Benzimidazole amine derivative, preparation method thereof and application of benzimidazole amine derivative as beta2-adrenergic receptor allosteric antagonist

Abstract

The invention belongs to the field of medicinal chemistry, and particularly discloses a benzimidazole amine derivative, a preparation method thereof and application of the benzimidazole amine derivative as a beta2-adrenergic receptor allosteric antagonist. According to the benzimidazole amine derivative disclosed by the invention, R1 is a hydrogen atom or a bromine atom; r2 is substituted benzoyl, substituted benzyl and the like. Pharmacological results show that the benzimidazole amine derivative disclosed by the invention has relatively good antagonistic activity on beta2AR, and the functional activity of isoprenaline (ISO) can be subjected to negative allosteric regulation.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and particularly relates to a benzimidazole amine derivative and a preparation method thereof and a method for preparing the same. 2 -Application of adrenergic receptor allosteric antagonists. Background Art

[0002] G-protein coupled receptors (GPCRs) are the largest family of cell membrane receptors in the human body. They are distributed in various tissues and organs of the human body and are involved in almost all life activities. GPCRs are a very important class of drug targets. More than one-third of clinical drugs approved by the US FDA exert their efficacy by regulating their behavior through GPCRs. β-adrenergic receptor (β-AR) is a class A GPCR, in which β 2 AR transmits signals to cells through the action of G proteins, mediating various physiological responses, including vasodilation and vasoconstriction, heart rate regulation, etc. (AJ Venkatakrishnan, et al. Nature, 2013, 494: 185-194).

[0003] The vast majority of β 2 AR drugs are orthosteric ligands, but orthosteric ligands have many side effects, so the development of β 2 AR allosteric modulator-related drugs have become an emerging trend. Summary of the invention

[0004] The purpose of the present invention is to provide a new benzimidazole derivative, so as to develop a heterocyclic derivative with stable chemical structure, high biological activity and more stable metabolism in vivo, as a β 2 Allosteric antagonists of AR provide a research basis for the creation of new drugs for diseases such as cardiovascular, asthma and cancer.

[0005] The present invention provides a benzimidazole amine derivative, the structure of which is shown in the following formula:

[0006]

[0007] R 1 =H,Br;

[0008] R 2 One of the following structural formulas:

[0009]

[0010] Furthermore, the benzimidazole amine derivative is specifically one of the following structural formulas:

[0011]

[0012] The present invention also provides a preparation method of the above-mentioned benzimidazole amine derivatives, which comprises: dissolving an acid and an activator in a solvent, adding different types of amines under an ice bath, and then adding an acid binding agent and an amide coupling agent and continuing to stir to room temperature to obtain the benzimidazole amine derivatives; wherein the solvent is N,N-dimethylformamide, the activator is 1-hydroxy-7-azabenzotriazole (HOAt), the acid binding agent is N-methylmorpholine (NMM), and the amide coupling agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI).

[0013] Furthermore, the preparation method specifically includes: dissolving 1 eq of substituted benzoic acid and 1.2 eq of HOAt in DMF, stirring for 10 min, and then adding benzimidazole amine; then, adding 0.7 eq of NMM at 0°C, stirring for 10 min, adding 1.2 eq of EDCI, maintaining stirring at 0°C for 1.5 h, and reacting at room temperature for 12 h; spin-drying DMF, extracting, and column-passing to obtain the benzimidazole amine derivative.

[0014] Furthermore, the benzimidazole amine derivative is specifically one of the following structural formulas:

[0015]

[0016] The present invention also provides a preparation method of the above-mentioned benzimidazole amine derivatives, which comprises: dissolving benzimidazole amine in a solvent to obtain a reaction system; adding benzaldehyde to the reaction system, and dripping a small amount of glacial acetic acid, reacting for 6 hours under a nitrogen environment to obtain a reaction liquid; then placing the reaction at 0°C, slowly adding a reducing agent to the reaction liquid, and returning the reaction to room temperature to react for 12 hours to obtain the benzimidazole amine derivatives; wherein the solvent is methanol and the reducing agent is sodium cyanoborohydride.

[0017] Furthermore, the preparation method specifically includes: dissolving 1eq of benzimidazole amine in methanol to obtain a reaction system; adding 1eq of substituted benzaldehyde to the reaction system, and dropwise adding 0.1eq of glacial acetic acid, reacting for 6 hours under a nitrogen environment to obtain a reaction liquid; then placing the reaction at 0°C, slowly adding 2.5eq of sodium cyanoborohydride to the reaction liquid, returning the reaction to room temperature for 12 hours to complete the reaction, adding water to quench, spinning methanol to dryness, extracting, and subjecting the obtained product to column chromatography to obtain the benzimidazole amine derivative.

[0018] The present invention also provides the benzimidazole amine derivatives as described above as β 2 -Application of adrenergic receptor allosteric antagonists.

[0019] The present invention also provides the benzimidazole amine derivatives as described above in the preparation of β 2 -Application of adrenaline receptor allosteric antagonists in the field of pharmaceutical preparations.

[0020] Furthermore, the benzimidazole derivatives are used as active ingredients and a pharmaceutically acceptable carrier to prepare a pharmaceutical preparation.

