was added. The mixture was stirred at room temperature for 4 h. The reaction was quenched with water, and the resulting mixture was extracted with ethyl acetate. The combined organic layers were washed with brine and dried over Na2SO4. The solid was filtered out. The filtrate was concentrated in vacuo. The resulting residue was purified by silica gel chromatography (0-40% EA / PE) to give (2S)-5-(2-oxoethyl)pyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester as a yellow oil. LC / MS: C 13 H 21 The calculated mass value of NO5 is 271.14, and the measured value is 272.15 [M+H] + ,216.05[M-C4H9] + .

[0271] Step 4: Synthesis of racemic (2R)-5-(4-ethoxy-2-hydroxy-4-oxobutyl)pyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester

[0272] To a solution of ethyl acetate (0.936 mL, 9.583 mmol, 4 eq.) in tetrahydrofuran (10 mL) was added 1 M lithium bis(trimethylsilyl)amide (9.583 mL, 9.583 mmol, 4 eq.) at -78 °C under N2 atmosphere. After stirring at the same temperature for 30 min, a solution of (2S)-5-(2-oxoethyl)pyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester (650 mg, 2.396 mmol, 1 eq.) in tetrahydrofuran (10 mL) was added and the mixture was stirred at -78 °C for 30 min. The reaction was quenched with saturated aqueous ammonium chloride solution and subsequently extracted with ethyl acetate (3 x 20 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated in vacuo. The obtained residue was purified by silica gel chromatography (0-45% ethyl acetate / petroleum ether) to give rac-(2R)-5-(4-ethoxy-2-hydroxy-4-oxobutyl)pyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester (747 mg, 86.751% yield) as a yellow oil. LC / MS: C 17 H 29 The calculated mass value of NO7 is 359, and the measured value is 382 [M+Na]+.

[0273] Step 5: Synthesis of 1-(tert-butyl) 2-methyl (2S)-5-(4-ethoxy-2,4-dioxobutyl)pyrrolidine-1,2-dicarboxylate

[0274] To a solution of 1-(tert-butyl)2-methyl (2S)-5-(4-ethoxy-2-hydroxy-4-oxobutyl)pyrrolidine-1,2-dicarboxylate (3.3 g, 9.2 mmol, 1.0 equiv) in acetonitrile (30 mL) was added 2-iodoylbenzoic acid (IBX) (10.3 g, 36.8 mmol, 4.0 equiv). The resulting mixture was stirred at 50 °C overnight. The solvent was removed and partitioned between EtOAc and brine. The organic layer was separated, dried and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (0-80% EtOAc / petroleum ether) to give 1-(tert-butyl)2-methyl (2S)-5-(4-ethoxy-2,4-dioxobutyl)pyrrolidine-1,2-dicarboxylate as a yellow oil. LC / MS: C 17 H 27 The calculated mass value of NO7 is 357.18, and the measured value is 380.15 [M+Na] + .

[0275] Step 6: Synthesis of methyl (3S)-5,7-dioxooctahydroindoleazine-3-carboxylate

[0276] To a solution of 1-(tert-butyl)2-methyl (2S)-5-(4-ethoxy-2,4-dioxobutyl)pyrrolidine-1,2-dicarboxylate (900 mg, 2.5 mmol, 1.0 eq.) in DCM (2.5 ml) was added HCl (in 1,4-dioxobutyl)pyrrolidine-1,2-dicarboxylate). The resulting mixture was stirred at room temperature for 1 h. The solvent was then removed in vacuo to give a yellow oil. The yellow oil was dissolved in toluene (5 mL) and sodium bicarbonate (2.1 g, 25.2 mmol, 10.0 equiv.) was added. The resulting mixture was stirred at 110 ° C overnight. The reaction was quenched with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated. The resulting residue was purified by silica gel chromatography (0-50% EtOAc / petroleum ether) to give (3S)-5,7-dioxooctahydroindolizine-3-carboxylic acid methyl ester as a yellow solid. LC / MS: C 10 H 13 The calculated mass value of NO4 is 211.08, and the measured value is 212.10 [M+H] + .

[0277] Step 7: Synthesis of methyl (3S)-5-oxo-7-(((trifluoromethyl)sulfonyl)oxy)-1,2,3,5,8,8a-hexahydroindoleazine-3-carboxylate

[0278] To a solution of (3S)-5,7-dioxooctahydroindolizine-3-carboxylic acid methyl ester (370 mg, 1.75 mmol, 1.0 equiv) in dichloromethane (5 mL) was added triethylamine (355 mg, 3.5 mmol, 2.0 equiv) and N,N-bis(trifluoromethylsulfonyl)aniline (751 mg, 2.1 mmol, 1.2 equiv). The resulting mixture was stirred at room temperature overnight. The mixture was then concentrated and the residue was purified by silica gel chromatography (0-80% EA / PE) to give (3S)-5-oxo-7-(((trifluoromethyl)sulfonyl)oxy)-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylic acid methyl ester as a yellow solid. LC / MS: C 11 H 12 The calculated mass value of F3NO6S is 343.03, and the measured value is 344.05 [M+H] + .

[0279] Step 8: Synthesis of methyl (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindoleazine-3-carboxylate

[0280] To (3S)-5-oxo-7-(((trifluoromethyl)sulfonyl)oxy)-1,2,3,5,8,8a-hexahydroindole-3-carboxylic acid methyl ester (350 mg, 1.02 mmol, 1.0 equiv), (6-amino-3-chloro-2-fluorophenyl)boronic acid (232 mg, 1.2 mmol, 1.2 equiv) and potassium carbonate (282 g, 2.04 mmol, 2.0 equiv) was added in 1,4-dihydro- [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (37 mg, 0.05 mmol, 0.1 equiv) was added to a solution in 2% paraffin / H2O (5 mL, 10 / 1). The resulting mixture was stirred at 80 °C under N2 for 2 h. The reaction was quenched with water and extracted with ethyl acetate. The combined organic layers were washed with brine and dried over Na2SO4. The solid was filtered out and the filtrate was concentrated under vacuum. The resulting residue was purified by silica gel chromatography (0-100% EtOAc / petroleum ether) to give (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindoleazine-3-carboxylic acid methyl ester as a yellow solid. LC / MS: C 16 H 16 The calculated mass value of ClFN2O3 is 338.08, and the measured mass value is 339.10 [M+H] + .

[0281] Step 9: Synthesis of (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindole-3-carboxylic acid

[0282] To a solution of (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindole-3-carboxylic acid methyl ester (210 mg, 0.62 mmol, 1.0 equiv) in THF / H2O / MeOH (5 mL, 3 / 1 / 1) was added lithium hydroxide (124 mg, 3.10 mmol, 5.0 equiv). The resulting mixture was stirred at room temperature for 1 h. The resulting mixture was adjusted to pH 4 with HCl, then extracted with ethyl acetate and washed with brine, dried and concentrated in vacuo to give (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindole-3-carboxylic acid as a yellow solid. LC / MS: C 15 H 14 The calculated mass value of ClFN2O3 is 324.07, and the measured value is 325.05 [M+H] + .

[0283] Example 2: Intermediate 2 Compound of Formula (VA)

[0284] (3S)-5,7-Dioxooctahydroindolizine-3-carboxylic acid methyl ester

[0285]

[0286] Step 1: Synthesis of methyl (2S)-5-allylpyrrolidine-2-carboxylate

[0287] (2S)-5-allylpyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester (10 g, 37.128 nmol, 1.0 eq.) was dissolved in HCl solution (90 mL, 10 eq.) (4 M 1,4-dicarboxylic acid 1-(tert-butyl) 2-methyl ester) The resulting mixture was kept under stirring at room temperature for 2 h. The solvent was removed under reduced pressure to give (2S)-5-allylpyrrolidine-2-carboxylic acid methyl ester, which was used in the next step without further purification.

[0288] Step 2: Synthesis of methyl (2S)-1-acryloyl-5-allylpyrrolidine-2-carboxylate

[0289] At -78 ° C, to a solution of (2S)-5-allylpyrrolidine-2-methyl carboxylate (6 g, 35.457 mmol, 1.0 equiv) in THF (200 mL), TEA (23 mL, 177.283 mmol, 5.0 equiv) was added, followed by acryloyl chloride (3.2 g, 35.457 mmol, 1.0 equiv). The resulting mixture was kept under stirring at room temperature for 40 min. The reaction was quenched with NH4Cl (aqueous solution), and the resulting layer was separated. The aqueous phase was extracted with ethyl acetate. The organic layer was dried over Na2SO4, filtered and concentrated. The resulting residue obtained was purified by silica gel chromatography (EtOAc / PE) to obtain (2S)-1-acryloyl-5-allylpyrrolidine-2-methyl carboxylate as a yellow oil.

[0290] Step 3: Synthesis of (3S)-5-oxo-1,2,3,5,8,8a-hexahydroindole-3-carboxylic acid methyl ester

[0291] Under N2, to a solution of (2S)-1-acryloyl-5-allylpyrrolidine-2-carboxylic acid methyl ester (7.2 g, 32.248 mmol, 1.0 equiv) in DCM (100 mL) was added Grubbs 2 generation catalyst (2.7 g, 3.225 mmol, 0.1 equiv). The resulting mixture was heated at 45 ° C for 10 h. The solvent was removed under reduced pressure, and the resulting residue was purified by silica gel column chromatography (EA / PE) to give (3S)-5-oxo-1,2,3,5,8,8a-hexahydroindole-3-carboxylic acid methyl ester as a brown oil.

[0292] Step 4: Synthesis of (3S)-7-hydroxy-5-oxo-octahydroindole-3-carboxylic acid methyl ester

[0293] To a solution of CuCl (0.102 g, 1.025 mmol, 0.2 eq.) in THF (100 mL) was added BINAP (0.638 g, 1.025 mmol, 0.2 eq.) under N2. The resulting mixture was kept under stirring at room temperature for 15 min. t-BuONa (0.098 g, 1.025 mmol, 0.2 eq.) was added to the mixture. After stirring for 30 min, 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (1.561 g, 6.147 mmol, 1.2 eq.) was added to the mixture, followed by (3S)-5-oxo-1,2,3,5,8,8a-hexahydroindole-3-carboxylic acid methyl ester (1 g, 5.123 mmol, 1.0 eq.). Then MeOH (0.328 g, 10.245 mmol, 2.0 equivalents) was added dropwise. The resulting mixture was stirred at room temperature for 16 h and then cooled to 0 ° C. H2O2 (6 mL, 51.226 mmol, 10 equivalents) was then added, and the resulting mixture was stirred for another 1 h. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH) to obtain (3S)-7-hydroxy-5-oxo-octahydroindole-3-methyl carboxylate as a brown oil.

[0294] Step 5: Synthesis of (3S)-5,7-dioxo-octahydroindole-3-carboxylic acid methyl ester

[0295] To a solution of (3S)-7-hydroxy-5-oxo-octahydroindolizine-3-carboxylic acid methyl ester (1 g) in DCM (100 mL) was added pyridinium chlorochromate (2 g, 9.38 mmol, 2.0 equiv.). The resulting mixture was stirred at room temperature for 16 h. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (EA / PE) to give (3S)-5,7-dioxo-octahydroindolizine-3-carboxylic acid methyl ester as a yellow oil.

[0296] Example 3: Intermediate 3 Compound of Formula (VI-A)

[0297] (3S,8aR)-5-oxo-7-(((trifluoromethyl)sulfonyl)oxy)-1,2,3,5,8,8a-hexahydroindole-3-carboxylic acid methyl ester

[0298]

[0299] Step 1: Synthesis of 7-benzyl 1-methyl (S)-2-((tert-butoxycarbonyl)amino)-5-oxopimelic acid

[0300] To a 10L 3-neck round bottom flask purged and maintained with an inert atmosphere of nitrogen was added a 1M LDA solution in THF (1132.2 mL, 1.00 equiv), solvent THF (3400.00 mL), followed by the dropwise addition of a solution of benzyl acetate (170.00 g, 1131.998 mmol, 1.00 equiv) in THF (850.0 mL) at -78°C with stirring. The resulting solution was stirred at -78°C for 30 min. To this solution was then added a solution of (2S)-5-oxopyrrolidine-1,2-dicarboxylic acid 1-tert-butyl 2-methyl ester (275.37 g, 1131.998 mmol, 1.00 equiv) in THF (850.0 mL) dropwise with stirring at -78°C. The resulting solution was kept overnight under stirring at room temperature and then quenched by adding 5000mL NH4Cl. The resulting solution was extracted with ethyl acetate (3×4000mL). The resulting mixture was washed with brine (1×1000mL). The resulting mixture was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by silica gel column, eluted with ethyl acetate / petroleum ether (1:9) to obtain (2S)-2-[(tert-butoxycarbonyl)amino]-5-oxopimelic acid 7-benzyl 1-methyl esters as yellow oil.

[0301] Step 2: Synthesis of methyl (S,Z)-5-(2-(benzyloxy)-2-oxoethylidene)pyrrolidine-2-carboxylate

[0302] To a 5000 mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen was added (2S)-2-[(tert-butoxycarbonyl)amino]-5-oxopimelic acid 7-benzyl 1-methyl ester (290.00 g, 1.00 equiv) and TFA (469.50 g, 6.00 equiv). The resulting solution was stirred at room temperature for 2 h and then concentrated in vacuo to remove most of the TFA. The pH value of the solution was adjusted to 8 with Na2CO3. The resulting solution was extracted with DCM (3×500 mL). The combined organic phases were washed with brine (1×500 mL). The mixture was dried over anhydrous sodium sulfate and concentrated in vacuo to give (2S,5Z)-5-[2-(benzyloxy)-2-oxoethylidene]pyrrolidine-2-carboxylic acid methyl ester as a semi-solid.

[0303] Step 3: Synthesis of 1-(tert-butyl)-2-methyl (2S,5R)-5-(2-(benzyloxy)-2-oxoethyl)pyrrolidine-1,2-dicarboxylate

[0304] Into a 5000 mL autoclave were placed methyl (2S,5Z)-5-[2-(benzyloxy)-2-oxoethylidene]pyrrolidine-2-carboxylate (175.00 g, 635.661 mmol, 1.00 equiv), MeOH (3500.00 mL), (Boc)2O (138.73 g, 635.661 mmol, 1.00 equiv) and PtO2 (43.30 g, 190.698 mmol, 0.30 equiv). The resulting mixture was reacted under a hydrogen atmosphere (15 atm). The resulting solution was stirred at 35 °C for 12 h. The precipitate was filtered off, and the filtrate was concentrated to give 1-(tert-butyl)2-methyl (2S,5R)-5-(2-(benzyloxy)-2-oxoethyl)pyrrolidine-1,2-dicarboxylate, which was used directly in the next step.

[0305] Step 4: Synthesis of methyl (2S,5R)-5-(2-(benzyloxy)-2-oxoethyl)pyrrolidine-2-carboxylate hydrochloride

[0306] Into a 5000 mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen was placed 1-(tert-butyl)-2-methyl-5-[2-(benzyloxy)-2-oxoethyl]pyrrolidine-1,2-dicarboxylate (244.00 g, 1.00 equiv), DCM (2440.00 mL) and 1,4-dicarboxylate containing HCl (gas). oxane (220.00 g, 4.000 mmol, 5.00 equiv). The resulting solution was stirred at room temperature for 3 h. The reaction was concentrated to give (2S,5R)-5-[2-(benzyloxy)-2-oxoethyl]pyrrolidine-2-carboxylic acid methyl ester hydrochloride as a yellow oil.

[0307] Step 5: Synthesis of methyl (2S,5R)-5-(2-(benzyloxy)-2-oxoethyl)-1-(3-methoxy-3-oxopropanoyl)pyrrolidine-2-carboxylate

[0308] Into a 5000 mL 3-necked round bottom flask purged and maintained with a nitrogen inert atmosphere was placed methyl (2S, 5R)-5-[2-(benzyloxy)-2-oxoethyl]pyrrolidine-2-carboxylate (197.00 g, 710.371 mmol, 1.00 equiv), DMF (1970.00 mL) and potassium 1-methyl 3-malonate (221.89 g, 1420.741 mmol, 2.00 equiv). This was followed by the addition of pyridine (140.48 g, 1775.927 mmol, 2.50 equiv) at 0°C. T3P (452.05 g, 1420.742 mmol, 2.00 equiv) was then added to the resulting mixture at 0°C. The resulting solution was stirred at 25°C for 12 h. The reaction was then quenched by adding water (2000 mL) and extracted with ethyl acetate (3×2000 mL). The combined organic phases were washed with brine (1×2000 mL). The mixture was dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by flash preparative HPLC using the following conditions (IntelFlash-1): column, C 18 Silica gel; mobile phase, ACN-H20=45, increased to ACN-H20=60 within 20 minutes; (2S,5R)-5-[2-(benzyloxy)-2-oxoethyl]-1-(3-methoxy-3-oxopropanoyl)pyrrolidine-2-carboxylic acid methyl ester was obtained as a yellow oil.

[0309] Step 6: Synthesis of 2-((2R,5S)-1-(3-methoxy-3-oxopropanoyl)-5-(methoxycarbonyl)pyrrolidin-2-yl)acetic acid

[0310] Into a 3000 mL round-bottom flask was placed (2S, 5R)-5-[2-(benzyloxy)-2-oxoethyl]-1-(3-methoxy-3-oxopropanoyl)pyrrolidine-2-carboxylic acid methyl ester (151.00 g), IPA (1510.00 mL) and Pd / C (15.10 g). The resulting suspension was stirred at room temperature under 2 atm H2 for 2 h. The precipitate was filtered off, and the filtrate was concentrated to give [(2R, 5S)-1-(3-methoxy-3-oxopropanoyl)-5-(methoxycarbonyl)pyrrolidin-2-yl]acetic acid as a yellow oil.

