CN109748914B - Pyridopyrimidine compound and application thereof - Google Patents

Abstract

The invention relates to a compound of formula I, an enantiomer, a racemate or a salt thereof, and application thereof in treating tumors.

Description

Pyridopyrimidine compound and application thereof

Technical Field

The invention relates to a pyridopyrimidine compound shown in a formula I and application thereof, in particular to application in tumor treatment.

Background

Malignant tumor has become one of the diseases with the highest morbidity and mortality in China. Radiation therapy is one of the main means of treating malignant tumors. The ideal purpose of radiotherapy is to radically treat malignant tumors and simultaneously reduce the damage of normal tissues as much as possible, thereby ensuring the long-term survival and the quality of life of patients. However, due to the heterogeneity of tumors and the individual variability, it is inevitable to cause various side effects to patients during the radiotherapy process, which also becomes a bottleneck limiting the application of radiotherapy technology.

Radiosensitizers refer to chemical substances that increase the sensitivity of tumor cells to radiation and increase the lethal effects of radiation when used in combination with radiation. Many compounds have been found to enhance the effects of radiating cells, such as halopyrimidines, nitroimidazoles, repair inhibitors, and the like, but most of them have limited application due to limited sensitizing effects or large toxic and side effects.

The radioprotectant is a compound which can reduce radiation damage and promote recovery of a certain chemical substance when the body or a certain biological system is given before or after the body or the certain biological system is irradiated by ionizing radiation. The first radioprotectants discovered were the sulfhydryl compounds. A radioprotectant that has proven effective at present is WR2721, amifostine. It can obviously reduce toxic side effect of radiotherapy, but it also has greater toxic side effect. The mechanism is to reduce the damage of free radicals to cells. Another class of anti-radiation side-effect drugs is to reduce radiation side-effects by anti-radiation inflammatory reactions.

In the radiotherapy of tumors, if the two opposite effects of the radiation protection and the radiation sensitization of the drug can be unified. That is, compounds that sensitize tumor cells while protecting normal tissues. This dual function drug would be ideal for improving the effectiveness of radiation therapy. At present, no compound exists, and the compound has radiosensitization and radioprotection effects and has small toxic and side effects. Therefore, it is urgent to develop new drugs that can not only increase the efficacy of radiotherapy, but also reduce the side effects of radiation.

Disclosure of Invention

An object of the present invention is to provide a pyridopyrimidine compound of formula I, an enantiomer, a racemate or a salt (especially a pharmaceutically acceptable salt) thereof:

wherein R is₁、R₂、R₃、R₄、n₁、n₂And all substituents are as defined in the detailed description.

Another object of the present invention is to provide a pyridopyrimidine compound of formula I-1, an enantiomer, a racemate or a salt thereof (especially a pharmaceutically acceptable salt), an enantiomer or a racemate:

wherein R is₂、R₃、R₄、R₅、R₆、R₇、n₁、n₂、n₃And all substituents are as defined in the detailed description.

Another object of the present invention is to provide a pyridopyrimidine compound of formula I-1-1, an enantiomer, a racemate or a salt thereof (especially a pharmaceutically acceptable salt), an enantiomer or a racemate:

wherein R is₂、R₃、R₄、R₅、R₆、R₇、n₁、n₂And n₃As defined in the detailed description.

Another object of the present invention is to provide a pyridopyrimidine compound of formula I-2, an enantiomer, a racemate or a salt thereof (especially a pharmaceutically acceptable salt), an enantiomer or a racemate:

wherein R is₂、R₃、R₄、R₈、R₉、R₁₀、R₁₁、R₁₂、U₁、U₂、U₃、U₄、U₅、n₁、n₂And n₄As defined in the detailed description.

The invention also provides a pharmaceutical composition, which comprises the compound of the formula I, enantiomer, racemate or pharmaceutically acceptable salt thereof as an active ingredient, and at least one pharmaceutically acceptable carrier.

The invention also provides application of the compound shown in the formula I, enantiomer, racemate or pharmaceutically acceptable salt thereof in preparing a medicament for treating tumors or cancers.

The present invention also provides a method of treating a tumor or cancer comprising administering to a subject a therapeutically effective amount of a compound of formula I, enantiomers, racemates or pharmaceutically acceptable salts thereof.

Drawings

FIG. 1 shows the cell growth inhibition rate of four different tumor cells in an MTT assay with the addition of an exemplary compound of the invention.

FIG. 2 shows the results of an annexin V-APC/PI double staining assay of exemplary compounds of the invention.

FIG. 3 shows a cross-sectional view of 8X 10⁵4T1-luc cells were injected subcutaneously and quantitative in vivo imaging (BLI) of tumor 4T1-luc activity in mice treated with the compound of the invention HW036(37.5mg/kg), the compound of the invention HW036(75mg/kg) and 5-Fu (20 mg/kg).

FIG. 4 shows a quantitative BLI for 4T1-luc activity, indicating that 37.5mg/kg and 75mg/kg of HW036 administration groups of the compound of the present invention have a stronger inhibitory effect on lung metastasis of breast cancer cells in mice than 5-Fu (20mg/kg)) administration groups.

FIG. 5 shows the results of CNE-2 cell proliferation toxicity assay (MTT assay) for HW086 compounds.

FIG. 6 shows the results of 4T1 cell proliferative toxicity assay (MTT method) for HW031 and HW086 compounds.

FIG. 7 shows the results of the apoptosis rate measured after treating liver cancer cells (SMMC-7721) with HW086 and HW031 at 20ug/ml for 24 hours.

Figures 8A-8B show in vivo imaging results of BABL/C mice in the first week after administration of HW086 and HW031, which significantly reduced lung metastatic tumor size of breast cancer in mice.

FIG. 9 shows the results of in vivo imaging of BABL/C mice 3 weeks after administration of HW086, HW086 significantly reduced the size of lung metastasis tumor of breast cancer in mice.

FIG. 10 shows the results of the in vivo inhibition of liver cancer by HW031 and HW086 on the H22 liver cancer model in ICR mice.

FIGS. 11-12 show the results of toxicity tests of HW031 and HW086 for their anti-cancer effects. FIG. 11 shows the change in body weight (g) of mice two weeks after administration of HW-086 and HW-031. The change of the body weight of mice in HW-086 and HW-031 administration groups has no significant difference compared with the control group. FIG. 12 shows that no significant impairment of hepatic and renal function was observed in HW-086 and HW-031 groups.

Detailed Description

The pyridopyrimidine compound, the enantiomer, the racemate or the salt (especially the pharmaceutically acceptable salt) thereof can effectively inhibit the proliferation of malignant tumor cells and has the effect of increasing radiotherapy.

One aspect of the present invention provides embodiment 1): a compound of formula I, enantiomers, racemates or salts thereof (especially pharmaceutically acceptable salts):

wherein the content of the first and second substances,

R₂represents S, O or NH;

R₃represents an alkyl group or an aryl group, wherein the alkyl group is optionally substituted with a substituent selected from: hydroxy, C_1-3Alkyl radical, C_1-3Alkoxy, amino, halogen or carboxyl (i.e., COOH), the aryl group optionally substituted with a substituent selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy;

R₄represents an alkyl group, an aryl group, OH, SH, or NH₂Wherein said alkyl is optionally substituted with a substituent selected from the group consisting of: hydroxy, C_1-3Alkyl radical, C_1-3Alkoxy, amino, halogen or carboxyl, said aryl being optionally substituted with a substituent selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy;

n₁represents an integer of 0, 1,2,3, 4, 5, 6,7,8,9 or 10;

n₂represents an integer of 0, 1,2,3, 4, 5, 6,7,8,9 or 10;

R₁represented by the following formula G₁Or G₂The group of (a):

wherein the content of the first and second substances,

R₅represents an alkyl group;

R₆represents aryl or heteroaryl, wherein said aryl is optionally substituted with a substituent selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy, and the heteroaryl is optionally substituted with a substituent selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy;

R₇represents aryl or heteroaryl, wherein said aryl is optionally substituted with a substituent selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy, and the heteroaryl is optionally substituted with a substituent selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy;

n₃represents 0, 1,2,3, 4, 5, 6,7,8,9 or 10;

R₈、R₉、R₁₀、R₁₁each independently represents halogen or hydrogen;

R₁₂represents S, O or NH;

n₄represents 0, 1,2,3, 4, 5, 6,7,8,9 or 10;

U₁represents NH or O; and

U₂、U₃、U₄、U₅the same or different, each independently represents CH or N.

In the present invention, the term "halogen" or "halogen atom", used alone or in combination, means fluorine, chlorine, bromine or iodine, and is preferably F, Cl or Br.

In the present invention, the term "alkyl" used alone or in combination refers to a straight or branched monovalent saturated hydrocarbon group consisting of carbon and hydrogen atoms. The term "C_x-C_yAlkyl "or" C_x-yAlkyl "(x and y are each an integer) means a straight or branched chain alkyl group containing x to y carbon atoms. In the present invention, the term "C" is used alone or in combination_1-10Alkyl "refers to a straight or branched chain alkyl group containing 1 to 10 carbon atoms. C of the invention_1-10The alkyl group is preferably C_1-9Alkyl, more preferably C_1-8Alkyl, still more preferably C_2-8Alkyl, more preferably C_1-7Alkyl, even more preferably C_1-6Alkyl radical, C_1-5Alkyl radical, C_1-4Alkyl, or C_1-3An alkyl group. Representative examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, or octyl. The term "C" used alone or in combination_1-3Alkyl "refers to a straight or branched chain alkyl group containing 1 to 3 carbon atoms. Representative examples include methyl, ethyl, n-propyl, isopropyl. In the present invention, the "alkyl group" is optionally substituted (preferably 1 to 3 times) with a substituent selected from the group consisting of: hydroxy, C_1-3Alkyl radical, C_1-3Alkoxy, amino, halogen or carboxyl.

In the present invention, the term "aryl" used alone or in combination refers to an aromatic hydrocarbon group containing 5 to 14 carbon atoms and optionally containing one or more fused rings, such as phenyl, naphthyl or fluorenyl. In the present invention, the "aryl" is optionally substituted (preferably 1 to 3 times) with a substituent selected from the group consisting of: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy.

In the present invention, the term "C" is used alone or in combination_1-3Alkoxy "refers to a straight or branched chain alkoxy group containing 1 to 3 carbon atoms and having the formula-O-C_1-3An alkyl group. C_1-3Representative examples of alkoxy groups include methoxy, ethoxy, n-propoxy, and isopropoxy. Methoxy and ethoxy are preferred.

In the present invention, the term "heteroaryl" used alone or in combination refers to a 5-to 10-membered monocyclic or bicyclic aromatic ring group containing 1 or more (e.g., 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3) heteroatoms independently selected from oxygen, nitrogen and sulfur. Representative examples of such heteroaryl groups include, but are not limited to, furyl, oxazolyl, isoxazolyl, oxadiazolyl, thienyl, thiazolyl, isothiazolyl, thiadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, indolyl, isoindolyl, phenylAnd furyl, isobenzofuryl, benzothienyl, indazolyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, benzo [2,1,3 ] benzo]Oxadiazolyl, benzo [2,1,3 ] s]Thiadiazolyl, benzo [1,2,3 ] s]Thiadiazolyl, quinolyl, isoquinolyl, naphthyridinyl, cinnolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, pyrazolo [1,5-a ]]Pyridyl, pyrazolo [1,5-a]Pyrimidinyl, imidazo [1,2-a ] -compounds]Pyridyl, 1H-pyrrolo [3,2-b ] s]Pyridyl, 1H-pyrrolo [2,3-b ] s]Pyridyl, 4H-fluoro [3,2-b ]]Pyrrolyl, pyrrolo [2,1-b ]]Thiazolyl and imidazo [2,1-b ]]A thiazolyl group. According to a definite definition, a heteroaryl group may be unsubstituted or substituted. Substituted heteroaryl means heteroaryl substituted 1-3 times with a substituent, wherein the substituent is preferably selected from C_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy.

Embodiment 2 of the present invention): relates to a compound of formula I as in embodiment 1), an enantiomer, a racemate or a salt (especially a pharmaceutically acceptable salt) thereof, wherein R₂Represents O;

R₃is represented by C_1-10Alkyl or aryl, wherein said C_1-10Alkyl is optionally substituted with a substituent selected from: hydroxy, C_1-3Alkyl radical, C_1-3Alkoxy, amino, halogen or carboxyl, and the aryl group is optionally substituted with a substituent selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy;

R₄is represented by C_1-10Alkyl, aryl, OH, SH or NH₂Wherein said C is_1-10Alkyl is optionally substituted with a substituent selected from: hydroxy, C_1-3Alkyl radical, C_1-3Alkoxy, amino, halogen or carboxyl, said aryl being optionally substituted with a substituent selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy;

n₁represents an integer of 0, 1,2,3, 4, 5, 6,7,8,9 or 10 (preferably 0, 1,2 or 3); and

n₂represents an integer of 01,2,3, 4 or 5 (preferably 0, 1,2 or 3).

Embodiment 3 of the present invention): relates to a compound of the formula I, an enantiomer, a racemate or a salt thereof (in particular a pharmaceutically acceptable salt) according to embodiment 1) or 2), wherein R₁Represented by the following formula G₁The group of (a):

wherein the content of the first and second substances,

R₅is represented by C_1-10An alkyl group;

R₆represents aryl or heteroaryl, wherein said aryl is optionally substituted with a substituent selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy, and the heteroaryl is optionally substituted with a substituent selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy;

R₇represents aryl or heteroaryl, wherein said aryl is optionally substituted with a substituent selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy, and the heteroaryl is optionally substituted with a substituent selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy; and

n₃represents 0, 1,2,3, 4, 5, 6,7,8,9 or 10 (preferably 0, 1,2 or 3).

Embodiment 4 of the present invention): relates to a compound of formula I as in embodiment 3), an enantiomer, a racemate or a salt (especially a pharmaceutically acceptable salt) thereof, wherein R₃、R₅Each independently represents C_1-8An alkyl group.

Embodiment 5 of the present invention): relates to a compound of formula I as in embodiment 3), an enantiomer, a racemate or a salt (especially a pharmaceutically acceptable salt) thereof, wherein R₃Represents a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, a,Neopentyl, hexyl, heptyl or octyl; and

R₅represents methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl or octyl.

