WO2021254254A1 - 一类基于有机砷的ii型丙酮酸激酶抑制剂及其制备方法和用途 - Google Patents

一类基于有机砷的ii型丙酮酸激酶抑制剂及其制备方法和用途 Download PDF

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WO2021254254A1
WO2021254254A1 PCT/CN2021/099500 CN2021099500W WO2021254254A1 WO 2021254254 A1 WO2021254254 A1 WO 2021254254A1 CN 2021099500 W CN2021099500 W CN 2021099500W WO 2021254254 A1 WO2021254254 A1 WO 2021254254A1
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zsq
substituted
unsubstituted
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cancer
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谭立
李盈
周少青
庄光磊
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中国科学院上海有机化学研究所
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Definitions

  • the invention relates to the field of medicine, in particular to a type of organic arsenic-based type II pyruvate kinase (PKM2) inhibitor, a preparation method thereof and its use for inhibiting tumors.
  • PPM2 organic arsenic-based type II pyruvate kinase
  • tumor cells mainly rely on glycolysis for sugar metabolism. Glycolysis not only provides ATP for the survival and proliferation of tumor cells, but also provides necessary intermediates for the synthesis of amino acids, nucleic acids and other macromolecules required by the disorderly and rapid proliferation of cancer cells.
  • Pyruvate kinase is the key rate-limiting enzyme in the glycolysis process.
  • pyruvate kinase has four isoforms: PKM1, PKM2, PKL and PKR.
  • PKM1 and PKM2 are two subtypes encoded by the same gene PKM. type.
  • PKM1 is mainly expressed in heart, brain and muscle tissues, while PKM2 is specifically and 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 isoenzymes, and during tumor formation, other isoenzymes disappear, and the PKM2 enzyme is highly expressed.
  • PKM2 plays an important role in the Warburg effect of tumors. Not only does it directly provide ATP to cancer cells, but its catalytic product, pyruvate, is also an important raw material for the energy-supplied tricarboxylic acid cycle or lactic acid in tumors. Therefore, PKM2 It 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.
  • PKM2 plays an extremely important role in tumor progression, and small molecule inhibitors that target PKM2 can treat or slow down cancer.
  • many of the reported PKM2 inhibitors have defects in activity or specificity.
  • Shikonin the most well-known PKM2 inhibitor, has been reported to inhibit chloride channels and inhibit multiple immune response pathways, which increases the risk of adverse reactions.
  • the purpose of the present invention is to provide a PKM2 inhibitor with high activity and high specificity.
  • 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;
  • the X 1 and X 2 are each independently selected from the following group: none, O, S, NR a , CR b R c ;
  • R a , R b and R c are each independently selected from: H or substituted or unsubstituted C1-C6 alkyl;
  • R 3 is selected from the following group: 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 ;
  • n 0, 1, 2, 3 or 4;
  • Each R 4 is independently selected from: H, halogen, D, OH, -NH 3 , -NO2, -CN, COOH, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy , -OCO- substituted or unsubstituted C1-C6 alkyl;
  • R 5 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 aromatic Groups, substituted or unsubstituted 5-10 membered heteroaryl groups with 1-3 heteroatoms selected from O, N and S, substituted or unsubstituted C3-C10 cycloalkyl groups, or substituted or unsubstituted ones with 3-10 membered heterocycloalkyl group with 1-3 heteroatoms selected from O, N and S, -C1-C4 alkylene-(O-CH 2 -CH 2 )m-NR f -CO-R i or
  • 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 group, substituted or unsubstituted 5-10 membered heteroaryl group with 1-4 heteroatoms selected from O, N and S;
  • 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, A substituted or unsubstituted 6-10 membered aryl group or a substituted or unsubstituted 5-10 membered heteroaryl group having 1-3 heteroatoms selected from O, N, S; wherein, the substitution refers to a group One or more of the hydrogen atoms are substituted by a substituent selected from the following group: halogen, -OH, -CN, -NH 2 , C1-C6 alkoxy, C1-C6 alkyl, C2-C6 alkenyl, C2 -C6 alkynyl, C1-C6 haloalkyl, -CO-NR f R g , -NR f R g ;
  • Each m is independently 0, 1, 2, 3, 4, or 5;
  • Each R i is 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, -L 2 -R 9 ;
  • L 2 is selected from the following group: substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C2-C6 alkenylene, substituted or unsubstituted C2-C6 alkynylene;
  • R 9 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, A substituted or unsubstituted 6-10 membered aryl group, a substituted or unsubstituted 5-10 membered heteroaryl group having 1-3 heteroatoms selected from O, N and S;
  • Each of R f and R g is independently selected from the following group: H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl or having 1-3 selected from O, N
  • each group is independently selected from the corresponding group in the compound of Table 1.
  • both X 1 and X 2 are O or both are S.
  • R 5 is Or substituted or unsubstituted group selected from the following group:
  • ring A is a substituted or unsubstituted group selected from the following group:
  • L 1 is connected to the N atom on ring A.
  • ring B is selected from the group consisting of substituted or unsubstituted 6-10 membered aryl groups, preferably, substituted or unsubstituted phenyl groups.
  • L 1 is -CH 2 -and ring B is a substituted or unsubstituted phenyl group.
  • L 2 is -C1-C4 methylene, preferably -CH 2 -.
  • R e is a substituted or unsubstituted group selected from the following group:
  • R e is a substituted or unsubstituted group selected from the following group:
  • the R e has a structure selected from the group consisting of:
  • the compound of formula I has the structure of formula Ia or Ib:
  • each R 9 is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • R i is -C4 alkyl
  • the compound of formula Ia has a structure of IIa:
  • the X 1 , X 2 , R 1 , R 2 , R 4 , A, B, L 1 and n are as defined above.
  • the compound is selected from the following group of compounds in Table 1:
  • 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-13-74, 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
  • the second aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: (a) the compound of formula I as described in the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof Isomer or tautomer, hydrate or solvate; and (b) a pharmaceutically acceptable carrier.
  • the third aspect of the present invention provides 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, a hydrate or a solvate thereof, or the like
  • the use of the pharmaceutical composition according to the second aspect of the invention is used to prepare a medicine or a preparation, and the pharmaceutical preparation is used for:
  • the drug or preparation is used to prevent and/or treat cancer by down-regulating and/or inhibiting the activity of PKM2.
  • the drug or preparation is a PKM2 inhibitor.
  • the tumor is a PKM2-dependent tumor.
  • the "PKM2-dependent tumor” refers to tumors with high expression of PKM2 in tumor cells and/or tumors that rely on PKM2 for glycolysis.
  • the tumor is selected from the group consisting of 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 endometrial cancer, Leukocyte 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, kidney cancer or bone cancer, glioblastoma, Mesothelioma, renal cell carcinoma, gastric cancer, sarcoma (Kaposi's sarcoma), choriocarcinoma, basal cell carcinoma of the skin, and testicular
  • the tumor is selected from the group consisting of 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.
  • the fourth aspect of the present invention provides a PKM2 inhibitor, which comprises: 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 thereof Isomer, hydrate or solvate.
  • the fifth aspect of the present invention provides a method for non-therapeutic down-regulation and/or inhibition of PKM2 activity in vitro, in the compound of formula I, or its stereoisomers or tautomers as described in the first aspect of the present invention , Or a pharmaceutically acceptable salt, hydrate or solvate thereof, or the pharmaceutical composition according to the second aspect of the present invention, culture cells to down-regulate and/or inhibit the activity of PKM2 in the cells.
  • the cell is a tumor cell.
  • the tumor is a PKM2-dependent tumor.
  • the effective concentration of the compound of formula I, or its stereoisomer or tautomer, or its pharmaceutically acceptable salt, hydrate or solvate is 0.01 nM/L- 1mM/L, preferably 0.1nM/L-500 ⁇ M/L, most preferably 1nM/L-100 ⁇ M/L.
  • the sixth aspect of the present invention provides a method for preventing and/or treating tumors by administering to a subject in need a therapeutically effective amount of the compound of formula I as described in the first aspect of the present invention, or its stereoisomers or tautomers Isomer, or a pharmaceutically acceptable salt, hydrate or solvate thereof, the pharmaceutical composition according to the second aspect of the present invention.
  • the subject is a mammal, preferably a human, rat, mouse, cat, dog, cow or horse.
  • the tumor is a PKM2-dependent tumor.
  • the seventh aspect of the present invention provides a preparation method of the compound of formula I as described in the first aspect of the present invention, which comprises the steps:
  • Step i Protect the arsenous acid functional group of A1 in an ester or amide manner
  • Step ii introducing the carbonyl-linked R 5 substituent into the amine group of A2 through a condensation reaction
  • Step iii Introduce R 3 substituent into the amine group of A3 through S N 2 substitution reaction;
  • X 1 , X 2 , R 1 , R 2 , R 4 , R 5 and n are as defined in the first aspect of the present invention.
  • the amine group of compound A2 is first introduced with the R 3 group, and then the carbonyl-linked R 5 substituent is introduced.
  • Figure 1 shows representative compounds and a control compound in vitro inhibitory concentrations of PKM2 - activity profile and half maximal inhibitory concentration (IC 50).
  • Figure 2 shows the inhibitory effects of representative compounds on the in vitro enzyme activity of PKM1 at different concentrations.
  • Figure 3 shows the mass spectrum of ZSQ13-83 covalently bound to the peptide chain where the cysteine 474 of the PKM2 protein is located.
  • Figure 4 shows ZSQ13-83 and the like, as well as different concentrations of a control compound to inhibit the growth of ovarian carcinoma cell line - activity profile and half maximal inhibitory concentration (IC 50).
  • Figure 5 shows the competitive inhibition of ZSQ13-74 covalently bound to PKM2 in PA-1 cells by representative compounds such as ZSQ13-83, ZSQ17-69 and ZSQ17-80 at a concentration of 10 ⁇ M.
  • Figure 6 shows the effect of ZSQ13-83 on the phosphorylation level of AMPK and ERK (p-AMPK, p-ERK) and the expression level of c-MYC in PA-1 cells at a concentration of 10 ⁇ M.
  • Figure 7 shows the protein enrichment level of the representative compound ZSQ13-83 biotin probe ZSQ13-74 at a concentration of 10 ⁇ M in 293T cells expressing PKM2 exogenously.
  • Figure 8 shows the effect of ZSQ13-83 on the glucose metabolism pathway in cancer cells; A shows the effect of ZSQ13-83 on the phosphorylation level of PA-1 extracellular acidification rate at different concentrations; B shows the effect of ZSQ13-83 on the phosphorylation level of the extracellular acidification rate of PA-1 The effect of different concentrations on the level of key metabolites in the glycolysis process of PA-1 cells.
  • Figure 9 shows the inhibitory effect of a representative compound ZSQ13-83 on the growth of subcutaneous tumors in HEY nude mice and its effect on the body weight of nude mice.
  • the compound of the present invention has high activity and high specificity for inhibiting PKM2, and is very suitable for preparing drugs for inhibiting tumors by inhibiting the PKM2 pathway.
  • the present invention has been completed on this basis.
  • the term “about” means that the value can vary from the recited value by no more than 1%.
  • the expression “about 100” includes all values between 99 and 101 (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the term “comprising” or “including (including)” can be open, semi-closed, and closed. In other words, the term also includes “substantially composed of” or “consisting of”.
  • alkyl by itself or as part of another substituent refers to a straight or branched chain hydrocarbon group having the specified number of carbon atoms (ie, C1-6 represents 1-6 carbons).
  • Alkyl groups include alkyl groups having 1, 2, 3, 4, 5, and 6 carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl and the like.
  • alkenyl refers to an unsaturated alkyl group having one or more double bonds. Typically, alkenyl includes alkenyl groups having 2, 3, 4, 5, and 6 carbon atoms. Similarly, the term “alkynyl” refers to an unsaturated alkyl group having one or more triple bonds. Typically, alkynyl groups include alkynyl groups having 2, 3, 4, 5, and 6 carbon atoms.
  • 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 homologues and isomers.
  • cycloalkyl refers to having the specified number of ring atoms (e.g., C 3-10 cycloalkyl, C 3-7 cycloalkyl) and fully saturated or having no more than one ring top Hydrocarbon ring with double bond.
  • cycloalkyl groups include cycloalkyl groups of 3, 4, 5, 6, 7, 8, 9 or 10 ring carbon atoms. It may be a monocyclic ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or similar groups. It can also be bicyclic and polycyclic hydrocarbon rings, such as bridged rings or spiro rings, such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane and the like.
  • heterocycloalkyl refers to a cycloalkyl group containing a specified number (such as 1, 2, or 3) 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 heterocycloalkyl group can be a monocyclic, bicyclic or polycyclic ring system. Typically, heterocycloalkyl groups include 3, 4, 5, 6, 7, 8, 9 or 10 ring atoms.
  • heterocycloalkyl groups 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.
  • the heterocycloalkyl group can be attached to the rest of the molecule via a ring carbon or a heteroatom.
  • cycloalkylalkyl and heterocycloalkylalkyl it is meant that the cycloalkyl or heterocycloalkyl is connected to the rest of the molecule through an alkyl or alkylene linker.
  • cyclobutylmethyl- is a cyclobutyl ring attached to the methylene linker of the rest of the molecule.
  • alkylene by itself or as part of another substituent refers to a divalent group derived from an alkane.
  • an alkylene group includes those having 1, 2, 3, 4, 5, 6 An alkylene group with three carbon atoms, such as -CH 2 -, -CH 2 CH 2 CH 2 CH 2 -.
  • alkenylene or “alkynylene” refers to an unsaturated form of "alkylene” with double or triple bonds, respectively.
  • alkenylene includes 2, 3, 4, 5, 6 Alkenylene groups having three carbon atoms
  • alkynylene groups include alkynylene groups having 2, 3, 4, 5, and 6 carbon atoms.
  • heteroalkyl refers to 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 aza The atoms can optionally be quaternized.
  • the heteroatoms O, N and S can be located in any internal position of the heteroalkyl group.
  • alkoxy or “alkyloxy”, “alkylamino”” or “alkylamino” are used in their conventional sense and refer to those attached to the molecule via an oxygen atom or nitrogen, respectively The remaining part of those alkyl groups.
  • the alkylamino group may be mono- or di-substituted.
  • the alkyl part can be the same or different, or And the nitrogen atom connected to each alkyl group to form a 3-7 membered ring. Therefore, the group represented by -NR f R g includes piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl (azetidinyl )Wait.
  • substituent 0
  • halo or halogen by itself or as part of another substituent refers to a fluorine, chlorine, bromine, or iodine atom.
  • terms such as “haloalkyl” are meant to include monohaloalkyl or polyhaloalkyl.
  • C 1-6 haloalkyl is meant to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl and the like.
  • aryl refers to a polyunsaturated (usually aromatic) hydrocarbon group, which can be a single ring or polycyclic (up to three rings) fused together or covalently linked
  • non-aryl Limiting examples include phenyl, naphthyl, and biphenyl.
  • heteroaryl refers to an aryl group (or ring) containing a specified number (such as 1, 2 or 3) of heteroatoms selected from O, N and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen The atoms are optionally quaternized.
  • heteroaryl groups include heteroaryl groups of 5, 6, 7, 8, 9 or 10 ring atoms. Heteroaryl groups can be attached to the rest of the molecule through heteroatoms.
  • heteroaryl groups include pyridinyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzene Benzotriazinyl (benzotriazinyl), purinyl, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzisoxazolyl, isobenzofuryl (isobenzofuryl), isoindolyl, medium Azindanyl, benzotriazinyl, thienopyridyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridine, benzothiazolyl, benzofuranyl, benzothienyl, indolyl, quine Linyl, isoquinolinyl, iso
  • the aforementioned terms (such as “alkyl”, “aryl” and “heteroaryl”) will include both substituted and unsubstituted forms of the specified groups.
  • the number of substituents can be 1, 2, 3, or 4.
  • each of R f and R g is independently selected from the following group: H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl or having 1-3 options
  • heteroatom is meant to include oxygen (O), nitrogen (N), and sulfur (S).
  • the bond from the substituent (usually the R group) to the center of the aromatic ring will be understood to mean a bond that provides a connection at any available vertex of the aromatic ring.
  • the description also includes a connection to a ring fused to an aromatic ring.
  • a bond drawn to the center of the indolebenzene moiety will represent a bond connected to any available vertex of the six- or five-membered ring portion of the indole. If for a cyclic group, the connection site is not specified, it means that it can be connected via any available vertex on the ring (including carbon atoms or heteroatoms).
  • each chiral carbon atom may optionally be R configuration or S configuration, or a mixture of R configuration and S configuration.
  • Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers, regioisomers and individual isomers (e.g., separated enantiomers) Body) should be included in the scope of the present invention.
  • the compounds provided herein have a defined stereochemistry (represented by R or S, or indicated by a dashed or wedge-shaped bond)
  • those skilled in the art will understand that those compounds are substantially free of other isomers (e.g., at least 80% , 90%, 95%, 98%, 99% and up to 100% free of other isomers).
  • 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 reactions), that is, a substance that has a reasonable benefit/risk ratio.
  • the term "therapeutically effective amount” refers to any amount of the drug as described below, when used alone or in combination with another therapeutic agent, the amount of the drug can promote the regression of the disease, which is manifested as disease symptoms Reduce the severity of the disease, increase the frequency and duration of the asymptomatic period of the disease, or prevent the disorder or disability caused by the disease.
  • the “therapeutically effective dose” of the drug of the present invention also includes the “preventively effective dose”.
  • the “preventively effective dose” is any amount of the drug as described below, when the amount of the drug is administered alone or in combination with another therapeutic agent.
  • a subject is at risk of developing a disease or suffering from a recurrence of the disease, the occurrence or recurrence of the disease can be suppressed.
  • solvate refers to a complex in which the compound of the present invention coordinates with solvent molecules to form a specific ratio.
  • hydrate refers to a complex formed by coordination of the compound of the present invention with water.
  • the invention also includes all suitable isotopic variants of the compounds of the invention.
  • Isotopic variants of the compounds of the present invention are defined as those in which at least one atom is replaced by an atom having the same atomic number but having an atomic mass different from the atomic mass commonly found in nature.
  • isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, and 17 respectively. O, 18 O, 35 S, 18 F and 36 Cl.
  • isotopic variants of the present invention for example, those in which radioisotopes (such as 3 H or 14 C) are incorporated, are used in drug and/or substrate tissue distribution studies. Tritiated, i.e., 3 H, and carbon-14, i.e., 14 C, isotopes are particularly preferred because they are easy to prepare and detect. Further, substitution with isotopes (e.g. deuterium, i.e., 2 H) substitution, may afford certain therapeutic advantages resulting from increased metabolic stability due to, for example, increased in vivo half-life or reduced dosage requirements and thus in some cases it may be preferred of. Isotopic variants of the compounds of the present invention can generally be prepared by conventional operations, for example, by using suitable reagents for appropriate isotopic variants, by an exemplary method or the preparation described in the experimental section below.
  • treating refers to reducing, delaying progression, attenuating, preventing, or maintaining an existing disease or condition (e.g., cancer). Treatment also includes curing one or more symptoms of the disease or condition, preventing its development, or alleviating to a certain degree.
  • the compound of the present invention refers to the compound of formula I, its pharmaceutically acceptable salt, or its stereoisomer or tautomer, or hydrate or solvate;
  • X 1 , X 2 , R 1 , R 2 , R 3 , R 4 , R 5 and n are as defined in the first aspect of the present invention.
  • pharmaceutically acceptable salt refers to a salt formed by a compound of the present invention and an acid or base suitable for use as a medicine.
  • Pharmaceutically acceptable salts include inorganic salts and organic salts.
  • a preferred class of salts are the salts of the compounds of this invention with acids.
  • Acids suitable for salt formation include, but are not limited to: hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid and other inorganic acids, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, Organic acids such as maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, toluenesulfonic acid, and benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.
  • a preferred class of salts are the salts of the compounds of this invention with bases.
  • Suitable bases for salt formation include, but are not limited to, inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and sodium phosphate, and organic bases such as ammonia, triethylamine, diethylamine, and piperazine.
  • inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and sodium phosphate
  • organic bases such as ammonia, triethylamine, diethylamine, and piperazine.
  • 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 methods for preparing these compounds may include (but are not limited to) the processes described below.
  • the compound of formula I of the present invention can be completed by the exemplary methods described in the following schemes and examples, and related publications used by those skilled in the art.
  • X 1 , X 2 , R 1 , R 2 , R 4 , R 5 and n are as defined above.
  • Step i Protect the arsenous acid functional group of A1 in an ester or amide manner, such as using ethanedithiol, ethylene glycol, etc. as a protective reagent.
  • Step ii Introduce the carbonyl-linked R 5 substituent into the amine group of A2 through a condensation reaction.
  • a condensation reaction for example, acid chloride, acid, acid anhydride, etc. (R 5 -CO-Cl, R 5 -CO-OH) can be used.
  • Step iii Introduce the R 3 substituent into the amine group of A3 by S N 2 substitution reaction, using R 3 -halogen, and react under basic conditions (such as sodium hydride).
  • Step i Protect the arsenous acid functional group of A1 in an ester or amide manner, and ethanedithiol, ethylene glycol, etc. can be used as a protective reagent.
  • Step ii Introduce R 3 substituent into the amine group of A2 by S N 2 substitution reaction or reductive amination reaction .
  • R 3 -halogen can be used and react under alkaline conditions (such as sodium hydride); or R 3 -can be used.
  • Formaldehyde reacts with the participation of reducing reagents (such as sodium triacetoxyborohydride).
  • Step iii The acylation reaction of the phenyl ring amine group of A3 introduces the R 5 group connected to the carbonyl group, for example, acid chloride, acid, acid anhydride, etc. (R 5 -CO-Cl, R 5 -CO-OH), etc. can be used; or Catalyzed by (but not limited to) phosphorus oxychloride reagent.
  • the above preparation method can also carry out the step of removing the protecting group as required.
  • 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 hexane, heptane, different heptane, petroleum ether, cyclopentane, different cyclohexane, benzene, toluene, Xylene, benzotrifluoride, halogenated benzenes such as chlorobenzene, fluorobenzene, dichlorobenzene and difluorobenzene, dichloromethane, chloroform, DMF, acetone, ethyl acetate, diethyl ether, tetrahydrofuran, DMSO, or combinations thereof.
  • the reaction in the method of the present invention can be carried out at any suitable temperature.
  • the reaction temperature may be about -78°C to about 100°C, or about -50°C to about 100°C, or about -25°C to about 50°C, or about -10°C to about 25°C, or about 0°C. °C to about 20°C. In some embodiments, the reaction temperature may be about 0°C to about 20°C.
  • the reactions in the above steps are all conventional reactions known to those skilled in the art. Unless otherwise specified, the reagents and raw material compounds used in the synthetic route are all commercially available, or those skilled in the art can prepare them by referring to known methods according to the structure of different compounds designed.
  • the compound of the present invention has excellent inhibitory activity on type II pyruvate kinase (PKM2), the compound of the present invention and its crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and containing the present invention
  • PKM2 type II pyruvate kinase
  • the pharmaceutical composition in which the compound is the main active ingredient can be used for the treatment, prevention and alleviation of tumors. More specifically, it is used to down-regulate 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.
  • the above-mentioned tumor is a PKM2-dependent tumor.
  • the PKM2-dependent tumors are tumors with high expression of PKM2 in cells and/or cells that rely on PKM2 for glycolysis.
  • the "high expression of PKM2” means that PKM2 in the cell accounts for more than 60% of the total pyruvate kinase, preferably more than 70%, 80%, 90% or 95%.
  • the "dependence on PKM2 for glycolysis” means that the cell mainly catalyzes the glycolysis process through PKM2. For example, ⁇ 60% of glycolysis in the cell is catalyzed by PKM2, preferably, ⁇ 70%, ⁇ 80%, ⁇ 90%, or ⁇ 95%.
