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WO2016127790A1 - 配体-细胞毒性药物偶联物、其制备方法及其应用 - Google Patents

配体-细胞毒性药物偶联物、其制备方法及其应用 Download PDF

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Publication number
WO2016127790A1
WO2016127790A1 PCT/CN2016/072129 CN2016072129W WO2016127790A1 WO 2016127790 A1 WO2016127790 A1 WO 2016127790A1 CN 2016072129 W CN2016072129 W CN 2016072129W WO 2016127790 A1 WO2016127790 A1 WO 2016127790A1
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group
compound
methoxy
formula
mmol
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PCT/CN2016/072129
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English (en)
French (fr)
Inventor
许建烟
章瑛
屈博磊
张富尧
于秀招
梁金栋
蒋贵阳
张连山
李昂
王亚里
Original Assignee
江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to MX2017010102A priority Critical patent/MX2017010102A/es
Priority to JP2017541769A priority patent/JP2018512377A/ja
Priority to KR1020177025385A priority patent/KR20170117473A/ko
Priority to EP16748591.1A priority patent/EP3251698A4/en
Priority to BR112017016503-1A priority patent/BR112017016503A2/zh
Priority to CA2976050A priority patent/CA2976050A1/en
Priority to US15/549,710 priority patent/US20180177890A1/en
Priority to RU2017131158A priority patent/RU2708461C2/ru
Priority to CN201680001840.7A priority patent/CN106794258B/zh
Priority to AU2016218840A priority patent/AU2016218840A1/en
Publication of WO2016127790A1 publication Critical patent/WO2016127790A1/zh

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Definitions

  • Chemotherapy remains one of the most important anti-cancer methods including surgery, radiation therapy, and targeted therapy. Although there are many types of highly potent cytotoxins, the difference between tumor cells and normal cells is small, which limits the clinical application of these antitumor compounds due to toxic side effects. While anti-tumor monoclonal antibodies are specific for tumor cell surface antigens, antibody drugs have become a front-line drug for anti-tumor therapy, but when antibodies are used as anti-tumor drugs alone, the efficacy is often unsatisfactory.
  • Antibody drug conjugates link monoclonal antibodies or antibody fragments to biologically active cytotoxins via stable chemical linker compounds, making full use of the specificity of antibodies for normal cell and tumor cell surface antigen binding. And the high efficiency of cytotoxin, while avoiding the defects of the former's low efficacy and excessive toxic side effects. This means that antibody drug conjugates bind precisely to tumor cells and reduce the effects on normal cells compared to traditional chemotherapeutic drugs.
  • ADC drugs mainly used murine monoclonal antibodies because human immune responses made it difficult for some drugs to reach the target.
  • the source of the antibody, the manner and number of linkers are not optimized.
  • T-DM1 trastuzumab emtansine
  • T-DM1 trastuzumab
  • trastuzumab trade name: Patients with advanced or metastatic breast cancer who are resistant to paclitaxel. with Both are targeted therapy for hematological tumors, and the tissue structure is relatively simple compared to solid tumors. It is the first ADC drug approved by the US FDA for the treatment of solid tumors.
  • the highly active mitotic inhibitor DM1 was ligated to Roche's trastuzumab with a stable thioether linker. On average, one trastuzumab binds approximately 3.5 DM1, trastuzumab The specific binding of breast cancer cells in the patient is mediated by intracellular endocytosis and cleavage of DM1. The concentration of DM1 in the cells is sufficient to cause the cells to die due to mitotic disorders, and the tumor foci disappear. T-DM1 has been retained Antibody-dependent inhibition of cell proliferation, while increasing the effects of potential cytotoxic drugs. Moreover, because its toxin is targeted for release in tumor cells, the drug side effects do not increase synchronously with increasing efficacy.
  • Pertuzumab (also known as 2C4, trade name Perjeta) is a recombinant humanized monoclonal antibody that is the first monoclonal antibody to be referred to as a "HER dimerization inhibitor.” By combining HER2 blocks the dimerization of HER2 with other HER receptors. Pertuzumab has been shown to inhibit tumor growth in models with high expression and low expression of prostate cancer.
  • trastuzumab (Trastuzumab, trade name Herceptin) is located in the sub-region IV of the extracellular domain of HER2, which inhibits the downstream signaling pathway. Pertuzumab is bound by the II domain (dimerization). The domain) effectively inhibits the heterodimerization of HER2.
  • trastuzumab only has a certain effect on cancer patients overexpressing HER2, especially breast cancer patients, while pertuzumab has the same target as trastuzumab, and both have the same internal
  • T-DM1 uses a random coupling of a cytotoxic drug to a free amino group of an antibody; The use of a cytotoxic drug and a free sulfhydryl group after reduction of the hinge region of the antibody is used. Both coupling methods yielded a mixture of inconsistent drug loadings. For example, although T-DM1 has an average drug loading of 3.5, the drug loading can be distributed from 0 to 8. Too low drug loading affects the efficacy of the ADC, while too high drug loading is more likely to cause the ADC drug to be recognized and destroyed by the tissue macrophage system due to excessive modification of the antibody.
  • ADC drug patents are WO2007008603, WO2013173393, WO2005081711, WO2013173391, WO2013173392, WO2013173393 and WO2012010287.
  • the present invention provides a novel ADC compound having a novel linkage method, a novel toxin combined with an antibody, thereby having a more beneficial effect.
  • the present invention provides a compound of the formula (PC-L-Dr) comprising an improved linking unit or a pharmaceutically acceptable salt or solvent compound thereof:
  • R, R 2 - R 7 are selected from the group consisting of a hydrogen atom, a halogen, a hydroxyl group, a cyano group, an alkyl group, an alkoxy group, and a cycloalkyl group;
  • At least one of R 8 to R 11 is selected from the group consisting of halogen, alkenyl, alkyl and cycloalkyl, the balance being a hydrogen atom;
  • any two of R 8 -R 11 form a cycloalkyl group, and the remaining two groups are selected from a hydrogen atom, an alkyl group and a cycloalkyl group;
  • R 12 -R 13 is selected from a hydrogen atom, an alkyl group or a halogen
  • R 14 is selected from aryl or heteroaryl, which is optionally further substituted with a substituent selected from the group consisting of a hydrogen atom, a halogen, a hydroxyl group, an alkyl group, an alkoxy group, and a cycloalkyl group;
  • Y is 1-8, preferably 2-5; y is a positive real number, which may be an integer or a decimal;
  • PC is a ligand
  • L is a linker unit.
  • a compound represented by the formula (PC-L-Dr) or a pharmaceutically acceptable salt or solvate thereof which is a compound represented by the formula (PC-LD) or a pharmaceutically acceptable salt or solvent compound thereof:
  • R 2 - R 14 are as defined in the formula (PC-L-Dr).
  • a compound of the formula (PC-L-Dr) or a pharmaceutically acceptable salt or solvate thereof which is represented by the formula (PC-L'-Dr) a compound or a pharmaceutically acceptable salt or solvent compound thereof:
  • R 15 is selected from a hydrogen atom, a halogen, a hydroxyl group, a cyano group, an alkyl group, an alkoxy group, and a cycloalkyl group;
  • R 16 is selected from the group consisting of alkyl, cycloalkyl, alkoxy and heterocyclic;
  • n 2-6, preferably 2-5;
  • n is 0-5, preferably 1-3;
  • PC, y, n, R, R 2 - R 14 are as defined in the formula (PC-L-Dr).
  • a compound represented by the formula (PC-L-Dr) or a pharmaceutically acceptable salt or solvate thereof which is represented by the formula (PC-L'-D) a compound or a pharmaceutically acceptable salt or solvent compound thereof:
  • R 15 , R 16 , m are as defined in the formula (PC-L'-Dr);
  • PC, y, n, R 2 - R 14 are as defined in the formula (PC-L-Dr).
  • a compound of the formula (PC-L'-D) or a pharmaceutically acceptable salt or solvate thereof which is of the formula (PC-L'-D1)
  • PC, y, n, m, R 2 - R 16 are as defined in the formula (PC-L'-D).
  • the ligand drug conjugate ADC compounds of the invention include, but are not limited to:
  • Typical ligand drug conjugate ADC compounds of the invention include, but are not limited to:
  • the present invention further provides a compound of the formula (Dr):
  • R, R 1 - R 7 are selected from the group consisting of a hydrogen atom, a halogen, a hydroxyl group, a cyano group, an alkyl group, an alkoxy group, and a cycloalkyl group;
  • At least one of R 8 to R 11 is selected from the group consisting of halogen, alkenyl, alkyl and cycloalkyl, the balance being a hydrogen atom;
  • any two of R 8 -R 11 form a cycloalkyl group, and the remaining two groups are selected from a hydrogen atom, an alkyl group and a cycloalkyl group;
  • R 12 -R 13 is selected from a hydrogen atom, an alkyl group or a halogen
  • R 14 is selected from an aryl or heteroaryl group, which is optionally further substituted with a substituent selected from a hydrogen atom, a halogen, a hydroxyl group, an alkyl group, an alkoxy group, and a cycloalkyl group.
  • a compound of the formula (Dr) is represented by the formula (D) compound of:
  • R 1 -R 14 is as defined in the formula (Dr).
  • R 1 -R 14 is as defined in the formula (D).
  • Typical prodrug compounds of the ligand drug conjugate ADCs of the invention include, but are not limited to:
  • the invention further provides a compound of the formula (L 1 -Dr):
  • n 2-6, preferably 2-5;
  • R, R 1 - R 7 are selected from the group consisting of a hydrogen atom, a halogen, a hydroxyl group, a cyano group, an alkyl group, an alkoxy group, and a cycloalkyl group;
  • At least one of R 8 to R 11 is selected from the group consisting of halogen, alkenyl, alkyl and cycloalkyl, the balance being a hydrogen atom;
  • R 12 -R 13 is selected from a hydrogen atom, an alkyl group or a halogen
  • R 14 is selected from an aryl or heteroaryl group, which is optionally further substituted with a substituent selected from a hydrogen atom, a halogen, a hydroxyl group, an alkyl group, an alkoxy group, and a cycloalkyl group.
  • n, R 2 - R 14 are as defined in the formula (L1 - Dr).
  • R 1 - R 12 are as defined in the formula (L 1 -D).
  • a typical prodrug compound (with a partial linker unit) of the ligand drug conjugate ADC of the invention can be used to prepare intermediates for the ligand drug conjugates of the invention, including, but not limited to, the following compounds:
  • the invention further provides a compound of the formula (PC-L 2 ):
  • a PC ligand preferably an antibody, more preferably Pertuzumab, Nimotuzumab or Trastuzumab;
  • R 15 is selected from a hydrogen atom, a halogen, a hydroxyl group, a cyano group, an alkyl group, an alkoxy group, and a cycloalkyl group;
  • R 16 is selected from the group consisting of alkyl, cycloalkyl, alkoxy and heterocyclic;
  • n is 0-5, preferably 1-3;
  • X is 0-5, preferably 1 ⁇ 3, and x is a positive real number, including decimals and integers.
  • the invention further provides a process for the preparation of a compound of the formula (PC-L 2 ) comprising the steps of:
  • RA is preferably sodium cyanoborohydride or triacetoxy boron Sodium hydride, more preferably sodium cyanoborohydride;
  • T is selected from H, tert-butyl, acetyl, n-propionyl, isopropionyl, triphenylmethyl, methoxymethyl, 2-(trimethylsilyl)ethoxymethyl, preferably H or acetyl ;
  • the (PC-L2-A) compound is preferably S-(3-carbonylpropyl) thioacetate
  • the invention further provides a process for the preparation of a compound of the formula (PC-L'-D), the process comprising:
  • PC-L2 A compound of the formula (PC-L2) is reacted with a compound of the formula (L1-D1) to give a compound of the formula (PC-L'-D);
  • PC, m, n, y, R 2 to R 16 are as defined in the formula (PC-L'-D).
  • the invention further provides a compound of the formula (D-A a):
  • R 8 -R 11 form a cycloalkyl group, and the remaining two groups are selected from a hydrogen atom, an alkyl group and a cycloalkyl group;
  • R 12 is selected from a hydrogen atom, an alkyl group or a halogen
  • P is a hydrogen atom or a protecting group, and the protecting group is preferably Boc, Bn, Cbz, most preferably Boc;
  • R a is selected from a hydroxyl group, an amino group, an alkoxy group, a cycloalkoxy group or an alkylamino group.
  • the present invention further provides a compound of the formula (D-Aa) which is a compound of the formula (D-A):
  • R 8 -R 12 ,P forms a cycloalkyl group, and the remaining two groups are selected from a hydrogen atom, an alkyl group and a cycloalkyl group;
  • R' is selected from a hydrogen atom, an alkyl group, and a cycloalkyl group.
  • the intermediate (D-Aa) or (D-A) of a typical prodrug compound of the ligand drug conjugate ADC of the present invention includes, but is not limited to:
  • the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a ligand-cytotoxic drug couple of the formula (PC-L-Dr), formula (PC-L'-D) and others as described above a complex, or a drug of the formula (Dr), formula (L1 - Dr), formula (D), formula (L1 - D), and other pharmaceutically acceptable salts or solvates thereof, And a pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention further provides a ligand-cytotoxic drug conjugate of the formula (PC-L-Dr), the formula (PC-L'-D) and others as described above, or as in the formula (Dr), Formula (L1 - Dr), Formula (D), Formula (L1 - D) and other pharmaceuticals as described above, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition as described above, in the preparation of a treatment
  • a medicament for mammalian cancer which is a cancer associated with HER2, HER3, EGFR expression.
  • the invention further relates to a method of treating cancer in a mammal, the method comprising administering to the mammal an effective amount of a formula (PC-L-Dr), a formula (PC-L'-D), and the like as described above.
  • a ligand-cytotoxic drug conjugate or a pharmaceutically acceptable salt or solvate thereof or as in the formula (Dr), formula (L1 ⁇ Dr), formula (D),
  • the present invention further provides a ligand-cytotoxic drug conjugate of the formula (PC-L-Dr), the formula (PC-L'-D) and others as described above, or as in the formula (Dr), Formula (L1 - Dr), Formula (D), Formula (L1 - D) and other pharmaceuticals as described above, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition as described above, in the preparation of a treatment
  • a medicament for mammalian cancer wherein the mammal is selected from the group consisting of breast cancer, ovarian cancer, gastric cancer, endometrial cancer, salivary gland cancer, lung cancer, colon cancer, kidney cancer, rectal cancer, thyroid cancer, Pancreatic cancer, prostate cancer, bladder cancer, acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma or
  • the invention further relates to a method of treating cancer in a mammal, the method comprising administering to the mammal an effective amount of a formula (PC-L-Dr), a formula (PC-L'-D), and the like as described above.
  • a ligand-cytotoxic drug conjugate or a pharmaceutically acceptable salt or solvate thereof or as in the formula (Dr), formula (L1-Dr), formula (D), formula (L1-D), and Further, a pharmaceutical composition or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising the same;
  • the mammal is preferably a human;
  • the cancer is selected from the group consisting of breast cancer, ovarian cancer, gastric cancer, and endometrial cancer , salivary gland cancer, lung cancer, colon cancer, kidney cancer, rectal cancer, thyroid cancer, pancreatic cancer, prostate cancer, bladder cancer, acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid le
  • the present invention binds the linking unit L containing a free sulfhydryl group to the amino group of the N-terminal amino group and/or the lysine residue of the antibody, the reduction reaction of the antibody hinge region can be avoided, thereby reducing the influence on the structure of the antibody itself. Moreover, the introduced carbon-nitrogen bond structure is stable, and it is not easy to be decomposed in the body circulation, and further, by controlling the reaction conditions, the drug loading amount can be distributed in the range of 0 to 5.
  • the applicant intends to include a formulation of the trade name product, a generic drug of the trade name product, and an active drug moiety.
  • alkyl refers to a saturated aliphatic hydrocarbon group which is a straight or branched chain group containing from 1 to 20 carbon atoms, preferably an alkyl group having from 1 to 12 carbon atoms, more preferably from 1 to 10 carbons.
  • the alkyl group of the atom is most preferably an alkyl group having 1 to 6 carbon atoms.
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1 ,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2- Methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3 - dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2 -methylhexyl, 3-methylhexyl, 4-methylhexyl,
  • lower alkyl groups having from 1 to 6 carbon atoms, non-limiting examples including methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl Base, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethyl Butyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl Base, 2,3-dimethylbutyl and the like.
  • the alkyl group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment, preferably one or more of the following groups independently selected from the group consisting of an alkane Base, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, fluorenyl, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, naphthenic Oxyl, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo.
  • an alkane Base alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, fluorenyl, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, naphthenic Oxyl, heterocycloal
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 carbon atoms, preferably from 3 to 12 carbon atoms, more preferably from 3 to 10 carbon atoms. One carbon atom, most preferably from 3 to 8 carbon atoms.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms wherein one or more ring atoms are selected from nitrogen, oxygen or S(O).
  • a hetero atom of m (where m is an integer of 0 to 2), but excluding the ring moiety of -OO-, -OS- or -SS-, the remaining ring atoms being carbon. It preferably contains 3 to 12 ring atoms, of which 1 to 4 are hetero atoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms.
  • Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl and the like.
  • Polycyclic heterocyclic groups include spiro, fused, and bridged heterocyclic groups.
  • the heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the parent structure is attached
  • the ring together is a heterocyclic group, non-limiting examples of which include:
  • the heterocyclic group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, fluorenyl, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, oxo.
  • aryl refers to a 6 to 14 membered all-carbon monocyclic or fused polycyclic ring (ie, a ring that shares a pair of adjacent carbon atoms) having a conjugated ⁇ -electron system, preferably 6 to 10 members, such as benzene. And naphthyl, preferably phenyl.
  • the aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring to which the parent structure is attached is an aryl ring, non-limiting examples of which include:
  • the aryl group may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, Alkylamino, halogen, fluorenyl, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycle Alkylthio group.
  • heteroaryl refers to a heteroaromatic system containing from 1 to 4 heteroatoms, from 5 to 14 ring atoms, wherein the heteroatoms are selected from the group consisting of oxygen, sulfur and nitrogen.
  • the heteroaryl group is preferably 5 to 10 members, more preferably 5 or 6 members, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetra Azolyl and the like.
  • the heteroaryl ring may be fused to an aryl, heterocyclic or cycloalkyl ring, wherein the ring to which the parent structure is attached is a heteroaryl ring, non-limiting examples of which include:
  • alkoxy refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl or cycloalkyl is as defined above.
  • alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy.
  • alkylamino refers to -N-(alkyl) and -N-(unsubstituted cycloalkyl), wherein alkyl or cycloalkyl is as defined above.
  • alkylamino groups include: methylamino, ethylamino, propylamino, butylamino, cyclopropylamino, cyclobutylamino, cyclopentylamino, cyclohexylamino.
  • the alkylamino group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkanethio Base, alkylamino, halogen, fluorenyl, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, Heterocycloalkylthio.
  • bond refers to a covalent bond represented by "-”.
  • hydroxy refers to an -OH group.
  • halogen means fluoro, chloro, bromo or iodo.
  • carboxylate group refers to -C(O)O(alkyl) or (cycloalkyl) wherein alkyl, cycloalkyl are as defined above.
  • “Optional” or “optionally” means that the subsequently described event or environment may, but need not, occur, including where the event or environment occurs or does not occur.
  • heterocyclic group optionally substituted by an alkyl group means that an alkyl group may be, but not necessarily, present, and the description includes the case where the heterocyclic group is substituted with an alkyl group and the case where the heterocyclic group is not substituted with an alkyl group. .
  • Substituted refers to one or more hydrogen atoms in the group, preferably up to 5, more preferably 1 to 3, hydrogen atoms, independently of each other, substituted by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art will be able to determine (by experiment or theory) substitutions that may or may not be possible without undue effort. For example, an amino group or a hydroxyl group having a free hydrogen may be unstable when combined with a carbon atom having an unsaturated (e.g., olefinic) bond.
  • pharmaceutical composition means a mixture comprising one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiological/pharmaceutically acceptable Carrier and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • pharmaceutically acceptable salt refers to a salt of a ligand-cytotoxic drug conjugate of the present invention which is safe and effective for use in a mammal, and which has the desired biological activity, the antibody of the present invention
  • the antibody drug-conjugated compound contains at least one amino group and thus can form a salt with an acid
  • non-limiting examples of the pharmaceutically acceptable salt include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, Citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, Malay An acid salt, a fumarate, a formate, a benzoate, a methanesulfonate, an ethanesulfonate, a besylate, a p-toluenesulfonate.
  • solvate means that a ligand-drug conjugated compound of the invention forms a pharmaceutically acceptable solvate with one or more solvent molecules, non-limiting examples of which include water, ethanol, acetonitrile, isopropyl Alcohol, DMSO, ethyl acetate.
  • ligand is a macromolecular compound that recognizes and binds to an antigen or receptor associated with a target cell.
  • the role of the ligand is to present the drug to a target cell population that binds to the ligand, including but not limited to protein hormones, lectins, growth factors, antibodies or other molecules capable of binding to cells.
  • the ligand is represented by Pc, and the ligand may form a linkage with a linking unit via a heteroatom on the ligand, preferably an antibody.
  • ligand-drug conjugate compound refers to a ligand attached to a biologically active cytotoxin via a stable chemical linker compound.
  • the ligand in the present invention is anti-total, and the "ligand-drug conjugate compound” is preferably an antibody drug conjugate (ADC), which means that the monoclonal antibody or antibody fragment is passed through a stable chemical linker compound and has Biologically active cytotoxins are linked.
  • ADC antibody drug conjugate
  • the term "antigen or receptor” allows the ligand to recognize and bind to a target cell.
  • Preferred in the present invention are ligands for cell surface antigens or receptors expressed on target cells and/or tissues of proliferative diseases such as cancer; non-limiting cell surface receptor embodiments are selected from HER2, HER3, HER4, CD20 , cell surface receptors of CD22, CD30, CD33, CD44, Lewis Y, CD56, CD105, VEGFR or GPNMB; most preferably cell surface receptors selected from HER2 or EGFR.
  • trastuzumab which specifically binds to the HER2 target
  • Pertuzumab which specifically binds to the HER2 target
  • nimotuzumab Nimotuzumab
  • trastuzumab which specifically binds to the HER2 target
  • Pertuzumab which specifically binds to the HER2 target
  • nimotuzumab Nimotuzumab
  • an “antibody” as used herein refers to any form of antibody that exhibits the desired biological activity. Thus, it is used in the broadest sense and specifically includes, but is not limited to, full length antibodies, antibody binding fragments or derivatives. Sources of antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, genetically engineered antibodies (eg, bispecific antibodies).
  • full length antibody refers to an immunoglobulin molecule (eg, IgM) comprising four polypeptide chains, ie, two heavy chains and two light chains, cross-linked to each other by disulfide bonds to form a multimer.
  • Each heavy chain comprises a heavy chain variable region (VH for short) and a heavy chain constant region, and the heavy chain constant region comprises three domains: CH1, CH2 and CH3.
  • Each light chain comprises a stretch of light chain variable region (VL) and a light chain constant region, and the light chain constant region comprises one domain (CL1).
  • the VH region and the VL region can be further divided into hypervariable regions, the term is complementarity determining regions (CDRs), and a more conserved domain interspersed between each complementarity determining region, called a framework region (FR).
  • CDRs complementarity determining regions
  • antibody-binding fragment or derivative includes any naturally occurring, enzymatically-derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex; typically includes a parent At least a portion of an antigen binding region or variable region (eg, one or more CDRs) of an antibody that retains at least some of the binding specificity of the parent antibody.
  • An "antibody binding fragment or derivative” may be derived from an antibody, for example by appropriate standard techniques including proteolysis or recombinant genetic engineering techniques, including manipulation and expression of a DNA expressing an antibody variable region and a partial constant region. The full length is modified.
  • Antibody-binding fragments or derivatives include, but are not limited to, (i) Fab fragments; (ii) F(ab') 2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) (vi) a dAb fragment; and (vii) a minimal recognition unit (eg, an isolated complementarity determining region (CDR)) that mimics the amino acid residue of the hypervariable region of the antibody.
  • CDR complementarity determining region
  • Other engineering molecules such as bivalent antibodies, trivalent antibodies, tetravalent antibodies, and minibodies are also within the scope of "antibody binding fragments or derivatives.”
  • a "Fab fragment” consists of a complete light and heavy chain VH and CH1 functional regions.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • the "Fc” region comprises two heavy chain fragments comprising the CH1 and CH2 domains of the antibody.
  • the two heavy chain fragments are held together by more than two disulfide bonds and by hydrophobic interactions of the CH3 domain.
  • Fab' fragment comprises a VH and CH1 functional region of a light and heavy chain, and also a region between the CH1 and CH2 domains such that a strand can be formed between the two heavy chains of the two Fab' fragments An inter-disulfide bond to form a F(ab')2 molecule.
  • the "F(ab')2 fragment” comprises two light chains and two heavy chains containing a partial constant region between the CH1 and CH2 domains such that an interchain disulfide bond is formed between the two heavy chains.
  • the F(ab')2 fragment consists of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.
  • An "Fv fragment” comprises a variable region VH functional region of a light or/and heavy chain.
  • the "Fc region” corresponds to the CH2 and CH3 functional regions of IgG, has no antigen-binding activity, and is a site where antibody molecules interact with effector molecules and cells.
  • the "hinge region” is used to link the Fab segment and the Fc segment of the antibody.
  • a bispecific fusion protein can be ligated to the Fc segment in the present invention.
  • the antibody of the present invention is preferably a specific antibody against a cell surface antigen on a target cell, and a non-limiting example is the following antibody: trastuzumab, which is a humanized anti-HER2 antibody for treating breast cancer, and is suitable for use.
  • trastuzumab which is a humanized anti-HER2 antibody for treating breast cancer, and is suitable for use.
  • trastuzumab also known as 2C4, trade name Perjeta
  • Pertuzumab is a recombinant humanized monoclonal antibody. It is the first monoclonal antibody called a "HER dimerization inhibitor" that blocks the dimerization of HER2 with other HER receptors by binding to HER2, thereby slowing tumor growth.
  • Pertuzumab has been shown to inhibit tumor growth in models with high expression and low expression of prostate cancer.
  • Pertuzumab has been approved by the US FDA for the treatment of HER2-positive metastatic breast cancer.
  • Nimotuzumab (trade name Taixinsheng) is a monoclonal antibody that targets the epidermal growth factor receptor (EGFR) and can be used to treat humanized monoclonal antibodies against malignant tumors.
  • EGFR epidermal growth factor receptor
  • EGFR is overexpressed in a variety of solid tumors, such as head and neck cancer, lung cancer, and colorectal cancer.
  • cytotoxic drug refers to a chemical molecule that has a strong disruption to its normal growth in tumor cells.
  • cytotoxic drugs can kill tumor cells at a sufficiently high concentration, but due to lack of specificity, while killing tumor cells, it also causes apoptosis of normal cells, leading to serious side effects.
  • the cytotoxic drug is represented as D/D1.
  • linker unit in the present invention is L, which refers to a chemical structural fragment or bond that is covalently linked at one end to the ligand and to the cytotoxic drug at the other end.
  • the structural unit of L in the present invention is as follows:
  • R15, R16, m, n are as defined in the formula (PC-L'-D).
  • drug loading refers to the average number of cytotoxic drugs loaded on each ligand in the molecule of formula (I), and may also be expressed as the ratio of the amount of drug to the amount of antibody, which may be in the range of each ligand. (Pc) is linked to 1-8 cytotoxic drugs (D). In the embodiment of the present invention, the drug loading amount is expressed as y, and can be coupled by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA test and HPLC characterization. The number of drug products per ADC molecule after the reaction.
  • y may be limited by the number of attachment sites.
  • the cytotoxic drug is coupled to the ⁇ -amino group of the N-terminal amino group and/or the lysine residue of the ligand through a linking unit, and generally, the coupling reaction can be coupled to the antibody.
  • the number of drug molecules will be less than the theoretical maximum.
  • the loading of the ligand cytotoxic drug conjugate can be controlled by the following non-limiting methods, including:
  • carrier refers to a system which changes the manner in which the drug enters the body and the distribution in the body, controls the release rate of the drug, and delivers the drug to the targeted organ.
  • Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and increase bioavailability.
  • Polymeric surfactants which can be used as carriers, can be self-assembled due to their unique amphiphilic structure to form aggregates of various forms, such as micelles, microemulsions, gels, liquid crystals, vesicles, etc. . These aggregates have the ability to entrap drug molecules while having good permeability to the membrane and can be used as an excellent drug carrier.
  • excipient is an addition to a pharmaceutical preparation other than the main drug, and may also be referred to as an excipient.
  • excipients such as adhesives, fillers, disintegrants, lubricants in tablets; semi-solid preparation ointments, matrix parts in creams; preservatives, antioxidants, flavoring agents, fragrances, and liquids in liquid preparations Solvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants and the like can be referred to as excipients.
  • the term "diluent” is also known as a filler and its primary use is to increase the weight and volume of the tablet. The addition of the diluent not only ensures a certain volume, but also reduces the dose deviation of the main component and improves the compression moldability of the drug.
  • an absorbent to absorb the oily substance so as to maintain a "dry” state to facilitate tableting.
  • the pharmaceutical composition may be in the form of a sterile injectable aqueous solution.
  • a sterile injectable aqueous solution Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • the sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oily phase.
  • the active ingredient is dissolved in a mixture of soybean oil and lecithin.
  • the oil solution is then added to a mixture of water and glycerin to form a microemulsion.
  • the injection or microemulsion can be injected into the bloodstream of the patient by a local injection. Alternatively, it is preferred to maintain a constant circulating concentration of the compound of the invention.
  • the solution and the microemulsion are administered in a manner.
  • a continuous intravenous delivery device can be used.
  • An example of such a device is the Deltec CADD-PLUS
  • the pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension for intramuscular and subcutaneous administration.
  • the suspension may be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension, such as a solution prepared in 1,3-butanediol, in a non-toxic parenterally acceptable diluent or solvent.
  • sterile fixed oils may conveniently be employed as a solvent or suspension medium. For this purpose, any blended fixed oil including synthetic mono- or diglycerides can be used.
  • fatty acids such as oleic acid can also be prepared as an injection.
  • reducing agent is a substance that loses electrons or has an electronic deviation in a redox reaction.
  • the reducing agent itself is also an antioxidant in a broad sense, is reductive, is oxidized, and its product is called an oxidation product.
  • the reducing agent is represented by RA, and non-limiting examples of reducing agents include: H 2 , carbon (C), carbon monoxide (CO), reduced iron powder (Fe), zinc powder (Zn), alkali Metals (usually Li, Na, K), other active metals (such as Mg, Al, Ca, La, etc.), stannous chloride (SnCl 2 ), oxalic acid, potassium borohydride (KBH 4 ), sodium borohydride ( NaBH 4 ), sodium cyanoborohydride (NaCNBH 3 ), sodium triacetoxyborohydride ((CH3COO) 3 BHNa), lithium aluminum hydride (LiAlH 4 ), hypophosphorous acid, sodium hypophosphite, sodium thiosulfate (Na 2 S 2 O 3 ), a preferred reducing agent of the invention is sodium cyanoborohydride or sodium triacetoxyborohydride.