[0021] The beneficial effects of the present invention are:

[0022] The new derivative synthesized by the present invention is firstly 2 The allosteric antagonist activity of the AR target has been significantly improved compared with the lead compound Cmpd-15; secondly, the newly synthesized derivative has a novel skeleton and a greatly simplified structure compared with the lead compound Cmpd-15, which simplifies the synthesis route, reduces the cost and difficulty of synthesis, and significantly improves the economic benefits of this type of compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The design idea of ​​the compound structure of the present invention;

[0024] Figure 2 Design ideas for amide bond flipping and amide bond reduction designed for benzimidazole series compounds;

[0025] Figure 3 This is a dose-response curve of ISO mediated by the benzimidazole amine derivative A101;

[0026] Figure 4 This is a dose-response curve of ISO mediated by the benzimidazole amine derivative A111. DETAILED DESCRIPTION

[0027] A few years ago, the inventors and collaborators reported the first beta 2 Cmpd-15 is an intracellular allosteric antagonist of AR (S. Ahn, et al. PNAS, 2017, 114: 1708-1713), but due to the poor solubility and low biological activity of Cmpd-15, the relative instability of its polypeptide structure will affect its drugability (KCMeng, et al. Bioorg. Med. Chem. 2018, 26: 2320-2330). Later, the inventors used the backbone migration strategy to replace the amide of the left fragment of the lead compound Cmpd-15 with a nitrogen-containing heterocycle, so that the compound has better solubility and pharmacological activity, and obtained a new type of structurally stable pyrazole skeleton (pyrazoleamides), and found that β-terminal amides with better activity than Cmpd-15 2 AR allosteric antagonist (X. Guo, et al. Bioorg. Med. Chem. 2024, 108: 117787).

[0028] On this basis, the inventors continued to optimize the structure of the lead through the strategies of bioisosteres, skeleton transitions, pharmacophore hybridization, and structural simplification, and designed and synthesized a series of new derivatives (Benzimidazole amides) with benzimidazole amide as the skeleton, whose activity is significantly better than Cmpd-15 (Chen Xin, et al. Chinese invention patent application number: CN202310937574.0, 2023). However, although this type of compound has considerable biological activity, the aniline structure it contains is easily oxidized by CYP450 enzymes, resulting in poor biological stability of this type of compound. The inventors recently conducted liver metabolic stability experiments on this type of compound and found that the half-life of benzimidazole derivatives was less than 30 minutes. In order to solve the stability problem and avoid the appearance of aniline structure in the compound, the inventors tried to flip the amide bond to form a new type A derivative (Type A). In addition, the inventors also optimized the amide bond into a carbon-nitrogen bond, that is, a new type B derivative (Type B), to avoid amide bond hydrolysis, so as to improve the metabolic stability of this type of compound, such as Figure 1 shown.

[0029] Benzimidazole is an important heterocyclic compound, and its chemical structure is composed of a benzene ring and an imidazole ring. In the previous research, the inventor found that a class of benzimidazole amide derivatives can be used as β 2 It is an allosteric antagonist of AR, and its biological activity is significantly stronger than that of the lead compound Cpmd-15. However, the metabolic stability of this type of compound is poor, with a half-life of only about half an hour, which is probably because its structure contains aniline structural units that are easily oxidized by CPY450 enzymes.

[0030] To this end, the present invention adopts two strategies to optimize the structure of benzimidazole amide derivatives: one is to flip the amide bond and change the aniline structural unit into a benzoyl structural unit, in order to avoid the oxidative metabolism directly related to aniline; the other is to change the flipped amide bond into a carbon-nitrogen bond, in order to avoid enzyme-catalyzed amide bond hydrolysis, such as Figure 2 shown.

[0031] The synthesis method of benzimidazole amide derivatives is as follows:

[0032]

[0033] The acid and activator are dissolved in a solvent, different types of amines are added under an ice bath, and then the acid-binding agent and amide coupling agent are added and stirred to room temperature. The solvent is N,N-dimethylformamide, the activator is 1-hydroxy-7-azabenzotriazole (HOAt), the acid-binding agent is N-methylmorpholine (NMM), and the amide coupling agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI).

[0034] The specific method is as follows: substituted benzoic acid (1 eq) and HOAt (1.2 eq) are dissolved in DMF, stirred for 10 min, and then benzimidazolamine is added. Then, NMM (0.7 eq) is added at 0°C, stirred for 10 min, EDCI (1.2 eq) is added, stirred at 0°C for 1.5 h, and reacted at room temperature for 12 h. DMF is spin-dried, extracted, and passed through a column to obtain compounds A101-A108.

[0035] Another method for synthesizing benzimidazole amine derivatives is as follows:

[0036]

[0037] Dissolve benzimidazole amine in a solvent, add benzaldehyde to the system, and drop a small amount of glacial acetic acid therein, react for 6 hours under a nitrogen environment, then place the reaction at 0°C, slowly add a reducing agent to the reaction solution, and react for 12 hours at room temperature. The solvent is methanol and the reducing agent is sodium cyanoborohydride.

[0038] The specific method is as follows: benzimidazole amine (1 eq) is dissolved in methanol, substituted benzaldehyde (1 eq) is added to the system, and a small amount of glacial acetic acid (0.1 eq) is added dropwise thereto, and the reaction is carried out under a nitrogen environment for 6 hours, and then the reaction is placed at 0°C, sodium cyanoborohydride (2.5 eq) is slowly added to the reaction solution, and the reaction is restored to room temperature for 12 hours to complete the reaction, and water is added to quench the reaction, the methanol is spin-dried, and the extraction is performed. The obtained product is subjected to column chromatography to obtain compounds A109-A122.

