WO2021115188A1 - 一种组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂及其制备方法和应用 - Google Patents

一种组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂及其制备方法和应用 Download PDF

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WO2021115188A1
WO2021115188A1 PCT/CN2020/133530 CN2020133530W WO2021115188A1 WO 2021115188 A1 WO2021115188 A1 WO 2021115188A1 CN 2020133530 W CN2020133530 W CN 2020133530W WO 2021115188 A1 WO2021115188 A1 WO 2021115188A1
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proteasome
room temperature
substituted
histone deacetylase
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French (fr)
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方浩
周易
侯旭奔
杨新颖
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山东大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds
    • C07F5/025—Boronic and borinic acid compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to a histone deacetylase, a proteasome dual target inhibitor and a pharmaceutically acceptable salt thereof, its stereoisomer, a preparation method thereof, a pharmaceutical composition and medical use, and belongs to the technical field of medicine.
  • the proteasome is an important part of the Ubiquitin-proteasome system (UPS). It is the main degradation pathway of misfolded proteins and other proteins in the process of protein synthesis. It participates in the growth and differentiation of cells. Important physiological and biochemical processes such as replication and repair, cell metabolism, and immune response. UPS mainly plays two roles: one is to maintain the quality of cells by breaking down abnormal or damaged proteins; the other is to control the basic life activities of cells by breaking down proteins with specific functions; the two ultimately ensure the normal functioning of tissues and organs. . Relevant studies have shown that the proteasome is related to many diseases including cancer. Proteasome inhibitors, such as bortezomib, have been approved by the US Food and Drug Administration for the treatment of multiple myeloma.
  • Histone deacetylases HDACs
  • HAT histone acetyltransferase
  • human HDACs The 18 members of the family can be divided into Zn 2+ dependent Class I (HDAC1,2,3,8), Class IIa (HDAC4,5,7,9), Class IIb (HDAC6,10), Class IV ( HDAC11) and NAD + dependent Class III (SIRT1-7).
  • the present invention provides a histone deacetylase and proteasome dual target inhibitor, as well as a preparation method and application.
  • the present invention further provides a pharmaceutical composition and medical use of the compound.
  • P 1 is N-hydroxyformamide or N-hydroxycinnamido substituted aryl, N-hydroxyformamide or N-hydroxycinnamido heteroaryl, N-hydroxyformamide or N- The aralkyl group of the hydroxycinnamido group, the heteroaralkyl group of the N-hydroxycinnamido group or the N-hydroxycinnamido group, or the cycloalkyl group of the N-hydroxyformamide group or the N-hydroxycinnamido group, 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 aforementioned aryl, heteroaryl, aralkyl, heteroaralkyl or cyclo
  • Substituent w is selected from hydroxyl, halogen, alkyl, trifluoromethyl, cyano, nitro, guanidino, amino, carboxy, nitro, monocyclic aryl containing 5 or 6 ring atoms or having 8-15 A bicyclic aryl group with one ring atom, a monocyclic heterocyclic aryl group with 5-6 ring atoms containing 1-2 heteroatoms;
  • P 2 is independently hydrogen, alkyl, cycloalkyl, alkaryl, aryl, 5-10 membered saturated or partially saturated heterocyclic or heteroaryl, wherein the aryl, aralkyl, alkaryl Or the ring part of the heterocyclic ring may be substituted by the above-mentioned substituent w;
  • Z 1 and Z 2 are independently an alkyl group, a hydroxyl group, an alkoxy group or an aryloxy group, or Z 1 and Z 2 together form a group derived from a dihydroxy compound which has a At least two hydroxyl groups separated by at least two connected atoms.
  • the chain or ring contains carbon atoms and optionally one or more heteroatoms.
  • the heteroatoms can be N, S or O.
  • P 1 is a meta or para substituted N-hydroxyformamide or N-hydroxycinnamamide substituted aryl group
  • P 2 is independently hydrogen, benzyl or 1H-indole-3-methyl
  • Z 1 and Z 2 are independently pinanediol.
  • the general formula I is a compound of the following structure:
  • alkyl in the present invention refers to a linear or branched alkyl group derived from an alkane containing 1-10 carbon atoms by removing one hydrogen atom, such as methyl, ethyl, n-propyl, isopropyl, n-propyl Butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1,2 -Dimethylpropyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-di Methylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-
  • cycloalkyl group in the present invention refers to a cyclic alkyl group derived from an alkane of 3 to 8 carbon atoms by removing one hydrogen atom, such as cyclopropyl, cyclobutyl, 1-methylcyclobutyl, cyclo Pentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.
  • cyclopropyl cyclobutyl
  • 1-methylcyclobutyl 1-methylcyclobutyl
  • cyclo Pentyl cyclohexyl
  • cycloheptyl cyclooctyl
  • C 4-7 cycloalkyl C 4-6 cycloalkyl and C 5-6 cycloalkyl
  • Examples of the "monoheterocyclic group” include: oxirane group, dioxanyl group, thiaziryl group, aziridine group, 2H-aziridine group, diaziridine group Group, 3H-diazepanyl, oxaziridinyl, oxetanyl, 1,2-dioxetanyl, thietane, 1,2-disulfide Cyclobutenyl, azetidinyl, 1,2-diazetidine, azetidinyl, 1,2-diazetidine, furanyl, tetrahydrofuranyl , Thienyl, 2,5-dihydrothienyl, tetrahydrothienyl, pyrrolyl, dihydropyrrolyl, pyrrolidinyl, 1,3-dioxolane, 1,3-dioxanyl Penten-2-onyl, 1,2-dithiolany
  • Aryl refers to a substituent containing an aromatic ring, such as a phenyl or benzyl group, which is optionally fused with a cycloalkyl group, which preferably has 4-7 ring atoms, more preferably 5 -6 ring atoms.
  • Preferred aryl groups contain 5-15 carbon atoms;
  • Heteroaryl is an aromatic heterocyclic ring, which can be a monocyclic or bicyclic group. They contain heteroaromatic groups containing one or more heteroatoms, preferably 1-3 heteroatoms, even more preferably 1-2 heteroatoms, said heteroatoms being independently selected from O, S and N.
  • Arylalkyl refers to an aryl group to which a C 1 -C 4 alkylene group is attached;
  • Alkyl refers to an aryl group to which a C 1 -C 4 alkylene group is attached
  • Heteroaralkyl refers to a heteroaryl group to which a C 1 -C 4 alkylene group is attached
  • the compound represented by the general formula I of the present invention can be prepared into a pharmaceutically acceptable salt by a known method, and the salt refers to a salt prepared by mixing the compound represented by the formula I with an acid or a base;
  • Suitable acid addition salts are formed from acids that form non-toxic salts.
  • Representative acid addition salts include, but are not limited to, acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, carbonic acid Hydrogen salt, butyrate, camphorate, camphorsulfonate, carbonate, citrate, digluconate, glycerophosphate, hemisulfate, enanthate, caproic acid Salt, formate, fumarate, gluconate, glucuronate, glutamate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isethionate), Lactate, maleate, malate, malonate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, grass Acid salt, palmitate, pamoate
  • the base addition salt can be used in the final isolation and purification process of the compound by mixing the carboxylic acid-containing part with an appropriate base (such as but not limited to the hydroxide, carbonate or bicarbonate salt of a pharmaceutically acceptable metal cation). ) Or prepared in situ by reacting with ammonia or organic primary, secondary or tertiary amines.
  • an appropriate base such as but not limited to the hydroxide, carbonate or bicarbonate salt of a pharmaceutically acceptable metal cation.
  • Pharmaceutically acceptable salts include, but are not limited to, alkali metal or alkaline earth metal-based cations, such as but not limited to lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and non-toxic quaternary ammonium and amine cations, including ammonium, tetrakis Methylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, etc.
  • Other representative organic amines that can be used to form base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, etc.;
  • Steps herein refer to all possible stereoisomeric forms of the compounds of the present invention or their physiological derivatives. Unless otherwise specified, the chemical nomenclature of the compounds involved in the present invention includes mixtures of all possible stereochemical forms. The mixtures contain all enantiomers and diastereomers of the basic structural molecule, as well as the single isomer form of the substantially pure compound. That is, it contains less than 10%, preferably less than 5%, especially less than 2%, and most preferably less than 1% of other isomers. The various stereoisomer forms of the peptoid compound of the present invention are obviously included in the scope of the present invention;
  • substituents described above may themselves be substituted by one or more substituents.
  • substituents include those listed in C. Hansch and A. Leo, Substituent Constants for Correlation Analysis in Chemistry and Biology (1979); preferred substituents include alkyl, alkenyl, alkoxy, and hydroxyl. , Nitro, amino, aminoalkyl, cyano, halogen, carboxy, thio, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, imino, hydroxyalkyl, aryloxy, aryl Alkyl group and its combination;
  • the compound of the present invention can be prepared into any pharmaceutical preparations by methods known in the art, and administered to patients in need of such treatment by oral, parenteral, rectal or pulmonary administration.
  • oral administration When used for oral administration, it can be prepared Conventional solid preparations, such as tablets, capsules, pills, granules, etc., can also be made into oral liquid preparations, such as oral solutions, oral suspensions, and syrups.
  • suitable fillers, binders, disintegrants, lubricants, etc. can be added.
  • parenteral administration it can be made into injections, including injections, sterile powders for injections, and concentrated solutions for injections.
  • parenteral administration it can be made into injections, including injections, sterile powders for injections, and concentrated solutions for injections.
  • an injection When preparing an injection, it can be produced by a conventional method in the existing pharmaceutical field.