  • mercapto protecting group means that when a chemical molecule containing both a thiol group and other groups participates in the reaction, the thiol group is protected when the reaction is completed in order to prevent the thiol group from being affected at a specific group.
  • the thiol protecting group is represented by T, and non-limiting examples of thiol protecting groups include: -tert-butyl, -acetyl, -n-propionyl, -isopropanoyl , -Triphenylmethyl, -methoxymethyl, 2-(trimethylsilyl)ethoxymethyl, the preferred sulfhydryl protecting group of the invention is acetyl.
  • a method for preparing a compound represented by the formula (PC-L-DR) of the present invention comprising:
  • a method for preparing a compound represented by the formula (PC-L-DR1) of the present invention comprising:
  • PC-L2 The compound of the formula (PC-L2) is reacted with a compound of the formula (L1-D1) in an acetonitrile solution, and subjected to desalting purification on a Sephadex G25 gel column to obtain a compound of the formula (PC-L-DR);
  • PC, m, n, y, R 2 to R 16 are as defined in the formula (PC-L-DR).
  • the structure of the compound is determined by nuclear magnetic resonance (NMR) or/and mass spectrometry (MS).
  • NMR chemical shift ( ⁇ ) is given in units of 10 -6 (ppm).
  • NMR was measured using a Bruker AVANCE-400 nuclear magnetic apparatus, and the solvent was deuterated dimethyl sulfoxide (DMSO-d 6 ), deuterated chloroform (CDCl 3 ), deuterated methanol (CD 3 OD), internal standard was four.
  • DMSO-d 6 dimethyl sulfoxide
  • CDCl 3 deuterated chloroform
  • CD 3 OD deuterated methanol
  • TMS Methyl silane
  • the measurement of the MS was carried out using a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).
  • ESI FINNIGAN LCQAd
  • Thin layer chromatography silica gel plate uses Yantai Yellow Sea HSGF254 or Qingdao GF254 silica gel plate.
  • the specification of silica gel plate used for thin layer chromatography (TLC) is 0.15mm ⁇ 0.2mm.
  • the specification for thin layer chromatography separation and purification is 0.4mm. ⁇ 0.5mm.
  • the known starting materials of the present invention may be synthesized by or according to methods known in the art, or may be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Dari Companies such as chemicals.
  • the reactions can be carried out under an argon atmosphere or a nitrogen atmosphere.
  • An argon atmosphere or a nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon having a volume of about 1 L.
  • the solution means an aqueous solution.
  • reaction temperature is room temperature and is 20 ° C to 30 ° C.
  • the progress of the reaction in the examples was monitored by thin layer chromatography (TLC).
  • TLC thin layer chromatography
  • the system used for the reaction was: A: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, C: petroleum ether And the ethyl acetate system, D: acetone, the volume ratio of the solvent is adjusted depending on the polarity of the compound.
  • Purification compounds using column chromatography eluent systems and thin layer chromatography developer systems include: A: dichloromethane and methanol systems, B: n-hexane and ethyl acetate systems, C: dichloromethane and acetone System, D: ethyl acetate and dichloromethane system, E: ethyl acetate and dichloromethane and n-hexane, F: ethyl acetate and dichloromethane and acetone, the volume ratio of the solvent is different depending on the polarity of the compound Adjustment, can also be adjusted by adding a small amount of alkaline or acidic reagents such as triethylamine and acetic acid.
  • the Q-TOF LC/MS uses the Agilent 6530 Accurate Mass Quadrupole-Time of Flight Mass Spectrometer and the Agilent 1290-Infinity Ultra Performance Liquid Chromatograph (Agilent Poroshell 300SB-C85 ⁇ m, 2.1 x 75mm column).
  • the known starting materials of the present invention can be synthesized by or according to methods known in the art, and the experimental methods in which no specific conditions are specified in the examples are usually carried out according to conventional conditions or according to the conditions recommended by the raw material or commodity manufacturer. .
  • Reagents without specific source are routine reagents purchased from the market.
  • transfected eukaryotic cells such as HEK293 cells (Life Technologies Cat. No. 11625019) were purified and expressed.
  • Pertuzumab which specifically binds to the HER2 target:
  • Nimotuzumab which specifically binds to EGFR targets:
  • Tetramethylurea hexafluorophosphate (97.4 mg, 0.256 mmol).
  • the reaction system was stirred at room temperature for 1 hour under an argon atmosphere. After the reaction was completed, 20 mL of water was added and stirred, and the layers were separated. The methylene chloride layer was washed with a saturated aqueous solution of sodium chloride (20 mL), dried over anhydrous sodium sulfate, filtered and evaporated.
  • the reaction system was stirred at room temperature for 2 hours under an argon atmosphere. After completion of the reaction, the reaction mixture was concentrated to dryness crystals crystals crystals crystals crystals crystals crystalssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssss
  • N,N-diisopropylethylamine (0.2 mL, 1.13 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium Fluorophosphate (103.3 mg, 0.271 mmol).
  • the reaction system was stirred at room temperature for 1 hour under an argon atmosphere. After the reaction was completed, 10 mL of water was added and stirred, and the layers were separated. The dichloromethane layer was washed with a saturated sodium chloride solution (10 mL) and dried over anhydrous sodium sulfate. Filtration and concentration of the filtrate under reduced pressure.
  • reaction solution was cooled to -75 ° C, and (2S,5S)-tert-butyl ester 2-formyl-5-methylpyrrolidin-1-carboxylic acid 3a (900 mg, 4.2 mmol, using the method disclosed in the patent application "US20120195857” was added.
  • the solution was dissolved in 5 mL of dichloromethane, stirred at -75 ° C for 1.5 hours, stirred at 0 ° C for 1.5 hours, and stirred at room temperature for 1 hour.
  • the reaction system was stirred at room temperature for 1 hour under an argon atmosphere. After completion of the reaction, 15 mL of dichloromethane was added and washed with water (6 mL ⁇ 2). The aqueous phase was extracted with methylene chloride (5 mL). Filtration and concentration of the filtrate under reduced pressure.
  • the starting material ((S)-2-amino-3-(2-fluorophenyl)propionic acid 7a 400 mg, 2.18 mmol, was prepared by a known method "Advanced Synthesis & Catalysis, 2012, 354 (17), 3327-3332”). It is dissolved in 10 mL of tert-butyl acetate, added with perchloric acid (300 mg (70%), 3.3 mmol), and stirred at room temperature for 16 hours. After the reaction is completed, 6 mL of water is added, and the organic phase is saturated with sodium hydrogencarbonate solution. (5 mL) Washing.
  • reaction solution was concentrated under reduced pressure, diluted with 5 mL of dichloromethane, and then 10mL of saturated sodium hydrogen carbonate solution was added and stirred for 10 minutes.
  • the system was layered and the aqueous layer was extracted with dichloromethane (5 mL ⁇ 3). The combined dichloromethane layers were washed with aq.
  • N,N-diisopropylethylamine (0.16 mL, 0.915 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium Fluorophosphate (84 mg, 0.22 mmol).
  • the reaction system was stirred at room temperature for 1 hour under an argon atmosphere. After the reaction was completed, 10 mL of water was added and stirred, and the layers were separated. The dichloromethane layer was washed with a saturated sodium chloride solution (10 mL) and dried over anhydrous sodium sulfate. Filtration and concentration of the filtrate under reduced pressure.
  • Triethylamine (0.98 mL, 7.08 mmol) was added dropwise, and boron di-n-butyl trifluoromethanesulfonate (6.65 mL, 6.65 mmol) was added dropwise thereto, and the mixture was stirred at 0 ° C for 50 minutes to cool the reaction mixture to - At 78 ° C, a pre-formed (S)-tert-butyl ester 2-formyl-4-methylene pyrrolidine-1-carboxylic acid 9b (1.43 g, 6.15 mmol) in dichloromethane was added and stirred at -78 ° C. After 2 hours, the mixture was stirred at 0 ° C for 1 hour and at room temperature for 1 hour.
  • the reaction system was stirred at room temperature for 2 hours under an argon atmosphere. After the completion of the reaction, the reaction mixture was washed with water, washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and filtered, and the filtrate was concentrated under reduced pressure.
  • the reaction system was stirred at room temperature for 2 hours under an argon atmosphere. After completion of the reaction, the reaction mixture was concentrated to dryness crystals crystals crystals crystals crystals crystals crystals crystalsssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssss ((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylglycol Acyl)-4-methylenepyrrolidin-2-yl)-3-methoxy-2-methylpropanamide)-3-phenylpropanoic acid 9i (375 mg, yellow oil). The next step is to react.
  • the starting material (S)-2-amino-3-(p-tolyl)propionic acid 15a 400 mg, 2.23 mmol, prepared by a known method "Organic & Biomolecular Chemistry, 2004, 2 (18), 2684-2691"
  • To 10 mL of t-butyl acetate the reaction system was added with perchloric acid (336.3 mg (70%), 3.34 mmol) under argon and stirred at room temperature for 16 hr. After the reaction is completed, 10 mL of water is added, and the liquid phase is separated, and the aqueous phase is saturated with sodium hydrogencarbonate solution.
  • the starting material (S)-2-amino-3-(thiophen-2-yl)propionic acid 16a 400 mg, 2.33 mmol, prepared by a known method "European Journal of Organic Chemistry, 2006, (5), 1113-1116").
  • the solution was dissolved in 10 mL of tert-butyl acetate.
  • the reaction was cooled to 0 ° C under argon, and perchloric acid (352 mg (70%), 3.5 mmol) was added dropwise, and the reaction was stirred at room temperature for 16 hours.
  • the starting material (S)-2-amino-3-(3-fluorophenyl)propionic acid 17a (549 mg, 3 mmol, prepared by a known method "Advanced Synthesis & Catalysis, 2012, 354 (17), 3327-3332") Dissolved in 15 mL of tert-butyl acetate, and the reaction system was added dropwise perchloric acid (450 mg (70%), 4.5 mmol) at 0 ° C under a nitrogen atmosphere, and the mixture was reacted at room temperature for 12 hours.
  • reaction solution was desalted by Sephadex G25 gel column (elution phase: 0.05 M PBS solution having a pH of 6.5), and filtered under a sterile condition through a 0.2 ⁇ m filter.
  • the title product 19 in PBS buffer (0.78 mg/mL, 20.0 mL) was stored frozen at 4 °C.
  • reaction solution was subjected to desalting purification on a Sephadex G25 gel column (elution phase: .05 M PBS solution having a pH of 6.5), and filtered under a sterile condition through a 0.2 ⁇ m filter to obtain a title product 21 in PBS buffer (1.31). Mg/mL, 12.5 mL), stored frozen at 4 °C.
  • reaction solution was subjected to desalting purification on a Sephadex G25 gel column (elution phase: 0.05 M PBS solution having a pH of 6.5), and filtered under a sterile condition through a 0.2 ⁇ m filter to obtain a title product 24 in PBS buffer (0.68 mg/ mL, 15.5 mL), stored frozen at 4 °C.
  • Q-TOF LC/MS characteristic peaks: 148094.99 (M Ab +0D), 149277.83 (M Ab +1D), 150343.15 (M Ab +2D), 151359.29 (M Ab +3D), 152478.14 (M Ab +4D), 153449.92 (M Ab +5D).
  • a solid sodium sulfite solid (1.47 g, 11.6 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 1 hour, and a small amount of water was added, and the organic layer was concentrated under reduced pressure. ⁇ 2).
  • the aqueous phase was added dropwise with hydrochloric acid to pH 3, and extracted with dichloromethane (40 mL ⁇ 3).
  • the organic phase was washed with water, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered.
  • the starting material (S)-2-amino-3-(3-chlorophenyl)propionic acid 31a (600 mg, 3 mmol) was dissolved in 15 mL of t-butyl acetate, and perchloric acid (450 mg) was added dropwise at 0 ° C under an argon atmosphere. (70%), 4.5 mmol), the reaction was completed at room temperature 12 hours. After completion of the reaction, 10 mL of water was added, and the organic phase was washed successively with 20 mL of 1N hydrochloric acid, and washed with saturated sodium hydrogen carbonate solution, and the aqueous phase was combined.
  • aqueous phase was added dropwise with a saturated aqueous solution of sodium hydrogencarbonate, and the mixture was adjusted to pH 8 to 9 and extracted with dichloromethane (100 mL ⁇ 2).
  • dichloromethane 100 mL ⁇ 2.
  • tert-Butyl ester 2-Amino-3-(3-chlorophenyl)propionic acid 31b 500 mg, oil).
  • the starting material (S)-2-amino-3-(o-methylphenyl)propanoic acid 34a (100 mg, 0.55 mmol, prepared by a known method "International Journal of Peptide & Protein Research, 1987, 30(1), 13-21”). It was dissolved in 2.5 mL of t-butyl acetate, and perchloric acid (107 mg (70%), 0.83 mmol) was added dropwise under an argon atmosphere, and the mixture was reacted at room temperature for 16 hours. After completion of the reaction, it was diluted with 5 mL of dichloromethane, and a saturated sodium hydrogencarbonate solution was added dropwise to adjust the pH to 8.
  • the G25 gel column was desalted and purified (elution phase: 0.05 M PBS solution at pH 6.5), and filtered through a 0.2 ⁇ m filter under sterile conditions to obtain the title product 36 in PBS buffer (3.51 mg/mL, 27.5 mL). Store frozen at 4 °C.
  • PBS buffer 200 mL
  • S-(3-carbonylpropyl)acetate 18a and sodium cyanoborohydride were added to give the title product 39b in PBS (about 75 mL).
  • the reaction solution was purified by desalting with a Sephadex G25 gel column (eluting phase: 0.05 M in PBS with a pH of 6.5) to give a crude title product 39 in PBS (2.96 mg/mL, 20.5 mL). 6 mL or so, and then desalted and purified on a Sephadex G25 gel column (elution phase: 0.05 M PBS solution at pH 6.5) to obtain the title product 39 in PBS buffer (4.25 mg/mL, 11.8 mL), and frozen at 4 ° C. Store.
  • the reaction solution was purified by desalting with a Sephadex G25 gel column (eluting phase: 0.05 M in PBS, pH 6.5) to give crude title product 40 in PBS (2.92 mg/mL, 20 mL).
  • the residue was purified by deionization on a Sephadex G25 gel column (elution phase: 0.05 M PBS solution at pH 6.5) to obtain the title product 40 in PBS buffer (4.25 mg/mL, 11.6 mL), and frozen at 4 ° C. Store.
  • the reaction solution was purified by desalting with a Sephadex G25 gel column (eluting phase: .05 M in PBS with a pH of 6.5) to give a crude title product 41 in PBS (2.96 mg/mL, 20 mL).
  • the mixture was purified by desalting with a Sephadex G25 gel column (elution phase: 0.05 M in PBS with a pH of 6.5) to obtain the title product 41 in PBS (4.33 mg/mL, 11 mL) and stored at 4 ° C.
  • the reaction solution was purified by desalting with a Sephadex G25 gel column (elution phase: 0.05 M in PBS with a pH of 6.5) to give the crude title product 42 in PBS (0.74 mg/mL, 10 mL). It was purified by desalting on a Sephadex G25 gel column (elution phase: 0.05 M in PBS with a pH of 6.5) to give the title product 42 in PBS (1.15 mg/mL, 6 mL) and stored at 4 ° C.
  • the reaction solution was purified by desalting with a Sephadex G25 gel column (eluting phase: 0.05 M in PBS with a pH of 6.5) to give a crude title product 43 in PBS (0.78 mg/mL, 9.5 mL). Further, it was subjected to desalting purification on a Sephadex G25 gel column (elution phase: 0.05 M in PBS with a pH of 6.5) to give the title product 43 in PBS (1.16 mg/mL, 6 mL) and stored at 4 ° C.
  • the starting material (R)-4-benzyl-3-propionyloxazol-2-one 44b (21 g, 90 mmol, obtained by a known method "Tetrahedron Letters, 1999, 40 (36), 6545-6547") Dissolved in 300 mL of dichloromethane, and cooled to 0 ° C under a nitrogen atmosphere.
  • the reaction solution was dropwise added with titanium tetrachloride (9.8 mL, 1.1 mmol) at 0 ° C, and the solution gradually changed from colorless to yellow, and a yellow solid appeared.
  • N,N-diisopropylethylamine (40 mL, 225 mmol) was added dropwise slowly, white fumes formed, the solution changed from yellow to reddish brown, stirred at 0 ° C for 1 hour, and the reaction solution was cooled to -78 ° C.
  • Add 50 mL of (2S,4S)-tert-butyl ester 4-fluoro-2-formylpyrrolidin-1-carboxylic acid 44a (21.27 g, 98 mmol, using a known method "Tetrahedron: Asymmetry, 2014, 25(3), A dichloromethane solution of 212-218 "prepared” was stirred at -78 °C for 1.5 hours. TLC monitors the completion of the reaction.
  • the aqueous phase was washed with diethyl ether (150 mL ⁇ 2), and then adjusted to pH 3 with 2N hydrochloric acid
  • the organic phase was combined, dried over anhydrous sodium sulfate, filtered, evaporated, evaporated, evaporated, evaporated,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, -1 -(tert-Butoxycarbonyl)-4-fluoropyrrolidin-2-yl)-3-methoxy-2-methylpropanoic acid 44e (8.0 g, pale yellow viscous liquid), yield 65.6% .
  • 6-(2,5-Dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid 4a (434 mg, 2.06 mmol) was dissolved in 20 mL of acetonitrile and N,N-diisopropyl was added.
  • reaction solution was purified by desalting on a Sephadex G25 gel column (elution phase: 0.05 M in PBS containing pH 6.5), and filtered through a 0.2 ⁇ m filter under sterile conditions to obtain the title product 48 in PBS buffer (0.75 mg/ mL, 19.8 mL), stored frozen at 4 °C.
  • the solution was purified by desalting on a Sephadex G25 gel column (elution phase: 0.05 M in PBS containing pH 6.5), and filtered through a 0.2 ⁇ m filter under sterile conditions to obtain the title product 49 in PBS buffer (0.75 mg/mL). , 19.8 mL), stored frozen at 4 °C.
  • Sephadex G25 gel column was desalted and purified (elution phase: pH 6.5 containing 0.05 M PBS solution), and filtered under a sterile condition through a 0.2 ⁇ m filter to obtain the title product 51 in PBS buffer (0.75 mg/mL, 19.5). mL), stored frozen at 4 °C.
  • Sephadex G25 gel column was desalted and purified (elution phase: pH 6.5 containing 0.05 M PBS solution), and filtered under a sterile condition through a 0.2 ⁇ m filter to obtain the title product 53 in PBS buffer (0.70 mg/mL, 20.5). mL), stored frozen at 4 °C.
  • Test Example 1 Cytotoxin Compound (D) compound inhibits tumor cell proliferation in vitro
  • the purpose of this experiment is to test the pharmaceutical compound of the general formula (D) of the present invention, for HepG2 tumor cells (human liver cancer cells, Chinese Academy of Sciences cell bank, catalog #TCHu 72) and A549 tumor cells (human lung adenocarcinoma cells, cell bank, article number #TCHu150) Inhibitory activity of proliferation in vitro.
  • the cells were treated in vitro with different concentrations of compounds. After 76 hours of culture, the cell proliferation was measured using CCK-8 (Cell Counting Kit-8, Dojindo, Cat. No.: CK04) reagent, and the in vitro activity of the compound was evaluated based on the IC50 value.
  • the method for in vitro proliferation inhibition test of HepG2 cells is exemplified for exemplifying the method for testing the in vitro proliferation inhibition activity of tumor cells in the present invention.
  • the method is equally applicable to, but not limited to, in vitro proliferation inhibition activity testing of other tumor cells.
  • the HepG2 single cell suspension was mixed, and the cell density was adjusted to 6 ⁇ 10 4 cells/ml with the cell culture solution, and the density-adjusted cell suspension was mixed, and added to a 96-well cell culture plate at 100 ⁇ l/well. The plate was incubated at 37 ° C for 18-20 hours in a 5% CO 2 incubator.
  • the compound was dissolved in DMSO (dimethyl sulfoxide, Shanghai Titan Technology Co., Ltd.) to prepare a storage solution having an initial concentration of 10 mM.
  • DMSO dimethyl sulfoxide, Shanghai Titan Technology Co., Ltd.
  • Example Plate 1 10 ⁇ l of 10 mM compound sample was added to each well of the first column of the U-bottom 96-well plate (Sample Plate 1). 90 ⁇ l of DMSO was added to the wells of each compound sample in the first column, that is, the stock solution was diluted 10-fold as a starting concentration point, followed by a 3-fold gradient dilution, and each compound was diluted by 10 concentration points. The dilution is a DMSO solution. Column 12 was added 60 ⁇ l of 100% DMSO. In column 11, 20 ⁇ l of 1 mM positive drug reference was added.
  • sample plate 2 A new U-bottom 96-well plate (sample plate 2) was taken, and each well sample in the sample plate 1 was diluted 20-fold with complete medium. A new U-bottom 96-well plate (sample plate 3) was then taken and the final sample of each well in sample plate 2 was subjected to a final 10-fold dilution.
  • the 96-well cell culture plate was taken out, placed in a microplate reader (PerkinElmer, VICTOR 3), and the absorbance at 450 nm was measured with a microplate reader.
  • the cell culture medium was also DMEM/F12 medium.
  • Test Example 2 In vitro proliferation inhibition test of tumor cell antibody conjugates of the present invention against HER2 target
  • the purpose of this experiment was to test the inhibitory activity of the antibody drug conjugate of the present invention against the HER2 target in vitro proliferation of SK-BR-3 tumor cells (human breast cancer cells, ATCC, Cat. No. HTB-30).
  • the cells were treated in vitro with different concentrations of compounds. After 76 hours of culture, the cell proliferation was measured using CCK-8 (Cell Counting Kit-8, Dojindo, Cat. No.: CK04) reagent, and the in vitro activity of the compound was evaluated based on the IC50 value.
  • CCK-8 Cell Counting Kit-8, Dojindo, Cat. No.: CK04
  • test cells were SK-BR-3, and the cell culture solution was McCoy's 5A medium (Gibco, Cat. No. 16600-108) containing 10% FBS. Test the relevant compounds and the results are shown in Table 2:
  • the antibody drug conjugates targeting the HER2 target of the present invention have significant proliferation inhibitory activity against SK-BR-3 cells.
  • Test Example 3 In vitro proliferation inhibition test of tumor cell conjugates of the present invention against EGFR targets against tumor cells
  • the purpose of this experiment was to test the inhibitory activity of the antibody conjugate of the present invention against EGFR target in vitro proliferation of HCC827 tumor cells (non-small cell lung cancer cells, Chinese Academy of Sciences cell bank, catalog #TCHu153).
  • the cells were treated in vitro with different concentrations of compounds. After 76 hours of culture, the cell proliferation was measured using CCK-8 (Cell Counting Kit-8, Dojindo, Cat. No.: CK04) reagent, and the in vitro activity of the compound was evaluated based on the IC50 value.
  • CCK-8 Cell Counting Kit-8, Dojindo, Cat. No.: CK04
  • test cells were HCC827, and the cell culture medium was RPMI1640 medium (Invitrogen), 10% (v/v) inactivated fetal bovine serum. Test the relevant compounds and the results are shown in Table 3:
  • the antibody drug conjugates targeting the EGFR target of the present invention have significant proliferation inhibitory activity against HCC827 cells.
  • the efficacy of the antibody cytotoxin conjugate of the present invention against nude mice xenografts of NCI-N87 cells was evaluated and compared.
  • Sample of the invention Compound 18, Compound 21, Compound 36.
  • Positive control Pertuzumab pertuzumab, Trastuzumab trastuzumab.
  • Preparation method all prepared with physiological saline.
  • BALB/cA-nude nude mice 6-7 weeks old, were purchased from Shanghai Slack Laboratory Animals LLC. Certificate No.: SCXK (Shanghai) 2012-0002. Feeding environment: SPF level.
  • a, b respectively represent length and width.

