CN110950897B

CN110950897B - Histone deacetylase, protease double-target inhibitor, preparation method and application thereof

Info

Publication number: CN110950897B
Application number: CN201911271691.8A
Authority: CN
Inventors: 方浩; 周易; 侯旭奔; 杨新颖
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2019-12-12
Filing date: 2019-12-12
Publication date: 2021-05-28
Anticipated expiration: 2039-12-12
Also published as: CN110950897A; WO2021115188A1

Abstract

The invention relates to a protein deacetylase, a proteasome double-target inhibitor, pharmaceutically acceptable salts thereof, stereoisomers thereof, a preparation method and application thereof, wherein the compound is shown as a general formula I, has better anti-histone deacetylase activity, anti-proteasome activity and anti-tumor cell proliferation activity, can be used for preparing a medicament for preventing or treating related mammal diseases caused by abnormal expression of histone deacetylase or abnormal proteasome, and also relates to pharmaceutical application of a composition containing the compound with the structure of the general formula I.

Description

Histone deacetylase, protease double-target inhibitor, preparation method and application thereof

Technical Field

The invention relates to a histone deacetylase, a protease double-target inhibitor, pharmaceutically acceptable salts thereof, stereoisomers thereof, a preparation method thereof, a pharmaceutical composition and medical application thereof, and belongs to the technical field of medicines.

Background

Proteasomes are important components of Ubiquitin-proteasome systems (UPS), which are major degradation pathways for hydrolysis of misfolded and other proteins during protein synthesis, and participate in cell growth, differentiation, DNA replication and repair, cellular metabolism, immune reactions, and other important physiological and biochemical processes. The UPS mainly serves two purposes: one is by breaking down abnormal or damaged proteins to maintain the quality of the cells; secondly, the basic life activity of the cells is controlled by decomposing proteins with specific functions; both of them finally ensure the normal function of tissues and organs. Related studies have shown that proteasomes are associated with a variety of diseases, including cancer, and inhibitors of proteasomes, such as bortezomib, have been approved by the U.S. food and drug administration for marketing for the treatment of multiple myeloma.

In the treatment of multiple myeloma, untreated patients tend to achieve better efficacy initially, but progression and relapse eventually inevitably occur. Bortezomib in combination with the Histone Deacetylase (HDACs) inhibitor panobinostat has been listed as a recommended treatment by the american society for hematology as a treatment for relapsed and refractory multiple myeloma. In addition, a great deal of research shows that histone deacetylase can reverse the drug resistance phenomenon of proteasome inhibitors of tumor cells.

Histone Deacetylases (HDACs) and Histone Acetyltransferases (HAT) maintain the balance of acetylation of Histone and non-Histone in the body, and 18 members of human HDACs family can be divided into Zn according to cellular location and homology difference²⁺Dependent classes I (HDAC1,2,3,8), Class IIa (HDAC4,5,7,9), Class IIb (HDAC6,10), Class IV (HDAC11) and NAD⁺Dependent Class III (SIRT 1-7).

In the research aiming at different types of tumors and other diseases, the combination of a proteasome inhibitor and a histone deacetylase inhibitor shows stronger synergistic action, but adverse factors such as pharmacokinetic mismatching, poor patient compliance, interaction between medicines and the like exist in the combination of multiple medicines. Therefore, the development of histone deacetylase and protease dual-target inhibitors has great significance.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a histone deacetylase, a protease double-target inhibitor, a preparation method and an application thereof.

The invention further provides a pharmaceutical composition and medical application of the compound.

The technical scheme of the invention is as follows:

one, histone deacetylase, protease double target point inhibitor

A histone deacetylase, a protease double-target inhibitor and pharmaceutically acceptable salts or stereoisomers thereof have a structure shown in a formula I:

wherein, P₁Is aryl substituted by N-hydroxycarbamido or N-hydroxycinnamamido, heteroaryl of N-hydroxycarbamido or N-hydroxycinnamamido, aralkyl of N-hydroxycarbamido or N-hydroxycinnamamido, heteroaralkyl of N-hydroxycarbamido or N-hydroxycinnamamido or cycloalkyl of N-hydroxycarbamido or N-hydroxycinnamamido, or N- (2-aminophenyl) carboxamido-substituted aryl, N- (2-aminophenyl) carboxamido-substituted heteroaryl, N- (2-aminophenyl) carboxamido-substituted aralkyl or N- (2-aminophenyl) carboxamido-substituted heteroaralkyl, the above aryl, heteroaryl, aralkyl, heteroaralkyl or cycloalkyl group may be substituted with an optional substituent w;

the substituent w is selected from hydroxyl, halogen, alkyl, trifluoromethyl, cyano, nitro, guanidino, amino, carboxyl, nitro, monocyclic aryl containing 5 or 6 ring atoms or bicyclic aryl containing 8 to 15 ring atoms, and monocyclic heterocyclic aryl containing 1 to 2 heteroatoms and having 5 to 6 ring atoms;

P₂independently hydrogen, alkyl, cycloalkyl, alkaryl, aryl, a 5-to 10-membered saturated or partially saturated heterocycle or heteroaryl, wherein the ring portion of said aryl, aralkyl, alkaryl or heterocycle may be substituted with the above substituent w;

Z₁and Z₂Independently is alkyl, hydroxy, alkoxy or aryloxy, or Z₁And Z₂Together form a group derived from a dihydroxy compound having at least two hydroxyl groups separated by at least two linking atoms in a chain or ring containing carbon atoms and optionally one or more heteroatoms which may be N, S or O.

Preferred according to the invention are those of the formula I:

P₁is meta-or para-substituted N-hydroxyformamide or N-hydroxycinnamic amide substituted aryl;

P₂independently hydrogen, benzyl or 1H-indol-3-methyl;

Z₁and Z₂Independently pinanediol.

According to a further preferred embodiment of the invention, the compounds of the general formula I are of the following structure:

Detailed Description

The terms and definitions used herein have the following meanings:

the "alkyl group" as used herein refers to a straight or branched alkyl group derived from an alkane having 1 to 10 carbon atoms by partially removing one hydrogen atom, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, 3-methylbutyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-dimethylbutyl, 2, 3-dimethylbutyl, 3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, etc, 1,1, 2-trimethylpropyl, 1,2, 2-trimethylpropyl, 1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl, heptyl, octyl, nonyl and decyl;

the "cycloalkyl" refers to a cyclic alkyl group derived from an alkane moiety of 3 to 8 carbon atoms by removing one hydrogen atom, such as cyclopropyl, cyclobutyl, 1-methylcyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. Preferably C_4-7Cycloalkyl radical, C_4-6Cycloalkyl and C_5-6A cycloalkyl group;

examples of such "mono-heterocyclic" are: oxiranyl, dioxiranepropyl, thietanyl, aziridinyl, 2H-aziridinyl, diazacyclopropyl, 3H-diazacyclopropenyl, oxazetank, oxetanyl, 1, 2-dioxetane, thietanyl, 1, 2-dithiocyclobutenyl, azetidinyl, 1, 2-diazacyclobutyl, azetidinyl, 1, 2-diazacyclobutenyl, furyl, tetrahydrofuryl, thienyl, 2, 5-dihydrothienyl, tetrahydrothienyl, pyrrolyl, dihydropyrrolyl, pyrrolidinyl, 1, 3-dioxolanyl, 1, 3-dioxol-2-onyl, 1, 2-dithiolyl, 1, 3-dithiolanyl, Imidazolyl, 4, 5-dihydroimidazolyl, imidazolidinyl, pyrazolyl, 4, 5-dihydropyrazolyl, pyrazolidinyl, oxazolyl, 4, 5-dihydrooxazolyl, isoxazolyl, 4, 5-dihydroisoxazolyl, 2, 3-dihydroisoxazolyl, 1,2, 3-oxadiazolyl, 1,2, 5-oxadiazolyl, thiazolyl, 4, 5-dihydrothiazolyl, isothiazolyl, 1,2, 3-thiadiazolyl, 1,2, 4-thiadiazolyl, 1,3, 4-thiadiazolyl, 1,2, 3-triazolyl, 1,2, 4-triazolyl, tetrazolyl, 2H-pyranyl, 2H-pyran-2-onyl, 3, 4-dihydro-2H-pyranyl, 4H-pyranyl, Tetrahydropyranyl, 4H-pyran-4-keto, pyridinyl, 2-pyridonyl, 4-pyridonyl, piperidinyl, 1, 4-dioxadienyl, 1, 4-dithiadienyl, 1, 4-oxacyclohexadienyl, 1, 4-dioxanyl, 1, 3-oxathianyl, 2H-1, 2-oxazinyl, 4H-1, 2-oxazinyl, 6H-1, 2-oxazinyl, 2H-1, 3-oxazinyl, 4H-1, 3-oxazinyl, 6H-1, 3-oxazinyl, 2H-1, 4-oxazinyl, 4H-1, 4-oxazinyl, 5, 6-dihydro-4H-1, 3-oxazinyl, morpholinyl, 2H-1, 3-thiazinyl, 4H-1, 3-thiazinyl, 5, 6-dihydro-4H-1, 3-thiazinyl, 6H-1, 3-thiazinyl, 2H-1, 4-thiazinyl, 4H-1, 4-thiazinyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl, 1,2, 3-triazinyl, 1,2, 4-triazinyl, 1,3, 5-triazinyl, 1,2,4, 5-tetrazinyl, oxepitrienyl, thiepintrienyl, 1, 4-dioxacyclooctatrienyl, azepintrienyl, 1, 2-diazacycloheptatrienyl, 1, 3-diazacyclotrienyl-trienyl, 1, 3-diazacyclo-trienyl, 1, 4-diazepanyl, azocyclotetraenyl, 1, 4-dihydro-1, 4-diazacyclooctenyl, and the like;

