CN109705015B

CN109705015B - Histone deacetylase inhibitor and preparation method and application thereof

Info

Publication number: CN109705015B
Application number: CN201811244713.7A
Authority: CN
Inventors: 李进; 吴波; 尚巳耘; 廖伟; 吕鹏; 农云宏; 陈伟
Original assignee: Hitgen Inc
Current assignee: Hitgen Inc
Priority date: 2017-10-25
Filing date: 2018-10-24
Publication date: 2022-05-24
Anticipated expiration: 2038-10-24
Also published as: CN109705015A

Abstract

The invention discloses a histone deacetylase inhibitor, a preparation method and application thereof, and discloses a compound shown in a formula I, or a crystal form thereof, or pharmaceutically acceptable salt thereof, or a solvate thereof, or a prodrug thereof, or a metabolite thereof. The novel compounds of formula I disclosed in the present invention show good deacetylationThe enzyme inhibitory activity provides a new medicinal possibility for clinically treating diseases related to abnormal histone deacetylase activity.

Description

Histone deacetylase inhibitor and preparation method and application thereof

Technical Field

The invention relates to a histone deacetylase inhibitor, a preparation method and application thereof.

Background

Inactivation of genes that control cell growth in the body is one of the hallmarks of tumorigenesis. Epigenetic mechanisms that cause gene inactivation mainly include DNA methylation, histone acetylation, and modifications of other components in chromatin higher order structures, which alter chromatin conformation, resulting in changes in gene transcription regulation, and dysregulation of gene transcription causing cell proliferation disorders, resulting in tumor production.

Histone acetylation plays a central role in transcriptional regulation in eukaryotic cells. Histone acetylation is regulated by a pair of functionally antagonistic proteases Histone Acetyltransferases (HATs) and Histone Deacetylases (HDACs). In normal cells, the pair of enzymes is in a state of dynamic equilibrium. In general, an increased acetylation level of histones is associated with an increased transcriptional activity of the gene, whereas an excessively low acetylation level is associated with an inhibition of gene expression (Forsberg EC et al. Bioessays, 2001, 23 (9): 820-. Studies have found that HDACs are overexpressed and recruited by transcription factors, leading to abnormal suppression of specific genes, leading to tumors and other diseases; inhibition of HDAC activity results in growth inhibition and apoptosis in many cancer cells (Somech R et al cancer Treat Rev, 2004, 30 (5): 461-472). Therefore, HDACs have become the newest and most popular target in the current field of antineoplastic drug development.

In humans, 18 HDACs have been identified and can be divided into four classes. Among these 11 HDACs, zinc is utilized as a cofactor and can be divided into four classes: class I (HDAC1, 2,3 and 8), class IIa (HDAC 4, 5, 7 and 9), class IIb (HDAC6 and 10), class IV (HDAC 11); another 7 HDACs are of class III, requiring NAD⁺As an additional cofactor (Bolden et al. Nat. Rev. drug, 2006, 5 (9): 769-). 784).

The mechanism of action of HDAC inhibitors is to modulate gene expression in the treatment of cancer by inhibiting HDAC, blocking the repression of gene expression due to dysfunction of HDAC recruitment, and altering chromatin structure by altering the degree of acetylation of histones. It has obvious curative effect on treating blood system tumor and solid tumor by inducing growth arrest, differentiation or apoptosis of tumor cell. HDAC inhibitors are tumor specific and cytotoxic to proliferating and quiescent variant cells, whereas normal cells are more than 10-fold tolerant to them and do not cause growth arrest and apoptosis in normal cells.

The HDAC inhibitors developed at present have certain problems in the aspects of anticancer activity, toxic and side effects, subtype selectivity and the like. Therefore, the development of a novel compound having histone deacetylase inhibitory activity is of great social and economic significance.

Disclosure of Invention

In order to solve the problems, the invention provides a histone deacetylase inhibitor, a preparation method and application thereof.

The invention provides a compound shown as a formula I, or a crystal form, a pharmaceutically acceptable salt, a solvate, a prodrug or a metabolite thereof:

wherein the content of the first and second substances,

is a single bond or a double bond;

n is 0 to 5;

x is N or CH;

y is NR¹Or none; wherein R is¹Selected from hydrogen or C₁～C₁₀Alkyl groups of (a);

L¹is selected from C₁～C₁₀Alkylene or none of (1);

the A ring is independently substituted by 0 to 3R²A substituted 5-to 10-membered aromatic ring, a 5-to 10-membered heteroaromatic ring; wherein each R is²Independently selected from halogen, hydroxy, amino, cyano, carboxy, trifluoromethyl, C₁～C₁₀Alkyl of (C)₁～C₁₀Alkoxy or C₁～C₁₀An alkylamino group of (a);

L²is selected from C₁～C₁₀Alkylene or none of (1);

ring B is independently substituted by 0 to 3R³Substituted 5-to 10-membered cycloalkane, 5-to 10-membered heterocyclic alkane, 5-to 10-membered cyclic olefin, 5-to 10-membered heterocyclic olefin, 5-to 10-membered aromatic ring, 5-to 10-membered heteroaromatic ring or none; wherein each R is³Independently selected from halogen, hydroxy, amino, cyano, carboxy, trifluoromethyl, C₁～C₁₀Alkyl of (C)₁～C₁₀Alkoxy or C₁～C₁₀An alkylamino group of (a);

L³is selected from C₁～C₁₀Alkylene or none of (1);

the C ring is independently substituted by 0 to 3R⁴Substituted 5-to 10-membered cycloalkane, 5-to 10-membered heterocyclic alkane, 5-to 10-membered cyclic olefin, 5-to 10-membered heterocyclic olefin, 5-to 10-membered aromatic ring, 5-to 10-membered heteroaromatic ring or none; wherein each R is⁴Independently selected from halogen, hydroxy, amino, cyano, carboxy, trifluoromethyl, C₁～C₁₀Alkyl of (C)₁～C₁₀Alkoxy or C₁～C₁₀An alkylamino group of (a).