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Abstract

提供了一种通式（I）为PC-L-Dr的配体-细胞毒性药物偶联物，其制备方法以及该配体-细胞毒性药物偶联物及含有其的药物组合物通过受体调节在制备治疗癌症的药物中的用途。

Description

配体-细胞毒性药物偶联物、其制备方法及其应用 技术领域
本发明涉及一类全新结构的配体-细胞毒性药物偶联物。具体地说，本发明涉及抗体-细胞毒性药物偶联物，也涉及本发明偶联物的制备方法、包含所述偶联物的药物组合物以及所述偶联物或药物组合物的用途。
背景技术
化疗依然是包括手术、放疗、以及靶向治疗法在内的最重要的抗癌手段之一。尽管高效细胞毒素的种类很多，但是肿瘤细胞和正常细胞之间差别很小，限制了这些抗肿瘤化合物由于毒副作用在临床上的广泛应用。而抗肿瘤单克隆抗体对于肿瘤细胞表面抗原的特异性，抗体药物已成为抗肿瘤治疗的前线药物，但单独使用抗体作为抗肿瘤药物时，疗效经常不尽人意。
抗体药物偶联物(antibody drug conjugate，ADC)把单克隆抗体或者抗体片段通过稳定的化学接头化合物与具有生物活性的细胞毒素相连，充分利用了抗体对正常细胞和肿瘤细胞表面抗原结合的特异性和细胞毒素的高效性，同时又避免了前者疗效偏低和后者毒副作用过大等缺陷。这也就意味着，与以往传统的化疗药物相比，抗体药物偶联物能精准地结合肿瘤细胞并降低将对正常细胞的影响。
早期的ADC药物主要使用鼠源的单抗，因为人类的免疫反应造成一部分药物难以到达标靶。其次，早期使用的包括阿霉素(doxorubicin)在内的效应分子的生物活性较低，限制了第一代抗体药物偶联物的疗效。除此之外，抗体的来源、接头连接的方式和数目也未得到优化。
2013年2月，
Figure PCTCN2016072129-appb-000001
(ado-trastuzumab emtansine，T-DM1)获得美国FDA批准，用于治疗HER2阳性同时对曲妥珠单抗(Trastuzumab，商品名：
Figure PCTCN2016072129-appb-000002
和紫杉醇有抗药性的晚期或转移性乳腺癌患者。
Figure PCTCN2016072129-appb-000003
和
Figure PCTCN2016072129-appb-000004
都是针对血液肿瘤进行靶向治疗，和固体肿瘤相比组织结构相对简单。
Figure PCTCN2016072129-appb-000005
是美国FDA批准的治疗固体肿瘤的第一个ADC药物。
Figure PCTCN2016072129-appb-000006
采用ImmunoGen的技术将高活性的有丝分裂抑制剂DM1用一个稳定的硫醚健接头连到罗氏公司的曲妥珠单抗上，平均一个曲妥珠单抗结合大约3.5个DM1，曲妥珠单抗在病人体内特异性结合乳腺癌细胞，被细胞内吞后在细胞内断裂释放出DM1，DM1在细胞内的聚集浓度足以导致细胞因有丝分裂障碍而死亡，肿瘤灶随之消退。T-DM1既保留了
Figure PCTCN2016072129-appb-000007
的抗体依赖性的细胞增殖抑制作用，同时又增加了潜在的细胞毒素药物的效应。而且因为其毒素靶向在肿瘤细胞内释放，药物毒副作用并没有随着疗效的增加而同步增大。
帕妥珠单抗(Pertuzumab，也被称作2C4，商品名Perjeta)是一种重组人源化单克隆抗体，它是第一个被称作“HER二聚化抑制剂”的单克隆抗体。通过结合 HER2阻滞了HER2与其它HER受体的二聚化作用。帕妥珠单抗已经被证实在HER2高表达及低表达的前列腺癌等模型上均有抑制肿瘤生长的作用。
与曲妥珠单抗(Trastuzumab,商品名Herceptin)结合位点位于HER2胞外区的近膜区IV亚域上抑制下游信号通路不同，帕妥珠单抗则是通过结合在II域(二聚化域)有效抑制HER2的异源二聚化反应。因此，曲妥珠单抗只能对HER2过表达的癌症患者尤其是乳腺癌患者有一定疗效，而帕妥珠单抗虽然和曲妥珠单抗作用同一靶点，且二者有同样的内吞特点，但因其作用机理不同，在抑制二聚化后可以切断由ErbB家族受体介导的信号通路，可能会有比单独阻断HER2信号通路更广泛的应用范围。
目前ADC药物的偶联技术主要有两种：T-DM1采用的是将细胞毒性药物与抗体的自由氨基随机偶联；而
Figure PCTCN2016072129-appb-000008
采用的是将细胞毒性药物和抗体铰链区还原后的自由巯基偶联。两种偶联方法都产出了载药量数不一致的混合物。如T-DM1虽然平均载药量是3.5，但载药量的分布可从0～8。过低的载药量影响了ADC的药效，而过高的载药量则更容易因对抗体的过分修饰而导致ADC药物被组织的巨噬细胞体系识别并破坏。这不仅减低了ADC的半衰期也因为毒素在非靶点组织的积累而增加毒性副作用；而
Figure PCTCN2016072129-appb-000009
用还原剂将抗体铰链区的二硫键进行还原，对抗体本身的稳定性产生一定影响。相关的ADC药物专利有WO2007008603、WO2013173393、WO2005081711、WO2013173391、WO2013173392、WO2013173393和WO2012010287。本发明提供一种有新型连接方式，新的毒素与抗体组合，从而有更有益效果的新的ADC化合物。
发明内容
为了改进配体，特别是抗体和药物的偶联效果，本发明提供了一种包含改进的连接单元的通式化合物(PC-L-Dr)或其药学上可接受的盐或溶剂化合物：
Figure PCTCN2016072129-appb-000010
其中：
R，R2-R7选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
R8-R11中至少一个选自卤素、烯基、烷基和环烷基，其余为氢原子；
或者R8-R11之中的任意两个形成环烷基，余下的两个基团任选自氢原子、烷基和环烷基；
R12-R13选自氢原子、烷基或卤素；
R14选自芳基或杂芳基，所述的芳基或杂芳基任选进一步被选自氢原子、卤素、羟基、烷基、烷氧基和环烷基的取代基所取代；
y为1-8，优选2-5；y为正实数，可以为整数或小数；
PC为配体；L为接头单元。
在本发明另一个优选方案中，一种通式(PC-L-Dr)所示的化合物或其药学上可接受的盐或溶剂化合物，其为通式(PC-L-D)所示的化合物或其药学上可接受的盐或溶剂化合物：
Figure PCTCN2016072129-appb-000011
其中，R2‐R14定义如通式(PC-L-Dr)中所定义。
在本发明另一个优选方案中，一种通式(PC-L-Dr)所示的化合物或其药学上可接受的盐或溶剂化合物，其为通式(PC-L’-Dr)所示的化合物或其药学上可接受的盐或溶剂化合物：
Figure PCTCN2016072129-appb-000012
其中：
R15选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
R16选自烷基、环烷基、烷氧基和杂环基；
n为2-6，优选2-5；
m为0-5，优选1-3；
PC，y，n，R，R2-R14如通式(PC-L-Dr)中所定义。
在本发明另一个优选方案中，一种通式(PC‐L‐Dr)所示的化合物或其药学上可接受的盐或溶剂化合物，其为通式(PC-L’-D)所示的化合物或其药学上可接受的盐或溶剂化合物：
Figure PCTCN2016072129-appb-000013
其中：
R15，R16，m如通式(PC-L’-Dr)中所定义；
PC，y，n，R2-R14如通式(PC-L-Dr)中所定义。
在本发明另一个优选方案中，一种通式(PC-L’-D)所示的化合物或其药学上可接受的盐或溶剂化合物，其为通式(PC-L’-D1)所示的化合物或其药学上可接受的盐或溶剂化合物：
Figure PCTCN2016072129-appb-000014
其中PC，y，n，m，R2-R16如通式(PC-L’-D)中所定义。
本发明的配体药物偶联物ADC化合物包括，但不限于：
Figure PCTCN2016072129-appb-000015
Figure PCTCN2016072129-appb-000016
Figure PCTCN2016072129-appb-000017
或其药学上可接受的盐或溶剂化合物。
在本发明另一个优选方案中，一种通式(PC‐L‐Dr)所示的化合物，其中PC为抗体，优选自帕妥珠单抗(Pertuzumab)、尼妥珠单抗(Nimotuzumab)和曲妥珠单抗(Trastuzumab)。
本发明的典型配体药物偶联物ADC化合物包括，但不限于：
Figure PCTCN2016072129-appb-000018
Figure PCTCN2016072129-appb-000019
Figure PCTCN2016072129-appb-000020
或其药学上可接受的盐或溶剂化合物。
本发明进一步提供一种通式(Dr)所示的化合物：
Figure PCTCN2016072129-appb-000021
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，可用于作为制备通式(PC‐L‐Dr)所示化合物的前药；
其中：
R，R1-R7选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
R8-R11中至少一个选自卤素、烯基、烷基和环烷基，其余为氢原子；
或者R8-R11之中的任意两个形成环烷基，余下的两个基团任选自氢原子、烷基和环烷基；
R12-R13选自氢原子、烷基或卤素；
R14选自芳基或杂芳基，所述的芳基或杂芳基任选进一步被且选自氢原子、卤素、羟基、烷基、烷氧基和环烷基的取代基所取代。
在本发明另一个优选方案中，一种通式(Dr)所示的化合物，其为通式(D)所示 的化合物：
Figure PCTCN2016072129-appb-000022
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其中：R1‐R14如通式(Dr)中所定义。
在本发明另一个优选方案中，一种通式(Dr)所示的化合物，其为通式(D1)所示的化合物：
Figure PCTCN2016072129-appb-000023
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其中：R1‐R14如通式(D)中所定义。
本发明的配体药物偶联物ADC的典型前药化合物包括，但不限于：
Figure PCTCN2016072129-appb-000024
Figure PCTCN2016072129-appb-000025
Figure PCTCN2016072129-appb-000026
Figure PCTCN2016072129-appb-000027
Figure PCTCN2016072129-appb-000028
本发明进一步提供一种通式(L1‐Dr)所示的化合物：
Figure PCTCN2016072129-appb-000029
可用于制备通式(PC-L’-D)的中间体，其中
n为2-6，优选2-5；
R，R1-R7选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
R8-R11中至少一个选自卤素、烯基、烷基和环烷基，其余为氢原子；
或者R8-R11之中的任意两个形成环烷基，余下的两个基团任选自氢原子、烷基和环烷基；
R12-R13选自氢原子、烷基或卤素；
R14选自芳基或杂芳基，所述的芳基或杂芳基任选进一步被且选自氢原子、卤素、羟基、烷基、烷氧基和环烷基的取代基所取代。
在本发明另一个优选方案中，一种通式(L1‐Dr)所示的化合物，其为通式(L1‐D)所示的化合物：
Figure PCTCN2016072129-appb-000030
其中n，R2‐R14如通式(L1‐Dr)中所定义。
在本发明另一个优选方案中，一种通式(L1‐D)所示的化合物，其为一种通式(L1‐D1)所示的化合物：
Figure PCTCN2016072129-appb-000031
其中n，R1‐R12如通式(L1‐D)中所定义。
本发明的配体药物偶联物ADC的典型前药化合物(带部分接头单元)，可用于制备本发明配体药物偶联物的中间体，包括，但不限于下列化合物：
Figure PCTCN2016072129-appb-000032
Figure PCTCN2016072129-appb-000033
Figure PCTCN2016072129-appb-000034
Figure PCTCN2016072129-appb-000035
本发明进一步提供一种通式(PC‐L2)所示的化合物：
Figure PCTCN2016072129-appb-000036
其中
PC配体，优选为抗体，更优选为帕妥珠单抗(Pertuzumab)、尼妥珠单抗(Nimotuzumab)或曲妥珠单抗(Trastuzumab)；
R15选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
R16选自烷基、环烷基、烷氧基和杂环基；
m为0-5，优选1-3；
X为0‐5，优选1‐3，x为正实数，包括小数和整数。
在本发明另一个优选方案中，一种通式(PC-L2)所示的化合物，其选自：
Figure PCTCN2016072129-appb-000037
本发明进一步提供一种制备如通式(PC-L2)所示的化合物的方法，其包括如 下步骤：
Figure PCTCN2016072129-appb-000038
1)向PC与通式(PC-L2-A)化合物中加入还原剂RA，进行反应，得到通式(PC-L2-B)化合物；RA优选为氰基硼氢化钠或三乙酰氧基硼氢化钠，更优选为氰基硼氢化钠；
2)向通式(PC-L2-B)化合物中加入脱保护剂脱去巯基保护基，得到通式(PC-L2)化合物；
T选自H、叔丁基、乙酰基、正丙酰基、异丙酰基、三苯基甲基、甲氧基甲基、2-(三甲硅烷基)乙氧甲基，优选为H或乙酰基；
(PC-L2-A)化合物优选为硫代乙酸S-(3-羰基丙基)酯；
其中PC，R15，R16，m，x如通式(PC-L2)中所定义。
本发明进一步提供一种制备通式(PC-L’-D)所示的化合物的方法，该方法包括：
Figure PCTCN2016072129-appb-000039
通式(PC-L2)化合物与通式(L1-D1)化合物反应，得到通式(PC-L’-D)化合物；
其中：PC，m，n，y，R2～R16如通式(PC-L’-D)中所定义。
本发明进一步提供一种通式(D‐A a)所示的化合物：
Figure PCTCN2016072129-appb-000040
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，可作为合成本发明的配体药物偶联物ADC的典型前药化合物的中间体，其中：
R8-R11之中的任意两个形成环烷基，余下的两个基团任选自氢原子、烷基和环烷基；
R12选自氢原子、烷基或卤素；
P为氢原子或保护基，所述的保护基优选Boc，Bn，Cbz，最优选Boc；
Ra选自羟基，氨基，烷氧基，环烷氧基或烷氨基。
本发明进一步提供一种通式(D‐Aa)所示的化合物，其为通式(D‐A)所示的化合物：
Figure PCTCN2016072129-appb-000041
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，可作为合成本发明的配体药物偶联物ADC的典型前药化合物的中间体，其中：
R8-R12，P之中的任意两个形成环烷基，余下的两个基团任选自氢原子、烷基和环烷基；
R’选自氢原子，烷基，环烷基。
本发明的配体药物偶联物ADC的典型前药化合物的中间体(D-Aa)或(D-A)包括，但不限于：
Figure PCTCN2016072129-appb-000042
Figure PCTCN2016072129-appb-000043
本发明进一步提供一种药物组合物，其含有治疗有效量的如通式(PC‐L‐Dr)、通式(PC‐L’‐D)及其它如上所述的配体-细胞毒性药物偶联物，或包含如通式(Dr)、通式(L1‐Dr)、通式(D)、通式(L1‐D)及其它如上所述药物、其可药用盐或溶剂合物，以及药学上可接受的载体、稀释剂或赋形剂。
本发明进一步提供将如通式(PC‐L‐Dr)、通式(PC‐L’‐D)及其它如上所述的配体-细胞毒性药物偶联物，或如通式(Dr)、通式(L1‐Dr)、通式(D)、通式(L1‐D)及其它如上所述的药物或其可药用盐或溶剂合物，或如上所述的药物组合物在制备治疗哺乳动物癌症的药物的用途，所述的癌症为与HER2，HER3，EGFR表达相关的癌症。
本发明进一步涉及一种治疗哺乳动物癌症的方法，该方法包括对于哺乳动物施用有效剂量的如通式(PC‐L‐Dr)、通式(PC‐L’‐D)及其它如上所述的配体‐细胞毒性药物偶联物或其可药用盐或溶剂合物，或如通式(Dr)、通式(L1‐Dr)、通式(D)、 通式(L1‐D)及其它如上所述的药物或其可药用盐或溶剂合物，或包含其的药物组合物；其中所述的癌症为与HER2，HER3，EGFR表达相关的癌症。
本发明进一步提供将如通式(PC‐L‐Dr)、通式(PC‐L’‐D)及其它如上所述的配体-细胞毒性药物偶联物，或如通式(Dr)、通式(L1‐Dr)、通式(D)、通式(L1‐D)及其它如上所述的药物或其可药用盐或溶剂合物，或如上所述的药物组合物在制备治疗哺乳动物癌症的药物的用途，其中所述哺乳动物为人，所述癌症选自乳腺癌、卵巢癌、胃癌、子宫内膜癌、唾液腺癌、肺癌、结肠癌、肾癌、直肠癌、甲状腺癌、胰腺癌、前列腺癌、膀胱癌、急性淋巴细胞白血病、急性髓细胞白血病、急性早幼粒细胞白血病、慢性髓细胞白血病、慢性淋巴细胞白血病、霍奇金淋巴瘤、非霍奇金淋巴瘤或复发性间变性大细胞淋巴瘤，优选乳腺癌症、霍奇金淋巴瘤或复发性间变性大细胞淋巴瘤；更优选为与HER2表达相关的乳腺癌。
本发明进一步涉及一种治疗哺乳动物癌症的方法，该方法包括对于哺乳动物施用有效剂量的如通式(PC-L-Dr)、通式(PC-L’-D)及其它如上所述的配体-细胞毒性药物偶联物或其可药用盐或溶剂合物，或如通式(Dr)、通式(L1-Dr)、通式(D)、通式(L1-D)及其它如上所述的药物或其可药用盐或溶剂合物，或包含其的药物组合物；其中所述哺乳动物优选为人；所述癌症选自乳腺癌、卵巢癌、胃癌、子宫内膜癌、唾液腺癌、肺癌、结肠癌、肾癌、直肠癌、甲状腺癌、胰腺癌、前列腺癌、膀胱癌、急性淋巴细胞白血病、急性髓细胞白血病、急性早幼粒细胞白血病、慢性髓细胞白血病、慢性淋巴细胞白血病、霍奇金淋巴瘤、非霍奇金淋巴瘤或复发性间变性大细胞淋巴瘤，优选为乳腺癌、霍奇金淋巴瘤或复发性间变性大细胞淋巴瘤；更优选为2+或更高水平过表达HER2的乳腺癌；最优选为与HER2表达相关的乳腺癌。
本发明在抗体的N端氨基和/或赖氨酸残基的氨基上连接本发明含有游离巯基的连接单元L后，可避免对抗体铰链区进行还原反应，从而减少对抗体自身结构的影响，并且引入的碳-氮键结构稳定，不易在体内循环中分解，进一步通过控制反应条件，可使载药量在0～5范围内分布。
发明详述
除非另有限定，本文所用的所有技术和科学术语均与本发明所属领域普通技术人员的通常理解一致。虽然也可采用与本文所述相似或等同的任何方法和材料实施或测试本发明，但本文描述了优选的方法和材料。描述和要求保护本发明时，依据以下定义使用下列术语。
当本发明中使用商品名时，申请人旨在包括该商品名产品的制剂、该商品名产品的非专利药和活性药物部分。
除非有相反陈述，在说明书和权利要求书中使用的术语具有下述含义。
术语“烷基”指饱和脂肪族烃基团，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子的烷基，更优选含有1至10个碳原子的烷基，最优选含有1至6个碳原子的烷基。非限制性实例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基、正壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2,2-二乙基戊基、正癸基、3,3-二乙基己基、2,2-二乙基己基，及其各种支链异构体等。更优选的是含有1至6个碳原子的低级烷基，非限制性实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基等。烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基。
术语“环烷基”指饱和或部分不饱和单环或多环环状烃取代基，环烷基环包含3至20个碳原子，优选包含3至12个碳原子，更优选包含3至10个碳原子，最优选包含3至8个碳原子。单环环烷基的非限制性实例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等；多环环烷基包括螺环、稠环和桥环的环烷基。
术语“杂环基”指饱和或部分不饱和单环或多环环状烃取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧或S(O)m(其中m是整数0至2)的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。优选包含3至12个环原子，其中1～4个是杂原子；更优选环烷基环包含3至10个环原子。单环杂环基的非限制性实例包括吡咯烷基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基等。多环杂环基包括螺环、稠环和桥环的杂环基。
所述杂环基环可以稠合于芳基、杂芳基或环烷基环上，其中与母体结构连接 在一起的环为杂环基，其非限制性实例包括：
Figure PCTCN2016072129-appb-000044
等。
杂环基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基。
术语“芳基”指具有共轭的π电子体系的6至14元全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，优选为6至10元，例如苯基和萘基，优选苯基。所述芳基环可以稠合于杂芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为芳基环，其非限制性实例包括：
Figure PCTCN2016072129-appb-000045
芳基可以是取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“杂芳基”指包含1至4个杂原子、5至14个环原子的杂芳族体系，其中杂原子选自氧、硫和氮。杂芳基优选为5至10元，更优选为5元或6元，例如呋喃基、噻吩基、吡啶基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、咪唑基、四唑基等。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环，其非限制性实例包括：
Figure PCTCN2016072129-appb-000046
杂芳基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“烷氧基”指-O-(烷基)和-O-(非取代的环烷基)，其中烷基或环烷基的定义如上所述。烷氧基的非限制性实例包括：甲氧基、乙氧基、丙氧基、丁氧基、环丙氧基、环丁氧基、环戊氧基、环己氧基。烷氧基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“烷氨基”指-N-(烷基)和-N-(非取代的环烷基)，其中烷基或环烷基的定义如上所述。烷氨基的非限制性实例包括：甲氨基、乙氨基、丙氨基、丁氨基、环丙氨基、环丁氨基、环戊氨基、环己氨基。烷氨基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“键”指用“—”表示的共价键。
术语“羟基”指-OH基团。
术语“卤素”指氟、氯、溴或碘。
术语“羧酸酯基”指‐C(O)O(烷基)或(环烷基)，其中烷基、环烷基如上所定义。“任选”或“任选地”意味着随后所描述的事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选被烷基取代的杂环基团”意味着烷基可以但不必须存在，该说明包括杂环基团被烷基取代的情形和杂环基团不被烷基取代的情形。
“取代的”指基团中的一个或多个氢原子，优选为最多5个，更优选为1～3个氢原子彼此独立地被相应数目的取代基取代。不言而喻，取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。
术语“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“可药用盐”是指本发明配体-细胞毒性药物偶联物的盐，这类盐用于哺乳动物体内时具有安全性和有效性，且具有应有的生物活性，本发明抗体-抗体药物偶联化合物至少含有一个氨基，因此可以与酸形成盐，可药用盐的非限制性实例包括：盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硫酸氢盐、柠檬酸盐、乙酸盐、琥珀酸盐、抗坏血酸盐、草酸盐、硝酸盐、梨酸盐、磷酸氢盐、磷酸二氢盐、水杨酸盐、柠檬酸氢盐、酒石酸盐、马来酸盐、富马酸盐、甲酸盐、苯甲酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐。
术语“溶剂合物”指本发明的配体-药物偶联化合物与一种或多种溶剂分子形成可药用的溶剂合物，溶剂分子的非限制性实例包括水、乙醇、乙腈、异丙醇、DMSO、乙酸乙酯。
术语“配体”是能识别和结合目标细胞相关的抗原或受体的大分子化合物。配体的作用是将药物呈递给与配体结合的目标细胞群，这些配体包括但不限于蛋白类激素、凝集素、生长因子、抗体或其他能与细胞结合的分子。在本发明实施方式中，配体表示为Pc，配体可通过配体上的杂原子与连接单元形成连接键，优选为抗体。
术语“配体-药物偶联化合物”，指配体通过稳定的化学接头化合物与具有生物活性的细胞毒素相连。本发明中配体优选为抗全，“配体-药物偶联化合物”优选为抗体药物偶联物(antibody drug conjugate，ADC)，指把单克隆抗体或者抗体片段通过稳定的化学接头化合物与具有生物活性的细胞毒素相连。
术语“抗原或受体”可供配体识别和结合目标细胞。本发明中优选针对在增生性疾病，例如癌症的靶细胞和/或组织上表达的细胞表面抗原或受体的配体；非限制性细胞表面受体实施例选自HER2，HER3，HER4，CD20，CD22，CD30，CD33，CD44，Lewis Y，CD56，CD105，VEGFR或GPNMB的细胞表面受体；最优选为选自HER2或EGFR的细胞表面受体。具体的优选的非限制性实施例为曲妥珠单抗(Trastuzumab)，可以和HER2靶标特异性结合；帕妥珠单抗(Pertuzumab)，可以和HER2靶标特异性结合；尼妥珠单抗(Nimotuzumab)，可以和EGFR靶标特异性结合。
本发明所述的“抗体”是指表现出所需生物学活性的任何形式的抗体。因此，它以最广义使用，具体地说，包括但不限于全长抗体，抗体结合片段或衍生物。抗体的来源包括但不限于单克隆抗体、多克隆抗体、基因工程抗体(例如双特异性抗体)。
术语“全长抗体”是指包含4条多肽链即2条重链和2条轻链通过二硫键相互交联形成多聚体的免疫球蛋白分子(例如IgM)。每条重链包含一段重链可变区(简称VH)和一段重链恒定区，重链恒定区包含3个结构域：CH1、CH2和CH3。每条轻链包含一段轻链可变区(简称VL)和一段轻链恒定区，轻链恒定区包含1个结构域(CL1)。VH区和VL区可进一步分为高变区，术语为互补决定区(CDRs)，各互补决定区之间穿插着更加保守的结构域，称为框架区(FR)。
术语“抗体结合片段或衍生物”包括任何一种自然发生的，酶催化获得的，合成的，或是通过基因工程得到的可与抗原特异性结合形成复合物的多肽或糖蛋白；通常包括亲本抗体的至少部分抗原结合区或可变区(例如一个或多个CDR)，其保留亲本抗体的至少某些结合特异性。“抗体结合片段或衍生物”可能由抗体衍生而来，例如通过适宜的标准技术包括蛋白水解或重组基因工程技术(包括对表达抗体可变区和部分恒定区的DNA进行操作和表达)对抗体全长进行改造而得。“抗 体结合片段或衍生物”包括但不限于：(i)Fab片段；(ii)F(ab’)2片段；(iii)Fd片段；(iv)Fv片段；(v)单链Fv(scFv)；(vi)dAb片段；和(vii)模拟抗体高变区氨基酸残基的最小识别单元(如一个分离的互补决定区(CDR))。其它工程分子如双价抗体、三价抗体、四价抗体和微抗体也在“抗体结合片段或衍生物”范围内。
“Fab片段”由一条完整的轻链和重链的VH和CH1功能区组成。Fab分子的重链不能与另一个重链分子形成二硫键。
“Fc”区包含含有抗体的CH1与CH2结构域的两个重链片段。两个重链片段通过两个以上的二硫键和通过CH3结构域的疏水作用保持在一起。
“Fab’片段”包含一条轻链和重链的VH和CH1功能区，还包含在CH1与CH2结构域之间的区域，以致于可在两个Fab’片段的两条重链之间形成链间二硫键，以形成F(ab’)2分子。
“F(ab’)2片段”包含二条轻链和含有CH1与CH2结构域之间的部分恒定区的两条重链，以致于在两条重链之间形成链间二硫键。因此，F(ab’)2片段由通过两条重链之间的二硫键保持在一起的两个Fab’片段组成。
“Fv片段”包含轻链或/和重链的可变区VH功能区。
“Fc区”相当于IgG的CH2和CH3功能区，无抗原结合活性，是抗体分子与效应分子和细胞相互作用的部位。
“铰链区”用于连接抗体的Fab段和Fc段。在本发明中可以将双特异性融合蛋白与Fc段连接。
本发明所述的抗体优选为针对靶细胞上细胞表面抗原的特异性抗体，非限制性实施例为以下抗体：曲妥珠单抗，是用于治疗乳腺癌的人源化抗HER2抗体，适用于治疗HER2过度表达的转移性乳腺癌。帕妥珠单抗(Pertuzumab，也被称作2C4，商品名Perjeta)，是一种重组人源化单克隆抗体。它是第一个被称作“HER二聚化抑制剂”的单克隆抗体，通过结合HER2阻滞了HER2与其它HER受体的二聚化作用，从而减缓了肿瘤的生长。帕妥珠单抗已经被证实在HER2高表达及低表达的前列腺癌等模型上均有抑制肿瘤生长的作用。帕妥珠单抗已被美国FDA批准用于治疗HER2阳性的转移性乳腺癌。尼妥珠单抗(Nimotuzumab，商品名泰欣生)，是一个以表皮生长因子受体(EGFR)为靶点的单抗药物，可用于治疗恶性肿瘤的人源化单克隆抗体。EGFR在多种实体瘤中过度表达，如头颈癌、肺癌、结直肠癌中，都存在EGFR过度表达现象。
术语“细胞毒性药物”是指在肿瘤细胞内具有较强破坏其正常生长的化学分子。细胞毒性药物原则上在足够高的浓度下都可以杀死肿瘤细胞，但是由于缺乏特异性，在杀伤肿瘤细胞的同时，也会导致正常细胞的凋亡，导致严重的副作用。在本发明的实施方式中，细胞毒性药物表示为D/D1。
术语“接头单元”在本发明中为L，指一端与配体共价连接而另一端与细胞毒性药物相连的化学结构片段或键。本发明中L的结构单元如：
Figure PCTCN2016072129-appb-000047
其中R15，R16，m，n如通式(PC‐L’‐D)通式中所定义。
术语“载药量”是指式(I)分子中每个配体上加载的细胞毒性药物平均数量，也可以表示为药物量和抗体量的比值，药物载量的范围可以是每个配体(Pc)连接1-8个细胞毒性药物(D)，在本发明的实施方式中，载药量表示为y，可用常规方法如UV/可见光光谱法，质谱，ELISA试验和HPLC特征鉴定偶联反应后每个ADC分子的药物品均数量。
在本发明中，y可能受连接位点数量的限制。本发明的一个实施方式中，细胞毒性药物通过连接单元偶联在配体的N端氨基和/或赖氨酸残基的ε-氨基上，一般地，偶联反应中能与抗体偶联的药物分子数将小于理论上的最大值。
可以用以下非限制性方法控制配体细胞毒性药物偶联物的载量，包括：
(1)控制连接试剂和单抗的摩尔比，
(2)控制反应时间和温度，
(3)选择不同的反应试剂。
常规的药物组合物的制备见中国药典。
术语“载体”用于本发明的药物，是指能改变药物进入人体的方式和在体内的分布、控制药物的释放速度并将药物输送到靶向器官的体系。药物载体释放和靶向系统能够减少药物降解及损失，降低副作用，提高生物利用度。如可作为载体的高分子表面活性剂由于其独特的两亲性结构，可以进行自组装，形成各种形式的聚集体，优选的实例如胶束、微乳液、凝胶、液晶、囊泡等。这些聚集体具有包载药物分子的能力，同时又对膜有良好的渗透性，可以作为优良的药物载体。
术语“赋形剂”是在药物制剂中除主药以外的附加物，也可称为辅料。如片剂中的黏合剂、填充剂、崩解剂、润滑剂；半固体制剂软膏剂、霜剂中的基质部分；液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂、着色剂等均可称为赋形剂。
术语“稀释剂”又称填充剂，其主要用途是增加片剂的重量和体积。稀释剂的加入不仅保证一定的体积大小，而且减少主要成分的剂量偏差，改善药物的压缩成型性等。当片剂的药物含有油性组分时，需加入吸收剂吸收油性物，使保持“干燥”状态，以利于制成片剂。如淀粉、乳糖、钙的无机盐、微晶纤维素等。
药物组合物可以是无菌注射水溶液形式。可在使用的可接受的溶媒和溶剂中有水、林格氏液和等渗氯化钠溶液。无菌注射制剂可以是其中活性成分溶于油相的无菌注射水包油微乳。例如将活性成分溶于大豆油和卵磷脂的混合物中。然后将油溶液加入水和甘油的混合物中处理形成微乳。可通过局部大量注射，将注射液或微乳注入患者的血流中。或者，最好按可保持本发明化合物恒定循环浓度的 方式给予溶液和微乳。为保持这种恒定浓度，可使用连续静脉内递药装置。这种装置的实例是Deltec CADD-PLUS.TM.5400型静脉注射泵。
药物组合物可以是用于肌内和皮下给药的无菌注射水或油混悬液的形式。可按已知技术，用上述那些适宜的分散剂或湿润剂和悬浮剂配制该混悬液。无菌注射制剂也可以是在无毒肠胃外可接受的稀释剂或溶剂中制备的无菌注射溶液或混悬液，例如1,3-丁二醇中制备的溶液。此外，可方便地用无菌固定油作为溶剂或悬浮介质。为此目的，可使用包括合成甘油单或二酯在内的任何调和固定油。此外，脂肪酸例如油酸也可以制备注射剂。
术语“还原剂”是在氧化还原反应里，失去电子或有电子偏离的物质。还原剂本身在广义上说也是抗氧化剂，具有还原性，被氧化，其产物叫氧化产物。在本发明的实施方式中，还原剂表示为RA，还原剂的非限制性实例包括：H2、碳(C)、一氧化碳(CO)、还原铁粉(Fe)、锌粉(Zn)、碱金属(常用的有Li、Na、K)、其他活泼金属(如Mg、Al、Ca、La等)、氯化亚锡(SnCl2)、草酸、硼氢化钾(KBH4)、硼氢化钠(NaBH4)、氰基硼氢化钠(NaCNBH3)、三乙酰氧基硼氢化钠((CH3COO)3BHNa)、氢化铝锂(LiAlH4)、次磷酸、次磷酸钠、硫代硫酸钠(Na2S2O3)，本发明优选的还原剂为氰基硼氢化钠或三乙酰氧基硼氢化钠。
术语“巯基保护基”指当同时含有巯基和其他基团的化学分子参与反应的情况下，为使反应只发生在特定的基团处，而避免巯基遭受影响，将巯基加以保护，当反应完成后脱除的基团，在本发明的实施方式中，巯基保护基表示为T，巯基保护基的非限制性实例包括：-叔丁基、-乙酰基、-正丙酰基、-异丙酰基、-三苯基甲基、-甲氧基甲基、-2-(三甲硅烷基)乙氧甲基，本发明优选的巯基保护基为乙酰基。
本发明的合成方法
为了完成本发明的合成目的，本发明采用如下的合成技术方案：
方案一：
本发明通式(PC-L-DR)所示的化合物的制备方法，该方法包括：
Figure PCTCN2016072129-appb-000048
方案二：
本发明通式(PC-L-DR1)所示的化合物的制备方法，该方法包括：
Figure PCTCN2016072129-appb-000049
通式(PC-L2)化合物与通式(L1-D1)化合物在乙腈溶液中反应，经Sephadex G25凝胶柱脱盐纯化后，得到通式(PC-L-DR)化合物；
其中：PC，m，n，y，R2～R16如通式(PC-L-DR)中所定义。
具体实施方式
以下结合实施例进一步描述本发明，但这些实施例并非限制本发明的范围。
本发明实施例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例
化合物的结构是通过核磁共振(NMR)或/和质谱(MS)来确定的。NMR化学位移(δ)以10-6(ppm)的单位给出。NMR的测定是用Bruker AVANCE-400核磁仪，测定溶剂为氘代二甲基亚砜(DMSO-d6)，氘代氯仿(CDCl3)，氘代甲醇(CD3OD)，内标为四甲基硅烷(TMS)。
MS的测定用FINNIGAN LCQAd(ESI)质谱仪(生产商：Thermo，型号：Finnigan LCQ advantage MAX)。
HPLC的测定使用安捷伦1200DAD高压液相色谱仪(Sunfire C18150×4.6mm色谱柱)和Waters 2695-2996高压液相色谱仪(Gimini C18150×4.6mm色谱柱)。
激酶平均抑制率及IC50值的测定用NovoStar酶标仪(德国BMG公司)。
薄层层析硅胶板使用烟台黄海HSGF254或青岛GF254硅胶板，薄层色谱法(TLC)使用的硅胶板采用的规格是0.15mm～0.2mm，薄层层析分离纯化产品采用的规格是0.4mm～0.5mm。
柱层析一般使用烟台黄海硅胶200～300目硅胶为载体。
本发明的已知的起始原料可以采用或按照本领域已知的方法来合成，或可购买自ABCR GmbH&Co.KG，Acros Organics，Aldrich Chemical Company，韶远化学科技(Accela ChemBio Inc)，达瑞化学品等公司。
实施例中无特殊说明，反应均能够在氩气氛或氮气氛下进行。
氩气氛或氮气氛是指反应瓶连接一个约1L容积的氩气或氮气气球。
实施例中无特殊说明，溶液是指水溶液。
实施例中无特殊说明，反应的温度为室温，为20℃～30℃。
实施例中pH＝6.5的PBS缓冲液的配制：取KH2PO48.5g，K2HPO4.3H2O 8.56g，NaCl 5.85g，EDTA 1.5g置于瓶中，定容至2L，超声波使其全部溶解，摇匀即得。
实施例中pH＝4.5的乙酸/乙酸钠缓冲液的配制：取9g无水乙酸钠置于瓶中，加入纯化水，定容至2L，摇匀后，加入醋酸钠4.9mL，摇匀即得。
实施例中磷酸盐缓冲液(pH＝7.0)的配制：0.2M的Na2HPO4 61mL中加入0.2M的NaH2PO4 39mL摇匀得0.2M pH＝7的缓冲液。
实施例中的反应进程的监测采用薄层色谱法(TLC)，反应所使用的展开剂的体系有：A：二氯甲烷和甲醇体系，B：正己烷和乙酸乙酯体系，C：石油醚和乙酸乙酯体系，D：丙酮，溶剂的体积比根据化合物的极性不同而进行调节。
纯化化合物采用的柱层析的洗脱剂的体系和薄层色谱法的展开剂体系包括：A：二氯甲烷和甲醇体系，B：正己烷和乙酸乙酯体系，C：二氯甲烷和丙酮体系，D：乙酸乙酯和二氯甲烷体系，E：乙酸乙酯和二氯甲烷和正己烷，F：乙酸乙酯和二氯甲烷和丙酮，溶剂的体积比根据化合物的极性不同而进行调节，也可以加入少量的三乙胺和醋酸等碱性或酸性试剂进行调节。
本发明部分化合物是通过Q-TOF LC/MS来表征的。Q-TOF LC/MS使用安捷伦6530精确质量数四级杆-飞行时间质谱仪和安捷伦1290-Infinity超高效液相色谱仪(安捷伦Poroshell 300SB-C85μm,2.1×75mm色谱柱)。
本发明的已知的起始原料可以采用或按照本领域已知的方法来合成，实施例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
一、作为抗体的中间体的制备：
以下抗体按抗体常规方法进行制备：如可进行载体构建后，转染真核细胞如HEK293细胞(Life Technologies Cat.No.11625019)，纯化表达。
抗体序列
(1)帕妥珠单抗(Pertuzumab)，可以和HER2靶标特异性结合：
轻链序列：
Figure PCTCN2016072129-appb-000050
Figure PCTCN2016072129-appb-000051
重链序列：
Figure PCTCN2016072129-appb-000052
(2)尼妥珠单抗(Nimotuzumab)，可以和EGFR靶标特异性结合：
轻链顺序：
Figure PCTCN2016072129-appb-000053
重链顺序：
Figure PCTCN2016072129-appb-000054
(3)曲妥珠单抗(Trastuzumab)，可以和HER2靶标特异性结合：
轻链顺序:
Figure PCTCN2016072129-appb-000055
                                                       
Figure PCTCN2016072129-appb-000056
重链顺序
Figure PCTCN2016072129-appb-000057
二、药物、药物连接体、配体药物偶联物(ADC)的制备
实施例1
(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸
Figure PCTCN2016072129-appb-000058
Figure PCTCN2016072129-appb-000059
第一步
(1S,3S,5S)-叔丁酯 3-((1R,2R)-1-羟基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-2-氮杂双环[3.1.0]己烷-2-羧酸
将原料(4R,5S)-4-甲基-5-苯基-3-丙酰基噁唑烷酮1b(1.96g，9.26mmol，采用公知的方法“Journal of the American Chemical Society,2003,125(50),15512-15520”制备而得)溶于25mL二氯甲烷中，氩气氛下，降温至0℃。反应液于0℃下滴加三乙胺(1.49mL，10.93mmol)，再滴加三氟甲磺酸二丁硼(9.7mL，9.72mmol)，于0℃下搅拌50分钟，干冰丙酮浴下将反应液降温至-75℃，加入(1S,3S,5S)-叔丁酯 3-甲酰基-2-氮杂双环[3.1.0]己烷-2-羧酸1a(2.16g，9.26mmol，采用专利申请“US20100249190”公开的方法制备而得)溶于7mL二氯甲烷的溶液，于-75℃下搅拌1.5小时，于0℃搅拌2小时，于室温搅拌1小时。反应结束后，加入36mL磷酸盐缓冲液(pH＝7.0)和甲醇(V/V＝1:3)的混合液。于0℃下加入36mL甲醇和双氧水(30％)(V/V＝2:1)的混合液，于室温搅拌1小时。减压浓缩除去有机相，加入少量水，用乙醚(50mL×3)萃取，依次用5％碳酸氢钠溶液，饱和氯化钠溶液(150mL)洗涤，用无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化残留物，得标题产物(1S,3S,5S)-叔丁酯 3-((1R,2R)-1-羟基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-2-氮杂双环[3.1.0]己烷-2-羧酸1c(2.4g，白色泡沫状固体)，产率58.5％。
MS m/z(ESI):345.1[M-100+1]
第二步
(1S,3S,5S)-叔丁酯 3-((1R,2R)-1-甲氧基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-2-氮杂双环[3.1.0]己烷-2-羧酸
将原料(1S,3S,5S)-叔丁酯 3-((1R,2R)-1-羟基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-2-氮杂双环[3.1.0]己烷-2-羧酸1c(1.4g，3.15mmol)溶于20mL二氯甲烷，加入1.4g碾碎的分子筛，氩气氛下，于0℃下加入1,8-双二甲氨基萘(1.75g，8.19mmol)，三甲基氧鎓四氟硼酸盐(1.16g，7.87mmol)，反应避光，于室温搅拌40小时。反应结束后，过滤，滤饼用二氯甲烷洗涤，滤液用饱和氯化铵溶液(50mL×4)洗去过量1,8-双二甲氨基萘，再用饱和氯化钠溶液(120mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化所得残余物，得到标题产物(1S,3S,5S)-叔丁酯 3-((1R,2R)-1-甲氧基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-2-氮杂双环[3.1.0]己烷-2-羧酸1d(400mg，白色固体)，产率27.8％。
MS m/z(ESI):459.4[M+1]
第三步
(2R,3R)-3-((1S,3S,5S)-2-(叔丁氧羰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酸
将原料(1S,3S,5S)-叔丁酯 3-((1R,2R)-1-甲氧基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-2-氮杂双环[3.1.0]己烷-2-羧酸1d(400mg，0.87mmol)溶于24mL四氢呋喃，氩气氛下，降温至0℃，缓慢滴加30％的双氧水(0.34mL/0.38g，3.31mmol)，再加入一水合氢氧化锂(62mg,，1.48mmol)，反应体系于室温反应20小时。反应结束后，向反应液中加入亚硫酸钠固体(440mg，3.48mmol)，于室温搅拌1小时，加入10mL水，减压浓缩掉有机相，所得残余物用二氯甲烷萃取(40mL×2)。水相在冰浴下滴加2N盐酸至反应液pH为3～4，用乙酸乙酯萃取(25mL×3)，乙酸乙酯层依次用水(50mL)，饱和氯化钠溶液(50mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得标题产物(2R,3R)-3-((1S,3S,5S)-2-(叔丁氧羰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酸1e(230mg，无色液体)，收率88.0％。
MS m/z(ESI):200.1[M-100+1]
第四步
(1S,3S,5S)-叔丁酯 3-((1R,2R)-3-((S)-1-叔丁氧基-1-羰基-3-苯丙基-2-基胺)-1-甲氧基-2-甲基-3-羰基丙基)-2-氮杂双环[3.1.0]己烷-2-羧酸
将原料(2R,3R)-3-((1S,3S,5S)-2-(叔丁氧羰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酸1e(100mg，0.334mmol)溶于6mL二氯甲烷和二甲基甲酰胺(V/V＝5:1)混合溶剂中，加入反应物(S)-叔丁酯 2-氨基-3-苯丙酸1f(73.9mg，0.334mmol，采用公知的方法“Tetrahedron:Asymmetry,2006,17(4),603-606”制备而得),再加入N,N-二异丙基乙基胺(0.29mL，67mmol)和2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(152.3mg，0.40mmol)。反应体系在氩气氛下，于室温下搅拌1小时。反应结束后，加10mL水搅拌，分层，二氯甲烷层用饱和氯化钠溶液(10mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化所得残余物，得到标题产物(1S,3S,5S)-叔丁酯 3-((1R,2R)-3-((S)-1-叔丁氧基-1-羰基-3-苯丙基-2-基胺)-1-甲氧基-2-甲基-3-羰基丙基)-2-氮杂双环[3.1.0]己烷-2-羧酸1g(140mg，无色粘稠液体)，收率83.7％。
MS m/z(ESI):503.3[M+1]
第五步
(S)-叔丁酯 2-((2R,3R)-3-((1S,3S,5S)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸
将原料(1S,3S,5S)-叔丁酯 3-((1R,2R)-3-((S)-1-叔丁氧基-1-羰基-3-苯丙基-2-基胺)-1-甲氧基-2-甲基-3-羰基丙基)-2-氮杂双环[3.1.0]己烷-2-羧酸1g(140mg，0.28 mmol)溶于2mL二氧六环中，加入5.6M的氯化氢二氧六环溶液(0.15mL，0.835mmol)后将反应体系密封。于室温搅拌8小时，置于0℃冰箱内12小时。反应结束后，依次加入3mL二氯甲烷，3mL水，3mL饱和碳酸氢钠溶液，搅拌10分钟。体系分层，二氯甲烷层用饱和氯化钠溶液(20mL×2)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，得到标题产品(S)-叔丁酯 2-((2R,3R)-3-((1S,3S,5S)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸1h(86mg，黄色固体)，收率76.7％。MS m/z(ESI):403.4[M+1]
第六步
(S)-叔丁酯 2-((2R,3R)-3-((1S,3S,5S)-2-((5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷基-14-酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸
将原料(S)-叔丁酯 2-((2R,3R)-3-((1S,3S,5S)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸1h(86mg，0.213mmol)，(5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷-14-羧酸1i(136mg，0.213mmol，采用专利申请“WO2013072813”公开的方法制备而得)溶于6mL二氯甲烷和二甲基甲酰胺(V/V＝5:1)混合溶剂中，加入N,N-二异丙基乙基胺(0.19mL，1.065mmol)和2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(97.4mg，0.256mmol)。反应体系在氩气氛下，于室温下搅拌1小时。反应结束后，加入20mL水搅拌，分层。二氯甲烷层用饱和氯化钠溶液(20mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化残留物，得到标题产品(S)-叔丁酯2-((2R,3R)-3-((1S,3S,5S)-2-((5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷基-14-酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸1j(120mg，白色泡沫状固体)，收率54.9％。
MS m/z(ESI):1023.1[M+1]
第七步
(S)-叔丁酯 2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸
将原料(S)-叔丁酯 2-((2R,3R)-3-((1S,3S,5S)-2-((5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷基-14-酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸1j(120mg，0.117mmol)溶于2mL二氯甲烷中，加入2mL二乙胺。反应体系在氩气氛下，于室温下搅拌2小时。反应结束后，将反应液减压浓缩，得到粗品标题产品(S)-叔丁酯 2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二 甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸1k(124mg，黄色液体)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):801.5[M+1]
第八步
(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸
将粗品(S)-叔丁酯 2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸1k(90mg，0.112mmol)溶于1mL二氧六环中，加入5.6M的氯化氢二氧六环溶液3mL，将反应体系密封，于室温下搅拌12小时。反应结束后，减压浓缩，用高效液相色谱法纯化所得残留物得标题产品(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸1(19mg，白色固体)，收率22.7％。
MS m/z(ESI):744.6[M+1]
1H NMR(400MHz,CD3OD):δ7.34-7.21(m,5H),4.76-4.70(m,2H),4.26-4.19(m,1H),4.14-4.06(m,1H),3.91-3.86(m,1H),3.85-3.77(m,1H),3.75-3.56(m,2H),3.44-3.10(m,9H),2.98-2.83(m,1H),2.71-2.57(m,4H),2.26-1.99(m,4H),1.92-1.77(m,1H),1.75-1.58(m,2H),1.49-1.27(m,4H),1.21-0.95(m,18H),0.93-0.79(m,4H),0.76-0.61(m,1H).
实施例2
(S)-3-(2-氯苯基)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)丙酸
Figure PCTCN2016072129-appb-000060
Figure PCTCN2016072129-appb-000061
第一步
(S)-叔丁酯 2-氨基-3-(2-氯苯基)丙酸
将原料(S)-2-氨基-3-(2-氯苯基)丙酸2a(400mg，2.0mmol，采用公知的方法“Journal of the American Chemical Society,1940,62,565-8”制备而得)溶于10mL乙酸叔丁酯，加入高氯酸(428mg(70％)，3.00mmol)，于室温下搅拌16小时。反应结束后，加入6mL水，分液，有机相用饱和碳酸氢钠溶液(3mL)洗涤。水相用饱和碳酸氢钠溶液调节至pH＝8，二氯甲烷(5mL×3)萃取，合并有机相，依次用水(3mL)，饱和氯化钠溶液(5mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得粗品标题产物(S)-叔丁酯 2-氨基-3-(2-氯苯基)丙酸2b(400mg，白色固体)，产品不经纯化直接进行下一步反应。
第二步
(1S,3S,5S)-叔丁酯 3-((1R,2R)-3-((S)-1-叔丁氧基-3-(2-氯苯基)-1-羰基丙基-2-基胺)-1-甲氧基-2-甲基-3-羰基丙基)-2-氮杂双环[3.1.0]己烷-2-羧酸
将原料(2R,3R)-3-((1S,3S,5S)-2-(叔丁氧羰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酸1e(110mg，0.367mmol)溶于6mL二氯甲烷和二甲基甲酰胺(V/V＝5:1)混合溶剂中，加入粗品(S)-叔丁酯 2-氨基-3-(2-氯苯基)丙酸2b(94mg，0.367mmol)，再加入N,N-二异丙基乙基胺(0.32mL，1.835mmol)和2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(168mg，0.44mmol)，反应体系在氩气氛下， 于室温下搅拌1.5小时。反应结束后，加10mL水搅拌，分层，有机相用饱和氯化钠溶液(10mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化所得残余物，得到标题产物(1S,3S,5S)-叔丁酯3-((1R,2R)-3-((S)-1-叔丁氧基-3-(2-氯苯基)-1-羰基丙基-2-基胺)-1-甲氧基-2-甲基-3-羰基丙基)-2-氮杂双环[3.1.0]己烷-2-羧酸2c(112mg，白色泡沫状固体)，收率56.8％。
MS m/z(ESI):537.3[M+1]
第三步
(S)-叔丁酯 2-((2R,3R)-3-((1S,3S,5S)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氯苯基)丙酸
将原料(1S,3S,5S)-叔丁酯 3-((1R,2R)-3-((S)-1-叔丁氧基-3-(2-氯苯基)-1-羰基丙基-2-基胺)-1-甲氧基-2-甲基-3-羰基丙基)-2-氮杂双环[3.1.0]己烷-2-羧酸2c(110mg，0.205mmol)溶于2mL二氧六环中，加入5.6M的氯化氢二氧六环溶液(0.13mL，0.717mmol)，氩气氛下，于室温搅拌1小时，置于0℃冰箱内6小时。反应结束后加入5mL二氯甲烷稀释，加入10mL饱和碳酸氢钠溶液，搅拌10分钟。体系分层，水层用5mL二氯甲烷萃取。合并二氯甲烷层，用饱和氯化钠溶液(20mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，得到粗品标题产品(S)-叔丁酯2-((2R,3R)-3-((1S,3S,5S)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氯苯基)丙酸2d(99mg，黄色液体)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):437.2[M+1]
第四步
(S)-叔丁酯 2-((2R,3R)-3-((1S,3S,5S)-2-((5S,8S,11S,12R)-11-仲丁基-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氯苯基)丙酸
将粗品(S)-叔丁酯 2-((2R,3R)-3-((1S,3S,5S)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氯苯基)丙酸2d(99mg，0.226mmol)，(5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷-14-羧酸1i(144.4mg，0.226mmol)溶于6mL二氯甲烷和二甲基甲酰胺(V/V＝5:1)混合溶剂中，加入N,N-二异丙基乙基胺(0.2mL，1.13mmol)和2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(103.3mg，0.271mmol)。反应体系在氩气氛下，于室温下搅拌1小时。反应结束后，加入10mL水搅拌，分层。二氯甲烷层用饱和氯化钠溶液(10mL)洗涤，无水硫酸钠干燥。过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化残留物，得到标题产品(S)-叔丁酯 2-((2R,3R)-3-((1S,3S,5S)-2-((5S,8S,11S,12R)-11-仲丁基-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氯苯基)丙酸2e(127mg， 白色泡沫状固体)，收率53.1％。
MS m/z(ESI):1056.4[M+1]
第五步
(S)-叔丁酯 3-(2-氯苯基)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)丙酸
将原料(S)-叔丁酯 2-((2R,3R)-3-((1S,3S,5S)-2-((5S,8S,11S,12R)-11-仲丁基-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氯苯基)丙酸2e(127mg，0.12mmol)溶于2mL二氯甲烷中，加入2mL二乙胺。反应体系在氩气氛下，于室温下搅拌3小时。反应结束后，将反应液减压浓缩，得到粗品标题产品(S)-叔丁酯 3-(2-氯苯基)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)丙酸2f(130mg，黄色粘稠物)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):834.5[M+1]
第六步
(S)-3-(2-氯苯基)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)丙酸
将粗品(S)-叔丁酯 3-(2-氯苯基)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)丙酸2f(100mg，0.12mmol)溶于1mL二氧六环中，加入5.6M的氯化氢二氧六环溶液3mL，氩气氛下，于室温下搅拌12小时。反应结束后，减压浓缩反应液，用高效液相色谱法纯化所得残留物，得标题产品(S)-3-(2-氯苯基)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)丙酸2(35mg，白色固体)，收率35.7％。
MS m/z(ESI):778.7[M+1]
1H NMR(400MHz,CD3OD):δ7.41-7.32(m,2H),7.31-7.16(m,2H),4.80-4.65(m,2H),4.22-4.13(m,1H),4.10-4.03(m,1H),3.98-3.91(m,1H),3.87-3.82(m,1H),3.73-3.63(m,2H),3.47-3.12(m,9H),3.09-3.01(m,1H),2.67-2.57(m,4H),2.24-2.11(m,3H),2.09-1.98(m,1H),1.89-1.67(m,3H),1.51-1.25(m,4H),1.20-0.93(m,18H),0.92-0.79(m,4H),0.75-0.66(m,1H).
实施例3
(S)-2-((2R,3R)-3-((2S,5S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸
Figure PCTCN2016072129-appb-000062
第一步
(2S,5S)-叔丁酯 2-((1R,2R)-1-羟基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-5-甲基吡咯烷-1-羧酸
将原料(4R,5S)-4-甲基-5-苯基-3-丙酰基噁唑烷酮1b(0.992g，4.2mmol)溶于20mL二氯甲烷中，氩气氛下，降温至0℃。反应液于0℃下滴加三乙胺(0.69mL，4.96mmol)，再滴加三氟甲磺酸二正丁基化硼(4.4mL，4.4mmol)，于0℃下搅拌50分钟，将反应液降温至-75℃，加入(2S,5S)-叔丁酯 2-甲酰基-5-甲基吡咯烷-1-羧酸3a(900mg，4.2mmol，采用专利申请“US20120195857”公开的方法制备而得)溶于5mL二氯甲烷的溶液，于-75℃下搅拌1.5小时，于0℃搅拌1.5小时，于室温搅拌1小时。于室温下加入36mL磷酸盐缓冲液(pH＝7.0)和甲醇(V/V＝1:3)的混合液。冷却至0℃，于0℃下加入36mL甲醇和双氧水(30％)(V/V＝2:1)的混合液，于室温搅拌1小时。反应结束后，减压浓缩除去有机相，加入15mL水。水相用乙醚(30mL×3)萃取，合并乙醚相，依次用5％碳酸氢钠溶液，水，饱和氯化钠溶液(150mL)洗涤， 用无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化残留物，得标题产物(2S,5S)-叔丁酯 2-((1R,2R)-1-羟基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-5-甲基吡咯烷-1-羧酸3b(600mg，白色固体)，产率33％。
第二步
(2S,5S)-叔丁酯 2-((1R,2R)-1-甲氧基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-5-甲基吡咯烷-1-羧酸
将原料(2S,5S)-叔丁酯 2-((1R,2R)-1-羟基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-5-甲基吡咯烷-1-羧酸3b(600mg，1.34mmol)溶于15mL二氯甲烷，加入1g磨成粉末的分子筛，氩气氛下，于0℃下加入1,8-双二甲氨基萘(740mg，3.45mmol)，三甲基氧鎓四氟硼酸盐(500mg，3.38mmol)，反应用锡箔纸包裹，于室温下反应38小时。反应结束后，过滤，滤饼用二氯甲烷洗涤。合并滤液，有机相用饱和氯化铵溶液(20mL×3)洗去过量1,8-双二甲氨基萘，再用饱和氯化钠溶液洗涤，无水硫酸钠干燥。过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化所得残余物，得到标题产物(2S,5S)-叔丁酯 2-((1R,2R)-1-甲氧基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-5-甲基吡咯烷-1-羧酸3c(200mg，白色固体)，产率32％。
第三步
(2R,3R)-3-((2S,5S)-1-(叔丁氧羰基)-5-甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酸
将原料(2S,5S)-叔丁酯 2-((1R,2R)-1-甲氧基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-5-甲基吡咯烷-1-羧酸3c(200mg，0.43mmol)溶于22mL四氢呋喃，氩气氛下，降温至0℃，缓慢滴加30％的双氧水(186mg，1.6mmol)，再加入一水合氢氧化锂(58mg，1.37mmol)，于0℃下反应10分钟后，撤去冰浴，反应体系于室温反应44小时。反应结束后，向反应液中加入亚硫酸钠固体(220mg，1.74mmol)，于室温搅拌1小时，加入15mL水。减压浓缩掉有机相，所得残余物用二氯甲烷萃取(20mL×2)。水相在冰浴下滴加2N盐酸至反应液pH为3～4，用乙酸乙酯萃取(20mL×3)，乙酸乙酯层依次用水，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得粗品标题产物(2R,3R)-3-((2S,5S)-1-(叔丁氧羰基)-5-甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酸3d(120mg，白色固体)，产品不经纯化直接进行下一步反应。
第四步
(2S,5S)-叔丁酯 2-((1R,2R)-3-((S)-1-叔丁氧基-1-羰基-3-苯丙基-2-基胺)-1-甲氧基-2-甲基-3-羰基丙基)-5-甲基吡咯烷-1-羧酸
将粗品(2R,3R)-3-((2S,5S)-1-(叔丁氧羰基)-5-甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酸3d(106mg，0.35mmol)溶于4.8mL二氯甲烷和二甲基甲酰胺(V/V＝5:1)混合溶剂中，加入(S)-叔丁酯 2-氨基-3-苯丙酸1f(80mg，0.36mmol)，反应体系在氩气 氛下，加入N,N-二异丙基乙基胺(0.30mL，1.74mmol)和2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(160mg，0.42mmol)，于室温下搅拌2小时。反应结束后，加10mL二氯甲烷，依次用水(5mL×2)、饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化所得残余物，得到标题产物(2S,5S)-叔丁酯 2-((1R,2R)-3-((S)-1-叔丁氧基-1-羰基-3-苯丙基-2-基胺)-1-甲氧基-2-甲基-3-羰基丙基)-5-甲基吡咯烷-1-羧酸3e(138mg，无色粘稠液体)，收率78％。
第五步
(S)-叔丁酯 2-((2R,3R)-3-甲氧基-2-甲基-3-((2S,5S)-5-甲基吡咯烷-2-基)丙酰基)-3-苯丙酸
将原料(2S,5S)-叔丁酯 2-((1R,2R)-3-((S)-1-叔丁氧基-1-羰基-3-苯丙基-2-基胺)-1-甲氧基-2-甲基-3-羰基丙基)-5-甲基吡咯烷-1-羧酸3e(150mg，0.267mmol)溶于2.2mL二氧六环中，加入4M的氯化氢二氧六环溶液(0.160mL，0.896mmol)后将反应体系密封。于室温搅拌7小时，置于4℃冰箱内16小时。反应结束后，将反应液减压浓缩，加入15mL二氯甲烷，冷却至0℃，滴加饱和碳酸氢钠溶液调至pH为8，分层，水相用二氯甲烷萃取(8mL×2)，合并有机相。有机相用饱和氯化钠溶液洗涤，无水硫酸钠干燥。过滤，滤液减压浓缩，得到标题产品(S)-叔丁酯2-((2R,3R)-3-甲氧基-2-甲基-3-((2S,5S)-5-甲基吡咯烷-2-基)丙酰基)-3-苯丙酸3f(80mg，无色粘稠固体)，收率74％。
第六步
(S)-叔丁酯 2-((2R,3R)-3-((2S,5S)-1-((5S,8S,11S,12R)-11-仲丁基-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷基)-5-甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸
将原料(S)-叔丁酯 2-((2R,3R)-3-甲氧基-2-甲基-3-((2S,5S)-5-甲基吡咯烷-2-基)丙酰基)-3-苯丙酸3f(80mg，0.198mmol)，(5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷-14-羧酸1i(136mg，0.213mmol)溶于4.8mL二氯甲烷和二甲基甲酰胺(V/V＝5:1)混合溶剂中，加入N,N-二异丙基乙基胺(0.170mL，0.98mmol)和2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(100mg，0.263mmol)。反应体系在氩气氛下，于室温下搅拌反应1小时。反应结束后，加入15mL二氯甲烷，用水(6mL×2)洗涤。水相用二氯甲烷萃取(5mL)，合并二氯甲烷相，用饱和氯化钠溶液洗涤，无水硫酸钠干燥。过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化残留物，得到标题产品(S)-叔丁酯 2-((2R,3R)-3-((2S,5S)-1-((5S,8S,11S,12R)-11-仲丁基-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷基)-5-甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸3g(168mg，白色泡沫状固体)，收率81％。
第七步
(S)-叔丁酯 2-((2R,3R)-3-((2S,5S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸
将原料(S)-叔丁酯 2-((2R,3R)-3-((2S,5S)-1-((5S,8S,11S,12R)-11-仲丁基-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷基)-5-甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸3g(167mg，0.16mmol)溶于2mL二氯甲烷中，加入2mL二乙胺。反应体系在氩气氛下，于室温下搅拌3小时。反应结束后，将反应液减压浓缩，得到粗品标题产品(S)-叔丁酯2-((2R,3R)-3-((2S,5S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸3h(180mg，黄色液体)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):802.6[M+1]
第八步
(S)-2-((2R,3R)-3-((2S,5S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸
将粗品(S)-叔丁酯 2-((2R,3R)-3-((2S,5S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸3h(130mg，0.16mmol)溶于1mL二氧六环中，加入3mL 5.6M的氯化氢二氧六环溶液，将反应体系密封，于室温下搅拌12小时。反应结束后，将反应液减压浓缩，用高效液相色谱法纯化所得残余物，得标题产品(S)-2-((2R,3R)-3-((2S,5S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸3(28mg，白色固体)，收率23％。
MS m/z(ESI):746.7[M+1]
1H NMR(400MHz,CD3OD):δ7.31-7.14(m,5H),4.79-4.67(m,2H),4.25-4.13(m,1H),4.10-3.97(m,2H),3.77-3.66(m,1H),3.60-3.52(m,1H),3.51-3.42(m,1H),3.41-3.12(m,7H),2.97-2.85(m,1H),2.67(d,3H),2.48-2.40(m,2H),2.30-2.02(m,4H),1.93-1.73(m,2H),1.70-1.55(m,1H),1.53-1.28(m,5H),1.26-1.11(m,7H),1.10-0.99(m,14H),0.98-0.83(m,4H).
实施例4
(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸
Figure PCTCN2016072129-appb-000063
第一步
6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)己酰氯
在原料6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)己酸4a(1.5g，7.10mmol，采用公知的方法“Journal of Medcinal Chemistry,2013,56(24),9955-9968”制备而得)中滴入一滴N,N-二甲基甲酰胺，氩气氛下，干冰浴降温后，缓慢滴入15mL草酰氯，滴加时剧烈搅拌，滴完于室温反应1小时。反应结束后，将反应液减压浓缩，用二氯甲烷溶解所得残留物，减压浓缩，得到粗品标题产物6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)己酰氯4b，产品不经纯化直接进行下一步反应。
第二步
(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲
基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸
将原料(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸1(17mg，0.023mmol)溶于1mL二氯甲烷中，加入N,N-二异丙基乙基胺(20μL，0.115mmol)，反应体系在氩气氛下，冰浴下滴加预制的6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)己酰氯4b(7.9mg,0.034mmol)的二氯甲烷溶液，滴加完毕后于室温反应5小时。反应结束后，加入10mL甲醇淬灭，将反应液减压浓缩，用高效液相色谱法纯化所得残留物，得标题产物(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸4(9mg，白色固体)，收率42％。
MS m/z(ESI):937.4[M+1]
1H NMR(400MHz,CD3OD):δ7.37-7.17(m,5H),6.84-6.79(m,2H),4.80-4.71(m,2H),4.69-4.56(m,2H),4.26-4.19(m,1H),4.14-4.07(m,1H),3.92-3.86(m,1H),3.84-3.78(m,1H),3.77-3.60(m,1H),3.55-3.47(m,2H),3.42-3.23(m,5H),3.18-3.12(m,2H),3.07-3.03(m,2H),3.02-2.82(m,2H),2.66-2.58(m,2H),2.54-2.46(m,1H),2.46-2.38(m,2H),2.30-2.14(m,2H),2.09-1.99(m,1H),1.90-1.78(m,1H),1.75-1.56(m,6H),1.48-1.28(m,6H),1.20-0.79(m,22H),0.77-0.69(m,1H).
实施例5
(S)-3-(2-氯苯基)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)丙酸
Figure PCTCN2016072129-appb-000064
将原料(S)-3-(2-氯苯基)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)丙酸2(18mg，0.023mmol)溶于1mL二氯甲烷中，加入N,N-二异丙基乙基胺(0.02mL，0.115mmol)，反应体系在氩气氛下，冰浴下滴加预制的6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)己酰氯4b(7.9mg，0.034mmol)的二氯甲烷溶液，滴加完毕后于室温反应4小时。反应结束后，加入5mL甲醇淬灭，将反应液减压浓缩，用高效液相色谱法纯化所得残留物，得标题产物(S)-3-(2-氯苯基)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)丙酸5(6mg，白色固体)，收率26.6％。
MS m/z(ESI):971.5[M+1]
1H NMR(400MHz,CD3OD):δ7.42-7.34(m,2H),7.33-7.19(m,2H),6.83-6.78(m,2H),4.83-4.70(m,2H),4.68-4.56(m,2H),4.24-4.16(m,1H),4.13-4.05(m,1H),4.03-3.95(m,1H),3.91-3.84(m,1H),3.76-3.65(m,1H),3.54-3.48(m,2H),3.47-3.18(m,5H),3.17-2.96(m,6H),2.67-2.58(m,2H),2.53-2.38(m,3H),2.28-2.18(m,2H),2.09-2.00(m, 1H),1.92-1.57(m,7H),1.51-1.28(m,6H),1.21-0.82(m,22H),0.78-0.69(m,1H).
实施例6
(S)-2-((2R,3R)-3-((2S,5S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸
Figure PCTCN2016072129-appb-000065
将原料(S)-2-((2R,3R)-3-((2S,5S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸3(18mg，0.024mmol)溶于1mL二氯甲烷中，加入N,N-二异丙基乙基胺(0.02mL，0.12mmol)，反应体系在氩气氛下，冰浴下滴加预制的6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)己酰氯4b(8.3mg,0.036mmol)的二氯甲烷溶液，于室温搅拌反应4小时。反应结束后，加入5mL甲醇淬灭，将反应液减压浓缩，用高效液相色谱法纯化所得残留物，得标题产物(S)-2-((2R,3R)-3-((2S,5S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸6(7mg，白色固体)，收率30.9％。
MS m/z(ESI):939.5[M+1]
1H NMR(400MHz,CD3OD):δ7.30-7.15(m,5H),6.83-6.78(m,2H),4.78-4.69(m,2H),4.69-4.56(m,2H),4.24-4.12(m,1H),4.10-3.96(m,2H),3.60-3.44(m,3H),3.41-3.22(m,4H),3.16-3.10(m,2H),3.07-3.02(m,2H),3.01-2.86(m,2H),2.52-2.38(m,4H),2.31-2.15(m,3H),2.09-1.99(m,1H),1.91-1.77(m,2H),1.71-1.56(m,5H),1.52-1.28(m,9H),1.26-1.14(m,6H),1.11-0.77(m,18H).