"aryl" means an aromatic ring-containing substituent, such as phenyl or benzyl, optionally fused with a cycloalkyl group, preferably having 4 to 7 ring atoms, more preferably having 5 to 6 ring atoms. Preferred aryl groups contain 5 to 15 carbon atoms;

"heteroaryl" is an aromatic heterocycle, which may be a monocyclic or bicyclic group. They contain an aromatic hetero group containing one or more heteroatoms, preferably 1 to 3 heteroatoms, even more preferably 1 to 2 heteroatoms, independently selected from O, S and N. "arylalkyl" means C₁-C₄An alkylene-linked aryl group;

"aralkyl" means C₁-C₄An alkylene-linked aryl group;

"Heteroaralkyl" means C₁-C₄An alkylene-linked heteroaryl;

the compound shown in the general formula I can be prepared into pharmaceutically acceptable salts by a known method, wherein the salts are prepared by mixing the compound shown in the general formula I with acid or alkali;

suitable acid addition salts are formed from acids which form non-toxic salts. Representative acid addition salts include, but are not limited to, acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, bicarbonate, butyrate, camphorate, camphorsulfonate, carbonate, citrate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, gluconate, glucuronate, glutamate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malate, malonate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, pectate (pectate), persulfate, 3-phenylpropionate, propionate, and mixtures thereof, Picrate (picrate), pivalate), propionate, sucrose, stearate, succinate, sulfate, tartrate, thiocyanate, phosphate, hydrogen phosphate, dihydrogen phosphate, p-toluenesulfonate, trifluoroacetate and undecanoate;

base addition salts can be prepared in situ during the final isolation and purification of the compounds by reacting the carboxylic acid containing moiety with an appropriate base such as, but not limited to, the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia or an organic primary, secondary or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali or alkaline earth metals, such as, but not limited to, lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like, as well as non-toxic quaternary ammonium and amine cations, including ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, and the like. Other representative organic amines useful for forming base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine and the like;

"stereoisomers" in this context means all possible stereoisomeric forms of the compounds of the invention or of their physiological derivatives. Unless otherwise indicated, the chemical designation of compounds referred to in this invention includes mixtures of all possible stereochemical forms, including all enantiomers and diastereomers of the basic structural molecule, as well as the substantially pure individual isomeric forms of the compounds, i.e., containing less than 10%, preferably less than 5%, particularly less than 2%, and most preferably less than 1% of the other isomers. Various stereoisomeric forms of the peptoid compounds of the present invention are expressly included within the scope of the present invention;

the compounds of formula I may also exist in other protected forms or derivatives, which forms are obvious to the skilled person and are intended to be included within the scope of the present invention;

the substituents described above may themselves be substituted by one or more substituents. Such substituents include those listed in c.hansch and a.leo, scientific Constants for Correlation Analysis in Chemistry and Biology (1979); preferred substituents include alkyl, alkenyl, alkoxy, hydroxy, nitro, amino, aminoalkyl, cyano, halogen, carboxy, thio, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, imino, hydroxyalkyl, aryloxy, arylalkyl, and combinations thereof;

the compounds of the present invention can be prepared into any pharmaceutical preparation by methods known in the art, and administered to a patient in need of such treatment by oral, parenteral, rectal or pulmonary administration, and when used for oral administration, the compounds can be prepared into conventional solid preparations such as tablets, capsules, pills, granules, etc., and also can be prepared into oral liquid preparations such as oral solutions, oral suspensions, syrups, etc. When the composition is formulated into oral preparations, appropriate filler, binder, disintegrating agent, lubricant, etc. can be added. For parenteral administration, it can be made into injection, including injection solution, sterile powder for injection and concentrated solution for injection. The injection can be prepared by conventional method in the existing pharmaceutical field, and can be prepared without adding additives or adding appropriate additives according to the properties of the medicine. For rectal administration, it can be made into suppository, etc. For pulmonary administration, it can be made into inhalant or spray;

the amount and frequency of administration of the compounds of the invention may be adjusted at the discretion of the clinician or pharmacist taking into account factors such as: the age, health and size of the patient, and the severity of the condition being treated. Generally, the total daily dose of the compounds of the present invention will range from about 0.1 to about 2000mg per day, although variations will occur if necessary depending on the purpose of the treatment, the patient and the route of administration. In one embodiment, the dose is from about 1 to about 200 mg/day, administered as a single dose or as 2-4 separate doses. In another embodiment, the dose is from about 10 to about 2000 mg/day, administered as a single dose or as 2-4 separate doses. In another embodiment, the dose is from about 100 to about 2000 mg/day, administered as a single dose or as 2-4 separate doses. In yet another embodiment, the dose is from about 500 to about 2000 mg/day, administered as a single dose or as 2-4 separate doses. When the compound of the present invention, its pharmaceutically acceptable salt, ester or solvate, or prodrug or isomer thereof is used in combination with other therapeutically active substances, they are administered simultaneously, separately or sequentially, and a pharmaceutical composition of single administration form can be prepared. The amount of the other therapeutically active substance used in combination may be based on the amount used clinically and may be appropriately selected depending on the administration subject, the administration route, the disease, the combination, and the like. There is no particular limitation on the administration form of the other therapeutically active substance as long as the compound of the present invention and the other therapeutically active substance are combined at the time of administration.

"pharmaceutical composition" refers to a preparation containing a therapeutically significant amount of an active agent, which is prepared in a form suitable for administration to a patient. Thus, the preparation does not contain any component or components in such amounts that a properly cautious medical practitioner finds the preparation unsuitable for administration to an ordinary subject. In many cases, such pharmaceutical compositions are sterile preparations.

The specific temperature range of "room temperature" referred to in the present invention is 20-30 ℃.

Preparation method of di, N- (2-aminophenyl) formamido substituted histone deacetylase and protease double-target inhibitor

Carrying out amide condensation reaction on the bortezomib intermediate I serving as a raw material and amino acid protected by Boc, and then removing a protecting group; on the other hand, substituted diacid monomethyl ester and o-phenylenediamine are subjected to amide condensation reaction, then methyl ester is removed, and amide condensation reaction is carried out on the substituted diacid monomethyl ester and the intermediate to obtain the histone deacetylase with N- (2-aminophenyl) formamido substitution and the protease double-target inhibitor.

The synthetic route is as follows:

wherein, P₂Wherein R is independently any of hydrogen, hydroxy, halogen, alkyl, trifluoromethyl, cyano, nitro, guanidino, amino, carboxyl, nitro, monocyclic aryl having 5 or 6 ring atoms, bicyclic aryl having 8 to 15 ring atoms, and monocyclic heterocyclyl having 1 to 2 heteroatom ring atoms of 5 to 6, and ring A is independently any of aryl, heteroaryl, aralkyl, heteroaralkyl, or cycloalkyl;

reaction reagents and reaction conditions: (a) 2-Boc-glycine substituted in position 2 with P2, TBTU, NMM, room temperature, 4-24 h; (b) hydrogen chloride saturated ethyl acetate at room temperature for 1-24 h; (c) o-phenylenediamine substituted by R at the 3-position or the 4-position, TBTU, NMM, keeping away from light and at room temperature for 8 hours; (d) lithium hydroxide, water/methanol at room temperature for 2-24 h; (e) TBTU, NMM, room temperature, 4-24 h.

The specific operation steps will be described in detail in the examples. The skilled in the art can modify the above steps to increase the yield, and they can design the synthetic route according to the basic knowledge in the art, such as selecting reactants, reaction solvent, reaction temperature, and can also improve the yield by using various Protecting Groups to avoid side reactions, and these conventional Protecting methods can be found in, for example, T.Green Protecting Groups in Organic Synthesis.

Preparation method of histone deacetylase and protease double-target inhibitor substituted by three, N-hydroxyformamide

Carrying out amide condensation reaction on the bortezomib intermediate I serving as a raw material and amino acid protected by Boc, and then removing a protecting group; on the other hand, substituted diacid monomethyl ester and O- (tetrahydro-2H-pyran-2-yl) hydroxylamine are subjected to amide condensation reaction, then methyl ester is removed, and the substituted diacid monomethyl ester and the intermediate are subjected to amide condensation reaction, and finally a tetrahydropyran protecting group is removed, so that the histone deacetylase and protease double-target inhibitor substituted by N-hydroxyformamide are obtained.

The synthetic route is as follows:

wherein P2 is as defined above for formula I, and ring A is independently any one of aryl, heteroaryl, aralkyl, heteroaralkyl or cycloalkyl;

reaction reagents and reaction conditions: (a) 2-Boc-glycine substituted in position 2 with P2, TBTU, NMM, room temperature, 4-24 h; (b) hydrogen chloride saturated ethyl acetate at room temperature for 1-24 h; (c) o- (tetrahydro-2H-pyran-2-yl) hydroxylamine, TBTU, NMM, in dark at room temperature for 4-24H; (d) lithium hydroxide, water/methanol at room temperature for 2-24 h; (e) TBTU, NMM, room temperature, 4-24 h; (f) hydrogen chloride saturated ethyl acetate at room temperature for 1-24 h.

Preparation method of tetra, N-hydroxycinnamic amide substituted histone deacetylase and protease double-target inhibitor

Carrying out amide condensation reaction on the bortezomib intermediate I serving as a raw material and amino acid protected by Boc, and then removing a protecting group; on the other hand, methyl formate with aldehyde group substitution reacts with malonic acid to obtain methyl formate substituted by cinnamic acid, then the methyl formate reacts with O- (tetrahydro-2H-pyran-2-yl) hydroxylamine to carry out amide condensation reaction, methyl ester is removed, the methyl formate reacts with the intermediate to carry out amide condensation reaction, and finally the tetrahydropyran protecting group is removed, so that histone deacetylase and protease double-target inhibitor with N-hydroxycinnamide substitution are obtained.