Preferably, n is 0-2;

and/or Y is NR¹Or none; wherein R is¹Selected from hydrogen or C₁～C₃Alkyl groups of (a); and/or, L¹Is selected from C₁～C₄Alkylene or none of (1);

and/or, the A ring is independently substituted by 0 to 3R²A substituted 6-to 10-membered aromatic ring, a 5-to 10-membered heteroaromatic ring; wherein each R is²Independently selected from halogen, hydroxy, amino, cyano, carboxy, trifluoromethyl, C₁～C₄Alkyl of (C)₁～C₄Alkoxy or C₁～C₄An alkylamino group of (a);

and/or, L²Is selected from C₁～C₃Alkylene or none of (1);

and/or, the B ring is independently substituted by 0-2R³Substituted 5-to 10-membered cycloalkane, 5-to 10-membered heterocyclic alkane, 5-to 10-membered cyclic olefin, 5-to 10-membered heterocyclic olefin, 6-to 10-membered aromatic ring, 5-to 10-membered heteroaromatic ring or none; wherein each R is³Independently selected from halogen, hydroxy, amino, cyano, carboxy, trifluoromethyl, C₁～C₃Alkyl of (C)₁～C₃Alkoxy or C₁～C₃An alkylamino group of (a);

and/or, L³Is selected from C₁～C₄Alkylene or none of (1);

and/or, the C ring is independently substituted by 0 to 3R⁴Substituted 5-to 10-membered cycloalkane, 5-to 10-membered heterocyclic alkane, 5-to 10-membered cyclic olefin, 5-to 10-membered heterocyclic olefin, 6-to 10-membered aromatic ring, 5-to 10-membered heteroaromatic ring or the like(ii) a Wherein each R is⁴Independently selected from halogen, hydroxy, amino, cyano, carboxy, trifluoromethyl, C₁～C₄Alkyl of (C)₁～C₄Alkoxy or C₁～C₄An alkylamino group of (a).

Further, the compounds of formula I are represented by formula IIa:

wherein n and L¹Ring A, ring L²Ring B, ring L³And ring C has the meaning indicated in claim 1.

Preferably, the compound of formula iia is:

further, the compounds of formula I are represented by formula IIb:

wherein n, ring A, L²Ring B, ring L³And ring C has the meaning indicated in claim 1.

Preferably, the compound of formula iib is:

further, the compounds of formula I are represented by formula IIc:

wherein the content of the first and second substances,

n, ring A, L²Ring B, ring L³And ring C has the meaning indicated in claim 1.

Preferably, the compound of formula iic is:

the application of the compound, or the crystal form, the pharmaceutically acceptable salt, the solvate, the prodrug and the metabolite of the compound in preparing HDAC inhibitor medicines.

Further, the HDAC inhibitor drug is a drug for diseases caused by abnormal HDAC activity.

Preferably, the drug for the disease caused by abnormal HDAC activity is a drug for a disease caused by abnormal HDAC6 activity.

Further, the disease is a cell proliferative disease, an autoimmune disease, inflammation, a neurodegenerative disease, a viral disease, or cancer.

The invention also discloses a pharmaceutical composition for inhibiting the activity of histone deacetylase, which is a preparation prepared from the compound, or a crystal form thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a prodrug thereof, or a metabolite thereof and pharmaceutically acceptable auxiliary materials.

Preferably, the formulation is an oral administration formulation, a sublingual administration formulation, a buccal administration formulation, a transdermal absorption formulation or an injection formulation.

The compounds and derivatives provided in the present invention may be named according to the IUPAC (international union of pure and applied chemistry) or CAS (chemical abstracts service, Columbus, OH) naming system.

Definitions of terms used in connection with the present invention: the initial definitions provided herein for a group or term apply to that group or term throughout the specification unless otherwise indicated; for terms not specifically defined herein, the meanings that would be given to them by a person skilled in the art are to be given in light of the disclosure and the context.

"substituted" means that a hydrogen atom in a molecule is replaced by a different atom or molecule.

Halogen is fluorine, chlorine, bromine or iodine.

The minimum and maximum values of the carbon atom content in the hydrocarbon group are indicated by a prefix, e.g. prefix (C)_a～C_b) Alkyl means any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, C₁～C ₁₀The alkyl group is a straight chain or branched chain alkyl group containing 1 to 10 carbon atoms;

C₁～C ₁₀the alkylene group is a straight-chain or branched alkylene group containing 1 to 10 carbon atoms; for example-CH₃Is methyl, -CH₂-is methylene.

C₁～C₁₀The alkylamino group of (a) means that an arbitrary position of a linear or branched alkyl group having 1 to 10 carbon atoms is substituted with an amino group.

The 5-to 10-membered aromatic ring is a single ring or a parallel ring having 5 to 10 carbon atoms.

The 5-to 10-membered heteroaromatic ring is a single ring or a parallel ring having 5 to 10 atoms such as C, O, S, N.

The ring A is independently substituted by 0 to 3R²0 to 3R's in a substituted 5-to 10-membered aromatic ring or 5-to 10-membered heteroaromatic ring²Substituted means 0 to 3 identical or different R²Independently selected from halogen, hydroxy, amino, cyano, carboxy, trifluoromethyl, C₁～C₁₀Alkyl of (C)₁～C₁₀Alkoxy or C₁～C₁₀An alkylamino group of (a). The B and C rings are similar and will not be explained here.

The 5-10 membered cycloalkane is a monocyclic or polycyclic hydrocarbon group having 5 to 10 carbon atoms.

The 5-to 10-membered heterocyclic alkane is a monocyclic or polycyclic hydrocarbon group containing at least one atom of 5 to 10 atoms selected from O, S, N.

The 5-10 membered cyclic olefin is a non-aromatic monocyclic or polycyclic hydrocarbon group of 5 to 10 carbon atoms, and may have a double bond at any position.