Embodiment 6 of the present invention): relates to a compound of formula I as in embodiment 3), an enantiomer, a racemate or a salt (especially a pharmaceutically acceptable salt) thereof, wherein R₆Represents an optionally substituted group selected from: phenyl, naphthyl, fluorenyl, furyl, oxazolyl, isoxazolyl, oxadiazolyl, thienyl, thiazolyl, isothiazolyl, thiadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, indolyl, isoindolyl, benzofuranyl, isobenzofuranyl, benzothienyl, indazolyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, benzo [2,1,3 ] benzo]Oxadiazolyl, benzo [2,1,3 ] s]Thiadiazolyl, benzo [1,2,3 ] s]Thiadiazolyl, quinolyl, isoquinolyl, naphthyridinyl, cinnolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, pyrazolo [1,5-a ]]Pyridyl, pyrazolo [1,5-a]Pyrimidinyl, imidazo [1,2-a ] -compounds]Pyridyl, 1H-pyrrolo [3,2-b ] s]Pyridyl, 1H-pyrrolo [2,3-b ] s]Pyridyl, 4H-fluoro [3,2-b ]]Pyrrolyl, pyrrolo [2,1-b ]]Thiazolyl or imidazo [2,1-b ]]Thiazolyl, wherein the substituents are selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy;

R₇represents an optionally substituted group selected from: phenyl, naphthyl, fluorenyl, furyl, oxazolyl, isoxazolyl, oxadiazolyl, thienyl, thiazolyl, isothiazolyl, thiadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, indolyl, isoindolyl, benzofuranyl, isobenzofuranyl, benzothienyl, indazolyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, benzo [2,1,3 ] benzo]Oxadiazole base, benzo [ alpha ], [ beta ], [ alpha ], [2,1,3]Thiadiazolyl, benzo [1,2,3 ] s]Thiadiazolyl, quinolyl, isoquinolyl, naphthyridinyl, cinnolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, pyrazolo [1,5-a ]]Pyridyl, pyrazolo [1,5-a]Pyrimidinyl, imidazo [1,2-a ] -compounds]Pyridyl, 1H-pyrrolo [3,2-b ] s]Pyridyl, 1H-pyrrolo [2,3-b ] s]Pyridyl, 4H-fluoro [3,2-b ]]Pyrrolyl, pyrrolo [2,1-b ]]Thiazolyl or imidazo [2,1-b ]]Thiazolyl, wherein the substituents are selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy.

Embodiment 7 of the present invention): relates to a compound of formula I as in embodiment 3), an enantiomer, a racemate or a salt (especially a pharmaceutically acceptable salt) thereof, wherein R₆Represents an optionally substituted thienyl group wherein the substituents are selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy.

Embodiment 8 of the present invention): relates to a compound of formula I as in embodiment 3), an enantiomer, a racemate or a salt (especially a pharmaceutically acceptable salt) thereof, wherein R₇Represents an optionally substituted naphthyl group, wherein the substituents are selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy.

Embodiment 9 of the present invention): relates to a compound of formula I, an enantiomer, a racemate or a salt thereof (especially a pharmaceutically acceptable salt) as in embodiment 1), which is also a compound of formula I-1

Wherein R is₂、R₃、R₄、R₅、R₆、R₇、n₁、n₂And n₃As defined in embodiment 1).

Embodiment 10 of the present invention): relates to a compound of the formula I, an enantiomer, a racemate or a salt thereof (in particular a pharmaceutically acceptable salt) according to embodiment 9), wherein

R₂Represents O, S or NH;

R₃、R₅each independently represents C_1-5An alkyl group;

R₄represents OH, SH or NH₂；

R₆Represents aryl or heteroaryl, wherein said aryl is optionally substituted with a substituent selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy, and the heteroaryl is optionally substituted with a substituent selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy;

R₇represents aryl or heteroaryl, wherein said aryl is optionally substituted with a substituent selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy, and the heteroaryl is optionally substituted with a substituent selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy; and

n₁、n₂、n₃each independently represents 0, 1,2,3, 4, 5, 6,7,8,9 or 10.

Embodiment 11 of the present invention): relates to a compound of the formula I, an enantiomer, a racemate or a salt thereof (in particular a pharmaceutically acceptable salt) according to embodiment 9) or 10), wherein R₆Represents an optionally substituted group selected from: phenyl, naphthyl, fluorenyl, furyl, oxazolyl, isoxazolyl, oxadiazolyl, thienyl, thiazolyl, isothiazolyl, thiadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, indolyl, isoindolyl, benzofuranyl, isobenzofuranyl, benzothienyl, indazolyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, benzo [2,1,3 ] benzo]Oxadiazolyl, benzo [2,1,3 ] s]Thiadiazolyl, benzo [1,2,3 ] s]Thiadiazolyl, quinolyl, isoquinolyl, naphthyridinyl, cinnolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, pyrazolo [1,5-a ]]Pyridyl, pyrazolo [1,5-a]Pyrimidinyl, imidazo [1,2-a ] -compounds]Pyridyl, 1H-pyrrolo [3,2-b ] s]A pyridyl group,1H-pyrrolo [2,3-b]Pyridyl, 4H-fluoro [3,2-b ]]Pyrrolyl, pyrrolo [2,1-b ]]Thiazolyl or imidazo [2,1-b ]]Thiazolyl, wherein the substituents are selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy;

R₇represents an optionally substituted group selected from: phenyl, naphthyl, fluorenyl, furyl, oxazolyl, isoxazolyl, oxadiazolyl, thienyl, thiazolyl, isothiazolyl, thiadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, indolyl, isoindolyl, benzofuranyl, isobenzofuranyl, benzothienyl, indazolyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, benzo [2,1,3 ] benzo]Oxadiazolyl, benzo [2,1,3 ] s]Thiadiazolyl, benzo [1,2,3 ] s]Thiadiazolyl, quinolyl, isoquinolyl, naphthyridinyl, cinnolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, pyrazolo [1,5-a ]]Pyridyl, pyrazolo [1,5-a]Pyrimidinyl, imidazo [1,2-a ] -compounds]Pyridyl, 1H-pyrrolo [3,2-b ] s]Pyridyl, 1H-pyrrolo [2,3-b ] s]Pyridyl, 4H-fluoro [3,2-b ]]Pyrrolyl, pyrrolo [2,1-b ]]Thiazolyl or imidazo [2,1-b ]]Thiazolyl, wherein the substituents are selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy.

Embodiment 12 of the present invention): relates to a compound of the formula I, an enantiomer, a racemate or a salt thereof (in particular a pharmaceutically acceptable salt) according to embodiment 9) or 10), wherein R₆Represents an optionally substituted thienyl group wherein the substituents are selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy.

Embodiment 13 of the present invention): relates to a compound of the formula I, an enantiomer, a racemate or a salt thereof (in particular a pharmaceutically acceptable salt) according to embodiment 9) or 10), wherein R₇Represents an optionally substituted naphthyl group, wherein the substituents are selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy.

Embodiment 14 of the present invention): relates to a compound of the formula I, an enantiomer, a racemate or a salt thereof (especially a pharmaceutically acceptable salt) as in embodiment 1), which is also a compound of the formula I-1-1

Wherein R is₂、R₃、R₄、R₅、R₆、R₇、n₁、n₂And n₃As defined in embodiment 1).

Embodiment 15 of the present invention): relates to a compound of the formula I, an enantiomer, a racemate or a salt thereof (in particular a pharmaceutically acceptable salt) according to embodiment 14), wherein

R₂Represents O, S or NH;

R₃、R₅each independently represents C_1-5An alkyl group;

R₄represents OH, SH or NH₂；

R₆Represents aryl or heteroaryl, wherein said aryl is optionally substituted with a substituent selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy, and the heteroaryl is optionally substituted with a substituent selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy;

R₇represents aryl or heteroaryl, wherein said aryl is optionally substituted with a substituent selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy, and the heteroaryl is optionally substituted with a substituent selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy; and

n₁、n₂、n₃each independently represents 0, 1,2,3, 4, 5, 6,7,8,9 or 10.

Embodiment 16 of the present invention): relates to a compound of the formula I, enantiomers, racemates or salts thereof (in particular pharmaceutically acceptable) as in embodiment 14) or 15)Salt) of (I), wherein R₆Represents an optionally substituted group selected from: phenyl, naphthyl, fluorenyl, furyl, oxazolyl, isoxazolyl, oxadiazolyl, thienyl, thiazolyl, isothiazolyl, thiadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, indolyl, isoindolyl, benzofuranyl, isobenzofuranyl, benzothienyl, indazolyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, benzo [2,1,3 ] benzo]Oxadiazolyl, benzo [2,1,3 ] s]Thiadiazolyl, benzo [1,2,3 ] s]Thiadiazolyl, quinolyl, isoquinolyl, naphthyridinyl, cinnolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, pyrazolo [1,5-a ]]Pyridyl, pyrazolo [1,5-a]Pyrimidinyl, imidazo [1,2-a ] -compounds]Pyridyl, 1H-pyrrolo [3,2-b ] s]Pyridyl, 1H-pyrrolo [2,3-b ] s]Pyridyl, 4H-fluoro [3,2-b ]]Pyrrolyl, pyrrolo [2,1-b ]]Thiazolyl or imidazo [2,1-b ]]Thiazolyl, wherein the substituents are selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy;

R₇represents an optionally substituted group selected from: phenyl, naphthyl, fluorenyl, furyl, oxazolyl, isoxazolyl, oxadiazolyl, thienyl, thiazolyl, isothiazolyl, thiadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, indolyl, isoindolyl, benzofuranyl, isobenzofuranyl, benzothienyl, indazolyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, benzo [2,1,3 ] benzo]Oxadiazolyl, benzo [2,1,3 ] s]Thiadiazolyl, benzo [1,2,3 ] s]Thiadiazolyl, quinolyl, isoquinolyl, naphthyridinyl, cinnolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, pyrazolo [1,5-a ]]Pyridyl, pyrazolo [1,5-a]Pyrimidinyl, imidazo [1,2-a ] -compounds]Pyridyl, 1H-pyrrolo [3,2-b ] s]Pyridyl, 1H-pyrrolo [2,3-b ] s]Pyridyl, 4H-fluoro [3,2-b ]]Pyrrolyl, pyrrolo [2,1-b ]]Thiazolyl or imidazo [2,1-b ]]Thiazolyl, wherein the substituents are selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, ammoniaA radical or a hydroxyl radical.

Embodiment 17 of the present invention): relates to a compound of the formula I, an enantiomer, a racemate or a salt thereof (in particular a pharmaceutically acceptable salt) according to embodiment 14) or 15), wherein R₆Represents an optionally substituted thienyl group wherein the substituents are selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy.

Embodiment 18 of the present invention): relates to a compound of the formula I, an enantiomer, a racemate or a salt thereof (in particular a pharmaceutically acceptable salt) according to embodiment 14) or 15), wherein R₇Represents an optionally substituted naphthyl group, wherein the substituents are selected from: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy.

Embodiment 19 of the present invention): relates to a compound of formula I as in embodiment 1), an enantiomer, a racemate or a salt (especially a pharmaceutically acceptable salt) thereof, wherein R₁Represented by the following formula G₂The group of (a):

wherein the content of the first and second substances,

n₄represents 0, 1,2,3, 4, 5, 6,7,8,9 or 10 (preferably 0, 1,2 or 3);

R₈、R₉、R₁₀、R₁₁each independently represents halogen or hydrogen;

R₁₂represents S, O or NH;

U₁represents NH or O; and

U₂、U₃、U₄、U₅the same or different, each independently represents CH or N.

Embodiment 20 of the present invention): relates to a compound of the formula I, an enantiomer, a racemate or a salt thereof (in particular a pharmaceutically acceptable salt) according to embodiment 1) or 19), wherein U₂And U₄Represents CH, and U₃And U₅Represents N.

Embodiment 21 of the present invention): relates to a compound of the formula I, an enantiomer, a racemate or a salt thereof (in particular a pharmaceutically acceptable salt) according to embodiment 1) or 19), wherein U₂、U₃And U₄Represents CH, and U₅Represents N.

Embodiment 22 of the present invention): relates to a compound of the formula I, an enantiomer, a racemate or a salt thereof (in particular a pharmaceutically acceptable salt) according to embodiment 1) or 19), wherein U₂、U₃、U₄And U₅Represents N.

Embodiment 23 of the present invention): relates to a compound of the formula I, an enantiomer, a racemate or a salt thereof (in particular a pharmaceutically acceptable salt) according to embodiment 1) or 19), wherein U₂Represents CH, and U₃、U₄And U₅Represents N.

Embodiment 24 of the present invention): relates to a compound of the formula I, an enantiomer, a racemate or a salt thereof (in particular a pharmaceutically acceptable salt) according to embodiment 1) or 19), wherein U₂、U₄And U₅Represents CH, and U₄Represents N.

Embodiment 25 of the present invention): relates to a compound of the formula I, an enantiomer, a racemate or a salt thereof (in particular a pharmaceutically acceptable salt) as in embodiment 1) or 2), which is also a compound of the formula I-2

Wherein R is₂、R₃、R₄、R₈、R₉、R₁₀、R₁₁、R₁₂、U₁、U₂、U₃、U₄、U₅、n₁、n₂And n₄As defined in embodiment 1.

Embodiment 26 of the present invention): relates to a compound of the formula I, an enantiomer, a racemate or a salt thereof (in particular a pharmaceutically acceptable salt) as in embodiment 25), wherein R₃Is represented by C_1-10Alkyl, phenyl, naphthyl or fluorenyl, wherein said C is_1-10Alkyl is optionally substituted with a substituent selected from: hydroxy, C_1-3Alkyl radical, C_1-3Alkoxy, amino, halogen or carboxyl, and the phenyl, naphthyl or fluorenyl group is optionally substituted with a substituent selected from the group consisting of: c_1-3Alkyl radical, C_1-3Alkoxy, halogen, amino or hydroxy.

Embodiment 27 of the present invention): relates to a compound of the formula I, an enantiomer, a racemate or a salt thereof (in particular a pharmaceutically acceptable salt) according to embodiment 25), wherein U₂And U₄Represents CH, and U₃And U₅Represents N.

Embodiment 28 of the present invention): relates to a compound of the formula I, an enantiomer, a racemate or a salt thereof (in particular a pharmaceutically acceptable salt) according to embodiment 25), wherein U₂、U₃And U₄Represents CH, and U₅Represents N.

Embodiment 29 of the present invention): relates to a compound of the formula I, an enantiomer, a racemate or a salt thereof (in particular a pharmaceutically acceptable salt) according to embodiment 25), wherein U₂、U₃、U₄And U₅Represents N.

Embodiment 30 of the present invention): relates to a compound of the formula I, an enantiomer, a racemate or a salt thereof (in particular a pharmaceutically acceptable salt) according to embodiment 25), wherein U₂Represents CH, and U₃、U₄And U₅Represents N.

Embodiment 31 of the present invention): relates to a compound of the formula I, an enantiomer, a racemate or a salt thereof (in particular a pharmaceutically acceptable salt) according to embodiment 25), wherein U₂、U₄And U₅Represents CH, and U₄Represents N.

Particularly preferred are the following compounds of formula I, enantiomers, racemates or salts thereof (especially pharmaceutically acceptable salts) as defined in the present invention:

9-hydroxy-2-methyl-3- (2- (methyl ((S) -3- (naphthalen-1-yloxy) -3- (thiophen-3-yl) propyl) amino) ethyl) -6,7,8, 9-tetrahydropyrido [1,2-a ] pyrimidin-4-one (compound No. HW031 thereof); and

9-hydroxy-2-methyl-3- (2- (methyl ((S) -3- (naphthalen-1-yloxy) -3- (thiophen-2-yl) propyl) amino) ethyl) -6,7,8, 9-tetrahydropyrido [1,2-a ] pyrimidin-4-one (compound No. HW 086).