The synthetic route is as follows:

reaction reagents and reaction conditions: (a)2 bit is P₂Substituted 2-Boc-glycine, TBTU, NMM, at room temperature, 4-24 h; (b) hydrogen chloride saturated ethyl acetate at room temperature for 1-24 h; (c) malonic acid, pyridine and N, N-dimethylformamide are heated and refluxed for 10 to 48 hours; (d) o- (tetrahydro-2H-pyran-2-yl) hydroxylamine, TBTU, NMM, in dark at room temperature for 4-24H; (e) lithium hydroxide, water/methanol at room temperature for 2-24 h; (f) TBTU, NMM, room temperature, 4-24 h; (g) hydrogen chloride saturated ethyl acetate at room temperature for 1-24 h.

Application of histone deacetylase and protease double-target inhibitor

The invention also provides application of the series of compounds in preparing medicaments for preventing or treating related mammal diseases caused by abnormal expression of histone deacetylase or abnormal function of proteasome, wherein the diseases comprise but are not limited to cancers, neurodegenerative diseases, inflammations, viral infections, diabetes and malaria.

In addition, the present invention also includes a pharmaceutical composition suitable for oral administration to a mammal comprising a compound of any of the above general formula I, a pharmaceutically acceptable carrier, optionally comprising one or more pharmaceutically acceptable excipients.

In addition, the present invention also includes a pharmaceutical composition suitable for parenteral administration to a mammal comprising a compound of any of the above general formula I, a pharmaceutically acceptable carrier, optionally comprising one or more pharmaceutically acceptable excipients.

Both aprotinin activity and cellular activity assays were performed to evaluate the biological activity of compounds in vitro.

The histone deacetylase inhibitory activity of the compound is evaluated by a fluorescence analysis method. In the evaluation of the inhibition of HDAC enzymatic activity by compounds in vitro, HDAC fluorogenic substrates (containing an acetylated lysine side chain-Boc-Lys (acetyl) -AMC) were incubated with samples containing HDAC activity (Hela nuclear extract) to deacetylate the substrate and activate the substrate. Boc-Lys-AMC was then hydrolyzed with pancreatin to generate AMC fluorophore and fluorescence intensity was measured at emission/excitation wavelengths (390nm/460 nm). The fluorescence intensity is proportional to the HDACs inhibition effect of the compound to be detected, and the Prism GraphPad software is used for processing data to calculate the inhibition rate and IC of different target compounds on the HDACs₅₀The value is obtained.

The compounds were evaluated for proteasome inhibitory activity using fluorescence analysis. In evaluating compounds for inhibition of proteasome activity in vitro, proteasome fluorescent substrates (Suc-LLVY-AMC) are incubated with proteasome to hydrolyze the substrates, release AMC fluorophores, and the fluorescence intensity is measured at the emission/excitation wavelength (390nm/460 nm). The fluorescence intensity is proportional to the proteasome inhibition effect of the compound to be detected, and the inhibition rate and IC of different target compounds on proteasomes are calculated by processing data with Prism GraphPad software₅₀The value is obtained.

The cell activity of the compound is tested by using MTT detection method, tumor cell suspension (human chronic myelogenous leukemia cell K562, human granulocyte leukemia cell KG1, human promyelocytic leukemia cell HL-60, acute T cell leukemia cell Jurkat, human multiple myeloma cell RPMI-8226, human colon cancer cell HCT-116 and prostate cancer cell line PC-3 are respectively inoculated in a 96-well plate, compounds with different concentrations diluted by culture medium are added into each well, MTT staining is carried out after 48 hours of incubation, the absorbance OD value of each well is measured at 490/570nm by a microplate reader after continuous 4 hours of incubation, and then the inhibition rate and IC are calculated₅₀And thereby determining the antiproliferative activity of the compound of interest.

The results of in vitro enzyme inhibition experiments show that the compound has better HDAC and proteasome inhibition activities. In-vitro anti-tumor cell proliferation experiments show that the inhibition activity of five compounds of ZY-2, ZY-11, ZY-12, ZY-13 and ZY-14 on various tumor cells is three times higher than that of a positive control drug SAHA, and the inhibition activity of ZY-2 and ZY-13 on bortezomib-resistant multiple myeloma cells KM3/BTZ is far beyond that of the positive drug. Therefore, part of the compounds in the invention have good HDACs inhibitory activity, proteasome inhibitory activity and anti-tumor cell proliferation activity, have a great development prospect, and can be used for guiding the discovery of novel efficient histone deacetylase, protease double-target inhibitors and anti-tumor active molecules.

The specific implementation mode is as follows:

the present invention will be further described with reference to the following examples, but is not limited thereto.

Example 1: tert-butyl ((S) -1- ((((R) -3-methyl-1- ((3aR,4R,6R,7aS) -5,5,7 a-trimethylhexahydro-4, 6-methylbenzo [ d ] [1,3,2] dioxaborolan-2-yl) butyl) amino) -1-oxo-3-phenylpropan-2-yl) carbamate (2a)

Boc-L-Phe (3.6g,13mmol) was dissolved in N, N-dimethylformamide under ice bath and TBTU (4.2g,13mmol), bortezomib intermediate I (4.56g,12mmol) and N-methylmorpholine (4ml,36mmol) were added. The reaction was stirred for 16 hours under nitrogen, poured into water and extracted 3 times with ethyl acetate (3X 100 mL). The combined organic phases were washed with 2% citric acid, 2% sodium bicarbonate, saturated brine, dried over magnesium sulfate and spin-dried to give a crude product. Chromatography on silica gel eluting with a gradient of ethyl acetate and petroleum ether (from 15% to 40%) afforded 6g (97%) of a colourless, clear oil 2 a. Yield: 95 percent.¹H NMR(400MHz,DMSO-d₆):δ8.85(brs,2H),7.35–7.07(m,5H),7.03(d,J＝8.3Hz,1H),4.28–4.18(m,1H),4.08(d,J＝7.1Hz,1H),2.91–2.72(m,3H),2.26–2.11(m,1H),2.05–1.98(m,1H),1.87–1.73(m,2H),1.68–1.49(m,2H),1.31–1.18(m,17H),0.81–0.80(m,9H).

Tert-butyl (2- ((((R) -3-methyl-1- ((3aR,4R,6R,7aS) -5,5,7 a-trimethylhexahydro-4, 6-methylbenzo [ d ] [1,3,2] dioxaborol-2-yl) butyl) amino) -2-oxyethyl) carbamate (2b)

Analogous to the synthesis of 2a, yield: 99 percent. A colorless oil.¹H NMR(400MHz,CDCl₃)δ6.51(s,1H),5.27(s,1H),4.33–4.26(m,1H),3.17(d,J＝5.5Hz,1H),2.80(d,J＝6.4Hz,1H),2.37–2.28(m,1H),2.19(ddd,J＝10.6,6.1,3.2Hz,1H),2.03(dd,J＝9.6,4.1Hz,2H),1.93–1.87(m,1H),1.87–1.80(m,1H),1.70–1.56(m,2H),1.48–1.43(m,10H),1.40(s,3H),1.32–1.23(m,5H),0.91–0.84(m,9H).

Tert-butyl ((S) -3- (1H-indol-3-yl) -1- (((R) -3-methyl-1- ((3aR,4R,6R,7aS) -5,5,7 a-trimethylhexahydro-4, 6-methoxybenzo [ d ] [1,3,2] dioxaborolan-2-yl) butyl) amino) -1-oxopropan-2-yl) carbamate (2c)

Analogous to the synthesis of 2a, yield: 85 percent. Brown powder, melting point: 174 to 178 ℃.¹H NMR(400MHz,DMSO-d₆)δ10.87(s,1H),9.05(d,J＝38.1Hz,1H),7.60(d,J＝8.0Hz,1H),7.33(d,J＝8.0Hz,1H),7.18(d,J＝14.4Hz,1H),7.06(t,J＝7.4Hz,1H),6.99(t,J＝7.4Hz,1H),6.91(d,J＝8.2Hz,1H),4.31(dd,J＝12.6,6.3Hz,1H),4.09(d,J＝8.4Hz,1H),3.00(qd,J＝14.7,7.2Hz,2H),2.22(p,J＝11.6Hz,1H),2.03(dt,J＝11.3,6.5Hz,1H),1.83(dt,J＝17.1,5.7Hz,2H),1.73–1.57(m,2H),1.38–1.18(m,18H),0.83(d,J＝6.5Hz,9H).

Example 2: (S) -1- ((((R) -3-methyl-1- ((3aR,4R,6R,7aS) -5,5,7 a-trimethylhexahydro-4, 6-methoxybenzo [ d ] [1,3,2] dioxaborol-2-yl) butyl) amino) -1-oxo-3-phenylpropane-2-aminium chloride (3a)

2a in ethyl acetate saturated with hydrogen chloride and stirred overnight to give pure white powder 3a, yield: 90%, melting point: 208 to 212 ℃.¹H NMR(400MHz,DMSO-d₆)δ8.55(s,1H),8.29(s,3H),7.38–7.20(m,5H),4.29(dd,J＝7.7,2.2Hz,1H),3.99(s,1H),3.00(d,J＝8.1Hz,2H),2.88(d,J＝5.0Hz,1H),2.35–2.24(m,1H),2.12(dd,J＝10.5,5.8Hz,1H),1.94(t,J＝5.5Hz,1H),1.85(s,1H),1.71(dt,J＝14.3,2.7Hz,1H),1.47(t,J＝6.5Hz,1H),1.36–1.15(m,9H),0.81–0.80(m,9H).

