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

A class of CDK inhibitors based on organic arsine and preparation method and use thereof

technical field

The invention belongs to the field of medicine, and in particular relates to a class of organoarsine-based cell cycle-dependent kinase (CDK) inhibitors and a preparation method and application thereof.

Background technique

In the past two decades, while chemotherapy drugs have been widely used in cancer research, the development and application of targeted drugs have also been in full swing. Among various proteins closely related to the occurrence and progression of cancer, multiple members of the kinase protein family have been verified as effective therapeutic targets, and dozens of targeted kinase inhibitors have also been approved for cancer treatment .

In 2014, Gray et al. reported the development of the first covalent CDK7 based on the cysteine residue Cys312 outside the ATP-binding pocket of the CDK7 kinase domain and on the C-terminal motif, using substituted acrylamide as the electrophilic group. Inhibitor THZ1. In addition to inhibiting CDK7, THZ1 also inhibits CDK12/13, which have cysteine-like residues. Through the inhibition of CDK7/12/13, THZ1 can effectively inhibit the activation of RNA polymerase II at low concentrations, thereby inhibiting the transcriptional initiation of superregulators in cancer cells, and finally effectively inhibiting the growth of cancer cells, and in T cells It has shown good efficacy in various tumor models such as acute lymphoblastic leukemia (T-ALL), small cell lung cancer (SCLC), neuroblastoma, and triple-negative breast cancer. However, THZ1 has significant off-target effects and its pharmacokinetic properties are not ideal.

In 2016, Gray et al. developed a highly specific CDK12/13 inhibitor THZ531 on the basis of THZ1, which can specifically induce apoptosis in some tumor cells, but it still has pharmacokinetic shortcomings. Including such as low bioavailability, short half-life and so on.

Based on THZ1, Syros has improved SY-1365 with better specificity, which is currently in the first phase of clinical trials for the treatment of solid tumors. Limitations in pharmacokinetic properties allow SY-1365 to be administered by intravenous injection.

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

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide CDK inhibitors with high selectivity, high safety, and/or better pharmacokinetic properties. The inhibitors of the present invention are a class of organoarsine-based CDK inhibitors.

The first aspect of the present invention provides a compound of formula I, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof;

in,

X ₁ , X ₂ are each independently selected from the group consisting of none, O, S, NR ₈ , CH ₂ ;

R _is selected from the group consisting of H, substituted or unsubstituted C1-C6 alkyl;

R ₁ and R ₂ are each 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 Substituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl; or R ₁ , R ₂ and adjacent X ₁ , X ₂ and As together form a substituted or unsubstituted 4- to 8-membered heterocycle , the heterocycle contains an As heteroatom and 0-3 heteroatoms selected from N, O and S;

R ₃ is 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;

n ₁ is 0, 1, 2, 3 or 4;

Each R is independently 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;

L ₁ , L ₂ and L ₃ are each independently selected from the group consisting of: none, -(Z)m-; and L ₁ and L ₂ are not both none; wherein each Z is independently selected from: C1-C6 alkylene base, -NR ₆ -, -NR ₆ -R ₇ -, -O-, -CO-, m is 1, 2, 3 or 4;

Each R is independently selected from the group consisting of H, substituted or unsubstituted C1 _- C4 alkyl;

R ₇ is substituted or unsubstituted C1-C8 alkylene;

A is selected from the group consisting of unsubstituted, 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 group consisting of substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-10 membered heteroaryl;

C is selected from the group consisting of H, OH, -N(Ra)Rb, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-10 membered heteroaryl, substituted or unsubstituted C3-C10 ring Alkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl;

Unless otherwise specified, the "substituted" means that the hydrogen atoms on the group are replaced by one or more (eg 2, 3, 4, etc.) substituents selected from the group consisting of halogen, deuterated, 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 the group consisting of: C1-C6 alkyl, C3- C8 cycloalkyl, C1-C6 amine, C6-C10 aryl, 5-10-membered heteroaryl with 1-3 heteroatoms selected from N, S and O, with 1-3 heteroatoms selected from N, 5-12 membered heterocyclyl of heteroatoms of S and O, -( _CH2 )-C6-C10 aryl, -( _CH2 )-(with 1-3 heteroatoms selected from N, S and O 5-10-membered heteroaryl), and the substituent is selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 alkynyl, C1-C6 alkoxy, oxo, -CN, -NH ₂ , -OH, C6-C10 aryl, C1-C6 amine, C2-C6 amido, 5-10-membered heteroaryl with 1-3 heteroatoms selected from N, S and O;

Wherein, Ra and Rb are each independently selected from the following group: H, substituted or unsubstituted C1-C4 alkyl; 3-10 membered heterocycloalkyl with 1, 2, 3) N heteroatoms and 0-2 heteroatoms selected from O, S.

In another preferred example, for R ₃ , the 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, R ₁ and R ₂ together form a substituted or unsubstituted -(CH ₂ )n ₂ - structure; wherein, n ₂ is 1, 2 or 3.

位于2、3或4位。In another preferred example,

in 2, 3 or 4 positions.

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

为

In another preferred example,

for

In another preferred example, in the compound, any one of R ₁ , R ₂ , R ₃ , R ₄ , n ₁ , L ₁ , L ₂ , L ₃ , A, B and C is in Table 1, respectively The corresponding group in the specific compound.

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

A second aspect of the present invention provides 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, the anticancer agents and/or immunosuppressive agents are selected from the group consisting of: PARP1/ 2 Inhibitors, chemotherapy drugs that induce DNA damage in cancer cells, DNA alkylating chemotherapy drugs, DNA or RNA synthesis inhibitors, EGFR, ALK or FGFR tyrosine receptor kinase inhibitors, KRAS, MEK or ERK tumor signaling pathways Inhibitors, tumor immunotherapy drugs (such as PD-1 antibody, PD-L ₁ antibody, etc.).

In another preferred embodiment, the pharmaceutical composition further comprises one or more anticancer agents and/or immunosuppressive agents, preferably, the anticancer agents and/or immunosuppressive agents are selected from the group consisting of: Panib, Lukapanib, Niraparib, Methotrexate, Capecitabine, Gemcitabine, Deoxyfluridine, Pemetrexed Disodium, Pazopanib, Imatinib, Errol tinib, lapatinib, gefitinib, vandetanib, herceptin, bevacizumab, rituximab, trastuzumab, paclitaxel, vinorelbine, docetaxel, Doxorubicin, Hydroxycamptothecin, Mitomycin, Epirubicin, Pirarubicin, Bleomycin, Letrozole, Tamoxifen, Fulvestrant, Triptorelin, Fluta Amine, leuprolide, anastrozole, ifosfamide, busulfan, cyclophosphamide, carmustine, nimustine, semustine, nitrogen mustard, melphalan, leucovorin, carboplatin , Cisplatin, Oxaliplatin, Triboplatin, Topotecan, Camptothecin, Topotecan, Everolimus, Sirolimus, Tetracarcinoma, 6-mercaptopurine, 6-thioguanine, sulfur Azathioprine, mycocin D, daunorubicin, doxorubicin, mitoxantrone, structocin, prucamycin, or aminolutamide.

The third aspect of the present invention provides a compound of formula I according to the first aspect of the present invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof Or the use of the pharmaceutical composition according to the second aspect of the present invention, for preparing a pharmaceutical composition for treating and/or preventing diseases or conditions related to the activity or expression of CDK12 and/or CDK13.

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 lymphoblastic leukemia (T-ALL), small cell lung cancer (SCLC), neuroblastoma, non-small cell lung cancer (NSCLC), colon cancer , acute myeloid 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, gastric cancer, esophagus cancer, bladder cancer, brain tumor, squamous cell carcinoma, peritoneal cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, esophageal adenocarcinoma, esophageal squamous cancer Cell carcinoma, carcinoma in situ, lymphoma, neurofibromatosis, thyroid cancer, bone cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glial tumor or sarcoma.

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

In another preferred embodiment, the cancer is non-CDK7 high expression type.

The fourth aspect of the present invention provides a preparation method of the compound of formula I as described in the first aspect of the present invention, comprising the steps:

(a) The compounds of formula A4 and A5 will be reacted in an inert solvent to form a compound of formula I:

In the formula,

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

X ₁ , X ₂ , R ₁ , R ₂ , R ₃ , R ₄ , L ₁ , L ₂ , L ₃ , A, B, C, and n1 are as defined in the first aspect of the present invention.

The fifth aspect of the present invention provides a preparation method of a compound of formula I, comprising the steps:

(b) reacting compounds of formula A6 and formula A7 in an inert solvent to form a compound of formula I:

In the formula,

The definitions of X ₁ , X ₂ , R ₁ , R ₂ , R ₃ , R ₄ , L ₁ , L ₂ , L ₃ , A, B, C, and n ₁ are as described in the first aspect of the present invention.

The sixth aspect of the present invention provides an intermediate compound, the intermediate compound has a structural formula selected from: A1, A2, A3 or A4:

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

X ₁ , X ₂ , R ₁ , R ₂ , R ₃ , R ₄ , L ₁ , L ₂ , L ₃ , A, B, C, and n1 are as defined in the first aspect of the present invention.

The seventh aspect of the present invention provides the use of a compound A1, A2, A3 or A4 for preparing compound I.

The eighth aspect of the present invention provides a CDK12 and/or CDK13 inhibitor, comprising:

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

A ninth aspect of the present invention provides a method for selectively inhibiting cell cycle-dependent kinases in vitro, comprising the steps of:

The cell cycle-dependent kinase is combined with a compound of formula I as described in the first aspect of the present invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof or as described herein. The pharmaceutical composition of the second aspect of the invention is contacted to selectively inhibit the 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 CDK13.

In another preferred embodiment, the "selectively inhibit" refers to the compound's inhibitory activity E1 on targeted CDKs (such as CDK12 and/or CDK13) and on non-targeted kinases (such as CDK7 or selected from the table The ratio of inhibitory activity E2 (E1/E2) of 4 other non-targeted kinases) is ≥2, preferably ≥5, more preferably ≥10.

In another preferred embodiment, the inhibitory activity E is the reciprocal of EC ₅₀ .

A tenth aspect of the present invention provides a method for inhibiting the growth or proliferation of tumor cells in vitro, comprising the steps of:

Tumor cells are combined with a compound of formula I as described in the first aspect of the present invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof or as in the second aspect of the present invention. The pharmaceutical composition of the aspect is contacted.

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

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.

Description of drawings

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

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

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

Figure 4 shows the plasma concentration-time profiles of representative compounds ZSQ5-38 and ZSQ8-36, along with pharmacokinetic parameters.

Figure 5 shows the inhibitory effects of representative compounds ZSQ5-38 and ZSQ8-36 on the growth and proliferation of various cell lines at a concentration of 1 μM.

Detailed ways

After extensive and in-depth research and extensive screening and testing, the inventors unexpectedly developed a class of compounds with high inhibitory activity on CDK12/13, but no or low inhibitory activity on CDK7. The compound of the present invention is an organoarsine-based cell cycle-dependent kinase inhibitor represented by formula I. In the structure of the compound of the present invention, the moiety is the part that covalently interacts with the active site of CDK12/13 , thus providing superior inhibitory activity and better specificity. The experimental results show that the compounds of the present invention have unexpectedly excellent specificity for targeting CDK12/13, high safety and few toxic and side effects. In addition, the compound of the present invention also has excellent pharmacokinetic properties, long half-life, and is suitable for medicine. The present invention has been completed on this basis.

the term

Unless otherwise defined, 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.

the term

Unless otherwise stated, 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 (ie, C1-8 represents 1-8 carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like .

Unless otherwise stated, 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 vinyl, 2-propenyl, crotyl, 2-prenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1, 4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl and higher homologs and isomers.

Unless otherwise stated, the term "cycloalkyl" refers to a hydrocarbon ring having the specified number of ring atoms (eg, _C3-10 cycloalkyl) and either fully saturated or having no more than one double bond between the ring tips. "Cycloalkyl" also refers to bicyclic and polycyclic hydrocarbon rings, such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, and the like.