[0311] Step 7: Synthesis of dimethyl (3S,8aR)-5,7-dioxooctahydroindolizine-3,6-dicarboxylate

[0312] Into a 5000 mL 3-neck round bottom flask purged and maintained with a nitrogen inert atmosphere, [(2R, 5S) -1- (3-methoxy-3-oxopropanoyl) -5- (methoxycarbonyl) pyrrolidin-2-yl] acetic acid (104.00 g, 362.031 mmol, 1.00 equiv), THF (3120.00 mL) and CDI (88.05 g, 543.047 mmol, 1.50 equiv) were placed. The resulting mixture was stirred at room temperature for 0.5 h. DBU (82.67 g, 543.047 mmol, 1.50 equiv) was then added to the resulting mixture. The mixture was stirred at room temperature for 2 hours and then quenched by adding water / ice (2000 mL). The pH of the solution was adjusted to pH 3 with HCl (2 mol / L). The resulting solution was extracted with DCM / MeOH=3:1 (3×1500 mL), dried over anhydrous sodium sulfate and concentrated to give (3S,8aR)-5,7-dioxo-hexahydroindoleazine-3,6-dicarboxylic acid 3,6-dimethyl ester as a light yellow oil.

[0313] Step 8: Synthesis of (3S,8aR)-5,7-dioxooctahydroindoleazine-3-carboxylic acid methyl ester

[0314] Into a 3000 mL round-bottom flask was placed (3S, 8aR) -5,7-dioxo-hexahydroindole-3,6-dicarboxylic acid 3,6-dimethyl ester (94.00 g, 1.00 equivalent) and AcOH (940.00 mL). The resulting solution was stirred at 110 ° C for 30 min. The resulting solution was extracted with DCM / MeOH = 2: 1 (3 × 2000 mL), dried over anhydrous sodium sulfate and concentrated to give (3S, 8aR) -5,7-dioxo-hexahydroindole-3-carboxylic acid methyl ester as a yellow oil.

[0315] Step 9: Synthesis of methyl (3S,8aR)-5-oxo-7-(((trifluoromethyl)sulfonyl)oxy)-1,2,3,5,8,8a-hexahydroindoleazine-3-carboxylate

[0316] Into a 3000mL round-bottom flask purged and maintained with a nitrogen inert atmosphere, (3S, 8aR)-5,7-dioxo-hexahydroindole-3-carboxylic acid methyl ester (55.00g, 1.00 equivalent), DCM (1100.00mL), DIEA (67.38g, 2.00 equivalents) and PhNTf2 (111.73g, 1.20 equivalents) were placed. The resulting solution was stirred at room temperature for 4h. The resulting mixture was washed with NaHCO3 (2×2000mL). The resulting mixture was concentrated in vacuo, and the residue was purified by silica gel with PE / EA=1 / 4 to obtain (3S, 8aR)-5-oxo-7-(trifluoromethanesulfonyloxy)-1,2,3,5,8,8a-hexahydro-1H-indole-3-carboxylic acid methyl ester as a white solid.

[0317] Example 4: Intermediate 4

[0318] (6-Amino-3-chloro-2-fluorophenyl)boronic acid

[0319]

[0320] Step 1: Synthesis of N-(4-chloro-3-fluorophenyl)-2,2,2-trifluoroacetamide

[0321] To a mixture of 4-chloro-3-fluoroaniline (20 g, 137.398 mmol, 1.00 equiv) and NaCO (24.7 g, 233.577 mmol, 1.70 equiv) in EtO (400 mL) was added TFAA (34.6 g, 164.878 mmol, 1.20 equiv) dropwise at -10 °C with stirring. The resulting mixture was allowed to warm to room temperature overnight, then diluted with hexanes (400 mL) and filtered. The filtrate was washed with saturated sodium bicarbonate solution (2 × 200 mL), brine (2 × 200 mL), dried over NaSO and filtered. The filtrate was concentrated to give N-(4-chloro-3-fluorophenyl)-2,2,2-trifluoroacetamide as a white solid.

[0322] Step 2: Synthesis of 6-amino-3-chloro-2-fluorophenylboronic acid

[0323] To a mixture of N-(4-chloro-3-fluorophenyl)-2,2,2-trifluoroacetamide (30 g, 124.188 mmol, 1.00 equiv) in THF (500 mL) was added n-BuLi (99 mL, 248.375 mmol, 2.00 equiv, 2.5 M in hexane) dropwise under a nitrogen inert atmosphere at -78 °C with stirring. The resulting mixture was stirred at -78 °C for 15 min and then allowed to warm to -50 °C. The resulting clear brown solution was cooled to -78 °C and then B(O-iPr)3 (51.3 g, 273.213 mmol, 2.20 equiv) was added dropwise. The mixture was stirred at -78 °C for 10 minutes and then allowed to warm to room temperature. The resulting orange suspension was stirred at room temperature for 4 h and then cooled in an ice bath and quenched with 1 M HCl (300 mL). The resulting mixture was stirred at room temperature overnight, then extracted with EtOAc (300 mL x 3), dried over Na2SO4 and filtered. The filtrate was concentrated, and the residue was recrystallized (EtOAc / PE: 1 / 10) to give 6-amino-3-chloro-2-fluorophenylboronic acid as a white solid.

[0324] Example 5: Compound of formula (IA)

[0325] (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one

[0326]

[0327] Step 1: Synthesis of 2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridine

[0328] To a solution of (3-fluoropyridin-2-yl)methanol (15.0 g, 118.002 mmol, 1.0 equiv) in DMF (200 mL) was added imidazole (16.1 g, 236.496 mmol, 2.0 equiv) followed by tert-butylchlorodimethylsilane (21.3 g, 141.320 mmol, 1.2 equiv). The mixture was stirred at room temperature overnight. The reaction was quenched with water (200 mL). The resulting mixture was extracted with ethyl acetate (3×400 mL) and then washed with water (3×200 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The resulting residue was purified by silica gel chromatography (0-30% PE / EA) to give 2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridine as a yellow oil. LC / MS: C 12 H 20The calculated mass value of FNOSi is 241.13, and the measured value is 242.10 [M+H] + .

[0329] Step 2: Synthesis of 2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoro-4-iodopyridine To a solution of 2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridine (24.0 g, 99.430 mmol, 1.0 eq.) in THF (400 mL) was added LDA (59.7 mL, 119.4 mmol, 1.2 eq.) at -78°C. The mixture was stirred at -78°C for 0.5 h. To the resulting mixture was then added a solution of iodine (30.3 g, 119.381 mmol, 1.2 eq.) in THF (60 mL) at -78°C. The mixture was stirred at -78°C for 3 h. The reaction was quenched with NH4Cl solution (200 mL) and extracted with ethyl acetate (3×400 mL). The combined extracts were washed with water, dried over Na2SO4, filtered and concentrated. The resulting residue was purified by silica gel chromatography (0-30% EA / PE) to give 2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoro-4-iodopyridine as a white solid. LC / MS: C 12 H 19 The calculated mass value of FINOSi is 367.03, and the measured value is 368.20 [M+H] + .

[0330] Step 3: Synthesis of 2-(((tert-butyldimethylsilyl)oxy)methyl)-4-(1-ethoxyvinyl)-3-fluoropyridine

[0331] 2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoro-4-iodopyridine (20.0 g, 54.455 mmol, 1.0 equiv) and bis(triphenylphosphine)palladium(II) chloride (3.8 g, 5.414 mmol, 0.1 equiv) were added to 1,4-dihydropyridine. Tributyl(1-ethoxyvinyl)stannane (29.5 g, 81.683 mmol, 1.5 equiv) was added to a solution of 1,2-dihydro-1,4-dimethyl-1-nitropropane (200 mL). The mixture was stirred at 90 °C for 2 h. After cooling to room temperature, the reaction was quenched with water (200 mL) and then extracted with ethyl acetate (3×300 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The resulting residue was purified by Al2O3 chromatography (0-50% ethyl acetate / petroleum ether) to give 2-(((tert-butyldimethylsilyl)oxy)methyl)-4-(1-ethoxyvinyl)-3-fluoropyridine as a yellow oil. LC / MS: C 16 H 26The calculated mass value of FNO2Si is 311.17, and the measured value is 312.15 [M+H] + .

[0332] Step 4: Synthesis of 2-bromo-1-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridin-4-yl)ethan-1-one

[0333] To a mixture of 2-(((tert-butyldimethylsilyl)oxy)methyl)-4-(1-ethoxyvinyl)-3-fluoropyridine (15 g, 48.159 mmol, 1.0 equiv) in THF (120 mL) and H2O (30 mL) was added NBS (8.6 g, 48.319 mmol, 1.0 equiv). The reaction was stirred at room temperature for 2 h. The resulting mixture was then diluted with EtOAc (700 mL), washed with brine (3×200 mL), dried over Na2SO4 and concentrated to give 2-bromo-1-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridin-4-yl)ethan-1-one as an off-white solid. LC / MS: C 14 H 21 The calculated mass value of BrFNO2Si is 361.05, and the measured value is 362.00 [M+H] + .

[0334] Step 5: Synthesis of 2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridin-4-yl)-2-oxoethyl (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylate

[0335] A mixture of (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindole-3-carboxylic acid (9.3 g, 28.64 mmol, 1.0 eq) and CsCO (5.6 g, 17.18 mmol, 0.6 eq) in DMF (90 mL) was stirred at 40 °C for 30 min. Then 2-bromo-1-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridin-4-yl)ethan-1-one (14.5 g, 40.09 mmol, 1.4 eq) was added and the mixture was stirred at room temperature for 4 h. The resulting mixture was diluted with EtOAc (900 mL), washed with water (3×150 mL), brine (2×150 mL), dried over NaSO and concentrated. The resulting residue was applied to a silica gel column (330 g, MeOH / DCM: 1 / 20) to give (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylic acid 2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridin-4-yl)-2-oxoethyl ester as a light yellow solid. LC / MS: C 29 H 34 The calculated mass value of ClF2N3O5Si is 605.19, and the measured value is 606.15 [M+H] + .

[0336] Step 6: Synthesis of (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-3-(5-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one

[0337] Under an inert atmosphere of nitrogen, a mixture of 2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridin-4-yl)-2-oxoethyl (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylate (15.0 g, 24.75 mmol, 1.0 equiv) and NH4OAc (19.1 g, 247.47 mmol, 10.0 equiv) in AcOH (30 mL) and toluene (300 mL) was stirred at 110° C. for 1 h. The resulting mixture was concentrated under reduced pressure. The residue was applied to a silica gel column (330 g, MeOH / DCM: 1 / 15) to give (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-3-(5-(2-((tert-butyldimethylsilyloxy)methyl)-3-fluoropyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one as a light yellow solid. LC / MS: C 29 H 34 The calculated mass value of ClF2N5O2Si is 585.21, and the measured value is 586.15 [M+H] + .

[0338] Step 7: Synthesis of (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one

[0339] A mixture of (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-3-(5-(2-((tert-butyldimethylsilyloxy)methyl)-3-fluoropyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one (8 g, 13.648 mmol, 1.00 equiv), TMSN3 (15.724 g, 136.484 mmol, 10.00 equiv) and trimethoxymethane (14.484 g, 136.484 mmol, 10.00 equiv) in AcOH (60 mL) was stirred at 55° C. overnight. The resulting mixture was concentrated under reduced pressure. The residue was applied onto a C18 reverse phase column (330 g, ACN / H2O (0.05% NH4HCO3): 5% >>> 35% >>> 40%) to give (3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindoleazine-5(1H)-one as a white solid. The white solid was further purified by preparative SFC (column: Exsil Chiral-NR, 250mm*30mm, 8μm; mobile phase A: CO2, mobile phase B: MeOH (0.1% 2M NH3-MeOH); flow rate: 100mL / min; gradient: 50% B; 220nm; RT1: 6.78; RT2: 8.45; injection volume: 3ml; number of runs: 37;) to give (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindoleazine-5(1H)-one as a white solid.

[0340] 1 H NMR (400MHz, DMSO-d6) δ12.25(s,1H),9.82(s,1H),8.32(d,J=5.0Hz,1H),7.95(t,J=8.7Hz,1 H),7.88(t,J=5.4Hz,1H),7.70(d,J=8.7Hz,1H),7.55(d,J=3.9Hz,1H),5.68(d,J=2.6Hz,1H), 5.26(t,J=5.9Hz,1H),5.02(d,J=8.7Hz,1H),4.61(dd,J=5.9,2.3Hz,2H),3.68-3.75(m,1H),2 .67-2.75(m,1H),2.52-2.54(m,1H),2.14-2.21(m,1H),2.05-2.11(m,1H),1.93-1.97(m,2H);19 F NMR (376MHz, DMSO-d6)δ-112.85,-130.14; LC / MS: C 24 H 19 The calculated mass value of ClF2N8O2 is 524.13, and the measured value is 525.10 [M+H] + .

[0341] Example 6: Compound of formula (IB)

[0342] (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl-4-d)-2,3,8,8a-tetrahydroindolizin-5(1H)-one

[0343]

[0344] Step 1: Synthesis of (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-4-iodo-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one

[0345] A mixture of (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one (180 mg, 0.343 mmol, 1.0 equiv) and NIS (77 mg, 0.342 mmol, 1.0 equiv) in DMF (8 mL) was stirred at room temperature for 2 h. The resulting mixture was diluted with EtOAc (80 mL), washed with water (4×20 mL), brine (2×20 mL), dried over Na2SO4 and concentrated. The residue was applied to a silica gel column (40 g, MeOH / DCM: 1 / 15) to give (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-4-iodo-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one as a light yellow solid. LC / MS: C 24 H 18 The calculated mass value of ClF2IN8O2 is 650.03, and the measured value is 650.95 [M+H] + .

[0346] Step 2: Synthesis of (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl-4-d)-2,3,8,8a-tetrahydroindolizin-5(1H)-one

[0347] A mixture of (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-4-iodo-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one (210 mg, 0.323 mmol, 1.0 equiv) and Zn (211 mg, 3.226 mmol, 10.0 equiv) in CDCOOD (8 mL) and D0 (4 mL) was stirred at room temperature for 10 min. The resulting mixture was filtered and the filtrate was concentrated. The residue was applied onto a C18 reverse phase column (330 g, ACN / H2O (0.05% NH4HCO3): 5% >>> 40% >>> 40%) to give (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl-4-d)-2,3,8,8a-tetrahydroindoleazine-5(1H)-one as a light yellow solid.

[0348] 1 H NMR(400MHz,DMSO-d6)δ12.30(br,1H),9.84(s,1H),8.30-8.40(m,1H),7.97(t ,J=7.9Hz,1H),7.89(t,J=5.4Hz,1H),7.71(dd,J=8.7,1.5Hz,1H),5.70(d,J=2. 7Hz,1H),5.13-5.50(m,1H),5.05(d,J=8.7Hz,1H),4.64(s,2H),3.65-3.83(m,1 H),2.62-2.83(m,1H),2.53-2.60(m,1H),2.05-2.30(m,2H),1.83-2.04(m,2H); 19 F NMR (376MHz, DMSO-d6)δ-112.51,-130.07; LC / MS: C 24 H 18 The calculated mass value of ClDF2N8O2 is 525.14, and the measured value is 526.15 [M+H] + .

[0349] Example 7A: Compound of formula (IC)

[0350] (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(4-(3-fluoro-2-(hydroxymethyl-d2)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one

[0351]

[0352] Step 1: Synthesis of (3-fluoropyridin-2-yl)methane-d2-ol

[0353] At 0 ° C, NaBD4 (4.0 g, 96.696 mmol, 3.0 equivalents) was added to a mixture of 3-fluoropicolinic acid methyl ester (5 g, 32.232 mmol, 1.00 equivalents) in EtOH (80 mL) in batches. The resulting mixture was stirred for 2 h at room temperature. The reaction was then quenched with a saturated ammonium chloride solution (60 mL). The EtOH solvent was removed by ROTOVAP evaporation under reduced pressure. The resulting mixture was then extracted with EtOAc (3 × 100 mL), dried over Na2SO4 and concentrated to give (3-fluoropyridin-2-yl)methane-d2-ol as a light yellow solid.

[0354] Step 2: Synthesis of 2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoropyridine

[0355] To a mixture of (3-fluoropyridin-2-yl)methane-d2-ol (3.2 g, 24.781 mmol, 1.0 eq.) and TBSCl (5.6 g, 37.172 mmol, 1.5 eq.) in DMF (60 mL) was added Et3N (7.5 g, 74.344 mmol, 3.0 eq.). The resulting mixture was then stirred at room temperature for 5 h. The resulting mixture was diluted with EtOAc (600 mL), washed with water (3×150 mL), brine (2×150 mL), dried over Na2SO4 and concentrated. The residue was applied to a silica gel column (80 g, EtOAc / PE: 1 / 3) to give 2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoropyridine as a light yellow solid. LC / MS: C 12 H 18 The calculated mass value of D2FNOSi is 243.14, and the measured value is 244.10 [M+H] + .

[0356] Step 3: Synthesis of 2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoro-4-iodopyridine

[0357] Under an inert atmosphere of nitrogen, to a mixture of 2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoropyridine (4.8 g, 19.721 mmol, 1.00 equiv) in THF (120 mL) at -78°C was added LDA (11.8 mL, 23.666 mmol, 1.2 equiv, 2M in THF), and the resulting mixture was stirred at -78°C for 30 min. A solution of I2 (5.5 g, 21.694 mmol, 1.1 equiv) in THF (25 mL) was then added to the resulting mixture at -78°C. The mixture was stirred at -78°C for 2 h. The resulting mixture was quenched with saturated ammonium chloride solution (200 mL), extracted with EtOAc (3×200 mL), dried over Na2SO4 and concentrated. The residue was applied onto a silica gel column (80 g, EtOAc / PE: 1 / 4) to give 2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoro-4-iodopyridine as a light yellow solid.

