WO2016197946A1 - 抗体药物偶联物、中间体、制备方法、药物组合物及应用 - Google Patents

抗体药物偶联物、中间体、制备方法、药物组合物及应用 Download PDF

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WO2016197946A1
WO2016197946A1 PCT/CN2016/085275 CN2016085275W WO2016197946A1 WO 2016197946 A1 WO2016197946 A1 WO 2016197946A1 CN 2016085275 W CN2016085275 W CN 2016085275W WO 2016197946 A1 WO2016197946 A1 WO 2016197946A1
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group
substituted
mmol
unsubstituted
etoac
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French (fr)
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向少云
刘世峰
杨鸿裕
马兴泉
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凯惠科技发展(上海)有限公司
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Priority to EP16806844.3A priority Critical patent/EP3308801A4/en
Priority to JP2018516616A priority patent/JP6802840B2/ja
Priority to US15/580,895 priority patent/US10449258B2/en
Publication of WO2016197946A1 publication Critical patent/WO2016197946A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5365—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/537—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines spiro-condensed or forming part of bridged ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/44—Antibodies bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68033—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a maytansine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
    • C07D498/18—Bridged systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes

Definitions

  • the invention particularly relates to antibody drug conjugates, intermediates (toxins with linkers), methods of preparation, pharmaceutical compositions and uses.
  • a typical antibody drug conjugate comprises a monoclonal antibody that binds to a cancer cell surface specific antigen.
  • These antibodies include proteins on the surface of immune system B cells and T cells, such as CD20, CD22, and human epidermal growth factor receptor 2 (Her2) and prostate specific membrane antigen (PSMA). These specific antibodies are linked to highly toxic drugs via a cleavable linker. Such drugs are designed to cause cancer cell death by inducing irreversible DNA damage or interfering with cell division.
  • ADCs antibody drug conjugates
  • the first antibody drug conjugate approved by the FDA is Melota (Gentuzumab/Ozomib) developed by Wyeth in the United States for the treatment of acute myeloid leukemia in 2000, but due to some adverse effects As well as security considerations, the drug was withdrawn 10 years after its launch. Since then, the technology for attaching antibodies to drugs has made great progress. Using a linker with a suitable biological half-life, the researchers were able to ensure that the antibody drug conjugate reached the target cell and reduced its side effects.
  • Kadcyla (trastuzumab emtansine, T-DM1) was approved for the treatment of metastatic breast cancer.
  • Kadcyla combines the antibody Herceptin, which treats breast cancer, with the toxin, which is licensed by ImmunoGen. The drug's 2015 sales were nearly $700 million.
  • Antibody drug conjugates are likely to be the next blockbuster drug to bring a sustainable source of income.
  • the synergy between biomedicine and clinical trials has led to the continued success of the antibody drug conjugate technology platform.
  • These common results indicate the use of humanized monoclonal antibody technology as a target-directed therapeutic administration effort.
  • most of the antibody drug conjugates in development have used the aristatins of Seattle Genetics or the maytansinoid of ImmunoGen as effective toxins.
  • companies such as Spirogen are also developing other toxins for the next generation of antibody drug conjugates, such as PBD.
  • Maytansinoid alkaloids were first isolated from East African shrubs, F. sylvestris (US 389611, JP 01006282, JP 63233986), and are a class of highly cytotoxic compounds as anticancer agents. Many of the maytansinoid derivatives prepared by people have high anticancer activity, which is 100 to 1000 times that of the traditional anticancer drug vinblastine or paclitaxel. (J. Med. Chem., 1978, 21, 31-37, Chem. Pharma. Bull., 1984, 3441-3451, J. Med. Chem. 2006, 49, 4392-4408, Chem. Pharm. Bull. 2004 , 52(1) 1-26, WO2011039721, WO2012/061590, WO2014/094453).
  • the maytansinoid analogs DM1 and DM4 are reported by Chari, R. V. J. et al. in the patents US Pat. No. 5,208,020, US Pat. No. 5,416,064 and J. Med. Chem, 2006, 49, 4392-4408.
  • Patents WO2004/103272, EP0425235 report conjugates of maytansin analogs DM1 and DM4 and their anticancer activity.
  • a typical antibody drug conjugate consists of a drug, a linker unit and an antibody.
  • the choice of specific antibodies and drugs depends on the particular disease and has a significant impact on the safety and efficacy of the conjugate.
  • the stability of the ligation unit and the method of coupling to the antibody play a decisive role in the development of the ADC drug.
  • Factors that determine the efficacy of antibody drug conjugates include the stability of the linker unit and its cleavage sensitivity, cell surface internalization, transport, and release of cytotoxin.
  • T-DM1 which readily degrades and releases toxins before endocytosis of antigen binding protein (Abu), causes side effects.
  • the ratio of the drug/antibody obtained by coupling with the antibody is low and the distribution changes are relatively large, and it is difficult to accurately control the efficacy and safety (WO2012/061590A1, WO201139721). Therefore, it is an urgent technical problem to find an antibody drug conjugate with high stability, good water solubility and high cytotoxin release efficiency, specificity, high cytotoxicity and good anticancer effect.
  • the technical problem to be solved by the present invention is to overcome the poor stability, low cleavage sensitivity of the antibody drug conjugate linking unit in the prior art, low cell cell surface internalization, transport and cytotoxic release efficiency, poor cytotoxicity, specificity
  • the invention relates to an antibody drug conjugate, an intermediate (toxin toxin with a linker), a preparation method, a pharmaceutical composition and application thereof.
  • the antibody drug conjugate of the invention has very high cytotoxicity, good anticancer effect and good market application prospect.
  • the present invention provides an antibody drug conjugate as shown in Formula IB,
  • Z is a nitrogen atom, Or an oxygen atom, when Z is an oxygen atom, R 1 does not exist; when Z 1 is Or a nitrogen atom, R 1 is hydrogen or a C 1 -C 4 alkyl group (the C 1 -C 4 alkyl group such as methyl, ethyl, propyl, butyl, isopropyl, isobutyl) Or a tert-butyl group, preferably a methyl group) or R 1 , Z and a C atom to which they are attached and R 6 or R 7 are bonded to form a 4-6 membered cyclic structure (preferably a six membered ring structure, said six membered ring) Shape structure );
  • R 2 is hydrogen or a halogen-substituted C 1 -C 4 alkyl group (the halogen described in the halogen-substituted C 1 -C 4 alkyl group may be fluorine, chlorine or bromine, the halogen substituted
  • the C 1 -C 4 alkyl group may be a halogen-substituted methyl group, a halogen-substituted ethyl group, a halogen-substituted propyl group, a halogen-substituted isopropyl group, a halogen-substituted butyl group, a halogen-substituted isobutyl group or a halogen substituent.
  • Tert-butyl group preferably halogen-substituted methyl group, said halogen-substituted methyl group preferred );
  • p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (preferably 0, 1 or 2);
  • n 0 or 1
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (preferably 0, 1 or 2);
  • Y1 is an oxygen atom, a chemical bond (preferably a single bond) or Wherein x2 is an integer from 1 to 24 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10);
  • q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (preferably 0, 1 or 2);
  • R 3 and R 4 are independently hydrogen, cyano or a substituted or unsubstituted C 1 -C 4 alkyl group (the unsubstituted C 1 -C 4 alkyl group such as methyl, ethyl, propyl, Butyl, isopropyl, isobutyl or tert-butyl, preferably methyl or isopropyl; substituted C 1 -C 4 alkyl such as substituted methyl, substituted ethyl, substituted C a substituted butyl group, a substituted isopropyl group, a substituted isobutyl group or a substituted tert-butyl group, preferably a substituted methyl group, said substituted methyl group being preferred
  • the substitution in the substituted or unsubstituted C 1 -C 4 alkyl group described in the above R 3 or R 4 means a C 1 -C 4 alkoxy group (the C 1 - Alkoxy groups of C 4 such
  • x is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (preferably 0, 1 or 2);
  • R 8 is a substituted or unsubstituted C 1 -C 4 alkyl group (the substituted or unsubstituted C 1 -C 4 alkyl group such as a substituted or unsubstituted methyl group, a substituted or unsubstituted ethyl group) , substituted or unsubstituted propyl, substituted or unsubstituted isopropyl, substituted or unsubstituted butyl, substituted or unsubstituted isobutyl or substituted or unsubstituted tert-butyl, preferably substituted or unsubstituted a propyl group, the substituted propyl group is preferred ), Y is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; y1 is 0 or 1; in the substituted or unsubstituted C 1 -C 4 alkyl group described in R 81 Said substitution means Substituted; t is 1 or 2
  • R 6 is hydrogen, a substituted or unsubstituted C 1 -C 12 alkyl group (the substituted or unsubstituted C 1 -C 12 alkyl group is preferably a substituted or unsubstituted C 1 -C 4 alkyl group;
  • the substituted or unsubstituted C 1 -C 4 alkyl group is preferably a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group or a t-butyl group, further preferably a methyl group) or r is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;
  • R 7 is hydrogen, a substituted or unsubstituted C 1 -C 12 alkyl group (the substituted or unsubstituted C 1 -C 12 alkyl group is preferably a substituted or unsubstituted C 1 -C 4 alkyl group;
  • the substituted or unsubstituted C 1 -C 4 alkyl group is preferably a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group or a t-butyl group, more preferably a methyl group, a substituted or unsubstituted C 1 - Alkoxy group of C 12 (the substituted or unsubstituted C 1 -C 12 alkoxy group is preferably a substituted or unsubstituted C 1 -C 4 alkoxy group; said substituted or unsubstituted C 1 -C 4 Alkoxy is preferably methoxy,
  • mAb represents a monoclonal antibody, such as Herceptin;
  • k is any of 1-8 (preferably any of 1-6), and represents the molar ratio (or ratio, DAR for short) of the drug in the brackets to the monoclonal antibody mAb;
  • the compound as shown in Formula IB is not the following compound:
  • Z is an oxygen atom or Y1 is an oxygen atom.
  • R 2 is a halogen-substituted C 1 -C 4 alkyl group; and R 3 and R 4 are independently hydrogen and cyanide; The group may be substituted or unsubstituted with a C 1 -C 4 alkyl group, but R 3 and R 4 are not hydrogen at the same time.
  • the antibody drug conjugate as shown in Formula IB is preferably an antibody drug conjugate as shown in Formula Ib or Ib1,
  • a butyl group an isobutyl group or a tert-butyl group, more preferably a methyl group, a substituted or unsubstituted C 1 -C 12 alkoxy group (the substituted or unsubstituted C 1 -C 12 alkoxy group is preferred) a substituted or unsubstituted C 1 -C 4 alkoxy group; the substituted or unsubstituted C 1 -C 4 alkoxy group is preferably a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group or a butoxy group.
  • mAb represents a monoclonal antibody, such as Herceptin;
  • k is any value from 1-8, indicating the molar ratio of the drug in the brackets to the monoclonal antibody mAb;
  • Y 2 is Where x2 is an integer from 1 to 24; Q2 is T1 is 0, 1, 2, 3, 4, 5 or 6;
  • the antibody drug conjugate of the formula IB is further preferably any of the following compounds:
  • the invention also provides an intermediate (toxin with a linker) as shown in Formula IA:
  • R 51 is H, C 1 -C 4 alkyl (the C 1 -C 4 alkyl group such as methyl, ethyl, propyl, butyl, isopropyl, isobutyl or tert-butyl, Preferred methyl), Or halogen (the halogen is preferably F, Cl, Br or I), y is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and R 9 is H or C 1 -C 4 Alkyl (the C 1 -C 4 alkyl group such as methyl, ethyl, propyl, butyl, isopropyl, isobutyl or tert-butyl).
  • Z is nitrogen, Or an oxygen atom, when Z is an oxygen atom, R 1 does not exist; when Z is Or a nitrogen atom, R 1 is a C 1 -C 4 alkyl group (the C 1 -C 4 alkyl group such as methyl, ethyl, propyl, butyl, isopropyl, isobutyl or tertiary) Butyl, preferably methyl) or R 1 , Z and the C atom to which they are attached and R 6 or R 7 are bonded to form a 4-6 membered cyclic structure (preferably a six membered ring structure, said six membered ring structure) E.g And R 2 is a halogen-substituted C 1 -C 4 alkyl group (the halogen described in the halogen-substituted C 1 -C 4 alkyl group may be fluorine, chlorine or bromine, said "halogen"
  • the C 1 is a C 1 -C 4 alkyl
  • Y 2 is Where x2 is an integer from 1 to 24; Q2 is R 52 is hydrogen or halogen (preferably F, Cl, Br or I).
  • the intermediate (toxin with a linker) as shown in Formula IA is further preferably any of the following compounds:
  • the carbon with * represents a chiral carbon atom or an achiral carbon, and its configuration may be R, S or a racemate.
  • the preparation method of the antibody drug conjugate as shown in Formula IB can be a conventional method and condition for the cross-linking reaction in the art, and can be specifically referred to the literature Gail D. Lewis Phillips, et al., Cancer. Res., 2008, 68, 9280 and Teemu T. Junttila, et al., Breast. Cancer. Res. Treat., 2011, 128, 347, the entire contents of which is hereby incorporated by reference.
  • the organic solvent is preferably one or more of an amide solvent, a sulfoxide solvent, and an ether solvent.
  • the amide solvent is preferably N,N dimethylformamide (DMF) and/or dimethylacetamide (DMA); and the sulfoxide solvent is preferably dimethyl sulfoxide (DMSO).
  • the ether solvent is preferably tetrahydrofuran.
  • the mass to volume ratio of the intermediate (toxin of the linker) IA to the organic solvent is preferably 0.1 mg/mL to 100 mg. /mL.
  • the pH 6-8 may be achieved by a buffer; the buffer solution is usually a low salt buffer, preferably a phosphate buffer solution.
  • a buffer solution of potassium phosphate and potassium dihydrogen phosphate or a buffer solution of boric acid buffer such as boric acid and sodium borate.
  • the temperature of the crosslinking reaction is preferably 4 ° C to 37 ° C (preferably room temperature).
  • the progress of the crosslinking reaction can be monitored by a conventional test method (such as TLC, HPLC or NMR) in the art, generally When the intermediate IA disappears, it is the end point of the reaction.
  • a conventional test method such as TLC, HPLC or NMR
  • the preparation method of the antibody drug conjugate as shown in Formula IB is preferably carried out under the protection of a protective gas, when the preparation method of the antibody drug conjugate as shown in Formula IB is carried out under protective gas protection
  • the protective gas is preferably nitrogen.
  • the method for preparing an antibody drug conjugate as shown in Formula IB preferably comprises the steps of: subjecting a monoclonal antibody (for example, Herceptin) to a buffer solution having a pH of 6-8 (for example, 7.5). The dialysis is carried out, and then the intermediate IA and an organic solvent are added to carry out the crosslinking reaction.
  • a monoclonal antibody for example, Herceptin
  • a buffer solution having a pH of 6-8 for example, 7.5.
  • the present invention also provides a method for preparing the antibody drug conjugate according to the formula Ib or Ib1, which comprises the steps of: an intermediate (with a linker in an organic solvent at a pH of 6-8) The toxin) Ia or the intermediate Ia1 is cross-linked with a monoclonal antibody (for example, Herceptin) to obtain the antibody drug conjugate Ib or Ib1;
  • a monoclonal antibody for example, Herceptin
  • the present invention also provides the use of the antibody drug conjugate as shown in Formula IB and/or the intermediate (linked toxin) of Formula IA for the preparation of a medicament for treating and/or preventing cancer. .
  • the invention also provides the use of one or more of the compounds of formula Ib, Ib1, Ia and Ia1 for the manufacture of a medicament for the treatment and/or prevention of cancer.
  • the cancer may be a cancer conventional in the art, including but not limited to breast cancer, lymphoma, lung cancer, liver cancer, colon cancer, head and neck cancer, bladder cancer, kidney cancer, esophageal cancer, gallbladder cancer.
  • the tumor cells of the cancer include, but are not limited to, Her2 positive human BT474 breast tumor cells, Her2 low expressed human MCF-7 breast tumor cells or human breast tumor MCF7 translocated with Her2 in MCF-7. -Her2 stabilized cell line.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody drug conjugate and/or intermediate IA as shown in Formula IB as previously described, and one or more pharmaceutically acceptable excipients.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody drug conjugate as shown in Formula Ib, an antibody drug conjugate as shown in Formula Ib1, Intermediate Ia and Intermediate Ia1 as previously described One or more, and one or more pharmaceutically acceptable excipients.
  • the pharmaceutically acceptable excipient refers to a conventional pharmaceutical excipient in the pharmaceutical field, and is an antibody drug conjugate of the present invention added to solve the moldability, effectiveness, stability and safety of the preparation.
  • All other conventional pharmaceutical materials such as diluents (such as sodium carboxymethyl starch), binders (such as povidone, etc.), disintegrants (such as microcrystalline cellulose, etc.), lubricants (such as stearic acid) Magnesium, micronized silica gel, etc.), as well as other adjuvants.
  • the above-mentioned excipients may be selected as needed, and the antibody drug conjugate of the present invention is formulated into a pharmaceutical preparation according to a conventional method in the art; the pharmaceutical preparations are various conventional dosage forms in the art, such as tablets, powders, pills, capsules. Agents, granules, oral liquids, dry suspensions or pills.
  • alkyl is a branched or straight-chain saturated aliphatic hydrocarbon group having the specified number of carbon atoms; as defined in the C 1 -C 4 alkyl group, it is included in a linear or branched structure. A group having 1, 2, 3 or 4 carbon atoms.
  • C 1 -C 4 alkyl specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl and the like.
  • alkoxy refers to a group formed by linking an alkyl group to an oxygen atom, that is, RO-, and R is an alkyl group.
  • halogen means fluorine, chlorine, bromine, iodine or hydrazine.
  • TBDPS means a tert-butyldiphenylsilyl group.
  • the reagents and raw materials used in the present invention are commercially available in the present invention, and the room temperature refers to an ambient temperature of 10 ° C to 35 ° C.
  • the positive progress of the present invention is that the present invention designs and synthesizes a series of novel antibody drug conjugates which are structurally stable and stable to acid and peptidase cathepsins.
  • the antibody drug conjugates designed and synthesized by the present invention use a stable ether bond. The connection is made to significantly improve water solubility and stability. Both in vivo and in vitro activity tests have shown that they have higher cytotoxicity, and some of the conjugates have better or nearer cytotoxicity than T-DM1.
  • the series of conjugate synthesis steps are simple, the yield is much higher than T-DM1, and it is also convenient for further structural modification to improve the physicochemical properties and biological activity of the conjugate, and has good market application. prospect.
  • Figure 1 is a graph showing growth inhibition of BT474 by T-CE-040 and T-DM1.
  • Figure 2 is a graph showing growth inhibition of MCF-7 by T-CE-040 and T-DM1.
  • Figure 3 is a graph showing growth inhibition of MCF-7-Her2 by T-CE-040 and T-DM1.
  • Figure 4 is a graph showing growth inhibition of BT474 by T-CE-063 and T-DM1.
  • Figure 5 is a graph showing growth inhibition of MCF-7 by T-CE-063 and T-DM1.
  • Figure 6 is a graph showing growth inhibition of MCF-7-Her2 by T-CE-063 and T-DM1.
  • the crude product 3 (13.2 g, 0.1 mol) from the previous step was dissolved in 100 mL of anhydrous methanol, and thiourea (15.2 g, 0.2 mol) was added.
  • the reaction solution was stirred at room temperature overnight, and the material was completely consumed by a TLC spot plate, and methanol was removed by distillation under reduced pressure. 200 mL of water was added to the residue, and extracted three times with dichloromethane (100 mL ⁇ 3).
  • n-Butyllithium (8.8 mL, 2.5 mol/mL, 22 mmol) was added to a 40 mL dry tetrahydrofuran solution cooled to -78 ° C under N2.
  • Acetonitrile (1.1 mL, 21.2 mmol) was dissolved in 10 mL dry THF.
  • the reaction solution was stirred at -78 ° C for half an hour.
  • Compound 4 (2.96 g, 20 mmol) was dissolved in 20 mL of tetrahydrofuran and added dropwise. After the addition was completed, the reaction solution was gradually warmed to room temperature and stirred for 3 hours. The reaction mixture was cooled to 0 ° C, EtOAc (EtOAc m.
  • reaction product of the previous step 5 (2.8 g, 15 mmol) was dissolved in 10 mL of ethanol, argon was applied three times, and 8M sodium hydroxide solution (4 mL, 32 mmol) was slowly added. The reaction solution was heated to reflux under argon overnight. The reaction solution was cooled to room temperature, diluted with hydrochloric acid (pH 2), and extracted twice with dichloromethane (50 mL ⁇ 3). The crude product was purified using EtOAc EtOAcjjjjjjj LCMS (ESI) m / z 209.1 (M + H) +.
  • N-Methyl-L-alanine (618 mg, 6.0 mmol) was dissolved in 15 mL of ethylene glycol dimethyl ether and 15 mL of water. Triethylamine (1.7 mL g, 12 mmol) was added and stirred vigorously. The reaction product of the previous step 8 (2.1 g, 6 mmol) was dissolved in 15 mL of ethylene glycol dimethyl ether and added dropwise over about 5 minutes.
  • the reaction solution was stirred at room temperature for 2 hours, the organic solvent was removed under reduced pressure, and 10 mL of water was added, and the mixture was adjusted to pH 2 with 1M diluted hydrochloric acid, and extracted three times with ethyl acetate (50 mL ⁇ 3), and the organic phase was combined and washed three times with saturated brine. (50 mL ⁇ 3), dried over anhydrous sodium sulfate and concentrated.
  • the crude product was purified using EtOAc EtOAc EtOAc EtOAc LCMS (ESI) m / z 340.1 (M + H) +.
  • CE-017 (30 mg, 0.034 mmol) was dissolved in 0.5 mL of ethyl acetate.
  • the reaction solution was stirred under a nitrogen atmosphere for 2 hours.
  • the mixture was extracted three times with ethyl acetate (20 mL ⁇ 3).
  • CE-023 The synthesis procedure of CE-023 is consistent with CE-024, using CE-016 as the starting material.
  • CE-024 (18 mg, 0.02 mmol) was dissolved in 2 mL of N,N-dimethylformamide and <RTIgt; The reaction was stirred room temperature for 12 hours, the reaction was filtered and the filtrate directly using preparative HPLC (CH 3 CN in H 2 O-0.05% TFA from 5% to 95%) to give product 13.7mg CE-005, as a white solid. The yield was 65%.
  • triphenylphosphine (12 g, 45.8 mmol) was dissolved in 200 mL of tetrahydrofuran, cooled in an ice water bath, and stirred. Diethyl azodicarboxylate (DEAD) (8.0 g, 45.8 mmol) was added and stirring was continued for 5 minutes.
  • DEAD Diethyl azodicarboxylate
  • Compound 4-2 (8.3 g, 36.2 mmol) in 50 mL of tetrahydrofuran and thioacetic acid (4.13 g, 54.3 mmol) were added. The reaction solution was allowed to warm to room temperature and stirred overnight.
  • the reaction product 5-2 (3.3 g, 11.4 mmol) in the previous step was dissolved in a mixed solution of 60 mL of methanol and 30 mL of water under a nitrogen atmosphere, and potassium carbonate (4 g, 28.9 mmol) was added. The reaction solution was stirred at room temperature for 2 hours under a nitrogen atmosphere. Methyl methylthiosulfonate (3.0 g, 23.8 mmol) was added. The reaction solution was stirred at room temperature for 2 hours under a nitrogen atmosphere.
  • reaction product of the previous step 6-2 160 mg, 0.549 mmol was dissolved in 5 mL of dry methylene chloride, and dicyclohexylcarbodiimide DCC (80 mg, 0.388 mmol) was added and stirred at room temperature for 30 min and filtered.
  • the compound 6-2-P 1 was used as a raw material, and the synthesis procedure of CE-029 was consistent with CE-011.
  • the compound 6-2-P 2 was used as a raw material, and the synthesis procedure of CE-030 was consistent with CE-011.
  • Hexane-1-carboxylic acid succinimide ester (20 mg, 0.06 mmol, SMCC). The reaction solution was stirred at room temperature overnight under a nitrogen atmosphere. Filtration and purification of the filtrate by preparative HPLC afforded 11.5 g of product, product s.
  • 2-Methylallyl alcohol 1-3 (10 g, 139 mmol) and imidazole (18.9 g, 278 mmol) were sequentially dissolved in 200 mL of dichloromethane, cooled to 0 ° C in ice water, and t-butyldiphenylchlorosilane was added portionwise. (57 g, 208 mmol).
  • the reaction solution was allowed to warm to room temperature and stirred for 2 hours. It was quenched by the addition of 100 mL of water and extracted three times with dichloromethane (100 mL ⁇ 3). The organic phase was combined and washed with brine (3 mL)
  • the crude product was purified by silica gel chromatography eluting elut elut elut elut elut
  • n-Butyllithium (2.5 M, 13 mL 32.1 mmol) was added to a solution of 100 mL of dry tetrahydrofuran cooled to -78 ° C under N2.
  • Acetonitrile (1.6 mL, 30.7 mmol) was dissolved in 10 mL dry tetrahydrofurane.
  • the reaction solution was stirred at -78 ° C for half an hour.
  • Compound 4-3 (10 g, 29.2 mmol) was dissolved in 20 mL of tetrahydrofuran and added dropwise. After the addition was completed, the reaction solution was gradually warmed to room temperature and stirred for 3 hours.
  • the reaction solution was cooled to 0 ° C, and the reaction was quenched slowly with dilute hydrochloric acid (0.5 M, 20 mL), and extracted three times with ethyl acetate (100 mL ⁇ 3). The organic phase was combined and washed three times with saturated brine (100 mL ⁇ 3) Dry over anhydrous sodium sulfate and concentrate.
  • the crude product was purified using silica gel chromatography eluting elut elut elut elut elut LCMS (ESI) m/z 384.2 (M+H) +
  • reaction product 5-3 (3 g, 7.8 mmol) from the previous step was dissolved in dichloromethane (30 mL), and methyl methylthiosulfonate (1.2 mL, 11.7 mmol) and triethylamine (1.6 mL, 11.7 mmol).
  • the reaction solution was stirred at room temperature overnight under a nitrogen atmosphere.
  • 1 mL of dilute hydrochloric acid 30 mL and 100 mL of dichloromethane were added, and the organic layer was separated, dried over anhydrous sodium sulfate and evaporated.
  • the crude product was purified by EtOAc EtOAcjjjjjjjjjj
  • reaction product 6-3 (3.2 g, 7.45 mmol) was dissolved in 30 mL of tetrahydrofuran, and then tetrabutylammonium fluoride (22 mL, 22 mmol, 1M in THF) was added. The reaction was stirred at room temperature overnight. The reaction was quenched by the dropwise addition of 1N EtOAc (3 mL). The crude product was purified by EtOAcjjjjjjjjjjjjj
  • reaction product 7-3 (1.18 g, 6.17 mmol) in the previous step was dissolved in 100 mL of dichloromethane, and trimethoxysulfonium tetrafluoroborate (1.83 g, 12.35 mmol) and 1,8-bismethylaminonaphthalene ( 2.7 g, 12.35 mmol).
  • the reaction was stirred at room temperature overnight. Filtration, washing with 30 mL of dichloromethane, and the filtrate was washed with 1M EtOAc EtOAc.
  • the crude product was purified by EtOAc EtOAcjjjjjjjjjjj
  • reaction product 9-3 (700 mg, 3.9 mmol) from the previous step was dissolved in dichloromethane (10 mL) and methyl methylthiosulfonate (0.5 mL, 14mmoL) and triethylamine (0.6mL, 4.3mmol). The reaction solution was stirred at room temperature overnight under a nitrogen atmosphere. After adding 1 mL of dilute hydrochloric acid (15 mL) and 50 mL of methylene chloride, the organic layer was separated, dried over anhydrous sodium sulfate, and evaporated. LCMS (ESI) m / z 225.1 (M + H) +.
  • reaction product 10-3 (1.2 g, 5.35 mmol) and HOSu (700 mg, 6.08 mmol) were dissolved in 20 mL of dichloromethane, and then EDC-HCl (1.2 g, 6.28 mmol) was added.
  • the reaction solution was stirred at room temperature for 2 hours. After adding 30 mL of water and ethyl acetate, the mixture was extracted three times (50 mL ⁇ 3).
  • the crude product was purified by silica gel chromatography eluting elut elut elut elut elut elut elut LCMS (ESI) m / z 339.0 (M + H) +.
  • N-methyl-L-alanine (560 mg, 5.28 mmol) was dissolved in 20 mL of ethylene glycol dimethyl ether and 20 mL of water. Triethylamine (1.5 mL, 10.56 mmol) was added and stirred vigorously. The reaction product of the previous step, 12-3 (1.7 g, 5.29 mmol), was dissolved in 20 mL of ethylene glycol dimethyl ether and added dropwise over about 5 minutes.
  • the reaction solution was stirred at room temperature for 3 hours, the organic solvent was removed under reduced pressure, 10 mL of water was added, and the mixture was adjusted to pH 3 with 1M diluted hydrochloric acid, and extracted three times with ethyl acetate (50 mL ⁇ 3), and the organic phase was combined and washed three times with saturated brine. (50 mL ⁇ 3), dried over anhydrous sodium sulfate and concentrated.
  • the crude product was purified by silica gel chromatography eluting elute LCMS (ESI) m / z310.0 ( M + H) +.
  • reaction product of the previous step 12-3 (360 mg, 1.165 mmol) was dissolved in 10 mL of methanol, and 0.5 mL of thionyl chloride was slowly dropped dropwise. The reaction solution was stirred at room temperature for 2 hr and then evaporated. 50 mL of ethyl acetate was added, and 20 mL of a saturated sodium hydrogencarbonate solution was added slowly, the organic phase was separated, dried over anhydrous sodium sulfate and evaporated.
  • CE-014, CE-015 synthesis steps are the same as CE-012, CE-013, using 14-P 2 as raw material.
  • CE-015 (40 mg, 0.047 mmol) was dissolved in 0.6 mL of ethyl acetate.
  • Dithiothreitol DTT (18 mg, 0.12 mmol) was dissolved in 1.2 mL of methanol, and a potassium phosphate buffer solution of pH 7.5 was added. mL.
  • the reaction solution was stirred under a nitrogen atmosphere for 2 hours.
  • Ethyl acetate was extracted three times (20 mL ⁇ 3), and the organic phase was combined and washed twice with saturated brine (20 mL ⁇ 3), dried over anhydrous sodium sulfate and evaporated.
  • CE-032 (30 mg, 0.037 mmol) was dissolved in 4 mL of N,N-dimethylformamide and EtOAc (24 mg, 0.074 mmol). The reaction solution was stirred overnight at room temperature, filtered and the filtrate directly using preparative HPLC (CH 3 CN in H 2 O-0.05% TFA from 5% to 95%) to give product 20.4mg CE-004, as a white solid. The yield was 54%. LCMS (ESI) m / z 1024.2 (M + H) +.
  • n-Butyllithium (2.5 M, 17.6 mL, 43.2 mmol) was added to a solution of 150 mL of dry tetrahydrofuran cooled to -78.
  • Acetonitrile (1.8 g, 44.8 mmol) was dissolved in 10 mL of dry tetrahydrofuran.
  • the reaction solution was stirred at -78 ° C for half an hour.
  • Compound 2-5 (4 g, 39.2 mmol) was dissolved in 20 mL of tetrahydrofuran and added dropwise. After the addition was completed, the reaction solution was gradually warmed to room temperature and stirred for 3 hours.
  • reaction product of the previous step 3-5 (2.86 g, 20 mmol) was dissolved in 20 mL of ethanol, argon was applied three times, and 8 M sodium hydroxide solution (10 mL, 80 mmol) was slowly added. The reaction solution was heated to reflux under argon overnight. The reaction mixture was cooled to room temperature, diluted with EtOAc EtOAc (EtOAc)EtOAc. The crude product was purified using EtOAc EtOAc EtOAc (EtOAc: LCMS (ESI) m / z163.1 ( M + H) +.
  • reaction product 4-5 (810 mg, 5 mmol) from the previous step was dissolved in 15 mL of dichloromethane under argon, and methyl methylthiosulfonate (0.65 mL, 18mmoL) and triethylamine (0.8 mL) were added. The reaction solution was stirred at room temperature under argon overnight. Add 1M of dilute hydrochloric acid 40 mL, and extract with ethyl acetate three times (50 mL ⁇ 3). The organic phase is combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate. One step reaction. LCMS (ESI) m / z 209.1 (M + H) +.
  • reaction product 5-5 (730 mg, 3.5 mmol) and HOSu (445 mg, 3.85 mmol) in the previous step were dissolved in 20 mL of methylene chloride, and EDC (800 mg, 4.2 mmol) was added.
  • the reaction solution was stirred at room temperature for 2 hours.
  • 30 mL of water and 50 mL of ethyl acetate were added, and the organic phase was separated, washed with saturated brine (3 mL) (50 mL ⁇ 3), dried over anhydrous sodium sulfate.
  • LCMS m / z 306.1 (M + H) +.
  • N-methyl-L-alanine (190 mg, 1.8 mmol) was dissolved in 10 mL of ethylene glycol dimethyl ether and 10 mL of water. Triethylamine (0.5 mL, 3.6 mmol) was added and stirred vigorously. The reaction product 6-5 (550 mg, 1.8 mmol) from the previous step was dissolved in 5 mL of ethylene glycol dimethyl ether and added dropwise over about 5 minutes.
  • the reaction solution was stirred at room temperature for 3 hours, the organic solvent was removed under reduced pressure, 10 mL of water was added, and the mixture was adjusted to pH 3 with 1M diluted hydrochloric acid, and extracted three times with ethyl acetate (3 ⁇ 50 mL), and the organic phase was combined and washed three times with saturated brine. (50 mL ⁇ 3), dried over anhydrous sodium sulfate and concentrated.
  • the crude product was purified by silica gel chromatography eluting elut elut elut elut elut eluting LCMS (ESI) m / z293.1 ( M + H) +.
  • the intermediate CE-017 was replaced with CE-022, and the subsequent reaction was carried out to obtain the compound CE-002.
  • N-hydroxysuccinimide (1.5 g, 12.8 mmol) was dissolved in 15 mL of N,N-dimethylformamide, cooled to 0 ° C, trifluoroacetic anhydride (1.8 mL, 12.8 mmol) was added and stirred 10 minute.
  • reaction product 2-6 (1.0 g, 3.2 mmol) in the previous step was dissolved in 15 mL of N,N-dimethylformamide, cooled to 0 ° C, stirred, and 2,4,6-trimethylpyridine (774.4 mg, 6.4 mmol), the above reaction solution was added. Stir to room temperature and stir overnight. The reaction was quenched by the addition of 1N EtOAc (EtOAc)EtOAc.
  • EtOAc 1N EtOAc
  • the crude product was purified by preparative HPLC (CH 3 CN in H 2 O-0.05% TFA from 5% to 95%) to give 752mg purified product 3-6 as a white solid, 80% yield.
  • reaction product 3-6 700 mg, 2.4 mmol was dissolved in 10 mL of dichloromethane and 3 mL of trifluoroacetic acid. The reaction mixture was stirred at room temperature for 4 hr. LCMS (ESI) m / z 195.2 (M + H) +.
  • reaction product 4-6 (300 mg, 1.6 mmol) was dissolved in 40 mL of acetonitrile, and triethylamine (315 mg, 3.2 mmol) and N,N'-disuccinimidyl carbonate (798 mg, 3.2 mmol). The reaction was stirred at room temperature overnight. Concentrated and the crude product purified by preparative HPLC (CH 3 CN in H 2 O-0.05% TFA from 5% to 95%) to give 188mg purified product 5-6 as a white solid, yield 35%. LCMS (ESI) m / z 336.2 (M + H) +.
  • DM1 N2'-deacetyl-N2'-(3-indolyl-1-oxopropyl) maytansin
  • reaction product 2-7 (19.6 g, 0.1 mol) and triethylamine (16.6 mL, 0.12 mol) in the previous step were dissolved in 150 mL of dichloromethane. Acetone was cooled to 0 ° C and methanesulfonyl chloride (8.5 mL, 0.11 mol) was added dropwise. The reaction solution was allowed to warm to room temperature and stirred for 2 hours. The reaction was quenched by the addition of 50 mL of EtOAc.
  • reaction solution was cooled to 0 ° C, and the reaction was quenched by slowly adding 20 mL of a saturated ammonium chloride solution, and 150 mL of water was added thereto, and ethyl acetate was extracted three times (100 mL ⁇ 3), and the organic phases were combined and washed three times (100 mL ⁇ 3).
  • the mixture was washed with saturated brine (100 mL) Purification by column chromatography on silica gel eluting EtOAc (EtOAc:EtOAc:EtOAc: LCMS (ESI) m/z 297.1 (M+H) +
  • reaction product 4-7 (12 g, 40 mmol) in the previous step was dissolved in 50 mL of methanol, and nitrogen was replaced three times, and 200 mg of 10% palladium carbon dry powder was added.
  • the reaction liquid was replaced with hydrogen three times, and stirred at room temperature overnight in a hydrogen atmosphere. Nitrogen was replaced by 3 times, 50 mL of dichloromethane was added, filtered, washed with dichloromethane, and the filtrate was concentrated to give 8.0 g of crude product as a pale yellow oil.
  • reaction product 5-7 (10.3 g, 50 mmol) and triethylamine (8.3 mL, 60 mmol) were dissolved in 80 mL of dichloromethane, and then cooled to 0 ° C, and methanesulfonyl chloride (4.4 mL, 55 mmol) was slowly added dropwise.
  • the reaction solution was allowed to warm to room temperature and stirred for 2 hours.
  • the reaction was quenched by the addition of 50 mL of water, and the organic phase was separated and washed with brine (3 mL) Dry and concentrate to give a crude product as a pale yellow oil.
