Preparation and Use of Targeted Delivery and Activated Immunostimulating Complexes

Abstract

The present invention provides the preparation and use of targeted delivery and activated immunostimulating complexes. Specifically, the present invention provides a compound of formula MI-SCA used as a linker, and a drug-conjugated pharmaceutical compound of formula MI-SCAD. The pharmaceutical compounds of the present invention have improved water solubility, reduced cytotoxicity, and enhanced pharmaceutical activity.

Description

[Technical field]

[0001] The present invention relates to anti-tumor pharmaceutical compounds, in particular to the preparation and use of targeted delivery and activated immune stimulating complexes. [Background technology]

[0002] Legumain was first identified in legume seeds as an asparaginyl endopeptidase belonging to the C13 family of cysteine ​​proteases. Legumain can process storage proteins during seed germination. It was subsequently discovered in parasites and mammals, including humans, proving that this protease is highly conserved. Legumain from pigs was first cloned and identified in 1997. Legumain is highly expressed in most solid cancers. The differential expression of legumain in normal and tumor tissues makes it an ideal therapeutic target for tumors. Legumain is an endopeptidase that specifically cleaves peptide bonds at the asparagine C-terminus of peptide chains under mildly acidic conditions. CN201210573744.3 discloses a method in which a peptide doxorubicin derivative activated by an aspartylase target releases Leu-doxorubicin compounds into tumors by legumain-mediated cleavage of the tetrapeptide group (linker). Summary of the Invention [Problem to be solved by the invention]

[0003] In the present invention, through compound screening and systematic research in biology, a chemically modified activation linker has been developed to further improve the activation efficiency. In addition, the chemically modified activation linker of the present invention enhances the selectivity of the drug conjugate to immune cells, creates enhanced immunotherapy properties in treatment, and enhances the synergistic effect of combination with PD-1 antibody.

[0004] The technical problem that the present invention seeks to solve is the creation of highly efficient and specifically selected linkers. [Means for solving the problem]

[0005] Previous studies have revealed that asparaginyl endopeptidase preferentially recognizes the substrate peptide sequence of a tetrapeptide and cleaves the amide bond between Asn and other residues. The idea of ​​the present invention to improve the activation efficiency is to first synthesize a large amount of structurally different compounds at both ends of a tripeptide (e.g., AAN), and then use these compounds to further elucidate the mechanism of action of asparaginyl endopeptidase. According to the crystal structure of asparaginyl endopeptidase (Figure 1), the active center of asparaginyl endopeptidase is located at the bottom of the entry hole, so the activation of the substrate peptide requires the active center of the enzyme close to the bottom. In the figure, C189 at the S1 position of asparaginyl endopeptidase attacks to cleave the Asn peptide chain, but the efficiency of enzyme cleavage is determined by the binding of the neighboring S2, S3, S4, and S1 positions to the substrate, and S2 and S3 correspond to the Ala-Ala amino acids of the substrate peptide. Considering the possible interactions and spatial hindrances when linking a compound such as doxorubicin with an MI group, this experiment further carried out extensive synthesis and screening of structures in which both ends of the substrate tripeptide were chemically modified. The synthesized compounds of different groups were linked to MI and doxorubicin, and the activation efficiency was screened under tumor tissue or asparaginyl endopeptidase conditions to optimize new compound complexes with mutual three-dimensional structural relationships. A schematic diagram of this structure is shown in Figure 2, which includes an MI group, a selective group S, an asparaginyl endopeptidase-cleaved tripeptide group C, an auxiliary group A, and a drug complex. The additional group of the present invention provides new functions. In addition to increasing the activation activity of asparaginyl endopeptidase against the bound drug compound (D), it also improves the physical properties and biological functions of the drug compound. The drug compounds provided by the present invention are hydrophilic with altered cell membrane permeability, making them ideal compounds for development as medicines. In addition, the pharmaceutical compound of formula (II) of the present invention has been found to be cell-selective in that it is specifically phagocytosed by tumor-associated macrophages and attacks or inhibits tumor-associated macrophages and MDSC cells, thereby alleviating the immunosuppressive effect of tumor-associated macrophages and promoting immunotherapy.The present invention also found that when doxorubicin is bound, the length of the S group affects the activation efficiency, and the longer the S chain length, the more difficult it is for the compound to bind to the enzyme, and the lower the activation efficiency due to spatial blockage.

[0006] Human serum albumin (HSA) is a small globular protein of 585 amino acids (66-69 kd) consisting of many charged residues (lysine, aspartic acid, coenzyme-free and carbohydrate-free groups, etc.) and small amounts of tryptophan and methionine residues. The compound of formula (II) of the present invention is conjugated to human serum albumin at the 34th cysteine ​​position as a polymeric drug. It has been experimentally shown that albumin covalently conjugated to the compound of formula (II) of the present invention or EMC-AANL-DOX has reduced toxicity, improved drug stability, and significantly improved therapeutic efficacy.

[0007] In summary, the linker of formula (I) and the drug compound of formula (II) of the present invention have improved activation efficiency, improved selectivity for immune cells, improved tissue selectivity, suitable water and lipid solubility and drug stability.

[0008] Accordingly, the present invention provides compounds of formula (I) (linkers) and drug compounds of formula (II) (conjugates) as described herein, as well as pharma-ceutically acceptable salts thereof.

[0009] The present invention also provides a platinum derivative having the structure of the following formula or a pharma- ceutically acceptable salt thereof: [ka]

[0010] The present invention also provides a pharmaceutical compound of formula (II) of the present invention, or a pharma- ceutically acceptable salt thereof, covalently bound to albumin, and EMC-AANL-DOX covalently bound to albumin; preferably, albumin is linked to the MI or EMC moiety of formula (II) via its cysteine ​​residue at position 36.

[0011] The present invention also provides a pharmaceutical composition, comprising a compound of formula (II) of the present invention or a pharma- ceutically acceptable salt thereof, a platinum derivative as described herein or a pharma- ceutically acceptable salt thereof, a drug compound of formula (II) of the present invention covalently bound to albumin or a pharma- ceutically acceptable salt thereof, or EMC-AANL-DOX covalently bound to albumin, and a pharma- ceutically acceptable carrier.

[0012] The present invention also provides for the preparation of a medicament comprising a compound of formula (II) or a pharma- ceutically acceptable salt thereof, a platinum derivative as described herein or a pharma- ceutically acceptable salt thereof, a pharmaceutical compound of formula (II) of the present invention covalently bound to albumin or a pharma- ceutically acceptable salt thereof, or EMC-AANL-DOX covalently bound to albumin or a pharma- ceutical acceptable salt thereof, for the treatment or prevention of the steatosis phenomenon of cancer, fatty liver (including alcoholic and non-alcoholic fatty liver), steatohepatitis, fatty liver disease, liver fibrosis, hepatitis, hepatocellular injury. Preferably, said cancer is a solid cancer or a hematological tumor, preferably a tumor of the bladder, brain, breast / chest, cervix, colon, rectum, esophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin, stomach, uterus, ovary, testis and hematological cancer.

[0013] The present invention also provides the use of the compounds according to formula (I) in the preparation of drugs having improved water solubility, reduced drug toxicity, improved drug efficacy and / or improved drug selectivity for immune cells, or in the preparation of drug molecules for drug delivery to the liver.

[0014] The present invention also provides the use of an EMC-AANL-DOX compound having the structure shown in the formula below, or a medicament comprising albumin conjugated thereto (preferably covalently bonded to the EMC moiety via the cysteine ​​residue at position 36 of albumin), in the preparation of a medicament for the treatment of hepatocellular carcinoma, and their use with an anti-PD-1 antibody and / or an anti-PD-L1 antibody in the preparation of a medicament for the combination treatment of a tumor. [ka]

[0015] The present invention also provides the use of a pharmaceutical compound of formula (II) of the present invention covalently bound to albumin or a pharma-ceutical acceptable salt thereof, a platinum derivative or a pharma-ceutical acceptable salt thereof as described herein, or EMC-AANL-DOX covalently bound to albumin or a pharma-ceutical acceptable salt thereof, in the preparation of a medicament for inhibiting immunosuppressive cells, inhibiting tumor-associated macrophages, inhibiting MDSC cells, inhibiting angiogenesis, promoting antitumor immunity and / or promoting T-lymphocyte proliferation.

[0016] The present invention also provides a pharmaceutical compound of formula (II) or a pharma- ceutically acceptable salt thereof, a platinum derivative as described herein or a pharma- ceutically acceptable salt thereof, of the present invention covalently linked to albumin or a pharma- ceutically acceptable salt thereof, or EMC-AANL-DOX covalently linked to an anti-PD-1 antibody and albumin or a pharma- ceutically acceptable salt thereof, in the preparation of a medicament for combination treatment of a tumor. [Brief description of the drawings]

[0017] [Figure 1] Diagram of the crystal structure of asparaginyl endopeptidase and its active substrate. [Diagram 2] Schematic diagram of targeted delivery and activated immunostimulating complex. [Diagram 3] Comparison of the enzymatic cleavage kinetics of preferred compounds. [Figure 4] Isolation of mouse bone marrow mononuclear cells and induction of M2 macrophage differentiation. [Diagram 5] Cytotoxicity test of compounds against CD8+ T cells. [Figure 6] Cytotoxicity testing of compounds against M2 macrophages. [Figure 7] Compound efficacy assay against HT1080 tumors. [Figure 8] EMC-AANL-DOX exhibits a high distribution profile in liver and liver cancer tissues. [Figure 9] QHL-087-DOX exhibits a high distribution profile in liver and hepatocellular carcinoma tissues. [Figure 10] Treatment of intrahepatic artery tumors with a combination of QHL-087-DOX and anti-PD-1 antibody. [Figure 11] We show that the combination of EMC-AANL-DOX and anti-PD-1 antibody is more effective than the combination of lenvatinib and anti-PD-1 antibody in treating liver in situ tumors. [Figure 12] Combination effects of HSA-EMC-AANL-DOX, QHL-087-DOX and anti-PD-1 antibody [Figure 13] In vitro cytotoxicity assay of N-CBP. [Figure 14] Cytotoxicity testing of HSA-QHL-095-N-CBP. [Figure 15] Efficacy of HSA-QHL-095-N-CBP as a single agent and in combination with anti-PD-1 antibodies. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The technical solutions of the present invention are further described below in conjunction with specific embodiments.