  • an additive When preparing an injection, it is not necessary to add an additive, or an appropriate additive can be added according to the nature of the drug. When used for rectal administration, it can be made into suppositories. When used for pulmonary administration, it can be made into inhalation or spray, etc.;
  • the dosage and frequency of administration of the compound of the present invention can be adjusted according to the judgment of the clinician or pharmacist, taking into consideration factors such as the following factors: the age, health condition and size of the patient, and the severity of the symptoms to be treated.
  • the total daily dose of the compound of the present invention ranges from about 0.1 to about 2000 mg per day, although it may vary if necessary, depending on the purpose of treatment, the patient, and the route of administration.
  • the dosage is about 1 to about 200 mg/day, administered in a single dose or in 2-4 divided doses.
  • the dosage is about 10 to about 2000 mg/day, administered in a single dose or in 2-4 divided doses.
  • the dosage is about 100 to about 2000 mg/day, administered in a single dose or in 2-4 divided doses. In yet another embodiment, the dosage is about 500 to about 2000 mg/day, administered in a single dose or in 2-4 divided doses.
  • the compound of the present invention its pharmaceutically acceptable salts, esters or solvates or their prodrugs or isomers are used in combination with other therapeutically active substances, they can be administered simultaneously, separately or sequentially, and can be made into a single administration Way of pharmaceutical composition.
  • the dosage of other therapeutically active substances used in combination can be based on the clinically used amount, and can be appropriately selected according to the administration object, administration route, disease, combination and the like. There are no special restrictions on the administration form of other therapeutically active substances, as long as the compound of the present invention and other therapeutically active substances are combined at the time of administration.
  • a “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. Therefore, the preparation does not contain any one component or multiple components in such an amount that a properly prudent medical practitioner finds that the preparation is not suitable for administration to ordinary subjects. In many cases, this pharmaceutical composition is a sterile preparation.
  • the specific temperature range of "room temperature” involved in the present invention is 20-30°C.
  • the synthetic route is as follows:
  • P 2 is the same as that described in the above formula I, and R is independently hydrogen, hydroxy, halogen, alkyl, trifluoromethyl, cyano, nitro, guanidino, amino, carboxy, nitro, containing 5 or Any one of a monocyclic aryl group with 6 ring atoms or a bicyclic aryl group with 8-15 ring atoms, or a monocyclic ring group with 5-6 ring atoms containing 1-2 heteroatoms, ring A is independent Any one of aryl, heteroaryl, aralkyl, heteroaralkyl, or cycloalkyl;
  • Reagents and reaction conditions (a) 2-Boc-aminoacetic acid substituted by P2 at position 2, TBTU, NMM, room temperature, 4-24h; (b) hydrogen chloride saturated ethyl acetate, room temperature, 1-24h; (c) Ortho-phenylenediamine substituted by R at position 3 or 4, TBTU, NMM, protected from light, room temperature, 8h; (d) lithium hydroxide, water/methanol, room temperature, 2-24h; (e) TBTU, NMM, room temperature, 4-24h.
  • the synthetic route is as follows:
  • the definition of P2 is the same as that described in the above formula I, and the A ring is independently any one of an aryl group, a heteroaryl group, an aralkyl group, a heteroaralkyl group or a cycloalkyl group;
  • Reagents and reaction conditions (a) 2-Boc-aminoacetic acid substituted by P2 at position 2, TBTU, NMM, room temperature, 4-24h; (b) hydrogen chloride saturated ethyl acetate, room temperature, 1-24h; (c) O-(Tetrahydro-2H-pyran-2-yl)hydroxylamine, TBTU, NMM, protected from light, room temperature, 4-24h; (d) Lithium hydroxide, water/methanol, room temperature, 2-24h; (e)TBTU , NMM, room temperature, 4-24h; (f) hydrogen chloride saturated ethyl acetate, room temperature, 1-24h.
  • the synthetic route is as follows:
  • the definition of P2 is the same as that described in the above formula I, and the A ring is independently any one of an aryl group, a heteroaryl group, an aralkyl group, a heteroaralkyl group or a cycloalkyl group;
  • Reagents and reaction conditions (a) 2 -Boc-aminoacetic acid substituted by P 2 at position 2, TBTU, NMM, room temperature, 4-24h; (b) hydrogen chloride saturated ethyl acetate, room temperature, 1-24h; (c ) Malonic acid, pyridine, N,N-dimethylformamide, heated to reflux, 10-48h; (d) O-(tetrahydro-2H-pyran-2-yl)hydroxylamine, TBTU, NMM, protected from light Room temperature, 4-24h; (e) Lithium hydroxide, water/methanol, room temperature, 2-24h; (f) TBTU, NMM, room temperature, 4-24h; (g) Hydrogen chloride saturated ethyl acetate, room temperature, 1-24h .
  • the present invention also provides the use of the series of compounds in the preparation of drugs for the prevention or treatment of related mammalian diseases caused by abnormal expression of histone deacetylase or abnormal proteasome function.
  • the diseases include but are not limited to cancer and neurodegeneration.
  • the present invention also includes a pharmaceutical composition suitable for oral administration to mammals, comprising any compound of the above general formula I, a pharmaceutically acceptable carrier, and optionally one or more pharmaceutically acceptable excipients.
  • a pharmaceutical composition suitable for oral administration to mammals comprising any compound of the above general formula I, a pharmaceutically acceptable carrier, and optionally one or more pharmaceutically acceptable excipients.
  • Shape agent any compound of the above general formula I, a pharmaceutically acceptable carrier, and optionally one or more pharmaceutically acceptable excipients.
  • the present invention also includes a pharmaceutical composition suitable for parenteral administration to mammals, comprising any compound of the above general formula I, a pharmaceutically acceptable carrier, and optionally one or more pharmaceutically acceptable Of excipients.
  • HDAC fluorescent substrate containing an acetylated lysine side chain-Boc-Lys(acetyl)-AMC
  • HDAC activity Hela cell nuclear extract
  • Boc-Lys-AMC was hydrolyzed with trypsin to produce AMC fluorophore, and the fluorescence intensity was measured at the emission wavelength/excitation wavelength (390nm/460nm). The fluorescence intensity is proportional to the HDACs inhibitory effect of the test compound.
  • Prism GraphPad software is used to process the data to calculate the HDACs inhibitory rate and IC 50 value of different target compounds.
  • Fluorescence analysis was used to evaluate the compound's inhibitory activity on the proteasome.
  • the proteasome fluorescent substrate Suc-LLVY-AMC
  • the AMC fluorophore measured at the emission wavelength/excitation wavelength (390nm/460nm) The fluorescence intensity.
  • the fluorescence intensity is proportional to the proteasome inhibitory effect of the test compound.
  • Prism GraphPad software is used to process the data to calculate the inhibition rate and IC 50 value of different target compounds on the proteasome.
  • the cell activity test of the compound uses MTT assay, tumor cell suspension (human chronic myeloid leukemia cell K562, human granulocytic leukemia cell KG1, human promyelocytic leukemia cell HL-60, acute T cell leukemia cell Jurkat, human multiple Myeloma cells RPMI-8226, human colon cancer cell line HCT-116, and prostate cancer cell line PC-3 were respectively inoculated into 96-well plates. Compounds of different concentrations diluted with culture medium were added to each well. After 48 hours of incubation, they were stained with MTT. After incubating for 4 hours, the absorbance OD value of each well was measured at 490/570nm with a microplate reader, and then the inhibition rate and IC 50 value were calculated to determine the anti-proliferative activity of the target compound.
  • tumor cell suspension human chronic myeloid leukemia cell K562, human granulocytic leukemia cell KG1, human promyelocytic leukemia cell HL-60, acute
  • 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). Under nitrogen protection, the reaction was continuously stirred for 16 hours, then poured into water, and extracted with ethyl acetate (3 ⁇ 100 mL) for 3 times. The organic phases were combined and washed with 2% citric acid, 2% sodium bicarbonate, saturated brine, dried with magnesium sulfate, and spin-dried to obtain a crude product.
  • Example 8 N1-((S)-3-(1H-indol-3-yl)-1-(((R)-3-methyl-1-((3aR, 4R, 6R, 7aS)- 5,5,7a-Trimethylhexahydro-4,6-methylbenzo[d][1,3,2]dioxaborolan-2-yl)butyl)amino)-1-oxopropane -2-yl)-N4-Hydroxyterephthalamide (ZY-7)
  • Target compound and positive control SAHA MS-275 stock solution (10mM, dissolved in dimethyl sulfoxide); HeLa cell nuclear extract; Boc-Lys-AMC Substrate; HDAC Buffer; Pancreatin; Trichostatin A (TSA, 0.3 mM, dissolved in dimethyl sulfoxide); 96-well plate; Thermo Varioskan Flash full-wavelength multifunctional microplate reader;
  • Tris dissolve it in slightly less than 480mL of distilled water, adjust the pH to 8 with concentrated hydrochloric acid, and then make up to 500mL with distilled water to obtain 1M Tris-HCl stock solution.
  • 1M Tris-HCl stock solution Take 7.5mL of 1M Tris-HCl stock solution, add 0.0365g of EDTA, 7.31g of NaCl, 50mL of glycerol, distilled water to dilute to 500mL to obtain HDAC buffer;
  • the substrate was dissolved in DMSO to prepare a 30mM stock solution, and then diluted with HDAC buffer to 300 ⁇ M, so that the content of DMSO was about 1%.