  • reaction product 7-7 (1.66 g, 6.3 mmol) from the previous step was dissolved in a mixed solvent of 20 mL of methanol and 10 mL of water, and lithium hydroxide (0.76 g, 31.5 mmol) was added.
  • the reaction solution was stirred at room temperature under nitrogen for 2 hr, and methyl methylthiosulfonate (0.95 g, 7.6 mmol) was added. The reaction was continued to stir at room temperature overnight.
  • 1-carboxylic acid succinimide ester (20 mg, 0.06 mmol) was stirred at room temperature under nitrogen overnight.
  • the reaction mixture was filtered and purified by preparative EtOAc EtOAc EtOAc LCMS (ESI) m / z 1075.4 (M + H) +.
  • reaction product 2-8 (3.9 g, 24.7 mmol) was dissolved in 40 mL of THF, cooled to 0 ° C, and 1M borane tetrahydrofuran solution (29 mL, 29 mmol) was added dropwise. The reaction solution was allowed to warm to room temperature and stirred for 2 hours. Reaction liquid cooling To 0 ° C, 3N sodium hydroxide solution (10 mL, 30 mmol) was added dropwise, 15 mL of 30% aqueous hydrogen peroxide was slowly added, and the reaction mixture was stirred at room temperature overnight. After adding 200 ml of diethyl ether, the organic phase was separated, washed with saturated brine (50 mL ⁇ 3), dried over anhydrous sodium sulfate and evaporated.
  • the crude product 4-8 (2.54 g, 10.0 mmol) was dissolved in 20 mL of N,N-dimethylformamide, and potassium thioacetate (2.3 g, 20 mmol) was added. The reaction mixture was stirred at room temperature. EtOAc (3 mL, EtOAc) The crude product was purified by EtOAc EtOAcjjjjjj LCMS (ESI) m/z 235.2 (M+H) +
  • reaction product of the previous step 5-8 (1.87 g, 8 mmol) was dissolved in 20 mL of methanol and 10 mL of water. To the solvent, lithium hydroxide (0.78 g, 32 mmol) was added. The reaction solution was stirred at room temperature under nitrogen for 2 hr, and methyl methylthiosulfonate (1.2 g, 9.6 mmol) was added. The reaction was continued to stir at room temperature overnight.
  • - Carboxylic acid succinimide ester 22 mg, 0.065 mmol
  • the reaction mixture was filtered, and purified by preparative HPLC to yield 9 g of product s.
  • reaction product 2-9 (8.6 g, 50 mmol) and triethylamine (8.3 mL, 60 mmol) were dissolved in 80 mL of dichloromethane, then cooled to 0 ° C, and methanesulfonyl chloride MsCl (4.3 mL, 55 mmol) was slowly added dropwise. .
  • the reaction solution was allowed to warm to room temperature and stirred for 2 hours.
  • the reaction was quenched by the addition of 50 mL of water, and the organic phase was separated, washed with brine (3 mL) .
  • LCMS (ESI) m / z251.1 ( M + H) +,
  • reaction product 4-9 (2.3 g, 10 mmol) from the previous step was dissolved in a mixed solvent of 25 mL of methanol and 10 mL of water under a nitrogen atmosphere, and lithium hydroxide (0.98 g, 40 mmol) was added. The reaction solution was stirred at room temperature under nitrogen for 2 hr, and methyl methylthiosulfonate (1.5 g, 12 mmol) was added. The reaction was continued to stir at room temperature overnight.
  • 1-carboxylic acid succinimide ester 22 mg, 0.065 mmol was stirred at room temperature overnight under nitrogen. The reaction mixture was filtered, and purified by preparative HPLC to yield 9.0 g of product s.
  • reaction product of the previous step 3-10 (23 g, 0.1 mol) and ethyl L-lactate (23.6 g, 0.2 mol) were dissolved in 150 mL of N,N-dimethylformamide, cooled to 0 ° C, and slowly added in portions.
  • Sodium hydrogen (8 g, 60% content, suspended in mineral oil, 0.2 mol). The reaction solution was allowed to warm to room temperature and stirred overnight.
  • reaction product 4-10 (11.3 g, 45 mmol) from the previous step was dissolved in 50 mL of methanol, and the nitrogen was replaced three times, and 200 mg of a 10% palladium carbon dry powder was added.
  • the reaction liquid was replaced with hydrogen three times, and stirred at room temperature overnight in a hydrogen atmosphere. Nitrogen was replaced by 3 times, 50 mL of dichloromethane was added, filtered, and washed with dichloromethane, and the filtrate was concentrated to give 7.2 g of crude product as a pale yellow oil.
  • reaction product 5-10 (6.5 g, 40 mmol) and triethylamine (6.6 mL, 48 mmol) were dissolved in 50 mL of dichloromethane, then cooled to 0 ° C, and methanesulfonyl chloride (3.5 mL, 44 mmol) was slowly added dropwise. The reaction solution was allowed to warm to room temperature and stirred for 2 hours. The reaction was quenched by the addition of 50 mL of EtOAc. EtOAc (EtOAc)
  • reaction product 7-10 (1.76 g, 8 mmol) from the previous step was dissolved in a mixed solvent of 20 mL of methanol and 10 mL of water, and lithium hydroxide (0.78 g, 32 mmol) was added.
  • the reaction solution was stirred at room temperature under nitrogen for 2 hr, and methyl methylthiosulfonate (1.2 g, 9.6 mmol) was added. The reaction was continued to stir at room temperature overnight.
  • - Carboxylic acid succinimide ester 22 mg, 0.065 mmol
  • the reaction solution was filtered and purified directly by preparative HPLC to give 12 mg of product CE-037, a white solid with a yield of 78%.
  • reaction product 2-11 (3.6 g, 21.4 mmol) and EDC-HCl (4.93 g, 25.7 mmol) from the previous step were dissolved in 50 mL of dichloromethane, and HOSU (2.96 g, 25.7 mol) was added.
  • the reaction solution was stirred at room temperature for 2 hours.
  • 50 mL of water and ethyl acetate the mixture was extracted three times (50 mL ⁇ 3), and the organic phase was combined and washed three times with saturated brine (50 mL ⁇ 3), dried over anhydrous sodium sulfate and evaporated.
  • the crude product was purified by silica gel chromatography eluting elut elut LCMS (ESI) m / z267.1 ( M + H) +.
  • reaction product 2-13 24 g, 0.1 mol
  • triethylamine 16.6 mL, 0.12 mol
  • methanesulfonyl chloride 8.5 mL, 0.11 mol
  • the reaction solution was allowed to warm to room temperature and stirred for 2 hours.
  • the reaction was quenched by the addition of 50 mL of water, and the organic phase was separated, and then washed with brine (3 mL).
  • reaction product 3-13 (15.9 g, 0.05 mol) and L-lactide ethyl ester (11.8 g, 0.1 mol) were dissolved in 80 mL of N,N-dimethylformamide, and cooled to 0 ° C.
  • Sodium hydrogen (4 g, 60% content, suspended in mineral oil, 0.1 mol) was added. The reaction solution was allowed to warm to room temperature and stirred overnight.
  • reaction solution was cooled to 0 ° C, and the reaction was quenched by slowly adding 20 mL of a saturated ammonium chloride solution, and 150 mL of water was added thereto, and ethyl acetate was extracted three times (100 mL ⁇ 3), and the organic phases were combined and washed three times (50 mL ⁇ 3).
  • the mixture was washed with saturated brine (50 mL)
  • the crude product was purified by silica gel column chromatography (EtOAc:EtOAc:EtOAc LCMS (ESI) m / z 341.7 (M + H) +,
  • reaction product 4-13 (13.6 g, 40 mmol) in the previous step was dissolved in 50 mL of methanol, and nitrogen was replaced three times, and 200 mg of 10% palladium carbon dry powder was added.
  • the reaction liquid was replaced with hydrogen three times, and stirred at room temperature overnight in a hydrogen atmosphere. Nitrogen was replaced by 3 times, 50 mL of dichloromethane was added, filtered, washed with dichloromethane, and the filtrate was concentrated to give a crude product (10.0 g) as a pale yellow oil.
  • reaction product 5-13 (2.5 g, 10 mmol) in the previous step was dissolved in 30 mL of acetone, cooled to 0 ° C in an ice water bath, and Jones reagent (3 g, 30 mmol) was slowly added dropwise, and the addition was completed in about 10 minutes.
  • the reaction mixture was stirred at room temperature, and the mixture was stirred overnight. The mixture was stirred and evaporated to dryness.
  • the product was a light yellow oil which was used directly in the next reaction.
  • reaction product 6-13 (264 mg, 1 mmol) from the previous step was dissolved in 10 mL of toluene under nitrogen, and the mixture was warmed to reflux, and 7 (264 mg, 1 mmol) was slowly added dropwise, and the reaction mixture was refluxed overnight.
  • the reaction product of the previous step 8-13 (1.83 g, 5.7 mmol) was dissolved in 50 mL of methanol and 20 mL of water, and K 2 CO 3 (4 g, 28.7 mmol) was added. The reaction was stirred at room temperature for 3 h. After the reaction of the raw materials was completed, methanol was removed under reduced pressure, 50 mL of water was added, 1N HCl was added dropwise to adjust the pH to 4-5, and ethyl acetate was extracted three times (50 mL ⁇ 3), and the organic phases were combined and washed with saturated brine (50 mL). The residue was dried over anhydrous sodium sulfate (MgSO4). LCMS (ESI) m / z 293.1 (M + H) +.
  • reaction product 10-13 (28 mg, 0.03 mmol) from the previous step was dissolved in dichloromethane (28 mL) and trifluoroacetic acid (4.2 ml) was slowly added. The reaction was stirred at room temperature for 2 h. After the reaction of the starting material was completed, the residue was evaporated to dryness.
  • reaction product 11-13 (18.3 mg, 0.023 mmol) from the previous step was dissolved in 2 mL DMF, and HOSU (13.2 mg, 0.11 mmol) and EDCI (21 mg, 0.11 mmol) were added. The reaction was stirred at room temperature for 2 h. Purification directly by preparative HPLC gave 2 mg of product, product s. LCMS (ESI) m / z 880.3 (M + H) +.
  • reaction product 1-14 (3.2 g, 10 mmol) from the previous step was dissolved in 20 mL of N,N-dimethylformamide, and sodium azide (715 mg, 11 mmol) was added. The reaction solution was stirred to 50 ° C and stirred overnight. The reaction solution was cooled to room temperature, and the reaction was quenched with 50 mL of water, and the mixture was extracted with ethyl acetate (3 mL) (30 mL ⁇ 3). The organic phase was combined and washed three times with saturated brine (30 mL ⁇ 3). The product was purified by silica gel column chromatography (EtOAcEtOAcEtOAcEtOAcEtOAc
  • reaction product 2-14 (2.2 g, 8 mmol) from the previous step was dissolved in 40 mL of tetrahydrofuran and 5 mL of water, and triphenylphosphine (2.62 g, 10 mmol) was added. The reaction was stirred at room temperature overnight. The tetrahydrofuran was removed under reduced pressure, and 20 mL of 1N diluted hydrochloric acid was added, and ethyl acetate was washed three times (20 mL ⁇ 3), and the aqueous phase was lyophilized to obtain 1.6 g of crude product 3-14 as a pale yellow oil.
  • the reaction product 3-14 (1.25 g, 5 mmol) in the previous step was dissolved in 20 mL of methanol and 5 mL of water, and lithium hydroxide monohydrate (840 mg, 20 mmol) was added. The reaction was stirred at room temperature for 3 h. After the reaction of the starting material was completed, the methanol was removed under reduced pressure, and then, 5 mL of a saturated sodium hydrogen carbonate solution and a 30 mL ice water bath were added and the mixture was cooled to 0 ° C, and Boc 2 O (1.3 g, 6 mmol) was added thereto, and the mixture was stirred at room temperature for 2 h.
  • the organic layer was purified by silica gel chromatography eluting elut elut elut LCMS (ESI) m / z322.3 ( M + H) +.
  • reaction product 2-15 (14.9 g, 0.04 mol) and triethylamine (6.64 mL, 0.048 mol) were dissolved in 80 mL of dichloromethane, cooled to 0 ° C, and methanesulfonyl chloride (3.4 mL, 0.044 mol) was added dropwise. ). The reaction solution was allowed to warm to room temperature and stirred for 2 hours. The reaction was quenched by the addition of 50 mL of water, and the organic layer was evaporated.
  • reaction product 3-15 (18 g, 0.04 mol) and L-lactide ethyl ester (9.44 g, 0.08 mol) were dissolved in 100 mL of N,N-dimethylformamide, cooled to 0 ° C, and slowly added in portions.
  • Sodium hydrogen (3.2 g, 60% content, suspended in mineral oil, 0.08 mol). The reaction solution was allowed to warm to room temperature and stirred for 4 h.
  • reaction solution was cooled to 0 ° C, and the reaction was quenched by slowly adding 10 mL of a saturated ammonium chloride solution, and 100 mL of water was added thereto, and ethyl acetate was extracted three times (100 mL ⁇ 3), and the organic phases were combined and washed three times (100 mL ⁇ 3).
  • the mixture was washed with saturated brine (100 mL)
  • the crude product was purified by silica gel column chromatography (EtOAc:EtOAc:EtOAc LCMS (ESI) m/z 473.1 (M+H) +
  • reaction product 4-15 (4.7 g, 10 mmol) in the previous step was dissolved in 50 mL of methanol, and the nitrogen was replaced three times, and 200 mg of 10% palladium carbon dry powder was added.
  • the reaction liquid was replaced with hydrogen three times, and stirred at room temperature overnight in a hydrogen atmosphere. Nitrogen was replaced by 3 times, 50 mL of dichloromethane was added, filtered, washed with dichloromethane, and the filtrate was concentrated to give 3.8 g of crude product as a pale yellow oil.
  • the reaction product of the previous step 6-15 (4.6 g, 10 mmol) was dissolved in 20 mL of N,N-dimethylformamide and sodium azide (715 mg, 11 mmol) was added. The reaction solution was stirred to 50 ° C and stirred overnight. The reaction solution was cooled to room temperature, and the reaction was quenched with 50 mL of water, and the mixture was extracted with ethyl acetate (3 mL) (30 mL ⁇ 3). The organic phase was combined and washed three times with saturated brine (30 mL ⁇ 3). The product was purified by silica gel column chromatography (EtOAc (EtOAc)
  • reaction product 7-15 (2.0 g, 5 mmol) from the previous step was dissolved in 30 mL of tetrahydrofuran and 5 mL of water, and triphenylphosphine (1.57 g, 6 mmol) was added. The reaction was stirred at room temperature overnight. The tetrahydrofuran was removed under reduced pressure, 20 mL of 1N diluted hydrochloric acid was added, and ethyl acetate was washed three times (20 mL ⁇ 3), and the aqueous phase was lyophilized to give 820 mg of crude product 8-15 as a pale yellow oil. LCMS (ESI) m / z 382.3 (M + H) +.
  • the reaction product of the previous step 8-15 (1.14 g, 3 mmol), was dissolved in 20 mL of methanol and 5 mL of water, and lithium hydroxide monohydrate (630 mg, 15 mmol) was added. The reaction was stirred at room temperature for 3 h. The reaction until starting material methanol was completely removed under reduced pressure, saturated sodium bicarbonate solution was added 3mL 30mL and was cooled to 0 deg.] C ice-water bath, was added Boc 2 O (785mg, 3.6mmol) , warmed to room temperature stirred for 2h.
  • the organic layer was purified by silica gel column chromatography eluting elut elut elut eluting LCMS (ESI) m / z 453.9 (M + H) +, 475.9 (M + Na) +.
  • the crude product 12-15 (12 mg, 0.015 mmol) was obtained fromjjjjjjjjjjjj The organic phase was extracted three times with dichloromethane (20 mL ⁇ 3), and the mixture was washed three times with saturated brine (20 mL ⁇ 3), dried over anhydrous sodium sulfate and concentrated to give a crude product. Yellow solid with a yield of 26%.
  • the crude product 12-15 (9 mg, 0.01 mmol) and the compound 9 (7 mg, 0.03 mmol) were dissolved in a mixed solvent of 1 mL of ethylene glycol dimethyl ether and 1 mL of water, and 0.2 mL of a saturated sodium hydrogencarbonate solution was added.
  • the reaction mixture was stirred at rt EtOAc EtOAc (EtOAc m. ) m/z 1000.4 (M–H 2 O) + .
  • reaction product 2-16 (17 g, 0.1 mol) and triethylamine (16.6 mL, 0.12 mol) in the previous step were dissolved in 150 mL of dichloromethane. Acetone was cooled to 0 ° C and methanesulfonyl chloride (8.5 mL, 0.11 mol) was added dropwise. The reaction solution was allowed to warm to room temperature and stirred for 2 hours. The reaction was quenched by the addition of 50 mL of water. The organic phase was separated, washed with brine (3 mL), dried over anhydrous sodium sulfate .
  • reaction product 3-16 (24.4 g, 0.1 mol) and L-ethyl lactate (20.8 g, 0.2 mol) in the previous step were dissolved in 150 mL of N,N-dimethylformamide and cooled to 0 ° C.
  • Sodium hydrogen (8 g, 60% content, suspended in mineral oil, 0.2 mol) was added. The reaction solution was allowed to warm to room temperature and stirred overnight.
  • reaction solution was cooled to 0 ° C, and the reaction was quenched by slowly adding 20 mL of a saturated ammonium chloride solution, and 150 mL of water was added thereto, and ethyl acetate was extracted three times (100 mL ⁇ 3), and the organic phases were combined and washed three times (100 mL ⁇ 3). The mixture was washed with saturated brine (100 mL) The crude product was purified by silica gel column chromatography (EtOAc:EtOAc:EtOAc LCMS (ESI) m / z 253.1 (M + H) +.
  • reaction product 4-16 (7.6 g, 30 mmol) in the previous step was dissolved in 50 mL of methanol, and the nitrogen was replaced three times, and 200 mg of 10% palladium carbon dry powder was added.
  • the reaction liquid was replaced with hydrogen three times, and stirred at room temperature overnight in a hydrogen atmosphere. Nitrogen was replaced by 3 times, 50 mL of dichloromethane was added, filtered, and washed with dichloromethane, and the filtrate was concentrated to give 4.8 g of crude product as a pale yellow oil.
  • the crude product 6-16 (2 g, 8.3 mmol) was dissolved in 20 mL of N,N-dimethylformamide, and potassium thioacetate (1.89 g, 16.6 mmol) was added. The reaction mixture was stirred at room temperature. EtOAc (3 mL, EtOAc) The crude product was purified by silica gel chromatography eluting elut elut elut LCMS (ESI) m / z 221.1 (M + H) +.
  • reaction product 7-16 (1.58 g, 7.2 mmol) from the previous step was dissolved in a mixed solvent of 20 mL of methanol and 10 mL of water, and lithium hydroxide (0.87 g, 36 mmol) was added. The reaction solution was stirred at room temperature under nitrogen for 2 hr, and methyl methylthiosulfonate (1.1 g, 8.7 mmol) was added. The reaction was continued to stir at room temperature overnight.
  • - Carboxylic acid succinimide ester 28 mg, 0.085 mmol
  • the reaction mixture was filtered and purified by preparative HPLC to afford 12 g of product of EtOAc (EtOAc)
  • reaction product of the previous step 2-17 (1.95 g, 8.7 mmol) was dissolved in 15 mL of THF, cooled to 0 ° C, and 1N borane tetrahydrofuran (10 mL, 10 mmol) was added dropwise with stirring. The reaction solution was allowed to warm to room temperature and stirred for 2 hours. The reaction solution was cooled to 0 ° C, 3N sodium hydroxide solution (3.4 mL, 10 mmol) was added dropwise, and 5 mL of 30% double was added slowly. Oxygen water, the reaction solution was allowed to warm to room temperature and stirred overnight. After adding 50 mL of diethyl ether, the organic phase was separated, washed with saturated brine (30 mL ⁇ 3), dried over anhydrous sodium sulfate, and evaporated.
  • the crude product 4-17 (2.16 g, 9 mmol) was dissolved in 20 mL of N,N-dimethylformamide, and potassium thioacetate (2.0 g, 18 mmol) was added. The reaction mixture was stirred at room temperature. EtOAc (3 mL, EtOAc) The crude product was purified by silica gel chromatography eluting elut elut elut LCMS (ESI) m / z 235.3 (M + H) +.
  • reaction product 5-17 (1.87 g, 8 mmol) of the previous step was dissolved in a mixed solvent of 20 mL of methanol and 10 mL of water under a nitrogen atmosphere, and lithium hydroxide (0.78 g, 32 mmol) was added. The reaction solution was stirred at room temperature under nitrogen for 2 hr, and methyl methylthiosulfonate (1.2 g, 9.6 mmol) was added. The reaction was continued to stir at room temperature overnight.
  • - Carboxylic acid succinimide ester (23.4 mg, 0.07 mmol), stirred at room temperature under nitrogen overnight. The reaction mixture was filtered, and purified by preparative HPLC to give 11.
  • the CE-051 synthesis step was consistent with CE-035.
  • Phosphorus oxychloride (7.7 mL, 83.4 mol) and DMF (39 ml) were added to a 25 mL flask, stirred at room temperature for 30 min, cooled to 0 ° C in an ice water bath, and compound 2-19 (6.5 g, 41.7 mmol) was dissolved in 39 mL DMF.
  • the mixture was slowly added dropwise to the flask, and the reaction solution was allowed to warm to room temperature and stirred overnight. After adding 100 mL of a saturated sodium hydrogencarbonate solution, the mixture was stirred for 10 hours, and ethyl acetate (50 mL ⁇ 3) was evaporated.
  • the reaction product of the previous step 8-19 (0.7 g, 3 mmol) was dissolved in a mixed solvent of 20 mL of methanol and 5 mL of water, and lithium hydroxide (0.36 g, 15 mmol) was added. The reaction solution was stirred at room temperature under nitrogen for 2 hr, and methyl methylthiosulfonate (0.57 g, 4.5 mmol) was added. The reaction was continued to stir at room temperature overnight.
  • Example 20 Synthesis route of CE-054, 055
  • reaction product 5-20 (436 mg, 2 mmol) and Pd(PPh 3 ) 4 (12 mg, 0.01 mmol) were placed in a dry Schlenk tube, argon was replaced three times, 15 mL of 1,4-dioxane was added, and the temperature was raised to 100 ° C. Stir for 1 hour. The reaction solution was cooled to room temperature, and then evaporated, mjjjjjj LCMS (ESI) m / z 175.7 (M + H) +.
  • reaction product of the previous step 6-20 (1.74 g, 10 mmol), was dissolved in 20 mL of THF, cooled to 0 ° C, and 1 M borane tetrahydrofuran solution (12 mL, 12 mmol) was added dropwise. The reaction solution was allowed to warm to room temperature and stirred for 2 hours. The reaction solution was cooled to 0 ° C, 3N sodium hydroxide solution (4 mL, 12 mmol) was added dropwise, and 6 mL of 30% aqueous hydrogen peroxide was slowly added, and the reaction mixture was stirred at room temperature overnight. After adding 80 ml of diethyl ether, the organic phase was separated, washed with saturated brine (30 mL ⁇ 3), dried over anhydrous sodium sulfate, and evaporated.
  • the reaction product of the previous step 9-20 (125 mg, 0.5 mmol) was dissolved in a mixed solvent of 3 mL of methanol and 2 mL of water, and lithium hydroxide monohydrate (210 g, 5 mmol) was added.
  • the reaction solution was stirred at room temperature for 2 hr under nitrogen atmosphere, and methyl methylthiosulfonate (126 mg, 1 mmol) was added.
  • the reaction was continued to stir at room temperature overnight.
  • After adding 5 mL of water and ethyl acetate the mixture was extracted three times (20 mL ⁇ 3), and the organic phase was combined and washed with brine (3 mL, 3), dried over anhydrous sodium sulfate and evaporated.
  • the crude product was purified by EtOAc EtOAcjjjjjj LCMS (ESI) m / z 241.7 (M + H) +.
  • the reaction product 2-21 (5 g, 11 mmol) in the previous step was dissolved in a mixed solvent of 50 mL of methanol and 5 mL of water, and lithium hydroxide (1.32 g, 55 mmol) was added.
  • the reaction solution was stirred at room temperature for 2 hours, methanol was removed under reduced pressure, and 50 mL of water was added thereto, and dichloromethane (50 mL ⁇ 3) was added, and the organic phase was combined, washed with saturated brine (3 mL) (50 mL ⁇ 3), dried over anhydrous sodium sulfate ,concentrate.
  • the crude product was purified by EtOAc EtOAcjjjjjj LCMS (ESI) m / z 345.2 (M + H) +.
  • reaction product of the previous step 3-21 (3.2 g, 9.3 mmol) and the compound M1 (3.2 g, 14 mmol) were dissolved in 100 mL of N,N-dimethylformamide, cooled to 0 ° C, and sodium hydrogen was slowly added in portions ( 0.75 g, 60% content, suspended in mineral oil, 18.6 mmol). The reaction solution was allowed to warm to room temperature and stirred for 3 h.
  • the reaction solution was cooled to 0 ° C, and the reaction was quenched by slowly adding 20 mL of a saturated ammonium chloride solution, and 50 mL of water was added thereto, and the mixture was extracted three times with dichloromethane (50 mL ⁇ 3), and the organic phases were combined and washed three times (100 mL ⁇ 3). The mixture was washed with saturated brine (100 mL)
  • the crude product was purified by silica gel column chromatography eluting elut elut elut elut elut elut LCMS (ESI) m / z479.2 ( M + H) +.
  • reaction product of the previous step 4-21 (1.4 g, 3 mmol) was dissolved in 20 ml of THF, and 1N TBAF in tetrahydrofuran (4.5 ml, 4.5 mmol). The reaction mixture was stirred at room temperature for 5 hours, and then added with 50 mL of water and ethyl acetate (50 mL ⁇ 3), and the organic phase was combined and washed three times with saturated brine (50 mL ⁇ 3), dried over anhydrous sodium sulfate and evaporated. The crude product was purified by EtOAcjjjjjjjj LCMS (ESI) m / z 241.1 (M + H) +.
  • reaction product of the previous step 5-21 (0.55 g, 2.3 mmol), was dissolved in 20 mL of acetone, cooled to 0 ° C in ice water, and the freshly prepared Jones reagent (0.46 g, 4.6 mmol) was slowly added dropwise, and the addition was completed in about 2 minutes.
  • the reaction solution was stirred to room temperature and stirred overnight.
  • the reaction was quenched by adding 1 mL of isopropyl alcohol.
  • reaction product of the previous step, 7-21 (0.5 g, 1.9 mmol), was dissolved in 20 mL of methanol, and nitrogen was replaced three times, and 30 mg of 10% palladium carbon dry powder was added.
  • the reaction liquid was replaced with hydrogen three times, and stirred at room temperature overnight in a hydrogen atmosphere. Nitrogen was replaced by 3 times, 20 mL of dichloromethane was added, filtered, and washed with dichloromethane, and the filtrate was concentrated to give a crude product (0.32 g) as a pale yellow oil.
  • LCMS (ESI) m / z 179.1 (M + H) +.
  • the reaction product 10-21 (170 mg, 0.72 mmol) from the previous step was dissolved in a mixed solvent of 10 mL of methanol and 5 mL of water under a nitrogen atmosphere, and lithium hydroxide (86 mg, 3.6 mmol) was added. The reaction solution was stirred at room temperature for 2 hr under nitrogen atmosphere, and methyl methylthiosulfonate (140 mg, 1.1 mmol) was added. The reaction was continued to stir at room temperature overnight. After adding 20 mL of water and ethyl acetate, the mixture was extracted three times (30 mL ⁇ 3), and the organic phase was combined and washed with brine (3 mL, 3), dried over anhydrous sodium sulfate and evaporated. The crude product was purified by EtOAc EtOAc EtOAc EtOAc LCMS (ESI) m / z 227.1 (M + H) +.
  • the crude product 3-22 (2.7 g, 10.0 mmol) was dissolved in 20 mL of N,N-dimethylformamide, and potassium thioacetate (2.3 g, 20 mmol) was added. The reaction mixture was stirred at room temperature. EtOAc (3 mL, EtOAc) The crude product was purified by silica gel chromatography eluting elut elut LCMS (ESI) m / z 248.2 (M + H) +.
  • reaction product of the previous step 4-22 (2 g, 8 mmol) was dissolved in a mixed solvent of 20 mL of methanol and 10 mL of water, and lithium hydroxide (0.78 g, 32 mmol) was added. The reaction solution was stirred at room temperature under nitrogen for 2 hr, and methyl methylthiosulfonate (1.2 g, 9.6 mmol) was added. The reaction was continued to stir at room temperature overnight.
  • the reaction product of the previous step 7-22 (15 mg, 0.02 mmol) was dissolved in a mixed solvent of 0.5 mL of ethyl acetate and 0.5 mL of methanol, and dithiothreitol DTT (15 mg, 0.10 mmol) was dissolved in 0.5 mL.
  • - Carboxylic acid succinimide ester (23 mg, 0.07 mmol), stirred at room temperature under nitrogen overnight.
  • the reaction mixture was filtered and purified by preparative EtOAc EtOAc EtOAc
  • Hexaethylene hexaethylene glycol (11.3 mL, 45 mmol) was dissolved in 80 mL of dry tetrahydrofuran, 50 mg of sodium metal was added, and stirred at room temperature until the sodium block was completely reacted, tert-butyl acrylate (4.35 mL, 30 mmol) was added and stirred at room temperature overnight. The tetrahydrofuran was removed under reduced pressure, and ethyl acetate (80 mL) was evaporated. Purification of 8 g of product 2-23 as a colorless oil, yield 65%. LCMS (ESI) m / z 432.9 (M + Na) +.
  • reaction product 2-23 (2.05 g, 5 mmol) and triethylamine (0.85 mL, 6 mmol) were dissolved in 30 mL of dichloromethane, cooled to 0 ° C, and methanesulfonyl chloride (0.45 mL, 5.5 mmol) was slowly added dropwise. .
  • the reaction solution was allowed to warm to room temperature and stirred for 2 hours.
  • the reaction was quenched by the addition of water (30 mL), EtOAc (EtOAc m.
  • reaction product 3-23 (2.5 g, 5 mmol) was dissolved in 20 mL of N,N-dimethylformamide, and sodium azide (390 mg, 6 mmol) was added. The reaction solution was stirred at 50 ° C for 4 hours. The reaction solution was cooled to room temperature, and the reaction was quenched with water (30 mL), EtOAc (EtOAc) (EtOAc) The product was purified by silica gel column chromatography (EtOAcEtOAcEtOAcEtOAcEtOAc LCMS (ESI) m / z 436.3 (M + H) +.
  • reaction product 4-23 (1.74 g, 4 mmol) from the previous step was dissolved in 30 mL of tetrahydrofuran and 5 mL of water, and triphenylphosphine (1.31 g, 5 mmol) was added. The reaction was stirred at room temperature overnight. The tetrahydrofuran was removed under reduced pressure, and 10 mL of 1N diluted hydrochloric acid was added, and ethyl acetate was washed three times (20 mL ⁇ 3), and the aqueous phase was lyophilized to obtain 1.5 g of crude product 5-23 as a pale yellow oil.
  • LCMS (ESI) m / z 410.3 (M + H) +.
  • the number of final conjugated CE-040 per Herceptin antibody was determined by absorbance at 252 nm and 280 nm of the conjugate and LC-MS detection, and the conjugate was tested for conjugate by polymerase.
  • the drug-to-antibody ratio (DAR) of CE-040 to Herceptin was determined by LC-MS to be 3.46:1.
  • 5 mM TCEP was added to give a ratio of TCEP to antibody of 2.5:1 (molar equivalent), reacted at 25 ° C for 2 hours and continuously stirred to mix the reactants.
  • CE-063 was added to give a ratio of CE-063 to antibody of 10:1 (molar equivalent), and DMSO was added to the DMAO in a total volume of 10%, and reacted at 25 ° C for 2 hours with constant stirring to mix the reactants.
  • the conjugate was then dialyzed against a phosphate buffered saline solution at pH 7.4 overnight, followed by filtration through a 0.22 micron filter. Store at 4 ° C.
  • the number of final conjugated CE-063 of each Herceptin antibody was determined by HIC detection and the conjugate was tested for conjugate by polymer.
  • the ratio of CE-063 to Herceptin (DAR) determined by HIC was 3.52:1.
  • antibody-conjugated drugs of the invention can be prepared by reference to Examples 24-25.
  • Her2-positive human BT474 breast tumor cells (abbreviated as BT474), Her2 low-expression human MCF-7 breast tumor cells (abbreviated as MCF-7), and MCF-7-expressing Her2 human breast tumor MCF7-Her2 stable cells Strain (MCF-7-Her2 for short)
  • MCF-7 Her2-positive human BT474 breast tumor cells
  • MCF-7 Her2 low-expression human MCF-7 breast tumor cells
  • MCF-7-Her2 Her2-positive human BT474 breast tumor cells
  • MCF-7 Her2 low-expression human MCF-7 breast tumor cells
  • MCF-7-Her2 stable cells Strain MCF-7-Her2 stable cells Strain
  • BT474, MCF7-Her2 and MCF-7 were digested with 0.25% (vol/vol) trypsin, the cells were exfoliated, then suspended in 100 ul of complete medium, and 2,000 cells were seeded in 96-well plates for culture. Incubate overnight at 37 ° C, then add 100 ul of T-CE-040 and T-CE-063 antibody drug conjugates with different concentration gradients and complete medium. Add 50ul after 120 hours Fluorescent cell activity assay reagent Luminescent, Promega) performs relative cell proliferation assays.
  • T-CE-040, T-CE-063 and T-DM1 tumor cell growth inhibition curves are shown in Figure 1 - Figure 6, where the abscissa indicates the drug concentration (ie ADC concentration); the ordinate indicates the cell survival rate ( %).
  • T-CE-040 can effectively inhibit the proliferation of the above three tumor cells, and the effect is comparable to or superior to T-DM1.

Abstract

抗体药物偶联物IB，使用醚键连接，提高了水溶性、稳定性、以及体内和体外细胞毒性。该抗体药物偶联物的中间体、药物组合物及应用。该抗体药物偶联物合成步骤简单，收率高。

Description

抗体药物偶联物、中间体、制备方法、药物组合物及应用
本申请要求申请日为2015年6月9日的中国专利申请CN201510313585.7的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明具体涉及抗体药物偶联物、中间体(带连接体的毒素)、制备方法、药物组合物及应用。
背景技术
典型的抗体药物偶联物(ADC)包含能够与癌细胞表面特异性抗原相结合的单克隆抗体。这些抗体包括免疫系统B细胞和T细胞表面的一些蛋白，比如CD20、CD22以及人表皮生长因子受体2(Her2)和前列腺特异性膜抗原(PSMA)。通过一个可裂解的连接单元将这些特异性抗体与强毒性药物进行连接。设计这样的药物通过诱导不可逆的DNA损伤或者干扰细胞分裂，进而导致癌细胞的死亡。
抗体药物偶联物(ADC)的作用机理是通过抗体识别并与特异性抗原结合，引发一系列反应，进而通过细胞内吞进入细胞质内，溶酶体酶将强毒性药物释放杀死癌细胞。相比传统化疗对正常组织的无差别损伤，通过靶向给药，可以使药物直接作用于癌细胞，减少了其对正常细胞的损害。
FDA批准上市的第一个抗体药物偶联物是美国惠氏2000年研发的用于治疗急性粒细胞性白血病的麦罗塔(吉妥珠单抗/奥佐米星)，但由于一些不良的效果以及处于安全方面的考虑，该药物在上市十年后被撤回。此后，将抗体和药物连接的技术取得了很大的进步。使用具有合适生物半衰期的连接子，研究人员得以确保抗体药物偶联物可以到达目标细胞从而减少其副作用。
2011年，美国FDA批准了Seattle Genetics的Adcetris(brentuximab vedotin)，用于治疗霍奇金氏淋巴瘤和系统性间变性大细胞淋巴瘤。该抗体药物偶联物在2012年第一季度的销售额高达$34，500，000并且收入持续在增加。预计在接下来的十年里面仅肿瘤治疗药品市场，抗体药物偶联物的使用将比过去增长50％左右。目前罗氏(Roche)和基因泰克(Genentech)一起有25个以上的抗体药物偶联物在研发中，其中有9个正在进行临床研究。
2013年2月第一个抗体药物偶联物潜在的重磅炸弹Kadcyla上市，Kadcyla(trastuzumab emtansine,T-DM1)被批准用于转移性乳腺癌的治疗。Kadcyla将治疗乳腺癌的抗体赫赛汀与ImmunoGen许可使用的毒素美坦新组合在一起。该药物的2015年销售额近7亿美元。
抗体药物偶联物很可能将成为下一个带来持续收入源的重磅炸弹级药物领域。生物医药和临床试验相互配合带来了抗体药物偶联物技术平台的持续成功。这些共同的结果表明使用人源化单克隆抗体技术作为靶标导向的治疗性给药的努力。目前为止，研发中的大部分抗体药物偶联物使用Seattle Genetics的auristatins或ImmunoGen的美登素类作为有效毒素。此外，Spirogen等公司也在为下一代抗体药物偶联物开发其他的毒素，比如PBD类。
美登素类生物碱首先是由东非灌木齿叶美登木(US 389611，JP 01006282，JP 63233986)中分离得到，作为抗癌试剂，是一类高度细胞毒性的化合物。人们制备得到的很多美登素类衍生物具有很高的抗癌活性，是传统抗癌药物长春碱或紫杉醇的100到1000倍。(J.Med.Chem.,1978,21,31-37,Chem.Pharma.Bull.,1984,3441-3451,J.Med.Chem.2006,49,4392-4408,Chem.Pharm.Bull.2004,52(1)1-26,WO2011039721,WO2012/061590,WO2014/094453).
