WO2022089449A1 - 一种特异性热休克蛋白90α亚型抑制剂制备及其应用 - Google Patents

一种特异性热休克蛋白90α亚型抑制剂制备及其应用 Download PDF

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WO2022089449A1
WO2022089449A1 PCT/CN2021/126517 CN2021126517W WO2022089449A1 WO 2022089449 A1 WO2022089449 A1 WO 2022089449A1 CN 2021126517 W CN2021126517 W CN 2021126517W WO 2022089449 A1 WO2022089449 A1 WO 2022089449A1
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cancer
alkenyl
alkyl
pharmaceutically acceptable
heat shock
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French (fr)
Inventor
宋淳
黄牛
张成城
武大雷
张永辉
张友忠
于晶
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JINAN CHENGCHENG BIOTECHNOLOGY Co Ltd
Shandong University
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JINAN CHENGCHENG BIOTECHNOLOGY Co Ltd
Shandong University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings

Definitions

  • the invention relates to the technical field of specific heat shock protein 90 ⁇ subtype inhibitors, in particular to the preparation and application of a specific heat shock protein 90 ⁇ subtype inhibitor.
  • the heat shock protein Hsp90 not only mediates the self-infinite proliferation of tumor cells without external stimulation, but also stabilizes multiple signaling pathways in the process of carcinogenesis. Therefore, the research on Hsp90 inhibitors is very important.
  • the heat shock protein inhibitors in the prior art have the following problems: the Hsp90 inhibitors currently entering the clinical research stage are not selective for Hsp90 ⁇ and Hsp90 ⁇ , and the inhibition of Hsp90 ⁇ will hinder normal life activities, which may appear in clinical tests. One of the important reasons for toxic side effects.
  • Hsp90 ⁇ and Hsp90 ⁇ have a high degree of sequence homology, and the N-terminal region where many inhibitor binding pockets are located differs by only two amino acids, and most residues are completely conserved, which limits the research on Hsp90 ⁇ subtype-selective inhibitors;
  • As an Hsp90 inhibitor mycin can promote cell differentiation and apoptosis, but the molecule contains benzoquinone fragments, which are highly toxic and have unsatisfactory pharmacokinetic properties; modified 17-AAG is effective against breast cancer, prostate cancer, colon cancer and Animal models of non-small cell lung cancer have antitumor activity, but still have strong toxic and side effects. After further modification, the hepatotoxicity is lower than that of similar compounds. But none of these compounds have the ability to distinguish between different subtypes of Hsp90.
  • Hsp90 selective inhibitors for different subtypes of Hsp90, such as the Grp94 inhibitor 4-Br-BnIm, have been used in animal models for the treatment of hereditary open-angle glaucoma. Its selectivity is more than 50 times greater than that of the ⁇ subtype, and it can be used as a tool compound for evaluating the subtype selectivity of compounds; however, these selective inhibitors also show different degrees of toxic and side effects in clinical tests, and there are also toxic effects on tumor cells. Problems such as weak inhibitory ability have led to the fact that although Hsp90 inhibitors have been studied as antitumor drugs for many years, no compounds have been successfully marketed so far.
  • the purpose of the present invention is to provide the preparation and application of a specific heat shock protein 90 ⁇ subtype inhibitor, the specific heat shock protein 90 ⁇ subtype inhibitor has less toxic and side effects, Can effectively inhibit tumor cell growth.
  • the present invention provides a specific heat shock protein 90 ⁇ subtype inhibitor or a pharmaceutically acceptable salt, solvate or hydrate thereof, which has the structure shown in formula I:
  • R 1 is selected from chlorine, fluorine, bromine, C 1 -C 3 alkyl or 1 to 6 fluorine substituted C 1 -C 3 alkyl;
  • R 2 is selected from chlorine, fluorine, bromine, C 1 -C 3 alkyl or 1 to 6 fluorine substituted C 1 -C 3 alkyl, -(C 1 -C 6 alkenyl)-OH, -(C 1 -C 6 alkyl)-OH, -O-(C 1 -C 6 alkyl), -O-(C 1 -C 6 alkenyl), -(C 1 -C 6 alkenyl)-O-( C 1 -C 6 alkyl), -(C 1 -C 6 alkenyl)-O-(C 1 -C 6 alkenyl)-(C 6 -C 10 aryl), -(C 1 -C 6 alkene) base)-O-(C 1 -C 6 alkenyl)-(C 3 -C 10 cycloalkyl), -(C 1 -C 6 alkenyl)-O-(C 1 -C 6 alkenyl)-( C 3
  • R is selected from oxygen, imino, sulfur or methylene
  • R 4 is selected from hydrogen group, C 1 -C 6 alkyl group, C 2 -C 8 alkenyl group, C 2 -C 8 alkynyl group, C 6 -C 14 aryl group, C 2 -C 9 hetero group Aryl, C 2 -C 9 isocycloalkyl or C 3 -C 8 cycloalkyl;
  • X is selected from nitrogen atoms or carbon atoms
  • Y is selected from nitrogen atom, sulfur atom, oxygen atom or carbon atom;
  • n is selected from 0, 1, 2, 3, 4 or 5.
  • the specific heat shock protein 90 ⁇ isoform inhibitor or a pharmaceutically acceptable salt, solvate or hydrate thereof, is of formula (I') or formula (II' ) shown in the structure:
  • R 1 is selected from chlorine, fluorine, bromine, C 1 -C 3 alkyl or 1 to 6 fluorine substituted C 1 -C 3 alkyl;
  • R 2 is selected from chlorine, fluorine, bromine, C 1 -C 3 alkyl or 1 to 6 fluorine substituted C 1 -C 3 alkyl, -(C 1 -C 6 alkenyl)-OH, -(C 1 -C 6 alkyl)-OH, -O-(C 1 -C 6 alkyl), -O-(C 1 -C 6 alkenyl), -(C 1 -C 6 alkenyl)-O-( C 1 -C 6 alkyl), -(C 1 -C 6 alkenyl)-O-(C 1 -C 6 alkenyl)-(C 6 -C 10 aryl), -(C 1 -C 6 alkene) base)-O-(C 1 -C 6 alkenyl)-(C 3 -C 10 cycloalkyl), -(C 1 -C 6 alkenyl)-O-(C 1 -C 6 alkenyl)-( C 3
  • R 4 is selected from hydrogen group, C 1 -C 6 alkyl group, C 2 -C 8 alkenyl group, C 2 -C 8 alkynyl group, C 6 -C 14 aryl group, C 2 -C 9 hetero group Aryl, C 2 -C 9 isocycloalkyl or C 3 -C 8 cycloalkyl;
  • n is selected from 1 or 2;
  • the specific heat shock protein 90 ⁇ subtype inhibitor or a pharmaceutically acceptable salt, solvate or hydrate thereof is selected from the following compounds:
  • the present invention provides a preparation method of the above-mentioned specific heat shock protein 90 ⁇ subtype inhibitor or a pharmaceutically acceptable salt, solvate or hydrate to prepare the compound of formula (I'):
  • the present invention provides yet another preparation method of the above-mentioned specific heat shock protein 90 ⁇ subtype inhibitor or a pharmaceutically acceptable salt, solvate or hydrate thereof, to prepare formula (II') Compound:
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned specific heat shock protein 90 ⁇ subtype inhibitor or a pharmaceutically acceptable salt, solvate or hydrate thereof.
  • the present invention provides a pharmaceutical preparation comprising the above-mentioned specific heat shock protein 90 ⁇ subtype inhibitor or a pharmaceutically acceptable salt, solvate or hydrate thereof, and a pharmaceutically acceptable Acceptable carrier or adjuvant;
  • the preparation is selected from oral preparations and parenteral preparations, and can be tablets, pills, capsules or injections.
  • the present invention provides the above-mentioned specific heat shock protein 90 ⁇ subtype inhibitor or a pharmaceutically acceptable salt, solvate or hydrate or the above-mentioned pharmaceutical composition in the preparation of inhibiting Hsp90 enzyme activity
  • the application comprises contacting the Hsp90 enzyme with the above-mentioned specific heat shock protein 90 ⁇ subtype inhibitor or a pharmaceutically acceptable salt, solvate or hydrate or the above-mentioned pharmaceutical composition.
  • the present invention provides the above-mentioned specific heat shock protein 90 ⁇ subtype inhibitor or a pharmaceutically acceptable salt, solvate or hydrate or the above-mentioned pharmaceutical composition in the preparation of prevention and/or Use in medicaments for the treatment of cancer or tumors.
  • the cancer or tumor includes but is not limited to bladder cancer, breast cancer, cervical cancer, colon cancer (eg colorectal cancer), esophageal cancer, head and neck cancer, liver cancer, lung cancer (eg small cell lung cancer and non-small cell lung cancer) ), melanoma, myeloma, neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, sarcoma (eg, osteosarcoma), skin cancer (eg, squamous cell carcinoma), stomach cancer, testicular cancer, thyroid cancer and uterine cancer.
  • bladder cancer eg colorectal cancer
  • lung cancer eg small cell lung cancer and non-small cell lung cancer
  • melanoma myeloma
  • neuroblastoma ovarian cancer
  • pancreatic cancer prostate cancer
  • kidney cancer sarcoma (eg, osteosarcoma)
  • skin cancer eg, squamous cell carcinoma
  • stomach cancer testicular cancer
  • thyroid cancer and
  • the present invention provides the above-mentioned specific heat shock protein 90 ⁇ subtype inhibitor or a pharmaceutically acceptable salt, solvate or hydrate or the above-mentioned pharmaceutical composition in the preparation of a cell growth inhibitor.
  • the use in medicine comprising contacting the cell with a specific heat shock protein 90 ⁇ subtype inhibitor or a pharmaceutically acceptable salt, solvate or hydrate thereof or the above-mentioned pharmaceutical composition.
  • the cells are tumor cells, and further preferably, the cells are mammalian tumor cells or human tumor cells.
  • the cells are cancer cells, further preferably, the cells are mammalian cancer cells or human cancer cells.
  • the cancer cells described in this application include, but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer (eg colorectal cancer), esophageal cancer, head and neck cancer, liver cancer, lung cancer (eg small cell lung cancer and non- small cell lung cancer), melanoma, myeloma, neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, sarcoma (eg, osteosarcoma), skin cancer (eg, squamous cell carcinoma), stomach cancer, testicular cancer , thyroid and uterine cancer cells.
