An allosteric modulator of adrenergic receptor function and methods of making and using the same

Abstract

This invention relates to the field of medicinal chemistry, specifically to an allosteric modulator of adrenaline receptor functional activity, its preparation method, and its application. The key technical points are as follows: An allosteric modulator of adrenaline receptor functional activity, characterized in that the allosteric modulator is a benzopyrazole amide compound. The preparation method is as follows: A derivative of 3-carboxylic acid benzopyrazole is added to a solvent, followed by the addition of an amine and HOAT, stirring, cooling, adding NMM, stirring, adding EDCI, and continuing the reaction. After the reaction is complete, extraction is performed, and the allosteric modulator is obtained by separation. The allosteric modulator of adrenaline receptor functional activity provided by this invention has high biological activity and good structural stability, providing a solid foundation for the development of new drugs for cardiovascular diseases, asthma, and cancer.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to an allosteric modulator of adrenaline receptor functional activity, its preparation method, and its application. Background Technology

[0002] G protein-coupled receptors (GPCRs) are currently the most studied and important drug targets, participating in almost all physiological, pathological, and pharmacological processes in humans. To date, the vast majority of GPCR drugs on the market are ortho-heterogeneous ligands, which have many adverse reactions and poor specificity. However, GPCR allosteric modulators have become a research hotspot in recent years due to their significant potential advantages. β2AR antagonists are classic GPCR drugs, playing a crucial role in the treatment of heart failure, hypertension, coronary artery disease, arrhythmia, angina pectoris, and other cardiovascular diseases, forming the cornerstone of cardiovascular disease treatment. Therefore, the development and design of β2AR allosteric antagonists are of great significance for the treatment of various cardiovascular diseases.

[0003] However, in existing technologies, negative allosteric modifiers typically suffer from poor water solubility and low biological activity, and their poor stability affects their drug-likeness. Furthermore, these compounds have complex structures, making their synthesis difficult. Summary of the Invention

[0004] The purpose of this invention is to develop an allosteric modulator of adrenergic receptor function, its preparation method, and its application. Using various substituted 3-carboxylic acid benzopyrazoles and various amines as raw materials, a series of various substituted benzopyrazole amide compounds were synthesized through amide coupling reactions. These compounds are allosteric antagonists of β2AR, exhibiting high biological activity and good structural stability, and can provide a solid foundation for the creation of new drugs for cardiovascular diseases, asthma, and cancer.

[0005] The above-mentioned technical objective of the present invention is achieved through the following technical solution:

[0006] This invention provides an allosteric modulator of adrenaline receptor functional activity. The allosteric modulator is a benzopyrazole amide compound with the following molecular structural formula:

[0007]

[0008] Among them, R 1 It is any one of hydrogen, alkyl, alkoxy, aryl, halogen, alkenyl, amino, hydroxyl, nitro, acyl, cyano or trifluoromethyl at any position on the benzene ring;

[0009] R 2 It is any one of hydrogen, alkyl, aryl, alkenyl or acyl;

[0010] R 3 It is any one of hydrogen, alkyl, alkoxy, aryl, or acyl;

[0011] R 4 It is any one of hydrogen, alkyl, alkoxy, aryl or acyl.

[0012] Furthermore, R 3 and R 4 It can also be linked with cycloalkyl or aryl groups.

[0013] Furthermore, R 2 It is any one of hydrogen, alkyl, or aryl.

[0014] Furthermore, R 3 It is any one of hydrogen, alkyl, aryl or acyl.

[0015] Furthermore, R 4 It is any one of hydrogen, alkyl, aryl or acyl.

[0016] This invention also provides a method for preparing an allosteric modulator of adrenaline receptor functional activity, as detailed below:

[0017]

[0018] Among them, R 1 It is any one of hydrogen, alkyl, alkoxy, aryl, halogen, alkenyl, amino, hydroxyl, nitro, acyl, cyano or trifluoromethyl at any position on the benzene ring;

[0019] R 2 It is any one of hydrogen, alkyl, aryl, alkenyl or acyl;

[0020] R 3 It is any one of hydrogen, alkyl, alkoxy, aryl, or acyl;

[0021] R 4 It is any one of hydrogen, alkyl, alkoxy, aryl or acyl.

[0022] Further, the preparation method is as follows: at room temperature, the derivative of 3-carboxylic acid benzopyrazole is added to a solvent, followed by the addition of amine and HOAT, and the mixture is stirred; the temperature is lowered, NMM is added, and the mixture is stirred; EDCI is added, and the reaction is continued. After the reaction is completed, the mixture is extracted and separated to obtain the allosteric modifier.

[0023] Furthermore, when NMM is added, the reaction system temperature is maintained between 0°C and 10°C.

[0024] Furthermore, when adding EDCI, the system temperature is maintained between 0°C and 10°C.

[0025] The present invention also provides an application of an allosteric modulator of adrenaline receptor functional activity, specifically, using the allosteric modulator to negatively regulate the functional activity of the β2AR endogenous ligand ISO.

[0026] In summary, the present invention has the following beneficial effects:

[0027] This invention provides a novel allosteric antagonist of β2AR, which is a benzopyrazole amide compound with the advantages of high biological activity, simple structure and simple synthetic route, and can provide a basis for creating new drugs for cardiovascular, asthma and cancer diseases. Attached Figure Description

[0028] Figure 1 This invention provides a synthetic route for benzopyrazole amide compounds;

[0029] Figure 2 This is the general structural formula of the benzopyrazole amide compounds provided by the present invention;

[0030] Figure 3 It is the ISO allosteric regulation curve mediated by compound W8; Detailed Implementation

[0031] To further illustrate the technical means and effects adopted by the present invention to achieve the intended purpose, the specific implementation methods, features and effects of an allosteric modulator of adrenaline receptor functional activity proposed according to the present invention, its preparation method and application are described in detail below.