在专利US 5208020,US,5416064和J.Med.Chem,2006,49,4392-4408中，Chari,R.V.J.等人报道了美登素类似物DM1和DM4。专利WO2004/103272,EP0425235报道了美登素类似物DM1和DM4的偶联物以及他们的抗癌活性。之后的一系列文章和专利中报道了许多美登素类似物的偶联物作为抗癌药物(WO 2011/039721,WO2012/061590,WO2014/134483,WO2014/134457,WO2014/194030,WO2014/134486,WO2014/094355,WO2015/000062,WO2014/145090,WO2013/173391)。
典型的抗体药物偶联物由药物、连接单元和抗体三部分组成。特异性抗体和药物的选择取决于特定疾病，并对偶联物的安全性和有效性有重要影响。而连接单元的稳定性以及偶联到抗体的方法对ADC药物的开发结果具有决定性的作用。决定抗体药物偶联物疗效的因素包括连接单元的稳定性及其断裂敏感性，细胞表面激发内化、转运和细胞毒素的释放。
Figure PCTCN2016085275-appb-000001
例如T-DM1，其容易在抗原结合蛋白(Abu)内吞之前过早降解并释放毒素，引起副作用。而且其与抗体偶联得到的药物/抗体比例较低而且分布变化比较大，药效和安全性精确控制的难度较大(WO2012/061590A1，WO201139721)。因此，寻找稳定性高,水溶性好的连接体，进而得到细胞毒素的释放效率高、特异性好、细胞毒性高、抗癌效果好的抗体药物偶联物是目前急需解决的技术问题。
发明内容
本发明所要解决的技术问题是为了克服现有技术中抗体药物偶联物连接单元的稳定性差、断裂敏感性低，细胞表面激发内化、转运和细胞毒素的释放效率低、胞毒性差，特异性差，副作用大等缺陷，而提供了一种抗体药物偶联物、中间体(带连接体的毒素毒素)、制备方法、药物组合物及应用。本发明的抗体药物偶联物，具有非常高的细胞毒性，抗癌效果好，具有良好的市场应用前景。
本发明提供了一种如式IB所示的抗体药物偶联物，
Figure PCTCN2016085275-appb-000002
其中：
Z为氮原子、
Figure PCTCN2016085275-appb-000003
或氧原子，当Z为氧原子时，R1不存在；当Z1为
Figure PCTCN2016085275-appb-000004
或氮原子时，R1为氢或C1～C4的烷基(所述的C1～C4的烷基例如甲基、乙基、丙基、丁基、异丙基、异丁基或叔丁基，优选甲基)或者R1、Z以及与它们连接的C原子和R6或R7连接成为4-6元环状结构(优选六元环状结构，所述的六元环状结构例如
Figure PCTCN2016085275-appb-000005
)；
R2为氢或卤素取代的C1～C4的烷基(所述的卤素取代的C1～C4的烷基中所述的卤素可以为氟、氯或溴，所述的卤素取代的C1～C4的烷基可以为卤素取代的甲基、卤素取代的乙基、卤素取代的丙基、卤素取代的异丙基、卤素取代的丁基、卤素取代的异丁基或卤素取代的叔丁基，优选卤素取代的甲基，所述的卤素取代的甲基优选
Figure PCTCN2016085275-appb-000006
)；
p为0、1、2、3、4、5、6、7、8、9或10(优选0、1或2)；
m为0或1；
n为0、1、2、3、4、5、6、7、8、9或10(优选0、1或2)；
Y1为氧原子、化学键(优选单键)或
Figure PCTCN2016085275-appb-000007
其中x2为1-24的整数(例如1、2、3、4、5、6、7、8、9或10)；
q为0、1、2、3、4、5、6、7、8、9或10(优选0、1或2)；
R3和R4独立地为氢、氰基或者取代或未取代C1～C4的烷基(所述的未取代的C1～C4的烷基例如甲基、乙基、丙基、丁基、异丙基、异丁基或叔丁基，优选甲基或异丙基；所述的取代的C1～C4的烷基例如取代的甲基、取代的乙基、取代的丙基、取代的丁基、取代的异丙基、取代的异丁基或取代的叔丁基，优选取代的甲基，所述的取代的甲基优选
Figure PCTCN2016085275-appb-000008
)，所述的R3或R4中所述的取代或未取代C1～C4的烷基中所述的取代是指被C1～C4的烷氧基(所述的C1～C4的烷氧基例如甲氧基、乙氧基、丙氧基、丁氧基、异丙氧基、异丁氧基或叔丁氧基，优选甲氧基)所取代；
x为0、1、2、3、4、5、6、7、8、9或10(优选0、1或2)；
Q1为
Figure PCTCN2016085275-appb-000009
Figure PCTCN2016085275-appb-000010
其中，R8为取代或未取代C1～C4的烷基(所述的取代或未取代的C1～C4的烷基例如取代或未取代的甲基、取代或未取代的乙基、取代或未取代的丙基、取代或未取代的异丙基、取代或未取代的丁基、取代或未取代的异丁基或者取代或未取代的叔丁基，优选取代或未取代的丙基，所述的取代的丙基优选
Figure PCTCN2016085275-appb-000011
)、
Figure PCTCN2016085275-appb-000012
y为1、2、3、4、5、6、7、8、9或10；y1为0或1；所述的R81中所述的取代或未取代C1～C4的烷基中所述的取代是指被
Figure PCTCN2016085275-appb-000013
所取代；t为1或2；t1为0、1、2、3、4、5或6；
R6为氢、取代或未取代C1～C12的烷基(所述的取代或未取代C1～C12的烷基优选取代或未取代C1～C4的烷基；所述的取代或未取代C1～C4的烷基优选甲基、乙基、丙基、异丙基、丁基、异丁基或叔丁基，进一步优选甲基)或
Figure PCTCN2016085275-appb-000014
r为1、2、3、4、5、6、7、8、9、10、11或12；
R7为氢、取代或未取代C1～C12的烷基(所述的取代或未取代C1～C12的烷基优选取代或未取代C1～C4的烷基；所述的取代或未取代C1～C4的烷基优选甲基、乙基、丙基、异丙基、丁基、异丁基或叔丁基，进一步优选甲基)、取代或未取代C1～C12的烷氧基(所述的取代或未取代C1～C12的烷氧基优选取代或未取代C1～C4的烷氧基；所述的取代或未取代C1～C4的烷氧基优选甲氧基、乙氧基、丙氧基、异丙氧基、丁氧基、异丁氧基或叔丁氧基，进一步优选甲氧基)或
Figure PCTCN2016085275-appb-000015
mAb表示单克隆抗体，例如赫赛汀；
k为1-8中的任一数值(优选1-6中的任一数值)，表示中括号内药物与单克隆抗体mAb的摩尔比(或比率，简称DAR)；
所述的如式IB所示的化合物不为下列化合物：
Figure PCTCN2016085275-appb-000016
在本发明另一优选实施方式中，Z为氧原子或者Y1为氧原子。
在本发明一优选实施方式中，当Z为氮原子时，p或q不同时为0，R2为卤素取代的C1～C4的烷基；R3和R4独立地为氢、氰基或者取代或未取代C1～C4的烷基，但R3和R4不同时为氢。
所述的如式IB所示的抗体药物偶联物优选如式Ib或Ib1所示的抗体药物偶联物，
Figure PCTCN2016085275-appb-000017
Figure PCTCN2016085275-appb-000018
如式Ib或Ib1所示的抗体药物偶联物中，Z为氮原子、
Figure PCTCN2016085275-appb-000019
或氧原子，当Z为氧原子时，R1不存在；当Z为
Figure PCTCN2016085275-appb-000020
或氮原子时，R1为氢或C1～C4的烷基(所述的C1～C4的烷基例如甲基、乙基、丙基、丁基、异丙基、异丁基或叔丁基，优选甲基)或者R1、Z以及与它们连接的C原子和R6或R7连接成为4-6元环状结构(优选六元环状结构，所述的六元环状结构例如
Figure PCTCN2016085275-appb-000021
)；R2为氢或卤素取代的C1～C4的烷基(所述的卤素取代的C1～C4的烷基中所述的卤素可以为氟、氯或溴，所述的卤素取代的C1～C4的烷基可以为卤素取代的甲基、卤素取代的乙基、卤素取代的丙基、卤素取代的异丙基、卤素取代的丁基、卤素取代的异丁基或卤素取代的叔丁基，优选卤素取代的甲基，所述的卤素取代的甲基优选
Figure PCTCN2016085275-appb-000022
)；p为0、1、2、3、4、5、6、7、8、9或10(优选0、1或2)；R6为氢、取代或未取代C1～C12的烷基(所述的取代或未取代C1～C12的烷基优选取代或未取代C1～C4的烷基；所述的“取代或未取代C1～C4的烷基”优选甲基、乙基、丙基、异丙基、丁基、异丁基或叔丁基，进一步优选甲基)或
Figure PCTCN2016085275-appb-000023
r为1、2、3、4、5、6、7、8、9、10、11或12；R7为氢、取代或未取代C1～C12的烷基(所述的取代或未取代C1～C12的烷基优选取代或未取代C1～C4的烷基；所述的取代或未取代C1～C4的烷基优选甲基、乙基、丙基、异丙基、丁基、异丁基或叔丁基，进一步优选甲基)、取代或未取代C1～C12的烷氧基(所述的取代或未取代C1～C12的烷氧基优选取代或未取代C1～C4的烷氧基；所述的取代或未取代C1～C4的烷氧基优选甲氧基、乙氧基、丙氧基、异丙氧基、丁氧 基、异丁氧基或叔丁氧基，进一步优选甲氧基)或
Figure PCTCN2016085275-appb-000024
mAb表示单克隆抗体，例如赫赛汀；k为1-8任一数值，表示中括号内药物与单克隆抗体mAb的摩尔比；
如式Ib所示的抗体药物偶联物中，m为0或1；n为0、1、2、3、4、5、6、7、8、9或10(优选0、1或2)；Y为氧原子或化学键(优选单键)；q为0、1、2、3、4、5、6、7、8、9或10(优选0、1或2)；R3和R4独立地为氢、氰基或者取代或未取代C1～C4的烷基(所述的未取代的C1～C4的烷基例如甲基、乙基、丙基、丁基、异丙基、异丁基或叔丁基，优选甲基或异丙基；所述的取代的C1～C4的烷基例如取代的甲基、取代的乙基、取代的丙基、取代的丁基、取代的异丙基、取代的异丁基或取代的叔丁基，优选取代的甲基，所述的取代的甲基优选
Figure PCTCN2016085275-appb-000025
)，所述的R3或R4中所述的取代或未取代C1～C4的烷基中所述的取代是指被C1～C4的烷氧基(所述的C1～C4的烷氧基例如甲氧基、乙氧基、丙氧基、丁氧基、异丙氧基、异丁氧基或叔丁氧基，优选甲氧基)所取代；x为0、1、2、3、4、5、6、7、8、9或10(优选0、1或2)；R8为取代或未取代C1～C4的烷基(所述的取代或未取代的C1～C4的烷基例如取代或未取代的甲基、取代或未取代的乙基、取代或未取代的丙基、取代或未取代的异丙基、取代或未取代的丁基、取代或未取代的异丁基或者取代或未取代的叔丁基，优选取代或未取代的丙基，所述的取代的丙基优选
Figure PCTCN2016085275-appb-000026
)、
Figure PCTCN2016085275-appb-000027
y为1、2、3、4、5、6、7、8、9或10；y1为0或1；所述的R8中所述的取代或未取代C1～C4的烷基中所述的取代是指被
Figure PCTCN2016085275-appb-000028
所取代；t为1或2；
如式Ib1所示的抗体药物偶联物中，Y2为
Figure PCTCN2016085275-appb-000029
其中x2为1-24的整数；Q2为
Figure PCTCN2016085275-appb-000030
t1为0、1、2、3、4、5或6；。
本发明中，所述的如式IB所示的抗体药物偶联物进一步优选如下任一化合物：
Figure PCTCN2016085275-appb-000031
Figure PCTCN2016085275-appb-000032
Figure PCTCN2016085275-appb-000033
Figure PCTCN2016085275-appb-000034
Figure PCTCN2016085275-appb-000035
本发明还提供了一种如式IA所示的中间体(带连接体的毒素)：
Figure PCTCN2016085275-appb-000036
如式IA所示的化合物中，各字母和基团的定义均同前所述；Q3为
Figure PCTCN2016085275-appb-000037
Figure PCTCN2016085275-appb-000038
t为1或2；t1为0、1、2、3、4、5或6；
R51为H、C1～C4的烷基(所述的C1～C4的烷基例如甲基、乙基、丙基、丁基、异丙基、异丁基或叔丁基，优选甲基)、
Figure PCTCN2016085275-appb-000039
Figure PCTCN2016085275-appb-000040
或卤素(所述的卤素优选F、Cl、Br或I)，y为1、2、3、4、5、6、7、8、9或10，R9为H或C1～C4的烷基(所述的C1～C4的烷基例如甲基、乙基、丙基、丁基、异丙基、异丁基或叔丁基)。
所述的如式IA所示的中间体，其优选如式Ia或Ia1所示的中间体(带链接体的毒素)：
Figure PCTCN2016085275-appb-000041
Figure PCTCN2016085275-appb-000042
如式Ia或Ia1所示的中间体中：Z为氮、
Figure PCTCN2016085275-appb-000043
或氧原子，当Z为氧原子时，R1不存在；当Z为
Figure PCTCN2016085275-appb-000044
或氮原子时，R1为C1～C4的烷基(所述的C1～C4的烷基例如甲基、乙基、丙基、丁基、异丙基、异丁基或叔丁基，优选甲基)或者R1、Z以及与它们连接的C原子和R6或R7连接成为4-6元环状结构(优选六元环状结构，所述的六元环状结构例如
Figure PCTCN2016085275-appb-000045
)；R2为卤素取代的C1～C4的烷基(所述的卤素取代的C1～C4的烷基中所述的卤素可以为氟、氯或溴，所述的“卤素取代的C1～C4的烷基”可以为卤素取代的甲基、卤素取代的乙基、卤素取代的丙基、卤素取代的异丙基、卤素取代的丁基、卤素取代的异丁基或卤素取代的叔丁基，优选卤素取代的甲基，所述的“卤素取代的甲基”优选
Figure PCTCN2016085275-appb-000046
)；p为0、1、2、3、4、5、6、7、8、9或10(优选0、1或2)；R6为氢或取代或未取代C1～C12的烷基(所述的“取代或未取代C1～C12的烷基”优选取代或未取代C1～C4的烷基；所述的“取代或未取代C1～C4的烷基”优选甲基、乙基、丙基、异丙基、丁基、异丁基或叔丁基，进一步优选甲基)或
Figure PCTCN2016085275-appb-000047
r为1、2、3、4、5、6、7、8、9、10、11或12；R7为氢，取代或未取代C1～C12的烷基(所述的“取代或未取代C1～C12的烷基”优选取代或未取代C1～C4的烷基；所述的“取代或未取代C1～C4的烷基”优选甲基、乙基、丙基、异丙基、丁基、异丁基或叔丁基，进一步优选甲基)、取代或未取代C1～C12的烷氧基(所述的“取代或未取代C1～C12的烷氧基”优选取代或未取代C1～C4的烷氧基；所述的“取代或未取代C1～C4的烷氧基”甲氧基、乙氧基、丙氧基、异丙氧基、丁氧基、异丁氧基或叔丁氧基，进一步优选甲氧基) 或者
Figure PCTCN2016085275-appb-000048
t为1或2；
如式Ia所示的中间体中：m为0或1；n为0、1、2、3、4、5、6、7、8、9或10(优选0、1或2)；Y为氧原子或化学键；q为0、1、2、3、4、5、6、7、8、9或10(优选0、1或2)；R3和R4独立地为氢、氰基、取代或未取代C1～C4的烷基(所述的“未取代的C1～C4的烷基”例如甲基、乙基、丙基、丁基、异丙基、异丁基或叔丁基，优选甲基或异丙基；所述的“取代的C1～C4的烷基”例如取代的甲基、取代的乙基、取代的丙基、取代的丁基、取代的异丙基、取代的异丁基或取代的叔丁基，优选取代的甲基，所述的“取代的甲基”优选
Figure PCTCN2016085275-appb-000049
)，所述的R3或R4中所述的“取代或未取代C1～C4的烷基”中所述的“取代”是指被C1～C4的烷氧基(所述的“C1～C4的烷氧基”例如甲氧基、乙氧基、丙氧基、丁氧基、异丙氧基、异丁氧基或叔丁氧基，优选甲氧基)所取代；R5为H、C1～C4的烷基(所述的“C1～C4的烷基”例如甲基、乙基、丙基、丁基、异丙基、异丁基或叔丁基，优选甲基)、
Figure PCTCN2016085275-appb-000050
Figure PCTCN2016085275-appb-000051
y为1、2、3、4、5、6、7、8、9或10，y1为0或1；R9为H或C1～C4的烷基(所述的“C1～C4的烷基”例如甲基、乙基、丙基、丁基、异丙基、异丁基或叔丁基)；
如式Ia1所示的中间体中：Y2为
Figure PCTCN2016085275-appb-000052
其中x2为1-24的整数；Q2为
Figure PCTCN2016085275-appb-000053
R52为氢或卤素(优选F、Cl、Br或I)。
本发明中，所述的如式IA所示的中间体(带连接体的毒素)进一步优选如下任一化合物：
Figure PCTCN2016085275-appb-000054
Figure PCTCN2016085275-appb-000055
Figure PCTCN2016085275-appb-000056
Figure PCTCN2016085275-appb-000057
Figure PCTCN2016085275-appb-000058
Figure PCTCN2016085275-appb-000059
Figure PCTCN2016085275-appb-000060
本发明中，带*的碳，表示手性碳原子或非手性碳，其构型可以为R、S或者消旋体。
本发明还提供了一种如式IB所示的抗体药物偶联物的制备方法，其包括以下步骤：有机溶剂中，pH为6-8条件下，将中间体(带连接体的毒素)IA与单克隆抗体(例如赫赛汀)进行交联，得到抗体药物偶联物IB即可；
Figure PCTCN2016085275-appb-000061
如式IA和如式IB所示的化合物中，各字母和基团的定义均同前所述。
所述的如式IB所示的抗体药物偶联物的制备方法，可以为本领域中该类交联反应的常规方法和条件，具体可参照文献Gail D.Lewis Phillips,et al.,Cancer.Res.,2008,68,9280以及Teemu T.Junttila,et al.,Breast.Cancer.Res.Treat.,2011,128,347，本申请在此引用该文献全文。
本发明中特别优选以下反应方法和条件：
在所述的如式IB所示的抗体药物偶联物的制备方法中，所述的有机溶剂优选酰胺类溶剂、亚砜类溶剂和醚类溶剂中的一种或多种。所述的酰胺类溶剂优选N,N二甲基甲酰胺(DMF)和/或二甲基乙酰胺(DMA)；所述的亚砜类溶剂优选二甲亚砜(DMSO)。所述的醚类溶剂优选四氢呋喃。
在所述的如式IB所示的抗体药物偶联物的制备方法中，所述的中间体(带连接体的毒素)IA与所述的有机溶剂的质量体积比优选0.1mg/mL-100mg/mL。
在所述的如式IB所示的抗体药物偶联物的制备方法中，所述的中间体IA与所述的单克隆抗体(例如赫赛汀)的摩尔比值优选1-10(例如6)，优选1-5。
在所述的如式IB所示的抗体药物偶联物的制备方法中，所述的pH6-8可以通过缓冲液实现；所述的缓冲溶液通常为低盐缓冲液，优选磷酸盐缓冲溶液，例如磷酸钾与磷酸二氢钾形成的缓冲溶液；或者硼酸缓冲溶液，例如硼酸和硼酸钠形成的缓冲溶液。在所述的如式Ib所示的抗体药物偶联物的制备方法中，所述的交联反应的温度优选4℃～37℃(优选室温)。
在所述的如式IB所示的抗体药物偶联物的制备方法中，所述的交联反应的进程可以采用本领域中的常规测试方法(如TLC、HPLC或NMR)进行监控，一般以中间体IA消失时为反应的终点。
所述的如式IB所示的抗体药物偶联物的制备方法优选在保护气体保护下进行，当所述的如式IB所示的抗体药物偶联物的制备方法在保护气体保护下进行时，所述的保护气体优选氮气。
所述的如式IB所示的抗体药物偶联物的制备方法，其优选包括以下步骤：在pH值为6-8(例如7.5)的缓冲溶液中，将单克隆抗体(例如赫赛汀)进行透析，然后加入中间体IA和有机溶剂，进行所述的交联反应，即可。
本发明还提供了所述的如式Ib或Ib1所示的抗体药物偶联物的制备方法，其包括以下步骤：有机溶剂中，pH为6-8条件下，将中间体(带连接体的毒素)Ia或中间体Ia1与单克隆抗体(例如赫赛汀)进行交联，得到抗体药物偶联物Ib或Ib1即可；
Figure PCTCN2016085275-appb-000062
如式Ia、Ia1、Ib和Ib1所示的化合物中，各字母和基团的定义均同前所述。
本发明还提供了所述的如式IB所示的抗体药物偶联物和/或如式IA所示的中间体(带连接体的毒素)在制备治疗和/或预防癌症的药物中的应用。本发明还提供了所述的如式Ib、Ib1、Ia和Ia1所示的化合物中的一种或多种在制备治疗和/或预防癌症的药物中的应用。
所述的应用中，所述的癌症可以为本领域中常规的癌症，包括但不限于乳腺癌、淋巴癌、肺癌、肝癌、结肠癌、头颈癌、膀胱癌、肾癌、食道癌、胆囊癌、卵巢癌、胰腺癌、胃癌、宫颈癌、甲状腺癌、前列腺癌、皮肤癌包括鳞状细胞癌；白细胞过多症、急性淋巴细胞性白血病、急性成淋巴细胞性白血病、B细胞淋巴瘤、T细胞淋巴瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、毛细胞淋巴瘤、伯基特淋巴瘤、急性和慢性髓细胞性白血病、骨髓增生异常综合征、前髓细胞白血病、纤维肉瘤、横纹肌肉瘤、星形细胞瘤、神经目细胞瘤、胶质瘤、神经鞘瘤、黑色素瘤、精原细胞瘤、畸胎癌、骨肉瘤、着色性干皮病、角质黄色瘤、甲状腺滤泡癌和卡波西肉瘤。
所述的应用中，所述的癌症的肿瘤细胞包括但不限于Her2阳性的人BT474乳腺肿瘤细胞、Her2低表达的人MCF-7乳腺肿瘤细胞或在MCF-7外转Her2的人乳腺肿瘤MCF7-Her2稳转细胞株。
本发明还提供了一种药物组合物，其包含如前所述的如式IB所示的抗体药物偶联物和/或中间体IA，以及一种或多种药学上可接受的辅料。
本发明还提供了一种药物组合物，其包含如前所述的如式Ib所示的抗体药物偶联物、如式Ib1所示的抗体药物偶联物、中间体Ia和中间体Ia1中的一种或多种，以及一种或多种药学上可接受的辅料。
本发明中，所述的药学上可接受的辅料是指药学领域常规的药用辅料，是为解决制剂的成型性、有效性、稳定性、安全性加入的除本发明的抗体药物偶联物以外的一切常规药用物料，如稀释剂(如羧甲淀粉钠等)、粘合剂(如聚维酮等)、崩解剂(如微晶纤维素等)、润滑剂(如硬脂酸镁、微粉硅胶等)、以及其它辅助剂。根据需要，可选择上述辅料，按本领域常规方法，将本发明的抗体药物偶联物制成药物制剂；所述的药物制剂为本领域各种常规剂型，如片剂、散剂、丸剂、胶囊剂、颗粒剂、口服液、干混悬剂或滴丸剂等。
本发明中，May是指
Figure PCTCN2016085275-appb-000063
本发明中，所述的术语烷基为包括具有指定碳原子数目的支链或直链的饱和脂肪族烃基；如在C1-C4烷基中定义为包括在直链或者支链结构中具有1、2、3或4个碳原子的基团。例如，C1-C4烷基具体包括甲基、乙基、正丙基、异丙基、正丁基、叔丁基、异丁基等。
本发明中，所述的术语烷氧基表示烷基与氧原子连接后的生成基团，即RO-，R为烷基。
本发明中，所述的确定了碳数范围的Cx1-y1的烷基(x1和y1为整数)，如C1-C4烷基，均表示未包含取代基的C1-C4烷基。
本发明中，所述的术语卤素表示氟、氯、溴、碘或砹。
本发明中，所述的术语氰基表示
Figure PCTCN2016085275-appb-000064
本发明中，TBDPS表示叔丁基二苯基硅基。
在不违背本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得本发明中，所述的室温指环境温度，为10℃～35℃。
本发明的积极进步效果在于：本发明设计合成了一系列结构新颖、对酸和肽酶组织蛋白酶稳定的抗体药物偶联物，本发明设计合成的抗体药物偶联物使用稳定性好的醚键进行连接，明显提高了水溶性和稳定性。体内和体外活性测试都表明其具有更高的细胞毒性，其中部分偶联物的细胞毒性优于或者接近T-DM1。此外，从化学合成的角度讲，该系列偶联物合成的步骤简单，收率远高于T-DM1，也便于进一步结构修饰以改善偶联物的理化性质以及生物活性，有良好的市场应用前景。
附图说明
图1为T-CE-040和T-DM1对BT474生长抑制曲线图。
图2为T-CE-040和T-DM1对MCF-7生长抑制曲线图。
图3为T-CE-040和T-DM1对MCF-7-Her2生长抑制曲线图。
图4为T-CE-063和T-DM1对BT474生长抑制曲线图。
图5为T-CE-063和T-DM1对MCF-7生长抑制曲线图。
图6为T-CE-063和T-DM1对MCF-7-Her2生长抑制曲线图。
具体实施方式
实施例1CE-016/017,CE-023/024,CE-005的合成路线
Figure PCTCN2016085275-appb-000065
实验步骤
化合物2的合成:
Figure PCTCN2016085275-appb-000066
称取3-氯-2-氯甲基丙烯(25g,0.2mol)溶于200mL无水甲醇中，分批加入甲醇钠(21.6g,0.4mol)。加热反应液回流过夜。将反应液冷却到室温，过滤，往滤液中加入250mL水，醋酸中和，用沸点30～60℃的石油醚萃取三次(150mL×3)，合并有机相，无水硫酸钠干燥，浓缩得到15g粗产品，为无色油状液体，直接用于下一步反应。
化合物3的合成:
Figure PCTCN2016085275-appb-000067
将上一步反应粗产物2(15.1g,0.13mol)溶于150mL二氯甲烷中，分批加入间氯过氧苯甲酸(33.54g,0.195mol)。反应液加热回流2小时。TLC点板确认反应完全，将反应液冷却到室温。30mL饱和硫代硫酸钠溶液加入搅拌半小时淬灭反应。使用二氯甲烷萃取3次(100mL×3)。合并有机相，无水硫酸钠干燥，浓缩得到12g粗产物3，为无色油状物，直接用于下一步反应。1H NMR(400MHz,CDCl3)δppm 2.57&2.54(dd,J1＝16.4Hz,J2＝11.2 Hz,4H),3.39(s,6H),2.78(s,2H).
化合物4的合成:
Figure PCTCN2016085275-appb-000068
将上一步的粗产物3(13.2g,0.1mol)溶于100mL无水甲醇中，加入硫脲(15.2g,0.2mol)。反应液室温搅拌过夜，TLC点板检测原料以完全消耗，减压蒸馏移除甲醇。往剩余物中加入200mL水，使用二氯甲烷萃取3次(100mL×3)。合并有机相，无水硫酸钠干燥，浓缩，粗产品使用硅胶柱层析(石油醚：乙酸乙酯10：1)得到9.3g产物4，为无色油状物，产率63％。1H NMR(400MHz,CDCl3)δppm 2.62&2.60(dd,J1＝16.0Hz,J2＝10.0Hz,4H),3.41(s,6H),2.40(s,2H).
化合物5的合成:
Figure PCTCN2016085275-appb-000069
氮气保护下，将正丁基锂(8.8mL,2.5mol/mL,22mmol)加入到冷却至-78℃的40mL干燥四氢呋喃溶液中。将乙腈(1.1mL,21.2mmol)溶于10mL干燥四氢呋喃加入。反应液在-78℃搅拌半小时。将化合物4(2.96g,20mmol)溶于20mL四氢呋喃中，并逐滴加入。加入完毕，反应液逐步升温至室温并搅拌3小时。将反应液冷却至0℃，逐滴缓慢加入稀盐酸(0.5M,10mL)淬灭反应，乙酸乙酯萃取3次(50mL×3)，合并有机相，无水硫酸钠干燥，浓缩。粗产品使用硅胶柱层析(石油醚/乙酸乙酯＝1:1)纯化得到产物2.23g，为浅黄色油状物，产率59％。1H NMR(400MHz,CDCl3)δppm 3.62(d,J＝9.2Hz,2H),3.51(d,J＝9.2Hz,2H),3.39(s,6H),2.81(t,J＝7.2Hz,2H),2.07(t,J＝7.2Hz,2H).
化合物6的合成:
Figure PCTCN2016085275-appb-000070
将上一步反应产物5(2.8g,15mmol)溶于10mL乙醇中，置换氩气三次，缓慢加入8M的氢氧化钠溶液(4mL,32mmol)。氩气保护下反应液升温回流过夜。反应液冷却至室 温，稀盐酸调PH＝2，二氯甲烷萃取3次(50mL×3)，合并有机相，无水硫酸钠干燥，浓缩。粗产物使用硅胶柱层析(石油醚/乙酸乙酯＝2：1)纯化得2.43g产品6，为浅黄色油状物，产率78％。LCMS(ESI)m/z 209.1(M+H)+.
化合物7的合成:
Figure PCTCN2016085275-appb-000071
将上一步反应产物6(2.08g,10mmol)溶于20mL乙醇中和10mL水中，加入甲基硫代磺酸甲酯(1.38g,11mmol)。氩气保护下反应液室温搅拌过夜。加入50mL水，乙酸乙酯萃取3次(50mL×3)，合并有机相，饱和食盐水洗涤3次(50mL×3)，无水硫酸钠干燥，浓缩得粗产物7，直接用于下一步反应。LCMS(ESI)m/z 255.1(M+H)+.
化合物8的合成:
Figure PCTCN2016085275-appb-000072
将上一步反应产物7(1g,4mmol)溶于2mL二氯甲烷中，N-羟基琥珀酰亚胺(HOSu)(0.50g,4.4mmol)and 1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC-HCl)(0.84g,4.4mmol)。反应液室温搅拌4小时。加入50mL水，乙酸乙酯萃取3次(50mL×3)，合并有机相，饱和食盐水洗涤3次(50mL×3)，无水硫酸钠干燥，浓缩。粗产物使用硅胶柱层析(石油醚/乙酸乙酯＝1:3)纯化得1.3g产物8，为黄色油状物，产率76％。LCMS(ESI)m/z352.1(M+H)+.
化合物9的合成:
Figure PCTCN2016085275-appb-000073
N-甲基-L-丙氨酸(618mg,6.0mmol)溶于15mL乙二醇二甲醚和15mL水中。加入三乙胺(1.7mL g,12mmol)，剧烈搅拌。将上一步反应产物8(2.1g,6mmol)溶于15mL乙二醇二甲醚中逐滴加入，约5分钟加完。反应液室温搅拌2小时，减压移除有机溶剂，加 入10mL水，使用1M的稀盐酸调PH＝2，乙酸乙酯萃取3次(50mL×3)，合并有机相，饱和食盐水洗涤3次(50mL×3)，无水硫酸钠干燥，浓缩。粗产物使用硅胶柱层析(石油醚/乙酸乙酯＝1:1)纯化得产物920mg，为黄色油状物，产率45％。LCMS(ESI)m/z 340.1(M+H)+.
化合物CE-016和CE-017的合成:
Figure PCTCN2016085275-appb-000074
将上一步产物9(122mg,0.36mmol)，二环己基碳二亚胺(DCC)(0.15g,0.72mmol)加入一个干燥的Schlenck管中，置换氩气3次，加入1mL二氯甲烷，搅拌。将美登醇(63mg,0.12mmol)溶于4mL干燥二氯甲烷中加入，然后加入1M的氯化锌乙醚溶液(0.72mL,0.72mmol)。反应液室温搅拌2h，缓慢加入0.3mL淬灭反应，加入15mL乙酸乙酯，过滤，乙酸乙酯洗涤。滤液无水硫酸钠干燥，浓缩。粗产物使用硅胶柱层析(二氯甲烷/甲醇＝40:1)分离纯化，得到两个异构体CE-016(25mg，P1)和CE-017(37mg，P2)，均为白色固体，产率24％+35％.LCMS(ESI)m/z 867.8(M+H)+。
HPLC(15min):CE-016,Rt＝10.680；CE-017,Rt＝10.621(Rt：保留时间)
Mobile Phase(流动相):A:water(水)(0.01％TFA)B:CAN(乙腈)(0.01％TFA)，％均为体积百分比
Gradient(梯度):0min 5％B,3min 5％B,10min 95％B,15min 95％B
Flow Rate(流速):1.2ml/min
Column:Eclipse XDB-C18,4.6*150mm,5um
Oven Temperature(柱温):40℃
CE-016:1H NMR(400MHz,CDCl3)δppm 6.85(s,1H),6.81(s,1H),6.44&6.42(dd,J1＝15.2Hz,J2＝11.2Hz,1H),6.31(d,J＝11.2Hz,1H),6.23(s,1H),5.90&5.87(dd,J1＝15.2Hz,J2＝9.2Hz,1H),5.23(q,J＝7.2Hz,1H),4.91(s,1H),4.86&484(dd,J1＝11.6Hz,J2＝2.8Hz,1H),4.32(t,J＝12.0Hz,1H),3.99(s,3H),3.55-3.40(m,5H),3.37(s,6H),3.34(s,3H),3.19(d,J＝12.8Hz,1H),3.17(s,3H),3.04(s,3H),2.86(d,J＝9.6Hz,1H),2.65&2.62(dd,J1＝14.4Hz,J2＝12.0Hz,1H),2.50(t,J＝8.0Hz,1H),2.40(s,3H),2.24-2.18(m,1H),2.06-1.92(m,2H),1.72(d,J＝13.6Hz,1H),1.68(s,3H),1.48(d,J＝7.6Hz,3H),1.50-1.42(m,1H),1.30(s,3H),1.28(s,3H),0.85(s,3H).
CE-017:1H NMR(400MHz,CDCl3)δppm 6.82(s,1H),6.75(d,J＝11.2Hz,1H),6.64(s, 1H),6.44&6.42(dd,J1＝15.2Hz,J2＝11.2Hz,1H),6.21(s,1H),5.70&5.68(dd,J1＝15.6Hz,J2＝8.8Hz,1H),5.41(q,J＝6.8Hz,1H),4.80&4.77(dd,J1＝12.0Hz,J2＝3.2Hz,1H),4.28(t,J＝11.2Hz,1H),3.98(s,3H),3.65(d,J＝12.8Hz,1H),3.50(d,J＝8.8Hz,1H),3.43(t,J＝8.8Hz,2H),3.40-3.30(m,2H),3.35(s,3H),3.26(s,3H),3.23(s,6H),3.10(d,J＝12.0Hz,1H),3.03(d,J＝9.2Hz,1H),2.84(s,3H),2.64-2.51(m,2H),2.64-2.51(m,2H),2.44-2.35(m,1H),2.30(s,3H),2.24-2.16(m,1H),2.10-1.89(m,2H),1.72(d,J＝13.6Hz,1H),1.64(s,3H),1.50-1.42(m,1H),1.30(s,3H),1.28(s,3H),0.85(s,3H).
化合物CE-024的合成:
Figure PCTCN2016085275-appb-000075
CE-017(30mg,0.034mmol)溶于0.5mL乙酸乙酯中，将二硫代苏糖醇(DTT)(13mg,0.084mmol)溶于0.9mL甲醇中加入，加入PH＝7.5的磷酸钾缓冲溶液2mL。反应液在氮气保护下搅拌2小时。加入PH＝6的磷酸钾缓冲溶液3.4mL淬灭反应。乙酸乙酯萃取3次(20mL×3)，合并有机相，饱和食盐水洗涤3次(20mL×3)，无水硫酸钠干燥，浓缩。粗产品使用制备HPLC(CH3CN in H2O-0.05％TFA from 5％to 95％)纯化得到22mg产物CE-024，白色固体，产率80％。LCMS(ESI)m/z 840.1(M+H)+.
化合物CE-023的合成:
Figure PCTCN2016085275-appb-000076
CE-023的合成步骤和CE-024一致，使用CE-016为原料。
化合物CE-005的合成:
Figure PCTCN2016085275-appb-000077
将CE-024(18mg,0.02mmol)溶于2mL N,N-二甲基甲酰胺中，加入SM1(14mg,0.04 mmol)。反应液室温搅拌12小时，反应液过滤，滤液直接使用制备HPLC(CH3CN in H2O-0.05％TFA from 5％to 95％)纯化得到13.7mg产物CE-005，为白色固体。产率65％。
LCMS(ESI)m/z 1076.8(M+Na)+.1H NMR(400MHz,CDCl3)δppm 6.85(s,1H),6.74(d,J＝9.4Hz,1H),6.63(s,1H),6.44&6.42(dd,J1＝15.2Hz,J2＝11.2Hz,1H),6.29(s,1H),5.70&5.68(dd,J1＝15.6Hz,J2＝8.8Hz,1H),5.42(q,J＝6.8Hz,1H),4.80&4.77(dd,J1＝12.0Hz,J2＝3.2Hz,1H),4.28(t,J＝11.2Hz,1H),3.98(s,3H),3.65(d,J＝12.8Hz,1H),3.50(d,J＝7.2Hz,1H),3.41(t,J＝8.8Hz,2H),3.40-3.30(m,2H),3.35(s,3H),3.25(s,3H),3.23(s,3H),3.22(s,3H),3.11(d,J＝12.0Hz,1H),3.03(d,J＝9.2Hz,1H),2.84(s,3H),2.64-2.51(m,2H),2.68(q,J＝7.2Hz,4H),2.63-2.50(m,2H),2.44-2.35(m,1H),2.19&2.16(dd,J1＝14.4Hz,J2＝3.2Hz,1H),2.08-1.99(m,3H),1.96-1.89(m,1H),1.72(d,J＝13.6Hz,1H),1.64(s,3H),1.50-1.42(m,2H),1.30(s,3H),1.29(s,3H),1.28(s,3H),0.80(s,3H).