  • 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, stomach 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 endometrial cancer, leukocyte 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, kidney cancer or bone cancer; or glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma (Kaposi's sarcoma), choriocarcinoma , Basal cell carcinoma of the skin and seminoma of the testis.
  • the compounds of the present invention can be administered alone or in combination with other therapeutic agents.
  • therapeutic agents include, but are not limited to, anti-cancer agents and/or immunosuppressive agents, such as anti-cancer agents and/or immunosuppressive agents selected from the group consisting of olaparib, lucapanib, niraparib , Methotrexate, capecitabine, gemcitabine, deoxyfluridine, pemetrexed disodium, pazopanib, imatinib, erlotinib, lapatinib, gefitinib , Vandetanib, Herceptin, Bevacizumab, Rituximab, Trastuzumab, Paclitaxel, Vinorelbine, Docetaxel, Doxorubicin, Hydroxycamptothecin, Mitosis Epirubicin, epirubicin, pirarubicin, bleomycin, letrozole, tamoxifen, fulvestrant
  • the compound of the present invention is administered to a subject suffering from cancer in combination with other traditional cancer treatments, for example, radiotherapy or surgery.
  • Radiotherapy is well known in the art and includes X-ray therapy, such as gamma radiation, and radiopharmaceutical therapy.
  • the therapeutic agent is a hypoxia-inducible factor proline hydroxylase (HIF-PH) inhibitor.
  • HIF-PH hypoxia-inducible factor proline hydroxylase
  • the compound of the present invention is used simultaneously or sequentially with other therapeutic agents as a combination treatment regimen in the same or separate formulations.
  • the pharmaceutical composition of the present invention contains 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.
  • the "safe and effective amount” refers to: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects.
  • the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per agent, and more preferably, contains 10-500 mg of the compound of the present invention per agent.
  • the "one dose" is a capsule or tablet.
  • “Pharmaceutically acceptable carrier” refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use, and must have sufficient purity and sufficiently low toxicity. "Compatibility” here means that the components in the composition can be blended with the compound of the present invention and between them without significantly reducing the efficacy of the compound.
  • pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, and 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, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • vegetable oils such as soybean oil, sesame oil, peanut oil, olive oil, etc.
  • polyols such as propylene glycol, glycerin, mannitol, sorbitol, etc.
  • emulsifiers such as emulsifiers
  • wetting agents such as sodium lauryl sulfate
  • administration method of the compound or pharmaceutical composition of the present invention is not particularly limited.
  • Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration .
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or mixed with the following ingredients: (a) fillers or compatibilizers, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectant, For example, glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) Absorption accelerators, for example, quaternary amine compounds; (g) wetting agents, such as cetyl alcohol and glycyl
  • Solid dosage forms such as tablets, sugar pills, capsules, pills and granules can be prepared with coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and the active compound or the release of the compound in such compositions may be released in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microcapsules with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • the liquid dosage form may contain inert diluents 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-Butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.
  • composition may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • suspending agents for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • composition for parenteral injection may contain physiologically acceptable sterile aqueous or non-aqueous 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.
  • the dosage forms of the compound of the present invention for topical administration include ointments, powders, patches, sprays and inhalants.
  • the active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.
  • 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 to 2000 mg/day, about 10 to about 1000 mg/day, about 10 to about 500 mg/day, about 10 to about 250 mg/day , About 10 to about 100 mg/day, or about 10 to about 50 mg/day.
  • the therapeutically effective dose will be given in one or more doses.
  • the specific dosage of the compound of the present invention for any specific patient will depend on various factors, for example, the age, gender, weight, general health, diet, individual response, administration time, and treatment of the patient to be treated. The severity of the disease, the activity of the specific compound administered, the dosage form, the mode of application and the concomitant drugs.
  • the therapeutically effective amount for a given situation can be determined by routine experiments and is within the ability and judgment of the clinician or physician.
  • the compound or composition will be administered in multiple doses based on the individual condition of the patient and in a manner that allows delivery of a therapeutically effective amount.
  • the present invention provides a new class of PKM2 inhibitors.
  • the compound of the present invention has high inhibitory activity and high selectivity to PKM2, low dosage during use, small side effects and high safety.
  • the compound of the present invention is an allosteric inhibitor, which is not easy to produce drug resistance; and the compound of the present invention can be covalently combined with PKM2, ensuring effective inhibition at low doses, and is particularly suitable for preparing medicines.
  • the compound of the present invention has a significant inhibitory effect on PKM2-dependent tumors.
  • 4-Aminophenylarsonic acid (108.5g, 500mmol) was dissolved in 300ml ethanol, and the mixed solution was heated to reflux. Phenylhydrazine (92ml, 1mol) was added dropwise (over 1h) to the mixed solution. During the addition, a large amount of nitrogen was generated. When the generation of nitrogen slowed down, continue to reflux and stir for 1.5h.
  • the mixed solution was concentrated by distillation under reduced pressure, sodium hydroxide solution (40 g dissolved in 400 ml of water) was added, and ether washed twice (400 ml).
  • ZSQ-1-18 (40 g, 199 mmol) was dissolved in 200 ml of absolute ethanol and heated to reflux. Then, ethanedithiol (20ml, 240mmol) was added dropwise to the mixed solution within 30min, and heating and stirring were continued for 30min. 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%.
  • N-methylpiperazine 66ul, 0.6mmol
  • DIPEA 99ul, 0.6mmol
  • ZSQ-5-4 76mg, 0.2mmol
  • ZSQ-6-27 (972 mg, 3.72 mmol) and palladium carbon (200 mg) were mixed in 20 ml of methanol, and mixed and stirred at room temperature under a hydrogen atmosphere overnight. After the reaction was completed, it was filtered with diatomaceous earth, washed with methanol, and the filtrate was concentrated and drained to obtain 870 mg of an oily product, which was directly carried to the next step without further purification.
  • ZSQ-6-20 (830 mg, 3.78 mmol) and palladium carbon (200 mg) were mixed in 15 ml of methanol, and mixed and stirred at room temperature under a hydrogen atmosphere overnight. After the reaction was completed, it was filtered with diatomaceous earth and washed with methanol. The filtrate was concentrated and drained to obtain 504 mg of a colorless oily product, which was directly carried to the next step without further purification.
  • ZSQ-6-57 (456mg, 1.0mmol) and benzyl bromide (178ul, 1.5mmol) were mixed and dissolved in 4ml of ultra-dry DMF, placed in an ice bath and mixed and stirred for 10 minutes.
  • NaH (60%, 1.2 mmol, 48 mg) was added to the mixed solution in batches, and after the addition, stirring was continued for 3 hours in an ice bath, and then moved to room temperature and stirred overnight.
  • the ZSQ-6-102 (53mg, 0.12mmol) , ZSQ-8-38 (27mg, 0.13mmol) and potassium carbonate (33mg, 0.24mmol) was dissolved in 2ml CH 3 CN was stirred in an ice-salt bath 2h. After the completion of the reaction, the reaction solution was concentrated, and was directly separated and purified by silica gel column chromatography, eluted with DCM/CH 3 OH(NH 3 ) (8:1), to obtain 13.3 mg of white solid, with a yield of 19%.
  • ZSQ-1-23 (4.3g, 16.6mmol), 1-tert-butoxycarbonyl-4-piperidinaldehyde (7.0g, 33.2mmol), sodium triacetoxyborohydride (7.0g, 33.2mmol), acetic acid (3.0ml, 49.8mmol) was mixed and dissolved in 100ml of 1,2-dichloroethane, placed at room temperature and mixed and stirred for 48h.
  • Phenylacetic acid (681mg, 5.0mmol), ethyl 2-hydroxyamino-2-iminoacetate (694mg, 5.25mmol), HATU (2.85g, 7.5mmol) and DIPEA (2.48ml, 15.0mmol) were dissolved in 50ml DCM Put it at room temperature and mix and stir for 6h.
  • ZSQ-16-18 (500mg, 2.0mmol) was dissolved in 5ml of dry DMF, heated to 140°C and mixed and stirred for 4h. After the reaction was completed, it was separated and purified by C18 reversed-phase column chromatography, and eluted with H 2 O/CH 3 CN (1:2) to obtain 399 mg of yellow oily product, with a yield of 86%.
  • Phenylacetic acid (1.36g, 10mmol), dimethylhydroxylamine hydrochloride (1.17g, 12mmol), HATU (4.56g, 12mmol) and DIPEA (4.96ml, 30mmol) were dissolved in 50ml DCM and placed at room temperature to mix and stir for 6h . After completion of the reaction washed with dilute hydrochloric acid solution (50ml), H 2 O ( 50ml), saturated NaCl solution (50ml), dried and extracted with 50ml DCM.
  • ZSQ-16-81 (1.7g, 9.48mmol) was dissolved in 30ml of dry THF, placed in an ice bath and stirred evenly. At 0°C, methylmagnesium bromide (1M tetrahydrofuran solution, 14.22ml, 14.22mmol) was added dropwise to the mixed solution within 30 minutes. After the addition, stirring was continued at 0°C for 1 hour and then placed at room temperature and stirred for 12 hours. After the reaction was completed, it was quenched with 2N hydrochloric acid solution (50ml), THF was distilled off under reduced pressure, extracted with EA, and washed twice with water (50ml).
  • Ethyl cyanoformate (1.0ml, 10.0mmol) was dissolved in 12ml of ultra-dry pyridine and placed in an ice bath and stirred evenly. Trifluoroacetic acid (0.74ml, 10mmol) was added dropwise, after stirring for 10min, sodium azide (702mg, 10.8mmol) was added, and the mixed solution was placed at 60°C and stirred for 48h. After the completion of the reaction, it was concentrated by distillation under reduced pressure, and it was used directly in the next step without purification.
  • ZSQ-12.91 (4.38g, 20.0mmol) was dissolved in 40ml of dry DCM, placed in an ice bath and stirred evenly, and triethylamine (4.17ml, 30.0mmol) was added.
  • 4-Toluenesulfonyl chloride (4.19g, 22.0mmol) was dissolved in 5ml of dry DCM, and added dropwise to the above mixed solution while keeping it in an ice bath. After the addition, it was moved to room temperature and stirred for 18h. After the reaction was completed, it was washed with 2N hydrochloric acid (50ml), water (50ml), saturated sodium chloride (50ml), and extracted with DCM (50ml).
  • ZSQ-13-30 34mg, 0.059mmol
  • ZSQ-13-62 32mg, 0.059mmol
  • potassium carbonate 24mg, 0.177mmol
  • ZSQ-19-88 was dissolved in 10ml DCM, stirred at room temperature, 2ml trifluoroacetic acid was added dropwise, and after the addition, stirring was continued at room temperature for 12h. After the completion of the reaction, it was concentrated to remove DCM, dissolved in THF, separated and purified by C18 reversed-phase column chromatography, eluted with H 2 O/CH 3 CN (4:1) to obtain 912 mg of white solid, with a yield of 65.85%
  • ZSQ-16-8 (24.7 g, 100 mmol) was dissolved in 100 ml methanol and heated to reflux. Then, ethanedithiol (10ml, 120mmol) was added dropwise to the mixed solution within 30min, and heating and stirring were continued for 30min. Subsequently, the mixed solution was concentrated, separated and purified by silica gel column chromatography, and eluted with PE/EA (10:1) to obtain a crude yellow solid product. It was dissolved in THF, and then separated and purified by C18 reversed-phase column chromatography, eluted with H 2 O/CH 3 CN (8:1), to obtain 9 g of a yellow solid product with a yield of 29.5%.
  • PKM2 protein can catalyze the reaction of one molecule of ADP and one molecule of phosphoenolpyruvate (PEP) to produce one molecule of ATP and one molecule of pyruvate in vitro.
  • PEP phosphoenolpyruvate
  • the inhibitory effect of the compound on the PKM2 enzyme activity can be calculated.
  • the biological test protocol used is: the test compound inhibits the catalytic activity of the PKM2 protein at a concentration of 2 ⁇ M, the "+” sign represents 30%-50% activity inhibition, and “++” represents 50%-70% activity inhibition. "+++” means 70-90% inhibition of activity, and “++++” means more than 90% inhibition of activity.
  • Compound number Inhibitory effect Compound number Inhibitory effect Compound number Inhibitory effect ZSQ-5-39 + ZSQ-9-32 ++ ZSQ-5-40 + ZSQ-9-33 ++ ZSQ-5-42 + ZSQ-9-34 ++ ZSQ-5-43 + ZSQ-9-35 ++ ZSQ-5-44 + ZSQ-9-36 + ZSQ-5-45 + ZSQ-9-37 + ZSQ-5-47 + ZSQ-11-8 ++ ZSQ-5-77 + ZSQ-11-9 ++ ZSQ-5-78 + ZSQ-11-11 ++ ZSQ-5-80 + ZSQ-11-12 ++ ZSQ-5-81 + ZSQ-11-31 ++ ZSQ-5-82 + ZSQ-11-32 ++ ZSQ-5-83 + ZSQ-11-34 ++ ZSQ-6-3 + ZSQ-11-37 ++ ZSQ-6-37 ++ ZSQ-11-79 +++ ZSQ-6-49 + ZSQ-11-80 +++
  • the inhibitory activities of representative compounds can be more accurately quantified and compared.
  • the arsenous acid functional group of the compound is protected with ethanedithiol, its covalent reaction activity will be greatly reduced, but it is beneficial to improve 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 activities can reflect their non-covalent binding ability to the PKM2 protein to a certain extent. The strengths and weaknesses.
  • the adopted biological test protocol is: titrate the half inhibitory concentration (IC 50 ) of representative compounds that inhibit PKM2 protease activity, and use the reported PKM2 inhibitors shikonin (shikonin) and PKM2-IN-1 as controls.
  • the compound was diluted in a 2-fold concentration gradient, and the final concentration to be tested was 9 points from 10 ⁇ M to 40 nM.
  • 5 ⁇ g/ml human recombinant PKM2 protein was incubated with the test compound at 4°C for 24 hours.
  • Add the enzyme reaction mixture to make the final reaction system contain 1mM ADP, 1mM PEP, 50mM MgCl 2 , and react at 30°C for 10 minutes.
  • Quickly add 20 ⁇ L of ATP-Glo reagent incubate at room temperature for 10 minutes, detect the intensity of chemiluminescence with a microplate reader, and calculate the concentration of ATP and the catalytic activity of PKM2 enzyme.
  • Figure 1 shows representative compounds and a control compound in vitro inhibitory concentrations of PKM2 - activity profile and half maximal inhibitory concentration (IC 50).
  • ZSQ13-83, ZSQ13-100, and ZSQ1-97 all exhibit an IC 50 of about 1 ⁇ M, and their inhibitory activity on PKM2 is significantly better than shikonin and PKM2-IN-1; and ZSQ1-94 also Has a very low onset concentration.
  • the adopted biological test protocol is to test the effect of the compound in inhibiting the catalytic activity of the PKM1 protein at a specific concentration, and use the reported PKM2 inhibitors shikonin and PKM2-IN-1 as controls.
  • Figure 2 shows the inhibitory effects of representative compounds on the in vitro enzyme activity of PKM1 at different concentrations.
  • the molecular weight and fragmentation peaks of each peptide of PKM2 protein treated by ZSQ13-83 can be tested and analyzed by protein spectroscopy.
  • the modification status of the side chain of amino acid residues can be detected, and the exact position of the covalent binding of ZSQ13-83 and PKM2 protein can be determined. point.
  • the adopted biological testing scheme is: using a liquid phase-tandem mass spectrometer to test the modification sites of the PKM2 protein at a specific concentration of the compound.
  • Figure 3 shows the mass spectrum of ZSQ13-83 covalently bound to the peptide chain where the cysteine 474 of the PKM2 protein is located.
  • the cell viability is calculated by detecting the chemiluminescence value, thereby obtaining the biological activity of the compound to inhibit the growth of cancer cells.
  • the adopted biological test protocol is: titration of representative compounds to inhibit the growth of ovarian cell lines PA-1, A2780 or SKOV3 cells at the half inhibitory concentration (IC 50 ), and use the reported PKM2 inhibitor shikonin (shikonin) and PKM2-IN-1, the PKM2 agonist TEPP-46, and the analog ZSQ20-4 with ZSQ13-100 arsenous acid functional group replaced with boric acid were used as controls.
  • Method Culture PA-1, A2780 or SKOV3 cells in vitro and grow to logarithmic growth phase. Collect the cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, adjust the cell concentration to 1.5 ⁇ 105/mL, and inoculate the cells into a 384-well plate , 40 ⁇ l per well. Add 5 ⁇ L each of compounds of different concentrations or DMSO to the corresponding wells, place in a cell incubator (37°C, 5% CO2) and incubate for 72 hours, add 15 ⁇ l Cell Titer-Glo solution to each well, incubate at room temperature for 30 minutes, and check the chemiluminescence value (luminescence) to measure the level of intracellular ATP. Take the unstimulated DMSO control well as 100% cell viability. Use Prism Graphpad statistical software to calculate compound IC50 value.
  • Figure 4 shows ZSQ13-83 and the like, as well as different concentrations of a control compound to inhibit the growth of ovarian carcinoma cell line - activity profile and half maximal inhibitory concentration (IC 50).
  • ZSQ12-54, ZSQ13-83, ZSQ17-69 and ZSQ17-86 exhibit nanomolar in PKM2 dependent on the PA-1 cell line A2780 and the IC 50, the boric acid control compound ZSQ20-4 completely inactive.
  • Shikonin has similar activity to ZSQ13-83 on PA-1 and A2780 cell lines, but also exhibits strong growth inhibitory activity on SKOV3 cell lines that do not depend on PKM2;
  • PKM2-IN-1 has weaker activity on PA-1 cell line
  • the PKM2 agonist TEPP-46 did not show obvious cytostatic activity.
  • ZSQ13-83 has better activity than PKM2-IN-1 in inhibiting the growth of PKM2-dependent cells, and its selectivity is better than shikonin.
  • the obtained ZSQ20-4 has no inhibitory activity, which suggests that the compound of the present invention inhibits PKM2 depending on the functional group containing arsenic atoms.
  • ZSQ13-83 can form a covalent bond with Cys474 near the allosteric site of PKM2, and then irreversibly bind to PKM2.
  • ZSQ13-74 is a biotin-labeled derivative of ZSQ13-83, which maintains the activity of the latter to inhibit PKM2. In theory, it can also form a covalent bond with PKM2 in the cell lysate, which can then be micro-linked by streptavidin. Beads (streptavidin beads) are enriched in PKM2.
  • ZSQ13-74 can no longer bind to PKM2 because the binding pocket is already occupied. Therefore, based on the competitive enrichment experiment of ZSQ13-74 and streptavidin microbeads, it can accurately determine whether the PKM2 inhibitor can irreversibly bind to PKM2 in living cells.
  • the adopted biological test protocol is: through living cell treatment and competitive binding pull-down test, it is tested whether the inhibitor of the present invention irreversibly binds to PKM2 in living cells.
  • Methods Culture PA-1 cells in vitro, after they grow to logarithmic growth phase, digest and collect the cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, and adjust the cell concentration to 1 ⁇ 106/mL.
  • a 12-well cell culture plate add 1ml of cells to each well, add 1 ⁇ L of DMSO solution (final concentration of 10 ⁇ M) of a representative drug with a concentration of 10mM to each well, control with DMSO, and place in a cell incubator (37°C, 5% CO2) After incubating for 4 hours, wash twice with pre-cooled PBS solution, add 200 ⁇ L of NP40 cell lysate and protease inhibitor to the well, and lyse at 4°C for 30 minutes.
  • Figure 5 shows the competitive inhibition of ZSQ13-74 covalently bound to PKM2 in PA-1 cells by representative compounds such as ZSQ13-83, ZSQ17-69 and ZSQ17-80 at a concentration of 10 ⁇ M.
  • ZSQ13-74 can effectively covalently bind to and enrich PKM2 in PA-1 cell lysate
  • ZSQ13-83, ZSQ17-69 and ZSQ17-80 can all covalently bind PKM2 protein in PA-1 living cells, so that ZSQ13-74 can no longer be effectively enriched, indicating that the combination of the compound of the present invention and PKM2 is covalent/ Irreversible inhibition.
  • the compounds of the present invention are PKM2 allosteric inhibitors, and allosteric inhibitors often require higher concentrations to effectively inhibit the target protein.
  • this example further proves that the PKM2 inhibitor of the present invention has a covalent inhibitory mechanism, which can enhance its binding force through covalent action, and then can perform allosteric inhibition at a lower concentration, ensuring that the PKM2 inhibitor has a covalent inhibition mechanism. Effective suppression.
  • the adopted biological test protocol is: treat PA-1 cells with ZSQ13-83, and test the phosphorylation level of AMPK and ERK in the cells, and the changes in the expression level of c-MYC.
  • Methods Culture PA-1 cells in vitro, after they grow to logarithmic growth phase, digest and collect the cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, and adjust the cell concentration to 1 ⁇ 106/mL.
  • a 12-well cell culture plate add 1ml of cells to each well, add 1 ⁇ L of DMSO solution of different concentrations of drugs to each well, control with DMSO, place it in a cell culture incubator (37°C, 5% CO 2 ) and incubate for 8 hours.
  • Figure 6 shows the effect of ZSQ13-83 on the phosphorylation level of AMPK and ERK (p-AMPK, p-ERK) and the expression level of c-MYC in PA-1 cells at a concentration of 1 ⁇ M.
  • ZSQ13-83 can effectively activate AMPK in PA-1 cells at a concentration of 1 ⁇ M, and the activation time point is about 12 hours later; after treatment with ZSQ13-83, ERK phosphorylation responds Acute upregulation, and then 12 hours later (after AMPK activation) again to the level before dosing; after 16 hours of treatment with ZSQ13-83, the protein expression level of c-MYC was also significantly inhibited;
  • ZSQ13-83 can effectively inhibit the growth of PA-1 cells by inhibiting PKM2 in PA-1 cells and at the same time interfering with PKM2-related energy supply and transcription pathways.
  • Activity Test Example 6 and Figure 5 show that inhibitors such as ZSQ13-83 can covalently bind to the PKM2 protein in cancer cells.
  • the enrichment level of PKM2 protein and other proteins can be compared based on the enrichment experiment of ZSQ13-74 and streptavidin beads.
  • the adopted biological test scheme is to test the selectivity of the diffracted biotin probe enriched protein of the inhibitor of the present invention through the covalent binding pull-down test.
  • Figure 7 shows the protein enrichment level of the representative compound ZSQ13-83 biotin probe ZSQ13-74 at a concentration of 10 ⁇ M in 293T cells expressing PKM2 exogenously.
  • Activity Test Example 8 and Figure 7 show that the PKM2 inhibitor ZSQ13-83 can promote the activation of AMPK signaling pathway in cancer cells, which means that ZSQ13-83 is likely to block the energy supply of cancer cells.
  • ZSQ13-83 is likely to block the energy supply of cancer cells.
  • the biological test protocol used is: treat PA-1 cells with ZSQ13-83, and test the glycolysis function of cancer cells based on the extracellular acidification rate (reflecting the level of lactic acid secretion), and use mass spectrometry metabolomics to test the key Relative levels of metabolites.
  • the PA-1 cells were cultured overnight in a 10 cm dish at a concentration of 4 ⁇ 10 6 /dish, and then each dish was added with different concentrations of ZSQ13-83, compared with DMSO, and placed in a cell incubator (37°C, 5% CO 2 ) Incubate for 12 hours.
  • the cells were collected, washed and suspended in 200 ⁇ L of distilled water, sonicated at 4°C for 10 minutes, then quickly frozen in liquid nitrogen for 1 minute, then returned to room temperature, and repeated freezing and thawing 4 times.
  • the supernatant was mixed with methanol/acetonitrile (1:1) in a ratio of 1:4, sonicated at 4°C for 10 minutes, frozen at -20°C for 1 hour, and centrifuged to collect the supernatant.
