Benzimidazole derivatives used as β2-adrenergic receptor antagonists

Abstract

The present invention belongs to the field of medicinal chemistry, and specifically discloses a benzimidazole derivative used as an allosteric antagonist of the β2-adrenergic receptor. The benzimidazole derivative of the present invention has a structure as shown in formula (I), wherein R1 is a hydrogen atom, a halogen atom, a nitro group, a methyl group, a methoxy group, a hydroxyl group, an amino group, etc.; R2 is an aliphatic amine, cyclopentylamine, cyclohexylamine, aniline, m-bromoaniline, m-bromobenzylamine, etc. Pharmacological results show that the benzimidazole derivative of the present invention has good antagonistic activity against β2AR and can negatively allosterically regulate the functional activity of isoproterenol.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and particularly relates to a benzimidazole derivative used as an allosteric antagonist of the β2-adrenergic receptor. Background Art

[0002] G-protein coupled receptors (GPCRs) are the largest family of cell membrane receptors in the human body, distributed in various tissues and organs of the human body, and are involved in almost all life activities. GPCRs are a very important drug target, and more than one-third of the clinically approved drugs by the FDA exert their efficacy by regulating their behavior. The β-adrenergic receptor (β-AR) is a class A GPCR. β2-AR transmits signals into cells through the action of G proteins, mediating various physiological responses, including vasodilation and vasoconstriction, heart rate regulation, etc. (Venkatakrishnan, A.J. et al. Nature, 2013, 494: 185-194). The vast majority of β2-AR drugs are orthosteric ligands, but orthosteric ligands have more side effects, so the development of drugs related to β2-AR allosteric modulators has become an emerging trend.

[0003] Previously, we first reported the first intracellular allosteric antagonist of the β2-adrenergic receptor, Cmpd-15 (Ahn S, et al. PNAS, 2017, 114: 1708-1713). However, due to the poor solubility and low biological activity of Cmpd-15, the relative instability of its polypeptide structure will affect its drugability (Meng KC, et al. Bioorg. Med. Chem., 2018, 26: 2320-2330). Therefore, we adopted a skeleton migration strategy to replace the amide of the left fragment of the original compound Cmpd-15 with a nitrogen-containing heterocycle, which may make the compound have better solubility and pharmacological activity, and obtain a new class of structurally stable heterocyclic skeletons (Chen Xin, et al. Chinese Patent Application No.: CN 202211403416.9, 2022).

[0004]

[0005] We applied the skeleton transition and pharmacophore hybridization strategies to connect the pharmacophores on the left, middle, and right sides of Cmpd-15 to the pyrazole ring respectively, forming pyrazole type derivatives. Given that the amide bond prepared from α-substituted phenylacetic acid is unstable and prone to hydrolysis, we tried to replace the amide bond with benzyl groups with different substituents. Previous studies in this research group found that after replacing the hydrogen on the pyrazole N with benzyl, the water solubility of this type of compound is poor. Therefore, for the purpose of structural simplification, we decided to remove the benzyl group and obtained pyrazole type derivatives.

[0006] Benzimidazole is a polycyclic aromatic heterocyclic compound formed by the fusion of benzene and imidazole, with the molecular formula C7H6N2. The benzimidazole ring is an aromatic heterocycle containing two nitrogen atoms. This special structure can form hydrogen bonds with enzymes and receptors in organisms, coordinate with metal ions, and undergo hydrophobic-hydrophobic and T-t interactions, etc. Benzimidazole and its derivatives containing an imidazole ring have important medicinal values in aspects such as anti-cancer, anti-hypertension, anti-parasitic, anti-fungal, anti-viral, analgesic and anti-inflammatory, anti-rheumatic, as histamine receptor antagonists and proton pump inhibitors, and anti-coagulation. Due to its special structure, physiological activity, and reactivity, etc., it has a very wide range of applications. It can be used to further simplify the structure of Cmpd-15 in order to obtain a new heterocyclic derivative with stable chemical structure, high biological activity, and improved water solubility. Summary of the Invention

[0007] Benzimidazole is an important heterocyclic compound, and its chemical structure is composed of two parts: a benzene ring and an imidazole ring. In the structure of benzimidazole, the benzene ring and the imidazole ring are connected together by a conjugated bond, forming a flat ring structure. Due to its special chemical structure, benzimidazole has a wide range of biological activities and is applied in many fields such as pharmaceuticals, textiles, and fungicides. Recent research has shown that benzimidazole and its derivatives have the characteristics of high efficiency and low toxicity in the antibacterial and anti-inflammatory processes of the human body. In order to try new skeletons, we introduced benzimidazole into our structure through principles such as structural simplification, and replaced the original pyridine ring with a benzimidazole ring. In order to further simplify the structure, the amine containing chiral factors in (S)-2-amino-3-(3-bromophenyl)-N-methylpropanamide was also replaced with an amine with a relatively simple structure and without chiral factors, as shown in the following formula. R1 is a hydrogen atom, a halogen atom, a nitro group, a methyl group, a methoxy group, etc.; R2 is an aliphatic amine, cyclopentylamine, cyclohexylamine, aniline, m-bromoaniline, m-bromobenzylamine, etc.

[0008]

[0009] The object of the present invention is to provide a new benzimidazole derivative to develop a new heterocyclic derivative with stable chemical structure, novel skeleton, high biological activity, and good subtype selectivity of the receptor as an allosteric antagonist of β2-AR, providing a solid foundation for the development of new drugs for diseases such as cardiovascular, asthma, and cancer.