[0032] This specific embodiment uses various substituted 3-carboxylic acid benzopyrazoles and various amines as raw materials to synthesize a series of various substituted benzopyrazole amide compounds via amide coupling reaction, the structural formulas of which are as follows: Figure 2 As shown in the figure. This specific embodiment confirms through cumulative experiments with GloSensor cAMP that the synthesized compounds are allotropic antagonists of β2AR and have high biological activity.

[0033] like Figure 1 As shown in this specific embodiment, the general synthetic steps for benzopyrazole amide compounds are as follows:

[0034] At room temperature, 3-carboxylic acid benzopyrazole was added to DMF, followed by amine and HOAT. After stirring at room temperature for 10 minutes, N-methylmorpholine (NMM) was added at 0°C and stirred at 0°C for 10 minutes. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) was added. The mixture was then reacted at 0°C for 9 hours. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain benzopyrazole amide compounds.

[0035] Example 1: An allosteric modulator of adrenaline receptor functional activity and its preparation method

[0036] The allosteric modulator of adrenaline receptor functional activity provided in this embodiment, (S)-N-(3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-1-phenyl-1H-indazole-3-carboxamide, has the following structural formula:

[0037]

[0038] Its preparation method is as follows:

[0039] At room temperature, 47.7 mg (0.2 mmol, 1 eq) of N-phenylindazole-3-carboxylic acid was added to N,N-dimethylformamide (2 ml), followed by 51.4 mg (0.2 mmol, 1 eq) of 2-amino-3-(3-bromophenyl)-N-methylpropionamide and 41 mg (0.3 mmol, 1.5 eq) of N-hydroxy-7-azabenzotriazole. After stirring at room temperature for 10 minutes, 18 μL (0.15 mmol, 0.75 eq) of N-methylmorpholine was added at 0 °C. After stirring at 0 °C for 10 minutes, 58 mg (0.3 mmol, 1.5 eq) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The reaction was then carried out at 0 °C for 9 hours. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound W1 (14 mg), a white solid, in 15% yield.

[0040] Its characterization data are as follows:

[0041] 1 H NMR (400MHz, CDCl3): δ8.38 (d, J = 8.2Hz, 1H), 7.74-7.72 (m, 3H), 7.60

[0042] -7.56(m,2H),7.49-7.43(m,3H),7.38-7.34(m,2H),7.26-7.23(m,2H),7.16(t,J=7.8Hz, 1H),4.87(t,J=7.0Hz,1H),3.22(dd,J=6.6,3.4Hz,2H),2.75(s,3H); HRMS(ESI)m / z:calcd for C 24 H 21 BrN4NaO2+ [M+Na] + 499.0740, found 499.0742.

[0043] Example 2: An allosteric modulator of adrenaline receptor functional activity and its preparation method

[0044] The allosteric modulator of adrenaline receptor functional activity provided in this embodiment, N-(3-fluorophenyl)-1-phenyl-1H-indole-3-carboxamide, has the following structural formula:

[0045]

[0046] Its preparation method is as follows:

[0047] At room temperature, 71.5 mg (0.3 mmol, 1 eq) of N-phenylindazole-3-carboxylic acid was added to N,N-dimethylformamide (3 ml), followed by 29 μL (0.3 mmol, 1 eq) of m-fluoroaniline and 61 mg (0.45 mmol, 1.5 eq) of N-hydroxy-7-azabenzotriazole. After stirring at room temperature for 10 minutes, 26.5 μL (0.23 mmol, 0.75 eq) of N-methylmorpholine was added at 0 °C. After stirring at 0 °C for 10 minutes, 87 mg (0.45 mmol, 1.5 eq) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The reaction was then carried out at 0 °C for 9 hours. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound W2 (77 mg), a white solid with a yield of 78%.

[0048] Its characterization data are as follows:

[0049] 1 H NMR (400MHz, CDCl3) δ9.01 (s, 1H), 8.52 (d, J = 8.2Hz, 1H), 7.80-7.71 (m, 4H), 7.63-7.5 8(m,2H),7.52-7.46(m,2H),7.41-7.36(m,2H),7.34-7.29(m,1H),6.87-6.82(m,1H).

[0050] Example 3: An allosteric modulator of adrenaline receptor functional activity and its preparation method

[0051] The allosteric modulator of adrenaline receptor functional activity provided in this embodiment, N-(3-chlorophenyl)-1-phenyl-1H-indazole-3-carboxamide, has the following structural formula:

[0052]

[0053] Its preparation method is as follows:

[0054] 71.5 mg (0.3 mmol, 1 eq) of N-phenylindazole-3-carboxylic acid was added to N,N-dimethylformamide (2 ml), followed by 32 μL (0.3 mmol, 1 eq) of m-chloroaniline and 61 mg (0.45 mmol, 1.5 eq) of N-hydroxy-7-azabenzotriazole. After stirring at room temperature for 10 minutes, 26.5 μL (0.23 mmol, 0.75 eq) of N-methylmorpholine was added at 0 °C. After stirring at 0 °C for 10 minutes, 87 mg (0.45 mmol, 1.5 eq) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The reaction was carried out at 0 °C for 9 hours. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound W3 (70 mg), a white solid, in 67% yield.

[0055] Its characterization data are as follows:

[0056] 1 H NMR (400MHz, CDCl3) δ9.01 (s, 1H), 8.52 (d, J = 8.2Hz, 1H), 7.80-7.71 (m, 4H), 7.63-7.5 8(m,2H),7.52-7.46(m,2H),7.41-7.36(m,2H),7.34-7.29(m,1H),6.87-6.82(m,1H).

