A benzimidazole amine derivative, a preparation method thereof and use thereof as a beta2-adrenergic receptor allosteric modulator

Abstract

The application belongs to the field of pharmaceutical chemistry, and specifically discloses a benzimidazole amine derivative, a preparation method thereof and application of the benzimidazole amine derivative as a beta2-adrenergic receptor allosteric antagonist. In the benzimidazole amine derivative, R1 is a hydrogen atom or a bromine atom; and R2 is a substituted benzoyl group or a substituted benzyl group. Pharmacological results show that the benzimidazole amine derivative has good antagonistic activity on beta2AR and can negatively allosterically regulate the functional activity of isoprenaline (ISO).

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical chemistry, and particularly relates to a benzimidazole amine derivative, a preparation method thereof and application thereof as a beta2-adrenergic receptor allosteric modulator. BACKGROUND

[0002] G-protein coupled receptors (GPCRs) are the largest family of cell membrane receptors in human body, which are distributed in all tissues and organs of human body and are involved in almost all life activities. GPCRs are a very important drug target, and more than one-third of the clinical drugs approved by the US FDA are through GPCR to regulate their behavior to play the drug efficacy. Beta-adrenergic receptor (beta-AR) is a class A GPCR, among which, beta2AR transmits signals to cells through G protein to mediate various physiological responses, including vasodilation and vasoconstriction, heart rate regulation, etc. (A.J. Venkatakrishnan, et al. Nature, 2013, 494: 185-194).

[0003] Most of the beta2AR drugs are orthosteric ligands, but orthosteric ligands have many side effects, so the development of beta2AR allosteric modulator related drugs has become a new trend. SUMMARY

[0004] The purpose of the present application is to provide a new benzimidazole derivative, so as to develop a heterocyclic derivative with stable chemical structure, high biological activity and stable in vivo metabolism, as a beta2AR allosteric antagonist, to provide a research basis for creating new drugs for cardiovascular, asthma and cancer diseases.

[0005] The present application provides a benzimidazole amine derivative, the structure of the benzimidazole amine derivative is as follows:

[0006]

[0007] R1=H, Br;

[0008] R2 is one of the following structural formulae:

[0009]

[0010] Further, the benzimidazole amine derivative is specifically one of the following structural formulae:

[0011]

[0012] The application also provides a preparation method of the above-mentioned benzimidazole amine derivative, which comprises the following steps: dissolving acid and an activating agent in a solvent, adding different types of amine under ice bath, continuously stirring until room temperature after adding an acid-binding agent and an amide coupling agent, and obtaining the benzimidazole amine derivative; wherein the solvent is N,N-dimethylformamide, the activating agent 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] Further, the preparation method specifically comprises the following steps: dissolving 1 eq of substituted benzoic acid and 1.2 eq of HOAt in DMF, adding benzimidazole amine after stirring for 10 min, adding 0.7 eq of NMM at 0 DEG C, stirring for 10 min, adding 1.2 eq of EDCI, stirring for 1.5 h at 0 DEG C, and reacting for 12 h at room temperature; spinning dry the DMF, extracting, and column chromatography, so as to obtain the benzimidazole amine derivative.

[0014] Further, the benzimidazole amine derivative specifically has one of the following structural formulas:

[0015]

[0016] The application also provides a preparation method of the above-mentioned benzimidazole amine derivative, which comprises the following steps: dissolving benzimidazole amine in a solvent to obtain a reaction system; adding benzaldehyde into the reaction system, and adding a small amount of glacial acetic acid dropwise, so as to obtain a reaction liquid under nitrogen environment and react for 6 h; then, the reaction is placed at 0 DEG C, a reducing agent is slowly added into the reaction liquid, the reaction is restored to room temperature and reacts for 12 h, so as to obtain the benzimidazole amine derivative; wherein the solvent is methanol, and the reducing agent is sodium cyanoborohydride.

[0017] Further, the preparation method specifically comprises the following steps: dissolving 1 eq of benzimidazole amine in methanol to obtain a reaction system; adding 1 eq of substituted benzaldehyde into the reaction system, and adding 0.1 eq of glacial acetic acid dropwise, so as to obtain a reaction liquid under nitrogen environment and react for 6 h; then, the reaction is placed at 0 DEG C, 2.5 eq of sodium cyanoborohydride is slowly added into the reaction liquid, the reaction is restored to room temperature and reacts for 12 h, the reaction is quenched by adding water, methanol is spun dry, extraction is performed, and column chromatography is performed on the obtained product, so as to obtain the benzimidazole amine derivative.

