WO2021164765A1 - 靶向递送和激活的免疫刺激性偶联复合物的制备和用途 - Google Patents

靶向递送和激活的免疫刺激性偶联复合物的制备和用途 Download PDF

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WO2021164765A1
WO2021164765A1 PCT/CN2021/077056 CN2021077056W WO2021164765A1 WO 2021164765 A1 WO2021164765 A1 WO 2021164765A1 CN 2021077056 W CN2021077056 W CN 2021077056W WO 2021164765 A1 WO2021164765 A1 WO 2021164765A1
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qhl
dox
asn
compound
add
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PCT/CN2021/077056
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French (fr)
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刘辰
刘源
王海洋
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亚飞（上海）生物医药科技有限公司
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Priority to US17/800,691 priority Critical patent/US20230414771A1/en
Priority to KR1020227032497A priority patent/KR20220143908A/ko
Priority to EP21756482.2A priority patent/EP4108675A4/en
Priority to AU2021222203A priority patent/AU2021222203A1/en
Priority to CA3167564A priority patent/CA3167564A1/en
Priority to JP2022549600A priority patent/JP2023515034A/ja
Publication of WO2021164765A1 publication Critical patent/WO2021164765A1/zh

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Definitions

  • the present invention relates to an anti-tumor drug compound, in particular to the preparation and use of an immunostimulatory coupling complex for targeted delivery and activation
  • Legumain was first identified as an asparagine endopeptidase in the seeds of legumes, and belongs to a member of the C13 family of cysteine proteases. Legumain can process storage proteins during seed germination. The subsequent discovery of Legumain in parasites and mammals including humans proved that the protease is highly conserved. Legumain from pigs was cloned and identified for the first time in 1997. Legumain is highly expressed in most solid tumors. The differential expression of Legumain in normal tissues and tumor tissues makes it an ideal target for tumor treatment. Legumain is an endopeptidase that can specifically cleave the C-terminal peptide bond of asparagine on the peptide chain under weak acid conditions.
  • CN 201210573744.3 discloses a polypeptide doxorubicin derivative with targeted activation of aspartase, which releases Leu-doxorubicin compounds in tumors by cleaving a tetrapeptide linker by Legumain.
  • the present invention develops a chemically modified activation linker (chemical modified linker) through further compound screening and biological system research, which can further enhance the activation efficiency.
  • the chemically modified activation linking arm of the present invention can enhance the selectivity of the coupled drug to immune cells, produce immunotherapeutic enhancement properties in treatment, and enhance the synergistic effect of the combination with PD-1 antibody.
  • the technical problem to be solved by the present invention is to create highly efficient and specific coupling arms.
  • Previous studies have found that asparagine endopeptidase preferentially recognizes the peptide sequence of the tetrapeptide substrate and cuts the amide bond between Asn and other residues.
  • the idea of improving the activation efficiency of the present invention is to firstly synthesize a large number of compounds with different structures at both ends of the tripeptide (such as AAN), and then use these compounds to further study the working mechanism of the asparagine endopeptidase.
  • this experiment further synthesized and screened a large number of chemically modified structures at both ends of the substrate tripeptide.
  • MI and doxorubicin we screen the activation efficiency under the conditions of tumor tissue or asparagine endonuclease to optimize the new structure-activity relationship.
  • Compound conjugate The schematic diagram of the structure is shown in Figure 2. It includes MI group, selective group S, tripeptide group C cleaved by asparagine endopeptidase, auxiliary group A and the conjugated drug .
  • the added groups of the present invention bring new functions: in addition to enhancing the activation activity of the asparagine endopeptidase to the coupled drug compound (D), the physical properties and biological functions of the drug compound are also improved.
  • the pharmaceutical compound provided by the present invention is hydrophilic, and its cell membrane permeability is changed, so it is the most suitable compound for developing medicine.
  • the present invention also found that the pharmaceutical compound of formula (II) of the present invention has cell selectivity, can be specifically engulfed by tumor-associated macrophages, attack or inhibit tumor-associated macrophages, MDSC cells, and thereby relieve tumor-associated macrophages. The suppressive effect of cells on immunity promotes immunotherapy.
  • the present invention also found that when doxorubicin is coupled, the length of the S group affects the activation efficiency. The longer the chain length of the S is, the combination of the compound and the enzyme is not conducive to the steric hindrance, and the activation efficiency is reduced.
  • Human serum albumin is a small spherical protein composed of 585 amino acids (66-69kd), with many charged residues (such as lysine, aspartic acid and groups without prosthetic groups or carbohydrates) ), there are a small amount of tryptophan or methionine residues.
  • the compound of formula (II) of the present invention is coupled to the cysteine 34 of human albumin as a macromolecular drug; through experiments, it is found that the compound of formula (II) of the present invention covalently coupled to albumin or EMC- AANL-DOX has reduced toxicity, improved drug stability and greatly improved therapeutic efficacy.
  • linking arm of formula (I) and the pharmaceutical compound of formula (II) of the present invention have enhanced activation efficiency, enhanced selectivity for immune cells, enhanced tissue selectivity, suitable water and fat solubility, and drug stability. sex.
  • the present invention provides a compound of formula (I) (linker) and a pharmaceutical compound (conjugate) of formula (II) as described herein, and a pharmaceutically acceptable salt thereof.
  • the present invention also provides a platinum derivative or a pharmaceutically acceptable salt thereof having the following structure:
  • the present invention also provides the pharmaceutical compound represented by formula (II) of the present invention or a pharmaceutically acceptable salt thereof covalently linked to albumin, and EMC-AANL-DOX covalently linked to albumin; preferably, albumin It is connected to the MI or EMC moiety of formula (II) through its 36-position cysteine residue.
  • the present invention also provides a pharmaceutical composition containing the compound of formula (II) of the present invention or a pharmaceutically acceptable salt thereof, the platinum derivative of the present invention or a pharmaceutically acceptable salt thereof, and The pharmaceutical compound represented by formula (II) of the present invention covalently linked to albumin or a pharmaceutically acceptable salt thereof, or EMC-AANL-DOX covalently linked to albumin or a pharmaceutically acceptable salt thereof, and a pharmaceutical Acceptable carrier.
  • the present invention also provides a compound of formula (II) or a pharmaceutically acceptable salt thereof, a platinum derivative of the present invention or a pharmaceutically acceptable salt thereof, and a compound of formula (II) of the present invention covalently linked to albumin
  • the drug compound or its pharmaceutically acceptable salt, or EMC-AANL-DOX covalently linked to albumin or its pharmaceutically acceptable salt is used in the preparation of treatment or prevention of cancer, fatty liver (including alcohol and non-alcoholic fat Liver), steatohepatitis, fatty liver disease, liver fibrosis, liver inflammation, liver cell injury and steatosis; preferably, the cancer is solid cancer or hematological tumor, preferably bladder, brain , Breast/breast, cervix, colon, rectum, esophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin, stomach, uterus, ovaries, testes and blood cancers.
  • the present invention also provides the application of the compound of formula (I) in enhancing the water solubility of the compound drug, reducing the toxicity of the drug, improving the curative effect of the drug, and/or referring to the selectivity of the drug to immune cells, or in the preparation of an improved water-soluble
  • the present invention also provides an EMC-AANL-DOX compound having the structure shown in the following formula or a drug coupled with albumin (preferably, the cysteine residue at position 36 of albumin is co-coated with the EMC moiety).
  • Valence connection in the preparation of drugs for the treatment of liver cancer, and its application with anti-PD-1 antibodies and/or anti-PD-L1 antibodies in the preparation of drugs for combined treatment of tumors:
  • the present invention also provides a compound of formula (II) or a pharmaceutically acceptable salt thereof, a platinum derivative of the present invention or a pharmaceutically acceptable salt thereof, and a compound of formula (II) of the present invention covalently linked to albumin
  • the pharmaceutical compound or its pharmaceutically acceptable salt, or EMC-AANL-DOX covalently linked to albumin or its pharmaceutically acceptable salt is used in the preparation of immunosuppressive cells, tumor-associated macrophages, and MDSC Cells, inhibit angiogenesis, promote anti-tumor immunity and/or promote the application of T lymphocyte proliferation drugs.
  • the present invention also provides a compound of formula (II) or a pharmaceutically acceptable salt thereof, a platinum derivative of the present invention or a pharmaceutically acceptable salt thereof, and a compound of formula (II) of the present invention covalently linked to albumin
  • the pharmaceutical compound or its pharmaceutically acceptable salt, or EMC-AANL-DOX covalently linked to albumin or its pharmaceutically acceptable salt and anti-PD-1 antibody are used in the preparation of a drug for the combined treatment of tumors application.
  • Figure 1 Asparagine Endopeptidase crystal structure and substrate diagram.
  • FIG. 1 Schematic diagram of targeted delivery and activation of immunostimulatory conjugate complexes.
  • Figure 4 Isolation of mouse bone marrow mononuclear cells and induced differentiation of M2 macrophages.
  • Figure 5 Cytotoxicity experiment of compounds on CD8+ T cells.
  • Figure 7 The therapeutic effect of the compound on HT1080 tumor.
  • FIG. 8 EMC-AANL-DOX has high distribution characteristics of liver and liver cancer tissues.
  • FIG. 9 QHL-087-DOX has high distribution characteristics of liver and liver cancer tissues.
  • Figure 10 QHL-087-DOX combined with anti-PD-1 antibody to treat liver tumor in situ.
  • Figure 11 The combination of EMC-AANL-DOX and anti-PD-1 antibody in the treatment of hepatoma in situ is more effective than the combination of lenvatinib and anti-PD-1 antibody.
  • Figure 12 Combined treatment effect of HSA-EMC-AANL-DOX, QHL-087-DOX and anti-PD-1 antibody
  • Figure 13 In vitro cytotoxicity experiment of N-CBP.
  • Figure 15 The therapeutic effect of HSA-QHL-095-N-CBP as a single agent and in combination with an anti-PD-1 antibody.
  • the present invention provides a compound having the structure represented by the following formula (I).
  • the compound can be used as a linking arm.
  • a drug of interest such as an anticancer compound
  • it can enhance the water solubility of the compound drug, reduce drug toxicity, and improve The efficacy of the drug, and/or the selectivity of the drug to immune cells:
  • MI is a maleimide group
  • S is a group that improves the efficiency or selectivity of enzyme cleavage
  • C is an amino acid linking arm that can be cleavable by a proteolytic enzyme
  • A is an auxiliary linking arm.
  • An exemplary MI is a maleimide group represented by the following formula:
  • the wavy line indicates the connection position with S.
  • S in formula (I) can be represented as S1-S2-S3, where S1 is selected from:
  • Rx does not exist or is selected from: C 1-6 alkylene, C 1-6 alkylene amino, C 1-6 alkylene carboxyl and C 1-6 alkylene carbonyl amino, and the wavy line indicates the phase The connection position of the adjacent part; S2 does not exist or is -[(CH 2 ) p O] q -, wherein p is an integer of 1-4, preferably 2, q is 0-15, preferably 1-15, more Preferably it is an integer of 2-6; S3 does not exist or is selected from polar amino acid residues, such as: Glu, Asp, Gly, Ala, Val, Leu, Ile, Met, Phe, Trp, Ser, Thr, Cys, Tyr, Asn, Gln, Lys, Arg and His, preferably Glu and Asp.
  • MI, S1, S2, S3, C, and A are connected to each other in any of the following ways:
  • the wavy line indicates the connection site to the adjacent site; preferably, S is connected to C through a group selected from:
  • S is -R 1 -[(CH 2 ) p O] q -R 2 -R 3 -, wherein R 1 is connected to MI, is absent or is selected from C 1-6 alkylene or C 1-6 alkylenecarbonylamino; R 2 is selected from C 1-6 alkylene; R 3 is selected from -C(O)O-, -NH-, -O- or -C(O)-R 4 , Wherein R 4 is selected from Glu, Asp, Gly, Ala, Val, Leu, Ile, Met, Phe, Trp, Ser, Thr, Cys, Tyr, Asn, Gln, Lys, Arg and His, and preferably Glu and The amino acid residue of Asp, and R 4 forms an amide bond with the -C(O)- through its amino group; p is an integer of 1-4; q is an integer of 0-15, preferably 1-15, more preferably 2-6 .
  • R 1 does not exist, p is 2 or 3, q is 1-15, preferably 2-6, R 2 is C 1-4 alkylene, R 3 is selected from -C(O)O-, -NH -And -O-. In some embodiments, preferably, R 1 is not present, q is 0, R 2 is C 1-6 alkylene, R 3 is -C(O)-R 4 , R 4 is preferably Glu and Asp, and R 4 forms an amide bond with the -C(O)- through its amino group.
  • R1 is C 1-6 alkylenecarbonylamino
  • p is 2 or 3
  • q is 1-15, preferably 2-6
  • R 2 is C 1-4 alkylene
  • R 3 is -C(O)-R 4
  • R 4 is preferably Glu and Asp
  • R 4 forms an amide bond with the -C(O)- through its amino group.
  • An exemplary MI-S is selected from:
  • C connected to any of the above MI-S is AAN, and A is any structure described below.
  • C is selected from the group expressing cleavage by asparagine endopeptidase in the tumor microenvironment, and the group contains an Asn residue.
  • C is X 1 X 2 X 3 , wherein X 1 is selected from L or D type Ala, Thr, Val, and Asn; X 2 is selected from L or D type Ala, Thr, Val, and Ile; X 3 It is Asn, preferably not D-Asn.
  • Exemplary C is selected from: Ala-Ala-Asn, Thr-Ala-Asn, Val-Ala-Asn, Asn-Ala-Asn, Thr-Thr-Asn, Val-Thr-Asn, Asn-Thr-Asn, Ala -Val-Asn, Thr-Val-Asn, Val-Val-Asn, Asn-Val-Asn, Ala-Ile-Asn, Thr-Ile-Asn, Val-Ile-Asn, Asn-Ile-Asn, Ala-Thr -Asn, D-Thr-L-Val-L-Asn, D-Thr-L-Ala-L-Asn, D-Ala-L-Val-L-Asn, L-Thr-D-Val-L-Asn , L-Thr-D-Ala-L-Asn, L-Thr-D-Val-L-Asn, L-Thr-D-Ala-
  • A is preferably selected from Leu, PABC-OH and PABC-NH 2 , and its structures are respectively shown in the following formulas:
  • the wavy line indicates the connection position with C.
  • S and A in the compound of formula (I) of the present invention are selected from any one of the following groups 1-137 [wherein "2peg” means -(CH 2 CH 2 O) 2 -, 3peg means- (CH 2 CH 2 O) 3 -, 4peg means -(CH 2 CH 2 O) 4 -, 6peg means -(CH 2 CH 2 O) 6 -), and so on]:
  • MI is a maleimide group
  • S and A are any group of QHL-001 to QHL-162
  • C is AAN.
  • the particularly preferred compound of formula (I) (linking arm) of the present invention is selected from: QHL-005, QHL-006, QHL-008, QHL-086, QHL-087, QHL-089, QHL-090, QHL-092, QHL -093, QHL-095, QHL-096, QHL-098, QHL-099, QHL-101, QHL-102, QHL-104, QHL-105, QHL-107, QHL-108, QHL-116, QHL-119 , QHL-138, QHL-140, QHL-141, QHL-143, QHL-144, QHL-146, QHL-147, QHL-150, QHL-153, QHL-154, QHL-155, QHL-156, QHL -157, QHL-158, QHL-159, QHL-160, QHL-161 and QHL-162, more preferably any one of QHL-086, QHL-087, QHL-089 and QHL-090.
  • the present invention provides a compound (conjugate) represented by the following formula (II) or a pharmaceutically acceptable salt thereof:
  • MI, S, C and A form the linker compound according to any embodiment of the present invention
  • D is a drug, preferably an anticancer compound.
  • the wavy line indicates the connection position with C and D.
  • it is connected to C through -NH-.
  • D is selected from Resimod, prednisone, triiodothyronine (T3), doxorubicin, daunorubicin, epirubicin, methotrexate, fludarabine, gemcitabine , Cytarabine, Melphalan, Nimustine, Mitoxantrone, Mitomycin, Camptothecin, 10-Hydroxycamptothecin, Topotecan, Fluorouridine, Deoxyfluridine, Etoposide, fludarabine, capecitabine, vincristine, epothilone B, paclitaxel, docetaxel, dabrafenib, dovetinib, motesinib, compound a, compound b And platinum derivatives shown in the following formula:
  • D is selected from daunorubicin, dovitinib, epirubicin, compound a, compound b, mitomycin, dabrafenib, motesinil, resimod, prednisone Songhe T3.
  • the compound of formula (I) (linking arm) of the present invention used for linking with these drugs (D) is selected from: QHL-005, QHL-006, QHL-008, QHL-086, QHL-087, QHL-089 , QHL-090, QHL-092, QHL-093, QHL-095, QHL-096, QHL-098, QHL-099, QHL-101, QHL-102, QHL-104, QHL-105, QHL-107, QHL -108, QHL-116, QHL-119, QHL-138, QHL-140, QHL-141, QHL-143, QHL-144, QHL-146, QHL-147, QHL-150, QHL-153, QHL-154 , QHL-155, QHL-156, QHL-157, QHL-158, QHL-159, QHL-160, QHL-161 and QHL-162, more preferably QHL-086, QHL-087, QHL- Either 089
  • a and D are connected in any of the following ways:
  • the wavy line indicates the connection part with the adjacent part.
  • a and D are connected by -CO-NH-, wherein the carbonyl group is connected to A or is part of A (for example, when A is Leu), and the amino group is connected to D or is part of D.
  • the position where the drug compound is connected to A does not affect the biological activity of the drug, for example, the connection position is far away from the active center of the drug compound.
  • the pharmaceutical compound of formula (II) according to the present invention is selected from:
  • the present invention also provides platinum derivatives, prodrugs thereof, or pharmaceutically acceptable salts thereof having the following structure:
  • the pharmaceutical composition of the present invention can be covalently coupled with albumin to form a new pharmaceutical compound. Therefore, the present invention also includes the pharmaceutical compound of formula (II) of the present invention covalently linked to albumin. Usually, albumin is connected to the MI of the link arm. In some embodiments, the present invention also includes EMC-AANL-DOX linked to albumin, its pharmaceutical composition and its application. The present invention is also a pharmaceutical compound of formula (II) of the present invention covalently linked to albumin or a pharmaceutically acceptable salt thereof.
  • the pharmaceutically acceptable salt may be various pharmaceutically acceptable salts known in the art, including inorganic and organic acid salts, such as hydrochloride, hydrobromide, phosphate, sulfate, and citric acid. Salt, lactate, tartrate, maleate, fumarate, mandelate and oxalate; and with alkalis such as sodium hydroxy, tris(hydroxymethyl)aminomethane (TRIS, tromethamine) ) And N-methylglucosamine to form inorganic and organic base salts.
  • inorganic and organic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, and citric acid. Salt, lactate, tartrate, maleate, fumarate, mandelate and oxalate; and with alkalis such as sodium hydroxy, tris(hydroxymethyl)aminomethane (TRIS, tromethamine) ) And N-methylglucosamine to form inorganic and organic base salts.
  • TMS tris
  • Exemplary preparation methods of the compounds of formula (I) and (II) of the present invention include:
  • Step 1 Preparation of tripeptide-PABC or tetrapeptide: coupling amino acid residues, and separating the formed tripeptide-PABC or tetrapeptide, namely C-A;
  • Step 2 Preparation of MI-S: select a compound suitable for the MI-S group, and perform condensation or cyclization to obtain MI-S with a carboxyl group at one end;
  • Step 3 Preparation of MI-S-C-A: The C-A obtained in step 1 and the MI-S obtained in step 2 are coupled via amino and carboxyl groups to obtain an intermediate (MI-S-C-A);
  • Step 4 Covalently bind the A-terminal carboxyl or hydroxyl activation product of the compound MI-S-C-A obtained in Step 3 to the amino group of the optional drug to form an immunostimulatory adriamycin coupling complex for targeted delivery and activation.
  • the synthetic route includes: using known chemical and biological recombinant coupling techniques to couple the amino acid residues suitable for the present invention with PABC, and then purify and isolate C-PABC with appropriate amino acid protecting groups ;
  • the reaction can be carried out in the presence of a condensing agent, a base, and a polar aprotic solvent. Then remove the protecting group to obtain C-PABC.
  • C-PABC is reacted with an acid or ester or acid chloride containing MI-S groups to form the MI-SCA represented by the formula (I) of the present invention, and then React with the pharmaceutical compound of interest or its salt in the presence of a condensing agent, a base, and a polar aprotic solvent to form the pharmaceutical compound represented by formula (II) of the present invention
  • Examples of the base used in the preparation method include, for example, organic bases such as triethylamine, pyridine, N,N-diisopropylethylamine, 4-dimethylaminopyridine, 1,2,2,6,6 -Pentamethylpiperidine, etc., or inorganic bases, such as sodium carbonate, potassium carbonate, sodium bicarbonate and potassium bicarbonate, etc.
  • Examples of the condensing agent used in the preparation method include, for example, HBTU, DMC, HATU, HOBT, DIC, DCC, EDCI, DEPBT, etc.
  • the solvent used in the preparation method may be any solvent as long as the The solvent itself is inert in the reaction and does not inhibit the reaction.
  • Such solvents include halogenated hydrocarbon solvents such as dichloromethane and chloroform, aromatic hydrocarbon solvents such as benzene and toluene, and aprotic solvents such as acetonitrile, N,N-dimethylformamide, and dimethyl Sulfoxide, etc., ester solvents, such as methyl acetate and ethyl acetate, ether solvents, such as tetrahydrofuran, or a mixture of these solvents.
  • the reaction in the preparation method can be carried out at a temperature ranging from 150°C under ice cooling.
  • the present invention includes a pharmaceutical composition containing the compound of the formula (II) of the present invention or a pharmaceutically acceptable salt thereof, or the platinum derivative of the present invention or a pharmaceutically acceptable salt thereof, Or it contains a compound of formula (II) covalently coupled to albumin or a pharmaceutically acceptable salt thereof, or contains EMC-AANL-DOX covalently coupled to albumin or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition may also contain pharmaceutically acceptable carriers or excipients.
  • the carrier or excipient may be various pharmaceutically acceptable carriers or excipients known in the art, and vary according to different pharmaceutical dosage forms or administration methods.
  • the pharmaceutical composition contains one or more of a solvent, a solubilizer/cosolvent, a pH adjuster, a lyophilized excipient, and an osmotic pressure adjuster.
  • Freeze-dried excipients suitable for use in the present invention include sugars (e.g. lactose, maltose, dextran, glucose, fructose), amino acids (e.g. arginine, lysine, histidine), mannitol, tartaric acid, maleic acid , Citric acid, sodium chloride and cyclodextrin (for example, hydroxypropyl ⁇ cyclodextrin, sulfobutyl ⁇ cyclodextrin) one or more.
  • sugars e.g. lactose, maltose, dextran, glucose, fructose
  • amino acids e.g. arginine, lysine, histidine
  • mannitol e.g. arginine, lysine, histidine
  • mannitol e.g. arginine, lysine, histidine
  • mannitol e.g. argin
  • the pH adjusting agent suitable for the present invention includes hydrochloric acid, phosphoric acid, sulfuric acid, carbonic acid, nitric acid, acetic acid, citric acid, DL-tartaric acid, D-tartaric acid, L-tartaric acid, sodium hydroxide, potassium hydroxide, meglumine
  • acetic acid ethylenediamine, triethylamine, arginine, lysine, histidine, sodium dihydrogen phosphate, and disodium hydrogen phosphate.
  • the solvent suitable for the present invention is preferably an organic solvent, including ethanol, propylene glycol, polyethylene glycol 300, polyethylene glycol 400, tert-butanol, glycerin, Tween, soybean oil, hydroxypropyl ⁇ cyclodextrin solution and sulfur One or more of the butyl ⁇ cyclodextrin solution.
  • organic solvent including ethanol, propylene glycol, polyethylene glycol 300, polyethylene glycol 400, tert-butanol, glycerin, Tween, soybean oil, hydroxypropyl ⁇ cyclodextrin solution and sulfur One or more of the butyl ⁇ cyclodextrin solution.
  • the osmotic pressure regulator suitable for the present invention includes one or more of glucose, sodium chloride, mannitol and sodium lactate.
  • Solubilizers/cosolvents suitable for use in the present invention include Tween 80, Tween 60, poloxamer, hydroxypropyl ⁇ cyclodextrin, polyethylene glycol (PEG), lithium dodecyl hydroxy stearate, sulfonbutane One or more of ⁇ -cyclodextrin, PVP, glycerin and polyoxyethylene castor oil.
  • the compound of the present invention or a pharmaceutically acceptable salt thereof is orally administered to a mammal every day, and the dosage is usually about 0.0025 to 50 mg/kg body weight, preferably about 0.01 to 10 mg/kg body weight. If a known anti-cancer drug or other treatment is administered at the same time, its dosage should be effective to achieve its intended purpose. The optimal dosage of these known anticancer drugs is well known to those skilled in the art.
  • the unit oral dose may include about 0.01 to 50 mg, preferably about 0.1 to 10 mg, of the compound of the present invention or a pharmaceutically acceptable salt thereof.
  • the unit dose can be administered one or more times, with one or more doses per day, each dose containing about 0.1 to 50 mg, suitably about 0.25 to 10 mg of the compound of the present invention or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition of the present invention can be prepared into any suitable dosage form, including but not limited to tablets, capsules, injections and the like.