[0039] The structure of the benzimidazole amine derivative is as follows:

[0040]

[0041] The present invention is described in detail below in conjunction with specific implementation modes.

[0042] The synthetic route of the benzimidazole amine derivatives provided by the present invention is as follows:

[0043] Example 1

[0044]

[0045] Preparation of Benzamide-1H-Benzimidazole-2-amine (A101)

[0046] Benzoic acid (122mg, 1.50mmol) was dissolved in DMF (4mL), HOAt (265mg, 1.95mmol) was added to the system and stirred for 10min, then compound 2-aminobenzimidazole (200mg, 1.50mmol) was added to the above system, N-methylmorpholine (134.7mL, 1.05mmol) was added thereto under ice bath and stirred for 10min, and finally EDCI (374mg, 1.95mmol) was added to the system, kept at 0°C for 1h, and returned to room temperature for 12h. TLC monitored the completion of the reaction, the reaction solution was concentrated under vacuum, the obtained oily liquid was extracted with ethyl acetate (15mL*3), the organic phase was washed with saturated brine, the organic phase was collected, anhydrous sodium sulfate was added to dry, and the obtained product was concentrated in vacuum. Column chromatography (petroleum ether: ethyl acetate = 4:1) gave 162mg of a white solid with a yield of 64%. 1 HNMR (400 MHz, DMSO-d 6 ): δ12.28(s,2H),8.16-8.12(m,2H),7.63-7.58(m,1H),7.53(t,J=7.4Hz,2H),7.46(dd,J=5.9,3.2Hz,2H),7.13(dq,J=6.7,3.5Hz,2H). 13 C NMR (101 MHz, DMSO-d 6 ): δ168.3,148.9,134.3,132.0,128.4,128.3,121.5,113.4.HRMS(ESI,m / z):Calcd.for C 14 H 11 N 3 O[M+H] + 238.09748,found:238.0982.

[0047] Example 2

[0048]

[0049] Preparation of N-(1H-benzimidazol-2-yl)-2-bromobenzamide (A102)

[0050] The preparation method is the same as that of Example 1, except that 2-bromobenzoic acid is used instead of benzoic acid to obtain a white solid with a yield of 62%. 1 H NMR (400 MHz, DMSO-d 6): δ12.30(s,2H),7.72(d,J=7.9Hz,1H),7.64(d,J=5.8Hz,1H),7.52-7.40(m,4H),7.12(dd,J=5.9,3.3Hz,2H). 13 C NMR (101 MHz, DMSO-d 6 ): δ168.0,138.0,132.9,131.7,129.3,127.7,121.6,119.2,113.9.

[0051] Example 3

[0052]

[0053] Preparation of N-(1H-benzimidazol-2-yl)-3-bromobenzamide (A103)

[0054] The preparation method is the same as that of Example 1, except that 3-bromobenzoic acid is used instead of benzoic acid to obtain a white solid with a yield of 67%. 1 H NMR (400 MHz, DMSO-d 6 ): δ12.44(s,2H),8.31(t,J=1.8Hz,1H),8.13(dt,J=7.8,1.3Hz,1H),7.76(ddd,J=8.0,2.1, 1.1Hz, 1H), 7.48 (d, J=7.9Hz, 1H), 7.44 (dd, J=5.9, 3.1Hz, 2H), 7.17 (dd, J=5.9, 3.1Hz, 2H). 13 C NMR (101 MHz, DMSO-d 6 ): δ169.1,138.4,134.2,132.2,131.1,130.6,127.4,122.1,121.6,112.6.HRMS(ESI,m / z):Calcd.for C 14 H 10 Bn 3 O[M+H] + 316.0080,found:316.0086.

[0055] Example 4

[0056]

[0057] Preparation of N-(1H-benzimidazol-2-yl)-4-bromobenzamide (A104)

[0058] The preparation method is the same as that of Example 1, except that 4-bromobenzoic acid is used instead of benzoic acid to obtain a white solid with a yield of 61%.1 H NMR (400 MHz, DMSO-d 6 ): δ12.39(s,2H),8.08(d,J=8.4Hz,2H),7.71(d,J=8.2Hz,2H),7.44(dd,J=5.7,3.0Hz,2H),7.15(dd,J=5.9,3.1Hz,2H). 13 C NMR (101 MHz, DMSO-d 6 ): δ169.1,150.3,134.8,132.8,131.3,130.5,125.6,121.9,112.8.

[0059] Example 5

[0060]

[0061] Preparation of N-(1H-benzimidazol-2-yl)-3-chlorobenzamide (A105)

[0062] The preparation method is the same as that of Example 1, except that 3-chlorobenzoic acid is used instead of benzoic acid to obtain a white solid with a yield of 65%. 1 H NMR (400 MHz, DMSO-d 6 ): δ12.45(s,2H),8.16(s,1H),8.09(d,J=7.8Hz,1H),7.62(d,J=8.0Hz,1H),7 .53(t,J=7.8Hz,1H),7.44(dd,J=5.9,3.1Hz,2H),7.16(dd,J=6.0,3.2Hz,2H). 13 C NMR (101 MHz, DMSO-d 6 ): δ169.1,150.7,138.2,133.1,132.3,131.4,130.3,128.2,127.1,122.2,112.7.

