The use of fused-ring heterocyclic compounds or their pharmaceutically acceptable salts in the preparation of drugs for treating Parkinson's disease

Abstract

This application discloses the use of a fused-ring heterocyclic compound, or a pharmaceutically acceptable salt thereof, in the preparation of a drug for treating Parkinson's disease. In this application, the fused-ring heterocyclic compound, or a pharmaceutically acceptable salt, stereoisomer, solvent compound, or prodrug thereof, having the structure shown in general formula (I), effectively inhibits the death of dopaminergic neurons in the SNc brain region during the progression of Parkinson's disease, reduces damage to dopaminergic neurons derived from sporadic PD patients, and improves the vitality of differentiated dopaminergic neurons, thereby treating or preventing Parkinson's disease. This invention opens up new application areas for fused-ring heterocyclic compounds, particularly in the preparation of drugs for treating Parkinson's disease.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of chemical pharmaceutical applications, and particularly to the use of fused-ring heterocyclic compounds or pharmaceutically acceptable salts thereof in the preparation of drugs for treating Parkinson's disease. Background Technology

[0002] Parkinson's disease (PD) is a common, prevalent, central nervous system degenerative disease affecting the elderly. The main pathological feature is the death of dopaminergic neurons in the substantia nigra pars compacta (SNc) of the midbrain, and the deposition of Lewy bodies due to α-synuclein tangles. This leads to a decrease in striatal dopamine (DA) levels, causing clinical symptoms primarily manifested as resting tremor, rigidity, bradykinesia, and postural and gait abnormalities, severely impacting patients' quality of life. Current clinical drug treatments for PD mainly include dopamine agonists such as levodopa, levodopa enhancers such as carbidopa and selegiline, and anticholinergic drugs such as trihexyphenidyl and benzalkonium chloride. The overall goal is to maintain the function of the dopaminergic nervous system and its balance relative to the cholinergic nervous system by supplementing dopamine, thereby achieving therapeutic effects.

[0003] However, this type of treatment is not effective in reducing the degeneration and death of SNc dopaminergic neurons, and long-term use leads to decreased efficacy and side effects such as gastrointestinal reactions, cardiovascular reactions, and psychiatric symptoms. Therefore, developing more effective PD treatments is of significant clinical importance.

[0004] Transient receptor potential (TRPM2) channels are non-selective cation channels that primarily allow calcium ion permeability. They are also receptors for oxidative stress and are expressed at relatively high levels in the nervous and immune systems. They can be activated by H2O2, reactive oxygen species, and oxidative stress metabolites. Currently, no researchers have found that TRPM2 may play a significant role in the death of dopaminergic neurons in the SNc brain region during the progression of Parkinson's disease (PD). Therefore, drugs targeting TRPM2 channels have the potential to play a role in the treatment of PD. Summary of the Invention

[0005] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of the compound of the present invention that is pharmaceutically acceptable and has the pharmacological activity of the parent compound. Such salts include: salts formed by addition to an inorganic acid or an organic acid, such as nitric acid, phosphoric acid, carbonic acid, etc.; organic acids such as propionic acid, hexanoic acid, cyclopentylpropionic acid, glycolic acid, pyruvic acid, gluconic acid, stearic acid, mucoacinic acid, etc.; or salts formed when an acidic proton present on the parent compound is replaced by a metal ion, such as an alkali metal ion or an alkaline earth metal ion; or coordination compounds formed with an organic base, such as ethanolamine, diethanolamine, triethanolamine, N-methylglucosamine, etc. The pharmaceutically acceptable salts of the present invention can be synthesized from a parent compound containing an acid radical or a base by conventional chemical methods. Generally, such salts are prepared by reacting these compounds in their free acid or base form with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture of both. Generally, non-aqueous media such as ethers, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. In addition to the salt form, the compounds provided by this invention also exist in prodrug form. The prodrugs of the compounds described herein readily undergo chemical changes under physiological conditions to be converted into the compounds of this invention. Furthermore, the prodrugs can be converted into the compounds of this invention in the in vivo environment via chemical or biochemical methods.

[0006] As used herein, the terms “heteroaryl,” “heteroaryl ring,” and “heteroaryl ring” are used interchangeably to refer to a monocyclic or fused polycyclic (i.e., sharing adjacent ring atom pairs, which may be CC or NC) group in which the ring atoms are substituted by at least one heteroatom independently selected from nitrogen, oxygen, or sulfur, wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen atom may optionally be quaternized. The term “five-membered heteroaryl” as used herein refers to a heteroaryl having five ring atoms, and the term “six-membered heteroaryl” refers to a heteroaryl having six ring atoms. The terms “five-membered heteroaryl” and “six-membered heteroaryl” as used herein do not limit the inclusion of the following forms:

[0007]

[0008] As used herein, the term "heterocyclic group" is a cyclic group in which at least one carbon atom of a monocyclic ring is replaced by a heteroatom, which is a non-carbon atom, preferably an N, O, or S atom.

[0009] The purpose of this invention is to provide the use of fused-ring heterocyclic compounds or pharmaceutically acceptable salts thereof in the preparation of drugs for treating Parkinson's disease.

[0010] To address the aforementioned technical problems, a first aspect of the present invention provides the use of a fused-ring heterocyclic compound, or a pharmaceutically acceptable salt thereof, in the preparation of a drug for treating Parkinson's disease, wherein the fused-ring heterocyclic compound has the structure shown in general formula (I):

[0011]

[0012] In the formula, R 1 For unsubstituted or at least one hydrogen atom is R 1-1 Substituted heteroaryl, unsubstituted, or at least one hydrogen atom is R 1-2 Substituted phenyl, wherein R 1-1 Halogen or C 1～6 Alkyl, R 1-2 Halogen, cyano, nitro, C 1～6 Alkyl, C 1～6 Alkoxy;

[0013] Indicates a single bond or a double bond, when When it is a single bond, R 2 Hydroxyl group, C 2～12 Acyloxy or C 1～6 alkoxy; when When it is a double bond, R 2 It is an oxygen atom;

[0014] R 3 Hydroxyl group, C 1～4 Alkyl, C 1～6 Alkoxy, Or a heterocyclic group containing at least one N, O, and / or S atom, wherein Ra and Rb are independently selected from hydrogen, C, and S, respectively. 1～4 Alkyl or C 1～4 Alkoxy;

[0015] X is a carbon atom or a nitrogen atom.

[0016] In some preferred embodiments, the R 1 For unsubstituted or at least one hydrogen atom is R 1-1 Substituted heteroaryl groups, wherein the unsubstituted or at least one hydrogen atom is R 1-1 The substituted heteroaryl group is either unsubstituted or has at least one hydrogen atom replaced by R. 1-1 Substituted five-membered heteroaryl or unsubstituted or at least one hydrogen atom is R 1-1 Substituted six-membered heteroaryl; wherein, the R 1-1 Halogen or C 1～6 Alkyl group, wherein the halogen is preferably fluorine, chlorine, bromine or iodine, wherein the C 1～6 Alkyl groups are preferably C 1～4 Alkyl groups (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl);

[0017] The unsubstituted or at least one hydrogen atom is R 1-1The substituted five-membered heteroaryl group is preferably an unsubstituted group containing at least one N, O, or S atom, or at least one hydrogen atom is replaced by R. 1-1 The substituted five-membered heteroaryl group is more preferably an unsubstituted group containing one O or S atom or at least one hydrogen atom replaced by R. 1-1 The substituted five-membered heteroaryl group is most preferably furanyl or thiophene, wherein the furanyl group is preferably... The thiophene group is preferably... (the R mentioned above) 1-1 The number of R can be one or more, when there are multiple R 1-1 At that time, the R mentioned 1-1 (Same or different);

[0018] The unsubstituted or at least one hydrogen atom is R 1-1 The substituted six-membered heteroaryl group is preferably an unsubstituted group containing at least one N, O, or S atom, or at least one hydrogen atom is replaced by R. 1-1 The substituted six-membered heteroaryl group is more preferably unsubstituted or has at least one hydrogen atom replaced by R. 1-1 The substituted pyridinyl group, preferably... (the R mentioned above) 1-1 The number of R can be one or more, when there are multiple R 1-1 At that time, the R mentioned 1-1 (Same or different);

[0019] Or, the R 1 For unsubstituted or at least one hydrogen atom is R 1-2 Substituted phenyl (R) 1-2 The number of R can be one or more, when there are multiple R 1-2 At that time, the R mentioned 1-2 (Same or different), the R 1-2 Halogen, cyano, nitro, C 1～6 Alkyl, C 1～6 alkoxy group; the halogen is preferably fluorine, chlorine, bromine or iodine; the C 1～6 Alkyl groups are preferably C 1～4 Alkyl groups (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl); the C 1～6 Alkoxy groups are preferably C 1～4 Alkyl groups (e.g., methoxy or ethoxy).

[0020] In some preferred solutions, Indicates a single bond, R 2 Hydroxyl group, C 2～12 Acyloxy or C 1～6 Alkoxy; the C 2～12 Acyloxy group is preferably C 2～8 Acyloxy group, the C 2～8Acyloxy group is Where Re is C 1～7 Alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, or heptyl); the C 1～6 Alkoxy groups are preferably C 1～4 Alkyl groups (e.g., methoxy, ethoxy, or propoxy);

[0021] or, It is a double bond, R 2 It is an oxygen atom.

[0022] In some preferred solutions, R 3 Hydroxyl group, C 1～4 Alkyl, C 1～6 Alkoxy, Or a heterocyclic group containing at least one N, O, or S atom, wherein Ra and Rb are independently selected from hydrogen, C, and S, respectively. 1～4 Alkyl or C 1～4 Alkoxy; the C 1～4 The alkyl group is preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl; the C 1～6 Alkoxy groups are preferably C 1～4 Alkyl groups (e.g., methoxy, ethoxy, or propoxy); the In this context, Ra and Rb are each preferably hydrogen, methyl, ethyl, methoxy, or ethoxy (e.g.: The heterocyclic group containing at least one N, O, and / or S atom is preferably a three-membered heterocyclic group containing at least one N, O, and / or S atom, a four-membered heterocyclic group containing at least one N, O, and / or S atom, a five-membered heterocyclic group containing at least one N, O, and / or S atom, or a six-membered heterocyclic group containing at least one N, O, and / or S atom. More preferably, it is a four-membered heterocyclic group containing at least one N and / or O atom, a five-membered heterocyclic group containing at least one N and / or O atom, or a six-membered heterocyclic group containing at least one N and / or O atom. Further preferably, it is a four-membered heterocyclic group containing one or two N and / or O atoms, a five-membered heterocyclic group containing one or two N and / or O atoms, or a six-membered heterocyclic group containing one or two N and / or O atoms. Most preferably, it is a four- to six-membered cyclic imine group (e.g., ). A six-membered heterocyclic group containing two nitrogen atoms, preferably Rc is selected from hydrogen or C. 1～4 Alkoxy groups (e.g.: Or a six-membered heterocyclic group containing one nitrogen atom and one oxygen atom (e.g.: ).

[0023] In some preferred embodiments, X is a carbon atom, and the fused-ring heterocyclic compound has the structure shown in general formula (II):

[0024]

[0025] In the formula, R 1 For unsubstituted or at least one hydrogen atom is R 1-1 Substituted heteroaryl, unsubstituted, or at least one hydrogen atom is R 1-2 Substituted phenyl, wherein R 1-1 Halogen or C 1～6 Alkyl, R 1-2 Halogen, cyano, nitro, C 1～6 Alkyl, C 1～6 Alkoxy;

[0026] Indicates a single bond or a double bond, when When it is a single bond, R 2 Hydroxyl group, C 2～12 Acyloxy or C 1～6 alkoxy; when When it is a double bond, R 2 It is an oxygen atom;

[0027] R 3 Hydroxyl group, C 1～4 Alkyl, C 1～6 Alkoxy, Or a heterocyclic group containing at least one N, O, and / or S atom, wherein Ra and Rb are independently selected from hydrogen, C, and S, respectively. 1～4 Alkyl or C 1～4 Alkyl group.

[0028] In some preferred embodiments, X is a carbon atom. The fused-ring heterocyclic compound has a structure represented by general formula (III), consisting of a single bond.

[0029]

[0030] In the formula, R 1 For unsubstituted or at least one hydrogen atom is R 1-2 Substituted phenyl, wherein R 1-2 Halogen, cyano, nitro, C 1～6 Alkyl, C 1～6 Alkoxy; preferably, R 1 It is an unsubstituted phenyl group;

[0031] R 2 Hydroxyl group, C 2～12 Acyloxy or C 1～6 Alkoxy; preferably, R 2 Hydroxyl group, C 2～8 Acyloxy or C 1～4 Alkoxy, the C2～8 Acyloxy group is Where Re is C 1～7 Alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, or heptyl); the C 1～4 The alkoxy group is preferably methoxy, ethoxy, or propoxy;

[0032] R 3 Hydroxyl group, C 1～4 Alkyl, C 1～6 alkoxy or Ra and Rb are independently selected from hydrogen and C, respectively. 1～4 Alkyl or C 1～4 Alkoxy; preferably, R 3 Hydroxyl group, C 1～4 alkoxy or Ra and Rb are independently selected from hydrogen or C, respectively. 1～4 Alkyl; more preferably, R 3 Hydroxyl, methoxy, ethoxy

[0033] In some preferred embodiments, X is a carbon atom. The fused-ring heterocyclic compound has a double bond and the structure shown in general formula (Ⅳ):

[0034]

[0035] In the formula, R 1 For unsubstituted or at least one hydrogen atom is R 1-1 Substituted heteroaryl, unsubstituted or R 1-2 Substituted phenyl, wherein R 1-1 Halogen or C 1～6 Alkyl, R 1-2 Halogen, cyano, nitro, C 1～6 Alkyl, C 1～6 Alkoxy;

[0036] R 3 Hydroxyl group, C 1～4 Alkyl, C 1～6 alkoxy groups, heterocyclic groups containing at least one N, O, and / or S atom, or Ra and Rb are independently selected from hydrogen and C, respectively. 1～4 Alkyl or C 1～4 Alkoxy; preferably, R 3 Hydroxyl group, C 1～4 Alkoxy groups, four-membered heterocyclic groups containing at least one N and / or O atom, five-membered heterocyclic groups containing at least one N and / or O atom, six-membered heterocyclic groups containing at least one N and / or O atom, or Ra and Rb are each independently selected from hydrogen, hydrogen, methyl, ethyl, methoxy, or ethoxy; more preferably, R 3 Hydroxyl, methoxy, ethoxy, propoxy, four- to six-membered cyclic imino groups (e.g.: ), six-membered heterocyclic groups containing two nitrogen atoms (e.g.: ) or a six-membered heterocyclic group containing one nitrogen atom and one oxygen atom (e.g.: ).

