Preparation and use of immunostimulatory coupled complexes for targeted delivery and activation

Abstract

The present invention provides the preparation and use of immunostimulatory coupling complexes for targeted delivery and activation. Specifically, the present invention provides a compound shown in formula M-S-C-A, which is used as a connecting arm, and a drug compound shown in formula M-S-C-A-D connected to a drug. The drug compound of the present invention has improved water solubility, reduced cytotoxicity, and enhanced drug activity.

Description

Technical Field

[0001] The present invention relates to an anti-tumor drug compound, and in particular to the preparation and use of an immunostimulatory coupling complex for targeted delivery and activation Background Art

[0002] Legumain was first identified as an asparagine endopeptidase in the seeds of leguminous plants and is a member of the C13 family of cysteine ​​proteases. During seed germination, legumain can process storage proteins. Later, the discovery of legumain in parasites and mammals including humans proved that the protease is highly conserved. In 1997, porcine legumain was cloned and identified for the first time. 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 a peptide chain endopeptidase that can specifically cleave the peptide bond at the C-terminus of asparagine on the peptide chain under weakly acidic conditions. CN201210573744.3 discloses a polypeptide doxorubicin derivative targeted for activation by aspartase, which releases Leu-doxorubicin compounds in tumors by cutting the tetrapeptide group (linker) by legumain. Summary of the invention

[0003] The present invention develops a chemically modified activation linker through further compound screening and biological systematic research, which can further enhance the activation efficiency. In addition, the chemically modified activation linker of the present invention can enhance the selectivity of the coupled drug to immune cells, produce immunotherapy enhancement properties during treatment, and enhance the synergistic therapeutic effect of combining with PD-1 antibodies.

[0004] The technical problem to be solved by the present invention is to create a coupling arm with high efficiency and specific selection. Previous studies have found that asparagine endopeptidase preferentially recognizes the substrate peptide sequence of the tetrapeptide and cuts the amide bond between Asn and other residues. The idea of ​​the present invention to improve the activation efficiency is: first, a large number of compounds with different structures are synthesized at both ends of the tripeptide (such as AAN), and then these compounds are used to further study the working mechanism of asparagine endopeptidase. According to the crystal structure of asparagine endopeptidase ( Figure 1), since the active center of asparagine endopeptidase is located at the bottom of the invaginate, the substrate peptide needs to be activated close to the enzyme active center at the bottom; in the figure, C189 at the S1 position of asparagine endopeptidase attacks and cuts off the Asn peptide chain, while the adjacent positions S2, S3, S4 and S1 combine with the substrate to determine the efficiency of enzyme cleavage, and S2 and S3 correspond to the Ala-Ala amino acids of the substrate peptide. Taking into account the possible interactions or spatial hindrances caused by connecting compounds such as doxorubicin and the MI group, this experiment further synthesized and screened chemically modified structures at both ends of the substrate tripeptide. After we connected the synthesized compounds with different groups with MI and doxorubicin, we screened the activation efficiency under the conditions of tumor tissue or asparagine endopeptidase, thereby optimizing and obtaining new compound couples with mutual structure-activity relationships. The schematic diagram of the structure is shown in Figure 2 , including MI group, selective group S, tripeptide group C cleaved by asparagine endopeptidase, auxiliary group A and coupled drug. The added group of the present invention brings new functions: in addition to enhancing the activation activity of asparagine endopeptidase on the coupled drug compound (D), the physical properties and biological functions of the drug compound are also improved. The drug compound provided by the present invention is hydrophilic, and its cell membrane permeability is changed, so it is the most suitable compound for drug development. In addition, the present invention also found that the drug 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 and MDSC cells, thereby relieving the inhibitory effect of tumor-associated macrophages on immunity and promoting immunotherapy. The present invention also found that when coupled with doxorubicin, the length of the S group affects the activation efficiency. The longer the chain length of S, the less favorable the binding of the compound to the enzyme due to steric hindrance, and the lower the activation efficiency.

[0005] Human serum albumin (HSA) is a small globular protein consisting of 585 amino acids (66-69 kd), with many charged residues (such as lysine, aspartic acid and groups without prosthetic groups or carbohydrates), and a small amount of tryptophan or methionine residues. The compound of formula (II) of the present invention is coupled to cysteine ​​at position 34 of human serum albumin to form a macromolecular drug; through experiments, it was found that the compound of formula (II) of the present invention or EMC-AANL-DOX covalently coupled to albumin has reduced toxicity, improved drug stability and greatly improved therapeutic efficacy.

[0006] In summary, the linker arm of formula (I) and the drug compound of formula (II) of the present invention have enhanced activation efficiency, enhanced selectivity for immune cells, enhanced tissue selectivity, suitable water solubility and lipid solubility, and drug stability.

[0007] Therefore, the present invention provides the compound (linker) of formula (I) and the drug compound (conjugate) represented by formula (II) as described herein and pharmaceutically acceptable salts thereof.

[0008] The present invention also provides a platinum derivative having the following structure or a pharmaceutically acceptable salt thereof:

[0009]

[0010] The present invention also provides a drug compound of 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 is linked to the MI or EMC portion of formula (II) via its cysteine ​​residue at position 36.

[0011] The present invention also provides a pharmaceutical composition, which contains a compound of formula (II) of the present invention or a pharmaceutically acceptable salt thereof, a platinum derivative of the present invention or a pharmaceutically acceptable salt thereof, a drug compound of 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 pharmaceutically acceptable carrier.

[0012] The present invention also provides the use of a compound of formula (II) or a pharmaceutically acceptable salt thereof, a platinum derivative or a pharmaceutically acceptable salt thereof according to the present invention, a drug compound of formula (II) of the present invention covalently linked to albumin or a pharmaceutically acceptable salt thereof, or EMC-AANL-DOX or a pharmaceutically acceptable salt thereof covalently linked to albumin in the preparation of a drug for treating or preventing cancer, fatty liver (including alcoholic and non-alcoholic fatty liver), fatty hepatitis, fatty liver disease, liver fibrosis, liver inflammation, fatty degeneration of liver cell damage; preferably, the cancer is a solid cancer or a blood tumor, preferably cancer of the bladder, brain, breast / mammary gland, cervix, colon, rectum, esophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin, stomach, uterus, ovary, testis and blood.

[0013] The present invention also provides the use of the compound described in formula (I) in enhancing the water solubility of compound drugs, reducing drug toxicity, improving drug efficacy, and / or increasing the selectivity of drugs for immune cells, or in preparing drugs with improved water solubility, reduced drug toxicity, improved drug efficacy, and / or improved drug selectivity for immune cells, or in preparing drug molecules for delivering drugs to the liver.

[0014] The present invention also provides an EMC-AANL-DOX compound having a structure shown in the following formula or a drug coupled with albumin (preferably, covalently linked to the EMC portion via the cysteine ​​residue at position 36 of albumin) for use in preparing a drug for treating liver cancer, and an anti-PD-1 antibody and / or an anti-PD-L1 antibody for use in preparing a drug for combined treatment of tumors:

[0015]

[0016] The present invention also provides the use of a compound of formula (II) or a pharmaceutically acceptable salt thereof, a platinum derivative or a pharmaceutically acceptable salt thereof according to the present invention, a drug compound of formula (II) of the present invention covalently linked to albumin or a pharmaceutically acceptable salt thereof, or EMC-AANL-DOX or a pharmaceutically acceptable salt thereof covalently linked to albumin in the preparation of a drug for inhibiting immunosuppressive cells, inhibiting tumor-associated macrophages, inhibiting MDSC cells, inhibiting angiogenesis, promoting anti-tumor immunity and / or promoting T lymphocyte proliferation.

[0017] The present invention also provides the use of a compound of formula (II) or a pharmaceutically acceptable salt thereof, a platinum derivative or a pharmaceutically acceptable salt thereof according to the present invention, a drug compound of formula (II) of the present invention covalently linked to albumin or a pharmaceutically acceptable salt thereof, or EMC-AANL-DOX or a pharmaceutically acceptable salt thereof covalently linked to albumin and an anti-PD-1 antibody in the preparation of a drug for combined treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Crystal structure and substrate map of asparagine endopeptidase.

[0019] Figure 2 : Schematic diagram of immunostimulatory conjugate complexes for targeted delivery and activation.

[0020] Figure 3 : Comparison of enzyme cleavage kinetics of preferred compounds.

[0021] Figure 4 :Isolation of mouse bone marrow mononuclear cells and induction of differentiation into M2 macrophages.

[0022] Figure 5 :Cytotoxicity experiment of compounds on CD8+T cells.

[0023] Figure 6 :Cytotoxicity experiment of compounds on M2 macrophages.

[0024] Figure 7 :Effectiveness experiment of the compound on HT1080 tumor.

[0025] Figure 8 : EMC-AANL-DOX has a high distribution characteristic in liver and liver cancer tissues.

[0026] Fig. 9 :QHL-087-DOX has a high distribution characteristic in liver and liver cancer tissues.

[0027] Fig.10 :QHL-087-DOX combined with anti-PD-1 antibody for the treatment of hepatic tumor in situ.

[0028] Fig.11 : The efficacy of EMC-AANL-DOX combined with anti-PD-1 antibody in the treatment of hepatic tumor in situ is better than that of lenvatinib combined with anti-PD-1 antibody.

[0029] Fig.12 :Combination therapy effects of HSA-EMC-AANL-DOX, QHL-087-DOX and anti-PD-1 antibody

[0030] Fig.13 :In vitro cytotoxicity experiment of N-CBP.

[0031] Fig.14 :HSA-QHL-095-N-CBP cytotoxicity experiment.

[0032] Fig.15 : The therapeutic effects of HSA-QHL-095-N-CBP as a single agent and in combination with anti-PD-1 antibodies. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0034] 1. Linker Compounds

[0035] The present invention provides a compound having a structure shown in the following formula (I), which can be used as a linker arm. When connected to a drug of interest (such as an anticancer compound), the compound can enhance the water solubility of the drug, reduce the toxicity of the drug, improve the efficacy of the drug, and / or increase the selectivity of the drug for immune cells:

[0036] MI-SCA (I)

[0037] In the formula, MI is a maleimide group; S is a group that improves the efficiency of enzymatic cleavage or improves the selectivity; C is an amino acid linker arm that can be cleaved by a proteolytic enzyme; and A is an auxiliary linker arm.

[0038] An exemplary MI is a maleimide group represented by the following formula:

[0039]

[0040] The wavy line indicates the connection position with S.

[0041] In some embodiments, S in formula (I) can be represented by S1-S2-S3, wherein S1 is selected from:

[0042]

[0043] Wherein, Rx is absent or selected from: C 1-6 Alkylene, C 1-6 Alkyleneamino, C 1-6 Alkylene carboxyl and C 1-6 Alkylenecarbonylamino, the wavy line indicates the connection position with the adjacent part; S2 is absent or -[(CH2) p O] q -, wherein p is an integer of 1-4, preferably 2, q is an integer of 0-15, preferably 1-15, more preferably 2-6; S3 is absent or 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.

[0044] It should be understood that at least one of S1, S2 and S3 exists.

[0045] Preferably, MI, S1, S2, S3, C and A are connected to each other by any of the following methods:

[0046]

[0047] Wherein, the wavy line represents the connection site with the adjacent site; preferably, S is connected to C through a group selected from the following:

[0048]

[0049] In some embodiments, S is -R1-[(CH2) p O] q -R2-R3-, wherein R1 is connected to MI, is absent or is selected from C 1-6 Alkylene or C 1-6 Alkylenecarbonylamino; R2 is selected from C 1-6Alkylene; R3 is selected from -C(O)O-, -NH-, -O- or -C(O)-R4, wherein R4 is an amino acid residue selected from Glu, Asp, Gly, Ala, Val, Leu, Ile, Met, Phe, Trp, Ser, Thr, Cys, Tyr, Asn, Gln, Lys, Arg and His, preferably Glu and Asp, and R4 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. Preferably, R1 is absent, p is 2 or 3, q ​​is 1-15, preferably 2-6, and R2 is C 1-4 Alkylene, R3 is selected from -C(O)O-, -NH- and -O-. In some embodiments, preferably, R1 is absent, q is 0, R2 is C 1-6 Alkylene, R3 is -C(O)-R4, R4 is preferably Glu and Asp, and R4 forms an amide bond with the -C(O)- through its amino group. 1-6 Alkylenecarbonylamino, p is 2 or 3, q ​​is 1-15, preferably 2-6, R2 is C 1-4 Alkylene, R3 is -C(O)-R4, R4 is preferably Glu and Asp, and R4 forms an amide bond with the -C(O)- through its amino group.

[0050] Exemplary MI-S are selected from:

[0051]

[0052]

[0053]

[0054] Preferably, C connected to any of the above MI-S is AAN, and A is any of the structures described below.

[0055] Preferably, in the compound of formula (I) of the present invention, C is selected from a group that is cleaved by asparagine endopeptidase expressed in the tumor microenvironment, and the group contains an Asn residue. In some embodiments, C is X1X2X3, wherein X1 is selected from L or D-type Ala, Thr, Val and Asn; X2 is selected from L or D-type Ala, Thr, Val and Ile; X3 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-I In some particularly preferred embodiments, C is AAN.

[0056] In the compound of formula (I) of the present invention, preferably A is selected from Leu, PABC-OH and PABC-NH2, and their structures are shown below:

[0057] (Leu), (PABC-OH), and (PABC-NH2);

[0058] The wavy line indicates the connection position with C.

[0059] In some embodiments, 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" represents -(CH2CH2O)2-, 3peg represents -(CH2CH2O)3-, 4peg represents -(CH2CH2O)4-, 6peg represents -(CH2CH2O)6-), and so on]:

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] Preferably, in the compound of formula (I) of the present invention, MI is a maleimide group, S and A are any one of the groups QHL-001 to QHL-162, and C is AAN.

[0067] Particularly preferred compounds of formula (I) of the present invention (linker arm) are 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, Any one of 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.

[0068] 2. Drug Compounds

[0069] The present invention provides a compound (conjugate) represented by the following formula (II) or a pharmaceutically acceptable salt thereof:

[0070] MI-SCAD (II)

[0071] In the formula, MI, S, C and A form the linker compound described in any embodiment of the present invention; D is a drug, preferably an anticancer compound.

[0072] In formula II, when A is used as a linking group, it is selected from:

[0073] Leu,

[0074] PABC-OH,

[0075] PABC-NH2,

[0076] The wavy lines indicate the connection positions with C and D. Preferably, the connection with C is via -NH-.

