CN110964034B - Pyrimidothiophene derivative, preparation method and medical application thereof - Google Patents

Abstract

The invention relates to pyrimidothiophene derivatives, a preparation method thereof and application thereof in medicines. Specifically, the invention relates to a pyrimidothiophene derivative shown in a general formula (I), a preparation method thereof, a pharmaceutical composition containing the derivative and application of the derivative as a therapeutic agent, in particular as an MCL-1 inhibitor, wherein each substituent group of the general formula (I) is defined as the same as that in the specification.

Description

Pyrimidothiophene derivative, preparation method and medical application thereof

Technical Field

The invention belongs to the field of medicines, and relates to a pyrimidothiophene derivative shown in a general formula (I), a preparation method thereof, a pharmaceutical composition containing the derivative, and application of the derivative as a therapeutic agent, in particular to an MCL-1 inhibitor.

Background

An important feature of tumor cells from normal cells is that apoptosis is inhibited, giving them a greater survival advantage. Apoptosis, also known as programmed death, can be divided into exogenous apoptosis and endogenous apoptosis. Wherein endogenous apoptosis is an important barrier to the development of cancer. BCL-2 family proteins are important regulators of endogenous apoptosis

BCL-2 family proteins are mainly present on mitochondrial membranes and can be classified into two major classes, anti-apoptotic proteins and pro-apoptotic proteins, according to their function. Anti-apoptotic proteins include BCL-2, BCL-XL, BCL-w and MCL-1. Pro-apoptotic proteins include Bax, Bak and BH3-only proteins. When Bax and Bak are activated, a polymer cavity is formed, which increases the permeability of the mitochondrial membrane of cells, promotes the release of cytochrome C and the like into the cytoplasm, and leads to cell death. The BH3-only protein comprises the BH3 domain only. In the state of cell survival, BH3-only proteins (e.g., Bim) bind to anti-apoptotic proteins. When the cell is stressed, the combination balance is broken, BH3-only protein is released to combine with BAX on mitochondria, BAX/BAK is promoted to form polymer, cytochrome C and SMAC are promoted to be released into cytoplasm, and downstream apoptosis pathway is activated.

Current clinical data indicate that MCL-1 is overexpressed in a variety of tumors, for example, MCL-1 overexpression is detected in 55% of breast cancer and 84% of lung cancer samples. In the multiple myeloma sample, MCL-1 expression is obviously improved along with the increase of the cancer deterioration degree, but BCL-2 expression is not changed. Furthermore, the expression level of MCL-1 is inversely correlated with the survival rate of the patients. High MCL-1 expression was observed with lower survival in both breast cancer and multiple myeloma patients. Therefore, MCL-1 is an important target for tumor treatment.

Novartis, Amgen and AstraZeneca have developed small molecule inhibitors against MCL-1, but are now in clinical stage, so further development of MCL-1 inhibitor drugs is needed.

Disclosure of Invention

The invention aims to provide a compound shown in a general formula (I):

or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof,

wherein:

ring a is heteroaryl or aryl;

R¹is alkyl or deuterated alkyl;

R²the same or different, and each is independently selected from hydrogen atom, halogen, alkyl, deuterated alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino or nitro;

R³is NR⁷R⁸Cycloalkyl or heterocyclyl; wherein said cycloalkyl and heterocyclyl are each independently optionally substituted with one or more substituents selected from the group consisting of halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl;

R⁴selected from the group consisting of halogen, alkyl, deuterated alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein said alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl are each independently optionally substituted with one or more substituents selected from the group consisting of halogen, alkyl, deuterated alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl;

R⁵the same or different, and each is independently selected from hydrogen atom, halogen, alkyl, deuterated alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino or nitro;

R⁶the same or different, and each is independently selected from hydrogen atom, halogen, alkyl, deuterated alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino or nitro;

R⁷and R⁸Are the same or different and are each independently selected from the group consisting of a hydrogen atom, an alkyl group, a deuterated alkyl group, a haloalkyl group, a cycloalkyl group, a heterocyclic group, an aryl group, or a heteroaryl group; wherein said alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each independently optionally substituted with one or more substituents selected from the group consisting of alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl;

or, said R⁷And R⁸Together with the attached nitrogen atom, form a heterocyclic group, wherein said heterocyclic group optionally contains 0 to 2 of the same or different heteroatoms selected from N, O and S in addition to 1 nitrogen atom, and said heterocyclic group is optionally substituted with one or more substituents selected from the group consisting of halogen, alkyl, deuterated alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl;

n is an integer of 1 to 6; preferably 1;

p is an integer from 1 to 6; preferably 2;

m is 0, 1,2 or 3;

s is 0, 1,2, 3 or 4;

t is 0, 1,2, 3 or 4.

In a preferred embodiment of the present invention, the compound represented by the general formula (I) is a compound represented by the general formula (II):

or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof,

wherein

G¹And G²Are the same or different and are each independently CH or N;

R¹、R²、R⁴、R⁷and R⁸As defined in formula (I).

In a preferred embodiment of the present invention, the compound represented by the general formula (I) wherein R is²Is halogen; chlorine is preferred.

In a preferred embodiment of the present invention, the compound represented by the general formula (I) is a compound represented by the general formula (III):

or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof,

wherein

G³Selected from NR⁹、CR¹⁰R¹¹Or O;

R⁹selected from alkyl, deuterated alkyl, haloalkyl or cycloalkyl;

R¹⁰and R¹¹The same or different, and each is independently selected from hydrogen atom, halogen, alkyl, deuterated alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino or nitro;

R¹and R⁴As defined in formula (I).

