Indole compound with effect of treating idiopathic pulmonary fibrosis as well as preparation method and application of indole compound

Abstract

The invention discloses an indole compound with an effect of treating idiopathic pulmonary fibrosis as well as a preparation method and application of the indole compound, belongs to the field of medicinal chemistry, and particularly relates to an indole compound shown in a general formula I or pharmaceutically acceptable salt of the indole compound and application of the indole compound serving as an HDAC6 inhibitor in the aspect of resisting idiopathic pulmonary fibrosis. The invention also relates to a pharmaceutical composition taking the indole compound or the pharmaceutically acceptable salt thereof as an active ingredient. The invention also relates to an administration dosage form of the compound containing at least one compound or salt thereof, wherein the dosage form is selected from one or more of the following dosage forms: tablets, capsules, injections, suppositories, patches, inhalable powder preparations, suspensions, emulsions or ointments. The invention provides a new thought for exploring medicines capable of effectively treating idiopathic pulmonary fibrosis, and has important clinical significance. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemistry, and specifically relates to an indole compound capable of treating idiopathic pulmonary fibrosis, a preparation method and application thereof. Background Art

[0002] Epigenetic regulation modulates many cellular processes and greatly influences key disease mechanisms. Histone deacetylases (HDACs) play a crucial role both as biomarkers and therapeutic targets due to their involvement in specific pathophysiological pathways. HDACs are a class of enzymes responsible for removing acetyl groups from histones, resulting in decreased gene transcription, and are primarily involved in chromatin-related biological processes. HDACs can deacetylate histones and non-histone substrates, including transcription factors such as p53 and Rb. They can also regulate key protein substrates such as α-tubulin, actin, and corticosterone, thereby affecting many cellular processes, namely apoptosis, cell signaling, and DNA repair, replication, and recombination.

[0003] Due to the limitations of pirfenidone and nintedanib in the treatment of idiopathic pulmonary fibrosis (IPF) and the lack of promising clinical drug candidates, unprecedented efforts are needed to enrich the available drug options for the treatment of IPF and other rare fibrotic diseases. Increasing evidence indicates that HDACs are involved in the initiation and progression of fibrosis in organs such as the lungs, heart, liver, and kidneys; preliminary studies in animal models have shown that HDAC inhibitors can improve various forms of fibrosis. Among them, the regulation of transforming growth factor β1 (TGF-β1) by HDAC6 (a tubulin-associated deacetylase) is of great significance in the pathogenesis and progression of fibrotic diseases through epithelial-mesenchymal transition (EMT): abnormal expression of HDAC6 activates TGF-β1, promotes Smad2 / 3 phosphorylation, and after forming polymers with Smad4, promotes the EMT process of alveolar epithelial cells and ultimately forms fibrotic lesions. One of the multiple mechanisms of action of the marketed drug pirfenidone is to inhibit the activity of TGF-β1 increased by HDAC6 in this process, thereby having a therapeutic effect on IPF.

[0004] Recent reports have shown that inhibition of HDAC6 by siRNA or tubacin can reduce TGF-β1-induced EMT markers and interfere with the response of Smad3 to TGF-β1. Since Smad3 is a core element of TGF-β1 signaling, its inactivation inhibits HDAC6-dependent α-tubulin deacetylation, which undoubtedly highlights the role of HDAC6 in EMT through the TGF-β1-Smad3 signaling pathway. Studies have demonstrated that the efficacy of pan-HDAC inhibitors (such as SAHA and panobinostat) in IPF and fibrotic lung is mainly based on the reduction of TGF-β1-induced fibroblast-myofibroblast differentiation and fibroblast proliferation. Recently, HDAC6 inhibitors have also been reported to protect mice from lung fibrosis by inhibiting TGF-β1-induced collagen expression and reducing Akt phosphorylation. This relevant pharmacological mechanism has now been confirmed by our immunohistochemical study of HDAC6 overexpression in IPF lung tissue.

[0005] There are many related literature reports on indole compounds, and such compounds have multiple biological activities. Based on a large number of literature surveys, the present invention designs and synthesizes a series of new indole compounds, and conducts anti-idiopathic pulmonary fibrosis activity tests, and unexpectedly finds that the indole compounds exhibit activity in inhibiting the occurrence and progression of idiopathic pulmonary fibrosis. Summary of the invention

[0006] The purpose of the present invention is to design and synthesize a new class of indole compounds based on the original work, and to test their anti-idiopathic pulmonary fibrosis activity. The present invention unexpectedly discovered that the indole compounds exhibit activity in resisting the occurrence and progression of idiopathic pulmonary fibrosis.

[0007] In order to achieve the purpose of the present invention, the following technical scheme is adopted:

[0008] In the first aspect, the present invention provides a novel indole compound having the following general formula I or a pharmaceutically acceptable salt or an optically active form thereof:

[0009]

[0010] Where: R 1 , R 2 , R 3 , R 4 , R 5 Each or both of them is any one of the following groups: (1) H atom, (2) hydroxyl group, (3) halogen atom, (4) cyano group, (5) trihalomethyl group, (6) C 1-6 Alkyl, C 3-8 Cycloalkyl, C 2-6 Alkenyl, C 2-6Alkynyl, C 1-6 Alkanesulfonylamino, C 1-18 Alkoxycarbonyl, C 1-18 Alkoxysulfonyl, C 1-6 Alkoxy, C 2-6 Alkenyloxy, C 2-6 Alkynyloxy, C 1-6 Alkylthio, C 2-6 Alkenylthio, C 2-6 Alkynylthio, C 1-6 Alkylsulfonyl, C 2-6 Alkenylsulfonyl, C 2-6 Alkynylsulfonyl, C 1-6 Alkylsulfinyl, C 2-6 Alkenylsulfinyl, C 2-6 Alkynylsulfinyl, C 3-8 Cycloalkenyl, the above groups are arbitrarily substituted by one or more substituents selected from H atoms, halogen atoms, hydroxyl groups, cyano groups, nitro groups and amino groups, (7) substituted carbonyl groups, which are arbitrarily selected from H atoms, hydroxyl groups, C 1-6 Alkyl, amino, C 1-6 Alkylamino, C 1-6 Alkoxy, C 1-6 Alkylthio and C 3-8 The cycloalkyl group is substituted by one or more substituents, (8) by various substituted amino groups, which are arbitrarily selected from H atoms, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkylcarbonyl, C 2-6 Alkenylcarbonyl and C 2-6 substituted by one or more substituents in the alkynylcarbonyl group, (9) substituted by various substituted phenyl groups, substituted by various substituted benzyl groups, substituted by various substituted benzyloxy groups, substituted by various substituted benzoyl groups, substituted by various substituted pyridine rings, substituted by various substituted pyrazole rings, substituted by various substituted pyrrole rings, substituted by various substituted pyrimidine rings, substituted by various substituted quinoline rings, substituted by various substituted isoquinoline rings, substituted by various substituted imidazole rings, substituted by various substituted morpholine rings, substituted by various substituted piperazine rings, substituted by various substituted pyridazine rings ring, a pyrazine ring substituted in various ways, a piperidine ring substituted in various ways, a thiophene ring substituted in various ways, a thiazole ring substituted in various ways, an isothiazole ring substituted in various ways, a benzothiazole ring substituted in various ways, a pyran ring substituted in various ways, an indole ring substituted in various ways, an oxazole ring substituted in various ways, an isoxazole ring substituted in various ways, a triazole ring substituted in various ways, a benzotriazole ring substituted in various ways, a furan ring substituted in various ways, wherein each of the above groups is arbitrarily selected from H atoms, halogen atoms, C 1-6 Alkyl, C 1-6 Alkylamino, C 3-7 Cycloalkyl, C1-6 Alkoxy, C 1-6 Alkanesulfonylamino, benzyloxycarbonyl, C 1-18 Alkoxycarbonyl, C 1-18 is substituted by one or more substituents selected from the group consisting of alkoxysulfonyl, trihalomethyl, hydroxyl, cyano and amino, (10) the adjacent R 1 With R 2 The halogens include fluorine, chlorine and bromine. Wherein X is any one of the following groups: (1) methylene, (2) nitrogen atom, (3) oxygen atom, (4) sulfur atom. Y is any one of the following groups: (1) methine, (2) nitrogen atom. n = 1 or 2.

[0011] As an optional mode, in the above-mentioned compound or its pharmaceutically acceptable salt or its optically active form, wherein R 1 , R 2 , R 3 , R 4 , R 5 Each or both of them is any one of the following groups: (1) H atom, (2) hydroxyl group, (3) halogen atom, (4) C 1-4 Alkyl, C 1-4 Alkoxy, each of the above groups is substituted by one or more substituents selected from H atoms, halogen atoms, hydroxyl groups and amino groups, (5) phenyl groups substituted by various substituents, benzyl groups substituted by various substituents, each of the above groups is substituted by one or more substituents selected from H atoms, halogen atoms, C 1-4 Alkyl, C 1-4 Alkylamino, C 1-4 Alkoxy, benzyloxycarbonyl, C 1-10 The alkylene group is substituted by one or more substituents selected from the group consisting of alkoxycarbonyl, trihalomethyl, hydroxymethyl, hydroxyl and amino. Wherein X is any one of the following groups: (1) methylene, (2) nitrogen atom, (3) oxygen atom. Y is any one of the following groups: (1) methine, (2) nitrogen atom. n=1 or 2.

[0012] As an optional mode, in the above-mentioned compound or its pharmaceutically acceptable salt or its optically active form, wherein R 1 , R 2 , R 3 , R 4 , R 5 Each or both of them is any one of the following groups: (1) H atom, (2) hydroxyl group, (3) fluorine, chlorine, bromine, (4) C 1-4 Alkyl, C 1-4 Alkoxy, the above groups are arbitrarily substituted by one or more substituents selected from H atoms, halogen atoms, hydroxyl groups and amino groups; (5) unsubstituted or substituted phenyl, benzyl, the substituents are selected from H atoms, halogen atoms, C1-4 Alkyl, C 1-4 Alkoxy, C 1-4 One or more of alkoxycarbonyl and trihalomethyl. Wherein X is any one of the following groups: (1) methylene, (2) nitrogen atom, (3) oxygen atom. Y is any one of the following groups: (1) methine, (2) nitrogen atom. n=1 or 2.

[0013] As an optional mode, in the above-mentioned compound or its pharmaceutically acceptable salt or its optically active form, wherein R 1 , R 2 , R 3 , R 4 , R 5 Each or all of them are any one of the following groups: H atom, hydroxyl group, bromine atom, chlorine atom, fluorine atom, methyl group, ethyl group, propyl group, n-butyl group, tert-butyl group, tert-butylmethyl group, methoxy group, ethoxy group, phenyl group, 2-methyl-phenyl group, 4-methylphenyl group, 4-methoxyphenyl group, 4-trifluoromethylphenyl group, 4-bromophenyl group, benzyl group, 4-fluorobenzyl group, 2,4-difluorobenzyl group, 3,4-difluorobenzyl group, 3,5-difluorobenzyl group, 3,4,5-trifluorobenzyl group. Wherein X is any one of the following groups: (1) methylene group, (2) oxygen atom. Y is any one of the following groups: (1) methine group, (2) nitrogen atom. n=1 or 2.

