CN112574255A - Organic arsine-based CDK inhibitor and preparation method and application thereof - Google Patents

Abstract

The invention provides an organic arsine-based CDK inhibitor and a preparation method and application thereof. Specifically, provided are compounds of formula I, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof; the preparation method and the application thereof, and the definition of each group in the formula is shown in the specification.

Description

Organic arsine-based CDK inhibitor and preparation method and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a cell Cycle Dependent Kinase (CDK) inhibitor based on organic arsine, and a preparation method and application thereof.

Background

During the last twenty years, while chemotherapy drugs have been widely used in cancer research, the development and application of targeted drugs has been active. Among the various proteins closely related to cancer development and progression, many members of the kinase protein family have been validated as effective therapeutic targets, and tens of targeted kinase inhibitors have also been approved for cancer therapy.

In 2014, Gray et al reported that the first covalent inhibitor THZ1 of CDK7 was developed based on the CDK7 kinase domain ATP binding to the extra-pocket, cysteine residue Cys312 on the C-terminal motif, using substituted acrylamide as electrophilic group. In addition to inhibiting CDK7, THZ1 also inhibits CDK12/13 with similar cysteine residues. Through the inhibition of CDK7/12/13, THZ1 can effectively inhibit the activation of RNA polymerase II under low concentration, further inhibit the transcription initiation of a super regulator in cancer cells, finally effectively inhibit the growth of the cancer cells, and show good curative effect in various tumor models such as T-cell acute lymphoblastic leukemia (T-ALL), Small Cell Lung Cancer (SCLC), neuroblastoma, triple negative breast cancer and the like. However, the off-target effect of THZ1 is significant and its pharmacokinetic properties are also not ideal.

In 2016, Gray et al developed a highly specific inhibitor THZ531 of CDK12/13 based on THZ1, which specifically induced apoptosis in some tumor cells, but still had pharmacokinetic drawbacks including, for example, low bioavailability, short half-life, etc.

Syros corporation improved upon THZ1 to obtain a more specific SY-1365, currently in the first phase of clinical trials for the treatment of solid tumors. The limitation of the pharmacological property allows SY-1365 to be administered by intravenous injection.

Therefore, there is an urgent need in the art to provide CDK inhibitors with high selectivity, safety, and/or better pharmacokinetic properties.

Disclosure of Invention

It is an object of the present invention to provide CDK inhibitors with high selectivity, safety and/or better pharmacokinetic properties. The inhibitors of the invention are a class of organic arsine based CDK inhibitors.

In a first aspect of the invention, there is provided a compound of formula I, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof;

wherein the content of the first and second substances,

X₁、X₂each independently selected from the group consisting of: none, O, S, NR₈、CH₂；

R₈Selected from the group consisting of: H. substituted or unsubstituted C1-C6 alkyl;

R₁、R₂each independently selected from the group consisting of: H. substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, -CO-Rd, wherein Rd is substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl; or R₁、R₂To adjacent X₁、X₂And As together form a substituted or unsubstituted 4 to 8 membered heterocyclic ring containing one As heteroatom and 0-3 heteroatoms selected from N, O and S;

R₃is H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C3-C10 cycloalkyl;

n₁is 0, 1, 2, 3 or 4;

each R is₄Independently selected from: H. d, OH, amino, nitro, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C3-C10 cycloalkyl;

L₁、L₂and L₃Each independently selected from the group consisting of: no, - (Z) m-; and L is₁And L₂Is not absent at the same time; wherein each Z is independently selected from: C1-C6 alkylene, -NR₆-,-NR₆-R₇-, -O-, -CO-, m is 1,2.3 or 4;

each R₆Independently selected from the group consisting of: H. substituted or unsubstituted C1-C4 alkyl;

R₇is a substituted or unsubstituted C1-C8 alkylene group;

a is selected from the following group: a non-substituted, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-10 membered heteroaryl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl;

b is selected from the following group: substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-10 membered heteroaryl;

c is selected from the following group: H. OH, -N (Ra) Rb, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-10 membered heteroaryl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl;

unless otherwise specified, "substituted" means that a hydrogen atom on a group is replaced with one or more (e.g., 2, 3, 4, etc.) substituents selected from the group consisting of: halogen, deuteration, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy, C1-C6 alkyl S (═ O)₂-, oxo (═ O), -CN, -OH, -n (ra) Rb, carboxyl, or a substituted or unsubstituted group selected from: C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 amino, C6-C10 aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, 5-12 membered heterocyclyl having 1-3 heteroatoms selected from N, S and O, - (CH) 6 alkyl₂) -C6-C10 aryl, - (CH)₂) - (5-to 10-membered heteroaryl having 1 to 3 heteroatoms selected from N, S and O), and the substituents are selected from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkynyl, C1-C6 alkoxy, oxo, -CN, -NH₂OH, -OH, C6-C10 aryl, C1-C6 amino, C2-C6 amido, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O;

wherein Ra and Rb are each independently selected from the group consisting of: H. substituted or unsubstituted C1-C4 alkyl; or Ra, Rb together with the nitrogen atom to which they are attached form a substituted or unsubstituted 3-10 membered heterocycloalkyl having at least 1 (e.g., 1, 2, 3) N heteroatom and 0-2 heteroatoms selected from O, S.

In another preferred embodiment, for R₃Said substituted substitution refers to having one or more substituents selected from group a: halogen, C1-C4 alkyl, C1-C4 haloalkyl, C3-C6 cycloalkyl, -OH, -N (Ra) Rb.

In another preferred embodiment, R₁、R₂Together form a substituted or unsubstituted- (CH)₂)n₂-a structure; wherein n is₂Is 1, 2 or 3.

In a further preferred embodiment of the method,

at position 2, 3 or 4.

In another preferred embodiment, the compound of formula I has the structure shown in I-a:

in a further preferred embodiment of the method,

is composed of

In another preferred embodiment, in the compound, R₁、R₂、R₃、R₄、n₁、L₁、L₂、L₃Any of A, B and C are each a group corresponding to the particular compound set forth in Table 1.

In another preferred embodiment, the compound of formula I is selected from the following table:

in a second aspect of the invention, there is provided a pharmaceutical composition comprising;

(a) a therapeutically effective amount of a compound of formula I as described in the first aspect of the invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof; and (b) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition further comprises one or more anticancer agents and/or immunosuppressive agents, preferably selected from the group consisting of: PARP1/2 inhibitor, chemotherapeutic agent for inducing DNA damage of cancer cells, DNA alkylating chemotherapeutic drug, DNA or RNA synthesis inhibitor, EGFR, ALK or FGFR tyrosine receptor kinase inhibitor, KRAS, MEK or ERK tumor signaling pathway inhibitor, tumor immunotherapy drug (such as PD-1 antibody, PD-L)₁Antibodies, etc.).

In another preferred embodiment, the pharmaceutical composition further comprises one or more anticancer agents and/or immunosuppressive agents, preferably selected from the group consisting of: olaparib, Lucapenib, Nilaparib, methotrexate, capecitabine, gemcitabine, doxifluridine, pemetrexed disodium, pazopanib, imatinib, erlotinib, lapatinib, gefitinib, vandetanib, herceptin, bevacizumab, rituximab, trastuzumab, paclitaxel, vinorelbine, docetaxel, doxorubicin, hydroxycamptothecin, mitomycin, epirubicin, pirarubicin, bleomycin, letrozole, tamoxifen, fulvestrant, triptorelin, flutamide, leuprolide, anastrozole, ifosfamide, busulfan, cyclophosphamide, carmustine, nimustine, semustine, mechlorethamine, maflange, oncoclonine, carboplatin, cisplatin, oxaliplatin, carboplatin, topotecan, camptothecin, topotecan, epirubicin, sirolimus, cerine, cerotide, 6-mercaptopurine, thioprine, 6-mercaptopurine, leupeptaib, 6-thioguanine, azathioprine, rhzomorph D, daunorubicin, doxorubicin, mitoxantrone, bleomycin, plicamycin, or aminoglutethimide.

In a third aspect of the invention, there is provided the use of a compound of formula I, as described in the first aspect of the invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or of a pharmaceutical composition as described in the second aspect of the invention, for the preparation of a pharmaceutical composition for the treatment and/or prevention of a disease or condition associated with the activity or amount of expression of CDK12 and/or CDK 13.

In another preferred embodiment, the disease or disorder is cancer.

In another preferred embodiment, the cancer is selected from the group consisting of: t-cell acute lymphocytic leukemia (T-ALL), Small Cell Lung Cancer (SCLC), neuroblastoma, non-small cell lung cancer (NSCLC), colon cancer, Acute Myelogenous Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), non-hodgkin's lymphoma (NHL), multiple myeloma, ovarian cancer, Ewing's sarcoma, skin cancer, prostate cancer, liver cancer, pancreatic cancer, stomach cancer, esophageal cancer, bladder cancer, brain tumor, squamous cell cancer, peritoneal cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, esophageal adenocarcinoma, esophageal squamous cell cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma, or sarcoma.

In another preferred embodiment, the cancer is a kinase-high-expression type cancer selected from the group consisting of: CDK12, CDK13, KRAS, or a combination thereof.

In another preferred embodiment, the cancer is non-CDK 7 high-expressing.

In a fourth aspect of the invention, there is provided a process for the preparation of a compound of formula I as described in the first aspect of the invention, comprising the steps of:

(a) reacting a compound of formula a4 and a compound of formula a5 in an inert solvent to form a compound of formula I:

in the formula (I), the compound is shown in the specification,

z is selected from: halogen, -OMs, -OTs, -OTf, -OAc or-OAr;

X₁、X₂、R₁、R₂、R₃、R₄、L₁、L₂、L₃a, B, C and n1 are as defined in the first aspect of the invention.

In a fifth aspect of the invention, there is provided a process for the preparation of a compound of formula I, comprising the steps of:

(b) reacting a compound of formula a6 and a compound of formula a7 in an inert solvent to form a compound of formula I:

in the formula (I), the compound is shown in the specification,

X₁、X₂、R₁、R₂、R₃、R₄、L₁、L₂、L₃a, B, C, and n₁Is as defined in the first aspect of the invention.

In a sixth aspect of the invention, there is provided an intermediate compound having a structure selected from: a1, a2, A3, or a4 structural formula:

wherein Z is selected from: H. halogen, -OMs, -OTs, -OTf, -OAc or-OAr;

X₁、X₂、R₁、R₂、R₃、R₄、L₁、L₂、L₃a, B, C and n1 are as defined in the first aspect of the invention.

In a seventh aspect of the invention, there is provided the use of a compound of a1, a2, A3 or a4 for the preparation of compound I.

In an eighth aspect of the invention, there is provided an inhibitor of CDK12 and/or CDK13, comprising:

a compound of formula I according to the first aspect of the invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition according to the second aspect of the invention.

In a ninth aspect of the invention, there is provided a method for selectively inhibiting a cell cycle dependent kinase in vitro comprising the steps of:

contacting a cell cycle dependent kinase with a compound of formula I as described in the first aspect of the invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, or with a pharmaceutical composition as described in the second aspect of the invention, thereby selectively inhibiting said cell cycle dependent kinase.

In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

In another preferred embodiment, the cell cycle dependent kinase CDK is selected from the group consisting of: CDK12, CDK13, or a combination thereof, preferably CDK12 and CDK 13.

In another preferred embodiment, said "selectively inhibiting" means that the ratio (E1/E2) of the inhibitory activity E1 of said compound against targeted CDKs (e.g. CDK12 and/or CDK13) to the inhibitory activity E2 against non-targeted kinases (e.g. CDK7 or other non-targeted kinases selected from Table 4) is ≧ 2, preferably ≧ 5, more preferably ≧ 10.

In another preferred embodiment, the inhibitory activity E is EC₅₀The reciprocal of (c).

In a tenth aspect of the invention, there is provided a method of inhibiting tumor cell growth or proliferation in vitro, comprising the steps of,

contacting a tumor cell with a compound of formula I as described in the first aspect of the invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, or a pharmaceutical composition as described in the second aspect of the invention.

In another preferred embodiment, the method is non-therapeutic, non-diagnostic.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the effect of representative compounds on phosphorylation of Ser2 and Ser5 sites of Pol II in H3122(A) or A549(B) cells.

FIG. 2 shows the competitive effect of representative compounds such as ZSQ5-38, ZSQ8-36, etc. on the covalent binding of CDK7 or 12 in THZ1-biotin to H3122 cells at a concentration of 1 μ M.

FIG. 3 shows the effect of representative compounds such as ZSQ8-36 and ZSQ14-66 at different concentrations on CDK7 or 12 catalyzing the phosphorylation modification of Pol II CTD Ser5 site.

FIG. 4 shows the time-course of concentration of representative compounds ZSQ5-38 and ZSQ8-36 in plasma, as well as the pharmacokinetic parameters.

FIG. 5 shows the inhibitory effect of representative compounds ZSQ5-38 and ZSQ8-36 on growth and proliferation of various cell lines at a concentration of 1 μ M.

Detailed Description

The present inventors have made extensive and intensive studies and, as a result, have unexpectedly developed a class of compounds having high inhibitory activity against CDK12/13, but no inhibitory activity or low inhibitory activity against CDK7, through extensive screening and testing. The compound is an organic arsine-based cell cycle dependent kinase inhibitor shown in a formula I, and in the structure of the compound, an organic arsine group (moiey) is a part which has covalent interaction with an active site of CDK12/13, so that excellent inhibitory activity and better specificity can be provided. The experimental result shows that the compound has the unexpected excellent specificity of targeting CDK12/13, and has high safety and little toxic and side effect. In addition, the compound also has excellent pharmacokinetic property and long half-life period, and is suitable for being used as a patent drug. The present invention has been completed based on this finding.

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Term(s) for

Unless otherwise indicated, the term "alkyl" by itself or as part of another substituent refers to a straight or branched chain hydrocarbon group having the indicated number of carbon atoms (i.e., C1-8 represents 1-8 carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

Unless otherwise indicated, the term "alkenyl" refers to an unsaturated alkyl group having one or more double bonds. Similarly, the term "alkynyl" refers to an unsaturated alkyl group having one or more triple bonds. Examples of such unsaturated alkyl groups include ethenyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2, 4-pentadienyl, 3- (1, 4-pentadienyl), ethynyl, 1-and 3-propynyl, 3-butynyl, and higher homologs and isomers.

Unless otherwise statedThe term "cycloalkyl" refers to a ring having the indicated number of ring atoms (e.g., C)_3-10Cycloalkyl) and a hydrocarbon ring that is fully saturated or has no more than one double bond between ring vertices. "cycloalkyl" also refers to bicyclic and polycyclic hydrocarbon rings, e.g. bicyclo [2.2.1]Heptane, bicyclo [2.2.2]Octane, and the like.

Unless otherwise indicated, the term "heterocycloalkyl" refers to a cycloalkyl group containing one to five heteroatoms selected from N, O and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. The heterocycloalkyl group can be a monocyclic, bicyclic, or polycyclic ring system. Non-limiting examples of heterocycloalkyl groups include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, 1, 4-dioxane, morpholine, thiomorpholine-S-oxide, thiomorpholine-S, S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine, and the like. The heterocycloalkyl group can be attached to the rest of the molecule via a ring carbon or a heteroatom. By terms such as cycloalkylalkyl and heterocycloalkylalkyl, it is meant that the cycloalkyl or heterocycloalkyl group is attached to the rest of the molecule through an alkyl or alkylene linker. For example, cyclobutylmethyl-is a cyclobutyl ring linked to a methylene linkage in the remainder of the molecule.