[0063] Example 6

[0064]

[0065] Preparation of N-(6-bromo-1H-benzimidazol-2-yl)benzamide (A106)

[0066] The preparation method is the same as that of Example 1, except that 2-aminobenzimidazole is replaced by 6-bromo-1H-benzimidazole-2-amine to obtain a white solid with a yield of 63%. 1 H NMR (400 MHz, DMSO-d 6): δ12.39(s,2H),8.12(d,J=7.9Hz,2H),7.66-7.61(m,2H),7.55(t,J=7.6Hz,2H),7.43(d,J=8.4Hz,1H),7.27(dd,J=8.5,2.1Hz,1H).

[0067] Example 7

[0068]

[0069] Preparation of N-(6-bromo-1H-benzimidazol-2-yl)-3-chlorobenzamide (A107)

[0070] The preparation method is the same as that of Example 6, except that 2-aminobenzimidazole in the step is replaced by 6-bromo-1H-benzimidazole-2-amine, and benzoic acid is replaced by 3-chlorobenzoic acid to obtain a white solid with a yield of 61%. 1 H NMR (400 MHz, DMSO-d 6 ): δ8.14(s,1H),8.06(d,J=7.8Hz,1H),7.69-7.62(m,2H),7.56(t,J=7.9Hz,1H),7.42(d,J=8.4Hz,1H),7.28(d,J=8.5Hz,1H). 13 C NMR (101 MHz, DMSO-d 6 ): δ136.21,133.38,132.11,130.56,128.20,127.12,124.51,113.73.

[0071] Example 8

[0072]

[0073] Preparation of N-(6-bromo-1H-benzimidazol-2-yl)-3-bromobenzamide (A108)

[0074] The preparation method is the same as that of Example 6, except that 2-aminobenzimidazole in the step is replaced by 6-bromo-1H-benzimidazole-2-amine, and benzoic acid is replaced by 3-bromobenzoic acid to obtain a white solid with a yield of 66%. 1 H NMR (400 MHz, DMSO-d 6): δ12.44(s,2H),8.30(d,J=2.1Hz,1H),8.10(d,J=7.8Hz,1H),7.81(d,J=7.9Hz,1H),7.63( s, 1H), 7.50 (td, J=7.9, 1.6Hz, 1H), 7.42 (dd, J=8.4, 1.6Hz, 1H), 7.29 (dt, J=8.6, 1.8Hz, 1H).

[0075] Example 9

[0076]

[0077] Preparation of N-benzyl-1H-benzimidazol-2-amine (A109)

[0078] 2-aminobenzimidazole (250mg, 1.88mmol) was dissolved in methanol, and then benzaldehyde (1.88mmol) was added to the system and a small amount of glacial acetic acid was added dropwise thereto. The reaction was allowed to react for 6h under nitrogen atmosphere, and then the reaction was placed at 0°C, and sodium cyanoborohydride (295mg, 4.69mmol) was slowly added to the reaction solution, and the reaction was returned to room temperature for 12h. The reaction was monitored by TLC, and after the reaction was completed, water was added to quench, and the reaction solution was concentrated under vacuum, extracted with ethyl acetate (15mL*3), and the organic phase was washed with saturated brine, the organic phase was collected, dried with anhydrous sodium sulfate, and concentrated under vacuum. The obtained product was subjected to column chromatography (petroleum ether: ethyl acetate = 1:4) to obtain 86.52mg of a white solid with a yield of 44%. 1 HNMR (400 MHz, DMSO-d 6 ): δ7.31(t,J=7.3Hz,2H),7.24(d,J=7.0Hz,1H),7.19(d,J=7.1Hz,2H),7.14(d,J=7.8Hz,1H), 7.04(d,J=7.7Hz,1H),6.92(t,J=7.5Hz,1H),6.81(t,J=7.5Hz,1H),6.59(s,2H),5.26(s,2H). 13 CNMR (101MHz, DMSO-d 6 ): δ155.1,143.0,137.3,134.2,128.5,127.3,127.0,120.5,118.1,114.8,107.9,44.7.HRMS(ESI,m / z):Calcd.for C 14 H 13 N 3 [M+H] + 224.1181, found:224.1187.

[0079] Example 10

[0080]

[0081] Preparation of N-(2-bromobenzyl)-1H-benzimidazol-2-amine (A110)

[0082] The preparation method is the same as that of Example 9, except that the benzaldehyde in the step is replaced by 2-bromobenzaldehyde to obtain a white solid with a yield of 41%. 1 H NMR (400 MHz, DMSO-d 6 ): δ7.7(dd,J=7.3,1.9Hz,1H),7.3–7.2(m,3H),6.9(t,J=7.5Hz,1H),6.9(d,J=7. 3Hz,1H),6.8(t,J=7.5Hz,1H),6.6(s,2H),6.4(dd,J=7.2,2.2Hz,1H),5.3(s,2H). 13 C NMR (101 MHz, DMSO-d 6 ): δ155.3,143.0,135.7,134.0,132.7,129.2,128.0,126.9,121.8,120.8,118.3,114.9,107.7,45.3.

