WO2022089449A1 - PREPARATION OF SPECIFIC HEAT SHOCK PROTEIN 90α SUBTYPE INHIBITOR AND USE THEREOF - Google Patents

Abstract

Disclosed are the preparation of a specific heat shock protein 90α subtype inhibitor and the use thereof. The inhibitor has the structure of formula I, wherein: R1 is selected from chlorine, fluorine, bromine, a C1-C3 alkyl, or a 1 to 6 fluorine substituted C1-C3; R2 is selected from chlorine, fluorine, bromine, a C1-C3 alkyl, a 1 to 6 fluorine substituted C1-C3 alkyl, -(C1-C6 alkenyl)-OH, -(C1-C6 alkyl)-OH, -O-(C1-C6 alkyl), -O-(C1-C6 alkenyl), -(C1-C6 alkenyl)-O-(C1-C6 alkyl), -(C1-C6 alkenyl)-O-(C1-C6 alkenyl)-(C6-C10 aryl), -(C1-C6 alkenyl)-O-(C1-C6 alkenyl)-(C3-C10 cycloalkyl), -(C1-C6 alkenyl)-O-(C1-C6 alkenyl)-(C3-C10 heterocycle), -(C1-C6 alkenyl)-O-(C2-C6 alkenyl), -O-(C1-C6 alkenyl)-(C3-C10 heterocycle), -O-(C2-C6 alkyl)-(C3-C10 heterocycle) or a C1-C8 alkoxy; R3 is selected from oxygen, imino, sulfur, or methylene; R4 is selected from a hydrogen group, a C1-C6 alkyl, a C2-C8 alkenyl or alkynyl, a C6-C14 aryl, a C2-C9 heteroaryl or isocycloalkyl, or a C3-C8 cycloalkyl; X: a nitrogen or carbon atom; Y is a nitrogen, sulfur, oxygen, or carbon atom; and n is selected from 0-5.

Description

Preparation and application of a specific heat shock protein 90α isoform inhibitor technical field

The invention relates to the technical field of specific heat shock protein 90α subtype inhibitors, in particular to the preparation and application of a specific heat shock protein 90α subtype inhibitor.

Background technique

The information disclosed in this Background section is only for enhancement of understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

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 Hsp90α and Hsp90β, and the inhibition of Hsp90β will hinder normal life activities, which may appear in clinical tests. One of the important reasons for toxic side effects. Hsp90α and Hsp90β 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 Hsp90α 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.

At present, 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 α 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.

SUMMARY OF THE INVENTION

In order to solve the technical problems existing in the prior art, the purpose of the present invention is to provide the preparation and application of a specific heat shock protein 90α subtype inhibitor, the specific heat shock protein 90α subtype inhibitor has less toxic and side effects, Can effectively inhibit tumor cell growth.

Specifically, the technical solution of the present invention is as follows:

In the first aspect of the present invention, the present invention provides a specific heat shock protein 90α subtype inhibitor or a pharmaceutically acceptable salt, solvate or hydrate thereof, which has the structure shown in formula I:

in:

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 -C ₁₀ heterocycle), -(C ₁ -C ₆ alkenyl)-O-(C ₂ -C ₆ alkenyl), -O-(C ₁ -C ₆ alkenyl)-(C ₃ -C ₁₀ heterocycle), -O-(C ₂ -C ₆ alkyl)-(C ₃ -C ₁₀ heterocycle) or C ₁ -C ₈ alkoxy;

_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.

In one or more embodiments, the specific heat shock protein 90α isoform inhibitor, or a pharmaceutically acceptable salt, solvate or hydrate thereof, is of formula (I') or formula (II' ) shown in the structure:

Preferably, 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 -C ₁₀ heterocycle), -(C ₁ -C ₆ alkenyl)-O-(C ₂ -C ₆ alkenyl), -O-(C ₁ -C ₆ alkenyl)-(C ₃ -C ₁₀ heterocycle), -O-(C ₂ -C ₆ alkyl)-(C ₃ -C ₁₀ heterocycle) or C ₁ -C ₈ alkoxy;

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;

Preferably, the specific heat shock protein 90α subtype inhibitor or a pharmaceutically acceptable salt, solvate or hydrate thereof is selected from the following compounds:

(Z)-1 ² -Amino-2 ⁴ ,2 ⁶ -dichloro-3-oxo-8-aza-1(4,6)-pyrrolo[2,1-f][1,2,4] Triazin-2(1,2)-benzocyclononaza-9-one;

(Z)-1 ² -Amino-2 ⁴ ,2 ⁶ -dichloro-3-oxo-7-nitro-1(4,6)-pyrrolo[2,1-f][1,2,4]tris oxazin-2(1,2)-benzocyclononaza-8-one;

(Z)-1 ² -Amino-2 ⁴ ,2 ⁶ -dichloro-3-oxo-8-aza-1(4,6)-pyrrolo[2,1-f][1,2,4] Triazin-2(1,3)-benzocyclononaza-9-one;