In a preferred embodiment of the present invention, the compound represented by the general formula (I) wherein R is¹Is C_1-6An alkyl group; preferably methyl.

In a preferred embodiment of the present invention, the compound represented by the general formula (I) wherein R is⁴Is phenyl, wherein said phenyl is optionally substituted with one or more substituents selected from the group consisting of halogen, alkyl, deuterated alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, and nitro.

Typical compounds of the invention include, but are not limited to:

or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention relates to a compound represented by the general formula (IA):

or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof,

wherein:

x is halogen;

R^ais an alkyl group;

ring A, R⁴～R⁶S, t and n are as defined in formula (I).

Typical compounds of formula (IA) of the present invention include, but are not limited to:

or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention relates to a method of preparing a compound of formula (I), the method comprising:

coupling a compound of formula (IA) with a compound of formula (IB) to give a compound of formula (I) wherein:

x is halogen; preferably bromine;

m is

R^aIs an alkyl group; superior foodSelecting ethyl;

ring A, R¹～R⁶S, t, m, p and n are as defined in formula (I).

Another aspect of the present invention relates to a method of preparing a compound represented by the general formula (II), the method comprising:

the compound of the general formula (IIA) and the compound of the general formula (IIB) are subjected to coupling reaction to obtain a compound of a general formula (II),

wherein:

x is halogen; preferably bromine;

m is

R^aIs an alkyl group; preferably ethyl;

G¹、G²、R¹、R²、R⁴、R⁷and R⁸As defined in formula (II).

Another aspect of the present invention relates to a method of preparing a compound of formula (III), comprising:

the compound of the general formula (IIIA) and the compound of the general formula (IIIB) are subjected to coupling reaction to obtain a compound of a general formula (III),

wherein:

x is halogen; preferably bromine;

m is

R^aIs an alkyl group; preferably ethyl;

R¹、R⁴and G³Such as a cartoonAs defined in formula (III).

Another aspect of the present invention relates to a method of preparing compound 1, comprising:

and carrying out coupling reaction on the compound 1g and the compound 1h to obtain the compound 1.

Another aspect of the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) of the present invention, or a tautomer, mesomer, racemate, enantiomer, or diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

The invention further relates to the use of a compound of formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for the manufacture of a medicament for the treatment or prevention of a MCL-1 mediated disease.

The invention further relates to the use of a compound of formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for the preparation of a medicament for inhibiting MCL-1.

The invention further relates to the use of a compound of general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for the preparation of a medicament for the treatment of tumors, autoimmune diseases and immune system disorders; wherein said tumor is preferably selected from the group consisting of bladder cancer, brain tumor, breast cancer, uterine cancer, cervical cancer, endometrial cancer, ovarian cancer, leukemia (such as chronic myeloid leukemia, chronic lymphoid leukemia, lymphoblastic leukemia, or acute myeloid leukemia), renal cancer, colon cancer, rectal cancer, colorectal cancer, esophageal cancer, liver cancer, stomach cancer, head and neck cancer, skin cancer, lymphoma, pancreatic cancer, melanoma, myeloma (such as multiple myeloma), bone cancer, neuroblastoma, glioma, sarcoma, lung cancer (such as non-small cell lung cancer or small cell lung cancer), thyroid cancer, and prostate cancer.

The present invention also relates to a method of inhibiting MCL-1 comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same.

The present invention also relates to a method for treating or preventing MCL-1 mediated diseases, which comprises administering to a patient in need thereof a therapeutically effective amount of a compound represented by the general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same.

The present invention also relates to a method for the treatment of tumors, autoimmune diseases and diseases of the immune system, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising same; wherein said tumor is preferably selected from the group consisting of bladder cancer, brain tumor, breast cancer, uterine cancer, cervical cancer, endometrial cancer, ovarian cancer, leukemia (such as chronic myeloid leukemia, chronic lymphoid leukemia, lymphoblastic leukemia, or acute myeloid leukemia), renal cancer, colon cancer, rectal cancer, colorectal cancer, esophageal cancer, liver cancer, stomach cancer, head and neck cancer, skin cancer, lymphoma, pancreatic cancer, melanoma, myeloma (such as multiple myeloma), bone cancer, neuroblastoma, glioma, sarcoma, lung cancer (such as non-small cell lung cancer or small cell lung cancer), thyroid cancer, and prostate cancer.

The invention further relates to a compound of general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for use as a medicament.

The invention also relates to a compound shown in the general formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer or a mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the compound, which is used as the MCL-1 inhibitor.

The invention also relates to a compound shown in the general formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer or a mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the compound, which treats or prevents MCL-1 mediated diseases.

The invention also relates to a compound shown in the general formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer or a mixture form thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the compound, which is used for treating tumors, autoimmune diseases and immune system diseases; wherein said tumor is preferably selected from the group consisting of bladder cancer, brain tumor, breast cancer, uterine cancer, cervical cancer, endometrial cancer, ovarian cancer, leukemia (such as chronic myeloid leukemia, chronic lymphoid leukemia, lymphoblastic leukemia, or acute myeloid leukemia), renal cancer, colon cancer, rectal cancer, colorectal cancer, esophageal cancer, liver cancer, stomach cancer, head and neck cancer, skin cancer, lymphoma, pancreatic cancer, melanoma, myeloma (such as multiple myeloma), bone cancer, neuroblastoma, glioma, sarcoma, lung cancer (such as non-small cell lung cancer or small cell lung cancer), thyroid cancer, and prostate cancer.

The active compound may be formulated so as to be suitable for administration by any suitable route, preferably in unit dose form, or in such a way that the patient may self-administer it in a single dose. The unit dose of a compound or composition of the invention may be expressed in the form of a tablet, capsule, cachet, bottle, powder, granule, lozenge, suppository, reconstituted powder or liquid.