[0019] I. Linker Compounds The present invention relates to a compound having a structure represented by the following formula (I), which can be used as a linker with a target drug (e.g., an anticancer drug) to increase the water solubility of the compound, reduce the toxicity of the drug, improve the efficacy of the drug, and / or provide selectivity for immune cells. MI-SCA (I)

[0020] In the formula, MI represents a maleimide group, S represents a group that improves the efficiency or selectivity of enzymatic cleavage, C represents an amino acid linker that can be cleaved by a protease, and A represents an auxiliary linker.

[0021] An exemplary MI is a maleimide group having the formula: [ka]

[0022] Here, the wavy line indicates the connection point with S.

[0023] In some embodiments, S in formula (I) can be represented as S1-S2-S3, where S1 is selected from the following: [ka]

[0024] wherein Rx is absent or selected from: 1-6 Alkylidene, C 1-6 Alkylideneamino, C 1-6 Alkylidene carboxyl and C 1-6 alkylidenecarbonylamino, the wavy lines indicate the points of attachment to the adjacent moiety; S2 is present or -[(CH2) p O] q -, where p is an integer from 1 to 4, preferably 2, and q is an integer from 0 to 15, preferably 1 to 15, more preferably 2 to 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, Glyn, Lys, Arg and His, preferably Glu and Asp.

[0025] It should be understood that at least one of S1, S2, S3 is present.

[0026] Preferably, MI, S1, S2, S3, C and A are connected to each other by any of the following:

[0027] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] ;

[0028] Here, the wavy line indicates the connection site to the adjacent site. Preferably, S is connected to C by a group selected from the group consisting of:

[0029] [ka] [ka] [ka]

[0030] In some embodiments, S is -R-[(CH) p O] q -R2-R3-, where R1 is linked to MI and is absent or C 1-6 Alkylidene or C 1-6 alkylidenecarbonylamino; R2 is selected from C 1-6R3 is selected from -C(O)O-, -NH-, -O- or -C(O)-R4, where R4 is an amino acid residue of 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 -C(O)- via its amino group; p is an integer of 1 to 4; q is an integer of 0 to 15, preferably an integer of 1 to 15, more preferably an integer of 2 to 6. Preferably, R1 is absent, p is 2 or 3, q ​​is 1 to 15, preferably 2 to 6, and R2 is C 1-4 alkylidene, R3 is selected from -C(O)O-, -NH-, and -O-. In some embodiments, preferably, R1 is absent, q is 0, and R2 is C 1-6 In some embodiments, R1 is preferably an alkylidene, 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 alkylidenecarbonylamino group, p is 2 or 3, q ​​is 1 to 15, preferably 2 to 6, R2 is C 1-4 R3 is alkylidene, R4 is -C(O)-R4, R4 is preferably Glu or Asp, and R4 forms an amide bond with this -C(O)- via its amino group.

[0031] Exemplary MI-S are selected from the following:

[0032] [ka]

[0033] [ka]

[0034] [ka]

[0035] [ka]

[0036] [ka]

[0037] Preferably, C connected to any of the above MI-S is an AAN, and A is any of the structures described below.

[0038] Preferably, in the compound of formula (I) of the present invention, C is selected from the group that exhibits asparaginyl endopeptidase cleavage in the tumor microenvironment and contains an Asn residue. In some embodiments, C is X1X2X3, where X1 is selected from L- or D-Ala, Thr, Val, and Asn; X2 is selected from L- or D-Ala, Thr, Val, and Ile; and X3 is Asn, preferably not D-Asn. Exemplary C are 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, Ala ... sn-Ile-Asn, Ala-Thr-Asn, D-Thr-L-Val-L-Asn, D-Thr-L-Ala-L-Asn, D-Ala-L-Val-L-Asn, L-Thr-D-Val-L-Asn, L-Thr-D-Ala-L-Asn, L-Ala-D-Val-L-Asn, D-Thr-D-Val-L-Asn, D-Thr-D-Ala-L-Asn, D-Ala-D-Val-L-Asn. In some particularly preferred embodiments, C is AAN.

[0039] In the compound of formula (I) of the present invention, A is preferably selected from Leu, PABC-OH and PABC-NH2, the structures of which are respectively shown in the following formulas.

[0040] [ka] (Leu) [ka] (PABC-OH) [ka] (PABC-NH2)

[0041] Here, the wavy line indicates the connection position with C.

[0042] In some embodiments, S and A in the compounds of formula (I) of the present invention are selected from the following groups 1 to 162 [In the formula, -(CH2CH2O)2- is 2peg, -(CH2CH2O)3- is 3peg, -(CH2CH2O)4- is 4peg, -(CH2CH2O)6- is 6peg] and the like.

[0043] [Table 1]

[0044] [Table 2]

[0045] [Table 3]

[0046] [Table 4]

[0047] [Table 5]

[0048] [Table 6]

[0049] Preferably, in the compound of formula (I) of the present invention, MI is a maleimide group, S and A are any of groups QHL-001 to QHL-162, and C is AAN.

[0050] Particularly preferred compounds (linkers) of formula (I) of the present invention are selected from the following: 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-1 19, 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.

[0051] ii. Drug Compounds The present invention provides a compound (complex) represented by the following formula (II) or a pharma- ceutically acceptable salt thereof: MI-SCAD (II)

[0052] wherein MI, S, C and A form a linker compound according to any embodiment of the present invention, and D is a drug, preferably an anti-cancer compound.

[0053] In Formula II, when A is used as a linking group, it is selected from the following:

[0054] [ka] Leu [ka] PABC-OH [ka] PABC-NH2

[0055] Here, the wavy lines indicate the positions of the connections to C and D. Preferably, the connection to C is via -NH-.

[0056] Preferably, D is rescimoto, prednisone, triiodothyronine (T3), doxorubicin, daunorubicin, epirubicin, methotrexate. , Ge The compound is selected from mucitabine, cytarabine, melphalan, nimustine, mitoxantrone, mitomycin, camptothecin, 10-hydroxycamptothecin, topotecan, fluorouracil, doxifluridine, etoposide, fludarabine, capecitabine, vincristine, epodilone B, paclitaxel, polyimpaclitaxel, darafenib, dovitinib, compound a, compound b, and a platinum derivative represented by the following formula:

[0057] Here, the compound a and the compound b have the following structures:

[0058] [ka] [ka]

[0059] More preferably, D is selected from daunorubicin, dovitinib, epirubicin, compound a, compound b, mitomycin, darafenib, motesanib, rescimoto, prednisone, and T3. Preferably, the compound (linker) of formula (I) of the present invention for linking to these drugs (D) is selected from the following: 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-109, QHL-200, QHL-201, QHL-202, QHL-203, QHL-204, QHL-205, QHL-207, QHL-208, QHL-209, QHL-300, QHL-301, QHL-302, QHL-303, QHL-304, QHL-305, QHL-307, QHL-308, QHL-309, QHL-401, QHL-402, QHL-403, QHL-404, QHL-405, QHL-406, QHL-407, QHL-408, QHL-409, QHL-501, QHL-502, QHL-503, QHL-504, QHL-505, QHL-506, QHL-507, QHL-508, QHL-509, QHL-601, QHL-602, QHL-603, QHL-6 16, 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.

[0060] Preferably, A is connected to D by any of the following means:

[0061] [ka]

[0062] The wavy lines indicate the connections with adjacent components.

[0063] More preferably, A and D are linked by -CO-NH-, with the carbonyl group being linked to or part of A (e.g., when A is Leu) and the amino group being linked to or part of D. Typically, the position of the drug compound bound to A does not affect the biological activity of the drug, such as being away from the active center of the drug compound.

[0064] Preferably, the pharmaceutical compounds of formula (II) according to the present invention are selected from:

[0065]

Table 7

[0066]

Table 8

[0067]

Table 9

[0068]

Table 10

[0069]

Table 11

[0070]

Table 12

[0071]

Table 13

[0072]

Table 14

[0073]

Table 15

[0074]

Table 16

[0075] In some embodiments of the present invention, the present invention also provides a platinum derivative having the following structure, a precursor thereof, or a pharma- ceutically acceptable salt thereof: [ka]

[0076] The pharmaceutical composition of the present invention can be covalently bound to albumin to form a novel pharmaceutical compound. Therefore, the present invention also includes the pharmaceutical compound of formula (II) of the present invention covalently bound to albumin. Typically, albumin is linked to the MI of the linker. In some embodiments, the present invention also includes EMC-AANL-DOX linked to albumin, its pharmaceutical composition, and its application. Also, the pharmaceutical compound of formula (II) of the present invention or its pharmaceutically acceptable salt is covalently bound to albumin.

[0077] The pharma- ceutically acceptable salts in the present invention may be various pharma- ceutically acceptable salts known in the art, including inorganic and organic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, citrate, lactate, tartrate, maleate, fumarate, amygdalate and oxalate; and inorganic and organic base salts formed with bases such as sodium hydroxide, tris(hydroxymethyl)aminomethane (TRIS, aminobutyrol), and N-methylglutamine.

[0078] III.Preparation method Exemplary methods for the preparation of compounds of formula (I) and (II) of the present invention include: Step 1: Preparation of tripeptide-PABC or tetrapeptide: coupling and isolating amino acid residues to obtain tripeptide-PABC or tetrapeptide, i.e., CA; Step 2: Preparation of MI-S: Select a compound suitable for the MI-S group and carry out condensation or cyclization to obtain MI-S with a carboxyl group at one end; 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); Step 4: The A-terminal carboxyl or hydroxyl activation product of the compound MI-SCA obtained in step 3 is covalently coupled to the amino group of any drug to form a targeted delivery and activated immunostimulatory doxorubicin conjugate.

[0079] When A is PABC-OH, the synthetic route is as follows: an amino acid residue suitable for the present invention is coupled to PABC using known chemical and biological recombinant coupling techniques, followed by purification and isolation to obtain C-PABC containing a suitable amino acid protecting group. The reaction can be carried out in the presence of a condensing agent, a base, and a polar aprotic solvent. The protecting group is then removed to obtain C-PABC. C-PABC can then be reacted with an acid or ester or chloride containing an MI-S group in the presence of a condensing agent, a base, and a polar aprotic solvent to obtain MI-SCA of formula (I) of the present invention, which can then be reacted with a desired drug compound or its salt in the presence of a condensing agent, a base, and a polar aprotic solvent to form a drug compound of formula (II) of the present invention.