(Z)-1 ² -Amino-2 ⁴ ,2 ⁶ -dichloro-3-oxo-7-nitro-1(4,6)-pyrrolo[2,1-f][1,2,4]tris oxazin-2(1,3)-benzocyclononaza-8-one;

(Z)-1 ² -Amino-2 ⁴ ,2 ⁶ -dichloro-3-oxo-8-nitro-1(4,6)-thieno[2,3-d]pyrimidine-2(1,2) - benzocyclononaza-9-one;

(Z)-1 ² -Amino-2 ⁴ ,2 ⁶ -dichloro-3-oxo-7-aza-1(4,6)-thieno[2,3-d]pyrimidine-2(1,2) - benzocyclononaza-8-one;

(Z)-1 ² -Amino-2 ⁴ ,2 ⁶ -dichloro-3-oxo-8-aza-1(4,6)-thieno[2,3-d]pyrimidine-2(1,3) - benzocyclononaza-9-one;

(Z)-1 ² -Amino-2 ⁴ ,2 ⁶ -dichloro-3-oxo-7-aza-1(4,6)-thieno[2,3-d]pyrimidine-2(1,3) - Benzocyclononaza-8-ones.

In the second aspect of the present invention, the present invention provides a preparation method of the above-mentioned specific heat shock protein 90α subtype inhibitor or a pharmaceutically acceptable salt, solvate or hydrate to prepare the compound of formula (I'):

by

Preparation of intermediates for starting materials

by

Preparation of intermediates for starting materials

Intermediates (5) and (11) react to generate compounds of formula (I');

Specifically, compound (16): (Z)-12 ^- amino-24,26-dichloro- ³ -oxo- ⁸ -aza-1(4,6)-pyrrolo[2,1-f ][1,2,4]triazine-2(1,2)-benzocyclononaza-9-one reaction process is shown below:

In the third aspect of the present invention, the present invention provides yet another preparation method of the above-mentioned specific heat shock protein 90α subtype inhibitor or a pharmaceutically acceptable salt, solvate or hydrate thereof, to prepare formula (II') Compound:

by

Preparation of intermediates for starting materials

by

Preparation of intermediates for starting materials

Intermediates (20) and (22) react to form compounds of formula (II');

Specifically, compound (27)(Z)-12 ^- amino-24,26-dichloro- ³ -oxo- ⁸ -aza-1(4,6)-thieno[2,3-d]pyrimidine The reaction process of -2(1,2)-benzocyclononaza-9-one is shown below:

In the fourth aspect of the present invention, the present invention provides a pharmaceutical composition comprising the above-mentioned specific heat shock protein 90α subtype inhibitor or a pharmaceutically acceptable salt, solvate or hydrate thereof.

In a fifth aspect of the present invention, the present invention provides a pharmaceutical preparation comprising the above-mentioned specific heat shock protein 90α 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.

In the sixth aspect of the present invention, the present invention provides the above-mentioned specific heat shock protein 90α 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 use of the medicament, the application comprises contacting the Hsp90 enzyme with the above-mentioned specific heat shock protein 90α subtype inhibitor or a pharmaceutically acceptable salt, solvate or hydrate or the above-mentioned pharmaceutical composition.

In the seventh aspect of the present invention, the present invention provides the above-mentioned specific heat shock protein 90α 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.

Preferably, 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.

In the eighth aspect of the present invention, the present invention provides the above-mentioned specific heat shock protein 90α 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 90α subtype inhibitor or a pharmaceutically acceptable salt, solvate or hydrate thereof or the above-mentioned pharmaceutical composition.

Preferably, the cells are tumor cells, and further preferably, the cells are mammalian tumor cells or human tumor cells.

Alternatively, the cells are cancer cells, further preferably, the cells are mammalian cancer cells or human cancer cells.

Preferably, 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.

Further preferably, 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.

Preferably, 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.

Preferably, 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 embodiment of the present invention has the following beneficial effects:

In the embodiment of the present invention, the specific heat shock protein 90α 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.

Detailed ways

Terms appearing in this application are defined as follows: for a specific term, if the meaning defined in this application is inconsistent with the meaning commonly understood by those skilled in the art, the meaning defined in this application shall prevail; if there is no definition in this application , then it has the meaning commonly understood by those skilled in the art.

The term "alkyl" in the present invention refers to a straight or branched chain saturated hydrocarbon group. The term "C _1-3 alkyl" refers to an alkyl group having 1-3 carbon atoms. The term "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.

The term "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. The term "C _2-8 alkenyl" refers to an alkenyl group having 2-8 carbon atoms, typically vinyl, propenyl, butenyl, pentenyl, hexenyl, and the like.

The term "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. The term "C _2-8 alkynyl" refers to an alkynyl group having 2-8 carbon atoms, typically ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.

The term "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.

The term "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.

The term "alkoxy," as used herein, 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.

The term "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.

The term "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. Examples of "heteroaryl" 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, benzotriazolyl, pyrazolopyridyl and the like.