[0037] In some preferred embodiments, the fused-ring heterocyclic compound is selected from any of the following structures:

[0038]

[0039] In some preferred embodiments, X is a nitrogen atom. The fused-ring heterocyclic compound has a structure represented by general formula (V), consisting of a single bond.

[0040]

[0041] In the formula, R 1 For unsubstituted or at least one hydrogen atom is R 1-2 Substituted phenyl, wherein R 1-2 Halogen, cyano, nitro, C 1～6 Alkyl, C 1～6 Alkoxy; preferably, R 1 An unsubstituted phenyl group or at least one hydrogen atom is halogenated or C 1～4 Alkyl-substituted phenyl; more preferably, R 1 It is an unsubstituted phenyl group;

[0042] R 2 Hydroxyl or C 1～6 Alkyl group; preferably hydroxyl group;

[0043] R 3 Hydroxyl group, C 1～4 Alkyl or C 1～6 Alkyl group; preferably C 1～6 Alkyloxy group; more preferably C 1～4 Alkoxy groups (e.g., ethoxy groups).

[0044] In some preferred embodiments, X is a nitrogen atom. The fused-ring heterocyclic compound has a double bond and the structure shown in general formula (VI):

[0045]

[0046] In the formula, R 1 For unsubstituted or at least one hydrogen atom is R 1-2Substituted phenyl, wherein R 1-2 Halogen, cyano, nitro, C 1～6 Alkyl, C 1～6 Alkoxy; preferably, R 1 An unsubstituted phenyl group or at least one hydrogen atom is halogenated or C 1～4 Alkyl-substituted phenyl;

[0047] R 3 Hydroxyl group, C 1～4 Alkyl, C 1～6 Alkyl groups, heterocyclic groups containing at least one nitrogen atom, or Ra and Rb are independently selected from hydrogen or C, respectively. 1～4 Alkyl; preferably, R 3 Hydroxyl group, C 1～4 Alkoxy (e.g., ethoxy), five- or six-membered ring imino (e.g., )or Ra and Rb are independently selected from hydrogen, methyl, or ethyl (e.g.: ).

[0048] In some preferred embodiments, the fused-ring heterocyclic compound has any of the following structures:

[0049]

[0050] A second aspect of the present invention provides a method for treating Parkinson's disease, the method comprising the step of administering to a Parkinson's disease patient an effective dose of the above-mentioned fused-ring heterocyclic compound or a pharmaceutically acceptable salt thereof.

[0051] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0052] The reagents and raw materials used in this invention are all commercially available.

[0053] The embodiments of the present invention have at least the following advantages over the prior art:

[0054] (1) This invention opens up new application areas for fused-ring heterocyclic compounds, especially in the preparation of drugs for treating Parkinson's disease.

[0055] (2) The fused-ring heterocyclic compound in this invention can effectively inhibit the death of dopaminergic neurons in the SNc brain region during the progression of Parkinson's disease, reduce damage to dopaminergic neurons from sporadic PD patients, and improve the vitality of differentiated dopaminergic neurons. Attached Figure Description

[0056] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative descriptions do not constitute a limitation on the embodiments.

[0057] Figure 1 This is a schematic diagram of the electrophysiological representative currents of the fused-ring heterocyclic compounds II-1, II-19 and II-36 described in Example 66 of the present invention inhibiting the TRPM2 channel current;

[0058] Figure 2 This is a schematic diagram of the cell viability detection results of DA-ergonomic neurons differentiated from induced pluripotent stem cells (iPSCs) derived from normal individuals and sporadic PD patients according to Embodiment 67 of the present invention;

[0059] Figure 3 This is a schematic diagram of the cell viability test results of dopaminergic neurons derived from induced pluripotent stem cells (iPSCs) of sporadic PD patients according to Embodiment 67 of the present invention after treatment with TRPM2 inhibitor II-23 and ACA;

[0060] Figure 4 This is the result of DA-ergonomic neurons derived from induced pluripotent stem cells (iPSCs) of sporadic PD patients as described in Example 67 of the present invention being treated with TRPM2 inhibitor II-23 and ACA, labeled with the Girk2 protein antibody of susceptible DA-ergonomic neurons, and stained with mitochondria using Mitotracker.

[0061] Figure 5 According to embodiment 67 of the present invention, Figure 4 A schematic diagram showing the statistical results of mitochondrial size in dopaminergic neurons induced by IPSC differentiation.

[0062] Figure 6 This is a schematic diagram of immunofluorescence staining of striatal tyrosine hydroxylase in wild-type and TRPM2 knockout mice after rotenone modeling according to Example 68 of the present invention.

[0063] Figure 7 This is a schematic diagram of the statistical results of the immunofluorescence intensity of mouse striatal tyrosine hydroxylase on the damaged side compared with the undamaged side in Example 68 of the present invention;

[0064] Figure 8 This is a schematic diagram of the statistical results of the rotating rod behavior experiment in Embodiment 68 of the present invention;

[0065] Figure 9 This is a schematic diagram of the results of the apomorphine-induced rotational behavioral experiment in the first week according to Embodiment 68 of the present invention;

[0066] Figure 10This is a schematic diagram of the results of the apomorphine-induced rotational behavioral experiment in the second week according to Embodiment 68 of the present invention. Detailed Implementation

[0067] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the various embodiments of the present invention will be described in detail below with reference to the embodiments. However, those skilled in the art will understand that many technical details have been presented in the various embodiments of the present invention to enable the reader to better understand this application. However, the technical solutions claimed in this application can be implemented even without these technical details and various changes and modifications based on the following embodiments.

[0068]

[0069] Example 1: Preparation of ethyl 5-benzoimide-7-carboxylate (II-1).

[0070] Step a1: Under nitrogen protection, pyrrole-2-carboxaldehyde (0.95 g, 10.0 mmol) and potassium carbonate (1.66 g, 12.0 mmol) were dissolved in 50 mL of anhydrous acetonitrile. Then, 2-bromo-1-phenylethane-1-one (2.3 g, 12.0 mmol) was added in portions. After complete addition, the mixture was heated to 60°C and reacted overnight. Thin-layer chromatography was used to monitor the complete reaction of the starting material. After cooling to room temperature, the potassium carbonate was removed by filtration, and the solvent was removed by vacuum distillation of the filtrate. The crude product was purified by silica gel column chromatography to obtain 1-(2-oxo-2-phenylethyl)-1H-pyrrole-2-carboxaldehyde as a white solid.

[0071] Step b: Under nitrogen protection, the compound 1-(2-oxo-2-phenylethyl)-1H-pyrrole-2-carboxaldehyde (852 mg, 4.0 mmol), ethyl propynate (470 mg, 4.8 mmol), and potassium carbonate (662 mg, 4.8 mmol) obtained in the previous step were dissolved in 25 mL of anhydrous N,N-dimethylformamide and reacted overnight at 80°C. After the reaction was monitored by thin-layer chromatography to ensure complete reaction of the starting materials, the mixture was cooled to room temperature. The reaction solution was extracted with 100 mL and 50 mL of ethyl acetate, and the aqueous phase was extracted twice with ethyl acetate (50 mL × 2). The ethyl acetate phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography to obtain a yellow solid (II-1, 63%). 1¹H NMR (500MHz, CDCl₃): δ 8.93 (m, 1H), 8.47 (d, J = 1.5Hz, 1H), 7.84 (dd, J = 8.0, 1.5Hz, 2H), 7.78 (d, J = 1.5Hz, 1H), 7.66 (tt, J = 7.5, 1.5Hz, 1H), 7.55 (t, J = 7.5Hz, 2H), 7.10 (dd, J = 4.0, 3.0Hz, 1H), 7.03 (dd, J = 4.0, 1.0Hz, 1H), 4.38 (q, J = 7.0Hz, 2H), 1.40 (t, J = 7.0Hz, 3H); HRMS (ESI): m / z predicted value C 19 H 17 NO3[M+H] + 294.1130, detected value 294.1132.

[0072] Example 2: Preparation of 5-benzoimide-7-carboxylic acid propyl ester (II-2).

[0073] According to the method of Example 1, propyl propargyl (538 mg, 4.8 mmol) was used instead of ethyl propargyl to obtain a yellow solid (II-2, 54%). 1 H NMR (500MHz, CDCl3): δ8.74(m,1H),8.42(d,J=1.0Hz,1H),7.76(dd,J= 7.5,2.0Hz,2H),7.64(d,J=2.0Hz,1H),7.53(tt,J=7.5,1.5Hz,1H),7.47(t,J=7.5Hz,2H),7 .08(dd,J=4.0,3.0Hz,1H),7.01(dd,J=4.0,1.0Hz,1H),4.33(q,J=7.0Hz,2H),1.85(m,2H), 1.04 (t, J=7.0Hz, 3H); HRMS (ESI): m / z predicted value C 18 H 15 NO3[M+H] + 308.1287, detected value 308.1295.

[0074] Example 3: Preparation of 1-(5-benzoimide-7-yl)ethane-1-one (II-3).

[0075] According to the method of Example 1, ethyl propargyl ester was replaced with propargyl 3-yn-2-one (326 mg, 4.8 mmol) to obtain a yellow solid (II-3, 57%). 1¹H NMR (500MHz, CDCl₃): δ 8.87 (m, 1H), 8.31 (m, 1H), 7.81 (dt, J = 7.5, 2.0Hz, 2H), 7.76 (d, J = 2.0Hz, 1H), 7.64 (tt, J = 7.0, 1.0Hz, 1H), 7.53 (tt, J = 7.0, 2.0Hz, 2H), 7.08 (dd, J = 4.5, 3.0Hz, 1H), 7.05 (dd, J = 4.5, 1.0Hz, 1H), 2.58 (s, 3H); HRMS (ESI): m / z predicted value C 18 H 15 NO3[M+H] + 264.1025, detected value 264.1030.

[0076] Example 4: Preparation of methyl 5-(4-fluorobenzoyl)indoleazine-7-carboxylic acid (II-4).

[0077] Step a2: Under nitrogen protection, pyrrole-2-carboxaldehyde was dissolved in anhydrous N,N-dimethylformamide. The reaction solution was cooled to 0°C in an ice bath, and then sodium hydride (0.29 g, 12.0 mmol) was added in batches. After complete addition, the reaction was carried out at 0°C for 30 minutes. Then, 2-bromo-1-(4-fluorophenyl)ethane-1-one (2.6 g, 12.0 mmol) dissolved in N,N-dimethylformamide was added dropwise. After the addition was complete, the temperature was raised to room temperature and the reaction was carried out overnight. After the reaction was monitored by thin-layer chromatography, saturated ammonium chloride solution was added dropwise to the reaction solution in an ice bath. After the addition was complete, the reaction solution was extracted with ethyl acetate and water. The aqueous phase was extracted twice with ethyl acetate. The ethyl acetate phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography to obtain a white solid, which was directly used in the next step of the reaction. This method is suitable for the synthesis of R. 1 M1 is an intermediate consisting of fluorinated benzene rings, furan rings, thiophene rings, and pyridine rings.

[0078] According to the method of Example 2, 2-bromo-1-(4-fluorophenyl)ethane-1-one (2.6 g, 12.0 mmol) was used instead of 2-bromo-1-phenylethane-1-one, and methyl propargylate (403 mg, 4.8 mmol) was used instead of ethyl propargylate to give a yellow solid (II-4, 45%). 1H NMR (500MHz, CDCl3): δ 8.86 (d, J = 2.5Hz, 1H), 8.47 (d, J = 1.5Hz, 1H), 7.89 (td, J = 5.5, 2.0Hz, 2H), 7.71 (d, J = 1.5Hz, 1H), 7.24 (t, J = 7.5Hz, 2H), 7.10 (q, J = 7.0Hz, 1H), 7.04 (dd, J = 4.0, 1.0Hz, 1H), 3.92 (s, 3H); HRMS (ESI): m / z predicted value C 17 H 12 FNO3[M+H] + 298.0897, detected value 298.0899.

[0079]

[0080] Example 5: Preparation of ethyl 5-(4-fluorobenzoyl)indoleazine-7-carboxylate (II-5).

[0081] According to the method of Example 4, methyl propargylate (346 mg, 4.8 mmol) was used instead of ethyl propargylate to obtain a yellow solid (II-5, 56%). 1 H NMR (500MHz, CDCl3): δ 8.86 (d, J = 2.5Hz, 1H), 8.47 (J = 1.5Hz, 1H), 7.88 (td, J = 5.5, 2.0Hz, 2H), 7.73 (J = 1.5Hz, 1H), 7.24 (t, J = 8.5Hz, 2H), 7.10 (dd, J = 4.5, 2.0Hz, 1H), 7.03 (dd, J = 4.5, 1.5Hz, 1H), 4.39 (q, J = 7.0Hz, 2H), 1.40 (t, J = 7.0Hz, 3H); HRMS (ESI): m / z predicted value C 18 H 14 FNO3[M+H] + 312.1036, detected value 312.1038.

[0082]

[0083] Example 6: Preparation of methyl 5-(3-fluorobenzoyl)indoleazine-7-carboxylic acid (II-6).