  • the pharmaceutical composition of the present invention can be administered by a route known in the art, such as oral, intravenous injection, intramuscular injection, and the like.
  • Tumor-associated macrophages M2 type
  • monocytes and inflammatory macrophages M1 type
  • the compound of the present invention can be activated and released in the presence of aspartate endopeptidase.
  • the asparagine peptide chain of the present invention is used.
  • the endonuclease-specifically activated conjugate can effectively reduce the toxicity of the connected drug, so that the final drug has new targeting, activation and metabolism properties, increasing the effect of treating tumors, and generating new tumor indications And have an effect on anti-tumor metastasis, resulting in a brand-new structure and function.
  • the present invention also found that the compound of formula (II) of the present invention can kill tumor-associated macrophages, weaken immunosuppressive cytokines in the microenvironment, and promote the immune enhancement of toxic CD8 cells. More importantly, these tumor microenvironment-releasing compounds are only activated locally in the tumor, unlike traditional chemotherapy drugs that can damage the overall immune system.
  • the tumor microenvironment-releasing compound and PD-1 (programmed death-1) inhibitory antibody (anti-PD-L1 antibody, commercially available, is currently considered to be a candidate drug for immunotherapy) have strong The synergistic therapeutic effect can solve the disadvantage that immunotherapy is difficult to use in combination with chemotherapy drugs.
  • the compound of the present invention can be used to treat or prevent Resimod, prednisone, T3, doxorubicin, daunorubicin, and epirubicin.
  • Various diseases that can be treated by tinib, motesani, compound a, compound b, and platinum compounds such as carboplatin, cisplatin, oxaliplatin
  • cancer ophthalmological diseases, liver diseases, and the like.
  • camptothecin can be used to treat or prevent malignant tumors, psoriasis, warts, acute/chronic leukemia and hepatosplenomegaly caused by schistosomiasis; 10-hydroxycamptothecin can be used to treat gastric cancer, Liver cancer, head and neck cancer and leukemia, etc.; Paclitaxel is mainly used to treat ovarian cancer and breast cancer.
  • Mitomycin can be used for Treatment of chronic lymphoma, chronic myelogenous leukemia, esophageal cancer, gastric cancer, colon cancer, rectal cancer, lung cancer, pancreatic cancer, liver cancer, cervical cancer, uterine body cancer, ovarian cancer, breast cancer, head and neck cancer, bladder cancer, malignant Fluid accumulation in the cavity and so on.
  • diseases that can be treated or prevented with the compound of the present invention, its pharmaceutically acceptable salt or pharmaceutical composition include, but are not limited to, bladder, brain, breast/breast, cervix, colon-rectum, esophagus, kidney, liver, lung , Nasopharynx, pancreas, prostate, skin, stomach, uterus, ovaries, testes and blood.
  • these cancers are selected from: liver cancer, kidney cancer, thyroid cancer, colorectal cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, rectal cancer, esophageal cancer, lung cancer (such as bronchial lung cancer, including undifferentiated small cell Sexual and non-small cell), nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, gastric cancer, uterine cancer, ovarian cancer, testicular cancer, blood cancer (such as chronic or acute leukemia, including lymphocytic and granulocytic leukemia) , Malignant lymphoma, cellulosarcoma, soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, Wilms tumor, neuroblastoma, thyroid cancer and head and neck squamous cell carcinoma.
  • lung cancer such as bronchial lung cancer, including undifferentiated small cell Sexual and non-small cell
  • the pharmaceutical compound represented by formula (II) of the present invention wherein D is mitomycin or a pharmaceutically acceptable salt thereof can also be used for the treatment or prevention of ophthalmic diseases, including the treatment or prevention of healing scars or Choroidal neovascularization, or inhibition of macrophages.
  • the pharmaceutical compound in which D is mitomycin represented by formula (II) or a pharmaceutically acceptable salt thereof can also be used to treat or prevent the sequelae of corneal transplantation, glaucoma, and pterygium surgery.
  • the compound or pharmaceutical composition of the present invention can also be used to prevent tumor metastasis, especially to prevent tumor metastasis to the lung.
  • the compound or pharmaceutical composition of the present invention can be used to prevent lung metastasis of breast cancer.
  • liver diseases of the present invention include but are not limited to fatty liver (including alcoholic and non-alcoholic fatty liver), steatohepatitis, fatty liver disease, liver fibrosis, liver inflammation, and liver cell damage.
  • the present invention includes methods for treating or preventing diseases (preferably cancers, ophthalmological diseases and liver diseases according to any embodiment of the present invention), including administering to a subject in need thereof a therapeutically or preventively effective amount of a compound of formula (II) of the present invention or A pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the compound of formula (II) of the present invention or a pharmaceutically acceptable salt thereof.
  • diseases preferably cancers, ophthalmological diseases and liver diseases according to any embodiment of the present invention
  • the present invention also includes a method for preventing tumor metastasis, including administering to a subject in need an effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the compound of the present invention or a pharmaceutically acceptable salt thereof.
  • Preventing tumor metastasis includes, but is not limited to, preventing tumor metastasis to lung and/or bone metastasis.
  • tumor-associated macrophages play an extremely important role in tumor-related inflammation.
  • TAM tumor-associated macrophages
  • EGF epidermal growth factor
  • the present invention also includes a method for inhibiting tumor-associated macrophages, including administering to a subject in need an effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof, or a drug containing the compound of the present invention or a pharmaceutically acceptable salt thereof combination.
  • tumor-associated macrophages By inhibiting tumor-associated macrophages, it can inhibit tumor growth, inhibit angiogenesis, and inhibit the infiltration and metastasis of cancer cells, and promote anti-tumor immunity, thereby achieving cancer prevention and/or treatment.
  • tumor-associated macrophages express aspartate endopeptidase and are of type M2.
  • the above method of the present invention can be used in combination with radiotherapy or immunotherapy known in the art.
  • the present invention also includes the compound of the present invention, the pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the present invention used in the above-mentioned various methods or uses.
  • the present invention also includes the use of the compound of the present invention or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of the present invention in the preparation of a medicament for the treatment or prevention of the above-mentioned diseases (such as cancer and cancer metastasis).
  • the present invention also includes the compound of the present invention, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the present invention for preparing tumor-associated macrophages, inhibiting tumor growth, inhibiting angiogenesis, inhibiting the infiltration and metastasis of cancer cells, and/or promoting anti-tumor-related macrophages. Use in drugs for tumor immunity.
  • the present invention also provides a method for reducing the toxic and side effects of anticancer compounds (especially the anticancer compounds described herein), the method comprising linking the anticancer compound with the linker compound represented by formula (I) of the present invention.
  • the treatment or prevention method of the present invention includes administering the compound or pharmaceutical composition of the present invention to a subject in need thereof.
  • Administration methods include, but are not limited to, oral, intravenous injection, intramuscular injection, and the like.
  • Subjects include mammals, especially humans.
  • the present invention also provides the use of EMC-AANL-DOX compound having the structure shown in the following formula or a drug coupled with albumin in the preparation of a drug for treating liver cancer:
  • reaction solution was evaporated under reduced pressure, and a small amount of DMF (180ml) was added to dissolve it. It was added dropwise to the stirring 3L of water. A light yellow solid precipitated out. After washing with water for 2-3 times, it was filtered with suction to collect the solid and dried in vacuo to obtain an off-white solid ( The yield is more than 90%).
  • reaction solution was evaporated under reduced pressure, and a small amount of DMF (180ml) was added to dissolve it. It was added dropwise to the stirring 3L of water. A light yellow solid precipitated out. After washing with water for 2-3 times, it was filtered with suction to collect the solid and dried in vacuo to obtain an off-white solid ( The yield is more than 90%).
  • the MI-S group in QHL-006, the synthetic route is as follows:
  • MI-S Intermediate-1 obtained in the above steps into a 100ml single-necked reaction flask, add 10ml of acetic anhydride and stir to dissolve, weigh out NaOAC (98.7mg, 1.216mmol), add slowly in batches, and heat up the oil bath to 110°C for 3h , LC-MS monitors the completion of the reaction of MI-S Intermediate-1, cool to room temperature, spin-dry the reaction solution, and purify by column to obtain MI-S Intermediate-2 (312, yield 70%).
  • the final product is prepared by a method similar to the synthesis of QHL-095-DOX, using different MI-S for connection (the preparation of MI-S refers to the synthesis process of MI-S in QHL-006-DOX).
  • the crude intermediate 4 obtained in the previous step was dissolved in N,N-dimethylformamide (200mL), and intermediate 2 (2.94g, 0.012mol), benzotriazole-N,N,N',N '-Tetramethylurea hexafluorophosphate (HBTU) (6.07g, 0.016mol), add N,N-diisopropylethylamine (2.6g, 0.02mol) after ice bath to 0°C, at room temperature After stirring overnight, the solvent was evaporated under reduced pressure.
  • HBTU benzotriazole-N,N,N',N '-Tetramethylurea hexafluorophosphate
  • the crude intermediate 4 obtained in the previous step was dissolved in N,N-dimethylformamide (200mL), and intermediate 2 (2.94g, 0.012mol), benzotriazole-N,N,N',N '-Tetramethylurea hexafluorophosphate (HBTU) (6.07g, 0.016mol), add N,N-diisopropylethylamine (2.6g, 0.02mol) after ice bath to 0°C, at room temperature After stirring overnight, the solvent was evaporated under reduced pressure.
  • HBTU benzotriazole-N,N,N',N '-Tetramethylurea hexafluorophosphate
  • QHL-090-Compound b 1249.66 1250 Light yellow solid 116mg QHL-087 Mitomycin 1014.05 1014 Gray blue solid powder 55mg QHL-090-mitomycin 1058.1 1058 Gray blue solid powder 78mg QHL-087-Dalafenib 1,199.28 1199 Light yellow solid 61mg QHL-090-Dalafenib 1243.33 1243 Light yellow solid 102mg QHL-087-Motesini 1097.23 1097 Light yellow solid 40mg QHL-090-Motesini 1053.18 1053 Light yellow solid 85mg QHL-140-Resimot 913.00 913 White solid 57mg QHL-086-Resimot 1030.11 1030 White solid 82mg QHL-089-Resimot 1074.16 1074 Off-white solid 49mg QHL-092-Resimot 1118.21 1118 Off-white solid 76mg QHL-095-Resimot 12
  • the present invention also provides the following comparative compounds, the structural formula is as follows:
  • Example 10 Preparation of human albumin-coupled HSA-EMC-AANL-DOX, HSA-QHL-087-DOX and HSA-QHL-087-N-CBP drugs
  • EMC-AANL-DOX, QHL-087-DOX and QHL-087-N-CBP wherein EMC-AANL-DOX is dissolved in DMSO, and QHL-087-DOX and QHL-087-N-CBP are dissolved in sterile water.
  • HSA is dissolved in sterile water.
  • the compound and HSA were combined with HSA at a ratio of 3:1 (4.8umol/mL, 1.6umol/mL), and reacted in a 37°C water bath for 3 hours.
  • the reaction solution was taken out, and the unbound compound was filtered using a pressurized ultrafiltration membrane, and diluted with physiological saline and filtered 3 Second, semi-finished products are obtained.
  • S-C-A is a chemically modified linker and shows high activation efficiency.
  • C selects AAN
  • the activation of different S-C-A linkers and control linkers is evaluated in the activation assay.
  • the sample compound was added to 100 ⁇ g of acidified human breast cancer (MDA-MB435) tumor tissue homogenate (pH 6.0) at a concentration of 1 mg/ml.
  • MDA-MB435 acidified human breast cancer
  • tumor tissue homogenate pH 6.0
  • the enzymes in the tumor tissue homogenate can be released and detected by HPLC to compare the activation efficiency of the tumor tissue to the linker.
  • the results are shown in Tables 2-1, 2-2, 2-3 and 2-4.
  • V(umoL/mL/min) C(umoL/mL)/120min
  • A. Washing of CD3/CD28 magnetic beads a. Shake and suspend the immunomagnetic beads in the vial (vortex for more than 30 seconds, or tilt and rotate for 5 minutes). b. Take out the required amount of immunomagnetic beads into a 1.5ml test tube, add 1ml 1640 containing serum and suspend it, vortex for more than 30s, or keep rolling for at least 5min.
  • a culture plate such as 6-well plate 1E6/ml, 2ml culture medium, keep the T cell density above 2E6/ml, but not more than 2E6/ml.
  • Example 14 Determination of the inhibitory effect of drugs on cell growth by MTT method
  • Example 13 After counting the cells in Example 13, adjust the cell concentration with culture medium and seed it on a 96-well culture plate with 100 ⁇ l cell suspension per well.
  • the seeding concentration of CD8+ T cells is 100,000 cells/well, and the seeding concentration of M2 macrophages It is 20000 cells/well.
  • Cell survival rate% (OD test-OD blank control)/(OD test control-OD blank control)*100%.
  • the survival rate (%) was calculated with Excel software, and Prism 5 software was used to draw the dose-response curve of the drug to cells. Each index was expressed by the mean value, and the coefficient of variation (CV) was used to evaluate the consistency of the data.
  • the maximum initial concentration of the drug to be tested is set to 14uM, and it is diluted in a 1:3 ratio into 9 dose groups (3 replicates in each group).
  • the concentration of DMSO in the drug hole is controlled at 0.1%, only the drug solvent (0.1% DMSO) is added as the test control group (Control), and the blank group (Blank) without cells and only medium (Blank), and then based on the following Methods Calculate the tumor cell survival rate (%) of each dose group relative to the control group (Control).
  • Example 15 Cytotoxicity screening of various drugs of the present invention on M2 macrophages
  • Example 14 the cytotoxicity screening experiment of some compounds was performed on M2 macrophages. Three holes were tested for each drug, and the following drugs were added to each hole of 10uM to test the inhibition rate of the relatively drug-free group. The experimental results are shown in Table 4.
  • Example 16 Comparison of the water solubility of the water-soluble and highly effective targeted activation of doxorubicin derivatives prepared in the examples of the present invention and the control compound
  • the compound prepared in the embodiment of the present invention the compound prepared above and the reference compound C1, C2, C3 and C4 were freeze-dried (-70°C).
  • the compounds were dissolved in different concentrations of water, and the water solubility was checked by observation and HPLC test (>95%). The results are shown in Table 4.
  • QHL-140-N-CBP QHL-140 >10mg/ml 89.5%
  • QHL-143-N-CBP QHL-143 >10mg/ml 78.4%
  • QHL-092-N-CBP QHL-092 >10mg/ml 75.8%
  • To QHL-104-N-CBP QHL-104 >20mg/ml
  • QHL-101-N-CBP QHL-101 >20mg/ml
  • QHL-146-N-CBP QHL-146 >
  • the test results show that under other conditions with the same structure, the 2peg group significantly improves water solubility, changing from insoluble in water to water soluble, and the solubility increases as the amount of PEG increases. Under the same conditions of PEG connection, increasing Glu and Asp can increase water solubility. Through the change of the group, the water solubility of the coupled drug changes, which will have a huge impact on the drug's blood vessel membrane and the permeability of the tumor cell membrane, thereby affecting the efficacy of the treatment.
  • the enhanced water solubility of the compound provides the necessary conditions for the druggability of the drug and the production of conjugated drugs.
  • Example 17 Study on the efficacy of C3, QHL-085-DOX, QHL-087-DOX, QHL-091-DOX, QHL-094-DOX injection in the HT1080 model of nude mice
  • Test purpose To investigate the anti-tumor efficacy of C3, QHL-085-DOX, QHL-087-DOX, QHL-091-DOX and QHL-94-DOX in mouse models during tumor treatment.
  • Test drugs C3, QHL-085-DOX, QHL-087-DOX, QHL-091-DOX and QHL-094-DOX are used as injections and diluted with normal saline to the corresponding concentration during the test.
  • HT1080 cells were purchased from ATCC and identified according to the instructions provided by ATCC. The cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO 2 . Passage every three days, and use cells within 15 generations.
  • Tumor generation 5 ⁇ 10 6 HT1080 cells were injected subcutaneously into the back of nude mice. When the tumor size reaches 100mm 3 , randomization will be performed. Then start treatment, and the day the treatment starts is recorded as the first day.
  • C3, QHL-085-DOX, QHL-087-DOX, QHL-091-DOX and QHL-094-DOX intravenous injection of drugs (IV).
  • C3, QHL-085-DOX, QHL-087-DOX, QHL-091-DOX and QHL-094-DOX were administered at a low dose and the same dose of 18umol/kg, respectively.
  • the control group was given saline. It is administered once a week for 3 weeks.
  • Example 18 C1, C2, C3, QHL-086-DOX, QHL-092-DOX, QHL-095-DOX, QHL-087-DOX, QHL-010-DOX, QHL-117-DOX injection in nude mice HT1080 Pharmacodynamic research in the model
  • Test purpose To investigate the anti-tumor efficacy of the above compounds in mouse models during tumor treatment.
  • Test drugs C1, C2, C3, and corresponding compound injections, diluted with physiological saline to the corresponding concentration during the test.
  • HT1080 cells were purchased from ATCC and identified according to the instructions provided by ATCC. The cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO 2 . Passage every three days, and use cells within 15 generations.
  • Tumor generation 5 ⁇ 10 6 HT1080 cells were injected subcutaneously into the back of nude mice. When the tumor size reaches 100mm 3 , randomization will be performed. Then start treatment, and the day the treatment starts is recorded as the first day.
  • the drug is injected intravenously (IV).
  • IV intravenously
  • the compounds shown in the table were administered at a low dose and the same dose of 36umol/kg.
  • the control group was given saline. It is administered once a week for 3 weeks.
  • Example 19 The tissue distribution study of QHL-087-DOX and EMC-AANL-DOX in orthotopic liver transplantation CT26 tumors.
  • Test purpose To study the tissue distribution of activating drugs in liver tumors.
  • Test animals BALB/c mice aged 6-8 weeks, all females.
  • CT26 cells were purchased and placed in ATCC, and the cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO 2 . Passage every three days, and use cells within 15 generations.
  • Tumor generation 5 ⁇ 10 6 CT26 cells were injected subcutaneously into the back of nude mice. When the tumor size reaches 800-1000mm 3 , randomization will be performed. Then the tumor tissue was extracted and cut into 100mm 3 tumor tissue pieces and transplanted into the liver of BALB/c mice in situ.
  • Example 20 Pharmacodynamic study of QHL-087-DOX in CT26 tumor of orthotopic liver transplantation.
  • Test purpose To study the efficacy of QHL-087-DOX, PD-1 and their combination in orthotopic transplantation of CT26 tumors.
  • Test drug QHL-087-DOX 18 micromol/kg, mouse PD-1 5 mg/kg.
  • CT26 tumor cells are from ATCC. The cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO 2 . Cells were passaged every three days, and cells within the 15th passage were used. 5 ⁇ 10 5 CT26 cancer cells were injected subcutaneously into the back of nude mice. After the tumor reached 800-1000 mm 3 , the mice were randomly divided into groups. Then the tumor tissue was extracted and cut into 100mm 3 tumor tissue pieces and transplanted into the liver of BALB/c mice in situ. One week later, when the orthotopic transplanted tumor grew up, the mice with orthotopic transplanted tumor were randomly divided into groups.
  • mice in a group are treated with drugs.
  • the day of treatment is the first day.
  • IV drugs are administered once a week for 3 weeks.
  • Mouse PD-1 antibody was injected intravenously (IV) twice a week for 3 weeks. See Figure 10 for grouping and test results.
  • Example 21 The effect of EMC-AANL-DOX (legubicin), lenvatinib and PD-1 in the treatment of liver cancer in situ
  • Test purpose EMC-AANL-DOX, lenvatinib and PD-1 combined with each other in the treatment of liver cancer in situ.
  • Test drugs EMC-AANL-DOX 18 micromol/kg, mouse PD-1 5 mg/kg.
  • CT26 tumor cells are from ATCC. The cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO 2 . Cells were passaged every three days, and cells within the 15th passage were used. 5 ⁇ 10 5 CT26 cancer cells were injected subcutaneously into the back of nude mice. After the tumor reached 800-1000 mm 3 , the mice were randomly divided into groups. Then the tumor tissue was extracted and cut into 100mm 3 tumor tissue pieces and transplanted into the liver of BALB/c mice in situ. One week later, when the orthotopic transplanted tumor grew up, the mice with orthotopic transplanted tumor were randomly divided into groups.
  • mice in a group were treated with drugs.
  • the day of treatment is the first day.
  • intravenous (IV) drugs are injected once a week for 3 weeks.
  • Mouse PD-1 antibody was injected intravenously (IV) twice a week for 3 weeks. See Figure 11 for grouping and test results.
  • Example 22 Study on the curative effect of some compound injections of the present invention on human liver cancer HepG2 cells in nude mice
  • Test purpose To study the anti-tumor efficacy of some of the compounds of the present invention in a mouse tumor treatment model.
  • Test drug The corresponding compound injection and control injection in the table, diluted with normal saline to the corresponding concentration during the test.
  • Human liver cancer HepG2 cells were purchased from ATCC and identified according to the instructions provided by ATCC. The cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO 2 . Passage every three days, and use cells within 15 generations.
  • Tumor generation 5 ⁇ 10 6 HepG2 cells were injected subcutaneously into the back of nude mice. When the tumor size reached 100mm3, randomized grouping was performed. Then start treatment, and the day the treatment starts is recorded as the first day.
  • the drug is injected intravenously (IV).
  • the compound and the control drug were administered at a dose of 54umol/kg, and DOX can only be used at a dose of 18umol/kg due to toxicity limitations.
  • the control group was given saline. It is administered once a week for 4 weeks.
  • EMC-AANL-DOX has a better effect on liver cancer. Compared with the use of the same molar dose of EMC-AANL-DOX, the compound of the present invention has a better effect on liver cancer. The therapeutic effect of growth is increased.
  • Example 23 Study on the efficacy of QHL-096-DOX, QHL-087-DOX, QHL-090-DOX, QHL-093-DOX, QHL-117-DOX in the treatment of CT26 tumor immune model
  • Test purpose To study the anti-tumor efficacy of the above compounds in immunotherapy in CT26 tumor model.
  • Test drugs QHL-096-DOX, QHL-087-DOX, QHL-090-DOX, QHL-093-DOX, QHL-117-DOX and control group, dose 36umol/k, mouse PD-1 antibody, 5mg/ kg.
  • Test animals BALB/c mice aged 6-8 weeks, all female mice.
  • CT26 cells were purchased and placed in ATCC, and the cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO 2 . Passage every three days, and use cells within 15 generations.
  • the treatment process The drug was administered at an equimolar dose of 36umol/kg.
  • the control group was given saline. It is administered once a week for three weeks.
  • Example 24 Study on the efficacy of QHL-087-DOX injection in a variety of tumor models
  • Test purpose To study the anti-tumor spectrum of QHL-087 through multiple tumor models in mice.
  • Test drug Inject QHL-087-DOX and dilute with normal saline to the corresponding concentration during the test.
  • the corresponding tumor cells were purchased from the American Type Culture Collection (ATCC) and were identified according to the specifications provided by ATCC. The cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO 2 . Cells were passaged every three days, and cells within the 15th passage were used.
  • ATCC American Type Culture Collection
  • QHL-087-DOX is administered at a dose of 36umol/kg.
  • the control group was given saline. It is administered once a week for three weeks.
  • Table 8 The therapeutic effect of QHL-087-DOX in a variety of tumor models
  • Example 25 Comparison of the solubility of HSA-EMC-AANL-DOX, HSA-QHL-087-DOX, and HSA-QHL-087-N-CBP with the control compound
  • the lyophilized products EMC-AANL-DOX, HSA-EMC-AANL-DOX, HSA-QHL-087-DOX and HSA-QHL-087-N-CBP prepared in the embodiment of the present invention are subpackaged in a sterile room, Reconstitute with water for injection.
  • HSA-EMC-AANL-DOX, HSA-QHL-087-DOX and HSA-QHL-087-N-CBP can be completely dissolved, as shown in Table 9.
  • HSA-EMC-AANL-DOX, HSA-QHL-087-DOX and HSA-QHL-087-N-CBP are used as macromolecular proteins Class drugs can be directly dissolved to high concentration with water for injection or physiological saline, without the use of irritating organic solvents required for EMC-AANL-DOX dissolution. Unlike EMC-AANL-DOX small molecule compound drugs that are insoluble in water, the change in solubility characteristics has a huge impact on the distribution and metabolism of the drug and the mode of action of the drug.
  • Example 26 Comparison of the solution stability of the water-soluble and highly effective targeted activation of doxorubicin derivatives prepared in the examples of the present invention and the control compound
  • EMC-AANL-DOX Accurately weigh the compounds EMC-AANL-DOX, HSA-EMC-AANL-DOX, QHL-087-DOX, HSA-QHL-087-DOX, QHL-087-N-CBP and HSA-QHL-087-N-CBP , Divide each 5.0mg sample in a sterile room, add 0.5ml sterile water for injection to prepare 10mg/ml mother liquor, EMC-AANL-DOX needs 50% ethanol to dissolve. Take 30ul mother liquor and add 570ul buffer solution with different pH value of 5.5 to prepare 0.5mg/ml sample solution.