The dosage of the compound or composition used in the methods of treatment of the present invention will generally vary with the severity of the disease, the weight of the patient and the relative efficacy of the compound. However, as a general guide, a suitable unit dose may be 0.1 to 1000 mg.

The pharmaceutical compositions of the invention may contain, in addition to the active compound, one or more adjuvants selected from the following: fillers (diluents), binders, wetting agents, disintegrants, excipients, and the like. Depending on the method of administration, the compositions may contain from 0.1 to 99% by weight of active compound.

The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Oral compositions may be prepared according to any method known in the art for preparing pharmaceutical compositions, and such compositions may contain one or more ingredients selected from the group consisting of: sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide a pleasant to the eye and palatable pharmaceutical preparation. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. The aqueous suspensions may also contain one or more preservatives, for example ethyl or n-propyl paraben, one or more coloring agents, one or more flavoring agents and one or more sweetening agents.

Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil. The oil suspension may contain a thickener. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable preparation.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent or one or more preservatives. Suitable dispersing or wetting agents and suspending agents are illustrative of the examples given above. Other excipients, for example sweetening, flavoring and coloring agents, may also be present. These compositions are preserved by the addition of an antioxidant such as ascorbic acid.

The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous solution. Among the acceptable vehicles or solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. The sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then treated to form a microemulsion by adding to a mixture of water and glycerol. The injection solution or microemulsion may be injected into the bloodstream of a patient by local bulk injection. Alternatively, it may be desirable to administer the solutions and microemulsions in a manner that maintains a constant circulating concentration of the compounds of the present invention. To maintain such a constant concentration, a continuous intravenous delivery device may be used. An example of such a device is an intravenous pump model Deltec CADD-PLUS. TM.5400.

The pharmaceutical compositions may be in the form of sterile injectable aqueous or oleaginous suspensions for intramuscular and subcutaneous administration. The suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a parenterally-acceptable, non-toxic diluent or solvent. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.

The compounds of the present invention may be administered in the form of suppositories for rectal administration. These pharmaceutical compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid in the rectum and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, glycerogelatin, hydrogenated vegetable oils, polyethylene glycols of various molecular weights and mixtures of fatty acid esters of polyethylene glycols.

As is well known to those skilled in the art, the dosage of a drug administered depends on a variety of factors, including, but not limited to: the activity of the particular compound employed, the age of the patient, the weight of the patient, the health condition of the patient, the behavior of the patient, the diet of the patient, the time of administration, the mode of administration, the rate of excretion, the combination of drugs, and the like; in addition, the optimal treatment regimen, such as the mode of treatment, the daily amount of compound (I) of the formula or the type of pharmaceutically acceptable salt, can be verified according to conventional treatment protocols.

Detailed description of the invention

Unless stated to the contrary, terms used in the specification and claims have the following meanings.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-dimethylpentyl, 2, 4-dimethylpentyl, 2-dimethylpentyl, 3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2, 3-dimethylhexyl, 2, 4-dimethylhexyl, 2, 5-dimethylhexyl, 2-dimethylhexyl, 3-dimethylhexyl, 4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-dimethylpentyl, 2-dimethylhexyl, 3-dimethylpentyl, 2-ethylhexyl, 3-dimethylhexyl, 2-ethylhexyl, 2-dimethylhexyl, 2-ethylhexyl, 2-dimethylhexyl, 2-dimethylhexyl, 2-dimethylhexyl, 2-ethylhexyl, 2-ethyl, 2-2, 2-2, 2-2, or, 2, 2-diethylpentyl, n-decyl, 3-diethylhexyl, 2-diethylhexyl, and various branched isomers thereof. More preferred are lower alkyl groups having 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl and the like. The alkyl group may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, preferably independently optionally substituted with one or more substituents selected from H atom, D atom, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl.

The term "alkoxy" refers to-O- (alkyl) and-O- (unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. The alkoxy group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more groups substituted with one or more substituents independently selected from H atom, D atom, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing from 3 to 20 carbon atoms, preferably from 3 to 12 carbon atoms, preferably from 3 to 10 carbon atoms, more preferably from 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like; polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups. Cycloalkyl groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, preferably independently optionally substituted with one or more substituents selected from hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl.

The term "heterocyclyl" means saturated or partially unsaturatedSaturated monocyclic or polycyclic cyclic hydrocarbon substituents containing from 3 to 20 ring atoms, wherein one or more of the ring atoms is selected from nitrogen, oxygen or S (O)_m(wherein m is an integer from 0 to 2) but excludes the ring moiety of-O-O-, -O-S-, or-S-S-, the remaining ring atoms being carbon. Preferably 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably 3 to 10 ring atoms, of which 1-4 is a heteroatom; more preferably from 5 to 6 ring atoms; of which 1 to 3 are heteroatoms. Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, tetrahydropyranyl, 1, 2.3.6-tetrahydropyridinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclic groups include spiro, fused and bridged heterocyclic groups.

The heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring to which the parent structure is attached is heterocyclyl, non-limiting examples of which include:

the heterocyclyl group may be substituted or unsubstituted and when substituted, the substituents may be substituted at any available point of attachment, preferably independently optionally substituted with one or more substituents selected from hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl.

The term "aryl" refers to a 6 to 14 membered all carbon monocyclic or fused polycyclic (i.e., rings which share adjacent pairs of carbon atoms) group having a conjugated pi-electron system, preferably 6 to 10 membered, such as phenyl and naphthyl. The aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring, non-limiting examples of which include:

aryl groups may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, preferably independently optionally substituted with one or more substituents selected from hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl.