  • SAHA vorinostat
  • MS-275 entinostat
  • M mole/liter
  • mM millimole/liter
  • ⁇ M micromol/liter
  • mL milliliter
  • ⁇ L microliter
  • IC 50 half inhibition Concentration
  • TSA Trichostatin A, HDAC inhibitor
  • Substrate 300 ⁇ MBoc-Lys-AMC solution
  • Enzyme solution HeLa cell nuclear extract diluted 80 times with HDAC buffer.
  • Target compounds with good HDAC and proteasome inhibitory activity were selected for in vitro inhibition of tumor cell proliferation experiments. The results are shown in Table 3.
  • Terminology description 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 monocyte Leukemia THP-1, human liver cancer cell HepG2.
  • Terminology description human multiple myeloma cells RPMI-8226, U266, KM3, bortezomib-resistant multiple myeloma cells KM3/BTZ.

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Abstract

本发明涉及一种蛋白去乙酰化酶、蛋白酶体双靶点抑制剂及其药学上可接受的盐、其立体异构体、其制备方法和应用，所述化合物具有如通式(I)所示，本发明化合物具有较好的抗组蛋白去乙酰化酶的活性、抗蛋白酶体活性和抗肿瘤细胞增殖的活性，可用于制备预防或治疗因组蛋白去乙酰化酶表达异常或蛋白酶体异常导致的相关哺乳动物疾病的药物，本发明还涉及含有通式I结构化合物的组合物的制药用途。

Description

一种组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂及其制备方法和应用 技术领域
本发明涉及一种组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂及其药学上可接受的盐、其立体异构体、其制备方法、药物组合物与医药用途，属于医药技术领域。
背景技术
蛋白酶体是泛素-蛋白酶体系统(Ubiquitin-proteasome system,UPS)的重要组成部分，它是蛋白质合成过程中错误折叠的蛋白和其他蛋白被水解的主要降解途径，参与细胞的生长、分化，DNA复制与修复、细胞代谢、免疫反应等重要生理生化过程。UPS主要起两方面的作用：一是通过分解异常或损伤的蛋白质以维持细胞的质量；二是通过分解特定功能的蛋白质来控制细胞的基本生命活动；两者最终保障组织和器官功能的正常发挥。相关研究表明，蛋白酶体与包括癌症在内的多种疾病相关，蛋白酶体的抑制剂，如硼替佐米，已经被美国食品药品监督管理局批准上市用于多发性骨髓瘤的治疗。
在多发性骨髓瘤的治疗中，未经治疗的患者起初往往能取得较好的疗效，但进展与复发最终不可避免的发生。硼替佐米与组蛋白去乙酰化酶(Histone deacetylases,HDACs)抑制剂帕比司他联用作为一种复发性和难治性多发性骨髓瘤的治疗方案已经被美国血液学会列为了推荐方案。另有大量研究表明，组蛋白去乙酰化酶可以逆转肿瘤细胞的蛋白酶体抑制剂耐药现象。
组蛋白去乙酰化酶(Histone deacetylases,HDACs)与组蛋白乙酰转移酶(histone acetyltransferase，HAT)共同维持机体的组蛋白和非组蛋白乙酰化的平衡，根据细胞定位和同源性差异，人体HDACs家族的18个成员，可以分为Zn 2+依赖型的Class I(HDAC1,2,3,8)、Class IIa(HDAC4,5,7,9)、Class IIb(HDAC6,10)、Class IV(HDAC11)和NAD +依赖型的Class III(SIRT1-7)。
在针对许多不同类型的肿瘤和其他疾病的研究中，蛋白酶体抑制剂与组蛋白去乙酰化酶抑制剂联用均表现出了较强的协同作用，但多药联用中存在药代动力学不匹配、患者依从性差、药物之间相互作用等不利因素。所以，开发组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂具有巨大的意义。
发明内容
针对现有技术的不足，本发明提供一种组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂及制备方法和应用。
本发明进一步还提供该化合物的药物组合物及医药用途。
本发明的技术方案如下：
一、组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂
一种组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂及其药学上可接受的盐或其立体异构体，具有如式I所示的结构：
Figure PCTCN2020133530-appb-000001
其中，P 1为N-羟基甲酰胺基或N-羟基肉桂酰胺基取代的芳基、N-羟基甲酰胺基或N-羟 基肉桂酰胺基的杂芳基、N-羟基甲酰胺基或N-羟基肉桂酰胺基的芳烷基、N-羟基甲酰胺基或N-羟基肉桂酰胺基的杂芳烷基或N-羟基甲酰胺基或N-羟基肉桂酰胺基的环烷基，或N-(2-氨基苯基)甲酰胺基取代的芳基、N-(2-氨基苯基)甲酰胺基取代的杂芳基、N-(2-氨基苯基)甲酰胺基取代的芳烷基或N-(2-氨基苯基)甲酰胺基取代的杂芳烷基，上述芳基、杂芳基、芳烷基、杂芳烷基或环烷基可被任意取代基w取代；
取代基w选自羟基、卤素、烷基、三氟甲基、氰基、硝基、胍基、氨基、羧基、硝基、含有5或6个环原子的单环芳基或具有8-15个环原子的双环芳基、含有1-2个杂原子的环原子数为5-6的单杂环芳基；
P 2独立地是氢、烷基、环烷基、烷芳基、芳基、5-10元饱和或部分饱和的杂环或杂芳基，其中所述芳基、芳烷基、烷芳基或杂环的环部分可被上述取代基w取代；
Z 1和Z 2独立地是烷基、羟基、烷氧基或芳氧基，或Z 1和Z 2一起形成由二羟基化合物衍生的基团，所述二羟基化合物在链或环中具有由至少两个相连原子分隔的至少两个羟基，所述链或环含有碳原子，和任意性可有可无的一个或多个杂原子，杂原子可以是N，S或O。
根据本发明优选的，通式I中：
P 1为间位或对位取代的N-羟基甲酰胺或N-羟基肉桂酰胺取代的芳基；
P 2独立的为氢、苄基或1H-吲哚-3-甲基；
Z 1和Z 2独立地是蒎烷二醇。
根据本发明进一步优选的，通式I是如下结构的化合物：
Figure PCTCN2020133530-appb-000002
发明详述
本文中所用的术语和定义含义如下：
本发明所述“烷基”指含有1～10个碳原子的烷烃部分去除一个氢原子衍生的直链或支链的烷基，如甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、异戊基、2-甲基丁基、3-甲基丁基、1,1-二甲基丙基、1,2-二甲基丙基、新戊基、1-乙基丙基、 正己基、异己基、2-甲基戊基、3-甲基戊基、4-甲基戊基、1,1-二甲基丁基、1,2-二甲基丁基、1,3-二甲基丁基、2,2-二甲基丁基、2,3-二甲基丁基、3,3-二甲基丁基、1-乙基丁基、2-乙基丁基、1,1,2-三甲基丙基、1,2,2-三甲基丙基、1-乙基-1-甲基丙基，1-乙基-2-甲基丙基，庚基，辛基，壬基和癸基；
本发明所述的“环烷基”是指3～8个碳原子的烷烃部分去除一个氢原子衍生的环状烷基，如环丙基、环丁基、1-甲基环丁基、环戊基、环己基、环庚基、环辛基等。优选C 4-7环烷基、C 4-6环烷基和C 5-6环烷基；