实施例7
(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
Figure PCTCN2016072129-appb-000066
第一步
(S)-叔丁酯 2-氨基-3-(2-氟苯基)丙酸
将原料((S)-2-氨基-3-(2-氟苯基)丙酸7a(400mg，2.18mmol，采用公知的方法“Advanced Synthesis&Catalysis,2012,354(17),3327-3332”制备而得)溶于10mL乙酸叔丁酯，加入高氯酸(300mg(70％)，3.3mmol)，于室温下搅拌16小时。反应完毕后加入6mL水，分液，有机相用饱和碳酸氢钠溶液(5mL)洗涤。水相用饱和碳酸氢钠溶液调节至pH＝8，二氯甲烷(5mL×3)萃取，合并有机相，依次用水(3mL)，饱和氯化钠溶液(5mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得粗品标题产物(S)-叔丁酯 2-氨基-3-(2-氟苯基)丙酸7b(390mg，黄色油状物)，产品不经纯化直接进行下一步反应。
第二步
(1S,3S,5S)-叔丁酯 3-((1R,2R)-3-(((S)-1-(叔丁氧基)-3-(2-氟苯基)-1-羰基丙基-2-基)氨基)-1-甲氧基-2-甲基-3-羰基丙基)-2-氮杂双环[3.1.0]己烷-2-羧酸
将原料(2R,3R)-3-((1S,3S,5S)-2-(叔丁氧羰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酸1e(100mg，0.334mmol)溶于6mL二氯甲烷和二甲基甲酰胺(V/V＝5:1)混合溶剂中，加入反应物粗品(S)-叔丁酯 2-氨基-3-(2-氟苯基)丙酸7b(80mg，0.334mmol)。再加入N,N-二异丙基乙基胺(0.29mL，1.67mmol)和2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(152.3mg，0.40mmol)。反应体系在氩气氛下，于室温搅拌1小时。反应结束后，加10mL水搅拌，分层，二氯甲烷层 用饱和氯化钠溶液(10mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。用硅胶柱色谱法以洗脱剂体系B纯化所得残余物，得到标题产物(1S,3S,5S)-叔丁酯3-((1R,2R)-3-(((S)-1-(叔丁氧基)-3-(2-氟苯基)-1-羰基丙基-2-基)氨基)-1-甲氧基-2-甲基-3-羰基丙基)-2-氮杂双环[3.1.0]己烷-2-羧酸7c(173mg，无色液体)，收率99.5％。MS m/z(ESI):521.2[M+1]
第三步
(S)-叔丁酯 2-((2R,3R)-3-((1S,3S,5S)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
将原料(1S,3S,5S)-叔丁酯 3-((1R,2R)-3-(((S)-1-(叔丁氧基)-3-(2-氟苯基)-1-羰基丙基-2-基)氨基)-1-甲氧基-2-甲基-3-羰基丙基)-2-氮杂双环[3.1.0]己烷-2-羧酸7c(173mg，0.33mmol)溶于2mL二氧六环中，加入5.6M的氯化氢二氧六环溶液(0.21mL，1.16mmol)，氩气氛下，于室温搅拌1小时，置于0℃冰箱内12小时。反应结束后，将反应液减压浓缩，加入5mL二氯甲烷稀释，加入10mL饱和碳酸氢钠溶液，搅拌10分钟。体系分层，水层用二氯甲烷萃取(5mL×3)。合并二氯甲烷层，用饱和氯化钠溶液(10mL)洗涤，无水硫酸钠干燥。过滤，滤液减压浓缩，得到粗品标题产品(S)-叔丁酯 2-((2R,3R)-3-((1S,3S,5S)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸7d(77mg，黄色液体)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):421.2[M+1]
第四步
(S)-叔丁酯 2-((2R,3R)-3-((1S,3S,5S)-2-((5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧-4,7,10-三氮杂十四烷基-14-酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
将粗品(S)-叔丁酯 2-((2R,3R)-3-((1S,3S,5S)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸7d(77mg，0.183mmol)，(5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷-14-羧酸1i(116.8mg，0.183mmol)溶于6mL二氯甲烷和二甲基甲酰胺(V/V＝5:1)混合溶剂中，加入N,N-二异丙基乙基胺(0.16mL，0.915mmol)和2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(84mg，0.22mmol)。反应体系在氩气氛下，于室温下搅拌1小时。反应结束后，加入10mL水搅拌，分层。二氯甲烷层用饱和氯化钠溶液(10mL)洗涤，无水硫酸钠干燥。过滤，滤液减压浓缩。用硅胶柱色谱法以洗脱剂体系B纯化残留物，得到标题产品(S)-叔丁酯 2-((2R,3R)-3-((1S,3S,5S)-2-((5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷基-14-酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙 酸7e(190.5mg，黄色粘稠物)，收率100％。
MS m/z(ESI):1040.6[M+1]
第五步
(S)-叔丁酯 2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
将原料(S)-叔丁酯 2-((2R,3R)-3-((1S,3S,5S)-2-((5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷基-14-酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸7e(190.5mg，0.183mmol)溶于1.5mL二氯甲烷中，加入2mL二乙胺。反应体系在氩气氛下，于室温搅拌3小时。反应结束后，将反应液减压浓缩，得到粗品标题产品(S)-叔丁酯 2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸7f(150mg，黄色粘稠物)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):818.5[M+1]
第六步
(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
将粗品(S)-叔丁酯 2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸7f(150mg，0.183mmol)溶于1mL二氧六环中，加入5.6M的氯化氢二氧六环溶液3mL，氩气氛下，于室温搅拌12小时。反应结束后，将反应液减压浓缩，用乙醚带旋溶剂。所得残余物用高效液相色谱法纯化得标题产品(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸7(28mg，白色粉末固体)，收率20％。
MS m/z(ESI):762.7[M+1]
1H NMR(400MHz,CD3OD):δ7.38-7.18(m,2H),7.13-7.01(m,2H),4.80-4.67(m,2H),4.30-4.15(m,1H),4.13-4.01(m,1H),3.96-3.83(m,2H),3.75-3.60(m,2H),3.42-3.11(m,9H),3.06-2.95(m,1H),2.70-2.58(m,4H),2.28-2.01(m,4H),1.88-1.70(m,3H),1.57-1.25(m,4H),1.22-0.95(m,18H),0.92-0.80(m,4H),0.78-0.65(m,1H).
实施例8
(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢 -1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
Figure PCTCN2016072129-appb-000067
将原料(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸7(25mg，0.033mmol)溶于3mL二氯甲烷中，加入N,N-二异丙基乙基胺(0.029mL，0.164mmol)，反应体系在氩气氛下，冰浴下滴加预制的6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)己酰氯4b(11.3mg，0.049mmol)的二氯甲烷溶液，于室温反应3小时。反应结束后，加入5mL水，搅拌20分钟，分液，有机层用无水硫酸钠干燥，过滤，滤液减压浓缩，残留物用高效液相色谱法纯化得标题产物(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸8(7mg，黄色粘稠物)，收率22.4％。MS m/z(ESI):955.4[M+1]
1H NMR(400MHz,CD3OD):δ7.36-7.30(m,1H),7.29-7.21(m,1H),7.17-7.02(m,2H),6.83-6.79(m,2H),4.81-4.71(m,2H),4.69-4.55(m,2H),4.25-4.15(m,1H),4.13-4.04(m,1H),3.96-3.85(m,2H),3.70-3.61(m,1H),3.55-3.46(m,3H),3.40-3.21(m,4H),3.18-3.10(m,2H),3.07-2.96(m,4H),2.67-2.56(m,2H),2.54-2.34(m,3H),2.29-2.17(m,2H),2.10-1.99(m,1H),1.89-1.57(m,7H),1.52-1.28(m,6H),1.21-1.11(m,4H),1.07-0.96(m,6H),0.95-0.81(m,12H),0.80-0.69(m,1H).
实施例9
(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰胺)-4-亚甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸
Figure PCTCN2016072129-appb-000068
第一步
(S)-叔丁酯 2-甲酰基-4-亚甲基吡咯烷-1-羧酸
将原料(S)-叔丁酯 2-(羟甲基)-4-亚甲基吡咯烷-1-羧酸9a(1.32g，6.19mmol,采用公知的方法“From Journal of Organic Chemistry,2003,68(10),3923-3931”制备而得)溶于15mL二氯甲烷中，降温至0℃。反应体系于氩气氛下，加入N,N-二异丙基乙基胺(5.38mL，30.9mmol)，二甲基亚砜(7.26g，92.9mmol)，加入三氧化硫-吡啶络合物(7.26g，92.9mmol)，反应液于0℃下反应3小时。反应结束后，加入磷酸盐缓冲液(pH＝7)淬灭反应，依次用水，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化残留物，得标题产物(S)-叔丁酯 2-甲酰基-4-亚甲基吡咯烷-1-羧酸9b(1.1g，淡黄色液体)，产率84.2％。
第二步
(S)-叔丁酯 2-((1R,2R)-1-羟基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-4-亚甲基吡咯烷-1-羧酸
将原料(4R,5S)-4-甲基-5-苯基-3-丙酰基噁唑烷酮1b(1.43g，6.15mmol)溶于30mL二氯甲烷中，氩气氛下，降温至0℃，滴加三乙胺(0.98mL，7.08mmol)，再滴加三氟甲磺酸二正丁基化硼(6.65mL，6.65mmol)，于0℃下搅拌50分钟，将反应液降温至-78℃，加入预制的(S)-叔丁酯 2-甲酰基-4-亚甲基吡咯烷-1-羧酸9b(1.43g，6.15mmol)的二氯甲烷溶液，于-78℃下搅拌2小时，于0℃搅拌1小时，于室温搅拌1小时。加入36mL磷酸盐缓冲液(pH＝7)和甲醇(V/V＝3:1)的混合液淬灭反应。于0℃下加入甲醇和双氧水(V/V＝1:2)的混合液，于室温搅拌1小时。反应结束后，旋除甲醇和有机相，残留水相用二氯甲烷萃取，合并有机相，依次用水洗涤，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化残留物，得标题产物(S)-叔丁酯 2-((1R,2R)-1-羟基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-4-亚甲基吡咯烷-1-羧酸9c(810mg，白色泡沫状固体)，产率30％。
MS m/z(ESI):354.2[M-100+1]
第三步
(S)-叔丁酯 2-((1R,2R)-1-甲氧基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-4-亚甲基吡咯烷-1-羧酸
将原料(S)-叔丁酯 2-((1R,2R)-1-羟基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-4-亚甲基吡咯烷-1-羧酸9c(810mg，1.82mmol)溶于15mL二氯甲烷，加入碾碎的分子筛，加入碳酸钾(1.25g，9.11mmol)，三氟甲磺酸甲酯(897.7mg，5.47mmol)，于室温搅拌反应12小时。反应结束后，将反应液过滤，滤饼用二氯甲烷洗涤，合并有机相，将有机相减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化所得残余物，得到标题产物(S)-叔丁酯 2-((1R,2R)-1-甲氧基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-4-亚甲基吡咯烷-1-羧酸9d(280mg，白色泡沫固体)，产率33.5％。
MS m/z(ESI):359.2[M-100+1]
第四步
(2R,3R)-3-((S)-1-(叔丁氧羰基)-4-亚甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酸
将原料(S)-叔丁酯 2-((1R,2R)-1-甲氧基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-4-亚甲基吡咯烷-1-羧酸9d(400mg，0.87mmol)溶于20mL四氢呋喃，加入一水合氢氧化锂(62.2mg,，1.484mmol)，反应体系在氩气氛下，降温至0℃，缓慢滴加30％的双氧水(112.7mg，3.31mmol)，反应体系于室温反应12小时。反应结束后，向反应液中加入亚硫酸钠(416mg，3.3mmol)，于室温搅拌1小时。将四氢呋喃旋干，残余物中加入水溶解，用二氯甲烷萃取。水相滴加稀盐酸调节反应液pH为3～4，用二氯甲烷萃取(30mL×3)，合并有机相，依次用水洗 涤，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得得粗品标题产物(2R,3R)-3-((S)-1-(叔丁氧羰基)-4-亚甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酸9e(230mg，无色液体)，产品不经纯化直接进行下一步。
MS m/z(ESI):298.2[M-1]
第五步
(S)-叔丁酯 2-((1R,2R)-3-(((S)-1-(叔丁氧基)-1-羰基-3-苯丙基-2-基)氨基)-1-甲氧基-2-甲基-3-羰基丙基)-4-亚甲基吡咯烷-1-羧酸
将粗品(2R,3R)-3-((S)-1-(叔丁氧羰基)-4-亚甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酸9e(220mg，0.735mmol)溶于6mL二氯甲烷中，加入反应物(S)-叔丁酯 2-氨基-3-苯丙酸1f(178.8mg，0.809mmol)。再加入N,N-二异丙基乙基胺(0.51mL，2.94mmol)和2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(363mg，0.956mmol)。反应体系在氩气氛下，于室温下反应3小时。反应结束后，反应液用水洗涤，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化所得残余物，得到标题产物(S)-叔丁酯 2-((1R,2R)-3-(((S)-1-(叔丁氧基)-1-羰基-3-苯丙基-2-基)氨基)-1-甲氧基-2-甲基-3-羰基丙基)-4-亚甲基吡咯烷-1-羧酸9f(271mg，白色固体)，收率66％。
MS m/z(ESI):503.3[M+1]
第六步
(S)-叔丁酯 2-((2R,3R)-3-甲氧基-2-甲基-3-((S)-4-亚甲基吡咯烷-2-基)丙酰胺)-3-苯丙酸
将原料(S)-叔丁酯 2-((1R,2R)-3-(((S)-1-(叔丁氧基)-1-羰基-3-苯丙基-2-基)氨基)-1-甲氧基-2-甲基-3-羰基丙基)-4-亚甲基吡咯烷-1-羧酸9f(270mg，0.537mmol)溶于4mL 1,4-二氧六环中，加入4M的氯化氢二氧六环溶液(0.335mL，1.881mmol)，于室温下搅拌1小时，置于0～4℃冰箱内12小时。将反应物减压浓缩，残留物用二氯甲烷溶解，滴加饱和碳酸氢钠溶液调节pH为8～9，分液，水相用二氯甲烷萃取。合并有机相，依次用水洗涤，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得到粗品标题产品(S)-叔丁酯 2-((2R,3R)-3-甲氧基-2-甲基-3-((S)-4-亚甲基吡咯烷-2-基)丙酰胺)-3-苯丙酸9g(210mg，淡黄色油状物)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):403.4[M+1]
第七步
(S)-叔丁酯 2-((2R,3R)-3-((S)-1-((5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷基-14-酰基)-4-亚甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸
将粗品(S)-叔丁酯 2-((2R,3R)-3-甲氧基-2-甲基-3-((S)-4-亚甲基吡咯烷-2-基)丙酰胺)-3-苯丙酸9g(210mg，0.521mmol)，(5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H- 芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷-14-羧酸1i(365.9mg，0.547mmol)溶于6mL二氯甲烷中，加入N,N-二异丙基乙基胺(0.4537mL，2.609mmol)和2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(257.8mg，0.678mmol)。反应体系在氩气氛下，室温下搅拌2小时。反应结束后，反应液依次用水洗涤，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化残留物，得到标题产品(S)-叔丁酯 2-((2R,3R)-3-((S)-1-((5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷基-14-酰基)-4-亚甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸9h(345mg，白色泡沫状固体)，收率64.7％。
MS m/z(ESI):1022.5[M+1]
第八步
(S)-叔丁酯 2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-4-亚甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸
将原料(S)-叔丁酯 2-((2R,3R)-3-((S)-1-((5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷基-14-酰基)-4-亚甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸9h(345mg，0.337mmol)溶于2mL二氯甲烷中，加入3mL二乙胺。反应体系在氩气氛下，于室温下搅拌2小时。反应结束后将反应液减压浓缩，得到粗品标题产品(S)-叔丁酯 2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-4-亚甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸9i(375mg，黄色油状物)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):800.5[M+1]
第九步
(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-4-亚甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸
将粗品(S)-叔丁酯 2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-4-亚甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸9i(370mg，0.462mmol)加入反应瓶中，加入4M的氯化氢二氧六环溶液7mL，将反应体系密封，于室温下搅拌12小时。反应结束后，将反应液减压浓缩，残留物用高效液相色谱法纯化得标题产物(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-4-亚甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸9(30mg，白色粉末)，收率11.9％。
MS m/z(ESI):744.7[M+1]
1H NMR(400MHz,CDCl3)δ10.57(s,1H),10.00(s,1H),8.41-8.26(m,2H),7.52-6.99(m,5H),5.34(s,1H)，5.02-4.95(m,4H),4.38-4.33(m,2H),4.16(m,2H),3.93-3.75(m,3H),3.49-3.07(m,6H),2.94-2.32(m,16H),2.30-2.01(m,4H),1.75-1.65(m,2H),1.32-0.83(m,16H).
实施例10
(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-4-亚甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸
Figure PCTCN2016072129-appb-000069
将原料(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-4-亚甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸9(22mg，0.0295mmol)溶于3mL二氯甲烷中，加入N,N-二异丙基乙基胺(19mg，0.1479mmol)，降温至0℃，滴加预制的6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)己酰氯4b(10.1mg，0.0443mmol)的二氯甲烷溶液，反应体系在氩气氛下，于室温反应2.5小时。反应结束后，加入甲醇淬灭，将反应液减压浓缩，用高效液相色谱法纯化所得残留物，得标题产物(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-4-亚甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸10(1.3mg，白色粘稠物)，收率4.6％。
MS m/z(ESI):937.9[M+1]
实施例11
(S)-2-((2R,3R)-3-((S)-5-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-氮杂螺环[2.4]庚烷-6-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
Figure PCTCN2016072129-appb-000070
第一步
(S)-叔丁酯 6-甲酰基-5-氮杂螺环[2.4]庚烷-5-羧酸
将原料(S)-叔丁酯 6-(羟甲基)-5-氮杂螺环[2.4]庚烷-5-羧酸11a(2.37g，10.4mmol,采用公知的方法“Bioorganic&Medicinal Chemistry Letters,2013,23(9),2653-2658”制备而得)溶于40mL二氯甲烷中，降温至0℃。反应体系在氩气氛下，加入N,N-二异丙基乙基胺(10.8mL，62.5mmol)，二甲基亚砜(12.2g，156.4mmol)，加入三氧化硫-吡啶络合物(6.63g，41.7mmol)，反应液于0℃反应3小时。反应结束后，加入磷酸盐缓冲液(pH＝7)淬灭反应，依次用水，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化残留物，得标题产物(S)-叔丁酯 6-甲酰基-5-氮杂螺环[2.4]庚烷-5-羧酸11b(2g，黄色液体)，产率88％。
第二步
(S)-叔丁酯 6-((1R,2R)-1-羟基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-5-氮杂螺环[2.4]庚烷-5-羧酸
将原料(4R,5S)-4-甲基-5-苯基-3-丙酰基噁唑烷酮1b(2g，8.88mmol)溶于35mL二氯甲烷中，氩气氛下，滴加三乙胺(1.42mL，10.2mmol)，降温至0℃，滴加三氟甲磺酸二正丁基化硼(9.59mL，9.59mmol)，于0℃搅拌50分钟，于78℃加入预制的(S)-叔丁酯 6-甲酰基-5-氮杂螺环[2.4]庚烷-5-羧酸11b(2g，8.88mmol)的二氯甲烷溶液，于-78℃下搅拌2小时，于0℃搅拌1小时，于室温搅拌1小时。反应结束后，加入磷酸盐缓冲液(pH＝7.0)和甲醇(V/V＝3:1)的混合液淬灭反应。于0℃加入甲醇和双氧水(V/V＝1:2)的混合液，于室温搅拌1小时。减压浓缩后，加水溶解残留物，用二氯甲烷萃取，合并有机相，依次用水洗涤，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化残留物，得标题产物(S)-叔丁酯 6-((1R,2R)-1-羟基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-5-氮杂螺环[2.4]庚烷-5-羧酸11c(1.8g，白色泡沫状固体)，产率44.2％。
MS m/z(ESI):459.4[M+1]
第三步
(S)-叔丁酯 6-((1R,2R)-1-甲氧基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-5-氮杂螺环[2.4]庚烷-5-羧酸
将原料(S)-叔丁酯 6-((1R,2R)-1-羟基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-5-氮杂螺环[2.4]庚烷-5-羧酸11c(1.8g，3.92mmol)溶于30mL二氯甲烷，加入碾碎的分子筛，反应体系在氩气氛下，加入碳酸钾(3.78g，27.48mmol)，三氟甲磺酸甲酯(3.22g，19.64mmol)，于室温下搅拌反应12小时。反应结束后，将反应液过滤，滤饼用二氯甲烷洗涤，合并有机相，将有机相减压浓缩，用硅胶柱色谱法以洗脱剂体系A纯化所得残余物，得到标题产物(S)-叔丁酯6-((1R,2R)-1-甲氧基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-5-氮杂螺环[2.4]庚烷-5-羧酸11d(930mg，无色油状物)，产率50.2％。
MS m/z(ESI):473.4[M+1]
第四步
(2R,3R)-3-((S)-5-(叔丁氧羰基)-5-氮杂螺环[2.4]庚烷-6-基)-3-甲氧基-2-甲基丙酸
将原料(S)-叔丁酯 6-((1R,2R)-1-甲氧基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)-5-氮杂螺环[2.4]庚烷-5-羧酸11d(1.03g，2.8mmol)溶于20mL四氢呋喃，加入一水合氢氧化锂(155mg,，3.7mmol)，反应体系在氩气氛下，滴加30％的双氧水(939mg，8.28mmol)，反应体系于室温反应12小时。反应结束后，向反应液中加入亚硫酸钠(1.04g，8.28mmol)，于室温搅拌1小时。将四氢呋喃旋干，残余物中加入水溶解，用二氯甲烷萃取。水相滴加稀盐酸调节反应液pH为3～4，用二氯甲烷萃取(30mL×5)，合并有机相，依次用水洗涤，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得得粗品标题产物(2R,3R)-3-((S)-5-(叔丁氧羰基)-5-氮杂螺环[2.4]庚烷-6-基)-3-甲氧基-2-甲基丙酸11e (700mg，无色粘稠液体)，产品不经纯化直接进行下一步。
MS m/z(ESI):314.4[M+1]
第五步
(S)-叔丁酯 6-((1R,2R)-3-(((S)-1-(叔丁氧基)-3-(2-氟苯基)-1-羰基丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-羰基丙基)-5-氮杂螺环[2.4]庚烷-5-羧酸
将粗品(2R,3R)-3-((S)-5-(叔丁氧羰基)-5-氮杂螺环[2.4]庚烷-6-基)-3-甲氧基-2-甲基丙酸11e(350mg，1.117mmol)溶于6mL二氯甲烷中，加入反应物(S)-叔丁酯2-氨基-3-(2-氟苯基)丙酸7b(267mg，1.117mmol)。反应体系在氩气氛下，加入N,N-二异丙基乙基胺(720mg，5.587mmol)和2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(509.8mg，1.341mmol)，于室温下反应2小时。反应结束后，反应液用水洗涤，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化所得残余物，得到标题产物(S)-叔丁酯6-((1R,2R)-3-(((S)-1-(叔丁氧基)-3-(2-氟苯基)-1-羰基丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-羰基丙基)-5-氮杂螺环[2.4]庚烷-5-羧酸11f(570mg，无色油状物)，收率95.3％。MS m/z(ESI):535.3[M+1]
第六步
(S)-叔丁酯 3-(2-氟苯基)-2-((2R,3R)-3-甲氧基-2-甲基-3-((S)-5-氮杂螺环[2.4]庚烷-6-基)丙酰胺)丙酸
将原料(S)-叔丁酯 6-((1R,2R)-3-(((S)-1-(叔丁氧基)-3-(2-氟苯基)-1-羰基丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-羰基丙基)-5-氮杂螺环[2.4]庚烷-5-羧酸11f(570mg，1.049mmol)溶于8mL 1,4-二氧六环中，加入4M的氯化氢二氧六环溶液(0.749mL，4.196mmol)，于室温下搅拌1小时，置于0～4℃冰箱内12小时。将反应物减压浓缩，残留物用二氯甲烷溶解，滴加饱和碳酸氢钠溶液调节pH为8～9，分液，水相用二氯甲烷萃取。合并有机相，依次用水洗涤，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得到粗品标题产品(S)-叔丁酯 3-(2-氟苯基)-2-((2R,3R)-3-甲氧基-2-甲基-3-((S)-5-氮杂螺环[2.4]庚烷-6-基)丙酰胺)丙酸11g(440mg，淡黄色油状物)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):435.4[M+1]
第七步
(S)-叔丁酯 2-((2R,3R)-3-((S)-5-((5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷基-14-酰基-5-氮杂螺环[2.4]庚烷-6-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
将粗品(S)‐叔丁酯 3‐(2‐氟苯基)‐2‐((2R,3R)‐3‐甲氧基‐2‐甲基‐3‐((S)‐5‐氮杂螺环[2.4]庚烷-6-基)丙酰胺)丙酸11g(440mg，1.013mmol)，(5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷-14-羧酸1i(645.8mg，1.013mmol)溶于10mL二氯甲烷中， 加入N,N-二异丙基乙基胺(0.88mL，5.06mmol)和2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(500.5mg，1.317mmol)。反应体系在氩气氛下，于室温下搅拌2小时。反应结束后，反应液依次用水洗涤，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化残留物，得到标题产品(S)‐叔丁酯 2‐((2R,3R)‐3‐((S)‐5‐((5S,8S,11S,12R)‐11‐((S)‐仲丁基)‐1‐(9H‐芴-9-基)‐5,8‐二异丙基‐12‐甲氧基‐4,10‐二甲基‐3,6,9‐三羰基‐2‐氧杂‐4,7,10‐三氮杂十四烷基-14-酰基‐5‐氮杂螺环[2.4]庚烷‐6‐基)‐3‐甲氧基‐2‐甲基丙酰胺)‐3‐(2‐氟苯基)丙酸11h(570mg，白色粉末)，收率53.4％。
MS m/z(ESI):1054.9[M+1]
第八步
(S)-叔丁酯 2-((2R,3R)-3-((S)-5-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-氮杂螺环[2.4]庚烷-6-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
将原料(S)‐叔丁酯 2‐((2R,3R)‐3‐((S)‐5‐((5S,8S,11S,12R)‐11‐((S)‐仲丁基)‐1‐(9H‐芴-9-基)‐5,8‐二异丙基‐12‐甲氧基‐4,10‐二甲基‐3,6,9‐三羰基‐2‐氧杂‐4,7,10‐三氮杂十四烷基-14-酰基‐5‐氮杂螺环[2.4]庚烷‐6‐基)‐3‐甲氧基‐2‐甲基丙酰胺)‐3‐(2‐氟苯基)丙酸11h(560mg，0.531mmol)溶于2mL二氯甲烷中，加入6mL二乙胺，于室温下搅拌2.5小时。反应结束后将反应液减压浓缩，得到粗品标题产品(S)-叔丁酯2-((2R,3R)-3-((S)-5-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-氮杂螺环[2.4]庚烷-6-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸11i(550mg，白色粘稠物)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):832.5[M+1]
第九步
(S)-2-((2R,3R)-3-((S)-5-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-氮杂螺环[2.4]庚烷-6-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
将粗品(S)-叔丁酯 2-((2R,3R)-3-((S)-5-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-氮杂螺环[2.4]庚烷-6-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸11i(450mg，0.541mmol)加入反应瓶中，加入4M的氯化氢二氧六环溶液7mL，将反应体系密封，于室温下搅拌反应12小时。反应结束后，将反应液减压浓缩，残留物用高效液相色谱法纯化得标题产物(S)-2-((2R,3R)-3-((S)-5-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-氮杂螺环[2.4]庚烷-6-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸11(403mg，白色固体)，收率96％。
MS m/z(ESI):776.7[M+1]
1H NMR(400MHz,CD3OD)δ7.30-7.22(m,2H),7.12-7.04(m,2H),4.72-4.68(m,2H), 4.13-4.07(m,2H),3.96-3.94(m,1H),3.70-3.66(m,2H),3.50-3.47(m,2H),3.40-3.37(m,3H),3.34-3.28(m,4H),3.26-3.22(m,2H),3.11(s,1H),3.05-2.91(m,2H),2.67-2.65(m,3H),2.57-2.43(m,2H),2.39-2.28(m,2H),2.25-2.16(m,3H),1.93-1.88(m,2H),1.55-1.43(m,2H),1.23-1.21(d,2H),1.16-1.08(m,3H),1.08-0.97(m,10H),0.89-0.83(m,3H),0.66-0.53(m,3H),0.46-0.43(m,2H).
实施例12
(S)-2-((2R,3R)-3-((S)-5-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-氮杂螺环[2.4]庚烷-6-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
Figure PCTCN2016072129-appb-000071
将原料(S)-2-((2R,3R)-3-((S)-5-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-氮杂螺环[2.4]庚烷-6-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸11(150mg，0.193mmol)溶于7mL二氯甲烷中，加入N,N-二异丙基乙基胺(87.3mg，0.677mmol)，反应体系在氩气氛下，降温至0℃，滴加预制的6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)己酰氯4b(57.6mg，0.251mmol)的二氯甲烷溶液，于室温反应2小时。反应结束后，加入甲醇淬灭，将反应液减压浓缩，用高效液相色谱法纯化所得残留物，得标题产物(S)-2-((2R,3R)-3-((S)-5-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-氮杂螺环[2.4]庚烷-6-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸12(14.7mg，白色粉末)，收率7.8％。
MS m/z(ESI):969.9[M+1]
1H NMR(400MHz,CD3OD)δ7.29-7.23(m,2H),7.10-7.04(m,2H),6.79-6.78(m,2H),4.69-4.54(m,3H),4.18-4.07(m,3H),3.98-3.92(m,1H),3.75-3.71(m,2H),3.50-3.47(m,3H),3.42-3.39(m,2H),3.34-3.32(m,5H),3.27-3.19(m,4H),3.09-2.95(m,5H),2.49-2.47(m,2H),2.41-2.36(m,2H),2.29-2.18(m,3H),2.09-2.02(m,2H),1.90-1.87(m,2H),1.63-1.59(m,4H),1.49(s,2H),1.32-1.28(m,3H),1.21-1.12(m,3H),1.00-0.81(m,12H),0.62-0.55(m,3H),0.46-0.40(m,2H).
实施例13
(S)-2-((2R,3R)-3-((S)-5-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-氮杂螺环[2.4]庚烷-6-基)-3-甲氧基-2-甲基丙酰胺)-3-苯基丙酸
Figure PCTCN2016072129-appb-000072
采用实施例11的合成路线，将第五步原料替换为(2R,3R)-3-((S)-5-(叔丁氧羰基)-5-氮杂螺环[2.4]庚烷-6-基)-3-甲氧基-2-甲基丙酸11e和(S)-叔丁酯 2-氨基-3-苯丙酯1f制得标题产物(S)-2-((2R,3R)-3-((S)-5-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-氮杂螺环[2.4]庚烷-6-基)-3-甲氧基-2-甲基丙酰胺)-3-苯基丙酸13(219mg，白色粉末)。
MS m/z(ESI):758.7[M+1]
1H NMR(400MHz,CD3OD)δ7.31-7.19(m,5H),4.70-4.68(m,2H),4.14-4.03(m,2H),3.95-3.93(m,1H),3.69-3.66(m,2H),3.48-3.45(m,2H),3.43-3.38(m,3H),3.34-3.29(m,4H),3.23-3.21(m,2H),3.11(s,1H),2.97-2.89(m,2H),2.67-2.65(m,3H),2.51-2.43(m,2H),2.39-2.27(m,2H),2.21-2.06(m,3H),1.88-1.82(m,2H),1.41-1.39(m,2H),1.23-1.21(d,2H),1.16-1.10(m,3H),1.08-0.98(m,10H),0.89-0.84(m,3H),0.63-0.52(m,3H),0.46-0.43(m,2H).