[0057] Example 4: An allosteric modulator of adrenaline receptor functional activity and its preparation method

[0058] The allosteric modulator of adrenaline receptor functional activity provided in this embodiment, N-(3-bromophenyl)-1-phenyl-1H-indazole-3-carboxamide, has the following structural formula:

[0059]

[0060] Its preparation method is as follows:

[0061] At room temperature, 36 mg (0.15 mmol, 1 eq) of N-phenylindazole-3-carboxylic acid was added to N,N-dimethylformamide (2 ml), followed by 17 μL (0.22 mmol, 1 eq) of m-bromoaniline and 30 mg (0.22 mmol, 1.5 eq) of N-hydroxy-7-azabenzotriazole. After stirring at room temperature for 10 minutes, 14 μL (0.12 mmol, 0.75 eq) of N-methylmorpholine was added at 0 °C. After stirring at 0 °C for 10 minutes, 43 mg (0.22 mmol, 1.5 eq) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The reaction was then carried out at 0 °C for 9 hours. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound W4 (40 mg) as a white solid with a yield of 68%.

[0062] Its characterization data are as follows:

[0063] 1 H NMR (400MHz, CDCl3): δ8.99 (s, 1H), 8.56 (d, J = 8.2Hz, 1H), 8.11 (t, J = 1.9Hz, 1H), 7.81-7 .76(m,3H),7.71-7.63(m,3H),7.57-7.50(m,2H),7.46-7.42(m,1H),7.31-7.28(m,2H).

[0064] Example 5: An allosteric modulator of adrenaline receptor functional activity and its preparation method

[0065] The allosteric modulator of adrenaline receptor functional activity provided in this embodiment, N-(3-bromophenyl)-1-methyl-1H-indazole-3-carboxamide, has the following structural formula:

[0066]

[0067] Its preparation method is as follows:

[0068] At room temperature, 81 mg (0.5 mmol, 1 eq) of N-methyl-indazole-3-carboxylic acid was added to N,N-dimethylformamide (3 ml), followed by 54.4 μL (0.5 mmol, 1 eq) of m-bromoaniline and 102 mg (0.75 mmol, 1.5 eq) of N-hydroxy-7-azabenzotriazole. After stirring at room temperature for 10 minutes, 43 μL (0.375 mmol, 0.75 eq) of N-methylmorpholine was added at 0 °C. After stirring at 0 °C for 10 minutes, 144 mg (0.75 mmol, 1.5 eq) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The reaction was then carried out at 0 °C for 9 hours. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound W5 (149 mg), a white solid with a yield of 90%.

[0069] Its characterization data are as follows:

[0070] 1 H NMR (400MHz, (CD3)2SO): δ10.59 (s, 1H), 8.27 (t, J = 2.0Hz, 1H), 8.24-8.21 (m, 1H), 7.8 9-7.86(m,1H),7.80-7.77(m,1H),7.53-7.48(m,1H),7.35-7.26(m,3H),4.20(s,3H). 13 C NMR (100MHz, (CD3)2SO): δ160.9,141.2,140.6,136.5,130.6,126.9,126.1,12 2.9,122.6,122.5,121.7,121.5,119.1,110.7,36.1ppm.HRMS(ESI)m / z:calcd forC 15 H 12 BrN3NaO + [M+Na] + 352.0056, found 352.0059.

[0071] Example 6: An allosteric modulator of adrenaline receptor functional activity and its preparation method

[0072] The allosteric modulator of adrenaline receptor functional activity provided in this embodiment is (S)-N-(3-(3-bromophenyl)-1-(methylamino)-1-oxopropionyl)-1-hydro-indole-3-carboxamide.

[0073] ((S)-N-(3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)-1H-indazole-3-carboxamide), with the following structural formula:

[0074]

[0075] Its preparation method is as follows:

[0076] At room temperature, 33 mg (0.2 mmol, 1 eq) of indazole-3-carboxylic acid was added to N,N-dimethylformamide (2 ml), followed by 51.4 mg (0.2 mmol, 1 eq) of 2-amino-3-(3-bromophenyl)-N-methylpropionamide and 41 mg (0.3 mmol, 1.5 eq) of N-hydroxy-7-azabenzotriazole. After stirring at room temperature for 10 minutes, 18 μL (0.15 mmol, 0.75 eq) of N-methylmorpholine was added at 0 °C. After stirring at 0 °C for 10 minutes, 58 mg (0.3 mmol, 1.5 eq) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The reaction was then carried out at 0 °C for 9 hours. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound W6 (57 mg), a white solid, with a yield of 71%.

[0077] Its characterization data are as follows:

[0078] 1 H NMR (400MHz, CDCl3) δ14.41(s,1H),9.95(d,J=8.3Hz,1H),8.24(d,J=5.1Hz,1H),8 .12(d,J=8.2Hz,1H),7.76(d,J=8.5Hz,1H),7.48(t,J=7.6Hz,1H),7.37(t,J=1.8Hz ,1H),7.29(t,J=7.6Hz,1H),7.23(d,J=7.7Hz,1H),7.18(dd,J=8.0,1.9Hz,1H),6.9 9(t,J=7.8Hz,1H),5.32(q,J=8.0Hz,1H),3.26-3.13(m,2H),2.95(d,J=4.6Hz,3H). 13 C NMR (100MHz, CDCl3) δ174.1,163.9,141.4,139.1,137.7,132.5,130.2,130.1,127 .8,127.1,122.9,122.5,121.9,111.2,55.4,37.6,26.6ppm.HRMS(ESI)m / z:calcd forC18 H 17 BrN4NaO2 + [M+Na] + 423.0427, found 423.0429.