  • bladder cancer eg colorectal cancer
  • lung cancer eg small cell lung cancer and non- small cell lung cancer
  • melanoma myeloma
  • neuroblastoma ovarian cancer
  • pancreatic cancer prostate cancer
  • kidney cancer sarcoma (eg, osteosarcoma)
  • skin cancer eg, squamous cell carcinoma
  • stomach cancer testicular cancer
  • the cancer cells are bladder cancer, squamous cell carcinoma, head and neck cancer, colorectal cancer, esophageal cancer, gastric cancer, gynecological cancer, pancreatic cancer, rectal cancer, breast cancer, prostate cancer, female genital cancer, skin cancer, Brain, genitourinary, lymphatic, gastric, laryngeal or lung cancer cells.
  • the cancer cells described in this application are metastatic cancer cells, including but not limited to bladder cancer, breast cancer, cervical cancer, colon cancer (eg colorectal cancer), esophagus cancer, head and neck cancer, liver cancer, lung cancer (eg small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, sarcoma (eg, osteosarcoma), skin cancer (eg, squamous cell carcinoma) ), gastric, testicular, thyroid and uterine cancers.
  • bladder cancer eg colorectal cancer
  • esophagus cancer eg head and neck cancer
  • liver cancer eg small cell lung cancer and non-small cell lung cancer
  • lung cancer eg small cell lung cancer and non-small cell lung cancer
  • melanoma myeloma
  • neuroblastoma ovarian cancer
  • pancreatic cancer prostate cancer
  • kidney cancer s
  • inhibition of cell growth can be measured, for example, by counting the number of cells contacted with the compound of interest, compared to otherwise identical cells not contacted with the compound, or by assaying comprising The size of the tumor in this cell.
  • the number of cells, as well as the size of the cells can be readily assessed using any method known in the art (eg, trypan blue exclusion and cell counting to determine the incorporation of 3H-thymus in nascent DNA into cells. pyrimidine deoxynucleosides).
  • the specific heat shock protein 90 ⁇ subtype inhibitor or a pharmaceutically acceptable salt, solvate or hydrate thereof is provided with little side effects and can effectively inhibit the growth of tumor cells.
  • alkyl in the present invention refers to a straight or branched chain saturated hydrocarbon group.
  • C 1-3 alkyl refers to an alkyl group having 1-3 carbon atoms.
  • C 1-6 alkyl refers to an alkyl group having 1-6, ie 1, 2, 3, 4, 5 or 6 carbon atoms, typically methyl, ethyl, n-propyl, isopropyl base, n-butyl, isobutyl, tert-butyl, pentyl and hexyl, etc.
  • the term "1 to 6 fluorine-substituted C1 - C3 alkyl group” in the present invention refers to a linear or branched saturated hydrocarbon group containing at least one fluorine atom.
  • the term “1 to 6 fluoro-substituted C1 - C3 alkyl” refers to mono- or polyfluoroalkyl having 1-3, ie 1, 2, 3 carbon atoms, typically fluoromethyl , difluoromethyl, trifluoromethyl, 1-fluoroethyl, 2-fluoroethyl, polyfluoroethyl, 1-fluoropropyl, 2-fluoropropyl, 3-fluoropropyl propyl, polyfluoropropyl, 1-fluoroisopropyl, 2-fluoroisopropyl, polyfluoroisopropyl, and the like.
  • alkenyl in the present invention refers to a straight or branched chain hydrocarbon group having at least one carbon-carbon double bond.
  • the alkenyl group has 2-8 carbon atoms.
  • C 2-8 alkenyl refers to an alkenyl group having 2-8 carbon atoms, typically vinyl, propenyl, butenyl, pentenyl, hexenyl, and the like.
  • alkynyl in the present invention refers to a straight or branched chain hydrocarbon group having at least one carbon-carbon triple bond.
  • the alkynyl group has 2-8 carbon atoms.
  • C 2-8 alkynyl refers to an alkynyl group having 2-8 carbon atoms, typically ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.
  • cycloalkyl in the present invention refers to a saturated cyclic hydrocarbon group having 3-8 carbon atoms and having a single ring or multiple condensed rings (including condensed and bridged ring systems), preferably having 3-8 carbon atoms atom.
  • Typical examples of "cycloalkyl” include, but are not limited to, monocyclic structures, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, etc.; and polycyclic structures, such as bicyclo[2.2.1] Heptyl, adamantyl, etc.
  • Preferred in the present invention are cycloalkyl groups having 3 to 8, ie 3, 4, 5, 6, 7 or 8 carbon atoms.
  • heterocyclyl in the present invention refers to a cycloalkyl group as defined herein containing 1, 2, 3 or 4 heteroatoms independently selected from N, O and S, preferably having 3-8, ie A heterocyclyl group of 3, 4, 5, 6, 7 or 8 ring atoms, more preferably a heterocyclyl group having 3 to 6, ie, 3, 4, 5 or 6 ring atoms.
  • Preferred heterocyclyl groups include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, morpholinyl, or piperazinyl, and the like.
  • alkoxy refers to the group alkyl-O-, wherein alkyl is as defined herein.
  • Typical examples of “alkoxy” include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexyloxy, 1,2-dimethylbutoxy, etc.
  • aryl in the present invention refers to a monocyclic, bicyclic or tricyclic aromatic hydrocarbon group having 5-14 carbon atoms, preferably 6-10, ie 6, 7, 8, 9 or 10 carbon atoms .
  • Examples of the aryl group in the present invention include phenyl, naphthyl and the like.
  • heteroaryl in the present invention refers to an aryl group as defined herein wherein at least one carbon atom is replaced by a heteroatom independently selected from N, O and S, preferably having 5-12, ie 5, 6, 7, 8, 9, 10, 11 or 12 ring atoms.
  • heteroaryl examples include, but are not limited to, thienyl, pyridyl, thiazolyl, isothiazolyl, furyl, pyrrolyl, triazolyl, imidazolyl, triazinyl, oxadiazolyl, oxazolyl, Isoxazolyl, pyrazolyl, tetrazolyl, thiadiazolyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, benzofuranyl, benzothienyl, thiadiazolyl, indole base, isoindolyl, pyridazinyl, pyrazinyl, pyrimidinyl, quinolinyl, phthalazinyl, quinoxalinyl, quinazolinyl, imidazopyridyl, thiazolopyridyl, imidazopyridazine base, pteridyl
  • salts refers to a salt of a compound of the present invention that is pharmaceutically acceptable and possesses the desired pharmacological activity of the parent compound.
  • Such salts include: acid addition salts with inorganic acids or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; organic acids such as acetic acid, propionic acid, Caproic acid, Cyclopentanoic acid, Glycolic acid, Pyruvic acid, Lactic acid, Malonic acid, Succinic acid, Malic acid, Maleic acid, Fumaric acid, Tartaric acid, Citric acid, Benzoic acid, Cinnamic acid, Mandelic acid, Methanesulfonic acid acid, ethanesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, camphorsulfonic acid, gluconic acid, glutamic acid, hydroxynaphth
  • solvate in the present invention refers to a substance formed by combining a compound of the present invention with a pharmaceutically acceptable solvent.
  • Pharmaceutically acceptable solvents include, but are not limited to, water, ethanol, acetic acid, and the like. Solvates include stoichiometric amounts and non-stoichiometric amounts of solvates, preferably hydrates.
  • the compounds of the present invention may be crystallized or recrystallized from water or various organic solvents, in which case various solvates may be formed.
  • the solvate of the present invention may be a hydrate.
  • the compounds of the present invention exist as isomers, such as stereoisomers (including enantiomers and diastereomers) and cis-trans isomers. Accordingly, when referring to the compounds of the present invention in this specification, the compounds of the present invention include the compounds of formula I and pharmaceutically acceptable salts, isomers, solvates and hydrates thereof. More specifically, the compounds of the present invention include their single enantiomers, mixtures of enantiomers or mixtures of diastereomers.
  • compound (1) (20 g, 0.12 mol) and sodium hydride (14.4 g, 0.36 mol) were dissolved in 250 ml of anhydrous toluene. After the addition, the temperature was raised to room temperature, stirred for 30 minutes, the resulting suspension was cooled to zero, and then iodine (72 g, 0.28 mol) was slowly added.
  • compound (4) (1.42 g, 3.8 mmol) was dissolved in 15 ml, cooled to minus 78 degrees, and boron trichloride solution (1.0 M in DCM, 15 ml, 15 mol) was added to the above reaction system. After the dropwise addition, the temperature was raised to room temperature for 3 h. Cool down to zero after 3 hours. 10 ml of methanol was added dropwise. After the dropwise addition was completed, the reaction was stirred for 1 h and concentrated to obtain a light yellow solid. 10% sodium acetate solution (20ml) and 50ml of ethyl acetate were added to the crude product, several layers were separated, washed with water (20ml*2) and saturated brine (20ml).
  • the synthesis method of compound (28) refers to the synthesis method of compound (16).
  • the synthesis method of compound (29) refers to the synthesis method of compound (16).
  • the synthesis method of compound (30) refers to the synthesis method of compound (16).
  • the intermediate compound (20 ) of the compound (27) of the present invention was synthesized by the following scheme 3 , wherein R1 is chlorine, R2 is chlorine, and R4 is oxygen:
  • potassium acetate (1.6 g, 16.3 mmol), (2.1 g, 5.4 mmol) compound (22), palladium acetate (450 mg, cat.) and double pinacol diboron (1.5 g, 5.7 mmol) were mixed ) was dissolved in 10 ml of DMF and reacted at 90 degrees for 18 hours. After the reaction, concentrated under reduced pressure, added 50 ml of ethyl acetate, washed three times with 30 ml of water, washed once with 30 ml of saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a light brown viscous substance.
  • EDC hydrochloride (245 mg, 1.3 mmol) EDC hydrochloride, (272 mg, 0.6 mmol) compound (26) and (0.27 ml, 1.9 mmol) triethylamine were dissolved in 5 ml DMF. Stir overnight at room temperature. After the reaction, 30 ml of ethyl acetate was added for extraction 3 times, the organic phases were combined, washed twice with 50 ml of water and 50 ml of saturated brine each, dried over anhydrous sodium sulfate, concentrated, and passed through a silica gel column (EA/PE, 0-50% ) to obtain 217 mg (80%) of a pale yellow solid.
  • EA/PE silica gel column
  • the synthesis method of compound (31) refers to the synthesis method of compound (27).
  • the synthesis method of compound (32) refers to the synthesis method of compound (27).
  • the synthesis method of compound (33) refers to the synthesis method of compound (27).
  • This example provides the determination of the enzyme level activity of the compound of formula I or a pharmaceutically acceptable salt, solvate or hydrate thereof: construction of a screening platform for human Hsp90 ⁇ /Hsp90 ⁇ inhibitors, applying the method of fluorescence polarization (FP), based on The principle is to calculate the fluorescence polarization values in the horizontal and vertical directions for correlation analysis by detecting the molecular weight changes before and after the interaction of fluorescein-labeled small molecules with other molecules. If the binding equilibrium between the fluorescently labeled small molecule and the macromolecule is established, it moves slowly when excited, and the measured fluorescence polarization value will increase.