[0014] As an optional mode, in the above-mentioned compound or its pharmaceutically acceptable salt or its optically active form, the compound is selected from:

[0015]

[0016] In the second aspect, a synthetic route for preparing the indole compounds of general formula I or pharmaceutically acceptable salts thereof or optically active forms or racemates, or diastereoisomer mixtures thereof is provided.

[0017] Using a tryptamine starting material, the amino group is first protected with Boc anhydride to obtain an intermediate M1. M1 undergoes a substitution reaction with a compound s2 (such as 4-bromomethylbenzoic acid methyl ester or 6-bromomethylnicotinate) under the action of NaH to obtain an intermediate M2. M2 removes the Boc protecting group under trifluoroacetic acid conditions to obtain an intermediate M3. Subsequently, M3 undergoes an acylation reaction with an acyl chloride raw material s3 in the presence of triethylamine to obtain an intermediate M4. M4 undergoes an aminolysis reaction with hydroxylamine hydrochloride under KOH conditions to obtain compounds A1 to A26, wherein R 1 , R 2 , R 3 , R 4 , R 5 , n, X, and Y are corresponding groups at corresponding positions of compounds A1 to A26.

[0018]

[0019] Reagents and solvents used in the synthesis routes of compounds A1 to A26: (a) Boc2O, TEA, THF; (b) NaH, DMF; (c) TFA, DCM; (d) TEA, DCM; (e) 1) NH2OH·HCl, NaOH, MeOH / THF, 2) HCl.

[0020] In a third aspect, the present invention provides a pharmaceutical composition comprising the compound described in the first aspect or a pharmaceutically acceptable salt thereof or an optically active form thereof and a pharmaceutically acceptable carrier.

[0021] In a fourth aspect, the present invention provides a pharmaceutical preparation, comprising the pharmaceutical composition described in the third aspect, wherein the dosage form of the pharmaceutical preparation is selected from one or more of the following: tablets, capsules, injections, suppositories, patches, inhalable powder preparations, suspensions, emulsions and ointments.

[0022] In a fifth aspect, the present invention provides use of the compound described in the first aspect or its pharmaceutically acceptable salt or optically active form thereof, or the pharmaceutical composition described in the third aspect, or the pharmaceutical preparation described in the fourth aspect in the preparation of an HDAC6 inhibitor.

[0023] In the sixth aspect, the present invention provides the use of the compound described in the first aspect or its pharmaceutically acceptable salt or its optically active form or the pharmaceutical composition described in the third aspect or the pharmaceutical preparation described in the fourth aspect in the preparation of anti-idiopathic pulmonary fibrosis drugs.

[0024] As an optional mode, in the above application, the anti-idiopathic pulmonary fibrosis drug has the activity of preventing the occurrence and progression of idiopathic pulmonary fibrosis.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention derived and designed a new class of substituted indole compounds and conducted anti-idiopathic pulmonary fibrosis activity tests, and unexpectedly found that the indole compounds showed activity against the occurrence and progression of idiopathic pulmonary fibrosis. The present invention provides a new idea for exploring drugs that can effectively treat idiopathic pulmonary fibrosis and has important clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a toxicity test of compound A21 of the present invention on HELF cells;

[0028] Figure 2The effect of the compound A21 of the present invention on the proliferation of HELF cells induced by TGF-β1; the data are expressed as the mean ± standard deviation of three independent experiments. ###p<0.001 (comparison between the model group and the control group), *p<0.05, **p<0.01 (comparison between Tubastatin A and the model group, comparison between A21 and the model group); NS indicates no significant difference;

[0029] Figure 3 This is a test of the inhibitory effect of the compound A21 of the present invention on the HDAC6 activity in HELF cells during TGF-β1-induced pulmonary fibrosis in vitro; the data are expressed as the mean ± standard deviation of three independent experiments: ###p<0.001 (comparison between the model group and the control group); *p<0.05, **p<0.01 (comparison between Tubastatin A and the model group, comparison between A21 and the model group); NS indicates no significant difference;

[0030] Figure 4 The effect of the compound A21 of the present invention on the expression changes of HDAC6-related substrate proteins in HELF cells during TGF-β1-induced pulmonary fibrosis in vitro; wherein, (A) is the expression of each protein in the detected cells, and (B) is the quantification result of the relative expression of the protein; the data are expressed as the mean ± standard deviation of three independent experiments: ###p<0.001 (comparison between the model group and the control group); **p<0.01, ***p<0.001 (comparison between SAHA and the model group, comparison between Tubastatin A and the model group, comparison between A21 and the model group); NS indicates no significant difference;

[0031] Figure 5 The effect of the compound A21 of the present invention on the expression of fibrosis-related substrates in HELF cells during TGF-β1-induced pulmonary fibrosis in vitro; wherein, (A) is the expression of COL1A1 and α-SMA, and (B) is the protein expression level of COL1A1 and α-SMA; the data are expressed as the mean ± standard deviation of three independent experiments: ###p<0.001 (comparison between model group and control group); **p<0.01, ***p<0.001 (comparison between Tubastatin A and model group, comparison between A21 and model group);

[0032] Figure 6The effect of the compound A21 of the present invention on the expression of p-Smad2, p-Smad3 and p-TGF-βR1 in HELF cells during TGF-β1-induced pulmonary fibrosis in vitro; wherein, (A) is the expression of p-Smad2 / 3 and p-TGF-βR1, and (B) is the protein expression level of p-Smad2 / 3 and p-TGF-βR1; the data are expressed as the mean ± standard deviation of three independent experiments: ###p<0.001 (comparison between model group and control group); **p<0.01, ***p<0.001 (comparison between Tubastatin A and model group, comparison between A21 and model group);

[0033] Figure 7 The effect of the compound A21 of the present invention on the expression of TGF-β1-related mRNA in HELF cells during TGF-β1-induced pulmonary fibrosis in vitro; ###p<0.001 (comparison between the model group and the control group); *p<0.05, **p<0.01 (comparison between Tubastatin A and the model group, comparison between A21 and the model group);

[0034] Figure 8 The weight changes of mice in each group during the test of the compound A21 of the present invention in the mouse pulmonary fibrosis model;

[0035] Fig. 9 The effect of the compound A21 of the present invention on the HDAC6 activity in the lung tissue of mice with pulmonary fibrosis model; ##p<0.01 (comparison between the model group and the control group); *p<0.05, **p<0.01 (comparison between A21 and the model group); NS indicates no significant difference;

[0036] Fig.10 The effect of the compound A21 of the present invention on the expression of TGF-β1 related mRNA in the lung tissue of mice with pulmonary fibrosis model; ###p<0.001 (comparison between the model group and the control group); **p<0.01, ***p<0.001 (comparison between PFD and the model group, comparison between A21 and the model group); NS indicates no significant difference;

[0037] Fig.11 The effect of compound A21 of the present invention on pathological changes in lung tissue of mice with pulmonary fibrosis model;

[0038] Fig.12The effects of the compound A21 of the present invention on the changes of relevant biomarkers in the lung tissue of mice with pulmonary fibrosis model; wherein, (A) is the effect of A21 on collagen deposition studied by Masson trichrome staining; (B) is the change of α-SMA expression in mouse lung tissue; (C) is the change of p-Smad2 expression in mouse lung tissue; (D) is the change of p-Smad3 expression in mouse lung tissue; ###p<0.001 (comparison between model group and control group), **p<0.01, ***p<0.001 (comparison between PFD and model group, comparison between A21 and model group). DETAILED DESCRIPTION

[0039] The present invention will be further described with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0040] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0041] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0042] Example 1: N-Hydroxy-4-((3-(2-acetamidoethyl)-1H-indol-1-yl)methyl)-benzamide (A1)

[0043] At room temperature, 3-(2-aminoethyl)indole (tryptamine 15g, 93.62mmol) was suspended in 150mL of anhydrous THF, triethylamine (15.59mL, 112.35mmol) was added, and after stirring evenly, Boc2O (24.52g, 112.35mmol) was slowly added in batches. After the addition was completed, it was stirred for 2h. The reaction liquid was clear and the reaction was monitored by TLC. After that, 5mL of water was added to quench the reaction, and the solvent was concentrated to obtain the off-white solid crude product of M1. After purification by column chromatography (PE:EA=5:1, v / v), the intermediate M1 was obtained as a white to light yellow shiny solid 22.55g, with a yield of 92.52%.

[0044] Under ice bath, the intermediate M1 (10 g, 38.41 mmol) obtained above was dissolved in 50 mL of anhydrous DMF, and NaH (3.07 g, 76.82 mmol) was slowly added in batches under stirring. After the addition was completed, it was stirred for 0.5 h. After that, methyl 4-bromomethylbenzoate (10.56 g, 46.09 mmol) was added to the reaction solution, and the reaction was moved to room temperature and stirred for 2 h. The reaction was monitored by TLC to complete. Subsequently, 300 mL of water was added to the reaction solution, and after stirring evenly, it was extracted with ethyl acetate (150 mL / time) until no product remained in the water layer, and the organic layers were combined, washed twice with saturated brine and dried over anhydrous sodium sulfate, and then filtered and concentrated to remove the solvent to obtain a brown solid crude product of M2, which was purified by column chromatography (PE: EA = 5: 1) to obtain 13.24 g of intermediate M2 as a white solid, with a yield of 84.38%.

[0045] Under ice bath, the intermediate M2 (13.24 g, 32.41 mmol) obtained above was dissolved in 200 mL of anhydrous DCM, and trifluoroacetic acid (72.23 mL, 972.34 mmol) was added dropwise under stirring. After the addition was completed, it was stirred for 0.5 h and the reaction was monitored by TLC. After concentrating to remove most of the solvent, 100 mL of water was added to the residue, and the pH was adjusted to 8-9 with saturated sodium bicarbonate solution under stirring. After that, the obtained mixture was extracted with ethyl acetate (150 mL / time) until there was no residue in the water layer, and the organic layers were combined, washed twice with saturated brine and dried over anhydrous sodium sulfate, filtered, concentrated to remove the solvent, and obtained the brown viscous crude product of M3, which was purified by column chromatography (DCM: MeOH = 15: 1) to obtain intermediate M3 as a yellow solid 8.62 g, yield: 86.25%.

[0046] Under ice bath, the intermediate M3 (1 g, 3.243 mmol) obtained above was dissolved in 20 mL of anhydrous DCM, triethylamine (0.90 mL, 6.486 mmol) was added, and then acetyl chloride (0.31 g, 3.891 mmol) was added dropwise under stirring. After the addition was completed, the mixture was stirred for 0.5 h, and the reaction was monitored by TLC to complete. After concentrating and removing most of the solvent, a pale yellow solid crude product of M4 was obtained, which was purified by column chromatography (PE: EA = 5: 1) to obtain 1.06 g of intermediate M4 as a white solid, with a yield of 93.28%.

