A pyrrolamide derivative, its preparation method, and its application as an allosteric antagonist of β2-adrenergic receptors.

Abstract

This invention belongs to the field of medicinal chemistry, specifically disclosing a pyrrolamide derivative, its preparation method, and its application as an allosteric antagonist of the β2-adrenergic receptor. The pyrrolamide derivative of this invention has the structure shown in Formula I, wherein R1 is ethyl, methyl, isopropyl, etc.; and R2 is a fatty amine, aniline, m-bromoaniline, benzylamine, etc. Pharmacological results show that the pyrrolamide derivative of this invention has good antagonistic activity against β2AR and can negatively allosterically regulate the functional activity of the ortho-agonist β2AR agonist isoproterenol (ISO).

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, and specifically relates to a pyrrolamide derivative, its preparation method, and its application as an allosteric antagonist of β2-adrenergic receptor. Background Technology

[0002] G protein-coupled receptors (GPCRs) are membrane receptor proteins located on the cell surface that translate extracellular signals into crucial physiological effects. Their endogenous ligands include odorants, hormones, neurotransmitters, chemokines, and a wide range of substances from photons, amines, carbohydrates, lipids, peptides to proteins. They are the most numerous and diverse of all cell surface receptors. GPCRs are widely distributed in various eukaryotes. They first bind to G proteins (guanine nucleotide-binding proteins), regulating the activity of corresponding intracellular enzymes and generating a second messenger. This second messenger then transmits the ligand signal from the extracellular space to the intracellular space, thereby participating in the regulation of related intracellular biological processes and fulfilling its function.

[0003] With the deepening research into the structure and function of GPCRs, an increasing number of GPCR-based targeted drugs are being developed and applied in the treatment of major diseases such as central nervous system diseases, diabetes, and cancer. GPCRs participate in numerous physiological activities and signal regulation processes within the body, forming a systematic signal transduction network. Abnormalities in GPCR-related signal transduction pathways or signal molecules can lead to imbalances in the body's internal environment, thereby triggering diseases such as rheumatoid arthritis, autoimmune diseases, liver damage, and cancer. Of the 826 human GPCRs, approximately 350 non-olfactory members are considered druggable, of which 165 are validated drug targets. Statistics show that 527 drugs approved by the Food and Drug Administration (FDA) and 1060 drug candidates currently in clinical trials target GPCRs.

[0004] Endogenous ligands, along with conventional agonists, antagonists, and inverse agonists, typically occupy orthoallic binding sites. These binding sites are highly conserved within the AGPCR family, and even more so among individual subtypes. This severely hinders the development of selective orthoallic agonists and antagonists. However, proteins, like receptors, ion channels, and enzymes, can possess additional allosteric binding sites. Allosteric modulators can enhance ligand subtype selectivity for GPCRs and improve drug efficacy. Furthermore, the low conservation of key amino acids involved in the allosteric regulation of GPCRs provides a structural basis for the development of more selective allosteric modulatory drugs.

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

[0006] However, Cmpd-15 is a poorly water-soluble dipeptide, 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 this invention is to provide a class of pyrrolamide derivatives to develop novel 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 development of new drugs for cardiovascular and cerebrovascular diseases, diabetes, and cancer.

[0008] This invention provides a pyrrolamide derivative, the structural formula of which is shown in Formula I:

[0009]

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

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

[0012]

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

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

[0015] Furthermore, when the amount of the pyrrolic carboxylic acid is 1 eq, 1-hydroxy-7-azabenzotriazole is 1.2 eq, different types of amine R2-NH2 is 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 pyrrolamide derivatives described above as allosteric antagonists of the β2-adrenergic receptor.

[0018] The present invention provides the use of the pyrrolamide derivatives described above in the preparation of β2-adrenergic receptor allosteric antagonist drugs.

[0019] Furthermore, the aforementioned pyrrolamide derivatives are used as active ingredients and prepared into pharmaceutical formulations using pharmaceutically acceptable carriers.