  • FP fluorescence polarization
  • the measured polarized light value decreased to calculate the polarization value of the sample (polarization value in mP).
  • the fluorescently labeled small molecule used in the present invention is GM-BODIPY (synthesized with reference to the synthetic method described in BMCL, 2003, 13, 3975-3978). Reactions were performed in 384-well black plates using reaction hydrophobin HFB buffer: 50 mM KCl, 5 mM MgCl2 , 20 mM Na2MoO4, 0.01% NP40, 0.1 mg/ml BGG, 2 mM DTT, pH 7.3.
  • the volume of the reaction system is 50ml, including 30nM Hsp90 ⁇ , 30Nm Hsp90 ⁇ , 5nM GM-BODIPY (geldanamycin) and the small molecule compound to be tested of the present invention (formula I compound) or DMSO (dimethyl sulfoxide), and DMSO does not exceed 2 ⁇ .
  • Set blank control group and 5nM GM-BODIPY negative control group, blank control wells only add HFB buffer, react at 4 degrees Celsius for 12-16 hours, use microplate reader to detect, the excitation wavelength of polarized light is 485/20nm, and the emission wavelength is 535/25nm, measured mP value.
  • the inhibition rate was calculated using the following formula:
  • the compound of formula I or its pharmaceutically acceptable salt, solvate or hydrate was determined by the method of fluorescence polarization (FP), and the inhibition rate of the compound at different concentrations was calculated, so as to complete the determination of the activity at the enzyme level .
  • FP fluorescence polarization
  • This example also provides the binding Kd of the compound of formula I or a pharmaceutically acceptable salt, solvate or hydrate thereof to Hsp90 ⁇ /Hsp90 ⁇ protein:
  • Intrinsic fluorescence measurements were performed with a SprectraMax M5 spectrophotometer. Test compounds were diluted to 20 ⁇ M in assay buffer pH 7.4 containing 20 mM Hepes and 50 mM NaCl. Recombinant Hsp90 ⁇ and Hsp90 ⁇ proteins were added to the KU675 solution, respectively, adjusted to design concentrations (0 to 100 ⁇ g/mL for Hsp90 ⁇ and 0 to 140 ⁇ g/mL for Hsp90 ⁇ ), and incubated for 30 minutes before measurement. All measurements were performed at 25°C and repeated three times. The excitation wavelength was 345 nm and the emission was monitored from 350 nm to 600 nm. Concentration-dependent binding curves were analyzed using nonlinear fitting using GraphPad Prism 5 software, and binding affinity (Kd) was determined accordingly.
  • the binding Kd value of the compound of formula I or its pharmaceutically acceptable salt, solvate or hydrate and Hsp90 ⁇ /Hsp90 ⁇ protein is obtained.
  • This example also provides a cellular level activity assay for a compound of formula I or a pharmaceutically acceptable salt, solvate or hydrate thereof:
  • the selected cell lines of the present invention include: HCT116, HT-29, MCF-7 , MDA-MB-231, H460, A549, ovca3, SKOV-3, pc-3, Hela, U87, Hep G2, Vero, B16, SGC-7901, HL-60, JAK3STAT5, K562/ADR, BEL7404, TE- 1. ZR-75-30, H1975, BT474, U266, K562, A375, Caki-1, SW620, MCF7, Molt-4, MDA-MB-435s,
  • Cell Counting Kit-8 (CCK-8) cytotoxicity screening assay.
  • Cell Counting Kit-8(CCK-8) is based on WST-8(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4 -disulfophenyl)-2H-tetrazolium monosodium salt)-based detection method, the specific steps are as follows: the tumor cell suspension is inoculated in a 96-well cell culture plate at a concentration of 5000/well, and cultured for 24 hours (37 °C, 5% CO 2 ).
  • the inhibition rate of the compound of formula I or its pharmaceutically acceptable salt, solvate or hydrate on tumor cell growth is obtained, and the compound of the present invention can effectively inhibit the growth of tumor cells.
  • This example also provides the selectivity of a compound of formula I, or a pharmaceutically acceptable salt, solvate or hydrate thereof, for the Hsp90 ⁇ isoform:
  • Cells (eg, NCI-H23, etc.) were collected in cold PBS and lysed with a lysis buffer containing a mammalian protein extraction reagent containing protease and phosphatase inhibitors for 1 hour on ice. Lysates were clarified at 15,000 g for 20 minutes at 4°C. Protein concentration was determined using the Qubit protein quantification kit according to the manufacturer's instructions (ThermoFisher). Equal amounts of proteins (2.5-20 ⁇ g) were electrophoresed in 10% acrylamide gels under reducing conditions, transferred to polyvinylidene fluoride membranes (PVDF), and immunoblotted with corresponding specific antibodies.
  • PVDF polyvinylidene fluoride membranes
  • Membranes were incubated with an appropriate horseradish peroxidase-conjugated secondary antibody and visualized with a chemiluminescent substrate. Data were first converted to 8-bit images in ImageJ, and then density measurements were performed with Image Studio Lite Ver 5.2 or Li-COR Odyssey Image Studio Ver 4.0. Controls were actin and DMSO.
  • the His6-tagged human Hsp90 ⁇ N-terminal domain (amino acids 1-218) was cloned into a modified pET vector, overexpressed in E. coli BL21DE3 cells and purified by Ni-NTA chromatography.
