Compound with high inhibitory activity on blood coagulation factor XIIa as well as pharmaceutical composition and application of compound

Abstract

The invention discloses a compound with high inhibitory activity on blood coagulation factor XIIa as well as a pharmaceutical composition and application of the compound. The structural formula of the compound is shown as a general formula (I) or (II). The benzo-seven-membered ring is adopted as a core structure, the benzo-seven-membered ring can well occupy an S1 pocket of the blood coagulation factor XIIa due to the large size and space flexibility, the affinity of molecules and a target spot is remarkably improved, and therefore the inhibition activity, selectivity and in-vitro anticoagulation and anti-inflammatory activity of the blood coagulation factor XIIa are improved, and the compound has good application prospects on the basis of avoiding or reducing the bleeding risk. The pharmaceutical composition is used for preventing and treating thrombus inflammation diseases, such as sepsis and atherosclerosis.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a compound with high inhibitory activity against coagulation factor XIIa, its pharmaceutical composition, and its uses. Background Technology

[0002] Thrombo-inflammation is prevalent in cardiovascular diseases, and coagulation factor XIIa, as a key target that simultaneously regulates coagulation and inflammatory signaling, has become an important direction for drug development. Atherosclerosis and sepsis are common thrombotic-inflammatory diseases. Currently, the development of small molecule inhibitors of coagulation factor XIIa is still in the early exploratory stage, and no candidate drugs have entered clinical trials. In preclinical studies, 1,2,4-triazole-5-amine covalent inhibitors are the most widely studied class of small molecule inhibitors of coagulation factor XIIa, but due to problems such as poor plasma stability, most compounds have failed to advance to the subsequent development stages.

[0003] Atherosclerosis is a chronic metabolic vascular disease. Coagulation factor XIIa, by activating the kallikrein-kinin system (KKS), catalyzes the production of bradykinin, increases vascular permeability, and promotes the secretion of pro-inflammatory factors, exacerbating plaque inflammation. Sepsis is a systemic inflammatory response syndrome triggered by infection, which can lead to organ dysfunction and even failure. Coagulation factor XIIa promotes thrombus formation by initiating intrinsic coagulation, while simultaneously activating the KKS and complement system, exacerbating sepsis symptoms. Targeting coagulation factor XIIa shows potential for treating atherosclerosis and sepsis.

[0004] Anticoagulation therapy is a major intervention for thrombotic diseases. Warfarin, the first classic oral anticoagulant to be clinically applied, has excellent oral bioavailability and is widely used for stroke prevention, but its therapeutic window is narrow and its bleeding risk is high. To overcome the limitations of traditional anticoagulants, novel oral anticoagulants (NOACs) such as dapigatran, rivaroxaban, and betrixaban have been approved for the treatment of thrombotic diseases; these drugs have better safety profiles.

[0005] The risk of bleeding can be reduced by inhibiting intrinsic coagulation factors without affecting the extrinsic coagulation pathway. Coagulation factor XIIa, as an initiator of the intrinsic coagulation pathway, has become a highly promising antithrombotic target.

[0006] Currently, no small molecule coagulation factor XIIa inhibitors have been reported to have entered clinical trials, but a series of patents for coagulation factor XIIa inhibitors have been published, including WO 2021032937, WO 2021032936, WO 2022175675, WO2022118016, WO 2018093695, and WO 2019211585. The efficacy of most compounds against thrombosis, thrombosis-inflammation, or other diseases in the body remains unknown, and covalent inhibitors of coagulation factor XIIa are unstable in plasma. The aforementioned studies indicate that FXIIa is an effective therapeutic target and has great potential for further research and development. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a compound with high inhibitory activity against coagulation factor XIIa, a pharmaceutical composition thereof, and its use. The benzo[7]7-membered ring structure improves the activity, selectivity, in vitro anticoagulant activity, and anti-inflammatory activity against coagulation factor XIIa.

[0008] The technical solution of this invention is as follows:

[0009] A compound with high inhibitory activity against coagulation factor XIIa, wherein the structural formula of the compound is shown in general formula (I) or (II) below:

[0010]

[0011] In general formulas (Ⅰ) and (Ⅱ), X is CH2, NH or O; Y is CH2, NH or O; W is NH or O; n1 is 1, 2, 3 or 4; n2 is 1, 2, 3 or 4;

[0012] 3-Pyrrolidone, Morpholine, Thiomorpholine, Thiomorpholinone, Morpholin-2-one, 2-oxa-6-azaspiro[3,3]heptane and 3-Methyl-4-piperidinone, Benzene ring, 2-Chlorophenyl, 3-Oxyphenyl, 3-Oxy-5-Oxyphenyl, 2-Cyanophenyl, 3-Cyanophenyl, 3-Ethoxyphenyl, 3-Ethylphenyl, 2-Oxyphenyl, 3-Oxyphenyl, 4-Oxyphenyl, 3-Isopropylphenyl, 3-Methoxyphenyl, 2-Methylphenyl, 3-Methylphenyl 3-Nitrophenyl, 3-Dioxomethoxyphenyl, 3-Difluoromethylphenyl, 2-Pyridyl, 4-Chloro-2-pyridyl, 5-Chloro-2-pyridyl, 5-Fluoro-2-pyridyl, 6-Fluoro-2-pyridyl, 4-Methyl-2-pyridyl, 6-Methoxy-3-pyridyl, 6-Methyl-2-pyridyl, 4-Trifluoromethyl-2-pyridyl, 3-Pyridyl, 6-Fluoro-3-pyridyl, 2-Furanyl, 2-Thienyl, 3-Thienyl, 2-Pyrazinyl and C l-3 One of the alkylaminosulfonyl groups; In this context, X' is CH or N, Y' is CH or N, m is 1, 2, 3, or 4, and R7 is selected from hydrogen, deuterium, halogens, and C. l-5 Alkyl, C l-5 Haloalkyl, C l-5 One of alkoxy, unsubstituted monocyclic alkyl, unsubstituted heterocyclic alkyl, substituted monocyclic alkyl, substituted heterocyclic alkyl, unsubstituted aromatic heterocycle and substituted aromatic heterocycle, wherein R8 is absent or selected from one of hydrogen, deuterium, halogen, hydroxyl, hydroxymethyl, methyl, isopropyl, methoxy, unsubstituted monocyclic alkyl, unsubstituted heterocyclic alkyl, substituted monocyclic alkyl and substituted heterocyclic alkyl; R9 in the formula either does not exist or is selected from one of the following: hydrogen, deuterium, halogen, hydroxyl, hydroxymethyl, methyl, isopropyl, methoxy, unsubstituted monocyclic alkyl, unsubstituted heterocyclic alkyl, substituted monocyclic alkyl, and substituted heterocyclic alkyl. 10 It does not exist or is selected from one of hydrogen, deuterium, halogen, hydroxyl, hydroxymethyl, methyl, isopropyl, methoxy, unsubstituted monocyclic alkyl, unsubstituted heterocyclic alkyl, substituted monocyclic alkyl and substituted heterocyclic alkyl;

