Tricyclic compound, preparation method therefor and use thereof, and pharmaceutical composition

Abstract

The present application relates to the technical field of pharmaceutical chemistry, and provides a tricyclic compound, a preparation method therefor and a use thereof, and a pharmaceutical composition. The tricyclic compound (having a structure represented by formula (1)) or a pharmaceutically acceptable salt thereof provided by the present application has the characteristic of improving mitochondrial function, and can be used for preventing and / or treating diseases related to mitochondrial dysfunction.

Description

Tricyclic compounds, preparation method and application thereof, and pharmaceutical composition

[0001] This application claims priority to the Chinese patent application No. 2025100680747, filed on January 16, 2025, and entitled "Tricyclic compounds, preparation method and application thereof, and pharmaceutical composition", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application relates to the technical field of medicinal chemistry, in particular to tricyclic compounds, preparation method and application thereof, and pharmaceutical composition. BACKGROUND

[0003] Mitochondria is an organelle that produces energy in cells, and its main function is to provide energy (ATP) required by cells. Mitochondrial disease is a series of diseases caused by abnormal function of mitochondria. Mitochondrial autophagy disorder, i.e., the function of aging or damaged mitochondria cannot be effectively cleared, is closely related to the occurrence and development of various nervous system degenerative diseases, mental diseases, muscle diseases and metabolic diseases. Some typical diseases are Friedreich ataxia, amyotrophic lateral sclerosis, Alzheimer's disease, muscle atrophy, Duchenne muscular dystrophy and Parkinson's disease.

[0004] Friedreich ataxia (FA) is a progressive autosomal recessive neurodegenerative disease. In most patients, a doublet of trinucleotides (GAA) in the first intron of the FXN gene is expanded, which in turn impairs transcription and significantly reduces the number of functional frataxin protein. Frataxin deficiency can cause a variety of pathological consequences such as interruption of iron-sulfur cluster biosynthesis, dysregulation of cellular iron, mitochondrial dysfunction and increased sensitivity to oxidative stress in vitro, and ultimately lead to the clinical symptoms of FA.

[0005] Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown etiology, mainly involving the cerebral cortex, brainstem and spinal motor neurons. Its main clinical manifestations are progressive skeletal muscle atrophy, weakness, fasciculation, bulbar palsy and pyramidal signs. Studies have shown that the pathogenesis of ALS involves a variety of pathological phenomena such as abnormal RNA processing, glutamate excitotoxicity, cytoskeletal arrangement disorder, mitochondrial dysfunction, viral infection, apoptosis, growth factor abnormalities and inflammatory response.

[0006] Alzheimer's disease (AD) is the most common neurodegenerative disease, and its clinical symptoms mainly include memory impairment, aphasia, apraxia, agnosia, impairment of visual-spatial skills, executive dysfunction, and personality and behavior changes, and other comprehensive dementia manifestations. Its pathology mainly includes brain tissue atrophy, neurofibrillary tangles, senile plaque formation, and massive amyloid deposition. The pathogenesis of AD is not clear, and the main pathogenesis hypotheses include Aβ toxicity hypothesis, abnormal metabolism of Tau protein, neuroinflammation, cholinergic damage, metal ion metabolism disorder, glycolipid metabolism disorder, free radical damage, neurovascular dysfunction, mitochondrial dysfunction, excitatory amino acid toxicity, viral infection, etc.

[0007] The aging process in humans involves a variety of changes such as gradual loss of function, decreased fertility, and increased mortality. Among all observations on humans (including health, nutrition, physical activity), the decline in muscle mass and function represents the most intense and significant process among all changes in the aging process, and has become an unavoidable key issue for aging intervention. Sarcopenia is a kind of aging-related muscle disease, which refers to the decline in skeletal muscle mass and muscle strength or physical function associated with aging. The pathology of sarcopenia is very complex, and various lifestyle factors can lead to the occurrence of the disease, including malnutrition and lack of exercise. In addition, muscle is a high-energy-demand tissue, and mitochondrial dysfunction associated with aging, malnutrition, and physical inactivity has been considered as its main pathological feature.

[0008] Duchenne Muscular Dystrophy (DMD) is commonly seen in male children and is a severe and progressive hereditary muscle atrophy disease caused by mutations in the gene encoding dystrophin. The mutation leads to a lack of muscle nutrition protein, which in turn promotes a pathological cascade involving structural defects in the muscle membrane, oxidative stress, mitochondrial dysfunction, chronic inflammation, muscle degradation, and impaired regeneration, and ultimately leads to death due to the involvement of respiratory muscles and myocardium.

[0009] Parkinson's disease (PD) is a common neurodegenerative disease, mainly seen in the elderly, and its prominent pathological changes are the degenerative death of dopaminergic neurons in the substantia nigra, a significant decrease in the content of dopamine (DA) in the striatum, and the presence of acidophilic inclusions in the cytoplasm of residual neurons in the substantia nigra. Mitochondrial dysfunction, proteinopathy, and oxidative stress are key factors in the pathogenesis of PD.

[0010] Urolithin A is a secondary metabolite of natural polyphenolic compound ellagitannin, which has various biological activities, such as antioxidant, anti-inflammatory, estrogen / androgen regulation, autophagy promotion, etc. In recent years, studies have shown that urolithin A has activity related to improving mitochondrial function, which is of great significance for the prevention and treatment of mitochondrial diseases. However, urolithin A has poor solubility and extremely low oral bioavailability, and is difficult to be developed into a drug. SUMMARY

[0011] The purpose of the present application is to provide a tricyclic compound and a preparation method and application thereof, and a pharmaceutical composition. The present application modifies the structure of urolithin A by chemical means, and the obtained tricyclic compound has good solubility, overcomes the problem of low bioavailability of urolithin A, and improves the drug performance. The tricyclic compound has the characteristics of improving mitochondrial function, and can be used for preventing and / or treating diseases related to mitochondrial function.

[0012] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0013] The present application provides a tricyclic compound or a pharmaceutically acceptable salt thereof, which has the structure shown in formula 1:

[0014] In formula 1, X1 and X2 are independently selected from -O-, -S-, -NR3- or -CH2-, and at least one of X1 and X2 is -CH2-;

[0015] R1 is selected from -H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C7 cycloalkyl, substituted or unsubstituted C3-C7 heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -C(=O)R4, -C(=O)OR5 or -C(=O)NR6R7;

[0016] is a 3-10 membered nitrogen-containing heterocycle;

[0017] n is an integer of 0-18;

[0018] R2 is independently selected from deuterium, halogen, amino, nitro, carbonyl, ester, carboxyl, sulfonic acid, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 amine, substituted or unsubstituted C3-C7 cycloalkyl, substituted or unsubstituted C3-C7 heterocycloalkyl; or R2 and the connected nitrogen-containing heterocycle together form a substituted or unsubstituted spiro ring, a substituted or unsubstituted bridged ring;

[0019] R3, R4, R5, R6and R7are independently selected from -H, deuterium, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C3-C7cycloalkyl, substituted or unsubstituted C3-C7heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl; or R6, R7and the nitrogen atom to which they are attached form a substituted or unsubstituted C3-C7heterocycloalkyl.

[0020] Preferably, X1and X2are -O- and -CH2-, respectively.

[0021] R1is selected from -H, -CH3, -C(=O)CH3, -C(=O)OC(CH3)3, -C(=O)N(CH3)2or

[0022] R2is selected from -CH3or -N(CH3)2, or R2and the nitrogen-containing heterocycle to which it is attached form an unsubstituted spirocycle or an unsubstituted bridged ring.

[0023] Preferably, R1is selected from -H, -CH3, -C(=O)CH3, -C(=O)OC(CH3)3, -C(=O)N(CH3)2or selected from

[0024] Preferably, R1is selected from -H, -CH3, -C(=O)CH3, -C(=O)OC(CH3)3, -C(=O)N(CH3)2or selected from

[0025] Preferably, the tricyclic compound is selected from any one of the following compounds:

[0026] Preferably, the pharmaceutically acceptable salt of the tricyclic compound includes a hydrochloride salt, a sulfate salt, a phosphate salt, a hydrobromide salt, a nitrate salt, a salicylate salt, a benzoate salt, a C1-C6aliphatic carboxylate salt, a C1-C6alkyl sulfonate salt, a benzenesulfonate salt, a p-toluenesulfonate salt, or a camphorsulfonate salt.

[0027] The present application provides a preparation method of the tricyclic compound of the above technical solution, comprising the following steps:

[0028] (I) when R1is -H, and the when the nitrogen-containing heterocycle does not contain an -NH- group, the preparation method of the tricyclic compound comprises the following steps:

[0029] mixing compound a, compound b, a basic reagent, and an organic solvent to perform a substitution reaction to obtain compound c;

[0030] performing a hydroxyl deprotection reaction on the compound c to obtain compound d, wherein R1of the compound d is -H and the tricyclic compound containing a nitrogen-containing heterocycle without a -NH- group;

[0031] The structural formulas of the compound a, the compound b and the compound c are shown in sequence as follows:

[0032] The Z is a hydroxyl protecting group, X1, X2, R2 and n are as defined in formula 1.

[0033] (II) when the R1 is -H, and the The preparation method of the tricyclic compound containing a nitrogen-containing heterocycle with a -NH- group comprises the following steps:

[0034] The compound a, the compound b', a base reagent and an organic solvent are mixed to perform a substitution reaction to obtain a compound c';

[0035] The compound c' is subjected to a hydroxyl deprotection reaction and an imino deprotection reaction to obtain a compound e, wherein the R1 of the compound e is -H and The tricyclic compound containing a nitrogen-containing heterocycle with a -NH- group;

[0036] The compound b' is different from the compound b in that the -NH- group of the nitrogen-containing heterocycle in the compound b' contains an imino protecting group; the compound c' is different from the compound c in that the -NH- group of the nitrogen-containing heterocycle in the compound c' contains an imino protecting group.

[0037] (III) when the R1 is not -H, the preparation method of the tricyclic compound comprises the following steps:

[0038] The compound d is prepared according to (I) or the compound e is prepared according to (II);

[0039] In the presence of a base reagent and an organic solvent, the compound d or the compound e is subjected to a substitution reaction with a compound f to obtain the tricyclic compound in which the R1 is not -H.

[0040] The compound f is R4C(=O)X3, R5OC(=O)X3, R6R7NC(=O)X3 or R8X3, the X3 is a halogen atom, and the R8 is a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C3-C7 cycloalkyl, a substituted or unsubstituted C3-C7 heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl.

[0041] The application provides an application of the tricyclic compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating a disease related to mitochondrial function.

[0042] Preferably, the disease associated with mitochondrial dysfunction comprises a nervous system disease, a muscle disease, a metabolic disease or aging.

[0043] Preferably, the nervous system disease comprises Alzheimer's disease, frontotemporal dementia, Friedreich's ataxia, amyotrophic lateral sclerosis, Parkinson's disease, spinocerebellar ataxia or Huntington's disease.

[0044] The muscle disease comprises sarcopenia or Duchenne muscular dystrophy.

[0045] The metabolic disease comprises obesity, diabetes or diabetic complications, the diabetic complications comprising diabetic nephropathy or diabetic eye disease.

[0046] The present application provides a pharmaceutical composition comprising at least one of the above technical solutions tricyclic compound and its pharmaceutically acceptable salt and pharmaceutically acceptable adjuvant.

[0047] The present application provides a tricyclic compound or its pharmaceutically acceptable salt. The present application modifies the structure of urolithin A by chemical means, and the obtained tricyclic compound has good solubility, overcomes the problem of low bioavailability of urolithin A, and improves its drug performance. The tricyclic compound described in the present application has the characteristics of improving mitochondrial function, and can be used for preventing and / or treating diseases related to mitochondrial function. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 is a graph showing the effect of XL-01 on the behavior test of AD model mice;

[0049] Figure 2 is a graph showing the effect of XL-01 on the motor function and muscle strength of naturally aging mice;

[0050] Figure 3 is a graph showing the effect of XL-01 on the inflammatory infiltration of gastrocnemius muscle of naturally aging mice;

[0051] Figure 4 is a graph showing the effect of XL-01 on the fibrosis level of gastrocnemius muscle of naturally aging mice;

[0052] Figure 5 is a graph showing the effect of XL-10 on the behavior test of PD model mice;

[0053] Figure 6 is a graph showing the effect of XL-18 on the behavior test of DMD model mice;

[0054] Figure 7 is a graph showing the effect of XL-18 on the pathological staining of gastrocnemius muscle of DMD model mice;

[0055] Figure 8 is a graph showing the effect of XL-18 on the behavior test of FA model mice;

[0056] Figure 9 shows the effects of XL-18 on gait and limb coordination in FA model mice.

[0057] Figure 10 shows the effect of XL-18 on the pathological changes of the cerebellar dentate gyrus and gastrocnemius muscle in FA model mice.

[0058] Figure 11 shows the effect of XL-18 on the expression level of FXN in the cerebellum tissue of FA model mice;

[0059] Figure 12 shows the effect of XL-18 on serum liver function indicators in FA model mice;

[0060] Figure 13 shows the effect of XL-18 on behavioral tests in ALS model mice.

[0061] Figure 14 shows the effects of XL-18 on gait and limb coordination in ALS model mice.

[0062] Figure 15 shows the effects of XL-18 on the wet weight ratio and pathological staining of gastrocnemius muscle in ALS model rats.

[0063] Figure 16 shows the effect of XL-18 on Nissl staining of the spinal cord of ALS model mice;

[0064] Figure 17 shows the immunohistochemical effects of XL-18 on GFAP in the spinal cord of ALS model mice.

[0065] Figure 18 shows the immunohistochemical effects of XL-18 on IBA1 in the spinal cord of ALS model mice. Detailed Implementation

[0066] This application provides a tricyclic compound or a pharmaceutically acceptable salt thereof, said tricyclic compound having the structure shown in Formula 1:

[0067] In Equation 1, X1 and X2 are independently selected from -O-, -S-, -NR3- or -CH2-, and at least one of X1 and X2 is -CH2-;

[0068] R1 is selected from -H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C7 cycloalkyl, substituted or unsubstituted C3-C7 heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -C(=O)R4, -C(=O)OR5 or -C(=O)NR6R7.