[0020] The beneficial effects of this invention are as follows:

[0021] Most of the novel pyrrole amide derivatives synthesized in this invention possess β2AR allosteric antagonistic activity. Compared with the lead compound Cmpd-15, compounds G1, G2, G6, and G16 exhibit significantly enhanced allosteric antagonistic activity against β2AR. The novel pyrrole derivatives synthesized in this invention have the advantages of simple structure, simple synthetic route, and readily available raw materials, and can provide a basis for the development of new drugs for cardiovascular diseases, asthma, and cancer. Attached Figure Description

[0022] Figure 1 Here is a structural diagram of Cmpd-15;

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

[0024] Figure 3 Design for pyrrole target compounds;

[0025] Figure 4 Synthetic routes for pyrrolamide derivatives;

[0026] Figure 5 ISO dose-response curve mediated by pyrrolamide derivative G1;

[0027] Figure 6 ISO dose-response curve mediated by pyrrolamide derivative G14. Detailed Implementation

[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 heterocyclic ring as its core backbone. The inventors initially used Cmpd-15 as a lead compound and applied a backbone skipping strategy, replacing the peptide structure of Cmpd-15 with a pyrazole ring. While keeping the 2-amino-N-methyl-3-bromophenylpropionamide and 2-cyclohexylphenylacetyl groups unchanged in Cmpd-15, the amino group in the 2-cyclohexylphenylacetyl group and the methylene group in the (4-carbamoyl)benzyl group were cyclized, fusing to form N-phenylacetylpyrazole. Since N-phenylacetyl is readily hydrolyzed and removed from the pyrazole backbone, the inventors replaced the phenylacetyl group with a benzyl group. In this way, the peptide backbone of Cmpd-15 was replaced by a more stable pyrazole backbone. The results showed that most 1-benzyl-3-aryl-pyrazole-5-carbamoamide derivatives exhibited stronger allosteric antagonistic activity against β2AR than Cmpd-15. However, the 1-benzyl-5-arylpyrazole-3-carboxamide derivatives exhibit poor allosteric antagonistic activity against β2AR, or even no allosteric antagonistic activity (X. Guo, et al. Bioorg. Med. Chem. 2024, 108: 117787).

[0029] Furthermore, such as Figure 2 As shown, the inventors simplified the structure of an active 1-benzyl-3-aryl-pyrazole-5-carboxamide derivative by removing the N-benzyl group. The resulting pyrazole derivatives allosterically antagonized the G-protein signaling pathway of β2AR and negatively regulated the agonistic effect of the endogenous ligand isoproterenol (ISO) on β2AR. cAMP accumulation experiments showed that most unsubstituted pyrazole derivatives exhibited significantly better allosteric antagonistic activity against β2AR than their lead compound Cmpd-15, and hold promise as potential precursor compounds for treating new vascular diseases (Chen Xin, et al., Chinese Invention Patent Publication No.: CN115745891A).

[0030] The structures of pyrazole amide derivatives show that the phenylalanine on the right, containing a chiral carbon atom, has significant steric hindrance, which may affect drug-like properties and also results in poor water solubility. Therefore, as... Figure 3 As shown, the inventors designed a method to replace the pyrazole ring with a pyrrole ring, and synthesized new pyrroleamide compounds by simply substituting aniline, benzylamine, or naphthylamine. The synthesized pyrroleamide compounds exhibited further improved activity compared to pyrazole derivatives.

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

[0032]

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

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

[0035]

[0036] The compounds of this invention are obtained by using pyrroles with different substituents as raw materials and undergoing amide coupling reactions with anilines and aliphatic amines 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] Synthetic routes such as Figure 4 As shown.

