A type II pyruvate kinase inhibitor based on organic arsenic and its preparation method and use

Abstract

The present invention provides an organoarsenic-based type II pyruvate kinase inhibitor, a preparation method and use thereof. Specifically, the PKM2 inhibitor of the present invention is a compound of formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer or tautomer, hydrate or solvate thereof; wherein X1, X2, R1, R2, R3, R4, R5 and n are as defined in the specification. The compound of the present invention has high inhibitory activity and high specificity against PKM2, and is very suitable for preparing a drug that inhibits tumors by inhibiting the PKM2 pathway.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a type II pyruvate kinase (PKM2) inhibitor based on organic arsenic, a preparation method thereof, and use thereof for inhibiting tumors. Background Art

[0002] Tumor cells mainly rely on glycolysis for sugar metabolism regardless of the presence or absence of oxygen. Glycolysis not only provides the required ATP for the survival and proliferation of tumor cells, but also provides essential intermediates for the synthesis of amino acids, nucleic acids and other macromolecules required for the disorderly and rapidly proliferating cancer cells.

[0003] Pyruvate kinase is a key rate-limiting enzyme in the glycolysis process. In the human body, pyruvate kinase has four isoforms: PKM1, PKM2, PKL and PKR, of which PKM1 and PKM2 are two subtypes encoded by the same gene PKM. PKM1 is mainly expressed in the heart, brain and muscle tissue, while PKM2 is specifically highly expressed in a variety of proliferating cells such as embryonic cells, adult stem cells and cancer cells. With the formation of the embryo, PKM2 is replaced by different isozymes, and during the formation of tumors, other isozymes disappear, and the PKM2 enzyme is highly expressed. Experiments have shown that PKM2 plays an important role in the Warburg effect of tumors. It not only directly provides ATP for cancer cells, but its catalytic product pyruvate is also an important raw material for the tricarboxylic acid cycle or lactic acid that supplies energy in tumors. Therefore, PKM2 is an essential factor for the rapid proliferation of tumor cells and is closely related to the malignancy of tumors, tumor metastasis and tumor development.

[0004] A large number of studies have confirmed that PKM2 plays an extremely important role in tumor progression, and small molecule inhibitors targeting PKM2 have the effect of treating or slowing down cancer. However, many of the PKM2 inhibitors reported so far have defects in activity or specificity. For example, Shikonin, the most well-known PKM2 inhibitor, has been reported to inhibit chloride channels and multiple immune response pathways, which increases the risk of adverse reactions.

[0005] Therefore, there is an urgent need in the art to provide more highly active and specific PKM2 inhibitors. Summary of the invention

[0006] The purpose of the present invention is to provide a PKM2 inhibitor with high activity and high specificity.

[0007] The first aspect of the present invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer or tautomer, hydrate or solvate thereof;

[0008]

[0009] Wherein, X1 and X2 are each independently selected from the following group: none, O, S, NR a , CR b R c ;

[0010] R a , R b and R c Each is independently selected from: H or substituted or unsubstituted C1-C6 alkyl;

[0011] R1 and R2 are each independently selected from the following group: H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl or -(C=O)-R d , where R d is a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C3-C8 cycloalkyl group; or R1, R2, together with the connected X1, X2 and As, form a substituted or unsubstituted 4- to 8-membered heterocyclic ring containing one As heteroatom and 0-3 heteroatoms selected from N, O and S;

[0012] R3 is selected from the group consisting of H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C3-C8 cycloalkyl or substituted or unsubstituted C1-C3 alkylene-R e ;

[0013] R e Selected from the group consisting of: -CO-NR f R g , substituted or unsubstituted 6-10 membered aryl, substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from O, N and S, substituted or unsubstituted C3-C10 cycloalkyl or substituted or unsubstituted 3-10 membered heterocycloalkyl having 1-3 heteroatoms selected from O, N and S; the substitution means that one or more hydrogen atoms on the group are replaced by a group selected from the following group: halogen, -OH, -CN, -NH2, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C1-C6 alkoxy, -(C=O)-substituted or unsubstituted C1-C6 alkyl, -NR h -(C=O)-substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl having 1-3 heteroatoms selected from O, N and S, -C1-C4 alkylene-(O-CH2-CH2)m-NR f -(C=O)-R i or -(C=O)-CH2(O-CH2-CH2)m-NRf -(C=O)-R i ;

[0014] n is 0, 1, 2, 3 or 4;

[0015] Each R4 is independently selected from: H, halogen, D, OH, -NH3, -NO2, -CN, COOH, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, -OCO-substituted or unsubstituted C1-C6 alkyl;

[0016] R5 is selected from the following group: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted 6-10 membered aryl, substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from O, N and S, substituted or unsubstituted C3-C10 cycloalkyl, or substituted or unsubstituted 3-10 membered heterocycloalkyl having 1-3 heteroatoms selected from O, N and S, -C1-C4 alkylene-(O-CH2-CH2)m-NR f -CO-R i or

[0017]

[0018] wherein Ring A is selected from the following group: substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl having 1-4 heteroatoms selected from O, N and S, substituted or unsubstituted 6-10 membered aryl, substituted or unsubstituted 5-10 membered heteroaryl having 1-4 heteroatoms selected from O, N and S;

[0019] L1 is selected from the group consisting of none, substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C2-C6 alkenylene, substituted or unsubstituted C2-C6 alkynylene or substituted or unsubstituted C1-C6 alkylene-(C=O)-;

[0020] Ring B is selected from the following group: substituted or unsubstituted C3-C10 cycloalkyl, or substituted or unsubstituted 3-10 membered heterocycloalkyl having 1-3 heteroatoms selected from O, N and S, substituted or unsubstituted 6-10 membered aryl, or substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from O, N, S; wherein the substitution means that one or more hydrogen atoms on the group are replaced by a substituent selected from the following group: halogen, -OH, -CN, -NH2, C1-C6 alkoxy, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, -CO-NR f R g 、-NR f R g ;

[0021] Each m is independently 0, 1, 2, 3, 4 or 5;

[0022] Each R i Independently selected from the following group: H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, -L2-R9;

[0023] Wherein, L2 is selected from the group consisting of substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C2-C6 alkenylene, substituted or unsubstituted C2-C6 alkynylene;

[0024] R9 is selected from the following group: substituted or unsubstituted C3-C10 cycloalkyl, or substituted or unsubstituted 3-10 membered heterocycloalkyl having 1-3 heteroatoms selected from O, N and S, substituted or unsubstituted 6-10 membered aryl, substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from O, N and S;

[0025] If not otherwise specified, the term "substituted" means that one or more hydrogen atoms on the group are replaced by a group selected from the group consisting of halogen, -OH, -CN, -NR f R g 、-NR f -CO-C1-C6 alkyl, -C1-C6 alkyl-CO-NR f R g , oxo (=O), C1-C6 alkyl, C1-C6 oxaalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, C1-C6 haloalkyl, -(C=O)-C1-C6 alkyl, C5-C10 aryl, benzyl, 3-8 membered cycloalkyl; and

[0026] Each R f and R g is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, or a 3-10 membered heterocycloalkyl having 1-3 heteroatoms selected from O, N and S, or R f With R g Together with the attached nitrogen atom, it forms a 3-7 membered cycloalkyl group having one nitrogen atom.

[0027] In another preferred embodiment, each group is independently selected from the corresponding groups in the compounds in Table 1.

[0028] In another preferred embodiment, Having a structure selected from the group consisting of:

[0029]

[0030] In another preferred embodiment, R5 is or a substituted or unsubstituted group selected from the following group:

[0031]

[0032] In another preferred embodiment, ring A is a substituted or unsubstituted group selected from the following group:

[0033]

[0034] In another preferred embodiment, L1 is connected to the N atom on ring A.

[0035] In another preferred embodiment, L1 is selected from the following group: -C1-C4 methylene, -CH2(C=O)-.

[0036] In another preferred embodiment, ring B is selected from the following group: substituted or unsubstituted 6-10 membered aryl groups, preferably substituted or unsubstituted phenyl groups.

[0037] In another preferred embodiment, L1 is -CH2- and ring B is a substituted or unsubstituted phenyl group.

[0038] In another preferred embodiment, L2 is -C1-C4 methylene, preferably, -CH2-.

[0039] In another preferred embodiment, R e is a substituted or unsubstituted group selected from the following group:

[0040]

[0041] In another preferred embodiment, R e is a substituted or unsubstituted group selected from the following group:

[0042]

[0043] wherein each Rj is independently selected from the following group: H, C1-C4 alkoxy, C1-C4 alkyl, C1-C4 haloalkyl, -(C=O)-C1-C4 alkyl, hydroxy-substituted C1-C4 alkyl, -C1-C4 alkylene-(O-CH2-CH2)m-NH-(C=O)-R i or -(C=O)-CH2(O-CH2-CH2)m-NH-(C=O)-R i , m and Ri are as defined above;

[0044] The substitution refers to that one or more hydrogen atoms on the ring carbon atoms of the group are replaced by a group selected from the group consisting of halogen, cyano, C1-C4 alkoxy, C1-C4 alkyl, -(C=O)-C1-C4 alkyl, hydroxy-substituted C1-C4 alkyl, and -NHCO-C1-C4 alkyl.

[0045] In another preferred embodiment, the R e Having a structure selected from the group consisting of: Wherein, Rj is independently selected from the following group: H, C1-C4 alkyl, C1-C4 haloalkyl, -(C=O)-C1-C4 alkyl, hydroxyl-substituted C1-C4 alkyl, preferably, Rj is selected from the following group: -CH3, -CH2CH3, -CH2CH2OH, -(C=O)CH3.

[0046] In another preferred embodiment, the compound of formula I has a structure of formula Ia or Ib:

[0047]

[0048] Wherein, X1, X2, R1, R2, R3, R4, R i , A, B, L1, n, m are as defined above.

[0049] In another preferred embodiment, each R9 is

[0050] In another preferred embodiment, the compound of formula Ia has structure IIa:

[0051]

[0052] Among them, R k Selected from the group consisting of C1-C4 alkyl, C1-C4 haloalkyl, -(C=O)-C1-C4 alkyl, hydroxy-substituted C1-C4 alkyl;

[0053] Said X1, X2, R1, R2, R4, A, B, L1 and n are as defined above.

[0054] In another preferred embodiment, R k Selected from the group consisting of -CH3, -CH2CH3, -CH2CH2OH, -(C=O)CH3.

[0055] In another preferred embodiment, the compound is selected from the following group of compounds in Table 1:

[0056] ZSQ-5-39、ZSQ-5-40、ZSQ-5-42、ZSQ-5-43、ZSQ-5-44、ZSQ-5-45、ZSQ-5-47、ZSQ-5-77、ZSQ-5-78、ZSQ-5-80、ZSQ-5-81、ZSQ-5-82、ZSQ-5-83、ZSQ-6-2、ZSQ-6-3、ZSQ-6-37、ZSQ-6-49、ZSQ-6-50、ZSQ-6-51、ZSQ-6-52、ZSQ-6-54、ZSQ-6-55、ZSQ-6-56、ZSQ-6-91、ZSQ-7-32、ZSQ-7-33、ZSQ-7-34、ZSQ-7-35、

[0057] ZSQ-7-36, ZSQ-7-37, ZSQ-7-86, ZSQ-7-106, ZSQ-8-32, ZSQ-8-33, ZSQ-8-49, ZSQ-8-50, ZSQ-9-19, ZSQ-9-24, ZSQ-9-25, ZSQ-9-26, ZSQ-9-27, ZSQ-9-28, ZSQ-9-30, ZSQ-9-31, ZSQ-9-32, ZSQ-9-33, ZSQ-9-34, ZSQ-9-35, ZSQ-9-36, ZSQ-9-37, ZSQ-10-101, ZSQ-10-104, ZSQ-11-8, ZSQ-11-9, ZSQ-11-11, ZSQ-11-12, ZSQ-11-31, ZSQ-11-32, ZSQ-11-34, ZSQ-11-37, ZSQ-11-79, ZSQ-11-80, ZSQ-11-101, ZSQ-11-104, ZSQ-12-15, ZSQ-12-54, ZSQ-12-66, ZSQ-12-67, ZSQ-12-70, ZSQ-12-73, ZSQ-12-76, ZSQ-12-80, ZSQ-12-84, ZSQ-12-85, ZSQ-13-74, ZSQ-13-83, ZSQ-13-96, ZSQ-13-97, ZSQ-13-98, ZSQ-13-99, ZSQ-13-100, ZSQ-13-101, ZSQ-17-69, ZSQ-17-74, ZSQ-17-75, ZSQ-17-79, ZSQ-17-80, ZSQ-17-81, ZSQ-17-86, ZSQ-17-87, ZSQ-20-29, ZSQ-20-30, AZY-5-37, AZY-5-26, AZY-4-31, AZY-4-30, AZY-4-36, ZSQ-1-78, ZSQ-1-68, ZSQ-1-97, ZSQ-1-94, ZSQ-13-34, ZSQ-13-37, ZSQ-13-63, ZSQ-13-102 and ZSQ-19-99.

[0058] In another preferred embodiment, the compound is selected from the following group of compounds in Table 1: ZSQ-7-35, ZSQ-8-33, ZSQ-11-79, ZSQ-11-80, ZSQ-12-15, ZSQ-12-67, ZSQ-12-70, ZSQ-13-96, ZSQ-13-98, ZSQ-17-74, ZSQ-17-75, ZSQ-17-79, ZSQ-17-80, ZSQ-17-81, ZSQ-12-54, ZSQ-12-80, ZSQ-13-98 Q-13-74, ZSQ-13-83, ZSQ-13-100, ZSQ-13-101, ZSQ-17-69, ZSQ-17-86, ZSQ-20-29 and ZSQ-20-30, preferably, ZSQ-12-54, ZSQ-12-80, ZSQ-13-74, ZSQ-13-83, ZSQ-13-100, ZSQ-13-101, ZSQ-17-69, ZSQ-17-86, ZSQ-20-29 and ZSQ-20-30.

[0059] The second aspect of the present invention provides a pharmaceutical composition, comprising: (a) a compound of formula I as described in the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, or a stereoisomer or tautomer, hydrate or solvate thereof; and (b) a pharmaceutically acceptable carrier.

[0060] The third aspect of the present invention provides the use of the compound of formula I as described in the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, or a stereoisomer or tautomer, hydrate or solvate thereof, or the pharmaceutical composition as described in the second aspect of the present invention, for preparing a drug or preparation, wherein the pharmaceutical preparation is used for:

[0061] (i) downregulating and / or inhibiting PKM2 activity;

[0062] (ii) prevention and / or treatment of tumors;

[0063] (iii) reducing and / or inhibiting the growth and / or proliferation of tumor cells;

[0064] (iv) reducing and / or inhibiting the metastasis of tumor cells;

[0065] (v) promoting apoptosis of tumor cells; and / or

[0066] (vi) Reduce and / or inhibit the glycolysis process of cells.

[0067] In another preferred embodiment, the drug or preparation is used to prevent and / or treat cancer by downregulating and / or inhibiting PKM2 activity.

[0068] In another preferred embodiment, the drug or preparation is a PKM2 inhibitor.

[0069] In another preferred embodiment, the tumor is a PKM2-dependent tumor.

[0070] In another preferred embodiment, the "PKM2-dependent tumor" refers to a tumor with high expression of PKM2 in tumor cells and / or a tumor that relies on PKM2 for glycolysis.

[0071] In another preferred embodiment, the tumor is selected from the following group: liver cancer, gastric cancer, breast cancer, pancreatic cancer, ovarian cancer (including human ovarian teratoma), laryngeal cancer (including laryngeal squamous cell carcinoma), prostate cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, endometrial cancer, brain cancer, bladder cancer, testicular cancer, head cancer, neck cancer, skin cancer (including melanoma and basal cell carcinoma), mesothelial cancer, white blood cell cancer (including lymphoma and leukemia), esophageal cancer, muscle cancer, connective tissue cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), adrenal cancer, thyroid cancer, kidney cancer or bone cancer, glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma (Kaposi sarcoma), choriocarcinoma, skin basal cell carcinoma and testicular seminoma.

[0072] In another preferred embodiment, the tumor is selected from the following group: liver cancer, gastric cancer, breast cancer, pancreatic cancer, ovarian cancer (including human ovarian teratoma), laryngeal cancer (including laryngeal squamous cell carcinoma), prostate cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, endometrial cancer, brain cancer, bladder cancer, testicular cancer, head cancer and neck cancer.

[0073] The fourth aspect of the present invention provides a PKM2 inhibitor, which includes: the compound of formula I as described in the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, or a stereoisomer or tautomer, hydrate or solvate thereof.