Unless otherwise stated, 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 quaternary Ammonized. Heterocycloalkyl can be a monocyclic, bicyclic or polycyclic ring system. Non-limiting examples of heterocycloalkyl include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyranone, tetrahydrofuran, tetrahydrothiophene, quinuclidine, etc. A heterocycloalkyl group can be attached to the remainder of the molecule through a ring carbon or a heteroatom. For terms such as cycloalkylalkyl and heterocycloalkylalkyl, it is meant that the cycloalkyl or heterocycloalkyl is attached to the remainder of the molecule through an alkyl or alkylene linker. For example, cyclobutylmethyl- is a cyclobutyl ring attached to a methylene linker on the rest of the molecule.

Unless otherwise stated, the term "alkylene" by itself or as part of another substituent refers to a _divalent group derived from an _alkane , _eg , _{-CH2CH2CH2CH2-} . Alkyl groups (or alkylene groups) typically have 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms (eg, 1, 2, 3, 4, 5, or 6) being preferred herein. "Lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, usually having 4 or fewer carbon atoms. Similarly, "alkenylene" or "alkynylene" refer to unsaturated forms of "alkylene" having double or triple bonds, respectively.

Unless otherwise indicated, the terms "alkoxy" or "alkyloxy", "alkylamino" or "alkylamino" and "alkylthio" or "alkylthio" (or thioalkoxy ) is used in its conventional sense to refer to those alkyl groups attached to the rest of the molecule via an oxygen atom, an amino group or a sulfur atom, respectively. In addition, for dialkylamino groups, the alkyl moieties may be the same or different, or the same as the respective The nitrogen atoms to which the alkyl groups are attached combine to form a 3-7 membered ring.

Unless stated otherwise, the term "halo" or "halogen" by itself or as part of another substituent refers to a fluorine, chlorine, bromine, or iodine atom. In addition, terms such as "haloalkyl" are meant to include monohaloalkyl or polyhaloalkyl. For example, the term "C _1-4 haloalkyl" is meant to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

Unless otherwise stated, the term "aryl" refers to a polyunsaturated (usually aromatic) hydrocarbon group, which may be monocyclic or polycyclic (up to three rings) fused together or covalently linked. The term "heteroaryl" refers to an aryl group (or ring) containing 1 to 5 heteroatoms selected from N, O, and S, which may be monocyclic or polycyclic fused together or covalently linked ( Bicyclic, up to tricyclic) wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of aryl groups include phenyl, naphthyl, and biphenyl, while non-limiting examples of heteroaryl groups include pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolyl, Quinoxalinyl, quinazolinyl, cinnoline, phthalazinyl, benzotriazin, purinyl, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzoyl Isoxazolyl, isobenzofuryl, isoindolyl, indolizine, benzotriazinyl, thienopyridyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridine , benzothiazolyl, benzofuranyl, benzothienyl, indolyl, quinolinyl, isoquinolinyl, isothiazolyl, pyrazolyl, indazolyl, pteridyl, imidazolyl, triazole oxazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furyl, thienyl and the like. 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 intended to include oxygen (O), nitrogen (N), sulfur (S), and silicon (Si).

For the compounds provided herein, a bond from a substituent (usually an R group) to the center of an aromatic ring (eg, benzene, pyridine, etc.) will be understood to mean a bond that provides a link at any available vertex of the aromatic ring. In some embodiments, the description also includes linkages on rings fused to aromatic rings. 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 moiety of the indole.

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

Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers, regioisomers, and individual isomers (eg, isolated enantiomers) body) shall be included within the scope of the present invention. When compounds provided herein have a defined stereochemistry (denoted as R or S, or indicated with a dashed line or a wedge bond), those compounds will be understood by those of skill in the art to be substantially free of other isomers (eg, at least 80% , 90%, 95%, 98%, 99% and up to 100% free of other isomers).

As used herein, unless otherwise specified, the term "substituted" refers to the replacement of one or more hydrogen atoms on a group with a substituent selected from the group consisting of halogen, deuterated, C1-C6 alkyl, C2-C6 alkene base, 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 _b , carboxyl, or a substituted or unsubstituted group selected from the group consisting of C1-C6 alkyl, C3-C8 cycloalkyl, C1- C6 amine group, C6-C10 aryl group, 5-10 membered heteroaryl group with 1-3 heteroatoms selected from N, S and O, 1-3 heteroatoms selected from N, S and O 5-12 membered heterocyclyl, -(CH ₂ )-C6-C10 aryl, -(CH ₂ )-(5-10 membered heteroaryl with 1-3 heteroatoms selected from N, S and O ), and the substituent is selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 alkynyl, C1-C6 alkoxy, oxo, -CN, -NH ₂ , -OH, C6-C10 Aryl, C1-C6 amine, C2-C6 amide, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O.

As used herein, the terms "containing", "comprising" or "including" mean that the various ingredients can be used together in the mixture or composition of the present 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 (eg, toxicity, irritation, and allergy), ie, with a reasonable benefit/risk ratio.

As used herein, the term "therapeutically effective dose" refers to any amount of a drug, as described below, that, when used alone or in combination with another therapeutic agent, promotes disease regression, which manifests as disease symptoms decrease the severity of disease, increase the frequency and duration of disease-free periods, or prevent impairment or disability resulting from the disease. A "therapeutically effective dose" of a drug of the present invention also includes a "prophylactically effective dose", which is any amount of the drug as described below, when the amount of the drug is administered alone or in combination with another therapeutic agent In a subject at risk of developing the disease or suffering from relapse of the disease, the occurrence or relapse of the disease can be inhibited.

As used herein, "pharmaceutically acceptable salts" refer to salts of compounds of the present invention with acids or bases that are suitable for use as pharmaceuticals. Pharmaceutically acceptable salts include inorganic and organic salts. A preferred class of salts are the salts of the compounds of the present invention with acids. Acids suitable for forming salts include, but are not limited to, inorganic acids such as hydrochloric, hydrobromic, hydrofluoric, sulfuric, nitric, phosphoric, formic, acetic, propionic, oxalic, malonic, succinic, fumaric, Maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenemethanesulfonic acid, benzenesulfonic acid and other organic acids; and acidic amino acids such as aspartic acid and glutamic acid. A preferred class of salts are the salts of the compounds of the present invention with bases. 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 from water or various organic solvents, in which case various solvates may be formed. Solvates of the present invention include stoichiometric solvates such as hydrates and the like, as well as compounds containing variable amounts of water formed when prepared by low pressure sublimation drying.

The present invention also includes all suitable isotopic variations of the compounds of the present invention. Isotopic variants of the compounds of the present invention are defined as those in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from that commonly found in nature. Examples of isotopes that may be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as 2H, ^3H , ^11C , ^13C , ^14C , ^15N , ¹⁷ , ^respectively O, ¹⁸ O, ³⁵ S, ¹⁸ F and ³⁶ Cl. Some isotopic variants of the present invention, eg, those into which radioactive isotopes (eg, ^{3H or14C} ) are incorporated, are used in drug and/or substrate tissue distribution studies. Tritiated, ie, ³ H, and carbon-14, ie, ¹⁴ C, isotopes are particularly preferred because of their ease of preparation and detection. Furthermore, substitution with isotopes (eg, deuterium, ie, ² H) may provide some therapeutic advantages resulting from increased metabolic stability, eg, increased in vivo half-life or reduced dosage requirements and may therefore be preferred in some cases of. Isotopic variants of the compounds of the present invention can generally be prepared by routine procedures, eg, by using the appropriate reagents for the appropriate isotopic variant, by the exemplified methods or the preparations described in the experimental section below.

表示与其他原子的连接。

Represents connections to other atoms.

As used herein, "treating" refers to alleviating, delaying progression, attenuating, preventing, or maintaining an existing disease or disorder (eg, cancer). Treatment also includes curing, preventing the development or alleviating to some extent one or more symptoms of a 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 "ZSQ5-38" and "ZSQ538" to refer to the ZSQ-5-38 compound, and the rules for using similar numbers are the same.

Active ingredient

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

X ₁ , X ₂ , R ₁ , R ₂ , R ₃ , R ₄ , L ₁ , L ₂ , L ₃ , A, B, C, and n1 are as defined in the first aspect of the present invention.

Preparation

The present invention provides methods for the preparation of compounds of formula I. The compounds of the present invention can be prepared by various synthetic operations. Exemplary preparation methods of these compounds can include, but are not limited to, the schemes described below.

Preferably, the compound of formula I of the present invention can be completed by the exemplary methods described in the following schemes and examples and the relevant published literature operations 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 arsenous acid functional group of A1 is protected in the form of ester or amide, and ethanedithiol, ethylene glycol, etc. can be used as protective reagents.

Step ii: Introducing _R3 substituent at the amine group of A2 by S _N2 substitution reaction, R3-halogen can be used, and the reaction is performed under basic conditions (eg, sodium hydride).

Step iii: the acylation reaction of the phenyl ring amino group of A3 to introduce the L1 group, which can be catalyzed by (but not limited to) phosphorus oxychloride reagent.

Step iv: Link the A functional group site of A5 to L ₁ of A4 through acylation reaction, urea-forming reaction or S _N 2 substitution.

The reactions in the above steps are all conventional reactions known to those skilled in the art. Unless otherwise specified, the reagents and starting compounds used in the synthetic route are commercially available, or prepared by those skilled in the art with reference to known methods according to the designed structures of different compounds.

Pharmaceutical compositions and methods of administration

Since the compounds of the present invention have excellent inhibitory activity against cell cycle-dependent kinases (CDKs), the compounds of the present invention and pharmaceutically acceptable salts, hydrates or solvates thereof, and medicines containing the compounds of the present invention as the main active ingredient The compositions can be used to treat, prevent and alleviate diseases or conditions associated with CDK activity or expression.

Preferably, the compounds of the present invention are useful in the treatment of cancer. Representative cancers include (but are not limited to): T-cell acute lymphoblastic leukemia (T-ALL), small cell lung cancer (SCLC), neuroblastoma, non-small cell lung cancer (NSCLC), colon cancer, acute myeloid 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, esophagus cancer, bladder cancer, brain tumor, squamous cell carcinoma, peritoneal cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, in situ Cancer, lymphoma, neurofibromatosis, 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 and a pharmacologically acceptable excipient or carrier within a safe and effective amount. The "safe and effective amount" refers to: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably 10-500 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.

)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。"Pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid filler or gel substances which are suitable for human use and which must be of sufficient purity and sufficiently low toxicity. "Compatibility" as used herein means that the components of the composition can be admixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid) , magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as

), wetting agents (such as sodium lauryl sulfate), colorants, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

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 (a) fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) Absorption accelerators such as quaternary amine compounds; (g) wetting agents such as cetyl alcohol and glyceryl monostearate; (h) adsorbents such as kaolin; and (i) lubricants such as talc, hard Calcium fatty acid, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared using coatings and shell materials, 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 in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be employed are polymeric substances and waxes. 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 compound, liquid dosage forms may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, and the like.

Besides 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 such as 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 non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms for topical administration of the compounds of this 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 that 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, (eg, PARP1/2 inhibitors (Olaparid, Rucaparib, or Niraparib, etc.), chemotherapeutic drugs that induce DNA damage in cancer cells (cis Platinum, carboplatin or oxaliplatin, etc.), DNA alkylating chemotherapy drugs (nimustine, etc.), DNA or RNA synthesis inhibitors (gemcitabine, etc.), tyrosine receptor kinases such as EGFR, ALK or FGFR Inhibitors, tumor signaling pathway inhibitors such as KRAS, MEK, or ERK, tumor immunotherapy drugs (PD- ₁ antibody, PD-L1 antibody, etc.); in certain embodiments, the compounds of the present invention are administered in combination to cancer subjects and other traditional cancer treatments, eg, 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 inhibitors of the invention are used concomitantly with, or sequentially with, other agents that are part of a combination therapy regimen, in the same or separate formulations.

Typical ranges for therapeutically effective doses of compounds of formula I or compositions of compounds of formula I will be: about 1-2000 mg/day, about 10- about 1000 mg/day, about 10- about 500 mg/day, about 10- 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 is to be understood, however, that the particular dosage of a compound of the present invention for any particular patient will depend on a variety of factors, eg, the age, sex, weight, general health, diet, individual response, time of administration, and treatment of the patient to be treated. disease severity, activity of the particular compound administered, dosage form, mode of application, and concomitant medications. 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 delivery of a therapeutically effective amount.

The main advantages of the present invention include:

1. For the first time in the compounds of the present invention, an organic arsine moiety is used as a moiety that covalently interacts with the active site of CDK12/13, thus providing excellent inhibitory activity and better specificity.