Unless otherwise indicated, the term "alkylene" by itself or as part of another substituent refers to a divalent radical derived from an alkane, e.g., -CH₂CH₂CH₂CH₂-. Alkyl (or alkylene) groups typically have 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms (e.g., 1, 2, 3, 4,5, or 6) being preferred herein. "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, typically having 4 or fewer carbon atoms. Similarly, "alkenylene" or "alkynylene" refers to an unsaturated form of "alkylene" having a double or triple bond, respectively.

Unless otherwise indicated, the terms "alkoxy" or "alkyloxy", "alkylamino" or "alkylthio" (or thioalkoxy) are used in their conventional sense to refer to those alkyl groups attached to the remainder of the molecule through an oxygen atom, an amino group, or a sulfur atom, respectively. Further, for dialkylamino groups, the alkyl moieties can be the same or different and can be combined with the nitrogen atom to which each alkyl group is attached to form a 3-7 membered ring.

Unless otherwise stated, the term "halo" or "halogen" by itself or as part of another substituent refers to a fluorine, chlorine, bromine, or iodine atom. Further, terms such as "haloalkyl" are meant to include monohaloalkyl or polyhaloalkyl. For example, the term "C_1-4Haloalkyl "is meant to include trifluoromethyl, 2,2, 2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

Unless otherwise indicated, the term "aryl" denotes a polyunsaturated (usually aromatic) hydrocarbon group which may be a single ring or multiple rings (up to three rings) which are fused together or linked covalently. The term "heteroaryl" refers to an aryl (or ring) containing 1 to 5 heteroatoms selected from N, O, and S, which may be monocyclic or polycyclic (bicyclic, up to tricyclic) fused together or covalently linked wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atoms are optionally quaternized. The heteroaryl group may be attached to the rest of the molecule through a heteroatom. Non-limiting examples of aryl groups include phenyl, naphthyl and biphenyl groups, while non-limiting examples of heteroaryl groups include pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzotriazinyl (benzotriazinyl), purinyl, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzisoxazolyl, isobenzofuranyl (isobenzofur), isoindolyl, indolizinyl, benzotriazinyl, thienopyridyl, thienopyrimidyl, pyrazolopyrimidinyl, imidazopyridine, benzothiazolyl, benzofuranyl, benzothienyl, indolyl, quinolinyl, isoquinolinyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furanyl, thienyl, and the like. The substituents for each of the above aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.

As used herein, the term "heteroatom" is meant to include oxygen (O), nitrogen (N), sulfur (S), and silicon (Si).

For the compounds provided herein, a bond from a substituent (typically an R group) to the center of an aromatic ring (e.g., benzene, pyridine, etc.) will be understood to refer to a bond that provides attachment at any available vertex of the aromatic ring. In some embodiments, the description also includes a link on a ring fused to the aromatic ring. For example, a bond drawn to the center of the indole benzene moiety would represent a bond to any available vertex of the six or five membered ring portion of the indole.

Unless otherwise specified, all occurrences of a compound in the present invention are intended to include all possible optical isomers, such as a single chiral compound, or a mixture of various chiral compounds (i.e., a racemate). In all compounds of the present invention, each chiral carbon atom may optionally be in the R configuration or the S configuration, or a mixture of the R configuration and the S configuration.

Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers, regioisomers and individual isomers (e.g., isolated enantiomers) are all intended to be included within the scope of the present invention. Where the compounds provided herein have a defined stereochemistry (denoted as R or S, or as indicated by a dashed or wedged bond), those compounds are understood by those skilled in the art to be substantially free of other isomers (e.g., at least 80%, 90%, 95%, 98%, 99% and up to 100% free of other isomers).

Herein, unless otherwise specified, the term "substituted" means that one or more hydrogen atoms on a group are replaced with a substituent selected from the group consisting of: halogen, deuteration, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy, C1-C6 alkyl S (═ O)₂-, oxo (═ O), -CN, -OH, -N (R)_a)R_bA carboxyl group, or a substituted or unsubstituted group selected from the group consisting of: C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 amino, C6-C10 aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, 5-membered heteroaryl having 1-3 heteroatoms selected from N, S and O12-membered heterocyclic group, - (CH)₂) -C6-C10 aryl, - (CH)₂) - (5-to 10-membered heteroaryl having 1 to 3 heteroatoms selected from N, S and O), and the substituents are selected from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkynyl, C1-C6 alkoxy, oxo, -CN, -NH₂OH, -C6-C10 aryl, -C1-C6 amino, -C2-C6 amido, -5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O.

As used herein, the terms "comprising," "including," or "including" mean that the various ingredients may be used together in a mixture or composition of the invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "comprising.

As used herein, the term "pharmaceutically acceptable" ingredient refers to a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio.

As used herein, the term "therapeutically effective dose" refers to any amount of a drug that, when used alone or in combination with another therapeutic agent, promotes disease regression as manifested by a decrease in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic phases of the disease, or the prevention of a disorder or disability resulting from the disease. The "therapeutically effective dose" of the drug of the present invention also includes a "prophylactically effective dose", which is any amount of the drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or suffering from a recurrence of a disease, inhibits the development or recurrence of the disease.

As used herein, "pharmaceutically acceptable salt" refers to a salt formed by a compound of the present invention with an acid or base that is suitable for use as a pharmaceutical. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is that formed by reacting a compound of the present invention with an acid. Suitable acids for forming the salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, etc., organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, phenylmethanesulfonic acid, benzenesulfonic acid, etc.; and acidic amino acids such as aspartic acid and glutamic acid. One preferred class of salts is that formed by reacting a compound of the present invention with a base. Suitable bases for salt formation include, but are not limited to: inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate and sodium phosphate, and organic bases such as ammonia, triethylamine, diethylamine and piperazine.

Some of the compounds of the present invention may be crystallized or recrystallized using water or various organic solvents, in which case various solvates may be formed. Solvates of the invention include stoichiometric solvates such as hydrates and the like, as well as compounds containing variable amounts of water formed when prepared by the low pressure sublimation drying method.

The invention also includes all suitable isotopic variations of the compounds of the invention. Isotopic variations of the compounds of the present invention are defined as those wherein at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as²H、³H、¹¹C、¹³C、¹⁴C、¹⁵N、¹⁷O、¹⁸O、³⁵S、¹⁸F and³⁶and (4) Cl. Some isotopic variations of the present invention, for example, in which a radioactive isotope is incorporated (e.g.³H or¹⁴C) Are used in drug and/or substrate tissue distribution studies. Tritiated, i.e.,³h, and carbon-14, i.e.,¹⁴c, isotopes are particularly preferred because of their ease of preparation and detection. In addition, the compounds are prepared with isotopes such as deuterium (i.e.,²H) may provide some therapeutic advantages resulting from increased metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements and may therefore be preferred in some circumstances. Isotopic variations of the compounds of the present invention can generally be prepared by conventional procedures, e.g., by employing appropriate reagents for the appropriate isotopic variations, by the procedures exemplified, or as described in the experimental section belowAnd (4) preparing.

Representing a connection to another atom.

As used herein, "treating" or "treatment" refers to alleviating, delaying progression, attenuating, preventing, or maintaining an existing disease or disorder (e.g., cancer). Treatment also includes curing, preventing the development of, or alleviating to some extent one or more symptoms of the disease or disorder.

As used herein, "patient" refers to an animal, preferably a mammal, more preferably a human. The term "mammal" refers to warm-blooded vertebrate mammals, including, for example, cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice, pigs, and humans.

As used herein, "ZSQ-5-38" is used interchangeably with "ZSQ 5-38" and "ZSQ 538" to refer to compounds ZSQ-5-38, with similar numbering using the same rules.

Active ingredient

As used herein, "a compound of the invention" refers to a compound of formula I, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof;

X₁、X₂、R₁、R₂、R₃、R₄、L₁、L₂、L₃a, B, C and n1 are as defined in the first aspect of the invention.

Preparation method

The present invention provides methods for the preparation of compounds of formula I, which may be prepared by a variety of synthetic procedures, and exemplary methods for the preparation of these compounds may include, but are not limited to, the schemes described below.

Preferably, the compounds of formula I of the present invention can be prepared by the following schemes and exemplary methods described in the examples and related disclosure procedures used by those skilled in the art.

In the specific operation process, the steps in the method can be expanded or combined as required.

Step i: the arsenious acid functional group of A1 is protected by an ester or amide, and ethanedithiol, ethylene glycol, or the like can be used as a protecting agent.

Step ii: by S _N2 substitution reaction introduces R into amine group of A2₃The substituents, R3-halogen, can be reacted under basic conditions (e.g., sodium hydride).

Step iii: acylation reaction is carried out on the benzene ring amine group of A3 to introduce an L1 group, which can be catalyzed by (but is not limited to) phosphorus oxychloride reagent.

Step iv: by acylation, urea-forming reaction or S _N2 substitution, the A functional group site of A5 with L of A4₁The connection is made.

The reactions in the above steps are conventional reactions known to those skilled in the art. Unless otherwise indicated, reagents and starting compounds used in the synthetic routes are either commercially available or prepared by one skilled in the art by reference to known methods based on the structure of the various compounds designed.

Pharmaceutical compositions and methods of administration

Since the compound of the present invention has excellent inhibitory activity against cell Cycle Dependent Kinases (CDKs), the compound of the present invention and a pharmaceutically acceptable salt, hydrate or solvate thereof, and a pharmaceutical composition containing the compound as a main active ingredient thereof are useful for treating, preventing and alleviating diseases or disorders associated with the activity or expression level of CDKs.

Preferably, the compounds of the present invention are useful for the treatment of cancer. Representative cancers include (but are not limited to): t-cell acute lymphocytic leukemia (T-ALL), Small Cell Lung Cancer (SCLC), neuroblastoma, non-small cell lung cancer (NSCLC), colon cancer, Acute Myelogenous Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), non-hodgkin's lymphoma (NHL), multiple myeloma, ovarian cancer, Ewing's sarcoma, skin cancer, prostate cancer, liver cancer, pancreatic cancer, stomach cancer, esophageal cancer, bladder cancer, brain tumor, squamous cell cancer, peritoneal cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, esophageal adenocarcinoma, esophageal squamous cell cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma, or sarcoma.

The pharmaceutical composition of the present invention comprises the compound of the present invention or a pharmacologically acceptable salt thereof in a safe and effective amount range and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000mg of a compound of the invention per dose, more preferably, 10-500mg of a compound of the invention per dose. Preferably, said "dose" is a capsule or tablet.

"pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of intermixing with and with the compounds of the present invention without significantly diminishing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), and the like

) Wetting agent (such as sodium lauryl sulfate), colorant, and flavoring agentAgents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be delayed in release in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.

In addition to these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms for topical administration of the compounds of the present invention include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds. Such as anticancer agents (for example, PARP1/2 inhibitors (such as Olaparid, Rucapenib, or Nilaparib), chemotherapeutic drugs inducing DNA damage in cancer cells (such as cisplatin, carboplatin, or oxaliplatin), DNA-alkylating chemotherapeutic drugs (such as nimustine), DNA or RNA synthesis inhibitors (such as gemcitabine), tyrosine receptor kinase inhibitors such as EGFR, ALK, or FGFR, tumor signaling pathway inhibitors such as KRAS, MEK, or ERK), and tumor immunotherapy drugs (PD-1 antibody, PD-L)₁Antibodies, etc.); in certain embodiments, a compound of the invention is administered to a subject with cancer in combination with other conventional cancer therapiesAn object, for example, radiation therapy or surgery. Radiation therapy is well known in the art and includes X-ray therapy, such as gamma radiation, and radiopharmaceutical therapy.

In certain embodiments, the CDK inhibitor of the present invention is administered in the same or separate formulations concurrently with, or sequentially with, other agents that are part of a combination therapeutic regimen.

The general range of therapeutically effective dosages for a compound of formula I or a composition of compounds of formula I will be: about 1 to 2000 mg/day, about 10 to about 1000 mg/day, about 10 to about 500 mg/day, about 10 to about 250 mg/day, about 10 to about 100 mg/day, or about 10 to about 50 mg/day. A therapeutically effective dose will be administered in one or more doses. It will be understood, however, that the specific dose of a compound of the invention for any particular patient will depend upon a variety of factors, such as the age, sex, body weight, general health, diet, individual response, time of administration, the severity of the condition to be treated, the activity of the specific compound administered, the dosage form, mode of application and concomitant drugs. A therapeutically effective amount for a given situation can be determined using routine experimentation and is within the ability and judgment of the clinician or physician. In any event, the compound or composition will be administered in multiple doses based on the individual condition of the patient and in a manner that allows for the delivery of a therapeutically effective amount.

The main advantages of the invention include:

1. the compounds of the present invention have for the first time organic arsine groups (moities) as part of the covalent interaction with the active site of CDK12/13, thus providing excellent inhibitory activity and better specificity.

2. The inhibition activity of CDK12, 13 is better than that of the existing inhibitor, and the growth and proliferation of cancer cells can be effectively inhibited.

3. The compound has the advantages of unobvious inhibition on other kinases in cells, high selectivity, good targeting property, good safety and no obvious toxic or side effect under effective concentration;

4. has ideal pharmacokinetic properties: the half-life is long, and the area under the drug concentration-time curve (AUC) is high.

5. The compound of the invention is combined with CDK12 and CDK13 in an irreversible manner, so that the drug effect is longer and the curative effect is more durable.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Example 1

Synthetic route to compound ZSQ-5-38

Synthesis of Compound (4-aminophenyl) arsinic acid (ZSQ-1-18)

4-Aminophenylarsonic acid (108.5g,500mmol) was dissolved in 300ml of ethanol, and the mixed solution was heated to reflux. Phenylhydrazine (92ml, 1mol) was added dropwise (over 1h) with a large amount of nitrogen evolved during the addition, and stirring was continued under reflux for 1.5h as nitrogen was slowed. The mixed solution was concentrated by distillation under reduced pressure, and a sodium hydroxide solution (40g in 400ml of water) and 400ml of diethyl ether were added. Separating, adding saturated ammonium chloride solution (400ml) into the water phase, stirring at 0 deg.C for 1 hr to obtain a large amount of white solid. Filtration gave a white needle-like solid which was dried in vacuo to give 40g of product in 40% yield.

Synthesis of Compound 4- (1,3, 2-Diazaarsolan-2-yl) Aniline (ZSQ-1-23)

ZSQ-1-18(40g, 199mmol) was dissolved in 200ml of absolute ethanol and heated to reflux. Then, ethanedithiol (20ml, 240mmol) was added dropwise to the mixed solution over 30min, and heating and stirring were continued for 30 min. Subsequently, the mixture was cooled in ice water and filtered to give a crude product, which was recrystallized from ethanol to give 42g of a white solid in 81% yield.

Synthesis of Compound N- (4- (1,3, 2-Diazaarsolan-2-yl) phenyl) -2-bromoacetamide (ZSQ-5-4)

ZSQ-1-23(1.03g, 4mmol) and triethylamine (0.83ml, 6mmol) were dissolved in 20ml of dry DCM solution and stirred at 0 ℃. Bromoacetyl bromide (0.38ml, 4.4mmol) was added dropwise over 15min and stirring was continued at 0 ℃ for 1 h. The mixture was diluted with DCM (20ml), washed successively with 2M dilute hydrochloric acid solution (30ml), water (50ml), saturated sodium bicarbonate solution (30ml), saturated sodium chloride solution (30ml), DCM extracted, the organic phases combined, dried over anhydrous sodium sulphate, filtered and concentrated. Purification by column chromatography on silica gel eluting with PE/EA (4:1) gave 1.02g of a yellow solid in 67% yield.

