Indole allylamine amide derivative as well as preparation method and application thereof

Abstract

The invention belongs to the field of medicinal chemistry, and relates to an indole allylamine amide derivative as well as a preparation method and application thereof. Functional activity screening of G protein dependent signaling pathways is carried out on all the synthesized compounds, and it is found that the new derivatives can be used for preparing beta2-adrenergic receptor allosteric antagonism regulator drugs. Trans-indole allylamine is used as a raw material, amide coupling is performed to obtain the novel indole allylamine amide derivative, and R1 is any one of phenyl, m-methylbenzene, m-methoxyphenyl, m-bromophenyl, m-chlorphenyl, m-fluorophenyl, o-methoxyphenyl, p-methoxyphenyl, naphthalene ring, oxazole, cyclohexyl, cyclopentyl, methyl, ethyl and isopropyl. A biological activity test result shows that the indole allylamine amide derivative disclosed by the invention has relatively good antagonistic activity on beta2AR and can be used for carrying out negative allosteric regulation on the functional activity of isoproterenol.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, and specifically relates to an indole-allylamine amide compound, its synthesis method, and its application as an allosteric antagonist regulator of β2-adrenergic receptors. Background Technology

[0002] G protein-coupled receptors (GPCRs) are one of the most critical families of membrane receptors in cell signal transduction, mediating the body's response to various external stimuli such as light, odor, hormones, neurotransmitters, and chemokines. Currently, over 500 drugs targeting GPCRs have been approved by the FDA, accounting for approximately one-third of all approved drugs, highlighting their central role in life sciences and drug development.

[0003] GPCRs consist of seven transmembrane helices (7TMs). Their extracellular region recognizes ligands and induces transmembrane helical conformational rearrangement, while their intracellular region recruits G proteins or β-arrestin, thereby achieving transmembrane signal transduction. GPCR activation is essentially an allosteric coupling process between ligand binding and effector protein recruitment. Therefore, elucidating the conformational changes of GPCRs is crucial for revealing their physiological functions and disease mechanisms, and for promoting precision drug design.

[0004] The β2-adrenergic receptor (β2AR) is the most thoroughly studied class A GPCR. Traditional drugs primarily target its orthographic binding site, but this pocket is highly conserved across adrenergic receptor subtypes, limiting the subtype selectivity and clinical safety of orthographic antagonists. Allosteric regulation theory proposes that GPCRs possess structurally diverse and less sequence-conserved allosteric sites, offering higher subtype selectivity. Compared to orthographic drugs, allosteric modulators regulate receptor activity by acting on differentiated structural domains, potentially reducing side effects, improving specificity, and decreasing the risk of drug resistance. Therefore, small-molecule antagonists based on β2AR allosteric sites are becoming an important research direction for GPCR-targeted drugs.

[0005] In 2017, our research group collaborated with scientists at Duke University to report the first small-molecule negative allosteric regulator of β2AR, compound 15 (Cmpd-15) (S. Ahn, et al. Proc. Natl. Acad. Sci. USA, 2017, 114: 1708-1713; X. Liu, et al. Nature, 2017, 548: 480-484). However, Cmpd-15 is a dipeptide compound with extremely poor water solubility, exhibiting a half-life of only 2 minutes in mouse liver stability tests, significantly affecting its drug-like properties. Therefore, this project used Cmpd-15 as a lead compound, employing skeletal transition strategies, structural simplification, and bioelectronic isosterism principles for drug design. The synthesized new compound was analyzed using GloSensor... TM The cAMP Accumulation assay was used to screen for bioactivity in classical signaling pathways (G protein signaling) (BF Binkowski, et al. ACS Chem. Biol., 2011, 6: 1193-1197). The aim was to obtain a series of novel indoleamide derivatives with stable and simplified structures, novel skeletons, enhanced allosteric activity, improved water solubility, and metabolic stability as allosteric antagonists of β2AR.

[0006] Previous research by our group revealed that cAMP accumulation experiments showed that most pyrazolamide derivatives exhibited significantly better allosteric antagonism against β2AR than the lead compound Cmpd-15, and the newly derived compounds showed significantly improved water solubility compared to Cmpd-15. However, the phenylalanine moiety on the right side of Cmpd-15 suffers from significant steric hindrance, which may affect the druggability of the compound (Chinese Invention Patent Publication No.: CN115894373A). Indole, an aromatic heterocyclic organic compound containing a six-membered benzene ring and a five-membered nitrogen-containing pyrrole ring, also known as benzopyrrole, plays an important role in drug design. It is widely distributed in nature and exhibits various pharmacological activities such as antidepressant, anticancer, and anti-inflammatory effects. Therefore, further optimization of this structure is of great significance. Summary of the Invention

[0007] The purpose of this invention is to provide a new indole derivative to develop target compounds with high biological activity, stable chemical structure, good water solubility, and novel skeleton, as new allosteric antagonists of β2AR, providing a new direction for the development of new drugs for cardiovascular and cerebrovascular diseases, diabetes and cancer.

[0008] This invention provides an indole-allylamine amide derivative, the structure of which is shown in Formula 1:

[0009] Formula 1; R1 = Me, Et, i-Pr, and one of the following structural formulas: .

[0010] Furthermore, the indoleallylamine amide derivative specifically comprises one of the following structural formulas: .