The 5-to 10-membered heterocyclic olefin is a nonaromatic monocyclic or polycyclic hydrocarbon group having at least one atom of 5 to 10 atoms selected from O, S, N, and a double bond may be present at any position.

The term "pharmaceutically acceptable" means that the carrier, cargo, diluent, adjuvant, and/or salt formed is generally chemically or physically compatible with the other ingredients comprising a pharmaceutical dosage form and physiologically compatible with the recipient.

The terms "salt" and "pharmaceutically acceptable salt" refer to acid and/or base salts of the above compounds or stereoisomers thereof, with inorganic and/or organic acids and bases, as well as zwitterionic (inner) salts, and also quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. The compound or a stereoisomer thereof may be obtained by appropriately (e.g., equivalently) mixing the above compound or a stereoisomer thereof with a predetermined amount of an acid or a base. These salts may form precipitates in the solution which are collected by filtration, or they may be recovered after evaporation of the solvent, or they may be prepared by reaction in an aqueous medium followed by lyophilization. The salt in the invention can be hydrochloride, sulfate, citrate, benzene sulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate of the compound.

In certain embodiments of the present invention, the invention includes isotopically-labeled compounds, which are intended to be identical to those recited herein, but wherein one or more atoms are replaced by another atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Isotopes which may be incorporated into compounds of formula (I) include hydrogen, carbon, nitrogen, oxygen, sulphur, i.e.²H,³H、¹³C、¹⁴C、¹⁵N、¹⁷O、¹⁸O、³⁵And S. The compounds of formula (I) and stereoisomers thereof, and pharmaceutically acceptable salts of the compounds and stereoisomers, containing the aforementioned isotopes and/or other atomic isotopes, are intended to includeAre within the scope of the invention.

In certain embodiments, one or more compounds of the present invention may be used in combination with each other. Alternatively, the compounds of the present invention may be used in combination with any other active agent for the preparation of a medicament or pharmaceutical composition for modulating cellular function or treating a disease. If a group of compounds is used, the compounds may be administered to the subject simultaneously, separately or sequentially.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, parenteral (intravenous, intramuscular, or subcutaneous), and topical administration.

The compound of the invention has the activities of inducing differentiation, immunoregulation, blocking cell cycle and promoting apoptosis and good HDAC6 subtype selectivity, aims to have better curative effect on various cancers, and simultaneously overcomes the toxic and side effects of the existing HDAC inhibitor, such as anemia, ischemic stroke, deep venous thrombosis, thrombocytopenia, emesis and the like.

The compounds of the present invention have HDAC inhibitory activity and are useful for the treatment of diseases associated with abnormal HDAC activity.

The invention has the beneficial effects that: the novel compound shown in the formula I shows good deacetylase inhibitory activity, can treat colon cancer, and provides a new medicinal possibility for clinically treating diseases related to abnormal histone deacetylase activity.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Detailed Description

The raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products.

Example 1 Synthesis of target products 1 to 7

The route for synthesizing the target product 1-7 is as follows:

1. synthesis of intermediate 1

(1) Synthesis of intermediate 1 a:

chlorosulfonyl isocyanate (11.8g,83.2mmol) was dissolved in dry dichloromethane (220mL), cooled to 0 deg.C, and a solution of 2-chloroethanol (6.70g,83.2mmol) in dichloromethane (220mL) was added dropwise under argon. After stirring the reaction for 30 minutes, a solution of aniline (8.53g,91.5mmol) and triethylamine (16.8 g,166mmol,23.1mL) in dichloromethane (220mL) was added slowly, the temperature was raised to room temperature and the reaction was stirred for 1 hour. The reaction was quenched by the addition of 0.2N hydrochloric acid (10mL) and the pH was adjusted to 2.0 with concentrated hydrochloric acid. The organic layer was separated, washed successively with 0.05mol/L hydrochloric acid and water, and dried by adding anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to give intermediate 1a (18.0g,74.3mmol, yield 89%).

(2) Synthesis of intermediates 1b to 1 g:

the intermediates 1b to 1g obtained from the raw materials in table 1 by the same synthesis method as the intermediate 1a and other conditions, and the yields thereof are shown in table 1; wherein, the molar amount of the raw material 1 corresponds to aniline used for preparing 1a, the raw material 2 corresponds to chlorosulfonyl isocyanate used for preparing 1a, the raw material 3 corresponds to 2-chloroethanol used for preparing 1a, and other raw materials or solvents which are not mentioned, reaction conditions thereof and the like are the same as those in the preparation 1 a.

TABLE 1 starting materials and final yields for the synthesis of intermediates 1b to 1g

2. Synthesis of intermediate 2

Tert-butyl 5-bromoindoline-1-carboxylate (5.05g,16.9mmol) and ethyl acrylate (17.0g,169mmol) were dissolved in DMF (100mL), and palladium acetate (760mg,3.39mmol), tris (o-methylphenyl) phosphorus (3.09g,10.2mmol) and triethylamine (5.14g,50.8mmol,7.04mL) were added and the reaction was stirred at 90 ℃ overnight. The reaction was quenched by addition of water (30mL), and the organic layer was separated, washed with water, and dried with anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and purified by column chromatography (eluent: petroleum ether: ethyl acetate 10:1 to 1:1) to give intermediate 2(3.64g,11.5mmol, yield 68%).

3. Synthesis of intermediate 3

Intermediate 2(1.90g,6.00mmol) was dissolved in tetrahydrofuran (20.0mL), and 37% concentrated hydrochloric acid (5.98mL) was added and the reaction stirred at room temperature for 4 hours. Sodium bicarbonate was added to adjust the pH to 7.0, dichloromethane was extracted, and the solvent was distilled off under reduced pressure to give crude intermediate 3 (1.50 g).