Synthesis of Compound 3- (2, 5-dichloropyrimidin-4-yl) -1H-indole (ZSQ-3-76)

Methylmagnesium bromide (1M in tetrahydrofuran, 100ml, 100mmol) was added dropwise to a solution of indole (11.7g, 100mmol) in THF (60ml) at 0 ℃ over 30 min. The solution was stirred at 0 ℃ for a further 30 min. 2,4, 5-trichloropyrimidine (5.73ml, 50mmol) was added dropwise to give a yellow solution. The ice bath was removed and the solution was stirred at room temperature for 1h to give a red solution. The mixed solution was warmed to 60 ℃ and stirred for 1.5 h. The mixture was cooled to room temperature, acetic acid (100ml) was added dropwise, water (100ml) and THF (20ml) were added, and the mixture was stirred further at 60 ℃ for 20min to give a biphasic solution. The layers were separated and petroleum ether (100ml) was added to the organic solution, resulting in crystallization of the solid. The solid was collected by filtration, washed with petroleum ether (20ml) and dried in vacuo to give 7.02g of a yellow solid in 53% yield.

Synthesis of the Compound 3- (2, 5-dichloropyrimidin-4-yl) -1- (phenylsulfonyl) -1H-indole (ZSQ-5-26)

ZSQ-3-76(5.28g, 20mmol), NaOH (1.2g, 30mmol) and Bu₄NHSO₄(5.8g, 10mmol) was dissolved in 100ml of dichloromethane and benzenesulfonyl chloride (3.84ml, 30mmol) was added dropwise at room temperature. The reaction mixture was stirred at room temperature for a further 3 h. The mixture was quenched with water (100ml) and extracted with DCM (50ml × 3). The combined organic layers were passed over anhydrous Na₂SO₄Drying, filtration, concentration and purification by column chromatography on silica gel eluting with DCM/EA (8:1) gave 6.79g of a white solid in 84% yield.

Synthesis of compound (R) -tert-butyl 3- ((5-chloro-4- (1- (phenylsulfonyl) -1H-indol-3-yl) pyrimidin-2-yl) amino) piperidine-1-carboxylate (ZSQ-8-30)

ZSQ-5-26(606mg, 1.5mmol), (R) -1-Boc-3-aminopiperidine (300mg, 1.5mmol) and DIPEA (0.74ml, 4.5mmol) were mixed and dissolved in 3ml of N-methylpyrrolidone. The mixed solution is heated and stirred for 3 hours at 135 ℃. Separating and purifying the cooled solution directly by C18 reverse phase column chromatography with H₂O/CH₃CN (1:9) gave 802mg of a yellowish brown powder in 94% yield.

Synthesis of compound (R) -5-chloro-4- (1- (phenylsulfonyl) -1H-indol-3-yl) -N- (piperidin-3-yl) pyrimidin-2-amine (ZSQ-8-31)

ZSQ-8-30(802mg, 1.4mmol) and 1ml trifluoroacetic acid were mixed in 5ml DCM and warmed to room temperatureStirring overnight. After the reaction is finished, the reaction solution is concentrated and separated and purified by C18 reverse phase column chromatography and then is treated with H₂O/CH₃CN (1:6) to give 620mg of a yellowish brown powder in 94% yield.

Synthesis of Compound (R) -N- (4- (1,3, 2-Diazaarsolan-2-yl) phenyl) -2- (3- ((5-chloro-4- (1- (phenylsulfonyl) -1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) acetamide (ZSQ-8-34)

ZSQ-8-31(140mg, 0.3mmol), DIPEA (0.15ml, 0.9mmol) and ZSQ-5-4(114mg, 0.3mmol) were dissolved in 5ml of THF solution at room temperature, and the mixture was stirred at room temperature for 4 h. Then washed with 2N dilute hydrochloric acid solution (20ml) and extracted with EA (3X 30ml) followed by washing with water (2X 20 ml). The organic phases were combined and washed with anhydrous Na₂SO₄Drying, filtration and concentration under reduced pressure, purification by column chromatography on silica gel eluting with DCM/EA (4:1) gave 209mg of a white powder in 91% yield.

Synthesis of compound (R) -N- (4- (1,3, 2-dithiaarsolan-2-yl) phenyl) -2- (3- ((5-chloro-4- (1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) acetamide (ZSQ-8-36)

ZSQ-8-34(209mg, 0.27mmol) and anhydrous potassium carbonate (113mg, 0.82mmol) were dissolved in a mixed solution of 5ml of anhydrous methanol and 5ml of anhydrous THF, and the mixture was stirred at room temperature for 3 hours. Adjusting pH to weak acidity with TFA after reaction, separating and purifying the reaction solution by C18 reversed phase column chromatography, and purifying with H₂O/CH₃CN (1:6) gave 123mg of a yellow powder in 73% yield.

Synthesis of Compound (R) - (4- (2- (3- ((5-chloro-4- (1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) acetamido) phenyl) arsinic acid (ZSQ-5-38)

ZSQ-8-36(123mg, 0.19mmol), mercuric perchlorate trihydrate (71mg, 0.157mmol) were dissolved in 3ml DMSO, and the mixture was stirred at room temperature for 10min, immediately subjected to separation and purification by C18 reverse phase column chromatography, and purified with H₂O/CH₃CN (1:4) to give 102mg of a yellow powder in 92% yield.

Example 2:

synthetic route to compounds ZSQ-5-70

Synthesis of Compound (3- (2- ((4- (1,3, 2-Diazaarsolan-2-yl) phenyl) amino) -2-oxoethyl) -phenyl) carbamic acid tert-butyl ester (ZSQ-5-57-1)

ZSQ-1-23(272mg, 1.05mmol), 3-tert-butoxycarbonylaminophenylacetic acid (251mg, 1mmol), HATU (570mg, 1.5mmol) and DIPEA (496ul, 3mmol) were dissolved in 5ml of DCM, and they were stirred at room temperature for 4 h. After the reaction is finished, H is used in sequence₂O (20 ml. times.2), saturated NaCl solution (20ml), extracted with 20ml DCM, the combined organic phases, dried over anhydrous sodium sulfate, filtered, concentrated and purified by column chromatography on silica gel eluting with PE/EA (3:1) to give 412mg of a white solid in 83% yield.

Synthesis of Compound N- (4- (1,3, 2-Diazaarsolan-2-yl) phenyl) -2- (3-aminophenyl) acetamide (ZSQ-5-57-2)

ZSQ-5-57-1(412mg, 0.83mmol) and 1ml trifluoroacetic acid were mixed in 5ml DCM and stirred overnight at RT. Reaction liquid after reactionConcentrating, separating and purifying by C18 reverse phase column chromatography, and purifying with H₂O/CH₃CN (1:7) gave 290mg of a white solid in 89% yield.

Synthesis of the compound N- (4- (1,3, 2-ditharsolan-2-yl) phenyl) -2- (3- ((5-chloro-4- (1- (phenylsulfonyl) -1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) acetamide (ZSQ-5-63)

ZSQ-5-26(202mg, 0.5mmol), ZSQ-5-57-2(196mg, 0.5mmol) and p-toluenesulfonic acid monohydrate (285mg, 1.5mmol) were mixed and dissolved in 2ml of N-methylpyrrolidone. The mixed solution is heated and stirred for 2h at 135 ℃. Separating and purifying the cooled solution directly by C18 reverse phase column chromatography with H₂O/CH₃CN (1:9) gave 86mg of a yellow powder in 22% yield.

Synthesis of the compound N- (4- (1,3, 2-ditharsolan-2-yl) phenyl) -2- (3- ((5-chloro-4- (1H-indol-3-yl) -pyrimidin-2-yl) amino) phenyl) acetamide (ZSQ-5-66)

ZSQ-5-63(86mg, 0.11mmol) and anhydrous potassium carbonate (47mg, 0.33mmol) were dissolved in a mixed solution of 3ml of anhydrous methanol and 3ml of anhydrous THF, and the mixture was stirred at room temperature for 4 hours. Adjusting pH to weak acidity with TFA after reaction, separating and purifying the reaction solution by C18 reversed phase column chromatography, and purifying with H₂O/CH₃CN (1:6) to give 31mg of a yellow powder in 45% yield.

Synthesis of the compound (4- (2- (3- ((5-chloro-4- (1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) acetamido) -phenyl) arsinic acid (ZSQ-5-70)

ZSQ-5-66(31mg, 0.05 m)mol), mercuric perchlorate trihydrate (18mg, 0.04mmol) in 2ml DMSO, the mixture stirred at room temperature for 10min, immediately separated and purified by C18 reverse phase column chromatography, H₂O/CH₃CN (1:4) to give 21mg of a yellow powder in 75% yield.

Example 3

Synthetic route to compound ZSQ-7-84

Synthesis of the Compound, tert-butyl 3- (((5-chloro-4- (1- (phenylsulfonyl) -1H-indol-3-yl) pyrimidin-2-yl) amino) methyl) piperidine-1-carboxylate (ZSQ-7-72)

ZSQ-5-26(606mg, 1.5mmol), 1-Boc-3-aminomethylpiperidine (354mg, 1.65mmol) and DIPEA (0.74ml, 4.5mmol) were mixed and dissolved in 3ml of N-methylpyrrolidone. The mixed solution is heated and stirred for 3 hours at 135 ℃. Separating and purifying the cooled solution directly by C18 reverse phase column chromatography with H₂O/CH₃CN (1:9) to give 630mg of a yellow solid in 72% yield.

Synthesis of compound 5-chloro-4- (1- (phenylsulfonyl) -1H-indol-3-yl) -N- (piperidin-3-ylmethyl) -pyrimidin-2-amine (ZSQ-7-76)

ZSQ-7-72(630mg, 1.08mmol) and 1ml trifluoroacetic acid were mixed in 5ml DCM and stirred overnight at RT. After the reaction is finished, the reaction solution is concentrated and separated and purified by C18 reverse phase column chromatography and then is treated with H₂O/CH₃CN (1:6) gave 501mg of a tan solid in 96% yield.

Synthesis of the compound N- (4- (1,3, 2-ditharsolan-2-yl) phenyl) -3- (((5-chloro-4- (1- (phenylsulfonyl) -1H-indol-3-yl) pyrimidin-2-yl) amino) methyl) piperidine-1-carboxamide (ZSQ-7-80)

ZSQ-1-23(130mg, 0.5mmol), bis (trichloromethyl) carbonate (59mg, 0.2mmol) were dissolved in 5ml THF, stirred at 0 deg.C, DIPEA (207ul, 1.25mmol) was added dropwise, and after addition the mixture was stirred for 1h at 0 deg.C. ZSQ-7-76(241mg, 0.5mmol) was added, after which stirring was continued at 0 ℃ for 0.5h and then allowed to warm to room temperature for 2 h. After completion of the reaction, the reaction mixture was washed successively with a 2N dilute hydrochloric acid solution (20ml), a saturated sodium bicarbonate solution (20ml), and a saturated sodium chloride solution (20ml), extracted with 40ml of DCM, and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by silica gel column chromatography eluting with PE/EA (2:1) gave 178mg of a white powder in 46% yield.

Synthesis of the compound N- (4- (1,3, 2-dithiaarsolan-2-yl) phenyl) -3- (((5-chloro-4- (1H-indol-3-yl) -pyrimidin-2-yl) amino) methyl) piperidine-1-carboxamide (ZSQ-7-83)

ZSQ-7-80(178mg, 0.23mmol) and anhydrous potassium carbonate (96mg, 0.69mmol) were dissolved in a mixed solution of 5ml of anhydrous methanol and 5ml of anhydrous THF, and the mixture was stirred at room temperature for 3 hours. Adjusting pH to weak acidity with TFA after reaction, separating and purifying the reaction solution by C18 reversed phase column chromatography, and purifying with H₂O/CH₃CN (1:7) gave 124mg of a yellow powder in 86% yield.

Synthesis of Compound (4- (3- (((5-chloro-4- (1H-indol-3-yl) pyrimidin-2-yl) amino) methyl) piperidine-1-carboxamido) phenyl) arsinic acid (ZSQ-7-84)

ZSQ-7-83(124mg, 0).2mmol), mercuric perchlorate trihydrate (71mg, 0.16mmol) in 3ml DMSO, the mixture stirred at room temperature for 10min, immediately separated and purified by C18 reverse phase column chromatography, H₂O/CH₃CN (1:4) to give 78mg of a yellow powder in 70% yield.

Example 4

Synthetic route to compound ZSQ-7-105

Synthesis of Compound 2-formyl-3-methylbutyronitrile (ZSQ-7-74)

A2M solution of lithium diisopropylamide (27.5ml, 55mmol) was mixed in 50ml of dry THF, stirred at-78 deg.C, isovaleronitrile (5.24ml, 50mmol) was added dropwise, stirring continued for 30min after addition was complete, ethyl formate (4.85ml, 60mmol) was mixed in 25ml of dry THF and added dropwise to the mixture at-78 deg.C (over 30min), stirring continued for 45min after addition was complete, allowed to warm to room temperature, and stirred for 14 h. After the reaction was complete, 10ml of H was added₂O quenching, concentrating the mixture, adjusting the pH to 3 by adding dilute hydrochloric acid solution, extracting with EA (20 ml. times.3), drying the organic phase with anhydrous sodium sulfate, filtering and concentrating, and draining to obtain 5g of crude product as brown oil.

Synthesis of Compound 4-isopropyl-1H-pyrazol-3-amine (ZSQ-7-81)

ZSQ-7-74(5g, 45mmol), hydrazine hydrate (3.27ml, 67.5mmol) and acetic acid (5ml, 89mmol) were mixed in 75ml of absolute ethanol, heated in a sealed tube and stirred for 16 h. After completion of the reaction, the mixed solution was concentrated to leave 1/3, which was washed with a saturated sodium bicarbonate solution (40ml) (made alkaline in pH), extracted with DCM (30 ml. times.2), and washed with a saturated sodium chloride solution (40 ml). The combined organic phases were dried over anhydrous sodium sulfate, the filtrate was concentrated and drained without further purification to give 6.7g of a yellow solid.

Synthesis of Compound 3-isopropylpyrazolo [1,5-a ] pyrimidine-5, 7-diol (ZSQ-7-85)

ZSQ-7-81(1.25g, 10mmol) was dissolved in a sealed tube containing 30ml of dried ethanol, 20% sodium ethoxide solution (3.74g, 11mmol) was added, the mixture was heated to reflux temperature, and stirred for 22 h. After the reaction, the reaction mixture was concentrated, diluted with water (20ml), adjusted to pH 3 with dilute hydrochloric acid solution, and a large amount of solid was precipitated, filtered and washed with water several times. Yellow powder was obtained and dried to give 1.29g of white powder with a yield of 66%.

Synthesis of Compound 5, 7-di-chloro-3-isopropylpyrazolo [1,5-a ] pyrimidine (ZSQ-7-88)

ZSQ-7-85(1.29g, 6.67mmol) and N, N-dimethylaniline (84ul, 0.67mmol) were mixed in a sealed tube containing 15ml of phosphorus oxychloride, and heated to 115 ℃ overnight with stirring under reflux. After completion of the reaction, the mixed solution was concentrated, and the remaining solution was dropped dropwise into ice water, and extracted with DCM several times, washed with a saturated sodium chloride solution (30ml), and the organic phases were combined. Dried over anhydrous sodium sulfate, filtered, concentrated and purified by column chromatography on silica gel eluting with PE/EA (4:1) to give 950mg of a yellow solid in 62% yield.