2. The inhibitory activity of CDK12 and 13 is better than that of the existing inhibitors, which can effectively inhibit the growth and proliferation of cancer cells.

3. The inhibition of other intracellular kinases is not obvious, the selectivity is high, the targeting is good, the safety is good, and there is no obvious toxic and side effects at the effective concentration;

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

5. The combination of the compound of the present invention with CDK12 and 13 is irreversible, and its drug effect lasts longer and its curative effect is more lasting.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the experimental methods without specific conditions are usually in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise specified.

Example 1

Synthetic route of compound ZSQ-5-38

Synthesis of Compound (4-aminophenyl) arsine (ZSQ-1-18)

4-Aminophenylarsinic acid (108.5 g, 500 mmol) was dissolved in 300 ml of ethanol, and the mixed solution was heated to reflux. Phenylhydrazine (92 ml, 1 mol) was added dropwise (over 1 h), a large amount of nitrogen was generated during the addition, and when the nitrogen generation slowed down, reflux stirring was continued for 1.5 h. The mixed solution was concentrated by distillation under reduced pressure, sodium hydroxide solution (40 g dissolved in 400 ml of water) and 400 ml of ether were added. Separation, adding saturated ammonium chloride solution (400 ml) to the aqueous phase and stirring at 0° C. for 1 h, a large amount of white solid was precipitated. The white needle-like solid was obtained by filtration, and dried in vacuo to obtain 40 g of the product with a yield of 40%.

Synthesis of Compound 4-(1,3,2-Dithiasopentane-2-yl)aniline (ZSQ-1-23)

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

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

ZSQ-1-23 (1.03 g, 4 mmol) and triethylamine (0.83 ml, 6 mmol) were dissolved in 20 ml of dry DCM solution and stirred at 0°C. Bromoacetyl bromide (0.38 ml, 4.4 mmol) was added dropwise over 15 min and stirring was continued for 1 h at 0 °C. 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), extracted with DCM, and the organic phases were combined, Dry over anhydrous sodium sulfate, filter and concentrate. It was separated and purified by silica gel column chromatography and eluted with PE/EA (4:1) to obtain 1.02 g of a yellow solid with a yield of 67%.

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

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

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

ZSQ-3-76 (5.28 g, 20 mmol), NaOH (1.2 g, 30 mmol) and _Bu4NHSO4 (5.8 g, ₁₀ mmol) were dissolved in 100 ml of dichloromethane, and benzenesulfonyl chloride (3.84 ml) was added dropwise at room temperature , 30 mmol). The reaction mixture was continued to stir at room temperature for 3 h. The mixture was quenched with water (100 ml) and extracted with DCM (50 ml x 3). The combined organic layers _were dried over anhydrous _Na2SO4 , filtered, concentrated, separated and purified by silica gel column chromatography, eluted with DCM/EA (8:1) to give 6.79 g of white solid in 84% yield.

Compound (R)-3-((5-Chloro-4-(1-(phenylsulfonyl)-1H-indol-3-yl)pyrimidin-2-yl)amino)piperidine-1-carboxylic acid tertiary Synthesis of Butyl Ester (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 in 3ml N-methylpyrrolidone middle. The mixed solution was heated and stirred at 135 °C for 3 h. The cooled solution was directly separated and purified by C18 reverse phase column chromatography, eluted with H ₂ O/CH ₃ CN (1:9), to obtain 802 mg of yellow-brown powder with a yield of 94%.

Compound (R)-5-Chloro-4-(1-(phenylsulfonyl)-1H-indol-3-yl)-N-(piperidin-3-yl)pyrimidin-2-amine (ZSQ-8 -31) synthesis

ZSQ-8-30 (802 mg, 1.4 mmol) and 1 ml of trifluoroacetic acid were mixed in 5 ml of DCM and stirred at room temperature overnight. After the completion of the reaction, the reaction solution was concentrated and separated and purified by C18 reverse phase column chromatography, eluted with H ₂ O/CH ₃ CN (1:6), to obtain 620 mg of yellow-brown powder with a yield of 94%.

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

ZSQ-8-31 (140 mg, 0.3 mmol), DIPEA (0.15 ml, 0.9 mmol) and ZSQ-5-4 (114 mg, 0.3 mmol) were dissolved in 5 ml of THF solution at room temperature, and the mixed solution was stirred at room temperature 4h. It was then washed with 2N dilute hydrochloric acid solution (20ml) and extracted with EA (3x30ml) followed by water (2x20ml). The organic phases were combined, dried _over anhydrous _Na2SO4 , filtered and concentrated under reduced pressure, separated and purified by silica gel column chromatography, eluted with DCM/EA (4:1) to give 209 mg of white powder, yield 91%.

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

ZSQ-8-34 (209 mg, 0.27 mmol) and anhydrous potassium carbonate (113 mg, 0.82 mmol) were dissolved in a mixed solution of 5 ml of anhydrous methanol and 5 ml of anhydrous THF, and stirred at room temperature for 3 h. After the reaction was completed, the pH was adjusted to weakly acidic with TFA, and the reaction solution was directly separated and purified by C18 reverse-phase column chromatography, and eluted with H ₂ O/CH ₃ CN (1:6) to obtain 123 mg of yellow powder with a yield of 73%.

Compound (R)-(4-(2-(3-((5-Chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)piperidin-1-yl)acetamido) Synthesis of Phenyl)arsine (ZSQ-5-38)

ZSQ-8-36 (123mg, 0.19mmol), mercuric perchlorate trihydrate (71mg, 0.157mmol) were dissolved in 3ml DMSO, the mixed solution was stirred at room temperature for 10min, and immediately separated and purified by C18 reverse phase column chromatography, Elution with _H2O / _CH3CN (1:4) gave 102 mg of a yellow powder in 92% yield.

Example 2:

Synthetic route of compound ZSQ-5-70

Compound (tert-butyl 3-(2-((4-(1,3,2-dithiazolan-2-yl)phenyl)amino)-2-oxoethyl)-phenyl)carbamic acid Synthesis of Esters (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 DCM and placed in Mix and stir at room temperature for 4h. After the reaction was completed, washed with H ₂ O (20ml×2) and saturated NaCl solution (20ml) in turn, extracted with 20ml of DCM, combined the organic phases, dried over anhydrous sodium sulfate, filtered, concentrated and separated and purified by silica gel column chromatography, Elution with PE/EA (3:1) gave a white solid 412 mg in 83% yield.

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

ZSQ-5-57-1 (412 mg, 0.83 mmol) and 1 ml of trifluoroacetic acid were mixed in 5 ml of DCM and stirred at room temperature overnight. After completion of the reaction, the reaction solution was concentrated and separated and purified by C18 reverse-phase column chromatography, eluted with H ₂ O/CH ₃ CN (1:7), to obtain 290 mg of white solid with a yield of 89%.

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

ZSQ-5-26 (202 mg, 0.5 mmol), ZSQ-5-57-2 (196 mg, 0.5 mmol) and p-toluenesulfonic acid monohydrate (285 mg, 1.5 mmol) were mixed and dissolved in 2 ml of N-methylpyrrolidone. The mixed solution was heated and stirred at 135 °C for 2 h. The cooled solution was directly separated and purified by C18 reverse phase column chromatography, eluting with H ₂ O/CH ₃ CN (1:9), to give 86 mg of yellow powder in 22% yield.

Compound N-(4-(1,3,2-Dithiarsinopentan-2-yl)phenyl)-2-(3-((5-chloro-4-(1H-indol-3-yl) )-pyrimidin-2-yl)amino)phenyl)acetamide (ZSQ-5-66) synthesis

ZSQ-5-63 (86 mg, 0.11 mmol) and anhydrous potassium carbonate (47 mg, 0.33 mmol) were dissolved in a mixed solution of 3 ml of anhydrous methanol and 3 ml of anhydrous THF, and stirred at room temperature for 4 h. After the reaction was completed, the pH was adjusted to weakly acidic with TFA, and the reaction solution was directly separated and purified by C18 reverse-phase column chromatography, and eluted with H ₂ O/CH ₃ CN (1:6) to obtain 31 mg of yellow powder with a yield of 45%.

Compound (4-(2-(3-((5-Chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)acetamido)-phenyl)arsinidine ( Synthesis of ZSQ-5-70)

ZSQ-5-66 (31mg, 0.05mmol), mercuric perchlorate trihydrate (18mg, 0.04mmol) were dissolved in 2ml DMSO, the mixed solution was stirred at room temperature for 10min, and immediately separated and purified by C18 reverse phase column chromatography, Elution with _H2O / _CH3CN (1:4) gave 21 mg of a yellow powder in 75% yield.

Example 3

Synthetic route of compound ZSQ-7-84

Compound 3-(((5-Chloro-4-(1-(phenylsulfonyl)-1H-indol-3-yl)pyrimidin-2-yl)amino)methyl)piperidine-1-carboxylic acid tertiary Synthesis of Butyl Ester (ZSQ-7-72)

ZSQ-5-26 (606 mg, 1.5 mmol), 1-Boc-3-aminomethylpiperidine (354 mg, 1.65 mmol) and DIPEA (0.74 ml, 4.5 mmol) were mixed in 3 ml of N-methylpyrrolidone. The mixed solution was heated and stirred at 135 °C for 3 h. The cooled solution was directly separated and purified by C18 reverse phase column chromatography, eluting with H ₂ O/CH ₃ CN (1:9) to give 630 mg of a yellow solid in 72% yield.

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

ZSQ-7-72 (630 mg, 1.08 mmol) and 1 ml of trifluoroacetic acid were mixed in 5 ml of DCM and stirred at room temperature overnight. After completion of the reaction, the reaction solution was concentrated and separated and purified by C18 reverse-phase column chromatography, eluted with H ₂ O/CH ₃ CN (1:6), to obtain 501 mg of a yellow-brown solid with a yield of 96%.

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

ZSQ-1-23 (130 mg, 0.5 mmol), bis(trichloromethyl)carbonate (59 mg, 0.2 mmol) were dissolved in 5 ml of THF, stirred at 0 °C, and DIPEA (207 ul, 1.25 mmol) was added gradually. It was added dropwise, and the mixed solution was stirred at 0° C. for 1 h after the addition. ZSQ-7-76 (241 mg, 0.5 mmol) was added, and after the addition, stirring was continued at 0° C. for 0.5 h, and then moved to room temperature and stirred for 2 h. After the reaction was completed, washed with 2N dilute hydrochloric acid solution (20ml), saturated sodium bicarbonate solution (20ml), saturated sodium chloride solution (20ml) in turn, extracted with 40ml DCM, combined organic phases were dried with anhydrous sodium sulfate, filtered and passed through concentrate. It was separated and purified by silica gel column chromatography and eluted with PE/EA (2:1) to obtain 178 mg of white powder with a yield of 46%.

Compound N-(4-(1,3,2-Dithiasopentan-2-yl)phenyl)-3-(((5-chloro-4-(1H-indol-3-yl)- Synthesis of Pyrimidine-2-yl)amino)methyl)piperidine-1-carboxamide (ZSQ-7-83)

ZSQ-7-80 (178 mg, 0.23 mmol) and anhydrous potassium carbonate (96 mg, 0.69 mmol) were dissolved in a mixed solution of 5 ml of anhydrous methanol and 5 ml of anhydrous THF, and mixed and stirred at room temperature for 3 h. After the reaction was completed, the pH was adjusted to weakly acidic with TFA, and the reaction solution was directly separated and purified by C18 reverse-phase column chromatography, and eluted with H ₂ O/CH ₃ CN (1:7) to obtain 124 mg of yellow powder with a yield of 86%.

Compound (4-(3-(((5-Chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)methyl)piperidine-1-carboxamido)phenyl)idene Synthesis of Arsinic Acid (ZSQ-7-84)

ZSQ-7-83 (124mg, 0.2mmol), mercuric perchlorate trihydrate (71mg, 0.16mmol) were dissolved in 3ml DMSO, the mixed solution was stirred at room temperature for 10min, immediately separated and purified by C18 reverse phase column chromatography, Elution with _H2O / _CH3CN (1:4) gave 78 mg of yellow powder in 70% yield.