[0010] The present invention provides a benzimidazole derivative, and its structure is shown in formula (I):

[0011]

[0012] R1 = H, F, Cl, Br, NO2, CH3, OCH3, OH, NH2;

[0013] R2 is one of the following structural formulas:

[0014]

[0015] The present invention also provides a preparation method of the above benzimidazole derivatives. Using 4-substituted o-phenylenediamine with different substituents, which is economically available and easy to obtain, as the starting material, it is cyclized under acidic conditions, and then oxidized by potassium permanganate to obtain acids with different substituents. The acids are coupled with (S)-2-amino-3-(3-bromophenyl)-N-methylpropanamide, m-bromoaniline, m-bromobenzylamine, aniline, cyclopentylamine, cyclohexylamine fragments to obtain a series of new benzimidazole derivatives of compound 15.

[0016] The synthetic route of benzimidazole derivatives is as follows:

[0017]

[0018] The specific synthesis method steps of benzimidazole derivatives are as follows:

[0019] The specific synthesis steps are as follows:

[0020] (1) Dissolve o-phenylenediamine 1 (1 eq) and glycolic acid (4 eq) in 6N dilute hydrochloric acid, and reflux for 2 - 3 h. Post-treatment: Cool to room temperature, adjust the pH to neutral with concentrated ammonia water under ice bath, filter to obtain benzimidazole alcohol 2.

[0021] (2) Dissolve alcohol 2 (1 eq) and NaOH (2 eq) in water, stir at 80 °C for 2 h, add KMnO4 (1.5 eq) in batches, and reflux for 10 h. After cooling, filter, take the filtrate, adjust the pH to 4 with dilute hydrochloric acid under ice bath, filter to obtain benzimidazole acid 3.

[0022] (3) Dissolve acid 3 and the activator in the solvent, add different types of amines under ice bath, then add the acid-binding agent and amide coupling agent and continue to stir to room temperature. Among them, 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). Dissolve compound 3 (1 eq) and HOAT (1.2 eq) in DMF, stir for 10 min, and add different amines. Then, at 0 °C, add NMM (0.7 eq), stir for 10 min, add EDCI (1.2 eq), keep stirring at 0 °C for 1.5 h, and react at room temperature for 12 h. Spin dry DMF, extract, and column chromatograph to obtain compound A1.

[0023] In this invention, the cumulative experiment of GloSensor cAMP was used to test the functional activity of target compounds on β2AR in the G-protein-dependent signaling pathway and to clarify whether the new compounds can allosterically regulate the functional activity of the endogenous ligand isoproterenol (ISO) of β2AR. The experimental results showed that most of the benzimidazole derivatives had good antagonistic activity against β2AR and could negatively allosterically regulate the functional activity of isoproterenol (ISO). Many of these compounds had significantly better allosteric antagonistic activity against β2AR than the lead compound Cmpd-15, such as A103, A106, A108, A112, A113, A114, A126, A127, A128, A129.

[0024] The structural formulas of the benzimidazole derivatives with β2AR antagonistic activity are as follows:

[0025]

[0026]

[0027] The beneficial effects of this invention are as follows:

[0028] The beneficial effects of this invention are mainly manifested in the following aspects. Firstly, the allosteric antagonistic activity of the newly synthesized derivatives against the β2-AR target has been significantly improved compared with the lead compound Cmpd-15. Secondly, the skeletons of the newly synthesized derivatives are novel, and the structures are greatly simplified compared with the lead compound Cmpd-15, which simplifies the synthesis route, greatly reduces the cost and difficulty of synthesis, and significantly improves the economic benefits of this type of compound. Brief Description of the Drawings

[0029] Figure 1 It is the dose-response curve graph mediated by the benzimidazole derivative A108 for ISO.

[0030] Figure 2 It is the dose-response curve graph mediated by the benzimidazole derivative A126 for ISO. Detailed Embodiments

[0031] The synthesis route of the benzimidazole derivatives is as follows:

[0032]

[0033] Example 1:

[0034]

[0035] Preparation of N,5-dimethyl-1H-benzimidazole-2-carboxamide A101

[0036] Step 1: Preparation of 5-Methyl-1H-benzoimidazole-2-methanol

[0037] Under an ice bath, 4-Methyl-1,2-phenylenediamine (1 g, 8.2 mmol) and glycolic acid (2.5 g, 32.8 mmol) were dissolved in 6N HCl (41 mL), and stirred under reflux at 120 °C for 12 h. After the reaction, the temperature was lowered to room temperature. Under an ice bath, concentrated ammonia water was added to adjust the pH to neutral. The mixture was filtered by suction, washed with water, and a pink solid compound 2, 1.1 g, with a yield of 79% was obtained.

[0038] Step 2: Preparation of 5-Methyl-1H-benzoimidazole-2-carboxylic acid

[0039] The crude product from Step 1 (1.1 g, 6.7 mmol) was dissolved in 34 mL of water, and NaOH solid (0.54 g, 13.5 mmol) was added. The mixture was heated and stirred at 80 °C for 2 h. Then, KMnO4 (1.6 g, 10.2 mmol) was added in batches, and the mixture was refluxed for 10 h. After the reaction, the temperature was lowered to room temperature. Under an ice bath, 6N HCl was used to adjust the pH to 4, and the mixture was filtered to obtain the solid, which was washed with water and purified by column chromatography (dichloromethane:methanol:formic acid = 10:1:0.1) to obtain 766 mg of a milky white solid compound 3, with a yield of 60%.