Synthesis of Compound N-benzyl-5-chloro-3-isopropylpyrazolo [1,5-a ] pyrimidin-7-amine (ZSQ-7-90)

ZSQ-7-88(950mg, 4.13mmol), DIPEA (1.36ml, 8.26mmol) and benzylamine (0.9ml, 8.26mmol) were dissolved in 50ml of anhydrous ethanol, heated to 85 ℃ and stirred overnight. After completion of the reaction, the reaction mixture was concentrated, washed with water (30 ml. times.2), extracted with EA (30ml), and washed with a saturated sodium chloride solution (30 ml). The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated and purified by silica gel column chromatography eluting with PE/EA (5:1) to give 1.16g of a white solid in 93% yield.

Synthesis of the Compound benzyl (5-chloro-3-isopropylpyrazolo [1,5-a ] pyrimidin-7-yl) -carbamic acid tert-butyl ester (ZSQ-7-94)

ZSQ-7-90(902mg, 3mmol), 4-dimethylaminopyridine (37mg, 0.3mmol) and di-tert-butyl dicarbonate (897ul, 3.9mmol) were mixed and dissolved in 15ml of THF, and the mixture was stirred at room temperature for 12 hours. After the reaction is finished, H is used₂O (20 ml. times.2), EA extraction (30ml) and then saturated sodium bicarbonate solution (30 ml). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and purified by column chromatography on silica gel eluting with PE/EA (8:1) to give 1.20g of a pale yellow flaky solid in 99% yield.

Synthesis of the Compound tert-butyl 3- (((7- (benzyl (tert-butoxycarbonyl) amino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-5-yl) amino) methyl) piperidine-1-carboxylate (ZSQ-7-96)

Compound ZSQ-7-94(400mg, 1mmol), 1-Boc-3-aminomethylpiperidine (236mg, 1.1mmol), tris (dibenzylideneacetone) dipalladium (46mg, 0.05mmol), 1,1 '-binaphthyl-2, 2' -bisdiphenylphosphine (93mg, 0.15mmol) and sodium tert-butoxide (106mg, 1.1mmol) were mixed in 3ml of toluene, heated to 95 ℃ under a nitrogen atmosphere and stirred for 16 h. After the reaction is finished, H is used₂O (30 ml. times.3), EA extraction (40ml) and then saturated sodium chloride solution (30 ml). The combined organic phases are dried over anhydrous sodium sulfate, filtered and concentrated, purified by column chromatography on silica gel, washed with PE/EA (5:1)Then, 554mg of yellowish brown powder was obtained in 95% yield.

Compound N⁷-benzyl-3-isopropyl-N⁵- (piperidin-3-ylmethyl) pyrazolo [1, 5-a)]Synthesis of pyrimidine-5, 7-diamine (ZSQ-7-98)

ZSQ-7-96(554mg, 0.96mmol) and 1ml trifluoroacetic acid were mixed in 5ml DCM and stirred overnight at RT. After the reaction is finished, the reaction solution is concentrated and separated and purified by C18 reverse phase column chromatography and then is treated with H₂O/CH₃CN (1:5) elution gave 360mg of a white solid in 99% yield.

Synthesis of the compound N- (4- (1,3, 2-dithiaarsolan-2-yl) phenyl) -3- (((7- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-5-yl) amino) methyl) piperidine-1-carboxamide (ZSQ-7-99)

ZSQ-1-23(130mg, 0.5mmol), bis (trichloromethyl) carbonate (59mg, 0.2mmol) were dissolved in 5ml THF, stirred at 0 deg.C, DIPEA (207ul, 1.25mmol) was added dropwise, and after addition the mixture was stirred for 1h at 0 deg.C. ZSQ-7-98(189mg, 0.5mmol) was added and after addition stirring was continued at 0 ℃ for 0.5h, after which time stirring was allowed to warm to room temperature for 2 h. After completion of the reaction, the reaction mixture was washed successively with a 2N dilute hydrochloric acid solution (20ml), a saturated sodium bicarbonate solution (20ml), and a saturated sodium chloride solution (20ml), extracted with 40ml of DCM, and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by column chromatography on silica gel eluting with DCM/EA (1:1) gave 162mg of a white powder in 49% yield.

Synthesis of Compound (4- (3- (((7- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-5-yl) amino) -methyl) piperidine-1-carboxamido) phenyl) arsinic acid (ZSQ-7-105)

ZSQ-7-99(80mg, 0.12mmol), mercuric perchlorate trihydrate (38mg, 0.08mmol) were dissolved in 2ml DMSO, the mixture was stirred at room temperature for 10min, and immediately subjected to separation and purification by C18 reverse phase column chromatography using H₂O/CH₃CN (1:5) elution gave 56mg of a white powder in 78% yield.

Example 5

Synthetic route to compound ZSQ-8-12

Synthesis of Compound benzyl 3- (((tert-butoxycarbonyl) amino) methyl) benzoate (ZSQ-7-104)

3- (N-Boc-aminomethyl) benzoic acid (1.0g, 4mmol) and potassium carbonate (1.10g, 8mmol) were dissolved in 10ml DMF and after stirring at room temperature for 20min benzyl bromide (522ul, 4.4mmol) was added dropwise and after addition stirring at room temperature was continued for 6 h. After the reaction is finished, H is used₂O (30 ml. times.3) and EA. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. After separation and purification by silica gel column chromatography, elution with PE/EA (3:1) gave 1.2g of a white solid with a yield of 88%.

Synthesis of Compound 3- (aminomethyl) benzyl benzoate (ZSQ-8-1)

ZSQ-7-104(1.2g, 3.5mmol) and 1ml trifluoroacetic acid were mixed in 5ml DCM and stirred overnight at RT. After the reaction is finished, the reaction solution is concentrated and separated and purified by C18 reverse phase column chromatography and then is treated with H₂O/CH₃CN (1:6) gave 800mg of a white solid in 95% yield.

Synthesis of the Compound benzyl 3- (((7- (benzyl (tert-butoxycarbonyl) amino) -3-isopropylpyrrolo [1,5-a ] pyrimidin-5-yl) amino) methyl) benzoate (ZSQ-8-3)

Compound ZSQ-7-94(200mg, 0.5mmol), ZSQ-8-1(133mg, 0.55mmol), tris (dibenzylideneacetone) dipalladium (23mg, 0.025mmol), 1,1 '-binaphthyl-2, 2' -bis-diphenylphosphine (47mg, 0.075mmol) and sodium tert-butoxide (58mg, 0.6mmol) were mixed in 2ml of toluene and heated to 95 ℃ under a nitrogen atmosphere and stirred for 16 h. After the reaction is finished, H is used₂O (30 ml. times.3), EA extraction (40ml) and then saturated sodium chloride solution (30 ml). The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated, and purified by column chromatography on silica gel eluting with PE/EA (4:1) to give 154mg of pale yellow powder in 51% yield.

Synthesis of the Compound 3- (((7- (benzyl (tert-butoxycarbonyl) amino) -3-isopropylpyrazolo [1,5-a ] -pyrimidin-5-yl) amino) methyl) benzoic acid (ZSQ-8-6)

ZSQ-8-3(154mg, 0.25mmol) and Pd/C (100mg) were mixed in 10ml of methanol, and the mixture was hydrogenated and stirred at room temperature for 6 hours. Filtering with diatomite, washing with methanol, concentrating, separating and purifying with C18 reversed phase column chromatography, and purifying with H₂O/CH₃CN (1:7) gave 116mg of product as a yellow oil, 90% yield

Synthesis of compound tert-butyl 5- ((3- ((4- (1,3, 2-dithiaarsolan-2-yl) phenyl) carbamoyl) benzyl) amino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-7-yl) (benzyl) carbamate (ZSQ-8-8)

ZSQ-8-6(116mg, 0.22mmol) and ZSQ-1-23(70mg, 0.27mmol) were dissolvedIn 3ml of dry pyridine, the mixed solution is placed at 0 ℃ and stirred, phosphorus oxychloride (42ul, 0.45mmol) is added dropwise to the mixed solution (more than 10min), after the addition, the mixed solution is stirred for 1h at 0 ℃ and then is moved to room temperature and stirred for 2 h. After completion of the reaction, the reaction mixture was acidified with a dilute hydrochloric acid solution, washed with water (30 ml. times.3), extracted with EA, and washed with a saturated sodium chloride solution. The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated. Separating and purifying with silica gel column chromatography, eluting with PE/EA (4:1) to obtain pyridinium, and eluting with DCM/CH₃OH(NH₃) (10:1) to give 92mg of a yellow powder in 54% yield.

Synthesis of the compound N- (4- (1,3, 2-Diazaarsolan-2-yl) phenyl) -3- (((7- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-5-yl) amino) methyl) benzamide (ZSQ-8-9)

ZSQ-8-8(92mg, 0.12mmol) and 0.5ml trifluoroacetic acid were mixed in 5ml DCM and stirred overnight at RT. After the reaction is finished, the reaction solution is concentrated and separated and purified by C18 reverse phase column chromatography and then is treated with H₂O/CH₃CN (1:7) elution gave 64.8mg of a white solid in 82% yield.

Synthesis of Compound (4- (3- (((7- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-5-yl) amino) methyl) benzoylamino) phenyl) arsinic acid (ZSQ-8-12)

ZSQ-8-9(64.8mg, 0.099mmol), mercuric perchlorate trihydrate (33mg, 0.074mmol) were dissolved in 2ml DMSO, and the mixture was stirred at room temperature for 10min, immediately subjected to C18 reverse phase column chromatography for separation and purification, and purified with H₂O/CH₃CN (1:5) was eluted to give 45mg of a white powder in 76% yield.

Example 6

Synthetic route to compound ZSQ-9-73

Synthesis of the Compound tert-butyl (R) -3- ((7- (benzyl (tert-butoxycarbonyl) amino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-5-yl) amino) piperidine-1-carboxylate (ZSQ-9-48)

Compound ZSQ-7-94(200mg, 0.5mmol), (R) -1-Boc-3-aminopiperidine (110mg, 0.55mmol), tris (dibenzylideneacetone) dipalladium (23mg, 0.025mmol), 1,1 '-binaphthyl-2, 2' -bisdiphenylphosphine (47mg, 0.075mmol) and sodium tert-butoxide (53mg, 0.55mmol) were mixed in 2ml of toluene and heated to 95 ℃ under a nitrogen atmosphere and stirred for 16 h. After the reaction is finished, H is used₂O (30 ml. times.3), EA extraction (40ml) and then saturated sodium chloride solution (30 ml). The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated, purified by column chromatography on silica gel eluting with PE/EA (3:1) to give 242.5mg of a reddish brown solid in 85% yield.

Compound (R) -N⁷-benzyl-3-isopropyl-N⁵- (piperidin-3-yl) pyrazolo [1,5-a]Synthesis of pyrimidine-5, 7-diamine (ZSQ-9-55)

ZSQ-9-48(242mg, 0.43mmol) and 0.8ml trifluoroacetic acid were mixed in 5ml DCM and stirred overnight at RT. After the reaction is finished, the reaction solution is concentrated and separated and purified by C18 reverse phase column chromatography and then is treated with H₂O/CH₃CN (1:5) elution gave 150mg of a white solid in 96% yield.

Synthesis of compound (R) -N- (4- (1,3, 2-dithiaarsolan-2-yl) phenyl) -2- (3- ((7- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-5-yl) amino) piperidin-1-yl) acetamide (ZSQ-9-60)

ZSQ-9-55(150mg, 0.41mmol), DIPEA (0.20ml, 1.23mmol) and ZSQ-5-4(155mg, 0.41mmol) were dissolved in 7ml of THF solution at room temperature, and the mixture was stirred at room temperature for 3 hours. Then washed with 2N dilute hydrochloric acid solution (20ml) and extracted with EA (3X 30ml) followed by washing with water (2X 20 ml). The organic phases were combined with Na₂SO₄Drying, filtration and concentration under reduced pressure, purification by column chromatography on silica gel eluting with PE/EA (1:1) gave 133.3mg of a yellow solid in 49% yield.

Synthesis of Compound (R) - (4- (2- (3- ((7- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-5-yl) amino) piperidin-1-yl) acetamido) phenyl) arsinic acid (ZSQ-9-73)

ZSQ-8-9(66mg, 0.1mmol) mercuric perchlorate trihydrate (36mg, 0.08mmol) is dissolved in 2ml DMSO, the mixed solution is stirred at room temperature for 10min, and the solution is immediately separated and purified by C18 reverse phase column chromatography, and H is used₂O/CH₃CN (1:5) elution gave 49.5mg of a yellow solid in 82% yield.

Example 7

Synthetic route to compound ZSQ-10-98

Synthesis of Compound tert-butyl (3- ((4- (1,3, 2-Diazaarsolan-2-yl) phenyl) carbamoyl) benzyl) carbamate (ZSQ-7-71)

ZSQ-1-23(1.03g, 4mmol), 3- (N-Boc-aminomethyl) benzoic acid (1.00g, 4mmol), HATU (3.04g, 8mmol) and DIPEA (1.98ml, 12mmol)mmol) was dissolved in 20ml DCM and left to stir at room temperature for 4 h. After the reaction is finished, H is used in sequence₂O (20 ml. times.2), saturated NaCl solution (20ml), extracted with 20ml DCM, the combined organic phases, dried over anhydrous sodium sulfate, filtered, concentrated and purified by column chromatography on silica gel eluting with PE/EA (4:1) to give 990mg of pale yellow powder, 50% yield.

Synthesis of Compound N- (4- (1,3, 2-Diazaarsolan-2-yl) phenyl) -3- (aminomethyl) benzamide (ZSQ-7-75)

ZSQ-7-71(990mg, 2.01mmol) and 1ml trifluoroacetic acid were mixed in 5ml DCM and stirred overnight at RT. After the reaction is finished, the reaction solution is concentrated and separated and purified by C18 reverse phase column chromatography and then is treated with H₂O/CH₃CN (1:5) was eluted to give 651mg of a white solid in 82% yield.

Synthesis of the compound N- (4- (1,3, 2-dithiaarsolan-2-yl) phenyl) -3- (((5-chloro-3-isopropylpyrazolo [1,5-a ] pyrimidin-7-yl) amino) methyl) benzamide (ZSQ-10-91)

ZSQ-7-88(318mg, 1.39mmol), DIPEA (0.46ml, 2.78mmol) and ZSQ-7-75(544mg, 1.39mmol) were dissolved in 6ml of absolute ethanol, heated to 85 ℃ and stirred overnight. After completion of the reaction, the reaction mixture was concentrated, washed with water (30 ml. times.2), extracted with EA (30ml), and washed with a saturated sodium chloride solution (30 ml). The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated and purified by silica gel column chromatography eluting with PE/EA (5:1) to give 588mg of a brown solid in 72% yield.

Synthesis of the Compound tert-butyl 3- ((4- (1,3, 2-Diazaarsolan-2-yl) phenyl) carbamoyl) benzyl) (5-chloro-3-isopropylpyrazolo [1,5-a ] pyrimidin-7-yl) carbamate (ZSQ-10-92)

ZSQ-10-91(588mg, 1mmol), 4-dimethylaminopyridine (24mg, 0.2mmol) and di-tert-butyl dicarbonate (300ul, 1.3mmol) were mixed and dissolved in 5ml of THF, and the mixture was stirred at room temperature for 12 hours. After the reaction is finished, H is used₂O (20 ml. times.2), EA extraction (30ml) and then saturated sodium bicarbonate solution (30 ml). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and purified by column chromatography on silica gel eluting with PE/EA (7:1) to give 524mg of a yellow solid in 76% yield.