Particularly preferred are the following compounds of formula I, enantiomers, racemates or salts thereof (especially pharmaceutically acceptable salts) as defined in the present invention:

3- (2- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperidin-1-yl) benzoyl) piperidin-1-yl) ethyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound number HW036 thereof);

3- (2- (4- (4-fluoro-2- (4- (6-fluoro-1H-indazol-3-yl) piperidin-1-yl) benzoyl) piperidin-1-yl) ethyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound number HW03601 thereof);

3- (2- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperazin-1-yl) benzoyl) piperidin-1-yl) ethyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound number HW 03602);

3- (2- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperazin-1-yl) benzoyl) piperazin-1-yl) ethyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound number HW03603 thereof);

3- (2- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperidin-1-yl) benzoyl) piperazin-1-yl) ethyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound number HW03604 thereof);

3- (3- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperidin-1-yl) benzoyl) piperazin-1-yl) propyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound number HW03605 thereof);

3- (3- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperazin-1-yl) benzoyl) piperazin-1-yl) propyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound number HW03606 thereof);

3- (3- (4- (4-fluoro-2- (4- (6-fluoro-1H-indazol-3-yl) piperazin-1-yl) benzoyl) piperazin-1-yl) propyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound number HW 03607);

3- (3- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperazin-1-yl) benzoyl) piperidin-1-yl) propyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound number HW03608 thereof);

3- (3- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperazin-1-yl) benzoyl) piperidin-1-yl) propyl) -9-hydroxy-2-phenyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound number HW 03609);

3- (3- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperazin-1-yl) benzoyl) piperazin-1-yl) propyl) -9-hydroxy-2-phenyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound number HW03610 thereof);

3- (3- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperidin-1-yl) benzoyl) piperazin-1-yl) propyl) -9-hydroxy-2-phenyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound number HW03611 thereof).

It will be appreciated that the compounds of formula (I) of the present invention may have a stereoconfiguration and thus may exist in more than one stereoisomeric form. The invention also relates to compounds having a stereoconfiguration in the form of substantially pure isomers, such as greater than 90% ee, e.g., about 95% ee or 97% ee, or greater than 99% ee, and mixtures, including racemic mixtures thereof. These isomers can be prepared using asymmetric synthesis (e.g., with chiral intermediates) or by chiral resolution.

In another aspect of the present invention, there is also provided a pharmaceutical composition comprising the compound of formula I, an enantiomer, a racemate or a pharmaceutically acceptable salt thereof as an active ingredient, and at least one pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent.

In one embodiment, the at least one additional therapeutic agent is for treating or preventing a tumor.

The compound of formula I, enantiomer, racemate or salt thereof (especially pharmaceutically acceptable salt) is suitable for being used as a tumor therapeutic agent. In another embodiment, the tumor is a malignant tumor.

Therefore, another object of the present invention relates to the use of the compounds of formula I, their enantiomers, racemates or salts thereof according to the present invention for the preparation of a medicament for the treatment of tumors. In another embodiment, the tumor is a malignant tumor.

The compound of formula I, enantiomer, racemate or salt thereof (especially pharmaceutically acceptable salt) is suitable for being used as a malignant tumor therapeutic agent.

The compound of formula I, enantiomer, racemate or salt thereof (especially pharmaceutically acceptable salt) is suitable for being used as a radiosensitizer of tumors. In another embodiment, the tumor is a malignant tumor.

The compound of formula I, enantiomer, racemate or salt thereof (especially pharmaceutically acceptable salt) is suitable for being used as a radioprotectant for tumors. In another embodiment, the tumor is a malignant tumor.

The compound of formula I, enantiomer, racemate or salt thereof (especially pharmaceutically acceptable salt) can be used as a radiosensitizer in the radiotherapy of tumors. In another embodiment, the tumor is a malignant tumor.

In one embodiment, the invention relates to the use of a compound of formula I according to the invention, its enantiomers, racemates or salts thereof for the preparation of radiosensitizers and/or radioprotectors for tumors.

In the present invention, the tumor is a malignant tumor.

In the present invention, the malignant tumor includes but is not limited to: breast cancer, breast cancer lung metastasis, nasopharyngeal carcinoma, cervical cancer, lung cancer, pancreatic cancer, lymphoma, liver cancer, and melanoma.

In the present invention, the malignant tumor is a lung metastasis of breast cancer.

The invention also relates to pharmaceutical compositions comprising as active ingredient at least one compound of formula I, an enantiomer, a racemate or a salt thereof, in particular a pharmaceutically acceptable salt thereof, and optionally pharmaceutical excipients such as carriers and/or diluents.

The compounds of formula I, enantiomers, racemates or pharmaceutically acceptable salts thereof according to the present invention can be used as medicaments in the form of pharmaceutical compositions for enteral or parenteral administration.

The compound of formula I, its enantiomer, racemate or a pharmaceutically acceptable salt thereof according to the present invention may be formulated into suitable formulations such as spray formulations, patches, tablets, capsules, dragees, troches, powders, granules, powder injections, or liquid formulations (e.g., suspensions, solutions, emulsions or syrups) according to a suitable route of administration, including, but not limited to, nasal administration, inhalation administration, topical administration, oral administration, oromucosal administration, rectal administration, pleural administration, peritoneal administration, vaginal administration, intramuscular administration, subcutaneous administration, transdermal administration, epidural administration, intrathecal administration, and intravenous administration.

In all embodiments of the invention, the salts or pharmaceutically acceptable salts of the compounds of formula I refer to non-toxic inorganic or organic acid and/or base addition salts. Examples include: sulfate, hydrochloride, citrate, maleate, sulfonate, citrate, lactate, tartrate, fumarate, phosphate, dihydrogen phosphate, pyrophosphate, metaphosphate, oxalate, malonate, benzoate, mandelate, succinate, glycolate, p-toluenesulfonate, etc.

Herein, the bond broken by the wavy line shows the point at which the depicted group is attached to the rest of the molecule. For example, R is illustrated below₁A group represented by:

said formula G₁The compound of (1) is partially represented byThe other moiety of the compound of formula I is attached through the N atom.

The term "pharmaceutically acceptable carrier" of the present invention refers to a pharmaceutically acceptable material, such as a filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent or encapsulating material, which carries or transports a compound useful in the present invention into a patient or is administered to a patient so that it can perform its intended function. Typically, such constructs are carried or transported from one organ or part of the body to another organ or part of the body. The carrier is compatible with the other ingredients of the formulation (including the compounds useful in the present invention) and not deleterious to the patient, and must be "acceptable". Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered gum tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; a surfactant phosphate buffer solution; and other non-toxic compatible materials used in pharmaceutical formulations.

The term "room temperature" according to the present invention refers to the temperature of the surrounding environment, e.g. a temperature of 20-30 ℃.

The term "treatment" or "treating" refers to administering a compound of formula I or a pharmaceutically acceptable salt thereof, as described herein, or a pharmaceutical composition comprising as an active ingredient a compound of formula I or a pharmaceutically acceptable salt thereof, to a subject to slow down (lessen) the development of an undesired disease or condition, such as a mycobacterial infection. Beneficial or desired clinical results of the present invention include, but are not limited to: alleviating symptoms, reducing the severity of the disease, stabilizing the state of the disease, delaying or slowing the progression of the disease, ameliorating or palliating the condition, and alleviating the disease.

The "therapeutically effective amount" of a compound of the invention depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of the cancer in the patient being treated. One skilled in the art will be able to determine the appropriate dosage based on these and other factors.

Examples

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The present invention may be practiced without some or all of these specific details. In other instances, well known process operations have not been described in detail in order not to unnecessarily obscure the present invention. While the invention will be described in conjunction with the specific embodiments, it will be understood that they are not intended to limit the invention to these embodiments.

The compounds of formula I can be prepared according to the following methods.

Abbreviations:

ADM Adriamycin

BnBr benzyl bromide

(Boc)₂Di-tert-butyl O dicarbonate

Boc tert-butyloxycarbonyl group

CbzCl benzyl chloroformate

DIPEA N, N-diisopropylethylamine

DMF N, N-dimethylformamide

FITC fluorescein isothiocyanate

5-FU 5-Fluorouracil

THF tetrahydrofuran

TFA trifluoroacetic acid

PI propidium iodide

PG protecting group

PBS phosphate buffer

MTT tetramethyl azozole salt

MTX methotrexate

EA Ethyl acetate

MeOH methanol

MRT mean residence time

DMSO dimethyl sulfoxide

TLC thin layer chromatography

DCM dichloromethane

The process for the preparation of the compounds of formula I according to the invention is described in more detail below, but these particular processes do not limit the invention in any way. The compounds of the present invention may also be conveniently prepared by optionally combining various synthetic methods described in the present specification or known in the art, and such combinations may be readily carried out by those skilled in the art to which the present invention pertains.

The reaction in the production method of the present invention is usually carried out at 0 ℃ to a state where the reaction system is under reflux, preferably at room temperature (e.g., 20 to 30 ℃) to reflux.

All temperatures are described in ℃. The compounds were characterized by 1H-NMR (400MHz) (Bruker Advance 400). Chemical shifts δ are listed in ppm relative to the solvent used; multiplicity is as follows: s is singlet, d is doublet, t is triplet, q is quartet, p is quintet, hex is hexamet, hep is heptat, m is multiplet, br. is broad, coupling constants are given in Hz. Alternatively, compounds were characterized by LC-MS (Sciex API 2000 with Agilent 1100 binary pump and DAD and ELSD or Agilent quadrupole MS 6140 with Agilent 1200 binary pump, DAD and ELSD).

Solvent and reagent treatments were as follows:

the solvents used in the reaction, such as THF, DCM, DMF, EtOH, MeOH, acetonitrile, etc., were purchased from the national pharmaceutical group; HPLC preparation used is preparative CH₃CN and deionized water; substrate A, substrate B, compound HW036-1, compound HW036-3, compound HW036-6, compound HW03601-3, compound HW03602-3, compound HW03603-1, compound HW03605-6, compound HW03606-3, compound HW03607-3, compound HW03609-6, compound HW031-A, compound HW031G, and the compound of formula VII, and the like can be prepared from manufacturersDirectly purchasing; other reagents and drugs were purchased from the manufacturer and used without specific reference.

General synthetic method

The present invention prepares the compound of formula I-1 according to scheme 1. The scheme 1 comprises the following steps:

scheme 1

R in scheme 1₂、R₃、R₄、R₅、R₆、R₇、n₁、n₂、n₃Each as defined above.

X in scheme 1 represents a leaving group such as halogen (e.g., fluorine, chlorine, bromine or iodine) Or Methylsulfonyl (OMs).

In scheme 1, in an inert solvent (e.g., acetone, tetrahydrofuran, N' N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile or ethanol, etc.) in an inorganic base (e.g., K)₂CO₃、Na₂CO₃DIPEA or Cs₂CO₃Etc.) or in the presence of an organic base, reacting substrate a and substrate B for a time period (e.g., 10 hours to 30 hours), thereby forming a compound of formula I-1.

The present invention prepares the compound of formula I-2 according to scheme 2. The scheme 2 comprises the following steps:

scheme 2

R in scheme 2₂、R₃、R₄、R₈、R₉、R₁₀、R₁₁、R₁₂、U₁、U₂、U₃、U₄、U₅、n₁、n₂、n₄Respectively as defined above.

X in scheme 2 represents a halogen, such as fluorine, chlorine, bromine or iodine.

PG in scheme 2 is a protecting group. The person skilled in the art will be able to select the appropriate protecting groups conventionally used, depending on the desired protecting group.

The compound of formula III is prepared in step 1 of scheme 2. In particular, in an inert solvent (such as acetone, tetrahydrofuran, N' -N-Dimethylformamide (DMF) or ethanol) in a base (such as K)₂CO₃、Na₂CO₃DIPEA or Cs₂CO₃) In the presence of a compound of formula II and a suitable protecting group (e.g. CbzCl, BnBr, (Boc)₂O, etc.) at 0 ℃ to room temperature for a period of time (e.g., 10 hours to 30 hours), thereby forming the compound of formula III.

The compound of formula V is prepared in step 2 of scheme 2. Specifically, the compound of formula III and the compound of formula IV are reacted in an inert solvent (such as methanol, isopropanol, DMF, THF or ethanol) in the presence of a base (such as sodium ethoxide, sodium methoxide, sodium hydride or sodium tert-butoxide, potassium tert-butoxide) or a suitable catalyst at 40 ℃ to 80 ℃ for a period of time (such as 10 hours to 30 hours) to form the compound of formula V.

The compound of formula VI is prepared in step 3 of scheme 2. Specifically, the compound of formula V is deprotected in an inert solvent (e.g., methanol, isopropanol, dichloromethane, acetonitrile, THF, or ethanol) in the presence of a base (e.g., potassium hydroxide, sodium hydroxide or sodium tert-butoxide, potassium tert-butoxide) or an acid (sulfuric acid, hydrochloric acid, benzenesulfonic acid, etc.) at room temperature for a period of time (e.g., 1 hour to 5 hours) to form the compound of formula VI.

The compound of formula I-2 is prepared in step 4 of scheme 2. Specifically, the compound of formula VI and the compound of formula VII are heated under reflux in an inert solvent (such as methanol, isopropanol, DMF, THF, acetonitrile or ethanol) in the presence of a base (such as sodium ethoxide, sodium methoxide, sodium hydride or sodium tert-butoxide, potassium tert-butoxide) or a suitable catalyst for a period of time (such as 10 hours to 30 hours) to form the compound of formula I-2.

Example 1: preparation of 3- (2- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperidin-1-yl) benzoyl) piperidin-1-yl) ethyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound HW036)

The synthetic route is as follows:

step 1-1: compound HW036-2 was prepared according to step 1 of scheme 2.

Specifically, at room temperature, to a 500 ml three-necked flask were added in the order: THF/water (100mL/100mL), HW036-1(20g,76mmol) and Na₂CO₃(10.5g,99 mmol). Then, the temperature is reduced to 0 ℃ by adopting an ice water bath external bath. Under stirring, (Boc) was added₂O (19.9g,91 mmol). The reaction was allowed to spontaneously warm to room temperature and stirred overnight. The organic solvent was concentrated under reduced pressure, the aqueous phase was extracted with ethyl acetate (3X 100mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to give HW036-2(23.2g, 94% yield) as a white solid which was used directly in the next step.

Step 1-2: compound HW036-4 was prepared according to step 2 of scheme 2.

Specifically, at room temperature, to a 500 ml three-necked flask were added in the order: 1, 4-dioxane (300mL), HW036-2(20g,61mmol), HW036-3(17.8g,73mmol) and K₂CO₃(10.9g,79 mmol). Then, the reaction system was stirred at 90 ℃ overnight. The reaction was brought to room temperature and then poured into 500 mL of water, stirred well, extracted with ethyl acetate (3X 300mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid which was washed with ethyl acetate slurry to give HW036-4(26.8g, 83% yield) as a white solid.¹H NMR(400MHz,CDCl₃)δ7.64(m,1H),7.29(m,2H),7.10(t,1H),6.80(m,2H),4.09(m,2H),3.63(m,1H),3.40(d,2H),3.20(m,1H),2.97(t,2H),2.82(m,2H),2.24(m,2H),2.15(m,2H),1.78(m,2H),1.61(m,2H),1.46(s,9H)；MS(ES⁺):m/z＝526.24(M+H)⁺。

Step 1-3: compound HW036-5 was prepared according to step 3 of scheme 2.