[0018] The application also provides the use of the above-mentioned benzimidazole amine derivative as a beta2-adrenergic receptor allosteric modulator.

[0019] The application also provides the use of the above-mentioned benzimidazole amine derivative in the field of preparing a beta2-adrenergic receptor allosteric modulator drug preparation.

[0020] Further, the benzimidazole derivative is prepared into a pharmaceutical preparation as an active ingredient together with a pharmaceutically acceptable carrier.

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

[0022] The new derivative synthesized first has significantly improved allosteric antagonistic activity on the β2AR target compared with the lead compound Cmpd-15; secondly, the new derivative has a novel skeleton, and compared with the lead compound Cmpd-15, the structure is greatly simplified, the synthetic route is simplified, the synthesis cost and difficulty are reduced, and the economic benefit of the compound can be obviously improved. BRIEF DESCRIPTION OF DRAWINGS

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

[0024] Figure 2 The design idea of the amide bond flip and the reduction of the amide bond designed from the benzimidazole series of compounds;

[0025] Figure 3 The ISO dose-response curve graph mediated by the benzimidazole amine derivative A101;

[0026] Figure 4 The ISO dose-response curve graph mediated by the benzimidazole amine derivative A111. DETAILED DESCRIPTION

[0027] A few years ago, the inventors and collaborators reported the first intracellular allosteric antagonist of β2AR, Cmpd-15 (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 (K.C. Meng, et al. Bioorg. Med. Chem. 2018, 26: 2320-2330). Later, the inventors used the scaffold 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 class of structurally stable pyrazole amides, and found a β2AR allosteric antagonist with better activity than Cmpd-15 (X. Guo, et al. Bioorg. Med. Chem. 2024, 108: 117787).

[0028] On this basis, the inventors continue to optimize the structure of the lead compound through the strategies of bioisosteric groups, skeleton transition, pharmacophore hybridization and structure simplification, and design and synthesize a series of new derivatives (Benzimidazole amides) with benzimidazole amide as the skeleton, which have significantly better activity than Cmpd-15 (Chen Xin, et al. Chinese invention patent application number: CN202310937574.0, 2023). However, although these compounds have considerable biological activity, the aniline structure contained therein is easily oxidized by CYP450 enzymes, resulting in poor biological stability of these compounds. The inventors recently conducted liver metabolic stability experiments on these compounds and found that the half-life of benzimidazole derivatives is less than 30 minutes. In order to solve the stability problem and avoid the presence of aniline structure in the compound, the inventors attempt to flip the amide bond to form a new derivative of Type A. In addition, the inventors also optimize the amide bond into a carbon-nitrogen bond, that is, a new derivative of Type B, to avoid amide bond hydrolysis and improve the metabolic stability of these compounds, as shown in Figure 1 . .

[0029] Benzimidazole is an important heterocyclic compound, whose chemical structure is composed of benzene ring and imidazole ring. In the inventors' previous research, a class of benzimidazole amide derivatives were found to act as allosteric antagonists of β2AR, and their biological activity was significantly better than that of the lead compound Cpmd-15. However, the metabolic stability of these compounds is poor, with a half-life of only about half an hour, which is likely due to the presence of aniline structure units in their structure that are easily oxidized by CPY450 enzymes. .

[0030] Therefore, the present application adopts two strategies to optimize the structure of benzimidazole amide derivatives: first, flip the amide bond to change the aniline structure unit to a benzoyl structure unit, with the aim of avoiding oxidation metabolism directly related to aniline; second, change the flipped amide bond to a carbon-nitrogen bond, with the aim of avoiding enzyme-catalyzed amide bond hydrolysis, as shown in Figure 2 . .

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

[0032] . . .

[0033] Dissolve the acid and the activating agent in a solvent, add different types of amines under ice bath, and then continue to stir until room temperature after adding the acid binding agent and the amide coupling agent. Among them, the solvent is N,N-dimethylformamide, the activating agent 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: the substituted benzoic acid (1 eq) and HOAt (1.2 eq) are dissolved in DMF, stirred for 10 min, then benzimidazole amine is added. Then, NMM (0.7 eq) is added at 0℃, stirred for 10 min, EDCI (1.2 eq) is added, stirred at 0℃ for 1.5 h, and reacted at room temperature for 12 h. The DMF is rotary evaporated, extracted, columned, and compound A101-A108 is obtained.

