An indoleacrylamide derivative, its preparation method and application

Abstract

This invention belongs to the field of medicinal chemistry and relates to an indole acrylate derivative, its preparation method, and its application. Through functional activity screening of all synthesized compounds for G protein-dependent signaling pathways, these new derivatives were found to act as allosteric antagonistic regulators of the β2-adrenergic receptor. This invention uses trans-indole acrylic acid with different substituents as a starting material, and amide coupling yields the following new indole acrylate derivatives, wherein R1 is a hydrogen atom, a halogen atom, a methyl group, or a methoxy group; and R2 is any one of phenyl, m-bromophenyl, m-chlorophenyl, m-fluorophenyl, m-cyanophenyl, m-methoxyphenyl, cyclohexyl, or piperidinyl. Bioactivity tests show that the indole acrylate derivatives of this invention exhibit good antagonistic activity against β2AR and can negatively allosterically regulate the functional activity of isoproterenol.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, and specifically relates to an indoleacrylamide compound, its synthesis method, and its application as an allosteric antagonist modulator of β2-adrenergic receptor. Background Technology

[0002] G protein-coupled receptors (GPCRs) are the largest family of cell signal transduction receptors in the human body and one of the most important drug targets. They participate in almost all life activities, regulating cellular responses to light, odor, hormones, neurotransmitters, chemokines, etc., and are of great significance for maintaining life and health. They are widely used in biological cell signal transduction research and drug screening and development. Currently, there are about 500 drugs targeting GPCRs, accounting for 34% of FDA-approved drugs. GPCR research has won 10 Nobel Prizes, which also illustrates its importance. GPCRs have a conserved structural feature: seven transmembrane helices (7TMs). These seven helices form a transmembrane domain that divides the receptor into an extracellular N-terminus, an intracellular C-terminus, three extracellular loops, and three intracellular loops. The extracellular region of GPCRs undergoes a conformational change upon binding to agonist signaling molecules (such as odorants and hormones), triggering transmembrane helical movement. Understanding the activation mechanism of GPCRs is essential for comprehending their physiological functions and the causes of related diseases, and it forms the basis for precision drug development. It has consistently been a core focus and cutting-edge research area in this field. GPCR activation refers to the recruitment of downstream effector proteins triggered by agonist binding. Essentially, GPCR activation involves the coupling and allosteric communication between extracellular agonist binding and intracellular downstream effector protein recruitment—a conformational transition of the receptor from an inactive to an activated state.

[0003] β2-adrenergic receptors (β2ARs) are a typical GPCR and have long been used as a model system for studying GPCR regulatory mechanisms. β2-receptor blockers, ortho-arc antagonists of β2ARs, are key cardiovascular drugs used to treat various diseases. Adrenergic receptors belong to a subfamily of type A GPCRs and play an important role in the sympathetic nervous system.

[0004] In the human body, there are nine subtypes of adrenaline receptors, and their sequences and structures are relatively conserved. Traditional drug development for GPCRs has mainly targeted the ortho-constitutional binding site (i.e., the site where the receptor's endogenous ligand binds). The high conserved nature of this site among different subtypes poses a significant challenge to the development of selective drugs. Summary of the Invention

[0005] The purpose of this invention is to prepare new indole acrylamide derivatives to develop target compounds with high biological activity, stable chemical structure, good water solubility, and novel skeleton, as new allosteric antagonists of β2AR.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides an indoleacrylamide derivative, the structure of which is shown in Formula 1:

[0008]

[0009] R1 = H, F, Cl, Br, CN, Me, OCH3;

[0010]

[0011] The present invention also provides a method for preparing indole acrylamide derivatives, the method comprising: dissolving trans-indole acrylic acid with different substituents in N,N-dimethylformamide (DMF), adding 1-hydroxy-7-azabenzotriazole (HOAt), stirring at room temperature for 10 min, adding N-methylmorpholine and aniline, aliphatic amine or heterocyclic amine with different substituents at 0°C, then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and then reacting at room temperature for several hours to obtain the indole acrylamide derivatives.

[0012] Furthermore, the trans-indole acrylic acid with different substituents includes (E3)-3-(4-bromo-1H-indole-3-yl)acrylic acid, (E3)-3-(5-bromo-1H-indole-3-yl)acrylic acid, and (E3)-3-(6-bromo-1H-indole-3-yl)acrylic acid.

[0013] Furthermore, the anilines with different substituents include m-bromoaniline, m-chloroaniline, and m-fluoroaniline; the aliphatic amines with different substituents include cyclohexylamine; and the heterocyclic amines with different substituents include piperidine and piperazine.

[0014] Furthermore, the preparation method specifically includes: dissolving 1 mmol of trans-indole propene with different substituents in N,N-dimethylformamide; adding 1.20 mmol of N-hydroxy-7-azabenzotriazole, 1.20 mmol of aniline, aliphatic amine or heterocyclic amine with different substituents, and 0.70 mmol of N-methylmorpholine under ice bath conditions; reacting for 10 min; then adding 1.20 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; and reacting at room temperature for 4 h; after the reaction is completed, the reaction solution is diluted with ethyl acetate, washed with water, and dried with anhydrous sodium sulfate; after removing the solvent by rotary evaporation, the obtained crude solid product is purified by silica gel column chromatography, eluted with eluent, the product after column chromatography is concentrated, and then filtered through dichloromethane to obtain the aforementioned new indole acrylate derivative.

[0015] Furthermore, the eluent is prepared from petroleum ether and ethyl acetate in a volume ratio of 1:1.

[0016] The present invention also provides the use of the indole acrylamide derivatives as described above as allosteric antagonists of the β2-adrenergic receptor (β2AR).

[0017] Furthermore, the indoleacrylamide derivatives are used to allosterically antagonize the G-protein signaling pathway of β2AR and negatively regulate the agonistic effect of the endogenous agonist isoproterenol (ISO) on β2AR.

[0018] Compared with the prior art, the beneficial effects of the present invention are at least as follows:

[0019] This invention provides novel indole acrylate amide derivatives and their preparation method. Through functional activity screening of all synthesized compounds for G protein-dependent signaling pathways, the new derivatives can serve as allosteric antagonistic modulators (NAMs) of the β2-adrenergic receptor. This invention uses trans-indole acrylic acid with different substituents as raw materials, and amide coupling yields the following novel indole acrylate amide derivatives, characterized by high bioactivity, stable chemical structure, good water solubility, and novel skeleton. R1 is a hydrogen atom, a halogen atom, a methyl group, or a methoxy group; R2 is any one of phenyl, m-bromophenyl, m-chlorophenyl, m-fluorophenyl, m-cyanophenyl, m-methoxyphenyl, cyclohexyl, or piperidinyl. Bioactivity tests show that the indole acrylate amide derivatives of this invention have good antagonistic activity against β2AR and can negatively allosterically regulate the functional activity of isoproterenol (ISO). These indole acrylate amide derivatives can serve as allosteric antagonists of β2AR, providing a useful reference for the structure-activity relationship and drug development studies of this class of compounds. Attached Figure Description

[0020] Figure 1For the design of pyrazole compounds in the early stage;

[0021] Figure 2 Design of target compounds of the indole acrylate class;

[0022] Figure 3 Synthesis of indoleacrylamide compounds L1-L27;

[0023] Figure 4 This describes a method for synthesizing M-class compounds;

[0024] Figure 5 The ISO dose-response curve is mediated. Detailed Implementation

[0025] In 1961, Jacques Monod of the Pasteur Institute in France... Jacob and Jean-Pierre Changeux first proposed the concept of allostery. Allosteric sites (regions that differ spatially and topologically from the orthogonal binding sites) have relatively high degrees of freedom, less evolutionary pressure, and a relatively high mutation frequency. Compared to orthogonal drugs targeting the active site, allosteric modulators exhibit higher selectivity and lower toxicity. Allosteric modulators bind outside the orthogonal ligand pocket of the receptor, and due to their relatively low conservatism at the binding site, they may possess better subtype selectivity. Their discovery provides new insights into obtaining highly selective drugs. Furthermore, orthogonal and allosteric modulators exert their effects based on different mechanisms of action, making it possible for allosteric modulators to overcome acquired resistance to orthogonal drugs during treatment and to achieve better selectivity and safety.

[0026] In 2017, the applicant collaborated with scientists at Duke University to report the first small-molecule negative allosteric modulator of β2AR, compound 15 (Cmpd-15, as shown in Formula 2) (Ahn S, et al. Proc. Natl. Acad. Sci. USA, 2017, 114:1708-1713; Liu X, et al. Nature, 2017, 548:480-484). However, Cmpd-15 is a dipeptide compound with poor water solubility and low biological activity, and its structure is relatively unstable, which may affect its druggability. Therefore, this project uses Cmpd-15 as a lead compound and employs skeletal transition strategies, structural simplification, and bioelectronic isosterism concepts for drug design. The synthesized new compound was analyzed using GloSensor... TMThe cAMPAccumulation assay was used to screen for the bioactivity of classical signaling pathways (G-protein signaling) (Binkowski BF et al. ACS Chem Biol. 2011; 6(11):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.

[0027]

[0028] Pyrazoles, indoles, and other heterocyclic compounds are common drug skeletons. Therefore, the inventors initially replaced the peptide structure of Cmpd-15 with a pyrazole skeleton, such as... Figure 1 As shown, keeping the (S)-2-amino-3-(3-bromophenyl)-N-methylpropionamide on the right side unchanged, a series of pyrazole derivatives were designed and synthesized (Chinese Invention Patent Publication No.: CN115745891A).