  • the supernatant was freeze-dried and re-dissolved in 100 ⁇ L methanol/acetonitrile (1:1). The supernatant was collected after sonication and centrifugation, and the relative level of metabolites was analyzed by a liquid phase-mass spectrometer.
  • Figure 8A shows the effect of ZSQ13-83 on the phosphorylation level of PA-1 extracellular acidification rate at different concentrations
  • Figure 8B shows the effect of ZSQ13-83 on the glycolysis process in PA-1 cells at different concentrations The impact of key metabolite levels.
  • the nude mouse tumor formation experiment is a method of studying tumors and their treatment in vivo, that is, injecting human tumor cells under the skin of nude mice to observe tumor growth and even the effects of anti-tumor drugs.
  • a blank dispensed solvent was used as a control, and the volume of subcutaneous tumors and the body weight of nude mice were monitored at the same time.
  • Ovarian cancer is the gynecological malignant tumor with the highest fatality rate, which is a serious hazard to women’s health. More than 70% of patients are in the middle and advanced stages when they are diagnosed. The clinical benefit has reached a plateau.
  • Method Use ovarian cancer cell HEY to inject 1X10 6 cells/mouse into the right dorsal side of nude mice, observe the growth of the subcutaneous tumor every day, and prepare for administration when the tumor grows to a suitable size.
  • the compound to be tested was dissolved in a dispensing solvent (0.5% hypromellose aqueous solution) on the same day to make a 10 mg/mL solution.
  • Nude mice injected with HEY cells were divided into administration group and blank group. The administration group was given 50mg/kg by gavage, while the control group was given a blank solvent by gavage, and the subcutaneous tumor volume was monitored every other day.
  • Figure 9 shows the inhibitory effect of a representative compound ZSQ13-83 on the growth of subcutaneous tumors in HEY nude mice and its effect on the body weight of nude mice. It can be seen from Figure 9 that at a dose of 50 mg/kg intragastrically, compound ZSQ13-83 can effectively slow down the growth rate of subcutaneous tumors of ovarian cancer cells (Figure 9A-C) without obvious toxic side effects (Figure 9D).

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Abstract

本发明提供了一种基于有机砷的II型丙酮酸激酶抑制剂及其制备方法和用途。具体地，本发明的PKM2抑制剂为式I化合物、或其药学上可接受的盐、或其立体异构体或互变异构体、水合物或溶剂化物；其中，X1、X2、R1、R2、R3、R4、R5和n如说明书所定义。本发明的化合物对PKM2具有高抑制活性和高特异性，非常适合用于制备通过抑制PKM2通路从而抑制肿瘤的药物。

Description

一类基于有机砷的II型丙酮酸激酶抑制剂及其制备方法和用途 技术领域
本发明涉及医药领域，具体涉及一类基于有机砷的II型丙酮酸激酶(PKM2)抑制剂及其制备方法和其用于抑制肿瘤的用途。
背景技术
肿瘤细胞无论氧气存在与否都主要依赖糖酵解进行糖代谢。糖酵解不仅为肿瘤细胞的生存、增殖提供所需ATP，同时为无序快速增殖的癌细胞提供合成所需的氨基酸、核酸等大分子的必需中间体。
丙酮酸激酶是糖酵解过程中的关键限速酶，人体中，丙酮酸激酶具有四种同工型：PKM1、PKM2、PKL和PKR，其中PKM1和PKM2是由同一基因PKM编码的两种亚型。PKM1主要表达于心脏大脑和肌肉组织，而PKM2特定地高表达于胚胎细胞、成体干细胞和癌细胞等多种增殖细胞中。随着胚胎的形成，PKM2被不同的同工酶替代，而在肿瘤形成过程中，其他同工酶消失，PKM2酶则高度表达。实验证明，PKM2在肿瘤瓦尔堡(Warburg)效应中扮演了重要角色，不但为癌细胞直接提供了ATP，其催化产物丙酮酸也是肿瘤中供能的三羧酸循环或乳酸的重要原料，所以PKM2是肿瘤细胞快速增殖的必需因子，与肿瘤的恶性程度、肿瘤的转移和肿瘤的发展等密切相关。
大量研究已经证实PKM2在肿瘤进展中扮演者极其重要的角色，靶向PKM2的小分子抑制剂具有治疗或减缓癌症的作用。然而，现已报道的PKM2抑制剂很多具有活性或特异性上的缺陷。如目前最为知名的PKM2抑制剂紫草素(Shikonin)，被报道会抑制氯离子通道，并且抑制多种免疫应答通路，这增加了其产生不良反应的风险。
因此，本领域急需提供更多高活性、高特异性的PKM2抑制剂。
发明内容
本发明的目的是提供一种高活性、高特异性的PKM2抑制剂。
本发明第一方面提供了一种式I化合物、或其药学上可接受的盐、或其立体异构体或互变异构体、水合物或溶剂化物；
Figure PCTCN2021099500-appb-000001
其中，所述X 1、X 2各自独立地选自下组：无、O、S、NR a、CR bR c；
R a、R b和R c各自独立的选自：H或取代或未取代地C1-C6烷基；
R 1、R 2各自独立地选自下组：H、取代或未取代的C1-C6烷基、取代或未取代的C3-C8环烷基或-(C＝O)-R d，其中，R d为取代或未取代的C1-C6烷基、取代或未取代的C3-C8环烷基；或R 1、R 2与相连的X 1、X 2和As共同形成取代或未取代的4至8元杂环，所述杂环含有一个As杂原子以及0-3个选自N、O和S的杂原子；
R 3选自下组：H、取代或未取代的C1-C6烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、取代或未取代的C3-C8环烷基或取代或未取代的C1-C3亚烷基-R e；
R e选自下组：-CO-NR fR g、取代或未取代的6-10元芳基、取代或未取代的具有1-3个选自O、N和S的杂原子的5-10元杂芳基、取代或未取代的C3-C10环烷基或取代或未取代的具有1-3个选自O、N和S的杂原子的3-10元杂环烷基；所述取代是 指基团上的一个或多个氢原子被选自下组的基团取代：卤素、-OH、-CN、-NH 2、取代或未取代的C1-C6烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、取代或未取代的C1-C6烷氧基、-(C＝O)-取代或未取代的C1-C6烷基、-NR h-(C＝O)-取代或未取代的C1-C6烷基、取代或未取代的C3-C10环烷基、取代或未取代的具有1-3个选自O、N和S的杂原子的3-10元杂环烷基、-C1-C4亚烷基-(O-CH 2-CH 2)m-NR f-(C＝O)-R i或-(C＝O)-CH 2(O-CH 2-CH 2)m-NR f-(C＝O)-R i；
n为0、1、2、3或4；
各个R 4独立地选自：H、卤素、D、OH、-NH 3、-NO2、-CN、COOH、取代或未取代的C1-C6烷基、取代或未取代的C1-C6烷氧基、-OCO-取代或未取代的C1-C6烷基；
R 5选自下组：取代或未取代的C1-C6烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、取代或未取代的6-10元芳基、取代或未取代的具有1-3个选自O、N和S的杂原子的5-10元杂芳基、取代或未取代的C3-C10环烷基、或取代或未取代的具有1-3个选自O、N和S的杂原子的3-10元杂环烷基、-C1-C4亚烷基-(O-CH 2-CH 2)m-NR f-CO-R i或
Figure PCTCN2021099500-appb-000002
其中，环A选自下组：取代或未取代的C3-C10环烷基、取代或未取代的具有1-4个选自O、N和S的杂原子的3-10元杂环烷基、取代或未取代的6-10元芳基、取代或未取代的具有1-4个选自O、N和S的杂原子的5-10元杂芳基；
L 1选自下组：无、取代或未取代的C1-C6亚烷基、取代或未取代的C2-C6亚烯基、取代或未取代的C2-C6亚炔基或取代或未取代的C1-C6亚烷基-(C＝O)-；
环B选自下组：取代或未取代的C3-C10环烷基、或取代或未取代的具有1-3个选自O、N和S的杂原子的3-10元杂环烷基、取代或未取代的6-10元芳基或取代或未取代的具有1-3个选自O、N、S的杂原子的5-10元杂芳基；其中，所述取代是指基团上的一个或多个氢原子被选自下组的取代基取代：卤素、-OH、-CN、-NH 2、C1-C6烷氧基、C1-C6烷基、C2-C6烯基、C2-C6炔基、C1-C6卤代烷基、-CO-NR fR g、-NR fR g；
各m独立地为0、1、2、3、4或5；
各R i独立地选自下组：H、取代或未取代的C1-C6烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、-L 2-R 9；
其中，L 2选自下组：取代或未取代的C1-C6亚烷基、取代或未取代的C2-C6亚烯基、取代或未取代的C2-C6亚炔基；
R 9选自下组：取代或未取代的C3-C10环烷基、或取代或未取代的具有1-3个选自O、N和S的杂原子的3-10元杂环烷基、取代或未取代的6-10元芳基、取代或未取代的具有1-3个选自O、N和S的杂原子的5-10元杂芳基；
如未特别说明，所述“取代”是指基团上的一个或多个氢原子被选自下组的基团取代：卤素、-OH、-CN、-NR fR g、-NR f-CO-C1-C6烷基、-C1-C6烷基-CO-NR fR g、氧代(＝O)、C1-C6烷基、C1-C6氧杂烷基、C2-C6烯基、C2-C6炔基、C1-C6烷氧基、C1-C6卤代烷基、-(C＝O)-C1-C6烷基、C5-C10芳基、苄基、3-8元环烷基；和
各R f和R g独立地选自下组：H、C1-C6烷基、C2-C6烯基、C2-C6炔基、C3-C8环烷基或具有1-3个选自O、N和S的杂原子的3-10元杂环烷基，或R f与R g与相连的氮原子一起形成具有一个氮原子的3-7元环烷基。
在另一优选例中，各基团独立的选自表1化合物中对应的基团。
在另一优选例中，X 1和X 2都为O或都为S。
在另一优选例中，
Figure PCTCN2021099500-appb-000003
具有选自下组的结构：
Figure PCTCN2021099500-appb-000004
在另一优选例中，R 5为
Figure PCTCN2021099500-appb-000005
或取代或未取代的选自下组的基团：
Figure PCTCN2021099500-appb-000006
在另一优选例中，环A为取代或未取代的选自下组的基团：
Figure PCTCN2021099500-appb-000007
在另一优选例中，L 1与环A上的N原子相连。
在另一优选例中，L 1选自下组：-C1-C4亚甲基、-CH 2(C＝O)-。
在另一优选例中，环B选自下组：取代或未取代的6-10元芳基，较佳地，取代或未取代的苯基。
在另一优选例中，L 1为-CH 2-且环B为取代或未取代的苯基。
在另一优选例中，L 2为-C1-C4亚甲基，较佳地，-CH 2-。
在另一优选例中，R e为取代或未取代的选自下组的基团：
Figure PCTCN2021099500-appb-000008
在另一优选例中，R e为取代或未取代的选自下组的基团：
Figure PCTCN2021099500-appb-000009
其中，各Rj独立地选自下组：H、C1-C4烷氧基、C1-C4烷基、C1-C4卤代烷基、-(C＝O)-C1-C4烷基、羟基取代的C1-C4烷基，-C1-C4亚烷基-(O-CH 2-CH 2)m-NH-(C＝O)-R i或-(C＝O)-CH 2(O-CH 2-CH 2)m-NH-(C＝O)-R i，m和Ri如上定义；
所述取代指基团的环碳原子上的一个或多个氢被选自下组的基团取代：卤素、氰基、C1-C4烷氧基、C1-C4烷基、-(C＝O)-C1-C4烷基、羟基取代的C1-C4烷基、-NHCO-C1-C4烷基。
在另一优选例中，所述R e具有选自下组的结构：
Figure PCTCN2021099500-appb-000010
其中，Rj独立地选自下组：H、C1-C4烷基、C1-C4卤代烷基、-(C＝O)-C1-C4烷基、羟基取代的C1-C4烷基，较佳地，Rj选自下组：-CH 3、-CH 2CH 3、-CH 2CH 2OH、-(C＝O)CH 3。
在另一优选例中，所述式I化合物具有式Ia或Ib的结构：
Figure PCTCN2021099500-appb-000011
其中，所述X 1、X 2、R 1、R 2、R 3、R 4、R i、A、B、L 1、n、m如上定义。
在另一优选例中，各R 9为
Figure PCTCN2021099500-appb-000012
在另一优选例中，R i为-C4烷基
Figure PCTCN2021099500-appb-000013
在另一优选例中，所述式Ia化合物具有IIa结构：
Figure PCTCN2021099500-appb-000014
其中，R k选自下组：C1-C4烷基、C1-C4卤代烷基、-(C＝O)-C1-C4烷基、羟基取代的C1-C4烷基；
所述X 1、X 2、R 1、R 2、R 4、A、B、L 1、n如上定义。
在另一优选例中，R k选自下组：-CH 3、-CH 2CH 3、-CH 2CH 2OH、-(C＝O)CH 3。
在另一优选例中，所述化合物选自表1中的下组化合物：
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、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和ZSQ-19-99。
在另一优选例中，所述化合物选自表1中的下组化合物：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-13-74、ZSQ-13-83、ZSQ-13-100、ZSQ-13-101、ZSQ-17-69、ZSQ-17-86、ZSQ-20-29和ZSQ-20-30，较佳地，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和ZSQ-20-30。
本发明第二方面提供了一种药物组合物，所述药物组合物包括：(a)如本发明第一方面所述的式I化合物、或其药学上可接受的盐、或其立体异构体或互变异构体、水合物或溶剂化物；和(b)药学上可接受的载体。
本发明第三方面提供了如本发明第一方面所述的式I化合物、或其药学上可接受的盐、或其立体异构体或互变异构体、水合物或溶剂化物或如本发明第二方面所述的药物组合物的用途，用于制备一药物或制剂，所述药物制剂用于：
(i)下调和/或抑制PKM2活性；
(ii)预防和/或治疗肿瘤；
(iii)降低和/或抑制肿瘤细胞的生长和/或增殖；
(iv)降低和/或抑制肿瘤细胞的转移；
(v)促进肿瘤细胞凋亡；和/或
(vi)降低和/或抑制细胞的糖酵解过程。
在另一优选例中，所述药物或制剂用于通过下调和/或抑制PKM2活性从而预防和/或治疗癌症。
在另一优选例中，所述药物或制剂为PKM2抑制剂。
在另一优选例中，所述肿瘤为PKM2依赖型肿瘤。
在另一优选例中，所述的“PKM2依赖型肿瘤”指肿瘤细胞中PKM2高表达的肿瘤和/或依赖PKM2进行糖酵解的肿瘤。
在另一优选例中，所述肿瘤选自下组：肝癌、胃癌、乳腺癌、胰腺癌、卵巢癌(包括人卵巢畸胎瘤)、喉癌(包括喉鳞状细胞癌)、前列腺癌、结肠癌、直肠癌、胰腺癌、宫颈癌、子宫内膜癌、脑癌、膀胱癌、睾丸癌、头癌、颈癌、皮肤癌(包括黑色素瘤和基底细胞癌)、间皮内膜癌、白血球癌(包括淋巴瘤和白血病)、食道癌、肌癌、结缔组织癌、肺癌(包括小细胞肺癌和非小细胞肺癌)、肾上腺癌、甲状腺、肾癌或骨癌、胶质母细胞瘤、间皮瘤、肾细胞癌、胃癌、肉瘤(卡波西肉瘤)、绒毛膜癌、皮肤基底细胞癌和睾丸精原细胞瘤。
在另一优选例中，所述肿瘤选自下组：肝癌、胃癌、乳腺癌、胰腺癌、卵巢癌(包括人卵巢畸胎瘤)、喉癌(包括喉鳞状细胞癌)、前列腺癌、结肠癌、直肠癌、胰腺癌、宫颈癌、子宫内膜癌、脑癌、膀胱癌、睾丸癌、头癌和颈癌。
本发明第四方面提供了一种PKM2抑制剂，所述抑制剂包括：如本发明第一方面所述的式I化合物、或其药学上可接受的盐、或其立体异构体或互变异构体、水合物或溶剂化物。
本发明第五方面提供了一种体外非治疗性的下调和/或抑制PKM2活性的方法，在如本发明第一方面所述的式I化合物、或其立体异构体或互变异构体、或其药学上可接受的盐、水合物或溶剂化物、如本发明第二方面所述的药物组合物存在下，培养细胞，从而在所述细胞中下调和/或抑制PKM2的活性。
在另一优选例中，所述细胞为肿瘤细胞。
在另一优选例中，所述肿瘤为PKM2依赖型肿瘤。
在另一优选例中，所述式式I化合物、或其立体异构体或互变异构体、或其药学上可接受的盐、水合物或溶剂化物的有效浓度为0.01nM/L-1mM/L，较佳地为0.1nM/L-500μM/L，最佳地为1nM/L-100μM/L。
本发明第六方面提供了一种预防和/或治疗肿瘤的方法，给有需要的对象施用治疗有效量的如本发明第一方面所述的式I化合物、或其立体异构体或互变异构体、或其药学上可接受的盐、水合物或溶剂化物、如本发明第二方面所述的药物组合物。
在另一优选例中，所述对象为哺乳动物，较佳地，人、大鼠、小鼠、猫、狗、牛或马。
在另一优选例中，所述肿瘤为PKM2依赖型肿瘤。
本发明第七方面，提供了如本发明第一方面所述式I化合物的制备方法，包括步骤：
步骤i：将A1的亚砷酸官能团以酯或酰胺的方式保护；
步骤ii：通过缩合反应在A2的胺基引入羰基连接的R 5取代基；和
步骤iii：通过S N2取代反应在A3的胺基引入R 3取代基；
Figure PCTCN2021099500-appb-000015
其中，所述X 1、X 2、R 1、R 2、R 4、R 5和n如本发明第一方面所定义。
在另一优选例中，化合物A2的胺基先进行R 3基团的引入，再进行羰基连接的R 5取代基的引入。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了代表性化合物及对照化合物体外抑制PKM2的浓度-活性曲线及其半数抑制浓度(IC 50)。
图2显示了代表性化合物不同浓度下对PKM1的体外酶活性的抑制效果。
图3显示了ZSQ13-83与PKM2蛋白474位半胱氨酸所在肽链共价结合后的质谱谱图。
图4显示了ZSQ13-83及其类似物、以及对照化合物抑制不同卵巢癌细胞株生长的浓度-活性曲线及其半数抑制浓度(IC 50)。
图5显示了代表性化合物如ZSQ13-83、ZSQ17-69和ZSQ17-80在10μM浓度下对ZSQ13-74共价结合PA-1细胞中PKM2的竞争性抑制。
图6显示了ZSQ13-83在10μM浓度下对PA-1细胞中AMPK和ERK的磷酸化水平(p-AMPK、p-ERK)、以及c-MYC表达水平的影响。
图7显示了代表性化合物ZSQ13-83的生物素探针ZSQ13-74在10μM浓度下，对外源表达PKM2的293T细胞中蛋白的富集水平。
图8显示了ZSQ13-83对癌细胞中葡萄糖代谢通路的影响；A显示了ZSQ13-83在不同浓度下，对PA-1细胞外酸化率的磷酸化水平的影响；B显示了ZSQ13-83在不同浓度下，对PA-1细胞中糖酵解过程关键代谢物水平的影响。
图9显示了代表性化合物ZSQ13-83对HEY裸鼠皮下肿瘤生长的抑制效果，以及对裸鼠体重的影响。
具体实施方式
本发明人经过广泛而深入的研究，通过大量筛选和测试，提供了一类基于有机砷的PKM2抑制剂。本发明的化合物具有抑制PKM2的高活性和高特异性，非常适合用于制备通过抑制PKM2通路从而抑制肿瘤的药物。在此基础上完成了本发明。
术语
除非另有定义，否则本文中所用的全部技术术语和科学术语均具有如本发明所属领域普通技术人员通常理解的相同含义。
如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
除非另有表述，术语“烷基”本身或作为另一取代基的一部分是指具有指定碳原子数的直链或支链烃基(即，C1-6表示1-6个碳，)典型地，烷基包括具有1、2、3、4、5、6个碳原子的烷基。烷基的例子包括甲基、乙基、正丙基、异丙基、正丁基、叔丁基、异丁基、仲丁基、正戊基、正己基等。
除非另有表述，术语“烯基”指具有一个或多个双键的不饱和烷基，典型地，烯基包括具有2、3、4、5、6个碳原子的烯基。类似地，术语“炔基”指具有一个或多个三键的不饱和烷基，典型地，炔基包括具有2、3、4、5、6个碳原子的炔基。此类不饱和烷基的例子包括乙烯基、2-丙烯基、巴豆基、2-异戊烯基、2-(丁二烯基)、2,4-戊二烯基、3-(1,4-戊二烯基)、乙炔基、1-和3-丙炔基、3-丁炔基和更高级的同系物和异构体。
除非另有表述，术语“环烷基”是指具有指定环原子数(例如，C 3-10环烷基，C3-7环烷基)并且完全饱和的或在环顶之间具有不超过一个双键的烃环。典型地，环烷基包括3、4、5、6、7、8、9或10个环碳原子的环烷基。其可以是单环，例如环丙基、环丁基、环戊基、环己基、或类似基团。也可以是双环和多环烃环，例如桥环或螺环，如双环[2.2.1]庚烷、双环[2.2.2]辛烷等。
除非另有表述，术语“杂环烷基”是指含有指定个数(如1、2或3个)选自O、N和S的杂原子的环烷基，其中氮和硫原子任选被氧化，且氮原子任选被季铵化。杂环烷基可以是单环、双环或多环体系。典型地，杂环烷基包括3、4、5、6、7、8、9或10个环原子的杂环烷基。杂环烷基的非限制性例子包括吡咯烷、咪唑烷、吡唑烷、丁内酰胺、戊内酰胺、咪唑烷酮、乙内酰脲、二氧戊环、苯邻二甲酰亚胺、哌啶、1,4-二噁烷、吗啉、硫代吗啉、硫代吗啉-S-氧化物、硫代吗啉-S,S-氧化物、哌嗪、吡喃、吡啶酮、3-吡咯啉、噻喃、吡喃酮、四氢呋喃、四氢噻吩、奎宁环等。杂环烷基可以经环碳或杂原子连接于分子的其余部分。对于诸如环烷基烷基和杂环烷基烷基的术语，是指环烷基或杂环烷基通过烷基或亚烷基连接体连接到分子的其余部分。例如，环丁基甲基-是连接到分子其余部分的亚甲基连接基上的环丁基环。
除非另有表述，术语“亚烷基”本身或作为另一取代基的一部分是指衍生自烷烃的二价基团，典型地，亚烷基包括具有1、2、3、4、5、6个碳原子的亚烷基，例如-CH 2-、-CH 2CH 2CH 2CH 2-。类似地，“亚烯基”或“亚炔基”分别指具有双键或三键的不饱和形式的“亚烷基”，典型地，亚烯基包括具有2、3、4、5、6个碳原子的亚烯基，亚炔基包括具有2、3、4、5、6个碳原子的亚炔基。
类似地，除非另有说明，术语“杂烷基”指含有指定数目的碳和1至3个选自O、N和S的杂原子，且其中氮和硫原子可选地被氧化，氮杂原子可任选地被季铵化。杂 原子O，N和S可以位于杂烷基的任何内部位置。
除非另有表述，术语"烷氧基"或“烷基氧基”、"烷胺基""或“烷基胺基”以其常规意义使用，指代分别经氧原子或氮连接于分子的其余部分的那些烷基。此外，所述烷胺基可以是单取代或双取代的。例如，甲胺基、乙胺基、丙胺基、异丙胺基、丁胺基、异丁胺基、叔丁胺基、二甲胺基、二乙胺基、二丙胺基、二异丙胺基、二丁胺基、二异丁胺基、二叔丁胺基等。双取代时烷基部分可以相同或不同，也可和与各烷基相连的氮原子组合形成3-7元环。因此，-NR fR g所示基团表示包括哌啶基、吡咯烷基、吗啉基、氮杂环丁烷基(azetidinyl)等。