所述“单杂环基”的实例有：环氧乙烷基、二氧杂环丙烷基、硫杂环丙烷基、氮杂环丙烷基、2H-氮杂环丙烷基、二氮杂环丙烷基、3H-二氮杂环丙烯基、氧氮杂环丙烷基、氧杂环丁烷基、1,2-二氧杂环丁烷基、硫杂环丁烷基、1,2-二硫杂环丁烯基、氮杂环丁烷基、1,2-二氮杂环丁烷基、氮杂环丁二烯基、1,2-二氮杂环丁烯基、呋喃基、四氢呋喃基、噻吩基、2,5-二氢噻吩基、四氢噻吩基、吡咯基、二氢吡咯基、吡咯烷基、1,3-二氧杂环戊烷基、1,3-二氧杂环戊烯-2-酮基、1,2-二硫杂环戊烯基、1,3-二硫杂环戊烷基、咪唑基、4,5-二氢咪唑基、咪唑烷基、吡唑基、4,5-二氢吡唑基、吡唑烷基、噁唑基、4,5-二氢噁唑基、异噁唑基、4,5-二氢异噁唑基、2,3-二氢异噁唑基、1,2,3-噁二唑基、1,2,5-噁二唑基、噻唑基、4,5-二氢噻唑基、异噻唑基、1,2,3-噻二唑基、1,2,4-噻二唑基、1,3,4-噻二唑基、1,2,3-三唑基、1,2,4-三唑基、四唑基、2H-吡喃基、2H-吡喃-2-酮基、3,4-二氢-2H-吡喃基、4H-吡喃基、四氢吡喃基、4H-吡喃-4-酮基、吡啶基、2-吡啶酮基、4-吡啶酮基、哌啶基、1,4-二氧杂环己二烯基、1,4-二硫杂环己二烯基、1,4-氧硫杂环己二烯基、1,4-二氧杂环己烷基、1,3-二氧杂环己烷基、1,3-氧硫杂环己烷基、2H-1,2-噁嗪基、4H-1,2-噁嗪基、6H-1,2-噁嗪基、2H-1,3-噁嗪基、4H-1,3-噁嗪基、6H-1,3-噁嗪基、2H-1,4-噁嗪基、4H-1,4-噁嗪基、5,6-二氢-4H-1,3-噁嗪基、吗啉基、2H-1,3-噻嗪基、4H-1,3-噻嗪基、5,6-二氢-4H-1,3-噻嗪基、6H-1,3-噻嗪基、2H-1,4-噻嗪基、4H-1,4-噻嗪基、哒嗪基、嘧啶基、吡嗪基、哌嗪基、1,2,3-三嗪基、1,2,4-三嗪基、1,3,5-三嗪基、1,2,4,5-四嗪基、氧杂环庚三烯基、硫杂环庚三烯基、1,4-二氧杂环辛三烯基、氮杂环庚三烯基、1,2-二氮杂环庚三烯基、1,3-二氮杂环庚三烯基、1,4-二氮杂环庚三烯基、氮杂环辛四烯基、1,4-二氢-1,4-二氮杂环辛三烯基等；
“芳基”是指含有芳香环的取代基，如苯基或苄基，其可选地与环烷基稠合，所述环烷基优选地具有4-7个环原子，更优选具有5-6个环原子。优选的芳基含有5-15个碳原子；
“杂芳基”是芳香杂环，可以是单环或双环基团。他们含有芳杂基含有一个或多个杂原子，优选为1-3个杂原子、甚至更优选1-2个杂原子，所述杂原子独立地选自O、S和N。“芳基烷基”是指C 1-C 4亚烷基连接的芳基；
“芳烷基”是指C 1-C 4亚烷基连接的芳基；
“杂芳烷基”是指C 1-C 4亚烷基连接的杂芳基；
本发明的通式I所示化合物可以通过公知的方法制成药学上可接受的盐，该盐是指式I所示化合物与酸或碱混合制成的盐；
适宜的酸加成盐是由形成无毒盐的酸形成。具有代表性的酸加成盐包括但不限于乙酸盐、己二酸盐、藻酸盐、柠檬酸盐、天冬氨酸盐、苯甲酸盐、苯磺酸盐、硫酸氢盐、碳酸氢盐、丁酸盐、樟脑酸盐、樟脑磺酸盐、碳酸盐、柠檬酸盐、二葡糖酸盐(digluconate)、甘油磷酸盐、半硫酸盐(hemisulfate)、庚酸盐、己酸盐、甲酸盐、富马酸盐、葡萄糖酸盐、葡萄糖醛酸盐、谷氨酸盐、盐酸盐、氢溴酸盐、氢碘酸盐、2-羟基乙磺酸盐(isethionate)、乳酸盐、马来酸盐、苹果酸盐、丙二酸盐、甲磺酸盐、烟酸盐、2-萘磺酸盐、烟酸盐(nicotinate)、硝酸盐、乳清酸盐、草酸盐、棕榈酸盐、扑酸盐、果胶酸盐(pectinate)、过硫酸盐、3-苯基丙酸盐、苦味酸盐(picrate)、三甲基乙酸盐(pivalate)、丙酸盐、蔗糖盐、硬脂酸盐、琥珀酸盐、硫酸盐、酒石酸盐、硫氰酸盐、磷酸盐、磷酸氢盐、磷酸二氢盐、对甲苯磺酸盐、三氟乙酸盐及十一酸盐；
碱加成盐可在化合物的最后分离和纯化过程中，通过使含有羧酸的部分与适当的碱(如但不限于药用可接受的金属阳离子的氢氧化物、碳酸盐或碳酸氢盐)或者与氨或有机伯胺、仲胺或叔胺反应原位制备。药用可接受的盐包括但不限于基于碱金属或碱土金属的阳离子，如但不限于锂、钠、钾、钙、镁和铝盐等，以及非毒性季氨和胺阳离子，包括铵、四甲基铵、四乙基铵、甲胺、二甲胺、三甲胺、三乙胺、二乙胺、乙胺等。其它可用于形成碱加成盐的代表性有机胺包括乙二胺、乙醇胺、二乙醇胺、哌啶、哌嗪等；
本文中的“立体异构体”是指本发明化合物或其生理上的衍生物所有可能的立体异构体的形式。除非特别指出，本发明中涉及的化合物的化学命名包括所有可能的立体化学形式的混合物，所属混合物包含基本结构分子的所有对映体和非对映体，以及基本纯净的化合物单个异构体形式，即其中含有低于10％，优选低于5％，特别是低于2％，最优选低于1％的其它异构体。本发明类肽化合物各种立体异构体形式均明显包含于本发明的范围内；
通式I化合物还能以其它被保护的形式或衍生物的形式存在，这些形式对本领域技术人员而言是显而易见的，均应该包含于本发明的范围内；
如上所述的取代基自身还可被一个或多个取代基取代。这样的取代基包括在C.Hansch和A.Leo,Substituent Constants for Correlation Analysis in Chemistry and Biology(1979)中列出的那些取代基；优选的取代基包括烷基、烯基、烷氧基、羟基、硝基、氨基、氨基烷基、氰基、卤素、羧基、硫基、芳基、环烷基、杂芳基、杂环烷基、亚氨基、羟烷基、芳基氧基、芳基烷基及其结合；
本发明化合物可以用本领域已知的方法制成任何药物制剂，以口服、肠胃外、直肠或经肺给药等方式施用于需要这种治疗的患者，用于口服给药时，可制成常规的固体制剂，如片剂、胶囊剂、丸剂、颗粒剂等，也可制成口服液体制剂，如口服溶液剂、口服混悬剂、糖浆剂等。制成口服制剂时，可以加入适宜的填充剂、粘合剂、崩解剂、润滑剂等。用于肠胃外给药时，可制成注射剂，包括注射液、注射用无菌粉末与注射用浓溶液。制成注射剂时，可采用现有制药领域中的常规方法生产，配制注射剂时，可以不加入附加剂，也可根据药物的性质加入适宜的附加剂。用于直肠给药时，可制成栓剂等。用于经肺给药时，可制成吸入剂或喷雾剂等；
本发明化合物的施用量和施用频率可以根据临床医生或药师的判断考虑例如以下的一些因素而作出调整：患者的年龄、健康状况和大小，以及待治疗病征的严重性。一般而言，本发明化合物的总日服剂量范围为每天约0.1至约2000mg，尽管必要时会有变化，这取决于治疗目的、患者和施用途径。在一个实施方案中，剂量为约1至约200mg/天，以单一剂量或以2-4个分离剂量给药。在另一个实施方案中，剂量为约10至约2000mg/天，以单一剂量或以2-4个分离剂量给药。在另一个实施方案中，剂量为约100至约2000mg/天，以单一剂量或以2-4个分离剂量给药。在又另一个实施方案中，剂量为约500至约2000mg/天，以单一剂量或以2-4个分离剂量给药。本发明的化合物、其药学上可接受的盐、酯或溶剂化物或它们的前药或异构体与其它治疗活性物质联合使用时，它们同时、分开或依次给药，可制成单一给药方式的药物组合物。联合使用的其它治疗活性物质的用药量可基于临床上所用的量，并可根据给药对象、给药途径、疾病、组合等适当选择。对其它治疗活性物质的给药形式没有特殊限制，只要在给药时将本发明的化合物和其它治疗活性物质组合即可。
“药物组合物(pharmaceutical composition)”是指含有治疗上显著量的活性药剂的制备物，其以适于给予患者的形式被制备。因此，所述制备物不含有这样量的任何一种组分或多种组分，即，适当谨慎的医疗实施者发现所述制备物不适于给予普通对象。在许多情况下，这种药物组合物是无菌制备物。
本发明中所涉及的“室温”具体的温度范围是20-30℃。
二、N-(2-氨基苯基)甲酰胺基取代的组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂的制备方法
以硼替佐米中间体I为原料，与Boc保护的氨基酸进行酰胺缩合反应，随后脱去保护基；另一方面，取代的二酸单甲酯与邻苯二胺进行酰胺缩合反应，再脱去甲酯，并与上述中间体再进行酰胺缩合反应，得到具有N-(2-氨基苯基)甲酰胺基取代的组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂。
合成路线如下：
Figure PCTCN2020133530-appb-000003
其中，P 2的定义同上述式I所述，R独立的为氢、羟基、卤素、烷基、三氟甲基、氰基、硝基、胍基、氨基、羧基、硝基、含有5或6个环原子的单环芳基或具有8-15个环原子的双环芳基、含有1-2个杂原子的环原子数为5-6的单杂环基中任意一个，A环独立的为芳基、杂芳基、芳烷基、杂芳烷基或环烷基中的任意一个；
反应试剂和反应条件：(a)2位被P2取代的2-Boc-氨基乙酸，TBTU，NMM，室温，4-24h；(b)氯化氢饱和乙酸乙酯，室温，1-24h；(c)3位或4位被R取代的邻苯二胺，TBTU， NMM，避光室温，8h；(d)氢氧化锂，水/甲醇，室温，2-24h；(e)TBTU，NMM，室温，4-24h。
具体操作步骤将在实施例中详细说明。本领域的技术人员可以对上述步骤进行变动以增加收率，他们可以根据本领域的基本知识设计合成路线，如选择反应物、反应溶剂、反应温度，也可以通过使用各种保护基以避免副反应的发生从而提高收率，这些常规的保护方法可以参见例如T.Green Protecting Groups in Organic Synthesis。
三、N-羟基甲酰胺取代的组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂的制备方法
以硼替佐米中间体I为原料，与Boc保护的氨基酸进行酰胺缩合反应，随后脱去保护基；另一方面，取代的二酸单甲酯与O-(四氢-2H-吡喃-2-基)羟胺胺进行酰胺缩合反应，再脱去甲酯，并与上述中间体再进行酰胺缩合反应，最终脱去四氢吡喃保护基，得到具有N-羟基甲酰胺取代的组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂。
合成路线如下：
Figure PCTCN2020133530-appb-000004
其中，P2的定义同上述式I所述，A环独立的为芳基、杂芳基、芳烷基、杂芳烷基或环烷基中的任意一个；