[0047] Under ice bath, sodium hydroxide (0.48 g, 12.10 mmol) was stirred and dissolved in aqueous hydroxylamine solution (50%, wt., 9.99 mL, 151.25 mmol), and then the intermediate M4 (1.06 g, 3.025 mmol) obtained above was dissolved in THF / MeOH (1:1, v / v, 30 mL) and added dropwise to the reaction solution. After the addition was completed, the mixture was stirred for 0.5 h and the reaction was monitored by TLC to complete. Subsequently, after concentrating to remove most of the solvent, 20 mL of water was added to the resulting mixture, and the pH was adjusted to neutral with 1 mol / L dilute hydrochloric acid under stirring, a white solid precipitated, and the mixture was extracted with ethyl acetate (50 mL / time) until no product remained in the aqueous layer. The organic layers were combined, washed twice with saturated brine and dried over anhydrous sodium sulfate, filtered, and concentrated to remove the solvent to obtain a yellow solid crude product of the final product A1, which was purified by column chromatography (DCM:MeOH=15:1, 0.5% AcOH) to obtain 0.51 g of the final product A1 as a light yellow solid, with a yield of 47.98%. 1 H NMR(600MHz,DMSO-d6)δ11.14(s,1H),9.04(s,1H),7.95(t,J=5.7Hz,1H),7.68–7 .65(m,2H),7.55(dd,J=7.9,1.3Hz,1H),7.38(d,J=8.2Hz,1H),7.32(s,1H),7.22( d,J=8.1Hz,2H),7.08(ddd,J=8.2,6.9,1.2Hz,1H),7.01(ddd,J=7.9,7.1,1.1Hz, 1H),5.40(s,2H),3.32(td,J=7.6,5.8Hz,2H),2.82(t,J=7.5Hz,2H),1.80(s,3H). 13 C NMR(151MHz,DMSO-d6)δ172.54,169.50,142.04,136.49,132.30,128.34,127.58(2C),127.36(2C), 127.06,121.77,119.17,119.13,112.54,110.49,49.05,25.55,23.16,21.56.ESI-HRMS:calcd.forC 20 H 21 N3O3Na[M+Na] + ,374.1481; found 374.1471.

[0048] Example 2: N-Hydroxy-4-((3-(2-propionamidoethyl)-1H-indol-1-yl)methyl)-benzamide (A2)

[0049] The preparation method of the compound of Example 2 is the same as that of Example 1, except that acetyl chloride is replaced by propionyl chloride in step 4 to obtain 0.82 g of brown solid powder with a yield of 64.39%. 1 H NMR (600MHz, DMSO-d6) δ11.12(s,1H),9.01(s,1H),7.85(t,J=5.8Hz,1H),7.66(d, J=8.0Hz,2H),7.56(d,J=7.8Hz,1H),7.37(d,J=8.2Hz,1H),7.30(s,1H),7.22(d,J =8.0Hz,2H),7.10–7.06(m,1H),7.01(t,J=7.4Hz,1H),5.40(s,2H),3.32(dd,J=7. 3,5.4Hz,2H),2.83(t,J=7.4Hz,2H),2.06(q,J=7.6Hz,2H),0.98(t,J=7.6Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ173.22,164.38,142.03,136.49,132.31,128.37,127.57(2C),127.37(2C),127 .04,121.77,119.20,119.11,112.59,110.47,49.06,39.81,29.03,25.57,10.41.ESI-HRMS:calcd.for C 21 H 24 N3O3[M+H] + ,366.1818; found 366.1824.

[0050] Example 3: N-Hydroxy-4-((3-(2-butyramidoethyl)-1H-indol-1-yl)methyl)-benzamide (A3)

[0051] The preparation method of the compound of Example 3 is the same as that of Example 1, except that butyryl chloride is used to replace acetyl chloride in step 4 to obtain 0.34 g of brown solid powder with a yield of 27.15%. 1H NMR (600MHz, DMSO-d6) δ11.15(s,1H),9.03(s,1H),7.88(t,J=5.8Hz,1H),7.69–7.63(m ,2H),7.56(d,J=7.8Hz,1H),7.37(d,J=8.2Hz,1H),7.30(s,1H),7.22(d,J=8.1Hz,2H),7 .08(ddd,J=8.3,7.0,1.2Hz,1H),7.03–6.99(m,1H),5.40(s,2H),3.35–3.31(m,2H),2. 82(t,J=7.4Hz,2H), 2.02(t,J=7.3Hz,2H), 1.49(h,J=7.4Hz,2H), 0.83(t,J=7.4Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ172.60,164.37,142.02,136.50,132.30,128.34,127.57(2C),127.37(2C),127.03 ,121.77,119.20,119.11,112.57,110.47,49.05,39.71,37.90,25.61,19.14,14.14.ESI-HRMS:calcd.for C 22 H 26 N3O3[M+H] + ,380.1974; found 380.1972.

[0052] Example 4: N-Hydroxy-4-((3-(2-propionamidoethyl)-5-methoxy-1H-indol-1-yl)methyl)-benzamide (A4)

[0053] The preparation method of the compound of Example 4 is the same as that of Example 1, except that 5-methoxytryptamine is used to replace tryptamine in step 1, and acetyl chloride is used to replace propionyl chloride in step 4 to obtain 0.49 g of white solid powder with a yield of 30.35%. 1H NMR(600MHz,DMSO-d6)δ11.14(s,1H),8.99(s,1H),7.85(t,J=5.7Hz,1H), 7.67–7.64(m,2H),7.27–7.24(m,2H),7.19(d,J=8.3Hz,2H),7.05(d,J=2.5 Hz,1H),6.72(dd,J=8.8,2.4Hz,1H),5.35(s,2H),3.75(s,3H),3.34–3.30( m,2H),2.79(t,J=7.4Hz,2H),2.06(q,J=7.6Hz,2H),0.98(t,J=7.6Hz,3H). 13 CNMR(151MHz,DMSO-d6)δ173.20,164.44,153.78,142.19,132.26,131.73,128.74,127.63,127.56(2C),12 7.28(2C),112.17,111.73,111.23,101.17,55.85,49.20,40.26,29.05,25.60,10.42.ESI-HRMS:calcd.for C 22 H 26 N3O4[M+H] + ,396.1923;found396.1927.

[0054] Example 5: N-Hydroxy-4-((3-(2-butyramidoethyl)-5-methoxy-1H-indol-1-yl)methyl)-benzamide (A5)

[0055] The preparation method of the compound of Example 5 is the same as that of Example 1, except that 5-methoxytryptamine is used to replace tryptamine in step 1, and acetyl chloride is used to replace butyryl chloride in step 4 to obtain 0.22 g of brown solid powder with a yield of 73.16%. 1 H NMR (600MHz, DMSO-d6) δ11.15(s,1H),9.01(s,1H),7.87(t,J=5.7Hz,1H),7.69–7. 64(m,2H),7.28–7.24(m,2H),7.20(d,J=8.3Hz,2H),7.06(d,J=2.4Hz,1H),6.73(dd ,J=8.8,2.4Hz,1H),5.35(s,2H),3.76(s,3H),3.33(td,J=7.5,5.7Hz,2H),2.79(t ,J=7.4Hz,2H),2.03(t,J=7.4Hz,2H),1.50(h,J=7.4Hz,2H),0.83(t,J=7.4Hz,3H).13 C NMR(151MHz,DMSO-d6)δ170.21,162.30,151.65,140.03,130.11,129.61,126.60,125.49,125.41(2C),125.1 4(2C),110.04,109.60,109.09,99.05,53.72,47.06,37.52,35.80,23.49,17.02,12.00.ESI-HRMS:calcd.for C 23 H 27 N3O4Na[M+Na] + ,432.1899; found 432.1894.

[0056] Example 6: N-Hydroxy-4-((3-(2-benzamidoethyl)-5-methoxy-1H-indol-1-yl)methyl)-benzamide (A6)

[0057] The preparation method of the compound of Example 6 is the same as that of Example 1, except that acetyl chloride is replaced by benzoyl chloride in step 4 to obtain 0.29 g of a white solid powder with a yield of 22.96%. 1 H NMR (600MHz, DMSO-d6) δ11.14(s,1H),9.00(s,1H),8.61(t,J=5.7Hz,1H),7.84(d,J=7.5 Hz,2H),7.64(d,J=8.1Hz,2H),7.62(d,J=7.9Hz,1H),7.53–7.50(m,1H),7.45(t,J=7.6H z,2H),7.39(d,J=8.2Hz,1H),7.36(s,1H),7.22(d,J=8.1Hz,2H),7.09(ddd,J=8.2,6.9, 1.2Hz,1H),7.02(t,J=7.4Hz,1H),5.41(s,2H),3.59–3.53(m,2H),2.97(t,J=7.5Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ166.62,164.45,142.04,136.53,135.16,132.30,131.51,128.71(2C),128.39,127.60(2C),1 27.58(2C),127.37(2C),127.11,121.80,119.25,119.15,112.55,110.51,49.05,40.52,25.50.ESI-HRMS:calcd.for C 25 H 24N3O3[M+H] + ,414.1818; found 414.1821.

[0058] Example 7: N-Hydroxy-4-((3-(2-(2-phenylacetamido)ethyl)-1H-indol-1-yl)methyl)benzamide (A7)

[0059] The preparation method of the compound of Example 7 is the same as that of Example 1, except that acetyl chloride is replaced by phenylacetyl chloride in step 4 to obtain 0.29 g of light brown solid powder with a yield of 41.33%. 1 H NMR (600MHz, DMSO-d6) δ11.14(s,1H),9.03(s,1H),8.15(t,J=5.8Hz,1H),7.66(d,J=8.0Hz,2H),7.55(d,J=7.9Hz,1H),7.37(d,J=8.2Hz,1 H),7.29–7.19(m,8H),7.08(t,J=7.6Hz,1H),7.01(t,J=7.4Hz,1H),5.38(s,2H),3.40(s,2H),3.34(q,J=6.8Hz,2H),2.83(t,J=7.3Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ172.57,164.40,142.00,136.98,136.49,132.31,129.48(2C),128.65(2C),128.32,127.58(2C), 127.36(2C),127.09,126.76,121.79,119.21,119.15,112.46,110.49,49.07,42.95,40.52,25.48.ESI-HRMS:calcd.for C 26 H 26 N3O3[M+H] + ,428.1974; found 428.1966.

[0060] Example 8: N-Hydroxy-4-((3-(2-(4-methylbenzamido)ethyl)-1H-indol-1-yl)methyl)benzamide (A8)

[0061] The preparation method of the compound of Example 8 is the same as that of Example 1, except that 4-methylbenzoyl chloride is used to replace acetyl chloride in step 4 to obtain 0.30 g of light brown solid powder with a yield of 16.72%. 1H NMR (600MHz, DMSO-d6) δ11.14(s,1H),9.00(s,1H),8.52(t,J=5.7Hz,1H),7.74(d,J=8.1 Hz,2H),7.64(d,J=8.3Hz,2H),7.62(d,J=7.9Hz,1H),7.38(d,J=8.2Hz,1H),7.35(s,1H) ,7.25(d,J=8.0Hz,2H),7.22(d,J=8.1Hz,2H),7.09(ddd,J=8.1,6.9,1.1Hz,1H),7.03–7 .00(m,1H),5.40(s,2H),3.55(td,J=7.7,5.5Hz,2H),2.96(t,J=7.5Hz,2H),2.35(s,3H). 13 C NMR(151MHz,DMSO-d6)δ166.48,164.45,142.03,141.33,136.52,132.36,132.30,129.24(2C),128.39,127.63(2C),127. 59(2C),127.36(2C),127.09,121.79,119.25,119.14,112.58,110.49,49.04,40.47,25.54,21.41.ESI-HRMS:calcd.for C 26 H 26 N3O3[M+H] + ,428.1974; found 428.1975.

