WO2021051720A1 - Class of anti-human egfr antibody drug conjugate and preparation method and application thereof - Google Patents

Class of anti-human egfr antibody drug conjugate and preparation method and application thereof Download PDF

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WO2021051720A1
WO2021051720A1 PCT/CN2020/000225 CN2020000225W WO2021051720A1 WO 2021051720 A1 WO2021051720 A1 WO 2021051720A1 CN 2020000225 W CN2020000225 W CN 2020000225W WO 2021051720 A1 WO2021051720 A1 WO 2021051720A1
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antibody
linker
mmae
human egfr
synthesis
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French (fr)
Chinese (zh)
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李卓荣
胡馨月
江海伦
刘睿
白炜琪
刘秀筠
苗庆芳
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中国医学科学院医药生物技术研究所
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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
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • 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
    • 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
    • A61K47/54—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 organic compound
    • A61K47/55—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 organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • 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
    • 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
    • A61K47/6873—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 an immunoglobulin; the antibody being an anti-idiotypic antibody
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to an anti-human epidermal growth factor receptor (EGFR) antibody drug conjugate (ADC), including a substance using the ADC, a preparation method and its application in the field of solid tumor treatment.
  • ADC anti-human epidermal growth factor receptor
  • the invention belongs to the field of biotechnology medicine.
  • EGFR epidermal growth factor receptor
  • EGFR epidermal growth factor receptor
  • Anti-EGFR antibody is the main targeted therapy, and its mechanism of action is mainly to compete with endogenous ligands to bind EGFR, inhibit the activation of tyrosine kinase, and promote the internalization of EGFR to produce anti-tumor effects.
  • antibody drug research has its own unique advantages and good market prospects, the curative effects of existing EGFR drugs are limited. In most cases targeting EGFR, long-term use leads to drug resistance.
  • ADC Antibody-drug conjugate
  • EGFR-ADC also has certain challenges. Among them, the two ADCs that target EGFR, IMGN-289 and AMG-595 are also due to toxicity (such as skin toxicity, gastrointestinal toxicity, etc.) or poor efficacy. The clinical research was forced to terminate, so the research opportunities and challenges of ADC targeting EGFR coexist.
  • ADC generally consists of three parts: antibody, warhead and linker.
  • SCT-200 antibody is a new type of fully human anti-EGFR monoclonal antibody targeted for the treatment of metastatic colorectal cancer (Patent Publication Number: CN101058609A). It is a high-affinity 100% human antibody obtained through screening of human phage library , Has an original sequence targeting EGFR.
  • the current research on the treatment of metastatic colorectal cancer and head and neck squamous cell carcinoma is in clinical phase II, and its clinical application may have better therapeutic effects and safety than chimeric antibodies and humanized antibodies.
  • the cytotoxicity of warhead molecules must be extremely high, and EC 50 is generally required to be less than 1 nmol/L.
  • MMAE is based on the active ingredient dolastatins 10 (dolastatins 10) in the natural marine product dolastatins extracted from the censored sea hare. It is a type of linear pentapeptide cytotoxic molecule and is used in a variety of human cancer cell lines. , IC 50 is 10 -9 ⁇ 10 -11 mol/L, which is 200 times the activity of vinblastine.
  • MMAE as an ADC warhead molecule has three main advantages: (1) high cytotoxicity; (2) N-terminal single methyl substitution, which can be connected to a linker; (3) bystander effect: it is especially beneficial for the treatment of solid tumors.
  • the linker connected to MMAE generally uses Val-Cit dipeptide type.
  • the combination with MMAE is called MC-VC-PABC-MMAE.
  • This combination is the most widely used Linker in ADC clinical trials.
  • Drug the listed ADC Brentuximab Vedotin (SGN-35, Adcetris) chose this kind of Linker-Drug combination to cleave it in cathepsin B, thereby completely releasing MMAE.
  • SGN-35 Adcetris
  • the p-aminobenzyloxycarbonyl spacer group is similar to an ester bond, and its stability in plasma needs to be further improved.
  • the present invention makes full use of the advantages of the SCT200 antibody, designs different types of linkers to connect with MMAE, and finally prepares a novel SCT200-Linker-MMAE antibody drug conjugate.
  • the prepared ADCs are comparable to SCT200 in terms of affinity and endocytosis, and can up-regulate the expression of tumor apoptosis-related proteins.
  • the ADCs of the present invention all exhibit stronger anti-tumor activity than SCT200.
  • the purpose of the present invention is to provide a type of anti-human EGFR antibody drug conjugate and its preparation method and anti-tumor application.
  • the anti-human EGFR antibody drug conjugate of the present invention has high efficiency targeting, high endocytosis activity, high affinity activity and high anti-tumor activity to EGFR-positive tumor tissues.
  • the present invention adopts the following technical means:
  • a type of anti-human EGFR antibody drug conjugate of the present invention has a structure shown in formula I:
  • M is the part of the spacer that can react with the cysteine sulfhydryl group of the antibody, and its structure is one of the following structures:
  • C is a part that can be hydrolyzed under the action of an enzyme, and is selected from the dipeptide sequence Val-Cit, Val-Ala, Val-Lys or Val-Arg; or the tripeptide sequence Ala-Val-Ala, Gly-Phe-Gly, Gly-Phe-Lys or Ala-Phe-Lys; or one of the tetrapeptide sequence Gly-Gly-Phe-Gly or Gly-Phe-Leu-Gly;
  • R is the self-decomposing part, and its structure is one of the following structures:
  • the antibody is SCT-200 monoclonal antibody.
  • the present invention also proposes a method for preparing the anti-human EGFR antibody conjugated drug, which includes: the Linker-MMAE having the structure of formula II and the interchain disulfide bond of the anti-human EGFR antibody undergo an addition reaction Preparing the anti-human EGFR antibody drug conjugate;
  • the method includes the following steps:
  • step (2) Add anti-human EGFR antibody to the reducing agent stock solution described in step (2), and incubate for 1-2h;
  • step (1) Add the linker-cytotoxic drug stock solution of step (1) to the reaction solution obtained in step (4), and incubate for 1-2 hours to prepare an anti-human EGFR antibody drug conjugate;
  • step (3) Add the L-Cys stock solution of step (3) to the reaction solution obtained in step (5) to stop the reaction.
  • the reducing agent in the step (2) is TCEP, and the molar amount of the anti-human EGFR antibody is used as a reference when mixing, and the molar amount of the reducing agent is 2-4 times the molar amount of the antibody.
  • the molar amount of the anti-human EGFR antibody is used as a reference when mixing in the step (5), and the molar amount of Linker-MMAE added is 4-8 times the molar amount of the antibody.
  • the reactions in the steps (4) and (5) are carried out under the protection of nitrogen, and the reaction temperature in the step (4) is 35-40°C, preferably 37°C.
  • the AKTA purifier protein purification system is used to collect the peak of the required antibody-drug conjugate; after the collection is completed, a 30kDa ultrafiltration tube is used for ultrafiltration Filter, centrifuge, concentrate, filter through a sterile filter membrane, and store at low temperature.
  • the present invention also proposes the use of the antibody-drug conjugate in the preparation of drugs for tumor targeted therapy, wherein the tumor is an EGFR-positive solid tumor, including squamous cell carcinoma, Esophageal cancer, nasopharyngeal cancer, lung cancer, breast cancer, pancreatic cancer, prostate cancer, head and neck cancer, colon cancer, etc.
  • a pharmaceutical composition for targeted tumor therapy using the antibody-drug conjugate as an active ingredient is also within the protection scope of the present invention, and the pharmaceutical composition contains a pharmaceutically effective amount of the present invention.
  • Antibody drug conjugates and pharmaceutically acceptable adjuvants are also within the protection scope of the present invention, and the pharmaceutical composition contains a pharmaceutically effective amount of the present invention.
  • the present invention prepares a series of Linker, which shows better plasma stability and enzyme cleavage rate, and reacts with MMAE in one step to prepare Linker-MMAE.
  • the present invention uses the SCT-200 fully humanized monoclonal antibody to couple with Linker-MMAE through the chemical method of reducing the disulfide bond between the antibody chains to construct a type of solid tumor therapy targeting EGFR.
  • Anti-human EGFR antibody drug conjugate Preferably, TCEP is used as a reducing agent.
  • the average drug-antibody coupling ratio (DAR) is prepared within the range of 4.0 ⁇ 0.5, and the quality is stable and controllable, with good repeatability and considerable yield.
  • the novel antibody drug conjugate SCT-200-Linker-MMAE proposed by the present invention does not affect the affinity, endocytic activity and targeting of the antibody, and better retains its biology Function;
  • IC 50 is in the nM level;
  • Western Blot experiment shows that compared to SCT-200, SCT-200-Linker-MMAE ADCs can Significantly inhibit the expression of related proteins in the process of tumor apoptosis; in the evaluation of in vivo activity, compared with SCT-200, SCT-200-Linker-MMAE ADCs have greatly enhanced tumor suppressing effect.
  • Figure 1 is a synthetic route diagram of Fmoc-dipeptide sequence
  • Figure 2 is a synthetic route diagram of Fmoc-AVA
  • Figure 3 is a synthetic route diagram of Fmoc-GFG
  • Figure 4 is a synthetic route diagram of Fmoc-GGFG
  • Figure 5 is a synthetic route diagram of Fmoc-GFLG
  • Figure 6 is a linker-MMAE synthesis route diagram of spacer and hydrolysis zone types
  • Figure 7 is a linker-MMAE synthesis route diagram of the self-decomposition zone type
  • Fig. 7A is a linker synthesis route diagram of the self-decomposition zone type
  • Fig. 7B is a synthesis route diagram of the Linker and MMAE condensation reaction of the self-decomposition zone type
  • Figure 8 is the HIC-HPLC spectrum of the spacer type SCT-200-Linker-MMAE
  • Figure 9 is the HIC-HPLC spectrum of the hydrolysis zone type SCT-200-Linker-MMAE
  • Figure 10 shows the HIC-HPLC spectrum of the self-decomposition zone type SCT-200-Linker-MMAE
  • Figure 11 is the ESI-MS spectrum of SCT-200-Linker-MMAE
  • Figure 12 is a graph of the affinity of SCT-200-Linker-MMAE ADCs measured by ELISA
  • Figure 13 is a diagram showing the endocytosis rate of SCT-200-Linker-MMAE ADCs measured by flow cytometry;
  • Figure 14 is a Western Blot determination of EGFR expression levels in different cells
  • Figure 15 is a diagram showing the influence of SCT200-Linker-MMAE ADCs on the expression of apoptosis-related proteins
  • Figure 16 shows the tumor inhibition curve (A) and weight change curve (B) of SCT200-Linker-MMAE ADCs on the A431 nude mouse xenograft tumor model.
  • the Buchner funnel is suction filtered until the filtrate is clear, and then filtered The cake is drained and dried in a vacuum drying oven. After drying, the white solid was ground, washed with ether for several times, and filtered with suction to obtain 1a, 15.1 g of the white solid, with a yield of 83%.
  • the 1a reaction product (7.6g, 15.3mmol) and PABOH (3.76g, 30.6mmol) were dissolved in 140mL CH 2 Cl 2 and 70 mL of CH 3 OH mixed solvent, and EEDQ (7.5 g, 30.6 mmol).
  • the synthesis method is the same as that of 4a, to obtain a yellow solid with a yield of 62%.
  • the solubility of the product in this step is not good, and it is directly washed with a mixed solvent of diethyl ether and dichloromethane and filtered by suction, and directly proceed to the next step of the reaction.
  • the Linker-MMAE to be tested is prepared as a 10mM DMSO stock solution, ready for use. Add 4 ⁇ L of DMSO stock solution to 996 ⁇ L of blank rat plasma, mix well and incubate in a 37°C water bath. Sampling 50 ⁇ L each at 0, 30, 60, 180, and 360 minutes to terminate the reaction. Two parallel samples at each reaction time point were stored at -20°C and processed after sampling. The sample was centrifuged to settle the protein, the supernatant was taken, and the concentration of MMAE was detected by LC-MS/MS, and the concentration unit (nM) was detected. Draw a curve of the release amount of MMAE released over time, and calculate the release rate of MMAE in plasma. The release rate is shown in Table 1.
  • Linker-MMAE linker-cytotoxic drug
  • step 4 Add the SCT-200 antibody to the reducing agent stock solution described in step 2), and incubate at 37°C for 1.5 hours under nitrogen protection; the molar amount of the reducing agent is 3 times the molar amount of the antibody;
  • step 5 Add the linker-cytotoxic drug stock solution of step 1) to the reaction solution of step 4), and incubate at 37°C for 1-2.5 hours under nitrogen protection to prepare the SCT-200 antibody anti-human EGFR antibody drug conjugate;
  • the molar amount of the linker-cytotoxic drug is 4-8 times the molar amount of the antibody;
  • step 6) Add the stock solution of step 3) to the reaction solution obtained in step 5) to stop the reaction.
  • the step of further purifying the obtained antibody-conjugated drug SCT-200-Linker-MMAE, preferably the AKTA purifier protein purification system, collect the required antibody-conjugated drug component peaks; after collection, use a 30kDa ultrafiltration tube for ultrafiltration Filter, centrifuge, concentrate, filter through a sterile filter membrane, and store at low temperature.
  • Flow rate 0.5mL/min; injection volume: 8 ⁇ L; analysis time: 17min; column temperature: 25°C; detection wavelength: 280nm and 248nm;
  • the liquid system adopts ACQUITY UPLC H-Class (Waters), the chromatographic column is ACQUITY UPLC Protein, BEH SEC, 1.7 ⁇ m, 2.1mm ⁇ 100mm, column temperature 25°C, mobile phase is 100mM ammonium acetate aqueous solution, isocratic elution, flow rate is 0.1ml/min.
  • the mass detection system is: Xevo G2-XS Qtof (Waters), the detection mode is positive ion, and the full scan mode.
  • MassLynx 4.1 Waters
  • the model parameter setting resolution is 2-3.5Da.
  • the minimum intensity ratio is set to 60%, and the output resolution is set to 1Da.
  • the algorithm iteration parameter is set to 20, and the result is shown in Figure 11.
  • Coating antigen Dilute the antigen with PBS to 2 ⁇ g/mL, add 50 ⁇ L coating to each well, and wrap it with fresh-keeping film at 4°C overnight. Wash the plate: Shake the liquid in the plate, add 200 ⁇ L of PBST/well, wash 3 times, shake for 3-5 min each time. Blocking: Add 1% BSA to each well, 200 ⁇ L/well, overnight at 4°C. Wash the plate: Shake the liquid in the plate, add 200 ⁇ L of PBST/well, wash 3 times, shake for 3-5 min each time. Add the sample to be tested, PBST as a diluent, add 50 ⁇ L to each well, 3 times in parallel for each concentration, and incubate at 37°C for 1h.
  • wash the plate spin dry the liquid in the plate, add 200 ⁇ L of PBST/well, wash 3 times, shake 3-5 min each time, and pat dry the ELISA plate.
  • Incubation of secondary antibody Add 50 ⁇ L of secondary antibody (diluted at 1:5000) to each well, and incubate at 37°C for 1 hour. Wash the plate: spin dry the liquid in the plate, add 200 ⁇ L of PBST/well, wash 3 times, shake 3-5 min each time, and pat dry the ELISA plate.
  • the results show that the results are shown in Figure 12.
  • the naked antibody SCT-200 and ADCs have a concentration-dependent affinity to the antigen, indicating that the affinity of the SCT200-Linker-MMAE ADCs prepared based on the cysteine coupling method is comparable to that of the antibody SCT-200
  • the affinity is similar, and the affinity of the original antigen and antibody is basically maintained.
  • the affinity between the antigen and the antibody comes from the ADC antibody itself, and the change of the linker basically has no effect on it.
  • KYSE520 cells were trypsinized, and the cells were pelleted by centrifugation at 4°C for 5 min. The cell resuspended concentration was 1 ⁇ 10 7 /mL. The cells were divided into 50 ⁇ L/tube, and each group had 3 samples in parallel.
  • Primary antibody binding Dilute the primary antibody sample with FACS staining buffer so that the final concentration of the primary antibody is 20 ⁇ g/mL. After incubating for 1 hour in an ice bath, the cells were washed with FACS staining buffer and centrifuged at 4°C for 5 minutes to remove the supernatant.
  • Antibody internalization add 200 ⁇ L of FACS staining buffer to each centrifuge tube and incubate at 37°C for 2h.
  • Flow cytometry was used to detect the internalization rate of SCT200-Linker-MMAE ADCs, and the internalization rate of each ADCs in KYSE520 cells was determined.
  • the ADCs were incubated at 4°C for 30min and 37°C for 2h, respectively.
  • the internalization level of the antibody bound to the cell surface was calculated by calculating the average fluorescence intensity (MFI) reduction level of the 37°C incubation sample compared to the 4°C incubation control.
  • MFI average fluorescence intensity
  • %MFI at time point t MFI of the sample incubated at 37°C ⁇ MFI of the sample incubated at 100/4°C;
  • the protein sample to be tested is loaded, and the protein loading amount is 30 ⁇ g/well.
  • the cells in the logarithmic growth phase were resuspended and counted by centrifugation, and seeded in 96 wells at a rate of 1 ⁇ 10 4 to 3 ⁇ 10 4 /well, and cultured at 37°C for 2 hours. Then, different concentrations of LR004-VC-MMAE (LR004 and MMAE are used as controls) were added, and 3 parallel holes were set for each drug concentration. After 72 hours of incubation at 37°C, add 20 ⁇ L of CCK8 reagent to each well and continue to incubate for 1-2 hours. Observe the color reaction, and measure the absorbance at 450nm with a microplate reader.
  • cell survival rate (additional group A450 value-blank group A450 value)/(control group) A450 value-blank group A450 value) ⁇ 100%.
  • the IC 50 value is calculated using SPSS software.
  • Example 11 The effect of SCT-200-Linker-MMAE ADCs on the expression of related proteins in the process of tumor apoptosis
  • SCT200-M-2, SCT200-C-2 and SCT200-C-4 were selected for the correlation study of apoptosis proteins based on the above experimental results.
  • the A431 cells were treated with the above-mentioned ADCs at 0.01, 0.1 and 1 ⁇ g/mL, 1 ⁇ g/mL SCT200 and 1 nM MMAE for 24 hours, and the samples were collected and tested by Western Blot.
  • the expression level of cleaved-PARP is different, the control and SCT200 bands are not obvious, the MMAE band is shallow, and ADCs 1 ⁇ g/mL can significantly increase the expression level of cleaved-PARP, and it is concentration-dependent.
  • ADCs can induce cell apoptosis through P-P53, caspase-3 and cleaved-PARP-dependent apoptotic pathways.
  • mice were injected with 5 ⁇ 10 6 /100 ⁇ L A431 cells subcutaneously into the right armpit.
  • the tumor volume of nude mice is about 110mm 3 , and they are randomly divided into 5 groups, which are the control group, SCT-200 (12mg/kg), SCT-200-M-1 (4mg/kg) , SCT-200-M-2 (4mg/kg), SCT-200-C-2 (4mg/kg) and SCT-200-C-4 (4mg/kg) each group of 6 nude mice, every 3 days
  • the tail vein was administered once for a total of 4 times.
  • the tumor suppression curve is shown in Figure 16.

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Abstract

Disclosed are a class of anti-human EGFR antibody drug conjugates and preparation method and application thereof. The anti-human EGFR antibody drug conjugate has the structure represented by formula (I). In the present invention, by means of using SCT-200 fully humanized monoclonal antibody, and coupling with Linker-MMAE by means of the chemical method of reducing the disulfide bonds between antibody chains, a class of anti-human EGFR antibody drug conjugates targeting EGFR for the treatment of solid tumors is constructed. The novel antibody drug conjugate SCT-200-Linker-MMAE provided by the present invention, in comparison with SCT-200 itself, does not affect the affinity, endocytic activity, and targeting of the antibody, better retains its biological function, and, in comparison with SCT-200, the activity is significantly improved, and can significantly inhibit the expression of related proteins during the process of tumor apoptosis, greatly improving the tumor inhibition effect.

Description

一类抗人EGFR抗体药物偶联物及其制备方法与应用A class of anti-human EGFR antibody drug conjugate and its preparation method and application 技术领域Technical field
本发明涉及抗人表皮生长因子受体(EGFR)抗体药物偶联物(ADC)，包括使用所述ADC的物质、制备方法及其在实体瘤治疗领域的应用。本发明属于生物技术药物领域。The present invention relates to an anti-human epidermal growth factor receptor (EGFR) antibody drug conjugate (ADC), including a substance using the ADC, a preparation method and its application in the field of solid tumor treatment. The invention belongs to the field of biotechnology medicine.
背景技术Background technique
EGFR(epidermal growth factor receptor)是肿瘤治疗中的一个很重要的靶点，其在多种上皮来源的实体瘤中都呈过表达，因此，研发针对EGFR家族受体的靶向药物，成为近年来抗肿瘤治疗的主要热点。抗EGFR抗体为主要的靶向疗法，其作用机制主要是与内源性配体竞争性结合EGFR，通过抑制酪氨酸激酶的激活，促进EGFR内化等作用产生抗肿瘤效果。虽然抗体药物研究有自身独特的优势和不错市场前景，但现有的EGFR药物疗效效果都有限，靶向EGFR绝大多数情况下，长时间使用导致耐药。耐药的主要原因有EGFR蛋白胞外段突变或者EGFR下游的信号通路突变等。抗体-药物偶联物(Antibody-drug conjugate，ADC)结合了抗体的靶向性与细胞毒药物的高效细胞毒作用等优点，成为目前靶向治疗癌症的有效策略之一，同时ADC可以弥补抗体药物的不足。以EGFR为靶点的ADC药物的研究，可克服EGFR抗体产生的耐药性，提高EGFR抗体的疗效。尤其是针对实体瘤的研究，临床及市场前景广阔。然而，目前尚未有以EGFR为靶点的ADC上市药物，临床在研的靶向于EGFR的ADC有AVID-100和ABT-414和ABBV-221。然而，EGFR-ADC的研发也有一定挑战性，其中以EGFR为靶点的两个ADC IMGN-289和AMG-595也因毒性(如皮肤毒性、胃肠道毒性等)或疗效不佳等问题在临床研究中被迫终止，所以EGFR为靶点的ADC的研究机遇与挑战并存。EGFR (epidermal growth factor receptor) is a very important target in tumor therapy, and it is overexpressed in a variety of solid tumors of epithelial origin. Therefore, the development of targeted drugs for EGFR family receptors has become a major issue in recent years. The main focus of anti-tumor therapy. Anti-EGFR antibody is the main targeted therapy, and its mechanism of action is mainly to compete with endogenous ligands to bind EGFR, inhibit the activation of tyrosine kinase, and promote the internalization of EGFR to produce anti-tumor effects. Although antibody drug research has its own unique advantages and good market prospects, the curative effects of existing EGFR drugs are limited. In most cases targeting EGFR, long-term use leads to drug resistance. The main reasons for drug resistance are mutations in the extracellular segment of the EGFR protein or mutations in the downstream signaling pathways of EGFR. Antibody-drug conjugate (ADC) combines the advantages of antibody targeting and high-efficiency cytotoxicity of cytotoxic drugs, and has become one of the current effective strategies for targeted cancer treatment. At the same time, ADC can compensate for antibodies. Insufficient medication. The study of ADC drugs targeting EGFR can overcome the drug resistance caused by EGFR antibodies and improve the efficacy of EGFR antibodies. Especially for solid tumor research, the clinical and market prospects are broad. However, there are currently no ADC drugs targeting EGFR on the market. The ADCs targeting EGFR in clinical research are AVID-100, ABT-414 and ABBV-221. However, the research and development of EGFR-ADC also has certain challenges. Among them, the two ADCs that target EGFR, IMGN-289 and AMG-595 are also due to toxicity (such as skin toxicity, gastrointestinal toxicity, etc.) or poor efficacy. The clinical research was forced to terminate, so the research opportunities and challenges of ADC targeting EGFR coexist.
ADC一般由抗体(antibody)、弹头分子(warhead)和连接子(linker)3个部分组成。SCT-200抗体是一种新型靶向治疗转移性结直肠癌的全人源抗 EGFR单克隆抗体(专利公布号：CN101058609A)，是通过人噬菌体库筛选获得的高亲合性100％人源抗体，具有靶向EGFR原创性序列，目前治疗转移性结直肠癌和头颈鳞癌的研究在临床II期，临床应用可能会比嵌合抗体和人源化抗体具有更好的治疗效果和安全性。弹头分子的细胞毒性必须极高，一般要求EC 50小于1nmol/L。目前，以MMAE作为弹头分子的ADC占有主导地位。MMAE是以截尾海兔中提取的天然海洋产物海兔毒素中的有效成分海兔毒素10(dolastatins 10)为先导物，是一类线性五肽细胞毒分子，在多种人癌症细胞系中，IC 50为10 -9～10 -11mol/L，为长春碱活性的200倍。MMAE作为ADC弹头分子的优势主要有三点：(1)高细胞毒性；(2)N端单甲基取代，可与连接子相连接；(3)旁观者效应：尤其是对实体瘤治疗有益。与MMAE连接的连接子一般选用缬氨酸-瓜氨酸(Val-Cit)二肽型，与MMAE组合全称为MC-VC-PABC-MMAE，该组合为ADC临床试验中使用最广泛的Linker-Drug，上市ADC Brentuximab Vedotin(SGN-35，Adcetris)就选择该类Linker-Drug组合，使其在组织蛋白酶B中裂解，从而完全释放出MMAE。然而，VC连接子结构类型较为单一，且从结构角度出发仍存在一些问题需要改进： ADC generally consists of three parts: antibody, warhead and linker. SCT-200 antibody is a new type of fully human anti-EGFR monoclonal antibody targeted for the treatment of metastatic colorectal cancer (Patent Publication Number: CN101058609A). It is a high-affinity 100% human antibody obtained through screening of human phage library , Has an original sequence targeting EGFR. The current research on the treatment of metastatic colorectal cancer and head and neck squamous cell carcinoma is in clinical phase II, and its clinical application may have better therapeutic effects and safety than chimeric antibodies and humanized antibodies. The cytotoxicity of warhead molecules must be extremely high, and EC 50 is generally required to be less than 1 nmol/L. Currently, ADCs with MMAE as the warhead molecule dominate. MMAE is based on the active ingredient dolastatins 10 (dolastatins 10) in the natural marine product dolastatins extracted from the censored sea hare. It is a type of linear pentapeptide cytotoxic molecule and is used in a variety of human cancer cell lines. , IC 50 is 10 -9 ～10 -11 mol/L, which is 200 times the activity of vinblastine. MMAE as an ADC warhead molecule has three main advantages: (1) high cytotoxicity; (2) N-terminal single methyl substitution, which can be connected to a linker; (3) bystander effect: it is especially beneficial for the treatment of solid tumors. The linker connected to MMAE generally uses Val-Cit dipeptide type. The combination with MMAE is called MC-VC-PABC-MMAE. This combination is the most widely used Linker in ADC clinical trials. Drug, the listed ADC Brentuximab Vedotin (SGN-35, Adcetris) chose this kind of Linker-Drug combination to cleave it in cathepsin B, thereby completely releasing MMAE. However, the type of VC linker structure is relatively single, and from the structural point of view, there are still some problems that need to be improved:
(1)间隔区部分：MC会慢慢水解，失去与巯基反应的特异性；MC与抗体连接后水解产物的稳定性有待进一步改善。(1) Spacer part: MC will slowly hydrolyze and lose the specificity of reaction with sulfhydryl groups; the stability of the hydrolysate after MC is connected with antibody needs to be further improved.
(2)酶水解部分：某些血浆酶(如羧酸酯酶1C)，可以部分地消化ADC产品中的Val-Cit结构，导致药物提前脱靶。(2) Enzymatic hydrolysis: certain plasma enzymes (such as carboxylesterase 1C) can partially digest the Val-Cit structure in ADC products, causing the drug to miss the target in advance.
(3)自分解部分：氨基苄氧羰基(p-aminobenzyloxycarbonyl)间隔基团类似于酯键，在血浆中的稳定性有待进一步改善。(3) Self-decomposing part: The p-aminobenzyloxycarbonyl spacer group is similar to an ester bond, and its stability in plasma needs to be further improved.