反应试剂和反应条件：(a)2位被P2取代的2-Boc-氨基乙酸，TBTU，NMM，室温，4-24h；(b)氯化氢饱和乙酸乙酯，室温，1-24h；(c)O-(四氢-2H-吡喃-2-基)羟胺，TBTU，NMM，避光室温，4-24h；(d)氢氧化锂，水/甲醇，室温，2-24h；(e)TBTU，NMM，室温，4-24h；(f)氯化氢饱和乙酸乙酯，室温，1-24h。
具体操作步骤将在实施例中详细说明。本领域的技术人员可以对上述步骤进行变动以增加收率，他们可以根据本领域的基本知识设计合成路线，如选择反应物、反应溶剂、反应温度，也可以通过使用各种保护基以避免副反应的发生从而提高收率，这些常规的保护方法可以参见例如T.Green Protecting Groups in Organic Synthesis。
四、N-羟基肉桂酰胺取代的组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂的制备方法
以硼替佐米中间体I为原料，与Boc保护的氨基酸进行酰胺缩合反应，随后脱去保护基；另一方面，具有醛基取代的甲酸甲酯与丙二酸反应，得到肉桂酸取代的甲酸单甲酯，再与O-(四氢-2H-吡喃-2-基)羟胺胺进行酰胺缩合反应，并脱去甲酯，并与上述中间体再进行酰胺缩合反应，最终脱去四氢吡喃保护基，得到具有N-羟基肉桂酰胺取代的组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂。
合成路线如下：
Figure PCTCN2020133530-appb-000005
其中，P2的定义同上述式I所述，A环独立的为芳基、杂芳基、芳烷基、杂芳烷基或环烷基中的任意一个；
反应试剂和反应条件：(a)2位被P 2取代的2-Boc-氨基乙酸，TBTU，NMM，室温，4-24h；(b)氯化氢饱和乙酸乙酯，室温，1-24h；(c)丙二酸，吡啶，N,N-二甲基甲酰胺，加热回流，10-48h；(d)O-(四氢-2H-吡喃-2-基)羟胺，TBTU，NMM，避光室温，4-24h；(e)氢氧化锂，水/甲醇，室温，2-24h；(f)TBTU，NMM，室温，4-24h；(g)氯化氢饱和乙酸乙酯，室温，1-24h。
具体操作步骤将在实施例中详细说明。本领域的技术人员可以对上述步骤进行变动以增加收率，他们可以根据本领域的基本知识设计合成路线，如选择反应物、反应溶剂、反应温度，也可以通过使用各种保护基以避免副反应的发生从而提高收率，这些常规的保护方法可以参见例如T.Green Protecting Groups in Organic Synthesis。
五、组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂的应用
本发明还提供了该系列化合物在制备预防或治疗因组蛋白去乙酰化酶表达异常或蛋白酶体功能异常导致的相关哺乳动物疾病药物中的应用，所述的疾病包括但不限于癌症、神经退行性疾病、炎症、病毒感染、糖尿病、疟疾。
此外，本发明还包括一种适于口服给予哺乳动物的药物组合物，包含上述通式I的任一化合物，药学上可接受的载体，任选包含一种或多种药学上可接受的赋形剂。
此外，本发明还包括一种适用于胃肠外给予哺乳动物的药物组合物，包括上述通式I的任一化合物，药学上可接受的载体，任选包含一种或多种药学上可接受的赋形剂。
进行抑酶活性和细胞活性两方面测试来评价化合物在体外的生物活性。
应用荧光分析法评价化合物对组蛋白去乙酰化酶抑制活性。在评价化合物体外抑制HDAC混酶活性时，HDAC荧光底物(含有一个乙酰化的赖氨酸侧链-Boc-Lys(acetyl)-AMC)用含HDAC活性的样本(Hela细胞核提取液)孵育，使底物去乙酰化，激活底物。之后用胰酶水解Boc-Lys-AMC，产生AMC荧光团，在发射波长/激发波长(390nm/460nm)测定 荧光强度。荧光强度与待测化合物的HDACs抑制作用成比例，运用Prism GraphPad software处理数据进而计算不同目标化合物对HDACs的抑制率和IC 50值。
应用荧光分析法评价化合物对蛋白酶体抑制活性。在评价化合物体外抑制蛋白酶体活性时，蛋白酶体荧光底物(Suc-LLVY-AMC)用含蛋白酶体孵育，使底物水解，释放AMC荧光团，在发射波长/激发波长(390nm/460nm)测定荧光强度。荧光强度与待测化合物的蛋白酶体抑制作用成比例，运用Prism GraphPad software处理数据进而计算不同目标化合物对蛋白酶体的抑制率和IC 50值。
化合物的细胞活性测试使用MTT检测法，肿瘤细胞悬液(人慢性髓原白血病细胞K562，人粒细胞白血病细胞KG1，人早幼粒白血病细胞HL-60，急性T细胞白血病细胞Jurkat，人多发性骨髓瘤细胞RPMI-8226，人结肠癌细胞HCT-116，前列腺癌细胞株PC-3分别接种于96孔板，每孔加入用培养基稀释的不同浓度的化合物，孵育48小时后用MTT染色，继续孵育4h后用酶标仪在490/570nm处测定每孔的吸光度OD值，然后计算出抑制率和IC 50值，从而确定目标化合物的抗增殖活性。
体外抑酶实验结果表明，本发明所述化合物具有较好的HDAC、蛋白酶体抑制活性。体外抗肿瘤细胞增殖实验表明，ZY-2、ZY-11、ZY-12、ZY-13、ZY-14五个化合物对多种肿瘤细胞的抑制活性超过阳性对照药SAHA的三倍，ZY-2、ZY-13对耐硼替佐米的多发性骨髓瘤细胞KM3/BTZ具有远超阳性药的抑制活性。由此可见本分明中的部分化合物拥有较好的HDACs抑制活性、蛋白酶体抑制活性和抗肿瘤细胞增殖活性，具有很大的开发前景，可用于指导发现新型高效的组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂和抗肿瘤活性分子。
具体实施方式：
下面结合实施例对本发明做进一步的说明，但不限于此。
实施例1：叔丁基((S)-1-((((R)-3-甲基-1-((3aR,4R,6R,7aS)-5,5,7a-三甲基六氢-4,6-甲基苯并[d][1,3,2]二氧杂硼醇-2-基)丁基)氨基)-1-氧代-3-苯基丙烷-2-基)氨基甲酸酯(2a)
Boc-L-Phe(3.6g,13mmol)冰浴下溶于N,N-二甲基甲酰胺，并加入TBTU(4.2g,13mmol)，硼替佐米中间体I(4.56g,12mmol)和N-甲基吗啉(4ml,36mmol)。氮气保护下，反应持续搅拌16小时后倒入水中，用乙酸乙酯提取3次(3×100mL)。合并有机相后用2％柠檬酸、2％碳酸氢钠、饱和食盐水洗，用硫酸镁干燥，旋干得到粗产品。经过乙酸乙酯、石油醚梯度(从15％到40％)洗脱的硅胶色谱分离得到6g(97％)的无色透明油状物2a。产率：95％。 1H NMR(400MHz,DMSO-d 6):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).
叔丁基(2-((((R)-3-甲基-1-((3aR，4R，6R，7aS)-5,5,7a-三甲基六氢-4,6-甲基苯并[d][1,3,2]二氧杂硼醇-2-基)丁基)氨基)-2-氧乙基)氨基甲酸酯(2b)
与2a的合成类似，产率：99％。无色油。 1H NMR(400MHz,CDCl 3)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).
叔丁基((S)-3-(1H-吲哚-3-基)-1-(((R)-3-甲基-1-((3aR，4R，6R，7aS)-5,5,7a-三甲基六氢-4,6-甲氧基苯并[d][1,3,2]二氧杂硼醇-2-基)丁基)氨基)-1-氧丙烷-2-基)氨基甲酸酯(2c)
与2a的合成类似，产率：85％。棕色粉末，熔点：174～178℃。 1H NMR(400MHz,DMSO-d 6)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).
实施例2：(S)-1-((((R)-3-甲基-1-((3aR，4R，6R，7aS)-5,5,7a-三甲基六氢-4,6-甲氧基苯并[d][1,3,2]二氧杂硼醇-2-基)丁基)氨基)-1-氧代-3-苯基丙烷-2-氯化铵(3a)
2a溶解于饱和氯化氢的乙酸乙酯，搅拌过夜得到纯的白色粉末3a，产率：90％，熔点：208～212℃。 1H NMR(400MHz,DMSO-d 6)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-甲基-1-((3aR，4R，6R，7aS)-5,5,7a-三甲基六氢-4,6-甲氧基苯并[d][1,3,2]二氧杂硼醇-2-基)丁基)氨基)-2-氧杂-1-胺氯化铵(3b)
与3a的合成类似，产率：98％，无色油状物。 1H NMR(400MHz,DMSO-d 6)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-吲哚-3-基)-1-(((R)-3-甲基-1-(((3aR，4R，6R，7aS)-5,5,7a-三甲基六氢-4，6-甲基苯并[d][1,3,2]二氧杂硼环-2-基)丁基)氨基)-1-氧丙烷-2-氯化铵(3c)
与3a的合成类似，产率：70％，棕色粉末，熔点：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).