4. Synthesis of intermediate 4

(1) Synthesis of intermediate 4 a:

intermediate 1a (1.30g,6.00mmol), intermediate 3(2.33g,9.60mmol) and triethylamine (1.82g,18.0mmol) were dissolved in acetonitrile (30.0mL) and the reaction was stirred at reflux for 16 h. The solvent was evaporated under reduced pressure and purified by column chromatography (eluent: petroleum ether: ethyl acetate 10:1 to 1:1) to give intermediate 4a (750mg,2.01mmol, yield 34%).

(2) Synthesis of intermediates 4b to 4 g:

respectively taking the intermediates 1 b-1 g and the intermediate 3 as raw materials, and obtaining the intermediates 4 b-4 g and the yield thereof respectively by the same synthesis method of the intermediate 4a under the other conditions, as shown in table 2; wherein other raw materials or solvents not mentioned and reaction conditions thereof and the like are the same as in the preparation 1 a.

TABLE 2 starting materials and final yields for the synthesis of intermediates 4b to 4g

5. Synthesis of target product

(1) Synthesis of target product 1:

intermediate 4a (750mg,2.01mmol) was dissolved in dichloromethane (5.00mL) and methanol (5.00mL), and sodium hydroxide (241mg,6.03mmol) and aqueous hydroxylamine (3.98mL,60.30mmol, 50% w/w) were added and the reaction stirred at room temperature for 20 hours. The solvent was evaporated under reduced pressure and purified by MPLC (eluent: water: acetonitrile 4:1 to 1:1) to obtain the objective compound 1(300mg,826 μmol, yield 41%).

MS(ESI)m/z 360(M+1)⁺

¹H NMR(400MHz,DMSO-d₆)δ10.67(s,1H),10.54(s,1H),8.99(s,1H),7.40–7.20(m,6H),7.15–7.08(m,2H),7.04(td,J＝7.2,1.2Hz,1H),6.30(d,J＝15.6Hz,1H),3.91(t,J＝8.4Hz,2H),2.99(t,J＝8.4Hz,2H).

(2) Synthesizing a target product 2-7:

respectively taking the intermediates 4 b-4 g as raw materials, and respectively obtaining target compounds 2-7 and the yield thereof by using the same synthesis method of the target compound 1 under the other conditions, as shown in Table 3; wherein other raw materials or solvents not mentioned and reaction conditions thereof and the like are the same as those in the production of the objective compound 1.

TABLE 3 Synthesis of the target Compoundsb～gRaw materials used and final yield

EXAMPLE 2 Synthesis of target Compounds 8 and 9

The synthesis routes of the target products 8 and 9 are as follows:

1. synthesis of intermediate 5

Synthesis of intermediates 5a and 5 b:

the intermediates 5a and 5b obtained using the starting materials in table 4 and the other conditions were the same as for the synthesis of intermediate 1a, and the structures and yields corresponding thereto are shown in table 4. Wherein, the molar amount of the raw material 1 corresponds to aniline used for preparing 1a, the raw material 2 corresponds to chlorosulfonyl isocyanate used for preparing 1a, the raw material 3 corresponds to 2-chloroethanol used for preparing 1a, and other raw materials or solvents which are not mentioned, reaction conditions thereof and the like are the same as those in the preparation 1 a.

TABLE 4 starting materials and final yields for the synthesis of intermediates 5a and 5b

2. Synthesis of intermediate 6

Synthesis of intermediates 6a and 6 b:

as shown in table 5, intermediates 6b and the yields thereof were obtained according to the method for synthesizing intermediate 4a in example 1, starting from intermediates 5a and 5b, respectively, and starting from raw material 2, respectively, as shown in table 5; wherein starting materials 5a, 5b correspond to 1a used in the preparation of intermediate 4a and starting material 2 corresponds to intermediate 3 used in the preparation of intermediate 4 a. Other raw materials or solvents not mentioned and reaction conditions thereof and the like are the same as in preparation 4a in example 1.

TABLE 5 starting materials and final yields for the synthesis of intermediates 6a and 6b

3. Synthesis of target compounds 8 and 9:

the intermediates 6a and 6b were used as starting materials, respectively, and the other conditions were the same as the synthesis method of the objective compound 1 in example 1, to obtain the objective compounds 8 and 9, respectively, and their yields, as shown in table 6; wherein other raw materials or solvents not mentioned and reaction conditions thereof and the like are the same as those in the production of the objective compound 1.

TABLE 6 starting materials and final yields for the syntheses of the target compounds 8 and 9

Example 3 Synthesis of target Compounds 10 to 15

The route for synthesizing the target product 10-15 is as follows:

1. synthesis of intermediate 7

2, 3-dihydro-1H-pyrrolo [2,3-b ] pyridine (5.00g,41.6mmol) was dissolved in DMF (140mL), and after N-bromosuccinimide (8.89g,49.9mmol) was added, the reaction was stirred at room temperature for 3 hours. Adding saturated sodium bicarbonate ice water solution, extracting with ethyl acetate, washing with saturated sodium chloride solution, adding anhydrous sodium sulfate, and drying. The solvent was evaporated under reduced pressure and purified by column chromatography (eluent: petroleum ether/ethyl acetate 10:1 to 1:1) to give intermediate 7(3.60g,18.1mmol, 43% yield).

2. Synthesis of intermediate 8:

intermediate 7(2.00g,10.1mmol), zinc cyanide (1.77g,15.1mmol) and tetrakis (triphenylphosphine) palladium (1.16g,1.00mmol) were dissolved in DMSO (30.0mL) and reacted under nitrogen at 125 ℃ with stirring for 8 hours. After cooling to room temperature, a saturated sodium bicarbonate solution was added and extracted with ethyl acetate. The solvent was evaporated under reduced pressure and purified by column chromatography (eluent: petroleum ether/ethyl acetate 10:1 to 1:1) to give intermediate 8(1.00g,6.89mmol, 69% yield).