为了克服上述问题，本发明充分采用SCT200抗体的优势，设计不同类型连接子与MMAE连接，最终制备得到新型SCT200-Linker-MMAE抗体药物偶联物。制备所得ADCs在亲和力方面和内吞方面与SCT200相当，能够上调肿瘤凋亡相关的蛋白的表达。在体内外多种实体瘤模型中，本发明的ADCs均体现了较SCT200更强的抗肿瘤活性。In order to overcome the above-mentioned problems, the present invention makes full use of the advantages of the SCT200 antibody, designs different types of linkers to connect with MMAE, and finally prepares a novel SCT200-Linker-MMAE antibody drug conjugate. The prepared ADCs are comparable to SCT200 in terms of affinity and endocytosis, and can up-regulate the expression of tumor apoptosis-related proteins. In a variety of solid tumor models in vivo and in vitro, the ADCs of the present invention all exhibit stronger anti-tumor activity than SCT200.
发明内容Summary of the invention
本发明的目的在于提供一类抗人EGFR抗体药物偶联物及其制备方法和抗肿瘤应用。本发明的一类抗人EGFR抗体药物偶联物对EGFR阳性的肿瘤组织具有高效靶向性、高内吞活性、高亲和活性以及高抗肿瘤活性。The purpose of the present invention is to provide a type of anti-human EGFR antibody drug conjugate and its preparation method and anti-tumor application. The anti-human EGFR antibody drug conjugate of the present invention has high efficiency targeting, high endocytosis activity, high affinity activity and high anti-tumor activity to EGFR-positive tumor tissues.
为了达到上述目的，本发明采用了以下技术手段：In order to achieve the above objective, the present invention adopts the following technical means:
本发明一类抗人EGFR抗体药物偶联物，其具有式I所示结构：A type of anti-human EGFR antibody drug conjugate of the present invention has a structure shown in formula I:
Figure PCTCN2020000225-appb-000001
Figure PCTCN2020000225-appb-000001
其中，among them,
所述的抗体为抗人EGFR抗体，z＝2～8；The antibody is an anti-human EGFR antibody, z=2-8;
M为能与抗体半胱氨酸巯基反应的间隔区部分，其结构为以下所示结构中的一种：M is the part of the spacer that can react with the cysteine sulfhydryl group of the antibody, and its structure is one of the following structures:
Figure PCTCN2020000225-appb-000002
Figure PCTCN2020000225-appb-000002
其中，n＝0，1，2，3，4或5，m＝1～10，12或24；Wherein, n = 0, 1, 2, 3, 4 or 5, m = 1-10, 12 or 24;
C为能够在酶的作用下发生水解的部分，选自二肽序列Val-Cit，Val-Ala，Val-Lys或Val-Arg；或三肽序列Ala-Val-Ala，Gly-Phe-Gly，Gly-Phe-Lys或Ala-Phe-Lys；或四肽序列Gly-Gly-Phe-Gly或Gly-Phe-Leu-Gly中的一种；C is a part that can be hydrolyzed under the action of an enzyme, and is selected from the dipeptide sequence Val-Cit, Val-Ala, Val-Lys or Val-Arg; or the tripeptide sequence Ala-Val-Ala, Gly-Phe-Gly, Gly-Phe-Lys or Ala-Phe-Lys; or one of the tetrapeptide sequence Gly-Gly-Phe-Gly or Gly-Phe-Leu-Gly;
R为自分解部分，其结构为以下所示结构中的一种：R is the self-decomposing part, and its structure is one of the following structures:
Figure PCTCN2020000225-appb-000003
Figure PCTCN2020000225-appb-000003
其中，优选的，所述的抗体为SCT-200单克隆抗体。Among them, preferably, the antibody is SCT-200 monoclonal antibody.
进一步的，本发明还提出了一种制备所述的抗人EGFR抗体偶联药物的方法，包括：将具有式II结构的Linker-MMAE与抗人EGFR抗体的链间二硫键发生加成反应制得所述的抗人EGFR抗体药物偶联物；Further, the present invention also proposes a method for preparing the anti-human EGFR antibody conjugated drug, which includes: the Linker-MMAE having the structure of formula II and the interchain disulfide bond of the anti-human EGFR antibody undergo an addition reaction Preparing the anti-human EGFR antibody drug conjugate;
Figure PCTCN2020000225-appb-000004
Figure PCTCN2020000225-appb-000004
其中，优选的，所述的方法包括以下步骤：Wherein, preferably, the method includes the following steps:
(1)将Linker-MMAE溶于二甲基亚砜，得到连接子-细胞毒药物储备液；(1) Dissolve Linker-MMAE in dimethyl sulfoxide to obtain a linker-cytotoxic drug stock solution;
(2)将还原剂溶于缓冲液中，配置还原剂储备液；(2) Dissolve the reducing agent in the buffer and configure the reducing agent stock solution;
(3)将L-Cys溶于缓冲液中，制备L-Cys储备液；(3) Dissolve L-Cys in buffer to prepare L-Cys stock solution;
(4)将抗人EGFR抗体加入步骤(2)所述的还原剂储备液中，孵育1-2h；(4) Add anti-human EGFR antibody to the reducing agent stock solution described in step (2), and incubate for 1-2h;
(5)向步骤(4)得到的反应液中加入步骤(1)的连接子-细胞毒药物储备液，孵育1-2h，制备抗人EGFR抗体药物偶联物；(5) Add the linker-cytotoxic drug stock solution of step (1) to the reaction solution obtained in step (4), and incubate for 1-2 hours to prepare an anti-human EGFR antibody drug conjugate;
(6)向步骤(5)得到的反应液中加入步骤(3)的L-Cys储备液，停止反应。(6) Add the L-Cys stock solution of step (3) to the reaction solution obtained in step (5) to stop the reaction.
其中，优选的，所述步骤(2)中还原剂为TCEP，混合时以抗人EGFR抗体的摩尔量为基准，还原剂的摩尔量为抗体摩尔量的2-4倍。Wherein, preferably, the reducing agent in the step (2) is TCEP, and the molar amount of the anti-human EGFR antibody is used as a reference when mixing, and the molar amount of the reducing agent is 2-4 times the molar amount of the antibody.
其中，优选的，所述步骤(5)中混合时以抗人EGFR抗体的摩尔量为基准，所加Linker-MMAE的摩尔量为抗体摩尔量的4-8倍。Wherein, preferably, in the step (5), the molar amount of the anti-human EGFR antibody is used as a reference when mixing in the step (5), and the molar amount of Linker-MMAE added is 4-8 times the molar amount of the antibody.
其中，优选的，在所述步骤(4)和步骤(5)中的反应在氮气保护下进行，步骤(4)中的反应温度为35～40℃，优选为37℃。Among them, preferably, the reactions in the steps (4) and (5) are carried out under the protection of nitrogen, and the reaction temperature in the step (4) is 35-40°C, preferably 37°C.
其中，优选的，还包括将得到的抗体药物偶联物进一步纯化的步骤，优选采用AKTA purifier蛋白质纯化系统，收集所需抗体偶联药物组分峰；收集完毕后，使用30kDa超滤管进行超滤离心，浓缩，经无菌滤膜过滤，低温储存。Among them, preferably, it also includes the step of further purifying the obtained antibody-drug conjugate. Preferably, the AKTA purifier protein purification system is used to collect the peak of the required antibody-drug conjugate; after the collection is completed, a 30kDa ultrafiltration tube is used for ultrafiltration Filter, centrifuge, concentrate, filter through a sterile filter membrane, and store at low temperature.
更进一步的，本发明还提出了所述的抗体药物偶联物在制备用于肿瘤靶向治疗的药物中的用途，其中，所述的肿瘤为EGFR阳性实体肿瘤，包括鳞状上皮细胞癌，食管癌，鼻咽癌，肺癌，乳腺癌，胰腺癌，前列腺癌，头颈癌，结肠癌等。Furthermore, the present invention also proposes the use of the antibody-drug conjugate in the preparation of drugs for tumor targeted therapy, wherein the tumor is an EGFR-positive solid tumor, including squamous cell carcinoma, Esophageal cancer, nasopharyngeal cancer, lung cancer, breast cancer, pancreatic cancer, prostate cancer, head and neck cancer, colon cancer, etc.
以所述的抗体药物偶联物为活性成分的用于肿瘤靶向治疗的药物组合物也在本发明的保护范围之内，所述的药物组合物含有药学上有效量的本发明所述的抗体药物偶联物及药学上允许的佐剂。A pharmaceutical composition for targeted tumor therapy using the antibody-drug conjugate as an active ingredient is also within the protection scope of the present invention, and the pharmaceutical composition contains a pharmaceutically effective amount of the present invention. Antibody drug conjugates and pharmaceutically acceptable adjuvants.
相较于现有技术，本发明的有益效果是：Compared with the prior art, the beneficial effects of the present invention are:
1、本发明制备了一系列Linker，体现了较好的血浆稳定性和酶切速率，与MMAE进行一步反应制得Linker-MMAE。1. The present invention prepares a series of Linker, which shows better plasma stability and enzyme cleavage rate, and reacts with MMAE in one step to prepare Linker-MMAE.
2、本发明通过利用SCT-200全人源化单克隆抗体，通过还原抗体链间二硫键的化学方法与Linker-MMAE偶联，构建了一类靶向于EGFR以实体瘤治疗为主的抗人EGFR抗体药物偶联物。优选TCEP作为还原剂，在对其反应条件进行一定优化之后，制备出平均药物抗体偶联比率(DAR)在4.0±0.5的范围内，且质量稳定可控，重复性好，收率可观。2. The present invention uses the SCT-200 fully humanized monoclonal antibody to couple with Linker-MMAE through the chemical method of reducing the disulfide bond between the antibody chains to construct a type of solid tumor therapy targeting EGFR. Anti-human EGFR antibody drug conjugate. Preferably, TCEP is used as a reducing agent. After certain optimization of the reaction conditions, the average drug-antibody coupling ratio (DAR) is prepared within the range of 4.0±0.5, and the quality is stable and controllable, with good repeatability and considerable yield.
3、本发明提出的新型抗体药物偶联物SCT-200-Linker-MMAE，相比与SCT-200本身，并未影响抗体的亲和力，内吞活性和靶向性，较好的保留其生物学功能；在体外活性评价中，相比于SCT-200，活性得到了明显的提高，IC 50均在nM级别；Western Blot实验表明，相比于SCT-200，SCT-200-Linker-MMAE ADCs能够显著抑制肿瘤凋亡过程中相关蛋白的表达；在体内活性评价中，相比于SCT-200，SCT-200-Linker-MMAE ADCs的抑瘤效果大大增强。 3. Compared with SCT-200 itself, the novel antibody drug conjugate SCT-200-Linker-MMAE proposed by the present invention does not affect the affinity, endocytic activity and targeting of the antibody, and better retains its biology Function; In the in vitro activity evaluation, compared with SCT-200, the activity has been significantly improved, IC 50 is in the nM level; Western Blot experiment shows that compared to SCT-200, SCT-200-Linker-MMAE ADCs can Significantly inhibit the expression of related proteins in the process of tumor apoptosis; in the evaluation of in vivo activity, compared with SCT-200, SCT-200-Linker-MMAE ADCs have greatly enhanced tumor suppressing effect.
附图说明Description of the drawings
图1为Fmoc-二肽序列合成路线图；Figure 1 is a synthetic route diagram of Fmoc-dipeptide sequence;
图2为Fmoc-AVA合成路线图；Figure 2 is a synthetic route diagram of Fmoc-AVA;
图3为Fmoc-GFG合成路线图；Figure 3 is a synthetic route diagram of Fmoc-GFG;
图4为Fmoc-GGFG合成路线图；Figure 4 is a synthetic route diagram of Fmoc-GGFG;
图5为Fmoc-GFLG合成路线图；Figure 5 is a synthetic route diagram of Fmoc-GFLG;
图6为间隔区和水解区类型的Linker-MMAE合成路线图；Figure 6 is a linker-MMAE synthesis route diagram of spacer and hydrolysis zone types;
图7为自分解区类型的Linker-MMAE合成路线图；Figure 7 is a linker-MMAE synthesis route diagram of the self-decomposition zone type;
其中，图7A为自分解区类型的Linker合成路线图；图7B为自分解区类型的Linker和MMAE缩合反应合成路线图；Among them, Fig. 7A is a linker synthesis route diagram of the self-decomposition zone type; Fig. 7B is a synthesis route diagram of the Linker and MMAE condensation reaction of the self-decomposition zone type;
图8为间隔区型SCT-200-Linker-MMAE的HIC-HPLC图谱；Figure 8 is the HIC-HPLC spectrum of the spacer type SCT-200-Linker-MMAE;
图9为水解区型SCT-200-Linker-MMAE的HIC-HPLC图谱；Figure 9 is the HIC-HPLC spectrum of the hydrolysis zone type SCT-200-Linker-MMAE;
图10为自分解区型SCT-200-Linker-MMAE的HIC-HPLC图谱；Figure 10 shows the HIC-HPLC spectrum of the self-decomposition zone type SCT-200-Linker-MMAE;
图11为SCT-200-Linker-MMAE的ESI-MS图谱；Figure 11 is the ESI-MS spectrum of SCT-200-Linker-MMAE;
图12为ELISA测定SCT-200-Linker-MMAE ADCs亲和力图；Figure 12 is a graph of the affinity of SCT-200-Linker-MMAE ADCs measured by ELISA;
图13为流式细胞法测定SCT-200-Linker-MMAE ADCs内吞速率图；Figure 13 is a diagram showing the endocytosis rate of SCT-200-Linker-MMAE ADCs measured by flow cytometry;
图14为Western Blot测定不同细胞EGFR表达水平图；Figure 14 is a Western Blot determination of EGFR expression levels in different cells;
图15为SCT200-Linker-MMAE ADCs对凋亡相关蛋白表达的影响图；Figure 15 is a diagram showing the influence of SCT200-Linker-MMAE ADCs on the expression of apoptosis-related proteins;
图16为SCT200-Linker-MMAE ADCs对A431裸鼠移植瘤模型的抑瘤曲线(A)以及体重变化曲线(B)。Figure 16 shows the tumor inhibition curve (A) and weight change curve (B) of SCT200-Linker-MMAE ADCs on the A431 nude mouse xenograft tumor model.
具体实施方式detailed description
下面结合具体实例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。The present invention will be further described below in conjunction with specific examples, and the advantages and characteristics of the present invention will become clearer with the description. However, these examples are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements fall within the protection scope of the present invention.
实施例1 Linker-MMAE的合成Example 1 Synthesis of Linker-MMAE
1.1 Fmoc-AA的合成1.1 Synthesis of Fmoc-AA
1.1.1 Fmoc-二肽的合成1.1.1 Synthesis of Fmoc-dipeptide
Fmoc-L-Val-OSu的合成Synthesis of Fmoc-L-Val-OSu
根据图1中Scheme S1步骤所示，将反应产物Fmoc-L-Val(10g，29.3mmol)和HoSu(3.7g，32.3mmol)溶于100mL THF中，冰浴条件下加入DCC(6.6g，32.3mmol)，加入完毕后，撤去冰浴，室温下搅拌反应过夜。反应完毕后(石油醚∶乙酸乙酯＝1∶1检测)，将反应液移至冰浴，待固体完全析出后抽滤，将滤液旋蒸至泡沫状，残余物即为产物。反应产物无需进一步纯化，直接用于下步反应。According to the Scheme S1 step shown in Figure 1, the reaction products Fmoc-L-Val (10g, 29.3mmol) and HoSu (3.7g, 32.3mmol) were dissolved in 100mL THF, and DCC (6.6g, 32.3mmol) was added under ice bath conditions. mmol). After the addition is complete, the ice bath is removed, and the reaction is stirred overnight at room temperature. After the completion of the reaction (petroleum ether: ethyl acetate = 1:1 detection), the reaction solution was transferred to an ice bath, after the solid was completely precipitated, filtered with suction, the filtrate was rotary evaporated to a foamy state, and the residue was the product. The reaction product does not require further purification and is directly used in the next step.
1a的合成Synthesis of 1a
根据图1中Scheme S1所示，将Fmoc-L-Val-OSu(16g，36.6mmol)先溶于90mL DME中，L-Cit(6.7g，38.5mmol)溶于NaHCO 3(3.2g，38.5mmol)的90mL水中成盐，再加入到反应体系中，50mL THF助溶，反应室温搅拌过夜至澄清。反应完毕后(二氯甲烷∶甲醇＝10∶1检测)，冰浴下加入与THF等体积的15％的柠檬酸水溶液，待反应产物白色固体完全析出，布氏漏斗抽滤至滤液澄清，滤饼抽干，真空干燥箱干燥。干燥完毕后，将白色固体研磨，用乙醚洗涤数次，抽滤，得1a，白色固体15.1g，收率为83％。分子式为C 26H 32N 4O 6，MS(ESI)m/z：497[M+H]+； 1H NMR(400MHz，DMSO-d 6)δ(ppm)：12.48(s，1H)，8.17(d，J＝7.4Hz，1H)，7.90(d，J＝7.5Hz，2H)，7.76(t，J＝7.1Hz，2H)，7.41(q，J＝7.7Hz，3H)，7.33(m，2H)，6.00(s，1H)，5.63(s，2H)，4.30-4.21(m，3H)，4.16(m，1H)，3.93(m，1H)，2.98-2.89(m，2H)，1.99(m，1H)，1.77-1.66(m，1H)，1.57(m，1H)，1.41(m，2H)，0.88(m，6H). According to Scheme S1 shown in Figure 1, Fmoc-L-Val-OSu (16g, 36.6mmol) was first dissolved in 90mL DME, L-Cit (6.7g, 38.5mmol) was dissolved in NaHCO 3 (3.2g, 38.5mmol) ) Salt was formed in 90 mL of water, and then added to the reaction system, 50 mL of THF was used to aid solubility, and the reaction was stirred at room temperature overnight until it became clear. After the reaction is completed (dichloromethane: methanol = 10:1 detection), 15% citric acid aqueous solution equal in volume with THF is added under ice bath. After the white solid of the reaction product is completely precipitated, the Buchner funnel is suction filtered until the filtrate is clear, and then filtered The cake is drained and dried in a vacuum drying oven. After drying, the white solid was ground, washed with ether for several times, and filtered with suction to obtain 1a, 15.1 g of the white solid, with a yield of 83%. The molecular formula is C 26 H 32 N 4 O 6 , MS (ESI) m/z: 497[M+H]+; 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 12.48 (s, 1H), 8.17 (d, J = 7.4 Hz, 1H), 7.90 (d, J = 7.5 Hz, 2H), 7.76 (t, J = 7.1 Hz, 2H), 7.41 (q, J = 7.7 Hz, 3H), 7.33 ( m, 2H), 6.00 (s, 1H), 5.63 (s, 2H), 4.30-4.21 (m, 3H), 4.16 (m, 1H), 3.93 (m, 1H), 2.98-2.89 (m, 2H) , 1.99 (m, 1H), 1.77-1.66 (m, 1H), 1.57 (m, 1H), 1.41 (m, 2H), 0.88 (m, 6H).
1b的合成Synthesis of 1b
根据图1中Scheme S1所示，合成方法同1a，得白色固体纯品，两步收率为37％。分子式为C 23H 26N 2O 5，MS(ESI)m/z：411[M+H]+； 1H NMR(400MHz，DMSO-d 6)δ(ppm)：12.48(s，1H)，8.23(d，J＝7.0Hz，1H)，7.90(d，J＝7.5Hz，2H)，7.76(m，2H)，7.42(m，3H)，7.33(m，2H)，4.34-4.16(m，4H)，3.90(dd，J ＝9.2Hz，7.1Hz，1H)，1.98(m，1H)，1.28(d，J＝7.2Hz，3H)，0.89(m，6H). 13C NMR(101MHz，DMSO-d6)δ(ppm)：174.45，171.44，156.52，144.26，141.15，128.09，127.51，125.87，120.55，66.14，60.22，47.92，47.13，30.97，19.61，18.70，17.56. According to Scheme S1 shown in Figure 1, the synthesis method is the same as 1a, and a pure white solid product is obtained. The two-step yield is 37%. The molecular formula is C 23 H 26 N 2 O 5 , MS (ESI) m/z: 411 [M+H]+; 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 12.48 (s, 1H), 8.23 (d, J = 7.0 Hz, 1H), 7.90 (d, J = 7.5 Hz, 2H), 7.76 (m, 2H), 7.42 (m, 3H), 7.33 (m, 2H), 4.34 to 4.16 (m , 4H), 3.90 (dd, J = 9.2 Hz, 7.1 Hz, 1H), 1.98 (m, 1H), 1.28 (d, J = 7.2 Hz, 3H), 0.89 (m, 6H). 13 C NMR (101MHz , DMSO-d6) δ (ppm): 174.45, 171.44, 156.52, 144.26, 141.15, 128.09, 127.51, 125.87, 120.55, 66.14, 60.22, 47.92, 47.13, 30.97, 19.61, 18.70, 17.56.
1.1.2 Fmoc-三肽的合成1.1.2 Synthesis of Fmoc-Tripeptide
1c的合成Synthesis of 1c
根据图2中Scheme S2所示，以2-CTC为树脂，经过固相合成，得白色固体，收率为89％。产物用乙醚洗涤、产物收率和纯度较高，无需进一步纯化。分子式为C 26H 31N 3O 6，MS(ESI)m/z：482[M+H] +； 1H NMR(400MHz，DMSO-d 6)δ(ppm)：12.41(s，1H)，8.26(d，J＝6.9Hz，1H)，7.90(d，J＝7.5H[z，2H)，7.72(m，2H)，7.66(d，J＝9.0Hz，1H)，7.57(d，J＝7.9Hz，1H)，7.42(m，2H)，7.34(m，2H)，4.28(d，J＝6.9Hz，2H)，4.22(dd，J＝8.3，6.1Hz，2H)，4.18(d，J＝7.0Hz，2H)，1.97(m，1H)，1.27(d，J＝7.3Hz，3H)，1.22(d，J＝7.2Hz，3H)，0.88(d，J＝6.8Hz，3H)，0.84(d，J＝6.8Hz，3H)； 13C NMR(101MHz，DMSO-d 6)δ(ppm)：174.40，172.74，171.03，156.07，144.34，141.16，128.08，127.55，125.74，125.70，120.56，66.05，57.36，50.52，47.91，47.10，31.53，19.55，18.68，18.37，17.47. According to Scheme S2 in Figure 2, using 2-CTC as the resin, solid phase synthesis was performed to obtain a white solid with a yield of 89%. The product is washed with ether, and the yield and purity of the product are high, and no further purification is required. The molecular formula is C 26 H 31 N 3 O 6 , MS (ESI) m/z: 482 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 12.41 (s, 1H), 8.26 (d, J = 6.9 Hz, 1H), 7.90 (d, J = 7.5H [z, 2H), 7.72 (m, 2H), 7.66 (d, J = 9.0 Hz, 1H), 7.57 (d, J =7.9Hz, 1H), 7.42(m, 2H), 7.34(m, 2H), 4.28(d, J=6.9Hz, 2H), 4.22(dd, J=8.3, 6.1Hz, 2H), 4.18(d , J=7.0Hz, 2H), 1.97(m, 1H), 1.27(d, J=7.3Hz, 3H), 1.22(d, J=7.2Hz, 3H), 0.88(d, J=6.8Hz, 3H ), 0.84 (d, J=6.8 Hz, 3H); 13 C NMR (101MHz, DMSO-d 6 ) δ (ppm): 174.40, 172.74, 171.03, 156.07, 144.34, 141.16, 128.08, 127.55, 125.74, 125.70, 120.56, 66.05, 57.36, 50.52, 47.91, 47.10, 31.53, 19.55, 18.68, 18.37, 17.47.
1d的合成Synthesis of 1d
根据图3中Scheme S3所示，得白色固体，收率为65％。产物用乙醚洗涤、产物收率和纯度较高，无需进一步纯化。分子式为C 28H 27N 3O 6，MS(ESI)m/z：502[M+H] +； 1H NMR(400MHz，DMSO-d 6)δ(ppm)：12.60(s，1H)，8.42(t，J＝5.9Hz，1H)，8.09(d，J＝8.5Hz，1H)，7.90(d，J＝7.5Hz，2H)，7.71(d，J＝7.5Hz，2H)，7.48(m，1H)，7.42(m，2H)，7.33(m，2H)，7.24(d，J＝4.4Hz，4H)，7.18(m，1H)，4.57(m，1H)，4.31-4.17(m，3H)，3.79(d，J＝5.9Hz，2H)，3.67(m，1H)，3.52(m，1H)，3.04(m，1H)，2.78(m，1H)； 13C NMR(101MHz，DMSO-d 6)δ(ppm)：171.87，171.52，144.29，141.16，138.21，129.65，128.49，128.09，127.54，126.71，125.73，120.56，66.20，54.13，47.06，43.71，41.10，38.25. According to Scheme S3 in Figure 3, a white solid was obtained with a yield of 65%. The product is washed with ether, and the yield and purity of the product are high, and no further purification is required. The molecular formula is C 28 H 27 N 3 O 6 , MS (ESI) m/z: 502[M+H] + ; 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 12.60 (s, 1H), 8.42 (t, J = 5.9 Hz, 1H), 8.09 (d, J = 8.5 Hz, 1H), 7.90 (d, J = 7.5 Hz, 2H), 7.71 (d, J = 7.5 Hz, 2H), 7.48 ( m, 1H), 7.42 (m, 2H), 7.33 (m, 2H), 7.24 (d, J=4.4 Hz, 4H), 7.18 (m, 1H), 4.57 (m, 1H), 4.31-4.17 (m , 3H), 3.79 (d, J = 5.9 Hz, 2H), 3.67 (m, 1H), 3.52 (m, 1H), 3.04 (m, 1H), 2.78 (m, 1H); 13 C NMR (101MHz, DMSO-d 6 )δ (ppm): 171.87, 171.52, 144.29, 141.16, 138.21, 129.65, 128.49, 128.09, 127.54, 126.71, 125.73, 120.56, 66.20, 54.13, 47.06, 43.71, 41.10, 38.25.
1.1.3 Fmoc-四肽的合成1.1.3 Synthesis of Fmoc-tetrapeptide
1e的合成Synthesis of 1e
根据图4中Scheme S4所示，得白色固体，收率为49％。产物用乙醚洗涤、产物收率和纯度较高，无需进一步纯化。分子式为C 30H 30N 4O 7，MS(ESI) m/z：559[M+H] +； 1H NMR(400MHz，DMSO-d 6)δ(ppm)：12.55(s，1H)，8.38(m，1H)，8.13(d，J＝8.6Hz，1H)，8.00(m，1H)，7.90(d，J＝7.5Hz，2H)，7.72(d，J＝7.5Hz，2H)，7.60(m，1H)，7.42(m，2H)，7.34(d，J＝7.4Hz，2H)，7.25(d，J＝4.3Hz，4H)，7.18(m，1H)，4.55(m，1H)，4.35-4.18(m，3H)，3.77(m，3H)，3.63(d，J＝6.3Hz，3H)，3.06(m，1H)，2.77(m，1H)； 13C NMR(101MHz，DMSO-d 6)δ(ppm)：171.84，171.51，169.79，168.92，144.31，141.17，129.62，128.52，128.09，127.55，126.72，125.71，120.58，66.22，54.28，47.08，43.92，42.23，41.12，38.12. According to Scheme S4 in Figure 4, a white solid was obtained with a yield of 49%. The product is washed with ether, and the yield and purity of the product are high, and no further purification is required. The molecular formula is C 30 H 30 N 4 O 7 , MS (ESI) m/z: 559 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 12.55 (s, 1H), 8.38 (m, 1H), 8.13 (d, J=8.6Hz, 1H), 8.00 (m, 1H), 7.90 (d, J=7.5Hz, 2H), 7.72 (d, J=7.5Hz, 2H), 7.60 (m, 1H), 7.42 (m, 2H), 7.34 (d, J = 7.4 Hz, 2H), 7.25 (d, J = 4.3 Hz, 4H), 7.18 (m, 1H), 4.55 (m, 1H) ), 4.35-4.18 (m, 3H), 3.77 (m, 3H), 3.63 (d, J = 6.3 Hz, 3H), 3.06 (m, 1H), 2.77 (m, 1H); 13 C NMR (101MHz, DMSO-d 6 )δ (ppm): 171.84, 171.51, 169.79, 168.92, 144.31, 141.17, 129.62, 128.52, 128.09, 127.55, 126.72, 125.71, 120.58, 66.22, 54.28, 47.08, 43.92, 42.23, 41.12, 38.12.