实施例3：4-((2-氨基苯基)氨基甲酰基)苯甲酸甲酯(5a)
对苯二甲酸单甲酯(4a,1.8g,10mmol)，邻苯二胺(1.08g,10mmol)，TBTU(3.5g,12mmol)在冰浴下溶于N,N-二甲基甲酰胺。加入Et 3N(1.5ml,30mmol)后避光搅拌8小时。反应结束后 倒入水中，用乙酸乙酯提取3次(3×100mL)。合并有机相后用2％柠檬酸、2％碳酸氢钠、饱和食盐水洗，用硫酸镁干燥，旋干得到粗产品。甲醇重结晶后得到精制的产品5a。产率：56％，熔点：190～194℃。 1H NMR(400MHz,CDCl 3)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-氨基苯基)氨基甲酰基)苯甲酸甲酯(5b)
与3a的合成类似，产率：56％，黄色固体，熔点：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).
实施例4：4-((2-氨基苯基)氨基甲酰基)苯甲酸(6a)
5a(1.52g,5mmol)与氢氧化锂(0.94g,20mmol)溶解在水(20mL)和甲醇(40mL)的混合溶液中搅拌8小时后旋去甲醇，盐酸调至pH＝7，用乙酸乙酯(3×20mL)萃取，硫酸镁干燥，旋干，即得到产物6a，产率75％，熔点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-氨基苯基)氨基甲酰基)苯甲酸(6b)
与6a的合成类似，产率：86％，黄色固体，熔点：200～204℃。 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).
实施例5：N1-(2-氨基苯基)-N4-((S)-1-((((R)-3-甲基-1-((3aR，4R，6R，7aS)-5,5,7a-三甲基六氢-4，6-甲基苯并[d][1,3,2]二氧杂硼醇-2-基)丁基)氨基)-1-氧代-3-苯基丙烷-2-基)对苯二甲酰胺(ZY-2)
3a(0.9g,2mmol)，6a(0.51g,2mmol)和TBTU(0.8g,2.2mmol)在冰浴下溶于N,N-二甲基甲酰胺，随后加入N-甲基吗啉(0.7ml,6mmol)。氮气保护下搅拌8小时后倒入水中，用乙酸乙酯(3×20mL)提取，合并有迹象用5％柠檬酸、饱和碳酸氢钠、饱和食盐水各洗一次，无水硫酸镁干燥后旋干即得到出产品。硅胶柱层析(乙酸乙酯：石油醚＝1:1)纯化得到黄色固体ZY-2，产率75％，熔点：160～162℃。 1H NMR(400MHz,DMSO-d 6)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-d 6)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 C 38H 48BN 4O 5 651.3719,found[M+H] +651.3745.
N1-((S)-3-(1H-吲哚-3-基)-1-(((R)-3-甲基-1-((3aR，4R，6R，7aS)-5,5,7a-三甲基六氢-4,6-甲氧基苯并[d][1,3,2]二氧杂硼-2-(丁基)氨基)氨基)-1-氧丙烷-2-基)-N4-(2-氨基苯基)对苯二甲酰胺(ZY-1)
合成同ZY-2类似，产率：68％，黄色固体，熔点：164～166℃。 1H NMR(400MHz,DMSO-d 6)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 C 40H 48BN 5O 5690.3828,found[M+H] +690.3867.
N1-((S)-3-(1H-吲哚-3-基)-1-(((R)-3-甲基-1-((3aR，4R，6R，7aS)-5,5,7a-三甲基六氢-4,6-亚甲基苯并[d][1,3,2]二氧杂硼-2-(丁基)氨基)氨基)-1-氧丙烷-2-基)-N3-(2-氨基苯基)间苯二甲酰胺(ZY-3)
合成同ZY-2类似，产率：56％，黄色固体，熔点：158～160℃。 1H NMR(400MHz,DMSO-d 6)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 C 40H 48BN 5O 5690.3828,found[M+H] +690.3834
N1-(2-氨基苯基)-N3-((S)-1-((((R)-3-甲基-1-((3aR，4R，6R，7aS)-5,5,7a-三甲基六氢-4，6-甲基苯并[d][1,3,2]二氧杂硼醇-2-基)丁基)氨基)-1-氧代-3-苯基丙烷-2-基)间苯二甲酰胺(ZY-4)
合成同ZY-2类似，产率：65％，黄色固体，熔点：160～163℃。 1H NMR(400MHz,DMSO-d 6)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 C 38H 47BN 4O 5 651.3719,found[M+H] +651.3586.
N1-(2-氨基苯基)-N3-(2-((((R)-3-甲基-1-(((3aR，4R，6R，7aS)-5,5,7a-三甲基六氢-4,6-甲基苯并[d][1,3,2]二氧杂硼环-2-基)丁基)氨基)-2-氧乙基)间苯二甲酰胺(ZY-5)
合成同ZY-2类似，产率：72％，黄色固体，熔点：170～172℃。 1H NMR(400MHz,DMSO-d 6)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 C 31H 41BN 4O 5 561.3248,found[M+H] +561.3120
N1-(2-氨基苯基)-N4-(2-((((R)-3-甲基-1-(((3aR，4R，6R，7aS)-5,5,7a-三甲基六氢-4,6-甲基苯并[d][1,3,2]二氧杂硼环-2-基)丁基)氨基)-2-氧乙基)对苯二甲酰胺(ZY-6)
合成同ZY-2类似，产率：46％，黄色固体，熔点：138～140℃。 1H NMR(400MHz,DMSO-d 6)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 C 31H 41BN 4O 5 561.3248,found[M+H] +561.3063.
实施例6：4-((((四氢-2H-吡喃-2-基)氧基)氨基甲酰基)苯甲酸甲酯(8a)
O-(四氢-2H-吡喃-2-基)羟胺(0.32g,3mmol)，4-(甲氧羰基)苯甲酸(0.49g,3mmol)和TBTU(1.3g,3.3mmol)冰浴下溶解在N,N-二甲基甲酰胺中，随后加入Et 3N(1ml,9mmol)，氮气保护下搅拌8小时，倒入水中，用乙酸乙酯(3×20mL)提取3次，合并有机相，用2％柠檬酸、饱和NaHCO 3溶液、饱和食盐水洗，无水硫酸镁干燥，旋干得粗产品。乙醚溶解后加入正己烷析晶，即得到白色方晶纯品8a，产率：68％，熔点：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).
3-((((四氢-2H-吡喃-2-基)氧基)氨基甲酰基)苯甲酸甲酯(8b)
合成与8a类似，产率：85％，白色方晶，熔点：186～188℃。 1H NMR(400MHz,DMSO-d 6)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).
实施例7：4-(((四氢-2H-吡喃-2-基)氧基)氨基甲酰基)苯甲酸(9a)
8a(0.8g,3mmol)和氢氧化锂(0.48g,9mmol)溶解在水(10mL)和甲醇(20mL)的混合溶液中，搅拌8小时后，旋去甲醇，pH调至4后，用乙酸乙酯提取，无水硫酸镁干燥，旋干即得到白色固体9a，产率：68％，熔点>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-(((四氢-2H-吡喃-2-基)氧基)氨基甲酰基)苯甲酸(9b)
合适与9a类似，产率：83％，白色固体，熔点：242～244℃。 1H NMR(400MHz,DMSO-d 6)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).
实施例8：N1-((S)-3-(1H-吲哚-3-基)-1-(((R)-3-甲基-1-((3aR，4R，6R，7aS)-5,5,7a-三甲基六氢-4,6-甲基苯并[d][1,3,2]二氧杂硼环-2-基)丁基)氨基)-1-氧丙烷-2-基)-N4-羟基对苯二甲酰胺(ZY-7)
3(0.9g,3mmol)，6(0.8g,3mmol)和TBTU(0.8g,3.6mmol)冰浴下溶于N,N-二甲基甲酰胺，加入NMM(4ml,36mmol)后氮气保护下搅拌8小时，倒入水中，用乙酸乙酯(3×20mL)提取3次，合并有机相，用2％柠檬酸、饱和NaHCO 3溶液、饱和食盐水洗，无水硫酸镁干燥，旋干得中间体。将此中间体溶解于饱和氯化氢的乙酸乙酯，搅拌5分钟后过滤，硅胶柱层析纯化，得产物白色固体ZY-7，产率：25％，熔点：180～184℃。 1H NMR(400MHz,DMSO-d 6)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 C 34H 43BN 4O 6 613.3317,found[M-H] -613.3321.
N1-羟基-N4-((S)-1-(((R)-3-甲基-1-(((3aR，4R，6R，7aS)-5,5,7，a-三甲基六氢-4,6-甲基苯并[d][1,3,2]二氧杂硼醇-2-基)丁基)氨基)-1-氧代-3-苯基丙烷-2-基)对苯二甲酰胺(ZY-8)
合成与ZY-7类似，产率：30％，白色固体，熔点：200～202℃。 1H NMR(400MHz,DMSO-d 6)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 C 32H 42BN 3O 6 574.3208,found[M-H] -574.3215.
N1-((S)-3-(1H-吲哚-3-基)-1-(((R)-3-甲基-1-((3aR，4R，6R，7aS)-5,5,7a-三甲基六氢-4,6-甲基苯并[d][1,3,2]二氧杂硼-2-(丁基)氨基)氨基)-1-氧丙烷-2-基)-N3-羟基间苯二甲酰胺(ZY-9)合成与ZY-7类似，产率：33％，紫色固体，熔点：188～190℃。 1H NMR(400MHz,DMSO-d 6)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 C 34H 43BN 4O 6 509.2817,found[M-H] -509.2811.