[0040] Step 3: Preparation of N,5-Dimethyl-1H-benzoimidazole-2-carboxamide

[0041] 5-Methyl-1H-benzoimidazole-2-carboxylic acid (200 mg, 1.13 mmol) and HOAT (185.54 mg, 1.36 mmol) were dissolved in 6 mL of DMF. After stirring for 10 min, methylamine hydrochloride (154 mg, 2.26 mmol) was added. Under an ice bath, N-methylmorpholine (0.092 mL, 0.79 mmol) was added and stirred for 10 min, then EDCI (261.4 mg, 1.36 mmol) was added. The mixture was stirred at 0 °C for 1 h and then reacted at room temperature for 12 h. After the reaction, DMF was evaporated under reduced pressure, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine and concentrated, and then purified by column chromatography (petroleum ether:ethyl acetate = 3:1) to obtain 150 mg of a white solid product, with a yield of 70%. 1 H NMR (400 MHz, DMSO-d6) δ 13.14 (s, 1H), 8.92 (s, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.31 (s, 1H), 7.06 (d, J = 8.4 Hz, 1H), 2.84 (s, 3H), 2.40 (s, 3H). MS (ESI, m / z): Calcd. for C 10 H 11 N3O [M+1]+ 190.

[0042] Example 2:

[0043]

[0044] Preparation of N-cyclopentyl-5-methyl-1H-benzoimidazole-2-carboxamide A102

[0045] The preparation method is the same as that of Example 1, except that cyclopentylamine is used instead of methylamine hydrochloride in Step 3, and a white solid is obtained with a yield of 68%. 1 H NMR(400MHz,DMSO-d6)δ13.10(s,1H),8.75(d,J=8.0Hz,1H),7.57-7.31(m,2H),7.09(s,1H),4.27(m,1H),2.41(s,3H),1.91-1.82(m,2H),1.73-1.49(m,6H).MS(ESI,m / z):Calcd.for C 14 H 17 N3O[M+1]+244.

[0046] Example 3:

[0047]

[0048] Preparation of N-cyclopentyl-5-methoxy-1H-benzoimidazole-2-carboxamide A103

[0049] The preparation method is the same as that of Example 1, except that 4-methoxyphenyl-1,2-diamine is used instead of 4-methylphenyl-1,2-diamine in Step 1 and cyclopentylamine is used instead of methylamine hydrochloride in Step 3, and a white solid is obtained with a yield of 65%. 1 HNMR(400MHz,DMSO-d6)δ13.08(s,1H),8.67(d,J=8.1Hz,1H),7.57-6.91(m,3H),4.26(q,J=7.3Hz,1H),3.79(s,3H),1.91-1.8x2(m,2H),1.72-1.50(m,6H).MS(ESI,m / z):Calcd.for C 14 H 17 N3O2[M+1]+260.

[0050] Example 4:

[0051]

[0052] Preparation of N-cyclohexyl-5-methyl-1H-benzoimidazole-2-carboxamide A104

[0053] The preparation method is the same as that of Example 1, except that cyclopentylamine is used instead of methylamine hydrochloride in Step 3, and a white solid is obtained with a yield of 65%. 1 H NMR(400MHz,DMSO-d6)δ13.19(s,1H),8.71(d,J=8.6Hz,1H),7.66(d,J=8.3Hz,1H),7.40(s,1H),7.16(d,J=8.3Hz,1H),3.88-3.84(m,1H),1.82-1.78(m,4H),1.66(s,1H),1.51-1.48(m,2H),1.37-1.34(m,2H),1.17-1.14(m,1H).MS(ESI,m / z):Calcd.forC 15 H 19 N3O[M+1]+258。

[0054] Example 5:

[0055]

[0056] Preparation of N-cyclohexyl-5-methoxy-1H-benzimidazole-2-carboxamide A105

[0057] The preparation method is the same as that of Example 1, except that 4-methoxyphenyl-1,2-diamine is used instead of 4-methylphenyl-1,2-diamine in Step 1, and cyclohexylamine is used instead of methylamine hydrochloride in Step 3, with a yield of 62%. 1 H NMR(400MHz,DMSO-d6)δ13.10(s,1H),8.55(d,J=8.5Hz,1H),7.54(s,1H),6.97-6.89(m,2H),3.78(s,3H),3.38(s,1H),1.80-1.69(m,4H),1.60-1.57(m,1H),1.48-1.25(m,4H),1.15-1.06(m,1H).MS(ESI,m / z):Calcd.for C 15 H 19 N3O2[M+1]+274。

[0058] Example 6:

[0059]

[0060] Preparation of N-phenyl-1H-benzimidazole-2-carboxamide A106

[0061] The preparation method is the same as that of Example 1, except that phenyl-1,2-diamine is used instead of 4-methylphenyl-1,2-diamine in Step 1, and aniline is used instead of methylamine hydrochloride in Step 3, and a white solid is obtained with a yield of 55%. 11H NMR (400 MHz, DMSO-d6) δ 13.46 (s, 1H), 10.92 (s, 1H), 7.95 (d, J = 8.0 Hz, 2H), 7.70 (s, 2H), 7.36 (d, J = 29.6 Hz, 4H), 7.13 (d, J = 14.8 Hz, 1H). MS (ESI, m / z): Calcd. for C 14 H 11 N3O [M+1]+ 238.