[0084] According to the method of Example 4, 2-bromo-1-(3-fluorophenyl)ethane-1-one (2.6 g, 12.0 mmol) was used instead of 2-bromo-1-(4-fluorophenyl)ethane-1-one to give a yellow solid (II-6, 61%). 1H NMR (500MHz, CDCl3): δ 8.95 (d, J = 2.0Hz, 1H), 8.50 (d, J = 1.5Hz, 1H), 7.77 (d, J = 1.5Hz, 1H), 7.60 (d, J = 7.5Hz, 1H), 7.54 (m, 2H), 7.36 (tdd, J = 8.5, 2.5, 1.0Hz, 1H), 7.12 (t, J = 6.5Hz, 1H), 7.06 (dd, J = 5.5Hz, 1H), 3.93 (s, 3H); HRMS (ESI): m / z predicted value C 17 H 12 FNO3[M+H] + 298.0897, detected value 298.0901.

[0085]

[0086] Example 7: Preparation of ethyl 5-(3-fluorobenzoyl)indoleazine-7-carboxylate (II-7).

[0087] According to the method of Example 4, 2-bromo-1-(3-fluorophenyl)ethane-1-one (2.6 g, 12.0 mmol) was used instead of 2-bromo-1-(4-fluorophenyl)ethane-1-one, and ethyl propynate (403 mg, 4.8 mmol) was used instead of methyl propynate to obtain a yellow solid (II-7, 58%). 1 ¹H NMR (500MHz, CDCl₃): δ 8.95 (d, J = 2.5Hz, 1H), 8.49 (d, J = 1.5Hz, 1H), 7.79 (d, J = 1.5Hz, 1H), 7.60 (dt, J = 7.5, 1.0Hz, 1H), 7.54 (m, 1H), 7.36 (tdd, J = 8.0, 2.5Hz, 1H), 7.12 (t, J = 7.0Hz, 1H), 7.06 (dd, J = 4.5, 1.0Hz, 1H), 4.39 (q, J = 7.0Hz, 2H), 1.40 (t, J = 7.0Hz, 3H); HRMS (ESI): m / z predicted value C 17 H 12 FNO3[M+H] + 312.1036, detected value 312.1039.

[0088]

[0089] Example 8: Preparation of methyl 5-(2-fluorobenzoyl)indoleazine-7-carboxylic acid (II-8).

[0090] According to the method of Example 4, 2-bromo-1-(2-fluorophenyl)ethane-1-one (2.6 g, 12.0 mmol) was used instead of 2-bromo-1-(4-fluorophenyl)ethane-1-one to give a yellow solid (II-8, 47%). 1 ¹H NMR (500MHz, CDCl₃): δ 9.24 (d, J = 2.0Hz, 1H), 8.51 (d, J = 1.0Hz, 1H), 7.79 (t, J = 1.5Hz, 1H), 7.59 (m, 2H), 7.33 (t, J = 1.5Hz, 1H), 7.24 (t, J = 9.0Hz, 1H), 7.15 (dd, J = 4.0, 2.5Hz, 1H), 7.08 (d, J = 4.0Hz, 1H), 3.90 (s, 3H); HRMS (ESI): m / z predicted value C 17 H 12 FNO3[M+H] + 298.0897, detected value 298.0898.

[0091]

[0092] Example 9: Preparation of ethyl 5-(3-chlorobenzoyl)indoleazine-7-carboxylate (II-9).

[0093] According to the method of Example 1, 2-bromo-1-(3-chlorophenyl)ethane-1-one (2.8 g, 12.0 mmol) was used instead of 2-bromo-1-phenylethane-1-one to give a yellow solid (II-9, 71%). 1 H NMR (500MHz, CDCl3): δ8.74(d,J=2.0Hz,1H), 8.26(d,J=2.0Hz,1H),7.57(d,J=1.5Hz,1H), 7.42(dd,J=7.5,1.5Hz,1H),7.36(m,2H),7.23 (dd,J=4.0,3.0Hz,1H),7.12(t,J=7.0Hz,1H),7.03(dd,J=4.5,1.0Hz,1H),4.17(q,J=7.0Hz,2H),1.41(t,J=7.0Hz,3H); HRMS(ESI):m / z predicted value C 17 H 12 ClNO3[M+H] + 328.0740, detected value 328.0745.

[0094]

[0095] Example 10: Preparation of ethyl 5-(3-bromobenzoyl)indoleazine-7-carboxylic acid (II-10).

[0096] According to the method of Example 1, 2-bromo-1-(3-bromophenyl)ethane-1-one (3.3 g, 12.0 mmol) was used instead of 2-bromo-1-phenylethane-1-one to give a yellow solid (II-10, 65%). 1 H NMR (500MHz, CDCl3): δ8.72(d,J=1.5Hz, 1H),8.24(d,J=2.0Hz,1H),7.56(m,1H),7.41(dd,J=7.0,2.0Hz,1H),7.34(m,2H),7.21(dd,J =4.0, 3.0Hz, 1H), 7.09 (t, J = 7.0Hz, 1H), 7.01 (dd, J = 4.5, 1.0Hz, 1H), 4.18 (q, J = 7.0Hz, 2H), 1.40 (t, J = 7.0Hz, 3H); HRMS (ESI): m / z predicted value C 17 H 12 BrNO3[M+H] + 372.0235, detected value 372.0238.

[0097]

[0098] Example 11: Preparation of ethyl 5-(3-methylbenzoyl)indoleazine-7-carboxylate (II-11).

[0099] According to the method of Example 1, 2-bromo-1-(m-tolyl)ethane-1-one (2.5 g, 12.0 mmol) was used instead of 2-bromo-1-phenylethane-1-one to give a yellow solid (II-11, 74%). 1 H NMR (500MHz, CDCl3): δ 8.61 (m, 1H), 8.18 (d, J = 2.0Hz, 1H), 7.49 (m, 1H), 7.35 (m, 3H), 7.17 (dd, J = 4.0, 3.0Hz, 1H), 7.08 (t, J = 7.0Hz, 1H), 7.02 (dd, J = 4.5, 1.5Hz, 1H), 4.21 (q, J = 7.0Hz, 2H), 1.41 (t, J = 7.0Hz, 3H); HRMS (ESI): m / z predicted value C 18 H 15 NO3[M+H] + 308.1287, detected value 308.1285.

[0100]

[0101] Example 12: Preparation of ethyl 5-(3-methoxybenzoyl)indoleazine-7-carboxylate (II-12).

[0102] According to the method of Example 1, 2-bromo-1-phenylethane-1-one (2.7 g, 12.0 mmol) was used instead of 2-bromo-1-phenylethane-1-one to give a yellow solid (II-12, 78%). 1 H NMR (500MHz, CDCl3): δ 8.67 (d, J = 2.0 Hz, 1H), 8.18 (d, J = 1.5 Hz, 1H), 7.47 (m, 1H), 7.36 (m, 2H), 7.21 (dd, J = 4.0, 3.0 Hz, 1H), 7.13 (t, J = 7.0 Hz, 1H), 7.04 (dd, J = 4.5, 1.0 Hz, 1H), 4.20 (q, J = 7.0 Hz, 2H), 3.64 (s, 3H), 1.41 (t, J = 7.0 Hz, 3H); HRMS (ESI): m / z predicted value C 19 H 17 NO4[M+H] + 324.1236, detected value 324.1238.

[0103]

[0104] Example 13: Preparation of methyl 5-(furan-2-carbonyl)indoleazine-7-carboxylic acid (II-13).

[0105] According to the method of Example 1, 2-bromo-1-(furan-2-yl)ethane-1-one (2.3 g, 12.0 mmol) was used instead of 2-bromo-1-phenylethane-1-one, and methyl propargylate (403 mg, 4.8 mmol) was used instead of ethyl propargylate to give a yellow solid (II-13, 65%). 1 H NMR (500MHz, CDCl3): δ8.79(d,J=3.0Hz,1H),8.47(d,J=1.0Hz,1H),8.19(d,J=1.5 Hz,1H),7.80(dd,J=1.5,0.5Hz,1H),7.35(dd,J=3.5,0.5Hz,1H),7.07(dd,J=4.5,3.0Hz,1 H), 7.01 (J=4.5, 1.0Hz, 1H), 6.68 (dd, J=4.0, 2.0Hz, 1H), 3.97 (s, 3H); HRMS (ESI): m / z predicted value C 15 H 11 NO4[M+H] + 270.0766, detected value 270.0768.

[0106]

[0107] Example 14: Preparation of ethyl 5-(furan-2-carbonyl)indoleazine-7-carboxylate (II-14).

[0108] According to the method of Example 1, 2-bromo-1-(furan-2-yl)ethane-1-one (2.3 g, 12.0 mmol) was used instead of 2-bromo-1-phenylethane-1-one to give a yellow solid (II-14, 57%). 1 H NMR (500MHz, CDCl3): δ8.72(d,J=3.0Hz, 1H),8.45(d,J=1.0Hz,1H),8.14(d,J=1.5Hz,1H),7.79(dd,J=1.5,0.5Hz,1H),7.42(dd,J= 3.5,0.5Hz,1H),7.14(dd,J=4.5,3.0Hz,1H),6.94(J=4.5,1.0Hz,1H),6.65(dd,J= 4.0, 2.0Hz, 1H), 4.37 (q, J=7.0Hz, 2H), 1.49 (t, J=7.0Hz, 3H); HRMS (ESI): m / z predicted value C 16 H 13 NO4[M+H] + 284.0923, detected value 284.0927.

[0109]

[0110] Example 15: Preparation of methyl 5-(thiophene-2-carbonyl)indoleazine-7-carboxylic acid (II-15).

[0111] According to the method of Example 1, 2-bromo-1-(thiophen-2-yl)ethane-1-one (2.4 g, 12.0 mmol) was used instead of 2-bromo-1-phenylethane-1-one, and methyl propargylate (403 mg, 4.8 mmol) was used instead of ethyl propargylate to give a yellow solid (II-15, 78%). 1H NMR (500MHz, CDCl3): δ8.65(m,1H),8.46(d,J=1.5Hz,1H),7.99(d,J=1.5Hz,1H),7.81 (dd,J=5.0,1.0Hz,1H),7.78(dd,J=4.0,1.0Hz,1H),7.24(dd,J=5.0,4.0Hz,1H),7.06 (dd, J=4.5, 3.0Hz, 1H), 7.00 (dd, J=4.5, 1.0Hz, 1H), 3.96 (s, 3H); HRMS (ESI): m / z predicted value C 15 H 11 NO3S[M+H] + 286.0538, detected value 286.0540.

[0112]

[0113] Example 16: Preparation of ethyl 5-(thiophene-2-carbonyl)indoleazine-7-carboxylate (II-16).

[0114] According to the method of Example 1, 2-bromo-1-(thiophen-2-yl)ethane-1-one (2.4 g, 12.0 mmol) was used instead of 2-bromo-1-phenylethane-1-one to give a yellow solid (II-16, 76%). 1 H NMR(500MHz, CDCl3): δ8.59(m,1H),8.37 (d,J=1.5Hz,1H),7.91(d,J=1.5Hz,1H),7.83(dd,J=4.5,1.5Hz,1H),7.75(dd,J=4.0,1.5Hz ,1H),7.21(dd,J=4.5,4.0Hz,1H),7.03(dd,J=4.5,3.0Hz,1H),6.97(dd,J=4.5,1.5Hz,1H), 4.35(q,J=7.0Hz,2H),1.47(t,J=7.0Hz,3H); HRMS(ESI):m / z predicted value C 16 H 13 NO3S[M+H] + 300.0694, detected value 300.0697.

[0115]

[0116] Example 17: Preparation of methyl 5-methylpyridinimide-7-carboxylic acid ester (II-17).

[0117] According to the method of Example 1, 2-bromo-1-(pyridin-2-yl)ethane-1-one (2.3 g, 12.0 mmol) was used instead of 2-bromo-1-phenylethane-1-one, and methyl propargylate (403 mg, 4.8 mmol) was used instead of ethyl propargylate to give a yellow solid (II-17, 78%). 1 H NMR (500MHz, CDCl3): 9.13 (m, 1H), 8.82 (m, 1H), 8.56 (d, J = 1.5 Hz, 1H), 8.23 ​​(d, J = 1.5 Hz, 1H), 8.04 (dt, J = 7.5, 1.5 Hz, 1H), 7.97 (ddd, J = 9.0, 7.0, 1.0 Hz, 1H), 7.54 (ddd, J = 8.5, 7.0, 1.0 Hz, 1H), 7.10 (dd, J = 4.5, 2.5 Hz, 1H), 7.03 (dd, J = 4.5, 1.0 Hz, 1H), 4.05 (s, 3H); HRMS (ESI): m / z predicted value C 16 H 12 N₂O₃[M+H] + 281.0926, detected value 281.0929.

[0118]

[0119] Example 18: Preparation of ethyl 5-methylpyridineimide-7-carboxylate (II-18).

[0120] According to the method of Example 1, 2-bromo-1-(pyridin-2-yl)ethane-1-one (2.3 g, 12.0 mmol) was used instead of 2-bromo-1-phenylethane-1-one to give a yellow solid (II-18, 76%). 1 H NMR (500MHz, CDCl3): δ 9.07 (m, 1H), 8.74 (m, 1H), 8.46 (d, J = 1.5Hz, 1H), 8.21 (d, J = 1.5Hz, 1H), 8.00 (dt, J = 8.0, 1.0Hz, 1H), 7.93 (ddd, J = 9.0, 7.5, 1.5Hz, 1H), 7.51 (ddd, J = 8.0, 6.5, 1.5Hz, 1H), 7.08 (dd, J = 4.5, 2.5Hz, 1H), 7.01 (dd, J = 4.5, 1.0Hz, 1H), 4.36 (q, J = 7.0Hz, 2H), 1.36 (t, J = 7.0Hz, 3H); HRMS (ESI): m / z predicted value C 17 H 14 N₂O₃[M+H] + 295.1083, detected value 295.1088.

[0121]

[0122] Example 19: Preparation of 5-benzoimide-7-carboxylic acid (II-19).