3. Synthesis of intermediate 9:

intermediate 8(500mg,3.44mmol) was dissolved in water (5.00mL), 98% sulfuric acid (5.00mL) was added slowly at room temperature, then the reaction was stirred to 110 ℃ for 5 hours. Cooling to room temperature and adjusting pH to 4-5. The solvent was distilled off under reduced pressure, methanol was added thereto, followed by filtration, and the solvent was distilled off under reduced pressure from the filtrate to give a crude intermediate 9 (700 mg).

4. Synthesis of intermediate 10:

intermediate 9(558mg,3.40mmol) was dissolved in methanol (10.0mL) and thionyl chloride (5.00mL) was added slowly at room temperature. After warming to 80 ℃ the reaction was stirred overnight. The solvent was evaporated under reduced pressure, ethyl acetate was added thereto, and the mixture was washed with a saturated sodium bicarbonate solution. The solvent was distilled off under reduced pressure to give a crude product (500mg) of intermediate 10.

5. Synthesis of intermediate 11

(1) Synthesis of intermediate 11 a:

intermediate 10(606mg,3.40mmol) was dissolved in dichloromethane (10.0mL) and pyridine (2.00mL), 4'- (piperidin-1-ylmethyl) - [1,1' -biphenyl ] -4-sulfonyl chloride (1.19g,3.40mmol) was added at 0 deg.C, and the reaction was stirred at room temperature overnight. The solvent was evaporated under reduced pressure and purified by column chromatography (eluent: petroleum ether/ethyl acetate 10:1 to 1:1) to give intermediate 11a (500mg,1.02mmol, yield 30%).

(2) Synthesis of intermediates 11b to 11 f:

the intermediates 11b to 11f obtained using the starting materials in table 7 and the synthesis method of the intermediate 11a under the other conditions, and the structures and yields corresponding thereto are shown in table 7. Wherein the molar amount of the raw material 1 corresponds to 4'- (piperidin-1-ylmethyl) - [1,1' -biphenyl ] -4-sulfonyl chloride used in the preparation 11a, the raw material 2 corresponds to the intermediate 10 used in the preparation 11a, and other raw materials, solvents, reaction conditions thereof, and the like which are not mentioned are the same as those in the preparation 11 a.

TABLE 7 starting materials and final yields for the synthesis of intermediates 11b to 11f

6. Synthesizing a target compound 10-15:

taking the intermediates 11b to 11f as raw materials, respectively, and obtaining target compounds 10 to 15 and the yields thereof respectively under the same conditions as the synthesis method of the target compound 1 in the example 1, as shown in table 6; wherein other raw materials or solvents not mentioned and reaction conditions thereof and the like are the same as those in the production of the objective compound 1.

TABLE 8 raw materials and final yields for the synthesis of target compounds 10-15

EXAMPLE 4 Synthesis of Compound of interest 16

The route for synthesizing the target product 16 is as follows:

1. synthesis of intermediate 12:

methyl 1H-indole-5-carboxylate (651mg,3.72mmol) was dissolved in tetrahydrofuran (10.0mL), sodium hydrogen (446mg,11.2mmol, 60% purity) was added at 0 deg.C, stirring at room temperature, 4'- (piperidin-1-ylmethyl) - [1,1' -biphenyl ] -4-sulfonyl chloride (1.30g,3.72mmol) was added after 1 hour of reaction, stirring at room temperature was continued, and the reaction was allowed to proceed overnight. The solvent was distilled off under reduced pressure to give crude intermediate 12(1.20 g).

2. Synthesis of intermediate 13:

intermediate 12(1.20g,2.46mmol) was dissolved in DMF (8.00mL) and water (2.00mL), lithium hydroxide monohydrate (309mg,7.37mmol) was added at room temperature, the reaction was allowed to warm to 50 ℃ and stirred for 4 h. The solvent was evaporated under reduced pressure and purified by preparative HPLC (eluent: water: acetonitrile 4:1 to 1:1) to give intermediate 13(300mg,632 μmol, yield 26%).

3. Synthesis of intermediate 14:

intermediate 13(270mg, 569. mu. mol), O- (tetrahydro-2H-pyran-2-yl) hydroxylamine (66.6mg, 569. mu. mol) and DIPEA (221mg,1.71mmol, 297. mu.L) were dissolved in DMF (5.00mL), and 2- (7-oxybenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (324mg, 853. mu. mol) was added thereto at 0 ℃ and stirred at room temperature for reaction overnight. Extraction with ethyl acetate, washing with water and a saturated sodium chloride solution in this order, evaporation of the solvent under reduced pressure, and purification by column chromatography (eluent: dichloromethane: methanol 50:1 to 10:1) gave intermediate 14(100mg,174 μmol, yield 31%).

4. Synthesis of target compound 16:

intermediate 14(100mg, 174. mu. mol) was dissolved in tetrahydrofuran (5.00mL), and the reaction was continued for 3 hours with stirring at room temperature by dropwise addition of 37% concentrated hydrochloric acid (1.00 mL). Adding saturated sodium carbonate solution to regulate pH to 7-8. The solvent was evaporated under reduced pressure and purified by preparative HPLC (eluent: water: acetonitrile 4:1 to 1:1) to give the compound of example 16 (50.0mg,94.1 μmol, yield 54%).

MS(ESI)m/z 490(M+1)⁺

¹H NMR(400MHz,DMSO-d6)δ11.29(s,1H),10.76(s,1H),8.11(d,J＝8.8Hz,2H),8.06–8.04(m,2H),7.96(d,J＝3.6Hz,1H),7.92(d,J＝8.8Hz,2H),7.82–7.68(m,5H),6.97(dd,J＝3.6,0.4Hz,1H),4.28(d,J＝5.2Hz,2H),3.27(d,J＝11.6Hz,2H),2.89–2.78(m,2H),1.92–1.58(m,5H),1.39–2.28(m,1H).

