Selective glutamine acyl cyclase isoenzyme inhibitor as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicinal chemistry, and discloses a selective glutaminyl cyclase isoenzyme inhibitor as well as a preparation method and application thereof. Wherein the structural general formula of the selective glutamine acyl cyclase isoenzyme inhibitor is # imgabs 0 # R, and the selective glutamine acyl cyclase isoenzyme inhibitor is selected from one of # imgabs 1 #. The glutamine acyl cyclase isoenzyme inhibitory activity of the selective glutamine acyl cyclase isoenzyme inhibitor is superior to that of glutamine acyl cyclase, and the selective glutamine acyl cyclase isoenzyme inhibitor has glutamine acyl cyclase isoenzyme selectivity; the risk of cross reaction with glutaminyl cyclase when glutaminyl cyclase isoenzyme is inhibited can be reduced, and side effects possibly brought by clinical application are reduced. Meanwhile, the preparation method of the selective glutamine acyl cyclase isoenzyme inhibitor is simple to operate, raw materials are easy to obtain, and the selective glutamine acyl cyclase isoenzyme inhibitor can be widely applied to preparation of drugs for treating diseases related to high specific expression of glutamine acyl cyclase isoenzyme.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and in particular to a selective glutaminyl cyclase isozyme inhibitor, a preparation method and application thereof. Background Art

[0002] Glutaminyl cyclase (QC) and its isoenzyme (isoglutaminylcyclase, isoQC) mainly catalyze the cyclization of N-terminal glutamate and glutamine of polypeptides / proteins to generate pyroglutamate (pGlu / pE), and have important biological functions such as changing the chemical structure of the N-terminus, regulating biological functions, and enhancing stability. QC and isoQC have similar structural characteristics and substrate selectivity, but QC abnormalities mainly induce neurodegenerative diseases, while isoQC specific activity upregulation is more related to the occurrence of inflammation, cancer, etc. (Chenshu X, YiNan W, Haiqiang W. Glutaminyl Cyclase, Diseases, and Development of Glutaminyl Cyclase Inhibitors. [J]. Journal of medicinal chemistry, 2021, 64 (10): 6549-6565.).

[0003] Given that isoQC and QC have similar highly conserved catalytic domains, reported QC / isoQC inhibitors usually have inhibitory effects on both QC and isoQC. Studies on isoQC also mostly use reported QC inhibitors. Existing QC / isoQC inhibitors mainly include: PBD150 and DPCI-28 of imidazoles, PQ912 and PQ529 of benzimidazoles, SEN177 and QP5038 of triazoles, etc. However, the QC inhibitory activity of these QC / isoQC inhibitors is generally better than their isoQC inhibitory activity. Only SEN177 (IC 50QC =0.15μM, IC 50 isoQC =0.05μM) showed that the inhibitory activity of isoQC was superior to that of QC. However, isoQC and QC are involved in different types of diseases. Direct application of non-selective QC / isoQC inhibitors to diseases induced by abnormal upregulation of isoQC may induce serious adverse reactions. Therefore, it is crucial to develop selective isoQC inhibitors to reduce the risk and side effects of cross-reactions between QC and isoQC. However, there is a lack of research reports on selective isoQC inhibitors.

[0004] Therefore, the existing technology still needs to be improved and developed. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a selective glutaminyl cyclase isozyme inhibitor and a preparation method and application thereof, aiming to solve the problem that the existing QC / isoQC inhibitors are not selective and will produce cross reactions and side effects with QC when applied to diseases induced by abnormal upregulation of isoQC.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect of the present invention, a selective glutaminyl cyclase isozyme inhibitor is provided. The general structural formula of the selective glutaminyl cyclase isozyme inhibitor is as follows:

[0008]

[0009] R is selected from one of the following substituents:

[0010]

[0011] Among them, R 1 Selected from hydrogen, C 1-6 Alkyl, C 5-7 One of aryl and benzyloxy;

[0012] R 2 and R 4 are each independently selected from hydrogen, halogen, nitro, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylamino, C 1-6 Fluoroalkyl, C 1-6 Fluoroalkoxy, C 1-6 Ester group, C 5-7 One of aryl and benzyloxy;

[0013] R 3 Selected from hydrogen, halogen, nitro, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylamino, C 1-6 Fluoroalkyl, C 1-6 Fluoroalkoxy, C 1-6 Ester group, C 5-7 Aryl, phenoxy, benzyloxy, C 7-10 Aryl substituted acyl, C 7-10 Aryl substituted ester group, C 7-10 Aryl substituted amide, C 7-10 One of the aryl-substituted amino groups;

[0014] R 5 and R 6are each independently selected from hydrogen or C 1-6 alkyl;

[0015] Among them, the C 5-7 The aryl group is unsubstituted or substituted by halogen, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Fluoroalkyl substitution.

[0016] The preferred technical solution, R 1 One selected from hydrogen, phenyl, benzyloxy;

[0017] R 2 and R 4 Each is independently selected from one of hydrogen, fluorine, nitro, methoxy, dimethylamino, trifluoromethyl, trifluoromethoxy, methyl acetate, phenyl, and benzyloxy;

[0018] R 3 One selected from hydrogen, fluorine, nitro, tert-butyl, methoxy, isobutoxy, dimethylamino, trifluoromethyl, trifluoromethoxy, methyl acetate, phenyl, benzyloxy, acetyl, benzoyl, benzoate, benzamide, 4-amino-2-(trifluoromethyl)phenyl, 3,4,5-trimethoxybenzamide, and 3-fluoroanilino;

[0019] R 5 and R 6 Each is independently selected from hydrogen or methyl.

[0020] In a preferred technical solution, the general structural formula of the selective glutaminyl cyclase isozyme inhibitor is as follows:

[0021]

[0022] Among them, R 3 One selected from the group consisting of hydrogen, nitro, methyl acetate, phenyl, benzoyl, benzoate, benzamide, 3,4,5-trimethoxybenzamide, and 3-fluoroaniline;

[0023] R 4 Selected from nitro or trifluoromethyl.

[0024] In a preferred technical solution, the selective glutaminyl cyclase isozyme inhibitor is selected from one of the following structures:

[0025]

[0026]

[0027]

[0028] The second aspect of the present invention provides a method for preparing the selective glutaminyl cyclase isozyme inhibitor as described in the first aspect, wherein the synthetic route of the selective glutaminyl cyclase isozyme inhibitor is:

[0029]

[0030]

[0031] A preferred technical solution, in the synthesis route of the selective glutaminyl cyclase isozyme inhibitor, the first reaction condition specifically includes: the reaction solvent is selected from one or more of acetone, acetonitrile, tetrahydrofuran, dioxane or N,N-dimethylformamide, the reaction time is 2 to 12 hours, the reaction temperature is 50 to 80°C, and the acid binding agent is selected from one or more of potassium carbonate, sodium carbonate, and triethylamine.