Specifically, at room temperature, to a 250ml three-necked flask were added in the order: ethanol (100mL), HW036-4(24g,46mmol) and 6N HCl or trifluoroacetic acid (50 mL). The reaction was then stirred at room temperature overnight. Distillation under reduced pressure gave HW036-5(21g, 100% yield) as a white solid. Used directly in the next step.

Step 1-4: compound HW036 was prepared according to step 3 of scheme 2.

Specifically, at room temperature, to a 100ml three-necked flask were added in the order: methanol (50mL), HW036-5(5g,10.8mmol), HW036-6(2.62g,10.8mmol) and N, N-diisopropylethylamine (4.2g,32.4 mmol). Then, the reaction system was heated to reflux and stirred overnight. The solvent was evaporated to dryness under reduced pressure, water (100mL) was added, extraction was performed with ethyl acetate (3 × 100mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid, and recrystallized from methanol to give HW036(2.5g, 36% yield) as a white solid.

¹H NMR(400MHz,CDCl₃)δ7.68(m,1H),7.29(m,2H),7.12(t,1H),6.80(m,2H),4.50(m,1H),4.31(brs,1H),3.92(m,2H),3.50(m,1H),3.37(d,2H),3.21(m,1H),3.08(d,2H),2.95(t,2H),2.74(m,2H),2.48(m,2H),2.35(m,4H),2.29-2.10(m,7H),1.95(m,1H),1.88-1.70(m,5H).C₃₅H₄₀F₂N₅LC-MS of O ([ M + H ]]⁺): calculated values: 632.3, found: 632.0.

example 2: preparation of 3- (2- (4- (4-fluoro-2- (4- (6-fluoro-1H-indazol-3-yl) piperidin-1-yl) benzoyl) piperidin-1-yl) ethyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound HW03601)

The synthetic route is as follows:

preparation of HW 036-2: the procedure was carried out according to step 1-1 in example 1.

Preparation of HW 03601-4: referring to step 1-2 of example 1, HW03601-4 was prepared using HW03601-3 and HW 036-2:

at room temperature, the mixture is put into a 500 ml three-mouth bottleAdding for the second time: 1, 4-dioxane (300mL), HW036-2(20g,61mmol), HW03601-3(18.6g,73mmol) and K₂CO₃(10.9g,79 mmol). Then, the reaction system was stirred at 90 ℃ overnight. The reaction was brought to room temperature and then poured into 500 mL of water, stirred well, extracted with ethyl acetate (3X 300mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid which was washed with ethyl acetate slurry to give HW03601-4(25.9g, 81% yield) as a white solid.

¹H NMR(400MHz,CDCl₃)δ12.4(s,1H),7.90(m,1H),7.79(m,1H),7.31(m,1H),6.91(m,2H),6.71(m,1H),3.51-3.62(m,4H),3.06-3.18(m,5H),2.76-2.81(m,1H),1.76-1.82(m,4H),1.58-1.72(m,4H),1.42(s,9H)；MS(ES⁺):m/z＝525.26(M+H)⁺。

Preparation of HW 03601-5: steps 1 to 3 of reference example 1

To a 250ml three-necked flask, at room temperature, were added in the following order: ethanol (100mL), HW03601-4(24.12g,46mmol) and 6N HCl or trifluoroacetic acid (50 mL). The reaction was then stirred at room temperature overnight. Distillation under reduced pressure gave HW03601-5 as a white solid (about 21g, 100% yield). Used directly in the next step.

Preparation of the target compound HW 03601: steps 1 to 4 of reference example 1

To a 100ml three-necked flask, at room temperature, were added in the following order: methanol (50mL), HW03601-5(5g,10.8mmol), HW036-6(2.62g,10.8mmol) and N, N-diisopropylethylamine (4.2g,32.4 mmol). Then, the reaction system was heated to reflux and stirred overnight. The solvent was evaporated to dryness under reduced pressure, water (100mL) was added, extraction was performed with ethyl acetate (3X 100mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid, and recrystallized from methanol to give the title compound HW03601(2.5g, 36% yield) as a white solid.

¹H NMR(400MHz,CDCl₃)δ12.5(s,1H),7.89-7.91(m,1H),7.76-7.72(m,1H),7.29-7.32(m,1H),6.89-6.92(m,2H),6.69-6.72(m,1H),5.50(br,1H),3.89-3.92(m,2H),3.39-3.42(m,3H),3.22(s,3H),3.10-3.15(m,5H),2.75-2.79(m,1H),2.49-2.52(m,4H),1.70-2.16(m,6H),1.43-1.68(m,8H).C₃₅H₄₀F₂N₆O₃LC-MS ([ M + H)]⁺): calculated values: 631.31, found: 631.31.

example 3: preparation of 3- (2- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperazin-1-yl) benzoyl) piperidin-1-yl) ethyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound HW03602)

The synthetic route is as follows:

preparation of HW 036-2: the procedure was carried out according to step 1-1 in example 1.

Preparation of HW 03602-4: referring to step 1-2 of example 1, HW03602-4 was prepared using HW03602-3 and HW 036-2:

to a 500 ml three-necked flask, at room temperature, were added in the following order: 1, 4-dioxane (300mL), HW036-2(20g,61mmol), HW03602-3(18.8g,73mmol) and K₂CO₃(10.9g,79 mmol). Then, the reaction system was stirred at 90 ℃ overnight. The reaction was brought to room temperature and then poured into 500 mL of water, stirred well, extracted with ethyl acetate (3X 300mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid which was washed with ethyl acetate slurry to give HW03602-4(25.3g, 79% yield) as a white solid.

¹H NMR(400MHz,CDCl₃)δ7.51-7.53(m,1H),7.39-7.41(m,1H),7.11-7.13(m,1H),6.89-6.96(m,2H),6.68-6.72(m,1H),3.51-3.59(m,8H),3.26-3.29(m,4H),3.03-3.06(m,1H),2.91-2.96(m,2H),2.62-2.70(m,2H),1.43(s,9H)；MS(ES⁺):m/z＝527.24(M+H)⁺。

Preparation of HW 03602-5: steps 1 to 3 of reference example 1

To a 250ml three-necked flask, at room temperature, were added in the following order: ethanol (100mL), HW03602-4(24.2g,46mmol) and 6N HCl or trifluoroacetic acid (50 mL). The reaction was then stirred at room temperature overnight. Distillation under reduced pressure gave HW03602-5 as a white solid (about 20g, 100% yield). Used directly in the next step.

Preparation of target compound HW 03602: steps 1 to 4 of reference example 1

To a 100ml three-necked flask, at room temperature, were added in the following order: methanol (50mL), HW03602-5(4.6g,10.8mmol), HW036-6(2.62g,10.8mmol) and N, N-diisopropylethylamine (4.2g,32.4 mmol). Then, the reaction system was heated to reflux and stirred overnight. The solvent was evaporated to dryness under reduced pressure, water (100mL) was added, extraction was performed with ethyl acetate (3 × 100mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid, and recrystallized from methanol to give HW03602(2.87g, 42% yield) as a white solid.

¹H NMR(400MHz,CDCl₃)δ7.76-7.79(m,1H),7.41-7.45(m,1H),7.12-7.16(m,1H),6.91-6.97(m,2H),6.71-6.73(m,1H),5.51(brs,1H),3.92-3.95(m,2H),3.41-3.57(m,7H),3.26-3.29(m,4H),3.22(s,3H),3.28-3.02(m,1H),2.48-2.52(m,4H),2.02-2.11(m,2H),1.71-1.89(m,6H),1.51-1.59(m,2H).C₃₄H₃₈F₂N₆O₄LC-MS ([ M + H)]⁺): calculated values: 633.29, found: 633.29.

example 4: preparation of 3- (2- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperazin-1-yl) benzoyl) piperazin-1-yl) ethyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound HW03603)

The synthetic route is as follows:

preparation of HW 03603-2: step 1-1 of reference example 1

To a 500 ml three-necked flask, at room temperature, were added in the following order: THF/water (100mL/100mL), HW03603-1(19.9g,76mmol) and Na₂CO₃(10.5g,99 mmol). Then, the temperature of the external bath is reduced to 0 ℃ by using ice water. Under stirring, (Boc) was added₂O (19.9g,91 mmol). Reaction system is naturalThe temperature was raised to room temperature and stirred overnight. The organic solvent was concentrated under reduced pressure, the aqueous phase was extracted with ethyl acetate (3X 100mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to give HW03603-2(22.6g, 91% yield) as a white solid which was used directly in the next step.

Preparation of HW 03603-4: steps 1-2 of reference example 1

To a 500 ml three-necked flask, at room temperature, were added in the following order: 1, 4-dioxane (300mL), HW03603-2(19.9g,61mmol), HW03602-3(18.8g,73mmol) and K₂CO₃(10.9g,79 mmol). Then, the reaction system was stirred at 90 ℃ overnight. The reaction was brought to room temperature and then poured into 500 mL of water, stirred well, extracted with ethyl acetate (3X 300mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid which was washed with ethyl acetate slurry to give HW03603-4(26.4g, 82% yield) as a white solid.

¹H NMR(400MHz,CDCl₃)δ7.78-7.80(m,1H),7.39-7.41(m,1H),7.11-7.12(m,1H),6.97-7.01(m,2H),6.80-6.81(m,1H),3.56-3.58(m,8H),3.28-3.34(m,8H),1.42(s,9H)；MS(ES⁺):m/z＝528.23(M+H)⁺。

Preparation of HW 03603-5: steps 1 to 3 of reference example 1

To a 250ml three-necked flask, at room temperature, were added in the following order: ethanol (100mL), HW03603-4(24.2g,46mmol) and 6N HCl or trifluoroacetic acid (50 mL). The reaction was then stirred at room temperature overnight. Distillation under reduced pressure gave HW03603-5 as a white solid (about 20g, 100% yield). Used directly in the next step.

Preparation of the target compound HW 03603: steps 1 to 4 of reference example 1

To a 100ml three-necked flask, at room temperature, were added in the following order: methanol (50mL), HW03603-5(4.6g,10.8mmol), HW036-6(2.62g,10.8mmol) and N, N-diisopropylethylamine (4.2g,32.4 mmol). Then, the reaction system was heated to reflux and stirred overnight. The solvent was evaporated to dryness under reduced pressure, water (100mL) was added, extraction was performed with ethyl acetate (3 × 100mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid, and recrystallized from methanol to give HW03603(3.35g, 49% yield) as a white solid.

¹H NMR(400MHz,CDCl3)δ7.78-7.80(m,1H),7.40-7.41(m,1H),7.12-7.13(m,1H),6.97-7.02(m,2H),6.80-6.81(m,1H),5.50(brs,1H),3.92-3.95(m,2H),3.503.57(m,5H),3.41-3.43(m,6H),3.28-3.30(m,4H),3.22(s,3H),2.52-2.55(m,4H),1.91-2.06(m,4H),1.82-1.84(m,1H),1.59-1.60(m,1H).C₃₃H₃₇F₂N₇O₄LC-MS ([ M + H)]⁺): measured value: 634.29, calculated as: 634.29.

example 5: preparation of 3- (2- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperidin-1-yl) benzoyl) piperazin-1-yl) ethyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound HW03604)

The synthetic route is as follows:

preparation of HW 03603-2: step 1-1 of reference example 1

To a 500 ml three-necked flask, at room temperature, were added in the following order: THF/water (100mL/100mL), HW03603-1(19.9g,76mmol) and Na₂CO₃(10.5g,99 mmol). Then, the temperature of the external bath is reduced to 0 ℃ by using ice water. Under stirring, (Boc) was added₂O (19.9g,91 mmol). The reaction was allowed to spontaneously warm to room temperature and stirred overnight. The organic solvent was concentrated under reduced pressure, the aqueous phase was extracted with ethyl acetate (3X 100mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to give HW03603-2(22.6g, 91% yield) as a white solid which was used directly in the next step.

Preparation of HW 03604-4: steps 1-2 of reference example 1

To a 500 ml three-necked flask, at room temperature, were added in the following order: 1, 4-dioxane (300mL), HW03603-2(19.9g,61mmol), HW036-3(18.7g,73mmol) and K₂CO₃(10.9g,79 mmol). Then, the reaction system is stirred at 90 DEG CStirring overnight. The reaction was brought to room temperature and then poured into 500 mL of water, stirred well, extracted with ethyl acetate (3X 300mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid which was washed with ethyl acetate slurry to give HW03604-4(24.4g, 76% yield) as a white solid.

¹H NMR(400MHz,CDCl₃)δ7.79-7.80(m,1H),7.41-7.42(m,1H),7.11-7.12(m,1H),6.97-7.01(m,2H),6.81-6.82(m,1H),3.59-3.41(m,4H),3.34-3.36(m,4H),3.03-3.11(m,4H),2.78-2.82(m,1H),1.72-1.74(m,2H),1.48-1.52(m,2H),1.42(s,9H)；MS(ES⁺):m/z＝527.24(M+H)⁺。

Preparation of HW 03604-5: steps 1 to 3 of reference example 1

To a 250ml three-necked flask, at room temperature, were added in the following order: ethanol (100mL), HW03604-4(24.2g,46mmol) and 6N HCl or trifluoroacetic acid (50 mL). The reaction was then stirred at room temperature overnight. Distillation under reduced pressure gave HW03604-5 as a white solid (about 20g, 100% yield). Used directly in the next step.

Preparation of the target compound HW 03604: steps 1 to 4 of reference example 1

To a 100ml three-necked flask, at room temperature, were added in the following order: methanol (50mL), HW03604-5(4.6g,10.8mmol), HW036-6(2.62g,10.8mmol) and N, N-diisopropylethylamine (4.2g,32.4 mmol). Then, the reaction system was heated to reflux and stirred overnight. The solvent was evaporated to dryness under reduced pressure, water (100mL) was added, extraction was performed with ethyl acetate (3 × 100mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid, and recrystallized from methanol to give HW03604 as a white solid (2.8g, 41% yield).

¹H NMR(400MHz,CDCl3)δ7.79-7.81(m,1H),7.39-7.41(m,1H),7.12-7.13(m,1H),6.97-7.01(m,2H),6.81-6.83(m,1H),5.50(brs,1H),3.92-3.94(m,2H),3.41-3.50(m,7H),3.22(s,3H),3.05-3.13(m,4H),2.76-2.79(m,1H),2.52-2.56(m,4H),2.01-2.06(m,3H),1.84-1.91(m,2H),1.59-1.74(m,3H),1.46-1.50(m,2H).C₃₄H₃₈F₂N₆O₄LC-MS ([ M + H)]⁺): measured in factThe value: 633.29, calculated as: 633.29.

example 6: preparation of 3- (3- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperidin-1-yl) benzoyl) piperazin-1-yl) propyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound HW03605)

The synthetic route is as follows:

preparation of HW 03603-2: step 1-1 of reference example 1

To a 500 ml three-necked flask, at room temperature, were added in the following order: THF/water (100mL/100mL), HW03603-1(19.9g,76mmol) and Na₂CO₃(10.5g,99 mmol). Then, the temperature of the external bath is reduced to 0 ℃ by using ice water. Under stirring, (Boc) was added₂O (19.9g,91 mmol). The reaction was allowed to spontaneously warm to room temperature and stirred overnight. The organic solvent was concentrated under reduced pressure, the aqueous phase was extracted with ethyl acetate (3X 100mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to give HW03603-2(22.6g, 91% yield) as a white solid which was used directly in the next step.