[0035] Another synthesis method of the benzimidazole amine derivative is as follows:

[0036]

[0037] The benzimidazole amine is dissolved in a solvent, benzaldehyde is added to the system, and a small amount of glacial acetic acid is added dropwise, and the reaction is carried out under nitrogen for 6 h, and then the reaction is placed at 0℃, and a reducing agent is slowly added to the reaction solution, and the reaction is restored to room temperature and reacted for 12 h. The solvent is methanol, and the reducing agent is sodium cyanoborohydride.

[0038] The specific method is: the 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, and the reaction is carried out under nitrogen for 6 h, and then the reaction is placed at 0℃, and sodium cyanoborohydride (2.5 eq) is slowly added to the reaction solution, and the reaction is restored to room temperature and reacted for 12 h. After the reaction is completed, water is added to quench, methanol is rotary evaporated, extracted, and the obtained product is column chromatographed to obtain compound A109-A122.

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

[0040]

[0041] The application will be described in detail below in combination with specific embodiments.

[0042] The synthesis route of the benzimidazole amine derivative provided by the application is as follows:

[0043] Example 1

[0044]

[0045] Preparation of benzamide-1H-benzimidazole-2-amine (A101)

[0046] Benzoic acid (122 mg, 1.50 mmol) was dissolved in DMF (4 mL), to this system HOAt (265 mg, 1.95 mmol) was added and stirred for 10 min, then compound 2-aminobenzimidazole (200 mg, 1.50 mmol) was added to the above system, N-methylmorpholine (134.7 mL, 1.05 mmol) was added to the system under ice bath and stirred for 10 min, finally EDCI (374 mg, 1.95 mmol) was added to the system, the reaction was kept at 0 °C for 1 h, then the reaction was kept at room temperature for 12 h. The reaction was monitored by TLC, the reaction solution was concentrated under vacuum, the obtained oily liquid was extracted with ethyl acetate (15 mL*3), the organic phase was washed with saturated brine again, the organic phase was collected, dried by adding anhydrous sodium sulfate, concentrated under vacuum, the obtained product was purified by column chromatography (petroleum ether: ethyl acetate = 4: 1) to obtain white solid 162 mg, the yield was 64%. 1 HNMR (400 MHz, DMSO-d6): δ 12.28 (s, 2H), 8.16-8.12 (m, 2H), 7.63-7.58 (m, 1H), 7.53 (t, J = 7.4 Hz, 2H), 7.46 (dd, J = 5.9, 3.2 Hz, 2H), 7.13 (dq, J = 6.7, 3.5 Hz, 2H). 13 C NMR (101 MHz, DMSO-d6): δ 168.3, 148.9, 134.3, 132.0, 128.4, 128.3, 121.5, 113.4. HRMS (ESI, m / z): Calcd. for C 14 H 11 N3O[M+H] + 238.09748, found: 238.0982.

[0047] Example 2

[0048]

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

[0050] Preparation method was the same as example 1, except that 2-bromobenzoic acid was used instead of benzoic acid, white solid was obtained, the yield was 62%. 1 H NMR (400 MHz, DMSO-d6): δ 12.30 (s, 2H), 7.72 (d, J = 7.9 Hz, 1H), 7.64 (d, J = 5.8 Hz, 1H), 7.52-7.40 (m, 4H), 7.12 (dd, J = 5.9, 3.3 Hz, 2H). 13C NMR (101 MHz, DMSO-d6): δ 168.0, 138.0, 132.9, 131.7, 129.3, 127.7, 121.6, 119.2, 113.9.

[0051] Example 3

[0052]

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

[0054] Preparation Method same as Example 1, except that 3-bromobenzoic acid was used instead of benzoic acid to give a white solid in 67% yield. 1 H NMR (400 MHz, DMSO-d6): δ 12.44 (s, 2H), 8.31 (t, J = 1.8 Hz, 1H), 8.13 (dt, J = 7.8, 1.3 Hz, 1H), 7.76 (ddd, J = 8.0, 2.1, 1.1 Hz, 1H), 7.48 (d, J = 7.9 Hz, 1H), 7.44 (dd, J = 5.9, 3.1 Hz, 2H), 7.17 (dd, J = 5.9, 3.1 Hz, 2H). 13 C NMR (101 MHz, DMSO-d6): δ 169.1, 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 BrN3O [M+H] + 316.0080, found: 316.0086.

