Pyrrole amide derivative, preparation method thereof and application of pyrrole amide derivative as beta2-adrenergic receptor allosteric antagonist

Abstract

The invention belongs to the field of medicinal chemistry, and particularly discloses a pyrrole amide derivative, a preparation method thereof and application of the pyrrole amide derivative as a beta2-adrenergic receptor allosteric antagonist. The structure of the pyrrole amide derivative is shown as a formula I. In the formula I, R1 is ethyl, methyl, isopropyl and the like; r2 is fatty amine, aniline, m-bromoaniline, benzylamine and the like. Pharmacological results show that the pyrrole amide derivative disclosed by the invention has relatively good antagonistic activity on beta2AR, and can perform negative allosteric regulation on the functional activity of isoprenaline (ISO) serving as a normal agonist of beta2AR.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a pyrrolamide derivative and a preparation method thereof and an application thereof as a β2-adrenaline receptor allosteric antagonist. Background Art

[0002] G protein-coupled receptors (GPCRs) are membrane receptor proteins present on the cell surface that convert extracellular signals into key physiological effects. Their endogenous ligands include odors, hormones, neurotransmitters, chemokines, etc., from photons, amines, carbohydrates, lipids, peptides to proteins. Their types and numbers are the most among all cell surface receptors. GPCRs are widely distributed in various eukaryotic organisms. They first bind to G proteins (guanylate-binding proteins), regulate the activity of corresponding enzymes in cells and produce second messengers. Then the second messengers transmit the ligand signals from outside the cells to inside the cells, thereby participating in regulating the relevant biological reaction processes in the cells and exerting their functions.

[0003] With the continuous in-depth study of GPCR structure and function, more and more targeted drugs based on GPCR structure have been developed and applied to the treatment of major diseases such as central nervous system diseases, diabetes, cancer, etc. GPCR participates in many physiological activities and signal regulation processes in the body, forming a systematic signal molecule conduction network. If the related signal transduction pathways or signal molecules of GPCR are abnormal, it will lead to an imbalance in the biological environment, thereby causing diseases such as rheumatoid arthritis, autoimmune diseases, liver damage, cancer, etc. Among the 826 human GPCRs, about 350 non-olfactory members are considered to be druggable, of which 165 are verified drug targets. Statistics show that 527 Food and Drug Administration (FDA)-approved drugs and 1,060 candidate drugs currently in clinical trials target GPCRs.

[0004] Endogenous ligands as well as conventional agonists, antagonists, and inverse agonists typically occupy the orthosteric binding site. This binding site is highly conserved within the AGPCR family and even more conserved between individual subtypes. This has severely hampered the development of selective orthosteric agonists and antagonists. However, proteins, like receptors, ion channels, and enzymes, can have additional allosteric binding sites. Allosteric modulators can enhance the subtype selectivity of ligands for GPCRs and improve drug efficacy. In addition, the key amino acids involved in allosteric regulation of GPCRs are less conserved, which provides a structural basis for the development of more selective allosteric modulators.

[0005] In 2017, the research group of Professor Brian Kobilka at Tsinghua University School of Medicine and the research group of Professor Robert Lefkowitz at Duke University reported the isolation of an allosteric modulator of β2 adrenergic receptor (β2AR) from a DNA-encoded small molecule library. This is the first intracellular allosteric antagonist Cmpd-15 (S. Ahn, et al. PNAS, 2017, 114: 1708-1713). It is the first small molecule negative allosteric modulator (NAM) for β2AR, showing negative synergy with agonists and positive synergy with inverse agonists. Biochemical experiments showed that Cmpd-15 may cross the cell membrane and bind to the intracellular location of β2AR.

[0006] However, Cmpd-15 is a dipeptide with poor water solubility, and the relative instability of its structure may affect its development performance. Therefore, it is necessary to redesign the structure. Summary of the invention

[0007] The purpose of the present invention is to provide a class of pyrrolamide derivatives to develop new heterocyclic derivatives with stable chemical structure, high biological activity, receptor subtype selectivity and good water solubility as allosteric modulators of β2AR, providing a new direction for the research and development of new drugs for cardiovascular and cerebrovascular diseases, diabetes and cancer diseases.

[0008] The present invention provides a pyrrolamide derivative, the structural formula of the pyrrolamide derivative is as shown in Formula I:

[0009]

[0010] Wherein, R1=H, Me, Et, i-Pr, Ph;

[0011] R2 is one of the following structural formulas:

[0012]

[0013] The present invention also provides a preparation method of pyrrole amide derivatives, which comprises: dissolving pyrrole carboxylic acid and 1-hydroxy-7-azabenzotriazole (HOAt) in DMF, adding different types of amines R2-NH2 (1 eq) under ice bath, then adding N-methylmorpholine (NMM) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and then continuing to stir to room temperature, and reacting at room temperature for several hours; spin-drying the DMF, extracting, and passing through a column to obtain the pyrrole amide derivatives.

[0014] Furthermore, the pyrrole carboxylic acid includes 5-ethyl-1H-pyrrole-2-carboxylic acid, 1H-pyrrole-2-carboxylic acid, 5-methyl-1H-pyrrole-2-carboxylic acid, 5-isopropyl-1H-pyrrole-2-carboxylic acid, 5-phenyl-1H-pyrrole-2-carboxylic acid, 4,5-dimethyl-1H-pyrrole-2-carboxylic acid, and 4-bromo-1H-pyrrole-2-carboxylic acid.