[0123] Step c: Dissolve compound II-1 (2.9 g, 10 mmol) in 30 mL of ethanol, add 10 mL of 2N sodium hydroxide solution, and react the reaction solution overnight at 80°C. After the reaction of the starting material is complete as monitored by thin-layer chromatography, remove the ethanol by vacuum distillation. Adjust the remaining solution to acidity with hydrochloric acid solution, and a yellow solid precipitates out. Filter the solution, wash the solid with water, and dry it to obtain a yellow solid (II-19, 95%). 1 H NMR (500MHz, CDCl3): δ 12.05 (s, 1H), 8.74 (d, J = 2.0Hz, 1H), 8.35 (J = 2.0Hz, 1H), 7.57 (m, 3H), 7.35 (dd, J = 7.5, 2.0Hz, 1H), 7.21 (t, J = 8.5Hz, 2H), 7.10 (dd, J = 4.5, 2.5Hz, 1H), 7.03 (dd, J = 4.5, 1.5Hz, 1H); HRMS (ESI): m / z predicted value C 16 H 11 NO3[M+H] + 266.0817, detected value 266.1818.

[0124]

[0125] Example 20: Preparation of 5-benzoimide-7-carboxamide (II-20).

[0126] Step d: Compound Ⅱ-19 (275 mg, 1 mmol), 1-hydroxybenzotriazole (64 mg, 0.3 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (229 mg, 1.2 mmol) were dissolved in 10 mL of anhydrous tetrahydrofuran. N,N-diisopropylethylamine (168 mg, 1.3 mmol) was added at room temperature, and the reaction was carried out for 8 hours at room temperature until the reaction was complete as monitored by thin-layer chromatography. Then, ammonium chloride (107 mg, 2 mmol) was added, and the reaction was carried out for 2 hours at room temperature. After the reaction of the starting materials was complete as monitored by thin-layer chromatography, the solvent was removed by vacuum distillation of the reaction solution. The crude product was purified by silica gel column chromatography to obtain a red solid (Ⅱ-20, 87%). 1HNMR (500 MHz, d6-DMSO): δ 8.75 (s, 1H), 8.50 (s, 1H), 8.10 (s, 1H), 7.82 (d, J = 7.0 Hz, 2H), 7.72 (t, J = 7.5 Hz, 1H), 7.62 (m, 3H), 7.44 (s, 1H), 7.11 (t, J = 3.5 Hz, 1H), 7.02 (d, J = 4.0 Hz, 1H); HRMS (ESI): m / z predicted value C 16 H 12 N₂O₂[M+H] + 265.0977, detected value 265.0979.

[0127]

[0128] Example 21: Preparation of 5-benzoyl-N-ethylindoleazine-7-carboxamide (II-21).

[0129] According to the method of Example 20, ethylamine (90 mg, 2.0 mmol) was used instead of ammonium chloride to obtain a yellow solid (II-21, 86%). 1 ¹H NMR (500MHz, CDCl₃): δ 8.82 (d, J = 2.0Hz, 1H), 8.01 (d, J = 1.5Hz, 1H), 7.81 (dd, J = 8.0, 1.0Hz, 2H), 7.62 (m, 2H), 7.52 (t, J = 7.5Hz, 2H), 7.05 (dd, J = 4.0, 2.5Hz, 1H), 6.91 (dd, J = 3.5, 1.0Hz, 1H), 6.01 (s, 1H), 3.47 (m, 2H), 1.24 (t, J = 7.0Hz, 3H); HRMS (ESI): m / z predicted value C 18 H 16 N₂O₂[M+H] + 293.1290, detected value 293.1292.

[0130]

[0131] Example 22: Preparation of 5-benzoyl-N,N-dimethylindoleazine-7-carboxamide (II-22).

[0132] According to the method of Example 20, dimethylamine (90 mg, 2 mmol) was used instead of ammonium chloride to obtain a yellow solid (II-22, 85%). 1¹H NMR (500MHz, CDCl₃): δ 8.82 (d, J = 2.0Hz, 1H), 7.82 (d, J = 1.5Hz, 1H), 7.79 (dd, J = 7.0, 1.5Hz, 2H), 7.60 (tt, J = 7.5, 1.0Hz, 1H), 7.50 (t, J = 8.0Hz, 2H), 7.29 (d, J = 1.5Hz, 1H), 7.00 (dd, J = 4.0, 2.5Hz, 1H), 6.86 (dd, J = 4.5, 1.0Hz, 1H), 3.08 (s, 6H). HRMS (ESI): m / z predicted value C 18 H 16 N₂O₂[M+H] + 293.1290, detected value 293.1292.

[0133]

[0134] Example 23: Preparation of 5-benzoyl-N,N-diethylindoleazine-7-carboxamide (II-23).

[0135] According to the method of Example 20, diethylamine (146 mg, 2 mmol) was used instead of ammonium chloride to obtain a yellow solid (II-23, 89%). 1 ¹H NMR (500MHz, CDCl₃): δ 8.81 (d, J = 3.0Hz, 1H), 7.79 (m, 3H), 7.61 (tt, J = 7.5, 1.0Hz, 1H), 7.50 (t, J = 8.0Hz, 2H), 7.23 (d, J = 2.0Hz, 1H), 7.04 (dd, J = 4.5, 3.0Hz, 1H), 6.84 (dd, J = 4.5, 1.5Hz, 1H), 3.43 (m, 4H), 1.19 (m, 6H); HRMS (ESI): m / z predicted value C 20 H 20 N₂O₂[M+H] + 321.1603, detected value 321.1606.

[0136]

[0137] Example 24: Preparation of 5-benzoyl-N-ethyl-N-methylindoleazine-7-carboxamide (II-24).

[0138] According to the method of Example 20, methyl ethylamine (118 mg, 2 mmol) was used instead of ammonium chloride to obtain a yellow solid (II-24, 88%). 1NMR (500MHz, CDCl3): δ 8.84 (d, J = 1.5Hz, 1H), 7.82 (m, 3H), 7.63 (t, J = 8.0Hz, 1H), 7.53 (t, J = 8.0Hz, 2H), 7.29 (m, 1H), 7.08 (dd, J = 4.0, 3.0Hz, 1H), 6.88 (dd, J = 3.5, 1.0Hz, 1H), 3.49 (m, 2H), 3.06 (m, 3H), 1.20 (m, 6H); HRMS (ESI): m / z predicted value C 19 H 18 N₂O₂[M+H] + 307.1447, detected value 307.1449.

[0139]

[0140] Example 25: Preparation of 5-benzoyl-N-methoxy-N-methylindoleazine-7-carboxamide (II-25).

[0141] According to the method of Example 20, N,O-dimethylhydroxylamine (122 mg, 2 mmol) was used instead of ammonium chloride to obtain a yellow solid (II-25, 85%). 1 NMR (500MHz, CDCl3): δ 8.82 (d, J = 1.5Hz, 1H), 7.79 (m, 3H), 7.58 (t, J = 8.0Hz, 1H), 7.49 (t, J = 8.0Hz, 2H), 7.27 (m, 1H), 7.05 (dd, J = 4.5, 3.0Hz, 1H), 6.85 (dd, J = 4.5, 1.5Hz, 1H), 3.59 (s, 2H), 3.36 (s, 3H); HRMS (ESI): m / z predicted value C 18 H 16 N₂O₃[M+H] + 309.1239, detected value 209.1243.

[0142]

[0143] Example 26: Preparation of aziridine-1-yl(5-benzoimide-7-yl) methyl ketone (II-26).

[0144] According to the method of Example 20, aziridine (122 mg, 2 mmol) was used instead of ammonium chloride to give a yellow solid (II-26, 77%). 1H NMR (500MHz, CDCl3): δ8.91(d,J=2.0Hz,1H),7.93(d,J=2.0Hz,1H),7.87(dd,J =7.5,2.0Hz,2H),7.67(tt,J=7.5,1.0Hz,1H),7.54(t,J=7.5Hz,2H),7.33(d,J=2.0Hz,1H ),7.07(dd,J=4.0,2.5Hz,1H),6.92(dd,J=4.5,1.5Hz,1H),4.06(m,4H),3.58(m,2H); HRMS (ESI):m / z predicted value C 19 H 16 N₂O₂[M+H] + 305.1290, detected value 305.1294.

[0145]

[0146] Example 27: Preparation of (5-benzoimide-7-yl)(pyrrolidine-1-yl) methyl ketone (II-27).

[0147] According to the method of Example 20, pyrrolidine (142 mg, 2 mmol) was used instead of ammonium chloride to give a yellow solid (II-27, 82%). 1 H NMR (500MHz, CDCl3): δ8.84(d,J=2.0Hz,1H),7.81(d,J=2.0Hz,1H),7.77(dd,J=8.0,1.5Hz,2H),7.59(t,J=7.5Hz,1H),7.43(t,J= 8.0Hz,2H),7.29(d,J=2.0Hz,1H),7.04(dd,J=4.0,2.5Hz,1H),6.87(dd,J=4.5,1.0Hz,1H),3.94(m,4H),2.17(m,4H); HRMS(ESI): m / z predicted value C 20 H 18 N₂O₂[M+H] + 319.1447, detected value 319.1451.

[0148]

[0149] Example 28: Preparation of (5-benzoimide-7-yl)(piperidin-1-yl)methyl ketone (II-28).

[0150] According to the method of Example 20, piperidine (170 mg, 2 mmol) was used instead of ammonium chloride to give a yellow solid (II-28, 84%). δ 8.79 (d, J = 2.0 Hz, 1H), 7.76 (d, J = 1.5 Hz, 1H), 7.62 (dd, J = 7.5, 2.0 Hz, 2H), 7.54 (t, J = 7.5 Hz, 1H), 7.40 (t, J = 7.5 Hz, 2H), 7.25 (m, 1H), 7.01 (dd, J = 4.0, 2.5 Hz, 1H), 6.84 (dd, J = 4.5, 1.5 Hz, 1H), 3.87 (m, 4H), 1.93 (m, 4H), 1.52 (m, 2H); HRMS (ESI): m / z predicted value C 21 H 20 N₂O₂[M+H] + 333.1603, detected value 333.1608.

[0151]

[0152] Example 29: Preparation of (5-benzoylindoleazine-7-yl)(piperazine-1-yl)methyl ketone (II-29).

[0153] According to the method of Example 20, piperazine (172 mg, 2 mmol) was used instead of ammonium chloride to obtain a yellow solid (II-29, 85%). 1 NMR (500MHz, CDCl3): δ8.81(d,J=2.0Hz,1H),7.76(m,3H),7.54(t,J=7.5Hz,1H),7.43(t, J=7.5Hz,2H),7.24(d,J=1.5Hz,1H),7.03(dd,J=4.5,3.0Hz,1H),6.82(dd,J =4.5,1.0Hz,1H),4.01(s,1H),3.62(m,4H),3.47(m,4H); HRMS(ESI):m / z predicted value C 20 H 19 N3O2[M+H] + 334.1556, detected value 334.1558.

[0154]

[0155] Example 30: Preparation of (5-benzoimide-7-yl)(morpholino) methyl ketone (II-30).

[0156] According to the method of Example 20, morpholine (174 mg, 2 mmol) was used instead of ammonium chloride to give a yellow solid (II-30, 81%). δ 8.82 (d, J = 1.5 Hz, 1H), 7.80 (d, J = 2.0 Hz, 1H), 7.68 (dd, J = 7.5, 2.0 Hz, 2H), 7.60 (t, J = 8.0 Hz, 1H), 7.45 (t, J = 8.0 Hz, 2H), 7.25 (d, J = 2.0 Hz, 1H), 7.07 (dd, J = 4.0, 2.5 Hz, 1H), 6.91 (dd, J = 4.5, 1.0 Hz, 1H), 4.05 (m, 4H), 3.93 (m, 4H); HRMS (ESI): m / z predicted value C 20 H 18 N₂O₃[M+H] + 335.1396, detected value 335.1399.

[0157]

[0158] Example 31: Preparation of (5-benzoimide-7-yl)(4-methylpiperazin-1-yl) methyl ketone (II-31).

[0159] According to the method of Example 20, 4-methylpiperazine (200 mg, 2 mmol) was used instead of ammonium chloride to give a yellow solid (II-31, 85%). δ 8.91 (d, J = 2.0 Hz, 1H), 7.87 (d, J = 1.5 Hz, 1H), 7.78 (dd, J = 8.0, 2.0 Hz, 2H), 7.69 (t, J = 7.5 Hz, 1H), 7.52 (t, J = 7.5 Hz, 2H), 7.31 (m, 1H), 7.11 (dd, J = 4.0, 2.5 Hz, 1H), 6.93 (dd, J = 4.5, 1.5 Hz, 1H), 3.72 (m, 4H), 2.93 (m, 4H), 2.78 (s, 3H); HRMS (ESI): m / z predicted value C 21 H 21 N3O2[M+H] + 348.1712, detected value 348.1712.

[0160]

[0161] Example 32: Preparation of N,N-diethyl-5-(2-fluorobenzoyl)indoleazine-7-carboxamide (II-32).

[0162] Step c: Dissolve compound II-8 (1.5 g, 5 mmol) in 15 mL of ethanol, add 5 mL of 2N sodium hydroxide solution, and react the reaction solution at 80°C overnight. After the reaction of the starting material is complete as monitored by thin-layer chromatography, remove the ethanol by vacuum distillation. Adjust the remaining solution to slightly acidic with hydrochloric acid solution, and a yellow solid precipitates out. Filter the solid, wash it with water, and dry it to obtain a yellow solid (1.3 g, 94%).

[0163] Step d: The yellow solid (283 mg, 1 mmol), 1-hydroxybenzotriazole (64 mg, 0.3 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (229 mg, 1.2 mmol) from the previous step were dissolved in 10 mL of anhydrous tetrahydrofuran. N,N-diisopropylethylamine (168 mg, 1.3 mmol) was added at room temperature, and the reaction was continued for 8 hours at room temperature until the reactants were completely reacted as monitored by thin-layer chromatography. Then, diethylamine (146 mg, 2 mmol) was added, and the reaction was continued for 2 hours at room temperature. After the reactants were completely reacted as monitored by thin-layer chromatography, the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography to obtain a red solid (II-32, 87%). 1 H NMR (500MHz, CDCl3): δ8.84(m,1H),7.81(d,J=1.5Hz,1H),7.55(dt,J=7.5,1.0Hz,1H),7.49(m, 2H),7.30(dddd,J=11.0,7.5,3.0,1.5Hz,1H),7.26(d,J=2.0Hz,1H),7.05(dd,J=4.5,3.0Hz ,1H),6.86(dd,J=4.5,1.5Hz,1H),3.45(q,J=7.0Hz,4H),1.12(t,J=7.0Hz,6H); HRMS(ESI): m / z predicted value C 20 H 19 FN2O2[M+H] + 339.1509, detected value 339.1510.