[0083] Embodiment 11

[0084]

[0085] Preparation of N-(3-bromobenzyl)-1H-benzimidazol-2-amine (A111)

[0086] The preparation method is the same as that of Example 9, except that the benzaldehyde in the step is replaced by 3-bromobenzaldehyde to obtain a white solid with a yield of 46%. 1 H NMR (400 MHz, DMSO-d 6 ): δ7.45(d,J=7.9Hz,1H),7.39(s,1H),7.28(t,J=7.8Hz,1H),7.16(t,J=7.8Hz,2H),7.08(d,J= 7.7Hz, 1H), 6.94 (td, J=7.5, 1.2Hz, 1H), 6.84 (td, J=7.5, 1.2Hz, 1H), 6.62 (s, 2H), 5.27 (s, 2H). 13 C NMR (101 MHz, DMSO-d 6): δ155.0,142.9,140.1,134.0,130.8,130.2,129.7,126.1,121.8,120.7,118.3,114.9,107.8,44.0.HRMS(ESI,m / z):Calcd.for C 14 H 12 Bn 3 [M+H] + 302.0288,found:302.0288.

[0087] Example 12

[0088]

[0089] Preparation of N-(4-bromobenzyl)-1H-benzimidazol-2-amine (A112)

[0090] The preparation method is the same as that of Example 9, except that the benzaldehyde in the step is replaced by 4-bromobenzaldehyde to obtain a white solid with a yield of 44%. 1 H NMR (400 MHz, DMSO-d 6 ): δ7.5(d,J=8.5Hz,2H),7.2-7.1(m,3H),7.0(d,J=7.7Hz,1H),6.9(t,J=7.5Hz,1H),6.8(t,J=7.5Hz,1H),6.6(s,2H),5.2(s,2H). 13 C NMR (101 MHz, DMSO-d 6 ): δ155.0,142.9,136.7,134.0,131.4,129.2,120.6,120.4,118.2,114.9,107.8,44.1.

[0091] Embodiment 13

[0092]

[0093] Preparation of N-(2-chlorobenzyl)-1H-benzimidazol-2-amine (A113)

[0094] The preparation method is the same as that of Example 9, except that the benzaldehyde in the step is replaced by 2-chlorobenzaldehyde to obtain a white solid with a yield of 43%. 1 H NMR (400 MHz, DMSO-d 6): δ7.52(d,J=7.8Hz,1H),7.29(t,J=7.1Hz,1H),7.20(dd,J=11.6,7.5Hz,2H),6.95(t,J=7.5Hz,1 H), 6.89 (d, J = 7.4Hz, 1H), 6.80 (t, J = 7.5Hz, 1H), 6.61 (s, 2H), 6.49 (d, J = 7.4Hz, 1H), 5.33 (s, 2H). 13 C NMR (101 MHz, DMSO-d 6 ): δ155.3,143.0,134.2,134.0,131.8,129.5,128.9,127.4,127.0,120.8,118.3,114.9,107.7,42.9.

[0095] Embodiment 14

[0096]

[0097] Preparation of N-(3-chlorobenzyl)-1H-benzimidazol-2-amine (A114)

[0098] The preparation method is the same as that of Example 9, except that the benzaldehyde in the step is replaced by 3-chlorobenzaldehyde to obtain a white solid with a yield of 41%. 1 H NMR (400 MHz, DMSO-d 6 ): δ7.37–7.30(m,2H),7.24(s,1H),7.14(t,J=7.0Hz,2H),7.08(d,J=7.7Hz, 1H), 6.94 (t, J = 7.5Hz, 1H), 6.83 (t, J = 7.5Hz, 1H), 6.61 (s, 2H), 5.28 (s, 2H). 13 C NMR (101 MHz, DMSO-d 6 ): δ155.0,142.9,139.9,134.1,133.2,130.5,127.3,126.8,125.7,120.7,118.3,114.9,107.8,44.1.HRMS(ESI,m / z):Calcd.for C 14 H 12 C1N 3 [M+H] + 258.0793,found:258.0800.

[0099] Embodiment 15

[0100]

[0101] Preparation of N-(4-chlorobenzyl)-1H-benzimidazol-2-amine (A115)

[0102] The preparation method is the same as that of Example 9, except that the benzaldehyde in the step is replaced by 4-chlorobenzaldehyde to obtain a white solid with a yield of 42%. 1 HNMR (400 MHz, DMSO-d 6 ): δ7.38(d,J=8.3Hz,2H),7.20(d,J=8.3Hz,2H),7.15(d,J=7.8Hz,1H),7.05(d,J= 7.7Hz,1H),6.93(t,J=7.6Hz,1H),6.82(t,J=7.5Hz,1H),6.62(s,2H),5.26(s,2H). 13 C NMR (101 MHz, DMSO-d 6 ): δ155.0,142.9,136.3,134.1,131.9,128.9,128.6,120.6,118.2,114.9,107.9,44.0.

[0103] Example 16

[0104]

[0105] Preparation of N-(2-fluorobenzyl)-1H-benzimidazol-2-amine A116

[0106] The preparation method is the same as that of Example 9, except that the benzaldehyde in the step is replaced by 2-fluorobenzaldehyde to obtain a white solid with a yield of 40%. 1 H NMR (400 MHz, DMSO-d 6 ): δ7.34-7.29(m,1H),7.23(t,J=9.3Hz,1H),7.16(d,J=7.8Hz,1H),7.09(t,J=7.5Hz,1H), 6.99(d,J=7.8Hz,1H),6.94(t,J=7.6Hz,1H),6.85–6.79(m,2H),6.60(s,2H),5.33(s,2H). 13 C NMR (101 MHz, DMSO-d 6 ): δ161.1,158.7,155.1,142.9,134.0,129.4,129.3,128.3,128.2,124.5, 124.5,124.0,123.8,120.6,118.2,115.5,115.3,114.8,107.7,39.1,39.1.