[0069] It consists of 3- to 10-membered nitrogen-containing heterocycles;

[0070] n is an integer from 0 to 18;

[0071] R2 is independently selected from deuterium, halogen, amino, nitro, carbonyl, ester, carboxyl, sulfonic acid, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 amino, substituted or unsubstituted C3-C7 cycloalkyl, substituted or unsubstituted C3-C7 heterocyclic alkyl; or R2 together with the attached nitrogen-containing heterocycle forms a substituted or unsubstituted spirocyclic ring or a substituted or unsubstituted bridged ring;

[0072] R3, R4, R5, R6, and R7 are independently selected from -H, deuterium, substituted or unsubstituted C1-C6 chain alkyl, substituted or unsubstituted C3-C7 cycloalkyl, substituted or unsubstituted C3-C7 heterocyclic alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl; or R6 and R7 together with the attached nitrogen atom form a substituted or unsubstituted C3-C7 heterocyclic alkyl.

[0073] In this application, X1 and X2 are independently selected from -O-, -S-, -NR3-, or -CH2-, and at least one of X1 and X2 is -CH2-. In this application, R3 is selected from -H, deuterium, substituted or unsubstituted C1-C6 alkyl groups, substituted or unsubstituted C3-C7 cycloalkyl groups, substituted or unsubstituted C3-C7 heterocycloalkyl groups, substituted or unsubstituted aryl groups, and substituted or unsubstituted heteroaryl groups. This application does not specifically limit the specific type of substituent in substituted C1-C6 alkyl groups, substituted C3-C7 cycloalkyl groups, substituted C3-C7 heterocycloalkyl groups, substituted aryl groups, and substituted heteroaryl groups. In this application, preferably, X1 and X2 are -O- and -CH2-, respectively; that is, when X1 is -CH2-, X2 is preferably -O-, and when X2 is -CH2-, X1 is preferably -O-.

[0074] In this application, R1 is selected from -H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C7 cycloalkyl, substituted or unsubstituted C3-C7 heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -C(=O)R4, -C(=O)OR5 or -C(=O)NR6R7. In this application, R4, R5, R6, and R7 are independently selected from -H, deuterium, substituted or unsubstituted C1-C6 alkyl chains, substituted or unsubstituted C3-C7 cycloalkyl chains, substituted or unsubstituted C3-C7 heterocyclic alkyl chains, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, or R6 and R7 together with the attached nitrogen atom to form substituted or unsubstituted C3-C7 heterocyclic alkyl chains. This application does not specifically limit the specific types of substituents in substituted C1-C6 alkyl chains, substituted C3-C7 cycloalkyl chains, substituted C3-C7 heterocyclic alkyl chains, substituted aryl groups, and substituted heteroaryl groups. In this application, R4 is preferably -CH3; R5 is preferably -C(CH3)3; R6 is preferably -CH3; R7 is preferably -CH3; or R6 and R7 together with the attached nitrogen atom form a substituted 6-membered heterocyclic alkyl group, wherein the substituent is preferably -CH3. Specifically, the group formed by R6 and R7 together with the attached nitrogen atom is preferably... In this application, specifically, R1 is preferably -H, -CH3, -C(=O)CH3, -C(=O)OC(CH3)3, -C(=O)N(CH3)2 or

[0075] In this application, the stated It is a 3- to 10-membered nitrogen-containing heterocycle, specifically a 3-membered, 4-membered, 5-membered, 6-membered, 7-membered, 8-membered, 9-membered, or 10-membered nitrogen-containing heterocycle; specifically, the... Selected from

[0076] In this application, R2 is a substituent on the nitrogen-containing heterocycle, and n represents the number of R2 groups on the nitrogen-containing heterocycle.

[0077] In this application, n is an integer from 0 to 18, specifically 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18.

[0078] In this application, R2 is independently selected from deuterium, halogen, amino, nitro, carbonyl, ester, carboxyl, sulfonic acid, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 amino, substituted or unsubstituted C3-C7 cycloalkyl, substituted or unsubstituted C3-C7 heterocyclic alkyl, or R2 together with the attached nitrogen-containing heterocycle to form a substituted or unsubstituted spirocycle or a substituted or unsubstituted bridged ring. This application does not specifically limit the specific types of substituents in substituted C1-C6 alkyl, substituted C1-C6 amino, substituted C3-C7 cycloalkyl, substituted C3-C7 heterocyclic alkyl, substituted spirocycle, and substituted bridged ring. In this application, the carbonyl group has the structural formula -C(=O)R9, where R9 is selected from -H, deuterium, substituted or unsubstituted C1-C6 alkyl groups, substituted or unsubstituted C3-C7 cycloalkyl groups, substituted or unsubstituted C3-C7 heterocyclic alkyl groups, substituted or unsubstituted aryl groups, and substituted or unsubstituted heteroaryl groups. This application does not specifically limit the specific type of substituents among substituted C1-C6 alkyl groups, substituted C3-C7 cycloalkyl groups, substituted C3-C7 heterocyclic alkyl groups, substituted aryl groups, and substituted heteroaryl groups. Specifically, in this application, R2 is preferably -CH3 or -N(CH3)2, or R2 forms an unsubstituted spirocyclic or unsubstituted bridged ring together with the attached nitrogen-containing heterocycle. Specifically, in this application, the... Preferred

[0079] In this application, the tricyclic compound is specifically selected from any one of the following compounds:

[0080] In this application, the pharmaceutically acceptable salts of the tricyclic compounds preferably include hydrochloride, sulfate, phosphate, hydrobromide, nitrate, salicylate, benzoate, C1-C6 fatty carboxylates, C1-C6 alkyl sulfonates, benzene sulfonates, p-toluene sulfonate, or camphor sulfonate; the C1-C6 fatty carboxylates of this application preferably include oxalate, maleate, fumarate, citrate, succinate, or tartrate.

[0081] This application provides a method for preparing the tricyclic compounds described in the above-mentioned technical solutions. The preparation method is specifically selected according to the specific structure of the tricyclic compounds, and will be described in detail below. Unless otherwise specified, all raw materials used in this application are commercially available products well-known to those skilled in the art or prepared using methods well-known to those skilled in the art.

[0082] In this application, specifically, (I) when R1 is -H, and the When the tricyclic compound is a nitrogen-containing heterocycle that does not contain a -NH- group, the preparation method of the tricyclic compound includes the following steps:

[0083] Compound a, compound b, a base reagent, and an organic solvent were mixed and subjected to a substitution reaction to obtain compound c.

[0084] Compound c was subjected to a hydroxyl deprotection reaction to obtain compound d, wherein compound d is a compound with R1 being -H and The tricyclic compound is a nitrogen-containing heterocycle that does not contain a -NH- group;

[0085] The structural formulas of compounds a, b, and c are shown below in sequence:

[0086] Z is a hydroxyl protecting group, X1, X2, R2 and n are defined as in Equation 1.

[0087] This application involves mixing compound a, compound b, a base reagent, and an organic solvent to conduct a substitution reaction to obtain compound c. In this application, Z in compound a is a hydroxyl protecting group, preferably including a benzyl protecting group (-Bn) or an oxycarbonyl protecting group (-Boc). In this application, the molar ratio of compound a (the preparation method of some compounds is detailed below) and compound b (the preparation method is detailed below) is preferably 1:0.5–2, more preferably 1:1–1.5. In this application, the base reagent is preferably a carbonate, more preferably potassium carbonate; the organic solvent is preferably acetonitrile; the preferred molar ratio of compound a, base reagent, and organic solvent is 1–1.5 mmol:1.5–3.5 mmol:15–22 mL, more preferably 1–1.5 mmol:2–3.28 mmol:20 mL. In this application, the temperature of the substitution reaction is preferably 75–85 °C, more preferably 80 °C; the preferred time is 5–7 h, more preferably 6 h. Following the substitution reaction, this application preferably extracts the resulting product system with ethyl acetate, dries the organic phase to anhydrous sodium sulfate, filters it, concentrates the filtrate, and then separates it by silica gel column chromatography to obtain compound c. In this application, the eluent used for silica gel column chromatography is preferably dichloromethane and methanol. The volume ratio of dichloromethane to methanol is selected according to actual needs, preferably 20–100:1, more preferably 50–80:1.

[0088] After obtaining compound c, this application subjects compound c to a hydroxyl deprotection reaction to obtain compound d, wherein compound d is the tricyclic compound with R1 being -H. This application preferably selects the appropriate hydroxyl deprotection reaction according to the specific type of the hydroxyl protecting group. Specifically, when the hydroxyl protecting group is -Bn, this application preferably removes it through a hydrogen reduction deprotection reaction; when the hydroxyl protecting group is -Boc, this application preferably removes it through an acid deprotection reaction. In this application, the hydrogen reduction deprotection reaction is preferably carried out in the presence of a hydrogenation catalyst, an organic solvent, and hydrogen. The hydrogenation catalyst is preferably palladium on carbon, and the amount used is not particularly required, as long as it can ensure the smooth progress of the reaction. The organic solvent is preferably a mixed solution of tetrahydrofuran and methanol, and the volume ratio of tetrahydrofuran to methanol is preferably 1:0.5 to 1.5, more preferably 1:1. The volume ratio of compound c to organic solvent is preferably 0.5 to 1 mmol: 15 to 25 mL, more preferably 0.5 to 1 mmol: 20 mL. The temperature of the hydrogen reduction deprotection reaction is preferably room temperature, and the time is preferably 10 to 15 h, more preferably 12 h. After the hydrogen reduction deprotection reaction, the obtained product system is preferably filtered through diatomaceous earth, and the filtrate is concentrated to obtain the tricyclic compound. In this application, the acid deprotection reaction is preferably carried out in the presence of an ethyl hydrochloride solution and an organic solvent; the concentration of hydrochloric acid in the ethyl hydrochloride solution is preferably 3-5 mol / L, more preferably 4 mol / L; the organic solvent is preferably methanol; the ratio of compound c, ethyl hydrochloride solution and organic solvent is preferably 0.2-0.5 mmol: 1-1.5 mL: 5-15 mL; the temperature of the acid deprotection reaction is preferably room temperature, and the time is preferably 1.5-2.5 h, more preferably 2 h; after the acid deprotection reaction, this application preferably concentrates the obtained product system, dilutes the concentrate with ethyl acetate, washes it with saturated sodium bicarbonate solution, dries it with anhydrous sodium sulfate, filters it, concentrates the filtrate and separates it by silica gel column chromatography to obtain the tricyclic compound. In this application, the eluent used for silica gel column chromatography is preferably dichloromethane and methanol. The volume ratio of dichloromethane to methanol is selected according to actual needs. The volume ratio of dichloromethane to methanol is preferably 20 to 100:1, and more preferably 30 to 50:1.

[0089] In this application, specifically, (II) when R1 is -H, and the When the tricyclic compound is a nitrogen-containing heterocycle containing a -NH- group, the preparation method of the tricyclic compound includes the following steps:

[0090] Compound a, compound b', a base reagent and an organic solvent were mixed and subjected to a substitution reaction to obtain compound c';

[0091] The compound c' is subjected to a hydroxyl deprotection reaction and an imino deprotection reaction to obtain compound e, wherein compound e is the tricyclic compound with R1 being -H;

[0092] The difference between compound b' and compound b is that compound b' contains an imino protecting group at the -NH- group corresponding to the nitrogen-containing heterocycle; the difference between compound c' and compound c is that compound c' contains an imino protecting group at the -NH- group corresponding to the nitrogen-containing heterocycle.

[0093] This application involves mixing compound a, compound b', a base reagent, and an organic solvent to conduct a substitution reaction, yielding compound c'. In this application, compound b' differs from compound b in that compound b' contains an imino protecting group at the -NH- group corresponding to the nitrogen-containing heterocycle; similarly, compound c' differs from compound c in that compound c' contains an imino protecting group at the -NH- group corresponding to the nitrogen-containing heterocycle. The imino protecting group is preferably a tert-butoxycarbonyl protecting group (-Boc), a benzyloxycarbonyl protecting group (-Cbz), a benzyl protecting group (-Bn), a p-methoxybenzyl protecting group (-PMB), or a 2,4-dimethoxybenzyl protecting group (-DMB), more preferably -Boc. In this application, the ratio of compound a to compound b' (the preparation method is detailed below), the specific types and amounts of the base reagent and organic solvent, the substitution reaction conditions, and the post-treatment methods are preferably consistent with the substitution reaction using compound b as a starting material described above, and will not be repeated here.

[0094] After obtaining compound c', this application subjects compound c' to a hydroxyl deprotection reaction and an imino deprotection reaction to obtain compound e, wherein compound e is the tricyclic compound with R1 being -H. In this application, when the hydroxyl protecting group is -Bn and the imino protecting group is -Boc, it is preferable to perform the hydroxyl deprotection reaction and the imino deprotection reaction sequentially, and more preferably to perform the hydrogen reduction deprotection reaction and the acid deprotection reaction sequentially. The specific reagents, reaction conditions, and post-treatment methods used are preferably consistent with the above-mentioned technical solutions and will not be repeated here. When both the hydroxyl protecting group and the imino protecting group are -Boc, it is preferable to perform the hydroxyl deprotection reaction and the imino deprotection reaction simultaneously, and more preferably to perform the acid deprotection reaction, that is, to simultaneously remove two -Boc groups through a single acid deprotection reaction. The specific reagents, reaction conditions, and post-treatment methods used are preferably consistent with the above-mentioned technical solutions and will not be repeated here.

[0095] In this application, specifically, (III) when R1 is not -H, the method for preparing the tricyclic compound includes the following steps:

[0096] Compound d was prepared according to (I) or compound e was prepared according to (II);

[0097] In the presence of an alkaline reagent and an organic solvent, compound d or compound e is subjected to a substitution reaction with compound f to obtain the tricyclic compound in which R1 is not -H;

[0098] The compound f is R4C(=O)X3, R5OC(=O)X3, R6R7NC(=O)X3 or R8X3, where X3 is a halogen atom and R8 is a substituted or unsubstituted C1-C6 chain alkyl, a substituted or unsubstituted C3-C7 cycloalkyl, a substituted or unsubstituted C3-C7 heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl.