1f的合成Synthesis of 1f
根据图5中Scheme S5所示，得白色固体，收率为75％。产物用乙醚洗涤、产物收率和纯度较高，无需进一步纯化。分子式为C 34H 38N 4O 7，MS(ESI)m/z：615[M+H] +； 1H NMR(400MHz，DMSO-d 6)δ(ppm)：12.54(s，1H)，8.18-8.07(m，2H)，8.01(d，J＝8.1，1H)，7.90(d，J＝7.5Hz，2H)，7.71(d，J＝7.4Hz，2H)，7.53(t，J＝6.1Hz，1H)，7.42(t，J＝7.4Hz，2H)，7.33(t，J＝7.4Hz，2H)，7.23(d，J＝4.4Hz，4H)，7.19-7.15(m，1H)，4.57(dt，J＝9.2Hz，4.6Hz，1H)，4.35(q，J＝7.8Hz，1H)，4.31-4.19(m，3H)，3.83-3.47(m，4H)，3.03(m，1H)，2.79(m，1H)，1.50(q，J＝8.2Hz，7.3Hz，3H)，0.86(m，6H)； 13C NMR(101MHz，DMSO-d 6)δ(ppm)：172.62，171.52，171.16，169.36，144.29，141.16，129.72，128.45，128.09，127.54，126.66，125.71，120.57，66.23，54.14，51.30，47.06，43.76，41.46，41.07，38.01，24.51，23.48，22.11. According to Scheme S5 in Figure 5, a white solid was obtained with a yield of 75%. The product is washed with ether, and the yield and purity of the product are high, and no further purification is required. The molecular formula is C 34 H 38 N 4 O 7 , MS (ESI) m/z: 615 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 12.54 (s, 1H), 8.18-8.07 (m, 2H), 8.01 (d, J = 8.1, 1H), 7.90 (d, J = 7.5 Hz, 2H), 7.71 (d, J = 7.4 Hz, 2H), 7.53 (t, J = 6.1Hz, 1H), 7.42(t, J=7.4Hz, 2H), 7.33(t, J=7.4Hz, 2H), 7.23(d, J=4.4Hz, 4H), 7.19-7.15(m, 1H) , 4.57 (dt, J = 9.2 Hz, 4.6 Hz, 1H), 4.35 (q, J = 7.8 Hz, 1H), 4.31-4.19 (m, 3H), 3.83-3.47 (m, 4H), 3.03 (m, 1H), 2.79 (m, 1H), 1.50 (q, J=8.2 Hz, 7.3 Hz, 3H), 0.86 (m, 6H); 13 C NMR (101 MHz, DMSO-d 6 ) δ (ppm): 172.62, 171.52,171.16,169.36,144.29,141.16,129.72,128.45,128.09,127.54,126.66,125.71,120.57,66.23,54.14,51.30,47.06,43.76,41.46,41.07,38.01,24.51,23.48,22.11.
1.2 Linker-MMAE的合成1.2 Synthesis of Linker-MMAE
2a的合成Synthesis of 2a
根据图6步骤所示，将1a反应产物(7.6g，15.3mmol)和PABOH(3.76g，30.6mmol)溶于140mL CH 2Cl 2和70mL的CH 3OH混合溶剂中，避光加入EEDQ(7.5g，30.6mmol)。室温下搅拌反应，反应完毕后(二氯甲烷∶甲醇＝20∶1检测)旋干反应液，残余物用乙醚洗涤，抽滤，得黄色固体8.4g，收率90％。分子式为C 33H 39N 5O 6，MS(ESI)m/z：602[M+H] +； 1H NMR(400MHz，DMSO-d 6)δ(ppm)：9.99(s，1H)，8.11(d，J＝7.5Hz，1H)，7.90(d，J＝7.6Hz，2H)，7.75(m，2H)，7.55(d，J＝8.2Hz，2H)，7.43(m，3H)，7.33(m，2H)，7.24(d，J＝8.1Hz，2H)，5.99(m，1H)，5.41(s，2H)，5.10(m，1H)，4.43(d，J＝5.5Hz，3H)，4.31(s，1H)，4.25(d，J＝6.3Hz，2H)，3.94(m，1H)，3.03-2.88(m，2H)，2.00(d，J＝9.0Hz，1H)，1.76-1.65(m，1H)，1.60(m，1H)，1.48-1.36(m，2H)， 0.92-0.85(m，6H). According to the steps shown in Figure 6, the 1a reaction product (7.6g, 15.3mmol) and PABOH (3.76g, 30.6mmol) were dissolved in 140mL CH 2 Cl 2 and 70 mL of CH 3 OH mixed solvent, and EEDQ (7.5 g, 30.6 mmol). The reaction was stirred at room temperature. After the reaction was completed (dichloromethane: methanol = 20:1 detection), the reaction solution was spin-dried, the residue was washed with ether, and filtered with suction to obtain 8.4 g of a yellow solid with a yield of 90%. The molecular formula is C 33 H 39 N 5 O 6 , MS (ESI) m/z: 602 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 9.99 (s, 1H), 8.11 (d, J = 7.5 Hz, 1H), 7.90 (d, J = 7.6 Hz, 2H), 7.75 (m, 2H), 7.55 (d, J = 8.2 Hz, 2H), 7.43 (m, 3H), 7.33(m, 2H), 7.24(d, J=8.1Hz, 2H), 5.99(m, 1H), 5.41(s, 2H), 5.10(m, 1H), 4.43(d, J=5.5Hz, 3H ), 4.31 (s, 1H), 4.25 (d, J = 6.3 Hz, 2H), 3.94 (m, 1H), 3.03-2.88 (m, 2H), 2.00 (d, J = 9.0 Hz, 1H), 1.76 -1.65(m, 1H), 1.60(m, 1H), 1.48-1.36(m, 2H), 0.92-0.85(m, 6H).
2b的合成Synthesis of 2b
根据图6步骤所示，1b(1.87g，0.0045mmol)和PABOH(1.12g，0.009mmol)溶于无水40mL四氢呋喃中，加入HATU(2.05g，0.0054mmol)和DIEA 1.5mL，室温搅拌反应。反应完毕后，减压蒸馏，硅胶柱层析纯化，二氯甲烷∶甲醇＝15∶1，得淡黄色固体1.3g，收率59％。分子式为C 30H 33N 3O 5，MS(ESI)m/z：516[M+H] +； 1H NMR(600MHz，DMSO-d 6)δ(ppm)：9.92(s，1H)，8.17(d，J＝7.1Hz，1H)，7.89(d，J＝7.5Hz，2H)，7.75(dd，J＝10.6Hz，7.5Hz，2H)，7.54(d，J＝8.1Hz，2H)，7.47-7.39(m，3H)，7.33(m，2H)，7.24(d，J＝8.2Hz，2H)，5.10(s，1H)，4.43(s，3H)，4.33-4.28(m，1H)，4.26-4.20(m，2H)，3.92(dd，J＝8.9Hz，7.1Hz，1H)，2.01-1.98(m，1H)，1.31(d，J＝7.1Hz，3H)，0.88(m，6H). According to the steps shown in Figure 6, 1b (1.87g, 0.0045mmol) and PABOH (1.12g, 0.009mmol) were dissolved in anhydrous 40mL tetrahydrofuran, HATU (2.05g, 0.0054mmol) and DIEA 1.5mL were added, and the reaction was stirred at room temperature. After the completion of the reaction, it was distilled under reduced pressure and purified by silica gel column chromatography, dichloromethane: methanol = 15:1, to obtain 1.3 g of light yellow solid, with a yield of 59%. The molecular formula is C 30 H 33 N 3 O 5 , MS (ESI) m/z: 516[M+H] + ; 1 H NMR (600MHz, DMSO-d 6 ) δ (ppm): 9.92 (s, 1H), 8.17(d, J=7.1Hz, 1H), 7.89(d, J=7.5Hz, 2H), 7.75(dd, J=10.6Hz, 7.5Hz, 2H), 7.54(d, J=8.1Hz, 2H) , 7.47-7.39 (m, 3H), 7.33 (m, 2H), 7.24 (d, J = 8.2 Hz, 2H), 5.10 (s, 1H), 4.43 (s, 3H), 4.33-4.28 (m, 1H) ), 4.26-4.20 (m, 2H), 3.92 (dd, J = 8.9 Hz, 7.1 Hz, 1H), 2.01-1.98 (m, 1H), 1.31 (d, J = 7.1 Hz, 3H), 0.88 (m , 6H).
2c的合成Synthesis of 2c
根据图6步骤所示，以1c和PABOH为原料，方法同2b，得淡黄色固体，收率83％。分子式为C 33H 38N 4O 6，MS(ESI)m/z：587[M+H] +； 1H NMR(400MHz，DMSO-d 6)δ(ppm)：9.96(s，1H)，7.89(d，J＝7.5Hz，2H)，7.85(d，J＝7.4Hz，1H)，7.72(t，J＝7.4Hz，1H)，7.58(m，2H)，7.42(t，J＝7.3Hz，2H)，7.35(t，J＝7.3Hz，2H)，7.24(dd，J＝8.5Hz，4.0Hz，2H)，6.29(s，1H)，4.43(s，2H)，4.29-4.10(m，2H)，2.04-1.96(m，2H)，1.46(m，1H)，1.31(m，4H)，1.25(m，5H)，1.18-1.14(m，2H)，0.91-0.83(m，6H). According to the steps shown in Figure 6, using 1c and PABOH as raw materials, the method is the same as that of 2b, and a light yellow solid is obtained with a yield of 83%. The molecular formula is C 33 H 38 N 4 O 6 , MS (ESI) m/z: 587 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 9.96 (s, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.85 (d, J = 7.4 Hz, 1H), 7.72 (t, J = 7.4 Hz, 1H), 7.58 (m, 2H), 7.42 (t, J = 7.3 Hz, 2H), 7.35(t, J=7.3Hz, 2H), 7.24(dd, J=8.5Hz, 4.0Hz, 2H), 6.29(s, 1H), 4.43(s, 2H), 4.29-4.10( m, 2H), 2.04-1.96 (m, 2H), 1.46 (m, 1H), 1.31 (m, 4H), 1.25 (m, 5H), 1.18-1.14 (m, 2H), 0.91-0.83 (m, 6H).
2d的合成2d synthesis
根据图6步骤所示，以1d和PABOH为原料，合成方法同2b，得淡黄色固体，收率74％。分子式为C 35H 34N 4O 6，MS(ESI)m/z：502[M+H] +； 1H NMR(400MHz，DMSO-d 6)δ(ppm)：9.78(s，1H)，8.55-8.46(m，1H)，8.20(d，J＝7.8Hz，1H)，7.90(d，J＝7.6Hz，2H)，7.70(d，J＝7.6Hz，2H)，7.59(d，J＝8.1Hz，2H)，7.54(d，J＝5.9Hz，1H)，7.43(d，J＝7.5Hz，2H)，7.32(t，J＝7.5Hz，2H)，7.26(d，J＝3.4Hz，4H)，6.56-6.50(m，1H)，5.11(s，1H)，4.54(m，1H)，4.43(s，2H)，4.26(m，1H)，4.22(m，1H)，4.02(d，J＝7.5Hz，1H)，3.92(m，2H)，3.73-3.67(m，1H)，3.57(m，1H)，3.41-3.36(m，1H)，3.07(m，1H)，2.90-2.82(m，1H)，1.11(m，1H). According to the steps shown in Figure 6, using 1d and PABOH as raw materials, the synthesis method is the same as that of 2b, and a light yellow solid is obtained with a yield of 74%. The molecular formula is C 35 H 34 N 4 O 6 , MS (ESI) m/z: 502[M+H] + ; 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 9.78 (s, 1H), 8.55-8.46 (m, 1H), 8.20 (d, J = 7.8 Hz, 1H), 7.90 (d, J = 7.6 Hz, 2H), 7.70 (d, J = 7.6 Hz, 2H), 7.59 (d, J = 8.1 Hz, 2H), 7.54 (d, J = 5.9 Hz, 1H), 7.43 (d, J = 7.5 Hz, 2H), 7.32 (t, J = 7.5 Hz, 2H), 7.26 (d, J = 3.4 Hz, 4H), 6.56-6.50 (m, 1H), 5.11 (s, 1H), 4.54 (m, 1H), 4.43 (s, 2H), 4.26 (m, 1H), 4.22 (m, 1H), 4.02 (d, J=7.5Hz, 1H), 3.92 (m, 2H), 3.73-3.67 (m, 1H), 3.57 (m, 1H), 3.41-3.36 (m, 1H), 3.07 (m, 1H), 2.90-2.82(m, 1H), 1.11(m, 1H).
2e的合成Synthesis of 2e
根据图6步骤所示，以1e和PABOH为原料，方法同2b，得淡黄色固体，收率78％。分子式为C 37H 37N 5O 7，MS(ESI)m/z：664[M+H] +； 1H NMR(400MHz，DMSO-d 6)δ(ppm)：9.83(m，1H)，8.47(t，J＝5.9Hz，1H)，8.24(d，J＝8.0Hz，1H)，8.10(q，J＝5.3Hz，1H)，7.89(d，J＝7.5Hz，2H)，7.71(d，J＝7.5Hz，1H)，7.58(d，J＝8.2Hz，2H)，7.42(m，2H)，7.37-7.31(m，2H)，7.27-7.23(m，6H)，7.19(m，1H)，6.29(s，1H)，5.12(s，1H)，4.54(m，1H)，4.44(s，2H)，4.30(d，J＝6.0Hz，1H)，4.23(s，1H)，3.89(m，1H)，3.84-3.77(m，1H)，3.65(d，J＝5.8Hz，2H)，3.12-3.06(m，1H)，2.89(s，2H)，2.74(s，1H)，1.24(d，J＝3.1Hz，2H). According to the steps shown in Figure 6, using 1e and PABOH as raw materials, the method is the same as that of 2b, and a light yellow solid is obtained with a yield of 78%. The molecular formula is C 37 H 37 N 5 O 7 , MS (ESI) m/z: 664[M+H] + ; 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 9.83 (m, 1H), 8.47 (t, J = 5.9 Hz, 1H), 8.24 (d, J = 8.0 Hz, 1H), 8.10 (q, J = 5.3 Hz, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.71 ( d, J = 7.5 Hz, 1H), 7.58 (d, J = 8.2 Hz, 2H), 7.42 (m, 2H), 7.37-7.31 (m, 2H), 7.27-7.23 (m, 6H), 7.19 (m , 1H), 6.29 (s, 1H), 5.12 (s, 1H), 4.54 (m, 1H), 4.44 (s, 2H), 4.30 (d, J = 6.0 Hz, 1H), 4.23 (s, 1H) , 3.89(m, 1H), 3.84-3.77(m, 1H), 3.65(d, J=5.8Hz, 2H), 3.12-3.06(m, 1H), 2.89(s, 2H), 2.74(s, 1H) ), 1.24(d, J=3.1Hz, 2H).
2f的合成Synthesis of 2f
根据图6步骤所示，以1f和PABOH为原料，方法同2b，得淡黄色固体，收率66％。分子式为C 41H 45N 5O 7，MS(ESI)m/z：720[M+H] +； 1H NMR(400MHz，DMSO-d 6)δ(ppm)：9.80(s，1H)，8.19(m，2H)，8.06(d，J＝8.1Hz，1H)，7.90(d，J＝7.5Hz，2H)，7.71(d，J＝7.5Hz，2H)，7.56(m，3H)，7.44-7.39(m，2H)，7.35-7.30(m，2H)，7.26(s，1H)，7.22(s，5H)，7.16(m，1H)，5.09(s，1H)，4.58(m，1H)，4.44(s，2H)，4.36-4.19(m，4H)，3.88(dd，J＝5.9Hz，1.8Hz，2H)，3.65(m，1H)，3.54(m，1H)，3.05(m，1H)，2.80(m，1H)，1.57(m，3H)，0.88(m，6H). According to the steps shown in Figure 6, using 1f and PABOH as raw materials, the method is the same as that of 2b, and a light yellow solid is obtained with a yield of 66%. The molecular formula is C 41 H 45 N 5 O 7 , MS (ESI) m/z: 720 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 9.80 (s, 1H), 8.19 (m, 2H), 8.06 (d, J = 8.1 Hz, 1H), 7.90 (d, J = 7.5 Hz, 2H), 7.71 (d, J = 7.5 Hz, 2H), 7.56 (m, 3H), 7.44-7.39 (m, 2H), 7.35-7.30 (m, 2H), 7.26 (s, 1H), 7.22 (s, 5H), 7.16 (m, 1H), 5.09 (s, 1H), 4.58 (m, 1H), 4.44(s, 2H), 4.36-4.19(m, 4H), 3.88(dd, J=5.9Hz, 1.8Hz, 2H), 3.65(m, 1H), 3.54(m, 1H), 3.05( m, 1H), 2.80 (m, 1H), 1.57 (m, 3H), 0.88 (m, 6H).
4a的合成Synthesis of 4a
根据图6步骤所示，3a(0.5g，1.3mmol)溶于18mL DMF中，加入MC-OSu(0.45g，1.4mmol)，室温搅拌反应过夜。反应完毕后(二氯甲烷∶甲醇＝15∶1检测)，旋干反应溶剂，残余物乙醚洗三遍后，抽滤得黄色固体0.69g，收率为92％。分子式为C 28H 40N 6O 7，MS(ESI)m/z：573[M+H] +； 1H NMR(400MHz，DMSO-d 6)δ(ppm)：9.91(s，1H)，8.07(d，J＝7.6Hz，1H)，7.82(d，J＝8.6Hz，1H)，7.55(d，J＝8.1Hz，2H)，7.23(d，J＝8.1Hz，2H)，7.01(s，2H)，5.99(m，1H)，5.42(s，2H)，5.10(m，1H)，4.43(d，J＝4.9Hz，2H)，4.20(m，1H)，3.00(m，2H)，2.17(m，2H)，1.97(m，1H)，1.71(m，1H)，1.64-1.57(m，1H)，1.54-1.43(m，6H)，1.20(m，2H)，1.10(m，1H)，0.84(m，6H). According to the step shown in Figure 6, 3a (0.5g, 1.3mmol) was dissolved in 18mL DMF, MC-OSu (0.45g, 1.4mmol) was added, and the reaction was stirred overnight at room temperature. After the completion of the reaction (dichloromethane: methanol = 15:1 detection), the reaction solvent was spin-dried, the residue was washed three times with ether, and then suction filtered to obtain 0.69 g of a yellow solid with a yield of 92%. The molecular formula is C 28 H 40 N 6 O 7 , MS (ESI) m/z: 573 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 9.91 (s, 1H), 8.07 (d, J = 7.6 Hz, 1H), 7.82 (d, J = 8.6 Hz, 1H), 7.55 (d, J = 8.1 Hz, 2H), 7.23 (d, J = 8.1 Hz, 2H), 7.01 ( s, 2H), 5.99 (m, 1H), 5.42 (s, 2H), 5.10 (m, 1H), 4.43 (d, J = 4.9 Hz, 2H), 4.20 (m, 1H), 3.00 (m, 2H) ), 2.17 (m, 2H), 1.97 (m, 1H), 1.71 (m, 1H), 1.64-1.57 (m, 1H), 1.54-1.43 (m, 6H), 1.20 (m, 2H), 1.10 ( m, 1H), 0.84 (m, 6H).
4b的合成Synthesis of 4b
根据图6步骤所示，以SMCC和3a为起始原料，合成方法同4a，得黄色固体，收率89％。分子式为C 30H 42N 6O 7，MS(ESI)m/z：599[M+H] +； 1H NMR (400MHz，DMSO-d 6)δ(ppm)：9.91(s，1H)，8.02(d，J＝7.5Hz，1H)，7.72(d，J＝8.7Hz，1H)，7.55(d，J＝8.1Hz，2H)，7.24(d，J＝8.2Hz，2H)，7.02(s，3H)，5.98(m，1H)，5.41(s，2H)，5.10(m，1H)，4.43(d，J＝5.6Hz，2H)，4.16(m，1H)，3.25(d，J＝7.1Hz，2H)，2.98(m，2H)，2.60(s，2H)，2.23(m，1H)，1.99(m，1H)，1.73-1.60(m，6H)，1.36-1.25(m，3H)，0.84(m，8H). According to the steps shown in Figure 6, using SMCC and 3a as starting materials, the synthesis method is the same as that of 4a, and a yellow solid is obtained with a yield of 89%. The molecular formula is C 30 H 42 N 6 O 7 , MS(ESI) m/z: 599[M+H] + ; 1 H NMR (400MHz, DMSO-d 6 )δ(ppm): 9.91(s, 1H), 8.02 (d, J = 7.5 Hz, 1H), 7.72 (d, J = 8.7 Hz, 1H), 7.55 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 8.2 Hz, 2H), 7.02 ( s, 3H), 5.98 (m, 1H), 5.41 (s, 2H), 5.10 (m, 1H), 4.43 (d, J = 5.6 Hz, 2H), 4.16 (m, 1H), 3.25 (d, J =7.1Hz, 2H), 2.98 (m, 2H), 2.60 (s, 2H), 2.23 (m, 1H), 1.99 (m, 1H), 1.73-1.60 (m, 6H), 1.36-1.25 (m, 3H), 0.84(m, 8H).
4c的合成Synthesis of 4c
根据图6步骤所示，以MC-Ph-OSu和3a为起始原料，合成方法同4a，得黄色固体，收率62％。此步产物溶解性不好，直接用乙醚和二氯甲烷混合溶剂洗涤、抽滤得到，直接进行下一步反应。According to the steps shown in Figure 6, using MC-Ph-OSu and 3a as starting materials, the synthesis method is the same as that of 4a, to obtain a yellow solid with a yield of 62%. The solubility of the product in this step is not good, and it is directly washed with a mixed solvent of diethyl ether and dichloromethane and filtered by suction, and directly proceed to the next step of the reaction.
4d的合成4d synthesis
根据图6步骤所示，以3b和SMCC为起始原料，合成方法同4a，得黄色固体，收率67％。分子式为C 27H 36N 4O 6，MS(ESI)m/z：513[M+H] +； 1H NMR(400MHz，DMSO-d 6)δ(ppm)：9.86(s，1H)，8.10(d，J＝6.8Hz，1H)，7.71(d，J＝8.6Hz，1H)，7.54(d，J＝7.9Hz，2H)，7.25(s，2H)，7.02(s，2H)，4.43(s，3H)，4.14(t，J＝7.7Hz，1H)，3.24(d，J＝6.9Hz，2H)，2.23(m，1H)，1.98(s，1H)，1.75-1.50(m，6H)，1.36-1.24(m，6H)，0.91-0.82(m，7H). According to the steps shown in Figure 6, with 3b and SMCC as starting materials, the synthesis method is the same as that of 4a, and a yellow solid is obtained with a yield of 67%. The molecular formula is C 27 H 36 N 4 O 6 , MS (ESI) m/z: 513 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 9.86 (s, 1H), 8.10 (d, J = 6.8 Hz, 1H), 7.71 (d, J = 8.6 Hz, 1H), 7.54 (d, J = 7.9 Hz, 2H), 7.25 (s, 2H), 7.02 (s, 2H), 4.43 (s, 3H), 4.14 (t, J = 7.7 Hz, 1H), 3.24 (d, J = 6.9 Hz, 2H), 2.23 (m, 1H), 1.98 (s, 1H), 1.75-1.50 (m , 6H), 1.36-1.24(m, 6H), 0.91-0.82(m, 7H).
4e的合成Synthesis of 4e
根据图6步骤所示，以3c和SMCC为起始原料，合成方法同4a，得黄色固体，收率63％。分子式为C 30H 41N 5O 7，MS(ESI)m/z：584[M+H] +； 1H NMR(400MHz，DMSO-d 6)δ(ppm)：9.96(m，1H)，8.06(m，1H)，7.59(d，J＝7.9Hz，1H)，7.55(d，J＝8.2Hz，1H)，7.23(d，J＝8.2Hz，2H)，7.01(s，1H)，5.12(s，1H)，4.43(s，3H)，4.34-4.26(m，1H)，4.16(m，1H)，3.24(m，1H)，2.44(s，1H)，2.12(m，1H)，2.03-1.96(m，2H)，1.71(m，2H)，1.62(m，2H)，1.30(d，J＝6.9Hz，3H)，1.24(s，5H)，1.19(m，3H)，0.87-0.81(m，8H). According to the steps shown in Figure 6, using 3c and SMCC as starting materials, the synthesis method is the same as that of 4a, and a yellow solid is obtained with a yield of 63%. The molecular formula is C 30 H 41 N 5 O 7 , MS (ESI) m/z: 584 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 9.96 (m, 1H), 8.06 (m, 1H), 7.59 (d, J = 7.9 Hz, 1H), 7.55 (d, J = 8.2 Hz, 1H), 7.23 (d, J = 8.2 Hz, 2H), 7.01 (s, 1H), 5.12(s, 1H), 4.43(s, 3H), 4.34 to 4.26(m, 1H), 4.16(m, 1H), 3.24(m, 1H), 2.44(s, 1H), 2.12(m, 1H) , 2.03-1.96 (m, 2H), 1.71 (m, 2H), 1.62 (m, 2H), 1.30 (d, J = 6.9 Hz, 3H), 1.24 (s, 5H), 1.19 (m, 3H), 0.87-0.81 (m, 8H).
4f的合成Synthesis of 4f
根据图6步骤所示，以3d和SMCC为起始原料，合成方法同4a，得黄色固体，收率71％。分子式为C 32H 37N 5O 7.MS(ESI)m/z：604[M+H] +； 1H NMR(400MHz，DMSO-d 6)δ(ppm)：9.85(s，1H)，8.69(s，1H)，8.29(d，J＝7.3Hz，1H)，8.14-8.08(m，1H)，7.59(d，J＝8.2Hz，2H)，7.25(d，J＝3.9Hz，6H)，5.14(s，1H)， 4.44(s，3H)，3.87(m，2H)，3.78-3.72(m，1H)，3.61-3.53(m，1H)，3.20(d，J＝7.2Hz，2H)，3.03(m，2H)，2.90-2.82(m，1H)，2.63-2.57(m，1H)，2.42(s，1H)，2.09-1.99(m，2H)，1.67(m，2H)，1.60(m，1H)，1.51(m，1H)，0.88(m，3H). According to the steps shown in Figure 6, using 3d and SMCC as starting materials, the synthesis method is the same as that of 4a, to obtain a yellow solid with a yield of 71%. The molecular formula is C 32 H 37 N 5 O 7 .MS (ESI) m/z: 604 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 9.85 (s, 1H), 8.69(s, 1H), 8.29(d, J=7.3Hz, 1H), 8.14-8.08(m, 1H), 7.59(d, J=8.2Hz, 2H), 7.25(d, J=3.9Hz, 6H ), 5.14 (s, 1H), 4.44 (s, 3H), 3.87 (m, 2H), 3.78-3.72 (m, 1H), 3.61-3.53 (m, 1H), 3.20 (d, J=7.2Hz, 2H), 3.03(m, 2H), 2.90-2.82(m, 1H), 2.63-2.57(m, 1H), 2.42(s, 1H), 2.09-1.99(m, 2H), 1.67(m, 2H) , 1.60 (m, 1H), 1.51 (m, 1H), 0.88 (m, 3H).
4g的合成4g synthesis
根据图6步骤所示，以3e和SMCC为起始原料，合成方法同4a，得黄色固体，收率72％。C 34H 40N 6O 8，MS(ESI)m/z：661[M+H] +； 1H NMR(400MHz，DMSO-d 6)δ(ppm)：9.77(s，1H)，8.38(t，J＝5.8Hz，1H)，8.18(d，J＝7.9Hz，1H)，8.04-7.98(m，2H)，7.57(d，J＝8.2Hz，2H)，7.26(d，J＝3.9Hz，4H)，7.19(q，J＝4.3Hz，1H)，7.01(s，2H)，5.11(t，J＝5.7Hz，1H)，4.54-4.47(m，2H)，4.44(d，J＝5.6Hz，2H)，3.88(m，2H)，3.83-3.74(m，2H)，3.66(d，J＝5.8Hz，2H)，3.23(s，2H)，3.11-3.06(m，1H)，2.87-2.81(m，1H)，2.11(m，1H)，1.78-1.73(m，2H)，1.65-1.60(m，2H)，1.26(m，3H)，0.90(m，3H). According to the steps shown in Figure 6, using 3e and SMCC as starting materials, the synthesis method is the same as that of 4a, and a yellow solid is obtained with a yield of 72%. C 34 H 40 N 6 O 8 , MS (ESI) m/z: 661 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 9.77 (s, 1H), 8.38 ( t, J = 5.8 Hz, 1H), 8.18 (d, J = 7.9 Hz, 1H), 8.04-7.98 (m, 2H), 7.57 (d, J = 8.2 Hz, 2H), 7.26 (d, J = 3.9 Hz, 4H), 7.19 (q, J = 4.3 Hz, 1H), 7.01 (s, 2H), 5.11 (t, J = 5.7 Hz, 1H), 4.54-4.47 (m, 2H), 4.44 (d, J =5.6Hz, 2H), 3.88(m, 2H), 3.83-3.74(m, 2H), 3.66(d, J=5.8Hz, 2H), 3.23(s, 2H), 3.11-3.06(m, 1H) , 2.87-2.81 (m, 1H), 2.11 (m, 1H), 1.78-1.73 (m, 2H), 1.65-1.60 (m, 2H), 1.26 (m, 3H), 0.90 (m, 3H).