[0032] The preferred technical solution, the second reaction condition specifically includes: carrying out in an aerobic environment, the reaction solvent is selected from one or more of methanol, ethanol, and dichloromethane, the reaction time is 5 to 24 hours, the reaction temperature is 10 to 50° C., the acid binding agent is selected from one or more of potassium carbonate, sodium carbonate, and triethylamine, and the catalyst is copper acetate or copper (I) thiophene-2-carboxylic acid.

[0033] In a preferred technical solution, the third reaction condition specifically includes: the reaction solvent is selected from one or more of acetone, dichloromethane, tetrahydrofuran, and dioxane, the reaction time is 0.5 to 2 hours, and the reaction temperature is 0 to 10°C.

[0034] The preferred technical solution is that the synthetic route of intermediate I is:

[0035]

[0036] The fourth reaction condition specifically includes: the reaction solvent is selected from one or more of methanol, ethanol, acetone, tetrahydrofuran, and dioxane, the reaction time is 2 to 8 hours, and the reaction temperature is 50 to 80° C.;

[0037] The fifth reaction conditions specifically include: the reaction solvent is water, the reaction time is 1 to 5 hours, and the reaction temperature is 80 to 100° C.;

[0038] The sixth reaction condition specifically includes: the reaction solvent is selected from one or more of methanol, ethanol, acetone, dichloromethane, tetrahydrofuran, and dioxane, the reaction time is 0.5 to 2 hours, and the reaction temperature is 10 to 50° C.;

[0039] The seventh reaction condition specifically includes: the reaction solvent is selected from one or more of methanol, ethanol, acetone, dichloromethane, tetrahydrofuran, and dioxane, the reaction time is 2 to 8 hours, and the reaction temperature is 20 to 80°C.

[0040] The third aspect of the present invention provides a use of the selective glutaminyl cyclase isozyme inhibitor as described in the first aspect in the preparation of a drug for treating diseases related to specific high expression of glutaminyl cyclase isozymes.

[0041] In a preferred technical solution, the disease associated with the specific high expression of glutaminyl cyclase isozyme is inflammation caused by high expression of pE-CCL2.

[0042] In a preferred technical solution, the disease associated with the specific high expression of glutaminyl cyclase isozymes is cancer caused by high expression of pE-CD47.

[0043] Beneficial effects: The present invention provides a selective glutaminyl cyclase isozyme inhibitor and its preparation method and application. The structural general formula of the selective glutaminyl cyclase isozyme inhibitor is R is selected from Compared with the prior art, the isoQC inhibitory activity of this type of selective glutaminyl cyclase isozyme inhibitor is better than the QC inhibitory activity, has isoQC selectivity, can reduce the risk of cross-reaction with QC when inhibiting isoQC, and reduce the side effects that may be caused by the clinical application of isoQC inhibitors. At the same time, the preparation method of this type of selective glutaminyl cyclase isozyme inhibitor is simple to operate and the raw materials are easily available, and it can be widely used in the preparation of drugs for treating diseases related to the specific high expression of glutaminyl cyclase isozymes. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is a schematic diagram of the molecular docking of existing isoQC inhibitors with isoQC and QC.

[0045] Figure 2 Schematic diagram of molecular docking of compound 1 with isoQC and QC.

[0046] Figure 3 Schematic diagram of molecular docking of compound 37 with isoQC and QC. DETAILED DESCRIPTION

[0047] The present invention provides a selective glutaminyl cyclase isozyme inhibitor and a preparation method and application thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below.

[0048] Abnormal upregulation of glutaminyl cyclase (QC) and its isoenzymes (isoglutaminylcyclase, isoQC) is closely related to the occurrence and development of a variety of complex diseases. Among them, QC abnormalities mainly induce neurodegenerative diseases, while isoQC specific activity upregulation is more related to the occurrence of inflammation, cancer, etc. Given that isoQC and QC have similar highly conserved catalytic domains, reported QC / isoQC inhibitors usually have inhibitory effects on both QC and isoQC, which makes the existing QC / isoQC inhibitors likely to induce serious adverse reactions when used. Therefore, it is crucial to develop selective isoQC inhibitors to reduce the risk of cross-reactions and side effects between QC and isoQC. However, no selective isoQC inhibitors have been reported so far.

[0049] The applicant of the present invention disclosed a structural formula in 2022 The glutaminyl cyclase isozyme inhibitor (patent number ZL202210526819.6) has a structure with methyltriazole-piperidine-sulfonyl-aryl-imino-aryl as the skeleton, which shows good inhibitory activity against isoQC. However, the inventors further tested and found that the structure has inhibitory activity against both isoQC and QC, and the QC inhibitory activity is generally better than the iosQC inhibitory activity, and cannot be used as a selective isoQC inhibitor.

[0050] In this regard, the inventor uses a specific structure (Comparative Example 1 of the present invention) as an example, the interaction mode between the structure and isoQC and QC is further analyzed. Figure 1 As shown in Figure A, in the isoQC structure, the 1st N atom of the methyltriazole group can form a coordination with the zinc ion at the bottom of the active pocket, the imino N atom can form a hydrogen bond with Ser236, and the benzene ring can form a π-type hydrogen bond with Pro233 and Glu325, so this structure has good isoQC inhibitory activity. Figure 1 As shown in B, in this structure, the 1st N atom of the methyltriazole group can form a coordination effect with the zinc ion at the bottom of the active pocket, and the benzene ring can form a π-π stacking effect with Phe325. Therefore, this structure has good QC inhibitory activity. Figure 1 It can be seen that the structure forms the same coordination with zinc ions at the bottom of the active pockets of isoQC and QC, but has different interactions outside the active pockets of isoQC and QC. Therefore, the key to designing selective isoQC inhibitors is to change the interaction of the structure with the amino acid residues outside the active pockets of isoQC and QC, that is, to increase its interaction with the amino acid residues outside the active pocket of isoQC, and / or reduce its interaction with the amino acid residues outside the active pocket of QC.

[0051] Based on this, an embodiment of the present invention provides a selective glutaminyl cyclase isozyme inhibitor, and the general structural formula of the selective glutaminyl cyclase isozyme inhibitor is as follows:

[0052]

[0053] R is selected from one of the following substituents:

[0054]

[0055] Among them, R 1 Selected from hydrogen, C 1-6 Alkyl, C 5-7 One of aryl and benzyloxy;

[0056] R 2 and R 4 are each independently selected from hydrogen, halogen, nitro, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylamino, C 1-6 Fluoroalkyl, C 1-6 Fluoroalkoxy, C 1-6 Ester group, C 5-7 One of aryl and benzyloxy;

[0057] R 3 Selected from hydrogen, halogen, nitro, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylamino, C 1-6 Fluoroalkyl, C 1-6 Fluoroalkoxy, C 1-6 Ester group, C 5-7 Aryl, phenoxy, benzyloxy, C 7-10 Aryl substituted acyl, C 7-10 Aryl substituted ester group, C 7-10 Aryl substituted amide, C 7-10 One of the aryl-substituted amino groups;

[0058] R 5 and R 6 are each independently selected from hydrogen or C 1-6 alkyl;

[0059] Among them, the C 5-7 The aryl group is unsubstituted or substituted by halogen, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Fluoroalkyl substitution.