[0099] After preparing compound d according to (I) or compound e according to (II), in the presence of an alkaline reagent and an organic solvent, compound d or compound e is subjected to a substitution reaction with compound f (X3 is a halogen atom, preferably -F, -Cl, -Br or -I) to obtain the tricyclic compound whose R1 is not -H. In this application, the molar ratio of compound d (or compound e) to compound f is preferably 0.5-1.5:0.7-3, more preferably 0.5-1.5:0.8-2.5. In this application, the alkaline reagent is preferably a carbonate, more preferably potassium carbonate; the organic solvent is preferably acetonitrile; the molar ratio of compound d (or compound e), alkaline reagent and organic solvent is preferably 0.5-1.5 mmol:0.8-3.5 mmol:15-25 mL, more preferably 0.5-1.5 mmol:1-3.28 mmol:20 mL. In this application, the temperature of the substitution reaction is preferably room temperature to reflux temperature; the time is preferably 5 to 7 hours, more preferably 6 hours. After the substitution reaction, the resulting product system is preferably washed sequentially with dilute hydrochloric acid (preferably 1 mol / L) and saturated sodium bicarbonate solution, dried with anhydrous sodium sulfate, filtered, and the filtrate is concentrated and separated by silica gel column chromatography to obtain the tricyclic compound. In this application, the eluent used for silica gel column chromatography is preferably dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is selected according to actual needs, preferably 20 to 100:1, more preferably 30 to 50:1.

[0100] In this application, it should be noted that when the hydroxyl protecting group in compound a is -Boc, and R1 in the target tricyclic compound to be prepared is -OC(=O)OC(CH3)3, i.e. -OBoc, then compound c prepared by the substitution reaction of compound a and compound b is the tricyclic compound with R1 being -OBoc. It is not necessary to perform a hydroxyl deprotection reaction to prepare compound d and further use compound f for substitution reaction, as shown in Example 22 of this application for the preparation of compound Ua-f02.

[0101] The preparation methods of some compounds a, b, and b' in this application are described in detail below.

[0102] In this application, specifically, when X1 is -CH2-, X2 is -O-, and Z is -Bn, compound a has the structure shown in Formula 4; when X1 is -O-, X2 is -CH2-, and Z is -Boc, compound a has the structure shown in Formula 5:

[0103] In this application, the method for preparing the compound with the structure shown in Formula 4 preferably includes the following steps:

[0104] Compound I, benzyl bromide, a base reagent, and an organic solvent were mixed to carry out a substitution reaction, yielding compound II;

[0105] Compound II, a base reagent, water, and an organic solvent were mixed and subjected to an ester hydrolysis reaction to obtain compound III.

[0106] Compound III, resorcinol, a base reagent, copper sulfate, and water were mixed to carry out a ring-closure reaction to obtain compound IV;

[0107] Compound IV, a reducing agent, and an organic solvent were mixed to carry out a reduction reaction, yielding the compound with the structure shown in Formula 4.

[0108] The structural formulas of compounds I, II, III, and IV are shown below in sequence:

[0109] This application involves mixing compound I, benzyl bromide, a base reagent, and an organic solvent to conduct a substitution reaction, yielding compound II. In this application, the molar ratio of compound I to benzyl bromide is preferably 10:18–25, more preferably 10:21. In this application, the base reagent is preferably a carbonate, more preferably potassium carbonate; the organic solvent is preferably N,N-dimethylformamide; the molar ratio of compound I, the base reagent, and the organic solvent is preferably 10 mmol:15–25 mmol:30–50 mL, more preferably 10 mmol:20 mmol:40 mL. In this application, the temperature of the substitution reaction is preferably 50–60 °C, more preferably 55 °C; the time is preferably 10–15 h, more preferably 12 h. After the substitution reaction, this application preferably extracts the resulting product system with a mixture of ethyl acetate and saturated ammonium chloride solution (the volume ratio of EA to saturated NH4Cl solution is preferably 2:1). The organic phase is dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated and separated by silica gel column chromatography to obtain compound II. In this application, the eluent used for silica gel column chromatography is preferably petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is selected according to actual needs. The volume ratio of petroleum ether to ethyl acetate is preferably 10:1.

[0110] After obtaining compound II, this application mixes compound II, a base reagent, water, and an organic solvent to perform an ester hydrolysis reaction to obtain compound III. In this application, the base reagent is preferably lithium hydroxide; the organic solvent is preferably tetrahydrofuran; the preferred ratio of compound II, the base reagent, water, and the organic solvent is 5 mmol:25–35 mmol:8–12 mL:15–25 mL, more preferably 5 mmol:30 mmol:10 mL:20 mL. In this application, the preferred temperature for the ester hydrolysis reaction is -2 to 2°C, more preferably 0°C; the preferred time is 3.5–4.5 h, more preferably 4 h. After the ester hydrolysis reaction, this application preferably warms the resulting product system to room temperature, extracts with diethyl ether, adjusts the aqueous phase to pH=1 with dilute hydrochloric acid (preferably 1 mol / L), then extracts with ethyl acetate, dries the organic phase with anhydrous sodium sulfate, filters, and concentrates the filtrate to obtain compound III, which can be directly used in the next reaction without further purification.

[0111] After obtaining compound III, this application mixes compound III, resorcinol, an alkaline reagent, copper sulfate, and water to carry out a ring-closure reaction to obtain compound IV. In this application, the molar ratio of compound III to resorcinol is preferably 4:7-9, more preferably 4:8. In this application, the alkaline reagent is preferably sodium hydroxide; the preferred ratio of compound III, alkaline reagent, and water is 4 mmol:15-25 mmol:35-55 mL, more preferably 4 mmol:19.92 mmol:45 mL; in this application, copper sulfate acts as a catalyst, and its amount is sufficient to ensure the smooth progress of the reaction. Preferably, the alkaline reagent and copper sulfate are dissolved separately in water to obtain an alkaline reagent solution and a copper sulfate solution. Compound III and resorcinol are added sequentially to the alkaline reagent solution, and the mixture is refluxed and stirred for 25-35 min (preferably 30 min). Then, an aqueous solution of copper sulfate (preferably 5 wt%) is added, and the mixture is refluxed for another 10-15 h (preferably 12 h). After the ring-closing reaction, the resulting product system is preferably filtered, the filter cake is washed with dilute hydrochloric acid (preferably 1 mol / L), and then dried under vacuum to obtain compound IV.

[0112] After obtaining compound IV, this application mixes compound IV, a reducing agent, and an organic solvent to carry out a reduction reaction, yielding a compound with the structure shown in Formula 4. In this application, the reducing agent is preferably sodium borohydride and boron trifluoride diethyl ether; the organic solvent is preferably tetrahydrofuran; the preferred ratio of compound IV, sodium borohydride, boron trifluoride diethyl ether, and the organic solvent is 1.5 mmol: 2.5–3.5 mmol: 3.8–4.5 mmol: 10–15 mL, more preferably 1.5 mmol: 3.14 mmol: 4.06 mmol: 10 mL. Preferably, this application dissolves compound IV and sodium borohydride in an organic solvent, adds boron trifluoride diethyl ether at -2–2°C (preferably 0°C), and then carries out the reduction reaction. In this application, the preferred temperature for the reduction reaction is 60–70°C, more preferably 66°C; the preferred time is 10–15 h, more preferably 12 h. Following the reduction reaction, this application preferably adds methanol and dilute hydrochloric acid (preferably 1 mol / L) sequentially to the resulting reaction system, cools to 45–55°C (preferably 50°C), and continues stirring for 25–35 min (preferably 30 min). Then, the pH is adjusted to neutral with sodium hydroxide solution (preferably 50 wt%), followed by extraction with ethyl acetate. The organic phase is dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated and separated by silica gel column chromatography to obtain the compound with the structure shown in Formula 4. In this application, the eluent used for silica gel column chromatography is preferably dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is selected according to actual needs, preferably 100:1.

[0113] In this application, the method for preparing the compound with the structure shown in Formula 5 preferably includes the following steps:

[0114] The compound with the structure shown in Formula 4, ditert-butyl dicarbonate, imidazole, a base reagent and an organic solvent were mixed and subjected to a hydroxyl Boc protection reaction to obtain compound VIII.

[0115] Compound VIII was subjected to hydrogen reduction and debenzylation to obtain the compound with the structure shown in Formula 5.

[0116] This application involves mixing the compound with the structure shown in Formula 4, di-tert-butyl dicarbonate, imidazole, a base reagent, and an organic solvent to perform a hydroxyl Boc protection reaction to obtain compound VIII. In this application, the molar ratio of the compound with the structure shown in Formula 4 to di-tert-butyl dicarbonate is preferably 2:3.5–4.5, more preferably 2:4. In this application, the base reagent is preferably an amine compound, more preferably triethylamine; the organic solvent is preferably dichloromethane; the molar ratio of the compound with the structure shown in Formula 4, imidazole, base reagent, and organic solvent is preferably 2 mmol:2.5–3.5 mmol:3.5–4.5 mmol:15–25 mL, more preferably 2 mmol:3 mmol:4 mmol:20 mL. In this application, the temperature of the hydroxyl Boc protection reaction is preferably room temperature, and the time is preferably 10–15 h, more preferably 12 h. After the hydroxyl Boc protection reaction, this application preferably extracts the resulting product system with ethyl acetate, dries the organic phase to anhydrous sodium sulfate, filters, concentrates the filtrate, and separates it by silica gel column chromatography to obtain compound VIII. In this application, the eluent used for silica gel column chromatography is preferably petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is selected according to actual needs, preferably 5:1.

[0117] After obtaining compound VIII, this application subjectes compound VIII to a hydrogen reduction debenzylation reaction to obtain the compound with the structure shown in Formula 5. In this application, the hydrogen reduction debenzylation reaction is preferably carried out in the presence of a hydrogenation catalyst, an organic solvent, and hydrogen. The hydrogenation catalyst is preferably palladium on carbon, and this application has no special requirements for its amount, as long as it can ensure the smooth progress of the reaction. The organic solvent is preferably a mixed solution of tetrahydrofuran and methanol, and the volume ratio of tetrahydrofuran to methanol is preferably 1:0.5 to 1.5, more preferably 1:1. The volume ratio of compound VIII to organic solvent is preferably 1.5 mmol:15 to 25 mL, more preferably 1.5 mmol:20 mL. The temperature of the hydrogen reduction deprotection reaction is preferably room temperature, and the time is preferably 10 to 15 h, more preferably 12 h. After the hydrogen reduction deprotection reaction, this application preferably filters the obtained product system through diatomaceous earth and concentrates the filtrate to obtain the compound with the structure shown in Formula 5.

[0118] In this application, the method for preparing compound b preferably includes the following steps:

[0119] A nitrogen heterocyclic compound, an acyl chloride reagent, pyridine, and an organic solvent were mixed and subjected to an acyl chloride reaction to obtain compound b.

[0120] The structural formula of the nitrogen heterocyclic compound is shown below:

[0121] In this application, the acyl chloride reagent is preferably triphosgene; the organic solvent is preferably dichloromethane; the preferred ratio of the nitrogen heterocyclic compound, acyl chloride reagent, pyridine, and organic solvent is 2 mmol: 1.8–2.2 mmol: 2.5–3.5 mmol: 15–25 mL, more preferably 2 mmol: 2 mmol: 3 mmol: 20 mL. In this application, the preferred temperature for the acyl chloride reaction is room temperature, and the preferred time is 50–70 min, more preferably 60 min. After the acyl chloride reaction, the resulting product system is preferably washed with dilute hydrochloric acid (preferably 1 mol / L), the organic phase is dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated to obtain compound b, which can be directly used in the next reaction without further purification.

[0122] In this application, the preparation method of compound b' is preferably the same as that of compound b, the only difference being that the nitrogen-containing heterocyclic compound used to prepare compound b' contains an imino protecting group at the -NH- group of the nitrogen-containing heterocycle. Specifically, in this application, compound b' is used as... For example, the preferred preparation method is as follows:

[0123] N-Boc-piperazine, an acyl chloride reagent, pyridine, and an organic solvent were mixed and subjected to an acyl chloride reaction to obtain compound b'.

[0124] In this application, the specific types of acyl chloride reagent and organic solvent, the specific amounts of N-Boc-piperazine, acyl chloride reagent, pyridine and organic solvent, as well as the acyl chloride reaction conditions and post-treatment methods are preferably consistent with the above technical solutions, and will not be repeated here.

[0125] This application also preferably provides a method for preparing a pharmaceutically acceptable salt of the aforementioned tricyclic compound. Taking tricyclic compound hydrochloride as an example, the method for preparing the tricyclic compound hydrochloride preferably includes the following steps:

[0126] A tricyclic compound, an ethyl acetate hydrochloride solution, and an organic solvent are mixed to carry out a salt formation reaction, yielding the tricyclic compound hydrochloride.

[0127] In this application, the concentration of hydrochloric acid in the ethyl hydrochloric acid solution is preferably 3-5 mol / L, more preferably 4 mol / L; the organic solvent is preferably methanol; the ratio of the tricyclic compound, the ethyl hydrochloric acid solution, and the organic solvent is preferably 0.2 mmol:0.5-1.5 mL:5-15 mL, more preferably 0.2 mmol:1 mL:10 mL; the salt-forming reaction is preferably carried out at room temperature for 4-6 hours, more preferably 5 hours. After the salt-forming reaction, the resulting product system is preferably filtered, and the filter cake is collected to obtain the tricyclic compound hydrochloride.

[0128] This application provides the use of the tricyclic compounds or pharmaceutically acceptable salts thereof described above in the preparation of medicaments for the prevention and / or treatment of diseases related to mitochondrial function. In this application, the diseases related to mitochondrial dysfunction may include neurological diseases, muscle diseases, metabolic diseases, or aging. In this application, the neurological diseases may include Alzheimer's disease (AD), frontotemporal dementia, Friedreich's ataxia, amyotrophic lateral sclerosis (ALS), Parkinson's disease, spinocerebellar ataxia, or Huntington's disease; the muscle diseases may include sarcopenia or Duchenne muscular dystrophy; the metabolic diseases may include obesity, diabetes, or diabetic complications, and the diabetic complications may include diabetic nephropathy or diabetic retinopathy.