[0011] The present invention also provides a method for preparing the above-mentioned indole-allylamine amide derivatives, the method comprising: dissolving trans-indole-allylamine protected by tert-butyloxycarbonyl in N,N-dimethylformamide (DMF), adding 1-hydroxy-7-azabenzotriazole (HOAt), stirring at room temperature for 10 min, adding N-methylmorpholine and benzoic acid, fatty acid or heterocyclic acid with different substituents at 0°C, then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), reacting at room temperature, extracting and purifying to obtain an intermediate; dissolving the intermediate in a mixed solution of methanol and water, adding potassium carbonate, reacting and heating under reflux for 2 hours, then evaporating the methanol to dryness, extracting to remove N,N-dimethylformamide, and purifying by silica gel column chromatography to obtain the indole-allylamine amide derivatives.

[0012] Furthermore, the trans-indolylamine protected by the tert-butoxycarbonyl group comprises (E)-3-(1H-indo-3-yl)prop-2-en-1-amine.

[0013] Furthermore, the benzoic acid, fatty acid, or heterocyclic acid with different substituents includes one of benzoic acid, 3-methylbenzoic acid, 3-methoxybenzoic acid, 3-bromobenzoic acid, 3-fluorobenzoic acid, 3-chlorobenzoic acid, 2-methoxybenzoic acid, 4-methoxybenzoic acid, 2-naphthoic acid, oxazol-5-carboxylic acid, cyclohexanecarboxylic acid, cyclopentanecarboxylic acid, formic acid, acetic acid, or isopropionic acid.

[0014] Further, the preparation method is carried out in the following proportions: (1) 1 eq of trans-indopropylamine protected by tert-butyloxycarbonyl is dissolved in N,N-dimethylformamide, 1.2 eq of 1-hydroxy-7-azabenzotriazole is added, and after stirring at room temperature for 10 min, 0.7 eq of N-methylmorpholine and 1.5 eq of benzoic acid, fatty acid or heterocyclic acid with different substituents are added at 0℃, and then 1.2 eq of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is added. After reacting at room temperature for 4 h, the mixture is extracted and purified to obtain the intermediate; (2) 1 eq of the intermediate is dissolved in a mixed solution of methanol and water, 1.2 eq of potassium carbonate is added, and after heating and refluxing for 2 hours, the methanol is evaporated to dryness, N,N-dimethylformamide is extracted and removed, and the mixture is purified by silica gel column chromatography to obtain the indopropylamine amide derivative.

[0015] Furthermore, the eluent used for purification in step (1) is prepared by mixing dichloromethane and anhydrous methanol in a volume ratio of 100:1.

[0016] Furthermore, after the reaction was completed as monitored by TLC, the reaction solution was diluted with ethyl acetate (50 mL), washed with water (50 mL × 3), and then dried with anhydrous sodium sulfate. After removing the solvent by rotary evaporation, the resulting crude solid product was purified by silica gel column chromatography using eluent [V(dichloromethane):V(anhydrous methanol) = 100:1]. The product after column chromatography was concentrated to obtain trans-indopropylamine amide derivatives.

[0017] The present invention provides the use of the indoleallylamine amide derivatives as described above for the preparation of β2-adrenergic receptor (β2AR) allosteric antagonist drugs.

[0018] Furthermore, the indole-allylamine amide derivatives are used to allosterically antagonize the G-protein signaling pathway of β2AR and negatively regulate the agonistic effect of the endogenous agonist isoproterenol on β2AR.

[0019] Furthermore, the aforementioned indoleallylamine amide derivatives are used as active ingredients and pharmaceutically acceptable carriers to prepare pharmaceutical formulations.

[0020] The beneficial effects of this invention are as follows: Most of the novel indole-allylamine amide derivatives synthesized in this invention exhibit β2AR allosteric antagonistic activity. Compared with the lead compound Cmpd-15, the compounds show significantly enhanced allosteric antagonistic activity against β2AR. These novel indole-allylamine amide 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

[0021] Figure 1 Design of indole-allylamine amide derivatives; Figure 2 Synthetic routes for indoleallylamine amide derivatives; Figure 3 The graph shows the linear regression equation for the solubility of JD3. Figure 4 For JD 15 Solubility linear regression equation graph; Figure 5 This is a plot of the ISO dose-response curve mediated by JD3; Figure 6 For JD 15 mediated ISO dose-response curve. Detailed Implementation

[0022] In view of the background technology, the inventors previously used the principles of skeletal transition, structural simplification, and bioelectronic isosterism to replace the pyrazole skeleton with an indole acrylic acid skeleton, and replaced the right-side phenylalanine moiety of Cmpd-15 with substituted aniline, aliphatic amine, and heterocyclic amine, designing and synthesizing a series of indole acrylic acid amide derivatives. They then studied the bioactivity of these new derivatives against β2-AR. The results showed that replacing the peptide core structure of Cmpd-15 with an indole skeleton yielded new compounds with better allosteric antagonistic activity. Specifically, when 3-indolepropionic acid was coupled with aniline, its antagonistic activity was only about half that of Cmpd-15, while when coupled with m-bromoaniline, its antagonistic activity was comparable to Cmpd-15, approximately 1.15 times that of Cmpd-15. This demonstrates that the antagonistic activity of indole derivatives without double bonds is either inferior to or comparable to Cmpd-15, highlighting the necessity of double bonds. (Chinese Invention Patent Publication No.: CN119330869A) The aniline structural unit in indoleacrylamide derivatives enhances the biological activity of these compounds to some extent, but also introduces some weaknesses, such as poor structural stability, increased toxicity, and metabolic problems. Furthermore, the aniline structure is easily oxidized by CYP450 enzymes, leading to poor biological stability in these compounds (CN119330869A). To improve the stability of lead compounds, such as... Figure 1 As shown, based on previous research on indole acrylamide derivatives, this invention attempts to flip the amide bond, change the basic structure to indole allylamine amide derivatives, design and synthesize a series of new derivatives, and study the bioactivity of the new derivatives on β2AR.