4h的合成4h synthesis
根据图6步骤所示，以3f和SMCC为起始原料，合成方法同4a，得黄色固体，收率68％。分子式为C 38H 48N 6O 8，MS(ESI)m/z：717[M+H] +； 1H NMR(400MHz，DMSO-d 6)δ(ppm)：9.77(s，1H)，8.22-8.13(m，2H)，8.00-7.94(m，2H)，7.57(d，J＝8.3Hz，2H)，7.26-7.19(m，6H)，7.01(m，2H)，5.11(t，J＝5.6Hz，1H)，4.54(m，1H)，4.44(m，2H)，4.31(m，1H)，3.87(d，J＝6.0Hz，2H)，3.69(m，1H)，3.54(m，1H)，3.24(d，J＝7.1Hz，2H)，3.04(m，1H)，2.80(m，1H)，2.08(m，2H)，1.70(m，2H)，1.64-1.58(m，3H)，1.54(m，2H)，1.26-1.16(m，3H)，0.88(m，8H). According to the steps shown in Figure 6, using 3f and SMCC as starting materials, the synthesis method is the same as that of 4a, and a yellow solid is obtained with a yield of 68%. The molecular formula is C 38 H 48 N 6 O 8 , MS (ESI) m/z: 717 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 9.77 (s, 1H), 8.22-8.13(m, 2H), 8.00-7.94(m, 2H), 7.57(d, J=8.3Hz, 2H), 7.26-7.19(m, 6H), 7.01(m, 2H), 5.11(t, J = 5.6 Hz, 1H), 4.54 (m, 1H), 4.44 (m, 2H), 4.31 (m, 1H), 3.87 (d, J = 6.0 Hz, 2H), 3.69 (m, 1H), 3.54 ( m, 1H), 3.24 (d, J = 7.1 Hz, 2H), 3.04 (m, 1H), 2.80 (m, 1H), 2.08 (m, 2H), 1.70 (m, 2H), 1.64-1.58 (m , 3H), 1.54 (m, 2H), 1.26-1.16 (m, 3H), 0.88 (m, 8H).
4i的合成Synthesis of 4i
根据图6步骤所示，以3b和MC-OSu为起始原料，合成方法同4a，得黄色固体，收率70％。分子式C 25H 34N 4O 6，MS(ESI)m/z：487[M+H] +； 1H NMR(400MHz，DMSO-d 6)δ(ppm)：9.84(s，1H)，8.13(d，J＝7.0Hz，1H)，7.81(d，J＝8.6Hz，1H)，7.53(d，J＝8.1Hz，2H)，7.23(d，J＝8.1Hz，2H)，7.00(s，2H)，5.08(s，1H)，4.40(m，3H)，4.17(t，J＝7.8Hz，1H)，2.14(m，2H)，1.96(m，1H)，1.49(m，4H)，1.35-1.14(m，7H)，0.84(m，6H). According to the steps shown in Figure 6, with 3b and MC-OSu as starting materials, the synthesis method is the same as that of 4a to obtain a yellow solid with a yield of 70%. Molecular formula C 25 H 34 N 4 O 6 , MS (ESI) m/z: 487[M+H] + ; 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 9.84 (s, 1H), 8.13 (d, J=7.0Hz, 1H), 7.81(d, J=8.6Hz, 1H), 7.53(d, J=8.1Hz, 2H), 7.23(d, J=8.1Hz, 2H), 7.00(s , 2H), 5.08 (s, 1H), 4.40 (m, 3H), 4.17 (t, J = 7.8 Hz, 1H), 2.14 (m, 2H), 1.96 (m, 1H), 1.49 (m, 4H) , 1.35-1.14 (m, 7H), 0.84 (m, 6H).
4j的合成Synthesis of 4j
根据图6步骤所示，以3c和MC-OSu为起始原料，合成方法同4a，得黄色固体，收率71％。分子式为C 28H 39N 5O 7，MS(ESI)m/z：558[M+H] +； 1H NMR (400MHz，DMSO-d 6)δ(ppm)：9.89(m，1H)，8.05(t，J＝6.8Hz，1H)，7.59(d，J＝8.2Hz，1H)，7.54(d，J＝8.2Hz，1H)，7.24(d，J＝8.0Hz，2H)，7.00(s，1H)，5.11(s，1H)，4.43(s，2H)，4.42-4.37(m，1H)，4.33(t，J＝7.1Hz，1H)，4.16(m，1H)，2.46(s，1H)，2.09(m，2H)，2.00(m，2H)，1.46(m，2H)，1.43-1.39(m，2H)，1.30(m，4H)，1.24(s，4H)，1.18(m，3H)，0.88-0.82(m，6H). According to the steps shown in Figure 6, using 3c and MC-OSu as starting materials, the synthesis method is the same as that of 4a, and a yellow solid is obtained with a yield of 71%. The molecular formula is C 28 H 39 N 5 O 7 , MS (ESI) m/z: 558[M+H] + ; 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 9.89 (m, 1H), 8.05 (t, J = 6.8 Hz, 1H), 7.59 (d, J = 8.2 Hz, 1H), 7.54 (d, J = 8.2 Hz, 1H), 7.24 (d, J = 8.0 Hz, 2H), 7.00 ( s, 1H), 5.11(s, 1H), 4.43(s, 2H), 4.42-4.37(m, 1H), 4.33(t, J=7.1Hz, 1H), 4.16(m, 1H), 2.46(s , 1H), 2.09 (m, 2H), 2.00 (m, 2H), 1.46 (m, 2H), 1.43-1.39 (m, 2H), 1.30 (m, 4H), 1.24 (s, 4H), 1.18 ( m, 3H), 0.88-0.82 (m, 6H).
4k的合成4k synthesis
根据图6步骤所示，以3d和MC-OSu为起始原料，合成方法同4a，得黄色固体，收率52％。分子式为C 30H 35N 5O 7，MS(ESI)m/z：578[M+H] +； 1H NMR(400MHz，DMSO-d 6)δ(ppm)：9.87(s，1H)，8.51(s，1H)，8.26(d，J＝8.0Hz，1H)，8.14(s，1H)，7.59(m，2H)，7.29-7.18(m，6H)，5.13(s，1H)，4.53-4.43(m，2H)，3.95-3.86(m，2H)，3.83-3.74(m，1H)，3.68(m，2H)，3.65-3.58(m，1H)，3.43(s，2H)，3.20(m，1H)，3.12-3.05(m，1H)，2.98(m，1H)，2.91-2.82(m，1H)，2.12(m，2H)，1.54-1.42(m，3H)，1.32-1.20(m，3H). According to the steps shown in Figure 6, using 3d and MC-OSu as starting materials, the synthesis method is the same as that of 4a, and a yellow solid is obtained with a yield of 52%. The molecular formula is C 30 H 35 N 5 O 7 , MS (ESI) m/z: 578 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 9.87 (s, 1H), 8.51(s, 1H), 8.26(d, J=8.0Hz, 1H), 8.14(s, 1H), 7.59(m, 2H), 7.29-7.18(m, 6H), 5.13(s, 1H), 4.53 -4.43(m, 2H), 3.95-3.86(m, 2H), 3.83-3.74(m, 1H), 3.68(m, 2H), 3.65-3.58(m, 1H), 3.43(s, 2H), 3.20 (m, 1H), 3.12-3.05 (m, 1H), 2.98 (m, 1H), 2.91-2.82 (m, 1H), 2.12 (m, 2H), 1.54-1.42 (m, 3H), 1.32-1.20 (m, 3H).
4l的合成Synthesis of 4l
根据图6步骤所示，以3e和MC-OSu为起始原料，合成方法同4a，得黄色固体，收率51％。分子式为C 32H 38N 6O 8，MS(ESI)m/z：635[M+H] +； 1H NMR(400MHz，DMSO-d 6)δ(ppm)：9.87(s，1H)，8.53(s，1H)，8.28(t，J＝8.5Hz，1H)，8.17-8.11(m，1H)，7.59(m，2H)，7.26(t，J＝6.0Hz，5H)，7.18(m，1H)，5.14(s，1H)，4.54-4.29(m，3H)，3.97-3.75(m，3H)，3.66(m，3H)，3.43(s，2H)，3.20(d，J＝4.0Hz，1H)，3.13-2.94(m，2H)，2.85(m，1H)，2.17-1.98(m，2H)，1.45(m，4H)，1.24(m，4H). According to the steps shown in Figure 6, using 3e and MC-OSu as starting materials, the synthesis method is the same as that of 4a, and a yellow solid is obtained with a yield of 51%. The molecular formula is C 32 H 38 N 6 O 8 , MS (ESI) m/z: 635[M+H] + ; 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 9.87 (s, 1H), 8.53(s, 1H), 8.28(t, J=8.5Hz, 1H), 8.17-8.11(m, 1H), 7.59(m, 2H), 7.26(t, J=6.0Hz, 5H), 7.18(m , 1H), 5.14(s, 1H), 4.54-4.29(m, 3H), 3.97-3.75(m, 3H), 3.66(m, 3H), 3.43(s, 2H), 3.20(d, J=4.0 Hz, 1H), 3.13-2.94 (m, 2H), 2.85 (m, 1H), 2.17-1.98 (m, 2H), 1.45 (m, 4H), 1.24 (m, 4H).
4m的合成4m synthesis
根据图6步骤所示，以3f和MC-OSu为起始原料，合成方法同4a，得黄色固体，收率62％。分子式为C 36H 46N 6O 8，MS(ESI)m/z：691[M+H] +； 1H NMR(400MHz，DMSO-d 6)δ(ppm)：9.77(s，1H)，8.16(t，J＝6.5Hz，2H)，8.02(m，2H)，7.60-7.55(m，2H)，7.27-7.22(m，6H)，7.18(dd，J＝6.1Hz，2.6Hz，1H)，7.01-6.99(m，1H)，5.11(s，1H)，4.54(m，1H)，4.44(s，2H)，4.30(m，1H)，3.90-3.85(m，2H)，3.71(m，1H)，3.56(m，1H)，3.05(m，1H)，2.79(m，1H)，2.08(d，J＝5.9Hz，2H)，1.62(s，1H)，1.55-1.44(m，7H)，1.22-1.15(m，3H)，0.88(m，7H). According to the steps shown in Figure 6, using 3f and MC-OSu as starting materials, the synthesis method is the same as that of 4a, and a yellow solid is obtained with a yield of 62%. The molecular formula is C 36 H 46 N 6 O 8 , MS (ESI) m/z: 691[M+H] + ; 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 9.77 (s, 1H), 8.16(t, J=6.5Hz, 2H), 8.02(m, 2H), 7.60-7.55(m, 2H), 7.27-7.22(m, 6H), 7.18(dd, J=6.1Hz, 2.6Hz, 1H ), 7.01-6.99(m, 1H), 5.11(s, 1H), 4.54(m, 1H), 4.44(s, 2H), 4.30(m, 1H), 3.90-3.85(m, 2H), 3.71( m, 1H), 3.56 (m, 1H), 3.05 (m, 1H), 2.79 (m, 1H), 2.08 (d, J = 5.9 Hz, 2H), 1.62 (s, 1H), 1.55-1.44 (m , 7H), 1.22-1.15 (m, 3H), 0.88 (m, 7H).
5a的合成Synthesis of 5a
根据图6步骤所示，将4a(0.69g，1.2mmol)溶于12mL DMF中，溶解 后加入(PNP) 2CO(1.1g，3.6mmol)和DIEA 0.5mL，室温搅拌下过夜。反应完毕后，旋干溶剂，硅胶柱分离纯化，分离条件为二氯甲烷∶甲醇＝20∶1，得产物为微黄色固体0.42g，收率为47％，mp：156-158℃。分子式为C 35H 43N 7O 11，HRMS[M+H] +(ESI)：found 738.3088，calcd 738.3093. 1H NMR(500MHz，DMSO-d 6)δ(ppm)：10.09(s，1H)，8.32(d，J＝9.1Hz，2H)，8.14(d，J＝7.1Hz，1H)，7.83(d，J＝8.6Hz，1H)，7.66(d，J＝8.4Hz，2H)，7.57(d，J＝9.1Hz，2H)，7.41(d，J＝8.6Hz，2H)，7.01(s，2H)，6.00(m，1H)，5.44(s，2H)，5.24(s，2H)，4.39(m，1H)，4.20(m，1H)，3.37(m，2H)，2.99(m，2H)，2.22-2.09(m，2H)，1.96(m，1H)，1.72(m，1H)，1.65-1.56(m，1H)，1.52-1.43(m，6H)，1.22-1.15(m，2H)，0.87-0.82(m，6H). According to the steps shown in Figure 6, 4a (0.69g, 1.2mmol) was dissolved in 12mL DMF, after dissolution, (PNP) 2 CO (1.1g, 3.6mmol) and DIEA 0.5mL were added, and stirred at room temperature overnight. After the reaction is completed, the solvent is spin-dried and the silica gel column is used for separation and purification. The separation condition is dichloromethane:methanol=20:1, and the product is 0.42 g of light yellow solid, the yield is 47%, mp: 156-158°C. The molecular formula is C 35 H 43 N 7 O 11 , HRMS[M+H] + (ESI): found 738.3088, calcd 738.3093. 1 H NMR (500MHz, DMSO-d 6 ) δ (ppm): 10.09 (s, 1H) , 8.32 (d, J = 9.1Hz, 2H), 8.14 (d, J = 7.1Hz, 1H), 7.83 (d, J = 8.6Hz, 1H), 7.66 (d, J = 8.4Hz, 2H), 7.57 (d, J = 9.1 Hz, 2H), 7.41 (d, J = 8.6 Hz, 2H), 7.01 (s, 2H), 6.00 (m, 1H), 5.44 (s, 2H), 5.24 (s, 2H) , 4.39 (m, 1H), 4.20 (m, 1H), 3.37 (m, 2H), 2.99 (m, 2H), 2.22-2.09 (m, 2H), 1.96 (m, 1H), 1.72 (m, 1H) ), 1.65-1.56 (m, 1H), 1.52-1.43 (m, 6H), 1.22-1.15 (m, 2H), 0.87-0.82 (m, 6H).
5b的合成Synthesis of 5b
根据图6步骤所示，以4b和(PNP) 2CO为起始原料，合成方法同5a，得黄色固体，收率66％，mp：171-173℃。分子式为C 37H 45N 7O 11，HRMS(ESI)：[M+H] +found 764.3243，calcd 764.3249. 1H NMR(400MHz，DMSO-d 6)δ(ppm)：10.12(s，1H)，8.38(d，J＝8.5Hz，1H)，8.33-8.31(m，2H)，8.29-8.25(m，1H)，8.13-8.08(m，1H)，7.99(d，J＝8.3Hz，2H)，7.65(s，2H)，7.59-7.56(m，2H)，7.47(d，J＝8.3Hz，2H)，7.42(d，J＝8.4Hz，2H)，7.22(s，2H)，6.88(d，J＝8.7Hz，1H)，6.00(m，2H)，5.43(s，2H)，5.25(s，2H)，4.44-4.36(m，2H)，3.07-3.02(m，2H)，2.97(m，2H)，2.81(s，1H)，2.16(m，1H)，1.72(d，J＝7.2Hz，1H)，1.64(m，1H)，1.51-1.44(m，2H)，1.41(m，2H)，0.96(m，6H). According to the steps shown in Figure 6, using 4b and (PNP) 2 CO as starting materials, the synthesis method is the same as that of 5a to obtain a yellow solid with a yield of 66%, mp: 171-173°C. The molecular formula is C 37 H 45 N 7 O 11 , HRMS (ESI): [M+H] + found 764.3243, calcd 764.3249. 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 10.12 (s, 1H) , 8.38(d, J=8.5Hz, 1H), 8.33-8.31(m, 2H), 8.29-8.25(m, 1H), 8.13-8.08(m, 1H), 7.99(d, J=8.3Hz, 2H ), 7.65 (s, 2H), 7.59-7.56 (m, 2H), 7.47 (d, J = 8.3 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.22 (s, 2H), 6.88 (d, J=8.7Hz, 1H), 6.00 (m, 2H), 5.43 (s, 2H), 5.25 (s, 2H), 4.44-4.36 (m, 2H), 3.07-3.02 (m, 2H), 2.97(m, 2H), 2.81(s, 1H), 2.16(m, 1H), 1.72(d, J=7.2Hz, 1H), 1.64(m, 1H), 1.51-1.44(m, 2H), 1.41 (m, 2H), 0.96 (m, 6H).
5c的合成Synthesis of 5c
根据图6步骤所示，以4c和(PNP) 2CO为起始原料，合成方法同5a，得黄色固体，收率59％，mp：148-152℃。分子式为C 36H 37N 7O 11，HRMS(ESI)：[M+H] +found 744.2620，calcd 744.9420. 1H NMR(400MHz，DMSO-d 6)δ(ppm)：10.09(d，J＝10.6Hz，1H)，8.32(d，J＝8.6Hz，1H)，8.12-8.06(m，1H)，7.72(d，J＝8.9Hz，1H)，7.66(d，J＝8.1Hz，2H)，7.58(d，J＝8.7Hz，2H)，7.42(d，J＝8.1Hz，2H)，7.02(s，2H)，5.99(d，J＝6.1Hz，1H)，5.42(s，2H)，5.25(s，1H)，4.39(q，J＝7.1Hz，1H)，4.17(t，J＝7.7Hz，1H)，3.25(d，J＝7.0Hz，2H)，3.05-2.94(m，2H)，2.23(m，1H)，1.99(m，1H)，1.72-1.60(m，5H)，1.28(m，2H)，0.84(m，6H). According to the steps shown in Figure 6, with 4c and (PNP) 2 CO as starting materials, the synthesis method is the same as that of 5a to obtain a yellow solid with a yield of 59%, mp: 148-152°C. The molecular formula is C 36 H 37 N 7 O 11 , HRMS (ESI): [M+H] + found 744.2620, calcd 744.9420. 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 10.09 (d, J= 10.6Hz, 1H), 8.32(d, J=8.6Hz, 1H), 8.12-8.06(m, 1H), 7.72(d, J=8.9Hz, 1H), 7.66(d, J=8.1Hz, 2H) , 7.58(d, J=8.7Hz, 2H), 7.42(d, J=8.1Hz, 2H), 7.02(s, 2H), 5.99(d, J=6.1Hz, 1H), 5.42(s, 2H) , 5.25 (s, 1H), 4.39 (q, J = 7.1 Hz, 1H), 4.17 (t, J = 7.7 Hz, 1H), 3.25 (d, J = 7.0 Hz, 2H), 3.05-2.94 (m, 2H), 2.23 (m, 1H), 1.99 (m, 1H), 1.72-1.60 (m, 5H), 1.28 (m, 2H), 0.84 (m, 6H).
5d的合成Synthesis of 5d
根据图6步骤所示，以4d和(PNP) 2CO为起始原料，合成方法同5a，得浅黄色固体，收率65％，mp：159-161℃。分子式为C 34H 39N 5O 10，HRMS(ESI)：[M+H] +found 678.2753，calcd 678.2769. 1H NMR(500MHz，DMSO-d 6)δ(ppm)：10.04(s，1H)，8.34-8.31(m，2H)，8.17(d，J＝6.8Hz，1H)，7.73(d，J＝8.6Hz，1H)，7.66-7.63(m，2H)，7.59-7.56(m，2H)，7.43-7.40(m，2H)，7.02(s，2H)，5.24(s，2H)，4.38(t，J＝7.0Hz，1H)，4.15(m，1H)，3.24(d，J＝7.1Hz，2H)，2.23(m，1H)，1.97(m，1H)，1.75-1.67(m，2H)，1.62(m，2H)，1.31(m，4H)，1.26-1.23(m，3H)，0.85(m，7H). According to the steps shown in Figure 6, using 4d and (PNP) 2 CO as starting materials, the synthesis method is the same as that of 5a to obtain a pale yellow solid with a yield of 65%, mp: 159-161°C. The molecular formula is C 34 H 39 N 5 O 10 , HRMS(ESI): [M+H] + found 678.2753, calcd 678.2769. 1 H NMR(500MHz, DMSO-d 6 )δ(ppm): 10.04(s, 1H) , 8.34-8.31 (m, 2H), 8.17 (d, J = 6.8 Hz, 1H), 7.73 (d, J = 8.6 Hz, 1H), 7.66-7.63 (m, 2H), 7.59-7.56 (m, 2H) ), 7.43-7.40 (m, 2H), 7.02 (s, 2H), 5.24 (s, 2H), 4.38 (t, J = 7.0 Hz, 1H), 4.15 (m, 1H), 3.24 (d, J = 7.1Hz, 2H), 2.23 (m, 1H), 1.97 (m, 1H), 1.75-1.67 (m, 2H), 1.62 (m, 2H), 1.31 (m, 4H), 1.26-1.23 (m, 3H) ), 0.85 (m, 7H).
5e的合成Synthesis of 5e
根据图6步骤所示，以4e和(PNP) 2CO为起始原料，合成方法同5a，得浅黄色固体，收率54％，mp：155-156℃。分子式为C 37H 44N 6O 11，HRMS(ESI)：[M+H] +found 749.3128，calcd 749.3141. 1H NMR(400MHz，DMSO-d 6)δ(ppm)：10.03(s，1H)，8.32(d，J＝8.8Hz，1H)，8.21(d，J＝7.0Hz，1H)，7.99(d，J＝7.4Hz，1H)，7.64(d，J＝8.2Hz，1H)，7.58(m，2H)，7.42(d，J＝8.1Hz，1H)，7.01(s，1H)，5.25(s，1H)，4.41(m，2H)，4.28(m，1H)，4.23-4.16(m，1H)，3.24(d，J＝7.2Hz，1H)，2.60(s，1H)，2.21-2.08(m，1H)，1.98(m，2H)，1.77-1.56(m，4H)，1.48(s，1H)，1.37-1.22(m，8H)，1.19(m，3H)，1.13-1.03(m，2H)，0.86(m，7H). According to the steps shown in Figure 6, 4e and (PNP) 2 CO were used as starting materials and the synthesis method was the same as that of 5a to obtain a light yellow solid with a yield of 54%, mp: 155-156°C. The molecular formula is C 37 H 44 N 6 O 11 , HRMS(ESI): [M+H] + found 749.3128, calcd 749.3141. 1 H NMR(400MHz, DMSO-d 6 )δ(ppm): 10.03(s, 1H) , 8.32 (d, J = 8.8 Hz, 1H), 8.21 (d, J = 7.0 Hz, 1H), 7.99 (d, J = 7.4 Hz, 1H), 7.64 (d, J = 8.2 Hz, 1H), 7.58 (m, 2H), 7.42 (d, J = 8.1 Hz, 1H), 7.01 (s, 1H), 5.25 (s, 1H), 4.41 (m, 2H), 4.28 (m, 1H), 4.23-4.16 ( m, 1H), 3.24 (d, J = 7.2 Hz, 1H), 2.60 (s, 1H), 2.21-2.08 (m, 1H), 1.98 (m, 2H), 1.77-1.56 (m, 4H), 1.48 (s, 1H), 1.37-1.22 (m, 8H), 1.19 (m, 3H), 1.13-1.03 (m, 2H), 0.86 (m, 7H).
5f的合成Synthesis of 5f
根据图6步骤所示，以4f和(PNP) 2CO为起始原料，合成方法同5a，得浅黄色固体，收率51％，mp：131-133℃。分子式为C 39H 40N 6O 11，HRMS(ESI)：[M+H] +found 769.2835，calcd 769.2828. 1H NMR(600MHz，DMSO-d 6)δ(ppm)：9.84(d，J＝8.7Hz，1H)，8.46(m，1H)，8.32(d，J＝8.6Hz，1H)，8.15(d，J＝7.7Hz，1H)，7.94(t，J＝6.0Hz，1H)，7.67(m，2H)，7.57(d，J＝8.6Hz，2H)，7.42(d，J＝8.5Hz，2H)，7.25(d，J＝5.0Hz，4H)，7.19(s，1H)，7.00(s，1H)，5.25(s，1H)，4.48(s，1H)，3.92(m，1H)，3.88-3.83(m，1H)，3.73(m，1H)，3.56(m，1H)，3.22(t，J＝6.7Hz，2H)，3.06(m，1H)，2.86-2.81(m，1H)，2.06(m，1H)，1.63(m，4H)，1.49(s，1H)，1.26-1.18(m，3H)，1.09(m，1H)，0.87(m，3H). According to the steps shown in Figure 6, using 4f and (PNP) 2 CO as starting materials, the synthesis method is the same as that of 5a to obtain a pale yellow solid, with a yield of 51%, mp: 131-133°C. The molecular formula is C 39 H 40 N 6 O 11 , HRMS (ESI): [M+H] + found 769.2835, calcd 769.2828. 1 H NMR (600MHz, DMSO-d 6 ) δ (ppm): 9.84 (d, J= 8.7 Hz, 1H), 8.46 (m, 1H), 8.32 (d, J = 8.6 Hz, 1H), 8.15 (d, J = 7.7 Hz, 1H), 7.94 (t, J = 6.0 Hz, 1H), 7.67 (m, 2H), 7.57 (d, J = 8.6 Hz, 2H), 7.42 (d, J = 8.5 Hz, 2H), 7.25 (d, J = 5.0 Hz, 4H), 7.19 (s, 1H), 7.00 (s, 1H), 5.25 (s, 1H), 4.48 (s, 1H), 3.92 (m, 1H), 3.88-3.83 (m, 1H), 3.73 (m, 1H), 3.56 (m, 1H), 3.22(t, J=6.7Hz, 2H), 3.06(m, 1H), 2.86-2.81(m, 1H), 2.06(m, 1H), 1.63(m, 4H), 1.49(s, 1H), 1.26 -1.18 (m, 3H), 1.09 (m, 1H), 0.87 (m, 3H).
5g的合成Synthesis of 5g
根据图6步骤所示，以4g和(PNP) 2CO为起始原料，合成方法同5a，得浅黄色固体，收率59％，mp：162-164℃。分子式为C 41H 43N 7O 12，HRMS(ESI)：[M+H] +found 826.3052，calcd 826.3043. 1H NMR(500MHz，DMSO-d 6)δ(ppm)： 9.95(s，1H)，8.43(t，J＝5.9Hz，1H)，8.34-8.31(m，2H)，8.22(d，J＝8.0Hz，1H)，8.03(m，2H)，7.69-7.67(m，2H)，7.60-7.56(m，2H)，7.46-7.42(m，2H)，7.27(d，J＝4.4Hz，4H)，7.19(m，1H)，7.01(s，2H)，5.26(s，2H)，4.51(m，1H)，3.90(m，2H)，3.77(m，1H)，3.68-3.58(m，3H)，3.23(d，J＝7.1Hz，2H)，3.08(m，1H)，2.84(m，1H)，2.11(m，1H)，1.78-1.71(m，2H)，1.65-1.58(m，2H)，1.51(m，1H)，1.31-1.21(m，2H)，0.93-0.85(m，2H). According to the steps shown in Figure 6, with 4g and (PNP) 2 CO as starting materials, the synthesis method is the same as that of 5a to obtain a pale yellow solid, with a yield of 59%, mp: 162-164°C. The molecular formula is C 41 H 43 N 7 O 12 , HRMS(ESI): [M+H] + found 826.3052, calcd 826.3043. 1 H NMR(500MHz, DMSO-d 6 )δ(ppm): 9.95(s, 1H) , 8.43(t, J=5.9Hz, 1H), 8.34-8.31(m, 2H), 8.22(d, J=8.0Hz, 1H), 8.03(m, 2H), 7.69-7.67(m, 2H), 7.60-7.56(m, 2H), 7.46-7.42(m, 2H), 7.27(d, J=4.4Hz, 4H), 7.19(m, 1H), 7.01(s, 2H), 5.26(s, 2H) , 4.51 (m, 1H), 3.90 (m, 2H), 3.77 (m, 1H), 3.68-3.58 (m, 3H), 3.23 (d, J = 7.1 Hz, 2H), 3.08 (m, 1H), 2.84 (m, 1H), 2.11 (m, 1H), 1.78-1.71 (m, 2H), 1.65-1.58 (m, 2H), 1.51 (m, 1H), 1.31-1.21 (m, 2H), 0.93- 0.85(m, 2H).