[0013] R2 is absent or selected from one of hydrogen, deuterium, hydroxyl, methyl, ethyl, isopropyl, methoxy, ethoxy, halogen, 4-piperidinone glycol condensate, 6-azaspiro[2.5]octane, N-methylpiperazine, N-ethylpiperazine, N-isopropylpiperazine, 1-(2-hydroxyethyl)piperazine, piperazine-1-ylmethanol, and 1-(2-methoxyethyl)piperazine;

[0014] R3 is absent or selected from one of hydrogen, deuterium, hydroxyl, halogen, methyl, ethyl and isopropyl;

[0015] Z is a carbon atom or a nitrogen atom; when Z is a carbon atom, R4 is selected from hydrogen, deuterium, halogen, methoxy, ethoxy, hydroxymethyl, C l-5 Alkyl, C l-5 Haloalkyl, C l-5 One of alkoxy, cyclopropane, cyclobutane, cyclopentane, cyclohexane, and cycloheptane; R4 is absent when Z is a nitrogen atom;

[0016] Ring A is selected from pyridine ring, pyrrole ring, pyrimidine ring, benzene ring, indole ring, quinoxalinyl, tetrahydroindole ring, piperazine ring, 2-hydroxypyridine, cyclohexene, 1-hydroxy-3-cyclohexene, 2-cyclohexen-1-one, quinolinyl, isoquinoline, 5,6,7,8-tetrahydroquinoline, etc. One of them; In the above, X” is CH2, NH, O or S; p is 1, 2, 3 or 4; g is 1, 2, 3 or 4; R5 is absent or is one of hydrogen, deuterium, halogen, hydroxyl, hydroxymethyl, methyl, ethyl, propyl, butyl, isopropyl, methoxy, trifluoromethyl, difluoromethyl, trifluoromethoxy and difluoromethoxy; R6 is absent or is one of hydrogen, deuterium, halogen, hydroxyl, hydroxymethyl, methyl, ethyl, propyl, butyl, isopropyl, methoxy, methylcyclopropane, prop-1-ylcyclopropane, but-2-yn-1-ol, but-2-yn-yne.

[0017] R1 is In this case, R7 is selected from one of unsubstituted monocyclic alkyl, unsubstituted heterocyclic alkyl, substituted monocyclic alkyl, substituted heterocyclic alkyl, unsubstituted aromatic ring and substituted aromatic ring, and R8 is selected from one of unsubstituted monocyclic alkyl, unsubstituted heterocyclic alkyl, substituted monocyclic alkyl and substituted heterocyclic alkyl.

[0018] In R7, the unsubstituted monocyclic alkyl group is selected from one of cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, and cyclooctane; in R7, the unsubstituted heterocyclic alkyl group and the substituted heterocyclic alkyl group are selected from one of ethylene oxide, oxetane, and oxetane; in R7, the substituted monocyclic alkyl group is selected from one of substituted cyclopropane, substituted cyclobutane, substituted cyclopentane, and substituted cyclohexane.

[0019] In the R7, when there is one substituent of the substituted monocyclic alkyl or substituted heterocyclic alkyl, the one substituent is substituted on any methylene group; when there are multiple substituents, the multiple substituents are substituted on any methylene group respectively. The substituents are independently alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro or cyano.

[0020] In R7, the unsubstituted aromatic ring is selected from one of benzene ring, pyrrole, thiophene, pyridine and furan;

[0021] In R7, the substituted aromatic ring is selected from one of 2-chlorophenyl, 3-oxophenyl, 3-oxo-5-oxophenyl, 2-cyanophenyl, 3-cyanophenyl, 3-ethoxyphenyl, 3-ethylphenyl, 2-oxophenyl, 3-oxophenyl, 4-oxophenyl, 3-isopropylphenyl, 3-methoxyphenyl, 2-methylphenyl, 3-methylphenyl, 3-nitrophenyl, 3-dioxomethoxyphenyl, 3-difluoromethylphenyl, 2-pyridyl, 4-chloro-2-pyridyl, 5-chloro-2-pyridyl, 5-fluoro-2-pyridyl, 6-fluoro-2-pyridyl, 4-methyl-2-pyridyl, 6-methoxy-3-pyridyl, 6-methyl-2-pyridyl, 4-trifluoromethyl-2-pyridyl, and 6-fluoro-3-pyridyl.