[0055] Example 4

[0056]

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

[0058] Preparation Method same as Example 1, except that 4-bromobenzoic acid was used instead of benzoic acid to give a white solid in 61% yield. 1 H NMR (400 MHz, DMSO-d6): δ 12.39 (s, 2H), 8.08 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 8.2 Hz, 2H), 7.44 (dd, J = 5.7, 3.0 Hz, 2H), 7.15 (dd, J = 5.9, 3.1 Hz, 2H). 13C NMR (101 MHz, DMSO-d6): δ 169.1, 150.3, 134.8, 132.8, 131.3, 130.5, 125.6, 121.9, 112.8.

[0059] Example 5

[0060]

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

[0062] Preparation Method same as Example 1, except that 3-chlorobenzoic acid was used instead of benzoic acid to give a white solid in 65% yield. 1 H NMR (400 MHz, DMSO-d6): δ 12.45 (s, 2H), 8.16 (s, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.53 (t, J = 7.8 Hz, 1H), 7.44 (dd, J = 5.9, 3.1 Hz, 2H), 7.16 (dd, J = 6.0, 3.2 Hz, 2H). 13 C NMR (101 MHz, DMSO-d6): δ 169.1, 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-lH-benzimidazol-2-yl)benzamide (A106)

[0066] Preparation Method same as Example 1, except that 2-aminobenzimidazole was replaced by 6-bromo-lH-benzimidazol-2-amine to give a white solid in 63% yield. 1 H NMR (400 MHz, DMSO-d6): δ 12.39 (s, 2H), 8.12 (d, J = 7.9 Hz, 2H), 7.66 - 7.61 (m, 2H), 7.55 (t, J = 7.6 Hz, 2H), 7.43 (d, J = 8.4 Hz, 1H), 7.27 (dd, J = 8.5, 2.1 Hz, 1H).

[0067] Example 7

[0068]

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

[0070] Preparation method is the same as example 6, except that 2-aminobenzimidazole in step is replaced by 6-bromo-lH-benzimidazol-2-amine, benzoic acid is replaced by 3-chlorobenzoic acid, to obtain white solid, yield is 61%. 1 H NMR (400 MHz, DMSO-d6): δ 8.14 (s, 1H), 8.06 (d, J = 7.8 Hz, 1H), 7.69-7.62 (m, 2H), 7.56 (t, J = 7.9 Hz, 1H), 7.42 (d, J = 8.4 Hz, 1H), 7.28 (d, J = 8.5 Hz, 1H). 13 C NMR (101 MHz, DMSO-d6): δ 136.21, 133.38, 132.11, 130.56, 128.20, 127.12, 124.51, 113.73.

[0071] Example 8

[0072]

[0073] Preparation of N-(6-bromo-lH-benzimidazol-2-yl)-3-chlorobenzamide (A107)

[0074] Preparation method is the same as example 6, except that 2-aminobenzimidazole in step is replaced by 6-bromo-lH-benzimidazol-2-amine, benzoic acid is replaced by 3-chlorobenzoic acid, to obtain white solid, yield is 61%. 1 H NMR (400 MHz, DMSO-d6): δ 8.14 (s, 1H), 8.06 (d, J = 7.8 Hz, 1H), 7.69-7.62 (m, 2H), 7.56 (t, J = 7.9 Hz, 1H), 7.42 (d, J = 8.4 Hz, 1H), 7.28 (d, J = 8.5 Hz, 1H).

[0075] Example 9

[0076]

[0077] Preparation of N-(6-bromo-lH-benzimidazol-2-yl)-3-chlorobenzamide (A107)

[0078] To a solution of 2-aminobenzimidazole (250 mg, 1.88 mmol) in methanol, benzaldehyde (1.88 mmol) was added and a few drops of glacial acetic acid was added drop wise. The reaction was stirred for 6 h under nitrogen atmosphere. The reaction was cooled to 0 °C and sodium cyanoborohydride (295 mg, 4.69 mmol) was added slowly to the reaction mixture. The reaction was stirred at room temperature for 12 h. The reaction was monitored by TLC and was quenched with water. The reaction mixture was concentrated under vacuum and extracted with ethyl acetate (15 mL*3). The organic layer was washed with saturated brine and the organic layer was collected, dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by column chromatography (petroleum ether: ethyl acetate = 1:4) to get 86.52 mg of white solid with 44% yield. 1 HNMR (400 MHz, DMSO-d6): δ 7.31 (t, J = 7.3 Hz, 2H), 7.24 (d, J = 7.0 Hz, 1H), 7.19 (d, J = 7.1 Hz, 2H), 7.14 (d, J = 7.8 Hz, 1H), 7.04 (d, J = 7.7 Hz, 1H), 6.92 (t, J = 7.5 Hz, 1H), 6.81 (t, J = 7.5 Hz, 1H), 6.59 (s, 2H), 5.26 (s, 2H). 13 CNMR (101 MHz, DMSO-d6): δ 155.1, 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 N3[M+H] + 224.1181, found: 224.1187.