[0129] In this application, the drug specifically includes at least one of the tricyclic compound and its pharmaceutically acceptable salt, as well as a pharmaceutically acceptable excipient. This application does not specifically limit the type of the pharmaceutically acceptable excipient; any excipient well known to those skilled in the art can be used. In this application, the content of the functional component (the tricyclic compound and / or its pharmaceutically acceptable salt) in the drug is preferably 0.1–99.9 wt%, more preferably 1–99 wt%, and even more preferably 50–80 wt%. This application does not specifically limit the dosage form of the drug; any dosage form well known to those skilled in the art can be used.

[0130] This application provides a pharmaceutical composition comprising at least one of the tricyclic compound and its pharmaceutically acceptable salt as described in the above-described technical solutions, as well as a pharmaceutically acceptable excipient. In this application, the types of pharmaceutically acceptable excipients, the content of the functional ingredient, and the range of applicable dosage forms are preferably consistent with the above-described solutions, and will not be repeated here.

[0131] The technical solutions of this application will be clearly and completely described below with reference to the embodiments therein. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0132] Example 1: Synthesis of compound US-a01

[0133] Compound I (2.15 g, 10 mmol) was dissolved in N,N-dimethylformamide (DMF, 40 mL), followed by the sequential addition of potassium carbonate (K₂CO₃, 2.76 g, 20 mmol) and benzyl bromide (BnBr, 2.49 mL, 21 mmol). The mixture was stirred at 55 °C for 12 h. After the reaction was complete, the mixture was extracted with a mixture of ethyl acetate and saturated ammonium chloride solution (EA to saturated NH₄Cl solution, volume ratio 2:1). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (eluting PE:EA = 10:1) to give compound II (2.49 g, 63%). 1 H NMR (400MHz, CDCl3) δ7.51(d,J=8.8Hz,1H),7.49-7.29(m,11H),6.93(dd,J=8.8,3.1Hz,1H),5.35(s,2H),5.04(s,2H).

[0134] Compound II (1.98 g, 5 mmol) was dissolved in a mixed solution of tetrahydrofuran and methanol (THF to MeOH volume ratio 2:1, 30 mL). An aqueous solution of lithium hydroxide (0.72 g, 30 mmol) (10 mL) was added at 0 °C, and stirring was continued for 4 h. After the reaction was complete, the mixture was brought to room temperature and extracted twice with diethyl ether (Et₂O). The aqueous phase was adjusted to pH 1 with dilute hydrochloric acid (1 mol / L) and then extracted twice with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain the crude product, compound III. No further purification was required, and it was used directly in the next reaction.

[0135] Sodium hydroxide (0.8 g, 19.92 mmol) was dissolved in water (40 mL), and compound III (1.22 g, 4 mmol) and resorcinol (0.88 g, 8 mmol) were added sequentially. The mixture was refluxed and stirred for 30 min. Then, an aqueous solution of copper sulfate (5 wt%, 5 mL) was added, and the mixture was refluxed for another 12 h. After the reaction was complete, the mixture was filtered, washed with dilute hydrochloric acid (1 mol / L), and dried under vacuum to obtain compound IV (0.58 g, 46%). 1H NMR(400MHz, DMSO-d6)δ7.96(d,J=8.0Hz,1H),7.70(d,J=9.9Hz,1H),7.61-7.55(m,1H),7.49( d,J=7.3Hz,2H),7.45-7.27(m,4H),6.38(d,J=8.7Hz,1H),6.18(d,J=1.6Hz,1H),5.20(s,2H).

[0136] Compound IV (500 mg, 1.5 mmol) and sodium borohydride (119 mg, 3.14 mmol) were dissolved in tetrahydrofuran (10 mL). Boron trifluoride diethyl ether (578 mg, 4.06 mmol) was added at 0 °C, and the reaction was carried out at 66 °C for 12 h. After the reaction was completed, methanol (1 mL) and dilute hydrochloric acid (1 mol / L) were added sequentially, and the mixture was stirred at 50 °C for 30 min. The pH was then adjusted to neutral with sodium hydroxide solution (50 wt%), extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (using DCM:MeOH = 100:1 as the eluent) to give compound V (406 mg, 85%). 1 H NMR (400MHz, CDCl3) δ7.54 (dd, J=8.5, 3.5Hz, 2H), 7.50-7.33 (m, 5H), 6.99 (dd, J=8.6, 2.6Hz, 1H), 6 .77(d,J=2.6Hz,1H),6.55(dd,J=8.4,2.5Hz,1H),6.50(d,J=2.5Hz,1H),5.11(s,2H),5.08(s,2H).

[0137] N-methylpiperazine (200 mg, 2 mmol) was dissolved in anhydrous dichloromethane (DCM, 20 mL), followed by the sequential addition of pyridine (Py, 0.24 mL, 3 mmol) and triphosgene (BTC, 592 mg, 2 mmol). The reaction was carried out at room temperature for 1 h. After the reaction was complete, the mixture was washed with dilute hydrochloric acid (1 mol / L), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. This crude product was then dissolved directly in acetonitrile (CH3CN, 20 mL), followed by the sequential addition of compound V (500 mg, 1.64 mmol) and potassium carbonate (453 mg, 3.28 mmol). The mixture was stirred at 80 °C for 6 h. After the reaction was complete, the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (using DCM:MeOH = 50:1 as the eluent) to obtain compound VI-01 (564 mg, 80%). 1H NMR (400MHz, CDCl3) δ7.60(d,J=8.5Hz,1H),7.57(d,J=8.6Hz,1H),7.41(m,4H),7.34(d,J=7.0Hz,1H),6.97(dd,J=8.6,2.4Hz, 1H),6.80(dd,J=8.4,2.4Hz,1H),6.75(d,J=2.4Hz,2H),5.09(s,2H),5.07(s,2H),3.64(m,4H),2.50-2.43(m,4H),2.34(s,3H).

[0138] Compound VI-01 (430 mg, 1 mmol) was dissolved in a mixed solution of tetrahydrofuran and methanol (THF to MeOH volume ratio 1:1, 20 mL), and palladium on carbon (Pd / C, Pd content 10 wt%, 2 g) was added. Air was replaced with nitrogen, followed by nitrogen replacement with hydrogen, and the reaction was stirred at room temperature for 12 h under a hydrogen atmosphere. After the reaction was complete, the mixture was filtered through diatomaceous earth, and the filtrate was concentrated to obtain compound US-a01 (306 mg, 90%). 1 H NMR (400MHz, DMSO-d6) δ9.71(s,1H),7.71(d,J=8.5Hz,1H),7.62(d,J=8.5Hz,1H),6.81-6.76(m,2H),6.71(d,J =2.4Hz,1H),6.66(d,J=2.5Hz,1H),5.05(s,2H),3.56(s,2H),3.43(s,2H),2.35(t,J=5.1Hz,4H),2.22(s,3H).

[0139] Example 2 Synthesis of compound US-a02

[0140] The preparation method was basically the same as in Example 1, except that 4-dimethylaminopiperidine was used instead of N-methylpiperazine to obtain compound US-a02 (327 mg, 89%). 1 H NMR (400MHz, DMSO-d6) δ9.78(s,1H),7.73(d,J=8.5Hz,1H),7.64(d,J=8.5Hz,1H),6.83-6.77(m,2H),6.73(d,J= 2.4Hz,1H),6.66(d,J=2.5Hz,1H),5.05(s,2H),4.33(m,4H),2.93(m,1H),2.78(s,6H),2.10(s,2H),1.71(s,2H).

[0141] Example 3 Synthesis of compound US-a03

[0142] The preparation method is basically the same as that in Example 1, except that N-methylperpiperazine is used instead of N-methylpiperazine to obtain compound US-a03 (300 mg, 85%). 1 H NMR(400MHz, DMSO-d6)δ9.93(s,1H),7.84(d,J=8.5Hz,1H),7.69(d,J=8.5Hz,1H),6.85(m,2H),6.79(d,J=2.4Hz,1H), 6.69(d,J=2.5Hz,1H),5.04(s,2H),3.73-3.60(m,2H),3.53-3.44(m,2H),2.67-2.48(m,5H),2.33(s,2H),1.87(m,2H).

[0143] Example 4: Synthesis of compound US-a04

[0144]

[0145] The preparation method was basically the same as in Example 1, except that (R)-octahydropyrrolo[1,2-A]pyrazine was used instead of N-methylpiperazine to obtain compound US-a04 (230 mg, 63%). 1 H NMR (400MHz, DMSO-d6) δ9.77(s,1H),7.73(d,J=8.5Hz,1H),7.64(d,J=8.6Hz,1H),6.83(m,2H),6.72(d,J=2.4Hz,1H),6.66(d,J=2.5Hz,1H),5. 03(s,2H),4.30-3.97(m,2H),3.24-2.91(m,3H),2.84-2.61(m,1H),2.1 7-2.08(m,2H),2.02-1.90(m,1H),1.87-1.61(m,3H),1.38-1.26(m,1H).

[0146] Example 5: Synthesis of compound US-b01

[0147] The synthesis steps of compound V are as described in Example 1.

[0148] N-Boc-piperazine (372 mg, 2 mmol) was dissolved in anhydrous dichloromethane (DCM, 20 mL), followed by the sequential addition of pyridine (Py, 0.24 mL, 3 mmol) and triphosgene (BTC, 592 mg, 2 mmol), and the reaction was carried out at room temperature for 1 h. After the reaction was completed, the mixture was washed with dilute hydrochloric acid (1 mol / L), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. This crude product was then directly dissolved in acetonitrile (CH3CN, 20 mL), followed by the sequential addition of compound V (304 mg, 1 mmol) and potassium carbonate (276 mg, 2 mmol), and the reaction was carried out at 80 °C with stirring for 6 h. After the reaction was completed, the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (using DCM:MeOH = 80:1 as the eluent) to obtain compound VI-05 (433 mg, 84%).

[0149] Compound VI-05 (258 mg, 0.5 mmol) was dissolved in a mixed solution of tetrahydrofuran and methanol (THF to MeOH volume ratio 1:1, 20 mL), and palladium on carbon (Pd / C, Pd content 10 wt%, 2 g) was added. Air was replaced with nitrogen, followed by nitrogen replacement, and the reaction was carried out under a hydrogen atmosphere at room temperature with stirring for 12 h. After the reaction was complete, the mixture was filtered through diatomaceous earth, and the filtrate was concentrated to obtain compound VII-05 (187 mg, 88%).

[0150] Compound VII-05 (85 mg, 0.2 mmol) was dissolved in methanol (10 mL), and ethyl acetate hydrochloride solution (4 mol / L, 1 mL) was added. The mixture was stirred at room temperature for 2 h. After the reaction was completed, the solution was concentrated, diluted with ethyl acetate, washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (using DCM:MeOH = 20:1 as the eluent) to give compound US-b01 (50 mg, 77%). 1 H NMR(400MHz,DMSO-d6)δ9.89(s,1H),7.91(d,J=8.5Hz,1H),7.84(d,J=8.5Hz,1H),7.01-6.87(m,2H ),6.88(d,J=2.4Hz,1H),6.75(d,J=2.5Hz,1H),5.08(s,2H),3.51(s,2H),3.38(s,2H),3.21(s,4H).

[0151] Example 6 Synthesis of compound US-b02

[0152] The preparation method is basically the same as that in Example 5, except that N-1-Boc-2-methylpiperazine is used instead of N-Boc-piperazine to obtain compound US-b02 (58 mg, 86%). 1 H NMR (400MHz, DMSO-d6) δ9.87(s,1H),7.72(d,J=8.6Hz,1H),7.65(d,J=8.6Hz,1H),6.84(m,2H),6.72(d,J =2.4Hz,1H),6.65(d,J=2.5Hz,1H),5.05(s,2H),4.49-4.37(m,1H),3.34-2.88(m,6H),1.33-1.27(m,3H).

[0153] Example 7 Synthesis of compound US-b03

[0154] The preparation method is basically the same as that in Example 5, except that N-Boc-piperazine is replaced with tert-butyl 4,7-diazaspiro[2.5]octane-4-carboxylate to obtain compound US-b03 (49 mg, 70%). 1 H NMR(400MHz, DMSO-d6)δ9.89(s,1H),7.73(d,J=8.5Hz,1H),7.65(d,J=8.6Hz,1H),6.83(m,2H),6.72(d,J= 2.4Hz,1H),6.64(d,J=2.5Hz,1H),5.02(s,2H),3.63(m,2H),3.52(s,2H),2.85-2.79(m,2H),0.43(s,4H).

[0155] Example 8 Synthesis of compound US-b04

[0156] The preparation method is basically the same as that in Example 5, except that (1S,4S)-2-Boc-2,5-diazabicyclo[2.2.1]heptane is used to replace N-Boc-piperazine to obtain compound US-b04 (50 mg, 37%). 1H NMR (400MHz, DMSO-d6) δ9.91(s,1H),7.71(d,J=8.6Hz,1H),7.62(d,J=8.6Hz,1H),6.79-6.75(m,2H),6.72(d,J=2.4Hz,1H),6.63(d,J=2.5Hz,1H ),5.03(s,2H),4.51(s,1H),4.30(s,1H),3.60(d,J=9.6Hz,1H),3.51-3 .14(m,2H),3.03-2.74(m,2H),1.83-1.67(m,1H),1.58(t,J=9.5Hz,1H).

[0157] Example 9: Synthesis of compound US-b05

[0158] The preparation method was basically the same as in Example 5, except that N-Boc-piperazine was replaced with cis-2-Boc-hexahydropyrrolo[3,4-c]pyrrole to obtain compound US-b05 (29 mg, 41%). 1 H NMR (400MHz, DMSO-d6) δ9.93(s,1H),7.78(d,J=8.5Hz,1H),7.65(d,J=8.5Hz,1H),6.83-6.77(m,2H),6.72(d,J=2.4Hz,1H),6.66(d,J= 2.5Hz,1H),5.05(s,2H),4.05-3.89(m,2H),3.58-3.48(m,2H),2.90(s,2H),2.81(s,2H),2.68(d,J=3.0Hz,1H),2.65(d,J=3.0Hz,1H).