[0022] In R8, the unsubstituted monocyclic alkyl group is selected from one of cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, and cyclooctane; the unsubstituted heterocyclic alkyl group and the substituted heterocyclic alkyl group are selected from one of ethylene oxide, oxetane, and oxetane; and the substituted monocyclic alkyl group is selected from one of substituted cyclopropane, substituted cyclobutane, substituted cyclopentane, and substituted cyclohexane.

[0023] R1 is In this case, R9 is selected from one of unsubstituted monocyclic alkyl, unsubstituted heterocyclic alkyl, substituted monocyclic alkyl, and substituted heterocyclic alkyl. 10 It is selected from one of unsubstituted monocyclic alkyl, unsubstituted heterocyclic alkyl, substituted monocyclic alkyl and substituted heterocyclic alkyl.

[0024] In R9, the unsubstituted monocyclic alkyl group is selected from one of cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, and cyclooctane; the unsubstituted heterocyclic alkyl group and the substituted heterocyclic alkyl group are selected from one of ethylene oxide, oxetane, and oxetane; the substituted monocyclic alkyl group is selected from one of substituted cyclopropane, substituted cyclobutane, substituted cyclopentane, and substituted cyclohexane.

[0025] The R mentioned 10 In this context, the unsubstituted monocyclic alkyl group is selected from one of cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, and cyclooctane; the unsubstituted heterocyclic alkyl group and the substituted heterocyclic alkyl group are selected from one of ethylene oxide, oxetane, and oxecyclopentane; the substituted monocyclic alkyl group is selected from one of substituted cyclopropane, substituted cyclobutane, substituted cyclopentane, and substituted cyclohexane.

[0026] The general formula (Ⅰ) is any one of the following expressions (Ⅰ-1) to (Ⅰ-10):

[0027]

[0028] In (Ⅰ-4) and (Ⅰ-5), * represents a chiral carbon atom.

[0029] A pharmaceutical composition comprising one or more of the said compound, stereoisomers of the compound, tautomers of the compound, and pharmaceutically acceptable salts thereof.

[0030] The compound or the pharmaceutical composition is used in the preparation of a medicament for inhibiting coagulation factor XIIa.

[0031] The aforementioned drug for inhibiting coagulation factor XIIa is used to prevent and treat diseases mediated by coagulation factor XIIa by inhibiting coagulation factor XIIa.

[0032] Stable isotope derivatives, metabolites, or prodrugs of the compounds may prevent and / or treat thrombosis and hereditary angioedema while avoiding or mitigating the side effects of existing anticoagulation therapies.

[0033] Advantages of this invention:

[0034] This invention employs a benzo[7]-ring structure, replacing the rigid ring structure near the S1 pocket of coagulation factor XIIa with a more flexible ring system. This significantly enhances the affinity between the molecule and the target, thereby improving the activity, selectivity, in vitro anticoagulant activity, and anti-inflammatory activity against coagulation factor XIIa. It exhibits excellent inhibitory effects on coagulation factor XIIa and, while avoiding the risk of bleeding, can be used to prevent and treat thrombotic inflammations such as sepsis and atherosclerosis. Detailed Implementation

[0035] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0036] Example 1

[0037] A compound with high inhibitory activity against coagulation factor XIIa has the following structural formula (I-1):

[0038]

[0039] The preparation method of the compound shown in formula (I-1) specifically includes the following steps:

[0040] (1) Synthesis of methyl 4-bromo-2,3-dihydroxybenzoate:

[0041]

[0042] 2,3-Dihydroxybenzoate (2 g, 1 equiv) was placed in a reaction flask and dissolved in dichloromethane (DCM) (15 ml). N-bromosuccinimide (NBS) (2.5 g, 1.2 equiv) was added in portions at 0 °C and stirred for 5 min. The reaction was then carried out at room temperature. The reaction was monitored by thin-layer chromatography (TLC). After the reaction was completed, the product was extracted with 1 M HCl and CH2Cl2. The organic phase was collected and column chromatography was used to obtain the target product 4-bromo-2,3-dihydroxybenzoate (1.02 g, white solid, yield 34.2%).

[0043] (2) Synthesis of methyl 6-bromo-2H,4H-spiro[benzo[b][1,4]dioxane-3,3'-oxacyclobutane]-9-carboxylate:

[0044]

[0045] 4-Bromo-2,3-dihydroxybenzoate methyl ester (1.02 g, 1 equiv) and cesium carbonate (4.03 g, 3 equiv) were placed in a bottle, dissolved in dimethylformamide (DMF, 15 ml), and then 1,1-bis(bromomethyl)cyclopropane (1.3 g, 1.5 equiv) was added dropwise. After stirring at room temperature for 5 min, the mixture was transferred to 90 °C for reaction. The reaction was monitored by thin-layer chromatography (TLC). After the reaction was completed, the product 6-bromo-2H,4H-spiro[benzo[b][1,4]dioxane-3,3'-oxetane]-9-carboxylate (white solid, yield 72%) was obtained by extraction and column chromatography.

[0046] (3) Synthesis of methyl (S)-6-(3-((tert-butoxycarbonyl)(methyl)amino)pyrrolidine-1-yl)-2H,4H-spiro[benzo[b][1,4]dioxane-3,1'-cyclopropane]-9-carboxylate:

[0047]

[0048] Methyl 6-bromo-2H,4H-spiro[benzo[b][1,4]dioxane-3,3'-oxetane]-9-carboxylate (1 g, 1 equiv), tert-butyl(S)-methyl(pyrrolidine-3-yl)carbamate (0.77 g, 1.2 equiv), palladium acetate (0.03 g, 0.05 equiv), cesium carbonate (2.1 g, 2 equiv), and 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (0.1 g, 0.05 equiv) were added. The mixture was placed in a reaction flask filled with N2, and toluene was added under N2 atmosphere. The mixture was stirred overnight at 100°C. The reaction was monitored by thin-layer chromatography (TLC). After the reaction was completed, the solvent was evaporated, and the product (S)-6-(3-((tert-butoxycarbonyl)(methyl)amino)pyrrolidine-1-yl)-2H,4H-spiro[benzo[b][1,4]dioxane-3,1'-cyclopropane]-9-carboxylic acid methyl ester (white solid, yield 50.0%) was obtained by extraction and column chromatography.