Example 4

Synthetic route of compound ZSQ-7-105

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

Mix 2M lithium diisopropylamide solution (27.5ml, 55mmol) in 50ml dry THF, stir at -78°C, add isovaleronitrile (5.24ml, 50mmol) dropwise, continue after adding Stir for 30min, mix ethyl formate (4.85ml, 60mmol) in 25ml of dry THF, add dropwise to the mixed solution (over 30min) at -78°C, continue stirring for 45min after adding, move to room temperature, stir 14h. After the reaction was completed, 10ml of H ₂ O was added to quench, the mixed solution was concentrated, diluted hydrochloric acid solution was added to adjust the pH to 3, and then extracted with EA (20ml×3), the organic phase was dried with anhydrous sodium sulfate, filtered and concentrated, and drained 5 g of crude product in the form of tan oil were obtained.

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

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

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

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

Synthesis of Compound 5,7-dichloro-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, heated to 115°C overnight with reflux stirring . After the reaction was completed, the mixed solution was concentrated, and the remaining solution was added dropwise to ice water, extracted several times with DCM, washed with saturated sodium chloride solution (30 ml), and the organic phases were combined. Dry with anhydrous sodium sulfate, filter, concentrate and separate and purify by silica gel column chromatography, eluted with PE/EA (4:1), to obtain 950 mg of yellow solid, yield 62%.

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

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

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

ZSQ-7-90 (902 mg, 3 mmol), 4-dimethylaminopyridine (37 mg, 0.3 mmol) and di-tert-butyl dicarbonate (897 ul, 3.9 mmol) were dissolved in 15 ml of THF, and the mixture was stirred at room temperature for 12 h. After the reaction was completed, it was washed with H ₂ O (20 ml×2), extracted with EA (30 ml), and washed with saturated sodium bicarbonate solution (30 ml). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and separated and purified by silica gel column chromatography, eluted with PE/EA (8:1), to obtain 1.20 g of a pale yellow flake solid with a yield of 99%.

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

Compound ZSQ-7-94 (400mg, 1mmol), 1-Boc-3-aminomethylpiperidine (236mg, 1.1mmol), tris(dibenzylideneacetone)dipalladium (46mg, 0.05mmol), 1, 1'-Binaphthalene-2,2'-bisdiphenylphosphine (93 mg, 0.15 mmol) and sodium tert-butoxide (106 mg, 1.1 mmol) were mixed in 3 ml of toluene, heated to 95 °C under nitrogen atmosphere and stirred for 16 h. After the reaction was completed, it was washed with H ₂ O (30 ml×3), extracted with EA (40 ml), and washed with saturated sodium chloride solution (30 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, separated and purified by silica gel column chromatography, and eluted with PE/EA (5:1) to obtain 554 mg of yellow-brown powder with a yield of 95%.

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

ZSQ-7-96 (554 mg, 0.96 mmol) and 1 ml of trifluoroacetic acid were mixed in 5 ml of DCM and stirred at room temperature overnight. After completion of the reaction, the reaction solution was concentrated and separated and purified by C18 reverse-phase column chromatography, eluted with H ₂ O/CH ₃ CN (1:5), to obtain 360 mg of white solid with a yield of 99%.

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

ZSQ-1-23 (130 mg, 0.5 mmol), bis(trichloromethyl)carbonate (59 mg, 0.2 mmol) were dissolved in 5 ml of THF, stirred at 0 °C, and DIPEA (207 ul, 1.25 mmol) was added gradually. It was added dropwise, and the mixed solution was stirred at 0° C. for 1 h after the addition. ZSQ-7-98 (189 mg, 0.5 mmol) was added, and after the addition, stirring was continued at 0° C. for 0.5 h, and then moved to room temperature and stirred for 2 h. After the reaction was completed, washed with 2N dilute hydrochloric acid solution (20ml), saturated sodium bicarbonate solution (20ml), saturated sodium chloride solution (20ml) in turn, extracted with 40ml DCM, combined organic phases were dried with anhydrous sodium sulfate, filtered and passed through concentrate. It was isolated and purified by silica gel column chromatography, eluted with DCM/EA (1:1), to give 162 mg of white powder in 49% yield.

Compound (4-(3-(((7-(benzylamino)-3-isopropylpyrazolo[1,5-a]pyrimidin-5-yl)amino)-methyl)piperidine-1- Synthesis of 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 mixed solution was stirred at room temperature for 10min, and immediately separated and purified by C18 reverse phase column chromatography, Elution with _H2O / _CH3CN (1:5) gave 56 mg of white powder in 78% yield.

Example 5

Synthetic route of compound ZSQ-8-12

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

3-(N-Boc-aminomethyl)benzoic acid (1.0 g, 4 mmol) and potassium carbonate (1.10 g, 8 mmol) were dissolved in 10 ml of DMF, and after stirring at room temperature for 20 min, benzyl bromide (522 ul, 4.4 mmol) was added to the solution. It was added dropwise, and stirring was continued at room temperature for 6 h after the addition. After the reaction was completed, it was washed with H ₂ O (30 ml×3) and extracted with EA. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. It was separated and purified by silica gel column chromatography and eluted with PE/EA (3:1) to obtain 1.2 g of white solid with a yield of 88%.

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

ZSQ-7-104 (1.2 g, 3.5 mmol) and 1 ml of trifluoroacetic acid were mixed in 5 ml of DCM and stirred at room temperature overnight. After the completion of the reaction, the reaction solution was concentrated and separated and purified by C18 reverse phase column chromatography, eluted with H ₂ O/CH ₃ CN (1:6), to obtain 800 mg of white solid with a yield of 95%.

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

Compound ZSQ-7-94 (200 mg, 0.5 mmol), ZSQ-8-1 (133 mg, 0.55 mmol), tris(dibenzylideneacetone)dipalladium (23 mg, 0.025 mmol), 1,1'-binaphthyl -2,2'-Bisdiphenylphosphine (47 mg, 0.075 mmol) and sodium tert-butoxide (58 mg, 0.6 mmol) were mixed in 2 ml of toluene, heated to 95 °C under nitrogen atmosphere and stirred for 16 h. After the reaction was completed, it was washed with H ₂ O (30 ml×3), extracted with EA (40 ml), and washed with saturated sodium chloride solution (30 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, separated and purified by silica gel column chromatography and eluted with PE/EA (4:1) to obtain 154 mg of pale yellow powder with a yield of 51%.

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

ZSQ-8-3 (154 mg, 0.25 mmol) and Pd/C (100 mg) were mixed in 10 ml of methanol, hydrogenated and stirred at room temperature for 6 h. After the reaction was completed, it was filtered with celite, washed with methanol, and the filtrate was concentrated and separated and purified by C18 reverse-phase column chromatography, eluted with H ₂ O/CH ₃ CN (1:7), to obtain 116 mg of yellow oily product with a yield of 90 %

Compound (5-((3-((4-(1,3,2-dithiasopentan-2yl)phenyl)carbamoyl)benzyl)amino)-3-isopropylpyrazolo Synthesis of tert-butyl [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 dissolved in 3ml of dry pyridine, the mixed solution was stirred at 0°C, phosphorus oxychloride (42ul, 0.45mmol) It was added dropwise to the mixed solution (over 10 min), and after the addition, stirring was continued at 0° C. for 1 h, and then moved to room temperature and stirred for 2 h. After the reaction was completed, it was acidified with dilute hydrochloric acid solution, washed with water (30ml×3), extracted with EA, and washed with saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. It was separated and purified by silica gel column chromatography, eluted with PE/EA (4:1) to wash out the pyridinium salt, and then eluted with DCM/CH ₃ OH(NH ₃ ) (10: 1) to obtain yellow powder 92 mg, yield 54 %.

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

ZSQ-8-8 (92 mg, 0.12 mmol) and 0.5 ml trifluoroacetic acid were mixed in 5 ml DCM and stirred at room temperature overnight. After completion of the reaction, the reaction solution was concentrated and separated and purified by C18 reverse-phase column chromatography, eluted with H ₂ O/CH ₃ CN (1:7), to obtain 64.8 mg of white solid with a yield of 82%.

Compound (4-(3-(((7-(benzylamino)-3-isopropylpyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)benzamido)benzene Synthesis of Arsinic Acid (ZSQ-8-12)

ZSQ-8-9 (64.8 mg, 0.099 mmol) and mercuric perchlorate trihydrate (33 mg, 0.074 mmol) were dissolved in 2 ml of DMSO, the mixed solution was stirred at room temperature for 10 min, and immediately separated and purified by C18 reverse-phase column chromatography, Elution with _H2O / _CH3CN (1:5) gave 45 mg of white powder in 76% yield.

Example 6

Synthetic route of compound ZSQ-9-73

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

Compound ZSQ-7-94 (200 mg, 0.5 mmol), (R)-1-Boc-3-aminopiperidine (110 mg, 0.55 mmol), tris(dibenzylideneacetone)dipalladium (23 mg, 0.025 mmol) , 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (47mg, 0.075mmol) and sodium tert-butoxide (53mg, 0.55mmol) were mixed in 2ml of toluene, heated to 95°C under nitrogen atmosphere and stirred 16h. After the reaction was completed, it was washed with H ₂ O (30 ml×3), extracted with EA (40 ml), and washed with saturated sodium chloride solution (30 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, separated and purified by silica gel column chromatography, and eluted with PE/EA (3:1) to obtain 242.5 mg of a reddish brown solid with a yield of 85%.

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

ZSQ-9-48 (242 mg, 0.43 mmol) and 0.8 ml of trifluoroacetic acid were mixed in 5 ml of DCM and stirred at room temperature overnight. After completion of the reaction, the reaction solution was concentrated and separated and purified by C18 reverse-phase column chromatography, eluted with H ₂ O/CH ₃ CN (1:5), to obtain 150 mg of white solid with a yield of 96%.

Compound (R)-N-(4-(1,3,2-Dithiasopentan-2-yl)phenyl)-2-(3-((7-(benzylamino)-3-iso Synthesis of propylpyrazolo[1,5-a]pyrimidin-5-yl)amino)piperidin-1-yl)acetamide (ZSQ-9-60)

ZSQ-9-55 (150 mg, 0.41 mmol), DIPEA (0.20 ml, 1.23 mmol) and ZSQ-5-4 (155 mg, 0.41 mmol) were dissolved in 7 ml of THF solution at room temperature, and the mixed solution was stirred at room temperature 3h. It was then washed with 2N dilute hydrochloric acid solution (20ml) and extracted with EA (3x30ml) followed by water (2x20ml). The organic phases were combined, dried _{over Na2SO4} , filtered and concentrated under reduced pressure, separated and purified by silica gel column chromatography, eluting with PE/EA (1:1) to give 133.3 mg of a yellow solid in 49% yield.

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

ZSQ-8-9 (66mg, 0.1mmol) trihydrate mercuric perchlorate (36mg, 0.08mmol) was dissolved in 2ml DMSO, the mixed solution was stirred at room temperature for 10min, immediately separated and purified by C18 reverse-phase column chromatography, using Elution with _H2O / _CH3CN (1:5) gave 49.5 mg of a yellow solid in 82% yield.

Example 7

Synthetic route of compound ZSQ-10-98

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

ZSQ-1-23 (1.03 g, 4 mmol), 3-(N-Boc-aminomethyl)benzoic acid (1.00 g, 4 mmol), HATU (3.04 g, 8 mmol) and DIPEA (1.98 ml, 12 mmol) were dissolved in In 20ml of DCM, the mixture was placed at room temperature and stirred for 4h. After the reaction was completed, washed with H ₂ O (20ml×2) and saturated NaCl solution (20ml) successively, extracted with 20ml of DCM, combined the organic phases, dried over anhydrous sodium sulfate, filtered, concentrated and separated and purified by silica gel column chromatography. Elution with PE/EA (4:1) gave a pale yellow powder 990 mg in 50% yield.

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

ZSQ-7-71 (990 mg, 2.01 mmol) and 1 ml of trifluoroacetic acid were mixed in 5 ml of DCM and stirred at room temperature overnight. After completion of the reaction, the reaction solution was concentrated and separated and purified by C18 reverse-phase column chromatography, eluted with H ₂ O/CH ₃ CN (1:5), to obtain 651 mg of white solid with a yield of 82%.