实施例14
(S)-2-((2R,3R)-3-((S)-5-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-氮杂螺环[2.4]庚烷-6-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸
Figure PCTCN2016072129-appb-000073
将原料(S)-2-((2R,3R)-3-((S)-5-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-氮杂螺环[2.4]庚烷-6-基)-3-甲氧基-2-甲基丙酰胺)-3-苯基丙酸13(120mg，0.158mmol)溶于5mL二氯甲烷中，加入N,N-二异丙基乙基胺(71.5mg，0.554mmol)，反应体系在氩气氛下，于0℃滴加预制的6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)己酰氯4b(47.1mg，0.2059mmol)的二氯甲烷溶液，于室温反应2小时。反应结束后，加入1mL甲醇，搅拌10分钟，将反应液减压浓缩，用高效液相色谱法纯化所得残留物，得标题产物(S)-2-((2R,3R)-3-((S)-5-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-氮杂螺环[2.4]庚烷-6-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸14(5.1mg，白色粉末)，收率3.39％。
MS m/z(ESI):951.9[M+1]
1H NMR(400MHz,CD3OD)δ7.29-7.18(m,5H),6.80-6.79(m,2H),4.75-4.61(m,3H),4.21-3.99(m,3H),3.94-3.91(m,1H),3.74-3.70(m,2H),3.51-3.44(m,3H),3.42-3.37(m,2H),3.34-3.28(m,5H),3.23-3.21(m,4H),3.10-2.86(m,5H),2.49-2.36(m,4H),2.32-2.17(m,3H),2.12-2.03(m,2H),1.88-1.80(m,2H),1.67-1.58(m,4H),1.49(s,2H),1.37-1.26(m,3H),1.22-1.13(m,3H),1.00-0.83(m,12H),0.63-0.51(m,3H),0.43-0.40(m,2H).
实施例15
(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(对-甲苯基)丙酸
Figure PCTCN2016072129-appb-000074
(S)-tert-butyl 2-amino-3-(p-tolyl)propanoate
(S)-叔丁酯 2-氨基-3-(对甲苯基)丙酸
将原料(S)-2-氨基-3-(对甲苯基)丙酸15a(400mg，2.23mmol，采用公知的方法“Organic&Biomolecular Chemistry,2004,2(18),2684-2691”制备而得)溶于10mL乙酸叔丁酯，反应体系在氩气氛下，加入高氯酸(336.3mg(70％)，3.34mmol)，于室温下搅拌16小时。反应完毕后加入10mL水，分液，水相用饱和碳酸氢钠溶液 调节pH＝8，二氯甲烷(5mL×3)萃取，合并有机相，用饱和氯化钠溶液(5mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得标题产物(S)-叔丁酯 2-氨基-3-(p-甲苯基)丙酸15b(370mg，白色固体)，收率70％。
采用实施例1的合成路线，将第四步原料替换为(2R,3R)-3-((1S,3S,5S)-2-(叔丁氧羰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-异丁酸1e和(S)-叔丁酯 2-氨基-3-(p-甲苯基)丙酸15b，制得标题产物(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(对-甲苯基)丙酸15(30mg，白色粉末)。
MS m/z(ESI):758.8[M+1]
1H NMR(400MHz,CD3OD):δ7.19-7.07(m,4H),4.82-4.68(m,2H),4.30-4.18(m,1H),4.15-4.05(m,1H),3.89-3.84(m,1H),3.83-3.76(m,1H),3.74-3.62(m,2H),3.47-3.12(m,9H),2.89-2.79(m,1H),2.70-2.59(m,4H),2.34-2.03(m,7H),1.91-1.75(m,1H),1.73-1.53(m,2H),1.50-1.24(m,4H),1.22-0.92(m,18H),0.90-0.79(m,4H),0.75-0.64(m,1H).
实施例16
(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(噻吩-2-基)丙酸
Figure PCTCN2016072129-appb-000075
第一步
(S)-叔丁酯 2-氨基-3-(噻吩-2-基)丙酸
将原料(S)-2-氨基-3-(噻吩-2-基)丙酸16a(400mg，2.33mmol，采用公知的方法“European Journal of Organic Chemistry,2006,(5),1113-1116”制备而得)溶于10mL乙酸叔丁酯，反应体系在氩气氛下，冷却至0℃，滴加高氯酸(352mg(70％)，3.5mmol)，于室温搅拌反应16小时。反应结束后，加入水，加饱和碳酸氢钠溶液调节pH为8～9，用二氯甲烷萃取，合并有机相，依次用水洗涤，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得粗品标题产物(S)-叔丁酯 2-氨基-3-(噻吩-2-基)丙酸16b(370mg，淡黄油状物)，产品不经纯化直接进行下一步反应。 MS m/z(ESI):228.3[M+1]
采用实施例1的合成路线，将第四步原料替换为(2R,3R)-3-((1S,3S,5S)-2-(叔丁氧羰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-异丁酸1e和(S)-叔丁酯 2-氨基-3-(噻吩-2-基)丙酸16b，制得标题产物(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(噻吩-2-基)丙酸16(18mg，白色粉末)。
MS m/z(ESI):527.6[M+1]
1H NMR(400MHz,CDCl3)δ11.00(s,1H),8.44(s,1H),7.17-7.15(d,1H),6.94-6.88(d,2H),4.97-4.95(m,2H),4.10-4.08(m,1H),3.85-3.83(m,2H),3.71-3.69(m,2H),3.52-3.50(m,2H),3.37-3.34(m,12H),3.06-3.04(m,2H),2.80-2.78(m,2H),2.3-2.26(m,4H),1.64-1.59(m,3H),1.48-1.46(m,2H),1.31-1.259(m,12H),1.08-0.98(m,8H),0.89-0.82(m,4H).
实施例17
(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(3-氟苯基)丙酸
Figure PCTCN2016072129-appb-000076
第一步
(S)-叔丁酯 2-氨基-3-(3-氟苯基)丙酸
将原料(S)-2-氨基-3-(3-氟苯基)丙酸17a(549mg，3mmol，采用公知的方法“Advanced Synthesis&Catalysis,2012,354(17),3327-3332”制备而得)溶于15mL乙酸叔丁酯，反应体系在氮气氛下，于0℃滴加高氯酸(450mg(70％)，4.5mmol)，滴完于室温反应12小时。反应完毕后加入30mL水，分液，有机相依次用20mL1N的盐酸洗涤，饱和碳酸氢钠溶液洗涤，合并水相。水相用饱和碳酸氢钠溶液调节pH为8～9，二氯甲烷(100mL×2)萃取，合并有机相，无水硫酸钠干燥，过滤，滤液减压浓缩得粗品标题产物(S)-叔丁酯 2-氨基-3-(3-氟苯基)丙酸17b(600mg，油状物)，产品不经纯化直接进行下一步反应。
采用实施例1的合成路线，将第四步原料替换为((2R,3R)-3-((1S,3S,5S)-2-(叔丁 氧羰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-异丁酸1e和(S)-叔丁酯 2-氨基-3-(3-氟苯基)丙酸17b，制得标题产物(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(3-氟苯基)丙酸17(25mg，白色粉末)。
MS m/z(ESI):762.8[M+1]
1H NMR(400MHz,CD3OD):δ7.29-7.21(m,1H),7.08-6.87(m,3H),4.81-4.73(m,1H),4.72-4.67(m,1H),4.61-4.53(m,1H),4.29-4.22(m,1H),4.15-4.06(m,1H),3.99-3.93(m,1H),3.75-3.58(m,2H),3.43-3.12(m,9H),2.99-2.90(m,1H),2.69-2.60(m,4H),2.30-1.97(m,4H),1.87-1.77(m,1H),1.63-1.53(m,1H),1.49-1.36(m,1H),1.18-0.92(m,22H),0.91-0.82(m,4H),0.81-0.71(m,1H).
实施例18
Figure PCTCN2016072129-appb-000077
第一步
将硫代乙酸S-(3-羰基丙基)酯18a(0.35mg，2.65μmol)，溶解于0.45mL乙腈溶液，备用；向帕妥珠单抗(Pertuzumab)pH＝4.5的乙酸/乙酸钠缓冲液(10.85mg/ml，4.5mL，0.488mmol)加入上述预制的硫代乙酸S-(3-羰基丙基)酯18a的乙腈溶液，然后滴加1.0mL的氰基硼氢化钠(7.06mg,112μmol)的水溶液，于25℃下振荡反应2小时。反应结束后用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)后得标题产物18b溶液，直接进行下一步反应。
第二步
向18b溶液(15.0mL)中加入0.45mL的2.0M盐酸羟胺溶液，于25℃下振荡反应30分钟后将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M 的PBS溶液)后得标题产物帕妥珠单抗-丙硫醇18c溶液(浓度1.65mg/ml，22.6mL)。
第三步
将化合物(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸4(1.09mg，1.16μmol)溶解于1.1mL乙腈中，加入帕妥珠单抗-丙硫醇溶液18c(1.65mg/mL，11.3mL)中，于25℃下振荡反应4小时后将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的含0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物18的PBS缓冲液(0.75mg/mL，19.5mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：148119.54(MAb+0D)、149331.45(MAb+1D)、150407.02(MAb+2D)、151297.79(MAb+3D)、152448.85(MAb+4D)、153782.23(MAb+5D)。
平均值：y＝2.0。
实施例19
Figure PCTCN2016072129-appb-000078
将化合物(S)-3-(2-氯苯基)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)丙酸5(1.39mg，1.43μmol)溶解于1.1mL乙腈中，加入帕妥珠单抗-丙硫醇溶液18c(1.65mg/mL，11.3mL)中，于25℃下振荡反应4小时后将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物19的PBS缓冲液(0.78mg/mL，20.0mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：148119.68(MAb+0D)、149308.79(MAb+1D)、150194.76(MAb+2D)、151354.52(MAb+3D)、152410.57(MAb+4D)、153375.31(MAb+5D)。
平均值：y＝1.9。
实施例20
Figure PCTCN2016072129-appb-000079
将化合物(S)-2-((2R,3R)-3-((2S,5S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸6(1.08mg，1.15μmol)溶解于1.25mL乙腈中，加入帕妥珠单抗-丙硫醇溶液18c(1.50mg/mL，12.5mL)中，于25℃下振荡反应4小时后将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物20的PBS缓冲液(0.74mg/mL，19.0mL)，于4℃冷冻储存。Q-TOF LC/MS:特征峰：Q-TOF LC/MS:148253.27(MAb+0D)、149263.59(MAb+1D)、150315.25(MAb+2D)、151334.45(MAb+3D)、152383.92(MAb+4D)、153446.37(MAb+5D)。
平均值：y＝2.2。
实施例21
Figure PCTCN2016072129-appb-000080
将化合物(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟 苯基)丙酸8(3.0mg，3.0μmol)溶解于1.0mL乙腈中，加入帕妥珠单抗-丙硫醇溶液18c(2.11mg/mL，10.0mL)中，于25℃下振荡反应4小时后将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物21的PBS缓冲液(1.31mg/mL，12.5mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：148312.73(MAb+0D)、149515.61(MAb+1D)、150459.55(MAb+2D)、151521.47(MAb+3D)、152580.02(MAb+4D)。
平均值：y＝1.7。
实施例22
Figure PCTCN2016072129-appb-000081
将化合物(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-4-亚甲基吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸10(1.50mg，1.60μmol)溶解于1.0ml乙腈中，加入帕妥珠单抗-丙硫醇溶液18c(2.11mg/mL，10.0mL)中，于25℃下振荡反应4小时后将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物22的PBS缓冲液(1.28mg/mL，13.0mL)，于4℃冷冻储存。Q-TOF LC/MS:特征峰：148411.82(MAb+0D)、149412.97(MAb+1D)、150468.08(MAb+2D)、151496.41(MAb+3D)、152580.37(MAb+4D)。
平均值：y＝2.1。
实施例23
Figure PCTCN2016072129-appb-000082
Figure PCTCN2016072129-appb-000083
将化合物(S)-2-((2R,3R)-3-((S)-5-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-氮杂螺环[2.4]庚烷-6-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸12(0.86mg，0.89μmol)溶解于0.6mL乙腈中，加入帕妥珠单抗-丙硫醇溶液18c(2.06mg/mL，6.0mL)中，于25℃下振荡反应4小时后将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物23的PBS缓冲液(0.70mg/mL，15mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：148092.94(MAb+0D)、149296.82(MAb+1D)、150339.86(MAb+2D)、151416.51(MAb+3D)、152516.25(MAb+4D)、153422.64(MAb+5D)。
平均值：y＝1.7。
实施例24
Figure PCTCN2016072129-appb-000084
将化合物(S)-2-((2R,3R)-3-((S)-5-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-5-氮杂螺环[2.4]庚烷-6-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸14(0.73mg，0.78μmol)溶解于0.6mL乙腈中，加入帕妥珠单抗-丙硫醇溶液18c(2.06mg/mL，6.0mL)中，于25℃下振荡反应4小时后将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物24的PBS缓冲液(0.68mg/mL，15.5mL)，于4℃冷冻储存。Q-TOF LC/MS:特征峰：148094.99(MAb+0D)、149277.83(MAb+1D)、150343.15(MAb+2D)、151359.29(MAb+3D)、152478.14(MAb+4D)、153449.92(MAb+5D)。
平均值：y＝1.6。
实施例25
(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
Figure PCTCN2016072129-appb-000085
第一步
(S)-叔丁酯 2-((1R,2R)-1-羟基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)吡咯烷-1-羧酸
将原料(4R,5S)-4-甲基-5-苯基-3-丙酰基噁唑烷酮1b(4.6g，20.1mmol)溶于80mL二氯甲烷中，在氩气氛下，降温至0℃。反应液于0℃下滴加三乙胺(3.2mL，23.1mmol)，再滴加三氟甲磺酸二正丁基化硼(20mL，20.7mmol)，于0℃下搅拌50分钟，于-75℃滴加5mL预制的(S)-叔丁酯 2-甲酰基吡咯烷-1-羧酸25a(4g，20.1mmol，采用公知的方法“Journal of the American Chemical Society,2011,133(42),16901-16910”制备而得)的二氯甲烷溶液，于-75℃下搅拌1小时，于0℃搅拌2小时，于室温搅拌1小时。反应液中加入60mL磷酸盐缓冲液(pH＝7.0)和甲醇(V/V＝1:3)的混合液。于0℃加入60mL甲醇和双氧水(30％)(V/V＝2:1)的混合液，于室温搅拌1小时。反应结束后，减压浓缩除去有机相，加入15mL水。水相用乙醚(30mL×3)萃取，合并乙醚相，依次用5％碳酸氢钠溶液，水，饱和氯化钠溶液洗涤，用无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化残留物，得标题产物(S)-叔丁酯 2-((1R,2R)-1-羟基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基 噁唑-3-基)-3-羰基丙基)吡咯烷-1-羧酸25b(2.26g，白色泡沫固体)，产率24.9％。MS m/z(ESI):333.3[M-100+1]
第二步
(S)-叔丁酯 2-((1R,2R)-1-甲氧基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)吡咯烷-1-羧酸
将原料(S)-叔丁酯 2-((1R,2R)-1-羟基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)吡咯烷-1-羧酸25b(2g，4.62mmol)溶于20mL二氯甲烷，加入2g磨成粉末的分子筛，在氩气氛下，于0℃加入1,8-双二甲氨基萘(2.57g，12mmol)，三甲基氧鎓四氟硼酸盐(1.71g，11.5mmol)，于室温搅拌17小时。反应结束后，过滤，滤饼用二氯甲烷洗涤，合并滤液，有机相用饱和氯化铵溶液(20mL×3)洗涤，再用饱和氯化钠溶液洗涤，无水硫酸钠干燥。过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化所得残余物，得到标题产物(S)-叔丁酯2-((1R,2R)-1-甲氧基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)吡咯烷-1-羧酸25c(1.3g，白色泡沫固体)，产率63％。
MS m/z(ESI):447.3[M+1]
第三步
(2R,3R)-3-((S)-1-(叔丁氧羰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酸
将原料(S)-叔丁酯 2-((1R,2R)-1-甲氧基-2-甲基-3-((4R,5S)-4-甲基-2-羰基-5-苯基噁唑-3-基)-3-羰基丙基)吡咯烷-1-羧酸25c(1.3g，2.9mmol)溶于80mL四氢呋喃，反应体系在氩气氛下，降温至0℃，缓慢滴加30％的双氧水(1.25g，11mmol)，再加入一水合氢氧化锂(207mg，4.95mmol)，于室温反应12小时。反应结束后，向反应液中加入亚硫酸钠固体(1.47g，11.6mmol)，于室温搅拌1小时，加入少量水，减压浓缩除去有机相，加入少量水溶解残留物，用二氯甲烷萃取(50mL×2)。水相滴加盐酸至pH为3，用二氯甲烷萃取(40mL×3)，有机相用水洗涤，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得粗品标题产物(2R,3R)-3-((S)-1-(叔丁氧羰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酸25d(870mg，无色油状)，产品不经纯化直接进行下一步反应。
第四步
(S)-叔丁酯 2-((1R,2R)-3-(((S)-1-(叔丁氧基)-3-(2-氟苯基)-1-羰基丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-羰基丙基)吡咯烷-1-羧酸
将粗品(2R,3R)-3-((S)-1-(叔丁氧羰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酸25d(100mg，0.368mmol)溶于6mL二氯甲烷与1.8mL二甲基甲酰胺中，加入反应物(S)-叔丁酯 2-氨基-3-(2-氟苯基)丙酸7b(97mg，0.405mmol)，反应体系在氩气氛下。再加入N,N-二异丙基乙基胺(237.8mg，2.844mmol)和2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(168.2mg，0.442mmol)，于室温搅拌2小时。反应结束后，加10mL二氯甲烷，依次用水，饱和氯化钠溶液洗涤，无水硫酸钠 干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化所得残余物，得到标题产物(S)-叔丁酯 2-((1R,2R)-3-(((S)-1-(叔丁氧基)-3-(2-氟苯基)-1-羰基丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-羰基丙基)吡咯烷-1-羧酸25e(157mg，无色油状)，收率83.9％。
MS m/z(ESI):509.3[M+1]
第五步
(S)-叔丁酯 3-(2-氟苯基)-2-((2R,3R)-3-甲氧基-2-甲基-3-((S)-吡咯烷-2-基)丙酰胺)丙酸
将原料(S)-叔丁酯 2-((1R,2R)-3-(((S)-1-(叔丁氧基)-3-(2-氟苯基)-1-羰基丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-羰基丙基)吡咯烷-1-羧酸25e(157mg，0.308mmol)溶于2mL二氧六环中，加入4M的氯化氢二氧六环溶液(0.193mL，1.08mmol)后将反应体系密封。于室温搅拌1小时，置于4℃冰箱内12小时。反应结束后，将反应液减压浓缩，加入二氯甲烷溶解残留物，用饱和碳酸氢钠溶液洗涤，水相用二氯甲烷萃取，合并有机相，用饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，得到粗品标题产品(S)-叔丁酯 3-(2-氟苯基)-2-((2R,3R)-3-甲氧基-2-甲基-3-((S)-吡咯烷-2-基)丙酰胺)丙酸25f(100mg，淡黄色油状)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):407.2[M-1]
第六步
(S)-叔丁酯 2-((2R,3R)-3-((S)-1-((5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧-4,7,10-三氮杂十四烷基-14-酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
将产品(S)-叔丁酯 3-(2-氟苯基)-2-((2R,3R)-3-甲氧基-2-甲基-3-((S)-吡咯烷-2-基)丙酰胺)丙酸25f(100mg，0.244mmol)，(5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷-14-羧酸1i(156.1mg，0.244mmol)溶于7.5mL二氯甲烷和二甲基甲酰胺(V/V＝4:1)混合溶剂中，加入N,N-二异丙基乙基胺(158mg，1.224mmol)，反应体系在氩气氛下，加入2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(121mg，0.318mmol)，于室温下搅拌1小时。反应结束后，加入二氯甲烷稀释，依次用水洗涤，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化残留物，得到标题产品(S)-叔丁酯 2-((2R,3R)-3-((S)-1-((5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷基-14-酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸25g(242mg，淡黄色油状)，收率96.5％。MS m/z(ESI):1028.4[M+1]
第七步
(S)-叔丁酯 2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
将原料(S)-叔丁酯 2-((2R,3R)-3-((S)-1-((5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧-4,7,10-三氮杂十四烷基-14-酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸25g(242mg，0.235mmol)溶于3mL二氯甲烷中，加入3mL二乙胺，于室温下搅拌3小时。反应结束后，将反应液减压浓缩，得到粗品标题产品(S)-叔丁酯2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸25h(250mg，黄色油状)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):806.7[M+1]
第八步
(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
将粗品(S)-叔丁酯 2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸25h(262mg，0.325mmol)置于反应瓶中，加入4M的氯化氢二氧六环溶液7mL，将反应体系密封，于室温下搅拌12小时。反应结束后，将反应液减压浓缩，用高效液相色谱法纯化所得残余物，得标题产品(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸25(50mg，白色固体)，收率28.4％。
MS m/z(ESI):750.7[M+1]
1H NMR(400MHz,CD3OD)δ7.35-7.30(m,1H),7.20-7.15(m,1H),7.09-6.09(m,2H),4.81-4.75(m,1H),4.70-4.64(m,1H),4.15-4.13(m,1H),4.05-4.03(m,1H),3.84-3.81(m,1H),3.69-3.64(m,1H),3.55-3.51(m,1H),3.48-3.13(m,13H),3.03-2.91(m,2H),2.66-2.53(m,3H),2.47-2.45(m,1H),2.40-2.33(m,2H),2.27-2.23(m,1H),2.17-2.11(m,2H),1.96-1.85(m,3H),1.63-1.40(m,4H),1.21-1.14(m,3H),1.09-0.94(m,12H),0.87-0.84(m,3H).
实施例26
(S)-2-((2R,3R)-3-((S)-1-((3R,4R,5S)-4-((S)-2-((R)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
Figure PCTCN2016072129-appb-000086
将原料(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸25(20mg，0.026mmol)溶于3mL二氯甲烷中，加入N,N-二异丙基乙基胺(13.7mg，0.106mmol)，降温至0℃，反应体系在氩气氛下，滴加预制的6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)己酰氯4b(9.1mg，0.040mmol)的二氯甲烷溶液，于室温反应2小时。反应结束后，加入甲醇淬灭，将反应液减压浓缩，残留物用高效液相色谱法纯化，得标题产物(S)-2-((2R,3R)-3-((S)-1-((3R,4R,5S)-4-((S)-2-((R)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸26(7mg，白色粉末)，收率28.5％。MS m/z(ESI):941.6[M-1]
1HNMR(400MHz,CD3OD)δ7.32-7.21(m,2H),7.10-7.00(m,2H),6.80-6.78(m,2H),4.75-4.55(m,3H),4.12-4.06(m,2H),3.93-3.89(m,1H),3.87-3.78(m,2H),3.69-3.63(m,1H),3.52-3.47(m,3H),3.44-3.28(m,3H),3.21-3.10(m,4H),3.04-2.96(m,4H),2.53-2.40(m,4H),2.35-2.18(m,2H),2.13-2.00(m,2H),1.94-1.75(m,4H),1.68-1.56(m,5H),1.37-1.27(m,4H),1.20-1.13(m,3H),1.05-0.81(m,18H).
实施例27
(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰基)-3-(2-甲氧基苯基)丙酸
Figure PCTCN2016072129-appb-000087
第一步
(S)-tert-butyl 2-amino-3-(2-methoxyphenyl)propanoate
(S)-叔丁酯 2-氨基-3-(2-甲氧基苯基)丙酸
将原料(S)-2-氨基-3-(2-甲氧基苯基)丙酸27a(250mg，1.28mmol，采用公知的方法“Chemical Communications(Cambridge,United Kingdom),2013,49(70),7744-766”制备而得)溶于7mL乙酸叔丁酯，反应体系在氩气氛下，加入高氯酸(270mg(70％)，1.88mmol)，于室温下搅拌16小时。反应结束后，加入10mL二氯甲烷，滴加饱和碳酸氢钠溶液调节pH为8，分液，水相用二氯甲烷(10mL×3)萃取，合并有机相。有机相用饱和氯化钠溶液(10mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得标题产物(S)-叔丁酯 2-氨基-3-(2-甲氧基苯基)丙酸27b(280mg，浅黄色油状物)，收率87％。
采用实施例25的合成路线，将第四步原料替换为(2R,3R)-3-((S)-1-(叔丁氧羰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酸25d和(S)-叔丁酯 2-氨基-3-(2-甲氧基苯基)丙酸27b，制得标题产物(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰基)-3-(2-甲氧基苯基)丙酸27(22mg，灰白色粉末)。
MS m/z(ESI):760.7[M-1]
实施例28
(S)-2-((2R,3R)-3-((S)-1-((3R,4R,5S)-4-((S)-2-((R)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-甲氧基苯基)丙酸
Figure PCTCN2016072129-appb-000088
将原料(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰基)-3-(2-甲氧基苯基)丙酸27(18mg，0.023mmol)溶于5mL二氯甲烷中，反应体系在氩气氛下，加入N,N-二异丙基乙基胺(12.19mg，0.29mmol)，于0℃滴加预制的6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)己酰氯4b(6.5mg，0.028mmol)的二氯甲烷溶液，于室温反应2小时。反应结束后，加入甲醇淬灭，将反应液减压浓缩， 残留物用高效液相色谱法纯化，得标题产物(S)-2-((2R,3R)-3-((S)-1-((3R,4R,5S)-4-((S)-2-((R)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-甲氧基苯基)丙酸28(4mg，白色固体)，收率18.2％。MS m/z(ESI):955.5[M+1]
实施例29
(S)-2-((2R,3R)-3-((S)-1-((3R,4R,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(对甲苯基)丙酸
Figure PCTCN2016072129-appb-000089
采用实施例25的合成路线，(2R,3R)-3-((S)-1-(叔丁氧羰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酸25d和(S)-叔丁酯 2-氨基-3-(对甲苯基)丙酸15b，制得标题产物(S)-2-((2R,3R)-3-((S)-1-((3R,4R,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(对甲苯基)丙酸29(20mg，白色粉末)。
MS m/z(ESI):746.6[M+1]
1H NMR(400MHz,CD3OD):δ7.17-7.05(m,4H),4.84-4.67(m,2H),4.27-4.15(m,1H),4.12-4.05(m,1H),3.89-3.82(m,1H),3.78-3.63(m,2H),3.57-3.47(m,1H),3.43-3.11(m,7H),2.92-2.79(m,1H),2.67(d,3H),2.52-2.44(m,1H),2.40-2.15(m,7H),2.12-2.01(m,1H),1.95-1.69(m,3H),1.67-1.49(m,2H),1.48-1.27(m,4H),1.23-1.12(m,5H),1.11-0.95(m,14H),0.94-0.84(m,3H).
实施例30
(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(对甲苯基)丙酸
Figure PCTCN2016072129-appb-000090
Figure PCTCN2016072129-appb-000091
将原料(S)-2-((2R,3R)-3-((S)-1-((3R,4R,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(对甲苯基)丙酸29(12mg，0.016mmol)溶于1mL二氯甲烷中，反应体系在氩气氛下，加入N,N-二异丙基乙基胺(0.014mL，0.08mmol)，于0℃滴加预制的6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)己酰氯4b(5.54mg，0.024mmol)的二氯甲烷溶液，于室温反应4小时。反应结束后，加入甲醇淬灭，将反应液减压浓缩，残留物用高效液相色谱法纯化，得标题产物(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(对甲苯基)丙酸30(4mg，白色固体)，收率26.5％。
MS m/z(ESI):939.4[M+1]
1H NMR(400MHz,CD3OD):δ7.22-7.03(m,4H),6.85-6.78(m,2H),4.83-4.55(m,3H),4.27-3.97(m,3H),3.90-3.62(m,3H),3.59-2.95(m,14H),2.94-2.79(m,1H),2.54-2.35(m,3H),2.34-2.12(m,5H),2.08-1.99(m,1H),1.93-1.72(m,3H),1.71-1.51(m,5H),1.48-1.24(m,8H),1.23-1.12(m,3H),1.11-0.78(m,17H).
实施例31
(S)-3-(3-氯苯基)-2-((2R,3R)-3-((S)-1-((3R,4R,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)丙酸
Figure PCTCN2016072129-appb-000092
第一步
(S)-tert-butyl 2-amino-3-(3-chlorophenyl)propanoate
(S)-叔丁酯 2-氨基-3-(3-氯苯基)丙酸
将原料(S)-2-氨基-3-(3-氯苯基)丙酸31a(600mg，3mmol)溶于15mL乙酸叔丁酯，在氩气氛下，于0℃滴加高氯酸(450mg(70％)，4.5mmol)，滴完于室温反应 12小时。反应完毕后加入10mL水，分液，有机相依次用20mL 1N的盐酸洗涤，饱和碳酸氢钠溶液洗涤，合并水相。水相滴加饱和碳酸氢钠溶液调节pH为8～9，二氯甲烷(100mL×2)萃取，合并有机相，无水硫酸钠干燥，过滤，滤液减压浓缩得粗品标题产物(S)-叔丁酯 2-氨基-3-(3-氯苯基)丙酸31b(500mg，油状物)，产品不经纯化直接进行下一步反应。
采用实施例25的合成路线，将第四步原料替换为(2R,3R)-3-((S)-1-(叔丁氧羰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酸25d和(S)-叔丁酯 2-氨基-3-(3-氯苯基)丙酸31b，制得标题产物(S)-3-(3-氯苯基)-2-((2R,3R)-3-((S)-1-((3R,4R,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)丙酸31(4mg，白色粉末)。
MS m/z(ESI):766.6[M+1]
1H NMR(400MHz,CD3OD):δ7.31-7.12(m,4H),4.73-4.51(m,2H),4.22-4.05(m,1H),3.92-3.83(m,1H),3.76-3.65(m,1H),3.62-3.51(m,1H),3.50-3.12(m,9H),3.09-2.98(m,1H),2.59-2.39(m,4H),2.38-2.24(m,1H),2.23-2.17(m,1H),2.16-2.01(m,3H),2.00-1.85(m,2H),1.84-1.76(m,1H),1.73-1.57(m,2H),1.43-1.27(m,5H),1.25-1.14(m,3H),1.10-0.95(m,13H),0.94-0.83(m,5H).