[0049] (4) Synthesis of methyl (S)-9-(3-(methylamino)pyrrolidone-1-yl)-2H,4H-spiro[benzo[b][1,4]dioxane-3,1'-cyclopropane]-6-carboxylate:

[0050]

[0051] Methyl (S)-6-(3-((tert-butoxycarbonyl)(methyl)amino)pyrrolidine-1-yl)-2H,4H-spiro[benzo[b][1,4]dioxane-3,1'-cyclopropane]-9-carboxylate (0.5 g, 1 equiv) was dissolved in DCM (15 ml), and trifluoroacetic acid (445 μl, 5 equiv) was slowly added dropwise. The mixture was stirred at room temperature, and the reaction was monitored by thin-layer chromatography (TLC). After the reaction was completed, the DCM in the reaction solution was removed by rotary evaporation. The target product (S)-9-(3-(methylamino)pyrrolidine-1-yl)-2H,4H-spiro[benzo[b][1,4]dioxane-3,1'-cyclopropane]-6-carboxylate (white solid, yield 92.3%) was obtained by extraction.

[0052] (5) Synthesis of (S)-9-(3-(isopropyl(methyl)amino)pyrrolidine-1-yl)-2H,4H-spiro[benzo[b][1,4]dioxane-3,1'-cyclopropane]-6-carboxylic acid methyl ester:

[0053]

[0054] Methyl (S)-9-(3-(methylamino)pyrrolidone-1-yl)-2H,4H-spiro[benzo[b][1,4]dioxane-3,1'-cyclopropane]-6-carboxylate (0.45 g, 1 equiv) was dissolved in methanol (MeOH), and acetone solution (810 μL, 7.5 equiv) was slowly added dropwise. The mixture was stirred for 5 min, and sodium cyanoborohydride (0.19 g, 2 equiv) was added in portions. The mixture was stirred overnight, and the reaction was monitored by thin-layer chromatography (TLC). After the reaction was completed, MeOH in the reaction solution was removed by rotary evaporation. The product (S)-9-(3-(isopropyl(methyl)amino)pyrrolidone-1-yl)-2H,4H-spiro[benzo[b][1,4]dioxane-3,1'-cyclopropane]-6-carboxylate (white solid, yield 55.4%) was obtained by extraction and column chromatography.

[0055] (6) Synthesis of (S)-9-(3-(isopropyl(methyl)amino)pyrrolidine-1-yl)-2H,4H-spiro[benzo[b][1,4]dioxane-3,1'-cyclopropane]-6-carboxylic acid:

[0056]

[0057] Methyl (S)-9-(3-(isopropyl(methyl)amino)pyrrolidine-1-yl)-2H,4H-spiro[benzo[b][1,4]dioxane-3,1'-cyclopropane]-6-carboxylic acid (0.4 g, 1 equiv) was dissolved in THF (10 ml) and MeOH (10 ml), and lithium hydroxide aqueous solution (0.22 g, 5 equiv) was slowly added dropwise. The mixture was stirred overnight at 60 °C. The reaction was monitored by thin-layer chromatography (TLC). After the reaction was completed, THF and MeOH in the reaction solution were removed by rotary evaporation. The product (S)-9-(3-(isopropyl(methyl)amino)pyrrolidine-1-yl)-2H,4H-spiro[benzo[b][1,4]dioxane-3,1'-cyclopropane]-6-carboxylic acid (white solid, yield 40%) was obtained by extraction and column chromatography.

[0058] (7) Synthesis of (S)-(5-amino-3-(quinolin-2-yl)-1H-1,2,4-triazol-1-yl)(6-(3-(isopropyl(methyl)amino)pyrrolidine-1-yl)-2H,4H-spiro[benzo[b][1,4]dioxane-3,1'-cyclopropane]-9-yl) ketone:

[0059]

[0060] (S)-9-(3-(isopropyl(methyl)amino)pyrrolid-1-yl)-2H,4H-spiro[benzo]

[0061] [b][1,4]dioxane-3,1'-cyclopropane]-6-carboxylic acid (300 mg, 1 equiv), 3-

[0062] (quinolin-2-yl)-1H-1,2,4-triazol-5-amine (176 mg, 1 equiv), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (192 mg, 1.2 equiv), and 4-dimethylaminopyridine (122 mg, 1.2 equiv) were added to a reaction flask filled with N2 atmosphere. DMF (5 mL) was then added and stirred for 1 h under ice bath conditions, followed by stirring at room temperature for 2 h. The reaction was monitored by thin-layer chromatography (TLC). After the reaction was completed, the solvent was evaporated, and the target compound (S)-(5-amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)(6-(3-(isopropyl(methyl)amino)pyrrolidine-1-yl)-2H,4H-spiro[benzo[b][1,4]dioxane-3,1'-cyclopropane]-9-yl) methyl ketone (yield 14%) was obtained by extraction and column chromatography. 1 H NMR (600MHz, DMSO-d6) δ8.44(d,J=8.5Hz,1H),8.12–8.05(m,2H),8.00(d,J=8.1Hz,1H),7.79(t,J=7.7Hz,1H),7.72(s,2H ),7.62(t,J=7.5Hz,1H),7.17(d,J=8.6Hz,1H),6.45(d,J=8.7Hz,1H),4.18(d,J=12.1Hz,1H),3.97(d,J=11.6Hz,1H),3.76 (d,J=11.6Hz,1H),3.59(d,J=12.2Hz,1H),3.55–3.48(m,2H),3.47–3.41(m,1H),3.33–3.28(m,1H),3.14–3.05(m,1H),3.0 2–2.95(m,1H),2.10(s,3H),2.07–2.04(m,1H),1.76–1.66(m,1H),0.98–0.93(m,6H),0.63–0.44(m,4H).HR-MS:m / z,(M+H) + cal.for C 31 H 36 N7O3 + ,554.2874; found,554.2875.