Preparation of HW 03604-4: steps 1-2 of reference example 1

To a 500 ml three-necked flask, at room temperature, were added in the following order: 1, 4-dioxane (300mL), HW03603-2(19.9g,61mmol), HW036-3(18.7g,73mmol) and K₂CO₃(10.9g,79 mmol). Then, the reaction system was stirred at 90 ℃ overnight. The reaction was brought to room temperature and then poured into 500 mL of water, stirred well, extracted with ethyl acetate (3X 300mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid which was washed with ethyl acetate slurry to give HW03604-4(24.4g, 76% yield) as a white solid.

¹H NMR(400MHz,CDCl₃)δ7.79-7.80(m,1H),7.41-7.42(m,1H),7.11-7.12(m,1H),6.97-7.01(m,2H),6.81-6.82(m,1H),3.59-3.41(m,4H),3.34-3.36(m,4H),3.03-3.11(m,4H),2.78-2.82(m,1H),1.72-1.74(m,2H),1.48-1.52(m,2H),1.42(s,9H)；MS(ES⁺):m/z＝527.24(M+H)⁺。

Preparation of HW 03604-5: steps 1 to 3 of reference example 1

To a 250ml three-necked flask, at room temperature, were added in the following order: ethanol (100mL), HW03604-4(24.2g,46mmol) and 6N HCl or trifluoroacetic acid (50 mL). The reaction was then stirred at room temperature overnight. Distillation under reduced pressure gave HW03604-5 as a white solid (about 20g, 100% yield). Used directly in the next step.

Preparation of the target compound HW 03605: steps 1 to 4 of reference example 1

To a 100ml three-necked flask, at room temperature, were added in the following order: methanol (50mL), HW03604-5(4.6g,10.8mmol), HW03605-6(2.76g,10.8mmol) and N, N-diisopropylethylamine (4.2g,32.4 mmol). Then, the reaction system was heated to reflux and stirred overnight. The solvent was evaporated to dryness under reduced pressure, water (100mL) was added, extraction was performed with ethyl acetate (3 × 100mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid, and recrystallized from methanol to give HW03605(2.58g, 37% yield) as a white solid.

¹H NMR(400MHz,CDCl3)δ7.79-7.80(m,1H),7.41-7.42(m,1H),7.12-7.13(m,1H),6.97-7.01(m,2H),6.81-6.82(m,1H),5.50(brs,1H),3.90-3.91(m,2H),3.43-3.50(m,5H),3.22(s,3H),3.04-3.13(m,4H),2.76-2.78(m,1H),2.41-2.52(m,8H),1.92-2.01(m,2H),1.74-1.84(m,3H),1.49-1.59(m,3H).C₃₅H₄₀F₂N₆O₄LC-MS ([ M + H)]⁺): measured value: 647.31, calculated as: 647.31.

example 7: preparation of 3- (3- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperazin-1-yl) benzoyl) piperazin-1-yl) propyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound HW03606)

The synthetic route is as follows:

preparation of HW 03603-2: step 1-1 of reference example 1

To a 500 ml three-necked flask, at room temperature, were added in the following order: THF/water (100mL/100mL), HW03603-1(19.9g,76mmol) and Na₂CO₃(10.5g,99 mmol). Then, the temperature of the external bath is reduced to 0 ℃ by using ice water. Under stirring, (Boc) was added₂O (19.9g,91 mmol). The reaction was allowed to spontaneously warm to room temperature and stirred overnight. The organic solvent was concentrated under reduced pressure, the aqueous phase was extracted with ethyl acetate (3X 100mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to give HW03603-2(22.6g, 91% yield) as a white solid which was used directly in the next step.

Preparation of HW 03606-4: steps 1-2 of reference example 1

To a 500 ml three-necked flask, at room temperature, were added in the following order: 1, 4-dioxane (300mL), HW03603-2(19.9g,61mmol), HW03606-3(18.8g,73mmol) and K₂CO₃(10.9g,79 mmol). Then, the reaction system was stirred at 90 ℃ overnight. The reaction was brought to room temperature and then poured into 500 mL of water, stirred well, extracted with ethyl acetate (3X 300mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid, which was washed with ethyl acetate slurry to give HW03606-4(27.7g, 86% yield) as a white solid.

¹H NMR(400MHz,CDCl₃)δ7.79-7.80(m,1H),7.41-7.42(m,1H),7.11-7.12(m,1H),6.97-7.01(m,2H),6.81-6.82(m,1H),3.59-3.41(m,4H),3.34-3.36(m,4H),3.03-3.11(m,4H),2.78-2.82(m,1H),1.72-1.74(m,2H),1.48-1.52(m,2H),1.42(s,9H)；MS(ES⁺):m/z＝527.24(M+H)⁺。

Preparation of HW 03606-5: steps 1 to 3 of reference example 1

To a 250ml three-necked flask, at room temperature, were added in the following order: ethanol (100mL), HW03606-4(24.3g,46mmol) and 6N HCl or trifluoroacetic acid (50 mL). The reaction was then stirred at room temperature overnight. Distillation under reduced pressure gave HW03606-5 (about 20g, 100% yield) as a white solid. Used directly in the next step.

Preparation of target compound HW 03606: steps 1 to 4 of reference example 1

To a 100ml three-necked flask, at room temperature, were added in the following order: methanol (50mL), HW03606-5(4.6g,10.8mmol), HW03605-6(2.76g,10.8mmol) and N, N-diisopropylethylamine (4.2g,32.4 mmol). Then, the reaction system was heated to reflux and stirred overnight. The solvent was evaporated to dryness under reduced pressure, water (100mL) was added, extraction was performed with ethyl acetate (3 × 100mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid, and recrystallized from methanol to give HW03606(1.43g, 31% yield) as a white solid.

¹H NMR(400MHz,CDCl3)δ7.79-7.81(m,1H),7.41-7.42(m,1H),7.12-7.13(m,1H),6.97-7.01(m,2H),6.81-6.82(m,1H),5.50(brs,1H),3.90-3.92(m,2H),3.43-3.50(m,5H),3.57-3.60(m,4H),3.26-3.28(m,4H),3.22(s,3H),2.41-2.52(m,8H),1.91-2.01(m,2H),1.83-1.85(m,1H),1.56-1.59(m,1H).C₃₄H₃₉F₂N₇O₄LC-MS ([ M + H)]⁺): measured value: 648.30, calculated as: 648.30.

example 8: preparation of 3- (3- (4- (4-fluoro-2- (4- (6-fluoro-1H-indazol-3-yl) piperazin-1-yl) benzoyl) piperazin-1-yl) propyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound HW03607)

The synthetic route is as follows:

preparation of HW 03603-2: step 1-1 of reference example 1

To a 500 ml three-necked flask, at room temperature, were added in the following order: THF/water (100mL/100mL), HW03603-1(19.9g,76mmol) and Na₂CO₃(10.5g,99 mmol). Then, the temperature of the external bath is reduced to 0 ℃ by using ice water. Under stirring, (Boc) was added₂O (19.9g,91 mmol). The reaction was allowed to spontaneously warm to room temperature and stirred overnight. Concentrating the organic solvent under reduced pressure, extracting the aqueous phase with ethyl acetate (3 × 100mL), combining the organic phases, washing with saturated salt water, drying over anhydrous sodium sulfate, filtering, and reducing the pressureDistillation under pressure gave HW03603-2(22.6g, 91% yield) as a white solid which was used directly in the next step.

Preparation of HW 03607-4: steps 1-2 of reference example 1

To a 500 ml three-necked flask, at room temperature, were added in the following order: 1, 4-dioxane (300mL), HW03603-2(19.9g,61mmol), HW03607-3(18.7g,73mmol) and K₂CO₃(10.9g,79 mmol). Then, the reaction system was stirred at 90 ℃ overnight. The reaction was brought to room temperature and then poured into 500 mL of water, stirred well, extracted with ethyl acetate (3X 300mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid which was washed with ethyl acetate slurry to give HW03607-4(27.7g, 78% yield) as a white solid.

¹H NMR(400MHz,CDCl₃)δ12.2(brs,1H),7.90-7.91(m,1H),7.79-7.80(m,1H),7.59-7.60(m,1H),7.19-7.20(m,1H),7.01-7.02(m,1H),6.81-6.82(m,1H),3.57-3.59(m,8H),3.28-3.34(m,8H),1.42(s,9H)；MS(ES+):m/z＝527.25(M+H)⁺。

Preparation of HW 03607-5: steps 1 to 3 of reference example 1

To a 250ml three-necked flask, at room temperature, were added in the following order: ethanol (100mL), HW03607-4(24.2g,46mmol) and 6N HCl or trifluoroacetic acid (50 mL). The reaction was then stirred at room temperature overnight. Distillation under reduced pressure gave HW03607-5 as a white solid (about 20g, 100% yield). Used directly in the next step.

Preparation of the target compound HW 03607: steps 1 to 4 of reference example 1

To a 100ml three-necked flask, at room temperature, were added in the following order: methanol (50mL), HW03607-5(4.6g,10.8mmol), HW03605-6(2.76g,10.8mmol) and N, N-diisopropylethylamine (4.2g,32.4 mmol). Then, the reaction system was heated to reflux and stirred overnight. The solvent was evaporated to dryness under reduced pressure, water (100mL) was added, extraction was performed with ethyl acetate (3 × 100mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid, and recrystallized from methanol to give HW03607(3.28g, 47% yield) as a white solid.

¹H NMR(400MHz,CDCl3)δ12.2(brs,1H),7.90-7.91(m,1H),7.79-7.80(m,1H),7.59-7.61(m,1H),7.18-7.19(m,1H),7.01-7.02(m,1H),6.81-6.82(m,1H),5.50(brs,1H),3.90-3.93(m,2H),3.43-3.57(m,9H),3.27-3.29(m,4H),3.22(s,3H),2.41-2.52(m,8H),1.53-1.59(m,3H),1.84-2.01(m,3H).C₃₄H₄₀F₂N₈O₃LC-MS ([ M + H)]⁺): measured value: 647.32, calculated as: 647.32.

example 9: preparation of 3- (3- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperazin-1-yl) benzoyl) piperidin-1-yl) propyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound HW03608)

The synthetic route is as follows:

preparation of HW 036-2: step 1-1 of reference example 1

To a 500 ml three-necked flask, at room temperature, were added in the following order: THF/water (100mL/100mL), HW036-1(19.9g,76mmol) and Na₂CO₃(10.5g,99 mmol). Then, the temperature of the external bath is reduced to 0 ℃ by using ice water. Under stirring, (Boc) was added₂O (19.9g,91 mmol). The reaction was allowed to spontaneously warm to room temperature and stirred overnight. The organic solvent was concentrated under reduced pressure, the aqueous phase was extracted with ethyl acetate (3X 100mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to give HW036-2(22.6g, 91% yield) as a white solid which was used directly in the next step.

Preparation of HW 03608-4: steps 1-2 of reference example 1

To a 500 ml three-necked flask, at room temperature, were added in the following order: 1, 4-dioxane (300mL), HW036-2(19.9g,61mmol), HW03606-3(18.8g,73mmol) and K₂CO₃(10.9g,79 mmol). Then, the reaction system was stirred at 90 ℃ overnight. The reaction system was brought to room temperature, then poured into 500 mL of water, stirred well, extracted with ethyl acetate (3X 300mL), the organic phases were combined, washed with saturated brine,dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid, which was washed with ethyl acetate slurry to give HW03608-4(22.8g, 71% yield) as a white solid.

¹H NMR(400MHz,CDCl₃)δ7.82-7.83(m,1H),7.41-7.43(m,1H),7.12-7.13(m,1H),6.91-6.96(m,2H),6.71-6.72(m,1H),3.57-3.59(m,8H),3.28-3.30(m,4H),3.05-3.06(m,1H),1.92-1.93(m,2H),1.65-1.67(m,2H),1.42(s,9H)；MS(ES⁺):m/z＝527.24(M+H)⁺。

Preparation of HW 03608-5: steps 1 to 3 of reference example 1

To a 250ml three-necked flask, at room temperature, were added in the following order: ethanol (100mL), HW03608-4(24.2g,46mmol) and 6N HCl or trifluoroacetic acid (50 mL). The reaction was then stirred at room temperature overnight. Distillation under reduced pressure gave HW03608-5 as a white solid (about 20g, 100% yield). Used directly in the next step.

Preparation of target compound HW 03608: steps 1 to 4 of reference example 1

To a 100ml three-necked flask, at room temperature, were added in the following order: methanol (50mL), HW03608-5(4.6g,10.8mmol), HW03605-6(2.76g,10.8mmol) and N, N-diisopropylethylamine (4.2g,32.4 mmol). Then, the reaction system was heated to reflux and stirred overnight. The solvent was evaporated to dryness under reduced pressure, water (100mL) was added, extraction was performed with ethyl acetate (3 × 100mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid, and recrystallized from methanol to give HW03608(2.72g, 39% yield) as a white solid.

¹H NMR(400MHz,CDCl3)δ7.79-7.81(m,1H),7.41-7.42(m,1H),7.12-7.13(m,1H),6.91-6.96(m,2H),6.71-6.72(m,1H),5.50(brs,1H),3.90-3.93(m,2H),3.50-3.57(m,5H),3.26-3.28(m,4H),3.22(s,3H),3.00(m,1H),2.41-2.51(m,8H),1.91-2.01(m,2H),1.77-1.84(m,3H),1.52-1.59(m,3H).C₃₅H₄₀F₂N₆O₄LC-MS ([ M + H)]⁺): measured value: 647.31.0, calculated as: 647.31.

example 10: preparation of 3- (3- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperazin-1-yl) benzoyl) piperidin-1-yl) propyl) -9-hydroxy-2-phenyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound HW03609)

The synthetic route is as follows:

preparation of HW 036-2: step 1-1 of reference example 1

To a 500 ml three-necked flask, at room temperature, were added in the following order: THF/water (100mL/100mL), HW036-1(19.9g,76mmol) and Na₂CO₃(10.5g,99 mmol). Then, the temperature of the external bath is reduced to 0 ℃ by using ice water. Under stirring, (Boc) was added₂O (19.9g,91 mmol). The reaction was allowed to spontaneously warm to room temperature and stirred overnight. The organic solvent was concentrated under reduced pressure, the aqueous phase was extracted with ethyl acetate (3X 100mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to give HW036-2(22.6g, 91% yield) as a white solid which was used directly in the next step.

Preparation of HW 03608-4: steps 1-2 of reference example 1

To a 500 ml three-necked flask, at room temperature, were added in the following order: 1, 4-dioxane (300mL), HW036-2(19.9g,61mmol), HW03606-3(18.8g,73mmol) and K₂CO₃(10.9g,79 mmol). Then, the reaction system was stirred at 90 ℃ overnight. The reaction was brought to room temperature and then poured into 500 mL of water, stirred well, extracted with ethyl acetate (3X 300mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid which was washed with ethyl acetate slurry to give HW03608-4(22.8g, 71% yield) as a white solid.