[0358] Step 4: Synthesis of 2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-4-(1-ethoxyvinyl)-3-fluoropyridine

[0359] Under a nitrogen inert atmosphere, 2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoro-4-iodopyridine (2.7 g, 7.311 mmol, 1.0 equiv), tributyl(1-ethoxyvinyl)stannane (5.3 g, 14.623 mmol, 2.0 equiv) and Pd(PPh3)4 (845 mg, 0.731 mmol, 0.1 equiv) were stirred in 1,4-dimethylsilylcarbonyl solution for 1 h. The mixture was stirred at 100° C. overnight in 2% paraffin (50 mL). The resulting mixture was concentrated and the residue was applied to a silica gel column (80 g, EtOAc / PE: 1 / 3) to give 2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-4-(1-ethoxyvinyl)-3-fluoropyridine as a yellow solid. LC / MS: C 16 H 24 The calculated mass value of D2FNO2Si is 313.18, and the measured value is 314.10 [M+H] + .

[0360] Step 5: Synthesis of 2-bromo-1-(2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoropyridin-4-yl)ethan-1-one

[0361] To a mixture of 2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-4-(1-ethoxyvinyl)-3-fluoropyridine (2.5 g, 7.975 mmol, 1.0 equiv) in THF (60 mL) and H2O (20 mL) was added NBS (1.4 g, 7.975 mmol, 1.0 equiv). The reaction was stirred at room temperature for 1 h. The resulting mixture was then diluted with EtOAc (500 mL), washed with brine (3×100 mL), dried over Na2SO4 and concentrated to give 2-bromo-1-(2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoropyridin-4-yl)ethan-1-one as a light yellow oil.

[0362] Step 6: Synthesis of 2-(2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoropyridin-4-yl)-2-oxoethyl (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylate

[0363] A mixture of (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylic acid (2.4 g, 7.391 mmol, 1.0 eq) and Cs2CO3 (1.4 g, 4.434 mmol, 0.6 eq) in DMF (60 mL) was stirred at 40°C for 30 min. Then 2-bromo-1-(2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoropyridin-4-yl)ethan-1-one (2.7 g, 7.391 mmol, 1.0 eq) was added and the mixture was stirred at room temperature for 4 h. The resulting mixture was then diluted with EtOAc (500 mL), washed with water (3×100 mL), brine (2×100 mL), dried over Na2SO4 and concentrated. The residue was applied to a silica gel column (80 g, MeOH / DCM: 1 / 20) to give (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylic acid 2-(2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoropyridin-4-yl)-2-oxoethyl ester as a yellow solid. LC / MS: C 29 H 32 The calculated mass value of ClD2F2N3O5Si is 607.20, and the measured value is 608.15 [M+H] + .

[0364] Step 7: Synthesis of (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-3-(4-(2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoropyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one

[0365] Under an inert atmosphere of nitrogen, a mixture of 2-(2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoropyridin-4-yl)-2-oxoethyl (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylate (3.5 g, 5.755 mmol, 1.0 equiv) and NH4OAc (3.5 g, 46.042 mmol, 8.0 equiv) in AcOH (10 mL) and toluene (100 mL) was stirred at 110° C. for 1 h. The resulting mixture was concentrated. The residue was applied to a silica gel column (80 g, MeOH / DCM: 1 / 15) to give (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-3-(4-(2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoropyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one as a light yellow solid. LC / MS: C 29 H 32 The calculated mass value of ClD2F2N5O2Si is 587.23, and the measured value is 588.15 [M+H] + .

[0366] Step 8: Synthesis of (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(4-(3-fluoro-2-(hydroxymethyl-d2)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one

[0367] A mixture of (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-3-(4-(2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoropyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one (2.8 g, 4.761 mmol, 1.0 equiv), TMSN3 (5.5 g, 47.606 mmol, 10.0 equiv) and trimethoxymethane (5.1 g, 47.606 mmol, 10.0 equiv) in AcOH (50 mL) was stirred at 55° C. overnight. The resulting mixture was concentrated. The residue was applied to C 18On a reverse phase column (330 g, ACN / H2O (0.05% NH4HCO3): 5% >>> 35% >>> 40%), (3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(4-(3-fluoro-2-(hydroxymethyl-d2)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindoleazine-5(1H)-one was obtained as a white solid. The white solid was further purified by preparative SFC (column: Exsil Chiral-NR, 250mm*30mm, 8μm; mobile phase A: CO2, mobile phase B: MeOH (0.5% 2M NH3-MeOH)--HPLC; flow rate: 100mL / min; gradient: 45% B; 220nm; RT1: 9.02; RT2: 11.5; injection volume: 4.8ml; number of runs: 13;) to give (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(4-(3-fluoro-2-(hydroxymethyl-d2)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindoleazine-5(1H)-one as a white solid.

[0368] 1 H NMR (400MHz, DMSO-d6) δ12.27(s,1H),9.85(s,1H),8.35(d,J=5.0Hz,1H),7.98(t,J=8. 0Hz,1H),7.90(t,J=5.4Hz,1H),7.72(dd,J=8.7,1.5Hz,1H),7.58(d,J=1.6Hz,1H),5.7 1(d,J=2.8Hz,1H),5.24(s,1H),5.05(d,J=8.7Hz,1H),3.65-3.84(m,1H),2.73(t,J=15 .4Hz,1H),2.54-2.62(m,1H),2.17-2.30(m,1H),2.06-2.16(m,1H),1.89-2.05(m,2H); 19 F NMR (376MHz, DMSO-d6)δ-112.85,-130.23; LC / MS: C 24 H 17 The calculated mass value of ClD2F2N8O2 is 526.14, and the measured mass value is 527.05 [M+H] + .

[0369] Example 7B: Compound of formula (IC)

[0370] (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(4-(3-fluoro-2-(hydroxymethyl-d2)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one

[0371]

[0372] Step 1: (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylic acid 2-(2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoropyridin-4-yl)-2-oxoethyl ester

[0373] A mixture of ((3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindole-3-carboxylic acid (260 mg, 0.80 mmol, 1.0 eq) and Cs2CO3 (156 mg, 0.48 mmol, 0.6 eq) in DMF (12 mL) was stirred at 40°C for 30 min. Then 2-bromo-1-(2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoropyridin-4-yl)ethan-1-one (437 mg, 1.20 mmol, 1.5 eq) was added to the mixture. The reaction mixture was stirred at room temperature for 4 h. The resulting mixture was then diluted with EA (120 mL), washed with water (3×30 mL), brine (2×20 mL), dried over Na2SO4 and concentrated. The residue was purified by flash column chromatography on silica gel (0→10% The product was purified by HPLC-MS / MS: 50 % MeOH / DCM to afford 2-(2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoropyridin-4-yl)-2-oxoethyl (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylate as a yellow solid. LC / MS: C 29 H 32 The calculated mass value of ClD2F2N3O5Si is 607.20, and the measured value is 608.10 [M+H] + .

[0374] Step 2: (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-3-(4-(3-fluoro-2-(hydroxymethyl-d2)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one

[0375] A mixture of 2-(2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoropyridin-4-yl)-2-oxoethyl (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindoleazine-3-carboxylate (280 mg, 0.46 mmol, 1.0 equiv) and NH4OAc (355 mg, 4.60 mmol, 10.0 equiv) in AcOH (3 mL) and toluene (30 mL) was stirred at 110°C for 1 h, then cooled to room temperature and concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography (0→9% MeOH / DCM) to give (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-3-(4-(3-fluoro-2-(hydroxymethyl-d2)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one as a yellow solid. LC / MS: C 23 H 18 The calculated mass value of ClD2F2N5O2 is 473.14, and the measured mass value is 474.05 [M+H] + .

[0376] Step 3: (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(4-(3-fluoro-2-(hydroxymethyl-d2)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one

[0377] A mixture of ((3S)-7-(6-amino-3-chloro-2-fluorophenyl)-3-(4-(3-fluoro-2-(hydroxymethyl-d2)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one (200 mg, 0.422 mmol, 1.00 equiv), TMSN3 (486 mg, 4.22 mmol, 10.0 equiv) and trimethoxymethane (448 mg, 4.22 mmol, 10.0 equiv) in AcOH (10 mL) was stirred at 50 °C for 4 h and then concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography (0→10% MeOH / DCM ) to give (3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(4-(3-fluoro-2-(hydroxymethyl-d2)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one as a light yellow solid, which was further purified by chiral HPLC to give (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(4-(3-fluoro-2-(hydroxymethyl-d2)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one as a white solid.

[0378] LC / MS: C 24 H 17 The calculated mass value of ClD2F2N8O2 is 526.14, and the measured value (ES, m / z) is 527.05 [M+H] + . 1 HNMR(400MHz,DMSO-d6)d 12.27(s,1H),9.85(s,1H),8.35(d,J=5.0Hz,1H),7.98(t,J=8.0Hz,1H),7.90(t, J=5.4Hz,1H),7.72(dd,J=8.7,1.5Hz,1H),7.58(d,J=1.6Hz,1H),5.71(d,J=2.8H z,1H),5.24(s,1H),5.05(d,J=8.7Hz,1H),3.65-3.84(m,1H),2.73(t,J=15.4Hz, 1H),2.54-2.62(m,1H),2.17-2.30(m,1H),2.06-2.16(m,1H),1.89-2.05(m,2H). 19 FNMR(376MHz,DMSO-d6)d-112.85,-130.23

[0379] Example 8: Compound of formula (ID)

[0380] (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one-8a-d

[0381]

[0382] Step 1: 2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridin-4-yl)-2-oxoethyl (3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylate 8a-d.

[0383] To a solution of (3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylic acid-8a-d (300 mg, 0.80 mmol, 1.0 eq) in CH3CN (10 mL) was added K2CO3 (142 mg, 1.03 mmol, 1.3 eq). After stirring at room temperature for 0.5 h, 2-bromo-1-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridin-4-yl)ethan-1-one (430 mg, 1.20 mmol, 1.5 eq) was added. The mixture was stirred at room temperature for 1 h. The resulting mixture was purified by silica gel column with MeOH / DCM (0→7%) to give 2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridin-4-yl)-2-oxoethyl (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylate-8a-d as a yellow oil. LC / MS: C 30 H 32 The calculated mass value of DClF2N6O5Si is 659.20, and the measured value (ES, m / z) is 660.15 [M+H] + .

[0384] Step 2: (3S)-3-(5-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridin-4-yl)-1H-imidazol-2-yl)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one-8a-d.

[0385] To a solution of 2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridin-4-yl)-2-oxoethyl (3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylate-8a-d (380 mg, 0.58 mmol, 1.0 equiv) in toluene (10 mL) and acetic acid (1 mL) was added ammonium acetate (665 mg, 8.63 mmol, 15.0 equiv). The mixture was stirred at 100° C. for 1 h. The solvent was removed in vacuo, and the residue was purified by silica gel column with MeOH / DCM (0→10%) to give (3S)-3-(5-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridin-4-yl)-1H-imidazol-2-yl)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one-8a-d as a yellow oil. LC / MS: C 30 H 32 The mass calculated value of DClF2N8O2Si is 639.22, and the measured value (ES, m / z) is 640.20 [M+H] + .

[0386] Step 3: (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one-8a-d.

[0387] To a solution of (3S)-3-(5-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridin-4-yl)-1H-imidazol-2-yl)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one-8a-d (280 mg, 0.44 mmol, 1.0 equiv) in THF (5 mL) was added triethylamine trihydrogen fluoride (1.3 mL). The mixture was stirred at 70° C. for 1 h. The solvent was removed in vacuo, and the residue was purified by C18 column [conditions: ACN-water-6.5 mM NH4HCO3+NH3H2O ​​(5→50%)] to give a second residue. The second residue was purified by chiral HPLC [column: (R,R) WHELK-O1, 4.6*50 mm, 3.5 μm; mobile phase: Hex (0.1% DEA): EtOH = 55:45; flow rate: 1 mL / min] to give (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindoleazine-5(1H)-one-8a-d as an off-white solid.

[0388] LC / MS: C 24 H 18 The calculated mass value of ClDF2N8O2 is 525.14, and the measured value (ES, m / z) is 526.10 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ12.25(s,1H),9.84(s,1H),8.34(d,J=5.1Hz,1H),7.94-8.00(m,1H ),7.86-7.90(m,1H),7.71(dd,J=8.6,1.5Hz,1H),7.57(d,J=3.8Hz,1H),5.70(d,J=2.7Hz, 1H),5.23-5.29(m,1H),5.04(d,J=8.7Hz,1H),4.63(dd,J=6.0,2.3Hz,2H),2.65-2.77(d,J =16.7Hz,1H),2.52-2.58(m,1H),2.16-2.29(m,1H),2.06-2.14(m,1H),2.90-2.02(m,2H). 19 F NMR (376MHz, DMSO-d6) δ-112.85,-130.16.

[0389] Example 9: Compound of formula (IE)

[0390] (3S,8aS)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one-8a-d

[0391]

[0392] The title compound was prepared according to the procedure as described above in Example 8 and the second fraction was isolated and lyophilized to give the title compound as a white solid.

[0393] LC / MS: C 24 H 18 The mass calculated value of DClF2N8O2 is 525.14, and the measured value (ES, m / z) is 526.10 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ12.52(s,1H),9.84(s,1H),8.34(d,J=5.1Hz,1H),7.94-8.01 (m,1H),7.85-7.92(m,1H),7.71(dd,J=8.7,1.5Hz,1H),7.62(d,J=3.7Hz,1H),5.70(d ,J=2.7Hz,1H),5.00-5.08(m,1H),4.64(d,J=2.2Hz,2H),2.42-2.46(m,1H),2.31-2.4 1(m,1H),2.20-2.30(m,1H),1.95-2.08(m,1H),1.64-1.78(m,1H),1.14-1.28(m,1H). 19 F NMR (376MHz, DMSO-d6) δ-113.06,-129.81.

[0394] Example 10: Compound of formula (IF)

[0395] (3S,8aR*)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl-d2)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one-8a-d

[0396]

[0397] Step 1: (2S)-5-methoxypyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester-5-d

[0398] To a solution of (2S)-5-hydroxypyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester-5-d (14 g, 56.85 mmol, 1.0 equiv) in MeOH (140 mL) was added p-toluenesulfonic acid monohydrate (2.7 g, 14.21 mmol, 0.25 equiv). The mixture was stirred at room temperature overnight. The solvent was removed in vacuo, and the residue was purified by silica gel column with EA / PE (0%-35%) to give (2S)-5-methoxypyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester-5-d as a yellow oil.

[0399] Step 2: 1-(tert-butyl)-2-methyl (2S)-5-((2,2-dimethyl-4-oxo-4H-1,3-dioxin-6-yl)methyl)pyrrolidine-1,2-dicarboxylate

[0400] To a solution of (2S)-5-methoxypyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester-5-d (10 g, 38.4 mmol, 1.0 equiv) and ((2,2-dimethyl-4-methylene-4H-1,3-dioxin-6-yl)oxy)trimethylsilane (14.0 g, 65.32 mmol, 1.7 equiv) in tert-butyl methyl ether (100 mL) was added BF3πEt2O (6.8 g, 47.91 mmol, 1.25 equiv) at -40 °C over 10 min. The resulting mixture was stirred at -78 °C for 40 min. The reaction was allowed to warm to ambient temperature overnight. The reaction was quenched with saturated sodium bicarbonate solution (50 mL). The resulting mixture was extracted with ethyl acetate (3×100 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (0-40% ethyl acetate / petroleum ether) to give 1-(tert-butyl)2-methyl-5-((2,2-dimethyl-4-oxo-4H-1,3-dioxin-6-yl)methyl)pyrrolidine-1,2-dicarboxylate-5-d as a yellow oil. LC / MS: C 18 H 26 The calculated mass value of DNO7 is 370.19, and the measured value is 393.20 [M+H] + .

[0401] Step 3: (2S)-5-((2,2-dimethyl-4-oxo-4H-1,3-dioxin-6-yl)methyl)pyrrolidine-2-carboxylic acid methyl ester-5-d

[0402] To a solution of 1-(tert-butyl)-2-methyl (2S)-5-((2,2-dimethyl-4-oxo-4H-1,3-dioxin-6-yl)methyl)pyrrolidine-1,2-dicarboxylate-5-d (8 g, 21.60 mmol, 1.0 equiv) in DCM (50 mL) was added TFA (10 mL). The mixture was stirred at room temperature for 1 h. The mixture was washed with NaHCO3 solution (2×10 mL) and NaCl solution (1×10 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated to give methyl (2S)-5-((2,2-dimethyl-4-oxo-4H-1,3-dioxin-6-yl)methyl)pyrrolidine-2-carboxylate-5-d as a yellow oil. LC / MS: C 13 H 18 The calculated mass value of DNO5 is 270.13, and the measured value is 213.05 [M+H-CO2Me] + .

[0403] Step 4: (3S)-5-oxo-7-(((trifluoromethyl)sulfonyl)oxy)-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylic acid methyl ester-8a-d

[0404] A mixture of (2S)-methyl 5-((2,2-dimethyl-4-oxo-4H-1,3-dioxin-6-yl)methyl)pyrrolidine-2-carboxylate-5-d (6 g, 22.20 mmol, 1.0 equiv) in toluene (60 mL) was stirred at 100° C. for 1 h. The solvent was removed in vacuo, and the residue was purified by silica gel column with MeOH / DCM (0%-20%) to give (3S)-methyl 5,7-dioxoocthydroindolizine-3-carboxylate-8a-d as a yellow oil.