[0063] Example 2

[0064] A compound with high inhibitory activity against coagulation factor XIIa has the following structural formula (II-1):

[0065]

[0066] The preparation method of the compound shown in formula (Ⅱ-1) specifically includes the following steps:

[0067] (1) The synthesis of methyl 4-bromo-2,3-dihydroxybenzoate is the same as in Example 1;

[0068] (2) Synthesis of methyl 6-bromo-2H,4H-spiro[benzo[b][1,4]dioxane-3,3'-oxacyclobutane]-9-carboxylate:

[0069]

[0070] Ethyl 4-bromo-2,3-dihydroxybenzoate (1.0 g, 1.00 equiv) was dissolved in dimethyl sulfoxide and stirred for 5 min. Potassium carbonate was added, and the solution color changed from transparent to yellow-green. Then, 3,3-bis(bromomethyl)oxetane (1.18 g, 1.20 equiv) was added, and the mixture was stirred at 100 °C for 1 h. After the reaction was completed, the solution was extracted, concentrated, and separated by column chromatography using a suitable polarity (PE:EA = 10:1) to obtain a white powder of methyl 6-bromo-2H,4H-spiro[benzo[b][1,4]dioxetane-3,3'-oxetane]-9-carboxylate (yield: 67.3%).

[0071] (3) Synthesis of methyl 9-(4-isopropylpiperazin-1-yl)-2H,4H-spiro[benzo[b][1,4]dioxane-3,3'-oxecyclobutane]-6-carboxylate:

[0072]

[0073] Methyl 6-bromo-2H,4H-spiro[benzo[b][1,4]dioxane-3,3'-oxetane]-9-carboxylate (860 mg, 1.00 equiv), isopropylpiperazine (504 mg, 1.50 equiv), palladium acetate (29 mg, 0.05 equiv), 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (82 mg, 0.05 equiv), and cesium carbonate (1.7 g, 2.00 equiv) were added to a nitrogen-containing container and stored. The system was filled with nitrogen atmosphere by multiple N2 substitutions, toluene solvent was quickly added, and the mixture was then transferred to an oil bath at 100°C and reacted for 12 hours. The product spot was monitored by TLC. After the reaction was completed, the mixture was extracted, evaporated to dryness and concentrated. The mixture was then separated by column chromatography using a suitable polarity (PE:EA = 4:1) to obtain methyl 9-(4-isopropylpiperazin-1-yl)-2H,4H-spiro[benzo[b][1,4]dioxane-3,3'-oxecyclobutane]-6-carboxylate (yield: 61.3%).

[0074] (4) Synthesis of 9-(4-isopropylpiperazin-1-yl)-2H,4H-spiro[benzo[b][1,4]dioxane-3,3'-oxecyclobutane]-6-carboxylic acid:

[0075]

[0076] Methyl 9-(4-isopropylpiperazin-1-yl)-2H,4H-spiro[benzo[b][1,4]dioxane-3,3'-oxetane]-6-carboxylic acid (500 mg, 1.00 equiv) was dissolved in a solution of THF:MeOH = 1:1. Lithium hydroxide aqueous solution (73 mg, 1.30 equiv) was added dropwise, causing the solution to change from clear to turbid. The reaction was allowed to proceed overnight, and the product spot was monitored by TLC. After the reaction was complete, THF and MeOH were evaporated, and 1 M HCl solution was added to adjust the pH to approximately 7. The aqueous solution was then evaporated, and the mixture was separated by appropriate polarity and subjected to column chromatography (DCM:MeOH = 20:1) to obtain a white powder of 9-(4-isopropylpiperazin-1-yl)-2H,4H-spiro[benzo[b][1,4]dioxane-3,3'-oxetane]-6-carboxylic acid (yield: 49.6%).

[0077] (5) Synthesis of (5-amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)(6-(4-isopropylpiperazin-1-yl)-2H,4H-spiro[benzo[b][1,4]dioxane-heptane-3,3'-oxetane]-9-yl) ketone:

[0078]

[0079] 9-(4-isopropylpiperazin-1-yl)-2H,4H-spiro[benzo[b][1,4]dioxane-3,3'-oxacyclobutane]-6-carboxylic acid (230 mg, 1.00 equiv), 3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine (98 mg, 1.20 equiv), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (146 mg, 1.2 equiv), and 4-dimethylaminopyridine (93 mg, 1.2 equiv) were weighed into a nitrogen storage container, and nitrogen was purged for 30 minutes. Add anhydrous DMF (5 mL) solvent, stir for 1 h in an ice bath, then stir for 3 h at room temperature. Monitor the product spot by TLC. After the reaction is complete, extract and concentrate by rotary evaporation, and then use a suitable polarity separation and purification method (DCM:MeOH = 20:1) to obtain the white target compound (5-amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)(6-(4-isopropylpiperazin-1-yl)-2H,4H-spiro[benzo[b][1,4]dioxane-heptane-3,3'-oxetane]-9-yl) methyl ketone (yield: 23.9%). 1 H NMR (600MHz, DMSO-d6) δ (ppm): 8.59 (d, J = 3.4Hz, 1H), 7.96 (d, J = 7.8Hz, 1H), 7.89(t,J=6.8Hz,1H),7.79(s,2H),7.45–7.42(m,1H),7.19(d,J=8.4Hz,1H) ,6.76(d,J=8.5Hz,1H),4.39(d,J=6.6Hz,2H),4.37(s,2H),4.32(d,J=6.5Hz ,2H),4.30(s,2H),3.05–3.02(m,5H),1.20–1.17(m,10H).HR-MS:m / z,(M+H) + cal.for C 26 H 32 N7O4 + ,506.2510; found,506.2507.