[0159] Example 10 Synthesis of compound US-c01

[0160] Compound US-a01 (170 mg, 0.5 mmol) was dissolved in acetonitrile (20 mL), and potassium carbonate (138 mg, 1 mmol) and dimethylcarbamoyl chloride (0.07 mL, 0.8 mmol) were added sequentially. The mixture was stirred under reflux for 6 h. After the reaction was completed, the mixture was washed successively with dilute hydrochloric acid (1 mol / L) and saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (using DCM:MeOH = 40:1 as the eluent) to obtain compound US-c01 (197 mg, 96%). 1H NMR (400MHz, CDCl3) δ7.59(d,J=8.6Hz,1H),7.55(d,J=8.6Hz,1H),6.98(dd,J=8.6,2.4Hz,1H),6.80(dd,J=8.4,2.3Hz,1H),6.75 (d,J=2.4Hz,2H),5.07(s,2H),3.73(t,J=5.1Hz,2H),3.62(t,J=5.0Hz,2H),3.13(s,3H),3.07(s,3H),2.51(s,4H),2.36(s,3H).

[0161] Example 11 Synthesis of compound US-CO2

[0162] The preparation method was basically the same as in Example 10, except that acetyl chloride was used instead of dimethylcarbamoyl chloride to obtain compound US-c02 (173 mg, 91%). 1 H NMR (400MHz, CDCl3) δ7.58(d,J=8.5Hz,1H),7.55(d,J=8.5Hz,1H),6.98(dd,J=8.6,2.4Hz,1H),6.80(dd,J=8.4,2.4Hz,1H ),6.75(d,J=2.4Hz,2H),5.07(s,2H),3.68(t,J=5.2Hz,2H),3.55(t,J=5.2Hz,2H),2.49(s,4H),2.30(s,3H),2.28(s,3H).

[0163] Example 12 Synthesis of compound US-CO3

[0164] Compound US-a01 (170 mg, 0.5 mmol) was dissolved in acetonitrile (20 mL), followed by the addition of potassium carbonate (138 mg, 1 mmol) and iodomethane (0.05 mL, 0.8 mmol) in sequence. The mixture was refluxed and stirred for 6 h. After the reaction was complete, the mixture was washed with dilute hydrochloric acid (1 mol / L), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (using DCM:MeOH = 50:1 as the eluent) to obtain compound US-c03 (147 mg, 83%). 1HNMR (400MHz, CDCl3) δ7.60(d,J=8.6Hz,1H),7.57(d,J=8.5Hz,1H),6.97(dd,J=8.6,2.4Hz,1H),6.80(dd,J=8.4, 2.4Hz,1H),6.75(d,J=2.4Hz,2H),5.09(s,2H),3.93(s,3H),3.72(s,2H),3.62(s,2H),2.49(s,2H),2.36(s,3H).

[0165] Example 13 Synthesis of compound US-d01

[0166] The synthesis steps of compound V are as described in Example 1.

[0167] Compound V (608 mg, 2 mmol) was dissolved in dichloromethane (20 mL), and imidazole (204 mg, 3 mmol), triethylamine (0.55 mL, 4 mmol), and di-tert-butyl dicarbonate (0.9 mL, 4 mmol) were added sequentially. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (using PE:EA = 5:1 as the eluent) to give compound VIII (783 mg, 97%). 1 HNMR (400MHz, CDCl3) δ7.54(d,J=8.5Hz,1H),7.50(d,J=8.6Hz,1H),7.40-7.24(m,5H),6.91(dd,J=8.6,2.5Hz,1H) ,6.78(dd,J=8.5,2.4Hz,1H),6.74(d,J=2.3Hz,1H),6.69(d,J=2.4Hz,1H),5.02(s,2H),5.01(s,2H),1.49(s,9H).

[0168] Compound VIII (606 mg, 1.5 mmol) was dissolved in a mixed solution of tetrahydrofuran and methanol (THF to MeOH volume ratio 1:1, 20 mL), and palladium on carbon (Pd / C, Pd content 10 wt%, 2 g) was added. Air was replaced with nitrogen, followed by nitrogen replacement, and the reaction was carried out under a hydrogen atmosphere at room temperature with stirring for 12 h. After the reaction was complete, the mixture was filtered through diatomaceous earth, and the filtrate was concentrated to obtain crude compound IX, which was used directly in the next reaction without further purification.

[0169] N-methylpiperazine (200 mg, 2 mmol) was dissolved in anhydrous dichloromethane (DCM, 20 mL), followed by the sequential addition of pyridine (Py, 0.24 mL, 3 mmol) and triphosgene (BTC, 592 mg, 2 mmol). The reaction was carried out at room temperature for 1 h. After the reaction was complete, the mixture was washed with dilute hydrochloric acid (1 mol / L), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. This crude product was then dissolved directly in acetonitrile (CH3CN, 20 mL), followed by the sequential addition of compound IX (471 mg, 1.5 mmol) and potassium carbonate (453 mg, 3.28 mmol). The mixture was stirred at 80 °C for 6 h. After the reaction was complete, the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (using DCM:MeOH = 50:1 as the eluent) to obtain compound X-01 (567 mg, 86%). 1 H NMR (400MHz, DMSO-d6) δ7.89(d,J=8.6Hz,1H),7.83(d,J=8.5Hz,1H),7.17(dd,J=8.5,2.4Hz,1H),7.11(d,J=2.4Hz,1H),6.92 (dd,J=8.5,2.4Hz,1H),6.86(d,J=2.4Hz,1H),5.16(s,2H),3.60(s,2H),3.45(s,2H),2.38(s,4H),2.24(s,3H),1.50(s,9H).

[0170] Compound X-01 (220 mg, 0.5 mmol) was dissolved in methanol (10 mL), and ethyl acetate hydrochloride solution (4 mol / L, 1.5 mL) was added. The mixture was stirred at room temperature for 2 h. After the reaction was completed, the solution was concentrated, diluted with ethyl acetate, washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (using DCM:MeOH = 30:1 as the eluent) to obtain compound US-d01 (148 mg, 87%). 1 H NMR (400MHz, DMSO-d6) δ9.66(s,1H),7.66(d,J=8.6Hz,1H),7.63(d,J=8.6Hz,1H),7.08(dd,J=8.4,2.5Hz,1H),7.02(d,J=2.5 Hz,1H),6.49(dd,J=8.4,2.4Hz,1H),6.35(d,J=2.4Hz,1H),5.03(s,2H),3.58(s,2H),3.43(s,2H),2.36(s,4H),2.22(s,3H).

[0171] Example 14 Synthesis of compound US-d02

[0172] The preparation method was basically the same as in Example 13, except that 4-dimethylaminopiperidine was used instead of N-methylpiperazine to obtain compound US-d02 (156 mg, 85%). 1 H NMR (400MHz, DMSO-d6) δ7.66(d,J=8.6Hz,1H),7.62(d,J=8.5Hz,1H),7.08(dd,J=8.6,2.5Hz,1H),7.02(d,J=2.5Hz,1H),6.49 (dd,J=8.5,2.4Hz,1H),6.35(d,J=2.4Hz,1H),5.04(s,2H),4.33(s,4H),2.91(m,1H),2.75(s,6H),2.11(s,2H),1.68(s,2H).

[0173] Example 15 Synthesis of compound US-d03

[0174] The preparation method was basically the same as in Example 13, except that N-methylperpiperazine was used instead of N-methylpiperazine to obtain compound US-d03 (136 mg, 77%). 1 H NMR (400MHz, DMSO-d6) δ9.68(s,1H),7.67(d,J=8.5Hz,1H),7.63(d,J=8.5Hz,1H),7.08(dd,J=8.5,2.5Hz,1H),7.02(d,J=2.5Hz,1H),6.49(d d,J=8.5,2.4Hz,1H),6.35(d,J=2.4Hz,1H),5.03(s,2H),3.72-3.60(m ,2H),3.58-3.46(m,2H),2.78-2.47(m,5H),2.34(s,2H),1.88(m,2H).

[0175] Example 16 Synthesis of compound US-d04

[0176] The preparation method was basically the same as in Example 13, except that (R)-octahydropyrrolo[1,2-A]pyrazine was used instead of N-methylpiperazine to obtain compound US-d04 (195 mg, 52%). 1H NMR (400MHz, DMSO-d6) δ7.65(d,J=8.6Hz,1H),7.61(d,J=8.5Hz,1H),7.08(dd ,J=8.6,2.5Hz,1H),7.02(d,J=2.5Hz,1H),6.46(dd,J=8.5,2.5Hz,1H),6.35(d ,J=2.5Hz,1H),5.02(s,2H),4.32-3.97(m,2H),3.16(s,3H),2.84-2.60(m,1H ),2.19-2.05(m,2H),2.02-1.89(m,1H),1.87-1.59(m,3H),1.38-1.25(m,1H).

[0177] Example 17 Synthesis of compound US-e01

[0178] The synthesis steps of compound IX are described in Example 13.

[0179] N-Boc-piperazine (372 mg, 2 mmol) was dissolved in anhydrous dichloromethane (DCM, 20 mL), followed by the sequential addition of pyridine (Py, 0.24 mL, 3 mmol) and triphosgene (BTC, 592 mg, 2 mmol), and the reaction was carried out at room temperature for 1 h. After the reaction was completed, the mixture was washed with dilute hydrochloric acid (1 mol / L), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. This crude product was then dissolved directly in acetonitrile (CH3CN, 20 mL), followed by the sequential addition of compound IX (314 mg, 1 mmol) and potassium carbonate (276 mg, 2 mmol), and the reaction was carried out at 80 °C with stirring for 6 h. After the reaction was completed, the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (using DCM:MeOH = 100:1 as the eluent) to obtain compound X-05 (499 mg, 95%).

[0180] Compound X-05 (105 mg, 0.2 mmol) was dissolved in methanol (10 mL), and ethyl acetate hydrochloride solution (4 mol / L, 1 mL) was added. The mixture was stirred at room temperature for 2 h. After the reaction was completed, the mixture was concentrated, diluted with ethyl acetate, and washed with saturated sodium bicarbonate solution. The washed product was then concentrated and separated by silica gel column chromatography (using DCM:MeOH = 20:1 as the eluent) to obtain compound US-e01 (60 mg, 93%). 1H NMR (400MHz, DMSO-d6) δ9.72(s,1H),7.69(d,J=8.5Hz,1H),7.64(d,J=8.5Hz,1H),7.08(dd,J=8.5,2.5Hz,1H),7.05(d ,J=2.4Hz,1H),6.51(dd,J=8.5,2.4Hz,1H),6.37(d,J=2.4Hz,1H),5.07(s,2H),3.53(s,2H),3.38(s,2H),3.18(s,4H).

[0181] Example 18 Synthesis of compound US-e02

[0182] The preparation method was basically the same as in Example 17, except that N-1-Boc-2-methylpiperazine was used instead of N-Boc-piperazine to obtain compound US-e02 (57 mg, 84%). 1 H NMR (400MHz, DMSO-d6) δ9.71(s,1H),7.69(d,J=8.6Hz,1H),7.65(d,J=8.5Hz,1H),7.08(dd,J=8.5,2.5Hz,1H),7.05(d,J=2.5Hz ,1H),6.51(dd,J=8.5,2.4Hz,1H),6.36(d,J=2.4Hz,1H),5.05(s,2H),4.49-4.36(m,1H),3.28-2.77(m,6H),1.31-1.23(m,3H).

[0183] Example 19 Synthesis of compound US-e03

[0184] The preparation method was basically the same as in Example 17, except that N-Boc-piperazine was replaced with tert-butyl 4,7-diazaspiro[2.5]octane-4-carboxylate to obtain compound US-e03 (33 mg, 47%). 1 H NMR (400MHz, DMSO-d6) δ9.74(s,1H),7.69(d,J=8.6Hz,1H),7.64(d,J=8.6Hz,1H),7.07(dd,J=8.5,2.5Hz,1H),7.05(d,J=2.5Hz ,1H),6.51(dd,J=8.5,2.4Hz,1H),6.37(d,J=2.4Hz,1H),5.04(s,2H),3.61(m,2H),3.51(s,2H),2.85-2.78(m,2H),0.45(s,4H).

[0185] Example 20 Synthesis of compound US-e04

[0186] The preparation method is basically the same as that in Example 17, except that (1S,4S)-2-BOC-2,5-diazabicyclo[2.2.1]heptane is used to replace N-Boc-piperazine to obtain compound US-e04 (42 mg, 63%). 1 H NMR(400MHz,DMSO-d6)δ9.68(s,1H),7.67(d,J=8.6Hz,1H),7.63(d,J=8.5Hz,1H) ,7.08(dd,J=8.5,2.5Hz,1H),7.02(d,J=2.5Hz,1H),6.49(dd,J=8.5,2.4Hz,1H),6 .35(d,J=2.4Hz,1H),5.03(s,2H),4.50(s,1H),4.33(s,1H),3.61(d,J=9.7Hz,1H ),3.52-3.33(m,2H),3.00-2.74(m,2H),1.85-1.66(m,1H),1.58(t,J=9.6Hz,1H).

[0187] Example 21 Synthesis of compound US-f01

[0188] 4-Methylpiperazine-1-formyl chloride hydrochloride (500 mg, 2.5 mmol) was dissolved in acetonitrile (CH3CN, 20 mL), and compound US-d01 (510 mg, 1.5 mmol) and potassium carbonate (453 mg, 3.28 mmol) were added sequentially. The mixture was stirred at 80 °C for 6 h. After the reaction was complete, the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (using DCM:MeOH = 20:1 as the eluent) to give compound X-01 (657 mg, 94%). 1 H NMR (400MHz, DMSO-d6) δ7.85(d,J=8.6Hz,1H),7.82(d,J=8.5Hz,1H),7.16(dd,J=8.4,2.5Hz,1H),7.10(d,J=2.4Hz,1H ),6.85(dd,J=8.5,2.4Hz,1H),6.78(d,J=2.3Hz,1H),5.14(s,2H),3.58(s,4H),3.44(s,4H),2.37(s,8H),2.23(s,6H).