[0107] Embodiment 17

[0108]

[0109] Preparation of N-(3-fluorobenzyl)-1H-benzimidazol-2-amine (A117)

[0110] The preparation method is the same as that of Example 9, except that the benzaldehyde in the step is replaced by 3-fluorobenzaldehyde to obtain a white solid with a yield of 39%. 1 HNMR (400 MHz, DMSO-d 6 ): δ7.36(q,J=7.7Hz,1H),7.15(d,J=7.8Hz,1H),7.07(d,J=7.4Hz,2H),7.00(d,J= 7.7Hz,2H),6.94(t,J=7.5Hz,1H),6.83(t,J=7.5Hz,1H),6.64(s,2H),5.28(s,2H). 13 C NMR (101 MHz, DMSO-d 6 ): δ163.4,161.0,155.0,142.9,140.3,140.2,134.1,130.7,130.6,123. 1,123.0,120.7,118.3,114.9,114.3,114.1,113.9,113.7,107.9,44.2.

[0111] Embodiment 18

[0112]

[0113] Preparation of N-(4-fluorobenzyl)-1H-benzimidazol-2-amine (A118)

[0114] The preparation method is the same as that of Example 9, except that the benzaldehyde in the step is replaced by 4-fluorobenzaldehyde to obtain a white solid with a yield of 41%. 1 HNMR (400 MHz, DMSO-d 6 ): δ7.28–7.22(m,2H),7.15(t,J=8.4Hz,3H),7.07(d,J=7.5Hz,1H),6.93(t,J=7.4Hz,1H),6.82(t,J=7.4Hz,1H),6.62(s,2H),5.24(s,2H). 13 CNMR (101MHz, DMSO-d 6 ): δ162.6,160.1,154.9,142.9,129.1,129.0,120.5,118.1,115.4,115.2,114.8,107.8,43.9.

[0115] Embodiment 19

[0116]

[0117] Preparation of N-(3-methylbenzyl)-1H-benzimidazol-2-amine (A119)

[0118] The preparation method is the same as that of Example 9, except that the benzaldehyde in the step is replaced by 3-methylbenzaldehyde to obtain a white solid with a yield of 43%. 1 HNMR (400 MHz, DMSO-d 6 ): δ7.21-7.12(m,2H),7.08–7.00(m,3H),6.97(d,J=7.7Hz,1H),6.95-6. 89(m,1H),6.81(t,J=7.5Hz,1H),6.58(s,2H),5.21(s,2H),2.24(s,3H). 13 C NMR (101 MHz, DMSO-d 6 ): δ155.1,142.9,137.7,137.2,134.2,128.5,128.0,127.6,124.1,120.5,118.1,114.8,107.9,44.7,21.1.

[0119] Embodiment 20

[0120]

[0121] Preparation of N-benzyl-6-bromo-1H-benzimidazol-2-amine (A120)

[0122] The preparation method is the same as that of Example 9, except that 2-aminobenzimidazole in the step is replaced by 6-bromo-1H-benzimidazole-2-amine to obtain a light yellow solid with a yield of 46%. 1 H NMR (400 MHz, DMSO-d 6 ): δ7.46(dd,J=7.9,2.1Hz,1H),7.38(d,J=1.9Hz,1H),7.31–7.24(m,2H),7.13(d,J=7.7Hz,1H ),7.08–7.03(m,1H),6.97(dd,J=8.3,1.9Hz,1H),6.82(d,J=3.0Hz,1H),5.28(d,J=6.2Hz,2H). 13 C NMR (101 MHz, DMSO-d 6): δ156.0,144.7,139.6,133.3,130.9,130.3,129.7,126.0,121.8,120.5,117.2,112.9,109.4,44.1.

[0123] Embodiment 21

[0124]

[0125] Preparation of 6-bromo-N-(3-bromobenzyl)-1H-benzimidazol-2-amine (A121)

[0126] The preparation method is the same as that of Example 9, except that 2-aminobenzimidazole in the step is replaced by 6-bromo-1H-benzimidazole-2-amine, and benzaldehyde is replaced by 3-bromobenzaldehyde to obtain a light yellow solid with a yield of 48%. 1 HNMR (400 MHz, DMSO-d 6 ): δ7.5(d,J=7.9Hz,1H),7.4(d,J=1.8Hz,1H),7.3(d,J=1.6Hz,1H),7.3(t,J= 7.8Hz,1H),7.1(d,J=7.7Hz,1H),7.1(d,J=2.4Hz,2H),6.8(s,2H),5.3(s,2H). 13 C NMR (101 MHz, DMSO-d 6 ): δ155.7,142.2,139.7,135.5,130.9,130.3,129.7,126.0,123.4,121.8,116.3,110.6,109.9,44.0.

[0127] Embodiment 22

[0128]

[0129] Preparation of 6-bromo-N-(3-chlorobenzyl)-1H-benzimidazol-2-amine (A122)

[0130] The preparation method is the same as that of Example 9, except that 2-aminobenzimidazole in the step is replaced by 6-bromo-1H-benzimidazole-2-amine, and benzaldehyde is replaced by 3-chlorobenzaldehyde to obtain a white solid with a yield of 42%. 1 HNMR (400 MHz, DMSO-d 6): δ7.3(d,J=7.4Hz,2H),7.3(d,J=1.9Hz,1H),7.2(s,1H),7.1–7.0(m,2H),7.0(dd,J=8.3,2.0Hz,1H),6.8(d,J=21.5Hz,2H),5.3(d,J=6.3Hz,2H). 13 C NMR (101 MHz, DMSO-d 6 ): δ156.0,144.8,139.4,133.3,133.2,130.6,127.4,126.8,125.6,120.5,117.2,112.9,109.4,44.2.HRMS(ESI,m / z):Calcd.for C 14 H 11 BrClN 3 [M+H] + 335.9898,found:335.9903.