[0077] Preferably, D is selected from resiquimod, prednisone, triiodothyronine (T3), doxorubicin, daunorubicin, epirubicin, methotrexate, fludarabine, gemcitabine, cytarabine, melphalan, nimustine, mitoxantrone, mitomycin, camptothecin, 10-hydroxycamptothecin, topotecan, floxuridine, doxifluridine, etoposide, fludarabine, capecitabine, vincristine, epothilone B, paclitaxel, docetaxel, dabrafenib, dovitinib, motesanib, compound a, compound b and platinum derivatives shown in the following formula:

[0078]

[0079] Wherein, the structures of compound a and compound b are as follows:

[0080]

[0081]

[0082] More preferably, D is selected from daunorubicin, dovitinib, epirubicin, compound a, compound b, mitomycin, dabrafenib, motesanib, resimod, prednisone and T3. Preferably, the compound of formula (I) of the present invention (linker) for connection 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-117, QHL-118, QHL-119, QHL-200, QHL-201, QHL-202, QHL-203, QHL-204, QHL-205, QHL-206, QHL-207, QHL-208, QHL-209, QHL-210, QHL-211, QHL-212, QHL-213, QHL-214, QHL-215, QHL-216, QHL-217, QHL-218, QHL-219, QHL-220, QHL-221 Any one of 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.

[0083] Preferably, A and D are connected by any of the following methods:

[0084]

[0085] The wavy lines represent the connection parts with the adjacent parts.

[0086] More preferably, A and D are connected by -CO-NH-, wherein the carbonyl group is connected to A or is a part of A (such as when A is Leu), and the amino group is connected to D or is a part of D. Generally, 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.

[0087] Preferably, the pharmaceutical compound of formula (II) of the present invention is selected from:

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097] In some embodiments of the present invention, the present invention also provides a platinum derivative having the following structure, a prodrug thereof, or a pharmaceutically acceptable salt thereof:

[0098]

[0099] 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 a pharmaceutical compound of formula (II) of the present invention covalently linked to albumin. Typically, albumin is linked to the MI of the connecting arm. In some embodiments, the present invention also includes EMC-AANL-DOX linked to albumin, a pharmaceutical composition thereof, and its use. The present invention also includes a pharmaceutical compound of formula (II) of the present invention covalently linked to albumin or a pharmaceutically acceptable salt thereof.

[0100] In the present invention, 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, citrate, lactate, tartrate, maleate, fumarate, mandelate and oxalate; and inorganic and organic base salts formed with bases such as sodium hydroxy, tris (hydroxymethyl) aminomethane (TRIS, tromethamine) and N-methylglucamine.

[0101] 3. Preparation method

[0102] Exemplary preparation methods of compounds of formula (I) and (II) of the present invention include:

[0103] Step 1: preparing tripeptide-PABC or tetrapeptide: coupling amino acid residues and separating the formed tripeptide-PABC or tetrapeptide, i.e. CA;

[0104] 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;

[0105] Step 3: Preparation of MI-SCA: CA obtained in step 1 and MI-S obtained in step 2 are coupled via amino and carboxyl groups to obtain an intermediate (MI-SCA);

[0106] Step 4: The carboxyl or hydroxyl activation product at the A-terminus of the compound MI-SCA obtained in step 3 is covalently bound to the amino group of an optional drug to form an immunostimulatory doxorubicin-coupled complex for targeted delivery and activation.

[0107] When A is PABC-OH, 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 purifying and isolating to obtain C-PABC containing 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, the protecting groups are removed to obtain C-PABC. Then, in the presence of a condensing agent, a base, and a polar aprotic solvent, C-PABC is reacted with an acid or ester or acyl chloride containing a MI-S group to form MI-SCA represented by formula (I) of the present invention, and then reacted with a drug compound of interest or a salt thereof in the presence of a condensing agent, a base, and a polar aprotic solvent to form a drug compound represented by formula (II) of the present invention.

[0108] 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 can be any solvent as long as 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, aprotic solvents such as acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, etc., ester solvents such as methyl acetate and ethyl acetate, etc., ether solvents such as tetrahydrofuran, or mixtures of these solvents. The reaction in the preparation method can be carried out at a temperature ranging from ice cooling to 150°C.

[0109] IV. Pharmaceutical Composition

[0110] The present invention includes a pharmaceutical composition, which contains a compound of formula (II) of the present invention or a pharmaceutically acceptable salt thereof, or a platinum derivative of the present invention or a pharmaceutically acceptable salt thereof, or a compound of formula (II) covalently linked to albumin or a pharmaceutically acceptable salt thereof, or EMC-AANL-DOX covalently coupled to albumin or a pharmaceutically acceptable salt thereof.

[0111] The pharmaceutical composition may further contain a pharmaceutically acceptable carrier or excipient. The carrier or excipient may be any pharmaceutically acceptable carrier or excipient known in the art, and may vary depending on the pharmaceutical dosage form or administration method.

[0112] In a specific embodiment, the pharmaceutical composition contains one or more of a solvent, a solubilizer / co-solvent, a pH adjuster, a lyophilization excipient, and an osmotic pressure regulator.

[0113] Freeze-drying excipients suitable for the present invention include one or more of carbohydrates (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 (e.g., hydroxypropyl β-cyclodextrin, sulfobutyl β-cyclodextrin).

[0114] The pH regulators suitable for use in the present invention include one or more of 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, maleic acid, ethylenediamine, triethylamine, arginine, lysine, histidine, sodium dihydrogen phosphate and disodium hydrogen phosphate.

[0115] The solvent suitable for the present invention is preferably an organic solvent, including one or more of ethanol, propylene glycol, polyethylene glycol 300, polyethylene glycol 400, tert-butyl alcohol, glycerol, Tween, soybean oil, hydroxypropyl β-cyclodextrin solution and sulfobutyl β-cyclodextrin solution.

[0116] The osmotic pressure regulators suitable for use in the present invention include one or more of glucose, sodium chloride, mannitol and sodium lactate.

[0117] Solubilizers / co-solvents suitable for use in the present invention include one or more of Tween 80, Tween 60, poloxamer, hydroxypropyl β-cyclodextrin, polyethylene glycol (PEG), lithium dodecanoate, sulfobutyl β-cyclodextrin, PVP, glycerol and polyoxyethylene castor oil.

[0118] Generally, the compound of the present invention or its pharmaceutically acceptable salt is orally administered to mammals daily, 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 anticancer drug or other treatment is administered at the same time, the 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.

[0119] 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 may be administered once or more, one or more times per day, each dose containing about 0.1 to 50 mg, conveniently about 0.25 to 10 mg of the compound of the present invention or a pharmaceutically acceptable salt thereof.

[0120] The pharmaceutical composition of the present invention can be prepared into any suitable dosage form, including but not limited to tablets, capsules, injections, etc. The pharmaceutical composition of the present invention can be administered by routes well known in the art, such as oral administration, intravenous injection, intramuscular injection, etc.

[0121] V. Uses of the Compounds and Pharmaceutical Compositions

[0122] The cytokines secreted by the tumor induce monocytes to transform into tumor-associated macrophages (TAMs), which can stimulate strong immunosuppression and directly help tumor cells to infiltrate and metastasize. The confirmation marker of tumor-associated macrophages (M2 type) is the expression of asparagine endopeptidase, which is different from monocytes and inflammatory macrophages (M1 type). The compound of the present invention can be activated and released under the condition of the presence of aspartic acid endopeptidase. Because the different parts of the conjugates specifically activated by asparagine endopeptidase have a huge impact on the functions of the final drug in terms of targeting, activation, stability, toxicity and efficacy, therefore, the conjugates specifically activated by asparagine endopeptidase of the present invention can effectively reduce the toxicity of the connected drug, so that the final drug has new targeting, activation and metabolic characteristics, increases the effect of treating tumors, and produces new tumor indications and anti-tumor metastasis, and produces a brand-new structure and function.

[0123] The present invention also found that the compound of formula (II) of the present invention has the immunopotency of killing tumor-associated macrophages, weakening immunosuppressive cytokines in the microenvironment and promoting toxic CD8 cells. More importantly, these tumor microenvironment-releasing compounds are only activated locally in the tumor, which is different from traditional chemotherapy drugs that damage the overall immune system. In the experiment, tumor microenvironment-releasing compounds and PD-1 (programmed death molecule 1, programmed death-1) inhibitory antibodies (anti-PD-L1 antibodies, commercially available, are currently considered candidate drugs with immunotherapy effects) have a strong synergistic therapeutic effect, which can solve the disadvantage that immunotherapy is difficult to combine with chemotherapy drugs.

[0124] Therefore, the compounds of the present invention, their pharmaceutically acceptable salts, or pharmaceutical compositions can be used to treat or prevent various diseases known in the art that can be treated with resiquimod, prednisone, T3, doxorubicin, daunorubicin, epirubicin, methotrexate, fludarabine, gemcitabine, cytarabine, melphalan, nimustine, mitoxantrone, mitomycin, camptothecin, 10-hydroxycamptothecin, topotecan, floxuridine, doxifluridine, etoposide, fludarabine, capecitabine, vincristine, epothilone B, paclitaxel, docetaxel, dabrafenib, dovitinib, motesanib, compound a, compound b, and platinum compounds (such as carboplatin, cisplatin, oxaliplatin) including cancer, ophthalmic diseases, liver diseases, etc.

[0125] For example, it is known in the art that 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, leukemia, etc.; paclitaxel is mainly used to treat ovarian cancer and breast cancer, and is also effective for lung cancer, colorectal cancer, melanoma, head and neck cancer, lymphoma, brain tumor, etc.; mitomycin can be used to treat chronic lymphoma, chronic myeloid 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 tumors, bladder tumors, malignant intracavitary effusion, etc.

[0126] Thus, for example, diseases that can be treated or prevented by the compounds of the present invention, their pharmaceutically acceptable salts or pharmaceutical compositions include, but are not limited to, cancers of the bladder, brain, breast / mammary gland, cervix, colon-rectum, esophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin, stomach, uterus, ovary, testis and blood. Specifically, these cancers are selected from the group consisting of liver cancer, kidney cancer, thyroid cancer, colorectal cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, rectal cancer, esophageal cancer, lung cancer (e.g., bronchial lung cancer, including undifferentiated small cell and non-small cell), nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, ovarian cancer, testicular cancer, blood cancer (e.g., chronic or acute leukemia, including lymphocytic and granulocytic leukemia), malignant lymphoma, fibrosarcoma, soft tissue sarcoma, osteogenic sarcoma, rhabdomyosarcoma, Ewing's sarcoma, Wilms' tumor, neuroblastoma, thyroid cancer and head and neck squamous cell carcinoma.

[0127] In a specific embodiment, 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 to treat or prevent ophthalmic diseases, including treating or preventing wound healing scars or choroidal neovascularization, or inhibiting macrophages. In other embodiments, the pharmaceutical compound represented by formula (II) wherein D is mitomycin, or a pharmaceutically acceptable salt thereof, can also be used to treat or prevent sequelae of corneal transplantation, glaucoma, pterygium surgery, etc.

[0128] The compound or pharmaceutical composition of the present invention can also be used to prevent tumor metastasis, especially to prevent tumor lung metastasis. In one embodiment, the compound or pharmaceutical composition of the present invention can be used to prevent breast cancer lung metastasis.

[0129] The liver diseases described in 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 fatty degeneration of liver cell damage.

[0130] Therefore, the present invention includes a method for treating or preventing a disease (preferably cancer, ophthalmic disease and liver disease according to any embodiment of the present invention), comprising administering to a subject in need thereof an effective amount of a compound of formula (II) of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing a compound of formula (II) of the present invention or a pharmaceutically acceptable salt thereof. In some embodiments, a platinum derivative or a pharmaceutically acceptable salt thereof according to the present invention, or a compound of formula (II) or a pharmaceutically acceptable salt thereof covalently linked to albumin, or EMC-AANL-DOX or a pharmaceutically acceptable salt thereof covalently coupled to albumin, or a pharmaceutical composition thereof, or each of them is administered.

[0131] The present invention also includes a method for preventing tumor metastasis, comprising administering an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing a compound of the present invention or a pharmaceutically acceptable salt thereof, to a subject in need thereof. Preventing tumor metastasis includes but is not limited to preventing tumor lung metastasis and / or bone metastasis.

[0132] Tumor-associated macrophages (TAMs), as a key inflammatory cell, play an extremely important role in tumor-related inflammation. In the tumor microenvironment, TAMs promote tumor development by affecting the biological characteristics of various aspects of the tumor. It secretes some molecules (such as EGF) to directly promote the growth of tumor cells and promote angiogenesis, thereby creating conditions for the infiltration and metastasis of cancer cells, and can also inhibit the acquired immune function. Therefore, the present invention also includes a method for inhibiting tumor-associated macrophages, comprising administering an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof to a subject in need, or a pharmaceutical composition containing a compound of the present invention or a pharmaceutically acceptable salt thereof. By inhibiting tumor-associated macrophages, tumor growth, angiogenesis, and infiltration and metastasis of cancer cells can be inhibited, and anti-tumor immunity can be promoted, thereby achieving the prevention and / or treatment of cancer. In a specific embodiment, tumor-associated macrophages express aspartate endopeptidase, which is M2 type.

[0133] The above method of the present invention can be used in combination with radiotherapy or immunotherapy known in the art.

[0134] Therefore, the present invention also includes the compounds of the present invention, pharmaceutically acceptable salts thereof, or pharmaceutical compositions of the present invention for use in the above-mentioned various methods or uses.

[0135] The present invention also includes the use of the compounds of the present invention or their pharmaceutically acceptable salts or the pharmaceutical compositions of the present invention in the preparation of drugs for treating or preventing the above-mentioned diseases (e.g., cancer and cancer metastasis). The present invention also includes the use of the compounds of the present invention or their pharmaceutically acceptable salts or the pharmaceutical compositions of the present invention in the preparation of drugs for inhibiting tumor-associated macrophages, inhibiting tumor growth, inhibiting angiogenesis, inhibiting the infiltration and metastasis of cancer cells and / or promoting anti-tumor immunity.

[0136] The present invention also provides a method for reducing the toxic side effects of an anticancer compound (especially the anticancer compound described herein), the method comprising connecting the anticancer compound to a linker compound represented by formula (I) of the present invention.

[0137] The treatment or prevention method of the present invention comprises administering the compound or pharmaceutical composition of the present invention to a subject in need thereof. The administration method includes but is not limited to oral administration, intravenous injection, intramuscular injection, etc. The subject includes mammals, especially humans.

[0138] In some embodiments, the present invention also provides the use of an EMC-AANL-DOX compound having a structure shown in the following formula or a drug coupled with albumin in the preparation of a drug for treating liver cancer:

[0139]

[0140] It should be understood that the present invention "contains", "includes" also includes "consists of", "is composed of". The sum of all weight percentages or volume percentages should be equal to 100%. The various reagents and products used in the examples are commercially available unless otherwise specified; for the methods involved, unless otherwise specified, they are implemented according to conventional techniques. The following examples are not intended to limit the scope of the present invention.