[0080] Examples of bases used in this preparation method include organic bases such as triethylamine, pyridine, N,N-diisopropylethylamine, 4-dimethylaminopyridine, 1,2,2,6,6-pentamethylpyridine, and inorganic bases such as sodium carbonate, potassium carbonate, sodium bicarbonate, and potassium bicarbonate. Examples of condensing agents used in this preparation method include HBTU, DMC, HATU, HOBT, DIC, DCC, EDCI, DEPBT, and the like. The solvent used in this preparation method may be any solvent as long as the solvent itself is inert to the reaction and does not inhibit the reaction. Examples of such solvents include halogenated hydrocarbon solvents such as methylene chloride and dichloromethane, aromatic hydrocarbon solvents such as benzene and toluene, aproton transfer solvents such as acetonitrile, N,N-dimethylformamide, and dimethylsulfoxide, ester solvents such as methyl acetate and ethyl acetate, ether solvents such as tetrahydrofuran, and mixtures thereof. The reaction in this preparation method can be carried out at a temperature ranging from ice-cooling to 150°C.

[0081] IV. Pharmaceutical Compositions The present invention comprises a pharmaceutical composition comprising a compound of the invention as set forth in formula (II) or a pharma- ceutically acceptable salt thereof, or a platinum derivative of the invention as described herein or a pharma- ceutically acceptable salt thereof, or a compound of formula (II) or a pharma- ceutically acceptable salt thereof covalently bound to albumin, or EMC-AANL-DOX or a pharma- ceutically acceptable salt thereof covalently bound to albumin.

[0082] The pharmaceutical composition may also include a pharma- ceutically acceptable carrier or excipient, which may be a variety of pharma- ceutically acceptable carriers or excipients known in the art and will vary depending on the dosage form or mode of administration of the drug.

[0083] In specific embodiments, the pharmaceutical composition contains one or more of a solvent, a solubilizer / co-solvent, a pH adjuster, a lyophilization excipient, and an osmolality adjuster.

[0084] Lyophilization excipients suitable for use in the present invention include one or more of sugars (e.g., lactose, maltose, dextrose, glucose, fructose), amino acids (e.g., arginine, lysine, histidine), mannitol, tartaric acid, maleic acid, citric acid, sodium chloride, and cyclodextrins (e.g., hydroxypropyl β-cyclodextrin, sulfobutyl β-cyclodextrin).

[0085] Examples of pH adjusters suitable for use in the present invention include one or more selected from 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, glucosamine, maleic acid, ethylenediamine, triethylamine, arginine, lysine, histidine, sodium dihydrogen phosphate, and disodium hydrogen phosphate.

[0086] The solvent suitable for use in the present invention is preferably an organic solvent consisting of one or more selected from 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.

[0087] Examples of osmotic pressure regulators that can be suitably used in the present invention include one or more of glucose, sodium chloride, mannitol, and sodium lactate.

[0088] Solubilizers / co-solvents suitable for use in the present invention may include one or more of Tween 80, Tween 60, Poloxamer, hydroxypropyl beta cyclodextrin, polyethylene glycol (PEG), lithium dodecahydroxystearate, sulfobutyl beta cyclodextrin, PVP, glycerol, and polyoxyethylene castor oil.

[0089] In general, the compound of the present invention or its pharma- ceutically acceptable salt is orally administered to a mammal daily in an amount usually ranging from about 0.0025 to 50 mg / kg body weight, preferably about 0.01 to 10 mg / kg body weight. When administered together with a known anticancer agent or in combination with other therapies, the dosage must be effective to achieve the intended purpose. The optimal dosage of these known anticancer agents is well known to those skilled in the art.

[0090] A unit oral dose may comprise about 0.01 to 50 mg, preferably about 0.1 to 10 mg, of the compound of the invention or a pharma- ceutically acceptable salt thereof. The unit dose may be administered one or more times daily, with each dose containing about 0.1 to 50 mg, preferably about 0.25 to 10 mg, of the compound of the invention or a pharma- ceutically acceptable salt thereof.

[0091] The pharmaceutical composition of the present invention can be prepared in any suitable dosage form, such as, but not limited to, tablets, capsules, injections, etc. The pharmaceutical composition of the present invention can be administered by any route known in the art, such as orally, intravenously, or intramuscularly.

[0092] V. Uses of the Compounds and Pharmaceutical Compositions Cytokines secreted from tumors convert monocytes into tumor-associated macrophages (TAMs), stimulating strong immunosuppression and directly contributing to tumor cell invasion and metastasis. A confirmatory marker that distinguishes tumor-associated macrophages (M2 type) from monocytes and inflammatory macrophages (M1 type) is the expression of asparaginyl endopeptidase. The compounds of the present invention can be activated for release in the presence of aspartic acid peptide chain endonuclease. Different parts of the specifically activated complex with asparaginyl endopeptidase have a significant impact on the final drug in terms of targeting, activation, stabilization, toxicity and efficacy, so the use of the specifically activated complex with asparaginyl endopeptidase of the present invention can effectively reduce the toxicity of the linked drug, and the final drug can take on new targeting, activation and metabolic properties, enhancing the efficacy of treatment against tumors, and generating new tumor adaptations and anti-tumor metastasis that create completely new structures and functions.

[0093] The present invention also reveals that the compound of formula (II) of the present invention has the ability to kill tumor-associated macrophages, attenuate immunosuppressive cytokines in the microenvironment, and promote immune enhancement of toxic CD8 cells. More importantly, these tumor microenvironment-neutralizing compounds are activated only locally in the tumor, unlike conventional chemotherapy agents that damage the entire immune system. The tumor microenvironment-neutralizing compounds and PD-1 (programmed death-1) inhibitor antibodies (currently, commercially available anti-PD-L1 antibodies are considered to be candidates for immunotherapy effects) have a strong synergistic effect in experiments, which can solve the disadvantage that immunotherapy is difficult to combine with chemotherapy agents.

[0094] Thus, the compounds of the present invention, their pharma- ceutically acceptable salts, or pharmaceutical compositions include those known in the art, such as rescimoto, prednisone, T3, doxorubicin, daunorubicin, epirubicin, methotrexate, fludarabine, gemcitabine, cytarabine, melphalan, nimustine, mitoxantrone, mitomycin, camptothecin, 10-hydroxycamptothecin, topotecan, fluorouracil, doxifluridine, etoposide, fludarabine, capecitabine, vincristine, epodione B, paclitaxel, polyene paclitaxel, darafenib, dovitinib, motesanib, compound a, compound b. platinum and platinum compounds (e.g., carboplatin, cisplatin, oxaliplatin) can treat various diseases, cancer, ophthalmic diseases, and liver diseases.

[0095] For example, camptothecin can be used to treat or prevent malignant tumors, psoriasis, warts, acute / chronic leukemia, and hepatosplenomegaly due to schistosomiasis; 10-hydroxycamptothecin can be used to treat gastric cancer, liver cancer, head and neck cancer, and leukemia; paclitaxel is mainly used for ovarian cancer and breast cancer, and is also effective against lung cancer, colon cancer, melanoma, head and neck cancer, lymphoma, and brain tumors; 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 cancer, ovarian cancer, breast cancer, head and neck tumors, bladder tumors, malignant intrathoracic effusion, etc.

[0096] Thus, for example, diseases that can be treated or prevented with the compounds of the present invention, their pharma- ceutically acceptable salts or pharmaceutical compositions include, but are not limited to, cancers of the bladder, brain, breast, cervix, colorectum, esophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin, stomach, uterus, ovary, testis and blood, specifically selected from the following cancers: liver, kidney, thyroid, colorectum, bladder, brain, breast, cervix, rectum, esophagus, lung (e.g., bronchopulmonary, both undifferentiated small cell and non-small cell), nasopharynx, pancreas, prostate, skin, stomach, uterus, ovary, testis, blood (including, e.g., chronic or acute leukemia, lymphocytic leukemia and granulocytic leukemia), malignant lymphoma, fibrous sarcoma, soft tissue sarcoma, osteogenic sarcoma, rhabdomyosarcoma, Ewing's sarcoma, nephroblastoma, neuroblastoma, thyroid cancer and head and neck squamous cell carcinoma.

[0097] In a specific embodiment, the pharmaceutical compound of the present invention having D as mitomycin represented by formula (II) or a pharma- ceutical acceptable salt thereof can be used for the treatment or prevention of wound healing scars or choroidal neovascularization, or for the treatment or prevention of ophthalmic diseases including the inhibition of macrophages. In another embodiment, the pharmaceutical compound of formula (II) having D as mitomycin or a pharma-ceutical acceptable salt thereof can be used for the treatment or prevention of sequelae of corneal transplantation, glaucoma, pterygium surgery, etc.

[0098] The compounds or pharmaceutical compositions of the present invention can also be used to inhibit tumor metastasis, particularly to inhibit lung metastasis of tumors. In one embodiment, the compounds or pharmaceutical compositions of the present invention can be used to inhibit lung metastasis from breast cancer.

[0099] Liver diseases described in the present invention include, but are not limited to, fatty liver (both alcoholic fatty liver and non-alcoholic fatty liver), steatohepatitis, fatty liver disease, hepatic fibrosis, liver inflammation, and hepatocyte damage phenomenon called steatosis.

[0100] Thus, the present invention relates to a method for treating or preventing a disease (preferably cancer, ophthalmological disease, and liver disease as described in any of the embodiments of the present invention), comprising administering to a subject in need of such treatment or prevention an effective amount of a compound of formula (II) of the present invention or a pharma- ceutical composition comprising a compound of formula (II) of the present invention or a pharma- ceutical salt thereof. In some embodiments, a platinum derivative according to the present invention or the present invention or a pharma- ceutical acceptable salt thereof, or a compound of formula (II) covalently bound to albumin or a pharma- ceutical acceptable salt thereof, or EMC-AANL-DOX covalently bound to albumin or a pharma- ceutical acceptable salt thereof, or their respective pharmaceutical compositions, is administered.