[0079] Example 7: An allosteric modulator of adrenaline receptor functional activity and its preparation method

[0080] The allosteric modulator of adrenaline receptor functional activity provided in this embodiment, N-(3-fluorophenyl)-1H-indazole-3-carboxamide, has the following structural formula:

[0081]

[0082] Its preparation method is as follows:

[0083] 81 mg (0.5 mmol, 1 eq) of indazole-3-carboxylic acid was added to N,N-dimethylformamide (3 ml), followed by 48.1 μL (0.5 mmol, 1 eq) of m-fluoroaniline and 102 mg (0.75 mmol, 1.5 eq) of N-hydroxy-7-azabenzotriazole. After stirring at room temperature for 10 minutes, 43 μL (0.375 mmol, 0.75 eq) of N-methylmorpholine was added at 0 °C. After stirring at 0 °C for 10 minutes, 144 mg (0.75 mmol, 1.5 eq) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The reaction was then carried out at 0 °C for 9 hours. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound W7 (80 mg), a white solid with a yield of 63%.

[0084] Its characterization data are as follows:

[0085] 1 H NMR (400MHz, (CD3)2SO): δ13.86 (s, 1H), 10.61 (s, 1H), 8.24 (d, J = 8.2Hz, 1H), 7.91-7.88 (m, 1H), 7.75 (d, J = 8.2Hz, 1H),7.68(d,J=8.4Hz,1H),7.47(t,J=7.4Hz,1H),7.38(q,J=7.8Hz,1H),7.31(t,J=7.6Hz,1H),6.94-6.89(m,1H). 13C NMR (100MHz, (CD3)2SO): δ163.3,161.3,160.9,141.4,140.8,140.7,138.0,130.3,130.2,126.9,1 22.6,121.8,121.5,116.0,116.0,111.0,110.0,109.7,107.0,106.8ppm.HRMS(ESI)m / z:calcdfor C 14 H 10 FN3NaO + [M+Na] + 278.0700, found 278.0705.

[0086] Example 8: An allosteric modulator of adrenaline receptor functional activity and its preparation method

[0087] The allosteric modulator of adrenaline receptor functional activity provided in this embodiment, N-(3-bromophenyl)-1H-indazole-3-carboxamide, has the following structural formula:

[0088]

[0089] Its preparation method is as follows:

[0090] At room temperature, 81 mg (0.5 mmol, 1 eq) of indazole-3-carboxylic acid was added to N,N-dimethylformamide (3 ml), followed by 52.9 μL (0.5 mmol, 1 eq) of m-bromoaniline and 102 mg (0.75 mmol, 1.5 eq) of N-hydroxy-7-azabenzotriazole. After stirring at room temperature for 10 minutes, 43 μL (0.375 mmol, 0.75 eq) of N-methylmorpholine was added at 0 °C. After stirring at 0 °C for 10 minutes, 144 mg (0.75 mmol, 1.5 eq) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The reaction was then carried out at 0 °C for 9 hours. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound W8 (62 mg) as a white solid with a yield of 78%.

[0091] Its characterization data are as follows:

[0092] 1H NMR (400MHz, (CD3)2SO): δ13.87(s,1H),10.59(s,1H),8.27(s,1H),8.23(d,J=8.1Hz,1H) ,7.89(dt,J=7.8,1.8Hz,1H),7.68(d,J=8.4Hz,1H),7.48-7.45(m,1H),7.33-7.26(m,3H); 13 C NMR (100MHz, DMSO): δ161.3,141.4,140.6,138.0,130.6,126.9,126.0,122.6,122.5,121.8,121.5,119.0,111.0ppm.HRMS(ESI)m / z:calcd for C 14 H 10 BrN3NaO + [M+Na] + 337.9900, found 337.9900.

[0093] Example 9: An allosteric modulator of adrenaline receptor functional activity and its preparation method

[0094] The allosteric modulator of adrenaline receptor functional activity provided in this embodiment, N-(3-chlorophenyl)-1H-indazole-3-carboxamide, has the following structural formula:

[0095]

[0096] Its preparation method is as follows:

[0097] At room temperature, 81 mg (0.5 mmol, 1 eq) of indazole-3-carboxylic acid was added to N,N-dimethylformamide (3 ml), followed by 52.9 μL (0.5 mmol, 1 eq) of m-chloroaniline and 102 mg (0.75 mmol, 1.5 eq) of N-hydroxy-7-azabenzotriazole. After stirring at room temperature for 10 minutes, 43 μL (0.375 mmol, 0.75 eq) of N-methylmorpholine was added at 0 °C. After stirring at 0 °C for 10 minutes, 144 mg (0.75 mmol, 1.5 eq) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The reaction was then carried out at 0 °C for 9 hours. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound W9 (49 mg), a white solid, with a yield of 36%.

[0098] Its characterization data are as follows:

[0099] 1 H NMR (400MHz, (CD3)2SO): δ13.87(s,1H),10.61(s,1H),8.24(d,J=8.0Hz,1H),8.13(s,1H),7.86(dd,J=8.1,2.1Hz, 1H),7.68(d,J=8.4Hz,1H),7.47(t,J=7.4Hz,1H),7.37(t,J=8.1Hz,1H),7.31(t,J=7.6Hz,1H),7.15-7.13(m,1H); 13 C NMR (100MHz, (CD3)2SO): δ161.3,141.4,140.5,138.0,133.0,130.3,126.9,123.1,122.6,121.8,121.5,119.6,118.6,111.0ppm.HRMS(ESI)m / z:calcd for C 14 H 10 ClN3NaO + [M+Na] + 294.0404, found294.0409.

[0100] Example 10: An allosteric modulator of adrenaline receptor functional activity and its preparation method

[0101] The allosteric modulator of adrenaline receptor functional activity provided in this embodiment, N-(3-methylphenyl)-1H-indole-3-carboxamide (N-(m-tolyl)-1H-indazole-3-carboxamide), has the following structural formula:

[0102]

[0103] Its preparation method is as follows:

[0104] At room temperature, 81 mg (0.5 mmol, 1 eq) of indazole-3-carboxylic acid was added to N,N-dimethylformamide (3 ml), followed by 53.6 μL (0.5 mmol, 1 eq) of 3-methylaniline and 102 mg (0.75 mmol, 1.5 eq) of N-hydroxy-7-azabenzotriazole. After stirring at room temperature for 10 minutes, 43 μL (0.375 mmol, 0.75 eq) of N-methylmorpholine was added at 0 °C. After stirring at 0 °C for 10 minutes, 144 mg (0.75 mmol, 1.5 eq) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The reaction was then carried out at 0 °C for 9 hours. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound W10 (95 mg), a pale yellow solid, with a yield of 76%.