[0141] Example 1: Synthesis of QHL-095-DOX

[0142] The synthesis process of QHL-095-DOX is as follows:

[0143]

[0144] 1. Synthesis of Intermediate 1

[0145] Take a dry and clean 2L reaction bottle, add THF 500ml, weigh Fmoc-Asn(Trt)-OH 80g, add it to the reaction bottle, stir to dissolve, add DEPBT 46.6g, stir at room temperature for 15 minutes, add PABC 16g, react at room temperature for 30 minutes, add DIPEA 45ml, ventilate with nitrogen for protection, react at room temperature for 3 hours, and monitor the reaction by TLC (Fmoc-Asn(Trt)-OH reaction is complete).

[0146] The reaction solution was evaporated under reduced pressure, a small amount of DMF (180 ml) was added to dissolve it, and the mixture was added dropwise to 3 L of stirring water to precipitate a light yellow solid. After washing with water 2-3 times, the solid was collected by suction filtration and dried in vacuo to obtain an off-white solid (yield of more than 90%).

[0147] 2. Synthesis of Intermediate 2

[0148] Add 500ml of THF and the off-white solid obtained in the previous step to a 2L single-mouth reaction bottle in sequence, stir to dissolve, cool to 0-5°C in an ice-salt bath, add 100ml of piperidine dropwise, add dropwise, gradually return to room temperature to react for 1h, and monitor the completion of the reaction by TLC. Evaporate the solvent under reduced pressure, add a small amount of DMF to dissolve, add dropwise to 2L of stirring water, stir mechanically for 30min, filter, repeat water washing 2-3 times, filter, add 800ml of methyl tert-butyl ether to the filter cake, stir for 30min, filter, add PE:EA=10:1 to the filter cake and wash twice, filter, collect the filter cake, and vacuum dry to obtain 80g of off-white solid with a purity of 70%.

[0149] 3. Synthesis of Intermediate 3

[0150] Into a dry and clean 250ml single-mouth reaction bottle, 50ml THF, 5.04g Boc-Ala-Ala-OH, and 3.89g DEPBT were added in sequence, and the reaction was carried out at room temperature for 10min. 2.6g NH2H2H2-Asn(Trt)-PABC was added, and nitrogen was ventilated for protection. The reaction was carried out at room temperature for 15min. 3.5ml DIPEA was added dropwise, and nitrogen was ventilated for protection. The reaction was carried out at room temperature for 3 hours. The solvent was evaporated under reduced pressure, and water was added to slurry 2-3 times. The mixture was filtered to obtain a light yellow solid (3.7g). The product was purified by column to obtain 2.0g of the product with a purity of 94.8% and a yield of 26.6%.

[0151] 4. Synthesis of Intermediate 4

[0152] 1.8 g of intermediate 3 was added to a 250 ml single-mouth reaction bottle, 28.5 ml of TFA was added, 1.5 ml of water was added dropwise, and the reaction was carried out at room temperature for 30 min. After the reaction was completed by monitoring by TLC, the solvent was evaporated under reduced pressure, methyl tert-butyl ether was added to slurry, and the solid was filtered to obtain a solid. The solid was dissolved in a solution of dioxane: water = 1:1, and 1N sodium hydroxide was added to adjust the pH to 13. The mixture was stirred at room temperature for 40 min, the solvent was evaporated under reduced pressure, and the sample was mixed with silica gel and passed through a column to obtain 450 mg of the product with a yield of 47.5%.

[0153] 5. Synthesis of MI-S intermediate

[0154] MI-S1 (338 mg, 2 mmol) and DEPBT (717.6 mg, 2.4 mmol) were added to a 100 ml single-mouth bottle, and DMF (15 ml) was added to dissolve, nitrogen was ventilated for protection, and the reaction was carried out at room temperature for 15 min. R3-b (819 mg, 2 mmol) was added, and after stirring and dissolving, the reaction was carried out at room temperature for 15 min. DIPEA 137 μl was added dropwise, nitrogen was ventilated for protection, and the reaction was carried out at room temperature for 3 h. The reaction of R3-a was completed by TLC monitoring, and the solvent was removed by distillation under reduced pressure. The crude product was dissolved in methanol and passed through a reverse phase high pressure column to obtain the intermediate of R3-1 (720 mg, yield: 64.3%).

[0155] 6. Synthesis of MI-S

[0156] The intermediate obtained in the previous step (720 mg, 1.28 mmol) was added to a 100 ml single-mouth reaction bottle, 15 ml of dichloromethane was added to dissolve, 5 ml of TFA was added dropwise, 0.25 ml of water was added dropwise, and the reaction was carried out at room temperature for 30 min. After the reaction was completed by TLC monitoring, the solvent was evaporated under reduced pressure, methyl tert-butyl ether was added to slurry, and suction was filtered to obtain a solid, and the sample was mixed with silica gel and passed through a reverse phase column to obtain 242 mg of the product. The yield was 37.5%.

[0157] 7. Synthesis of Intermediate 5

[0158] Intermediate 4 (150 mg, 0.395 mmol) and EMC-6Peg-COOH (239 mg, 0.474 mmol) were added to a 100 ml single-necked bottle, and DMF (15 ml) was added to dissolve, nitrogen was ventilated for protection, and the reaction was carried out at room temperature for 15 min. 137 μl of DIPEA was added dropwise, nitrogen was ventilated for protection, and the reaction was carried out at room temperature for 3 h. The reaction of intermediate 4 was completed after TLC monitoring, and the solvent was removed by distillation under reduced pressure. The crude product was dissolved in methanol and passed through a reverse phase high pressure column to obtain intermediate 5 (95 mg, yield: 21%).

[0159] 8. Synthesis of Intermediate 6

[0160] To a 100 ml single-mouth reaction bottle, 25 ml of DMF, intermediate 5 (300 mg, 0.346 mmol), and Bis-PNP (316 mg, 1.04 mmol) were added in sequence, nitrogen was ventilated for protection, and the reaction was carried out at room temperature for 15 min. 258 μl of DIPEA was added dropwise, nitrogen was ventilated for protection, and the reaction was carried out at room temperature for 3 h. HPLC monitoring showed that the remaining raw material was 7%, the reaction was terminated, the solvent was evaporated under reduced pressure, and the product was purified by column to obtain 150 mg of the product with a yield of 42%.

[0161] 9. Synthesis of the final product QHL-095-DOX

[0162] To a 100 mL reaction bottle, 84 mg of doxorubicin hydrochloride (1.0 eq, 0.145 mmol) and 150 mg of intermediate 6 (1.0 eq, 0.145 mmol) were added, and the mixture was reacted at room temperature for 15 min under nitrogen protection. 75 μl of DIPEA was added dropwise, and the mixture was reacted at room temperature for 4 hours. The solvent was evaporated under reduced pressure, and the crude product was dissolved in methanol and passed through a reverse phase high pressure column to obtain QHL-095-DOX (49 mg red solid, yield: 23.8%).

[0163] Example 2: Synthesis of QHL-116-DOX

[0164]

[0165] 1. Synthesis of Intermediate 1

[0166] Take a dry and clean 2L reaction bottle, add THF 500ml, weigh Fmoc-Asn(Trt)-OH 80g, add it to the reaction bottle, stir to dissolve, add DEPBT 46.6g, stir at room temperature for 15 minutes, add PABC 16g, react at room temperature for 30 minutes, add DIPEA 45ml, ventilate with nitrogen for protection, react at room temperature for 3 hours, and monitor the reaction by TLC (Fmoc-Asn(Trt)-OH reaction is complete).

[0167] The reaction solution was evaporated under reduced pressure, a small amount of DMF (180 ml) was added to dissolve it, and the mixture was added dropwise to 3 L of stirring water to precipitate a light yellow solid. After washing with water 2-3 times, the solid was collected by suction filtration and dried in vacuo to obtain an off-white solid (yield of more than 90%).

[0168] 2. Synthesis of Intermediate 2

[0169] Add 500ml of THF and the off-white solid obtained in the previous step to a 2L single-mouth reaction bottle, stir to dissolve, cool to 0-5°C in an ice-salt bath, add 100ml of piperidine dropwise, and after the addition is complete, gradually return to room temperature to react for 1h. The reaction is monitored by TLC. The solvent is evaporated under reduced pressure, a small amount of DMF is added to dissolve, and the mixture is added dropwise to 2L of stirring water, mechanically stirred for 30min, filtered, and washed with water 2-3 times, filtered, 800ml of methyl tert-butyl ether is added to the filter cake, stirred for 30min, filtered, and the filter cake is washed twice with PE:EA=10:1, filtered, and the filter cake is collected. After vacuum drying, 80g of off-white solid is obtained with a purity of 70%.

[0170] 3. Synthesis of Intermediate 3

[0171] Into a dry and clean 250ml single-mouth reaction bottle, 50ml THF, 5.04g Boc-Ala-Ala-OH, and 3.89g DEPBT were added in sequence, and the reaction was carried out at room temperature for 10min. 2.6g NH2H2H2-Asn(Trt)-PABC was added, and nitrogen was ventilated for protection. The reaction was carried out at room temperature for 15min. 3.5ml DIPEA was added dropwise, and nitrogen was ventilated for protection. The reaction was carried out at room temperature for 3 hours. The solvent was evaporated under reduced pressure, and water was added to slurry 2-3 times. The mixture was filtered to obtain a light yellow solid (3.7g). The product was purified by column to obtain 2.0g of the product with a purity of 94.8% and a yield of 26.6%.

[0172] 4. Synthesis of Intermediate 4

[0173] 1.8 g of intermediate 3 was added to a 250 ml single-mouth reaction bottle, 28.5 ml of TFA was added, 1.5 ml of water was added dropwise, and the reaction was carried out at room temperature for 30 min. After the reaction was completed by monitoring by TLC, the solvent was evaporated under reduced pressure, methyl tert-butyl ether was added to slurry, and the solid was filtered to obtain a solid. The solid was dissolved in a solution of dioxane: water = 1:1, and 1N sodium hydroxide was added to adjust the pH to 13. The mixture was stirred at room temperature for 40 min, the solvent was evaporated under reduced pressure, and the sample was mixed with silica gel and passed through a column to obtain 450 mg of the product with a yield of 47.5%.

[0174] 5. Synthesis of Intermediate 5

[0175] Weigh Fmoc-Glu(OAll)-COOH (1.554 g, 3.79 mmol), add 10 ml of a mixed solution of DCM and THF to dissolve, add 2.72 ml of HOtBu dropwise under stirring, and after the addition is complete, ventilate under N2 protection, react at room temperature for 16 hours, monitor the reaction by TLC, evaporate the solvent under reduced pressure, and pass the sample through a silica gel column to obtain 1.4 g of the product, with a yield of 79.5%.

[0176] 6. Synthesis of Intermediate 6

[0177] In a dry and clean 250ml single-mouth reaction bottle, 10ml of THF and the intermediate 5 (1.4g, 3mmol) obtained in the previous step were added in sequence. After stirring and dissolving, the mixture was cooled to 0-5°C in an ice-salt bath, and 3ml of piperidine was added dropwise. After the addition was complete, the mixture was gradually heated to room temperature and reacted for 2 hours. The reaction was completed after monitoring by TLC. The solvent was evaporated under reduced pressure, and the sample was purified by mixing with silica gel. The eluate containing the product was collected and dried under vacuum to constant weight to obtain 583mg of the product with a yield of 80%.

[0178] 7. Synthesis of Intermediate 7

[0179] Into a dry and clean 250ml single-mouth reaction bottle, 15ml THF, 583mg intermediate 6, 932.8mg DEPBT were added in sequence, and the reaction was carried out at room temperature for 10min. Then, 506.4mg maleimidocaproic acid was added, and nitrogen was ventilated for protection. The reaction was carried out at room temperature for 15min. 1.3ml DIPEA was added dropwise, and nitrogen was ventilated for protection. The reaction was carried out at room temperature for 3 hours. The solvent was evaporated under reduced pressure, and water was added to slurry 2-3 times. The product was filtered to obtain a light yellow solid (800mg). The product was purified by column to obtain 628mg of the product with a purity of 94.8% and a yield of 59.9%.

[0180] 8. Synthesis of Intermediate 8

[0181] In a dry and clean 100ml single-mouth reaction bottle, 10ml of dichloromethane and 872mg of intermediate 7 were added in sequence. After stirring evenly, 3ml of TFA was added dropwise and reacted at room temperature for 2 hours. The reaction of the raw materials was completed after monitoring by TLC. The solvent was evaporated under reduced pressure in vacuo, and methyl tert-butyl ether was added for pulping. The solid was filtered to obtain a solid, and the sample was purified by mixing with silica gel. The eluate containing the product was collected and dried under reduced pressure in vacuo to constant weight to obtain 459mg of the product with a yield of 60.3%.

[0182] 9. Synthesis of Intermediate 9

[0183] In a dry and clean 250ml single-mouth reaction bottle, 15ml THF, 459mg intermediate 8, and 434mg DEPBT were added in sequence, and the reaction was carried out at room temperature for 10min. Then 457.8mg intermediate 4 was added, and nitrogen was ventilated for protection. The reaction was carried out at room temperature for 15min. 627μl DIPEA was added dropwise. After the addition was completed, nitrogen was ventilated for protection. The reaction was carried out at room temperature for 3 hours. The solvent was evaporated under reduced pressure, and water was added to slurry 2-3 times. The mixture was filtered to obtain a light yellow solid (750mg). The product was purified by column to obtain 655mg, with a yield of 63.2%.

[0184] 10. Synthesis of Intermediate 10

[0185] To a 100 ml single-mouth reaction bottle, 25 ml of DMF, intermediate 9 (655 mg, 0.88 mmol), and Bis-PNP (804 mg, 2.64 mmol) were added in sequence, nitrogen was ventilated for protection, and the reaction was carried out at room temperature for 15 min. 258 μl of DIPEA was added dropwise, nitrogen was ventilated for protection, and the reaction was carried out at room temperature for 3 h. HPLC monitoring showed that the remaining raw material was 7%, the reaction was terminated, the solvent was evaporated under reduced pressure, and the product was purified by column to obtain 335 mg of the product with a yield of 42%.

[0186] 11. Synthesis of Intermediate 11

[0187] To a 100 mL reaction bottle, 214.3 mg of doxorubicin hydrochloride (1.0 eq, 0.369 mmol) and 335 mg of intermediate 10 (1.0 eq, 0.369 mmol) were added, and the mixture was reacted at room temperature for 15 min under nitrogen protection. 190 μl of DIPEA was added dropwise, and the mixture was reacted at room temperature for 4 hours. The solvent was evaporated under reduced pressure, and the crude product was dissolved in methanol and passed through a reverse phase high pressure column to obtain intermediate 11 (115 mg of a red solid, yield: 23.8%).