[0079] Example 10

[0080]

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

[0082] Preparation method same as example 9, except that benzaldehyde in the step was replaced by 2-bromobenzaldehyde to get white solid with 41% yield. 1H NMR (400 MHz, DMSO-d6): δ 7.7 (dd, J = 7.3, 1.9 Hz, 1H), 7.3 - 7.2 (m, 3H), 6.9 (t, J = 7.5 Hz, 1H), 6.9 (d, J = 7.3 Hz, 1H), 6.8 (t, J = 7.5 Hz, 1H), 6.6 (s, 2H), 6.4 (dd, J = 7.2, 2.2 Hz, 1H), 5.3 (s, 2H). 13 C NMR (101 MHz, DMSO-d6): δ 155.3, 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] Example 11

[0084]

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

[0086] Preparation method same as Example 9, except that benzaldehyde in step was replaced by 3-bromobenzaldehyde to give white solid with yield of 46%. 1 H NMR (400 MHz, DMSO-d6): δ 7.45 (d, J = 7.9 Hz, 1H), 7.39 (s, 1H), 7.28 (t, J = 7.8 Hz, 1H), 7.16 (t, J = 7.8 Hz, 2H), 7.08 (d, J = 7.7 Hz, 1H), 6.94 (td, J = 7.5, 1.2 Hz, 1H), 6.84 (td, J = 7.5, 1.2 Hz, 1H), 6.62 (s, 2H), 5.27 (s, 2H). 13 C NMR (101 MHz, DMSO-d6): δ 155.0, 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 BrN3[M+H] + 302.0288, found: 302.0288.

[0087] Example 12

[0088]

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

[0090] The preparation method is the same as in Example 9, except that benzaldehyde in the step is replaced with 4-bromobenzaldehyde, resulting in a white solid with a yield of 44%. 1 H NMR (400MHz, DMSO-d6): 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 (101MHz, DMSO-d6): 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] Example 13

[0092]

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

[0094] The preparation method is the same as in Example 9, except that benzaldehyde in the step is replaced with 2-chlorobenzaldehyde, resulting in a white solid with a yield of 43%. 1 H NMR (400MHz, DMSO-d6): 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,1H),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 (101MHz, DMSO-d6): 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] Example 14

[0096]

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

[0098] Preparation method same as example 9, except that benzaldehyde in step was replaced by 3-chlorobenzaldehyde to give white solid in 41% yield. 1 H NMR (400 MHz, DMSO-d6): δ 7.37 - 7.30 (m, 2H), 7.24 (s, 1H), 7.14 (t, J = 7.0 Hz, 2H), 7.08 (d, J = 7.7 Hz, 1H), 6.94 (t, J = 7.5 Hz, 1H), 6.83 (t, J = 7.5 Hz, 1H), 6.61 (s, 2H), 5.28 (s, 2H). 13 C NMR (101 MHz, DMSO-d6): δ 155.0, 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 ClN3[M+H] + 258.0793, found: 258.0800.

[0099] Example 15

[0100]

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

[0102] Preparation method same as example 9, except that benzaldehyde in step was replaced by 4-chlorobenzaldehyde to give white solid in 42% yield. 1 H NMR (400 MHz, DMSO-d6): δ 7.38 (d, J = 8.3 Hz, 2H), 7.20 (d, J = 8.3 Hz, 2H), 7.15 (d, J = 7.8 Hz, 1H), 7.05 (d, J = 7.7 Hz, 1H), 6.93 (t, J = 7.6 Hz, 1H), 6.82 (t, J = 7.5 Hz, 1H), 6.62 (s, 2H), 5.26 (s, 2H). 13 C NMR (101 MHz, DMSO-d6): δ 155.0, 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)-lH-benzimidazol-2-amine (A116)

