CN112574255B - Organic arsine-based CDK inhibitor and preparation method and application thereof - Google Patents

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

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CN112574255B
CN112574255B CN201910925781.8A CN201910925781A CN112574255B CN 112574255 B CN112574255 B CN 112574255B CN 201910925781 A CN201910925781 A CN 201910925781A CN 112574255 B CN112574255 B CN 112574255B
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CN112574255A (en
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谭立
李盈
周少青
庄光磊
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Shanghai Institute of Organic Chemistry of CAS
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Abstract

The invention provides an organic arsine-based CDK inhibitor, and a preparation method and application thereof. In particular, compounds of formula I, or stereoisomers or tautomers thereof, or pharmaceutically acceptable salts, hydrates or solvates thereof, are provided; and the preparation method and application thereof, wherein the definition of each group in the formula is shown in the specification.

Description

Organic arsine-based CDK inhibitor and preparation method and application thereof
Technical Field
The invention belongs to the field of medicines, and in particular relates to an organic arsine-based cell Cycle Dependent Kinase (CDK) inhibitor, a preparation method and application thereof.
Background
In the past twenty years, the development and application of targeted drugs have also been well-developed while chemotherapeutic drugs have been widely used in cancer research. Of the various proteins closely related to carcinogenesis and exacerbation, several members of the kinase protein family have been identified as effective therapeutic targets, and tens of targeted kinase inhibitors have also been approved for cancer treatment.
In 2014, gray et al reported that the first covalent inhibitor of CDK7, THZ1, was developed based on cysteine residue Cys312 on the C-terminal motif outside the ATP binding pocket of the CDK7 kinase domain, using a substituted acrylamide as electrophilic group. In addition to inhibiting CDK7, THZ1 also inhibits CDK12/13, which has a cysteine-like residue. Through inhibiting CDK7/12/13, THZ1 can effectively inhibit the activation of RNA polymerase II at low concentration, further inhibit the transcription initiation of superregulators in cancer cells, finally effectively inhibit the growth of the cancer cells, and has good curative effects in various tumor models such as T cell acute lymphoblastic leukemia (T-ALL), small Cell Lung Cancer (SCLC), neuroblastoma, triple negative breast cancer and the like. However, THZ1 has significant off-target effects and its pharmacokinetic properties are also not ideal.
In 2016 Gray et al developed a highly specific CDK12/13 inhibitor THZ531 based on THZ1, which was able to specifically induce apoptosis in some tumor cells, but which still had pharmacokinetic disadvantages including, for example, low bioavailability, short half-life, etc.
Syros improves on the basis of THZ1 to obtain SY-1365 with better specificity, and is currently in the first stage of clinical trials for treating solid tumors. Limitations in drug substitution properties have led to SY-1365 being administered by intravenous injection.
Accordingly, there is a great need in the art to provide CDK inhibitors that are highly selective, highly safe, and/or have better pharmacokinetic properties.
Disclosure of Invention
It is an object of the present invention to provide CDK inhibitors that are highly selective, highly safe, and/or have better pharmacokinetic properties. The inhibitors of the invention are a class of organic arsine based CDK inhibitors.
In a first aspect of the invention, there is provided a compound of formula I, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof;
Wherein,
Each X 1、X2 is independently selected from the group consisting of: none, O, S, NR 8、CH2;
R 8 is selected from the group consisting of: H. a substituted or unsubstituted C1-C6 alkyl group;
R 1、R2 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 C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl; or R 1、R2 taken together with the adjacent X 1、X2 and As forms a substituted or unsubstituted 4 to 8 membered heterocyclic ring containing one 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 4 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、L2 and L 3 are each independently selected from the group consisting of: none, - (Z) m-; and L 1 and L 2 are not both absent; wherein each Z is independently selected from: C1-C6 alkylene, -NR 6-,-NR6-R7 -, -O-, -CO-, m is 1, 2, 3 or 4;
Each R 6 is independently selected from the group consisting of: H. a substituted or unsubstituted C1-C4 alkyl group;
r 7 is a substituted or unsubstituted C1-C8 alkylene group;
A is selected from the group consisting of: an unsubstituted, substituted or unsubstituted C6-C10 aryl, a substituted or unsubstituted 5-10 membered heteroaryl, a substituted or unsubstituted C3-C10 cycloalkyl, a substituted or unsubstituted 3-10 membered heterocycloalkyl;
B is selected from the group consisting of: a substituted or unsubstituted C6-C10 aryl, a 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 cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl;
Unless otherwise specified, the term "substituted" refers to a radical in which a hydrogen atom is replaced with one or more (e.g., 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 amino, C6-C10 aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, 5-12 membered heterocyclyl having 1-3 heteroatoms selected from N, S and O, - (CH 2) -C6-C10 aryl, - (CH 2) - (5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O), and said substituents are 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 amino, C2-C6 amido, 5-to 10-membered heteroaryl having 1-3 heteroatoms selected from N, S and O;
Wherein Ra and Rb are each independently selected from the group consisting of: H. a substituted or unsubstituted C1-C4 alkyl group; or Ra, rb, together with the nitrogen atom to which they are attached, form a substituted or unsubstituted 3-to 10-membered heterocycloalkyl having at least 1 (e.g., 1, 2, 3) N heteroatoms and 0-2 heteroatoms selected from O, S.
In another preferred embodiment, for R 3, the substitution means 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、R2 together form a substituted or unsubstituted- (CH 2)n2 -structure) wherein n 2 is 1, 2 or 3.
In a further preferred embodiment of the present invention,At positions 2, 3 or 4.
In another preferred embodiment, the compound of formula I has the structure shown in I-a:
in a further preferred embodiment of the present invention, For/>
In another preferred embodiment, any of R 1、R2、R3、R4、n1、L1、L2、L3, A, B and C in the compounds described are each a group corresponding to a particular compound described in Table 1.
In another preferred embodiment, the compound of formula I is selected from the following table:
In a second aspect of the present invention, there is provided a pharmaceutical composition comprising;
(a) A therapeutically effective amount of a compound of formula I according to the first aspect of the invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof; and (b) a pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition further comprises one or more anti-cancer agents and/or immunosuppressives, preferably selected from the group consisting of: PARP1/2 inhibitors, chemotherapeutics inducing DNA damage to cancer cells, DNA alkylating chemotherapeutics, DNA or RNA synthesis inhibitors, EGFR, ALK or FGFR tyrosine receptor kinase inhibitors, KRAS, MEK or ERK tumor signaling pathway inhibitors, tumor immunotherapeutic drugs (e.g. PD-1 antibodies, PD-L 1 antibodies, etc.).
In another preferred embodiment, the pharmaceutical composition further comprises one or more anti-cancer agents and/or immunosuppressives, preferably selected from the group consisting of: oxipa, lu Kapa, nilapamide, methotrexate, capecitabine, gemcitabine, doxifluridine, pemetrexed disodium, pazopanib, imatinib, erlotinib, lapatinib, gefitinib, vandetanib, herceptin, bevacizumab, rituximab, trastuzumab, paclitaxel, vinorelbine, docetaxel, doxorubicin, hydroxycamptothecin, mitomycin, epirubicin, pirarubicin, bleomycin, letrozole, tamoxifen, fulvestrant, trazosin, fluzamide, leuprolide, amoxazole, cyclophosphamide, carmustine, nimustine, semustine, nitrogen, marflange, flufluvaldeconing, carboplatin, cisplatin, topotecan, camptothecine, topotecan, evericin, sequoyitol, 6-daptomycin, amitraz, 6-mercaptomycin, thiomycin, amitraz, and other drugs.
In a third aspect the present invention provides the use of a compound of formula I as described in the first aspect of the invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition as described in the second aspect of the invention, for the manufacture of a pharmaceutical composition for the treatment and/or prophylaxis of a disease or condition associated with CDK12 and/or CDK13 activity or expression.
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 Myelogenous Leukemia (AML), chronic Lymphocytic Leukemia (CLL), non-hodgkin's lymphoma (NHL), multiple myeloma, ovarian cancer, ewing's sarcoma, skin cancer, prostate cancer, liver cancer, pancreatic cancer, gastric cancer, esophageal 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, carcinoma in situ, lymphoma, neurofibromatosis, thyroid cancer, bone cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma, or sarcoma.
In another preferred embodiment, the cancer is a kinase highly expressed cancer selected from the group consisting of: CDK12, CDK13, KRAS, or a combination thereof.
In another preferred embodiment, the cancer is non-CDK 7 high expression.
In a fourth aspect of the present invention, there is provided a process for the preparation of a compound of formula I according to the first aspect of the present invention, comprising the steps of:
(a) Reacting the compounds of formula A4 and formula A5 in an inert solvent to form a compound of formula I:
In the method, in the process of the invention,
Z is selected from: halogen, -OMs, -OTs, -OTf, -OAc or-OAr;
X1、X2、R1、R2、R3、R4、L1、L2、L3、A、B、C、 And n1 is as defined in the first aspect of the invention.
In a fifth aspect of the present invention, there is provided a process for the preparation of a compound of formula I comprising the steps of:
(b) Reacting the compounds of formula A6 and formula A7 in an inert solvent to form a compound of formula I:
In the method, in the process of the invention,
X1、X2、R1、R2、R3、R4、L1、L2、L3、A、B、C、 And n 1 are as defined in the first aspect of the invention.
In a sixth aspect of the invention, there is provided an intermediate compound having a structure selected from: a1, A2, A3 or A4 has the structural formula:
Wherein Z is selected from: H. halogen, -OMs, -OTs, -OTf, -OAc or-OAr;
X1、X2、R1、R2、R3、R4、L1、L2、L3、A、B、C、 And n1 is as defined in the first aspect of the invention.
In a seventh aspect of the invention there is provided the use of a compound A1, A2, A3 or A4 for the preparation of compound I.
In an eighth aspect of the invention there is provided an inhibitor of CDK12 and/or CDK13 comprising:
A compound of formula I according to the first aspect of the invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition according to the second aspect of the invention.
In a ninth aspect of the invention, there is provided a method of selectively inhibiting a cell cycle dependent kinase in vitro comprising the steps of:
contacting a cell cycle dependent kinase with a compound of formula I, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, as described in the first aspect of the invention, or a pharmaceutical composition according to the second aspect of the invention, thereby selectively inhibiting 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, said "selectively inhibiting" means that the ratio (E1/E2) of the inhibitory activity E1 of said compound against a targeted CDK (e.g. CDK12 and/or CDK 13) to the inhibitory activity E2 against a non-targeted kinase (e.g. CDK7 or other non-targeted kinase selected from Table 4) is ≡2, preferably ≡5, more preferably ≡10.
In another preferred embodiment, the inhibitory activity E is the reciprocal of EC 50.
In a tenth aspect of the present invention, there is provided a method of inhibiting the growth or proliferation of tumour cells in vitro comprising the steps of,
Contacting a tumor cell with a compound of formula I as described in the first aspect of the invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition as described in the second aspect of the invention.
In another preferred embodiment, the method is non-therapeutic and non-diagnostic.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows the effect of representative compounds on Ser2 and Ser5 site phosphorylation of Pol II in H3122 (A) or A549 (B) cells.
FIG. 2 shows the competitive effect of representative compounds such as ZSQ-38, ZSQ8-36, etc. on covalent binding of THZ1-biotin to CDK7 or 12 in H3122 cells at a concentration of 1. Mu.M.
FIG. 3 shows the effect of representative compounds such as ZSQ-36 and ZSQ-66 on CDK7 or 12 catalytic Pol II CTD Ser5 site phosphorylation modification at various concentrations.
Fig. 4 shows concentration time curves of representative compounds ZSQ-38 and ZSQ8-36 in plasma, as well as pharmacokinetic parameters.
FIG. 5 shows the inhibitory effect of representative compounds ZSQ-38 and ZSQ-36 on growth proliferation of various cell lines at a concentration of 1. Mu.M.
Detailed Description
The present inventors have conducted extensive and intensive studies and, through extensive screening and testing, unexpectedly developed a class of compounds having high inhibitory activity against CDK12/13, but no or low inhibitory activity against CDK 7. The compounds of the invention are organic arsine-based cell cycle dependent kinase inhibitors of formula I, and in the structure of the compounds of the invention, the organic arsine group (moeity) is the moiety that covalently interacts with the active site of CDK12/13, thus providing excellent inhibitory activity and better specificity. Experimental results show that the compound has unexpected excellent specificity of targeting CDK12/13, and is high in safety and less in toxic and side effects. In addition, the compound of the invention has excellent pharmacokinetic property and long half life, and is suitable for patent medicine. The present invention has been completed on the basis of this finding.
Terminology
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Terminology
Unless otherwise stated, the term "alkyl" by itself or as part of another substituent refers to a straight or branched hydrocarbon radical having the indicated number of carbon atoms (i.e., C1-8 represents 1-8 carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, 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-isopentenyl, 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 (e.g., C 3-10 cycloalkyl) and either fully saturated or having no more than one double bond between ring vertices. "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 indicated, the term "heterocycloalkyl" refers to cycloalkyl groups containing one to five heteroatoms selected from N, O and S, where the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. Heterocycloalkyl groups may be monocyclic, bicyclic or polycyclic ring systems. Non-limiting examples of heterocycloalkyl groups include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidone, hydantoin, dioxolane, phthalimide, piperidine, 1, 4-dioxane, morpholine, thiomorpholine-S-oxide, thiomorpholine-S, S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine, and the like. Heterocycloalkyl groups can be attached to the remainder of the molecule via a ring carbon or heteroatom. Terms such as cycloalkylalkyl and heterocyclylalkyl refer to a cycloalkyl or heterocycloalkyl group attached to the remainder of the molecule through an alkyl or alkylene linker. For example, cyclobutylmethyl-is a cyclobutyl ring attached to the methylene linker of 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, such as-CH 2CH2CH2CH2 -. Alkyl (or alkylene) groups typically have 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms (e.g., 1, 2, 3, 4, 5, or 6) being preferred in the present invention. "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, typically having 4 or fewer carbon atoms. Similarly, "alkenylene" or "alkynylene" refer to an unsaturated form of "alkylene" having a double or triple bond, respectively.
Unless otherwise stated, the terms "alkoxy" or "alkyloxy", "alkylamino", "or" alkylamino "and" alkylthio "or" alkylthio "(or thioalkoxy) are used in their conventional sense to refer to those alkyl groups attached to the rest of the molecule via an oxygen atom, amino group, or sulfur atom, respectively. In addition, for dialkylamino groups, the alkyl moieties can be the same or different and can be combined with the nitrogen atom to which each alkyl group is attached to form a 3-7 membered ring.