¹H NMR(400MHz,CDCl₃)δ7.82-7.83(m,1H),7.41-7.43(m,1H),7.12-7.13(m,1H),6.91-6.96(m,2H),6.71-6.72(m,1H),3.57-3.59(m,8H),3.28-3.30(m,4H),3.05-3.06(m,1H),1.92-1.93(m,2H),1.65-1.67(m,2H),1.42(s,9H)；MS(ES⁺):m/z＝527.24(M+H)⁺。

Preparation of HW 03608-5: steps 1 to 3 of reference example 1

To a 250ml three-necked flask, at room temperature, were added in the following order: ethanol (100mL), HW03608-4(24.2g,46mmol) and 6N HCl or trifluoroacetic acid (50 mL). The reaction was then stirred at room temperature overnight. Distillation under reduced pressure gave HW03608-5 as a white solid (about 20g, 100% yield). Used directly in the next step.

Preparation of target compound HW 03609: steps 1 to 4 of reference example 1

To a 100ml three-necked flask, at room temperature, were added in the following order: methanol (50mL), HW03608-5(4.6g,10.8mmol), HW03609-6(3.44g,10.8mmol) and N, N-diisopropylethylamine (4.2g,32.4 mmol). Then, the reaction system was heated to reflux and stirred overnight. The solvent was evaporated to dryness under reduced pressure, water (100mL) was added, extraction was performed with ethyl acetate (3 × 100mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid, and recrystallized from methanol to give HW03609(3.52g, 46% yield) as a white solid.

¹H NMR(400MHz,CDCl₃)δ8.02-8.03(m,2H),7.76-7.78(m,1H),7.41-7.46(m,4H),7.12-7.13(m,1H),6.91-6.97(m,2H),6.71-6.72(m,1H),5.50(brs,1H),3.90-3.93(m,2H),3.50-3.57(m,5H),3.28-3.30(m,4H),3.00(m,1H),2.41-2.51(m,8H),1.91-2.01(m,2H),1.77-1.84(m,3H),1.52-1.59(m,3H).C₄₀H₄₂F₂N₆O₄LC-MS ([ M + H)]⁺): measured value: 709.32, calculated as: 709.32.

example 11: preparation of 3- (3- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperazin-1-yl) benzoyl) piperazin-1-yl) propyl) -9-hydroxy-2-phenyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound HW03610)

The synthetic route is as follows:

preparation of HW 03603-2: step 1-1 of reference example 1

To a 500 ml three-necked flask, at room temperature, were added in the following order: THF/water (100mL/100mL), HW03603-1(19.9g,76mmol) and Na₂CO₃(10.5g,99 mmol). Then, the temperature of the external bath is reduced to 0 ℃ by using ice water. Under stirring, (Boc) was added₂O (19.9g,91 mmol). The reaction was allowed to spontaneously warm to room temperature and stirred overnight. The organic solvent was concentrated under reduced pressure, the aqueous phase was extracted with ethyl acetate (3X 100mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to give HW03603-2(22.6g, 91% yield) as a white solid which was used directly in the next step.

Preparation of HW 03610-4: steps 1-2 of reference example 1

To a 500 ml three-necked flask, at room temperature, were added in the following order: 1, 4-dioxane (300mL), HW03603-2(19.9g,61mmol), HW03606-3(18.8g,73mmol) and K₂CO₃(10.9g,79 mmol). Then, the reaction system was stirred at 90 ℃ overnight. The reaction was brought to room temperature and then poured into 500 mL of water, stirred well, extracted with ethyl acetate (3X 300mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid which was washed with ethyl acetate slurry to give HW03610-4(25.1g, 78% yield) as a white solid.

¹H NMR(400MHz,CDCl₃)δ7.79-7.80(m,1H),7.41-7.42(m,1H),7.12-7.13(m,1H),6.97-7.01(m,2H),6.81-6.82(m,1H),3.57-3.59(m,8H),3.28-3.34(m,8H),1.42(s,9H)；MS(ES⁺):m/z＝528.23(M+H)⁺。

Preparation of HW 03610-5: steps 1 to 3 of reference example 1

To a 250ml three-necked flask, at room temperature, were added in the following order: ethanol (100mL), HW03610-4(24.3g,46mmol) and 6N HCl or trifluoroacetic acid (50 mL). The reaction was then stirred at room temperature overnight. Distillation under reduced pressure gave HW03610-5 as a white solid (about 20g, 100% yield). Used directly in the next step.

Preparation of target compound HW 03610: steps 1 to 4 of reference example 1

To a 100ml three-necked flask, at room temperature, were added in the following order: methanol (50mL), HW03610-5(4.6g,10.8mmol), HW03609-6(3.44g,10.8mmol) and N, N-diisopropylethylamine (4.2g,32.4 mmol). Then, the reaction system was heated to reflux and stirred overnight. The solvent was evaporated to dryness under reduced pressure, water (100mL) was added, extraction was performed with ethyl acetate (3 × 100mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid, and recrystallized from methanol to give HW03610(3.13g, 41% yield) as a white solid.

¹H NMR(400MHz,CDCl3)δ8.02-8.03(m,2H),7.77-7.79(m,1H),7.41-7.46(m,4H),7.12-7.13(m,1H),6.91-6.97(m,2H),6.71-6.72(m,1H),5.50(brs,1H),3.90-3.93(m,2H),3.51-3.57(m,5H),3.27-3.28(m,4H),3.00-3.01(m,1H),2.41-2.51(m,8H),1.91-2.01(m,2H),1.77-1.84(m,3H),1.52-1.59(m,3H).C₄₀H₄₂F₂N₆O₄LC-MS ([ M + H)]⁺): measured value: 709.32.0, calculated as: 709.32.

example 12: preparation of 3- (3- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperidin-1-yl) benzoyl) piperazin-1-yl) propyl) -9-hydroxy-2-phenyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one (compound HW03611)

The synthetic route is as follows:

preparation of HW 03603-2: step 1-1 of reference example 1

To a 500 ml three-necked flask, at room temperature, were added in the following order: THF/water (100mL/100mL), HW03603-1(19.9g,76mmol) and Na₂CO₃(10.5g,99 mmol). Then, the temperature of the external bath is reduced to 0 ℃ by using ice water. Under stirring, (Boc) was added₂O (19.9g,91 mmol). The reaction was allowed to spontaneously warm to room temperature and stirred overnight. The organic solvent was concentrated under reduced pressure, the aqueous phase was extracted with ethyl acetate (3X 100mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to give HW03603-2(22.6g, 91% yield) as a white solid which was used directly in the next step.

Preparation of HW 03611-4: steps 1-2 of reference example 1

At room temperature, the mixture was added to a 500 ml three-necked flask:1, 4-dioxane (300mL), HW03603-2(19.9g,61mmol), HW036-3(18.7g,73mmol) and K₂CO₃(10.9g,79 mmol). Then, the reaction system was stirred at 90 ℃ overnight. The reaction was brought to room temperature and then poured into 500 mL of water, stirred well, extracted with ethyl acetate (3X 300mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid, which was washed with ethyl acetate slurry to give HW03611-4(22.5g, 70% yield) as a white solid.

¹H NMR(400MHz,CDCl₃)δ7.79-7.80(m,1H),7.41-7.42(m,1H),7.12-7.13(m,1H),6.97-7.01(m,2H),6.81-6.82(m,1H),3.59-3.60(m,4H),3.34-3.36(m,4H),3.04-3.14(m,4H),2.76-2.78(m,1H),1.72-1.73(m,2H),1.49-1.50(m,2H),1.42(s,9H)；MS(ES⁺):m/z＝527.24(M+H)⁺。

Preparation of HW 03611-5: steps 1 to 3 of reference example 1

To a 250ml three-necked flask, at room temperature, were added in the following order: ethanol (100mL), HW03611-4(24.3g,46mmol) and 6N HCl or trifluoroacetic acid (50 mL). The reaction was then stirred at room temperature overnight. Distillation under reduced pressure gave HW03611-5 as a white solid (about 20g, 100% yield). Used directly in the next step.

Preparation of the target compound HW 03611: steps 1 to 4 of reference example 1

To a 100ml three-necked flask, at room temperature, were added in the following order: methanol (50mL), HW03611-5(4.6g,10.8mmol), HW03609-6(3.44g,10.8mmol) and N, N-diisopropylethylamine (4.2g,32.4 mmol). Then, the reaction system was heated to reflux and stirred overnight. The solvent was evaporated to dryness under reduced pressure, water (100mL) was added, extraction was performed with ethyl acetate (3 × 100mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure to give a solid, and recrystallized from methanol to give HW03611(2.52g, 33% yield) as a white solid.

¹H NMR(400MHz,CDCl3)δ8.01-8.02(m,2H),7.79-7.80(m,1H),7.41-7.46(m,4H),7.12-7.13(m,1H),6.97-7.01(m,2H),6.80-6.81(m,1H),5.50(brs,1H),3.90-3.92(m,2H),3.43-3.50(m,5H),3.04-3.14(m,4H),2.76-2.78(m,1H),2.41-2.52(m,8H),1.91-2.01(m,2H),1.74-1.84(m,3H),1.49-1.59(m,5H).C₄₀H₄₂F₂N₆O₄LC-MS ([ M + H)]⁺): measured value: 709.32, calculated as: 709.32.

example 13: preparation of 9-hydroxy-2-methyl-3- (2- (methyl ((S) -3- (naphthalen-1-yloxy) -3- (thiophen-3-yl) propyl) amino) ethyl) -6,7,8, 9-tetrahydropyrido [1,2-a ] pyrimidin-4-one (compound HW031)

The synthetic route is as follows:

step 1: preparation of the compound HW-031B.

Specifically, 60g (0.476mol) of 3-acetylthiophene (HW-031A), 21g (0.7mol) of paraformaldehyde, 50g (0.613mol) of dimethylamine hydrochloride, 140mL of isopropanol and 5mL of concentrated hydrochloric acid are stirred and mixed, then heated to reflux (82-83 ℃) for reaction for 6 hours, and after the reaction is finished, the mixture is cooled to 0 ℃ overnight. The precipitated solid was filtered, and the obtained solid was washed with 50mL of cold ethanol and dried in vacuo at 60 ℃ overnight to give HW-031B (60.5g,0.275mol, yield: 57.8%) as white crystals.

¹H NMR(400MHz,CDCl₃):δ7.64(m,1H),7.29(m,2H),7.10(t,1H),6.80(m,2H),4.09(m,2H),3.63(m,1H),3.40(d,2H),3.20(m,1H),2.97(t,2H),2.82(m,2H),2.24(m,2H),2.15(m,2H),1.78(m,2H),1.61(m,2H),1.46(s,9H)；MS(ES⁺):m/z＝526.24(M+H)⁺。

Step 2: preparation of the compound HW-031C.

Specifically, HW-031B (45g,0.205moL) and 200mL absolute ethanol are mixed with stirring, then the pH is adjusted to 12 by 50% sodium hydroxide (14mL), and NaBH is added in portions under ice bath₄(9g, 237mmol, exotherm, control addition rate, maintain temperature<After completion of the addition at 30 ℃ for 4 hours at room temperature, 28mL of acetone was added (exothermic, ice-water cooled). Stirring is continued for half an hour, and then concentration is carried out under reduced pressure to dryness, so as to obtain HW-031C which is directly used in the next step.

And step 3: preparation of the compound HW-031D.

Specifically, 450mL of methyl tert-butyl ether and 200mL of water were added to the HW-031C obtained in the previous step, the pH was adjusted to 1-1.5 with concentrated hydrochloric acid (35mL), the mixture was stirred for 10min, and then the pH was adjusted to 12 with 50% sodium hydroxide (0 mL). Separating an organic layer, extracting an aqueous layer once by using 100mL of methyl tert-butyl ether, combining the organic layers, washing once by using 150mL of water, concentrating to 250mL under reduced pressure, heating to reflux, slowly dropwise adding 50mL of anhydrous ethanol solution of (S) - (+) -mandelic acid (34.2g,0.225mol), continuing refluxing for 1 hour, then cooling to room temperature (20-30 ℃), stirring for 1 hour, filtering out a precipitated solid, washing a filter cake (changing from red to white) by using 50mL of methyl tert-butyl ether, and drying in vacuum at 50 ℃ to obtain 21.41g of a white solid, which is mandelate of HW-031D.

Dissolving mandelate salt of HW-031D in 400mL water, adding ethyl acetate 400mL, adjusting pH to 11 with 50% sodium hydroxide, separating organic layer, extracting water layer with 100mL × 2 ethyl acetate, combining organic layers, washing with brine 100mL, drying with anhydrous sodium sulfate, filtering, and concentrating under reduced pressure to dryness to obtain white solid about 20 g. Washed with 100mL brine, dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure to give about 20g of a white solid. Recrystallization from 40mL of methyl tert-butyl ether (heating, refluxing, dissolution), cooling overnight, no solid precipitated, rubbing the flask wall with a spatula, and filtration yielded HW-031D (13.58g,0.073mol, yield: 35.8%).

And 4, step 4: preparation of the compound HW-031E.

Dissolving a compound HW-031D (13.58g, 73.3mmol) in DMSO (150mL), dissolving in water, adding sodium hydrogen (3.36g, 77.0mmol) in batches, stirring for 0.5 hour after the addition is finished, adding 4.32g (0.025mmol) of potassium p-methylbenzoate, continuing stirring for half an hour, dropwise adding 1-fluoronaphthalene (13.1g,90.0mmol), heating to 50 ℃, reacting for half an hour, heating to 60 ℃, reacting for 4-6 hours, cooling an organic phase after the reaction is finished, and washing with brine (100 mL). Drying with anhydrous sodium sulfate, filtering, adding oxalic acid 12g, stirring for crystallization (1-2 hr), filtering, drying the solid at 60 deg.C overnight, and recrystallizing with 80-100 ml ethanol to obtain white solid HW-031E (22g,54.0mmol, yield: 75%).

And 5: preparation of the compound HW-031F.

HW-031E (22g,54.0mmol), toluene 175mL, water 175mL are mixed with stirring, concentrated ammonia water 20mL is added, stirring is carried out for 1 hour, the organic layer is separated, washed with brine 100mL, dried and filtered with anhydrous sodium sulfate, and the next step is directly carried out.

DIPEA (1.5mL) was added, the temperature was raised to 55 ℃ and phenyl chloroformate (9.45g, 60.0mmol) was added dropwise. After dropping, the mixture reacts for 1 hour at 55 ℃, and 1 percent NaHCO is added₃Aqueous solution (185mL), stirred for half an hour, the organic layer was separated and washed successively with 75mL 0.5N HCl,75mL 1% NaHCO₃The aqueous solution was washed with 75mL of brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. DMSO (200mL) was added, the temperature was raised to 50 ℃ and 9g of 50mL NaOH solution was added dropwise. After the dripping is finished, the mixture is subjected to oil bath at 50 ℃ and is subjected to heat preservation reaction overnight. Disappearance of starting material was detected by TLC (MeOH: EA ═ 1: 4). After cooling to room temperature, the reaction mixture was poured into 150mL of ice water, extracted with ethyl acetate (200 mL. times.3), the organic layers were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated to 150mL, oxalic acid (5.9g,66.0mmol) was added, stirred for 1 hour, filtered to give a white solid, rotary evaporated to dryness, and recrystallized from methanol/ethyl acetate (100mL:100 mL). Cooled to room temperature, filtered, washed with a little ethyl acetate and dried to obtain the oxalate salt of HW-031F as a white solid (14.76g, 38.0mmol, yield: 70.3%).