实施例32
(S)-2-((2R,3R)-3-((S)-1-((3R,4R,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(3-氟苯基)丙酸
Figure PCTCN2016072129-appb-000093
采用实施例25的合成路线，将第四步原料替换为(2R,3R)-3-((S)-1-(叔丁氧羰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酸25d和(S)-叔丁酯 2-氨基-3-(3-氟苯基)丙酸17b，制得标题产物(S)-2-((2R,3R)-3-((S)-1-((3R,4R,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(3-氟苯基)丙酸32(33.5mg，白色粉末)。
MS m/z(ESI):750.7[M+1]
1H NMR(400MHz,CD3OD)δ7.31-7.28(m,1H),7.08-6.95(m,3H),4.77-4.69(m,2H),4.09-3.94(m,3H),3.69-3.66(m,2H),3.48-3.44(m,2H),3.38-3.36(m,3H),3.34-3.28(m,6H),3.26-3.19(m,2H),3.13(m,1H),3.01-2.91(m,2H),2.67-2.65(m,2H),2.54-2.47(m,2H),2.34-2.28(m,2H),2.18-2.00(m,2H),1.98-1.77(m,2H),1.55-1.42(m,2H),1.08-0.98(m,18H),0.88-0.83(m,3H).
实施例33
(S)-3-(2,4-二氯苯基)-2-((2R,3R)-3-((S)-1-((3R,4R,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)丙酸
Figure PCTCN2016072129-appb-000094
第一步
(S)-tert-butyl 2-amino-3-(2,4-dichlorophenyl)propanoate
(S)-叔丁酯 2-氨基-3-(2,4-二氯苯基)丙酸
将原料(S)-2-氨基-3-(2,4-二氯苯基)丙酸33a(1.3g，5.57mmol，采用公知的方法“International Journal of Peptide&Protein Research,1987,30(1),13-21”制备而得)溶于10mL乙酸叔丁酯，反应体系在氩气氛下，于0℃滴加高氯酸(1.2g(70％)，8.36mmol)，滴完于室温反应12小时。反应结束后，加入二氯甲烷稀释，滴加饱和碳酸氢钠溶液至pH为8～9，分液，水相用二氯甲烷萃取，合并有机相，依次用水洗涤，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得粗品标题产物(S)-叔丁酯 2-氨基-3-(2,4-二氯苯基)丙酸33b(3.4g，淡黄油状物)，产品不经纯化直接进行下一步反应。
采用实施例25的合成路线，将第四步原料替换为(2R,3R)-3-((S)-1-(叔丁氧羰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酸25d和(S)-叔丁酯 2-氨基-3-(2,4-二氯苯基)丙酸33b，制得标题产物(S)-3-(2,4-二氯苯基)-2-((2R,3R)-3-((S)-1-((3R,4R,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)丙酸33(23mg，白色粉末)。
MS m/z(ESI):800.6[M+1]
1H NMR(400MHz,CD3OD)δ7.43-7.42(m,1H),7.26-7.23(m,2H),4.75-4.67(m,2H),4.14-4.04(m,2H),4.00-3.98(m,1H),3.91-3.89(m,1H),3.68-3.67(m,3H),3.39-3.26(m,12H),3.21-3.12(m,3H),2.67-2.64(m,3H),2.50-2.46(m,3H),2.31-2.28(m,2H),2.17-2.15(m,2H),2.02-2.00(m,4H),1.90-1.88(m,2H),1.74-1.72(m,2H),1.39-1.37(m,2H),1.06-0.98(m,12H).
实施例34
(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁 酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(邻甲苯基)丙酸
Figure PCTCN2016072129-appb-000095
第一步
(S)-tert-butyl 2-amino-3-(o-tolyl)propanoate
(S)-叔丁酯 2-氨基-3-(邻甲苯基)丙酸
将原料(S)-2-氨基-3-(o-甲苯基)丙酸34a(100mg，0.55mmol，采用公知的方法“International Journal of Peptide&Protein Research,1987,30(1),13-21”制备而得)溶于2.5mL乙酸叔丁酯，在氩气氛下，滴加高氯酸(107mg(70％)，0.83mmol)，于室温反应16小时。反应结束后，加入5mL二氯甲烷稀释，滴加饱和碳酸氢钠溶液调节pH为8。分液，水相用二氯甲烷(5mL×3)萃取，合并有机相，依次用水洗涤，饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得粗品标题产物(S)-叔丁酯 2-氨基-3-(邻甲苯基)丙酸34b(140mg，无色油状物)，产品不经纯化直接进行下一步反应。
采用实施例25的合成路线，将第四步原料替换为(2R,3R)-3-((S)-1-(叔丁氧羰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酸25d和(S)-叔丁酯 2-氨基-3-(邻甲苯基)丙酸34b，制得标题产物(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(邻甲苯基)丙酸34(40mg，白色粉末)。
MS m/z(ESI):746.8[M+1]
1H NMR(400MHz,CD3OD)δ8.41-8.39(d,1H),8.19-8.17(d,1H),7.18-7.05(m,4H),4.80-4.78(d,1H),4.71-4.69(d,1H),3.86-3.84(m,1H),3.76-3.72(m,1H),3.69-3.63(m,2H),3.48-3.42(d,2H),3.38-3.23(m,5H),3.20-3.12(m,4H),2.98-2.87(m,2H),2.66-2.65(d,3H),2.53-2.50(d,1H),2.46-2.44(d,1H),2.41-2.26(m,4H),2.21-2.14(m,2H),2.10-2.04(m,1H),1.87-1.85(m,2H),1.77-1.74(m,1H),1.62-1.53(m,2H),1.33-1.28(m,3H),1.23-1.21(d,2H),1.16-1.14(d,2H),1.08-0.96(m,14H),0.90-0.84(m,3H).
实施例35
(S)-2-((2R,3R)-3-((S)-1-((3R,4R,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰基)丁酰基)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(噻 吩-2-基)丙酸
Figure PCTCN2016072129-appb-000096
采用实施例25的合成路线，将第四步原料替换为(2R,3R)-3-((S)-1-(叔丁氧羰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酸25d和(S)-叔丁酯 2-氨基-3-(噻吩-2-基)丙酸16b，制得标题产物(S)-2-((2R,3R)-3-((S)-1-((3R,4R,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰基)丁酰基)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(噻吩-2-基)丙酸35(2.6mg，白色粉末)。
1H NMR(400MHz,CD3OD)δ7.21-7.19(m,1H),6.91-6.90(m,2H),4.80-4.65(m,2H),4.16-4.08(m,2H),3.93-3.90(m,1H),3.70-3.66(m,2H),3.55-3.52(m,1H),3.48-3.45(m,2H),3.40-3.26(m,6H),3.25-3.13(m,4H),2.96-2.82(t,1H),2.69-2.65(d,3H),2.52-2.47(m,2H),2.39-2.29(m,1H),2.21-2.14(m,2H),1.92-(s,3H),1.63-1.61(m,2H),1.40-1.29(m,3H),1.23-1.14(m,3H),1.10-0.98(m,13H),0.88-0.85(t,3H).
实施例36
Figure PCTCN2016072129-appb-000097
将化合物(S)-2-((2R,3R)-3-((S)-1-((3R,4R,5S)-4-((S)-2-((R)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸26(5.45mg，5.78μmol)溶解于1.2mL乙腈中，加入帕妥珠单抗-丙硫醇溶液18c(7.56mg/mL，12mL)中，于25℃下振荡反应4小时后将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物36的PBS缓冲液(3.51mg/mL，27.5mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：148094.39(MAb+0D)、149111.06(MAb+1D)、150167.12(MAb+2D)、151188.93(MAb+3D)、152243.46(MAb+4D)、153272.96(MAb+5D)。
平均值：y＝1.9
实施例37
Figure PCTCN2016072129-appb-000098
将化合物(S)-2-((2R,3R)-3-((S)-1-((3R,4R,5S)-4-((S)-2-((R)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-甲氧基苯基)丙酸28(1.75mg，1.83μmol)溶解于0.9mL乙腈中，加入帕妥珠单抗-丙硫醇溶液18c(2.31mg/mL，9.0mL)中，于25℃下振荡反应6小时后将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物37的PBS缓冲液(1.35mg/mL，13.0mL)，于4℃冷冻储存。Q-TOF LC/MS:特征峰：148093.25(MAb+0D)、149281.78(MAb+1D)、150474.33(MAb+2D)、151372.72(MAb+3D)、152373.24(MAb+4D)、153469.40(MAb+5D)。
平均值：y＝2.3。
实施例38
Figure PCTCN2016072129-appb-000099
将化合物(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(对甲苯基)丙酸30(1.07mg，1.14μmol)溶解于1.25mL乙腈中，加入帕妥珠单抗-丙硫醇溶液18c(1.5mg/mL，12.5mL)中，于25℃下振荡反应4.5小时后将反应液用Sephadex G25凝胶柱脱盐 纯化(洗脱相：pH为6.5的0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物38的PBS缓冲液(0.74mg/mL，17.5mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：148247.08(MAb+0D)、149265.32(MAb+1D)、150276.10(MAb+2D)、151334.50(MAb+3D)、152392.21(MAb+4D)、153388.72(MAb+5D)。
平均值：y＝2.4。
实施例39
Figure PCTCN2016072129-appb-000100
第一步
将硫代乙酸S-(3-羰基丙基)酯18a(2.44mg，18.5μmol)，溶解于3.0mL乙腈溶液，备用；向尼妥珠单抗pH＝4.3的乙酸/乙酸钠缓冲液(10.22mg/ml，30mL，0.204mmol)加入上述预制的硫代乙酸S-(3-羰基丙基)酯18a的乙腈溶液，然后滴加1.2mL的氰基硼氢化钠(49.86mg,793μmol)的水溶液，于25℃下振荡反应2小时。将反应液先用含10％乙腈的PBS缓冲液(250mL)通过30KDa超滤包进行纯化，再改用pH＝6.5的PBS缓冲液(200mL)通过30KDa超滤包进行纯化，除去未反应的硫代乙酸S-(3-羰基丙基)酯18a以及氰基硼氢化钠，得到标题产物39b的PBS缓冲溶液(约75mL)，直接进行下一步反应。
第二步
向39b的PBS缓冲溶液(75.0mL)中加入2.0mL的2.0M盐酸羟胺溶液，加毕，置于水浴振荡器，于25℃下振荡反应30分钟，停止反应。将反应液用pH＝6.5的PBS缓冲液通过30KDa超滤包进行纯化，得到标题产物尼妥珠单抗-丙硫醇39c的PBS缓冲溶液(浓度5.38mg/ml，55mL)。
第三步
将化合物(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲 氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸4(4.48mg，4.78μmol)溶解于1.1mL乙腈中，加入尼妥珠单抗-丙硫醇PBS缓冲溶液39c(5.38mg/mL，11mL)中，置于水浴振荡器中，于25℃下振荡反应4小时后停止反应。
将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)，得到粗品标题产物39的PBS缓冲液(2.96mg/mL，20.5mL)，进一步离心浓缩至6mL左右，再用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)，得到标题产物39的PBS缓冲液(4.25mg/mL，11.8mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：150188.68(MAb+0D)、151234.76(MAb+1D)、152248.46(MAb+2D)、153419.10(MAb+3D)、154312.17(MAb+4D)、155358.48(MAb+5D)。
平均值：y＝2.2。
实施例40
Figure PCTCN2016072129-appb-000101
将化合物(S)‐2‐((2R,3R)‐3‐((1S,3S,5S)‐2‐((3R,4S,5S)‐4‐((S)‐2‐((S)‐2‐(6‐(2,5‐二羰基‐2,5‐二氢‐1H‐吡咯‐1‐基)‐N‐甲基己酰胺)‐3‐甲基丁酰胺)‐N,3‐二甲基丁酰胺)‐3‐甲氧基‐5‐甲基庚酰基)‐2‐氮杂双环[3.1.0]己烷‐3‐基)‐3‐甲氧基‐2‐甲基丙酰胺)‐3‐(2‐氟苯基)丙酸8(4.32mg，4.52μmol)溶解于1.1mL乙腈中，加入尼妥珠单抗‐丙硫醇PBS缓冲溶液39c(5.38mg/mL，11mL)中，置于水浴振荡器中，于25℃下振荡反应4小时后停止反应。
将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)，得到粗品标题产物40的PBS缓冲液(2.92mg/mL，20mL)，进一步离心浓缩至5.5mL左右，再用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)，得到标题产物40的PBS缓冲液(4.25mg/mL，11.6mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：150186.98(MAb+0D)、151374.09(MAb+1D)、152287.22(MAb+2D)、153353.26(MAb+3D)、154501.80(MAb+4D)、155575.57(MAb+5D)。
平均值：y＝2.2。
实施例41
Figure PCTCN2016072129-appb-000102
将化合物(S)‐2‐((2R,3R)‐3‐((S)‐1‐((3R,4R,5S)‐4‐((S)‐2‐((R)‐2‐(6‐(2,5‐二羰基‐2,5‐二氢‐1H‐吡咯‐1‐基)‐N‐甲基己酰胺)‐3‐甲基丁酰胺)‐N,3‐二甲基丁酰胺)‐3‐甲氧基‐5‐甲基庚酰基)吡咯烷‐2‐基)‐3‐甲氧基‐2‐甲基丙酰胺)‐3‐(2‐氟苯基)丙酸26(4.45mg，4.72μmol)溶解于1.1mL乙腈中，加入尼妥珠单抗‐丙硫醇PBS缓冲溶液39c(5.38mg/mL，11mL)中，置于水浴振荡器中，于25℃振荡反应4小时后停止反应。
将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的.05M的PBS溶液)，得到粗品标题产物41的PBS缓冲液(2.96mg/mL，20mL)，进一步离心浓缩至6mL左右，再用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)，得到标题产物41的PBS缓冲液(4.33mg/mL，11mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：150186.05(MAb+0D)、151362.44(MAb+1D)、152261.90(MAb+2D)、153438.29(MAb+3D)、154339.30(MAb+4D)、155511.23(MAb+5D)。
平均值：y＝2.3。
实施例42
Figure PCTCN2016072129-appb-000103
将化合物(S)‐2‐((2R,3R)‐3‐((S)‐5‐((3R,4S,5S)‐4‐((S)‐2‐((S)‐2‐(6‐(2,5‐二羰基‐2,5‐二氢 ‐1H‐吡咯‐1‐基)‐N‐甲基己酰胺)‐3‐甲基丁酰胺)‐N,3‐二甲基丁酰胺)‐3‐甲氧基‐5‐甲基庚酰基)‐5‐氮杂螺环[2.4]庚烷‐6‐基)‐3‐甲氧基‐2‐甲基丙酰胺)‐3‐(2‐氟苯基)丙酸12(0.56mg，0.58μmol)溶解于0.42mL乙腈中，加入尼妥珠单抗‐丙硫醇溶液39c(2.06mg/mL，4.2mL)中，置于水浴振荡器中，于25℃振荡反应5小时后停止反应。
将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)，得到粗品标题产物42的PBS缓冲液(0.74mg/mL，10mL)，进一步离心浓缩，再用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)，得到标题产物42的PBS缓冲液(1.15mg/mL，6mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：150188.42(MAb+0D)、151387.82(MAb+1D)、152472.27(MAb+2D)、153528.33(MAb+3D)。
平均值：y＝1.0。
实施例43
Figure PCTCN2016072129-appb-000104
将化合物(S)‐2‐((2R,3R)‐3‐((S)‐5‐((3R,4S,5S)‐4‐((S)‐2‐((S)‐2‐(6‐(2,5‐二羰基‐2,5‐二氢‐1H‐吡咯‐1‐基)‐N‐甲基己酰胺)‐3‐甲基丁酰胺)‐N,3‐二甲基丁酰胺)‐3‐甲氧基‐5‐甲基庚酰基)‐5‐氮杂螺环[2.4]庚烷‐6‐基)‐3‐甲氧基‐2‐甲基丙酰胺)‐3‐苯丙酸14(0.60mg，0.63μmol)溶解于0.42mL乙腈中，加入尼妥珠单抗‐丙硫醇溶液39c(2.06mg/mL，4.2mL)中，置于水浴振荡器中，于25℃下振荡反应5小时后停止反应。
将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)，得到粗品标题产物43的PBS缓冲液(0.78mg/mL，9.5mL)，进一步离心浓缩，再用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)，得到标题产物43的PBS缓冲液(1.16mg/mL，6mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：150188.39(MAb+0D)、151251.46(MAb+1D)、152442.90(MAb+2D)、153507.73(MAb+3D)。
平均值：y＝1.0。
实施例44
(S)-2-((2R,3R)-3-((2S,4S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-4-氟吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯基丙酸
Figure PCTCN2016072129-appb-000105
第一步
(2S,4S)-叔丁酯 2-((1R,2R)-3-((R)-4-苄基-2-羰基噁唑-3-基)-1-羟基-2-甲基-3-羰基丙基)-4-氟吡咯烷-1-羧酸
将原料(R)-4-苄基-3-丙酰基噁唑-2-酮44b(21g，90mmol，采用公知的方法“Tetrahedron Letters,1999,40(36),6545-6547”制备而得)溶于300mL二氯甲烷中，氮气氛下，降温至0℃。反应液于0℃下滴加四氯化钛(9.8mL，1.1mmol)，溶液由无色逐渐变为黄色，并且有黄色固体出现。再缓慢滴加N,N-二异丙基乙基胺(40mL，225mmol)，有白烟生成，溶液由黄色变为红棕色，于0℃下搅拌1小时，将反应液降温至-78℃，加入50mL(2S,4S)-叔丁酯 4-氟-2-甲酰基吡咯烷-1-羧酸44a(21.27g，98mmol，采用公知的方法“Tetrahedron:Asymmetry,2014,25(3),212-218”制备而得)的二氯甲烷溶液，-78℃下搅拌1.5小时。TLC监测反应的完成情况。反应完毕后，加入200mL碳酸氢钠溶液(5％)，分液，水相用二氯甲烷萃取(300mL×2)，合并有机相，依次用水(200mL)、饱和氯化钠溶液(200mL)洗涤，无水硫酸 镁干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化所得残余物，得标题产物(2S,4S)-叔丁酯 2-((1R,2R)-3-((R)-4-苄基-2-羰基噁唑-3-基)-1-羟基-2-甲基-3-羰基丙基)-4-氟吡咯烷-1-羧酸44c(19g，淡黄色固体)，收率43.2％。
MS m/z(ESI):351.06[M-100+1]
第二步
(2R,3R)-3-((2S,4S)-1-(叔丁氧羰基)-4-氟吡咯烷-2-基)-3-羟基-2-甲基丙酸
将原料(2S,4S)-叔丁酯 2-((1R,2R)-3-((R)-4-苄基-2-羰基噁唑-3-基)-1-羟基-2-甲基-3-羰基丙基)-4-氟吡咯烷-1-羧酸44c(19g，42mmol)溶于200mL四氢呋喃与50mL水中，氮气氛下，降温至0℃，缓慢滴加30％的双氧水(17.2mL，147mmol)，再加入80mL一水合氢氧化锂(2.86g，68mmol)溶液，反应体系于0℃反应5小时。向反应液中加入100mL亚硫酸钠(21.2g，168mmol)溶液，于25℃反应16小时。反应结束后，减压浓缩除去有机相，所得残留物用二氯甲烷洗涤(200mL×3)；水相用1N盐酸调节pH为3左右，用乙酸乙酯萃取(200mL×4)。合并有机相，减压浓缩，所得残留物用350mL碳酸氢钠溶液(5％)溶解，用二氯甲烷洗涤(200mL×2)，水相用2N盐酸调节pH为3左右，用乙酸乙酯萃取(200mL×5)，合并有机相，用无水硫酸钠干燥，过滤，滤液减压浓缩，得粗品标题产物(2R,3R)-3-((2S,4S)-1-(叔丁氧羰基)-4-氟吡咯烷-2-基)-3-羟基-2-甲基丙酸44d(11.6g，淡黄色粘稠液体)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):191.47[M-100+1]
第三步
(2R,3R)-3-((2S,4S)-1-(叔丁氧羰基)-4-氟吡咯烷-2-基)-3-甲氧基-2-甲基丙酸
将原料(2R,3R)-3-((2S,4S)-1-(叔丁氧羰基)-4-氟吡咯烷-2-基)-3-羟基-2-甲基丙酸44d(11.6g，39.8mmol)溶于200mL四氢呋喃中，氮气氛下，在0℃下加入碘甲烷(91g，640mmol)，分批加入氢化钠(7.32g(60％)，183mmol)，于0℃下反应48小时。反应结束后，加入500mL冰水淬灭反应，加入乙酸乙酯萃取(150mL)。水相用乙醚(150mL×2)洗涤，用2N盐酸调节pH为3，再用乙酸乙酯萃取(250mL×3)。合并有机相，用无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化所得残余物，得到标题产物(2R,3R)-3-((2S,4S)-1-(叔丁氧羰基)-4-氟吡咯烷-2-基)-3-甲氧基-2-甲基丙酸44e(8.0g，淡黄色粘稠液体)，产率65.6％。
MS m/z(ESI):205.68[M-100+1]
第四步
(2S,4S)-叔丁酯 4-氟-2-((1R,2R)-1-甲氧基-3-(((S)-1-甲氧基-1-羰基-3-苯基丙烷-2-基)氨基)-2-甲基-3-羰基丙基)吡咯烷-1-羧酸
将原料(2R,3R)-3-((2S,4S)-1-(叔丁氧羰基)-4-氟吡咯烷-2-基)-3-甲氧基-2-甲基丙酸44e(580mg，1.9mmol)溶于15mL乙腈中，加入(S)-2-氨基-3-苯基丙酸甲酯盐酸盐44f(480mg，2.2mmol，采用公知的方法“Journal of Heterocyclic Chemistry, 2013,50(2),320-325”制备而得)，再加入N,N-二异丙基乙基胺(1.42mL，8mmol)和2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(1.07g，2.8mmol)，反应体系于室温下反应12小时。TLC显示反应完全，停止反应，加入40mL乙酸乙酯稀释，依次用饱和氯化铵溶液(20mL)、饱和氯化钠溶液(20mL)洗涤，用无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化所得残余物，得到标题产物(2S,4S)-叔丁酯 4-氟-2-((1R,2R)-1-甲氧基-3-(((S)-1-甲氧基-1-羰基-3-苯基丙烷-2-基)氨基)-2-甲基-3-羰基丙基)吡咯烷-1-羧酸44g(750mg，白色固体)，收率83.2％。
MS m/z(ESI):222.62[M-100+1]
第五步
(S)-2-((2R,3R)-3-((2S,4S)-4-氟吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯基丙酸甲酯 三氟乙酸盐
将原料(2S,4S)-叔丁酯 4-氟-2-((1R,2R)-1-甲氧基-3-(((S)-1-甲氧基-1-羰基-3-苯基丙烷-2-基)氨基)-2-甲基-3-羰基丙基)吡咯烷-1-羧酸44g(738mg，1.58mmol)溶于15mL二氯甲烷中，逐滴加入三氟乙酸(3mL，30mmol)，于室温反应12小时。TLC检测反应完全后，将反应液减压浓缩得粗品标题产物(S)-2-((2R,3R)-3-((2S,4S)-4-氟吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯基丙酸甲酯 三氟乙酸盐44h(1.24g，黄色粘稠液体)，产品不经纯化直接进行下一步反应。MS m/z(ESI):236.39[M+1]
第六步
(S)-2-((2R,3R)-3-((2S,4S)-1-((5S,8S,11S,12R)-11-((S)-仲丁基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-1-苯基-2-氧杂-4,7,10-三氮杂十四烷-14-酰基-4-氟吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯基丙酸甲酯
将原料(S)-2-((2R,3R)-3-((2S,4S)-4-氟吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯基丙酸甲酯 三氟乙酸盐44h(572mg，1.56mmol)，(5S,8S,11S,12R)-11-((S)-仲丁基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-1-苯基-2-氧杂-4,7,10-三氮杂十四烷-14-羧酸44i(868mg，1.56mmol，采用专利申请“WO2007008848”公开的方法制备而得)溶于15mL乙腈中，加入N,N-二异丙基乙基胺(2.0mL，12mmol)和2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(890mg，2.34mmol)，反应体系于室温下反应12小时。TLC显示反应完全，停止反应，加入50mL乙酸乙酯稀释，依次用饱和氯化铵溶液(30mL)、饱和氯化钠溶液(30mL)洗涤，用无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B和A纯化所得残余物，得到标题产品(S)-2-((2R,3R)-3-((2S,4S)-1-((5S,8S,11S,12R)-11-((S)-仲丁基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-1-苯基-2-氧杂-4,7,10-三氮杂十四烷-14-酰基-4-氟吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯基丙酸甲酯44j(1.4g，白色泡沫状固体)，收率99.9％。
MS m/z(ESI):898.99[M+1]
第七步
(S)-2-((2R,3R)-3-((2S,4S)-1-((5S,8S,11S,12R)-11-((S)-仲丁基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-1-苯基-2-氧杂-4,7,10-三氮杂十四烷-14-酰基)-4-氟吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯基丙酸
将原料(S)-2-((2R,3R)-3-((2S,4S)-1-((5S,8S,11S,12R)-11-((S)-仲丁基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-1-苯基-2-氧杂-4,7,10-三氮杂十四烷-14-酰基-4-氟吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯基丙酸甲酯44j(180mg，0.2mmol)溶于0.5mL甲醇、0.5mL四氢呋喃与0.5mL水中，冷却至0℃，加入一水合氢氧化锂(34mg，0.8mmol)，于23℃下反应2小时。TLC及Ms显示反应完毕，加入2mL水稀释，用1N盐酸调节pH为4.5左右，用乙酸乙酯萃取(5mL×3)，合并有机相，用无水硫酸镁干燥，过滤，滤液减压浓缩得粗品标题产品(S)-2-((2R,3R)-3-((2S,4S)-1-((5S,8S,11S,12R)-11-((S)-仲丁基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-1-苯基-2-氧杂-4,7,10-三氮杂十四烷-14-酰基)-4-氟吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯基丙酸44k(160mg，淡黄色泡沫状固体)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):883.73[M-1]
第八步
(S)-2-((2R,3R)-3-((2S,4S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-4-氟吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯基丙酸
将粗品(S)-2-((2R,3R)-3-((2S,4S)-1-((5S,8S,11S,12R)-11-((S)-仲丁基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-1-苯基-2-氧杂-4,7,10-三氮杂十四烷-14-酰基)-4-氟吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯基丙酸44k(160mg，0.2mmol)溶于10mL无水乙醇中，加入30mg钯碳(10％)，将反应体系用氢气置换三次，于23℃反应15小时。TLC显示反应完全，通过硅藻土过滤除去钯碳，滤液减压浓缩得残留物120mg，用高效液相色谱法纯化所得残留物得标题产品(S)-2-((2R,3R)-3-((2S,4S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-4-氟吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯基丙酸44(29mg，白色固体)，收率21.5％。
MS m/z(ESI):749.94[M+1]
1H NMR(400MHz,CD3OD)δ7.29-7.14(m,5H),4.81-4.71(m,2H),4.18-4.13(m,2H),4.05-4.03(m,2H),3.84-3.81(m,1H),3.63-3.60(m,1H),3.58-3.52(m,1H),3.48-3.13(m,14H),3.00-2.91(m,1H),2.66-1.81(m,11H),1.63-1.38(m,2H),1.21-1.10(m,5H),1.09-0.94(m,14H),0.91-0.82(m,3H).
实施例45
(S)-2-((2R,3R)-3-((2S,4S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-4-氟吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯基丙酸
Figure PCTCN2016072129-appb-000106
将原料(S)-2-((2R,3R)-3-((2S,4S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-4-氟吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯基丙酸44(1.0g，1.34mmol)溶于20mL乙腈中，加入N,N-二异丙基乙基胺(0.53mL，3.0mmol)，2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(560mg，1.48mmol)，反应体系于23℃反应30分钟，加入6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)己酸4a(300mg，1.41mmol)。待TLC显示反应完全后，停止反应，加入50mL乙酸乙酯，减压浓缩，用硅胶柱色谱法先后以洗脱剂体系B和A纯化所得残余物，得粗品标题产物1.0g，然后用高效液相色谱法纯化所得残留物，得标题产物(S)-2-((2R,3R)-3-((2S,4S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-4-氟吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯基丙酸45(140mg，白色固体)，收率11％。
MS m/z(ESI):942.67[M+1]
1H NMR(400MHz,CD3OD)δ7.34-7.14(m,5H),6.83-6.80(m,2H),5.20-5.10(m,1H),5.01-4.96(m,1H),4.78-4.57(m,2H),4.18-4.01(m,2H),4.00-3.80(m,1H),3.56-3.41(m,5H),3.35-3.24(m,10H),3.19-2.90(m,4H),2.60-2.40(m,4H),2.39-2.00(m,4H),1.93-1.58(m,6H),1.50-1.10(m,7H),1.09-0.82(m,21H).