5h的合成5h synthesis
根据图6步骤所示，以4h和(PNP) 2CO为起始原料，合成方法同5a，得浅黄色固体，收率59％，mp：126-127℃。分子式为C 45H 51N 7O 12，HRMS(ESI)：[M+H] +found 882.3698，calcd 882.3629. 1H NMR(400MHz，DMSO-d 6)δ(ppm)：9.93(s，1H)，8.32(d，J＝8.8Hz，2H)，8.19(d，J＝7.1Hz，2H)，8.00-7.92(m，2H)，7.67(d，J＝8.2Hz，2H)，7.57(d，J＝8.8Hz，2H)，7.43(d，J＝8.2Hz，2H)，7.25-7.17(m，5H)，7.01(s，2H)，5.26(s，2H)，4.54(m，1H)，4.30(q，J＝7.9，7.5Hz，1H)，3.89(d，J＝6.0Hz，2H)，3.69(m，1H)，3.54(m，1H)，3.23(d，J＝7.0Hz，2H)，3.04(m，1H)，2.80(m，1H)，2.08(m，1H)，1.70(m，2H)，1.60(m，3H)，1.53(m，2H)，1.23(m，2H)，1.10(t，J＝7.0Hz，1H)，0.91(m，4H)，0.86(m，4H). According to the steps shown in Figure 6, with 4h and (PNP) 2 CO as starting materials, the synthesis method is the same as that of 5a to obtain a pale yellow solid with a yield of 59%, mp: 126-127°C. The molecular formula is C 45 H 51 N 7 O 12 , HRMS(ESI): [M+H] + found 882.3698, calcd 882.3629. 1 H NMR(400MHz, DMSO-d 6 )δ(ppm): 9.93(s, 1H) , 8.32 (d, J = 8.8 Hz, 2H), 8.19 (d, J = 7.1 Hz, 2H), 8.00-7.92 (m, 2H), 7.67 (d, J = 8.2 Hz, 2H), 7.57 (d, J = 8.8 Hz, 2H), 7.43 (d, J = 8.2 Hz, 2H), 7.25-7.17 (m, 5H), 7.01 (s, 2H), 5.26 (s, 2H), 4.54 (m, 1H), 4.30 (q, J = 7.9, 7.5 Hz, 1H), 3.89 (d, J = 6.0 Hz, 2H), 3.69 (m, 1H), 3.54 (m, 1H), 3.23 (d, J = 7.0 Hz, 2H) ), 3.04 (m, 1H), 2.80 (m, 1H), 2.08 (m, 1H), 1.70 (m, 2H), 1.60 (m, 3H), 1.53 (m, 2H), 1.23 (m, 2H) , 1.10 (t, J = 7.0 Hz, 1H), 0.91 (m, 4H), 0.86 (m, 4H).
5i的合成Synthesis of 5i
根据图6步骤所示，以4i和(PNP) 2CO为起始原料，合成方法同5a，得浅黄色固体，收率63％，mp：155-157℃。分子式为C 32H 37N 5O 10，HRMS(ESI)：[M+H] +found 652.2598，calcd 652.2613. 1H NMR(400MHz，DMSO-d 6)δ(ppm)：10.02(s，1H)，8.32(d，J＝8.8Hz，2H)，8.19(d，J＝6.9Hz，1H)，7.82(d，J＝8.6Hz，1H)，7.65(d，J＝8.2Hz，2H)，7.58(d，J＝9.0Hz，2H)，7.42(d，J＝8.2Hz，2H)，7.01(s，2H)，5.25(s，2H)，4.40(m，1H)，4.18(t，J＝7.7Hz，1H)，2.15(m，2H)，1.96(m，1H)，1.49(m，4H)，1.32(m，4H)，1.19(m，2H)，0.86(m，7H). According to the steps shown in Figure 6, using 4i and (PNP) 2 CO as starting materials, the synthesis method is the same as that of 5a to obtain a pale yellow solid, with a yield of 63%, mp: 155-157°C. The molecular formula is C 32 H 37 N 5 O 10 , HRMS(ESI): [M+H] + found 652.2598, calcd 652.2613. 1 H NMR(400MHz, DMSO-d 6 )δ(ppm): 10.02(s, 1H) , 8.32 (d, J = 8.8 Hz, 2H), 8.19 (d, J = 6.9 Hz, 1H), 7.82 (d, J = 8.6 Hz, 1H), 7.65 (d, J = 8.2 Hz, 2H), 7.58 (d, J = 9.0 Hz, 2H), 7.42 (d, J = 8.2 Hz, 2H), 7.01 (s, 2H), 5.25 (s, 2H), 4.40 (m, 1H), 4.18 (t, J = 7.7Hz, 1H), 2.15 (m, 2H), 1.96 (m, 1H), 1.49 (m, 4H), 1.32 (m, 4H), 1.19 (m, 2H), 0.86 (m, 7H).
5j的合成Synthesis of 5j
根据图6步骤所示，以4j和(PNP) 2CO为起始原料，合成方法同5a，得浅黄色固体，收率60％，mp：134-135℃。分子式为C 35H 42N 6O 11，HRMS(ESI)：[M+H] +found 723.2980，calcd 723.29843. 1H NMR(600MHz，DMSO-d 6)δ(ppm)：10.00(s，1H)，8.30一8.27(m，1H)，8.15(d，J＝6.8Hz，1H)，7.99(d，J＝7.4Hz，1H)，7.67-7.60(m，2H)，7.54(d，J＝8.7Hz，1H)，7.39(m，1H)，6.96(m，1H)，5.22(m，1H)， 4.33(m，2H)，4.18-4.08(m，1H)，3.59(m，1H)，3.11(m，1H)，2.10-2.03(m，2H)，1.96(m，1H)，1.44(q，J＝7.6Hz，3H)，1.28(d，J＝6.5Hz，2H)，1.26-1.20(m，11H)，1.15(d，J＝7.5Hz，3H)，0.86-0.79(m，5H). According to the steps shown in Figure 6, using 4j and (PNP) 2 CO as starting materials, the synthesis method is the same as that of 5a to obtain a pale yellow solid, with a yield of 60%, mp: 134-135°C. The molecular formula is C 35 H 42 N 6 O 11 , HRMS (ESI): [M+H] + found 723.2980, calcd 723.29843. 1 H NMR (600MHz, DMSO-d 6 ) δ (ppm): 10.00 (s, 1H) , 8.30-8.27 (m, 1H), 8.15 (d, J = 6.8 Hz, 1H), 7.99 (d, J = 7.4 Hz, 1H), 7.67-7.60 (m, 2H), 7.54 (d, J = 8.7 Hz, 1H), 7.39 (m, 1H), 6.96 (m, 1H), 5.22 (m, 1H), 4.33 (m, 2H), 4.18-4.08 (m, 1H), 3.59 (m, 1H), 3.11 (m, 1H), 2.10-2.03 (m, 2H), 1.96 (m, 1H), 1.44 (q, J = 7.6 Hz, 3H), 1.28 (d, J = 6.5 Hz, 2H), 1.26-1.20 ( m, 11H), 1.15 (d, J = 7.5 Hz, 3H), 0.86-0.79 (m, 5H).
5k的合成5k synthesis
根据图6步骤所示，以4k和(PNP) 2CO为起始原料，得浅黄色固体，收率63.2％，mp：138-141℃。分子式为C 37H 38N 6O 11，HRMS(ESI)：[M+H] +found 743.2655，calcd 743.2671. 1H NMR(600MHz，DMSO-d 6)δ(ppm)：9.84(s，1H)，8.43(t，J＝5.8Hz，1H)，8.30-8.26(m，2H)，8.20-8.16(m，1H)，8.00-7.96(m，1H)，7.66(m，2H)，7.54(d，J＝9.0Hz，2H)，7.39(d，J＝8.3Hz，2H)，7.23(d，J＝4.6Hz，4H)，7.16(m，1H)，6.95(m，1H)，5.22(s，2H)，4.47(m，1H)，3.90(m，1H)，3.87-3.81(m，1H)，3.72(m，1H)，3.56(m，1H)，3.31(d，J＝8.0Hz，2H)，3.05(m，1H)，2.81(m，1H)，2.03(m，2H)，1.45-1.37(m，4H)，1.13(m，3H). According to the steps shown in Figure 6, using 4k and (PNP) 2 CO as starting materials, a light yellow solid was obtained with a yield of 63.2%, mp: 138-141°C. The molecular formula is C 37 H 38 N 6 O 11 , HRMS(ESI): [M+H] + found 743.2655, calcd 743.2671. 1 H NMR(600MHz, DMSO-d 6 )δ(ppm): 9.84(s, 1H) , 8.43 (t, J = 5.8 Hz, 1H), 8.30-8.26 (m, 2H), 8.20-8.16 (m, 1H), 8.00-7.96 (m, 1H), 7.66 (m, 2H), 7.54 (d , J=9.0Hz, 2H), 7.39(d, J=8.3Hz, 2H), 7.23(d, J=4.6Hz, 4H), 7.16(m, 1H), 6.95(m, 1H), 5.22(s , 2H), 4.47 (m, 1H), 3.90 (m, 1H), 3.87-3.81 (m, 1H), 3.72 (m, 1H), 3.56 (m, 1H), 3.31 (d, J = 8.0 Hz, 2H), 3.05 (m, 1H), 2.81 (m, 1H), 2.03 (m, 2H), 1.45-1.37 (m, 4H), 1.13 (m, 3H).
5l的合成Synthesis of 5l
根据图6步骤所示，以4l和(PNP) 2CO为起始原料，得浅黄色固体，收率60％，mp：144-147℃。分子式为C 39H 41N 7O 12，HRMS(ESI)：[M+H] +found 800.2856，calcd 800.2886. 1H NMR(500MHz，DMSO-d 6)δ(ppm)：9.95(s，1H)，8.48-8.42(m，1H)，8.35-8.30(m，2H)，8.22(m，1H)，8.13-8.07(m，2H)，7.68(d，J＝8.3Hz，2H)，7.60-7.56(m，2H)，7.44(d，J＝8.6Hz，2H)，7.27(d，J＝4.5Hz，4H)，7.22-7.17(m，1H)，7.01-6.99(m，2H)，5.25(s，2H)，4.52(m，1H)，3.97-3.85(m，2H)，3.78(m，1H)，3.66(m，3H)，3.08(m，1H)，2.90-2.82(m，2H)，2.11(m，2H)，1.47(m，5H)，1.19(m，2H). According to the steps shown in Figure 6, using 4l and (PNP) 2 CO as starting materials, a light yellow solid was obtained with a yield of 60%, mp: 144-147°C. The molecular formula is C 39 H 41 N 7 O 12 , HRMS(ESI): [M+H] + found 800.2856, calcd 800.2886. 1 H NMR(500MHz, DMSO-d 6 )δ(ppm): 9.95(s, 1H) , 8.48-8.42 (m, 1H), 8.35-8.30 (m, 2H), 8.22 (m, 1H), 8.13-8.07 (m, 2H), 7.68 (d, J=8.3Hz, 2H), 7.60-7.56 (m, 2H), 7.44 (d, J = 8.6 Hz, 2H), 7.27 (d, J = 4.5 Hz, 4H), 7.22-7.17 (m, 1H), 7.01-6.99 (m, 2H), 5.25 ( s, 2H), 4.52 (m, 1H), 3.97-3.85 (m, 2H), 3.78 (m, 1H), 3.66 (m, 3H), 3.08 (m, 1H), 2.90-2.82 (m, 2H) , 2.11 (m, 2H), 1.47 (m, 5H), 1.19 (m, 2H).
5m的合成5m synthesis
根据图6步骤所示，以4m和(PNP) 2CO为起始原料，得浅黄色固体，收率54％，mp：134-136℃。分子式为C 43H 49N 7O 12，HRMS(ESI)：[M+H] +found 856.3679，calcd 856.3512. 1H NMR(400MHz，DMSO-d 6)δ(ppm)：9.92(s，1H)，8.31(d，J＝8.4Hz，2H)，8.24-8.12(m，2H)，8.02(m，2H)，7.67(d，J＝8.0Hz，2H)，7.57(d，J＝8.6Hz，2H)，7.43(d，J＝8.2Hz，2H)，7.23(s，5H)，6.99(s，2H)，5.25(s，2H)，4.54(s，1H)，4.29(m，1H)，3.98-3.82(m，2H)，3.76-3.65(m，1H)，3.63-3.50(m，1H)，3.05(m，1H)，2.79(m，1H)，2.07(m，2H)，1.68-1.35(m，8H)，1.14(m，3H)，0.88(m，6H). According to the steps shown in Figure 6, using 4m and (PNP) 2 CO as starting materials, a light yellow solid was obtained with a yield of 54%, mp: 134-136°C. The molecular formula is C 43 H 49 N 7 O 12 , HRMS (ESI): [M+H] + found 856.3679, calcd 856.3512. 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 9.92 (s, 1H) , 8.31(d, J=8.4Hz, 2H), 8.24-8.12(m, 2H), 8.02(m, 2H), 7.67(d, J=8.0Hz, 2H), 7.57(d, J=8.6Hz, 2H), 7.43 (d, J = 8.2 Hz, 2H), 7.23 (s, 5H), 6.99 (s, 2H), 5.25 (s, 2H), 4.54 (s, 1H), 4.29 (m, 1H), 3.98-3.82 (m, 2H), 3.76-3.65 (m, 1H), 3.63-3.50 (m, 1H), 3.05 (m, 1H), 2.79 (m, 1H), 2.07 (m, 2H), 1.68- 1.35 (m, 8H), 1.14 (m, 3H), 0.88 (m, 6H).
M-1的合成Synthesis of M-1
根据图6步骤所示，5a(93mg，0.125mmol)和MMAE(60mg，0.083mmol)溶于4mL DMF中，依次加入HoBt(10mg，0.074mmol)和0.5mL吡啶，室温下搅拌反应过夜。反应产物使用制备液相进行分离纯化。色谱条件如下：A相为H2O+0.08％TFA，B相为乙腈+0.08％TFA，0-35min B相65％等梯度洗脱，收集合有产物纯品的流份，冻干得产物80mg，收率为47％，mp：134-136℃。分子式为C 68H 105N 11O 15，HRMS(ESI)：[M+H] +found 1316.7885，calcd 1316.7864。HPLC：t R＝5.838min，purity 99.2％(归一化法). 1H NMR(500MHz，DMSO-d 6)δ(ppm)：10.05(s，1H)，7.88(d，J＝7.6Hz，2H)，7.73(m，2H)，7.56(m，2H)，7.41(dd，J＝7.6，4.1Hz，2H)，7.37-7.21(m，7H)，7.16(m，2H)，5.16-4.95(m，4H)，4.77-4.69(m，1H)，4.69-4.59(m，1H)，4.55-4.49(m，2H)，4.46-4.38(m，2H)，4.37-4.17(m，5H)，4.09-3.94(m，3H)，3.93-3.89(m，1H)，3.80-3.75(m，1H)，3.34-3.29(m，1H)，3.28-3.21(m，4H)，3.19(m，3H)，3.11(s，2H)，3.03(m，2H)，2.98-2.95(m，1H)，2.96-2.91(m，1H)，2.86(m，3H)，2.40(m，2H)，2.32-2.24(m，2H)，2.16-2.04(m，3H)，2.04-1.91(m，3H)，1.86-1.65(m，5H)，1.63-1.43(m，5H)，1.41-1.27(m，3H)，1.09-0.94(m，6H)，0.94-0.69(m，21H). According to the steps shown in Figure 6, 5a (93mg, 0.125mmol) and MMAE (60mg, 0.083mmol) were dissolved in 4mL DMF, HoBt (10mg, 0.074mmol) and 0.5mL pyridine were added in sequence, and the reaction was stirred overnight at room temperature. The reaction product is separated and purified using the preparation liquid phase. The chromatographic conditions are as follows: Phase A is H2O+0.08% TFA, Phase B is acetonitrile + 0.08% TFA, 0-35min Phase B is 65% isocratic elution, collect the fraction containing the pure product, freeze-dry the product 80mg, The yield was 47%, mp: 134-136°C. The molecular formula is C 68 H 105 N 11 O 15 , HRMS (ESI): [M+H] + found 1316.7885, calcd 1316.7864. HPLC: t R =5.838min, purity 99.2% (normalized method). 1 H NMR (500MHz, DMSO-d 6 )δ(ppm): 10.05(s, 1H), 7.88(d, J=7.6Hz, 2H), 7.73 (m, 2H), 7.56 (m, 2H), 7.41 (dd, J=7.6, 4.1 Hz, 2H), 7.37-7.21 (m, 7H), 7.16 (m, 2H), 5.16 to 4.95 (m, 4H), 4.77-4.69 (m, 1H), 4.69-4.59 (m, 1H), 4.55-4.49 (m, 2H), 4.46-4.38 (m, 2H), 4.37-4.17 (m, 5H) , 4.09-3.94 (m, 3H), 3.93-3.89 (m, 1H), 3.80-3.75 (m, 1H), 3.34-3.29 (m, 1H), 3.28-3.21 (m, 4H), 3.19 (m, 3H), 3.11(s, 2H), 3.03(m, 2H), 2.98-2.95(m, 1H), 2.96-2.91(m, 1H), 2.86(m, 3H), 2.40(m, 2H), 2.32 -2.24 (m, 2H), 2.16-2.04 (m, 3H), 2.04-1.91 (m, 3H), 1.86-1.65 (m, 5H), 1.63-1.43 (m, 5H), 1.41-1.27 (m, 3H), 1.09-0.94 (m, 6H), 0.94-0.69 (m, 21H).
M-2的合成Synthesis of M-2
根据图6步骤所示，以5b和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率41％，mp：149-151℃。分子式为C 70H 107N 11O 15，HRMS(ESI)：[M+H] +found 1342.8050，calcd 1342.8021.HPLC：t R＝5.964min，purity 97.2％(归一化法)。 1H NMR(600MHz，DMSO-d 6)δ(ppm)：9.94(d，J＝12.8Hz，1H)，8.28-8.24(m，1H)，8.00(d，J＝7.5Hz，1H)，7.86(d，J＝8.7Hz，1H)，7.67(dd，J＝8.7，3.0Hz，1H)，7.61-7.59(m，1H)，7.54(m，2H)，7.33-7.26(m，3H)，7.23(m，2H)，7.14(m，1H)，6.97(d，J＝4.4Hz，2H)，6.15-5.95(m，2H)，5.08-4.93(m，3H)，4.74-4.68(m，1H)，4.63-4.57(m，1H)，4.46(d，J＝5.9Hz，1H)，4.40(m，1H)，4.35(m，2H)，4.23(m，2H)，4.12(m，2H)，3.95(m，4H)，3.76-3.74(m，1H)，3.59-3.49(m，2H)，3.4-3.41(m，1H)，3.28(d，J＝10.1Hz，1H)，3.21(d，J＝8.4Hz，4H)，3.16(m，3H)，3.09(s，2H)，3.04-2.90(m，4H)，2.88-2.79(m，3H)，2.38(m，1H)，2.22(m，2H)，2.09(m，2H)，1.95(m，2H)，1.77(m，2H)，1.72-1.62(m，4H)，1.61-1.55(m，2H)，1.54-1.45(m，3H)，1.41-1.39(m，1H)，1.34-1.31(m，1H)，1.29-1.20(m，3H)，1.01(d，J＝6.6Hz，2H)， 0.98(d，J＝6.6Hz，2H)，0.95(d，J＝6.6Hz，1H)，0.90(m，2H)，0.88-0.66(m，21H). According to the steps shown in Figure 6, using 5b and MMAE as starting materials, the synthesis method is the same as that of M-1 to obtain a pure white solid product with a yield of 41%, mp: 149-151°C. The molecular formula is C 70 H 107 N 11 O 15 , HRMS (ESI): [M+H] + found 1342.8050, calcd 1342.8021. HPLC: t R =5.964 min, purity 97.2% (normalization method). 1 H NMR (600MHz, DMSO-d 6 ) δ (ppm): 9.94 (d, J=12.8Hz, 1H), 8.28-8.24 (m, 1H), 8.00 (d, J=7.5Hz, 1H), 7.86 (d, J = 8.7 Hz, 1H), 7.67 (dd, J = 8.7, 3.0 Hz, 1H), 7.61-7.59 (m, 1H), 7.54 (m, 2H), 7.33-7.26 (m, 3H), 7.23 (m, 2H), 7.14 (m, 1H), 6.97 (d, J = 4.4Hz, 2H), 6.15-5.95 (m, 2H), 5.08-4.93 (m, 3H), 4.74-4.68 (m, 1H), 4.63-4.57 (m, 1H), 4.46 (d, J=5.9 Hz, 1H), 4.40 (m, 1H), 4.35 (m, 2H), 4.23 (m, 2H), 4.12 (m, 2H) ), 3.95 (m, 4H), 3.76-3.74 (m, 1H), 3.59-3.49 (m, 2H), 3.4-3.41 (m, 1H), 3.28 (d, J = 10.1Hz, 1H), 3.21 ( d, J=8.4Hz, 4H), 3.16 (m, 3H), 3.09 (s, 2H), 3.04-2.90 (m, 4H), 2.88-2.79 (m, 3H), 2.38 (m, 1H), 2.22 (m, 2H), 2.09 (m, 2H), 1.95 (m, 2H), 1.77 (m, 2H), 1.72-1.62 (m, 4H), 1.61-1.55 (m, 2H), 1.54-1.45 (m , 3H), 1.41-1.39 (m, 1H), 1.34-1.31 (m, 1H), 1.29-1.20 (m, 3H), 1.01 (d, J = 6.6 Hz, 2H), 0.98 (d, J = 6.6 Hz, 2H), 0.95 (d, J = 6.6 Hz, 1H), 0.90 (m, 2H), 0.88-0.66 (m, 21H).
M-3的合成Synthesis of M-3
根据图6步骤所示，以5c和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率35％，mp：170-173℃。分子式为C 69H 99N 11O 15，HRMS(ESI)：[M+H] +found 1322.7418，calcd 1322.7395.HPLC：t R＝5.853min，purity 98.7％(归一化法)。 1H NMR(600MHz，DMSO-d 6)δ(ppm)：10.01(d，J＝11.6Hz，1H)，8.35(d，J＝8.4Hz，1H)，8.23(d，J＝7.5Hz，1H)，7.95(d，J＝8.2Hz，2H)，7.56(m，2H)，7.43(d，J＝8.1Hz，2H)，7.33-7.22(m，5H)，7.19(s，1H)，5.96(s，1H)，5.39(s，2H)，5.10-4.92(m，3H)，4.49-4.33(m，4H)，4.27-4.18(m，2H)，3.96(m，3H)，3.55(m，3H)，3.18(m，7H)，3.09(s，2H)，3.04-2.90(m，4H)，2.83(m，3H)，2.38(m，1H)，2.23(m，1H)，2.10(m，3H)，1.95(m，2H)，1.83-1.65(m，5H)，1.59(m，2H)，1.55-1.41(m，3H)，1.35(m，2H)，1.24(m，3H)，1.04-0.90(m，12H)，0.87-0.71(m，16H). According to the steps shown in Figure 6, with 5c and MMAE as starting materials, the synthesis method is the same as M-1 to obtain a pure white solid product with a yield of 35%, mp: 170-173°C. The molecular formula is C 69 H 99 N 11 O 15 , HRMS (ESI): [M+H] + found 1322.7418, calcd 1322.7395. HPLC: t R =5.853 min, purity 98.7% (normalization method). 1 H NMR (600MHz, DMSO-d 6 ) δ (ppm): 10.01 (d, J = 11.6 Hz, 1H), 8.35 (d, J = 8.4 Hz, 1H), 8.23 (d, J = 7.5 Hz, 1H ), 7.95 (d, J = 8.2 Hz, 2H), 7.56 (m, 2H), 7.43 (d, J = 8.1 Hz, 2H), 7.33-7.22 (m, 5H), 7.19 (s, 1H), 5.96 (s, 1H), 5.39 (s, 2H), 5.10-4.92 (m, 3H), 4.49-4.33 (m, 4H), 4.27-4.18 (m, 2H), 3.96 (m, 3H), 3.55 (m , 3H), 3.18 (m, 7H), 3.09 (s, 2H), 3.04-2.90 (m, 4H), 2.83 (m, 3H), 2.38 (m, 1H), 2.23 (m, 1H), 2.10 ( m, 3H), 1.95 (m, 2H), 1.83-1.65 (m, 5H), 1.59 (m, 2H), 1.55-1.41 (m, 3H), 1.35 (m, 2H), 1.24 (m, 3H) , 1.04-0.90 (m, 12H), 0.87-0.71 (m, 16H).
C-1的合成Synthesis of C-1
根据图6步骤所示，以5d和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率40％，mp：166-168℃。分子式为C 67H 101N 9O 14，HRMS(ESI)：[M+H] +found 1256.7564，calcd 1256.7541.HPLC：t R＝9.658min，purity 99.9％(归一化法). 1H NMR(600MHz，DMSO-d 6)δ(ppm)：9.89(d，J＝12.3Hz，1H)，8.26-8.24(m，1H)，8.08(d，J＝6.9Hz，1H)，8.01-7.99(m，1H)，7.88-7.86(m，1H)，7.67(d，J＝8.3Hz，1H)，7.61-7.59(m，1H)，7.54(d，J＝7.4Hz，2H)，7.29(m，3H)，7.23(m，2H)，7.14(q，J＝7.8Hz，1H)，6.97(d，J＝3.5Hz，2H)，5.00(m，3H)，4.71(m，1H)，4.48-4.44(m，1H)，4.40(d，J＝6.7Hz，1H)，4.35(m，1H)，4.23(m，2H)，4.11(t，J＝7.7Hz，1H)，3.96(m，4H)，3.76-3.74(m，1H)，3.59-3.49(m，6H)，3.45-3.43(m，1H)，3.31-3.28(m，1H)，3.21(d，J＝8.4Hz，4H)，3.16(m，3H)，3.09(s，1H)，3.01(m，1H)，2.94(s，1H)，2.85(d，J＝6.4Hz，1H)，2.81(d，J＝6.2Hz，1H)，2.38(m，1H)，2.22(m，2H)，2.11-2.05(m，2H)，1.93(m，2H)，1.82-1.73(m，2H)，1.67(m，3H)，1.61-1.55(m，2H)，1.52-1.45(m，2H)，1.26(d，J＝7.4Hz，4H)，1.22(m，2H)，0.98(m，6H)，0.86-0.72(m，21H). According to the steps shown in Figure 6, using 5d and MMAE as starting materials, the synthesis method is the same as that of M-1 to obtain a pure white solid product with a yield of 40%, mp: 166-168°C. The molecular formula is C 67 H 101 N 9 O 14 , HRMS (ESI): [M+H] + found 1256.7564, calcd 1256.7541. HPLC: t R =9.658min, purity 99.9% (normalized method). 1 H NMR( 600MHz, DMSO-d 6 )δ(ppm): 9.89(d, J=12.3Hz, 1H), 8.26-8.24(m, 1H), 8.08(d, J=6.9Hz, 1H), 8.01-7.99(m , 1H), 7.88-7.86 (m, 1H), 7.67 (d, J = 8.3 Hz, 1H), 7.61-7.59 (m, 1H), 7.54 (d, J = 7.4 Hz, 2H), 7.29 (m, 3H), 7.23 (m, 2H), 7.14 (q, J = 7.8 Hz, 1H), 6.97 (d, J = 3.5 Hz, 2H), 5.00 (m, 3H), 4.71 (m, 1H), 4.48- 4.44 (m, 1H), 4.40 (d, J = 6.7 Hz, 1H), 4.35 (m, 1H), 4.23 (m, 2H), 4.11 (t, J = 7.7 Hz, 1H), 3.96 (m, 4H) ), 3.76-3.74(m, 1H), 3.59-3.49(m, 6H), 3.45-3.43(m, 1H), 3.31-3.28(m, 1H), 3.21(d, J=8.4Hz, 4H), 3.16(m, 3H), 3.09(s, 1H), 3.01(m, 1H), 2.94(s, 1H), 2.85(d, J=6.4Hz, 1H), 2.81(d, J=6.2Hz, 1H ), 2.38 (m, 1H), 2.22 (m, 2H), 2.11-2.05 (m, 2H), 1.93 (m, 2H), 1.82-1.73 (m, 2H), 1.67 (m, 3H), 1.61- 1.55(m, 2H), 1.52-1.45(m, 2H), 1.26(d, J=7.4Hz, 4H), 1.22(m, 2H), 0.98(m, 6H), 0.86-0.72(m, 21H) .