[0074] The fifth aspect of the present invention provides an in vitro non-therapeutic method for down-regulating and / or inhibiting PKM2 activity, wherein cells are cultured in the presence of a compound of formula I as described in the first aspect of the present invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition as described in the second aspect of the present invention, thereby down-regulating and / or inhibiting PKM2 activity in the cells.

[0075] In another preferred embodiment, the cells are tumor cells.

[0076] In another preferred embodiment, the tumor is a PKM2-dependent tumor.

[0077] In another preferred embodiment, the effective concentration of the compound of formula I, or its stereoisomer or tautomer, or its pharmaceutically acceptable salt, hydrate or solvate is 0.01nM / L-1mM / L, preferably 0.1nM / L-500μM / L, and most preferably 1nM / L-100μM / L.

[0078] The sixth aspect of the present invention provides a method for preventing and / or treating tumors, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula I as described in the first aspect of the present invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition as described in the second aspect of the present invention.

[0079] In another preferred embodiment, the subject is a mammal, preferably a human, a rat, a mouse, a cat, a dog, a cow or a horse.

[0080] In another preferred embodiment, the tumor is a PKM2-dependent tumor.

[0081] In a seventh aspect, the present invention provides a method for preparing the compound of formula I as described in the first aspect of the present invention, comprising the steps of:

[0082] Step i: protecting the arsenite functional group of A1 in the form of ester or amide;

[0083] Step ii: introducing a carbonyl-linked R5 substituent at the amine group of A2 by condensation reaction; and

[0084] Step iii: Through S N 2 Substitution reaction introduces R3 substituent at the amine group of A3;

[0085]

[0086] Wherein, X1, X2, R1, R2, R4, R5 and n are as defined in the first aspect of the present invention.

[0087] In another preferred embodiment, the R3 group is first introduced into the amine group of compound A2, and then the R5 substituent connected to the carbonyl group is introduced.

[0088] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 The concentration-activity curves of representative compounds and control compounds for inhibiting PKM2 in vitro and their half inhibitory concentrations (IC 50 ).

[0090] Figure 2 The inhibitory effects of representative compounds at different concentrations on the in vitro enzyme activity of PKM1 are shown.

[0091] Figure 3 The mass spectrometry spectrum of ZSQ13-83 covalently bound to the peptide chain where cysteine ​​474 of PKM2 protein is located is shown.

[0092] Figure 4 The concentration-activity curves and half inhibitory concentrations (IC50) of ZSQ13-83 and its analogs, as well as control compounds, for inhibiting the growth of different ovarian cancer cell lines are shown. 50 ).

[0093] Figure 5 Competitive inhibition of covalent binding of ZSQ13-74 to PKM2 in PA-1 cells by representative compounds such as ZSQ13-83, ZSQ17-69, and ZSQ17-80 at 10 μM concentration is shown.

[0094] Figure 6 Shown are the effects of ZSQ13-83 at a concentration of 10 μM on the phosphorylation levels of AMPK and ERK (p-AMPK, p-ERK), and the expression level of c-MYC in PA-1 cells. DETAILED DESCRIPTION

[0095] The inventors have conducted extensive and in-depth research, and through a large number of screenings and tests, provided a class of PKM2 inhibitors based on organic arsenic. The compounds of the present invention have high activity and high specificity in inhibiting PKM2, and are very suitable for preparing drugs that inhibit tumors by inhibiting the PKM2 pathway. On this basis, the present invention was completed.

[0096] the term

[0097] 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.

[0098] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0099] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0100] Unless otherwise indicated, the term "alkyl" by itself or as part of another substituent refers to a straight or branched chain hydrocarbon radical having the specified number of carbon atoms (i.e., C1-6 means 1-6 carbons, preferably 1-3 carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, and the like.

[0101] 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.

[0102] Unless otherwise indicated, the term "cycloalkyl" refers to a group having the specified number of ring atoms (e.g., C 3-10 The term "cycloalkyl" refers to a hydrocarbon ring which is fully saturated or has no more than one double bond between the ring tops. It can be a monocyclic ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or similar groups. It can also be a bicyclic and polycyclic hydrocarbon ring, such as a bridged ring or a spirocyclic ring, such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, etc.

[0103] Unless otherwise stated, the term "heterocycloalkyl" refers to a cycloalkyl containing a specified number of heteroatoms selected from O, N and S, wherein nitrogen and sulfur atoms are optionally oxidized, and nitrogen atoms are optionally quaternized. Heterocycloalkyl can be a monocyclic, bicyclic or polycyclic system. Non-limiting examples of heterocycloalkyl include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S, S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine, etc. Heterocycloalkyl can be connected to the rest of the molecule via a ring carbon or a heteroatom. For terms such as cycloalkylalkyl and heterocycloalkylalkyl, it is meant that cycloalkyl or heterocycloalkyl is connected to the rest of the molecule via an alkyl or alkylene linker. For example, cyclobutylmethyl- is a cyclobutyl ring attached to a methylene linkage to the rest of the molecule.

[0104] Unless otherwise indicated, the term "alkylene" by itself or as part of another substituent refers to a divalent radical derived from an alkane, for example -CH2-, -CH2CH2CH2CH2-. Similarly, "alkenylene" or "alkynylene" refer to unsaturated forms of "alkylene" having double or triple bonds, respectively.

[0105] Similarly, unless otherwise indicated, the term "heteroalkyl" refers to a group containing the specified number of carbons and 1 to 3 heteroatoms selected from O, N and S, and wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized. The heteroatoms O, N and S may be located at any interior position of the heteroalkyl group.

[0106] Unless otherwise indicated, the term "alkoxy" or "alkyloxy", "alkylamino" or "alkylamino" is used in its conventional sense to refer to those alkyl groups attached to the rest of the molecule via an oxygen atom or nitrogen, respectively. In addition, the alkylamino group can be monosubstituted or disubstituted. For example, methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, tert-butylamino, dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, ditert-butylamino, etc. When disubstituted, the alkyl portions can be the same or different, and can also be combined with the nitrogen atom attached to each alkyl group to form a 3-7 membered ring. Therefore, -NR f R g The groups shown are meant to include piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl, and the like.

[0107] Unless otherwise indicated, the term "acyl", used alone or as part of another group, refers to a group in which both substituents are substituted with a substituent = O on the carbon closest to the point of attachment of the group (e.g., -(C=O)CH3 (or -CO-CH3), -(C=O)CH2CH3, etc.). In addition, the term "carbonylalkylene" or "alkylenecarbonyl" includes those groups that are attached to the rest of the molecule through a carbonyl carbon atom or an alkylene carbon atom, such as methylenecarbonyl includes the forms "-(C=O)-CH2-" and "-CH2-(C=O)-".

[0108] Unless otherwise indicated, the term "halo" or "halogen" by itself or as part of another substituent refers to a fluorine, chlorine, bromine, or iodine atom. In addition, terms such as "haloalkyl" are meant to include monohaloalkyl or polyhaloalkyl. For example, the term "C 1-6 "Haloalkyl" is meant to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0109] Unless otherwise indicated, the term "aryl" means a polyunsaturated (usually aromatic) hydrocarbon radical which may be a single ring or multiple rings (up to three) fused together or linked covalently. Non-limiting examples of aryl include phenyl, naphthyl and biphenyl.

[0110] The term "heteroaryl" refers to an aryl group (or ring) containing the specified number of heteroatoms selected from O, N and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. The heteroaryl group can be attached to the rest of the molecule through a heteroatom. Non-limiting examples of heteroaryl groups include pyridinyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzotriazinyl, purinyl, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzisoxazolyl, isobenzofuryl, isoindolyl, indolizinyl, benzotriazinyl, thienopyridinyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridine, benzothiazolyl, benzofuranyl, benzothiophenyl, indolyl, quinolyl, isoquinolyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furanyl, thienyl, and the like.

[0111] In some embodiments, the above terms (such as "alkyl", "aryl" and "heteroaryl") will include substituted and unsubstituted forms of the specified groups, and the number of substituents can be 1, 2, 3 or 4. Unless otherwise specified, the "substituted" means that one or more hydrogen atoms on the group are replaced by a group selected from the group consisting of halogen, -OH, -CN, -NR f R g , -NRa-CO-C1-C6 alkyl, -C1-C6 alkyl-CO-NR f R g , oxo (=O), C1-C6 alkyl, C1-C6 oxoalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, C1-C6 haloalkyl, -(C=O)-C1-C6 alkyl, C5-C10 aryl, benzyl, 3-8 membered cycloalkyl. Unless otherwise specified, each R f and R g is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, or a 3-10 membered heterocycloalkyl having 1-3 heteroatoms selected from O, N and S, or R f With R g Together with the attached nitrogen atom, it forms a 3-7 membered cycloalkyl group having one nitrogen atom.

[0112] As used herein, the term "heteroatom" is intended to include oxygen (O), nitrogen (N), and sulfur (S).

[0113] For 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 mean a bond providing a connection at any available vertex of the aromatic ring. In some embodiments, this description also includes connections on a ring fused to an aromatic ring. For example, a bond drawn to the center of an indole benzene moiety will represent a bond connected to any available vertex of a six-membered or five-membered ring moiety of indole.

[0114] Unless otherwise specified, in the present invention, all compounds appearing are intended to include all possible optical isomers, such as single chiral compounds, or mixtures of various chiral compounds (i.e., racemates). Among all compounds of the present invention, each chiral carbon atom can be optionally in R configuration or S configuration, or a mixture of R configuration and S configuration.

[0115] Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers, regioisomers and individual isomers (e.g., isolated enantiomers) are all intended to be included within the scope of the present invention. When compounds provided herein have defined stereochemistry (expressed as R or S, or indicated by 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).

[0116] As used herein, the term "pharmaceutically acceptable" ingredient refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic response), ie, at a reasonable benefit / risk ratio.

[0117] As used herein, the term "therapeutically effective amount" refers to any amount of a drug as described below, which, when used alone or in combination with another therapeutic agent, can promote disease regression, which is manifested as a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of disorders or disabilities caused by the disease. The "therapeutically effective dose" of the drug of the present invention also includes a "prophylactic effective dose", which is any amount of a drug as described below, which, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or suffering from a recurrence of the disease, can inhibit the occurrence or recurrence of the disease.

[0118] As used herein, the term "solvate" refers to a complex in which the compound of the present invention is coordinated with solvent molecules to form a specific ratio.

[0119] As used herein, the term "hydrate" refers to a complex formed by coordination of a compound of the present invention with water.

[0120] The present invention also includes all suitable isotopic variations of the compounds of the present invention. Isotopic variations of the compounds of the present invention are defined as those in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, for example 2 H. 3 H. 11 C. 13 C. 14 C. 15 N. 17 O. 18 O. 35 S. 18 F and 36 Cl. Some isotopic variants of the invention, for example, wherein a radioactive isotope (e.g. 3 H or 14 C) are used in drug and / or substrate tissue distribution studies. 3 H, and carbon-14, i.e., 14 C, isotopes are particularly preferred because they are easy to prepare and detect. In addition, isotopes (such as deuterium, i.e., 2 H) substitution may provide some therapeutic advantages caused by increased metabolic stability, for example, increased half-life in vivo or reduced dosage requirements and may therefore be preferred in some cases. Isotopic variations of the compounds of the invention can generally be prepared by conventional procedures, for example, using suitable reagents of appropriate isotopic variations, by the illustrative methods or the preparation described in the experimental section below.

[0121] Indicates the connection site with other atoms.

[0122] As used herein, "treat" means to lessen, slow the progression, attenuate, prevent, or maintain an existing disease or condition (eg, cancer). Treatment also includes curing, preventing the development of, or alleviating to some extent, one or more symptoms of a disease or condition.

[0123] Active ingredients

[0124] As used herein, "compound of the present invention" refers to a compound of formula I, a pharmaceutically acceptable salt thereof, or a stereoisomer or tautomer, or a hydrate or solvate thereof;

[0125]

[0126] wherein X1, X2, R1, R2, R3, R4, R5 and n are as defined in the first aspect of the present invention.

[0127] As used herein, "pharmaceutically acceptable salts" refer to salts formed by the compounds of the present invention and acids or bases that are suitable for use as medicines. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts is the salts formed by the compounds of the present invention and acids. Suitable acids for forming salts include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, 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, benzenesulfonic acid, and organic acids such as aspartic acid and glutamic acid. A preferred class of salts is the salts formed by the compounds of the present invention and bases. Suitable bases for forming salts include, but are not limited to, inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, sodium phosphate, and organic bases such as ammonia water, triethylamine, diethylamine, and piperazine.

[0128] Preparation method

[0129] The present invention provides methods for preparing compounds of formula I. The compounds of the present invention can be prepared by a variety of synthetic operations. Exemplary preparation methods of these compounds may include (but are not limited to) the processes described below.

[0130] Preferably, the compounds of formula I of the present invention can be prepared by the exemplary methods described in the following schemes and examples and the relevant public literature operations used by those skilled in the art.

[0131] During the specific operation process, the steps in the method can be expanded or combined as needed.

[0132] Solution 1: includes the following steps:

[0133]

[0134] Wherein, X1, X2, R1, R2, R4, R5 and n are as defined above.

[0135] More specifically, the steps include:

[0136] Step i: Protect the arsenite functional group of A1 in the form of ester or amide, such as using ethanedithiol, ethylene glycol, etc. as a protecting agent.

[0137] Step ii: Introduce a carbonyl-linked R5 substituent into the amine group of A2 by condensation reaction, such as using acyl chloride, acid, acid anhydride, etc. (R5-CO-Cl, R5-CO-OH), etc.

[0138] Step iii: Through S N 2 Substitution reaction: To introduce R3 substituent into the amine group of A3, R3-halogen can be used under alkaline conditions (such as sodium hydride).

[0139] Scenario 2:

[0140]

[0141] Step i: Protect the arsenite functional group of A1 in the form of ester or amide, and ethanedithiol, ethylene glycol, etc. can be used as a protecting agent.

[0142] Step ii: Through S N 2 Substitution reaction or reductive amination reaction to introduce R3 substituent into the amine group of A2 can use R3-halogen under alkaline conditions (such as sodium hydride); or can use R3-formaldehyde in the presence of a reducing agent (such as sodium triacetoxyborohydride).

[0143] Step iii: Acylation reaction is carried out on the phenyl ring amine group of A3 to introduce a carbonyl-linked R5 group, such as using acyl chloride, acid, acid anhydride, etc. (R5-CO-Cl, R5-CO-OH), etc.; or catalyzed by (but not limited to) phosphorus oxychloride reagent.

[0144] The above preparation method may further include a step of removing the protecting group as required.

[0145] Any suitable inert solvent can be used in the method of the present invention. Representative inert solvents include, but are not limited to, pentane, different pentanes, hexane, different hexanes, heptane, different heptanes, petroleum ether, cyclopentane, different cyclohexanes, benzene, toluene, xylene, trifluorotoluene, halogenated benzenes such as chlorobenzene, fluorobenzene, dichlorobenzene and difluorobenzene, methylene chloride, chloroform, DMF, acetone, ethyl acetate, diethyl ether, tetrahydrofuran, DMSO, or a combination thereof.

[0146] The reaction in the method of the present invention can be carried out at any suitable temperature. For example, the reaction temperature can be from about -78°C to about 100°C, or from about -50°C to about 100°C, or from about -25°C to about 50°C, or from about -10°C to about 25°C, or from about 0°C to about 20°C. In some embodiments, the reaction temperature can be from about 0°C to about 20°C. The reactions in the above steps are conventional reactions known to those skilled in the art. Unless otherwise specified, the reagents and raw materials used in the synthetic route can be commercially available, or those skilled in the art can prepare them with reference to known methods according to the designed different compound structures.

[0147] Pharmaceutical compositions and methods of administration

[0148] Since the compounds of the present invention have excellent inhibitory activity on type II pyruvate kinase (PKM2), the compounds of the present invention and their crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredients can be used to treat, prevent and alleviate tumors. More specifically, they are used to downregulate or inhibit the PKM2 activity of tumor cells, thereby inhibiting the glycolysis process, thereby achieving the effect of inhibiting tumors. Therefore, the compounds of the present invention can be used to prepare PKM2 inhibitors.

[0149] Preferably, the above-mentioned tumor is a PKM2-dependent tumor. In the present invention, the PKM2-dependent tumor is a tumor with high expression of PKM2 in the cell and / or a cell that relies on PKM2 for glycolysis. The "high expression of PKM2" means that PKM2 accounts for more than 60% of the total pyruvate kinase in the cell, preferably more than 70%, more than 80%, more than 90% or more than 95%. The "dependence on PKM2 for glycolysis" means that the cell mainly catalyzes the glycolysis process through PKM2, for example, ≥60% of the glycolysis in the cell is catalyzed by PKM2, preferably ≥70%, ≥80%, ≥90%, or ≥95%.