[0188] 12. Synthesis of the final product

[0189] To a 100 mL reaction flask, 15 ml of THF, intermediate 11 (115 mg, 0.0877 mmol), tri-n-butyltin hydride (76 mg, 0.2631 mmol) were added in sequence, and the reaction solution was saturated with nitrogen. Tetrakis(triphenylphosphine)palladium(0) (14.2 mg, 0.012 mmol) was then added, and the mixture was stirred at room temperature overnight. Monitored by TLC until the conversion was complete. The contents of the flask were then filtered through diatomaceous earth, and the residue was washed with THF. The filtrate was concentrated under reduced pressure. The crude product obtained was purified by column to obtain 100 mg (yield: 90%) of the target compound.

[0190] Example 3: Synthesis of N-CBP

[0191]

[0192] 1. Synthesis of Intermediate 1

[0193] 300 mg of the raw material was added into a 100 ml three-necked flask, and 15 ml of THF / ETOH (4:1) was added to dissolve it. The temperature was cooled to -5°C-0°C in an ice-salt bath. 210 mg of LiOH (5 ml) aqueous solution was added dropwise to the flask in batches under stirring and temperature control. After the addition, the temperature was naturally raised to react for 1 hour. The reaction liquid was sent to HPLC. The raw material reacted completely, and the temperature was controlled at -5°C-0°C. The pH of the reaction liquid was adjusted to 3-4 with 1 mol / L HCl. The temperature was controlled at 25-30°C and the solvent was removed by rotation to obtain the crude intermediate 1, which was directly used in the next step.

[0194] 2. Synthesis of Intermediate 2

[0195] Add 15 ml of 1 mol / L dioxane hydrochloride solution to intermediate 1, stir at room temperature for 1 h, send the reaction solution to HPLC, and if intermediate 1 is completely reacted, remove the solvent by rotation at 25-30 °C to obtain a crude intermediate 2, which is directly used for the next step.

[0196] 3. Synthesis of Intermediate 3

[0197] The crude intermediate 2 was added to a 100 ml three-necked flask, and 20 ml of dioxane was added to dissolve it. The temperature was cooled to -5-0°C in an ice-salt bath. 159 mg of sodium carbonate aqueous solution (pH about 8) was added dropwise to the flask under temperature control. Under nitrogen protection and temperature control at -5-0°C, 311 mg of Fmoc-Cl dioxane solution was added dropwise. After the addition was completed, the temperature was naturally raised to react for 1 hour. The product was sent to HPLC for detection. The reaction of intermediate 2 was complete. The solvent was removed by rotation. The crude product was mixed with silica gel and passed through a reverse phase medium pressure column to obtain 107 mg of intermediate 3.

[0198] 4. Synthesis of Intermediate 4

[0199] Add 107 mg of intermediate 3 into a 50 ml single-necked bottle, add 15 ml of methanol to dissolve, cool to -20°C with liquid nitrogen, and drop 302 ul of tetrabutylammonium hydroxide (25% methanol solution) into the bottle. After the dropwise addition, heat naturally to react for 1 hour. This reaction solution is the reserve solution 1.

[0200] 140.8 mg of diiododiammineplatinum was added to a 50 ml single-mouth bottle, and 10 ml of ultrapure water was added to dissolve it. The solution was heated to 50°C and protected from light. Under nitrogen protection, 49.5 mg of silver nitrate aqueous solution was added dropwise to the bottle. After the reaction for 15 minutes, 49.5 mg of silver nitrate aqueous solution was continued to be added dropwise to the bottle. After the reaction was completed for 15 minutes, the reaction solution was filtered with a filter membrane, and the filtrate was transferred to a 100 ml single-mouth bottle. At room temperature, the reserve solution 1 was added dropwise to the bottle. After the addition was completed, the solution was replaced with nitrogen three times. The reaction solution was transferred to an oil bath and heated to 50°C. The solution was protected from light and reacted overnight (generally 16 hours). The reaction was stopped, the reaction solution was centrifuged, and the supernatant was directly passed through a high-pressure reverse phase column. The prepared solution was lyophilized to obtain 79 mg of intermediate 4 with a yield of 45.7%.

[0201] 5. Synthesis of N-CBP

[0202] 5 mg of intermediate 4 was added to a 10 ml single-mouth bottle, and 2 ml of MeOH / ACN (1:1) was added and stirred to dissolve. 2 ul of DBU was dropped into the reaction solution at room temperature. The reaction was protected by nitrogen for half an hour. HPLC detection showed that the reaction of intermediate 4 was complete. The reaction solution was dropped into 6 ml of methyl tert-butyl ether to precipitate an off-white solid. The solution was centrifuged and the supernatant was removed. The solid was dissolved in water / tert-butyl alcohol and passed through a column to obtain 1.8 mg of the product N-CBP.

[0203] Example 4: Synthesis of QHL-140-N-CBP

[0204]

[0205] 1. Synthesis of Intermediate 1

[0206] 500 mg of the raw material was added into a 100 ml three-necked flask, 10 ml of DCM was added to dissolve, the temperature was lowered to -5°C-0°C, 5 ml of TFA was added dropwise with stirring, and after reacting for 1 h, HPLC was used to monitor the complete reaction of the raw material, and the solvent in the reaction solution was removed by rotation, and the remaining oily substance was intermediate 1.

[0207] 2. Synthesis of Intermediate 2

[0208] Intermediate 1 and 1.15 g of raw material Fmoc-AAN-PABC-PNP were added to a 100 ml single-mouth bottle, dissolved with 20 ml of DMF, protected by nitrogen, activated for 10 min under stirring, 0.87 ml of DIPEA was added dropwise to the reaction bottle, reacted for 0.5 h, and sent to HPLC for detection. The raw material Fmoc-AAN-PABC-PNP was completely reacted, and the DMF in the reaction solution was removed by rotation. The crude product was dissolved in water / DMF and passed through a high-pressure reverse phase column to obtain 975 mg of intermediate 2, with a yield of 78.6%.

[0209] 3. Synthesis of Intermediate 3

[0210] 400 mg of intermediate 2 was added into a 250 ml three-necked flask, 35 ml of THF / ETOH (4:1) was added to dissolve, the temperature was cooled to -5°C-0°C with an ice-salt bath, the temperature was controlled at -5°C-0°C, 202 mg of LiOH aqueous solution was added dropwise in batches, after the dropwise addition, the temperature was controlled to react for 3 hours, and the mixture was sent to HPLC for detection. The intermediate 2 was completely reacted, the temperature was controlled at -5°C-0°C, the pH of the reaction solution was adjusted to 6-7 with 1 mol / L HCL, the solvent was removed by rotation at 25°C-30°C, the crude product was slurried twice with methyl tert-butyl ether, the solid was dissolved with methanol / water, and passed through a high-pressure reverse phase column to obtain 230 mg of intermediate 3 with a yield of 86.7%.

[0211] 4. Synthesis of Intermediate 4

[0212] 235 mg of intermediate 3 and 222 mg of EMC-OSU were added to a 100 ml single-mouth bottle, 30 ml of DMF was added and stirred to dissolve, and the mixture was heated to 50°C. The reaction was carried out under nitrogen protection overnight (generally 16 hours), and the mixture was sent to HPLC for detection. The reaction of intermediate 3 was complete, and DMF was removed by rotation. The crude product was dissolved in methanol / water and passed through a high-pressure reverse phase column to obtain 200 mg of intermediate 4, with a yield of 53.6%.

[0213] 5. Synthesis of the final product QHL-140-N-CBP

[0214] Add 200 mg of intermediate 4 into a 100 ml single-necked bottle, add 20 ml of methanol to dissolve, cool to -20°C with liquid nitrogen, add 279 ul of tetrabutylammonium hydroxide (25% methanol solution) dropwise into the bottle, and naturally heat to react for 1 hour after the addition. This reaction solution is the reserve solution 1.

[0215] 130 mg of diiododiammineplatinum was added to a 100 ml single-mouth bottle, and 30 ml of ultrapure water was added to dissolve it. The solution was heated to 50°C and protected from light. 46 mg of silver nitrate aqueous solution was added dropwise to the bottle under nitrogen protection. After the reaction for 15 minutes, 46 mg of silver nitrate aqueous solution was continued to be added dropwise to the bottle. After the reaction was completed for 15 minutes, the reaction solution was filtered with a filter membrane, and the filtrate was transferred to a 250 ml single-mouth bottle. At room temperature, the reserve solution 1 was added dropwise to the bottle. After the dropwise reaction was completed, the solution was replaced with nitrogen three times. The reaction solution was transferred to an oil bath and heated to 50°C. The solution was protected from light and reacted overnight (generally 16 hours). The reaction solution was centrifuged, and the supernatant was directly passed through a high-pressure reverse phase column. The prepared solution was lyophilized to obtain 90 mg of the product QHL-140-N-CBP with a yield of 34.5%.

[0216] Example 5: Synthesis of QHL-086-N-CBP

[0217]

[0218] 1. Synthesis of Intermediate 1

[0219] 500 mg of the raw material was added into a 100 ml three-necked flask, 10 ml of DCM was added to dissolve, the temperature was lowered to -5°C-0°C, 5 ml of TFA was added dropwise with stirring, and after reacting for 1 h, HPLC was used to monitor the complete reaction of the raw material, and the solvent in the reaction solution was removed by rotation, and the remaining oily substance was intermediate 1.

[0220] 2. Synthesis of Intermediate 2

[0221] Intermediate 1 and 1.15 g of raw material Fmoc-AAN-PABC-PNP were added to a 100 ml single-mouth bottle, dissolved with 20 ml of DMF, protected by nitrogen, activated for 10 min under stirring, 0.87 ml of DIPEA was added dropwise to the reaction bottle, reacted for 0.5 h, and sent to HPLC for detection. The raw material Fmoc-AAN-PABC-PNP was completely reacted, and the DMF in the reaction solution was removed by rotation. The crude product was dissolved in water / DMF and passed through a high-pressure reverse phase column to obtain 975 mg of intermediate 2 with a yield of 78.6%.

[0222] 3. Synthesis of Intermediate 3

[0223] 400 mg of intermediate 2 was added into a 250 ml three-necked flask, 35 ml of THF / ETOH (4:1) was added to dissolve, the temperature was cooled to -5°C-0°C with an ice-salt bath, the temperature was controlled at -5°C-0°C, 202 mg of LiOH aqueous solution was added dropwise in batches, after the dropwise addition, the temperature was controlled to react for 3 hours, and the solution was sent to HPLC for detection. The intermediate 2 was completely reacted, the temperature was controlled at -5°C-0°C, the pH of the reaction solution was adjusted to 6-7 with 1 mol / L HCL, the solvent was removed by rotation at 25°C-30°C, the crude product was slurried twice with methyl tert-butyl ether, the solid was dissolved with methanol / water, and passed through a high-pressure reverse phase column to obtain 235 mg of intermediate 3 with a yield of 88.6%.

[0224] 4. Synthesis of Intermediate 4

[0225] 89 mg of EMC-2Peg-OH was added to a 100 ml single-mouth bottle, dissolved in DMF, 97 mg of DEPBT was added to the bottle, and activated by stirring at room temperature for 1 hour. 95 ul of DEPBT was added to the bottle, and continued to stir for 1 hour. Then, 150 mg of DMF solution of intermediate 3 was added to the bottle in batches, and stirred at room temperature for reaction. HPLC detection was performed. After the reaction was completed, DMF was removed by rotation, and the crude product was dissolved in water / methanol and passed through a reverse phase high pressure column to obtain 88 mg of the product, with a yield of 37.6%.

[0226] 5. Synthesis of the final product QHL-086-N-CBP

[0227] Add 88 mg of intermediate 4 into a 50 ml single-mouth bottle, add 10 ml of methanol to dissolve, cool to -20°C with liquid nitrogen, add 106 ul of tetrabutylammonium hydroxide (25% methanol solution) dropwise into the bottle, and naturally heat to react for 1 hour after the addition. This reaction solution is the reserve solution 1.

[0228] 49 mg of diiododiammineplatinum was added to a 50 ml single-mouth bottle, and 10 ml of ultrapure water was added to dissolve it. The solution was heated to 50° C. and protected from light. Under nitrogen protection, 17 mg of silver nitrate aqueous solution was added dropwise to the bottle. After reacting for 15 minutes, 17 mg of silver nitrate aqueous solution was continued to be added dropwise to the bottle. After reacting for 15 minutes, the reaction solution was filtered with a filter membrane, and the filtrate was transferred to a 100 ml single-mouth bottle. At room temperature, the reserve solution 1 was added dropwise to the bottle. After the addition was completed, the solution was replaced with nitrogen three times. The reaction solution was transferred to an oil bath and heated to 50° C. The solution was reacted overnight (generally 16 hours) in the dark. The solution was sent to HPLC for detection. About 20% of the intermediate 4 was not completely reacted. The reaction was stopped, the reaction solution was centrifuged, and the supernatant was directly passed through a high-pressure reverse phase column. The prepared solution was lyophilized to obtain 54 mg of the product QHL-086-N-CBP with a yield of 48.6%.

[0229] Example 6: Synthesis of QHL-095-N-CBP

[0230]

[0231] 1. Synthesis of Intermediate 1

[0232] 500 mg of the raw material was added into a 100 ml three-necked flask, 10 ml of DCM was added to dissolve, the temperature was lowered to -5°C-0°C, 5 ml of TFA was added dropwise with stirring, and after reacting for 1 h, HPLC was used to monitor the complete reaction of the raw material, and the solvent in the reaction solution was removed by rotation, and the remaining oily substance was intermediate 1.

[0233] 2. Synthesis of Intermediate 2

[0234] Intermediate 1 and 1.15 g of raw material Fmoc-AAN-PABC-PNP were added to a 100 ml single-mouth bottle, dissolved with 20 ml of DMF, protected by nitrogen, activated for 10 min under stirring, 0.87 ml of DIPEA was added dropwise to the reaction bottle, reacted for 0.5 h, and sent to HPLC for detection. The raw material Fmoc-AAN-PABC-PNP was completely reacted, and the DMF in the reaction solution was removed by rotation. The crude product was dissolved in water / DMF and passed through a high-pressure reverse phase column to obtain 975 mg of intermediate 2, with a yield of 78.6%.

[0235] 3. Synthesis of Intermediate 3

[0236] 400 mg of intermediate 2 was added into a 250 ml three-necked flask, 35 ml of THF / ETOH (4:1) was added to dissolve, the temperature was cooled to -5°C-0°C with an ice-salt bath, the temperature was controlled at -5°C-0°C, 202 mg of LiOH aqueous solution was added dropwise in batches, after the dropwise addition, the temperature was controlled to react for 3 hours, and the solution was sent to HPLC for detection. The intermediate 2 was completely reacted, the temperature was controlled at -5°C-0°C, the pH of the reaction solution was adjusted to 6-7 with 1 mol / L HCL, the solvent was removed by rotation at 25°C-30°C, the crude product was slurried twice with methyl tert-butyl ether, the solid was dissolved with methanol / water, and passed through a high-pressure reverse phase column to obtain 235 mg of intermediate 3 with a yield of 88.6%.