[0105] Its characterization data are as follows:

[0106] 1 H NMR (400MHz, (CD3)2SO): δ13.87(s,1H),10.61(s,1H),8.24(d,J=8.0Hz,1H),8.13(s,1H),7.86(dd,J=8.1,2.1Hz, 1H),7.68(d,J=8.4Hz,1H),7.47(t,J=7.4Hz,1H),7.37(t,J=8.1Hz,1H),7.31(t,J=7.6Hz,1H),7.15-7.13(m,1H); 13 C NMR (100MHz, (CD3)2SO): δ161.3,141.4,140.5,138.0,133.0,130.3,126.9,123.1,122.6,121.8,121.5,119.6,118.6,111.0ppm.HRMS(ESI)m / z:calcd for C 14 H 10 ClN3NaO + [M+Na] + 294.0404, found294.0409.

[0107] Example 11: An allosteric modulator of adrenaline receptor functional activity and its preparation method

[0108] The allosteric modulator of adrenaline receptor functional activity provided in this embodiment, N-(3,5-dibromophenyl)-1H-indazole-3-carboxamide, has the following structural formula:

[0109]

[0110] Its preparation method is as follows:

[0111] At room temperature, 81 mg (0.5 mmol, 1 eq) of indazole-3-carboxylic acid was added to N,N-dimethylformamide (3 ml), followed by 125.5 mg (0.5 mmol, 1 eq) of 3,5-dibromoaniline and 102 mg (0.75 mmol, 1.5 eq) of N-hydroxy-7-azabenzotriazole. After stirring at room temperature for 10 minutes, 43 μL (0.375 mmol, 0.75 eq) of N-methylmorpholine was added at 0 °C. After stirring at 0 °C for 10 minutes, 144 mg (0.75 mmol, 1.5 eq) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The reaction was then carried out at 0 °C for 9 hours. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound W11 (17 mg), a white solid, in 9% yield.

[0112] Its characterization data are as follows:

[0113] 1 H NMR (400MHz, (CD3)2SO) δ13.93 (s, 1H), 10.75 (s, 1H), 8.24 (d, J = 1.8Hz, 2H), 8.22 (d, J = 8.1Hz, 1H),7.68(d,J=8.4Hz,1H),7.52(d,J=1.6Hz,1H),7.47(t,J=7.4Hz,1H),7.31(t,J=7.6Hz,1H). 13 C NMR (100MHz, (CD3)2SO) δ161.4,141.7,141.4,137.6,127.9,126.9,122.8,122.3,121.8,121.4,121.4,111.1ppm.

[0114] Example 12: An allosteric modulator of adrenaline receptor functional activity and its preparation method

[0115] The allosteric modulator of adrenaline receptor functional activity provided in this embodiment, N-benzyl-1H-indazole-3-carboxamide, has the following structural formula:

[0116]

[0117] Its preparation method is as follows:

[0118] At room temperature, 81 mg (0.5 mmol, 1 eq) of indazole-3-carboxylic acid was added to N,N-dimethylformamide (3 ml), followed by 54.4 μL (0.5 mmol, 1 eq) of benzylamine and 102 mg (0.75 mmol, 1.5 eq) of N-hydroxy-7-azabenzotriazole. After stirring at room temperature for 10 minutes, 43 μL (0.375 mmol, 0.75 eq) of N-methylmorpholine was added at 0 °C. After stirring at 0 °C for 10 minutes, 144 mg (0.75 mmol, 1.5 eq) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The reaction was then carried out at 0 °C for 9 hours. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound W12 (117 mg), a white solid with a yield of 93%.

[0119] Its characterization data are as follows:

[0120] 1 H NMR (400MHz, (CD3)2SO): δ13.64(s,1H),9.02(t,J=6.4Hz,1H),8.19(d,J=8.1Hz,1H),7.62(d,J=8.4Hz,1H),7.43-7.39(m,1H),7.37 -7.35(m,2H),7.33-7.29(m,2H),7.26-7.20(m,2H),4.51(d,J=6.4Hz,2H); 13 C NMR (100MHz, (CD3)2SO): δ162.4,141.1,140.0,138.3,128.3,127.3,126.7,126.5,122.1,121.6,121.6,110.7,41.9ppm.HRMS(ESI)m / z:calcd for C 15 H 13 N3NaO + [M+Na] + 274.0951, found 274.0957.

[0121] Example 13: An allosteric modulator of adrenaline receptor functional activity and its preparation method

[0122] The allosteric modulator of adrenaline receptor functional activity provided in this embodiment, N-methyl-1H-indazole-3-carboxamide, has the following structural formula:

[0123]

[0124] Its preparation method is as follows:

[0125] At room temperature, 81 mg (0.5 mmol, 1 eq) of indazole-3-carboxylic acid was added to N,N-dimethylformamide (3 ml), followed by 33.8 mg (0.5 mmol, 1 eq) of methylamine hydrochloride and 102 mg (0.75 mmol, 1.5 eq) of N-hydroxy-7-azabenzotriazole. After stirring at room temperature for 10 minutes, 86.5 μL (0.75 mmol, 0.75 eq) of N-methylmorpholine was added at 0 °C. After stirring at 0 °C for 10 minutes, 144 mg (0.75 mmol, 1.5 eq) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The reaction was then carried out at 0 °C for 9 hours. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound W13 (75 mg), a white solid with a yield of 86%.