[0029] Previous research by the inventors revealed that cAMP accumulation experiments showed that most pyrazolamide derivatives exhibited significantly better allosteric antagonism against β2AR than their lead compound Cmpd-15, and the water solubility of the newly derived compound was significantly improved compared to Cmpd-15. However, the phenylalanine moiety on the right side of Cmpd-15 suffers from significant steric hindrance, which may affect the drug-likeness of the compound (Chinese Invention Patent Publication No.: CN115894373A). Therefore, structural optimization is necessary. Indole is an aromatic heterocyclic organic compound containing a six-membered benzene ring and a five-membered nitrogen-containing pyrrole ring, hence also known as benzopyrrole, and is a crucial component in drug design. It is widely distributed in nature and exhibits broad-spectrum pharmacological activities, such as antidepressant, anticancer, and anti-inflammatory effects. This invention utilizes the principles of skeletal transition, structural simplification, and bioisosterism to replace the pyrazole skeleton with an indole acrylate skeleton, and to replace the right-side phenylalanine moiety of Cmpd-15 with substituted aniline, aliphatic amine, and heterocyclic amine, thereby designing and synthesizing a series of indole acrylate amide derivatives, such as... Figure 2 As shown, the bioactivity of the new derivative on β2AR was investigated.

[0030] In view of this, the present invention uses trans-indole acrylic acid 1 with different substituents as raw materials, and through amino-acyl coupling with substituted aniline, aliphatic amine and heterocycle 2 respectively, obtains a series of new indole acrylic amide derivatives, such as... Figure 3 As shown.

[0031] The specific steps of the synthetic method for indoleamide derivatives provided by this invention are as follows:

[0032] Compound 1 (1 mmol) was dissolved in N,N-dimethylformamide (DMF). Under ice bath conditions, N-hydroxy-7-azabenzotriazole (HOAT) (1.20 mmol), compound 2 (1.20 mmol), and N-methylmorpholine (NMM) (0.70 mmol) were added. After reacting for 10 min, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (1.20 mmol) was added, and the reaction was continued at room temperature for 4 h. 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 dried over anhydrous sodium sulfate. After removing the solvent by rotary evaporation, the resulting crude solid product was purified by silica gel column chromatography using a elution of [V(petroleum ether):V(ethyl acetate) = 1:1]. The concentrated product was then filtered through a suitable amount of dichloromethane to obtain a new indoleacrylamide derivative (hereinafter referred to as target compound L), as shown in Table 1.

[0033] Among them, compound 1 is a trans-indole propene with different substituents;

[0034] Compound 2 is aniline, aliphatic amine or heterocyclic amine with different substituents.

[0035] Table 1. Structures of novel indoleacrylamide derivatives

[0036]

[0037]

[0038]

[0039]

[0040] To investigate the effect of double bonds in indoleacrylamide on the bioactivity of its derivatives, the inventors synthesized compounds M1 and M2, which do not contain double bonds, such as... Figure 4 As shown, the specific synthesis method is the same as that for the L-series derivatives.

[0041] The present invention will now be described in detail with reference to specific embodiments.

[0042] The specific preparation method of the indole acrylate derivatives provided by this invention is as follows.

[0043] Example 1:

[0044] Preparation of (E3)-(1H-indol-3-yl)-N-phenylacrylamide (L1)

[0045] Trans-1H-3-indole-acrylic acid (250 mg, 1.34 mmol) was dissolved in DMF in a 25 mL round-bottom flask. HOAt (218 mg, 1.60 mmol) was added and the reaction proceeded for 10 min. Then, aniline (146 μL, 1.20 mmol) and NMM (104 μL, 0.94 mmol) were added under ice bath conditions, and the reaction proceeded for another 10 min. Finally, EDCI (307 mg, 1.60 mmol) was added, and the reaction proceeded 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(petroleum ether):V(ethyl acetate) = 15:1]. The concentrated crude product was then filtered through dichloromethane to obtain the target compound L1 as a pale yellow solid powder, with a yield of 49%. 1 HNMR (400MHz, DMSO-d6): δ11.69(s,1H),10.04(s,1H),7.99(d,J=7.1Hz,1H),7.88–7.76(m,2H),7.73(d,J=8.0Hz, 2H),7.49(d,J=7.2Hz,1H),7.33(t,J=7.3Hz,2H),7.26–7.15(m,2H),7.03(t,J=7.4Hz,1H),6.85(d,J=15.7Hz,1H). 13 C NMR (100MHz, DMSO-d6): δ165.1,139.9,137.6,134.9,131.2,128.9,124.9,122. 8,122.5,120.6,120.1,118.9,116.0,112.5,112.3.HRMS(ESI,m / z):Calcd.for C 17 H 14 N₂O[M+Na] + 285.0998; found: 285.1003.

[0046] Example 2:

[0047] Preparation of (E3)-N-(3-bromophenyl)-3-(1H-indole-3-yl)acrylamide (L2)

[0048] Trans-1H-3-indole-acrylic acid (300 mg, 1.60 mmol) was dissolved in DMF in a 25 mL round-bottom flask. HOAt (261 mg, 1.92 mmol) was added and the reaction proceeded for 10 min. Then, m-bromoaniline (0.35 mL, 3.20 mmol) and NMM (0.12 mL, 1.12 mmol) were added under ice bath conditions and the reaction proceeded for another 10 min. Finally, EDCI (368 mg, 1.20 mmol) was added and the reaction proceeded 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 using a 1:1 eluent of petroleum ether and ethyl acetate. The concentrated crude product was then filtered through dichloromethane to give the target compound L2 as a yellow solid, with a yield of 89%. 1 H NMR (400MHz, DMSO-d6): δ11.73(s,1H),10.20(s,1H),8.16(s,1H),8.00–7.91(m,1H),7.88(s,1H),7.81(d,J=15.6 Hz,1H),7.55(d,J=8.2Hz,1H),7.51-7.44(m,1H),7.29(t,J=8.0Hz,1H),7.25-7.18(m,3H),6.79(d,J=15.7Hz,1H). 13 C NMR (100MHz, DMSO-d6): δ165.4,141.5,137.6,135.6,131.7,130.9,125.4,124.9,122.5,121.8,121.2,120.7,120.1,117.6,115.4,112.6,112.2.

[0049] Example 3:

[0050] Preparation of (E3)-N-(3-chlorophenyl)-3-(1H-indole-3-yl)acrylamide (L3)

[0051] Trans-1H-3-indole-acrylic acid (250 mg, 1.34 mmol) was dissolved in DMF in a 25 mL round-bottom flask. HOAt (218 mg, 1.60 mmol) was added and the reaction proceeded for 10 min. Then, m-chloroaniline (0.17 mL, 1.60 mmol) and NMM (104 μL, 0.94 mmol) were added under ice bath conditions, and the reaction proceeded for another 10 min. Finally, EDCI (307 mg, 1.60 mmol) was added, and the reaction proceeded 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(petroleum ether):V(ethyl acetate) = 15:1]. The concentrated crude product was then filtered through dichloromethane to give the target compound L3 as a yellow solid, with a yield of 47%. 1 H NMR (300MHz, DMSO-d6): δ11.70(s,1H),10.21(s,1H),7.97(d,J=10.6Hz,2H),7.83(t,J=10.8Hz,2H),7.50 (t,J=8.3Hz,2H),7.36(t,J=8.0Hz,1H),7.29–7.18(m,2H),7.09(d,J=8.0Hz,1H),6.80(d,J=15.7Hz,1H). 13 C NMR (75MHz, DMSO-d6): δ165.4,141.4,137.6,135.6,133.2,131.6,130.5,124.9,122.5,122.4,120.7,120.1,118.4,117.3,115.5,112.5,112.2.

[0052] Example 4:

[0053] Preparation of (E3)-N-(3-fluorophenyl)-3-(1H-indole-3-yl)acrylamide (L4)

[0054] Trans-1H-3-indole-acrylic acid (250 mg, 1.34 mmol) was dissolved in DMF in a 25 mL round-bottom flask. HOAt (218 mg, 1.60 mmol) was added and the reaction proceeded for 10 min. Then, 3-fluoroaniline (0.15 mL, 1.60 mmol) and NMM (100 μL, 0.94 mmol) were added under ice bath conditions, and the reaction proceeded for another 10 min. Finally, EDCI (307 mg, 1.60 mmol) was added, and the reaction proceeded 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(petroleum ether):V(ethyl acetate) = 15:1]. The concentrated crude product was then filtered through dichloromethane to give the target compound L4 as a yellow solid, with a yield of 73%. 1 HNMR (400MHz, DMSO-d6): δ11.75(s,1H),10.31(s,1H),7.99(d,J=7.1Hz,1H),7.88(s,1H),7 .85-7.64(m,2H),7.50(d,J=7.3Hz,1H),7.38(s,2H),7.29–7.18(m,2H),6.93–6.79(m,2H). 13 C NMR (100MHz, DMSO-d6): δ165.4,141.7,141.6,137.6,135.5,131.6,130.5,130.4,124.9,122.5,1 20.7,120.1,115.5,114.7,112.5,112.2,109.3,109.1,105.8,105.6.HRMS(ESI,m / z):Calcd.for C 17 H 13 FN2O[M+Na] + 303.0904; found: 303.0908.