[0237] 4. Synthesis of Intermediate 4

[0238] 180 mg of intermediate 3 and 240 mg of EMC-6Peg-OSU were added to a 100 ml single-mouth bottle, 20 ml of DMF was added and stirred to dissolve, the mixture was heated to 50°C, and the reaction was carried out overnight (generally 16 hours) under nitrogen protection. The reaction was sent to HPLC for detection. The intermediate 3 was completely reacted, and the DMF was removed by rotation. The crude product was dissolved in methanol / water and passed through a high-pressure reverse phase column to obtain 234 mg of intermediate 4, with a yield of 69.2%.

[0239] 5. Synthesis of the final product QHL-095-N-CBP

[0240] Add 234 mg of intermediate 4 into a 100 ml single-necked bottle, add 15 ml of methanol to dissolve, cool to -20°C with liquid nitrogen, add 234 ul of tetrabutylammonium hydroxide (25% methanol solution) dropwise into the bottle, and naturally heat to react for 1 hour after the addition. This reaction solution is the reserve solution 1.

[0241] Add 109 mg of diiododiammineplatinum to a 100 ml single-mouth bottle, add 20 ml of ultrapure water to dissolve, heat to 50°C, protect from light, add 38 mg of silver nitrate aqueous solution to the bottle under nitrogen protection, react for 15 minutes, continue to add 38 mg of silver nitrate aqueous solution to the bottle, react for 15 minutes after the dropwise addition, filter the reaction solution with a filter membrane, transfer the filtrate to a 250 ml single-mouth bottle, add reserve solution 1 to the bottle at room temperature, replace the dropwise addition with nitrogen three times, transfer the reaction solution to an oil bath and heat to 50°C, react overnight (generally 16 hours) in the dark, centrifuge the reaction solution, directly pass the supernatant through a high-pressure reverse phase column, and freeze-dry the preparation solution to obtain 138 mg of the final product with a yield of 48%.

[0242] Example 7: Synthesis of QHL-006-DOX

[0243] The synthesis route of MI-S group in QHL-006 is as follows:

[0244]

[0245] 1. Synthesis of MI-S intermediate-1 in QHL-006-DOX

[0246] Take a dry and clean 100ml single-mouth reaction bottle, weigh maleic anhydride (245mg, 2.5mmol), add 10ml of dichloromethane and stir to dissolve, weigh NH2H2H2-3Peg-COOtBu (624mg, 2.25mmol), react at room temperature for 6 hours, LC-MS monitors the completion of the maleic anhydride reaction, spin-dry the reaction solution, mix the sample with silica gel and pass it through a column to obtain MI-S intermediate-1 (456mg, yield 48.6%).

[0247] 2. Synthesis of MI-S intermediate-2 in QHL-006-DOX

[0248] 456 mg of MI-S intermediate-1 obtained in the above step was added to a 100 ml single-mouth reaction bottle, 10 ml of acetic anhydride was added and stirred to dissolve, NaOAC (98.7 mg, 1.216 mmol) was weighed and slowly added in batches, the oil bath was heated to 110° C. and reacted for 3 h. LC-MS monitored that the reaction of MI-S intermediate-1 was complete, cooled to room temperature, the reaction liquid was spin-dried, and purified by column to obtain MI-S intermediate-2 (312, yield 70%).

[0249] 3. Synthesis of MI-S in QHL-006-DOX

[0250] The MI-S intermediate-2 (312 mg, 0.87 mmol) obtained in the previous step was added to a 100 ml single-mouth reaction bottle, 10 ml of dichloromethane was added to dissolve, 2 ml of TFA was added dropwise, 0.15 ml of water was added dropwise, and the reaction was carried out at room temperature for 30 min. After the reaction was completed by TLC monitoring, the solvent was evaporated under reduced pressure, methyl tert-butyl ether was added to slurry, and suction was filtered to obtain a solid, and the sample was mixed with silica gel and passed through a reverse phase column to obtain 196 mg of the product. The yield was 75%.

[0251] The final product was 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).

[0252] Example 8: Synthesis of QHL-096-DOX

[0253]

[0254] 1) Synthesis of intermediate 1

[0255] N-Benzyloxycarbonyl-L-alanine (100 g, 0.45 mol) was dissolved in dry N,N-dimethylformamide (3 L), and 1-hydroxybenzotriazole (72.6 g, 0.54 mol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (103.3 g, 0.54 mol) were added under stirring. After stirring for 1 hour, L-alanine methyl ester (46.2 g, 0.45 mol) and N,N-dimethylformamide were added dropwise under ice bath at 0 °C. A solution of isopropylethylamine (173.8 g, 1.34 mol) in N,N-dimethylformamide (1 L) was added dropwise and stirred at room temperature for 10 hours. The solvent was evaporated under reduced pressure, and the crude product was dissolved in dichloromethane (2 L), washed with saturated ammonium chloride solution, water and saturated sodium chloride solution in sequence. The organic phase was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the crude product was recrystallized from ethyl acetate / petroleum ether to obtain the pure product of intermediate 1 (101 g white solid, yield: 73.1%).

[0256] 2) Synthesis of Intermediate 2

[0257] Intermediate 1 (100 g, 0.34 mol) was dissolved in a mixed solution of tetrahydrofuran (2 L) and water (1 L), cooled to 0°C, 1 mol / L lithium hydroxide solution (400 mL) was added dropwise, and the reaction was stirred for 10 hours. Concentrated hydrochloric acid was added dropwise to neutralize until the pH was <6, and tetrahydrofuran was evaporated under reduced pressure. The remaining aqueous phase was extracted with dichloromethane (1 L×3), and the organic phase was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure to obtain intermediate 2 (88 g white solid, yield: 92.2%).

[0258] 3) Synthesis of Intermediate 3

[0259] In a three-necked flask, dissolve L-leucine tert-butyl ester (22.4 g, 0.1 mol) and N-Fmoc-N'-trityl asparagine (59.6 g, 0.1 mol) in N,N-dimethylformamide (1000 mL). Add 1-hydroxybenzotriazole (14.85 g, 0.11 mol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (23 g, 0.12 mol) under stirring. After cooling to 0°C with an ice bath, add N,N-diisopropylethylamine (25.8 g, 0.2 mol) was added, and after stirring for 10 hours, the solvent was evaporated under reduced pressure. The crude product was dissolved in chloroform (1000 ml), washed with saturated ammonium chloride solution, saturated sodium chloride solution and water in sequence, and the organic phase was dried over anhydrous sodium sulfate. After filtering, the solvent was evaporated under reduced pressure to obtain a crude product, which was purified by recrystallization (by volume ratio, dichloromethane: ethyl acetate = 1:1) to obtain intermediate 3 (42.4 g white solid, yield: 55.4%).

[0260] 4) Synthesis of intermediate 4

[0261] Intermediate 3 (7.65 g, 0.01 mol) was dissolved in a mixed solution of dichloromethane (100 mL) and N,N-dimethylformamide (100 mL), piperidine (40 ml) was added, and after stirring at room temperature for 5 hours, the solvent was evaporated under reduced pressure, and then placed in a vacuum drying oven under high vacuum to remove a small amount of piperidine to obtain intermediate 4 as a light yellow solid, which was directly used in the next step without purification.

[0262] 5) Synthesis of Intermediate 5

[0263] The crude intermediate 4 obtained in the previous step was dissolved in N,N-dimethylformamide (200 mL), and intermediate 2 (2.94 g, 0.012 mol) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (6.07 g, 0.016 mol) were added. The mixture was cooled to 0°C and then N,N-diisopropylethylamine (2.6 g, 0.02 mol) was added. The mixture was stirred at room temperature overnight, and the solvent was evaporated under reduced pressure. The residue was dissolved in chloroform (100 ml), washed with saturated ammonium chloride solution and saturated sodium chloride solution in turn, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The crude product was chromatographed on a silica gel column to obtain intermediate 5 (3.1 g white solid, total yield of two steps: 37.8%).

[0264] 6) Synthesis of Intermediate 6

[0265] Cbz-AAN(trt)-L-Otbu (3.00 g, 3.65 mmol) was dissolved in methanol (100 mL), 10% palladium carbon (0.3 g) was added, hydrogen was introduced, and the reaction was stirred at room temperature and pressure for 4 hours. The palladium carbon was filtered out and washed with methanol. The filtrate and washings were combined, and the solvent was evaporated under reduced pressure to obtain intermediate 6 (2.38 g white solid, yield: 95.2%).

[0266] 7) Synthesis of Intermediate 7

[0267] Add intermediate 6 (2.38 g, 3.4 mmol) and EMC-6Peg-OSu (2.4 g, 4.08 mmol) into a 250 ml single-mouth bottle, add DMF (30 ml) to dissolve, and heat to 50 ° C for 6 h. Remove the solvent by vacuum distillation, dissolve the crude product in methanol, and pass through a reverse phase high pressure column to obtain intermediate 7 (2.5 g, yield: 63.2%).

[0268] 8) Synthesis of Intermediate 8

[0269] Intermediate 7 (1.00 g, 0.852 mmol) was dissolved in DCM (20 mL), and trifluoroacetic acid (10 ml) was added dropwise at room temperature. The mixture was stirred for 2 h and the reaction solution was monitored by HPLC. After the reaction of intermediate 1 was completed, the solvent was removed by distillation under reduced pressure. The crude product was washed twice with methyl tert-butyl ether, and the solid was dissolved in methanol. The solid was passed through a reverse phase high pressure column to obtain intermediate 8 (721 mg white solid, yield: 96.8%).

[0270] 9) Synthesis of the final product QHL-096-DOX

[0271] 63 mg of doxorubicin hydrochloride (1.0 eq), 95 mg of intermediate 8 (1 eq), 39 mg of DEPBT (1.2 eq) and 10 mL of DMF were added to a 100 mL reaction bottle. 60 ul of DIPEA (3 eq) was added to the reaction mixture under nitrogen protection. After reacting at room temperature for 4 hours, the solvent was evaporated under reduced pressure, and the crude product was dissolved in methanol and passed through a reverse phase high pressure column to obtain QHL-096-DOX (52 mg of red solid, yield: 34.2%). 7) Synthesis of intermediate 7

[0272] Intermediate 6 (1.00 g, 1.46 mmol) was dissolved in DCM (20 mL), and trifluoroacetic acid (10 ml) was added dropwise at room temperature. The reaction was stirred for 2 h, and the reaction solution was monitored by HPLC. The reaction of intermediate 1 was complete, and the solvent was removed by distillation under reduced pressure. The crude product was washed twice with methyl tert-butyl ether, and the solid was dissolved in methanol. The solid was passed through a reverse phase high pressure column to obtain intermediate 7 (546 mg white solid, yield: 96.8%).

[0273] Example 9: Synthesis of QHL-117-DOX

[0274] The synthetic route of QHL-117 is as follows:

[0275]

[0276] 1) Synthesis of intermediate 1

[0277] N-Benzyloxycarbonyl-L-alanine (100 g, 0.45 mol) was dissolved in dry N,N-dimethylformamide (3 L), and 1-hydroxybenzotriazole (72.6 g, 0.54 mol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (103.3 g, 0.54 mol) were added under stirring. After stirring for 1 hour, L-alanine methyl ester (46.2 g, 0.45 mol) and N,N-dimethylformamide were added dropwise under ice bath at 0 °C. A solution of isopropylethylamine (173.8 g, 1.34 mol) in N,N-dimethylformamide (1 L) was added dropwise and stirred at room temperature for 10 hours. The solvent was evaporated under reduced pressure, and the crude product was dissolved in dichloromethane (2 L), washed with saturated ammonium chloride solution, water and saturated sodium chloride solution in sequence. The organic phase was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the crude product was recrystallized from ethyl acetate / petroleum ether to obtain the pure product of intermediate 1 (101 g white solid, yield: 73.1%).

[0278] 2) Synthesis of Intermediate 2

[0279] Intermediate 1 (100 g, 0.34 mol) was dissolved in a mixed solution of tetrahydrofuran (2 L) and water (1 L), cooled to 0°C, 1 mol / L lithium hydroxide solution (400 mL) was added dropwise, and the reaction was stirred for 10 hours. Concentrated hydrochloric acid was added dropwise to neutralize until the pH was <6, and tetrahydrofuran was evaporated under reduced pressure. The remaining aqueous phase was extracted with dichloromethane (1 L×3), and the organic phase was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure to obtain intermediate 2 (88 g white solid, yield: 92.2%).

[0280] 3) Synthesis of Intermediate 3

[0281] In a three-necked flask, dissolve L-leucine tert-butyl ester (22.4 g, 0.1 mol) and N-Fmoc-N'-trityl asparagine (59.6 g, 0.1 mol) in N,N-dimethylformamide (1000 mL). Add 1-hydroxybenzotriazole (14.85 g, 0.11 mol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (23 g, 0.12 mol) under stirring. After cooling to 0°C with an ice bath, add N,N-diisopropylethylamine (25.8 g, 0.2 mol) was added, and after stirring for 10 hours, the solvent was evaporated under reduced pressure. The crude product was dissolved in chloroform (1000 ml), washed with saturated ammonium chloride solution, saturated sodium chloride solution and water in sequence, and the organic phase was dried over anhydrous sodium sulfate. After filtering, the solvent was evaporated under reduced pressure to obtain a crude product, which was purified by recrystallization (by volume ratio, dichloromethane: ethyl acetate = 1:1) to obtain intermediate 3 (42.4 g white solid, yield: 55.4%).

[0282] 4) Synthesis of intermediate 4

[0283] Intermediate 3 (7.65 g, 0.01 mol) was dissolved in a mixed solution of dichloromethane (100 mL) and N,N-dimethylformamide (100 mL), piperidine (40 ml) was added, and after stirring at room temperature for 5 hours, the solvent was evaporated under reduced pressure, and then placed in a vacuum drying oven under high vacuum to remove a small amount of piperidine to obtain intermediate 4 as a light yellow solid, which was directly used in the next step without purification.

[0284] 5) Synthesis of Intermediate 5

[0285] The crude intermediate 4 obtained in the previous step was dissolved in N,N-dimethylformamide (200 mL), and intermediate 2 (2.94 g, 0.012 mol) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (6.07 g, 0.016 mol) were added. The mixture was cooled to 0°C and then N,N-diisopropylethylamine (2.6 g, 0.02 mol) was added. The mixture was stirred at room temperature overnight, and the solvent was evaporated under reduced pressure. The residue was dissolved in chloroform (100 ml), washed with saturated ammonium chloride solution and saturated sodium chloride solution in turn, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The crude product was chromatographed on a silica gel column to obtain intermediate 5 (3.1 g white solid, total yield of two steps: 37.8%).