Unless otherwise stated, the term "halo" or "halogen" by itself or as part of another substituent refers to a fluorine, chlorine, bromine, or iodine atom. Furthermore, 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-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 a single ring or multiple rings (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 multiple rings (bicyclic, up to tricyclic) fused together or covalently linked wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atoms are optionally quaternized. Heteroaryl groups may be attached to the remainder of the molecule through heteroatoms. Non-limiting examples of aryl groups include phenyl, naphthyl, and biphenyl, while non-limiting examples of heteroaryl groups include pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzotriazinyl (benzotriazin yl), purinyl, benzimidazolyl, benzopyrazolyl, benzotriazole, benzisoxazolyl, isobenzofuranyl (isobenzofur yl), isoindolyl, indolizinyl, benzotriazinyl, thienopyridinyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridine, benzothiazolyl, benzofuranyl, benzothienyl, indolyl, quinolinyl, isoquinolinyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furanyl, thienyl, and the like. The substituents for each of the above aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.
As used herein, the term "heteroatom" is intended to include oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).
For the compounds provided herein, a bond from a substituent (typically an R group) to the center of an aromatic ring (e.g., benzene, pyridine, etc.) will be understood to refer to a bond that provides a connection 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 will represent a bond to any available vertex of the six-or five-membered ring portion of the indole.
Unless otherwise indicated, all compounds present in the present invention are intended to include all possible optical isomers, such as single chiral compounds, or mixtures of various chiral compounds (i.e., racemates). Among all the compounds of the invention, each chiral carbon atom may optionally be in the R configuration or in the S configuration, or in a mixture of R and S configurations.
Certain compounds of the invention possess an asymmetric carbon atom (optical center) or double bond; racemates, diastereomers, geometric isomers, regioisomers and individual isomers (e.g., isolated enantiomers) are all intended to be included within the scope of the present invention. When compounds provided herein have a defined stereochemistry (denoted R or S, or indicated with dashed or wedge-shaped bonds), those compounds will be understood by those skilled in the art to be substantially free of other isomers (e.g., at least 80%,90%,95%,98%,99% and up to 100% free of other isomers).
In this context, the term "substituted" means that one or more hydrogen atoms on the group are replaced by 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 (R a)Rb, carboxy, or a substituted or unsubstituted group selected from the group consisting of C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 amino, C6-C10 aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, - (CH 2) -C6-C10 aryl, - (CH 2) - (5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O), and said substituent is selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 alkoxy, -CN, -C6 alkoxy, C6-C6 alkenyl, 5-C6 aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, - (CH 2) -C6-C10 aryl, - (CH 2) - (5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O.
As used herein, the terms "comprising," "including," or "comprising" mean that the various ingredients can be used together in a mixture or composition of the invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "containing.
As used herein, the term "pharmaceutically acceptable" ingredient refers to a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio.
As used herein, the term "therapeutically effective dose" refers to any amount of a drug that, when used alone or in combination with another therapeutic agent, promotes disease regression as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free symptoms, or prevention of a disorder or disability caused by the disease. "therapeutically effective dose" of a drug of the present invention also includes a "prophylactically effective dose," which is any amount of a drug that, when administered alone or in combination with another therapeutic agent, inhibits the occurrence or recurrence of a disease in a subject at risk of developing or suffering from recurrence of the disease.
As used herein, "pharmaceutically acceptable salts" refers to salts of the compounds of the invention with acids or bases that are suitable for use as medicaments. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is the salts of the compounds of the present invention with acids. Suitable salts forming acids include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, and the like; acidic amino acids such as aspartic acid and glutamic acid. One preferred class of salts is 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 water, 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 the low pressure sublimation drying process.
The invention also includes all suitable isotopic variations of the compounds of the present invention. Isotopic variations of 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 the atomic mass usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as 2H、3H、11C、13C、14C、15N、17O、18O、35S、18F and 36 Cl, respectively. Some isotopic variants of the present invention, for example, those into which a radioisotope (e.g., 3 H or 14 C) is incorporated, are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., 3 H, and carbon-14, i.e., 14 C, isotopes are particularly preferred because they are easy to prepare and detect. Furthermore, substitution with isotopes (e.g., deuterium, i.e., 2 H) may afford certain therapeutic advantages resulting from increased metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and therefore may be preferred in some circumstances. Isotopic variations of the compounds of the present invention can generally be prepared by conventional procedures, for example, by exemplary methods or by using suitable reagents for the appropriate isotopic variations described in the experimental section below.
Indicating a connection to other atoms.
As used herein, "treating" refers to alleviating, slowing progression, attenuating, preventing, or maintaining an existing disease or disorder (e.g., cancer). Treatment also includes curing, preventing the development of, or alleviating to some extent, one or more symptoms of the disease or disorder.
As used herein, "patient" refers to an animal, preferably a mammal, more preferably a human. The term "mammal" refers to a warm-blooded vertebrate mammal, 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", "ZSQ538" to denote ZSQ-5-38 compounds, with similar numbering usage rules being the same.
Active ingredient
As used herein, "a compound of the invention" refers to a compound of formula I, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof;
X1、X2、R1、R2、R3、R4、L1、L2、L3、A、B、C、 And n1 is as defined in the first aspect of the invention.
Preparation method
The present invention provides methods for preparing compounds of formula I, where the compounds of the present invention may be prepared by a variety of synthetic procedures, and exemplary methods for preparing these compounds may include, but are not limited to, the schemes described below.
Preferably, the compounds of formula I of the present invention can be accomplished by the exemplary methods described in the schemes and examples below, as well as by related published literature procedures used by those skilled in the art.
During specific operations, steps in the method may be expanded or combined as desired.
Step i: as the protective agent, ethylene dithiol, ethylene glycol, etc. can be used by protecting the arsenite functional group of A1 with an ester or amide.
Step ii: r 3 substituent is introduced into the amino group of A2 through S N substitution reaction, and R3-halogen can be used for reaction under alkaline conditions (such as sodium hydride).
Step iii: the acylation reaction of the benzenoid group at A3 introduces the L1 group, which can be catalyzed by (but is not limited to) phosphorus oxychloride reagent.
Step iv: the a functional site of A5 is linked to L 1 of A4 by an acylation reaction, a urea formation reaction or S N substitution.
The reactions in each of the above steps are conventional reactions known to those skilled in the art. Reagents and starting compounds used in the synthetic routes are commercially available or can be prepared by one skilled in the art with reference to known methods depending on the different compound structures designed, unless otherwise specified.
Pharmaceutical compositions and methods of administration
Because the compounds of the present invention have excellent inhibitory activity against cell Cycle Dependent Kinase (CDK), the compounds of the present invention, and pharmaceutically acceptable salts, hydrates or solvates thereof, and pharmaceutical compositions containing the compounds of the present invention as a main active ingredient are useful for treating, preventing and alleviating diseases or disorders associated with CDK activity or expression level.
Preferably, the compounds of the present invention are useful for the treatment of cancer. Representative cancers include (but are not limited to): t-cell acute lymphoblastic leukemia (T-ALL), small Cell Lung Cancer (SCLC), neuroblastoma, non-small cell lung cancer (NSCLC), colon cancer, acute Myelogenous Leukemia (AML), chronic Lymphocytic Leukemia (CLL), non-hodgkin's lymphoma (NHL), multiple myeloma, ovarian cancer, ewing's sarcoma, skin cancer, prostate cancer, liver cancer, pancreatic cancer, gastric cancer, esophageal 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, carcinoma in situ, 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 compositions of the present invention comprise a safe and effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical compositions contain 1-2000mg of the compound of the invention per dose, more preferably 10-500mg of the compound of the invention per dose. Preferably, the "one dose" is a capsule or tablet.
"Pharmaceutically acceptable carrier" means: one or more compatible solid or liquid filler or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "compatible" as used herein means that the components of the composition are capable of blending with and between the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, and the like), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, and the like), polyols (e.g., propylene glycol, glycerol, mannitol, sorbitol, and the like), emulsifiers (e.g.) Wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizing agents, antioxidants, preservatives, pyrogen-free water and the like.
The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.
Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) Fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders, for example, hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, e.g., glycerin; (d) Disintegrants, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent, such as paraffin; (f) an absorption accelerator, e.g., a quaternary amine compound; (g) Wetting agents, such as cetyl alcohol and glycerol monostearate; (h) an adsorbent, for example, kaolin; and (i) a lubricant, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.
Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. The active compound may also be in the form of microcapsules with one or more of the above excipients, if desired.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of these substances and the like.
In addition to these inert diluents, the compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar-agar or mixtures of these substances, and the like.
Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.
The compounds of the invention may be administered alone or in combination with other pharmaceutically acceptable compounds. Such as anti-cancer agents, (e.g., PARP1/2 inhibitors (olapanib (Olaparid), lu Kapa b, or nippanib (Niraparib), etc.), chemotherapeutics inducing DNA damage to cancer cells (cisplatin, carboplatin, or oxaliplatin, etc.), DNA alkylating chemotherapeutics (nimustine, etc.), inhibitors of DNA or RNA synthesis (gemcitabine, etc.), tyrosine receptor kinase inhibitors such as EGFR, ALK, or FGFR, tumor signaling pathway inhibitors such as KRAS, MEK, or ERK, tumor immunotherapeutic agents (PD-1 antibodies, PD-L 1 antibodies, etc.), in certain embodiments, the compounds of the invention are administered in combination with other conventional cancer therapies, e.g., radiation therapy or surgery.
In certain embodiments, the CDK inhibitor of the invention is used in the same or separate formulations either concurrently with, or sequentially with, other agents that are part of a combination therapeutic regimen.
The general range of therapeutically effective doses for a compound of formula I or a composition of a compound of formula I will be: about 1 to 2000 mg/day, about 10 to about 1000 mg/day, about 10 to about 500 mg/day, about 10 to about 250 mg/day, about 10 to about 100 mg/day, or about 10 to about 50 mg/day. A therapeutically effective dose will be administered in one or more doses. However, it will be appreciated that the particular dosage of a compound of the invention for any particular patient will depend on a variety of factors, such as the age, sex, weight, general health, diet, individual response of the patient to be treated, the time of administration, the severity of the disease to be treated, the activity of the particular compound administered, the dosage form, the mode of application and concomitant medication. The therapeutically effective amount for a given situation can be determined by routine experimentation and is within the ability and judgment of a clinician or physician. In any event, the compound or composition will be administered in multiple doses based on the individual condition of the patient and in a manner that allows for the delivery of a therapeutically effective amount.
The main advantages of the invention include:
1. the compound of the present invention has an organic arsine group (moeity) as a moiety which is covalently reacted with the active site of CDK12/13 for the first time, thus providing excellent inhibitory activity and better specificity.
2. The inhibiting activity of CDK12, 13 is superior to the existing inhibitor, and can effectively inhibit the growth and proliferation of cancer cells.
3. The inhibitor has no obvious inhibition to other kinases in cells, high selectivity, good targeting property, good safety and no obvious toxic or side effect under the effective concentration;
4. has ideal pharmacokinetic properties: the half-life is long and the area under the drug concentration-time curve (AUC) is high.
5. The combination of the compound and CDK12 and 13 is irreversible combination, the efficacy duration is longer, and the curative effect is longer.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
Example 1
Synthetic route to Compounds ZSQ-38
Synthesis of Compound (4-aminophenyl) arsinic acid (ZSQ-1-18)
4-Aminophenylarsonic acid (108.5 g,500 mmol) was dissolved in 300ml ethanol and the mixed solution was heated to reflux. Phenylhydrazine (92 ml,1 mol) was added dropwise (over 1 h) with a large amount of nitrogen generated during the addition, and when the nitrogen generation was slowed down, stirring under reflux was continued for 1.5h. The mixture was concentrated by distillation under reduced pressure, and sodium hydroxide solution (40 g in 400ml of water) was added thereto, followed by 400ml of diethyl ether. The solution was separated, a saturated ammonium chloride solution (400 ml) was added to the aqueous phase, and the mixture was stirred at 0℃for 1 hour, whereby a large amount of white solid was precipitated. Filtration gave a white needle-like solid which was dried in vacuo to give 40g of product in 40% yield.
Synthesis of Compound 4- (1, 3, 2-dithioarsin-2-yl) aniline (ZSQ-1-23)
ZSQ-1-18 (40 g, 199mmol) was dissolved in 200ml absolute ethanol and heated to reflux. Then, ethanedithiol (20 ml,240 mmol) was added dropwise to the mixed solution over 30min, and heating and stirring were continued for 30min. Subsequently, the mixture was cooled in ice water and filtered to give a crude product, which was recrystallized from ethanol to give 42g of white solid in 81% yield.
Synthesis of the Compound N- (4- (1, 3, 2-dithioarsin pent-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 20ml of dry DCM and stirred at 0deg.C. Bromoacetyl bromide (0.38 ml,4.4 mmol) was added dropwise over 15min and stirring was continued for 1h at 0 ℃. The mixture was diluted with DCM (20 ml), washed with 2M dilute hydrochloric acid solution (30 ml), water (50 ml), saturated sodium bicarbonate solution (30 ml), saturated sodium chloride solution (30 ml) in this order, extracted with DCM, the organic phases combined, dried over anhydrous sodium sulfate, filtered and concentrated. Purification by column chromatography on silica eluting with PE/EA (4:1) afforded 1.02g of a yellow solid in 67% yield.
Synthesis of Compound 3- (2, 5-dichloropyrimidin-4-yl) -1H-indole (ZSQ-3-76)
Methyl magnesium bromide (1M in tetrahydrofuran, 100ml,100 mmol) was added dropwise to a solution of indole (11.7 g,100 mmol) in THF (60 ml) at 0deg.C over 30min. The solution was stirred for a further 30min at 0 ℃.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 1h to give a red solution. The mixed solution was warmed to 60 ℃ and stirred for an additional 1.5h. 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 stirred at 60 ℃ for 20min to give a two-phase solution. Layering and adding petroleum ether (100 ml) to the organic solution resulted in crystallization of the solid. The solid was collected by filtration, washed with petroleum ether (20 ml) and dried under vacuum to give 7.02g 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 Bu 4NHSO4 (5.8 g,10 mmol) were dissolved in 100ml of dichloromethane and benzenesulfonyl chloride (3.84 ml,30 mmol) was added dropwise at room temperature. The reaction mixture was stirred at room temperature for 3h. The mixture was quenched with water (100 ml) and extracted with DCM (50 ml. Times.3). The combined organic layers were dried over anhydrous Na 2SO4, filtered, concentrated and purified by column chromatography on silica gel eluting with DCM/EA (8:1) to give 6.79g of a white solid in 84% yield.