[0150] When the glycolysis process of cancer cells is inhibited, it will slow down or inhibit the growth, proliferation, and metastasis of cancer cells and accelerate the apoptosis of cancer cells.

[0151] Preferably, the compounds of the present invention can be used to prevent and / or treat cancer. Representative cancers include (but are not limited to): liver cancer, gastric cancer, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, endometrial cancer, brain cancer, bladder cancer, testicular cancer, head cancer, neck cancer, skin cancer (including melanoma and basal cell carcinoma), mesothelial cancer, white blood cell cancer (including lymphoma and leukemia), esophageal cancer, myoma, connective tissue cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), adrenal cancer, thyroid, kidney cancer or bone cancer; or glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma (Kaposi's sarcoma), choriocarcinoma, skin basal cell carcinoma and testicular seminoma.

[0152] The compounds of the present invention may be administered alone or in combination with other therapeutic agents. Such therapeutic agents include, but are not limited to, anticancer agents and / or immunosuppressants, such as anticancer agents and / or immunosuppressants selected from the group consisting of olaparib, rucaparib, niraparib, methotrexate, capecitabine, gemcitabine, doxifluridine, pemetrexed disodium, pazopanib, imatinib, erlotinib, lapatinib, gefitinib, vandetanib, Herceptin, bevacizumab, rituximab, trastuzumab, paclitaxel, vinorelbine, docetaxel, doxorubicin, hydroxycamptothecin, mitomycin, epirubicin, pirarubicin, bleomycin, letrozole, tamoxifen, fulvestrant, triptorelin, flutamide, leuprolide, anastrozole, ifosfamide, busulfan, cyclophosphamide, carmustine, nimustine, Semustine, nitrogen mustard, melphalan, leuprorelin, carboplatin, cisplatin, oxaliplatin, cisplatin, topotax, camptothecin, topotecan, everolimus, sirolimus, temozolomide, 6-mercaptopurine, 6-thioguanine, azathioprine, streptomycin D, daunorubicin, doxorubicin, mitoxantrone, bleomycin, plicamycin or aminoglutethimide; such as nivolumab, pembrolizumab, ipilimumab, avelumab, durvalumab, atezolizumab or pidilizumab, or a combination thereof.

[0153] In certain embodiments, a compound of the invention is administered to a subject with cancer in combination with other conventional cancer treatments, such as radiation therapy or surgery. Radiation therapy is well known in the art and includes X-ray therapy, such as gamma radiation, and radiopharmaceutical therapy.

[0154] In another preferred embodiment, the therapeutic agent is a hypoxia-inducible factor prolyl hydroxylase (HIF-PH) inhibitor.

[0155] In certain embodiments, the compounds of the present invention are administered simultaneously with other therapeutic agents as a combination therapy regimen, in the same or separate formulations, or administered sequentially.

[0156] The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, and more preferably, contains 10-500 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.

[0157] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0158] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administrations include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0159] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0160] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

[0161] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0162] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0163] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.

[0164] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0165] Dosage forms for topical administration of the compounds of the invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0166] The general range of the therapeutically effective dose of the compound of formula I or the composition of the compound of formula I will be: about 1-2000mg / day, about 10-about 1000mg / day, about 10-about 500mg / day, about 10-about 250mg / day, about 10-about 100mg / day, or about 10-about 50mg / day. The therapeutically effective dose will be given with one or more doses. However, it should be understood that the specific dose of the compound of the present invention for any particular patient will depend on multiple factors, for example, the age, sex, body weight, general health, diet, individual response of the patient to be treated, the activity, dosage form, application mode and concomitant drug of the specific compound of the time, the severity of the disease to be treated, the administration. The therapeutically effective amount of a given situation can be determined by routine experiments, and within the scope of the clinician or physician's ability and judgment. In any case, the compound or composition will be given with multiple doses based on the individual situation of the patient and in a manner that allows the delivery of a therapeutically effective amount.

[0167] The main advantages of the present invention include:

[0168] 1. The present invention provides a new class of PKM2 inhibitors.

[0169] 2. The compounds of the present invention have high inhibitory activity and high selectivity against PKM2, require low dosage when used, and have little side effects.

[0170] 3. The compounds of the present invention are allosteric inhibitors and are not prone to drug resistance; moreover, the compounds of the present invention can covalently bind to PKM2, ensuring effective inhibition at low doses, and are particularly suitable for drug development.

[0171] 4. The compounds of the present invention have a significant inhibitory effect on PKM2-dependent tumors.

[0172] The present invention will be further described below in conjunction with specific implementation. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0173] Preparation Example

[0174] Example 1

[0175] Synthesis route of compound ZSQ-5-77

[0176]

[0177] Synthesis of compound (4-aminophenyl)arsonous acid (ZSQ-1-18)

[0178]

[0179] 4-Aminophenylarsonic acid (108.5 g, 500 mmol) was dissolved in 300 ml of ethanol, and the mixed solution was heated to reflux.

[0180] Phenylhydrazine (92ml, 1mol) was added dropwise (over 1h) to the mixed solution. A large amount of nitrogen was generated during the addition. When the generation of nitrogen slowed down, reflux stirring was continued for 1.5h. The mixed solution was concentrated by vacuum distillation, sodium hydroxide solution (40g dissolved in 400ml water) was added, and ether was washed twice (400ml). Liquid separation, saturated ammonium chloride solution (400ml) was added to the aqueous phase and stirred at 0℃ for 1h, and a large amount of white solid precipitated. A white needle-shaped solid was obtained by filtration and vacuum drying to obtain 40g of product with a yield of 40%.

[0181] Synthesis of Compound 4-(1,3,2-dithiarsolan-2-yl)aniline(ZSQ-1-23)

[0182]

[0183] ZSQ-1-18 (40 g, 199 mmol) was dissolved in 200 ml of anhydrous ethanol and heated to reflux. Then, ethanedithiol (20 ml, 240 mmol) was added dropwise to the mixed solution within 30 min, and heating and stirring were continued for 30 min. Subsequently, the mixture was cooled in ice water and filtered to obtain a crude product, which was recrystallized from ethanol to obtain 42 g of a white solid with a yield of 81%.

[0184] Synthesis of tert-butyl(S)-2-((4-(1,3,2-dithiarsolan-2-yl)phenyl)carbamoyl)pyrrolidine-1-carboxylate(ZSQ-5-67)

[0185]

[0186] Dissolve ZSQ-1-23 (518 mg, 2 mmol), Boc-L-proline (430 mg, 2 mmol), HATU (1.14 g, 3 mmol) and DIPEA (0.92 ml, 6 mmol) in 6 ml DCM and stir at room temperature for 3 h. After the reaction is completed, wash with H2O (20 ml × 2), saturated NaCl solution (20 ml), and extract with 20 ml DCM. Combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, separate and purify by silica gel column chromatography, and elute with PE / EA (3:1) to obtain 850 mg of yellow solid with a yield of 93%.

[0187] Synthesis of compound (S)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)pyrrolidine-2-carboxamide (ZSQ-5-68)

[0188]

[0189] ZSQ-5-67 (850 mg, 1.86 mmol) and 1 ml of trifluoroacetic acid were mixed in 5 ml of DCM and stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:4), and 680 mg of yellow solid was obtained with a yield of 95%.

[0190] Synthesis of compound (S)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-1-acetylpyrrolidine-2-carboxamide (ZSQ-5-73)

[0191]

[0192] ZSQ-5-68 (356 mg, 1.0 mmol) and DIPEA (0.5 ml, 3 mmol) were mixed in 5 ml DCM, and acetic anhydride (0.14 ml, 1.5 mmol) was added dropwise and stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated and separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:7), and 288 mg of yellow solid was obtained, with a yield of 72%.

[0193] Synthesis of compound (S)-(4-(1-acetylpyrrolidine-2-carboxamido)phenyl)arsonous acid (ZSQ-5-77)

[0194]

[0195] ZSQ-5-73 (79.6 mg, 0.20 mmol) and mercuric perchlorate trihydrate (72 mg, 0.16 mmol) were dissolved in 3 ml DMSO, and the mixed solution was stirred at room temperature for 10 min, and immediately separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:4), and the crude product was purified by HPLC to obtain 43 mg of white solid, with a yield of 63%. M / Z: Exact Mass: 340.04, (LC-MS): Found 341.0.

[0196] Example 2

[0197] Synthesis route of compound ZSQ-5-40

[0198]

[0199] Synthesis of the compound methyl(1s,4s)-4-acetamidocyclohexane-1-carboxylate(ZSQ-5-24)

[0200]

[0201] Cis-4-aminocyclohexanecarboxylic acid methyl ester hydrochloride (968 mg, 5.0 mmol) and DIPEA (2.48 ml, 15 mmol) were mixed in 25 ml DCM, and acetic anhydride (0.14 ml, 1.5 mmol) was added dropwise and stirred at room temperature for 6 h. After the reaction was completed, the reaction solution was concentrated and separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:3), and 892 mg of white solid was obtained, with a yield of 89%.

[0202] Synthesis of the compound methyl(1s,4s)-4-(N-methylacetamido)cyclohexane-1-carboxylate(ZSQ-5-28)

[0203]

[0204] ZSQ-5-24 (1.17 g, 5 mmol) and iodomethane (10 mmol, 0.62 ml) were mixed and dissolved in 15 ml of ultra-dry THF, and mixed and stirred for 10 min in an ice bath. NaH (60%, 10 mmol, 400 mg) was added to the mixed solution in batches, and after the addition, the mixture was stirred for 3 h in an ice bath, and then moved to room temperature and stirred overnight. After the reaction was completed, water was added to quench, and the reaction solution was concentrated by vacuum distillation after acidification with 2N hydrochloric acid. After separation and purification by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:2), 482 mg of light yellow liquid was obtained, with a yield of 45%.

[0205] Synthesis of compound (1s,4s)-4-(N-methylacetamido)cyclohexane-1-carboxylic acid (ZSQ-5-30)

[0206]

[0207] Compound ZSQ-5-28 (2.42 mmol, 482 mg) was dissolved in a mixed solution of 3 ml THF and 3 ml H2O, and lithium hydroxide monohydrate (4.84 mmol, 203 mg) was added, and the mixture was stirred at room temperature for 3 h. After the reaction was completed, 2N hydrochloric acid was added to acidify the mixture, and the THF was concentrated to remove the THF. The mixture was separated and purified by C18 reverse phase column chromatography, and eluted with H2O / CH3CN (1:4) to obtain 426 mg of light yellow powder with a yield of 88%.

[0208] Synthesis of compound (1s,4s)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-4-(N-methylacetamido)-cyclo-hexane-1-carboxamide (ZSQ-5-32)

[0209]

[0210] ZSQ-5-30 (99 mg, 0.5 mmol), ZSQ-1-23 (129 mg, 0.5 mmol), HATU (285 mg, 1.5 mmol) and DIPEA (0.24 ml, 3 mmol) were dissolved in a mixed solution of 3 ml DCM and 3 ml DMF, and stirred at room temperature for 3 h. After the reaction was completed, it was washed with H2O (20 ml × 2), saturated NaCl solution (20 ml), and extracted with 20 ml DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, separated and purified by silica gel column chromatography, and eluted with DCM / EA (1:1) to obtain 77 mg of white solid with a yield of 35%.

[0211] Synthesis of compound (4-((1s,4s)-4-(N-methylacetamido)cyclohexane-1-carboxamido)phenyl)arsonous acid (ZSQ-5-40)

[0212]

[0213] ZSQ-5-32 (77 mg, 0.17 mmol) and mercuric perchlorate trihydrate (71 mg, 0.15 mmol) were dissolved in 3 ml DMSO, and the mixed solution was stirred at room temperature for 10 min, and immediately separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:3), and the crude product was purified by HPLC to obtain 50 mg of white solid, with a yield of 76%. M / Z: Exact Mass: 382.09, (LC-MS): Found 383.1.

[0214] Example 3

[0215] Synthesis route of compound ZSQ-5-42

[0216]

[0217] Synthesis of Compound N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-2-bromoacetamide(ZSQ-5-4)

[0218]

[0219] ZSQ-1-23 (1.03 g, 4 mmol) and triethylamine (0.83 ml, 6 mmol) were dissolved in 20 ml of dry DCM solution and stirred at 0 ° C. Bromoacetyl bromide (0.38 ml, 4.4 mmol) was added dropwise over 15 min and continued to stir at 0 ° C for 1 h. The mixture was diluted with DCM (20 ml), washed with 2N dilute hydrochloric acid (30 ml), water (50 ml), saturated sodium bicarbonate solution (30 ml), saturated sodium chloride solution (30 ml) in sequence, and extracted with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. Purification was separated and purified by silica gel column chromatography and eluted with PE / EA (4:1) to obtain 1.02 g of a yellow solid with a yield of 67%.

[0220] Synthesis of Compound N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-2-(4-methylpiperazin-1-yl)acetamide (ZSQ-5-10)

[0221]

[0222] At room temperature, N-methylpiperazine (66ul, 0.6mmol), DIPEA (99ul, 0.6mmol) and ZSQ-5-4 (76mg, 0.2mmol) were dissolved in 1ml THF solution, and the mixed solution was stirred at room temperature for 3h. The reaction solution was concentrated under reduced pressure, separated and purified by C18 reverse phase column chromatography, and eluted with H2O / CH3CN (1:4) to obtain 57mg white solid, with a yield of 71%.

[0223] Synthesis of compound (4-(2-(4-methylpiperazin-1-yl)acetamido)phenyl)arsonous acid (ZSQ-5-42)

[0224]

[0225] ZSQ-5-10 (34 mg, 0.10 mmol) and mercuric perchlorate trihydrate (42 mg, 0.09 mmol) were dissolved in 3 ml DMSO, and the mixed solution was stirred at room temperature for 10 min, and immediately separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:9), and the crude product was purified by HPLC to obtain 23 mg of white solid, with a yield of 67%. M / Z: Exact Mass: 341.07, (LC-MS): Found 342.1.

[0226] Example 4

[0227] Synthesis route of compound ZSQ-6-3

[0228]

[0229] Synthesis of Compound 4-(1,3,2-dithiarsolan-2-yl)-N-methylaniline(ZSQ-5-87)

[0230]

[0231] ZSQ-1-23 (1.03 g, 4.0 mmol) and iodomethane (0.37 ml, 6 mmol) were mixed and dissolved in 20 ml of ultra-dry THF, and mixed and stirred for 10 min under an ice bath. NaH (60%, 4.2 mmol, 168 mg) was added to the mixed solution in batches, and after the addition, stirring was continued for 3 h under an ice bath, and then moved to room temperature and stirred overnight. After the reaction was completed, 30 ml of water was added to quench, the reaction solution was concentrated, extracted with EA (30 ml), and then washed with saturated sodium chloride solution (30 ml). The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated, separated and purified by silica gel column chromatography, and eluted with PE / EA (1:8) to obtain 425 mg of white solid with a yield of 39%.

[0232] Synthesis of Compound N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-N-methylbutyramide(ZSQ-5-102)

[0233]

[0234] Dissolve ZSQ-5-87 (136 mg, 0.5 mmol) and triethylamine (173 ul, 1.25 mmol) in 8 ml of dry DCM solution and stir at 0 ° C. Add n-butyryl chloride (57 ul, 0.55 mmol) dropwise over 5 min and continue stirring at 0 ° C for 3 h. The reaction solution is washed with water (20 ml), saturated sodium chloride solution (20 ml), and then extracted with DCM (15 ml). The combined organic phases are dried over anhydrous sodium sulfate, filtered, concentrated, separated and purified by silica gel column chromatography, and eluted with PE / EA (1:3) to obtain 96 mg of yellow solid with a yield of 56%.

[0235] Synthesis of compound (4-(N-methylbutyramido)phenyl)arsonous acid (ZSQ-6-3)

[0236]

[0237] ZSQ-5-102 (48 mg, 0.14 mmol) and mercuric perchlorate trihydrate (57 mg, 0.12 mmol) were dissolved in 2 ml DMSO, and the mixed solution was stirred at room temperature for 10 min, and immediately separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:3), and the crude product was purified by HPLC to obtain 28 mg of white solid, with a yield of 70%. M / Z: Exact Mass: 285.03, (LC-MS): Found 286.10.