[0055] Example 5:

[0056] Preparation of (E3)-3-(1H-indol-3-yl)-N-(m-tolyl)acrylamide (L5)

[0057] Trans-1H-3-indole-acrylic acid (250 mg, 1.34 mmol) was dissolved in DMF in a 25 mL round-bottom flask. HOAt (218 mg, 1.60 mmol) was added and the reaction proceeded for 10 min. Then, m-methylaniline (172 μL, 1.60 mmol) and NMM (103.90 μL, 0.94 mmol) were added under ice bath conditions, and the reaction proceeded for another 10 min. Finally, EDCI (307 mg, 1.60 mmol) was added, and the reaction proceeded 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 using a 1:1 eluent of petroleum ether and ethyl acetate. The concentrated crude product was then filtered through dichloromethane to give the target compound L5 as a pale yellow solid, with a yield of 96%. 1 H NMR (400MHz, DMSO-d6): δ11.68(s,1H),9.96(s,1H),7.99(d,J=7.4Hz,1H),7.85(s,1H),7.80(d,J=15.6Hz ,1H),7.58(s,1H),7.56–7.46(m,2H),7.28–7.16(m,3H),6.87(d,J=5.6Hz,1H),6.84(s,1H),2.30(s,3H). 13 C NMR (100MHz, DMSO-d6): δ165.2,165.1,139.8,139.7,138.1,137.6,137.4,134.8,131.2,131.1,128.8,1 25.0,124.9,123.7,122.5,120.7,120.2,119.5,119.4,116.2,116.1,112.5,112.5,112.3,112.3,21.4.

[0058] Example 6:

[0059] Preparation of (E3)-N-(3-cyanophenyl)-3-(1H-indol-3-yl)acrylamide (L6)

[0060] Trans-1H-3-indole-acrylic acid (250 mg, 1.34 mmol) was dissolved in DMF in a 25 mL round-bottom flask. HOAt (218 mg, 1.60 mmol) was added and the reaction proceeded for 10 min. Then, 3-aminobenzonitrile (0.29 mL, 2.67 mmol) and NMM (100 μL, 0.94 mmol) were added under ice bath conditions and the reaction proceeded for another 10 min. Finally, EDCI (307 mg, 1.60 mmol) was added and the reaction proceeded 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(petroleum ether):V(ethyl acetate) = 15:1]. The concentrated crude product was then filtered through dichloromethane to give the target compound L6 as a yellow solid, with a yield of 37%. 1 HNMR (400MHz, DMSO-d6): δ11.72(s,1H),10.35(s,1H),8.27(s,1H),7.98–7.92(m,1H),7.89(d,J=2.8Hz,1 H),7.88–7.80(m,2H),7.55(t,J=7.8Hz,1H),7.52–7.46(m,2H),7.29-7.18(m,2H),6.79(d,J=15.6Hz,1H). 13 C NMR(100MHz,DMSO-d6)δ165.6,140.6,137.6,135.9,131.8,130.3,126.3,124.9,123.4,1 22.5,121.5,120.7,120.0,118.9,115.1,112.5,112.1,111.6.HRMS(ESI,m / z):Calcd.for C 18 H 13 N3O[M+Na] + 310.0951 found: 310.0955.

[0061] Example 7:

[0062] Preparation of (E3)-3-(1H-indol-3-yl)-N-(3-methoxyphenyl)acrylamide (L7)

[0063] Trans-1H-3-indole-acrylic acid (250 mg, 1.34 mmol) was dissolved in DMF in a 25 mL round-bottom flask. HOAT (218 mg, 1.60 mmol) was added and reacted for 10 min. Then, 3-methoxyaniline (0.18 mL, 1.60 mmol) and NMM (100 μL, 0.94 mmol) were added under ice bath conditions and reacted for another 10 min. Finally, EDCI (307 mg, 1.60 mmol) was added and the reaction was carried out 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(petroleum ether):V(ethyl acetate) = 10:1]. The concentrated crude product was then filtered with dichloromethane to give the target compound L7 as a pale yellow solid, with a yield of 72%. 1H NMR (400MHz, DMSO-d6): δ11.70(s,1H),10.06(s,1H),7.99(d,J=7.5Hz,1H),7.90–7.77(m,2H) ,7.55–7.43(m,2H),7.32–7.16(m,4H),6.85(d,J=15.7Hz,1H),6.66–6.58(m,1H),3.75(s,3H). 13 C NMR (100MHz, DMSO-d6): δ165.1,159.6,141.0,137.5,134.9,131.3,129.6,124.9,122.4, 120.6,120.1,116.0,112.5,112.2,111.3,108.2,104.7,79.2.HRMS(ESI,m / z):Calcd.for C 18 H 16 N₂O₂[M+Na] + 315.1104; found: 315.1112.

[0064] Example 8:

[0065] Preparation of (E3)-3-(4-bromo-1H-indol-3-yl)-N-phenylacrylamide (L8)

[0066] 4-Bromo-1H-indole-3-carboxaldehyde (770 mg, 3.44 mmol) was dissolved in pyridine in a 50 mL round-bottom flask, and malonic acid (358 mg, 3.44 mmol) was added. Piperidine was used as a catalyst, and the reaction was carried out under nitrogen protection at room temperature for 10 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 silica gel column chromatography, using [eluent]. [V(petroleum ether):V(ethyl acetate) = 10:1], concentrated column chromatography yielded 280.20 mg of crude product. Then, 100 mg of the acid was dissolved in DMF in a 25 mL round-bottom flask, and HOAt (61 mg, 0.45 mmol) was added. After reacting for 10 min, aniline (34 μL, 0.38 mmol) and NMM (30 μL, 0.26 mmol) were added under ice bath conditions, and the reaction was carried out for 10 min. Then, EDCI (86 mg, 0.45 mmol) was added, and the reaction was carried out at room temperature for 4 h. After the reaction was completed by TLC monitoring, the product 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(petroleum ether):V(ethyl acetate) = 8:1]. The crude product from the concentrated column chromatography was then filtered with dichloromethane to give 92.60 mg of the target compound L8 yellow solid, with a yield of 93%. 1 HNMR (400MHz, DMSO-d6): δ11.95(s,1H),10.07(s,1H),7.93(s,1H),7.87(d,J=15.2Hz,1H),7.74(d,J=8.0Hz,2H),7.33( t,J=8.1Hz,3H),7.18(td,J=8.0,5.2Hz,1H),7.05(t,J=7.4Hz,1H),6.94(dd,J=11.8,7.8Hz,1H),6.73(d,J=15.6Hz,1H). 13 C NMR (100MHz, DMSO-d6): δ164.5,139.8,138.0,133.6,128.8,126.7,124.7,123. 9,123.0,123.0,119.1,117.2,112.9,112.5,112.1.HRMS(ESI,m / z):Calcd.for C 17 H 13 BrN2O[M+Na] + 363.0103; found: 363.0109.

[0067] Example 9:

[0068] Preparation of (E3)-3-(4-bromo-1H-indol-3-yl)-N-(3-fluorophenyl)acrylamide (L9)

[0069] 4-Bromo-1H-indole-3-carboxaldehyde (770 mg, 3.44 mmol) was dissolved in pyridine in a 50 mL round-bottom flask, and malonic acid (358 mg, 3.44 mmol) was added. Piperidine was used as a catalyst, and the reaction was carried out under nitrogen protection at room temperature for 10 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 the eluent being [V(petroleum ether):V(ethyl acetate)]. =10:1], concentrated column chromatography yielded crude product (E3)-3-(4-bromo-1H-indol-3-yl)acrylic acid 280.20 mg, then 91 mg of the acid was dissolved in DMF in a 25 mL round-bottom flask, HOAt (56 mg, 0.41 mmol) was added and reacted for 10 min, then 3-fluoroaniline (33 μL, 0.34 mmol) and NMM (26 μL, 0.24 mmol) were added under ice bath conditions and reacted for 10 min, then EDCI (79 mg, 0.41 mmol) was added and reacted at room temperature for 4 h. After the reaction was monitored by TLC, the product 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(petroleum ether):V(ethyl acetate) = 8:1]. The crude product obtained by concentrated column chromatography was then filtered with dichloromethane to give 88 mg of the target compound L9 as a yellow solid, with a yield of 97%. 1 H NMR (400MHz, Methanol-d4): δ8.82(d,J=15.6Hz,1H),7.88(s,1H),7.66(dt,J=11.4,2.2Hz,1H),7.42(d,J=4.4 Hz,1H),7.36–7.31(m,2H),7.31–7.27(m,1H),7.04(t,J=7.9Hz,1H),6.85-6.76(m,1H),6.54(d,J=15.5Hz,1H). 13C NMR(100MHz,Methanol-d4)δ167.9,165.5,163.1,142.2,142.1,139.6,137.3,131.2,131.1,127.2,12 6.1,125.8,124.0,116.4,114.7,114.6,112.5,111.2,111.0,108.1,107.8.HRMS(ESI,m / z):Calcd.for C 17 H 12 BrFN2O[M+Na] + 381.0009; found: 381.0014.

[0070] Example 10:

[0071] (E3)-3-(4-bromo-1H-indol-3-yl)-N-cyclohexylacrylamide (L 10 Preparation of )

[0072] Similar to Example 9, (E3)-3-(4-bromo-1H-indole-3-yl)acrylic acid was first synthesized. Then, 280 mg of this acid was dissolved in DMF in a 25 mL round-bottom flask. HOAt (172 mg, 1.26 mmol) was added, and the reaction proceeded for 10 min. Cyclohexylamine (120 μL, 1.05 mmol) and NMM (82 μL, 0.74 mmol) were added under ice bath conditions, and the reaction proceeded for 10 min. EDCI (242 mg, 1.26 mmol) was then added, and the reaction proceeded 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 using [V(dichloromethane V(methanol) = 30:1] as eluent. The concentrated crude product was then filtered with dichloromethane to obtain the target compound L. 10 199 mg of yellow solid powder, yield 75%. 1 HNMR (400MHz, DMSO-d6): δ11.90(s,1H),8.36(s,1H),7.84(d,J=27.8Hz,2H),7.38(d, J=73.4Hz,2H),7.04(s,1H),6.39(s,1H),3.66(s,1H),2.14–1.45(m,5H),1.20(s,6H). 13C NMR (100MHz, DMSO-d6): δ164.7,137.9,131.6,125.9,124.4,123.9,122.8,118.0,112.9,112.7,112.0,47.5,32.7,25.3,24.7.HRMS(ESI,m / z):Calcd.for C 17 H 19 BrN2O[M+Na] + 369.0573; found: 369.0570.