[0015] Further, when the amount of pyrrole carboxylic acid used is 1 eq, 1-hydroxy-7-azabenzotriazole is 1.2 eq, different types of amines R2-NH2 are 1 eq, N-methylmorpholine is 0.7 eq, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1.2 eq.

[0016] Furthermore, the reaction time at room temperature is 12 hours.

[0017] The present invention also provides the use of the above-mentioned pyrrolamide derivatives as β2-adrenaline receptor allosteric antagonists.

[0018] The present invention provides the use of the above-mentioned pyrrolamide derivatives in the preparation of β2-adrenaline receptor allosteric antagonists.

[0019] Furthermore, the pyrrolamide derivatives are used as active ingredients and a pharmaceutically acceptable carrier to prepare a pharmaceutical preparation.

[0020] The beneficial effects of the present invention are:

[0021] Most of the new derivatives of pyrrole amides synthesized by the present invention have β2AR allosteric antagonist activity. Compared with the lead compound Cmpd-15, the allosteric antagonist activity of compounds G1, G2, G6, and G16 on β2AR is significantly improved. The new derivatives of pyrrole synthesized by the present invention have the advantages of simple structure, simple synthesis route, and easy-to-obtain raw materials, and can provide a basis for the creation of new drugs for cardiovascular, asthma and cancer diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the structural diagram of Cmpd-15;

[0023] Figure 2 For the design of early pyrazole compounds;

[0024] Figure 3 Design of pyrrole target compounds;

[0025] Figure 4 It is a synthetic route for pyrrolamide derivatives;

[0026] Figure 5 This is a dose-response curve of ISO mediated by the pyrrolamide derivative G1;

[0027] Figure 6 This is a dose-response curve of ISO mediated by the pyrrolamide derivative G14. DETAILED DESCRIPTION

[0028] The structure of Cmpd-15 is as follows Figure 1 As shown, the relative instability of its structure may affect its development performance. Therefore, it is necessary to redesign the structure and use a heterocycle as its core skeleton. In the early stage, the inventor used Cmpd-15 as a lead compound and applied a skeleton hopping strategy to replace the polypeptide structure of Cmpd-15 with a pyrazole ring. While keeping the 2-amino-N-methyl-3-bromophenylpropionamide and 2-cyclohexylphenylacetyl groups in Cmpd-15 unchanged, the amino group in the 2-cyclohexylphenylacetyl group and the methylene group in the (4-formamido)benzyl group were cyclized and fused to form N-phenylacetylpyrazole. Since N-phenylacetyl is easily hydrolyzed and removed from the pyrazole skeleton, the inventor replaced the phenylacetyl group with a benzyl group. In this way, the polypeptide skeleton of Cmpd-15 was replaced by a more stable pyrazole skeleton. The results showed that most 1-benzyl-3-aryl-pyrazole-5-carboxamide derivatives showed stronger allosteric antagonist activity against β2AR than Cmpd-15. However, 1-benzyl-5-arylpyrazole-3-carboxamide derivatives have poor allosteric antagonist activity against β2AR, or even no allosteric antagonist activity (X. Guo, et al. Bioorg. Med. Chem. 2024, 108: 117787).

[0029] Furthermore, if Figure 2 As shown in Figure 1, the inventors simplified the structure of active 1-benzyl-3-aryl-pyrazole-5-carboxamide derivatives. After removing the benzyl group on N, the obtained pyrazole derivatives all have allosteric antagonism on the G-protein signaling pathway of β2AR, and can negatively regulate the agonism of endogenous ligand isoproterenol (ISO) on β2AR. The results of cAMP accumulation experiments show that the allosteric antagonism of most unsubstituted pyrazole derivatives on β2AR is significantly better than the functional activity of its lead compound Cmpd-15, and is expected to be used as a lead compound for the treatment of neovascular diseases (Chen Xin, et al., China Invention Patent Publication No.: CN115745891A).

[0030] From the structure of pyrazole amide derivatives, it can be seen that the phenylalanine on the right side containing a chiral carbon atom has a large steric hindrance, which may affect the drugability, and there is still a problem of poor water solubility. Figure 3 As shown, the inventors designed to replace the pyrazole ring with a pyrrole ring, and to synthesize new pyrrole amide compounds with simple substituted aniline, benzylamine or naphthylamine. The synthesized pyrrole amide compounds have further improved activity compared to pyrazole derivatives.

[0031] The present invention provides a pyrrolamide derivative, the structure of which is shown in Formula I:

[0032]

[0033] Wherein, R1=H, Me, Et, i-Pr, Ph;

[0034] R2 is one of the following structural formulas:

[0035]

[0036] The compound of the invention uses pyrrole with different substituents as raw materials, and undergoes amide coupling reaction with aniline and aliphatic amine with different substituents to finally obtain a series of new pyrrole amide derivatives.

[0037] Table 1 shows the structures of pyrrolamide compounds

[0038]

[0039]

[0040] Synthesis route such as Figure 4 shown.

[0041] The specific synthesis steps of pyrrolamide derivatives are as follows:

[0042] Pyrrole carboxylic acid 1 (1 eq) and 1-hydroxy-7-azabenzotriazole (HOAt) (1.2 eq) were dissolved in DMF, and different types of amines R2-NH2 (1 eq) were added under ice bath, and then N-methylmorpholine (NMM) (0.7 eq) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) (1.2 eq) were added and stirred to room temperature. The mixture was reacted at room temperature for 12 h. DMF was spin-dried, extracted, and passed through a column to obtain pyrrole amide derivative 2.