[0131] Biological activity test

[0132] The cAMP accumulation assay was used to test the effect of target compounds on β 2 AR functional activity and elucidation of whether the new compound is β 2 Negative allosteric modulator (NAM) of AR. The accumulation level of cAMP was mainly tested using GloSensor, a bioluminescence-based biosensor that can directly detect intracellular cAMP (Promega). Briefly described as follows, HEK 293T cells were seeded in 6-well plates, with 4×105 cells per well. The next day, β 2 AR and pGloSensor-22FcAMP plasmids were transfected into HEK 293T cells simultaneously. After 48 h, CO 2 The transfected cells were washed with independent culture medium and then incubated with 2% v / v GloSensor cAMP reagent stock solution (dissolved in CO containing 10% FBS). 2 The cells were incubated with the balance solution of the culture medium (independent culture medium). After incubation at 37°C for 1 hour and then at room temperature for 1 hour, the bioluminescent signal was detected by a multifunctional microplate reader until a steady-state baseline signal was obtained. Then, different concentration gradients of the new derivative and the control compound Cmpd-15 were added to the cells, and the positive control ISO (final concentration 1nM-100μM) was added after incubation at 37°C for 30 minutes. The changes in bioluminescence were read with a microplate reader.

[0133] Allosteric antagonistic activity screening

[0134] Using cAMP accumulation experiment, different concentration gradients of isoproterenol (ISO) were used as negative control (final concentration gradient was 1nM-100μM), and the lead compound Cmpd-15 (final concentration was 50μM) was used as positive control to compare the allosteric antagonist activity of the newly synthesized benzimidazole derivatives (final concentration was 50μM) with that of the lead compound Cmpd-15. The test results in Table 1 show that the synthesized compounds have an allosteric antagonist activity against β 2 -Adrenaline receptors have different degrees of allosteric antagonism. Generally, when the amine structure is phenylalanine, there is a certain activity when the left side is an electron-donating group. Except for A103, A105, A106, A107, A108, and A121, most compounds have allosteric antagonism activity, and many of them have allosteric antagonism activity on β 2 The allosteric antagonist activity of AR is significantly better than that of the lead compound Cmpd-15, such as A110, A111, A112, A113, and A114. Among them, the allosteric antagonist activity of A101, A111, A112, and A114 is more than 2.20 times that of the lead compound, and the allosteric antagonist activity of A110 is 2.53 times that of the lead compound.

[0135] Table 1 Statistical comparison of the allosteric activity of benzimidazole amine derivatives and the activity of Cmpd-15

[0136]

[0137]

[0138] Note: a value represents the blocking activity relative to Cmpd-15; “-” means the compound has no allosteric antagonistic activity

[0139] Allosteric antagonism mechanism studies

[0140] The GloSensor cAMP accumulation assay was used to further investigate whether this class of compounds could allosterically regulate β 2 The functional activity of ISO, an endogenous ligand of -AR, further confirmed that this class of compounds is β 2 -AR negative allosteric modulator (NAM). Taking the benzimidazole amine derivative A101 as an example (such as Figure 3As shown in the figure, when the concentration of compound A101 reaches 30μM, the curve of ISO shows a significant downward shift and almost reaches the maximum downward shift limit of the dose-effect regulation of ISO functional activity, which indicates that the IC50 value of compound A101 may be between 15μM and 30μM, which is specifically manifested as a sudden drop in the concentration curve. When the concentration of A101 increases from 30.0μM to 120.0μM, the downward shift of the concentration curve of ISO is very weak. This phenomenon explains that with the increase of the concentration of compound A101, the concentration-dependent curve of ISO shows a limited downward shift, indicating that the benzimidazole amine derivative A101 can effectively negatively allosterically regulate β 2 The functional activity of ISO, the endogenous ligand of -AR, is allosterically regulated, which is consistent with the reported allosteric antagonistic regulation mechanism. The other benzimidazole amine derivatives represent the ISO concentration dependence curve of A111 allosteric regulation (such as Figure 4 The results of A101 are consistent with those of the newly synthesized benzimidazole amine derivatives. 2 -Negative allosteric modulator (NAM) of AR.

[0141] Liver metabolic stability studies

[0142] The inventors chose human and mouse liver microsomes to evaluate the in vitro metabolic stability of Cmpd-15 and benzimidazole amine derivatives A103 and A111. The incubation mixture consisted of 0.1M PBS (pH 7.4), NADPH (2mM), liver microsomes (0.2mg / mL) and the test compound (1μM). The half-life (T 1 / 2 ) and hepatic extraction ratio (Eh) are two main parameters used to evaluate the metabolic stability of test compounds. The hepatic extraction ratio (Eh) refers to the proportion of the compound that passes through the liver (usually due to metabolism). The extraction rate can generally be divided into high (>70%), medium (30%-70%) and low (<30%) according to the proportion of the compound removed during a single pass through the liver.