Example 5 Synthesis of target Compounds 17 to 19

The route for synthesizing the target products 17-19 is as follows:

1. synthesis of intermediate 15:

4-bromobenzene-1-sulfonyl chloride and indoline-5-carboxylic acid methyl ester are taken as raw materials, and the other conditions are the same as the synthesis method of the intermediate 11a, so as to obtain an intermediate 15. Wherein the molar amount of 4-bromobenzene-1-sulfonyl chloride corresponds to 4'- (piperidine-1-ylmethyl) - [1,1' -biphenyl ] -4-sulfonyl chloride used in preparation 11a, and the methyl indoline-5-carboxylate corresponds to intermediate 10 used in preparation 11a, and other raw materials, solvents, reaction conditions thereof and the like which are not mentioned are the same as those in preparation 11 a.

2. Synthesis of intermediate 16:

intermediate 15(2.20g,4.66mmol) was dissolved in dioxane (30.0mL), bis (pinacolato) diboron (4.73g,18.6mmol), potassium acetate (457mg,4.66mmol) and [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (3.41g,4.66mmol) were added at room temperature, the reaction was allowed to warm to 100 ℃ and stirred for 3 hours. After the solvent was distilled off under reduced pressure, water (50ml) and ethyl acetate (50ml) were added, the aqueous layer was extracted 2 times with ethyl acetate (100ml), the organic layers were combined and dried over anhydrous sodium sulfate, and after the solvent was distilled off under reduced pressure, column chromatography (eluent: petroleum ether/ethyl acetate 10:1 to 1:1) was performed to obtain intermediate 16(1.50g,2.89mmol, yield 62%).

3. Synthesis of intermediate 17

(1) Synthesis of intermediate 17 a:

intermediate 16(300mg, 677. mu. mol) was dissolved in dioxane (10.0mL), potassium carbonate (280mg,2.03mmol), [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (49.5mg, 67.7. mu. mol) and 2- (4-bromophenyl) -N, N-dimethylethylamine (154mg, 677. mu. mol) were added at room temperature, and the reaction was stirred after being raised to 100 ℃ for 3 hours. After the solvent was distilled off under reduced pressure, water (50ml) and ethyl acetate (50ml) were added, the aqueous layer was extracted 2 times with ethyl acetate (100ml), the organic layers were combined and dried over anhydrous sodium sulfate, and after the solvent was distilled off under reduced pressure, column chromatography (eluent: petroleum ether/ethyl acetate 10:1 to 1:1) was performed to obtain intermediate 17a (200mg,431 μmol, yield 64%).

(2) Synthesis of intermediates 17b and 17 c:

the intermediates 17b and 17c obtained using the starting materials in table 9 and the synthesis of intermediate 17a under the same conditions and with the corresponding structures and yields are shown in table 9. Wherein the molar amount of the raw material 1 corresponds to 2- (4-bromophenyl) -N, N-dimethylethylamine used for the preparation 17a, the raw material 2 corresponds to the intermediate 16 used for the preparation 17a, and other raw materials, solvents, reaction conditions thereof, and the like which are not mentioned are the same as those in the preparation 17 a.

Table 9 starting materials and final yields for the synthesis of intermediates 17b and 17c

4. Synthesis of target Compounds 17 to 19

Taking the intermediates 17a to 17c as raw materials, respectively, and obtaining target compounds 17 to 19 and the yields thereof respectively under the same conditions as the synthesis method of the target compound 1 in the example 1, as shown in table 10; wherein other raw materials or solvents not mentioned and reaction conditions thereof and the like are the same as those in the production of the objective compound 1.

TABLE 10 raw materials and final yields for the synthesis of the target compounds 17-19

EXAMPLE 6 Synthesis of target Compound 20

The route for synthesizing the target product 20 is as follows:

1. synthesis of intermediate 18:

intermediate 15(298mg,753 μmol) was dissolved in toluene (10.0mL), piperidine (192mg,2.26mmol), cesium carbonate (318mg,979 μmol), (+ -) -2,2 '-bis- (diphenylphosphino) -1,1' -binaphthyl (23.5mg,37.7 μmol) and palladium acetate (8.44mg,37.7 μmol) were added at room temperature, and the reaction was stirred at 100 ℃ under nitrogen blanket for 3 hours. The solvent was distilled off under reduced pressure, ethyl acetate (50.0ml) and water (50.0ml) were added to the residue, the aqueous layer was separated, and the mixture was extracted with ethyl acetate (100ml) 2 times, and the organic layers were combined and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and then purified by column chromatography (eluent: petroleum ether/ethyl acetate ═ 10:1 to 1:1) to give intermediate 18(199mg,497 μmol, yield 66%).

2. Synthesis of target compound 20:

using the intermediate 18 as a raw material, and obtaining a target compound 20 and a yield thereof under the same conditions as the synthesis method of the target compound 1 in the example 1; wherein other raw materials or solvents not mentioned and reaction conditions thereof and the like are the same as those in the production of the objective compound 1. MS (ESI) M/z 402(M +1)⁺

¹HNMR(400MHz,DMSO+D₂O):δ7.62-7.57(m,3H),7.51(s,1H),7.44(d,J＝8.4Hz,1H),6.98(d,J＝9.2Hz,2H),3.86(t,J＝8.4Hz,2H),3.31(s,4H),2.96(t,J＝8.4Hz,2H),1.57-1.53(m,6H).

Example 7 Synthesis of target Compounds 21 to 23

The route for synthesizing the target product 21-23 is as follows:

1. synthesis of intermediate 19

(1) Synthesis of intermediate 19 a:

n, N-dimethyl-1- (4-phenylphenyl) methylamine (5.00g,23.7mmol) was dissolved in dichloromethane (30.0mL), chlorosulfonic acid (27.6g,237mmol) was added at 0 deg.C, and the reaction was stirred for additional 3 hours. The solvent was distilled off under reduced pressure, ethyl acetate (50.0ml) and water (50.0ml) were added to the residue, the aqueous layer was separated, and the mixture was extracted with ethyl acetate (100ml) 2 times, and the organic layers were combined and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and purified by column chromatography (eluent: dichloromethane: water 50:1 to 10:1) to give intermediate 19a (3.00g,10.3mmol, yield 44%).