[0062] Example 7:

[0063]

[0064] Preparation of 5-methyl-N-phenyl-1H-benzimidazole-2-carboxamide A107

[0065] The preparation method was the same as that of Example 1, except that aniline was used instead of methylamine hydrochloride in Step 3, and a white solid was obtained with a yield of 55%. 1 1H NMR (400 MHz, DMSO-d6) δ 13.45 (s, 1H), 11.09 (d, J = 10.5 Hz, 1H), 8.25 (s, 1H), 7.93 (d, J = 7.3 Hz, 1H), 7.68 - 7.57 (m, 1H), 7.49 - 7.38 (m, 1H), 7.33 (d, J = 7.5 Hz, 2H), 7.19 - 7.12 (m, 1H), 2.43 (s, 3H). MS (ESI, m / z): Calcd. for C 15 H 13 N3O [M+1]+ 252.

[0066] Example 8:

[0067]

[0068] Preparation of 5-methoxy-N-phenyl-1H-benzimidazole-2-carboxamide A108

[0069] The preparation method was the same as that of Example 1, except that 4-methoxybenzene-1,2-diamine was used instead of 4-methylbenzene-1,2-diamine in Step 1 and aniline was used instead of methylamine hydrochloride in Step 3, and a white solid was obtained with a yield of 58%. 11H NMR (400 MHz, DMSO-d6) δ 13.32 (s, 1H), 10.79 (s, 1H), 7.92 (d, J = 8.0 Hz, 2H), 7.61 (s, 1H), 7.37 (t, J = 7.7 Hz, 2H), 7.12 (t, J = 7.4 Hz, 1H), 7.05 (s, 1H), 6.96 (d, J = 9.1 Hz, 1H), 3.82 (s, 3H). MS (ESI, m / z): Calcd. for C 15 H 13 N3O2 [M+1]+ 268.

[0070] Example 9:

[0071]

[0072] Preparation of 5-fluoro-N-phenyl-1H-benzimidazole-2-carboxamide A109

[0073] The preparation method is the same as that of Example 1, except that in Step 1, 4-fluoro-1,2-diamine is used instead of 4-methylbenzene-1,2-diamine, and in Step 3, aniline is used instead of methylamine hydrochloride, to obtain a white solid with a yield of 52%. 1 1H NMR (400 MHz, DMSO-d6) δ 9.67 (t, J = 6.4 Hz, 1H), 7.67 - 7.63 (m, 2H), 7.55 (s, 1H), 7.44 (d, J = 7.9 Hz, 1H), 7.36 - 7.29 (m, 3H), 4.49 (d, J = 6.2 Hz, 2H). MS (ESI, m / z): Calcd. for C 14 H 10F N3O [M+1]+ 256.

[0074] Example 10:

[0075]

[0076] Preparation of 5-chlorophenyl-1H-benzimidazole-2-carboxamide A110

[0077] The preparation method is the same as that of Example 1, except that in Step 1, 4-chloro-1,2-diamine is used instead of 4-methylbenzene-1,2-diamine, and in Step 3, aniline is used instead of methylamine hydrochloride, to obtain a white solid with a yield of 52%. 11H NMR (400 MHz, DMSO-d6) δ 13.66 (s, 1H), 10.99 (s, 1H), 7.94 (d, J = 8.0 Hz, 2H), 7.68 (s, 2H), 7.39 - 7.35 (m, 3H), 7.13 (t, J = 7.4 Hz, 1H). MS (ESI, m / z): Calcd. for C 14 H 10 ClN3O [M+1]+ 272.

[0078] Example 11:

[0079]

[0080] Preparation of 5-bromo-N-phenyl-1H-benzoimidazole-2-carboxamide A111

[0081] The preparation method was the same as that of Example 1, except that 4-bromo-1,2-diamine was used instead of 4-methylbenzene-1,2-diamine in Step 1, and aniline was used instead of methylamine hydrochloride in Step 3, to obtain a white solid with a yield of 55%. 1 1H NMR (400 MHz, DMSO-d6) δ 13.53 (s, 1H), 11.00 (s, 1H), 7.93 (d, J = 8.0 Hz, 3H), 7.64 (s, 1H), 7.48 (d, J = 8.6 Hz, 1H), 7.38 (t, J = 7.7 Hz, 2H), 7.14 (t, J = 7.4 Hz, 1H). MS (ESI, m / z): Calcd. for C 14 H 10 BrN3O [M+1]+ 316.

[0082] Example 12:

[0083]

[0084] Preparation of 5-nitro-N-phenyl-1H-benzoimidazole-2-carboxamide A112

[0085] The preparation method was the same as that of Example 1, except that 4-nitrobenzene-1,2-diamine was used instead of 4-methylbenzene-1,2-diamine in Step 1, and aniline was used instead of methylamine hydrochloride in Step 3, to obtain a white solid with a yield of 52%. 11H NMR (400 MHz, DMSO-d6) δ 14.10 (s, 1H), 11.10 (s, 1H), 8.52 (s, 1H), 8.21 (d, J = 8.9 Hz, 1H), 7.93 (d, J = 7.9 Hz, 2H), 7.82 (d, J = 8.3 Hz, 1H), 7.39 (d, J = 7.7 Hz, 2H), 7.15 (t, J = 7.4 Hz, 1H). MS (ESI, m / z): Calcd. for C 14 H 10 N4O3 [M+1]+ 283.