Compound N-(4-(1,3,2-Dithiasopentan-2-yl)phenyl)-3-((((5-Chloro-3-isopropylpyrazolo[1,5- a] Synthesis of pyrimidin-7-yl)amino)methyl)benzamide (ZSQ-10-91)

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

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

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

Compound (3-((4-(1,3,2-Dithiasopentan-2-yl)phenyl)carbamoyl)benzyl)(5-(benzylamino)-3-isopropyl Synthesis of tert-butyl pyrazolo[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'-binaphthalene-2, 2'-Bisdiphenylphosphine (37 mg, 0.06 mmol) and sodium tert-butoxide (46 mg, 0.48 mmol) were mixed in 2 ml of toluene, heated to 95° C. and stirred for 16 h under nitrogen atmosphere. After the reaction was completed, it was washed with H ₂ O (30 ml×3), extracted with EA (40 ml), and washed with saturated sodium chloride solution (30 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, separated and purified by silica gel column chromatography, and eluted with PE/EA (3:1) to obtain 40 mg of white powder with a yield of 13%.

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

ZSQ-10-93 (40 mg, 0.05 mmol) and 0.5 ml trifluoroacetic acid were mixed in 5 ml DCM and stirred at room temperature overnight. After the completion of the reaction, the reaction solution was concentrated and separated and purified by C18 reverse phase column chromatography, eluted with H ₂ O/CH ₃ CN (1:7), to obtain 23 mg of white solid with a yield of 69%.

Compound (4-(3-(((5-(benzylamino)-3-isopropylpyrazolo[1,5-a]pyrimidin-7-yl)amino)methyl)benzamido)benzene Synthesis of Arsinic Acid (ZSQ-10-98)

ZSQ-10-95 (23mg, 0.035mmol), mercuric perchlorate trihydrate (12mg, 0.028mmol) were dissolved in 2ml DMSO, the mixed solution was stirred at room temperature for 10min, immediately separated and purified by C18 reverse-phase column chromatography, using Elution with _H2O / _CH3CN (1:5) gave 17 mg of a white solid in 85% yield.

Example 8

Synthetic route of compound ZSQ-12-3

Compound (R)-3-((5-Chloro-3-isopropylpyrazolo[1,5-a]pyrimidin-7-yl)amino)piperidine-1-carboxylate tert-butyl ester (ZSQ-11 -64) synthesis

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

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

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

Compound (R)-3-((5-(benzylamino)-3-isopropylpyrazolo[1,5-a]pyrimidin-7-yl)(tert-butoxycarbonyl)amino)piperidine- Synthesis of tert-butyl 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'-Bisdiphenylphosphine (93 mg, 0.15 mmol) and sodium tert-butoxide (115 mg, 1.2 mmol) were mixed in 5 ml of toluene, heated to 95° C. and stirred for 16 h under nitrogen atmosphere. After the reaction was completed, it was washed with H ₂ O (30 ml×3), extracted with EA (40 ml), and washed with saturated sodium chloride solution (30 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, separated and purified by silica gel column chromatography, and eluted with PE/EA (3:1) to obtain 524 mg of a reddish-brown solid with a yield of 93%.

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

ZSQ-11-94 (524 mg, 0.93 mmol) and 1 ml of trifluoroacetic acid were mixed in 6 ml of DCM and stirred at room temperature overnight. After completion of the reaction, the reaction solution was concentrated and separated and purified by C18 reverse-phase column chromatography, eluted with H ₂ O/CH ₃ CN (1:5), to obtain 322 mg of white solid with a yield of 95%.

Compound (R)-N-(4-(1,3,2-Dithiasopentan-2-yl)phenyl)-2-(3-((5-(benzylamino)-3-iso Synthesis of propylpyrazolo[1,5-a]pyrimidin-7-yl)amino)piperidin-1-yl)acetamide (ZSQ-11-104)

ZSQ-11-96 (182 mg, 0.5 mmol), K ₂ CO ₃ (166 mg, 1.2 mmol) and ZSQ-5-4 (190 mg, 0.5 mmol) were dissolved in 4 ml of CH ₃ CN solution at room temperature, and the solutions were mixed Stir at room temperature for 4 h. It was then washed with 2N dilute hydrochloric acid solution (20ml) and extracted with EA (3x30ml) followed by water (2x20ml). The organic phases were combined, dried _{over Na2SO4} , filtered and concentrated under reduced pressure, separated and purified by silica gel column chromatography eluting with DCM/EA (4:1) to give 205 mg of white powder in 62% yield.

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

ZSQ-11-104 (66mg, 0.10mmol), mercuric perchlorate trihydrate (36mg, 0.08mmol) were dissolved in 3ml DMSO, the mixed solution was stirred at room temperature for 10min, and immediately separated and purified by C18 reverse phase column chromatography, Elution with _H2O / _CH3CN (1:5) gave 49 mg of a white solid in 81% yield.

Example 9

Synthetic route of compound ZSQ-13-92

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

Methylmagnesium bromide (1 M in tetrahydrofuran, 70 ml, 70 mmol) was added dropwise to a solution of indole (8.26 g, 70 mmol) in THF (50 ml) at 0°C over 30 min. The solution was continued to stir at 0 °C for 30 min. 2,4-Dichloro-5-iodopyrimidine (2.74 g, 35 mmol) was added dropwise to give a yellow solution. The ice bath was removed and the solution was stirred at room temperature for 1 h to give a red solution. The mixed solution was heated to 60 °C and stirred for 1.5 h. The mixture was cooled to room temperature and acetic acid (70 ml) was added dropwise. Water (70 ml) and THF (30 ml) were added and the mixture was continued to stir at 60°C for 20 min, resulting in a biphasic solution. The layers were separated, and petroleum ether (100 ml) was added to the organic solution, causing the solid to crystallize. The solid was collected by filtration, washed with petroleum ether (20 ml), and dried in vacuo to give 2.5 g 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) were dissolved in 20ml DCM and benzenesulfonyl chloride (576ul, 4.5mmol) was added dropwise at room temperature ). The reaction mixture was continued to stir at room temperature for 4 h. The mixture was quenched with water (30ml) and extracted with DCM (20ml x 3). The combined organic layers _were dried over anhydrous _Na2SO4 , filtered, concentrated, separated and purified by silica gel column chromatography, eluting with DCM/EA (4:1) to give 850 mg of white solid in 57% yield.

Compound 3-(2-Chloro-5-((trimethylsilyl)ethynyl)pyrimidin-4-yl)-1-(phenylsulfonyl)-1H-indole (ZSQ-13-77) synthesis

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

Compound (R)-3-((4-(1-(phenylsulfonyl)-1H-indol-3-yl)-5-((trimethylsilyl)ethynyl)pyrimidin-2-yl ) Amino) piperidine-1-carboxylate tert-butyl ester (ZSQ-13-86) synthesis

ZSQ-13-77 (380 mg, 0.817 mmol), (R)-1-Boc-3-aminopiperidine (172 mg, 0.858 mmol) and DIPEA (0.4 ml, 2.451 mmol) were mixed and dissolved in 3 ml of N-methylpyrrolidone middle. The mixed solution was heated and stirred at 135 °C for 3 h. The cooled solution was directly separated and purified by C18 reverse phase column chromatography, eluting with H ₂ O/CH ₃ CN (1:9), to give 448 mg of brown powder in 87% yield.

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

ZSQ-13-86 (448 mg, 0.71 mmol) and 1 ml of trifluoroacetic acid were mixed in 6 ml of DCM and stirred at room temperature overnight. After the completion of the reaction, the reaction solution was concentrated and separated and purified by C18 reverse phase column chromatography, eluted with H ₂ O/CH ₃ CN (1:5), to obtain 105 mg of a yellow-brown solid with a yield of 32%.

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

ZSQ-13-87 (105 mg, 0.23 mmol), DIPEA (0.11 ml, 0.69 mmol) and ZSQ-5-4 (91 mg, 0.24 mmol) were dissolved in 4 ml of THF solution at room temperature, and the mixed solution was stirred at room temperature 4h. It was then washed with 2N dilute hydrochloric acid solution (20ml) and extracted with EA (3x30ml) followed by water (2x20ml). The organic phases were combined, dried _{over Na2SO4} , filtered and concentrated under reduced pressure, separated and purified by silica gel column chromatography eluting with DCM/EA (2:1) to give 75 mg of white powder in 43% yield.

Compound (R)-N-(4-(1,3,2-Dithiasopentan-2-yl)phenyl)-2-(3-((5-ethynyl-4-(1H-indium) Synthesis of dol-3-yl)pyrimidin-2-yl)amino)piperidin-1-yl)acetamide (ZSQ-13-91)

ZSQ-13-88 (75 mg, 0.1 mmol) and anhydrous potassium carbonate (41 mg, 0.3 mmol) were dissolved in a mixed solution of 2 ml of anhydrous methanol and 2 ml of anhydrous THF, and stirred at room temperature for 4 h. After the reaction was completed, the pH was adjusted to weakly acidic with TFA, and the reaction solution was directly separated and purified by C18 reverse-phase column chromatography, eluted with H ₂ O/CH ₃ CN (1:6), to obtain 52 mg of yellow powder with a yield of 85%.

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) Synthesis

ZSQ-13-91 (52mg, 0.08mmol), mercuric perchlorate trihydrate (30mg, 0.06mmol) were dissolved in 3ml DMSO, the mixed solution was stirred at room temperature for 10min, and immediately separated and purified by C18 reverse phase column chromatography, Elution with _H2O / _CH3CN (1:5) gave 40 mg of a yellow solid in 88% yield.

Example 10

Synthetic route of compound ZSQ-14-23

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

Compound ZSQ-1-23 (1.03 mg, 4 mmol), 3-bromopropyne (379 ul, 4.4 mmol) and anhydrous potassium carbonate (829 mg, 6.0 mmol) were mixed in 8 ml of DMF solution and stirred at room temperature for 12 h. After the reaction was completed, it was washed with H ₂ O (30 ml×3) and extracted with EA (30 ml). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and separated and purified by silica gel column chromatography, eluted with PE/EA (7:1), to obtain 850 mg of yellow solid, yield 72%

Compound N-(4-(1,3,2-Dithiasopentan-2-yl)phenyl)-2-bromo-N-(prop-2-yn-1-yl)acetamide (ZSQ- 14-10) synthesis

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

Compound (R)-N-(4-(1,3,2-dithiasopentan-2-yl)phenyl)-2-(3-((5-chloro-4-(1-(benzene) (ylsulfonyl)-1H-indol-3-yl)pyrimidin-2-yl)amino)piperidin-1-yl)-N-(prop-2-yn-1-yl)acetamide (ZSQ-14- 13) Synthesis

ZSQ-14-10 (93 mg, 0.20 mmol), DIPEA (0.10 ml, 0.60 mmol) and ZSQ-5-4 (88 mg, 0.21 mmol) were dissolved in 2 ml of THF solution at room temperature, and the mixed solution was stirred at room temperature 4h. It was then washed with 2N dilute hydrochloric acid solution (20ml) and extracted with EA (3x30ml) followed by water (2x20ml). The organic phases were combined, dried _{over Na2SO4} , filtered and concentrated under reduced pressure, separated and purified by silica gel column chromatography eluting with DCM/EA (3:1) to give 95 mg of yellow solid in 59% yield.

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

ZSQ-14-13 (95 mg, 0.12 mmol) and anhydrous potassium carbonate (66 mg, 0.48 mmol) were dissolved in a mixed solution of 2 ml of anhydrous methanol and 2 ml of anhydrous THF, and stirred at room temperature for 4 h. After the reaction was completed, the pH was adjusted to weakly acidic with TFA, and the reaction solution was directly separated and purified by C18 reverse-phase column chromatography, and eluted with H ₂ O/CH ₃ CN (1:5) to obtain 50 mg of yellow powder with a yield of 63%.

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

ZSQ-14-18 (50mg, 0.07mmol), mercuric perchlorate trihydrate (27mg, 0.06mmol) were dissolved in 3ml DMSO, the mixed solution was stirred at room temperature for 10min, and immediately separated and purified by C18 reverse phase column chromatography, Elution with _H2O / _CH3CN (1:5) gave 35 mg of a yellow solid in 83% yield.