[0101] The present invention also includes a method for stopping tumor metastasis, comprising administering to a subject in need thereof an effective amount of a compound of the present invention or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of the present invention or a pharma- ceutically acceptable salt thereof. Stopping tumor metastasis includes, but is not limited to, stopping lung and / or bone metastasis of a tumor.

[0102] Tumor-associated macrophages (TAMs) play a crucial role in tumor-associated inflammation as key inflammatory cells. In the tumor microenvironment, TAMs promote tumor development by influencing various aspects of tumor biology. They secrete molecules (such as EGF) that directly promote tumor cell proliferation, promote angiogenesis, prepare conditions for cancer cell invasion and metastasis, and inhibit adaptive immune motility. Thus, the present invention also encompasses a method for suppressing tumor-associated macrophages, comprising administering to a desired subject an effective amount of the compound of the present invention or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound of the present invention or a pharma- ceutically acceptable salt thereof. By suppressing tumor-associated macrophages, tumor growth inhibition, angiogenesis, and cancer cell invasion and metastasis can be suppressed, anti-tumor immunity can be promoted, and cancer can be prevented and / or treated. In one specific embodiment, tumor-associated macrophages express M2-type aspartic acid peptide chain endonuclease.

[0103] The above methods of the invention can be used in combination with radiation therapy or immunotherapy known in the art.

[0104] Thus, the present invention also includes the compounds of the present invention, pharma- ceutically acceptable salts thereof, or pharmaceutical compositions of the present invention, for use in the various methods or applications described above.

[0105] The present invention is also directed to the use of the compound of the present invention or a pharma- ceutically acceptable salt thereof, or the pharmaceutical composition of the present invention, in the preparation of a medicament for the treatment or prevention of the above-mentioned diseases, such as cancer and cancer metastasis.The present invention also includes the use of the compound of the present invention or a pharma- ceutically acceptable salt thereof, or the pharmaceutical composition of the present invention, in the preparation of a medicament for inhibiting tumor-associated macrophages, suppressing tumor growth, suppressing angiogenesis, suppressing cancer cell invasion and metastasis, and / or promoting anti-tumor immunity.

[0106] The present invention also provides a method for reducing the toxic effect of an anti-cancer compound, particularly an anti-cancer compound as described herein, said method comprising linking an anti-cancer compound to a linker compound of formula (I) of the present invention.

[0107] The therapeutic or prophylactic 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 administration, intramuscular administration, etc. The subject is a mammal, particularly a human. In some embodiments, the present invention also provides the use of an EMC-AANL-DOX compound having the structure shown in the following formula, or an albumin-bound drug thereof, in the preparation of a medicament for the treatment of hepatocellular carcinoma. [ka]

[0108] In the present invention, "comprises" and "comprises" are understood to include "consisting of" and "consisting of." All weight or volume percentages total 100%. The various reagents and products used in the present embodiments are commercially available unless otherwise specified, and the methods are carried out in accordance with conventional techniques unless otherwise specified. It should be noted that the following embodiments are not intended to limit the scope of the present invention. EXAMPLES

[0109] Example 1: Synthesis of QHL-095-DOX The synthesis of QHL-095-DOX is shown below.

[0110] [ka]

[0111] [ka]

[0112] 1. Synthesis of intermediate 1 Add 500 ml of THF to a dry, clean 2 L reaction flask, weigh out 80 g of Fmoc-Asn(Trt)-OH, add to the flask, stir to dissolve, add 46.6 g of DEPBT, stir at room temperature for 15 minutes, add 16 g of PABC, react at room temperature for 30 minutes, add 45 ml of DIPEA, protect by nitrogen substitution, react at room temperature for 3 hours, and monitor the completion of the reaction by TLC (Fmoc-Asn(Trt)-OH has reacted completely).

[0113] The reaction liquid was removed by evaporation under reduced pressure, a small amount of DMF (180 ml) was added to dissolve the product, and the product was added dropwise to 3 L of water with stirring to precipitate a pale yellow solid. After washing with water 2 to 3 times, the solid was extracted and filtered, collected, and dried in vacuum to obtain an off-white solid (yield 90% or more).

[0114] 2. Synthesis of intermediate 2 The white solid obtained in the previous step was added to a 2L single-necked reaction flask in order to 500ml of THF, stirred and dissolved, cooled to 0-5°C in an ice-salt bath, dropped with 100ml of piperidine, and gradually returned to room temperature after the dropwise addition, reacted for 1 hour, and monitored by TLC. The solvent was removed by vacuum evaporation, a small amount of DMF was added to dissolve, 2L of water was dropped while stirring, mechanically stirred for 30min, extracted and filtered, washed with water 2-3 times in duplicate, extracted and filtered, 800ml of methyl tert-butyl ether was added to the filter cake, stirred for 30min, extracted and filtered, added PE:EA=10:1 to the filter cake, washed twice, extracted and filtered, the filter cake was collected and dried in vacuum to obtain 80g of white solid, with a purity of 70%.

[0115] 3. Synthesis of intermediate 3 In a dry and clean 250ml one-neck reaction flask, add 50ml of nuTHF, 5.04g of Boc-Ala-Ala-OH, and 3.89g of DEPBT in sequence, react at room temperature for 10 minutes, protect with nitrogen substitution, add 2.6g of NH2H2H2-Asn(Trt)-PABC, react at room temperature for 15 minutes, and add 3.5ml of DIPEA dropwise while protecting with nitrogen. React at room temperature for 3 hours, remove the solvent by vacuum evaporation, add water and beat 2-3 times, extract and filter to obtain 3.7g of light yellow solid. Furthermore, 2.0g of product was obtained by column purification (purity 94.8%, yield 26.6%).

[0116] 4. Synthesis of intermediate 4 1.8g of intermediate 3 was placed in a 250ml one-necked reaction flask, 28.5ml of TFA was added, 1.5ml of water was added dropwise, and the mixture was reacted at room temperature for 30 minutes. The reaction was monitored by TLC, and the solvent was evaporated under reduced pressure. Methyl tert-butyl ether was added, and the mixture was beaten, extracted, and filtered to obtain a solid. A 1:1 dioxane:water solution was added to dissolve the mixture, and the pH was adjusted to 13 with 1N sodium hydroxide. The mixture was stirred at room temperature for 40 minutes, and then the mixture dissolved in sodium hydroxide was added. The solvent was removed by evaporation under reduced pressure, silica gel was added, and the mixture was passed through a column to obtain 450mg of the product in a yield of 47.5%.

[0117] 5. Synthesis of MI-S intermediate MI-S1 (338 mg, 2 mmol) and DEPBT (717.6 mg, 2.4 mmol) were added to a 100 ml one-neck flask, DMF (15 ml) was added to dissolve, the mixture was protected by nitrogen substitution, and the mixture was reacted at room temperature for 15 minutes. R3-b (819 mg, 2 mmol) was added to dissolve the mixture, the mixture was reacted at room temperature for 15 minutes, and DIPEA was added dropwise. 137 μl was added, and the mixture was reacted at room temperature for 3 hours while protecting the mixture by nitrogen substitution. The reaction of R3-a was monitored by TLC, the solvent was removed by vacuum distillation, and the crude product was dissolved in methanol and passed through a reverse phase high pressure column to obtain the intermediate R3-1 (720 mg, yield: 64.3%).

[0118] 6. Synthesis of MI-S The intermediate (720 mg, 1.28 mmol) obtained in the above step was added to a 100 ml one-neck flask, dissolved in 15 ml of dichloromethane, and then 5 ml of TFA and 0.25 ml of water were added dropwise to react at room temperature for 30 minutes, and the reaction was monitored by TLC until completion. The solvent was removed by evaporation under reduced pressure, and methyl tert-butyl ether was added and the mixture was beaten, extracted and filtered, and solid silica gel was added and passed through a reverse phase column to obtain 242 mg of the product. The yield was 37.5%.

[0119] 7. Synthesis of intermediate 5 Intermediate 4 (150 mg, 0.395 mmol) and EMC-6Peg-COOH (239 mg, 0.474 mmol) were added to a 100 ml one-neck flask, and DMF (15 ml) was added to dissolve the mixture while protecting the mixture with nitrogen. After reacting for 15 minutes at room temperature, 137 μl of DIPEA was added dropwise while protecting the mixture with nitrogen, and the reaction was carried out at room temperature for 3 hours. The reaction was monitored by TLC. Intermediate 4 was reacted, and the solvent was removed by distillation under reduced pressure. The crude product was dissolved in methanol and passed through a reversed-phase high-pressure column to obtain intermediate 5 (95 mg, yield: 21%).

[0120] 8. Synthesis of intermediate 6 Sequentially, 25 ml of DMF, intermediate 5 (300 mg, 0.346 mmol), and Bis-PNP (316 mg, 1.04 mmol) were added to a 100 ml one-necked reaction flask, and the mixture was reacted at room temperature for 15 minutes while protected by nitrogen substitution. Then, 258 μl of DIPEA was added dropwise, and the mixture was reacted at room temperature for 3 hours while protected by nitrogen substitution. The reaction was terminated at the timing when 7% of the raw material remained, and the solvent was removed by evaporation under reduced pressure. 150 mg of the product was obtained by column purification in a yield of 42%.

[0121] 9. Synthesis of the final product QHL-095-DOX In a 100mL reaction flask, 84mg (1.0eq, 0.145mmol) of doxorubicin hydrochloride and 150mg (1.0eq, 0.145mmol) of intermediate 6 were added, and the mixture was reacted at room temperature for 15 minutes under nitrogen protection. Then, 75μl of DIPEA was added dropwise, and the mixture was reacted at room temperature for 4 hours. The solvent was removed by evaporation under reduced pressure, and the crude product was dissolved in methanol and passed through a reversed-phase high-pressure column to obtain QHL-095-DOX (49mg red solid, yield 23.8%).