The term "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. Heteroaryl is preferably 5 to 10 membered, more preferably 5 or 6 membered, for example furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring joined together with the parent structure is a heteroaryl ring, non-limiting examples of which include:

heteroaryl groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, preferably independently optionally substituted with one or more substituents selected from hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl.

The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

The term "deuterated alkyl" refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.

The term "hydroxy" refers to an-OH group.

The term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group, wherein alkyl is as defined above.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "hydroxy" refers to an-OH group.

The term "amino" refers to the group-NH₂。

The term "cyano" refers to — CN.

The term "nitro" means-NO₂。

The term "carbonyl" refers to C ═ O.

The term "carboxy" refers to-C (O) OH.

The term "carboxylate" refers to-C (O) O (alkyl) or-C (O) O (cycloalkyl), wherein alkyl, cycloalkyl are as defined above.

The invention also includes various deuterated forms of the compounds of formula (I). Each available hydrogen atom attached to a carbon atom may be independently replaced by a deuterium atom. The person skilled in the art is able to synthesize the deuterated forms of the compounds of the formula (I) with reference to the relevant literature. Commercially available deuterated starting materials can be used in preparing the deuterated forms of the compounds of formula (I), or they can be synthesized using conventional techniques using deuterated reagents including, but not limited to, deuterated boranes, trideuteroborane tetrahydrofuran solutions, deuterated lithium aluminum hydrides, deuterated iodoethanes, deuterated iodomethanes, and the like.

"optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "a heterocyclic group optionally substituted with an alkyl" means that an alkyl may, but need not, be present, and the description includes the case where the heterocyclic group is substituted with an alkyl and the heterocyclic group is not substituted with an alkyl.

"substituted" means that one or more, preferably up to 5, more preferably 1 to 3, hydrogen atoms in the group are independently substituted with a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and that the person skilled in the art is able to determine (experimentally or theoretically) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups having free hydrogen may be unstable in combination with carbon atoms having unsaturated (e.g., olefinic) bonds.

"pharmaceutical composition" means a mixture containing one or more compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof in admixture with other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

"pharmaceutically acceptable salts" refers to salts of the compounds of the present invention which are safe and effective for use in the body of a mammal and which possess the requisite biological activity.

Synthesis of the Compounds of the invention

In order to achieve the purpose of the invention, the invention adopts the following technical scheme:

scheme one

The invention relates to a method for preparing a compound shown as a general formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereoisomer or a mixture form thereof, or a pharmaceutically acceptable salt form thereof, which comprises the following steps:

carrying out coupling reaction on a compound of a general formula (IA) and a compound of a general formula (IB) in the presence of a catalyst under alkaline conditions to obtain a compound of a general formula (I);

wherein:

x is halogen; preferably bromine;

m is

R^aIs an alkyl group; preferably ethyl;

ring A, R¹～R⁶S, t, m, p and n are as defined in formula (I).

The reagents that provide basic conditions include organic bases including, but not limited to, triethylamine, N-diisopropylethylamine, N-butyllithium, lithium diisopropylamide, lithium bistrimethylsilylamide, potassium acetate, sodium t-butoxide, potassium t-butoxide, and sodium N-butoxide, and inorganic bases including, but not limited to, sodium bicarbonate, potassium bicarbonate, sodium hydride, potassium phosphate, sodium carbonate, potassium acetate, cesium carbonate, sodium hydroxide, and lithium hydroxide; preferably cesium carbonate;

such catalysts include, but are not limited to, palladium on carbon, tetrakis-triphenylphosphine palladium, palladium dichloride, palladium acetate, dichlorobis [ di-tert-butyl- (4-dimethylaminophenyl) phosphine ] palladium (II), bis (dibenzylideneacetone) palladium, chloro (2-dicyclohexylphosphino-2 ',4',6 '-triisopropyl-1, 1' -biphenyl) [2- (2 '-amino-1, 1' -biphenyl) ] palladium, [1,1 '-bis (diphenylphosphino) ferrocene ] dichloropalladium or tris (dibenzylideneacetone) dipalladium, preferably dichlorobis [ di-tert-butyl- (4-dimethylaminophenyl) phosphine ] palladium (II), [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium;

the above reaction is preferably carried out in a solvent including, but not limited to: acetic acid, methanol, ethanol, N-butanol, tert-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, N-hexane, dimethyl sulfoxide, 1, 4-dioxane, ethylene glycol dimethyl ether, water or N, N-dimethylformamide, and mixtures thereof.

Scheme two

The invention relates to a method for preparing a compound shown as a general formula (II) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer or a mixture thereof, or a pharmaceutically acceptable salt thereof, which comprises the following steps:

reacting a compound of a general formula (IIA) with a compound of a general formula (IIB) in the presence of a catalyst under an alkaline condition to obtain a compound of a general formula (II);

wherein:

x is halogen; preferably bromine;

m is

R^aIs an alkyl group; preferably ethyl;

G¹、G²、R¹、R²、R⁴、R⁷and R⁸As defined in formula (II).