[0405] To a solution of (3S)-5,7-dioxooctahydroindolizine-3-carboxylic acid methyl ester-8a-d (1 g, 4.71 mmol, 1.0 equiv) and triethylamine (1.4 g, 14.14 mmol, 3.0 equiv) in DCM (10 mL) at 0°C was added 1,1,1-trifluoro-N-phenyl-N-((trifluoromethyl)sulfonyl)methanesulfonamide (1.7 g, 4.71 mmol). The mixture was stirred at room temperature for 1 h. The reaction was quenched with water (20 mL). The resulting mixture was extracted with DCM (1×50 mL) and washed with water and NaCl solution (30 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (0-20% ethyl acetate / petroleum ether) to give (3S)-methyl 5-oxo-7-(((trifluoromethyl)sulfonyl)oxy)-1,2,3,5,8,8a-hexahydroindoleazine-3-carboxylate-8a-d as a yellow oil. LC / MS: C 11 H 11 The calculated mass value of DF3NO6S is 344.04, and the measured value is 345.00 [M+H] + .

[0406] Step 5: (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylic acid methyl ester-8a-d

[0407] To (3S)-5-oxo-7-(((trifluoromethyl)sulfonyl)oxy)-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylic acid methyl ester-8a-d (1 g, 2.91 mmol, 1.0 equiv) in 1,4-dihydro- To a solution of 1,4-dihydro-2-nitropropene (15 mL) and HO (2 mL) was added (6-amino-3-chloro-2-fluorophenyl)boronic acid (1.1 g, 5.81 mmol, 1.0 eq), followed by KCO (803 mg, 5.81 mmol, 2.0 eq), Pd(dppf)Cl (237 mg, 0.29 mmol, 0.1 eq). The resulting mixture was kept under nitrogen and stirred at 90 °C for 2 h. The reaction was quenched with saturated ammonium chloride solution (20 mL). The resulting mixture was extracted with ethyl acetate (3×20 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (0-20% MEOH / DCM) to give (3S)-methyl 7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylate-8a-d as a brown solid. LC / MS: C 16 H 15The calculated mass value of ClDFN2O3 is 339.09, and the measured value is 340.05 [M+H] + .

[0408] Step 6: (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylic acid-8a-d

[0409] To a solution of (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindole-3-carboxylic acid methyl ester-8a-d (1.6 g, 4.71 mmol, 1.0 equiv) in tetrahydrofuran (20 mL) and water (4 mL) was added LiOH (169 mg, 7.06 mmol, 1.5 equiv). The reaction was stirred at room temperature for 1 h. The pH was adjusted to 2-5 with 2M HCl. The resulting mixture was extracted with ethyl acetate (2×50 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated to give (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindole-3-carboxylic acid-8a-d as a brown solid. LC / MS: C 15 H 13 The calculated mass value of ClDFN2O3 is 325.07, and the measured value is 326.20 [M+H] + .

[0410] Step 7: (3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylic acid-8a-d

[0411] To a solution of (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindole-3-carboxylic acid-8a-d (1.6 g, 4.91 mmol, 1.0 equiv) in AcOH (6 mL) was added TMSN3 (3 ml) and trimethoxymethane (3 mL). The mixture was stirred at room temperature overnight. The solvent was removed in vacuo, and the residue was purified by C18 column with CH3CN / water (5%-35%) to give (3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindole-3-carboxylic acid-8a-d as a yellow solid. LC / MS: C 16 H 12 The calculated mass value of ClDFN5O3 is 378.08, and the measured value is 379.05 [M+H] + .

[0412] Step 8: (3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylic acid 2-(2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoropyridin-4-yl)-2-oxoethyl ester-8a-d

[0413] To a solution of (3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylic acid-8a-d (300 mg, 0.80 mmol, 1.0 eq) in CH3CN (10 mL) was added K2CO3 (142 mg, 1.03 mmol, 1.3 eq). After stirring at room temperature for 0.5 h, 2-bromo-1-(2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoropyridin-4-yl)ethan-1-one (432 mg, 1.19 mmol, 1.5 eq) was then added. The mixture was stirred at room temperature for 1 h. The resulting mixture was purified by silica gel chromatography (0-7% MeOH / DCM) to afford 2-(2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoropyridin-4-yl)-2-oxoethyl (3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylate-8a-d as a yellow oil. LC / MS: C 30 H 30 The calculated mass value of ClD3F2N6O5Si is 661.21, and the measured value is 662.15 [M+H] + .

[0414] Step 9: (3S)-3-(5-(2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoropyridin-4-yl)-1H-imidazol-2-yl)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one-8a-d

[0415] To a solution of 2-(2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoropyridin-4-yl)-2-oxoethyl (3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylate-8a-d (280 mg, 0.42 mmol, 1.0 equiv) in toluene (10 mL) and glacial acetic acid (1 mL) was added ammonium acetate (489 mg, 6.34 mmol, 15.0 equiv). The mixture was stirred at 100° C. for 1 h. The solvent was removed in vacuo and the residue was purified by silica gel column with MeOH / DCM (0%-10%) to give (3S)-3-(5-(2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoropyridin-4-yl)-1H-imidazol-2-yl)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one-8a-d as a yellow oil. LC / MS: C 30 H 30 The calculated mass value of ClD3F2N8O2Si is 641.23, and the measured value is 642.20 [M+H] + .

[0416] Step 10: (3S,8aR*)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl-d2)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one-8a-d

[0417] To a solution of (3S)-3-(5-(2-(((tert-butyldimethylsilyl)oxy)methyl-d2)-3-fluoropyridin-4-yl)-1H-imidazol-2-yl)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-2,3,8,8a-tetrahydroindolizine-5(1H)-one-8a-d (140 mg, 0.22 mmol, 1.0 equiv) in THF (4 mL) was added triethylamine trihydrogen fluoride (1 mL). The mixture was stirred at 70° C. for 1 h. The solvent was removed in vacuo and the residue was purified by C18 column [conditions: ACN-water-6.5 mM NH4HCO3+NH3H2O ​​(5%-50%)] to give a residue. The residue was purified by chiral HPLC [column: (R,R)-Whelk-O1, 4.6*50 mm, 3.5 μm; mobile phase A: Hex (0.1% DEA): EtOH = 50:50, mobile phase B:; flow rate: 1 mL / min] to give (3S,8aR*)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl-d2)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindoleazine-5(1H)-one-8a-d as an off-white solid.

[0418] LC / MS: C 24 H 16 The calculated mass value of D3ClF2N8O2 is 527.15, and the measured value (ES, m / z) is 642.20 [M+H] + . 1 HNMR(400MHz,DMSO-d6)(400MHz,DMSO-d6)δ12.29(s,1H),9.84(s,1H),8.34(d,J=5 .1Hz,1H),7.93-8.02(m,1H),7.84-7.92(m,1H),7.70-7.75(m,1H),7.58(d,J=4.0H z,1H),5.69(d,J=2.7Hz,1H),5.12-5.32(m,1H),5.04(d,J=8.7Hz,1H),2.63-2.80( m,1H),2.50-2.56(m,1H),2.17-2.31(m,1H),2.05-2.15(m,1H),1.90-2.01(m,2H). 19 F-NMR: (376MHz, DMSO-d6)δ-112.86,-130.13.

[0419] Example 11: Compound of formula (IG)

[0420] (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one-1,1,8,8,8a-d5

[0421]

[0422] Step 1: (3S)-2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridin-4-yl)-2-oxoethyl 7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylate-1,1,8,8,8a-d5.

[0423] A mixture of (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylic acid-1,1,8,8,8a-d5 (120 mg, 0.36 mmol, 1.0 eq.) and Cs2CO3 (71 mg, 0.22 mmol, 0.6 eq.) in DMF (8 mL) was stirred at room temperature for 30 min. Then 2-bromo-1-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridin-4-yl)ethan-1-one (185 mg, 0.51 mmol, 1.4 eq.) was added to the mixture. The reaction mixture was stirred at room temperature for 4 h. The resulting mixture was then diluted with EtOAc (120 mL), washed with water (3×20 mL), brine (2×20 mL), dried over Na2SO4 and concentrated. The residue was purified by silica gel flash column chromatography (40 g, MeOH / DCM: 1 / 20) to give 2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridin-4-yl)-2-oxoethyl (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylate-1,1,8,8,8a-d5 as a yellow solid. LC / MS: C 29 H 29 The calculated mass value of ClD5F2N3O5Si is 610.22, and the measured value (ES, m / z) is 611.15 [M+H] + .

[0424] Step 2: (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-3-(5-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one-1,1,8,8,8a-d5.

[0425] Under an inert atmosphere of nitrogen, a mixture of 2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridin-4-yl)-2-oxoethyl (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindoleazine-3-carboxylate-1,1,8,8,8a-d5 (130 mg, 0.21 mmol, 1.0 equiv) and NH4OAc (164 mg, 2.13 mmol, 10.0 equiv) in AcOH (2 mL) and toluene (20 mL) was stirred at 110°C for 30 min and then concentrated. The residue was purified by silica gel flash column chromatography (40 g, MeOH / DCM: 1 / 15) to give (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-3-(5-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizine-5(1H)-one-1,1,8,8,8a-d5 as a yellow solid. LC / MS: C 29 H 29 The mass calculated value of D5ClF2N5O2Si is 590.25, and the measured value (ES, m / z) is 591.30 [M+H] + .

[0426] Step 3: (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one-1,1,8,8,8a-d5.

[0427] A mixture of (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-3-(5-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one-1,1,8,8,8a-d5 (80 mg, 0.14 mmol, 1.0 equiv), TMSN3 (156 mg, 1.35 mmol, 10.0 equiv) and trimethoxymethane (143 mg, 1.35 mmol, 10.0 equiv) in AcOH (8 mL) was stirred at 55° C. overnight. The solvent was removed under reduced pressure and the residue was applied to a C18 reverse phase column (330 g, ACN / H2O (0.05% NH4HCO3): 5→50%), (3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizine-5(1H)-one-1,1,8,8,8a-d5 was obtained as a white solid. Further purification by preparative chiral HPLC afforded (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one-1,1,8,8,8a-d5 as a white solid.

[0428] LC / MS: C 24 H 14 The calculated mass value of D5ClF2N8O2 is 529.16, and the measured value (ES, m / z) is 530.10 [M+H] + . 1 HNMR(400MHz,DMSO-d6)(400MHz,DMSO-d6)δ12.26(s,1H),9.84(s,1H),8.34(d,J=5.0Hz,1H),7.90-8.00(m,1H),7.90(t,J=5.4Hz,1H),7.75-7. 85(m,1H),7.51-7.62(m,1H),5.70(s,1H),5.27(t,J=6.0Hz,1H),5.04(d ,J=8.7Hz,1H),4.58-4.69(m,2H),2.13-2.30(m,1H),1.84-2.00(m,1H). 19 F-NMR: (376MHz, DMSO-d6)δ-112.86,-130.15.

[0429] Example 12: Compound of formula (IH)

[0430] (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl-d2)pyridin-4-yl)-1H-imidazol-2-yl-4-d)-2,3,8,8a-tetrahydroindolizin-5(1H)-one

[0431]

[0432] To a mixture of (3R, 8aS) -7- (3-chloro-2-fluoro-6- (1H-tetrazolyl-1-yl) phenyl) -3- (5- (3-fluoro-2- (hydroxymethyl-d2) pyridin-4-yl) -4-iodo-1H-imidazol-2-yl) -2,3,8,8a-tetrahydroindolazine-5 (1H) -one (25 mg, 0.038 mmol) and Zn powder (25 mg, 0.38 mmol) was added 1.5 g of CD3COOD (1.5 g), and the resulting mixture was stirred at room temperature for 4 h. The precipitate was then filtered off. The filtrate was concentrated under reduced pressure, and the residue was purified by Gilson HPLC to give 3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl-d2)pyridin-4-yl)-1H-imidazol-2-yl-4-d)-2,3,8,8a-tetrahydroindolizine-5(1H)-one as a light yellow solid.

[0433] C 24 H 19 The LC / MS calculated value of ClF2N8O2 was 527.1 and the found value was 528.3 (MH + ); 1 H NMR (400MHz, methanol-d4) δ9.62(s,1H),8.56(s,1H),8.46(s,1H),7.85(dd,J=7.83,8.80Hz,1H),7.58(dd,J=1.71,8.56Hz,1H),5.77-5.85(m,1H),5.21 -5.30(m,1H),3.96-4.13(m,1H),2.75-2.88(m,1H),2.66-2.73(m,1H),2. 35-2.45(m,1H),2.27-2.33(m,1H),2.16-2.24(m,1H),2.02-2.12(m,1H).

[0434] Example 13: Compound of formula (IJ)

[0435] (3R,8aS)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl-d)phenyl)-3-(4-(3-fluoro-2-(hydroxymethyl-d2)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one

[0436]

[0437] To a solution of (3S,8aR)-7-[3-chloro-2-fluoro-6-(tetrazol-1-yl)phenyl]-3-[4-[2-[dideuterio(hydroxy)methyl]-3-fluoro-4-pyridinyl]-1H-imidazol-2-yl]-2,3,8,8a-tetrahydro-1H-indolizin-5-one (35 mg, 0.07 mmol) in CD3OD was added NaHCO3 in an NMR tube and the mixture was stirred for 2 h. 1 The sample was analyzed by H NMR. Within a few minutes, the acidic proton in the tetrazole ring was exchanged with deuterium to give (3R,8aS)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl-d)phenyl)-3-(4-(3-fluoro-2-(hydroxymethyl-d2)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one.

[0438] C 24 H 16 The LC / MS calculated value of D3ClF2N8O2 was 527.1, and the found value was 528.3 (MH + ). 1 H NMR (400MHz, methanol-d4) δ8.02 (dd, J=8.31, 9.78Hz, 1H), 7.78-7.88 (m, 1H), 7.54 -7.61(m,1H),7.14(d,J=3.42Hz,1H),6.49(dd,J=1.96,8.31Hz,1H),5.73(d, J=2.93Hz,1H),5.16(d,J=8.80Hz,1H),3.84-3.96(m,1H),2.89-3.04(m,1H) ,2.56-2.65(m,1H),2.28-2.39(m,1H),2.16-2.26(m,2H),1.99-2.12(m,1H).

[0439] Example 14: Compound of formula (IK)

[0440] (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizine-5(1H)-one-8,8,8a-d3

[0441]

[0442] Step 1: (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one-8,8,8a-d3

[0443] To a solution of 2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoropyridin-4-yl)-2-oxoethyl (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindoleazine-3-carboxylate-8,8,8a-d3 (0.51 g, 0.837 mmol, 1.0 equiv) in toluene (10 mL) were added NH4OAc (0.65 g, 8.4 mmol, 10 equiv) and AcOH (0.5 mL), and the resulting mixture was stirred at 100 °C for 1 h. The mixture was then concentrated and purified by C18 reverse phase chromatography (80 g, MeCN / H2O (0.05% CF3COOH): 0>45%) to give (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizine-5(1H)-one-8,8,8a-d3 (0.24 g, 60.4% yield) as a yellow solid. LC / MS: C 23 H 17 The calculated mass value of ClD3F2N5O2 is 474.15, and the measured value is 476.20 [M+H] + .

[0444] Step 2: (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one-8,8,8a-d3

[0445] A mixture of (3S)-7-(6-amino-3-chloro-2-fluorophenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one-8,8,8a-d3 (0.22 g, 0.463 mmol, 1.0 equiv), trimethoxymethane (2 mL), trimethylsilylazide (2 mL) and acetic acid (2 mL) was stirred at room temperature for 2 h. The solvent was evaporated in vacuo to give (3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one-8,8,8a-d3. The racemic product was separated by chiral HPLC (column: CHIRALPAK IF-3, 4.6*50 mm, 3 μm; mobile phase: MtBE (0.1% DEA):EtOH=70:30; flow rate: 1 mL / min) to give (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindoleazine-5(1H)-one-8,8,8a-d3 as a white solid.

[0446] LC / MS: C 24 H 16 The calculated mass value of ClD3F2N8O2 is 527.15, and the measured mass value is 528.10 [M+H] + . 1 H NMR(400MHz,DMSO-d6)d 12.27(s,1H),9.85(s,1H),8.30–8.40(m,1H),7.94-8.01(m,1H),7.86-7.93(m,1H),7.69-7.75(m,1H),7.54-7.60(m,1H) ,5.70(s,1H),5.28(t,J=6.0Hz,1H),5.08–5.01(m,1H),4.64(dd,J=5.9,2.3Hz,2H),2.14-2.28(m,1H),1.91-2.00(m,2H).

[0447] Example 15: Compound of formula (II-A)

[0448] 4-(2-((3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-yl)-1H-imidazol-5-yl)-3-fluoropicolinic acid

[0449]

[0450] To a mixture of (3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-3-(5-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)-1H-imidazol-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one (1.2 g, 2.286 mmol, 1.00 equiv) in DCM (60 mL) and H2O (30 mL) was added TEMPO (179 mg, 1.146 mmol, 0.50 equiv) and PhI(OAc)2 (2.2 g, 6.830 mmol, 3.0 equiv). The reaction mixture was stirred at room temperature for 2 h. The resulting mixture was concentrated and the residue was applied to C 18 On a reverse phase column (330 g, ACN / H2O (0.05% NH4HCO3): 5% >>> 23% >>> 25%), 4-(2-((3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizin-3-yl)-1H-imidazol-5-yl)-3-fluoropicolinic acid was obtained as a white solid.