C-2的合成Synthesis of C-2
根据图6步骤所示，以5e和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率54％，mp：137-139℃。分子式为C 70H 106N 10O 15，HRMS(ESI)：[M+H]+ found 1327.79309，calcd 1327.79119.HPLC：t R＝8.952min，purity 99.4％(归一化法). 1H NMR(600MHz，DMSO-d 6)δ(ppm)：9.94(d，J＝11.8Hz，1H)，8.29(t，J＝9.5Hz，1H)，8.18(d，J＝6.9Hz，1H)，8.05(t，J＝7.7Hz，1H)，7.97(d，J＝7.4Hz，1H)，7.89(d，J＝8.7Hz，1H)，7.62(d，J＝8.6Hz，1H)，7.59-7.52(m，2H)，7.36-7.24(m，5H)，7.19-7.14(m，1H)，7.01(s，1H)，5.14-4.92(m，3H)，4.74(s，1H)，4.63(s，1H)，4.50(m，1H)，4.46-4.35(m，2H)，4.30-4.23(m，2H)，4.17(t，J＝7.5Hz，1H)，3.98(m，3H)，3.91-3.62(m，9H)，3.57(m，2H)，3.49-3.45(m，1H)，3.38(q，J＝7.1Hz，1H)，3.31(m，1H)，3.26-3.22(m，4H)，3.19(m，3H)，3.12(s，1H)，3.04(m，1H)，2.97(s，1H)，2.88(d，J＝7.5Hz，1H)，2.84(d，J＝7.2Hz，1H)，2.41(m，1H)，2.26(m，1H)，2.11(m，4H)，1.96(m，2H)，1.84＝1.67(m，5H)，1.62(m，2H)，1.58-1.45(m，3H)，1.27(m，5H)，1.18(d，J＝7.1Hz，2H)，1.08(s，1H)，1.04(d，J＝6.7Hz，2H)，1.01(d，J＝6.6Hz，2H)，0.98(d，J＝6.6Hz，1H)，0.92-0.74(m，19H). According to the steps shown in Figure 6, using 5e and MMAE as starting materials, the synthesis method is the same as that of M-1, to obtain a pure white solid product with a yield of 54%, mp: 137-139°C. The molecular formula is C 70 H 106 N 10 O 15 , HRMS (ESI): [M+H]+ found 1327.79309, calcd 1327.79119. HPLC: t R =8.952min, purity 99.4% (normalized method). 1 H NMR( 600MHz, DMSO-d 6 )δ(ppm): 9.94(d, J=11.8Hz, 1H), 8.29(t, J=9.5Hz, 1H), 8.18(d, J=6.9Hz, 1H), 8.05( t, J = 7.7 Hz, 1H), 7.97 (d, J = 7.4 Hz, 1H), 7.89 (d, J = 8.7 Hz, 1H), 7.62 (d, J = 8.6 Hz, 1H), 7.59-7.52 ( m, 2H), 7.36-7.24 (m, 5H), 7.19-7.14 (m, 1H), 7.01 (s, 1H), 5.14-4.92 (m, 3H), 4.74 (s, 1H), 4.63 (s, 1H), 4.50 (m, 1H), 4.46-4.35 (m, 2H), 4.30-4.23 (m, 2H), 4.17 (t, J = 7.5 Hz, 1H), 3.98 (m, 3H), 3.91-3.62 (m, 9H), 3.57 (m, 2H), 3.49-3.45 (m, 1H), 3.38 (q, J=7.1 Hz, 1H), 3.31 (m, 1H), 3.26-3.22 (m, 4H), 3.19(m, 3H), 3.12(s, 1H), 3.04(m, 1H), 2.97(s, 1H), 2.88(d, J=7.5Hz, 1H), 2.84(d, J=7.2Hz, 1H ), 2.41 (m, 1H), 2.26 (m, 1H), 2.11 (m, 4H), 1.96 (m, 2H), 1.84=1.67 (m, 5H), 1.62 (m, 2H), 1.58-1.45 ( m, 3H), 1.27 (m, 5H), 1.18 (d, J = 7.1 Hz, 2H), 1.08 (s, 1H), 1.04 (d, J = 6.7 Hz, 2H), 1.01 (d, J = 6.6 Hz, 2H), 0.98 (d, J=6.6 Hz, 1H), 0.92-0.74 (m, 19H).
C-3的合成Synthesis of C-3
根据图6步骤所示，以5f和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率52％，mp：147-148℃。分子式为C 72H 102N 10O 15，HRMS(ESI)：[M+H]+ found 1347.7619，calcd 1347.7599.HPLC：t R＝8.957min，purity 99.8％(归一化法). 1H NMR(600MHz，DMSO-d 6)δ(ppm)：9.79(d，J＝5.8Hz，1H)，8.45(d，J＝5.9Hz，1H)，8.31(d，J＝9.0Hz，1H)，8.13(d，J＝8.1Hz，1H)，8.05(d，J＝9.2Hz，1H)，7.93(d，J＝6.1Hz，1H)，7.89(d，J＝8.6Hz，1H)，7.64-7.57(m，2H)，7.31(d，J＝7.8Hz，3H)，7.28-7.22(m，5H)，7.17(m，2H)，7.00(s，1H)，5.40(s，1H)，5.05(m，3H)，4.74(s，1H)，4.63(s，1H)，4.48(t，J＝7.1Hz，2H)，4.42(m，1H)，4.27(m，1H)，3.99(m，2H)，3.87(m，2H)，3.72(m，2H)，3.56(m，2H)，3.51-3.34(m，5H)，3.31(m，1H)，3.24(s，2H)，3.22(d，J＝6.8Hz，3H)，3.20(s，1H)，3.17(s，1H)，3.12(s，1H)，3.06(m，2H)，2.97(s，1H)，2.90-2.79(m，4H)，2.41(m，1H)，2.26(m，1H)，2.15-2.03(m，3H)，1.98(m，2H)，1.83-1.71(m，3H)，1.71-1.65(m，2H)，1.61-1.55(m，2H)，1.54-1.47(m，2H)，1.30(m，1H)，1.27-1.17(m，2H)，1.09(t，J＝7.0Hz，1H)，1.04(d，J＝6.6Hz，1H)，0.99(m，4H)，0.88(d，J＝6.1Hz，2H)，0.81(m，16H). According to the steps shown in Figure 6, using 5f and MMAE as starting materials, the synthesis method is the same as that of M-1 to obtain a pure white solid product with a yield of 52%, mp: 147-148°C. The molecular formula is C 72 H 102 N 10 O 15 , HRMS (ESI): [M+H]+ found 1347.7619, calcd 1347.7599. HPLC: t R =8.957min, purity 99.8% (normalized method). 1 H NMR( 600MHz, DMSO-d 6 )δ(ppm): 9.79(d, J=5.8Hz, 1H), 8.45(d, J=5.9Hz, 1H), 8.31(d, J=9.0Hz, 1H), 8.13( d, J = 8.1 Hz, 1H), 8.05 (d, J = 9.2 Hz, 1H), 7.93 (d, J = 6.1 Hz, 1H), 7.89 (d, J = 8.6 Hz, 1H), 7.64-7.57 ( m, 2H), 7.31 (d, J = 7.8 Hz, 3H), 7.28-7.22 (m, 5H), 7.17 (m, 2H), 7.00 (s, 1H), 5.40 (s, 1H), 5.05 (m , 3H), 4.74 (s, 1H), 4.63 (s, 1H), 4.48 (t, J = 7.1 Hz, 2H), 4.42 (m, 1H), 4.27 (m, 1H), 3.99 (m, 2H) , 3.87 (m, 2H), 3.72 (m, 2H), 3.56 (m, 2H), 3.51-3.34 (m, 5H), 3.31 (m, 1H), 3.24 (s, 2H), 3.22 (d, J =6.8Hz, 3H), 3.20 (s, 1H), 3.17 (s, 1H), 3.12 (s, 1H), 3.06 (m, 2H), 2.97 (s, 1H), 2.90-2.79 (m, 4H) , 2.41 (m, 1H), 2.26 (m, 1H), 2.15-2.03 (m, 3H), 1.98 (m, 2H), 1.83-1.71 (m, 3H), 1.71-1.65 (m, 2H), 1.61 -1.55(m, 2H), 1.54-1.47(m, 2H), 1.30(m, 1H), 1.27-1.17(m, 2H), 1.09(t, J=7.0Hz, 1H), 1.04(d, J =6.6Hz, 1H), 0.99 (m, 4H), 0.88 (d, J = 6.1Hz, 2H), 0.81 (m, 16H).
C-4的合成Synthesis of C-4
根据图6步骤所示，以5g和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率43％，mp：140-141℃。分子式为C 74H 105N 11O 16，HRMS(ESI)：[M+H]+ found 1404.7839，calcd 1404.7814.HPLC：t R＝8.618min，purity 99.2％(归一化法). 1H NMR(600MHz，DMSO-d 6)δ(ppm)：9.82(d，J＝5.4Hz，1H)，8.35(d，J＝5.9Hz，1H)，8.14(d，J＝7.1Hz，1H)，7.97(m，2H)，7.86(d，J＝8.6Hz，1H)，7.58(m，2H)，7.28(d，J＝7.6Hz，3H)，7.22(d，J＝4.5Hz，5H)，7.14(m，2H)，6.97(s，2H)，5.03(m，3H)，4.72(s，1H)，4.61(s，1H)，4.52-4.35(m，4H)，4.24(m，2H)，4.03-3.89(m，3H)，3.84(m，2H)，3.77-3.70(m，2H)，3.65-3.51(m，4H)，3.44(q，J＝7.8Hz，1H)，3.29(m，1H)，3.21(d，J＝6.3Hz，5H)，3.16(m，3H)，3.11-3.01(m，3H)，2.95(s，1H)，2.86(d，J＝8.8Hz，2H)，2.82(m，2H)，2.38(m，1H)，2.24(m，1H)，2.09(m，3H)，1.95(m，2H)，1.75(m，5H)，1.58(m，2H)，1.54-1.43(m，3H)，1.30-1.18(m，4H)，0.98(m，6H)，0.90-0.69(m，18H). According to the steps shown in Figure 6, with 5g and MMAE as starting materials, the synthesis method is the same as M-1 to obtain a pure white solid product with a yield of 43%, mp: 140-141°C. The molecular formula is C 74 H 105 N 11 O 16 , HRMS (ESI): [M+H]+ found 1404.7839, calcd 1404.7814. HPLC: t R =8.618min, purity 99.2% (normalized method). 1 H NMR( 600MHz, DMSO-d 6 )δ(ppm): 9.82(d, J=5.4Hz, 1H), 8.35(d, J=5.9Hz, 1H), 8.14(d, J=7.1Hz, 1H), 7.97( m, 2H), 7.86 (d, J = 8.6 Hz, 1H), 7.58 (m, 2H), 7.28 (d, J = 7.6 Hz, 3H), 7.22 (d, J = 4.5 Hz, 5H), 7.14 ( m, 2H), 6.97 (s, 2H), 5.03 (m, 3H), 4.72 (s, 1H), 4.61 (s, 1H), 4.52-4.35 (m, 4H), 4.24 (m, 2H), 4.03 -3.89(m, 3H), 3.84(m, 2H), 3.77-3.70(m, 2H), 3.65-3.51(m, 4H), 3.44(q, J=7.8Hz, 1H), 3.29(m, 1H) ), 3.21 (d, J = 6.3 Hz, 5H), 3.16 (m, 3H), 3.11-3.01 (m, 3H), 2.95 (s, 1H), 2.86 (d, J = 8.8 Hz, 2H), 2.82 (m, 2H), 2.38 (m, 1H), 2.24 (m, 1H), 2.09 (m, 3H), 1.95 (m, 2H), 1.75 (m, 5H), 1.58 (m, 2H), 1.54- 1.43 (m, 3H), 1.30-1.18 (m, 4H), 0.98 (m, 6H), 0.90-0.69 (m, 18H).
C-5的合成Synthesis of C-5
根据图6步骤所示，以5h和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率40.8％，mp：156-159℃。分子式为C 78H 113N 11O 16，HRMS(ESI)：[M+H]+ found 1460.84619，calcd 1460.84395.HPLC：t R＝9.837min，purity 96.648％(归一化法). 1H NMR(500MHz，DMSO-d 6)δ(ppm)：9.88(d，J＝4.2Hz，1H)，8.19(t，J＝8.7Hz，3H)，8.02-7.87(m，3H)，7.60(d，J＝9.1Hz，3H)，7.38-7.13(m，12H)，5.04(m，2H)，4.69(m，1H)，4.51(m，3H)，4.34-4.23(m，2H)，3.99(m，3H)，3.87(d，J＝5.8Hz，6H)，3.82-3.62(m，2H)，3.53(m，3H)，3.31-3.17(m，9H)，3.12(s，2H)，3.08-2.93(m，3H)，2.83(m，4H)，2.41(m，1H)，2.26(m，1H)，2.16-1.89(m，5H)，1.85-1.65(m，4H)，1.63-1.44(m，8H)，1.34-1.18(m，4H)，1.01(m，6H)，0.90-0.76(m，22H). According to the steps shown in Figure 6, with 5h and MMAE as starting materials, the synthesis method is the same as M-1 to obtain a pure white solid product with a yield of 40.8%, mp: 156-159°C. The molecular formula is C 78 H 113 N 11 O 16 , HRMS (ESI): [M+H]+ found 1460.84619, calcd 1460.84395. HPLC: t R = 9.837min, purity 96.648% (normalized method). 1 H NMR( 500MHz, DMSO-d 6 )δ(ppm): 9.88(d, J=4.2Hz, 1H), 8.19(t, J=8.7Hz, 3H), 8.02-7.87(m, 3H), 7.60(d, J =9.1Hz, 3H), 7.38-7.13 (m, 12H), 5.04 (m, 2H), 4.69 (m, 1H), 4.51 (m, 3H), 4.34-4.23 (m, 2H), 3.99 (m, 3H), 3.87(d, J=5.8Hz, 6H), 3.82-3.62(m, 2H), 3.53(m, 3H), 3.31-3.17(m, 9H), 3.12(s, 2H), 3.08-2.93 (m, 3H), 2.83 (m, 4H), 2.41 (m, 1H), 2.26 (m, 1H), 2.16-1.89 (m, 5H), 1.85-1.65 (m, 4H), 1.63-1.44 (m , 8H), 1.34-1.18 (m, 4H), 1.01 (m, 6H), 0.90-0.76 (m, 22H).
C-6的合成Synthesis of C-6
根据图6步骤所示，以5i和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率43％，mp：130-132℃。分子式为C 65H 99N 9O 14，HRMS(ESI)：[M+H]+ found 1230.7406，calcd 1230.7384.HPLC：t R＝9.069min，Purity 97.9％(归一化法). 1H NMR(600MHz，DMSO-d 6)δ(ppm)：9.90(d，J＝11.4Hz，1H)，8.27(d，J＝10.1Hz，1H)，8.13(d，J＝6.9Hz，1H)，8.02(d，J＝8.0Hz，1H)，7.86(d，J＝8.7Hz，1H)，7.78(d，J＝8.6Hz，1H)，7.60(d，J＝8.4Hz，1H)，7.54(t，J＝6.0Hz，2H)， 7.32-7.21(m，5H)，7.14(m，1H)，6.97(s，1H)，5.35(m，1H)，5.08-4.94(m，2H)，4.74-4.58(m，1H)，4.49-4.44(m，1H)，4.38(m，2H)，4.24(m，1H)，4.14(t，J＝7.8Hz，1H)，4.01-3.90(m，2H)，3.55(m，1H)，3.44(q，J＝7.6Hz，1H)，3.34(q，J＝7.5，7.0Hz，3H)，3.28(s，1H)，3.21(m，3H)，3.17(s，1H)，3.15(s，1H)，3.09(s，2H)，3.01(m，1H)，2.95(s，1H)，2.83(m，3H)，2.38(m，1H)，2.24(m，1H)，2.11(m，4H)，1.94(m，2H)，1.80-1.67(m，3H)，1.48(m，6H)，1.27(d，J＝7.2Hz，3H)，1.21(s，1H)，1.15(m，2H)，1.06(t，J＝7.4Hz，1H)，1.02(d，J＝6.7Hz，2H)，0.98(d，J＝6.6Hz，3H)，0.95(d，J＝6.7Hz，2H)，0.93-0.88(m，1H)，0.86(s，1H)，0.85-0.70(m，21H). According to the steps shown in Figure 6, with 5i and MMAE as starting materials, the synthesis method is the same as M-1 to obtain a pure white solid product with a yield of 43%, mp: 130-132°C. The molecular formula is C 65 H 99 N 9 O 14 , HRMS (ESI): [M+H]+ found 1230.7406, calcd 1230.7384. HPLC: t R ＝9.069min, Purity 97.9% (normalized method). 1 H NMR( 600MHz, DMSO-d 6 )δ(ppm): 9.90(d, J=11.4Hz, 1H), 8.27(d, J=10.1Hz, 1H), 8.13(d, J=6.9Hz, 1H), 8.02( d, J = 8.0 Hz, 1H), 7.86 (d, J = 8.7 Hz, 1H), 7.78 (d, J = 8.6 Hz, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.54 (t, J=6.0Hz, 2H), 7.32-7.21(m, 5H), 7.14(m, 1H), 6.97(s, 1H), 5.35(m, 1H), 5.08-4.94(m, 2H), 4.74-4.58 (m, 1H), 4.49-4.44 (m, 1H), 4.38 (m, 2H), 4.24 (m, 1H), 4.14 (t, J=7.8Hz, 1H), 4.01-3.90 (m, 2H), 3.55 (m, 1H), 3.44 (q, J = 7.6 Hz, 1H), 3.34 (q, J = 7.5, 7.0 Hz, 3H), 3.28 (s, 1H), 3.21 (m, 3H), 3.17 (s , 1H), 3.15 (s, 1H), 3.09 (s, 2H), 3.01 (m, 1H), 2.95 (s, 1H), 2.83 (m, 3H), 2.38 (m, 1H), 2.24 (m, 1H), 2.11 (m, 4H), 1.94 (m, 2H), 1.80-1.67 (m, 3H), 1.48 (m, 6H), 1.27 (d, J = 7.2 Hz, 3H), 1.21 (s, 1H) ), 1.15 (m, 2H), 1.06 (t, J = 7.4 Hz, 1H), 1.02 (d, J = 6.7 Hz, 2H), 0.98 (d, J = 6.6 Hz, 3H), 0.95 (d, J =6.7Hz, 2H), 0.93-0.88 (m, 1H), 0.86 (s, 1H), 0.85-0.70 (m, 21H).
C-7的合成Synthesis of C-7
根据图6步骤所示，以5i和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率50％，mp：137-139℃。分子式为C 68H 104N 10O 15，HRMS(ESI)：[M+H]+ found 1301.7769，calcd 1301.7756.HPLC：t R＝8.818min，purity 97.7％(归一化法). 1H NMR(600MHz，DMSO-d 6)δ(ppm)：9.97-9.90(m，1H)，8.30(d，J＝8.3Hz，1H)，8.16(d，J＝6.8Hz，1H)，8.08-7.99(m，2H)，7.89(d，J＝8.7Hz，1H)，7.64(m，2H)，7.56(t，J＝6.4Hz，1H)，7.36-7.24(m，5H)，7.16(m，1H)，7.00(s，1H)，5.13-4.94(m，3H)，4.78-4.71(m，1H)，4.63(s，1H)，4.53-4.47(m，1H)，4.45-4.36(m，2H)，4.32(d，J＝7.2Hz，1H)，4.26(m，1H)，4.18(t，J＝7.5Hz，1H)，4.13(t，J＝7.5Hz，1H)，3.98(m，2H)，3.78(m，1H)，3.56(s，9H)，3.38(m，3H)，3.31(m，1H)，3.24(m，3H)，3.19(m，3H)，3.12(s，1H)，3.04(m，1H)，2.97(s，1H)，2.86(m，3H)，2.41(m，1H)，2.26(m，1H)，2.10(m，4H)，1.98(m，2H)，1.77(m，3H)，1.49(m，5H)，1.29(d，J＝6.9Hz，3H)，1.18(m，4H)，1.09(t，J＝7.0Hz，1H)，1.04(m，2H)，1.01(d，J＝6.7Hz，2H)，0.98(d，J＝6.7Hz，1H)，0.91-0.73(m，19H). According to the steps shown in Figure 6, using 5i and MMAE as starting materials, the synthesis method is the same as M-1 to obtain a pure white solid product with a yield of 50%, mp: 137-139°C. The molecular formula is C 68 H 104 N 10 O 15 , HRMS (ESI): [M+H]+ found 1301.7769, calcd 1301.7756. HPLC: t R =8.818min, purity 97.7% (normalized method). 1 H NMR( 600MHz, DMSO-d 6 )δ(ppm): 9.97-9.90(m, 1H), 8.30(d, J=8.3Hz, 1H), 8.16(d, J=6.8Hz, 1H), 8.08-7.99(m , 2H), 7.89 (d, J = 8.7 Hz, 1H), 7.64 (m, 2H), 7.56 (t, J = 6.4 Hz, 1H), 7.36-7.24 (m, 5H), 7.16 (m, 1H) , 7.00(s, 1H), 5.13-4.94(m, 3H), 4.78-4.71(m, 1H), 4.63(s, 1H), 4.53-4.47(m, 1H), 4.45-4.36(m, 2H) , 4.32(d, J=7.2Hz, 1H), 4.26(m, 1H), 4.18(t, J=7.5Hz, 1H), 4.13(t, J=7.5Hz, 1H), 3.98(m, 2H) , 3.78 (m, 1H), 3.56 (s, 9H), 3.38 (m, 3H), 3.31 (m, 1H), 3.24 (m, 3H), 3.19 (m, 3H), 3.12 (s, 1H), 3.04 (m, 1H), 2.97 (s, 1H), 2.86 (m, 3H), 2.41 (m, 1H), 2.26 (m, 1H), 2.10 (m, 4H), 1.98 (m, 2H), 1.77 (m, 3H), 1.49 (m, 5H), 1.29 (d, J = 6.9 Hz, 3H), 1.18 (m, 4H), 1.09 (t, J = 7.0 Hz, 1H), 1.04 (m, 2H) , 1.01 (d, J = 6.7 Hz, 2H), 0.98 (d, J = 6.7 Hz, 1H), 0.91-0.73 (m, 19H).
C-8的合成Synthesis of C-8
根据图6步骤所示，以5k和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率40％，mp：126-126℃。分子式为C 70H 100N 10O 15，HRMS(ESI)：[M+H] +found 1321.7466，calcd 1321.7442.HPLC：t R＝8.824min，purity 98.6％(归一化法). 1H NMR(600MHz，DMSO-d 6)δ(ppm)：9.82(s，1H)，8.44(s，1H)，8.31(s，1H)，8.18(s，1H)，8.06(s，1H)，7.99(s，1H)，7.89(d，J＝8.6Hz，1H)，7.61(d，J＝9.0Hz，2H)，7.36-7.23(m，8H)，7.17(m，2H)，6.99(s，1H)，5.06(m，3H)，4.74(m，1H)，4.63(s，1H)，4.49(d，J＝7.7Hz，2H)，4.42(m，1H)，4.27(m，1H)，3.93(m，5H)， 3.80-3.70(m，2H)，3.62-3.53(m，2H)，3.47(s，1H)，3.41-3.31(m，3H)，3.24(m，3H)，3.19(m，2H)，3.12(s，2H)，3.07(m，2H)，2.97(s，1H)，2.85(m，4H)，2.41(m，1H)，2.26(m，1H)，2.08(m，5H)，1.84-1.68(m，3H)，1.59-1.39(m，6H)，1.31(s，1H)，1.24(s，1H)，1.16(t，J＝7.9Hz，2H)，1.09(t，J＝7.2Hz，2H)，1.01(m，6H)，0.82(m，16H). According to the steps shown in Figure 6, using 5k and MMAE as starting materials, the synthesis method is the same as M-1 to obtain a pure white solid product with a yield of 40%, mp: 126-126°C. The molecular formula is C 70 H 100 N 10 O 15 , HRMS(ESI): [M+H] + found 1321.7466, calcd 1321.7442. HPLC: t R =8.824min, purity 98.6% (normalized method). 1 H NMR( 600MHz, DMSO-d 6 )δ(ppm): 9.82(s, 1H), 8.44(s, 1H), 8.31(s, 1H), 8.18(s, 1H), 8.06(s, 1H), 7.99(s , 1H), 7.89 (d, J = 8.6 Hz, 1H), 7.61 (d, J = 9.0 Hz, 2H), 7.36-7.23 (m, 8H), 7.17 (m, 2H), 6.99 (s, 1H) , 5.06 (m, 3H), 4.74 (m, 1H), 4.63 (s, 1H), 4.49 (d, J = 7.7 Hz, 2H), 4.42 (m, 1H), 4.27 (m, 1H), 3.93 ( m, 5H), 3.80-3.70 (m, 2H), 3.62-3.53 (m, 2H), 3.47 (s, 1H), 3.41-3.31 (m, 3H), 3.24 (m, 3H), 3.19 (m, 2H), 3.12 (s, 2H), 3.07 (m, 2H), 2.97 (s, 1H), 2.85 (m, 4H), 2.41 (m, 1H), 2.26 (m, 1H), 2.08 (m, 5H) ), 1.84-1.68(m, 3H), 1.59-1.39(m, 6H), 1.31(s, 1H), 1.24(s, 1H), 1.16(t, J=7.9Hz, 2H), 1.09(t, J=7.2Hz, 2H), 1.01 (m, 6H), 0.82 (m, 16H).
C-9的合成Synthesis of C-9
根据图6步骤所示，以5l和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率49％，mp：154-157℃。分子式为C 72H 103N 11O 16，HRMS(ESI)：[M+H] +found 1378.7682，calcd 1378.7657.HPLC：t R＝8.538min，purity 99.7％(归一化法). 1H NMR(600MHz，DMSO-d 6)δ(ppm)：9.84(d，J＝5.5Hz，1H)，8.37(t，J＝5.9Hz，1H)，8.31(d，J＝9.3Hz，1H)，8.14(t，J＝6.7Hz，1H)，8.05(t，J＝5.7Hz，1H)，8.02(m，1H)，7.87(d，J＝8.6Hz，1H)，7.63-7.55(m，2H)，7.34-7.27(m，3H)，7.23(dd，J＝8.0，4.0Hz，5H)，7.15(dd，J＝8.8，5.4Hz，2H)，6.96(s，1H)，5.11-4.93(m，3H)，4.74-4.67(m，1H)，4.65-4.57(m，1H)，4.47(m，2H)，4.44-4.37(m，1H)，4.24(m，1H)，3.97(m，2H)，3.91(m，2H)，3.84(m，4H)，3.74(m，2H)，3.65-3.63(m，2H)，3.59(d，J＝5.5Hz，1H)，3.58-3.52(m，3H)，3.33(t，J＝7.1Hz，2H)，3.28(m，1H)，3.21(m，4H)，3.17(s，1H)，3.14(s，1H)，3.09(s，1H)，3.04(m，1H)，2.95(s，1H)，2.86(m，1H)，2.84-2.78(m，2H)，2.38(m，1H)，2.23(m，1H)，2.08(m，3H)，2.04-1.90(m，2H)，1.81-1.66(m，3H)，1.55-1.40(m，6H)，1.31-1.24(m，1H)，1.21(d，J＝5.0Hz，1H)，1.16(m，2H)，1.06(t，J＝7.0Hz，1H)，1.01(d，J＝6.6Hz，2H)，0.98(d，J＝6.6Hz，3H)，0.95(d，J＝6.6Hz，1H)，0.87-0.71(m，16H). According to the steps shown in Figure 6, using 51 and MMAE as starting materials, the synthesis method is the same as that of M-1 to obtain a pure white solid product with a yield of 49%, mp: 154-157°C. The molecular formula is C 72 H 103 N 11 O 16 , HRMS (ESI): [M+H] + found 1378.7682, calcd 1378.7657. HPLC: t R =8.538min, purity 99.7% (normalized method). 1 H NMR( 600MHz, DMSO-d 6 )δ(ppm): 9.84(d, J=5.5Hz, 1H), 8.37(t, J=5.9Hz, 1H), 8.31(d, J=9.3Hz, 1H), 8.14( t, J = 6.7 Hz, 1H), 8.05 (t, J = 5.7 Hz, 1H), 8.02 (m, 1H), 7.87 (d, J = 8.6 Hz, 1H), 7.63-7.55 (m, 2H), 7.34-7.27 (m, 3H), 7.23 (dd, J=8.0, 4.0 Hz, 5H), 7.15 (dd, J=8.8, 5.4 Hz, 2H), 6.96 (s, 1H), 5.11-4.93 (m, 3H), 4.74-4.67(m, 1H), 4.65-4.57(m, 1H), 4.47(m, 2H), 4.44-4.37(m, 1H), 4.24(m, 1H), 3.97(m, 2H) , 3.91 (m, 2H), 3.84 (m, 4H), 3.74 (m, 2H), 3.65-3.63 (m, 2H), 3.59 (d, J=5.5Hz, 1H), 3.58-3.52 (m, 3H) ), 3.33(t, J=7.1Hz, 2H), 3.28(m, 1H), 3.21(m, 4H), 3.17(s, 1H), 3.14(s, 1H), 3.09(s, 1H), 3.04 (m, 1H), 2.95 (s, 1H), 2.86 (m, 1H), 2.84-2.78 (m, 2H), 2.38 (m, 1H), 2.23 (m, 1H), 2.08 (m, 3H), 2.04-1.90(m, 2H), 1.81-1.66(m, 3H), 1.55-1.40(m, 6H), 1.31-1.24(m, 1H), 1.21(d, J=5.0Hz, 1H), 1.16( m, 2H), 1.06 (t, J = 7.0 Hz, 1H), 1.01 (d, J = 6.6 Hz, 2H), 0.98 (d, J = 6.6 Hz, 3H), 0.95 (d, J = 6.6 Hz, 1H), 0.87-0.71 (m, 16H).