[0122] Example 2: Synthesis of QHL-116-DOX

[0123] [ka]

[0124] [ka]

[0125] 1. Synthesis of intermediate 1 Add 500 ml of THF to a dry, clean 2 L reaction flask, weigh out 80 g of Fmoc-Asn(Trt)-OH, add to the flask, stir to dissolve, add 46.6 g of DEPBT, stir at room temperature for 15 minutes, add 16 g of PABC, react at room temperature for 30 minutes, add 45 ml of DIPEA, protect by nitrogen substitution, react at room temperature for 3 hours, and monitor the completion of the reaction by TLC (Fmoc-Asn(Trt)-OH has reacted completely).

[0126] The reaction liquid was removed by evaporation under reduced pressure, a small amount of DMF (180 ml) was added to dissolve the product, and the product was added dropwise to 3 L of water with stirring to precipitate a pale yellow solid. After washing with water 2 to 3 times, the solid was extracted and filtered, collected, and dried in vacuum to obtain an off-white solid (yield 90% or more).

[0127] 2. Synthesis of intermediate 2 In a 2L one-necked reaction flask, add 500ml of THF to the white solid obtained in the previous step, dissolve by stirring, cool to 0-5°C in an ice-salt bath, add 100ml of piperidine, gradually return to room temperature after the dropwise addition, react for 1 hour, and monitor by TLC. Remove the solvent by vacuum evaporation, add a small amount of DMF to dissolve, drop into 2L of water while stirring, mechanically stir for 30min, extract and filter, wash with water 2-3 times in duplicate, extract and filter, add 800ml of methyl tert-butyl ether to the filter cake, stir for 30min, extract and filter, add PE:EA=10:1 to the filter cake, wash twice, extract and filter, collect the filter cake, and vacuum dry to obtain 80g of white solid, with a purity of 70%.

[0128] 3. Synthesis of intermediate 3 In a dry and clean 250ml one-neck reaction flask, add 50ml of nuTHF, 5.04g of Boc-Ala-Ala-OH, and 3.89g of DEPBT in sequence, react at room temperature for 10 minutes, protect with nitrogen substitution, add 2.6g of NH2H2H2-Asn(Trt)-PABC, react at room temperature for 15 minutes, and add 3.5ml of DIPEA dropwise while protecting with nitrogen. React at room temperature for 3 hours, remove the solvent by vacuum evaporation, add water and beat 2-3 times, extract and filter to obtain 3.7g of light yellow solid. Furthermore, 2.0g of product was obtained by column purification (purity 94.8%, yield 26.6%).

[0129] 4. Synthesis of intermediate 4 1.8g of intermediate 3 was placed in a 250ml one-necked reaction flask, 28.5ml of TFA was added, 1.5ml of water was added dropwise, and the mixture was reacted at room temperature for 30 minutes. The reaction was monitored by TLC, and the solvent was evaporated under reduced pressure. Methyl tert-butyl ether was added, and the mixture was beaten, extracted, and filtered to obtain a solid. A 1:1 dioxane:water solution was added to dissolve the mixture, and the pH was adjusted to 13 with 1N sodium hydroxide. The mixture was stirred at room temperature for 40 minutes, and then the mixture dissolved in sodium hydroxide was added. The solvent was removed by evaporation under reduced pressure, silica gel was added, and the mixture was passed through a column to obtain 450mg of the product in a yield of 47.5%.

[0130] 5. Synthesis of intermediate 5 Weigh out Fmoc-Glu(OAll)-COOH (1.554g, 3.79mmol), add 10ml of a mixture of DCM and THF to dissolve, and add 2.72ml of HOtBu dropwise while stirring. Protect with N2 substitution, react at room temperature for 16 hours, and monitor the reaction by TLC. Remove the solvent by evaporation under reduced pressure, add silica gel and pass through a column to obtain 1.4g of product in 79.5% yield.

[0131] 6. Synthesis of intermediate 6 In a dry, clean 250ml one-necked reaction flask, add 10ml of THF and intermediate 5 (1.4g, 3mmol) obtained in the previous step, stir to dissolve, cool to 0-5℃ in an ice-salt bath, add 3ml of piperidine dropwise, gradually warm to room temperature, react for 2 hours, and monitor the reaction by TLC. Remove the solvent by evaporation under reduced pressure, purify with silica gel, collect the eluent containing the product, and dry to constant weight under reduced pressure to obtain 583mg of product in 80% yield.

[0132] 7. Synthesis of intermediate 7 In a dry and clean 250ml one-neck reaction flask, add 15ml THF, 583mg intermediate 6, and 932.8mg DEPBT in sequence, react at room temperature for 10 minutes, add 506.4mg maleimidohexanoic acid, protect with nitrogen substitution, react at room temperature for 15 minutes, add 1.3ml DIPEA dropwise, protect with nitrogen substitution, react at room temperature for 3 hours, remove the solvent by vacuum evaporation, add water and beat 2-3 times, extract and filter to obtain 800mg of light yellow solid. Further, obtain 628mg of product by column purification (purity 94.8%, yield 59.9%).

[0133] 8. Synthesis of intermediate 8 In a dry, clean 100ml one-necked reaction flask, add 10ml of dichloromethane and 872mg of intermediate 7 in sequence, stir evenly, add 3ml of TFA dropwise, react at room temperature for 2 hours, and monitor the reaction by TLC until completion. Remove the solvent by vacuum evaporation, add methyl tert-butyl ether to beat, extract, filter and obtain a solid. Add silica gel to purify, collect the eluent containing the product, and dry under vacuum to a constant weight to obtain 459mg of product, yield 60.3%.

[0134] 9. Synthesis of intermediate 9 In a dry and clean 250ml one-necked reaction flask, add 15ml THF, 459mg intermediate 8, and 434mg DEPBT in sequence, react at room temperature for 10 minutes, add 457.8mg intermediate 4, protect with nitrogen substitution, react at room temperature for another 15 minutes, add 627ml DIPEA dropwise, and react at room temperature for 3 hours while protecting with nitrogen substitution, remove the solvent by evaporation under reduced pressure, add water and beat 2-3 times, extract and filter to obtain 750mg of light yellow solid. Column purification gave 655mg of product with a yield of 63.2%.

[0135] 10. Synthesis of intermediate 10 Sequentially, 25 ml of DMF, intermediate 9 (655 mg, 0.88 mmol), and Bis-PNP (804 mg, 2.64 mmol) were added to a 100 ml one-necked reaction flask, and the mixture was reacted at room temperature for 15 minutes while protected by nitrogen substitution. Then, 258 μl of DIPEA was added dropwise, and the mixture was reacted at room temperature for 3 hours while protected by nitrogen substitution. The reaction was terminated at the timing when 7% of the raw material remained, and the solvent was removed by evaporation under reduced pressure. 335 mg of the product was obtained by column purification in a yield of 42%.

[0136] 11. Synthesis of intermediate 11 In a 100mL reaction flask, 214.3mg (1.0eq, 0.369mmol) of doxorubicin hydrochloride and 335mg (1.0eq, 0.369mmol) of intermediate 10 were added and reacted at room temperature for 15 minutes under nitrogen protection. 190μl of DIPEA was added dropwise and reacted at room temperature for 4 hours. The solvent was removed by evaporation under reduced pressure, and the crude product was dissolved in methanol and passed through a reverse phase high pressure column to obtain intermediate 11 (115mg red solid, yield: 23.8%).

[0137] 12. Synthesis of the final product In a 100 mL single-necked reaction flask, 15 mL of THF, intermediate 11 (115 mg, 0.0877 mmol), and tri-n-butyltin hydrogen (76 mg, 0.2631 mmol) were added in that order, and the reaction solution was aerated with nitrogen. Protect Tetrakis(triphenylphosphine)palladium(0) (14.2 mg, 0.012 mmol) was then added and the mixture was stirred at room temperature overnight. The mixture was monitored by TLC until the conversion was complete. The flask contents were then filtered through diatomaceous earth and the residue was washed with THF. The filtrate was concentrated under reduced pressure. The resulting crude product was purified by column to give 100 mg of the desired compound (yield: 90%).

[0138] Example 3: Synthesis of N-CBP

[0139] [ka]

[0140] 1. Synthesis of intermediate 1 300mg of raw material was added to a 100ml three-neck flask, dissolved in 15ml of THF / ETOH (4:1), cooled to -5℃~0℃ in an ice-salt bath, and 210mg of LiOH (5ml) aqueous solution was added dropwise to the temperature-controlled flask under stirring, and naturally warmed for 1 hour after the addition. The reaction solution was sent to HPLC to completely react the raw material, and the temperature was controlled to -5℃~0℃, and the pH of the reaction solution was adjusted to 3-4 with 1mol / LHCl. The pH of the reaction solution was adjusted to 3-4, and the solvent was removed at a temperature of 25-30℃ to obtain the crude intermediate 1, which was used directly in the next step.

[0141] 2. Synthesis of intermediate 2 Add 15ml of 1mol / L dioxane hydrochloride solution to intermediate 1 and stir at room temperature for 1 hour. Then, send the reaction solution to HPLC to complete the reaction of intermediate 1. Remove the solvent at 25-30°C to obtain the crude product of intermediate 2, which can be used for the next step.

[0142] 3. Synthesis of intermediate 3 Place the crude intermediate 2 in a 100ml three-neck flask, add 20ml of dioxane, cool to -5 to 0°C in an ice-salt bath, add 159mg of sodium carbonate aqueous solution (pH about 8) dropwise to the flask while controlling the temperature under nitrogen protection, add 311mg of Fmoc-Cl solution in dioxane dropwise, warm naturally for 1 hour, and detect by HPLC. Intermediate 2 reacted completely, remove the solvent, add silica gel, and pass through a reverse-phase medium pressure column to obtain 107mg of intermediate 3.

[0143] 4. Synthesis of intermediate 4 Add 107 mg of intermediate 3 to a 50 ml single-neck flask, dissolve in 15 ml of methanol, cool to -20°C with liquid nitrogen, add 302 ul of tetrabutylammonium hydroxide (25% methanol solution) dropwise to the flask, and then warm naturally for 1 hour. This reaction solution is called preliminary solution 1.