Example 11

Synthetic route of compound ZSQ-15-99

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

Mix 2M lithium diisopropylamide solution (27.5ml, 55mmol) in 50ml of dry THF, stir at -78°C, add butyronitrile (4.35ml, 50mmol) dropwise, continue stirring after adding For 30min, ethyl formate (4.85ml, 60mmol) was mixed with 25ml of dry THF, and added dropwise to the mixed solution (over 30min) at -78°C. After adding, stirring was continued for 45min, then moved to room temperature and stirred for 14h . After the reaction was completed, 10ml of H ₂ O was added to quench, the mixed solution was concentrated, diluted hydrochloric acid solution was added to adjust the pH to 3, and then extracted with EA (20ml×3), the organic phase was dried with anhydrous sodium sulfate, filtered and concentrated, and drained 4.82g of yellow oily crude product was obtained

Synthesis of compound 4-ethyl-1H-pyrazol-3-amine (ZSQ-15-1)

ZSQ-14-104 (4.82 g, 49.6 mmol), hydrazine hydrate (3.61 ml, 74.4 mmol) and acetic acid (5.67 ml, 99.2 mmol) were mixed in 75 ml absolute ethanol, heated and stirred in a sealed tube for 16 h. After the completion of the reaction, the mixed solution was concentrated and the remaining 1/3 was washed with saturated sodium bicarbonate solution (40ml) (adjusted to PH to be basic), extracted with DCM (30ml×2), and washed with saturated sodium chloride solution (40ml) ). The combined organic phases were dried over anhydrous sodium sulfate, and the filtrate was concentrated to dryness without further purification to obtain 4.88 g 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 dry ethanol, 20% sodium ethoxide solution (16g, 47.5mmol) was added, the mixed solution was heated to reflux temperature and stirred for 22h. After the completion of the reaction, the reaction solution was concentrated, diluted with water (50 ml), and the pH was adjusted to about 3 with dilute hydrochloric acid solution, a large amount of solid was precipitated, filtered and washed with water for many times. A yellow powder was obtained, and 2.98 g of white powder was obtained after drying.

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, heated to 115°C overnight with reflux stirring . After the reaction was completed, the mixed solution was concentrated, and the remaining solution was added dropwise to ice water, extracted several times with DCM, washed with saturated sodium chloride solution (30 ml), and the organic phases were combined. Dry with anhydrous sodium sulfate, filter, concentrate and separate and purify by silica gel column chromatography, eluted with PE/EA (5:1), to obtain 2.33 g of white needle powder with a yield of 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 absolute ethanol, heated to 85°C and stirred overnight . After completion of the reaction, the reaction solution was concentrated, washed with water (30ml×2), extracted with EA (30ml), and washed with saturated sodium chloride solution (30ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, separated and purified by silica gel column chromatography, eluted with PE/EA (5:1) to obtain 631 mg of white solid with a yield of 99%.

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 (631 mg, 2 mmol), 4-dimethylaminopyridine (48 mg, 0.4 mmol) and di-tert-butyl dicarbonate (598 ul, 2.6 mmol) were mixed and dissolved in 8 ml of THF, and the mixture was stirred at room temperature for 12 h. After the reaction was completed, it was washed with H ₂ O (20 ml×2), extracted with EA (30 ml), and washed with saturated sodium bicarbonate solution (30 ml). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and separated and purified by silica gel column chromatography, eluted with PE/EA (8:1), to obtain 830 mg of yellow solid with a yield of 99%.

Compound (3-ethyl-5-(2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7-yl)(4-methoxybenzyl) ) Synthesis of tert-butyl carbamate (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'-bisdiphenylphosphine (93 mg, 0.15 mmol) and sodium tert-butoxide (106 mg, 1.1 mmol) were mixed in 3 ml of toluene, heated to 95 °C under nitrogen atmosphere and stirred for 16 h. After the reaction was completed, it was washed with H ₂ O (30 ml×3), extracted with EA (40 ml), and washed with saturated sodium chloride solution (30 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, separated and purified by silica gel column chromatography, and eluted with PE/EA (8:1) to obtain 350 mg of red solid with a yield of 68%.

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

Compound ZSQ-15-97 (283 mg, 0.69 mmol) was dissolved in a mixed solution of 2 ml of concentrated hydrochloric acid and 2 ml of DCM, and stirred at room temperature for 6 h. After completion of the reaction, the reaction solution was concentrated, separated and purified by silica gel column chromatography, and eluted with DCM/CH ₃ OH(NH ₃ ) (5:1) to obtain 115 mg of white solid with a yield of 57%.

Compound N-(4-(1,3,2-Dithiasopentan-2-yl)phenyl)-2-((3-ethyl-5-(2-(2-hydroxyethyl)piperidine) Synthesis of pyridin-1-yl)pyrazolo[1,5-a]pyrimidin-7-yl)amino)acetamide (ZSQ-15-98)

ZSQ-15-97 (115 mg, 0.4 mmol), potassium carbonate (138 mg, 1.0 mmol), potassium iodide (33 mg, 0.2 mmol) and ZSQ-5-4 (228 mg, 0.6 mmol) were dissolved in 1.5 ml CH at room temperature ₃ CN/1.5ml DMF solution, the mixed solution was stirred at room temperature for 4h. It was then acidified with TFA and isolated and purified by C18 reverse phase column chromatography eluting with _H2O / _CH3CN (1:9) to give 153 mg of a pink solid in 65% yield.

Compound (4-(2-((3-ethyl-5-(2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7-yl)amino Synthesis of )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 mixed solution was stirred at room temperature for 10min, and immediately separated and purified by C18 reverse phase column chromatography, Elution with _H2O / _CH3CN (1:5) gave 28 mg of a yellow solid in 53% yield.

Example 12

Synthetic route of compound ZSQ-14-66

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

1-Fluoro-2-nitrobenzene (14g, 100mmol), isopropanethiol (10.2ml, 110mmol), anhydrous potassium carbonate (27.6g, 200mmol) were mixed and dissolved in 100ml of anhydrous DMF, and placed at 110°C Stir for 12h. After the completion of the reaction, the reaction solution was concentrated under reduced pressure, washed with 100 ml of water and 100 ml of saturated sodium chloride solution in turn, and extracted with EA. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The product was separated and purified by silica gel column chromatography and eluted with PE/EA (7:1) to obtain 18 g of yellow oily product with a yield of 91%.

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

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

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

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

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

NaH (600 mg, 15 mmol) was dissolved in dry 60 ml DMF and stirred at 0°C. ZSQ-14-48 (1.99 g, 10 mmol) was dissolved in 5 ml of dried DMF, and added dropwise to the above mixed solution (over 10 min). After adding and stirring at 0°C for 0.5 h, 2,4,5-trichloropyrimidine (2.29 ml, 20 mmol) was added dropwise, stirred for 1 h, and then moved to room temperature and stirred overnight. After the reaction was completed, 20 ml of water was added to quench, and DMF was removed by distillation under reduced pressure. Wash with 100 ml of water, 100 ml of saturated sodium bicarbonate solution, 100 ml of saturated sodium chloride solution, and extract with EA. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. It was separated and purified by silica gel column chromatography and eluted with PE/EA (3:1) to obtain 1.2 g of white solid with a yield of 35%.

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

ZSQ-14-49 (173mg, 0.5mmol), (R)-1-Boc-3-aminopiperidine (100mg, 0.5mmol) and DIPEA (248ul, 1.5mmol) were mixed in 2ml N-methylpyrrolidone . The mixed solution was heated and stirred at 135 °C for 3 h. The cooled solution was directly separated and purified by C18 reverse phase column chromatography, eluting with H ₂ O/CH ₃ CN (1:9) to give 232 mg of pale yellow solid in 91% yield.

Compound (R)-5-Chloro-N4-(2-(isopropylsulfonyl)phenyl) ^-N2- (piperidin- ³ -yl)pyrimidine-2,4-diamine (ZSQ-14- 58) Synthesis

ZSQ-14-54 (232 mg, 0.45 mmol) and 1 ml of trifluoroacetic acid were mixed in 5 ml of DCM and stirred at room temperature overnight. After the completion of the reaction, the reaction solution was concentrated and separated and purified by C18 reverse phase column chromatography, eluted with H ₂ O/CH ₃ CN (3:1), to obtain 180 mg of pale yellow solid with a yield of 97%.

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

ZSQ-14-58 (82 mg, 0.2 mmol), DIPEA (0.13 ml, 0.8 mmol) and ZSQ-5-4 (76 mg, 0.2 mmol) were dissolved in 3 ml of THF solution at room temperature, and the mixed solution was stirred at room temperature 4h. It was then washed with 2N dilute hydrochloric acid solution (20ml) and extracted with EA (3x30ml) followed by water (2x20ml). The organic phases were combined, dried _over anhydrous _Na2SO4 , filtered and concentrated under reduced pressure, separated and purified by silica gel column chromatography, eluted with DCM/EA (3:1) to give 79 mg of yellow solid in 56% yield.

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

ZSQ-14-60 (36mg, 0.05mmol), mercuric perchlorate trihydrate (18mg, 0.04mmol) were dissolved in 2ml DMSO, the mixed solution was stirred at room temperature for 10min, and immediately separated and purified by C18 reverse phase column chromatography, Elution with _H2O / _CH3CN (1:3) gave 24 mg of a yellow solid in 75% yield.

Synthesis Scheme 13

Synthetic route of compound ZSQ-16-91

Synthesis of Compound (4-Hydroxy-3-nitrophenyl) arsine (ZSQ-16-8)

3-Nitro-4-hydroxyphenylarsinic acid (26.3 g, 100 mmol) was dissolved in 80 ml of methanol, and the mixed solution was heated to reflux. Phenylhydrazine (19.6 ml, 200 mol) was added dropwise (over 1 h), a large amount of nitrogen was generated during the addition, and when the nitrogen generation slowed down, reflux stirring was continued for 1.5 h. The mixed solution was concentrated by distillation under reduced pressure, sodium hydroxide solution (12 g dissolved in 200 ml of water) and 200 ml of ether were added. Separation, adding 2N dilute hydrochloric acid solution to the aqueous phase, stirring (100ml) for 1h, and extracting with EA (200ml×3). The organic phase was dried with anhydrous sodium sulfate, concentrated and dried by oil pump, and the next reaction was carried out without purification.

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

ZSQ-16-8 (24.7 g, 100 mmol) was dissolved in 100 ml methanol and heated to reflux. Then, ethanedithiol (10 ml, 120 mmol) was added dropwise to the mixed solution over 30 min, and heating and stirring were continued for 30 min. The mixed solution was then concentrated, separated and purified by silica gel column chromatography, and eluted with PE/EA (10:1) to obtain a yellow solid crude product. It was dissolved in THF, separated and purified by C18 reverse-phase column chromatography, and eluted with H ₂ O/CH ₃ CN (8:1) to obtain 9 g of a yellow solid product with a yield of 29.5%.

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

ZSQ-16-14 (9.3 g, 30 mmol) was dissolved in a mixed solution of ethanol/ethyl acetate (10:1, 100 ml), stannous chloride (13.5 g, 60 mmol) was added, and the mixed solution was placed at 75 ° C to reflux Stir for 48h. After the reaction was completed, the reaction solution was concentrated, washed with water (100×3 ml), and extracted with EA (100 ml). The organic phase was dried with anhydrous sodium sulfate, concentrated and separated and purified by silica gel column chromatography, eluted with PE/EA (8:1), to obtain 340 mg of a yellow solid crude product with a yield of 4%.

Synthesis of compound (tert-butyl 5-(1,3,2-dithiasopentan-2-yl)-2-hydroxyphenyl)carbamate (ZSQ-16-1)

ZSQ-16-90 (120 mg, 0.72 mmol), and di-tert-butyl dicarbonate (200 ul, 0.87 mmol) were mixed and dissolved in 4 ml of THF, and the mixture was stirred at room temperature for 24 h. After the reaction was completed, it was washed with H ₂ O (50 ml×2) and extracted with EA (50 ml). The organic phase was dried with anhydrous sodium sulfate, filtered and concentrated, and separated and purified by silica gel column chromatography, eluted with PE/EA (6:1), to obtain 120 mg of white solid with a yield of 43%.

Synthesis of compound (tert-butyl 5-(1,3,2-dithiasopentane-2-yl)-2-methoxyphenyl)carbamate (ZSQ-16-68)

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

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

ZSQ-16-68 (90 mg, 0.23 mmol) and 0.3 ml trifluoroacetic acid were mixed in 3 ml DCM and stirred at room temperature overnight. After the completion of the reaction, the reaction solution was concentrated and separated and purified by C18 reverse phase column chromatography, eluted with H ₂ O/CH ₃ CN (1:6), to obtain 52 mg of white solid with a yield of 78%.