[0060] Specifically, the existing structure Although disclosed as an isoQC inhibitor, actual tests have found that its QC inhibition activity is superior to isoQC inhibition activity and it does not have isoQC selectivity. In the embodiment of the present invention, the rigid sulfonyl group in the existing structure is replaced with a flexible alkyl group to obtain a new structure with a methyltriazole-piperidine-alkyl-aryl-imino-aryl group as the skeleton.

[0061] Compound 1 Take the new structure as an example to analyze the interaction mode between isoQC and QC. Figure 2 As shown in Figure A, in the new structure, the 1st N atom of the methyltriazole group can form a coordination with the zinc ion at the bottom of the active pocket, and the imino N atom can form a hydrogen bond with Ser236, ensuring the isoQC inhibitory activity of this structure. Figure 2 As shown in B, in the new structure, the 1st N atom of the methyltriazole group can form a coordination with the zinc ion at the bottom of the active pocket, but the flexible alkyl group allows the group located outside the QC active pocket to rotate more freely and no longer interact with the amino acid residues outside the QC active pocket due to conformational restrictions. Therefore, the iosQC inhibitory activity of the new structure is better than that of QC, and it has isoQC selectivity.

[0062] Furthermore, in the embodiment of the present invention, the terminal aromatic group is subjected to R 1 ~R 6 By modifying the isoQC structure with a variety of different substituents, we screened out substituents that can interact with the outside of the isoQC active pocket, thereby improving the isoQC selectivity and isoQC inhibitory activity of the new structure.

[0063] In some embodiments, R 1 One selected from hydrogen, phenyl, benzyloxy;

[0064] R 2 and R 4 Each is independently selected from one of hydrogen, fluorine, nitro, methoxy, dimethylamino, trifluoromethyl, trifluoromethoxy, methyl acetate, phenyl, and benzyloxy;

[0065] R 3 One selected from hydrogen, fluorine, nitro, tert-butyl, methoxy, isobutoxy, dimethylamino, trifluoromethyl, trifluoromethoxy, methyl acetate, phenyl, benzyloxy, acetyl, benzoyl, benzoate, benzamide, 4-amino-2-(trifluoromethyl)phenyl, 3,4,5-trimethoxybenzamide, and 3-fluoroanilino;

[0066] R 5 and R 6 Each is independently selected from hydrogen or methyl.

[0067] In some embodiments, the selective glutaminyl cyclase isozyme inhibitor has the following general structural formula:

[0068]

[0069] Among them, R 3 One selected from the group consisting of hydrogen, nitro, methyl acetate, phenyl, benzoyl, benzoate, benzamide, 3,4,5-trimethoxybenzamide, and 3-fluoroaniline;

[0070] R 4 is selected from nitro or trifluoromethyl; preferably, R 4 For nitro.

[0071] Specifically, the inventors found through testing that: 4 When it is a strong electron-withdrawing group, it can improve the selectivity of the compound for isoQC; in particular, R 4 When the nitro group is present, the selectivity of the compound for isoQC is significantly improved.

[0072] Compound 37 This further defines the new structure and the mode of action of isoQC and QC. Figure 3 As shown in Figure A, the new structure further defines that in isoQC, the 1st N atom of the methyltriazole group can form a coordination with the zinc ion at the bottom of the active pocket, the nitro O atom can form a hydrogen bond with Pro233 and Arg241, and the ester O atom can form a hydrogen bond with Arg241, so this structure has good isoQC inhibitory activity. Figure 3 As shown in B, in the further defined new structure, in QC, the N atom No. 1 of the methyltriazole group can form a coordination effect with the zinc ion at the bottom of the active pocket, and the nitro O atom can form a hydrogen bond with Phe325, but the formation of this hydrogen bond hinders the π-π stacking effect between the benzene ring and Phe325. Therefore, the QC inhibitory activity of compound 37 is reduced relative to the QC inhibitory activity of the existing isoQC inhibitor in Comparative Example 1. With the dual advantages of increasing the interaction with the amino acid residues outside the isoQC active pocket and reducing the interaction with the amino acid residues outside the QC active pocket, the iosQC inhibitory activity of the further defined new structure is significantly better than the QC inhibitory activity, more than 10 times.

[0073] In some embodiments, the selective glutaminyl cyclase isozyme inhibitor is selected from one of the following structures:

[0074]

[0075]

[0076]

[0077] The embodiment of the present invention also provides a method for preparing the selective glutaminyl cyclase isozyme inhibitor as described above. The synthetic route of the selective glutaminyl cyclase isozyme inhibitor is as follows:

[0078]

[0079]

[0080] In some embodiments, in the synthesis route of the selective glutaminyl cyclase isozyme inhibitor, the first reaction condition specifically includes: the reaction solvent is selected from one or more of acetone, acetonitrile, tetrahydrofuran, dioxane or N,N-dimethylformamide, the reaction time is 2 to 12 hours, the reaction temperature is 50 to 80°C, and the acid binding agent is selected from one or more of potassium carbonate, sodium carbonate, and triethylamine.

[0081] In some embodiments, the second reaction condition specifically includes: carrying out in an aerobic environment, the reaction solvent is selected from one or more of methanol, ethanol, and dichloromethane, the reaction time is 5 to 24 hours, the reaction temperature is 10 to 50° C., the acid binding agent is selected from one or more of potassium carbonate, sodium carbonate, and triethylamine, and the catalyst is copper acetate or copper (I) thiophene-2-carboxylic acid.

[0082] Furthermore, in some embodiments, in the second reaction condition, the reaction is carried out in an aerobic environment, using oxygen in the air through an open reaction or adding 2,2,6,6-tetramethylpiperidinyl oxide as an oxidant.

[0083] In some embodiments, the third reaction condition specifically includes: the reaction solvent is selected from one or more of acetone, dichloromethane, tetrahydrofuran, and dioxane, the reaction time is 0.5 to 2 hours, and the reaction temperature is 0 to 10°C.