[0080] The compounds of Examples 3-33 were prepared according to the steps described below. The structural formulas of the compounds prepared in Examples 3-33 are shown in Table 1 below.

[0081] Table 1

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] Enzyme activity test:

[0089] The enzyme activity of coagulation factor XIIa was tested on the compounds prepared in each example according to the following method: First, the test compound (the compound prepared in each example) was dissolved in dimethyl sulfoxide (DMSO) for later use. The coagulation factor XIIa enzyme and its corresponding specific coagulation factor XIIa substrate were then taken out and diluted with pre-cooled CaCl2-free buffer to the required stock solution, aliquoted, and stored at -20°C. Before the experiment, the enzyme and substrate were mixed in a buffer solution (buffer solution preparation: Tris was dissolved in ultrapure water, pH was adjusted to 7.4 with concentrated HCl, NaCl and bovine serum albumin (BSA) were added sequentially, and CaCl2 was added if necessary; after bringing to volume, sterilization was performed), and the reaction was started at room temperature. During the reaction, the coagulation factor XIIa enzyme catalyzed the hydrolysis of the substrate, and the absorbance (OD value) was measured over time at a wavelength of 405 nm using a microplate reader. The enzyme activity was calculated based on the slope of the OD value-time curve (i.e., the reaction rate).

[0090] Specific experimental procedures: 75 μL of coagulation factor XIIa enzyme solution at a specific concentration was added to a blank 96-well plate. 2 μL of DMSO (negative control) was added using a pipette. The plate was incubated at room temperature for 10 min. Then, substrate solutions of different concentrations were added, and the absorbance at 405 nM was immediately monitored dynamically using a microplate reader. The optimal substrate concentration was calculated based on the substrate hydrolysis rate. Next, 75 μL of coagulation factor XIIa enzyme and 2 μL of a concentration gradient of the test compound (DMSO final concentration <1%) were added to the reaction wells. An equal volume of DMSO was added to the negative control wells. The plate was incubated at room temperature for 10 min, and then the optimal substrate concentration was added to start the reaction. The OD value at 405 nM absorbance was immediately measured over time. The obtained data were fitted using GraphPad Prism software to calculate the inhibitory effect of the compound on the enzyme, and the IC50 was obtained through nonlinear regression analysis. 50 Values, IC values ​​corresponding to the compounds prepared in each embodiment. 50 The values ​​are shown in Table 2 below.

[0091] The inhibition experiments of the remaining proteases (plasma kallikrein Pkal, coagulation factor XIa, thrombin, coagulation factor Xa, chymotrypsin and trypsin) were conducted in accordance with the above method. The reaction system was adjusted according to the optimal conditions for each enzyme, and the inhibitory effect of the compound was calculated.

[0092] Table 2

[0093]

[0094]

[0095]

[0096] In Table 2, A represents IC. 50 It is 0.1-1 nM; B represents IC 50 1-10 nM; C represents IC 50 10-50 nM; D indicates IC 50 50-100nM; E indicates IC 50 The concentration is 100-200 nM. Table 2 shows the IC50 values ​​of the compounds obtained in the various embodiments of the present invention. 50 All are less than 100 nm, showing good inhibitory activity against coagulation factor XIIa.

[0097] Enzyme selectivity analysis:

[0098] To characterize the selectivity of the compounds, the inhibitory activities of the compounds (prepared in Examples 1, 3-6, 9-11, 24, 25, 28-33) against serine proteases such as thrombin, PKal, FXa, FXIa, Trypsin, and Chymotrypsin were tested. The test results (IC50) were analyzed. 50 See Table 3 below. Overall, the various compounds showed 1000 times greater selectivity for coagulation factor XIIa (see Table 2) than for Pkal, FXa, FXIa, Trypsin, or Chymotrypsin, and 100 times greater selectivity for coagulation factor XIIa (see Table 2) than for thrombin, demonstrating excellent enzyme selectivity.

[0099] Table 3

[0100]

[0101]

[0102] Plasma stability test:

[0103] The test compound (the compound obtained in Examples 1 and 32) was diluted to an appropriate concentration, and the compound was incubated with sodium citrate anticoagulant plasma at 37°C. aPTT was measured at 20, 40, 150, 180, 210, and 240 min. The anticoagulant effect of the compound in plasma did not significantly decrease over time.

[0104] In vitro anti-inflammatory experiment:

[0105] Enzyme activity (IC)50 Compounds with a concentration less than 10 nM (compounds prepared in Examples 1, 3, 28, and 31-33) were diluted with DMSO to the corresponding concentrations to a final concentration of 10 μM. RAW264.7 macrophages were added to 12-well plates for culture and growth, with regular medium changes. When the cell adhesion coverage reached 80%, LPS (lipopolysaccharide can induce an inflammatory response in cells) was added, followed by the addition of the aforementioned compounds and culturing for 12 hours. After incubation, the cells were processed by removing the culture medium and washing once with PBS. Lysis buffer was then added, and the solution was pipetted until homogeneous. The corresponding volume of chloroform was then added, and the mixture was vigorously stirred. Shake for 30 seconds, centrifuge, collect the upper transparent liquid, add isopropanol at a 1:1 volume ratio, and shake again to mix; incubate overnight at -20℃ to precipitate RNA, centrifuge at 12000 rpm at 4℃ for 10 min, remove the supernatant, add 75% ethanol solution to the EP tube, shake to mix, centrifuge, then replace with 100% ethanol, shake to mix, centrifuge, remove the ethanol with a vacuum pump, and place the lower solid in a clean bench to air dry for 25 min, at which point the RNA gradually turns white; add 60℃ sterilized ddH2O to the dried RNA to dissolve the RNA, and determine the RNA concentration of each sample using NanoDrop.