[0143] For Cmpd-15, its half-life (T 1 / 2 ) had a half-life of 2.19 min in mouse liver microsomes and 1.53 min in human liver microsomes, and had a high clearance rate in mouse liver (Eh: 98.60%) and human liver (Eh: 99.3%), as shown in Table 2.

[0144] Table 2 Comparison of stability of compounds A103 and A111

[0145]

[0146] These results show that the inventor's lead compound Cmpd-15 has very poor metabolic stability, while the half-life of the newly synthesized benzimidazole amine amide bond compound A103 is 9.60min in mouse liver microsomes and 13.0min in human liver microsomes, which is much more stable than Cmpd-15. When the inventor reduced the amide bond of the compound to a carbon-nitrogen bond, the stability of the compound was significantly improved. Under the same other conditions, the half-life of the A111 compound was 15.5min in mouse liver microsomes, but the half-life in human liver microsomes reached 173min. It can be seen that the half-life of the carbon-nitrogen bond compound A111 is significantly improved compared with the half-life of the amide bond compound A103, and the stability of the compound is significantly improved.

[0147] In summary, the present invention uses the GloSensor cAMP accumulation experiment to test the effect of the target compound on the G protein-dependent signaling pathway on β 2 AR functional activity and to elucidate whether new compounds can allosterically modulate β 2 The functional activity of AR endogenous ligand isoproterenol (ISO). The experimental results showed that most benzimidazole derivatives had no significant effect on β 2 AR has good antagonist activity and can negatively allosterically regulate the functional activity of isoproterenol (ISO). Among them, except A103, A105, A106, A107, A108, and A121, all compounds have good antagonist activity against β 2 AR has allosteric antagonist activity, and its activity is significantly better than that of the lead compound Cmpd-15.

[0148] Those skilled in the art can understand that the above-mentioned embodiments are specific examples for implementing the present application, and in practical applications, various changes can be made to them in form and detail without departing from the spirit and scope of the present application. Any person skilled in the art can make their own changes and modifications without departing from the spirit and scope of the present application, so the scope of protection of the present application shall be based on the scope defined in the claims.

Claims

1. A benzimidazole amine derivative, characterized in that: The structure of the benzimidazole amine derivative is shown below: R1=H, Br; R2 is one of the following structural formulas:

2. The benzimidazole amine derivative according to claim 1, characterized in that: The benzimidazole amine derivative is specifically one of the following structural formulas:

3. The method for preparing the benzimidazole amine derivatives according to claim 2, characterized in that: The preparation method comprises: dissolving an acid and an activator in a solvent, adding different types of amines in an ice bath, and then adding an acid binding agent and an amide coupling agent and continuing to stir to room temperature to obtain the benzimidazole amine derivative; wherein the solvent is N,N-dimethylformamide, the activator is 1-hydroxy-7-azabenzotriazole, the acid binding agent is N-methylmorpholine, and the amide coupling agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

4. The method for preparing the benzimidazole amine derivatives according to claim 3, characterized in that: The preparation method specifically comprises: dissolving 1 eq of substituted benzoic acid and 1.2 eq of HOAt in DMF, stirring for 10 min, and then adding benzimidazole amine; then, adding 0.7 eq of NMM at 0°C, stirring for 10 min, adding 1.2 eq of EDCI, maintaining stirring at 0°C for 1.5 h, and reacting at room temperature for 12 h; spin-drying the DMF, extracting, and column-passing to obtain the benzimidazole amine derivative.

5. The benzimidazole amine derivative according to claim 1, characterized in that: The benzimidazole amine derivative is specifically one of the following structural formulas:

6. The method for preparing the benzimidazole amine derivatives according to claim 5, characterized in that: The preparation method comprises: dissolving benzimidazole amine in a solvent to obtain a reaction system; adding benzaldehyde to the reaction system, and dripping a small amount of glacial acetic acid, reacting for 6 hours under a nitrogen environment to obtain a reaction liquid; then placing the reaction at 0°C, slowly adding a reducing agent to the reaction liquid, and returning the reaction to room temperature to react for 12 hours to obtain the benzimidazole amine derivative; wherein the solvent is methanol and the reducing agent is sodium cyanoborohydride.

7. The method for preparing the benzimidazole amine derivatives according to claim 6, characterized in that: The preparation method specifically comprises: dissolving 1 eq of benzimidazole amine in methanol to obtain a reaction system; adding 1 eq of substituted benzaldehyde to the reaction system, and dripping 0.1 eq of glacial acetic acid, reacting for 6 hours under a nitrogen environment to obtain a reaction solution; then placing the reaction at 0°C, slowly adding 2.5 eq of sodium cyanoborohydride to the reaction solution, returning the reaction to room temperature to react for 12 hours to complete the reaction, adding water to quench, spinning methanol to dryness, extracting, and subjecting the obtained product to column chromatography to obtain the benzimidazole amine derivative.

8. Use of the benzimidazole amine derivative according to any one of claims 1, 2 or 5 as a β2-adrenaline receptor allosteric antagonist.

9. Use of the benzimidazole amine derivative according to any one of claims 1, 2 or 5 in the preparation of β2-adrenaline receptor allosteric antagonist pharmaceutical preparations.

10. The use according to claim 9, characterized in that: The benzimidazole derivatives are used as active ingredients and a pharmaceutically acceptable carrier to prepare a pharmaceutical preparation.

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