[0023] The compounds of this invention are obtained by amide coupling reactions with acids of different substituents using indoleallylamine as a starting material, ultimately yielding a series of novel trans-indoleallylamine amide derivatives, the structures of which are shown in Formula 1: Formula 1. The specific structures of indole-allylamine amide derivatives are shown in Table 1.

[0024] Table 1 shows the specific structures of indole-allylamine amide derivatives.

[0025] Synthetic routes for indole-allylamine amide derivatives are as follows: Figure 2 As shown.

[0026] Step 1: Potassium phthalimide (10.80 mmol, 1 eq) was dissolved in DMF, then tetrabutylammonium bromide (0.32 mmol, 0.03 eq) was added, followed by the slow addition of allyl chloride (10.80 mmol, 1 eq). After the reaction was monitored by TLC, the reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (50 mL) and water (50 mL × 3) to remove DMF. The organic phase was collected, dried over anhydrous sodium sulfate, and then concentrated by rotary evaporation to obtain the crude product. The crude product was purified by silica gel column chromatography with eluent [V(petroleum ether):V(EtOAc) = 8:1] to give compound 2 as a white solid. 1 H NMR (400 MHz, DMSO) δ 7.93 –7.76 (m, 4H), 5.87 (ddt, J = 16.3, 11.2, 5.1 Hz, 1H), 5.13 (s, 1H), 5.09 (d, J = 6.8 Hz, 1H), 4.17 (d, J = 6.6 Hz, 2H). Step 2: 1-Boc-3-bromoindole (6.12 mmol, 1 eq) and compound 2 were dissolved in Et3N (12.24 mmol, 2 eq), followed by the addition of Pd(OAc)2 (0.06 mmol, 0.01 eq) and tri-o-tolylphosphine (0.12 mmol, 0.02 eq). Diatomaceous earth was washed with EtOAc until the product was clean, then evaporated and dried by rotary evaporation. The mixture was extracted three times with 50 mL EtOAc and 50 mL water, and the organic phase was collected, dried over sodium sulfate, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography using a silica gel column chromatography eluent [V(petroleum ether):V(EtOAc) = 80:1] to obtain compound 3. 1 H NMR (400 MHz, DMSO) δ 8.08 (d, J = 8.3 Hz, 1H), 7.90 (dd, J= 5.7, 3.1Hz, 2H), 7.88 – 7.84 (m, 3H), 7.79 (s, 1H), 7.33 (d, J = 8.2 Hz, 1H), 7.26 (t, J = 7.5 Hz, 1H), 4.39 (d, J = 5.7 Hz, 2H), 1.60 (s, 9H). Step 3: Compound 3 (1.09 mmol, 1 eq) was dissolved in anhydrous methanol, and 64% hydrazine hydrate (2.18 mmol, 2 eq) was added. The mixture was heated under reflux at 70 °C overnight. After the reaction was completed by TLC monitoring, the mixture was filtered through diatomaceous earth, the product was washed with anhydrous methanol, and the filtrate was collected to obtain compound 4. 1 H NMR (400 MHz, DMSO) δ 8.08 (d, J =7.9 Hz, 1H), 7.86 (d, J = 7.1 Hz, 1H), 7.72 (s, 1H), 7.36 (t, J = 7.7 Hz, 1H), 7.29 (t, J = 7.6 Hz, 1H), 6.64 (d, J = 16.3 Hz, 1H), 6.45 (dt, J = 16.0, 5.4 Hz,1H), 3.35 (s, 2H), 1.63 (s, 9H). Step 4: Compound 4 (0.64 mmol, 1 eq) was dissolved in 4 mL of DMF, HOAt (0.77 mmol, 1.2 eq) was added and stirred for 10 min, followed by benzoic acid, fatty acid, or heterocyclic acid with different substituents (0.96 mmol, 1.5 eq), then NMM (0.45 mmol, 0.7 eq), and the mixture was stirred in an ice bath for 10 min. EDCI (0.77 mmol, 1.2 eq) was then added, and the reaction mixture was kept at room temperature for 4 h. After the reaction was completed by TLC monitoring, the mixture was extracted three times with ethyl acetate (50 mL) and water (50 mL × 3) to remove DMF. The organic phase was collected, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude product was purified by column chromatography with eluent [V(DCM):V(MeOH) = 100:1] to give intermediate 5.