[0238] Example 5

[0239] Synthesis route of compound ZSQ-6-52

[0240]

[0241] Synthesis of Compound (1s,4s)-4-((tert-butoxycarbonyl)amino)cyclohexane-1-carboxylic acid (ZSQ-6-10)

[0242]

[0243] 4-Aminocyclohexane-1-carboxylic acid (2.86 g, 20 mmol) and di-tert-butyl dicarbonate (6.9 ml, 30 mmol) were mixed and dissolved in 40 ml DCM, and triethylamine (8.3 ml, 60 mmol) was added dropwise, and the mixture was stirred at room temperature for 24 h. After the reaction was completed, the mixture was directly concentrated by vacuum distillation and the next step was directly started.

[0244] Synthesis of compound benzyl(1s,4s)-4-((tert-butoxycarbonyl)amino)cyclohexane-1-carboxylate(ZSQ-6-13-1)

[0245]

[0246] ZSQ-6-10 (20 mmol) and potassium carbonate (5.5 g, 40 mmol) were dissolved in 20 ml DMF. After stirring at room temperature for 20 min, benzyl bromide (2.37 ml, 20 mmol) was added dropwise. After the addition, stirring was continued at room temperature overnight. After the reaction was completed, it was filtered with diatomaceous earth and washed with DCM. The filtrate was concentrated and separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:7), and 1.6 g of brown liquid was obtained, with a total yield of 24%.

[0247] Synthesis of enzyl(1s,4s)-4-aminocyclohexane-1-carboxylate(ZSQ-6-13-2)

[0248]

[0249] ZSQ-6-13-1 (1.6 g, 4.8 mmol) and 2 ml of trifluoroacetic acid were mixed in 10 ml of DCM and stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated and separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:5), and 1.04 g of colorless oily product was obtained with a yield of 93%.

[0250] Synthesis of compound benzyl(1s,4s)-4-(dimethylamino)cyclohexane-1-carboxylate(ZSQ-6-27)

[0251]

[0252] ZSQ-6-13-2 (933 mg, 4 mmol), paraformaldehyde (242 mg, 8 mmol), triacetoxyborane

[0253] Sodium hydride (1.69 g, 8 mmol) and acetic acid (0.96 ml, 16 mmol) were mixed and dissolved in 40 ml of 1,2-dichloroethane and stirred at room temperature for 48 h. After the reaction was completed, the mixture was filtered through diatomaceous earth and washed with methanol. The filtrate was concentrated and separated and purified by C18 reverse phase column chromatography and eluted with H2O / CH3CN (1:6) to obtain 972 mg of colorless oily product with a yield of 93%.

[0254] Synthesis of compound (1s,4s)-4-(dimethylamino)cyclohexane-1-carboxylic acid (ZSQ-6-31)

[0255]

[0256] ZSQ-6-27 (972 mg, 3.72 mmol) and palladium carbon (200 mg) were mixed in 20 ml methanol and stirred overnight at room temperature under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered through diatomaceous earth, washed with methanol, and the filtrate was concentrated and dried to obtain 870 mg of an oily product, which was directly used for the next step without further purification.

[0257] Synthesis of compound (1s,4s)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-4-(dimethylamino)cyclo-hexane-1-carboxamide(ZSQ-6-35)

[0258]

[0259] ZSQ-6-31 (342 mg, 2.0 mmol), ZSQ-1-23 (518 mg, 2.0 mmol), HATU (1.14 g, 3.0 mmol) and DIPEA (992 ul, 6.0 mmol) were dissolved in 4 ml DCM and stirred at room temperature for 6 h. After the reaction was completed, it was washed with H2O (20 ml×2) and saturated NaCl solution (20 ml) in sequence, extracted with 20 ml DCM, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, separated and purified by silica gel column chromatography, and eluted with DCM / CH3OH(NH3) (5:1) to obtain 486 mg of white solid with a yield of 59%.

[0260] Synthesis of compound (4-((1s,4s)-4-(dimethylamino)cyclohexane-1-carboxamido)phenyl)arsonous acid (ZSQ-6-52)

[0261]

[0262] ZSQ-6-35 (70 mg, 0.17 mmol) and mercuric perchlorate trihydrate (61 mg, 0.13 mmol) were dissolved in 2 ml DMSO, and the mixed solution was stirred at room temperature for 10 min, and immediately separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:3), and the crude product was purified by HPLC to obtain 52 mg of white solid, with a yield of 86%. M / Z: Exact Mass: 354.09, (LC-MS): Found 355.10.

[0263] Example 6

[0264] Synthesis route of compound ZSQ-6-37

[0265]

[0266] Synthesis of compound 3-benzyl 1-(tert-butyl)(R)-pyrrolidine-1,3-dicarboxylate(ZSQ-5-84)

[0267]

[0268] (R)-1-Boc-3-carboxypyrrolidine (1.07 g, 5.0 mmol) and potassium carbonate (2.07 g, 15 mmol) were dissolved in 20 ml of DMF. After stirring at room temperature for 20 min, benzyl bromide (653 ul, 5.5 mmol) was added dropwise. After the addition, stirring was continued at room temperature for 3 h. After the reaction was completed, the mixture was filtered with diatomaceous earth and washed with DCM. The filtrate was concentrated and separated and purified by C18 reverse phase column chromatography and eluted with H2O / CH3CN (1:7) to obtain 1.35 g of light yellow liquid with a total yield of 89%.

[0269] Synthesis of compound benzyl(R)-pyrrolidine-3-carboxylate(ZSQ-5-89)

[0270]

[0271] ZSQ-5-84 (1.35 g, 4.42 mmol) and 2 ml of trifluoroacetic acid were mixed in 10 ml of DCM and stirred at room temperature. After the reaction was completed, the reaction solution was concentrated and separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (4:1), and 843 mg of light yellow oily product was obtained, with a yield of 93%.

[0272] Synthesis of compound benzyl(R)-1-methylpyrrolidine-3-carboxylate(ZSQ-6-20)

[0273]

[0274] ZSQ-5-89 (410 mg, 2 mmol), paraformaldehyde (120 mg, 4 mmol), sodium triacetoxyborohydride (847 mg, 4 mmol), acetic acid (0.48 ml, 8 mmol) were mixed and dissolved in 10 ml 1,2-dichloroethane, and stirred at room temperature for 48 h. After the reaction was completed, it was filtered through diatomaceous earth, washed with methanol, and the filtrate was concentrated and separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (4:1), and 333 mg of colorless oily product was obtained with a yield of 76%.

[0275] Synthesis of compound (R)-1-methylpyrrolidine-3-carboxylic acid (ZSQ-6-25)

[0276]

[0277] ZSQ-6-20 (830 mg, 3.78 mmol) and palladium carbon (200 mg) were mixed in 15 ml of methanol and stirred overnight at room temperature under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered through diatomaceous earth, washed with methanol, and the filtrate was concentrated and dried to obtain 504 mg of a colorless oily product, which was directly carried out to the next step without further purification.

[0278] Synthesis of compound (R)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-1-methylpyrrolidine-3-carboxamide (ZSQ-6-28)

[0279]

[0280] ZSQ-6-25 (258 mg, 2.0 mmol), ZSQ-1-23 (518 mg, 2.0 mmol), HATU (1.14 g, 3.0 mmol) and DIPEA (992 ul, 6.0 mmol) were dissolved in 4 ml DCM and stirred at room temperature for 6 h. After the reaction was completed, it was washed with H2O (20 ml×2) and saturated NaCl solution (20 ml) in sequence, extracted with 20 ml DCM, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, separated and purified by silica gel column chromatography, and eluted with DCM / CH3OH(NH3) (4:1) to obtain 130 mg of yellow solid with a yield of 17%.

[0281] Synthesis of compound (R)-(4-(1-methylpyrrolidine-3-carboxamido)phenyl)arsonous acid (ZSQ-6-37)

[0282]

[0283] ZSQ-6-28 (45 mg, 0.12 mmol) and mercuric perchlorate trihydrate (44 mg, 0.10 mmol) were dissolved in 2 ml DMSO, and the mixed solution was stirred at room temperature for 10 min, and immediately separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:4), and the crude product was purified by HPLC to obtain 25 mg white powder, with a yield of 67%. M / Z: Exact Mass: 312.05, (LC-MS): Found 313.0.

[0284] Example 7

[0285] Synthesis route of compound ZSQ-7-37

[0286]

[0287] Synthesis of tert-butyl(R)-3-((4-(1,3,2-dithiarsolan-2-yl)phenyl)carbamoyl)pyro-lidine-1-carboxylate(ZSQ-6-57)

[0288]

[0289] (R)-1-Boc-3-carboxylpyrrolidine (645 mg, 3.0 mmol), ZSQ-1-23 (854 mg, 3.3 mmol), HATU (1.71 g, 4.5 mmol) and DIPEA (1.49 ml, 9.0 mmol) were dissolved in 10 ml DCM and stirred at room temperature for 3 h. After the reaction was completed, it was washed with H2O (40 ml × 2), saturated NaCl solution (40 ml), and extracted with 40 ml DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, separated and purified by silica gel column chromatography, and eluted with DCM / CH3OH(NH3) (20:1) to obtain 1.25 g of yellow solid with a yield of 91%.

[0290] Synthesis of tert-butyl(R)-3-((4-(1,3,2-dithiarsolan-2-yl)phenyl)(benzyl)carbamoyl)pyrolidine-1-carboxylate(ZSQ-6-97)

[0291]

[0292] Dissolve ZSQ-6-57 (456 mg, 1.0 mmol) and benzyl bromide (178 ul, 1.5 mmol) in 4 ml of ultra-dry DMF, place in an ice bath and stir for 10 min. Add NaH (60%, 1.2 mmol, 48 mg) to the mixed solution in batches, continue stirring for 3 h in an ice bath after addition, and then move to room temperature and stir overnight. After the reaction is completed, add water to quench, extract with EA (20 ml), then wash with 2N hydrochloric acid solution and saturated NaCl solution in turn, combine the organic phases, dry over anhydrous Na2SO4, filter and concentrate the filtrate, separate and purify by silica gel column chromatography, and elute with PE / EA (1:4) to obtain 151 mg of white solid with a yield of 27%.

[0293] Synthesis of compound (R)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-N-benzylpyrrolidine-3-carboxamide (ZSQ-6-102)

[0294]

[0295] ZSQ-6-97 (766 mg, 1.40 mmol) and 1 ml of trifluoroacetic acid were mixed in 5 ml of DCM and stirred at room temperature for 12 h. After the reaction was completed, the reaction solution was concentrated and separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:1), and 586 mg of white solid was obtained with a yield of 94%.

[0296] Synthesis of compound (R)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-N-benzyl-1-methylpyrrolidine-3-carboxamide (ZSQ-7-8)

[0297]

[0298] ZSQ-6-102 (130 mg, 0.29 mmol), paraformaldehyde (17 mg, 0.58 mmol), sodium triacetoxyborohydride (123 mg, 0.58 mmol), acetic acid (66 ul, 1.16 mmol) were mixed and dissolved in 2 ml of 1,2-dichloroethane, and stirred at room temperature for 48 hours. After the reaction was completed, it was directly concentrated and purified by silica gel column chromatography, eluted with DCM / CH3OH(NH3) (10:1), and 70 mg of white solid was obtained, with a yield of 52%.

[0299] Synthesis of compound (R)-(4-(N-benzyl-1-methylpyrrolidine-3-carboxamido)phenyl)arsonous acid (ZSQ-7-37)

[0300]

[0301] ZSQ-7-8 (70 mg, 0.15 mmol) and mercuric perchlorate trihydrate (55 mg, 0.12 mmol) were dissolved in 2 ml DMSO, and the mixed solution was stirred at room temperature for 10 min, and immediately separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (2:1), and the crude product was purified by HPLC to obtain 47 mg white powder, with a yield of 77%. M / Z: Exact Mass: 402.09, (LC-MS): Found 402.8.

[0302] Example 8

[0303] Synthesis route of compound ZSQ-7-86

[0304]

[0305] Synthesis of Compound 2-((4-(1,3,2-dithiarsolan-2-yl)phenyl)amino)-N,N-dimethylacetamide(ZSQ-6-81)

[0306]

[0307] ZSQ-1-23 (259 mg, 1.0 mmol), 2-bromo-N,N-dimethylacetamide (135 ul, 1.2 mmol) and

[0308] Potassium carbonate (276 mg, 2.0 mmol) was dissolved in 3 ml CH3CN and stirred at room temperature for 18 h. After the reaction was completed, it was washed with water (20 ml × 2) and extracted with EA (20 ml). The organic phase was dried over anhydrous Na2SO4, filtered and the filtrate was concentrated, separated and purified by silica gel column chromatography, eluted with DCM / EA (1:2), and a white powder of 244 mg was obtained with a yield of 71%.

[0309] Synthesis of compound (R)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-N-(2-(dimethylamino)-2-oxoethyl)-1-methylpyrrolidine-3-carboxamide (ZSQ-7-51)

[0310]

[0311] ZSQ-6-25 (230 mg, 1.7 mmol) and ZSQ-6-81 (400 mg, 1.2 mmol) were dissolved in 6 ml of dry pyridine, and the mixed solution was stirred for 10 min in an ice-salt bath. Phosphorus oxychloride (130 ul, 1.4 mmol) was added dropwise to the mixed solution (over 10 min), and continued to stir at 0°C for 3 h after the addition. After the reaction was completed, it was directly separated and purified by silica gel column chromatography, first eluted with PE / EA (0-70%) to flush out pyridine, and then eluted with DCM / CH3OH (NH3) (4:1) to obtain a crude product, which was separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (2:1), and 131 mg of white solid was obtained, with a yield of 24%.

[0312] Synthesis of compound (R)-(4-(N-(2-(dimethylamino)-2-oxoethyl)-1-methylpyrrolidine-3-carboxamido)phenyl)arsonous acid (ZSQ-7-86)

[0313]

[0314] ZSQ-7-51 (23 mg, 0.05 mmol) and mercuric perchlorate trihydrate (18 mg, 0.04 mmol) were dissolved in 2 ml DMSO, and the mixed solution was stirred at room temperature for 10 min, and immediately separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (4:1), and the crude product was purified by HPLC to obtain 11 mg of white powder, with a yield of 55%. M / Z: Exact Mass: 397.10, (LC-MS): Found 397.8.

[0315] Example 9

[0316] Synthesis route of compound ZSQ-7-106

[0317]

[0318] Synthesis of compound (R)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-N,1-dibenzylpyrrolidine-3-carboxamide (ZSQ-7-102)

[0319]

[0320] ZSQ-6-102 (89 mg, 0.2 mmol) and potassium carbonate (55 mg, 0.4 mmol) were dissolved in 2 ml CH3CN, stirred in an ice-salt bath for 20 min, and then benzyl bromide (24 ul, 0.2 mmol) was added dropwise (over 10 min), and continued to stir in an ice-salt bath for 2 h. After the reaction was completed, the reaction solution was concentrated and directly separated and purified by silica gel column chromatography, eluted with DCM / CH3OH(NH3) (10:1), and 21 mg of white solid was obtained, with a yield of 20%.

[0321] Synthesis of compound (R)-(4-(N,1-dibenzylpyrrolidine-3-carboxamido)phenyl)arsonousacid(ZSQ-7-106)

[0322]

[0323] ZSQ-7-102 (21 mg, 0.04 mmol) and mercuric perchlorate trihydrate (14 mg, 0.03 mmol) were dissolved in 1 ml of DMSO, and the mixed solution was stirred at room temperature for 10 min, and immediately separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (2:1), and the crude product was purified by HPLC to obtain 9 mg of white powder, with a yield of 48%. M / Z: Exact Mass: 478.12, (LC-MS): Found 478.9.

[0324] Example 10

[0325] Synthesis route of compound ZSQ-8-50

[0326]

[0327] Synthesis of compound 2-bromo-1-morpholinoethan-1-one (ZSQ-8-38)

[0328]

[0329] Bromoacetyl bromide (174ul, 2mmol) was dissolved in 15ml of ultra-dry DCM, placed in an ice-salt bath and stirred, morpholine (348ul, 4mmol) was added dropwise to the mixed solution, and continued to stir at 0°C for 30min after the addition, and then moved to room temperature and stirred for 1h. After the reaction was completed, it was washed with saturated ammonium chloride solution (30ml), extracted twice with ether (30ml), the organic phase was dried with anhydrous sodium sulfate, and the filtrate was concentrated and drained to obtain 387mg of yellow oily liquid, with a yield of 94%.