[0451] 1 H NMR(400MHz,DMSO-d6)δ12.30(br.,1H),9.85(s,1H),8.28(d,J=8.0Hz,1H),7.9 3-8.01(m,1H),7.84-7.92(m,1H),7.71(d,J=8.7Hz,1H),7.55(d,J=4.0Hz,1H), 5.69(d,J=2.7Hz,1H),5.05(d,J=8.8Hz,1H),3.65-3.85(m,1H),2.63-2.86(m,1 H),2.52-2.62(m,1H),2.15-2.19(m,1H),2.05-2.14(m,1H),1.89-2.05(m,2H); 19 F NMR (376MHz, DMSO-d6)δ-112.81,-127.49; LC / MS: C 24 H 17 The calculated mass value of ClF2N8O3 is 538.11, and the measured value is 539.05 [M+H] + ;

[0452] Example 16: Compound of formula (III-A)

[0453] 4-(2-((3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizin-3-yl)-1H-imidazol-5-yl)-3-fluoro-2-(hydroxymethyl)pyridine 1-oxide

[0454]

[0455] Step 1: 2-(((tert-Butyldimethylsilyl)oxy)methyl)-4-(1-ethoxyvinyl)-3-fluoropyridine

[0456] 2-(((tert-Butyldimethylsilyl)oxy)methyl)-3-fluoro-4-iodopyridine (0.80 g, 2.178 mmol, 1.00 equiv), tributyl(1-ethoxyvinyl)stannane (1.180 g, 3.267 mmol, 1.50 equiv) and Pd(PPh3)4 (0.252 g, 0.218 mmol, 0.10 equiv) were prepared in 1,4-dimethylsilylcarbonyl. The mixture was stirred at 100 °C in 2% ethanol (10 mL) and the mixture was stirred at 100 °C for 4 h. The reaction was diluted with water and extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate and concentrated to give 2-(((tert-butyldimethylsilyl)oxy)methyl)-4-(1-ethoxyvinyl)-3-fluoropyridine as a light yellow solid. LC / MS: C 16 H 26 The calculated mass value of FNO2Si is 311.2, and the measured value is 312.2 [M+H] + .

[0457] Step 2: 1-(3-Fluoro-2-(hydroxymethyl)pyridin-4-yl)ethan-1-one

[0458] To a solution of 2-(((tert-butyldimethylsilyl)oxy)methyl)-4-(1-ethoxyvinyl)-3-fluoropyridine (2.5 g, 8.027 mmol, 1 eq.) in THF (15 mL) was added 2M HCl (20 mL). The mixture was stirred at room temperature for 5 h, and then the pH was adjusted to 8-10 with NaHCO3 solution. The resulting mixture was extracted three times with EA. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo to give 1-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)ethan-1-one as a light yellow solid.

[0459] Step 3: 4-Acetyl-3-fluoro-2-(hydroxymethyl)pyridine 1-oxide

[0460] To a solution of 1-(3-fluoro-2-(hydroxymethyl)pyridin-4-yl)ethan-1-one (1.25 g, 7.390 mmol, 1 eq.) in DCM (20 mL) was added m-CPBA (2.550 g, 14.780 mmol, 2.0 eq.). The reaction mixture was stirred at room temperature for 2 h and then quenched with NaHCO3 solution. The resulting mixture was extracted three times with DCM. The combined organic layers were dried over Na2SO4 and concentrated in vacuo. The residue was applied to a silica gel column with EA / PE (0-100%) to give 4-acetyl-3-fluoro-2-(hydroxymethyl)pyridine 1-oxide as a white solid. LC / MS: The mass calculated for C8H8FNO3 was 185.05 and the found value was 186.05 [M+H] +

[0461] Step 4: 2-(Acetoxymethyl)-4-(2-bromoacetyl)-3-fluoropyridine 1-oxide

[0462] To a solution of 4-acetyl-3-fluoro-2-(hydroxymethyl)pyridine 1-oxide (0.9 g, 4.861 mmol, 1 eq) in acetic acid (20 mL) was added a solution of hydrogen bromide in acetic acid (2.384 g, 9.722 mmol, 2 eq) followed by the slow addition of pyridinium tribromide (1.477 g, 4.618 mmol, 0.95 equiv). The reaction mixture was stirred at room temperature for 2 h and then concentrated in vacuo. The residue was mixed with water and EA. The organic phase was collected, dried over Na2SO4 and concentrated in vacuo to give 2-(acetoxymethyl)-4-(2-bromoacetyl)-3-fluoropyridine 1-oxide as a light yellow oil. LC / MS: C 10 The calculated mass value of H9BrFNO4 is 304.97, and the measured value is 306.00 [M+H] + ,308.00[M+2+H] + .

[0463] Step 5: 2-(Acetoxymethyl)-4-(2-(((3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carbonyl)oxy)acetyl)-3-fluoropyridine 1-oxide

[0464] To a solution of (3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carboxylic acid (0.01 g, 0.026 mmol, 1.0 equiv) in MeCN (1 mL) was added potassium carbonate (0.007 g, 0.053 mmol, 2.0 equiv). After the mixture was stirred for 30 min, 2-(acetoxymethyl)-4-(2-bromoacetyl)-3-fluoropyridine 1-oxide (0.012 g, 0.040 mmol, 1.5 equiv) was added. The reaction mixture was stirred at room temperature for 2 h. LCMS showed that 2-(acetoxymethyl)-4-(2-(((3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carbonyl)oxy)acetyl)-3-fluoropyridine 1-oxide was produced, and the reaction mixture was used in the next step without further isolation or purification. LC / MS: C 26 H 21 The calculated mass value of ClF2N6O7 is 602.11, and the measured value is 603.15 [M+H] + .

[0465] Step 6: 2-(Acetoxymethyl)-4-(2-((3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizin-3-yl)-1H-imidazol-5-yl)-3-fluoropyridine 1-oxide

[0466] To a solution of 2-(acetoxymethyl)-4-(2-(((3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizine-3-carbonyl)oxy)acetyl)-3-fluoropyridine 1-oxide (0.34 g, 0.564 mmol, 1.0 equiv) in toluene (15 mL) under N2 was added ammonium acetate (0.870 g, 11.290 mmol, 20 equiv) followed by acetic acid (0.5 mL). The reaction mixture was stirred at 100 °C for 1 h, then cooled to room temperature and concentrated in vacuo. The residue was purified by silica gel chromatography with MeOH / DCM (0-15%) to give 2-(acetoxymethyl)-4-(2-((3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizin-3-yl)-1H-imidazol-5-yl)-3-fluoropyridine 1-oxide as a light yellow oil. LC / MS: C 26 H 21The calculated mass value of ClF2N8O4 is 582.13, and the measured value is 583.10 [M+H] +

[0467] Step 7: 4-(2-((3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizin-3-yl)-1H-imidazol-5-yl)-3-fluoro-2-(hydroxymethyl)pyridine 1-oxide

[0468] To a solution of 2-(acetoxymethyl)-4-(2-((3S)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindolizin-3-yl)-1H-imidazol-5-yl)-3-fluoropyridine 1-oxide (0.16 g, 0.274 mmol, 1 eq) in acetonitrile (10 mL) was added LiBr (0.119 g, 1.372 mmol, 5 eq), triethylamine (0.278 g, 2.745 mmol, 10 eq) and water (0.25 mL). The reaction mixture was stirred at 60 °C for 5 h and then concentrated in vacuo. The residue was purified by (column: YMC-Actus Triart C18 ExRS, 30 mm×150 mm, 5 μm; mobile phase A: water (10 mmol NH4HCO3), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 20B to 30B in 12 min) to give 4-(2-((3S,8aR)-7-(3-chloro-2-fluoro-6-(1H-tetrazol-1-yl)phenyl)-5-oxo-1,2,3,5,8,8a-hexahydroindoleazine-3-yl)-1H-imidazol-5-yl)-3-fluoro-2-(hydroxymethyl)pyridine 1-oxide as a white solid.

[0469] LC / MS: C 30 H 19 The calculated mass value of ClF4N6O4 is 540.12, and the measured value is 541.15 [M+H] + . 1H NMR (400MHz, methanol-d4)d 9.57(s,1H),8.29(d,J=6.9Hz,1H),8.10(t,J=7.9Hz,1H),7.81(dd,J=8.7, 7.6Hz,1H),7.60(d,J=4.1Hz,1H),7.55(dd,J=8.7,1.6Hz,1H),5.76(d,J=2 .7Hz,1H),5.16(d,J=8.6Hz,1H),4.94(d,J=3.2Hz,2H),3.88-4.01(m,1H), 2.72-2.89(m,1H),2.56-2.69(m,1H),2.14-2.39(m,3H),1.97-2.12(m,1H); 19 F NMR (376 MHz, methanol-d4) d -113.62, -124.02.

[0470] Those skilled in the art will recognize that additional stereoisomers (e.g., enantiomers, diastereomers, etc.) of compounds of Formula (I), (II), (III), (IV), (V), and (VI) may be prepared as described herein by selecting and appropriately substituting enantiomerically and / or diastereomerically enriched (e.g., ≥90% ee, preferably ≥98% ee) reagents or starting materials, and / or by preparing the corresponding racemic compounds followed by chiral separation by, for example, preparative chiral supercritical fluid chromatography (SFC) using isocratic process conditions and stacked injections for optimal collection efficiency, by crystallization of salts using chiral resolving agents, kinetic resolution, or other known methods.

[0471] Biological Examples

[0472] Biological Example 1: Factor XIa Inhibition Assay

[0473] Using fluorophore-quencher pairs for peptide substrates

[0474] The assay based on fluorescence intensity is used to monitor the inhibition of factor XIa. The peptide substrate 5Fam-KLTRAETV-K5Tamra (purchased from New England Peptide) was selected based on the FXI sequence. The conversion of the zymogen FIX to its activated form FIXa occurs by the proteolytic cleavage of FXIa at two sites Arg146 and Arg180. The custom peptide used in this assay is based on the Arg146 cleavage site. The peptide substrate is designed with a fluorophore-quencher pair, wherein the fluorescence is quenched until FXIa cleaves the 8-mer peptide after the Arg residue. The substrate K M Fitting to the substrate inhibition model, where k cat =0.86s -1, K M =12.4μM, K i = 61.6 μM, with enzyme efficiency, and k cat / K M =69523M -1 s -1 .

[0475] The Factor XIa FLINT assay is used with the following 5Fam-KLTRAETV-K5Tamra assay buffer: 50mM Tris, pH 7.5, 100mM NaCl, 5mM CaCl2, 0.1mg / mL BSA, 0.03% CHAPS is used. The assay buffer is prepared by freshly mixing all ingredients. First, 2X 5Fam-KLTRAETV-K5Tamra peptide substrate is prepared in 100% DMSO, and then 10mM is diluted to 3mM in 100% DMSO. The assay buffer is then added directly to the 3mM substrate stock solution to prepare a 30μM 2X working concentration (15μM final concentration). For a 200pM working stock solution (100pM final concentration), a 2X Factor XIa stock solution is prepared by diluting a 6.562μM stock solution in 1X assay buffer.

[0476] Test compounds were run in 11 point, 3-fold serial dilutions with a final top compound concentration of 100 nM. The final DMSO in the assay was 2% DMSO. FXIa and compound were pre-incubated for 30 minutes before substrate was added to initiate the reaction. The assay was linear over 30 minutes using 100 pM FXIa. More specifically, the assay was run as follows:

[0477] Dispense 100 nL of 1 mM test compound into a black 384-well non-binding Greiner BioOne 784900 plate to give a 10 μM final concentration and 1% DMSO final concentration;

[0478] Dispense 5 μL of 1X Assay Buffer into column 24 (low control) and 5 μL of 2X Factor XIa solution into columns 1-23 (column 23 high control);

[0479] Centrifuge the plate at 500 rpm for 1 min.

[0480] Pre-incubate the plate at room temperature for 30 minutes and cover the plate;

[0481] Dispense 5 μL of 2X 5Fam-KLTRAETV-K5Tamra peptide substrate across the plate (columns 1-24);

[0482] · Centrifuge the plate with the cover plate at 500 rpm for 1 min;

[0483] • Plates were read using a fluorescence module F1 485mm / 520mm with dynamic monitoring of fluorescence intensity at room temperature on a BMG PHERAStar.

[0484] Each compound tested generated a percentage of inhibition (IC 50 ) curves, and the data were analyzed using GeneData Screener using a 4-parameter logistic fit. Relative fluorescence unit (RFU) values ​​were normalized to percent inhibition using the following equation:

[0485] Inhibition % = ((HC-LC) - (Compound-LC) / (HC-LC)) * 100

[0486] Where LC-Low Control = Average signal of no Factor XIa or 100% inhibition of Factor XIa; HC-High Control = Average signal of Factor XIa + 5Fam-KLTRAETV-K5Tamra peptide substrate containing DMSO only.

[0487] An 11-point dose-response curve for the test compound was generated using GENDATA to determine the IC based on the following equation 50 value:

[0488] Y=Bottom+(Top-Bottom) / (1+10^((logIC 50 -X)*HillSlope))

[0489] Where Y is the % inhibition in the presence of X inhibitor concentration, Top = high control = average signal of Factor XIa + 5Fam-KLTRAETV-K5Tamra peptide substrate containing only DMSO; Bottom = low control - average signal of no Factor XIa or 100% inhibition of Factor XIa; Hillslope-Hill coefficient; and IC50 = the concentration of compound that has 50% inhibition relative to the top / high control.

[0490] Biological Example 2: Kallikrein Inhibition Assay

[0491] Utilizing quenched AMC peptide substrate

[0492] A fluorescence intensity-based assay was used to monitor inhibition of human plasma kallikrein. The peptide substrate, Z-Gly-Pro-Arg-AMC (purchased from Bachem; catalog #I-1150) was used based on its relatively low kallikrein K M This enables the assay to be run at lower substrate concentrations to control background fluorescence. The titration data were fitted to generate K M =40μM, k cat =0.76s-1 and k cat / K M =18932M -1 s -1 The Michaelis-Menten equation was used to determine the kinetic parameters of the substrate.

[0493] The Kallikrein FLINT assay was used with the following Z-Gly-Pro-Arg-AMC assay buffer: 50 mM Tris, pH 7.5, 100 mM NaCl, 5 mM CaCl2, 0.1 mg / mL BSA, 0.03% CHAPS. The assay buffer was prepared by freshly mixing all ingredients. The 2X Z-Gly-Pro-Arg-AMC peptide substrate was prepared by diluting a 10 mM stock solution into 1X assay buffer to achieve a 100 μM working concentration (50 μM final concentration). For a 4 nM working stock solution (2 nM final concentration), a 2X Kallikrein stock solution was prepared by diluting a 14.76 μM stock solution in 1X assay buffer.

[0494] Test compounds were run in 11-point, 3-fold serial dilutions with a final top compound concentration of 1 μM. Final DMSO in assay: 2% DMSO. 2 nM Kallikrein and test compounds were pre-incubated for 30 minutes before the addition of 50 μM substrate to initiate the reaction. The assay was linear over 30 minutes using 2 nM Kallikrein. More specifically, the assay was run as follows:

[0495] Dispense 100 nL of 1 mM test compound into a black 384-well non-binding Greiner BioOne 784900 plate to give a 10 μM final concentration and 1% DMSO final concentration;

[0496] Dispense 5 μL of 1X Assay Buffer into column 24 (low control) and 5 μL of 2X Human Kallikrein Solution into columns 1-23 (column 23 high control);

[0497] Centrifuge the plate at 500 rpm for 1 min.

[0498] Pre-incubate the plate at room temperature for 30 minutes and cover the plate;

[0499] Dispense 5 μL of 2X Z-Gly-Pro-Arg-AMC peptide substrate across the plate (columns 1-24);

[0500] · Centrifuge the plate with the “cover” plate at 500 rpm for 1 min;

[0501] • Plates were read using a fluorescence module F1 340mm / 440mm with dynamic monitoring of fluorescence intensity at room temperature on a BMG PHERAStar.

[0502] Each compound tested generated a percentage of inhibition (IC 50 ) curves, and the data were analyzed using GeneData Screener using a 4-parameter logistic fit. Relative fluorescence unit (RFU) values ​​were normalized to percent inhibition using the following equation:

[0503] Inhibition % = ((HC-LC) - (Compound-LC) / (HC-LC)) * 100

[0504] Where LC-low control = average signal of human kallikrein or 100% inhibition of human kallikrein; HC-high control = average signal of human kallikrein + Z-Gly-Pro-Arg-AMC peptide substrate containing DMSO only.

[0505] An 11-point dose-response curve for the test compound was generated using GENDATA to determine the IC based on the following equation 50 value:

[0506] Y=Bottom+(Top-Bottom) / (1+10^((logIC 50 -X)*HillSlope))

[0507] where Y is the % inhibition in the presence of X inhibitor concentration, Top = average signal of high control = human kallikrein + Z-Gly-Pro-Arg-AMC peptide substrate containing DMSO alone; Bottom = average signal of low control – no human kallikrein or 100% inhibition of human kallikrein; Hillslope – Hill coefficient; and IC 50 = concentration of compound that gives 50% inhibition relative to top / high control.

[0508] Representative compounds of the present invention were tested according to the procedures described in Biological Example 1 and Biological Example 2 above, and the results are listed in Table B-1 below.