[0084] In some embodiments, the synthetic route of intermediate I is:

[0085]

[0086] The fourth reaction condition specifically includes: the reaction solvent is selected from one or more of methanol, ethanol, acetone, tetrahydrofuran, and dioxane, the reaction time is 2 to 8 hours, and the reaction temperature is 50 to 80° C.;

[0087] The fifth reaction conditions specifically include: the reaction solvent is water, the reaction time is 1 to 5 hours, and the reaction temperature is 80 to 100° C.;

[0088] The sixth reaction condition specifically includes: the reaction solvent is selected from one or more of methanol, ethanol, acetone, dichloromethane, tetrahydrofuran, and dioxane, the reaction time is 0.5 to 2 hours, and the reaction temperature is 10 to 50° C.;

[0089] The seventh reaction condition specifically includes: the reaction solvent is selected from one or more of methanol, ethanol, acetone, dichloromethane, tetrahydrofuran, and dioxane, the reaction time is 2 to 8 hours, and the reaction temperature is 20 to 80°C.

[0090] The embodiment of the present invention also provides a use of the selective glutaminyl cyclase isozyme inhibitor as described above in the preparation of a drug for treating diseases related to specific high expression of glutaminyl cyclase isozymes.

[0091] In some embodiments, the disease associated with specific high expression of glutaminyl cyclase isozymes is inflammation caused by high expression of pE-CCL2.

[0092] In some embodiments, the disease associated with specific high expression of glutaminyl cyclase isozymes is cancer caused by high expression of pE-CD47.

[0093] The present invention will be further described below by means of specific examples.

[0094] Example 1: Preparation of Intermediate I

[0095]

[0096] Synthesis of tert-butyl 4-(2-(methylcarbamoyl)hydrazine-1-carbonyl)piperidine-1-carboxylate (Intermediate i): tert-butyl 4-(hydrazinecarbonyl)piperidine-1-carboxylate (10.62 mmol, 1 equiv) was dissolved in ethanol (20 mL), methyl isothiocyanate (15.51 mmol, 1.5 equiv) was added, and the mixture was reacted at 70°C for 3 h. After the reaction was completed, the mixture was allowed to stand and cool, and a white solid precipitated. The mixture was filtered, washed with water, and dried to obtain 2.97 g of a white solid with a yield of 88.52%.

[0097] Synthesis of tert-butyl 4-(4-methyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate (Intermediate ii): tert-butyl 4-(2-((methyl-2-azepinyl)carbonylthio)hydrazine-1-carbonyl)piperidine-1-carboxylate (9.42mmol, 1equiv) was dissolved in water (20mL), sodium hydroxide (18.85mmol, 2equiv) was added, and the mixture was reacted at 100°C for 2h. After the reaction was completed, the mixture was allowed to stand and cool, and 10% hydrochloric acid (14mL) was added dropwise. A white solid precipitated, which was filtered, washed with water, and dried to obtain 2.40g of a white solid with a yield of 85.34%.

[0098] Synthesis of tert-butyl 4-(4-methyl-4H-1,2,4-triazol-3-yl)piperidine-1-carboxylate (Intermediate iii): tert-butyl 4-(4-methyl-5-thio-4,5-dihydro-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate (8.04 mmol, 1 equiv) was dissolved in dichloromethane (20 mL), and glacial acetic acid (6 mL) and 30% hydrogen peroxide solution (2 mL) were added, and the mixture was reacted at 25° C. for 0.5 h. After the reaction was completed, 10% sodium hydroxide solution was added to quench the reaction, and the mixture was extracted with dichloromethane three times. The organic phases were combined, dried over anhydrous sodium sulfate, and 1.55 g of a colorless oily liquid was obtained by spin drying, with a yield of 72.53%.

[0099] Synthesis of 4-(4-methyl-4H-1,2,4-triazol-3-yl)piperidine (Intermediate I): tert-butyl 4-(4-methyl-4H-1,2,4-triazol-3-yl)piperidine-1-carboxylate (5.83mmol, 1equiv) was dissolved in methanol (20mL), trifluoroacetic acid (2mL) was added, and the mixture was reacted at 70°C for 4h. After the reaction was completed, the mixture was allowed to stand and cool, sodium hydroxide solution was added to adjust the pH to 8, the mixture was spin-dried, and the mixture was purified by silica gel flash chromatography with a mobile phase of methanol: ammonia water = 100:1 to obtain 0.68g of a white solid with a yield of 70.98%.

[0100] Example 2: Preparation of Compound 1

[0101]

[0102] Synthesis of 3-(bromomethyl)aniline: (3-aminophenyl)methanol (10.56mmol, 1equiv) was dissolved in acetone (20mL) from which water had been distilled off, and phosphorus tribromide (30.16mmol, 3equiv) was added, and the mixture was reacted in an ice bath for 0.5h. After the reaction was completed, the reaction solution was added dropwise to a 10% sodium hydroxide solution to quench, extracted three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and dried by spin drying. The organic phases were purified by silica gel flash chromatography, and the mobile phase was n-hexane: ethyl acetate: ammonia water = 100: 50: 1, to obtain a yellow oily liquid.

[0103] Synthesis of 3-((4-(4-methyl-4H-1,2,4-triazol-3-yl)piperidin-1-yl)methyl)aniline (Intermediate IV): 4-(4-methyl-4H-1,2,4-triazol-3-yl)piperidine (Intermediate I prepared in Example 1, 3.01 mmol, 1 equiv) was dissolved in acetone (20 mL) from which water was distilled off, potassium carbonate (6.01 mmol, 2 equiv) was added, stirred at 25°C for 30 min, 3-(bromomethyl)aniline (4.52 mmol, 1.5 equiv) was added, and the mixture was reacted at 70°C for 4 h. After the reaction was completed, the mixture was extracted with ethyl acetate three times, the organic phases were combined, dried over anhydrous sodium sulfate, spin-dried, and purified by silica gel flash chromatography, with the mobile phase being ethyl acetate: methanol: ammonia water = 100:20:1, to obtain a white solid.

[0104] Synthesis of 3-((4-(4-methyl-4H-1,2,4-triazol-3-yl)piperidin-1-yl)methyl)-N-phenylaniline (1): 3-((4-(4-methyl-4H-1,2,4-triazol-3-yl)piperidin-1-yl)methyl)aniline (1.71 mmol, 1 equiv) and phenylboronic acid (2.56 mmol, 1.5 equiv) were dissolved in dichloromethane (20 mL), and copper acetate monohydrate (CuAc, 3.42 mmol, 2 equiv), potassium carbonate (3.45 mmol, 2 equiv) and 2,2,6,6-tetramethylpiperidinoxide (TEMPO, 1.72 mmol, 1 equiv) were added, and the reaction was carried out at 25°C for 12 h. After the reaction was completed, the product was extracted with ethyl acetate three times, the organic phases were combined, dried over anhydrous sodium sulfate, spin-dried, and purified by silica gel flash chromatography with the mobile phase being ethyl acetate: methanol: ammonia water = 100: 20: 1 to obtain a white solid with a total yield of 11%.