  • Example 27 HSA-EMC-AANL-DOX, HSA-QHL-087-DOX and HSA-QHL-087-N-CBP activation efficiency test
  • EMC-AANL-DOX is dissolved in solvent (50% water for injection + 50% alcohol), HSA-EMC-AANL-DOX, HSA-QHL-087-DOX and HSA-QHL-087-N-CBP are dissolved in water for injection uniformly. And diluted 10 times with water to 1 mg/ml.
  • 1 mg/ml sample compound is added to 100 micrograms of acidified tumor tissue homogenate (pH 6.0) at 37 degrees, the enzyme in the tumor tissue homogenate can cause the release of doxorubicin, The reduction of compounds and the increase of doxorubicin can be detected by HPLC to compare the activation efficiency of drugs in tumor tissues.
  • Table 11 The activation ratio of EMC-AANL-DOX, HSA-EMC-AANL-DOX, HSA-QHL-087-DOX, and HSA-QHL-087-N-CBP in different tumor tissue homogenates (%)
  • Example 28 EMC-AANL-DOX, HSA-EMC-AANL-DOX, QHL-087-DOX, HSA-QHL-087-DOX, QHL-087-N-CBP and HSA-QHL-087-N-CBP Toxicity determination
  • Test purpose To understand the acute toxicity of the drug organism of the present invention by measuring the MTD experiment of intravenous medication in mice.
  • Test drug EMC-AANL-DOX is dissolved in solvent (50% water for injection, 50% alcohol), HSA-EMC-AANL-DOX, QHL-087-DOX, HSA-QHL-087-DOX, QHL-087-N- CBP and HSA-QHL-087-N-CBP are dissolved in water for injection, and diluted with normal saline to the corresponding dose during the test.
  • mice BALB/C mice (purchased from Shanghai Slack Laboratory Animal Co., Ltd.), weighing 19-21 g, all females.
  • mice weighing 19-21 g, all females were randomly divided into 7 groups according to their body weight, with 6 mice in each group.
  • EMC-AANL-DOX, HSA-EMC-AANL-DOX, QHL-087-DOX, HSA-QHL-087-DOX, QHL-087-N-CBP and HSA- QHL-087-N-CBP were randomly divided into 7 groups according to their body weight, with 6 mice in each group.
  • Table 12 EMC-AANL-DOX, HSA-EMC-AANL-DOX, QHL-087-DOX, HSA-QHL-087-DOX, QHL-087-N-CBP and HSA- QHL-087-N-CBP.
  • a control experiment of the saline group and the paclitaxel group injection commercially available, Beijing Yuekang was carried out, and the administration volume of each mouse was 0.2ml.
  • Table 12 Comparison of the mortality results of test mice receiving different doses of compound injections, saline and paclitaxel injections
  • Example 29 Combined therapeutic effect of HSA-EMC-AANL-DOX, HSA-QHL-087-DOX and anti-PD-1 antibody
  • Test purpose To compare the therapeutic effects of EMC-AANL-DOX, HSA-QHL-087-DOX and anti-PD-1 antibody.
  • Test drugs HSA-EMC-AANL-DOX and HSA-QHL-087-DOX, the doses were 18 ⁇ mol/kg; mouse PD-1 antibody, 5 mg/kg.
  • Test animals BALB/c mice aged 6-8 weeks, all female mice.
  • CT26 cells were purchased and placed in ATCC, and the cells were cultured in DMEM medium containing 10% fetal bovine serum at 37° C. and 5% CO 2 . Passage every three days, and use cells within 15 generations. 5 ⁇ 10 6 CT26 cancer cells were injected subcutaneously into mice. The mice were injected with the drug 3 times, once a week, and the anti-PD-1 antibody twice a week, a total of 8 times.
  • Example 30 Determination of the inhibitory effect of N-CBP and HSA-QHL-095-N-CBP on tumor cell growth by MTT method
  • the cell concentration was adjusted with culture medium, and the cells were seeded on a 96-well culture plate with 100 ⁇ l cell suspension per well, 100,000 CD8+ T cells/well, and 20,000 CT26 tumor cells/well. Place the 96-well culture plate in a 37°C, carbon dioxide (5%) incubator overnight for 24 hours. After 24 hours, add 100ul of cell culture medium containing different concentrations of drugs to the 96-well culture plate, and set the control wells (0.1% DMSO) that do not add drugs but only add the corresponding drug solvents, and set the adjustment that only add medium without cells. Zero hole (Blank).
  • Example 31 The therapeutic effect of HSA-QHL-095-N-CBP as a single agent and combined with anti-PD-1 antibody
  • Test purpose To compare the therapeutic effects of carboplatin, HSA-QHL-095-N-CBP, and anti-PD-1 antibody.
  • Test drug carboplatin, HSA-QHL-095-N-CBP, dose 18 ⁇ mol/kg, mouse PD-1 antibody, 5mg/kg.
  • Test animals BALB/c mice aged 6-8 weeks, all female mice.
  • CT26 cells were purchased and placed in ATCC, and the cells were cultured in DMEM medium containing 10% fetal bovine serum at 37° C. and 5% CO 2 . Passage every three days, and use cells within 15 generations. 5 ⁇ 10 6 CT26 cancer cells were injected subcutaneously into mice.
  • Treatment process Inject the corresponding drug once a week for 3 weeks; anti-PD-1 antibody is administered once a week for 4 weeks.
  • Example 32 Influence of inflammatory response in non-alcoholic fatty liver (NAFLD) model mice.
  • mice were randomly divided into normal group (standard feed), model group (high-fat feed), simvastatin group (positive control, 3mg/kg) and drug group dose group (50mg/kg), each group 6 only.
  • the mice in the normal group were fed with standard diets, and the mice in the other groups were fed with high-fat diets to induce NAFLD models.
  • mice in each group were given IV doses of 48umol/kg drugs twice a week for a total of 8 weeks. 12h after the last administration, the automatic biochemical analyzer measured the serum biochemical indicators: high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein (LDL-C). The results are shown in Table 13.
  • HDL-C high-density lipoprotein cholesterol
  • LDL-C low-density lipoprotein

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Abstract

一种靶向递送和激活的免疫刺激性偶联复合物的制备和用途。具体而言，提供式MI-S-C-A所示的化合物，用做连接臂，以及式MI-S-C-A-D所示的连接了药物的药物化合物。该药物化合物具有改善的水溶性、降低的细胞毒性、增强的药物活性。

Description

靶向递送和激活的免疫刺激性偶联复合物的制备和用途 技术领域
本发明涉及一种抗肿瘤药物化合物，具体地，涉及一种靶向递送和激活的免疫刺激性偶联复合物的制备和用途
背景技术
Legumain首先在豆科植物种子中被鉴定为天冬酰胺内肽酶，属于半胱氨酸蛋白酶C13家族的成员。在种子萌发过程中Legumain可处理储藏蛋白。之后Legumain在寄生虫和包括人类的哺乳动物中的发现证明该蛋白酶具有很高的保守性。1997年首次克隆并鉴定猪源Legumain。多数实体瘤中高表达Legumain。Legumain在正常组织和肿瘤组织的差异表达使其成为一个理想的治疗肿瘤的靶点。Legumain是一种肽链内肽酶，能在弱酸性条件下特异性剪切肽链上天冬酰胺C端的肽键。CN 201210573744.3公开了天冬氨酸酶靶向激活的多肽阿霉素衍生物，通过Legumain切割四肽基团(linker)而在肿瘤中释放Leu-阿霉素化合物。
发明内容
本发明通过进一步化合物筛选和生物学的系统研究，开发出化学修饰的激活连接臂(chemical modified linker)，其能进一步增强了激活效率。此外，本发明的化学修饰的激活连接臂能增强偶联药物对免疫细胞的选择性，在治疗中产生免疫治疗增强特性，并增强与PD-1抗体联合的协同疗效。
本发明所要解决的技术问题在于创造高效和特异性选择的偶联臂。之前的研究发现天冬酰胺肽链内切酶优选识别四肽的底物肽序列，并切断Asn与其他残基之间的酰胺键。本发明提高激活效率的思路是：首先通过在三肽(如AAN)两端合成大量结构不同的化合物，然后用这些化合物进一步研究天冬酰胺肽链内切酶的工作机理。根据天冬酰胺肽链内切酶晶体结构作图(图1)，由于天冬酰胺肽链内切酶的活中心位于内陷的底部，因此底物肽被激活需要接近底部的酶活性中心；图中天冬酰胺肽链内切酶的S1位置的C189攻击切断Asn肽链，而相邻位置S2、S3、S4和S1与底物相结合决定了酶切的效率，S2、S3对应于底物肽的Ala-Ala氨基酸。考虑到连接阿霉素 等化合物和MI基团可能带来的相互作用或空间阻碍，本试验进一步在底物三肽两端大量合成和筛选化学修饰结构。我们将合成的不同基团的化合物与MI和阿霉素连接后，通过肿瘤组织或天冬酰胺肽链内切酶的条件下进行激活效率的筛选，从而优化得到新的具有相互构效关系的化合物偶联体，该结构的示意图见图2，包括MI基团，选择性基团S，天冬酰胺肽链内切酶切割的三肽基团C，辅助基团A和被偶联的药物。本发明增加的基团带来了新的功能：除了增强天冬酰胺肽链内切酶对偶联的药物化合物(D)的激活活性外，还改善了药物化合物的物理性质和生物功能。本发明提供的药物化合物是亲水性的，其细胞膜通透性改变，因此是最适合开发成药的化合物。此外，本发明还发现，本发明式(II)药物化合物具有细胞选择性，能特异性地被肿瘤相关巨噬细胞吞噬，攻击或抑制肿瘤相关巨噬细胞、MDSC细胞，从而解除肿瘤相关巨噬细胞对免疫的抑制作用，促进免疫治疗。本发明还发现，当偶联阿霉素时，对S基团得长短影响激活效率，S的链长越长，由于空间位阻的关系反而不利于化合物与酶的结合，激活效率降低。
人血清白蛋白(HSA)是小的球形蛋白质，由585个氨基酸组成(66-69kd)，有许多带电的残基(例如赖氨酸、天冬氨酸和没有辅基的基团或者碳水化合物)，有少量的色氨酸或者甲硫丁氨酸残基。本发明式(II)化合物在与人血白蛋白偶联的34位半胱氨酸偶联为大分子药物；通过实验发现，白蛋白共价偶联的本发明式(II)化合物或EMC-AANL-DOX，具有降低的毒性，提高的药物稳定性和大大提高的治疗疗效。
综上所述，本发明的式(I)连接臂和式(II)药物化合物具有增强的激活效率，对免疫细胞的选择性增强，组织选择性增强，适宜的水溶性和脂溶性以及药物稳定性。
因此，本发明提供本文所述式(I)的化合物(连接臂)和式(II)所示的药物化合物(偶联物)及其药学上可接受的盐。
本发明还提供有下式结构的铂衍生物或其药学上可接受的盐：
Figure PCTCN2021077056-appb-000001
本发明还提供与白蛋白共价连接的本发明式(II)所示的药物化合物或其药学上可接受的盐，以及与白蛋白共价连接的EMC-AANL-DOX；优选地，白蛋白通过其36位的半胱氨酸残基与式(II)的MI或EMC部分连接。
本发明还提供一种药物组合物，所述药物组合物含有本发明式(II)化合物或其药学上可接受的盐、本发明所述的铂衍生物或其药学上可接受的盐、与白蛋白共价连 接的本发明式(II)所示的药物化合物或其药学上可接受的盐、或与白蛋白共价连接的EMC-AANL-DOX或其药学上可接受的盐，以及药学上可接受的载体。
本发明还提供式(II)化合物或其药学上可接受的盐、本发明所述的铂衍生物或其药学上可接受的盐、与白蛋白共价连接的本发明式(II)所示的药物化合物或其药学上可接受的盐、或与白蛋白共价连接的EMC-AANL-DOX或其药学上可接受的盐在制备治疗或预防癌症、脂肪肝(包括酒精和非酒精性脂肪肝)、脂肪性肝炎、脂肪性肝病、肝脏纤维化、肝部炎症、肝细胞损伤的脂肪变性现象的药物中的用途；优选地，所述癌症为实体癌或血液肿瘤，优选为膀胱、脑、乳房/乳腺、宫颈、结肠、直肠、食管、肾、肝、肺、鼻咽、胰腺、前列腺、皮肤、胃、子宫、卵巢、睾丸和血液部位的癌症。
本发明还提供式(I)所述的化合物在增强化合物药物水溶性、降低药物毒性、提高药物疗效、和/或提到药物对免疫细胞的选择性中的应用，或在制备具有改善的水溶性、降低的药物毒性、提高的药物疗效、和/或提高的药物对免疫细胞的选择性的药物中的应用，或在制备将药物递送到肝脏的药物分子中的应用。
本发明还提供具有下式所示结构的EMC-AANL-DOX化合物或其偶联了白蛋白的药物(优选地，通过白蛋白第36位的半胱氨酸残基与所述EMC部分进行共价连接)在制备治疗肝癌的药物中的应用，以及其与抗PD-1抗体和/或抗PD-L1抗体在制备用于联合治疗肿瘤的药物中的应用：
Figure PCTCN2021077056-appb-000002
本发明还提供式(II)化合物或其药学上可接受的盐、本发明所述的铂衍生物或其药学上可接受的盐、与白蛋白共价连接的本发明式(II)所示的药物化合物或其药学上可接受的盐、或与白蛋白共价连接的EMC-AANL-DOX或其药学上可接受的盐在制备抑制免疫抑制性细胞、抑制肿瘤相关巨噬细胞、抑制MDSC细胞、抑制血管新生、促进抗肿瘤免疫和/或促进T淋巴细胞增殖的药物中的应用。
本发明还提供式(II)化合物或其药学上可接受的盐、本发明所述的铂衍生物或其药学上可接受的盐、与白蛋白共价连接的本发明式(II)所示的药物化合物或其药学上可接受的盐、或与白蛋白共价连接的EMC-AANL-DOX或其药学上可接受的盐与抗PD-1抗体在制备用于联合治疗肿瘤的药物中的应用。
附图说明
图1：天冬酰胺肽链内切酶晶体结构和作用底物图。
图2：靶向递送和激活的免疫刺激性偶联复合物示意图。
图3：优选化合物的酶切动力学比较。
图4：小鼠骨髓单个核细胞分离及M2巨噬细胞的诱导分化。
图5：化合物对CD8+T细胞的细胞毒实验。
图6：化合物对M2巨噬细胞的细胞毒实验。
图7：化合物对HT1080肿瘤的疗效实验。
图8：EMC-AANL-DOX具有肝脏和肝癌组织的高分布特征。
图9：QHL-087-DOX具有肝脏和肝癌组织的高分布特征。
图10：QHL-087-DOX与抗PD-1抗体联和治疗肝原位瘤。
图11：EMC-AANL-DOX与抗PD-1抗体联合治疗肝原位瘤疗效优于仑伐替尼与联合抗PD-1抗体联合治疗。
图12：HSA-EMC-AANL-DOX，QHL-087-DOX与抗PD-1抗体联合治疗效果
图13：N-CBP的体外细胞毒性实验。
图14：HSA-QHL-095-N-CBP细胞毒实验。
图15：HSA-QHL-095-N-CBP的单药以及与抗PD-1抗体联合治疗效果。
具体实施方式
下面结合具体实施例对本发明的技术方案做进一步的说明。
一、连接臂化合物
本发明提供具有下式(I)所示结构的化合物，该化合物可用作连接臂，当与感兴趣的药物(如抗癌化合物)连接时，可增强化合物药物水溶性、降低药物毒性、提高药物疗效、和/或提到药物对免疫细胞的选择性：
MI-S-C-A    (I)
式中，MI为马来酰亚胺基；S为提高酶切效率或提高选择性的基团；C为蛋白水解酶可断裂的氨基酸连接臂；和A为辅助连接臂。
示例性的MI为下式所示的马来酰亚胺基团：
Figure PCTCN2021077056-appb-000003
其中，波浪线表示与S的连接位置。
在一些实施方案中，式(I)中的S可表示为S1-S2-S3，其中S1选自：
Figure PCTCN2021077056-appb-000004
其中，Rx不存在或选自：C 1-6亚烷基，C 1-6亚烷基氨基、C 1-6亚烷基羧基和C 1-6亚烷基羰基氨基，波浪线表示与相邻部分的连接位置；S2不存在或为-[(CH 2) pO] q-，其中，p为1-4的整数、优选为2，q为0-15、优选为1-15、更优选为2-6的整数；S3不存在或选自极性氨基酸残基，如：Glu、Asp、Gly、Ala、Val、Leu、Ile、Met、Phe、Trp、Ser、Thr、Cys、Tyr、Asn、Gln、Lys、Arg和His，优选Glu和Asp。
应理解，S1、S2和S3中至少存在一个。
优选地，MI、S1、S2、S3、C和A相互之间通过以下任一方式连接：
Figure PCTCN2021077056-appb-000005
其中，波浪线表示与相邻部位的连接部位；优选地，S通过选自以下的基团与C连接：
Figure PCTCN2021077056-appb-000006
在一些实施方案中，S为-R 1-[(CH 2) pO] q-R 2-R 3-，其中，R 1与MI连接，不存在或选自C 1-6亚烷基或C 1-6亚烷基羰基氨基；R 2选自C 1-6亚烷基；R 3选自-C(O)O-、-NH-、-O-或-C(O)-R 4，其中，R 4为选自Glu、Asp、Gly、Ala、Val、Leu、Ile、Met、Phe、Trp、Ser、Thr、Cys、Tyr、Asn、Gln、Lys、Arg和His，且优选Glu和Asp的氨基酸残基，且R 4通过其氨基与该-C(O)-形成酰胺键；p为1-4的整数；q为0-15、优选1-15、更优选2-6的整数。优选地，R 1不存在，p为2或3，q为1-15、优选2-6，R 2为C 1-4亚烷基，R 3选自-C(O)O-、-NH-和-O-。在一些实施方案中，优选地，R 1不存在，q为0，R 2为C 1-6亚烷基，R 3为-C(O)-R 4，R 4优选为Glu和Asp，且R 4通过其氨基与该-C(O)-形成酰胺键。在一些实施方案中，优选地，R1为C 1-6亚烷基羰基氨基，p为2或3，q为1-15、优选2-6，R 2为C 1-4亚烷基，R 3为-C(O)-R 4，R 4优选为Glu 和Asp，且R 4通过其氨基与该-C(O)-形成酰胺键。
示例性的MI-S选自：
Figure PCTCN2021077056-appb-000007
Figure PCTCN2021077056-appb-000008
Figure PCTCN2021077056-appb-000009
优选地，与上述任一MI-S相连的C为AAN，A为下文所述的任一结构。
优选地，本发明式(I)化合物中，C选自肿瘤微环境中表达天冬酰胺肽链内切酶断裂的基团，且该基团中包含Asn残基。在一些实施方案中，C为X 1X 2X 3，其中X 1选自L或D型Ala、Thr、Val和Asn；X 2选自L或D型Ala、Thr、Val和Ile；X 3为Asn，优选不为D-Asn。示例性的C选自：Ala-Ala-Asn，Thr-Ala-Asn，Val-Ala-Asn，Asn-Ala-Asn，Thr-Thr-Asn，Val-Thr-Asn，Asn-Thr-Asn，Ala-Val-Asn，Thr-Val-Asn，Val-Val-Asn，Asn-Val-Asn，Ala-Ile-Asn，Thr-Ile-Asn，Val-Ile-Asn，Asn-Ile-Asn，Ala-Thr-Asn，D-Thr-L-Val-L-Asn，D-Thr-L-Ala-L-Asn，D-Ala-L-Val-L-Asn，L-Thr-D-Val-L-Asn，L-Thr-D-Ala-L-Asn，L-Ala-D-Val-L-Asn，D-Thr-D-Val-L-Asn，D-Thr-D-Ala-L-Asn，D-Ala-D-Val-L-Asn。在一些特别优选的实施方案中，C为AAN。
本发明式(I)化合物中，优选A选自为Leu、PABC-OH和PABC-NH 2，其结构分别如下式所示：
Figure PCTCN2021077056-appb-000010
其中，波浪线表示与C的连接位置。
在一些实施方案中，本发明式(I)化合物中S和A选自以下组1-137中的任意一组[式中“2peg”表示-(CH 2CH 2O) 2-，3peg表示-(CH 2CH 2O) 3-，4peg表示-(CH 2CH 2O) 4-，6peg表示-(CH 2CH 2O) 6-)，依次类推]：
Figure PCTCN2021077056-appb-000011
Figure PCTCN2021077056-appb-000012
Figure PCTCN2021077056-appb-000013
Figure PCTCN2021077056-appb-000014
Figure PCTCN2021077056-appb-000015
Figure PCTCN2021077056-appb-000016
优选地，本发明式(I)化合物中，MI为马来酰亚胺基，S和A为QHL-001到QHL-162中的任一组，C为AAN。