[0189] Example 22 Synthesis of compound US-f02

[0190] The synthetic route of compound US-f02 is the same as that of intermediate compound X-01 in Example 13 (567 mg, 86%). 1 H NMR (400MHz, DMSO-d6) δ7.89(d,J=8.6Hz,1H),7.83(d,J=8.6Hz,1H),7.17(dd,J=8.4,2.4Hz,1H),7.11(d,J=2.3Hz,1H),6.9 2(dd,J=8.5,2.4Hz,1H),6.86(d,J=2.4Hz,1H),5.16(s,2H),3.63-3.42(m,4H),2.45-2.33(m,4H),2.24(s,3H),1.50(s,9H).

[0191] Example 23 Synthesis of compound US-f03

[0192] Compound US-d01 (170 mg, 0.5 mmol) was dissolved in acetonitrile (20 mL), and potassium carbonate (138 mg, 1 mmol) and acetyl chloride (0.07 mL, 0.8 mmol) were added sequentially. The mixture was stirred at room temperature for 6 h. After the reaction was completed, the mixture was washed successively with dilute hydrochloric acid (1 mol / L) and saturated sodium bicarbonate solution, then dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (using DCM:MeOH = 30:1 as the eluent) to obtain compound US-f03 (169 mg, 89%). 1 H NMR (400MHz, DMSO-d6) δ7.87(d,J=8.5Hz,1H),7.83(d,J=8.5Hz,1H),7.16(dd,J=8.5,2.4Hz,1H),7.11(d,J=2.4Hz,1H ),6.89(dd,J=8.5,2.4Hz,1H),6.85(d,J=2.4Hz,1H),5.13(s,2H),3.56(s,4H),2.42(s,4H),2.33(s,3H),2.25(s,3H).

[0193] Example 24 Synthesis of compound US-f04

[0194] Compound US-d01 (170 mg, 0.5 mmol) was dissolved in acetonitrile (20 mL), followed by the addition of potassium carbonate (138 mg, 1 mmol) and iodomethane (0.05 mL, 0.8 mmol). The mixture was refluxed (80 °C) and stirred for 6 h. After the reaction was complete, the mixture was washed successively with dilute hydrochloric acid (1 mol / L) and saturated sodium bicarbonate solution, then dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (using DCM:MeOH = 30:1 as the eluent) to obtain compound US-f04 (164 mg, 93%). 1 H NMR (400MHz, DMSO-d6) δ7.87(d,J=8.6Hz,1H),7.83(d,J=8.5Hz,1H),7.16(dd,J=8.5,2.4Hz,1H),7.11(d,J=2.4Hz,1H),6 .89(dd,J=8.5,2.4Hz,1H),6.85(d,J=2.4Hz,1H),5.17(s,2H),3.81(s,3H),3.61-3.45(s,4H),2.42(s,4H),2.23(s,3H).

[0195] Example 25 Synthesis of compound XL-01 (US-a01 hydrochloride)

[0196] Compound US-a01 (68 mg, 0.2 mmol) was dissolved in methanol (10 mL), and ethyl acetate hydrochloride solution (4 mol / L, 1 mL) was added. The mixture was stirred at room temperature for 5 h. After the reaction was completed, the mixture was filtered, and the filter cake was collected to give a white solid compound XL-01 (74 mg, 91%). 1 H NMR (400MHz, DMSO-d6) δ11.04(s,1H),9.78(s,1H),7.74(d,J=8.6Hz,1H),7.62(d,J=8.6Hz,1H),6.84-6.78(m,2H), 6.77(d,J=2.4Hz,1H),6.67(d,J=2.5Hz,1H),5.06(s,2H),4.21(s,2H),3.59-3.42(m,4H),3.15(s,2H),2.80(s,3H).

[0197] Following the method of Example 25, the hydrochlorides of the compounds in Examples 2 to 24 were prepared.

[0198] Test Example 1: Water Solubility Test

[0199] To determine the water solubility of the compounds in this application, the following compounds were dissolved in water at room temperature, and their solubility was observed. The results are shown in Table 1. As can be seen from Table 1, the water solubility of the compounds provided in this application is significantly improved compared to urolithin A.

[0200] Table 1. Water solubility test results of the compounds in this application.

[0201] Test Example 2: Cell Viability Test

[0202] The cell viability assessment method in this test case is as follows:

[0203] Mitophagy: Mouse neuroblastoma-derived N2a cells were passaged in confocal microscopy dishes, the culture medium was discarded, and the cells were washed twice with HBBS. Mitophagy dye (100 nmol / L) was prepared in advance according to the manufacturer's instructions, and the cells were incubated at 37°C for 30 min in the dark. After staining, the cells were gently washed twice with HBBS, ensuring complete darkness throughout the process, and the cells were treated for 24 h as designed. After the treatment, the cells were washed three times with HBBS and treated in the dark. Lyso dye (1 μmol / L) was prepared in advance and incubated at 37°C for 30 min. After staining, the cells were gently washed twice with PBS, stained with Hoechst for 10 min, and the colocalization of mitochondrial autophagosomes was analyzed using a confocal fluorescence microscope.

[0204] Mitochondrial ATP content detection: Lysis buffer (200 μL) was added to N2a cells, and the cells were homogenized using an ultrasonic homogenizer. After lysis, a portion of the sample was boiled for 2 min to fully release ATP. The remaining sample was centrifuged for 20 min (4℃, 12000g), and the supernatant was collected for BCA protein quantification. Standard curve preparation: Concentration gradients of 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, and 10 μM were set. ATP detection working solution (100 μL) was added to a 96-well black plate and incubated at room temperature for 3–5 min. Then, 20 μL of sample or standard was added to each well, and the mixture was quickly mixed with a pipette. The RLU value was measured using a chemiluminescence analyzer. ATP levels were calculated in nmol / mg protein.

[0205] The specific test results are shown in Table 2.

[0206] Table 2 Results of cell viability test

[0207] Test Example 3: Pharmacokinetic Study

[0208] This test case specifically selected two representative compounds (XL-01: US-a01 hydrochloride; XL-18: US-f01 hydrochloride) for pharmacokinetic studies, and the specific steps are as follows:

[0209] 1. Test drugs: Comparative compound UA (urolithiasis A, 50 mg / kg) and example compounds XL-01 (US-a01 hydrochloride, 82.64 mg / kg) and XL-18 (US-f01 hydrochloride, 118.3 mg / kg).

[0210] 2. Experimental animals: 15 male ICR mice, SPF grade.

[0211] 3. Drug administration and sample collection: Animals were fasted overnight (12h) before drug administration, with free access to water. They were weighed before drug administration, and the dosage was calculated based on their body weight. The drug was administered orally via gavage, and the animals were fed 4 hours after drug administration.

[0212] Blank blood was collected before drug administration, and blood was collected at predetermined time points after drug administration: 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, and 24h. Blood was collected via the jugular vein or other suitable method, with 0.03mL of blood collected per sample. Heparin sodium was used for anticoagulation, and the samples were placed on ice after collection.

[0213] 4. Sample processing: After blood sample collection, place it on ice and centrifuge to separate plasma within 1 hour (centrifugation conditions: 6800g, 6min, 2-8℃). Plasma samples should be stored at -80℃ before analysis.

[0214] 5. Pharmacokinetic Analysis: Pharmacokinetic parameters, namely AUC, were calculated using Phoenix WinNonlin based on plasma drug concentration data at different time points. (0-∞) T 1 / 2 C max T max and MRT (0-∞) .

[0215] The specific test results are shown in Table 3. As can be seen from Table 3, both compounds in this application exhibit superior pharmacokinetic properties compared to UA. Specifically, both compounds in this application are absorbed and reach peak concentration more rapidly, and their plasma concentrations and areas under the curve (AUC) are significantly higher than those of UA, demonstrating better in vivo exposure characteristics and overcoming the problem of low bioavailability of UA.

[0216] Table 3 Pharmacokinetic parameters

[0217] Test Example 4: The therapeutic effect of compound XL-01 on AD

[0218] 1. Test drug: Compound XL-01 (US-a01 hydrochloride) and positive control drug memantine.

[0219] 2. Experimental animals: SPF-grade 5*FAD mice and C57BL / 6J mice, 2 months old.

[0220] 3. Animal grouping and administration: After one week of acclimatization, mice were divided into 5 groups of 10-12 mice each. Administered the drug by gavage once daily for 8 weeks. Specific grouping and administration details were as follows: normal control group (C57BL / 6J mice, saline), model group (5*FAD mice, saline), low-dose group (5*FAD mice, XL-01, 18 mg / kg), high-dose group (5*FAD mice, XL-01, 72 mg / kg), and positive control group (5*FAD mice, memantine, 5 mg / kg).

[0221] 4. Evaluation methods: Behavioral tests were conducted on mice at the end of the drug administration period. The specific behavioral experiments are as follows:

[0222] New Object Recognition: Mice were placed individually in open behavior boxes and allowed to explore freely for 5 minutes. After exploration, the mice were removed and returned to their cages. The behavior boxes were cleaned of feces and urine, and wiped with 75% alcohol to eliminate odor. Two identical objects were placed in two opposite areas of the behavior box, and the mice were again placed in the box to explore for 5 minutes. After each experiment, the behavior box was thoroughly cleaned. One of the objects was replaced, and the mice were placed in the box again to explore for 5 minutes. After each experiment, the behavior box was thoroughly cleaned. An animal motion tracking system was used to record the time spent exploring the objects and calculate the percentage of time spent exploring the new object.

[0223] Y-maze experiment: An animal was placed at the end of any arm of a Y-maze, facing the maze wall, and allowed to explore freely for 8 minutes. Thereafter, the animal was placed on the same arm each time. A video system recorded the animal's behavioral changes over 8 minutes, recording the following indicators: time to enter the new arm, total exploration time, and calculating the ratio. After each mouse was tested, the maze was wiped with alcohol to avoid leaving mouse odor that could affect subsequent experiments.

[0224] 5. The experimental results are as follows:

[0225] Figure 1 shows the effect of XL-01 on behavioral tests in AD model mice. A) Percentage of time spent exploring new objects in the new object recognition test; B) Percentage of time spent exploring new arms in the Y-maze test; n=10, data are expressed as mean ± SEM; **p<0.01 vs WT; #p<0.05, ##p<0.01 vs 5*FAD. The results showed that compared with the normal control group, the time spent exploring new objects and new arms in the model group mice was significantly reduced, indicating impaired memory. After drug treatment, the time spent exploring new objects and new arms in the low-dose group, high-dose group, and positive drug group was significantly increased, indicating that XL-01 can effectively improve the memory ability of 5*FAD mice.

[0226] Test case 5XL-01's anti-aging effects and therapeutic effects on sarcopenia

[0227] 1. Test drugs: Comparative compound UA (urolithin A) and example compound XL-01 (US-a01 hydrochloride).

[0228] 2. Experimental animals: 20 SPF-grade C57BL / 6J mice, 2 months old; 50 SPF-grade C57 mice, 18.5 months old.

[0229] 3. Animal grouping and administration: Mice were randomly divided into 7 groups of 10 mice each. Administered the medication via gavage. The specific groupings and administration methods were as follows: Young control group (young C57 mice, saline), Young administration group (young C57 mice, XL-01, 72 mg / kg), Natural aging group (aged C57 mice, saline), Low-dose group (aged C57 mice, XL-01, 18 mg / kg), Medium-dose group (aged C57 mice, XL-01, 36 mg / kg), High-dose group (aged C57 mice, XL-01, 72 mg / kg), and UA control group (aged C57 mice, UA, 10 mg / kg).

[0230] 4. Assessment method: Each group was given the corresponding dose of drug or saline by gavage for 10 weeks, followed by behavioral tests and pathological examinations.

[0231] Specifically as follows:

[0232] Pole Climbing Test: The pole climbing test is mainly used to assess the motor and coordination abilities of an animal's limbs. A homemade wooden pole (50cm long, 1cm in diameter) is wrapped in gauze to increase friction. The pole is placed vertically against a horizontal table, and a mouse is placed headfirst at the top of the pole. The mouse crawls down the pole independently without external force, and the time taken to climb from the top to the bottom platform is recorded. Before drug administration, the mice are trained for 3 days, 3 times a day, and unfit mice are eliminated. After the formal experiment begins, tests are conducted every two weeks, with 15 seconds as the cutoff value for each test; any result exceeding 15 seconds is recorded. Each experiment is repeated 3 times, and the average crawling time for each mouse is recorded and calculated.

[0233] Grasping force test: The gripping force test is mainly used to assess the limb muscle strength of animals. A mouse is gently placed in the center of a gripping force meter, and its tail is gently pulled to encourage the mouse to grasp the gripping plate with its limbs. Then, the mouse's tail is pulled backward at a constant speed until the mouse's forelimbs release the gripping plate. The maximum value displayed on the gripping force meter before the mouse releases the gripping plate is recorded. Each mouse is tested three times, with a 30-minute interval between each test. The maximum value from the three tests is taken as the evaluation value.

[0234] Suspension test: The grip strength of mice was evaluated using an inverted grid suspension test. Each mouse was placed in the center of a square wire grid with sides of 21 cm (line width: 0.1 cm, spacing: 0.5 cm). The grid was tapped to induce the mouse to grip, and then the grid was slowly inverted. The suspension time of the mouse on the grid was recorded. After 56 days of treatment, all mice were trained twice a day for 3 days, with 90 seconds as the cutoff value. The experiment was repeated 3 times for each mouse, and the average suspension time was recorded and calculated.

[0235] HE staining: Sections were routinely dewaxed to water, hematoxylin stained the nuclei for 5 min, tap water was used for blueing for 10 min, eosin stained for 3 min, and finally dehydrated with ethanol (95% v / v) for 10 s, followed by dehydration three times with anhydrous ethanol for 10 s each time; xylene was used for clearing three times for 5 min each time. The sections were then mounted with neutral resin.