[0106] Reverse transcription PCR: Calculate the volume based on the RNA concentration measured in the previous step, prepare cDNA, and perform reverse transcription PCR.

[0107] qPCR quantitative analysis: Using the cDNA obtained above as a template, the reaction system is as follows: upstream primer (1μL), downstream primer (1μL), 2×SYBR (10μL), ddH2O (7μL), cDNA (2μL).

[0108] After adding the above reagents, centrifuge, shake, and add to 96-well plates according to the modeling group. Cover and seal with membrane, mix, centrifuge, and then perform qPCR reaction. Finally, statistically analyze the changes in mRNA expression levels of inflammatory factors IL-6 and IL-1β.

[0109] All of the above compounds showed certain anti-inflammatory effects; among them, the compounds obtained in Examples 1 and 33 showed significant anti-inflammatory inhibitory effects in IL-6 and IL-1β.

[0110] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A compound with high inhibitory activity against coagulation factor XIIa, characterized in that: The structural formula of the compound is shown in general formula (I) or (II) below: In general formulas (Ⅰ) and (Ⅱ), X is CH2, NH or O; Y is CH2, NH or O; W is NH or O; n1 is 1, 2, 3 or 4; n2 is 1, 2, 3 or 4; R1 is selected from Hydrogen, deuterium, halogens, hydroxymethyl, C l-3 Alkyl, C l-3 Alkoxy, aziridine hepta-4-one, 3-pyrrolidone, morpholine, thiomorpholine, thiomorpholinone, morpholine-2-one, 2-oxa-6-azaspiro[3,3]heptane and 3-methyl-4-piperidinone, benzene ring, 2-chlorophenyl, 3-oxophenyl, 3-oxo-5-oxophenyl, 2-cyanophenyl, 3-cyanophenyl, 3-ethoxyphenyl, 3-ethylphenyl, 2-oxophenyl, 3-oxophenyl, 4-oxophenyl, 3-isopropylphenyl, 3-methoxyphenyl, 2-methylphenyl, 3 -Methylphenyl, 3-nitrophenyl, 3-dioxomethoxyphenyl, 3-difluoromethylphenyl, 2-pyridyl, 4-chloro-2-pyridyl, 5-chloro-2-pyridyl, 5-fluoro-2-pyridyl, 6-fluoro-2-pyridyl, 4-methyl-2-pyridyl, 6-methoxy-3-pyridyl, 6-methyl-2-pyridyl, 4-trifluoromethyl-2-pyridyl, 3-pyridyl, 6-fluoro-3-pyridyl, 2-furanyl, 2-thienyl, 3-thienyl, 2-pyrazinyl and C l-3 One of the alkylaminosulfonyl groups; In this context, X' is CH or N, Y' is CH or N, m is 1, 2, 3, or 4, and R7 is selected from hydrogen, deuterium, halogens, and C. l-5 Alkyl, C l-5 Haloalkyl, C l-5 One of alkoxy, unsubstituted monocyclic alkyl, unsubstituted heterocyclic alkyl, substituted monocyclic alkyl, substituted heterocyclic alkyl, unsubstituted aromatic heterocycle and substituted aromatic heterocycle, wherein R8 is absent or selected from one of hydrogen, deuterium, halogen, hydroxyl, hydroxymethyl, methyl, isopropyl, methoxy, unsubstituted monocyclic alkyl, unsubstituted heterocyclic alkyl, substituted monocyclic alkyl and substituted heterocyclic alkyl; R9 in the formula either does not exist or is selected from one of the following: hydrogen, deuterium, halogen, hydroxyl, hydroxymethyl, methyl, isopropyl, methoxy, unsubstituted monocyclic alkyl, unsubstituted heterocyclic alkyl, substituted monocyclic alkyl, and substituted heterocyclic alkyl. 10 It does not exist or is selected from one of hydrogen, deuterium, halogen, hydroxyl, hydroxymethyl, methyl, isopropyl, methoxy, unsubstituted monocyclic alkyl, unsubstituted heterocyclic alkyl, substituted monocyclic alkyl and substituted heterocyclic alkyl; R2 is absent or selected from one of hydrogen, deuterium, hydroxyl, methyl, ethyl, isopropyl, methoxy, ethoxy, halogen, 4-piperidinone glycol condensate, 6-azaspiro[2.5]octane, N-methylpiperazine, N-ethylpiperazine, N-isopropylpiperazine, 1-(2-hydroxyethyl)piperazine, piperazine-1-ylmethanol, and 1-(2-methoxyethyl)piperazine; R3 is absent or selected from one of hydrogen, deuterium, hydroxyl, halogen, methyl, ethyl and isopropyl; Z is a carbon atom or a nitrogen atom; when Z is a carbon atom, R4 is selected from hydrogen, deuterium, halogen, methoxy, ethoxy, hydroxymethyl, C l-5 Alkyl, C l-5 Haloalkyl, C l-5 One of alkoxy, cyclopropane, cyclobutane, cyclopentane, cyclohexane, and cycloheptane; R4 is absent when Z is a nitrogen atom; Ring A is selected from pyridine ring, pyrrole ring, pyrimidine ring, benzene ring, indole ring, quinoxalinyl, tetrahydroindole ring, piperazine ring, 2-hydroxypyridine, cyclohexene, 1-hydroxy-3-cyclohexene, 2-cyclohexen-1-one, quinolinyl, isoquinoline, 5,6,7,8-tetrahydroquinoline, etc. One of them; In the above, X” is CH2, NH, O or S; p is 1, 2, 3 or 4; g is 1, 2, 3 or 4; R5 is absent or is one of hydrogen, deuterium, halogen, hydroxyl, hydroxymethyl, methyl, ethyl, propyl, butyl, isopropyl, methoxy, trifluoromethyl, difluoromethyl, trifluoromethoxy and difluoromethoxy; R6 is absent or is one of hydrogen, deuterium, halogen, hydroxyl, hydroxymethyl, methyl, ethyl, propyl, butyl, isopropyl, methoxy, methylcyclopropane, prop-1-ylcyclopropane, but-2-yn-1-ol, but-2-yn-yne.