Synthesis of tert-butyl Compound (R) -3- ((5-chloro-4- (1- (phenylsulfonyl) -1H-indol-3-yl) pyrimidin-2-yl) amino) piperidine-1-carboxylate (ZSQ-8-30)
ZSQ-5-26 (316 mg,1.5 mmol), (R) -1-Boc-3-aminopiperidine (300 mg,1.5 mmol) and DIPEA (0.74 ml,4.5 mmol) were mixed and dissolved in 3ml N-methylpyrrolidone. The mixed solution was heated at 135℃and stirred for 3h. The cooled solution was directly purified by C18 reverse phase column chromatography eluting with H 2O/CH3 CN (1:9) to give 802mg of a yellowish brown powder in 94% yield.
Synthesis of the Compound (R) -5-chloro-4- (1- (phenylsulfonyl) -1H-indol-3-yl) -N- (piperidin-3-yl) pyrimidin-2-amine (ZSQ-8-31)
ZSQ-8-30 (803 mg,1.4 mmol) and 1ml trifluoroacetic acid were mixed in 5ml DCM and stirred overnight at room temperature. After the completion of the reaction, the reaction mixture was concentrated and purified by C18 reverse phase column chromatography, eluting with H 2O/CH3 CN (1:6), to give 620mg of a yellowish brown powder in 94% yield.
Synthesis of the Compound (R) -N- (4- (1, 3, 2-dithioarsin-2-yl) phenyl) -2- (3- ((5-chloro-4- (1- (phenylsulfonyl) -1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) acetamide (ZSQ-8-34)
ZSQ-8-31 (140 mg,0.3 mmol), DIPEA (0.15 ml,0.9 mmol) and ZSQ-5-4 (114 mg,0.3 mmol) were dissolved in 5ml THF at room temperature and the mixed solution stirred at room temperature for 4h. Then washed with 2N dilute hydrochloric acid solution (20 ml) and extracted with EA (3X 30 ml), followed by washing with water (2X 20 ml). The combined organic phases were dried over anhydrous Na 2SO4, filtered and concentrated under reduced pressure, separated and purified by column chromatography on silica gel eluting with DCM/EA (4:1) to give 209mg of a white powder in 9 1% yield.
Synthesis of the Compound (R) -N- (4- (1, 3, 2-dithioarsin-2-yl) phenyl) -2- (3- ((5-chloro-4- (1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) acetamide (ZSQ-8-36)
ZSQ-8-34 (209 mg,0.27 mmol) of anhydrous potassium carbonate (113 mg,0.82 mmol) was dissolved in a mixed solution of 5ml of anhydrous methanol and 5ml of anhydrous THF, and the mixture was stirred at room temperature for 3 hours. After the reaction was completed, pH was adjusted to weak acidity with TFA, and the reaction mixture was directly purified by C18 reverse phase column chromatography, eluting with H 2O/CH3 CN (1:6), to give 123mg of a yellow powder in 73% yield.
Synthesis of Compound (R) - (4- (2- (3- ((5-chloro-4- (1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) acetamido) phenyl) arsinic acid (ZSQ-5-38)
ZSQ-8-36 (123 mg,0.19 mmol), mercuric perchlorate trihydrate (71 mg,0.157 mmol) was dissolved in 3ml DMSO, the mixed solution was stirred at room temperature for 10min, immediately purified by C18 reverse phase column chromatography eluting with H 2O/CH3 CN (1:4) to give 102mg of yellow powder in 92% yield.
Example 2:
Synthetic route to Compounds ZSQ-70
Synthesis of tert-butyl (ZSQ-5-57-1) carbamate, the compound (3- (2- ((4- (1, 3, 2-dithioarsin-2-yl) phenyl) amino) -2-oxoethyl) -phenyl)
ZSQ-1-23 (272 mg,1.05 mmol), 3-t-butoxycarbonylaminophenylacetic acid (251 mg,1 mmol), HATU (560 mg,1.5 mmol) and DIPEA (496 ul,3 mmol) were dissolved in 5ml DCM and left to stir at room temperature for 4h. After the reaction was completed, it was washed with H 2 O (20 ml. Times.2), saturated NaCl solution (20 ml), extracted with 20ml DCM, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated and purified by column chromatography on silica gel eluting with PE/EA (3:1) to give 412mg of a white solid in 83% yield.
Synthesis of the Compound N- (4- (1, 3, 2-dithioarsin-2-yl) phenyl) -2- (3-aminophenyl) acetamide (ZSQ-5-57-2)
ZSQ-5-57-1 (412 mg,0.83 mmol) and 1ml trifluoroacetic acid were mixed in 5ml DCM and stirred overnight at room temperature. After the completion of the reaction, the reaction mixture was concentrated and purified by C18 reverse phase column chromatography, eluting with H 2O/CH3 CN (1:7), to give 290mg of a white solid in 89% yield.
Synthesis of the Compound N- (4- (1, 3, 2-dithioarsin-2-yl) phenyl) -2- (3- ((5-chloro-4- (1- (phenylsulfonyl) -1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) acetamide (ZSQ-5-63)
ZSQ-5-26 (202 mg,0.5 mmol), ZSQ-5-57-2 (196 mg,0.5 mmol) and p-toluenesulfonic acid monohydrate (284 mg,1.5 mmol) were mixed and dissolved in 2ml of N-methylpyrrolidone. The mixed solution was heated at 135℃and stirred for 2h. The cooled solution was directly purified by C18 reverse phase column chromatography eluting with H 2O/CH3 CN (1:9) to give 86mg of a yellow powder in 22% yield.
Synthesis of the Compound N- (4- (1, 3, 2-dithioarsin-2-yl) phenyl) -2- (3- ((5-chloro-4- (1H-indol-3-yl) -pyrimidin-2-yl) amino) phenyl) acetamide (ZSQ-5-66)
ZSQ-5-63 (86 mg,0.11 mmol) in a mixed solution of anhydrous potassium carbonate (47 mg,0.33 mmol) in 3ml of anhydrous methanol and 3ml of anhydrous THF was stirred at room temperature for 4 hours. After the reaction was completed, pH was adjusted to weak acidity with TFA, and the reaction mixture was directly purified by C18 reverse phase column chromatography, eluting with H 2O/CH3 CN (1:6), to give 31mg of a yellow powder in 45% yield.
Synthesis of Compound (4- (2- (3- ((5-chloro-4- (1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) acetamido) -phenyl) arsinic acid (ZSQ-5-70)
ZSQ-5-66 (31 mg,0.05 mmol), mercuric perchlorate trihydrate (18 mg,0.04 mmol) was dissolved in 2ml DMSO and the mixed solution stirred at room temperature for 10min, immediately purified by C18 reverse phase column chromatography eluting with H 2O/CH3 CN (1:4) to give 21mg of yellow powder in 75% yield.
Example 3
Synthetic route to Compounds ZSQ-7-84
Synthesis of tert-butyl (ZSQ-7-72) piperidine-1-carboxylate (3- (((5-chloro-4- (1- (phenylsulfonyl) -1H-indol-3-yl) pyrimidin-2-yl) amino) methyl) compound
ZSQ-5-26 (316 mg,1.5 mmol), 1-Boc-3-aminomethylpiperidine (354 mg,1.65 mmol) and DIPEA (0.74 ml,4.5 mmol) were mixed and dissolved in 3ml N-methylpyrrolidone. The mixed solution was heated at 135℃and stirred for 3h. The cooled solution was directly purified by C18 reverse phase column chromatography eluting with H 2O/CH3 CN (1:9) to give 630mg of a yellow solid in 72% yield.
Synthesis of the Compound 5-chloro-4- (1- (phenylsulfonyl) -1H-indol-3-yl) -N- (piperidin-3-ylmethyl) -pyrimidin-2-amine (ZSQ-7-76)
ZSQ-7-72 (630 mg,1.08 mmol) and 1ml trifluoroacetic acid were mixed in 5ml DCM and stirred overnight at room temperature. After the completion of the reaction, the reaction mixture was concentrated and purified by C18 reverse phase column chromatography, eluting with H 2O/CH3 CN (1:6), to give 501mg of a tan solid in 96% yield.
Synthesis of the Compound N- (4- (1, 3, 2-dithioarsin-2-yl) phenyl) -3- (((5-chloro-4- (1- (phenylsulfonyl) -1H-indol-3-yl) pyrimidin-2-yl) amino) methyl) piperidine-1-carboxamide (ZSQ-7-80)
ZSQ-1-23 (130 mg,0.5 mmol) of bis (trichloromethyl) carbonate (59 mg,0.2 mmol) was dissolved in 5ml THF and stirred at 0deg.C, DIPEA (207 ul,1.25 mmol) was added dropwise and the mixed solution after addition was stirred at 0deg.C for 1h. ZSQ-7-76 (241 mg,0.5 mmol) was added, and after addition was completed stirring was continued at 0deg.C for 0.5h, and then was allowed to stir at room temperature for 2h. After completion of the reaction, washed successively with 2N dilute hydrochloric acid solution (20 ml), saturated sodium bicarbonate solution (20 ml), saturated sodium chloride solution (20 ml), extracted with 40ml DCM, the combined organic phases dried over anhydrous sodium sulfate, filtered and concentrated. Purification by column chromatography on silica eluting with PE/EA (2:1) gave 178mg of a white powder in 46% yield.
Synthesis of the Compound N- (4- (1, 3, 2-dithioarsin-2-yl) phenyl) -3- (((5-chloro-4- (1H-indol-3-yl) -pyrimidin-2-yl) amino) methyl) piperidine-1-carboxamide (ZSQ-7-83)
ZSQ-7-80 (178 mg,0.23 mmol) of anhydrous potassium carbonate (96 mg,0.69 mmol) was dissolved in a mixed solution of 5ml of anhydrous methanol and 5ml of anhydrous THF, and the mixture was stirred at room temperature for 3 hours. After the reaction was completed, pH was adjusted to weak acidity with TFA, and the reaction mixture was directly purified by C18 reverse phase column chromatography, eluting with H 2O/CH3 CN (1:7), to give 124mg of a yellow powder in 86% yield.
Synthesis of Compound (4- (3- (((5-chloro-4- (1H-indol-3-yl) pyrimidin-2-yl) amino) methyl) piperidine-1-carboxamido) phenyl) arsinic acid (ZSQ-7-84)
ZSQ-7-83 (124 mg,0.2 mmol), mercuric perchlorate trihydrate (71 mg,0.16 mmol) was dissolved in 3ml DMSO and the mixed solution stirred at room temperature for 10min, immediately purified by C18 reverse phase column chromatography eluting with H 2O/CH3 CN (1:4) to give 78mg of yellow powder in 70% yield.
Example 4
Synthetic route to Compound ZSQ-7-105
Synthesis of Compound 2-formyl-3-methylbutanenitrile (ZSQ-7-74)
2M lithium diisopropylamide solution (27.5 ml,55 mmol) was mixed in 50ml of dry THF, stirred at-78℃and isovaleronitrile (5.24 ml,50 mmol) was added dropwise, stirring was continued for 30min after addition was completed, ethyl formate (4.85 ml,60 mmol) was mixed in 25ml of dry THF, added dropwise to the mixed solution (over 30 min) at-78℃and stirring was continued for 45min, and then the mixture was allowed to move to room temperature and stirred for 14h. After completion of the reaction, 10ml of H 2 O was added to quench, the mixed solution was concentrated, diluted hydrochloric acid solution was added to adjust pH to 3, then extracted with EA (20 ml. Times.3), and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and dried by suction to give 5g of a tan oily crude product.
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 75ml absolute ethanol, heated in a sealed tube and stirred for 16h. After completion of the reaction, the mixture was concentrated to leave 1/3, washed with saturated sodium bicarbonate solution (40 ml) (pH was adjusted to alkaline), extracted with DCM (30 ml. Times.2), and washed with saturated sodium chloride solution (40 ml). The combined organic phases were dried over anhydrous sodium sulfate, and the filtrate was concentrated to dryness without further purification to give 6.7g of a yellow solid.
Synthesis of the 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 lock tube containing 30ml of dry ethanol, 20% sodium ethoxide solution (3.74 g,11 mmol) was added, the mixed solution was heated to reflux temperature and stirred for 22h. After the completion of the reaction, the reaction mixture was concentrated, diluted with water (20 ml), adjusted to pH 3 with a dilute hydrochloric acid solution, and a large amount of solids were precipitated, filtered and washed with water several times. A yellow powder was obtained, which after drying gave 1.29g of a white powder with a yield of 66%.
Synthesis of the Compound 5, 7-di-chloro-3-isopropylpyrazolo [1,5-a ] pyrimidine (ZSQ-7-88)
ZSQ-7-85 (1.29 g,6.67 mmol) and N, N-dimethylaniline (84 ul,0.67 mmol) were mixed in a sealed tube containing 15ml of phosphorus oxychloride, heated to 115℃overnight and stirred under reflux. After completion of the reaction, the mixed solution was concentrated, the remaining solution was dropwise added to ice water, extracted with DCM a plurality of times, washed with saturated sodium chloride solution (30 ml), and the organic phases were combined. Drying over anhydrous sodium sulfate, filtering, concentrating and purifying by silica gel column chromatography eluting with PE/EA (4:1) to give 950mg of yellow solid in 62% yield.
Synthesis of the 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 50ml absolute ethanol, heated to 85℃and stirred overnight. After completion of the reaction, the reaction mixture was concentrated, washed with water (30 ml. Times.2), extracted with EA (30 ml), and washed with a saturated sodium chloride solution (30 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated and purified by column chromatography on silica gel eluting with PE/EA (5:1) to give 1.16g of a white solid in 93% yield.
Synthesis of 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 mixed and dissolved in 15ml THF and stirred at room temperature for 12h. After completion of the reaction, the reaction mixture was washed with H 2 O (20 ml. Times.2), extracted with EA (30 ml) and washed with saturated sodium hydrogencarbonate solution (30 ml). The organic phase was dried over anhydrous sodium sulfate, concentrated by filtration and purified by column chromatography on silica gel eluting with PE/EA (8:1) to give 1.20g of pale yellow flaky solid with 99% yield.