  • the tag was cleaved using TEV protease, followed by a second Ni-NTA chromatography to remove the TEV and his6 tag moieties.
  • the effluent containing the cleaved protein was concentrated and further purified by Superdex 200 size exclusion chromatography in 20 mM Tris-HCl, 150 mM NaCl (pH 7.8).

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Abstract

一种特异性热休克蛋白90α亚型抑制剂制备及其应用，具有式I结构，其中：R1选自氯、氟、溴、C1-C3的烷基或1至6的氟取代C1-C3；R2选自氯、氟、溴、C1-C3的烷基或1至6的氟取代C1-C3的烷基、-(C1-C6烯基)-OH、-(C1-C6烷基)-OH、-O-(C1-C6烷基)、-O-(C1-C6烯基)、-(C1-C6烯基)-O-(C1-C6烷基)、-(C1-C6烯基)-O-(C1-C6烯基)-(C6-C10芳基)、-(C1-C6烯基)-O-(C1-C6烯基)-(C3-C10环烷基)、-(C1-C6烯基)-O-(C1-C6烯基)-(C3-C10杂环)、-(C1-C6烯基)-O-(C2-C6烯基)、-O-(C1-C6烯基)-(C3-C10杂环)、-O-(C2-C6烷基)-(C3-C10杂环)或C1-C8烷氧基；R3选自氧、亚氨基、硫、亚甲基；R4选自氢基、C1-C6的烷基、C2-C8的烯基或炔基、C6-C14的芳基、C2-C9的杂芳基或异环烷基、C3-C8的环烷基；X：氮或碳原子；Y为氮、硫、氧或碳原子；n选自0-5。

Description

一种特异性热休克蛋白90α亚型抑制剂制备及其应用 技术领域
本发明涉及特异性热休克蛋白90α亚型抑制剂技术领域，具体涉及一种特异性热休克蛋白90α亚型抑制剂制备及其应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
热休克蛋白Hsp90既介导肿瘤细胞在无外界刺激情况下的自我无限增殖，又能稳定癌变过程中的多条信号通路，因此针对Hsp90的抑制剂研究非常重要。
现有技术中的热休克蛋白抑制剂存在以下问题：目前进入临床研究阶段的Hsp90抑制剂都对Hsp90α和Hsp90β没有选择性，而对Hsp90β的抑制会妨碍正常生命活动，这可能是临床测试中出现毒副作用的重要原因之一。Hsp90α和Hsp90β具有高度的序列同源性,许多抑制剂结合口袋所在的N末端区域只有两个氨基酸不同，且多数残基完全保守，使得Hsp90α亚型选择性抑制剂的研究受到限制；格尔德霉素作为Hsp90抑制剂，能促使细胞分化和凋亡，但分子中含有苯醌片断，毒性大且药动学性质不理想；经修饰得17-AAG，对乳腺癌、前列腺癌、结肠癌和非小细胞型肺癌的动物模型均具有抑瘤活性，但仍具有较强毒副作用，经进一步修饰肝毒性低于同类化合物。但是这些化合物都不具有区分不同亚型Hsp90的能力。
现阶段中针对Hsp90不同亚型的选择性抑制剂如Grp94抑制剂4-Br-BnIm被应用于治疗遗传性开角型青光眼的动物模型中，最新报道的Hsp90β选择性抑制剂KUNB31对β亚型的选择性比α亚型大50多倍，可以作为评价化合物亚型选择性的工具化合物；但是这些选择性抑制剂在临床测试中也都表现出不同程度的毒副作用，并且也存在对肿瘤细胞抑制能力弱等问题，导致虽然Hsp90抑制剂作为抗肿瘤药物的研究已进行多年，但是至今仍没有化合物成功上市。
发明内容
为了解决现有技术中存在的技术问题，本发明的目的是提供一种特异性热休克蛋白90α亚型抑制剂制备及其应用，所述特异性热休克蛋白90α亚型抑制剂毒副作用小，能够有效抑制肿瘤细胞生长。
具体地，本发明的技术方案如下所述：
在本发明的第一方面，本发明提供一种特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物，其具有式I所示结构：
Figure PCTCN2021126517-appb-000001
其中：
R 1选自氯、氟、溴、C 1-C 3的烷基或1至6的氟取代C 1-C 3的烷基；
R 2选自氯、氟、溴、C 1-C 3的烷基或1至6的氟取代C 1-C 3的烷基、-(C 1-C 6烯基)-OH、-(C 1-C 6烷基)-OH、-O-(C 1-C 6烷基)、-O-(C 1-C 6烯基)、-(C 1-C 6烯基)-O-(C 1-C 6烷基)、-(C 1-C 6烯基)-O-(C 1-C 6烯基)-(C 6-C 10芳基)、-(C 1-C 6烯基)-O-(C 1-C 6烯基)-(C 3-C 10环烷基)、-(C 1-C 6烯基)-O-(C 1-C 6烯基)-(C 3-C 10杂环)、-(C 1-C 6烯基)-O-(C 2-C 6烯基)、-O-(C 1-C 6烯基)-(C 3-C 10杂环)、-O-(C 2-C 6烷基)-(C 3-C 10杂环)或C 1-C 8烷氧基；
R 3选自氧、亚氨基、硫或亚甲基；
R 4选自氢基、C 1-C 6的烷基、C 2-C 8的烯基、C 2-C 8的炔基、C 6-C 14的芳基、C 2-C 9的杂芳基、C 2-C 9的异环烷基或C 3-C 8的环烷基；
X选自氮原子或碳原子；
Y选自氮原子、硫原子、氧原子或碳原子；
n选自0，1，2，3，4或5。
在一种或多种实施方式中，所述特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物，其具有式(I’)或式(II’)所示结构：
Figure PCTCN2021126517-appb-000002
优选地，R 1选自氯、氟、溴、C 1-C 3的烷基或1至6的氟取代C 1-C 3的烷基；
R 2选自氯、氟、溴、C 1-C 3的烷基或1至6的氟取代C 1-C 3的烷基、-(C 1-C 6烯基)-OH、-(C 1-C 6烷基)-OH、-O-(C 1-C 6烷基)、-O-(C 1-C 6烯基)、-(C 1-C 6烯基)-O-(C 1-C 6烷基)、-(C 1-C 6烯基)-O-(C 1-C 6烯基)-(C 6-C 10芳基)、-(C 1-C 6烯基)-O-(C 1-C 6烯基)-(C 3-C 10环烷基)、-(C 1-C 6烯基)-O-(C 1-C 6烯基)-(C 3-C 10杂环)、-(C 1-C 6烯基)-O-(C 2-C 6烯基)、-O-(C 1-C 6烯基)-(C 3-C 10杂环)、-O-(C 2-C 6烷基)-(C 3-C 10杂环)或C 1-C 8烷氧基；
R 4选自氢基、C 1-C 6的烷基、C 2-C 8的烯基、C 2-C 8的炔基、C 6-C 14的芳基、C 2-C 9的杂芳基、C 2-C 9的异环烷基 或C 3-C 8的环烷基；
n选自1或2；
优选地，所述特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物选自下列化合物：
(Z)-1 2-氨-2 4,2 6-二氯-3-氧-8-氮杂-1(4,6)-吡咯并[2,1-f][1,2,4]三嗪-2(1,2)-苯并环非氮杂-9-酮；
(Z)-1 2-氨基-2 4,2 6-二氯-3-氧-7-氮-1(4,6)-吡咯并[2,1-f][1,2,4]三嗪-2(1,2)-苯并环非氮杂-8-酮；
(Z)-1 2-氨-2 4,2 6-二氯-3-氧-8-氮杂-1(4,6)-吡咯并[2,1-f][1,2,4]三嗪-2(1,3)-苯并环非氮杂-9-酮；
(Z)-1 2-氨基-2 4,2 6-二氯-3-氧-7-氮-1(4,6)-吡咯并[2,1-f][1,2,4]三嗪-2(1,3)-苯并环非氮杂-8-酮；
(Z)-1 2-氨基-2 4,2 6-二氯-3-氧-8-氮-1(4,6)-噻吩并[2,3-d]嘧啶-2(1,2)-苯并环非氮杂-9-酮；
(Z)-1 2-氨基-2 4,2 6-二氯-3-氧-7-氮-1(4,6)-噻吩并[2,3-d]嘧啶-2(1,2)-苯并环非氮杂-8-酮；
(Z)-1 2-氨基-2 4,2 6-二氯-3-氧-8-氮-1(4,6)-噻吩并[2,3-d]嘧啶-2(1,3)-苯并环非氮杂-9-酮；
(Z)-1 2-氨基-2 4,2 6-二氯-3-氧-7-氮-1(4,6)-噻吩并[2,3-d]嘧啶-2(1,3)-苯并环非氮杂-8-酮。
在本发明的第二方面，本发明提供一种上述特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物的制备方法制备式(I’)化合物：
以
Figure PCTCN2021126517-appb-000003
为原料制备中间体
Figure PCTCN2021126517-appb-000004
以
Figure PCTCN2021126517-appb-000005
为原料制备中间体
Figure PCTCN2021126517-appb-000006
中间体(5)和(11)反应生成式(I’)化合物；
具体地，化合物(16)：(Z)-1 2-氨-2 4,2 6-二氯-3-氧-8-氮杂-1(4,6)-吡咯并[2,1-f][1,2,4]三嗪-2(1,2)-苯并环非氮杂-9-酮反应过程如下所示：
Figure PCTCN2021126517-appb-000007
Figure PCTCN2021126517-appb-000008
在本发明的第三方面，本发明提供又一种上述特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物的制备方法，制备式(II’)化合物：
以
Figure PCTCN2021126517-appb-000009
为原料制备中间体
Figure PCTCN2021126517-appb-000010
以
Figure PCTCN2021126517-appb-000011
为原料制备中间体
Figure PCTCN2021126517-appb-000012
中间体(20)和(22)反应生成式(II’)化合物；
具体地，化合物(27)(Z)-1 2-氨基-2 4,2 6-二氯-3-氧-8-氮-1(4,6)-噻吩并[2,3-d]嘧啶-2(1,2)-苯并环非氮杂-9-酮反应过程如下所示：
Figure PCTCN2021126517-appb-000013
Figure PCTCN2021126517-appb-000014
在本发明的第四方面，本发明提供一种药物组合物，其包含上述特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物。
在本发明的第五方面，本发明提供一种药物制剂，其包含上述特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物，，以及药学上可接受的载体或辅料；所述制剂选自口服制剂和肠胃外给药制剂，可以为片剂、丸剂、胶囊或注射剂。
在本发明的第六方面，本发明提供一种上述特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物或上述药物组合物在制备抑制Hsp90酶活性的药物中的应用，所述应用包括使Hsp90酶与上述特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物或上述药物组合物在接触。
在本发明的第七方面，本发明提供一种上述特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物或上述药物组合物在制备预防和/或治疗癌症或肿瘤的药物中的应用。