Synthesis of the Compound tert-butyl 3- ((4- (1,3, 2-Diazaarsolan-2-yl) phenyl) carbamoyl) benzyl) (5- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-7-yl) carbamate (ZSQ-10-93)

Compound ZSQ-10-93(274mg, 0.4mmol), benzylamine (52ul, 0.48mmol), tris (dibenzylideneacetone) dipalladium (18mg, 0.02mmol), 1,1 '-binaphthyl-2, 2' -bis-diphenylphosphine (37mg, 0.06mmol) and sodium tert-butoxide (46mg, 0.48mmol) were mixed in 2ml toluene and heated to 95 ℃ under a nitrogen atmosphere and stirred for 16 h. After the reaction is finished, H is used₂O (30 ml. times.3), EA extraction (40ml) and then saturated sodium chloride solution (30 ml). The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated, purified by column chromatography on silica gel eluting with PE/EA (3:1) to give 40mg of a white powder with a yield of 13%.

Synthesis of the compound N- (4- (1,3, 2-Diazaarsolan-2-yl) phenyl) -3- (((5- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-7-yl) amino) methyl) benzamide (ZSQ-10-95)

ZSQ-10-93(40mg, 0.05mmol) and 0.5ml trifluoroacetic acid were mixed in 5ml DCM and stirred overnight at RT. Reaction ofAfter completion, the reaction mixture was concentrated and purified by C18 reverse phase column chromatography using H₂O/CH₃CN (1:7) gave 23mg of a white solid in 69% yield.

Synthesis of Compound (4- (3- (((5- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-7-yl) amino) methyl) benzoylamino) phenyl) arsinic acid (ZSQ-10-98)

ZSQ-10-95(23mg, 0.035mmol), mercuric perchlorate trihydrate (12mg, 0.028mmol) were dissolved in 2ml DMSO, the mixture was stirred at room temperature for 10min, immediately subjected to separation and purification by C18 reverse phase column chromatography, and purified with H18₂O/CH₃CN (1:5) elution gave 17mg of a white solid in 85% yield.

Example 8

Synthetic route to compound ZSQ-12-3

Synthesis of compound (R) -tert-butyl 3- ((5-chloro-3-isopropylpyrazolo [1,5-a ] pyrimidin-7-yl) amino) piperidine-1-carboxylate (ZSQ-11-64)

ZSQ-7-88(345mg, 1.5mmol), DIPEA (0.49ml, 3.0mmol) and (R) -1-Boc-3-aminopiperidine (450mg, 2.25mmol) were dissolved in 6ml of anhydrous ethanol, heated to 85 ℃ and stirred overnight. After completion of the reaction, the reaction mixture was concentrated, washed with water (30 ml. times.2), extracted with EA (30ml), and washed with a saturated sodium chloride solution (30 ml). The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated and purified by silica gel column chromatography eluting with PE/EA (4:1) to give 571mg of white powder with a yield of 96%.

Synthesis of the Compound tert-butyl (R) -3- ((tert-butoxycarbonyl) (5-chloro-3-isopropylpyrazolo [1,5-a ] pyrimidin-7-yl) amino) piperidine-1-carboxylate (ZSQ-11-89)

ZSQ-10-64(571mg, 1.45mmol), 4-dimethylaminopyridine (35mg, 0.20mmol) and di-tert-butyl dicarbonate (434ul, 1.89mmol) were mixed and dissolved in 6ml of THF, and the mixture was stirred at room temperature for 12 hours. After the reaction is finished, H is used₂O (20 ml. times.2), EA extraction (30ml) and then saturated sodium bicarbonate solution (30 ml). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and purified by column chromatography on silica gel eluting with PE/EA (8:1) to give 670mg of a white solid in 93% yield.

Synthesis of the Compound tert-butyl (R) -3- ((5- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-7-yl) (tert-butoxycarbonyl) amino) piperidine-1-carboxylate (ZSQ-11-94)

Compound ZSQ-11-89(494mg, 1.0mmol), benzylamine (131ul, 1.2mmol), tris (dibenzylideneacetone) dipalladium (46mg, 0.05mmol), 1,1 '-binaphthyl-2, 2' -bis-diphenylphosphine (93mg, 0.15mmol) and sodium tert-butoxide (115mg, 1.2mmol) were mixed in 5ml of toluene and heated to 95 ℃ under a nitrogen atmosphere and stirred for 16 h. After the reaction is finished, H is used₂O (30 ml. times.3), EA extraction (40ml) and then saturated sodium chloride solution (30 ml). The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated, and purified by column chromatography on silica gel eluting with PE/EA (3:1) to give a reddish brown solid 524mg, 93% yield.

Compound (R) -N⁵-benzyl-3-isopropyl-N⁷- (piperidin-3-yl) pyrazolo [1,5-a]Synthesis of pyrimidine-5, 7-diamine (ZSQ-11-96)

ZSQ-11-94 (5)24mg, 0.93mmol) and 1ml of trifluoroacetic acid are mixed in 6ml of DCM and stirred overnight at room temperature. After the reaction is finished, the reaction solution is concentrated and separated and purified by C18 reverse phase column chromatography and then is treated with H₂O/CH₃CN (1:5) elution gave 322mg of a white solid in 95% yield.

Synthesis of compound (R) -N- (4- (1,3, 2-dithiaarsolan-2-yl) phenyl) -2- (3- ((5- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-7-yl) amino) piperidin-1-yl) acetamide (ZSQ-11-104)

ZSQ-11-96(182mg, 0.5mmol), K, was added at room temperature₂CO₃(166mg, 1.2mmol) and ZSQ-5-4(190mg, 0.5mmol) were dissolved in 4ml CH₃In CN solution, the mixed solution was stirred at room temperature for 4 hours. Then washed with 2N dilute hydrochloric acid solution (20ml) and extracted with EA (3X 30ml) followed by washing with water (2X 20 ml). The organic phases were combined with Na₂SO₄Drying, filtration and concentration under reduced pressure, purification by column chromatography on silica gel eluting with DCM/EA (4:1) gave 205mg of a white powder in 62% yield.

Synthesis of Compound (R) - (4- (2- (3- ((5- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-7-yl) amino) piperidin-1-yl) acetamido) phenyl) arsinic acid (ZSQ-12-3)

ZSQ-11-104(66mg, 0.10mmol), mercuric perchlorate trihydrate (36mg, 0.08mmol) were dissolved in 3ml DMSO, and the mixture was stirred at room temperature for 10min, immediately subjected to separation and purification by C18 reverse phase column chromatography, and purified with H₂O/CH₃CN (1:5) elution gave 49mg of a white solid in 81% yield.

Example 9

Synthetic route to compound ZSQ-13-92

Synthesis of Compound 3- (2-chloro-5-iodopyrimidin-4-yl) -1H-indole (ZSQ-12-59)

Methylmagnesium bromide (1M in tetrahydrofuran, 70ml, 70mmol) was added dropwise to a solution of indole (8.26g, 70mmol) in THF (50ml) at 0 ℃ over 30 min. The solution was stirred at 0 ℃ for a further 30 min. 2, 4-dichloro-5-iodopyrimidine (2.74g, 35mmol) was added dropwise to give a yellow solution. The ice bath was removed and the solution was stirred at room temperature for 1h to give a red solution. The mixed solution was warmed to 60 ℃ and stirred for 1.5 h. The mixture was cooled to room temperature, and acetic acid (70ml) was added dropwise. Water (70ml) and THF (30ml) were added and the mixture was stirred further at 60 ℃ for 20min to give a biphasic solution. The layers were separated and petroleum ether (100ml) was added to the organic solution, resulting in crystallization of the solid. The solid was collected by filtration, washed with petroleum ether (20ml) and dried in vacuo to give 2.5g of a yellow solid in 20% yield.

Synthesis of Compound 3- (2-chloro-5-iodopyrimidin-4-yl) -1- (phenylsulfonyl) -1H-indole (ZSQ-13-76)

ZSQ-13-59(1.06g, 3mmol), NaOH (180mg, 4.5mmol) and Bu₄NHSO₄(871mg, 1.5mmol) was dissolved in 20ml DCM and benzenesulfonyl chloride (576ul, 4.5mmol) was added dropwise at room temperature. The reaction mixture was stirred at room temperature for a further 4 h. The mixture was quenched with water (30ml) and extracted with DCM (20ml × 3). The combined organic layers were passed over anhydrous Na₂SO₄Drying, filtration, concentration and purification by column chromatography on silica gel eluting with DCM/EA (4:1) gave 850mg of a white solid in 57% yield.

Synthesis of the compound 3- (2-chloro-5- ((trimethylsilyl) ethynyl) pyrimidin-4-yl) -1- (phenylsulfonyl) -1H-indole (ZSQ-13-77)

Compound ZSQ-13-76(850mg, 1.72mmol), trimethylsilylacetylene (364ul, 2.58mmol), cuprous iodide (32mg, 0.17mmol), 1,1' -bisdiphenylphosphinoferrocene palladium dichloride (73mg, 0.10mmol) and triethylamine (478ul, 3.44mmol) were mixed in 10ml of dry tetrahydrofuran solution and heated to 50 ℃ under a nitrogen atmosphere and stirred for 12 h. After completion of the reaction, the reaction mixture was washed with 2M dilute hydrochloric acid solution (30 ml. times.2), extracted with DCM (50ml), and then washed with water (30ml) and saturated sodium chloride solution (30ml) in this order. The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated, and purified by column chromatography on silica gel eluting with PE/EA (4:1) to give 380mg of a white solid in 47% yield.

Synthesis of compound tert-butyl 3- ((4- (1- (phenylsulfonyl) -1H-indol-3-yl) -5- ((trimethylsilyl) ethynyl) pyrimidin-2-yl) amino) piperidine-1-carboxylate (ZSQ-13-86)

ZSQ-13-77(380mg, 0.817mmol), (R) -1-Boc-3-aminopiperidine (172mg, 0.858mmol) and DIPEA (0.4ml, 2.451mmol) were mixed and dissolved in 3ml of N-methylpyrrolidone. The mixed solution is heated and stirred for 3 hours at 135 ℃. Separating and purifying the cooled solution directly by C18 reverse phase column chromatography with H₂O/CH₃CN (1:9) gave 448mg of a brown powder in 87% yield.

Synthesis of compound (R) -5-ethynyl-4- (1- (phenylsulfonyl) -1H-indol-3-yl) -N- (piperidin-3-yl) pyrimidin-2-amine (ZSQ-13-87)

ZSQ-13-86(448mg, 0.71mmol) and 1ml trifluoroacetic acidMix in 6ml DCM and stir at rt overnight. After the reaction is finished, the reaction solution is concentrated and separated and purified by C18 reverse phase column chromatography and then is treated with H₂O/CH₃CN (1:5) gave 105mg of a tan solid in 32% yield.

Synthesis of compound (R) -N- (4- (1,3, 2-dithiaarsolan-2-yl) phenyl) -2- (3- ((5-ethynyl-4- (1- (phenylsulfonyl) -1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) acetamide (ZSQ-13-88)

ZSQ-13-87(105mg, 0.23mmol), DIPEA (0.11ml, 0.69mmol) and ZSQ-5-4(91mg, 0.24mmol) were dissolved in 4ml of THF solution at room temperature, and the mixture was stirred at room temperature for 4 hours. Then washed with 2N dilute hydrochloric acid solution (20ml) and extracted with EA (3X 30ml) followed by washing with water (2X 20 ml). The organic phases were combined with Na₂SO₄Drying, filtration and concentration under reduced pressure, purification by column chromatography on silica gel eluting with DCM/EA (2:1) gave 75mg of a white powder in 43% yield.

Synthesis of compound (R) -N- (4- (1,3, 2-dithiaarsolan-2-yl) phenyl) -2- (3- ((5-ethynyl-4- (1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) acetamide (ZSQ-13-91)

ZSQ-13-88(75mg, 0.1mmol) and anhydrous potassium carbonate (41mg, 0.3mmol) were dissolved in a mixed solution of 2ml of anhydrous methanol and 2ml of anhydrous THF, and the mixture was stirred at room temperature for 4 hours. Adjusting pH to weak acidity with TFA after reaction, separating and purifying the reaction solution by C18 reversed phase column chromatography, and purifying with H₂O/CH₃CN (1:6) to give 52mg of a yellow powder in 85% yield.

Synthesis of Compound (R) - (4- (2- (3- ((5-ethynyl-4- (1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) acetamido) phenyl) arsinic acid (ZSQ-13-92)

ZSQ-13-91(52mg, 0.08mmol), mercuric perchlorate trihydrate (30mg, 0.06mmol) were dissolved in 3ml DMSO, and the mixture was stirred at room temperature for 10min, immediately subjected to C18 reverse phase column chromatography for separation and purification, and purified with H₂O/CH₃CN (1:5) gave 40mg of a yellow solid in 88% yield.

Example 10

Synthetic route to compound ZSQ-14-23

Synthesis of Compound 4- (1,3, 2-Diazaarsolan-2-yl) -N- (prop-2-yn-1-yl) aniline (ZSQ-14-7)

Compound ZSQ-1-23(1.03mg, 4mmol), 3-bromopropyne (379ul, 4.4mmol) and anhydrous potassium carbonate (829mg, 6.0mmol) were mixed in 8ml of DMF solution, and the mixture was stirred at room temperature for 12 hours. After the reaction is finished, H is used₂O (30 ml. times.3) and EA (30 ml). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and purified by column chromatography on silica gel eluting with PE/EA (7:1) to give 850mg of yellow solid in 72% yield

Synthesis of Compound N- (4- (1,3, 2-Diazaarsolan-2-yl) phenyl) -2-bromo-N- (prop-2-yn-1-yl) acetamide (ZSQ-14-10)

ZSQ-14-7(850mg, 2.86mmol) and DIPEA (7.15ml, 3.14mmol) were dissolved in 20ml of dry DCM solution and stirred at 0 ℃. Bromoacetyl bromide (0.27ml, 3.14mmol) was added dropwise over 15min and stirring was continued at 0 ℃ for 1 h. The mixture was diluted with DCM (20ml), which was washed successively with a 2N diluted hydrochloric acid solution (30ml), water (50ml), a saturated sodium bicarbonate solution (30ml), and a saturated brine (30ml), dried over anhydrous sodium sulfate, and the solvent was removed by filtration. Purification by column chromatography on silica gel eluting with PE/EA (1:4) gave 338mg as a yellow solid in 28% yield.

Synthesis of compound (R) -N- (4- (1,3, 2-dithiaarsolan-2-yl) phenyl) -2- (3- ((5-chloro-4- (1- (phenylsulfonyl) -1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) -N- (prop-2-yn-1-yl) acetamide (ZSQ-14-13)

ZSQ-14-10(93mg, 0.20mmol), DIPEA (0.10ml, 0.60mmol) and ZSQ-5-4(88mg, 0.21mmol) were dissolved in 2ml of THF solution at room temperature, and the mixture was stirred at room temperature for 4 hours. Then washed with 2N dilute hydrochloric acid solution (20ml) and extracted with EA (3X 30ml) followed by washing with water (2X 20 ml). The organic phases were combined with Na₂SO₄Drying, filtration and concentration under reduced pressure, purification by column chromatography on silica eluting with DCM/EA (3:1) gave 95mg of a yellow solid in 59% yield.