Synthesis of tert-butyl Compound 3- (((7- (benzyl (tert-butoxycarbonyl) amino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-5-yl) amino) methyl) piperidine-1-carboxylate (ZSQ-7-96)
Compounds ZSQ-7-94 (400 mg,1 mmol), 1-Boc-3-aminomethylpiperidine (236 mg,1.1 mmol), tris (dibenzylideneacetone) dipalladium (46 mg,0.05 mmol), 1 '-binaphthyl-2, 2' -bisdiphenylphosphine (93 mg,0.15 mmol) and sodium t-butoxide (106 mg,1.1 mmol) were mixed in 3ml toluene and heated to 95℃under nitrogen and stirred for 16h. After completion of the reaction, the reaction mixture was washed with H 2 O (30 ml. Times.3), extracted with EA (40 ml) and washed with a saturated sodium chloride solution (30 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, separated and purified by column chromatography on silica gel eluting with PE/EA (5:1) to give 554mg of yellow-brown powder in 95% yield.
Synthesis of Compound N 7 -benzyl-3-isopropyl-N 5 - (piperidin-3-ylmethyl) pyrazolo [1,5-a ] pyrimidine-5, 7-diamine (ZSQ-7-98)
ZSQ-7-96 (554 mg,0.96 mmol) and 1ml trifluoroacetic acid were mixed in 5ml DCM and stirred overnight at room temperature. After the completion of the reaction, the reaction mixture was concentrated and purified by C18 reverse phase column chromatography, eluting with H 2O/CH3 CN (1:5), to give 360mg of a white solid in 99% yield.
Synthesis of the Compound N- (4- (1, 3, 2-dithioarsin-2-yl) phenyl) -3- (((7- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-5-yl) amino) methyl) piperidine-1-carboxamide (ZSQ-7-99)
ZSQ-1-23 (130 mg,0.5 mmol) of bis (trichloromethyl) carbonate (59 mg,0.2 mmol) was dissolved in 5ml THF and stirred at 0deg.C, DIPEA (207 ul,1.25 mmol) was added dropwise and the mixed solution after addition was stirred at 0deg.C for 1h. ZSQ-7-98 (189 mg,0.5 mmol) was added and after the addition was completed stirring was continued at0℃for 0.5h and then brought to room temperature for stirring for 2h. After completion of the reaction, washed successively with 2N dilute hydrochloric acid solution (20 ml), saturated sodium bicarbonate solution (20 ml), saturated sodium chloride solution (20 ml), extracted with 40ml DCM, the combined organic phases dried over anhydrous sodium sulfate, filtered and concentrated. Purification by column chromatography on silica eluting with DCM/EA (1:1) gave 162mg of a white powder in 49% yield.
Synthesis of Compound (4- (3- (((7- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-5-yl) amino) -methyl) piperidine-1-carboxamido) phenyl) arsinic acid (ZSQ-7-105)
ZSQ-7-99 (80 mg,0.12 mmol), mercury perchlorate trihydrate (38 mg,0.08 mmol) was dissolved in 2ml DMSO, the mixed solution was stirred at room temperature for 10min, immediately purified by C18 reverse phase column chromatography eluting with H 2O/CH3 CN (1:5) to give 56mg of white powder in 78% yield.
Example 5
Synthetic route to Compound ZSQ-8-12
Synthesis of benzyl Compound 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 10ml DMF and after stirring at room temperature for 20min benzyl bromide (522. Mu.l, 4.4 mmol) was added dropwise and stirring at room temperature was continued for 6h after addition. After completion of the reaction, the reaction mixture was washed with H 2 O (30 ml. Times.3) and extracted with EA. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. Purification by column chromatography on silica eluting with PE/EA (3:1) afforded 1.2g of a white solid in 88% yield.
Synthesis of benzyl 3- (aminomethyl) benzoate (ZSQ-8-1)
ZSQ-7-104 (1.2 g,3.5 mmol) and 1ml trifluoroacetic acid were mixed in 5ml DCM and stirred overnight at room temperature. After the completion of the reaction, the reaction mixture was concentrated and purified by C18 reverse phase column chromatography, eluting with H 2O/CH3 CN (1:6), to give 800mg of a white solid in 95% yield.
Synthesis of benzyl (ZSQ-8-3) benzoate of Compound 3- (((7- (benzyl (tert-butoxycarbonyl) amino) -3-isopropylpyrrolo [1,5-a ] pyrimidin-5-yl) amino) methyl)
Compounds 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 '-binaphthyl-2, 2' -bisdiphenylphosphine (47 mg,0.075 mmol) and sodium t-butoxide (58 mg,0.6 mmol) were mixed in 2ml toluene and heated to 95℃under nitrogen and stirred for 16h. After completion of the reaction, the reaction mixture was washed with H 2 O (30 ml. Times.3), extracted with EA (40 ml) and washed with a saturated sodium chloride solution (30 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, separated and purified by column chromatography on silica gel eluting with PE/EA (4:1) to give 154mg of a pale yellow powder in 51% yield.
Synthesis of Compound 3- (((7- (benzyl (tert-butoxycarbonyl) amino) -3-isopropylpyrazolo [1,5-a ] -pyrimidin-5-yl) amino) methyl) benzoic acid (ZSQ-8-6)
ZSQ-8-3 (154 mg,0.25 mmol) and Pd/C (100 mg) were mixed in 10ml of methanol and stirred for 6h at room temperature. After the reaction was completed, the mixture was filtered through celite, washed with methanol, and the filtrate was concentrated and purified by reverse phase column chromatography on C18, eluting with H 2O/CH3 CN (1:7), to give 116mg of the product as a yellow oil in 90% yield
Synthesis of tert-butyl (ZSQ-8-8) carbamate, the compound (5- ((3- ((4- (1, 3, 2-dithioarsine pent-2 yl) phenyl) carbamoyl) benzyl) amino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-7-yl) (benzyl)
ZSQ-8-6 (116 mg,0.22 mmol) and ZSQ-1-23 (70 mg,0.27 mmol) were dissolved in 3ml of dry pyridine, the mixed solution was stirred at 0deg.C, phosphorus oxychloride (42 ul,0.45 mmol) was added dropwise to the mixed solution (over 10 min), stirring was continued at 0deg.C for 1h after the addition was completed, and then the solution was moved to room temperature and stirred for 2h. After the completion of the reaction, the mixture was acidified with a dilute hydrochloric acid solution, washed with water (30 ml. Times.3), extracted with EA and washed with a saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. Purification by column chromatography on silica gel eluting with PE/EA (4:1) to give the pyridinium salt, followed by DCM/CH 3OH(NH3) (10:1) gave 92mg as a yellow powder in 54% yield.
Synthesis of the Compound N- (4- (1, 3, 2-dithioarsin-2-yl) phenyl) -3- (((7- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-5-yl) amino) methyl) benzamide (ZSQ-8-9)
ZSQ-8-8 (92 mg,0.12 mmol) and 0.5ml trifluoroacetic acid were mixed in 5ml DCM and stirred overnight at room temperature. After the completion of the reaction, the reaction mixture was concentrated and purified by C18 reverse phase column chromatography, eluting with H 2O/CH3 CN (1:7), to give 64.8mg of a white solid in 82% yield.
Synthesis of Compound (4- (3- (((7- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-5-yl) amino) methyl) benzoylamino) phenyl) arsinic acid (ZSQ-8-12)
ZSQ-8-9 (64.8 mg,0.099 mmol), mercuric perchlorate trihydrate (33 mg,0.074 mmol) was dissolved in 2ml DMSO, the mixed solution was stirred at room temperature for 10min, immediately purified by C18 reverse phase column chromatography eluting with H 2O/CH3 CN (1:5) to give 45mg of white powder in 76% yield.
Example 6
Synthetic route to Compounds ZSQ-9-73
Synthesis of tert-butyl (ZSQ-9-48) piperidine-1-carboxylate as Compound (R) -3- ((7- (benzyl (tert-butoxycarbonyl) amino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-5-yl) amino)
Compounds 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 '-binaphthyl-2, 2' -bisdiphenylphosphine (47 mg,0.075 mmol) and sodium tert-butoxide (53 mg,0.55 mmol) were mixed in 2ml toluene and heated to 95℃under nitrogen atmosphere and stirred for 16h. After completion of the reaction, the reaction mixture was washed with H 2 O (30 ml. Times.3), extracted with EA (40 ml) and washed with a saturated sodium chloride solution (30 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, separated and purified by column chromatography on silica gel eluting with PE/EA (3:1) to give 242.5mg of a reddish brown solid in 85% yield.
Synthesis of the Compound (R) -N 7 -benzyl-3-isopropyl-N 5 - (piperidin-3-yl) pyrazolo [1,5-a ] pyrimidine-5, 7-diamine (ZSQ-9-55)
ZSQ-9-48 (242 mg,0.43 mmol) and 0.8ml trifluoroacetic acid were mixed in 5ml DCM and stirred overnight at room temperature. After the completion of the reaction, the reaction mixture was concentrated and purified by C18 reverse phase column chromatography, eluting with H 2O/CH3 CN (1:5), to give 150mg of a white solid in 96% yield.
Synthesis of the Compound (R) -N- (4- (1, 3, 2-dithioarsin-2-yl) phenyl) -2- (3- ((7- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-5-yl) amino) piperidin-1-yl) acetamide (ZSQ-9-60)
ZSQ-9-55 (150 mg,0.41 mmol), DIPEA (0.20 ml,1.23 mmol) and ZSQ-5-4 (155 mg,0.41 mmol) were dissolved in 7ml THF at room temperature and the mixture was stirred at room temperature for 3h. Then washed with 2N dilute hydrochloric acid solution (20 ml) and extracted with EA (3X 30 ml), followed by washing with water (2X 20 ml). The combined organic phases were dried over Na 2SO4, filtered and concentrated under reduced pressure, separated and purified by column chromatography on silica gel eluting with PE/EA (1:1) to give 133.3mg of a yellow solid in 49% yield.
Synthesis of Compound (R) - (4- (2- (3- ((7- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-5-yl) amino) piperidin-1-yl) acetamido) phenyl) arsinic acid (ZSQ-9-73)
ZSQ-8-9 (66 mg,0.1 mmol) of mercury perchlorate trihydrate (36 mg,0.08 mmol) was dissolved in 2ml of DMSO, the mixed solution was stirred at room temperature for 10min, immediately purified by C18 reverse phase column chromatography eluting with H 2O/CH3 CN (1:5) to give 49.5mg of a yellow solid in 82% yield.
Example 7
Synthetic route to Compounds ZSQ-10-98
Synthesis of tert-butyl carbamate (ZSQ-7-71) of Compound (3- ((4- (1, 3, 2-dithioarsine pent-2-yl) phenyl) carbamoyl) benzyl)
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 20ml DCM and left to stir at room temperature for 4h. After the reaction was completed, it was washed with H 2 O (20 ml. Times.2), saturated NaCl solution (20 ml), extracted with 20ml DCM, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography, eluting with PE/EA (4:1) to give 990mg of pale yellow powder in 50% yield.
Synthesis of the Compound N- (4- (1, 3, 2-dithioarsin-2-yl) phenyl) -3- (aminomethyl) benzamide (ZSQ-7-75)
ZSQ-7-71 (990 mg,2.01 mmol) and 1ml trifluoroacetic acid were mixed in 5ml DCM and stirred overnight at room temperature. After the completion of the reaction, the reaction solution was concentrated and purified by C18 reverse phase column chromatography, eluting with H 2O/CH3 CN (1:5), to give 651mg of a white solid in 82% yield.
Synthesis of the Compound N- (4- (1, 3, 2-dithioarsin-2-yl) phenyl) -3- (((5-chloro-3-isopropylpyrazolo [1,5-a ] 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 6ml absolute ethanol, heated to 85℃and stirred overnight. After completion of the reaction, the reaction mixture was concentrated, washed with water (30 ml. Times.2), extracted with EA (30 ml), and washed with a saturated sodium chloride solution (30 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated and purified by column chromatography on silica gel eluting with PE/EA (5:1) to give 588mg of brown solid in 72% yield.
Synthesis of tert-butyl (ZSQ-10-92) carbamate of Compound (3- ((4- (1, 3, 2-dithioarsin-2-yl) phenyl) carbamoyl) benzyl) (5-chloro-3-isopropylpyrazolo [1,5-a ] pyrimidin-7-yl)
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 5ml THF and stirred at room temperature for 12h. After completion of the reaction, the reaction mixture was washed with H 2 O (20 ml. Times.2), extracted with EA (30 ml) and washed with saturated sodium hydrogencarbonate solution (30 ml). The organic phase was dried over anhydrous sodium sulfate, concentrated by filtration and purified by column chromatography on silica gel eluting with PE/EA (7:1) to give 524mg of yellow solid in 76% yield.
Synthesis of tert-butyl (ZSQ-10-93) carbamate of the compound (3- ((4- (1, 3, 2-dithioarsen-2-yl) phenyl) carbamoyl) benzyl) (5- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-7-yl)
Compounds ZSQ-10-93 (274 mg,0.4 mmol), benzylamine (52 ul,0.48 mmol), tris (dibenzylideneacetone) dipalladium (18 mg,0.02 mmol), 1 '-binaphthyl-2, 2' -bisdiphenylphosphine (37 mg,0.06 mmol) and sodium t-butoxide (46 mg,0.48 mmol) were mixed in 2ml toluene and heated to 95℃under nitrogen atmosphere and stirred for 16h. After completion of the reaction, the reaction mixture was washed with H 2 O (30 ml. Times.3), extracted with EA (40 ml) and washed with a saturated sodium chloride solution (30 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, separated and purified by column chromatography on silica gel eluting with PE/EA (3:1) to give 40mg of a white powder in 13% yield.
Synthesis of the Compound N- (4- (1, 3, 2-dithioarsin-2-yl) phenyl) -3- (((5- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-7-yl) amino) methyl) benzamide (ZSQ-10-95)
ZSQ-10-93 (40 mg,0.05 mmol) and 0.5ml trifluoroacetic acid were mixed in 5ml DCM and stirred overnight at room temperature. After the completion of the reaction, the reaction mixture was concentrated and purified by C18 reverse phase column chromatography, eluting with H 2O/CH3 CN (1:7), to give 23mg of a white solid in 69% yield.
Synthesis of Compound (4- (3- (((5- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-7-yl) amino) methyl) benzoylamino) phenyl) arsinic acid (ZSQ-10-98)
ZSQ-10-95 (23 mg,0.035 mmol), mercury perchlorate trihydrate (12 mg,0.028 mmol) was dissolved in 2ml DMSO and the mixed solution stirred at room temperature for 10min, immediately purified by C18 reverse phase column chromatography eluting with H 2O/CH3 CN (1:5) to give 17mg of a white solid in 85% yield.