[0126] Its characterization data are as follows:

[0127] 1 H NMR(400MHz, (CD3)2SO) δ13.56(s,1H),8.38(d,J=4.9Hz,1H),8.19(d,J=8.1Hz,1H),7 .60(d,J=8.4Hz,1H),7.42-7.38(m,1H),7.23(t,J=7.6Hz,1H),2.82(d,J=4.7Hz,3H). 13 C NMR (100MHz, (CD3)2SO) δ162.9,141.1,138.5,126.5,122.0,121.7,121.5,110.7,25.6ppm..

[0128] Example 14: An allosteric modulator of adrenaline receptor functional activity and its preparation method

[0129] The allosteric modulator of adrenaline receptor functional activity provided in this embodiment, N-isopropyl-1H-indazole-3-carboxamide, has the following structural formula:

[0130]

[0131] Its preparation method is as follows:

[0132] 81 mg (0.5 mmol, 1 eq) of indazole-3-carboxylic acid was added to N,N-dimethylformamide (3 ml), followed by 42.8 μL (0.5 mmol, 1 eq) of isopropylamine and 102 mg (0.75 mmol, 1.5 eq) of N-hydroxy-7-azabenzotriazole. After stirring at room temperature for 10 minutes, 43 μL (0.375 mmol, 0.75 eq) of N-methylmorpholine was added at 0 °C. After stirring at 0 °C for 10 minutes, 144 mg (0.75 mmol, 1.5 eq) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The reaction was then carried out at 0 °C for 9 hours. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound W14 (84 mg), a white solid with a yield of 83%.

[0133] Its characterization data are as follows:

[0134] 1 H NMR(400MHz, (CD3)2SO) δ13.55(s,1H),8.19(d,J=7.9Hz,1H),8.11(d,J=8.3Hz,1H),7.60(d,J= 8.4Hz,1H),7.42-7.38(m,1H),7.22(t,J=7.9Hz,1H),4.24-4.12(m,1H),1.19(d,J=6.6Hz,6H). 13 C NMR (100MHz, (CD3)2SO) δ161.4,141.1,138.5,126.5,122.0,121.7,121.6,110.7,40.2,22.4ppm..

[0135] Example 15: An allosteric modulator of adrenaline receptor functional activity and its preparation method

[0136] The allosteric modulator of adrenaline receptor functional activity provided in this embodiment, N-isopropyl-1H-indazole-3-carboxamide, has the following structural formula:

[0137]

[0138] Its preparation method is as follows:

[0139] 81 mg (0.5 mmol, 1 eq) of indazole-3-carboxylic acid was added to N,N-dimethylformamide (3 ml), followed by 52.9 μL (0.5 mmol, 1 eq) of cyclohexylamine and 102 mg (0.75 mmol, 1.5 eq) of N-hydroxy-7-azabenzotriazole. After stirring at room temperature for 10 minutes, 43 μL (0.375 mmol, 0.75 eq) of N-methylmorpholine was added at 0 °C. After stirring at 0 °C for 10 minutes, 144 mg (0.75 mmol, 1.5 eq) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The reaction was then carried out at 0 °C for 9 hours. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound W15 (98 mg), a white solid, in 81% yield.

[0140] Its characterization data are as follows: 1 H NMR (400MHz, CD3OD): δ8.21(d,J=8.2Hz,1H),7.57-7.55(m,1H),7.43-7.39(m,1H),7.26-7.22(m,1H),3.96-3.90(m,1H) ),2.01-1.98(m,2H),1.84-1.80(m,2H),1.70-1.65(m,1H),1.50-1.35(m,4H),1.30-1.23(m,1H).HRMS(ESI)m / z:calcd forC 14 H 17 N3NaO + [M+Na] + 266.1264, found 266.1271.

[0141] Example 16: An allosteric modulator of adrenaline receptor functional activity and its preparation method

[0142] The allosteric modulator of adrenaline receptor functional activity provided in this embodiment, 5-bromo-N-(3-bromophenyl)-1H-indazole-3-carboxamide, has the following structural formula:

[0143]

[0144] Its preparation method is as follows:

[0145] At room temperature, 120.5 mg (0.5 mmol, 1 eq) of 5-bromoindazole-3-carboxylic acid was added to N,N-dimethylformamide (3 ml), followed by 54.5 μL (0.5 mmol, 1 eq) of m-bromoaniline and 102 mg (0.75 mmol, 1.5 eq) of N-hydroxy-7-azabenzotriazole. After stirring at room temperature for 10 minutes, 43 μL (0.375 mmol, 0.75 eq) of N-methylmorpholine was added at 0 °C. After stirring at 0 °C for 10 minutes, 144 mg (0.75 mmol, 1.5 eq) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The reaction was then carried out at 0 °C for 9 hours. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound W16 (70 mg), a white solid, in 36% yield.

[0146] Its characterization data are as follows:

[0147] 1 H NMR (400MHz, (CD3)2SO): δ14.07 (s, 1H), 10.66 (s, 1H), 8.37 (d, J = 1.8Hz, 1H), 8.25 (t, J = 2.0Hz, 1H) ,7.87(dt,J=7.7,1.8Hz,1H),7.67(d,J=8.8Hz,1H),7.59(dd,J=8.8,1.9Hz,1H),7.34-7.26(m,2H); 13 C NMR (100MHz, (CD3)2SO): δ160.8,140.4,140.1,137.5,130.6,129.7,126.2,123.6,123.4,122.6,121.5,119.1,115.2,113.3ppm.HRMS(ESI)m / z:calcd for C 14 H9Br2N3NaO + [M+Na] + 415.9004, found 415.9001.