实施例2CE-011,CE-038,CE-041的合成路线
Figure PCTCN2016085275-appb-000078
Figure PCTCN2016085275-appb-000079
往N-甲基-L-丙氨酸1-2(10g,0.097mol)的500mL乙醇悬浊液中加入氢氧化钾(5.5g,0.098mol)。反应液室温搅拌1小时直至氢氧化钾完全溶解。加入4,4,4-三氟巴豆酸乙酯2-2(20g,0.119mmol)。反应液升温至35℃搅拌7天。反应液冷却至0℃，逐滴加入10mL浓盐酸，室温搅拌1小时，减压浓缩。加入500mL乙酸乙酯，过滤，100mL乙酸乙酯洗涤，滤液浓缩得到12.5g粗产品，收率48％。LCMS(ESI)m/z 272.1(M+H)+.
化合物4-2的合成
Figure PCTCN2016085275-appb-000080
上一步产物3-2(12.5g,46.1mmol)溶于150mL四氢呋喃，冷至0℃，分批小量加入硼氢化锂(2.0g,90.9mmol)。反应液室温搅拌3小时，冰水浴冷却，逐滴缓慢加入10mL甲醇直至没有气泡冒出，继续搅拌2小时。加入300mL乙酸乙酯和饱和300mL氯化铵溶液，有机相分离，无水硫酸钠干燥，浓缩得到8.3g粗产品4-2，为浅黄色油状物。LCMS(ESI)m/z 230.0(M+H)+.
化合物5-2的合成
Figure PCTCN2016085275-appb-000081
氮气保护下，三苯基磷(12g,45.8mmol)溶于200mL四氢呋喃，冰水浴冷却，搅拌。加入偶氮二甲酸二乙酯(DEAD)(8.0g,45.8mmol)，继续搅拌5分钟。依次加入化合物4-2(8.3g,36.2mmol)的50mL四氢呋喃溶液和硫代乙酸(4.13g,54.3mmol)。反应液升至室温搅拌过夜。往反应液中加入300mL乙酸乙酯和200mL饱和氯化铵溶液。分出有机相，浓缩。粗产物硅胶柱层析(石油醚/乙酸乙酯体积比3：1)纯化得3.3g产物5-2，为浅黄色油状物，产率32％。LCMS(ESI)m/z 288.0(M+H)+ .
化合物6-2的合成
Figure PCTCN2016085275-appb-000082
氮气保护下，将上一步反应产物5-2(3.3g,11.4mmoL)溶于60mL甲醇和30mL水的混合溶液中，加入碳酸钾(4g,28.9mmol)。反应液氮气保护下室温搅拌2小时。加入甲基硫代磺酸甲酯(3.0g,23.8mmol)。氮气保护下反应液室温搅拌2小时。加入200mL水和200mL二氯甲烷，分出有机相，水相用1M的稀盐酸调PH至4到5，乙酸乙酯萃取(150mL×3)，合并有机相，饱和食盐水洗涤(100mL)，无水硫酸镁干燥，浓缩得2.0g粗产物，为浅黄色油状物。手性制备HPLC(AD-H,4.6×250mm,5um,flow rate:CO2+MeOH＝2.7+0.3)分离得到两个异构体6-2-P1和6-2-P2。LCMS(ESI)m/z 292.1(M+H)+。
6-2-P1:Rt＝1.72,70％；900mg,1H NMR(400MHz,CDCl3)δppm 3.65(q,J＝7.2Hz,1H),3.51(q,J＝7.2Hz,1H),2.92-2.74(m,2H),2.51(s,3H),2.42(s,3H),2.11-2.05(m,2H),1.41(d,J＝7.2Hz,3H).
6-2-P2:Rt＝2.38,30％；200mg,1H NMR(400MHz,CDCl3)δppm 3.65(q,J＝7.2Hz,1H),3.43(q,J＝7.2Hz,1H),2.89-2.75(m,2H),2.46(s,3H),2.41(s,3H),2.13-2.08(m,2H),1.43(d,J＝7.2Hz,3H).
化合物CE-011的合成
Figure PCTCN2016085275-appb-000083
上一步反应产物6-2(160mg,0.549mmol)溶于5mL干燥的二氯甲烷中，加入二环己基碳二酰亚胺DCC(80mg,0.388mmol)，室温搅拌30分钟，过滤。
氩气保护下，美登醇(41mg,0.0726mmol)溶于3mL干燥DMF中，加入Zn(HDMS)2(0.5mL,0.74mmol)，室温搅拌。将上述滤液加入反应液中，室温搅拌2小时，加入20mL饱和碳酸氢钠溶液和30mL乙酸乙酯。分出有机相20mL饱和食盐水洗涤，无水硫酸钠干燥，浓缩。粗产品用制备HPLC纯化得到10mg产物CE-011，为白色固体，产率17％。LCMS(ESI)m/z 838.2(M+H)+。
化合物CE-029的合成
Figure PCTCN2016085275-appb-000084
使用化合物6-2-P1为原料，CE-029的合成步骤和CE-011一致。
1H NMR(400MHz,CDCl3)δppm 6.84(s,2H),6.42&6.38(dd,J1＝15.6Hz,J2＝8.0Hz,1H),6.35(s,1H),6.14(d,J＝11.6Hz,1H),5.49&5.46(dd,J1＝15.6Hz,J2＝6.4Hz,1H),5.35(t,J＝4.8Hz,1H),4.92&4.91(dd,J1＝12.0Hz,J2＝2.8Hz,1H),4.28(t,J＝12.8Hz,1H),3.99(s,3H),3.59(q,J＝6.8Hz,2H),3.50(s,1H),3.49(d,J＝6.4Hz,1H),3.37(s,3H),3.22(d,J＝12.8Hz,1H),3.13(s,3H),2.89-2.84(m,1H),2.81(d,J＝8.8Hz,1H),2.57(t,J＝12.0Hz,1H),2.44-2.42(m,3H),2.36(s,3H),2.29-2.27(m,2H),2.08-1.99(m,3H),1.68(s,3H),1.58-1.65(m,2H),1.45(d,J＝6.8Hz,3H),1.30(d,J＝7.2Hz,3H),0.84(s,3H).
化合物CE-030的合成
Figure PCTCN2016085275-appb-000085
使用化合物6-2-P2为原料，CE-030的合成步骤和CE-011一致。
1H NMR(400MHz,CDCl3)δppm 6.83(s,1H),6.74(s,1H),6.44(d,J＝11.2Hz,1H),6.40(t,J＝6.4Hz,1H),6.15(d,J＝10.4Hz,1H),5.49&5.48(dd,J1＝13.6Hz,J2＝8.8Hz,1H),4.88&4.87(dd,J1＝12.0Hz,J2＝2.8Hz,1H),4.28(t,J＝12.8Hz,1H),3.99(s,3H),3.61(q,J＝6.8Hz,1H),3.51(d,J＝9.2Hz,2H),3.35(s,3H),3.22(d,J＝12.8Hz,1H),3.16(s,3H),2.93-2.84(m,2H),2.77(d,J＝8.8Hz,1H),2.53(t,J＝12.0Hz,1H),2.44(s,3H),2.43(s,3H),2.36-2.10(m,1H),2.29-2.27(m,2H),2.08-1.99(m,3H),1.68(s,3H),1.58-1.65(m,2H),1.45(d,J＝6.8Hz,3H),1.30(d,J＝7.2Hz,3H),0.84(s,3H).
化合物7-2的合成
Figure PCTCN2016085275-appb-000086
CE-029(12mg,0.015mmol)溶于1mL乙酸乙酯和1mL甲醇的混合溶剂中，将二硫代苏糖醇DTT(18mg,0.117mmol)溶于0.5mL PH＝7.5的磷酸钾缓冲溶液中加入。反应液在氮气保护下搅拌3小时。加入PH＝6.0的磷酸钾缓冲溶液1mL淬灭反应。乙酸乙酯萃取3次(10mL×3)，合并有机相，饱和食盐水洗涤3次(5mL)，无水硫酸钠干燥，浓缩。粗产品使用制备HPLC(CH3CN in H2O-0.05％TFA from 5％to 95％)纯化得到10mg产物7-2，白色固体，产率85％。LCMS(ESI)m/z792.2(M+H)+。
化合物CE-038的合成
Figure PCTCN2016085275-appb-000087
氮气保护下，上一步反应产物7-2(10mg,0.012mmol)溶于1.5mL四氢呋喃中，加入PH＝6.0的磷酸钾缓冲溶液1.5mL和4-(N-马来酰亚胺甲基)环己烷-1-羧酸琥珀酰亚胺酯(20mg,0.06mmol，SMCC)。反应液氮气保护下室温搅拌过夜。过滤，滤液由制备HPLC纯化得11.5mg产品CE-038，为白色固体，产率85％。
LCMS(ESI)m/z1125.9(M+H)+,1H NMR(400MHz,CDCl3)δppm 6.84(s,1H),6.80(s,1H),6.56(d,J＝8.8Hz,1H),6.44&6.42(dd,J1＝11.6Hz,J2＝8.8Hz,1H),6.15(d,J＝8.8Hz,1H),5.52&5.50(dd,J1＝11.2Hz,J2＝6.4Hz,1H),4.88-4.84(m,1H),4.29(t,J＝8.4Hz,1H),3.99(s,3H),3.95&3.94(dd,J1＝7.2Hz,J2＝2.8Hz,1H),3.53-3.48(m,4H),3.41-3.37(m,2H),3.37(s, 3H),3.26-3.19(m,2H),3.13(d,J＝4.8Hz,1H),3.13(s,3H),2.85-2.77(m,5H),2.59(t,J＝8.8Hz,2H),2.46(s,3H),2.29-2.15(m,8H),1.83-1.78(m,3H),1.68(s,3H),1.63(d,J＝10.8Hz,1H),1.54(d,J＝10.8Hz,1H),1.45(t,J＝6.8Hz,3H),1.30(t,J＝7.2Hz,3H),1.30(t,J＝7.2Hz,3H),1.10(q,J＝10.4Hz,2H),0.84(s,3H).
化合物CE-041的合成
Figure PCTCN2016085275-appb-000088
将化合物7-2(10mg,0.012mmol)溶于2mL N,N-二甲基甲酰胺中，加入CE-L-019N-琥珀酰亚胺4-(2-二硫代吡啶)丁酰酯(6.8mg,0.018mmol)。反应液氮气保护下室温搅拌过夜，过滤，滤液直接使用制备HPLC(CH3CN in H2O-0.05％TFA from 5％to 95％)纯化得到9.6mg产物CE-041，为白色固体，产率80％。
LCMS(ESI)m/z1007.3(M+H)+,1H NMR(400MHz,CDCl3)δppm 6.84(s,2H),6.80(s,1H),6.43(dd,J1＝12.4Hz,J2＝8.8Hz,1H),6.15(d,J＝8.8Hz,1H),5.52&5.51(dd,J1＝12.8Hz,J2＝6.8Hz,1H),4.90(d,J＝9.6Hz,1H),4.31(t,J＝8.8Hz,1H),3.99(s,3H),3.61(q,J＝5.6Hz,2H),3.50(d,J＝7.2Hz,2H),3.37(s,3H),3.22(d,J＝12.8Hz,1H),3.13(s,3H),2.85-2.77(m,12H),2.59(t,J＝8.8Hz,2H),2.42(s,3H),2.29(d,J＝8.4Hz,1H),2.19-2.16(m,2H),2.00-2.08(m,2H),1.69(s,3H),1.64(d,J＝10.8Hz,1H),1.45(d,J＝6.8Hz,3H),1.30(d,J＝7.2Hz,3H),0.84(s,3H).
实施例3CE-012,013,014,015,032,004的合成路线
Figure PCTCN2016085275-appb-000089
Figure PCTCN2016085275-appb-000090
2-甲基烯丙醇1-3(10g,139mmol)和咪唑(18.9g,278mmol)依次溶于200mL二氯甲烷中，冰水浴冷至0℃，分批加入叔丁基二苯基氯硅烷(57g,208mmol)。反应液升至室温搅拌2小时。加入100mL水淬灭，二氯甲烷萃取3次(100mL×3)。合并有机相，饱和食盐水洗3次(50mL×3)，无水硫酸钠干燥，浓缩。粗产物由硅胶柱层析(石油醚/乙酸乙酯＝10:1)纯化得35g产物2-3，为浅黄色固体，产率81％。
1H NMR(400MHz,CDCl3)δppm 7.70-7.68(m,4H),7.42-7.35(m,6H),5.13(s,1H),4.85(d,J＝6.4Hz,1H),4.07(s,2H),1.68(s,3H),1.07(s,9H).
化合物3-3的合成:
Figure PCTCN2016085275-appb-000091
将上一步反应粗产物2-3(30g,96mmol)溶于600mL二氯甲烷中，加入碳酸钾(24.9g,144mmol)，室温搅拌1小时。分批加入间氯过氧苯甲酸(26.4g,192mmol)。反应液室温搅拌过夜。150mL饱和硫代硫酸钠溶液加入搅拌半小时淬灭反应。使用二氯甲烷萃取3次(100mL×3)。合并有机相，饱和食盐水洗3次(100mL×3)，无水硫酸钠干燥，浓缩。粗产物经硅胶柱层析(石油醚/乙酸乙酯＝10:1)纯化得22.8g产物3-3，产率72％。
1H NMR(400MHz,CDCl3)δppm 7.68(d,J＝6.0Hz,4H),7.42-7.36(m,6H),3.66(s,2H),2.71(d,J＝4.8Hz,1H),2.58(d,J＝4.8Hz,1H),1.37(s,3H),1.06(s,9H).
化合物4-3的合成:
Figure PCTCN2016085275-appb-000092
将上一步的粗产物3-3(20g,62mmol)溶于200mL无水甲醇中，加入硫脲(48g,124mmol)。反应液回流搅拌1小时，TLC点板检测原料以完全消耗，冷却至室温，减压移除甲醇。往剩余物中加入200mL水，使用乙酸乙酯萃取3次(100mL×3)。合并有机相，饱和食盐水洗涤3次(100mL×3)，无水硫酸钠干燥，浓缩，粗产品使用硅胶柱层析(石油醚：乙酸乙酯10：1)得到14.8g产物，为无色油状物，产率70％。
1H NMR(400MHz,CDCl3)δppm 7.67-7.64(m,4H),7.43-7.34(m,6H),3.88(d,J＝10.4Hz,1H),3.57(d,J＝10.4Hz,1H),2.30(d,J＝18.4Hz,2H),1.68(s,3H),1.07(s,9H).
化合物5-3的合成:
Figure PCTCN2016085275-appb-000093
氮气保护下，将正丁基锂(2.5M,13mL 32.1mmol)加入到冷却至-78℃的100mL干燥四氢呋喃溶液中。将乙腈(1.6mL,30.7mmol)溶于10mL干燥四氢呋喃加入。反应液在-78℃搅拌半小时。将化合物4-3(10g,29.2mmol)溶于20mL四氢呋喃中，并逐滴加入。加入完毕，反应液逐步升温至室温并搅拌3小时。将反应液冷却至0℃，逐滴缓慢加入稀盐酸(0.5M,20mL)淬灭反应，乙酸乙酯萃取3次(100mL×3)，合并有机相，饱和食盐水洗3次(100mL×3)，无水硫酸钠干燥，浓缩。粗产品使用硅胶柱层析(石油醚/乙酸乙酯＝1:1)纯化得到产物9.2g，为浅黄色油状物，产率82％。LCMS(ESI)m/z 384.2(M+H)+
化合物6-3的合成:
Figure PCTCN2016085275-appb-000094
将上一步反应产物5-3(3g,7.8mmol)溶于30mL二氯甲烷中，加入甲基硫代磺酸甲酯(1.2mL,11.7mmoL)和三乙胺(1.6mL,11.7mmol)。氮气保护下反应液室温搅拌过夜。加入1M稀盐酸30mL和100mL二氯甲烷，分出有机相，无水硫酸钠干燥，浓缩。粗产物经硅胶柱层析(石油醚/乙酸乙酯＝10:1)纯化得2.93g产品6-3，为浅黄色固体，产率87％。
LCMS(ESI)m/z 451.9(M+H)+,1HNMR(400MHz,CDCl3)δ7.65～7.62(m,4H),7.45～7.38(m,6H),3.57(s,2H),2.43～2.37(m,2H),2.25(s,3H),2.14～2.03(m,2H),1.27(s,3H), 1.08(s,9H).
化合物7-3的合成:
Figure PCTCN2016085275-appb-000095
上一步反应产物6-3(3.2g,7.45mmoL)溶于30mL四氢呋喃中，加入四丁基氟化铵(22mL,22mmol,1M in THF)。反应液室温搅拌过夜。缓慢加入1N的稀盐酸300mL淬灭反应，加入200mL乙酸乙酯，分出有机相，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝2:1)纯化得1.18g产物7-3，为无色油状物，产率83％。
LCMS(ESI)m/z 192.1(M+H)+,1HNMR(400MHz,CDCl3)δ3.57(s,3H),2.59～2.42(m,2H),2.43(s,3H),2.10～1.94(m,2H),1.30(s,3H).
化合物8-3的合成:
Figure PCTCN2016085275-appb-000096
上一步反应产物7-3(1.18g,6.17mmoL)溶于100mL二氯甲烷中，加入三甲氧基鎓四氟化硼盐(1.83g,12.35mmoL)和1,8-双甲胺基萘(2.7g,12.35mmol)。反应液室温搅拌过夜。过滤，30mL二氯甲烷洗涤，滤液用1M稀盐酸150mL洗涤，分出有机相，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝8:1)纯化得820mg化合物8-3，为黄色油状物，产率65％。
LCMS(ESI)m/z 205.9(M+H)+,1HNMR(400MHz,CDCl3)δ3.37～3.31(m,5H),2.56～2.42(m,2H),2.41(s,3H),2.08-2.03(m,2H),1.30(s,3H).
化合物9-3的合成:
Figure PCTCN2016085275-appb-000097
将上一步反应产物8-3(760mg,3.7mmol)溶于8mL乙醇中，置换氩气三次，缓慢加入8M的氢氧化钠溶液(4mL,32mmol)。氩气保护下反应液升温回流过夜。反应液冷却至室温，稀盐酸调PH＝2，二氯甲烷萃取3次(50mL×3)，合并有机相，无水硫酸钠干燥，浓缩得粗产物700mg，为黄色油状物，直接用于下一步反应。1HNMR(400MHz,CDCl3)δ3.37～3.31(m,5H),2.61～2.13(m,2H),2.03～1.91(m,2H),1.30(s,3H).
化合物10-3的合成:
Figure PCTCN2016085275-appb-000098
将上一步反应产物9-3(700mg,3.9mmol)溶于10mL二氯甲烷中，加入甲基硫代磺酸甲酯(0.5mL,14mmoL)和三乙胺(0.6mL,4.3mmol)。氮气保护下反应液室温搅拌过夜。加入1M稀盐酸15mL和50mL二氯甲烷，分出有机相，无水硫酸钠干燥，浓缩得1.2g粗产物10-3，为黄色油状物，直接用于下一步反应。LCMS(ESI)m/z 225.1(M+H)+.
化合物11-3的合成:
Figure PCTCN2016085275-appb-000099
将上一步反应产物10-3(1.2g,5.35mmol)和HOSu(700mg,6.08mmoL)溶于20mL二氯甲烷中，加入EDC-HCl(1.2g,6.28mmol)。反应液室温搅拌2小时。加入30mL水，乙酸乙酯萃取3次(50mL×3)，合并有机相，饱和食盐水洗(50mL)，无水硫酸钠干燥，浓缩。粗产物使用硅胶柱层析(石油醚/乙酸乙酯＝1:3)得产物1.7g，为黄色油状物，直接用于下一步反应。LCMS(ESI)m/z 339.0(M+H)+.
化合物12-3的合成:
Figure PCTCN2016085275-appb-000100
N-甲基-L-丙氨酸(560mg,5.28mmol)溶于20mL乙二醇二甲醚和20mL水中。加入三乙胺(1.5mL,10.56mmol)，剧烈搅拌。将上一步反应产物12-3(1.7g,5.29mmol)溶于20mL乙二醇二甲醚中逐滴加入，约5分钟加完。反应液室温搅拌3小时，减压移除有机溶剂，加入10mL水，使用1M的稀盐酸调PH＝3，乙酸乙酯萃取3次(50mL×3)，合并有机相，饱和食盐水洗涤3次(50mL×3)，无水硫酸钠干燥，浓缩。粗产物使用硅胶柱层析(石油醚 /乙酸乙酯/醋酸＝50:50:0.5)得产物460mg，为黄色油状物，产率25％。LCMS(ESI)m/z310.0(M+H)+.
化合物13-3-P1,13-3-P2的合成:
Figure PCTCN2016085275-appb-000101
上一步反应产物12-3(360mg,1.165mmol)溶于10mL甲醇，逐滴缓慢滴入0.5mL二氯亚砜，。反应液室温搅拌2小时，减压浓缩。加入50mL乙酸乙酯，缓慢加入20mL饱和碳酸氢钠溶液，分离有机相，无水硫酸钠干燥，浓缩。粗产品经手性制备(AD-H,4.6×250mm,CO2+MeOH＝2.55+0.45,P1:Rt＝2.19；P2:Rt＝2.77)HPLC纯化得119mg产物13-3-P1和130mg产物13-3-P2。LCMS(ESI)m/z 324.1(M+H)+.
化合物14-3-P2的合成:
Figure PCTCN2016085275-appb-000102
上一步反应产物13-3-P2(119mg,0.0368mmoL)溶于5mL甲醇和5mL水的混合溶液中加入氢氧化锂(50mg,2.08mmol)。反应液室温搅拌1.5小时。加入1M稀盐酸酸化调PH到3。乙酸乙酯萃取3次(50mL×3)，合并有机相，饱和食盐水(30mL)洗涤，无水硫酸钠干燥，浓缩得106mg产物14-3-P2，为无色油状物，产率94％，LCMS(ESI)m/z 310.0(M+H)+。
化合物14-3-P1的合成:
Figure PCTCN2016085275-appb-000103
14-3-P1的合成步骤和14-3-P2相同，使用13-3-P1为原料.
化合物CE-012,013的合成:
Figure PCTCN2016085275-appb-000104
将上一步产物14-3-P1(106mg,0.343mmol)，DCC(290mg,1.40mmol)加入一个干燥的Schlenck管中，置换氩气3次，加入2mL二氯甲烷，搅拌。将美登醇(96.5mg,0.171mmol)溶于8mL干燥二氯甲烷中加入，然后加入1M的氯化锌乙醚溶液(1mL,1mmol)。反应液室温搅拌2h，缓慢加入饱和碳酸氢钠溶液1mL淬灭反应，加入30mL乙酸乙酯，过滤，乙酸乙酯洗涤。滤液无水硫酸钠干燥，浓缩。粗产物使用硅胶柱层析(二氯甲烷/甲醇＝50:1至40:1)分离得到两个异构体CE-012(45mg，P1)和CE-013(40mg，P2)，均为白色固体，产率31％+28％.LCMS(ESI)m/z 838.3(M+H)+.
HPLC(15min):CE-012,Rt＝8.063；CE-013,Rt＝8.142
Mobile Phase:A:Water(0.01％TFA)B:CAN(0.01％TFA)
Gradient:0min 5％B,7min 95％B,15min 95％B
Flow Rate:1.0ml/min
Column:Xbridge C18,4.6*150mm,3.5um
Oven Temperature:40℃
CE-012:1H NMR(400MHz,CDCl3)δppm 6.85(s,1H),6.79(s,1H),6.44&6.41(dd,J1＝15.2Hz,J2＝10.8Hz,1H),6.26(d,J＝10.8Hz,1H),6.25(s,1H),5.86&5.83(dd,J1＝15.2Hz,J2＝9.2Hz,1H),5.17(q,J＝7.2Hz,1H),5.00(s,1H),4.94&4.91(dd,J1＝12.0Hz,J2＝3.2Hz,1H),4.33(t,J＝10.4Hz,1H),3.99(s,3H),3.52(d,J＝13.2Hz,1H),3.43(d,J＝8.8Hz,1H),3.38(s,3H),3.35(s,3H),3.20(d,J＝12.8Hz,1H),3.17(s,3H),3.04(s,3H),2.82(d,J＝9.2Hz,1H),2.66&2.63(dd,J1＝14.8Hz,J2＝12.0Hz,1H),2.51-2.46(m,2H),2.41(s,3H),2.24-2.18(m,1H),2.04-1.88(m,2H),1.76(d,J＝13.6Hz,1H),1.69(s,3H),1.55(s,2H),1.49(d,J＝7.2Hz,3H),1.30-1.35(m,1H),1.30(s,3H),1.28(s,3H),0.86(s,3H).
CE-013:1H NMR(400MHz,CDCl3)δppm 6.82(s,1H),6.74(d,J＝10.8Hz,1H),6.63(s,1H),6.44&6.41(dd,J1＝15.2Hz,J2＝10.8Hz,1H),6.21(s,1H),5.70&5.66(dd,J1＝15.2Hz,J2＝9.2Hz,1H),5.40(q,J＝7.2Hz,1H),4.79&4.78(dd,J1＝12.0Hz,J2＝3.2Hz,1H),4.28(t,J＝12.0Hz,1H),3.98(s,3H),3.65(d,J＝12.4Hz,1H),3.50(d,J＝8.8Hz,1H),3.35(s,3H),3.29(d,J＝2.8Hz,2H),3.26(s,3H),3.23(s,3H),3.11(d,J＝12.8Hz,1H),3.03(d,J＝10.0Hz,1H),2.85(s,3H),2.62&2.59(dd,J1＝14.8Hz,J2＝12.0Hz,1H),2.52-2.38(m,2H),2.30(s,3H),2.19&2.16(dd,J1＝14.4Hz,J2＝3.5Hz,1H),2.06-1.88(m,2H),1.64(s,3H),1.55(s,2H),1.50-1.42 (m,1H),1.20-1.30(m,1H),1.30(d,J＝2.8Hz,3H),1.28(d,J＝2.8Hz,3H),1.22(s,3H),0.80(s,3H).
化合物CE-014,015的合成:
Figure PCTCN2016085275-appb-000105
CE-014，CE-015的合成步骤和CE-012，CE-013相同，使用14-P2为原料.
CE-014:1H NMR(400MHz,CDCl3)δppm 6.85(s,1H),6.81(s,1H),6.43&6.40(dd,J1＝15.2Hz,J2＝11.2Hz,1H),6.28(d,J＝10.8Hz,1H),6.24(s,1H),5.88&5.85(dd,J1＝15.2Hz,J2＝9.6Hz,1H),5.19(q,J＝7.6Hz,1H),4.99(s,1H),4.91&4.88(dd,J1＝12.0Hz,J2＝3.2Hz,1H),4.32(t,J＝10.4Hz,1H),4.00(s,3H),3.52(d,J＝13.2Hz,1H),3.43(d,J＝9.2Hz,1H),3.39(s,3H),3.38(s,3H),3.35(s,3H),3.20(d,J＝12.8Hz,1H),3.17(s,3H),3.04(s,3H),2.84(d,J＝9.2Hz,1H),2.66&2.63(dd,J1＝14.8Hz,J2＝12.0Hz,1H),2.57-2.45(m,2H),2.41(s,3H),2.24-2.18(m,1H),2.04-1.88(m,2H),1.74(d,J＝13.6Hz,1H),1.69(s,3H),1.49(d,J＝7.2Hz,3H),1.38-1.30(m,1H),1.30(s,3H),1.28(s,3H),0.86(s,3H).
CE-015:1H NMR(400MHz,CDCl3)δppm 6.82(s,1H),6.74(d,J＝10.8Hz,1H),6.64(s,1H),6.44&6.41(dd,J1＝15.2Hz,J2＝10.8Hz,1H),6.23(s,1H),5.71&5.67(dd,J1＝15.2Hz,J2＝9.2Hz,1H),5.41(q,J＝7.2Hz,1H),4.80&4.77(dd,J1＝12.0Hz,J2＝3.2Hz,1H),4.28(t,J＝12.0Hz,1H),3.98(s,3H),3.65(d,J＝12.4Hz,1H),3.50(d,J＝8.8Hz,1H),3.35(s,3H),3.30(d,J＝2.8Hz,2H),3.26(s,3H),3.22(s,3H),3.11(d,J＝12.8Hz,1H),3.03(d,J＝10.0Hz,1H),2.85(s,3H),2.64-2.50(m,2H),2.36-2.28(m,1H),2.33(s,3H),2.19&2.16(dd,J1＝14.4Hz,J2＝3.5Hz,1H),2.06-1.88(m,2H),1.64(s,3H),1.55(s,2H),1.50-1.42(m,1H),1.35-1.25(m,1H),1.30(d,J＝2.8Hz,3H),1.28(d,J＝2.8Hz,3H),1.19(s,3H),0.80(s,3H).
化合物CE-032的合成:
Figure PCTCN2016085275-appb-000106
CE-015(40mg,0.047mmol)溶于0.6mL乙酸乙酯中，将二硫代苏糖醇DTT(18mg,0.12mmol)溶于1.2mL甲醇中加入，加入PH＝7.5的磷酸钾缓冲溶液2.7mL。反应液在氮气保护下搅拌2小时。加入PH＝6.0的磷酸钾缓冲溶液10.2mL淬灭反应。乙酸乙酯萃 取3次(20mL×3)，合并有机相，饱和食盐水洗涤3次(20mL×3)，无水硫酸钠干燥，浓缩。粗产品使用制备HPLC(CH3CN in H2O-0.05％TFA from 5％to 95％)纯化得到22mg产物CE-032，白色固体，产率80％。LCMS(ESI)m/z 808.2(M+H)+.
化合物CE-004的合成：
Figure PCTCN2016085275-appb-000107
将CE-032(30mg,0.037mmol)溶于4mLN,N-二甲基甲酰胺中，加入SM1(24mg,0.074mmol)。反应液室温搅拌过夜，过滤，滤液直接使用制备HPLC(CH3CN in H2O-0.05％TFA from 5％to 95％)纯化得到20.4mg产物CE-004，为白色固体。产率54％。LCMS(ESI)m/z 1024.2(M+H)+.
1H NMR(400MHz,CDCl3)δppm 6.84(s,1H),6.73(d,J＝11.2Hz,1H),6.63(s,1H),6.44&6.42(dd,J1＝15.2Hz,J2＝11.2Hz,1H),6.29(s,1H),5.70&5.68(dd,J1＝15.2Hz,J2＝9.2Hz,1H),5.41(q,J＝6.8Hz,1H),4.78&4.78(dd,J1＝11.6Hz,J2＝2.8Hz,1H),4.28(q,J＝10.4Hz,1H),3.98(s,3H),3.65(d,J＝13.2Hz,1H),3.50(d,J＝8.8Hz,1H),3.36(s,3H),3.28(d,J＝3.2Hz,2H),3.25(s,3H),3.22(s,3H),3.12(d,J＝8.4Hz,1H),3.03(d,J＝10.0Hz,1H),2.84(s,6H),2.70(q,J＝7.6Hz,4H),2.64-2.49(m,2H),2.36-2.28(m,1H),2.18&2.17(dd,J1＝14.4Hz,J2＝2.8Hz,1H),2.10-1.98(m,4H),1.94-1.86(m,1H),1.64(s,3H),1.58(d,J＝13.2Hz,1H),1.50-1.42(m,1H),1.30(s,3H),1.28(s,3H),1.30-1.24(m,1H),1.19(s,3H),0.80(s,3H).
实施例4CE-018和CE-019的合成路线
Figure PCTCN2016085275-appb-000108
实验步骤
化合物3-4的合成:
Figure PCTCN2016085275-appb-000109
化合物1-4(2.0g,7.2mmol)溶于80mL水和20mL甲醇的混合溶液中，调PH到8，加入苄胺(1.53g,14mmol)。反应液氮气保护下室温搅拌1小时。加入甲基硫代磺酸甲酯(1.18g,9.4mmol)，反应液氮气保护下室温搅拌过夜。减压移除甲醇，加入二氯甲烷萃取(80mL)，分出水相，使用2N的稀盐酸调PH＝2，使用乙酸乙酯萃取三次(50mL×3)，合并有机相，水洗(50mL)，饱和食盐水洗(50mL)，无水硫酸钠干燥，浓缩得粗产物1.1g，为黄色油状物，直接用于下一步反应。
LCMS(ESI)m/z 206(M+H)+.1H NMR(400MHz,CD3OD)δppm 2.51(s,3H),2.46-2.42(m,2H),2.19-2.05(m,2H),1.56(s,3H).
化合物4-4的合成:
Figure PCTCN2016085275-appb-000110
将上一步反应产物3-4(1.8g,8.78mmol)和HOSu(1.51g,13mmoL)溶于20mL二氯甲烷中，加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)(2.51g,13mmol)。反应液室温搅拌2小时。加入50mL水，乙酸乙酯萃取3次(50mL×3)，合并有机相，饱和食盐水洗涤3次(50mL×3)，无水硫酸钠干燥，浓缩得2.2g为黄色油状物，直接用于下一步反应。LCMS(ESI)m/z 303.1(M+H)+.
化合物5-4的合成:
Figure PCTCN2016085275-appb-000111
N-甲基-L-丙氨酸(900mg,8.74mmol)溶于20mL N,N-二甲基甲酰胺中。加入三乙胺(0.5mL,3.5mmol)，剧烈搅拌。将上一步反应产物4-4(2.2g,7.28mmol)溶于5mL N,N-二甲基甲酰胺中逐滴加入，约5分钟加完。反应液室温搅拌过夜。加入1M的稀盐酸调PH＝3，乙酸乙酯萃取3次(50mL×3)，合并有机相，饱和食盐水洗涤3次(50mL×3)，无水硫酸钠 干燥，浓缩。粗产物使用硅胶柱层析(石油醚/乙酸乙酯/乙酸＝50:50:0.5)得产物1.2g，为黄色油状物，产率57％。
LCMS(ESI)m/z 291.1(M+H)+.1H NMR(400MHz,CD3OD)δppm 5.03(q,J＝7.2Hz,1H),3.05(s,3H),2.72-2.68(m,2H),2.63(s,3H),2.36-2.15(m,2H),1.71(s,3H),1.43(d,J＝7.2Hz,3H).
化合物6-4的合成:
Figure PCTCN2016085275-appb-000112
将上一步产物5-4(130mg,0.45mmol)，DCC(130mg,0.45mmol))加入一个干燥的Schlenck管中，置换氩气3次，加入1mL二氯甲烷，搅拌。将美登醇(84.0mg,0.15mmol)溶于5mL干燥二氯甲烷中加入，然后逐滴加入1M的氯化锌乙醚溶液(1mL,1mmol)。反应液室温搅拌2h，缓慢加入5mL饱和碳酸氢钠溶液淬灭反应，加入30mL乙酸乙酯，分出有机相，无水硫酸钠干燥，浓缩。粗产物使用制备硅胶板(二氯甲烷/甲醇＝20:1)分离得到两个异构体CE-018(15mg，P1)和CE-019(15mg，P2)，均为白色固体，产率12％+12％.LCMS(ESI)m/z 837.2(M+H)+.
HPLC(15min):CE-018,Rt＝7.812；CE-019,Rt＝7.656
Mobile Phase:A:Water(0.01％TFA)B:ACN(0.01％TFA)
Gradient:0min 5％B,7min 95％B,15min 95％B
Flow Rate:1.0ml/min
Column:Xbridge C18,4.6*150mm,3.5um
Oven Temperature:40℃
CE-018:1H NMR(400MHz,CDCl3)δppm 6.85(s,1H),6.79(s,1H),6.45&6.43(dd,J1＝15.2Hz,J2＝10.8Hz,1H),6.37(s,1H),6.24(d,J＝10.8Hz,1H),5.83&5.81(dd,J1＝15.2Hz,J2＝9.2Hz,1H),5.23-5.18(m,1H),4.95(d,J＝10.0Hz,1H),4.29(t,J＝10.4Hz,1H),3.99(s,3H),3.52(d,J＝12.8Hz,1H),3.45(d,J＝9.2Hz,1H),3.35(s,3H),3.20(d,J＝12.8Hz,1H),3.18(s,3H),3.05(d,J＝5.6Hz,3H),2.81(d,J＝10.0Hz,1H),2.69-2.58(m,2H),2.63(s,3H),2.36-2.15(m,2H),2.09-2.02(m,2H),1.74(d,J＝14.4Hz,3H),1.76-1.69(m,1H),1.69(s,3H),1.52(d,J＝7.2Hz,3H),1.49-1.40(m,1H),1.35-1.30(m,1H),1.28(d,J＝6.4Hz,3H),0.86(s,3H).