[0286] 6) Synthesis of Intermediate 6

[0287] Cbz-AAN(trt)-L-Otbu (3.00 g, 3.65 mmol) was dissolved in methanol (100 mL), 10% palladium carbon (0.3 g) was added, hydrogen was introduced, and the reaction was stirred at room temperature and pressure for 4 hours. The palladium carbon was filtered out and washed with methanol. The filtrate and washings were combined, and the solvent was evaporated under reduced pressure to obtain intermediate 6 (2.38 g white solid, yield: 95.2%).

[0288] 7) Synthesis of Intermediate 7

[0289] Into a dry and clean 250ml single-mouth reaction bottle, 15ml THF, (2.387g, 3.4mmol) intermediate 6, 1.35g DEPBT were added in sequence, and the reaction was carried out at room temperature for 10min. EMC-Glu(OAll)-COOH (1.3g, 3.4mmol) was added, and nitrogen was ventilated for protection. The reaction was carried out at room temperature for 15min. DIPEA 1.8ml was added dropwise. After the addition was completed, nitrogen was ventilated for protection. The reaction was carried out at room temperature for 3 hours. The solvent was evaporated under reduced pressure, and water was added to slurry 2-3 times. The product was filtered to obtain a light yellow solid (700mg). The product was purified by column to obtain 2.2g of the product with a yield of 63.2%.

[0290] 8) Synthesis of Intermediate 8

[0291] Intermediate 7 (1.53 g, 1.46 mmol) was dissolved in DCM (20 mL), and trifluoroacetic acid (10 ml) was added dropwise at room temperature. The reaction was stirred for 2 h, and the reaction solution was monitored by HPLC. The reaction of intermediate 1 was complete, and the solvent was removed by distillation under reduced pressure. The crude product was washed twice with methyl tert-butyl ether, and the solid was dissolved in methanol. The solid was passed through a reverse phase high pressure column to obtain intermediate 8 (928 mg white solid, yield: 84.8%).

[0292] 9) Synthesis of Intermediate 9

[0293] Add 510.4 mg doxorubicin hydrochloride (1.0 eq, 0.88 mmol) and 659 mg of intermediate 8 (1.0 eq, 0.88 mmol) to a 100 mL reaction bottle, react at room temperature for 15 min under nitrogen protection, add 78 μl of DIPEA dropwise, react at room temperature for 4 hours, evaporate the solvent under reduced pressure, dissolve the crude product in methanol, and pass through a reverse phase high pressure column to obtain intermediate 9 (258 mg red solid, yield: 23.8%). 10) Synthesis of the final product

[0294] THF 15ml, intermediate 9 (258mg, 0.202mmol), n-butyltin hydrogen (175.7mg, 0.606mmol) were added to a 100mL reaction bottle in sequence, and the reaction solution was saturated with nitrogen. Tetrakis(triphenylphosphine)palladium(0) (32.7mg, 0.028mmol) was then added, and the mixture was stirred at room temperature overnight. Monitored by TLC until the conversion was complete. The contents of the flask were then filtered through diatomaceous earth, and the residue was washed with THF. The filtrate was concentrated under reduced pressure. The crude product obtained was purified by column to obtain 224mg (yield: 90%) of the target compound.

[0295] The other compounds in Table 1 below were prepared by methods similar to those of Examples 1-2 and 4-9 by using different MI, S, C, A and D parts.

[0296] The compounds were confirmed by mass spectrometry (MS) and their molecular weights are shown in Table 1, which are consistent with the calculated molecular weights based on their structures.

[0297] Table 1

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307] The present invention also provides the following comparative compounds, the structural formula of which is as follows:

[0308] Compound C1: Doxorubicin

[0309]

[0310] Compound C2: AANL-DOX

[0311]

[0312] Compound C3: EMC-AANL-DOX

[0313]

[0314] Compound C4: Peg-AANL-DOX

[0315]

[0316] Example 10: Preparation of human albumin-coupled HSA-EMC-AANL-DOX, HSA-QHL-087-DOX and HSA-QHL-087-N-CBP drugs

[0317] Prepare EMC-AANL-DOX, QHL-087-DOX and QHL-087-N-CBP, where 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 is combined with HSA at a ratio of 3:1 (4.8umol / mL, 1.6umol / mL), reacted in a water bath at 37°C for 3h, the reaction solution is taken out, the unbound compound is filtered using a pressurized ultrafiltration membrane, and diluted with physiological saline and filtered 3 times to obtain a semi-finished product. Use, for example, chromatography methods to separate human albumin-coupled doxorubicin antitumor drugs, such as DEAE ion exchange, gel filtration and hydroxyapatite chromatography. The semi-finished product is promptly packaged, frozen and freeze-dried. The freeze-drying process of the product can be formulated according to the performance characteristics of the machine, but the preparation quality and storage quality of the product should be guaranteed to meet the requirements. The experiment compared the binding of legubicin to HSA at different ratios and times. The results showed that EMC-AANL-DOX, QHL-087-DOX and QHL-087-N-CBP bound to HSA at a ratio of 3:1 and bound in a 37°C water bath for 3 hours. The HSA binding rates were 62%, 99.6% and 99.7%, respectively.

[0318] Example 11: Selection of chemically modified linker arms to obtain optimized activation efficiency

[0319] Compared with the natural peptide sequence joint cleaved by Legumain, SCA is a chemically modified joint and shows high activation efficiency. When C selects AAN, the activation of different SCA joints and control joints is evaluated in the activation assay. Use SCA conjugates to dissolve and dilute them tenfold to a concentration of 0.1mM / ml. At 37°C, the sample compound was added to 100μg of acidified human breast cancer (MDA-MB435) tumor tissue homogenate (pH 6.0) at a concentration of 1mg / ml. The enzyme in the tumor tissue homogenate can be released and detected by HPLC, thereby comparing the activation efficiency of the tumor tissue to the joint. The results are shown in Tables 2-1, 2-2, 2-3 and 2-4.

[0320] Table 2-1

[0321]

[0322]

[0323] Table 2-2

[0324]

[0325] Table 2-3

[0326]

[0327]

[0328] Compared with the natural peptide sequence linker cleaved by Legumain, the effect of D-type tripeptide on activation efficiency was detected, and S1 in MI-S was -CH2CH2-CONH-, and S2: 2peg. The results are shown in Tables 2-4 below.

[0329] Table 2-4

[0330] C A Activation efficiency (%) 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

[0331] From the data in the table, we can see that when S and A are designed to be highly activated, when the variables are different tripeptides, different amino acid selections and configurations have an impact on the activation efficiency, especially D-Asn causes the loss of activation ability, while the activation activity is still present when the amino acids at the other two positions are adjusted to the D-type.

[0332] Example 12: Comparison of Enzyme Cleavage Kinetic Rates of Preferred Compounds

[0333] Accurately weigh 10 mg of each of C3, QHL-087-DOX, QHL-090-DOX, QHL-093-DOX, QHL-094-DOX, QHL-093-DOX and QHL-096-DOX samples, add appropriate amount of water to prepare 4 umol / mL sample stock solution, and gradually dilute with water to the sample solutions of each concentration in Table 3 below; measure 20 ul of sample solutions of different concentrations respectively, add 80 ul of Legumain, and bathe in a 37°C water bath; take out after 2 hours of water bath, inject 10 ul, and detect by HPLC; read the area of ​​each corresponding product, calculate the product concentration according to the linear equation of the product, and substitute into the formula to obtain the corresponding V:

[0334] V(umoL / mL / min)=C(umoL / mL) / 120min

[0335] By plotting V against [C], we can obtain the intercept Km / Vmax and the intersection of the straight line and the x-axis, which is -Km. [C] is the concentration of each substrate, that is, the concentration of the sample solution, in umoL / mL.

[0336] Table 3

[0337] <![CDATA[[C1]]]> <![CDATA[[C2]]]> <![CDATA[[C3]]]> <![CDATA[[C4]]]> <![CDATA[[C5]]]> <![CDATA[[C6]]]> <![CDATA[[C7]]]> 2 1.6 0.8 0.7 0.35 0.175 0.0875

[0338] The experimental results are as follows Figure 3 As shown, under other structurally consistent conditions: the 2PEG group of QHL-087 significantly improves the activation efficiency, but as the number of PEG increases, the efficiency decreases. Compared with the same conditions under which the 6PEG group is connected: H2PABC-NH2H2 replaces leu and significantly improves the activation efficiency.

[0339] Embodiment 13:

[0340] Isolation and culture of mouse spleen and CD8+T cells, isolation of mouse bone marrow mononuclear cells and induction of differentiation of M2 macrophages

[0341] 1. Mouse Spleen Cell Isolation

[0342] 1) Take the spleen of a C57BL / 6 mouse, place it on a culture dish with a 40uM mesh (on ice), add about 10mL of normal saline, and gently grind it with a sterile syringe core.

[0343] 2) Transfer the ground cell suspension into a 50 mL centrifuge tube, add about 5 mL of physiological saline to rinse the culture dish, transfer it into a 50 mL centrifuge tube, and combine the suspension.

[0344] 3) Centrifuge the cell suspension at 1000r / min for 10min, discard the supernatant, resuspend with an appropriate volume of saline, add 3 times the volume of ammonium chloride red blood cell lysis buffer, and pipette evenly. After lysis on ice for about 10min, add 10mL of saline to terminate lysis, and centrifuge at 1000r / min for 5min.

[0345] 4) After centrifugation, discard the supernatant, add 10 mL of physiological saline, pipette evenly, centrifuge at 1000 r / min for 5 min, repeat this operation once, and then resuspend the cells in 10% RMPI 1640 medium for subsequent culture or 0.5% BSA for subsequent T cell sorting.

[0346] 2. CD8+ T cell sorting

[0347] The mouse spleen cells isolated above were resuspended to 1E8 / mL, 100ul Miltenyibiotec CD8a (Ly-2) microBeads were added to each 1E8 cells and mixed evenly, incubated at 4 degrees in the dark for 15 minutes, 5-10 times the volume of PBS was added, mixed thoroughly and washed, centrifuged at 300g for 5 minutes, the supernatant was removed, the washing was repeated once, and the cells were resuspended to 2E8 / mL for column separation, and the cell suspension was placed on the magnetic plate LS column (LS column was pre-washed with a wash buffer (pH7.2 PBS + 0.5% BSA + 2mM After the cell suspension slowly flows through the LS column and the CD8+T cells are bound to the magnetic particles in the LS column, the LS column is washed with a washing buffer of 3 times the volume of the cell suspension. After the washing, the LS column is taken out from under the magnet plate and placed in a 15mL centrifuge tube. 5mL of washing buffer is added to the LS column, and then the LS column core is used to quickly squeeze and elute the bound cells in the LS into the centrifuge tube, and all the cells that pass through the column are collected, the obtained cells are centrifuged to remove the supernatant, and the washing is repeated once with the washing buffer, and the cells are resuspended in an appropriate volume of 10% RMPI1640 culture medium, and the cells are counted for use.

[0348] 3. Activation and expansion of CD8+ T positive cells

[0349] A. Washing of CD3 / CD28 magnetic beads: a. Shake and suspend the immunomagnetic beads in the small tube (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 of 1640 containing serum and suspend well, vortex for more than 30 seconds, or keep rolling for at least 5 minutes.

[0350] B. Activation of T cells: a. Inoculate the appropriate number of cells in the culture plate (e.g. 1E6 / ml for a 6-well plate, 2ml of culture medium, keep the T cell density above 2E6 / ml, but not more than 2E6 / ml). b. Add the washed magnetic beads to make the ratio of magnetic beads to cells = 1:1. c. Place in an incubator and culture for 3 days.

[0351] C. Expansion: a. The culture medium does not need to be replaced within 3 days. On the 3rd day, add 30U / ml IL-2 (can be increased appropriately according to actual conditions), and change the culture medium at the same time (the number of cells will approximately double after 48 hours, and the cell size and morphology should be monitored in real time). b. After 4-5 days of stimulation, remove the magnetic beads (avoid over-activation), move the cells up and down 5-10 times, avoid bubbles and turbulence, so as to separate the cells and the magnetic beads. Collect the cells into a 1.5ml tube, place them on a magnet for 1 minute until the magnetic beads are attached to the tube wall, move the cells into another tube to remove the magnetic beads as completely as possible, and continue to culture the cells with the magnetic beads in the culture medium with 30U / ml IL-2 (can be increased appropriately according to actual conditions) and monitor their status, proliferation and vitality).

[0352] 4. Isolation of Mouse Bone Marrow Mononuclear Cells and Induction of Differentiation of M2 Macrophages

[0353] Under sterile conditions, the bilateral femurs and tibias of 2 C57BL / 6 mice were removed, the epiphyses were cut open in the clean bench, and a 5mL sterile syringe was used to extract serum-free MEM culture medium to gently rinse the bone marrow cavity repeatedly for 4 times, and all the cell suspensions were collected; centrifuged at 1000r / min for 10min, the supernatant was discarded to obtain the cell pellet, and an appropriate volume of serum-free MEM culture medium was resuspended, and after repeated blowing and uniformity, 40uM filter was filtered, and 3 times the volume of red blood cell lysis solution was added, and lysis was carried out on ice for 10min; centrifuged at 1000r / min for 5min, the supernatant was discarded to obtain the cell pellet, and the cell pellet was washed twice with serum-free MEM culture medium and collected; the cells were resuspended with an appropriate volume of MEM complete culture medium containing 10% FBS and 1% PS by volume, and the cell count was used for subsequent differentiation; 100uL, 20,000 cells / well were inoculated in a 96-well cell culture plate, and 100ng M-CSF was used to differentiate M2 macrophages. The cells were cultured in an incubator at 37°C and 5% CO2 for 7 days to induce differentiation. The morphology of the cells was observed. Figure 4 , M2 macrophages were differentiated from monocytes, DCs, and GM-macrophages and confirmed as M2 macrophages.

[0354] Example 14: MTT method to determine the inhibitory effect of drugs on cell growth

[0355] After counting the cells in Example 13, the cell concentration was adjusted with the culture medium and inoculated on a 96-well culture plate, with 100 μl of cell suspension per well, the inoculation concentration of CD8+T cells was 100,000 cells / well, and the inoculation concentration of M2 macrophages was 20,000 cells / well. The 96-well culture plate was placed in a 37°C, carbon dioxide (5%) incubator and cultured overnight for 24 hours. After 24 hours, 100ul of cell culture fluid containing different concentrations of drugs was added to the 96-well culture plate, and control wells (0.1% DMSO) without drugs and only with the corresponding drug solvent were set, as well as zeroing wells (Blank) with only culture medium and no cells. Three parallel wells were set for each group, and the plate was then placed in a 37°C, carbon dioxide (5%) incubator for 48 hours. After 48 hours, 20 μl of MTT (at a concentration of 5 mg / ml) was added to each well and incubated for 4 hours. Then the culture medium was gently aspirated, and 150 μl of DMSO was added to each well as a solvent for dissolution. After dissolution, the absorbance at 490 nm was measured using an enzyme-labeled instrument.