The term "pharmaceutically acceptable salt" in the present invention 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, hydroxynaphthoic acid, salicylic acid, stearic acid, etc.; ions, such as salts formed when alkali metal ions or alkaline earth metal ions are substituted; or coordination compounds formed with organic bases such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, and the like.

The term "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.

Those skilled in the art will appreciate that 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.

The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.

For all of the following examples, standard procedures and purification methods known to those skilled in the art can be used. All temperatures are expressed in °C (degrees Celsius) unless otherwise indicated. All reactions were performed at room temperature unless otherwise indicated. The synthetic methods described in the following Examples 1 to 4 are intended to illustrate the available chemical methods by use of specific examples, and are not intended to represent the scope of the present invention.

Example 1

(Z)-1 ² -Amino-2 ⁴ ,2 ⁶ -dichloro-3-oxo-8-aza-1(4,6)-pyrrolo[2,1-f][1,2,4] Preparation of triazine-2(1,2)-benzocyclononaza-9-one (compound 16):

Intermediates of compounds of _formula (16) of the present invention, wherein R1 is chlorine, R2 is chlorine, and R4 is oxygen, _were synthesized by the following scheme ₁ :

Synthesis of compound (2):

Under nitrogen protection and zero degrees, 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. Then, the temperature was raised to room temperature, and after stirring the reaction for 24 hours, the reaction mixture was sequentially quenched with 250 ml of 1N hydrochloric acid solution, extracted with 300 ml of ethyl acetate, washed with 100 ml of saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate overnight. , concentrated, and purified by silica gel column (EA/PE, 0-20%) to obtain 24 g (70%) of white solid.

Synthesis of compound (3)

Compound (2) (6.7g, 23mmol), potassium carbonate (38g, 27.6mmol) were dissolved in 100ml of anhydrous acetonitrile, bromine (3ml, 25mmol) was added dropwise to the above reaction solution, after the dropwise addition, refluxed 18h. After the reaction was completed, it was cooled to room temperature, 250 ml was added, and the aqueous phase was extracted with dichloromethane (100 ml). The organic phases were combined, washed with 2N sodium hydroxide (100ml), washed with water (100ml*2), washed with saturated brine (100ml), dried over anhydrous sodium sulfate, concentrated, and passed through a silica gel column (EA/PE, 0-30%) , yielding 7 g (80%) of a yellow solid.

Synthesis of compound (4):

Under nitrogen protection, (7.7 g, 22 mmol) compound (3), pinacol diboronate (5.7 ml, 44 mmol) and triethylamine (9.2 ml, 66 mmol) were dissolved in 50 g of anhydrous dioxane solution, Then, palladium acetate (270 mg, 1.1 mmol) and (850 mg, 2.2 mmol) biphenyl-2-yl-dicyclohexylphosphine were added to the reaction system, and the reaction was carried out at 80 degrees for one and a half hours. After the reaction was completed, it was cooled, and 50 ml of ethyl acetate was added to the reaction solution, followed by washing with 25 ml of saturated ammonium chloride, 25 ml of water, and 25 ml of saturated brine. Dry over anhydrous sodium sulfate and concentrate. Silica gel column (EA/PE, 0-50%) gave 3.6 g (46%) of brown solid.

Synthesis of compound (5):

Under nitrogen protection, 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). Dry over anhydrous sodium sulfate and concentrate. The obtained crude product was washed with n-hexane, suction filtered, and dried to obtain 930 mg (85%) of a light brown solid. ¹ H-NMR: (400 MHz, MeOD) δ 6.88-6.89 (d, 1H), 6.73-6.74 (d, 1H), 1.30 (s, 12H).

The compound of formula (16) of the present invention was synthesized by the following scheme 2, wherein R ₃ is oxygen, R ₄ is hydrogen, X is nitrogen atom, and Y is methylene group:

Synthesis of compound (7):

(9.1 g, 80 mmol) ethyl isocyanate and (18.4 g, 120 mmol) 1,8-diazabicycloundec-7-ene (DBU) were dissolved in 400 ml of tetrahydrofuran. Under stirring conditions, 160 mmol of p-type methanol was added to the reaction system, and the temperature was raised to 50 degrees to react for 5 hours. After the reaction was completed, it was cooled to room temperature, 1000 ml of ethyl acetate was added, washed with 300 ml of saturated sodium bicarbonate, 300 ml of saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain 6.1 g of a pale yellow solid. The next reaction was carried out directly without purification.

¹ H-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).

Synthesis of compound (8):

Compound (7) (9.0 g, 42.7 mmol), ammonium chloride (6.9 g, 130.2 mmol) were dissolved in 2N sodium hydroxide solution (42.9 ml, 85.8 mmol) and 600 ml of methyl tert-butyl ether, and under stirring, 120 ml of concentrated ammonia water and 90 ml of sodium hypochlorite aqueous solution were added, and the reaction was stirred at room temperature for 6 hours. After the reaction is completed, filter, add 1500 ml of ethyl acetate to the filtrate, wash with 500 ml of saturated sodium bicarbonate and 500 ml of saturated brine, dry over anhydrous sodium sulfate, and concentrate to obtain 7.5 g (77%) of a pale yellow solid without purification. Proceed directly to the next reaction. ¹ H-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).