Step 6: preparation of the compound HW-031.

HW-031F (14.76G, 38.0mmol), potassium carbonate (15.7G,114mmol), HW-031G (11.0G,45.6mmol), 18-crown-6 (0.2G) are added to 200ml tetrahydrofuran sequentially, heated to reflux, stirred for 2-6 hours. The solvent was evaporated to dryness, diluted with 200mL of water, then extracted with ethyl acetate (200mL × 3), the organic phases were combined, washed with saturated brine (100mL × 2), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to give the crude product, which was recrystallized sequentially from ethyl acetate and ethanol to give a pale yellow to white solid (15.6g,31mmol, yield: 82%).

¹H-NMR(300MHz,CDCl₃) Delta 8.365-8.341 (q, J-2.4 Hz,1H), 7.781-7.758 (q, J-2.4 Hz,1H), 7.494-7.453 (sext, J-2.6 Hz,2H), 7.372-7.351 (d, J-6.3 Hz,1H), 7.270-7.220 (m,2H, CDCl encapsulation)₃Residual H), 7.113-7.099 (dd, J)₁＝0.6Hz,J₂＝3.0Hz,1H),6.738～6.708(dd,J₁＝3.3Hz,J₂＝2.4Hz,1H),5.622～5.577(dt,J₁＝3.9Hz,J₂＝2.1Hz,1H),4.461～4.377(hept,J₁＝4.4Hz,J₂＝3.3Hz,J₃4.8Hz,1H),4.169(m,1H, Baotong), 3.940-3.803 (m,2H), 2.772-2.660 (m,4H), 2.630-2.540 (m,2H), 2.440-2.300 (m,5H), 2.267-2.194 (m,4H), 2.116-2.037 (m,1H), 1.930-1.833 (m,1H), 1.763-1.655 (m,1H).

LCMS(m/z)m+1＝504.1

Example 14: preparation of 9-hydroxy-2-methyl-3- (2- (methyl ((S) -3- (naphthalen-1-yloxy) -3- (thiophen-2-yl) propyl) amino) ethyl) -6,7,8, 9-tetrahydropyrido [1,2-a ] pyrimidin-4-one (compound HW086)

The synthetic route is as follows:

10.00g of duloxetine hydrochloride and 12.35g of the compound [ M ] were placed in a 250mL round-bottom single-neck flask, 120mL of anhydrous DMF and 28.92g of powdered anhydrous potassium carbonate and 4.66g of sodium iodide were added, the mixture was replaced with nitrogen three times, and the mixture was stirred in a 65 ℃ oil bath overnight. TLC sampling showed complete reaction and no starting material. The reaction solution changed color from brown to dark gray. Poured into 600mL of water, EA (100 mL. times.3) extracted, the organic phase was checked again by TLC to find that a considerable amount of the starting material had not reacted, the organic phases were combined, washed with 150mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated to dryness, and then supplemented again with 8.87g of the compound [ M ], 18.21g of potassium carbonate, 3.77g of sodium iodide, 100mL of DMF, and 3 times replaced with nitrogen, followed by stirring again overnight in a 65 ℃ oil bath. TLC detection showed a little more starting material, which was poured into 600mL of water, EA (100 mL. times.3) extracted, combined organic phases, washed with 150mL of brine, dried over anhydrous sodium sulfate, filtered, concentrated and applied to a silica gel column, eluting the desired product with DCM/MeOH (30/1 to 12/1 gradient) as eluent to give about 5.1g of pure product and about 6.5g of less polar impurities. The yield thereof was found to be 72%.

¹H-NMR(CDCl₃ 400MHz)δ8.351～8.328(q,J＝3.2Hz,1H),7.784～7.743(dt,J＝3.2Hz,1H),7.488～7.446(dt,J＝3.4Hz,2H),7.388～7.367(d,J＝8.4Hz,1H),7.290～7.245(dt,J₁＝2.4Hz,J₂5.6Hz,1H, peak of residual inclusion solvent), 7.202-7.186 (dd, J₁＝1.2Hz,J₂＝4.0Hz,1H),7.039～7.030(d,J＝3.6Hz,1H),6.931～6.910(dd,J₁＝4.0Hz,J₂＝1.2Hz,1H),6.860～6.827(dd,J₁＝4.2Hz,J₂＝2.0Hz,1H),5.767～5.720(dd,J₁＝5.2Hz,J₂＝7.6Hz,1H),4.453～4.413(dd,J₁＝6.4Hz,J₂＝3.6Hz,0.5H),4.392～4.352(dd,J₁＝6.4Hz,J₂3.6Hz,0.5H), 3.907-3.850 (m,2H),2,949(s,0.35H, -OH split), 2.879(0.35H, -OH split), 2.722-2.608 (m,4H), 2.599-2.491 (m,2H), 2.450-2.382 (m,1H), 2.356-2.350 (d, J-2.4 Hz,3H), 2.324-2.251 (m,1H), 2.228-2.216 (d, J-4.8 Hz,3H), 2.109-2.015 (m,1H), 1.950-1.786 (m,2H), 1.750-1.641 (m,1H).

LCMS(m/z)m+1＝504.2.

Antitumor Activity test of the Compound of the present invention

Firstly, the in vitro anti-tumor effect of the compound is evaluated by adopting an MTT method.

Preparation of main reagent

PBS buffer (pH 7.4): dissolving PBS powder (Beijing China fir Jinqiao biotechnology Co., Ltd.) in double distilled water, diluting to a constant volume of 2000mL, adjusting pH, autoclaving, and storing in a refrigerator at 4 deg.C

RPMI1640 medium: RPMI-1640 medium (Hyclone) contained 10% fetal bovine serum (Hyclone) and 1% diabody (penicillin and streptomycin) (Hyclone), mixed well and stored in a refrigerator at 4 ℃ until use.

MTT solution: MTT powder (Sigma, USA) is dissolved by PBS buffer solution to prepare a solution with the concentration of 5mg/mL, and the solution is filtered, subpackaged and stored in a refrigerator at-20 ℃ in a dark place for later use.

Preparation of 2.5% pancreatin: 2.5g of pancreatin powder (Gibco) was taken, added to 100mL of 1 XPBS, dissolved, filtered, dispensed, and stored in a refrigerator at-20 ℃ in the dark for later use.

Preparation of 0.25% EDTA: taking 0.25g of EDTA powder, adding 100mL of 1 XPBS, dropwise adding NaOH solution for assisting dissolution, filtering, subpackaging, and storing in a refrigerator at the temperature of-20 ℃ in a dark place for later use.

Preparation of digestive juice: 1 XPBS 40mL, respectively adding 5mL 2.5% pancreatin and 5mL 0.25% EDTA, mixing, at 4 ℃ refrigerator storage for use.

Preparation of compound HW036 mother liquor of example 1 of the present invention: 4.8mg of the compound HW036 of example 1 of the present invention was dissolved in 1mL of DMSO and stored in a freezer at-20 ℃ until use.

Cell lines and culture conditions

CNE-2 human nasopharyngeal carcinoma cells, Hela human cervical carcinoma cells, 4T1 murine breast cancer cells, and B16 murine melanoma cells were all obtained from ATCC. These cells were cultured in RPMI1640 medium (Hyclone) containing 10% (v/v) fetal bovine serum (Hyclone) and 100U/ml penicillin-streptomycin, respectively, and the cells were incubated at 37 ℃ with 5% CO₂Culturing in a saturated humidity incubator, and taking cells in logarithmic growth phase for experiment.

Experimental methods

Respectively placing CNE-2 human nasopharyngeal carcinoma cells, Hela human cervical carcinoma cells, 4T1 murine breast cancer cells and B16 murine melanoma cells in 10% fetal calf serum RPMI1640 culture solution at 37 deg.C and 5% CO₂Culturing in a saturated humidity incubator. Under aseptic conditions, cells in logarithmic growth phase were taken approximately 3X 10 cells per well³Inoculating each cell on 96-well culture plate, setting three multiple wells with 100 μ L of each well, adding 100 μ L of culture medium into blank control group, standing at 37 deg.C and 5% CO₂Culturing in a saturated humidity incubator for 24 h. After the cells are attached to the wall, 100. mu.L of different concentrations of HW036 in DMSO (to make the final concentrations of the drugs 0, 3, 6, 12, 24. mu.g/mL respectively) is added to the drug group, 100. mu.L of equal concentration DMSO in the culture solution is added to the negative control group, 100. mu.L of the culture solution is added to the blank group, and 100. mu.L of 40. mu.g/mL MTX (or 5. mu.g/mL doxorubicin) is added to the positive control group. After 24, 48, 72h, 20. mu.L MTT solution (5mg/mL) was added to each well, the culture was continued for 4h in an incubator, the supernatant was removed, 100. mu.L DMSO was added, and the mixture was shaken for 10min to facilitate the pellet to dissolve sufficiently. Detecting with enzyme-linked immunosorbent assay (ELISA) detector, setting wavelength at 570nm, measuring light absorption (OD) value of each well, and calculating cell growth according to formulaLong inhibition, IC calculated according to GraphPad Prism software₅₀。

Wherein, OD₁Absorbance, OD, for blank set₂Absorbance, OD, of the negative group₃The absorbance of the drug group.

MTT method results show that the compound HW036 of example 1 of the invention has stronger proliferation inhibition effect on CNE-2, HeLa, 4T1 and B16 cells (see figure 1 and Table 1).

TABLE 1 IC of Compounds of the examples of the invention on tumor cells CNE-2, HeLa, 4T1 and B16₅₀Value of

As can be seen from fig. 1 and table 1, after the compound HW036 of example 1 of the present invention at different concentrations acts on four tumor cells, namely CNE-2 nasopharyngeal carcinoma cells, HeLa cervical carcinoma cells, 4T1 breast carcinoma cells and B16 melanoma cells, the growth inhibition rate of the tumor cells is significantly increased, the survival rate is significantly decreased, and an obvious dose-effect relationship is presented. After the compound HW036 of the embodiment 1 of the invention acts on the tumor cells for 24 hours, 48 hours and 72 hours, the tumor growth inhibition rate is obviously increased and the survival rate is obviously reduced along with the prolonging of time, thus showing an obvious aging relationship.

Second, annexin V-APC/PI double staining method for detecting apoptosis rate of 4T1 cells

Digesting 4T1 cells in logarithmic growth phase with 2.5% pancreatin (containing 0.025% EDTA), collecting cell suspension, centrifuging, removing supernatant, and adjusting cell concentration to 1 × 10⁵Inoculating the cells/mL in a six-well plate, adding different concentrations of HW036 (mother solution of 2mg/mL dissolved in DMSO; diluted with culture medium to concentrations of 0, 3, 6, 12 and 24 μ g/mL) of the compound of the invention example 1 after adhering to the wall, acting for 48h, collecting stock culture solution, washing with PBS, digesting with pancreatin, collecting cells, mixing with the previous culture solution, separating at 1000r/min for 5minRemoving supernatant from the heart, washing with cold PBS for 2 times, adding 100 μ L Annexin V Binding Buffer (Annexin V Binding Buffer), 5 μ L Annexin V-APC and 30 μ L Pyridine Iodide (PI), mixing, and incubating at room temperature in dark for 15 min; mu.L of annexin V binding buffer was added to each tube, filtered and subjected to flow cytometry for 1 h. Compensation and quadrants were established simultaneously, including unlabeled cells, cells labeled with PI only (30. mu.L), and cells labeled with Annexin V-APC only (5. mu.L). The result takes Annexin V positive/PI negative as early apoptosis and Annexin V positive/PI positive as late apoptosis. The results are shown in fig. 2 and table 2.

SPSS13.0 statistical software is used for experimental data processing, and the comparison adopts paired t test, and the difference of P <0.05 and P <0.01 has statistical significance.

TABLE 2 apoptosis rate of 4T1 tumor cells by HW036, a compound of the present invention

Note: p <0.05 compared to control group

The control group (0. mu.g/mL HW036) of 4T1 cells showed 4.3% late apoptosis from Table 2 and FIG. 2; the late apoptosis rate of the 3 mu g/mL group of cells is 10.6 percent; the late apoptosis rate of the 6 mu g/mL group of cells is 21.7 percent; the late apoptosis rate of the 12 mu g/mL group cells is 24.2%; the late apoptosis rate of 24 ug/mL group cells was 48.5%. Compared with a control group, the group with the concentration of the compound HW036 of 6, 12 and 24 mug/mL in the embodiment 1 of the invention has statistical significance (P < 0.05), which indicates that the compound HW036 of the embodiment 1 of the invention has the apoptosis promoting effect on 4T1 cells.

Thirdly, the effect of the exemplified compound in inhibiting the lung metastasis tumor of the breast cancer of the mouse

1. Preparation of the principal agent

Preparation of a compound HW036 solution of the invention: accurately weighing a certain amount of the compound HW036, adding ethanol to dissolve the compound HW036, adding propylene glycol and PEG400, uniformly mixing, and diluting with 0.9% NaCl to prepare the compound HW036 solution with the concentration of 3.75mg/mL and 7.5 mg/mL. Each milliliter of HW036 solution of the compound of the invention contains 5% ethanol, 20% propylene glycol, 10% PEG400 and 65% 0.9% NaCl, and is filtered by a disposable 0.22 μm sterile filter and stored at 4 ℃ for later use.

Preparation of 5-Fu solution (5-fluorouracil): diluting 5-Fu injection with normal saline to make the final concentration of the medicine 2.5mg/mL, filtering with a disposable 0.22 μm sterile filter, and storing at 4 deg.C for use.

Solvent: a solution containing 5% ethanol, 20% propylene glycol, 10% PEG400 and 65% 0.9% NaCl was prepared, filtered through a disposable 0.22 μm sterile filter, and stored at 4 ℃ for further use.

Preparing a D-Luciferin (D-fluorescein) solution: weighing a certain amount of D-Luciferin powder, dissolving with sterile 1 XPBS to prepare 15mg/mL D-Luciferin solution, and storing at-20 ℃ for later use.

2. Experimental methods

2.1 Experimental groups and dosing regimens

Collecting firefly luciferase-labeled 4T1-luc mouse breast cancer cells at logarithmic growth phase, digesting the cells with 0.25% trypsin-0.025% EDTA, collecting cell suspension, centrifuging, removing supernatant, resuspending with 1 × PBS, and diluting to 4 × 10⁶one/mL. Inoculating 0.2mL of cell suspension to Balb/c mice (Balb/c female mice, 6 weeks old, weight between 18-22 g) through tail vein, and inoculating 8 × 10 cells to each Balb/c mouse⁵4T1-luc cells, which were co-inoculated with 32 cells. Balb/c mice were randomly divided into four groups of 8 mice each, 24h after tail vein inoculation of 4T1-luc cells. A blank control group (containing 5% ethanol, 20% propylene glycol, 10% PEG400, and 65% 0.9% NaCl) was set up; a positive control group (25mg/kg 5-Fu); two dose groups (37.5mg/kg, 75mg/kg) of the compound HW036 group of the invention were orally administered 0.2mL per day. The administration was started after the grouping, and the administration was continued for 4 weeks, during which the body weight, the number of days to live, the death time, and the like of the mice were observed.