[0106] The preparation method is the same as that in Example 9, except that 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-d6): δ 7.34-7.29 (m, 1H), 7.23 (t, J = 9.3 Hz, 1H), 7.16 (d, J = 7.8 Hz, 1H), 7.09 (t, J = 7.5 Hz, 1H), 6.99 (d, J = 7.8 Hz, 1H), 6.94 (t, J = 7.6 Hz, 1H), 6.85-6.79 (m, 2H), 6.60 (s, 2H), 5.33 (s, 2H). 13 C NMR (101 MHz, DMSO-d6): δ 161.1, 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] Example 17

[0108]

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

[0110] The preparation method is the same as that in Example 9, except that benzaldehyde in the step is replaced by 3-fluorobenzaldehyde to obtain a white solid with a yield of 39%. 1 H NMR (400 MHz, DMSO-d6): δ 7.36 (q, J = 7.7 Hz, 1H), 7.15 (d, J = 7.8 Hz, 1H), 7.07 (d, J = 7.4 Hz, 2H), 7.00 (d, J = 7.7 Hz, 2H), 6.94 (t, J = 7.5 Hz, 1H), 6.83 (t, J = 7.5 Hz, 1H), 6.64 (s, 2H), 5.28 (s, 2H). 13 C NMR (101 MHz, DMSO-d6): δ 163.4, 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] Example 18

[0112]

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

[0114] Preparation method is the same as example 9, except that benzaldehyde in step is replaced by 4-fluorobenzaldehyde to obtain white solid with yield of 41%. 1 HNMR (400 MHz, DMSO-d6): δ 7.28 - 7.22 (m, 2H), 7.15 (t, J = 8.4 Hz, 3H), 7.07 (d, J = 7.5 Hz, 1H), 6.93 (t, J = 7.4 Hz, 1H), 6.82 (t, J = 7.4 Hz, 1H), 6.62 (s, 2H), 5.24 (s, 2H). 13 CNMR (101 MHz, DMSO-d6): δ 162.6, 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] Example 19

[0116]

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

[0118] Preparation method is the same as example 9, except that benzaldehyde in step is replaced by 3-methylbenzaldehyde to obtain white solid with yield of 43%. 1 HNMR (400 MHz, DMSO-d6): δ 7.21 - 7.12 (m, 2H), 7.08 - 7.00 (m, 3H), 6.97 (d, J = 7.7 Hz, 1H), 6.95 - 6.89 (m, 1H), 6.81 (t, J = 7.5 Hz, 1H), 6.58 (s, 2H), 5.21 (s, 2H), 2.24 (s, 3H). 13 CNMR (101 MHz, DMSO-d6): δ 155.1, 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] Example 20

[0120]

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

[0122] Preparation method is the same as example 9, except that 2-aminobenzimidazole in step is replaced by 6-bromo-lH-benzimidazol-2-amine, to get a light yellow solid, yield of 46%. 1 H NMR (400 MHz, DMSO-d6): δ 7.46 (dd, J = 7.9, 2.1 Hz, 1H), 7.38 (d, J = 1.9 Hz, 1H), 7.31 - 7.24 (m, 2H), 7.13 (d, J = 7.7 Hz, 1H), 7.08 - 7.03 (m, 1H), 6.97 (dd, J = 8.3, 1.9 Hz, 1H), 6.82 (d, J = 3.0 Hz, 1H), 5.28 (d, J = 6.2 Hz, 2H). 13 C NMR (101 MHz, DMSO-d6): δ 156.0, 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] Example 21

[0124]

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

[0126] Preparation method is the same as example 9, except that 2-aminobenzimidazole in step is replaced by 6-bromo-lH-benzimidazol-2-amine, benzaldehyde is replaced by 3-bromobenzaldehyde, to get a light yellow solid, yield of 48%. 1 H NMR (400 MHz, DMSO-d6): δ 7.46 (dd, J = 7.9, 2.1 Hz, 1H), 7.38 (d, J = 1.9 Hz, 1H), 7.31 - 7.24 (m, 2H), 7.13 (d, J = 7.7 Hz, 1H), 7.08 - 7.03 (m, 1H), 6.97 (dd, J = 8.3, 1.9 Hz, 1H), 6.82 (d, J = 3.0 Hz, 1H), 5.28 (d, J = 6.2 Hz, 2H). 13 C NMR (101 MHz, DMSO-d6): δ 156.0, 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.