Synthesis of compound (R) -N- (4- (1,3, 2-dithiaarsolan-2-yl) phenyl) -2- (3- ((5-chloro-4- (1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) -N- (prop-2-yn-1-yl) acetamide (ZSQ-14-18)

ZSQ-14-13(95mg, 0.12mmol) and anhydrous potassium carbonate (66mg, 0.48mmol) were dissolved in a mixed solution of 2ml of anhydrous methanol and 2ml of anhydrous THF, and the mixture was stirred at room temperature for 4 hours. Adjusting pH to weak acidity with TFA after reaction, separating and purifying the reaction solution by C18 reversed phase column chromatography, and purifying with H₂O/CH₃CN (1:5) elution gave 50mg of a yellow powder in 63% yield.

Synthesis of Compound (R) - (4- (2- (3- ((5-chloro-4- (1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) -N- (prop-2-yn-1-yl) acetamido) phenyl) arsinic acid (ZSQ-14-23)

ZSQ-14-18(50mg, 0.07mmol), mercury perchlorate trihydrate (27mg, 0.06mmol) were dissolved in 3ml DMSO, and the mixture was stirred at room temperature for 10min, immediately subjected to C18 reverse phase column chromatography for separation and purification, and purified with H₂O/CH₃CN (1:5) gave 35mg of a yellow solid in 83% yield.

Example 11

Synthetic route to compound ZSQ-15-99

Synthesis of Compound 2-formylbutyronitrile (ZSQ-14-104)

A2M solution of lithium diisopropylamide (27.5ml, 55mmol) was mixed in 50ml of dry THF, stirred at-78 deg.C, butyronitrile (4.35ml, 50mmol) was added dropwise, stirring was continued for 30min after addition, ethyl formate (4.85ml, 60mmol) was mixed in 25ml of dry THF, added dropwise to the mixture at-78 deg.C (over 30min), stirring was continued for 45min after addition, and the mixture was allowed to warm to room temperature and stirred for 14 h. After the reaction was complete, 10ml of H was added₂Quenching with O, concentrating the mixture, adjusting pH to 3 with dilute hydrochloric acid solution, extracting with EA (20 ml. times.3), drying the organic phase with anhydrous sodium sulfate, filtering and concentrating, and draining to obtain 4.82g of crude product as yellow oil

Synthesis of Compound 4-Ethyl-1H-pyrazol-3-amine (ZSQ-15-1)

ZSQ-14-104(4.82g, 49.6mmol), hydrazine hydrate (3.61ml, 74.4mmol) and acetic acid (5.67ml, 99.2mmol) were mixed in 75ml of absolute ethanol, heated in a sealed tube and stirred for 16 h. After completion of the reaction, the mixed solution was concentrated to leave 1/3, which was washed with a saturated sodium bicarbonate solution (40ml) (made alkaline in pH), extracted with DCM (30 ml. times.2), and washed with a saturated sodium chloride solution (40 ml). The combined organic phases were dried over anhydrous sodium sulfate, the filtrate was concentrated and drained without further purification to give 4.88g of a yellow solid.

Synthesis of Compound 3-Ethylpyrazolo [1,5-a ] pyrimidine-5, 7-diol (ZSQ-15-2)

ZSQ-15-1(4.88g, 43mmol) was dissolved in a sealed tube containing 80ml of dried ethanol, 20% sodium ethoxide solution (16g, 47.5mmol) was added, and the mixture was heated to reflux temperature and stirred for 22 h. After the reaction, the reaction mixture was concentrated, diluted with water (50ml), adjusted to pH 3 with dilute hydrochloric acid solution, and a large amount of solid was precipitated, filtered and washed with water several times. Yellow powder was obtained and dried to obtain 2.98g of white powder.

Synthesis of Compound 5, 7-dichloro-3-ethylpyrazolo [1,5-a ] pyrimidine (ZSQ-15-4)

ZSQ-15-2(2.98g, 16.6mmol) and N, N-dimethylaniline (250ul, 2.01mmol) were mixed in a sealed tube containing 45ml of phosphorus oxychloride, and heated to 115 ℃ overnight with stirring under reflux. After completion of the reaction, the mixed solution was concentrated, and the remaining solution was dropped dropwise into ice water, and extracted with DCM several times, washed with a saturated sodium chloride solution (30ml), and the organic phases were combined. Drying with anhydrous sodium sulfate, filtering, concentrating, separating and purifying by silica gel column chromatography, eluting with PE/EA (5:1) to obtain white needle powder 2.33g, yield 65%.

Synthesis of Compound 5-chloro-3-ethyl-N- (4-methoxybenzyl) pyrazolo [1,5-a ] pyrimidin-7-amine (ZSQ-15-72)

ZSQ-15-4(432mg, 2.0mmol), DIPEA (4.96ml, 3.0mmol) and 4-methoxybenzylamine (0.52ml, 4.0mmol) were dissolved in 8ml of anhydrous ethanol, heated to 85 ℃ and stirred overnight. After completion of the reaction, the reaction mixture was concentrated, washed with water (30 ml. times.2), extracted with EA (30ml), and washed with a saturated sodium chloride solution (30 ml). The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated and purified by silica gel column chromatography eluting with PE/EA (5:1) to give 631mg of a white solid in 99% yield.

Synthesis of Compound (5-chloro-3-ethylpyrazolo [1,5-a ] pyrimidin-7-yl) (4-methoxybenzyl) carbamic acid tert-butyl ester (ZSQ-15-74)

ZSQ-15-72(631mg, 2mmol), 4-dimethylaminopyridine (48mg, 0.4mmol) and di-tert-butyl dicarbonate (598ul, 2.6mmol) were mixed and dissolved in 8ml THF, and the mixture was stirred at room temperature for 12 h. After the reaction is finished, H is used₂O (20 ml. times.2), EA extraction (30ml) and then saturated sodium bicarbonate solution (30 ml). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and purified by column chromatography on silica gel eluting with PE/EA (8:1) to give 830mg of a yellow solid in 99% yield.

Synthesis of Compound (3-Ethyl-5- (2- (2-hydroxyethyl) piperidin-1-yl) pyrazolo [1,5-a ] pyrimidin-7-yl) (4-methoxybenzyl) carbamic acid tert-butyl ester (ZSQ-15-83)

Compound ZSQ-15-74(416mg, 1.0mmol), 2-piperidineethanol (165mg, 1.0mmol), tris (dibenzylideneacetone) dipalladium (46mg, 0.05mmol), 1,1' -binaphthyl-2,2' -bis-diphenylphosphine (93mg, 0.15mmol) and sodium tert-butoxide (106mg, 1.1mmol) were mixed in 3ml of toluene and heated to 95 ℃ under a nitrogen atmosphere and stirred for 16 h. After the reaction is finished, H is used₂O (30 ml. times.3), EA extraction (40ml) and then saturated sodium chloride solution (30 ml). The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated, purified by column chromatography on silica gel eluting with PE/EA (8:1) to give 350mg of a red solid in 68% yield.

Synthesis of Compound 2- (1- (7-amino-3-ethylpyrazolo [1,5-a ] pyrimidin-5-yl) piperidin-2-yl) ethan-1-ol (ZSQ-15-97)

Compound ZSQ-15-97(283mg, 0.69mmol) was dissolved in a mixed solution of 2ml concentrated hydrochloric acid and 2ml DCM and stirred at room temperature for 6 h. After the reaction is finished, the reaction solution is concentrated, separated and purified by silica gel column chromatography and added with DCM/CH₃OH(NH₃) (5:1) to give 115mg of a white solid in a yield of 57%.

Synthesis of the compound N- (4- (1,3, 2-ditharsolan-2-yl) phenyl) -2- ((3-ethyl-5- (2- (2-hydroxyethyl) piperidin-1-yl) pyrazolo [1,5-a ] pyrimidin-7-yl) amino) acetamide (ZSQ-15-98)

ZSQ-15-97(115mg, 0.4mmol), potassium carbonate (138mg, 1.0mmol), potassium iodide (33mg, 0.2mmol) and ZSQ-5-4(228mg, 0.6mmol) were dissolved in 1.5ml CH at room temperature₃CN/1.5ml DMF solution, the mixture was stirred at room temperature for 4 h. Then acidifying with TFA, separating and purifying by C18 reversed phase column chromatography, and purifying with H₂O/CH₃CN (1:9) gave 153mg of a pink solid in 65% yield.

Synthesis of Compound (4- (2- ((3-Ethyl-5- (2- (2-hydroxyethyl) piperidin-1-yl) pyrazolo [1,5-a ] pyrimidin-7-yl) amino) acetamido) phenyl) arsinic acid (ZSQ-15-99)

ZSQ-15-98(50mg, 0.10mmol), mercuric perchlorate trihydrate (36mg, 0.08mmol) were dissolved in 2ml DMSO, the mixture was stirred at room temperature for 10min, and immediately subjected to separation and purification by C18 reverse phase column chromatography using H₂O/CH₃CN (1:5) gave 28mg of a yellow solid in 53% yield.

Example 12

Synthetic route to compound ZSQ-14-66

Synthesis of Compound isopropyl (2-nitrophenyl) sulfane (ZSQ-14-27)

1-fluoro-2-nitrobenzene (14g, 100mmol), isopropylmercaptan (10.2ml, 110mmol) and anhydrous potassium carbonate (27.6g, 200mmol) were mixed and dissolved in 100ml anhydrous DMF and stirred at 110 ℃ for 12 h. After completion of the reaction, the reaction solution was concentrated under reduced pressure, washed with 100ml of water and 100ml of a saturated sodium chloride solution in this order, and extracted with EA. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The product was isolated and purified by column chromatography on silica gel eluting with PE/EA (7:1) to give 18g of a yellow oily product in 91% yield.

Synthesis of Compound 1- (isopropylsulfonyl) -2-Nitrobenzene (ZSQ-14-35)

Compound ZSQ-14-27(9.85g, 50mmol) was dissolved in 100ml of methanol and stirred at 0 ℃. Potassium peroxymonosulfonate (15.37g, 250mmol) was dissolved in 100ml of water, added dropwise to the mixed solution at 0 ℃ and after addition was continued stirring at 0 ℃ for 1h and then moved to room temperature and stirred for 24 h. After completion of the reaction, methanol was removed by distillation under reduced pressure, washed successively with 50ml of water, 50ml of a saturated sodium chloride solution, and extracted with EA. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give 8.3g of a bright yellow oily liquid in 72% yield.

Synthesis of Compound 2- (isopropylsulfonyl) aniline (ZSQ-14-48)

Compound ZSQ-14-35(2.29g, 10mmol) was dissolved in 20ml of methanol, Pd/C (200mg) was added, and the mixture was stirred at room temperature under a hydrogen atmosphere for 12 hours. After completion of the reaction, it was filtered through celite and washed with DCM. The filtrate was concentrated and pumped to dryness by an oil pump to give 1.99g of a white solid with a yield of 99%.

Synthesis of Compound 2, 5-dichloro-N- (2- (isopropylsulfonyl) phenyl) pyrimidin-4-amine (ZSQ-14-49)

NaH (600mg, 15mmol) was dissolved in dry 60ml DMF and stirred at 0 ℃. ZSQ-14-48(1.99g, 10mmol) was dissolved in 5ml of dry DMF and added dropwise to the above mixed solution (over 10 min). After the addition was continued at 0 ℃ for 0.5h, 2,4, 5-trichloropyrimidine (2.29ml, 20mmol) was added dropwise, stirred for 1h and then allowed to warm to room temperature overnight with stirring. After the reaction was completed, 20ml of water was added to quench, and DMF was removed by concentration by distillation under reduced pressure. Washed successively with 100ml of water, 100ml of saturated sodium bicarbonate solution, 100ml of saturated sodium chloride solution and extracted with EA. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. After separation and purification by silica gel column chromatography and elution with PE/EA (3:1), 1.2g of white solid is obtained with the yield of 35%.

Synthesis of compound tert-butyl (R) -3- ((5-chloro-4- ((2- (isopropylsulfonyl) phenyl) amino) pyrimidin-2-yl) amino) piperidine-1-carboxylate (ZSQ-14-54)

ZSQ-14-49(173mg, 0.5mmol), (R) -1-Boc-3-aminopiperidine (100mg, 0.5mmol) and DIPEA (248ul, 1.5mmol) were mixed and dissolved in 2ml of N-methylpyrrolidone. The mixed solution is heated and stirred for 3 hours at 135 ℃. Separating and purifying the cooled solution directly by C18 reverse phase column chromatography with H₂O/CH₃CN (1:9) elution gave 232mg of a pale yellow solid in 91% yield.

Compound (R) -5-chloro-N⁴- (2- (isopropylsulfonyl) phenyl) -N²Synthesis of- (piperidin-3-yl) pyrimidine-2, 4-diamine (ZSQ-14-58)

ZSQ-14-54(232mg, 0.45mmol) and 1ml trifluoroacetic acid were mixed in 5ml DCM and stirred overnight at RT. After the reaction is finished, the reaction solution is concentrated and separated and purified by C18 reverse phase column chromatography and then is treated with H₂O/CH₃CN (3:1) gave 180mg of a pale yellow solid in 97% yield.

Synthesis of compound (R) -N- (4- (1,3, 2-dithiaarsolan-2-yl) phenyl) -2- (3- ((5-chloro-4- ((2- (isopropyl-sulfonyl) phenyl) amino) pyrimidin-2-yl) amino) piperidin-1-yl) acetamide (ZSQ-14-60)

ZSQ-14-58(82mg, 0.2mmol), DIPEA (0.13ml, 0.8mmol) and ZSQ-5-4(76mg, 0.2mmol) were dissolved in 3ml of THF solution at room temperature, and the mixture was stirred at room temperature for 4 hours. Then washed with 2N dilute hydrochloric acid solution (20ml) and extracted with EA (3X 30ml) followed by washing with water (2X 20 ml). The organic phases were combined and washed with anhydrous Na₂SO₄Drying, filtration and concentration under reduced pressure, purification by column chromatography on silica eluting with DCM/EA (3:1) gave 79mg of a yellow solid in 56% yield.

Synthesis of Compound (R) - (4- (2- (3- ((5-chloro-4- ((2- (isopropylsulfonyl) phenyl) amino) pyrimidin-2-yl) amino) piperidin-1-yl) acetamido) phenyl) arsinic acid (ZSQ-14-66)

ZSQ-14-60(36mg, 0.05mmol), mercuric perchlorate trihydrate (18mg, 0.04mmol) were dissolved in 2ml DMSO, and the mixture was stirred at room temperature for 10min, immediately subjected to C18 reverse phase column chromatography for separation and purification, and purified with H₂O/CH₃CN (1:3) gave 24mg of a yellow solid in 75% yield.

Synthesis scheme 13

Synthetic route to compounds ZSQ-16-91

Synthesis of Compound (4-hydroxy-3-nitrophenyl) arsinic acid (ZSQ-16-8)

3-Nitro-4-hydroxyphenylarsonic acid (26.3g,100mmol) was dissolved in 80ml of methanol, and the mixed solution was heated to reflux. Phenylhydrazine (19.6ml, 200mol) was added dropwise (over 1h) with a large amount of nitrogen evolved during the addition, and stirring was continued under reflux for 1.5h as nitrogen was slowed. The mixed solution was concentrated by distillation under reduced pressure, and a sodium hydroxide solution (12g in 200ml of water) and 200ml of diethyl ether were added. The layers were separated, and the aqueous phase was stirred (100ml) with 2N dilute hydrochloric acid solution and extracted with EA (200 ml. times.3). The organic phase was dried over anhydrous sodium sulfate, concentrated and pumped to dryness by an oil pump, and the next reaction was carried out without purification.

