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WO2022089449A1 - PRÉPARATION D'UN INHIBITEUR DU SOUS-TYPE 90α DE PROTÉINE DE CHOC THERMIQUE SPÉCIFIQUE ET SON UTILISATION - Google Patents

PRÉPARATION D'UN INHIBITEUR DU SOUS-TYPE 90α DE PROTÉINE DE CHOC THERMIQUE SPÉCIFIQUE ET SON UTILISATION Download PDF

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Publication number
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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Prior art keywords
cancer
alkenyl
alkyl
pharmaceutically acceptable
heat shock
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English (en)
Chinese (zh)
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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Priority claimed from CN202111242267.8A external-priority patent/CN115124550B/zh
Application filed by JINAN CHENGCHENG BIOTECHNOLOGY Co Ltd, Shandong University filed Critical JINAN CHENGCHENG BIOTECHNOLOGY Co Ltd
Publication of WO2022089449A1 publication Critical patent/WO2022089449A1/fr
Anticipated expiration legal-status Critical
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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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  • Veterinary Medicine (AREA)
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Abstract

L'invention concerne la préparation d'un inhibiteur du sous-type 90α de protéine de choc thermique spécifique et son utilisation. L'inhibiteur a la structure de formule I, dans laquelle : R1 est choisi parmi le chlore, le fluor, le brome, un alkyle en C1-C3, ou un alkyle en C1-C3 substitué par du fluor en position 1 à 6 ; R2 est choisi parmi le chlore, le fluor, le brome, un alkyle en C1-C3, un alkyle en C1-C3 substitué par du fluor en position 1 à 6, -(C1-C6 alcényl)-OH, -(C1-C6 alkyl)-OH, -O-(C1-C6 alkyl), -O-(C1-C6 alcényl), -(C1-C6 alcényl)-O-(C1-C6 alkyl), -(C1-C6 alcényl)-O-(C1-C6 alcényl)-(C6-C10 aryl), -(C1-C6 alcényl)-O-(C1-C6 alcényl)-(C3-C10 cycloalkyl), -(C1-C6 alcényl)-O-(C1-C6 alcényl)-(C3-C10 hétérocycle), -(C1-C6 alcényl)-O-(C2-C6 alcényl), -O-(C1-C6 alcényl)-(C3-C10 hétérocycle), -O-(C2-C6 alkyl)-(C3-C10 hétérocycle) ou a C1-C8 alcoxy ; R3 est choisi parmi oxygène, imino, soufre ou méthylène ; R4 est choisi parmi un groupe hydrogène, un alkyle en C1-C6, un alcényle ou un alcynyle en C2-C8, un aryle en C6-C14, un hétéroaryle en C2-C9 ou un isocycloalkyle, ou un cycloalkyle en C3-C8 ; X représente un atome d'azote ou de carbone ; Y représente un atome d'azote, de soufre, d'oxygène ou de carbone ; et n est choisi parmi 0 à 5.
PCT/CN2021/126517 2020-10-26 2021-10-26 PRÉPARATION D'UN INHIBITEUR DU SOUS-TYPE 90α DE PROTÉINE DE CHOC THERMIQUE SPÉCIFIQUE ET SON UTILISATION Ceased WO2022089449A1 (fr)

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CN202111242267.8A CN115124550B (zh) 2020-10-26 2021-10-25 一种特异性热休克蛋白90α亚型抑制剂制备及其应用

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