2- ((((R) -3-methyl-1- ((3aR,4R,6R,7aS) -5,5,7 a-trimethylhexahydro-4, 6-methoxybenzo [ d ] [1,3,2] dioxaborol-2-yl) butyl) amino) -2-oxa-1-aminium chloride (3b)

Synthesis of and 3aSimilarly, yield: 98% colorless oil.¹H NMR(400MHz,DMSO-d₆)δ8.48(d,J＝5.2Hz,1H),8.10(s,3H),4.29(d,J＝7.2Hz,1H),3.54(s,2H),3.05–2.99(m,1H),2.32–2.25(m,1H),2.14(dd,J＝10.4,5.8Hz,1H),1.95(t,J＝5.5Hz,1H),1.85(s,1H),1.49–1.38(m,1H),1.35–1.16(m,9H),0.85–0.83(m,9H).

(S) -3- (1H-indol-3-yl) -1- (((R) -3-methyl-1- (((3aR, 4R,6R,7aS) -5,5,7 a-trimethylhexahydro-4, 6-methylbenzo [ d ] [1,3,2] dioxaborolan-2-yl) butyl) amino) -1-oxopropane-2-ammonium chloride (3c)

Analogous to the synthesis of 3a, yield: 70%, brown powder, melting point: 130-134 ℃.¹H NMR(400MHz,DMSO)δ11.04(s,1H),8.62(d,J＝4.7Hz,1H),8.21(s,3H),7.71(d,J＝7.8Hz,1H),7.37(d,J＝8.0Hz,1H),7.22(d,J＝1.9Hz,1H),7.06(dt,J＝31.7,7.1Hz,2H),4.30(d,J＝7.2Hz,1H),3.97(d,J＝5.1Hz,1H),3.14(ddd,J＝36.0,14.6,7.0Hz,2H),2.98–2.90(m,1H),2.30(dd,J＝12.7,10.3Hz,1H),2.12(dd,J＝10.0,5.4Hz,1H),1.94(dd,J＝12.4,6.8Hz,1H),1.85(s,1H),1.72(d,J＝14.2Hz,1H),1.61–1.49(m,1H),1.38–1.19(m,9H),0.90–0.76(m,9H).

Example 3: 4- ((2-aminophenyl) carbamoyl) benzoic acid methyl ester (5a)

Monomethyl terephthalate (4a,1.8g,10mmol), o-phenylenediamine (1.08g,10mmol), TBTU (3.5g,12mmol) were dissolved in N, N-dimethylformamide on ice. Et was added₃After N (1.5ml,30mmol), the mixture was stirred for 8 hours in the dark. After the reaction, the mixture was poured into water and extracted 3 times with ethyl acetate (3X 100 mL). The combined organic phases were washed with 2% citric acid, 2% sodium bicarbonate, saturated brine, dried over magnesium sulfate and spin-dried to give a crude product. The methanol is recrystallized to obtain a refined product 5 a. Yield: 56%, melting point: 190-194 ℃.¹H NMR(400MHz,CDCl₃)δ8.16(d,J＝7.9Hz,2H),7.99(dd,J＝15.1,8.3Hz,2H),7.37(d,J＝7.9Hz,1H),7.12(dd,J＝8.7,5.3Hz,1H),6.87(t,J＝6.3Hz,2H),3.97(s,3H),3.86(s,1H).

3- ((2-aminophenyl) carbamoyl) benzoic acid methyl ester (5b)

Analogous to the synthesis of 3a, yield: 56%, yellow solid, melting point: 184-186℃。¹H NMR(400MHz,DMSO)δ9.89(s,1H),8.57(s,1H),8.27(d,J＝7.8Hz,1H),8.15(d,J＝7.8Hz,1H),7.68(t,J＝7.8Hz,1H),7.16(d,J＝7.4Hz,1H),7.03–6.95(m,1H),6.79(dd,J＝8.0,1.2Hz,1H),6.61(dd,J＝10.9,4.0Hz,1H),4.95(s,2H),3.91(s,3H).

Example 4: 4- ((2-aminophenyl) carbamoyl) benzoic acid (6a)

Dissolving 5a (1.52g,5mmol) and lithium hydroxide (0.94g,20mmol) in a mixed solution of water (20mL) and methanol (40mL), stirring for 8 hours, removing methanol by spinning, adjusting the pH to 7 with hydrochloric acid, extracting with ethyl acetate (3 × 20mL), drying with magnesium sulfate, and spinning to obtain the product 6a, wherein the yield is 75%, and the melting point is 210-212 ℃.¹H NMR(400MHz,DMSO)δ9.82(s,1H),8.07(q,J＝8.4Hz,4H),7.17(d,J＝7.1Hz,1H),7.01–6.94(m,1H),6.78(dd,J＝8.0,1.1Hz,1H),6.60(t,J＝7.5Hz,1H),5.05(s,2H).

3- ((2-aminophenyl) carbamoyl) benzoic acid (6b)

Analogous to the synthesis of 6a, yield: 86%, yellow solid, melting point: 200 to 204 ℃.¹H NMR(400MHz,DMSO)δ9.87(s,1H),8.54(s,1H),8.20(d,J＝7.7Hz,1H),8.12(d,J＝7.8Hz,1H),7.63(t,J＝7.7Hz,1H),7.16(d,J＝7.3Hz,1H),7.02–6.93(m,1H),6.78(dd,J＝8.0,1.2Hz,1H),6.64–6.54(m,1H),5.02(s,2H).

Example 5: n1- (2-aminophenyl) -N4- ((S) -1- ((((R) -3-methyl-1- ((3aR,4R,6R,7aS) -5,5,7 a-trimethylhexahydro-4, 6-methylbenzo [ d ] [1,3,2] dioxaborolan-2-yl) butyl) amino) -1-oxo-3-phenylpropan-2-yl) terephthalamide (ZY-2)

3a (0.9g,2mmol), 6a (0.51g,2mmol) and TBTU (0.8g,2.2mmol) were dissolved in N, N-dimethylformamide under ice bath, followed by addition of N-methylmorpholine (0.7ml,6 mmol). Stirring for 8 hours under nitrogen protection, pouring into water, extracting with ethyl acetate (3X 20mL), mixing the two signs, washing with 5% citric acid, saturated sodium bicarbonate and saturated saline solution once respectively, drying with anhydrous magnesium sulfate, and spin-drying to obtain the product. Purification by silica gel column chromatography (ethyl acetate: petroleum ether ═ 1:1) afforded ZY solid ZY-2, yield 75%, melting point: 160-162 ℃.¹H NMR(400MHz,DMSO-d₆)δ9.76(s,1H),9.03(d,J＝3.0Hz,1H),8.87(d,J＝8.4Hz,1H),8.01(t,J＝13.4Hz,2H),7.90(d,J＝8.3Hz,2H),7.34(d,J＝7.2Hz,2H),7.26(dd,J＝13.6,6.0Hz,2H),7.22–7.13(m,2H),7.01–6.93(m,1H),6.78(d,J＝6.9Hz,1H),6.60(t,J＝7.5Hz,1H),4.94(s,1H),4.90–4.80(m,1H),4.12(d,J＝6.8Hz,1H),3.08(dd,J＝12.2,8.6Hz,2H),2.60(t,J＝6.0Hz,1H),2.22(dd,J＝12.5,10.0Hz,1H),2.07–2.00(m,1H),1.83(dd,J＝16.2,10.5Hz,2H),1.62(td,J＝14.2,9.2Hz,2H),1.36–1.19(m,10H),0.85(m,10H)；¹³C NMR(101MHz,DMSO-d₆)δ174.04,166.11,165.16,143.73,138.08,137.55,136.70,129.71,128.58,128.13,127.81,127.27,127.13,126.88,123.45,116.65,116.54,83.54,76.33,53.20,52.14,38.15,37.61,36.52,29.34,27.58,26.40,25.30,24.38,23.51,22.62.HRMS(ESI⁺):m/z calculated for C₃₈H₄₈BN₄O₅651.3719,found[M+H]⁺651.3745.

N1- ((S) -3- (1H-indol-3-yl) -1- (((R) -3-methyl-1- ((3aR,4R,6R,7aS) -5,5,7 a-trimethylhexahydro-4, 6-methoxybenzo [ d ] [1,3,2] dioxaboro-2- (butyl) amino) -1-oxopropan-2-yl) -N4- (2-aminophenyl) terephthalamide (ZY-1)

The synthesis is similar to ZY-2, yield: 68%, yellow solid, melting point: 164-166 ℃.¹H NMR(400MHz,DMSO-d₆)δ10.89–10.80(m,1H),10.29–10.00(m,1H),9.76(d,J＝11.2Hz,1H),9.20–9.07(m,1H),8.74(d,J＝8.2Hz,1H),8.04(dt,J＝18.4,9.5Hz,3H),7.91(d,J＝8.1Hz,2H),7.69(d,J＝8.0Hz,1H),7.38–6.95(m,7H),6.78(dd,J＝8.0,1.5Hz,1H),6.60(t,J＝7.4Hz,1H),4.88(dd,J＝19.0,11.3Hz,3H),4.12(dd,J＝8.6,2.1Hz,1H),3.21(d,J＝7.5Hz,2H),2.59(s,1H),2.22(t,J＝11.0Hz,1H),2.02(t,J＝12.2Hz,1H),1.83(dt,J＝25.3,6.9Hz,2H),1.65(dd,J＝13.5,7.2Hz,2H),1.40–1.15(m,9H),0.94–0.70(m,9H).¹³C NMR(101MHz,DMSO)δ174.91,166.15,165.14,143.77,137.52,136.68,136.51,132.00,129.14,128.11,127.86,124.49,123.42,121.42,118.88,116.65,116.54,111.84,110.07,83.20,76.16,65.50,60.24,52.40,52.25,38.14,36.72,30.48,29.47,27.84,27.63,26.44,25.38,24.43,23.50,22.75,21.24,19.13,14.56,14.03.HRMS(ESI⁺):m/z calculated for C₄₀H₄₈BN₅O₅690.3828,found[M+H]⁺690.3867.