[0356] Calculate the cell survival rate and the half inhibitory concentration of the drug on the cell. Cell survival rate % = (OD test-OD blank control) / (OD test control-OD blank control) * 100%. The survival rate (%) was calculated using Excel software, and the dose-response curve of the drug on the cell was drawn using Prism 5 software. Each index was expressed as a mean value, and the coefficient of variation (CV) was used to evaluate the consistency of the data.

[0357] According to the experimental schematic diagram and the setting of cell drug addition concentration in the above experimental method, the maximum starting concentration of the drug to be tested was set to 14uM, and it was gradiently diluted at a ratio of 1:3 into 9 dose groups (3 replicates in each group). The concentration of drug solvent (DMSO) in all drug addition wells was controlled at 0.1%. Only drug solvent (0.1% DMSO) was added as the experimental control group (Control), and a blank group (Blank) with no cells and only culture medium was added. Then, the tumor cell survival rate (%) of each dose group relative to the control group (Control) was calculated according to the following method.

[0358] Cell survival rate of each dose group (%) = (OD dose group - OD blank group) / (OD 0.1% DMSO - OD blank group) * 100%

[0359] The experimental results are as follows Figure 5 and 6 As shown. Under other structurally consistent conditions, QHL-087-DOX has significantly improved cytotoxicity against M2 macrophages, but weaker cytotoxicity against CD8+T cells, compared with C3 (i.e., EMC-AANL-DOX), thereby producing selectivity for immunosuppressive cells.

[0360] Example 15: Screening of cytotoxicity of various drugs of the present invention on M2 macrophages

[0361] The cytotoxicity screening experiment of some compounds on M2 macrophage inhibition was carried out according to the method of Example 14. Three wells were tested for each drug, and 10 uM of the following drugs were added to each well to test the inhibition rate relative to the drug-free group. The experimental results are shown in Table 4.

[0362] Example 16: Comparison of water solubility of water-soluble, highly efficient, targeted activated doxorubicin derivatives prepared in the present invention and control compounds

[0363] The compounds prepared according to the examples of the present invention, the compounds prepared above and the reference compounds C1, C2, C3 and C4 were freeze-dried (-70°C). The compounds were dissolved in water of different concentrations and the water solubility was checked by observation and HPLC test (>95%). The results are shown in Table 4.

[0364] Table 4: Water solubility test data of screened drugs and M2 macrophage inhibition rate

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373] The test results show that under other structurally consistent conditions, the 2PEG group significantly improves water solubility, from insoluble to water soluble, and the solubility increases with the increase in the number of PEGs. Under the same PEG connection conditions, increasing Glu and Asp can increase water solubility. Through the change of the group, the water solubility of the conjugated drug changes, which will have a huge impact on the drug's blood vessel membrane and the permeability to the tumor cell membrane, thereby affecting the efficacy of the treatment. The enhanced water solubility of the compound provides the necessary conditions for the drug's drugability and the production of conjugated drugs.

[0374] Example 17: Efficacy study of C3, QHL-085-DOX, QHL-087-DOX, QHL-091-DOX, and QHL-094-DOX injections in nude mouse HT1080 model

[0375] Objective: To investigate the antitumor efficacy of C3, QHL-085-DOX, QHL-087-DOX, QHL-091-DOX and QHL-94-DOX in mouse models during tumor treatment.

[0376] Experimental drugs: C3, QHL-085-DOX, QHL-087-DOX, QHL-091-DOX and QHL-094-DOX were used as injections and diluted to the corresponding concentrations with normal saline during the experiment.

[0377] Methods and Results:

[0378] 1. Animals: 6-8 week old nude mice, all female.

[0379] 2. Preparation of Tumor Model

[0380] 1) 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, 5% CO2. The cells were subcultured every three days, and cells within the 15th generation were used.

[0381] 2) Tumor generation: 5×10 6 HT1080 cells were injected subcutaneously into the back of nude mice. When the tumor size reached 100 mm 3 The patients were randomly divided into groups at 1:10 pm and 1:15 pm, and then the treatment started, and the day of treatment start was recorded as day 1.

[0382] 3) Treatment process

[0383] According to the clinical application of C3, QHL-085-DOX, QHL-087-DOX, QHL-091-DOX and QHL-094-DOX, drugs were injected intravenously (IV). C3, QHL-085-DOX, QHL-087-DOX, QHL-091-DOX and QHL-094-DOX were administered at low doses and the same dose of 18umol / kg, respectively. The control group was given normal saline. The administration was continued once a week for 3 weeks.

[0384] 4) Results and discussion: Grouping and experimental results are as follows Figure 7 As shown, compared with the equimolar dose low-dose treatment group, the tumor inhibition effects of the 4peg and 2peg groups were enhanced successively.

[0385] Example 18: Pharmacodynamic study of C1, C2, C3, QHL-086-DOX, QHL-092-DOX, QHL-095-DOX, QHL-087-DOX, QHL-010-DOX, QHL-117-DOX injections in nude mouse HT1080 model

[0386] Purpose of the experiment: To investigate the anti-tumor efficacy of the above compounds in mouse models during tumor treatment.

[0387] Test drugs: C1, C2, C3, and corresponding compound injections, diluted with physiological saline to corresponding concentrations during testing.

[0388] Methods and Results:

[0389] 1. Animals: 6-8 week old nude mice, all female.

[0390] 2. Preparation of Tumor Model

[0391] 1) 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, 5% CO2. The cells were subcultured every three days, and cells within the 15th generation were used.

[0392] 2) Tumor generation: 5×10 6 HT1080 cells were injected subcutaneously into the back of nude mice. When the tumor size reached 100 mm 3 The patients were randomly divided into groups at 1:10 pm and 1:15 pm, and then the treatment started, and the day of treatment start was recorded as day 1.

[0393] 3) Treatment process

[0394] According to the clinical application of the corresponding compound, the drug was injected intravenously (IV). The compounds shown in the table were administered at a low dose and the same dose of 36umol / kg. The control group was given normal saline. The administration was continued once a week for 3 weeks.

[0395] 5) Grouping and test results are shown in Table 5.

[0396] Table 5: Treatment of nude mouse tumors by C1, C2, C3 and some compounds of the present invention and mitomycin

[0397]

[0398] 5) Results and discussion: As shown in Table 5, compared with the equimolar dose AANL-DOX treatment group, high-dose treatment with QHL-086-DOX, QHL-092-DOX, QHL-095-DOX, QHL-087-DOX, QHL-010-DOX, and QHL-117-DOX greatly improved the inhibition of tumor growth, and the tumor disappeared, achieving a cure effect.

[0399] Example 19: Study on the tissue distribution of QHL-087-DOX and EMC-AANL-DOX in orthotopically transplanted CT26 tumors in the liver.

[0400] Purpose of the experiment: To study the tissue distribution of activating drugs in liver tumors.

[0401] Experimental animals: BALB / c mice, 6-8 weeks old, all female.

[0402] Preparation of tumor model:

[0403] 1) CT26 cells were purchased from ATCC and cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells were subcultured every three days, and cells within the 15th generation were used.

[0404] 2) Tumor generation: 5×106 CT26 cells were injected subcutaneously into the back of nude mice. When the tumor size reached 800-1000mm 3 The tumor tissues were then extracted and cut into 100 mm 3 The tumor tissue pieces were orthotopically transplanted into the liver of BALB / c mice.

[0405] 3) Administration process: After 14 days, when the orthotopic transplanted tumors grew larger, a group of 36 mice with orthotopic transplanted tumors were treated with drugs. Then different tissues were collected at 1, 6, 12, 24, 36, and 72 hours to detect the concentration of doxorubicin released in different tissues. AUClast h*nmol / g was calculated, and the mean and SEM were as follows: Figure 8 and 9 shown.

[0406] 4) Results and discussion: Figure 8 and 9 As shown, the active doxorubicin of QHL-087-DOX and EMC-AANL-DOX is mainly distributed in the liver and liver orthotopic tumors. The previously published use of EMC-AANL-DOX is for breast cancer treatment. Through further research, it was found that QHL-087-DOX has the characteristics of delivering more drugs to the liver, and the activation of liver orthotopic tumors leads to higher exposure to doxorubicin. Compared with EMC-AANL-DOX, QHL-087-DOX has enhanced exposure to doxorubicin in orthotopic liver cancer delivery and activation.

[0407] Example 20: Study on the efficacy of QHL-087-DOX in orthotopic liver transplanted CT26 tumors.

[0408] Objective: To investigate the efficacy of QHL-087-DOX, PD-1 and their combination in orthotopically transplanted CT26 tumors.

[0409] Experimental drugs: QHL-087-DOX 18 μmol / kg, mouse PD-1 5 mg / kg.

[0410] Animals: BALB / c mice, 6-8 weeks old, all female.

[0411] 1) Generation of tumor model: CT26 tumor cells were obtained from ATCC. The cells were cultured in DMEM containing 10% fetal bovine serum at 37°C and 5% CO2. The 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. The tumor reached 800-1000mm 3 Afterwards, the mice were randomly divided into groups. The tumor tissues were then extracted and cut into 100 mm 3The tumor tissue pieces were orthotopically transplanted into the liver of BALB / c mice. One week later, when the orthotopically transplanted tumors grew larger, the mice with orthotopically transplanted tumors were randomly divided into groups.

[0412] 2) Treatment process: 6 mice in one group were treated with drugs. The day of treatment was day 1. According to the clinical application of QHL-087-DOX, drugs were injected intravenously (IV) once a week for 3 weeks. Mouse PD-1 antibody was injected intravenously (IV) twice a week for 3 weeks. Grouping and test results are attached Fig.10 .

[0413] 3) Results and discussion: It was discovered for the first time that the combination of QHL-087-DOX and PD-1 has an immune synergistic effect on liver tumors in situ. The combination of QHL-087-DOX and PD-1 has better efficacy than QHL-087-DOX alone and has the characteristics of immunotherapy.

[0414] Example 21: Effect of EMC-AANL-DOX (legubicin), lenvatinib and PD-1 combined with each other in the treatment of hepatocellular carcinoma in situ

[0415] Purpose of the experiment: To investigate the effect of EMC-AANL-DOX, lenvatinib and PD-1 combined in the treatment of hepatocellular carcinoma in situ.

[0416] Experimental drugs: EMC-AANL-DOX 18 μmol / kg, mouse PD-1 5 mg / kg.

[0417] Animals: BALB / c mice, 6-8 weeks old, all female.

[0418] Generation of tumor model: CT26 tumor cells were obtained from ATCC. The cells were cultured in DMEM containing 10% fetal bovine serum at 37°C and 5% CO2. The 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. The tumor reached 800-1000mm 3 Afterwards, the mice were randomly divided into groups. The tumor tissues were then extracted and cut into 100 mm 3 The tumor tissue pieces were orthotopically transplanted into the liver of BALB / c mice. One week later, when the orthotopically transplanted tumors grew larger, the mice with orthotopically transplanted tumors were randomly divided into groups.

[0419] Treatment process: 6 mice in one group were treated with drugs. The day of treatment was day 1. According to the clinical application of EMC-AANL-DOX, drugs were injected intravenously (IV) once a week for 3 weeks. Mouse PD-1 antibody was injected intravenously (IV) twice a week for 3 weeks. Grouping and test results are attached Fig.11 .

[0420] Results and discussion: For the first time, it was found that the combination of EMC-AANL-DOX and PD-1 was more effective than the combination of lenvatinib and PD-1 in the treatment of hepatic in situ tumors. It was also found that the combination of EMC-AANL-DOX and lenvatinib had a stronger efficacy than the single-drug treatment of each.

[0421] Example 22: Study on the efficacy of injection of some compounds of the present invention on nude mouse human liver cancer HepG2 cells

[0422] Experimental purpose: To study the anti-tumor efficacy of some compounds of the present invention in a mouse tumor treatment model.

[0423] Test drugs: injections of corresponding compounds and control group injections in the table, diluted to corresponding concentrations with physiological saline during the test.

[0424] Methods and Results:

[0425] 1. Experimental animals: 6-8 week old nude mice, all female.

[0426] 2. Preparation of Tumor Model

[0427] 1) Human hepatoma 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, 5% CO2. The cells were subcultured every three days, and cells within the 15th generation were used.

[0428] 2) Tumor generation: 5×10 6 HepG2 cells were subcutaneously injected into the back of nude mice. When the tumor size reached 100 mm3, they were randomly divided into groups. Then treatment began, and the day of treatment was recorded as day 1.

[0429] 3) Treatment process

[0430] According to the clinical application of the corresponding compound, the drug was injected intravenously (IV). The compound and the control group were administered at a dose of 54umol / kg, and DOX could only be administered at a dose of 18umol / kg due to toxicity restrictions. The control group was given normal saline. The drug was administered once a week for 4 weeks.

[0431] 6) Grouping and test results are shown in Table 6.

[0432] Table 6: Effects of some compounds of the present invention and the control group on tumor treatment in nude mice

[0433]

[0434]

[0435] 5) Results and discussion: As shown in Table 6, EMC-AANL-DOX has a good effect on liver cancer. Compared with EMC-AANL-DOX at the same molar dose, the preferred compounds of the present invention have increased therapeutic effects on tumor growth.

[0436] Example 23: Study on the efficacy of QHL-096-DOX, QHL-087-DOX, QHL-090-DOX, QHL-093-DOX, and QHL-117-DOX in the treatment of CT26 tumor immune model

[0437] Purpose of the experiment: To study the anti-tumor efficacy of the above compounds in immunotherapy in the CT26 tumor model.

[0438] Experimental 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.

[0439] Experimental animals: 6-8 week old BALB / c mice, all female.

[0440] Preparation of tumor model:

[0441] 1) CT26 cells were purchased from ATCC and cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells were subcultured every three days, and cells within the 15th generation were used.

[0442] 2) Tumor formation. 5×10 6 The corresponding cells were injected subcutaneously into the back of nude mice. The tumors reached at least 100 mm 3 After that, the mice were randomly divided into groups. Then the treatment started, and the day when the treatment started was the first day.

[0443] 3) Treatment course. The drug was administered at an equimolar dose of 36umol / kg. The control group was given normal saline. The drug was administered once a week for three weeks.

[0444] 4) Tumor CD8 + T cell analysis. The tumor tissue was homogenized, and the individual cells in the tumor were filtered, separated and washed twice with buffer, and then incubated with antibodies labeled with leukocyte common antigen CD45-PE and CD8-FITC for 1 hour at room temperature. The cells were washed twice with phosphate buffer containing 1% fetal bovine serum, and then the ratio of T lymphocyte antigen (CD8) positive cells among leukocyte common antigen (CD45) positive cells was analyzed by flow cytometry. An increase in the ratio indicates an increase in T lymphocytes, and therefore an improvement in the animal's immunity to tumors.

[0445] 4) Grouping and test results are shown in Table 7.