[0062] Example 9: N-hydroxy-4-((3-(2-(4-methoxybenzamido)ethyl)-1H-indol-1-yl)methyl)benzamide (A9)

[0063] The preparation method of the compound of Example 9 is the same as that of Example 1, except that 4-methoxybenzoyl chloride is used to replace acetyl chloride in step 4 to obtain 0.097 g of yellow solid powder with a yield of 6.82%. 1H NMR (600MHz, DMSO-d6) δ11.13(s,1H),9.00(s,1H),8.45(t,J=5.7Hz,1H),7.83–7.80( m,2H),7.66–7.63(m,2H),7.61(d,J=7.9Hz,1H),7.38(d,J=8.2Hz,1H),7.34(s,1H),7 .22(d,J=8.1Hz,2H),7.09(ddd,J=8.2,6.9,1.2Hz,1H),7.04–7.00(m,1H),6.99–6.97 (m,2H),5.41(s,2H),3.81(s,3H),3.54(td,J=7.6,5.7Hz,2H),2.95(t,J=7.5Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ166.10,164.45,161.90,142.04,136.53,132.28,129.40(2C),128.38,127.58(2C),127.38,127. 35(2C),127.08,121.78,119.26,119.13,113.89(2C),112.62,110.49,55.79,49.03,40.45,25.61.ESI-HRMS:calcd.for C 26 H 26 N3O4[M+H] + ,444.1923; found 444.1924.

[0064] Example 10: N-Hydroxy-4-((3-(2-(4-trifluoromethylbenzamido)ethyl)-1H-indol-1-yl)methyl)benzamide (A10)

[0065] The preparation method of the compound of Example 10 is the same as that of Example 1, except that 4-trifluoromethylbenzoyl chloride is used to replace acetyl chloride in step 4 to obtain 0.2531 g of white solid powder with a yield of 18.04%. 1H NMR (600MHz, DMSO-d6) δ11.14(s,1H),9.00(s,1H),8.86(t,J=5.7Hz,1H),8.03(d,J=8 .1Hz,2H),7.85(d,J=8.2Hz,2H),7.65(d,J=8.3Hz,2H),7.62(d,J=7.8Hz,1H),7.39(d ,J=8.2Hz,1H),7.36(s,1H),7.22(d,J=8.2Hz,2H),7.09(ddd,J=8.3,6.9,1.2Hz,1H), 7.02(t,J=7.3Hz,1H),5.41(s,2H),3.58(td,J=7.5,5.6Hz,2H),2.99(t,J=7.5Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ164.36,163.34,140.95,137.80,135.44,131.23,130.40(q,J=31.4Hz),127.45(2C),127.29,126.50(2C),126.28(2C) ,126.07,124.70(q,J=3.9Hz,2C),123.37(d,J=271.9Hz),120.74,118.13,118.09,111.34,109.45,47.98,39.65,24.29.ESI-HRMS:calcd.for C 26 H 22 F3N3NaO3[M+Na] + ,504.1511; found 504.1517.

[0066] Example 11: N-hydroxy-4-((3-(2-pivalamidoethyl)-1H-indol-1-yl)methyl)benzamide (A11)

[0067] The preparation method of the compound of Example 11 is the same as that of Example 1, except that pivaloyl chloride is used to replace acetyl chloride in step 4 to obtain 0.5218 g of white solid powder with a yield of 40.63%. 1H NMR (600MHz, DMSO-d6) δ11.14(s,1H),9.00(s,1H),7.66(d,J=8.3Hz,2H),7.59(d d,J=7.8,1.2Hz,1H),7.53(t,J=5.7Hz,1H),7.37(d,J=8.2Hz,1H),7.28(s,1H),7 .23(d,J=8.2Hz,2H),7.08(ddd,J=8.2,6.9,1.2Hz,1H),7.01(ddd,J=7.9,7.0,1. 0Hz, 1H), 5.40 (s, 2H), 3.32 (t, J = 7.4Hz, 2H), 2.83 (t, J = 7.4Hz, 2H), 1.06 (s, 9H). 13 C NMR(151MHz,DMSO-d6)δ177.73,164.43,142.05,136.49,132.29,129.38,127.57(2C),127.38(2C),127.0 1,121.75,119.28,119.07,112.65,110.45,49.03,40.51,38.42,27.93(3C),25.45.ESI-HRMS:calcd.for C 23 H 27 N3NaO3[M+Na] + ,416.1950; found 416.1955.

[0068] Example 12: N-Hydroxy-4-((3-(2-(3,3-dimethylbutyramido)ethyl)-1H-indol-1-yl)methyl)benzamide (A12)

[0069] The preparation method of the compound of Example 12 is the same as that of Example 1, except that 3,3-dimethylbutyryl chloride is used to replace acetyl chloride in step 4 to obtain 0.3434 g of light brown solid powder with a yield of 29.53%. 1 H NMR(600MHz,DMSO-d6)δ11.14(s,1H),9.00(s,1H),7.84(t,J=5.7Hz,1H),7.6 8–7.65(m,2H),7.56(d,J=7.8Hz,1H),7.37(d,J=8.2Hz,1H),7.31(s,1H),7.2 3(d,J=8.2Hz,2H),7.08(ddd,J=8.2,6.9,1.2Hz,1H),7.03–7.00(m,1H),5.40 (s,2H),3.34(m,2H,-CH2-),2.82(t,J=7.5Hz,2H),1.94(s,2H),0.94(s,9H).13 C NMR(151MHz,DMSO-d6)δ171.17,164.43,142.03,136.50,132.31,128.32,127.58(2C),127.38(2C),127.02,1 21.78,119.20,119.11,112.57,110.47,49.39,49.06,39.64,30.82,30.18(3C),25.69.ESI-HRMS:calcd.for C 24 H 29 N3NaO3[M+Na] + ,430.2107;found430.2113.

[0070] Example 13: N-Hydroxy-4-((3-(2-(4-bromobenzamido)ethyl)-1H-indol-1-yl)methyl)benzamide (A13)

[0071] The preparation method of the compound of Example 13 is the same as that of Example 1, except that 4-bromobenzoyl chloride is used to replace acetyl chloride in step 4 to obtain 0.3111 g of light brown solid powder with a yield of 70.56%. 1 H NMR (600MHz, DMSO-d6) δ11.15(s,1H),9.01(s,1H),8.71(t,J=5.7Hz,1H),7.80–7. 77(m,2H),7.68–7.66(m,2H),7.66–7.63(m,2H),7.61(d,J=7.8Hz,1H),7.38(d,J= 8.2Hz,1H),7.35(s,1H),7.22(d,J=8.1Hz,2H),7.09(ddd,J=8.2,6.9,1.2Hz,1H), 7.03–7.00(m,1H),5.41(s,2H),3.55(td,J=7.6,5.7Hz,2H),2.96(t,J=7.5Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ165.66,164.41,142.02,136.51,134.23,131.91,131.75(2C),129.76(2C),128.37,127.57(2 C),127.36(2C),127.12,125.27,121.80,119.21,119.16,112.47,110.52,49.05,40.64,25.42.ESI-HRMS:calcd.forC 25 H 22 BrN3NaO3[M+Na]+ ,514.0742;found514.0740.

[0072] Example 14: N-Hydroxy-4-((3-(2-benzamidoethyl)-5-methoxy-1H-indol-1-yl)methyl)-benzamide (A14)

[0073] The preparation method of the compound of Example 14 is the same as that of Example 1, except that 5-methoxytryptamine is used to replace tryptamine in step 1, and acetyl chloride is used to replace benzoyl chloride in step 4 to obtain 0.3203 g of white solid powder with a yield of 24.58%. 1 H NMR (600MHz, DMSO-d6) δ11.14(s,1H),9.00(s,1H),8.59(t,J=5.7Hz,1H),7.85–7.82( m,2H),7.66–7.62(m,2H),7.53–7.50(m,1H),7.45(dd,J=8.3,6.9Hz,2H),7.31(s,1H), 7.26(d,J=8.9Hz,1H),7.19(d,J=8.1Hz,2H),7.09(d,J=2.4Hz,1H),6.72(dd,J=8.8,2. 4Hz,1H),5.36(s,2H),3.72(s,3H),3.55(td,J=7.5,5.7Hz,2H),2.94(t,J=7.4Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ166.60,164.45,153.80,142.17,135.16,132.25,131.75,131.51,128.79,128.70(2C),127.70,1 27.61(2C),127.56(2C),127.29(2C),112.21,111.79,111.26,101.18,55.78,49.19,40.52,25.50.ESI-HRMS:calcd.for C 26 H 25 N3NaO4[M+Na] + ,466.1743;found466.1749.

[0074] Example 15: N-Hydroxy-4-((3-(2-(2-phenylacetamido)ethyl)-5-methoxy-1H-indol-1-yl)methyl)-benzamide (A15)

[0075] The preparation method of the compound of Example 15 is the same as that of Example 1, except that 5-methoxytryptamine is used to replace tryptamine in step 1, and acetyl chloride is used to replace phenylacetyl chloride in step 4 to obtain 0.0875 g of a white solid powder with a yield of 5.86%. 1 H NMR (600MHz, DMSO-d6) δ11.14(s,1H),8.99(s,1H),8.13(t,J=5.7Hz,1H),7.67–7.64(m,2H),7.28–7.18(m,9H),7.05(d,J =2.4Hz,1H),6.72(dd,J=8.8,2.4Hz,1H),5.34(s,2H),3.75(s,3H),3.40(s,2H),3.36–3.32(m,2H),2.80(t,J=7.3Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ170.46,164.44,153.81,142.16,136.98,132.26,131.73,129.47(2C),128.69,128.65(2C),127.68, 127.57(2C),127.27(2C),126.76,112.01,111.76,111.25,101.17,55.87,49.22,42.97,40.52,25.50.ESI-HRMS:calcd.for C 27 H 28 N3O4[M+H] + ,458.2080; found 458.2084.

[0076] Example 16: N-Hydroxy-4-((3-(2-(4-methylbenzamido)ethyl)-5-methoxy-1H-indol-1-yl)methyl)-benzamide (A16)

[0077] The preparation method of the compound of Example 16 is the same as that of Example 1, except that 5-methoxytryptamine is used to replace tryptamine in step 1, and 4-methylbenzoyl chloride is used to replace acetyl chloride in step 4 to obtain 0.3901 g of a white solid powder with a yield of 13.15%. 1H NMR (600MHz, DMSO-d6) δ11.13(s,1H),9.00(s,1H),8.50(t,J=5.8Hz,1H),7.74( d,J=7.9Hz,2H),7.63(d,J=8.2Hz,2H),7.30(s,1H),7.25(t,J=8.2Hz,3H),7.19 (d,J=8.1Hz,2H),7.09(d,J=2.4Hz,1H),6.72(dd,J=8.8,2.4Hz,1H),5.35(s,2H ),3.72(s,3H),3.53(td,J=7.5,5.7Hz,2H),2.93(t,J=7.4Hz,2H),2.35(s,3H). 13 C NMR(151MHz,DMSO-d6)δ166.47,164.45,153.79,142.16,141.33,132.36,132.25,131.75,129.23(2C),128.80,127.67,127. 63(2C),127.56(2C),127.28(2C),112.23,111.79,111.25,101.18,55.78,49.19,40.45,25.54,21.40.ESI-HRMS:calcd.for C 27 H 28 N3O4[M+H] + ,458.2080; found 458.2082.