Synthesis of Compound 4- (1,3, 2-Diazaarsolan-2-yl) -2-nitrophenol (ZSQ-16-14)

ZSQ-16-8(24.7g, 100mmol) was dissolved in 100ml of methanol and heated to reflux. Then, ethanedithiol (10ml, 120mmol) was added dropwise to the mixed solution over 30min, and heating and stirring were continued for 30 min. The mixture was then concentrated and purified by column chromatography on silica gel eluting with PE/EA (10:1) to give the crude product as a yellow solid. Dissolving with THF, separating and purifying with C18 reverse phase column chromatography, and purifying with H₂O/CH₃CN (8:1) gave 9g of product as a yellow solid in 29.5% yield.

Synthesis of Compound 2-amino-4- (1,3, 2-Diazaarsolan-2-yl) phenol (ZSQ-16-90)

ZSQ-16-14(9.3g, 30mmol) is dissolved in a mixed solution of ethanol/ethyl acetate (10:1, 100ml), stannous chloride (13.5g, 60mmol) is added, and the mixed solution is placed at 75 ℃ and stirred under reflux for 48 h. After completion of the reaction, the reaction mixture was concentrated, washed with water (100X 3ml), and extracted with EA (100 ml). The organic phase was dried over anhydrous sodium sulfate, concentrated and purified by column chromatography on silica gel eluting with PE/EA (8:1) to give 340mg of a crude yellow solid in 4% yield.

Synthesis of Compound (5- (1,3, 2-Diazaarsolan-2-yl) -2-hydroxyphenyl) carbamic acid tert-butyl ester (ZSQ-16-1)

ZSQ-16-90(120mg, 0.72mmol) and di-tert-butyl dicarbonate (200. mu.l, 0.87mmol) were mixed and dissolved in 4ml of THF, and the mixture was stirred at room temperature for 24 hours. After the reaction is finished, H is used₂O (50 ml. times.2) and EA (50 ml). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and purified by column chromatography on silica gel eluting with PE/EA (6:1) to give 120mg of a white solid in 43% yield.

Synthesis of Compound (5- (1,3, 2-Diazaarsolan-2-yl) -2-methoxyphenyl) carbamic acid tert-butyl ester (ZSQ-16-68)

Compound ZSQ-16-1(120mg, 0.32mmol), methyl iodide (24ul, 0.38mmol), anhydrous potassium carbonate (88mg, 0.64mmol), and potassium iodide (10mg, 0.06mmol) were mixed in 2ml of acetonitrile, and the mixture was stirred at room temperature. After the reaction is finished, the reaction solution is directly concentrated, separated and purified by silica gel column chromatography, and eluted by PE/EA (6:1) to obtain 90mg of white solid with the yield of 72 percent.

Synthesis of Compound 5- (1,3, 2-Diazaarsolan-2-yl) -2-methoxyaniline (ZSQ-16-76)

ZSQ-16-68(90mg, 0.23mmol) and 0.3ml trifluoroacetic acid were mixed in 3ml DCM and stirred overnight at RT. After the reaction is finished, the reaction solution is concentrated and separated and purified by C18 reverse phase column chromatography and then is treated with H₂O/CH₃CN (1:6) elution gave 52mg of a white solid in 78% yield.

Synthesis of Compound N- (5- (1,3, 2-Diazaarsolan-2-yl) -2-methoxyphenyl) -2-bromoacetamide (ZSQ-16-86)

ZSQ-16-76(289mg, 1.0mmol) and DIPEA (0.41ml, 2.5mmol) were dissolved in 5ml of dry DCM solution and stirred at 0 ℃. Bromoacetyl bromide (0.1ml, 1.1mmol) was added dropwise over 10min and stirring was continued for 1h at 0 ℃ before moving to room temperature and stirring for 2 h. The mixture was diluted with DCM (20ml), washed with water (50ml), extracted with DCM, the organic phases combined, dried over anhydrous sodium sulphate, filtered and concentrated. Purification by column chromatography on silica gel eluting with PE/EA (5:1) afforded 110mg of a yellow solid in 27% yield.

Synthesis of compound (R) -N- (5- (1,3, 2-dithiaarsolan-2-yl) -2-methoxyphenyl) -2- (3- ((5-chloro-4- (1- (phenylsulfonyl) -1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) acetamide (ZSQ-16-87)

ZSQ-16-68(110mg, 0.26mmol), DIPEA (0.13ml, 0.80mmol) and ZSQ-5-4(138mg, 0.29mmol) were dissolved in 3ml of THF solution at room temperature, and the mixture was stirred at room temperature for 4 h. Then washed with 2N dilute hydrochloric acid solution (20ml) and extracted with EA (3X 30ml) followed by washing with water (2X 20 ml). The organic phases were combined with Na₂SO₄Drying, filtration and concentration under reduced pressure, purification by column chromatography on silica eluting with DCM/EA (3:1) gave 177mg of a white solid in 83% yield.

Synthesis of compound (R) -N- (5- (1,3, 2-dithiaarsolan-2-yl) -2-methoxyphenyl) -2- (3- ((5-chloro-4- (1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) acetamide (ZSQ-16-88)

ZSQ-16-87(177mg, 0.22mmol) and anhydrous potassium carbonate (92mg, 0.66mmol) were dissolved in a mixed solution of 1.5ml of anhydrous methanol and 1.5ml of anhydrous THF, and the mixture was stirred at room temperature for 4 hours. Adjusting pH to weak acidity with TFA after reaction, separating and purifying the reaction solution by C18 reversed phase column chromatography, and purifying with H₂O/CH₃CN (1:8) gave 121mg of a pale yellow solid in 84% yield.

Synthesis of Compound (R) - (3- (2- (3- ((5-chloro-4- (1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) acetamido) -4-methoxyphenyl) arsinic acid (ZSQ-16-91)

ZSQ-16-88(66mg, 0.1mmol), mercuric perchlorate trihydrate (36mg, 0.08mmol) were dissolved in 2ml DMSO, and the mixture was stirred at room temperature for 10min, immediately subjected to separation and purification by C18 reverse phase column chromatography, and purified with H₂O/CH₃CN (1:5) gave 46mg of a yellow solid in 76% yield.

TABLE 1

Activity test example 1 CDK inhibitor test for inhibitory activity against growth of cancer cells

The bioassay protocol used was: the effect of the compound on the cell growth activity of the acute lymphoblastic leukemia cell line Jurkat and the non-small cell lung cancer cell line H3122.

In order to verify the growth inhibition effect of the compound on cancer cells at a cellular level, Jurkat cells (blood cancer, suspension type) and H3122 cells (solid tumor, adherent type) are selected, and the cell viability is calculated by detecting chemiluminescence values, so that the biological activity of the compound for inhibiting the growth of the cancer cells is obtained.

The method comprises the following steps: culturing Jurkat or H3122 cells in vitro, growing to logarithmic growth phase, collecting cells, centrifuging at 1000rpm for 5min, discarding supernatant, and adjusting cell concentration to 2.5 × 10⁵(Jurkat) or 1.5X 10⁵mL (H3122), cells were seeded into 384 well plates at 40 μ l per well. mu.L each of the compounds or DMSO was added to the corresponding wells at different concentrations, and the wells were placed in a cell culture chamber (37 ℃ C., 5% CO)₂) After 72h incubation, 15. mu.l of Cell Titer-Glo solution was added to each well, incubated at room temperature for 30min, and chemiluminescence (luminescence) was measured to measure intracellular ATP levels. Cell viability was 100% for unstimulated DMSO control wells. Calculation of Compound IC Using Prism Graphpad statistical software₅₀The value is obtained.

TABLE 2 CDK inhibitors test results for growth inhibitory Activity on cancer cell lines

Activity test example 2 CDK inhibitor test for growth inhibitory activity against non-small cell lung cancer cell lines

The bioassay protocol used was: influence of the compound on the growth activity of each of a plurality of non-small cell lung cancer cell lines.

In order to verify the growth inhibition effect of the representative compound on non-small cell lung cancer cell lines which depend on different driving genes for growth and survival at the cellular level, H3122, H1299, H1975, H2077, H358, EBC-1, H23, PC9 and A549 cells are respectively selected, the cell viability is calculated by detecting chemiluminescence values, and the biological activity of the compound for inhibiting the growth of the cancer cells is obtained and compared with reported CDK inhibitors THZ1 and THZ 531.

The method comprises the following steps: culturing in vitro various non-small cell lung cancer cells, growing to logarithmic growth phase, digesting and collecting cells, centrifuging at 1000rpm for 5min, discarding supernatant, and adjusting cell concentration to 1.5 × 10⁵Cells were seeded into 384 well plates at 40. mu.l per well. mu.L each of the compounds or DMSO was added to the corresponding wells at different concentrations, and the wells were placed in a cell culture chamber (37 ℃ C., 5% CO)₂) After 72h incubation, 15. mu.l of Cell Titer-Glo solution was added to each well, incubated at room temperature for 30min, and chemiluminescence (luminescence) was measured to measure intracellular ATP levels. Cell viability was 100% for unstimulated DMSO control wells. Calculation of Compound IC Using Prism Graphpad statistical software₅₀The value is obtained.

TABLE 3 measurement results of the growth inhibitory Activity of CDK inhibitors on different non-small cell Lung cancer cell lines

As can be seen from table 3, representative compounds of the present invention exhibited growth inhibitory activity comparable to or even better than THZ531 on a variety of non-small cell lung cancer cell lines.

Activity test example 3 Effect of CDK inhibitors on CDK7/12/13 kinase Activity in cancer cells

In many cancer cells, RNA polymerase II (pol II) promotes MYC, RUNX by forming transcription complexes with transcription factors such as CDK7, 12, 13, and BRD4 via super enhancers₁High expression of oncogenes, and further maintain the survival and proliferation of cancer cells. In Pol II transcription complexes, CDK7, 12, 13 are collectively responsible for sustained phosphorylation of Ser5 site in Pol II C-terminal domain (CTD) repeats to ensure normal initiation of Pol II transcription programs; CDK12, 13 was responsible for the sustained phosphorylation of the Ser2 site in Pol II CTD repeats to ensure proper transcriptional extension and DNA damage repair. Thus, when CDK12, 13 was inhibited simultaneously, the phosphorylation level of Ser2 site would be inhibited; when CDK7, 12, 13 was simultaneously inhibited, the level of phosphorylation at Ser5 site would also be inhibited.

Experimental conditions and procedures: culturing H3122 or A549 cells in vitro, growing to logarithmic growth phase, digesting and collecting cells, centrifuging at 1000rpm for 5min, discarding supernatant, and adjusting cell concentration to 1 × 10⁶and/mL. In a 12-well cell culture plate, 1ml of cells is added into each well, 1 mu L of DMSO solution of drugs with different concentrations is added into each well, THZ1, THZ531 or DMSO is used as a control, the cell culture plate is placed in a cell culture box (37 ℃, 5% CO2) for culture for 8 hours, then precooled PBS solution is used for washing twice, the solution is sucked off, 200 mu L of RIPA cell lysate, protease inhibitor and phosphatase inhibitor are added into the wells, the wells are transferred into a sample tube and placed in a4 ℃ shaking table for lysis for 30min, and then centrifugation is carried out at 15000rpm and 4 ℃ for 15min, and cell lysate of supernatant is taken. Protein content of each group was measured using BCA protein quantification kit and adjusted to a final volume of 100. mu.L using PIPA lysate. And (3) carrying out western-blot identification on the sample.

Western-blot: to 100. mu.L of cell lysate, 25. mu.L of 5X protein loading buffer was added and heated at 95 ℃ for 10 min. After the sample had cooled, it was run on SDS-PAGE (9%) gel 60V and after 30min was switched to 120V until the front band ran to the bottom of the gel. The proteins in the gel were transferred to a PC membrane with a pore size of 0.2. mu.L using a turbo semi-dry transfer system at a constant current of 0.2A for 80 minutes. The transferred PC membrane was blocked in 5% skimmed milk powder (TBST solution) for 2h and incubated with the corresponding primary antibody at 4 ℃ for 12 h. TBST washing was performed 3 times for 10min each. The corresponding secondary antibody was incubated for 2h at room temperature. TBST was washed three times for 10min each. ECL luminescence was used for incubation and detection of the luminescent signal.

FIG. 1 shows the effect of representative compounds on phosphorylation of Ser2 and Ser5 sites of Pol II in H3122(A) or A549(B) cells. The experimental results show that:

ZSQ5-38, ZSQ8-36 and ZSQ9-75 and the like can effectively inhibit the phosphorylation of a Ser2 site at a concentration of 1 mu M, but not inhibit the phosphorylation of a Ser5 site;

ZSQ5-38, ZSQ17-22, etc., exhibit superior inhibitory activity over THZ 531;

THZ1 inhibited phosphorylation at both sites.

This indicates that compounds ZSQ5-38, ZSQ8-36, and ZSQ9-75 were effective in inhibiting the kinase activity of CDK12, 13 in cancer cells, but not CDK 7.

Activity test example 4 irreversible binding of CDK inhibitors to CDKs in H3122 cells

In this example, CDK inhibitors of the present invention were tested for irreversible binding to CDK7, 12 or 13.

Covalent inhibitors such as THZ1 or THZ531 are capable of forming covalent bonds with specific cysteine residues in the binding pocket of CDK7, 12 or 13 and thereby irreversibly binding CDK7, 12 or 13. THZ1-biotin, a reported derivative of THZ1, is also capable of forming covalent bonds with CDK7, 12 and 13 in cell lysates, which in turn can enrich CDK7, 12 and 13 with streptavidin microbeads (streptavidin beads). When a THZ1, THZ531 or ZSQ series CDK inhibitor binds irreversibly to CDK7, 12 or 13, THZ1-biotin can no longer bind to CDK7, 12 or 13 because the binding pocket is occupied. Therefore, based on a competition enrichment experiment with THZ1-biotin and streptavidin microbeads, it was possible to accurately determine whether CDK inhibitors bound irreversibly to CDK7, 12 or 13.

Experimental conditions and procedures: culturing H3122 cells in vitro, growing to logarithmic growth phase, digesting and collecting cells, centrifuging at 1000rpm for 5min, discarding supernatant, and adjusting cell concentration to 1 × 10⁶and/mL. In 12-well cell culture plates, 1ml of cells was added per well, 100nL of a 10mM concentration DMSO solution of a representative drug (final concentration 1 μ M) was added per well, and after incubation for 4 hours in a cell incubator (37 ℃, 5% CO2) with THZ1, THZ531 or DMSO control, washed twice with pre-cooled PBS solution, 200 μ L NP40 cell lysate and protease inhibitor were added to the wells and lysed for 30 minutes at 4 ℃. The lysate was collected, centrifuged at 15000rpm at 4 ℃ for 15min, the supernatant was transferred to a sample tube, THZ1-biotin (final concentration 1. mu.M) was added, and the tube was incubated overnight at 4 ℃. Add 10. mu.L streptavidin beads and let stand at room temperature for binding for 2 hours. After washing the microbeads 10 times with 1% NP40 cell lysate, 20. mu.L of 2% SDS lysate was added for lysis, and the samples were boiled at 95 ℃ for 10 minutes, and subjected to western-blot identification.