N1-羟基-N3-((S)-1-(((R)-3-甲基-1-(((3aR，4R，6R，7aS)-5,5,7，a-三甲基六氢-4,6-甲基苯并[d][1,3,2]二氧杂硼醇-2-基)丁基)氨基)-1-氧代-3-苯基丙烷-2-基)间苯二甲酰胺(ZY-10)
合成与ZY-7类似，产率：45％，白色固体，熔点：160～164℃。 1H NMR(400MHz,DMSO-d 6)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 C 32H 42BN 3O 6 574.3208,found[M-H] -574.3203.
实施例9：(E)-3-(4-(甲氧羰基)苯基)丙烯酸(11a)
对醛基苯甲酸甲酯(0.42g,0.5mmol)，丙二酸(0.94g,1.5mmol)，吡啶(0.2mL)和N,N-二甲基甲酰胺(10mL)的混合物在氮气保护下加热到90℃搅拌10小时，反应完毕后用60mL水淬灭，冰浴下过滤固体得到产物白色晶体11a。产率：95％，熔点：175～179℃。 1H NMR(400MHz,DMSO-d 6)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-(甲氧羰基)苯基)丙烯酸(11b)
合成与11a类似，产率：100％，白色固体，熔点：246～248℃。 1H NMR(400MHz,DMSO-d 6)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)-4-(3-氧代-3-((((四氢-2H-吡喃-2-基)氧基)氨基)丙-1-烯-1-基)苯甲酸甲酯(12a)
合成与8a类似，产率：90％，白色固体，熔点132～134℃。 1H NMR(400MHz,DMSO-d 6)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)-3-(3-氧代-3-((((四氢-2H-吡喃-2-基)氧基)氨基)丙-1-烯-1-基)苯甲酸甲酯(12b)
合成与8a类似，产率：68％，白色固体，熔点186～188℃。 1H NMR(400MHz,DMSO-d 6)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-氧代-3-((((四氢-2H-吡喃-2-基)氧基)氨基)丙-1-烯-1-基)苯甲酸(13a)
合成与9a类似，产率：76％，白色固体，熔点：184～186℃。 1H NMR(400MHz,DMSO-d 6)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-氧代-3-((((四氢-2H-吡喃-2-基)氧基)氨基)丙-1-烯-1-基)苯甲酸(13b)
合成与9a类似，产率：25％，白色固体，熔点>250℃。 1H NMR(400MHz,DMSO-d 6)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-(羟氨基)-3-氧代丙-1-烯-1-基)-N-(2-(((((R)-3-甲基-1-((3aR，4R，6R，7aS)-5,5,7a- 三甲基六氢-4,6-甲氧基苯并[d][1,3,2]二氧杂硼醇-2-基)丁基)氨基)-2-氧乙基)苯甲酰胺(ZY-11)
合成与ZY-7类似，产率：20％，黄色固体，熔点：186～188℃。 1H NMR(400MHz,DMSO-d 6)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 C 27H 38BN 3O 6 613.3320,found[M-H] -613.3317.
N-((S)-3-(1H-吲哚-2-基)-1-(((R)-3-甲基-1-((3aR，4R，6R，7aS)-5,5,7a-三甲基六氢-4,6-甲基苯并[d][1,3,2]二氧杂硼烷基-2-基)丁基)氨基)-1-氧丙烷-2-基)-3-((E)-3-(羟基氨基)-3-氧代丙-1-烯-1-基)苯甲酰胺(ZY-12)
合成与ZY-7类似，产率：32％，棕色固体，熔点：182～184℃。 1H NMR(400MHz,DMSO-d 6)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 C 27H 38BN 3O 6 639.3474,found[M-H] -639.3476.
3-(((E)-3-(羟基氨基)-3-氧代丙-1-烯-1-基)-N-(2-(((((R)-3-甲基-1-((3aR，4R，6R，7aS)-5,5,7a-三甲基六氢-4,6-甲氧基苯并[d][1,3,2]二氧杂硼醇-2-基)丁基)氨基)-2-氧乙基)苯甲酰胺(ZY-13)
合成与ZY-7类似，产率：25％，黄色固体，熔点：160～162℃。 1H NMR(400MHz,DMSO-d 6)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 C 27H 38BN 3O 6 510.2895,found [M-H] -510.2891.
N-((S)-3-(1H-吲哚-2-基)-1-(((R)-3-甲基-1-((3aR，4R，6R，7aS)-5,5,7a-三甲基六氢-4,6-甲氧基苯并[d][1,3,2]二氧杂硼烷基-2-基)丁基)氨基)-1-氧丙烷-2-基)-4-((E)-3-(羟基氨基)-3-氧代丙-1-烯-1-基)苯甲酰胺(ZY-14)
合成与ZY-7类似，产率：28％，橘黄色固体，熔点：186～190℃。 1H NMR(400MHz,DMSO-d 6)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 C 27H 38BN 3O 6 639.3474,found[M-H] -639.3479.
目标化合物活性评价
实验例1 目标化合物对组蛋白去乙酰化酶抑制，结果见表1
1.[材料]
目标化合物和阳性对照SAHA、MS-275的储备液(10mM，溶于二甲基亚砜)；海拉细胞核提取物；Boc-Lys-AMC Substrate；HDAC Buffer；胰酶；Trichostatin A(TSA，0.3mM，溶于二甲基亚砜)；96孔板；Thermo Varioskan Flash全波长多功能酶标仪；
2.[方法]
(1)HDAC buffer的配制
称取60.57g的Tris，溶于略少于480mL的蒸馏水中，用浓盐酸调pH＝8后再用蒸馏水补齐至500mL即得1M Tris-HCl储备液。取7.5mL的1M Tris-HCl储备液，加入0.0365g的EDTA，7.31g的NaCl，50mL甘油，用蒸馏水定容至500mL即得HDAC buffer；
(2)Trypsin solution的配制
取25mL 1M Tris-HCl储备液，加入2.92g的NaCl，用蒸馏水定容，临用前再加入适量的胰酶和TSA(使胰酶的浓度为10mg/mL,TSA的浓度为2μM)。
(3)底物溶液的配制
用DMSO溶解底物配制成30mM的储备液，然后用HDAC buffer稀释至300μM，使得DMSO的含量为1％左右。
(4)酶液的稀释
用HDAC buffer将酶液按1：80的比例稀释。
(5)化合物溶液的配制
用HDAC buffer将化合物(待测化合物和阳性对照药SAHA)稀释成5×终浓度
(6)100％和空白的配制与测定
①100％溶液的配制与测定：
50μL HDAC buffer与10μL酶液混合，5min后加入40μL底物在37℃下反应0.5h，然后加入100μL Trypsin solution终止上述反应，并在37℃下反应20min后在(390nm/460nm)测定荧光强度，即得100％吸收；
②空白溶液的配制与测定：
60μL HDAC buffer加入40μL底物后在37℃下反应1h
加入100μL Trypsin solution溶液，并在37℃下反应1h后在(390nm/460nm)测定荧光强度，即得空白吸收；
(7)化合物抑制HDACs活性的测定:
50μL含有药物的HDAC buffer与10μL酶液混合预先孵育5min，加入40μL底物后，在37℃下反应1h，然后再加入100μL Trypsin solution终止上述反应，并在37℃下反应1h后，在(390nm/460nm)测定荧光强度；
(8)利用软件和公式计算抑制率和IC 50值
术语说明：
SAHA：伏立诺他；MS-275：恩替诺他；M：摩尔/升；mM：毫摩尔/升；μM：微摩尔/升；mL：毫升；μL：微升；IC 50：半数抑制浓度；TSA：Trichostatin A，HDAC抑制剂；底物：300μMBoc-Lys-AMC溶液；酶液：海拉细胞核提取物用HDAC buffer稀释80倍得到的溶液。
表1 目标化合物体外抑制HDACs实验结果
Figure PCTCN2020133530-appb-000006
Figure PCTCN2020133530-appb-000007
a表中数据为三次实验的平均值，“±”后的数值表示标准偏差
实验例2 目标化合物对蛋白酶体抑制，结果见表2
1.[材料]
目标化合物和阳性对照硼替佐米的储备液(10mM，溶于二甲基亚砜)；重组人20s蛋白酶体；Suc-LLVY-AMC；蛋白酶体Buffer；96孔板；Thermo Varioskan Flash全波长多功能酶标仪；硼替佐米(bortezomib)。
2.[方法]
与HDACs酶活类似，区别在于无需进行胰酶孵育，加底物孵育后直接进行读板。
表2 目标化合物体外抑制蛋白酶体实验结果
Figure PCTCN2020133530-appb-000008
a表中数据为三次实验的平均值，“±”后的数值表示标准偏差
实验例3 目标化合物抑制肿瘤细胞增殖实验
选取HDAC和蛋白酶体抑制活性较好的目标化合物进行体外抑制肿瘤细胞增殖实验，结果见表3
1.[材料]
HepG2,Mcf-7,K562，KG1,THP-1，HL-60，Jurkat，RPMI-8226,KM3，KM3/BTZ，U266四甲基偶氮唑盐MTT，10％胎牛血清(美国Hyclone公司)，2.5g·L-1胰蛋白酶(美国Gibco公司)，改良型RPMI1640培养基(美国Hyclone公司)，阳性对照药SAHA，Bortezomib，96孔板；