[0330] Synthesis of compound (R)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-N-benzyl-1-(2-morpholino-2-oxoethyl)pyrrolidine-3-carboxamide (ZSQ-8-41)

[0331]

[0332] ZSQ-6-102 (53 mg, 0.12 mmol), ZSQ-8-38 (27 mg, 0.13 mmol) and potassium carbonate (33 mg, 0.24 mmol) were dissolved in 2 ml CH3CN and stirred in an ice-salt bath for 2 h. After the reaction was completed, the reaction solution was concentrated and directly separated and purified by silica gel column chromatography, eluted with DCM / CH3OH(NH3) (8:1), and 13.3 mg of white solid was obtained, with a yield of 19%.

[0333] Synthesis of compound (R)-(4-(N-benzyl-1-(2-morpholino-2-oxoethyl)pyrrolidine-3-carboxamido)phenyl)arsonous acid (ZSQ-8-50)

[0334]

[0335] ZSQ-8-41 (67 mg, 0.12 mmol) and mercuric perchlorate trihydrate (42 mg, 0.09 mmol) were dissolved in 2 ml DMSO, and the mixed solution was stirred at room temperature for 10 min, and immediately separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (2:1), and the crude product was purified by HPLC to obtain 42 mg white powder, with a yield of 70%. M / Z: Exact Mass: 515.14, (LC-MS): Found 515.9.

[0336] Embodiment 11

[0337] Synthesis route of compound ZSQ-12-66

[0338]

[0339] Synthesis of compound benzyl(R)-1-(2-(dimethylamino)-2-oxoethyl)pyrrolidine-3-carboxylate(ZSQ-12-41)

[0340]

[0341] ZSQ-5-89 (3.19 g, 10 mmol), 2-bromo-N,N-dimethylacetamide (1.66 g, 10 mmol) and potassium carbonate (4.15 g, 30 mmol) were dissolved in 50 ml CH3CN, stirred in an ice-salt bath for 3 h, and then moved to room temperature and stirred overnight. After the reaction was completed, it was filtered through diatomaceous earth, rinsed with DCM, and the filtrate was concentrated and dried to obtain 4.8 g of yellow oily crude product, which was used directly in the next step without further purification.

[0342] Synthesis of compound (R)-1-(2-(dimethylamino)-2-oxoethyl)pyrrolidine-3-carboxylic acid (ZSQ-12-58)

[0343]

[0344] ZSQ-12-41 (1.0 g, 3.44 mmol) and palladium carbon (200 mg) were mixed in 15 ml of methanol and stirred overnight at room temperature under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered through diatomaceous earth, washed with methanol, and the filtrate was concentrated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (9:1), and 602 mg of colorless oily product was obtained with a yield of 87%.

[0345] Synthesis of tert-butyl 3-(((4-(1,3,2-dithiarsolan-2-yl)phenyl)amino)methyl)pyro-lidine-1-carboxylate (ZSQ-11-38)

[0346]

[0347] ZSQ-1-23 (1.03 g, 4 mmol), 1-Boc-3-pyrrolidine carboxaldehyde (1.59 g, 8 mmol), sodium triacetoxyborohydride (1.69 g, 8 mmol), acetic acid (0.96 ml, 16 mmol) were mixed and dissolved in 30 ml 1,2-dichloroethane, and stirred at room temperature for 48 h. After the reaction was completed, it was washed with saturated sodium bicarbonate solution (50 ml), H2O (50 ml), saturated sodium chloride solution (50 ml), and extracted with DCM (30 ml). The organic phases were combined and dried over anhydrous sodium sulfate. After the filtrate was concentrated, it was directly separated and purified by silica gel column chromatography, eluted with PE / EA (4:1). The crude product was further separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:8), and 1.0 g of light yellow solid was obtained with a yield of 57%.

[0348] Synthesis of compound tert-butyl 3-(((R)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-1-(2-(dimethyl-amino)-2-oxoethyl)pyrrolidine-3-carboxamido)methyl)pyrrolidine-1-carboxylate(ZSQ-12-61-1)

[0349]

[0350] ZSQ-11-38 (442 mg, 1.0 mmol) and ZSQ-12-58 (200 mg, 1.0 mmol) were dissolved in 5 ml of dry pyridine, and the mixed solution was stirred for 10 min in an ice-salt bath. Phosphorus oxychloride (140 ul, 1.5 mmol) was added dropwise to the mixed solution (over 10 min), and continued to stir at 0°C for 3 h after the addition. After the reaction was completed, it was directly separated and purified by silica gel column chromatography, first eluted with PE / EA (0-70%) to flush out pyridine, and then eluted with DCM / CH3OH (NH3) (4:1) to obtain a crude product, which was separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:6), and 223 mg of white solid was obtained, with a yield of 35.7%.

[0351] Synthesis of compound (3R)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-1-(2-(dimethylamino)-2-oxoethyl)-N-(pyrrolidin-3-ylmethyl)pyrrolidine-3-carboxamide (ZSQ-12-61-2)

[0352]

[0353] ZSQ-12-61-1 (233 mg, 0.37 mmol) and 0.5 ml trifluoroacetic acid were mixed in 4 ml DCM and stirred at room temperature for 12 h. After the reaction was completed, the reaction solution was concentrated and separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (2:1), and 190 mg of white solid was obtained with a yield of 97%.

[0354] Synthesis of compound (3R)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-N-((1-acetylpyrrolidin-3-yl)methyl)-1-(2-(dimethylamino)-2-oxoethyl)pyrrolidine-3-carboxamide (ZSQ-12-64)

[0355]

[0356] ZSQ-12-61-2 (105 mg, 0.2 mmol) and DIPEA (0.1 ml, 0.6 mmol) were mixed in 3 ml DCM, and acetic anhydride (28 ul, 0.3 mmol) was added dropwise and stirred at room temperature for 3 h. After the reaction was completed, the reaction solution was concentrated and separated and purified by silica gel column chromatography, eluted with DCM / CH3OH(NH3) (9:1), and 59.4 mg of yellow solid was obtained, with a yield of 52%.

[0357] Synthesis of compound (4-((3R)-N-((1-acetylpyrrolidin-3-yl)methyl)-1-(2-(dimethylamino)-2-oxoethyl)pyrrolidine-3-carboxamido)phenyl)arsonous acid (ZSQ-12-66)

[0358]

[0359] ZSQ-12-64 (59 mg, 0.10 mmol) and mercuric perchlorate trihydrate (38 mg, 0.08 mmol) were dissolved in 2 ml DMSO, and the mixed solution was stirred at room temperature for 10 min, and immediately separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (2:1), and the crude product was purified by HPLC to obtain 39 mg white powder, with a yield of 77%. M / Z: Exact Mass: 508.17, (LC-MS): Found 509.10.

[0360] Example 12

[0361] Synthesis route of compound ZSQ-13-100

[0362] Synthesis of compound benzyl(R)-1-benzylpyrrolidine-3-carboxylate(ZSQ-13-52)

[0363]

[0364] Dissolve ZSQ-5-89 (2.14 g, 6.7 mmol) and potassium carbonate (2.31 g, 16.75 mmol) in 60 ml CH3CN, place in an ice-salt bath and mix and stir, dissolve benzyl bromide (796 ul, 6.7 mmol) in 7 ml CH3CN and add dropwise to the mixed solution, stir in an ice-salt bath for 3 h, then move to room temperature and stir for 3 h. After the reaction is completed, filter with diatomaceous earth, rinse with DCM, and concentrate the filtrate to dryness to obtain a yellow oily crude product, which is used directly in the next step without further purification.

[0365] Synthesis of compound (R)-1-benzylpyrrolidine-3-carboxylic acid (ZSQ-13-54)

[0366]

[0367] Compound ZSQ-13-52 (6.7 mmol) was dissolved in a mixed solution of 5 ml THF and 5 ml H2O, and lithium hydroxide monohydrate (20.1 mmol, 843 mg) was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, 2N hydrochloric acid was added to acidify, and THF was removed by concentration. The mixture was separated and purified by C18 reverse phase column chromatography, and eluted with H2O / CH3CN (9:1) to obtain 323 mg of a light yellow oily product with a combined yield of 23%.

[0368] Synthesis of tert-butyl 4-(((4-(1,3,2-dithiarsolan-2-yl)phenyl)amino)methyl)piperidine-1-carboxylate (ZSQ-13-48)

[0369]

[0370] ZSQ-1-23 (4.3 g, 16.6 mmol), 1-tert-butyloxycarbonyl-4-piperidinecarboxaldehyde (7.0 g, 33.2 mmol), sodium triacetoxyborohydride (7.0 g, 33.2 mmol), acetic acid (3.0 ml, 49.8 mmol) were mixed and dissolved in 100 ml 1,2-dichloroethane, and stirred at room temperature for 48 h. After the reaction was completed, the pH was adjusted to alkaline with saturated sodium bicarbonate solution (50 ml), washed with H2O (100 ml), saturated sodium chloride solution (100 ml), extracted with DCM (100 ml), the organic phases were combined, dried with anhydrous sodium sulfate, and the filtrate was concentrated and directly separated and purified by silica gel column chromatography, eluted with PE / EA (4:1), and the crude product was further separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:9), and 3.0 g of light yellow powder was obtained with a yield of 40%.

[0371] Synthesis of tert-butyl(R)-4-((N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-1-benzylpyrro-lidine-3-carboxamido)methyl)piperidine-1-carboxylate(ZSQ-13-59)

[0372]

[0373] ZSQ-13-48 (790 mg, 1.73 mmol) and ZSQ-13-54 (323 mg, 1.57 mmol) were dissolved in 5 ml of dry pyridine, and the mixed solution was stirred for 10 min in an ice-salt bath. Phosphorus oxychloride (292 ul, 2.0 mmol) was added dropwise to the mixed solution (over 10 min), and continued to stir at 0°C for 3 h after the addition. After the reaction was completed, it was directly separated and purified by silica gel column chromatography, first eluted with PE / EA (0-70%) to flush out pyridine, and then eluted with DCM / CH3OH (NH3) (9:1) to obtain a crude product, which was separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:6), and 397 mg of yellow solid was obtained, with a yield of 39%.

[0374] Synthesis of compound (R)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-1-benzyl-N-(piperidin-4-ylmethyl)pyrrolidine-3-carboxamide (ZSQ-13-62)

[0375]

[0376] ZSQ-13-59 (397 mg, 0.62 mmol) and 0.5 ml trifluoroacetic acid were mixed in 5 ml DCM and stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated and separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (5:2), and 308 mg of yellow solid was obtained, with a yield of 91%.

[0377] Synthesis of compound (R)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-1-benzyl-N-((1-ethylpiperidin-4-yl)methyl)pyrrolidine-3-carboxamide (ZSQ-13-83)

[0378]

[0379] ZSQ-13-62 (81 mg, 0.15 mmol), iodoethane (11 ul, 0.135 mmol) and potassium carbonate (62 mg, 0.45 mmol) were dissolved in 3 ml CH3CN, stirred in an ice-salt bath for 3 h, and then moved to room temperature and stirred for 1 h. After the reaction was completed, the reaction solution was concentrated and directly separated and purified by silica gel column chromatography, eluted with DCM / CH3OH(NH3) (4:1), and 21.6 mg of yellow solid was obtained, with a yield of 25%.

[0380] Synthesis of compound (R)-(4-(1-benzyl-N-((1-ethylpiperidin-4-yl)methyl)pyrrolidine-3-carboxamido)phenyl)arsonous acid (ZSQ-13-100)

[0381]

[0382] ZSQ-13-83 (35 mg, 0.061 mmol) and mercuric perchlorate trihydrate (22 mg, 0.049 mmol) were dissolved in 2 ml DMSO, and the mixed solution was stirred at room temperature for 10 min, and immediately separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (2:1), and the crude product was purified by HPLC to obtain 18 mg white powder, with a yield of 58%. M / Z: Exact Mass: 513.20, (LC-MS): Found 514.50.

[0383] Example 13

[0384] Synthesis route of compound ZSQ-17-79

[0385]

[0386] Synthesis of compound ethyl(E)-2-(hydroxyimino)-2-(2-phenylacetamido)acetate(ZSQ-16-18)

[0387]

[0388] Phenylacetic acid (681 mg, 5.0 mmol), ethyl 2-hydroxyamino-2-iminoacetate (694 mg, 5.25 mmol), HATU (2.85 g, 7.5 mmol) and DIPEA (2.48 ml, 15.0 mmol) were dissolved in 50 ml DCM and stirred at room temperature for 6 h. After the reaction was completed, the mixture was washed with H2O (50 ml×2) and saturated NaCl solution (50 ml), extracted with DCM (50 ml), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, separated and purified by silica gel column chromatography, and eluted with PE / EA (1:4) to obtain 1.18 g of white solid with a yield of 94%.

[0389] Synthesis of compound ethyl 5-benzyl-1,2,4-oxadiazole-3-carboxylate (ZSQ-16-22)

[0390]

[0391] Dissolve ZSQ-16-18 (500 mg, 2.0 mmol) in 5 ml of dry DMF, heat to 140°C and stir for 4 h. After the reaction is completed, separate and purify it by C18 reverse phase column chromatography, eluting with H2O / CH3CN (1:2) to obtain 399 mg of yellow oily product with a yield of 86%.

[0392] Synthesis of compound 5-benzyl-1,2,4-oxadiazole-3-carboxylic acid (ZSQ-16-33)

[0393]

[0394] Compound ZSQ-16-22 (399 mg, 1.71 mmol) was dissolved in a mixed solution of 4 ml THF and 4 ml H2O, and lithium hydroxide monohydrate (5.15 mmol, 216 mg) was added, and the mixture was stirred at room temperature for 4 h. After the reaction was completed, 2N hydrochloric acid was added to acidify the mixture, and the THF was concentrated to remove the THF. The mixture was separated and purified by C18 reverse phase column chromatography, and eluted with H2O / CH3CN (4:1) to obtain 286 mg of a white solid with a combined yield of 82%.

[0395] Synthesis of compound tert-butyl 4-((N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-5-benzyl-1,2,4-oxadiazole-3-carboxamido)methyl)piperidine-1-carboxylate (ZSQ-17-51)

[0396]

[0397] ZSQ-13-48 (456 mg, 1.0 mmol) and ZSQ-16-33 (204 mg, 1.0 mmol) were dissolved in 5 ml of dry pyridine, and the mixed solution was stirred for 10 min in an ice-salt bath. Phosphorus oxychloride (140 ul, 1.5 mmol) was added dropwise to the mixed solution (over 10 min), and continued to stir at 0°C for 3 h after the addition. After the reaction was completed, it was directly separated and purified by silica gel column chromatography, first eluted with PE / EA (0-70%) to flush out pyridine, and then eluted with DCM / CH3OH (NH3) (9:1) to obtain a crude product, which was separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:3), and 274 mg of yellow solid was obtained, with a yield of 43%.

[0398] Synthesis of Compound N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-5-benzyl-N-(piperidin-4-ylmethyl)-1,2,4-oxadiazole-3-carboxamide (ZSQ-17-72)

[0399]

[0400] ZSQ-17-51 (274 mg, 0.43 mmol) and 0.5 ml trifluoroacetic acid were mixed in 5 ml DCM and stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated and separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:1), and 229 mg of yellow solid was obtained, with a yield of 98%.

[0401] Synthesis of Compound N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-5-benzyl-N-((1-ethylpiperidin-4-yl)methyl)-1,2,4-oxadiazole-3-carboxamide (ZSQ-17-75)

[0402]

[0403] ZSQ-17-72 (229 mg, 0.42 mmol), iodoethane (34 ul, 0.42 mmol) and potassium carbonate (174 mg, 1.26 mmol) were dissolved in 2 ml CH3CN, stirred in an ice-salt bath for 3 h, and then moved to room temperature and stirred for 1 h. After the reaction was completed, the reaction solution was concentrated and directly separated and purified by silica gel column chromatography, eluted with DCM / CH3OH(NH3) (9:1), and 107 mg of yellow solid was obtained, with a yield of 45%.

[0404] Synthesis of compound (4-(5-benzyl-N-((1-ethylpiperidin-4-yl)methyl)-1,2,4-oxadiazole-3-carboxamido)phenyl)arsonous acid (ZSQ-17-79)

[0405]

[0406] ZSQ-17-75 (57 mg, 0.10 mmol) and mercuric sulfate (60 mg, 0.20 mmol) were dissolved in 2 ml DMSO, the mixed solution was heated to 60°C and stirred for 30 min, and immediately separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (2:5), and the crude product was purified by HPLC to obtain 33 mg white powder, with a yield of 64%. M / Z: Exact Mass: 512.14, (LC-MS): Found 513.0.