[0509] Table B-1: FXIa and plasma kallikrein biological activities

[0510] Determination Compound formula result <![CDATA[Factor XIa IC 50 > (IA) 1.8nM <![CDATA[Factor XIa IC 50 > (IB) 2.9nM <![CDATA[Factor XIa IC 50 > (IC) 2.2nM <![CDATA[Factor XIa IC 50 > (ID) 5.7nM <![CDATA[Factor XIa IC 50 > (IE) 22.5nM <![CDATA[Factor XIa IC 50 > (IF) 5.1nM <![CDATA[Factor XIa IC 50 > (IG) 3.9nM <![CDATA[Factor XIa IC 50 > (IH) 3.2nM <![CDATA[Factor XIa IC 50 > (IJ) 4.1nM <![CDATA[Factor XIa IC 50 > (IK) 5.0nM <![CDATA[Factor XIa IC 50 > (II-A) 0.3nM <![CDATA[Factor XIa IC 50 > (III-A) 45.4nM Determination Compound formula result <![CDATA[Plasma kallikrein (PK) IC 50 > (IA) 13nM <![CDATA[Plasma kallikrein (PK) IC 50 > (IB) 25nM <![CDATA[Plasma kallikrein (PK) IC 50 > (IC) 17nM <![CDATA[Plasma kallikrein (PK) IC 50 > (ID) 43nM <![CDATA[Plasma kallikrein (PK) IC 50 > (IE) 72nM <![CDATA[Plasma kallikrein (PK) IC 50 > (IF) 34nM <![CDATA[Plasma kallikrein (PK) IC 50 > (IG) 33nM <![CDATA[Plasma kallikrein (PK) IC 50 > (IH) 27nM <![CDATA[Plasma kallikrein (PK) IC 50 > (IJ) 32nM <![CDATA[Plasma kallikrein (PK) IC 50 > (IK) 9.7nM <![CDATA[Plasma kallikrein (PK) IC 50 > (II-A) 11nM <![CDATA[Plasma kallikrein (PK) IC 50 > (III-A) 420nM

[0511] Biological Example 3

[0512] Study on Reactive Metabolites in Human Liver Microsomes

[0513] The bioactivation of the compound of formula (IA) in HLM was evaluated using GSH and KCN as trapping agents. The compound was incubated at 37°C for 60 min in human liver microsomes supplemented with NADPH, KCN and GSH. Incubation was performed with a total volume of 1 mL with 1 mg / mL protein, 10 μM substrate, 1 mM GSH (unlabeled and labeled with 3:1) and 1 mM KCN ​​(unlabeled and labeled with 3:1). Nefazodone (10 μM) was incubated under the same conditions as the positive control. After incubation, protein precipitation was performed by mixing with 5X volume of acetonitrile to extract metabolites. Protein was precipitated by centrifugation (3000 rpm, 10 minutes, 4°C), and the supernatant was transferred to a clean test tube and evaporated to dryness under a gentle stream of nitrogen. The resulting residue was reconstituted in 300 μL of a solvent mixture of water:acetonitrile (8:2 v / v) containing 0.1% formic acid and then filtered using a 0.45 μm nylon filter at 10,000×g for 3 minutes at room temperature. The filtrate was transferred to a 96-well plate for liquid chromatography / tandem mass spectrometry (LC-MS / MS) analysis.

[0514] LC-MS / MS analysis: Agilent 1260 Infinity UHPLC was connected to a Bruker Q-TOF. The mass spectrometer was operated in positive electrospray ionization mode. The column was Phenomenex, Luna C8 (150×2.0 mm ID, 3 μm). The mobile phase was A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in acetonitrile. A linear gradient with a flow rate of 0.2 mL / min was used throughout the analysis as follows:

[0515] Time (min) Acetonitrile% 0 5 5 5 20 95 24 95 24.5 5 30 5

[0516] The compound of formula (IA) was tested for reactive metabolites according to the HLM assay procedure described above and did not form GSH adducts or KCN adducts.

[0517] Biological Example 4

[0518] In vitro metabolism of cryopreserved human hepatocytes

[0519] Human hepatocytes used for in vitro metabolism of compounds of formula (IA) were purchased from Bioreclamation IVT (Westbury, NY); cryopreserved male human hepatocytes (lot number: UYG).

[0520] Hepatocyte incubation

[0521] The cryopreserved hepatocytes were rapidly thawed on ice and the vials were placed in a 37°C water bath for approximately 90 seconds and then immediately added to pre-warmed hepatocyte thawing medium. The cells were resuspended in the medium and then centrifuged at 700×g for 7 minutes at room temperature. The supernatant was discarded and the cells were resuspended in 2 mL of Krebs-Henseleit buffer containing 12.5 mM HEPES pH 7.4 (KHB). Cell viability and yield were determined by trypan blue exclusion. The hepatocytes were then diluted to 2×10 in KHB. 6 The cells were then added to a concentration of 10 cells / mL (500 μL / well in a 10 mL glass tube). The tubes were placed on a rotary shaker in a humidified incubator supplied with 5% CO2 and allowed to acclimate at 37°C for 15 minutes before use. The compound of formula (IA) or the positive control diclofenac (10 μM) in KHB was then added to the hepatocytes to achieve a final concentration of 10 μM and 1×10 6 The cells were incubated at a cell concentration of 10 cells / mL (1000 μL final culture volume). The cells were incubated for zero or 3 hours. At the end of the incubation period, the cells were quenched with 6 volumes of ice-cold acetonitrile containing 0.02% formic acid, vortexed and sonicated (5 min). The precipitated protein was precipitated by centrifugation (3000 rpm, 10 minutes, 4°C), and the supernatant was transferred to a borosilicate tube and evaporated to dryness under a gentle stream of nitrogen.

[0522] Sample preparation

[0523] The residue obtained after evaporation was reconstituted in 250 μL of water:acetonitrile (9:1 v / v) containing 0.01% formic acid, sonicated and vortexed to help dissolve the drug derivative material, and then filtered at 10,000×g for 10 minutes at room temperature using a 0.45 μ nylon filter. The filtrate was transferred to a 96-well plate for liquid chromatography / ultraviolet / mass spectrometry (LC-UV / MS) analysis.

[0524] LC / UV / MS (Liquid Chromatography / Ultraviolet / Mass Spectrometry)

[0525] LTQ / Orbitrap (Thermo Scientific, Inc., Bremen, Germany) was used in single ion lock mass mode at a resolution of 15,000 for full scan MS and 10,000 for MS 2(also known as MS / MS or tandem mass spectrometry) was performed with data-dependent accurate mass measurements at a resolution of 7,500. Chromatographic separation of unchanged drug and metabolites was achieved using a Kinetex phenyl-hexyl column (150×2.1 mm ID, 1.7 μm) from Phenomenex, Inc. (Torrance, CA, US), maintained at 35° C. and eluted with a nonlinear gradient consisting of water (solvent A) and acetonitrile containing 0.1% formic acid (solvent B).

[0526] Quantification of unchanged drug and its metabolites was performed by integrating the individual chromatographically separated UV peaks and providing percentages of the total peak area.

[0527] result :

[0528] The compound of formula (IA) was tested according to the in vitro metabolite assay procedure as described above. The compound of formula (IA) exhibited low metabolic turnover in human hepatocytes, with three oxidative metabolites and two glucuronide metabolites produced from the incubation. Diclofenac was used as a positive control for the study, and the metabolic turnover of diclofenac from human hepatocytes was 89%, indicating that the incubation system was effective. The (3-fluoropyridin-2-yl)methanol moiety was the major metabolic soft spot for the compound of formula (IA). The carboxylic acid metabolite, compound of formula (II-A)

[0529]

[0530] It is a metabolite in human hepatocytes.

[0531] Biological Example 5: PK Study

[0532] Animals: Male Sprague-Dawley rats (7-8 weeks old, weighing 250 g-300 g) were placed in plastic cages with free access to standard rat diet (Keaoxieli, Beijing, China) and water. The animals were maintained at a temperature of 20-25°C, a 12-hour light / dark cycle, and a relative humidity of 40%-70% before the experiment.

[0533] Pharmacokinetics in rats: The rats were pre-treated by jugular vein cannulation 2-3 days before the study. The test compound (dissolved in PEG400 / water (1:1) or suspended in 0.5% HPMC) was orally administered to 3 rats via oral gavage at a dose of 5 mg / kg or 10 mg / kg, and the test compound (dissolved in 20% HPbCD or PEG400 / water (1:1)) was injected into the tail vein of 3 rats at a dose of 1 mg / kg or 2 mg / kg.

[0534] Blood samples (approximately 0.2 ml each) were collected from the jugular vein at 0 minutes (as pre-dose), 5 minutes (only for IV administration), 15 minutes, 30 minutes, 60 minutes, 120 minutes, 240 minutes, 360 minutes, 480 minutes and 1440 minutes after administration. Immediately after obtaining each blood sample, a 0.2 ml volume of heparinized 0.9% NaCl injectable solution (50 U / mL) was used to rinse each cannula to prevent blood clotting and compensate for fluid loss. The blood samples were separated by centrifugation, and approximately 0.1 ml plasma aliquots were stored at -80°C until analysis.

[0535] LC-MS / MS Analysis: Analytes were determined by liquid chromatography coupled to tandem mass spectrometry (LC-MS / MS) method. The LC-MS / MS system consisted of a Shimadzu HPLC system (Kyoto, Japan) and an API5500 triple quadrupole mass spectrometer (AB Sciex, Toronto, Canada) equipped with a turbo electrospray ionization (ESI) source. The LC conditions were as follows: column, Synergi 2.5μ Polar-RP 100A (50*3.0 mm) (Phenomenex, Torrance, CA); gradient elution with 5% acetonitrile in water containing 0.1% formic acid or 95% acetonitrile in water containing 0.1% formic acid at 0.5 mL / min; and injection volume, 10 μL. Multiple reaction monitoring (MRM) method was selected for quantitative analysis. The optimized transitions for analytes and internal standards were Q1→Q3 and M1→M3, respectively. The instrument parameters were set as follows: ion spray voltage: 5500 V; curtain gas: 30 psi; nebulizer gas: 50 psi; vortex gas: 50 psi; collision gas: 10 psi; temperature: 450°C.

[0536] Data Analysis: Pharmacokinetic parameters such as the total area under the plasma concentration-time curve from time 0 to infinity (AUC 0–t ) and terminal half-life were determined directly from experimental data using non-compartmental analysis (WinNonlin). max ) and reach C max Time (T max ).

[0537] Results: The rat PK of the compound of formula (IA) was measured according to the above assay procedure and the results are listed in Table B-2 below:

[0538] Table B-2: Rat PK of compounds of formula (IA)

[0539] IV clearance 19.6ml / min / kg <![CDATA[t 1 / 2 (IV)]]> 3.25h (preparation vehicle: 20% HPbCD, 2 mg / kg) <![CDATA[Oral C max > 3767ng / ml <![CDATA[t 1 / 2 (AFTER)]]> 2.66h F 53.4% ​​(formulation vehicle: 0.5% HPMC, 10 mg / kg)

[0540] Biological Example 6: hERG Assay

[0541] HERG (human ether-à-go-go related gene) encodes a protein with a function similar to the rapidly activating delayed rectifier K in cardiac myocytes. + Current (I Kr ) of the potassium channel. Kr The current contributes to the K phase responsible for the repolarization phase of the cardiac action potential. + Current. Blockage of this current can prolong the action potential duration and lead to long QT-syndrome. The development of QT prolongation can lead to the occurrence of ventricular arrhythmias such as torsades de pointes, which can lead to sudden death.

[0542] Experimental Procedure: The effects of test compounds on HERG currents were investigated at the single-cell level using the single-electrode whole-cell voltage clamp technique.

[0543] Cells: Human embryonic kidney cell line (HEK293) stably transfected with HERG was used. The cells were kept in culture continuously. Before use, the cells were seeded into small culture dishes coated with poly-L-lysine. The cells were tested 1-2 days after seeding.

[0544] Solutions: The electrolyte solution contained (in mM) 150 NaCl, 4 KCl, 5 glucose, 10 HEPES, 1.8 CaCl2, and 1 MgCl2 (pH 7.4 with NaOH). The pipette solution contained (in mM) 120 KCl, 5 EGTA, 10 HEPES, 4 MgATP, 0.5 CaCl2, and 2 MgCl2 (pH 7.2 with KOH). The test compounds were dissolved in DMSO to give 10 -2 M or 10 -1 Stock solutions of M. Control (= electrolyte + DMSO) and test solutions (= electrolyte + DMSO + test compound) contained 0.3% or 0.03% DMSO. Solutions were applied to the cells under study using a Y-tube system, allowing rapid changes of solution near the cells (less than 0.5 s).

[0545] Electrophysiological measurements: The membrane current of cells was measured at different membrane potentials using the patch clamp technique using an EPC-9 patch clamp amplifier. The holding potential was -80 mV. HERG current (K +-Selective outward current) is determined as the maximum tail current at -40mV after 2 seconds depolarization to +60mV. The pulse cycle rate is 15s. Before each test pulse, a short pulse (0.5s) from the holding potential to -60mV is given to determine the (linear) leakage current. After setting up the full-cell configuration, a 5-minute equilibrium period allows the cell to be perfused internally with a pipette solution. Thereafter, a test pulse is given for 5 minutes to quantify the HERG current under control conditions. While continuing the pulse scheme, perfusion is switched from the control solution to a solution containing the test compound. The effect of the test compound is measured after 5 minutes of drug administration. Each cell tests one to three concentrations of the test compound.

[0546] Measurements: HERG current was determined as the maximum tail current at -40 mV following a 2 sec depolarization to +60 mV from a holding potential of -80 mV.

[0547] Even under control conditions, the amplitude of HERG current also gradually decreases over time. In order to accurately quantify the blocking degree of test compounds, the continuous reduction of HERG current must be considered. Therefore, the time course of HERG current is exponentially fitted to the initial period of 5 minutes in the control solution, and extrapolated to the remainder of the experiment. If test compounds are not provided, these extrapolations provide estimated current amplitudes. In order to determine the blocking degree of test compounds, the ratio between the measured current and the fitted current in the control solution extrapolated to the same time point is calculated.

[0548] Standard conditions: Test compounds were dissolved in DMSO (final DMSO concentration: 0.3% or 0.03%). The effects of test compounds on HERG currents were measured 5 minutes after drug application.

[0549] All or None Criteria: If a decrease of more than 5% in the HERG current could be observed, the R-compound was considered to (partially) block the HERG current.

[0550] Results: Compounds of formula (IA) were tested in the hERG assay according to the above procedure and measured to show an IC of >30 μM 50 .

[0551] Biological Example 7: AMES II Assay

[0552] The purpose of this study was to evaluate the ability of compounds and / or their metabolites to induce reversion mutations in genes of histidine-requiring strains of Salmonella typhimurium in the absence and presence of a mammalian metabolic activation system to generate histidine-independent strains of these bacteria. The Ames reversion mutation test has been shown to be a rapid and sensitive indicator of mutagenic activity for a wide range of chemical classes.

[0553] The mutagenic potential was assessed by using an in-house adaptation of the Ames II mutagenicity assay kit from Xenometrics (Boulder, CA, USA).The strains TA98 and pooled TA (a combination of 6 strains) used in this study enable the detection of frameshift mutations and base pair substitutions, respectively.

[0554] Experimental Procedures: Test compounds were dissolved in DMSO and handled at all times in a manner that reduced the risk of any accidental exposure to personnel and the environment.

[0555] In the in vitro gene reverse mutation test, the effect is determined by exposing the mutant strain of Salmonella typhimurium to different concentrations of test compounds. Generally, Salmonella typhimurium is able to synthesize the histidine required for its growth, but the mutant strain used in this test cannot play this function. When a large number of these bacteria are exposed to mutagens, a reverse mutation to the original histidine-independent form occurs in a certain proportion of the bacterial population. These colonies (revertants) are easy to detect due to their ability to grow on histidine-deficient medium. Since many compounds do not play their mutagenic effects until they are metabolized by unavailable enzyme systems in bacterial cells, the test compounds and bacteria are incubated in the presence of supplemented liver parts taken from animals treated with compounds known to induce higher levels of enzyme activity in advance. The bacterial reverse mutation test uses a strain of Salmonella typhimurium that requires amino acids to detect point mutations, which involve substitutions, additions or deletions of one or more DNA base pairs. The principle of this bacterial reverse mutation test is that it detects mutations, which restore the mutations present in the test strain and restore the functional ability of bacteria to synthesize essential amino acids. Revertant bacteria are detected by their ability to grow in the absence of the amino acid required by the parent test strain. Bacterial reverse mutation tests have been shown to be sensitive, fast and accurate indicators of mutagenic activity of multiple chemical categories. In this study, Salmonella typhimurium strain TA98 and mixed TA were used. They are histidine-dependent (his-) and are reverted to histidine prototrophy (his+) by mutation. These strains have additional mutations that increase their sensitivity to mutagens. In bacteria, the major damage caused by almost all mutagens to DNA is repaired by excision and repair systems, so only a small portion of potential mutagenic damage is expressed. The uvrB mutation is a deletion in the uvrB gene, which causes a defective DNA excision repair system, making the strain more sensitive to mutagens. The uvrB deletion extends through the bio gene, so that bacteria also need biotin to grow. The rfa mutation (depth roughness) affects the cell membrane, making the normal lipopolysaccharide (LPS) cell wall defective. The rfa mutation increases the permeability of the cell wall to macromolecules (such as those containing macrocyclic systems), otherwise these macromolecules will be excluded by the normal cell wall. Strains containing plasmid pKM101 are reverted by many mutagens that are weakly or not detected at all by the parent strain carrying the non-plasmid. The pKM101 plasmid increases chemical and spontaneous mutagenesis by enhancing the error-prone DNA repair system.

[0556] The genotypes of S. typhimurium used in this assay are listed below in Table B-3.

[0557] Table B-3: Genotypes of Salmonella Typhimurium strains

[0558]

[0559] Test strains were prepared from fresh overnight Oxoid nutrient broth cultures using frozen strains stored below -70°C. Bacterial cultures for the studies were obtained by inoculating Oxoid nutrient broth with frozen cultures and incubating overnight at 37°C.

[0560] The in vitro metabolic activation system consists of rat liver enzymes (S9-fraction) and an energy production system (core) containing nicotinamide adenine dinucleotide phosphate (NADP) and glucose-6-phosphate (G-6-P). Briefly, the enzymes are contained in 9000×g supernatant from liver homogenates prepared from adult male Wistar rats treated with 500 mg / kg of Aroclor 1254 five days before sacrifice. Treatment with Aroclor 1254 is used to induce mixed function oxidases capable of metabolizing chemicals into more active forms. The S9 fraction is kept frozen at about -70°C until use. The S9 fraction is thawed immediately before use and combined with the core to form the activation system (S9-mix) described in Table B-4 below.