[0144] 140.8 mg of cis-diamminedichloroplatinum (CDDP) was added to a 50 ml single-neck flask, dissolved in 10 ml of ultrapure water, and heated to 50 ° C. Shielded from light, 49.5 mg of silver nitrate aqueous solution was added dropwise to the flask under nitrogen protection. After reacting for 15 minutes, 49.5 mg of silver nitrate aqueous solution was added dropwise, and after reacting for another 15 minutes, the reaction solution was membrane filtered, the filtrate was transferred to a 100 ml single-neck flask, and spare solution 1 was added dropwise to it at room temperature, and nitrogen replacement was performed three times. The resulting reaction solution was transferred to an oil bath, heated to 50 ° C, and left overnight (usually 16 hours) in the dark. The reaction was stopped, the reaction solution was centrifuged, the supernatant was directly passed through a high-pressure reverse phase column, and the preparation solution was freeze-dried to obtain 79 mg of intermediate 4 in a yield of 45.7%.

[0145] 5. Synthesis of N-CBP 5 mg of intermediate 4 was placed in a 10 ml single-neck flask, 2 ml MeOH / ACN (1:1) was added and dissolved by stirring, 2 ul DBU was added dropwise to the reaction solution at room temperature, and the mixture was protected with nitrogen for 30 minutes and detected by HPLC. When intermediate 4 was completely reacted, the reaction solution was added dropwise to 6 ml methyl tert-butyl ether to precipitate a whitish solid, which was centrifuged to remove the supernatant, and the solid was dissolved in water / tert-butyl alcohol and passed through a column to obtain 1.8 mg of N-CBP.

[0146] Example 4: Synthesis of QHL-140-N-CBP

[0147] [ka]

[0148] 1. Synthesis of intermediate 1 Put 500 mg of the raw material into a 100 ml three-neck flask, add 10 ml of DCM to dissolve, cool to -5°C to 0°C, add 5 ml of TFA dropwise while stirring, react for 1 hour, monitor the complete reaction of the raw material by HPLC, remove the solvent from the reaction solution, and the remaining oily substance is intermediate 1.

[0149] 2. Synthesis of intermediate 2 Intermediate 1 and 1.15g of the raw material Fmoc-AAN-PABC-PNP were added to a 100ml one-neck flask, dissolved in 20ml of DMF, protected with nitrogen, and activated under stirring for 10 minutes. 0.87ml of DIPEA was then added dropwise to the reaction flask, reacted for 0.5 hours, and detected by HPLC. DMF was removed from the reaction solution, and the crude product was dissolved in water / DMF and passed through a high-pressure reverse phase column to obtain 975mg of intermediate 2, with a yield of 78.6%.

[0150] 3. Synthesis of intermediate 3 Add 400mg of intermediate 2 to a 250ml three-neck flask, add 35ml of THF / ETOH (4:1) to dissolve, cool to -5℃~0℃ in an ice-salt bath, control the temperature to -5℃~0℃, add 202mg of LiOH aqueous solution dropwise, react for 3 hours under temperature control, send to HPLC for monitoring, intermediate 2 reacts completely, control the temperature to -5℃~0℃, and control the reaction solution pH 6~7 with 1mol / L. Adjust the reaction solution to pH 6-7 with 1mol / L HCL, and remove the solvent at 25℃-30℃. The crude product was triturated twice with methyl tert-butyl ether, and the solid was dissolved in methanol / water and passed through a high-pressure reverse phase column to obtain 230mg of intermediate 3 with a yield of 86.7%.

[0151] 4. Synthesis of intermediate 4 235 mg of intermediate 3 and 222 mg of EMC-OSU were added to a 100 ml single-neck flask, 30 ml of DMF was added and stirred to dissolve, heated to 50°C and purged with nitrogen overnight (usually 16 hours), and then sent to HPLC for detection. The reaction of intermediate 3 was completed, and the DMF was removed and the crude product was dissolved in methanol / water and passed through a high-pressure reverse phase column to obtain 200 mg of intermediate 4 in a yield of 53.6%.

[0152] 5. Synthesis of the Final Product QHL-140-N-CBP Add 200 mg of intermediate 4 to a 100 ml single-neck flask, dissolve in 20 ml of methanol, cool to -20°C with liquid nitrogen, add 279 ul of tetrabutylammonium hydroxide (25% methanol solution) dropwise to the flask, and then warm naturally for 1 hour. This reaction solution is called preliminary solution 1.

[0153] 130 mg of cis-diamminedichloroplatinum (CDDP) was added to a 100 ml single-neck flask, dissolved in 30 ml of ultrapure water, and heated to 50 ° C. Shielded from light, 46 mg of silver nitrate aqueous solution was added dropwise to the flask under nitrogen protection. After 15 minutes of reaction, 46 mg of silver nitrate aqueous solution was added dropwise, and after another 15 minutes of reaction, the reaction solution was membrane filtered, the filtrate was transferred to a 250 ml single-neck flask, and spare solution 1 was added dropwise to it at room temperature, and nitrogen replacement was performed three times. The resulting reaction solution was transferred to an oil bath, heated to 50 ° C, and left overnight (usually 16 hours) in the dark. The reaction solution was centrifuged, the supernatant was passed directly through a high-pressure reverse phase column, and the preparation was freeze-dried to obtain the product QHL-140-N-CBP, 90 mg, in a yield of 34.5%.

[0154] Example 5: Synthesis of QHL-086-N-CBP

[0155] [ka]

[0156] 1. Synthesis of intermediate 1 Put 500 mg of the raw material into a 100 ml three-neck flask, add 10 ml of DCM to dissolve, cool to -5°C to 0°C, add 5 ml of TFA dropwise while stirring, react for 1 hour, monitor the complete reaction of the raw material by HPLC, remove the solvent from the reaction solution, and the remaining oily substance is intermediate 1.

[0157] 2. Synthesis of intermediate 2 Intermediate 1 and 1.15g of the raw material Fmoc-AAN-PABC-PNP were added to a 100ml single-neck flask, dissolved in 20ml DMF, protected with nitrogen, and activated with stirring for 10min. 0.87ml DIPEA was added dropwise to the reaction flask, reacted for 0.5h, and detected by HPLC. The raw material Fmoc-AAN-PABC-PNP was completely reacted. The DMF in the reaction solution was removed, and the crude product was dissolved in water / DMF and then passed through a high-pressure reverse phase column to obtain 975mg of intermediate 2 with a yield of 78.6%.

[0158] 3. Synthesis of intermediate 3 Add 400mg of intermediate 2 to a 250ml three-neck flask, add 35ml of THF / ETOH (4:1) to dissolve, cool to -5℃~0℃ in an ice-salt bath, control the temperature to -5℃~0℃, add 202mg of LiOH aqueous solution dropwise, react for 3 hours under temperature control, send to HPLC for monitoring, intermediate 2 reacts completely, control the temperature to -5℃~0℃, and control the reaction solution pH 6~7 with 1mol / L. Adjust the reaction solution to pH 6-7 with 1mol / L HCL, and remove the solvent at 25℃-30℃. The crude product was triturated twice with methyl tert-butyl ether, and the solid was dissolved in methanol / water and passed through a high-pressure reverse phase column to obtain 235mg of intermediate 3 with a yield of 88.6%.

[0159] 4. Synthesis of intermediate 4 89mg EMC-2Peg-OH was added to a 100ml single-neck flask, DMF was dissolved, 97mg DEPBT was added, and the mixture was stirred at room temperature for 1 hour to activate the product. 95ul DEPBT was then added dropwise to the flask, stirring was continued for 1 hour, and then 150mg DMF solution of intermediate 3 was added dropwise. After addition, the mixture was stirred at room temperature and detected by HPLC. After the reaction, the DMF was spun off, and the crude product was dissolved in water / methanol and passed through a high-pressure reverse phase column, yielding 88mg of the product in a yield of 37.6%.

[0160] 5. Synthesis of the Final Product QHL-086-N-CBP Add 88 mg of intermediate 4 to a 50 ml single-neck flask, dissolve in 10 ml of methanol, cool to -20°C with liquid nitrogen, add 106 ul of tetrabutylammonium hydroxide (25% methanol solution) dropwise to the flask, and then warm naturally for 1 hour. This reaction solution is called preliminary solution 1.

[0161] Add 49 mg of cis-diamminedichloroplatinum (CDDP) to a 50 ml single-neck flask, dissolve in 10 ml of ultrapure water, and heat to 50 ° C. Under the protection of nitrogen, add 17 mg of silver nitrate solution dropwise to the flask in the dark. After reacting for 15 minutes, add 17 mg of silver nitrate solution dropwise, and react for another 15 minutes, then filter the reaction solution through a membrane, transfer the filtrate to a 100 ml single-neck flask, add spare solution 1 dropwise to it at room temperature, and perform nitrogen replacement three times. The resulting reaction solution was transferred to an oil bath, heated to 50 ° C, and left overnight (usually 16 hours) in the dark. HPLC method was performed, and about 20% of intermediate 4 did not react completely. The reaction was stopped, the reaction solution was centrifuged, and the supernatant was directly passed through a high-pressure reverse phase column, and the preparation was freeze-dried to obtain the product QHL-086-N-CBP, 54 mg, in a yield of 48.6%.

[0162] Example 6: Synthesis of QHL-095-N-CBP

[0163] [ka]

[0164] 1. Synthesis of intermediate 1 Put 500 mg of the raw material into a 100 ml three-neck flask, add 10 ml of DCM to dissolve, cool to -5°C to 0°C, add 5 ml of TFA dropwise while stirring, react for 1 hour, monitor the complete reaction of the raw material by HPLC, remove the solvent from the reaction solution, and the remaining oily substance is intermediate 1.

[0165] 2. Synthesis of intermediate 2 Intermediate 1 and 1.15 g of the raw material Fmoc-AAN-PABC-PNP were added to a 100 ml one-neck flask, dissolved in 20 ml of DMF, protected with nitrogen, and activated under stirring for 10 minutes. Then, 0.87 ml of DIPEA was added dropwise to the reaction vessel and reacted for 0.5 hours, and detection was performed by HPLC. The raw material Fmoc-AN-PABC-PNP reaction was completed, and DMF was removed from the reaction solution. 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%.