CE-019:1H NMR(400MHz,CDCl3)δppm 6.83(s,1H),6.72&6.69(dd,J1＝15.6Hz,J2＝2.8Hz,1H),6.61(s,1H),6.45&6.42(dd,J1＝15.6Hz,J2＝10.8Hz,1H),6.27(s,1H),5.69-5.62(m,1H),5.45-5.36(m,1H),4.78(d,J＝10.8Hz,1H),4.28(t,J＝10.8Hz,1H),3.99(s,3H), 3.63&3.61(dd,J1＝12.4Hz,J2＝9.2Hz,1H),3.51(d,J＝9.2Hz,1H),3.36(s,3H),3.23(d,J＝9.2Hz,3H),3.15(t,J＝11.6Hz,3H),3.03(d,J＝5.6Hz,3H),2.88(s,3H),2.69-2.58(m,2H),2.55(d,J＝22.0Hz,3H),2.40-2.05(m,2H),1.76-1.69(m,1H),1.64(s,3H),1.58(d,J＝8.0Hz,1H),1.60-1.40(m,1H),1.32-1.28(m,7H),0.80(s,3H).
实施例5CE-021和CE-022的合成路线
Figure PCTCN2016085275-appb-000113
实验步骤
化合物2-5的合成:
Figure PCTCN2016085275-appb-000114
将原料1-5(10g,116mmol)溶于50mL无水甲醇中，加入硫脲(18g,232mmol)。反应液加热至50℃搅拌24小时，TLC点板检测原料已完全消耗。反应液冷却至室温，加入100m水，使用沸点为30℃～60℃的石油醚萃取3次(50mL×3)。合并有机相，饱和食盐水洗3次(50mL×3)，无水硫酸钠干燥，浓缩，粗产品使用硅胶柱层析(沸点为30℃～60℃石油醚)得到4.7g产物2-5，为无色油状物，产率40％。1H NMR(400MHz,CDCl3)δppm2.71(m,J＝10.0Hz,1H),2.47(d,J＝6.4Hz,1H),2.18(d,J＝6.0Hz,1H),1.38-1.24(m,1H),1.09(t,J＝10.0Hz,6H).
化合物3-5的合成:
Figure PCTCN2016085275-appb-000115
氮气保护下，将正丁基锂(2.5M,17.6mL,43.2mmol)加入到冷却至-78℃的150mL干燥四氢呋喃溶液中。将乙腈(1.8g,44.8mmol)溶于10mL干燥四氢呋喃加入。反应液在-78℃搅拌半小时。将化合物2-5(4g,39.2mmol)溶于20mL四氢呋喃中，并逐滴加入。加 入完毕，反应液逐步升温至室温并搅拌3小时。将反应液冷却至0℃，逐滴缓慢加入稀盐酸(0.5M,20mL)淬灭反应，乙酸乙酯萃取3次(100mL×3)，合并有机相，饱和食盐水洗3次(100mL×3)，无水硫酸钠干燥，浓缩得3.8g粗产品，为浅黄色油状物，直接用于下一步反应，产率68％。LCMS(ESI)m/z144.1(M+H)+.
化合物4-5的合成:
Figure PCTCN2016085275-appb-000116
将上一步反应产物3-5(2.86g,20mmol)溶于20mL乙醇中，置换氩气三次，缓慢加入8M的氢氧化钠溶液(10mL,80mmol)。氩气保护下反应液升温回流过夜。反应液冷却至室温，稀盐酸调PH＝2，二氯甲烷萃取3次(50mL×3)，合并有机相，无水硫酸钠干燥，浓缩。粗产物使用硅胶柱层析(石油醚/乙酸乙酯＝2:1)纯化得2.4g产品4-5，为浅黄色油状物，产率75％。LCMS(ESI)m/z163.1(M+H)+。1H NMR(400MHz,d6-DMSO)δppm12.07(br,1H),3.35(br,1H),2.73-2.66(m,1H),2.49-2.28(m,2H),2.04-2.00(m,1H),1.93-1.77(m,1H),1.60-1.50(m,1H),0.92(d,J＝6.8Hz,3H),0.86(d,J＝6.8Hz,3H).
化合物5-5的合成:
Figure PCTCN2016085275-appb-000117
氩气保护下将上一步反应产物4-5(810mg,5mmol)溶于15mL二氯甲烷中，加入甲基硫代磺酸甲酯(0.65mL,18mmoL)和三乙胺(0.8mL)。氩气保护下反应液室温搅拌过夜。加入1M的稀盐酸40mL，乙酸乙酯萃取3次(50mL×3)，合并有机相，饱和食盐水洗涤(50mL)，无水硫酸钠干燥，浓缩得780mg粗产物5-5，直接用于下一步反应。LCMS(ESI)m/z 209.1(M+H)+.
化合物6-5的合成:
Figure PCTCN2016085275-appb-000118
将上一步反应产物5-5(730mg,3.5mmol)和HOSu(445mg,3.85mmoL)溶于20mL二 氯甲烷中，加入EDC(800mg,4.2mmol)。反应液室温搅拌2小时。加入30mL水和50mL乙酸乙酯，分出有机相，饱和食盐水洗涤3次(50mL×3)，无水硫酸钠干燥，浓缩得578mg粗产物6-5，为黄色油状物，直接用于下一步反应。LCMS(ESI)m/z 306.1(M+H)+.
化合物7-5的合成:
Figure PCTCN2016085275-appb-000119
N-甲基-L-丙氨酸(190mg,1.8mmol)溶于10mL乙二醇二甲醚和10mL水中。加入三乙胺(0.5mL,3.6mmol)，剧烈搅拌。将上一步反应产物6-5(550mg,1.8mmol)溶于5mL乙二醇二甲醚中逐滴加入，约5分钟加完。反应液室温搅拌3小时，减压移除有机溶剂，加入10mL水，使用1M的稀盐酸调PH＝3，乙酸乙酯萃取3次(3×50mL)，合并有机相，饱和食盐水洗涤3次(50mL×3)，无水硫酸钠干燥，浓缩。粗产物使用硅胶柱层析(石油醚/乙酸乙酯/乙酸＝50:50:0.5)得260mg产物7-5，为黄色油状物，产率50％。LCMS(ESI)m/z293.1(M+H)+。
化合物CE-021和CE-022的合成
Figure PCTCN2016085275-appb-000120
将上一步产物7-5(40mg,0.138mmol)，DCC(76mg,0.368mmol)加入一个干燥的Schlenck管中，置换氩气3次，加入1mL二氯甲烷，搅拌。将美登醇(26mg,0.046mmol)溶于4mL干燥二氯甲烷中加入，然后加入1M的氯化锌乙醚溶液(0.3mL,0.3mmol)。反应液室温搅拌2h，缓慢加入0.5mL淬灭反应，加入20mL乙酸乙酯，过滤，乙酸乙酯洗涤。滤液无水硫酸钠干燥，浓缩。粗产物使用硅胶柱层析(二氯甲烷/甲醇＝50:1至40:1)分离得到两个异构体CE-021(6mg，P1)和CE-022(12mg，P2)，均为白色固体，产率18.5％+36.7％。LCMS(ESI)m/z 840.9(M+H)+。
HPLC(15min):CE-021,Rt＝11.197；CE-022,Rt＝11.292&11.345
Mobile Phase:A:water(0.01％TFA)B:CAN(0.01％TFA)
Gradient:0min 5％B,3min 5％B,10min 95％B,15min 95％B
Flow Rate:1.2ml/min
Column:Eclipse XDB-C18,4.6×150mm,5um
Oven Temperature:40℃
CE-021:1H NMR(400MHz,CDCl3)δppm 6.85(s,1H),6.79(s,1H),6.44(t,J＝12.8Hz,1H),6.29(d,J＝6.8Hz,1H),6.24(t,d,J＝6.8Hz,1H),5.88-5.80(m,1H),5.18-5.08(m,1H),4.98-4.90(m,1H),4.32(t,J＝10.4Hz,1H),3.99(s,3H),3.51(d,J＝12.8Hz,1H),3.44(d,J＝8.8Hz,1H),3.34(s,3H),3.20(d,J＝12.8Hz,1H),3.17(s,3H),3.05(d,J＝5.6Hz,3H),2.82(t,J＝10.0Hz,1H),2.69-2.57(m,4H),2.41(s,3H),2.23-2.20(m,1H),2.09-2.02(m,2H),1.76-1.69(m,1H),1.69(s,3H),1.50(d,J＝7.2Hz,3H),1.49-1.40(m,1H),1.30(s,3H),1.28(s,3H),1.03(d,J＝6.0Hz,6H),0.86(s,3H).
CE-022:1H NMR(400MHz,CDCl3)δppm 6.82(s,1H),6.76&6.75(dd,J1＝11.2Hz,J2＝6.4Hz,1H),6.64(d,J＝4.0Hz,1H),6.44&6.41(dd,J1＝14.8Hz,J2＝11.2Hz,1H),6.22(s,1H),5.72-5.63(m,1H),5.43-5.37(m,1H),4.81-4.76(m,1H),4.28(t,J＝11.2Hz,1H),3.99(s,3H),3.67&3.65(dd,J1＝12.4Hz,J2＝8.0Hz,1H),3.50(d,J＝10.4Hz,1H),3.35(s,3H),3.30(d,J＝7.2Hz,1H),3.23(s,3H),3.11(d,J＝13.2Hz,1H),3.04(d,J＝10.0Hz,1H),2.85(s,3H),2.70-2.33(m,4H),2.30(d,J＝6.4Hz,3H),2.23-2.16(m,1H),2.04-1.89(m,2H),1.71-1.65(m,1H),1.64(s,3H),1.60-1.55(m,2H),1.51-1.43(m,1H),1.30(d,J＝6.8Hz,3H),1.27(s,3H),0.99-0.88(m,6H),0.80(s,3H).
按照实施例1化合物CE-005的制备方法，将中间体CE-017替换为CE-022，进行后续反应，即可得到化合物CE-002。
实施例6CE-033的合成路线
Figure PCTCN2016085275-appb-000121
实验步骤
化合物2-6的合成:
Figure PCTCN2016085275-appb-000122
将化合物1-6(2.0g,9.4mmol)溶于20mL N,N-二甲基甲酰胺中，加入三乙胺(1.9g,18.8mmol)和马来酸酐(1.8g,18.8mmol)。反应液室温搅拌过夜。加入50mL水，乙酸乙酯萃取3次(50mL×3)，合并有机相，饱和食盐水洗3次(50mL×3)，无水硫酸钠干燥，浓缩。粗产品经制备HPLC(CH3CN in H2O-0.05％TFA from 5％to 95％)纯化得1g产物2-6，为黄色油状物，产率76％。LCMS(ESI)m/z 312.2(M+H)+。
化合物3-6的合成:
Figure PCTCN2016085275-appb-000123
将N-羟基丁二酰亚胺(1.5g,12.8mmol)溶于N,N-二甲基甲酰胺15mL中，冷却至0℃，加入三氟乙酸酐(1.8mL,12.8mmol)，搅拌10分钟。
上一步反应产物2-6(1.0g,3.2mmol)溶于N,N-二甲基甲酰胺15mL中，冷至0℃，搅拌，加入2,4,6-三甲基吡啶(774.4mg,6.4mmol)，加入上述反应液。升至室温搅拌过夜。加入1N的稀盐酸20mL淬灭反应，二氯甲烷萃取3次(30mL×3)，合并有机相，1N稀盐酸洗涤3次(20mL×3)，无水硫酸钠干燥，浓缩。粗产品经制备HPLC(CH3CN in H2O-0.05％TFA from 5％to 95％)纯化得752mg产物3-6，为白色固体，产率80％。LCMS(ESI)m/z 295.1(M+H)+。
化合物4-6的合成:
Figure PCTCN2016085275-appb-000124
上一步反应产物3-6(700mg,2.4mmol)溶于10mL二氯甲烷中加入3mL三氟乙酸。 反应液室温搅拌4小时，浓缩得442mg粗产品，为黄色油状物，产率95％，直接用于下一步反应。LCMS(ESI)m/z 195.2(M+H)+。
化合物5-6的合成:
Figure PCTCN2016085275-appb-000125
上一步反应产物4-6(300mg,1.6mmol)溶于40mL乙腈中，加入三乙胺(315mg,3.2mmol)和N,N'-二琥珀酰亚胺基碳酸酯(798mg,3.2mmol)。反应液室温搅拌过夜。浓缩，粗产品经制备HPLC(CH3CN in H2O-0.05％TFA from 5％to 95％)纯化得188mg产物5-6，为白色固体，产率35％。LCMS(ESI)m/z 336.2(M+H)+.
化合物CE-033的合成:
Figure PCTCN2016085275-appb-000126
氩气保护下，将DM1(N2'-去乙酰基-N2'-(3-巯基-1-氧代丙基)美登素)(37mg,0.05mmol)溶于2mL四氢呋喃中，加入PH＝6.0的磷酸钾缓冲溶液2mL和上一步反应产物5-6(20mg,0.06mmol)。反应液氩气保护下室温搅拌过夜。过滤，滤液直接进行制备纯化得28mg产品CE-033，为白色固体，产率51％。LCMS(ESI)m/z 1094.8(M+Na)+.
1H NMR(400MHz,CDCl3)δppm 12.30(br,1H),6.84(s,1H),6.68(t,J＝7.2Hz,1H),6.64(d,J＝7.2Hz,1H),6.42(t,J＝12.8Hz,1H),6.24(s,1H),5.70-5.61(m,1H),5.37(q,J＝6.8Hz,1H),4.79(d,J＝12.0Hz,1H),4.28(t,J＝10.4Hz,1H),4.21-4.05(m,2H),3.99(s,3H),3.76-3.64(m,3H),3.49(d,J＝8.8Hz,1H),3.42(d,J＝7.2Hz,1H),3.35(d,J＝2.4Hz,3H),3.32(d,J＝6.4Hz,1H),3.20(d,J＝2.8Hz,3H),3.19-3.08(m,6H),3.02(d,J＝9.6Hz,1H),3.01-2.93(m,2H),2.85(d,J＝2.8Hz,3H),2.82(s,3H),2.68-2.56(m,2H),2.43-2.33(m,1H),2.18(d,J＝14.4Hz,1H),1.95-1.85(m,1H),1.70(s,3H),1.59-1.55(m,1H),1.50-1.43(m,1H),1.35-1.23(m,10H),0.80(s, 3H).
实施例7CE-028,034的合成路线
Figure PCTCN2016085275-appb-000127
实验步骤
化合物2-7的合成:
Figure PCTCN2016085275-appb-000128
250mL茄形瓶中加入化合物1-7(64g,0.6mol)，搅拌下加入氢氧化钾(11.2g,0.2mol)。将该悬浊液加热至90℃，搅拌至氢氧化钾完全溶解。苄溴(34g,23.6mL,0.2mol)逐滴缓慢加入。反应液升温至110℃，搅拌过夜。反应液冷却至室温，加入800mL水，乙酸乙酯萃取3次(150mL×3)，合并有机相，水洗3次(150mL×3)，饱和食盐水洗(150mL)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯10:1至3:1)纯化得30g产品2-7，为无色油状物，产率76％。LCMS(ESI)m/z 197.1(M+H)+
化合物3-7的合成:
Figure PCTCN2016085275-appb-000129
上一步反应产物2-7(19.6g,0.1mol)和三乙胺(16.6mL,0.12mol)溶于150mL二氯甲 烷中，冷却至0℃，逐滴加入甲烷磺酰氯(8.5mL,0.11mol)。反应液升至室温搅拌2小时。加入50mL水淬灭反应，分出有机相，饱和食盐水洗3次(50mL×3)，无水硫酸钠干燥，浓缩得27g粗产品3-7，为黄色油状物，直接用于下一步反应。
化合物4-7的合成:
Figure PCTCN2016085275-appb-000130
将上一步反应产物3-7(27.4g,0.1mol)和L-乳酸乙酯(23.6g,0.2mol)溶于N,N-二甲基甲酰胺150mL中，冷却至0℃，分批缓慢加入钠氢(8g,60％含量,悬浮于矿物油中,0.2mol)。反应液升至室温搅拌过夜。将反应液冷至0℃，逐滴缓慢加入20mL饱和氯化铵溶液淬灭反应，加入150mL水，乙酸乙酯萃取3次(100mL×3)，合并有机相，水洗3次(100mL×3)，饱和食盐水洗(100mL)，无水硫酸钠干燥，浓缩。粗产品硅胶柱层析(石油醚/乙酸乙酯＝10:1至2:1)纯化得12g产品4-7，为浅黄色油状物，产率40％。LCMS(ESI)m/z 297.1(M+H)+,
化合物5-7的合成:
Figure PCTCN2016085275-appb-000131
上一步反应产物4-7(12g,40mmol)溶于50mL甲醇中，置换氮气3次，加入200mg10％的钯碳干粉。反应液置换氢气3次，氢气氛中室温搅拌过夜。置换氮气3次，加入50mL二氯甲烷，过滤，二氯甲烷洗涤，滤液浓缩，得粗产品8.0g，为浅黄色油状物，直接用于下一步反应。LCMS(ESI)m/z 207.1(M+H)+,230.1(M+Na)+.
化合物6-7的合成:
Figure PCTCN2016085275-appb-000132
上一步反应产物5-7(10.3g,50mmol)和三乙胺(8.3mL,60mmol)溶于80mL二氯甲烷中，冷却至0℃，逐滴缓慢加入甲烷磺酰氯(4.4mL,55mmol)。反应液升至室温搅拌2小时。加入50mL水淬灭反应，分出有机相，饱和食盐水洗涤3次(50mL×3)，无水硫酸钠 干燥，浓缩得粗产品，为浅黄色油状物，直接用于下一步反应。
化合物7-7的合成:
Figure PCTCN2016085275-appb-000133
上一步反应粗产物6-7(2g,7.0mmol)溶于N,N-二甲基甲酰胺20mL中，加入硫代乙酸钾(1.6g,14mmol)。反应液室温搅拌过夜，加入30mL水，乙酸乙酯萃取3次(50mL×3)，合并有机相，水洗3次(30mL×3)，饱和食盐水洗(50mL)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析纯化得1.66g产品7-7，为棕色油状物，产率90％。LCMS(ESI)m/z 265.1(M+H)+,
化合物8-7的合成:
Figure PCTCN2016085275-appb-000134
氮气保护下，将上一步反应产物7-7(1.66g,6.3mmol)溶于20mL甲醇和10mL水的混合溶剂中，加入氢氧化锂(0.76g,31.5mmol)。反应液在氮气保护下室温搅拌2小时，加入甲基硫代磺酸甲酯(0.95g,7.6mmol)。反应液继续在室温下搅拌过夜。加入50mL水，乙酸乙酯萃取3次(50mL×3)，合并有机相，饱和食盐水洗3次(50mL×3)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝1:2)纯化得0.91g产物8-7，为浅黄色油状物，产率60％。LCMS(ESI)m/z 241.0(M+H)+,263.0(M+Na)+,
化合物CE-028的合成:
Figure PCTCN2016085275-appb-000135
将上一步产物8-7(80mg,0.34mmol)，DCC(148mg,0.72mmol)和DMAP(29mg,0.24mmol)加入一个干燥的Schlenck管中，置换氩气3次，加入1mL二氯甲烷，搅拌。将美登醇(63mg,0.12mmol)溶于4mL干燥二氯甲烷中加入。反应液室温搅拌2h，缓慢加入0.3mL水淬灭反应，加入15mL乙酸乙酯，过滤，乙酸乙酯洗涤。滤液无水硫酸钠干燥，浓缩。粗产物使用制备HPLC分离得到28mg产物CE-028，为白色固体，产率30％。 LCMS(ESI)m/z 787.2(M+H)+.
1H NMR(400MHz,CDCl3)δppm 6.83(d,J＝10.8Hz,2H),6.44&6.42(dd,J1＝15.2Hz,J2＝10.8Hz,1H),6.22(s,1H),6.19(d,J＝11.2Hz,1H),5.57&5.55(dd,J1＝14.8Hz,J2＝8.4Hz,1H),4.93(d,J＝12.0Hz,1H),4.27(t,J＝10.8Hz,1H),4.18(q,J＝6.8Hz,1H),3.99(s,3H),3.78(t,J＝6.4Hz,3H),3.69-3.64(m,1H),3.67(s,3H),3.51(t,J＝9.2Hz,2H),3.36(s,3H),3.20(d,J＝12.8Hz,1H),3.15(s,3H),2.92(d,J＝6.4Hz,3H),2.58(t,J＝14.0Hz,1H),2.43(s,3H),2.30-2.25(m,1H),2.01(q,J＝6.8Hz,1H),1.69(s,3H),1.52(d,J＝7.2Hz,3H),1.53-1.50(m,1H),1.29(d,J＝7.2Hz,3H),0.83(s,3H).
化合物9-7的合成:
Figure PCTCN2016085275-appb-000136
CE-028(12mg,0.015mmol)溶于0.5mL乙酸乙酯和0.5mL甲醇的混合溶剂中，将二硫代苏糖醇DTT(18mg,0.117mmol)溶于0.5mL PH＝7.5的磷酸钾缓冲溶液中加入。反应液在氮气保护下搅拌3小时。加入PH＝6的磷酸钾缓冲溶液1mL淬灭反应。乙酸乙酯萃取3次(5mL×3)，合并有机相，饱和食盐水洗涤(5mL)，无水硫酸钠干燥，浓缩。粗产品使用制备HPLC(CH3CN in H2O-0.05％TFA from 5％to 95％)纯化得到8.9mg产物9-7，为白色固体，产率80％。LCMS(ESI)m/z 741.3(M+H)+
化合物CE-034的合成:
Figure PCTCN2016085275-appb-000137
上一步反应产物9-7(8.9mg,0.012mmol)溶于1.5mL四氢呋喃中，加入PH＝6的磷酸钾缓冲溶液1.5mL和4-(N-马来酰亚胺甲基)环己烷-1-羧酸琥珀酰亚胺酯(20mg,0.06mmol)，氮气保护下室温搅拌过夜。反应液过滤，直接由制备HPLC纯化得11mg产品CE-034，为白色固体，产率85％。LCMS(ESI)m/z 1075.4(M+H)+.
1H NMR(400MHz,CDCl3)δppm 6.82(d,J＝17.2Hz,2H),6.42(d,,J＝15.2Hz,2H),6.19(d,J＝14.4Hz,1H),5.56&5.54(dd,J1＝14.8Hz,J2＝8.4Hz,1H),4.91(d,J＝14.4Hz,1H),4.27(t,J＝10.8Hz,1H),4.15(q,J＝6.8Hz,1H),3.99(s,3H),3.83-3.76(m,3H),3.66(s,3H),3.50(t, J＝9.2Hz,2H),3.40(d,J＝12.0Hz,2H),3.35(s,3H),3.21-3.15(m,3H),3.15(s,3H),2.91-2.83(m,3H),2.83(s,3H),2.62-2.50(m,3H),2.25-2.14(m,3H),1.81-1.70(m,4H),1.68(s,3H),1.58-1.45(m,6H),1.29(d,J＝7.2Hz,6H),1.08(d,J＝12.0Hz,2H),0.83(s,3H).
实施例8CE-027,035的合成路线
Figure PCTCN2016085275-appb-000138
实验步骤
化合物2-8的合成:
Figure PCTCN2016085275-appb-000139
化合物1-8(9.7mL,84mmol)和烯丙基溴(9.3mL,108mmol)溶于200mL乙醚中，加入氧化银(21g,92.4mmol)。将该悬浊液加热回流过夜。反应液冷却至室温，过滤，乙酸乙酯洗涤，滤液浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯10:1)纯化得3.9g产品2-8，为无色油状物，产率30％。
1H NMR(400MHz,CDCl3)δppm 5.97-5.87(m,1H),5.29(d,J＝16.8Hz,1H),5.20(d,J＝10.0Hz,1H),4.25-4.12(m,3H),4.04-3.92(m,2H),1.42(d,J＝6.8Hz,3H),1.29(t,J＝7.2Hz,3H).
化合物3-8的合成:
Figure PCTCN2016085275-appb-000140
上一步反应产物2-8(3.9g,24.7mmol)溶于40mL四氢呋喃中，冷至0℃，搅拌下逐滴加入1M硼烷四氢呋喃溶液(29mL,29mmol)。反应液升至室温搅拌2小时。反应液冷却 至0℃，逐滴加入3N的氢氧化钠溶液(10mL,30mmol)，缓慢加入15mL30％含量的双氧水，反应液升至室温搅拌过夜。加入200mL乙醚，分出有机相，饱和食盐水洗(50mL×3)，无水硫酸钠干燥，浓缩得1.6g粗产品3-8，为浅黄色油状物，直接用于下一步反应。
1H NMR(400MHz,CDCl3)δppm 4.27-4.17(m,2H),3.96(q,J＝7.2Hz,1H),3.89-3.71(m,2H),3.68-3.61(m,2H),2.82(s,1H),1.93-1.75(m,2H),1.40(d,J＝7.2Hz,3H),1.27(t,J＝6.8Hz,3H).
化合物4-8的合成:
Figure PCTCN2016085275-appb-000141
上一步反应产物3-8(7.04g,40mmol)和三乙胺(6.6mL,48mmol)溶于50mL二氯甲烷中，冷却至0℃，逐滴缓慢加入甲烷磺酰氯(3.5mL,44mmol)。反应液升至室温搅拌2小时。加入50mL水淬灭反应，分出有机相，饱和食盐水洗涤3次(50mL×3)，无水硫酸钠干燥，浓缩得粗产品，为浅黄色油状物，直接用于下一步反应。LCMS(ESI)m/z 255.1(M+H)+。
化合物5-8的合成:
Figure PCTCN2016085275-appb-000142
上一步反应粗产物4-8(2.54g,10.0mmol)溶于N,N-二甲基甲酰胺20mL中，加入硫代乙酸钾(2.3g,20mmol)。反应液室温搅拌过夜，加入30mL水，乙酸乙酯萃取3次(50mL×3)，合并有机相，水洗3次(30mL×3)，饱和食盐水洗(50mL)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝1:1)纯化得2.0g产品5-8，为棕色油状物，产率86％。LCMS(ESI)m/z 235.2(M+H)+,
化合物7-8的合成:
Figure PCTCN2016085275-appb-000143
氮气保护下，将上一步反应产物5-8(1.87g,8mmol)溶于20mL甲醇和10mL水的混 合溶剂中，加入氢氧化锂(0.78g,32mmol)。反应液在氮气保护下室温搅拌2小时，加入甲基硫代磺酸甲酯(1.2g,9.6mmol)。反应液继续在室温下搅拌过夜。加入50mL水，乙酸乙酯萃取3次(50mL×3)，合并有机相，饱和食盐水洗3次(50mL×3)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝1:2)纯化得1.18g产物7-8，为浅黄色油状物，产率70％。
LCMS(ESI)m/z197.0(M+H)+,211.1(M+Na)+,1H NMR(400MHz,CDCl3)δppm 4.02(q,J＝7.2Hz,1H),3.70-3.60(m,2H),2.82(t,J＝7.2Hz,2H),2.41(s,3H),2.05-2.01(m,2H),1.47(d,J＝6.8Hz,3H).
化合物CE-027的合成:
Figure PCTCN2016085275-appb-000144
将上一步产物7-8(63mg,0.30mmol)，DCC(124mg,0.6mmol)和DMAP(24mg,0.2mmol)加入一个干燥的Schlenck管中，置换氩气3次，加入1mL二氯甲烷，搅拌。将美登醇(56mg,0.10mmol)溶于4mL干燥二氯甲烷中加入。反应液室温搅拌2h，缓慢加入0.3mL水淬灭反应，加入15mL乙酸乙酯，过滤，乙酸乙酯洗涤。滤液无水硫酸钠干燥，浓缩。粗产物使用制备HPLC分离得到23mg产物CE-027，为白色固体，产率30％。
LCMS(ESI)m/z 757.3(M+H)+.1H NMR(400MHz,CDCl3)δppm 6.96(s,1H),6.84(s,1H),6.48&6.44(dd,J1＝21.2Hz,J2＝6.8Hz,1H),6.23(s,1H),6.18(d,J＝6.8Hz,1H),5.48&5.45(dd,J1＝14.8Hz,J2＝8.4Hz,1H),4.92(d,J＝12.0Hz,1H),4.25(t,J＝13.2Hz,1H),4.13(q,J＝6.8Hz,1H),3.99(s,3H),3.65(q,J＝6.8Hz,1H),3.56-3.43(m,3H),3.36(s,3H),3.21(d,J＝12.8Hz,1H),3.17(s,3H),3.06(s,1H),2.87(d,J＝8.4Hz,1H),2.80(t,J＝7.2Hz,1H),2.58&2.55(dd,J1＝19.2Hz,J2＝8.4Hz,1H),2.37(s,3H),2.36-2.20(m,2H),2.03-1.96(m,3H),1.68(s,3H),1.47(d,J＝6.8Hz,3H),1.38-1.30(m,1H),1.30(d,J＝7.2Hz,3H),0.84(s,3H).
化合物8-8的合成:
Figure PCTCN2016085275-appb-000145
CE-027(15mg,0.02mmol)溶于0.5mL乙酸乙酯和0.5mL甲醇的混合溶剂中，将二硫代苏糖醇DTT(18mg,0.117mmol)溶于0.5mL PH＝7.5的磷酸钾缓冲溶液中加入。反应 液在氮气保护下搅拌3小时。加入PH＝6的磷酸钾缓冲溶液1mL淬灭反应。乙酸乙酯萃取3次(5mL×3)，合并有机相，饱和食盐水洗涤(5mL)，无水硫酸钠干燥，浓缩。粗产品使用制备HPLC(CH3CN in H2O-0.05％TFA from 5％to 95％)纯化得到11mg产物8-8，为白色固体，产率77％。LCMS(ESI)m/z711.3(M+H)+.
化合物CE-035的合成:
Figure PCTCN2016085275-appb-000146
上一步反应产物8-8(9mg,0.013mmol)溶于1.5mL四氢呋喃中，加入PH＝6的磷酸钾缓冲溶液1.5mL和4-(N-马来酰亚胺甲基)环己烷-1-羧酸琥珀酰亚胺酯(22mg,0.065mmol)，氮气保护下室温搅拌过夜。反应液过滤，直接由制备HPLC纯化得9mg产品CE-035，为白色固体，产率66％。LCMS(ESI)m/z 1045.4(M+H)+.1H NMR(400MHz,CDCl3)δppm 6.93(d,J＝7.6Hz,1H),6.84(s,1H),6.43&6.41(dd,J1＝21.2Hz,J2＝7.2Hz,1H),6.30(s,1H),6.20&6.18(dd,J1＝13.2Hz,J2＝6.8Hz,1H),5.47&5.45(dd,J1＝13.2Hz,J2＝7.2Hz,1H),4.92-4.88(m,1H),4.26(q,J＝7.2Hz,1H),4.12(q,J＝6.8Hz,1H),3.99(s,3H),3.75(q,J＝6.8Hz,1H),3.66(q,J＝7.2Hz,1H),3.55-3.43(m,3H),3.38(d,J＝6.4Hz,3H),3.36(s,3H),3.23-3.16(m,2H),3.18(s,3H),3.10-2.94(m,1H),2.90-2.83(m,3H),2.83(s,3H),2.57-2.53(m,3H),2.27(d,J＝14.4Hz,1H),2.16(d,J＝13.6Hz,2H),1.98-1.90(m,1H),1.80-1.78(m,2H),1.68(s,3H),1.56-1.51(m,4H),1.47-1.42(m,4H),1.28(d,J＝8.4Hz,3H),1.07(q,J＝8.4Hz,3H),0.83(s,3H).
实施例9CE-036的合成路线
Figure PCTCN2016085275-appb-000147
实验步骤
化合物2-9的合成:
Figure PCTCN2016085275-appb-000148
化合物1-9(9.30g,50mmol)溶于50mL四氢呋喃中，冷至0℃，搅拌下逐滴加入1M硼烷二甲硫醚复合物的四氢呋喃溶液(75mL,75mmol)。反应液升至室温搅拌过夜。反应液冷却至0℃，逐滴加入50mL甲醇淬灭反应，室温搅拌1小时，减压浓缩，剩余物经硅胶柱层析(石油醚/乙酸乙酯＝1:1)纯化得4.3g产品2-9，为无色油状物，产率50％。LCMS(ESI)m/z173.1(M+H)+
化合物3-9的合成:
Figure PCTCN2016085275-appb-000149
上一步反应产物2-9(8.6g,50mmol)和三乙胺(8.3mL,60mmol)溶于80mL二氯甲烷中，冷却至0℃，逐滴缓慢加入甲烷磺酰氯MsCl(4.3mL,55mmol)。反应液升至室温搅拌2小时。加入50mL水淬灭反应，分出有机相，饱和食盐水洗涤3次(50mL×3)，无水硫酸钠干燥，浓缩得粗产品3-9，为浅黄色油状物，直接用于下一步反应。LCMS(ESI)m/z251.1(M+H)+,
化合物4-9的合成:
Figure PCTCN2016085275-appb-000150
上一步反应粗产物3-9(4g,16.0mmol)溶于N,N-二甲基甲酰胺50mL中，加入硫代乙 酸钾(3.45g,30mmol)。反应液室温搅拌过夜，加入30mL水，乙酸乙酯萃取3次(50mL×3)，合并有机相，水洗3次(30mL×3)，饱和食盐水洗(50mL)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝1:1)纯化得2.9g产品4-9，为棕色油状物，产率85％。LCMS(ESI)m/z 231.1(M+H)+,
化合物6-9的合成:
Figure PCTCN2016085275-appb-000151
氮气保护下，将上一步反应产物4-9(2.3g,10mmol)溶于25mL甲醇和10mL水的混合溶剂中，加入氢氧化锂(0.98g,40mmol)。反应液在氮气保护下室温搅拌2小时，加入甲基硫代磺酸甲酯(1.5g,12mmol)。反应液继续在室温下搅拌过夜。加入50mL水，乙酸乙酯萃取3次(50mL×3)，合并有机相，饱和食盐水洗3次(50mL×3)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝1:2)纯化得1.43g产物6-9，为浅黄色油状物，产率65％。LCMS(ESI)m/z 221.0(M+H)+,243.1(M+Na)+.
化合物CE-031的合成:
Figure PCTCN2016085275-appb-000152
将上一步产物6-9(53mg,0.24mmol)，DCC(99mg,0.48mmol)和DMAP(19mg,0.16mmol)加入一个干燥的Schlenck管中，置换氩气3次，加入1mL二氯甲烷，搅拌。将美登醇(45mg,0.08mmol)溶于4mL干燥二氯甲烷中加入。反应液室温搅拌过夜，缓慢加入0.5mL水淬灭反应，加入20mL乙酸乙酯，过滤，乙酸乙酯洗涤。滤液无水硫酸钠干燥，浓缩。粗产物使用制备HPLC分离得到18mg产物CE-031，为白色固体，产率30％.
LCMS(ESI)m/z 767.3.0(M+H)+,1H NMR(400MHz,CDCl3)δppm 6.83(d,J＝6.8Hz,2H),6.46&6.44(dd,J1＝15.6Hz,J2＝10.8Hz,1H),6.35(s,1H),6.14(d,J＝10.8Hz,1H),5.44&5.42(dd,J1＝16.0Hz,J2＝8.4Hz,1H),4.86&4.85(dd,J1＝12.0Hz,J2＝3.2Hz,1H),4.26 (t,J＝10.4Hz,1H),3.99(s,3H),3.51(s,1H),3.48(d,J＝3.6Hz,1H),3.37(s,3H),3.23(d,J＝12.8Hz,1H),3.15(s,3H),2.90(d,J＝10.8Hz,1H),2.75-2.63(m,2H),2.55(t,J＝13.6Hz,1H),2.42(s,3H),2.34-2.02(m,5H),1.84-1.65(m,4H),1.70(s,3H),1.54-1.44(m,3H),1.47(d,J＝6.8Hz,3H),1.29-1.09(m,3H),0.84(s,3H).
化合物8-9的合成:
Figure PCTCN2016085275-appb-000153
CE-031(15mg,0.02mmol)溶于0.5mL乙酸乙酯和0.5mL甲醇的混合溶剂中，将二硫代苏糖醇DTT(18mg,0.117mmol)溶于0.5mL PH＝7.5的磷酸钾缓冲溶液中加入。反应液在氮气保护下搅拌3小时。加入PH＝6的磷酸钾缓冲溶液1mL淬灭反应。乙酸乙酯萃取3次(5mL×3)，合并有机相，饱和食盐水洗涤(5mL)，无水硫酸钠干燥，浓缩。粗产品使用制备HPLC(CH3CN in H2O-0.05％TFA from 5％to 95％)纯化得到11mg产物8-9，为白色固体，产率78％。LCMS(ESI)m/z 721.3(M+H)+.
化合物CE-036的合成:
Figure PCTCN2016085275-appb-000154
上一步反应产物8-9(9.0mg,0.013mmol)溶于1.5mL四氢呋喃中，加入PH＝6的磷酸钾缓冲溶液1.5mL和4-(N-马来酰亚胺甲基)环己烷-1-羧酸琥珀酰亚胺酯(22mg,0.065mmol)，氮气保护下室温搅拌过夜。反应液过滤，直接由制备HPLC纯化得9.0mg产品CE-036，为白色固体，产率64％。
LCMS(ESI)m/z 1055.4(M+H)+.1H NMR(400MHz,CDCl3)δppm 6.83(d,J＝16.0Hz,2H),6.47-6.39(m,2H),6.15&6.13(dd,J1＝10.0Hz,J2＝4.8Hz,1H),5.43&5.41(dd,J1＝15.2Hz,J2＝8.8Hz,1H),4.86(t,J＝9.2Hz,1H),4.26(t,J＝11.2Hz,1H),3.99(s,3H),3.69(d,J＝8.4Hz,1H),3.50-3.36(m,4H),3.37(s,3H),3.23(d,J＝12.8Hz,1H),3.14(s,3H),2.90-2.77(m,6H), 2.58-2.51(m,3H),2.29(q,J＝11.2Hz,1H),2.22-1.90(m,10H),1.85(s,3H),1.70-1.50(m,8H),1.27(d,J＝6.8Hz,3H),1.20-1.00(m,5H),0.83(s,3H).