Example 8
Synthetic route to Compound ZSQ-12-3
Synthesis of tert-butyl (ZSQ-11-64) piperidine-1-carboxylate, a compound (R) -3- ((5-chloro-3-isopropylpyrazolo [1,5-a ] pyrimidin-7-yl) amino)
ZSQ-7-88 (345 mg,1.5 mmol), DIPEA (0.49 ml,3.0 mmol) and (R) -1-Boc-3-aminopiperidine (450 mg,2.25 mmol) were dissolved in 6ml absolute ethanol, heated to 85℃and stirred overnight. After completion of the reaction, the reaction mixture was concentrated, washed with water (30 ml. Times.2), extracted with EA (30 ml), and washed with a saturated sodium chloride solution (30 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated and purified by column chromatography on silica gel eluting with PE/EA (4:1) to give 571mg of white powder in 96% yield.
Synthesis of tert-butyl (ZSQ-11-89) piperidine-1-carboxylate, the compound (R) -3- ((tert-butoxycarbonyl) (5-chloro-3-isopropylpyrazolo [1,5-a ] pyrimidin-7-yl) amino)
ZSQ-10-64 (571 mg,1.45 mmol), 4-dimethylaminopyridine (35 mg,0.20 mmol) and di-tert-butyl dicarbonate (433 ul,1.89 mmol) were mixed and dissolved in 6ml THF and stirred at room temperature for 12h. After completion of the reaction, the reaction mixture was washed with H 2 O (20 ml. Times.2), extracted with EA (30 ml) and washed with saturated sodium hydrogencarbonate solution (30 ml). The organic phase was dried over anhydrous sodium sulfate, concentrated by filtration and purified by column chromatography on silica gel eluting with PE/EA (8:1) to give 670mg of a white solid in 93% yield.
Synthesis of tert-butyl Compound (R) -3- ((5- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-7-yl) (tert-butoxycarbonyl) amino) piperidine-1-carboxylate (ZSQ-11-94)
Compounds ZSQ-11-89 (495 mg,1.0 mmol), benzylamine (131 ul,1.2 mmol), tris (dibenzylideneacetone) dipalladium (46 mg,0.05 mmol), 1 '-binaphthyl-2, 2' -bisdiphenylphosphine (93 mg,0.15 mmol) and sodium t-butoxide (115 mg,1.2 mmol) were mixed in 5ml toluene and heated to 95℃under nitrogen atmosphere and stirred for 16h. After completion of the reaction, the reaction mixture was washed with H 2 O (30 ml. Times.3), extracted with EA (40 ml) and washed with a saturated sodium chloride solution (30 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, separated and purified by column chromatography on silica gel eluting with PE/EA (3:1) to give 524mg of a reddish brown solid in 93% yield.
Synthesis of the Compound (R) -N 5 -benzyl-3-isopropyl-N 7 - (piperidin-3-yl) pyrazolo [1,5-a ] pyrimidine-5, 7-diamine (ZSQ-11-96)
ZSQ-11-94 (524 mg,0.93 mmol) and 1ml trifluoroacetic acid were mixed in 6ml DCM and stirred overnight at room temperature. After the completion of the reaction, the reaction mixture was concentrated and purified by C18 reverse phase column chromatography, eluting with H 2O/CH3 CN (1:5), to give 322mg of a white solid in 95% yield.
Synthesis of the Compound (R) -N- (4- (1, 3, 2-dithioarsin-2-yl) phenyl) -2- (3- ((5- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-7-yl) amino) piperidin-1-yl) acetamide (ZSQ-11-104)
ZSQ-11-96 (182 mg,0.5 mmol), K 2CO3 (166 mg,1.2 mmol) and ZSQ-5-4 (190 mg,0.5 mmol) were dissolved in 4ml CH 3 CN solution at room temperature and the mixed solution was stirred at room temperature for 4h. Then washed with 2N dilute hydrochloric acid solution (20 ml) and extracted with EA (3X 30 ml), followed by washing with water (2X 20 ml). The combined organic phases were dried over Na 2SO4, filtered and concentrated under reduced pressure, purified by column chromatography on silica gel eluting with DCM/EA (4:1) to give 205mg of a white powder in 62% yield.
Synthesis of Compound (R) - (4- (2- (3- ((5- (benzylamino) -3-isopropylpyrazolo [1,5-a ] pyrimidin-7-yl) amino) piperidin-1-yl) acetamido) phenyl) arsinic acid (ZSQ-12-3)
ZSQ-11-104 (66 mg,0.10 mmol), mercury perchlorate trihydrate (36 mg,0.08 mmol) was dissolved in 3ml DMSO and the mixed solution stirred at room temperature for 10min, immediately purified by C18 reverse phase column chromatography eluting with H 2O/CH3 CN (1:5) to give 49mg of a white solid in 81% yield.
Example 9
Synthetic route to compound ZSQ-13-92
Synthesis of Compound 3- (2-chloro-5-iodopyrimidin-4-yl) -1H-indole (ZSQ-12-59)
Methyl magnesium bromide (1M in tetrahydrofuran, 70ml,70 mmol) was added dropwise to a solution of indole (8.26 g,70 mmol) in THF (50 ml) at 0deg.C over 30min. The solution was stirred for a further 30min at 0 ℃.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 1h to give a red solution. The mixed solution was warmed to 60 ℃ and stirred for an additional 1.5h. 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 stirred at 60℃for 20min to give a two-phase solution. Layering and adding petroleum ether (100 ml) to the organic solution resulted in crystallization of the solid. The solid was collected by filtration, washed with petroleum ether (20 ml) and dried under vacuum to give 2.5g of a yellow solid in 20% yield.
Synthesis of Compound 3- (2-chloro-5-iodopyrimidin-4-yl) -1- (phenylsulfonyl) -1H-indole (ZSQ-13-76)
ZSQ-13-59 (1.06 g,3 mmol), naOH (180 mg,4.5 mmol) and Bu 4NHSO4 (871 mg,1.5 mmol) were dissolved in 20ml DCM and benzenesulfonyl chloride (576 ul,4.5 mmol) was added dropwise at room temperature. The reaction mixture was stirred at room temperature for a further 4h. The mixture was quenched with water (30 ml) and extracted with DCM (20 ml. Times.3). The combined organic layers were dried over anhydrous Na 2SO4, filtered, concentrated, and purified by column chromatography on silica gel eluting with DCM/EA (4:1) to give 850mg of a white solid in 57% yield.
Synthesis of Compound 3- (2-chloro-5- ((trimethylsilyl) ethynyl) pyrimidin-4-yl) -1- (phenylsulfonyl) -1H-indole (ZSQ-13-77)
Compounds ZSQ-13-76 (850 mg,1.72 mmol), trimethylsilylacetylene (264 ul,2.58 mmol), cuprous iodide (32 mg,0.17 mmol), 1' -bis-diphenylphosphino ferrocene palladium dichloride (73 mg,0.10 mmol) and triethylamine (178 ul,3.44 mmol) were mixed in 10ml of dry tetrahydrofuran solution and heated to 50℃under nitrogen atmosphere and stirred for 12h. After completion of the reaction, the reaction mixture was washed with 2M dilute hydrochloric acid (30 ml. Times.2), extracted with DCM (50 ml), and washed successively with water (30 ml) and saturated sodium chloride solution (30 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, separated and purified by column chromatography on silica gel eluting with PE/EA (4:1) to give 380mg of white solid in 47% yield.
Synthesis of tert-butyl (ZSQ-13-86) piperidine-1-carboxylate (R) -3- ((4- (1- (phenylsulfonyl) -1H-indol-3-yl) -5- ((trimethylsilyl) ethynyl) pyrimidin-2-yl) amino)
ZSQ-13-77 (380 mg,0.817 mmol), (R) -1-Boc-3-aminopiperidine (172 mg,0.858 mmol) and DIPEA (0.4 ml, 2.457 mmol) were mixed and dissolved in 3ml N-methylpyrrolidone. The mixed solution was heated at 135℃and stirred for 3h. The cooled solution was directly purified by C18 reverse phase column chromatography eluting with H 2O/CH3 CN (1:9) to give 448mg of brown powder in 87% yield.
Synthesis of the Compound (R) -5-ethynyl-4- (1- (phenylsulfonyl) -1H-indol-3-yl) -N- (piperidin-3-yl) pyrimidin-2-amine (ZSQ-13-87)
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ZSQ-13-86 (4478 mg,0.71 mmol) and 1ml trifluoroacetic acid were mixed in 6ml DCM and stirred overnight at room temperature. After the completion of the reaction, the reaction mixture was concentrated and purified by C18 reverse phase column chromatography, eluting with H 2O/CH3 CN (1:5), to give 105mg of a tan solid in 32% yield.
Synthesis of the Compound (R) -N- (4- (1, 3, 2-dithioarsin-2-yl) phenyl) -2- (3- ((5-ethynyl-4- (1- (phenylsulfonyl) -1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) acetamide (ZSQ-13-88)
ZSQ-13-87 (105 mg,0.23 mmol), DIPEA (0.11 ml,0.69 mmol) and ZSQ-5-4 (91 mg,0.24 mmol) were dissolved in 4ml THF at room temperature and the mixture stirred at room temperature for 4h. Then washed with 2N dilute hydrochloric acid solution (20 ml) and extracted with EA (3X 30 ml), followed by washing with water (2X 20 ml). The combined organic phases were dried over Na 2SO4, filtered and concentrated under reduced pressure, purified by column chromatography on silica gel eluting with DCM/EA (2:1) to give 75mg of a white powder in 43% yield.
Synthesis of the Compound (R) -N- (4- (1, 3, 2-dithioarsin-2-yl) phenyl) -2- (3- ((5-ethynyl-4- (1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) acetamide (ZSQ-13-91)
ZSQ-13-88 (75 mg,0.1 mmol) of anhydrous potassium carbonate (41 mg,0.3 mmol) was dissolved in a mixed solution of 2ml of anhydrous methanol and 2ml of anhydrous THF, and the mixture was stirred at room temperature for 4 hours. After the reaction was completed, the pH was adjusted to weak acidity with TFA, and the reaction mixture was directly purified by C18 reverse phase column chromatography, eluting with H 2O/CH3 CN (1:6), to give 52mg of a yellow powder in 85% yield.
Synthesis of Compound (R) - (4- (2- (3- ((5-ethynyl-4- (1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) acetamido) phenyl) arsinic acid (ZSQ-13-92)
ZSQ-13-91 (52 mg,0.08 mmol), mercuric perchlorate trihydrate (30 mg,0.06 mmol) was dissolved in 3ml DMSO and the mixed solution stirred at room temperature for 10min, immediately purified by C18 reverse phase column chromatography eluting with H 2O/CH3 CN (1:5) to give 40mg of a yellow solid in 88% yield.
Example 10
Synthetic route to Compound ZSQ-14-23
Synthesis of the Compound 4- (1, 3, 2-dithioarsin-2-yl) -N- (prop-2-yn-1-yl) aniline (ZSQ-14-7)
Compounds 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 8ml DMF and stirred at room temperature for 12h. After completion of the reaction, the reaction mixture was washed with H 2 O (30 ml. Times.3) and extracted with EA (30 ml). The organic phase was dried over anhydrous sodium sulfate, concentrated by filtration and purified by column chromatography on silica gel eluting with PE/EA (7:1) to give 850mg of yellow solid in 72% yield
Synthesis of the Compound N- (4- (1, 3, 2-dithioarsin-2-yl) phenyl) -2-bromo-N- (prop-2-yn-1-yl) acetamide (ZSQ-14-10)
ZSQ-14-7 (850 mg,2.86 mmol) and DIPEA (7.15 ml,3.14 mmol) were dissolved in 20ml of dry DCM and stirred at 0deg.C. Bromoacetyl bromide (0.27 ml,3.14 mmol) was added dropwise over 15min and stirring was continued for 1h at 0 ℃. The mixture was diluted with DCM (20 ml), washed with 2N diluted hydrochloric acid solution (30 ml), water (50 ml), saturated sodium bicarbonate solution (30 ml), saturated brine (30 ml) and dried over anhydrous sodium sulfate, and the solvent was removed by filtration. Purification by column chromatography on silica eluting with PE/EA (1:4) afforded 338mg as a yellow solid in 28% yield.
Synthesis of the Compound (R) -N- (4- (1, 3, 2-dithioarsin-2-yl) phenyl) -2- (3- ((5-chloro-4- (1- (phenylsulfonyl) -1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) -N- (prop-2-yn-1-yl) acetamide (ZSQ-14-13)
ZSQ-14-10 (93 mg,0.20 mmol), DIPEA (0.10 ml,0.60 mmol) and ZSQ-5-4 (88 mg,0.21 mmol) were dissolved in 2ml THF at room temperature and the mixed solution stirred at room temperature for 4h. Then washed with 2N dilute hydrochloric acid solution (20 ml) and extracted with EA (3X 30 ml), followed by washing with water (2X 20 ml). The combined organic phases were dried over Na 2SO4, filtered and concentrated under reduced pressure, purified by column chromatography on silica gel eluting with DCM/EA (3:1) to give 95mg of a yellow solid in 59% yield.
Synthesis of the Compound (R) -N- (4- (1, 3, 2-dithioarsin-2-yl) phenyl) -2- (3- ((5-chloro-4- (1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) -N- (prop-2-yn-1-yl) acetamide (ZSQ-14-18)
ZSQ-14-13 (95 mg,0.12 mmol) of anhydrous potassium carbonate (66 mg,0.48 mmol) was dissolved in a mixed solution of 2ml of anhydrous methanol and 2ml of anhydrous THF, and the mixture was stirred at room temperature for 4 hours. After the completion of the reaction, pH was adjusted to weak acidity with TFA, and the reaction mixture was directly purified by C18 reverse phase column chromatography, eluting with H 2O/CH3 CN (1:5), to give 50mg of a yellow powder in 63% yield.
Synthesis of Compound (R) - (4- (2- (3- ((5-chloro-4- (1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) -N- (prop-2-yn-1-yl) acetamido) phenyl) arsinic acid (ZSQ-14-23)
ZSQ-14-18 (50 mg,0.07 mmol), mercuric perchlorate trihydrate (27 mg,0.06 mmol) was dissolved in 3ml DMSO and the mixed solution stirred at room temperature for 10min, immediately purified by C18 reverse phase column chromatography eluting with H 2O/CH3 CN (1:5) to give 35mg of a yellow solid in 83% yield.