[0027] Step 5: Intermediate 5 (0.41 mmol, 1 eq) was dissolved in a mixture of methanol and water (3:1), and potassium carbonate (0.49 mmol, 1.2 eq) was added. The mixture was refluxed at 80 °C for 2 hours. After the reaction was completed by TLC monitoring, methanol was evaporated under pressure. The residue was extracted three times with ethyl acetate (50 mL) and water (50 mL x 3) to remove DMF. The organic phase was collected, dried over anhydrous sodium sulfate, and the crude product was purified by silica gel column chromatography with petroleum ether and ethyl acetate (8:1) as the eluent to obtain the target compound, namely the indole-allylamine amide derivative described in this invention.

[0028] For the specific structures of compounds 2, 3, 4, and intermediate 5, please refer to [link / reference needed]. Figure 2 As shown.

[0029] The present invention will now be described in detail with reference to specific embodiments.

[0030] Example 1

[0031] Preparation of (E)-N-(3-(1H-indol-3-yl)allyl)benzamide (JD1): (E)-3-(1H-indol-3-yl)prop-2-en-1-amine (169 mg, 0.64 mmol, 1 eq) was dissolved in 4 mL of DMF. HOAt (0.77 mmol, 1.2 eq) was added and stirred for 10 min. Benzoic acid (0.96 mmol, 1.5 eq) and NMM (0.45 mmol, 0.7 eq) were added under ice bath conditions, and the reaction was carried out for 10 min. Then, EDCI (0.77 mmol, 1.2 eq) was added, and the reaction was maintained at room temperature for 4 h. After the reaction was completed as monitored by TLC, the mixture was extracted three times with ethyl acetate (50 mL) and water (50 mL × 3) to remove DMF. The organic phase was collected, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography with eluent [V(dichloromethane):V(anhydrous methanol) = 100:1] to give intermediate 5a, a pale yellow solid, in 51% yield. Intermediate 5a (0.41 mmol) was dissolved in a mixture of methanol and water (3:1), and potassium carbonate (1.2 eq) was added. The reaction was heated to 80 °C and refluxed for 2 hours. After the reaction was completed as monitored by TLC, the methanol was evaporated to dryness, and the crude product in the flask was extracted three times with ethyl acetate (50 mL) and water (50 mL × 3) to remove DMF. The organic phase was collected, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was purified by silica gel column chromatography with eluent [V(petroleum ether):V(ethyl acetate) = 8:1] to give the target compound JD1 as a white solid with a yield of 49%. 1H NMR (400MHz, DMSO-d6): δ 11.18 (s, 1H), 8.75 (t, J = 5.8 Hz, 1H), 7.92 (d, J = 7.4 Hz, 2H), 7.78 (d, J = 7.9 Hz, 1H), 7.57–7.43 (m, 4H), 7.39 (d, J = 8.0 Hz, 1H), 7.09 (dt, J = 26.0, 7.3 Hz, 2H), 6.72 (d, J = 16.2 Hz, 1H), 6.30 – 6.12 (m,1H), 4.10 (t, J = 5.9 Hz, 2H). Example 2

[0032] Preparation of (E)-N-(3-(1H-indol-3-yl)allyl)-3-methylbenzamide (JD2): The preparation method is the same as in Example 1, except that 3-methylbenzoic acid is used instead of benzoic acid, and the product is a white solid with a yield of 61%. 1 H NMR (400 MHz, DMSO-d6): δ 11.18 (s, 1H), 8.75 (t, J = 5.8 Hz, 1H), 7.92 (d, J = 7.4 Hz, 2H), 7.78 (d, J = 7.9 Hz, 1H), 7.57 – 7.43 (m, 4H), 7.39(d, J = 8.0 Hz, 1H), 7.09 (dt, J = 26.0, 7.3 Hz, 2H), 6.72 (d, J = 16.2 Hz,1H), 6.30 – 6.12 (m, 1H), 4.10 (t, J = 5.9 Hz, 2H). Example 3

[0033] Preparation of (E)-N-(3-(1H-indol-3-yl)allyl)-3-methoxybenzamide (JD3): The preparation method is the same as in Example 1, except that 3-methoxybenzoic acid is used instead of benzoic acid. The product is a white solid with a yield of 63%. 1 H NMR (400 MHz, DMSO-d6): δ 11.20 (s, 1H), 8.75 (t, J = 5.6 Hz, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.55 – 7.45 (m, 3H), 7.38 (t, J = 7.8 Hz, 2H),7.16 – 7.02 (m, 3H), 6.72 (d, J = 16.1 Hz, 1H), 6.20 (dt, J = 16.1, 6.1 Hz, 1H), 4.10 (t, J = 5.9 Hz, 2H), 3.81 (s, 3H). 13 C NMR (101 MHz, DMSO-d6): δ166.12, 159.62, 137.32, 136.45, 129.85, 125.58, 125.55, 124.87, 121.97,119.96, 119.89, 119.86, 117.47, 113.28, 112.81, 112.27, 55.69, 42.55, 21.22,14.54. Example 4

[0034] Preparation of (E)-N-(3-(1H-indol-3-yl)allyl)-3-bromobenzamide (JD4): The preparation method is the same as in Example 1, except that 3-bromobenzoic acid is used instead of benzoic acid. The product is a white solid with a yield of 68%. 1 H NMR (400 MHz, DMSO-d6): δ 11.19 (s, 1H), 8.87 (t, J = 5.5 Hz, 1H),8.10 (s, 1H), 7.92 (d, J = 7.5 Hz, 1H), 7.76 (dd, J= 20.5, 7.9 Hz, 2H), 7.54 –7.35 (m, 3H), 7.09 (dt, J = 26.1, 7.2 Hz, 2H), 6.72 (d, J = 16.1 Hz, 1H), 6.19(dd, J = 15.9, 6.1 Hz, 1H), 4.09 (t, J = 5.9 Hz, 2H). Example 5