[0073] Example 11:

[0074] (E3)-3-(4-bromo-1H-indol-3-yl)-1-(piperidin-1-yl)prop-2-en-1-one (L) 11 Preparation of )

[0075] Similar to Example 10, (E3)-3-(4-bromo-1H-indol-3-yl)acrylic acid was first synthesized. Then, 300 mg of this acid was dissolved in DMF in a 25 mL round-bottom flask. HOAt (155 mg, 1.36 mmol) was added, and the reaction was carried out for 10 min. Cyclohexylamine (112 μL, 1.13 mmol) and NMM (88 μL, 0.79 mmol) were added under ice bath conditions, and the reaction was carried out for 10 min. Then, EDCI (260 mg, 1.36 mmol) was added, and the reaction was carried out 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 using [V(dichloromethane V(methanol) = 40:1] as the eluent. The crude product from the concentrated column chromatography was then filtered with dichloromethane to obtain the target compound L. 11 145 mg of yellow solid powder, yield 55%. 1 HNMR (400MHz, DMSO-d6): δ12.00(s,1H),8.49(d,J=15.2Hz,1H),8.24(s,1H),7.46(d,J=8.1Hz,1H),7 .29(d,J=7.6Hz,1H),7.10–7.03(m,1H),7.01(s,1H),3.58(s,4H),1.60(d,J=5.8Hz,2H),1.49(s,4H). 13C NMR(100MHz, DMSO-d6): δ164.9,137.8,134.4,127.2,124.5,123.8,122.7,112.9,112.8,112.7,111.9,24.3.HRMS(ESI,m / z):Calcd.for C 16 H 17 BrN2O[M+Na] + 355.0416; found: 355.0414.

[0076] Example 12:

[0077] (E3)-3-(4-chloro-1H-indol-3-yl)-N-phenylacrylamide (L) 12 Preparation of )

[0078] 4-Chloro-1H-indole-3-carboxaldehyde (970 mg, 5.40 mmol) was dissolved in pyridine in a 50 mL round-bottom flask, and malonic acid (562 mg, 5.40 mmol) was added. Piperidine was used as a catalyst, and the reaction was carried out under nitrogen protection at room temperature for 10 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 silica gel column chromatography with the eluent [V(dichloromethane):V(methanol)]. The crude product (E3)-3-(4-chloro-1H-indol-3-yl)acrylic acid (530 mg) was obtained by concentrated column chromatography [V(petroleum ether):V(ethyl acetate) = 6:1]. 147 mg of this acid was dissolved in DMF in a 25 mL round-bottom flask, and HOAt (108 mg, 0.79 mmol) was added. After reacting for 10 min, aniline (60 μL, 0.66 mmol) and NMM (50 μL, 0.46 mmol) were added under ice bath conditions, and the reaction was continued for 10 min. Then, EDCI (151 mg, 0.79 mmol) was added, and the reaction was continued 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 silica gel column chromatography with eluent [V(petroleum ether):V(ethyl acetate) = 6:1]. The crude product obtained by concentrated column chromatography was then filtered with dichloromethane to obtain the target compound L. 12 135.60 mg of yellow solid was produced, with a yield of 92%. 1H NMR (400MHz, DMSO-d6): δ12.02(s,1H),10.07(s,1H),8.44(d,J=15.5Hz,1H),8.01(s,1H),7.73(d,J=7.9Hz,2 H),7.48-7.41(m,1H),7.32(t,J=7.8Hz,2H),7.19–7.09(m,2H),7.03(t,J=7.4Hz,1H),6.63(d,J=15.5Hz,1H). 13 CNMR (100MHz, DMSO-d6): δ164.5,139.8,138.2,133.8,128.8,126.6,124.8,123 .0,122.7,122.6,121.3,119.2,117.4,112.0,111.7.HRMS(ESI,m / z):Calcd.for C 17 H 13 ClN2O[M+Na] + 319.0608; found: 319.0610.

[0079] Example 13:

[0080] (E3)-N-(3-bromophenyl)-3-(4-chloro-1H-indole-3-yl)acrylamide (L 13 Preparation of )

[0081] Similar to Example 12 above, (E3)-3-(4-chloro-1H-indol-3-yl)acrylic acid was first synthesized. Then, 200 mg of this acid was dissolved in DMF in a 25 mL round-bottom flask. HOAt (147 mg, 1.08 mmol) was added, and the reaction proceeded for 10 min. Under ice bath conditions, m-bromoaniline (98 μL, 0.90 mmol) and NMM (70 μL, 0.63 mmol) were added, and the reaction proceeded for 10 min. Finally, EDCI (207 mg, 1.08 mmol) was added, and the reaction proceeded 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 using [V(petroleum ether:V(ethyl acetate) = 8:1] as eluent. The concentrated crude product was then filtered through dichloromethane to obtain the target compound L. 13 75.30 mg of yellow solid powder, yield 22%. 1HNMR (400MHz, DMSO-d6): δ12.03(s,1H),10.22(s,1H),8.44(d,J=15.5Hz,1H),8.12(s,1H),8.03(s,1H),7.57(d,J=8.4Hz,1H ),7.45(dd,J=6.6,2.5Hz,1H),7.28(t,J=8.0Hz,1H),7.22(dt,J=8.2,1.5Hz,1H),7.19–7.11(m,2H),6.56(d,J=15.6Hz,1H). 13 C NMR (100MHz, DMSO-d6): δ 164.7, 141.3, 138.1, 134.5, 130.7, 126.9, 125.5, 124.7, 122.7, 122.5, 121.7, 121.4, 121.4, 117.8, 116.7, 111.9, 111.7. Example 14:

[0082] (E3)-3-(4-chloro-1H-indol-3-yl)-N-(3-fluorophenyl)acrylamide (L 14 Preparation of )

[0083] Similar to Example 12 above, (E3)-3-(4-chloro-1H-indol-3-yl)acrylic acid was first synthesized. Then, 131 mg of this acid was dissolved in DMF in a 25 mL round-bottom flask. HOAt (97 mg, 0.71 mmol) was added, and the reaction was carried out for 10 min. Then, 3-fluoroaniline (57 μL, 0.59 mmol) and NMM (46 μL, 0.41 mmol) were added under ice bath conditions, and the reaction was carried out for 10 min. Finally, EDCI (136 mg, 0.71 mmol) was added, and the reaction was carried out 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(petroleum ether):V(ethyl acetate) = 8:1]. The concentrated crude product was then filtered with dichloromethane to obtain the target compound L. 14 67.30 mg of yellow solid powder, yield 53%. 1H NMR (400MHz, DMSO-d6): δ12.04(s,1H),10.27(s,1H),8.45(d,J=15.5Hz,1H),8.04(d,J=2.8Hz,1H),7.77(d,J=12 .2Hz,1H),7.51-7.41(m,1H),7.41-7.28(m,2H),7.20-7.10(m,2H),6.86(t,J=7.4Hz,1H),6.59(d,J=15.5Hz,1H). 13 C NMR (100MHz, DMSO-d6): δ164.7,163.4,141.6,141.4,138.1,134.4,130.4,130.3,126.9,124.7,122. 8,122.6,121.4,116.8,114.9,111.9,111.7,109.5,109.3,106.0,105.8.HRMS(ESI,m / z):Calcd.for C 17 H 12 ClFN2O[M+Na] + 337.0514; found: 337.0513.

[0084] Example 15:

[0085] (E3)-3-(4-fluoro-1H-indol-3-yl)-N-phenylacrylamide (L 15 Preparation of )

[0086] 4-Fluoro-1H-indole-3-carboxaldehyde (960 mg, 5.88 mmol) was dissolved in pyridine in a 50 mL round-bottom flask, and malonic acid (612 mg, 5.88 mmol) was added. Piperidine was used as a catalyst, and the reaction was carried out under nitrogen protection at room temperature for 10 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 silica gel column chromatography with the eluent [V(dichloromethane):V(methanol = 40:1]. Concentration column chromatography yielded 650 mg of crude product (E3)-3-(4-fluoro-1H-indole-3-yl)acrylic acid. 50 mg of this acid was then dissolved in DMF in 2 mL of silica gel. In a 5 mL round-bottom flask, HOAt (40 mg, 0.29 mmol) was added and reacted for 10 min. Then, aniline (22 μL, 0.24 mmol) and NMM (18 μL, 0.17 mmol) were added under ice bath conditions and reacted for another 10 min. Finally, EDCI (56 mg, 0.29 mmol) was added and the mixture was reacted 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(petroleum ether):V(ethyl acetate) = 8:1]. The concentrated crude product was then filtered through dichloromethane to obtain the target compound L. 15 42.5 mg of yellow solid powder, yield 85%. 1 H NMR (400MHz, DMSO-d6): δ11.93(s,1H),10.06(s,1H),7.87(t,J=16.3Hz,2H),7.72(d,J=8.0Hz,2H),7.31(t,J=8.2 Hz,3H),7.17(td,J=8.0,5.02Hz,1H),7.03(t,J=7.4Hz,1H),6.92(dd,J=11.8,7.8Hz,1H),6.71(d,J=15.6Hz,1H). 13 C NMR (100MHz, DMSO-d6): δ164.8,157.4,154.9,139.9,139.9,133.9,129.2,128. 8,123.1,123.0,122.9,119.2,117.7,117.6,113.7,111.0,108.9,105.8,105.6.