[0086] Example 13:

[0087]

[0088] Preparation of N-(3-bromophenyl)-1H-benzoimidazole-2-carboxamide A113

[0089] The preparation method was the same as that of Example 1, except that in Step 1, phenyl-1,2-diamine was used instead of 4-methylphenyl-1,2-diamine, and in Step 3, 3-bromoaniline was used instead of methylamine hydrochloride, to obtain a white solid with a yield of 52%. 1 1H NMR (400 MHz, DMSO-d6) δ 13.55 (s, 1H), 11.14 (s, 1H), 8.26 (s, 1H), 7.96 - 7.93 (m, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.37 - 7.31 (m, 4H). MS (ESI, m / z): Calcd. for C 14 H 10 BrN3O [M+1]+ 316.

[0090] Example 14:

[0091]

[0092] Preparation of N-(3-bromophenyl)-5-methyl-1H-benzoimidazole-2-carboxamide A114

[0093] The preparation method was the same as that of Example 1, except that in Step 1, 4-methylphenyl-1,2-diamine was used instead of 4-methylphenyl-1,2-diamine, and in Step 3, 3-bromoaniline was used instead of methylamine hydrochloride, to obtain a white solid with a yield of 52%. 11H NMR (400 MHz, DMSO-d6) δ 13.32 (s, 1H), 10.84 (s, 1H), 7.93 (d, J = 7.9 Hz, 2H), 7.37 (t, J = 7.8 Hz, 3H), 7.13 (t, J = 7.3 Hz, 2H), 2.45 (s, 3H). MS (ESI, m / z): Calcd. for C 15 H 12 BrN3O [M+1]+ 330.

[0094] Example 15:

[0095]

[0096] Preparation of N-(3-bromophenyl)-5-methoxy-1H-benzimidazole-2-carboxamide A115

[0097] The preparation method is the same as that of Example 1, except that in Step 1, 4-methoxyphenyl-1,2-diamine is used instead of 4-methylphenyl-1,2-diamine, and in Step 3, 3-bromoaniline is used instead of methylamine hydrochloride, to obtain a white solid with a yield of 52%. 1 1H NMR (400 MHz, DMSO-d6) δ 13.35 (s, 1H), 11.02 (s, 1H), 8.25 (s, 1H), 7.93 (d, J = 11.3 Hz, 1H), 7.69 (s, 1H), 7.32 (d, J = 23.3 Hz, 2H), 6.99 (d, J = 17.6 Hz, 2H), 3.82 (s, 3H). MS (ESI, m / z): Calcd. for C 15 H 12 BrN3O2 [M+1]+ 346.

[0098] Example 16:

[0099]

[0100] Preparation of N-(3-bromophenyl)-5-fluoro-1H-benzimidazole-2-carboxamide A116

[0101] The preparation method is the same as that of Example 1, except that in Step 1, 4-fluoro-1,2-diamine is used instead of 4-methylphenyl-1,2-diamine, and in Step 3, 3-bromoaniline is used instead of methylamine hydrochloride, to obtain a white solid with a yield of 52%. 11H NMR (400 MHz, DMSO-d6) δ 13.59 (s, 1H), 11.13 (s, 1H), 8.25 (s, 1H), 7.94 - 7.91 (m, 1H), 7.72 - 7.69 (m, 1H), 7.45 (d, J = 9.2 Hz, 1H), 7.36 - 7.33 (m, 2H), 7.23 - 7.18 (m, 1H). MS (ESI, m / z): Calcd. for C 14 H9BrFN3O [M + 1]+ 333。

[0102] Example 17:

[0103]

[0104] Preparation of N-(3-bromophenyl)-5-chloro-1H-benzimidazole-2-carboxamide A117

[0105] The preparation method was the same as that of Example 1, except that 4-chloro-1,2-diamine was used instead of 4-methylbenzene-1,2-diamine in Step 1, and 3-bromoaniline was used instead of methylamine hydrochloride in Step 3, to obtain a white solid with a yield of 52%. 1 1H NMR (400 MHz, DMSO-d6) δ 13.67 (s, 1H), 11.19 (s, 1H), 8.23 (s, 1H), 7.93 (d, J = 6.4 Hz, 1H), 7.71 (s, 2H), 7.38 - 7.33 (m, 3H). MS (ESI, m / z): Calcd. for C 14 H9BrClN3O [M + 1]+ 349。

[0106] Example 18:

[0107]

[0108] Preparation of 5-bromo-N-(3-bromophenyl)-1H-benzimidazole-2-carboxamide A118

[0109] The preparation method was the same as that of Example 1, except that 4-bromo-1,2-diamine was used instead of 4-methylbenzene-1,2-diamine in Step 1, and 3-bromoaniline was used instead of methylamine hydrochloride in Step 3, to obtain a white solid with a yield of 52%. 1 1H NMR (400 MHz, DMSO-d6) δ 13.70 (s, 1H), 11.21 (s, 1H), 7.94 - 7.91 (m, 2H), 7.55 (s, 1H), 7.34 - 7.32 (m, 2H). MS (ESI, m / z): Calcd. for C 14 H9Br2N3O [M + 1]+ 395。

[0110] Example 19:

[0111]

[0112] Preparation of N-(3-bromophenyl)-5-nitro-1H-benzoimidazole-2-carboxamide A119

[0113] The preparation method was the same as that of Example 1, except that 4-nitro-1,2-diamine was used instead of 4-methylbenzene-1,2-diamine in Step 1, and 3-bromoaniline was used instead of methylamine hydrochloride in Step 3 to obtain a white solid with a yield of 52%. 1 H NMR(400MHz,DMSO-d6)δ14.12(s,1H),11.30(s,1H),8.51(s,1H),8.22-8.19(m,2H),7.94-7.80(m,2H),7.36-7.33(m,2H).MS(ESI,m / z):Calcd.for C 14 H9BrN4O3[M+1]+360.