[0035] Preparation of (E)-N-(3-(1H-indol-3-yl)allyl)-3-fluorobenzamide (JD5): The preparation method is the same as in Example 1, except that 3-fluorobenzoic acid is used instead of benzoic acid. The product is a white solid with a yield of 54%. 1 H NMR (400 MHz, DMSO-d6): δ 11.18 (s, 1H), 8.75 (t, J = 5.5 Hz, 1H), 7.92 (d, J = 7.4 Hz, 2H), 7.78 (d, J = 7.9 Hz, 1H), 7.54 – 7.47 (m, 3H), 7.39(d, J = 8.1 Hz, 1H), 7.09 (dt, J = 26.0, 7.3 Hz, 2H), 6.72 (d, J = 16.0 Hz, 1H), 6.20 (dt, J = 16.2, 5.8 Hz, 1H), 4.11 (d, J = 5.8 Hz, 2H). Example 6

[0036] Preparation of (E)-N-(3-(1H-indol-3-yl)allyl)-3-chlorobenzamide (JD6): The preparation method is the same as in Example 1, except that 3-chlorobenzoic acid is used instead of benzoic acid. The product is a white solid with a yield of 62%. 1 H NMR (400 MHz, DMSO-d6): δ 8.14 – 8.07 (m, 1H), 7.92 (d, J= 7.9 Hz, 1H), 7.76 (dd, J = 20.8, 8.0 Hz, 2H), 7.51 – 7.36 (m, 3H), 7.09 (dt, J = 25.9, 7.3 Hz, 2H), 6.72 (d, J = 16.0 Hz, 1H), 6.18 (dt, J = 15.8, 6.1 Hz, 1H), 4.09(t, J = 5.8 Hz, 2H). Example 7

[0037] Preparation of (E)-N-(3-(1H-indol-3-yl)allyl)-2-methoxybenzamide (JD7): The preparation method is the same as in Example 1, except that 2-methoxybenzoic acid is used instead of benzoic acid. The product is a white solid with a yield of 65%. 1 H NMR (400 MHz, MeOD): δ 7.94 (dd, J = 7.8, 1.9 Hz, 1H), 7.81 (dd, J = 7.7, 1.3 Hz, 1H), 7.50 (ddd, J = 8.8, 7.3, 1.9 Hz, 1H), 7.36 (dd, J = 8.0,1.3 Hz, 1H), 7.31 (s, 1H), 7.17 – 7.12 (m, 2H), 7.09 – 7.04 (m, 2H), 6.83(dt, J = 16.1, 1.7 Hz, 1H), 6.26 (dt, J = 16.0, 6.3 Hz, 1H), 4.21 (ddd, J = 6.2,4.0, 1.5 Hz, 2H), 3.97 (s, 3H). Example 8

[0038] Preparation of (E)-N-(3-(1H-indol-3-yl)allyl)-4-methoxybenzamide (JD8): The preparation method is the same as in Example 1, except that 4-methoxybenzoic acid is used instead of benzoic acid. The product is a white solid with a yield of 55%. 1H NMR (400 MHz, MeOD): δ 7.87 – 7.83 (m, 2H), 7.35 (dt, J = 8.1,1.0 Hz, 1H), 7.29 (s, 1H), 7.09 (dddd, J = 23.6, 8.1, 7.0, 1.2 Hz, 3H), 6.99 –6.96 (m, 2H), 6.79 (dt, J = 16.0, 1.5 Hz, 1H), 6.25 (dt, J = 16.0, 6.4 Hz, 1H), 4.16 (dd, J = 6.4, 1.5 Hz, 2H), 3.83 (s, 3H). Example 9

[0039] Preparation of (E)-N-(3-(1H-indol-3-yl)allyl)-2-naphthylcarboxamide (JD9): The preparation method was the same as in Example 1, except that 2-naphthoic acid was used instead of benzoic acid to obtain a white solid with a yield of 67%. ¹H NMR (400 MHz, MeOD): δ 8.43 (s, 1H), 8.01 – 7.91 (m, 5H), 7.83 (d, J = 7.8 Hz, 1H), 7.57 (ddd, J = 7.3, 5.2, 1.8 Hz, 3H), 7.37 – 7.31 (m, 2H), 7.16– 7.04 (m, 3H), 6.85 (d, J = 15.9 Hz, 1H), 6.34 – 6.27 (m, 1H), 4.24 (dd, J = 6.5, 1.4 Hz, 2H). Example 10

[0040] (E)-N-(3-(1H-indol-3-yl)allyl)oxazol-5-carboxamide (JD) 10 Preparation of ) The preparation method is the same as in Example 1, except that oxazole-5-carboxylic acid is used instead of benzoic acid to obtain a white solid with a yield of 55%. 1H NMR (400 MHz, MeOD): δ 8.32 (s, 1H), 7.81 – 7.76 (m, 1H), 7.75 (s,1H), 7.38 – 7.33 (m, 1H), 7.30 (s, 1H), 7.09 (dddd, J = 23.8, 8.2, 7.1, 1.2Hz, 3H), 6.80 (dt, J = 15.8, 1.5 Hz, 1H), 6.21 (dt, J = 15.9, 6.5 Hz, 1H), 4.18– 4.13 (m, 2H). Example 11