Example 11
Synthetic route to Compound ZSQ-15-99
Synthesis of Compound 2-formylbutyronitrile (ZSQ-14-104)
2M lithium diisopropylamide solution (27.5 ml,55 mmol) was mixed in 50ml dry THF, stirred at-78℃and butyronitrile (4.35 ml,50 mmol) was added dropwise, stirring continued for 30min after addition was completed, ethyl formate (4.85 ml,60 mmol) was mixed in 25ml dry THF, added dropwise to the mixed solution at-78℃over 30min, stirring continued for 45min after addition was continued and then brought to room temperature, stirred for 14h. After completion of the reaction, 10ml of H 2 O was added to quench the reaction mixture, the mixture was concentrated, diluted hydrochloric acid solution was added to adjust pH to 3, then extracted with EA (20 ml. Times.3), the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and dried by suction to give 4.82g of a crude product as a yellow oil
Synthesis of the 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 75ml absolute ethanol, heated in a closed tube and stirred for 16h. After completion of the reaction, the mixture was concentrated to leave 1/3, washed with saturated sodium bicarbonate solution (40 ml) (pH was adjusted to alkaline), extracted with DCM (30 ml. Times.2), and washed with saturated sodium chloride solution (40 ml). The combined organic phases were dried over anhydrous sodium sulfate, and the filtrate was concentrated to dryness without further purification to give 4.88g of a yellow solid.
Synthesis of the Compound 3-ethylpyrazolo [1,5-a ] pyrimidine-5, 7-diol (ZSQ-15-2)
ZSQ-15-1 (4.88 g,43 mmol) was dissolved in a sealed tube containing 80ml of dry ethanol, 20% sodium ethoxide solution (16 g,47.5 mmol) was added, the mixed solution was heated to reflux temperature and stirred for 22h. After the completion of the reaction, the reaction mixture was concentrated, diluted with water (50 ml), adjusted to pH 3 with a dilute hydrochloric acid solution, and a large amount of solids were precipitated, filtered and washed with water several times. A yellow powder was obtained, and after drying, 2.98g of a white powder was obtained.
Synthesis of the Compound 5, 7-dichloro-3-ethylpyrazolo [1,5-a ] pyrimidine (ZSQ-15-4)
ZSQ-15-2 (2.98 g,16.6 mmol) and N, N-dimethylaniline (250 ul,2.01 mmol) were mixed in a sealed tube containing 45ml of phosphorus oxychloride, heated to 115℃overnight and stirred under reflux. After completion of the reaction, the mixed solution was concentrated, the remaining solution was dropwise added to ice water, extracted with DCM a plurality of times, washed with saturated sodium chloride solution (30 ml), and the organic phases were combined. Drying over anhydrous sodium sulfate, filtering, concentrating, separating and purifying by silica gel column chromatography, eluting with PE/EA (5:1) to obtain white needle-like powder 2.33g, with a yield of 65%.
Synthesis of the Compound 5-chloro-3-ethyl-N- (4-methoxybenzyl) pyrazolo [1,5-a ] pyrimidin-7-amine (ZSQ-15-72)
ZSQ-15-4 (433 mg,2.0 mmol), DIPEA (4.96 ml,3.0 mmol) and 4-methoxybenzylamine (0.52 ml,4.0 mmol) were dissolved in 8ml absolute ethanol, heated to 85℃and stirred overnight. After completion of the reaction, the reaction mixture was concentrated, washed with water (30 ml. Times.2), extracted with EA (30 ml), and washed with a saturated sodium chloride solution (30 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated and purified by column chromatography on silica gel eluting with PE/EA (5:1) to give 631mg of white solid in 99% yield.
Synthesis of tert-butyl (ZSQ-15-74) carbamate, the compound (5-chloro-3-ethylpyrazolo [1,5-a ] pyrimidin-7-yl) (4-methoxybenzyl)
ZSQ-15-72 (8231 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 8ml THF and stirred at room temperature for 12h. After completion of the reaction, the reaction mixture was washed with H 2 O (20 ml. Times.2), extracted with EA (30 ml) and washed with saturated sodium hydrogencarbonate solution (30 ml). The organic phase was dried over anhydrous sodium sulfate, concentrated by filtration and purified by column chromatography on silica gel eluting with PE/EA (8:1) to give 830mg of a yellow solid in 99% yield.
Synthesis of tert-butyl (ZSQ-15-83) carbamate, the compound (3-ethyl-5- (2- (2-hydroxyethyl) piperidin-1-yl) pyrazolo [1,5-a ] pyrimidin-7-yl) (4-methoxybenzyl)
Compounds ZSQ-15-74 (416 mg,1.0 mmol), 2-piperidineethanol (165 mg,1.0 mmol), tris (dibenzylideneacetone) dipalladium (46 mg,0.05 mmol), 1 '-binaphthyl-2, 2' -bisdiphenylphosphine (93 mg,0.15 mmol) and sodium t-butoxide (106 mg,1.1 mmol) were mixed in 3ml toluene and heated to 95℃under nitrogen atmosphere and stirred for 16h. After completion of the reaction, the reaction mixture was washed with H 2 O (30 ml. Times.3), extracted with EA (40 ml) and washed with a saturated sodium chloride solution (30 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, separated and purified by column chromatography on silica gel eluting with PE/EA (8:1) to give 350mg of red solid in 68% yield.
Synthesis of Compound 2- (1- (7-amino-3-ethylpyrazolo [1,5-a ] pyrimidin-5-yl) piperidin-2-yl) ethan-1-ol (ZSQ-15-97)
Compound ZSQ-15-97 (283 mg,0.69 mmol) was dissolved in a mixture of 2ml concentrated hydrochloric acid and 2ml DCM and stirred at room temperature for 6h. After the reaction was completed, the reaction solution was concentrated, separated and purified by silica gel column chromatography, eluting with DCM/CH 3OH(NH3 (5:1) to give 115mg of a white solid with a yield of 57%.
Synthesis of the Compound N- (4- (1, 3, 2-dithioarsin-2-yl) phenyl) -2- ((3-ethyl-5- (2- (2-hydroxyethyl) piperidin-1-yl) pyrazolo [1,5-a ] pyrimidin-7-yl) amino) acetamide (ZSQ-15-98)
ZSQ-15-97 (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 1.5ml CH 3 CN/1.5ml DMF at room temperature, and the mixed solution was stirred at room temperature for 4h. Acidification with TFA followed by separation and purification by C18 reverse phase column chromatography eluting with H 2O/CH3 CN (1:9) afforded 153mg of a pink solid in 65% yield.
Synthesis of Compound (4- (2- ((3-ethyl-5- (2- (2-hydroxyethyl) piperidin-1-yl) pyrazolo [1,5-a ] pyrimidin-7-yl) amino) acetamido) phenyl) arsinic acid (ZSQ-15-99)
ZSQ-15-98 (50 mg,0.10 mmol), mercuric perchlorate trihydrate (36 mg,0.08 mmol) was dissolved in 2ml DMSO and the mixed solution stirred at room temperature for 10min, immediately purified by C18 reverse phase column chromatography eluting with H 2O/CH3 CN (1:5) to give 28mg of a yellow solid in 53% yield.
Example 12
Synthetic route to Compound ZSQ-14-66
Synthesis of isopropyl (2-nitrophenyl) sulfane (ZSQ-14-27) compound
1-Fluoro-2-nitrobenzene (14 g,100 mmol), isopropyl mercaptan (10.2 ml,110 mmol), anhydrous potassium carbonate (27.6 g,200 mmol) were mixed and dissolved in 100ml anhydrous DMF and stirred at 110℃for 12h. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, washed with 100ml of water, 100ml of saturated sodium chloride solution, and extracted with EA. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. Purification by column chromatography on silica gel eluting with PE/EA (7:1) afforded 18g as a yellow oil with 91% yield.
Synthesis of Compound 1- (isopropylsulfonyl) -2-nitrobenzene (ZSQ-14-35)
Compound ZSQ-14-27 (9.85 g,50 mmol) was dissolved in 100ml methanol and stirred at 0deg.C. Potassium peroxomonosulphonate (15.37 g,250 mmol) was dissolved in 100ml of water and added dropwise to the mixture at 0℃and after the addition was completed, stirring was continued at 0℃for 1h and then the mixture was allowed to stand at room temperature for 24h. After completion of the reaction, methanol was distilled off under reduced pressure, and then washed with 50ml of water, 50ml of a saturated sodium chloride solution, and extracted with EA. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give 8.3g of a bright yellow oily liquid in 72% yield.
Synthesis of Compound 2- (isopropylsulfonyl) aniline (ZSQ-14-48)
Compound ZSQ-14-35 (2.29 g,10 mmol) was dissolved in 20ml methanol, pd/C (200 mg) was added and stirred at room temperature under a hydrogen atmosphere for 12h. After completion of the reaction, it was filtered through celite and washed with DCM. The filtrate was concentrated and pumped down by an oil pump to give 1.99g of a white solid with a yield of 99%.
Synthesis of Compound 2, 5-dichloro-N- (2- (isopropylsulfonyl) phenyl) pyrimidin-4-amine (ZSQ-14-49)
NaH (600 mg,15 mmol) was dissolved in dry 60ml DMF and stirred at 0deg.C. ZSQ-14-48 (1.99 g,10 mmol) was dissolved in 5ml of dry DMF and added dropwise to the above mixed solution (over 10 min). After the addition was completed and stirring was continued at 0℃for 0.5 hours, 2,4, 5-trichloropyrimidine (2.29 ml,20 mmol) was added dropwise, and after stirring for 1 hour, it was allowed to move to room temperature overnight and stirred. After the completion of the reaction, 20ml of water was added thereto, and DMF was removed by concentration under reduced pressure. This was washed successively with 100ml of water, 100ml of saturated sodium bicarbonate solution, 100ml of saturated sodium chloride solution, and extracted with EA. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. Purification by column chromatography on silica gel eluting with PE/EA (3:1) afforded 1.2g of a white solid in 35% yield.
Synthesis of tert-butyl (ZSQ-14-54) piperidine-1-carboxylate, a compound (R) -3- ((5-chloro-4- ((2- (isopropylsulfonyl) phenyl) amino) pyrimidin-2-yl) amino)
ZSQ-14-49 (173 mg,0.5 mmol), (R) -1-Boc-3-aminopiperidine (100 mg,0.5 mmol) and DIPEA (248 ul,1.5 mmol) were mixed and dissolved in 2ml N-methylpyrrolidone. The mixed solution was heated at 135℃and stirred for 3h. The cooled solution was directly purified by C18 reverse phase column chromatography eluting with H 2O/CH3 CN (1:9) to give 232mg of a pale yellow solid in 91% yield.
Synthesis of Compound (R) -5-chloro-N 4 - (2- (isopropylsulfonyl) phenyl) -N 2 - (piperidin-3-yl) pyrimidine-2, 4-diamine (ZSQ-14-58)
ZSQ-14-54 (232 mg,0.45 mmol) and 1ml trifluoroacetic acid were mixed in 5ml DCM and stirred overnight at room temperature. After the completion of the reaction, the reaction mixture was concentrated and purified by C18 reverse phase column chromatography, eluting with H 2O/CH3 CN (3:1), to give 180mg of a pale yellow solid in 97% yield.
Synthesis of the Compound (R) -N- (4- (1, 3, 2-dithioarsin-2-yl) phenyl) -2- (3- ((5-chloro-4- ((2- (isopropyl-sulfonyl) phenyl) amino) pyrimidin-2-yl) amino) piperidin-1-yl) acetamide (ZSQ-14-60)
ZSQ-14-58 (82 mg,0.2 mmol), DIPEA (0.13 ml,0.8 mmol) and ZSQ-5-4 (76 mg,0.2 mmol) were dissolved in 3ml THF at room temperature and the mixture stirred at room temperature for 4h. Then washed with 2N dilute hydrochloric acid solution (20 ml) and extracted with EA (3X 30 ml), followed by washing with water (2X 20 ml). The combined organic phases were dried over anhydrous Na 2SO4, filtered and concentrated under reduced pressure, separated and purified by column chromatography on silica gel eluting with DCM/EA (3:1) to give 79mg of a yellow solid in 56% yield.
Synthesis of Compound (R) - (4- (2- (3- ((5-chloro-4- ((2- (isopropylsulfonyl) phenyl) amino) pyrimidin-2-yl) amino) piperidin-1-yl) acetamido) phenyl) arsinic acid (ZSQ-14-66)
ZSQ-14-60 (36 mg,0.05 mmol), mercuric perchlorate trihydrate (18 mg,0.04 mmol) was dissolved in 2ml DMSO and the mixed solution stirred at room temperature for 10min, immediately purified by C18 reverse phase column chromatography eluting with H 2O/CH3 CN (1:3) to give 24mg of a yellow solid in 75% yield.
Synthesis scheme 13
Synthetic route to Compound ZSQ-16-91
Synthesis of Compound (4-hydroxy-3-nitrophenyl) arsinic acid (ZSQ-16-8)
3-Nitro-4-hydroxyphenylarsonic acid (26.3 g,100 mmol) was dissolved in 80ml methanol and the mixed solution was heated to reflux. Phenylhydrazine (19.6 ml,200 mol) was added dropwise (over 1 h), with a large amount of nitrogen being generated during the addition, and reflux stirring was continued for 1.5h when nitrogen generation was slowed down. The mixture was concentrated by distillation under reduced pressure, and sodium hydroxide solution (12 g in 200ml of water) and 200ml of diethyl ether were added. The mixture was separated, and a 2N diluted hydrochloric acid solution (100 ml) was added to the aqueous phase and stirred for 1h, followed by extraction with EA (200 ml. Times.3). The organic phase was dried over anhydrous sodium sulfate, concentrated, pumped out by an oil pump, and the next reaction was carried out without purification.
Synthesis of Compound 4- (1, 3, 2-dithioarsin-2-yl) -2-nitrophenol (ZSQ-16-14)
ZSQ-16-8 (24.7 g,100 mmol) was dissolved in 100ml methanol and heated to reflux. Then, ethanedithiol (10 ml,120 mmol) was added dropwise to the mixed solution over 30min, and heating and stirring were continued for 30min. The mixed solution was then concentrated, separated and purified by column chromatography on silica gel eluting with PE/EA (10:1) to give a crude yellow solid. Dissolution with THF and further purification by C18 reverse phase column chromatography eluting with H 2O/CH3 CN (8:1) afforded 9g of the product as a yellow solid in 29.5% yield.
Synthesis of Compound 2-amino-4- (1, 3, 2-dithioarsin-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 stirred at 75℃under reflux for 48h. After completion of the reaction, the reaction mixture was concentrated, washed with water (100X 3 ml), and extracted with EA (100 ml). The organic phase was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography on silica gel eluting with PE/EA (8:1) to give 340mg of crude yellow solid in 4% yield.