[0105] 1 H NMR (500MHz, DMSO) δ8.31(s,1H),8.15(s,1H),7.27-7.14(m,3H),7.09-7.04(m,3H),6.96(dd,J=8.0,1.6Hz,1H),6.81(t,J=7.3Hz,1H),6. 76(d,J=7.5Hz,1H),3.59(s,3H),3.44(s,2H),2.91(d,J=11.5Hz,2H),2.77(tt,J=11.4,3.8Hz,1H),2.12-2.03(m,2H),1.87-1.70(m,4H). 13C NMR (126MHz, DMSO) δ156.93,145.00,143.93,143.80,139.97,129.60,129.34,120.66, 120.04,117.47,117.16,115.53,62.96,53.29,31.71,30.57,30.49.HRMS(ESI):calcd for C 21 H 25 N5 347.2110, found [M+H] + 348.2176.HPLC purity=98.0%.

[0106] Example 3: Preparation of Compound 20

[0107]

[0108] Synthesis of 3-(4-(4-methyl-4H-1,2,4-triazol-3-yl)piperidin-1-yl)methyl)-N-(3-nitrophenyl)aniline (20): According to the synthesis method of compound 1 in Example 2, 3-nitrophenylboronic acid and intermediate IV were reacted to obtain compound 20 as a yellow solid with an overall yield of 33%.

[0109] 1 H NMR(500MHz,DMSO-d6)δ8.75(s,1H),8.31(s,1H),7.80(t,J=2.2Hz,1H),7.58( dd,J=7.5,1.7Hz,1H),7.49-7.39(m,2H),7.28(t,J=7.7Hz,1H),7.15(s,1H),7. 09-7.04(m,1H),6.92(d,J=7.5Hz,1H),3.59(s,3H),3.49(s,2H),2.92(d,J=11 .5Hz,2H),2.77(m,1H),2.14-2.05(m,2H),1.84(d,J=10.7Hz,2H),1.76(m,2H). 13 C NMR(126MHz,DMSO-d6)δ156.44,148.74,145.45,144.54,141.48,139.84,130.47,129.21,122.32,1 21.53,119.06,117.07,113.07,108.59,62.25,52.76,31.30,31.22,30.10,30.02.HRMS(ESI):calcd forC 21 H 24N6O2 392.1961, found [M+H] + 393.2068.HPLC purity=95%.

[0110] Example 4: Preparation of Compound 31

[0111]

[0112] Synthesis of (3-(4-(4-methyl-4H-1,2,4-triazol-3-yl)piperidin-1-yl)methyl)phenyl)boronic acid (Intermediate II): (3-(bromomethyl)phenyl)boronic acid (1 mmol, 1 equiv) and 4-(4-methyl-4H-1,2,4-triazol-3-yl)piperidine (Intermediate I prepared in Example 1, 1 mmol, 1 equiv) were dissolved in acetonitrile (10 mL), potassium carbonate (2 mmol, 2 equiv) was added, and the mixture was reacted at 80° C. for 12 h. After the reaction was completed, the mixture was filtered, the filtrate was collected, and the solvent was removed by distillation under reduced pressure to obtain a white solid.

[0113] Synthesis of N-(3-((4-(4-methyl-4H-1,2,4-triazol-3-yl)piperidin-1-yl)methyl)phenyl)-4-nitro-3-(trifluoromethyl)aniline (31): (3-(4-(4-methyl-4H-1,2,4-triazol-3-yl)piperidin-1-yl)methyl)phenyl)boric acid (0.28 mmol, 2 equiv) and 4-nitro-3-(trifluoromethyl)aniline (0.14 mmol, 1 equiv) were dissolved in dichloromethane (5 mL), and copper (I) thiophene-2-carboxylic acid (CuTC, 0.07 mmol, 0.5 equiv) and potassium carbonate (0.28 mmol, 2 equiv) were added, oxygen was introduced, and the reaction was carried out at 25°C for 12 h. After the reaction, dichloromethane was removed by distillation under reduced pressure, and the mixture was extracted with ethyl acetate three times. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel flash chromatography, with the mobile phase being ethyl acetate: methanol: ammonia water = 100: 5: 1. The crude product was further purified by high performance liquid chromatography (HPLC), with the mobile phase being methanol: water (0.1% ammonia water) = 65: 35, to obtain a yellow solid with a total yield of 11%.

[0114] 1H NMR (500MHz, DMSO-d6) δ9.58(s,1H),8.32(s,1H),8.13(d,J=9.1Hz,1H),7.44-7.33(m,2H),7.31-7.22(m,2H),7.18(dd,J=7.8, 2.2Hz,1H),7.11(d,J=7.6Hz,1H),3.59(s,5H),2.97(d,J=19.4Hz,2H),2.80(s,1H),2.13(d,J=48.9Hz,2H),1.93-1.71(m,4H). 13 CNMR(126MHz,DMSO-d6)δ156.34,149.65,144.61,144.59,139.38,136.10,129.61,124.96-124.74(m),123. 42,126.00-120.78(m),119.75,112.96,δ52.59,52.56,48.61,30.04,29.85,29.82,29.03.HRMS(ESI):calcd for C 22 H 23 F3N6O2 460.1835, found [M+H] + 461.1997.HPLC purity=94%.

[0115] Example 5: Preparation of Compound 33

[0116]

[0117] Synthesis of 2-nitro-[1,1'-biphenyl]-4-amine: 4-bromo-3-nitroaniline (1 mmol, 1 equiv) and phenylboronic acid (2 mmol, 2 equiv) were dissolved in a mixed solution (10 mL) of acetonitrile: saturated potassium carbonate aqueous solution = 1:1, and a catalyst [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium (II) dichloromethane complex (DPPF, 0.06 mmol, 0.06 equiv) was added. The reaction system was protected by nitrogen and reacted at 100°C for 3 h. After the reaction, the mixture was extracted with ethyl acetate three times, the organic phases were combined, dried over anhydrous sodium sulfate, the solvent was removed by distillation under reduced pressure, and purified by silica gel flash chromatography. The mobile phase was n-hexane: ethyl acetate: ammonia water = 100: 5: 1 to obtain a yellow solid.

[0118] Synthesis of N-(3-((4-(4-methyl-4H-1,2,4-triazol-3-yl)piperidin-1-yl)methyl)phenyl)-2-nitro-[1,1'-biphenyl]-4-amine (33): According to the synthesis method of compound 31 in Example 4, 2-nitro-[1,1'-biphenyl]-4-amine was reacted with intermediate II to obtain compound 33 as a yellow solid with a total yield of 23%.