[0043] The present invention is described in detail below in conjunction with specific implementation modes.

[0044] Example 1

[0045]

[0046] Preparation of 5-ethyl-N-phenyl-1H-pyrrole-2-carboxamide (G1)

[0047] 5-Ethyl-1H-pyrrole-2-carboxylic acid (69 mg, 0.48 mmol, 1 eq) and HOAt (79 mg, 0.58 mmol, 1.2 eq) were dissolved in DMF (3 mL), stirred for 10 min, then m-fluoroaniline (43 μL, 0.48 mmol, 1 eq) was added, NMM (37 μL, 0.34 mmol, 0.7 eq) was added under ice bath, stirred for 5 min, then EDCI (111 mg, 0.58 mmol, 1.2 eq) was added, stirred to room temperature. The mixture was reacted at room temperature for 12 h. The mixture was extracted with ethyl acetate, the organic phase was concentrated and separated by column chromatography to obtain compound G1 (80 mg, yield 78%) as a light yellow solid. 1 H NMR (300MHz, CDCl3) δ: 9.96 (s, 1H), 7.62 (s, 1H), 7.58 (s, 2H), 7.34 (t, J = 7.7Hz, 2H), 7.11 (t, J = 7.4 Hz,1H),6.65(t,J=3.5Hz,1H),5.99(t,J=3.4Hz,1H),2.65(q,J=7.6Hz,2H),1.23(t,J=7.7Hz,3H). 13 C NMR(101MHz,CD3OD)δ:162.03,141.19,140.09,129.69,125.54,124.75,121.84,113.48,107.50,21.73,14.27.HRMS(ESI,positive):Calcd.for C 13 H 14 N2O[M+Na] + 237.0998,found:237.1002.

[0048] Example 2

[0049]

[0050] Preparation of 5-ethyl-N-(3-fluorophenyl)-1H-pyrrole-2-carboxamide (G2)

[0051] 5-Ethyl-1H-pyrrole-2-carboxylic acid (50 mg, 0.36 mmol, 1 eq) and HOAt (67 mg, 0.43 mmol, 1.2 eq) were dissolved in DMF (2 mL), stirred for 10 min, and then aniline (35 μL, 0.36 mmol, 1 eq) was added. NMM (29 μL, 0.25 mmol, 0.7 eq) was added under ice bath, and then EDCI (95 mg, 0.43 mmol, 1.2 eq) was added after stirring for 5 min. The mixture was stirred to room temperature and reacted at room temperature for 12 h. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound G2 (60 mg, yield 72%) as a light yellow solid. 1 H NMR (400MHz, DMSO-d6) δ: 11.45 (s, 1H), 9.79 (s, 1H), 7.73 (d, J = 12.0Hz, 1H), 7.49 (d, J = 8.3Hz, 1H), 7.33 (q, J=7.8Hz,1H),6.99(s,1H),6.83(t,J=8.5Hz,1H),5.91(s,1H),2.58(q,J=7.6Hz,2H),1.16(t,J=7.6Hz,3H). 13 C NMR (101MHz, DMSO-d6) δ: 163.42, 161.03, 159.36 (d, J = 5.0Hz), 141.40 (d, J = 11.2Hz), 139.89 ,139.73,130.22(d,J=9.7Hz),20.34(d,J=4.2Hz),13.96.HRMS(ESI,positive):Calcd.forC 13 H 13 FN2O[M+Na] + 255.0904,found:255.0914.

[0052] Example 3

[0053]

[0054] Preparation of N-(3-chlorophenyl)-5-ethyl-1H-pyrrole-2-carboxamide (G3)

[0055] 5-Ethyl-1H-pyrrole-2-carboxylic acid (150 mg, 1.08 mmol, 1 eq) and HOAt (176 mg, 1.3 mmol, 1.2 eq) were dissolved in DMF (5 mL), stirred for 10 min, then m-chloroaniline (114 μL, 1.08 mmol, 1 eq) was added, NMM (84 μL, 0.76 mmol, 0.7 eq) was added under ice bath, stirred for 5 min, then EDCI (250 mg, 1.3 mmol, 1.2 eq) was added, stirred to room temperature, and reacted at room temperature for 12 h. Extracted with ethyl acetate, the organic phase was concentrated and separated by column chromatography to obtain compound G3 (139 mg, yield 52%) as a brown solid. 1 H NMR (400MHz, CDCl3) δ: 10.33 (s, 1H), 7.84 (t, J = 2.0Hz, 1H), 7.70 (s, 1H), 7.42-7.40 (m, 1H), 7.26 (t, J = 8.3Hz ,1H),7.11–7.09(m,1H),6.72–6.70(m,1H),6.03–6.01(m,1H),2.70(q,J=7.6Hz,2H),1.25(t,J=7.6Hz,3H). 13 C NMR (101MHz, CDCl3) δ: 159.55, 141.11, 139.31, 134.73, 130.01, 124.03, 123.86, 120.16, 117.98, 111.06, 107.10, 21.07, 13.60.