[0127] Example 22

[0128]

[0129] 6-Bromo-N-(3-chlorobenzyl)-lH-benzimidazol-2-amine (A122) was prepared according to the procedure described in Example 9, except that 2-aminobenzimidazole was replaced by 6-bromo-lH-benzimidazol-2-amine and benzaldehyde was replaced by 3-chlorobenzaldehyde to give a white solid in 42% yield.

[0130] Preparation Method was the same as Example 9, except that 2-aminobenzimidazole was replaced by 6-bromo-lH-benzimidazol-2-amine and benzaldehyde was replaced by 3-chlorobenzaldehyde to give a white solid in 42% yield. 1 HNMR (400 MHz, DMSO-d6): δ 7.3 (d, J = 7.4 Hz, 2H), 7.3 (d, J = 1.9 Hz, 1H), 7.2 (s, 1H), 7.1 - 7.0 (m, 2H), 7.0 (dd, J = 8.3, 2.0 Hz, 1H), 6.8 (d, J = 21.5 Hz, 2H), 5.3 (d, J = 6.3 Hz, 2H). 13 C NMR (101 MHz, DMSO-d6): δ 156.0, 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 BrClN3[M+H] + 335.9898, found: 335.9903.

[0131] Biological activity test

[0132] The cAMP accumulation assay was used to test the functional activity of the target compounds on β2AR and to clarify whether the new compounds are negative allosteric modulators (NAM) of β2AR. The cAMP accumulation level was tested mainly by using GloSensor, a bioluminescence-based biosensor that can directly detect intracellular cAMP (Promega). Briefly, HEK 293T cells were seeded in 6-well plates at 4 x 105cells per well. The next day, β2AR and pGloSensor-22F cAMP plasmids were co-transfected into HEK 293T cells using FuGene transfection reagent (Promega). After 48 h, the transfected cells were washed with CO2-independent medium and incubated with equilibration solution containing 2% v / v GloSensor cAMP reagent stock solution (dissolved in CO2-independent medium containing 10% FBS). After incubation at 37 °C for 1 h and then at room temperature for 1 h, the bioluminescence signal was detected using a multifunctional microplate reader until a stable baseline signal was obtained. Then, different concentrations of the new derivatives and the control compound Cmpd-15 were added to the cells, and after incubation at 37 °C for 30 min, the positive control ISO (final concentration 1 nM-100 μΜ) was added. The change in bioluminescence was read using the microplate reader.

[0133] Screening of allosteric antagonistic activity

[0134] The cAMP accumulation assay was used to test the functional activity of the target compounds on β2AR and to clarify whether the new compounds are negative allosteric modulators (NAM) of β2AR. The cAMP accumulation level was tested mainly by using GloSensor, a bioluminescence-based biosensor that can directly detect intracellular cAMP (Promega). Briefly, HEK 293T cells were seeded in 6-well plates at 4 x 105cells per well. The next day, β2AR and pGloSensor-22F cAMP plasmids were co-transfected into HEK 293T cells using FuGene transfection reagent (Promega). After 48 h, the transfected cells were washed with CO2-independent medium and incubated with equilibration solution containing 2% v / v GloSensor cAMP reagent stock solution (dissolved in CO2-independent medium containing 10% FBS). After incubation at 37 °C for 1 h and then at room temperature for 1 h, the bioluminescence signal was detected using a multifunctional microplate reader until a stable baseline signal was obtained. Then, different concentrations of the new derivatives and the control compound Cmpd-15 were added to the cells, and after incubation at 37 °C for 30 min, the positive control ISO (final concentration 1 nM-100 μΜ) was added. The change in bioluminescence was read using the microplate reader.

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

[0136]

[0137]

[0138] Note: a Values represent the blocking activity relative to Cmpd-15; "-" indicates that the compound has no allosteric antagonistic activity

[0139] Allosteric antagonistic mechanism study

[0140] Further study on whether the compounds can allosterically modulate the functional activity of the endogenous ligand ISO of β2-AR through GloSensor cAMP accumulation experiment, further confirm that the compounds are negative allosteric modulators (NAM) of β2-AR. Take benzimidazole amine derivative A101 as an example (as shown in Figure 3 When the concentration of compound A101 reaches 30 μM, the ISO concentration curve shows a large downward shift and almost reaches the maximum downward shift lower limit of the functional activity of ISO, which indicates that the IC50 value of compound A101 may be between 15 μM and 30 μM, which is manifested as a sharp drop in the concentration curve. When the concentration of A101 increases from 30.0 μM to 120.0 μM, the degree of downward shift of the ISO concentration curve is very weak. This phenomenon illustrates that with the increase of the concentration of compound A101, the concentration-dependent curve of ISO shows a limited downward shift, indicating that benzimidazole amine derivative A101 can effectively negatively allosterically modulate the functional activity of the endogenous ligand ISO of β2-AR. This allosteric modulation phenomenon is consistent with the reported allosteric antagonistic modulation mechanism. The ISO concentration-dependent curve of the rest of the benzimidazole amine derivatives represented by A111 (as shown in Figure 4 ) is consistent with the results of A101, that is, the newly synthesized benzimidazole amine derivatives are all negative allosteric modulators (NAM) of β2-AR.