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

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

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

ZSQ-16-68 (110 mg, 0.26 mmol), DIPEA (0.13 ml, 0.80 mmol) and ZSQ-5-4 (138 mg, 0.29 mmol) were dissolved in 3 ml of THF solution at room temperature, and the mixed solution was stirred at room temperature 4h. It was then washed with 2N dilute hydrochloric acid solution (20ml) and extracted with EA (3x30ml) followed by water (2x20ml). The organic phases were combined, dried _{over Na2SO4} , filtered and concentrated under reduced pressure, separated and purified by silica gel column chromatography, eluted with DCM/EA (3:1), to give 177 mg of white solid powder in 83% yield.

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

ZSQ-16-87 (177 mg, 0.22 mmol) and anhydrous potassium carbonate (92 mg, 0.66 mmol) were dissolved in a mixed solution of 1.5 ml of anhydrous methanol and 1.5 ml of anhydrous THF, and stirred at room temperature for 4 h. After the reaction was completed, the pH was adjusted to weakly acidic with TFA, and the reaction solution was directly separated and purified by C18 reverse-phase column chromatography, eluted with H ₂ O/CH ₃ CN (1:8), to obtain 121 mg of pale yellow solid with a yield of 84% .

Compound (R)-(3-(2-(3-((5-Chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)piperidin-1-yl)acetamido) Synthesis of -4-Methoxyphenyl) arsine (ZSQ-16-91)

ZSQ-16-88 (66mg, 0.1mmol), mercuric perchlorate trihydrate (36mg, 0.08mmol) were dissolved in 2ml DMSO, the mixed solution was stirred at room temperature for 10min, and immediately separated and purified by C18 reverse phase column chromatography, Elution with _H2O / _CH3CN (1:5) gave 46 mg of a yellow solid in 76% yield.

Table 1

Activity test example 1 CDK inhibitor to cancer cell growth inhibitory activity test

The adopted biological test protocol is: the effect of compounds on the growth activity of acute lymphoblastic leukemia cell line Jurkat and non-small cell lung cancer cell line H3122.

In order to verify the growth inhibitory effect of the compounds of the present invention on cancer cells at the cellular level, Jurkat cells (blood cancer, suspension type) and H3122 cells (solid tumor, adherent type) were selected, and cell viability was calculated by detecting chemiluminescence values , thereby deriving the biological activity of the compound to inhibit the growth of cancer cells.

Methods: Jurkat or H3122 cells were cultured in vitro. After growing to the logarithmic growth phase, the cells were collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell concentration was adjusted to 2.5×10 ⁵ /mL (Jurkat) or 1.5×10 ⁵ /mL (H3122 ), cells were seeded into 384-well plates, 40 μl per well. Add 5 μL of different concentrations of compounds or DMSO to the corresponding wells, place them in a cell incubator (37°C, 5% CO ₂ ) for 72 hours, add 15 μl of Cell Titer-Glo solution to each well, incubate at room temperature for 30 minutes, and detect chemiluminescence luminescence to measure intracellular ATP levels. Control wells with unstimulated DMSO were taken as 100% cell viability. Compound _IC50 values were calculated using Prism Graphpad statistical software.

Table 2 The test results of CDK inhibitors on the growth inhibition activity of cancer cell lines

Activity test example 2 CDK inhibitor growth inhibitory activity test of non-small cell lung cancer cell line

The adopted biological test protocol is: the effect of compounds on the respective growth activities of various non-small cell lung cancer cell lines.

In order to verify the growth inhibitory effect of the representative compounds in the present invention on the growth and survival of non-small cell lung cancer cell lines dependent on different driver genes at the cellular level, H3122, H1299, H1975, H2077, H358, EBC-1, H23, In PC9 and A549 cells, the cell viability was calculated by detecting the chemiluminescence value, and the biological activity of the compounds in inhibiting the growth of cancer cells was obtained, and compared with the reported CDK inhibitors THZ1 and THZ531.

Methods: Various non-small cell lung cancer cells were cultured in vitro. After growing to the logarithmic growth phase, the cells were digested and collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the cell concentration was adjusted to 1.5×10 ⁵ /mL, and the cells were inoculated into 384 wells plate, 40 μl per well. Add 5 μL of different concentrations of compounds or DMSO to the corresponding wells, place them in a cell incubator (37°C, 5% CO ₂ ) for 72 hours, add 15 μl of Cell Titer-Glo solution to each well, incubate at room temperature for 30 minutes, and detect chemiluminescence luminescence to measure intracellular ATP levels. Control wells with unstimulated DMSO were taken as 100% cell viability. Compound _IC50 values were calculated using PrismGraphpad statistical software.

Table 3 The test results of CDK inhibitors on the growth inhibitory activity of different non-small cell lung cancer cell lines

It can be seen from Table 3 that the representative compounds of the present invention exhibited comparable or even better growth inhibitory activity than THZ531 on various non-small cell lung cancer cell lines.

Activity Test Example 3 Effects of CDK Inhibitors on CDK7/12/13 Kinase Activity in Cancer Cells

In a variety of cancer cells, RNA polymerase II (Pol II) forms transcription complexes with transcription factors such as CDK7, 12, 13, and BRD4 through super-enhancers to promote the high expression of oncogenes such as MYC and RUNX ₁ , thereby maintaining cancer. Cell survival and proliferation. In the Pol II transcription complex, CDK7, 12, and 13 are jointly responsible for the continuous phosphorylation of the Ser5 site in the Pol II C-terminal domain (CTD) repeat to ensure the normal initiation of the Pol II transcription program; while CDK12, 13 It is responsible for the continuous phosphorylation of the Ser2 site in the Pol IICTD repeat to ensure the correct process of transcription elongation and DNA damage repair. Therefore, when CDK12 and 13 are simultaneously inhibited, the phosphorylation level of Ser2 site will be inhibited; when CDK7, 12, and 13 are inhibited simultaneously, the phosphorylation level of Ser5 site will also be inhibited.

Experimental conditions and process: H3122 or A549 cells were cultured in vitro, and after growing to the logarithmic growth phase, the cells were digested and collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell concentration was adjusted to 1×10 ⁶ /mL. In a 12-well cell culture plate, add 1 ml of cells to each well, add 1 μL of DMSO solutions of different concentrations of drugs to each well, and control with THZ1, THZ531 or DMSO, and place them in a cell incubator (37°C, 5% CO2) for 8 hours after culturing. , washed twice with pre-cooled PBS solution, aspirated the solution, added 200 μL of RIPA cell lysate, protease inhibitor and phosphatase inhibitor to the wells, transferred to the sample tube and placed it in a 4°C shaker for 30min lysis, 15000rpm for 4 Centrifuge at °C for 15 min, and take the supernatant cell lysate. Use the BCA protein quantification kit to detect the protein content of each group, and use PIPA lysis buffer to adjust the protein amount to make the final volume 100 μL. Samples were identified by western-blot.

Western-blot: Add 25μL of 5X protein loading buffer to 100μL of cell lysate, and heat at 95°C for 10min. After the samples were cooled, electrophoresis was performed using SDS-PAGE (9%) gel at 60V, and then switched to 120V after 30 min until the leading band was electrophoresed to the bottom of the gel. Using a turbo semi-dry transfer system with a constant current of 0.2 A for 80 minutes, the proteins in the gel were transferred to a PC membrane with a pore size of 0.2 μL. The transferred PC membrane was placed in 5% nonfat dry milk (TBST solution) for blocking for 2h, and incubated with the corresponding primary antibody at 4°C for 12h. Washed with TBST for 3 times, 10 min each time. Incubate with the corresponding secondary antibody for 2 h at room temperature. Washed with TBST three times, 10 min each time. Incubate with ECL luminescent solution and detect the luminescent signal.

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

Compounds such as ZSQ5-38, ZSQ8-36 and ZSQ9-75 can effectively inhibit Ser2 phosphorylation at a concentration of 1 μM, but not Ser5 phosphorylation;

Compounds such as ZSQ5-38 and ZSQ17-22 showed better inhibitory activity than THZ531;

THZ1 inhibits phosphorylation at both sites simultaneously.

This indicates that compounds such as ZSQ5-38, ZSQ8-36 and ZSQ9-75 can effectively inhibit the kinase activities of CDK12 and 13 in cancer cells, but not CDK7.

Activity Test Example 4 Irreversible binding of CDK inhibitors to CDKs in H3122 cells

In this example, it was tested whether the CDK inhibitors of the invention bind irreversibly to CDK7, 12 or 13.

Covalent inhibitors such as THZ1 or THZ531 can form covalent bonds with specific cysteine residues in the binding pockets of CDK7, 12 or 13, which in turn bind irreversibly to CDK7, 12 or 13. The reported THZ1-biotin, a derivative of THZ1, can also form covalent bonds with CDK7, 12, and 13 in cell lysates, and then CDK7, 12, and 13 can be separated by streptavidin beads. enrichment. When THZ1, THZ531 or ZSQ series CDK inhibitors irreversibly bind to CDK7, 12 or 13, THZ1-biotin can no longer bind to CDK7, 12 or 13 because the binding pocket has been occupied. Therefore, based on competitive enrichment experiments of THZ1-biotin and streptavidin microbeads, it is possible to accurately determine whether CDK inhibitors irreversibly bind to CDK7, 12 or 13.

Experimental conditions and process: H3122 cells were cultured in vitro, and after growing to the logarithmic growth phase, the cells were digested and collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell concentration was adjusted to 1×10 ⁶ /mL. In a 12-well cell culture plate, add 1 ml of cells to each well, and add 100 nL of 10 mM DMSO solution (final concentration of 1 μM) of a representative drug to each well, and place it in a cell incubator (37°C, After culturing in 5% CO2) for 4 hours, wash twice with pre-cooled PBS solution, add 200 μL of NP40 cell lysate and protease inhibitor to the well, and lyse at 4°C for 30 minutes. The lysate was collected, centrifuged at 15,000 rpm at 4°C for 15 min, the supernatant was transferred to a sample tube, THZ1-biotin (final concentration of 1 μM) was added, and incubated at 4°C overnight. Add 10 μL of streptavidin beads and allow to bind for 2 hours at room temperature. The microbeads were washed 10 times with 1% NP40 cell lysate, then lysed by adding 20 μL of 2% SDS lysate, boiled at 95°C for 10 minutes, loaded, and identified by western-blot.

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

Experimental results show that:

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

While 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 μM, but hardly affect THZ1-biotin Covalent binding to CDK7, compared with THZ531, compounds such as ZSQ5-38, ZSQ8-36, ZSQ9-75 and ZSQ17-22 have less effect on CDK7 and higher selectivity;

This indicates that ZSQ compounds can inhibit the kinase activities of CDK12 and 13 in cancer cells efficiently, with high specificity and irreversibly.

Activity Test Example 5 Effects of CDK inhibitors on the in vitro kinase activities of CDK7 and 12

Both CDK7 and 12 proteins can catalyze the phosphorylation of Ser5 site of Pol II CTD in vitro. Therefore, when the kinase activity of CDK7 or 12 is inhibited, the phosphorylation modification of Ser5 site will be weakened or lost.

Experimental conditions and process: 293T cells overexpressed 3XFlag-CDK7 or 3X-Flag-CDK12, washed twice with pre-cooled PBS solution after 48 hours, added 1 mL of RIPA buffer containing protease inhibitors, and lysed at 4°C for 1 hour. The lysate was collected, centrifuged at 15,000 rpm at 4°C for 15 min, the supernatant was transferred to a sample tube, 20 μL of anti-Flag affinity gel was added, and incubated at 4°C overnight. The gel was washed 5 times with RIPAbuffer, and then washed 3 times with kinase buffer (50mM HEPES pH7.4, 50mM KCl, 10mM MgCl ₂ ), and 100 μL of kinase buffer was added to mix well for subsequent in vitro kinase reaction. Add 10 μL of gel mix, different concentrations of compounds to each well, and incubate overnight at 4°C. Then 1 μg of bacterially expressed and purified GST-Pol II CTD protein and 100 μM ATP were added. After shaking reaction at 37°C for 1 hour, 4% SDS loading buffer was added to stop the reaction, and the reaction was terminated by cooking at 95°C for 10 minutes, and then identified by western-blot.