实施例10CE-026,037的合成路线
Figure PCTCN2016085275-appb-000155
实验步骤
化合物2-10的合成:
Figure PCTCN2016085275-appb-000156
250mL茄形瓶中加入化合物1-10(37.2g,0.6mol)，搅拌下加入氢氧化钾KOH(11.2g,0.2mol)。将该悬浊液加热至90℃，搅拌至氢氧化钾完全溶解。苄溴(34g,23.6mL,0.2mol)逐滴缓慢加入。反应液升温至110℃，搅拌过夜。反应液冷却至室温，加入800mL水，乙酸乙酯萃取3次(150mL×3)，合并有机相，水洗3次(150mL×3)，饱和食盐水洗(150mL)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯10:1至3:1)纯化得22g产品2-10，为无色油状物，产率72％。LCMS(ESI)m/z153.1(M+H)+,
化合物3-10的合成:
Figure PCTCN2016085275-appb-000157
上一步反应产物2-10(15.2g,0.1mol)和三乙胺(16.6mL,0.12mol)溶于150mL二氯甲烷中，冷却至0℃，逐滴加入甲烷磺酰氯(8.5mL,0.11mol)。反应液升至室温搅拌2小时。加入50mL水淬灭反应，分出有机相，饱和食盐水洗3次(50mL×3)，无水硫酸钠干燥，浓缩得23g粗产品3-10，为黄色油状物，直接用于下一步反应。
化合物4-10的合成:
Figure PCTCN2016085275-appb-000158
将上一步反应产物3-10(23g,0.1mol)和L-乳酸乙酯(23.6g,0.2mol)溶于N,N-二甲基甲酰胺150mL中，冷却至0℃，分批缓慢加入钠氢(8g,60％含量,悬浮于矿物油中,0.2mol)。反应液升至室温搅拌过夜。将反应液冷至0℃，逐滴缓慢加入20mL饱和氯化铵溶液淬灭反应，加入150mL水，乙酸乙酯萃取3次(100mL×3)，合并有机相，水洗3次(100mL×3)，饱和食盐水洗(100mL)，无水硫酸钠干燥，浓缩。粗产品硅胶柱层析(石油醚/乙酸乙酯＝10:1至2:1)纯化得11.3g产品4-10，为浅黄色油状物，产率45％。LCMS(ESI)m/z 253.1(M+H)+。
化合物5-10的合成:
Figure PCTCN2016085275-appb-000159
上一步反应产物4-10(11.3g,45mmol)溶于50mL甲醇中，置换氮气3次，加入200mg10％的钯碳干粉。反应液置换氢气3次，氢气氛中室温搅拌过夜。置换氮气3次，加入50mL二氯甲烷，过滤，二氯甲烷洗涤，滤液浓缩，得粗产品7.2g，为浅黄色油状物，直接用于下一步反应。LCMS(ESI)m/z164.1(M+H)+,186.1(M+Na)+.
化合物6-10的合成:
Figure PCTCN2016085275-appb-000160
上一步反应产物5-10(6.5g,40mmol)和三乙胺(6.6mL,48mmol)溶于50mL二氯甲烷中，冷却至0℃，逐滴缓慢加入甲烷磺酰氯(3.5mL,44mmol)。反应液升至室温搅拌2小时。加入50mL水淬灭反应，分出有机相，饱和食盐水洗涤3次(50mL×3)，无水硫酸钠干燥，浓缩得粗产品，为浅黄色油状物，直接用于下一步反应。
化合物7-10的合成:
Figure PCTCN2016085275-appb-000161
上一步反应粗产物6-10(2g,7.0mmol)溶于N,N-二甲基甲酰胺20mL中，加入硫代乙酸钾(1.6g,14mmol)。反应液室温搅拌过夜，加入30mL水，乙酸乙酯萃取3次(50mL×3)，合并有机相，水洗3次(30mL×3)，饱和食盐水洗(50mL)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析纯化得2.0g产品7-10，为棕色油状物，产率90％。LCMS(ESI)m/z 221.1(M+H)+。
化合物9-10的合成:
Figure PCTCN2016085275-appb-000162
氮气保护下，将上一步反应产物7-10(1.76g,8mmol)溶于20mL甲醇和10mL水的混合溶剂中，加入氢氧化锂(0.78g,32mmol)。反应液在氮气保护下室温搅拌2小时，加入甲基硫代磺酸甲酯(1.2g,9.6mmol)。反应液继续在室温下搅拌过夜。加入50mL水，乙酸乙酯萃取3次(50mL×3)，合并有机相，饱和食盐水洗3次(50mL×3)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝1:2)纯化得1.01g产物9-10，为浅黄色油状物，产率65％。LCMS(ESI)m/z197.0(M+H)+，219.0(M+Na)+.
化合物CE-026的合成:
Figure PCTCN2016085275-appb-000163
将上一步产物9-10(65mg,0.33mmol)，DCC(136mg,0.66mmol)和DMAP(27mg,0.22mmol)加入一个干燥的Schlenck管中，置换氩气3次，加入1mL二氯甲烷，搅拌。 将美登醇(62mg,0.11mmol)溶于4mL干燥二氯甲烷中加入。反应液室温搅拌2h，缓慢加入0.3mL水淬灭反应，加入15mL乙酸乙酯，过滤，乙酸乙酯洗涤。滤液无水硫酸钠干燥，浓缩。粗产物使用制备HPLC分离得到29mg产物CE-026，为白色固体，产率35％。
LCMS(ESI)m/z 744.3(M+H)+.1H NMR(400MHz,CDCl3)δppm 6.83(d,J＝8.0Hz,2H),6.46&6.44(dd,J1＝15.2Hz,J2＝10.8Hz,1H),6.21(s,1H),6.18(d,J＝11.6Hz,1H),5.55&5.53(dd,J1＝15.2Hz,J2＝9.2Hz,1H),4.93&4.92(dd,J1＝12.0Hz,J2＝2.8Hz,1H),4.28(t,J＝10.4Hz,1H),4.10(q,J＝9.2Hz,1H),3.99(s,3H),3.96-3.92(m,1H),3.74-3.67(m,1H),3.53(d,J＝4.8Hz,1H),3.50(s,1H),3.36(s,3H),3.29(s,1H),3.19(d,J＝13.6Hz,1H),3.16(s,3H),3.04(s,1H),2.94-2.89(m,3H),2.59(t,J＝12.0Hz,1H),2.44(s,3H),2.25-2.21(m,1H),2.05-2.00(m,1H),1.70(s,3H),1.54-1.44(m,1H),1.52(d,J＝7.2Hz,3H),1.40(d,J＝6.8Hz,1H),1.30(d,J＝7.2Hz,3H),1.29-1.09(m,2H),0.83(s,3H).
化合物10-10的合成:
Figure PCTCN2016085275-appb-000164
CE-026(15mg,0.02mmol)溶于0.5mL乙酸乙酯和0.5mL甲醇的混合溶剂中，将二硫代苏糖醇DTT(18mg,0.117mmol)溶于0.5mL PH＝7.5的磷酸钾缓冲溶液中加入。反应液在氮气保护下搅拌3小时。加入PH＝6.0的磷酸钾缓冲溶液1mL淬灭反应。乙酸乙酯萃取3次(5mL×3)，合并有机相，饱和食盐水洗涤(5mL)，无水硫酸钠干燥，浓缩。粗产品使用制备HPLC(CH3CN in H2O-0.05％TFA from 5％to 95％)纯化得到12mg产物10-10，为白色固体，产率86％。LCMS(ESI)m/z697.3(M+H)+.
化合物CE-037的合成:
Figure PCTCN2016085275-appb-000165
上一步反应产物10-10(9mg,0.013mmol)溶于1.5mL四氢呋喃中，加入PH＝6的磷酸钾缓冲溶液1.5mL和4-(N-马来酰亚胺甲基)环己烷-1-羧酸琥珀酰亚胺酯(22mg,0.065mmol)，氮气保护下室温搅拌过夜。反应液过滤，直接由制备HPLC纯化得12mg产品 CE-037，为白色固体，产率78％。
LCMS(ESI)m/z 1031.4(M+H)+.1H NMR(400MHz,CDCl3)δppm 6.81(d,J＝8.0Hz,2H),6.46&6.44(dd,J1＝15.2Hz,J2＝10.8Hz,1H),6.44(s,1H),6.18(d,J＝7.6Hz,1H),5.59-5.51(m,1H),4.97(d,J＝12.4Hz,1H),4.26(q,J＝10.4Hz,1H),4.13(t,J＝6.4Hz,1H),3.99(s,3H),3.99-3.96(m,2H),3.53-3.47(m,2H),3.40-3.37(m,2H),3.35(d,J＝13.6Hz,3H),3.35-3.30(m,2H),3.21-3.15(m,2H),3.13(s,3H),2.92-2.82(m,2H),2.82(s,3H),2.60-2.58(m,3H),2.25-2.15(m,3H),1.70(s,3H),1.70-1.60(m,2H),1.54-1.44(m,3H),1.52(d,J＝7.2Hz,3H),1.30-1.26(m,6H),1.19-1.09(m,2H),0.83(s,3H).
实施例11CE-039,040,043的合成路线
Figure PCTCN2016085275-appb-000166
实验步骤
化合物2-11的合成
Figure PCTCN2016085275-appb-000167
马来酸酐(5g,51mmol,1eq)和β-氨基丙酸(4.54g,51mmol,1eq)溶于80mL乙酸中。氮气保护下反应液加热回流搅拌过夜。反应液冷却到室温，减压移除乙酸，剩余物经硅胶柱层析(二氯甲烷)纯化得3.6g产品2-11，为白色粉末，产率42％。LCMS(ESI)m/z170.1(M+H)+.
化合物CE-L-055的合成
Figure PCTCN2016085275-appb-000168
将上一步反应产物2-11(3.6g,21.4mmol)和EDC-HCl(4.93g,25.7mmol)溶于50mL二氯甲烷中，加入HOSu(2.96g,25.7mol)。反应液室温搅拌2小时。加入50mL水，乙酸乙酯萃取3次(50mL×3)，合并有机相，饱和食盐水洗涤3次(50mL×3)，无水硫酸钠干燥，浓缩。粗产物使用硅胶柱层析(石油醚/乙酸乙酯＝1:3)得产物4.55g，为黄色油状物，产率80％。LCMS(ESI)m/z267.1(M+H)+.
化合物4-11的合成
Figure PCTCN2016085275-appb-000169
化合物3-11(4.55g,18.7mmol)溶于10mL水中，加入碳酸氢钠(1.71g,20.4mmol)，搅拌。将CE-L-055(4.55g,17mmol)溶于30mL乙二醇二甲醚中逐滴缓慢加入。反应液室温搅拌2小时。加入50mL水，用1M稀盐酸调PH3～4。乙酸乙酯萃取10次(50mL×10)，合并有机相，无水硫酸钠干燥，浓缩得粗产品，直接用于下一步反应。LCMS(ESI)m/z417.2(M+H)+.
化合物CE-L-075的合成
Figure PCTCN2016085275-appb-000170
将上一步反应产物4-11(1.0g,2.4mmol)和EDC-HCl(0.55g,2.88mmol)溶于25mL二氯甲烷中，加入HOSu(0.33g,2.88mmol)。反应液室温搅拌2小时。加入50mL水，乙 酸乙酯萃取3次(50mL×3)，合并有机相，饱和食盐水洗涤3次(50mL×3)，无水硫酸钠干燥，浓缩。粗产物使用制备HPLC纯化得产物330mg，为无色油状物，产率27％。LCMS(ESI)m/z514.2(M+H)+.
化合物CE-039的合成
Figure PCTCN2016085275-appb-000171
化合物10-10(20mg,0.03mmol)溶于2mL N,N-二甲基甲酰胺中，加入PH＝6的磷酸钾缓冲溶液0.1mL和CE-L-075(77mg,0.15mmol)，氮气保护下室温搅拌过夜。反应液过滤，直接由制备HPLC纯化得18mg产品CE-040，为白色固体，产率50％。
LCMS(ESI)m/z1232.3(M+H)+,627.8(M/2+H)+.1H NMR(400MHz,CDCl3)δppm7.13(br,1H),6.77(s,1H),6.72(s,1H),6.70(s,1H),6.38&6.36(dd,J1＝12.4Hz,J2＝8.8Hz,1H),6.12(d,J＝9.2Hz,1H),5.55-5.48(m,1H),4.92(t,J＝8.4Hz,1H),4.25(q,J＝7.2Hz,1H),4.07(t,J＝6.8Hz,1H),3.92(s,3H),3.88-3.76(m,4H),3.65(s,14H),3.58-3.42(m,7H),3.35(s,3H),3.22-3.18(m,4H),3.15(s,3H),3.00-2.88(m,4H),2.85(s,3H),2.56(t,J＝7.6Hz,1H),2.50(t,J＝7.2Hz,2H),2.16(d,J＝12.8Hz,1H),1.63(s,3H),1.44(d,J＝8.4Hz,3H),1.29(d,J＝8.4Hz,3H),1.23-1.18(m,6H),0.77(s,3H).
化合物CE-040的合成
Figure PCTCN2016085275-appb-000172
化合物9-7(20mg,0.027mmol)溶于2mL N,N-二甲基甲酰胺中，加入PH＝6的磷酸钾缓冲溶液0.1mL和CE-L-075(70mg,0.14mmol)，氮气保护下室温搅拌过夜。反应液过滤，直接由制备HPLC纯化得15mg产品CE-040，为白色固体，产率44％。
LCMS(ESI)m/z627.8(M/2+H)+.1H NMR(400MHz,CDCl3)δppm 6.85(s,1H),6.79(s,1H),6.44(t,J＝12.8Hz,1H),6.19(t,J＝9.2Hz,1H),5.60-5.55(m,1H),4.92(t,J＝8.4Hz,1H),4.29(q,J＝7.6Hz,1H),4.14(t,J＝6.8Hz,1H),3.99(s,3H),3.87-3.76(m,6H),3.66(s,16H),3.55-3.42(m,9H),3.35(s,3H),3.20(t,J＝10.8Hz,2H),3.15(s,3H),2.92(t,J＝6.8Hz,2H),2.90-2.85(m,6H),2.60-2.50(m,4H),2.30-2.20(m,2H),1.69(s,3H),1.50(d,J＝8.4Hz,3H),1.28(d,J＝8.4Hz,6H),0.83(s,3H).
化合物CE-043的合成
Figure PCTCN2016085275-appb-000173
化合物8-8(21mg,0.03mmol)溶于2mL N,N-二甲基甲酰胺中，加入PH＝6的磷酸钾缓冲溶液0.1mL和CE-L-075(77mg,0.15mmol)，氮气保护下室温搅拌过夜。反应液过滤，直接由制备HPLC纯化得18mg产品CE-043，为白色固体，产率49％。
LCMS(ESI)m/z 1246.4(M+Na)+,612.8(M/2+Na)+,.1H NMR(400MHz,CDCl3)δppm6.84(s,1H),6.79(s,1H),6.44(t,J＝8.8Hz,1H),6.18(d,J＝8.8Hz,1H),5.59-5.56(m,1H),4.95(t,J＝9.2Hz,1H),4.32(q,J＝7.6Hz,1H),4.05(q,J＝6.8Hz,1H),3.99(s,3H),3.88-3.76(m,6H),3.65(s,16H),3.58-3.42(m,8H),3.35(s,3H),3.22-3.18(m,2H),3.15(s,3H),3.00-2.88(m,6H),2.85(s,3H),2.56(t,J＝7.6Hz,2H),2.50(t,J＝7.2Hz,2H),2.16(d,J＝12.8Hz,1H),1.98-1.90(m,2H),1.69(s,3H),1.48(d,J＝8.4Hz,3H),1.29(d,J＝8.4Hz,3H),0.83(s,3H).
实施例12XDCE-M-001、XDCE-M-002的合成路线
Figure PCTCN2016085275-appb-000174
实验步骤
化合物2-12的合成
Figure PCTCN2016085275-appb-000175
化合物1-12(1.77g,10mmol)溶于5mL水中，加入碳酸氢钠(0.92g,11mmol)，搅拌。将CE-L-055(2.51g,9.5mmol)溶于15mL乙二醇二甲醚中逐滴缓慢加入。反应液室温搅拌2小时。LCMS显示反应完全后直接用反相柱纯化(TFA0.05％水溶液)得产物1.0g，为无色油状物，产率48％。LCMS(ESI)m/z 329.1(M+H)+.
化合物CE-L-071的合成
Figure PCTCN2016085275-appb-000176
将上一步反应产物2-12(1.0g,3.0mmol)和EDC-HCl(0.69g,3.6mmol)溶于25mL二氯甲烷中，加入HOSu(0.41g,3.6mmol)。反应液室温搅拌2小时。LCMS显示反应完全后，浓缩，粗产物用反相柱纯化(TFA0.05％水溶液)得产物0.32g。为无色油状物，产率25％。LCMS(ESI)m/z 426.3(M+H)+.
化合物4-12的合成
Figure PCTCN2016085275-appb-000177
化合物3-12(2.21g,10mmol)溶于5mL水中，加入碳酸氢钠(0.94g,11mmol)，搅拌。将CE-L-055(2.52g,9.5mmol)溶于15mL乙二醇二甲醚中逐滴缓慢加入。反应液室温搅拌2小时。LCMS显示反应完全后直接用反相柱纯化(TFA0.05％水溶液)得产物1.5g，为无色油状物，产率42％。LCMS(ESI)m/z 373.2(M+H)+.
化合物CE-L-073的合成
Figure PCTCN2016085275-appb-000178
将上一步反应产物4-12(1.0g,2.7mmol)和EDC-HCl(0.62g,3.24mmol)溶于25mL 二氯甲烷中，加入HOSu(0.37g,3.24mmol)。反应液室温搅拌2小时。LCMS显示反应完全后，浓缩，粗产物用反相柱纯化(TFA0.05％水溶液)得产物0.31g。为无色油状物，产率25％。LCMS(ESI)m/z 470.2(M+H)+.
化合物XDCE-M-001的合成
Figure PCTCN2016085275-appb-000179
化合物9-7(40mg,0.054mmol)溶于4mL N,N-二甲基甲酰胺中，加入PH＝6的磷酸钾缓冲溶液0.2mL和CE-L-071(115mg,0.27mmol)，氮气保护下室温搅拌过夜。反应液过滤，直接由制备HPLC纯化得37mg产品XDCE-M-001，为白色固体，产率59％。
LCMS(ESI)m/z627.8(M/2+H)+.1H NMR(400MHz,CDCl3)δppm 6.87(br,1H),6.85(s,1H),6.78(s,1H),6.74(br,1H),6.41(t,J＝13.2Hz,1H),6.19(d,J＝10.8Hz,1H),5.61-5.53(m,1H),4.92(d,J＝11.2Hz,1H),4.30(q,J＝10.4Hz,1H),4.15(q,J＝7.6Hz,1H),3.99(s,3H),4.04-3.96(m,1H),3.87-3.76(m,6H),3.70-3.60(m,6H),3.56-3.42(m,9H),3.36(s,3H),3.20(d,J＝10.8Hz,2H),3.15(s,3H),2.92-2.85(m,8H),2.62-2.50(m,4H),2.23(d,J＝14.4Hz,1H),1.69(s,3H),1.51-1.48(m,3H),1.30-1.25(m,6H),0.83(s,3H).
化合物XDCE-M-002的合成
Figure PCTCN2016085275-appb-000180
化合物9-7(40mg,0.054mmol)溶于4mL N,N-二甲基甲酰胺中，加入PH＝6的磷酸钾缓冲溶液0.2mL和CE-L-073(127mg,0.27mmol)，氮气保护下室温搅拌过夜。直接由制备HPLC纯化得34mg产品XDCE-M-002，为白色固体，产率52％。
LCMS(ESI)m/z627.8(M/2+H)+.1H NMR(400MHz,CDCl3)δppm 7.08(br,1H),6.86-6.81(m,1H),6.84(s,1H),6.79(s,1H),6.44(dd,J1＝15.2Hz,J2＝10.8Hz,1H),6.19(d,J＝10.8Hz,1H),5.60-5.55(m,1H),4.92(t,J＝10.8Hz,1H),4.29(q,J＝7.6Hz,1H),4.13(t,J＝6.8Hz,1H),4.05-3.95(m,1H),3.99(s,3H),3.87-3.76(m,6H),3.70-3.60(m,10H),3.56-3.42(m,10H),3.36(s,3H),3.20(d,J＝10.8Hz,2H),3.15(s,3H),2.92-2.85(m,8H),2.62-2.50(m,4H),2.23(d,J＝14.4Hz,1H),1.69(s,3H),1.51-1.48(m,3H),1.30-1.25(m,6H),0.83(s,3H).
实施例13CE-045的合成路线
Figure PCTCN2016085275-appb-000181
实验步骤
化合物2-13的合成
Figure PCTCN2016085275-appb-000182
500mL茄形瓶中加入化合物1-13(135g,0.9mol)，搅拌下加入氢氧化钾(16.8g,0.3mol)。将该悬浊液加热至90℃，搅拌至氢氧化钾完全溶解。苄溴(35.6mL,0.3mol)逐滴缓慢加入。反应液升温至110℃，搅拌过夜。反应液冷却至室温，加入1000mL水，乙酸乙酯萃取3次(200mL×3)，合并有机相，水洗3次(150mL×3)，饱和食盐水洗(150mL)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯10:1至1:1)纯化得50g产品2-13，为浅黄色油状物，产率69％。LCMS(ESI)m/z 240.3(M+H)+.
化合物3-13的合成
Figure PCTCN2016085275-appb-000183
上一步反应产物2-13(24g,0.1mol)和三乙胺(16.6mL,0.12mol)溶于150mL二氯甲烷中，冷却至0℃，逐滴加入甲烷磺酰氯(8.5mL,0.11mol)。反应液升至室温搅拌2小时。加入50mL水淬灭反应，分出有机相，饱和食盐水洗3次(50mL×3)，无水硫酸钠干燥，浓缩得30g粗产品3-13，为黄色油状物，直接用于下一步反应。
化合物4-13的合成
Figure PCTCN2016085275-appb-000184
将上一步反应产物3-13(15.9g,0.05mol)和L-乳酸乙酯(11.8g,0.1mol)溶于N,N-二甲基甲酰胺80mL中，冷却至0℃，分批缓慢加入钠氢(4g,60％含量,悬浮于矿物油中,0.1mol)。反应液升至室温搅拌过夜。将反应液冷至0℃，逐滴缓慢加入20mL饱和氯化铵溶液淬灭反应，加入150mL水，乙酸乙酯萃取3次(100mL×3)，合并有机相，水洗3次(50mL×3)，饱和食盐水洗(50mL)，无水硫酸钠干燥，浓缩。粗产品硅胶柱层析(石油醚/乙酸乙酯＝10:1至2:1)纯化得6.5g产品4-13，为浅黄色油状物，产率38％。LCMS(ESI)m/z 341.7(M+H)+,
化合物5-13的合成
Figure PCTCN2016085275-appb-000185
上一步反应产物4-13(13.6g,40mmol)溶于50mL甲醇中，置换氮气3次，加入200mg10％的钯碳干粉。反应液置换氢气3次，氢气氛中室温搅拌过夜。置换氮气3次，加入50mL二氯甲烷，过滤，二氯甲烷洗涤，滤液浓缩，得粗产品10.0g，为浅黄色油状物，直接用于下一步反应。LCMS(ESI)m/z 251.1(M+H)+。
化合物6-13的合成
Figure PCTCN2016085275-appb-000186
上一步反应产物5-13(2.5g,10mmol)溶于30mL丙酮中，冰水浴冷却至0℃，逐滴缓慢加入Jones试剂(3g,30mmol),约10分钟滴加完毕。反应液升至室温搅拌过夜，加入2mL异丙醇淬灭反应，减压移除丙酮，剩余物二氯甲烷萃取3次(50mL×3)，合并有机相，无水硫酸钠干燥，浓缩得粗产品，为浅黄色油状物，直接用于下一步反应。LCMS(ESI)m/z265.7(M+H)+。
化合物8-13的合成
Figure PCTCN2016085275-appb-000187
氮气保护下，将上一步反应产物6-13(264mg,1mmol)溶于10mL甲苯中，升温至回流，逐滴缓慢加入7(264mg,1mmol)，反应液回流过夜。将反应液冷却至室温，减压浓缩，粗产品硅胶柱层析(石油醚/乙酸乙酯＝2:1)纯化得240mg产品8-13，为无色油状物，产率75％。LCMS(ESI)m/z 321.7(M+H)+,1H NMR(400MHz,CDCl3)δppm 4.21(q,J＝6.8Hz,2H),4.03(s,2H),3.76-3.67(m,8H),3.61-3.58(m,1H),1.48(s,9H),1.41(d,J＝5.6Hz,3H),1.29(t,J＝6.0Hz,3H).
化合物9-13的合成
Figure PCTCN2016085275-appb-000188
上一步反应产物8-13(1.83g,5.7mmol)溶于50mL甲醇和20mL水中，加入K2CO3(4g,28.7mmol)。反应液室温搅拌3h。待原料反应完全后减压移除甲醇，加入50mL水，缓慢滴加1N HCl调节PH值至4-5，乙酸乙酯萃取3次(50mL×3)，合并有机相，饱和食盐水洗(50mL)，无水硫酸钠干燥，浓缩，粗产品硅胶柱层析(石油醚/乙酸乙酯＝1：1)纯化得1.0g产品9-13，为浅黄色油状物，产率60％。LCMS(ESI)m/z 293.1(M+H)+。
化合物10-13的合成
Figure PCTCN2016085275-appb-000189
将上一步产物9-13(105mg,0.36mmol)，DCC(148mg,0.72mmol)和DMAP(29mg,0.24mmol)加入一个干燥的Schlenck管中，置换氩气3次，加入1mL二氯甲烷，搅拌。将美登醇(63mg,0.12mmol)溶于2mL干燥二氯甲烷中加入。反应液室温搅拌2h，缓慢加入0.3mL水淬灭反应，加入15mL乙酸乙酯，过滤，乙酸乙酯洗涤。滤液无水硫酸钠干燥，浓缩。粗产物使用制备HPLC分离得到28mg产物10-13，为白色固体，产率30％。LCMS(ESI)m/z 838.2(M+H)+.
化合物11-13的合成
Figure PCTCN2016085275-appb-000190
将上一步反应产物10-13(28mg,0.03mmol)溶于28mL二氯甲烷中，缓慢加入三氟醋酸(4.2ml)。反应室温搅拌2h。待原料反应完后减压浓缩得粗产品11-13 18.3mg，为浅黄色油状物，直接用于下一步反应。
化合物CE-045的合成
Figure PCTCN2016085275-appb-000191
将上一步反应产物11-13(18.3mg,0.023mmol)溶于2mL DMF中，加入HOSu(13.2mg,0.11mmol)和EDCI(21mg,0.11mmol)。反应室温搅拌2h。直接由制备HPLC纯化得2mg产品CE-045，为白色固体，产率10％。LCMS(ESI)m/z 880.3(M+H)+.
实施例14CE-047、CE-050的合成路线
Figure PCTCN2016085275-appb-000192
Figure PCTCN2016085275-appb-000193
实验步骤
化合物1-14的合成
Figure PCTCN2016085275-appb-000194
5-13(2.5g,10mmol)和三乙胺(1.7mL,12mmol)溶于30mL二氯甲烷中，冷却至0℃，逐滴加入甲烷磺酰氯(0.85mL,11mmol)。反应液升至室温搅拌2小时。加入30mL水淬灭反应，分出有机相，饱和食盐水洗3次(30mL×3)，无水硫酸钠干燥，浓缩得3.2g粗产品1-14，为浅黄色油状物，直接用于下一步反应。
化合物2-14的合成
Figure PCTCN2016085275-appb-000195
上一步反应产物1-14(3.2g,10mmol)溶于20mL N,N-二甲基甲酰胺中，加入叠氮化钠(715mg,11mmol)。反应液升至50℃搅拌过夜。将反应液冷却至室温，加入50mL水淬灭反应，乙酸乙酯萃取3次(30mL×3)，合并有机相，饱和食盐水洗3次(30mL×3)，无水硫酸钠干燥，浓缩得粗产品，硅胶柱层析(石油醚/乙酸乙酯＝2:1)纯化得2.2g产品2-14，为浅黄色油状物，产率80％。
化合物3-14的合成
Figure PCTCN2016085275-appb-000196
上一步反应产物2-14(2.2g,8mmol)溶于40mL四氢呋喃和5mL水中，加入三苯基磷(2.62g,10mmol)。反应液室温搅拌过夜。减压移除四氢呋喃，加入1N稀盐酸20mL， 乙酸乙酯洗涤3次(20mL×3)，水相冻干得1.6g粗产品3-14，为浅黄色油状物。LCMS(ESI)m/z 250.7(M+H)+.
化合物5-14的合成
Figure PCTCN2016085275-appb-000197
上一步反应产物3-14(1.25g,5mmol)溶于20mL甲醇和5mL水中，加入一水合氢氧化锂(840mg,20mmol)。反应液室温搅拌3h。待原料反应完全后减压移除甲醇，加入饱和碳酸氢钠溶液5mL和30mL冰水浴冷却至0℃，加入Boc2O(1.3g,6mmol)，升至室温搅拌2h。反应液水洗两次(20mL×2)，合并水相加入0.5M KHSO4溶液淬灭反应调节PH＝3-4，二氯甲烷萃取3次(20mL×3)，合并有机相，无水硫酸钠干燥，浓缩，剩余物硅胶柱层析(二氯甲烷/甲醇＝30:1)纯化得950mg产品5-14，为浅黄色油状物，产率59％。LCMS(ESI)m/z322.3(M+H)+.
化合物6-14的合成
Figure PCTCN2016085275-appb-000198
将上一步产物5-14(77mg,0.24mmol)，DCC(132mg,0.64mmol)和DMAP(20mg,0.16mmol)加入一个干燥的Schlenck管中，置换氩气3次，加入1mL二氯甲烷，搅拌。将美登醇(45mg,0.08mmol)溶于3mL干燥二氯甲烷中加入。反应液室温搅拌过夜，缓慢加入0.3mL水淬灭反应并搅拌15分钟，加入15mL乙酸乙酯，过滤，乙酸乙酯洗涤。滤液无水硫酸钠干燥，浓缩。粗产物使用制备HPLC分离得到32mg产物6-14，为浅黄色固体，产率46％。LCMS(ESI)m/z 868.7(M+H)+.
化合物7-14的合成
Figure PCTCN2016085275-appb-000199
上一步反应产物6-14(17mg,0.02mmol)溶于4mL干燥的二氯甲烷中，冰水浴冷却至0℃，逐滴缓慢加入0.4mL三氟乙酸，逐步升至室温搅拌1小时，LCMS检测原料已完全转化，常温减压移除二氯甲烷和三氟乙酸得15mg粗产品7-14，为黄色固体，直接用于下一步反应。LCMS(ESI)m/z 768.7(M+H)+.
化合物CE-047的合成
Figure PCTCN2016085275-appb-000200
上一步反应粗产物7-14(15mg,0.02mmol)悬浮于2mL饱和碳酸氢钠溶液中，加入化合物8(15.5mg,0.1mmol)，反应液室温搅拌过夜。二氯甲烷萃取3次(20mL×3)，合并有机相，饱和食盐水洗3次(20mL×3)，无水硫酸钠干燥，浓缩得粗产品，制备HPLC纯化得2.7mg产品CE-047，为浅黄色固体，产率16％。
LCMS(ESI)m/z 848.7(M+H)+.
1H NMR(400MHz,MeOD)δppm 7.06(s,1H),6.81(s,1H),6.55&6.53(dd,J1＝12.4Hz,J2＝8.8Hz,1H),6.38(d,J＝8.8Hz,1H),6.21-6.15(m,2H),5.51&5.49(dd,J1＝12.4Hz,J2＝7.2Hz,1H),4.73(d,J＝8.0Hz,1H),4.15-4.18(m,2H),3.88(s,3H),3.72-3.64(m,1H),3.62-3.53(m,10H),3.49-3.46(m,2H),3.41(t,J＝4.8Hz,2H),3.26(s,3H),3.04(s,3H),2.69(d,J＝8.0Hz,1H),2.56(t,J＝10.0Hz,1H),2.11(d,J＝10.0Hz,1H),1.64(s,3H),1.50-1.42(m,2H),1.40(d,J＝5.6Hz,3H),1.14(d,J＝5.6Hz,3H),0.78(s,3H).
化合物CE-050的合成
Figure PCTCN2016085275-appb-000201
上一步反应粗产品7-14(15mg,0.02mmol)和化合物9(14mg,0.06mmol)溶于2mL乙二醇二甲醚和2mL水的混合溶剂中，加入0.2mL饱和碳酸氢钠溶液。反应液室温搅拌过夜，二氯甲烷萃取(20mL×3)，合并有机相，无水硫酸钠干燥，浓缩，粗产品由制备HPLC纯化得6mg产品CE-050，为浅黄色固体，产率34％。LCMS(ESI)m/z 910.2(M+Na)+.
1H NMR(400MHz,CDCl3)δppm 7.24(br,1H),6.84(s,1H),6.78(s,1H),6.44(s,1H),6.44&6.42(dd,J1＝12.0Hz,J2＝8.0Hz,1H),6.17(d,J＝8.8Hz,1H),5.58&5.56(dd,J1＝12.4Hz,J2＝7.2Hz,1H),4.93(d,J＝8.0Hz,1H),4.30(t,J＝8.8Hz,1H),4.19-4.11(m,1H),4.09(s,2H),3.99(s,3H),3.80-3.74(m,1H),3.70-3.60(m,10H),3.52-3.46(m,4H),3.35(s,3H),3.20(d,J＝10.4Hz,1H),3.16(s,3H),2.89(d,J＝8.0Hz,1H),2.59(t,J＝10.0Hz,1H),2.23(d,J＝10.0Hz,1H),1.68(s,3H),1.66(d,J＝10.4Hz,1H),1.51(d,J＝5.6Hz,3H),1.29(d,J＝5.6Hz,3H),0.83(s,3H).
实施例15CE-046、CE-052a的合成路线
Figure PCTCN2016085275-appb-000202
实验步骤
化合物2-15的合成
Figure PCTCN2016085275-appb-000203
250mL茄形瓶中加入化合物1-15(70.5g,0.25mol)，搅拌下加入氢氧化钾(5.6g,0.1mol)。将该悬浊液加热至90℃，搅拌至氢氧化钾完全溶解。苄溴(17g,11.8mL,0.1mol)逐滴缓慢加入。反应液升温至110℃，搅拌过夜。反应液冷却至室温，加入800mL水，乙酸乙酯萃取3次(150mL×3)，合并有机相，水洗3次(150mL×3)，饱和食盐水洗(150mL)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯10:1至1:1)纯化得21g产品2-15，为无色油状物，产率57％。LCMS(ESI)m/z 373.3(M+H)+.
化合物3-15的合成
Figure PCTCN2016085275-appb-000204
上一步反应产物2-15(14.9g,0.04mol)和三乙胺(6.64mL,0.048mol)溶于80mL二氯甲烷中，冷却至0℃，逐滴加入甲烷磺酰氯(3.4mL,0.044mol)。反应液升至室温搅拌2小时。加入50mL水淬灭反应，分出有机相，饱和食盐水洗3次(50mL×3)，无水硫酸钠干燥，浓缩得18g粗产品3-15，为黄色油状物，直接用于下一步反应。
化合物4-15的合成
Figure PCTCN2016085275-appb-000205
将上一步反应产物3-15(18g,0.04mol)和L-乳酸乙酯(9.44g,0.08mol)溶于N,N-二甲基甲酰胺100mL中，冷却至0℃，分批缓慢加入钠氢(3.2g,60％含量,悬浮于矿物油中,0.08mol)。反应液升至室温搅拌4h。将反应液冷至0℃，逐滴缓慢加入10mL饱和氯化铵溶液淬灭反应，加入100mL水，乙酸乙酯萃取3次(100mL×3)，合并有机相，水洗3次(100mL×3)，饱和食盐水洗(100mL)，无水硫酸钠干燥，浓缩。粗产品硅胶柱层析(石油醚/乙酸乙酯＝10:1至1:1)纯化得5g产品4-15，为浅黄色油状物，产率26％。LCMS(ESI)m/z 473.1(M+H)+,
化合物5-15的合成
Figure PCTCN2016085275-appb-000206
上一步反应产物4-15(4.7g,10mmol)溶于50mL甲醇中，置换氮气3次，加入200mg 10％的钯碳干粉。反应液置换氢气3次，氢气氛中室温搅拌过夜。置换氮气3次，加入50mL二氯甲烷，过滤，二氯甲烷洗涤，滤液浓缩，得粗产品3.8g，为浅黄色油状物，直接用于下一步反应。LCMS(ESI)m/z 383.1(M+H)+.
化合物6-15的合成
Figure PCTCN2016085275-appb-000207
上一步反应产物5-15(3.8g,10mmol)和三乙胺(1.7mL,12mmol)溶于30mL二氯甲烷中，冷却至0℃，逐滴缓慢加入甲烷磺酰氯(0.9mL,11mmol)。反应液升至室温搅拌2小时。加入50mL水淬灭反应，分出有机相，饱和食盐水洗涤3次(50mL×3)，无水硫酸钠干燥，浓缩得粗产品，为浅黄色油状物，直接用于下一步反应。
化合物7-15的合成
Figure PCTCN2016085275-appb-000208
上一步反应产物6-15(4.6g,10mmol)溶于20mL N,N-二甲基甲酰胺中，加入叠氮化钠(715mg,11mmol)。反应液升至50℃搅拌过夜。将反应液冷却至室温，加入50mL水淬灭反应，乙酸乙酯萃取3次(30mL×3)，合并有机相，饱和食盐水洗3次(30mL×3)，无水硫酸钠干燥，浓缩得粗产品，硅胶柱层析(石油醚/乙酸乙酯＝1:1)纯化得3.2g产品7-15，为浅黄色油状物，产率80％。
化合物8-15的合成
Figure PCTCN2016085275-appb-000209
上一步反应产物7-15(2.0g,5mmol)溶于30mL四氢呋喃和5mL水中，加入三苯基磷(1.57g,6mmol)。反应液室温搅拌过夜。减压移除四氢呋喃，加入1N稀盐酸20mL，乙酸乙酯洗涤3次(20mL×3)，水相冻干得820mg粗产品8-15，为浅黄色油状物。LCMS(ESI)m/z 382.3(M+H)+.