[0078] Example 17: N-Hydroxy-4-((3-(2-(4-methoxybenzamido)ethyl)-5-methoxy-1H-indol-1-yl)methyl)-benzamide (A17)

[0079] The preparation method of the compound of Example 17 is the same as that of Example 1, except that 5-methoxytryptamine is used to replace tryptamine in step 1, and 4-methoxybenzoyl chloride is used to replace acetyl chloride in step 4 to obtain 0.6986 g of white solid powder with a yield of 44.69%. 1H NMR (600MHz, DMSO-d6) δ11.14(s,1H),9.00(s,1H),8.44(t,J=5.7Hz,1H),7.82(d,J =8.8Hz,2H),7.66–7.62(m,2H),7.30(s,1H),7.26(d,J=8.9Hz,1H),7.19(d,J=8.1H z,2H),7.09(d,J=2.5Hz,1H),6.99–6.96(m,2H),6.72(dd,J=8.9,2.4Hz,1H),5.36( s,2H),3.81(s,3H),3.72(s,3H),3.53(td,J=7.5,5.6Hz,2H),2.92(t,J=7.4Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ166.10,164.47,161.91,153.81,142.20,132.25,131.77,129.42(2C),128.80,127.68,127.57(2C), 127.38,127.29(2C),113.89(2C),112.28,111.79,111.25,101.19,55.79,49.18,49.07,40.44,25.59.ESI-HRMS:calcd.for C 27 H 28 N3O5[M+H] + ,474.2029; found 474.2035.

[0080] Example 18: N-Hydroxy-4-((3-(2-(4-trifluoromethylbenzamido)ethyl)-5-methoxy-1H-indol-1-yl)methyl)-benzamide (A18)

[0081] The preparation method of the compound of Example 18 is the same as that of Example 1, except that 5-methoxytryptamine is used to replace tryptamine in step 1, and 4-trifluoromethylbenzoyl chloride is used to replace acetyl chloride in step 4 to obtain 0.2483 g of white solid powder with a yield of 7.69%. 1H NMR (600MHz, DMSO-d6) δ11.14(d,J=2.0Hz,1H),9.00(d,J=2.0Hz,1H),8.86(t,J=5.7 Hz,1H),8.03(d,J=8.1Hz,2H),7.84(d,J=8.2Hz,2H),7.66–7.63(m,2H),7.32(s,1H), 7.26(d,J=8.8Hz,1H),7.21–7.18(m,2H),7.09(d,J=2.4Hz,1H),6.73(dd,J=8.8,2.4 Hz,1H),5.36(s,2H),3.71(s,3H),3.57(td,J=7.6,5.7Hz,2H),2.95(t,J=7.4Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ165.43,164.44,153.82,142.17,138.89,132.27 ,131.76,131.49(d,J=32.4Hz),128.77,128.54(2C),127.75,127.56(2C) ,127.29(2C),125.78(t,J=4.0Hz,2C),124.60(d,J=317.4Hz),112.06,111.80,111.29,101.16,55.77,49.20,40.70,25.37.ESI-HRMS:calcd.for C 27 H 25 F3N3O4[M+H] + ,512.1797; found 512.1796.

[0082] Example 19: N-Hydroxy-4-((3-(2-pivalamidoethyl)-5-methoxy-1H-indol-1-yl)methyl)-benzamide (A19)

[0083] The preparation method of the compound of Example 19 is the same as that of Example 1, except that 5-methoxytryptamine is used to replace tryptamine in step 1, and acetyl chloride is used to replace pivaloyl chloride in step 4 to obtain 0.0638 g of yellow solid powder with a yield of 4.93%. 1H NMR (600MHz, DMSO-d6) δ11.13(s,1H),8.99(s,1H),7.67–7.63(m,2H),7.51(t,J=5.7Hz,1H),7.26–7.23(m,2H),7.19(d,J=8.1Hz, 2H),7.07(d,J=2.5Hz,1H),6.72(dd,J=8.8,2.4Hz,1H),5.35(s,2H),3.76(s,3H),3.33(s,2H),2.79(t,J=7.4Hz,2H),1.06(s,9H). 13 C NMR(151MHz,DMSO-d6)δ177.70,164.42,153.77,142.19,132.24,131.73,128.78,127.61,127.54(2C),127.2 8(2C),112.18,111.69,111.21,101.26,55.89,49.18,40.52,38.42,27.94(3C),25.45.ESI-HRMS:calcd.for C 24 H 29 N3NaO4[M+Na] + ,446.2056; found 446.2062.

[0084] Example 20: N-Hydroxy-4-((3-(2-(3,3-dimethylbutyramido)ethyl)-5-methoxy-1H-indol-1-yl)methyl)-benzamide (A20)

[0085] The preparation method of the compound of Example 20 is the same as that of Example 1, except that 5-methoxytryptamine is used to replace tryptamine in step 1, and 3,3-dimethylbutyryl chloride is used to replace acetyl chloride in step 4 to obtain 0.2249 g of yellow solid powder with a yield of 15.80%. 1 H NMR (600MHz, DMSO-d6) δ11.14(s,1H),8.99(s,1H),7.82(t,J=5.7Hz,1H),7.67–7.64(m,2H),7.25(d,J=8.9Hz,2H),7.19(d,J=8.1Hz,2H),7.0 5(d,J=2.4Hz,1H),6.72(dd,J=8.8,2.4Hz,1H),5.35(s,2H),3.76(s,3H),3.35–3.31(m,2H),2.78(t,J=7.4Hz,2H),1.94(s,2H),0.94(s,9H). 13CNMR(151MHz,DMSO-d6)δ171.16,164.43,153.77,142.16,132.26,131.75,128.70,127.62,127.56(2C),127.29( 2C),112.16,111.71,111.23,101.20,55.87,49.41,49.21,39.53,30.82,30.19(3C),25.69.ESI-HRMS:calcd.for C 25 H 32 N3O4[M+H] + ,438.2393; found 438.2401.

[0086] Example 21: N-Hydroxy-4-((3-(2-(4-bromobenzamido)ethyl)-5-methoxy-1H-indol-1-yl)methyl)-benzamide (A21)

[0087] The preparation method of the compound of Example 21 is the same as that of Example 1, except that 5-methoxytryptamine is used to replace tryptamine in step 1, and 4-bromobenzoyl chloride is used to replace acetyl chloride in step 4 to obtain 0.2483 g of brown solid powder with a yield of 7.69%. 1 H NMR (600MHz, DMSO-d6) δ11.13(s,1H),9.00(s,1H),8.70(t,J=5.7Hz,1H),7.78( d,J=8.1Hz,2H),7.67(d,J=8.2Hz,2H),7.64(d,J=8.1Hz,2H),7.30(s,1H),7.26( d,J=8.9Hz,1H),7.19(d,J=7.9Hz,2H),7.08(d,J=2.4Hz,1H),6.72(dd,J=8.8,2. 4Hz, 1H), 5.35 (s, 2H), 3.71 (s, 3H), 3.53 (q, J = 6.9Hz, 2H), 2.93 (t, J = 7.4Hz, 2H). 13 C NMR(151MHz,DMSO-d6)δ165.64,164.39,153.80,142.14,134.22,132.28,131.74(3C),129.76(2C),128.77,127.70, 127.53(2C),127.28(2C),125.26,112.11,111.79,111.28,101.14,55.78,49.20,40.53,25.42.ESI-HRMS:calcd.for C 26 H 24 BrN3NaO4[M+Na]+ ,544.0848; found 544.0854.

[0088] Example 22: N-Hydroxy-4-((3-(2-(2-(4-fluorophenyl)acetamido)ethyl)-5-methoxy-1H-indol-1-yl)methyl)-benzamide (A22)

[0089] The preparation method of the compound of Example 22 is the same as that of Example 1, except that 5-methoxytryptamine is used to replace tryptamine in step 1, and 4-fluorophenylacetyl chloride is used to replace acetyl chloride in step 4 to obtain 0.3560 g of brown solid powder with a yield of 35.17%. 1 H NMR (600MHz, DMSO-d6) δ11.14(s,1H),9.03(s,1H),8.15(t,J=5.7Hz,1H),7.65 (d,J=8.2Hz,2H),7.25(dt,J=8.5,2.7Hz,3H),7.21(s,1H),7.18(d,J=8.1Hz,2 H),7.10–7.06(m,2H),7.04(d,J=2.4Hz,1H),6.72(dd,J=8.8,2.4Hz,1H),5.34 (s,2H),3.75(s,3H),3.39(s,2H),3.33(d,J=6.2Hz,2H),2.79(t,J=7.3Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ170.36,164.34,161.45(d,J=242.6Hz),153.79,133 .12,133.10,131.74,131.26(d,J=7.8Hz,2C),130.06(d,J=23.4Hz),128.69 ,127.69,127.52(2C),127.25(2C),115.32(d,J=21.8Hz,2C),111.99,111.7 4,111.25,101.18,55.86,49.21,41.94,40.52,25.49.ESI-HRMS:calcd.forC 27 H 26 FN3NaO4[M+Na] + ,498.1805; found 498.1803.

[0090] Example 23: N-Hydroxy-4-((3-(2-(2-(3,4-difluorophenyl)acetamido)ethyl)-5-methoxy-1H-indol-1-yl)methyl)-benzamide (A23)

[0091] The preparation method of the compound of Example 23 is the same as that of Example 1, except that 5-methoxytryptamine is used to replace tryptamine in step 1, and 3,4-difluorophenylacetyl chloride is used to replace acetyl chloride in step 4 to obtain 0.7369 g of white solid powder with a yield of 50.72%. 1 H NMR (600MHz, DMSO-d6) δ11.14(s,1H),9.01(s,1H),8.18(t,J=5.7Hz,1H),7.67–7. 63(m,2H),7.34–7.29(m,1H),7.29–7.26(m,1H),7.26(d,J=8.8Hz,1H),7.23(s,1H) ,7.19(d,J=8.1Hz,2H),7.05(dd,J=6.9,3.3Hz,2H),6.73(dd,J=8.8,2.4Hz,1H),5 .35(s,2H),3.75(s,3H),3.41(s,2H),3.34(q,J=7.3Hz,2H),2.80(t,J=7.3Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ169.84,164.43,153.81,149.49(dd,J=244.7,12.7Hz),148.71(dd ,J=243.7,12.5Hz),142.14,135.77–133.87(m),132.27,131.75,128.70,127.70,127.56( 2C),127.25(2C),126.25(dd,J=6.4,3.4Hz),118.41(d,J=17.2Hz),117.49(d,J=16.8Hz), 111.98,111.74,111.25,101.20,55.87,49.22,41.71,39.95,25.46.ESI-HRMS:calcd.forC 27 H 25 F2N3NaO4[M+Na] + ,516.1711; found 516.1716.