[0056] Example 4

[0057]

[0058] Preparation of N-(3-bromophenyl)-5-ethyl-1H-pyrrole-2-carboxamide (G4)

[0059] 5-Ethyl-1H-pyrrole-2-carboxylic acid (72 mg, 0.52 mmol, 1 eq) and HOAt (71 mg, 0.62 mmol, 1.2 eq) were dissolved in DMF (3 mL), stirred for 10 min, then m-bromoaniline (47 μL, 0.52 mmol, 1 eq) was added, NMM (34 μL, 0.36 mmol, 0.7 eq) was added under ice bath, stirred for 5 min, then EDCI (100 mg, 0.62 mmol, 1.2 eq) was added, stirred to room temperature, and reacted at room temperature for 12 h. Extracted with ethyl acetate, the organic phase was concentrated and separated by column chromatography to obtain compound G4 (52 mg, yield 34%) as a light brown solid. 1H NMR (400MHz, CDCl3) δ: 10.27 (s, 1H), 7.95 (t, J = 2.0 Hz, 1H), 7.66 (s, 1H), 7.45 (d, J = 8.0, 1H), 7.24–7. 16(m,2H),6.68(t,J=3.1Hz,1H),6.00(t,J=3.3Hz,1H),2.67(q,J=7.6Hz,2H),1.23(t,J=7.6Hz,3H). 13 C NMR (101MHz, CDCl3) δ: 159.54, 141.10, 139.46, 130.31, 126.97, 123.86, 122.99, 122.75, 118.50, 111.08, 107.12, 21.08, 13.60.

[0060] Example 5

[0061]

[0062] Preparation of 5-ethyl-N-(m-tolyl)-1H-pyrrole-2-carboxamide (G5)

[0063] 5-Ethyl-1H-pyrrole-2-carboxylic acid (100 mg, 0.72 mmol, 1 eq) and HOAt (118 mg, 0.86 mmol, 1.2 eq) were dissolved in DMF (4 mL), stirred for 10 min, then m-methylaniline (77 μL, 0.72 mmol, 1 eq) was added, NMM (56 μL, 0.50 mmol, 0.7 eq) was added under ice bath, stirred for 5 min, then EDCI (166 mg, 0.86 mmol, 1.2 eq) was added, stirred to room temperature, and reacted at room temperature for 12 h. Extracted with ethyl acetate, the organic phase was concentrated and separated by column chromatography to obtain compound G5 (148 mg, yield 90%) as a light brown solid. 1 H NMR(300MHz, CDCl3)δ:9.99(s,1H),7.56(s,1H),7.46(s,1H),7.41–7.38(m,1H),7.22(t,J=7.8Hz,1H),6.93(d,J=7 .5Hz,1H),6.63(t,J=3.8,2.6Hz,1H),5.99(t,J=3.4,1H),2.66(q,J=7.6Hz,2H),2.35(s,3H),1.23(t,J=7.6Hz,3H). 13C NMR(101MHz, CDCl3)δ:159.64,140.46,138.93,138.07,128.85,124.84,124.37,120 .79,117.25,110.52,106.79,21.57,20.98,13.67.HRMS(ESI,positive):Calcd.forC 14 H 16 N2O[M+Na] + 251.1155,found:251.1164.

[0064] Example 6

[0065]

[0066] Preparation of 5-ethyl-N-(3-methoxyphenyl)-1H-pyrrole-2-carboxamide (G6)

[0067] 5-Ethyl-1H-pyrrole-2-carboxylic acid (100 mg, 0.72 mmol, 1 eq) and HOAt (118 mg, 0.86 mmol, 1.2 eq) were dissolved in DMF (4 mL), stirred for 10 min, then m-methoxyaniline (81 μL, 0.72 mmol, 1 eq) was added, NMM (56 μL, 0.50 mmol, 0.7 eq) was added under ice bath, stirred for 5 min, then EDCI (166 mg, 0.86 mmol, 1.2 eq) was added, stirred to room temperature, and reacted at room temperature for 12 h. Extracted with ethyl acetate, the organic phase was concentrated and separated by column chromatography to obtain compound G6 (109 mg, yield 62%) as a light brown solid. 1 H NMR(400MHz, CDCl3)δ:9.81(s,1H),7.59(s,1H),7.36(t,J=2.2Hz,1H),7.23(t,J=8.1Hz,1H),7.10–7.07( m,1H),6.69–6.64(m,2H),5.99(t,J=3.0Hz,1H),3.81(s,3H),2.65(q,J=7.6Hz,2H),1.23(t,J=7.6Hz,3H). 13 C NMR(101MHz, CDCl3)δ:160.24,159.59,140.46,139.36,129.75,124.27,112.34,110 .63,109.79,106.93,105.91,55.39,21.01,13.60.HRMS(ESI,positive):Calcd.for C 14 H 16N2O2[M+Na] + 267.1104,found:267.1112.

[0068] Example 7

[0069]

[0070] Preparation of 5-ethyl-N-(2-methoxyphenyl)-1H-pyrrole-2-carboxamide (G7)

[0071] 5-Ethyl-1H-pyrrole-2-carboxylic acid (100 mg, 0.72 mmol, 1 eq) and HOAt (118 mg, 0.86 mmol, 1.2 eq) were dissolved in DMF (4 mL), stirred for 10 min, then o-methylaniline (77 μL, 0.72 mmol, 1 eq) was added, NMM (56 μL, 0.50 mmol, 0.7 eq) was added under ice bath, stirred for 5 min, then EDCI (166 mg, 0.86 mmol, 1.2 eq) was added, stirred to room temperature, and reacted at room temperature for 12 h. Extracted with ethyl acetate, the organic phase was concentrated and separated by column chromatography to obtain compound G7 (121 mg, yield 69%) as a light green solid. 1 HNMR(400MHz, CDCl3)δ:10.03(s,1H),8.47(dd,J=7.8,1.8Hz,1H),8.26(s,1H),7.06–7.02(m,1H),7.00–6.96(m,1H),6.91(d d,J=7.9,1.5Hz,1H),6.66(t,,J=3.1Hz,1H),6.00(t,J=3.2Hz,1H),3.92(s,3H),2.69(q,J=7.6Hz,2H),1.25(t,J=7.6Hz,3H). 13 C NMR (101MHz, CDCl3) δ: 158.35, 146.77, 139.34, 126.90, 123.61, 122.07, 119.86, 118.52, 109.30, 108.84, 105.53, 54.72, 19.89, 12.71.