[0148] Example 17: An allosteric modulator of adrenaline receptor functional activity and its preparation method

[0149] The allosteric modulator of adrenaline receptor functional activity provided in this embodiment, 5-bromo-N-(phenyl)-1H-indole-3-carboxamide, has the following structural formula:

[0150]

[0151] Its preparation method is as follows:

[0152] At room temperature, 120.5 mg (0.5 mmol, 1 eq) of 5-bromoindazole-3-carboxylic acid was added to N,N-dimethylformamide (3 ml), followed by 45.6 μL (0.5 mmol, 1 eq) of aniline and 102 mg (0.75 mmol, 1.5 eq) of N-hydroxy-7-azabenzotriazole. After stirring at room temperature for 10 minutes, 43 μL (0.375 mmol, 0.75 eq) of N-methylmorpholine was added at 0 °C. After stirring at 0 °C for 10 minutes, 144 mg (0.75 mmol, 1.5 eq) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The reaction was then carried out at 0 °C for 9 hours. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound W17 (70 mg), a white solid with a yield of 44%.

[0153] Its characterization data are as follows:

[0154] 1 H NMR(400MHz, (CD3)2SO) δ14.02(s,1H),10.44(s,1H),8.38(d,J=1.8Hz,1H),7.90(d,J=7.6Hz,2H) ,7.67(d,J=8.8Hz,1H),7.58(dd,J=8.9,1.9Hz,1H),7.35(t,J=7.7Hz,2H),7.10(t,J=7.4Hz,1H). 13 C NMR (100MHz, (CD3)2SO) δ160.6,140.1,138.8,137.8,129.6,128.6,123.7,123.6,123.4,120.4,115.0,113.2ppm..

[0155] Example 18: An allosteric modulator of adrenaline receptor functional activity and its preparation method

[0156] The allosteric modulator of adrenaline receptor functional activity provided in this embodiment, 5-methyl-N-phenyl-1H-indole-3-carboxamide (5-bromo-N-phenyl-1H-indazole-3-carboxamide), has the following structural formula:

[0157]

[0158] Its preparation method is as follows:

[0159] At room temperature, 88.8 mg (0.5 mmol, 1 eq) of 5-methylindazole-3-carboxylic acid was added to N,N-dimethylformamide (3 ml), followed by 45.6 μL (0.5 mmol, 1 eq) of aniline and 102 mg (0.75 mmol, 1.5 eq) of N-hydroxy-7-azabenzotriazole. After stirring at room temperature for 10 minutes, 43 μL (0.375 mmol, 0.75 eq) of N-methylmorpholine was added at 0 °C. After stirring at 0 °C for 10 minutes, 144 mg (0.75 mmol, 1.5 eq) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The reaction was then carried out at 0 °C for 9 hours. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound W18 (100 mg) as a yellow solid with a yield of 80%.

[0160] Its characterization data are as follows:

[0161] 1 H NMR (400MHz, (CD3)2SO): δ13.68(s,1H),10.30(s,1H),8.03(s,1H),7.92-7.90(m,2H),7.56(d,J =8.6Hz,1H),7.35(t,J=7.5Hz,2H),7.29(dd,J=8.6,1.6Hz,1H),7.10-7.06(m,1H),2.45(s,3H); 13 C NMR (100MHz, (CD3)2SO): δ161.1,140.0,139.0,137.7,131.5,128.8,128.6,123.4,122.2,120.5,120.2,110.6,21.2ppm.HRMS(ESI)m / z:calcd forC 15 H 13 N3NaO + [M+Na] + 274.0951, found 274.0957.

[0162] Example 19: An allosteric modulator of adrenaline receptor functional activity and its preparation method

[0163] The allosteric modulator of adrenaline receptor functional activity provided in this embodiment, 6-bromo-N-phenyl-1H-indole-3-carboxamide, has the following structural formula:

[0164]

[0165] Its preparation method is as follows:

[0166] At room temperature, 120.5 mg (0.5 mmol, 1 eq) of 6-bromoindazole-3-carboxylic acid was added to N,N-dimethylformamide (3 ml), followed by 45.6 μL (0.5 mmol, 1 eq) of aniline and 102 mg (0.75 mmol, 1.5 eq) of N-hydroxy-7-azabenzotriazole. After stirring at room temperature for 10 minutes, 43 μL (0.375 mmol, 0.75 eq) of N-methylmorpholine was added at 0 °C. After stirring at 0 °C for 10 minutes, 144 mg (0.75 mmol, 1.5 eq) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The reaction was then carried out at 0 °C for 9 hours. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound W19 (130 mg), a white solid with a yield of 83%.

[0167] Its characterization data are as follows:

[0168] 1 H NMR (400MHz, (CD3)2SO): δ13.93(s,1H),10.42(s,1H),8.16(d,J=8.6Hz,1H),7.91(d,J=5.7H z,2H),7.88(s,1H),7.43(dd,J=8.6,1.6Hz,1H),7.35(t,J=7.7Hz,2H),7.09(t,J=7.4Hz,1H); 13 C NMR(100MHz,(CD3)2SO)δ160.6,142.1,138.8,138.7,128.6,125.7,123.6,123.4,120.8,120.4,120.3,113.6ppm.HRMS(ESI)m / z:calcd forC 14 H 10 BrN3NaO + [M+Na] + 337.9900, found 337.9906.

[0169] Example 20: An allosteric modulator of adrenaline receptor functional activity and its preparation method

[0170] The allosteric modulator of adrenaline receptor functional activity provided in this embodiment, 5-chloro-N-phenyl-1H-indole-3-carboxamide, has the following structural formula:

[0171]

[0172] Its preparation method is as follows:

[0173] At room temperature, 98.3 mg (0.5 mmol, 1 eq) of 5-chloroindazole-3-carboxylic acid was added to N,N-dimethylformamide (3 ml), followed by 45.6 μL (0.5 mmol, 1 eq) of aniline and 102 mg (0.75 mmol, 1.5 eq) of N-hydroxy-7-azabenzotriazole. After stirring at room temperature for 10 minutes, 43 μL (0.375 mmol, 0.75 eq) of N-methylmorpholine was added at 0 °C. After stirring at 0 °C for 10 minutes, 144 mg (0.75 mmol, 1.5 eq) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The reaction was then carried out at 0 °C for 9 hours. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound W20 (101 mg), a white solid with a yield of 75%.