[0561] Table B-4: AMES activation system (S9-mix)

[0562] Components Final concentration NADP 4mM G-6-P 5mM Mg 8mM KCl 33mM Phosphate buffer, pH 7.4 0.1M S9 30％

[0563] Standard conditions: Test compound concentrations and S9-mix were prepared just before use. In the presence of bacteria, test compounds, negative controls and positive controls at all concentration levels were inoculated in triplicate in 24-well plates. After a 90-min incubation (37°C) step (orbital shaker, 250 rpm), 2.75 ml of indicator culture medium was added to each well, followed by a 50 μl volume transfer to a 384-well plate according to a fixed pattern. The 384-well plate was incubated at 37°C for 48 h in the dark. After incubation, the plate was analyzed by scoring the number of positive wells (visible yellow or bacterial colonies at the bottom of the well). Information on possible precipitation of the compound in the well was also recorded. Individual plate counts were directly entered into a computer through bacterial strains and metabolic activation systems, which allowed each plate count to be correctly presented relative to the plate count of the control or compound.

[0564] All or None Criteria: The number of yellow wells on the solvent control plate is taken as the mutation background level for the test. Reversion values ​​are considered positive if the following conditions are met: (a) the average number of revertants produced by the test compound at one or more concentration levels with one strain (TA98 or mixed) increases by at least three-fold compared to the simultaneous solvent control. Due to the nature of the strain, low spontaneous mutation rates can sometimes result in extremely low numbers of revertant wells in the solvent control. In this case, historical background levels must be considered; and (b) a dose-effect relationship is observed.

[0565] These criteria apply in most cases. If equivocal results are obtained, more testing is necessary to assess the mutagenic potential of the test compound. If a test compound produces a positive response in a single test that cannot be reproduced in additional runs, the initial positive test data are discounted.

[0566] Control experiments: Before a test is considered valid, a positive control is tested and shows at least a three-fold increase in the number of revertant wells over the solvent control value for both the mixed strain and TA98.

[0567] PIR-Observations: Recorded as follows:

[0568] PIR1 = No increase in revertant colonies

[0569] PIR2 = equivocal increase (no clear dose response or reproducibility)

[0570] PIR3 = clear dose-related and reproducible increase in revertant colonies.

[0571] Reference compounds: The positive control preparations were 2-aminoanthracene for metabolic activation experiments and 2-nitrofluorene and 4-nitroquinoline-N-oxide for experiments without metabolic activation, as listed in Table B-5 below.

[0572] Table B-5: AMES Reference Compounds

[0573] Compound CAS Number Solvents Final concentration S9 strain 2-Aminoanthracene 613-13-8 DMSO 5.0μg / ml + All strains 2-Nitrofluorene 607-57-8 DMSO 1.0μg / ml - TA98 4-Nitroquinoline-N-oxide 56-57-5 DMSO 1.0μg / ml - Mixed TA

[0574] The control product was handled at all times in a manner that reduced the risk of any accidental exposure to personnel and the environment. A positive control product solution was prepared immediately prior to use and a final 2% DMSO solvent solution would be present in each culture. The stability of the positive control product in solution was unknown, but an increase in the number of revertant colonies within the expected range demonstrated biological stability.

[0575] Results: The compound of formula (IA) was tested according to the above procedure and was determined to be negative in the AMES II assay.

[0576] Biological Example 8: In vitro micronucleus assay

[0577] The in vitro micronucleus test (MNT) is a genotoxicity test for detecting micronuclei (MN) in the cytoplasm of interphase cells. Micronuclei can be derived from a centromere chromosome fragment (i.e., lacking a centromere) or an entire chromosome that cannot migrate to the poles in the late stage of cell division. The ability of compounds and / or their metabolites to induce chromosome / genomic aberrations in the TK6 human lymphoblastoid cell line is evaluated in the absence and presence of a mammalian metabolic activation system. Human TK6 cells are often used for in vitro micronucleus testing, and it is worth noting that these cells have a wild-type p53 state, a feature speculated by some groups that may help reduce the frequency of false positive results. It is evaluated by manual and / or semi-automated microscopic analysis. It has been shown that in vitro MNT is a sensitive test for evaluating the genotoxic potential of chemical and physical agents in primary cells and cell lines. MNs have the same morphology as interphase nuclei, except that they are smaller than the main nucleus in the cell, so they are called "micronuclei".

[0578] The in vitro micronucleus test using TK6 cells is a recognized regulatory genotoxicity test. Studies involving the micronucleus assay in TK6 were designed to meet the requirements of current international guidelines published by the OECD 487 test guideline and ICH S2(R1).

[0579] Reference compounds: Positive control reagents are used simultaneously to ensure the responsiveness of the test system and to demonstrate the potential of the metabolic system to convert the promutagen into an active metabolite, but do not provide a standard for comparison with the test items. The positive control chemicals used for each treatment condition (treatment time and metabolic activation) in this test are shown in Table B-5 below. The solvent used for each positive control chemical and the final test concentration under each condition are listed in Table B-6 below.

[0580] Table B-6: Positive Control

[0581]

[0582]

[0583] A simultaneous negative (vehicle) control containing dimethyl sulfoxide (DMSO); CAS No. 67-68-5) (final DMSO concentration was 1%).

[0584] Principle of determination: Cell culture is exposed to test compounds with and without exogenous metabolic activation sources. Negative, solvent / vehicle and positive controls are included in all tests at the same time. During or after exposure to the test compound, the cells are allowed to grow enough to damage chromosomes or spindles to cause micronuclei to form in interphase cells. The presence of micronuclei in interphase cells harvested and stained is analyzed. It is important to prove that the analyzed cells may undergo cell division during or after exposure to the test compound. For all schemes, it is important to prove that cell proliferation occurs in control and treated cultures, and to assess the degree of cytotoxicity and / or leukocytosis induced by the test compound in cultures scored for micronuclei. The total duration allows cells to complete at least one division, and therefore the induced lesions are expressed as micronuclei (i.e., 1-5-2.0 cell doublings should occur).

[0585] The in vitro micronucleus test was performed using two treatment regimens: three hours in the presence of a rat liver S9-based metabolic activation system (S9 mix) and approximately 24 hours in the absence of S9 mix. A second test was performed as needed to confirm the results of the first.

[0586] Equipment used in the assay: freezer (approximately -10°C to -30°C); freezer (approximately -80°C); refrigerator (approximately 2°C to 8°C); centrifuge (capable of centrifuging 96-well plates); CO2-regulated 37°C incubator; Z1 Coulter counter; Shandon Cytospin 4Thermo; vortex mixer; ultrasonic bath (Grant Water Bath OLS 200); microscope (AxioImager 2 - Zeiss); and microscope (GXML3201 - GX microscope)

[0587] Test-specific materials used in the assay: Cell line TK6 (ATTC CRL-8015); R10P medium; containing RPMI1640+Glutamax TM –I(Gibco TM61870044) + horse inactivated serum (Gibco 16050-122) + penicillin / streptomycin (Gibco 15140-122) + sodium pyruvate (Gibco 11360-039) + pluronic acid (Gibco 24040-032) culture medium; centrifuge tubes (15ml / 50ml)-5mL round bottom tubes (Corning / falcon 352054); micropipette / multichannel cell culture flask T75 (Falcon, BD-Catalog No. 353136); 96-well plate U-bottom, sterile, polystyrene (Star Lab E2996-1700); airtight sealing film (Sigma Aldrich / Merck2380059); 96-well flat bottom plate, sterile with lid-96-well plate V-bottom, sterile, polystyrene (Thermo Fisher Scientific 249662);DMSO(Fisher BP231-100)

[0588] Methods and Procedures: Test compounds were dissolved in DMSO (dimethyl sulfoxide). Vortex mixing, sonication, and warming to 37 ± 2°C were used as needed to aid dissolution. Test compounds were stored at room temperature in the dark. After preparation in DMSO and use in initial experiments, the remaining prepared test compounds were stored in a ventilated storage cabinet with desiccant in the dark at room temperature.

[0589] Test compound formulations were prepared immediately prior to dosing according to the relevant Gentronix standard operating procedures. Briefly, a stock solution of the test compound in a solvent was prepared and then diluted in the same solvent to prepare the desired concentration. The dose used for any subsequent testing was based on the results in the initial testing.

[0590] The TK6 cell line was purchased from the European Collection of Certified Cell Cultures. It was then used to generate an internal master stock of TK6 cells, and from this a working stock was generated. All stocks were stored in liquid nitrogen. The cells were maintained in logarithmic phase and passaged every 1-4 days in RPMI1640 containing 10% heat-inactivated horse serum (Gibco, Life Technologies, UK), antibiotics and Pluronic F68.

[0591] TK6 culture wells were treated with 2.2 μl of test compound solution or positive control solution; negative control cultures were treated with the same volume of solvent. 218 μl of cells were added to the wells. The final total volume was 220 μl.

[0592] Determination procedure :

[0593] Day 1: TK6 cells were seeded at a density of approximately 200,000 cells / ml in T75 culture flasks (5% CO2 at 37°C).

[0594] Day 2: On the day of treatment, cultures were prepared at a density of 200,000 cells / ml in flat-bottom 96-well plates. Cells were treated with at least 24 concentrations of the test compound, repeated 3 times (final concentration of DMSO was 1%). Simultaneous negative, vehicle and positive controls were included. At least 2 possible treatment designs were performed. Cells were treated for 3 hours in the presence of the rat metabolic activation system, after which the cultures were centrifuged (in U-bottom 96-well plates), washed with culture medium, and then resuspended with fresh culture medium for a post-treatment recovery period (in U-bottom 96-well plates). Cells were counted with a Z1 Coulter counter. Continuous treatment (23 hours-28 hours) was performed without metabolic activation.

[0595] Mammalian liver mitochondrial post fraction (S9 homogenate) for metabolic activation was prepared from male Sprague Dawley rats induced with Aroclor1254 and purchased from Molecular Toxicology Inc. (MOLTOX, USA). The S9 batches were stored frozen (-80°C) and thawed just before use. The sterility, protein content, cytochrome P450-catalyzed enzyme activity, and the ability to convert known premutagens into bacterial mutagens were certified by the manufacturer for each batch. The S9 fraction requires exogenous cofactors for activation. The cofactors used consisted of an NADPH regeneration system purchased from Apollo. Cofactor reagents were prepared in R10P medium as follows: (a) 7.0 g / L of glucose-6-phosphate (molecular weight 260.14), the final solution was 25 mM; and (b) 3.8 g / L of NADP (molecular weight 743.41), the final solution was 5 mM.

[0596] Equal amounts of the two solutions were mixed and adjusted to pH 7.2 with 1 M aqueous sodium hydroxide solution. The solutions were filter sterilized and approximately 5.5 mL aliquots were dispensed into suitable containers. All solutions were stored at approximately -80°C and used within six months.

[0597] The S9 homogenate was adjusted to a protein content of 30 mg / mL and then added to the culture medium. The final concentration of liver homogenate (S9) in the test system was 2.0%:

[0598] Day 3: Cytotoxicity profile: After treatment, the number of viable cells was determined by counting cells in duplicate wells using a Z1 Coulter Counter to calculate the population doublings (PD) and relative population doublings (RPD):

[0599] PD=[log(Ni / N0)] / log 2

[0600] Where N0: baseline counts at time zero; Ni: cell concentration at a given time point (i, end of incubation). For treated cultures, PD is expressed as a percentage of the number of PD in the simultaneous vehicle control.

[0601] RPD = [number of PD in treated culture / number of PD in control culture] × 100

[0602] Based on the obtained cytotoxicity profile, the test compound concentration (each treatment group) was selected for MN analysis. The goal was to select at least one concentration that covered 55 ± 5% cytotoxicity. If no cytotoxicity or precipitation was observed, the highest test concentration was selected to correspond to 1 mM or 500 μg / ml, whichever was lower (ICH S2 (R1), 2011). When solubility is a limiting factor, the maximum concentration is the lowest concentration at which minimal precipitation is visible in the culture, provided that there is no interference with the scoring. The assessment of precipitation was performed by optical microscopy at the end of the treatment.

[0603] Microscopic analysis: After treatment, cells were centrifuged (cytospin) onto slides, fixed in methanol and stained with acridine orange (manual analysis) or DAPI (semi-automated analysis). For all selected test conditions (test compounds and positive controls), cell slides were made from separate cultures: 2 cultures were used for microscopic analysis.

[0604] Microscopic analysis (of blinded coded cell slides) was performed to determine the number of micronucleated cells (MNCs). The presence of micronuclei was assessed in at least 2000 mononuclear cells per test condition (at least 1000 cells per culture; 2 cultures per concentration). As a measure of genotoxicity, the number of MNCs was used to calculate the percentage of MNCs:

[0605] MNC% = [number of MNCs / number of monocytes analyzed] × 100

[0606] In addition, the fold increase in MNC in treated cultures compared to simultaneous vehicle controls was calculated:

[0607] Fold increase in MNC = % MNC in treated culture / % MNC in VC culture

[0608] For semi-automatic analysis, the images obtained were also manually evaluated (Metafer 4.0.8 and Neon 1.1.6) to exclude false positives. To confirm the results obtained, specific concentrations were verified by manual microscopic analysis. Manual microscopic analysis allowed quantification of additional cellular effects, such as the induction of binucleated (BN), multinucleated (POLY) and apoptotic (APO) cells; endpoints that are not usually quantified by semi-automatic analysis.

[0609] Microscope scoring criteria: An appropriate slide area (i.e., not too thin or densely spread) is selected by the analyst. Cells should not overlap, and the cytoplasm must be clearly visible around the nucleus. Suspected MNs are confirmed or rejected according to the following criteria: (a) the MN diameter is less than one-third of the diameter of the main nucleus and is located within the intact cytoplasm of the cell; (b) the MN stains evenly, the same color and in the same focal plane as the main nucleus; (c) the MN is non-refractive and spherical with well-defined outlines; and (d) the MN is not connected or connected to the main nucleus. Be careful not to score binucleated cells with irregular shapes or large differences in the size of the two nuclei; binucleated cells should not be confused with poorly diffused multinucleated cells. Multinucleated cells are analyzed for micronuclei.

[0610] Test Acceptance Criteria: Treatment conditions are considered acceptable for evaluation of genotoxic effects if the following conditions are met: (a) at least 1.5 cell population doublings occur in vehicle control cultures; (b) the frequency of MNCs in vehicle control cultures is close to the frequency within the acceptable range of values ​​from the historical control database and / or published values ​​from Gentronix; (c) the positive control compound induces a biologically significant increase in MNC frequency (statistically significant and outside the historical vehicle control range) above the concurrent vehicle control value; (d) the maximum concentration reaches (i) approximately 55 ± 5% of the cytotoxicity limit (RPD = 45 ± 5%), (ii) the solubility limit or (iii) 500 μg / ml or 1 mM, whichever is lower. Note that the test acceptance criteria are not absolute and other extenuating factors may enter into the final acceptability decision. Experiments are repeated independently as needed to meet the acceptance criteria.

[0611] Test Evaluation Criteria: If the test acceptance criteria are met, the results are evaluated using the following criteria based on the OECD 487 Test Guideline (2016):

[0612] Clearly positive (score 3): (a) At least one tested concentration induces a statistically significant (Fisher's exact test, p-value < 0.05) increase compared to the simultaneous vehicle control. (b) The increase in MNC is dose-related in at least one experimental condition when assessed by a trend test (Cochran Armitage test, p-value < 0.05). (c) Any statistically significant increase in MNC is outside the distribution of the laboratory's historical vehicle control data (95% confidence limits based on Poisson). Test compounds that meet all of the above criteria in at least one experimental condition (treatment group) are considered capable of inducing biologically relevant chromosome breakage and / or chromosome gains or losses in this system.

[0613] Clearly negative (score 1): (a) None of the tested concentrations induces a statistically significant increase compared to the simultaneous vehicle control (Fisher's exact test). (b) There is no concentration-related increase in MNC assessed by trend testing (p-value < 0.05). (c) All results are within the distribution of historical vehicle control data (95% control limits). Test compounds that meet all of the above criteria are considered incapable of inducing chromosome breakage and / or gain or loss in this test system.

[0614] Equivocal (score 2): Test compounds that meet only some of the above criteria for a clear positive or clear negative response are evaluated through expert judgment and / or further investigation (e.g., analysis of more cells from existing experiments or repeating the test with an adjusted concentration range). In rare cases, even after further investigation, the data set does not allow for a positive or negative conclusion, and the result is considered equivocal.

[0615] Uncertain (score 0): The test acceptance criteria are not met and further testing is required before final compound classification.

[0616] Note that clear positive or clear negative results are not required for verification. The aforementioned criteria are not absolute, and other extenuating factors may enter into the final evaluation decision. Trend tests were considered valid only for those experiments in which statistically significant MN induction was observed.

[0617] Results: The compound of formula (IA) was tested according to the in vitro MNT assay procedure described above and was negative (score of 1).

[0618] Biological Example 9

[0619] Using IC 50 Estimation of TDI potential using the offset method

[0620] In order to determine the TDI potential of a specific P450 isozyme, the test compound is pre-incubated with HLM in a certain concentration range (usually 0.04 μM to 10 μM) in the presence of the cofactor NADPH for 30 minutes. After pre-incubation, a probe substrate known to be specifically metabolized to a determined metabolite by the isoform under study is then added at a fixed concentration. After a predetermined incubation period (e.g., 10 minutes for CYP3A4 testosterone assay), the reaction is terminated by adding a suitable organic solvent (usually acetonitrile) containing an internal standard. The sample is centrifuged before analysis by LC-MS / MS analysis, and the IC50 TDI value is calculated using curve fitting. It is compared with the reversible inhibition IC50 (IC50 Rev) obtained when the pre-incubation step is performed in the absence of the test compound. The leftward shift of the IC50 after pre-incubation (i.e., inhibition increases) indicates the TDI potential of the isozyme. The IC50 offset value is calculated as the ratio of IC50 Rev / IC50 TDI.