In each group of Balb/c mice, at three time points of 7 days, 21 days and 28 days after administration, 20g of mice were intraperitoneally injected with 0.2mL of D-Luciferin solution (15mg/mL), and after 10min of inhalation anesthesia, the lung metastasis of breast cancer of each mouse at different time points was observed by an IVIS (acute respiratory syndrome) small animal living body optical imaging system, and the results are shown in FIGS. 3 and 4.

The number of absorbed photons is in direct proportion to the growth number and activity of tumor cells, and the larger the number of absorbed photons is, the faster the growth of the tumor cells is, the larger the tumor volume is, and the weaker the anti-tumor efficacy of the medicine is; the smaller the number of absorbed photons, the more the tumor cell growth is inhibited, the smaller the tumor volume, and the stronger the anti-tumor effect of the drug. From fig. 3 and 4, it can be seen that the 5-Fu (20mg/kg) administration groups absorbed light with a smaller number of photons than the blank control group at the 7 th and 28 th days after administration and the 37.5mg/kg and 75mg/kg HW036 groups at the 7 th, 21 th and 28 th days after administration, and had statistical significance compared with the blank control group (P <0.05 and P <0.01), and the results showed that the 5-Fu (20mg/kg), 37.5mg/kg and 75mg/kg HW036 administration groups had the effect of inhibiting lung metastasis of breast cancer cells in mice. The inhibiting effect of 75mg/kg of the compound HW036 administration group on the lung metastasis of the breast cancer cells of the mice is stronger than that of 37.5mg/kg of the compound HW036 administration group; on the 28 th day after administration, 37.5mg/kg and 75mg/kg of the compound HW036 administration groups of the present invention absorbed light with a number of less than 5-Fu (20mg/kg), indicating that the 37.5mg/kg and 75mg/kg of the compound HW036 administration groups of the present invention have a stronger inhibitory effect on lung metastasis of breast cancer cells in mice than the 5-Fu (20mg/kg)) administration groups.

3. Mortality of mice in different dosing groups

The number of deaths of each group of mice after administration was recorded separately and the mortality of each group is shown in table 3 below. The results showed that the mortality rate of the 5-Fu (25mg/kg) group was similar to that of the blank control group, and the 5-Fu administered for a long period of time was toxic to the mice according to the knowledge known in the art, and therefore, the death of the mice was probably related to the toxicity of the 5-Fu. The mortality rate of the compound HW036(75mg/kg) and the compound HW036(37.5mg/kg) administration groups is lower than that of the blank control group and the 5-Fu group, and probably because the compound HW036 plays a role in inhibiting the lung metastasis tumor of breast cancer of the mice and reduces the death risk of pulmonary embolism of the mice caused by pulmonary nodules, the mortality rate of the mice is reduced, the survival rate of the mice is improved, and toxic and side effects are reduced.

Table 3 mortality of mice treated with different drugs (n ═ 8)

4. Weight change of mice in different administration groups

The body weight changes of the mice in each group before and after administration are shown in the following table 4, and the results show that the body weight changes of the mice in the compound HW036(37.5mg/kg), the compound HW036(75mg/kg) and the 5-Fu (20mg/kg) administration groups have no significant difference compared with the blank control group (P > 0.05).

Table 4 weights of mice treated with different drugs (n ═ 8)

Note: compared with the control, P >0.05, no significant difference

Fourth, HW031 and HW086 cytotoxicity assays for CNE-2 cells and 4T1 cell proliferation (MTT method)

Experimental methods

CNE-2 resistant nasopharyngeal carcinoma cells (1000/100. mu.l) and 4T1 breast cancer cells (1000/100. mu.l) were respectively and uniformly plated on each 96-well cell culture plate, 100. mu.l of the compounds HW031 and HW086 (mother solution 2mg/mL, dissolved in DMSO) of the present invention at final drug concentrations of 3.25, 6.25, 12.5 and 25. mu.g/mL were added, and the control group was administered with the same amount of solvent and cultured for another 72 hours. 20 μ l (5mg/ml) of MTT solution was added 4 hours before termination of the experiment and after 4 hours the supernatant was centrifuged (1500 rpm/3 min). MTT, A converted to purple by dissolution in 100. mu.l DMSO₅₇₀MTT concentration (representing the amount of cell survival) was measured. The results are shown in FIGS. 5-6. The results show that: HW086 and HW031 were able to significantly inhibit tumor cell growth compared to the control group (photograph), and were dose-response related.

Fifthly, the annexin V-APC/PI double staining method is used for detecting the apoptosis rate of the liver cancer cell (SMMC-7721) treated by the compound HW031 and HW086

Experimental methods and principles the apoptosis rate of 4T1 cells was examined by annexin V-APC/PI double staining method of HW036 as the second item.

Hepatoma cells (1000/100. mu.l) in logarithmic growth phase (SMMC-7721) were spread evenly on cell culture plates, 100. mu.l of each of the compounds HW031 and HW086 (2 mg/mL stock solution in DMSO) were added to the plates to final drug concentrations of 3.25, 6.25, 12.5, 25. mu.g/mL (equivalent amounts of solvent given to control group) and incubation was continued for 24 hours. Staining with anti-apoptotic antibody (Annexin V/PI). The results show that: as shown in fig. 7, compared with the control group nc (a), the compounds HW031 and HW086 of the present invention have significant pro-apoptotic effect on SMMC-7721 hepatoma cells.

Sixthly, HW031 and HW086 inhibiting mouse breast cancer lung metastasis tumor effect

Experimental methods

Will be 1 × 10⁶4T1-Luc mouse breast cancer cells (firefly enzyme-stably transfected breast cancer cells) are inoculated to Balb/c mice (Balb/c female mice, 6 weeks old, 18-22 g in weight) through tail veins. And (4) grouping randomly. The control group (NC group) was treated with a solvent, and the HW031 administration group and the HW086 administration group were orally administered at a dose of 150mg/kg body weight, every other day, and continuously administered for three weeks, respectively. In vivo imaging is carried out on the 7 th, 21 th and 28 th days after Balb/c mice are dosed to monitor the lung metastasis condition of the breast cancer of the mice in vivo through an IVIS small animal in-vivo optical imaging system, wherein 10 mg of firefly essence (15mg/mL of D-Luciferin solution) is injected into the mice 5 minutes before the in vivo imaging is carried out, and the number of photons emitted by the firefly essence in the tumors is obtained by in vivo imaging immediately after inhalation anesthesia, so that the number represents the size of the tumors.

The results are shown in FIGS. 8A-8B, and FIG. 9. The results show that: the inhibitory effect of the compounds HW031 and HW086 of the invention on lung metastasis of breast cancer cells of mice is stronger than that of a control group.

Seven, HW031 and HW086 in vivo liver cancer inhibition effect on H22 liver cancer model of ICR mice

Will be 1 × 10⁶Liver cancer cells were injected subcutaneously into both sides of the abdomen of mice, and the next day, 150mg/kg body weight of HW031 and HW086 were orally administered, respectively, and the tumor volume (volume ═ width) was measured every three days using a single solvent in the control group²)/2). The results are shown in FIG. 10. ResultsShows that: compared with the control group, the HW031 and HW086 of the invention can inhibit the growth of liver cancer tumor.

Eighthly, toxicity test for detecting HW031 and HW086

Toxicity tests are referred to conventional toxicity test methods (e.g., methods as described in Yuanberjun's Experimental methods and techniques of drug toxicology (chemical industry Press, 2007)). In the toxicity test, HW031 and HW086 may be used in 5-10 times the anticancer dose of 150 mg/kg.

Balb/c mice were randomly grouped by body weight, and a control group (administered with the solvent DMSO), a HW031 group (1500mg/kg dose), and a HW086 group (1500mg/kg dose) were established. The medicine is orally taken once after grouping. The change in body weight of each group of mice was observed and recorded for 2 weeks after administration, and at the end of the experiment, a hematological test, a biochemical test, and the like were performed. The results are shown in FIGS. 11 and 12.

FIG. 11 shows that there was no significant change in body weight between mice in the group of compound HW031 of the invention and HW086 of the invention compared to the control group.

As can be seen from fig. 12, no significant hematological changes were observed in the compound HW031 group and HW086 group, with no significant impairment of hepatic and renal function. There was no decline in WBC leukocyte counts, indicating that bone marrow was not suppressed (a mild elevation might indicate a tumor inflammatory response). HW-086 and HW-031 have no effect on lymphocyte percentage (LYM%) and number (LYM #); has no influence on the number of RBC (red blood cells); no effect on liver and kidney functions (creatinine means kidney function; alanine aminotransferase means liver cell damage); no effect on protein synthesis and fat metabolism (see total protein and triglyceride data results); no effect on lipid metabolism (see results for total cholesterol and very low density lipoprotein data); has no effect on glucose metabolism.

The invention is not limited by the embodiments shown and described above, but may be varied within the scope of the claims.

Claims (22)

1. A compound of formula I:

wherein the content of the first and second substances,

R₂represents O;

R₃is represented by C_1-3Alkyl or phenyl;

R₄represents OH;

n₁represents an integer of 0 or 1;

n₂represents an integer of 1;

R₁represented by the following formula G₁Or G₂The group of (a):

wherein the content of the first and second substances,

R₅is represented by C_1-3An alkyl group;

R₆represents a thienyl group;

R₇represents a naphthyl group;

n₃represents an integer of 1;

R₈、R₉、R₁₀、R₁₁each independently represents halogen or hydrogen;

R₁₂represents O;

n₄represents an integer of 1;

U₁represents NH or O; and

U₂、U₃、U₄、U₅each independently represents CH or N.

2. A compound of formula I or a salt thereof as claimed in claim 1 wherein R is₁Represented by the following formula G₁The group of (a):

wherein the content of the first and second substances,

R₅is represented by C_1-3An alkyl group;

R₆represents a thienyl group;

R₇represents a naphthyl group; and

n₃represents the integer 1.

3. A compound of formula I or a salt thereof as claimed in claim 2 wherein R is₃、R₅Each independently represents a methyl group.

4. A compound of formula I according to claim 1 or a salt thereof, which is also a compound of formula I-1

Wherein

R₂Represents O;

R₃、R₅each independently represents C_1-3An alkyl group;

R₄represents OH;

R₆represents a thienyl group;

R₇represents a naphthyl group; and

n₁、n₂、n₃each independently represents an integer of 1.

5. A compound of formula I or a salt thereof as claimed in claim 4 wherein R is₃、R₅Each independently represents a methyl group.

6. A compound of formula I according to claim 1, or a salt thereof, which is also a compound of formula I-1-1

Wherein

R₂Represents O;

R₃、R₅each independently represents C_1-3An alkyl group;

R₄represents OH;

R₆represents a thienyl group;

R₇represents a naphthyl group; and

n₁、n₂、n₃each independently represents an integer of 1.

7. A compound of formula I or a salt thereof as claimed in claim 6 wherein R is₃、R₅Each independently represents a methyl group.

8. A compound of formula I or a salt thereof according to claim 1,

R₁represented by the following formula G₂The group of (a):

wherein the content of the first and second substances,

n₄represents an integer of 1;

R₈、R₉、R₁₀、R₁₁each independently represents halogen or hydrogen;

R₁₂represents O;

U₁represents NH or O; and

U₂、U₃、U₄、U₅each independently represents CH or N.

9. The compound of formula I or a salt thereof as claimed in claim 1 or 8, wherein U is₂And U₄Represents CH, and U₃And U₅Represents N.

10. The compound of formula I or a salt thereof as claimed in claim 1 or 8, wherein U is₂、U₃And U₄Represents CH, and U₅Represents N.

11. The compound of formula I or a salt thereof as claimed in claim 1 or 8, wherein U is₂、U₃、U₄And U₅Represents N.

12. The compound of formula I or a salt thereof as claimed in claim 1 or 8, wherein U is₂Represents CH, and U₃、U₄And U₅Represents N.

13. The compound of formula I or a salt thereof as claimed in claim 1 or 8, wherein U is₂、U₄And U₅Represents CH, and U₄Represents N.

14. A compound of formula I according to claim 1, or a salt thereof, selected from the following:

9-hydroxy-2-methyl-3- (2- (methyl ((S) -3- (naphthalen-1-yloxy) -3- (thiophen-3-yl) propyl) amino) ethyl) -6,7,8, 9-tetrahydropyrido [1,2-a ] pyrimidin-4-one; and

9-hydroxy-2-methyl-3- (2- (methyl ((S) -3- (naphthalen-1-yloxy) -3- (thiophen-2-yl) propyl) amino) ethyl) -6,7,8, 9-tetrahydropyrido [1,2-a ] pyrimidin-4-one.

15. A compound of formula I according to claim 1, or a salt thereof, selected from the following:

3- (2- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperidin-1-yl) benzoyl) piperidin-1-yl) ethyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one;

3- (2- (4- (4-fluoro-2- (4- (6-fluoro-1H-indazol-3-yl) piperidin-1-yl) benzoyl) piperidin-1-yl) ethyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one;

3- (2- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperazin-1-yl) benzoyl) piperidin-1-yl) ethyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one;

3- (2- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperazin-1-yl) benzoyl) piperazin-1-yl) ethyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one;

3- (2- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperidin-1-yl) benzoyl) piperazin-1-yl) ethyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one;

3- (3- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperidin-1-yl) benzoyl) piperazin-1-yl) propyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one;

3- (3- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperazin-1-yl) benzoyl) piperazin-1-yl) propyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one;

3- (3- (4- (4-fluoro-2- (4- (6-fluoro-1H-indazol-3-yl) piperazin-1-yl) benzoyl) piperazin-1-yl) propyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one;

3- (3- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperazin-1-yl) benzoyl) piperidin-1-yl) propyl) -9-hydroxy-2-methyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one;

3- (3- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperazin-1-yl) benzoyl) piperidin-1-yl) propyl) -9-hydroxy-2-phenyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one;

3- (3- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperazin-1-yl) benzoyl) piperazin-1-yl) propyl) -9-hydroxy-2-phenyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one; and

3- (3- (4- (4-fluoro-2- (4- (6-fluorobenzo [ d ] isoxazol-3-yl) piperidin-1-yl) benzoyl) piperazin-1-yl) propyl) -9-hydroxy-2-phenyl-6, 7,8, 9-tetrahydro-4H-pyrido [1,2-a ] pyrimidin-4-one.

16. A pharmaceutical composition comprising, as active ingredient, a compound of formula I as defined in any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.

17. The pharmaceutical composition of claim 16, further comprising at least one additional therapeutic agent.

18. The pharmaceutical composition of claim 17, wherein the at least one additional therapeutic agent is for treating or preventing a tumor.

19. Use of a compound of formula I as defined in any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of tumours.

20. The use of claim 19, wherein the tumor is a malignant tumor.

21. The use of claim 20, wherein the malignancy is selected from: breast cancer, breast cancer lung metastasis, nasopharyngeal carcinoma, cervical cancer, pancreatic cancer, lymphoma, melanoma, liver cancer, and lung cancer.

22. The use of claim 20 or 21, wherein the malignancy is a breast cancer lung metastasis.

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