[0174] Its characterization data are as follows:

[0175] 1 H NMR(400MHz, (CD3)2SO) δ14.02(s,1H),10.43(s,1H),8.22(d,J=2.1Hz,1H),7.90(d,J=8.0Hz,2H) ,7.72(d,J=8.9Hz,1H),7.48(dd,J=8.8,2.0Hz,1H),7.35(t,J=7.7Hz,2H),7.10(t,J=7.4Hz,1H). 13 C NMR (100MHz, (CD3)2SO) δ160.6,139.9,138.8,138.0,128.7,127.2,127.0,123.6,122.7,120.5,120.4,112.9ppm.

[0176] Performance testing

[0177] 1. Bioactivity screening

[0178] (1) GloSensor TM cAMP Assay measures cAMP levels:

[0179] cAMP is a key signaling molecule in many G protein-coupled receptors. The accumulation level of cAMP is mainly measured using GloSensor—a bioluminescent biosensor (Promega) that can directly detect intracellular cAMP.

[0180] The principle is to insert a cAMP-binding domain at the N-terminus and C-terminus of firefly luciferase using genetic engineering technology, which puts the enzyme in an inactive state. When cAMP binds to the cAMP-binding domain, the enzyme is activated, thereby oxidizing the substrate luciferin to produce bioluminescence.

[0181] The cAMP accumulation experiment was used to test the functional activity of the target compound for β2AR and to clarify whether the new compound is a negative allosteric modulator (NAM) of β2AR.

[0182] The specific method is as follows: HEK293T cells were seeded into 6-well plates, with 4 × 10⁵ cells per well. The next day, β2AR and pGloSensor-22F cAMP plasmids were simultaneously transfected into HEK293T cells using FuGene transfection reagent (Promega). After 48 hours, the transfected cells were washed with CO₂-independent medium and then incubated with equilibrated medium containing 2% v / v GloSensor cAMP reagent stock solution (dissolved in CO₂-independent medium containing 10% FBS).

[0183] After incubation at 37°C for 1 hour, followed by incubation at room temperature for 1 hour, the bioluminescence signal was detected using a multi-mode microplate reader until a steady-state baseline signal was obtained. Then, different concentration gradients of the novel derivative and the control compound Cmpd-15 were added to the cells, and after incubation at 37°C for 30 minutes, the positive control ISO (final concentration 1 nM–100 μM) was added. Changes in biofluorescence were read using a microplate reader.

[0184] The structural formula of compound Cmpd-15 is as follows:

[0185]

[0186] Using the GloSensor cAMP accumulation assay, with isoproterenol (ISO) at different concentration gradients as positive controls (final concentration 1 nM–100 μM) and compound Cmpd-15 (final concentration 50 μM) as a reference control, the allosteric antagonistic activity of the allosteric modifier provided in this invention (final concentration 50 μM) was compared with that of compound Cmpd-15. Table 1 shows that all compounds provided in this invention exhibit allosteric antagonistic activity against β2AR, with compound W8 showing the highest activity, 4.17 times that of Cmpd-15.

[0187] Table 1. List of allosteric activities of the synthesized compounds relative to Cmpd-15

[0188]

[0189]

[0190] (2) Study on allosteric antagonistic mechanism

[0191] This invention also used a cAMP accumulation assay to further investigate the allosteric antagonistic mechanism, testing whether the target compounds could allosterically regulate the functional activity of the β2AR endogenous ligand ISO to determine whether they are negative allosteric regulators of β2AR (NAM). Specifically, the W8 compound provided by this invention was first added to cells at concentrations in multiples (1 nM to 100 μM), and incubated at 37°C for 30 min. Then, different concentration gradients of the positive control ISO (final concentration 1 nM to 100 μM) were added to the cells, and the change in biofluorescence was tested to see whether it exhibited a concentration-dependent, limited downward shift. The specific experimental steps were the same as the Glosensor cAMP accumulation assay described above. The experimental results are shown below. Figure 3 The results showed that the target compound W8 could negatively allosterically regulate the functional activity of the β2AR endogenous ligand ISO.

[0192] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been shown above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. Use of a benzopyrazole carboxamide compound for the manufacture of allosteric modulators of adrenergic receptor functional activity, characterized in that, The molecular structural formula of the benzopyrazole amide compounds is as follows: ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； The allosteric modulator is used to negatively modulate the functional activity of the β2AR endogenous ligand ISO.

2. The application according to claim 1, characterized in that, The preparation method of the benzopyrazole amide compound is as follows: ； Wherein, R1 is any one of H, 5-Br, 6-Br, 5-Me, and 5-Cl; R2 is any one of H, Me, and Ph; -NR3R4 can be any of the following structures: ； ； ； ； ； ； ； ； ； ； ； 。 3. The application according to claim 2, characterized in that, At room temperature, a 3-carboxylic acid benzopyrazole derivative was added to the solvent DMF, followed by the addition of an amine and HOAT. The mixture was stirred, cooled, and then NMM was added. The mixture was stirred again, and then EDCI was added. The reaction was continued until the reaction was complete. The mixture was then extracted and separated to obtain the benzopyrazole amide compound.

4. The application according to claim 3, characterized in that, When NMM is added, the temperature of the reaction system is maintained at 0℃~10℃.

5. The application according to claim 3, characterized in that, When adding EDCI, the system temperature is maintained between 0°C and 10°C.

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