[0092] Example 24: N-Hydroxy-4-((3-(2-(2-(2,4-difluorophenyl)acetamido)ethyl)-5-methoxy-1H-indol-1-yl)methyl)-benzamide (A24)

[0093] The preparation method of the compound of Example 24 is the same as that of Example 1, except that 5-methoxytryptamine is used to replace tryptamine in step 1, and 2,4-difluorophenylacetyl chloride is used to replace acetyl chloride in step 4 to obtain 0.1654 g of brown solid powder with a yield of 11.38%. 1H NMR(600MHz,DMSO-d6)δ11.05(s,1H),9.03(s,1H),8.17(s,1H),7.65(d,J=7 .9Hz,2H),7.32(q,J=8.1Hz,1H),7.26(d,J=8.3Hz,2H),7.18(t,J=8.1Hz,3H ),7.04(s,1H),6.99(d,J=8.7Hz,1H),6.72(d,J=8.8Hz,1H),5.35(s,2H),3. 75(s,3H),3.45(s,2H),3.35(dd,J=14.0,7.6Hz,2H),2.81(t,J=7.5Hz,2H). 13 C NMR (151MHz, DMSO-d6) δ169.22, 164.31, 161.61 (dd, J=244.6, 12.1Hz), 160.95 (dd, J=246.7 ,11.8Hz),153.81,133.16(dd,J=9.7,5.9Hz),132.33,131.74,129.38,128.69,127.69,127 .51(2C),127.26(2C),121.20–119.19(m),111.97,111.74,111.54(dd,J=20.8,3.8Hz),111 .26,103.94(t,J=26.0Hz),101.16,55.86,49.23,40.52,35.18,25.49.ESI-HRMS:calcd.for C 27 H 25 F2N3NaO4[M+Na] + ,516.1711; found 516.1714.

[0094] Example 25: N-Hydroxy-4-((3-(2-(2-(3,5-difluorophenyl)acetamido)ethyl)-5-methoxy-1H-indol-1-yl)methyl)-benzamide (A25)

[0095] The preparation method of the compound of Example 25 is the same as that of Example 1, except that 5-methoxytryptamine is used to replace tryptamine in step 1, and 3,5-difluorophenylacetyl chloride is used to replace acetyl chloride in step 4 to obtain 0.4270 g of a white solid powder with a yield of 29.39%. 1H NMR(600MHz,DMSO-d6)δ11.13(s,1H),8.99(s,1H),8.20(t,J=5.7Hz,1H),7.67–7.63 (m,2H),7.25(d,J=8.9Hz,1H),7.23(s,1H),7.19(d,J=8.1Hz,2H),7.08(tt,J=9.5,2. 4Hz, 1H), 7.05 (d, J=2.4Hz, 1H), 6.96 (qd, J=6.3, 3.1Hz, 2H), 6.72 (dd, J=8.9, 2.4Hz, 1H), 5.34 (s, 2H), 3.75 (s, 3H), 3.46 (s, 2H), 3.37–3.33 (m, 2H), 2.81 (t, J = 7.3Hz, 2H). 13 C NMR(151MHz,DMSO-d6)δ169.38,164.43,162.59(dd,J=245.8,13.1Hz,2C),1 53.81,142.13,141.55–141.06(m),132.27,131.74,128.69,127.70,127.57( 2C),127.26(2C),113.06–112.45(m,2C),111.96,111.75,111.27,102.34(t, J=25.9Hz),101.17,55.86,49.22,42.24,40.52,25.46.ESI-HRMS:calcd.for C 27 H 25 F2N3NaO4[M+Na] + ,516.1711; found 516.1719.

[0096] Example 26: N-Hydroxy-4-((3-(2-(2-(3,4,5-trifluorophenyl)acetamido)ethyl)-5-methoxy-1H-indol-1-yl)methyl)-benzamide (A26)

[0097] The preparation method of the compound of Example 26 is the same as that of Example 1, except that 5-methoxytryptamine is used to replace tryptamine in step 1, and 3,4,5-trifluorophenylacetyl chloride is used to replace acetyl chloride in step 4 to obtain 0.3550 g of light brown solid powder with a yield of 23.65%. 1H NMR(600MHz,DMSO-d6)δ11.10(s,1H),9.03(s,1H),8.22–8.11(m,1H),7.65(d ,J=8.1Hz,2H),7.25(d,J=8.6Hz,2H),7.18(d,J=8.1Hz,2H),7.15(dd,J=9.0, 6.7Hz,2H),7.03(d,J=2.5Hz,1H),6.72(dd,J=8.9,2.4Hz,1H),5.35(s,2H),3 .74(s,3H),3.43(s,2H),3.34(dd,J=13.3,6.6Hz,2H),2.80(t,J=7.3Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ169.29,164.33,153.79,150.30(ddd,J=246.9,9.6,4.3Hz ,2C),142.08,137.98(dt,J=246.8,15.5Hz),134.27–134.05(m),132.30,131.73, 128.71,127.73,127.52(2C),127.23(2C),114.12(dd,J=16.9,3.9Hz,2C),111.94 ,111.71,111.25,101.17,55.85,49.21,41.62,40.52,25.43.ESI-HRMS:calcd.for C 27 H 24 F3N3NaO4[M+Na] + ,534.1617; found 534.1622.

[0098] Example 27: Pharmacological studies of the product of the present invention

[0099] Pharmacological tests have demonstrated that the compounds of the present invention have therapeutic activity for idiopathic pulmonary fibrosis.

[0100] (1) Inhibitory activity and selectivity test of compounds A1-A26 against HDAC6

[0101] The inhibitory activity of compounds A1-A26 against HDAC6 and their selectivity against other HDACs subtypes were tested using substrate-enzyme binding assay.

[0102] Recombinant human HDAC1, 2, 3, 4, 6, 7 and 11 proteins were purchased from BPS Bioscience, USA. All reactions were performed in the black half of a 96-well microplate. Recombinant HDAC proteins were diluted to 1 μg / mL with assay buffer, and Boc-Lys(Ac)-AMC (for HDAC1, 2, 3 and 6) and Boc-Lys(Tfa)-AMC (for HDAC4, 7 and 11) were used as substrates. HDACs were mixed with 20 μL of samples of different concentrations on a 96-well plate, and then 20 μL of fluorescent substrate was added. After incubation at 37°C for 120 min, 50 μL of developer was added to each well and incubated at 37°C for 30 min to develop the fluorescent signal. The fluorescence intensity of each well was measured under the conditions of an excitation wavelength of 355 nm and an emission wavelength of 460 nm. Enzyme binding inhibition can be observed by comparing the fluorescence intensity. The test results are shown in Tables 1 and 2.

[0103] Table 1 Inhibitory activity test of compounds A1-A26 on HDAC6

[0104]

[0105]

[0106] *Positive control drug.

[0107] Table 2 Selectivity test of preferred active compounds A15 and A21 for HDACs subtypes

[0108]

[0109] *Values ​​are expressed as the means±SD of three independent experiments.

[0110] (2) Toxicity test of compound A21 on HELF cells

[0111] HELF cells were cultured in MEM medium containing 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin, with a pH value of 7.23. The cells used in the above experiments were cultured in a 37°C constant temperature incubator (conditions: 5% CO2, saturated humidity).

[0112] MTT assay to detect the inhibitory effect of drugs on cell proliferation

[0113] The cells were subcultured to the logarithmic growth phase, and after normal digestion with 0.25% trypsin, they were prepared into a cell suspension using a medium without FBS or containing 10% FBS. 4The cells were inoculated in a 96-well plate at a cell density of 100 μL / mL. After culturing the cells for 12 hours, the original liquid in the wells was aspirated, and the culture medium containing gradient dilution drugs was immediately added. The control group was replaced with a culture medium containing an equal amount of DMSO, and then the cells were placed in an incubator for continued culture. After 48 hours, the culture medium in the 96-well plate was aspirated, and 100 μL of culture medium containing 10% MTT (5 mg / mL) was immediately added, and the cells were placed in the incubator again for 4 hours. After the incubation, the original liquid in the well was discarded and 150 μL DMSO was immediately added. The cell culture plate was placed on a shaker for 10 minutes, and the absorbance (OD) value was detected by setting the wavelength to 492 nm with the aid of an enzyme marker.

[0114] Finally, the cell proliferation inhibition rate was calculated (the formula is as follows).

[0115]

[0116] The test results are as follows Figure 1 As shown, compound A21 and Tubastatin A are toxic to HELF cells at higher concentrations and in a dose-dependent manner. The maximum concentration at which both have little effect on HELF cell survival (80% survival rate) is 10 μM, so the maximum concentration of the subsequent in vitro pharmacology study of A21 in the present invention is 10 μM.

[0117] (3) Effect of compound A21 on TGF-β1-induced HELF cell proliferation

[0118] The method is the same as (2).

[0119] The test results are as follows Figure 2 As shown, Tubastatin A was used as a positive control drug, and the MTT method was used to study the proliferation toxicity of compound A21 on human embryonic lung fibroblasts (HELF). Through the analysis of the experimental results, compound A21 can significantly inhibit the proliferation of HELF cell lines induced by TGF-β1 in a dose-dependent manner, reaching a level similar to that of the positive control drug Tubastatin A at a dose of 10μM.

[0120] (4) Inhibition test of compound A21 on HDAC6 activity in HELF cells during TGF-β1-induced pulmonary fibrosis in vitro

[0121] After the cells in the culture flask are digested normally, they are inoculated into a 6-well plate and placed in an incubator for 12 hours until they adhere to the wall. The original liquid in the well is removed with a pipette and the culture medium containing gradient dilution drugs is added. After 48 hours of cell culture, the cells are taken out and placed on an ice box and the surface is washed with PBS buffer. 50 μL of the pre-prepared cell lysis solution is evenly added to each well. After incubation on the ice box for 10 minutes, the fully lysed cells are collected with a scraper and centrifuged in a 4°C high-speed centrifuge for 15 minutes (14000rpm). After centrifugation, the supernatant is quickly transferred to a 0.5mL EP tube using a pipette. Remember not to touch the precipitate. The sample concentration is quantified using a BCA kit. The sample protein concentration is uniformly diluted with cell lysis buffer, and then 30 μL of protein sample and 20 μL of Ac-Lys(Ac)-AMC fluorescent substrate (final concentration of 400 μM per well) are added to each well of the black 96-well plate, and incubated at a constant temperature of 37°C for 120 minutes. Then, use a pipette to draw 50 μL of stop solution and add it to each well. Continue incubating at 37°C for 15 min to stop the reaction. Use an enzyme-labeled instrument to detect the intensity of the fluorescence generated during the reaction (Ex: 380 nm, Em: 460 nm).

[0122] The experimental results are as follows Figure 3 As shown, with the increase of A21 concentration (2.5μM, 5μM, 10μM), HDAC6 activity gradually decreased, indicating that A21 has a dose-dependent inhibitory effect on the increased activity of HDAC6 in the process of TGF-β1-induced fibrosis.

[0123] (5) Effect of compound A21 on the expression of HDAC6-related substrate proteins in HELF cells during TGF-β1-induced pulmonary fibrosis in vitro

[0124] 1) Total protein extraction and protein concentration quantification

[0125] Add 1% PMSF to the RIPA high-efficiency lysis buffer in advance and prepare it before use. Place the cells on an ice box and wash them with PBS. After removing the liquid in the wells with a pipette, evenly add 100 μL of the pre-prepared lysis buffer and incubate on the ice box for 10 minutes. Use a scraper to scrape the cells in the culture dish and collect them into a pre-cooled EP (1.5mL) tube, then perform high-speed centrifugation at 4°C for 15 minutes (12000rpm). After centrifugation, transfer the supernatant into a pre-cooled 0.5mL EP tube and quantify the supernatant using a BCA kit.