The reagents that provide basic conditions include organic bases including, but not limited to, triethylamine, N-diisopropylethylamine, N-butyllithium, lithium diisopropylamide, lithium bistrimethylsilylamide, potassium acetate, sodium t-butoxide, potassium t-butoxide, and sodium N-butoxide, and inorganic bases including, but not limited to, sodium bicarbonate, potassium bicarbonate, sodium hydride, potassium phosphate, sodium carbonate, potassium acetate, cesium carbonate, sodium hydroxide, and lithium hydroxide; preferably cesium carbonate;

such catalysts include, but are not limited to, palladium on carbon, tetrakis-triphenylphosphine palladium, palladium dichloride, palladium acetate, dichlorobis [ di-tert-butyl- (4-dimethylaminophenyl) phosphine ] palladium (II), bis (dibenzylideneacetone) palladium, chloro (2-dicyclohexylphosphino-2 ',4',6 '-triisopropyl-1, 1' -biphenyl) [2- (2 '-amino-1, 1' -biphenyl) ] palladium, [1,1 '-bis (diphenylphosphino) ferrocene ] dichloropalladium or tris (dibenzylideneacetone) dipalladium, preferably dichlorobis [ di-tert-butyl- (4-dimethylaminophenyl) phosphine ] palladium (II), [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium;

the above reaction is preferably carried out in a solvent including, but not limited to: acetic acid, methanol, ethanol, N-butanol, tert-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, N-hexane, dimethyl sulfoxide, 1, 4-dioxane, ethylene glycol dimethyl ether, water or N, N-dimethylformamide, and mixtures thereof.

Scheme three

The invention relates to a method for preparing a compound shown as a general formula (III) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer or a mixture thereof, or a pharmaceutically acceptable salt thereof, which comprises the following steps:

reacting a compound of a general formula (IIIA) with a compound of a general formula (IIIB) in the presence of a catalyst under an alkaline condition to obtain a compound of a general formula (III),

wherein:

x is halogen; preferably bromine;

m is

R^aIs an alkyl group; preferably ethyl;

R¹、R⁴and G³As defined in formula (III).

The reagents that provide basic conditions include organic bases including, but not limited to, triethylamine, N-diisopropylethylamine, N-butyllithium, lithium diisopropylamide, lithium bistrimethylsilylamide, potassium acetate, sodium t-butoxide, potassium t-butoxide, and sodium N-butoxide, and inorganic bases including, but not limited to, sodium bicarbonate, potassium bicarbonate, sodium hydride, potassium phosphate, sodium carbonate, potassium acetate, cesium carbonate, sodium hydroxide, and lithium hydroxide; preferably cesium carbonate;

such catalysts include, but are not limited to, palladium on carbon, tetrakis-triphenylphosphine palladium, palladium dichloride, palladium acetate, dichlorobis [ di-tert-butyl- (4-dimethylaminophenyl) phosphine ] palladium (II), bis (dibenzylideneacetone) palladium, chloro (2-dicyclohexylphosphino-2 ',4',6 '-triisopropyl-1, 1' -biphenyl) [2- (2 '-amino-1, 1' -biphenyl) ] palladium, [1,1 '-bis (diphenylphosphino) ferrocene ] dichloropalladium or tris (dibenzylideneacetone) dipalladium, preferably dichlorobis [ di-tert-butyl- (4-dimethylaminophenyl) phosphine ] palladium (II), [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium;

the above reaction is preferably carried out in a solvent including, but not limited to: acetic acid, methanol, ethanol, N-butanol, tert-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, N-hexane, dimethyl sulfoxide, 1, 4-dioxane, ethylene glycol dimethyl ether, water or N, N-dimethylformamide, and mixtures thereof.

Scheme four

A process for the preparation of a compound of compound 1 of the present invention, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, comprising the steps of:

compound 1g and compound 1h are reacted in the presence of a catalyst, preferably dichlorobis [ di-tert-butyl- (4-dimethylaminophenyl) phosphine ] palladium (II), under basic conditions (cesium carbonate) to give compound 1.

The reagents that provide basic conditions include organic bases including, but not limited to, triethylamine, N-diisopropylethylamine, N-butyllithium, lithium diisopropylamide, lithium bistrimethylsilylamide, potassium acetate, sodium t-butoxide, potassium t-butoxide, and sodium N-butoxide, and inorganic bases including, but not limited to, sodium bicarbonate, potassium bicarbonate, sodium hydride, potassium phosphate, sodium carbonate, potassium acetate, cesium carbonate, sodium hydroxide, and lithium hydroxide; preferably cesium carbonate;

such catalysts include, but are not limited to, palladium on carbon, tetrakis-triphenylphosphine palladium, palladium dichloride, palladium acetate, dichlorobis [ di-tert-butyl- (4-dimethylaminophenyl) phosphine ] palladium (II), bis (dibenzylideneacetone) palladium, chloro (2-dicyclohexylphosphino-2 ',4',6 '-triisopropyl-1, 1' -biphenyl) [2- (2 '-amino-1, 1' -biphenyl) ] palladium, [1,1 '-bis (diphenylphosphino) ferrocene ] dichloropalladium or tris (dibenzylideneacetone) dipalladium, preferably dichlorobis [ di-tert-butyl- (4-dimethylaminophenyl) phosphine ] palladium (II), [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium;

the above reaction is preferably carried out in a solvent including, but not limited to: acetic acid, methanol, ethanol, N-butanol, tert-butanol, toluene, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, N-hexane, dimethyl sulfoxide, 1, 4-dioxane, ethylene glycol dimethyl ether, water or N, N-dimethylformamide, and mixtures thereof.

Detailed Description

The present invention will be further described with reference to the following examples, which are not intended to limit the scope of the present invention.

Examples

The structure of the compounds is determined by Nuclear Magnetic Resonance (NMR) or Mass Spectrometry (MS). NMR was measured using a Bruker AVANCE-400 NMR spectrometer using deuterated dimethyl sulfoxide (DMSO-d6) and deuterated chloroform (CDCl)₃) Deuterated methanol (CD)₃OD), internal standard Tetramethylsilane (TMS), chemical shift is 10^-6(ppm) is given as a unit.