Synthesis of compound (9):

(4.8 g, 21.4 mmol) of compound (8) and (39.4 g, 342.4 mmol) of chloromethylamidine hydrochloride were dissolved in 400 ml of DMSO, and the temperature was raised to 150 degrees and stirred for 1 hour. After the reaction was completed, it was cooled to room temperature, 700 ml of ethyl acetate was added, washed with 300 ml of saturated sodium bicarbonate and 300 ml of saturated brine, and the organic phase was dried over anhydrous sodium sulfate and concentrated. Crystallization yielded 2.5 g (53%) of an off-white powdery solid.

¹ H-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).

Synthesis of compound (10):

Compound (9) (5 g, 22.5 mmol) and thionyl chloride (3.45 ml, 10.9 mol) were dissolved in 100 ml of dichloromethane and reacted under reflux for 3 hours. After the reaction was completed, it was cooled to room temperature, 1000 ml of dichloromethane was added, washed with 500 ml of saturated sodium bicarbonate and 500 ml of saturated brine, dried over anhydrous sodium sulfate, and concentrated. Crystallization yielded 6.6 g (93%) of an off-white solid.

¹ H-NMR: (400 MHz, DMSO-D ₆ ) δ 1.28 (d, J=5.81 Hz, 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).

Synthesis of compound (11):

Compound (9) (10 g, 36.1 mmol) was dissolved in phosphorus oxychloride (170 ml), and the reaction was stirred at 100 degrees for 30 minutes. After the color of the reaction solution changed to uniform brown, the heating was continued for 30 minutes. After the reaction, the reaction flask was placed in ice water and rapidly cooled to zero degrees, and then the Ph was adjusted to weakly alkaline 7 with 2N aqueous sodium hydroxide solution. A white suspension was formed, filtered, washed with 500 ml of ethyl acetate, the filtrate was washed with brine, several layers were dried over anhydrous sodium sulfate, concentrated, and passed through a silica gel column (EA/PE, 0-30%) to obtain 6 g of a pale yellow solid (59%).

¹ H-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).

Synthesis of compound (12):

Under nitrogen protection, sodium carbonate (3.5 g, 33.0 mmol), compound (10) (2.3 g, 8.3 mmol) and tetrakis(triphenylphosphine)palladium (959 mg, 0.8 mmol) were dissolved in 100 ml of anhydrous dioxane in the ring. The reaction was stirred at 80 degrees overnight. After the reaction was completed, after cooling to room temperature, 100 ml of water was added, and extraction was performed three times with 200 ml of ethyl acetate. The organic phases were combined, washed with 500 ml of saturated brine, dried over anhydrous sodium sulfate, concentrated, and passed through a silica gel column (EA/PE, 0-50%) to obtain 2.7 g (79%) of a gray solid.

¹ H-NMR: (400 MHz, DMSO-D ₆ ) δ 1.28 (d, J=5.81 Hz, 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). Synthesis of compound (13):

Under nitrogen protection, cesium carbonate (6.4 g, 20 mmol) and compound (12) (5.0 g, 11.8 mmol) and 6-chloro-n-hexyl-1-amine (2.1 g, 15.4 mmol) were dissolved in 40 ml of anhydrous DMF , and the reaction was stirred at 100 degrees for 3 hours. After the reaction was completed, the mixture was cooled to room temperature and concentrated under reduced pressure to remove the solvent. DCM (200ml) was added, and the organic phase was washed with water (200ml*4) and saturated brine (200ml). It was dried over anhydrous sodium sulfate, concentrated, and passed through a silica gel column (DCM/MeOH, 0-5%) to obtain 3.7 g (60%) of a light brown viscous substance.

Synthesis of compound (14):

(12 ml, 48 mmol) 4M hydrochloric acid in dioxane, (1.6 g, 3.2 mmol) compound (13) was mixed with 10 ml methanol and the reaction was stirred at room temperature overnight. After the reaction, excess hydrochloric acid was neutralized with saturated sodium carbonate, and then the organic solvent was removed by concentration under reduced pressure. 50 ml of ethyl acetate was added to the system for extraction twice, the organic phases were combined, washed with 100 ml of saturated brine, dried over anhydrous sodium sulfate, concentrated, and passed through a silica gel column (DCM/MeOH, 0-5%) to obtain a gray solid 1.3 g (90%).

Synthesis of compound (15):

Compound (14) (930 mg, 2 mmol) was dissolved in 40 ml of ethanol, and 2N aqueous sodium hydroxide solution (8 ml, 16 mmol) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the pH was adjusted to neutrality with 2N hydrochloric acid. It was extracted twice with 50 ml of ethyl acetate, and the organic phases were combined, washed with 100 ml of saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain 740 mg (85%) of a gray solid. Proceed directly to the next reaction.