[0166] 3. Synthesis of intermediate 3 Add 400mg of intermediate 2 to a 250ml three-neck flask, add 35ml of THF / ETOH (4:1) to dissolve, cool to -5℃~0℃ in an ice-salt bath, control the temperature to -5℃~0℃, add 202mg of LiOH aqueous solution dropwise, react for 3 hours under temperature control, send to HPLC for monitoring, intermediate 2 reacts completely, control the temperature to -5℃~0℃, and control the reaction solution pH 6~7 with 1mol / L. Adjust the reaction solution to pH 6-7 with 1mol / L HCL, and remove the solvent at 25℃-30℃. The crude product was triturated twice with methyl tert-butyl ether, and the solid was dissolved in methanol / water and passed through a high-pressure reverse phase column to obtain 235mg of intermediate 3 with a yield of 88.6%.

[0167] 4. Synthesis of intermediate 4 180mg of intermediate 3 and 240mg of EMC-6Peg-OSU were placed in a 100ml single-neck flask, 20ml of DMF was added, stirred and dissolved, heated to 50℃, protected with nitrogen overnight (usually 16 hours), and detected by HPLC. Intermediate 3 was completely reacted, DMF was removed, and the crude product was dissolved in methanol / water and passed through a high-pressure reverse phase column to obtain 234mg of intermediate 4 in a yield of 69.2%.

[0168] 5. Synthesis of the Final Product QHL-095-N-CBP Add 234 mg of intermediate 4 to a 100 ml single-neck flask, dissolve in 15 ml of methanol, cool to -20°C with liquid nitrogen, add 234 ul of tetrabutylammonium hydroxide (25% methanol solution) dropwise to the flask, and then warm naturally for 1 hour. This reaction solution is called preliminary solution 1.

[0169] 109 mg of cis-diamminedichloroplatinum (CDDP) was added to a 100 ml single-neck flask, dissolved in 20 ml of ultrapure water, and heated to 50 ° C. 38 mg of silver nitrate aqueous solution was added dropwise to the flask under nitrogen protection in the dark. After reacting for 15 minutes, 38 mg of silver nitrate aqueous solution was added dropwise, and after reacting for another 15 minutes, the reaction solution was membrane filtered, the filtrate was transferred to a 250 ml single-neck flask, and spare solution 1 was added dropwise to it at room temperature, and nitrogen replacement was performed three times. The resulting reaction solution was transferred to an oil bath, heated to 50 ° C, and left overnight (usually 16 hours) in the dark. The reaction solution was centrifuged, the supernatant was passed directly through a high-pressure reverse phase column, and the preparation was freeze-dried to obtain the final product, 138 mg, with a yield of 48%.

[0170] Example 7: Synthesis of QHL-006-DOX The synthetic route for the MI-S group of QHL-006 is shown below.

[0171] [ka]

[0172] 1. Synthesis of MI-S intermediate-1 of QHL-006-DOX Maleic anhydride (245 mg, 2.5 mmol) was weighed out into a dry, clean 100 ml single-neck reaction flask, 10 ml of dichloromethane was added, and the mixture was stirred to dissolve. NH2H2H2-3Peg-COOtBu (624 mg, 2.25 mmol) was weighed out and reacted at room temperature for 6 hours. Completion of the maleic anhydride reaction was monitored by LC-MS, and the reaction solution was spin-dried, silica gel was added, and the mixture was passed through a column to obtain MI-S intermediate-1 (456 mg, yield 48.6%).

[0173] 2. Synthesis of MI-S intermediate-2 of QHL-006-DOX Add 456 mg of MI-S intermediate-1 obtained by the above procedure to a 100 ml one-necked reaction flask, add 10 ml of acetic anhydride and stir to dissolve, weigh out NaOAC (98.7 mg, 1.216 mmol) and slowly add it all at once, heat to 110 ° C in an oil bath and react for 3 hours, monitor the reaction of MI-S intermediate-1 with LC-MS, cool to room temperature, spin-dry the reaction solution, and perform column purification. The reaction solution was cooled to room temperature, spun off, and purified with a column to obtain MI-S intermediate-2 (312, yield 70%).

[0174] 3. Synthesis of MI-S contained in QHL-006-DOX The MI-S intermediate-2 (312 mg, 0.87 mmol) obtained in the previous step was added to a 100 ml one-necked reaction flask, 10 ml of dichloromethane was added to dissolve it, 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, and the reaction was monitored by TLC. The solvent was removed by evaporation under reduced pressure, methyl tert-butyl ether was added to beat, and the mixture was extracted and filtered, and the solid, silica gel, was added and passed through a reverse phase column to obtain 196 mg of the product. The yield was 75%.

[0175] The final product was prepared in a similar manner to that used for the synthesis of QHL-095-DOX, using a different MI-S for linkage (see the preparation procedure of MI-S for QHL-006-DOX for the preparation of MI-S).

[0176] Example 8: Synthesis of QHL-096-DOX

[0177] [ka]

[0178] 1) Synthesis of intermediate 1 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 with stirring. After 1 hour of stirring, the temperature was lowered to 0°C in an ice bath and L-alanine methyl ester (46.2 g, 0.45 mol) and N,N-diisopropyl ether were added. Add dropwise a solution of ethylamine (173.8g, 1.34mol) in N,N-dimethylformamide (1L), and after completion of addition, stir at room temperature for 10 hours, remove the solvent by evaporation under reduced pressure, dissolve the crude product in dichloromethane (2L), wash successively with saturated ammonium chloride solution, water and saturated sodium chloride solution, dry the organic phase with anhydrous sodium sulfate, remove the solvent by evaporation under reduced pressure, and then recrystallize the crude product with ethyl acetate / petroleum ether to obtain intermediate 1 (101g white solid, yield: 73.1%).

[0179] 2) Synthesis of intermediate 2 Intermediate 1 (100g, 0.34mol) was dissolved in a mixture of tetrahydrofuran (2L) and water (1L), cooled to 0℃, 1mol / L lithium hydroxide solution (400mL) was added dropwise, stirred for 10 hours, neutralized with concentrated hydrochloric acid to pH<6, evaporated under reduced pressure to remove tetrahydrofuran, the remaining aqueous phase was extracted with dichloromethane (1L×3), and the organic phase was dried over anhydrous sodium sulfate. The organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure to obtain intermediate 2 (88g white solid, yield: 92.2%).

[0180] 3) Synthesis of intermediate 3 In a three-necked flask, L-leucine tert-butyl ester (22.4 g, 0.1 mol) and N-Fmoc-N'-triphenylmethylaspartic acid (59.6 g, 0.1 mol) were dissolved in N,N-dimethylformamide (1000 mL), and 1-hydroxybenzotriazole (14.85 g, 0.11 mol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (23 g, 0.12 mol) were added with stirring. After cooling in an ice bath at 0°C, the N,N-diisopropyl Propylethylamine (25.8 g, 0.2 mol) was added, and the mixture was stirred for 10 hours. The solvent was then removed by evaporation under reduced pressure. The crude product was dissolved in chloroform (1000 ml) and washed successively with saturated ammonium chloride solution, saturated sodium chloride solution and water. The organic phase was dried over anhydrous sodium sulfate and filtered. The crude product obtained by removing the solvent by evaporation under reduced pressure was purified by recrystallization (calculated by volume ratio: dichloromethane: ethyl acetate = 1: 1) to obtain intermediate 3 (42.4 g, white solid, yield: 55.4%).

[0181] 4) Synthesis of intermediate 4 Intermediate 3 (7.65 g, 0.01 mol) was dissolved in a mixture of dichloromethane (100 mL) and N,N-dimethylformamide (100 mL), piperidine (40 ml) was added, and the mixture was stirred at room temperature for 5 hours. The solvent was then evaporated under reduced pressure, and then the mixture was dried under high vacuum in a vacuum oven to remove a small amount of piperidine to give intermediate 4 as a light yellow solid, which was used in the next step without purification.

[0182] 5) Synthesis of intermediate 5 The crude product of intermediate 4 obtained in the previous step was dissolved in N,N-dimethylformamide (200 mL), intermediate 2 (2.94 g, 0.012 mol), benzotriazole-N,N,N',N'-tetramethyluroniumhexafluorophosphate (HBTU) (6.07 g, 0.016 mol) were added, and after ice bath, N,N-disopropylethylamine (2.6 g, 0.02 mol) was added to 0 ° C. 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 evaporated, and the obtained crude product was subjected to silica gel column chromatography to obtain intermediate 5 (3.1 g white solid, total yield in two steps: 37.8%).

[0183] 6) Synthesis of intermediate 6 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 gas was introduced, and the mixture was stirred at room temperature and normal pressure for 4 hours. The palladium carbon was removed by filtration and washed with methanol. The filtrate and washings were combined, and the solvent was removed by evaporation under reduced pressure to obtain intermediate 6 (2.38 g white solid, yield: 95.2%).

[0184] 7) Synthesis of intermediate 7 Intermediate 6 (2.38 g, 3.4 mmol) and EMC-6Peg-OSu (2.4 g, 4.08 mmol) were added to a 250 ml single-neck flask, dissolved in DMF (30 ml), and reacted for 6 hours by heating to 50°C. The solvent was removed by distillation under reduced pressure, and the crude product was dissolved in methanol and passed through a reversed-phase high-pressure column to obtain intermediate 7 (2.5 g, yield: 63.2%).

[0185] 8) Synthesis of intermediate 8 Intermediate 7 (1.00g, 0.852mmol) was dissolved in DCM (20mL), (10ml) was added dropwise at room temperature, the reaction solution was stirred for 2 hours, and the reaction solution was sent to HPLC for monitoring. Intermediate 1 was completely reacted, and the solvent was removed by distillation under reduced pressure. After the crude product was washed twice with methyl tert-butyl ether, the solid was dissolved in methanol and passed through a reversed-phase high-pressure column to obtain intermediate 8 (721mg white solid, yield: 96.8%).

[0186] 9) Synthesis of the final product QHL-096-DOX In a 100mL reaction flask, 63mg (1.0eq) of doxorubicin hydrochloride, 895mg (1eq) of intermediate, 39mg (1.2eq) of DEPBT and 10mL of DMF were added, and under nitrogen protection, 60ul (3eq) of DIPEA was added to the reaction mixture and reacted at room temperature for 4 hours. The solvent was evaporated under reduced pressure, and the crude product was dissolved in methanol, and the reversed phase crude product was dissolved in methanol and passed through a reversed phase column to obtain QHL-096-DOX (52mg red solid, yield: 34.2%).