MS was determined using a FINNIGAN LCQAD (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).

HPLC was carried out using an Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18150X 4.6mm column) and a Waters 2695-2996 high pressure liquid chromatograph (Gimini C18150X 4.6mm column).

High Performance liquid preparation A preparative chromatograph was used from Waters2767, Waters 2767-SQ Detector 2, Shimadzu LC-20AP and Gilson-281.

The thin layer chromatography silica gel plate adopts HSGF254 of tobacco yellow sea or GF254 of Qingdao, the specification of the silica gel plate used by Thin Layer Chromatography (TLC) is 0.15 mm-0.2 mm, and the specification of the thin layer chromatography separation and purification product is 0.4 mm-0.5 mm.

The column chromatography generally uses 200-300 mesh silica gel of Taiwan yellow sea as a carrier.

Known starting materials of the present invention can be synthesized by or according to methods known in the art, or can be purchased from companies such as ABCR GmbH & Co.KG, Acros Organnics, Aldrich Chemical Company, Shao Yuan Chemical technology (Accela ChemBio Inc), Darri Chemicals, and the like.

In the examples, the reaction was carried out under an argon atmosphere or a nitrogen atmosphere unless otherwise specified.

An argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to a balloon of argon or nitrogen with a volume of about 1L.

The hydrogen atmosphere refers to a reaction flask connected with a hydrogen balloon with a volume of about 1L.

The pressure hydrogenation reaction used a hydrogenation apparatus of Parr 3916EKX type and a hydrogen generator of Qinglan QL-500 type or a hydrogenation apparatus of HC2-SS type.

The hydrogenation reaction was usually evacuated and charged with hydrogen and repeated 3 times.

The microwave reaction was carried out using a CEM Discover-S908860 type microwave reactor.

In the examples, the solution in the reaction is an aqueous solution unless otherwise specified.

In the examples, the reaction temperature was room temperature unless otherwise specified.

The room temperature is the optimum reaction temperature, and the temperature range is 20-30 ℃.

The progress of the reaction in the examples was monitored by Thin Layer Chromatography (TLC) using a developing solvent system of: a: dichloromethane and methanol system, B: n-hexane and ethyl acetate, and the volume ratio of the solvent is adjusted according to the polarity of the compound.

The system of eluents for column chromatography and developing agents for thin layer chromatography used for purifying compounds include: a: dichloromethane and methanol system, B: the volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of triethylamine, an acidic or basic reagent and the like can be added for adjustment.

Example 1

(R) -2- (((S) -6- (bicyclo [4.2.0] octan-1 (6),2, 4-trien-3-yl) -5- (3-chloro-2-methyl-4- (2- (4-methylpiperazin-1-yl) ethoxy) phenyl) thieno [2,3-d ] pyrimidin-4-yl) oxy) -3- (2- ((2- (2-methoxyphenyl) pyrimidin-4-yl) methoxy) phenyl) propanoic acid 1

First step of

2- (bicyclo [4.2.0] octan-1 (6),2, 4-trien-3-yl) -4,4,5, 5-tetramethyl-1, 3, 2-dioxaborole cyclopentane 1c

4-4-bromo-1, 2-dihydrobutylbenzene 1a (1.00g, 5.46mmol, available from Pebax), pinacol diborate 1b (1.67g, 6.58mmol), [1,1 '-bis (diphenylphosphino) ferrocene ] dichloropalladium (400mg, 0.55mmol), 1,1' -bis (diphenylphosphino) ferrocene (400mg, 0.55mmol) and potassium acetate (1.61g, 16.41mmol) were dissolved in 20mL dioxane, displaced with argon three times, and the reaction solution was heated to 90 ℃ under argon atmosphere and stirred for reaction for 16 hours. The reaction was cooled to room temperature, concentrated under reduced pressure to remove the solvent, and the resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 1c (1.10g), yield: 87 percent.

¹H NMR(400MHz,CDCl₃):δ7.71(d,1H),7.53(s,1H),7.10(d,1H),3.22(s,4H),1.38(s,12H).

Second step of

6- (bicyclo [4.2.0] oct-1 (6),2, 4-trien-3-yl) -5-bromo-4-chlorothieno [2,3-d ] pyrimidine 1e

1c (730mg,3.17mmol), 5-bromo-4-chloro-6-iodothieno [2,3-d ] pyrimidine 1d (1.00g,2.66mmol, prepared by the method disclosed in patent application "WO 201715224"), [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (200mg, 0.27mmol) and cesium carbonate (2.60g, 7.98mmol) were dissolved in 12mL of a mixed solution of dioxane and water (V: V ═ 5:1), replaced with argon three times, and the reaction solution was heated to 90 ℃ under an argon atmosphere and stirred for reaction for 3.0 hours. The reaction solution was cooled to room temperature, the solvent was removed by concentration under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 1e (420mg), yield: 44 percent.

MS m/z(ESI):352.9[M+1]

The third step

(R) -2- ((6- (bicyclo [4.2.0] oct-1 (6),2, 4-trien-3-yl) -5-bromothieno [2,3-d ] pyrimidin-4-yl) oxygen

1g of ethyl 3- (2- ((2- (2-methoxyphenyl) pyrimidin-4-yl) methoxy) phenyl) propionate

Ethyl 1e (360mg, 1.02mmol), (R) -2-hydroxy-3- (2- ((2- (2-methoxyphenyl) pyrimidin-4-yl) methoxy) phenyl) propanoate, 1f (390mg, 1.02mmol, prepared by the method disclosed in the patent application "WO 2016207225"), and cesium carbonate (500mg, 1.50mmol) were dissolved in a 25mL round-bottomed flask, 5.0mL of t-butanol was added, and the reaction was heated to 36 ℃ and stirred for 16 hours. The reaction solution was cooled to room temperature, filtered, the filter cake was washed with ethyl acetate, the filtrate was concentrated under reduced pressure to remove the solvent, and the resulting residue was purified by silica gel column chromatography with eluent system B to obtain the title product 1g (130mg), yield: 18 percent.