优选的，所述癌症或肿瘤包括但不限于膀胱癌、乳癌、子宫颈癌、结肠癌(例如结肠直肠癌)、食管癌、头颈癌、肝癌、肺癌(例如小细胞肺癌和非-小细胞肺癌)、黑素瘤、骨髓瘤、成神经细胞瘤、卵巢癌、胰腺癌、前列腺癌、肾癌、肉瘤(例如骨肉瘤)、皮肤癌(例如鳞状细胞癌)、胃癌、睾丸癌、甲状腺癌和子宫癌。
在本发明的第八方面，本发明提供一种上述特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物或上述药物组合物在制备抑制细胞生长的药物中的应用，该应用包括使所述细胞与特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物或上述药物组合物接触。
优选的，所述细胞是肿瘤细胞，进一步优选的，所述细胞是哺乳动物肿瘤细胞或人肿瘤细胞。
或者，所述细胞是癌细胞，进一步优选的，所述细胞是哺乳动物癌细胞或人癌细胞。
优选的，该应用中所述的癌细胞包括但不限于膀胱癌、乳癌、子宫颈癌、结肠癌(例如结肠直肠癌)、食管癌、头颈癌、肝癌、肺癌(例如小细胞肺癌和非-小细胞肺癌)、黑素瘤、骨髓瘤、成神经细胞瘤、卵巢癌、胰腺癌、前列腺癌、肾癌、肉瘤(例如骨肉瘤)、皮肤癌(例如鳞状细胞癌)、胃癌、睾丸癌、甲状腺癌和子宫癌的细胞。
进一步优选的，所述癌细胞是膀胱癌、鳞状细胞癌、头颈癌、结肠直肠癌、食管癌、胃癌、妇科癌、胰腺癌、直肠癌、乳癌、前列腺癌、女阴癌、皮肤癌、脑癌、生殖泌尿道癌、淋巴系统癌、胃癌、喉癌或肺癌的细胞。
优选的，该应用中所述的癌细胞是转移性癌细胞，包括但不限于膀胱癌、乳癌、子宫颈癌、结肠癌(例如结肠直肠癌)、食管癌、头颈癌、肝癌、肺癌(例如小细胞肺癌和非-小细胞肺癌)、黑素瘤、骨髓瘤、成神经细胞瘤、卵巢癌、胰腺癌、前列腺癌、肾癌、肉瘤(例如骨肉瘤)、皮肤癌(例如鳞状细胞癌)、胃癌、睾丸癌、甲状腺癌和子宫癌的细胞。
优选的，细胞生长的抑制作用可以被计量，计量方式是通过，例如，对与感兴趣的化合物接触的细胞数进行计数，与其它方面相同但不与该化合物接触的细胞进行比较，或者测定包含该细胞的肿瘤的大小。细胞数以及细胞的大小可以容易地使用本领域已知的任何方法进行评价(例如，台盼蓝排除法(trypan blue exclusion)和细胞计数，测定掺入到细胞中的新生DNA中的3H-胸腺嘧啶脱氧核苷)。
本发明的具体实施方式具有以下有益效果：
本发明实施方式中提供特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物毒副作用小，能够有效抑制肿瘤细胞生长。
具体实施方式
本申请中出现的术语定义如下：对于特定的术语，如果本申请中定义的含义与本领域技术人员通常理解的含义不一致，则以本申请中定义的含义为准；如果在本申请中没有定义，则其具有本领域技术人员通常理解的含义。
本发明中的术语“烷基”是指直链或支链饱和烃基。术语“C 1-3烷基”是指具有1-3个碳原子的烷基。术语“C 1-6烷基”是指具有1-6，即1、2、3、4、5或6个碳原子的烷基，典型地为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、戊基和己基等。
本发明中的术语“1至6的氟取代C 1-C 3烷基”是指至少含有一个氟原子的直链或支链饱和烃基。术语“1至6的氟取代C 1-C 3烷基”是指具有1-3，即1、2、3个碳原子的单氟代或多氟代烷基，典型地为氟代甲基、二氟代甲基、三氟甲基、1-氟代乙基、2-氟代乙基、多氟代乙基、1-氟代丙基、2-氟代丙基、3-氟代丙基、多氟代丙基、1-氟代异丙基、2-氟代异丙基、多氟代异丙基等。
本发明中的术语“烯基”是指直链或支链的具有至少一个碳碳双键的烃基。所述烯基具有2-8个碳原子。术语“C 2-8烯基”是指具有2-8碳原子的烯基，典型地为乙烯基、丙烯基、丁烯基、戊烯基和己烯基等。
本发明中的术语“炔基”是指直链或支链的具有至少一个碳碳叁键的烃基。所述炔基具有2-8个碳原子。术语“C 2-8炔基”是指具有2-8个碳原子的炔基，典型地为乙炔基、丙炔基、丁炔基、戊炔基、己炔基等。
本发明中的术语“环烷基”是指具有3-8个碳原子并且具有单环或多个稠合环(包括稠合和桥连环系)的饱和环状烃基，优选具有3-8碳原子。“环烷基”的典型实例包括但不限于单环结构，诸如环丙基，环丁基，环戊基，环己基，环辛基等；和多环结构，诸如二环[2.2.1]庚基，金刚烷基等。本发明中优选的是具有3-8，即3、4、5、6、7或8个碳原子的环烷基。
本发明中的术语“杂环基”是指包含1、2、3或4个独立地选自N、O和S的杂原子的如本文所定义的环烷基，优选具有3-8，即3、4、5、6、7或8个环原子的杂环基，更优选具有3-6，即3、4、5或6个环原子的杂环基。优选的杂环基包括但不限于吡咯烷基、四氢呋喃基、四氢噻吩基、哌啶基、吗啉基或哌嗪基等。
本文所用的术语“烷氧基”是指基团烷基-O-，其中烷基如本文中所定义。“烷氧基”的典型实例包括但不限于甲氧基，乙氧基，正丙氧基，异丙氧基，正丁氧基，叔丁氧基，仲丁氧基，正戊氧基，正己氧基，1,2-二甲基丁氧基等。
本发明中的术语“芳基”是指具有5-14个碳原子的单环、二环或三环芳族烃基，优选具有6-10，即6、7、8、9或10个碳原子。本发明中的芳基的实例包括苯基、萘基等。
本发明中的术语“杂芳基”是指至少一个碳原子被独立地选自N、O和S的杂原子替代的如本文所定义的芳基，优选具有5-12，即5、6、7、8、9、10、11或12个环原子。“杂芳基”的实例包括但不限于噻吩基、吡啶基、噻唑基、异噻唑基、呋喃基、吡咯基、三唑基、咪唑基、三嗪基、噁二唑基、噁唑基、异噁唑基、吡唑基、四唑基、噻二唑基、苯并咪唑基、苯并噁唑基、苯并噻唑基、苯并呋喃基、苯并噻吩基、硫茚基、吲哚基、异吲哚基、哒嗪基、吡嗪基、嘧啶基、喹啉基、酞嗪基、喹喔啉基、喹唑啉基、咪唑并吡啶基、噻唑并吡啶基、咪唑并哒嗪基、蝶啶基、苯并三唑基、吡唑并吡啶基等。
本发明中的术语“药学上可接受的盐”是指在制药上可接受的并且具有母体化合物的所需药理学活性的本发明化合物的盐。这类盐包括：与无机酸或与有机酸形成的酸加成的盐，所述的无机酸诸如盐酸，氢溴酸，硫酸，硝酸，磷酸等；所述的有机酸诸如乙酸，丙酸，己酸，环戊丙酸，乙醇酸，丙酮酸，乳酸，丙二酸，琥珀酸，苹果酸，马来酸，富马酸，酒石酸，柠檬酸，苯甲酸，肉桂酸，扁桃酸，甲磺酸，乙磺酸，苯磺酸，萘磺酸，樟脑磺酸，葡糖酸，谷氨酸，羟基萘甲酸，水杨酸，硬脂酸等；或在母体化合物上存在的酸性质子被金属离子，例如碱金属离子或碱土金属离子取代时形成的盐；或与有机碱形成的配位化合物，所述的有机碱诸如乙醇胺，二乙醇胺，三乙醇胺，N-甲基葡糖胺等。
本发明中的术语“溶剂合物”是指本发明化合物与制药上可接受的溶剂结合形成的物质。制药上可接受的溶剂包括但不限于水，乙醇，乙酸等。溶剂合物包括化学计算量的溶剂合物和非化学计算量的溶剂合物，优选为水合物。本发明的化合物可以用水或各种有机溶剂结晶或重结晶，在这种情况下，可能形成各种溶剂合物。本发明的溶剂合物可以为水合物。
本领域的技术人员能够理解，本发明的化合物存在异构体，例如立体异构体(包括对映异构体和非对映异构体)和顺反异构体。因此，本说明书中当提及本发明的化合物时，本发明化合物包括所述式I的化合物及 其药学上可接受的盐、异构体、溶剂合物和水合物。更具体地，本发明的化合物包括其单一对映异构体、对映异构体的混合物或非对映异构体的混合物。
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
对于以下所有的实施例，可以使用本领域技术人员已知的标准操作和纯化方法。除非另有指明，所有温度以℃(摄氏度)表示。所有反应均是在室温下进行的，除非另有指明。在以下实施例1至4中描述的合成方法欲意通过使用具体实例来举例说明可用的化学方法，并且不表示本发明的范围。
实施例1
(Z)-1 2-氨-2 4,2 6-二氯-3-氧-8-氮杂-1(4,6)-吡咯并[2,1-f][1,2,4]三嗪-2(1,2)-苯并环非氮杂-9-酮(化合物16)的制备：
通过以下方案1合成了本发明式(16)化合物的中间体，其中R 1为氯，R 2为氯，R 4为氧：
Figure PCTCN2021126517-appb-000015
化合物(2)的合成：
在氮气保护及零度下，将化合物(1)(20g，0.12mol)和氢化钠(14.4g，0.36mol)溶于250ml无水甲苯中。加完后，升温至室温，搅拌30分钟，将生成的悬浮液降温至零度，然后慢慢加入碘(72g，0.28mol)。然后，升温至室温，搅拌反应24小时后，依次用250ml的1N盐酸溶液淬灭反应混合物，用300ml乙酸乙酯萃取，分层后，用100ml饱和氯化钠水溶液洗，无水硫酸钠干燥过夜，浓缩，硅胶柱纯化(EA/PE,0-20％),得到白色固体24g(70％)。
化合物(3)的合成
将化合物(2)(6.7g，23mmol)，碳酸钾(38g，27.6mmol)溶于100ml无水乙腈中，将溴卞(3ml，25mmol)滴加到上述反应液中，滴加完毕后，回流18h。反应结束后，冷却至室温，加入250ml，用二氯甲烷(100ml)萃取水相。合并有机相，用2N的氢氧化钠(100ml)洗，水洗(100ml*2)，饱和食盐水洗(100ml)，无水硫酸钠干燥，浓缩，过硅胶柱(EA/PE,0-30％)，得到黄色固体7g(80％)。
化合物(4)的合成：
氮气保护下，将(7.7g，22mmol)化合物(3)，联硼酸频那醇酯(5.7ml，44mmol)和三乙胺(9.2ml，66mmol)溶于50无水二氧六环溶液中，然后加入醋酸钯(270mg，1.1mmol)和(850mg，2.2mmol)联苯-2-基-二环己基膦加入到反应体系中，80度下反应一个半小时。反应结束后，冷却，反应液加入50ml乙酸乙酯，随后用25ml饱和氯化铵，25ml水，25ml饱和食盐水洗涤。无水硫酸钠干燥，浓缩。过硅胶柱(EA/PE,0-50％)，得到棕色固体3.6g(46％)。
化合物(5)的合成：
在氮气保护下，将化合物(4)(1.42g，3.8mmol)溶于15ml，降温至零下78度，将三氯化硼溶液(1.0M in DCM，15ml，15mol)加入上述反应体系中。滴加完毕后，升温至室温反应3h。3h后降温至零度。滴加10ml甲醇，滴加完毕后，搅拌反应1h，浓缩，得浅黄色固体。向粗品中加入10％的醋酸钠溶液(20ml)，乙酸乙酯50ml，分离有几层，用水(20ml*2)，饱和食盐水洗(20ml)。无水硫酸钠干燥，浓缩。得到的粗品用正己烷洗涤，抽滤，干燥，得到浅棕色固体930mg(85％)。 1H-NMR：(400MHz，MeOD)δ6.88-6.89(d,1H),6.73-6.74(d,1H),1.30(s,12H)。
通过以下方案2合成了本发明式(16)化合物，其中R 3为氧，R 4为氢，X为氮原子，Y为亚甲基：
Figure PCTCN2021126517-appb-000016
化合物(7)的合成：
将(9.1g，80mmol)异氰酸乙酸乙酯和(18.4g，120mmol)1,8-二氮杂二环十一碳-7-烯(DBU)溶于400ml四氢呋喃中。在搅拌条件下，向反应体系中加入160mmol的p型甲醇，升温至50度反应5小时。反应结束后，冷却至室温，加入1000ml乙酸乙酯，用300ml饱和碳酸氢钠洗，300ml饱和食盐水洗，无水硫酸钠干燥，浓缩，得到6.1g淡黄色固体。不经纯化直接进行下一步反应。
1H-NMR：(400MHz，DMSO-D 6)δ1.52-1.58(t,6H),2.44-2.54(m,4H),4.33(br,1H),7.10(s,1H),8.24(s,1H)。
化合物(8)的合成：
将(9.0g，42.7mmol)化合物(7)，氯化铵(6.9g，130.2mmol)溶于2N氢氧化钠溶液(42.9ml，85.8mmol)和600ml甲基叔丁基醚，搅拌条件下，加入120ml浓氨水和90ml次氯酸钠水溶液，室温下搅拌反应6小时。反应结束后，过滤，向滤液中加入1500ml乙酸乙酯，分别用500ml饱和碳酸氢钠，500ml饱和食盐水洗，无水硫酸钠干燥，浓缩，得到淡黄色固体7.5g(77％)，不经纯化直接进行下一步反应。 1H-NMR：(400MHz，DMSO-D 6)δ1.56-1.62(t,6H),2.48-2.58(m,4H),4.50(br,2H),7.24(s,1H),8.34(s,1H)。