本发明特别优选的式(I)化合物(连接臂)选自：QHL-005、QHL-006、QHL-008、QHL-086、QHL-087、QHL-089、QHL-090、QHL-092、QHL-093、QHL-095、QHL-096、QHL-098、QHL-099、QHL-101、QHL-102、QHL-104、QHL-105、QHL-107、QHL-108、QHL-116、QHL-119、QHL-138、QHL-140、QHL-141、QHL-143、QHL-144、QHL-146、QHL-147、QHL-150、QHL-153、QHL-154、QHL-155、QHL-156、QHL-157、QHL-158、QHL-159、QHL-160、QHL-161和QHL-162中的任意一个，更优选为QHL-086、QHL-087、QHL-089和QHL-090中的任意一个。
二、药物化合物
本发明提供下式(II)所示的化合物(偶联物)或其药学上可接受的盐：
MI-S-C-A-D    (II)
式中，MI、S、C和A形成本发明任意实施方案所述的连接臂化合物；D为药物，优选为抗癌化合物。
式II中，当A作为连接基团时，其选自：
Figure PCTCN2021077056-appb-000017
其中，波浪线表示与C和D的连接位置。优选地，通过-NH-与C连接。
优选地，D选自雷西莫特、泼尼松、三碘甲状腺原氨酸(T3)、阿霉素、柔红霉素、表阿霉素、甲氨蝶呤、氟达拉滨、吉西他滨、阿糖胞苷、美法仑、尼莫司汀、米托蒽醌、丝裂霉素、喜树碱、10-羟基喜树碱、拓扑替康、氟脲苷、去氧氟尿苷、依托泊苷、氟达拉滨、卡培他滨、长春新碱、埃坡霉素B、紫杉醇、多烯紫杉醇、达拉非尼、多韦替尼、莫特塞尼、化合物a、化合物b和下式所示铂类衍生物：
Figure PCTCN2021077056-appb-000018
其中，所述化合物a和化合物b的结构如下：
Figure PCTCN2021077056-appb-000019
Figure PCTCN2021077056-appb-000020
更优选地，D选自柔红霉素、多韦替尼、表阿霉素、化合物a、化合物b、丝裂霉素、达拉非尼、莫特塞尼、雷西莫特、泼尼松和T3。优选地，用于与这些药物(D)连接的本发明式(I)化合物(连接臂)选自：QHL-005、QHL-006、QHL-008、QHL-086、QHL-087、QHL-089、QHL-090、QHL-092、QHL-093、QHL-095、QHL-096、QHL-098、QHL-099、QHL-101、QHL-102、QHL-104、QHL-105、QHL-107、QHL-108、QHL-116、QHL-119、QHL-138、QHL-140、QHL-141、QHL-143、QHL-144、QHL-146、QHL-147、QHL-150、QHL-153、QHL-154、QHL-155、QHL-156、QHL-157、QHL-158、QHL-159、QHL-160、QHL-161和QHL-162中的任意一个，更优选为QHL-086、QHL-087、QHL-089和QHL-090中的任意一个。
优选地，A与D之间通过以下任一方式连接：
Figure PCTCN2021077056-appb-000021
其中，波浪线表示与相邻部位的连接部位。
更优选地，A与D之间通过-CO-NH-方式连接，其中，该羰基与A连接或者为A的一部分(如当A为Leu时)，该氨基与D连接或者为D的一部分。通常，药物化合物与A连接的位置不影响到药物的生物学活性，例如连接位置远离药物化合物的活性中心。
优选地，本发明所述的式(II)药物化合物选自：
Figure PCTCN2021077056-appb-000022
Figure PCTCN2021077056-appb-000023
Figure PCTCN2021077056-appb-000024
Figure PCTCN2021077056-appb-000025
Figure PCTCN2021077056-appb-000026
Figure PCTCN2021077056-appb-000027
Figure PCTCN2021077056-appb-000028
Figure PCTCN2021077056-appb-000029
Figure PCTCN2021077056-appb-000030
在本发明的一些实施方案中，本发明还提供具有下式结构的铂衍生物、其前药或其药学上可接受的盐：
Figure PCTCN2021077056-appb-000031
本发明的药物组合物可与白蛋白共价偶联形成新的药物化合物。因此，本发明也包括与白蛋白共价连接的本发明式(II)药物化合物。通常，白蛋白与连接臂的MI连接。在一些实施方案中，本发明还包括与白蛋白连接的EMC-AANL-DOX、其药物组合物及其应用。本发明也与白蛋白共价连接的本发明式(II)药物化合物的或其药学上可接受的盐。
本发明中，药学上可接受的盐可以是本领域周知的各种药学上可接受的盐，包括 无机和有机酸盐，例如盐酸盐、氢溴酸盐、磷酸盐、硫酸盐、柠檬酸盐、乳酸盐、酒石酸盐、马来酸盐、富马酸盐、扁桃酸盐和草酸盐；以及与碱例如钠羟基、三(羟基甲基)胺基甲烷(TRIS，胺丁三醇)和N-甲基葡糖胺形成的无机和有机碱盐。
三、制备方法
本发明式(I)和(II)化合物的示例性制备方法包括：
步骤1：制备三肽-PABC或四肽：偶联氨基酸残基，并分离得到所形成的三肽-PABC或四肽，即C-A；
步骤2：制备MI-S：选取适合MI-S基团的化合物，进行缩合或者环合，得到一端带有羧基的MI-S；
步骤3：制备MI-S-C-A：将步骤1所得C-A和步骤2所得MI-S通过氨基、羧基偶联得到中间体(MI-S-C-A)；
步骤4：将步骤3所得到的化合物MI-S-C-A的A端的羧基或羟基活化产物与可选药物的氨基共价结合，形成靶向递送和激活的免疫刺激性阿霉素偶联复合物。
当A是PABC-OH时，合成路线包括：运用已知的化学、生物学重组偶联技术偶联适用于本发明的氨基酸残基与PABC，然后纯化分离得到含有适当氨基酸保护基的C-PABC；反应可在缩合剂、碱、极性非质子溶剂的存在下进行。然后脱除保护基，获得C-PABC。之后在缩合剂、碱、极性非质子溶剂的存在下使C-PABC与包含MI-S基团的酸或酯或酰氯反应，形成本发明式(I)所示的MI-S-C-A，然后再在缩合剂、碱、极性非质子溶剂的存在下与感兴趣的药物化合物或其盐反应，形成本发明式(II)所示的药物化合物
在该制备方法中所使用的碱的例子包括例如有机碱，如三乙胺、吡啶、N，N-二异丙基乙胺、4-二甲氨基吡啶、1，2，2，6，6-五甲基哌啶等，或无机碱，如碳酸钠、碳酸钾、碳酸氢钠和碳酸氢钾等。在该制备方法中所使用的缩合剂的例子包括，如HBTU、DMC、HATU、HOBT、DIC、DCC、EDCI、DEPBT等，在该制备方法中所使用的溶剂可以是任何一种溶剂，只要该溶剂本身在反应中是惰性的，且不会抑制反应就可以。这样的溶剂包括卤代烃溶剂，如二氯甲烷和氯仿等，芳族烃类溶剂，如苯和甲苯等，非质子传递溶剂，如乙腈、N，N-二甲基甲酰胺、二甲基亚砜等，酯类溶剂，如乙酸甲酯和乙酸乙酯等，醚类溶剂，如四氢呋喃，或这些溶剂的混合物。该制备方法中的反应可在冰冷却下至150℃的温度范围内进行。
四、药物组合物
本发明包括药物组合物，该药物组合物含有本发明式(II)所述的化合物或其药学上可接受的盐，或含有本发明所述的铂衍生物或其药学上可接受的盐，或含有与白蛋白共价连接的式(II)化合物或其药学上可接受的盐，或含有与白蛋白共价偶联的EMC-AANL-DOX或其药学上可接受的盐。
药物组合物中还可含有药学上可接受的载体或赋形剂。载体或赋形剂可以是本领域周知的各种药学上可接受的载体或赋形剂，并依药物剂型或施用方式不同而不同。
在一具体实施例中，药物组合物中含有溶媒、增溶剂/助溶剂、pH调节剂、冻干赋形剂和渗透压调节剂中的一种或多种。
适用于本发明的冻干赋形剂包括糖类(例如乳糖、麦芽糖、右旋糖酐、葡萄糖、果糖)、氨基酸(例如精氨酸、赖氨酸、组氨酸)、甘露醇、酒石酸、马来酸、柠檬酸、氯化钠和环糊精(例如羟丙基β环糊精、磺丁基β环糊精)中的一种或多种。
适用于本发明的pH调节剂包括盐酸、磷酸、硫酸、碳酸、硝酸、醋酸、枸橼酸、DL-酒石酸、D-酒石酸、L-酒石酸、氢氧化钠、氢氧化钾、葡甲胺、马来酸、乙二胺、三乙胺、精氨酸、赖氨酸、组氨酸、磷酸二氢钠和磷酸氢二钠中的一种或多种。
适用于本发明的溶媒优选为有机溶媒，包括乙醇、丙二醇、聚乙二醇300、聚乙二醇400、叔丁醇、甘油、吐温、大豆油、羟丙基β环糊精溶液和磺丁基β环糊精溶液中的一种或多种。
适用于本发明的渗透压调节剂包括葡萄糖、氯化钠、甘露醇和乳酸钠中的一种或多种。
适用于本发明的增溶剂/助溶剂包括吐温80、吐温60、波洛沙姆、羟丙基β环糊精、聚乙二醇(PEG)、十二羟基硬脂酸锂、磺丁基β环糊精、PVP、甘油和聚氧乙烯蓖麻油中的一种或多种。
一般情况下，对哺乳动物每天口服给予本发明化合物或其药学上可接受的盐，药量通常为约0.0025到50毫克/公斤体重，最好是约0.01到10毫克/公斤体重。如果同时施用一个已知的抗癌药物或施与其它治疗，其剂量应可有效地实现其预期的目的。这些已知的抗癌药物的最佳剂量是本领域技术人员所熟知的。
单位口服剂量可以包括约0.01到50毫克，最好是约0.1到10毫克的本发明化合物或其药学上可接受的盐。单位剂量可给予一次或多次，每天为一剂或多剂，每剂含有约0.1到50毫克，合宜地约0.25到10毫克的本发明化合物或其药学上可接受的盐。
本发明药物组合物可以被制备成任何合适的剂型，包括但不限于片剂、胶囊、注射剂等。可通过本领域周知的途径给予本发明的药物组合物，例如口服、静脉注射、肌肉内注射等。
五、化合物和药物组合物的用途
肿瘤分泌的细胞因子诱导单核细胞转化为肿瘤相关巨噬细胞(TAM)，肿瘤相关巨噬细胞能够刺激产生强烈的免疫抑制，并能直接帮助肿瘤细胞浸润和转移。肿瘤相关巨噬细胞(M2型)区别于单核细胞和炎症型巨噬细胞(M1型)的确认标记是天冬酰胺肽链内切酶的表达。本发明的化合物能在天冬氨酸肽链内切酶存在的条件下被激活释放。由于天冬酰胺肽链内切酶特异性激活的偶联体的不同部分对最终药物在靶向、激活、稳定、毒性和药效等功能产生巨大影响，因此，使用本发明天冬酰胺肽链内切酶特异性激活的偶联体能够有效降低被连接药物的毒性，使最终药物带有了新的靶向、激活和代谢特性，增加了治疗肿瘤的效果，并产生了新的肿瘤适应症和对抗肿瘤转移产生作用，产生了全新的结构和功能。
本发明还发现，本发明式(II)化合物具有杀伤肿瘤相关巨噬细胞，减弱微环境中免疫抑制的细胞因子和促进毒性CD8细胞的免疫增强现象。更重要的是，这些肿瘤微环境释放性化合物只在肿瘤局部激活，不同于传统化疗药物会损伤整体免疫系统。在实验中肿瘤微环境释放性化合物和PD-1(程序性死亡分子1，programmed death-1)抑制抗体(抗PD-L1抗体，市售，是目前认为具有免疫治疗效果的候选药物)具有强烈协同治疗作用，能够解决免疫治疗很难和化疗药物结合使用的弊端。
因此，本发明化合物、其药学上可接受的盐、或药物组合物可用来治疗或预防本领域已知雷西莫特、泼尼松、T3、阿霉素、柔红霉素、表阿霉素、甲氨蝶呤、氟达拉滨、吉西他滨、阿糖胞苷、美法仑、尼莫司汀、米托蒽醌、丝裂霉素、喜树碱、10-羟基喜树碱、拓扑替康、氟脲苷、去氧氟尿苷、依托泊苷、氟达拉滨、卡培他滨、长春新碱、埃坡霉素B、紫杉醇、多烯紫杉醇、达拉非尼、多韦替尼、莫特塞尼、化合物a、化合物b、以及铂化合物(如卡铂、顺铂、奥沙利铂)能够治疗的各种疾病，包括癌症、眼科疾病和肝脏疾病等。
例如，本领域已知，喜树碱可用来治疗或预防恶性肿瘤、银屑病、疣、急/慢性白血病以及血吸虫病引起的肝脾肿大等；10-羟基喜树碱可用于治疗胃癌、肝癌、头颈部癌及白血病等；紫杉醇主要用来治疗卵巢癌和乳腺癌，对肺癌、大肠癌、黑色素瘤、头颈部癌、淋巴瘤、脑瘤等也有疗效；丝裂霉素可用于治疗慢性淋巴瘤，慢性骨 髓性白血病，食管癌、胃癌，结肠癌、直肠癌，肺癌，胰癌，肝癌，子宫颈癌，子宫体癌，卵巢癌，乳癌，头颈部肿瘤，膀胱肿瘤，恶性腔内积液等。
因此，例如，可用本发明化合物、其药学上可接受的盐或药物组合物治疗或预防的疾病包括但不限于膀胱、脑、乳房/乳腺、宫颈、结肠-直肠、食管、肾、肝、肺、鼻咽、胰腺、前列腺、皮肤、胃、子宫、卵巢、睾丸和血液等部位的癌症。具体而言，这些癌症选自：肝癌、肾癌、甲状腺癌、结肠直肠癌、膀胱癌、脑癌、乳腺癌、宫颈癌、直肠癌、食管癌、肺癌(例如支气管肺癌，包括未分化小细胞性和非小细胞性)、鼻咽癌、胰腺癌、前列腺癌、皮肤癌、胃癌、子宫癌、卵巢癌、睾丸癌、血癌(例如慢性或急性白血病，包括淋巴细胞性和粒细胞性白血病)、恶性淋巴瘤、纤维素肉瘤、软组织肉瘤、成骨肉瘤、横纹肌肉瘤、尤文肉瘤、肾母细胞瘤、神经母细胞瘤、甲状腺癌和头颈部鳞癌。
在一个具体实施例中，本发明式(II)所示的D为丝裂霉素的药物化合物或其药学上可接受的盐还可用于治疗或预防眼科疾病，包括治疗或预防伤愈疤痕或脉络膜新生血管，或抑制巨噬细胞。在其它实施例中，式(II)所示的D为丝裂霉素的药物化合物或其药学上可接受的盐还可用于治疗或预防角膜移植、青光眼、翼状胬肉手术的后遗症等。
本发明的化合物或药物组合物还可用于阻止肿瘤转移，尤其是阻止肿瘤肺转移。在一个实施例中，本发明的化合物或药物组合物可用于阻止乳腺癌肺转移。
本发明所述的肝脏疾病包括但不限于脂肪肝(包括酒精和非酒精性脂肪肝)、脂肪性肝炎、脂肪性肝病、肝脏纤维化、肝部炎症、肝细胞损伤的脂肪变性现象。
因此，本发明包括疾病(优选为本发明任一实施方案所述的癌症、眼科疾病和肝脏疾病)治疗或预防方法，包括给予需要的对象治疗或预防有效量的本发明式(II)化合物或其药学上可接受的盐，或含有本发明式(II)化合物或其药学上可接受的盐的药物组合物。在一些实施方案中，给与或本发明所述的铂衍生物或其药学上可接受的盐，或与白蛋白共价连接的式(II)化合物或其药学上可接受的盐，或与白蛋白共价偶联的EMC-AANL-DOX或其药学上可接受的盐，或者它们各自的药物组合物。
本发明还包括阻止肿瘤转移的方法，包括给予需要的对象有效量的本发明化合物或其药学上可接受的盐，或含有本发明化合物或其药学上可接受的盐的药物组合物。阻止肿瘤转移包括但不限于阻止肿瘤肺转移和/或骨转移。
肿瘤相关巨噬细胞(TAM)作为一种关键的炎性细胞，在肿瘤相关炎症中扮演极其重要的角色。在肿瘤微环境中，TAM通过影响肿瘤各方面的生物学特性来促进 肿瘤发展。它分泌一些分子(如EGF)来直接促进肿瘤细胞的生长，促进血管新生，从而为癌细胞的浸润和转移创造条件，同时还能抑制获得性免疫行使功能。因此，本发明还包括抑制肿瘤相关巨噬细胞的方法，包括给予需要的对象有效量的本发明化合物或其药学上可接受的盐，或含有本发明化合物或其药学上可接受的盐的药物组合物。通过抑制肿瘤相关巨噬细胞，可抑制肿瘤生长、抑制血管新生，并抑制癌细胞的浸润和转移，促进抗肿瘤免疫，从而实现癌症的预防和/或治疗。在一个具体实施例中，肿瘤相关巨噬细胞表达天冬氨酸肽链内切酶，为M2型。
本发明上述方法可与本领域已知的放疗或免疫疗法联用。
因此，本发明还包括用于上述各种方法或用途的本发明化合物、其药学上可接受的盐，或本发明药物组合物。
本发明也包括本发明化合物或其药学上可接受的盐或本发明药物组合物在制备治疗或预防上述疾病(例如癌症及癌症转移)用的药物中的用途。本发明还包括本发明化合物或其药学上可接受的盐或本发明药物组合物在制备抑制肿瘤相关巨噬细胞、抑制肿瘤生长、抑制血管新生、抑制癌细胞的浸润和转移和/或促进抗肿瘤免疫用的药物中的用途。
本发明还提供一种降低抗癌化合物(尤其是本文所述的抗癌化合物)毒副作用的方法，所述方法包括将该抗癌化合物与本发明式(I)所示的连接臂化合物连接。
本发明的治疗或预防方法包括将本发明的化合物或药物组合物施与有此需要的对象。施用方法包括但不限于口服、静脉注射、肌肉注射等。对象包括哺乳动物，尤其是人。
在一些实施方案中，本发明还提供具有下式所示结构的EMC-AANL-DOX化合物或其偶联了白蛋白的药物在制备治疗肝癌的药物中的应用：
Figure PCTCN2021077056-appb-000032
应理解，本发明“含有”、“包括”也包括“由……组成”、“由……构成”。所有重量百分比或体积百分比之和应等于100％。实施例中使用到的各种试剂和产品，除非另有说明，否则为市售产品；对于所涉及的方法，除非另有说明，否则按常规技术实施。下述实施例并非是对本发明范围的限制。
实施例1：QHL-095-DOX的合成
QHL-095-DOX的合成过程如下所示：
Figure PCTCN2021077056-appb-000033
1.中间体1的合成
取干燥洁净2L反应瓶，加入THF 500ml，称取Fmoc-Asn(Trt)-OH 80g，加入反 应瓶中，搅拌溶解，加入DEPBT 46.6g，室温搅拌15分钟，加入PABC 16g，室温反应30分钟后，加入DIPEA 45ml，氮气换气保护，室温反应3小时，TLC监测反应毕(Fmoc-Asn(Trt)-OH反应完全)。
减压蒸除反应液，加少量DMF(180ml)溶解，滴加至搅拌中的3L水中，析出淡黄色固体，水洗2-3次后，抽滤，收集固体，真空干燥，得类白色固体(收率90％以上)。
2.中间体2的合成
将THF 500ml，上步所得类白色固体依次加入到2L单口反应瓶中，搅拌溶解，冰盐浴降温至0-5℃，滴加哌啶100ml，滴加完毕，逐渐恢复至室温反应1h，TLC监测反应完毕。减压蒸除溶剂，加少量DMF溶解，滴加至搅拌中得2L水中，机械搅拌30min，抽滤，重复水洗2-3次，抽滤，滤饼加甲基叔丁基醚800ml，搅拌30min，抽滤，滤饼加PE∶EA＝10∶1洗2次，抽滤，收集滤饼，真空干燥后，得类白色固体80g，纯度70％。
3.中间体3的合成
在干燥洁净的250ml单口反应瓶中依次加入50ml THF，5.04g Boc-Ala-Ala-OH，3.89g DEPBT，室温反应10min，加入2.6g NH 2H 2H 2-Asn(Trt)-PABC，氮气换气保护，室温反应15min，滴加DIPEA 3.5ml，氮气换气保护，室温反应3小时，减压蒸除溶剂，加水打浆2-3次，抽滤，得浅黄色固体，3.7g，过柱纯化，得产品2.0g，纯度94.8％，收率26.6％。
4.中间体4的合成
将1.8g中间体3加入250ml单口反应瓶中，加入TFA 28.5ml，滴加水1.5ml，室温反应30min，TLC监测反应毕，减压蒸除溶剂，加甲基叔丁基醚打浆，抽滤，得固体，加二氧六环∶水＝1∶1溶液溶解，加1N氢氧化钠调节pH值至13，室温搅拌40min，减压蒸除溶剂，硅胶拌样过柱，得产品450mg，收率47.5％。
5.MI-S中间体的合成
将MI-S1(338mg，2mmol)和DEPBT(717.6mg，2.4mmol)加入100ml单口瓶中，加入DMF(15ml)溶解，氮气换气保护，室温反应15min，加入R3-b(819mg，2mmol)，搅拌溶解后，室温反应15min，滴加DIPEA 137μl，氮气换气保护，室温反应3h，TLC监测R3-a反应完毕，减压蒸馏旋除溶剂，粗品用甲醇溶解，过反相高压柱得R3-1的中间体(720mg，收率：64.3％)。
6.MI-S的合成
将上步所得中间体(720mg，1.28mmol)加入100ml单口反应瓶中，加二氯甲烷15ml溶解，滴加TFA5ml，滴加水0.25ml，室温反应30min，TLC监测反应毕，减压蒸除溶剂，加甲基叔丁基醚打浆，抽滤，得固体，硅胶拌样过反相柱，得产品242mg。收率37.5％。
7.中间体5的合成
将中间体4(150mg，0.395mmol)和EMC-6Peg-COOH(239mg，0.474mmol)加入100ml单口瓶中，加入DMF(15ml)溶解，氮气换气保护，室温反应15min，滴加DIPEA 137μl，氮气换气保护，室温反应3h，TLC监测中间体4反应完毕，减压蒸馏旋除溶剂，粗品用甲醇溶解，过反相高压柱得中间体5(95mg，收率：21％)。
8.中间体6的合成
依次向100ml单口反应瓶中加入DMF 25ml，中间体5(300mg，0.346mmol)，Bis-PNP(316mg，1.04mmol)，氮气换气保护，室温反应15min，滴加DIPEA 258μl，氮气换气保护，室温反应3h，HPLC监测原料剩余7％，终止反应，减压蒸除溶剂，过柱纯化，得产品150mg，收率42％。
9.终产物QHL-095-DOX的合成
向100mL反应瓶中加入84mg盐酸阿霉素(1.0eq，0.145mmol)，150mg的中间体6(1.0eq，0.145mmol)，氮气保护下室温反应15min，滴加DIPEA 75μl，室温下反应4小时后，减压蒸除溶剂，粗品用甲醇溶解，过反相高压柱得QHL-095-DOX(49mg红色固体，收率：23.8％)。
实施例2：QHL-116-DOX的合成
Figure PCTCN2021077056-appb-000034
1.中间体1的合成
取干燥洁净2L反应瓶，加入THF 500ml，称取Fmoc-Asn(Trt)-OH 80g，加入反应瓶中，搅拌溶解，加入DEPBT 46.6g，室温搅拌15分钟，加入PABC 16g，室温反 应30分钟后，加入DIPEA 45ml，氮气换气保护，室温反应3小时，TLC监测反应毕(Fmoc-Asn(Trt)-OH反应完全)。
减压蒸除反应液，加少量DMF(180ml)溶解，滴加至搅拌中的3L水中，析出淡黄色固体，水洗2-3次后，抽滤，收集固体，真空干燥，得类白色固体(收率90％以上)。
2.中间体2的合成
将THF 500ml，上步所得类白色固体依次加入到2L单口反应瓶中，搅拌溶解，冰盐浴降温至0-5℃，滴加哌啶100ml，滴加完毕，逐渐恢复至室温反应1h，TLC监测反应完毕。减压蒸除溶剂，加少量DMF溶解，滴加至搅拌中的2L水中，机械搅拌30min，抽滤，重复水洗2-3次，抽滤，滤饼加甲基叔丁基醚800ml，搅拌30min，抽滤，滤饼加PE∶EA＝10∶1洗2次，抽滤，收集滤饼，真空干燥后，得类白色固体80g，纯度70％。
3.中间体3的合成
在干燥洁净的250ml单口反应瓶中依次加入50ml THF，5.04g Boc-Ala-Ala-OH，3.89g DEPBT，室温反应10min，加入2.6g NH 2H 2H 2-Asn(Trt)-PABC，氮气换气保护，室温反应15min，滴加DIPEA3.5ml，氮气换气保护，室温反应3小时，减压蒸除溶剂，加水打浆2-3次，抽滤，得浅黄色固体，3.7g，过柱纯化，得产品2.0g，纯度94.8％，收率26.6％。
4.中间体4的合成
将1.8g中间体3加入250ml单口反应瓶中，加入TFA 28.5ml，滴加水1.5ml，室温反应30min，TLC监测反应毕，减压蒸除溶剂，加甲基叔丁基醚打浆，抽滤，得固体，加二氧六环∶水＝1∶1溶液溶解，加1N氢氧化钠调节pH值至13，室温搅拌40min，减压蒸除溶剂，硅胶拌样过柱，得产品450mg，收率47.5％。
5.中间体5合成
称取Fmoc-Glu(OAll)-COOH(1.554g，3.79mmol)，加入DCM和THF的混合溶液10ml溶解，搅拌下，滴加HOtBu 2.72ml，滴加完毕，N 2换气保护，室温下反应16小时，TLC监测反应毕，减压蒸除溶剂，硅胶拌样过柱，得产品1.4g，收率：79.5％。
6.中间体6的合成
在干燥洁净的250ml单口反应瓶中依次加入10mlTHF，上步所得中间体5(1.4g，3mmol)，搅拌溶解后，冰盐浴降温至0-5℃，滴加哌啶3ml，滴加完毕，逐渐升温至室温，反应2小时，TLC监测反应完毕，减压蒸除溶剂，硅胶拌样纯化，收集含 产品的洗脱液，真空减压干燥至恒重，得产品583mg，收率80％。
7.中间体7的合成
在干燥洁净的250ml单口反应瓶中依次加入15ml THF，583mg中间体6，932.8mg DEPBT，室温反应10min，加入506.4mg马来酰亚胺己酸，氮气换气保护，室温反应15min，滴加DIPEA 1.3ml，氮气换气保护，室温反应3小时，减压蒸除溶剂，加水打浆2-3次，抽滤，得浅黄色固体，800mg，过柱纯化，得产品628mg，纯度94.8％，收率59.9％。
8.中间体8的合成
在干燥洁净的100ml单口反应瓶中，依次加入二氯甲烷10ml，872mg中间体7，搅拌均匀后，滴加3ml的TFA，室温反应2小时，TLC监测原料反应完毕，真空减压蒸除溶剂，加甲基叔丁基醚打浆，抽滤，得固体，硅胶拌样纯化，收集含产品的洗脱液，真空减压干燥至恒重，得产品459mg，收率60.3％。
9.中间体9的合成
在干燥洁净的250ml单口反应瓶中依次加入15ml THF，459mg中间体8，434mg DEPBT，室温反应10min，加入457.8mg中间体4，氮气换气保护，室温反应15min，滴加DIPEA 627微升，加毕，氮气换气保护，室温反应3小时，减压蒸除溶剂，加水打浆2-3次，抽滤，得浅黄色固体，750mg，过柱纯化，得产品655mg，收率63.2％。
10.中间体10的合成
依次向100ml单口反应瓶中加入DMF 25ml，中间体9(655mg，0.88mmol)，Bis-PNP(804mg，2.64mmol)，氮气换气保护，室温反应15min，滴加DIPEA 258μl，氮气换气保护，室温反应3h，HPLC监测原料剩余7％，终止反应，减压蒸除溶剂，过柱纯化，得产品335mg，收率42％。
11.中间体11的合成
向100mL反应瓶中加入214.3mg盐酸阿霉素(1.0eq，0.369mmol)，335mg的中间体10(1.0eq，0.369mmol)，氮气保护下室温反应15min，滴加DIPEA 190μl，室温下反应4小时后，减压蒸除溶剂，粗品用甲醇溶解，过反相高压柱得中间体11(115mg红色固体，收率：23.8％)。
12.终产物的合成
向100mL反应瓶中依次加入THF 15ml，中间体11(115mg，0.0877mmol)，三正丁基锡氢(76mg，0.2631mmol)，反应溶液用氮气饱和。然后加入四(三苯基膦)钯(0)(14.2mg，0.012mmol)，并将混合物在室温下搅拌过夜。通过TLC监测， 直至转化完全。然后将烧瓶内容物通过硅藻土过滤，并将残余物用THF洗涤。滤液在减压下浓缩。得到的粗品过柱纯化，得到100mg(收率：90％)目标化合物。
实施例3：N-CBP的合成
Figure PCTCN2021077056-appb-000035
1.中间体1的合成
将原料300mg加入100ml三口瓶中，加15ml THF/ETOH(4∶1)溶解，冰盐浴降温至-5℃-0℃，搅拌控温下往瓶中分批滴加210mg LiOH(5ml)水溶液，滴毕自然升温反应1h，反应液送HPLC，原料反应完全，控温-5℃-0℃，用1mol/L HCl调节反应液pH至3-4，控温25-30℃旋除溶剂得中间体1粗品，直接用于下一步。
2.中间体2的合成
往中间体1中加入15ml 1mol/L的盐酸二氧六环溶液，室温搅拌反应1h，反应液送HPLC，中间体1反应完全，控温25-30℃旋除溶剂，得中间体2粗品，直接下一步。
3.中间体3的合成
将中间体2粗品加入100ml三口瓶中，加20ml二氧六环溶解，冰盐浴降温至-5-0℃，控温往瓶中滴加159mg碳酸钠水溶液(pH约8)，氮气保护，控温-5-0℃下， 滴加311mg Fmoc-Cl的二氧六环溶液，滴毕，自然升温反应1h，送HPLC检测，中间体2反应完全，旋除溶剂，粗品硅胶拌样过反相中压柱得107mg中间体3.