[0164]

[0165] Example 33: Preparation of (5-(2-fluorobenzoyl)indoleazine-7-yl)(pyrrolidine-1-yl)methyl ketone (II-33).

[0166] According to the method of Example 32, pyrrolidine (142 mg, 2 mmol) was used instead of diethylamine to give a yellow solid (II-33, 81%). 1H NMR (500MHz, CDCl3): δ8.87(m,1H),7.83(d,J=2.0Hz,1H),7.57(dt,J=8.0,1.5 Hz,1H),7.52(m,2H),7.33(1H,dddd,J=11.5,8.0,3.5,2.0Hz),7.29(1H,d,J=1.5Hz),7.08 (1H,dd,J=4.5,2.5Hz),6.89(1H,dd,J=4.5,1.0Hz),3.57(m,4H),1.82(m,4H); HRMS(ESI): m / z predicted value C 20 H 17 FN2O2[M+H] + 337.1352, detected value 337.1356.

[0167]

[0168] Example 34: Preparation of N,N-diethyl-5-pyridinimide-7-carboxamide (II-34).

[0169] According to the method of Example 32, compound II-9 was replaced with compound II-9 by using compound II-17 (1.4 g, 5 mmol) to obtain a yellow solid (II-34, 89%). 1 H NMR (500MHz, CDCl3): 9.17 (m, 1H), 8.85 (m, 1H), 8.61 (d, J = 2.0Hz, 1H), 8.27 (d, J=2.0Hz,1H),8.09(dt,J=8.0,2.0Hz,1H),7.99(ddd,J=8.5,7.5,2.0Hz,1H),7.56(ddd,J= 8.5,7.0,1.5Hz,1H),7.13(dd,J=4.5,2.5Hz,1H),7.05(dd,J=4.5,1.5Hz,1H),3.34(m,4H), 1.24(m,6H); HRMS(ESI):m / z predicted value C 19 H 19 N3O2[M+H] + 322.1556, detected value 322.1557.

[0170]

[0171] Example 35: Preparation of (5-benzoimide-7-yl)(4-methylpiperazin-1-yl) methyl ketone (II-35).

[0172] According to the method of Example 32, compound II-9 was replaced with compound II-17 (1.4 g, 5 mmol) and diethylamine was replaced with pyrrolidine (142 mg, 2 mmol) to obtain a yellow solid (II-35, 85%). 1 H NMR(500MHz,CDCl3):9.11(m,1H),8.76 (m,1H),8.57(m,1H),8.23(d,J=2.0Hz,1H),8.04(dt,J=7.5,2.0Hz,1H),7.94(ddd,J=8.0, 7.0,1.5Hz,1H),7.52(ddd,J=8.0,7.0,1.5Hz,1H),7.10(dd,J=4.5,2.5Hz,1H),7.01(dd,J= 4.5,1.0Hz,1H),3.49(m,4H)1.74(m,4H); HRMS(ESI):m / z predicted value C 19 H 17 N3O2[M+H] + 320.1399, detected value 320.1403.

[0173]

[0174] Example 36: Preparation of (5-pyridyllinpolyazine-7-yl)(piperazine-1-yl)methyl ketone (II-36).

[0175] According to the method of Example 32, compound II-9 was replaced with compound II-17, and diethylamine was replaced with piperazine (172 mg, 2 mmol), to obtain a yellow solid (II-36, 73%). 1 H NMR (500MHz, CDCl3): δ9.05(m,1H),8.71(m,1H),8.54(d,J=1.5Hz,1H),8.18(d,J=2.0Hz,1H),7.99(dt,J=7.5,2.0Hz, 1H),7.91(ddd,J=8.5,7.0,1.5Hz,1H),7.48(ddd,J=8.5,7.5,1.5Hz,1H),7.07(dd,J=4.5,2.5Hz,1H),6.98(dd,J=4.5, 1.5Hz,1H),4.13(s,1H),3.54(m,4H),3.14(m,4H); HRMS(ESI):m / z predicted value C 19 H 18 N4O2[M+H] + 335.1508, detected value 335.1510.

[0176]

[0177] Example 37: Preparation of (5-benzoimide-7-yl)(piperazin-1-yl)methyl ketone hydrochloride (II-37).

[0178] Step e: Dissolve compound II-29 (33 mg, 0.1 mmol) in diethyl ether, add hydrochloric acid methanol solution, react at room temperature for 2 hours until the reaction of the starting material is complete as monitored by thin-layer chromatography, and filter to obtain a yellow solid (II-37, 98%). 1 NMR (500MHz, CDCl3): δ 10.43 (s, 2H), 9.04 (d, J = 1.5Hz, 1H), 7.94 (m, 3H), 7.73 (t, J = 7.0Hz, 1H), 7.61 (t, J = 7.0Hz, 2H), 7.42 (d, J = 2.0Hz, 1H), 7.13 (dd, J = 4.5, 3.0Hz, 1H), 6.95 (dd, J = 4.5, 1.5Hz, 1H), 4.43 (m, 4H), 3.76 (m, 4H); HRMS (ESI): m / z predicted value C 20 H 20 ClN3O2[M] + 334.1556, detected value 334.1560.

[0179]

[0180] Example 38: Preparation of N,N-diethyl-5-pyridineimide-7-carboxamide hydrochloride (II-38).

[0181] According to the method of Example 37, compound II-29 was replaced with compound II-34 (36 mg, 0.1 mmol) to give a yellow solid (II-38, 87%). 1 H NMR (500MHz, CDCl3): 11.54 (1H, s), 9.31 (d, J = 2.0Hz, 1H), 8.95 (m, 2H), 8.46(d,J=2.0Hz,1H),8.19(t,J=8.0Hz,1H),8.13(ddd,J=8.0,7.0,2.0Hz,1H),7.72(ddd,J =8.0,7.5,1.5Hz,1H),7.23(dd,J=4.5,3.0Hz,1H),7.09(dd,J=4.5,1.0Hz,1H),3.54(m,4H), 1.37(m,6H); HRMS(ESI):m / z predicted value C 19 H 20 ClN3O2[M] + 322.1556, detected value 322.1559.

[0182]

[0183] Example 39: Preparation of (5-methylpyridinimide-7-yl)(pyrrolidine-1-yl)methyl ketone hydrochloride (II-39).

[0184] According to the method of Example 37, compound II-29 was replaced with compound II-35 (32 mg, 0.1 mmol) to obtain a yellow solid (II-39, 92%). 1 ¹H NMR (500MHz, CDCl₃): 11.27 (1H, s), 9.27 (m, 1H), 8.85 (m, 1H), 8.67 (m, 1H), 8.17 (m, 2H), 8.04 (ddd, J = 8.5, 7.0, 1.5Hz, 1H), 7.59 (ddd, J = 8.0, 7.0, 1.5Hz, 1H), 7.19 (dd, J = 4.5, 3.0Hz, 1H), 7.05 (d, J = 4.0Hz, 1H), 3.65 (m, 4H), 1.86 (m, 4H); HRMS (ESI): m / z predicted value C 19 H 18 ClN3O2[M] + 320.1399, detected value 320.1340.

[0185]

[0186] Example 40: Preparation of (5-pyridyllindoazine-7-yl)(piperazine-1-yl)methyl ketone hydrochloride (II-40).

[0187] According to the method of Example 37, compound II-29 was replaced with compound II-36 (36 mg, 0.1 mmol) to give a yellow solid (II-40, 91%). 1 H NMR(500MHz, CDCl3):10.94(1H,s),9.16(m,1H),8.87(m,1H),8.62(d, J=2.0Hz,1H),8.26(m,1H),8.05(t,J=8.0Hz,1H),7.98(ddd,J=8.0,7.5,1.5Hz,1H),7.54(d dd,J=8.0,7.5,1.5Hz,1H),7.11(dd,J=4.5,2.5Hz,1H),7.02(dd,J=4.5,1.0Hz,1H),5.34(s, 1H),3.78(m,4H),3.47(m,4H); HRMS(ESI):m / z predicted value C 19 H 19 ClN4O2[M]+ 335.1508, detected value 335.1511.

[0188]

[0189] Example 41: Preparation of 1-(5-(hydroxy(phenyl)methyl)indoleazine-7-yl)prop-1-one (II-41).

[0190] Step f: Under nitrogen protection, compound II-1 (293 mg, 1 mmol) was dissolved in anhydrous methanol. The reaction solution was kept in an ice bath at 0°C. Sodium borohydride was added to the reaction solution in batches. The reaction was carried out at 0°C for 2 hours. After the reaction of the starting material was monitored by thin-layer chromatography, the reaction solution was quenched with saturated ammonium chloride solution in an ice bath. The solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography to obtain a white solid (II-41, 97%). 1 ¹H NMR (500MHz, CDCl₃): δ 8.24 (d, J = 1.5Hz, 1H), 7.47 (dt, J = 8.0, 1.5Hz, 2H), 7.37 (m, 5H), 6.83 (dd, J = 4.0, 3.0Hz, 1H), 6.79 (dd, J = 4.5, 1.5Hz, 1H), 6.04 (d, J = 4.0Hz, 1H), 4.38 (q, J = 7.0Hz, 2H), 2.70 (d, J = 3.0Hz, 1H), 1.42 (t, J = 7.0Hz, 3H); HRMS (ESI): m / z predicted value C 18 H 17 NO3[M+H] + 296.1287, detected value 296.1290.

[0191]

[0192] Example 42: Preparation of 5-(hydroxy(phenyl)methylindoleazine-7-carboxylic acid (II-42)).

[0193] According to the method of Example 41, compound II-1 was replaced with compound II-1 (265 mg, 1 mmol) to give a white solid (II-42, 94%). 1H NMR (500MHz, CDCl3): δ12.72(s,1H),8.14(d,J=1.0Hz,1H),7.63(s,1H),7.42(d, J=7.0Hz,2H),7.36(t,J=7.0Hz,2H),7.30(tt,J=7.0,1.0Hz,1H),6.86(dd,J=3.5,2.0Hz,1 H),6.82(dd,J=4.0,1.0Hz,1H),6.38(d,J=4.0Hz,1H),6.01(d,J=5.0Hz,1H),3.34(s,1H); HRMS(ESI):m / z predicted value C 16 H 13 NO3[M+H] + 268.0974, detected value 268.0979.

[0194]

[0195] Example 43: Preparation of N-ethyl-5-(hydroxy(phenyl)methyl)indoleazine-7-carboxamide (II-43).

[0196] According to the method of Example 41, compound II-1 was replaced with compound II-21 (293 mg, 1 mmol) to give a white solid (II-43, 95%). 1 H NMR (500MHz, CDCl3): δ8.01(d,J=1.5Hz,1H),7.48(dt,J=7.0Hz,2H),7.43(d,J =2.0Hz,1H),7.36(t,J=7.0Hz,2H),7.30(t,J=7.0Hz,1H),7.21(d,J=1.5Hz,1H),6.78(dd,J= 4.0,3.0Hz,1H),6.71(dd,J=4.0,0.5Hz,1H),6.04(s,1H),4.66(s,1H),3.44(q,J=7.0Hz,2H), 1.26 (t, J=7.0Hz, 3H); HRMS (ESI): m / z predicted value C 18 H 18 N₂O₂[M+H] + 295.1447, detected value 295.1448.

[0197]

[0198] Example 44: Preparation of 5-(hydroxy(phenyl)methyl)-N,N-dimethylindoleazine-7-carboxamide (II-44).

[0199] According to the method of Example 41, compound II-1 was replaced with compound II-22 (293 mg, 1 mmol) to give a white solid (II-44, 97%). 1 ¹H NMR (500MHz, CDCl₃): δ 7.50 (d, J = 1.5Hz, 1H), 7.41 (dd, J = 7.0, 2.0Hz, 2H), 7.34 (m, 3H), 7.29 (d, J = 2.0Hz, 1H), 6.77 (m, 2H), 6.59 (dd, J = 4.0, 1.0Hz, 1H), 5.97 (s, 1H), 3.36 (s, 1H), 3.06 (s, 6H); HRMS (ESI): m / z predicted value C 18 H 18 N₂O₂[M+H] + 295.1447, detected value 295.1451.

[0200]

[0201] Example 45: Preparation of N,N-diethyl-5-(hydroxy(phenyl)methyl)indoleazine-7-carboxamide (II-45).

[0202] According to the method of Example 41, compound II-1 was replaced with compound II-23 (321 mg, 1 mmol) to give a white solid (II-45, 96%). 1 H NMR (500MHz, CDCl3): δ7.44 (d, J=1.0Hz, 1H), 7.40 (dd, J=7.5, 1.5Hz, 2H), 7.32 (m,3H),7.26(m,1H),6.75(dd,J=4.0,3.0Hz,1H),6.71(s,1H),6.56(dd,J=4.0,1 .0Hz,1H),5.95(s,1H),3.57(s,1H),3.42(m,4H)1.16(m,6H); HRMS(ESI):m / z predicted value C 20 H 22 N₂O₂[M+H] + 323.1760, detected value 323.1763.

[0203]

[0204] Example 46: Preparation of N-ethyl-5-(hydroxy(phenyl)methyl)-N-methylindoleazine-7-carboxamide (II-46).

[0205] According to the method of Example 41, compound II-1 was replaced with compound II-25 (307 mg, 1 mmol) to give a white solid (II-46, 97%). 1 ¹H NMR (500MHz, CDCl₃): δ 7.51 (s, 1H), 7.44 (d, J = 7.0Hz, 2H), 7.34 (m, 5H), 6.78 (m, 2H), 6.62 (d, J = 4.0Hz, 1H), 6.00 (s, 1H), 3.46 (m, 2H), 3.05 (s, 3H), 1.20 (m, 3H); HRMS (ESI): m / z predicted value C 19 H 20 N₂O₂[M+H] + 309.1603, detected value 309.1606.