2. The compound with high inhibitory activity against coagulation factor XIIa according to claim 1, characterized in that: R1 is In this case, R7 is selected from one of unsubstituted monocyclic alkyl, unsubstituted heterocyclic alkyl, substituted monocyclic alkyl, substituted heterocyclic alkyl, unsubstituted aromatic ring and substituted aromatic ring, and R8 is selected from one of unsubstituted monocyclic alkyl, unsubstituted heterocyclic alkyl, substituted monocyclic alkyl and substituted heterocyclic alkyl.

3. The compound with high inhibitory activity against coagulation factor XIIa according to claim 2, characterized in that: In R7, the unsubstituted monocyclic alkyl group is selected from one of cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, and cyclooctane; in R7, the unsubstituted heterocyclic alkyl group and the substituted heterocyclic alkyl group are selected from one of ethylene oxide, oxetane, and oxetane; in R7, the substituted monocyclic alkyl group is selected from one of substituted cyclopropane, substituted cyclobutane, substituted cyclopentane, and substituted cyclohexane. In the R7, when there is one substituent of the substituted monocyclic alkyl or substituted heterocyclic alkyl, the one substituent is substituted on any methylene group; when there are multiple substituents, the multiple substituents are substituted on any methylene group respectively. The substituents are independently alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro or cyano. In R7, the unsubstituted aromatic ring is selected from one of benzene ring, pyrrole, thiophene, pyridine and furan; In R7, the substituted aromatic ring is selected from one of 2-chlorophenyl, 3-oxophenyl, 3-oxo-5-oxophenyl, 2-cyanophenyl, 3-cyanophenyl, 3-ethoxyphenyl, 3-ethylphenyl, 2-oxophenyl, 3-oxophenyl, 4-oxophenyl, 3-isopropylphenyl, 3-methoxyphenyl, 2-methylphenyl, 3-methylphenyl, 3-nitrophenyl, 3-dioxomethoxyphenyl, 3-difluoromethylphenyl, 2-pyridyl, 4-chloro-2-pyridyl, 5-chloro-2-pyridyl, 5-fluoro-2-pyridyl, 6-fluoro-2-pyridyl, 4-methyl-2-pyridyl, 6-methoxy-3-pyridyl, 6-methyl-2-pyridyl, 4-trifluoromethyl-2-pyridyl, and 6-fluoro-3-pyridyl. In R8, the unsubstituted monocyclic alkyl group is selected from one of cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, and cyclooctane; the unsubstituted heterocyclic alkyl group and the substituted heterocyclic alkyl group are selected from one of ethylene oxide, oxetane, and oxetane; and the substituted monocyclic alkyl group is selected from one of substituted cyclopropane, substituted cyclobutane, substituted cyclopentane, and substituted cyclohexane.

4. The compound with high inhibitory activity against coagulation factor XIIa according to claim 1, characterized in that: R1 is In this case, R9 is selected from one of unsubstituted monocyclic alkyl, unsubstituted heterocyclic alkyl, substituted monocyclic alkyl, and substituted heterocyclic alkyl. 10 It is selected from one of unsubstituted monocyclic alkyl, unsubstituted heterocyclic alkyl, substituted monocyclic alkyl and substituted heterocyclic alkyl.

5. A compound with high inhibitory activity against coagulation factor XIIa according to claim 4, characterized in that: In R9, the unsubstituted monocyclic alkyl group is selected from one of cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, and cyclooctane; the unsubstituted heterocyclic alkyl group and the substituted heterocyclic alkyl group are selected from one of ethylene oxide, oxetane, and oxetane; the substituted monocyclic alkyl group is selected from one of substituted cyclopropane, substituted cyclobutane, substituted cyclopentane, and substituted cyclohexane. The R mentioned 10 In this context, the unsubstituted monocyclic alkyl group is selected from one of cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, and cyclooctane; the unsubstituted heterocyclic alkyl group and the substituted heterocyclic alkyl group are selected from one of ethylene oxide, oxetane, and oxecyclopentane; the substituted monocyclic alkyl group is selected from one of substituted cyclopropane, substituted cyclobutane, substituted cyclopentane, and substituted cyclohexane.

6. A compound with high inhibitory activity against coagulation factor XIIa according to claim 1, characterized in that: The general formula (Ⅰ) is any one of the following expressions (Ⅰ-1) to (Ⅰ-10): In (Ⅰ-4) and (Ⅰ-5), * represents a chiral carbon atom.

7. A pharmaceutical composition, characterized in that: Includes one or a mixture of the compounds of claim 1, stereoisomers of the compounds, tautomers of the compounds, and pharmaceutically acceptable salts thereof.

8. Use of the compound of claim 1 or the pharmaceutical composition of claim 7 in the preparation of a medicament for inhibiting coagulation factor XIIa.

9. The use according to claim 7, characterized in that: The aforementioned drug for inhibiting coagulation factor XIIa is used to prevent and treat diseases mediated by coagulation factor XIIa by inhibiting coagulation factor XIIa.

10. The use according to claim 7, characterized in that: Stable isotope derivatives, metabolites, or prodrugs of the compounds may prevent and / or treat thrombosis and hereditary angioedema while avoiding or mitigating the side effects of existing anticoagulation therapies.

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