2.[方法]
常规培养细胞，收集对数生长的细胞进行实验；用含胎牛血清10％的RPMI1640培养基将对数生长期的细胞稀释至4×10 4个·mL -1后接种于96孔板中(每孔加100μL)，不加细胞的作为空白孔，之后放于恒温孵育箱(37℃，5％二氧化碳)中培养8小时；加入用培养基配制的目标化合物溶液和阳性药(SAHA)溶液，不加药的作为100％孔，于恒温孵育箱(37℃,5％ 二氧化碳)中培养48小时后加入30μL MTT，四小时后除去孔中液体(悬浮细胞需离心)，加150μL DMSO，最后于恒温摇床中振摇10min用酶标仪于570nm波长处测定每孔的吸光度值，计算抑制率和IC 50值；
100％孔平均OD值-实验孔平均OD值100％孔平均OD值-空白孔平均OD值
表3.目标化合物进行体外抑制肿瘤细胞增殖实验结果
Figure PCTCN2020133530-appb-000009
a表中数据为三次实验的平均值，“±”后的数值表示标准偏差
术语说明：人慢性髓原白血病细胞K562，人乳腺癌细胞Mcf-7，人早幼粒白血病细胞HL-60，急性T细胞白血病细胞Jurkat，人多发性骨髓瘤细胞RPMI-8226，人单核细胞白血病THP-1，人肝癌细胞HepG2。
表4.目标化合物进行体外抑制多发性骨髓瘤细胞增殖实验结果
Figure PCTCN2020133530-appb-000010
a表中数据为三次实验的平均值，“±”后的数值表示标准偏差
术语说明：人多发性骨髓瘤细胞RPMI-8226、U266、KM3，耐硼替佐米的多发性骨髓瘤细胞KM3/BTZ。
结论：
这些化合物具有较好的HDAC抑制活性和蛋白酶体抑制活性，测试的化合物均表现出了较好的抗肿瘤细胞增殖活性，未来，可深入进行活性研究，开发出更有活性的化合物用于制备预防和治疗因组蛋白去乙酰化酶表达异常或蛋白酶体异常而导致的相关哺乳动物的疾病。

Claims (10)

  1. 一种组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂及其药学上可接受的盐或其立体异构体，其特征在于，具有如式I所示的结构：
    Figure PCTCN2020133530-appb-100001
    其中，P 1为N-羟基甲酰胺基或N-羟基肉桂酰胺基取代的芳基、N-羟基甲酰胺基或N-羟基肉桂酰胺基的杂芳基、N-羟基甲酰胺基或N-羟基肉桂酰胺基的芳烷基、N-羟基甲酰胺基或N-羟基肉桂酰胺基的杂芳烷基或N-羟基甲酰胺基或N-羟基肉桂酰胺基的环烷基，或N-(2-氨基苯基)甲酰胺基取代的芳基、N-(2-氨基苯基)甲酰胺基取代的杂芳基、N-(2-氨基苯基)甲酰胺基取代的芳烷基或N-(2-氨基苯基)甲酰胺基取代的杂芳烷基，上述芳基、杂芳基、芳烷基、杂芳烷基或环烷基可被任意取代基w取代；
    取代基w选自羟基、卤素、烷基、三氟甲基、氰基、硝基、胍基、氨基、羧基、硝基、含有5或6个环原子的单环芳基或具有8-15个环原子的双环芳基、含有1-2个杂原子的环原子数为5-6的单杂环芳基；
    P 2独立地是氢、烷基、环烷基、烷芳基、芳基、5-10元饱和或部分饱和的杂环或杂芳基，其中所述芳基、芳烷基、烷芳基或杂环的环部分可被上述取代基w取代；
    Z 1和Z 2独立地是烷基、羟基、烷氧基或芳氧基，或Z 1和Z 2一起形成由二羟基化合物衍生的基团，所述二羟基化合物在链或环中具有由至少两个相连原子分隔的至少两个羟基，所述链或环含有碳原子，和任意性可有可无的一个或多个杂原子，杂原子是N，S或O。
  2. 如权利要求1所述的一种组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂，其特征在于，通式I中：
    P 1为间位或对位取代的N-羟基甲酰胺或N-羟基肉桂酰胺取代的芳基；
    P 2独立的为氢、苄基或1H-吲哚-3-甲基；
    Z 1和Z 2独立地是蒎烷二醇。
  3. 如权利要求1所述的一种组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂，其特征在于，通式I是如下结构的化合物：
    Figure PCTCN2020133530-appb-100002
  4. 如权利要求1中所述具有N-(2-氨基苯基)甲酰胺基取代的组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂的制备方法，包括步骤：
    以硼替佐米中间体I为原料，与Boc保护的氨基酸进行酰胺缩合反应，随后脱去保护基；另一方面，取代的二酸单甲酯与邻苯二胺进行酰胺缩合反应，再脱去甲酯，并与上述中间体再进行酰胺缩合反应，得到具有N-(2-氨基苯基)甲酰胺基取代的组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂；
    合成路线如下：
    Figure PCTCN2020133530-appb-100003
    其中，P 2的定义同权利要求1中式I所述，R独立的为氢、羟基、卤素、烷基、三氟甲基、氰基、硝基、胍基、氨基、羧基、硝基、含有5或6个环原子的单环芳基或具有8-15个环原子的双环芳基、含有1-2个杂原子的环原子数为5-6的单杂环基中任意一个，A环独立的为芳基、杂芳基、芳烷基、杂芳烷基或环烷基中的任意一个；
    反应试剂和反应条件：(a)2位被P2取代的2-Boc-氨基乙酸，TBTU，NMM，室温，4-24h；(b)氯化氢饱和乙酸乙酯，室温，1-24h；(c)3位或4位被R取代的邻苯二胺，TBTU，NMM，避光室温，8h；(d)氢氧化锂，水/甲醇，室温，2-24h；(e)TBTU，NMM，室温，4- 24h。
  5. 如权利要求1中所述具有N-羟基甲酰胺取代的组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂的制备方法，包括步骤：
    以硼替佐米中间体I为原料，与Boc保护的氨基酸进行酰胺缩合反应，随后脱去保护基；另一方面，取代的二酸单甲酯与O-(四氢-2H-吡喃-2-基)羟胺胺进行酰胺缩合反应，再脱去甲酯，并与上述中间体再进行酰胺缩合反应，最终脱去四氢吡喃保护基，得到具有N-羟基甲酰胺取代的组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂；
    合成路线如下：
    Figure PCTCN2020133530-appb-100004
    其中，P2的定义同权利要求1中式I所述，A环独立的为芳基、杂芳基、芳烷基、杂芳烷基或环烷基中的任意一个；
    反应试剂和反应条件：(a)2位被P2取代的2-Boc-氨基乙酸，TBTU，NMM，室温，4-24h；(b)氯化氢饱和乙酸乙酯，室温，1-24h；(c)O-(四氢-2H-吡喃-2-基)羟胺，TBTU，NMM，避光室温，4-24h；(d)氢氧化锂，水/甲醇，室温，2-24h；(e)TBTU，NMM，室温，4-24h；(f)氯化氢饱和乙酸乙酯，室温，1-24h。
  6. 如权利要求1中所述具有N-羟基肉桂酰胺取代的组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂的制备方法，包括步骤：
    以硼替佐米中间体I为原料，与Boc保护的氨基酸进行酰胺缩合反应，随后脱去保护基；另一方面，具有醛基取代的甲酸甲酯与丙二酸反应，得到肉桂酸取代的甲酸单甲酯，再与O-(四氢-2H-吡喃-2-基)羟胺胺进行酰胺缩合反应，并脱去甲酯，并与上述中间体再进行酰胺缩合反应，最终脱去四氢吡喃保护基，得到具有N-羟基肉桂酰胺取代的组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂；
    合成路线如下：
    Figure PCTCN2020133530-appb-100005
    其中，P2的定义同权利要求1中式I所述，A环独立的为芳基、杂芳基、芳烷基、杂芳烷基或环烷基中的任意一个；
    反应试剂和反应条件：(a)2位被P 2取代的2-Boc-氨基乙酸，TBTU，NMM，室温，4-24h；(b)氯化氢饱和乙酸乙酯，室温，1-24h；(c)丙二酸，吡啶，N,N-二甲基甲酰胺，加热回流，10-48h；(d)O-(四氢-2H-吡喃-2-基)羟胺，TBTU，NMM，避光室温，4-24h；(e)氢氧化锂，水/甲醇，室温，2-24h；(f)TBTU，NMM，室温，4-24h；(g)氯化氢饱和乙酸乙酯，室温，1-24h。
  7. 权利要求1-3任一项所述的组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂，或其药学上可接受的盐或其立体异构体在制备预防或治疗因组蛋白去乙酰化酶表达异常或蛋白酶体功能异常导致的相关哺乳动物疾病药物中的应用，所述的疾病包括但不限于癌症、神经退行性疾病、炎症、病毒感染、糖尿病或疟疾。
  8. 一种适用于口服给予哺乳动物的药物组合物，包含权利要求的1-3任一项所述的组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂、其药学上可接受的盐或其立体异构体和一种或多种药学上可接受载体或赋形剂。
  9. 一种适用于胃肠外给予哺乳动物的药物组合物，包含权利要求1-3任一项所述的组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂、其药学上可接受的盐或其立体异构体和一种或多种药学上可接受的载体或赋形剂。
  10. 一种药物组合物，其特征在于，含有权利要求1-3任一项所述的组蛋白去乙酰化酶、蛋白酶体双靶点抑制剂，或其药学上可接受的盐或其立体异构体和一种或多种治疗活性物质，所述治疗活性物质选自其他类的组蛋白去乙酰化酶活性异常表达抑制剂，抗肿瘤药物，类固醇类激素或者化疗剂。
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