化合物9-15的合成
Figure PCTCN2016085275-appb-000210
上一步反应产物8-15(1.14g,3mmol)溶于20mL甲醇和5mL水中，加入一水合氢氧化锂(630mg,15mmol)。反应液室温搅拌3h。待原料反应完全后减压移除甲醇，加入饱和碳酸氢钠溶液3mL和30mL冰水浴冷却至0℃，加入Boc2O(785mg,3.6mmol)，升至室温搅拌2h。反应液水洗两次(20mL×2)，合并水相加入0.5M KHSO4溶液淬灭反应调节PH＝3-4，二氯甲烷萃取3次(20mL×3)，合并有机相，无水硫酸钠干燥，浓缩，剩余物硅胶柱层析(二氯甲烷/甲醇＝20:1)纯化得650mg产品10-15，为浅黄色油状物，产率48％。LCMS(ESI)m/z 453.9(M+H)+,475.9(M+Na)+.
化合物11-15的合成:12786-191
Figure PCTCN2016085275-appb-000211
将上一步产物10-15(136mg,0.3mmol)，DCC(165mg,0.8mmol)和DMAP(24mg,0.2mmol)加入一个干燥的Schlenck管中，置换氩气3次，加入1mL二氯甲烷，搅拌。将美登醇(57mg,0.1mmol)溶于4mL干燥二氯甲烷中加入。反应液室温搅拌2h，缓慢加入0.3mL水淬灭反应，加入15mL乙酸乙酯，过滤，乙酸乙酯洗涤。滤液无水硫酸钠干燥，浓缩。粗产物使用制备HPLC分离得到26mg产物11-15，为浅黄色固体，产率26％。LCMS(ESI)m/z 1000.7(M+H)+.
化合物12-15的合成
Figure PCTCN2016085275-appb-000212
上一步反应产物11-15(20mg,0.02mmol)溶于4mL干燥的二氯甲烷中，冰水浴冷却至0℃，逐滴缓慢加入0.4mL三氟乙酸，逐步升至室温搅拌1小时，LCMS检测原料已完全转化， 常温减压移除二氯甲烷和三氟乙酸得14mg粗产品12-15，为浅黄色固体，直接用于下一步反应。LCMS(ESI)m/z 900.7(M+H)+.
化合物CE-046的合成
Figure PCTCN2016085275-appb-000213
上一步反应粗产物12-15(12mg,0.015mmol)悬浮于2mL饱和碳酸氢钠溶液中，加入化合物8(15.5mg,0.1mmol)，反应液室温搅拌过夜。二氯甲烷萃取3次(20mL×3)，合并有机相，饱和食盐水洗3次(20mL×3)，无水硫酸钠干燥，浓缩得粗产品，制备HPLC纯化得4mg产品CE-046，为浅黄色固体，产率26％。
LCMS(ESI)m/z 980.4(M+H)+.
1H NMR(400MHz,CDCl3)δppm 6.78(s,1H),6.66(s,1H),6.36&6.33(dd,J1＝12.4Hz,J2＝8.8Hz,2H),6.16(d,J＝10.8Hz,1H),6.11(d,J＝10.2Hz,1H),5.60&5.58(dd,J1＝12.0Hz,J2＝6.8Hz,1H),5.01(d,J＝8.0Hz,1H),4.40-4.30(m,2H),3.93(s,3H),3.77-3.68(m,2H),3.62-3.53(m,24H),3.43(d,J＝10.8Hz,2H),3.27(s,3H),3.12(d,J＝10.4Hz,1H),3.09(s,3H),2.64(d,J＝5.6Hz,1H),2.55(t,J＝10.0Hz,1H),2.15-2.10(m,1H),1.93(t,J＝8.0Hz,1H),1.62(s,3H),1.46(d,J＝5.6Hz,3H),1.18(s,3H),0.79(s,3H).
化合物CE-052a的合成
Figure PCTCN2016085275-appb-000214
上一步反应粗产品12-15(9mg,0.01mmol)和化合物9(7mg,0.03mmol)溶于1mL乙二醇二甲醚和1mL水的混合溶剂中，加入0.2mL饱和碳酸氢钠溶液。反应液室温搅拌过夜，二氯甲烷萃取(20mL×3)，合并有机相，无水硫酸钠干燥，浓缩，粗产品由制备HPLC纯化得4.8mg产品CE-052，为浅黄色固体，LCMS(ESI)m/z 1000.4(M–H2O)+.
1H NMR(400MHz,CDCl3)δppm 7.37(br,1H),6.85(s,1H),6.78(s,1H),6.68(s,1H),6.44&6.42(dd,J1＝12.0Hz,J2＝9.6Hz,1H),6.18(d,J＝8.4Hz,1H),5.60&5.58(dd,J1＝12.0Hz,J2＝6.8Hz,1H),4.95(d,J＝8.8Hz,1H),4.32(t,J＝8.0Hz,1H),4.19-4.11(m,1H),4.08(s,2H),3.99(s,3H),3.85-3.74(m,1H),3.70-3.60(m,25H),3.51(d,J＝9.2Hz,2H),3.46(d,J＝6.8 Hz,1H),3.35(s,3H),3.20(d,J＝10.0Hz,1H),3.15(s,3H),2.86(d,J＝5.6Hz,1H),2.60(t,J＝10.0Hz,1H),2.22(d,J＝11.2Hz,1H),1.68(s,3H),1.67(d,J＝10.4Hz,1H),1.51(d,J＝5.6Hz,3H),1.28(d,J＝5.6Hz,3H),0.84(s,3H).
实施例16CE-048的合成路线
Figure PCTCN2016085275-appb-000215
实验步骤
化合物2-16的合成
Figure PCTCN2016085275-appb-000216
250mL茄形瓶中加入化合物1-16(30g,0.39mol)，搅拌下加入氢氧化钾(8.1g,0.14mol)。将该悬浊液加热至90℃，搅拌至氢氧化钾完全溶解。苄溴(22g,0.13mol)逐滴缓慢加入。反应液升温至110℃，搅拌过夜。反应液冷却至室温，加入200mL水，乙酸乙酯萃取3次(100mL×3)，合并有机相，水洗3次(150mL×3)，饱和食盐水洗(100mL)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯10:1至3:1)纯化得17g产品2-10，为无色油状物，产率80％。LCMS(ESI)m/z167.1(M+H)+.
化合物3-16的合成
Figure PCTCN2016085275-appb-000217
上一步反应产物2-16(17g,0.1mol)和三乙胺(16.6mL,0.12mol)溶于150mL二氯甲 烷中，冷却至0℃，逐滴加入甲烷磺酰氯(8.5mL,0.11mol)。反应液升至室温搅拌2小时。加入50mL水淬灭反应，分出有机相，饱和食盐水洗3次(50mL×3)，无水硫酸钠干燥，浓缩得24.4g粗产品3-16，为黄色油状物，直接用于下一步反应。
化合物4-16的合成
Figure PCTCN2016085275-appb-000218
将上一步反应产物3-16(24.4g,0.1mol)和L-乳酸乙酯(20.8g,0.2mol)溶于N,N-二甲基甲酰胺150mL中，冷却至0℃，分批缓慢加入钠氢(8g,60％含量,悬浮于矿物油中,0.2mol)。反应液升至室温搅拌过夜。将反应液冷至0℃，逐滴缓慢加入20mL饱和氯化铵溶液淬灭反应，加入150mL水，乙酸乙酯萃取3次(100mL×3)，合并有机相，水洗3次(100mL×3)，饱和食盐水洗(100mL)，无水硫酸钠干燥，浓缩。粗产品硅胶柱层析(石油醚/乙酸乙酯＝10:1至2:1)纯化得7.1g产品4-16，为浅黄色油状物，产率30％。LCMS(ESI)m/z 253.1(M+H)+。
化合物5-16的合成
Figure PCTCN2016085275-appb-000219
上一步反应产物4-16(7.6g,30mmol)溶于50mL甲醇中，置换氮气3次，加入200mg 10％的钯碳干粉。反应液置换氢气3次，氢气氛中室温搅拌过夜。置换氮气3次，加入50mL二氯甲烷，过滤，二氯甲烷洗涤，滤液浓缩，得粗产品4.8g，为浅黄色油状物，直接用于下一步反应。LCMS(ESI)m/z163.1(M+H)+,185.1(M+Na)+.
化合物6-16的合成
Figure PCTCN2016085275-appb-000220
上一步反应产物5-16(4.8g,30mmol)和三乙胺(5.0mL,36mmol)溶于50mL二氯甲烷中，冷却至0℃，逐滴缓慢加入甲烷磺酰氯(2.64mL,33mmol)。反应液升至室温搅拌2小时。加入50mL水淬灭反应，分出有机相，饱和食盐水洗涤3次(50mL×3)，无水硫酸钠干燥，浓缩得粗产品，为浅黄色油状物，直接用于下一步反应。
化合物7-16的合成
Figure PCTCN2016085275-appb-000221
上一步反应粗产物6-16(2g,8.3mmol)溶于N,N-二甲基甲酰胺20mL中，加入硫代乙酸钾(1.89g,16.6mmol)。反应液室温搅拌过夜，加入30mL水，乙酸乙酯萃取3次(50mL×3)，合并有机相，水洗3次(30mL×3)，饱和食盐水洗(50mL)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析纯化得1.58g产品7-16，为棕色油状物，产率86％。LCMS(ESI)m/z 221.1(M+H)+。
化合物9-16的合成
Figure PCTCN2016085275-appb-000222
氮气保护下，将上一步反应产物7-16(1.58g,7.2mmol)溶于20mL甲醇和10mL水的混合溶剂中，加入氢氧化锂(0.87g,36mmol)。反应液在氮气保护下室温搅拌2小时，加入甲基硫代磺酸甲酯(1.1g,8.7mmol)。反应液继续在室温下搅拌过夜。加入50mL水，乙酸乙酯萃取3次(50mL×3)，合并有机相，饱和食盐水洗3次(50mL×3)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝1:2)纯化得0.98g产物9-16，为浅黄色油状物，产率70％。LCMS(ESI)m/z 197.0(M+H)+，219.0(M+Na)+.
化合物10-16的合成
Figure PCTCN2016085275-appb-000223
将上一步产物9-10(66.6mg,0.34mmol)，DCC(148mg,0.72mmol)和DMAP(29mg,0.24mmol)加入一个干燥的Schlenck管中，置换氩气3次，加入1mL二氯甲烷，搅拌。将美登醇(65mg,0.12mmol)溶于2mL干燥二氯甲烷中加入。反应液室温搅拌2h，缓慢加入0.3mL水淬灭反应，加入15mL乙酸乙酯，过滤，乙酸乙酯洗涤。滤液无水硫酸钠干燥，浓缩。粗产物使用制备HPLC分离得到25.6mg产物10-16，为白色固体，产率30％。LCMS(ESI)m/z 743.3(M+H)+.
化合物11-16的合成
Figure PCTCN2016085275-appb-000224
10-16(15mg,0.02mmol)溶于0.5mL乙酸乙酯和0.5mL甲醇的混合溶剂中，将二硫代苏糖醇DTT(25mg,0.16mmol)溶于0.5mL PH＝7.5的磷酸钾缓冲溶液中加入。反应液在氮气保护下搅拌3小时。加入PH＝6.0的磷酸钾缓冲溶液1mL淬灭反应。乙酸乙酯萃取3次(5mL×3)，合并有机相，饱和食盐水洗涤(5mL)，无水硫酸钠干燥，浓缩。粗产品使用制备HPLC(CH3CN in H2O-0.05％TFA from 5％to 95％)纯化得到12mg产物11-16，为白色固体，产率85％。LCMS(ESI)m/z 697.2(M+H)+.
化合物CE-048的合成
Figure PCTCN2016085275-appb-000225
上一步反应产物11-16(12mg,0.017mmol)溶于1.5mL四氢呋喃中，加入PH＝6的磷酸钾缓冲溶液1.5mL和4-(N-马来酰亚胺甲基)环己烷-1-羧酸琥珀酰亚胺酯(28mg,0.085mmol)，氮气保护下室温搅拌过夜。反应液过滤，直接由制备HPLC纯化得12mg产品CE-048，为白色固体，产率70％。
LCMS(ESI)m/z 1031.3(M+H)+.
1H NMR(400MHz,CDCl3)δppm 6.85(s,1H),6.80(d,J＝5.6Hz,1H),6.55(s,1H),6.45&6.43(dd,J1＝12.4Hz,J2＝8.8Hz,1H),6.20(d,J＝7.2Hz,1H),5.59-5.53(m,1H),4.92(d,J＝9.6Hz,1H),4.28(q,J＝8.4Hz,1H),4.19(s,2H),3.99(s,3H),3.80-3.74(m,1H),3.72-3.68(m,2H),3.52(d,J＝13.2Hz,1H),3.50(d,J＝10.0Hz,1H),3.39(d,J＝5.6Hz,2H),3.37(s,3H),3.21(d,J＝10.8Hz,1H),3.17(s,3H),3.18-3.13(m,1H),3.06-3.00(m,1H),2.93-2.86(m,3H),2.82(s,3H),2.61-2.52(m,3H),2.25-2.15(m,3H),2.01-1.93(m,2H),1.82-1.73(m,3H),1.68(s,3H),1.61-1.46(m,4H),1.30-1.26(m,4H),1.08(q,J＝10.4Hz,2H),0.83(s,3H).
实施例17CE-049的合成路
Figure PCTCN2016085275-appb-000226
实验步骤
化合物2-17的合成
Figure PCTCN2016085275-appb-000227
将1-17(4.5g,38mmol)和烯丙基溴(6.6mL,76mmol)溶于N,N-二甲基甲酰胺50mL中，冷却至0℃，分批缓慢加入钠氢(3.04g,60％含量,悬浮于矿物油中,76mmol)。反应液升至室温搅拌过夜。将反应液冷至0℃，逐滴缓慢加入20mL饱和氯化铵溶液淬灭反应，加入50mL水，乙酸乙酯萃取3次(50mL×3)，合并有机相，水洗3次(50mL×3)，饱和食盐水洗(50mL)，无水硫酸钠干燥，浓缩。粗产品硅胶柱层析(石油醚/乙酸乙酯＝10:1至2:1)纯化得1.95g产品2-17，为浅黄色油状物，产率30％。LCMS(ESI)m/z 159.2(M+H)+。
化合物3-17的合成
Figure PCTCN2016085275-appb-000228
上一步反应产物2-17(1.95g,8.7mmol)溶于15mL四氢呋喃中，冷至0℃，搅拌下逐滴加入1N硼烷四氢呋喃溶液(10mL,10mmol)。反应液升至室温搅拌2小时。反应液冷却至0℃，逐滴加入3N的氢氧化钠溶液(3.4mL,10mmol)，缓慢加入5mL30％含量的双 氧水，反应液升至室温搅拌过夜。加入50mL乙醚，分出有机相，饱和食盐水洗(30mL×3)，无水硫酸钠干燥，浓缩得1.94g粗产品3-17，为浅黄色油状物，直接用于下一步反应。
化合物4-17的合成
Figure PCTCN2016085275-appb-000229
上一步反应产物3-17(1.94g,10mmol)和三乙胺(1.7mL,12mmol)溶于20mL二氯甲烷中，冷却至0℃，逐滴缓慢加入甲烷磺酰氯(0.9mL,11mmol)。反应液升至室温搅拌2小时。加入50mL水淬灭反应，分出有机相，饱和食盐水洗涤3次(50mL×3)，无水硫酸钠干燥，浓缩得粗产品，为浅黄色油状物2.46g，直接用于下一步反应。LCMS(ESI)m/z255.3(M+H)+。
化合物5-17的合成
Figure PCTCN2016085275-appb-000230
上一步反应粗产物4-17(2.16g,9mmol)溶于N,N-二甲基甲酰胺20mL中，加入硫代乙酸钾(2.0g,18mmol)。反应液室温搅拌过夜，加入30mL水，乙酸乙酯萃取3次(50mL×3)，合并有机相，水洗3次(30mL×3)，饱和食盐水洗(50mL)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝1:1)纯化得1.7g产品5-17，为棕色油状物，产率84％。LCMS(ESI)m/z 235.3(M+H)+。
化合物7-17的合成
Figure PCTCN2016085275-appb-000231
氮气保护下，将上一步反应产物5-17(1.87g,8mmol)溶于20mL甲醇和10mL水的混合溶剂中，加入氢氧化锂(0.78g,32mmol)。反应液在氮气保护下室温搅拌2小时，加入甲基硫代磺酸甲酯(1.2g,9.6mmol)。反应液继续在室温下搅拌过夜。加入50mL水，乙酸乙酯萃取3次(50mL×3)，合并有机相，饱和食盐水洗3次(50mL×3)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝1:2)纯化得1.17g产物7-17，为浅 黄色油状物，产率65％。
LCMS(ESI)m/z 225.3(M+H)+,247.3(M+Na)+,
化合物8-17的合成
Figure PCTCN2016085275-appb-000232
将上一步产物7-17(74mg,0.33mmol)，DCC(136mg,0.66mmol)和DMAP(27mg,0.22mmol)加入一个干燥的Schlenck管中，置换氩气3次，加入1mL二氯甲烷，搅拌。将美登醇(62mg,0.11mmol)溶于2mL干燥二氯甲烷中加入。反应液室温搅拌2h，缓慢加入0.3mL水淬灭反应，加入15mL乙酸乙酯，过滤，乙酸乙酯洗涤。滤液无水硫酸钠干燥，浓缩。粗产物使用制备HPLC分离得到30mg产物8-17，为白色固体，产率35％。
LCMS(ESI)m/z 771.3(M+H)+.
化合物9-17的合成
Figure PCTCN2016085275-appb-000233
8-17(16mg,0.02mmol)溶于0.5mL乙酸乙酯和0.5mL甲醇的混合溶剂中，将二硫代苏糖醇DTT(18mg,0.117mmol)溶于0.5mL PH＝7.5的磷酸钾缓冲溶液中加入。反应液在氮气保护下搅拌3小时。加入PH＝6的磷酸钾缓冲溶液1mL淬灭反应。乙酸乙酯萃取3次(5mL×3)，合并有机相，饱和食盐水洗涤(5mL)，无水硫酸钠干燥，浓缩。粗产品使用制备HPLC(CH3CN in H2O-0.05％TFA from 5％to 95％)纯化得到12mg产物8-8，为白色固体，产率80％。LCMS(ESI)m/z725.3(M+H)+.
化合物CE-049的合成
Figure PCTCN2016085275-appb-000234
上一步反应产物9-17(10mg,0.014mmol)溶于1.5mL四氢呋喃中，加入PH＝6的磷酸钾缓冲溶液1.5mL和4-(N-马来酰亚胺甲基)环己烷-1-羧酸琥珀酰亚胺酯(23.4mg,0.07mmol)，氮气保护下室温搅拌过夜。反应液过滤，直接由制备HPLC纯化得11.4mg产品CE-049，为白色固体，产率78％。
LCMS(ESI)m/z 1059.3(M+H)+.
1H NMR(400MHz,CDCl3)δppm 6.85(s,1H),6.82(d,J＝5.6Hz,1H),6.59(s,1H),6.42&6.40(dd,J1＝12.4Hz,J2＝8.8Hz,1H),6.15(d,J＝8.4Hz,1H),5.55&5.53(dd,J1＝12.4Hz,J2＝3.6Hz,1H),4.88-4.80(m,1H),4.32(q,J＝8.4Hz,1H),3.99(s,3H),3.83-3.76(m,1H),3.56-3.47(m,4H),3.39(d,J＝5.6Hz,2H),3.37(s,3H),3.23-3.17(m,2H),3.15(s,3H),3.07-2.94(m,1H),2.93-2.84(m,3H),2.83(s,3H),2.63-2.56(m,4H),2.32-2.26(m,2H),2.17(d,J＝9.6Hz,2H),2.01-1.93(m,2H),1.82-1.73(m,4H),1.68(s,3H),1.60-1.40(m,2H),1.55(d,J＝4.0Hz,3H),1.44(s,3H),1.30(d,J＝4.8Hz,3H),1.08(q,J＝10.0Hz,2H),0.81(s,3H).
实施例18CE-051的合成路线
Figure PCTCN2016085275-appb-000235
使用D-乳酸乙酯为原料，CE-051合成步骤和CE-035一致。
LCMS(ESI)m/z 1045.4(M+H)+.
1H NMR(400MHz,CDCl3)δppm 6.84(s,1H),6.80(s,1H),6.56(s,1H),6.46&6.42(dd,J1＝12.4Hz,J2＝8.8Hz,1H),6.18(d,J＝8.0Hz,1H),5.55&5.54(dd,J1＝12.8Hz,J2＝7.2Hz,1H),4.92(t,J＝6.4Hz,1H),4.28(t,J＝8.4Hz,1H),4.07-4.01(m,1H),3.99(s,3H),3.80-3.69(m,2H),3.60-3.54(m,1H),3.53-3.46(m,2H),3.39(d,J＝5.6Hz,2H),3.36(s,3H),3.21(d,J＝11.6Hz,2H),3.15(s,3H),3.06-2.98(m,1H),2.92-2.86(m,2H),2.82(s,3H),2.61-2.52(m,3H),2.28-2.21(m,1H),2.16(d,J＝9.6Hz,2H),2.01-1.93(m,1H),1.82-1.73(m,3H),1.68(s,3H),1.60-1.40(m,2H),1.55(q,J＝8.8Hz,2H),1.49(d,J＝6.0Hz,3H),1.30(s,3H),1.26(s,3H),1.08(q,J＝10.0Hz,2H),0.84(s,3H).
实施例19CE-053的合成路线
Figure PCTCN2016085275-appb-000236
实验步骤
化合物2-19的合成
Figure PCTCN2016085275-appb-000237
将化合物1-19(5g,44mmol)溶于80mL二氯甲烷中，依次加入三乙胺(7.4mL,52.8mol)和醋酸酐(4.95mL,52.8mol)，室温搅拌过夜，待反应完全后，加入100mL水，分液，水相再用二氯甲烷萃取2次(50mL×2)，合并有机相，饱和食盐水洗涤(5mL)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝5:1)纯化得6.5g产物2-19，为无色油状物，产率95％。LCMS(ESI)m/z 157.1(M+H)+.
化合物3-19的合成
Figure PCTCN2016085275-appb-000238
在25mL烧瓶中加入三氯氧磷(7.7mL,83.4mol)和DMF(39ml)，常温搅拌30min后，冰水浴冷却至0℃，将化合物2-19(6.5g,41.7mmol)溶于39mL DMF中缓慢滴加至烧瓶中，反应液升至室温搅拌过夜。加入100mL饱和碳酸氢钠溶液，持续搅拌10小时，乙酸乙酯萃取(50mL×3)，合并有机相，饱和食盐水洗涤(50mL)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝1:1)纯化得6.5g产物3-19，为无色油状物，产率85％。LCMS(ESI)m/z 185.1(M+H)+.
化合物4-19的合成
Figure PCTCN2016085275-appb-000239
取上一步产物3-19(2g,11mmol)溶于乙腈：叔丁醇：水＝2：2：1的混合溶剂55ml中，加入磷酸二氢钠(3.96g,33ml)和浓度为30％的过氧化氢溶液(1.8mL,55mmol)，混合液室温搅拌30分钟后，加入次氯酸钠(6g,66mmol)，反应常温搅拌2小时，加入50mL水，并用10％的盐酸溶液调节PH至5-6，二氯甲烷萃取(50mL×3)，饱和食盐水洗涤(30mL)，无水硫酸钠干燥，浓缩得粗产品，为浅黄色油状物1.5g，直接用于下一步反应。LCMS(ESI)m/z 201.1(M+H)+。
化合物5-19的合成
Figure PCTCN2016085275-appb-000240
将上一步反应产物4-19(1.5g,7.5mmol)溶于甲醇(20mL)和水(5mL)的混合溶剂中，加入氢氧化锂(0.36g,15mmol)，反应常温搅拌2小时后，加入20mL水，并用1N盐酸溶液调节PH至5-6，二氯甲烷萃取(50mL×3)，饱和食盐水洗涤(30mL)，无水硫酸钠干燥，浓缩得粗产品，为浅黄色油状物1.2g，直接用于下一步反应。LCMS(ESI)m/z 161.1(M+H)+。
化合物6-19的合成
Figure PCTCN2016085275-appb-000241
将上一步反应产物5-19(1.2g,7.5mmol)溶于甲醇(20ml)中，冰水浴条件下缓慢滴加氯化亚砜(1.1mL,15mmol)，反应升至常温搅拌过夜，加入50mL水淬灭反应，乙酸乙酯萃取(50mL×3)，饱和食盐水洗涤(5mL)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝3:1)纯化得1.2g产物6-19，为无色油状物，产率63％。LCMS(ESI)m/z 175.1(M+H)+.
化合物7-19的合成
Figure PCTCN2016085275-appb-000242
将上一步反应产物6-19(1.2g,6.9mmol)和三乙胺(1.2mL,8.3mmol)溶于20mL二氯甲烷中，冷却至0℃，逐滴缓慢加入甲烷磺酰氯(0.68mL,8.3mmol)。反应液升至室温搅拌2小时。加入50mL水淬灭反应，分出有机相，饱和食盐水洗涤3次(50mL×3)，无水硫酸钠干燥，浓缩得1.5g粗产品，为浅黄色油状物，直接用于下一步反应。LCMS(ESI)m/z 253.1(M+H)+。
化合物8-19的合成
Figure PCTCN2016085275-appb-000243
上一步反应粗产物7-19(1.5g,6.3mmol)溶于N,N-二甲基甲酰胺20mL中，加入硫代乙酸钾(1.12g,9.5mmol)。反应液室温搅拌过夜，加入30mL水，乙酸乙酯萃取3次(50mL×3)，合并有机相，水洗3次(30mL×3)，饱和食盐水洗(50mL)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝3:1)纯化得700mg产品8-19，为棕色油状物，产率51％。LCMS(ESI)m/z 233.1(M+H)+.
化合物9-19的合成
Figure PCTCN2016085275-appb-000244
氮气保护下，将上一步反应产物8-19(0.7g,3mmol)溶于20mL甲醇和5mL水的混合溶剂中，加入氢氧化锂(0.36g,15mmol)。反应液在氮气保护下室温搅拌2小时，加入甲基硫代磺酸甲酯(0.57g,4.5mmol)。反应液继续在室温下搅拌过夜。加入50mL水，乙酸乙酯萃取3次(50mL×3)，合并有机相，饱和食盐水洗3次(50mL×3)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝1:1)纯化得0.45g产物10-19，为浅黄色油状物，产率67％。LCMS(ESI)m/z 223.1(M+H)+.
化合物CE-053的合成
Figure PCTCN2016085275-appb-000245
将上一步产物10-19(70mg,0.315mmol)，DCC(130mg,0.63mmol)和DMAP(25mg,0.21mmol)加入一个干燥的Schlenck管中，置换氩气3次，加入1mL二氯甲烷，搅拌。将美登醇(60mg,0.105mmol)溶于2mL干燥二氯甲烷中加入。反应液室温搅拌2h，缓慢加入0.3mL水淬灭反应，加入15mL乙酸乙酯，过滤，乙酸乙酯洗涤。滤液无水硫酸钠干燥，浓缩。粗产物使用制备HPLC分离得到40mg产物CE-053，为白色固体，产率48％。
LCMS(ESI)m/z 768.7(M+H)+.
1H NMR(400MHz,CDCl3)δppm 6.84(s,1H),6.79(t,J＝11.6Hz,1H),6.49-6.41(m,1H),6.36(d,J＝8.8Hz,1H),6.24-6.13(m,1H),5.59-5.38(m,1H),4.91&4.85(d,J＝7.6Hz,1H),4.31-4.16(m,2H),3.99(s,3H),3.65-3.45(m,5H),3.37(d,J＝8.4Hz,2H),3.23-3.18(m,2H),3.17(s,3H),3.00-2.54(m,6H),2.43(s,3H),2.23-2.15(m,2H),1.80-1.60(m,2H),1.69(s,3H),1.50-1.42(m,2H),1.40-1.33(m,1H),1.30(d,J＝4.8Hz,3H),0.81(s,3H).
实施例20CE-054、055的合成路线
Figure PCTCN2016085275-appb-000246
实验步骤
化合物2-20的合成
Figure PCTCN2016085275-appb-000247
原料1-20(6g,50mmol)和咪唑(4.08g,60mmol)溶于80mL四氢呋喃中，冰水浴冷却至0℃，分批缓慢加入TBDPSCl(14g,51mmol)，升至室温搅拌过夜。加入20mL饱和氯化铵溶液，二氯甲烷萃取，合并有机相，无水硫酸钠干燥，浓缩得18g粗产品2-20，直接用于下一步反应。LCMS(ESI)m/z 358.1(M+H)+.
化合物2-20的合成
Figure PCTCN2016085275-appb-000248
上一步反应产物2-20(18g,50mmol)溶于60mL乙腈中，加入氧化银(9.3g,75mmol)，搅拌下加入碘甲烷(9.3mL,150mmol)。反应液室温搅拌过夜，硅藻土过滤，二氯甲烷洗涤，滤液浓缩，剩余物硅胶柱层析(石油醚/乙酸乙酯＝5:1)，得12g产品3-20，为浅黄色油状物,产率65％。LCMS(ESI)m/z 372.1(M+H)+.
化合物2-20的合成
Figure PCTCN2016085275-appb-000249
上一步反应产物3-20(5.6g,15mmol)溶于20mL四氢呋喃中，加入四丁基氟化铵(30mL,1N,30mmol)。反应液室温搅拌过夜，浓缩，剩余物硅胶柱层析(二氯甲烷/甲醇＝20:1)，得2g产品4-20，为浅黄色油状物，产率80％。LCMS(ESI)m/z 134.7(M+H)+.1H NMR(400MHz,CDCl3)δppm 3.93-3.87(m,2H),3.83-3.79(m,1H),3.79(s,1H),3.50(s,1H).
化合物2-20的合成
Figure PCTCN2016085275-appb-000250
上一步反应产物4-20(402mg,3mmol)和DMAP(440mg,3.6mmol)溶于15mL二氯甲 烷中，冰水浴冷却至0℃，加入氯甲酸烯丙酯(0.38mL,36mmol)。反应液室温搅拌2小时，浓缩，剩余物硅胶柱层析(石油醚/乙酸乙酯＝5:1)，得500mg产品5-20，为浅黄色油状物，产率76％。LCMS(ESI)m/z 219.7(M+H)+.
化合物2-20的合成
Figure PCTCN2016085275-appb-000251
上一步反应产物5-20(436mg,2mmol)和Pd(PPh3)4(12mg,0.01mmol)于干燥Schlenk管中，置换氩气三次，加入15mL1,4-二氧六环，升温至100℃搅拌1小时。反应液冷却至室温，浓缩，剩余物硅胶柱层析(石油醚/乙酸乙酯＝3:1)，得160mg产品6-20，为浅黄色油状物，产率46％。LCMS(ESI)m/z 175.7(M+H)+.
化合物2-20的合成
Figure PCTCN2016085275-appb-000252
上一步反应产物6-20(1.74g,10mmol)溶于20mL四氢呋喃中，冷至0℃，拌下逐滴加入1M硼烷四氢呋喃溶液(12mL,12mmol)。反应液升至室温搅拌2小时。反应液冷却至0℃，逐滴加入3N的氢氧化钠溶液(4mL,12mmol)，缓慢加入6mL 30％含量的双氧水，反应液升至室温搅拌过夜。加入80mL乙醚，分出有机相，饱和食盐水洗(30mL×3)，无水硫酸钠干燥，浓缩得1.2g粗产品7-20，为浅黄色油状物，直接用于下一步反应。
化合物2-20的合成
Figure PCTCN2016085275-appb-000253
上一步反应产物7-20(960mg,5mmol)和三乙胺(0.83mL,6mmol)溶于50mL二氯甲烷中，冷却至0℃，逐滴缓慢加入甲烷磺酰氯(0.44mL,5.5mmol)。反应液升至室温搅拌2小时。加入50mL水淬灭反应，分出有机相，饱和食盐水洗涤3次(30mL×3)，无水硫酸钠干燥，浓缩得1.3g粗产品8-20，为浅黄色油状物，直接用于下一步反应。LCMS(ESI)m/z 271.7(M+H)+。
化合物2-20的合成
Figure PCTCN2016085275-appb-000254
上一步反应粗产物8-20(270mg,1mmol)溶于N,N-二甲基甲酰胺5mL中，加入硫代乙酸钾(228mg,2mmol)。反应液室温搅拌过夜，加入20mL水，乙酸乙酯萃取3次(20mL×3)，合并有机相，水洗3次(20mL×3)，饱和食盐水洗(20mL)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝5:1)纯化得170mg产品9-20，为棕色油状物，产率68％。LCMS(ESI)m/z 251.7(M+H)+,1H NMR(400MHz,CDCl3)δppm 3.97(t,J＝3.6Hz,1H),3.79(s,3H),3.75-3.68(m,2H),3.58-3.49(m,2H),3.47(s,3H),2.92(t,J＝5.6Hz,2H),2.32(s,3H),1.87-1.81(m,2H).
化合物2-20的合成
Figure PCTCN2016085275-appb-000255
氮气保护下，将上一步反应产物9-20(125mg,0.5mmol)溶于3mL甲醇和2mL水的混合溶剂中，加入一水合氢氧化锂(210g,5mmol)。反应液在氮气保护下室温搅拌2小时，加入甲基硫代磺酸甲酯(126mg,1mmol)。反应液继续在室温下搅拌过夜。加入5mL水，乙酸乙酯萃取3次(20mL×3)，合并有机相，饱和食盐水洗3次(20mL×3)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝1:2)纯化得80mg产物11-20，为浅黄色油状物，产率67％。LCMS(ESI)m/z 241.7(M+H)+.
化合物CE-054的合成
Figure PCTCN2016085275-appb-000256
将上一步产物11-20(58mg,0.24mmol)，DCC(132mg,0.64mmol)和DMAP(20mg,0.16mmol)加入一个干燥的Schlenck管中，置换氩气3次，加入1mL二氯甲烷，搅拌。将美登醇(46mg,0.08mmol)溶于4mL干燥二氯甲烷中加入。反应液室温搅拌2h，缓慢加入0.3mL水淬灭反应，加入15mL乙酸乙酯，过滤，乙酸乙酯洗涤。滤液无水硫酸钠干燥，浓缩。粗产物使用制备HPLC分离纯化得到2个异构体：CE-054(P1)，25mg为 白色固体；CE-055(P2)，13mg，为浅黄色固体。LCMS(ESI)m/z 787.7(M+H)+。
HPLC(15min):CE-054,Rt＝10.58min；CE-055,Rt＝10.75min
Mobile Phase:A:water(0.01％TFA)B:ACN(0.01％TFA)
Gradient:0min 5％B,3min 5％B,10min 95％B,15min 95％B
Flow Rate:1.2ml/min
Column:Eclipse XDB-C18,4.6×150mm,5um
Oven Temperature:40℃
CE-054:1H NMR(400MHz,CDCl3)δppm 6.89(s,1H),6.86(s,1H),6.46&6.44(dd,J1＝12.0Hz,J2＝9.2Hz,1H),6.27(s,1H),6.23(d,J＝8.4Hz,1H),5.59&5.57(dd,J1＝12.4Hz,J2＝7.2Hz,1H),4.92(d,J＝7.6Hz,1H),4.27(t,J＝9.2Hz,1H),4.14-4.12(m,1H),3.99(s,3H),3.78-3.76(m,1H),3.65(t,J＝7.2Hz,2H),3.52(t,J＝10.0Hz,1H),3.52(s,3H),3.44-3.40(m,1H),3.37(s,3H),3.20(d,J＝14.0Hz,1H),3.19(s,3H),2.89(d,J＝7.6Hz,1H),2.81(t,J＝6.0Hz,2H),2.60(t,J＝10.0Hz,1H),2.39(s,3H),2.25&2.23(dd,J1＝8.8Hz,J2＝3.2Hz,1H),1.94(d,J＝9.2Hz,2H),1.69(s,3H),1.62(d,J＝10.4Hz,2H),1.30(d,J＝4.8Hz,3H),1.20-1.12(m,2H),0.83(s,3H).
CE-055:1H NMR(400MHz,CDCl3)δppm 7.13(s,1H),6.84(s,1H),6.45&6.43(dd,J1＝12.4Hz,J2＝9.2Hz,1H),6.29(s,1H),6.26(d,J＝9.2Hz,1H),5.57&5.55(dd,J1＝11.2Hz,J2＝7.2Hz,1H),4.90(d,J＝7.6Hz,1H),4.37&4.36(dd,J1＝7.2Hz,J2＝3.2Hz,1H),4.24(t,J＝8.8Hz,1H),3.99(s,3H),3.78-3.76(m,1H),3.71-3.60(m,2H),3.59(d,J＝11.2Hz,1H),3.50(d,J＝9.2Hz,1H),3.50-3.40(m,2H),3.37(s,6H),3.18(d,J＝14.0Hz,1H),3.16(s,3H),2.88(d,J＝7.6Hz,1H),2.83(t,J＝5.6Hz,2H),2.59(t,J＝10.0Hz,1H),2.42(s,3H),2.25&2.23(dd,J1＝8.8Hz,J2＝3.2Hz,1H),1.94(d,J＝9.2Hz,2H),1.68(s,3H),1.62(d,J＝10.4Hz,1H),1.29(d,J＝4.8Hz,3H),1.20-1.12(m,2H),0.84(s,3H).