[0407] Embodiment 14

[0408] Synthesis route of compound ZSQ-17-80

[0409]

[0410] Synthesis of compound ethyl 5-benzyl-1-methyl-1H-pyrazole-3-carboxylate (ZSQ-17-66)

[0411]

[0412] Dissolve N-methyl-L-phenylalanine (1.79g, 10mmol) and sodium nitrite (759mg, 11mmol) in a mixed solution of DCM:H2O (30ml:30ml), place in an ice bath and stir evenly. Slowly add 1ml of 12N hydrochloric acid to the mixed solution, and continue stirring at 0℃ for 2h after the addition. After the reaction is completed, wash three times with H2O (50ml) and extract with DCM (50ml). The combined organic phases are dried over anhydrous Na2SO4, filtered, and the filtrate is concentrated and dried to obtain a white solid. The white solid is then dissolved in acetic anhydride (20ml), ethyl propiolate (1.5ml, 15mmol) is added, and the mixed solution is placed at 120℃ and refluxed with stirring for 12h. After the reaction is completed, wash three times with water (50ml) and extract with EA. The organic phase is dried over anhydrous Na2SO4, filtered, and the filtrate is concentrated

[0413] The residue was separated and purified by silica gel column chromatography using PE / EA (1:2) as eluent to obtain 1.03 g of a yellow oily product with a yield of 42%.

[0414] Synthesis of compound 5-benzyl-1-methyl-1H-pyrazole-3-carboxylic acid (ZSQ-17-68)

[0415]

[0416] Compound ZSQ-17-66 (366 mg, 1.5 mmol) was dissolved in a mixed solution of 2 ml THF and 2 ml H2O, and lithium hydroxide monohydrate (126 mg, 3.0 mmol) was added, and the mixture was stirred at room temperature for 4 h. After the reaction was completed, 2N hydrochloric acid was added to acidify, and THF was removed by concentration. The mixture was separated and purified by C18 reverse phase column chromatography, and eluted with H2O / CH3CN (4:1) to obtain 290 mg of a white solid with a combined yield of 89%.

[0417] Synthesis of the compound tert-butyl 4-((N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-5-benzyl-1-methyl-1H-pyrazole-3-carboxamido)methyl)piperidine-1-carboxylate (ZSQ-17-44)

[0418]

[0419] ZSQ-13-48 (456 mg, 1.0 mmol) and ZSQ-17-68 (216 mg, 1.0 mmol) were dissolved in 5 ml of dry pyridine, and the mixed solution was stirred for 10 min in an ice-salt bath. Phosphorus oxychloride (140 ul, 1.5 mmol) was added dropwise to the mixed solution (over 10 min), and continued to stir at 0°C for 3 h after the addition. After the reaction was completed, it was directly separated and purified by silica gel column chromatography, first eluted with PE / EA (0-70%) to flush out pyridine, and then eluted with DCM / CH3OH (NH3) (9:1) to obtain a crude product, which was separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:3), and 168 mg of yellow solid was obtained, with a yield of 26%.

[0420] Synthesis of Compound N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-5-benzyl-1-methyl-N-(piperidin-4-ylmethyl)-1H-pyrazole-3-carboxamide (ZSQ-17-71)

[0421]

[0422] ZSQ-17-44 (168 mg, 0.26 mmol) and 0.5 ml trifluoroacetic acid were mixed in 5 ml DCM and stirred at room temperature for 12 h. After the reaction was completed, the reaction solution was concentrated and separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:1), and 137 mg of yellow solid was obtained with a yield of 96%.

[0423] Synthesis of compound N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-5-benzyl-N-((1-ethylpiperidin-4-yl)methyl)-1-methyl-1H-pyrazole-3-carboxamide (ZSQ-17-74)

[0424]

[0425] ZSQ-17-72 (137 mg, 0.25 mmol), iodoethane (20 ul, 0.25 mmol) and potassium carbonate (102 mg, 0.75 mmol) were dissolved in 2 ml CH3CN, stirred in an ice-salt bath for 3 h, and then moved to room temperature and stirred for 1 h. After the reaction was completed, the reaction solution was concentrated and directly separated and purified by silica gel column chromatography, eluted with DCM / CH3OH(NH3) (9:1), and 52 mg of yellow solid was obtained, with a yield of 36%.

[0426] Synthesis of compound (4-(5-benzyl-N-((1-ethylpiperidin-4-yl)methyl)-1-methyl-1H-pyrazole-3-carboxamido)phenyl)arsonous acid (ZSQ-17-80)

[0427]

[0428] ZSQ-17-74 (29 mg, 0.05 mmol) and mercuric sulfate (30 mg, 0.10 mmol) were dissolved in 2 ml DMSO, the mixed solution was heated to 60°C and stirred for 30 min, and immediately separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (2:5), and the crude product was purified by HPLC to obtain 13 mg of white powder, with a yield of 50%. M / Z: Exact Mass: 524.18, (LC-MS): Found 525.0.

[0429] Embodiment 15

[0430] Synthesis route of compound ZSQ-17-81

[0431]

[0432] Synthesis of Compound N-methoxy-N-methyl-2-phenylacetamide (ZSQ-16-81)

[0433]

[0434] Phenylacetic acid (1.36 g, 10 mmol), dimethylhydroxylamine hydrochloride (1.17 g, 12 mmol), HATU (4.56 g, 12 mmol) and DIPEA (4.96 ml, 30 mmol) were dissolved in 50 ml DCM and stirred at room temperature for 6 h. After the reaction was completed, it was washed with dilute hydrochloric acid solution (50 ml), H2O (50 ml), and saturated NaCl solution (50 ml) in sequence, and extracted with 50 ml DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, separated and purified by silica gel column chromatography, and eluted with PE / EA (2:5) to obtain 1.7 g of colorless liquid with a yield of 95%.

[0435] Synthesis of Compound 1-phenylpropan-2-one (ZSQ-16-89)

[0436]

[0437] Dissolve ZSQ-16-81 (1.7 g, 9.48 mmol) in 30 ml of dry THF and stir evenly in an ice bath. Add methylmagnesium bromide (1M tetrahydrofuran solution, 14.22 ml, 14.22 mmol) dropwise to the mixed solution at 0°C within 30 min. After the addition, continue stirring at 0°C for 1 h and then stir at room temperature for 12 h. After the reaction is completed, quench with 2N hydrochloric acid solution (50 ml), remove THF by vacuum distillation, extract with EA, and wash twice with water (50 ml). Combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate the filtrate, separate and purify it by silica gel column chromatography, and elute with PE / EA (2:1) to obtain 1.15 g of colorless liquid with a yield of 90%.

[0438] Synthesis of compound ethyl 2,4-dioxo-5-phenylpentanoate (ZSQ-16-97)

[0439]

[0440] ZSQ-16-89 (670 mg, 5.0 mmol) and diethyl oxalate (815 ul, 6.0 mmol) were dissolved in 10 ml of ultra-dry ethanol and stirred evenly in an ice bath. 20% sodium ethoxide solution (1.87 g, 5.5 mmol) was added dropwise to the mixed solution. After the addition, the mixture was stirred for 1 h in an ice bath and then moved to room temperature and stirred for 24 h. After the reaction was completed, 2N hydrochloric acid solution was quenched (20 ml), extracted with EA (50 ml), and then washed with water (30 ml) and saturated sodium chloride solution (30 ml) in turn. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography, eluted with PE / EA (2:1) to obtain 870 mg of a yellow oily product with a yield of 74%.

[0441] Synthesis of compound ethyl 5-benzylisoxazole-3-carboxylate (ZSQ-17-64)

[0442]

[0443] ZSQ-16-97 (468 mg, 2 mmol) and hydroxylamine hydrochloride (210 mg, 3 mmol) were mixed and dissolved in 7 ml of ultra-dry ethanol, heated to 90 ° C and stirred for 12 h. After the reaction was completed, the reaction solution was concentrated, washed with water (20 ml), saturated sodium chloride solution (20 ml), extracted with EA (40 ml), and the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated and purified by silica gel column chromatography, eluted with PE / EA (2:5), and 302 mg of light yellow oily product was obtained with a yield of 65%.

[0444] Synthesis of compound 5-benzylisoxazole-3-carboxylic acid (ZSQ-17-46)

[0445]

[0446] Compound ZSQ-17-64 (231 mg, 1.0 mmol) was dissolved in a mixed solution of 2 ml THF and 2 ml H2O, and lithium hydroxide monohydrate (126 mg, 3.0 mmol) was added, and the mixture was stirred at room temperature for 4 h. After the reaction was completed, a small amount of dilute hydrochloric acid was added to acidify, and THF was removed by concentration. The mixture was separated and purified by C18 reverse phase column chromatography, and eluted with H2O / CH3CN (4:1) to obtain 200 mg of a white solid with a combined yield of 98%.

[0447] Synthesis of tert-butyl 4-((N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-5-benzylisoxazole-3-carboxamido)methyl)piperidine-1-carboxylate (ZSQ-17-49)

[0448]

[0449] ZSQ-13-48 (456 mg, 1.0 mmol) and ZSQ-17-46 (203 mg, 1.0 mmol) were dissolved in 5 ml of dry pyridine, and the mixed solution was stirred for 10 min in an ice-salt bath. Phosphorus oxychloride (140 ul, 1.5 mmol) was added dropwise to the mixed solution (over 10 min), and continued to stir at 0°C for 3 h after the addition. After the reaction was completed, it was directly separated and purified by silica gel column chromatography, first eluted with PE / EA (0-70%) to flush out pyridine, and then eluted with DCM / CH3OH (NH3) (9:1) to obtain a crude product, which was separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:3), and 432 mg of yellow solid was obtained, with a yield of 67%.

[0450] Synthesis of Compound N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-5-benzyl-N-(piperidin-4-ylmethyl)isoxazole-3-carboxamide (ZSQ-17-67)

[0451]

[0452] ZSQ-17-49 (432 mg, 0.66 mmol) and 1 ml of trifluoroacetic acid were mixed in 6 ml of DCM and stirred at room temperature for 12 h. After the reaction was completed, the reaction solution was concentrated and separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:1), and 336 mg of yellow viscous product was obtained with a yield of 94%.

[0453] Synthesis of Compound N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-5-benzyl-N-((1-ethylpiperidin-4-yl)methyl)isoxazole-3-carboxamide (ZSQ-17-69)

[0454]

[0455] ZSQ-17-67 (200 mg, 0.369 mmol), iodoethane (30 ul, 0.369 mmol) and potassium carbonate (153 mg, 1.107 mmol) were dissolved in 3 ml CH3CN, stirred in an ice-salt bath for 3 h, and then moved to room temperature and stirred for 1 h. After the reaction was completed, the reaction solution was concentrated and directly separated and purified by silica gel column chromatography, eluted with DCM / CH3OH(NH3) (9:1), and 76 mg of yellow solid was obtained, with a yield of 36%.

[0456] Synthesis of compound (4-(5-benzyl-N-((1-ethylpiperidin-4-yl)methyl)isoxazole-3-carboxamido)phenyl)arsonous acid (ZSQ-17-81)

[0457]

[0458] ZSQ-17-69 (48 mg, 0.084 mmol) and mercuric sulfate (50 mg, 0.168 mmol) were dissolved in 2 ml DMSO, the mixed solution was heated to 60°C and stirred for 30 min, and immediately separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (2:5), and the crude product was purified by HPLC to obtain 23 mg white powder, with a yield of 53%. M / Z: Exact Mass: 511.15, (LC-MS): Found 512.0.

[0459] Example 16

[0460] Synthesis route of compound ZSQ-17-87

[0461]

[0462] Synthesis of compound ethyl 2H-tetrazole-5-carboxylate (ZSQ-13-16)

[0463]

[0464] Dissolve ethyl cyanoformate (1.0 ml, 10.0 mmol) in 12 ml of ultra-dry pyridine and stir evenly in an ice bath. Add trifluoroacetic acid (0.74 ml, 10 mmol) dropwise, stir for 10 min, then add sodium azide (702 mg, 10.8 mmol), and stir the mixed solution at 60 ° C for 48 h. After the reaction is completed, concentrate by vacuum distillation and use it directly in the next step without purification.

[0465] Synthesis of compound ethyl 2-benzyl-2H-tetrazole-5-carboxylate (ZSQ-13-24)

[0466]

[0467] ZSQ-13-16 (284 mg, 2 mmol), benzyl bromide (237 ul, 2 mmol) and potassium carbonate (415 mg, 6 mmol) were dissolved in 6 ml DMF and stirred at room temperature for 18 h. After the reaction was completed, the mixture was filtered with diatomaceous earth, rinsed with DCM, and the filtrate was concentrated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (2:1), and 86 mg of brown solid was obtained with a yield of 18%.

[0468] Synthesis of compound 2-benzyl-2H-tetrazole-5-carboxylic acid (ZSQ-17-45)

[0469]

[0470] Compound ZSQ-13-24 (116 mg, 0.5 mmol) was dissolved in a mixed solution of 2 ml THF and 2 ml H2O, and lithium hydroxide monohydrate (63 mg, 1.5 mmol) was added, and the mixture was stirred at room temperature for 4 h. After the reaction was completed, a small amount of dilute hydrochloric acid was added to acidify, and THF was removed by concentration. The mixture was separated and purified by C18 reverse phase column chromatography, and eluted with H2O / CH3CN (4:1) to obtain 79 mg of a white solid with a combined yield of 77%.

[0471] Synthesis of compound tert-butyl 4-((N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-2-benzyl-2H-tetrazole-5-carboxamido)methyl)piperidine-1-carboxylate (ZSQ-17-48)

[0472]

[0473] ZSQ-13-48 (228 mg, 0.5 mmol) and ZSQ-17-45 (102 mg, 0.5 mmol) were dissolved in 4 ml of dry pyridine, and the mixed solution was stirred for 10 min in an ice-salt bath. Phosphorus oxychloride (70 ul, 0.75 mmol) was added dropwise to the mixed solution (over 10 min), and continued to stir at 0°C for 3 h after the addition. After the reaction was completed, it was directly separated and purified by silica gel column chromatography, first eluted with PE / EA (0-70%) to flush out pyridine, and then eluted with DCM / CH3OH (NH3) (9:1) to obtain a crude product, which was separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:3), and 136 mg of yellow solid was obtained, with a yield of 42%.

[0474] Synthesis of Compound N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-2-benzyl-N-(piperidin-4-ylmethyl)-2H-tetrazole-5-carboxamide(ZSQ-17-85)

[0475]

[0476] ZSQ-17-48 (136 mg, 0.21 mmol) and 0.5 ml trifluoroacetic acid were mixed in 4 ml DCM and stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated and separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:1), and 100 mg of yellow solid product was obtained with a yield of 88%.

[0477] Synthesis of Compound N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-2-benzyl-N-((1-ethylpiperidin-4-yl)methyl)-2H-tetrazole-5-carboxamide(ZSQ-17-86)

[0478]

[0479] ZSQ-17-85 (100 mg, 0.18 mmol), iodoethane (15 ul, 0.18 mmol) and potassium carbonate (75 mg, 0.54 mmol) were dissolved in 2 ml CH3CN, stirred in an ice-salt bath for 3 h, and then moved to room temperature and stirred for 1 h. After the reaction was completed, the reaction solution was concentrated and directly separated and purified by silica gel column chromatography, eluted with DCM / CH3OH(NH3) (7:1), and 46 mg of yellow solid was obtained, with a yield of 45%.

[0480] Synthesis of compound (4-(2-benzyl-N-((1-ethylpiperidin-4-yl)methyl)-2H-tetrazole-5-carbox-amido)phenyl)arsonous acid (ZSQ-17-87)

[0481]

[0482] ZSQ-17-86 (20 mg, 0.035 mmol) and mercuric sulfate (21 mg, 0.070 mmol) were dissolved in 2 ml DMSO, the mixed solution was heated to 60°C and stirred for 30 min, and immediately separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (2:5), and the crude product was purified by HPLC to obtain 8.6 mg white powder, with a yield of 48%. M / Z: Exact Mass: 512.15, (LC-MS): Found 513.0.

[0483] Embodiment 17

[0484] Synthesis route of compound ZSQ-13-37

[0485]

[0486] Synthesis of Compound 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate(ZSQ-10-70)

[0487]

[0488] Tetraethylene glycol (50g, 262mmol) was dissolved in 60ml dry DCM, placed in an ice bath and stirred evenly, and triethylamine (5.46ml, 39.3mmol) was added. 4-Toluenesulfonyl chloride (5.0g, 26.2mmol) was dissolved in 20ml dry DCM, added dropwise to the above mixed solution under an ice bath, and stirred at room temperature for 18h after addition. After the reaction was completed, 2N hydrochloric acid (100ml), water (100ml), saturated sodium chloride (100ml) were used to wash, and DCM (50ml) was used to extract, the organic phases were combined and dried over anhydrous sodium sulfate, and the filtrate was spin-dried and separated and purified by silica gel column chromatography, eluted with PE / EA (1:9), and 8.35g of light yellow oily product was obtained, with a yield of 91%.