N1- ((S) -3- (1H-indol-3-yl) -1- (((R) -3-methyl-1- ((3aR,4R,6R,7aS) -5,5,7 a-trimethylhexahydro-4, 6-methylenebenzo [ d ] [1,3,2] dioxaboro-2- (butyl) amino) -1-oxopropan-2-yl) -N3- (2-aminophenyl) isophthalamide (ZY-3)

The synthesis is similar to ZY-2, yield: 56%, yellow solid, melting point: 158-160 ℃.¹H NMR(400MHz,DMSO-d₆)δ10.85(d,J＝2.5Hz,1H),9.75(s,1H),9.19(t,J＝3.3Hz,1H),8.77(d,J＝8.2Hz,1H),8.41(t,J＝1.9Hz,1H),8.11(d,J＝7.9Hz,1H),7.99(dt,J＝7.8,1.4Hz,1H),7.68(t,J＝7.3Hz,1H),7.65–7.54(m,1H),7.36–7.13(m,3H),7.11–6.88(m,3H),6.79(dd,J＝8.0,1.4Hz,1H),6.68–6.53(m,1H),5.08–4.75(m,3H),4.11(dd,J＝8.6,2.2Hz,1H),3.21(d,J＝7.2Hz,2H),2.66–2.52(m,1H),2.27–2.16(m,1H),2.10–1.99(m,1H),1.82(dt,J＝21.5,4.9Hz,2H),1.71–1.54(m,2H),1.43–1.09(m,9H),0.94–0.56(m,9H).¹³C NMR(101MHz,DMSO)δ174.89,166.31,165.42,143.64,136.52,135.31,134.46,130.98,128.70,127.61,127.17,124.46,123.56,121.41,118.87,118.80,116.69,116.58,111.82,110.05,83.24,76.17,52.23,38.14,36.71,31.44,29.47,27.62,26.44,25.37,24.43,23.50,23.47,22.74,14.45.HRMS(ESI⁺):m/z calculated for C₄₀H₄₈BN₅O₅ 690.3828,found[M+H]⁺690.3834

N1- (2-aminophenyl) -N3- ((S) -1- ((((R) -3-methyl-1- ((3aR,4R,6R,7aS) -5,5,7 a-trimethylhexahydro-4, 6-methylbenzo [ d ] [1,3,2] dioxaborolan-2-yl) butyl) amino) -1-oxo-3-phenylpropan-2-yl) isophthalamide (ZY-4)

The synthesis is similar to ZY-2, yield: 65%, yellow solid, melting point: 160-163 ℃.¹H NMR(400MHz,DMSO-d₆)δ9.73(s,1H),8.94(d,J＝3.2Hz,1H),8.80(d,J＝8.4Hz,1H),8.38(s,1H),8.10(d,J＝7.7Hz,1H),7.97(d,J＝7.9Hz,1H),7.61–7.56(m,1H),7.33(d,J＝7.2Hz,2H),7.26(t,J＝7.5Hz,2H),7.21–7.14(m,2H),7.03–6.95(m,1H),6.79(dd,J＝8.0,1.2Hz,1H),6.61(t,J＝7.5Hz,1H),4.99–4.75(m,3H),4.12(dd,J＝8.5,1.8Hz,1H),3.06(h,J＝9.4,8.7Hz,2H),2.65–2.57(m,1H),2.26–2.16(m,1H),2.03(dt,J＝10.3,6.2Hz,1H),1.88–1.74(m,2H),1.62(td,J＝13.7,7.2Hz,2H),1.34–1.19(m,9H),0.92–0.67(m,9H).¹³C NMR(101MHz,DMSO)δ174.00,166.29,165.44,143.61,138.04,135.33,134.51,130.95,130.64,129.70,128.71,128.61,128.53,127.53,127.13,127.06,126.88,123.59,116.72,116.61,83.60,76.36,53.22,52.11,38.14,37.71,36.50,29.34,27.57,26.40,25.29,24.37,23.52,22.60.HRMS(ESI⁺):m/z calculated for C₃₈H₄₇BN₄O₅ 651.3719,found[M+H]⁺651.3586.

N1- (2-aminophenyl) -N3- (2- ((((R) -3-methyl-1- (((3aR, 4R,6R,7aS) -5,5,7 a-trimethylhexahydro-4, 6-methylbenzo [ d ] [1,3,2] dioxaboron-2-yl) butyl) amino) -2-oxyethyl) isophthalamide (ZY-5)

The synthesis is similar to ZY-2, yield: 72%, yellow solid, melting point: 170-172 ℃.¹H NMR(400MHz,DMSO-d₆)δ10.18(d,J＝7.7Hz,1H),9.78(s,1H),9.07–8.95(m,2H),8.49(s,1H),8.26–8.01(m,3H),7.73–7.57(m,2H),7.37–7.27(m,1H),7.18(dd,J＝8.0,1.5Hz,1H),6.98(ddd,J＝8.4,7.3,1.5Hz,1H),6.79(dd,J＝8.0,1.5Hz,1H),6.61(td,J＝7.5,1.5Hz,1H),4.94(s,2H),4.08–4.03(m,2H),2.58(s,1H),2.24–2.15(m,1H),2.06–1.96(m,1H),1.87–1.81(m,1H),1.78(dq,J＝5.7,2.8Hz,1H),1.74–1.66(m,1H),1.62(dt,J＝13.8,2.8Hz,1H),1.36–1.17(m,10H),0.89–0.77(m,10H).¹³C NMR(101MHz,DMSO)δ172.65,166.59,165.49,135.15,134.40,131.08,130.79,128.85,127.63,127.23,127.13,117.78,83.21,76.05,60.23,52.21,41.02,38.14,36.70,29.50,28.95,27.62,26.41,25.33,24.42,23.39,23.36,22.86,21.24,14.56.HRMS(ESI⁺):m/z calculated for C₃₁H₄₁BN₄O₅ 561.3248,found[M+H]⁺561.3120

N1- (2-aminophenyl) -N4- (2- (((R) -3-methyl-1- (((3aR, 4R,6R,7aS) -5,5,7 a-trimethylhexahydro-4, 6-methylbenzo [ d ] [1,3,2] dioxaborolan-2-yl) butyl) amino) -2-oxyethyl) terephthalamide (ZY-6)

The synthesis is similar to ZY-2, yield: 46% yellow solidMelting point: 138-140 ℃.¹H NMR(400MHz,DMSO-d₆)δ9.79(s,1H),9.10–8.98(m,2H),8.08(d,J＝8.3Hz,2H),8.00(d,J＝8.4Hz,2H),7.17(d,J＝7.2Hz,1H),7.02–6.95(m,1H),6.79(dd,J＝8.0,1.1Hz,1H),6.60(t,J＝7.1Hz,1H),4.95(s,2H),4.06(dd,J＝5.8,3.2Hz,3H),2.56(t,J＝6.3Hz,1H),2.24–2.16(m,1H),2.04–1.98(m,1H),1.86–1.76(m,2H),1.75–1.68(m,1H),1.63(d,J＝13.8Hz,1H),1.36–1.20(m,9H),0.88–0.78(m,9H).¹³C NMR(101MHz,DMSO)δ172.57,166.45,165.16,143.74,137.60,137.02,136.62,128.22,127.85,127.28,127.13,123.47,116.66,116.56,83.26,76.10,65.39,52.22,41.01,38.15,36.67,36.25,29.48,27.63,26.41,25.34,24.41,24.12,23.39,22.86,15.64.HRMS(ESI⁺):m/z calculated for C₃₁H₄₁BN₄O₅ 561.3248,found[M+H]⁺561.3063.

Example 6: 4- ((((tetrahydro-2H-pyran-2-yl) oxy) carbamoyl) benzoic acid methyl ester (8a)

O- (tetrahydro-2H-pyran-2-yl) hydroxylamine (0.32g,3mmol), 4- (methoxycarbonyl) benzoic acid (0.49g,3mmol) and TBTU (1.3g,3.3mmol) were dissolved in N, N-dimethylformamide under ice bath, followed by addition of Et₃N (1mL,9mmol), stirring under nitrogen for 8 hours, pouring into water, extracting 3 times with ethyl acetate (3X 20mL), combining the organic phases, adding 2% citric acid, saturated NaHCO₃Washing the solution with saturated salt water, drying with anhydrous magnesium sulfate, and spin-drying to obtain a crude product. Dissolving diethyl ether, adding n-hexane for crystallization to obtain a white square crystal pure product 8a, wherein the yield is as follows: 68%, melting point: 190-194 ℃.¹H NMR(400MHz,DMSO)δ11.87(s,1H),8.05(d,J＝8.3Hz,2H),7.89(d,J＝8.3Hz,2H),5.02(s,1H),4.05(d,J＝11.3Hz,1H),3.88(s,3H),3.53(d,J＝11.1Hz,1H),2.89(s,1H),1.73(s,3H),1.55(s,3H).

Methyl 3- ((((tetrahydro-2H-pyran-2-yl) oxy) carbamoyl) benzoate (8b)

Synthesis analogous to 8a, yield: 85%, white cristobalite, melting point: 186-188 ℃.¹H NMR(400MHz,DMSO-d₆)δ11.89(s,1H),8.36(t,J＝1.7Hz,1H),8.13(dt,J＝7.8,1.4Hz,1H),8.04(dt,J＝7.9,1.4Hz,1H),7.65(t,J＝7.8Hz,1H),5.03(t,J＝2.7Hz,1H),4.07(td,J＝12.3,10.0,6.1Hz,1H),3.90(s,3H),3.58–3.52(m,1H),1.73(dt,J＝7.0,3.5Hz,3H),1.56(dq,J＝8.5,4.7Hz,3H).