实施例21CE-056的合成路线
Figure PCTCN2016085275-appb-000257
实验步骤
化合物2-21的合成
Figure PCTCN2016085275-appb-000258
将化合物1-21(3g,14.3mmol)溶于50mL二氯甲烷中，加入TBDPSCl(4.32g,15.7mmol)和咪唑(1.07g,15.7mmol)，反应液常温搅拌2小时，加入50mL水，二氯甲烷萃取(50mL×3)，合并有机相，饱和食盐水洗3次(50mL×3)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝5:1)纯化得5g产物2-21，为无色油状物，产率78％。LCMS(ESI)m/z 449.2(M+H)+.
化合物3-21的合成
Figure PCTCN2016085275-appb-000259
上一步反应产物2-21(5g,11mmol)溶于50mL甲醇和5mL水的混合溶剂中，加入氢氧化锂(1.32g,55mmol)。反应液置于常温下搅拌2小时，减压移除甲醇，加入50mL水，二氯甲烷萃取(50mL×3)，合并有机相，饱和食盐水洗3次(50mL×3)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝3:1)纯化得3.2g产物3-21，为无色油状物，产率83％。LCMS(ESI)m/z 345.2(M+H)+.
化合物4-21的合成
Figure PCTCN2016085275-appb-000260
将上一步反应产物3-21(3.2g,9.3mmol)和化合物M1(3.2g,14mmol)溶于N,N-二甲基甲酰胺100mL中，冷却至0℃，分批缓慢加入钠氢(0.75g,60％含量,悬浮于矿物油中,18.6mmol)。反应液升至室温搅拌3h。将反应液冷至0℃，逐滴缓慢加入20mL饱和氯化铵溶液淬灭反应，加入50mL水，二氯甲烷萃取3次(50mL×3)，合并有机相，水洗3次(100mL×3)，饱和食盐水洗(100mL)，无水硫酸钠干燥，浓缩。粗产品硅胶柱层析(石油醚/乙酸乙酯＝5:1)纯化得1.4g产品4-21，为浅黄色油状物，产率31.5％。LCMS(ESI)m/z479.2(M+H)+。
化合物5-21的合成
Figure PCTCN2016085275-appb-000261
上一步反应产物4-21(1.4g,3mmol)溶于20ml四氢呋喃中，加入1N的TBAF四氢呋喃溶液(4.5ml,4.5mmol)。反应液置于常温下搅拌5小时，加入50mL水，乙酸乙酯萃取(50mL×3)，合并有机相，饱和食盐水洗3次(50mL×3)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝3:1)纯化得0.55g产物5-21，为无色油状物，产率73％。LCMS(ESI)m/z 241.1(M+H)+.
化合物6-21的合成
Figure PCTCN2016085275-appb-000262
上一步反应产物5-21(0.55g,2.3mmol)溶于20mL丙酮中，冰水浴冷却至0℃，逐滴缓慢加入新制的Jones试剂(0.46g,4.6mmol),约2分钟滴加完毕。反应液升至室温搅拌过夜，加入1mL异丙醇淬灭反应，减压移除丙酮，加入50ml水，二氯甲烷萃取3次(50mL×3)，合并有机相，饱和食盐水洗3次(50mL×3)，无水硫酸钠干燥，浓缩得粗产品，为无色油状物，直接用于下一步反应。LCMS(ESI)m/z 255.1(M+H)+。
化合物7-21的合成
Figure PCTCN2016085275-appb-000263
将上一步反应产物6-21(0.5g,2mmol)溶于甲醇(20ml)中，冰水浴条件下缓慢滴加氯化亚砜(0.48g,4mmol)，反应升至常温搅拌过夜，加入50ml水淬灭反应，乙酸乙酯萃取(50mL×3)，饱和食盐水洗涤(50mL)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝3:1)纯化得0.5g产物7-21，为无色油状物，产率81％。LCMS(ESI)m/z269.1(M+H)+.
化合物8-21的合成
Figure PCTCN2016085275-appb-000264
上一步反应产物7-21(0.5g,1.9mmol)溶于20mL甲醇中，置换氮气3次，加入30mg 10％的钯碳干粉。反应液置换氢气3次，氢气氛中室温搅拌过夜。置换氮气3次，加入20mL二氯甲烷，过滤，二氯甲烷洗涤，滤液浓缩，得粗产品0.32g，为浅黄色油状物，直接用于下一步反应。LCMS(ESI)m/z 179.1(M+H)+.
化合物9-21的合成
Figure PCTCN2016085275-appb-000265
将上一步反应产物8-21(0.32g,1.8mmol)和三乙胺(0.3mL,2.1mmol)溶于20mL二氯甲烷中，冷却至0℃，逐滴缓慢加入甲烷磺酰氯(0.17mL,2.1mmol)。反应液升至室温搅拌2小时。加入20mL水淬灭反应，分出有机相，饱和食盐水洗涤3次(50mL×3)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝3:1)纯化得250mg产物9-21，为无色油状物，产率52％。LCMS(ESI)m/z 257.1(M+H)+。
化合物10-21的合成
Figure PCTCN2016085275-appb-000266
上一步反应粗产物9-21(250mg,1mmol)溶于N,N-二甲基甲酰胺5mL中，加入硫代乙酸钾(120mg,1.5mmol)。反应液室温搅拌过夜，加入20mL水，乙酸乙酯萃取3次(20mL×3)，合并有机相，水洗3次(20mL×3)，饱和食盐水洗(20mL)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝3:1)纯化得170mg产品10-21，为棕色油状物，产率74％。LCMS(ESI)m/z 237.1(M+H)+。
化合物12-21的合成
Figure PCTCN2016085275-appb-000267
氮气保护下，将上一步反应产物10-21(170mg,0.72mmol)溶于10mL甲醇和5mL水的混合溶剂中，加入氢氧化锂(86mg,3.6mmol)。反应液在氮气保护下室温搅拌2小时，加入甲基硫代磺酸甲酯(140mg,1.1mmol)。反应液继续在室温下搅拌过夜。加入20mL水，乙酸乙酯萃取3次(30mL×3)，合并有机相，饱和食盐水洗3次(20mL×3)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝1:1)纯化得100mg产物12-21，为浅黄色油状物，产率61％。LCMS(ESI)m/z 227.1(M+H)+.
实化合物CE-056的合成
Figure PCTCN2016085275-appb-000268
将上一步产物12-21(100mg,0.44mmol)，DCC(173mg,0.84mmol)和DMAP(34mg,0.28mmol)加入一个干燥的Schlenck管中，置换氩气3次，加入1mL二氯甲烷，搅拌。将美登醇(79mg,0.14mmol)溶于2mL干燥二氯甲烷中加入。反应液室温搅拌2h，缓慢加入0.3mL水淬灭反应，加入15mL乙酸乙酯，过滤，乙酸乙酯洗涤。滤液无水硫酸钠干燥，浓缩。粗产物使用制备HPLC分离得到3mg产物CE-056，为白色固体，产率3％。
LCMS(ESI)m/z 773.3(M+H)+。
实施例22CE-057的合成路线
Figure PCTCN2016085275-appb-000269
实验步骤
化合物2-22的合成
Figure PCTCN2016085275-appb-000270
乙二醇(7.6mL,0.15mol)溶于100mL干燥四氢呋喃中，加入100mg金属钠，室温搅拌至钠块完全反应，加入丙烯酸叔丁酯(14.5mL,0.1mol)，室温搅拌过夜。减压移除四氢呋喃，往剩余物中加入100mL乙酸乙酯，水洗3次(50mL×3)，无水硫酸钠干燥，浓缩，粗产品硅胶柱层析(石油醚/乙酸乙酯＝1:1)纯化得14g产品2-22，为无色油状物，产率73％。LCMS(ESI)m/z 191.1(M+H)+。
化合物3-22的合成
Figure PCTCN2016085275-appb-000271
上一步反应产物2-22(7.6g,40mmol)和三乙胺(6.6mL,48mmol)溶于50mL二氯甲烷中，冷却至0℃，逐滴缓慢加入甲烷磺酰氯(3.5mL,44mmol)。反应液升至室温搅拌2小时。加入50mL水淬灭反应，分出有机相，饱和食盐水洗涤3次(50mL×3)，无水硫酸钠干燥，浓缩得粗产品，为浅黄色油状物，直接用于下一步反应。LCMS(ESI)m/z 269.1 (M+H)+。
化合物4-22的合成
Figure PCTCN2016085275-appb-000272
上一步反应粗产物3-22(2.7g,10.0mmol)溶于N,N-二甲基甲酰胺20mL中，加入硫代乙酸钾(2.3g,20mmol)。反应液室温搅拌过夜，加入30mL水，乙酸乙酯萃取3次(50mL×3)，合并有机相，水洗3次(30mL×3)，饱和食盐水洗(50mL)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝2:1)纯化得2.0g产品4-22，为棕色油状物，产率86％。LCMS(ESI)m/z 248.2(M+H)+.
化合物6-22的合成
Figure PCTCN2016085275-appb-000273
氮气保护下，将上一步反应产物4-22(2g,8mmol)溶于20mL甲醇和10mL水的混合溶剂中，加入氢氧化锂(0.78g,32mmol)。反应液在氮气保护下室温搅拌2小时，加入甲基硫代磺酸甲酯(1.2g,9.6mmol)。反应液继续在室温下搅拌过夜。加入50mL水，乙酸乙酯萃取3次(50mL×3)，合并有机相，饱和食盐水洗3次(50mL×3)，无水硫酸钠干燥，浓缩。粗产品经硅胶柱层析(石油醚/乙酸乙酯＝1:2)纯化得1.2g产物6-22，为浅黄色油状物，产率75％。
LCMS(ESI)m/z197.0(M+H)+
化合物7-22的合成
Figure PCTCN2016085275-appb-000274
将上一步产物6-22(47mg,0.24mmol)，DCC(132mg,0.64mmol)和DMAP(20mg,0.16mmol)加入一个干燥的Schlenck管中，置换氩气3次，加入1mL二氯甲烷，搅拌。将美登醇(45mg,0.08mmol)溶于4mL干燥二氯甲烷中加入。反应液室温搅拌2h，缓慢加入0.3mL水淬灭反应，加入15mL乙酸乙酯，过滤，乙酸乙酯洗涤。滤液无水硫酸钠 干燥，浓缩。粗产物使用制备HPLC分离得到30mg产物7-22，为白色固体，产率50％。
化合物8-22的合成
Figure PCTCN2016085275-appb-000275
上一步反应产物7-22(15mg,0.02mmol)溶于0.5mL乙酸乙酯和0.5mL甲醇的混合溶剂中，将二硫代苏糖醇DTT(15mg,0.10mmol)溶于0.5mL PH＝7.5的磷酸钾缓冲溶液中加入。反应液在氮气保护下搅拌3小时。加入PH＝6的磷酸钾缓冲溶液1mL淬灭反应。乙酸乙酯萃取3次(5mL×3)，合并有机相，饱和食盐水洗涤(5mL)，无水硫酸钠干燥，浓缩。粗产品使用制备HPLC(CH3CN in H2O-0.05％TFA from 5％to 95％)纯化得到11mg产物8-22，为白色固体，产率77％。LCMS(ESI)m/z 697.3(M+H)+.
化合物CE-057的合成
Figure PCTCN2016085275-appb-000276
上一步反应产物8-22(10mg,0.014mmol)溶于1.5mL四氢呋喃中，加入PH＝6的磷酸钾缓冲溶液1.5mL和4-(N-马来酰亚胺甲基)环己烷-1-羧酸琥珀酰亚胺酯(23mg,0.07mmol)，氮气保护下室温搅拌过夜。反应液过滤，直接由制备HPLC纯化得9.5mg产品CE-057，为白色固体，产率66％。
LCMS(ESI)m/z 1030.6(M+H)+.
1H NMR(400MHz,CDCl3)δppm 6.84(s,1H),6.78(d,J＝10.8Hz,1H),6.47&6.43(dd,J1＝15.2Hz,J2＝11.2Hz,1H),6.39(s,1H),6.19(d,J＝10.8Hz,1H),5.57&5.54(dd,J1＝15.2Hz,J2＝8.8Hz,1H),4.93-4.88(m,1H),4.29-4.21(m,1H),3.99(s,3H),3.92-3.85(m,1H),3.84-3.68(m,4H),3.55-3.47(m,2H),3.39(t,J＝6.8Hz,2H),3.35(s,3H),3.30-3.18(m,2H),3.15(d,J＝17.2Hz,3H),3.10-2.94(m,1H),2.94-2.67(m,4H),2.83(s,3H),2.61-2.47(m,3H),2.16(d,J＝11.2Hz,2H),1.98-1.90(m,1H),1.80-1.78(m,2H),1.68(s,3H),1.64-1.45(m,4H),1.32-1.25(m,4H),1.08(q,J＝8.4Hz,3H),0.84(s,3H).
实施例23化合物CE-063的合成路线
Figure PCTCN2016085275-appb-000277
实验步骤
化合物2-23的合成
Figure PCTCN2016085275-appb-000278
五缩六乙二醇(11.3mL,45mmol)溶于80mL干燥四氢呋喃中，加入50mg金属钠，室温搅拌至钠块完全反应，加入丙烯酸叔丁酯(4.35mL,30mmol)，室温搅拌过夜。减压移除四氢呋喃，往剩余物中加入80mL乙酸乙酯，水洗3次(50mL×3)，无水硫酸钠干燥，浓缩，粗产品硅胶柱层析(二氯甲烷/甲醇＝20:1)纯化得8g产品2-23，为无色油状物，产率65％。LCMS(ESI)m/z 432.9(M+Na)+。
化合物3-23的合成
Figure PCTCN2016085275-appb-000279
上一步反应产物2-23(2.05g,5mmol)和三乙胺(0.85mL,6mmol)溶于30mL二氯甲烷中，冷却至0℃，逐滴缓慢加入甲烷磺酰氯(0.45mL,5.5mmol)。反应液升至室温搅拌2小时。加入30mL水淬灭反应，分出有机相，饱和食盐水洗涤3次(30mL×3)，无水硫酸钠干燥，浓缩得粗产品，为浅黄色油状物，直接用于下一步反应。
化合物4-23的合成
Figure PCTCN2016085275-appb-000280
上一步反应产物3-23(2.5g,5mmol)溶于20mL N,N-二甲基甲酰胺中，加入叠氮化钠(390mg,6mmol)。反应液升至50℃搅拌4小时。将反应液冷却至室温，加入30mL水淬灭反应，乙酸乙酯萃取3次(30mL×3)，合并有机相，饱和食盐水洗3次(30mL×3)，无水硫酸钠干燥，浓缩得粗产品，硅胶柱层析(石油醚/乙酸乙酯＝1:1)纯化得1.8g产品4-23，为浅黄色油状物，两步反应产率84％。LCMS(ESI)m/z 436.3(M+H)+.
化合物5-23的合成
Figure PCTCN2016085275-appb-000281
上一步反应产物4-23(1.74g,4mmol)溶于30mL四氢呋喃和5mL水中，加入三苯基磷(1.31g,5mmol)。反应液室温搅拌过夜。减压移除四氢呋喃，加入1N稀盐酸10mL，乙酸乙酯洗涤3次(20mL×3)，水相冻干得1.5g粗产品5-23，为浅黄色油状物。LCMS(ESI)m/z 410.3(M+H)+.
化合物6-23的合成
Figure PCTCN2016085275-appb-000282
上一步反应粗产物5-23(818mg,2mmol)溶于4mL干燥的二氯甲烷中，冰水浴冷却至0℃，逐滴缓慢加入0.8mL三氟乙酸，逐步升至室温搅拌1小时，LCMS检测原料已完全转化，常温减压移除二氯甲烷和三氟乙酸得700mg粗产品6-23，为浅黄色固体，直接用于下一步反应。LCMS(ESI)m/z 354.2(M+H)+.
化合物7-23的合成
上一步反应粗产物6-23(530mg,1.5mmol)加入饱和碳酸氢钠溶液3mL和30mL二氯甲烷，冰水浴冷却至0℃，加入Boc2O(436mg,2mmol)，升至室温搅拌2h。反应液水洗两 次(20mL×2)，合并水相加入0.5M KHSO4溶液淬灭反应调节PH＝3-4，二氯甲烷萃取3次(20mL×3)，合并有机相，无水硫酸钠干燥，浓缩，剩余物硅胶柱层析(二氯甲烷/甲醇＝20:1)纯化得380mg产品7-23，为浅黄色油状物，产率56％。LCMS(ESI)m/z 453.9(M+H)+,475.9(M+Na)+.
化合物8-23的合成
Figure PCTCN2016085275-appb-000284
将上一步产物7-23(136mg,0.3mmol)，DCC(165mg,0.8mmol)和DMAP(24mg,0.2mmol)加入一个干燥的Schlenck管中，置换氩气3次，加入1mL二氯甲烷，搅拌。将美登醇(57mg,0.1mmol)溶于4mL干燥二氯甲烷中加入。反应液室温搅拌2h，缓慢加入0.3mL水淬灭反应，加入15mL乙酸乙酯，过滤，乙酸乙酯洗涤。滤液无水硫酸钠干燥，浓缩。粗产物使用制备HPLC分离得到26mg产物8-23，为浅黄色固体，产率26％。LCMS(ESI)m/z 1000.7(M+H)+.
1H NMR(400MHz,CDCl3)δppm 6.84(s,1H),6.78(s,1H),6.44&6.42(dd,J1＝12.4Hz,J2＝8.4Hz,1H),6.40(s,1H),6.21(d,J＝8.8Hz,1H),5.63&5.61(dd,J1＝12.4Hz,J2＝6.8Hz,1H),5.16(br,1H),4.92(d,J＝9.2Hz,1H),4.26(t,J＝8.8Hz,1H),3.99(s,3H),3.84-3.72(m,2H),3.71-3.60(m,20H),3.55-3.51(m,2H),3.47(d,J＝6.8Hz,1H),3.35(s,3H),3.20(d,J＝10.4Hz,1H),3.16(s,3H),2.83-2.78(m,2H),2.72-2.66(m,1H),2.64(t,J＝10.0Hz,1H),2.54-2.49(m,1H),2.18(t,J＝11.2Hz,1H),1.93(d,J＝7.6Hz,2H),1.74-1.69(m,2H),1.68(s,3H),1.64(d,J＝11.2Hz,1H),1.44(s,9H),1.27(d,J＝7.6Hz,3H),0.85(s,3H).
化合物9-23的合成
Figure PCTCN2016085275-appb-000285
上一步反应产物8-23(30mg,0.03mmol)溶于4mL干燥的二氯甲烷中，冰水浴冷却至0℃，逐滴缓慢加入0.4mL三氟乙酸，逐步升至室温搅拌1小时，LCMS检测原料已完全转化，常温减压移除二氯甲烷和三氟乙酸得20mg粗产品9-23，为浅黄色固体，直接用于下一步反应。LCMS(ESI)m/z 900.7(M+H)+.
化合物CE-063的合成
Figure PCTCN2016085275-appb-000286
化合物9-23(20mg,0.022mmol)和CE-L-055(12mg,0.044mmol)悬浮于5mL水中，加入饱和碳酸氢钠溶液0.2mL，反应液室温搅拌2小时。粗产品直接由制备HPLC纯化得5mg产品CE-063，为白色固体，产率21％。LCMS(ESI)m/z 1073.3(M+Na)+.
1H NMR(400MHz,CDCl3)δppm 7.01(br,1H),6.84(s,1H),6.77(s,1H),6.69(s,2H),6.51(s,1H),6.44&6.42(dd,J1＝12.4Hz,J2＝8.4Hz,1H),6.21(d,J＝8.4Hz,1H),5.63&5.61(dd,J1＝12.4Hz,J2＝7.2Hz,1H),4.92(d,J＝9.2Hz,1H),4.26(t,J＝8.8Hz,1H),3.99(s,3H),3.84(t,J＝5.6Hz,2H),3.81-3.74(m,2H),3.70-3.60(m,22H),3.56-3.52(m,3H),3.47(d,J＝7.2Hz,1H),3.44-3.39(m,2H),3.35(s,3H),3.20(d,J＝10.4Hz,1H),3.16(s,3H),2.85-2.78(m,2H),2.72-2.66(m,1H),2.53(d,J＝6.0Hz,3H),2.24-2.15(m,2H),1.68(s,3H),1.64(d,J＝11.2Hz,1H),1.50-1.43(m,1H),1.27(s,3H),0.85(s,3H).
实施例24T-CE-040的制备
Figure PCTCN2016085275-appb-000287
参考Gail D.Lewis Phillips,et al.,Cancer.Res.,2008,68,9280以及Teemu T.Junttila,et al.,Breast.Cancer.Res.Treat.,2011,128,347等文献，实施例中制备的抗HER2抗体(抗人类表皮生长因子受体2抗体，Herceptin)用溶液A(100mM磷酸盐缓冲液，pH＝7.5)透析过夜，用溶液A稀释透析液至10mg/mL。加入CE-040，使CE-040与抗体的比率为6:1(摩尔当量)。然后加入DMF至DMF占总体积的30％，25℃反应3.5小时并不断 搅拌使反应物混匀。过量的试剂和小分子药物通过装有Sephadex G-25(GE货号：17-0031-01)的凝胶过滤层析柱除去，用pH＝5.0的琥珀酸溶液预先平衡层析柱，经纯化获得T-CE-040。然后将抗体药物偶联物用pH＝5.0的琥珀酸溶液透析过夜，之后用0.22微米的过滤器过滤，4℃保存。每个Herceptin抗体最终共轭CE-040的数目通过共轭物在252nm和280nm处的吸光值和LC-MS检测来确定，再通过SEC检测偶联物是否含有多聚体。通过LC-MS确定CE-040与赫赛汀Herceptin的药物抗体比率(DAR)为3.46:1。
实施例25T-CE-063的制备
Figure PCTCN2016085275-appb-000288
实施例中制备的抗HER2抗体(抗人类表皮生长因子受体2抗体，Herceptin)用溶液A(25mM硼酸钠缓冲液，25mM氯化钠，1m MDTPA，PH＝7.0)透析过夜，并稀释至5mg/mL。加入5mM TCEP，使TCEP与抗体的比率为2.5:1(摩尔当量)，25℃反应2小时并不断搅拌使反应物混匀。然后加入CE-063，使CE-063与抗体的比率为10:1(摩尔当量)，并加入DMSO至DMAO占总体积为10％，25℃反应2小时并不断搅拌使反应物混匀。过量的试剂和小分子药物通过装有Sephadex G-25(GE货号：17-0031-01)的凝胶过滤层析柱除去，用pH＝7.4的磷酸缓冲盐溶液预先平衡层析柱，经纯化获得T-CE-063。然后将偶联物用pH为7.4的磷酸缓冲盐溶液透析过夜，之后用0.22微米的过滤器过滤， 4℃保存。每个赫赛汀(Herceptin)抗体最终共轭CE-063的数目通过HIC检测来确定，再通过SEC检测偶联物是否含有多聚体。通过HIC测定的CE-063与Herceptin的比率(DAR)为3.52:1。
参照实施例24-25，可制备得到本发明其他抗体偶联药物。
生物活性测试
T-CE-040和T-CE-063抗体药物偶联物生物活性测试
使用Her2阳性的人BT474乳腺肿瘤细胞(简称BT474)，Her2低表达的人MCF-7乳腺肿瘤细胞(简称MCF-7)，以及在MCF-7外转Her2的人乳腺肿瘤MCF7-Her2稳转细胞株(简称MCF-7-Her2)(参照文献：Teemu T.Junttila,et al.,Breast.Cancer.Res.Treat.,2011,128,347和Jeffrey J.Wallin et al.,Clin.Cancer.Res.,2012,18,3901公开的制备重组细胞方法进行制备，本申请在此引用该文献全文)。评价了T-CE-040和T-CE-063抗体药物偶联物对肿瘤细胞的生长抑制。BT474，MCF7-Her2和MCF-7用0.25％(体积/体积)的胰蛋白酶消化，使细胞剥离，然后悬浮于100ul完全培养基，2,000个细胞接种于96孔板进行培养。37℃过夜贴壁生长，然后加入100ul含有不同浓度梯度的T-CE-040和T-CE-063抗体药物偶联物以及完全培养基。120小时后加入50ul
Figure PCTCN2016085275-appb-000289
萤光细胞活性检测试剂(
Figure PCTCN2016085275-appb-000290
Luminescent，Promega)进行相对细胞增殖分析。T-CE-040、T-CE-063与T-DM1对肿瘤细胞生长抑制曲线图具体见图1-图6，其中，横坐标表示药物浓度(即ADC浓度)；纵坐标表示细胞存活率(％)。由图1-图6可知，T-CE-040能有效的抑制上述三种肿瘤细胞的增殖，其效果与T-DM1相当或更优。
按照上述相同操作，测试其他化合物的生物活性，结果如下表所示：
Figure PCTCN2016085275-appb-000291
Figure PCTCN2016085275-appb-000292
Figure PCTCN2016085275-appb-000293
Figure PCTCN2016085275-appb-000294
Figure PCTCN2016085275-appb-000295
Figure PCTCN2016085275-appb-000296
Figure PCTCN2016085275-appb-000297
Figure PCTCN2016085275-appb-000298
Figure PCTCN2016085275-appb-000299
Figure PCTCN2016085275-appb-000300
Figure PCTCN2016085275-appb-000301
Figure PCTCN2016085275-appb-000302
Figure PCTCN2016085275-appb-000303
Figure PCTCN2016085275-appb-000304
Figure PCTCN2016085275-appb-000305
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (14)

  1. 一种如式IB所示的抗体药物偶联物，
    Figure PCTCN2016085275-appb-100001
    其中：
    Z为氮原子、
    Figure PCTCN2016085275-appb-100002
    或氧原子，当Z1为氧原子时，R1不存在；当Z为
    Figure PCTCN2016085275-appb-100003
    或氮原子时，R1为氢或C1～C4的烷基或者R1、Z以及与它们连接的C原子和R6或R7连接成为4-6元环状结构；
    R2为氢或卤素取代的C1～C4的烷基；
    p为0、1、2、3、4、5、6、7、8、9或10；
    m为0或1；
    n为0、1、2、3、4、5、6、7、8、9或10；
    Y1为氧原子、化学键或
    Figure PCTCN2016085275-appb-100004
    其中x2为1-24的整数；
    q为0、1、2、3、4、5、6、7、8、9或10；
    R3和R4独立地为氢、氰基或者取代或未取代C1～C4的烷基，所述的R3或R4中所述的取代或未取代C1～C4的烷基中所述的取代是指被C1～C4的烷氧基所取代；
    x为0、1、2、3、4、5、6、7、8、9或10；
    Q1为
    Figure PCTCN2016085275-appb-100005
    Figure PCTCN2016085275-appb-100006
    其中，R8为取代或未取代C1～C4的烷基、
    Figure PCTCN2016085275-appb-100007
    Figure PCTCN2016085275-appb-100008
    y为1、2、3、4、5、6、7、8、9或10；y1为0或1；所述的R8中所述的“取代或未取代C1～C4的烷基”中所述的取代是指被
    Figure PCTCN2016085275-appb-100009
    所取代；t为1或2；t1为0、1、2、3、4、5或6；
    R6为氢、取代或未取代C1～C12的烷基或
    Figure PCTCN2016085275-appb-100010
    r为1、2、3、4、5、6、7、8、9、10、11或12；
    R7为氢、取代或未取代C1～C12的烷基、取代或未取代C1～C12的烷氧基或
    Figure PCTCN2016085275-appb-100011
    mAb表示单克隆抗体；
    k为1-8中的任一数值；
    所述的如式IB所示的化合物不为下列化合物：
    Figure PCTCN2016085275-appb-100012
  2. 根据权利要求1所述的如式IB所示的抗体药物偶联物，其特征在于，
    当所述的R1为C1～C4的烷基时，所述的C1～C4的烷基为甲基、乙基、丙基、丁基、 异丙基、异丁基或叔丁基；
    和/或，
    当所述的R1、Z以及与它们连接的C原子和R6或R7连接成为4-6元环状结构时，所述的4-6元环状结构为六元环状结构，所述的六元环状结构优选
    Figure PCTCN2016085275-appb-100013
    Figure PCTCN2016085275-appb-100014
    和/或，
    当所述的R2为卤素取代的C1～C4的烷基时，所述的卤素取代的C1～C4的烷基中所述的卤素为氟、氯或溴；
    和/或，
    当所述的R2为卤素取代的C1～C4的烷基时，所述的卤素取代的C1～C4的烷基为卤素取代的甲基、卤素取代的乙基、卤素取代的丙基、卤素取代的异丙基、卤素取代的丁基、卤素取代的异丁基或卤素取代的叔丁基；
    和/或，
    所述的p为0、1或2；
    和/或，
    所述的n为0、1或2；
    和/或，
    所述的q为0、1或2；
    和/或，当Y1为化学键时，所述的化学键为单键，
    和/或，当Y1为
    Figure PCTCN2016085275-appb-100015
    时，所述的
    Figure PCTCN2016085275-appb-100016
    中，x2为1、2、3、4、5、6、7、8、9或10；
    和/或，
    当所述的R3和R4独立地为取代或未取代C1～C4的烷基时，所述的未取代的C1～C4的烷基为甲基、乙基、丙基、丁基、异丙基、异丁基或叔丁基；
    和/或，
    当所述的R3和R4独立地为取代或未取代C1～C4的烷基时，所述的取代的C1～C4的烷基为取代的甲基、取代的乙基、取代的丙基、取代的丁基、取代的异丙基、取代的异丁基或取代的叔丁基；
    和/或，
    当所述的R3和R4中所述的取代或未取代C1～C4的烷基中所述的取代是指被C1～C4的烷氧基所取代时，所述的C1～C4的烷氧基为甲氧基、乙氧基、丙氧基、丁氧基、异丙氧基、异丁氧基或叔丁氧基；
    和/或，
    所述的x为0、1或2；
    和/或，
    当Q1为
    Figure PCTCN2016085275-appb-100017
    时，所述的
    Figure PCTCN2016085275-appb-100018
    中R8为取代或未取代C1～C4的烷基时，所述的取代或未取代的C1～C4的烷基为取代或未取代的甲基、取代或未取代的乙基、取代或未取代的丙基、取代或未取代的异丙基、取代或未取代的丁基、取代或未取代的异丁基或者取代或未取代的叔丁基；
    和/或，
    当所述的R6为取代或未取代C1～C12的烷基时，所述的取代或未取代C1～C12的烷基为取代或未取代C1～C4的烷基；
    和/或，
    当所述的R7为取代或未取代C1～C12的烷基时，所述的取代或未取代C1～C12的烷基为取代或未取代C1～C4的烷基；
    和/或，
    当所述的R7为取代或未取代C1～C12的烷氧基时，所述的取代或未取代C1～C12的烷氧基为取代或未取代C1～C4的烷氧基；
    mAb为单克隆抗体，例如赫赛汀。
  3. 根据权利要求2所述的如式IB所示的抗体药物偶联物，其特征在于，
    当所述的R2为卤素取代的甲基时，所述的卤素取代的甲基为
    Figure PCTCN2016085275-appb-100019
    和/或，
    当所述的R3和R4独立地为取代的甲基时，所述的取代的甲基为
    Figure PCTCN2016085275-appb-100020
    和/或，
    当Q1为
    Figure PCTCN2016085275-appb-100021
    时，所述的
    Figure PCTCN2016085275-appb-100022
    中所述的R8为取代的丙基时，所述的取代的丙基为
    Figure PCTCN2016085275-appb-100023
    和/或，
    当所述的R6为取代或未取代C1～C4的烷基时，所述的取代或未取代C1～C4的烷基为甲基、乙基、丙基、异丙基、丁基、异丁基或叔丁基；
    和/或，
    当所述的R7为取代或未取代C1～C4的烷基时，所述的取代或未取代C1～C4的烷基为甲基、乙基、丙基、异丙基、丁基、异丁基或叔丁基；
    和/或，
    当所述的R7为取代或未取代C1～C4的烷氧基时，所述的取代或未取代C1～C4的烷氧基为甲氧基、乙氧基、丙氧基、异丙氧基、丁氧基、异丁氧基或叔丁氧基。
  4. 根据权利要求1所述的如式IB所示的抗体药物偶联物，其特征在于，其为如式Ib或Ib1所示的抗体药物偶联物，
    Figure PCTCN2016085275-appb-100024
    如式Ib或Ib1所示的抗体药物偶联物中，各字母和基团的定义均同权利要求1-3任一项所述；Y为氧原子或化学键；Y2为
    Figure PCTCN2016085275-appb-100025
    其中x2为1-24的整数；Q2为
    Figure PCTCN2016085275-appb-100026
    t1为0、1、2、3、4、5或6。
  5. 根据权利要求1-4中至少一项所述的如式IB所示的抗体药物偶联物，其特征在于，其为下列任一化合物：
    Figure PCTCN2016085275-appb-100027
    Figure PCTCN2016085275-appb-100028
    Figure PCTCN2016085275-appb-100029
    Figure PCTCN2016085275-appb-100030
    Figure PCTCN2016085275-appb-100031
  6. 一种如式IA所示的中间体：
    Figure PCTCN2016085275-appb-100032
    如式IA所示的化合物中，各字母和基团的定义均同权利要求1-3任一项所述；Q3为
    Figure PCTCN2016085275-appb-100033
    t为1或2；t1为0、1、2、3、4、5或6；
    R51为H、C1～C4的烷基、
    Figure PCTCN2016085275-appb-100034
    Figure PCTCN2016085275-appb-100035
    或卤素，y为1、2、3、4、5、6、7、8、9或10，R9为H或C1～C4的烷基。
  7. 根据权利要求6所述的如式IA所示的中间体，其特征在于，
    当所述的R51为C1～C4的烷基时，所述的C1～C4的烷基为甲基、乙基、丙基、丁基、异丙基、异丁基或叔丁基；
    和/或，
    当R51为卤素时，所述的卤素为F、Cl、Br或I；
    和/或，
    当所述的R9为C1～C4的烷基时，所述的C1～C4的烷基为甲基、乙基、丙基、丁基、异丙基、异丁基或叔丁基。
  8. 根据权利要求6所述的如式IA所示的中间体，其特征在于，其为如式Ia或Ia1所示的中间体：
    Figure PCTCN2016085275-appb-100036
    Figure PCTCN2016085275-appb-100037
    其中，如式Ia和如式Ia1所示的中间体中各字母和基团的定义均同权利要求6或7所述；
    如式Ia所示的中间体中，R5为H、C1～C4的烷基(例如甲基、乙基、丙基、丁基、异丙基、异丁基或叔丁基)、
    Figure PCTCN2016085275-appb-100038
    Figure PCTCN2016085275-appb-100039
    y为1、2、3、4、5、6、7、8、9或10，y1为0或1；R9为H或C1～C4的烷基，R9中所述的C1～C4的烷基优选甲基、乙基、丙基、丁基、异丙基、异丁基或叔丁基；
    如式Ia1所示的中间体中，R52为氢或卤素，所述的卤素优选F、Cl、Br或I。
  9. 根据权利要求6-8中至少一项所述的如式IA所示的中间体，其特征在于，其为下列任一化合物：
    Figure PCTCN2016085275-appb-100040
    Figure PCTCN2016085275-appb-100041
    Figure PCTCN2016085275-appb-100042
    Figure PCTCN2016085275-appb-100043
    Figure PCTCN2016085275-appb-100044
    Figure PCTCN2016085275-appb-100045
    Figure PCTCN2016085275-appb-100046
  10. 一种如权利要求1-5任一项所述的如式IB所示的抗体药物偶联物的制备方法，其特征在于，其包括以下步骤：有机溶剂中，pH为6-8条件下，将中间体IA与单克隆抗体进行交联，得到抗体药物偶联物IB即可；
    Figure PCTCN2016085275-appb-100047
    如式IA和如式IB所示的化合物中，各字母和基团的定义均同权利要求1-9任一项所述。
  11. 根据权利要求1-5中至少一项所述的如式IB所示的抗体药物偶联物和/或根据权利要求6-9中至少一项所述的如式IA所示的中间体在制备治疗和/或预防癌症的药物中的应用。
  12. 根据权利要求11所述的应用，其特征在于，所述的癌症为乳腺癌、淋巴癌、肺癌、肝癌、结肠癌、头颈癌、膀胱癌、肾癌、食道癌、胆囊癌、卵巢癌、胰腺癌、胃癌、宫颈癌、甲状腺癌、前列腺癌、皮肤癌包括鳞状细胞癌；白细胞过多症、急性淋巴细胞性白血病、急性成淋巴细胞性白血病、B细胞淋巴瘤、T细胞淋巴瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、毛细胞淋巴瘤、伯基特淋巴瘤、急性和慢性髓细胞性白血病、骨髓增生异常综合征、前髓细胞白血病、纤维肉瘤、横纹肌肉瘤、星形细胞瘤、神经目细胞瘤、胶质瘤、神经鞘瘤、黑色素瘤、精原细胞瘤、畸胎癌、骨肉瘤、着色性干皮病、角质黄色瘤、甲状腺滤泡癌或卡波西肉瘤。
  13. 根据权利要求11或12所述的应用，其特征在于，所述的癌症的肿瘤细胞为Her2阳性的人BT474乳腺肿瘤细胞、Her2低表达的人MCF-7乳腺肿瘤细胞或在MCF-7外转Her2的人乳腺肿瘤MCF7-Her2稳转细胞株。
  14. 一种药物组合物，其包含根据权利要求1-5中至少一项所述的如式IB所示的抗体药物偶联物和/或根据权利要求6-9中至少一项所述的如式IA所示的中间体，以及一种或多种药学上可接受的辅料。
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