[0236] Masson staining: Sections were routinely dewaxed to water, stained with hematoxylin for 5–10 min, then differentiated with acidic ethanol differentiation solution, soaked in distilled water for 1 min to regain blue color, stained with Ponceau S and fuchsin for 10 min, washed with weak acid working solution for 1 min, washed with phosphomolybdic acid solution for 2 min, washed with weak acid working solution for 1 min, then stained with aniline blue staining solution for 2 min, washed with prepared weak acid working solution for 1 min. Finally, dehydrated with 95% ethanol for 10 s, dehydrated with anhydrous ethanol 3 times for 10 s each time; cleared with xylene 3 times for 5 min each time. Mounted with neutral resin.

[0237] 5. The experimental results are as follows:

[0238] The behavioral test results are shown in Figure 2. Figure 2 shows the effects of XL-01 on motor function and muscle strength in naturally aging mice (an animal model of sarcopenia). A) Pole climbing test, B) Suspension test, C) Grasp test; n=8, data are expressed as mean ± SEM; * p<0.05, ** p<0.01, *** p<0.001. The results showed that compared with the young group, the aging group mice had impaired limb balance, weakened limb strength, and decreased grip strength (as shown in Figure 2); after gavage treatment with XL-01, the limb balance, limb strength, grip strength, motor ability, and coordination of the aging mice were restored; the UA gavage group also showed a trend of improvement (as shown in Figure 2).

[0239] Figures 3 and 4 show the effects of XL-01 on the levels of inflammatory infiltration and fibrosis in the gastrocnemius muscle of naturally aged mice. HE staining (Figure 3) and Masson staining (Figure 4) showed that the aged mice had higher levels of inflammatory infiltration and fibrosis in the gastrocnemius muscle compared with the young group, and oral administration of XL-01 could improve the levels of muscle inflammation and fibrosis in aged mice.

[0240] In conclusion, XL-01 can significantly enhance and improve the motor function of aging mice and has a significant effect on improving muscle inflammation and fibrosis in aging mice, indicating that XL-01 has the potential efficacy in treating sarcopenia.

[0241] Test Case 6 XL-10's therapeutic effect on PD

[0242] 1. Test drug: XL-10 (US-d01 hydrochloride) and positive control compound L-DOPA (levodopa).

[0243] 2. Experimental animals: ordinary grade SD rats, 2-3 months old.

[0244] 3. Animal grouping and administration: After successful rat modeling, rats were randomly divided into 4 groups, with 10-12 rats in each group. The rats were administered the drug via gavage once daily for 2 weeks. The specific grouping and administration were as follows: Model group (6-OHDA model rats, physiological saline), Low-dose group (6-OHDA model rats, XL-10, 36 mg / kg), High-dose group (6-OHDA model rats, XL-10, 72 mg / kg), and Positive control group (6-OHDA model rats, l-DOPA, 25 mg / kg).

[0245] 4. Evaluation methods: Behavioral tests were performed on mice at the end of the drug administration period. The specific experimental details are as follows:

[0246] Rotation experiment: One week after the rats were successfully modeled with 6-OHDA, 0.1% apomorphine (APO; 1 mL / kg) was injected subcutaneously into the neck to induce unidirectional rotation behavior towards the healthy side. The rotation of the rats towards the contralateral side was recorded using a PD model rotation detector. The rotation was recorded once every 360°, and each experiment lasted for 30 minutes.

[0247] 5. The experimental results are as follows:

[0248] Figure 5 shows the effect of XL-10 on behavioral tests in PD model rats, representing the number of contralateral rotations in the rotation experiment; n=8, data are expressed as mean ± SEM; *p<0.05; **p<0.01 vs Before. Behavioral tests showed that XL-10 can effectively reduce the number of rotations in APO-induced 6-OHDA Parkinson's disease rats. After successful 6-OHDA modeling in the four groups of rats, corresponding drug treatments were administered. It was found that the number of rotations in the model group rats increased significantly, indicating faster disease progression; compared with the model group, the number of rotations in the low- and high-dose XL-10 groups rats was significantly reduced after drug treatment, indicating that XL-10 can improve the disease development in 6-OHDA Parkinson's disease rats.

[0249] The therapeutic effect of test case 7XL-18 on DMD

[0250] 1. Test drug: XL-18 (US-f01 hydrochloride) and positive control compound UA (urolithin A).

[0251] 2. Experimental animals: 24 male SPF-grade C57BL / 10J mice and 40 male Dmdem3Cd4 / Gpt mice, aged 26–28 weeks.

[0252] 3. Animal grouping and administration: After one week of acclimatization feeding, animals were randomly divided into 7 groups and administered the medication orally. The specific groupings and administration methods were as follows: Normal control group (C57BL / 10J mice, saline), Normal administration group (C57BL / 10J mice, XL-18, 48 mg / kg), Model group (Dmdem3Cd4 / Gpt mice, saline), Low-dose group (Dmdem3Cd4 / Gpt mice, XL-18, 24 mg / kg), Medium-dose group (Dmdem3Cd4 / Gpt mice, XL-18, 24 mg / kg), and Medium-dose group (Dmdem3Cd4 / Gpt mice, XL-18, 24 mg / kg). A Mice (XL-18, 48 mg / kg), high-dose group (Dmdem3Cd4 / Gpt mice, XL-18, 96 mg / kg), and model positive drug group (Dmdem3Cd4 / Gpt mice, UA, 24 mg / kg).

[0253] 4. Evaluation Methods: Mice were administered orally for 8 weeks, once daily. Behavioral and pathological examinations were performed at the end of the treatment period, using the following methods:

[0254] Pole climbing experiment: See Test Example 5 for experimental procedures.

[0255] Endurance Test: The exercise endurance of mice was assessed by measuring the number of electric shocks received on an uphill treadmill. Exercise program settings: initial speed 9 cm / s, incline angle 5°, acceleration 3 cm / s / 12 min. Mice that could not keep up with the treadmill speed received an electric shock. A mouse was considered exhausted if it received 5 shocks / min for two consecutive minutes. The training phase lasted 8 weeks, 3 days a week. After the training phase, the formal experiment began, and the number of electric shocks was recorded.

[0256] Grip test: See Test Example 5 for experimental procedures.

[0257] Suspension test: See Test Example 5 for experimental procedures.

[0258] HE staining: See Test Example 5 for experimental procedures.

[0259] Masson staining: See Test Example 5 for experimental procedures.

[0260] Immunohistochemistry: Sections were routinely dewaxed to water, then immersed in antigen retrieval solution (1X), heated in a microwave oven on medium heat for 10 min, washed three times with PBS (pH=7.4) for 3 min each time; incubated with hydrogen peroxide blocking solution for 15 min; washed three times with PBS on a shaker for 10 min each time; incubated with protease blocking solution BSA (containing 0.3% Triton-100) for 1 h; incubated directly with the corresponding diluted primary antibody working solution at room temperature for 1 h; then transferred to a 4°C refrigerator overnight; removed the overnight sections and equilibrated at room temperature for 1 h, then washed four times with PBS on a shaker for 10 min each time; incubated with an appropriate amount of secondary antibody working solution for 1 h; washed three times with PBS on a shaker for 5 min each time; stained the treated sections in DAB working solution for 1–10 min; washed twice with PBS; dehydrated sequentially with 75%, 85%, 95%, and 100% ethanol for 3 min each time; cleared twice with xylene for 5 min each time; mounted with neutral resin.

[0261] 5. The experimental results are as follows:

[0262] Figure 6 shows the effects of XL-18 on behavioral tests in DMD model mice: A) pole climbing test, B) treadmill endurance test, C) gripping test, and D) suspension test; n=8, data are expressed as mean ± SEM; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. The results showed that compared with the control group, the model group mice exhibited impaired motor function, with significant decreases in limb coordination, limb strength, and endurance, demonstrating clear differences. After treatment with XL-18, the mice's limb balance, limb strength, coordination, and exercise endurance all recovered to some extent. However, in the suspension test, although all treatment groups showed a trend of increased limb strength, possibly with a dose-dependent relationship, only the high-dose XL-18 group showed a significant difference (p<0.01).

[0263] Figure 7 shows the pathological staining results of XL-18 on the gastrocnemius muscle of DMD model mice: A) HE staining, B) Masson staining, and C) immunohistochemical staining. HE staining results showed that, compared with the control group, the gastrocnemius muscle bundles in the model group mice were smaller, more scattered, and irregular in shape, with some muscle fibers showing atrophy. The model group also showed cell nucleus aggregation, possibly due to excessive aggregation of inflammatory cells in this area, manifesting as inflammatory infiltration. After XL-18 treatment, inflammatory infiltration was significantly reduced, but the muscle bundle morphology did not change significantly. Masson staining results showed that there were a large number of collagen fibers between the muscle bundles in the gastrocnemius muscle of the model group mice. These collagen fibers wrapped around the muscle fibers, causing muscle fiber necrosis, indicating that the fibrosis of the gastrocnemius muscle in the model group mice had severely eroded normal muscle fibers. After XL-18 treatment, the low-dose group still showed collagen fiber erosion of muscle fibers, while the high-dose group showed a significant reduction in collagen fibers between muscle bundles, and the muscle fibers remained intact without necrosis. Immunohistochemical staining results showed that severe fibronectin aggregation occurred in some muscle fiber regions of the model group mice, indicating that the region had relatively severe fibrosis, leading to muscle fiber damage. After treatment with XL-18, the area of ​​fibronectin aggregation in the high-dose group was significantly reduced, indicating that XL-18 effectively alleviated muscle fiber damage caused by fibrosis.

[0264] In conclusion, XL-18 improved the motor function and muscle atrophy in DMD mice, indicating that XL-18 has the potential to treat DMD.

[0265] The therapeutic effect of test case 8XL-18 on FA

[0266] 1. Test drug: XL-18 (US-f01 hydrochloride).

[0267] 2. Experimental animals: Common grade C57BL / 10J mice and YG8R mice (FXN) -(Gene mutation type).

[0268] 3. Animal grouping and administration: After one week of acclimatization, mice were randomly divided into 3 groups, with 10-12 male mice in each group. Mice were administered the drug by gavage at 32 weeks of age, once a day, for 8 weeks. The specific grouping and administration were as follows: control group (C57BL / 10J mice, saline), model group (YG8R mice, saline), and model drug administration group (YG8R mice, XL-18, 96 mg / kg).

[0269] 4. Assessment methods: Behavioral tests and pathological examinations were performed at the end of the drug administration period. Specific methods are as follows:

[0270] Pole climbing experiment: See Test Example 5 for experimental procedures.

[0271] Balance Beam Test: The balance beam test is primarily used to assess the motor balance and coordination abilities of mice. Each mouse starts walking from the same starting point and, without external force, crosses a self-made, narrow wooden beam (length: 100 cm, width: 2.2 cm) to reach a safe platform. Mice are tested at the end of the drug administration period. The test is repeated three times for each mouse, and the average time is recorded and calculated.

[0272] Grip test: See Test Example 5 for experimental procedures.

[0273] Suspension test: See Test Example 5 for experimental procedures.

[0274] Gait detection: The natural gait of mice was acquired through a gait tracking and analysis system, and gait detection and analysis were performed. The entire gait test was conducted in a quiet, dark environment. During the training phase, mice were repeatedly allowed to walk across a glass walkway (50cm long) to adapt to the environment. During the formal test, the mice were placed at the entrance of the walkway and made to walk forward. Walking data was collected using a high-speed gigabit Ethernet camera and filtered and analyzed by the system software WalkAnalysator. If the mouse paused or turned back during walking, the measurement had to be repeated.

[0275] HE staining: See Test Example 5 for experimental procedures.

[0276] Western Blot: Mouse cerebellar tissue was lysed with RIPA buffer, sonicated, and centrifuged at 12,000×g for 15 min at 4°C. The supernatant was collected, and protein concentration was quantified using a BCA protein assay kit. After adding an appropriate amount of loading buffer, the sample was heated at 100°C for 5 min to completely denature the proteins. Proteins were then vertically separated according to molecular weight using SDS-polyacrylamide gel electrophoresis at an appropriate concentration. The separated proteins were transferred to a 0.2 μm nitrocellulose membrane by constant current electroporation. The membrane was then sequentially blocked at room temperature with 5% skim milk powder blocking buffer prepared by TBST, washed with TBST, incubated with primary antibody overnight at 4°C, washed with TBST, incubated with secondary antibody at room temperature for 1 h, and washed with TBST. Finally, the membrane was wetted with ECL high-sensitivity chemiluminescence buffer and imaged using a pre-cooled gel imaging system. The results were analyzed using ImageJ to determine the grayscale information of the bands in the images.

[0277] 5. The experimental results are as follows:

[0278] The behavioral test results are shown in Figures 8 and 9. Figure 8 shows the effect of XL-18 on the behavioral tests of the FA model mice: A) pole climbing test, B) balance beam test, C) grip strength test, D) suspension test; n=10, data are expressed as mean ± SEM; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Figure 9 shows the effect of XL-18 on the gait and limb coordination of the FA model mice: A) mouse gait trajectory diagram, B)~G) gait test statistical results; n=10, data are expressed as mean ± SEM; *p<0.05. The results in Figure 8 show that, compared with the normal group, the model group mice had impaired limb balance, decreased limb grip strength, and decreased exercise endurance. Eight weeks after XL-18 administration, limb balance and grip strength improved to some extent, and exercise endurance also recovered to some degree. Figure 9 shows that XL-18 treatment increased stride length and swing speed in the model group mice, and reduced footprint dragging area and walking cycle. This indicates that XL-18 has significant efficacy in YG8R mice and can effectively improve the symptoms of Friedreich ataxia.

[0279] Figure 10 shows the effects of XL-18 on the pathological changes of the cerebellar dentate gyrus and gastrocnemius muscle in FA model mice. A) and B) correspond to the cerebellar dentate gyrus, and C) and D) correspond to the gastrocnemius muscle. Cerebellar HE staining results showed that, compared with the control group, the number of Purkinje cells in the model group was reduced, while the number of Purkinje cells in the treatment group was increased, and the cells became rounder and fuller, indicating that XL-18 may have a certain restorative effect on Purkinje cells. Gastrocnemius muscle HE staining results showed that the muscle fibers in the model group were loosely arranged, and the cross-sectional area of ​​the muscle fibers was reduced, indicating that the XL-18 treatment group could alleviate the myofibrosis in the model group to some extent.