[0489] Synthesis of Compound 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-ol(ZSQ-12-91)

[0490]

[0491] Dissolve ZSQ-10-70 (7.21 g, 20.7 mmol) in 70 ml DMF, carefully add sodium azide (1.6 g, 24.8 mmol), and stir the mixed solution at 60° C. for 12 h. After the reaction is completed, directly concentrate the reaction solution, drain it, and proceed to the next step without purification.

[0492] Synthesis of Compound 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate(ZSQ-12-95)

[0493]

[0494] ZSQ-12.91 (4.38 g, 20.0 mmol) was dissolved in 40 ml of dry DCM, placed in an ice bath and stirred evenly, and triethylamine (4.17 ml, 30.0 mmol) was added. 4-Toluenesulfonyl chloride (4.19 g, 22.0 mmol) was dissolved in 5 ml of dry DCM, added dropwise to the above mixed solution under an ice bath, and stirred at room temperature for 18 h after the addition. After the reaction was completed, it was washed with 2N hydrochloric acid (50 ml), water (50 ml), and saturated sodium chloride (50 ml) in turn, and extracted with DCM (50 ml). The organic phases were combined and dried over anhydrous sodium sulfate, and the filtrate was separated and purified by silica gel column chromatography after being spin-dried, and eluted with PE / EA (1:6) to obtain 4.5 g of light yellow oily product with a combined yield of 58%.

[0495] Synthesis of Compound 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (ZSQ-13-28)

[0496]

[0497] Compound ZSQ-12-95 (560 mg, 1.5 mmol) was dissolved in a mixed solution of 4 ml THF and 1 ml H2O, triphenylphosphine (787 mg, 3.0 mmol) was added, and the mixture was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated, separated and purified by C18 reverse phase column chromatography, and eluted with H2O / CH3CN (3:1) to obtain 372 mg of a yellow solid with a yield of 71%.

[0498] Synthesis of Compound 13-oxo-17-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9-trioxa-12-azaheptadecyl 4-methylbenzenesulfonate (ZSQ-13-30)

[0499]

[0500] ZSQ-13-28 (186 mg, 0.536 mmol), (+) biotin-N-succinimidyl ester (183 mg, 0.536 mmol) and DIPEA (266 ul, 1.608 mmol) were dissolved in 3 ml of DCM and stirred at room temperature for 5 h. After the reaction was completed, it was directly separated and purified by silica gel column chromatography and eluted with DCM / CH3OH(NH3) (7:1) to obtain 264 mg of light yellow solid with a yield of 71%.

[0501] Compound (R)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-1-benzyl-N-((1-(13-oxo-17-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3 Synthesis of ,4-d]imidazol-4-yl)-3,6,9-trioxa-12-azahepta-decyl)piperidin-4-yl)methyl)pyrrolidine-3-carboxamide (ZSQ-13-34)

[0502]

[0503] ZSQ-13-30 (34 mg, 0.059 mmol), ZSQ-13-62 (32 mg, 0.059 mmol) and potassium carbonate (24 mg, 0.177 mmol) were dissolved in 2 ml of DMF, and the mixed solution was heated to 80° C. and stirred for 3 h.

[0504] After the reaction was completed, the product was directly purified by HPLC to obtain 17 mg of white powder with a yield of 30%.

[0505] Compound (4-((R)-1-benzyl-N-((1-(13-oxo-17-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9-trioxa-12-azaheptadecyl)piperidin-4-yl)methyl)pyrrolidi Synthesis of ne-3-carboxamido)phenyl)arsonous acid(ZSQ-13-37)

[0506]

[0507] ZSQ-13-34 (17 mg, 0.018 mmol) and mercuric perchlorate trihydrate (7 mg, 0.014 mmol) were dissolved in 1 ml of DMSO, and the mixed solution was stirred at room temperature for 10 min, and immediately separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (2:1), and then purified by HPLC to obtain 7.2 mg of white powder with a yield of 45%. M / Z: Exact Mass: 886.36, (LC-MS): Found 887.30.

[0508] Embodiment 18

[0509] Synthesis route of compound ZSQ-19-99

[0510]

[0511] Synthesis of the compound tert-butyl 14-hydroxy-3,6,9,12-tetraoxatetradecanoate (ZSQ-19-86)

[0512]

[0513] Dissolve tetraethylene glycol (38.84 g, 200 mmol) in 50 ml of anhydrous THF and stir under an ice bath. Dissolve NaH (1.6 g, 40 mmol) in 20 ml of anhydrous THF and add dropwise to the mixed solution (over 30 min). Continue stirring at 0°C for 30 min after the addition. Add tert-butyl bromoacetate (7.8 ml, 40 mmol) dropwise to the mixed solution. Move to room temperature and stir for 3 h after the addition. After the reaction is completed, concentrate the reaction solution, wash with H2O and saturated NaCl solution, and extract with EA. Dry the organic phase with anhydrous sodium sulfate, filter and concentrate, and pump dry with an oil pump. No further purification is performed to obtain 6.3 g of light yellow liquid, which is directly used for the next step of reaction.

[0514] Synthesis of the compound tert-butyl 14-(tosyloxy)-3,6,9,12-tetraoxatetradecanoate (ZSQ-19-87)

[0515]

[0516] The product ZSQ-19-86 (6.16 g, 20 mmol) of the previous step was dissolved in 50 ml of dry DCM, triethylamine (4.16 mmol, 30 mmol) was added, and stirred evenly at room temperature. p-Methylsulfonyl chloride (4.58 g, 24 mmol) was dissolved in 10 ml of ultra-dry DCM, added dropwise to the mixed solution, and stirred overnight at room temperature after the addition. After the reaction was completed, the reaction solution was concentrated by vacuum distillation, washed with dilute hydrochloric acid solution, H2O, and saturated NaCl solution in turn, extracted with EA, the organic phases were combined and dried over anhydrous sodium sulfate, filtered and concentrated, separated and purified by silica gel column chromatography, and eluted with DCM / EA (3:1) to obtain 5.127 g of yellow oily liquid with a yield of 55%.

[0517] Synthesis of the compound tert-butyl 14-azido-3,6,9,12-tetraoxatetradecanoate (ZSQ-19-88)

[0518]

[0519] ZSQ-19-87 (2.3 g, 5 mmol) and NaN3 (390 mg, 6 mmol) were dissolved in 20 ml of anhydrous DMF, heated to 60°C and stirred overnight. After the reaction was completed, DMF was removed by distillation under reduced pressure to obtain a yellow solid which was directly used for the next step without purification.

[0520] Synthesis of compound 14-azido-3,6,9,12-tetraoxatetradecanoic acid (ZSQ-19-89)

[0521]

[0522] Dissolve ZSQ-19-88 in 10 ml DCM, stir at room temperature, add 2 ml trifluoroacetic acid dropwise, and continue stirring at room temperature for 12 hours. After the reaction is completed, concentrate to remove DCM, dissolve in THF, separate and purify by C18 reverse phase column chromatography, and elute with H2O / CH3CN (4:1) to obtain 912 mg of white solid, with a yield of 65.85%.

[0523] Synthesis of compound (4-hydroxy-3-nitrophenyl)arsonous acid (ZSQ-16-8)

[0524]

[0525] Dissolve 3-nitro-4-hydroxyphenylarsenic acid (26.3g, 100mmol) in 80ml methanol, and heat the mixed solution to reflux. Add phenylhydrazine (19.6ml, 200mol) dropwise (over 1h). A large amount of nitrogen is generated during the addition. When the nitrogen generation slows down, continue to reflux and stir for 1.5h. The mixed solution is concentrated by vacuum distillation, and sodium hydroxide solution (12g dissolved in 200ml water) and 200ml of ether are added. Separate the liquids, add 2N dilute hydrochloric acid (100ml) solution to the aqueous phase, stir for 1h, and extract with EA (200ml×3). The organic phase is dried over anhydrous sodium sulfate, concentrated, and pumped dry by an oil pump. The next step of reaction is not purified.

[0526] Synthesis of Compound 4-(1,3,2-dithiarsolan-2-yl)-2-nitrophenol(ZSQ-16-14)

[0527]

[0528] ZSQ-16-8 (24.7 g, 100 mmol) was dissolved in 100 ml of methanol and heated to reflux. Then, ethanedithiol (10 ml, 120 mmol) was added dropwise to the mixed solution within 30 min, and heating and stirring were continued for 30 min. The mixed solution was then concentrated, separated and purified by silica gel column chromatography, and eluted with PE / EA (10:1) to obtain a yellow solid crude product. Dissolved in THF, separated and purified by C18 reverse phase column chromatography, and eluted with H2O / CH3CN (8:1) to obtain 9 g of yellow solid product with a yield of 29.5%.

[0529] Synthesis of Compound 2-amino-4-(1,3,2-dithiarsolan-2-yl)phenol(ZSQ-16-90)

[0530]

[0531] 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 refluxed at 75°C with stirring for 12 h. After the reaction was completed, the reaction solution was concentrated and separated and purified by silica gel column chromatography, eluted with PE / EA (8:1) to obtain a yellow solid crude product, which was dissolved in THF and separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:8) to obtain 4.83 g of yellow solid, with a yield of 58.54%.

[0532] Synthesis of Compound 5-(1,3,2-dithiarsolan-2-yl)-2-methoxyaniline (ZSQ-19-92)

[0533]

[0534] ZSQ-16-90 (2.74 g, 10 mmol), iodomethane (747 ul, 12 mmol), potassium iodide (332 mg, 2 mmol) and potassium carbonate (2.76 g, 20 mmol) were dissolved in 40 ml DMF and stirred at room temperature for 24 h. After the reaction was completed, the reaction solution was concentrated and directly separated and purified by silica gel column chromatography, eluted with PE / EA (4:1) to obtain a crude product, which was then separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:8) to obtain 1.27 g of yellow solid, with a yield of 44%.

[0535] Synthesis of Compound N-(5-(1,3,2-dithiarsolan-2-yl)-2-methoxyphenyl)-14-azido-3,6,9,12-tetra-oxatetradecanamide (ZSQ-19-90)

[0536]

[0537] ZSQ-19-92 (189 mg, 0.65 mmol), ZSQ-19-89 (217 mg, 0.78 mmol), HATU (370 mg, 0.98 mmol) and DIPEA (322 ul, 1.95 mmol) were dissolved in 40 ml DCM and stirred at room temperature for 12 h. After the reaction was completed, it was directly separated and purified by silica gel column chromatography, eluted with DCM / CH3OH(NH3) (10:1), and the crude product was further separated and purified by C18 reverse phase column chromatography, eluted with H2O / CH3CN (1:3) to obtain 45 mg of white solid, with a yield of 13%.

[0538] Synthesis of Compound N-(5-(1,3,2-dithiarsolan-2-yl)-2-methoxyphenyl)-14-amino-3,6,9,12-tetra-oxatetradecanamide (ZSQ-19-97)

[0539]

[0540] Compound ZSQ-19-90 (45 mg, 0.08 mmol) was dissolved in a mixed solution of 2 ml THF and 0.5 ml H2O, triphenylphosphine (43 mg, 0.16 mmol) was added, and stirred at room temperature overnight. After the reaction was completed, the mixture was directly concentrated and dried, and used in the next step without purification.

[0541] Synthesis of the compound N-(5-(1,3,2-dithiarsolan-2-yl)-2-methoxyphenyl)-14-(5-((4R)-2-oxohexa-hydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)-3,6,9,12-tetraoxatetradecanamide (ZS Q-19-99)

[0542]

[0543] The crude product of ZSQ-19-97, (+) biotin-N-succinimidyl ester (27 mg, 0.08 mmol) and DIPEA (40 ul, 0.24 mmol) were dissolved in 2 ml of DCM and stirred at room temperature for 6 h. After the reaction was completed, it was directly spin-dried and purified by HPLC to obtain 12 mg of white powder, with a combined yield of 20%. M / Z: Exact Mass: 748.16, (LC-MS): Found 749.10.

[0544] Using a method similar to the above example, the synthesized compounds are shown in Table 1:

[0545] Table 1 Compounds

[0546]

[0547]

[0548]

[0549]

[0550]

[0551]

[0552]

[0553]

[0554]

[0555] Table 2 Hydrogen spectrum and mass spectrum data of compounds

[0556]

[0557]

[0558]

[0559]

[0560]

[0561]

[0562]

[0563]

[0564]

[0565]

[0566]

[0567]

[0568] Activity Test Example 1 Inhibitory Effect of Inhibitors on PKM2 Enzyme Activity in Vitro

[0569] PKM2 protein can catalyze the reaction of one molecule of ADP and one molecule of PEP in vitro to generate one molecule of ATP and one molecule of pyruvate. By pre-treating PKM2 protein with a compound and then detecting the concentration of ATP generated by its catalysis, the inhibitory effect of the compound on PKM2 enzyme activity can be inferred.

[0570] The biological test scheme adopted is: the effect of the test compound on inhibiting the catalytic activity of PKM2 protein at a concentration of 2 μM, the "+" sign represents 30%-50% activity inhibition, "++" represents 50%-70% activity inhibition, "+++" represents 70-90% activity inhibition, and "++++" represents more than 90% activity inhibition.

[0571] Methods: 5 μg / ml human recombinant PKM2 protein was incubated with the test compound (final concentration 1 μM and 10 μM) at 4°C for 24 hours. The enzyme reaction mixture was added to make the final reaction system contain 1mM ADP, 1mM PEP, 50mM MgCl2, and reacted at 30°C for 10 minutes. 20 μL ATP-Glo ​​reagent was quickly added and incubated at room temperature for 10 minutes. The intensity of chemiluminescence was detected by a microplate reader to calculate the concentration of generated ATP and the catalytic activity of PKM2 enzyme.

[0572] Table 3 Inhibitory effect of inhibitors on PKM2 enzyme activity in vitro

[0573]

[0574]

[0575] Activity Test Example 2 Half-maximal inhibitory concentration of inhibitor on PKM2 in vitro enzyme activity

[0576] By testing the relative inhibitory activity of compounds against PKM2 at different concentrations in vitro, the inhibitory activity of representative compounds can be quantified and compared more accurately. After the arsenite functional group of the compound is protected with ethanedithiol, its covalent reaction activity will be greatly reduced, but it is beneficial to enhance the specific binding of the compound to the target protein. ZSQ1-94 and ZSQ13-83 are the protected forms of the early probe ZSQ1-97 and the optimized compound ZSQ13-100, respectively. The comparison of their inhibitory activity can, to a certain extent, reflect the strength of their non-covalent binding ability to the PKM2 protein.

[0577] The biological assay protocol used was to titrate the half inhibitory concentration (IC) of representative compounds to inhibit PKM2 protease activity. 50 ), and the reported PKM2 inhibitors shikonin and PKM2-IN-1 were used as controls.

[0578] Methods: The compound was diluted in a 2-fold concentration gradient, and the final concentration to be tested ranged from 10μM to 40nM for a total of 9 points. 5μg / ml human recombinant PKM2 protein was incubated with the test compound at 4℃ for 24 hours. The enzyme reaction mixture was added to make the final reaction system contain 1mMADP, 1mM PEP, 50mM MgCl2, and reacted at 30℃ for 10 minutes. 20μL ATP-Glo ​​reagent was quickly added, incubated at room temperature for 10 minutes, and the intensity of chemiluminescence was detected by a microplate reader to calculate the concentration of generated ATP and the catalytic activity of PKM2 enzyme.

[0579] Figure 1 The concentration-activity curves of representative compounds and control compounds for inhibiting PKM2 in vitro and their half inhibitory concentrations (IC 50 ).

[0580] from Figure 1 It can be seen that ZSQ13-83, ZSQ13-100, and ZSQ1-97 all showed an IC of about 1 μM. 50 , and its inhibitory activity on PKM2 is significantly better than that of shikonin and PKM2-IN-1; and ZSQ1-94 also has a very low effective concentration.

[0581] Activity Test Example 3 Inhibitory Effect of Inhibitors on PKM1 Enzyme Activity in Vitro

[0582] By testing the relative inhibitory activity of the compounds against PKM1 at different concentrations in vitro, the specificity of representative compounds between PKM2 and PKM1 can be compared.

[0583] The biological test scheme used was: the effect of the test compound on inhibiting the catalytic activity of PKM1 protein at a specific concentration was tested, and the reported PKM2 inhibitors shikonin and PKM2-IN-1 were used as controls.