Synthesis of compound (16):

Combine (245 mg, 1.3 mmol) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, (280 mg, 0.6 mmol) compound (15) and (0.27 ml, 1.9 mmol) three Ethylamine was dissolved in 5ml 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 209 mg (80%) of an off-white solid.

¹ H-NMR of compound (16): (400 MHz; DMSO-d6) δ 0.97-2.01 (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.

Example 2

(Z)-1 ² -Amino-2 ⁴ ,2 ⁶ -dichloro-3-oxo-7-nitro-1(4,6)-pyrrolo[2,1-f][1,2,4]tris Preparation of oxazine-2(1,2)-benzocyclononaza-8-one (compound 28):

The synthesis method of compound (28) refers to the synthesis method of compound (16).

¹ H-NMR of compound (28): (400 MHz; DMSO-d6) δ 0.99-2.02 (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.

Example 3

(Z)-1 ² -Amino-2 ⁴ ,2 ⁶ -dichloro-3-oxo-8-aza-1(4,6)-pyrrolo[2,1-f][1,2,4] Preparation of triazine-2(1,3)-benzocyclononaza-9-one (compound 29):

The synthesis method of compound (29) refers to the synthesis method of compound (16).

¹ H-NMR of compound (29): (400 MHz; DMSO-d6) δ 0.98-2.03 (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.

Example 4

(Z)-1 ² -Amino-2 ⁴ ,2 ⁶ -dichloro-3-oxo-7-nitro-1(4,6)-pyrrolo[2,1-f][1,2,4]tris Preparation of oxazine-2(1,3)-benzocyclononaza-8-one (compound 30):

The synthesis method of compound (30) refers to the synthesis method of compound (16).

¹ H-NMR of compound (30): (400 MHz; DMSO-d6) δ 0.99-2.02 (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.

Example 5

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:

Synthesis of compound (18):

Compound (17) (15.6g, 75mmol), potassium carbonate (12g, 87mmol) were dissolved in 200ml of anhydrous acetonitrile, bromine (9ml, 76mmol) was added dropwise to the above reaction solution, after the dropwise addition, refluxed for 18 Hour. After the reaction was completed, it was cooled to room temperature, 500 ml of water was added, and the aqueous phase was extracted with dichloromethane (200 ml). The organic phases were combined, washed with 2N sodium hydroxide (200ml), washed with water (200ml*2), washed with saturated brine (200ml), dried over anhydrous sodium sulfate, concentrated, and passed through a silica gel column (EA/PE, 0-30%) , to obtain 21.6 g (96%) of a yellow solid.

Synthesis of compound (19):

Iron powder (11 g, 188 mmol) and compound (18) (10.8 g, 36 mmol) were dissolved in (150 mL) acetic acid and water (75 mL), and the reaction was stirred at 85 degrees for 90 minutes. After the reaction was completed, suction filtration while still hot, the reaction solution was cooled to room temperature, 300 ml of water was added, and extracted three times with 100 ml of DCM. The combined organic phase was washed with 2M sodium hydroxide, once with water, once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a light brown solid (9.5 g, 99%).

¹ H-N MR (400 MHz, CDCl ₃ ) δ 5.20 (s, 2H), 7.34-7.48 (m, 5H), 7.56 (s, 1H), 7.58 (s, 1H).

Synthesis of compound (20):

Compound (19) (16.2 g, 60 mmol) was dissolved in 240 ml of acetic acid, then (6.0 M, 60 mL) hydrochloric acid was added to the reaction system, and the reaction system was cooled to zero. (4.8 g, 69.5 mmol) sodium nitrite was dissolved in 40 ml of water, and slowly added dropwise to the above reaction system, the temperature of the reaction system was lower than 5 degrees. After the dropwise addition was completed, the reaction was stirred for 30 minutes. After 30 minutes, (20 g, 120 mmol) potassium iodide and (4 g, 16 mmol) iodine were dissolved in 200 ml of water, and then added dropwise to the above reaction system, and the reaction was stirred for 90 minutes. After the reaction, add 800 ml of water, extract three times with 250 ml of DCM, combine the organic phases, quench iodine with 250 ml of 10% (w/v) sodium thiosulfate, wash once with 250 ml of 2M sodium hydroxide, and wash three times with 500 ml of water, and 200 ml of saturated brine It was washed once, dried over anhydrous sodium sulfate, and concentrated to obtain 21 g (90%) of a light brown solid.

¹ H-N MR (400 MHz; CDCl ₃ ) δ 5.10 (s, 2H), 7.33-7.48 (m, 7H).