[0041] (E)-N-(3-(1H-indol-3-yl)allyl)cyclohexaneformamide (JD) 11 Preparation of ) The preparation method is the same as in Example 1, except that cyclohexanecarboxylic acid is used instead of benzoic acid to obtain a white solid with a yield of 79%. H NMR (400 MHz, MeOD): δ 11.17 (s, 1H), 7.99 (t, J = 5.6 Hz, 1H), 7.74 (d, J = 7.9 Hz, 1H), 7.45 (d, J = 2.5 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.18– 7.00 (m, 2H), 6.63 (d, J = 16.0 Hz, 1H), 6.07 (dt, J = 16.0, 6.1 Hz, 1H), 4.02 (d, 2H), 3.33 – 3.25 (m, 1H), 1.74 – 1.65 (m, 2H), 1.51 – 1.41 (dd, 2H),1.06 (t, J = 7.3 Hz, 6H).13C NMR (101 MHz, MeOD) δ 176.04, 137.63, 129.49,127.88, 126.63, 124.00, 122.28, 120.18, 120.04, 116.28, 111.25, 44.59, 43.28,29.57, 25.69, 25.59. Example 12

[0042] (E)-N-(3-(1H-indol-3-yl)allyl)cyclopentaneformamide (JD) 12 Preparation of ) The preparation method is the same as in Example 1, except that cyclopentanecarboxylic acid is used instead of benzoic acid to obtain a white solid with a yield of 66%. 1 H NMR (400 MHz, DMSO-d6): δ 11.17 (s, 1H), 7.99 (t, J = 5.6 Hz, 1H), 7.74(d, J = 7.9 Hz, 1H), 7.45 (d, J = 2.5 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.14 –7.02 (m, 2H), 6.63 (d, J = 16.0 Hz, 1H), 6.07 (dt, J = 16.0, 6.1 Hz, 1H), 3.88– 3.81 (m, 2H), 2.59 (d, J = 7.8 Hz, 1H), 1.67 – 1.60 (m, 4H). Example 13

[0043] (E)-N-(3-(1H-indol-3-yl)allyl)methylformamide (JD) 13 Preparation of ) The preparation method is the same as in Example 1, except that formic acid is used instead of benzoic acid to obtain a white solid product with a yield of 58%. 1 HNMR (400 MHz, MeOD): δ 7.79 (d, J = 7.9 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.29(s, 1H), 7.14 (t, J = 7.5 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 6.74 (d, J = 15.9Hz, 1H), 6.14 (dt, J = 15.8, 6.5 Hz, 1H), 3.96 (d, J = 6.4 Hz, 2H), 2.00 (s, 3H). Example 14

[0044] (E)-N-(3-(1H-indol-3-yl)allyl)ethylformamide (JD) 14 Preparation of ) The preparation method is the same as in Example 1, except that acetic acid is used instead of benzoic acid to obtain a white solid product with a yield of 67%. 1 HNMR (400 MHz, MeOD): δ 7.94 – 7.90 (m, 1H), 7.50 – 7.46 (m, 1H), 7.42 (s,1H), 7.23 (dddd, J = 24.3, 8.2, 7.1, 1.2 Hz, 3H), 6.87 (dt, J = 15.8, 1.5 Hz, 1H), 6.28 (dt, J = 15.9, 6.5 Hz, 1H), 4.10 (ddd, J = 6.4, 4.3, 1.5 Hz, 2H),2.39 (q, J = 7.6 Hz, 2H), 1.30 (t, J = 7.6 Hz, 3H). Example 15

[0045] (E)-N-(3-(1H-indol-3-yl)allyl)isopropylformamide (JD) 15 Preparation of ) The preparation method is the same as in Example 1, except that isopropionic acid is used instead of benzoic acid to obtain a white solid with a yield of 71%. 1 H NMR (400 MHz, MeOD): δ 7.78 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H),7.27 (s, 1H), 7.12 (t, J = 7.5 Hz, 1H), 7.09 – 7.03 (m, 1H), 6.72 (d, J = 15.9Hz, 1H), 6.13 (dt, J = 15.7, 6.4 Hz, 1H), 3.95 (d, J = 6.2 Hz, 2H), 2.48 (hept, J = 7.2 Hz, 1H), 1.14 (d,J = 6.9 Hz, 6H). Bioactivity test cAMP is a key signaling molecule in many G protein-coupled receptors. The accumulation level of cAMP is mainly measured using the GloSensor method, a bioluminescent cAMP biosensor (Promega). Healthy HEK293T cells were seeded in 6-well plates or 35 mm cell culture dishes and incubated at 37°C with 5% CO2 for 24 h to allow cell adhesion. Then, the β2-adrenergic receptor plasmid and pGloSensor™-22FcAMP plasmid were transfected into the cells using transfection reagents and incubated at 37°C with 5% CO2 for 24 h to allow the target gene transcription and expression. Finally, the transfected cells were evenly seeded into 96-well plates and incubated at 37°C with 5% CO2 for 24 h. Finally, the old culture medium in the 96-well plate was discarded, and the cells were washed once with fresh culture medium. Then, a balanced culture medium containing GloSensor™ cAMP Reagent, serum, and CO2-independent medium was added, and the cells were incubated at 37°C in a 5% CO2 incubator for 1-2 hours or until a stable background signal was obtained. Isoproterenol (ISO) was dissolved in DMSO, sterilized, filtered, and used as the stock solution of the compound, diluted with culture medium to different concentration gradients (the concentration of DMSO in the prepared compound was less than or equal to 0.1%). The prepared compound was quickly added to the 96-well plate containing HEK293T cells to begin drug stimulation. The bioluminescent signal rose rapidly after the addition of the compound using a multi-mode microplate reader. When the signal value reached its peak and stopped rising, a 50 µM concentration of the compound was immediately added. The bioluminescent signal was collected immediately using a multi-mode microplate reader, and the bioluminescent signal value increased with increasing compound concentration. Finally, the data were processed using GraphPad Prism8 software, with concentration on the x-axis and signal value on the y-axis to obtain the dose-response curve of the allosteric modulator.