(2) Synthesis of intermediates 19b and 19 c:

intermediates 19b and 19c obtained from the starting materials in table 11 under otherwise the same conditions as the synthesis of intermediate 19a, and their corresponding structures and yields are shown in table 11. Wherein the molar amount of the starting materials corresponds to that of N, N-dimethyl-1- (4-phenylphenyl) methylamine used in the preparation of 19a, and other starting materials, solvents, reaction conditions thereof, and the like which are not mentioned are the same as those in the preparation of 19 a.

TABLE 11 starting materials and final yields for the syntheses of intermediates 19b and 19c

2. Synthesis of intermediate 20

(1) Synthesis of intermediate 20 a:

intermediate 19a (3.00g,10.3mmol) was dissolved in thionyl chloride (30.0mL) and the reaction was stirred at reflux for 3 hours. The solvent was distilled off under reduced pressure, ethyl acetate (10.0ml) and water (10.0ml) were added to the residue, the aqueous layer was separated, and the mixture was extracted with ethyl acetate (10.0ml) for 2 times, and the organic layers were combined and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and purified by column chromatography (eluent: dichloromethane: water 50:1 to 10:1) to give intermediate 20a (2.00g,6.46mmol, yield 63%).

(2) Synthesis of intermediates 20b and 20 c:

intermediates 20b and 20c obtained using intermediates 19b and 19c as starting materials, respectively, under the same conditions as the synthesis of intermediate 20a, and the corresponding structures and yields are shown in table 12. Wherein the molar amount of the starting materials corresponds to the intermediate 19a used in the preparation 20a, and other starting materials, solvents, reaction conditions thereof, and the like which are not mentioned are the same as those in the preparation 20 a.

Table 12 starting materials and final yields for the synthesis of intermediates 20b and 20c

3. Synthesis of intermediate 21

(1) Synthesis of intermediate 21 a:

intermediate 20a (2.00g,6.46mmol) was dissolved in dichloromethane (30.0mL), and methyl 1H-indole-5-carboxylate (1.14g,6.46mmol) and triethylamine (1.31g,12.9mmol) were added at room temperature, and the reaction was stirred under reflux for 3 hours. The solvent was distilled off under reduced pressure, ethyl acetate (50.0ml) and water (50.0ml) were added to the residue, the aqueous layer was separated, and the mixture was extracted with ethyl acetate (100ml) 2 times, and the organic layers were combined and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and purified by column chromatography (eluent: dichloromethane: water 50:1 to 10:1) to give intermediate 21a (300mg,666 μmol, yield 10%).

(2) Synthesis of intermediates 21b and 21 c:

intermediates 21b and 21c obtained from intermediates 20b and 20c, respectively, and their corresponding structures and yields are shown in table 13, except that the synthesis of intermediate 21a is performed under the same conditions. Wherein the molar amount of the raw material 1 corresponds to the intermediate 20a used in the preparation 21a, the molar amount of the raw material 1 corresponds to the methyl 1H-indole-5-carboxylate used in the preparation 21a, and other raw materials, solvents, reaction conditions thereof, and the like which are not mentioned are the same as those in the preparation 21 a.

Table 13 starting materials and final yields for the synthesis of intermediates 21b and 21c

4. Synthesizing a target compound 21-23:

the intermediates 21a to 21c were used as raw materials, and the other conditions were the same as the synthesis method of the target compound 1 in example 1, to obtain the target compounds 21 to 23 and the yields thereof, respectively, as shown in table 14; wherein other raw materials or solvents not mentioned and reaction conditions thereof and the like are the same as those in the preparation of the objective compound 1.

TABLE 14 raw materials and final yields for the synthesis of target compounds 21-23

To illustrate the advantageous effects of the present invention, the present invention provides the following test examples:

test example 1 HDAC1 and HDAC6 enzymatic detection method

The HDAC inhibitory activity of the compounds of the present invention was measured using a homogeneous time-resolved fluorescence (HTRF) method.

Enzyme buffer (50mM Tris-HCl pH 8.0,137mM NaCl,2.7mM KCl,1mM MgCl)₂0.01% Tween20) to make up solutions of the compounds at different concentrations. A detection mixture of Streptavidin XL-665(Cisbio Bioassays #610SAXLA) and anti-H3K9me0-Eu (K) (Cisbio Bioassays #61KB0KAE) was formulated using detection buffer (Cisbio Bioassays #62 SDBRDD).

mu.L of the compound solution was added to the reaction plate, and 2. mu.L of the HDAC solution (terminal HDAC 1: 30 ng/plate; HDAC 6: 70 ng/plate) was added and incubated at room temperature for 10 minutes. Mu.l of LHistone H3(1-21) lysine 9acetylated biotinylated peptide (Anaspec # AS-64361) was added and the membrane was incubated at 37 ℃ for 60 min. 10 μ L of the assay mixture was added, incubated at room temperature for 1 hour, and the fluorescent signal was read using a multifunctional microplate reader (Envision 2104). Determining the inhibitory effect of the compound from the data obtained and plotting it against the compound concentration to obtain a concentration response curve, fitting the IC to a four parameter model₅₀The value is obtained.

The enzyme activities of HDAC1 and HDAC6 were measured for the compounds prepared in the examples according to the above-mentioned methods, and the results are shown in Table 15, in which the IC of each compound was determined₅₀Sorted by description, in table 15:

"+" denotes IC₅₀Greater than 500 nM;

"+ +" denotes IC₅₀Greater than 100nM and less than 500 nM;

"+ + + +" denotes IC₅₀Less than 100nM

Inhibitory Activity of Table 15 Compounds on HDAC1 and HDAC6

Target compound	Active (HDAC1)	Active (HDAC6)	Target compound	Active (HDAC1)	Activity (HDAC6)
						1	+++	+++	2	+++	+++
3	+++	+++	4	+++	+++
						5	+++	+++	6	+++	+++
7	+++	+++	8	++	+++
						9	++	+++	10	+++	+++
11	ND	ND	12	ND	ND
						13	ND	ND	14	ND	ND
15	ND	ND	16	ND	ND
						17	++	+++	18	+	++
19	++	+++	20	+	++
						21	++	+++	22	++	+++
23	+	+++

ND: the data is being detected and analyzed.