[0041] The specific steps for synthesizing pyrrolamide derivatives are as follows:

[0042] Pyrroleic acid 1 (1 eq) and 1-hydroxy-7-azabenzotriazole (HOAt) (1.2 eq) were dissolved in DMF. Different types of amines, R2-NH2 (1 eq), were added under ice bath conditions, followed by N-methylmorphorline (NMM) (0.7 eq) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (1.2 eq), and the mixture was stirred until room temperature was reached. The reaction was carried out at room temperature for 12 h. The DMF was evaporated to dryness, extracted, and column-sected to obtain pyrroleamide derivative 2.

[0043] The present invention will now be described in detail with reference to specific embodiments.

[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). After stirring for 10 min, m-fluoroaniline (43 μL, 0.48 mmol, 1 eq) was added, followed by NMM (37 μL, 0.34 mmol, 0.7 eq) under ice bath conditions. After stirring for 5 min, EDCI (111 mg, 0.58 mmol, 1.2 eq) was added, and the mixture was stirred to room temperature. The reaction was allowed to proceed 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 give compound G1 (80 mg, 78% yield) as a pale 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). After stirring for 10 min, aniline (35 μL, 0.36 mmol, 1 eq) was added, followed by NMM (29 μL, 0.25 mmol, 0.7 eq) under ice bath conditions. After stirring for 5 min, EDCI (95 mg, 0.43 mmol, 1.2 eq) was added, and 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 give compound G2 (60 mg, 72% yield) as a pale 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). After stirring for 10 min, 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 conditions. After stirring for 5 min, EDCI (250 mg, 1.3 mmol, 1.2 eq) was added. 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 give compound G3 (139 mg, 52% yield) 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). After stirring for 10 min, m-bromoaniline (47 μL, 0.52 mmol, 1 eq) was added, followed by NMM (34 μL, 0.36 mmol, 0.7 eq) under ice bath conditions. After stirring for 5 min, EDCI (100 mg, 0.62 mmol, 1.2 eq) was added, and 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 give compound G4 (52 mg, 34% yield), 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). After stirring for 10 min, 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 conditions. After stirring for 5 min, EDCI (166 mg, 0.86 mmol, 1.2 eq) was added. 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 give compound G5 (148 mg, 90% yield) 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). After stirring for 10 min, 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 conditions. After stirring for 5 min, EDCI (166 mg, 0.86 mmol, 1.2 eq) was added. 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 give compound G6 (109 mg, 62% yield) 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). After stirring for 10 min, o-methylaniline (77 μL, 0.72 mmol, 1 eq) was added, followed by NMM (56 μL, 0.50 mmol, 0.7 eq) under ice bath conditions. After stirring for 5 min, EDCI (166 mg, 0.86 mmol, 1.2 eq) was added, and 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 give compound G7 (121 mg, 69% yield), 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). After stirring 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 conditions. After stirring for 5 min, EDCI (166 mg, 0.86 mmol, 1.2 eq) was added. 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 give compound G8 (144 mg, 82% yield) as a pale 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). After stirring 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 conditions. After stirring for 5 min, EDCI (104 mg, 0.60 mmol, 1.2 eq) was added. 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 give compound G9 (90 mg, yield 82%) as a pale 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). After stirring 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 conditions. After stirring for 5 min, EDCI (250 mg, 1.3 mmol, 1.2 eq) was added. 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 give compound G10 (147 mg, 52% yield) 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] Example 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). After stirring for 10 min, aniline (47 μL, 0.54 mmol, 1 eq) was added, followed by NMM (41 μL, 0.38 mmol, 0.7 eq) under ice bath conditions. After stirring for 5 min, EDCI (120 mg, 0.65 mmol, 1.2 eq) was added, and the mixture was stirred to room temperature. The reaction was allowed to proceed for 12 h at room temperature. The mixture was then extracted with ethyl acetate, and the organic phase was concentrated and separated by column chromatography to give compound G11 (97 mg, 96% yield) 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). After stirring for 10 min, 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 conditions. After stirring for 5 min, EDCI (120 mg, 0.65 mmol, 1.2 eq) was added, and the mixture was stirred to room temperature. The reaction was allowed to proceed 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 give compound G12 (120 mg, 84% yield) as a pale 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). After stirring for 10 min, aniline (44 μL, 0.48 mmol, 1 eq) was added. NMM (37 μL, 0.34 mmol, 0.7 eq) was added under ice bath conditions. After stirring for 5 min, EDCI (111 mg, 0.58 mmol, 1.2 eq) was added, and the mixture was stirred to room temperature. The reaction was allowed to proceed 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 give compound G13 (80 mg, 83% yield) 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] Example 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). After stirring for 10 min, aniline (35 μL, 0.38 mmol, 1 eq) was added, followed by NMM (30 μL, 0.27 mmol, 0.7 eq) under ice bath conditions. After stirring for 5 min, EDCI (87 mg, 0.45 mmol, 1.2 eq) was added, and 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 give compound G14 (30 mg, 35% yield) 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] Example 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). After stirring for 10 min, aniline (24 μL, 0.27 mmol, 1 eq) was added, followed by NMM (21 μL, 0.19 mmol, 0.7 eq) under ice bath conditions. After stirring for 5 min, EDCI (62 mg, 0.32 mmol, 1.2 eq) was added, and 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 give compound G15 (40 mg, 57% yield) 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). After stirring for 10 min, aniline (65 μL, 0.72 mmol, 1 eq) was added, followed by NMM (56 μL, 0.50 mmol, 0.7 eq) under ice bath conditions. After stirring for 5 min, EDCI (166 mg, 0.86 mmol, 1.2 eq) was added, and 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 give compound G16 (70 mg, 45% yield) 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] Example 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). After stirring for 10 min, aniline (95 μL, 1.05 mmol, 1 eq) was added. NMM (82 μL, 0.74 mmol, 0.7 eq) was added under ice bath conditions. After stirring for 5 min, EDCI (242 mg, 1.26 mmol, 1.2 eq) was added. 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 give compound G17 (96 mg, 35% yield) 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) GloSensor™ cAMP Assay measures cAMP content:

[0115] cAMP is a key signaling molecule in many G protein-coupled receptors. The accumulation level of cAMP is primarily measured using GloSensor, a bioluminescence-based biosensor (Promega) that directly detects intracellular cAMP. The principle is that a cAMP-binding domain is inserted at the N- and C-termini of firefly luciferase through genetic engineering, rendering the enzyme inactive. When cAMP binds to the cAMP-binding domain, the enzyme is activated, thereby oxidizing the substrate luciferin to produce bioluminescence.

[0116] 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.

[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 simultaneously transfected into HEK293T cells using FuGene transfection reagent (Promega). After 48 h, the transfected cells were washed with CO₂-independent medium and then incubated with equilibration medium containing 2% v / v GloSensor cAMP stock solution (dissolved in CO₂-independent medium containing 10% FBS). Incubation was performed at 37°C for 1 h, followed by incubation at room temperature for 1 h. Bioluminescence signals were detected using a multi-mode 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 after incubation at 37°C for 30 min, the positive control ISO (final concentration 1 nM–100 μM) was added. Changes in biofluorescence were read using a microplate reader.

[0118] Generally, when there is no substituent at the fifth position of the pyrrole ring, the allosteric activity of the compound is poor. When the substituent at the fifth position is an electron-donating group (methyl or ethyl), the allosteric activity is higher than that of the lead compound Cmpd-15, with ethyl substitution showing higher activity than methyl. The allosteric activity is highest when the fifth position is an electron-withdrawing group (isopropyl), 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. The highest activity is observed when the amine structure is aniline. When aniline has an electron-withdrawing group, the activity decreases as the electronegativity of the electron-withdrawing group increases. When the substitution is with an electron-donating group, the activity is generally higher than that of Cmpd-15. In particular, the substitution at different positions on aniline also has a significant impact on the activity; meta-substitution shows the highest activity, followed by ortho-substitution, while para-substitution shows no activity. When the amine structure is naphthylamine or cyclohexylamine, the allosteric activity is generally moderate.