C-10的合成Synthesis of C-10
根据图6步骤所示，以5m和MMAE为起始原料，合成方法同M-1，得白色固体纯品，收率37％，mp：117-119℃。分子式为C 76H 111N 11O 16，HRMS(ESI)：[M+H] +found 1434.8298，calcd 1434.8283.HPLC：t R＝9.649min，purity 96.4％(归一化法). 1H NMR(500MHz，DMSO-d 6)δ(ppm)：9.88(s，1H)，8.21-8.01(m，5H)，7.60(d，J＝8.1Hz，3H)，7.34-7.15(m，12H)，7.01(s，2H)，5.04(m，2H)，4.79-4.59(m，1H)，4.56-4.39(m，3H)，4.28(m，2H)，3.98(m，3H)，3.87(d，J＝5.8Hz，3H)，3.79(m，2H)，3.70(m，2H)，3.61-3.45(m，3H)，3.40-3.29(m，3H)，3.21(m，8H)，3.12(s，2H)，3.04(m，2H)，2.98(s，1H)，2.87(m，3H)，2.78(m，1H)，2.41(m，1H)，2.26(m，1H)， 2.13-1.96(m，5H)，1.83-1.70(m，3H)，1.48(m，7H)，1.32-1.17(m，3H)，1.06-0.98(m，5H)，0.84(m，22H). According to the steps shown in Figure 6, with 5m and MMAE as starting materials, the synthesis method is the same as M-1 to obtain a pure white solid product with a yield of 37%, mp: 117-119°C. The molecular formula is C 76 H 111 N 11 O 16 , HRMS (ESI): [M+H] + found 1434.8298, calcd 1434.8283. HPLC: t R =9.649min, purity 96.4% (normalized method). 1 H NMR( 500MHz, DMSO-d 6 )δ(ppm): 9.88(s, 1H), 8.21-8.01(m, 5H), 7.60(d, J=8.1Hz, 3H), 7.34-7.15(m, 12H), 7.01 (s, 2H), 5.04 (m, 2H), 4.79-4.59 (m, 1H), 4.56-4.39 (m, 3H), 4.28 (m, 2H), 3.98 (m, 3H), 3.87 (d, J =5.8Hz, 3H), 3.79 (m, 2H), 3.70 (m, 2H), 3.61-3.45 (m, 3H), 3.40-3.29 (m, 3H), 3.21 (m, 8H), 3.12 (s, 2H), 3.04(m, 2H), 2.98(s, 1H), 2.87(m, 3H), 2.78(m, 1H), 2.41(m, 1H), 2.26(m, 1H), 2.13-1.96(m , 5H), 1.83-1.70 (m, 3H), 1.48 (m, 7H), 1.32-1.17 (m, 3H), 1.06-0.98 (m, 5H), 0.84 (m, 22H).
15a的合成Synthesis of 15a
根据图7步骤所示，以2-CTC resin为固相载体，采用Fmoc保护方法，先将最后一个氨基酸Pro的氨基与树脂进行取代反应上载，顺序连接氨基酸，最后脱除Fmoc，与MC-OSu进行一步缩合，最后用三氟乙酸(TFA)将连接子从树脂上全部裂解上来，乙醚沉淀洗涤，抽滤，收率73％，mp：85-87℃。分子式为C 28H 43N 7O 9，HRMS(ESI)：[M+H] +found 622.3166，calcd 622.3195. 1H NMR(400MHz，DMSO-d 6)δ(ppm)：12.41(s，1H)，8.01-7.91(m，2H)，7.78(d，J＝8.9Hz，1H)，7.00(s，2H)，6.00(s，1H)，4.30(m，1H)，4.24(m，1H)，4.17(m，1H)，3.80(m，1H)，3.50(m，2H)，3.37(t，J＝7.1Hz，2H)，2.94(t，J＝6.8Hz，2H)，2.23-2.04(m，4H)，2.01-1.81(m，4H)，1.72-1.61(m，2H)，1.48(m，5H)，1.41-1.33(m，2H)，1.18(m，2H)，1.09(m，1H)，0.81(m，6H). According to the steps shown in Figure 7, using 2-CTC resin as the solid phase carrier, using the Fmoc protection method, the amino group of the last amino acid Pro is substituted with the resin and uploaded, the amino acids are sequentially connected, and finally Fmoc is removed and MC-OSu is removed. Carry out one-step condensation, and finally use trifluoroacetic acid (TFA) to completely cleave the linker from the resin, precipitate and wash with ether, and filter with suction. The yield is 73%, mp: 85-87°C. The molecular formula is C 28 H 43 N 7 O 9 , HRMS(ESI): [M+H] + found 622.3166, calcd 622.3195. 1 H NMR(400MHz, DMSO-d 6 )δ(ppm): 12.41(s, 1H) , 8.01-7.91(m, 2H), 7.78(d, J=8.9Hz, 1H), 7.00(s, 2H), 6.00(s, 1H), 4.30(m, 1H), 4.24(m, 1H), 4.17(m, 1H), 3.80(m, 1H), 3.50(m, 2H), 3.37(t, J=7.1Hz, 2H), 2.94(t, J=6.8Hz, 2H), 2.23-2.04(m , 4H), 2.01-1.81 (m, 4H), 1.72-1.61 (m, 2H), 1.48 (m, 5H), 1.41-1.33 (m, 2H), 1.18 (m, 2H), 1.09 (m, 1H) ), 0.81 (m, 6H).
15b的合成Synthesis of 15b
根据图7步骤所示，以2-CTC resin为固相载体，采用Fmoc保护方法，先将最后一个氨基酸Pro的氨基与树脂进行取代反应上载，顺序连接氨基酸，最后脱除Fmoc，与MC-OSu进行一步缩合，最后用三氟乙酸(TFA)将连接子从树脂上全部裂解上来，乙醚沉淀洗涤，抽滤，收率67％，mp：140＝143℃。分子式为C 29H 45N 7O 9，HRMS(ESI)：[M+H] +found 636.3323，calcd 636.3352. 1H NMR(400MHz，DMS0-d 6)δ(ppm)：12.40(s，1H)，8.00(d，J＝7.2Hz，1H)，7.89(d，J＝7.8Hz，1H)，7.80(d，J＝8.8Hz，1H)，7.01(s，2H)，6.16-5.93(m，1H)，4.48(m，1H)，4.31-4.11(m，3H)，3.62(m，1H)，3.50(m，1H)，3.37(t，J＝7.2Hz，2H)，2.94(s，2H)，2.13(m，3H)，1.89(m，4H)，1.61(m，1H)，1.54-1.30(m，7H)，1.27-1.06(m，7H)，0.81(m，6H). According to the steps shown in Figure 7, using 2-CTC resin as the solid phase carrier, using the Fmoc protection method, the amino group of the last amino acid Pro is substituted with the resin and uploaded, the amino acids are sequentially connected, and finally Fmoc is removed and MC-OSu is removed. A one-step condensation is carried out, and finally the linker is completely cleaved from the resin with trifluoroacetic acid (TFA), the ether is precipitated and washed, and filtered with suction. The yield is 67%, mp: 140=143°C. The molecular formula is C 29 H 45 N 7 O 9 , HRMS (ESI): [M+H] + found 636.3323, calcd 636.3352. 1 H NMR (400MHz, DMS0-d 6 ) δ(ppm): 12.40(s, 1H) , 8.00 (d, J = 7.2 Hz, 1H), 7.89 (d, J = 7.8 Hz, 1H), 7.80 (d, J = 8.8 Hz, 1H), 7.01 (s, 2H), 6.16-5.93 (m, 1H), 4.48 (m, 1H), 4.31-4.11 (m, 3H), 3.62 (m, 1H), 3.50 (m, 1H), 3.37 (t, J = 7.2Hz, 2H), 2.94 (s, 2H) ), 2.13 (m, 3H), 1.89 (m, 4H), 1.61 (m, 1H), 1.54-1.30 (m, 7H), 1.27-1.06 (m, 7H), 0.81 (m, 6H).
R-1的合成Synthesis of R-1
根据图7步骤所示，将连接子15a与MMAE进行缩合，得到白色纯品，收率35％，mp：129-131℃。分子式为C 67H 108N 12O 15，HRMS(ESI)：[M+H] +found 1321.8131，calcd 1321.8129.HPLC：t R＝7.535min，purity 97.0％(归一化法). 1H NMR(600MHz，DMSO-d 6)δ(ppm)：8.51(d，J＝9.9Hz，1H)，7.97-7.81(m，3H)， 7.77(t，J＝5.9Hz，1H)，7.62(d，J＝8.4Hz，1H)，7.28(d，J＝7.7Hz，2H)，7.24(d，J＝7.7Hz，2H)，7.15(t，J＝7.8Hz，1H)，6.98(d，J＝4.5Hz，1H)，5.86(m，1H)，5.44-5.29(m，3H)，4.92(m，1H)，4.76-4.68(m，1H)，4.66-4.55(m，1H)，4.49(m，2H)，4.40(m，1H)，4.26(m，1H)，4.15(m，1H)，4.09-3.89(m，4H)，3.72(m，2H)，3.52(m，3H)，3.42(m，1H)，3.35(d，J＝7.2Hz，3H)，3.29(s，1H)，3.25-3.14(m，6H)，3.10(d，J＝3.6Hz，1H)，3.05-2.98(m，2H)，2.95(t，J＝8.9Hz，2H)，2.89(m，2H)，2.79(m，1H)，2.42-2.35(m，1H)，2.28-2.21(m，1H)，2.12(m，5H)，1.94(m，3H)，1.74(m，4H)，1.61(m，2H)，1.46(m，7H)，1.31(m，3H)，1.15(m，2H)，1.06(m，2H)，0.98(m，5H)，0.78(m，21H). According to the steps shown in Figure 7, the linker 15a was condensed with MMAE to obtain a pure white product with a yield of 35%, mp: 129-131°C. The molecular formula is C 67 H 108 N 12 O 15 , HRMS (ESI): [M+H] + found 1321.8131, calcd 1321.8129. HPLC: t R ＝7.535min, purity 97.0% (normalized method). 1 H NMR( 600MHz, DMSO-d 6 )δ(ppm): 8.51(d, J=9.9Hz, 1H), 7.97-7.81(m, 3H), 7.77(t, J=5.9Hz, 1H), 7.62(d, J =8.4Hz, 1H), 7.28(d, J=7.7Hz, 2H), 7.24(d, J=7.7Hz, 2H), 7.15(t, J=7.8Hz, 1H), 6.98(d, J=4.5 Hz, 1H), 5.86 (m, 1H), 5.44-5.29 (m, 3H), 4.92 (m, 1H), 4.76-4.68 (m, 1H), 4.66-4.55 (m, 1H), 4.49 (m, 2H), 4.40 (m, 1H), 4.26 (m, 1H), 4.15 (m, 1H), 4.09-3.89 (m, 4H), 3.72 (m, 2H), 3.52 (m, 3H), 3.42 (m , 1H), 3.35 (d, J = 7.2 Hz, 3H), 3.29 (s, 1H), 3.25-3.14 (m, 6H), 3.10 (d, J = 3.6 Hz, 1H), 3.05-2.98 (m, 2H), 2.95 (t, J = 8.9 Hz, 2H), 2.89 (m, 2H), 2.79 (m, 1H), 2.42-2.35 (m, 1H), 2.28-2.21 (m, 1H), 2.12 (m , 5H), 1.94 (m, 3H), 1.74 (m, 4H), 1.61 (m, 2H), 1.46 (m, 7H), 1.31 (m, 3H), 1.15 (m, 2H), 1.06 (m, 2H), 0.98 (m, 5H), 0.78 (m, 21H).
R-2的合成Synthesis of R-2
根据图7步骤所示，将连接子15bH与MMAE进行缩合，得到白色纯品，收率30％，mp：124-125℃。分子式为C 68H 110N 12O 15，HRMS(ESI)：[M+H] +found 1335.8256，calcd 1335.8286.HPLC：t R＝7.621min，purity 98.9％(归一化法). 1H NMR(600MHz，DMSO-d 6)δ(ppm)：8.53(d，J＝8.4Hz，1H)，7.99(q，J＝6.7Hz，1H)，7.94-7.89(m，1H)，7.87(d，J＝7.0Hz，1H)，7.80(d，J＝8.8Hz，1H)，7.65(d，J＝8.4Hz，1H)，7.31(t，J＝5.0Hz，2H)，7.27(d，J＝7.5Hz，2H)，7.18(t，J＝7.6Hz，1H)，7.01(s，1H)，5.89(d，J＝6.7Hz，1H)，5.76(s，1H)，5.40(m，3H)，4.96(m，1H)，4.74(m，1H)，4.64(m，1H)，4.58-4.38(m，4H)，4.27-4.21(m，1H)，4.16(m，1H)，3.99(m，2H)，3.76(m，1H)，3.66(m，1H)，3.57(m，1H)，3.49(m，2H)，3.38(d，J＝6.5Hz，3H)，3.31(m，1H)，3.24(m，3H)，3.20(m，2H)，3.16(m，2H)，3.12(s，1H)，3.07-3.01(m，2H)，2.97(m，1H)，2.92(d，J＝6.5Hz，2H)，2.76(m，1H)，2.45-2.38(m，1H)，2.30-2.23(m，1H)，2.22-2.06(m，5H)，1.99(m，3H)，1.91-1.68(m，5H)，1.63(m，2H)，1.49(m，7H)，1.32(m，3H)，1.17(m，4H) According to the steps shown in Figure 7, the linker 15bH was condensed with MMAE to obtain a pure white product with a yield of 30%, mp: 124-125°C. The molecular formula is C 68 H 110 N 12 O 15 , HRMS(ESI): [M+H] + found 1335.8256, calcd 1335.8286. HPLC: t R =7.621min, purity 98.9% (normalized method). 1 H NMR( 600MHz, DMSO-d 6 ) δ (ppm): 8.53 (d, J = 8.4 Hz, 1H), 7.99 (q, J = 6.7 Hz, 1H), 7.94-7.89 (m, 1H), 7.87 (d, J =7.0Hz, 1H), 7.80(d, J=8.8Hz, 1H), 7.65(d, J=8.4Hz, 1H), 7.31(t, J=5.0Hz, 2H), 7.27(d, J=7.5 Hz, 2H), 7.18 (t, J = 7.6 Hz, 1H), 7.01 (s, 1H), 5.89 (d, J = 6.7 Hz, 1H), 5.76 (s, 1H), 5.40 (m, 3H), 4.96 (m, 1H), 4.74 (m, 1H), 4.64 (m, 1H), 4.58-4.38 (m, 4H), 4.27-4.21 (m, 1H), 4.16 (m, 1H), 3.99 (m, 2H), 3.76 (m, 1H), 3.66 (m, 1H), 3.57 (m, 1H), 3.49 (m, 2H), 3.38 (d, J = 6.5 Hz, 3H), 3.31 (m, 1H), 3.24 (m, 3H), 3.20 (m, 2H), 3.16 (m, 2H), 3.12 (s, 1H), 3.07-3.01 (m, 2H), 2.97 (m, 1H), 2.92 (d, J = 6.5Hz, 2H), 2.76 (m, 1H), 2.45-2.38 (m, 1H), 2.30-2.23 (m, 1H), 2.22-2.06 (m, 5H), 1.99 (m, 3H), 1.91-1.68 (m, 5H), 1.63 (m, 2H), 1.49 (m, 7H), 1.32 (m, 3H), 1.17 (m, 4H)
实施例2 Linker-MMAE的体外血浆稳定性检测Example 2 In vitro plasma stability test of Linker-MMAE
待测Linker-MMAE配制成10mM的DMSO储备液，待用。4μL DMSO储备液加入996μL空白大鼠血浆，混合均匀后置于37℃水浴孵育。分别于0、30、60、180、360min各取样50μL，终止反应，每个反应时间点两个平行样品，置于-20℃冻存，待取样完成后统一进行处理。样品进行离心沉降蛋白处理，取上清液，使用LC-MS/MS检测MMAE的浓度，检测浓度单位(nM)。 绘制释放MMAE释放量随时间变化的曲线，并计算血浆中MMAE释放速率，释放速率如表1所示。The Linker-MMAE to be tested is prepared as a 10mM DMSO stock solution, ready for use. Add 4μL of DMSO stock solution to 996μL of blank rat plasma, mix well and incubate in a 37℃ water bath. Sampling 50μL each at 0, 30, 60, 180, and 360 minutes to terminate the reaction. Two parallel samples at each reaction time point were stored at -20°C and processed after sampling. The sample was centrifuged to settle the protein, the supernatant was taken, and the concentration of MMAE was detected by LC-MS/MS, and the concentration unit (nM) was detected. Draw a curve of the release amount of MMAE released over time, and calculate the release rate of MMAE in plasma. The release rate is shown in Table 1.
结果表明，间隔区部分M-2最好(K＝4.0nM/hr)，其次是M-3(K＝10.9nM/hr)和M-1(K＝22.6nM/hr)。马来酰亚胺环N上α位连苯基和β位连环己基与N接链状烷基相比，对Linker-MMAE整体的稳定性有了提高，且β位连接环己基的Linker-MMAE最好。二肽水解位点：M-2(K＝4.0nM/hr)稳定性略好于C-1(K＝8.6nM/hr)。三肽水解位点：C-3(K＝8.77nM/hr)稳定性略好于C-2(K＝13.2nM/hr)。四肽水解位点：C-4(K＝14.7nM/hr)的稳定性好于C-5(K＝97.9nM/hr)，且C-5的稳定性最差。氨基酸数对血浆稳定性的影响不存在一定规律性。自分解部分Linker-MMAE在血浆中的稳定性较好，都好于M-1(K＝22.6nM/hr)，且R-2(K＝1.31nM/hr)好于R-1(K＝8.8nM/hr)。The results show that the spacer part M-2 is the best (K=4.0nM/hr), followed by M-3 (K=10.9nM/hr) and M-1 (K=22.6nM/hr). Compared with the N-linked chain alkyl group, the α-position phenyl group and β-position cyclohexyl group on the maleimide ring N have improved the overall stability of Linker-MMAE, and the β-position Linker-MMAE with cyclohexyl group the best. Dipeptide hydrolysis site: M-2 (K=4.0nM/hr) has a slightly better stability than C-1 (K=8.6nM/hr). Tripeptide hydrolysis site: C-3 (K=8.77nM/hr) has a slightly better stability than C-2 (K=13.2nM/hr). Tetrapeptide hydrolysis site: C-4 (K=14.7nM/hr) has better stability than C-5 (K=97.9nM/hr), and C-5 has the worst stability. There is no regularity in the influence of amino acid number on plasma stability. The stability of the self-decomposing part of Linker-MMAE in plasma is better, all better than M-1 (K=22.6nM/hr), and R-2 (K=1.31nM/hr) is better than R-1 (K= 8.8nM/hr).
表1 Linker-MMAE血浆中MMAE释放速率常数Table 1 MMAE release rate constant in Linker-MMAE plasma
Figure PCTCN2020000225-appb-000005
Figure PCTCN2020000225-appb-000005
实施例3 Linker-MMAE的酶切释放速率实验Example 3 Linker-MMAE enzyme digestion release rate experiment
待测Linker-MMAE配制成10mM的DMSO储备液，待用。将10μL储备液加入2490μL 100mM L-Cys的PBS缓冲液(pH＝6)，配制成浓度为0.04mM的缓冲工作液。组织蛋白酶B，购买浓度为1.52mM，用pH＝6的PBS缓冲液稀释成0.4mM的蛋白酶溶液。1000μL浓度为0.04mM的化合物缓冲 工作液与10μL浓度为0.4mM的蛋白酶溶液混合(底物与蛋白酶摩尔比为1∶10)，置于37℃水浴孵育。分别于0、10、60、120、240、360分钟，终止反应，每个反应时间点两个平行样品。使用LC-MS/MS检测MMAE的浓度，检测浓度单位(nM)。The Linker-MMAE to be tested is prepared as a 10mM DMSO stock solution, ready for use. Add 10 μL of stock solution to 2490 μL 100 mM L-Cys PBS buffer (pH=6) to prepare a working buffer solution with a concentration of 0.04 mM. Cathepsin B, purchased at a concentration of 1.52mM, diluted with pH=6 PBS buffer solution to 0.4mM protease solution. 1000 μL of compound buffer working solution with a concentration of 0.04 mM was mixed with 10 μL of a protease solution with a concentration of 0.4 mM (the molar ratio of substrate to protease was 1:10), and incubated in a water bath at 37°C. The reaction was terminated at 0, 10, 60, 120, 240, 360 minutes, and two parallel samples were taken at each reaction time point. Use LC-MS/MS to detect the concentration of MMAE, and detect the concentration unit (nM).
结果如表2所示，M-2水解释放最快(K＝32.3nM/hr)，其次是C-4(K＝32.1nM/hr)和C-5(K＝29.8nM/hr)，C-1(K＝25.0nM/hr)，C-2(K＝12.4nM/hr)和C-3(K＝7.4nM/hr)。自分解型Linker-MMAE经过组织蛋白酶B孵育后未检测到MMAE信号，表明R-1和R-2经水解不能完全释放出MMAE。The results are shown in Table 2. The hydrolysis release of M-2 is the fastest (K=32.3nM/hr), followed by C-4 (K=32.1nM/hr) and C-5 (K=29.8nM/hr), C -1 (K=25.0nM/hr), C-2 (K=12.4nM/hr) and C-3 (K=7.4nM/hr). After self-degrading Linker-MMAE was incubated with cathepsin B, no MMAE signal was detected, indicating that R-1 and R-2 could not completely release MMAE after hydrolysis.
表2 Linker-MMAE在组织蛋白酶B中的MMAE释放速率Table 2 MMAE release rate of Linker-MMAE in cathepsin B
Figure PCTCN2020000225-appb-000006
Figure PCTCN2020000225-appb-000006
实施例4 SCT-200-Linker-MMAE ADCs的制备Example 4 Preparation of SCT-200-Linker-MMAE ADCs
1)将Linker-MMAE(即连接子-细胞毒药物)溶于二甲基亚砜，得到连接子-细胞毒药物储备液；1) Dissolve Linker-MMAE (linker-cytotoxic drug) in dimethyl sulfoxide to obtain linker-cytotoxic drug stock solution;
2)将还原剂TCEP溶于缓冲液中，配置还原剂储备液；2) Dissolve the reducing agent TCEP in the buffer, and configure the reducing agent stock solution;
3)将L-Cys溶于缓冲液中，制备L-Cys储备液；3) Dissolve L-Cys in buffer to prepare L-Cys stock solution;
4)将SCT-200抗体加入步骤2)所述还原剂储备液，氮气保护下37℃孵育1.5h；还原剂的摩尔量为抗体摩尔量的3倍；4) Add the SCT-200 antibody to the reducing agent stock solution described in step 2), and incubate at 37°C for 1.5 hours under nitrogen protection; the molar amount of the reducing agent is 3 times the molar amount of the antibody;
5)将步骤4)所述反应液中加入步骤1)的连接子-细胞毒药物储备液，氮气保护下37℃孵育1-2.5h，制备SCT-200抗体抗人EGFR抗体药物偶联物；连接子-细胞毒药物的摩尔量为抗体摩尔量的4-8倍；5) Add the linker-cytotoxic drug stock solution of step 1) to the reaction solution of step 4), and incubate at 37°C for 1-2.5 hours under nitrogen protection to prepare the SCT-200 antibody anti-human EGFR antibody drug conjugate; The molar amount of the linker-cytotoxic drug is 4-8 times the molar amount of the antibody;
6)将步骤5)得到的反应液中加入步骤3)的储备液，停止反应。6) Add the stock solution of step 3) to the reaction solution obtained in step 5) to stop the reaction.
7)将得到的抗体偶联药物SCT-200-Linker-MMAE进一步纯化的步骤，优选AKTA purifier蛋白质纯化系统，收集所需抗体偶联药物组分峰；收集完毕后，使用30kDa超滤管进行超滤离心，浓缩，经无菌滤膜过滤，低温储存。7) The step of further purifying the obtained antibody-conjugated drug SCT-200-Linker-MMAE, preferably the AKTA purifier protein purification system, collect the required antibody-conjugated drug component peaks; after collection, use a 30kDa ultrafiltration tube for ultrafiltration Filter, centrifuge, concentrate, filter through a sterile filter membrane, and store at low temperature.
实施例5 SCT-200-Linker-MMAE ADCs的HIC-HPLC检测Example 5 HIC-HPLC detection of SCT-200-Linker-MMAE ADCs
疏水层析液相色谱(HIC-HPLC)检测Hydrophobic chromatography liquid chromatography (HIC-HPLC) detection
色谱条件如下：The chromatographic conditions are as follows:
色谱系统：安捷伦1200Chromatography system: Agilent 1200
色谱柱：TSKgel Butul-NPR column(TOSOH)，2.5μm；4.6×36mmColumn: TSKgel Butul-NPR column (TOSOH), 2.5μm; 4.6×36mm
流动相：Mobile phase:
A：1.5mol/L硫酸铵和25mmol/L磷酸钠水溶液(pH＝6.95)A: 1.5mol/L ammonium sulfate and 25mmol/L sodium phosphate aqueous solution (pH=6.95)
B：75％(V/V)25mmol/L(pH＝6.95)的磷酸钠水溶液和25％(V/V)的异丙醇混合溶液B: 75% (V/V) 25mmol/L (pH=6.95) sodium phosphate aqueous solution and 25% (V/V) isopropanol mixed solution
流速：0.5mL/min；进样体积：8μL；分析时间：17min；柱温：25℃；检测波长：280nm和248nm；Flow rate: 0.5mL/min; injection volume: 8μL; analysis time: 17min; column temperature: 25°C; detection wavelength: 280nm and 248nm;
梯度：A相100％→0％0-15min，A相100％15.01min-17.00min，加权如表3所示，平均DAR(药物抗体偶联比率)用峰面积百分比和偶联药物个数来计算，所得图谱如图8，图9和图10所示。Gradient: Phase A 100%→0% 0-15min, Phase A 100% 15.01min-17.00min, weighted as shown in Table 3, the average DAR (drug antibody coupling ratio) is calculated by the peak area percentage and the number of coupled drugs By calculation, the resulting maps are shown in Figure 8, Figure 9 and Figure 10.
表3 SCT-200-Linker-MMAE ADCs各组分比例及平均DARTable 3 SCT-200-Linker-MMAE ADCs component ratio and average DAR
Figure PCTCN2020000225-appb-000007
Figure PCTCN2020000225-appb-000007
Figure PCTCN2020000225-appb-000008
Figure PCTCN2020000225-appb-000008
实施例6 SCT-200-Linker-MMAE ADCs的ESI-MS检测Example 6 ESI-MS detection of SCT-200-Linker-MMAE ADCs
取ADC或裸抗样品100ug，加入PNGaseF 500U 1ul，37℃酶切过夜，获得待测样品。Take 100ug of ADC or naked antibody sample, add 500U 1ul of PNGaseF, digest it overnight at 37°C to obtain the sample to be tested.