除非另有表述，术语“酰基”，单独或作为另一基团的一部分使用，是指其中在最接近该基团的连接点的碳上两个取代基的被取代基＝O取代(例如-(C＝O)CH 3(或-CO-CH 3),-(C＝O)CH 2CH 3等)。另外，术语“羰基亚烷基”或“亚烷基羰基”包括通过羰基碳原子或亚烷基碳原子连接至分子其余部分的那些基团，如亚甲基羰基包括“-(C＝O)-CH 2-”和“-CH 2-(C＝O)-”的形式。
除非另有表述，术语“卤代”或“卤素”本身或作为另一取代基的一部分是指氟、氯、溴、或碘原子。此外，诸如“卤代烷基”等术语表示包括单卤代烷基或多卤代烷基。例如，术语“C 1-6卤代烷基”表示包括三氟甲基、2,2,2-三氟乙基、4-氯丁基、3-溴丙基等。
除非另有表述，术语“芳基”表示多不饱和的(通常芳香性)的烃基，其可以是单环或稠合在一起或共价连接的多环(最多三环)，芳基的非限制性例子包括苯基、萘基和联苯基。
术语"杂芳基"是指含有指定个数(如1、2或3个)选自O、N和S的杂原子的芳基(或环)，其中氮和硫原子任选被氧化，氮原子任选被季铵化。典型地，杂芳基包括5、6、7、8、9或10个环原子的杂芳基。杂芳基可通过杂原子连接于分子的其余部分。杂芳基的非限制性例子包括吡啶基、哒嗪基、吡嗪基、嘧啶基、三嗪基、喹啉基、喹喔啉基、喹唑啉基、噌啉基、酞嗪基、苯并三嗪基(benzotriazinyl)、嘌呤基、苯并咪唑基、苯并吡唑基、苯并三唑基、苯并异噁唑基、异苯并呋喃基(isobenzofuryl)、异吲哚基、中氮茚基、苯并三嗪基、噻吩并吡啶基、噻吩并嘧啶基、吡唑并嘧啶基、咪唑并吡啶、苯并噻唑基、苯并呋喃基、苯并噻吩基、吲哚基、喹啉基、异喹啉基、异噻唑基、吡唑基、吲唑基、蝶啶基、咪唑基、三唑基、四唑基、噁唑基、异噁唑基、噻二唑基、吡咯基、噻唑基、呋喃基、噻吩基等等。
在一些实施例中，上述术语(如“烷基”，“芳基”和“杂芳基”)将包括指定基团的取代和未取代形式，取代基个数可以为1、2、3或4。如未特别说明，所述“取代”是指基团上的一个或多个氢原子被选自下组的基团取代：卤素、-OH、-CN、-NR fR g、-NRa-CO-C1-C6烷基、-C1-C6烷基-CO-NR fR g、氧代(＝O)、C1-C6烷基、C1-C6氧杂烷基、C2-C6烯基、C2-C6炔基、C1-C6烷氧基、C1-C6卤代烷基、-(C＝O)-C1-C6烷基、C5-C10芳基、苄基、3-8元环烷基。除非特别说明，各R f和R g独立地选自下组：H、C1-C6烷基、C2-C6烯基、C2-C6炔基、C3-C8环烷基或具有1-3个选自O、N和S的杂原子的3-10元杂环烷基，或R f与R g与相连的氮原子一起形成具有一个氮原子的3-7元环烷基。
如本文所用，术语“杂原子”意在包括氧(O)、氮(N)和硫(S)。
对于本文提供的化合物，
Figure PCTCN2021099500-appb-000016
表示与其他原子的连接位点。从取代基(通常为R基团)到芳香环(例如苯，吡啶等)的中心的键将被理解为是指在芳香环的任何可用顶点提供连接的键。在一些实施例中，该描述也包括稠合在芳环上的环上的连接。例如，绘制到吲哚苯部分的中心的键将表示与吲哚的六元或五元环部分的任何可用顶点连接的键。如果对于一个环状基团，没有指明其连接位点，则表示可以通过环上任何可用顶点连接(包括碳原子或杂原子)。
除非特别说明，本发明中，所有出现的化合物均意在包括所有可能的光学异构体，如单一手性的化合物，或各种不同手性化合物的混合物(即外消旋体)。本发明的所有化合物之中，各手性碳原子可以任选地为R构型或S构型，或R构型和S构型的混合物。
本发明的某些化合物拥有不对称碳原子(光学中心)或双键；消旋体、非对映体、几何异构体、区域异构体和单独的异构体(例如，分离的对映体)均应包括在本发明范围内。当本文提供的化合物具有确定的立体化学(表示为R或S，或具有虚线或楔形键指明)时，被本领域技术人员将理解那些化合物为基本上不含其他异构体(例如至少80％，90％，95％，98％，99％和至多100％不含其他异构体)。
如本文所用，术语“药学上可接受的”成分是指适用于人和/或动物而无过度不良副反应(如毒性、刺激和变态反应)，即有合理的效益/风险比的物质。
如本文所用，术语“治疗有效量”是指药物的任何如下所述的量，当单独使用或与另一种治疗剂组合使用该量的药物时，可促进疾病消退，疾病消退表现为疾病症状的严重度降低、无疾病症状期的频率和持续时间增加、或者防止由患病导致的障碍或失能。本发明药物的“治疗有效剂量”也包括“预防有效剂量”，“预防有效剂量”是药物的任何如下所述的量，当将该量的药物单独施用或者与另一种治疗剂组合施用于具有发生疾病的风险或者遭受疾病复发的受试者时，可抑制疾病的发生或复发。
如本文所用，术语“溶剂合物”是指本发明化合物与溶剂分子配位形成特定比例的配合物。
如本文所用，术语“水合物”是指本发明化合物与水进行配位形成的配合物。
本发明还包括本发明化合物的所有合适的同位素变体。本发明化合物的同位素变体被定义为其中至少一个原子被具有相同原子数但原子质量不同于自然界中常见的原子质量的原子替代的那些。可并入本发明化合物的同位素的实例包括氢、碳、氮、氧、磷、硫、氟和氯的同位素，分别例如 2H、 3H、 11C、 13C、 14C、 15N、 17O、 18O、 35S、 18F和 36Cl。本发明的一些同位素变体，例如，其中并入放射性同位素(例如 3H或 14C)的那些，被用于药物和/或底物组织分布研究。氚代的，即， 3H，和碳-14，即， 14C，同位素是特别优选的，因为它们易于制备和检测。此外，用同位素(例如氘，即， 2H)的取代，可提供由增加的代谢稳定性引起的一些治疗优势，例如，增加的体内半衰期或降低的剂量需求并因此在一些情况下可能是优选的。本发明化合物的同位素变体通常可通过常规操作制备，例如使用适当的同位素变体的合适试剂，通过示例性的方法或下文实验部分中描述的制备。
如本文所用，“治疗”是指减轻、延缓进展、衰减、预防，或维持现有疾病或病症(例如癌症)。治疗还包括将疾病或病症的一个或多个症状治愈、预防其发展或减轻到某种程度。
活性成分
如本文所用，“本发明化合物”指式I化合物、其药学上可接受的盐、或其立体异构体或互变异构体、或水合物或溶剂化物；
Figure PCTCN2021099500-appb-000017
其中，X 1、X 2、R 1、R 2、R 3、R 4、R 5和n如本发明第一方面定义。
如本文所用，“药学上可接受的盐”指本发明化合物与酸或碱所形成的适合用作药物的盐。药学上可接受的盐包括无机盐和有机盐。一类优选的盐是本发明化合物与酸形成的盐。适合形成盐的酸包括但并不限于：盐酸、氢溴酸、氢氟酸、硫酸、硝酸、磷酸等无机酸，甲酸、乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、苦味酸、甲磺酸、苯甲磺酸，苯磺酸等有机酸；以及天冬氨酸、谷氨酸等酸性氨基酸。一类优选的盐是本发明化合物与碱形成的盐。适合形成盐的碱包括但并不限于：氢氧化钠、氢氧化钾、碳酸钠、碳酸氢钠、磷酸钠等无机碱，氨水、三乙胺、二乙胺、哌嗪等有机碱。
制备方法
本发明提供了式I化合物的制备方法，本发明中的化合物可以通过多种合成操作制备，这些化合物的示例性制备方法可以包括(但不限于)下文所述的流程。
较佳地，本发明式I化合物可以通过以下方案及实施例中所述的示例性方法以及本领域技术人员所用的相关公开文献操作完成。
在具体操作过程中，可以根据需要对方法中的步骤进行扩展或合并。
方案1：包括步骤：
Figure PCTCN2021099500-appb-000018
其中，所述X 1、X 2、R 1、R 2、R 4、R 5和n如上定义。
更具体地，包括步骤：
步骤i：将A1的亚砷酸官能团以酯或酰胺的方式进行保护，如使用乙二硫醇、乙二醇等作为保护试剂。
步骤ii：通过缩合反应在A2的胺基引入羰基连接的R 5取代基，如可以使用酰氯、酸、酸酐等(R 5-CO-Cl、R 5-CO-OH)等。
步骤iii：通过S N2取代反应在A3的胺基引入R 3取代基，可以使用R 3-卤素，在碱性条件下(如氢化钠)反应。
方案2：
Figure PCTCN2021099500-appb-000019
步骤i：将A1以的亚砷酸官能团以酯或酰胺的方式保护，可以使用乙二硫醇、乙二醇等作为保护试剂。
步骤ii：通过S N2取代反应或还原胺化反应在A2的胺基引入R 3取代基，可以使用R 3-卤素，在碱性条件下(如氢化钠)反应；或可以使用R 3-甲醛，在还原试剂(如三乙酰氧基硼氢化钠)的参与下反应。
步骤iii：在A3的苯环胺基发生酰化反应引入羰基连接的R 5基团，如可以使用酰氯、酸、酸酐等(R 5-CO-Cl、R 5-CO-OH)等；或通过(但并不限于)三氯氧磷试剂催化。
上述制备方法还可根据需要进行保护基的脱除步骤。
在本发明的方法中可以使用任何合适的惰性溶剂。代表性的惰性溶剂包括但不限于戊烷、不同的戊烷、己烷、不同的己烷、庚烷、不同的庚烷、石油醚、环戊烷、不同的环己烷、苯、甲苯、二甲苯、三氟甲苯，卤代苯如氯苯、氟苯、二氯苯和二氟苯，二氯甲烷、氯仿、DMF、丙酮、乙酸乙酯、二乙醚、四氢呋喃、DMSO，或其组合。
本发明的方法中的反应可以在任何合适的温度下进行。例如，反应温度可以是约-78℃至约100℃，或者为约-50℃至约100℃，或者为约-25℃至约50℃，或约-10℃至约25℃，或约0℃至约20℃。在一些实施方案中，反应温度可以是约0℃至约20℃。以上各步骤中的反应均是本领域技术人员已知的常规反应。如无特殊说明，合成路线中所使用的试剂和原料化合物均可市购得到，或本领域技术人员根据所设计的不同化合物结构参考已知方法制备得到。
药物组合物和施用方法
由于本发明化合物具有优异的对II型丙酮酸激酶(PKM2)的抑制活性，因此本发明化合物及其晶型，药学上可接受的无机或有机盐，水合物或溶剂合物，以及含有本发明化合物为主要活性成分的药物组合物可用于治疗、预防以及缓解肿瘤。更具体地，用于下调或抑制肿瘤细胞的PKM2活性，从而抑制糖酵解过程，从而达到抑制肿瘤的效果。因此本发明的化合物可用于制备PKM2抑制剂。
优选地，上述肿瘤为PKM2依赖型肿瘤。在本发明中，所述PKM2依赖型肿瘤为细胞内PKM2高表达的肿瘤和/或依赖PKM2进行糖酵解过程的细胞。所述“PKM2高表达”指细胞中PKM2占总丙酮酸激酶的60％以上，较佳地，70％以上、80％以上、90％以上或95％以上。所述“依赖PKM2进行糖酵解”指细胞主要通过PKM2催化糖酵解过程，例如细胞内≥60％的糖酵解是通过PKM2催化的，较佳地，≥70％、≥80％、≥90％、或≥95％。
当癌症细胞的糖酵解过程受到抑制时，会减缓或抑制癌细胞的生长、增殖、转移，加速癌细胞的凋亡。
优选地，本发明化合物可用于预防和/或治疗癌症。代表性的癌症包括(但并不限于)：肝癌、胃癌、乳腺癌、胰腺癌、卵巢癌、前列腺癌、结肠癌、直肠癌、胰腺癌、宫颈癌、子宫内膜癌、脑癌、膀胱癌、睾丸癌、头癌、颈癌、皮肤癌(包括黑色素瘤和基底细胞癌)、间皮内膜癌、白血球癌(包括淋巴瘤和白血病)、食道癌、肌癌、结缔组织癌、肺癌(包括小细胞肺癌和非小细胞肺癌)、肾上腺癌、甲状腺、肾癌或骨癌；或胶质母细胞瘤、间皮瘤、肾细胞癌、胃癌、肉瘤(卡波西肉瘤)、绒毛膜癌、皮肤基底细胞癌和睾丸精原细胞瘤。
本发明化合物可以单独给药，或者与其他治疗剂联合给药。这样的治疗剂包括，但不限于，抗癌剂和/或免疫抑制剂，如选自下组的抗癌剂和/或免疫抑制剂：奥拉帕尼、卢卡帕尼、尼拉帕尼、甲氨蝶呤、卡培他滨、吉西他滨、去氧氟尿苷、培美曲塞二钠、帕唑帕尼、伊马替尼、埃罗替尼、拉帕替尼、吉非替尼、凡德他尼、赫赛汀、贝伐单抗、利妥昔单抗、曲妥珠单抗、紫杉醇、长春瑞滨、多西他赛、多柔比星、羟基喜树碱、丝裂霉素、表柔比星、吡柔比星、博来霉素、来曲唑、他莫西芬、氟维司群、曲谱瑞林、氟他胺、亮丙瑞林、阿那曲唑、异环磷酰胺、白消安、环磷酰胺、卡莫司汀、尼莫司汀、司莫司汀、氮芥、马法兰、瘤可宁、卡铂、顺铂、奥沙利铂、络铂、拓扑特肯、喜树碱、拓扑替康、依维莫司、西罗莫斯、特癌适、6-巯基嘌呤、6-硫鸟嘌呤、硫唑嘌呤、菌素D、柔红霉素、阿霉素、米托蒽醌、争光霉素、普卡霉素或氨鲁米特；如纳武单抗(nivolumab)、派姆单抗(pembrolizumab)、伊匹单抗(ipilimumab)、阿维鲁单抗(avelumab)、度伐单抗(durvalumab)、阿特朱单抗(atezolizumab)或皮地利珠单抗(pidilizumab)，或其组合。
在某些实施方式中，向患癌对象联合给予本发明的化合物和其它传统癌治疗物，例如，放疗或手术。放疗是本领域熟知的并且包括X射线治疗，例如伽马放射，和放射药物治疗。
在另一优选例中，所述治疗剂为缺氧诱导因子脯氨酸羟化酶(HIF-PH)抑制剂。
在某些实施方式中，本发明的化合物在同一或分开的制剂中与作为联合治疗方案的其它治疗剂同时使用，或依次使用。
本发明的药物组合物包含安全有效量范围内的本发明化合物或其药理上可接受的盐及药理上可以接受的赋形剂或载体。其中“安全有效量”指的是：化合物的量足以明显改善病情，而不至于产生严重的副作用。通常，药物组合物含有1-2000mg本发明化合物/剂，更佳地，含有10-500mg本发明化合物/剂。较佳地，所述的“一剂”为一个胶囊或药片。
“药学上可接受的载体”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的化合物以及它们之间相互掺和，而不明显降低化合物的药效。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如
Figure PCTCN2021099500-appb-000020
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
本发明化合物或药物组合物的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、瘤内、直肠、肠胃外(静脉内、肌肉内或皮下)、和局部给药。
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性化合物与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、 蔗糖和阿拉伯胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性化合物或化合物的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性化合物也可与上述赋形剂中的一种或多种形成微胶囊形式。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性化合物外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。
除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
除了活性化合物外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
用于局部给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
式I化合物或式I化合物的组合物的治疗有效剂量的一般范围将是：约1-2000mg/天、约10-约1000mg/天、约10-约500mg/天、约10-约250mg/天、约10-约100mg/天，或约10-约50mg/天。治疗有效剂量将以一个或多个剂量给予。然而，应理解，对于任何特定患者的本发明化合物的特定剂量将取决于多种因素，例如，待治疗的患者的年龄、性别、体重、一般健康状况、饮食、个体响应，给予时间、待治疗的疾病的严重性、施用的具体化合物的活性、剂型、应用模式和伴用药物。给定情况的治疗有效量能用常规实验测定，并在临床医生或医师能力和判断范围内。在任何情况中，所述化合物或组合物将基于患者的个体情况以多个剂量给予并以允许递送治疗有效量的方式给予。
本发明的主要优点包括：
1.本发明提供了一类新的PKM2抑制剂。
2.本发明的化合物对PKM2具有高抑制活性和高选择性，在使用时用药量低，且副作用小，安全性高。
3.本发明的化合物为变构型抑制剂，不易产生耐药性；而且本发明的化合物能够与PKM2进行共价结合，确保了低剂量下的有效抑制，特别适合成药。
4.本发明的化合物对PKM2依赖型肿瘤具有显著的抑制效果。
下面结合具体实施，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照 常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
制备实施例
实施例1
化合物ZSQ-5-77的合成路线
Figure PCTCN2021099500-appb-000021
化合物(4-aminophenyl)arsonous acid(ZSQ-1-18)的合成
Figure PCTCN2021099500-appb-000022
将4-氨基苯胂酸(108.5g，500mmol)溶于300ml乙醇中，混合溶液加热至回流。将苯肼(92ml，1mol)逐滴加入(超过1h)混合溶液中，加入过程中有大量氮气产生，当氮气产生减缓时，继续回流搅拌1.5h。混合溶液经过减压蒸馏浓缩，加入氢氧化钠溶液(40g溶于400ml水中)，乙醚洗两遍(400ml)。分液，水相加入饱和氯化铵溶液(400ml)置于0℃下搅拌1h，有大量白色固体析出。过滤得白色针状固体，真空干燥得40g产物，产率40％。
化合物4-(1,3,2-dithiarsolan-2-yl)aniline(ZSQ-1-23)的合成
Figure PCTCN2021099500-appb-000023
将ZSQ-1-18(40g，199mmol)溶解在200ml无水乙醇中并加热至回流。然后，在30min内将乙二硫醇(20ml，240mmol)滴加到该混合溶液中，并继续加热和搅拌30min。随后，将混合物在冰水中冷却并过滤，得到粗产物，将其从乙醇中重结晶， 得到白色固体42g，产率81％。
化合物tert-butyl(S)-2-((4-(1,3,2-dithiarsolan-2-yl)phenyl)carbamoyl)pyrrolidine-1-carboxylate(ZSQ-5-67)的合成
Figure PCTCN2021099500-appb-000024
将ZSQ-1-23(518mg，2mmol)，Boc-L-脯氨酸(430mg，2mmol)，HATU(1.14g，3mmol)和DIPEA(0.92ml，6mmol)溶于6ml DCM中，置于室温混合搅拌3h。反应完成后依次用H 2O(20ml×2)，饱和NaCl溶液(20ml)洗涤，用20ml DCM萃取。合并有机相，用无水硫酸钠干燥，过滤，浓缩后经过硅胶柱层析分离纯化，用PE/EA(3:1)洗脱，得到黄色固体850mg，产率93％。
化合物(S)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)pyrrolidine-2-carboxamide(ZSQ-5-68)的合成
Figure PCTCN2021099500-appb-000025
将ZSQ-5-67(850mg，1.86mmol)和1ml三氟乙酸混合于5ml DCM中，室温过夜搅拌。反应完成后反应液浓缩并经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:4)洗脱，得到680mg黄色固体，产率95％。
化合物(S)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-1-acetylpyrrolidine-2-carboxamide(ZSQ-5-73)的合成
Figure PCTCN2021099500-appb-000026
将ZSQ-5-68(356mg，1.0mmol)，DIPEA(0.5ml，3mmol)混合于5ml DCM中，逐滴加入乙酸酐(0.14ml，1.5mmol)后室温过夜搅拌。反应完成后反应液浓缩并经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:7)洗脱，得到288mg黄色固体，产率72％。
化合物(S)-(4-(1-acetylpyrrolidine-2-carboxamido)phenyl)arsonous acid(ZSQ-5-77)的合成
Figure PCTCN2021099500-appb-000027
将ZSQ-5-73(79.6mg，0.20mmol)，三水合高氯酸汞(72mg，0.16mmol)溶于3ml  DMSO中，混合溶液在室温下搅拌10min，立即经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:4)洗脱，得到粗产物再经过HPLC纯化得43mg白色固体，产率63％。M/Z:Exact Mass:340.04，(LC-MS):Found 341.0。
实施例2
化合物ZSQ-5-40的合成路线
Figure PCTCN2021099500-appb-000028
化合物methyl(1s,4s)-4-acetamidocyclohexane-1-carboxylate(ZSQ-5-24)的合成
Figure PCTCN2021099500-appb-000029
将顺-4-氨基环己甲酸甲酯盐酸盐(968mg，5.0mmol)，DIPEA(2.48ml，15mmol)混合于25ml DCM中，逐滴加入乙酸酐(0.14ml，1.5mmol)后室温搅拌6h。反应完成后反应液浓缩并经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:3)洗脱，得到892mg白色固体，产率89％。
化合物methyl(1s,4s)-4-(N-methylacetamido)cyclohexane-1-carboxylate(ZSQ-5-28)的合成
Figure PCTCN2021099500-appb-000030
将ZSQ-5-24(1.17g，5mmol)，碘甲烷(10mmol，0.62ml)混合溶于15ml超干的THF中，置于冰浴下混合搅拌10min。将NaH(60％，10mmol，400mg)分批加入混合溶液中，加完后冰浴下继续搅拌3h后移至室温搅拌过夜。待反应完成后加水淬灭，用2N盐酸酸化后减压蒸馏浓缩反应液，经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:2)洗脱，得到淡黄色液体482mg，产率45％。
化合物(1s,4s)-4-(N-methylacetamido)cyclohexane-1-carboxylic acid(ZSQ-5-30)的合成
Figure PCTCN2021099500-appb-000031
将化合物ZSQ-5-28(2.42mmol，482mg)溶于3ml THF、3ml H 2O的混合溶液中，加入一水合氢氧化锂(4.84mmol，203mg)，置于室温搅拌3h。反应完成后加入2N盐酸酸化，浓缩除去THF，经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:4)洗脱，得到淡黄色粉末426mg，产率88％。
化合物(1s,4s)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-4-(N-methylacetamido)-cyclo-hexane-1-carboxamide(ZSQ-5-32)的合成
Figure PCTCN2021099500-appb-000032
将ZSQ-5-30(99mg，0.5mmol)，ZSQ-1-23(129mg，0.5mmol)，HATU(285mg，1.5mmol)和DIPEA(0.24ml，3mmol)溶于3ml DCM、3ml DMF混合溶液中，置于室温混合搅拌3h。反应完成后依次用H 2O(20ml×2)，饱和NaCl溶液(20ml)洗涤，用20ml DCM萃取。合并有机相，用无水硫酸钠干燥，过滤，浓缩后经过硅胶柱层析分离纯化，用DCM/EA(1:1)洗脱，得到白色固体77mg，产率35％。
化合物(4-((1s,4s)-4-(N-methylacetamido)cyclohexane-1-carboxamido)phenyl)arsonous acid(ZSQ-5-40)的合成
Figure PCTCN2021099500-appb-000033
将ZSQ-5-32(77mg，0.17mmol)，三水合高氯酸汞(71mg，0.15mmol)溶于3ml DMSO中，混合溶液在室温下搅拌10min，立即经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:3)洗脱，得到粗产物再经过HPLC纯化得50mg白色固体，产率76％。M/Z:Exact Mass:382.09，(LC-MS):Found 383.1。
实施例3
化合物ZSQ-5-42的合成路线
Figure PCTCN2021099500-appb-000034
化合物N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-2-bromoacetamide(ZSQ-5-4)的合成
Figure PCTCN2021099500-appb-000035