[0584] Methods: 3 μg / ml human recombinant PKM1 protein was incubated with the test compound at 4°C for 24 hours. The enzyme reaction mixture was added to make the final reaction system contain 1mM ADP, 1mM PEP, 50mM MgCl2, and reacted at 30°C for 10 minutes. 20μL ATP-Glo ​​reagent was quickly added and incubated at room temperature for 10 minutes. The intensity of chemiluminescence was detected by a microplate reader to calculate the concentration of generated ATP and the catalytic activity of PKM2 enzyme.

[0585] Figure 2 The inhibitory effects of representative compounds at different concentrations on the in vitro enzyme activity of PKM1 are shown.

[0586] from Figure 2 It can be seen that at a high concentration of 10 μM, ZSQ13-83 only weakly inhibited PKM1, and ZSQ13-100 inhibited the PKM1 enzyme activity to about half, while shikonin almost completely inhibited PKM1. This indicates that the compounds of the present invention have excellent selectivity for PKM2.

[0587] Activity Test Example 4 Covalent Binding Site of Inhibitor and PKM2 Protein

[0588] By testing and analyzing the molecular weight and fragmentation peaks of each peptide segment of the PKM2 protein treated with ZSQ13-83 by protein spectrum, the modification state of the side chains of the amino acid residues can be detected, and then the exact site where ZSQ13-83 covalently binds to the PKM2 protein can be determined.

[0589] The biological test scheme adopted is: using liquid chromatography-tandem mass spectrometry to test the modification sites of the compounds on the PKM2 protein at a specific concentration.

[0590] Methods: 10 μL of recombinant human PKM2 pure protein with a concentration of approximately 500 ng / μl was prepared in 20 mM Tris HCl, pH 8.0, 10% Glycerol, and 2% DMS. ZSQ13-83 was added to a final concentration of 1 μM and mixed, and shaken overnight at 4°C. The mixture was then reduced with TCEP at a final concentration of 10 mM, blocked with iodopropionamide at a final concentration of 22 mM, digested with trypsin overnight, and analyzed by liquid chromatography-tandem mass spectrometry. Finally, the molecular weight of the peptide in the primary mass spectrometry data was retrieved to confirm the modification state of the thiol side chain of the cysteine ​​residue in the peptide chain, and the attribution of the peptide chain in the PKM2 protein was determined by comparing the secondary mass spectrometry data.

[0591] Figure 3 The mass spectrometry spectrum of ZSQ13-83 covalently bound to the peptide chain where cysteine ​​474 of PKM2 protein is located is shown.

[0592] from Figure 3 It can be seen that at a concentration of 1 μM, ZSQ13-83 specifically reacted covalently with the sulfhydryl side chain of cysteine ​​at position 474 of the PKM2 protein, and then added to the peptide containing the cysteine. Therefore, the inhibitory mechanism of ZSQ13-83 and its analogs on PKM2 is a covalent mechanism. Since Cys474 is located at the allosteric site of PKM2, the above results suggest that the compounds of the present invention are allosteric inhibitors. Allosteric inhibitors are less affected by changes in the concentration of enzyme substrates because they do not compete directly with enzyme substrates; at the same time, allosteric sites are often not prone to drug resistance mutations that are prone to occur in catalytic sites.

[0593] Activity Test Example 5: Growth Inhibitory Activity Test of PKM2 Inhibitors on Different Ovarian Cancer Cell Lines

[0594] In order to verify the inhibitory effect of the compounds of the present invention on the growth of cancer cells at the cellular level, PA-1 human ovarian teratoma cells, A2780 human ovarian adenocarcinoma cells (PKM2-dependent) and SKOV3 ovarian adenocarcinoma cells (PKM2-independent) were selected, and the cell viability was calculated by detecting the chemiluminescence value, thereby obtaining the biological activity of the compounds in inhibiting the growth of cancer cells.

[0595] The bioassay protocol used was to titrate the half inhibitory concentration (IC50) of representative compounds to inhibit the growth of ovarian cell lines PA-1, A2780 or SKOV3 cells. 50 ), and the reported PKM2 inhibitors shikonin and PKM2-IN-1, the PKM2 agonist TEPP-46, and the analog ZSQ20-4 in which the arsenite functional group of ZSQ13-100 was replaced by boronic acid were used as controls.

[0596] Methods: PA-1, A2780 or SKOV3 cells were cultured in vitro and grown to the logarithmic growth phase. The cells were collected and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the cell concentration was adjusted to 1.5×105 / mL. The cells were inoculated into 384-well plates with 40 μl per well. Different concentrations of compounds or DMSO were added to the corresponding wells, and 5 μL of each was placed in a cell culture incubator (37°C, 5% CO2) for 72 h. After that, 15 μl of Cell Titer-Glo solution was added to each well and incubated at room temperature for 30 min. The chemiluminescence value (luminescence) was detected to measure the intracellular ATP level. The unstimulated DMSO control well was taken as 100% cell viability. The IC50 value of the compound was calculated using PrismGraphpad statistical software.

[0597] Figure 4 The concentration-activity curves and half inhibitory concentrations (IC50) of ZSQ13-83 and its analogs, as well as control compounds, for inhibiting the growth of different ovarian cancer cell lines are shown. 50 ).

[0598] from Figure 4 It can be seen that:

[0599] ZSQ12-54, ZSQ13-83, ZSQ17-69, and ZSQ17-86 exhibit nanomolar ICs in PKM2-dependent PA-1 and A2780 cell lines 50 , while the boronic acid control compound ZSQ20-4 was completely inactive.

[0600] Shikonin has similar activity to ZSQ13-83 in PA-1 and A2780 cell lines, but also exhibits strong growth inhibitory activity in the PKM2-independent SKOV3 cell line;

[0601] PKM2-IN-1 is less active than ZSQ13-83 in the PA-1 cell line;

[0602] The PKM2 agonist TEPP-46 showed no significant cell growth inhibitory activity.

[0603] Therefore, ZSQ13-83 is more active than PKM2-IN-1 and more selective than shikonin in inhibiting the growth of PKM2-dependent cells.

[0604] After the arsenic atom was replaced by a boron atom, the resulting ZSQ20-4 had no inhibitory activity, which suggests that the inhibition of PKM2 by the compounds of the present invention depends on the functional group containing an arsenic atom.

[0605] Activity Test Example 6 Irreversible Binding of Inhibitors to PKM2 in PA-1 Cells

[0606] Active test cases and Figure 3 It shows that inhibitors such as ZSQ13-83 can form a covalent bond with Cys474 near the allosteric site of PKM2, and then bind to PKM2 irreversibly. ZSQ13-74 is a derivative of ZSQ13-83 modified with a biotin label, which maintains the latter's activity in inhibiting PKM2. In theory, it can also form a covalent bond with PKM2 in cell lysate, and then enrich PKM2 through streptavidin beads. When the ZSQ series of PKM2 inhibitors bind to PKM2 irreversibly, ZSQ13-74 can no longer bind to PKM2 because the binding pocket has been occupied. Therefore, based on the competitive enrichment experiment of ZSQ13-74 and streptavidin beads, it can accurately determine whether PKM2 inhibitors can bind to PKM2 irreversibly in living cells.

[0607] The biological test scheme adopted is: through living cell treatment and competitive binding pull-down test, whether the inhibitor of the present invention binds irreversibly to PKM2 in living cells is tested.

[0608] Methods: PA-1 cells were cultured in vitro and grown to the logarithmic growth phase. The cells were digested and collected, centrifuged at 1000rpm for 5min, and the supernatant was discarded to adjust the cell concentration to 1×106 / mL. In a 12-well cell culture plate, 1ml of cells were added to each well, and 1μL of a 10mM DMSO solution of a representative drug was added to each well (final concentration 10μM). DMSO was used as a control. After culturing in a cell culture incubator (37℃, 5% CO2) for 4 hours, the cells were washed twice with pre-cooled PBS solution, and 200μL of NP40 cell lysis buffer and protease inhibitors were added to the wells. The cells were lysed at 4℃ for 30 minutes. The lysate was collected and centrifuged at 15000rpm for 15min at 4℃. The supernatant was transferred to a sample tube, and ZSQ13-74 (final concentration 100nM) was added. The cells were incubated at 4℃ overnight. 10μL of streptavidin microbeads were added and allowed to bind at room temperature for 2 hours. The microbeads were washed 10 times with 1% NP40 cell lysis buffer, lysed by adding 20 μL 2% SDS lysis buffer, boiled at 95°C for 10 minutes, loaded and subjected to western-blot identification.

[0609] Figure 5 Competitive inhibition of covalent binding of ZSQ13-74 to PKM2 in PA-1 cells by representative compounds such as ZSQ13-83, ZSQ17-69, and ZSQ17-80 at 10 μM concentration is shown.

[0610] from Figure 5 It can be seen that:

[0611] ZSQ13-74 can effectively covalently bind to and enrich PKM2 in PA-1 cell lysate;

[0612] The expression level of PKM1 protein in PA-1 cell lysate was low, and ZSQ13-74 could not enrich it;

[0613] ZSQ13-83, ZSQ17-69 and ZSQ17-80 can all covalently bind to PKM2 protein in living PA-1 cells, making it impossible for ZSQ13-74 to effectively enrich it, indicating that the binding of the compounds of the present invention to PKM2 is covalent / irreversible inhibition.

[0614] As can be seen from Example 4, the compounds of the present invention are allosteric inhibitors of PKM2, and allosteric inhibitors often require higher concentrations to effectively inhibit the target protein. Surprisingly, this example further proves that the PKM2 inhibitor of the present invention has a covalent inhibition mechanism, which can enhance its binding force through covalent action, and then can perform allosteric inhibition at lower concentrations, ensuring effective inhibition at low concentrations.

[0615] Activity test example 7 Effect of ZSQ13-83 on cancer cell signaling pathways

[0616] In many cancer cells, insufficient energy supply will lead to the activation of the AMPK signaling pathway, thereby inhibiting other signaling pathways that promote cell survival and proliferation (such as the ERK pathway), and ultimately promoting apoptosis. When PKM2 in cancer cells is inhibited, its direct ATP supply during glycolysis and the supply of pyruvate, the energy raw material, may be inhibited, thereby promoting the increase in AMPK phosphorylation levels and cancer cell apoptosis. It has also been reported that PKM2 also acts as a transcriptional regulator in some cancer cells, positively regulating the expression of the cancer-promoting factor c-MYC. Therefore, detecting the key links in these cancer cell signaling pathways will help understand the specific downstream mechanisms of PKM2 inhibitors in suppressing cancer.

[0617] The biological test scheme adopted was: PA-1 cells were treated with ZSQ13-83, and the changes in the phosphorylation levels of AMPK and ERK, and the expression levels of c-MYC in the cells were tested.

[0618] Methods: PA-1 cells were cultured in vitro, grown to the logarithmic growth phase, digested and collected, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the cell concentration was adjusted to 1×106 / mL. In a 12-well cell culture plate, 1ml of cells were added to each well, and 1μL of DMSO solution with different concentrations of drugs was added to each well. DMSO was used as a control. After culturing in a cell culture incubator (37℃, 5% CO2) for 8 hours, the cells were washed twice with pre-cooled PBS solution, the solution was aspirated, 200μL of RIPA cell lysis buffer, protease inhibitors and phosphatase inhibitors were added to the wells, transferred to sample tubes, placed in a 4℃ shaker for lysis for 30min, centrifuged at 15000rpm at 4℃ for 15min, and the supernatant of cell lysate was taken. The protein content of each group was detected using the BCA protein quantification kit, and the protein amount was adjusted using the PIPA lysis buffer to make the final volume 100μL. The samples were identified by western-blot.

[0619] Figure 6 Shown are the effects of ZSQ13-83 at a concentration of 10 μM on the phosphorylation levels of AMPK and ERK (p-AMPK, p-ERK), and the expression level of c-MYC in PA-1 cells.

[0620] from Figure 6 It can be seen that ZSQ13-83 can effectively activate AMPK in PA-1 cells at a concentration of 1 μM, and the activation time point is about 12 hours;

[0621] After treatment with ZSQ13-83, ERK phosphorylation was upregulated in a stress-induced manner, and then returned to the pre-drug level 12 hours later (i.e., after AMPK activation);

[0622] After 16 h of ZSQ13-83 treatment, the protein expression level of c-MYC was also significantly suppressed;

[0623] This suggests that ZSQ13-83 inhibits PKM2 in PA-1 cells and interferes with PKM2-related energy supply and transcription pathways, ultimately effectively inhibiting the growth of PA-1 cells.

[0624] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof; in, Having a structure selected from the group consisting of: R3 is selected from the following group: H, substituted or unsubstituted C1-C6 alkyl, or substituted or unsubstituted C1-C3 alkylene-R e ; where R e is a substituted or unsubstituted group selected from the following group: Wherein, each Rj is independently selected from the following group: H, C1-C4 alkyl, C1-C4 haloalkyl, -(C=O)-C1-C4 alkyl, hydroxy-substituted C1-C4 alkyl; the substitution refers to that one or more hydrogen atoms on the ring carbon atoms of the group are replaced by a group selected from the following group: halogen, cyano, C1-C4 alkoxy, C1-C4 alkyl, -(C=O)-C1-C4 alkyl, hydroxy-substituted C1-C4 alkyl, -NHCO-C1-C4 alkyl; n is 0, 1, 2, 3 or 4; Each R4 is independently selected from: H, halogen, D, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy; R5 is a substituted or unsubstituted group selected from the following group: Wherein, ring A is a substituted or unsubstituted group selected from the following group: L1 is selected from the group consisting of none, -C1-C4 methylene or -CH2(C=O)-; Ring B is selected from the following group: or substituted or unsubstituted 3-10 membered heterocycloalkyl having 1-3 heteroatoms selected from O, N and S, substituted or unsubstituted 6-10 membered aryl; wherein the substitution means that one or more hydrogen atoms on the group are replaced by a substituent selected from the following group: halogen, C1-C6 alkoxy, C1-C6 alkyl, C1-C6 haloalkyl, -NR f R g ; If not otherwise specified, the substitution means that one or more hydrogen atoms on the group are replaced by a group selected from the group consisting of halogen, -OH, -CN, -NR f R g 、-C1-C3 alkyl-CO-NR f R g , C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and -(C=O)-C1-C6 alkyl; and Each R f and R g Independently selected from the group consisting of H, C1-C6 alkyl.

2. The compound according to claim 1, characterized in that L1 is -CH2- and Ring B is a substituted or unsubstituted phenyl group.

3. The compound according to claim 1, characterized in that The R e Having a structure selected from the group consisting of: Wherein, Rj is independently selected from the following group: H, C1-C4 alkyl, C1-C4 haloalkyl, -(C=O)-C1-C4 alkyl, hydroxy-substituted C1-C4 alkyl.

4. The compound according to claim 1, characterized in that The compound of formula I has the structure of formula Ia: Wherein, X1, X2, R1, R2, R3, R4, A, B, L1 and n are as defined in claim 1.

5. The compound according to claim 4, characterized in that The compound of formula Ia has the structure IIa: Among them, R k Selected from the group consisting of C1-C4 alkyl, C1-C4 haloalkyl, -(C=O)-C1-C4 alkyl, hydroxy-substituted C1-C4 alkyl; Said X1, X2, R1, R2, R4, A, B, L1 and n are as defined in claim 1.

6. A compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The compound is selected from the group consisting of:

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (a) a compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof; and (b) a pharmaceutically acceptable carrier.

8. Use of the compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or the pharmaceutical composition according to claim 7 for preparing a medicament or a preparation, wherein the medicament or preparation is used for: (i) downregulating and / or inhibiting PKM2 activity; (ii) prevention and / or treatment of tumors; (iii) reducing and / or inhibiting the growth and / or proliferation of tumor cells; (iv) reducing and / or inhibiting the metastasis of tumor cells; (v) promoting apoptosis of tumor cells; and / or (vi) Reduce and / or inhibit the glycolysis process of cells.

9. The use according to claim 8, characterized in that The medicine or preparation is used to prevent and / or treat tumors, and the tumor is selected from the following group: liver cancer, gastric cancer, breast cancer, pancreatic cancer, ovarian cancer, laryngeal cancer, prostate cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, endometrial cancer, brain cancer, bladder cancer, testicular cancer, head cancer, neck cancer, skin cancer, mesothelial cancer, white blood cell cancer, esophageal cancer, muscle cancer, connective tissue cancer, lung cancer, adrenal cancer, thyroid, kidney cancer or bone cancer, glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma, choriocarcinoma, skin basal cell carcinoma and testicular seminoma.

10. An in vitro non-therapeutic method for down-regulating and / or inhibiting PKM2 activity, characterized in that: Cultivating cells in the presence of the compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or the pharmaceutical composition according to claim 7, thereby downregulating and / or inhibiting the activity of PKM2 in the cells.

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