液质联用参数设置：LC/MS parameter settings:
液相系统采用ACQUITY UPLC H-Class(Waters)，色谱柱为ACQUITY UPLC Protein，BEH SEC，
Figure PCTCN2020000225-appb-000009
1.7μm，2.1mm×100mm，柱温25℃，流动相为100mM乙酸铵水溶液，等度洗脱，流速为0.1ml/min。质谱检测系统为：Xevo G2-XS Qtof(Waters)，检测模式为正离子，全扫描方式。 The liquid system adopts ACQUITY UPLC H-Class (Waters), the chromatographic column is ACQUITY UPLC Protein, BEH SEC,
Figure PCTCN2020000225-appb-000009
1.7μm, 2.1mm×100mm, column temperature 25℃, mobile phase is 100mM ammonium acetate aqueous solution, isocratic elution, flow rate is 0.1ml/min. The mass detection system is: Xevo G2-XS Qtof (Waters), the detection mode is positive ion, and the full scan mode.
数据采集采用MassLynx 4.1(Waters)软件，并经过MaxEnt I去卷积处理。模型参数设置分辨率为2-3.5Da。最小强度比率设置60％，输出分辨率设置为1Da。算法迭代参数设置为20，结果如图11所示。MassLynx 4.1 (Waters) software is used for data collection, and is processed by MaxEnt I deconvolution. The model parameter setting resolution is 2-3.5Da. The minimum intensity ratio is set to 60%, and the output resolution is set to 1Da. The algorithm iteration parameter is set to 20, and the result is shown in Figure 11.
实施例7 SCT-200-Linker-MMAE ADCs的亲和活性检测Example 7 SCT-200-Linker-MMAE ADCs Affinity Activity Detection
包被抗原：用PBS稀释抗原至2μg/mL，每孔加入50μL包被，保鲜膜包好4℃过夜。洗板：板内液体甩干，加入PBST 200μL/孔，洗3次，每次摇3-5min。封闭：每孔加入1％的BSA，200μL/孔，4℃过夜。洗板：板内液体甩干，加入PBST 200μL/孔，洗3次，每次摇3-5min。加入待测样品，PBST做稀释液，每孔加入50μL，每个浓度平行3次，37℃孵育1h。洗板：板内液体甩干，加入PBST 200μL/孔，洗3次，每次摇3-5min，拍干ELISA板子。孵二抗：每孔加50μL二抗(1∶5000稀释)，37℃孵育1h。洗板：板内液体甩干，加入PBST 200μL/孔，洗3次，每次摇3-5min，拍干ELISA板子。每孔加TMB显色液(避光反应)100μL，室温避光显色15-20min。 终止反应：每孔加入2M的H 2SO 4100μL，由蓝色变为黄色，终止反应。酶标仪在405nM波长测定OD值，加终止液后立即进行检测。 Coating antigen: Dilute the antigen with PBS to 2μg/mL, add 50μL coating to each well, and wrap it with fresh-keeping film at 4℃ overnight. Wash the plate: Shake the liquid in the plate, add 200μL of PBST/well, wash 3 times, shake for 3-5 min each time. Blocking: Add 1% BSA to each well, 200μL/well, overnight at 4°C. Wash the plate: Shake the liquid in the plate, add 200μL of PBST/well, wash 3 times, shake for 3-5 min each time. Add the sample to be tested, PBST as a diluent, add 50μL to each well, 3 times in parallel for each concentration, and incubate at 37°C for 1h. Wash the plate: spin dry the liquid in the plate, add 200μL of PBST/well, wash 3 times, shake 3-5 min each time, and pat dry the ELISA plate. Incubation of secondary antibody: Add 50μL of secondary antibody (diluted at 1:5000) to each well, and incubate at 37°C for 1 hour. Wash the plate: spin dry the liquid in the plate, add 200μL of PBST/well, wash 3 times, shake 3-5 min each time, and pat dry the ELISA plate. Add 100μL of TMB color developing solution (light-proof reaction) to each well, and color-developing at room temperature for 15-20min. Stop the reaction: add 100μL of 2M H 2 SO 4 to each well, change from blue to yellow, and stop the reaction. The OD value was measured by the microplate reader at the wavelength of 405nM, and the detection was performed immediately after adding the stop solution.
结果显示，结果如图12所示，裸抗体SCT-200和ADCs与抗原亲和力呈现浓度依赖，说明基于半胱氨酸偶联的方法制备的SCT200-Linker-MMAE ADCs的亲和力与抗体SCT-200的亲和力相近，基本保持原有抗原抗体的亲和力。抗原抗体之间的亲和力来于ADC抗体本身，连接子的改变对其基本不产生影响。The results show that the results are shown in Figure 12. The naked antibody SCT-200 and ADCs have a concentration-dependent affinity to the antigen, indicating that the affinity of the SCT200-Linker-MMAE ADCs prepared based on the cysteine coupling method is comparable to that of the antibody SCT-200 The affinity is similar, and the affinity of the original antigen and antibody is basically maintained. The affinity between the antigen and the antibody comes from the ADC antibody itself, and the change of the linker basically has no effect on it.
实施例8 SCT-200-Linker-MMAE ADCs的内吞活性检测Example 8 Detection of endocytic activity of SCT-200-Linker-MMAE ADCs
收集细胞：KYSE520细胞用胰酶消化，4℃离心5min沉淀细胞，细胞重悬浓度为1×10 7/mL。将细胞50μL/管分装，每组平行3个样品。一抗结合：使用FACS染色缓冲液稀释一抗样品，使一抗终浓度为20μg/mL。冰浴孵育1h后，用FACS染色缓冲液洗涤细胞，4℃离心5min去上清。抗体内化：每个离心管加入200μL FACS染色缓冲液，在37℃孵育2h，同时设置4℃的样品管作为阴性对照。加入2mL冰浴FACS染色缓冲液终止内化。标记二抗：使用Alexa Fluor 488标记的羊抗人IgG(H+L)二抗，4℃避光孵育30min。加入FACS染色缓冲液洗涤细胞，4℃离心5min沉淀细胞去上清，重复洗涤两次。将处理好的细胞重悬于500μL冰浴PBS，流式细胞仪检测荧光强度。 Collect the cells: KYSE520 cells were trypsinized, and the cells were pelleted by centrifugation at 4°C for 5 min. The cell resuspended concentration was 1×10 7 /mL. The cells were divided into 50μL/tube, and each group had 3 samples in parallel. Primary antibody binding: Dilute the primary antibody sample with FACS staining buffer so that the final concentration of the primary antibody is 20μg/mL. After incubating for 1 hour in an ice bath, the cells were washed with FACS staining buffer and centrifuged at 4°C for 5 minutes to remove the supernatant. Antibody internalization: add 200μL of FACS staining buffer to each centrifuge tube and incubate at 37°C for 2h. At the same time, set a sample tube at 4°C as a negative control. Add 2mL ice bath FACS staining buffer to stop internalization. Labeled secondary antibody: Use Alexa Fluor 488 labeled goat anti-human IgG (H+L) secondary antibody, incubate at 4°C for 30 minutes in the dark. Add FACS staining buffer to wash the cells, centrifuge at 4°C for 5 min to pellet the cells to remove the supernatant, and repeat the washing twice. The processed cells were resuspended in 500 μL ice-bath PBS, and the fluorescence intensity was measured by flow cytometry.
流式细胞法检测SCT200-Linker-MMAE ADCs的内化速率，测定了KYSE520细胞中各个ADCs的内化速率。分别将ADCs在4℃孵育30min和37℃孵育2h，细胞表面结合抗体的内化水平通过计算37℃孵育样品相对于4℃孵育对照品的平均荧光强度(MFI)降低水平得出。Flow cytometry was used to detect the internalization rate of SCT200-Linker-MMAE ADCs, and the internalization rate of each ADCs in KYSE520 cells was determined. The ADCs were incubated at 4°C for 30min and 37°C for 2h, respectively. The internalization level of the antibody bound to the cell surface was calculated by calculating the average fluorescence intensity (MFI) reduction level of the 37°C incubation sample compared to the 4°C incubation control.
t时间点的％MFI＝37℃孵育样品的MFI×100/4℃孵育样品的MFI；%MFI at time point t=MFI of the sample incubated at 37°C×MFI of the sample incubated at 100/4°C;
t时间点的内化百分比＝100-t时间点％MFIPercentage of internalization at time t=100-%MFI at time t
结果如图13和表4所示，连接子的改变对于内吞速率没有明显的影响。The results are shown in Figure 13 and Table 4. The change of the linker has no significant effect on the endocytosis rate.
表4 SCT200-Linker-MMAE ADCs的内化速率Table 4 Internalization rate of SCT200-Linker-MMAE ADCs
Figure PCTCN2020000225-appb-000010
Figure PCTCN2020000225-appb-000010
实施例9 Western Blot实验检测肿瘤细胞EGFR表达水平Example 9 Western Blot experiment to detect tumor cell EGFR expression level
(1)样品制备和细胞蛋白抽提，细胞在直径60mm细胞培养皿中生长至密度80％-90％，PBS清洗细胞两次后，加入150μL含有蛋白酶抑制剂和磷酸酶抑制剂的蛋白裂解液，刮下细胞，4℃、12000rpm离心20min，离心完成后取上清。(1) Sample preparation and cell protein extraction. Cells are grown to a density of 80%-90% in a cell culture dish with a diameter of 60mm. After washing the cells twice with PBS, add 150μL of protein lysis solution containing protease inhibitors and phosphatase inhibitors , Scrape the cells, centrifuge at 4°C, 12000rpm for 20min, take the supernatant after centrifugation is complete.
(2)BCA法蛋白定量后用蛋白裂解液调整蛋白浓度，加入5×上样缓冲液，99℃水浴锅煮10min后，-20℃保存。(2) After protein quantification by BCA method, adjust the protein concentration with protein lysate, add 5× loading buffer, boil in a water bath at 99°C for 10 minutes, and store at -20°C.
(3)Western Blot实验(3) Western Blot experiment
上样：待检测蛋白样品上样，蛋白上样量为30μg/孔。两侧分别加入6μL和3μL蛋白Marker；电泳：通过预染蛋白marker和溴酚蓝的位置来确定电泳停止时间，约70min；湿转法转膜：转膜条件为390mA恒流70min，使 用0.45μm孔径的PVDF膜；封闭：将转好蛋白的膜完全浸在5％脱脂牛奶-TBST中室温轻摇1h；一抗孵育：用5％脱脂牛奶-TBST稀释一抗，EGFR和β-actin抗体都按照1∶1000稀释，在4℃孵育过夜；洗膜：回收一抗，-20℃保存。在TBST中洗膜5次，每次5min；二抗孵育：用5％脱脂奶粉-TBST稀释二抗，山羊抗兔IgG(H+L)和山羊抗小鼠IgG(H+L)抗体按照1∶10000稀释，室温轻摇2h。洗膜：TBST洗膜5次，每次5min。曝光：将ECL发光液加至膜上后，避光反应30s-1min后曝光条带。Loading: The protein sample to be tested is loaded, and the protein loading amount is 30μg/well. Add 6μL and 3μL protein markers on both sides; electrophoresis: determine the stop time of electrophoresis by pre-stained protein marker and bromophenol blue position, about 70min; wet transfer method: transfer membrane condition is 390mA constant current 70min, use 0.45μm PVDF membrane with pore size; blocking: completely immerse the protein-transferred membrane in 5% skimmed milk-TBST at room temperature and gently shake for 1 hour; primary antibody incubation: dilute the primary antibody with 5% skimmed milk-TBST, both EGFR and β-actin antibodies Dilute 1:1000 and incubate overnight at 4°C; wash membrane: recover the primary antibody and store at -20°C. Wash the membrane 5 times in TBST for 5 minutes each time; secondary antibody incubation: dilute the secondary antibody with 5% skimmed milk powder-TBST, goat anti-rabbit IgG (H+L) and goat anti-mouse IgG (H+L) according to 1 ：Dilute at 10000, shake gently at room temperature for 2h. Wash the membrane: Wash the membrane 5 times with TBST, 5 min each time. Exposure: After adding ECL luminescent liquid to the film, react for 30s-1min in the dark before exposing the strips.
实验结果如图14所示，A431，FaDu，AsPc-1，HCC827，KYSE520，KYSR450为EGFR表达细胞株，Raji和Daudi为EGFR不表达细胞株。The experimental results are shown in Figure 14. A431, FaDu, AsPc-1, HCC827, KYSE520, KYSR450 are EGFR expressing cell lines, and Raji and Daudi are EGFR non-expressing cell lines.
实施例10 SCT-200-Linker-MMAE ADCs的体外抗肿瘤活性测定Example 10 In vitro anti-tumor activity determination of SCT-200-Linker-MMAE ADCs
取对数生长期的细胞离心重悬计数，并以1×10 4至3×10 4/孔接种于96孔中，37℃培养2h。然后，加入不同浓度的LR004-VC-MMAE(LR004和MMAE作为对照品)，每个药物浓度设置3个平行孔。37℃孵育72h后，每孔加入20μL CCK8试剂继续培养1-2h。观察颜色反应，并用酶标仪测定450nm处的吸光值。实验过程中分别设置空白组(不合细胞)及阴性对照组(无药物处理)，按下列公式计算细胞的存活率：细胞存活率＝(加药组A450值-空白组A450值)/(对照组A450值-空白组A450值)×100％。IC 50值计算使用SPSS软件。 The cells in the logarithmic growth phase were resuspended and counted by centrifugation, and seeded in 96 wells at a rate of 1×10 4 to 3×10 4 /well, and cultured at 37°C for 2 hours. Then, different concentrations of LR004-VC-MMAE (LR004 and MMAE are used as controls) were added, and 3 parallel holes were set for each drug concentration. After 72 hours of incubation at 37°C, add 20μL of CCK8 reagent to each well and continue to incubate for 1-2 hours. Observe the color reaction, and measure the absorbance at 450nm with a microplate reader. During the experiment, a blank group (without cells) and a negative control group (without drug treatment) were set up, and the cell survival rate was calculated according to the following formula: cell survival rate = (additional group A450 value-blank group A450 value)/(control group) A450 value-blank group A450 value)×100%. The IC 50 value is calculated using SPSS software.
结果表明，如表5和表6所示，在细胞实验中，SCT-200-Linker-MMAE ADCs体现了较强的抗肿瘤活性，且对EGFR不表达细胞株没有抗肿瘤作用，展现了其靶向选择性。The results showed that, as shown in Table 5 and Table 6, in cell experiments, SCT-200-Linker-MMAE ADCs showed strong anti-tumor activity, and had no anti-tumor effect on cell lines that do not express EGFR, showing its target To selective.
表5 SCT200-Linker-MMAE ADC四株肿瘤细胞抗肿瘤活性(IC 50，nM) Table 5 Anti-tumor activity of four SCT200-Linker-MMAE ADC tumor cells (IC 50 , nM)
Figure PCTCN2020000225-appb-000011
Figure PCTCN2020000225-appb-000011
Figure PCTCN2020000225-appb-000012
Figure PCTCN2020000225-appb-000012
表6 SCT200-Linker-MMAE ADC另外四株肿瘤细胞抗肿瘤活性(IC 50，nM) Table 6 SCT200-Linker-MMAE ADC other four tumor cell lines anti-tumor activity (IC 50 , nM)
Figure PCTCN2020000225-appb-000013
Figure PCTCN2020000225-appb-000013
Figure PCTCN2020000225-appb-000014
Figure PCTCN2020000225-appb-000014
实施例11 SCT-200-Linker-MMAE ADCs对肿瘤凋亡过程中相关蛋白表达的影响Example 11 The effect of SCT-200-Linker-MMAE ADCs on the expression of related proteins in the process of tumor apoptosis
检测SCT200-Linker-MMAE ADCs对肿瘤凋亡相关蛋白的表达的影响，根据上述实验结果选择了SCT200-M-2，SCT200-C-2和SCT200-C-4进行凋亡蛋白相关性研究。将A431细胞分别经0.01，0.1和1μg/mL的上述ADCs、1μg/mL SCT200和1nM MMAE处理24h，收集样品经Western Blot检测。To detect the influence of SCT200-Linker-MMAE ADCs on the expression of tumor apoptosis-related proteins, SCT200-M-2, SCT200-C-2 and SCT200-C-4 were selected for the correlation study of apoptosis proteins based on the above experimental results. The A431 cells were treated with the above-mentioned ADCs at 0.01, 0.1 and 1 μg/mL, 1 μg/mL SCT200 and 1 nM MMAE for 24 hours, and the samples were collected and tested by Western Blot.
在细胞周期中，p53的调节功能主要体现在G1和G2/M期，与转录激活作用密切相关，结果如图15所示，在所有被检测样品中，MMAE与ADCs对p-p53均有上调作用；当DNA受到损伤时，可以使caspase-3活化(cleaved-caspase-3)和PARP失活，MMAE(1nM)可活化caspase-3，SCT200(1μg/mL)活化效果不明显，但是ADCs可以明显上调cleaved-caspase-3，并且呈浓度依赖；PARP为DNA损伤修复酶，当细胞凋亡时导致PARP失活，被cleaved-caspase-3剪切为cleaved-PARP。cleaved-PARP表达水平有所不同，control和SCT200条带不明显，MMAE的条带较浅，而ADCs 1μg/mL可以明显上调cleaved-PARP表达水平，并且呈浓度依赖。结果表明，ADCs可以通过P-P53，caspase-3和cleaved-PARP依赖的凋亡通路诱导细胞凋亡。In the cell cycle, the regulatory function of p53 is mainly reflected in the G1 and G2/M phases, which are closely related to transcriptional activation. The results are shown in Figure 15. In all tested samples, both MMAE and ADCs up-regulated p-p53. Effect; when DNA is damaged, it can activate caspase-3 (cleaved-caspase-3) and inactivate PARP, MMAE (1nM) can activate caspase-3, SCT200 (1μg/mL) has no obvious activation effect, but ADCs can Cleaved-caspase-3 is significantly up-regulated and is concentration-dependent; PARP is a DNA damage repair enzyme, which causes PARP inactivation when cells are apoptosis, and is cleaved-cleaved-PARP by cleaved-caspase-3. The expression level of cleaved-PARP is different, the control and SCT200 bands are not obvious, the MMAE band is shallow, and ADCs 1μg/mL can significantly increase the expression level of cleaved-PARP, and it is concentration-dependent. The results show that ADCs can induce cell apoptosis through P-P53, caspase-3 and cleaved-PARP-dependent apoptotic pathways.
实施例12 SCT-200-Linker-MMAE ADCs对A431裸鼠移植瘤模型的药效学评价Example 12 Pharmacodynamic evaluation of SCT-200-Linker-MMAE ADCs on A431 nude mouse xenograft tumor model
6-8周BALB/c裸鼠右侧腋下皮下注射5×10 6/100μL的A431细胞。当肿瘤长至第七天时，裸鼠肿瘤体积约为110mm 3，随机分为5组，分别为对照组，SCT-200(12mg/kg)，SCT-200-M-1(4mg/kg)，SCT-200-M-2(4mg/kg)，SCT-200-C-2(4mg/kg)和SCT-200-C-4(4mg/kg)每组6只裸鼠，每隔3 天尾静脉给药一次，共给药4次，肿瘤抑制曲线如图16所示。结果显示，四组ADC组均以4mg/kg的剂量给药显示出了较强的抑瘤效果，与对照组和SCT-200相比具有显著性差异(P＜0.01)。另外，SCT200-M-2(4mg/kg)，SCT200-C-2(4mg/kg)和SCT200-C-1(4mg/kg)三组与SCT200-M-1(4mg/kg)组抗肿瘤效果较强，且抗肿瘤效果相当，没有显著性差异。 At 6-8 weeks, BALB/c nude mice were injected with 5×10 6 /100 μL A431 cells subcutaneously into the right armpit. When the tumor grows to the seventh day, the tumor volume of nude mice is about 110mm 3 , and they are randomly divided into 5 groups, which are the control group, SCT-200 (12mg/kg), SCT-200-M-1 (4mg/kg) , SCT-200-M-2 (4mg/kg), SCT-200-C-2 (4mg/kg) and SCT-200-C-4 (4mg/kg) each group of 6 nude mice, every 3 days The tail vein was administered once for a total of 4 times. The tumor suppression curve is shown in Figure 16. The results showed that the four ADC groups all showed a strong anti-tumor effect at a dose of 4 mg/kg, which was significantly different from the control group and SCT-200 (P<0.01). In addition, SCT200-M-2 (4mg/kg), SCT200-C-2 (4mg/kg) and SCT200-C-1 (4mg/kg) three groups and SCT200-M-1 (4mg/kg) group anti-tumor The effect is strong, and the anti-tumor effect is equivalent, and there is no significant difference.

Claims (10)

  1. 一类抗人EGFR抗体药物偶联物，其特征在于，所述的抗人EGFR抗体药物偶联物具有式I所示结构：A type of anti-human EGFR antibody drug conjugate, characterized in that the anti-human EGFR antibody drug conjugate has a structure shown in formula I:
    Figure PCTCN2020000225-appb-100001
    Figure PCTCN2020000225-appb-100001
    其中，among them,
    所述的抗体为抗人EGFR抗体，z＝2～8；The antibody is an anti-human EGFR antibody, z=2-8;
    M为能与抗体半胱氨酸巯基反应的间隔区部分，其结构为以下所示结构中的一种：M is the part of the spacer that can react with the cysteine sulfhydryl group of the antibody, and its structure is one of the following structures:
    Figure PCTCN2020000225-appb-100002
    Figure PCTCN2020000225-appb-100002
    其中，n＝0，1，2，3，4或5，m＝1～10，12或24；Wherein, n = 0, 1, 2, 3, 4 or 5, m = 1-10, 12 or 24;
    C为能够在酶的作用下发生水解的部分，选自二肽序列Val-Cit，Val-Ala，Val-Lys或Val-Arg；或三肽序列Ala-Val-Ala，Gly-Phe-Gly，Gly-Phe-Lys或Ala-Phe-Lys；或四肽序列Gly-Gly-Phe-Gly或Gly-Phe-Leu-Gly中的一种；C is a part that can be hydrolyzed under the action of an enzyme, and is selected from the dipeptide sequence Val-Cit, Val-Ala, Val-Lys or Val-Arg; or the tripeptide sequence Ala-Val-Ala, Gly-Phe-Gly, Gly-Phe-Lys or Ala-Phe-Lys; or one of the tetrapeptide sequence Gly-Gly-Phe-Gly or Gly-Phe-Leu-Gly;
    R为自分解部分，其结构为以下所示结构中的一种：R is the self-decomposing part, and its structure is one of the following structures:
    Figure PCTCN2020000225-appb-100003
    Figure PCTCN2020000225-appb-100003
  2. 如权利要求1所述的一类抗人EGFR抗体药物偶联物，其特征在于，所述的抗体为SCT-200单克隆抗体。The anti-human EGFR antibody drug conjugate according to claim 1, wherein the antibody is SCT-200 monoclonal antibody.
  3. 一种制备权利要求1所述的抗人EGFR抗体偶联药物的方法，其特征在于，包括：将具有式II结构的Linker-MMAE与抗人EGFR抗体的链间二硫键发生加成反应制得所述的抗人EGFR抗体药物偶联物；A method for preparing an anti-human EGFR antibody conjugated drug according to claim 1, characterized in that it comprises: the Linker-MMAE with the structure of formula II and the interchain disulfide bond of the anti-human EGFR antibody undergo an addition reaction. To obtain the anti-human EGFR antibody drug conjugate;
    Figure PCTCN2020000225-appb-100004
    Figure PCTCN2020000225-appb-100004
  4. 如权利要求3所述的方法，其特征在于，包括以下步骤：The method according to claim 3, characterized by comprising the following steps:
    (1)将Linker-MMAE溶于二甲基亚砜，得到连接子-细胞毒药物储备液；(1) Dissolve Linker-MMAE in dimethyl sulfoxide to obtain a linker-cytotoxic drug stock solution;
    (2)将还原剂溶于缓冲液中，配置还原剂储备液；(2) Dissolve the reducing agent in the buffer and configure the reducing agent stock solution;
    (3)将L-Cys溶于缓冲液中，制备L-Cys储备液；(3) Dissolve L-Cys in buffer to prepare L-Cys stock solution;
    (4)将抗人EGFR抗体加入步骤(2)所述的还原剂储备液中，孵育1-2h；(4) Add anti-human EGFR antibody to the reducing agent stock solution described in step (2), and incubate for 1-2h;
    (5)向步骤(4)得到的反应液中加入步骤(1)的连接子-细胞毒药物储备液，孵育1-2h，制备抗人EGFR抗体药物偶联物；(5) Add the linker-cytotoxic drug stock solution of step (1) to the reaction solution obtained in step (4), and incubate for 1-2 hours to prepare an anti-human EGFR antibody drug conjugate;
    (6)向步骤(5)得到的反应液中加入步骤(3)的L-Cys储备液，停止反应。(6) Add the L-Cys stock solution of step (3) to the reaction solution obtained in step (5) to stop the reaction.
  5. 根据权利要求4所述的方法，其特征在于，所述步骤(2)中还原剂为TCEP，混合时以抗人EGFR抗体的摩尔量为基准，还原剂的摩尔量为抗体摩尔量的2-4倍。The method according to claim 4, wherein the reducing agent in the step (2) is TCEP, and the molar amount of the anti-human EGFR antibody is used as a benchmark when mixing, and the molar amount of the reducing agent is 2 to the molar amount of the antibody. 4 times.
  6. 根据权利要求34所述的方法，其特征在于，所述步骤(5)中混合时以抗人EGFRSCT-200抗体的摩尔量为基准，所加Linker-MMAE连接子-细胞毒药物的摩尔量为抗体摩尔量的4-8倍。The method according to claim 34, wherein in the step (5), the molar amount of the anti-human EGFR SCT-200 antibody is used as the benchmark when mixing in the step (5), and the molar amount of the Linker-MMAE linker-cytotoxic drug added is 4-8 times the molar amount of antibody.
  7. 根据权利要求4所述的方法，其特征在于，在所述步骤(4)和步骤(5)中的反应在氮气保护下进行，步骤(4)中的反应温度为35～40℃，优选为37℃。The method according to claim 4, wherein the reaction in the step (4) and step (5) is carried out under the protection of nitrogen, and the reaction temperature in the step (4) is 35-40°C, preferably 37°C.
  8. 根据权利要求4所述的方法，其特征在于，还包括将得到的抗体药物偶联物进一步纯化的步骤，优选采用AKTA purifier蛋白质纯化系统，收集所需抗体偶联药物组分峰；收集完毕后，使用30kDa超滤管进行超滤离心，浓缩，经无菌滤膜过滤，低温储存。The method according to claim 4, characterized in that it further comprises the step of further purifying the obtained antibody-drug conjugates, preferably using the AKTA purifier protein purification system to collect the peaks of the required antibody-drug conjugates; after the collection is complete , Use a 30kDa ultrafiltration tube for ultrafiltration and centrifugation, concentrate, filter through a sterile membrane, and store at low temperature.
  9. 权利要求1或2所述的抗体药物偶联物在制备用于肿瘤靶向治疗的药物中的用途，其中，所述的肿瘤为EGFR阳性实体肿瘤，包括鳞状上皮细胞癌，食管癌，鼻咽癌，肺癌，乳腺癌，胰腺癌，前列腺癌，头颈癌，结肠癌等。The use of the antibody-drug conjugate of claim 1 or 2 in the preparation of a drug for targeted tumor therapy, wherein the tumor is an EGFR-positive solid tumor, including squamous cell carcinoma, esophageal cancer, and nose Pharyngeal cancer, lung cancer, breast cancer, pancreatic cancer, prostate cancer, head and neck cancer, colon cancer, etc.
  10. 以权利要求1或2所述的抗体药物偶联物为活性成分的用于肿瘤靶向治疗的药物组合物，其特征在于，含有药学上有效量的权利要求1或2所述的抗体药物偶联物及药学上允许的佐剂。A pharmaceutical composition for targeted tumor therapy using the antibody-drug conjugate of claim 1 or 2 as an active ingredient, characterized in that it contains a pharmaceutically effective amount of the antibody-drug conjugate of claim 1 or 2 Conjugates and pharmaceutically acceptable adjuvants.
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