将ZSQ-1-23(1.03g，4mmol)和三乙胺(0.83ml，6mmol)溶于20ml干燥的DCM溶液中，置于0℃下搅拌。逐滴加入溴乙酰溴(0.38ml，4.4mmol)超过15min，并在0℃下继续搅拌1h。将混合物用DCM(20ml)稀释，用2N稀盐酸(30ml)，水(50ml)，饱和碳酸氢钠溶液(30ml)，饱和氯化钠溶液(30ml)依次洗涤，DCM萃取。合并有机相，经无水硫酸钠干燥，过滤并浓缩。硅胶柱层析分离纯化，用PE/EA(4:1)洗脱，得到黄色固体1.02g，产率67％。
化合物N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-2-(4-methylpiperazin-1-yl)acetamide(ZSQ-5-10)的合成
Figure PCTCN2021099500-appb-000036
在室温下，将N-甲基哌嗪(66ul，0.6mmol)，DIPEA(99ul，0.6mmol)和ZSQ-5-4(76mg，0.2mmol)溶于1ml THF溶液中，混合溶液在室温下搅拌3h。将反应液减压浓缩，经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:4)洗脱，得到57mg白色固体，产率71％。
化合物(4-(2-(4-methylpiperazin-1-yl)acetamido)phenyl)arsonous acid(ZSQ-5-42)的合成
Figure PCTCN2021099500-appb-000037
将ZSQ-5-10(34mg，0.10mmol)，三水合高氯酸汞(42mg，0.09mmol)溶于3ml DMSO中，混合溶液在室温下搅拌10min，立即经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:9)洗脱，得到粗产物再经过HPLC纯化得23mg白色固体，产率67％。M/Z:Exact Mass:341.07，(LC-MS):Found 342.1。
实施例4
化合物ZSQ-6-3的合成路线
Figure PCTCN2021099500-appb-000038
化合物4-(1,3,2-dithiarsolan-2-yl)-N-methylaniline(ZSQ-5-87)的合成
Figure PCTCN2021099500-appb-000039
将ZSQ-1-23(1.03g，4.0mmol)，碘甲烷(0.37ml，6mmol)混合溶于20ml超干的THF中，置于冰浴下混合搅拌10min。将NaH(60％，4.2mmol，168mg)分批加入混合溶液中，加完后冰浴下继续搅拌3h后移至室温搅拌过夜。待反应完成后加水30ml淬灭，浓缩反应液，用EA(30ml)萃取，再用饱和氯化钠溶液(30ml)洗。合并有机相用无水硫酸钠干燥，过滤并浓缩，经过硅胶柱层析分离纯化，用PE/EA(1:8)洗脱，得到白色固体425mg，产率39％。
化合物N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-N-methylbutyramide(ZSQ-5-102)的合成
Figure PCTCN2021099500-appb-000040
将ZSQ-5-87(136mg，0.5mmol)和三乙胺(173ul，1.25mmol)溶于8ml干燥的DCM溶液中，置于0℃下搅拌。逐滴加入正丁酰氯(57ul，0.55mmol)超过5min，并在0℃下继续搅拌3h。反应液依次经过水(20ml)，饱和氯化钠溶液(20ml)洗，再用DCM(15ml)萃取。合并有机相用无水硫酸钠干燥，过滤，浓缩后经硅胶柱层析分离纯化，用PE/EA(1:3)洗脱，得到黄色固体96mg，产率56％。
化合物(4-(N-methylbutyramido)phenyl)arsonous acid(ZSQ-6-3)的合成
Figure PCTCN2021099500-appb-000041
将ZSQ-5-102(48mg，0.14mmol)，三水合高氯酸汞(57mg，0.12mmol)溶于2ml DMSO中，混合溶液在室温下搅拌10min，立即经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:3)洗脱，得到粗产物再经过HPLC纯化得28mg白色固体，产率70％。M/Z:Exact Mass:285.03，(LC-MS):Found 286.10。
实施例5
化合物ZSQ-6-52的合成路线
Figure PCTCN2021099500-appb-000042
化合物(1s,4s)-4-((tert-butoxycarbonyl)amino)cyclohexane-1-carboxylic acid(ZSQ-6-10)的合成
Figure PCTCN2021099500-appb-000043
将4-氨基环己烷-1-羧酸(2.86g，20mmol)，二碳酸二叔丁酯(6.9ml，30mmol)混合溶于40ml DCM中，逐滴加入三乙胺(8.3ml，60mmol)，室温混合搅拌24h。反应完成后直接经过减压蒸馏浓缩直接开下一步。
化合物benzyl(1s,4s)-4-((tert-butoxycarbonyl)amino)cyclohexane-1-carboxylate(ZSQ-6-13-1)的合成
Figure PCTCN2021099500-appb-000044
将ZSQ-6-10(20mmol)和碳酸钾(5.5g，40mmol)溶于20ml DMF中，在室温下搅拌20min后将苄溴(2.37ml，20mmol)逐滴加入，加完后继续室温过夜搅拌。反应完成后用硅藻土过滤，DCM洗，滤液浓缩后经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:7)洗脱，得到棕色液体1.6g，合计产率24％。
化合物enzyl(1s,4s)-4-aminocyclohexane-1-carboxylate(ZSQ-6-13-2)的合成
Figure PCTCN2021099500-appb-000045
将ZSQ-6-13-1(1.6g，4.8mmol)和2ml三氟乙酸混合于10ml DCM中，室温过夜搅拌。反应完成后反应液浓缩并经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:5)洗脱，得到1.04g无色油状产物，产率93％。
化合物benzyl(1s,4s)-4-(dimethylamino)cyclohexane-1-carboxylate(ZSQ-6-27)的合成
Figure PCTCN2021099500-appb-000046
将ZSQ-6-13-2(933mg，4mmol)，多聚甲醛(242mg，8mmol)，三乙酰氧基硼氢化钠(1.69g，8mmol)，乙酸(0.96ml，16mmol)混合溶于40ml 1,2-二氯乙烷中，置于室温混合搅拌48h。待反应完成后经硅藻土过滤，用甲醇洗，滤液浓缩后经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:6)洗脱，得到无色油状产物972mg，产率93％。
化合物(1s,4s)-4-(dimethylamino)cyclohexane-1-carboxylic acid(ZSQ-6-31)的合成
Figure PCTCN2021099500-appb-000047
将ZSQ-6-27(972mg，3.72mmol)，钯碳(200mg)混合于20ml甲醇中，在氢气气氛下室温混合搅拌过夜。待反应完成后用硅藻土过滤，甲醇洗，滤液浓缩抽干得油状产物870mg，未进一步纯化直接进行下一步。
化合物(1s,4s)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-4-(dimethylamino)cyclo-hexane-1-carboxamide(ZSQ-6-35)的合成
Figure PCTCN2021099500-appb-000048
将ZSQ-6-31(342mg，2.0mmol)，ZSQ-1-23(518mg，2.0mmol)，HATU(1.14g，3.0mmol)和DIPEA(992ul，6.0mmol)溶于4ml DCM中，置于室温混合搅拌6h。反应完成后依次用H 2O(20ml×2)，饱和NaCl溶液(20ml)洗涤，用20ml DCM萃取，合并有机相，用无水硫酸钠干燥，过滤，浓缩后经过硅胶柱层析分离纯化，用DCM/CH 3OH(NH 3)(5:1)洗脱，得到白色固体486mg，产率59％。
化合物(4-((1s,4s)-4-(dimethylamino)cyclohexane-1-carboxamido)phenyl)arsonous acid(ZSQ-6-52)的合成
Figure PCTCN2021099500-appb-000049
将ZSQ-6-35(70mg，0.17mmol)，三水合高氯酸汞(61mg，0.13mmol)溶于2ml DMSO中，混合溶液在室温下搅拌10min，立即经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:3)洗脱，得到粗产物再经过HPLC纯化得52mg白色固体，产率86％。M/Z:Exact Mass:354.09，(LC-MS):Found 355.10。
实施例6
化合物ZSQ-6-37的合成路线
Figure PCTCN2021099500-appb-000050
化合物3-benzyl 1-(tert-butyl)(R)-pyrrolidine-1,3-dicarboxylate(ZSQ-5-84)的合成
Figure PCTCN2021099500-appb-000051
将(R)-1-Boc-3-羧基吡咯烷(1.07g，5.0mmol)和碳酸钾(2.07g，15mmol)溶于20ml DMF中，在室温下搅拌20min后将苄溴(653ul，5.5mmol)逐滴加入，加完后继续室温搅拌3h。反应完成后用硅藻土过滤，DCM洗，滤液浓缩后经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:7)洗脱，得到淡黄色液体1.35g，合计产率89％。
化合物benzyl(R)-pyrrolidine-3-carboxylate(ZSQ-5-89)的合成
Figure PCTCN2021099500-appb-000052
将ZSQ-5-84(1.35g，4.42mmol)和2ml三氟乙酸混合于10ml DCM中，室温混合搅拌。反应完成后反应液浓缩并经过C18反相柱层析分离纯化，用H 2O/CH 3CN(4:1)洗脱，得到843mg淡黄色油状产物，产率93％。
化合物benzyl(R)-1-methylpyrrolidine-3-carboxylate(ZSQ-6-20)的合成
Figure PCTCN2021099500-appb-000053
将ZSQ-5-89(410mg，2mmol)，多聚甲醛(120mg，4mmol)，三乙酰氧基硼氢化钠(847mg，4mmol)，乙酸(0.48ml，8mmol)混合溶于10ml 1,2-二氯乙烷中，置于室温混合搅拌48h。待反应完成后经硅藻土过滤，用甲醇洗，滤液浓缩后经过C18反相柱层析分离纯化，用H 2O/CH 3CN(4:1)洗脱，得到无色油状产物333mg，产率76％。
化合物(R)-1-methylpyrrolidine-3-carboxylic acid(ZSQ-6-25)的合成
Figure PCTCN2021099500-appb-000054
将ZSQ-6-20(830mg，3.78mmol)，钯碳(200mg)混合于15ml甲醇中，在氢气气氛下室温混合搅拌过夜。待反应完成后用硅藻土过滤，甲醇洗，滤液浓缩抽干得无色油状产物504mg，未进一步纯化直接进行下一步。
化合物(R)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-1-methylpyrrolidine-3-carboxamide(ZSQ-6-28)的合成
Figure PCTCN2021099500-appb-000055
将ZSQ-6-25(258mg，2.0mmol)，ZSQ-1-23(518mg，2.0mmol)，HATU(1.14g，3.0mmol)和DIPEA(992ul，6.0mmol)溶于4ml DCM中，置于室温混合搅拌6h。反应完成后依次用H 2O(20ml×2)，饱和NaCl溶液(20ml)洗涤，用20ml DCM萃取，合并有机相，用无水硫酸钠干燥，过滤，浓缩后经过硅胶柱层析分离纯化，用DCM/CH 3OH(NH 3)(4:1)洗脱，得到黄色固体130mg，产率17％。
化合物(R)-(4-(1-methylpyrrolidine-3-carboxamido)phenyl)arsonous acid(ZSQ-6-37)的合成
Figure PCTCN2021099500-appb-000056
将ZSQ-6-28(45mg，0.12mmol)，三水合高氯酸汞(44mg，0.10mmol)溶于2ml DMSO中，混合溶液在室温下搅拌10min，立即经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:4)洗脱，得到粗产物再经过HPLC纯化得到25mg白色粉末，产率67％。M/Z:Exact Mass:312.05，(LC-MS):Found 313.0。
实施例7
化合物ZSQ-7-37的合成路线
Figure PCTCN2021099500-appb-000057
化合物tert-butyl(R)-3-((4-(1,3,2-dithiarsolan-2-yl)phenyl)carbamoyl)pyro-lidine-1-carboxylate(ZSQ-6-57)的合成
Figure PCTCN2021099500-appb-000058
将(R)-1-Boc-3-羧基吡咯烷(645mg，3.0mmol)，ZSQ-1-23(854mg，3.3mmol)，HATU(1.71g，4.5mmol)和DIPEA(1.49ml，9.0mmol)溶于10ml DCM中，置于室温混合搅拌3h。反应完成后依次用H 2O(40ml×2)，饱和NaCl溶液(40ml)洗涤，用40ml DCM萃取。合并有机相，用无水硫酸钠干燥，过滤，浓缩后经过硅胶柱层析分离纯化，用DCM/CH 3OH(NH 3)(20:1)洗脱，得到黄色固体1.25g，产率91％。
化合物tert-butyl(R)-3-((4-(1,3,2-dithiarsolan-2-yl)phenyl)(benzyl)carbamoyl)pyrolidine-1-carboxylate(ZSQ-6-97)的合成
Figure PCTCN2021099500-appb-000059
将ZSQ-6-57(456mg，1.0mmol)，溴化苄(178ul，1.5mmol)混合溶于4ml超干的DMF中，置于冰浴下混合搅拌10min。将NaH(60％，1.2mmol，48mg)分批加入混合溶液中，加完后冰浴下继续搅拌3h后移至室温搅拌过夜。待反应完成后加水淬灭，用EA(20ml)萃取，再依次2N盐酸溶液，饱和NaCl溶液洗，合并有机相经无水Na 2SO 4干燥，过滤并浓缩滤液，经过硅胶柱层析分离纯化，用PE/EA(1:4)洗脱，得到151mg白色固体，产率27％。
化合物(R)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-N-benzylpyrrolidine-3-carboxamide(ZSQ-6-102)的合成
Figure PCTCN2021099500-appb-000060
将ZSQ-6-97(766mg，1.40mmol)和1ml三氟乙酸混合于5ml DCM中，室温混合搅拌12h。反应完成后反应液浓缩并经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:1)洗脱，得到586mg白色固体，产率94％。
化合物(R)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-N-benzyl-1-methylpyrrolidine-3-carboxamide(ZSQ-7-8)的合成
Figure PCTCN2021099500-appb-000061
将ZSQ-6-102(130mg，0.29mmol)，多聚甲醛(17mg，0.58mmol)，三乙酰氧基硼氢化钠(123mg，0.58mmol)，乙酸(66ul，1.16mmol)混合溶于2ml 1,2-二氯乙烷中，置于室温混合搅拌48h。待反应完成后直接浓缩经过硅胶柱层析分离纯化，用DCM/CH 3OH(NH 3)(10:1)洗脱，得到70mg白色固体，产率52％。
化合物(R)-(4-(N-benzyl-1-methylpyrrolidine-3-carboxamido)phenyl)arsonous acid(ZSQ-7-37)的合成
Figure PCTCN2021099500-appb-000062
将ZSQ-7-8(70mg，0.15mmol)，三水合高氯酸汞(55mg，0.12mmol)溶于2ml DMSO中，混合溶液在室温下搅拌10min，立即经过C18反相柱层析分离纯化，用H 2O/CH 3CN(2:1)洗脱，得到粗产物再经过HPLC纯化得到47mg白色粉末，产率77％。M/Z:Exact Mass:402.09，(LC-MS):Found 402.8。
实施例8
化合物ZSQ-7-86的合成路线
Figure PCTCN2021099500-appb-000063
化合物2-((4-(1,3,2-dithiarsolan-2-yl)phenyl)amino)-N,N-dimethylacetamide(ZSQ-6-81)的合成
Figure PCTCN2021099500-appb-000064
将ZSQ-1-23(259mg，1.0mmol)，2-溴-N,N-二甲基乙酰胺(135ul，1.2mmol)和碳酸钾(276mg，2.0mmol)溶于3ml CH 3CN中，在室温下混合搅拌18h。反应完成后用水(20ml×2)洗，EA(20ml)萃取。有机相经过无水Na 2SO 4干燥，过滤并浓缩滤液，经过硅胶柱层析分离纯化，用DCM/EA(1:2)洗脱，得到白色粉末244mg，产率71％。
化合物(R)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-N-(2-(dimethylamino)-2-oxoethyl)-1-methylpyrrolidine-3-carboxamide(ZSQ-7-51)的合成
Figure PCTCN2021099500-appb-000065
将ZSQ-6-25(230mg，1.7mmol)，ZSQ-6-81(400mg，1.2mmol)溶于6ml干燥的吡啶中，混合溶液置于冰盐浴下搅拌10min，氧氯化磷(130ul，1.4mmol)被逐滴加入到混合溶液中(超过10min)，加完后继续0℃搅拌3h。反应完成后直接经过硅胶柱层析分离纯化，先用PE/EA(0-70％)洗脱冲出吡啶，再用DCM/CH 3OH(NH 3)(4:1)洗脱，得到粗产物经过C18反相柱层析分离纯化，用H 2O/CH 3CN(2:1)洗脱，得到131mg白色固体，产率24％。
化合物(R)-(4-(N-(2-(dimethylamino)-2-oxoethyl)-1-methylpyrrolidine-3-carboxamido)phenyl)arsonous acid(ZSQ-7-86)的合成
Figure PCTCN2021099500-appb-000066
将ZSQ-7-51(23mg，0.05mmol)，三水合高氯酸汞(18mg，0.04mmol)溶于2ml DMSO中，混合溶液在室温下搅拌10min，立即经过C18反相柱层析分离纯化，用H 2O/CH 3CN(4:1)洗脱，得到粗产物再经过HPLC纯化得到11mg白色粉末，产率55％。M/Z:Exact Mass:397.10，(LC-MS):Found 397.8。
实施例9
化合物ZSQ-7-106的合成路线
Figure PCTCN2021099500-appb-000067
化合物(R)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-N,1-dibenzylpyrrolidine-3-carboxamide(ZSQ-7-102)的合成
Figure PCTCN2021099500-appb-000068
将ZSQ-6-102(89mg，0.2mmol)和碳酸钾(55mg，0.4mmol)溶于2ml CH 3CN中，在冰盐浴中搅拌20min后将苄溴(24ul，0.2mmol)逐滴加入(超过10min)，加完后继续冰盐浴中搅拌2h。反应完成后将反应液浓缩，直接经过硅胶柱层析分离纯化，用DCM/CH 3OH(NH 3)(10:1)洗脱，得到21mg白色固体，产率20％。
化合物(R)-(4-(N,1-dibenzylpyrrolidine-3-carboxamido)phenyl)arsonous acid(ZSQ-7-106)的合成
Figure PCTCN2021099500-appb-000069
将ZSQ-7-102(21mg，0.04mmol)，三水合高氯酸汞(14mg，0.03mmol)溶于1ml DMSO中，混合溶液在室温下搅拌10min，立即经过C18反相柱层析分离纯化，用H 2O/CH 3CN(2:1)洗脱，得到粗产物再经过HPLC纯化得到9mg白色粉末，产率48％。M/Z:Exact Mass:478.12，(LC-MS):Found 478.9。
实施例10
化合物ZSQ-8-50的合成路线
Figure PCTCN2021099500-appb-000070
化合物2-bromo-1-morpholinoethan-1-one(ZSQ-8-38)的合成
Figure PCTCN2021099500-appb-000071
将溴乙酰溴(174ul，2mmol)溶于15ml超干的DCM中，置于冰盐浴中搅拌，吗啉(348ul，4mmol)被逐滴加入混合溶液中，加完后继续0℃搅拌30min后移至室温搅拌1h。反应完成后用饱和氯化铵溶液(30ml)洗，乙醚(30ml)萃取两次，有机相用无水硫酸钠干燥，滤液浓缩抽干得黄色油状液体387mg，产率94％。
化合物(R)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-N-benzyl-1-(2-morpholino-2-oxoethyl)pyrrolidine-3-carboxamide(ZSQ-8-41)的合成
Figure PCTCN2021099500-appb-000072
将ZSQ-6-102(53mg，0.12mmol)，ZSQ-8-38(27mg，0.13mmol)和碳酸钾(33mg，0.24mmol)溶于2ml CH 3CN中，在冰盐浴中搅拌2h。反应完成后将反应液浓缩，直接经过硅胶柱层析分离纯化，用DCM/CH 3OH(NH 3)(8:1)洗脱，得到13.3mg白色固体，产率19％。
化合物(R)-(4-(N-benzyl-1-(2-morpholino-2-oxoethyl)pyrrolidine-3-carboxamido)phenyl)arsonous acid(ZSQ-8-50)的合成
Figure PCTCN2021099500-appb-000073
将ZSQ-8-41(67mg，0.12mmol)，三水合高氯酸汞(42mg，0.09mmol)溶于2ml DMSO中，混合溶液在室温下搅拌10min，立即经过C18反相柱层析分离纯化，用H 2O/CH 3CN(2:1)洗脱，得到粗产物再经过HPLC纯化得到42mg白色粉末，产率70％。M/Z:Exact Mass:515.14，(LC-MS):Found 515.9。
实施例11
化合物ZSQ-12-66的合成路线
Figure PCTCN2021099500-appb-000074
化合物benzyl(R)-1-(2-(dimethylamino)-2-oxoethyl)pyrrolidine-3-carboxylate(ZSQ-12-41)的合成
Figure PCTCN2021099500-appb-000075
将ZSQ-5-89(3.19g，10mmol)，2-溴-N,N-二甲基乙酰胺(1.66g，10mmol)和碳酸钾(4.15g，30mmol)溶于50ml CH 3CN中，在冰盐浴中搅拌3h后移至室温搅拌过夜。反应完成后用硅藻土过滤，DCM冲洗，滤液浓缩抽干得黄色油状粗产物4.8g，未进一步纯化直接用于下一步。
化合物(R)-1-(2-(dimethylamino)-2-oxoethyl)pyrrolidine-3-carboxylic acid(ZSQ-12-58)的合成
Figure PCTCN2021099500-appb-000076
将ZSQ-12-41(1.0g，3.44mmol)，钯碳(200mg)混合于15ml甲醇中，在氢气气氛下室温混合搅拌过夜。待反应完成后用硅藻土过滤，甲醇洗，滤液浓缩经过C18反相柱层析分离纯化，用H 2O/CH 3CN(9:1)洗脱，得无色油状产物602mg，产率87％。
化合物tert-butyl 3-(((4-(1,3,2-dithiarsolan-2-yl)phenyl)amino)methyl)pyro-lidine-1-carboxylate(ZSQ-11-38)的合成
Figure PCTCN2021099500-appb-000077