[0072] Example 8

[0073]

[0074] Preparation of 5-ethyl-N-(4-methoxyphenyl)-1H-pyrrole-2-carboxamide (G8)

[0075] 5-Ethyl-1H-pyrrole-2-carboxylic acid (100 mg, 0.72 mmol, 1 eq) and HOAt (118 mg, 0.86 mmol, 1.2 eq) were dissolved in DMF (4 mL), stirred for 10 min, p-methylaniline (77 μL, 0.72 mmol, 1 eq) was added, NMM (56 μL, 0.50 mmol, 0.7 eq) was added under ice bath, stirred for 5 min, EDCI (166 mg, 0.86 mmol, 1.2 eq) was added, stirred to room temperature, and reacted at room temperature for 12 h. Extracted with ethyl acetate, the organic phase was concentrated and separated by column chromatography to obtain compound G8 (144 mg, yield 82%) as a light yellow solid. 1 H NMR(400MHz,DMSO-d6)δ:11.36(s,1H),9.51(s,1H),7.64-7.60(m,2H),6.92–6.90(m,2H),6.89 -6.88(m,1H),5.88(t,J=3.0Hz,1H),3.73(s,3H),2.57(q,J=7.6Hz,2H),1.16(t,J=7.6Hz,3H). 13 C NMR (100MHz, DMSO-d6) δ: 159.06, 155.02, 138.92, 132.63, 124.71, 121.47, 113.77, 111.38, 105.77, 55.18, 20.32, 14.01.

[0076] Example 9

[0077]

[0078] Preparation of N-cyclohexyl-5-ethyl-1H-pyrrole-2-carboxamide (G9)

[0079] 5-Ethyl-1H-pyrrole-2-carboxylic acid (70 mg, 0.50 mmol, 1 eq) and HOAt (83 mg, 0.60 mmol, 1.2 eq) were dissolved in DMF (3 mL), stirred for 10 min, cyclohexylamine (58 μL, 0.50 mmol, 1 eq) was added, NMM (39 μL, 0.35 mmol, 0.7 eq) was added under ice bath, stirred for 5 min, EDCI (104 mg, 0.60 mmol, 1.2 eq) was added, stirred to room temperature, and reacted at room temperature for 12 h. Extracted with ethyl acetate, the organic phase was concentrated and separated by column chromatography to obtain compound G9 (90 mg, yield 82%) as a light yellow solid. 1H NMR(300MHz, CDCl3)δ:9.92(s,1H),6.44(t,J=3.2Hz,1H),5.91(t,J=3.3Hz,1H),5.69(d,J=8.3Hz,1H),3.98–3.86(m,1H ),2.65(q,J=7.5Hz,2H),2.02–1.97(m,2H),1.78-1.71(m,2H),1.61–1.41(m,1H),1.45–1.32(m,2H),1.27-1.14(m,6H). 13 CNMR (101MHz, CDCl3) δ160.72,139.00,124.61,108.90,106.15,48.21,33.53,25.67,25.13,20.97,13.87.

[0080] Example 10

[0081]

[0082] Preparation of 5-ethyl-N-(2-naphthyl)-1H-pyrrole-2-carboxamide (G10)

[0083] 5-Ethyl-1H-pyrrole-2-carboxylic acid (150 mg, 1.08 mmol, 1 eq) and HOAt (176 mg, 1.3 mmol, 1.2 eq) were dissolved in DMF (5 mL), stirred for 10 min, 2-naphthylamine (146 μL, 1.08 mmol, 1 eq) was added, NMM (84 μL, 0.76 mmol, 0.7 eq) was added under ice bath, stirred for 5 min, EDCI (250 mg, 1.3 mmol, 1.2 eq) was added, stirred to room temperature, and reacted at room temperature for 12 h. Extracted with ethyl acetate, the organic phase was concentrated and separated by column chromatography to obtain compound G10 (147 mg, yield 52%) as a white solid. 1 H NMR(400MHz, DMSO-d6)δ:11.43(s,1H),9.81(s,1H),8.35(s,1H),7.90–7.77(m,4H),7.46(t,J=7.6H z,1H),7.38(t,J=7.6Hz,1H),7.04(s,1H),5.94(s,1H),2.61(q,J=7.7Hz,2H),1.19(t,J=7.6Hz,3H). 13C NMR(101MHz,DMSO-d6)δ:159.38,139.44,137.27,133.47,129.57,128.07,127.45 ,127.17,126.29,124.55,124.34,120.78,115.58,112.04,105.96,20.30,13.92.