[0126] 2) Sample preparation

[0127] Add protein loading buffer (5×) to the extracted protein supernatant in proportion, boil it in a water bath for 10 minutes, and store it at -20°C after it cools to room temperature. Avoid repeated freezing and thawing of the sample.

[0128] 3) SDS-PAGE gel electrophoresis

[0129] a. Gel preparation: according to the preparation method of different concentrations of gel, add the corresponding solvents in order in a clean centrifuge tube, mix well and immediately add to the installed gel plate, then evenly add isopropyl alcohol to flatten the gel surface. After the gel solidifies, absorb the isopropyl alcohol. Use the same method to prepare the upper layer of gel, and after adding the glass gel plate, quickly insert the comb into the middle of the glass plate and make sure no bubbles are generated. After the upper layer of gel solidifies, transfer the gel plate to the electrophoresis instrument, pour in 1× electrophoresis solution, and evenly pull out the comb vertically and make sure that the gel does not deform.

[0130] b. Electrophoresis: According to the quantified protein concentration, the total amount of protein sample loaded in each well is the same (10-30μg), so as to calculate the actual amount of each sample. After the protein sample is loaded, the voltage of the electrophoresis instrument is set to 60V and run for 40 minutes, then adjusted to 90V and continued to run for about 60 minutes, until bromophenol blue is observed at the bottom of the glass gel plate and the electrophoresis is terminated.

[0131] c. Transfer: Use wet transfer method, place the gel and PVDF membrane between the filter papers on both sides, wrap a layer of sponge on the outside of the filter paper, and remove the bubbles between the layers. Put the fixed electrotransfer clip into the electrotransfer instrument, put a small ice bag in the gap, slowly pour in 1× electrotransfer solution, then set the voltage of the electrotransfer instrument to 100V, and perform electrotransfer at a low temperature of 4℃. The transfer time can be flexibly adjusted according to the different molecular weights of the target protein.

[0132] d. Antibody incubation: After transfer, place the PVDF membrane in the pre-prepared blocking solution and block on a shaker for 1 hour. Wash with 1×TBST and incubate with the diluted primary antibody at 4°C overnight. Wash the membrane the next day and add the diluted secondary antibody and continue incubating on a shaker at room temperature for 1 hour.

[0133] e. Development: Using a gel imaging system, evenly add ECL luminescent liquid onto the PVDF membrane and perform relevant development operations on the protein bands.

[0134] The test results are as follows Figure 4 As shown in the results, A21 could effectively inhibit the expression of AC-α-tubulin and AC-histone H3 in HELF cells induced by TGF-β1, which means that the potential anti-fibrotic effect of A21 is achieved by inhibiting HDAC6 and affecting its related signaling pathways.

[0135] (6) Effect of compound A21 on the expression of fibrosis-related substrates in HELF cells during TGF-β1-induced pulmonary fibrosis in vitro

[0136] The test method is the same as (5).

[0137] The test results are as follows Figure 5 As shown, with the increase of A21 concentration (2.5μM, 5μM, 10μM), the expression levels of COL1A1 and α-SMA gradually decreased, indicating that A21 has a dose-dependent inhibitory effect on the expression of pulmonary fibrosis-related proteins.

[0138] (7) Effect of compound A21 on the expression of p-Smad2 and p-Smad3 in HELF cells during TGF-β1-induced pulmonary fibrosis in vitro

[0139] The test method is the same as (5).

[0140] The test results are as follows Figure 6 As shown in the results, A21 significantly inhibited the phosphorylation of Smad2, Smad3 and TGF-βR1 induced by TGF-β1, indicating that A21 can exert anti-fibrotic effects by inhibiting the TGF-β-related signaling pathway downstream of HDAC6. A21 can significantly reduce the expression of p-Smad2, p-Smad3 and p-TGF-βR1 at three doses of 2.5μM, 5μM and 10μM, and showed a dose-dependent manner.

[0141] (8) Effect of compound A21 on the expression of TGF-β1-related mRNA in HELF cells during TGF-β1-induced pulmonary fibrosis in vitro

[0142] Total RNA was isolated from HELF cells using Trizol reagent (Takara, Japan). Reverse transcription-PCR was performed using an RT-PCR kit (Takara, Japan) according to the manufacturer's instructions. The specificity of the amplified products was confirmed by melting curve analysis. All reactions were performed in triplicate. As a measure of the relative expression change between parental and resistant samples, ΔΔCt values ​​were calculated and converted to approximate fold change values ​​(2 -ΔΔct ).

[0143] The test results are as follows Figure 7 As shown in the figure, when A21 was added to the TGF-β1-treated model at doses of 2.5μM, 5μM and 10μM, the expression of TGF-β1mRNA was significantly reduced. A21 can significantly reduce the expression of TGF-β1mRNA at all three doses, and the reduction effect is more significant with the increase of dose, showing dose dependence.

[0144] (9) Body weight changes of mice in each group during the in vivo pulmonary fibrosis model test

[0145] Construction of pulmonary fibrosis model:

[0146] Thirty C57BL / 6J mice were randomly divided into five groups: control group, BLM (bleomycin hydrochloride for injection) model group, pirfenidone (PFD) group, A21 low-dose group, and A21 high-dose group.

[0147] The mice were anesthetized with 1% sodium pentobarbital and hung on a mouse stand. The tongue of the mouse was gently pulled out with tweezers. When a small hole with alternating light and dark appeared in the trachea, a 22G trocar was inserted (the ups and downs of the water column indicated that the trocar had been inserted into the trachea). BLM (3.5 mg / kg) was then slowly pushed in with a syringe, and the mice were rotated left and right so that BLM was evenly distributed in the mouse lungs. The tracheal depth of the mouse was 38 mm for mice weighing 15-19 g and 39 mm for mice weighing 20-25 g.

[0148] Drug preparation: Compound A21 was first added with 1% DMSO, followed by physiological saline, and ultrasonicated for 5 minutes. Pirfenidone (PFD) was directly dissolved in physiological saline.

[0149] Dosage: The PFD group, A21 low-dose group and A21 high-dose group were given 100 mg / kg, 50 mg / kg and 100 mg / kg, respectively. The remaining groups were given solvent without A21.

[0150] Administration: Oral administration, once a day for 28 consecutive days.

[0151] The test results are as follows Figure 8 As shown in the figure, the weight loss of mice in the BLM+PFD group was smaller, and the weight was restored in the later stage of the experiment, indicating that PFD has a certain mitigating effect on BLM-induced weight loss. The weight change trend of mice in the BLM+A21 group was similar to that of the BLM+PFD group, with a smaller weight loss and weight recovery in the later stage of the experiment. In particular, the weight recovery of the 100 mg / kg dose of A21 group was more obvious, indicating that A21 has a potential inhibitory effect on BLM-induced pulmonary fibrosis.

[0152] (10) Effect of compound A21 on HDAC6 activity in lung tissue of pulmonary fibrosis model mice

[0153] Take an appropriate amount of lung tissue and put it into a 1.5mL EP tube. Add 1mL RIPA lysis buffer for every 0.1g tissue, use an ultrasonic disruptor to homogenize the tissue, and centrifuge at 12000rpm for 15min. Then take the supernatant into a 0.5mL EP tube and quantify the protein concentration to 5mg / mL. Add 30μL protein sample and 20μL Boc-Lys(Ac)-AMC fluorescent substrate to each well of the ELISA plate and incubate at 37℃ for 120min. Add 50μL stop solution to the well and continue incubation at 37℃ for 15min. Detect the fluorescence intensity generated by the ELISA instrument.

[0154] The test results are as follows Fig. 9 As shown, A21 can effectively reduce the activity of HDAC6 in the lung tissue of mice with bleomycin-induced pulmonary fibrosis model, and this effect is enhanced with the increase of A21 dose.

[0155] (11) Effect of compound A21 on the expression of TGF-β1-related mRNA in lung tissue of mice with pulmonary fibrosis model

[0156] RNA extraction: Place lung tissue in a pre-cooled mortar, pour liquid nitrogen and add 0.5 mL Trizol to the mortar for grinding. Transfer the ground powder to a 1.5 mL EP tube and add 0.5 mL Trizol. Let stand at room temperature for 5 minutes and centrifuge at 12000 rpm at 4°C for 5 minutes.

[0157] After centrifugation, take the supernatant and repeat the above step (10).

[0158] The test results are as follows Fig.10 As shown in the results, A21 can effectively reduce the expression of TGF-β1-related mRNA in the lung tissue of mice with pulmonary fibrosis induced by bleomycin, and this effect is enhanced with the increase of A21 dose. This indicates that A21 exerts its anti-fibrotic effect by inhibiting the TGF-β1 signaling pathway in vivo.

[0159] (12) Effect of compound A21 on pathological changes in lung tissue of mice with pulmonary fibrosis model

[0160] Preparation of paraffin sections and dewaxing and rehydration:

[0161] 1) Dehydration: Place the lung tissue obtained by perfusion into a tissue embedding box and place it in running water overnight. Dehydrate the tissue by soaking it in 70%, 80% and 90% ethanol solutions for 1 hour each, and 95% and 100% ethanol solutions for 2 hours and 1 hour respectively.

[0162] 2) Transparency: Place in xylene I and II for 15 min each;

[0163] 3) Wax dipping: Place the dehydrated and transparent lung tissue into melted paraffin and dip it into wax;

[0164] 4) Embedding: embedding using a biological tissue embedding machine;

[0165] 5) Cut the embedded tissue into 5 μm thick paraffin sections using a paraffin slicer and store them in a dry place;

[0166] Dewaxing and rehydration: soak in xylene I and xylene II for 15 min each → soak in anhydrous ethanol I and anhydrous ethanol II for 5 min each → soak in 95% ethanol, 85% ethanol, and 75% ethanol for 5 min → rinse in running water for 2 min.

[0167] H&E staining:

[0168] 1) Bake lung slices in an oven at 60°C for 1 h;

[0169] 2) Dewaxing and rehydrating according to the above method;

[0170] 3) Hematoxylin staining: Place lung sections in hematoxylin for 30 seconds and then rinse with running water for 2 minutes;

[0171] 4) Hydrochloric acid alcohol differentiation: Place the slices in hematoxylin differentiation solution for 2-3 times and then rinse with running water for 2 minutes;

[0172] 5) Blueing: Place the lung slices in the blueing solution and extract twice, then rinse with running water for 2 minutes;

[0173] 6) Eosin staining: Place the lung sections in eosin staining solution for 40 seconds and then rinse with running water for 2 minutes;

[0174] 7) Soak the washed lung slices in anhydrous ethanol II and anhydrous ethanol I for 2 minutes each, then soak them in xylene II and xylene I for 10 minutes each;

[0175] 8) After sealing with neutral gum, observe and photograph using an upright microscope.