4.中间体4的合成
将107mg中间体3加入50ml单口瓶中，加15ml甲醇溶解，用液氮降温至-20℃，往瓶中滴加302ul四丁基氢氧化铵(25％甲醇溶液)，滴毕自然升温反应1h，此反应液为备用液1。
将140.8mg二碘二氨合铂加入50ml单口瓶中，加超纯水10ml溶解，加热至50℃，避光，氮气保护下往瓶中滴加49.5mg硝酸银水溶液，反应15min后，继续往瓶中滴加49.5mg硝酸银水溶液，滴毕反应15min后，反应液用滤膜过滤，滤液转移至100ml单口瓶中，室温下，往瓶中滴加备用液1，滴毕，氮气置换三次，反应液转移至油浴中加热至50℃，避光下反应过夜(一般为16h)，停止反应，反应液离心，上清液直接过高压反相柱，制备液冻干得79mg中间体4，收率45.7％。
5.N-CBP的合成
将5mg中间体4加入10ml单口瓶中，加入2ml MeOH/ACN(1∶1)搅拌溶解，室温下往反应液中滴入2ul DBU，氮气保护反应半小时，HPLC检测，中间体4反应完全，将反应液滴入6ml甲基叔丁基醚中，析出类白色固体，离心，除去上清液，固体用水/叔丁醇溶解过柱得1.8mg产品N-CBP。
实施例4：QHL-140-N-CBP的合成
Figure PCTCN2021077056-appb-000036
1.中间体1的合成
将原料500mg加入100ml三口瓶中，加入10ml DCM溶解，降温至-5℃-0℃，搅拌下滴入5ml TFA，反应1h后，HPLC监测原料反应完全，旋除反应液中的溶剂，剩余油状物为中间体1。
2.中间体2的合成
将中间体1和1.15g原料Fmoc-AAN-PABC-PNP加入100ml单口瓶中，用20ml DMF溶解，氮气保护，搅拌下活化10min，往反应瓶中滴加0.87ml DIPEA，反应0.5h，送HPLC检测，原料Fmoc-AAN-PABC-PNP反应完全，旋除反应液中的DMF，粗品 用水/DMF溶解后，过高压反相柱得975mg中间体2，收率：78.6％。
3.中间体3的合成
将400mg中间体2加入250ml三口瓶中，加入THF/ETOH(4∶1)35ml溶解，用冰盐浴降温至-5℃-0℃，控温-5℃-0℃，分批滴加202mg LiOH水溶液，滴毕，控温反应3h，送HPLC检测，中间体2反应完全，控温-5℃-0℃，用1mol/L的HCL调节反应液PH至6-7，在25℃-30℃下，旋除溶剂，粗品用甲基叔丁基醚打浆两次后，固体用甲醇/水溶解，过高压反相柱，得230mg中间体3，收率86.7％。
4.中间体4的合成
将235mg中间体3和222mg EMC-OSU加入100ml单口瓶中，加30ml DMF搅拌溶解，加热至50℃，氮气保护反应过夜(一般是16h)，送HPLC检测，中间体3反应完全，旋除DMF，粗品用甲醇/水溶解，过高压反相柱得200mg中间体4，收率53.6％。
5.终产物QHL-140-N-CBP的合成
将200mg中间体4加入100ml单口瓶中，加20ml甲醇溶解，用液氮降温至-20℃，往瓶中滴加279ul四丁基氢氧化铵(25％甲醇溶液)，滴毕自然升温反应1h，此反应液为备用液1。
将130mg二碘二氨合铂加入100ml单口瓶中，加超纯水30ml溶解，加热至50℃，避光，氮气保护下往瓶中滴加46mg硝酸银水溶液，反应15min后，继续往瓶中滴加46mg硝酸银水溶液，滴毕反应15min后，反应液用滤膜过滤，滤液转移至250ml单口瓶中，室温下，往瓶中滴加备用液1，滴毕，氮气置换三次，反应液转移至油浴中加热至50℃，避光下反应过夜(一般为16h)，反应液离心，上清液直接过高压反相柱，制备液冻干得90mg产物QHL-140-N-CBP，收率34.5％。
实施例5：QHL-086-N-CBP的合成
Figure PCTCN2021077056-appb-000037
1.中间体1的合成
将原料500mg加入100ml三口瓶中，加入10ml DCM溶解，降温至-5℃-0℃，搅拌下滴入5ml TFA，反应1h后，HPLC监测原料反应完全，旋除反应液中的溶剂，剩余油状物为中间体1。
2.中间体2的合成
将中间体1和1.15g原料Fmoc-AAN-PABC-PNP加入100ml单口瓶中，用20ml DMF溶解，氮气保护，搅拌下活化10min，往反应瓶中滴加0.87ml DIPEA，反应0.5h，送HPLC检测，原料Fmoc-AAN-PABC-PNP反应完全，旋除反应液中的DMF，粗品用水/DMF溶解后，过高压反相柱得975mg中间体2，收率78.6％。
3.中间体3的合成
将400mg中间体2加入250ml三口瓶中，加入THF/ETOH(4∶1)35ml溶解，用冰盐浴降温至-5℃-0℃，控温-5℃-0℃，分批滴加202mg LiOH水溶液，滴毕，控 温反应3h，送HPLC检测，中间体2反应完全，控温-5℃-0℃，用1mol/L的HCL调节反应液PH至6-7，在25℃-30℃下，旋除溶剂，粗品用甲基叔丁基醚打浆两次后，固体用甲醇/水溶解，过高压反相柱，得235mg中间体3，收率88.6％。
4.中间体4的合成
将89mg EMC-2Peg-OH加入100ml单口瓶中，DMF溶解，往瓶中加入97mg DEPBT，室温搅拌活化1h，往瓶中滴加95ul DEPBT，继续搅拌1h后，往瓶中分批滴加150mg中间体3的DMF溶液，滴毕室温搅拌反应，HPLC检测。反应完毕，旋除DMF，粗品用水/甲醇溶解过反相高压柱得产品88mg，收率37.6％。
5.终产物QHL-086-N-CBP的合成
将88mg中间体4加入50ml单口瓶中，加10ml甲醇溶解，用液氮降温至-20℃，往瓶中滴加106ul四丁基氢氧化铵(25％甲醇溶液)，滴毕自然升温反应1h，此反应液为备用液1。
将49mg二碘二氨合铂加入50ml单口瓶中，加超纯水10ml溶解，加热至50℃，避光，氮气保护下往瓶中滴加17mg硝酸银水溶液，反应15min后，继续往瓶中滴加17mg硝酸银水溶液，滴毕反应15min后，反应液用滤膜过滤，滤液转移至100ml单口瓶中，室温下，往瓶中滴加备用液1，滴毕，氮气置换三次，反应液转移至油浴中加热至50℃，避光下反应过夜(一般为16h)，送HPLC检测，约20％的中间体4未反应完全，停止反应，反应液离心，上清液直接过高压反相柱，制备液冻干得54mg产物QHL-086-N-CBP，收率48.6％。
实施例6：QHL-095-N-CBP的合成
Figure PCTCN2021077056-appb-000038
1.中间体1的合成
将原料500mg加入100ml三口瓶中，加入10ml DCM溶解，降温至-5℃-0℃，搅拌下滴入5ml TFA，反应1h后，HPLC监测原料反应完全，旋除反应液中的溶剂，剩余油状物为中间体1。
2.中间体2的合成
将中间体1和1.15g原料Fmoc-AAN-PABC-PNP加入100ml单口瓶中，用20ml DMF溶解，氮气保护，搅拌下活化10min，往反应瓶中滴加0.87ml DIPEA，反应0.5h，送HPLC检测，原料Fmoc-AAN-PABC-PNP反应完全，旋除反应液中的DMF，粗品用水/DMF溶解后，过高压反相柱得975mg中间体2，收率：78.6％。
3.中间体3的合成
将400mg中间体2加入250ml三口瓶中，加入THF/ETOH(4∶1)35ml溶解，用冰盐浴降温至-5℃-0℃，控温-5℃-0℃，分批滴加202mg LiOH水溶液，滴毕，控温反应3h，送HPLC检测，中间体2反应完全，控温-5℃-0℃，用1mol/L的HCL调节反应液PH至6-7，在25℃-30℃下，旋除溶剂，粗品用甲基叔丁基醚打浆两次后，固体用甲醇/水溶解，过高压反相柱，得235mg中间体3，收率88.6％。
4.中间体4的合成
将180mg中间体3和240mg EMC-6Peg-OSU加入100ml单口瓶中，加20ml DMF搅拌溶解，加热至50℃，氮气保护反应过夜(一般是16h)，送HPLC检测，中间体 3反应完全，旋除DMF，粗品用甲醇/水溶解，过高压反相柱得234mg中间体4，收率69.2％。
5.终产物QHL-095-N-CBP的合成
将234mg中间体4加入100ml单口瓶中，加15ml甲醇溶解，用液氮降温至-20℃，往瓶中滴加234ul四丁基氢氧化铵(25％甲醇溶液)，滴毕自然升温反应1h，此反应液为备用液1。
将109mg二碘二氨合铂加入100ml单口瓶中，加超纯水20ml溶解，加热至50℃，避光，氮气保护下往瓶中滴加38mg硝酸银水溶液，反应15min后，继续往瓶中滴加38mg硝酸银水溶液，滴毕反应15min后，反应液用滤膜过滤，滤液转移至250ml单口瓶中，室温下，往瓶中滴加备用液1，滴毕，氮气置换三次，反应液转移至油浴中加热至50℃，避光下反应过夜(一般为16h)，反应液离心，上清液直接过高压反相柱，制备液冻干得138mg终产物，收率48％。
实施例7：QHL-006-DOX的合成
QHL-006中MI-S基团，合成路线如下所示：
Figure PCTCN2021077056-appb-000039
1.QHL-006-DOX中MI-S中间体-1的合成
取干燥洁净100ml单口反应瓶，称取马来酸酐(245mg，2.5mmol)，加二氯甲烷10ml搅拌溶解，称取NH 2H 2H 2-3Peg-COOtBu(624mg，2.25mmol)，室温反应6小时，LC-MS监测马来酸酐反应完毕，旋干反应液，硅胶拌样过柱，得MI-S中间体-1(456mg，收率48.6％)。
2.QHL-006-DOX中MI-S中间体-2的合成
将上述步骤所得456mg MI-S中间体-1加入100ml单口反应瓶中，加入乙酸酐10ml搅拌溶解，称取NaOAC(98.7mg，1.216mmol)，分批缓慢加入，油浴升温至110℃反应3h，LC-MS监测MI-S中间体-1反应完毕，冷却至室温，旋干反应液，过 柱纯化得MI-S中间体-2(312，收率70％)。
3.QHL-006-DOX中MI-S的合成
将上步所得MI-S中间体-2(312mg，0.87mmol)加入到100ml单口反应瓶中，加二氯甲烷10ml溶解，滴加TFA 2ml，滴加水0.15ml，室温反应30min，TLC监测反应毕，减压蒸除溶剂，加甲基叔丁基醚打浆，抽滤，得固体，硅胶拌样过反相柱，得产品196mg。收率75％。
终产物采用与QHL-095-DOX合成类似的方法，用不同的MI-S进行连接(MI-S的制备参照QHL-006-DOX中MI-S的合成过程)制备得到。
实施例8：QHL-096-DOX的合成
Figure PCTCN2021077056-appb-000040
1)中间体1的合成
将N-苄氧羰基-L-丙氨酸(100g，0.45mol)溶于干燥的N，N-二甲基甲酰胺(3L)中，搅拌下加入1-羟基苯并三氮唑(72.6g，0.54mol)和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(103.3g，0.54mol)，搅拌反应1小时后，冰浴至0℃下滴加L-丙氨酸甲酯(46.2g，0.45mol)和N，N-二异丙基乙基胺(173.8g，1.34mol)的N，N-二甲基甲酰胺(1L)溶液，滴加完毕后在室温下搅拌10小时，减压蒸除溶剂，粗产品溶于二氯甲烷(2L)，依次用饱和氯化铵溶液、水和饱和氯化钠溶液洗涤，有机相 用无水硫酸钠干燥，减压蒸除溶剂后粗产物经乙酸乙酯/石油醚重结晶后得到纯品为中间体1(101g白色固体，收率：73.1％)。
2)中间体2的合成
将中间体1(100g，0.34mol)溶于四氢呋喃(2L)和水(1L)的混合溶液中，冷却至0℃下滴加1摩尔/升氢氧化锂溶液(400mL)，搅拌反应10小时，滴加浓盐酸中和至PH＜6，减压蒸除四氢呋喃，剩余水相用二氯甲烷(1L×3)萃取，有机相经无水硫酸钠干燥，减压蒸干得到中间体2(88g白色固体，收率：92.2％)。
3)中间体3的合成
在三颈瓶中将L-亮氨酸叔丁酯(22.4g，0.1mol)，N-Fmoc-N’-三苯甲基天冬酰胺(59.6g，0.1mol)溶于N，N-二甲基甲酰胺(1000mL)中，搅拌下加入1-羟基苯并三氮唑(14.85g，0.11mol)和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(23g，0..12mol)，冰浴至0℃下后，加入N，N-二异丙基乙基胺(25.8g，0.2mol)，搅拌10小时后，减压蒸出溶剂，粗产品溶于氯仿(1000ml)，依次用饱和氯化铵溶液、饱和氯化钠溶液及水洗涤，有机相用无水硫酸钠干燥，过滤后减压蒸出溶剂得到的粗产品经重结晶(按体积比计，二氯甲烷∶乙酸乙酯＝1∶1)纯化后得到中间体3(42.4g白色固体，收率：55.4％)。
4)中间体4的合成
将中间体3(7.65g，0.01mol)溶于二氯甲烷(100mL)和N，N-二甲基甲酰胺(100mL)的混合溶液中，加入哌啶(40ml)，室温下搅拌5小时后，减压蒸出溶剂，然后置于真空干燥箱高真空干燥除去少量的哌啶，得到中间体4，为淡黄色固体，未经纯化直接用于下一步。
5)中间体5的合成
将上步所得中间体4粗品溶于N，N-二甲基甲酰胺(200mL)中，加入中间体2(2.94g，0.012mol)、苯并三氮唑-N，N，N′，N′-四甲基脲六氟磷酸酯(HBTU)(6.07g，0.016mol)，冰浴至0℃下后加入N，N-二异丙基乙基胺(2.6g，0.02mol)，室温下搅拌过夜，减压蒸除溶剂，残余物溶于氯仿(100ml)，依次用饱和氯化铵溶液、饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤后，蒸除溶剂，所得粗产品经硅胶柱层析后得到中间体5(3.1g白色固体，二步总收率：37.8％)。
6)中间体6的合成
将Cbz-AAN(trt)-L-Otbu(3.00g，3.65mmol)溶于甲醇(100mL)中，加入10％钯炭(0.3g)，通入氢气，常温常压下搅拌反应4小时，过滤除去钯炭，用甲醇洗涤， 合并滤液和洗液，减压蒸除溶剂得到中间体6(2.38g白色固体，收率：95.2％)。
7)中间体7的合成
将中间体6(2.38g，3.4mmol)和EMC-6Peg-OSu(2.4g，4.08mmol)加入250ml单口瓶中，加入DMF(30ml)溶解，加热至50℃反应6h。减压蒸馏旋除溶剂，粗品用甲醇溶解，过反相高压柱得中间体7(2.5g，收率：63.2％)。
8)中间体8的合成
将中间体7(1.00g，0.852mmol)溶于DCM(20mL)中，室温下，滴加三氟乙酸(10ml)，搅拌反应2h，反应液送HPLC监测，中间体1反应完全，减压蒸馏旋除溶剂，粗品用甲基叔丁基醚洗两次，固体用甲醇溶解，过反相高压柱得中间体8(721mg白色固体，收率：96.8％)。
9)终产物QHL-096-DOX的合成
向100mL反应瓶中加入63mg盐酸阿霉素(1.0eq)，95mg的中间体8(1eq)，39mg的DEPBT(1.2eq)和10mL的DMF，氮气保护下向反应混合物中加入60ul的DIPEA(3eq)，室温下反应4小时后，减压蒸除溶剂，粗品用甲醇溶解，过反相高压柱得QHL-096-DOX(52mg红色固体，收率：34.2％)。7)中间体7的合成
将中间体6(1.00g，1.46mmol)溶于DCM(20mL)中，室温下，滴加三氟乙酸(10ml)，搅拌反应2h，反应液送HPLC监测，中间体1反应完全，减压蒸馏旋除溶剂，粗品用甲基叔丁基醚洗两次，固体用甲醇溶解，过反相高压柱得中间体7(546mg白色固体，收率：96.8％)。
实施例9：QHL-117-DOX的合成
QHL-117的合成路线如下：
Figure PCTCN2021077056-appb-000041
1)中间体1的合成
将N-苄氧羰基-L-丙氨酸(100g，0.45mol)溶于干燥的N，N-二甲基甲酰胺(3L)中，搅拌下加入1-羟基苯并三氮唑(72.6g，0.54mol)和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(103.3g，0.54mol)，搅拌反应1小时后，冰浴至0℃下滴加L-丙氨酸甲酯(46.2g，0.45mol)和N，N-二异丙基乙基胺(173.8g，1.34mol)的N，N-二 甲基甲酰胺(1L)溶液，滴加完毕后在室温下搅拌10小时，减压蒸除溶剂，粗产品溶于二氯甲烷(2L)，依次用饱和氯化铵溶液、水和饱和氯化钠溶液洗涤，有机相用无水硫酸钠干燥，减压蒸除溶剂后粗产物经乙酸乙酯/石油醚重结晶后得到纯品为中间体1(101g白色固体，收率：73.1％)。
2)中间体2的合成
将中间体1(100g，0.34mol)溶于四氢呋喃(2L)和水(1L)的混合溶液中，冷却至0℃下滴加1摩尔/升氢氧化锂溶液(400mL)，搅拌反应10小时，滴加浓盐酸中和至PH＜6，减压蒸除四氢呋喃，剩余水相用二氯甲烷(1L×3)萃取，有机相经无水硫酸钠干燥，减压蒸干得到中间体2(88g白色固体，收率：92.2％)。
3)中间体3的合成
在三颈瓶中将L-亮氨酸叔丁酯(22.4g，0.1mol)，N-Fmoc-N’-三苯甲基天冬酰胺(59.6g，0.1mol)溶于N，N-二甲基甲酰胺(1000mL)中，搅拌下加入1-羟基苯并三氮唑(14.85g，0.11mol)和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(23g，0..12mol)，冰浴至0℃下后，加入N，N-二异丙基乙基胺(25.8g，0.2mol)，搅拌10小时后，减压蒸出溶剂，粗产品溶于氯仿(1000ml)，依次用饱和氯化铵溶液、饱和氯化钠溶液及水洗涤，有机相用无水硫酸钠干燥，过滤后减压蒸出溶剂得到的粗产品经重结晶(按体积比计，二氯甲烷∶乙酸乙酯＝1∶1)纯化后得到中间体3(42.4g白色固体，收率：55.4％)。
4)中间体4的合成
将中间体3(7.65g，0.01mol)溶于二氯甲烷(100mL)和N，N-二甲基甲酰胺(100mL)的混合溶液中，加入哌啶(40ml)，室温下搅拌5小时后，减压蒸出溶剂，然后置于真空干燥箱高真空干燥除去少量的哌啶，得到中间体4，为淡黄色固体，未经纯化直接用于下一步。
5)中间体5的合成
将上步所得中间体4粗品溶于N，N-二甲基甲酰胺(200mL)中，加入中间体2(2.94g，0.012mol)、苯并三氮唑-N，N，N′，N′-四甲基脲六氟磷酸酯(HBTU)(6.07g，0.016mol)，冰浴至0℃下后加入N，N-二异丙基乙基胺(2.6g，0.02mol)，室温下搅拌过夜，减压蒸除溶剂，残余物溶于氯仿(100ml)，依次用饱和氯化铵溶液、饱和氯化钠溶液洗涤，无水硫酸钠干燥，过滤后，蒸除溶剂，所得粗产品经硅胶柱层析后得到中间体5(3.1g白色固体，二步总收率：37.8％)。
6)中间体6的合成
将Cbz-AAN(trt)-L-Otbu(3.00g，3.65mmol)溶于甲醇(100mL)中，加入10％钯炭(0.3g)，通入氢气，常温常压下搅拌反应4小时，过滤除去钯炭，用甲醇洗涤，合并滤液和洗液，减压蒸除溶剂得到中间体6(2.38g白色固体，收率：95.2％)。
7)中间体7的合成
在干燥洁净的250ml单口反应瓶中依次加入15ml THF，(2.387g，3.4mmol)中间体6，1.35gDEPBT，室温反应10min，加入(1.3g，3.4mmol)EMC-Glu(OAll)-COOH，氮气换气保护，室温反应15min，滴加DIPEA 1.8ml，加毕，氮气换气保护，室温反应3小时，减压蒸除溶剂，加水打浆2-3次，抽滤，得浅黄色固体，700mg，过柱纯化，得产品2.2g，收率63.2％。
8)中间体8的合成
将中间体7(1.53g，1.46mmol)溶于DCM(20mL)中，室温下，滴加三氟乙酸(10ml)，搅拌反应2h，反应液送HPLC监测，中间体1反应完全，减压蒸馏旋除溶剂，粗品用甲基叔丁基醚洗两次，固体用甲醇溶解，过反相高压柱得中间体8(928mg白色固体，收率：84.8％)。
9)中间体9的合成
向100mL反应瓶中加入510.4mg盐酸阿霉素(1.0eq，0.88mmol)，659mg的中间体8(1.0eq，0.88mmol)，氮气保护下室温反应15min，滴加DIPEA 78μl，室温下反应4小时后，减压蒸除溶剂，粗品用甲醇溶解，过反相高压柱得中间体9(258mg红色固体，收率：23.8％)。10)终产物的合成
向100mL反应瓶中依次加入THF 15ml，中间体9(258mg，0.202mmol)，正丁基锡氢(175.7mg，0.606mmol)，反应溶液用氮气饱和。然后加入四(三苯基膦)钯(0)(32.7mg，0.028mmol)，并将混合物在室温下搅拌过夜。通过TLC监测，直至转化完全。然后将烧瓶内容物通过硅藻土过滤，并将残余物用THF洗涤。滤液在减压下浓缩。得到的粗品过柱纯化，得到224mg(收率：90％)目标化合物。
下表1中的其他化合物采用与实施例1-2、4-9类似的方法，通过使用不同的MI、S、C、A和D部分制备得到。
通过质谱(MS)验证化合物，并且它们的分子量显示在表1中，其与基于它们的结构的计算的分子量一致。
表1
化合物编号 分子量 MS 性状 产量
QHL-001-DOX 1143.16 1143 红色固体 71mg
QHL-002-DOX 1187.22 1187 红色固体 49mg
QHL-003-DOX 1231.27 1231 红色固体 112mg
QHL-004-DOX 1319.37 1319 红色固体 93mg
QHL-005-DOX 1144.15 1144 红色固体 37mg
QHL-006-DOX 1188.21 1188 红色固体 46mg
QHL-007-DOX 1232.26 1232 红色固体 158mg
QHL-008-DOX 1320.36 1320 红色固体 102mg
QHL-009-DOX 1152.17 1152 红色固体 34mg
QHL-010-DOX 1196.23 1196 红色固体 28mg
QHL-011-DOX 1240.28 1240 红色固体 18mg
QHL-012-DOX 1328.38 1328 红色固体 31mg
QHL-013-DOX 1272.27 1272 红色固体 180mg
QHL-014-DOX 1316.33 1316 红色固体 105mg
QHL-015-DOX 1360.38 1360 红色固体 214mg
QHL-016-DOX 1448.48 1448 红色固体 54mg
QHL-017-DOX 1273.26 1273 红色固体 189mg
QHL-018-DOX 1317.32 1317 红色固体 167mg
QHL-019-DOX 1361.37 1361 红色固体 102mg
QHL-020-DOX 1449.47 1449 红色固体 81mg
QHL-021-DOX 1281.28 1281 红色固体 106mg
QHL-022-DOX 1325.34 1325 红色固体 97mg
QHL-023-DOX 1369.39 1369 红色固体 139mg
QHL-024-DOX 1457.49 1457 红色固体 76mg
QHL-025-DOX 1258.24 1258 红色固体 143mg
QHL-026-DOX 1302.3 1302 红色固体 125mg
QHL-027-DOX 1346.35 1346 红色固体 136mg
QHL-028-DOX 1434.45 1434 红色固体 121mg
QHL-029-DOX 1259.23 1259 红色固体 223mg
QHL-030-DOX 1303.29 1303 红色固体 184mg
QHL-031-DOX 1347.34 1347 红色固体 98mg
QHL-032-DOX 1435.44 1435 红色固体 131mg
QHL-033-DOX 1267.25 1267 红色固体 135mg
QHL-034-DOX 1311.31 1311 红色固体 154mg
QHL-035-DOX 1355.36 1355 红色固体 164mg
QHL-036-DOX 1443.46 1443 红色固体 182mg
QHL-037-DOX 1343.35 1343 红色固体 155mg
QHL-038-DOX 1344.34 1344 红色固体 169mg
QHL-039-DOX 1352.36 1352 红色固体 156mg
QHL-040-DOX 1329.32 1329 红色固体 231mg
QHL-041-DOX 1330.31 1330 红色固体 143mg
QHL-042-DOX 1338.33 1338 红色固体 157mg
QHL-043-DOX 1387.41 1387 红色固体 241mg
QHL-044-DOX 1388.4 1388 红色固体 185mg
QHL-045-DOX 1396.42 1396 红色固体 174mg
QHL-046-DOX 1373.38 1373 红色固体 169mg
QHL-047-DOX 1374.37 1374 红色固体 64mg
QHL-048-DOX 1382.39 1382 红色固体 105mg
QHL-049-DOX 1431.46 1431 红色固体 98mg
QHL-050-DOX 1432.45 1432 红色固体 216mg
QHL-051-DOX 1440.47 1440 红色固体 198mg
QHL-052-DOX 1417.43 1417 红色固体 183mg
QHL-053-DOX 1418.42 1418 红色固体 175mg
QHL-054-DOX 1426.44 1426 红色固体 168mg