[0084] Embodiment 11

[0085]

[0086] Preparation of N-phenyl-1H-pyrrole-2-carboxamide (G11)

[0087] 1H-pyrrole-2-carboxylic acid (60 mg, 0.54 mmol, 1 eq) and HOAt (85 mg, 0.65 mmol, 1.2 eq) were dissolved in DMF (3 mL), stirred for 10 min, and then aniline (47 μL, 0.54 mmol, 1 eq) was added. NMM (41 μL, 0.38 mmol, 0.7 eq) was added under ice bath, and then EDCI (120 mg, 0.65 mmol, 1.2 eq) was added after stirring for 5 min, and stirred to room temperature. The mixture was reacted at room temperature for 12 h, extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound G11 (97 mg, yield 96%) as a white solid. 1 H NMR(400MHz,CD3OD)δ:7.66–7.62(m,2H),7.34–7.29(m,2H),7.09(ddt,J=8.6,7.3,1.2Hz, 1H), 7.02(dd,J=3.8,1.4Hz,1H), 6.97(dd,J=2.6,1.4Hz,1H), 6.22(dd,J=3.8,2.6Hz,1H). 13 C NMR(100MHz,CD3OD)δ:139.90,129.72,127.06,124.95,123.65,121.95,112.58,110.45.HRMS(ESI,positive):Calcd.for C 11 H 10 N2O[M+H] + 187.0866,found:187.0865.

[0088] Example 12

[0089]

[0090] Preparation of N-(3-bromophenyl)-1H-pyrrole-2-carboxamide (G12)

[0091] 1H-pyrrole-2-carboxylic acid (60 mg, 0.54 mmol, 1 eq) and HOAt (85 mg, 0.65 mmol, 1.2 eq) were dissolved in DMF (3 mL), stirred for 10 min, then m-bromoaniline (59 μL, 0.54 mmol, 1 eq) was added, NMM (41 μL, 0.38 mmol, 0.7 eq) was added under ice bath, stirred for 5 min, then EDCI (120 mg, 0.65 mmol, 1.2 eq) was added, and stirred to room temperature. The mixture was reacted at room temperature for 12 h. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound G12 (120 mg, yield 84%) as a light yellow solid. 1 H NMR(400MHz,CD3OD)δ:8.01-8.00(m,1H),7.60-7.57(m,1H),7.24-7.19(m,2H),7. 03(dd,J=3.8,1.4Hz,1H), 6.99(dd,J=2.5,1.4Hz,1H), 6.22(dd,J=3.8,2.5Hz,1H). 13 C NMR(100MHz,CD3OD)δ:161.80,141.69,131.23,127.47,126.79,124.27,124.00,123.20,120.01,112.83,110.53.HRMS(ESI,positive):Calcd.forC 11 H9BrN2O[M+Na] + 286.9790,found:286.9795.

[0092] Example 13

[0093]

[0094] Preparation of 5-methyl-N-phenyl-1H-pyrrole-2-carboxamide (G13)

[0095] 5-Methyl-1H-pyrrole-2-carboxylic acid (60 mg, 0.48 mmol, 1 eq) and HOAt (76 mg, 0.58 mmol, 1.2 eq) were dissolved in DMF (3 mL), stirred for 10 min, and then aniline (44 μL, 0.48 mmol, 1 eq) was added. NMM (37 μL, 0.34 mmol, 0.7 eq) was added under ice bath, and then EDCI (111 mg, 0.58 mmol, 1.2 eq) was added after stirring for 5 min, and stirred to room temperature. The mixture was reacted at room temperature for 12 h. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound G13 (80 mg, yield 83%) as a light brown solid. 1 H NMR (400MHz, CD3OD) δ7.64–7.60(m,2H),7.32–7.27(m,2H),7.09-7.04(m,1H),6.91(d,J=3.7Hz,1H),5.91(d,J=3.7Hz,1H),2.27(s,3H). 13 C NMR(100MHz,CD3OD)δ161.95,140.06,134.69,129.68,125.62,124.75,121.84,113.58,109.13,12.84.HRMS(ESI,positive):Calcd.for C 12 H 12 N2O[M+Na] + 223.0842,found:223.0847.

[0096] Embodiment 14

[0097]

[0098] Preparation of 5-isopropyl-N-phenyl-1H-pyrrole-2-carboxamide (G14)

[0099] 5-isopropyl-1H-pyrrole-2-carboxylic acid (58 mg, 0.38 mmol, 1 eq) and HOAt (62 mg, 0.45 mmol, 1.26 eq) were dissolved in DMF (3 mL), stirred for 10 min, and then aniline (35 μL, 0.38 mmol, 1 eq) was added. NMM (30 μL, 0.27 mmol, 0.7 eq) was added under ice bath, and then EDCI (87 mg, 0.45 mmol, 1.2 eq) was added after stirring for 5 min. The mixture was stirred to room temperature and reacted at room temperature for 12 h. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound G14 (30 mg, yield 35%) as a white solid. 1H NMR (400MHz, CDCl3) δ9.66 (s, 1H), 7.67–7.50 (m, 3H), 7.34 (t, J = 7.4Hz 2H), 7.11 (t, J = 7.4Hz, 1H), 6.65 (t, J = 2.5Hz, 1H), 6.00 (t, J = 3.3Hz, 1H), 2.95 (m, 1H), 1.25 (d, J = 7.0Hz, 6H).