实施例46
(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(4-氟苯基)丙酸
Figure PCTCN2016072129-appb-000107
第一步
(S)-叔丁酯 2-((1R,2R)-3-(((S)-3-(4-氟苯基)-1-甲氧基-1-羰基丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-羰基丙基)吡咯烷-1-羧酸
将(2R,3R)-3-((S)-1-(叔丁氧羰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酸25d(1.0g，3.5mmol)溶于15mL乙腈中，加入(S)-2-氨基-3-(4-氟苯基)丙酸甲酯盐酸盐46a(820mg，4mmol，采用公知的方法“Tetrahedron,2003,59(21),3719-3727”制备而得)，再加入N,N-二异丙基乙基胺(2.7mL，15mmol)和2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(1.9g，5.0mmol)，反应体系于23℃反应12小时。TLC显示反应完全，停止反应，加入50mL乙酸乙酯稀释，依次用饱和氯化铵溶液(30mL)、饱和氯化钠溶液(30mL)洗涤，用无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以洗脱剂体系B纯化所得残余物，得到标题产物(S)-叔丁酯 2-((1R,2R)-3-(((S)-3-(4-氟苯基)-1-甲氧基-1-羰基丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-羰基丙基)吡咯烷-1-羧酸46b(1.35g，白色固体)，收率82.6％。
MS m/z(ESI):366.46[M-100-1]
第二步
(S)-3-(4-氟苯基)-2-((2R,3R)-3-甲氧基-2-甲基-3-((S)-吡咯烷-2-基)丙酰胺)丙酸甲酯 三氟乙酸盐
将原料(S)-叔丁酯 2-((1R,2R)-3-(((S)-3-(4-氟苯基)-1-甲氧基-1-羰基丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-羰基丙基)吡咯烷-1-羧酸46b(1.34g，2.87mmol)溶于20mL二氯甲烷中，逐滴加入三氟乙酸(3mL，30mmol)，于23℃反应12小时。TLC 检测反应完全后，将反应液减压浓缩得粗品标题产物(S)-3-(4-氟苯基)-2-((2R,3R)-3-甲氧基-2-甲基-3-((S)-吡咯烷-2-基)丙酰胺)丙酸甲酯 三氟乙酸盐46c(1.92g，黄色粘稠液体)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):366.85[M+1]
第三步
(S)-2-((2R,3R)-3-((S)-1-((5S,8S,11S,12R)-11-((S)-仲丁基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-1-苯基-2-氧杂-4,7,10-三氮杂十四烷基-14-酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(4-氟苯基)丙酸甲酯
将原料(S)-3-(4-氟苯基)-2-((2R,3R)-3-甲氧基-2-甲基-3-((S)-吡咯烷-2-基)丙酰胺)丙酸甲酯 三氟乙酸盐46c(367mg，1.0mmol)，(5S,8S,11S,12R)-11-((S)-仲丁基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-1-苯基-2-氧杂-4,7,10-三氮杂十四烷-14-羧酸44i(549mg，1.0mmol)溶于10mL乙腈中，加入N,N-二异丙基乙基胺(1.24mL，7mmol)和2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(570mg，1.5mmol)，反应体系于23℃下反应12小时。TLC显示反应完全，停止反应，加入30mL乙酸乙酯稀释，依次用饱和氯化铵溶液(20mL)、饱和氯化钠溶液(20mL)洗涤，用无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法先后以洗脱剂体系B和A纯化所得残留物，得到标题产品(S)-2-((2R,3R)-3-((S)-1-((5S,8S,11S,12R)-11-((S)-仲丁基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-1-苯基-2-氧杂-4,7,10-三氮杂十四烷基-14-酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(4-氟苯基)丙酸甲酯46d(760mg，白色泡沫状固体)，收率84.6％。
MS m/z(ESI):898.10[M+1]
第四步
(S)-2-((2R,3R)-3-((S)-1-((5S,8S,11S,12R)-11-((S)-仲丁基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-1-苯基-2-氧杂-4,7,10-三氮杂十四烷基-14-酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(4-氟苯基)丙酸
将原料(S)-2-((2R,3R)-3-((S)-1-((5S,8S,11S,12R)-11-((S)-仲丁基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-1-苯基-2-氧杂-4,7,10-三氮杂十四烷基-14-酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(4-氟苯基)丙酸甲酯46d(180mg，0.2mmol)溶于0.5mL甲醇、0.5mL四氢呋喃与0.5mL水中，冷却至0℃，加入一水合氢氧化锂(34mg，0.8mmol)，于23℃下反应2小时。TLC及Ms显示反应完毕，加入2mL水稀释，用1N盐酸调节pH为4.5左右，用乙酸乙酯萃取(5mL×3)，合并有机相，用无水硫酸镁干燥，过滤，滤液减压浓缩得粗品标题产品(S)-2-((2R,3R)-3-((S)-1-((5S,8S,11S,12R)-11-((S)-仲丁基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-1-苯基-2-氧杂-4,7,10-三氮杂十四烷基-14-酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(4-氟苯基)丙酸46e(160mg，白色固体)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):883.78[M-1]
第五步
(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(4-氟苯基)丙酸
将粗品(S)-2-((2R,3R)-3-((S)-1-((5S,8S,11S,12R)-11-((S)-仲丁基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-1-苯基-2-氧杂-4,7,10-三氮杂十四烷基-14-酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(4-氟苯基)丙酸46e(160mg，0.2mmol)溶于10mL无水乙醇中，加入30mg钯碳(10％)，将反应体系用氢气置换三次，于室温反应15小时。TLC显示反应完全，通过硅藻土过滤除去钯碳，滤液减压浓缩得残留物130mg，用高效液相色谱法纯化所得残留物得标题产品(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(4-氟苯基)丙酸46(34mg，白色粉末)，收率25.2％。
MS m/z(ESI):749.94[M+1]
1H NMR(400MHz,CD3OD)δ7.31-7.24(m,2H),7.02-6.92(m,2H),4.48-4.58(m,2H),4.22-4.11(m,1H),3.85-3.40(m,4H),3.39-3.04(m,14H),3.00-2.90(m,1H),2.66-2.40(m,5H),2.39-2.02(m,4H),1.99-1.75(m,3H),1.73-1.24(m,3H),1.20-1.10(m,4H),1.09-0.95(m,16H),0.94-0.80(m,3H).
实施例47
(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(4-氟苯基)丙酸
Figure PCTCN2016072129-appb-000108
将6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)己酸4a(434mg，2.06mmol)溶于20mL乙腈中，加入N,N-二异丙基乙基胺(844mL，6.55mmol)，2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(781mg，1.87mmol)，反应体系于23℃反应30分钟，加入(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲 基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(4-氟苯基)丙酸46(1.4g，1.87mmol)。待TLC显示完全后，停止反应，加入50mL乙酸乙酯稀释，减压浓缩，用硅胶柱色谱法先后以洗脱剂体系B和A纯化所得残余物，得标题产物粗品1.2g，然后用高效液相色谱法纯化所得残留物，得标题产物(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(4-氟苯基)丙酸47(670mg，白色固体)，收率37.9％。
MS m/z(ESI):944.54[M+1]
1H NMR(400MHz,CD3OD)δ12.80(br.s,1H),8.56-8.52(m,1H),8.42-8.20(m,1H),8.18-8.10(m,1H),7.30-7.18(m,2H),7.07-6.75(m,4H),4.64-4.34(m,3H),4.08-3.94(m,1H),3.80-3.50(m,2H),3.46-2.70(m,15H),2.50-1.80(m,6H),1.73-1.38(m,7H),1.30-1.10(m,12H),1.09-0.62(m,20H).
实施例48
Figure PCTCN2016072129-appb-000109
将化合物(S)-2-((2R,3R)-3-((2S,4S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-4-氟吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯基丙酸45(1.1mg，1.16μmol)溶解于1.1mL乙腈中，加入尼妥珠单抗-丙硫醇PBS缓冲液39c(1.63mg/mL，11.4mL)中，于25℃下振荡反应4小时后将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的含0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物48的PBS缓冲液(0.75mg/mL，19.8mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：150186.5(MAb+0D)、151364.1(MAb+1D)、152262.3(MAb+2D)、153435.7(MAb+3D)、154499.6(MAb+4D)、155427.5(MAb+5D)。
平均值：y＝2.0。
实施例49
Figure PCTCN2016072129-appb-000110
将化合物(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(4-氟苯基)丙酸47(1.1mg，1.16μmol)溶解于1.1mL乙腈中，加入尼妥珠单抗-丙硫醇PBS缓冲液39c(1.63mg/mL，11.4mL)中，于25℃下振荡反应4小时后将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的含0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物49的PBS缓冲液(0.75mg/mL，19.8mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：150185.5(MAb+0D)、151364.7(MAb+1D)、152261.2(MAb+2D)、153436.2(MAb+3D)、154499.9(MAb+4D)、155428.6(MAb+5D)。。
平均值：y＝2.0。
实施例50
Figure PCTCN2016072129-appb-000111
将化合物(S)-2-((2R,3R)-3-((2S,4S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-4-氟吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯基丙酸45(1.09mg，1.16μmol)溶解于1.1mL乙腈中，加入帕妥珠单抗-丙硫醇溶液18c(1.65mg/mL，11.3mL)中，于25℃下振荡反应4小时后将反应液用Sephadex G25凝胶 柱脱盐纯化(洗脱相：pH为6.5的含0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物50的PBS缓冲液(0.75mg/mL，19.5mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：148095.6(MAb+0D)、149111.5(MAb+1D)、150165.3(MAb+2D)、151184.7(MAb+3D)、152255.2MAb+4D)、153297.5(MAb+5D)。
平均值：y＝2.0。
实施例51
Figure PCTCN2016072129-appb-000112
将化合物(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(4-氟苯基)丙酸47(1.09mg，1.16μmol)溶解于1.1mL乙腈中，加入帕妥珠单抗-丙硫醇溶液47c(1.65mg/mL，11.3mL)中，于25℃下振荡反应4小时后将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的含0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物51的PBS缓冲液(0.75mg/mL，19.5mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：148096.2(MAb+0D)、149112.2(MAb+1D)、150165.4(MAb+2D)、151184.8(MAb+3D)、152255.1(MAb+4D)、153297.6(MAb+5D)。
平均值：y＝2.0。
实施例52
Figure PCTCN2016072129-appb-000113
Figure PCTCN2016072129-appb-000114
第一步
将硫代乙酸S-(3-羰基丙基)酯18a(0.35mg，2.65μmol)，溶解于0.45mL乙腈溶液，备用；向曲妥珠单抗pH＝4.5的乙酸/乙酸钠缓冲液(10.0mg/ml，4.5mL，0.304μmol)加入上述预制的硫代乙酸S-(3-羰基丙基)酯18a的乙腈溶液，然后滴加1.0mL的氰基硼氢化钠(7.06mg,112μmol)溶液，于25℃下振荡反应2小时。反应结束后用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)后得标题产物52b溶液，直接进行下一步反应。
第二步
向52b溶液(约15.0mL)中加入0.45mL的2.0M盐酸羟胺溶液，于25℃下振荡反应30分钟后将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)后得标题产物曲妥珠单抗-丙硫醇52c溶液(浓度1.65mg/ml，22.6mL)。
第三步
将化合物(S)-2-((2R,3R)-3-((2S,4S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-4-氟吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-苯基丙酸45(1.1mg，1.2μmol)溶解于1.1mL乙腈中，加入曲妥珠单抗-丙硫醇溶液52c(1.65mg/mL，11.3mL)中，于25℃下振荡反应4小时后将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的含0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物52的PBS缓冲液(0.72mg/mL，20mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：148062.9(MAb+0D)、149235.2(MAb+1D)、150259.8(MAb+2D)、151268.2(MAb+3D)、152341.9(MAb+4D)、153356.4(MAb+5D)。
平均值：y＝2.0。
实施例53
Figure PCTCN2016072129-appb-000115
将化合物(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)吡咯烷-2-基)-3-甲氧基-2-甲基丙酰胺)-3-(4-氟苯基)丙酸47(1.12mg，1.2μmol)溶解于1.1mL乙腈中，加入曲妥珠单抗-丙硫醇溶液52c(1.65mg/mL，11.3mL)中，于25℃下振荡反应4小时后将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的含0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物53的PBS缓冲液(0.70mg/mL，20.5mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：148065.2(MAb+0D)、149244.6(MAb+1D)、150254.9(MAb+2D)、151280.9(MAb+3D)、152301.6(MAb+4D)、153457.9(MAb+5D)。
平均值：y＝2.0。
生物学评价
测试例1：细胞毒素通式(D)化合物对肿瘤细胞体外增殖抑制测试
一、测试目的
本实验的目的是为了检测本发明通式(D)药物化合物，对HepG2肿瘤细胞(人肝癌细胞，中科院细胞库，货号#TCHu 72)和A549肿瘤细胞(人肺腺癌细胞中科院细胞库，货号#TCHu150)体外增殖的抑制活性。以不同浓度的化合物体外处理细胞，经76小时培养后，采用CCK-8(Cell Counting Kit-8，Dojindo，货号：CK04)试剂对细胞的增值进行检测，根据IC50值评价该化合物的体外活性。
二、实验方法
下面以对HepG2细胞体外增殖抑制测试方法为例，用于举例说明本发明中测试本发明化合物对肿瘤细胞进行体外增殖抑制活性测试的方法。本方法同样适用于，但不限于对其它肿瘤细胞进行体外增殖抑制活性测试。
1、细胞准备。取对数生长期的HepG2细胞，用PBS(磷酸缓冲液，ThermoFisher)洗涤1次之后，加入2-3ml胰蛋白酶(0.25％Trypsin-EDTA(1x)，Gibico,Life Technologies公司)消化2-3min，待细胞消化完全后，加入10-15ml 细胞培养液(DMEM/F12培养基，Invitrogen；10％(v/v)灭活的胎牛血清)，将经过消化的HepG2细胞洗脱下来，1000rpm离心3min，弃上清，接着加入10-20ml细胞培养液将细胞重悬，制成单细胞悬液。
2、细胞铺板。将HepG2单细胞悬液混匀，用细胞培养液调整活细胞密度至6×104cells/ml，将密度调整过后的细胞悬液混匀，以100μl/孔加入96孔细胞培养板。将培养板置于37℃，5％CO2恒温培养箱内培养18-20小时。
3、化合物准备
用DMSO(二甲基亚砜，上海泰坦科技股份有限公司)溶解化合物，配制成初始浓度为10mM的存储液。
加入10μl 10mM的化合物样品至U型底96孔板(样品板1)第一列各孔。，在第一列各化合物样品孔内加入90μl DMSO，即对原液稀释10倍，作为起始浓度点，接下来进行3倍梯度稀释，每种化合物稀释10个浓度点。稀释液为DMSO溶液。第12列加入60μl 100％DMSO。第11列加入20μl 1mM的阳性药物参考品。
取一新的U型底96孔板(样品板2)，用完全培养基对样品板1内的各孔样品进行20倍稀释。接着再取一新的U型底96孔板(样品板3)，将样品板2内的各孔样品进行最终的10倍稀释。
4、加样操作。细胞预培养完成后，取出96孔细胞培养板，弃上清。以100μl/孔，将96孔样品板3中的各样品稀释液依次加入到96孔细胞培养板内。各化合物样品每种浓度点作2复孔检测。加样操作不应超过30min。完成加样操作后，将96孔细胞培养板置于37℃，5％CO2二氧化碳恒温培养箱中培养76小时左右。
5、显色操作。取出96孔细胞培养板，向各孔中以10μl/孔加入CCK-8显色液。轻轻拍板混匀10次以上，将细胞培养板置于37℃，5％CO2二氧化碳恒温培养箱继续孵育2.0小时。
6、读板操作。取出96孔细胞培养板，置于酶标仪(PerkinElmer，VICTOR 3)中，用酶标仪测定在450nm处的吸光度。
按照以上步骤，测试本发明药物化合物对A549肿瘤细胞体外增殖的抑制活性。细胞培养液同样为DMEM/F12培养基。
三、数据分析
用Microsoft Excel，Graphpad Prism 5对数据进行处理分析。实施例结果参见表1。
表1
Figure PCTCN2016072129-appb-000116
Figure PCTCN2016072129-appb-000117
测试例2：针对HER2靶标的本发明抗体药物偶联物对肿瘤细胞的体外增殖抑制测试
本实验的目的是为了检测本发明针对HER2靶标的抗体药物偶联物，对SK-BR-3肿瘤细胞(人乳腺癌细胞，ATCC，货号HTB‐30)体外增殖的抑制活性。以不同浓度的化合物体外处理细胞，经76小时培养后，采用CCK-8(Cell Counting Kit-8，Dojindo，货号：CK04)试剂对细胞的增值进行检测，根据IC50值评价该化合物的体外活性。
按照测试例1的测试方法，测试细胞为SK-BR-3，细胞培养液为含10％FBS的McCoy's 5A培养基(Gibco，货号16600‐108)。对相关化合物进行测试，得到结果见表2:
表2
Figure PCTCN2016072129-appb-000118
Figure PCTCN2016072129-appb-000119
结论：本发明针对HER2靶标的抗体药物偶联物对SK-BR-3细胞具有明显的增殖抑制活性。
测试例3：针对EGFR靶标的本发明抗体药物偶联物对肿瘤细胞的体外增殖抑制测试
本实验的目的是为了检测本发明针对EGFR靶标的抗体药物偶联物，对HCC827肿瘤细胞(非小细胞肺癌细胞，中科院细胞库，货号#TCHu153)体外增殖的抑制活性。以不同浓度的化合物体外处理细胞，经76小时培养后，采用CCK-8(Cell Counting Kit-8，Dojindo，货号：CK04)试剂对细胞的增值进行检测，根据IC50值评价该化合物的体外活性。
按照测试例1的测试方法，测试细胞为HCC827，细胞培养液为RPMI1640培养基(Invitrogen)，10％(v/v)灭活的胎牛血清。对相关化合物进行测试，得到结果见表3:
表3
Figure PCTCN2016072129-appb-000120
结论：本发明针对EGFR靶标的抗体药物偶联物对HCC827细胞具有明显的增殖抑制活性。
测试例4：NCI-N87抑瘤率实验
一、试验目的
评价并比较本发明抗体细胞毒素偶联物对NCI-N87细胞(HER2过表达的人胃癌细胞，ATCC，CRL-5822)裸小鼠移植瘤的疗效。
二、受试药物
本发明样品：化合物18，化合物21，化合物36。
阳性对照：Pertuzumab帕妥珠单抗，Trastuzumab曲妥珠单抗。
配制方法：均用生理盐水配制。
三.试验动物
BALB/cA-nude裸小鼠，6-7周，雌性，购自上海斯莱克实验动物有限责任公司。合格证号：SCXK(沪)2012-0002。饲养环境：SPF级。
四、试验步骤
裸小鼠皮下接种人胃癌NCI-N87细胞，待肿瘤生长至100-200mm3后，将动物随机分组(D0)。给药剂量和给药方案见表4。每周测2-3次瘤体积，称鼠重，记录数据。肿瘤体积(V)计算公式为：
V＝1/2×a×b2
其中：a、b分别表示长、宽。
T/C(％)＝(T-T0)/(C-C0)×100％其中T、C为实验结束时的肿瘤体积；T0、C0为实验开始时的肿瘤体积。
表4.化合物(18、21、36)对人胃癌NCI-N87裸小鼠移植瘤的疗效
Figure PCTCN2016072129-appb-000121
D0:第一次给药时间；Pertuzumab和Trastuzumab:首剂加倍；P值指与溶剂对照组相比；采用Student’s t检验。实验开始时小鼠数目：对照组n＝12，治疗组n＝6。
五、试验结果
本发明典型化合物显著抑制HER2高表达胃癌NCI-N87裸小鼠皮下移植瘤的生长，并且荷瘤小鼠对以上药物均能较好耐受。
测试例5：EGFRV3抗体及包含其的ADC对HCC827移植瘤裸小
鼠疗效测试
一、试验目的
本实验以Nude-nude裸小鼠为受试动物，评价本发明ADC化合物39、40多剂量腹腔注射给药后，对人非小细胞肺癌HCC827移植瘤裸小鼠的疗效。
本次实验结果显示，发明ADC化合物39、40多剂腹腔注射给药一次后观察抑瘤效果，并给药后第38天时结束实验，ADC化合物39(0.05mg/mouse)的抑瘤率为62.78％，与空白组相比差异有统计学意义(p<0.05)；ADC化合物40(0.025mg/mouse)的抑瘤率为49.20％，与空白组相比差异不显著；ADC化合物40(0.05mg/mouse)的抑瘤率为86.8％，与空白组相比有显著差异(p<0.01)；ADC化合物40(0.1mg/mouse)的抑瘤率为93.41％；与空白组相比差异有统计学意义(p<0.05)
二、受试药物及材料
1、受试药物
本发明ADC化合物：ADC化合物39，ADC化合物40，
2、配制方法：均用PBS稀释配制。
3、试验动物
Nude-nude裸小鼠，SPF，16-20g，♀，购自上海西普尔·必凯实验动物有限责任公司。合格证号：SCXK(沪)2008－0016。
三、试验方法
裸小鼠实验室环境适应三天后，进行肿瘤细胞移植。在裸小鼠右肋部皮下接种HCC827细胞(4×106+50％matrigel/mouse)，接种后第21天，肿瘤长至209.41±25.93mm3(d1)开始给药。给药剂量及方法具体见表5。
每周测定2次移植瘤体积，称量裸小鼠体重并记录数据。
使用Excel统计软件：平均值以avg计算；SD值以STDEV计算；SEM值以STDEV/SQRT计算；组间差异P值以TTEST计算。
肿瘤体积(V)计算公式为：V＝1/2×L长×L短 2
相对体积(RTV)＝VT/V0
抑瘤率(％)＝(CRTV-TRTV)/CRTV(％)
其中V0、VT分别为实验开始时及实验结束时的肿瘤体积。CRTV、TRTV分别为实验结束时的空白对照组(Blank)及实验组的相对肿瘤体积。
表5.受试抗体对HCC827裸小鼠移植瘤的疗效
Figure PCTCN2016072129-appb-000122
四、试验结果
本次实验结果显示抑瘤效果至第38天，ADC化合物39(0.05mg/mouse)的抑瘤率为62.78％，与空白组相比差异有统计学意义(p<0.05)；ADC化合物40(0.025mg/mouse)的抑瘤率为49.20％，与空白组相比差异无统计学意义(p＞0.05)；ADC化合物40(0.05mg/mouse)的抑瘤率为86.8％，ADC化合物40(0.1mg/mouse) 的抑瘤率为93.41％，与空白组相比均有显著差异(p<0.01)；此外，ADC化合物40三个不同剂量组的抑瘤效果具剂量依赖关系。

Claims (29)

  1. 一种通式(PC-L-Dr)所示的化合物或其药学上可接受的盐或溶剂化合物：
    Figure PCTCN2016072129-appb-100001
    其中：
    R，R2-R7选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
    R8-R11中至少一个选自卤素、烯基、烷基和环烷基，其余为氢原子；
    或者R8-R11之中的任意两个形成环烷基，余下的两个基团任选自氢原子、烷基和环烷基；
    R12-R13选自氢原子、烷基或卤素；
    R14选自芳基或杂芳基，所述的芳基或杂芳基任选进一步被选自氢原子、卤素、羟基、烷基、烷氧基和环烷基的取代基所取代；
    y为1-8，优选2-5；
    PC为配体；L为接头单元。
  2. 根据权利要求1所述的通式(PC-L-Dr)所示的化合物或其药学上可接受的盐或溶剂化合物，其为通式(PC-L-D)所示的化合物或其药学上可接受的盐或溶剂化合物：
    Figure PCTCN2016072129-appb-100002
    其中，R2‐R14定义如权利要求1中所述。
  3. 根据权利要求1所述的通式(PC-L-Dr)所示的化合物或其药学上可接受的盐或溶剂化合物，其为通式(PC-L’-Dr)所示的化合物或其药学上可接受的盐或溶剂化合物：
    Figure PCTCN2016072129-appb-100003
    其中：
    R15选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
    R16选自烷基、环烷基、烷氧基和杂环基；
    n为2-6，优选2-5；
    m为0-5，优选1-3；
    PC，y，n，R，R2-R14如权利要求1中所定义。
  4. 根据权利要求1所述的通式(PC-L-Dr)所示的化合物或其药学上可接受的盐或溶剂化合物，其为通式(PC-L’-D)所示的化合物或其药学上可接受的盐或溶剂化合物：
    Figure PCTCN2016072129-appb-100004
    其中：
    R15，R16，m如权利要求3中所定义；
    PC，y，n，R2-R14如权利要求1中所定义。
  5. 根据权利要求2所述的通式(PC-L’-D)所示的化合物或其药学上可接受的盐或溶剂化合物，其为通式(PC-L’-D1)所示的化合物：
    Figure PCTCN2016072129-appb-100005
    其中PC，y，n，m，R2-R16如权利要求2中所定义。
  6. 根据权利要求1至5任一项所述的通式(PC-L-Dr)所示的化合物或其药学上可接受的盐或溶剂化合物，其选自：
    Figure PCTCN2016072129-appb-100006
    其中PC，y如权利要求1所定义。
  7. 如下所述的配体药物偶联物或其药学上可接受的盐或溶剂化合物，其选自：
    Figure PCTCN2016072129-appb-100007
    其中PC、y如权利要求1所述。
  8. 根据权利要求1至7中任一所述的通式(PC‐L‐Dr)所示的化合物或其药学上可接受的盐或溶剂化合物，其中PC为抗体，优选自帕妥珠单抗(Pertuzumab)、尼妥珠单抗(Nimotuzumab)和曲妥珠单抗(Trastuzumab)。
  9. 根据权利要求1至8任一项所述的通式(PC-L-Dr)所示的化合物或其药学上可接受的盐或溶剂化合物，其选自：
    Figure PCTCN2016072129-appb-100008
    其中y如权利要求1所述。
  10. 如下所述的配体药物偶联物或其药学上可接受的盐或溶剂化合物，其选自：
    Figure PCTCN2016072129-appb-100009
    Figure PCTCN2016072129-appb-100010
    Figure PCTCN2016072129-appb-100011
    其中y如权利要求1所述。
  11. 一种通式(Dr)所示的化合物：
    Figure PCTCN2016072129-appb-100012
    或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其中：
    R，R1-R7选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
    R8-R11中至少一个选自卤素、烯基、烷基和环烷基，其余为氢原子；
    或者R8-R11之中的任意两个形成环烷基，余下的两个基团任选自氢原子、烷基和环烷基；
    R14选自芳基或杂芳基，所述的芳基或杂芳基任选进一步被选自氢原子、卤素、羟基、烷基、烷氧基和环烷基的取代基所取代；
    R12-R13选自氢原子、烷基或卤素。
  12. 根据权利要求11所述的通式(Dr)所示的化合物，其为通式(D1)所示的化合物：
    Figure PCTCN2016072129-appb-100013
    或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其中：R1‐R14如权利要求11中所定义。
  13. 根据权利要求11所述的通式(Dr)所示的化合物，其为通式(D1)所示的化合物：
    Figure PCTCN2016072129-appb-100014
    或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其中：R1‐R14如权利要求11中所定义。
  14. 根据权利要求11至13任一项所述的通式(Dr)所示的化合物，其选自：
    Figure PCTCN2016072129-appb-100015
    Figure PCTCN2016072129-appb-100016
  15. 如下所示的化合物，其选自：
    Figure PCTCN2016072129-appb-100017
  16. 一种通式(L1‐Dr)所示的化合物：
    Figure PCTCN2016072129-appb-100018
    其中
    n为2-6，优选2-5；
    R，R2-R7选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
    R8-R11中至少一个选自卤素、烯基、烷基和环烷基，其余为氢原子；
    或者R8-R11之中的任意两个形成环烷基，余下的两个基团任选自氢原子、烷基和环烷基；
    R12-R13选自氢原子、烷基或卤素；
    R14选自芳基或杂芳基，所述的芳基或杂芳基任选进一步被选自氢原子、卤素、羟基、烷基、烷氧基和环烷基的取代基所取代。
  17. 根据权利要求16所述的通式(L1‐Dr)所示的化合物，其为通式(L1‐D)所示 的化合物：
    Figure PCTCN2016072129-appb-100019
    其中n，R2‐R14如权利要求16中所定义。
  18. 根据权利要求16或17所述的通式(L1-D)所示的化合物，其选自：
    Figure PCTCN2016072129-appb-100020
  19. 制备如权利要求10所述的配体药物偶联物的中间体化合物，其选自：
    Figure PCTCN2016072129-appb-100021
  20. 一种通式(PC‐L2)所示的化合物：
    Figure PCTCN2016072129-appb-100022
    其中
    PC配体，优选为抗体，更优选为帕妥珠单抗(Pertuzumab)、尼妥珠单抗(Nimotuzumab)或曲妥珠单抗(Trastuzumab)；
    R15选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
    R16选自烷基、环烷基和杂环基；
    m为0-5，优选1-3；
    X为0‐5，优选1‐3。
  21. 根据权利要求20所述的通式(PC-L2)所示的化合物，其选自：
    Figure PCTCN2016072129-appb-100023
  22. 一种制备如通式(PC-L2)所示的化合物的方法，其包括如下步骤：
    Figure PCTCN2016072129-appb-100024
    1)向PC与通式(PC-L2-A)化合物中加入还原剂RA，进行反应，得到通式(PC-L2-B)化合物；RA优选为氰基硼氢化钠或三乙酰氧基硼氢化钠；
    2)向通式(PC-L2-B)化合物中加入脱保护剂脱去巯基保护基，得到通式(PC-L2)化合物；
    T选自H、叔丁基、乙酰基、正丙酰基、异丙酰基、三苯基甲基、甲氧基甲基、2-(三甲硅烷基)乙氧甲基，优选为H或乙酰基；
    其中PC，R15，R16，m，x如权利要求20中所定义。
  23. 一种制备根据权利要求4所述的通式(PC-L’-D)所示的化合物的方法，该方法包括：
    Figure PCTCN2016072129-appb-100025
    通式(PC-L2)化合物与通式(L1-D1)化合物在反应，得到通式(PC-L’-D)化合物；
    其中：PC，m，n，y，R2～R16如权利要求3中所定义。
  24. 一种通式(D‐Aa)所示的化合物：
    Figure PCTCN2016072129-appb-100026
    或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其中：
    R8-R11之中的任意两个形成环烷基，余下的两个基团任选自氢原子、烷基和环烷基；
    R12选自氢原子、烷基或卤素；
    P为氢原子或保护基，所述的保护基优选Boc，Bn，Cbz，最优选Boc；
    Ra选自羟基，氨基，烷氧基，环烷氧基或烷氨基。
  25. 根据权利要求24所述的通式(D‐Aa)所示的化合物，其为通式(D‐A)所示的化合物：
    Figure PCTCN2016072129-appb-100027
    或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其中：
    P，R8-R12，如权利要求24中所定义；
    R’选自氢原子，烷基，环烷基。
  26. 根据权利要求24所述的通式(D‐A)所示的化合物，为如下所示的化合物：
    Figure PCTCN2016072129-appb-100028
  27. 一种药物组合物，其含有治疗有效量的根据权利要求1-10任意一项所述的配体-细胞毒性药物偶联物，或根据权利要求11-19中所述药物、其可药用盐或溶剂合物，以及药学上可接受的载体、稀释剂或赋形剂。
  28. 根据权利要求1-10中任意一项所述的配体-细胞毒性药物偶联物，或根据权利要求11-19中任意一项所述的药物或其可药用盐或溶剂合物，或根据权利要求27所述的药物组合物在制备治疗哺乳动物癌症的药物的用途，所述的癌症为与HER2，HER3，EGFR表达相关的癌症。
  29. 根据权利要求1‐10中任意一项所述的配体‐细胞毒性药物偶联物，或根据权利要求11‐19中任意一项所述的药物或其可药用盐或溶剂合物，或根据权利要求27所述的药物组合物在制备治疗哺乳动物癌症的药物的用途，其中所述哺乳动物为人，所述癌症选自乳腺癌、卵巢癌、胃癌、子宫内膜癌、唾液腺癌、肺癌、结肠癌、肾癌、直肠癌、甲状腺癌、胰腺癌、前列腺癌、膀胱癌、急性淋巴细胞白血病、急性髓细胞白血病、急性早幼粒细胞白血病、慢性髓细胞白血病、慢性淋巴细胞白血病、霍奇金淋巴瘤、非霍奇金淋巴瘤或复发性间变性大细胞淋巴瘤，优选乳腺癌症、霍奇金淋巴瘤或复发性间变性大细胞淋巴瘤；更优选为乳腺癌。
PCT/CN2016/072129 2015-02-15 2016-01-26 配体-细胞毒性药物偶联物、其制备方法及其应用 WO2016127790A1 (zh)

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WO2020200196A1 (zh) 2019-04-01 2020-10-08 江苏恒瑞医药股份有限公司 抗Claudin18.2抗体及其应用
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WO2020200196A1 (zh) 2019-04-01 2020-10-08 江苏恒瑞医药股份有限公司 抗Claudin18.2抗体及其应用
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WO2022078425A1 (zh) 2020-10-14 2022-04-21 江苏恒瑞医药股份有限公司 抗her3抗体和抗her3抗体药物偶联物及其医药用途
WO2023000131A1 (zh) 2021-07-19 2023-01-26 烟台迈百瑞国际生物医药股份有限公司 一种负载双毒素的抗体药物偶联物及其应用
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