QHL-055-DOX 1519.56 1520 红色固体 156mg
QHL-056-DOX 1520.55 1521 红色固体 141mg
QHL-057-DOX 1528.57 1529 红色固体 139mg
QHL-058-DOX 1505.53 1506 红色固体 145mg
QHL-059-DOX 1506.52 1507 红色固体 182mg
QHL-060-DOX 1514.54 1515 红色固体 163mg
QHL-061-DOX 1357.38 1357 红色固体 196mg
QHL-062-DOX 1358.37 1358 红色固体 175mg
QHL-063-DOX 1366.39 1366 红色固体 154mg
QHL-064-DOX 1343.35 1343 红色固体 139mg
QHL-065-DOX 1344.34 1344 红色固体 28mg
QHL-066-DOX 1352.36 1352 红色固体 18mg
QHL-067-DOX 1401.44 1401 红色固体 31mg
QHL-068-DOX 1402.43 1402 红色固体 164mg
QHL-069-DOX 1410.45 1410 红色固体 84mg
QHL-070-DOX 1387.41 1387 红色固体 115mg
QHL-071-DOX 1388.4 1388 红色固体 54mg
QHL-072-DOX 1396.42 1396 红色固体 189mg
QHL-073-DOX 1445.49 1445 红色固体 167mg
QHL-074-DOX 1446.48 1446 红色固体 102mg
QHL-075-DOX 1454.5 1455 红色固体 81mg
QHL-076-DOX 1431.46 1431 红色固体 106mg
QHL-077-DOX 1432.45 1432 红色固体 97mg
QHL-078-DOX 1440.47 1440 红色固体 139mg
QHL-079-DOX 1533.59 1534 红色固体 76mg
QHL-080-DOX 1534.58 1535 红色固体 143mg
QHL-081-DOX 1542.6 1543 红色固体 125mg
QHL-082-DOX 1519.56 1520 红色固体 136mg
QHL-083-DOX 1520.55 1521 红色固体 121mg
QHL-084-DOX 1528.57 1529 红色固体 223mg
QHL-085-DOX 1214.24 1214 红色固体 184mg
QHL-086-DOX 1215.23 1215 红色固体 74mg
QHL-087-DOX 1223.25 1223 红色固体 121mg
QHL-088-DOX 1258.3 1258 红色固体 157mg
QHL-089-DOX 1259.29 1259 红色固体 84mg
QHL-090-DOX 1267.31 1267 红色固体 164mg
QHL-091-DOX 1302.35 1302 红色固体 182mg
QHL-092-DOX 1303.34 1303 红色固体 155mg
QHL-093-DOX 1311.36 1311 红色固体 169mg
QHL-094-DOX 1390.45 1390 红色固体 156mg
QHL-095-DOX 1391.44 1391 红色固体 49mg
QHL-096-DOX 1399.46 1399 红色固体 52mg
QHL-097-DOX 1228.27 1228 红色固体 157mg
QHL-098-DOX 1229.26 1229 红色固体 137mg
QHL-099-DOX 1237.28 1237 红色固体 49mg
QHL-100-DOX 1272.33 1272 红色固体 67mg
QHL-101-DOX 1273.32 1273 红色固体 71mg
QHL-102-DOX 1281.34 1281 红色固体 49mg
QHL-103-DOX 1316.38 1316 红色固体 86mg
QHL-104-DOX 1317.37 1317 红色固体 93mg
QHL-105-DOX 1325.39 1325 红色固体 37mg
QHL-106-DOX 1404.48 1404 红色固体 46mg
QHL-107-DOX 1405.47 1405 红色固体 158mg
QHL-108-DOX 1413.49 1413 红色固体 102mg
QHL-109-DOX 1184.17 1184 红色固体 34mg
QHL-110-DOX 1185.16 1185 红色固体 28mg
QHL-111-DOX 1193.18 1193 红色固体 38mg
QHL-112-DOX 1170.14 1170 红色固体 31mg
QHL-113-DOX 1171.13 1171 红色固体 104mg
QHL-114-DOX 1179.15 1179 红色固体 170mg
QHL-115-DOX 1226.25 1226 红色固体 118mg
QHL-116-DOX 1227.24 1227 红色固体 100mg
QHL-117-DOX 1235.26 1235 红色固体 224mg
QHL-118-DOX 1212.22 1212 红色固体 167mg
QHL-119-DOX 1213.21 1213 红色固体 102mg
QHL-120-DOX 1221.23 1221 红色固体 81mg
QHL-121-DOX 1163.18 1163 红色固体 106mg
QHL-122-DOX 1177.22 1177 红色固体 97mg
QHL-123-DOX 1205.28 1205 红色固体 139mg
QHL-124-DOX 1219.31 1219 红色固体 76mg
QHL-125-DOX 1219.31 1219 红色固体 143mg
QHL-126-DOX 1237.34 1237 红色固体 125mg
QHL-127-DOX 1239.3 1239 红色固体 136mg
QHL-128-DOX 1278.33 1278 红色固体 121mg
QHL-129-DOX 1193.22 1193 红色固体 64mg
QHL-130-DOX 1207.25 1207 红色固体 184mg
QHL-131-DOX 1209.28 1209 红色固体 164mg
QHL-132-DOX 1269.32 1269 红色固体 144mg
QHL-133-DOX 1220.25 1220 红色固体 104mg
QHL-134-DOX 1234.28 1234 红色固体 95mg
QHL-135-DOX 1234.32 1234 红色固体 164mg
QHL-136-DOX 1262.33 1262 红色固体 182mg
QHL-137-DOX 1243.29 1243 红色固体 155mg
QHL-140-N-CBP 970.86 971 淡黄色固体 90mg
QHL-143-N-CBP 942.8 943 淡黄色固体 41mg
QHL-095-N-CBP 1264.17 1264 淡黄色固体 138mg
QHL-092-N-CBP 1176.07 1176 淡黄色固体 88mg
QHL-089-N-CBP 1132.01 1132 淡黄色固体 43mg
QHL-107-N-CBP 1278.2 1278 淡黄色固体 55mg
QHL-104-N-CBP 1204.12 1204 淡黄色固体 150mg
QHL-101-N-CBP 1146.04 1146 淡黄色固体 142mg
QHL-098-N-CBP 1101.99 1102 淡黄色固体 95mg
QHL-146-N-CBP 914.75 915 淡黄色固体 116mg
QHL-086-N-CBP 1087.96 1088 淡黄色固体 54mg
QHL-086-多韦替尼 1108.16 1108 淡黄色固体 65mg
QHL-089-多韦替尼 1152.21 1152 淡黄色固体 86mg
QHL-086-表阿霉素 1259.24 1259 红色固体 78mg
QHL-089-表阿霉素 1303.3 1303 红色固体 90mg
QHL-086-化合物a 1223.62 1224 淡黄色固体 134mg
QHL-089-化合物a 1267.67 1268 淡黄色固体 45mg
QHL-086-化合物b 1241.61 1242 淡黄色固体 98mg
QHL-089-化合物b 1285.66 1286 淡黄色固体 41mg
QHL-086-丝裂霉素 1050.05 1050 灰蓝固体粉末 85mg
QHL-089-丝裂霉素 1094.1 1094 灰蓝固体粉末 58mg
QHL-086-达拉非尼 1235.28 1235 淡黄色固体 73mg
QHL-089-达拉非尼 1279.33 1279 淡黄色固体 81mg
QHL-086-莫特塞尼 1133.23 1133 淡黄色固体 118mg
QHL-089-莫特塞尼 1089.18 1089 淡黄色固体 44mg
QHL-138-N-CBP 934.86 935 淡黄色固体 95mg
QHL-141-N-CBP 906.8 907 淡黄色固体 53mg
QHL-096-N-CBP 1228.17 1228 淡黄色固体 70mg
QHL-093-N-CBP 1140.07 1140 淡黄色固体 135mg
QHL-090-N-CBP 1096.01 1096 淡黄色固体 90mg
QHL-108-N-CBP 1242.2 1242 淡黄色固体 37mg
QHL-105-N-CBP 1168.12 1168 淡黄色固体 65mg
QHL-102-N-CBP 1110.04 1110 淡黄色固体 136mg
QHL-099-N-CBP 1065.99 1066 淡黄色固体 41mg
QHL-144-N-CBP 878.75 879 淡黄色固体 118mg
QHL-087-N-CBP 1051.96 1052 淡黄色固体 53mg
QHL-087-多韦替尼 1072.16 1072 淡黄色固体 38mg
QHL-090-多韦替尼 1116.21 1116 淡黄色固体 82mg
QHL-087-表阿霉素 1223.24 1223 红色固体 73mg
QHL-090-表阿霉素 1267.3 1267 红色固体 69mg
QHL-087-化合物a 1187.62 1188 淡黄色固体 117mg
QHL-090-化合物a 1231.67 1232 淡黄色固体 115mg
QHL-087-化合物b 1205.61 1206 淡黄色固体 115mg
QHL-090-化合物b 1249.66 1250 淡黄色固体 116mg
QHL-087丝裂霉素 1014.05 1014 灰蓝固体粉末 55mg
QHL-090-丝裂霉素 1058.1 1058 灰蓝固体粉末 78mg
QHL-087-达拉非尼 1199.28 1199 淡黄色固体 61mg
QHL-090-达拉非尼 1243.33 1243 淡黄色固体 102mg
QHL-087-莫特塞尼 1097.23 1097 淡黄色固体 40mg
QHL-090-莫特塞尼 1053.18 1053 淡黄色固体 85mg
QHL-140-雷西莫特 913.00 913 白色固体 57mg
QHL-086-雷西莫特 1030.11 1030 白色固体 82mg
QHL-089-雷西莫特 1074.16 1074 灰白色固体 49mg
QHL-092-雷西莫特 1118.21 1118 灰白色固体 76mg
QHL-095-雷西莫特 1206.32 1206 灰白色固体 47mg
QHL-005-雷西莫特 959.03 959 白色固体 41mg
QHL-006-雷西莫特 1003.08 1003 白色固体 47mg
QHL-008-雷西莫特 1135.24 1135 白色固体 45mg
QHL-147-雷西莫特 1399.56 1400 白色固体 91mg
QHL-116-雷西莫特 1042.12 1042 白色固体 85mg
QHL-119-雷西莫特 1028.09 1028 灰白色固体 95mg
QHL-140-泼尼松 956.33 956 灰白色固体 65mg
QHL-086-泼尼松 1073.44 1073 灰白色固体 75mg
QHL-089-泼尼松 1117.49 1117 白色固体 76mg
QHL-092-泼尼松 1161.54 1162 白色固体 80mg
QHL-095-泼尼松 1249.65 1250 白色固体 57mg
QHL-005-泼尼松 1002.36 1002 白色固体 80mg
QHL-006-泼尼松 1046.41 1046 白色固体 88mg
QHL-008-泼尼松 1178.57 1179 白色固体 81mg
QHL-147-泼尼松 1442.89 1443 白色固体 86mg
QHL-116-泼尼松 1085.45 1085 白色固体 40mg
QHL-119-泼尼松 1071.42 1071 灰白色固体 37mg
QHL-150-T3 1100.44 1100 灰白色固体 68mg
QHL-157-T3 1217.55 1218 灰白色固体 95mg
QHL-158-T3 1261.6 1262 灰白色固体 86mg
QHL-159-T3 1305.65 1306 灰白色固体 76mg
QHL-160-T3 1393.76 1394 灰白色固体 55mg
QHL-153-T3 1146.47 1146 灰白色固体 76mg
QHL-154-T3 1190.52 1191 灰白色固体 80mg
QHL-155-T3 1322.68 1323 白色固体 91mg
QHL-156-T3 1587 1587 白色固体 42mg
QHL-161-T3 1229.56 1230 白色固体 87mg
QHL-162-T3 1215.53 1216 白色固体 65mg
本发明还提供下列对比化合物，结构式如下：
化合物C1：Doxorubicin
Figure PCTCN2021077056-appb-000042
化合物C2：AANL-DOX
Figure PCTCN2021077056-appb-000043
化合物C3：EMC-AANL-DOX
Figure PCTCN2021077056-appb-000044
化合物C4：Peg-AANL-DOX
Figure PCTCN2021077056-appb-000045
实施例10：制备人血白蛋白偶联的HSA-EMC-AANL-DOX，HSA-QHL-087-DOX和HSA-QHL-087-N-CBP药物
配制EMC-AANL-DOX，QHL-087-DOX和QHL-087-N-CBP，其中EMC-AANL-DOX使用DMSO溶解，QHL-087-DOX和QHL-087-N-CBP用无菌水溶解。HSA用无菌水溶解。化合物与HSA以3∶1(4.8umol/mL，1.6umol/mL)结合，于37℃水浴3h反应，取出反应液，利用加压超滤膜过滤没结合化合物，并加生理盐水稀释并过滤3次，获得半成品。使用例如层析方法来分离人血白蛋白偶联的阿霉素抗肿瘤药物，诸如DEAE离子交换、凝胶过滤和羟基磷灰石层析。半成品及时分装、旋冻、冻干。制品的冻干工艺可根据机器性能特点制定，但应保证制品制备质量及保存质量符合要求。实验比较了不同比例、不同时间莱古比星与HSA结合情况，结果表明，EMC-AANL-DOX，QHL-087-DOX和QHL-087-N-CBP与HSA以3∶1结合，于37℃水浴3h结合，HSA结合率分别为62％，99.6％和99.7％。
实施例11：选择化学修饰连接臂获得优化的激活效率
与通过Legumain裂解的天然肽序列接头相比，S-C-A是化学修饰的接头，并显示出高活化效率。当C选择AAN时，在活化测定中评估不同的S-C-A接头和对照接头的活化。使用S-C-A缀合物溶解并将它们稀释十倍至0.1mM/ml的浓度。在37℃下，将样品化合物以1mg/ml的浓度添加到100μg酸化的人乳腺癌(MDA-MB435)肿瘤组织匀浆(pH6.0)中。肿瘤组织匀浆中的酶可以释放并通过HPLC检测，从而比较了肿瘤组织对接头的激活效率。结果示于表2-1，2-2，2-3和2-4。
表2-1
Figure PCTCN2021077056-appb-000046
Figure PCTCN2021077056-appb-000047
表2-2
Figure PCTCN2021077056-appb-000048
表2-3
Figure PCTCN2021077056-appb-000049
Figure PCTCN2021077056-appb-000050
与通过Legumain裂解的天然肽序列接头相比，检测D型三肽对激活效率的影响，器重他，MI-S中S1为-CH 2CH 2-CONH-，S2：2peg。结果如下表2-4所示。
表2-4
C A 激活效率(％)
Ala-Ala-Asn Leu 96.8
Thr-Ala-Asn Leu 90.2
Val-Ala-Asn Leu 78.9
D-Thr-L-Val-L-Asn Leu 73.5
D-Thr-L-Ala-L-Asn Leu 89.6
D-Ala-L-Val-L-Asn Leu 93.5
L-Thr-D-Val-L-Asn Leu 90.6
L-Thr-D-Ala-L-Asn Leu 72.4
L-Ala-D-Val-L-Asn Leu 83.4
D-Thr-D-Val-L-Asn Leu 66.1
D-Thr-D-Ala-L-Asn Leu 78.4
D-Ala-D-Val-L-Asn Leu 61.5
L-Ala-L-Val-D-Asn Leu 22.4
由表格数据可知，S和A设计为高激活的条件下，变量为不同三肽时，不同氨基酸选择及构型对激活效率有影响，特别是D-Asn导致激活能力丢失，而其他两个位置的氨基酸调整为D-型时仍有激活活性。
实施例12：优选化合物的酶切动力学速率比较
精密称取C3，QHL-087-DOX，QHL-090-DOX，QHL-093-DOX，QHL-094-DOX，QHL-093-DOX和QHL-096-DOX样品各10mg，加适量的水配置成4umol/mL的样品 储备溶液，并加入水逐步稀释成下表3中各浓度的样品溶液；分别量取不同浓度的样品溶液20ul，加入80ul Legumain，37℃水浴锅水浴；水浴2h后取出，10ul进样，HPLC检测；读出各对应产物的面积，根据产物的线性方程计算出产物浓度，代入公式可得到对应的V：
V(umoL/mL/min)＝C(umoL/mL)/120min
用V对[C]作图，可得到截距Km/Vmax和直线与x轴的交点，为-Km，[C]为各底物浓度，即样品溶液浓度，单位为umoL/mL。
表3
[C 1] [C 2] [C 3] [C 4] [C 5] [C 6] [C 7]
2 1.6 0.8 0.7 0.35 0.175 0.0875
实验结果如图3所示，在其他结构一致的条件下：QHL-087的2PEG基团显著提高激活效率，但随着PEG数量的增加反而效率降低。比较在6PEG基团连接的相同条件下：H 2PABC-NH 2H 2替换leu显著提高激活效率。
实施例13：
小鼠脾脏及CD8+T细胞的分离及培养和小鼠骨髓单个核细胞分离及M2巨噬细胞的诱导分化
1.小鼠脾脏细胞分离
1)取C57BL/6小鼠脾脏，置于放有40uM筛网的培养皿上(冰浴)，加入10mL左右生理盐水，用无菌注射器芯轻轻研磨。
2)将研磨后的细胞悬液移入50mL离心管内，加入5mL左右生理盐水冲洗培养皿，移入50mL离心管内，合并悬液。
3)1000r/min离心细胞悬液10min，弃掉上清液，适量体积生理盐水重悬，加入3倍体积氯化铵红细胞裂解液，吹打均匀。冰上裂解10min左右后，加入10mL生理盐水终止裂解，1000r/min离心5min。
4)离心后，弃掉上清液，加入10mL生理盐水，吹打均匀，1000r/min离心5min，重复此操作一次，然后重悬细胞于10％RMPI 1640培养基中用于后续培养或0.5％BSA中，进行后续T细胞分选。
2.CD8+T细胞分选
将上述分离所得小鼠脾细胞，重悬至1E8/mL，每1E8细胞加入100ul Miltenyi biotec CD8a(Ly-2)microBeads混合均匀，4度避光孵育15分钟，加入5-10倍体积PBS， 充分混匀洗涤，300g离心5分钟，去上清，重复清洗一次，重悬细胞至2E8/mL待上柱分离，将细胞悬液置于磁体板上LS柱(LS柱预先用冲洗缓冲液(pH7.2 PBS+0.5％BSA+2mM EDTA)平衡)，待细胞悬液缓慢流过LS柱且CD8+T细胞结合于LS柱中磁颗粒后，用3倍体积细胞悬液的洗涤缓冲液洗涤LS柱子，洗涤结束后将LS柱子从磁体板下取出，置于15mL离心管，向LS柱中加入5mL冲洗缓冲液，然后利用LS柱芯快速挤压洗脱LS中结合细胞于离心管中，收集所有通过柱子的细胞，将所得细胞离心去上清，用冲洗缓冲液重复清洗一次，用适当体积10％RMPI1640培养基重悬细胞，细胞计数待用。
3.CD8+T阳性细胞的激活和扩增
A、CD3/CD28磁珠的洗涤：a、将小管内的免疫磁珠摇匀悬浮(漩涡30s以上，或倾斜旋转5min)。b、取出所需量的免疫磁珠到1.5ml试管内，加入1ml含血清的1640并混悬好，漩涡30s以上，或保持滚动至少5min。
B、T细胞的激活：a、适宜细胞数量接种于培养板中(如6孔板1E6/ml，2ml培养液，保持T细胞密度2E6/ml以上，但不要超过2E6/ml)。b、加入已洗好的磁珠，使磁珠∶细胞(数量比)＝1∶1。c、放入培养箱培养3天。
C、扩增：a、3天内培养液不需要更换，第3天时加30U/mlIL-2(可根据实际适当增加)，同时换培养基(细胞在48h后数量大约会增殖1倍，实时监测细胞大小与形态)。b、4-5天刺激后移去磁珠(避免过度激活)，上下移动细胞5-10次，避免有气泡和湍流，以使细胞和磁珠分离。收集细胞到1.5ml管内，放到磁体上1min至磁珠到管壁上，细胞移到另外的管内以尽量完全除去磁珠，移去磁珠的细胞继续于加有30U/mlIL-2(可根据实际适当增加)的培养基中培养并监测其状态、增殖和活力)。
4、小鼠骨髓单个核细胞分离及M2巨噬细胞的诱导分化
无菌条件下取出2只C57BL/6小鼠双侧股骨和胫骨，超净台中剪开干骺端，用5mL无菌注射器，抽取无血清MEM培养液反复轻柔冲洗骨髓腔4次，收集所有细胞悬液；1000r/min离心10min，弃上清得细胞沉淀，适当体积无血清MEM培养液重悬，反复吹散及均匀后，40uM滤网过滤，加入3倍体积的红细胞裂解液，冰上裂解10min；1000r/min离心5min，弃上清得细胞沉淀，无血清MEM培养基清洗2次后收集细胞沉淀；用适当体积用含体积分数10％的FBS、1％PS的MEM完全培养液重悬细胞，细胞计数待后续分化；接种100uL，20000个细胞/孔于96孔细胞培养板中，培养液中加入100ng M-CSF用于M2巨噬细胞分化，于37℃，体积分数5％CO2培育箱静置培养诱导分化7天，观察细胞形态。诱导的细胞形态参考图4，M2巨噬细胞 与单核细胞，DC和GM-巨噬细胞差异，确认为M2巨噬细胞。
实施例14：MTT法测定药物对细胞生长的抑制作用
实施例13的细胞计数后用培养基调整细胞浓度，接种于96孔培养板上，每孔100μl细胞悬液，CD8+T细胞的接种浓度为100000个细胞/孔，M2巨噬细胞的接种浓度为20000个细胞/孔。将96孔培养板置于37℃，二氧化碳(5％)培养箱中过夜培养24小时。24小时后，96孔培养板加入含有不同浓度药物的细胞培养液100ul，并设置不加药物只加相应药物溶剂的对照孔(0.1％DMSO)，以及只加培养基不含细胞的调零孔(Blank)。每组设3个平行孔，然后将板子置于37℃，二氧化碳(5％)培养箱中培养48小时。48小时后，每孔加入20μl MTT(浓度为5mg/ml)，继续孵育4h。然后将培养液轻轻吸出，每孔加入150μl DMSO作溶剂溶解，溶解后用酶标仪测定490nm处的吸光度。
计算细胞的存活率及药物对细胞的半数抑制浓度。细胞存活率％＝(OD试验-OD空白对照)/(OD试验对照-OD空白对照)*100％。存活率(％)用Excel软件进行计算，用Prism 5软件绘制药物对细胞的剂量-响应曲线，各指标用均值来表示，变异系数(CV)评估数据的一致性。
依据上述实验方法中试验示意图以及细胞加药浓度设置，将待测药物的最大起始浓度设为14uM，以1∶3比例进行梯度稀释成9个剂量组(每组3个重复)，所有加药孔的药物溶剂(DMSO)浓度均控制在0.1％，只加药物溶剂(0.1％DMSO)作为试验对照组(Control)，以及未加细胞只加培养基的空白组(Blank)，然后依据以下方法计算各剂量组相对于对照组(Control)的肿瘤细胞存活率(％)。
各剂量组细胞存活率(％)＝(OD剂量组-OD空白组)/(OD 0.1％DMSO-OD空白组)*100％
实验结果如图5和6所示。在其他结构一致的条件下，相对C3(即EMC-AANL-DOX)，QHL-087-DOX具有显著提高的针对M2巨噬细胞的细胞毒性，而对CD8+T细胞毒性较弱，从而可产生对免疫抑制细胞的选择性。
实施例15：本发明多种药物对M2巨噬细胞的细胞毒性筛选
按实施例14的方法对M2巨噬细胞抑制实行部分化合物的细胞毒性筛选实验。 每种药物检测3孔，每孔药物加入10uM下列药物，检测相对无药组的抑制率，实验结果见表4。
实施例16：本发明实施例制备的水溶性高效靶向激活的阿霉素衍生物等与对照化合物的水溶性比较