[0621] The assay was performed on a fully automated Tecan robotic system. Sample analysis was performed via LC-MS / MSMS using a stably labeled internal standard of the probe substrate metabolite.

[0622] The final incubation conditions are listed in Table B-7 below:

[0623] Table B-7: Incubation conditions, TDI potential determination

[0624]

[0625] Results: CYP3A4 (testosterone): Compounds of formula (IA) were tested according to the TDI potential assay as described above under standard solvent conditions. Without pre-incubation, the IC 50 The measured IC 50 The IC 50 The offset is 0.70.

[0626] Biological Example 10

[0627] Human Liver Microsome CYP450 Mixture Inhibition Assay

[0628] The mixture DDI assay is used to evaluate the potential of test compounds to cause drug-drug interactions via inhibition of clinically important P450 isoforms (CYP1A2, 2C8, 2C9, 2C19, 2D6, and 3A4) in early discovery. Discrimination of the inhibitory potential of test compounds to each isoform is achieved by using a mixture of specific probe substrates known to be selectively metabolized to defined metabolites by the isoform under study. For example, dextromethorphan is known to be demethylated only by the 2D6 isoform. Co-incubation of the probe substrates with the test compounds over a range of inhibitor concentrations in human liver microsomes (HLMs) allows simultaneous calculation of the IC of multiple CYP isoforms. 50 values, and thus provide higher throughput compared to traditional single-probe assays.

[0629] Methods and procedures: The final protein concentration in the incubation was 0.2 mg / ml and the incubation time was 10 minutes. The final total solvent in the incubation was 0.15% DMSO and 0.8% acetonitrile. The assay was performed automatically on a Beckman Biomek FX automated liquid handler with multichannel pipetting tools (8 and 96 channels), on deck reagent cooling capabilities and an integrated automated Cytomat incubator (37°C) with a plate shaking facility (Kendro).

[0630] Test compounds were provided as 5 mM DMSO stock solutions. 0.1 M phosphate buffer (pH 7.4) was prepared in house using Na2HPO4π12H2O and KH2PO4.

[0631] Frozen human liver microsomes (HLM) were thawed at 37°C with periodic stirring until the entire volume was thawed and then kept on ice. Based on the protein concentration of the HLM batch, the suspension was further diluted in assay buffer and probe substrate mix (substrate for each CYP isoform) to give a final protein concentration of 0.2 mg / mL.

[0632] Probe substrates were prepared separately in assay buffer (phenacetin, tolbutamide, S-mephenytoin, dextromethorphan) or water (amodiaquine, midazolam). They were combined with HLM and assay buffer (at appropriate concentrations for each substrate) to give the final enzyme / substrate mixture.

[0633] The final total solvent in the incubation was 0.15% DMSO and 0.8% acetonitrile.NADPH (5.27 mM) was prepared as a solution in assay buffer (room temperature).A quenching solution containing deuterated analogs of each of the seven probe substrate metabolites was dissolved in acetonitrile at appropriate concentrations.

[0634] Assay procedure: Samples for incubation were prepared by combining an appropriate volume of enzyme / substrate mixture (80 μl) and a stock solution of the test or reference inhibitor (1 μl solution added from the dilution series). After a pre-incubation period (typically 10 minutes), the reaction was initiated by adding NADPH solution (19 μl, 5.27 mM). The samples were incubated at 37°C for 10 minutes. The cultures were quenched by adding 1.6 volumes of refrigerated quench solution. The cultures were then centrifuged at 4000 rpm for 10 minutes at 4°C. A defined volume of supernatant (S / N) was transferred to a separate container and diluted with water (S / N: water = 60 μL / 180 μL). Each sample was injected onto a UPLC / MS system using an LC / MS method that selectively measures all seven probe substrate metabolites (and associated deuterated internal standards) simultaneously.

[0635] Calculation and formula: The activity percentage is calculated as

[0636] Activity % = (Ri / R) * 100

[0637] Where Ri and R are the metabolite to internal standard peak area ratios in the presence and absence of TC, respectively. The percent activity data were then plotted against log(TC concentration) and a curve was fit to the data using the data reduction tool within 3DX to return the IC 50 Specifically, the algorithm used in the tool is based on a sigmoid four-parameter relationship: response (% activity) as a function of concentration, where the four parameters are IC 50 , Hill slope, baseline and range, and the relationship between them is:

[0638] Response = Range / (1 + (Concentration / IC50)^Hill) + Baseline.

[0639] The best fit line using the above relationship was achieved using the nonlinear fitting algorithm of Marquardt-Levenberg. The curve generated from the seven single-point concentrations was used to calculate the IC 50 .

[0640] Results: Compounds of formula (IA) were tested according to the TDI potential assay procedure described above, wherein the IC against human liver microsomes was measured. 50 The values ​​are listed in Table B-8 below.

[0641] Table B-8: Human Liver Microsome Inhibition

[0642] HLM <![CDATA[Calculated IC 50 > 1A2 <![CDATA[IC 50 >20μM<!-- 73 --> ]]> 2C19 <![CDATA[IC 50 >20μM]]> 2C8 <![CDATA[IC 50 >20μM]]> 2C9 <![CDATA[IC 50 >20μM]]> 2D6 <![CDATA[IC 50 >20μM]]> 3A4 <![CDATA[IC 50 :14.4μM]]>

[0643] Biological Example 11: Seahorse Assay

[0644] The aim of this assay was to investigate the effect of compounds of formula (IA) on mitochondrial respiration in HepG2 cells using an Agilent Seahorse XFe96 analyzer. Cell viability was also determined in HepG2 cells in parallel with the Seahorse assay.

[0645] Measurement of mitochondrial respiration in HepG2 cells: Compound doses were prepared in 100% DMSO and subsequently diluted in assay medium to reach a final solvent concentration of 0.10%. HepG2 cells were seeded in Seahorse plates (Agilent Technologies, Santa Clara, California) at a density of 40,000 cells / well in EMEM containing 10% HI FBS and 1X Pen / Strep cell culture overnight. The culture was washed twice with an assay medium containing Seahorse XF basal medium (Agilent, part number 103334-100), 16mM glutamine, 5.5mM glucose and 8mM pyruvate. After washing, 175 μl of assay medium was added, and the plate was placed in a 37°C (no CO2) incubator to start the assay. According to the manufacturer's protocol, mitochondrial respiration was measured on an Agilent Seahorse XFe96 analyzer using the Seahorse Mito Stress test assay, quantified by oxygen consumption rate (OCR). Briefly, to assess the specific effects of compounds on mitochondrial function, each test compound (at concentrations of 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM, and 100 μM) or vehicle control (0.1% DMSO) was added, followed by the sequential addition of three mitochondrial modulators, each of which alters a unique metabolic parameter of the cells. Respectively, first 2 μM oligomycin (a complex V (ATP synthase) inhibitor), followed by 1 μM FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) (an uncoupling agent), and finally 0.5 μM rotenone / antimycin A combination (complex I and III inhibitors).

[0646] Cell viability measurement: HepG2 cells were seeded onto black wall 96-well plates at a density of 40,000 cells / well in EMEM containing 10% HI FBS and 1X Pen / Strep and allowed to attach overnight. The next day, test compounds were prepared and diluted in EMEM. After one hour of incubation with the test article, cell viability was assessed using the Cell Titer Fluor assay (Promega, catalog number G6081) according to the manufacturer's protocol. Viability assays monitor the activity of cathepsins (ubiquitous proteases found in living cells).

[0647] Data analysis: Mitochondrial SRC (spare respiration capacity) was calculated by subtracting the basal respiration rate from the maximum respiration rate. SRC values ​​were automatically generated by a report generator (Agilent Technologies-Version 4.0). All experiments were performed in triplicate, and the mean and standard deviation of each test concentration were calculated. All data were normalized to vehicle control.

[0648] IC 50 Values ​​were determined by nonlinear regression and fitting the dose-response curves to a two-parameter equation with a variable slope (Hill coefficient):

[0649] Y = [100 / (1+10^([logTC50-X]*Hill coefficient))]

[0650] Where Y is the normalized response between 0% and 100% and X is log(inhibitor concentration). All analyses were performed using GraphPadPrism 7.0 (San Diego, California).

[0651] Results: The compound of formula (IA) was tested according to the Seahorse assay procedure described above and showed 50 >100 μM had no direct effect on mitochondrial respiration, as measured by spare respiration. There was also no effect on cell viability, IC 50 >100μM.

[0652] Biological Example 12

[0653] Rabbit AV shunt (thrombosis) measurement and in vitro clotting time

[0654] Male New Zealand White rabbits (NZW) were purchased from Charles River Lab (CRL) with a weight range of 2.45 kg-3.22 kg and approximately 10-14 weeks of age. Animals were housed in standard rabbit cages, one animal per cage, and maintained in an animal room at 22°C with a 12 h daylight cycle. Standard rabbit diet and water were provided, as well as enriched fruit and carrots. No green leaves were provided to avoid interference with anticoagulant studies.

[0655] The rabbit AV shunt model was established as described in the literature (Wong, PC et al., Nonpeptide factor Xa inhibitors III: effects of DPC423, an orally-active pyrazole antithromboticagent, on arterial thrombosis in rabbits, J Pharmacol Exp Ther, 2002; pp. 993-1000, Vol. 303(3)). Rabbits were randomly divided into different experimental groups, and the group size (n = 2-5) varied according to the availability of the test compound.

[0656] Ketamine hydrochloride (20.0mg / kg-50.0mg / kg, IM) and xylazine (2.0mg / kg-10.0mg / kg, IM) were used for anesthesia induction. During the study, subsequent anesthesia maintenance was provided as needed (i.e., approximately every 30 minutes-50 minutes) with ketamine hydrochloride (at 22.7mg / kg-25.0mg / kg / ~0.5 hour, IM) and xylazine (at 2.27mg / kg-5.0mg / kg / ~0.5 hour, IM). Alternatively, rabbits were induced and subsequently maintained with isoflurane gas anesthetic (2-4% isoflurane and an O2 flow rate of 0.9L / min-1.2L / min via a nose cone).

[0657] After anesthesia, a 2 cm incision was made on each thigh to expose the femoral artery and vein from the opposite thigh, respectively. The vein and artery were cannulated with PE-100 tubing. After the vascular cannulation, the rabbit was allowed to equilibrate for 20 minutes. The AV shunt device was then connected between the femoral artery (one thigh) and femoral vein (the other thigh) cannulas. The shunt device consisted of an external TYGON piece (length, 8 cm; inner diameter, 7.9 mm) and an internal piece (length, 2.5 cm; inner diameter, 4.8 mm). The shunt also contained an 8 cm long 2-0 silk thread as a trigger for thrombosis. Forty (40) minutes after the start of blood flow through the shunt, the shunt was disconnected, and the silk thread and clot were removed and weighed.

[0658] The following test compounds and vehicles were used in the rabbit AV shunt studies:

[0659] A vehicle of the compound of formula (IA) and 35% hydroxypropyl β-cyclodextrin (HPβCD) in 10 mM phosphate buffer (pH 7);

[0660] · 70 / 20 / 10 PEG / water / ethanol vehicle.

[0661] Dosing solutions were prepared freshly in vehicle at different doses each day.Vehicle or test compounds were administered using intravenous administration via the marginal auricular vein 20 minutes prior to the start of AV shunt thrombosis studies (initial or loading dose) followed by continuous infusion over 40 minutes (maintenance dose).

[0662] Statistical analysis: All data are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism software (v8.0). Significance was defined as p < 0.05 as determined by one-way ANOVA. Tukey's test was used for multiple comparisons.

[0663] result :

[0664] The reduction in thrombus weight for the compound of formula (IA) compared to vehicle is listed below in Table B-9.

[0665] Table B-9: Thrombin Weight

[0666]

[0667]

[0668] In vitro coagulation assay :

[0669] The following coagulation reagents were purchased from Diagnostica Stago (Parsippany, NJ): Thrombin 10 (Cat. No. 00611, Batch No. 251851) for thrombin time (TT), Cl 5 (Catalog No.: 00666, Lot No.: 01504), for activated partial thromboplastin time (aPTT) 5 (Catalog No.: 00597, Batch No.: 112768) and CaCl2 (0.025 M, Catalog No.: 00367, Batch No.: 254158). Coagulometer STA rt4 (Diagnotica Stago) was used for in vitro coagulation time analysis.

[0670] Blood samples (1 mL each) were collected in 3.2% sodium citrate (BD Biosciences, Franklin Lakes, NJ) before and after shunt establishment from the femoral artery cannula and centrifuged at 2500 g for 15 min at 20° C. The resulting platelet-poor plasma (PPP) was transferred to a clean tube and stored at 80° C. until use.

[0671] Coagulation assays were performed with PPP in a 4-channel coagulometer STA rt4 (Diagnotica Stago). For aPTT analysis, 50 μl of CK pre-set reagents were incubated with 50 μl of PPP in a cuvette at 37° C. for 3 min, and coagulation was initiated by adding 50 μl of 25 mM CaCl2. The coagulation start time was recorded as the coagulation time.

[0672] Statistical analysis: All data are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism software (v8.0). Significance was defined as p < 0.05 as determined by one-way ANOVA. Tukey's test was used for multiple comparisons.

[0673] result :

[0674] The onset of coagulation measured for compounds of Formula (IA) compared to vehicle is listed below in Table B-10 and Table B-11.

[0675] Table B-10: Individual rabbit ex vivo aPTT coagulation onset times

[0676]

[0677]

[0678] a The test time point is after the initial administration of

[0679] Table B-11: In vitro aPTT coagulation onset time

[0680]

[0681] a The test time point is after the initial administration of

[0682] Preparation Example 1

[0683] Solid Oral Dosage Forms - Hypothetical Examples

[0684] As a specific embodiment of the oral composition, 100 mg of the compound of formula (IA) prepared in Example 5 above or the compound of formula (II-A) prepared in Example 13 above is formulated with enough finely divided lactose to provide a total amount of 580 mg to 590 mg to fill a size 0 hard gel capsule.

[0685] While the above description illustrates the principles of the invention by providing embodiments thereof, it should be understood that the practice of the invention encompasses all general variations, changes and / or modifications within the scope of the following claims and their equivalents.

[0686] Throughout this application, various publications are cited. The disclosures of these publications are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the invention pertains.

Claims

1. A compound of formula (I) or a stereoisomer, a deuterated isotope or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, which has the formula (IA) or a stereoisomer, a deuterated isotope or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1, wherein the compound is selected from (a) Compounds of formula (IB) (b) Compounds of formula (IC) (c) Compounds of formula (ID) (d) Compounds of formula (IE) (e) Compounds of formula (IF) (f) Compounds of formula (IG) (h) Compounds of formula (IH) (j) Compounds of formula (IJ) (k) Compounds of formula (IK) or a stereoisomer, a deuterated isotope or a pharmaceutically acceptable salt thereof.

4. A compound of formula (II) or a stereoisomer, a deuterated isotope or a pharmaceutically acceptable salt thereof.

5. The compound according to claim 4, which has the formula (II-A) or a stereoisomer, a deuterated isotope or a pharmaceutically acceptable salt thereof.

6. A compound of formula (III) or a stereoisomer, a deuterated isotope or a pharmaceutically acceptable salt thereof.

7. The compound according to claim 6, which has the formula (III-A) or a stereoisomer, a deuterated isotope or a pharmaceutically acceptable salt thereof.

8. A compound selected from the group consisting of (a) Compound of formula (IV-A) or a stereoisomer, deuterated isotope or salt thereof; (b) Compounds of formula (VA) or a stereoisomer, deuterated isotope or salt thereof; and (c) a compound of formula (VI-A) or a stereoisomer, a deuterated isotope or a salt thereof.

9. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound according to claim 1, 2, 3, 4, 5, 6 or 7.

10. Use of a therapeutically effective amount of a compound according to claim 1, 2, 3, 4, 5, 6 or 7 for the preparation of a medicament for the treatment or prevention of (a) a thromboembolic disorder treatable or preventable by inhibition of factor XIa; or (c) a disease or condition in which plasma kallikrein activity is involved, wherein the disease or condition in which plasma kallikrein activity is involved is selected from the group consisting of: impaired visual acuity, diabetic macular edema, hereditary angioedema, diabetes, nephropathy, cardiomyopathy, neuropathy, inflammation, septic shock, hypotension, cancer, adult respiratory distress syndrome and disseminated intravascular coagulation.

11. The use according to claim 10, wherein the thromboembolic disorder is selected from the group consisting of an arterial cardiovascular thromboembolic disorder, a venous cardiovascular thromboembolic disorder, an arterial cerebrovascular thromboembolic disorder and a venous cerebrovascular thromboembolic disorder.

12. The use according to claim 10, wherein the thromboembolic disorder is selected from the group consisting of unstable angina, acute coronary syndrome, first myocardial infarction, recurrent myocardial infarction, ischemic sudden death, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, thrombophlebitis, arterial embolism, cerebral embolism, renal embolism, pulmonary embolism and thrombosis caused by artificial valves or other implants, cardiopulmonary bypass, hemodialysis or other surgeries in which blood is exposed to artificial surfaces that promote thrombosis.

13. The use according to claim 10, wherein the thromboembolic disorder is selected from the group consisting of deep vein thrombosis, coronary artery thrombosis, cerebral artery thrombosis and thrombosis caused by an indwelling catheter or stent.

14. Use according to claim 10, wherein the thromboembolic disorder is selected from the group consisting of hereditary angioedema (HAE) and diabetic macular edema (DME).

15. The use according to claim 10, wherein the impaired visual acuity is diabetic retinopathy.

16. The use according to claim 10, wherein the inflammation is selected from the group consisting of pancreatitis, inflammatory bowel disease and arthritis.

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