Compound (27) of the present invention, wherein _R3 is oxygen, R4 is hydrogen, X is carbon atom, and Y is sulfur atom, was synthesized by, for example, the following scheme ₄ :

Synthesis of compound (22):

Compound (21) (2.5 g, 13 mmol) and potassium carbonate (4.5 g, 32.6 mmol) were dissolved in 80 ml of acetonitrile, and (2.86 mL, 26 mmol) of ethyl mercaptoacetate was added with stirring. The reaction was stirred at 85 degrees for 5 hours. After the reaction was completed, it was cooled to room temperature. After suction filtration, the obtained solid was washed with 25 ml of acetonitrile, then washed with 100 ml of water, and dried in vacuo to obtain 2.8 g (83%) of a pale yellow solid.

¹ H-N MR(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 ).

Synthesis of compound (23):

Under nitrogen protection, 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.

Under nitrogen protection, add 80 ml of dioxane, (1.3 g, 5 mmol) compound (20), 4 ml of 2M potassium phosphate solution, tetrakis(triphenylphosphine) palladium (cat.), and react at 100 degrees for 3 Hour. After the reaction was completed, it was cooled to room temperature, 40 ml of ethyl acetate was added, the organic phase was washed with 25 ml of saturated brine and dried over anhydrous sodium sulfate, and passed through a silica gel column (EA/PE=0-1/30) to obtain 1.1 g (45%) of a white solid. ).

¹ H-N MR (400MHz; DMSO-d6) δ 1.28 (t, 3H, J=7.1 Hz), 4.29 (q, 2H, J=7.1 Hz), 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).

Synthesis of compound (24):

Under nitrogen protection, compound (23) (1.8 g, 3.8 mmol) was dissolved in 15 ml of DCM, 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). Dry over anhydrous sodium sulfate and concentrate. The obtained crude product was washed with n-hexane, suction filtered, and dried to obtain 1.0 g (70%) of a pale yellow solid.

Synthesis of compound (25):

Under nitrogen protection, cesium carbonate (6.4 g, 20 mmol) and compound (24) (4.5 g, 11.8 mmol) and 6-chloro-n-hexyl-1-amine (2.1 g, 15.4 mmol) were dissolved in 40 ml of anhydrous DMF , and the reaction was stirred at 100 degrees for 3 hours. After the reaction was completed, the mixture was cooled to room temperature and concentrated under reduced pressure to remove the solvent. DCM (200ml) was added, and the organic phase was washed with water (200ml*4) and saturated brine (200ml). It was dried over anhydrous sodium sulfate, concentrated, and passed through a silica gel column (DCM/MeOH, 0-5%) to obtain 3.4 g (60%) of a light brown viscous substance.

Synthesis of compound (26):

Compound (25) (964 mg, 2 mmol) was dissolved in 40 ml of ethanol, and 2N aqueous sodium hydroxide solution (8 ml, 16 mmol) was added. The reaction was stirred at room temperature overnight. After the reaction, pH was adjusted to neutrality with 2N hydrochloric acid. It was extracted twice with 50 ml of ethyl acetate, the organic phases were combined, washed with 100 ml of saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain 772 mg (85%) of a pale yellow solid. Proceed directly to the next reaction.

Synthesis of compound (27):

(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.

¹ H-NMR of compound (27): (400 MHz; DMSO-d6) δ 0.97-2.01 (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.

Example 6

(Z)-1 ² -Amino-2 ⁴ ,2 ⁶ -dichloro-3-oxo-7-aza-1(4,6)-thieno[2,3-d]pyrimidine-2(1,2) - Preparation of benzocyclononaza-8-one (compound 31):

The synthesis method of compound (31) refers to the synthesis method of compound (27).

¹ H-NMR of compound (31): (400 MHz; DMSO-d6) δ 1.01-2.03 (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.

Example 7

(Z)-1 ² -Amino-2 ⁴ ,2 ⁶ -dichloro-3-oxo-8-aza-1(4,6)-thieno[2,3-d]pyrimidine-2(1,3) - Preparation of benzocyclononaza-9-one (compound 32):

The synthesis method of compound (32) refers to the synthesis method of compound (27).

¹ H-NMR of compound (32): (400 MHz; DMSO-d6) δ 0.98-2.01 (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.

Example 8

(Z)-1 ² -Amino-2 ⁴ ,2 ⁶ -dichloro-3-oxo-7-aza-1(4,6)-thieno[2,3-d]pyrimidine-2(1,3) - Preparation of benzocyclononaza-8-one (compound 33):

The synthesis method of compound (33) refers to the synthesis method of compound (27).

¹ H-NMR of compound (33): (400 MHz; DMSO-d6) δ 1.01-2.03 (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.

Example 9

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 Hsp90α/Hsp90β 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. If the binding between the fluorescently labeled small molecule and the macromolecule is replaced by other ligands, its rotation and flipping speed in the free state will become faster, and the emitted light will be depolarized relative to the excitation light plane. 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 Hsp90α, 30Nm Hsp90β, 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:

(without compound mP-with compound mP)/(without compound mP-negative control mP) x 100%

After calculating the inhibition rate of the compound at different concentrations, calculate the _IC50 of the compound.

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 .