Example 7: 4- (((tetrahydro-2H-pyran-2-yl) oxy) carbamoyl) benzoic acid (9a)

8a (0.8g,3mmol) and lithium hydroxide (0.48g,9mmol) were dissolved in a mixed solution of water (10mL) and methanol (20mL), stirred for 8 hours, the methanol was removed by spinning, the pH was adjusted to 4, and then extracted with ethyl acetate, dried over anhydrous magnesium sulfate, and spin-dried to obtain white solid 9a, yield: 68% melting point>250℃。¹H NMR(400MHz,DMSO)δ13.27(s,1H),11.83(s,1H),8.02(d,J＝8.3Hz,2H),7.86(d,J＝8.2Hz,2H),5.01(s,1H),4.06(s,1H),3.53(d,J＝11.2Hz,1H),1.73(s,3H),1.55(s,3H).

3- (((tetrahydro-2H-pyran-2-yl) oxy) carbamoyl) benzoic acid (9b)

Suitably similar to 9a, yield: 83%, white solid, melting point: 242 to 244 ℃.¹H NMR(400MHz,DMSO-d₆)δ13.26(s,1H),11.85(s,1H),8.38–8.32(m,1H),8.10(dt,J＝7.8,1.5Hz,1H),8.01(dt,J＝7.8,1.6Hz,1H),7.62(t,J＝7.8Hz,1H),5.02(d,J＝2.6Hz,1H),4.06(t,J＝9.6Hz,1H),3.54(dt,J＝11.2,3.6Hz,1H),1.73(h,J＝3.4,2.6Hz,3H),1.56(q,J＝6.1,5.0Hz,3H).

Example 8: n1- ((S) -3- (1H-indol-3-yl) -1- (((R) -3-methyl-1- ((3aR,4R,6R,7aS) -5,5,7 a-trimethylhexahydro-4, 6-methylbenzo [ d ] [1,3,2] dioxaboron-2-yl) butyl) amino) -1-oxopropan-2-yl) -N4-hydroxyterephthalamide (ZY-7)

3(0.9g,3mmol), 6(0.8g,3mmol) and TBTU (0.8g,3.6mmol) were dissolved in N, N-dimethylformamide under ice bath, NMM (4mL,36mmol) was added and stirred under nitrogen for 8 hours, poured into water, extracted 3 times with ethyl acetate (3X 20mL), the organic phases were combined, 2% citric acid, saturated NaHCO₃The solution was washed with saturated brine, dried over anhydrous magnesium sulfate, and spin-dried to obtain an intermediate. Dissolving the intermediate in ethyl acetate of saturated hydrogen chloride, stirring for 5 minutes, filtering, and purifying by silica gel column chromatography to obtain a white solid ZY-7, wherein the yield is as follows: 25%, melting point: 180-184 ℃.¹H NMR(400MHz,DMSO-d₆)δ13.23(s,1H),11.33(d,J＝1.8Hz,1H),10.84(d,J＝2.4Hz,1H),9.15(dd,J＝12.5,2.6Hz,3H),8.72(d,J＝8.2Hz,1H),8.02–7.64(m,8H),7.35–6.92(m,6H),5.76(s,1H),4.86(d,J＝7.4Hz,1H),4.10(dd,J＝8.6,2.2Hz,1H),3.18(dd,J＝7.9,6.2Hz,3H),2.57(d,J＝5.6Hz,1H),2.25–2.18(m,1H),2.12–1.93(m,3H),1.84(d,J＝5.8Hz,3H),1.65(d,J＝13.7Hz,2H),1.36(d,J＝10.0Hz,1H),1.30–1.19(m,9H),0.87–0.80(m,9H).¹³C NMR(101MHz,DMSO)δ166.15,163.87,136.51,129.79,128.00,127.61,127.20,124.45,121.41,118.79,110.06,83.18,76.14,52.25,38.14,36.71,29.46,27.63,25.37,24.43,23.47,22.77.HRMS(ESI^-):m/z calculated for C₃₄H₄₃BN₄O₆ 613.3317,found[M-H]^-613.3321.

N1-hydroxy-N4- ((S) -1- (((R) -3-methyl-1- (((3aR, 4R,6R,7aS) -5,5,7, a-trimethylhexahydro-4, 6-methylbenzo [ d ] [1,3,2] dioxaborolan-2-yl) butyl) amino) -1-oxo-3-phenylpropan-2-yl) terephthalamide (ZY-8)

Synthesis analogous to ZY-7, yield: 30%, white solid, melting point: 200 to 202 ℃.¹H NMR(400MHz,DMSO-d₆)δ11.32(d,J＝1.8Hz,1H),9.13(d,J＝1.8Hz,1H),8.97(d,J＝3.3Hz,1H),8.80(d,J＝8.4Hz,1H),7.89–7.71(m,4H),7.37–7.30(m,2H),7.26(t,J＝7.6Hz,2H),7.21–7.14(m,1H),4.82(td,J＝9.1,5.3Hz,1H),4.12(dd,J＝8.7,2.1Hz,1H),3.05(qd,J＝13.4,7.3Hz,2H),2.60(d,J＝3.2Hz,1H),2.27–2.17(m,1H),2.06–1.99(m,1H),1.85–1.79(m,2H),1.64(dt,J＝14.0,2.8Hz,2H),1.29–1.17(m,9H),1.14(s,1H),0.89–0.79(m,9H).¹³C NMR(101MHz,DMSO)δ174.08,166.11,163.90,138.06,136.60,135.70,129.67,128.59,127.95,127.22,126.88,83.47,76.27,72.70,68.18,54.19,53.14,52.12,38.14,37.55,36.54,30.38,29.34,28.44,28.38,27.58,26.39,25.29,24.49,24.39,23.50,22.63.HRMS(ESI^-):m/z calculated for C₃₂H₄₂BN₃O₆ 574.3208,found[M-H]^-574.3215.

N1- ((S) -3- (1H-indol-3-yl) -1- (((R) -3-methyl-1- ((3aR,4R,6R,7aS) -5,5,7 a-trimethylhexahydro-4, 6-methylbenzo [ d ] [1,3,2] dioxaboro-2- (butyl) amino) -1-oxopropan-2-yl) -N3-hydroxyisophthalamide (ZY-9)

Synthesis analogous to ZY-7, yield: 33%, purple solid, melting point: 188-190 ℃.¹H NMR(400MHz,DMSO-d₆)δ10.86(s,1H),9.01(d,J＝3.3Hz,1H),8.76(d,J＝8.4Hz,1H),7.82(d,J＝7.8Hz,2H),7.63(d,J＝8.1Hz,2H),7.47(d,J＝15.8Hz,1H),7.35–7.21(m,5H),7.17(t,J＝7.2Hz,1H),6.56(d,J＝15.8Hz,1H),4.82(td,J＝8.9,5.6Hz,1H),4.13–4.07(m,1H),3.06(dq,J＝11.1,6.2,4.3Hz,2H),2.63–2.54(m,1H),2.21(dd,J＝13.7,8.8Hz,1H),2.02(dd,J＝10.4,6.1Hz,1H),1.85–1.75(m,2H),1.62(t,J＝10.4Hz,2H),1.23(d,J＝10.8Hz,9H),0.85–0.79(m,9H).¹³C NMR(101MHz,DMSO)δ174.26,166.20,138.10,137.69,134.77,129.69,128.59,128.51,127.70,126.88,121.33,83.41,76.24,53.11,52.13,40.72,39.36,38.12,37.55,36.56,29.35,27.58,26.40,25.29,24.39,23.50,22.63.HRMS(ESI^-):m/z calculated for C₃₄H₄₃BN₄O₆ 509.2817,found[M-H]^-509.2811.

N1-hydroxy-N3- ((S) -1- (((R) -3-methyl-1- (((3aR, 4R,6R,7aS) -5,5,7, a-trimethylhexahydro-4, 6-methylbenzo [ d ] [1,3,2] dioxaborolan-2-yl) butyl) amino) -1-oxo-3-phenylpropan-2-yl) isophthalamide (ZY-10)

Synthesis analogous to ZY-7, yield: 45%, white solid, melting point: 160-164 ℃.¹H NMR(400MHz,DMSO-d₆)δ11.26(s,1H),9.11(s,1H),8.95(t,J＝3.6Hz,1H),8.79(d,J＝8.4Hz,1H),8.20(t,J＝1.8Hz,1H),7.88(ddt,J＝26.0,7.8,1.4Hz,2H),7.52(t,J＝7.8Hz,1H),7.37–7.14(m,5H),4.84(td,J＝8.9,5.6Hz,1H),4.12(dd,J＝8.6,2.1Hz,1H),3.14–2.98(m,2H),2.60(ddd,J＝8.7,6.7,3.0Hz,1H),2.22(ddd,J＝16.4,7.6,5.1Hz,1H),2.09–1.96(m,1H),1.89–1.75(m,2H),1.69–1.52(m,2H),1.31–1.22(m,9H),1.02(d,J＝6.1Hz,1H),0.87–0.74(m,9H).¹³C NMR(101MHz,DMSO)δ136.50,128.54,124.47,121.41,118.79,111.83,110.06,76.09,52.26,39.31,38.13,36.74,29.47,27.62,26.42,25.37,24.43,23.48,22.76.HRMS(ESI^-):m/z calculated for C₃₂H₄₂BN₃O₆574.3208,found[M-H]^-574.3203.