[0119] Table 2 lists the results of the comparison of allosteric activities of the synthesized compounds relative to Cmpd-15.

[0120]

[0121] "-" indicates that the compound does not have allosteric antagonistic activity.

[0122] (2) Study on allosteric antagonistic mechanism

[0123] 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 G1 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. Taking G1 as an example (e.g....), Figure 5As shown in the figure, when the concentration of compound G1 reaches 30 μM, the ISO curve shows a significant downward shift, almost reaching the lower limit of the dose-dependent regulation of ISO activity. This indicates that the IC50 value of compound A101 is likely between 15 μM and 30 μM, specifically manifested as a sharp drop in the concentration curve. When the concentration of G1 increases from 60 μM to 120 μM, the downward shift of the ISO concentration curve is very slight. This phenomenon explains that with increasing G1 concentration, the ISO concentration-dependent curve shows a limited downward shift, indicating that the pyrrolamide derivative G1 can effectively negatively allosterically regulate the activity of the β2AR endogenous ligand ISO. This allosteric regulation phenomenon is consistent with previously reported allosteric antagonistic regulatory mechanisms. Other pyrrolamide derivatives, such as G14, allosterically regulate the ISO concentration-dependent curves (e.g., Figure 6 As shown in the figure, the results are consistent with those of G1, that is, the newly synthesized pyrrolamide derivatives are all negative allosteric modulators (NAM) of β2AR.

[0124] This invention uses GloSensor cAMP accumulation experiments to test the functional activity of target compounds on β2AR in the G protein-dependent signaling pathway and to elucidate whether the new compounds can allosterically regulate the functional activity of the endogenous β2AR ligand isoproterenol (ISO). The experimental results show that compounds G1, G2, G5, G6, G13, G14, G15, and G16 in the table have good antagonistic activity against β2AR and can be used as allosteric antagonists of β2AR.

[0125] Those skilled in the art will understand that the above-described embodiments are specific examples of implementing this application, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of this application. Any person skilled in the art can make their own modifications and alterations without departing from the spirit and scope of this application; therefore, the scope of protection of this application should be determined by 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: Formula I; Where R1 = Et, i - Pr; R2 is one of the following structural formulas: 。 2. The method for preparing the pyrrolamide derivative according to claim 1, characterized in that, The preparation method includes: dissolving pyrrolic acid and 1-hydroxy-7-azabenzotriazole in DMF, adding different types of amines R2-NH2 under ice bath conditions, then adding N-methylmorpholine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, stirring continuously to room temperature, and reacting at room temperature for several hours; evaporating the DMF, extracting, and passing through a column to obtain the pyrrolic amide derivative.

3. The method for preparing pyrrolamide derivatives according to claim 2, characterized in that, The pyrrolic carboxylic acids include 5-ethyl-1H-pyrrolic-2-carboxylic acid and 5-isopropyl-1H-pyrrolic-2-carboxylic acid.

4. The method for preparing the pyrrolamide derivative according to claim 2, characterized in that, The amount of pyrrolic acid is 1 eq, 1-hydroxy-7-azabenzotriazole is 1.2 eq, different types of amine R2-NH2 is 1 eq, N-methylmorpholine is 0.7 eq, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1.2 eq.

5. The method for preparing the pyrrolamide derivative according to claim 2, characterized in that, The reaction time at room temperature was 12 hours.

6. The use of the pyrrolamide derivatives as described in claim 1 in the preparation of β2-adrenergic receptor allosteric antagonist drugs.

7. The application according to claim 6, characterized in that, The aforementioned pyrrolamide derivatives are used as active ingredients and pharmaceutically acceptable carriers to prepare pharmaceutical formulations.

Images