按照本发明实施例制备的化合物，将以上制备的化合物和参考化合物C1，C2，C3和C4冷冻干燥(-70℃)。将化合物溶解在不同浓度的水中，通过观察和HPLC测试(＞95％)来检查水溶性。结果如表4所示。
表4：筛选药物水溶性试验数据及M2巨噬细胞抑制率
化合物编号 连接臂 溶解度 M2巨噬细胞抑制率
DOX / ＜1mg/ml 12.6％
AANL-DOX / ＜1mg/ml 16.9％
EMC-AANL-DOX / ＜5mg/ml 34.6％
PEG-AANL-DOX / ＜5mg/ml 10.5％
QHL-001-DOX QHL-001 ＞10mg/ml 84.5％
QHL-002-DOX QHL-002 ＞10mg/ml  
QHL-003-DOX QHL-003 ＞15mg/ml 75.8％
QHL-004-DOX QHL-004 ＞20mg/ml 63.7％
QHL-005-DOX QHL-005 ＞10mg/ml  
QHL-006-DOX QHL-006 ＞10mg/ml  
QHL-007-DOX QHL-007 ＞15mg/ml  
QHL-008-DOX QHL-008 ＞20mg/ml  
QHL-009-DOX QHL-009 ＞10mg/ml  
QHL-010-DOX QHL-010 ＞10mg/ml  
QHL-011-DOX QHL-011 ＞15mg/ml  
QHL-012-DOX QHL-012 ＞20mg/ml  
QHL-013-DOX QHL-013 ＞20mg/ml 78.4％
QHL-014-DOX QHL-014 ＞20mg/ml  
QHL-015-DOX QHL-015 ＞25mg/ml  
QHL-016-DOX QHL-016 ＞30mg/ml  
QHL-017-DOX QHL-017 ＞20mg/ml  
QHL-018-DOX QHL-018 ＞20mg/ml  
QHL-019-DOX QHL-019 ＞25mg/ml  
QHL-020-DOX QHL-020 ＞30mg/ml  
QHL-021-DOX QHL-021 ＞20mg/ml 68.6％
QHL-022-DOX QHL-022 ＞20mg/ml  
QHL-023-DOX QHL-023 ＞25mg/ml  
QHL-024-DOX QHL-024 ＞30mg/ml  
QHL-025-DOX QHL-025 ＞20mg/ml  
QHL-026-DOX QHL-026 ＞20mg/ml  
QHL-027-DOX QHL-027 ＞25mg/ml  
QHL-028-DOX QHL-028 ＞30mg/ml  
QHL-029-DOX QHL-029 ＞20mg/ml 46.4％
QHL-030-DOX QHL-030 ＞20mg/ml  
QHL-031-DOX QHL-031 ＞25mg/ml  
QHL-032-DOX QHL-032 ＞30mg/ml  
QHL-033-DOX QHL-033 ＞20mg/ml  
QHL-034-DOX QHL-034 ＞20mg/ml  
QHL-035-DOX QHL-035 ＞25mg/ml  
QHL-036-DOX QHL-036 ＞30mg/ml  
QHL-037-DOX QHL-037 ＞20mg/ml 87.8％
QHL-038-DOX QHL-038 ＞20mg/ml 76.4％
QHL-039-DOX QHL-039 ＞20mg/ml 78.9％
QHL-040-DOX QHL-040 ＞20mg/ml 46.7％
QHL-041-DOX QHL-041 ＞20mg/ml  
QHL-042-DOX QHL-042 ＞20mg/ml  
QHL-043-DOX QHL-043 ＞20mg/ml  
QHL-044-DOX QHL-044 ＞20mg/ml  
QHL-045-DOX QHL-045 ＞20mg/ml  
QHL-046-DOX QHL-046 ＞20mg/ml  
QHL-047-DOX QHL-047 ＞20mg/ml  
QHL-048-DOX QHL-048 ＞20mg/ml  
QHL-049-DOX QHL-049 ＞25mg/ml  
QHL-050-DOX QHL-050 ＞25mg/ml  
QHL-051-DOX QHL-051 ＞25mg/ml  
QHL-052-DOX QHL-052 ＞25mg/ml  
QHL-053-DOX QHL-053 ＞25mg/ml  
QHL-054-DOX QHL-054 ＞25mg/ml  
QHL-055-DOX QHL-055 ＞25mg/ml  
QHL-056-DOX QHL-056 ＞25mg/ml  
QHL-057-DOX QHL-057 ＞25mg/ml  
QHL-058-DOX QHL-058 ＞25mg/ml  
QHL-059-DOX QHL-059 ＞25mg/ml  
QHL-060-DOX QHL-060 ＞25mg/ml  
QHL-061-DOX QHL-061 ＞15mg/ml  
QHL-062-DOX QHL-062 ＞15mg/ml  
QHL-063-DOX QHL-063 ＞15mg/ml  
QHL-064-DOX QHL-064 ＞15mg/ml  
QHL-065-DOX QHL-065 ＞15mg/ml  
QHL-066-DOX QHL-066 ＞15mg/ml  
QHL-067-DOX QHL-067 ＞15mg/ml  
QHL-068-DOX QHL-068 ＞15mg/ml  
QHL-069-DOX QHL-069 ＞25mg/ml  
QHL-070-DOX QHL-070 ＞25mg/ml  
QHL-071-DOX QHL-071 ＞25mg/ml  
QHL-072-DOX QHL-072 ＞25mg/ml  
QHL-073-DOX QHL-073 ＞25mg/ml  
QHL-074-DOX QHL-074 ＞25mg/ml  
QHL-075-DOX QHL-075 ＞25mg/ml  
QHL-076-DOX QHL-076 ＞25mg/ml  
QHL-077-DOX QHL-077 ＞25mg/ml  
QHL-078-DOX QHL-078 ＞25mg/ml  
QHL-079-DOX QHL-079 ＞25mg/ml 87.5％
QHL-080-DOX QHL-080 ＞25mg/ml 87.7％
QHL-081-DOX QHL-081 ＞30mg/ml 78.5％
QHL-082-DOX QHL-082 ＞30mg/ml  
QHL-083-DOX QHL-083 ＞30mg/ml  
QHL-084-DOX QHL-084 ＞30mg/ml  
QHL-085-DOX QHL-085 ＞10mg/ml 92.2％
QHL-086-DOX QHL-086 ＞15mg/ml 92.9％
QHL-087-DOX QHL-087 ＞10mg/ml 99.8％
QHL-088-DOX QHL-088 ＞10mg/ml 98.4％
QHL-089-DOX QHL-089 ＞15mg/ml 82.2％
QHL-090-DOX QHL-090 ＞10mg/ml 72.9％
QHL-091-DOX QHL-091 ＞20mg/ml 63.8％
QHL-092-DOX QHL-092 ＞25mg/ml 94.4％
QHL-093-DOX QHL-093 ＞20mg/ml 77.4％
QHL-094-DOX QHL-094 ＞20mg/ml 72.6％
QHL-095-DOX QHL-095 ＞20mg/ml 74.7％
QHL-096-DOX QHL-096 ＞20mg/ml 70.4.％
QHL-097-DOX QHL-097 ＞20mg/ml  
QHL-098-DOX QHL-098 ＞10mg/ml  
QHL-099-DOX QHL-099 ＞20mg/ml  
QHL-100-DOX QHL-100 ＞25mg/ml  
QHL-101-DOX QHL-101 ＞20mg/ml  
QHL-102-DOX QHL-102 ＞10mg/ml  
QHL-103-DOX QHL-103 ＞15mg/ml  
QHL-104-DOX QHL-104 ＞20mg/ml  
QHL-105-DOX QHL-105 ＞10mg/ml  
QHL-106-DOX QHL-106 ＞20mg/ml  
QHL-107-DOX QHL-107 ＞25mg/ml  
QHL-108-DOX QHL-108 ＞20mg/ml  
QHL-109-DOX QHL-109 ＞10mg/ml 54.8％
QHL-110-DOX QHL-110 ＞15mg/ml 46.8％
QHL-111-DOX QHL-111 ＞10mg/ml  
QHL-112-DOX QHL-112 ＞10mg/ml  
QHL-113-DOX QHL-113 ＞15mg/ml  
QHL-114-DOX QHL-114 ＞15mg/ml  
QHL-115-DOX QHL-115 ＞10mg/ml  
QHL-116-DOX QHL-116 ＞15mg/ml  
QHL-117-DOX QHL-117 ＞10mg/ml  
QHL-118-DOX QHL-118 ＞15mg/ml  
QHL-119-DOX QHL-119 ＞15mg/ml  
QHL-120-DOX QHL-120 ＞10mg/ml  
QHL-121-DOX QHL-121 ＞10mg/ml  
QHL-122-DOX QHL-122 ＞10mg/ml  
QHL-123-DOX QHL-123 ＞2mg/ml  
QHL-124-DOX QHL-124 ＞5mg/ml  
QHL-125-DOX QHL-125 ＞2mg/ml  
QHL-126-DOX QHL-126 ＞2mg/ml  
QHL-127-DOX QHL-127 ＞10mg/ml  
QHL-128-DOX QHL-128 ＞2mg/ml  
QHL-129-DOX QHL-129 ＞5mg/ml  
QHL-130-DOX QHL-130 ＞2mg/ml  
QHL-131-DOX QHL-131 ＞10mg/ml  
QHL-132-DOX QHL-132 ＞2mg/ml  
QHL-133-DOX QHL-133 ＞2mg/ml  
QHL-134-DOX QHL-134 ＞5mg/ml  
QHL-135-DOX QHL-135 ＞5mg/ml  
QHL-136-DOX QHL-136 ＞2mg/ml  
QHL-137-DOX QHL-137 ＞5mg/ml  
QHL-140-N-CBP QHL-140 ＞10mg/ml 89.5％
QHL-143-N-CBP QHL-143 ＞10mg/ml 78.4％
QHL-095-N-CBP QHL-095 ＞10mg/ml 68.7％
QHL-092-N-CBP QHL-092 ＞10mg/ml 75.8％
QHL-089-N-CBP QHL-089 ＞10mg/ml 97.4％
QHL-107-N-CBP QHL-107 ＞10mg/ml  
QHL-104-N-CBP QHL-104 ＞20mg/ml  
QHL-101-N-CBP QHL-101 ＞20mg/ml  
QHL-098-N-CBP QHL-098 ＞20mg/ml  
QHL-146-N-CBP QHL-146 ＞20mg/ml  
QHL-086-N-CBP QHL-086 ＞20mg/ml  
QHL-086-多韦替尼 QHL-086 ＞20mg/ml  
QHL-089-多韦替尼 QHL-089 ＞20mg/ml  
QHL-086-表阿霉素 QHL-086 ＞20mg/ml  
QHL-089-表阿霉素 QHL-089 ＞20mg/ml  
QHL-086-化合物a QHL-086 ＞20mg/ml  
QHL-089-化合物a QHL-089 ＞20mg/ml  
QHL-086-化合物b QHL-086 ＞20mg/ml  
QHL-089-化合物b QHL-089 ＞20mg/ml  
QHL-086-丝裂霉素 QHL-086 ＞20mg/ml  
QHL-089-丝裂霉素 QHL-089 ＞20mg/ml  
QHL-086-达拉非尼 QHL-086 ＞20mg/ml  
QHL-089-达拉非尼 QHL-089 ＞20mg/ml  
QHL-086-莫特塞尼 QHL-086 ＞20mg/ml  
QHL-089-莫特塞尼 QHL-089 ＞20mg/ml  
QHL-138-N-CBP QHL-138 ＞20mg/ml  
QHL-141-N-CBP QHL-141 ＞20mg/ml  
QHL-096-N-CBP QHL-096 ＞20mg/ml  
QHL-093-N-CBP QHL-093 ＞10mg/ml  
QHL-090-N-CBP QHL-090 ＞10mg/ml  
QHL-108-N-CBP QHL-108 ＞10mg/ml  
QHL-105-N-CBP QHL-105 ＞10mg/ml  
QHL-102-N-CBP QHL-102 ＞10mg/ml  
QHL-099-N-CBP QHL-099 ＞10mg/ml  
QHL-144-N-CBP QHL-144 ＞10mg/ml  
QHL-087-N-CBP QHL-087 ＞10mg/ml  
QHL-087-多韦替尼 QHL-087 ＞10mg/ml  
QHL-090-多韦替尼 QHL-090 ＞10mg/ml  
QHL-087-表阿霉素 QHL-087 ＞10mg/ml  
QHL-090-表阿霉素 QHL-090 ＞10mg/ml  
QHL-087-化合物a QHL-087 ＞10mg/ml  
QHL-090-化合物a QHL-090 ＞10mg/ml  
QHL-087-化合物b QHL-087 ＞10mg/ml  
QHL-090-化合物b QHL-090 ＞10mg/ml  
QHL-087丝裂霉素 QHL-087 ＞10mg/ml  
QHL-090-丝裂霉素 QHL-090 ＞10mg/ml  
QHL-087-达拉非尼 QHL-087 ＞10mg/ml  
QHL-090-达拉非尼 QHL-090 ＞10mg/ml  
QHL-087-莫特塞尼 QHL-087 ＞10mg/ml  
QHL-090-莫特塞尼 QHL-090 ＞10mg/ml  
QHL-140-雷西莫特 QHL-140 ＞10mg/ml  
QHL-086-雷西莫特 QHL-086 ＞10mg/ml  
QHL-089-雷西莫特 QHL-089 ＞10mg/ml  
QHL-092-雷西莫特 QHL-092 ＞10mg/ml  
QHL-095-雷西莫特 QHL-095 ＞10mg/ml  
QHL-005-雷西莫特 QHL-005 ＞10mg/ml  
QHL-006-雷西莫特 QHL-006 ＞10mg/ml  
QHL-008-雷西莫特 QHL-008 ＞10mg/ml  
QHL-147-雷西莫特 QHL-147 ＞10mg/ml  
QHL-116-雷西莫特 QHL-116 ＞10mg/ml  
QHL-119-雷西莫特 QHL-119 ＞10mg/ml  
QHL-140-泼尼松 QHL-140 ＞10mg/ml  
QHL-086-泼尼松 QHL-086 ＞10mg/ml  
QHL-089-泼尼松 QHL-089 ＞10mg/ml  
QHL-092-泼尼松 QHL-092 ＞10mg/ml  
QHL-095-泼尼松 QHL-095 ＞10mg/ml  
QHL-005-泼尼松 QHL-005 ＞10mg/ml  
QHL-006-泼尼松 QHL-006 ＞10mg/ml  
QHL-008-泼尼松 QHL-008 ＞10mg/ml  
QHL-147-泼尼松 QHL-147 ＞10mg/ml  
QHL-116-泼尼松 QHL-116 ＞10mg/ml  
QHL-119-泼尼松 QHL-119 ＞10mg/ml  
QHL-150-T3 QHL-150 ＞10mg/ml  
QHL-157-T3 QHL-157 ＞10mg/ml  
QHL-158-T3 QHL-158 ＞10mg/ml  
QHL-159-T3 QHL-159 ＞10mg/ml  
QHL-160-T3 QHL-160 ＞10mg/ml  
QHL-153-T3 QHL-153 ＞10mg/ml  
QHL-154-T3 QHL-154 ＞10mg/ml  
QHL-155-T3 QHL-155 ＞10mg/ml  
QHL-156-T3 QHL-156 ＞10mg/ml  
QHL-161-T3 QHL-161 ＞10mg/ml  
QHL-162-T3 QHL-162 ＞10mg/ml  
试验结果表明，在其他结构一致的条件下，2peg基团显著提水溶性，由不溶解于水变为水溶，且随着PEG数量的增加而溶解性提高。在PEG连接相同条件下，增加Glu和Asp可增加水溶性。通过基团的变化，偶联药物水溶性发生变化，从而对药物的出血管膜和对肿瘤细胞膜通透性都会有巨大影响，从而影响治疗的药效。化合物水溶性增强，为药物的成药性和偶联药物的生产提供了必要条件。
实施例17：C3，QHL-085-DOX，QHL-087-DOX，QHL-091-DOX，QHL-094-DOX注射剂在裸鼠HT1080模型中的药效研究
试验目的：调查在肿瘤治疗过程中，C3，QHL-085-DOX，QHL-087-DOX，QHL-091-DOX和QHL-94-DOX在小鼠模型中抗肿瘤药效。
试验药物：C3，QHL-085-DOX，QHL-087-DOX，QHL-091-DOX和QHL-094-DOX作为注射剂，试验时用生理盐水稀释至相应浓度。
方法和结果：
1.动物：6-8周龄的裸鼠，全为雌性。
2.肿瘤模型的制备
1)HT1080细胞从ATCC购买，并按照ATCC提供的说明书进行鉴定。细胞在包含10％胎牛血清的DMEM培养基中于37℃，5％CO 2中培养。每三天进行一次传代，并使用15代以内的细胞。
2)肿瘤的产生：5×10 6的HT1080细胞皮下注射至裸鼠的背部。当肿瘤大小达到100mm 3时，进行随机分组。然后开始治疗，开始治疗的当天记为第1天。
3)治疗过程
根据C3，QHL-085-DOX，QHL-087-DOX，QHL-091-DOX和QHL-094-DOX的临床应用，静脉注射药物(IV)。以低剂量和相同剂量的18umol/kg分别施用C3，QHL-085-DOX，QHL-087-DOX，QHL-091-DOX和QHL-094-DOX。对照组给予生理盐水。每周一次给药，持续3周。
4)结果与讨论：分组和试验结果如图7所示，与等摩尔剂量低剂量治疗组相比，4peg和2peg组抑瘤效果依次增强。
实施例18：C1，C2，C3，QHL-086-DOX，QHL-092-DOX，QHL-095-DOX，QHL-087-DOX，QHL-010-DOX，QHL-117-DOX注射剂在裸鼠HT1080模型中的药效研究
试验目的：调查在肿瘤治疗过程中上述化合物在小鼠模型中抗肿瘤药效。
试验药物：C1，C2，C3，及对应化合物注射剂，试验时用生理盐水稀释至相应浓度。
方法和结果：
1.动物：6-8周龄的裸鼠，全为雌性。
2.肿瘤模型的制备
1)HT1080细胞从ATCC购买，并按照ATCC提供的说明书进行鉴定。细胞在包含10％胎牛血清的DMEM培养基中于37℃，5％CO 2中培养。每三天进行一次传代，并使用15代以内的细胞。
2)肿瘤的产生：5×10 6的HT1080细胞皮下注射至裸鼠的背部。当肿瘤大小达到100mm 3时，进行随机分组。然后开始治疗，开始治疗的当天记为第1天。
3)治疗过程
根据相应化合物的临床应用，静脉注射药物(IV)。以低剂量和相同剂量的36umol/kg施用表格中所示的化合物。对照组给予生理盐水。每周一次给药，持续3周。
5)分组和试验结果见表5。
表5：C1，C2，C3及本发明部分化合物和丝裂霉素对裸鼠肿瘤的治疗
Figure PCTCN2021077056-appb-000051
5)结果与讨论：如表5所示，与等摩尔剂量AANL-DOX治疗组相比，QHL-086-DOX，QHL-092-DOX，QHL-095-DOX，QHL-087-DOX，QHL-010-DOX，QHL-117-DOX高剂量治疗极大地提高了对肿瘤的生长抑制，肿瘤消失，达到了治愈的效果。
实施例19：QHL-087-DOX和EMC-AANL-DOX在肝脏原位移植CT26肿瘤中的组织分布研究。
试验目的：研究肝肿瘤的激活性药物组织分布。
试验动物：6-8周龄的BALB/c小鼠，全为雌性。
制备肿瘤模型：
1)CT26细胞购置于ATCC，细胞在包含10％胎牛血清的DMEM培养基中于37℃，5％CO 2中培养。每三天进行一次传代，并使用15代以内的细胞。
2)肿瘤的产生：5×10 6的CT26细胞皮下注射至裸鼠的背部。当肿瘤大小达到800-1000mm 3时，进行随机分组。然后提取肿瘤组织并切成100mm 3的肿瘤组织块并原位移植到BALB/c小鼠肝脏中。
3)给药过程：14天后，当原位移植肿瘤长大时，对一组36只原位移植肿瘤小鼠进行药物治疗。然后在1、6、12、24、36、72hr收集不同的组织以检测在不同组织中释放的阿霉素的浓度。计算出AUClast h*nmol/g，均值和SEM如图8和9所示。
4)结果与讨论：如图8和9所示，QHL-087-DOX和EMC-AANL-DOX的活性阿霉素主要分布在肝脏和肝脏原位肿瘤中。之前EMC-AANL-DOX的公布的用途是用于乳腺癌治疗，通过进一步研究发现QHL-087-DOX具有将更多药物递送到肝脏的特性，而在肝脏原位肿瘤的激活使得阿霉素暴露量更高。相对EMC-AANL-DOX，QHL-087-DOX的原位肝癌递送和激活阿霉素暴露量增强。
实施例20：原位肝移植CT26肿瘤中QHL-087-DOX的药效研究。
试验目的：研究QHL-087-DOX，PD-1及其组合在原位移植CT26肿瘤中的疗效。
试验药物：QHL-087-DOX 18微摩尔/千克，小鼠PD-1 5毫克/千克。
动物：6-8周大的BALB/c小鼠，均为雌性。
1)肿瘤模型的产生：CT26肿瘤细胞来自ATCC。将细胞在含有10％胎牛血清的DMEM培养液中于37℃和5％CO 2培养。每三天传代细胞，并使用第15代内的细胞。将5×10 5CT26癌细胞皮下注射到裸鼠的背部。肿瘤达到800-1000mm 3后，将小鼠随机分组。然后提取肿瘤组织并切成100mm 3的肿瘤组织块并原位移植到BALB/c小鼠肝脏中。1周后，当原位移植肿瘤长大时，将原位移植肿瘤的小鼠随机分组。
2)治疗过程：一组中的6只小鼠用药物治疗。治疗当天为第1天。根据QHL-087-DOX的临床应用，每周一次静脉注射(IV)药物，持续3周。每周两次静脉内注射(IV)小鼠PD-1抗体，持续3周。分组和测试结果见附图10。
3)结果与讨论：首次发现QHL-087-DOX与PD-1联和治疗具有对肝原位瘤的免疫协同效果，QHL-087-DOX与PD-1联和相对QHL-087-DOX单药疗效更好，并具有免疫治疗的特性。
实施例21：EMC-AANL-DOX(莱古比星，legubicin)，仑伐替尼和PD-1相互联合治疗原位肝癌的效果
试验目的：EMC-AANL-DOX，仑伐替尼和PD-1相互联合治疗原位肝癌的效果。
试验药物：EMC-AANL-DOX 18微摩尔/千克，小鼠PD-1 5毫克/千克。
动物：6-8周大的BALB/c小鼠，均为雌性。
肿瘤模型的产生：CT26肿瘤细胞来自ATCC。将细胞在含有10％胎牛血清的DMEM培养液中于37℃和5％CO 2培养。每三天传代细胞，并使用第15代内的细胞。将5×10 5CT26癌细胞皮下注射到裸鼠的背部。肿瘤达到800-1000mm 3后，将小鼠随机分组。然后提取肿瘤组织并切成100mm 3的肿瘤组织块并原位移植到BALB/c小鼠肝脏中。1周后，当原位移植肿瘤长大时，将原位移植肿瘤的小鼠随机分组。
治疗过程：一组中的6只小鼠用药物治疗。治疗当天为第1天。根据EMC-AANL-DOX的临床应用，每周静脉注射一次(IV)药物，持续3周。每周两次静脉内注射(IV)小鼠PD-1抗体，持续3周。分组和测试结果见附图11。
结果与讨论：首次发现EMC-AANL-DOX与PD-1联和治疗肝原位瘤疗效优于仑伐替尼与联合PD-1联合治疗，同时发现EMC-AANL-DOX与仑伐替尼联合治疗比各自单药治疗疗效增强。
实施例22：本发明部分化合物注射液对裸鼠人肝癌HepG2细胞疗效的研究
试验目的：研究本发明部分化合物在小鼠肿瘤治疗模型中的抗肿瘤功效。
试验药物：表格中对应化合物注射剂和对照组注射剂，试验时用生理盐水稀释至相应浓度。
方法和结果：
1.试验动物：6-8周龄的裸鼠，全为雌性。
2.肿瘤模型的制备
1)人肝癌HepG2细胞从ATCC购买，并按照ATCC提供的说明书进行鉴定。细胞在包含10％胎牛血清的DMEM培养基中于37℃，5％CO 2中培养。每三天进行一次传代，并使用15代以内的细胞。
2)肿瘤的产生：5×10 6的HepG2细胞皮下注射至裸鼠的背部。当肿瘤大小达到100mm3时，进行随机分组。然后开始治疗，开始治疗的当天记为第1天。
3)治疗过程
根据相应化合物的临床应用，静脉注射药物(IV)。化合物和对照组药物以54umol/kg的剂量给药，DOX因为毒性限制只能采用18umol/kg的剂量。对照组给予生理盐水。每周一次给药，持续4周。
6)分组和试验结果见表6。