Synthesis of tert-butyl (ZSQ-16-1) carbamate, a compound (5- (1, 3, 2-dithioarsin-2-yl) -2-hydroxyphenyl)
ZSQ-16-90 (120 mg,0.72 mmol) and di-tert-butyl dicarbonate (200 ul,0.87 mmol) were mixed and dissolved in 4ml THF and stirred at room temperature for 24h. After completion of the reaction, the reaction mixture was washed with H 2 O (50 ml. Times.2) and extracted with EA (50 ml). The organic phase was dried over anhydrous sodium sulfate, concentrated by filtration and purified by column chromatography on silica gel eluting with PE/EA (6:1) to give 120mg of a white solid in 43% yield.
Synthesis of tert-butyl Compound (5- (1, 3, 2-dithioarsin-2-yl) -2-methoxyphenyl) carbamate (ZSQ-16-68)
Compound ZSQ-16-1 (120 mg,0.32 mmol), methyl iodide (24 ul,0.38 mmol), anhydrous potassium carbonate (88 mg,0.64 mmol), and potassium iodide (10 mg,0.06 mmol) were mixed in 2ml acetonitrile and stirred at room temperature. After the reaction is completed, the reaction solution is directly concentrated, separated and purified by silica gel column chromatography, and eluted by PE/EA (6:1) to obtain 90mg of white solid with the yield of 72 percent.
Synthesis of Compound 5- (1, 3, 2-dithioarsin-2-yl) -2-methoxyaniline (ZSQ-16-76)
ZSQ-16-68 (90 mg,0.23 mmol) and 0.3ml trifluoroacetic acid were mixed in 3ml DCM and stirred overnight at room temperature. After the completion of the reaction, the reaction mixture was concentrated and purified by C18 reverse phase column chromatography, eluting with H 2O/CH3 CN (1:6), to give 52mg of a white solid in 78% yield.
Synthesis of Compound N- (5- (1, 3, 2-dithioarsin-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 5ml of dry DCM and stirred at 0deg.C. Bromoacetyl bromide (0.1 ml,1.1 mmol) was added dropwise over 10min and stirring was continued at 0℃for 1h and then brought to room temperature for 2h. The mixture was diluted with DCM (20 ml), washed with water (50 ml), extracted with DCM, the organic phases combined, dried over anhydrous sodium sulfate, filtered and concentrated. Purification by column chromatography on silica eluting with PE/EA (5:1) afforded 110mg as a yellow solid in 27% yield.
Synthesis of the Compound (R) -N- (5- (1, 3, 2-dithioarsin-2-yl) -2-methoxyphenyl) -2- (3- ((5-chloro-4- (1- (phenylsulfonyl) -1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) acetamide (ZSQ-16-87)
ZSQ-16-68 (110 mg,0.26 mmol), DIPEA (0.13 ml,0.80 mmol) and ZSQ-5-4 (138 mg,0.29 mmol) were dissolved in 3ml THF at room temperature and the mixture was stirred at room temperature for 4h. Then washed with 2N dilute hydrochloric acid solution (20 ml) and extracted with EA (3X 30 ml), followed by washing with water (2X 20 ml). The combined organic phases were dried over Na 2SO4, filtered and concentrated under reduced pressure, separated and purified by column chromatography on silica gel eluting with DCM/EA (3:1) to give 177mg of a white solid powder in 83% yield.
Synthesis of the Compound (R) -N- (5- (1, 3, 2-dithioarsin-2-yl) -2-methoxyphenyl) -2- (3- ((5-chloro-4- (1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) acetamide (ZSQ-16-88)
ZSQ-16-87 (177 mg,0.22 mmol) of anhydrous potassium carbonate (92 mg,0.66 mmol) was dissolved in a mixed solution of 1.5ml of anhydrous methanol and 1.5ml of anhydrous THF, and the mixture was stirred at room temperature for 4 hours. After the reaction was completed, the pH was adjusted to weak acidity with TFA, and the reaction mixture was directly purified by C18 reverse phase column chromatography, eluting with H 2O/CH3 CN (1:8), to give 121mg of a pale yellow solid in 84% yield.
Synthesis of Compound (R) - (3- (2- (3- ((5-chloro-4- (1H-indol-3-yl) pyrimidin-2-yl) amino) piperidin-1-yl) acetamido) -4-methoxyphenyl) arsinic acid (ZSQ-16-91)
ZSQ-16-88 (66 mg,0.1 mmol), mercuric perchlorate trihydrate (36 mg,0.08 mmol) was dissolved in 2ml DMSO and the mixed solution stirred at room temperature for 10min, immediately purified by C18 reverse phase column chromatography eluting with H 2O/CH3 CN (1:5) to give 46mg of a yellow solid in 76% yield.
TABLE 1
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Activity test example 1 CDK inhibitor test for growth inhibitory Activity against cancer cells
The adopted biological test scheme is as follows: effect of the compounds on cell growth activity of acute lymphoblastic leukemia cell line Jurkat and non-small cell lung cancer cell line H3122.
To verify the growth inhibitory effect of the compounds of the present invention on cancer cells at the cellular level, jurkat cells (hematological cancer, suspension type) and H3122 cells (solid tumor, adherent type) were selected, and cell viability was calculated by detecting chemiluminescent values, thereby obtaining the biological activity of the compounds to inhibit the growth of cancer cells.
The method comprises the following steps: jurkat or H3122 cells were cultured in vitro, grown to logarithmic growth phase, cells were collected, centrifuged at 1000rpm for 5min, the supernatant was discarded, the cell concentration was adjusted to 2.5X10 5/mL (Jurkat) or 1.5X10 5/mL (H3122), and cells were seeded into 384 well plates at 40. Mu.l per well. After adding 5. Mu.L of each of the compound or DMSO at different concentrations to the corresponding wells, and incubating in a cell incubator (37 ℃ C., 5% CO 2) for 72 hours, 15. Mu. L CELL TITER-Glo solution was added to each well, incubated at room temperature for 30 minutes, and the chemiluminescent value (luminescence) was measured to measure intracellular ATP levels. The unstimulated DMSO control wells were 100% cell viability. Compound IC 50 values were calculated using PRISM GRAPHPAD statistical software.
Table 2 CDK results of test for inhibition of growth inhibitory Activity of inhibitors on cancer cell lines
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Activity test example 2 CDK test of inhibitor for growth inhibitory Activity against non-Small cell Lung cancer cell lines
The adopted biological test scheme is as follows: influence of the Compounds on the growth activity of each of a plurality of non-small cell lung cancer cell lines.
To verify the growth inhibitory effect of representative compounds of the present invention on non-small cell lung cancer cell lines depending on the growth survival of different driving genes at the cellular level, H3122, H1299, H1975, H2077, H358, EBC-1, H23, PC9 and a549 cells were selected, respectively, and cell viability was calculated by detecting chemiluminescence values, thereby obtaining the biological activity of the compounds for inhibiting the growth of cancer cells, and comparing with the reported CDK inhibitors THZ1 and THZ 531.
The method comprises the following steps: various non-small cell lung cancer cells were cultured in vitro, grown to logarithmic growth phase, digested and collected, centrifuged at 1000rpm for 5min, the supernatant was discarded, the cell concentration was adjusted to 1.5X10 5/mL, and the cells were inoculated into 384-well plates, 40. Mu.l per well. After adding 5. Mu.L of each of the compound or DMSO at different concentrations to the corresponding wells, and incubating in a cell incubator (37 ℃ C., 5% CO 2) for 72 hours, 15. Mu. L CELL TITER-Glo solution was added to each well, incubated at room temperature for 30 minutes, and the chemiluminescent value (luminescence) was measured to measure intracellular ATP levels. The unstimulated DMSO control wells were 100% cell viability. Compound IC 50 values were calculated using PRISM GRAPHPAD statistical software.
Table 3 CDK results of test of inhibitor Activity against growth inhibition on different non-Small cell lung cancer cell lines
As can be seen from Table 3, the representative compounds of the present invention exhibited growth inhibitory activity comparable to or even better than THZ531 on various non-small cell lung cancer cell lines.
Activity test example 3 influence of CDK inhibitors on CDK7/12/13 kinase Activity in cancer cells
In various cancer cells, RNA polymerase II (Pol II) forms a transcription complex with transcription factors such as CDK7, 12, 13 and BRD4 through super enhancers, promotes high expression of oncogenes such as MYC, RUNX 1 and the like, and further maintains survival and proliferation of cancer cells. In the Pol II transcription complex, CDKs 7, 12, 13 are collectively responsible for sustained phosphorylation of the Ser5 site in the Pol II C-terminal domain (CTD) repeat sequence to ensure the normal initiation of the Pol II transcription procedure; CDKs 12, 13 are responsible for sustained phosphorylation of the Ser2 site in the Pol II CTD repeat to ensure proper transcription elongation process and DNA damage repair. Thus, when CDK12, 13 is inhibited simultaneously, the level of phosphorylation of the Ser2 site will be inhibited; when CDK7, 12, 13 is simultaneously inhibited, the level of phosphorylation of the Ser5 site will also be inhibited.
Experimental conditions and process: h3122 or A549 cells were cultured in vitro, after growth to logarithmic growth phase, the cells were digested and collected, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the cell concentration was adjusted to 1X 10 6/mL. In a 12-well cell culture plate, 1ml of a DMSO solution of different concentrations of the drug was added to each well, THZ1, THZ531 or DMSO was used as a control, and after culturing in a cell incubator (37 ℃ C., 5% CO 2) for 8 hours, the cells were washed twice with a pre-chilled PBS solution, the solution was aspirated, 200. Mu.L of RIPA cell lysate, protease inhibitor and phosphatase inhibitor were added to the wells, transferred to a sample tube, and after shaking for 30 minutes at 4 ℃ C., centrifugation was performed at 15000rpm for 15 minutes at 4 ℃ C. And the supernatant was taken. Each group of proteins was assayed for content using BCA protein quantification kit and the amount of protein was adjusted using PIPA lysate to a final volume of 100 μl. And carrying out western-blot identification on the sample.
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Western-blot: to 100. Mu.L of the cell lysate was added 25. Mu.L of 5 Xprotein loading buffer and heated at 95℃for 10min. After the sample cooled, electrophoresis was performed using SDS-PAGE (9%) gel 60V, and after 30min, switching to 120V until the leading band was run to the bottom of the gel. Using a turbo semi-dry transfer system, constant current 0.2A was transferred for 80 minutes, and proteins in the gel were transferred to a PC membrane with a pore size of 0.2. Mu.L. The transferred PC membrane was blocked with 5% nonfat milk powder (TBST solution) for 2h and incubated with the corresponding primary antibody for 12h at 4 ℃. TBST was washed 3 times for 10min each. Incubate with the corresponding secondary antibody for 2h at room temperature. TBST was washed three times for 10min each. The ECL luminescence was used to incubate and detect the luminescence signal.
FIG. 1 shows the effect of representative compounds on Ser2 and Ser5 site phosphorylation of Pol II in H3122 (A) or A549 (B) cells. The experimental results show that:
ZSQ5-38, ZSQ8-36, ZSQ9-75 and other compounds can effectively inhibit the phosphorylation of the Ser2 site but not the Ser5 site at a concentration of 1. Mu.M;
ZSQ5-38, ZSQ17-22, etc. exhibit inhibitory activity superior to THZ 531;
THZ1 inhibits phosphorylation at both sites.
This suggests that ZSQ-38, ZSQ8-36, ZSQ9-75, and the like compounds are effective in inhibiting kinase activity of CDK12, 13 in cancer cells, but not CDK7.
Activity test example 4 irreversible binding of CDK inhibitor to CDK in H3122 cells
In this example, the CDK inhibitors of the invention are tested for irreversible binding to CDK7, 12 or 13.
Covalent inhibitors such as THZ1 or THZ531 are capable of forming covalent bonds with specific cysteine residues in the CDK7, 12 or 13 binding pocket and thereby irreversibly bind to CDK7, 12 or 13. THZ1-biotin has been reported to be a derivative of THZ1 and also to form covalent bonds with CDKs 7, 12 and 13 in cell lysates, and CDKs 7, 12 and 13 can be enriched by streptavidin microbeads (STREPTAVIDIN BEADS). When the THZ1, THZ531 or ZSQ series CDK inhibitor binds irreversibly to CDK7, 12 or 13, THZ1-biotin cannot bind to CDK7, 12 or 13 any more because the binding pocket is occupied. Thus, based on competition enrichment experiments of THZ1-biotin and streptavidin microbeads, it was possible to accurately determine whether CDK inhibitors bind irreversibly to CDK7, 12 or 13.
Experimental conditions and process: h3122 cells were cultured in vitro, grown to logarithmic growth phase, digested and collected cells, centrifuged at 1000rpm for 5min, the supernatant discarded, and the cell concentration was adjusted to 1X 10 6/mL. In a 12-well cell culture plate, 1ml of cells were added per well, 100nL of a DMSO solution (final concentration 1. Mu.M) of a representative drug at a concentration of 10mM was added per well, and the cells were incubated in a cell incubator (37 ℃ C., 5% CO 2) with THZ1, THZ531 or DMSO control for 4 hours, and then washed twice with a pre-chilled PBS solution, 200. Mu.L of NP40 cell lysate and a protease inhibitor were added to the wells, and lysed at 4 ℃ for 30 minutes. Lysates were collected, centrifuged at 15000rpm at 4℃for 15min, the supernatant was transferred to a sample tube, THZ1-biotin (final concentration 1. Mu.M) was added, and incubated at 4℃overnight. 10. Mu.L of streptavidin beads were added and allowed to bind for 2 hours at room temperature. After washing the microbeads with 1% NP40 cell lysate for 10 times, 20. Mu.L of 2% SDS lysate was added for lysis, and the mixture was boiled at 95℃for 10 minutes and loaded for western-blot identification.
FIG. 2 shows the competitive effect of representative compounds such as ZSQ-38, ZSQ8-36, etc. on covalent binding of THZ1-biotin to CDK7 or 12 in H3122 cells at a concentration of 1. Mu.M.
The experimental results show that:
THZ1 simultaneously inhibits covalent binding of THZ1-biotin to CDKs 7, 12 and 13; THZ531 has slight inhibition of CDK 7;
While ZSQ5-38, ZSQ-36, ZSQ9-75, ZSQ-22 and other compounds can completely or almost completely inhibit covalent binding of THZ1-biotin with CDK12 or 13 at a concentration of 1 mu M, but hardly affect covalent binding of THZ1-biotin with CDK7, ZSQ-38, ZSQ-8-36, ZSQ9-75, ZSQ-22 and other compounds have smaller effect on CDK7 and higher selectivity than THZ 531;
this demonstrates that ZSQ compounds are capable of highly potent, highly specific, irreversible inhibition of kinase activity of CDK12, 13 in cancer cells.