[0087] Example 16:

[0088] (E3)-3-(4-methyl-1H-indol-3-yl)-N-phenylacrylamide (L16 Preparation of )

[0089] 4-Methyl-1H-indole-3-carboxaldehyde (1000 mg, 6.28 mmol) was dissolved in pyridine in a 100 mL round-bottom flask, and malonic acid (1307 mg, 12.56 mmol) was added. Piperidine was used as a catalyst, and the reaction was carried out under nitrogen protection at room temperature for 10 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 silica gel column chromatography with the eluent [V(dichloromethane):V(methanol)]. =50:1], concentrated column chromatography yielded crude product (E3)-3-(4-methyl-1H-indol-3-yl)acrylic acid 736.10 mg, then 150.00 mg of the acid was dissolved in DMF in a 25 mL round-bottom flask, HOAt (121 mg, 0.89 mmol) was added and reacted for 10 min, then aniline (68 μL, 0.75 mmol) and NMM (58 μL, 0.53 mmol) were added under ice bath conditions and reacted for 10 min, then EDCI (171 mg, 0.89 mmol) was added and reacted at room temperature for 4 h. After the reaction was 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(petroleum ether):V(ethyl acetate) = 6:1]. The crude product obtained by concentrated column chromatography was then filtered with dichloromethane to obtain the target compound L. 16 76.60 mg of yellow solid, yield 51%. ¹H NMR (400 MHz, DMSO-d⁶): δ 11.47 (s, ¹H), 9.85 (s, ¹H), 7.95 (d, J = 15.4 Hz, ¹H), 7.67 (s, ¹H), 7.55 (d, J = 8.0 Hz, 2H), 7.17–7.06 (m, 3H), 6.84 (t, J = 7.5 Hz, 2H), 6.67 (d, J = 7.2 Hz, 1H), 6.39 (d, J = 15.4 Hz, 1H). 13 CNMR (100MHz, DMSO-d6): δ164.7,139.9,136.8,135.2,129.8,128.8,125.0,124.8, 122.9,122.1,121.9,119.1,116.8,113.2,110.3,21.1.HRMS(ESI,m / z):Calcd.for C 18 H 16 N₂O[M+Na] + 299.1155; found: 299.1156.

[0090] Example 17:

[0091] (E3)-N-(3-bromophenyl)-3-(4-methyl-1H-indole-3-yl)acrylamide (L 17 Preparation of )

[0092] Similar to Example 16 above, (E3)-3-(4-methyl-1H-indol-3-yl)acrylic acid was first synthesized. Then, 200 mg of this acid was dissolved in DMF in a 25 mL round-bottom flask. HOAt (162 mg, 1.19 mmol) was added, and the reaction was carried out for 10 min. Then, m-bromoaniline (108 μL, 0.99 mmol) and NMM (78 μL, 0.69 mmol) were added under ice bath conditions, and the reaction was carried out for 10 min. Finally, EDCI (228 mg, 1.19 mmol) was added, and the reaction was carried out 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 using [V(dichloromethane:V(ethyl acetate) = 60:1] as the eluent. The concentrated crude product was then filtered with dichloromethane to obtain the target compound L. 17 90 mg of yellow solid powder, yield 26%. 1 H NMR (400MHz, DMSO-d6): δ11.69(s,1H),10.19(s,1H),8.14(d,J=15.6Hz,2H),7.89(d,J=2.7Hz,1H),7.57(d,J=8.0Hz,1H), 7.32–7.18(m,2H),7.22(d,J=8.0Hz,1H),7.03(t,J=7.5Hz,1H),6.87(d,J=7.1Hz,1H),6.52(d,J=15.4Hz,1H),2.68(s,3H). 13 C NMR (100MHz, DMSO-d6): δ164.9,141.4,136.8,135.9,130.7,129.8,125.4,1 25.3,124.8,122.1,122.0,121.7,121.4,117.8,116.0,113.0,110.3,20.9.

[0093] Example 18:

[0094] (E3)-3-(5-bromo-1H-indol-3-yl)-N-phenylacrylamide (L) 18 Preparation of )

[0095] 5-Bromo-1H-indole-3-carboxaldehyde (2000 mg, 8.93 mmol) was dissolved in pyridine in a 100 mL round-bottom flask, and malonic acid (1394 mg, 13.40 mmol) was added. Piperidine was used as a catalyst, and the reaction was carried out under nitrogen protection at room temperature for 10 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 silica gel column chromatography, with the eluent being [V(dichloromethane):V [Methanol = 40:1], concentrated column chromatography yielded crude product (E3)-3-(5-bromo-1H-indol-3-yl)acrylic acid 771 mg. 133 mg of this acid was dissolved in DMF in a 25 mL round-bottom flask, and HOAt (82 mg, 0.60 mmol) was added. After reacting for 10 min, aniline (46 μL, 0.50 mmol) and NMM (39 μL, 0.35 mmol) were added under ice bath conditions, and the reaction was continued for 10 min. Then, EDCI (115 mg, 0.60 mmol) was added, and the reaction was continued 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(petroleum ether):V(ethyl acetate) = 6:1]. The crude product from the concentrated column chromatography was then filtered with dichloromethane to obtain the target compound L. 18 98 mg of pale yellow solid powder was produced, with a yield of 74%. 1 H NMR (400MHz, DMSO-d6): δ11.87(s,1H),10.04(s,1H),8.11(d,J=1.8Hz,1H),7.91(s,1H),7.78–7.71(m,2 H),7.70(s,1H),7.46(d,J=8.6Hz,1H),7.38–7.30(m,3H),7.04(t,J=7.3Hz,1H),6.77(d,J=15.8Hz,1H). 13 C NMR (100MHz, DMSO-d6): δ164.9,139.8,136.3,134.2,132.7,128.9,126.5,125.0,122.9,122.3,119.0,116.5,114.5,113.5,111.9.

[0096] Example 19:

[0097] (E3)-3-(5-bromo-1H-indol-3-yl)-N-(3-fluorophenyl)acrylamide (L 19 Preparation of )

[0098] Similar to Example 18 above, (E3)-3-(5-bromo-1H-indol-3-yl)acrylic acid was first synthesized. Then, 133 mg of this acid was dissolved in DMF in a 25 mL round-bottom flask. HOAt (74 mg, 0.54 mmol) was added, and the reaction proceeded for 10 min. Under ice bath conditions, 3-fluoroaniline (43 μL, 0.45 mmol) and NMM (35 μL, 0.32 mmol) were added, and the reaction proceeded for 10 min. Finally, EDCI (104 mg, 0.54 mmol) was added, and the reaction proceeded 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 using a 6:1 eluent ratio of petroleum ether to ethyl acetate. The concentrated crude product was then filtered through dichloromethane to obtain the target compound L. 19 81.20 mg of white solid powder, yield 50%. 1 H NMR (400MHz, DMSO-d6): δ12.04(s,1H),10.44(s,1H),8.11(d,J=2.0Hz,1H),7.91(s,1H),7.84-7.70(m,2H ),7.46(d,J=8.6Hz,1H),7.42-7.35(m,2H),7.35-7.31(m,1H),6.91-6.82(m,1H),6.80(d,J=15.6Hz,1H).

[0099] Example 20:

[0100] (E3)-N-(3-bromophenyl)-3-(5-fluoro-1H-indole-3-yl)acrylamide (L 20 Preparation of )

[0101] 5-Fluoro-1H-indole-3-carboxaldehyde (973 mg, 5.96 mmol) was dissolved in pyridine in a 100 mL round-bottom flask, and malonic acid (930 mg, 1.50 mmol) was added. Piperidine was used as a catalyst, and the reaction was carried out under nitrogen protection at room temperature for 10 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 silica gel column chromatography with the following eluent: [V(dichloromethane):V(methanol) = ... [60:1], concentrated column chromatography yielded 856.20 mg of crude product (E3)-3-(5-fluoro-1H-indol-3-yl)acrylic acid. 150 mg of this acid was then dissolved in DMF in a 25 mL round-bottom flask. HOAt (120 mg, 0.88 mmol) was added and reacted for 10 min. Then, m-bromoaniline (79 μL, 0.73 mmol) and NMM (57 μL, 0.51 mmol) were added under ice bath conditions and reacted for 10 min. Finally, EDCI (169 mg, 0.88 mmol) was added and reacted at room temperature for 4 h. After the reaction was 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(petroleum ether):V(ethyl acetate) = 6:1]. The crude product obtained by concentrated column chromatography was then filtered with dichloromethane to obtain the target compound L. 20 125 mg of yellow solid powder, yield 83%. 1 H NMR (400MHz, DMSO-d6): δ11.81(s,1H),10.14(s,1H),8.11(s,1H),7.94(s,1H),7.77(d,J=15.8Hz,1H),7.69(d,J=10. 2Hz,1H),7.58–7.41(m,2H),7.30(t,J=7.9Hz,1H),7.35-7.18(m,1H),7.09(t,J=9.2,Hz,1H),6.73(d,J=15.8Hz,1H). 13 C NMR (100MHz, DMSO-d6): δ165.2,141.4,135.0,134.1,133.2,130.8,125.4,125. 1,125.0,121.7,121.2,117.6,115.7,113.5,112.3,110.6,110.4,105.2,105.0.