将ZSQ-1-23(1.03g，4mmol)，1-Boc-3-吡咯烷甲醛(1.59g，8mmol)，三乙酰氧基硼氢化钠(1.69g，8mmol)，乙酸(0.96ml，16mmol)混合溶于30ml 1,2-二氯乙烷中，置于室温混合搅拌48h。待反应完成后依次用饱和碳酸氢钠溶液(50ml)，H 2O(50ml)，饱和氯化钠溶液(50ml)洗，DCM(30ml)萃取。合并有机相，用无水硫酸钠干燥，滤液浓缩后直接经过硅胶柱层析分离纯化，用PE/EA(4:1)洗脱，粗产物进一步经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:8)洗脱，得到1.0g淡黄色固体，产率57％。
化合物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)的合成
Figure PCTCN2021099500-appb-000078
将ZSQ-11-38(442mg，1.0mmol)，ZSQ-12-58(200mg，1.0mmol)溶于5ml干燥的吡啶中，混合溶液置于冰盐浴下搅拌10min，氧氯化磷(140ul，1.5mmol)被逐滴加入到混合溶液中(超过10min)，加完后继续0℃搅拌3h。反应完成后直接经过硅胶柱层析分离纯化，先用PE/EA(0-70％)洗脱冲出吡啶，再用DCM/CH 3OH(NH 3)(4:1)洗脱，得到粗产物经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:6)洗脱，得到223mg白色固体，产率35.7％。
化合物(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)的合成
Figure PCTCN2021099500-appb-000079
将ZSQ-12-61-1(233mg，0.37mmol)和0.5ml三氟乙酸混合于4ml DCM中，室温混合搅拌12h。反应完成后反应液浓缩并经过C18反相柱层析分离纯化，用H 2O/CH 3CN(2:1)洗脱，得到190mg白色固体，产率97％。
化合物(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)的合成
Figure PCTCN2021099500-appb-000080
将ZSQ-12-61-2(105mg，0.2mmol)，DIPEA(0.1ml，0.6mmol)混合于3ml DCM中，逐滴加入乙酸酐(28ul，0.3mmol)后室温搅拌3h。反应完成后反应液浓缩，经过硅胶柱层析分离纯化，用DCM/CH 3OH(NH 3)(9:1)洗脱，得黄色固体59.4mg，产率52％。
化合物(4-((3R)-N-((1-acetylpyrrolidin-3-yl)methyl)-1-(2-(dimethylamino)-2-oxoethyl)pyrrolidine-3-carboxamido)phenyl)arsonous acid(ZSQ-12-66)的合成
Figure PCTCN2021099500-appb-000081
将ZSQ-12-64(59mg，0.10mmol)，三水合高氯酸汞(38mg，0.08mmol)溶于2ml DMSO中，混合溶液在室温下搅拌10min，立即经过C18反相柱层析分离纯化，用H 2O/CH 3CN(2:1)洗脱，得到粗产物再经过HPLC纯化得到39mg白色粉末，产率77％。M/Z:Exact Mass:508.17，(LC-MS):Found 509.10。
实施例12
化合物ZSQ-13-100的合成路线
Figure PCTCN2021099500-appb-000082
化合物benzyl(R)-1-benzylpyrrolidine-3-carboxylate(ZSQ-13-52)的合成
Figure PCTCN2021099500-appb-000083
将ZSQ-5-89(2.14g，6.7mmol)和碳酸钾(2.31g，16.75mmol)溶于60ml CH 3CN中，置于冰盐浴中混合搅拌，溴化苄(796ul，6.7mmol)溶于7ml CH 3CN中逐滴加入到混合溶液中，冰盐浴中搅拌3h后移至室温搅拌3h。反应完成后用硅藻土过滤，DCM冲洗，滤液浓缩抽干得黄色油状粗产物，未进一步纯化直接用于下一步。
化合物(R)-1-benzylpyrrolidine-3-carboxylic acid(ZSQ-13-54)的合成
Figure PCTCN2021099500-appb-000084
将化合物ZSQ-13-52(6.7mmol)溶于5ml THF、5ml H 2O的混合溶液中，加入一水合氢氧化锂(20.1mmol，843mg)，置于室温搅拌6h。反应完成后加入2N盐酸酸化，浓缩除去THF，经过C18反相柱层析分离纯化，用H 2O/CH 3CN(9:1)洗脱，得到淡黄色油状产物323mg，合并产率23％。
化合物tert-butyl 4-(((4-(1,3,2-dithiarsolan-2-yl)phenyl)amino)methyl)piperidine-1-carboxylate(ZSQ-13-48)的合成
Figure PCTCN2021099500-appb-000085
将ZSQ-1-23(4.3g，16.6mmol)，1-叔丁氧羰基-4-哌啶甲醛(7.0g，33.2mmol)，三乙酰氧基硼氢化钠(7.0g，33.2mmol)，乙酸(3.0ml，49.8mmol)混合溶于100ml 1,2-二氯乙烷中，置于室温混合搅拌48h。待反应完成后依次用饱和碳酸氢钠溶液(50ml) 调PH至碱性，H 2O(100ml)，饱和氯化钠溶液(100ml)洗，DCM(100ml)萃取，合并有机相，用无水硫酸钠干燥，滤液浓缩后直接经过硅胶柱层析分离纯化，用PE/EA(4:1)洗脱，粗产物进一步经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:9)洗脱，得到3.0g淡黄色粉末，产率40％。
化合物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)的合成
Figure PCTCN2021099500-appb-000086
将ZSQ-13-48(790mg，1.73mmol)，ZSQ-13-54(323mg，1.57mmol)溶于5ml干燥的吡啶中，混合溶液置于冰盐浴下搅拌10min，氧氯化磷(292ul，2.0mmol)被逐滴加入到混合溶液中(超过10min)，加完后继续0℃搅拌3h。反应完成后直接经过硅胶柱层析分离纯化，先用PE/EA(0-70％)洗脱冲出吡啶，再用DCM/CH 3OH(NH 3)(9:1)洗脱得到粗产物，经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:6)洗脱，得到397mg黄色固体，产率39％。
化合物(R)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-1-benzyl-N-(piperidin-4-ylmethyl)pyrrolidine-3-carboxamide(ZSQ-13-62)的合成
Figure PCTCN2021099500-appb-000087
将ZSQ-13-59(397mg，0.62mmol)和0.5ml三氟乙酸混合于5ml DCM中，室温混合搅拌过夜。反应完成后反应液浓缩并经过C18反相柱层析分离纯化，用H 2O/CH 3CN(5:2)洗脱，得到308mg黄色固体，产率91％。
化合物(R)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-1-benzyl-N-((1-ethylpiperidin-4-yl)methyl)pyrrolidine-3-carboxamide(ZSQ-13-83)的合成
Figure PCTCN2021099500-appb-000088
将ZSQ-13-62(81mg，0.15mmol)，碘乙烷(11ul，0.135mmol)和碳酸钾(62mg，0.45mmol)溶于3ml CH 3CN中，在冰盐浴中搅拌3h后移至室温搅1h。反应完成后将反应液浓缩，直接经过硅胶柱层析分离纯化，用DCM/CH 3OH(NH 3)(4:1)洗脱，得黄色固体21.6mg，产率25％。
化合物(R)-(4-(1-benzyl-N-((1-ethylpiperidin-4-yl)methyl)pyrrolidine-3-carboxamido)phenyl)arsonous acid(ZSQ-13-100)的合成
Figure PCTCN2021099500-appb-000089
将ZSQ-13-83(35mg，0.061mmol)，三水合高氯酸汞(22mg，0.049mmol)溶于2ml DMSO中，混合溶液在室温下搅拌10min，立即经过C18反相柱层析分离纯化，用H 2O/CH 3CN(2:1)洗脱，得到粗产物再经过HPLC纯化得到18mg白色粉末，产率58％。M/Z:Exact Mass:513.20，(LC-MS):Found 514.50。
实施例13
化合物ZSQ-17-79的合成路线
Figure PCTCN2021099500-appb-000090
化合物ethyl(E)-2-(hydroxyimino)-2-(2-phenylacetamido)acetate(ZSQ-16-18)的合成
Figure PCTCN2021099500-appb-000091
将苯乙酸(681mg，5.0mmol)，2-羟胺基-2-亚氨基乙酸乙酯(694mg，5.25mmol)，HATU(2.85g，7.5mmol)和DIPEA(2.48ml，15.0mmol)溶于50ml DCM中，置于室温混合搅拌6h。反应完成后依次用H 2O(50ml×2)，饱和NaCl溶液(50ml)洗涤，用DCM(50ml)萃取，合并有机相，用无水硫酸钠干燥，过滤，浓缩后经过硅胶柱层析分离纯化，用PE/EA(1:4)洗脱，得到白色固体1.18g，产率94％。
化合物ethyl 5-benzyl-1,2,4-oxadiazole-3-carboxylate(ZSQ-16-22)的合成
Figure PCTCN2021099500-appb-000092
将ZSQ-16-18(500mg，2.0mmol)溶于5ml干燥的DMF中，加热到140℃混合搅拌4h。反应完成后经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:2)洗脱，得黄色油状产物399mg，产率86％。
化合物5-benzyl-1,2,4-oxadiazole-3-carboxylic acid(ZSQ-16-33)的合成
Figure PCTCN2021099500-appb-000093
将化合物ZSQ-16-22(399mg，1.71mmol)溶于4ml THF、4ml H 2O的混合溶液中，加入一水合氢氧化锂(5.15mmol，216mg)，置于室温搅拌4h。反应完成后加入2N盐酸酸化，浓缩除去THF，经过C18反相柱层析分离纯化，用H 2O/CH 3CN(4:1)洗脱，得到白色固体286mg，合并产率82％。
化合物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)的合成
Figure PCTCN2021099500-appb-000094
将ZSQ-13-48(456mg，1.0mmol)，ZSQ-16-33(204mg，1.0mmol)溶于5ml干燥的吡啶中，混合溶液置于冰盐浴下搅拌10min，氧氯化磷(140ul，1.5mmol)被逐滴加入到混合溶液中(超过10min)，加完后继续0℃搅拌3h。反应完成后直接经过硅胶柱层析分离纯化，先用PE/EA(0-70％)洗脱冲出吡啶，再用DCM/CH 3OH(NH 3)(9:1)洗脱得到粗产物，经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:3)洗脱，得到274mg黄色固体，产率43％。
化合物N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-5-benzyl-N-(piperidin-4-ylmethyl)-1,2,4-oxadiazole-3-carboxamide(ZSQ-17-72)的合成
Figure PCTCN2021099500-appb-000095
将ZSQ-17-51(274mg，0.43mmol)和0.5ml三氟乙酸混合于5ml DCM中，室温混合搅拌过夜。反应完成后反应液浓缩并经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:1)洗脱，得到229mg黄色固体，产率98％。
化合物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)的合成
Figure PCTCN2021099500-appb-000096
将ZSQ-17-72(229mg，0.42mmol)，碘乙烷(34ul，0.42mmol)和碳酸钾(174mg，1.26mmol)溶于2ml CH 3CN中，在冰盐浴中搅拌3h后移至室温搅1h。反应完成后将反应液浓缩，直接经过硅胶柱层析分离纯化，用DCM/CH 3OH(NH 3)(9:1)洗脱，得黄色固体107mg，产率45％。
化合物(4-(5-benzyl-N-((1-ethylpiperidin-4-yl)methyl)-1,2,4-oxadiazole-3-carboxamido)phenyl)arsonous acid(ZSQ-17-79)的合成
Figure PCTCN2021099500-appb-000097
将ZSQ-17-75(57mg，0.10mmol)，硫酸汞(60mg，0.20mmol)溶于2ml DMSO中，混合溶液加热到60℃搅拌30min，立即经过C18反相柱层析分离纯化，用H 2O/CH 3CN(2:5)洗脱，得到粗产物再经过HPLC纯化得到33mg白色粉末，产率64％。M/Z:Exact Mass:512.14，(LC-MS):Found 513.0。
实施例14
化合物ZSQ-17-80的合成路线
Figure PCTCN2021099500-appb-000098
化合物ethyl 5-benzyl-1-methyl-1H-pyrazole-3-carboxylate(ZSQ-17-66)的合成
Figure PCTCN2021099500-appb-000099
将N-甲基-L-苯丙氨酸(1.79g，10mmol)和亚硝酸钠(759mg，11mmol)溶于DCM:H 2O(30ml:30ml)混合溶液中，置于冰浴中搅拌均匀。缓慢向混合溶液中滴加1ml 12N盐酸，加完后继续0℃搅拌2h。反应完成后用H 2O(50ml)洗三次，DCM(50ml)萃取。合并有机相经过无水Na 2SO 4干燥，过滤，滤液浓缩抽干，为白色固体。之后将白色固体溶于乙酸酐(20ml)中，加入丙炔酸乙酯(1.5ml，15mmol)，混合溶液置于120℃中回流搅拌12h。反应完成后用水(50ml)洗三次，EA萃取。有机相经过无水Na 2SO 4干燥，过滤，滤液浓缩
经过硅胶柱层析分离纯化，用PE/EA(1:2)洗脱，得黄色油状产物1.03g，产率42％。
化合物5-benzyl-1-methyl-1H-pyrazole-3-carboxylic acid(ZSQ-17-68)的合成
Figure PCTCN2021099500-appb-000100
将化合物ZSQ-17-66(366mg，1.5mmol)溶于2ml THF、2ml H 2O的混合溶液中，加入一水合氢氧化锂(126mg，3.0mmol)，置于室温搅拌4h。反应完成后加入2N盐酸酸化，浓缩除去THF，经过C18反相柱层析分离纯化，用H 2O/CH 3CN(4:1)洗脱，得到白色固体290mg，合并产率89％。
化合物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)的合成
Figure PCTCN2021099500-appb-000101
将ZSQ-13-48(456mg，1.0mmol)，ZSQ-17-68(216mg，1.0mmol)溶于5ml干燥的吡啶中，混合溶液置于冰盐浴下搅拌10min，氧氯化磷(140ul，1.5mmol)被逐滴加入到混合溶液中(超过10min)，加完后继续0℃搅拌3h。反应完成后直接经过硅胶柱层析分离纯化，先用PE/EA(0-70％)洗脱冲出吡啶，再用DCM/CH 3OH(NH 3)(9:1)洗脱得到粗产物，经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:3)洗脱，得到168mg黄色固体，产率26％。
化合物N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-5-benzyl-1-methyl-N-(piperidin-4-ylmethyl)-1H-pyrazole-3-carboxamide(ZSQ-17-71)的合成
Figure PCTCN2021099500-appb-000102
将ZSQ-17-44(168mg，0.26mmol)和0.5ml三氟乙酸混合于5ml DCM中，室温混合搅拌12h。反应完成后反应液浓缩并经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:1)洗脱，得到137mg黄色固体，产率96％。
化合物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)的合成
Figure PCTCN2021099500-appb-000103
将ZSQ-17-72(137mg，0.25mmol)，碘乙烷(20ul，0.25mmol)和碳酸钾(102mg，0.75mmol)溶于2ml CH 3CN中，在冰盐浴中搅拌3h后移至室温搅1h。反应完成后将反应液浓缩，直接经过硅胶柱层析分离纯化，用DCM/CH 3OH(NH 3)(9:1)洗脱，得黄色固体52mg，产率36％。
化合物(4-(5-benzyl-N-((1-ethylpiperidin-4-yl)methyl)-1-methyl-1H-pyrazole-3-carboxamido)phenyl)arsonous acid(ZSQ-17-80)的合成
Figure PCTCN2021099500-appb-000104
将ZSQ-17-74(29mg，0.05mmol)，硫酸汞(30mg，0.10mmol)溶于2ml DMSO中，混合溶液加热到60℃搅拌30min，立即经过C18反相柱层析分离纯化，用H 2O/CH 3CN(2:5)洗脱，得到粗产物再经过HPLC纯化得到13mg白色粉末，产率50％。M/Z:Exact Mass:524.18，(LC-MS):Found 525.0。
实施例15
化合物ZSQ-17-81的合成路线
Figure PCTCN2021099500-appb-000105
化合物N-methoxy-N-methyl-2-phenylacetamide(ZSQ-16-81)的合成
Figure PCTCN2021099500-appb-000106
将苯乙酸(1.36g，10mmol)，二甲羟胺盐酸盐(1.17g，12mmol)，HATU(4.56g，12mmol)和DIPEA(4.96ml，30mmol)溶于50ml DCM中，置于室温混合搅拌6h。反应完成后依次用稀盐酸溶液(50ml)，H 2O(50ml)，饱和NaCl溶液(50ml)洗涤，用50ml DCM萃取。合并有机相，用无水硫酸钠干燥，过滤，浓缩后经过硅胶柱层析分离纯化，用PE/EA(2:5)洗脱，得到无色液体1.7g，产率95％。
化合物1-phenylpropan-2-one(ZSQ-16-89)的合成
Figure PCTCN2021099500-appb-000107
将ZSQ-16-81(1.7g，9.48mmol)溶于30ml干燥的THF中，置于冰浴中搅拌均匀。在0℃下，30min内将甲基溴化镁(1M的四氢呋喃溶液，14.22ml，14.22mmol)滴加到混合溶液中。加完后0℃下继续搅拌1h后置于室温搅拌12h。待反应完成后用2N盐酸溶液淬灭(50ml)，减压蒸馏除去THF，用EA萃取，再用水(50ml)洗两次。合并有机相用无水硫酸钠干燥，过滤，滤液浓缩后经过硅胶柱层析分离纯化，用PE/EA(2:1)洗脱，得到无色液体1.15g，产率90％。
化合物ethyl 2,4-dioxo-5-phenylpentanoate(ZSQ-16-97)的合成
Figure PCTCN2021099500-appb-000108
将ZSQ-16-89(670mg，5.0mmol)，草酸二乙酯(815ul，6.0mmol)溶于10ml超干乙醇中，置于冰浴中搅拌均匀。20％乙醇钠溶液(1.87g，5.5mmol)被逐滴加入到混合溶液中，加完后冰浴中继续搅拌1h后移至室温搅拌24h。待反应完成后2N盐酸溶液淬灭(20ml)，用EA(50ml)萃取，再依次用水(30ml)，饱和氯化钠溶液(30ml)洗。合并有机相用无水硫酸钠干燥，过滤，滤液浓缩后经过硅胶柱层析分离纯化，用PE/EA(2:1)洗脱，得到黄色油状产物870mg，产率74％。
化合物ethyl 5-benzylisoxazole-3-carboxylate(ZSQ-17-64)的合成
Figure PCTCN2021099500-appb-000109
将ZSQ-16-97(468mg，2mmol)，盐酸羟胺(210mg，3mmol)混合溶于7ml超干乙醇中，加热到90℃混合搅拌12h。反应完成后浓缩反应液，依次用水(20ml)，饱和氯化钠溶液(20ml)洗，EA(40ml)萃取，合并有机相用无水硫酸钠干燥，过滤，滤液浓缩后经过硅胶柱层析分离纯化，用PE/EA(2:5)洗脱，得到淡黄色油状产物302mg，产率65％。
化合物5-benzylisoxazole-3-carboxylic acid(ZSQ-17-46)的合成
Figure PCTCN2021099500-appb-000110
将化合物ZSQ-17-64(231mg，1.0mmol)溶于2ml THF、2ml H 2O的混合溶液中，加入一水合氢氧化锂(126mg，3.0mmol)，置于室温搅拌4h。反应完成后加入少量稀盐酸酸化，浓缩除去THF，经过C18反相柱层析分离纯化，用H 2O/CH 3CN(4:1)洗脱，得到白色固体200mg，合并产率98％。
化合物tert-butyl 4-((N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-5-benzylisoxazole-3-carboxamido)methyl)piperidine-1-carboxylate(ZSQ-17-49)的合成
Figure PCTCN2021099500-appb-000111
将ZSQ-13-48(456mg，1.0mmol)，ZSQ-17-46(203mg，1.0mmol)溶于5ml干燥的吡啶中，混合溶液置于冰盐浴下搅拌10min，氧氯化磷(140ul，1.5mmol)被逐滴加入到混合溶液中(超过10min)，加完后继续0℃搅拌3h。反应完成后直接经过硅胶柱层析分离纯化，先用PE/EA(0-70％)洗脱冲出吡啶，再用DCM/CH 3OH(NH 3)(9:1)洗脱得到粗产物，经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:3)洗脱，得到432mg黄色固体，产率67％。
化合物N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-5-benzyl-N-(piperidin-4-ylmethyl) isoxazole-3-carboxamide(ZSQ-17-67)的合成
Figure PCTCN2021099500-appb-000112
将ZSQ-17-49(432mg，0.66mmol)和1ml三氟乙酸混合于6ml DCM中，室温混合搅拌12h。反应完成后反应液浓缩并经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:1)洗脱，得到336mg黄色粘稠状产物，产率94％。
化合物N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-5-benzyl-N-((1-ethylpiperidin-4-yl)methyl)isoxazole-3-carboxamide(ZSQ-17-69)的合成
Figure PCTCN2021099500-appb-000113
将ZSQ-17-67(200mg，0.369mmol)，碘乙烷(30ul，0.369mmol)和碳酸钾(153mg，1.107mmol)溶于3ml CH 3CN中，在冰盐浴中搅拌3h后移至室温搅1h。反应完成后将反应液浓缩，直接经过硅胶柱层析分离纯化，用DCM/CH 3OH(NH 3)(9:1)洗脱，得黄色固体76mg，产率36％。
化合物(4-(5-benzyl-N-((1-ethylpiperidin-4-yl)methyl)isoxazole-3-carboxamido)phenyl)arsonous acid(ZSQ-17-81)的合成
Figure PCTCN2021099500-appb-000114
将ZSQ-17-69(48mg，0.084mmol)，硫酸汞(50mg，0.168mmol)溶于2ml DMSO中，混合溶液加热到60℃搅拌30min，立即经过C18反相柱层析分离纯化，用H 2O/CH 3CN(2:5)洗脱，得到粗产物再经过HPLC纯化得到23mg白色粉末，产率53％。M/Z:Exact Mass:511.15，(LC-MS):Found 512.0。