[0100] Embodiment 15

[0101]

[0102] Preparation of N,5-diphenyl-1H-pyrrole-2-carboxamide (G15)

[0103] 5-phenyl-1H-pyrrole-2-carboxylic acid (50 mg, 0.27 mmol, 1 eq) and HOAt (44 mg, 0.32 mmol, 1.26 eq) were dissolved in DMF (3 mL), stirred for 10 min, and then aniline (24 μL, 0.27 mmol, 1 eq) was added. NMM (21 μL, 0.19 mmol, 0.7 eq) was added under ice bath, and then EDCI (62 mg, 0.32 mmol, 1.2 eq) was added after stirring for 5 min, and stirred to room temperature, and reacted at room temperature for 12 h. Extracted with ethyl acetate, the organic phase was concentrated and separated by column chromatography to obtain compound G15 (40 mg, yield 57%) as a white solid. 1 H NMR (400MHz, CDCl3) δ10.19(s,1H),7.69(s,1H),7.60(d,J=8.0Hz,2H),7.54(d,J=7.0Hz,2H),7.29(t ,J=7.8Hz,2H),7.24–7.19(m,3H),7.09(t,J=7.4Hz,1H),6.77(t,J=3.2Hz,1H),6.53(t,J=3.4Hz,1H). 13 C NMR (101MHz, CDCl3) δ159.31,137.91,136.84,131.42,129.23,129.03,127.65,126.52,124.89,124.35,120.19,111.50,107.80.

[0104] Example 16

[0105]

[0106] Preparation of 4,5-dimethyl-N-phenyl-1H-pyrrole-2-carboxamide (G16)

[0107] 4,5-Dimethyl-1H-pyrrole-2-carboxylic acid (100 mg, 0.72 mmol, 1 eq) and HOAt (118 mg, 0.86 mmol, 1.2 eq) were dissolved in DMF (4 mL), stirred for 10 min, and then aniline (65 μL, 0.72 mmol, 1 eq) was added. NMM (56 μL, 0.50 mmol, 0.7 eq) was added under ice bath, and then EDCI (166 mg, 0.86 mmol, 1.2 eq) was added after stirring for 5 min, and stirred to room temperature, and reacted at room temperature for 12 h. Extracted with ethyl acetate, the organic phase was concentrated and separated by column chromatography to obtain compound G16 (70 mg, yield 45%) as a white solid. 1 H NMR(400MHz, CDCl3)δ9.78(s,1H),7.61(s,1H),7.59(s,1H),7.45(s,1H),7.34(t,J =7.9Hz,2H),7.11(t,J=7.4Hz,1H),5.81(d,J=2.9Hz,1H),2.44(s,3H),2.23(s,3H). 13 C NMR (101MHz, CDCl3) δ160.17,138.26,132.23,129.13,124.04,121.58,121.29,120.17,111.64,13.76,12.98.

[0108] Embodiment 17

[0109]

[0110] Preparation of 4-bromo-N-phenyl-1H-pyrrole-2-carboxamide (G17)

[0111] 4-Bromo-1H-pyrrole-2-carboxylic acid (50 mg, 1.05 mmol, 1 eq) and HOAt (172 mg, 1.26 mmol, 1.26 eq) were dissolved in DMF (8 mL), stirred for 10 min, and then aniline (95 μL, 1.05 mmol, 1 eq) was added. NMM (82 μL, 0.74 mmol, 0.7 eq) was added under ice bath, and then EDCI (242 mg, 1.26 mmol, 1.2 eq) was added after stirring for 5 min. The mixture was stirred to room temperature and reacted at room temperature for 12 h. The mixture was extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to obtain compound G17 (96 mg, yield 35%) as a white solid. 1H NMR (400MHz, CD3OD) δ7.65-7.61(m,2H),7.34–7.29(m,2H),7.11-7.08(m,1H),7.03(d,J=1.6Hz,1H),6.98(d,J=1.6Hz,1H). 13 C NMR (101MHz, CD3OD) δ160.66,139.64,129.75,127.70,125.18,123.47,121.95,113.97,97.65.

[0112] Performance Testing

[0113] 1. Bioactivity screening

[0114] (1) GloSensorTM cAMP Assay measures cAMP content:

[0115] cAMP is a key signaling molecule for many G protein-coupled receptors. The accumulation level of cAMP is mainly tested using GloSensor, a bioluminescence-based biosensor (Promega) that can directly detect intracellular cAMP. The principle is to insert a cAMP binding domain into the N-terminus and C-terminus of firefly luciferase through genetic engineering technology to make 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.

[0116] The cAMP accumulation assay was used to test the functional activity of the target compounds on β2AR and to elucidate whether the new compounds are negative allosteric modulators (NAMs) of β2AR.

[0117] 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 transfected into HEK293T cells simultaneously using FuGene transfection reagent (Promega). After 48 hours, the transfected cells were washed with CO2-independent medium and incubated with a balancing 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 hour and then at room temperature for 1 hour, the bioluminescent signal was detected using a multifunctional microplate reader until a steady-state baseline signal was obtained. Then, different concentration gradients of the new derivative and the control compound Cmpd-15 were added to the cells, and the positive control ISO (final concentration 1nM to 100μM) was added after incubation at 37°C for 30 minutes. The changes in bioluminescence were read using a microplate reader.