MS m/z(ESI):723.2[M+1]

The fourth step

(R) -2- (((S) -6- (bicyclo [4.2.0] octan-1 (6),2, 4-trien-3-yl) -5- (3-chloro-2-methyl-4- (2- (4-methylpiperazin-1-yl) ethoxy) phenyl) thieno [2,3-d ] pyrimidin-4-yl) oxy) -3- (2- ((2- (2-methoxyphenyl) pyrimidin-4-yl) methoxy) phenyl) propanoic acid 1

1g (100mg, 0.14mmol), 1- (2- (2-chloro-3-methyl-4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenoxy) ethyl) -4-methylpiperazine 1h (80mg, 0.20mmol, prepared by the method disclosed in patent application "WO 201715224"), cesium carbonate (130mg,0.40mmol) and dichlorobis [ di-tert-butyl- (4-dimethylaminophenyl) phosphine ] palladium (II) (10mg,0.01mmol) were dissolved in 3.0mL dioxane and 0.6mL water, replaced with argon three times, and the reaction solution was heated to 60 ℃ under an argon atmosphere and stirred for 3.0 hours. The reaction solution was cooled to room temperature, lithium hydroxide monohydrate (100mg,2.38mmol) was added to the reaction solution, the reaction was stirred for 30 minutes, the solvent was removed by concentration under reduced pressure, and the obtained residue was purified by high performance liquid chromatography (waters2767, elution system: 10mmol/L ammonium acetate, water, acetonitrile) to obtain the title product 1(30mg), yield: 24 percent.

MS m/z(ESI):883.1[M+1]

¹H NMR(400MHz,CD₃OD):δ8.87(d,1H),8.46(s,1H),7.94(d,1H),7.62-7.58(m,2H),7.49(dd,1H),7.16(d,1H),7.14-7.06(m,4H),6.94-6.87(m,3H),7.73(dd,1H),6.27(d,1H),5.62-5.60(m,1H),5.29-5.20(m,2H),4.26(s,2H),3.84(s,3H),3.48(d,1H),3.36(d,1H),3.13-3.10(m,4H),2.93-2.86(m,9H),2.60(s,4H),1.79(s,3H).

Test example:

biological evaluation

Test example 1 binding assay of the compounds of the present invention to MCL-1 protein.

The following method was used to determine the binding ability of the compounds of the present invention to MCL-1 protein. The experimental method is briefly described as follows:

first, experimental material and instrument

His-MCL-1 protein (Shanghai Hengrui medicine Co., Ltd., NA)

2. Biotin-labeled Bim protein (R & D, 3526/1)

3. Europium cryptate-labeled anti-6 His antibody (cisbio, 61HI2KLA)

4. Affinity streptomycin linkage XL665(cisbio, 611SAXLA)

5. Binding buffer (cisbio, 62DLBDDF)

6. Detection buffer (cisbio, 62DB1FDG)

7. Enzyme mark instrument (BMG, PHERASta)

Second, the experimental procedure

MCL-1 inhibitors bind to the MCL-1 protein to prevent binding of MCL-1 to the Bim protein. This experiment evaluates the binding capacity of MCL-1 inhibitors to the MCL-1 protein by detecting the binding of MCL-1 and Bim proteins by means of HTRF, and the activity of the compounds is evaluated on the basis of the Ki size.

His and biotin are respectively marked on the peptide segments of the human recombinant protein MCL-1 (sequence 171-327) and Bim (sequence 51-76). 0.1nM His-MCL-1, 2.5nM bio-Bim and different concentrations of small molecule compounds (first 10uM, 3 fold gradient diluted 11 concentrations in binding buffer) were mixed and incubated at room temperature for 2 hours, followed by addition of 0.5nM europium cryptate anti-6 His antibody and 1.25nM affinity streptomycin linked to 665 XL (diluted in assay buffer). After 2 hours incubation at room temperature, fluorescence signals at 620nm and 665nm were detected using PHERAStar. Data were processed using GraphPad software.

Third, experimental data

The binding capacity of the compounds of the invention to the MCL-1 protein can be determined by the above assay and the Ki values determined are shown in Table 1.

Table 1 Ki binding of the compounds of the invention to the MCL-1 protein.

Example numbering	Ki/nM	Max Inhibition(％)
			1	1.1	94

And (4) conclusion: the compound has stronger binding capacity with MCL-1 protein, and can well inhibit the binding of MCL-1 and Bim protein.