实施例16
化合物ZSQ-17-87的合成路线
Figure PCTCN2021099500-appb-000115
化合物ethyl 2H-tetrazole-5-carboxylate(ZSQ-13-16)的合成
Figure PCTCN2021099500-appb-000116
将氰基甲酸乙酯(1.0ml，10.0mmol)溶于12ml超干吡啶中，置于冰浴中搅拌均匀。逐滴加入三氟乙酸(0.74ml，10mmol)，搅拌10min后加入叠氮化钠(702mg，10.8mmol)，混合溶液置于60℃搅拌48h。反应完成后经减压蒸馏浓缩，未进行纯化直接用于下一步。
化合物ethyl 2-benzyl-2H-tetrazole-5-carboxylate(ZSQ-13-24)的合成
Figure PCTCN2021099500-appb-000117
将ZSQ-13-16(284mg，2mmol)，溴化苄(237ul，2mmol)和碳酸钾(415mg，6mmol)溶于6ml DMF中，置于室温下混合搅拌18h。反应完成后用硅藻土过滤，DCM冲洗，滤液浓缩，经过C18反相柱层析分离纯化，用H 2O/CH 3CN(2:1)洗脱，得到86mg褐色固体，产率18％。
化合物2-benzyl-2H-tetrazole-5-carboxylic acid(ZSQ-17-45)的合成
Figure PCTCN2021099500-appb-000118
将化合物ZSQ-13-24(116mg，0.5mmol)溶于2ml THF、2ml H 2O的混合溶液中，加入一水合氢氧化锂(63mg，1.5mmol)，置于室温搅拌4h。反应完成后加入少量稀盐酸酸化，浓缩除去THF，经过C18反相柱层析分离纯化，用H 2O/CH 3CN(4:1)洗脱，得到白色固体79mg，合并产率77％。
化合物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)的合成
Figure PCTCN2021099500-appb-000119
将ZSQ-13-48(228mg，0.5mmol)，ZSQ-17-45(102mg，0.5mmol)溶于4ml干燥的吡啶中，混合溶液置于冰盐浴下搅拌10min，氧氯化磷(70ul，0.75mmol)被逐滴加入到混合溶液中(超过10min)，加完后继续0℃搅拌3h。反应完成后直接经过硅胶柱层析分离纯化，先用PE/EA(0-70％)洗脱冲出吡啶，再用DCM/CH 3OH(NH 3)(9:1)洗脱得到粗产物，经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:3)洗脱，得到136mg黄色固体，产率42％。
化合物N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-2-benzyl-N-(piperidin-4-ylmethyl)-2H-tetrazole-5-carboxamide(ZSQ-17-85)的合成
Figure PCTCN2021099500-appb-000120
将ZSQ-17-48(136mg，0.21mmol)和0.5ml三氟乙酸混合于4ml DCM中，室温混合搅拌过夜。反应完成后反应液浓缩并经过C18反相柱层析分离纯化，用H 2O/CH 3CN(1:1)洗脱，得到100mg黄色固体产物，产率88％。
化合物N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-2-benzyl-N-((1-ethylpiperidin-4-yl)methyl)-2H-tetrazole-5-carboxamide(ZSQ-17-86)的合成
Figure PCTCN2021099500-appb-000121
将ZSQ-17-85(100mg，0.18mmol)，碘乙烷(15ul，0.18mmol)和碳酸钾(75mg，0.54mmol)溶于2ml CH 3CN中，在冰盐浴中搅拌3h后移至室温搅1h。反应完成后将反应液浓缩，直接经过硅胶柱层析分离纯化，用DCM/CH 3OH(NH 3)(7:1)洗脱，得黄色固体46mg，产率45％。
化合物(4-(2-benzyl-N-((1-ethylpiperidin-4-yl)methyl)-2H-tetrazole-5-carbox-amido)phenyl)arsonous acid(ZSQ-17-87)的合成
Figure PCTCN2021099500-appb-000122
将ZSQ-17-86(20mg，0.035mmol)，硫酸汞(21mg，0.070mmol)溶于2ml DMSO中，混合溶液加热到60℃搅拌30min，立即经过C18反相柱层析分离纯化，用H 2O/CH 3CN(2:5)洗脱，得到粗产物再经过HPLC纯化得到8.6mg白色粉末，产率48％。M/Z:Exact Mass:512.15，(LC-MS):Found 513.0。
实施例17
化合物ZSQ-13-37的合成路线
Figure PCTCN2021099500-appb-000123
化合物2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (ZSQ-10-70)的合成
Figure PCTCN2021099500-appb-000124
将三缩四乙二醇(50g，262mmol)溶于60ml干燥的DCM中，置于冰浴中搅拌均匀，加入三乙胺(5.46ml，39.3mmol)。将4-甲苯磺酰氯(5.0g，26.2mmol)溶于20ml干燥DCM中，保持冰浴下逐滴加入到上述混合溶液中，加完后移至室温搅拌18h。待反应完成后，依次用2N盐酸(100ml)、水(100ml)，饱和氯化钠(100ml)洗，用DCM(50ml)萃取，合并有机相并用无水硫酸钠干燥，滤液旋干后经过硅胶柱层析分离纯化，用PE/EA(1:9)洗脱，得8.35g淡黄色油状产物，产率91％。
化合物2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-ol(ZSQ-12-91)的合成
Figure PCTCN2021099500-appb-000125
将ZSQ-10-70(7.21g，20.7mmol)溶于70ml DMF中，小心加入叠氮化钠(1.6g，24.8mmol)，混合溶液置于60℃搅拌12h。反应完成后直接浓缩反应液，抽干不纯化直接进行下一步。
化合物2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate(ZSQ-12-95)的合成
Figure PCTCN2021099500-appb-000126
将ZSQ-12.91(4.38g，20.0mmol)溶于40ml干燥的DCM中，置于冰浴中搅拌均匀，加入三乙胺(4.17ml，30.0mmol)。将4-甲苯磺酰氯(4.19g，22.0mmol)溶于5ml干燥DCM中，保持冰浴下逐滴加入到上述混合溶液中，加完后移至室温搅拌18h。待反应完成后，依次用2N盐酸(50ml)、水(50ml)，饱和氯化钠(50ml)洗，用DCM(50ml)萃取。合并有机相并用无水硫酸钠干燥，滤液旋干后经过硅胶柱层析分离纯化，用PE/EA(1:6)洗脱，得4.5g淡黄色油状产物，合并产率58％。
化合物2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate(ZSQ-13-28)的合成
Figure PCTCN2021099500-appb-000127
将化合物ZSQ-12-95(560mg，1.5mmol)溶于4ml THF、1ml H 2O的混合溶液中，加入三苯基膦(787mg，3.0mmol)，置于室温搅拌过夜。反应完成后将反应液浓缩，经过C18反相柱层析分离纯化，用H 2O/CH 3CN(3:1)洗脱，得黄色固体372mg，产率71％。
化合物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)的合成
Figure PCTCN2021099500-appb-000128
将ZSQ-13-28(186mg，0.536mmol)，(+)生物素-N-琥珀酰亚胺基酯(183mg，0.536mmol)和DIPEA(266ul，1.608mmol)溶于3ml DCM中，置于室温混合搅拌5h。反应完成后直接经过硅胶柱层析分离纯化，用DCM/CH 3OH(NH 3)(7:1)洗脱，得淡黄色固体264mg，产率71％。
化合物(R)-N-(4-(1,3,2-dithiarsolan-2-yl)phenyl)-1-benzyl-N-((1-(13-oxo-17-((3aR, 4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9-trioxa-12-azahepta-decyl)piperidin-4-yl)methyl)pyrrolidine-3-carboxamide(ZSQ-13-34)的合成
Figure PCTCN2021099500-appb-000129
将ZSQ-13-30(34mg，0.059mmol)，ZSQ-13-62(32mg，0.059mmol)和碳酸钾(24mg，0.177mmol)溶于2ml DMF中，混合溶液升温到80℃搅拌3h。
反应完成后直接经过HPLC纯化得到17mg白色粉末，产率30％。
化合物(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)pyrrolidine-3-carboxamido)phenyl)arsonous acid(ZSQ-13-37)的合成
Figure PCTCN2021099500-appb-000130
将ZSQ-13-34(17mg，0.018mmol)，三水合高氯酸汞(7mg，0.014mmol)溶于1ml DMSO中，混合溶液在室温下搅拌10min，立即经过C18反相柱层析分离纯化，用H 2O/CH 3CN(2:1)洗脱，再经过HPLC纯化得到7.2mg白色粉末，产率45％。M/Z:Exact Mass:886.36，(LC-MS):Found 887.30。
实施例18
化合物ZSQ-19-99的合成路线
Figure PCTCN2021099500-appb-000131
化合物tert-butyl 14-hydroxy-3,6,9,12-tetraoxatetradecanoate(ZSQ-19-86)的合成
Figure PCTCN2021099500-appb-000132
将三缩四乙二醇(38.84g，200mmol)溶于50ml无水THF中，置于冰浴下搅拌。NaH(1.6g，40mmol)溶于20ml无水THF中，逐滴加入混合溶液中(超过30min)，加完后0℃继续搅拌30min。向混合溶液中逐滴加入溴乙酸叔丁酯(7.8ml，40mmol)，加完后移至室温搅拌3h。反应完成后将反应液浓缩，分别用H 2O，饱和NaCl溶液洗，用EA萃取。有机相用无水硫酸钠干燥，过滤并浓缩，油泵抽干，没有进一步纯化，得淡黄色液体6.3g，直接进行下一步反应。
化合物tert-butyl 14-(tosyloxy)-3,6,9,12-tetraoxatetradecanoate(ZSQ-19-87)的合成
Figure PCTCN2021099500-appb-000133
将上一步产物ZSQ-19-86(6.16g，20mmol)溶于50ml干燥DCM中，加入三乙胺(4.16mmol，30mmol)，室温下搅拌均匀。对甲基本磺酰氯(4.58g，24mmol)溶于10ml超干DCM中，逐滴加入到混合溶液中，加完后室温过夜搅拌。反应完成后减压蒸馏浓缩反应液，依次用稀盐酸溶液，H 2O，饱和NaCl溶液洗，EA萃取，有机相合并经无水硫酸钠干燥，过滤并浓缩，经过硅胶柱层析分离纯化，用DCM/EA(3:1)洗脱，得到黄色油状液体5.127g，产率55％。
化合物tert-butyl 14-azido-3,6,9,12-tetraoxatetradecanoate(ZSQ-19-88)的合成
Figure PCTCN2021099500-appb-000134
将ZSQ-19-87(2.3g，5mmol)，NaN 3(390mg，6mmol)溶于20ml无水DMF中，加热到60℃搅拌过夜。反应完成后减压蒸馏除去DMF，得黄色固体未进行纯化直接开下一步。
化合物14-azido-3,6,9,12-tetraoxatetradecanoic acid(ZSQ-19-89)的合成
Figure PCTCN2021099500-appb-000135
将ZSQ-19-88溶于10ml DCM中，置于室温下搅拌，逐滴加入2ml三氟乙酸，加完后室温继续搅拌12h。反应完成后浓缩除去DCM，用THF溶解，经过C18反相柱层析分离纯化，用H 2O/CH 3CN(4:1)洗脱，得到912mg白色固体，产率65.85％
化合物(4-hydroxy-3-nitrophenyl)arsonous acid(ZSQ-16-8)的合成
Figure PCTCN2021099500-appb-000136
将3-硝基-4-羟基苯砷酸(26.3g,100mmol)溶于80ml甲醇中，混合溶液加热至回流。将苯肼(19.6ml，200mol)逐滴加入(超过1h)，加入过程中有大量氮气产生，当氮气产生减缓时，继续回流搅拌1.5h。混合溶液经过减压蒸馏浓缩，加入氢氧化钠溶液(12g溶于200ml水中)，乙醚200ml。分液，水相加入2N的稀盐酸(100ml)溶液搅拌1h，用EA萃取(200ml×3)。有机相用无水硫酸钠干燥后浓缩经油泵抽干，没有纯化进行下一步反应。
化合物4-(1,3,2-dithiarsolan-2-yl)-2-nitrophenol(ZSQ-16-14)的合成
Figure PCTCN2021099500-appb-000137
将ZSQ-16-8(24.7g，100mmol)溶解在100ml甲醇中并加热至回流。然后，在30min内将乙二硫醇(10ml，120mmol)滴加到该混合溶液中，并继续加热和搅拌30min。随后将混合溶液浓缩，经过硅胶柱层析分离纯化，用PE/EA(10:1)洗脱，得黄色固体粗产物。用THF溶解，再经过C18反相柱层析分离纯化，用H 2O/CH 3CN(8:1)洗脱，得到黄色固体产物9g，产率29.5％。
化合物2-amino-4-(1,3,2-dithiarsolan-2-yl)phenol(ZSQ-16-90)的合成
Figure PCTCN2021099500-appb-000138
将ZSQ-16-14(9.3g，30mmol)溶于乙醇/乙酸乙酯(10:1，100ml)的混合溶液中，加入氯化亚锡(13.5g，60mmol)，混合溶液置于75℃回流搅拌12h。反应完成后，将反应液浓缩后经过硅胶柱层析分离纯化，用PE/EA(8:1)洗脱，得黄色固体粗产物，用THF溶解，经C18反相柱层析分离纯化，用H 2O/CH 3CN(1:8)洗脱，得到4.83g黄色固体，产率58.54％。
化合物5-(1,3,2-dithiarsolan-2-yl)-2-methoxyaniline(ZSQ-19-92)的合成
Figure PCTCN2021099500-appb-000139
将ZSQ-16-90(2.74g，10mmol)，碘甲烷(747ul，12mmol)，碘化钾(332mg，2mmol)和碳酸钾(2.76g，20mmol)溶于40ml DMF中，室温混合搅拌24h。反应完成后将反应液浓缩，直接经过硅胶柱层析分离纯化，用PE/EA(4:1)洗脱，得到粗产物再经C18反相柱层析分离纯化，用H 2O/CH 3CN(1:8)洗脱，得到1.27g黄色固体，产率44％
化合物N-(5-(1,3,2-dithiarsolan-2-yl)-2-methoxyphenyl)-14-azido-3,6,9,12-tetra-oxatetradecanamide(ZSQ-19-90)的合成
Figure PCTCN2021099500-appb-000140
将ZSQ-19-92(189mg，0.65mmol)，ZSQ-19-89(217mg，0.78mmol)，HATU(370mg，0.98mmol)和DIPEA(322ul，1.95mmol)溶于40ml DCM中，置于室温混合搅拌12h。反应完成后直接经过硅胶柱层析分离纯化，用DCM/CH 3OH(NH 3)(10:1)洗脱，粗产物再经C18反相柱层析分离纯化，用H 2O/CH 3CN(1:3)洗脱得白色固体45mg，产率13％。
化合物N-(5-(1,3,2-dithiarsolan-2-yl)-2-methoxyphenyl)-14-amino-3,6,9,12-tetra-oxatetradecanamide(ZSQ-19-97)的合成
Figure PCTCN2021099500-appb-000141
将化合物ZSQ-19-90(45mg，0.08mmol)溶于2ml THF、0.5ml H 2O的混合溶液中，加入三苯基膦(43mg，0.16mmol)，置于室温搅拌过夜。反应完成后直接浓缩抽干，未进行纯化直接用于下一步。
化合物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(ZSQ-19-99)的合成
Figure PCTCN2021099500-appb-000142
将ZSQ-19-97粗品，(+)生物素-N-琥珀酰亚胺基酯(27mg，0.08mmol)和DIPEA(40ul，0.24mmol)溶于2ml DCM中，置于室温混合搅拌6h。反应完成后直接旋干经过HPLC纯化得到12mg白色粉末，合并产率20％。M/Z:Exact Mass:748.16，(LC-MS):Found 749.10。
使用和上述实施例类似的方法，合成的化合物如表1所示：
表1 化合物
Figure PCTCN2021099500-appb-000143
Figure PCTCN2021099500-appb-000144
Figure PCTCN2021099500-appb-000145
Figure PCTCN2021099500-appb-000146
Figure PCTCN2021099500-appb-000147
Figure PCTCN2021099500-appb-000148
Figure PCTCN2021099500-appb-000149
Figure PCTCN2021099500-appb-000150
Figure PCTCN2021099500-appb-000151
表2 化合物的氢谱及质谱数据
Figure PCTCN2021099500-appb-000152
Figure PCTCN2021099500-appb-000153
Figure PCTCN2021099500-appb-000154
Figure PCTCN2021099500-appb-000155
Figure PCTCN2021099500-appb-000156
Figure PCTCN2021099500-appb-000157
Figure PCTCN2021099500-appb-000158
Figure PCTCN2021099500-appb-000159
Figure PCTCN2021099500-appb-000160
Figure PCTCN2021099500-appb-000161
Figure PCTCN2021099500-appb-000162
Figure PCTCN2021099500-appb-000163
活性测试例1 抑制剂对PKM2体外酶活性的抑制效果
PKM2蛋白能在体外催化一分子ADP与一分子磷酸烯醇丙酮酸(PEP)反应生成一分子ATP与一分子丙酮酸。通过化合物预先处理PKM2蛋白，再检测其催化生成ATP 的浓度，即可推算出化合物对PKM2酶活的抑制效果。
所采用的的生物测试方案为：测试化合物在2μM浓度下抑制PKM2蛋白的催化活性的效果，“+”号代表30％-50％活性抑制，“++”代表50％-70％活性抑制，“+++”代表70-90％活性抑制，“++++”代表90％以上活性抑制。
方法：5μg/ml人源重组PKM2蛋白与待测化合物(终浓度1μM和10μM)4℃孵育24小时。加入酶反应混合液，使最终反应体系中含1mM ADP、1mM PEP、50mM MgCl 2,30℃反应10分钟。迅速加入20μL ATP-Glo试剂，室温孵育10分钟，用酶标仪检测化学发光的强度，计算生成ATP的浓度及PKM2酶催化活性。
表3 抑制剂对PKM2体外酶活性的抑制效果
化合物编号 抑制效果 化合物编号 抑制效果
ZSQ-5-39 + ZSQ-9-32 ++
ZSQ-5-40 + ZSQ-9-33 ++
ZSQ-5-42 + ZSQ-9-34 ++
ZSQ-5-43 + ZSQ-9-35 ++
ZSQ-5-44 + ZSQ-9-36 +
ZSQ-5-45 + ZSQ-9-37 +
ZSQ-5-47 + ZSQ-11-8 ++
ZSQ-5-77 + ZSQ-11-9 ++
ZSQ-5-78 + ZSQ-11-11 ++
ZSQ-5-80 + ZSQ-11-12 ++
ZSQ-5-81 + ZSQ-11-31 ++
ZSQ-5-82 + ZSQ-11-32 ++
ZSQ-5-83 + ZSQ-11-34 ++
ZSQ-6-3 + ZSQ-11-37 ++
ZSQ-6-37 ++ ZSQ-11-79 +++
ZSQ-6-49 + ZSQ-11-80 +++
ZSQ-6-50 + ZSQ-11-101 ++
ZSQ-6-51 + ZSQ-11-104 ++
ZSQ-6-52 + ZSQ-12-15 +++
ZSQ-6-54 + ZSQ-12-54 ++++
ZSQ-6-55 ++ ZSQ-12-66 ++
ZSQ-6-56 + ZSQ-12-67 +++
AZY-5-26 ++ ZSQ-12-70 +++
AZY-5-37 ++ ZSQ-12-73 ++
ZSQ-6-91 ++ ZSQ-12-76 ++
ZSQ-7-32 ++ ZSQ-12-80 ++++
ZSQ-7-33 ++ ZSQ-12-84 +
ZSQ-7-34 ++ ZSQ-12-85 +
ZSQ-7-35 +++ ZSQ-13-74 ++++
ZSQ-7-36 ++ ZSQ-13-83 ++++
ZSQ-7-37 ++ ZSQ-13-96 +++
ZSQ-7-86 ++ ZSQ-13-97 ++
ZSQ-7-106 ++ ZSQ-13-98 +++
ZSQ-8-32 ++ ZSQ-13-99 ++
ZSQ-8-33 +++ ZSQ-13-100 ++++
ZSQ-8-49 ++ ZSQ-13-101 ++++
ZSQ-8-50 ++ ZSQ-17-69 ++++
ZSQ-9-19 ++ ZSQ-17-74 +++
ZSQ-9-24 ++ ZSQ-17-75 +++
ZSQ-9-25 ++ ZSQ-17-79 +++
ZSQ-9-26 ++ ZSQ-17-80 +++
ZSQ-9-27 ++ ZSQ-17-81 +++
ZSQ-9-28 ++ ZSQ-17-86 ++++
ZSQ-9-30 ++ ZSQ-20-29 ++++
ZSQ-9-31 ++ ZSQ-20-30 ++++
活性测试例2 抑制剂对PKM2体外酶活性的半数抑制浓度
通过测试化合物在体外不同浓度下对PKM2的相对抑制活性，可以更精确地对代表性化合物的抑制活性进行定量和比较。将化合物的亚砷酸官能团以乙二硫醇保护后，其共价反应活性会大幅下降，但有利于提升化合物对靶蛋白的特异性结合。ZSQ1-94与ZSQ13-83分别是早期探针ZSQ1-97与优化后化合物ZSQ13-100的被保护形式，他们之间抑制活性的比较能够在一定程度上反应出各自与PKM2蛋白非共价结合能力的强弱。
所采用的的生物测试方案为：滴定代表性化合物抑制PKM2蛋白酶活的半数抑制浓度(IC 50)，并使用已报道的PKM2抑制剂紫草素(shikonin)和PKM2-IN-1作为对照。
方法：化合物以2倍浓度梯度稀释，待测终浓度从10μM至40nM共9个点。5μg/ml人源重组PKM2蛋白与待测化合物4℃孵育24小时。加入酶反应混合液，使最终反应体系中含1mM ADP、1mM PEP、50mM MgCl 2,30℃反应10分钟。迅速加入20μL ATP-Glo试剂，室温孵育10分钟，用酶标仪检测化学发光的强度，计算生成ATP的浓度及PKM2酶催化活性。
图1显示了代表性化合物及对照化合物体外抑制PKM2的浓度-活性曲线及其半数抑制浓度(IC 50)。
从图1可以看出：ZSQ13-83和ZSQ13-100、ZSQ1-97都展现出约1μM的IC 50，对PKM2的抑制活性明显优于紫草素和PKM2-IN-1；而ZSQ1-94也具有很低的起效浓度。
活性测试例3 抑制剂对PKM1体外酶活性的抑制效果
通过测试化合物在体外不同浓度下对PKM1的相对抑制活性，可以对代表性化合物在PKM2和PKM1之间的特异性进行比较。
所采用的的生物测试方案为：测试化合物在特定浓度下抑制PKM1蛋白的催化活性的效果，并使用已报道的PKM2抑制剂紫草素(shikonin)和PKM2-IN-1作为对照。
方法：3μg/ml人源重组PKM1蛋白与待测化合物4℃孵育24小时。加入酶反应混合液，使最终反应体系中含1mM ADP、1mM PEP、50mM MgCl 2,30℃反应10分钟。迅速加入20μL ATP-Glo试剂，室温孵育10分钟，用酶标仪检测化学发光的强度，计算生成ATP的浓度及PKM2酶催化活性。
图2显示了代表性化合物不同浓度下对PKM1的体外酶活性的抑制效果。
从图2可以看出，在10μM的高浓度下，ZSQ13-83仅微弱地抑制了PKM1，ZSQ13-100将PKM1酶活性抑制至一半左右，与此相对，紫草素几乎完全抑制PKM1。这说明本发明的化合物具有优异的针对PKM2的选择性。