化合物(9)的合成：
将(4.8g，21.4mmol)化合物(8)和(39.4g，342.4mmol)氯甲基脒盐酸盐溶于400ml DMSO中，升温至150度搅拌反应1小时。反应结束后，冷却至室温，加入乙酸乙酯700ml，用300ml饱和碳酸氢钠和300ml饱和食盐水洗涤，无水硫酸钠干燥有机相，浓缩。结晶，得到灰白色粉末状固体2.5g(53％)。
1H-NMR：(400MHz，DMSO-D 6)δ1.55-1.58(t,3H),2.48-2.58(m,2H),6.55(br,2H),7.13(s,1H),8.34(s,1H),10.22(s,1H)。
化合物(10)的合成：
将化合物(9)(5g，22.5mmol)，氯化亚砜(3.45ml，10.9mol)溶于100ml二氯甲烷中，回流反应3小时。反应结束后，冷却至室温，加入二氯甲烷1000ml，用500ml饱和碳酸氢钠和500ml饱和食盐水洗，无水硫酸钠干燥，浓缩。结晶得到灰白色固体6.6g(93％)。
1H-NMR：(400MHz，DMSO-D 6)δ1.28(d,J＝5.81Hz,6H),1.52-1.58(t,3H),2.45-2.55(m,2H),4.56-4.89(m,1H),7.12(s,1H),8.35(s,1H),10.23(s,1H)。
化合物(11)的合成：
将(10g，36.1mmol)化合物(9)溶于三氯氧磷(170ml)，在100度下搅拌反应30分钟，待反应液的颜色变为均匀褐色后继续加热30分钟。反应结束后，将反应瓶放置于冰水中快速冷却到零度，然后用2N的氢氧化钠水溶液调节Ph至弱碱性7。生成白色悬浮液，过滤，用500ml乙酸乙酯洗涤，滤液用盐水洗涤，有几层用无水硫酸钠干燥，浓缩，过硅胶柱(EA/PE,0-30％)，得到淡黄色固体6g(59％)。
1H-NMR：(400MHz，DMSO-D 6)δ1.28(d,J＝5.81Hz,6H),1.52-1.58(t,3H),2.45-2.55(m,2H),7.12(s,1H),8.35(s,1H),10.23(s,1H)。
化合物(12)的合成：
氮气保护下，将碳酸钠(3.5g，33.0mmol)，化合物(10)(2.3g，8.3mmol)和四(三苯基膦)钯(959 mg，0.8mmol)溶于100ml无水二氧六环中。80度下搅拌反应过夜。反应结束后，冷却至室温后，加入100ml水，用乙酸乙酯200ml萃取三次。合并有机相，用500ml饱和食盐水洗，无水硫酸钠干燥，浓缩，过硅胶柱(EA/PE,0-50％)，得到灰色固体2.7g(79％)。
1H-NMR：(400MHz，DMSO-D 6)δ1.28(d,J＝5.81Hz,6H),1.52-1.58(t,3H),2.45-2.55(m,2H),4.56-4.89(m,1H),6.95(d,J＝1.77Hz,1H),7.08-7.21(m,1H),7.32(s,1H),8.35(s,1H),10.67(s,1H)。化合物(13)的合成：
氮气保护下，将碳酸铯(6.4g，20mmol)和化合物(12)(5.0g，11.8mmol)和6-氯-正己基-1-胺(2.1g，15.4mmol)溶于40ml无水DMF中，100度下搅拌反应3小时。反应结束后，冷却至室温，减压浓缩除去溶剂。加入DCM(200ml)，有机相用水(200ml*4)洗，饱和食盐水洗(200ml)。无水硫酸钠干燥，浓缩，过硅胶柱(DCM/MeOH,0-5％)，得到浅棕色粘稠状物3.7g(60％)。
化合物(14)的合成：
将(12ml，48mmol)4M盐酸的二氧六环溶液，(1.6g，3.2mmol)化合物(13)与10ml甲醇混合并在室温下搅拌反应过夜。反应结束后，用饱和碳酸钠中和过量的盐酸，然后减压浓缩除去有机溶剂。向体系中加入乙酸乙酯50ml萃取两次，合并有机相后，用100ml饱和食盐水洗涤，无水硫酸钠干燥，浓缩，过硅胶柱(DCM/MeOH,0-5％)，得到灰色固体1.3g(90％)。
化合物(15)的合成：
将(930mg，2mmol)化合物(14)溶于40ml乙醇中，加入2N氢氧化钠水溶液(8ml，16mmol)。室温搅拌反应过夜。反应结束后，用2N的盐酸调节pH至中性。用乙酸乙酯50ml萃取两次，合并有机相后，用100ml饱和食盐水洗，无水硫酸钠干燥，浓缩，得到灰色固体740mg(85％)。直接进行下一步反应。
化合物(16)的合成：
将(245mg，1.3mmol)1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，(280mg，0.6mmol)化合物(15)和(0.27ml，1.9mmol)三乙胺溶于5ml DMF。室温搅拌过夜。反应结束后，加入30ml乙酸乙酯萃取3次，合并有机相，用50ml水及50ml饱和食盐水各洗2次，无水硫酸钠干燥，浓缩，过硅胶柱(EA/PE,0-50％)，得灰白色固体209mg(80％)。
化合物(16)的 1H-NMR：(400MHz；DMSO-d6)δ0.97-2.01(m,4H),2.37-2.46(m,2H),3.67-3.77(m,2H),6.97(d,1H),7.12-7.19(m,1H),7.33(s,1H),8.33(s,1H)；
LC-MS：391.63。
实施例2
(Z)-1 2-氨基-2 4,2 6-二氯-3-氧-7-氮-1(4,6)-吡咯并[2,1-f][1,2,4]三嗪-2(1,2)-苯并环非氮杂-8-酮(化合物28)的制备：
Figure PCTCN2021126517-appb-000017
化合物(28)的合成方法参考化合物(16)的合成方法。
化合物(28)的 1H-NMR：(400MHz；DMSO-d6)δ0.99-2.02(m,2H),2.37-2.46(m,2H),3.67-3.73(m,2H),6.98(d,1H),7.12-7.19(m,1H),7.33(s,1H),8.33(s,1H)；
LC-MS：377.67。
实施例3
(Z)-1 2-氨-2 4,2 6-二氯-3-氧-8-氮杂-1(4,6)-吡咯并[2,1-f][1,2,4]三嗪-2(1,3)-苯并环非氮杂-9-酮(化合物29)的制备：
Figure PCTCN2021126517-appb-000018
化合物(29)的合成方法参考化合物(16)的合成方法。
化合物(29)的 1H-NMR：(400MHz；DMSO-d6)δ0.98-2.03(m,4H),2.37-2.47(m,2H),3.70-3.77(m,2H),6.96(d,1H),7.12-7.17(m,1H),7.35(s,1H),8.36(s,1H)；
LC-MS：391.7。
实施例4
(Z)-1 2-氨基-2 4,2 6-二氯-3-氧-7-氮-1(4,6)-吡咯并[2,1-f][1,2,4]三嗪-2(1,3)-苯并环非氮杂-8-酮(化合物30)的制备：
Figure PCTCN2021126517-appb-000019
化合物(30)的合成方法参考化合物(16)的合成方法。
化合物(30)的 1H-NMR：(400MHz；DMSO-d6)δ0.99-2.02(m,2H),2.37-2.46(m,2H),3.67-3.73(m,2H),6.98(d,1H),7.12-7.19(m,1H),7.34(s,1H),8.32(s,1H)；
LC-MS：377.71。
实施例5
通过以下方案3合成了本发明化合物(27)的中间体化合物(20)，其中R 1为氯，R 2为氯，R 4为氧：
Figure PCTCN2021126517-appb-000020
化合物(18)的合成：
将化合物(17)(15.6g，75mmol)，碳酸钾(12g，87mmol)溶于200ml无水乙腈中，将溴卞(9ml，76mmol)滴加到上述反应液中，滴加完毕后，回流18小时。反应结束后，冷却至室温，加入500ml水，用二氯甲烷(200ml)萃取水相。合并有机相，用2N的氢氧化钠(200ml)洗，水洗(200ml*2)，饱和食盐水洗(200ml)，无水硫酸钠干燥，浓缩，过硅胶柱(EA/PE,0-30％)，得到黄色固体21.6g(96％)。
化合物(19)的合成：
铁粉(11g,188mmol)和化合物(18)(10.8g,36mmol)溶于(150mL)醋酸和水(75mL)中，85度下搅拌反应90分钟。反应结束后，趁热抽滤，反应液冷却至室温，加入水300ml，用DCM 100ml萃取三次。结合有机相用2M的氢氧化钠洗一洗，水洗一次，饱和食盐水洗一次,无水硫酸钠干燥，浓缩，得到浅棕色固体(9.5g,99％)。
1H-Ν ΜR(400MHz,CDCl 3)δ5.20(s,2H),7.34-7.48(m,5H),7.56(s,1H),7.58(s,1H)。
化合物(20)的合成：
将(16.2g,60mmol)化合物(19)溶于240ml醋酸中，然后将(6.0M,60mL)盐酸加入反应体系中，反应体系冷却至零度。将(4.8g,69.5mmol)亚硝酸钠溶于40ml水，缓慢滴加到上述反应体系中，反应体系的温度低于5度。滴加完毕后，搅拌反应30分钟。30分钟后，将(20g,120mmol)碘化钾和(4g,16mmol)碘溶于200ml水，然后滴加到上述反应体系中，搅拌反应90分钟。反应结束后，加入水800ml，用250mlDCM萃取三次，合并有机相，用250ml10％(w/v)硫代硫酸钠淬灭碘，2M的氢氧化钠250ml洗一次水500ml洗三次，饱和食盐水200ml洗一次，无水硫酸钠干燥，浓缩，得到浅棕色固体21g(90％)。
1H-Ν ΜR(400MHz；CDCl 3)δ5.10(s,2H),7.33-7.48(m,7H).
通过例如以下方案4合成了本发明化合物(27)，其中R 3为氧，R 4为氢，X为碳原子，Y为硫原子：
Figure PCTCN2021126517-appb-000021
化合物(22)的合成：
将(2.5g,13mmol)化合物(21)和(4.5g,32.6mmol)碳酸钾溶于80ml乙腈中，搅拌下加入(2.86mL,26mmol)巯基乙酸乙酯。85度条件下搅拌反应5小时。反应结束后，冷却至室温。抽滤，得到的固体用25ml乙腈洗涤，然后用100ml水洗涤，真空干燥，得到浅黄色固体2.8g(83％)。
1H-Ν ΜR(400MHz；DMSO-d6)δ2.09(t,3H,J＝7.0Hz),4.31(q,2H,J＝7.0Hz),7.70(brs,2H),7.78 (s,1H).
化合物(23)的合成：
氮气保护下，将醋酸钾(1.6g,16.3mmol)，(2.1g,5.4mmol)化合物(22)，醋酸钯(450mg,cat.)和双联频哪醇基二硼(1.5g,5.7mmol)溶于10mlDMF，90度反应18小时。反应结束后，减压浓缩，加入乙酸乙酯50ml，水30ml洗三次，饱和食盐水30ml洗一次，无水硫酸钠干燥，浓缩得到浅棕色粘稠状物。
氮气保护下，向其中加入80ml二氧六环，(1.3g,5mmol)化合物(20),2M磷酸钾溶液4ml，四(三苯基膦)钯(cat.)，然后在100度下反应3小时。反应结束后，冷却至室温，加入40ml乙酸乙酯，有机相用25ml饱和食盐水洗无水硫酸钠干燥，过硅胶柱(EA/PE＝0-1/30)，得到白色固体1.1g(45％)。
1H-Ν ΜR(400MHz；DMSO-d6)δ1.28(t,3H,J＝7.1Hz),4.29(q,2H,J＝7.1Hz),5.22(s,2H),7.32-7.37(m,1H),7.38-7.43(m,3H),7.44-7.47(m,2H),7.48(s,1H),7.52(brs,2H),7.84(s,1H).