[0102] Example 21:

[0103] (E3)-N-(3-bromophenyl)-3-(5-methyl-1H-indole-3-yl)acrylamide (L 21 Preparation of )

[0104] 5-Methyl-1H-indole-3-carboxaldehyde (977 mg, 6.14 mmol) was dissolved in pyridine in a 100 mL round-bottom flask, and malonic acid (958 mg, 9.21 mmol) was added. Piperidine was used as a catalyst, and the reaction was carried out under nitrogen protection at room temperature for 10 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 silica gel column chromatography with the following eluent: [V(dichloromethane):V(methanol) = ... [60:1], concentrated column chromatography yielded 666 mg of crude product (E3)-3-(5-methyl-1H-indol-3-yl)acrylic acid. 153 mg of this acid was dissolved in DMF in a 25 mL round-bottom flask, HOAt (124 mg, 0.91 mmol) was added, and the reaction was carried out for 10 min. Then, m-bromoaniline (83 μL, 0.76 mmol) and NMM (59 μL, 0.53 mmol) were added under ice bath conditions, and the reaction was carried out for 10 min. Finally, EDCI (174.45 mg, 0.91 mmol) was added, and the reaction was carried out at room temperature for 4 h. After the reaction was 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(petroleum ether):V(ethyl acetate) = 6:1]. The crude product obtained by concentrated column chromatography was then filtered with dichloromethane to obtain the target compound L. 21 192.60 mg of yellow solid powder, yield 37%. 1 H NMR (400MHz, DMSO-d6): δ11.63(s,1H),10.21(s,1H),8.17(s,1H),7.88–7.79(m,2H),7.76(s,1H),7.60(d,J=8.1Hz,1 H),7.39(d,J=8.3Hz,1H),7.31(t,J=8.0Hz,1H),7.23(d,J=8.0Hz,1H),7.08(d,J=8.3Hz,1H),6.76(d,J=15.6Hz,1H). 13C NMR (100MHz, DMSO-d6): δ165.4,141.5,135.9,135.9,131.9,130.8,129.5,125.3,125.1, 124.0,121.7,121.2,119.8,117.6,114.9,112.1,111.8,21.5.HRMS(ESI,m / z):Calcd.for C 18 H 15 BrN2O[M+Na] + 377.0260; found: 377.0259.

[0105] Example 22:

[0106] (E3)-3-(6-bromo-1H-indol-3-yl)-N-phenylacrylamide (L 22 Preparation of )

[0107] 6-Bromo-1H-indole-3-carboxaldehyde (972 mg, 3.98 mmol) was dissolved in pyridine in a 100 mL round-bottom flask, and malonic acid (622 mg, 5.97 mmol) was added. Piperidine was used as a catalyst, and the reaction was carried out under nitrogen protection at room temperature for 10 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 silica gel column chromatography, with the eluent being [V(dichloromethane):V(methane)]. The crude product (E3)-3-(6-bromo-1H-indol-3-yl)acrylic acid was obtained by concentrated column chromatography [V(petroleum ether):V(ethyl acetate) = 60:1]. 720 mg of the crude product was then obtained by concentrated column chromatography. 180 mg of this acid was dissolved in DMF in a 25 mL round-bottom flask, and 110 mg (0.81 mmol) of HOAt was added. After reacting for 10 min, aniline (62 μL, 0.68 mmol) and NMM (53 μL, 0.48 mmol) were added under ice bath conditions, and the reaction was continued for 10 min. Then, 155 mg (0.81 mmol) of EDCI was added, and the reaction was continued 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 silica gel column chromatography with eluent [V(petroleum ether):V(ethyl acetate) = 8:1]. The crude product obtained by concentrated column chromatography was then filtered with dichloromethane to obtain the target compound L. 22 211 mg of white solid, yield 91%. 1H NMR (400MHz, DMSO-d6): δ11.78(s,1H),10.03(s,1H),7.90(d,J=8.6Hz,1H),7.88(s,1H),7.76(d,J=9.7Hz,1H ),7.72–7.68(m,2H),7.67(d,J=1.8Hz,1H),7.40-7.27(m,3H),7.04(t,J=7.3Hz,1H),6.81(d,J=15.8Hz,1H). 13 C NMR (100MHz, DMSO-d6): δ164.8,139.8,138.4,134.1,131.9,128.8,123.9,123.3,122.9,121.6,118.9,116.9,115.1,115.1,112.4.

[0108] Example 23:

[0109] (E3)-3-(6-bromo-1H-indol-3-yl)-N-(3-bromophenyl)acrylamide (L 23 Preparation of )

[0110] 6-Bromo-1H-indole-3-carboxaldehyde (972 mg, 3.98 mmol) was dissolved in pyridine in a 100 mL round-bottom flask, and malonic acid (622 mg, 5.97 mmol) was added. Piperidine was used as a catalyst, and the reaction was carried out under nitrogen protection at room temperature for 10 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 silica gel column chromatography, with the eluent being [V(dichloromethane):V(methane)]. The crude product (E3)-3-(6-bromo-1H-indol-3-yl)acrylic acid was obtained by concentrated column chromatography [V(petroleum ether:V(ethyl acetate) = 8:1], 720 mg of which was dissolved in DMF in 25 mL round-bottom flask. HOAt (116 mg, 0.85 mmol) was added and the reaction was carried out for 10 min. Then, aniline (77 μL, 0.71 mmol) and NMM (55 μL, 0.50 mmol) were added under ice bath conditions and the reaction was carried out for 10 min. EDCI (163 mg, 0.85 mmol) was then added and the reaction was carried out 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 silica gel column chromatography with eluent [V(petroleum ether:V(ethyl acetate) = 8:1]. The crude product obtained by concentrated column chromatography was then filtered with dichloromethane to obtain the target compound L. 23160 mg of yellow solid powder, yield 54%. 1 H NMR (400MHz, DMSO-d6): δ11.92(s,1H),10.21(s,1H),8.11(s,1H),7.95(s,2H),7.79(d,J=15.1 Hz,1H),7.54(d,J=8.0Hz,1H),7.47(d,J=7.1Hz,1H),7.35–7.11(m,3H),6.81(d,J=15.6Hz,1H). 13 C NMR (100MHz, DMSO-d6): δ165.1,141.4,138.4,134.9,132.3,130.9,125.5,123.9,123. 4,121.7,121.6,121.2,117.6,116.2,115.1,115.1,112.3.HRMS(ESI,m / z):Calcd.for C 17 H 12 Br2N2O[M+Na] + 440.9208; found: 440.9203.

[0111] Example 24:

[0112] (E3)-3-(6-chloro-1H-indol-3-yl)-N-phenylacrylamide (L 24 Preparation of )

[0113] 6-Chloro-1H-indole-3-carboxaldehyde (990 mg, 5.51 mmol) was dissolved in pyridine in a 100 mL round-bottom flask, and malonic acid (1032 mg, 9.90 mmol) was added. Piperidine was used as a catalyst, and the reaction was carried out under nitrogen protection at room temperature for 10 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 silica gel column chromatography with the eluent [V(dichloromethane):V(methanol)]. The crude product (E3)-3-(6-chloro-1H-indol-3-yl)acrylic acid was obtained by concentrated column chromatography [V(petroleum ether:V(ethyl acetate) = 6:1]]. 250 mg of this acid was dissolved in DMF in a 25 mL round-bottom flask, and HOAt (185 mg, 1.36 mmol) was added. After reacting for 10 min, aniline (103 μL, 1.13 mmol) and NMM (88 μL, 0.79 mmol) were added under ice bath conditions, and the reaction was continued for 10 min. Then, EDCI (260 mg, 1.36 mmol) was added, and the reaction was continued 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 silica gel column chromatography with eluent [V(petroleum ether:V(ethyl acetate) = 6:1]. The crude product obtained by concentrated column chromatography was then filtered with dichloromethane to obtain the target compound L. 24 374.80 mg of yellow solid powder, yield 88%. 1 HNMR (400MHz, DMSO-d6): δ11.77(s,1H),10.03(s,1H),7.95(d,J=8.6Hz,1H),7.89(s,1H),7.81–7.64(m,3H ),7.54(s,1H),7.33(t,J=7.7Hz,2H),7.25(d,J=8.7Hz,1H),7.04(t,J=7.2Hz,1H),6.82(d,J=15.4Hz,1H). 13 C NMR (100MHz, DMSO-d6): δ164.9,139.8,138.0,134.2,132.0,128.9,127.1,123.7,123.0,121.3,120.8,119.0,116.9,112.4,112.2.

[0114] Example 25:

[0115] (E3)-N-(3-bromophenyl)-3-(6-chloro-1H-indole-3-yl)acrylamide (L 25 Preparation of )

[0116] Similar to Example 24, (E3)-3-(6-chloro-1H-indol-3-yl)acrylic acid was first synthesized. Then, 198 mg of this acid was dissolved in DMF in a 25 mL round-bottom flask. HOAt (146 mg, 1.07 mmol) was added, and the reaction proceeded for 10 min. Under ice bath conditions, m-bromoaniline (97 μL, 0.89 mmol) and NMM (69 μL, 0.62 mmol) were added, and the reaction proceeded for 10 min. Finally, EDCI (205 mg, 1.07 mmol) was added, and the reaction proceeded 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 using [V(petroleum ether:V(ethyl acetate) = 8:1] as the eluent. The concentrated crude product was then filtered through dichloromethane to obtain the target compound L. 25 158.30 mg of yellow solid powder was produced, with a yield of 71%. 1 H NMR (400MHz, DMSO-d6): δ11.91(s,1H),10.45(s,1H),8.14(s,1H),7.98(d,J=8.6Hz,1H),7.90(s,1H),7.77(d,J= 15.7Hz,1H),7.60(d,J=8.2Hz,1H),7.54(s,1H),7.28(t,J=8.0Hz,1H),7.24-7.17(m,2H),6.87(d,J=15.7Hz,1H). 13 C NMR (100MHz, DMSO-d6): δ165.2,141.5,138.0,134.7,132.3,130.8,127.0,125.4,123. 6,121.7,121.4,121.2,120.8,117.7,116.4,112.3,112.1.HRMS(ESI,m / z):Calcd.forC 17 H 12 BrClN2O[M+Na] + 396.9714; found: 396.9709.