[0114] Example 20:

[0115]

[0116] Preparation of N-(3-bromobenzyl)-5-methyl-1H-benzoimidazole-2-carboxamide A120

[0117] The preparation method was the same as that of Example 1, except that 4-methyl-1,2-diamine was used instead of 4-methylbenzene-1,2-diamine in Step 1, and 3-bromobenzylamine was used instead of methylamine hydrochloride in Step 3 to obtain a white solid with a yield of 52%. 1 H NMR(400MHz,DMSO-d6)δ13.15(s,1H),9.57(s,1H),7.55(s,1H),7.50(dd,J=8.4,4.4Hz,1H),7.44(d,J=7.8Hz,1H),7.36-7.27(m,3H),7.12(s,1H),4.48(d,J=6.4Hz,2H),2.42(s,3H).MS(ESI,m / z):Calcd.for C 16 H 14 BrN3O[M+1]+344.

[0118] Example 21:

[0119]

[0120] Preparation of N-(3-bromobenzyl)-5-methoxy-1H-benzoimidazole-2-carboxamide A121

[0121] The preparation method was the same as that of Example 1, except that 4-methoxy-1,2-diamine was used instead of 4-methylbenzene-1,2-diamine in Step 1, and 3-bromobenzylamine was used instead of methylamine hydrochloride in Step 3, obtaining a white solid with a yield of 52%. 1 H NMR(400MHz,DMSO-d6)δ13.15(s,1H),9.52(s,1H),7.59-6.90(m,7H),4.49(s,2H),3.79(s,3H).MS(ESI,m / z):Calcd.for C 16 H 14 BrN3O2[M+1]+360.

[0122] Example 22:

[0123]

[0124] Preparation of N-(3-bromobenzyl)-5-fluoro-1H-benzoimidazole-2-carboxamide A122

[0125] The preparation method was the same as that of Example 1, except that 4-fluoro-1,2-diamine was used instead of 4-methylbenzene-1,2-diamine in Step 1, and 3-bromobenzylamine was used instead of methylamine hydrochloride in Step 3, obtaining a white solid with a yield of 52%. 1H NMR(400MHz,DMSO-d6)δ13.39(s,1H),9.63(t,J = 6.4Hz,1H),7.66-7.61(m,1H),7.55(s,1H),7.45(d,J = 7.8Hz,1H),7.36-7.27(m,3H),7.18(t,J = 8.9Hz,1H),4.49(d,J = 6.3Hz,2H).MS(ESI,m / z):Calcd.for C 15 H 11 BrFN3O[M+1]+348..

[0126] Example 23:

[0127]

[0128] Preparation of N-(3-bromobenzyl)-5-chloro-1H-benzoimidazole-2-carboxamide A123

[0129] The preparation method was the same as that of Example 1, except that 4-chloro-1,2-diamine was used instead of 4-methylbenzene-1,2-diamine in Step 1, and 3-bromobenzylamine was used instead of methylamine hydrochloride in Step 3, obtaining a white solid with a yield of 52%. 11H NMR (400 MHz, DMSO-d6) δ 12.98 (s, 1H), 9.67 (t, J = 6.4 Hz, 1H), 7.70–7.61 (m, 2H), 7.55 (s, 1H), 7.45 (d, J = 6.3 Hz, 1H), 7.36 - 7.27 (m, 3H), 4.49 (d, J = 6.2 Hz, 2H). MS (ESI, m / z): Calcd. for C 15 H 11 BrClN3O [M+1]+ 363.

[0130] Example 24:

[0131]

[0132] Preparation of N-(3-bromobenzyl)-5-bromo-1H-benzimidazole-2-carboxamide A124

[0133] The preparation method was the same as that of Example 1, except that 4-bromo-1,2-diamine was used instead of 4-methylbenzene-1,2-diamine in Step 1, and 3-bromobenzylamine was used instead of methylamine hydrochloride in Step 3, to obtain a white solid with a yield of 52%. 1 1H NMR (400 MHz, DMSO-d6) δ 9.70 (t, J = 6.4 Hz, 1H), 7.82 (s, 1H), 7.59 (d, J = 8.7 Hz, 1H), 7.55 (s, 1H), 7.44 (dd, J = 8.7, 1.9 Hz, 2H), 7.35 (d, J = 7.8 Hz, 1H), 7.28 (t, J = 7.7 Hz, 1H), 4.49 (d, J = 6.4 Hz, 2H). MS (ESI, m / z): Calcd. for C 15 H 11 Br2N3O [M+1]+ 409.