FIG. 2 shows the competitive effect of representative compounds such as ZSQ5-38, ZSQ8-36, etc. on the covalent binding of CDK7 or 12 in THZ1-biotin to H3122 cells at a concentration of 1 μ M.

The experimental results show that:

THZ1 simultaneously inhibited covalent binding of THZ1-biotin to CDK7, 12 and 13; THZ531 had slight inhibition of CDK 7;

compounds such as ZSQ5-38, ZSQ8-36, ZSQ9-75 and ZSQ17-22 can completely or almost completely inhibit the covalent binding of THZ1-biotin to CDK12 or 13 at a concentration of 1 μ M, but hardly affect the covalent binding of THZ1-biotin to CDK7, and compounds such as ZSQ5-38, ZSQ8-36, ZSQ9-75 and ZSQ17-22 have smaller influence and higher selectivity on CDK7 than THZ 531;

this demonstrates that ZSQ compounds are capable of inhibiting CDK12, 13 kinase activity in cancer cells with high efficiency, high specificity, and irreversibly.

Activity test example 5 Effect of CDK inhibitors on CDK7, 12 in vitro kinase Activity

Both CDK7 and CDK12 proteins were able to catalyze the phosphorylation of the Ser5 site of Pol II CTD in vitro, therefore, when the kinase activity of CDK7 or 12 was inhibited, the phosphorylation modification of the Ser5 site would be reduced or lost.

Experimental conditions and procedures: 293T cells overexpress 3XFLAG-CDK7 or 3X-Flag-CDK12, and after 48 hours are washed twice with pre-cooled PBS solution, 1mL RIPA buffer containing protease inhibitors is added, and lysis is carried out at 4 ℃ for 1 hour. The lysate was collected, centrifuged at 15000rpm at 4 ℃ for 15min, the supernatant was transferred to a sample tube, 20. mu.L of anti-Flag affinity gel was added, and incubated overnight at 4 ℃. The gel was washed 5 times with RIPAbuffer and then with kinase buffer (50mM HEPES pH7.4, 50mM KCl,10mM MgCl)₂) Washing for 3 times, adding 100 mu L kinase buffer, and mixing uniformly for subsequent in vitro kinase reaction. Add 10. mu.L of gel mix, varying concentrations of compound to each well and incubate overnight at 4 ℃. Adding 1 μ g of bacterial expression purified GST-Pol II CTD protein and 100 μ M ATP, oscillating at 37 ℃ for 1 hour, adding 4% SDS loading buffer to terminate the reaction, boiling at 95 ℃ for 10 minutes, and identifying by western-blot.

FIG. 3 shows the effect of representative compounds such as ZSQ8-36 and ZSQ14-66 at different concentrations on CDK7 or 12 catalyzing the phosphorylation modification of Pol II CTD Ser5 site.

The experimental results show that:

ZSQ5-38, ZSQ8-36 and the like can completely or nearly completely inhibit the phosphorylation modification of Pol II CTD Ser5 site catalyzed by CDK12 under low concentration, but does not influence the phosphorylation modification of Pol II CTD Ser5 site catalyzed by CDK7, and the phenotype is consistent with THZ 531;

in contrast, THZ1 inhibited both CDK7 and 12 catalytic substrate phosphorylation modification;

ZSQ14-66 showed significant inhibition of phosphorylation of both CDK7 and 12 catalytic substrates at 250nM concentration, while ZSQ14-60 partially inhibited phosphorylation of CDK7 catalytic substrate at 500nM concentration.

This demonstrates that multiple ZSQ compounds are capable of efficiently and specifically backing up the kinase activity of CDK 12.

Activity test example 6 selectivity of CDK inhibitors for kinase groups

The bioassay protocol used was: effect of compounds on kinase group in vitro enzyme activity.

To verify the selectivity of the compounds of the invention for the kinase group at the protein level, representative compounds ZSQ5-36, ZSQ8-36 were selected, using P³³The isotope labeled ATP and the activity of the radioactive kinase are tested to obtain the inhibition level of the compound on the phosphorylation of the substrates catalyzed by 370 kinase proteins.

The method comprises the following steps: and (4) testing the activity of a radioactive kinase group. The compound and protein are mixed and pre-incubated for 1 hour at room temperature, and then P is added³³ATP was reacted for 2 hours, and the reaction mixture was spotted on P81 ion exchange chromatography paper to detect kinase activity by membrane filtration. The final concentration tested for the compound was 1 μ M, and the activity of the drug-treated group was calculated as 100% kinase activity in the DMSO-alone group, with 2 replicates in the wells.

TABLE 4 selectivity of CDK inhibitors for 370 different kinases

As can be seen from table 4, representative compounds inhibited the enzymatic activity of only a few kinases to below 50% at a concentration of 1 μ M; in contrast, representative compounds were able to completely inhibit the kinase activity of CDK12 at a concentration of 500nM (activity test example 5).

Studies have shown that THZ531 binds to different kinases at 1 μ M concentration as shown in table 5, and the Score (Score) indicates the percentage of proteins that do not bind to the compound (i.e. proteins whose kinase activity is not inhibited), it can be seen that THZ531 binds strongly to kinases such as RSK2, STK16, etc., while the compounds of the present invention have substantially no effect on the activity of these kinases, suggesting that the compounds of the present invention have a kinase group selectivity different from that of the existing CDK12/13 inhibitors, and may have better selectivity and safety in application.

TABLE 5

Activity test example 7 pharmacokinetic Properties of CDK inhibitors

In this example, drug metabolism was tested in vivo in mice, and the selectivity of the compounds of the present invention for the kinase group was verified in vivo.

The method comprises the following steps; representative compounds ZSQ5-38, ZSQ8-36 were selected and tested for their pharmacokinetic properties in mice (n-3) using a single administration of i.p. (10 mg/kg).

FIG. 4 shows the time-course of concentration of representative compounds ZSQ5-38 and ZSQ8-36 in plasma, as well as the pharmacokinetic parameters.

The results showed that ZSQ5-38 and ZSQ8-36 peaked rapidly in mice after a single administration, were able to maintain a moderate drug concentration stably, and exhibited long half-lives (T of ZSQ5-38 and ZSQ 8-36)_1/217.3 and 9.8).

In addition, in the completed in vivo test, no significant toxic side effects have been observed, and no necrosis has been observed in normal tissues or cells of mice, suggesting that the compounds of the present invention have high safety.

Activity test example 8 growth inhibitory Activity of CDK inhibitors on various Lung cancer cell lines with KRAS mutations

In this example, the growth inhibitory effect of representative compounds ZSQ5-38 and ZSQ8-36 of the invention on KRAS-dependent growth survival or lung cancer cell lines with KRAS-activating point mutations was demonstrated at the cellular level. Respectively selecting 17 cells such as H441, H460 and the like, and observing the cell clone number by a crystal violet staining method, thereby obtaining the biological activity of the compound for inhibiting the growth of cancer cells.

The method comprises the following steps: culturing lung cancer cells in vitro, growing to logarithmic growth phase, digesting and collecting cells, centrifuging at 1000rpm for 5min, discarding supernatant, and adjusting cell concentration to 1.5 × 10⁵mL, cells were seeded into 12-well plates, 1mL per well. mu.L each of the compounds or DMSO was added to the corresponding wells at different concentrations, and the wells were placed in a cell culture chamber (37 ℃ C., 5% CO)₂) After one week of medium incubation (during which the medium was changed once), 4% PFA solution was added to each well for 20 minutes, 0.1% crystal violet solution was added for staining for 20 minutes, PBS washed twice, and scanned for imaging with 100% cell viability in unstimulated DMSO control wells.

FIG. 5 shows that representative compounds ZSQ5-38 and ZSQ8-36 completely or effectively inhibited growth and proliferation of most cell lines at 1. mu.M concentration, and several cell lines, SW1573, H460, etc., at 300nM concentration.

Discussion of the related Art

Among the various proteins closely related to cancer development and progression, multiple members of the kinase protein family have been identified as effective therapeutic targets. Many of the validated or potential kinase targets have reactive sulfydryl near the enzyme catalytic site, thereby providing possibility for the development of covalent kinase inhibitors. While CDK7, 12 and 13 in the cell Cycle Dependent Kinase (CDK) family are the kinase targets of this class.

Cdk (cyclin dependent kinases) family kinases are over ten members of the serine/threonine protein kinase family, some of which are key kinases involved in cell cycle regulation. CDKs can be classified into two main categories according to their functions: CDK1, 2,4 and 6 are involved in cell cycle regulation; CDK7-13 is involved in transcriptional regulation.

CDK7-cyclin H, CDK8-cyclin C, CDK9-cyclin T and CDK12/13-cyclin K regulate initiation (initiation) or extension (elongation) of a transcription process by regulating phosphorylation of RNA polymerase II, so that the initiation or extension (elongation) of the transcription process is promoted to promote the initiation or extension of the transcription process to highly express various cancer-promoting genes (such as MYC, RUNX and the like) through a super-enhancer (super-enhancer), and further the growth and survival of cancer cells are maintained.

CDK12 is an important component for regulating and controlling DNA damage repair in cancer cells, and inactivation of CDK12 can produce synergistic effect with DNA damage repair inhibitors (such as PARP inhibitor olaparib), and can enhance infiltration of immune cells in solid tumors, thereby promoting the therapeutic effect of tumor immunotherapy.

CDK13 is a homologous protein of CDK12, with a structure very similar to CDK12, with only a few amino acid residues different from CDK12, whose known function is identical to CDK 12.

CDK7 is an important transcription factor that regulates the initiation of transcription of oncogenes, while regulating the normal functioning of the cell cycle. Literature reports that inhibition of CDK7 may have an adverse effect on the differentiation of normal cells.

The present invention provides compounds of formula I which selectively inhibit CDK12 and/or 13, but not or weakly inhibit CDK 7. Studies have shown that simultaneous inhibition of CDK7, 12, 13 blocks both activation and extension of transcription, whereas inhibition of CDK12, 13 alone affects only the extension of the transcriptional process. The side effect of inhibiting multiple CDKs on the growth and survival of normal cells is large. In cancer cells, the genes themselves are often unstable and more dependent on DNA damage repair mechanisms, so selective inhibition of CDK12, 13 is sufficient to promote apoptosis in tumor cells.

On the other hand, the conventional triple inhibitor THZ1 of CDK7/12/13 inhibits a plurality of kinases besides three CDKs, and causes toxic and side effects; the improved SY-1365 mainly inhibits CDK7, and has weak inhibition effect on CDK12 and CDK 13; while the dual CDK12/13 inhibitor, which does not inhibit or substantially does not inhibit CDK7, has a significantly better target selectivity than the triple inhibitor (see activity test example 6), tumors of different gene subtypes are also sensitive to different than CDK7 inhibitors.

The CDK inhibitor disclosed by the invention is an optimally obtained targeted inhibitor, in the structure of the compound disclosed by the invention, an organic arsine group (moiey) is a part which has covalent interaction with an active site of CDK12/13, so that excellent inhibitory activity and better specificity can be provided, and from experimental data, the compound disclosed by the invention is specifically bound to specific kinases such as CDK12 and CDK13, is not bound or basically bound to CDK7 or other more than 300 tested kinases, and has good selectivity among different cell strains, so that the compound has safety far better than that of the existing arsines.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (10)

1. A compound of formula I, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof;

wherein the content of the first and second substances,

X₁、X₂each of which isIndependently selected from the group consisting of: none, O, S, NR₈、CH₂；

R₈Selected from the group consisting of: H. substituted or unsubstituted C1-C6 alkyl;

R₁、R₂each independently selected from the group consisting of: H. substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, -CO-Rd, wherein Rd is substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl; or R₁、R₂To adjacent X₁、X₂And As together form a substituted or unsubstituted 4 to 8 membered heterocyclic ring containing one As heteroatom and 0-3 heteroatoms selected from N, O and S;

R₃is H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C3-C10 cycloalkyl;

n₁is 0, 1, 2, 3 or 4;

each R is₄Independently selected from: H. d, OH, amino, nitro, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C3-C10 cycloalkyl;

L₁、L₂and L₃Each independently selected from the group consisting of: no, - (Z) m-; and L is₁And L₂Is not absent at the same time; wherein each Z is independently selected from: C1-C6 alkylene, -NR₆-,-NR₆-R₇-, -O-, -CO-, and m is 1, 2, 3 or 4;

each R₆Independently selected from the group consisting of: H. substituted or unsubstituted C1-C4 alkyl;

R₇is a substituted or unsubstituted C1-C8 alkylene group;

a is selected from the following group: a non-substituted, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-10 membered heteroaryl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl;

b is selected from the following group: substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-10 membered heteroaryl;

c is selected from the following group: H. OH, -N (Ra) Rb, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-10 membered heteroaryl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl;

unless otherwise specified, "substituted" means that a hydrogen atom on a group is replaced with one or more (e.g., 2, 3, 4, etc.) substituents selected from the group consisting of: halogen, deuteration, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy, C1-C6 alkyl S (═ O)₂-, oxo (═ O), -CN, -OH, -n (ra) Rb, carboxyl, or a substituted or unsubstituted group selected from: C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 amino, C6-C10 aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, 5-12 membered heterocyclyl having 1-3 heteroatoms selected from N, S and O, - (CH) 6 alkyl₂) -C6-C10 aryl, - (CH)₂) - (5-to 10-membered heteroaryl having 1 to 3 heteroatoms selected from N, S and O), and the substituents are selected from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkynyl, C1-C6 alkoxy, oxo, -CN, -NH₂OH, -OH, C6-C10 aryl, C1-C6 amino, C2-C6 amido, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O;

wherein Ra and Rb are each independently selected from the group consisting of: H. substituted or unsubstituted C1-C4 alkyl; or Ra, Rb together with the nitrogen atom to which they are attached form a substituted or unsubstituted 3-10 membered heterocycloalkyl having at least 1 (e.g., 1, 2, 3) N heteroatom and 0-2 heteroatoms selected from O, S.

2. The compound of claim 1,

at position 2, 3 or 4.

3. The compound of claim 1,

is composed of

4. The compound of claim 1, wherein the compound of formula I is selected from the following table:

5. a pharmaceutical composition, comprising;

(a) a therapeutically effective amount of a compound of formula I according to claim 1, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof; and (b) a pharmaceutically acceptable carrier.

6. Use of a compound of formula I according to claim 1, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or of a pharmaceutical composition according to claim 5, for the preparation of a pharmaceutical composition for the treatment and/or prevention of a disease or condition associated with the activity or amount of expression of CDK12 and/or CDK 13.

7. The use of claim 6, wherein the disease or condition is cancer.

8. A process for the preparation of a compound of formula I according to claim 1, comprising the steps of:

(a) reacting a compound of formula a4 and a compound of formula a5 in an inert solvent to form a compound of formula I:

in the formula (I), the compound is shown in the specification,

z is selected from: halogen, -OMs, -Ots, -Otf, -Oac or-OAr;

X₁、X₂、R₁、R₂、R₃、R₄、L₁、L₂、L₃a, B, C, and n1 are as defined in claim 1.

9. An intermediate compound having a structure selected from the group consisting of: a1, a2, A3, or a4 structural formula:

wherein Z is selected from: H. halogen, -OMs, -OTs, -OTf, -OAc or-OAr;

X₁、X₂、R₁、R₂、R₃、R₄、L₁、L₂、L₃a, B, C, and n1 are as defined in claim 1.

10. An inhibitor of CDK12 and/or CDK13 comprising:

a compound of formula I according to claim 1, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition according to claim 5.

Images