[0446] Table 7: Effects of corresponding compounds and control group on tumor inhibition and immune activation

[0447]

[0448]

[0449] 5) Results and discussion: C3, QHL-096-DOX, QHL-087-DOX, QHL-090-DOX, QHL-093-DOX, QHL-117-DOX combined with PD-1 have improved therapeutic effects compared to single drugs and can cure tumors. QHL-090-dabrafenib, QHL-090-dabrafenib combined with PD-1 also have certain synergistic effects.

[0450] Example 24: Study on the efficacy of QHL-087-DOX injection in various tumor models

[0451] Purpose of the experiment: To study the anti-tumor spectrum of QHL-087 using multiple tumor models in mice.

[0452] Test drug: QHL-087-DOX was injected and diluted to the corresponding concentration with normal saline during the test.

[0453] Methods and Results:

[0454] 1. Animals: 6-8 week old female nude mice.

[0455] 2. Generation of Tumor Models

[0456] 1) The corresponding tumor cells were purchased from the American Type Culture Collection (ATCC) and identified according to the specifications provided by ATCC. The cells were cultured in DMEM culture medium containing 10% fetal bovine serum at 37°C and 5% CO2. The cells were passaged every three days, and cells within the 15th passage were used.

[0457] 2) Tumor formation. 5×10 6 The corresponding cells were injected subcutaneously into the back of nude mice. The tumors reached at least 100 mm 3 After that, the mice were randomly divided into groups. Then the treatment started, and the day when the treatment started was the first day.

[0458] 3) Treatment course. According to the clinical application of QHL-087-DOX, QHL-087-DOX was administered at a dose of 36umol / kg. The control group was given normal saline. The administration was continued once a week for three weeks.

[0459] 4) Grouping and test results are shown in Table 8.

[0460] Table 8: The therapeutic effect of QHL-087-DOX in various tumor models

[0461] Group Tumor cells Tumor inhibition rate (day 26) Human Breast Cancer MDA-MB435 91.5％ Human ovarian cancer SK-OV-3 78.7％ Human colon cancer HT-29 85.3％ Human chronic leukemia K562 79.4％ Human colon cancer HT1080 90.5％ Human pancreatic cancer Panc-1 75.7％ Human non-small cell lung cancer A549 75.8％ Human liver cancer Hepg2 100％ Human kidney cancer OS-RC-2 87.4％

[0462] 5) Results and discussion: QHL-087-DOX showed excellent efficacy in multiple tumor models, indicating that the drug has a broad anti-tumor spectrum.

[0463] Example 25: Solubility comparison of HSA-EMC-AANL-DOX, HSA-QHL-087-DOX, and HSA-QHL-087-N-CBP with control compounds

[0464] The lyophilized products EMC-AANL-DOX, HSA-EMC-AANL-DOX, HSA-QHL-087-DOX and HSA-QHL-087-N-CBP prepared in the examples of the present invention were packaged in a sterile room and reconstituted with water for injection. HSA-EMC-AANL-DOX, HSA-QHL-087-DOX and HSA-QHL-087-N-CBP were completely dissolved, as shown in Table 9.

[0465] Table 9

[0466] Compound Water for injection EMC-AANL-DOX Insoluble HSA-EMC-AANL-DOX Dissolved, 5mg / ml HSA-QHL-087-DOX Dissolved, 35mg / ml HSA-QHL-087-N-CBP Dissolved, 35mg / ml

[0467] As can be seen from Table 9, the solubility of the compounds is further improved after coupling with human albumin. HSA-EMC-AANL-DOX, HSA-QHL-087-DOX and HSA-QHL-087-N-CBP, as macromolecular protein drugs, can be directly dissolved to high concentrations with injection water or saline, without the need for the irritating organic solvents required for the dissolution of EMC-AANL-DOX. Unlike the water-insoluble EMC-AANL-DOX small molecule compound drug, changes in solubility properties have a huge impact on the distribution, metabolism and mode of action of the drug.

[0468] Example 26: Comparison of solution stability of water-soluble, highly efficient, targeted activated doxorubicin derivatives prepared in the present invention and control compounds

[0469] 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 accurately, and pack 5.0 mg of each sample in a sterile room, add 0.5 ml of sterile water for injection to make a 10 mg / ml mother solution, and 50% ethanol is required to dissolve EMC-AANL-DOX. Take 30ul of the mother solution, add 570ul of buffer solution with different pH values ​​of 5.5, and make a 0.5 mg / ml sample solution. After the sample is clarified, place it in a 25℃ / 37℃ water bath, take a sample at 8h, and use HPLC and electrophoresis to detect the content of the sample relative to 0 hours, and the solution stability data of different compounds can be obtained. The results are shown in Table 10.

[0470] Table 10: Solution stability data

[0471] Compound 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％

[0472] It can be seen from the data in the above table that under the conditions of 25°C and pH = 5.5, the stability of the albumin-coupled compounds increases, which is more obvious for QHL-087-N-CBP and HSA-QHL-087-N-CBP.

[0473] Example 27: Activation efficiency test of HSA-EMC-AANL-DOX, HSA-QHL-087-DOX and HSA-QHL-087-N-CBP

[0474] EMC-AANL-DOX was dissolved using a solvent (50% water for injection + 50% alcohol), HSA-EMC-AANL-DOX, HSA-QHL-087-DOX and HSA-QHL-087-N-CBP were uniformly dissolved using water for injection, and diluted 10 times with water to 1 mg / ml. In the experiment of the present invention, 1 mg / ml of sample compound was 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 the compound and the increase of doxorubicin can be detected by HPLC to compare the activation efficiency of the drug in the tumor tissue. Through screening, it was found that the compounds of the present invention, EMC-AANL-DOX, HSA-EMC-AANL-DOX, HSA-QHL-087-DOX, and HSA-QHL-087-N-CBP were connected to have the highest activation efficiency among the screened compounds. The results are shown in Table 11.

[0475] Table 11: Compound 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

[0476]

[0477] Example 28: Toxicity Assays of EMC-AANL-DOX, HSA-EMC-AANL-DOX, QHL-087-DOX, HSA-QHL-087-DOX, QHL-087-N-CBP and HSA-QHL-087-N-CBP

[0478] Purpose of the experiment: To understand the acute toxicity of the drug of the present invention by determining the MTD experiment of intravenous administration in mice.

[0479] Experimental drugs: EMC-AANL-DOX was 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 were dissolved in water for injection and diluted to the corresponding dose with normal saline during the experiment.

[0480] Animals: Primary BALB / C mice (purchased from Shanghai Slake Laboratory Animal Co., Ltd.), weighing 19-21 g, all female.

[0481] Methods and Results: Thirty-six BALB / C mice weighing 19-21 g were randomly divided into seven groups according to their weight, with six 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 injected intravenously at the doses shown in Table 12. A control experiment was also conducted with a saline group and a paclitaxel group injection (commercially available, Beijing Yuekang), with a volume of 0.2 ml per mouse. The animals were observed for 17 consecutive days, and the animals were observed daily to see if they had erect hair, messy and dull, lethargic, hunched over, or overreacted, and the weight and death were recorded. Blood samples were collected on the 3rd, 5th, and 14th days for complete blood counts, and the heart, liver, kidney, lung, spleen, and pancreas were dissected on the 14th day for HE staining observation.

[0482] Table 12: Comparison of mortality results of mice receiving different doses of compound injections, saline, and paclitaxel injections

[0483] Group Dosage (μmol / kg) Animals Number of deaths (individuals) 1 Normal saline 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

[0484] Results and discussion: When the animals were injected with HSA-EMC-AANL-DOX, HSA-QHL-087-DOX and HSA-QHL-087-N-CBP, there was no evidence of erected hair, matted luster, lethargy, hunchback, overreaction or death, indicating that the toxicity of albumin-conjugated drugs was significantly lower than that of unconjugated drugs.

[0485] Example 29: Combination therapy effect of HSA-EMC-AANL-DOX, HSA-QHL-087-DOX and anti-PD-1 antibody

[0486] Purpose of the experiment: To compare the therapeutic effects of EMC-AANL-DOX, HSA-QHL-087-DOX and anti-PD-1 antibody combination therapy.

[0487] Experimental drugs: HSA-EMC-AANL-DOX and HSA-QHL-087-DOX, with doses of 18 μmol / kg respectively; mouse PD-1 antibody, 5 mg / kg.

[0488] Experimental animals: 6-8 week old BALB / c mice, all female.

[0489] Preparation of tumor model: CT26 cells were purchased from ATCC and cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells were subcultured every three days and cells within the 15th generation were used. 5×10 6 CT26 cancer cells were injected subcutaneously into mice. The mice were injected with the drug three times, once a week, and the anti-PD-1 antibody twice a week, for a total of eight times.

[0490] Results and discussion: The experimental results are shown in Fig.12 , the therapeutic effect of HSA-QHL-087-DOX combined with anti-PD-1 antibody is improved compared with HSA-EMC-AANL-DOX, and the cure rate is higher.

[0491] Example 30: MTT assay to determine the inhibitory effect of N-CBP and HSA-QHL-095-N-CBP on tumor cell growth

[0492] After counting the isolated and cultured cells, the cell concentration was adjusted with the culture medium, and the cells were inoculated on a 96-well culture plate, with 100 μl of cell suspension per well, 100,000 cells / well for CD8+T cells, and 20,000 cells / well for CT26 tumor cells. The 96-well culture plate was placed in a 37°C, carbon dioxide (5%) incubator for overnight culture for 24 hours. After 24 hours, 100 ul of cell culture solution containing different concentrations of drugs was added to the 96-well culture plate, and a control well (0.1% DMSO) without adding drugs but only adding the corresponding drug solvent was set, and a zeroing well (Blank) without adding culture medium and cells was set. Three parallel wells were set for each group, and then the plate was placed in a 37°C, carbon dioxide (5%) incubator for 48 hours. After 48 hours, 20 μl of MTT (concentration of 5 mg / ml) was added to each well and incubated for another 4 hours. Then the culture medium was gently aspirated, and 150 μl of DMSO was added to each well as a solvent for dissolution. After dissolution, the absorbance at 490 nm was measured using an enzyme-labeled instrument.

[0493] Results and discussion: The experimental results are shown in Fig.13 and 14 The in vitro cytotoxicity of N-CBP is stronger than that of carboplatin and oxaliplatin, but weaker than that of cisplatin ( Fig.13 ); HSA-QHL-095-N-CBP has a reduced in vitro cytotoxicity relative to N-CBP and carboplatin ( Fig.14 ).

[0494] Example 31: Effects of HSA-QHL-095-N-CBP as a single agent and in combination with anti-PD-1 antibodies

[0495] Purpose: To compare the therapeutic effects of carboplatin, HSA-QHL-095-N-CBP, and combination therapy with anti-PD-1 antibodies.

[0496] Experimental drugs: carboplatin, HSA-QHL-095-N-CBP, dose 18 μmol / kg, mouse PD-1 antibody, 5 mg / kg.

[0497] Experimental animals: 6-8 week old BALB / c mice, all female.

[0498] Preparation of tumor model: CT26 cells were purchased from ATCC and cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells were subcultured every three days and cells within the 15th generation were used. 5×10 6 CT26 cancer cells were injected subcutaneously into mice.

[0499] Treatment course: Inject the corresponding drug once a week for 3 weeks; anti-PD-1 antibody is administered once a week for 4 weeks.

[0500] Results and discussion: The experimental results are shown in Fig.15 , HSA-QHL-095-N-CBP equimolar dose was significantly better than carboplatin, and the therapeutic effect of HSA-QHL-095-N-CBP combined with anti-PD-1 antibody was improved compared with single drug, achieving a cure effect.

[0501] Example 32: Effects of inflammatory response in non-alcoholic fatty liver disease (NAFLD) model mice.

[0502] Experimental method: C57 mice were randomly divided into a normal group (standard feed), a model group (high-fat feed), a simvastatin group (positive control, 3 mg / kg) and a drug group (50 mg / kg), with 6 mice in each group. The mice in the normal group were fed with a standard feed, and the mice in the other groups were fed with a high-fat feed to induce the NAFLD model. At the same time as the modeling, each group of mice was IV-administered with a corresponding dose of 48umol / kg of the drug twice a week for a total of 8 weeks. 12 hours after the last administration, the serum biochemical indicators: high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein (LDL-C) were measured by an automatic biochemical analyzer. The results are shown in Table 13.

[0503] Table 13

[0504]

[0505]

[0506] Results: Compared with the normal group, the HDL-C content in the serum of the model group was significantly reduced to 66.2%, and the LDL-C content was significantly increased to 135.6% (P<0.05). The QHL-158-T3 and QHL-159-T3 treatment groups had a better effect in restoring HDL-C and LDL-C to normal levels.

Claims

1. A compound having a structure represented by the following formula (I): MI-SCA(I) In the formula, MI-S is selected from: In the formula, -CO in the above structural formulae 5 to 8 represents -C(=O)-, which and -C(=O)- in the above structural formulae 1 to 4 represent the position where MI-S is connected to C; C is Ala-Ala-Asn; A is: in, The wavy line indicates where A and C are linked; Among them, S and C are connected in the following way:

2. The compound according to claim 1, characterized in that The MI-S is selected from:

3. A conjugate represented by the following formula (II) or a pharmaceutically acceptable salt thereof: MI-SCAD(II) In the formula, MI-S, C and A are as described in any one of claims 1-2; D is selected from doxorubicin, epirubicin, mitomycin, dabrafenib, dovitinib, motesanib, And the following compounds a and b:

4. The conjugate or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that: A and D are connected in the following way: The wavy lines represent the connection parts with the adjacent parts.

5. A conjugate selected from the following or a pharmaceutically acceptable salt thereof:

6. A conjugate formed by covalently conjugating the conjugate according to any one of claims 3 to 5 with albumin, or a pharmaceutically acceptable salt thereof.

7. A pharmaceutical composition comprising the conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 3 to 5, or the conjugate or a pharmaceutically acceptable salt thereof according to claim 6, and a pharmaceutically acceptable carrier.

8. Use of the conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 3 to 5, or the conjugate or a pharmaceutically acceptable salt thereof according to claim 6 in the preparation of a medicament for treating or preventing cancer.

9. The use according to claim 8, characterized in that The cancer is a solid cancer or a blood tumor.

10. The use according to claim 9, characterized in that The cancer is of the bladder, brain, breast, mammary gland, cervix, colon, rectum, esophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin, stomach, uterus, ovary, testis, or blood.

11. Use of the conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 3 to 5, or the conjugate or a pharmaceutically acceptable salt thereof according to claim 6 in the preparation of a medicament for inhibiting immunosuppressive cells, inhibiting tumor-associated macrophages, inhibiting MDSC cells, inhibiting angiogenesis, promoting anti-tumor immunity and / or promoting T lymphocyte proliferation.

12. Use of the conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 3 to 5, or the conjugate or a pharmaceutically acceptable salt thereof according to claim 6 and an anti-PD-1 antibody in the preparation of a medicament for combined treatment of tumors.

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