[0206]

[0207] Example 47: Preparation of ethyl 5-(acetoxy(phenyl)methyl)indoleazine-7-carboxylate (II-47).

[0208] Step g1: Under nitrogen protection, compound II-41 (296 mg, 1 mmol) and triethylamine (122, 1.2 mmol) were dissolved in anhydrous dichloromethane. After the reaction solution was cooled to 0°C, acetyl chloride (94 mg, 1.2 mmol) was added dropwise. After the addition was complete, the reaction was carried out at room temperature for 8 hours. After the reaction of the starting materials was monitored by thin-layer chromatography, 2 mL of saturated ammonium chloride solution was added dropwise to the reaction solution under ice bath. After the addition was complete, the reaction solution was extracted with dichloromethane (15 mL) and water (15 mL). The aqueous phase was extracted twice with dichloromethane (15 mL). The dichloromethane phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography to obtain a pale yellow solid (II-47, 82%). 1 ¹H NMR (500MHz, CDCl₃): δ 8.28 (d, J = 1.0Hz, 1H), 7.44 (m, 2H), 7.39 (m, 3H), 7.34 (d, J = 2.5Hz, 1H), 7.27 (d, J = 1.0Hz, 1H), 7.19 (s, 1H), 6.86 (dd, J = 4.0, 3.0Hz, 1H), 6.83 (dd, J = 4.5, 1.0Hz, 1H), 4.40 (q, J = 7.0Hz, 2H), 2.24 (s, 3H), 1.43 (t, J = 7.0Hz, 3H); HRMS (ESI): m / z predicted value C 20 H 19 NO4[M+H] + 338.1392, detected value 338.1395.

[0209]

[0210] Example 48: Preparation of ethyl 5-(butyryloxy)(phenyl)methyl)indoleazine-7-carboxylate (II-48).

[0211] According to the method of Example 47, butyryl chloride (127 mg, 1.2 mmol) was used instead of acetyl chloride to give a pale yellow solid (II-48, 77%). 1 ¹H NMR (500MHz, CDCl₃): δ 8.28 (s, 1H), 7.39 (m, 6H), 7.27 (s, 1H), 7.21 (s, 1H), 6.86 (dd, J = 4.0, 2.5Hz, 1H), 6.83 (dd, J = 3.5, 0.5Hz, 1H), 4.40 (m, 2H), 2.40 (t, J = 7.5Hz, 2H), 1.70 (m, 2H), 1.40 (t, J = 7.0Hz, 3H), 0.97 (t, J = 7.0Hz, 3H); HRMS (ESI): m / z predicted value C 22 H 23 NO4[M+H] + 366.1705, detected value 366.1707.

[0212]

[0213] Example 49: Preparation of ethyl 5-((octyloxy)(phenyl)methyl)indoleazine-7-carboxylate (II-49).

[0214] According to the method of Example 47, octanoyl chloride (194 mg, 1.2 mmol) was used instead of acetyl chloride to give a pale yellow solid (II-49, 64%). 1 ¹H NMR (500MHz, CDCl₃): δ 8.26 (d, J = 1.0Hz, 1H), 7.43 (m, 2H), 7.36 (m, 3H), 7.31 (d, J = 2.0Hz, 1H), 7.25 (s, 1H), 7.18 (s, 1H), 6.84 (dd, J = 4.0, 2.5Hz, 1H), 6.81 (dd, J = 4.0, 1.0Hz, 1H), 4.37 (t, J = 7.5Hz, 2H), 2.37 (t, J = 7.5Hz, 2H), 1.64 (m, 2H), 1.38 (m, 9H), 0.87 (t, J = 7.0Hz, 3H); HRMS (ESI): m / z predicted value C 25 H 29 NO4[M+H] + 408.2175, detected value 408.2177.

[0215]

[0216] Example 50: Preparation of (7-(diethylcarbamoyl)indole-5-yl)(phenyl)acetic acid methyl ester (II-50).

[0217] According to the method of Example 47, compound II-41 was replaced with compound II-45 (322 mg, 1 mmol) to give a pale yellow solid (II-50, 79%). 1 H NMR (500MHz, CDCl3): δ7.46 (d, J = 1.5 Hz, 1H), 7.42 (dd, J = 7.0, 2.0 Hz, 2H), 7.34 (m, 3H), 7.21 (d, J = 2.0 Hz, 1H), 6.77 (dd, J = 4. 0,3.0Hz,1H),6.73(s,1H),6.57(dd,J=4.0,1.0Hz,1H),5.96(s,1H),3.41(m,4H),3.07(s,3H),1.19(m,6H); HRMS(ESI):m / z Predicted valueC 22 H 24 N₂O₃[M+H] + 365.1865, detected value 365.1866.

[0218]

[0219] Example 51: Preparation of methyl (7-(diethylcarbamoyl)indoleaza-5-yl)(phenyl)butyrate (II-51).

[0220] According to the method of Example 47, compound II-41 was replaced with compound II-45 (322 mg, 1 mmol) and acetyl chloride was replaced with butyryl chloride (127 mg, 1.2 mmol) to give a pale yellow solid (II-51, 72%). 1 ¹H NMR (500MHz, CDCl₃): δ 7.43 (m, 1H), 7.39 (d, J = 7.0Hz, 2H), 7.31 (m, 3H), 7.19 (d, J = 2.0Hz, 1H), 6.75 (dd, J = 4.0, 2.5Hz, 1H), 6.71 (s, 1H), 6.54 (dd, J = 4.0, 1.5Hz, 1H), 5.93 (s, 1H), 3.41 (m, 4H), 2.40 (t, J = 7.5Hz, 2H), 1.70 (m, 2H), 1.21 (m, 6H), 0.97 (t, J = 7.0Hz, 3H); HRMS (ESI): m / z predicted value C 24 H 28 N₂O₃[M+H]+ 393.2178, detected value 393.2183.

[0221]

[0222] Example 52: Preparation of (7-(diethylcarbamoyl)indole-5-yl)(phenyl)octanoate methyl ester (II-52).

[0223] According to the method of Example 47, compound II-41 was replaced with compound II-45 (322 mg, 1.0 mmol) and acetyl chloride (194 mg, 1.2 mmol) (127 mg, 1.2 mmol) was replaced with octanoyl chloride to give a pale yellow solid (II-52, 72%). 1 H NMR (500MHz, CDCl3): δ7.42 (d, J=2.0Hz, 1H), 7.37 (dd, J=7.0, 1.5Hz, 2H), 7.28 (m, 3H), 7.18(d,J=2.0Hz,1H),6.72(dd,J=4.0,2.5Hz,1H),6.69(s,1H),6.51(dd,J=4.0,1.0Hz,1H) ,5.91(s,1H),3.37(m,4H),2.35(t,J=7.5Hz,2H),1.31(m,15H),0.92(t,J=7.0Hz,3H); HRMS (ESI):m / z predicted value C 28 H 36 N₂O₃[M+H] + 449.2804, detected value 449.2805.

[0224]

[0225] Example 53: Preparation of (5-benzoimide-7-yl)(4-methylpiperazin-1-yl) methyl ketone (II-53).

[0226] Step g2: Under nitrogen protection, compound II-41 (30 mg, 0.1 mmol) was dissolved in N,N-dimethylformamide (2 mL). After the reaction solution was cooled to 0°C, sodium hydride (5 mg, 0.12 mmol) was added. After reacting at 0°C for 30 minutes, iodomethane (17 mg, 0.12 mmol) was added dropwise. After the addition was complete, the reaction was carried out at room temperature for 8 hours. After the reaction of the starting material was monitored by thin-layer chromatography, 2 mL of saturated ammonium chloride solution was added dropwise to the reaction solution under ice bath. After the addition was complete, the reaction solution was extracted with 5 mL of ethyl acetate and 5 mL of water. The aqueous phase was extracted twice with 5 mL of ethyl acetate. The ethyl acetate phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography to obtain a pale yellow solid (II-53, 85%). 1 ¹H NMR (500MHz, CDCl₃): δ 8.14 (m, 1H), 7.36 (t, J = 8.0Hz, 2H), 7.27 (m, 5H), 6.71 (dd, J = 4.0, 2.5Hz, 1H), 6.68 (dd, J = 4.5, 1.0Hz, 1H), 5.68 (s, 1H), 4.35 (q, J = 7.0Hz, 2H), 3.31 (s, 3H), 1.41 (t, J = 7.0Hz, 3H); HRMS (ESI): m / z predicted value C 19 H 19 NO3[M+H] + 310.1443, detected value 310.1447.

[0227]

[0228] Example 54: Ethyl 5-(phenyl(propoxy)methylindoleazine-7-carboxylic acid).

[0229] According to the method of Example 53, iodopropane (20 mg, 0.12 mmol) was used instead of iodomethane (II-54, 65%). δ 8.26 (d, J = 2.0 Hz, 1H), 7.35 (t, J = 8.0 Hz, 2H), 7.29 (d, J = 8.0 Hz, 2H), 7.25 (m, 3H), 6.74 (dd, J = 4.0, 2.5 Hz, 1H), 6.63 (dd, J = 4.5, 1.5 Hz, 1H), 5.72 (s, 1H), 4.37 (q, J = 7.0 Hz, 2H), 3.2 (t, J = 7.0 Hz, 2H), 1.52 (m, 2H), 1.41 (t, J = 7.0 Hz, 3H), 0.94 (t, J = 7.0 Hz, 3H); HRMS (ESI): m / z predicted value C 21 H 23 NO3[M+H] +338.1756, detected value 338.1758.

[0230]

[0231] Example 55: Preparation of methyl 5-benzoylimidazo[1,2-a]pyridine-7-carboxylic acid (ⅠII-1).

[0232] Step h: Under nitrogen protection, imidazole (680 mg, 10 mmol) and potassium carbonate (1.7 mg, 12 mmol) were added in anhydrous acetonitrile (30 mL), followed by the addition of 2-bromo-1-phenylethane-1-one (2.4 g, 12 mmol) in portions. After complete addition, the mixture was heated to 60°C and reacted overnight. Thin-layer chromatography was used to monitor the complete reaction of the starting material. The mixture was then cooled to room temperature, and potassium carbonate was removed by filtration. The solvent in the filtrate was removed by vacuum distillation. The crude product was purified by silica gel column chromatography, and the resulting white solid was used directly in the next step of the reaction.

[0233] Step i: Under nitrogen protection, the product from the previous step, 2-(1H-imidazol-1-yl)-1-phenylethane-1-one (930 mg, 5 mmol), was dissolved in anhydrous dichloromethane (30 mL), and then phosphorus oxychloride (912 mg, 6 mmol) was added. The reaction solution was kept in an ice bath at 0°C. N,N-dimethylformamide (438 mg, 6 mmol) was slowly added to the reaction solution. The reaction was carried out at 0°C for 2 hours. After the reaction was complete as monitored by thin-layer chromatography, the reaction solution was extracted with 50 mL of ethyl acetate and 50 mL of water. The aqueous phase was extracted twice with 50 mL of ethyl acetate. The ethyl acetate phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography to obtain a white solid.

[0234] Step j: Under nitrogen protection, the product obtained in the previous step, 1-(2-oxo-2-phenylethyl)-1H-imidazol-2-aminocarboxaldehyde (428 mg, 2 mmol), methyl propynate (202 mg, 2.4 mmol), and potassium carbonate (331 mg, 2.4 mmol) were dissolved in anhydrous N,N-dimethylformamide (6 mL). The reaction was carried out overnight at 80°C. After the reaction was monitored by thin-layer chromatography to ensure complete reaction of the starting materials, the mixture was cooled to room temperature. The reaction solution was extracted with 15 mL of ethyl acetate and 15 mL of water. The aqueous phase was extracted twice with 15 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography to obtain a yellow solid (III-1, 74%). 1H NMR (500MHz, CDCl3): δ 8.91 (s, 1H), 8.61 (d, J = 1.0 Hz, 1H), 7.99 (d, J = 1.0 Hz, 1H), 7.95 (d, J = 1.5 Hz, 1H), 7.83 (dt, J = 7.0, 1.5 Hz, 2H), 7.68 (tt, J = 7.5, 1.0 Hz, 1H), 7.56 (t, J = 7.5 Hz, 2H), 3.95 (s, 3H); HRMS (ESI): m / z predicted value C 16 H 12 N₂O₃[M+H] + 282.0926, detected value 282.0929.

[0235]

[0236] Example 56: Preparation of methyl 5-(4-chlorobenzoyl)imidazo[1,2-a]pyridine-7-carboxylic acid (ⅠII-2).

[0237] According to the method of Example 55, 2-bromo-1-phenylethane-1-one (2.8 g, 12 mmol) was substituted for 2-bromo-1-phenylethane-1-one to give a yellow solid (III-2, 67%). 1 ¹H NMR (500MHz, CDCl₃): δ 8.93 (d, J = 1.5Hz, 1H), 8.64 (d, J = 1.0Hz, 1H), 8.01 (d, J = 1.0Hz, 1H), 7.97 (d, J = 1.5Hz, 1H), 7.86 (d, J = 7.0Hz, 2H), 7.57 (d, J = 7.5Hz, 2H), 3.92 (3H, s); HRMS (ESI): m / z predicted value C 16 H 11 ClN2O3[M+H] + 315.0536, detected value 315.0538.

[0238]

[0239] Example 57: Preparation of methyl 5-(4-methylbenzoyl)imidazo[1,2-a]pyridine-7-carboxylic acid (ⅠII-3).