[0141] Liver metabolic stability study

[0142] The inventors selected 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 0.1 M PBS (pH 7.4), NADPH (2 mM), liver microsomes (0.2 mg / mL), and test compound (1 μM). Half-life (T 1 / 2 ) and liver extraction ratio (Eh) are two main parameters for assessing the metabolic stability of test compounds. Liver extraction ratio (Eh) refers to the proportion of the compound passing through the liver (usually due to metabolism). According to the proportion of the compound removed during one pass through the liver, the extraction rate can be generally divided into high (> 70%), medium (30%-70%), and low (< 30%).

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

[0144] Table 2 Stability comparison of compounds A103, A111

[0145]

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

[0147] In summary, the GloSensor cAMP accumulation experiment is used to test the functional activity of the target compound on the G protein-dependent signal pathway of β2AR and to clarify whether the new compound can allosterically regulate the functional activity of the β2AR endogenous ligand isoproterenol (ISO). The experimental results show that most of the benzimidazole derivatives have good antagonistic activity on β2AR and can negatively allosterically regulate the functional activity of isoproterenol (ISO). Among them, compounds other than A103, A105, A106, A107, A108, and A121 have allosteric antagonistic activity on β2AR, and their activity is significantly better than 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 actual application, various changes can be made in form and details without departing from the spirit and scope of the present application. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be limited by the scope defined in the claims.

Claims

1. The application of a benzimidazole amine derivative in the preparation of β2-adrenergic receptor allosteric antagonist pharmaceutical formulations, characterized in that, The structure of the benzimidazole amine derivative is shown in the following formula: R1 = H, Br; R2 is one of the following structural formulas: 。 2. The application according to claim 1, characterized in that, The benzimidazole amine derivative specifically comprises one of the following structural formulas: 。 3. The application according to claim 2, characterized in that, The preparation method of the benzimidazole amine derivative includes: dissolving an acid and an activator in a solvent, adding different types of amines under ice bath conditions, 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 application according to claim 3, characterized in that, 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, at 0 °C, adding 0.7 eq of NMM, stirring for 10 min, adding 1.2 eq of EDCI, stirring at 0 °C for 1.5 h, and reacting at room temperature for 12 h; evaporating the DMF, extracting, and passing through a column to obtain the benzimidazole amine derivative.

5. The application according to claim 1, characterized in that, The benzimidazole amine derivative specifically comprises one of the following structural formulas: 。 6. The application according to claim 5, characterized in that, The preparation method includes: dissolving benzimidazole amine in a solvent to obtain a reaction system; adding benzaldehyde to the reaction system and adding a small amount of glacial acetic acid dropwise, reacting under nitrogen atmosphere for 6 hours to obtain a reaction solution; then placing the reaction at 0°C, slowly adding a reducing agent to the reaction solution, and restoring the reaction to room temperature for 12 hours to obtain the benzimidazole amine derivative; wherein the solvent is methanol and the reducing agent is sodium cyanoborohydride.

7. The application according to claim 6, characterized in that, The preparation method specifically includes: dissolving 1 eq of benzimidazole amine in methanol to obtain a reaction system; adding 1 eq of substituted benzaldehyde to the reaction system and adding 0.1 eq of glacial acetic acid dropwise, reacting under nitrogen atmosphere for 6 h to obtain a reaction solution; then placing the reaction at 0 °C, slowly adding 2.5 eq of sodium cyanoborohydride to the reaction solution, restoring the reaction to room temperature for 12 h to complete the reaction, quenching with water, evaporating methanol, extracting, and then performing column chromatography to obtain the benzimidazole amine derivative.

8. The application according to claim 1, characterized in that, The aforementioned benzimidazole amine derivatives are used as active ingredients and pharmaceutically acceptable carriers to prepare pharmaceutical formulations.

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