This example also provides the binding Kd of the compound of formula I or a pharmaceutically acceptable salt, solvate or hydrate thereof to Hsp90α/Hsp90β protein:

Intrinsic fluorescence measurements were performed with a SprectraMax M5 spectrophotometer. Test compounds were diluted to 20 μM in assay buffer pH 7.4 containing 20 mM Hepes and 50 mM NaCl. Recombinant Hsp90α and Hsp90β proteins were added to the KU675 solution, respectively, adjusted to design concentrations (0 to 100 μg/mL for Hsp90α and 0 to 140 μg/mL for Hsp90β), 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.

Through this example, the binding Kd value of the compound of formula I or its pharmaceutically acceptable salt, solvate or hydrate and Hsp90α/Hsp90β 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:

This example provides the anticancer effect of the compound of formula I or a pharmaceutically acceptable salt, solvate or hydrate thereof in a common tumor cell line recommended by the National Cancer Institute (see 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) is the core development, 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, HO-8910.

The growth inhibition of tumor cells was detected by 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 ). Add different concentrations of the drug to be tested for 6-48 hours, then add CCK-8 solution, set it in an incubator (37°C, 5% CO ₂ ) for a warm bath until color development (1-4 hours), and measure it with a microplate reader at a wavelength of 450 nm. optical density (OD value). The results were recorded, and the inhibition rates of the compounds provided herein (compounds 1-142 in Table 1) on tumor cell growth at different concentrations and times were calculated. The inhibition rate of drug on tumor cell growth was calculated according to the following formula: cell growth inhibition rate=(1-average OD value of experimental group/average OD value of control group)×100%). The experiment was repeated three times.

Through this example, 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 Hsp90β 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 μg) were electrophoresed in 10% acrylamide gels under reducing conditions, transferred to polyvinylidene fluoride membranes (PVDF), and immunoblotted with corresponding specific antibodies. 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 Hsp90β 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). Mix 15 mg/mL of Hsp90βNT with each inhibitor to form protein inhibitor complexes at a final drug concentration of 1.5-2.0 mM and incubate at 4°C for 1 hour. Cocrystal droplets were formed with a 1:1 ratio of protein to drug in 30% PEG 8000, 0.2M sodium acetate and 0.1 sodium cacodylate (pH 6.5) buffer at room temperature. Crystals appeared within 1-2 days and were obtained in a low temperature buffer containing 20% glycerol, which was added to the buffer, each with 2 mM of inhibitor.

Table 1. Biological activity data of target compounds

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1. A specific heat shock protein 90α subtype inhibitor or a pharmaceutically acceptable salt, solvate or hydrate thereof, which has the structure shown in formula I:

   

   in:

   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 -C ₁₀ heterocycle), -(C ₁ -C ₆ alkenyl)-O-(C ₂ -C ₆ alkenyl), -O-(C ₁ -C ₆ alkenyl)-(C ₃ -C ₁₀ heterocycle), -O-(C ₂ -C ₆ alkyl)-(C ₃ -C ₁₀ heterocycle) or C ₁ -C ₈ alkoxy;

   _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.
2. The specific heat shock protein 90α isoform inhibitor or a pharmaceutically acceptable salt, solvate or hydrate thereof according to claim 1, wherein the specific heat shock protein 90α isoform inhibitor or Its pharmaceutically acceptable salt, solvate or hydrate has the structure represented by formula (I') or formula (II'):

   

   Preferably, 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 -C ₁₀ heterocycle), -(C ₁ -C ₆ alkenyl)-O-(C ₂ -C ₆ alkenyl), -O-(C ₁ -C ₆ alkenyl)-(C ₃ -C ₁₀ heterocycle), -O-(C ₂ -C ₆ alkyl)-(C ₃ -C ₁₀ heterocycle) or C ₁ -C ₈ alkoxy;

   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.
3. The specific heat shock protein 90α isoform inhibitor or a pharmaceutically acceptable salt, solvate or hydrate thereof according to claim 1, wherein the specific heat shock protein 90α isoform inhibitor or Its pharmaceutically acceptable salts, solvates or hydrates are selected from the following compounds:

   (Z)-1 ² -Amino-2 ⁴ ,2 ⁶ -dichloro-3-oxo-8-aza-1(4,6)-pyrrolo[2,1-f][1,2,4] Triazin-2(1,2)-benzocyclononaza-9-one;

   (Z)-1 ² -Amino-2 ⁴ ,2 ⁶ -dichloro-3-oxo-7-nitro-1(4,6)-pyrrolo[2,1-f][1,2,4]tris oxazin-2(1,2)-benzocyclononaza-8-one;

   (Z)-1 ² -Amino-2 ⁴ ,2 ⁶ -dichloro-3-oxo-8-aza-1(4,6)-pyrrolo[2,1-f][1,2,4] Triazin-2(1,3)-benzocyclononaza-9-one;

   (Z)-1 ² -Amino-2 ⁴ ,2 ⁶ -dichloro-3-oxo-7-nitro-1(4,6)-pyrrolo[2,1-f][1,2,4]tris oxazin-2(1,3)-benzocyclononaza-8-one;