[0119] 1 H NMR (500MHz, DMSO-d6) δ8.80 (s, 1H), 8.31 (s, 1H), 7.49 (d, J = 2.1Hz, 1H), 7. 42(t,J=7.3Hz,2H),7.36(m,3H),7.29(t,J=7.8Hz,3H),7.17(s,1H),7.10( d,J=7.9Hz,1H),6.93(d,J=7.5Hz,1H),3.59(s,3H),3.50(s,2H),2.92(d,J =11.6Hz,2H),2.79-2.74(m,1H),2.09(d,J=9.8Hz,2H),1.88-1.72(m,4H). 13 C NMR(126MHz,DMSO-d6)δ156.44,149.50,144.53,141.39,139.88,132.64,129.22,128.66,127.71,1 27.45,122.31,119.05,117.02,109.61,62.24,52.76,34.03,31.29,30.09,30.02.HRMS(ESI):calcd for C 27 H 28 N6O468.2274, found [M+H] + 469.2367.HPLC purity=97%.

[0120] Example 6: Preparation of Compound 36

[0121]

[0122] Synthesis of methyl 4-(3-(4-(4-methyl-4H-1,2,4-triazol-3-yl)piperidin-1-yl)methyl)phenyl)amino)-2-nitrobenzoate (36): According to the synthesis method of compound 1 in Example 2, compound 36 was prepared by reacting 4-methoxycarbonyl-3-nitrophenylboronic acid with intermediate IV as a yellow solid with a total yield of 18%.

[0123] 1H NMR (500MHz, DMSO-d6) δ9.24(s,1H),8.31(s,1H),7.75(d,J=8.7Hz,1H),7.33(t,J=7.7Hz,1H),7.27(d,J=2.3Hz,1H),7.24-7.11(m,3H), 7.03(d,J=7.5Hz,1H),3.76(s,3H),3.59(s,3H),3.51(s,2H),2.91(d,J=11.5Hz,2H),2.77(m,1H),2.15-2.05(m,2H),1.88-1.70(m,4H). 13 C NMR (126MHz, DMSO-d6) δ163.85,159.07,151.41,148.81,140.01,139.85,132.22,129.24,123.7 9,120.70,118.75,115.82,111.88,108.13,61.98,52.63,52.30,29.98,29.93.HRMS(ESI):calcd for C 23 H 26 N6O4 450.2016,found[M+H] + 451.2068.HPLC purity=94%.

[0124] Example 7: Preparation of Compound 37

[0125]

[0126] Synthesis of (3-nitro-4-(phenoxycarbonyl)phenyl)boronic acid: 4-bromo-2-nitrobenzoic acid (1 mmol, 1 equiv), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.25 mmol, 1.25 equiv), and 4-dimethylaminopyridine (DMAP, 0.25 mmol, 0.25 equiv) were dissolved in dichloromethane (10 mL). After complete dissolution, phenol (1.1 mmol, 1.1 equiv) was added and reacted at 25° C. for 24 h. After the reaction was completed, dichloromethane was extracted three times, the organic phases were combined, dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and recrystallized from n-hexane to obtain a yellow-green solid.

[0127] Synthesis of phenyl 4-(3-(4-(4-methyl-4H-1,2,4-triazol-3-yl)piperidin-1-yl)methyl)phenyl)amino)-2-nitrobenzoate (37): According to the synthesis method of compound 1 in Example 2, compound 37 was prepared by reacting (3-nitro-4-(phenoxycarbonyl)phenyl)boric acid with intermediate IV as a yellow solid with a total yield of 21%.

[0128] 1 H NMR(500MHz,DMSO-d6)δ9.38(s,1H),8.31(s,1H),7.96(d,J=8.7Hz,1H),7.50-7.43(m,2H),7.39-7.25(m,4H),7.24-7.12(m,4H),7.05(dd,J =15.1,7.6Hz,1H),3.59(s,3H),3.53(d,J=8.3Hz,2H),2.92(d,J=11.3Hz,2H),2.83-2.74(m,1H),2.12(t,J=10.6Hz,2H),1.89-1.70(m,4H). 13 C NMR (126MHz, DMSO-d6) δ162.18,156.41,151.84,150.25,149.45,144.55,139.72,133.07,132.35,129.66,129.41,126.2 0,124.11,121.54,120.92,118.98,115.88,110.81,108.39,62.03,52.72,52.44,31.15,30.06,30.02.HRMS(ESI):calcd for C 28 H 28 N6O4512.2172, found [M+H] + 513.2331.HPLC purity=98%.

[0129] Example 8: Preparation of Compound 38

[0130]

[0131] Synthesis of 4-((3-(4-(4-methyl-4H-1,2,4-triazol-3-yl)piperidin-1-yl)methyl)phenyl)amino)-2-nitro-N-benzamide (38): According to the synthesis method of compound 37 in Example 7, 4-bromo-2-nitrobenzoic acid and aniline were reacted to obtain (3-nitro-4-(phenylcarbamoyl)phenyl)boric acid, and then (3-nitro-4-(phenylcarbamoyl)phenyl)boric acid and intermediate IV were reacted to obtain compound 38 as a yellow solid with a total yield of 24%.

[0132] 1H NMR(500MHz,DMSO-d6)δ10.48(s,1H),9.01(s,1H),8.31(s,1H),7.66(d,J=7.9Hz, 2H),7.60(d,J=8.4Hz,1H),7.54(d,J=2.3Hz,1H),7.33(q,J=7.9Hz,4H),7.18(s,1H ),7.14-7.06(m,2H),6.99(d,J=7.6Hz,1H),3.59(s,3H),3.51(s,2H),2.92(d,J=1 1.5Hz,2H),2.78(td,J=7.5,3.8Hz,1H),2.11(d,J=13.6Hz,2H),1.89-1.71(m,4H). 13 CNMR(126MHz,DMSO-d6)δ163.99,156.43,148.74,146.42,144.54,140.76,140.02,139.13,130.61,129.33,128.73,1 23.64,123.02,121.65,119.61,119.57,117.89,117.69,109.23,62.21,52.76,31.18,30.09,30.02.HRMS(ESI):calcd for C 28 H 29 N7O3 511.2332, found [M+H] + 512.2374.HPLC purity=90%.

[0133] Comparative Example 1: Preparation of existing isoQC inhibitors

[0134]

[0135] Synthesis of 3-aminobenzenesulfonyl chloride: 3-aminobenzenesulfonic acid (10.21 mmol, 1 equiv) was dissolved in dichloromethane (20 mL) from which water had been redistilled, and thionyl chloride (30.15 mmol, 3 equiv) and 2 drops of N,N-dimethylformamide were added, and the mixture was reacted at 25°C for 12 h. After the reaction, the mixture was dried by rotation and used directly in the next step without purification.

[0136] Synthesis of 3-((4-(4-methyl-4H-1,2,4-triazol-3-yl)piperidin-1-yl)sulfonyl)aniline: 4-(4-methyl-4H-1,2,4-triazol-3-yl)piperidine (intermediate I prepared in Example 1, 3.02 mmol, 1 equiv) was dissolved in acetone (20 mL) from which water was distilled off, triethylamine (6.04 mmol, 2 equiv) was added, stirred at 25°C for 30 min, 3-aminobenzenesulfonyl chloride (9.07 mmol, 3 equiv) was added, and the mixture was reacted at 70°C for 4 h. After the reaction was completed, the mixture was extracted with ethyl acetate three times, the organic phases were combined, dried over anhydrous sodium sulfate, spin-dried, and purified by silica gel flash chromatography with a mobile phase of ethyl acetate: methanol: ammonia water = 100:20:1 to obtain a white solid.