Test example 2 cell proliferation test

The following method was performed by measuring intracellular ATP content based on IC₅₀Size the inhibitory effect of the compounds of the present application on AMO-1, NCI-H929, MOLP-8 and MV-4-11 cell proliferation was evaluated. The experimental method is briefly described as follows:

first, experimental material and instrument

AMO-1, human bone marrow plasmacytoma (Nanjing Kebai, CBP60242)

NCI-H929, human myeloma cells (ATCC, CRL-9068)

MV-4-11, human acute monocytic leukemia cells (ATCC, CRL-9591)

MOLP-8, human multiple myeloma cells (Nanjing Kebai, CBP60562)

5. Fetal bovine serum (GIBCO,10099)

6.RPMI1640(GE，SH30809.01)

7.IMDM(Gibco，12440053)

8.2-mercaptoethanol (sigma, 60-24-2)

9.CellTite(Promega，G7573)

10.96 well cell culture plate (corning, 3903)

11. Trypan blue solution (Sigma, T8154-100ML)

12. Enzyme mark instrument (BMG, PHERASta)

13. Cell counter (Shanghai Rui Yu biological science and technology Co., Ltd., IC1000)

Second, the experimental procedure

AMO-1 and molp-8 cells are cultured in RPMI1640 medium containing 20% FBS, NCI-H929 cells are cultured in RPMI1640 medium containing 10% FBS and 2-mercaptoethanol, MV-4-11 cells are cultured in IMDM medium containing 10% FBS for 2-3 passages in a week, and the passage ratio is 1:4 or 1: 6. During passage, the cells are transferred into a centrifuge tube, centrifuged at 1200rpm for 3 minutes, the residual liquid of the supernatant culture medium is discarded, and fresh culture medium is added to resuspend the cells. 90 μ L of cell suspension was added to a 96-well cell culture plate at a density of 1.33X 10⁵Cells/ml, only 100. mu.L of complete medium was added to the periphery of the 96-well plate. The plates were incubated in an incubator for 24 hours (37 ℃, 5% CO)₂)。

The test samples were diluted to 20mM in DMSO and sequentially diluted 4-fold to 9 concentrations, and blank and control wells were set. mu.L of the test compound solution prepared to a gradient concentration was added to 95. mu.L of fresh medium. Then, 10. mu.L of the above-mentioned drug-containing medium solution was added to the plate. The plates were incubated in an incubator for 3 days (37 ℃, 5% CO)₂). 50 μ L of CellTiter-Glo reagent was added to each well of a 96-well cell culture plate, and left at room temperature in the dark for 5-10min, and the chemiluminescent signal values were read in the PHERAStar and the data were processed using GraphPad software.

Third, experimental data

Inhibition of AMO-1, NCI-H929, MOLP-8 and MV-4-11 cell proliferation by the compounds of the present invention can be carried out by the above assayDetermination of the measured IC₅₀The values are shown in Table 2.

TABLE 2 IC of the compounds of the invention for inhibition of AMO-1, NCI-H929, MOLP-8 and MV-4-11 cell proliferation₅₀The value is obtained.

And (4) conclusion: the compound has good cell proliferation inhibition effect on AMO-1, NCI-H929, MOLP-8 and MV-4-11.

Claims (14)

1.A compound of the general formula (III):

or a pharmaceutically acceptable salt thereof,

wherein:

G³selected from NR⁹、CR¹⁰R¹¹And O;

R¹is C_1-6Alkyl or deuterated C_1-6An alkyl group;

R⁴is 6-to 10-membered aryl or 5-to 10-membered heteroaryl, wherein said 6-to 10-membered aryl and 5-to 10-membered heteroaryl are each independently optionally selected from halogen, C_1-6Alkyl, deuterated C_1-6Alkyl radical, C_1-6Alkoxy and halo C_1-6Substituted by one or more substituents in the alkyl group;

R⁹is selected from C_1-6Alkyl, deuterated C_1-6Alkyl, halo C_1-6Alkyl and 3 to 6 membered cycloalkyl;

R¹⁰and R¹¹Are the same or different and are each independently selected from the group consisting of hydrogen, halogen, C_1-6Alkyl, deuterated C_1-6Alkyl radical, C_1-6Alkoxy and halo C_1-6An alkyl group.

2. A compound of formula (III) according to claim 1, which isR described in (1)¹Is C_1-6An alkyl group.

3. A compound of formula (III) according to claim 2, wherein R is¹Is methyl.

4. A compound of formula (III) according to any one of claims 1 to 3, wherein R is⁴Is phenyl, wherein said phenyl is optionally selected from halogen, C_1-6Alkyl, deuterated C_1-6Alkyl radical, C_1-6Alkoxy and halo C_1-6Alkyl is substituted by one or more substituents.

5. A compound of general formula (III) according to any one of claims 1 to 3, selected from:

6. a compound of formula (IIIA):

or a pharmaceutically acceptable salt thereof,

wherein:

x is halogen;

R^ais C_1-6An alkyl group;

R⁴as defined in claim 1.

7. A compound of formula (IIIA) according to claim 6, selected from:

8. a process for the preparation of a compound of formula (III) according to claim 1, which process comprises:

the compound of the general formula (IIIA) and the compound of the general formula (IIIB) are subjected to coupling reaction to obtain a compound of a general formula (III),

wherein:

x is halogen;

m is

R^aIs C_1-6An alkyl group;

R¹、R⁴and G³As defined in claim 1.

9. A process for preparing compound 1 according to claim 5, comprising:

and carrying out coupling reaction on the compound 1g and the compound 1h to obtain the compound 1.

10. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (III) according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

11. Use of a compound of formula (III) according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 10 for the preparation of a medicament for inhibiting MCL-1.

12. Use of a compound of formula (III) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 10 for the preparation of a medicament for the treatment or prevention of a MCL-1 mediated disease.

13. Use of a compound of formula (III) according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 10 for the preparation of a medicament for the treatment of tumors, autoimmune diseases and immune system disorders.

14. The use according to claim 13, wherein the tumor is selected from the group consisting of bladder cancer, brain tumor, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, leukemia, kidney cancer, colorectal cancer, esophageal cancer, liver cancer, gastric cancer, head and neck cancer, skin cancer, lymphoma, pancreatic cancer, melanoma, myeloma, bone cancer, neuroblastoma, glioma, sarcoma, lung cancer, thyroid cancer, and prostate cancer.