[0240] According to the method of Example 55, 2-bromo-1-phenylethane-1-one (2.5 g, 12 mmol) was substituted for 2-bromo-1-phenylethane-1-one to give a yellow solid (III-3, 82%). 1¹H NMR (500MHz, CDCl₃): δ 8.87 (m, 1H), 8.54 (d, J = 1.5Hz, 1H), 7.94 (d, J = 1.5Hz, 1H), 7.91 (d, J = 1.5Hz, 1H), 7.79 (d, J = 7.0Hz, 2H), 7.51 (d, J = 7.0Hz, 2H), 3.96 (3H, s); HRMS (ESI): m / z predicted value C 17 H 14 N₂O₃[M+H] + 295.1083, detected value 295.1088.

[0241]

[0242] Example 58: Preparation of ethyl 5-benzoylimidazo[1,2-a]pyridine-7-carboxylate (ⅠII-4).

[0243] According to the method of Example 55, ethyl propargyl ester (235 mg, 2.4 mmol) was used instead of methyl propargyl ester to obtain a yellow solid (III-4, 58%). 1 H NMR (500MHz, CDCl3): δ 8.89 (s, 1H), 8.57 (d, J = 1.0Hz, 1H), 7.96 (d, J = 1.0Hz, 1H), 7.94 (d, J = 1.5Hz, 1H), 7.80 (dt, J = 7.0, 1.5Hz, 2H), 7.66 (tt, J = 7.0, 1.5Hz, 1H), 7.54 (t, J = 7.0Hz, 2H), 4.17 (q, J = 7.0Hz, 2H), 1.41 (t, J = 7.0Hz, 3H); HRMS (ESI): m / z predicted value C 17 H 14 N₂O₃[M+H] + 295.1083, detected value 295.1085.

[0244]

[0245] Example 59: Preparation of 5-benzoimide azo[1,2-a]pyridine-7-carboxylic acid (ⅠII-5).

[0246] Step k: Dissolve compound III-4 (588 mg, 2 mmol) in 6 mL of ethanol, add 2 N sodium hydroxide solution (2 mL), stir the reaction solution at 80 °C overnight, monitor the reaction of the starting material by thin-layer chromatography until the reaction is complete, remove the ethanol by vacuum distillation, adjust the remaining solution to acidity with hydrochloric acid solution, a yellow solid precipitates, filter, wash the solid with water and dry to obtain a yellow solid (III-5, 94%). 1H NMR (500MHz, CDCl3): δ 12.27 (1H, s), 8.87 (s, 1H), 8.54 (s, 1H), 7.93 (d, J = 1.5Hz, 1H), 7.91 (d, J = 1.5Hz, 1H), 7.78 (dt, J = 7.0, 1.0Hz, 2H), 7.63 (tt, J = 7.0, 1.0Hz, 1H), 7.52 (t, J = 7.0Hz, 2H); HRMS (ESI): m / z predicted value C 15 H 10 N₂O₃[M+H] + 267.0770, detected value 267.0771.

[0247]

[0248] Example 60: Preparation of 5-benzoyl-N,N-dimethylimidazolium[1,2-a]pyridine-7-carboxamide (ⅠII-6).

[0249] Step 1: Compound III-5 (53 mg, 0.2 mmol), 1-hydroxybenzotriazole (13 mg, 0.06 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (46 mg, 0.24 mmol) were dissolved in 2 mL of anhydrous tetrahydrofuran. N,N-diisopropylethylamine (34 mg, 0.26 mmol) was added at room temperature, and the reaction was carried out for 8 hours at room temperature until the reaction of III-5 was complete as monitored by thin-layer chromatography. Then, dimethylamine (11 mg, 0.24 mmol) was added, and the reaction was carried out for 2 hours at room temperature. After the reaction of the starting materials was complete as monitored by thin-layer chromatography, the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography to obtain a yellow solid (III-6, 86%). 1 H NMR (500MHz, CDCl3): δ 8.87 (d, J = 2.0Hz, 1H), 8.55 (d, J = 1.5Hz, 1H), 7.92 (d, J = 1.5Hz, 1H), 7.87 (d, J = 1.5Hz, 1H), 7.73 (dt, J = 7.5, 1.5Hz, 2H), 7.67 (tt, J = 7.5, 1.5Hz, 1H), 7.50 (t, J = 7.0Hz, 2H), 3.27 (s, 6H); HRMS (ESI): m / z predicted value C 17 H 15 N3O2[M+H] + 294.1243, detected value 294.1245.

[0250]

[0251] Example 61: Preparation of 5-benzoyl-N-ethylimidazo[1,2-a]pyridine-7-carboxamide (ⅠII-7).

[0252] According to the method of Example 60, ethylamine (11 mg, 0.24 mmol) was used instead of dimethylamine to obtain a yellow solid (III-7, 88%). 1 ¹H NMR (500MHz, CDCl₃): δ 8.85 (m, 1H), 8.56 (d, J = 1.5Hz, 1H), 7.94 (d, J = 1.0Hz, 1H), 7.86 (d, J = 1.0Hz, 1H), 7.74 (dt, J = 7.5, 1.5Hz, 2H), 7.65 (tt, J = 7.0, 1.5Hz, 1H), 7.50 (t, J = 7.5Hz, 2H), 5.85 (m, 1H), 3.47 (m, 2H), 1.24 (t, J = 7.0Hz, 3H); HRMS (ESI): m / z predicted value C 17 H 15 N3O2[M+H] + 294.1243, detected value 294.1247.

[0253]

[0254] Example 62: Preparation of 5-benzoyl-N,N-diethylimidazolium[1,2-a]pyridine-7-carboxamide (ⅠII-8).

[0255] According to the method of Example 60, diethylamine (18 mg, 0.24 mmol) was used instead of dimethylamine to obtain a yellow solid (III-8, 79%). 1 ¹H NMR (500MHz, CDCl₃): δ 8.86 (m, 1H), 8.54 (d, J = 1.5Hz, 1H), 7.91 (d, J = 1.5Hz, 1H), 7.89 (d, J = 1.5Hz, 1H), 7.74 (dt, J = 7.0, 1.0Hz, 2H), 7.65 (tt, J = 7.0, 1.0Hz, 1H), 7.48 (t, J = 7.0Hz, 2H), 3.56 (m, 4H), 1.14 (m, 6H); HRMS (ESI): m / z predicted value C 19 H 19 N3O2[M+H] + 322.1556, detected value 322.1557.

[0256]

[0257] Example 63: Preparation of (5-benzoylimidazo[1,2-a]pyridin-7-yl)(pyrrolidine-1-yl)methyl ketone (ⅠII-9).

[0258] According to the method of Example 60, pyrrolidine (17 mg, 0.24 mmol) was used instead of dimethylamine to give a yellow solid (III-9, 73%). 1 H NMR (500MHz, CDCl3): δ 8.86 (d, J = 1.5 Hz, 1H), 8.56 (d, J = 1.5 Hz, 1H), 7.93 (d, J = 1.5 Hz, 1H), 7.87 (d, J = 1.5 Hz, 1H), 7.72 (dt, J = 7.0, 1.0 Hz, 2H), 7.63 (tt, J = 7.0, 1.0 Hz, 1H), 7.50 (t, J = 7.0 Hz, 2H), 3.63 (m, 4H), 1.43 (m, 4H); HRMS (ESI): m / z predicted value C 19 H 17 N3O2[M+H] + 320.1399, detected value 320.1402.

[0259]

[0260] Example 64: Preparation of (5-benzoylimidazolium[1,2-a]pyridin-7-yl)(piperidin-1-yl)methyl ketone (ⅠⅡ-10).

[0261] According to the method of Example 60, replacing dimethylamine with piperidine (20 mg, 0.24 mmol) yielded a yellow solid (III-10, 77%). 1 ¹H NMR (500MHz, CDCl₃): δ 8.89 (m, 1H), 8.55 (d, J = 1.0Hz, 1H), 7.93 (d, J = 1.5Hz, 1H), 7.90 (d, J = 1.5Hz, 1H), 7.72 (dt, J = 7.0, 1.0Hz, 2H), 7.67 (tt, J = 7.0, 1.0Hz, 1H), 7.50 (t, J = 7.0Hz, 2H), 3.74 (m, 4H), 1.95 (m, 4H), 1.47 (m, 2H); HRMS (ESI): m / z predicted value C 20 H 19 N3O2[M+H] + 334.1556, detected value 334.1558.

[0262]

[0263] Example 65: Preparation of ethyl 5-(hydroxy(phenyl)methylimidazo[1,2-a]pyridine-7-carboxylate (ⅠII-11).

[0264] Step m: Under nitrogen protection, compound III-4 (294 mg, 1 mmol) was dissolved in anhydrous methanol (4 mL). The reaction solution was kept in an ice bath at 0°C. Sodium borohydride (152 mg, 4 mmol) was added to the reaction solution in portions. The reaction was carried out at 0°C for 2 hours. After the reaction was completed by thin-layer chromatography, the reaction solution was quenched with 5 mL of saturated ammonium chloride solution in an ice bath. The solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography to obtain a white solid (III-11, 93%). 1 H NMR (500 MHz, CDCl3): δ 8.37 (m, 1H), 7.51 (d, J = 1.5 Hz, 1H), 7.28 (m, 2H), 7.21 (dt, J = 7.0, 1.5 Hz, 2H), 7.14 (tt, J = 7.0, 1.5 Hz, 1H), 7.03 (t, J = 7.0 Hz, 2H), 5.83 (s, 1H), 4.52 (s, 1H), 4.15 (q, J = 7.0 Hz, 2H), 1.40 (t, J = 7.0 Hz, 3H); HRMS (ESI): m / z predicted value C 17 H 16 N₂O₃[M+H] + 297.1239, detected value 297.1243.

[0265] Example 66: Biological Evaluation

[0266] The fused-ring heterocyclic compound of this invention has an inhibitory effect on the TRPM2 channel. Its pharmacological activity was determined using whole-cell patch-clamp technique, and the corresponding steps are as follows:

[0267] 1) Cell preparation

[0268] HEK293 cells stably expressing the human TRPM2 channel were incubated in DMEM / F-12 medium containing 10% bovine serum, 50 units / mL penicillin, and 50 mg / mL streptomycin at 5% CO2 and 37°C.

[0269] 2) Electrophysiological testing

[0270] Cells were preserved in extracellular fluid (147mM NaCl, 2mM KCl, 1mM MgCl2, 2mM CaCl2, 10mM HEPES, 13mM glucose, pH 7.4) before testing. The electrode was filled with electrode internal fluid (147mM NaCl, 0.05mM EGTA, 1mM MgCl2, 10mM HEPES, 0.5mM ADPR, pH 7.3), and the resistance was maintained at 3–5 MΩ.

[0271] ADPR was administered intracellularly via electrodes, with a concentration of 500 μM used to activate TRPM2. Once a steady current was detected, the extracellular fluid was perfused with a corresponding concentration of fused-ring heterocyclic compound II-1 for at least 60 seconds. Changes in current magnitude were recorded using a voltage ramp mode, with the voltage changing from -100 mV to +100 mV over 500 ms. The normal clamping voltage was 0 mV, and each TRPM lasted 5 seconds. Finally, the extracellular fluid was replaced with a solution at pH 5.0 to block the TRPM2 current. The electrophysiological current test results are shown in Table 1, and the obtained electrophysiological current graphs are shown in [Table 1]. Figure 1 .

[0272] Similarly, other fused-ring heterocyclic compounds (II-2 to II-54, III-1 to III-11) were administered in extracellular fluid at corresponding concentrations, and electrophysiological tests were performed using the same method. The results are shown in Table 1. Representative electrophysiological current maps showing the inhibition of TRPM2 channel current by some fused-ring heterocyclic compounds (II-9, II-19, and II-36) are shown in Table 1. Figure 1 .

[0273] Table 1 shows the IC50 values ​​for TRPM2 inhibition by some fused-ring heterocyclic compounds. 50 data

[0274]

[0275] Example 67: Using TRPM2 inhibitor II-23 and ACA (a non-specific TRPM2 inhibitor) to improve damage to dopaminergic neurons differentiated from iPSCs derived from PD patients.

[0276] Induced pluripotent stem cells (iPSCs) derived from normal individuals and sporadic PD patients were induced to differentiate into mature dopaminergic neurons and then treated with the TRPM2 inhibitors ACA and II-23. The drug treatment methods are as follows:

[0277] a. Calculate the experimental materials, and preparation should begin on day 8 of dopaminergic neuron differentiation. Culture dishes should first be treated overnight with 33 μg / mL PDL, then rinsed with sterile water. After drying, add 2 μg / mL 1a minin and incubate overnight. Both PDL and 1a minin can be recycled and reused.

[0278] b. Collect differentiated cells using Accutase digestion, count them using a cell counting chamber, and then dilute them to 500,000 / mL using maturation medium (with Y-27632 added). For fluorescence confocal imaging, seed 2 mL / well using a confocal dish; for cell viability assay, seed 100 μL / well using a 96-well plate. Mark day 10 as the starting point. On day 11, replace the medium with one without Y-27632, changing the medium every two days.

[0279] C. When the cells have been cultured for 15 days, prepare the drug to the final concentration using the culture medium. The concentration of ACA is 0.5 μM and the working concentration of II-23 is 0.6 μM. Change the drug-containing culture medium every four days.

[0280] The experimental results are as follows:

[0281] 1) Improved viability of differentiated dopaminergic neurons. Using Cell Titter Glo (CTG kit, Promega), 100 μL / well of CTG reaction solution was added to a 96-well plate and incubated at room temperature for 10 minutes. Fluorescence intensity was read and statistically analyzed using a microplate reader. Results of cell viability testing of dopaminergic neurons differentiated from induced pluripotent stem cells (iPSCs) derived from normal individuals and sporadic PD patients are shown below. Figure 2 The results of cell viability assays of dopaminergic neurons derived from induced pluripotent stem cells (iPSCs) differentiated from sporadic PD patients after treatment with TRPM2 inhibitor II-23 and ACA are shown in the figure. Figure 3 .

[0282] The results showed that, compared with normal individuals, the dopaminergic neurons differentiated from sporadic PD patients had significantly reduced cell viability, which was restored after treatment with II-23 and ACA.

[0283] 2) Reduce the excessive mitochondrial fusion morphology of differentiated DA neurons from PD patients.