化合物(24)的合成：
在氮气保护下，将化合物(23)(1.8g，3.8mmol)溶于15ml DCM，降温至零下78度，将三氯化硼溶液(1.0M in DCM，15ml，15mol)加入上述反应体系中。滴加完毕后，升温至室温反应3h。3h后降温至零度。滴加10ml甲醇，滴加完毕后，搅拌反应1h，浓缩，得浅黄色固体。向粗品中加入10％的醋酸钠溶液(20ml)，乙酸乙酯50ml，分离有几层，用水(20ml*2)，饱和食盐水洗(20ml)。无水硫酸钠干燥，浓缩。得到的粗品用正己烷洗涤，抽滤，干燥，得到浅黄色固体1.0g(70％)。
化合物(25)的合成：
氮气保护下，将碳酸铯(6.4g，20mmol)和化合物(24)(4.5g，11.8mmol)和6-氯-正己基-1-胺(2.1g，15.4mmol)溶于40ml无水DMF中，100度下搅拌反应3小时。反应结束后，冷却至室温，减压浓缩除去溶剂。加入DCM(200ml)，有机相用水(200ml*4)洗，饱和食盐水洗(200ml)。无水硫酸钠干燥，浓缩，过硅胶柱(DCM/MeOH,0-5％)，得到浅棕色粘稠状物3.4g(60％)。
化合物(26)的合成：
将(964mg，2mmol)化合物(25)溶于40ml乙醇中，加入2N氢氧化钠水溶液(8ml，16mmol)。室温搅拌反应过夜。反应结束后，用2N的盐酸调节Ph至中性。用乙酸乙酯50ml萃取两次，合并有机相后，用100ml饱和食盐水洗，无水硫酸钠干燥，浓缩，得到浅黄色固体772mg(85％)。直接进行下一步反应。
化合物(27)的合成：
将(245mg，1.3mmol)EDC盐酸盐，(272mg，0.6mmol)化合物(26)和(0.27ml，1.9mmol)三乙胺溶于5ml DMF。室温搅拌过夜。反应结束后，加入30ml乙酸乙酯萃取3次，合并有机相，用50ml水及50ml饱和食盐水各洗2次，无水硫酸钠干燥，浓缩，过硅胶柱(EA/PE,0-50％)，得浅黄色固体217mg(80％)。
化合物(27)的 1H-NMR：(400MHz；DMSO-d6)δ0.97-2.01(m,4H),2.35-2.43(m,2H),3.71-3.79(m,2H),4.25(b,2H),6.96(d,1H),7.33(s,1H),7.88(s,1H).；
LC-MS：409.07。
实施例6
(Z)-1 2-氨基-2 4,2 6-二氯-3-氧-7-氮-1(4,6)-噻吩并[2,3-d]嘧啶-2(1,2)-苯并环非氮杂-8-酮(化合物31)的制备：
Figure PCTCN2021126517-appb-000022
化合物(31)的合成方法参考化合物(27)的合成方法。
化合物(31)的 1H-NMR：(400MHz；DMSO-d6)δ1.01-2.03(m,2H),2.33-2.41(m,2H),3.73-3.79(m,2H),4.25(b,2H),6.95(d,1H),7.33(s,1H),7.89(s,1H)；
LC-MS：395.05。
实施例7
(Z)-1 2-氨基-2 4,2 6-二氯-3-氧-8-氮-1(4,6)-噻吩并[2,3-d]嘧啶-2(1,3)-苯并环非氮杂-9-酮(化合物32)的制备：
Figure PCTCN2021126517-appb-000023
化合物(32)的合成方法参考化合物(27)的合成方法。
化合物(32)的 1H-NMR：(400MHz；DMSO-d6)δ0.98-2.01(m,4H),2.37-2.45(m,2H),3.73-3.79(m,2H),4.26(b,2H),6.96(d,1H),7.35(s,1H),7.89(s,1H)；
LC-MS：409.11。
实施例8
(Z)-1 2-氨基-2 4,2 6-二氯-3-氧-7-氮-1(4,6)-噻吩并[2,3-d]嘧啶-2(1,3)-苯并环非氮杂-8-酮(化合物33)的制备：
Figure PCTCN2021126517-appb-000024
化合物(33)的合成方法参考化合物(27)的合成方法。
化合物(33)的 1H-NMR：(400MHz；DMSO-d6)δ1.01-2.03(m,2H),2.33-2.41(m,2H),3.73-3.79(m,2H),4.25(b,2H),6.95(d,1H),7.37(s,1H),7.89(s,1H)；
LC-MS：395.09。
实施例9
本实施例提供了式I化合物或其药学上可接受的盐、溶剂合物或水合物的酶水平活性测定：构建人Hsp90α/Hsp90β抑制剂的筛选平台，应用荧光偏振(FP)的方法，基于的原理是通过检测荧光素标记的小分子与其他分子相互作用前后分子量的变化，计算水平方向及垂直方向的荧光偏振值作相关分析。如果被荧光标记小分子与大分子之间的结合平衡建立后，它受激发时运动慢，测得的荧光偏振光值会增高。如果荧光标记小分子与大分子之间的结合被其他配基取代，它在游离状态下的旋转和翻转速度会变快，发射光相对于激发光平面将去偏振化，测得的偏振光值降低，从而计算出样品的偏振值(偏振值单位mP)。
本发明中应用的荧光标记小分子是GM-BODIPY(参考BMCL，2003，13，3975-3978所述的合成方法合成)。反应在384孔黑板中进行，所用的反应疏水蛋白HFB缓冲液：50mM KCl，5mM MgCl 2，20mM Na 2MoO 4，0.01％NP40，0.1mg/ml BGG，2mM DTT，pH 7.3。反应体系体积50ml，包括30nM Hsp90α,30Nm Hsp90β，5nM GM-BODIPY(格尔德霉素)和本发明待测小分子化合物(式I化合物)或DMSO(二甲基亚枫)，DMSO均不超过2‰。设空白对照组和5nM GM-BODIPY阴性对照组，空白对照孔只加入HFB缓冲液，4摄氏度下反应12-16小时，采用酶标仪检测，偏振光的激发波长为485/20nm，发射波长为535/25nm，测得mP值。应用如下公式计算抑制率：
(不加化合物mP-加化合物mP)/(不加化合物mP-阴性对照mP)x 100％
计算出不同浓度的化合物的抑制率后，计算该化合物的IC 50。
用荧光偏振(FP)的方法测定了式I化合物或其药学上可接受的盐、溶剂合物或水合物，计算出了不同浓度下所述化合物的抑制率，从而完成酶水平上活性的测定。
本实施例还提供了式I化合物或其药学上可接受的盐、溶剂合物或水合物与Hsp90α/Hsp90β蛋白的结合Kd：
用SprectraMax M5分光光度计进行固有荧光测量。用含有20mM Hepes和50mM NaCl的pH 7.4的测定缓冲液中将待测化合物稀释至20μM。在KU675溶液中分别加入重组Hsp90α和Hsp90β蛋白，调整至设计浓 度(Hsp90α为0至100μg/mL，Hsp90β为0至140μg/mL)，并在测量前孵育30分钟。所有测量均在25℃进行并重复三次。激发波长为345nm，并且从350nm至600nm监测发射。使用GraphPad Prism 5软件使用非线性拟合分析浓度依赖性结合曲线，并相应地确定结合亲和力(Kd)。
通过本实施例，得出了式I化合物或其药学上可接受的盐、溶剂合物或水合物与Hsp90α/Hsp90β蛋白的结合Kd值。
本实施例还提供了式I化合物或其药学上可接受的盐、溶剂合物或水合物的细胞水平活性测定：
本实施例提供了式I化合物或其药学上可接受的盐、溶剂合物或水合物的抗癌效应，以美国国立癌症研究所推荐的常见肿瘤细胞株(参见Monks A Feasibility of a High-Flux Anticancer Drug Screen Using a Diverse Panel of Cultured Human Tumor Cell Lines.J.Natl.Cancer Inst.1991 83：757-766)为核心展开，本发明所选用的细胞株包括：HCT116、HT-29、MCF-7、MDA-MB-231、H460、A549、ovca3、SKOV-3、pc-3、Hela、U87、Hep G2、Vero、B16、SGC-7901、HL-60、JAK3STAT5、K562/ADR、BEL7404、TE-1、ZR-75-30、H1975、BT474、U266、K562、A375、Caki-1、SW620、MCF7、Molt-4、MDA-MB-435s、HO-8910。
肿瘤细胞的生长抑制检测采用Cell Counting Kit-8(CCK-8)细胞毒性筛查实验。Cell Counting Kit-8(CCK-8)是以WST-8(2-(2-甲氧基-4-硝基苯基)-3-(4-硝基苯基)-5-(2,4-二磺基苯基)-2H-四唑单钠盐)为基础的检测方法，具体步骤如下：将肿瘤细胞悬液以5000/孔浓度接种于96孔细胞培养板中，培养24小时(37℃，5％CO 2)。加入不同浓度的待测药物处理6-48小时，再加入CCK-8溶液，置培养箱(37℃，5％CO 2)温浴至显色(1-4小时)，酶标仪测定450nm波长下的光密度(OD值)。记录结果，计算本文提供的化合物(表1中的化合物1-142)在不同浓度、时间下对肿瘤细胞生长的抑制率。按以下公式计算药物对肿瘤细胞生长的抑制率：细胞生长抑制率＝(1-实验组平均OD值/对照组平均OD值)×100％)。实验重复3次。
通过本实施例得出了式I化合物或其药学上可接受的盐、溶剂合物或水合物对肿瘤细胞生长的抑制率，本发明的化合物能够有效抑制肿瘤细胞生长。
本实施例还提供了式I化合物或其药学上可接受的盐、溶剂合物或水合物对Hsp90β亚型的选择性：
将细胞(如NCI-H23等)收集于冷PBS中，并在冰上用含有蛋白酶和磷酸酶抑制剂的哺乳动物蛋白提取试剂的裂解缓冲液裂解1小时。裂解物在4℃以15,000g澄清20分钟。按照制造商的说明书(ThermoFisher)使用Qubit蛋白定量测定试剂盒测定蛋白质浓度。在还原条件下将等量的蛋白质(2.5-20μg)在10％丙烯酰胺凝胶中电泳，转移至聚偏氟乙烯膜(PVDF)，并用相应的特异性抗体进行免疫印迹。将膜与合适的辣根过氧化物酶标记的二抗一起温育，用化学发光底物显影观察。首先在ImageJ中将数据转换为8-bit图像，然后用Image Studio Lite Ver 5.2或Li-COR Odyssey Image Studio Ver 4.0进行密度测量。对照为肌动蛋白及DMSO。