[0176] The test results are as follows Fig.11 As shown, A21 at two doses (50 mg / kg and 100 mg / kg) can significantly improve the pathological changes in lung tissue of mice with bleomycin-induced pulmonary fibrosis. A high dose of A21 (100 mg / kg) showed a more obvious protective effect, indicating that it has a potential therapeutic effect in the treatment of pulmonary fibrosis.

[0177] (13) Effects of Compound A21 on the changes of relevant biomarkers in lung tissue of mice with pulmonary fibrosis model

[0178] 1) Dewaxing and rehydrating according to the method of (12);

[0179] 2) Antigen repair: Prepare a sodium citrate buffer solution with pH=6 using citric acid and sodium citrate in a beaker. Place the lung slices in the beaker and heat them in a microwave oven for 20 min (high heat for 7 min, low heat for 13 min). After cooling at room temperature, wash them three times with PBS solution, each time for 5 min.

[0180] 3) Block endogenous catalase: draw a circle around the lung slice with a histochemical pen, add H2O2 in the kit to the lung slice, place it in a wet box and incubate it at room temperature for 10-15 minutes, and wash it with PBS three times, 5 minutes each time;

[0181] 4) Blocking: Prepare a blocking solution containing 0.3% Triton X-100, add the blocking solution dropwise, and place the slices in a humidified box and incubate in a 37°C oven for 1 hour;

[0182] 5) Primary antibody incubation: shake off the blocking solution, add the prepared primary antibody to the lung sections and incubate in a 4°C refrigerator overnight;

[0183] 6) Secondary antibody incubation: The next day, remove the wet box, rewarm and wash with PBST solution for 5×5 min. Take the HRP-labeled IgG secondary antibody in the immunohistochemistry kit, add it to the lung section, place it in a wet box and incubate it in a 37°C oven for 1 hour, and wash it with PBST solution 5 times, 5 min each time;

[0184] 7) DAB reaction: Prepare fresh DAB solution and add it dropwise to the lung slices for reaction. After the color changes to slightly yellow, rinse in running water for 5 minutes.

[0185] 8) Hematoxylin counterstaining: Place lung sections in hematoxylin for 30 seconds and then rinse with running water for 5 minutes;

[0186] 9) Hydrochloric acid alcohol differentiation: Place the lung slices in hematoxylin differentiation solution for extraction and then rinse with running water for 5 minutes;

[0187] 10) Sealing: Place lung tissue sections in 75%, 85%, and 95% alcohol for 5 minutes each, then place them in xylene I and II for 15 minutes each, and add a small amount of neutral gum to seal the sections;

[0188] 11) Photography: Use an upright microscope to observe and photograph.

[0189] The test results are as follows Fig.12 As shown in the results, A21 can significantly reduce the deposition of collagen fibers at both doses and inhibit the expression of TGF-β signaling pathway-related substrates in the process of pulmonary fibrosis in mice in a dose-dependent manner. The high dose of A21 (100 mg / kg) has a more significant effect, indicating that it has a potential therapeutic effect on pulmonary fibrosis.

[0190] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention belong to the scope of the present invention and its equivalent technologies, the present invention is also intended to include these changes and variations.

Claims

1. An indole compound represented by general formula I or a pharmaceutically acceptable salt thereof or an optically active form thereof, characterized in that: Where: R 1 , R 2 , R 3 , R 4 , R 5 Each or both of them is any one of the following groups: (1) H atom, (2) hydroxyl group, (3) halogen atom, (4) cyano group, (5) trihalomethyl group, (6) C 1-6 Alkyl, C 3-8 Cycloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkanesulfonylamino, C 1-18 Alkoxycarbonyl, C 1-18 Alkoxysulfonyl, C 1-6 Alkoxy, C 2-6 Alkenyloxy, C 2-6 Alkynyloxy, C 1-6 Alkylthio, C 2-6 Alkenylthio, C 2-6 Alkynylthio, C 1-6 Alkylsulfonyl, C 2-6 Alkenylsulfonyl, C 2-6 Alkynylsulfonyl, C 1-6 Alkylsulfinyl, C 2-6 Alkenylsulfinyl, C 2-6 Alkynylsulfinyl, C 3-8 Cycloalkenyl, the above groups are arbitrarily substituted by one or more substituents selected from H atoms, halogen atoms, hydroxyl groups, cyano groups, nitro groups and amino groups, (7) substituted carbonyl groups, which are arbitrarily selected from H atoms, hydroxyl groups, C 1-6 Alkyl, amino, C 1-6 Alkylamino, C 1-6 Alkoxy, C 1-6 Alkylthio and C 3-8 The cycloalkyl group is substituted by one or more substituents, (8) by various substituted amino groups, which are arbitrarily selected from H atoms, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkylcarbonyl, C 2-6 Alkenylcarbonyl and C 2-6 substituted by one or more substituents in the alkynylcarbonyl group, (9) substituted by various substituted phenyl groups, substituted by various substituted benzyl groups, substituted by various substituted benzyloxy groups, substituted by various substituted benzoyl groups, substituted by various substituted pyridine rings, substituted by various substituted pyrazole rings, substituted by various substituted pyrrole rings, substituted by various substituted pyrimidine rings, substituted by various substituted quinoline rings, substituted by various substituted isoquinoline rings, substituted by various substituted imidazole rings, substituted by various substituted morpholine rings, substituted by various substituted piperazine rings, substituted by various substituted pyridazine rings ring, a pyrazine ring substituted in various ways, a piperidine ring substituted in various ways, a thiophene ring substituted in various ways, a thiazole ring substituted in various ways, an isothiazole ring substituted in various ways, a benzothiazole ring substituted in various ways, a pyran ring substituted in various ways, an indole ring substituted in various ways, an oxazole ring substituted in various ways, an isoxazole ring substituted in various ways, a triazole ring substituted in various ways, a benzotriazole ring substituted in various ways, a furan ring substituted in various ways, wherein each of the above groups is arbitrarily selected from H atoms, halogen atoms, C 1-6 Alkyl, C 1-6 Alkylamino, C 3-7 Cycloalkyl, C 1-6 Alkoxy, C 1-6 Alkanesulfonylamino, benzyloxycarbonyl, C 1-18 Alkoxycarbonyl, C 1-18 is substituted by one or more substituents selected from the group consisting of alkoxysulfonyl, trihalomethyl, hydroxyl, cyano and amino, (10) the adjacent R 1 With R 2 are connected to form an alkylenedioxy group; halogen includes fluorine, chlorine and bromine; X is any one of the following groups: (1) methylene, (2) nitrogen atom, (3) oxygen atom, (4) sulfur atom; Y is any one of the following groups: (1) methine, (2) nitrogen atom; n=1 or 2.

2. The indole compound or its pharmaceutically acceptable salt or its optically active form according to claim 1, characterized in that: R 1 , R 2 , R 3 , R 4 , R 5 Each or both of them is any one of the following groups: (1) H atom, (2) hydroxyl group, (3) halogen atom, (4) C 1-4 Alkyl, C 1-4 Alkoxy, each of the above groups is substituted by one or more substituents selected from H atoms, halogen atoms, hydroxyl groups and amino groups, (5) phenyl groups substituted by various substituents, benzyl groups substituted by various substituents, each of the above groups is substituted by one or more substituents selected from H atoms, halogen atoms, C 1-4 Alkyl, C 1-4 Alkylamino, C 1-4 Alkoxy, benzyloxycarbonyl, C 1-10 The alkylene group is substituted by one or more substituents selected from the group consisting of alkoxycarbonyl, trihalomethyl, hydroxymethyl, hydroxyl and amino. X is any one of the following groups: (1) methylene, (2) nitrogen atom, (3) oxygen atom.

3. The indole compound or its pharmaceutically acceptable salt or its optically active form according to claim 2, characterized in that: R 1 , R 2 , R 3 , R 4 , R 5 Each or both of them is any one of the following groups: (1) H atom, (2) hydroxyl group, (3) fluorine, chlorine, bromine, (4) C 1-4 Alkyl, C 1-4 Alkoxy, the above groups are arbitrarily substituted by one or more substituents selected from H atoms, halogen atoms, hydroxyl groups and amino groups, (5) unsubstituted or substituted phenyl, benzyl, the substituents are selected from H atoms, halogen atoms, C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 One or more of an alkoxycarbonyl group and a trihalomethyl group; X is any one of the following groups: (1) a methylene group, (2) a nitrogen atom, or (3) an oxygen atom.

4. The indole compound or its pharmaceutically acceptable salt or its optically active form according to claim 3, characterized in that: R 1 , R 2 , R 3 , R 4 , R 5 Each or all of them is any one of the following groups: H atom, hydroxyl group, bromine atom, chlorine atom, fluorine atom, methyl group, ethyl group, propyl group, n-butyl group, tert-butyl group, tert-butylmethyl group, methoxy group, ethoxy group, phenyl group, 2-methyl-phenyl group, 4-methylphenyl group, 4-methoxyphenyl group, 4-trifluoromethylphenyl group, 4-bromophenyl group, benzyl group, 4-fluorobenzyl group, 2,4-difluorobenzyl group, 3,4-difluorobenzyl group, 3,5-difluorobenzyl group, 3,4,5-trifluorobenzyl group; X is any one of the following groups: (1) methylene group, (2) oxygen atom.

5. The indole compound or its pharmaceutically acceptable salt or its optically active form according to claim 1, characterized in that: The indole compound is selected from:

6. The method for preparing the indole compound or its pharmaceutically acceptable salt or its optically active form according to claim 5, characterized in that: The following steps are involved: Using a tryptamine starting material, the amino group is first protected with Boc anhydride to obtain an intermediate M1. M1 is subjected to a substitution reaction with a compound s2, wherein s2 is 4-bromomethylbenzoic acid methyl ester or 6-bromomethylnicotinate, under the action of NaH to obtain an intermediate M2. M2 is deprotected from the Boc protecting group under trifluoroacetic acid conditions to obtain an intermediate M3. Subsequently, M3 is subjected to an acylation reaction with an acyl chloride raw material s3 in the presence of triethylamine to obtain an intermediate M4. M4 is subjected to an aminolysis reaction with hydroxylamine hydrochloride under KOH conditions to obtain compounds A1 to A26, wherein R 1 , R 2 , R 3 , R 4 , R 5 , n, X, and Y are corresponding groups at corresponding positions of compounds A1 to A26; 7. A pharmaceutical composition, characterized in that The invention comprises the indole compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof or an optically active form thereof, and a pharmaceutically acceptable carrier; the dosage form is selected from one or more of the following: tablets, capsules, injections, suppositories, patches, inhalable powder preparations, suspensions, emulsions and ointments.

8. A pharmaceutical preparation, characterized in that A pharmaceutical composition comprising the pharmaceutical composition of claim 7, wherein the dosage form of the pharmaceutical preparation is selected from one or more of the following: tablets, capsules, injections, suppositories, patches, inhalable powder preparations, suspensions, emulsions or ointments.

9. Use of the indole compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof or an optically active form thereof, or the pharmaceutical composition according to claim 7 or the pharmaceutical preparation according to claim 8 in the preparation of an HDAC6 inhibitor.

10. Use of the indole compound according to any one of claims 1 to 5 or its pharmaceutically acceptable salt or optically active form thereof, the pharmaceutical composition according to claim 7 or the pharmaceutical preparation according to claim 8 in the preparation of an anti-idiopathic pulmonary fibrosis drug.

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