Example 9: (E) -3- (4- (methoxycarbonyl) phenyl) acrylic acid (11a)

A mixture of methyl p-aldehyde benzoate (0.42g,0.5mmol), malonic acid (0.94g,1.5mmol), pyridine (0.2mL) and N, N-dimethylformamide (10mL) was heated to 90 ℃ under nitrogen blanket and stirred for 10 hours, after the reaction was complete, quenched with 60mL water, and the solid was filtered under ice bath to give the product as white crystals 11 a. Yield: 95%, melting point: 175 to 179 ℃.¹H NMR(400MHz,DMSO-d₆)δ12.58(s,1H),8.02–7.93(m,2H),7.90–7.80(m,2H),7.64(d,J＝16.1Hz,1H),6.66(d,J＝16.1Hz,1H),3.87(s,3H).

(E) -3- (3- (methoxycarbonyl) phenyl) acrylic acid (11b)

Synthesis analogous to 11a, yield: 100%, white solid, melting point: 246-248 ℃.¹H NMR(400MHz,DMSO-d₆)δ12.52(s,1H),8.19(t,J＝1.8Hz,1H),8.00(ddt,J＝11.0,7.9,1.5Hz,2H),7.67(d,J＝16.1Hz,1H),7.58(t,J＝7.8Hz,1H),6.62(d,J＝16.0Hz,1H),3.88(s,3H).

(E) -methyl 4- (3-oxo-3- ((((tetrahydro-2H-pyran-2-yl) oxy) amino) prop-1-en-1-yl) benzoate (12a)

Synthesis analogous to 8a, yield: 90 percent and white solid with a melting point of 132-134 ℃.¹H NMR(400MHz,DMSO-d₆)δ11.34(s,1H),8.02–7.97(m,2H),7.73(d,J＝8.0Hz,2H),7.55(d,J＝15.8Hz,1H),6.64(d,J＝15.9Hz,1H),4.93(d,J＝3.4Hz,1H),3.96(d,J＝11.0Hz,1H),3.87(s,3H),3.64–3.50(m,1H),1.72–1.49(m,6H).

(E) -methyl 3- (3-oxo-3- ((((tetrahydro-2H-pyran-2-yl) oxy) amino) prop-1-en-1-yl) benzoate (12b)

Synthesis analogous to 8a, yield: 68 percent of white solid, and the melting point of 186-188 ℃.¹H NMR(400MHz,DMSO-d₆)δ11.27(s,1H),8.15(t,J＝1.8Hz,1H),8.00–7.95(m,1H),7.86(d,J＝7.7Hz,1H),7.57(q,J＝7.5Hz,2H),6.63(d,J＝15.9Hz,1H),3.94(d,J＝10.8Hz,1H),3.88(s,3H),3.59–3.50(m,1H),1.70–1.55(m,6H).

(E) -4- (3-oxo-3- ((((tetrahydro-2H-pyran-2-yl) oxy) amino) prop-1-en-1-yl) benzoic acid (13a)

Synthesis and9a analogously, yield: 76%, white solid, melting point: 184-186 ℃.¹H NMR(400MHz,DMSO-d₆)δ13.06(s,1H),11.33(s,1H),7.97(d,J＝8.2Hz,2H),7.70(d,J＝8.0Hz,2H),7.55(d,J＝15.9Hz,1H),6.69–6.57(m,1H),4.93(s,1H),3.98(d,J＝9.2Hz,1H),3.54(dd,J＝10.3,5.7Hz,1H),1.71–1.55(m,6H).

(E) -3- (3-oxo-3- ((((tetrahydro-2H-pyran-2-yl) oxy) amino) prop-1-en-1-yl) benzoic acid (13b)

Synthesis analogous to 9a, yield: 25% white solid, melting point>250℃。¹H NMR(400MHz,DMSO-d₆)δ11.26(s,1H),8.14(d,J＝1.9Hz,1H),7.94(d,J＝7.7Hz,1H),7.82(d,J＝7.7Hz,1H),7.61–7.51(m,2H),6.62(d,J＝15.9Hz,1H),4.93(d,J＝3.4Hz,1H),3.95(d,J＝10.5Hz,1H),3.59–3.49(m,1H),1.71–1.55(m,6H).

4- (((E) -3- (hydroxyamino) -3-oxoprop-1-en-1-yl) -N- (2- (((((R) -3-methyl-1- ((3aR,4R,6R,7aS) -5,5,7 a-trimethylhexahydro-4, 6-methoxybenzo [ d ] [1,3,2] dioxaborocan-2-yl) butyl) amino) -2-oxyethyl) benzamide (ZY-11)

Synthesis analogous to ZY-7, yield: 20%, yellow solid, melting point: 186-188 ℃.¹H NMR(400MHz,DMSO-d₆)δ11.26(s,1H),10.83(s,1H),8.71(d,J＝8.2Hz,1H),8.22(s,1H),7.89(dd,J＝30.8,7.9Hz,2H),7.67(d,J＝7.8Hz,1H),7.52(t,J＝7.8Hz,1H),7.30(d,J＝8.0Hz,1H),7.21(d,J＝2.4Hz,1H),7.02(dt,J＝27.0,7.3Hz,2H),4.87(q,J＝7.5Hz,1H),4.11(d,J＝8.5Hz,1H),2.22(t,J＝11.4Hz,2H),2.03(s,1H),1.91–1.74(m,2H),1.71–1.53(m,2H),1.43–0.93(m,9H),0.94–0.41(m,9H).¹³C NMR(101MHz,DMSO)δ174.92,166.22,164.18,136.50,134.55,133.39,130.44,129.97,128.78,127.58,126.83,124.42,121.41,118.85,111.81,110.05,83.19,76.14,52.24,38.17,38.14,29.47,27.62,26.43,25.37,24.44,23.48,22.76..HRMS(ESI^-):m/z calculated for C₂₇H₃₈BN₃O₆ 613.3320,found[M-H]^-613.3317.

N- ((S) -3- (1H-indol-2-yl) -1- (((R) -3-methyl-1- ((3aR,4R,6R,7aS) -5,5,7 a-trimethylhexahydro-4, 6-methylbenzo [ d ] [1,3,2] dioxaboryl-2-yl) butyl) amino) -1-oxopropan-2-yl) -3- ((E) -3- (hydroxyamino) -3-oxoprop-1-en-1-yl) benzamide (ZY-12)

Synthesis analogous to ZY-7, yield: 32%, brown solid, melting point: 182 to 184 ℃.¹H NMR(400MHz,DMSO-d₆)δ10.83(d,J＝6.9Hz,2H),9.12(d,J＝22.4Hz,2H),8.69(d,J＝8.0Hz,1H),8.01(d,J＝14.8Hz,1H),7.73(dd,J＝34.7,7.7Hz,3H),7.54–7.42(m,2H),7.35–7.19(m,2H),7.07–6.94(m,2H),6.53(d,J＝15.8Hz,1H),5.76(s,1H),4.86(q,J＝7.8,7.4Hz,1H),4.11(d,J＝8.3Hz,1H),3.19(d,J＝7.5Hz,2H),2.22(t,J＝11.6Hz,1H),2.03(s,1H),1.81(dd,J＝21.1,6.7Hz,2H),1.65(d,J＝13.4Hz,2H),1.39–1.15(m,8H),0.94–0.69(m,9H).¹³C NMR(101MHz,DMSO)δ174.92,166.44,162.99,136.51,129.36,128.82,127.60,126.77,124.46,121.42,120.52,118.85,118.80,111.84,110.09,83.20,76.15,68.18,52.34,52.24,30.37,29.45,27.83,27.62,26.43,25.60,25.37,24.42,23.46,22.89,22.78.HRMS(ESI^-):m/z calculated for C₂₇H₃₈BN₃O₆ 639.3474,found[M-H]^-639.3476.

3- (((E) -3- (hydroxyamino) -3-oxoprop-1-en-1-yl) -N- (2- (((((R) -3-methyl-1- ((3aR,4R,6R,7aS) -5,5,7 a-trimethylhexahydro-4, 6-methoxybenzo [ d ] [1,3,2] dioxaborocan-2-yl) butyl) amino) -2-oxyethyl) benzamide (ZY-13)

Synthesis analogous to ZY-7, yield: 25%, yellow solid, melting point: 160-162 ℃.¹H NMR(400MHz,DMSO-d₆)δ13.67(s,1H),8.94(s,1H),8.21(t,J＝5.7Hz,1H),7.98(d,J＝8.4Hz,1H),7.72(d,J＝8.3Hz,1H),7.54(t,J＝7.6Hz,1H),7.41(t,J＝7.6Hz,1H),4.97(d,J＝6.3Hz,1H),4.28(s,1H),4.05(d,J＝8.1Hz,2H),3.81(d,J＝5.9Hz,2H),2.21(dd,J＝26.9,14.7Hz,2H),2.02(dd,J＝9.3,6.2Hz,2H),1.88–1.76(m,5H),1.74–1.58(m,3H),1.50(dd,J＝13.7,5.4Hz,1H),1.37–1.18(m,9H),0.89–0.78(m,9H).¹³C NMR(101MHz,DMSO)δ174.11,166.41,162.98,138.06,135.27,129.69,128.60,126.89,120.53,100.51,83.49,76.29,52.12,38.13,36.53,29.33,27.57,26.40,25.79,25.29,24.38,23.48,22.64.HRMS(ESI^-):m/z calculated for C₂₇H₃₈BN₃O₆ 510.2895,found[M-H]^-510.2891.