[0134] Example 25:

[0135]

[0136] Preparation of N-(3-bromobenzyl)-5-nitro-1H-benzimidazole-2-carboxamide A125

[0137] The preparation method was the same as that of Example 1, except that 4-nitro-1,2-diamine was used instead of 4-methylbenzene-1,2-diamine in Step 1, and 3-bromobenzylamine was used instead of methylamine hydrochloride in Step 3, to obtain a white solid with a yield of 52%. 11H NMR (400 MHz, DMSO-d6) δ 14.01 (s, 1H), 9.85 (t, J = 6.4 Hz, 1H), 8.52 (s, 1H), 8.20 (dd, J = 9.0, 2.3 Hz, 1H), 7.80 (d, J = 9.1 Hz, 1H), 7.56 (s, 1H), 7.45 (d, J = 7.9 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.30 (t, J = 7.7 Hz, 1H), 4.51 (d, J = 6.4 Hz, 2H). MS (ESI, m / z): Calcd. for C 15 H 11 BrN4O3 [M+1]+ 375.

[0138] Example 26:

[0139]

[0140] Preparation of N-(3-fluorophenyl)-5-methyl-1H-benzimidazole-2-carboxamide A126

[0141] The preparation method was the same as that of Example 1, except that 4-methyl-1,2-diamine was used instead of 4-methylbenzene-1,2-diamine in Step 1, and 3-fluoroaniline was used instead of methylamine hydrochloride in Step 3, to obtain a white solid with a yield of 52%. 1 1H NMR (400 MHz, DMSO-d6) δ 13.37 (d, J = 7.9 Hz, 1H), 11.11 (d, J = 10.1 Hz, 1H), 7.88 (d, J = 11.8 Hz, 1H), 7.79 (d, J = 8.2 Hz, 1H), 7.69 - 7.58 (m, 1H), 7.47 - 7.37 (m, 2H), 7.20 - 7.13 (m, 1H), 6.98 - 6.94 (m, 1H), 2.45 (s, 3H). MS (ESI, m / z): Calcd. for C 15 H 12 FN3O [M+1]+ 270.

[0142] Example 27:

[0143]

[0144] Preparation of N-(3-fluorophenyl)-5-methoxy-1H-benzimidazole-2-carboxamide A127

[0145] The preparation method was the same as that of Example 1, except that 4-methoxy-1,2-diamine was used instead of 4-methylbenzene-1,2-diamine in Step 1, and 3-fluoroaniline was used instead of methylamine hydrochloride in Step 3, to obtain a white solid with a yield of 52%. 11H NMR (400 MHz, DMSO-d6) δ 13.36 (s, 1H), 11.04 (s, 1H), 7.90 - 7.86 (m, 1H), 7.79 - 7.77 (m, 1H), 7.68 - 7.66 (d, J = 9.0 Hz, 1H), 7.43 - 7.37 (m, 1H), 7.00 (s, 1H), 6.98 - 6.92 (m, 2H), 3.81 (s, 3H). MS (ESI, m / z): Calcd. for C 15 H 12 FN3O2 [M + 1]+ 286。

[0146] Example 28:

[0147]

[0148] Preparation of N-(3-chlorophenyl)-5-methyl-1H-benzimidazole-2-carboxamide A128

[0149] The preparation method is the same as that of Example 1, except that in Step 1, 4-methyl-1,2-diamine is used instead of 4-methylbenzene-1,2-diamine, and in Step 3, m-chloroaniline is used instead of methylamine hydrochloride, to obtain a white solid with a yield of 52%. 1 1H NMR (400 MHz, DMSO-d6) δ 13.35 (s, 1H), 11.02 (s, 1H), 8.25 (s, 1H), 7.93 (d, J = 11.3 Hz, 1H), 7.69 (s, 1H), 7.32 (d, J = 23.3 Hz, 2H), 6.99 (d, J = 17.6 Hz, 2H), 3.82 (s, 3H). MS (ESI, m / z): Calcd. for C 15 H 12 ClN3O [M + 1]+ 286。

[0150] Example 29:

[0151]

[0152] Preparation of N-(3-chlorophenyl)-5-methoxy-1H-benzimidazole-2-carboxamide A129

[0153] The preparation method is the same as that of Example 1, except that in Step 1, 4-chloromethoxy-1,2-diamine is used instead of 4-methylbenzene-1,2-diamine, and in Step 3, m-chloroaniline is used instead of methylamine hydrochloride, to obtain a white solid with a yield of 52%. 11H NMR (400 MHz, DMSO-d6) δ 13.34 (s, 1H), 11.03 (s, 1H), 8.10 (t, J = 2.1 Hz, 1H), 7.91 - 7.88 (m, 1H), 7.67 (d, J = 8.9 Hz, 1H), 7.39 (t, J = 8.1 Hz, 1H), 7.19 - 7.17 (m, 1H), 7.0 - 6.9 (m, 2H), 3.82 (s, 3H). MS (ESI, m / z): Calcd. for C 15 H 12 ClN3O2 [M+1]+ 302.

[0154] Example 30:

[0155]

[0156] Preparation of N-(3,5-dibromophenyl)-5-methyl-1H-benzimidazole-2-carboxamide A130

[0157] The preparation method was the same as that of Example 1, except that 4-methyl-1,2-diamine was used instead of 4-methylbenzene-1,2-diamine in Step 1, and 3,5-dibromoaniline was used instead of methylamine hydrochloride in Step 3, obtaining a white solid with a yield of 52%. MS (ESI, m / z): Calcd. for C 15 H 11 Br2N3O [M+1]+ 409.