[0117] Example 26:

[0118] (E3)-3-(7-bromo-1H-indol-3-yl)-N-phenylacrylamide (L) 26 Preparation of )

[0119] 7-Bromo-1H-indole-3-carboxaldehyde (972 mg, 3.98 mmol) was dissolved in pyridine in a 100 mL round-bottom flask, and malonic acid (622 mg, 5.97 mmol) was added. Piperidine was used as a catalyst, and the reaction was carried out under nitrogen protection at room temperature for 10 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 silica gel column chromatography, with the eluent being [V(dichloromethane):V(methane)]. [E3)-3-(7-bromo-1H-indol-3-yl)acrylic acid (60:1) was concentrated and column chromatography was used to obtain 460 mg of crude product (E3)-3-(7-bromo-1H-indol-3-yl)acrylic acid. 230 mg of the acid was then dissolved in DMF in a 25 mL round-bottom flask. HOAt (140 mg, 1.03 mmol) was added and reacted for 10 min. Then, aniline (78 μL, 0.86 mmol) and NMM (67 μL, 0.60 mmol) were added under ice bath conditions and reacted for 10 min. Finally, EDCI (198 mg, 1.03 mmol) was added and reacted at room temperature for 4 h. After the reaction was 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 using [V(dichloromethane:V(methanol) = 60:1] as eluent. The crude product from the concentrated column chromatography was then filtered with dichloromethane to obtain the target compound L. 26 229 mg of white solid powder, yield 78%. 1 H NMR (400MHz, DMSO-d6): δ11.89(s,1H),10.04(s,1H),7.98(d,J=8.0Hz,1H),7.92(s,1H),7.77(d,J=15.8Hz,1H),7.71(d,J=8 .0Hz,2H),7.47(d,J=7.6Hz,1H),7.33(t,J=7.8Hz,2H),7.17(t,J=7.8Hz,1H),7.04(t,J=7.4Hz,1H),6.86(d,J=15.8Hz,1H). 13 C NMR (100MHz, DMSO-d6): δ164.8,139.8,135.8,134.2,131.9,128.9,126.6,125.1,123.0,121.9,119.5,119.0,117.3,113.4,105.2.

[0120] Example 27:

[0121] (E3)-3-(7-bromo-1H-indol-3-yl)-N-(3-bromophenyl)acrylamide (L27 Preparation of )

[0122] 7-Bromo-1H-indole-3-carboxaldehyde (972 mg, 3.98 mmol) was dissolved in pyridine in a 100 mL round-bottom flask, and malonic acid (622 mg, 5.97 mmol) was added. Piperidine was used as a catalyst, and the reaction was carried out under nitrogen protection at room temperature for 10 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 silica gel column chromatography with the eluent [V(dichloromethane):V(methanol)]. The crude product (E3)-3-(7-bromo-1H-indol-3-yl)acrylic acid was obtained by concentrated column chromatography [V(petroleum ether:V(ethyl acetate) = 8:1]]. 230 mg of this acid was dissolved in DMF in a 25 mL round-bottom flask, and HOAt (140 mg, 1.03 mmol) was added. After reacting for 10 min, m-bromoaniline (94 μL, 0.86 mmol) and NMM (67 μL, 0.60 mmol) were added under ice bath conditions, and the reaction was continued for 10 min. Then, EDCI (198 mg, 1.03 mmol) was added, and the reaction was continued 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 silica gel column chromatography using [V(petroleum ether:V(ethyl acetate) = 8:1]]. The crude product obtained by concentrated column chromatography was then filtered with dichloromethane to obtain the target compound L. 27 332.90 mg of white solid powder, yield 92%. 1 H NMR (400MHz, DMSO-d6): δ11.92(s,1H),10.21(s,1H),8.11(s,1H),7.96(d,J=7.8Hz,2H),7.79(d,J=15.8Hz,1H),7.54(d,J=8 .2Hz,1H),7.47(d,J=7.7Hz,1H),7.30(t,J=8.0Hz,1H),7.23(d,J=8.1Hz,1H),7.17(t,J=7.7Hz,1H),6.81(d,J=15.8Hz,1H). 13 C NMR (100MHz, DMSO-d6): δ165.0,141.3,135.7,134.8,132.2,130.8,126.5,125.4,125.1,122.0,121.7,121.2,119.4,117.6,116.6,113.2,105.1.

[0123] Example 28:

[0124] Preparation of 3-(1H-indol-3-yl)-N-phenylpropionamide (M1)

[0125] 3-(1H-indol-3-yl)propionic acid (500 mg, 2.64 mmol) was dissolved in DMF in a 25 mL round-bottom flask. HOAT (432 mg, 1.20 mmol) was added and reacted for 10 min. Then, aniline (0.29 mL, 3.17 mmol) and NMM (206 μL, 1.85 mmol) were added under ice bath conditions and reacted for 10 min. Finally, EDCI (608 mg, 3.17 mmol) was added and the reaction was carried out 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(petroleum ether):V(ethyl acetate) = 10:1]. The crude product was obtained by concentrated column chromatography, and then the target compound M1 was obtained as a white solid with a yield of 72%. 1H NMR (400MHz, DMSO-d6): δ10.78(s,1H),9.93(s,1H),7.61(s,1H)7.59–7.55(m,2H),7.33(d,J=8.1Hz,1H),7.28(t,J=8. 0Hz, 2H), 7.13 (d, J = 2.4Hz, 1H), 7.06 (t, J = 7.2Hz, 1H), 7.04-6.94 (m, 2H), 3.02 (t, J = 7.4Hz, 2H), 8.05 (t, J = 7.4Hz, 2H). 13 C NMR (100MHz, DMSO-d6): δ171.1,139.4,136.3,128.7,127.1,123.0,122.2,121 .0,119.1,118.5,118.3,113.8,111.4,37.3,20.9.HRMS(ESI,m / z):Calcd.for C 17 H 16 N₂O[M+Na] + 287.1155; found: 287.1157.

[0126] Example 29: Preparation of N-(3-bromophenyl)-3-(1H-indol-3-yl)propionamide (M2)

[0127] 3-(1H-indol-3-yl)propionic acid (500 mg, 2.64 mmol) was dissolved in DMF in a 25 mL round-bottom flask. HOAt (432 mg, 1.20 mmol) was added and the reaction proceeded for 10 min. Then, m-bromoaniline (0.35 mL, 3.17 mmol) and NMM (206 μL, 1.85 mmol) were added under ice bath conditions and the reaction proceeded for another 10 min. Finally, EDCI (608 mg, 3.17 mmol) was added and the reaction proceeded 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 using a elution of [V(petroleum ether):V(ethyl acetate) = 10:1]. Concentration column chromatography yielded the crude product, which in turn gave the target compound M2 as a white solid with a yield of 89%. 1 HNMR (400MHz, DMSO-d6): δ10.81(s,1H),10.13(s,1H),8.02(s,1H),7.58(d,J=7.9Hz,1H),7.51(d,J=7.8Hz,1H),7.36(d,J=8.1Hz,1H ),7.29–7.19(m,2H),7.15(d,J=2.3Hz,1H),7.08(t,J=7.4Hz,1H),6.99(t,J=7.6Hz,1H),3.05(t,J=7.6Hz,2H),2.72(t,J=7.6Hz,2H). 13 C NMR (100MHz, DMSO-d6): δ171.4,140.9,136.2,130.7,127.0,125.6,122.2,121.6,121 .4,121.0,118.3,118.2,117.7,113.5,111.4,37.3,20.7.HRMS(ESI,m / z):Calcd.forC 17 H 15 BrN2O[M+Na] + 365.0260; found: 365.0266.

[0128] Bioactivity test

[0129] 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 were transfected using transfection reagents. TM The -22FcAMP plasmid was transfected into cells and incubated at 37°C with 5% CO2 for 24 hours to allow for the transcription and expression of the target gene. The transfected cells were then seeded evenly into 96-well plates and incubated at 37°C with 5% CO2 for 24 hours. Finally, the old culture medium in the 96-well plates was discarded, the cells were washed once with fresh culture medium, and then GloSensor was added. TM The cAMP Reagent, serum, and CO2-independent medium 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 for the compound, then diluted with culture medium to different concentration gradients (the DMSO concentration in the prepared compound was less than or equal to 0.1%). The prepared compound was rapidly added to 96-well plates containing HEK293T cells to begin stimulation. A rapid rise in the bioluminescent signal after the addition of the compound was observed using a multi-mode microplate reader. When the signal value reached its peak and stopped increasing, a 50 μM concentration of the compound was immediately added. Simultaneously, the bioluminescent signal was collected using the multi-mode microplate reader; the bioluminescent signal value increased with increasing compound concentration. Finally, the data were processed using GraphPadPrism8 software, with concentration on the x-axis and signal value on the y-axis to obtain a dose-response curve for the allosteric modulator.

[0130] Table 2. Statistical table of the functional activities of new indole derivatives

[0131]

[0132]

[0133]

[0134] Note: "+" indicates the presence of the corresponding activity, and "-" indicates the absence of the corresponding activity.

[0135] Table 3. Comparison of the activities of the new allosteric antagonistic derivatives with Cmpd-15.

[0136]

[0137]

[0138] Note: The value of 'a' represents the blocking activity relative to Cmpd-15.

[0139] In the Glosensor cAMP cumulative assay results, the new compounds L1, L2, L4, L6, L7, L8, L9, and L... 10 L 11 L 12 L 14 L 15 L 16 L 23 L 24 L 25 L 26 L 27 M1 and M2 both exhibit β2AR allosteric antagonistic activity, while the remaining new compounds do not (see Table 2). Most of the compounds with allosteric antagonistic activity mentioned above have better or comparable activity than Cmpd-15. Among them, the compound with the best activity is L... 10 and L 11 The allosteric antagonistic activity was significantly improved compared to Cmpd-15, with compounds L1, L4, L7, L6, L9, and L... 14 L 16 L 26 The allosteric antagonistic activity of L is also significantly improved compared to the lead compound Cmpd-15. 23 L 24 The allosteric antagonistic activity of M2 is comparable to that of Cmpd-15, while the activities of the other compounds are not as good as those of Cmpd-15, as shown in Table 3.