[0046] Table 2. Comparison of allosteric antagonistic activities between indole-allylamine amide derivatives and Cmpd-15 provided in the embodiments of the present invention.

[0047] Note: a The numerical values ​​are expressed as blocking activity relative to Cmpd-15. In the Glosensor cAMP cumulative assay results, the new compounds JD1~JD 15 All of them exhibit β2AR allosteric antagonism. Most of the compounds with allosteric antagonism mentioned above have better or comparable activity than Cmpd-15. Among them, JD3 and JD are the most active compounds. 15The allosteric antagonistic activity was significantly improved compared to Cmpd-15, and compounds JD2 and JD... 11 The allosteric antagonistic activity of the compound was slightly higher than that of the lead compound Cmpd-15, while the activity of the other compounds was not as good as that of Cmpd-15, as shown in Table 2.

[0048] liver metabolic stability study In this invention, human and mouse liver microsomes were selected to evaluate L7 (CN 119330869A) and indole-allylamine amide derivatives JD3 and JD. 15 The in vitro metabolic stability was assessed. The incubation mixture consisted of 0.1 M PBS (pH 7.4), NADPH (2 mM), liver microsomes (0.2 mg / mL), and the test compound (1 μM). The half-life (T0) was determined. 1 / 2 Extraction rate (Eh) and liver extraction ratio (Eh) are two key parameters used to assess the metabolic stability of test compounds. The liver extraction ratio (Eh) refers to the proportion of a compound that passes through the liver (usually due to metabolism). Based on the proportion of the compound removed during a single liver pass, extraction rates are generally categorized as high (>70%), medium (30%-70%), and low (<30%).

[0049] Table 3 Compounds JD3, JD 15 Stability Comparison

[0050] As can be seen from Table 3, JD3 compared to JD 15 Greater stability and longer half-life. Therefore, compared with isopropyl, meta-substituted benzyloxy groups have higher metabolic stability in organisms (especially in liver microsomes) due to their aromatic conjugated system, stronger steric hindrance, and more stable ether bonds, and thus a longer half-life.

[0051] Solubility Experiment In this invention, the compounds JD3 and JD, which are the most active among indole-allylamine amide derivatives, were selected. 15 To conduct a solubility experiment, the maximum absorption wavelength of the compound was first measured to be 335 nm, followed by measurements of JD3 and JD. 15 Standard curves were obtained by measuring the absorbance values ​​of different concentrations at the maximum absorption wavelength, and then JD3 and JD were prepared. 15 The saturated solution was analyzed, and its absorbance was measured and substituted into the linear regression equation to calculate JD3 and JD. 15 Solubility.

[0052] Plotting concentration (µM)³ on the x-axis and absorbance (Abs) on the y-axis, the linear regression equation is y = 0.0157x + 0.0276, R0. 2=0.9957, the standard curve was obtained, and then the absorbance of the saturated solution of the compound was measured. Substituting the values ​​into the linear regression equation, the absorbance was found to be 2.092 Abs, and the solubility was 131.1 µM. (The remaining text appears to be incomplete and possibly contains errors.) Figure 3 .

[0053] Plotting concentration (µM)³ on the x-axis and absorbance (Abs) on the y-axis, the linear regression equation is y = 0.0056x + 0.0244, R0. 2 =0.9904, thus obtaining JD 15 The standard curve was obtained, and the absorbance of a saturated solution of the compound was measured. Substituting the results into a linear regression equation yielded an absorbance of 1.383 Abs and a solubility of 242.6 µM. Figure 4 .