[0158] Example 31:

[0159]

[0160] Preparation of N-(3,5-dibromophenyl)-5-methoxy-1H-benzimidazole-2-carboxamide A131

[0161] The preparation method was the same as that of Example 1, except that 4-methoxy-1,2-diamine was used instead of 4-methylbenzene-1,2-diamine in Step 1, and 3,5-dibromoaniline was used instead of methylamine hydrochloride in Step 3, obtaining a white solid with a yield of 52%. 1 1H NMR (400 MHz, DMSO-d6) δ 13.35 (s, 1H), 11.17 (s, 1H), 8.24 (s, 2H), 7.67 (d, J = 8.9 Hz, 1H), 7.55 - 7.55 (m, 1H), 6.99 (d, J = 2.5 Hz, 1H), 6.96 - 6.93 (m, 1H), 3.82 (s, 3H). MS (ESI, m / z): Calcd. for C 15 H 11 Br2N3O2 [M+1]+ 425.

[0162] Bioactivity test

[0163] The cAMP accumulation experiment was used to test the functional activity of the target compound against β2AR and to elucidate whether the new compound is a negative allosteric modulator (NAM) of β2AR. The cAMP accumulation level was mainly measured using GloSensor, a bioluminescence-based biosensor that directly detects intracellular cAMP (Promega). Briefly, HEK 293T cells were seeded into 6-well plates at 4×10 5 cells per well. The next day, β2AR and pGloSensor-22FcAMP 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 then incubated with a balanced 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 multimode microplate reader until a steady-state baseline signal was obtained. Then, new derivatives and the control compound Cmpd-15 at different concentration gradients were added to the cells, and after incubation at 37 °C for 30 min, the positive control ISO (final concentration 1 nM - 100 μM) was added. The changes in bioluminescence were read using a microplate reader.

[0164] Allosteric antagonistic activity screening

[0165] Using the cAMP accumulation experiment, isoproterenol (ISO) at different concentration gradients was used as the negative control (the final concentration gradient was 1 nM - 100 μM), and the lead compound Cmpd-15 (final concentration of 50 μM) was used as the positive control to compare the allosteric antagonistic activity of the newly synthesized benzimidazole derivatives (final concentration of 50 μM) with that of the lead compound Cmpd-15. The test results in Table 1 showed that all the synthesized compounds had varying degrees of allosteric antagonistic effects on the β2-adrenergic receptor. Generally, when the amine structure was phenylalanine, there was certain activity when there was an electron-donating group on the left. When the amine structure was aliphatic amine, cyclopentylamine, cyclohexylamine, or m-bromobenzylamine, the activity was generally average or even inactive. When the amine structure was aniline or m-fluoroaniline, and there was an electron-donating group in the acid structure, the activity was generally better. Except for A101, A102, A104, A120, A122, A131, most of the compounds had allosteric antagonistic activity, and many of them had significantly better allosteric antagonistic activity against β2AR than the lead compound Cmpd-15, such as A103, A106, A112, A113, A114, A127, A128, A129, A130. Among them, the allosteric antagonistic activity of A114 was 1.94 times that of the lead compound, and the allosteric antagonistic activities of A108 and A126 were more than 3 times that of the lead compound.

[0166]

[0167] Table 1 Statistical table of comparison results of allosteric activities of benzimidazole derivatives and activities of Cmpd-15

[0168]

[0169]

[0170] Note: a The value represents the blocking activity relative to Cmpd-15; "-" indicates that the compound has no allosteric antagonistic activity

[0171] Study on allosteric antagonistic mechanism

[0172] Through the GloSensor cAMP accumulation experiment, further study was conducted on whether this type of compound could allosterically regulate the functional activity of the endogenous ligand ISO of β2-AR, and further confirm that this type of compound is a negative allosteric modulator (NAM) of β2-AR. Taking the benzimidazole derivative A108 as an example (as Figure 1 shown), when the concentration of compound A108 reached 25 μM, the curve of ISO showed a large downward shift and almost reached the lower limit of the maximum downward shift of the dose-effect regulation of ISO functional activity, indicating that the IC of compound A108 50The value may be between 12.5 μM and 25 μM, specifically manifested as a sharp drop in the concentration curve. When the concentration of A108 increases from 50.0 μM to 100.0 μM, the degree of downward shift of the concentration curve of ISO is very weak. This phenomenon illustrates that as the concentration of compound A108 increases, the concentration-dependent curve of ISO shows a limited downward shift, indicating that benzimidazole derivative A108 can effectively allosterically regulate the functional activity of the endogenous ligand ISO of β2-AR in a negative manner. This allosteric regulation phenomenon is consistent with the reported allosteric antagonistic regulation mechanism. The concentration-dependent curve of ISO allosterically regulated by the other benzimidazole derivative representative A126 (as Figure 2 shown) is consistent with the results of A108, that is, the newly synthesized benzimidazole derivatives are all negative allosteric modulators (NAMs) of β2-AR.

Claims

1. Use of a benzimidazole derivative represented by formula (I) in the preparation of a β2-adrenaline receptor antagonist, characterized in that: The formula (I) is as follows: ， R1=H,F,Cl,Br,NO2,CH3,OCH3,OH,NH2; R2 is one of the following structural formulas: 。 2. The use according to claim 1, characterized in that: The structure of the benzimidazole derivatives is as follows: ， ， 。 3. The use according to claim 1, characterized in that: The benzimidazole derivatives shown in formula (I) are used as active ingredients and pharmaceutically acceptable carriers to prepare pharmaceutical preparations.

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