[0137] Synthesis of 3-((4-(4-methyl-4H-1,2,4-triazol-3-yl)piperidin-1-yl)sulfonyl)-N-phenylaniline: 3-((4-(4-methyl-4H-1,2,4-triazol-3-yl)piperidin-1-yl)sulfonyl)aniline (1.56 mmol, 1 equiv) and phenylboronic acid (3.09 mmol, 2 equiv) were dissolved in dichloromethane (20 mL), and copper acetate monohydrate (3.14 mmol, 2 equiv), triethylamine (3.14 mmol, 2 equiv) and 2,2,6,6-tetramethylpiperidinoxide (1.53 mmol, 1 equiv) were added and reacted at 25°C for 12 h. After the reaction was completed, the product was extracted with ethyl acetate three times, the organic phases were combined, dried over anhydrous sodium sulfate, spin-dried, and purified by silica gel flash chromatography with the mobile phase being ethyl acetate: methanol: ammonia water = 100: 20: 1 to obtain a white solid with a total yield of 10%.

[0138] 1 H NMR (500MHz, DMSO) δ8.64(s,1H),8.31(s,1H),7.47(t,J=8.0Hz,1H),7.39-7.28(m,4H),7.18-7.10(m,3H),6.95(t,J=7.3Hz,1 H),3.64(d,J=11.8Hz,2H),3.55(s,3H),2.89(tt,J=11.0,3.6Hz,1H),2.57-2.51(m,2H),2.03-1.87(m,2H),1.82-1.65(m,2H). 13C NMR (126MHz, DMSO) δ156.15,145.24,145.12,142.36,136.59,130.76,129.90,121.8 0,119.80,118.67,117.97,114.20,46.10,30.46,30.19,29.69.HRMS(ESI):calcdfor C 20 H 23 N5O2S 397.1572, found [M+H] + 398.2267.HPLC purity=99%.

[0139] Application Example 1: iosQC and QC Inhibitory Activity Test

[0140] 1. Reagent preparation

[0141] (1) HEPES buffer (pH = 7.0): Weigh 0.596 g of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) powder, add 90 mL of deionized water to dissolve, adjust the pH to 7.0 with 1 M NaOH, and then make up to 100 mL. Store at 4°C.

[0142] (2) PAP buffer (pH 9.0): Weigh 1.211 g of tris (hydroxymethyl)aminomethane (Tris) and 0.077 g of dithiothreitol (DTT), add 90 mL of deionized water to dissolve, adjust the pH to 9.0, and then make up to 100 mL. Store at -20°C.

[0143] (3) Preparation of compounds: Weigh the solid powder of the compound to be tested, add dimethyl sulfoxide (DMSO) to prepare a mother solution with a concentration of 10 mM, and store at -20°C. During the experiment, dilute the compound to 1.5625 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM, 400 μM, 800 μM, 1600 μM, etc. according to the required concentration of the specific compound.

[0144] (4) isoQC solution: The concentration of BCA quantitative protein is 0.50 mg / mL and stored at -80°C. During the experiment, add HEPES buffer and dilute 500 times to 1.0 μg / mL, mix well and place on ice for later use.

[0145] (5) QC solution: BCA quantitative protein concentration is 0.75 mg / mL, stored at -80°C. During the experiment, add HEPES buffer to dilute 1500 times to 0.5 μg / mL, mix well and place on ice for later use.

[0146] (6) Gln-AMC solution: 66 mM 2-amino-N1-(4-methyl-2-oxo-4a,8a-dihydro-2H-chromen-7-yl) glutaramide (Gln-AMC), stored at -20°C. Dilute 330 times to 200 μM with HEPES buffer during the experiment, mix well and place on ice for later use.

[0147] (7) PAP solution: 3 mg / mL pyroglutamyl peptidase (PAP), stored at -80°C. Dilute 300 times to 10 μg / mL with PAP buffer during the experiment, mix well, and place on ice for later use.

[0148] 2. isoQC inhibitory activity test

[0149] The isoQC inhibitory activity test used the existing isoQC inhibitor of comparative example 1 as a positive control, DMSO as a negative control, and the group without isoQC as a blank control. Each compound was set to 8 concentration gradients, 3 replicates, and repeated 3 times. The specific operation is as follows:

[0150] (1) Add 10 μL of compound diluted with HEPES buffer or 10 μL of DMSO diluted the same multiple with HEPES buffer or 50 μL of HEPES buffer (blank control) to a black 96-well plate.

[0151] (2) Except for the blank control, 40 μL of isoQC solution was added to the remaining samples and mixed at 25°C, 300 rpm for 2 min.

[0152] (3) Add 50 μL of Gln-AMC solution and mix at 25°C, 300 rpm for 20 min.

[0153] (4) Add 100 μL of PAP solution and mix at 25°C, 300 rpm for 60 min.

[0154] (5) Use single wavelength scanning, excitation wavelength (EX) / emission wavelength (EM) = 360 nm / 445 nm.

[0155] Inhibition rate (%) = (DMSO-Cpd) / (DMSO-Blank) × 100%, where DMSO = the fluorescence value of the DMSO group; Cpd = the fluorescence value of the compound group; Blank = the fluorescence value of the group without enzyme addition.

[0156] 3. QC inhibitory activity test

[0157] The QC inhibitory activity test used the existing isoQC inhibitor of comparative example 1 as a positive control, DMSO as a negative control, and the group without QC as a blank control. Each compound was set to 8 concentration gradients, 3 replicates, and repeated 3 times. The specific operation is as follows:

[0158] (1) Add 10 μL of compound diluted with HEPES buffer or 10 μL of DMSO diluted the same multiple with HEPES buffer or 50 μL of HEPES buffer (blank control) to a black 96-well plate.

[0159] (2) Except for the blank control, 40 μL of QC solution was added to the remaining samples and mixed at 25°C, 300 rpm for 2 min.

[0160] (3) Add 50 μL of Glu-AMC solution and mix at 25°C, 300 rpm for 10 min.

[0161] (4) Add 100 μL of PAP solution and mix at 25°C, 300 rpm for 60 min.

[0162] (5) Use single wavelength scanning, EX / EM = 360nm / 445nm.

[0163] Inhibition rate (%) = (DMSO-Cpd) / (DMSO-Blank) × 100%, where DMSO = the fluorescence value of the DMSO group; Cpd = the fluorescence value of the compound group; Blank = the fluorescence value of the group without enzyme addition.

[0164] The test results are shown in Table 1.