活性测试例4 抑制剂与PKM2蛋白的共价结合位点
通过蛋白质谱手段测试分析经ZSQ13-83处理的PKM2蛋白各肽段的分子量及碎片峰，可以检测氨基酸残基侧链的修饰状态，进而可以确定出ZSQ13-83与PKM2蛋白共价结合的确切位点。
所采用的的生物测试方案为：利用液相-二级质谱联用仪测试化合物在特定浓度 下对PKM2蛋白的修饰位点。
方法：10μL浓度大约为500ng/μl的重组人源PKM2纯蛋白，溶剂为20mM Tris HCl,pH 8.0,10％Glycerol,2％DMS。加入终浓度为1μM的ZSQ13-83并混合，4℃条件下，震荡过夜。再经终浓度10mM的TCEP还原，终浓度22mM的碘代丙酰胺进行封闭，胰蛋白酶消化过夜，再经由液相-二级质谱联用仪测试分析。最后，对一级质谱数据中的多肽分子量进行检索，以确认肽链中半胱氨酸残基巯基侧链所发生的修饰状态，并通过二级质谱数据比对以确定肽链在PKM2蛋白中的归属。
图3显示了ZSQ13-83与PKM2蛋白474位半胱氨酸所在肽链共价结合后的质谱谱图。
从图3可以看出：在1μM浓度下，ZSQ13-83特异地与PKM2蛋白474位的半胱氨酸的巯基侧链发生了共价反应，进而加成到了含有该半胱氨酸的肽段上，同时水解脱除一分子乙二硫醇。因此，ZSQ13-83及其类似物对PKM2的抑制机制属于共价型机制。由于Cys474位于PKM2的变构位点，上述结果提示本发明的化合物属于变构型抑制剂。变构型抑制剂因为不与酶底物直接竞争，受酶底物浓度的变化影响较小；同时，变构位点往往不易发生催化位点易产生的耐药性突变。
活性测试例5 PKM2抑制剂对不同卵巢癌细胞株生长抑制活性测试
为了在细胞水平上验证本发明中的化合物对癌细胞的生长抑制作用，选取PA-1人卵巢畸胎瘤细胞和A2780人卵巢腺癌细胞(依赖于PKM2)和SKOV3卵巢腺癌细胞(不依赖于PKM2)，通过检测化学发光值来计算细胞活力，从而得出化合物抑制癌细胞生长的生物活性。
所采用的的生物测试方案为：滴定代表性化合物抑制卵巢细胞株PA-1、A2780或SKOV3细胞生长的半数抑制浓度(IC 50)，并使用已报道的PKM2抑制剂紫草素(shikonin)和PKM2-IN-1，PKM2激动剂TEPP-46，以及将ZSQ13-100亚砷酸官能团替代为硼酸的类似物ZSQ20-4作为对照。
方法：体外培养PA-1、A2780或SKOV3细胞，生长至对数生长期后，收集细胞，1000rpm离心5min，弃上清，调整细胞浓度至1.5×105/mL，将细胞接种至384孔板中，每孔40μl。在相应的孔中加入不同浓度的化合物或DMSO各5μL，放置细胞培养箱(37℃，5％CO2)中培养72h后，每孔加入15μl Cell Titer-Glo溶液，室温孵育30min，检测化学发光值(luminescence)以衡量细胞内ATP水平。以未刺激DMSO对照孔为100％细胞活力。运用Prism Graphpad统计软件计算化合物IC50值。
图4显示了ZSQ13-83及其类似物、以及对照化合物抑制不同卵巢癌细胞株生长的浓度-活性曲线及其半数抑制浓度(IC 50)。
从图4可以看出：
ZSQ12-54，ZSQ13-83、ZSQ17-69和ZSQ17-86在依赖于PKM2的PA-1和A2780细胞株上展现出纳摩尔级别的IC 50，而硼酸对照化合物ZSQ20-4完全无活性。
紫草素在PA-1和A2780细胞株上和ZSQ13-83活性相近，然而在不依赖于PKM2的SKOV3细胞株上也展现出较强的生长抑制活性；
PKM2-IN-1相对ZSQ13-83在PA-1细胞株上活性较弱；
PKM2激动剂TEPP-46没有表现出明显的细胞生长抑制活性。
因此，ZSQ13-83在对依赖PKM2的细胞生长抑制上，其活性优于PKM2-IN-1，且选择性优于紫草素。
将砷原子替代为硼原子后，所得ZSQ20-4不具备抑制活性，这提示本发明化合物对PKM2的抑制依赖于含砷原子的官能团。
活性测试例6 抑制剂与PA-1细胞中PKM2的不可逆结合
活性测试例4和图3表明，ZSQ13-83等抑制剂能够与PKM2变构位点附近的Cys474形成共价键，进而与PKM2不可逆地结合。ZSQ13-74是ZSQ13-83修饰了生物素标记的衍生物，保持了后者抑制PKM2的活性，理论上也能与细胞裂解液中的PKM2形成共价键，进而可通过链霉亲和素微珠(streptavidin beads)富集PKM2。当ZSQ系列PKM2抑制剂与PKM2不可逆地结合后，因结合口袋已被占据，ZSQ13-74便不能再与PKM2结合。因此，基于ZSQ13-74和链霉亲和素微珠的竞争富集实验，能准确地测定PKM2抑制剂是否能在活细胞中与PKM2不可逆地结合。
所采用的的生物测试方案为：通过活细胞处理、竞争结合拉低试验，测试本发明中的抑制剂是否与活细胞中的PKM2发生不可逆地结合。
方法：体外培养PA-1细胞，生长至对数生长期后，消化并收集细胞，1000rpm离心5min，弃上清，调整细胞浓度至1×106/mL。在12孔细胞培养板中，每孔加入1ml细胞，每孔加入1μL浓度为10mM的代表性药物的DMSO溶液(终浓度10μM)，以DMSO对照，放置细胞培养箱(37℃，5％CO2)中培养4小时后，使用预冷PBS溶液清洗两遍，向孔中加入200μL NP40细胞裂解液和蛋白酶抑制剂，4℃裂解30分钟。收集裂解液，以15000rpm 4℃离心15min，将上清液转移至样品管中，加入ZSQ13-74(终浓度为100nM)，置于4℃孵育过夜。加入10μL链霉亲和素微珠，置于室温结合2小时。微珠用1％NP40细胞裂解液洗10遍后，加入20μL 2％SDS裂解液裂解，95℃煮10分钟，上样，进行western-blot鉴定。
图5显示了代表性化合物如ZSQ13-83、ZSQ17-69和ZSQ17-80在10μM浓度下对ZSQ13-74共价结合PA-1细胞中PKM2的竞争性抑制。
从图5可以看出：
ZSQ13-74能够有效地共价结合并富集PA-1细胞裂解液中的PKM2；
PA-1细胞裂解液中的PKM1蛋白表达水平较低，ZSQ13-74也不能对其富集；
ZSQ13-83、ZSQ17-69和ZSQ17-80均能在PA-1活细胞中共价结合PKM2蛋白，使得ZSQ13-74不能再对其有效富集，说明本发明的化合物与PKM2的结合是共价/不可逆抑制。
从实施例4可知，本发明的化合物为PKM2变构型抑制剂，而变构抑制剂往往需要较高浓度才能有效抑制靶蛋白。令人惊讶地，本实施例进一步证明了本发明的PKM2抑制剂具有共价抑制机制，可通过共价作用增强其结合力，进而可以在较低浓度进行变构抑制，确保了低浓度下的有效抑制。
活性测试例7 代表性化合物ZSQ13-83对癌细胞信号通路的影响
在多种癌细胞中，能量供应的不足将导致AMPK信号通路的激活，进而抑制其他促进细胞生存增殖的信号通路(例如ERK通路)，最终促使凋亡的发生。当癌细胞中的PKM2被抑制时，其在糖酵解过程中直接的ATP供应以及供能原料丙酮酸的供应都可能被抑制，进而促使AMPK磷酸化水平的上升以及癌细胞凋亡。另有报道，PKM2在某些癌细胞中还作为转录调控因子，正向调控促癌因子c-MYC的表达。因此，检测这些癌细胞信号通路中的关键环节，有助于理解PKM2抑制剂抑癌的具体下游机制。
所采用的的生物测试方案为：通过ZSQ13-83处理PA-1细胞，并测试细胞中AMPK和ERK的磷酸化水平、以及c-MYC表达水平的变化。
方法：体外培养PA-1细胞，生长至对数生长期后，消化并收集细胞，1000rpm离心5min，弃上清，调整细胞浓度至1×106/mL。在12孔细胞培养板中，每孔加入1ml细胞，每孔加入1μL不同浓度药物的DMSO溶液，以DMSO对照，放置细胞培养箱(37℃，5％CO 2)中培养8小时后，使用预冷PBS溶液清洗两遍，吸去溶液，向 孔中加入200μL RIPA细胞裂解液、蛋白酶抑制剂和磷酸酶抑制剂，转移至样品管中置于4℃摇床裂解30min后，15000rpm 4℃离心15min，取上清的细胞裂解液。使用BCA蛋白定量试剂盒检测每组蛋白含量，并使用RIPA裂解液调齐蛋白量，使终体积为100μL。样品进行western-blot鉴定。
图6显示了ZSQ13-83在1μM浓度下对PA-1细胞中AMPK和ERK的磷酸化水平(p-AMPK、p-ERK)、以及c-MYC表达水平的影响。
从图6可以看出，ZSQ13-83在1μM浓度下即能有效地激活PA-1细胞中的AMPK，激活的时间点约为12小时后；经ZSQ13-83处理后，ERK的磷酸化发生应激性上调，然后再12小时后(即AMPK激活后)再次回落至加药前水平；经ZSQ13-83处理16小时后，c-MYC的蛋白表达水平也被明显抑制；
这提示ZSQ13-83通过抑制PA-1细胞中PKM2，同时干扰了PKM2相关的供能和转录通路，最终能够有效抑制PA-1细胞的生长。
活性测试例8 抑制剂在癌细胞中对PKM2的选择性
活性测试例6和图5表明，ZSQ13-83等抑制剂能够与癌细胞中的PKM2蛋白发生共价结合。为验证抑制剂在癌细胞中与PKM2蛋白结合的特异性，可基于ZSQ13-74和链霉亲和素微珠的富集实验，比较PKM2蛋白与其他蛋白的富集水平。
所采用的的生物测试方案为：通过共价结合拉低试验，测试本发明中的抑制剂衍射化的生物素探针富集蛋白的选择性。
方法：体外培养外源表达PKM2的293T细胞，生长至对数生长期后，消化并收集细胞，1000rpm离心5min，弃上清，加入NP40细胞裂解液和蛋白酶抑制剂，4℃裂解30分钟。收集裂解液，以15000rpm 4℃离心15min，将上清液转移至样品管中，以BCA法定量蛋白浓度。取含有1mg的上清液，加入ZSQ13-74(终浓度为10μM)，置于4℃孵育过夜。加入10μL链霉亲和素微珠，置于室温结合2小时。微珠用1％NP40细胞裂解液洗10遍后，加入20μL 2％SDS裂解液裂解，95℃煮10分钟，上样，进行凝胶电泳和银染。
图7显示了代表性化合物ZSQ13-83的生物素探针ZSQ13-74在10μM浓度下，对外源表达PKM2的293T细胞中蛋白的富集水平。
从图7可以看出：相比非特异性的探针ZSQ1-97，ZSQ13-74显著地富集细胞裂解液中的PKM2蛋白，但没有明显富集其他蛋白的现象。这说明本发明所优化的ZSQ13-83等抑制剂对细胞中PKM2蛋白具有出色的选择性，因而在应用中不容易导致脱靶效应和副作用。
活性测试例9 代表性化合物ZSQ13-83对癌细胞中葡萄糖代谢通路的影响
活性测试例8和图7表明，PKM2抑制剂ZSQ13-83能够促进癌细胞中AMPK信号通路的激活，意味着ZSQ13-83很可能阻断了癌细胞的能量供应。通过测试癌细胞的糖酵解功能，以及丙酮酸、乳酸、ATP和ADP等糖酵解过程中关键的代谢物水平，能够直接反映出ZSQ13-83等抑制剂阻断癌细胞能量供应的能力。
所采用的的生物测试方案为：通过ZSQ13-83处理PA-1细胞，并基于细胞外酸化率(反应乳酸分泌水平)测试表征癌细胞的糖酵解功能，同时利用质谱代谢组学手段测试关键代谢物的相对水平。
方法：将PA-1细胞以2×10 4/孔的浓度在384孔板中培养过夜，然后每孔加入1μL不同浓度的ZSQ13-83，以DMSO对照，放置细胞培养箱(37℃，5％CO 2)中培养12小时。使用含有1mM谷氨酰胺的XF培养基(pH值为7.4)置换原有培养基，在细胞培养箱(37℃，无CO 2)中培养1小时后，转移至Seahorse XF生物能量测试仪中进行测试。测试过程中，逐次添加葡萄糖(10mM)、寡霉素(oligomycin)(1μM) 和2-脱氧-d-葡萄糖(2-DG)(50mM)，然后检测细胞外酸化率的水平变化。
将PA-1细胞以4×10 6/皿的浓度在10厘米皿中培养过夜，然后每皿加入不同浓度的ZSQ13-83，以DMSO对照，放置细胞培养箱(37℃，5％CO 2)中培养12小时。收集细胞，清洗并悬浮于200μL蒸馏水中，4℃下超声10分钟，然后在液氮中速冻1分钟，再回复至室温，并重复冻融4次。收集冻融后的细胞裂解液，15000rpm 4℃离心15min，取180μL上清的细胞裂解液，以BCA法定量蛋白浓度。将上清液与甲醇/乙腈(1:1)以1:4比例混合，在4℃下超声10分钟，-20℃下冷冻1小时，再离心收集上清。将上清冷冻干燥后，重新溶解于100μL甲醇/乙腈(1:1)中，经超声、离心后收集上清，通过液相-质谱联用仪进行代谢物相对水平分析。
图8A显示了ZSQ13-83在不同浓度下，对PA-1细胞外酸化率的磷酸化水平的影响；图8B则显示了ZSQ13-83在不同浓度下，对PA-1细胞中糖酵解过程关键代谢物水平的影响。
从图8A可以看出，在加入葡萄糖后，PA-1细胞的乳酸分泌水平显著上升，并在加入oligomycin抑制氧化磷酸化之后进一步提升，最后在加入糖酵解抑制剂2-DG后完全回落。而ZSQ13-83在5μM浓度下，显著地抑制了PA-1细胞所被上调的乳酸分泌水平。从图8B则可看出，ZSQ13-83在5μM浓度下，显著地抑制了PA-1细胞中的丙酮酸水平，以及ATP与ADP的比例。
这揭示PA-1细胞正常的生长和糖代谢相当地依赖于糖酵解通路。而ZSQ13-83通过抑制PA-1细胞中PKM2，有效地抑制了PKM2所介导的糖酵解过程，限制了细胞内ATP水平以及能量供应，最终有效地抑制了癌细胞的生长。
活性测试例10 代表性化合物ZSQ13-83对裸鼠皮下瘤生长的影响
裸鼠成瘤实验作为一种体内研究肿瘤及其治疗的方法，即在裸鼠皮下注入人源肿瘤细胞，观察肿瘤生长甚至抗肿瘤药物的效果。测试代表性化合物ZSQ13-83对人源卵巢癌细胞皮下瘤生长的影响，使用空白配药溶剂作为对照，同时监测皮下瘤体积和裸鼠体重。卵巢癌是致死率最高的妇科恶性肿瘤，严重危害女性健康，70％以上患者确诊时即已处于中晚期；长期以来标准治疗方案是肿瘤减灭手术配合铂类为基础的系统性联合化疗，病人临床获益已经达到平台期。近年来领域主要方向是针对其生物学特性合理设计靶向治疗策略，目前基于合成致死机理的PARP抑制剂得到应用，如奥拉帕利、尼拉帕利、维利帕尼等小分子药物。但其适应症仅限于具有同源重组缺陷的卵巢癌，且不同患者反应存在明显差异，因此卵巢癌临床治疗亟需更多新的分子靶标和靶向药物。
方法：使用卵巢癌细胞HEY在裸鼠右背侧注射1X10 6个细胞/只，每天观察皮下肿瘤生长，待肿瘤长到合适体积时，准备给药。待测化合物当天溶解于配药溶剂(0.5％羟丙甲纤维素水溶液)，制成10mg/mL溶液。将注射HEY细胞的裸鼠均分为给药组和空白组，给药组按每只50mg/kg灌胃给药，同时对照组灌胃空白配药溶剂，每隔一天监测皮下肿瘤体积。
图9显示了代表性化合物ZSQ13-83对HEY裸鼠皮下肿瘤生长的抑制效果，以及对裸鼠体重的影响。从图9可以看出，在50mg/kg灌胃剂量下，化合物ZSQ13-83能够有效减缓卵巢癌细胞皮下肿瘤的生长速度(图9A-C)，且没有明显的毒副作用(图9D)。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种式I化合物、或其药学上可接受的盐、或其立体异构体或互变异构体、水合物或溶剂化物；
    Figure PCTCN2021099500-appb-100001
    其中，所述X 1、X 2各自独立地选自下组：无、O、S、NR a、CR bR c；
    R a、R b和R c各自独立的选自：H或取代或未取代地C1-C6烷基；
    R 1、R 2各自独立地选自下组：H、取代或未取代的C1-C6烷基、取代或未取代的C3-C8环烷基或-(C＝O)-R d，其中，R d为取代或未取代的C1-C6烷基、取代或未取代的C3-C8环烷基；或R 1、R 2与相连的X 1、X 2和As共同形成取代或未取代的4至8元杂环，所述杂环含有一个As杂原子以及0-3个选自N、O和S的杂原子；
    R 3选自下组：H、取代或未取代的C1-C6烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、取代或未取代的C3-C8环烷基或取代或未取代的C1-C3亚烷基-R e；
    R e选自下组：-CO-NR fR g、取代或未取代的6-10元芳基、取代或未取代的具有1-3个选自O、N和S的杂原子的5-10元杂芳基、取代或未取代的C3-C10环烷基或取代或未取代的具有1-3个选自O、N和S的杂原子的3-10元杂环烷基；所述取代是指基团上的一个或多个氢原子被选自下组的基团取代：卤素、-OH、-CN、-NH 2、取代或未取代的C1-C6烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、取代或未取代的C1-C6烷氧基、-(C＝O)-取代或未取代的C1-C6烷基、-NR h-(C＝O)-取代或未取代的C1-C6烷基、取代或未取代的C3-C10环烷基、取代或未取代的具有1-3个选自O、N和S的杂原子的3-10元杂环烷基、-C1-C4亚烷基-(O-CH 2-CH 2)m-NR f-(C＝O)-R i或-(C＝O)-CH 2(O-CH 2-CH 2)m-NR f-(C＝O)-R i；
    n为0、1、2、3或4；
    各个R 4独立地选自：H、卤素、D、OH、-NH 3、-NO2、-CN、COOH、取代或未取代的C1-C6烷基、取代或未取代的C1-C6烷氧基、-OCO-取代或未取代的C1-C6烷基；
    R 5选自下组：取代或未取代的C1-C6烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、取代或未取代的6-10元芳基、取代或未取代的具有1-3个选自O、N和S的杂原子的5-10元杂芳基、取代或未取代的C3-C10环烷基、或取代或未取代的具有1-3个选自O、N和S的杂原子的3-10元杂环烷基、-C1-C4亚烷基-(O-CH 2-CH 2)m-NR f-CO-R i或
    Figure PCTCN2021099500-appb-100002
    其中，环A选自下组：取代或未取代的C3-C10环烷基、取代或未取代的具有1-4个选自O、N和S的杂原子的3-10元杂环烷基、取代或未取代的6-10元芳基、取代或未取代的具有1-4个选自O、N和S的杂原子的5-10元杂芳基；
    L 1选自下组：无、取代或未取代的C1-C6亚烷基、取代或未取代的C2-C6亚烯基、取代或未取代的C2-C6亚炔基或取代或未取代的C1-C6亚烷基-(C＝O)-；
    环B选自下组：取代或未取代的C3-C10环烷基、或取代或未取代的具有1-3个选自O、N和S的杂原子的3-10元杂环烷基、取代或未取代的6-10元芳基或取代或未取代的具有1-3个选自O、N、S的杂原子的5-10元杂芳基；其中，所述取代是指基团上的一个或多个氢原子被选自下组的取代基取代：卤素、-OH、-CN、-NH 2、C1-C6烷氧基、C1-C6烷基、C2-C6烯基、C2-C6炔基、C1-C6卤代烷基、-CO-NR fR g、-NR fR g；
    各m独立地为0、1、2、3、4或5；
    各R i独立地选自下组：H、取代或未取代的C1-C6烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基、-L 2-R 9；
    其中，L 2选自下组：取代或未取代的C1-C6亚烷基、取代或未取代的C2-C6亚烯基、取代或未取代的C2-C6亚炔基；
    R 9选自下组：取代或未取代的C3-C10环烷基、或取代或未取代的具有1-3个选自O、N和S的杂原子的3-10元杂环烷基、取代或未取代的6-10元芳基、取代或未取代的具有1-3个选自O、N和S的杂原子的5-10元杂芳基；
    如未特别说明，所述“取代”是指基团上的一个或多个氢原子被选自下组的基团取代：卤素、-OH、-CN、-NR fR g、-NR f-CO-C1-C6烷基、-C1-C6烷基-CO-NR fR g、氧代(＝O)、C1-C6烷基、C1-C6氧杂烷基、C2-C6烯基、C2-C6炔基、C1-C6烷氧基、C1-C6卤代烷基、-(C＝O)-C1-C6烷基、C5-C10芳基、苄基、3-8元环烷基；和
    各R f和R g独立地选自下组：H、C1-C6烷基、C2-C6烯基、C2-C6炔基、C3-C8环烷基或具有1-3个选自O、N和S的杂原子的3-10元杂环烷基，或R f与R g与相连的氮原子一起形成具有一个氮原子的3-7元环烷基。
  2. 如权利要求1所述的化合物，其特征在于，
    Figure PCTCN2021099500-appb-100003
    具有选自下组的结构：
    Figure PCTCN2021099500-appb-100004
  3. 如权利要求1所述的化合物，其特征在于，R 5为
    Figure PCTCN2021099500-appb-100005
    或取代或未取代的选自下组的基团：
    Figure PCTCN2021099500-appb-100006
  4. 如权利要求1所述的化合物，其特征在于，环A为取代或未取代的选自下组的基团：
    Figure PCTCN2021099500-appb-100007
  5. 如权利要求1所述的化合物，其特征在于，L 1为-CH 2-且环B为取代或未取代的苯基。
  6. 如权利要求1所述的化合物，其特征在于，R e为取代或未取代的选自下组的基团：
    Figure PCTCN2021099500-appb-100008
    其中，各Rj独立地选自下组：H、C1-C4烷氧基、C1-C4烷基、C1-C4卤代烷基、-(C＝O)-C1-C4烷基、羟基取代的C1-C4烷基，-C1-C4亚烷基-(O-CH 2-CH 2)m-NH-(C＝O)-R i或-(C＝O)-CH 2(O-CH 2-CH 2)m-NH-(C＝O)-R i，m和Ri如上定义；
    所述取代指基团的环碳原子上的一个或多个氢被选自下组的基团取代：卤素、氰基、C1-C4烷氧基、C1-C4烷基、-(C＝O)-C1-C4烷基、羟基取代的C1-C4烷基、-NHCO-C1-C4烷基。
  7. 如权利要求1所述的化合物，其特征在于，所述R e具有选自下组的结构：
    Figure PCTCN2021099500-appb-100009
    其中，Rj独立地选自下组：H、C1-C4烷基、C1-C4卤代烷基、-(C＝O)-C1-C4烷基、羟基取代的C1-C4烷基，较佳地，Rj选自下组：-CH 3、-CH 2CH 3、-CH 2CH 2OH、-(C＝O)CH 3。
  8. 如权利要求1所述的化合物，其特征在于，所述式I化合物具有式Ia或Ib的结构：
    Figure PCTCN2021099500-appb-100010
    其中，所述X 1、X 2、R 1、R 2、R 3、R 4、R i、A、B、L 1、n、m如权利要求1所定义。
  9. 如权利要求8所述的化合物，其特征在于，所述式Ia化合物具有IIa结构：
    Figure PCTCN2021099500-appb-100011
    其中，R k选自下组：C1-C4烷基、C1-C4卤代烷基、-(C＝O)-C1-C4烷基、羟基取代的C1-C4烷基；
    所述X 1、X 2、R 1、R 2、R 4、A、B、L 1、n如权利要求1所定义。
  10. 如权利要求1所述的化合物，其特征在于，所述化合物选自表1中的下组化合物：
    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、
    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和ZSQ-19-99。
  11. 一种药物组合物，其特征在于，所述药物组合物包括：(a)如权利要求1所述的式I化合物、或其药学上可接受的盐、或其立体异构体或互变异构体、水合物或溶剂化物；和(b)药学上可接受的载体。
  12. 如权利要求1所述的式I化合物、或其药学上可接受的盐、或其立体异构体或互变异构体、水合物或溶剂化物或包含其的药物组合物的用途，用于制备一药物或制剂，所述药物制剂用于：
    (i)下调和/或抑制PKM2活性；
    (ii)预防和/或治疗肿瘤；
    (iii)降低和/或抑制肿瘤细胞的生长和/或增殖；
    (iv)降低和/或抑制肿瘤细胞的转移；
    (v)促进肿瘤细胞凋亡；和/或
    (vi)降低和/或抑制细胞的糖酵解过程。
  13. 如权利要求11所述的用途，其特征在于，所述肿瘤选自下组：肝癌、胃癌、乳腺癌、胰腺癌、卵巢癌(包括人卵巢畸胎瘤)、喉癌(包括喉鳞状细胞癌)、前列腺癌、结肠癌、直肠癌、胰腺癌、宫颈癌、子宫内膜癌、脑癌、膀胱癌、睾丸癌、头癌、颈癌、皮肤癌(包括黑色素瘤和基底细胞癌)、间皮内膜癌、白血球癌(包括淋巴瘤和白血病)、食道癌、肌癌、结缔组织癌、肺癌(包括小细胞肺癌和非小细胞肺癌)、肾上腺癌、甲状腺、肾癌或骨癌、胶质母细胞瘤、间皮瘤、肾细胞癌、胃癌、肉瘤(卡波西肉瘤)、绒毛膜癌、皮肤基底细胞癌和睾丸精原细胞瘤。
  14. 一种下调和/或抑制PKM2活性的方法，其特征在于，包括步骤：在如权利要求1所述的式I化合物、或其药学上可接受的盐、或其立体异构体或互变异构体、水合物或溶剂化物或包含其的的药物组合物存在下，培养细胞，从而在所述细胞中下调和/或抑制PKM2的活性。
  15. 如权利要求1所述式I化合物的制备方法，其特征在于，包括步骤：
    步骤i：将A1的亚砷酸官能团以酯或酰胺的方式保护；
    步骤ii：通过缩合反应在A2的胺基引入羰基连接的R 5取代基；和
    步骤iii：通过S N2取代反应在A3的胺基引入R 3取代基；
    Figure PCTCN2021099500-appb-100012
    其中，所述X 1、X 2、R 1、R 2、R 4、R 5和n如权利要求1所定义。
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