[0140] This invention designed and synthesized L-type and M-type compounds. The L-type compounds have a trans-indole propenyl group substituted at the 3-position of the indole, while the M-type compounds lack a double bond. As shown in Table 3, among all synthesized indoleamide compounds, compound L... 10 and L 11 The allosteric antagonistic activity is the best, about 2.65 times that of Cmpd-15. The common feature is that the trans-indole propenyl group on the left is 4-Br substituted at the 3 position of indole, which is an electron-withdrawing substitution; the cyclohexylamine and piperidine on the right are both aliphatic amines.

[0141] Most L-type compounds exhibit allosteric antagonistic activity. The strongest allosteric activity, approximately 2.65 times that of Cmpd-15, occurs when trans-3-indoleacrylic acid with a bromine atom at the 4-position of R1 is coupled with aliphatic amines such as cyclohexylamine and piperidine. Other allosteric activities include: ① trans-3-indoleacrylic acid with a bromine, chlorine, or methyl atom at the 4-position of R1 coupled with aniline or m-fluoroaniline; ② trans-3-indoleacrylic acid with a bromine atom at the 7-position of R1 coupled with aniline; ③ trans-3-indoleacrylic acid with an H atom substituent in R1 coupled with aniline or m-fluoroaniline. The antagonistic activities are all relatively strong, about 1.6 times that of Cmpd-15. When trans-3-indole acrylic acid with bromine or chlorine atom substitution at R1 is coupled with aniline or m-bromoaniline, or when 3-indole acrylic acid with H atom substitution at R1 is coupled with m-bromoaniline, the allosteric activity is comparable to that of Cmpd-15. When trans-3-indole acrylic acid with H atom substitution at R1 is coupled with 3-aminobenzyl nitrile, or when trans-3-indole acrylic acid with bromine atom substitution at R1 is coupled with m-bromoaniline, the allosteric antagonistic activity is weaker and not as good as that of Cmpd-15. The inventors discovered that when trans-3-indoleacrylic acid with electron-withdrawing or electron-donating groups substituted at the 6 or 7 positions is coupled with aniline, its antagonistic activity is comparable to or stronger than that of Cmpd-15. When R1 is at the 4 position, whether trans-3-indoleacrylic acid with electron-withdrawing or electron-donating groups is coupled with aliphatic or aromatic amines, its antagonistic activity is significantly better than that of Cmpd-15, thus yielding compounds with better activity.

[0142] Furthermore, in class M compounds, the indolepropyl group is substituted at the 3-position of indole. When 3-indolepropionic acid is coupled with aniline, its antagonistic activity is only about half that of Cmpd-15, while when coupled with m-bromoaniline, its antagonistic activity is comparable to Cmpd-15, approximately 1.15 times that of Cmpd-15. This demonstrates that indole derivatives without double bonds exhibit antagonistic activity that is either inferior to or comparable to Cmpd-15, thus highlighting the necessity of double bonds.

[0143] The cAMP accumulation assay was used to test whether the target compounds could allosterically modulate the functional activity of the endogenous ligand ISO of β2AR. As shown in Table 3, among the new compounds, L1, L2, L4, L6, L7, L8, L9, and L... 10 L 11 L 12 L 14 L 15 L 16 L 23 L 24 L 25 L 26 L 27 M1 and M2 are both negative allosteric regulators of β2AR, with the latter being the most significant at the same concentration (50 μM). 10 and L 11The pharmacological activity was 2.6 times that of the lead compound (Cmpd-15). Furthermore, the presence of active compounds at concentrations in multiples was used to further confirm whether this class of compounds were negative allosteric modulators of β2AR. The following are two compounds with good activity (e.g., L). 10 L 11 The ISO allosteric antagonism curves of L8 and the allosteric antagonism mechanism diagram of L8 are shown. Taking the indoleamide compound L8 as an example... Figure 5 As shown, when the compound concentration increases to a certain level, the ISO curve exhibits a significant downward shift, reaching the lower limit of the dose-response regulation of ISO functional activity. Further increases in compound concentration almost no longer cause the ISO dose-response curve to shift downward. Therefore, the IC50 of L8... 50 The concentration values ​​are likely between 30 μM and 60 μM, and the concentration curve shows a sharp drop, indicating that the newly synthesized indoleamide derivative is a relatively active negative allosteric antagonist of the β2-adrenergic receptor. This allosteric regulation phenomenon is consistent with previously reported allosteric antagonistic regulatory mechanisms.

[0144] In summary, these results demonstrate that replacing the peptide core structure of Cmpd-15 with an indole skeleton can yield new compounds with superior allosteric antagonistic activity. This provides direction for lead compound skeleton transitions, structural simplification, and new drug development. In particular, when an aliphatic amine is attached to the trans-3-indoleacrylic acid ring with R1 at the 4-position, the antagonistic activity is significantly enhanced. Furthermore, when an aromatic amine is attached, the antagonistic activity of trans-3-indoleacrylic acid at the 4-position, whether electron-withdrawing or electron-donating, is stronger than that of Cmpd-15. Therefore, this study 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.

[0145] 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. An indoleacrylamide derivative, characterized in that, The structure of the indoleacrylamide derivative is shown in Formula 1: Formula 1 R1 = F, Cl, Br, CN, Me, OCH3; R2= 。 2. An indoleacrylamide derivative, characterized in that, The indoleacrylamide derivatives are compounds L1, L2, L4, L6, L7, L8, L9, and L... 10 L 11 L 12 L 14 L 15 L 16 L 23 L 24 L 25 L 26 L 27 ; Compound L1 is (E3)-(1H-indol-3-yl)-N-phenylacrylamide; compound L2 is (E3)-N-(3-bromophenyl)-3-(1H-indol-3-yl)acrylamide; compound L4 is (E3)-N-(3-fluorophenyl)-3-(1H-indol-3-yl)acrylamide; compound L6 is (E3)-N-(3-cyanophenyl)-3-(1H-indol-3-yl)acrylamide; compound L7 is (E3)-3-(1H-indol-3-yl)-N-(3-methoxyphenyl)acrylamide; compound L8 is (E3)-3-(4-bromo-1H-indol-3-yl)-N-phenylacrylamide; compound L9 is (E3)-3-(4-bromo-1H-indol-3-yl)-N-(3-fluorophenyl)acrylamide; compound L... 10 It is (E3)-3-(4-bromo-1H-indol-3-yl)-N-cyclohexylacrylamide; compound L 11 (E3)-3-(4-bromo-1H-indol-3-yl)-1-(piperidin-1-yl)prop-2-en-1-one; compound L 12 It is (E3)-3-(4-chloro-1H-indol-3-yl)-N-phenylacrylamide; compound L 14 It is (E3)-3-(4-chloro-1H-indol-3-yl)-N-(3-fluorophenyl)acrylamide; compound L 15 It is (E3)-3-(4-fluoro-1H-indol-3-yl)-N-phenylacrylamide; compound L 16 It is (E3)-3-(4-methyl-1H-indol-3-yl)-N-phenylacrylamide; compound L 23 It is (E3)-3-(6-bromo-1H-indol-3-yl)-N-(3-bromophenyl)acrylamide; compound L 24 It is (E3)-3-(6-chloro-1H-indol-3-yl)-N-phenylacrylamide; compound L 25 It is (E3)-N-(3-bromophenyl)-3-(6-chloro-1H-indol-3-yl)acrylamide; compound L 26 It is (E3)-3-(7-bromo-1H-indol-3-yl)-N-phenylacrylamide; compound L 27 It is (E3)-3-(7-bromo-1H-indol-3-yl)-N-(3-bromophenyl)acrylamide.

3. The method for preparing indoleacrylamide derivatives according to claim 1, characterized in that, The preparation method includes: dissolving trans-indole acrylic acid with different substituents in N,N-dimethylformamide, adding 1-hydroxy-7-azabenzotriazole, stirring at room temperature for 10 min, adding N-methylmorpholine and aniline, aliphatic amine or heterocyclic amine with different substituents at 0°C, then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and then reacting at room temperature to obtain the indole acrylic acid amide derivatives; The trans-indole acrylic acid with different substituents is (E3)-3-(4-bromo-1H-indole-3-yl)acrylic acid, (E3)-3-(5-bromo-1H-indole-3-yl)acrylic acid, and (E3)-3-(6-bromo-1H-indole-3-yl)acrylic acid; The aniline with different substituents is m-chloroaniline; the aliphatic amine with different substituents is cyclohexylamine; and the heterocyclic amine with different substituents is piperidine.

4. The method for preparing indoleacrylamide derivatives according to claim 3, characterized in that, The preparation method specifically includes: dissolving 1 mmol of trans-indole acrylic acid with different substituents in N,N-dimethylformamide; adding 1.20 mmol of 1-hydroxy-7-azabenzotriazole, 1.20 mmol of aniline, aliphatic amine or heterocyclic amine with different substituents, and 0.70 mmol of N-methylmorpholine under ice bath conditions; reacting for 10 min; then adding 1.20 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; and reacting at room temperature for 4 h; after the reaction is complete, the reaction solution is diluted with ethyl acetate, washed with water, and dried with anhydrous sodium sulfate; after removing the solvent by rotary evaporation, the obtained crude solid product is purified by silica gel column chromatography, eluted with eluent, the product after column chromatography is concentrated, and then filtered through dichloromethane to obtain the indole acrylic acid amide derivative.

5. The method for preparing indoleacrylamide derivatives according to claim 4, characterized in that, The eluent is prepared by mixing petroleum ether and ethyl acetate in a volume ratio of 1:

1.

6. The use of the indoleacrylamide 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 indole acrylamide 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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