[0280] Figure 11 shows the effect of XL-18 on FXN expression in the cerebellum of FA model mice. A) Western blot analysis of FXN protein expression level; B) Statistical graph of FXN protein expression level; n=10, data are expressed as mean ± SEM; **p<0.01. The results show that XL-18 treatment for 8 weeks significantly increased the content of the coagulant protein FXN in the cerebellum of mice.

[0281] Figure 12 shows the effects of XL-18 on serum liver function indicators in FA model mice. A) Hydrogen peroxide, B) Nitric oxide, C) Malondialdehyde, D) High-density lipoprotein cholesterol, E) Aspartate aminotransferase / alanine aminotransferase; n=10, data are expressed as mean ± SEM; *p<0.05; **p<0.01; ***p<0.001. The results showed that after 8 weeks of treatment with XL-18, the levels of hydrogen peroxide and nitric oxide in the cerebellum of mice were significantly reversed, and the level of malondialdehyde, an indicator of lipid peroxidation, was reduced. Blood biochemical indicators showed that XL-18 treatment for 8 weeks improved liver function in mice.

[0282] In conclusion, XL-18 can effectively improve the motor function of mice, has a significant therapeutic effect on Purkinje cells and muscle fibrosis damage in YG8R mice, and can increase the expression of human FXN and improve liver function, indicating that XL-18 has the potential efficacy in treating Friedreich ataxia.

[0283] Test case 9XL-18's therapeutic effect on ALS

[0284] 1. Test drug: Compound XL-18 (US-f01 hydrochloride) and positive control compound riluzole.

[0285] 2. Experimental animals: 20 SPF-grade C57BL / 10J male mice, SOD1 G93A Forty-six male mice, 11 weeks old.

[0286] 3. Animal grouping and administration: After 2 weeks of acclimatization feeding, mice were randomly divided into 7 groups. The specific grouping and administration were as follows: normal control group (C57BL / 10J mice, physiological saline), normal administration group (C57BL / 10J mice, XL-18, 48 mg / kg), model group (SOD1...). G93A Mice, saline, low-dose group (SOD1) G93A Mice, XL-18, 24 mg / kg, medium dose group (SOD1) G93A Mice, XL-18, 48 mg / kg, high-dose group (SOD1) G93A Mice, XL-18, 96 mg / kg, and the model group given the positive drug (SOD1) G93A Mice, riluzole, 10 mg / kg).

[0287] 4. Assessment Methods: Each group was administered the appropriate dose of medication or saline via gavage once daily for 6 weeks, followed by behavioral testing and pathological examination. Specific methods are as follows:

[0288] Pole climbing experiment: See Test Example 5 for experimental procedures.

[0289] Suspension test: See Test Example 5 for experimental procedures.

[0290] Roulette test: The roulette test is mainly used to assess the endurance, balance, and coordination of mice. During the training phase, mice were placed on a roulette fatigue tester with the rotation speed set to a maximum of 30 rpm. All mice were trained twice a day for 5 minutes each time, for a total of 3 days. The formal experiment was repeated 3 times, with at least 1 hour intervals between each test. The average time spent on the roulette for each mouse was recorded and calculated.

[0291] Gait detection: See Test Example 8 for experimental procedures.

[0292] HE staining: See Test Example 5 for experimental procedures.

[0293] Nissl staining: The sections were routinely dewaxed to water, stained with Nissl staining solution for 10 min, and washed with distilled water; washed with 70% ethanol for color separation; dehydrated with 75%, 85%, 95%, and 100% ethanol in sequence, 3 min each time; cleared twice with xylene, 5 min each time; mounted with neutral resin; and scanned for pathology using an automatic slide scanner.

[0294] Immunohistochemistry: See Test Example 6 for experimental procedures.

[0295] 5. The experimental results are as follows:

[0296] The behavioral test results are shown in Figures 13 and 14. Figure 13 shows the effect of XL-18 on the behavioral tests of ALS model mice. A corresponds to the pole climbing test, B to the hanging test, C to the spinner test-time, and D to the spinner test-distance. Figure 14 shows the effect of XL-18 on the gait and limb coordination of ALS model mice. A) Swing speed of the left forelimb, B) Swing speed of the left hindlimb, C) Swing speed of the right forelimb, and D) Swing speed of the right hindlimb. n = 8–10, and data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. The results show that compared with the control group, the model group mice exhibited impaired motor function, with significant decreases in limb coordination, limb strength, endurance, and limb swing speed, demonstrating obvious differences. This indicates that after treatment with XL-18, the mice's limb coordination, limb strength, endurance, and limb swing speed were significantly improved.

[0297] Figure 15 shows the effects of XL-18 on the wet weight ratio and pathological staining of the gastrocnemius muscle in ALS model mice. A is a representative image of the hind limb muscles of mice, B is a representative image of the gastrocnemius muscle stained with HE, and C is the result of the wet weight ratio of the gastrocnemius muscle of mice; n=7, data are expressed as mean ± SEM; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Compared with the WT group, the terminal SOD1 G93A The wet weight ratio of the gastrocnemius muscle in the model mice was significantly lower (P<0.0001). Compared with the model group mice, the wet weight ratio of the muscle in each group showed a slight increasing trend after treatment with different doses of XL-18 and the positive control drug riluzole, indicating that XL-18 can effectively reduce muscle loss and alleviate muscle atrophy in mice. HE staining results showed that compared with the normal control group, the gastrocnemius muscle cells in the model group mice were of varying size and morphology, with atrophied cell edges exhibiting elliptical and irregular polygonal shapes, sparse cell arrangement, and enlarged intercellular spaces; inflammatory infiltrating cells proliferated in the muscle cells, and a large number of nuclear aggregates appeared. Compared with the SOD1 model group mice, after treatment with different doses of XL-18 and the positive control drug, the gastrocnemius muscle tissue and morphology recovered to a certain extent, the muscle cell arrangement was more regular, the proportion of atrophied cells decreased, the cell morphology tended to be normal, and the nuclear aggregates and inflammatory cell infiltration area were significantly reduced. The results indicate that XL-18 can improve the morphology (size and arrangement) of gastrocnemius muscle fibers in SOD1 mice and delay the atrophy process of the gastrocnemius muscle.

[0298] Figure 16 shows the effect of XL-18 on Nissl staining of the spinal cord in ALS model mice. A is a representative Nissl staining map, and B is a statistical map of motor neurons; n=7, data are expressed as mean ± SEM; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. The results showed that the anterior horn neurons of the spinal cord in the normal control group mice exhibited a high density distribution and clearly visible neuronal morphology and processes. In contrast, the number of neurons in the model group mice was reduced (P<0.0001), and the neurons underwent vacuolar degeneration, with cell body atrophy and a significant decrease in volume. Compared with the SOD1 model group mice, the number of motor neurons in the anterior horn of the spinal cord recovered after treatment with different doses of XL-18 and positive control drugs.

[0299] The effects of XL-18 on the immunohistochemical activity of GFAP and IBA1 in the spinal cord of ALS model mice are shown in Figures 17 and 18. In Figure 17, A is a representative immunohistochemical image of GFAP, and B is a statistical graph of astrocytes; in Figure 18, A is a representative immunohistochemical image of IBA1, and B is a statistical graph of microglia. Data are expressed as mean ± SEM; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. The results show that the number of GFAP and IBA1 positive cells in the spinal cord of normal control mice is relatively limited, the staining is relatively mild, and they exhibit a scattered distribution. Compared with normal WT mice, the SOD1 model group showed increased positive expression in the spinal cord (GFAP: P<0.0001; IBA1: P<0.001), with deeper staining, denser cell density, and spider-like protrusions around the cell bodies. Compared with the SOD1 model group, the number of astrocytes and microglia in the XL-18 administration group and the positive drug group was reduced to some extent, with significant differences observed in the medium-dose (GFAP: P<0.01; IBA1: P<0.05) and high-dose (GFAP: P<0.001; IBA1: P<0.05) groups. These results indicate that XL-18 can reduce the number of activated astrocytes and microglia in the spinal cord, protect ALS neurons, and delay the progression of ALS.

[0300] In conclusion, XL-18 can effectively improve SOD1 G93A The study observed that XL-18 improved the motor function of mice and also showed significant therapeutic effects on muscle inflammation, spinal cord nerve inflammation, and spinal cord neuron loss, suggesting that XL-18 may have potential therapeutic efficacy for ALS.

[0301] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. A tricyclic compound or a pharmaceutically acceptable salt thereof, said tricyclic compound having the structure shown in Formula 1: In Equation 1, X1 and X2 are independently selected from -O-, -S-, -NR3- or -CH2-, and at least one of X1 and X2 is -CH2-; R1 is selected from -H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C7 cycloalkyl, substituted or unsubstituted C3-C7 heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -C(=O)R4, -C(=O)OR5 or -C(=O)NR6R7. It consists of 3- to 10-membered nitrogen-containing heterocycles; n is an integer from 0 to 18; The n R2s are independently selected from deuterium, halogen, amino, nitro, carbonyl, ester, carboxyl, sulfonic acid, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 amino, substituted or unsubstituted C3-C7 cycloalkyl, substituted or unsubstituted C3-C7 heterocyclic alkyl; or the R2s together with the attached nitrogen-containing heterocycle form a substituted or unsubstituted spirocyclic ring or a substituted or unsubstituted bridged ring; R3, R4, R5, R6, and R7 are independently selected from -H, deuterium, substituted or unsubstituted C1-C6 chain alkyl, substituted or unsubstituted C3-C7 cycloalkyl, substituted or unsubstituted C3-C7 heterocyclic alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl; or R6 and R7 together with the attached nitrogen atom form a substituted or unsubstituted C3-C7 heterocyclic alkyl.

2. The tricyclic compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, X1 and X2 are -O- and -CH2-, respectively; The R1 is selected from -H, -CH3, -C(=O)CH3, -C(=O)OC(CH3)3, -C(=O)N(CH3)2 or The R2 is selected from -CH3 or -N(CH3)2, or R2 together with the attached nitrogen-containing heterocycle forms an unsubstituted spiro ring or an unsubstituted bridged ring; The Selected from 3. The tricyclic compound or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that, The Selected from 4. The tricyclic compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, characterized in that, The tricyclic compound is selected from any one of the following compounds:

5. The tricyclic compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, Pharmaceutically acceptable salts of the tricyclic compounds include hydrochloride, sulfate, phosphate, hydrobromide, nitrate, salicylate, benzoate, C1-C6 fatty carboxylates, C1-C6 alkyl sulfonates, benzene sulfonates, p-toluene sulfonate, or camphor sulfonate.

6. A method for preparing the tricyclic compound according to any one of claims 1 to 5, comprising the following steps: (I) When R1 is -H, and the When the tricyclic compound is a nitrogen-containing heterocycle that does not contain a -NH- group, the preparation method of the tricyclic compound includes the following steps: Compound a, compound b, a base reagent, and an organic solvent were mixed and subjected to a substitution reaction to obtain compound c. Compound c was subjected to a hydroxyl deprotection reaction to obtain compound d, wherein compound d is a compound with R1 being -H and The tricyclic compound is a nitrogen-containing heterocycle that does not contain a -NH- group; The structural formulas of compounds a, b, and c are shown below in sequence: Z is a hydroxyl protecting group, X1, X2, R2 and n are defined as in Equation 1; (II) When R1 is -H, and the When the tricyclic compound is a nitrogen-containing heterocycle containing a -NH- group, the preparation method of the tricyclic compound includes the following steps: Compound a, compound b', a base reagent and an organic solvent were mixed and subjected to a substitution reaction to obtain compound c'; Compound c' is subjected to a hydroxyl deprotection reaction and an imine deprotection reaction to yield compound e, wherein compound e is R1-H and The tricyclic compound being a nitrogen-containing heterocycle containing an -NH- group; The difference between compound b' and compound b is that compound b' contains an imino protecting group at the -NH- group corresponding to the nitrogen-containing heterocycle; the difference between compound c' and compound c is that compound c' contains an imino protecting group at the -NH- group corresponding to the nitrogen-containing heterocycle. (III) When R1 is not -H, the preparation method of the tricyclic compound includes the following steps: Compound d was prepared according to (I) or compound e was prepared according to (II); In the presence of an alkaline reagent and an organic solvent, compound d or compound e is subjected to a substitution reaction with compound f to obtain the tricyclic compound in which R1 is not -H; The compound f is R4C(=O)X3, R5OC(=O)X3, R6R7NC(=O)X3 or R8X3, where X3 is a halogen atom and R8 is a substituted or unsubstituted C1-C6 chain alkyl, a substituted or unsubstituted C3-C7 cycloalkyl, a substituted or unsubstituted C3-C7 heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl.

7. A method for preventing and / or treating diseases related to mitochondrial dysfunction using the tricyclic compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, characterized in that, Prevention and / or treatment are achieved by administering tricyclic compounds or their pharmaceutically acceptable salts.

8. The use of the tricyclic compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of diseases related to mitochondrial dysfunction.

9. The application according to claim 8, characterized in that, The diseases associated with mitochondrial dysfunction include neurological disorders, muscle diseases, metabolic diseases, or aging.

10. The application according to claim 9, characterized in that, The neurological disorders mentioned include Alzheimer's disease, frontotemporal dementia, Friedreich's ataxia, amyotrophic lateral sclerosis, Parkinson's disease, spinocerebellar ataxia, or Huntington's disease.

11. The application according to claim 9, characterized in that, The muscle diseases mentioned include sarcopenia or Duchenne muscular dystrophy.

12. The application according to claim 9, characterized in that, The metabolic diseases include obesity, diabetes, or complications of diabetes, including diabetic nephropathy or diabetic retinopathy.

13. A pharmaceutical composition comprising at least one of the tricyclic compounds of any one of claims 1 to 5 and a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

14. The pharmaceutical composition according to claim 13, characterized in that, The content of the tricyclic compound and its pharmaceutically acceptable salt in the pharmaceutical composition is 0.1 to 99.9 wt%.

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