Experiments show that the compound has good deacetylase inhibitory activity and can be effectively used for treating diseases with abnormal histone deacetylase activity.

Test example 2 cell growth inhibition assay

HCT-116 cells were seeded in 12-well plates in logarithmic growth phase. After the cells adhered overnight, the compounds were added to treat the cells for 24 hours, respectively. The cells were harvested and lysed in SDS lysate on ice. And (3) performing SDS-PAGE electrophoresis on the cell lysate, and transferring the protein to a PVDF membrane by using a wet transfer system. After 5% skim milk blocking solution prepared in TBST solution (100mM Tris-HCl pH 7.2-7.4, 0.9% NaCl, 0.2% Tween-20) was added, the shaker was blocked for 60 minutes at room temperature. The membrane was placed in primary antibody diluted in antibody diluent (5% skim milk) overnight at 4 ℃. Three washes with TBST solution at room temperature for 10 minutes each. A secondary antibody labeled with a near infrared label was added thereto, and the mixture was gently shaken on a shaker at room temperature for 1 hour. And washing the solution with TBST for three times, and acquiring a fluorescence signal value in an Odyssey CLx near-infrared two-color fluorescence imaging system. Determining the inhibitory effect of the compound from the data and plotting it against the compound concentration to obtain a concentration response curve, fitting the EC according to a four parameter model₅₀The value is obtained.

The compounds prepared in the examples were tested for inhibition of cell growth according to the methods described above and the results are shown in Table 16, where the EC for each compound was determined₅₀Sorted by description, in table 16:

"+" indicates EC₅₀Greater than 50 μ M;

"+ +" indicates EC₅₀Greater than 10 μ M and less than 50 μ M; (ii) a

"+ + + +" denotes EC₅₀10 μ M smaller;

inhibitory Activity of Compounds of Table 16 on HCT-116 cells

Target compound	Activity of	Target compound	Activity of	Target compound	Activity of	Target compound	Activity of
								1	+++	2	+++	3	+++	4	+++
5	+++	6	+++	7	+++	8	+++
								9	++	10	+++	11	+++	12	++
13	+++	14	+++	15	ND	16	+++
								17	+++	18	+++	19	+++	20	+++
21	+++	22	+++	23	++

ND: the data is being detected and analyzed.

Experiments show that the compound has good inhibitory activity on HCT-116 cells and can be used as a medicine for treating colon cancer.

In conclusion, the novel compound shown in the formula I shows good deacetylase inhibitory activity, and provides a new medicinal possibility for clinically treating diseases related to abnormal histone deacetylase activity.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof: the compound of formula I is represented by formula IIa:

（Ⅱa）

wherein the content of the first and second substances,

n is 0;

L¹is selected from C₁~C₁₀Alkylene or none of (1);

the A ring is independently substituted by 0 to 3R²A substituted 6-to 10-membered aromatic ring; wherein each R is²Independently selected from C₁~C₁₀Alkyl groups of (a);

L²is selected from C₁~C₁₀Alkylene or none of (1);

ring B is independently substituted by 0 to 3R³Substituted 5-to 10-membered heterocyclic alkanes, 6-to 10-membered aromatic rings; wherein the 5-to 10-membered heterocycloalkane is a monocyclic ring of 5 to 10 atoms containing one N; each R³Independently selected from C₁~C₁₀Alkyl groups of (a);

L³is selected from C₁~C₁₀Alkylene or none of (1);

the C ring is independently substituted by 0-3R⁴Substituted 5-to 10-membered heterocyclic alkane or nothing; wherein the 5-to 10-membered heterocycloalkane is a monocyclic ring of 5 to 10 atoms containing one N; each R⁴Independently selected from C₁~C₁₀Alkyl group of (1).

2. The compound of claim 1, wherein:

L¹is selected from C₁~C₄Alkylene or none of (1); each R²Independently selected from C₁~C₄Alkyl groups of (a); l is²Is selected from C₁~C₃Alkylene or none of (1); each R³Independently selected from C₁~C₃Alkyl groups of (a); l is³Is selected from C₁~C₄Alkylene or none of (1); each R⁴Independently selected from C₁~C₄Alkyl group of (1).

3. The compound of claim 2, wherein: the compound of formula IIa is:

or

。

4. Use of a compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the HDAC inhibitor class.

5. Use according to claim 4, characterized in that: the HDAC inhibitor medicine is a medicine for diseases caused by abnormal activity of HDAC.

6. Use according to claim 5, characterized in that: the medicine for the disease caused by the abnormal activity of the HDAC is the medicine for the disease caused by the abnormal activity of the HDAC 6.

7. Use according to claim 6, characterized in that: the disease is a cell proliferative disease, an autoimmune disease, an inflammation, a neurodegenerative disease, a viral disease, or a cancer.

8. Use according to claim 7, characterized in that: the cancer is colon cancer, esophageal cancer, gastric cancer, liver cancer, nasopharyngeal cancer, brain tumor, lung cancer, breast cancer, cervical cancer or leukemia.

9. A pharmaceutical composition for inhibiting histone deacetylase activity, comprising: the compound or the pharmaceutically acceptable salt thereof as an active ingredient and pharmaceutically acceptable auxiliary materials.

10. The pharmaceutical composition of claim 9, wherein: the preparation is oral administration preparation, sublingual administration preparation, buccal administration preparation, transdermal absorption preparation or injection preparation.