将His6标记的人Hsp90βN-末端结构域(氨基酸1-218)克隆到修饰的pET载体中，在大肠杆菌BL21DE3细胞中过表达并通过Ni-NTA色谱纯化。使用TEV蛋白酶切割标签，然后第二次使用Ni-NTA色谱以去除TEV和his6标签部分。浓缩含有切割后蛋白的流出物，并通过Superdex 200排阻色谱在20mM Tris-HCl，150mM  NaCl(pH7.8)中进一步纯化。混合15mg/mL的Hsp90βNT与每种抑制剂，在1.5-2.0mM最终药物浓度下形成蛋白质抑制剂复合物，并在4℃下孵育1小时。室温下，用1：1的蛋白质与药物在30％PEG 8000，0.2M乙酸钠和0.1二甲胂酸钠(pH 6.5)缓冲液中形成共结晶滴。1-2天内出现晶体，在含有20％甘油的低温缓冲液中获得，上述低温缓冲液添加至缓冲液中，各自加抑制剂2mM。
表1.目标化合物的生物活性数据
Figure PCTCN2021126517-appb-000025
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物，其具有式I所示结构：
    Figure PCTCN2021126517-appb-100001
    其中：
    R 1选自氯、氟、溴、C 1-C 3的烷基或1至6的氟取代C 1-C 3的烷基；
    R 2选自氯、氟、溴、C 1-C 3的烷基或1至6的氟取代C 1-C 3的烷基、-(C 1-C 6烯基)-OH、-(C 1-C 6烷基)-OH、-O-(C 1-C 6烷基)、-O-(C 1-C 6烯基)、-(C 1-C 6烯基)-O-(C 1-C 6烷基)、-(C 1-C 6烯基)-O-(C 1-C 6烯基)-(C 6-C 10芳基)、-(C 1-C 6烯基)-O-(C 1-C 6烯基)-(C 3-C 10环烷基)、-(C 1-C 6烯基)-O-(C 1-C 6烯基)-(C 3-C 10杂环)、-(C 1-C 6烯基)-O-(C 2-C 6烯基)、-O-(C 1-C 6烯基)-(C 3-C 10杂环)、-O-(C 2-C 6烷基)-(C 3-C 10杂环)或C 1-C 8烷氧基；
    R 3选自氧、亚氨基、硫或亚甲基；
    R 4选自氢基、C 1-C 6的烷基、C 2-C 8的烯基、C 2-C 8的炔基、C 6-C 14的芳基、C 2-C 9的杂芳基、C 2-C 9的异环烷基或C 3-C 8的环烷基；
    X选自氮原子或碳原子；
    Y选自氮原子、硫原子、氧原子或碳原子；
    n选自0，1，2，3，4或5。
  2. 如权利要求1所述的特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物，其特征在于，所述特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物具有式(I’)或式(II’)所示结构：
    Figure PCTCN2021126517-appb-100002
    优选地，R 1选自氯、氟、溴、C 1-C 3的烷基或1至6的氟取代C 1-C 3的烷基；
    R 2选自氯、氟、溴、C 1-C 3的烷基或1至6的氟取代C 1-C 3的烷基、-(C 1-C 6烯基)-OH、-(C 1-C 6烷基)-OH、-O-(C 1-C 6烷基)、-O-(C 1-C 6烯基)、-(C 1-C 6烯基)-O-(C 1-C 6烷基)、-(C 1-C 6烯基)-O-(C 1-C 6烯基)-(C 6-C 10芳基)、-(C 1-C 6烯基)-O-(C 1-C 6烯基)-(C 3-C 10环烷基)、-(C 1-C 6烯基)-O-(C 1-C 6烯基)-(C 3-C 10杂环)、-(C 1-C 6烯基)-O-(C 2-C 6烯基)、-O-(C 1-C 6烯基)-(C 3-C 10杂环)、-O-(C 2-C 6烷基)-(C 3-C 10杂环)或C 1-C 8烷氧基；
    R 4选自氢基、C 1-C 6的烷基、C 2-C 8的烯基、C 2-C 8的炔基、C 6-C 14的芳基、C 2-C 9的杂芳基、C 2-C 9的异环烷基或C 3-C 8的环烷基；
    n选自1或2。
  3. 如权利要求1所述的特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物，其特征在于，所述特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物选自下列化合物：
    (Z)-1 2-氨-2 4,2 6-二氯-3-氧-8-氮杂-1(4,6)-吡咯并[2,1-f][1,2,4]三嗪-2(1,2)-苯并环非氮杂-9-酮；
    (Z)-1 2-氨基-2 4,2 6-二氯-3-氧-7-氮-1(4,6)-吡咯并[2,1-f][1,2,4]三嗪-2(1,2)-苯并环非氮杂-8-酮；
    (Z)-1 2-氨-2 4,2 6-二氯-3-氧-8-氮杂-1(4,6)-吡咯并[2,1-f][1,2,4]三嗪-2(1,3)-苯并环非氮杂-9-酮；
    (Z)-1 2-氨基-2 4,2 6-二氯-3-氧-7-氮-1(4,6)-吡咯并[2,1-f][1,2,4]三嗪-2(1,3)-苯并环非氮杂-8-酮；
    (Z)-1 2-氨基-2 4,2 6-二氯-3-氧-8-氮-1(4,6)-噻吩并[2,3-d]嘧啶-2(1,2)-苯并环非氮杂-9-酮；
    (Z)-1 2-氨基-2 4,2 6-二氯-3-氧-7-氮-1(4,6)-噻吩并[2,3-d]嘧啶-2(1,2)-苯并环非氮杂-8-酮；
    (Z)-1 2-氨基-2 4,2 6-二氯-3-氧-8-氮-1(4,6)-噻吩并[2,3-d]嘧啶-2(1,3)-苯并环非氮杂-9-酮；
    (Z)-1 2-氨基-2 4,2 6-二氯-3-氧-7-氮-1(4,6)-噻吩并[2,3-d]嘧啶-2(1,3)-苯并环非氮杂-8-酮。
  4. 一种权利要求1-3任一所述特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物的制备方法，其特征在于，所述制备方法为：
    以
    Figure PCTCN2021126517-appb-100003
    为原料制备中间体
    Figure PCTCN2021126517-appb-100004
    以
    Figure PCTCN2021126517-appb-100005
    为原料制备
    Figure PCTCN2021126517-appb-100006
    中间体(5)和(11)反应生成式(I’)化合物。
  5. 一种权利要求1-3任一所述特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物的制备方法，其特征在于，所述制备方法为：
    以
    Figure PCTCN2021126517-appb-100007
    为原料制备中间体
    Figure PCTCN2021126517-appb-100008
    以
    Figure PCTCN2021126517-appb-100009
    为原料制备中间体
    Figure PCTCN2021126517-appb-100010
    中间体(20)和(22)反应生成式(II’)化合物。
  6. 一种药物组合物，其特征在于，所述药物组合物包含权利要求1所述特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物。
  7. 一种药物制剂，其特征在于，所述药物制剂包含权利要求1所述特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物，以及药学上可接受的载体或辅料。
  8. 权利要求1所述特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物或权利要求2所述药物组合物在制备抑制Hsp90酶活性的药物中的应用；优选的，所述应用包括使Hsp90酶与权利要求1所述特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合 物或水合物或权利要求2所述药物组合物接触。
  9. 一种权利要求1所述特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物或权利要求2所述药物组合物在制备预防和/或治疗癌症或肿瘤的药物中的应用，
    优选的，所述癌症或肿瘤包括膀胱癌、乳癌、子宫颈癌、结肠癌、食管癌、头颈癌、肝癌、肺癌(例如小细胞肺癌和非-小细胞肺癌)、黑素瘤、骨髓瘤、成神经细胞瘤、卵巢癌、胰腺癌、前列腺癌、肾癌、肉瘤、皮肤癌、胃癌、睾丸癌、甲状腺癌和子宫癌；
    结肠癌进一步优选为结肠直肠癌；肺癌进一步优选为小细胞肺癌或非小细胞肺癌；肉瘤进一步优选为骨肉瘤、皮肤癌进一步优选为鳞状细胞癌。
  10. 一种权利要求1所述特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物或权利要求2所述药物组合物在制备抑制细胞生长的药物中的应用，优选的，所述应用包括使细胞与权利要求1所述细胞与特异性热休克蛋白90α亚型抑制剂或其药学上可接受的盐、溶剂合物或水合物或权利要求2所述药物组合物接触；
    优选的，所述细胞是肿瘤细胞，进一步优选的，所述细胞是哺乳动物肿瘤细胞或人肿瘤细胞；
    或者，所述细胞是癌细胞，进一步优选的，所述细胞是哺乳动物癌细胞或人癌细胞；
    进一步优选地，所述癌细胞包括膀胱癌、乳癌、子宫颈癌、结肠癌、食管癌、头颈癌、肝癌、肺癌、黑素瘤、骨髓瘤、成神经细胞瘤、卵巢癌、胰腺癌、前列腺癌、肾癌、肉瘤、皮肤癌、胃癌、睾丸癌、甲状腺癌和子宫癌的细胞；
    结肠癌进一步优选为结肠直肠癌；肺癌进一步优选为小细胞肺癌或非小细胞肺癌；肉瘤进一步优选为骨肉瘤、皮肤癌进一步优选为鳞状细胞癌；
    进一步优选的，所述癌细胞是膀胱癌、鳞状细胞癌、头颈癌、结肠直肠癌、食管癌、胃癌、妇科癌、胰腺癌、直肠癌、乳癌、前列腺癌、女阴癌、皮肤癌、脑癌、生殖泌尿道癌、淋巴系统癌、胃癌、喉癌或肺癌的细胞；
    进一步优选地，所述癌细胞是转移性癌细胞，包括膀胱癌、乳癌、子宫颈癌、结肠癌、食管癌、头颈癌、肝癌、肺癌、黑素瘤、骨髓瘤、成神经细胞瘤、卵巢癌、胰腺癌、前列腺癌、肾癌、肉瘤、皮肤癌、胃癌、睾丸癌、甲状腺癌和子宫癌的细胞；
    结肠癌进一步优选为结肠直肠癌；肺癌进一步优选为小细胞肺癌或非小细胞肺癌；肉瘤进一步优选为骨肉瘤、皮肤癌进一步优选为鳞状细胞癌。
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