Activity test example 5 influence of CDK inhibitors on CDK7, 12 in vitro kinase Activity
Both CDK7 and 12 proteins catalyze the phosphorylation of the Ser5 site of Pol II CTD in vitro, and therefore, when the kinase activity of CDK7 or 12 is inhibited, the phosphorylation modification of the Ser5 site will be attenuated or lost.
Experimental conditions and process: after 293T cells overexpressed 3XFlag-CDK7 or 3X-Flag-CDK12 for 48 hours, they were washed twice with pre-chilled PBS solution, 1mL of RIPA buffer containing protease inhibitor was added and lysed at 4℃for 1 hour. The lysate was collected, centrifuged at 15000rpm at 4℃for 15min, the supernatant was transferred to a sample tube, 20. Mu.L of anti-Flag affinity gel was added, and incubated at 4℃overnight. The gel was washed 5 times with RIPAbuffer times and 3 times with a kinase buffer (50mM HEPES pH7.4, 50mM KCl,10mM MgCl 2), and 100. Mu.L of the kinase buffer was added and mixed well for the subsequent in vitro kinase reaction. mu.L of gel mixture was added to each well, and incubated overnight at 4℃at different concentrations of compound. Then 1. Mu.g of the purified GST-Pol II CTD protein expressed by the bacteria and 100. Mu.M ATP were added, and after 1 hour of shaking reaction at 37℃the reaction was terminated by adding 4%SDS loading buffer thereto, and the mixture was boiled at 95℃for 10 minutes, and the western-blot was identified.
FIG. 3 shows the effect of representative compounds such as ZSQ-36 and ZSQ-66 on CDK7 or 12 catalytic Pol II CTD Ser5 site phosphorylation modification at various concentrations.
The experimental results show that:
ZSQ5-38, ZSQ8-36 and other compounds can completely or almost completely inhibit CDK12 from catalyzing phosphorylation modification of Pol II CTD Ser5 site at low concentration, but do not influence CDK7 to catalyze phosphorylation modification of Pol II CTD Ser5 site, consistent with THZ531 phenotype;
in contrast, THZ1 inhibits both CDK7 and 12 catalytic substrate phosphorylation modifications;
ZSQ14-66 significantly inhibited the phosphorylation of both CDK7 and 12 catalytic substrates at 250nM concentration, while ZSQ-60 partially inhibited the phosphorylation of CDK7 catalytic substrates at 500nM concentration.
This suggests that a number of ZSQ compounds are capable of high-potency, specific kinase activity of CDK 12.
Activity test example 6 CDK Selectivity of inhibitors to kinase set
The adopted biological test scheme is as follows: effects of the compounds on the in vitro enzymatic activity of the kinase group.
To verify the selectivity of the compounds of the invention to the kinase group at the protein level, representative compounds ZSQ-36, ZSQ-36 were selected and the inhibition levels of the compounds on the phosphorylation of 370 kinase protein catalytic substrates were obtained using P 33 isotopically labeled ATP and radioactive kinase activity assay.
The method comprises the following steps: activity test of radioactive kinase group. The compound and the protein are mixed and pre-incubated for 1 hour at room temperature, then P 33 -ATP is added for reaction for 2 hours, the reaction mixture is spotted on P81 ion exchange chromatography paper, and the kinase activity is detected by a membrane filtration method. The final concentration of the compound was 1 μm and 2 replicates were used to calculate the dosing treatment group activity with 100% kinase activity for the single DMSO dosing group.
Table 4 CDK inhibitors selectivity for 370 different kinases
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As can be seen from table 4, the representative compounds inhibited the enzymatic activity of only a few kinases to below 50% at a concentration of 1 μm; in contrast, the representative compounds were able to completely inhibit kinase activity of CDK12 at a concentration of 500nM (Activity test example 5).
Studies show that THZ531 binds to different kinases at a concentration of 1. Mu.M as shown in Table 5, and the Score (Score) represents the percentage of protein not bound to the compound (i.e., protein with uninhibited kinase activity), and it can be seen that THZ531 binds strongly to kinases such as RSK2, STK16, etc., whereas the compounds of the invention have substantially no effect on the activity of these kinases, suggesting that the compounds of the invention have a different kinase set selectivity than the existing CDK12/13 inhibitors, possibly with better selectivity and safety in use.
TABLE 5
Pharmacokinetic properties of the Activity test example 7 CDK inhibitor
In this example, drug metabolism was tested in mice and the selectivity of the compounds of the invention for the kinase group was verified in vivo.
The method is as follows; compounds ZSQ-38, ZSQ-36 were selected and tested for their pharmacokinetic properties in mice (n=3) using a single administration of intraperitoneal injection (10 mg/kg).
Fig. 4 shows concentration time curves of representative compounds ZSQ-38 and ZSQ8-36 in plasma, as well as pharmacokinetic parameters.
The results showed that ZSQ-38 and ZSQ-36 peaked rapidly in mice after a single dose, and were able to stably maintain moderate drug concentrations, and exhibited long half-lives (17.3 and 9.8 for T 1/2 of ZSQ5-38 and ZSQ-36).
In addition, in the completed in vivo test, no obvious toxic or side effect was observed, nor was necrosis observed in normal tissues or cells of mice, suggesting that the compounds of the present invention have high safety.
Activity test example 8 CDK test of growth inhibitory Activity of inhibitors against various lung cancer cell lines with KRAS mutations
In this example, the growth inhibitory effect of representative compounds ZSQ-38 and ZSQ-36 of the present invention on lung cancer cell lines that either survive on the growth of the KRAS gene or have KRAS activating point mutations was demonstrated at the cellular level. 17 cells such as H441 and H460 are selected respectively, and the clone number of the cells is observed by a crystal violet staining means, so that the biological activity of the compound for inhibiting the growth of cancer cells is obtained.
The method comprises the following steps: after growing various lung cancer cells in vitro to logarithmic growth phase, the cells were digested and collected, centrifuged at 1000rpm for 5min, the supernatant was discarded, the cell concentration was adjusted to 1.5X10 5/mL, and the cells were inoculated into 12-well plates at 1mL per well. After 5 μl of each of the compounds or DMSO at different concentrations was added to the corresponding wells, the wells were incubated in a cell incubator (37 ℃ C., 5% CO 2) for one week (with medium change) and fixed for 20 minutes with 4% PFA solution added to each well, stained with 0.1% crystal violet solution for 20 minutes, washed twice with PBS, and scanned to give 100% cell viability for non-stimulated DMSO control wells.
FIG. 5 shows that representative compounds ZSQ-38 and ZSQ-36 were able to completely or effectively inhibit the growth and proliferation of most cell lines at a concentration of 1. Mu.M, and several cell lines such as SW1573, H460, etc. were able to completely or effectively inhibit the growth and proliferation at a concentration of 300 nM.
Discussion of the invention
Among the various proteins closely related to carcinogenesis, exacerbation, a number of members of the kinase protein family have been identified as effective therapeutic targets. Many of the identified or potential kinase targets have reactive sulfhydryl groups near their enzyme catalytic sites, thus providing the possibility for the development of covalent kinase inhibitors. Whereas CDKs 7, 12 and 13 in the cell Cycle Dependent Kinase (CDK) family are targets for this class of kinases.
The CDK (cyclin dependent kinases) family of kinases are ten or more members of the serine/threonine protein kinase family, some of which are key kinases involved in cell cycle regulation. Depending on the CDKs function, it can be divided into two main categories: CDKs 1,2, 4 and 6 are involved in cell cycle regulation; CDK7-13 is involved in transcriptional regulation.
CDK7-cyclin H, CDK8-cyclin C, CDK9-cyclin T and CDK12/13-cyclin K regulate initiation (initiation) or extension (elongation) of transcription by regulating phosphorylation of RNA polymerase II, which causes the latter to highly express various oncogenes (such as MYC, RUNX, etc.) via super-enhancer (super-enhancer), thereby maintaining growth and survival of cancer cells.
CDK12 is an important component for regulating DNA damage repair in cancer cells, and the inactivation of CDK12 can produce synergistic effect with DNA damage repair inhibitors (such as PARP inhibitor Olaparib), and can enhance infiltration of immune cells in solid tumors, thereby promoting curative effect of tumor immunotherapy.
CDK13 is a homologous protein to CDK12, with a structure very similar to CDK12, and only a few amino acid residues differ from CDK12, with known functions equivalent to CDK 12.
CDK7 is an important transcription factor that regulates the initiation of oncogene transcription, while regulating the normal operation of the cell cycle. The literature reports that inhibition of CDK7 may have an adverse effect on the differentiation of normal cells.
The present invention provides compounds of formula I that selectively inhibit CDK12 and/or 13, while having no or weak inhibition of CDK 7. Studies have shown that inhibition of CDK7, 12, 13 simultaneously blocks activation and extension of transcription simultaneously, whereas inhibition of CDK12, 13 alone affects only extension of the transcription process. While inhibiting multiple CDKs has greater side effects on normal cell growth survival. For cancer cells, which are generally inherently genetically unstable and more dependent on the mechanism of DNA damage repair, selective inhibition of CDKs 12, 13 is sufficient to promote apoptosis in tumor cells.
On the other hand, the existing CDK7/12/13 triple inhibitor THZ1 inhibits not only the three CDKs but also many other kinases, thereby causing toxic and side effects; the modified SY-1365 mainly inhibits CDK7, and has weak inhibition effect on CDK12 and 13; whereas dual inhibitors of CDK12/13, which do not inhibit or substantially inhibit CDK7, have significantly better targeting selectivity than triple inhibitors (see activity test example 6), tumors of different genetic subtypes are also sensitive to them differently than CDK7 inhibitors.
The CDK inhibitor disclosed by the invention is a target inhibitor obtained in an optimized way, and in the structure of the compound disclosed by the invention, an organic arsine group (moeity) is a part which is subjected to covalent action with an active site of CDK12/13, so that excellent inhibitory activity and better specificity can be provided, and from experimental data, the CDK inhibitor disclosed by the invention specifically binds to specific kinases such as CDK12, CDK13 and the like, does not bind or basically does not bind to CDK7 or other 300 kinases tested, and has good selectivity among different cell lines, so that the safety of arsine compounds is far better than that of the prior arsine compounds.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims (10)

1. A compound of formula I, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt thereof;
Wherein,
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R 3 is H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl;
n 1 is 1 or 2;
Each R 4 is independently selected from: H. d, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy;
L 1、L2 and L 3 are each independently selected from the group consisting of: none, - (Z) m-; and L 1 and L 2 are not both absent; wherein each Z is independently selected from: C1-C6 alkylene, -NR 6-,-NR6-R7 -, m is 1,2,3 or 4;
Each R 6 is independently selected from the group consisting of: H. a substituted or unsubstituted C1-C4 alkyl group;
r 7 is a substituted or unsubstituted C1-C8 alkylene group;
a is selected from the group consisting of: an unsubstituted, substituted or unsubstituted phenyl group, a substituted or unsubstituted C3-C6 cycloalkyl group, a substituted or unsubstituted 3-6 membered heterocycloalkyl group;
b is selected from the group consisting of: substituted or unsubstituted 5-10 membered heteroaryl;
C is selected from the group consisting of: a substituted or unsubstituted phenyl group, a substituted or unsubstituted 5-10 membered heteroaryl group, a substituted or unsubstituted 3-10 membered heterocycloalkyl group;
wherein the term "substituted" refers to a group wherein a hydrogen atom is replaced with one or more substituents selected from the group consisting of: halogen, deuterated, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy, C1-C6 alkyl S (=o) 2 -, oxo (=o), -CN, -OH, carboxyl, or substituted C1-C6 alkyl and said substituents are selected from the group consisting of: oxo, -CN, -NH 2, -OH, C2-C6-amide.
2. The compound of claim 1, wherein R 3 is H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl; by "substituted" is meant that a hydrogen atom on the group is replaced by one or more substituents selected from the group consisting of: halogen, deuterated, C1-C6 alkyl;
n 1 is 1 or 2;
Each R 4 is independently selected from: H. d, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy;
L 1、L2 and L 3 are each independently selected from the group consisting of: none, - (Z) m-; and L 1 and L 2 are not both absent; wherein each Z is independently selected from: C1-C6 alkylene, -NR 6-,-NR6-R7 -, m is 1,2,3 or 4;
Each R 6 is independently selected from the group consisting of: H. unsubstituted C1-C4 alkyl;
R 7 is unsubstituted C1-C4 alkylene;
A is selected from the group consisting of: unsubstituted phenyl, unsubstituted cyclohexyl, unsubstituted piperidinyl;
B is selected from the group consisting of: substituted or unsubstituted pyrimidinyl or pyrazolopyrimidinyl, said "substitution" meaning that a hydrogen atom on the group is substituted with one or more substituents selected from the group consisting of: halogen, deuterated, C1-C6 alkyl;
C is selected from the group consisting of: substituted or unsubstituted phenyl, substituted or unsubstituted indolyl, substituted or unsubstituted piperidinyl; by "substituted" is meant that a hydrogen atom on the group is replaced by one or more substituents selected from the group consisting of: halogen, deuterated, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkyl S (=O) 2 -, -OH substituted C1-C6 alkyl.
3. A compound according to claim 1 wherein,At positions 2, 3 or 4.
4. The compound of claim 1, wherein the compound of formula I has the structure of I-a:
5. the compound of claim 1, wherein the compound of formula I is selected from the following table:
6. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises;
(a) A therapeutically effective amount of a compound of formula I as defined in claim 1, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier.
7. Use of a compound of formula I as defined in claim 1, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined in claim 6, for the manufacture of a pharmaceutical composition for the treatment and/or prophylaxis of a disease or condition associated with CDK12 and/or CDK13 activity or expression.
8. The use according to claim 7, wherein the disease or condition is cancer.
9. A process for the preparation of a compound of formula I according to claim 1, comprising the steps of:
(a) Reacting the compounds of formula A4 and formula A5 in an inert solvent to form a compound of formula I:
In the method, in the process of the invention,
Z is selected from: halogen, -OMs, -Ots, -Otf, -Oac or-OAr;
X1、X2、R1、R2、R3、R4、L1、L2、L3、A、B、C、 And n1 is as defined in claim 1.
10. A CDK12 and/or CDK13 inhibitor comprising:
A compound of formula I according to claim 1, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 6.
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