   (Z)-1 ² -Amino-2 ⁴ ,2 ⁶ -dichloro-3-oxo-8-nitro-1(4,6)-thieno[2,3-d]pyrimidine-2(1,2) - benzocyclononaza-9-one;

   (Z)-1 ² -Amino-2 ⁴ ,2 ⁶ -dichloro-3-oxo-7-aza-1(4,6)-thieno[2,3-d]pyrimidine-2(1,2) - benzocyclononaza-8-one;

   (Z)-1 ² -Amino-2 ⁴ ,2 ⁶ -dichloro-3-oxo-8-aza-1(4,6)-thieno[2,3-d]pyrimidine-2(1,3) - benzocyclononaza-9-one;

   (Z)-1 ² -Amino-2 ⁴ ,2 ⁶ -dichloro-3-oxo-7-aza-1(4,6)-thieno[2,3-d]pyrimidine-2(1,3) - Benzocyclononaza-8-ones.
4. A preparation method of the specific heat shock protein 90α subtype inhibitor described in any one of claims 1-3 or a pharmaceutically acceptable salt, solvate or hydrate thereof, wherein the preparation method is:

   by

   

   Preparation of intermediates for starting materials

   

   by

   

   Prepared for raw materials

   

   Intermediates (5) and (11) react to form compounds of formula (I').
5. A preparation method of the specific heat shock protein 90α subtype inhibitor described in any one of claims 1-3 or a pharmaceutically acceptable salt, solvate or hydrate thereof, wherein the preparation method is:

   by

   

   Preparation of intermediates for starting materials

   

   by

   

   Preparation of intermediates for starting materials

   

   Intermediates (20) and (22) react to yield compounds of formula (II').
6. A pharmaceutical composition, characterized in that, the pharmaceutical composition comprises the specific heat shock protein 90α subtype inhibitor according to claim 1 or a pharmaceutically acceptable salt, solvate or hydrate thereof.
7. A pharmaceutical preparation, characterized in that the pharmaceutical preparation comprises the specific heat shock protein 90α subtype inhibitor according to claim 1 or a pharmaceutically acceptable salt, solvate or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.
8. Application of the specific heat shock protein 90α subtype inhibitor according to claim 1 or a pharmaceutically acceptable salt, solvate or hydrate thereof or the pharmaceutical composition according to claim 2 in the preparation of a medicine for inhibiting Hsp90 enzyme activity Preferably, described application comprises making Hsp90 enzyme and the described specific heat shock protein 90α subtype inhibitor of claim 1 or its pharmaceutically acceptable salt, solvate or hydrate or the described medicine combination of claim 2 object contact.
9. A specific heat shock protein 90α subtype inhibitor according to claim 1 or a pharmaceutically acceptable salt, solvate or hydrate thereof or the pharmaceutical composition according to claim 2 in the preparation of preventing and/or treating cancer or the application of oncology drugs,

   Preferably, the cancer or tumor includes bladder cancer, breast cancer, cervical cancer, colon 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, skin cancer, stomach cancer, testicular cancer, thyroid cancer and uterine cancer;

   Colon cancer is more preferably colorectal cancer; lung cancer is more preferably small cell lung cancer or non-small cell lung cancer; sarcoma is more preferably osteosarcoma, and skin cancer is more preferably squamous cell carcinoma.
10. A specific heat shock protein 90α subtype inhibitor according to claim 1 or a pharmaceutically acceptable salt, solvate or hydrate thereof or the pharmaceutical composition according to claim 2 in the preparation of a medicine for inhibiting cell growth. Application, preferably, the application comprises making the cell of claim 1 and the specific heat shock protein 90α isoform inhibitor or a pharmaceutically acceptable salt, solvate or hydrate or the cell of claim 2 pharmaceutical composition contact;

    Preferably, the cells are tumor cells, further preferably, the cells are mammalian tumor cells or human tumor cells;

    Alternatively, the cells are cancer cells, further preferably, the cells are mammalian cancer cells or human cancer cells;

    Further preferably, the cancer cells include bladder cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, head and neck cancer, liver cancer, lung cancer, melanoma, myeloma, neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer cancer, kidney cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer and uterine cancer;

    Colon cancer is more preferably colorectal cancer; lung cancer is more preferably small cell lung cancer or non-small cell lung cancer; sarcoma is more preferably osteosarcoma, and skin cancer is more preferably squamous cell carcinoma;

    Further preferably, 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;

    Further preferably, the cancer cells are metastatic cancer cells, including bladder cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, head and neck cancer, liver cancer, lung cancer, melanoma, myeloma, neuroblastoma, ovary cancer, pancreatic cancer, prostate cancer, kidney cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer and uterine cancer;

    Colon cancer is more preferably colorectal cancer; lung cancer is more preferably small cell lung cancer or non-small cell lung cancer; sarcoma is more preferably osteosarcoma, and skin cancer is more preferably squamous cell carcinoma.