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

Experimental results show that:

Compounds such as ZSQ5-38 and ZSQ8-36 can completely or almost completely inhibit the phosphorylation modification of Pol II CTD Ser5 site catalyzed by CDK12 at low concentrations, but do not affect the phosphorylation modification of Pol II CTD Ser5 site catalyzed by CDK7. , consistent with the THZ531 phenotype;

In contrast, THZ1 simultaneously inhibited the phosphorylation of CDK7 and 12 catalytic substrates;

ZSQ14-66 significantly inhibited the phosphorylation of both CDK7 and 12 catalytic substrates at a concentration of 250 nM, while ZSQ14-60 partially inhibited the phosphorylation of CDK7 catalytic substrates at a concentration of 500 nM.

This indicates that multiple ZSQ compounds can efficiently and specifically target the kinase activity of CDK12.

Activity Test Example 6 Selectivity of CDK Inhibitors on Kinome

The bioassay protocol employed was the effect of compounds on the in vitro enzymatic activity of the kinome.

In order to verify the selectivity of the compounds of the present invention to the kinome at the protein level, the representative compounds ZSQ5-36 and ZSQ8-36 were selected, and the P ³³ isotope-labeled ATP and radioactive kinase activity test methods were used to obtain the compounds to 370 kinds of kinases. Inhibition level of phosphorylation of protein-catalyzed substrates.

Methods: Radioactive kinome activity assay. Compounds were mixed with protein and pre-incubated at room temperature for 1 hour, and then P ³³ -ATP was added to react for 2 hours. The reaction mixture was spotted on P81 ion-exchange chromatography paper, and the kinase activity was detected by membrane filtration. The final test concentration of the compound was 1 μM, 2 sub-wells were repeated, and the activity of the drug-added treatment group was calculated by taking the single-adding DMSO group as 100% kinase activity.

Table 4 Selectivity of CDK inhibitors against 370 different kinases

As can be seen from Table 4, the representative compounds only inhibited the enzymatic activities of a few kinases below 50% at a concentration of 1 μM; in contrast, the representative compounds were able to completely inhibit the kinase activity of CDK12 at a concentration of 500 nM ( Activity Test Example 5).

Studies have shown that the binding ability of THZ531 to different kinases at a concentration of 1 μM is shown in Table 5, and the score (Score) represents the percentage of proteins that are not bound to the compound (that is, proteins whose kinase activity is not inhibited). Kinases such as STK16 have strong binding, and the compounds of the present invention have little effect on the activity of these kinases, which suggests that the compounds of the present invention have different kinome selectivity from the existing CDK12/13 inhibitors. Possibly with better selectivity and security.

table 5

Activity Test Example 7 Pharmacokinetic Properties of CDK Inhibitors

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

The method is as follows; the representative compounds ZSQ5-38 and ZSQ8-36 are selected and administered by intraperitoneal injection (10 mg/kg) to test the pharmacokinetic properties of the compounds in mice (n=3).

Figure 4 shows the plasma concentration-time profiles of representative compounds ZSQ5-38 and ZSQ8-36, along with pharmacokinetic parameters.

The results showed that ZSQ5-38 and ZSQ8-36 rapidly reached the peak value in mice after a single dose, and could stably maintain a moderate drug concentration, and exhibited a long half-life (the T of ZSQ5-38 and ZSQ8-36). _1/2 is 17.3 and 9.8).

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

Activity Test Example 8 Growth Inhibitory Activity Test of CDK Inhibitors on Various Lung Cancer Cell Lines with KRAS Mutations

In this example, the representative compounds ZSQ5-38 and ZSQ8-36 of the present invention were verified at the cellular level to inhibit the growth of KRAS gene-dependent growth and survival of lung cancer cell lines with KRAS activating point mutations. 17 kinds of cells such as H441 and H460 were selected respectively, and the number of cell clones was observed by crystal violet staining, so as to obtain the biological activity of the compounds in inhibiting the growth of cancer cells.

Methods: Various lung cancer cells were cultured in vitro. After growing to the logarithmic growth phase, the cells were digested and collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the cell concentration was adjusted to 1.5×10 ⁵ /mL, and the cells were seeded into 12-well plates. 1ml per well. Add 5 μL of different concentrations of compounds or DMSO to the corresponding wells, place them in a cell culture incubator (37°C, 5% CO ₂ ) for one week (replace the medium once during the period), add 4% PFA solution to each well for fixation for 20 After 20 minutes, 0.1% crystal violet solution was added for staining for 20 minutes, PBS was washed twice, and the cells were scanned and imaged. The unstimulated DMSO control well was regarded as 100% cell viability.

Figure 5 shows that the representative compounds ZSQ5-38 and ZSQ8-36 can completely or effectively inhibit the growth and proliferation of most cell lines at a concentration of 1 μM, and can completely or effectively inhibit several cell lines such as SW1573 and H460 at a concentration of 300 nM growth and proliferation.

discuss

Among various proteins closely related to the occurrence and progression of cancer, many members of the kinase protein family have been verified as effective therapeutic targets. Many verified or potential kinase targets have reactive thiol groups near their enzyme catalytic sites, which provide the possibility for the development of covalent kinase inhibitors. CDK7, 12 and 13 in the cell cycle-dependent kinase (CDK) family belong to this class of kinase targets.

CDK (cyclin dependent kinases) family kinases are more than a dozen members of the serine/threonine protein kinase family, some of which are key kinases involved in cell cycle regulation. According to the different functions of CDKs, they can be mainly divided into two categories: CDK1, 2, 4 and 6 are involved in cell cycle regulation; CDK7-13 are involved in transcriptional regulation.

CDK7-cyclin H, CDK8-cyclin C, CDK9-cyclin T and CDK12/13-cyclin K all regulate the initiation or elongation of the transcription process by regulating the phosphorylation of RNA polymerase II, and promote the subsequent Through super-enhancer (super-enhancer) high expression of a variety of cancer-promoting genes (such as MYC, RUNX, etc.), and then maintain the growth and survival of cancer cells.

CDK12 is an important component in regulating DNA damage repair in cancer cells. Inactivation of CDK12 can synergize with DNA damage repair inhibitors (such as PARP inhibitor olaparib), and can also enhance the infiltration of immune cells in solid tumors - thereby promoting the efficacy of tumor immunotherapy.

CDK13 is a homologous protein of CDK12, its structure is very similar to CDK12, only a few amino acid residues are different from CDK12, and its known function is equivalent to CDK12.

CDK7 is an important transcription factor that regulates the transcription initiation of oncogenes and regulates the normal operation of the cell cycle. It has been reported in the literature that inhibition of CDK7 may have adverse effects on the differentiation of normal cells.

The present invention provides compounds of formula I that selectively inhibit CDK12 and/or 13, with no or weak inhibition of CDK7. Studies have shown that inhibiting CDK7, 12, and 13 at the same time will block both transcriptional activation and elongation, while only inhibiting CDK12 and 13 will only affect the elongation of the transcriptional process. Simultaneous inhibition of multiple CDKs has greater adverse effects on the growth and survival of normal cells. For cancer cells, they are usually genetically unstable and more dependent on DNA damage repair mechanisms, so selective inhibition of CDK12 and 13 is sufficient to induce tumor cell apoptosis.

On the other hand, the existing CDK7/12/13 triple inhibitor THZ1 not only inhibits these three CDKs, but also inhibits many other kinases, resulting in toxic and side effects; the improved SY-1365 mainly inhibits CDK7, and inhibits CDK12, 13 The inhibitory effect of CDK12/13 is weaker; CDK12/13 dual inhibitors that do not inhibit or basically do not inhibit CDK7 have significantly better target selectivity than triple inhibitors (see activity test example 6), and tumors of different genetic subtypes are sensitive to it Sex is also different from CDK7 inhibitors.

The CDK inhibitor of the present invention is a targeted inhibitor obtained by optimization. In the structure of the compound of the present invention, the organic arsine group (moiety) is the part that covalently interacts with the active site of CDK12/13, so it can provide Excellent inhibitory activity and better specificity. From the experimental data, the present invention specifically binds to specific kinases such as CDK12 and CDK13, and does not bind or basically does not bind to CDK7 or more than 300 other tested kinases. Moreover, it has shown good selectivity between different cell lines, so it has a safety far superior to the existing arsine compounds.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

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

in, X ₁ , X ₂ are each independently selected from the group consisting of none, O, S, NR ₈ , CH ₂ ; R _is selected from the group consisting of H, substituted or unsubstituted C1-C6 alkyl; R ₁ and R ₂ are each 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 Substituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl; or R ₁ , R ₂ and adjacent X ₁ , X ₂ and As together form a substituted or unsubstituted 4- to 8-membered heterocycle , the heterocycle contains an As heteroatom and 0-3 heteroatoms selected from N, O and S; R ₃ is 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; n ₁ is 0, 1, 2, 3 or 4; Each R is independently 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; L ₁ , L ₂ and L ₃ are each independently selected from the group consisting of: none, -(Z)m-; and L ₁ and L ₂ are not both none; wherein each Z is independently selected from: C1-C6 alkylene base, -NR ₆ -, -NR ₆ -R ₇ -, -O-, -CO-, m is 1, 2, 3 or 4; Each R is independently selected from the group consisting of H, substituted or unsubstituted C1 _- C4 alkyl; R ₇ is substituted or unsubstituted C1-C8 alkylene; A is selected from the group consisting of unsubstituted, 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 group consisting of substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-10 membered heteroaryl; C is selected from the group consisting of H, OH, -N(Ra)Rb, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-10 membered heteroaryl, substituted or unsubstituted C3-C10 ring Alkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl; Unless otherwise specified, the "substituted" means that the hydrogen atoms on the group are replaced by one or more (eg 2, 3, 4, etc.) substituents selected from the group consisting of halogen, deuterated, 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 the group consisting of: C1-C6 alkyl, C3- C8 cycloalkyl, C1-C6 amine, C6-C10 aryl, 5-10-membered heteroaryl with 1-3 heteroatoms selected from N, S and O, with 1-3 heteroatoms selected from N, 5-12 membered heterocyclyl of heteroatoms of S and O, -( _CH2 )-C6-C10 aryl, -( _CH2 )-(with 1-3 heteroatoms selected from N, S and O 5-10-membered heteroaryl), and the substituent is selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 alkynyl, C1-C6 alkoxy, oxo, -CN, -NH ₂ , -OH, C6-C10 aryl, C1-C6 amine, C2-C6 amido, 5-10-membered heteroaryl with 1-3 heteroatoms selected from N, S and O; Wherein, Ra and Rb are each independently selected from the following group: H, substituted or unsubstituted C1-C4 alkyl; 3-10 membered heterocycloalkyl with 1, 2, 3) N heteroatoms and 0-2 heteroatoms selected from O, S. 2. The compound of claim 1, wherein

in 2, 3 or 4 positions. 3. The compound of claim 1, wherein

for

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

5. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises; (a) a therapeutically effective amount of a compound of formula I as claimed in claim 1, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof; and (b) A pharmaceutically acceptable carrier. 6. A compound of formula I as claimed in claim 1, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof or as claimed in claim 5 Use of the pharmaceutical composition for preparing a pharmaceutical composition for treating and/or preventing diseases or conditions related to the activity or expression of CDK12 and/or CDK13. 7. The use of claim 6, wherein the disease or disorder is cancer. 8. a preparation method of formula I compound as claimed in claim 1, is characterized in that, comprises the step: (a) The compounds of formula A4 and A5 will be reacted in an inert solvent to form a compound of formula I:

In the formula, Z is selected from: halogen, -OMs, -Ots, -Otf, -Oac or -OAr; X ₁ , X ₂ , R ₁ , R ₂ , R ₃ , R ₄ , L ₁ , L ₂ , L ₃ , A, B, C, and n1 are as defined in claim 1 . 9. An intermediate compound having a structural formula selected from: A1, A2, A3 or A4:

Wherein, Z is selected from: H, halogen, -OMs, -OTs, -OTf, -OAc or -OAr; X ₁ , X ₂ , R ₁ , R ₂ , R ₃ , R ₄ , L ₁ , L ₂ , L ₃ , A, B, C, and n1 are as defined in claim 1 . 10. A CDK12 and/or CDK13 inhibitor comprising: The compound of formula I as claimed in claim 1, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition as claimed in claim 5.

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