[0054] Further investigation using GloSensorcAMP accumulation experiments revealed whether these compounds could allosterically modulate the functional activity of the endogenous β2AR ligand ISO, further confirming that these compounds are negative allosteric modulators (NAM) of β2AR. Taking the indole-allylamine amide derivative JD3 as an example (e.g.) Figure 5 As shown in the figure, when the concentration of compound JD3 reaches 50 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 JD3 is likely between 25 μM and 50 μM, specifically manifested as a sharp drop in the concentration curve. When the concentration of JD3 increases from 50.0 μM to 100.0 μM, the downward shift of the ISO concentration curve is very slight. This phenomenon explains that with increasing concentration of compound JD3, the concentration-dependent curve of ISO shows a limited downward shift, indicating that the indole-allylamine amide derivative JD3 can effectively negatively allosterically regulate the functional activity of the β2AR endogenous ligand ISO. This allosteric regulation phenomenon is consistent with previously reported allosteric antagonistic regulatory mechanisms. Other indole-allylamine amide derivatives represent JD... 15 Allosteric regulation of ISO concentration dependence curves (e.g.) Figure 6 (As shown) This is consistent with the results of JD3, that is, the newly synthesized indole-allylamine amide derivatives are all negative allosteric modulators of β2AR. In summary, this invention replaces the peptide core structure of Cmpd-15 with an indole skeleton, uses trans-indole allylamine as a raw material, and flips the amide bond to obtain a new indole allylamine amide derivative with better allosteric antagonistic activity, wherein R1 is any one of phenyl, m-methylbenzene, m-methoxyphenyl, m-bromophenyl, m-chlorophenyl, m-fluorophenyl, o-methoxyphenyl, p-methoxyphenyl, naphthyl ring, oxazole, cyclohexyl, cyclopentyl, methyl, ethyl, and isopropyl.

[0055] All synthesized compounds underwent functional activity screening and bioactivity testing for G protein-dependent signaling pathways. Results showed that the indoleallylamine amide derivatives of this invention exhibit good antagonistic activity against β2AR and can negatively allosterically regulate the functional activity of isoproterenol, making them suitable for use in the preparation of β2-adrenergic receptor allosteric antagonist drugs. This provides direction for lead compound skeletal transitions, structural simplification, and new drug development, and is of great significance for expanding the structural types and simplification of lead compounds, exploring more potent β2-adrenergic receptor allosteric antagonists, and developing new drugs.

[0056] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail can be made without departing from the spirit and scope of the present invention. Any person skilled in the art can make their own modifications and alterations without departing from the spirit and scope of the present invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. An indoleallylamine amide derivative, characterized in that, The structure of the indole-allylamine amide derivative is shown in Formula 1: Formula 1; R1 = Me, Et, i-Pr, and one of the following structural formulas: 。 2. The indoleallylamine amide derivative according to claim 1, characterized in that, The indole-allylamine amide derivative specifically comprises one of the following structural formulas: 。 3. A method for preparing an indoleallylamine amide derivative according to claim 1, characterized in that, The preparation method includes: dissolving trans-indolylamine protected by tert-butyloxycarbonyl in N,N-dimethylformamide, adding 1-hydroxy-7-azabenzotriazole, stirring at room temperature for 10 min, adding N-methylmorpholine and benzoic acid, fatty acid or heterocyclic acid with different substituents at 0℃, then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, reacting at room temperature, and extracting and purifying to obtain the intermediate; The intermediate was dissolved in a mixed solution of methanol and water, potassium carbonate was added, the reaction was heated under reflux for 2 hours, the methanol was evaporated to dryness, N,N-dimethylformamide was removed by extraction, and the product was purified by silica gel column chromatography to obtain the indole-allylamine amide derivative.

4. The method for preparing indoleallylamine amide derivatives according to claim 3, characterized in that, The trans-indolylamine protected by the tert-butoxycarbonyl group includes (E)-3-(1H-indo-3-yl)prop-2-en-1-amine.

5. The method for preparing indoleallylamine amide derivatives according to claim 3, characterized in that, The benzoic acid, fatty acid, or heterocyclic acid with different substituents includes one of benzoic acid, 3-methylbenzoic acid, 3-methoxybenzoic acid, 3-bromobenzoic acid, 3-fluorobenzoic acid, 3-chlorobenzoic acid, 2-methoxybenzoic acid, 4-methoxybenzoic acid, 2-naphthoic acid, oxazol-5-carboxylic acid, cyclohexanecarboxylic acid, cyclopentanecarboxylic acid, formic acid, acetic acid, or isopropionic acid.

6. The method for preparing indoleallylamine amide derivatives according to claim 3, characterized in that, The preparation method specifically uses the following proportions: (1) Dissolve 1 eq of trans-indolylamine in N,N-dimethylformamide, add 1.2 eq of 1-hydroxy-7-azabenzotriazole, stir at room temperature for 10 min, add 0.7 eq of N-methylmorpholine and 1.5 eq of benzoic acid, fatty acid or heterocyclic acid with different substituents at 0℃, then add 1.2 eq of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, react at room temperature for 4 h, extract and purify to obtain the intermediate; (2) Dissolve the 1 eq intermediate in a mixed solution of methanol and water, add 1.2 eq potassium carbonate, heat the reaction under reflux for 2 hours, evaporate the methanol to dryness, extract to remove N,N-dimethylformamide, and purify by silica gel column chromatography to obtain the indoleallylamine amide derivative.

7. The method for preparing indoleacrylamide derivatives according to claim 6, characterized in that, The eluent used for purification in step (1) was prepared by mixing dichloromethane and anhydrous methanol in a volume ratio of 100:

1.

8. The use of an indoleallylamine amide derivative according to claim 1 as a drug for preparing an allosteric antagonist of the β2-adrenergic receptor.

9. The application according to claim 8, characterized in that, The indole-allylamine amide derivatives are used to allosterically antagonize the G-protein signaling pathway of β2AR and negatively regulate the agonistic effect of the endogenous agonist isoproterenol on β2AR.

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