表6：本发明中部分化合物和对照组对裸鼠肿瘤治疗的影响
Figure PCTCN2021077056-appb-000052
Figure PCTCN2021077056-appb-000053
5)结果与讨论：如表6所示，EMC-AANL-DOX对肝癌有较好的作用，本发明的优选化合物与使用相同摩尔剂量的EMC-AANL-DOX相比，本发明的化合物对肿瘤生长的治疗作用增加。
实施例23：QHL-096-DOX，QHL-087-DOX，QHL-090-DOX，QHL-093-DOX，QHL-117-DOX在CT26肿瘤免疫模型治疗中的药效研究
试验目的：研究上述化合物在CT26肿瘤模型中免疫治疗的抗肿瘤药效。
试验药物：QHL-096-DOX，QHL-087-DOX，QHL-090-DOX，QHL-093-DOX，QHL-117-DOX和对照组，剂量36umol/k，鼠源PD-1抗体，5mg/kg。
试验动物：6-8周龄BALB/c小鼠，全为雌鼠。
制备肿瘤模型：
1)CT26细胞购置于ATCC，细胞在包含10％胎牛血清的DMEM培养基中于37℃，5％CO 2中培养。每三天进行一次传代，并使用15代以内的细胞。
2)肿瘤的产生。将5×10 6个相应的细胞皮下注射到裸鼠的背部。肿瘤达到至少100mm 3后，将小鼠随机分组。然后开始治疗，治疗开始的当天是第一天。
3)治疗过程。药物以等摩尔剂量36umol/kg的剂量施用。对照组给予生理盐水。每周给药一次，持续三周。
4)肿瘤CD8+T细胞分析。将肿瘤组织匀浆，过滤肿瘤中的各个细胞，分离并用缓冲液洗涤两次，然后在室温下用白细胞共同抗原CD45-PE和CD8-FITC标记的抗体培养1小时。用含有1％胎牛血清的磷酸盐缓冲液洗涤细胞两次，然后通过流式细胞术分析白细胞共同抗原(CD45)阳性细胞中T淋巴细胞抗原(CD8)阳性细胞的比例。比率的增加表明T淋巴细胞增加，因此动物对肿瘤的免疫力得到改善。
4)分组和试验结果见表7。
表7：对应化合物与对照组对肿瘤抑制和免疫激活的影响
组别 动物数量 肿瘤体积(mm 3) 抑瘤率％ CD8∶CD45(％)
    第28天 第28天  
生理盐水 6 1887.6±646.8 0 5.2
PD-1 6 1574.6±474.5 16.6％ 6.1
C3：EMC-AANL-DOX   624.5±313.6 66.9％ 8.9
QHL-096-DOX 6 347.7±207.1 81.6％ 11.8
QHL-087-DOX 6 214.8±134.2 88.6％ 12.5
QHL-090-DOX 6 335.7±257.8 82.2％ 15.2
QHL-093-DOX 6 323.7±242.8 82.9％ 11.3
QHL-117-DOX 6 306.4±197.8 83.8％ 9.5
C3+PD-1 6 74.3±45.8 96.1％ 11.7
QHL-087-达拉非尼 6 678.4±348.7 64.0％ 6.4
QHL-090-达拉非尼 6 589.7±247.4 68.7％ 7.4
QHL-140-雷西莫特 6 342.8±112.9 81.9％ 15.4
QHL-096-DOX+PD-1 6 44.3±25.6 97.7％ 18.4
QHL-087-DOX+PD-1 6 0 100％ 19.7
QHL-090-DOX+PD-1 6 0 100％ 21.7
QHL-093-DOX+PD-1 6 0 100％ 18.4
QHL-117-DOX+PD-1 6 64.6±42.6 96.6％ 20.2
QHL-087-达拉非尼+PD-1 6 178.6±67.4 90.6％ 13.2
QHL-090-达拉非尼+PD-1 6 104.7±78.4 94.5％ 17.8
QHL-140-雷西莫特+PD-1 6 59.7±32.7 96.9％ 19.4
5)结果与讨论：C3，QHL-096-DOX，QHL-087-DOX，QHL-090-DOX，QHL-093-DOX，QHL-117-DOX联合PD-1的治疗效果相对单药提高，可治愈肿瘤。QHL-090-达拉非尼，QHL-090-达拉非尼与PD-1合并治疗也有一定的协同疗效。
实施例24：QHL-087-DOX注射液在多种肿瘤模型中的疗效研究
试验目的：通过小鼠多种肿瘤模型研究QHL-087的抗肿瘤谱。
试验药物：注射QHL-087-DOX，试验时用生理盐水稀释至相应浓度。
方法和结果：
1.动物：6-8周龄的裸鼠，均为雌性。
2.肿瘤模型的产生
1)相应的肿瘤细胞购自美国典型培养物保藏中心(ATCC)，并根据ATCC提供的规范进行鉴定。将细胞在含有10％胎牛血清的DMEM培养液中于37℃和5％CO 2培养。每三天传代细胞，并使用第15代内的细胞。
2)肿瘤的产生。将5×10 6个相应的细胞皮下注射到裸鼠的背部。肿瘤达到至少100mm 3后，将小鼠随机分组。然后开始治疗，治疗开始的当天是第一天。
3)治疗过程。根据QHL-087-DOX的临床应用，QHL-087-DOX以36umol/kg的剂量施用。对照组给予生理盐水。每周给药一次，持续三周。
4)分组和测试结果见表8。
表8：QHL-087-DOX在多种肿瘤模型中的治疗效果
组别 肿瘤细胞 抑瘤率(第26天)
人乳腺癌 MDA-MB435 91.5％
人卵巢癌 SK-OV-3 78.7％
人结肠癌 HT-29 85.3％
人类慢性白血病 K562 79.4％
人结肠癌 HT1080 90.5％
人胰腺癌 Panc-1 75.7％
人类非小细胞肺癌 A549 75.8％
人肝癌 Hepg2 100％
人肾癌 OS-RC-2 87.4％
5)结果与讨论：QHL-087-DOX在多种肿瘤模型中显示出优异的疗效，表明该药物具有广泛的抗肿瘤谱。
实施例25：HSA-EMC-AANL-DOX，HSA-QHL-087-DOX，和HSA-QHL-087-N-CBP与对照化合物的溶解性比较
将本发明实施例制得的冻干品EMC-AANL-DOX，HSA-EMC-AANL-DOX，HSA-QHL-087-DOX和HSA-QHL-087-N-CBP在无菌室进行分装，用注射用水复溶。HSA-EMC-AANL-DOX，HSA-QHL-087-DOX和HSA-QHL-087-N-CBP均能够完全溶解，如表9所示。
表9
化合物 注射用水
EMC-AANL-DOX 不溶
HSA-EMC-AANL-DOX 溶解，5mg/ml
HSA-QHL-087-DOX 溶解，35mg/ml
HSA-QHL-087-N-CBP 溶解，35mg/ml
由表9可看出，人血白蛋白偶联化合物后使得溶解性进一步提高，HSA-EMC-AANL-DOX，HSA-QHL-087-DOX和HSA-QHL-087-N-CBP作为大分子蛋白类药物可用注射用水或生理盐水直接溶解到高浓度，而无需使用EMC-AANL-DOX溶解所需的刺激性的有机溶剂。与不溶于水的EMC-AANL-DOX小分子化合物药物不同，溶解特性的变化对药物的分布代谢和药物作用方式都有巨大影响。
实施例26：本发明实施例制备的水溶性高效靶向激活的阿霉素衍生物与对照化合物的溶液稳定性比较
分别精确称量化合物EMC-AANL-DOX，HSA-EMC-AANL-DOX，QHL-087-DOX，HSA-QHL-087-DOX，QHL-087-N-CBP和HSA-QHL-087-N-CBP，在无菌室进行分装各5.0mg样品，加0.5ml灭菌注射用水配置成10mg/ml母液，EMC-AANL-DOX溶解需要50％的乙醇。取30ul母液，加入570ul不同PH值5.5的缓冲溶液，配置成0.5mg/ml样品溶液。待样品澄清后，放置于25℃/37℃水浴中，8H取样用HPLC和电泳检测样品相对0小时的含量，即可得到不同化合物的溶液稳定性数据。结果如表10所示。
表10：溶液稳定性数据
化合物 8hr
EMC-AANL-DOX 94.3％
HSA-EMC-AANL-DOX 97.8％
QHL-087-DOX 98.2％
HSA-QHL-087-DOX 100.2％
QHL-087-N-CBP 85.3％
HSA-QHL-087-N-CBP 99.8％
由上表数据可知，25℃、pH＝5.5条件下，白蛋白偶联化合物稳定性增加，对于QHL-087-N-CBP和HSA-QHL-087-N-CBP更为明显。
实施例27：HSA-EMC-AANL-DOX，HSA-QHL-087-DOX和HSA-QHL-087-N-CBP激活效率试验
EMC-AANL-DOX使用溶剂(50％注射用水+50％酒精)溶解，HSA-EMC-AANL-DOX，HSA-QHL-087-DOX和HSA-QHL-087-N-CBP使用注射用水统一溶解，并用水稀释10倍到1毫克/毫升。在本发明的实验中，在37度温度下向100微克酸化的肿瘤组织匀浆(pH6.0)中加入1毫克/毫升的样品化合物，肿瘤组织匀浆中的酶能够导致释放阿霉素，通过HPLC能够检测化合物的减少和阿霉素的增加而比较药物在肿瘤组织中的激活效率，通过筛选发现本发明的化合物EMC-AANL-DOX，HSA-EMC-AANL-DOX，HSA-QHL-087-DOX，和HSA-QHL-087-N-CBP连接在筛选的化合物中具有最高的激活效率。结果如表11所示。
表11：不同肿瘤组织匀浆中的EMC-AANL-DOX，HSA-EMC-AANL-DOX，HSA-QHL-087-DOX，和HSA-QHL-087-N-CBP的化合物激活比例(％)
Figure PCTCN2021077056-appb-000054
实施例28：EMC-AANL-DOX，HSA-EMC-AANL-DOX，QHL-087-DOX，HSA-QHL-087-DOX，QHL-087-N-CBP和HSA-QHL-087-N-CBP的毒性测定
试验目的：通过测定小鼠静脉用药MTD实验，了解本发明药物生物的急性毒性。
试验药物：EMC-AANL-DOX使用溶剂(50％注射用水，50％酒精)溶解，HSA-EMC-AANL-DOX，QHL-087-DOX，HSA-QHL-087-DOX，QHL-087-N-CBP和HSA-QHL-087-N-CBP使用注射用水溶解，试验时用生理盐水稀释到相应剂量。
动物：一级巴比赛(BALB/C)小鼠(购自上海斯莱克实验动物有限责任公司)，体重19-21g，全为雌性。
方法和结果：受试BALB/C小鼠36只，体重19-21g，全为雌性，按体重随机分为7组，每组6只。如表12所示剂量，分别一次性静脉注射EMC-AANL-DOX，HSA-EMC-AANL-DOX，QHL-087-DOX，HSA-QHL-087-DOX，QHL-087-N-CBP和HSA-QHL-087-N-CBP。并进行生理盐水组、紫杉醇组注射液(市售，北京悦康)的对照试验，每个小鼠给药体积0.2ml。连续观察17天，每日观察动物是否出现立毛树立、糟乱无光泽、昏睡、弯腰驼背、过激反应等，记录体重和死亡情况。在第3、5、14天采血样进行全血球计数，在第14天解剖动物采取心脏、肝脏、肾脏、肺、脾脏、胰腺HE染色观察。
表12：受试小鼠分别接受不同剂量的化合物注射液与生理盐水、紫杉醇注射液的死亡率结果对照
组别   剂量(微摩/公斤) 动物(只) 死亡数(只)
1 生理盐水 0 10 0
2 EMC-AANL-DOX 38.4 10 8
3 HSA-EMC-AANL-DOX 38.4 10 0
4 QHL-087-DOX 38.4 10 4
5 HSA-QHL-087-DOX 38.4 10 0
6 QHL-087-N-CBP 19.2 10 7
7 HSA-QHL-087-N-CBP 19.2 10 0
结果与讨论：本发明注射HSA-EMC-AANL-DOX，HSA-QHL-087-DOX和HSA-QHL-087-N-CBP时，动物没有出现立毛树立、糟乱无光泽、昏睡、弯腰驼背、过激反应和死亡情况，表明白蛋白偶联药物的毒性比非偶联药物显著降低。
实施例29：HSA-EMC-AANL-DOX，HSA-QHL-087-DOX与抗PD-1抗体联合治疗效果
试验目的：比较研究EMC-AANL-DOX，HSA-QHL-087-DOX与抗PD-1抗体联合治疗效果。
试验药物：HSA-EMC-AANL-DOX和HSA-QHL-087-DOX，剂量分别为18μmol/kg；鼠源PD-1抗体，5mg/kg。
试验动物：6-8周龄BALB/c小鼠，全为雌鼠。
制备肿瘤模型：CT26细胞购置于ATCC，细胞在包含10％胎牛血清的DMEM培养基中于37℃，5％CO 2中培养。每三天进行一次传代，并使用15代以内的细胞。将5×10 6CT26癌细胞皮下注射给小鼠。给小鼠注射药物3次，每星期一次，抗PD-1抗体一周两次，共8次。
结果与讨论：试验结果见图12，HSA-QHL-087-DOX联合抗PD-1抗体的治疗效果相对HSA-EMC-AANL-DOX提高，治愈率更高。
实施例30：MTT法测定N-CBP以及HSA-QHL-095-N-CBP对肿瘤细胞生长的抑制作用
将分离及培养细胞计数后用培养基调整细胞浓度，将细胞接种于96孔培养板上，每孔100μl细胞悬液，CD8+T细胞100000个细胞/孔，CT26肿瘤细胞20000个细胞/孔。将96孔培养板置于37℃，二氧化碳(5％)培养箱中过夜培养24小时。待24小时后，96孔培养板加入含有不同浓度药物的细胞培养液100ul，并设置不加药物只加相应药物溶剂的对照孔(0.1％DMSO)，同时设置只加培养基不含细胞的调零孔(Blank)。每组设3个平行孔，然后将板子置于37℃，二氧化碳(5％)培养箱中培养48小时。48小时后，每孔加入20μl MTT(浓度为5mg/ml)，继续孵育4h。然后将培养液轻轻吸出，每孔加入150μl DMSO作溶剂溶解，溶解后用酶标仪测定490nm处的吸光度。
结果与讨论：试验结果见图13和14。N-CBP的体外细胞毒强于卡铂和奥沙利铂，弱于顺铂(图13)；HSA-QHL-095-N-CBP相对N-CBP和卡铂的体外细胞毒毒性降低(图14)。
实施例31：HSA-QHL-095-N-CBP的单药以及与抗PD-1抗体联合治疗效果
试验目的：比较研究卡铂，HSA-QHL-095-N-CBP，以及与抗PD-1抗体联合治疗效果。
试验药物：卡铂，HSA-QHL-095-N-CBP，剂量18μmol/kg，鼠源PD-1抗体，5mg/kg。
试验动物：6-8周龄BALB/c小鼠，全为雌鼠。
制备肿瘤模型：CT26细胞购置于ATCC，细胞在包含10％胎牛血清的DMEM培养基中于37℃，5％CO 2中培养。每三天进行一次传代，并使用15代以内的细胞。将5×10 6CT26癌细胞皮下注射给小鼠。
治疗过程：注射对应药物每周一次，持续3周；抗PD-1抗体每周给药一次，持续4周。
结果与讨论：试验结果见图15，HSA-QHL-095-N-CBP等摩尔剂量显著优于卡铂，HSA-QHL-095-N-CBP联合抗PD-1抗体的治疗效果相对单药提高，获得治愈效果。
实施例32：非酒精性脂肪肝(NAFLD)模型小鼠炎症反应的影响。
实验方法：将C57小鼠随机分为正常组(标准饲料)、模型组(高脂饲料)、辛伐他汀组(阳性对照，3mg/kg)和药物组剂量组(50mg/kg)，每组6只。正常组小鼠给予标准饲料喂养，其余各组小鼠给予高脂饲料喂养以诱导NAFLD模型。造模同时，各组小鼠IV相应剂量的48umol/kg药物，每一周给药2次，共计给药8周。末次给药后12h，全自动生化分析仪测定其血清生化指标：高密度脂蛋白胆固醇(HDL-C)，低密度脂蛋白(LDL-C)。结果如表13所示。
表13
Figure PCTCN2021077056-appb-000055
Figure PCTCN2021077056-appb-000056
结果：与正常组比较，模型组血清中HDL-C含量显著降低为66.2％，LDL-C含量均显著升高为135.6％(P＜0.05)；QHL-158-T3和QHL-159-T3治疗组HDL-C，LDL-C恢复正常水平效果较好。

Claims (20)

  1. 具有下式(I)所示结构的化合物：
    MI-S-C-A  (I)
    式中，
    MI为马来酰亚胺基；
    S为提高酶切效率或提高选择性的基团；
    C为蛋白水解酶可断裂的氨基酸连接臂；和
    A为辅助连接臂。
  2. 如权利要求1所述的化合物，其特征在于，MI为下式所示的马来酰亚胺基团：
    Figure PCTCN2021077056-appb-100001
    其中，波浪线表示与S的连接位置。
  3. 如权利要求1所述的化合物，其特征在于，S表示为S1-S2-S3，其中S1选自：
    Figure PCTCN2021077056-appb-100002
    其中，Rx不存在或选自：C 1-6亚烷基，C 1-6亚烷基氨基、C 1-6亚烷基羧基和C 1-6亚烷基羰基氨基，波浪线表示与相邻部分的连接位置；S2不存在或为-[(CH 2) pO] q-，其中，p为1-4的整数、优选为2，q为0-15、优选为1-15、更优选为2-6的整数；S3不存在或选自极性氨基酸残基，如：Glu、Asp、Gly、Ala、Val、Leu、Ile、Met、Phe、Trp、Ser、Thr、Cys、Tyr、Asn、Gln、Lys、Arg和His，优选Glu和Asp；
    其中，S通过选自以下的基团与C连接：
    Figure PCTCN2021077056-appb-100003
    其中，波浪线表示与相邻部位的连接部位；
    且其中，S1、S2和S3中至少存在一个。
  4. 如权利要求1所述的化合物，其特征在于，S为-R 1-[(CH 2) pO] q-R 2-R 3-，其中，R 1与MI连接，不存在或选自C 1-6亚烷基或C 1-6亚烷基羰基氨基；R 2选自C 1-6亚烷基；R 3选自-C(O)O-、-NH-、-O-或-C(O)-R 4，其中，R 4为选自Glu、Asp、Gly、Ala、Val、Leu、Ile、Met、Phe、Trp、Ser、Thr、Cys、Tyr、Asn、Gln、Lys、Arg和His，且优 选Glu和Asp的氨基酸残基，且R 4通过其氨基与该-C(O)-形成酰胺键；p为1-4的整数；q为0-15、优选1-15、更优选2-6的整数。
  5. 如权利要求1-4中任一项所述的化合物，其特征在于，MI、S1、S2、S3、C和A相互之间通过以下任一方式连接：
    Figure PCTCN2021077056-appb-100004
    其中，波浪线表示与相邻部位的连接部位。
  6. 如权利要求1所述的化合物，其特征在于，MI-S选自：
    Figure PCTCN2021077056-appb-100005
    Figure PCTCN2021077056-appb-100006
    Figure PCTCN2021077056-appb-100007
  7. 如权利要求1所述的化合物，其特征在于，C选自肿瘤微环境中表达天冬酰胺肽链内切酶断裂的基团，且该基团中包含Asn残基。
  8. 如权利要求7所述的化合物，其特征在于，C为X 1X 2X 3，其中X 1选自L或D型Ala、Thr、Val和Asn；X 2选自L或D型Ala、Thr、Val和Ile；X 3为Asn，优选不为D-Asn；
    优选地，C选自：Ala-Ala-Asn，Thr-Ala-Asn，Val-Ala-Asn，Asn-Ala-Asn，Thr-Thr-Asn，Val-Thr-Asn，Asn-Thr-Asn，Ala-Val-Asn，Thr-Val-Asn，Val-Val-Asn，Asn-Val-Asn，Ala-Ile-Asn，Thr-Ile-Asn，Val-Ile-Asn，Asn-Ile-Asn，Ala-Thr-Asn，D-Thr-L-Val-L-Asn，D-Thr-L-Ala-L-Asn，D-Ala-L-Val-L-Asn，L-Thr-D-Val-L-Asn，L-Thr-D-Ala-L-Asn，L-Ala-D-Val-L-Asn，D-Thr-D-Val-L-Asn，D-Thr-D-Ala-L-Asn，D-Ala-D-Val-L-Asn。
  9. 如权利要求1所述的化合物，其特征在于，A选自：
    Figure PCTCN2021077056-appb-100008
    其中，波浪线表示与C的连接位置。
  10. 如权利要求1所述的化合物，其特征在于，所述S和A选自以下组QHL-001到QHL-162中的任意一组：
    Figure PCTCN2021077056-appb-100009
    Figure PCTCN2021077056-appb-100010
    Figure PCTCN2021077056-appb-100011
    Figure PCTCN2021077056-appb-100012
    Figure PCTCN2021077056-appb-100013
    Figure PCTCN2021077056-appb-100014
    优选地，C为AAN。
  11. 下式(II)所示的偶联物或其药学上可接受的盐：
    MI-S-C-A-D  (II)
    式中，MI、S、C和A如权利要求1-10中任一项所述；D为药物，优选为抗癌化合物；
    更优选地，D选自阿霉素、柔红霉素、表阿霉素、甲氨蝶呤、氟达拉滨、吉西他滨、阿糖胞苷、美法仑、尼莫司汀、米托蒽醌、丝裂霉素、喜树碱、10-羟基喜树碱、拓扑替康、氟脲苷、去氧氟尿苷、依托泊苷、氟达拉滨、卡培他滨、长春新碱、埃坡霉素B、紫杉醇、多烯紫杉醇、达拉非尼、多韦替尼、莫特塞尼、泼尼松、三碘甲状腺原氨酸、雷西莫特、下式所示铂类衍生物：
    Figure PCTCN2021077056-appb-100015
    以及以下化合物a和化合物b：
    Figure PCTCN2021077056-appb-100016
    优选地，A与D之间通过以下任一方式连接：
    Figure PCTCN2021077056-appb-100017
    其中，波浪线表示与相邻部位的连接部位。
  12. 如权利要求11所述的偶联物或其药学上可接受的盐，其特征在于，所述化合物选自：
    Figure PCTCN2021077056-appb-100018
    Figure PCTCN2021077056-appb-100019
    Figure PCTCN2021077056-appb-100020
    Figure PCTCN2021077056-appb-100021
    Figure PCTCN2021077056-appb-100022
    Figure PCTCN2021077056-appb-100023
    Figure PCTCN2021077056-appb-100024
    Figure PCTCN2021077056-appb-100025
    Figure PCTCN2021077056-appb-100026
    Figure PCTCN2021077056-appb-100027
    Figure PCTCN2021077056-appb-100028
  13. 具有下式结构的铂衍生物、其前药或药学上可接受的盐：
    Figure PCTCN2021077056-appb-100029
  14. 权利要求11或12所述的偶联物与白蛋白共价偶联形成的偶联物或其药学上可接受的盐。
  15. 一种药物组合物，其含有权利要求11或12所述的偶联物或其药学上可接受的盐、权利要求13所述的铂衍生物或其药学上可接受的盐、或权利要求14所述的偶联物或其药学上可接受的盐，以及药学上可接受的载体。
  16. 权利要求11或12所述的偶联物或其药学上可接受的盐、权利要求13所述的铂衍生物或其药学上可接受的盐、或权利要求14所述的偶联物或其药学上可接受的盐在制备治疗或预防癌症、脂肪肝(包括酒精和非酒精性脂肪肝)、脂肪性肝炎、脂肪性肝病、肝脏纤维化、肝部炎症、肝细胞损伤的脂肪变性现象的药物中的用途；优选地，所述癌症为实体癌或血液肿瘤，优选为膀胱、脑、乳房/乳腺、宫颈、结肠、直肠、食管、肾、肝、肺、鼻咽、胰腺、前列腺、皮肤、胃、子宫、卵巢、睾丸和血液部位的癌症。
  17. 权利要求1-10中任一项所述的化合物在增强化合物药物水溶性、降低药物毒性、提高药物疗效、和/或提到药物对免疫细胞的选择性中的应用，或在制备具有改善的水溶性、降低的药物毒性、提高的药物疗效、和/或提高的药物对免疫细胞的选择性的药物中的应用，或在制备将药物递送到肝脏的药物分子中的应用。
  18. 具有下式所示结构的EMC-AANL-DOX化合物或其偶联了白蛋白的药物在制备肝癌治疗药物中的应用，或在与抗PD-1抗体在制备用于联合治疗肿瘤的药物中的应用：
    Figure PCTCN2021077056-appb-100030
  19. 权利要求11或12所述的偶联物或其药学上可接受的盐、权利要求13所述的铂衍生物或其药学上可接受的盐、或权利要求14所述的偶联物或其药学上可接受的盐在制备抑制免疫抑制性细胞、抑制肿瘤相关巨噬细胞、抑制MDSC细胞、抑制血管新生、促进抗肿瘤免疫和/或促进T淋巴细胞增殖的药物中的应用。
  20. 权利要求11或12所述的偶联物或其药学上可接受的盐、权利要求13所述的铂衍生物或其药学上可接受的盐、或权利要求14所述的偶联物或其药学上可接受的盐与抗PD-1抗体在制备用于联合治疗肿瘤的药物中的应用。
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