[0165] Table 1 IosQC and QC inhibitory activity test results of compounds

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172] In Table 1, it can be seen from the test results of compound 1 and comparative example 1 that after the rigid sulfonyl group in the existing isoQC inhibitor is replaced with a flexible alkyl group, the selectivity of the obtained new structure is reversed, from the original QC inhibition activity being better than the isoQC inhibition activity to the isoQC inhibition activity being better than the QC inhibition activity. It can be seen from the test results of compounds 1 to 42 and comparative example 2 that the isoQC selectivity of the compounds of the present invention is close to or exceeds the existing QC inhibitors.

[0173] In summary, the present invention provides a selective glutaminyl cyclase isozyme inhibitor and its preparation method and application. The general structural formula of the selective glutaminyl cyclase isozyme inhibitor is R is selected from This type of selective glutaminyl cyclase isozyme inhibitor has better isoQC inhibition activity than QC inhibition activity, has isoQC selectivity, can reduce the risk of cross-reaction with QC when inhibiting isoQC, and reduces the side effects that may be caused by the clinical application of isoQC inhibitors. At the same time, the preparation method of this type of selective glutaminyl cyclase isozyme inhibitor is simple to operate and the raw materials are easily available, and it can be widely used in the preparation of drugs for the treatment of diseases related to the specific high expression of glutaminyl cyclase isozymes.

[0174] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A selective glutaminyl cyclase isozyme inhibitor, characterized in that: The general structural formula of the selective glutaminyl cyclase isozyme inhibitor is as follows: R is selected from one of the following substituents: Among them, R 1 Selected from hydrogen, C 1-6 Alkyl, C 5-7 One of aryl and benzyloxy; R 2 and R 4 are each independently selected from hydrogen, halogen, nitro, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylamino, C 1-6 Fluoroalkyl, C 1-6 Fluoroalkoxy, C 1-6 Ester group, C 5-7 One of aryl and benzyloxy; R 3 Selected from hydrogen, halogen, nitro, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylamino, C 1-6 Fluoroalkyl, C 1-6 Fluoroalkoxy, C 1-6 Ester group, C 5-7 Aryl, phenoxy, benzyloxy, C 7-10 Aryl substituted acyl, C 7-10 Aryl substituted ester group, C 7-10 Aryl substituted amide, C 7-10 One of the aryl-substituted amino groups; R 5 and R 6 are each independently selected from hydrogen or C 1-6 alkyl; Among them, the C 5-7 The aryl group is unsubstituted or substituted by halogen, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Fluoroalkyl substitution.

2. The selective glutaminyl cyclase isozyme inhibitor according to claim 1, characterized in that R 1 One selected from hydrogen, phenyl, benzyloxy; R 2 and R 4 Each is independently selected from one of hydrogen, fluorine, nitro, methoxy, dimethylamino, trifluoromethyl, trifluoromethoxy, methyl acetate, phenyl, and benzyloxy; R 3 One selected from hydrogen, fluorine, nitro, tert-butyl, methoxy, isobutoxy, dimethylamino, trifluoromethyl, trifluoromethoxy, methyl acetate, phenyl, benzyloxy, acetyl, benzoyl, benzoate, benzamide, 4-amino-2-(trifluoromethyl)phenyl, 3,4,5-trimethoxybenzamide, and 3-fluoroanilino; R 5 and R 6 Each is independently selected from hydrogen or methyl.

3. The selective glutaminyl cyclase isozyme inhibitor according to claim 1, characterized in that: The general structural formula of the selective glutaminyl cyclase isozyme inhibitor is as follows: Among them, R 3 One selected from the group consisting of hydrogen, nitro, methyl acetate, phenyl, benzoyl, benzoate, benzamide, 3,4,5-trimethoxybenzamide, and 3-fluoroaniline; R 4 Selected from nitro or trifluoromethyl.

4. The selective glutaminyl cyclase isozyme inhibitor according to claim 1, characterized in that: The selective glutaminyl cyclase isozyme inhibitor is selected from one of the following structures:

5. A method for preparing a selective glutaminyl cyclase isozyme inhibitor according to any one of claims 1 to 4, characterized in that: The synthetic route of the selective glutaminyl cyclase isozyme inhibitor is:

6. The preparation method according to claim 5, characterized in that: In the synthetic route of the selective glutaminyl cyclase isozyme inhibitor, the first reaction condition specifically includes: the reaction solvent is selected from one or more of acetone, acetonitrile, tetrahydrofuran, dioxane or N,N-dimethylformamide, the reaction time is 2 to 12 hours, the reaction temperature is 50 to 80° C., and the acid-binding agent is selected from one or more of potassium carbonate, sodium carbonate, and triethylamine; And / or, the second reaction condition specifically includes: carrying out the reaction under an aerobic environment, the reaction solvent is selected from one or more of methanol, ethanol, and dichloromethane, the reaction time is 5 to 24 hours, the reaction temperature is 10 to 50° C., the acid binding agent is selected from one or more of potassium carbonate, sodium carbonate, and triethylamine, and the catalyst is copper acetate or copper (I) thiophene-2-carboxylic acid; And / or, the third reaction condition specifically includes: the reaction solvent is selected from one or more of acetone, dichloromethane, tetrahydrofuran, and dioxane, the reaction time is 0.5 to 2 hours, and the reaction temperature is 0 to 10°C.

7. The preparation method according to claim 5, characterized in that: The synthetic route of intermediate Ⅰ is: The fourth reaction condition specifically includes: the reaction solvent is selected from one or more of methanol, ethanol, acetone, tetrahydrofuran, and dioxane, the reaction time is 2 to 8 hours, and the reaction temperature is 50 to 80° C.; The fifth reaction conditions specifically include: the reaction solvent is water, the reaction time is 1 to 5 hours, and the reaction temperature is 80 to 100° C.; The sixth reaction condition specifically includes: the reaction solvent is selected from one or more of methanol, ethanol, acetone, dichloromethane, tetrahydrofuran, and dioxane, the reaction time is 0.5 to 2 hours, and the reaction temperature is 10 to 50° C.; The seventh reaction condition specifically includes: the reaction solvent is selected from one or more of methanol, ethanol, acetone, dichloromethane, tetrahydrofuran, and dioxane, the reaction time is 2 to 8 hours, and the reaction temperature is 20 to 80°C.

8. Use of the selective glutaminyl cyclase isozyme inhibitor according to any one of claims 1 to 4 in the preparation of a drug for treating diseases associated with specific high expression of glutaminyl cyclase isozymes.

9. The use according to claim 8, characterized in that: The disease associated with the specific high expression of glutaminyl cyclase isozyme is inflammation induced by high expression of pE-CCL2.

10. The use according to claim 8, characterized in that: The disease associated with the specific high expression of glutaminyl cyclase isozymes is cancer induced by high expression of pE-CD47.

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