N- ((S) -3- (1H-indol-2-yl) -1- (((R) -3-methyl-1- ((3aR,4R,6R,7aS) -5,5,7 a-trimethylhexahydro-4, 6-methoxybenzo [ d ] [1,3,2] dioxaboryl-2-yl) butyl) amino) -1-oxopropan-2-yl) -4- ((E) -3- (hydroxyamino) -3-oxoprop-1-en-1-yl) benzamide (ZY-14)

Synthesis analogous to ZY-7, yield: 28%, orange solid, melting point: 186-190 ℃.¹H NMR(400MHz,DMSO-d₆)δ10.83(d,J＝2.5Hz,2H),9.14(d,J＝3.0Hz,1H),8.63(d,J＝8.2Hz,1H),7.82(d,J＝8.2Hz,2H),7.65(dd,J＝20.0,8.0Hz,3H),7.47(d,J＝16.0Hz,1H),7.36–7.19(m,2H),7.07–6.96(m,2H),6.54(d,J＝15.9Hz,1H),5.00–4.80(m,1H),4.14–4.08(m,1H),3.19(d,J＝7.4Hz,2H),2.57(dd,J＝8.2,2.8Hz,1H),2.30–1.99(m,3H),1.91–1.75(m,3H),1.65(dt,J＝12.8,5.9Hz,2H),1.41–1.17(m,11H),0.89–0.80(m,9H).¹³C NMR(101MHz,DMSO)δ174.97,169.69,166.23,138.12,136.52,134.80,128.54,127.68,127.62,124.46,121.39,118.85,118.78,111.83,110.09,83.19,83.08,76.16,72.71,68.19,54.21,52.33,52.27,30.39,29.47,27.83,27.64,26.44,25.61,25.39,24.43,23.48,22.83,22.77.HRMS(ESI^-):m/z calculated for C₂₇H₃₈BN₃O₆ 639.3474,found[M-H]^-639.3479.

Evaluation of Activity of target Compound

Experimental example 1 target Compound inhibits Histone deacetylase, and the results are shown in Table 1

1.[ Material ]

Stock solutions (10mM in DMSO) of target compound and positive controls SAHA, MS-275; hela cell nucleus extract; Boc-Lys-AMC Substrate; HDAC Buffer; pancreatin; trichostatin A (TSA, 0.3mM in dimethyl sulfoxide); a 96-well plate; thermo Varioskan Flash full-wavelength multifunctional enzyme-labeling instrument;

[ method ]

(1) Formulation of HDAC buffer

60.57g of Tris was weighed and dissolved in slightly less than 480mL of distilled water, adjusted to pH 8 with concentrated HCl and then made up with 500mL of distilled water to obtain a 1M Tris-HCl stock solution. Taking 7.5mL of 1M Tris-HCl stock solution, adding 0.0365g of EDTA, 7.31g of NaCl and 50mL of glycerol, and diluting to 500mL with distilled water to obtain HDAC buffer;

(2) preparation of Trypsin solution

25mL of a 1M Tris-HCl stock solution was taken, 2.92g of NaCl was added, the volume was fixed with distilled water, and an appropriate amount of pancreatin and TSA was added immediately before use (the pancreatin concentration was 10mg/mL, and the TSA concentration was 2. mu.M).

(3) Preparation of substrate solution

The substrate was dissolved in DMSO to prepare a 30mM stock solution, which was then diluted to 300. mu.M with HDAC buffer so that the DMSO content was about 1%.

(4) Dilution of enzyme solutions

The enzyme solution was mixed with HDAC buffer at a ratio of 1: 80 in proportion.

(5) Preparation of Compound solutions

Compounds (test compound and positive control SAHA) were diluted to 5 Xfinal concentration with HDAC buffer

(6) Formulation and determination of 100% and blank

Preparation and measurement of 100% solution:

mixing 50 μ L HDAC buffer with 10 μ L enzyme solution, adding 40 μ L substrate after 5min, reacting at 37 deg.C for 0.5h, adding 100 μ L Trypsin solution to terminate the reaction, reacting at 37 deg.C for 20min, and measuring fluorescence intensity at 390nm/460nm to obtain 100% absorption;

preparing and measuring a blank solution:

60 μ L of HDAC buffer was reacted at 37 ℃ for 1h after adding 40 μ L of substrate

Adding 100 μ L of Trypsin solution, reacting at 37 deg.C for 1 hr, and measuring fluorescence intensity at 390nm/460nm to obtain blank absorption;

(7) determination of the inhibition of hdac activity by compounds:

mixing 50 μ L of HDAC buffer containing drug with 10 μ L of enzyme solution, incubating for 5min, adding 40 μ L of substrate, reacting at 37 deg.C for 1h, adding 100 μ L of Trypsin solution to terminate the reaction, reacting at 37 deg.C for 1h, and measuring fluorescence intensity (390nm/460 nm);

(8) calculating the inhibition ratio and IC by software and formula₅₀Value of

Description of terms:

SAHA: vorinostat; MS-275: entinostat; m: mol/l; and (mM): millimoles per liter; μ M: micromole/liter; mL: ml; μ L: microliter; IC (integrated circuit)₅₀: half maximal inhibitory concentration; TSA: trichostatin a, an HDAC inhibitor; substrate: 300 μ M Boc-Lys-AMC solution; enzyme solution: the Hela cell nucleus extract was diluted 80-fold with HDAC buffer to obtain a solution.

TABLE 1 Experimental results for in vitro inhibition of HDACs by the target Compounds

^aThe data in the table are the average of three experiments, and the values after "+ -" indicate the standard deviation

Experimental example 2 inhibition of proteasome by the objective Compound, the results are shown in Table 2

1.[ Material ]

Stock solutions of target compound and positive control bortezomib (10mM, in dimethyl sulfoxide); recombinant human 20s proteasome; Suc-LLVY-AMC; proteasome Buffer; a 96-well plate; thermo Varioskan Flash full-wavelength multifunctional enzyme-labeling instrument; bortezomib (bortezomib).

[ method ]

Similar to the activity of HDACs, the difference is that the plate reading is directly carried out after substrate incubation without pancreatin incubation.

TABLE 2 results of in vitro proteasome inhibition experiments with target compounds

EXAMPLE 3 inhibition of tumor cell proliferation assay by target Compound

The results of in vitro tumor cell proliferation inhibition experiments by selecting the target compounds with better HDAC and proteasome inhibition activities are shown in Table 3

1.[ Material ]

HepG2, Mcf-7, K562, KG1, THP-1, HL-60, Jurkat, RPMI-8226, KM3, KM3/BTZ, U266 tetramethylazoazolium salt MTT, 10% fetal bovine serum (Hyclone, USA), 2.5 g.L-1 trypsin (Gibco, USA), modified RPMI1640 medium (Hyclone, USA), positive control SAHA, Bortezomib, 96-well plate;

[ method ]

Culturing cells in a conventional mode, and collecting cells with logarithmic growth for experiment; cells in logarithmic growth phase were diluted to 4X 10 with RPMI1640 medium containing 10% fetal bovine serum⁴each.mL^-1Then inoculating the cells into a 96-well plate (adding 100 mu L of the cells into each well), taking the cells without the cells as blank wells, and then culturing the cells in a constant-temperature incubator (37 ℃, 5% carbon dioxide) for 8 hours; adding target compound solution and positive drug (SAHA) solution prepared with culture medium, culturing in constant temperature incubator (37 deg.C, 5% carbon dioxide) for 48 hr, adding 30 μ L MTT, removing liquid in the well (suspension cells need centrifugation) after four hr, adding 150 μ L DMSO, shaking in constant temperature shaking table for 10min, measuring absorbance value of each well with microplate reader at 570nm wavelength, and calculating inhibition rate and IC₅₀A value;

TABLE 3 results of in vitro tumor cell proliferation inhibition experiments with target compounds

The data in table a are the average values of three experiments, and the values after "+ -" represent standard deviation

Description of terms: human chronic myelogenous leukemia cell K562, human breast cancer cell Mcf-7, human promyelocytic leukemia cell HL-60, acute T cell leukemia cell Jurkat, human multiple myeloma cell RPMI-8226, human monocytic leukemia THP-1 and human liver cancer cell HepG 2.

TABLE 4 results of in vitro inhibition of multiple myeloma cell proliferation assay with target compounds

Description of terms: human multiple myeloma cells RPMI-8226, U266 and KM3, and bortezomib-resistant multiple myeloma cells KM 3/BTZ.

And (4) conclusion:

the compounds have good HDAC inhibitory activity and proteasome inhibitory activity, the tested compounds show good anti-tumor cell proliferation activity, and in the future, active research can be deeply carried out to develop more active compounds for preparing and preventing related mammal diseases caused by abnormal expression of histone deacetylase or abnormal proteasome.

Claims

1. A histone deacetylase, a proteasome dual-target inhibitor and pharmaceutically acceptable salts thereof are characterized by having a structure shown as a formula I:

wherein, P₁Is composed of

P₂Is hydrogen, benzyl or

Z₁Z2 independently of one another represent hydroxy, or Z1 and Z₂Together with boron atoms to form

2. A histone deacetylase, a proteasome dual-target inhibitor and pharmaceutically acceptable salts thereof are characterized by having the following structures:

3. the use of a histone deacetylase, proteasome dual target inhibitor, or a pharmaceutically acceptable salt thereof according to any one of claims 1-2 in the preparation of a medicament for preventing or treating a disease in a mammal associated with abnormal expression of histone deacetylase or abnormal function of proteasome.

4. A pharmaceutical composition suitable for oral administration to a mammal comprising a histone deacetylase, a proteasome dual target inhibitor, a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients according to any one of claims 1-2.

5. A pharmaceutical composition suitable for parenteral administration to a mammal comprising a histone deacetylase, a proteasome dual target inhibitor, a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients according to any one of claims 1-2.

6. A pharmaceutical composition comprising the histone deacetylase, the proteasome dual-target inhibitor, or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2 and one or more therapeutically active substances selected from the group consisting of other inhibitors of abnormal expression of histone deacetylase activity, antitumor agents, steroid hormones and chemotherapeutic agents.