[0118] In general, when there is no substituent at the fifth position of the pyrrole ring, the allosteric activity of the compound is poor. When the fifth position substituent is an electron-donating methyl or ethyl group, the allosteric activity is higher than that of the lead compound Cmpd-15, and the activity of ethyl substitution is higher than that of methyl. When the fifth position is an electron-withdrawing isopropyl, the allosteric activity is the highest, which is more than twice that of Cmpd-15. When the fifth position is substituted with a benzene ring, the allosteric activity is slightly higher than that of Cmpd-15. When the structure of the amine is aniline, the activity is the highest. When there is an electron-withdrawing group on the aniline, the activity decreases as the electronegativity of the electron-withdrawing group increases. When it is substituted with an electron-donating group, the activity is generally higher than that of Cmpd-15. In particular, substitution at different positions of aniline also has a significant effect on the activity. The meta-position has the highest activity, followed by the ortho-position, and the para-position has no activity. When the structure of the amine is naphthylamine and cyclohexylamine, the allosteric activity is generally average.

[0119] Table 2 is a list of the comparison results of the allosteric activity of the synthesized compounds relative to Cmpd-15

[0120]

[0121] “-” indicates that the compound has no allosteric antagonism.

[0122] (2) Allosteric antagonism mechanism studies

[0123] The present invention also uses the cAMP accumulation test to conduct further allosteric antagonism mechanism research, testing whether the target compounds can allosterically regulate the functional activity of the β2AR endogenous ligand ISO to determine whether they are β2AR negative allosteric modulators (NAM). Specifically, first add the G1 compound provided by the present invention in a multiple relationship of concentration to the cells, the concentration is (1nM~100μM), and after incubation at 37°C for 30 minutes, add different concentration gradients of positive control ISO (final concentration 1nM~100μM) to the cells to test whether the change in bioluminescence at this time shows a concentration-dependent limited downward trend. The specific experimental steps are the same as the above Glosensor cAMP accumulation test. Take G1 as an example (such as Figure 5As shown in the figure, when the concentration of compound G1 reaches 30 μM, the curve of ISO shows a significant downward shift and almost reaches the maximum downward limit of the dose-dependent regulation of ISO functional activity. This indicates that the IC50 value of compound A101 may be between 15 μM and 30 μM, which is specifically manifested as a sudden drop in the concentration curve. When the concentration of G1 increases from 60 μM to 120 μM, the downward shift of the concentration curve of ISO is very weak. This phenomenon explains that with the increase in the concentration of compound G1, the concentration-dependent curve of ISO shows a limited downward shift, indicating that the pyrrolamide derivative G1 can effectively negatively allosterically regulate the functional activity of the β2AR endogenous ligand ISO. This allosteric regulation phenomenon is consistent with the reported allosteric antagonistic regulation mechanism. The ISO concentration-dependent curves of the other pyrrolamide derivatives G14 allosterically regulated (as shown in the figure) Figure 6 The results of G1 are consistent with those of G1, that is, the newly synthesized pyrrolamide derivatives are all negative allosteric modulators (NAMs) of β2AR.

[0124] The present invention uses the GloSensor cAMP accumulation experiment to test the functional activity of the target compound on the β2AR in the G protein-dependent signaling pathway 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 the compounds G1, G2, G5, G6, G13, G14, G15, and G16 in the table have good antagonistic activity on β2AR and can be used as allosteric antagonists of β2AR.

[0125] Those skilled in the art can understand that the above-mentioned embodiments are specific examples for implementing the present application, and in practical applications, various changes can be made to them in form and detail without departing from the spirit and scope of the present application. Any person skilled in the art can make their own changes and modifications without departing from the spirit and scope of the present application, so the scope of protection of the present application shall be based on the scope defined in the claims.

Claims

1. A pyrrolamide derivative, characterized in that: The structural formula of the pyrrolamide derivatives is shown in Formula I: Wherein, R1=H, Me, Et, i-Pr, Ph; R2 is one of the following structural formulas:

2. The method for preparing the pyrrolamide derivatives according to claim 1, characterized in that: The preparation method comprises: dissolving pyrrole carboxylic acid and 1-hydroxy-7-azabenzotriazole (HOAt) in DMF, adding different types of amines R2-NH2 (1 eq) under ice bath, then adding N-methylmorpholine (NMM) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), stirring continuously to room temperature, and reacting at room temperature for several hours; spin-drying the DMF, extracting, and column-passing to obtain the pyrrole amide derivatives.

3. The method for preparing pyrrolamide derivatives according to claim 2, characterized in that: The pyrrole carboxylic acid includes 5-ethyl-1H-pyrrole-2-carboxylic acid, 1H-pyrrole-2-carboxylic acid, 5-methyl-1H-pyrrole-2-carboxylic acid, 5-isopropyl-1H-pyrrole-2-carboxylic acid, 5-phenyl-1H-pyrrole-2-carboxylic acid, 4,5-dimethyl-1H-pyrrole-2-carboxylic acid, and 4-bromo-1H-pyrrole-2-carboxylic acid.

4. The method for preparing the pyrrolamide derivatives according to claim 2, characterized in that: When the amount of the pyrrole carboxylic acid used is 1 eq, 1-hydroxy-7-azabenzotriazole is 1.2 eq, different types of amines R2-NH2 are 1 eq, N-methylmorpholine is 0.7 eq, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1.2 eq.

5. The method for preparing pyrrolamide derivatives according to claim 2, characterized in that: The reaction time at room temperature is 12 hours.

6. Use of the pyrrolamide derivatives according to claim 1 as β2-adrenaline receptor allosteric antagonists.

7. Use of the pyrrolamide derivatives according to claim 1 in the preparation of β2-adrenaline receptor allosteric antagonists.

8. The use according to claim 7, characterized in that: The pyrrolamide derivatives are used as active ingredients and a pharmaceutically acceptable carrier to prepare a pharmaceutical preparation.

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