[0284] Dopamine-positive neurons were labeled with an antibody against Girk2, a marker molecule for dopaminergic neurons susceptible to SNc, and cellular mitochondria were labeled with the mitochondrial dye mitotracker. Figure 4Specific method: Dissolve Mito-tracker Red (25mM) in mature DA neuron culture medium (1:500, working concentration 50uM). Add probe solution at 700μL per dish. Incubate at 37℃, 30% CO2 for 90-120 minutes. Then add 700μL of 4% PFA (1:1 with culture medium) and fix for 10 minutes. Remove the PFA-culture medium mixture and add 700μL of 4% PFA for post-fixation for 10 minutes. Subsequently, block: remove PFA, wash twice with 1×PBS, and block with 1% BSA (with 0.1% Triton added) at room temperature for 3 hours (blocking solution preparation: 0.5g BSA, diluted to 50mL with PBS, filtered through a 0.45μm PVDF membrane, and finally add 50μL Triton, shake well, and pre-cool before use). Next, primary antibody labeling was performed (human Girk2 primary antibody was dissolved in the above blocking buffer (recommended ratio 1:800–1:500), antibody was added at 400 μL per dish, and incubated overnight at 4°C) and secondary antibody labeling was performed (after removing the primary antibody, washing three times with 1×PBS, dissolving the secondary antibody in the above blocking buffer (recommended ratio 1:1000), antibody was added at 400 μL per dish, and incubated overnight at 4°C). Finally, fluorescence imaging was performed: after removing the secondary antibody, washing three times with 1×PBS, 50% Glycerol was added for fluorescence protection, and imaging was then performed using a Zeiss LSM880 laser confocal microscope. See [link to documentation]. Figure 4 Further investigation Figure 4 The size of mitochondria was statistically analyzed, and the results are shown in [the table below]. Figure 5 .

[0285] The results showed that mitochondrial excessive fusion occurred in dopaminergic neurons derived from PD patients. This phenomenon could be reversed by using TRPM2 inhibitor II-23 and ACA, indicating that TRPM2 inhibitors can alleviate the state of excessive mitochondrial fusion in the SPD control group.

[0286] Example 68: In vivo study of TRPM2 gene knockout-induced damage and behavioral changes induced by rotenone using TRPM2-KO mice (i.e., TRPM2 gene knockout mice).

[0287] 1) Stereotactic injection of the brain

[0288] C57-WT and TRPM-2-KO mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (0.06 mL / 10 g body weight) and then fixed on a stereotaxic instrument. The top of the head was disinfected with povidone-iodine, the hair on the surface of the head was trimmed, and the head was disinfected again with povidone-iodine. A midsagittal incision was made, and the skin on both sides of the incision was clamped with arterial clamps. The skin was wiped with cotton swabs soaked in 30% H2O2 to expose the anterior fontanelle. According to the mouse brain atlas, the position was determined (right forebrain medial fasciculus: AP-1.2 mm ML 1.1 mm DV 5.1 mm; right substantia nigra: AP-3.1 mm ML 1.2 mm DV 4.5 mm). A micro-perforated dental drill was used to make a hole, and a micro-syringe was used to slowly insert the needle (rotenone: 2.5 μg (2.5 μL, 1 μL / min); solvent: (2.5 μL, 1 μL / min)). After the procedure, the animal was sutured and placed in a 37°C constant temperature incubator. Once it regained consciousness, it was returned to its cage and allowed to eat freely. For three days post-surgery, it was given sugar water or diluted antibiotic solution, and wet food was added to its cage. 0.3 mL of normal saline was injected subcutaneously before surgery to prevent dehydration during the procedure. Erythromycin eye ointment was applied to its eyes, and rectal temperature was measured using a thermometer. During the entire procedure, a warming pad was used to keep it warm.

[0289] 2) Behavioral testing

[0290] 2.1 Apomorphine-induced rotational behavioral experiment

[0291] On days 7 and 14 after rotenone-induced modeling, apomorphine-induced rotational behavior was assessed. First, the animals were placed in the test area to acclimatize to their surroundings for 10 minutes. Then, apomorphine solution (APO, 0.5 mg / kg) was injected subcutaneously into the neck and back to induce rotational behavior. One complete 360° rotation was counted as one revolution, and the number of revolutions recorded within one hour was tracked. The entire testing process was conducted in a quiet environment. The results of the apomorphine-induced rotational behavior experiment are shown below. Figure 9 and Figure 10 .

[0292] 2.2 Rotating Rod Experiment

[0293] The rotarod test is a behavioral assessment of motor coordination. The length of time an animal remains on the rotarod measures its balance, coordination, physical condition, and movement. The longer a mouse remains on the rotarod, the better its motor coordination. Statistical results of the rotarod behavioral test are shown below. Figure 7 .

[0294] Adaptation period: Place the mouse in the center of the rotarod with its head facing inward and its tail at a 45-degree angle. The training adaptation period is in uniform speed mode, with the rotarod rotating at 5 rpm, 1 minute per mouse, three times a day.

[0295] Experimental period: After placing the mice on the rotundus for 2 minutes to acclimatize, the uniform acceleration experimental mode was activated, with a rotation speed of 5-30 rpm for 5 minutes. After the experiment started, the mice would run or fall off the rotundus continuously. After 5 minutes, the rotundus would automatically stop. The system could record the time the mice stayed on the rotundus. A non-continuous measurement method was used, with measurements taken again after a two-hour interval. A total of three measurements were taken and the average value was taken.

[0296] 3) Immunohistochemistry

[0297] After cardiac perfusion and fixation in mice, brain tissue was removed and fixed in 4% PFA for 24 hours. Following dehydration with 30% sucrose for three days, striatal brain slices (20 μm / slice) were obtained using a cryostat. One slice was retained every six slices for immunohistochemical staining of one specific indicator. After staining, the tissue was observed, quantified, and photographed under a microscope. The staining results are shown below. Figure 6 The optical density values ​​of TH staining in the striatum were quantitatively analyzed using ImageJ image analysis software. The results are shown in [Figure number missing]. Figure 7 .

[0298] The specific steps for immunofluorescence staining (floating method) are as follows: Brain slices are washed with PBS (0.1 mol / L, pH 7.4), then incubated with blocking and perforation solution (4% BSA dissolved in 1% PBST) at room temperature on a horizontal shaker for 2 hours. Without washing, the slices are incubated overnight at 4°C on a horizontal shaker in a chromatography refrigerator (TH: 1:5000, catalog number Sigma: MAB318). The next day, all brain slices are warmed to room temperature for 2 hours, washed with PBS (5 minutes × 3), incubated with fluorescent mouse secondary antibody in the dark for 2 hours, washed with PBS (5 minutes × 3), transferred to glass slides, air-dried, and mounted with approximately 60 μL of DAPI mounting medium.

[0299] 4) Results

[0300] like Figure 6 and Figure 7 As shown, tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine synthesis. In the mouse rotenone group (Rot), the number of TH-positive fibers in the striatum of the unilaterally lesioned substantia nigra and the lesioned medial forebrain was significantly reduced compared to the control group. However, in the TRPM2-KO rotenone model group, the number of TH-positive fibers in the striatum was significantly increased compared to the WT rotenone model group, suggesting that TRPM2 gene knockout can protect against the loss of TH-positive nerve fibers in the striatum induced by rotenone, thereby improving Parkinson's disease. Figure 8 As shown, the residence time on the rotator in the WT-ROT group was significantly lower than that in the WT control group, while the residence time in the rotator in the TRPM2-KO-ROT group was significantly higher than that in the WT-ROT group. Figure 9 and 10As shown, in the apomorphine-induced spinning behavior experiment, the number of spins per hour for TRPM2-KO-ROT was significantly lower than that for WT-ROT. These behavioral experiments combined indicate that TRPM2-KO can protect against rotenone-induced behavioral changes.

[0301] The above experiments show that the fused-ring heterocyclic compound of the present invention has an inhibitory effect on the TRPM2 channel. Inhibition of the TRPM2 channel can reduce the excessive mitochondrial fusion morphology of differentiated dopaminergic neurons from PD patients and improve the damage of dopaminergic neurons differentiated from iPSCs from PD patients.

[0302] 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 may be made without departing from the spirit and scope of the present invention.

Claims

1. The use of a fused-ring heterocyclic compound, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating Parkinson's disease, said fused-ring heterocyclic compound having the structure shown in general formula (I): I In the formula, R 1 For unsubstituted or at least one hydrogen atom is R 1-1 Substituted five- or six-membered heteroaryl groups, wherein the five-membered heteroaryl group is furanyl or thiophene, and the six-membered heteroaryl group contains a pyridinyl group; or R 1 For unsubstituted or at least one hydrogen atom is R 1-2 Substituted phenyl; wherein, R 1-1 Halogen or C 1~6 Alkyl, R 1-2 Halogen, cyano, nitro, C 1~6 Alkyl, C 1~6 Alkoxy; Indicates a single bond or a double bond, when When it is a single bond, R 2 Hydroxyl group, C 2~12 Acyloxy or C 1~6 alkoxy; when When it is a double bond, R 2 It is an oxygen atom; R 3 Hydroxyl group, C 1~4 Alkyl, C 1~6 Alkoxy, aziridine, pyrrolidinyl, piperidinyl, piperazine, or morpholinyl or Ra and Rb are independently selected from hydrogen and C, respectively. 1~4 Alkyl or C 1~4 Alkoxy; X is a carbon atom or a nitrogen atom.

2. The application according to claim 1, characterized in that, The R 1-1 It is fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl; and The R 1-2 It can be fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, or ethoxy. And / or, Representing a single bond, the R 2 Hydroxyl, methoxy, ethoxy, propoxy or Where Re is C 1~7 alkyl; or, It is a double bond, R 2 It is an oxygen atom; And / or, R 3 The compounds are hydroxyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, C 1~4 Alkoxy, aziridine, pyrrolidinyl, piperidinyl, piperazine, morpholinyl , , , , or .

3. The application according to claim 1, characterized in that, The fused-ring heterocyclic compound has the structure shown in general formula (II): II In the formula, R 1 For unsubstituted or at least one hydrogen atom is R 1-1 Substituted five- or six-membered heteroaryl groups, wherein the five-membered heteroaryl group is furanyl or thiophene, and the six-membered heteroaryl group is pyridinyl; or R 1 For unsubstituted or at least one hydrogen atom is R 1-2 Substituted phenyl; wherein, R 1-1 Halogen or C 1~6 Alkyl, R 1-2 Halogen, cyano, nitro, C 1~6 Alkyl, C 1~6 Alkoxy; Indicates a single bond or a double bond, when When it is a single bond, R 2 Hydroxyl group, C 2~12 Acyloxy or C 1~6 alkoxy; when When it is a double bond, R 2 It is an oxygen atom; R 3 Hydroxyl group, C 1~4 Alkyl, C 1~6 Alkoxy, aziridine, pyrrolidinyl, piperidinyl, piperazine, or morpholinyl or Ra and Rb are independently selected from hydrogen and C, respectively. 1~4 Alkyl or C 1~4 Alkyl group.

4. The application according to claim 3, characterized in that, The fused-ring heterocyclic compound has the structure shown in general formula (III): III In the formula, R 1 For unsubstituted or at least one hydrogen atom is R 1-2 Substituted phenyl, wherein R 1-2 Halogen, cyano, nitro, C 1~6 Alkyl, C 1~6 Alkoxy; R 2 Hydroxyl group, C 2~12 Acyloxy or C 1~6 Alkoxy; R 3 Hydroxyl group, C 1~4 Alkyl, C 1~6 alkoxy or Ra and Rb are independently selected from hydrogen and C, respectively. 1~4 Alkyl or C 1~4 Alkyl group.

5. The application according to claim 3, characterized in that, The fused-ring heterocyclic compound has the structure shown in general formula (Ⅳ): Ⅳ In the formula, R 1 For unsubstituted or at least one hydrogen atom is R 1-1 Substituted five- or six-membered heteroaryl groups, wherein the five-membered heteroaryl group is furanyl or thiophene, and the six-membered heteroaryl group is pyridinyl; or R 1 For unsubstituted or at least one hydrogen atom is R 1-2 Substituted phenyl; wherein, R 1-1 Halogen or C 1~6 Alkyl, R 1-2 Halogen, cyano, nitro, C 1~6 Alkyl, C 1~6 Alkoxy; R 3 Hydroxyl group, C 1~4 Alkyl, C 1~6 Alkoxy, aziridine, pyrrolidinyl, piperidinyl, piperazine, morpholinyl or Ra and Rb are independently selected from hydrogen and C, respectively. 1~4 Alkyl or C 1~4 Alkyl group.

6. The application according to any one of claims 1 to 5, characterized in that, The fused-ring heterocyclic compound is selected from any of the following structures: 。 7. The application according to claim 1, characterized in that, In the fused-ring heterocyclic compound, X is a nitrogen atom.

8. The application according to claim 7, characterized in that, The fused-ring heterocyclic compound has the structure shown in general formula (V): Ⅴ In the formula, R 1 For unsubstituted or at least one hydrogen atom is R 1-2 Substituted phenyl, wherein R 1-2 Halogen, cyano, nitro, C 1~6 Alkyl, C 1~6 Alkoxy; R 2 Hydroxyl or C 1~6 Alkoxy; R 3 Hydroxyl group, C 1~4 Alkyl or C 1~6 Alkyl group.

9. The application according to claim 7, characterized in that, The fused-ring heterocyclic compound has the structure shown in general formula (VI): Ⅵ In the formula, R 1 For unsubstituted or at least one hydrogen atom is R 1-2 Substituted phenyl, wherein R 1-2 Halogen, cyano, nitro, C 1~6 Alkyl, C 1~6 Alkoxy; R 3 Hydroxyl group, C 1~4 Alkyl, C 1~6 Alkoxy, aziridine, pyrrolidinyl, piperidinyl, piperazine, morpholinyl or Ra and Rb are independently selected from hydrogen or C, respectively. 1~4 alkyl.

10. The application according to any one of claims 1-2 and 7-9, characterized in that, The fused-ring heterocyclic compound is selected from any of the following structures: 。

Images