[0188] Example 9: Synthesis of QHL-117-DOX The synthetic route of QHL-117 is as follows.

[0189] [ka]

[0190] [ka]

[0191] 1) Synthesis of intermediate 1 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 with stirring. After 1 hour of stirring, the temperature was lowered to 0°C in an ice bath and L-alanine methyl ester (46.2 g, 0.45 mol) and N,N-diisopropyl ether were added. Add dropwise a solution of ethylamine (173.8g, 1.34mol) in N,N-dimethylformamide (1L), and after completion of addition, stir at room temperature for 10 hours, remove the solvent by evaporation under reduced pressure, dissolve the crude product in dichloromethane (2L), wash successively with saturated ammonium chloride solution, water and saturated sodium chloride solution, dry the organic phase with anhydrous sodium sulfate, remove the solvent by evaporation under reduced pressure, and then recrystallize the crude product with ethyl acetate / petroleum ether to obtain intermediate 1 (101g white solid, yield: 73.1%).

[0192] 2) Synthesis of intermediate 2 Intermediate 1 (100g, 0.34mol) was dissolved in a mixture of tetrahydrofuran (2L) and water (1L), cooled to 0℃, 1mol / L lithium hydroxide solution (400mL) was added dropwise, stirred for 10 hours, neutralized with concentrated hydrochloric acid to pH<6, evaporated under reduced pressure to remove tetrahydrofuran, the remaining aqueous phase was extracted with dichloromethane (1L×3), and the organic phase was dried over anhydrous sodium sulfate. The organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure to obtain intermediate 2 (88g white solid, yield: 92.2%).

[0193] 3) Synthesis of intermediate 3 In a three-necked flask, L-leucine tert-butyl ester (22.4 g, 0.1 mol) and N-Fmoc-N'-triphenylmethylaspartic acid (59.6 g, 0.1 mol) were dissolved in N,N-dimethylformamide (1000 mL), and 1-hydroxybenzotriazole (14.85 g, 0.11 mol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (23 g, 0.12 mol) were added with stirring. After cooling in an ice bath at 0°C, the N,N-diisopropyl Propylethylamine (25.8 g, 0.2 mol) was added, and the mixture was stirred for 10 hours. The solvent was then removed by evaporation under reduced pressure. The crude product was dissolved in chloroform (1000 ml) and washed successively with saturated ammonium chloride solution, saturated sodium chloride solution and water. The organic phase was dried over anhydrous sodium sulfate and filtered. The crude product obtained by removing the solvent by evaporation under reduced pressure was purified by recrystallization (calculated by volume ratio: dichloromethane: ethyl acetate = 1: 1) to obtain intermediate 3 (42.4 g, white solid, yield: 55.4%).

[0194] 4) Synthesis of intermediate 4 Intermediate 3 (7.65 g, 0.01 mol) was dissolved in a mixture of dichloromethane (100 mL) and N,N-dimethylformamide (100 mL), piperidine (40 ml) was added, and the mixture was stirred at room temperature for 5 hours. The solvent was then evaporated under reduced pressure, and then the mixture was dried under high vacuum in a vacuum oven to remove a small amount of piperidine to give intermediate 4 as a light yellow solid, which was used in the next step without purification.

[0195] 5) Synthesis of intermediate 5 The intermediate 4 crude product obtained in the above step was dissolved in N,N-dimethylformamide (200mL), intermediate 2 (2.94g, 0.012mol), benzotriazole-N,N,N',N'-tetramethylurethane hexafluorophosphate (HBTU) (6.07g, 0.016mol) were added, and the mixture was cooled to 0°C in an ice bath, followed by addition of N,N-diisopropylethylamine (2.6g, 0.02mol), and the mixture was stirred overnight at room temperature. The solvent was removed by evaporation under reduced pressure, and the residue was dissolved in chloroform (100ml), washed successively with saturated ammonium chloride solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The crude product obtained was layered on a silica gel column to obtain intermediate 5 (3.1g white solid, two-step total yield: 37.8%).

[0196] 6) Synthesis of intermediate 6 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 gas was introduced, and the mixture was stirred at room temperature and normal pressure for 4 hours. The palladium carbon was removed by filtration and washed with methanol. The filtrate and washings were combined, and the solvent was removed by evaporation under reduced pressure to obtain intermediate 6 (2.38 g white solid, yield: 95.2%).

[0197] 7) Synthesis of intermediate 7 In a dry and clean 250ml one-necked reaction flask, add 15ml THF, intermediate 6 (2.387g, 3.4mmol), and 1.35g DEPBT in sequence, react at room temperature for 10 minutes, add EMC-Glu(OAll)-COOH (1.3g, 3.4mmol), protect with nitrogen substitution, react at room temperature for 15 minutes, add 1.8ml DIPEA dropwise, and react at room temperature for 3 hours while protecting with nitrogen substitution, remove the solvent by evaporation under reduced pressure, add water and beat 2-3 times, extract and filter to obtain 700mg of light yellow solid. Column purification gave 2.2g of product with a yield of 63.2%.

[0198] 8) Synthesis of intermediate 8 Intermediate 7 (1.53 g, 1.46 mmol) was dissolved in DCM (20 mL), trifluoroacetic acid (10 ml) was added dropwise at room temperature, and the reaction mixture was stirred for 2 hours and monitored by HPLC. 7 After the reaction was completed, the solvent was removed by distillation under reduced pressure, the crude product was washed twice with methyl tert-butyl ether, the solid was dissolved in methanol, and passed through a reversed-phase high-pressure column to obtain intermediate 8 (928 mg white solid, yield: 84.8%).

[0199] 9) Synthesis of intermediate 9 In a 100 mL reaction flask, 10.4 mg (1.0 eq, 0.88 mmol) of doxorubicin hydrochloride 5 and 659 mg (1.0 eq, 0.88 mmol) of intermediate 8 were added and reacted at room temperature for 15 minutes under nitrogen protection. 78 μl of DIPEA was added dropwise. The reaction was allowed to proceed for 4 hours, and the solvent was removed by evaporation under reduced pressure. The crude product was dissolved in methanol and passed through a reversed-phase high-pressure column to obtain intermediate 9 (258 mg red solid, yield: 23.8%).

[0200] 10) Synthesis of the final product In a 100 mL reaction flask, add THF, intermediate 9 (258 mg, 0.202 mmol), and n-butyltin hydride (175.7 mg, 0.606 mmol) in that order, and then purge the reaction solution with nitrogen. Protect After the reaction, the reaction solution was discarded. Tetrakis(triphenylphosphine)palladium(0) (32.7 mg, 0.028 mmol) was then added and the mixture was stirred at room temperature overnight. The mixture was monitored by TLC until the conversion was complete. The flask contents were then filtered through diatomaceous earth and the residue was washed with THF. The filtrate was concentrated under reduced pressure. The resulting crude product was purified by column chromatography to give 224 mg of the desired compound (yield: 90%).

[0201] Other compounds in Table 1 below were obtained in the same manner as in Examples 1-2 and 4-9, except that the MI, S, C, A and D fractions were changed.

[0202] These compounds were identified by mass spectrometry (MS), and their molecular weights were as shown in Table 1, which was confirmed to be consistent with the molecular weights calculated based on the structures.

[0203] [Table 17]

[0204] [Table 18]

[0205] [Table 19]

[0206] [Table 20]

[0207] [Table 21]

[0208] [Table 22]

[0209] [Table 23]

[0210] [Table 24]

[0211] The present invention also provides comparative compounds of the following structural formulas:

[0212] [ka] Compound C1: Doxorubicin [ka] Compound C2:AANL-DOX [ka] Compound C3:EMC-AANL-DOX [ka] Compound C4: Peg-AANL-DOX

[0213] Example 10: Preparation of human albumin-bound HSA-EMC-AANL-DOX, HSA-QHL-087-DOX and HSA-QHL-087-N-CBP drugs Formulation of EMC-AANL-DOX, QHL-087-DOX and QHL-087-N-CBP (EMC-AANL-DOX was dissolved in DMSO, and QHL-087-DOX and QHL-087-N-CBP were dissolved in sterile water). HSA was dissolved in sterile water. The compounds were mixed with HSA in a ratio of 3:1 (4.8umol / mL, 1.6umol / mL) and reacted in a water bath at 37°C for 3 hours. The reaction solution was removed, and the unbound compounds were filtered through a pressurized ultrafiltration membrane, and diluted and filtered with saline three times to obtain the semi-finished product. Chromatographic methods such as DEAE ion exchange, gel filtration and hydroxyapatite chromatography are used to isolate human albumin-bound doxorubicin antitumor drugs. The semi-finished product is quickly divided into small portions, spin-frozen and freeze-dried. The freeze-drying process of the product can be developed according to the performance characteristics of the machine, but it is necessary to ensure that the quality of the product preparation and storage meets the requirements. In the experiment, the binding of legubicin to HSA was compared at different ratios and for different times. The results showed that EMC-AANL-DOX, QHL-087-DOX and QHL-087-N-CBP were bound to HSA at a ratio of 3:1 in a water bath at 37°C, and the HSA binding rates were 62%, 99.6% and 99.7%, respectively.

[0214] Example 11: Selection of chemically modified linkers for optimized activation efficiency SCA is a chemically modified conjugate, which shows high activation efficiency compared to conjugates of natural peptide sequences that are degraded by legumain. When C is selected for AAN, the activation of different SCA joints and control joints is evaluated by activation assay. SCA adducts were dissolved using and diluted 10-fold to a concentration of 0.1 mM / ml. Sample compounds were added to 100 μg of acidified human breast cancer (MDA-MB435) tumor tissue homogenate (pH 6.0) at a concentration of 1 mg / ml at 37 °C. The enzyme was released from the homogenate of tumor tissue and detected by HPLC, allowing the activation efficiency of the joints by tumor tissue to be compared. The results are shown in Tables 2-1, 2-2, 2-3, and 2-4.

[0215] [Table 25]

[0216] [Table 26]

[0217] [Table 27]

[0218] The effect of D-type tripeptides on activation efficiency was examined in comparison with the junctions of the natural peptide sequence degraded by legumain. S1 of MI-S was -CH2CH2-CONH-, S2: 2peg. The results are shown in Table 2-4 below.

[0219] [Table 28]

[0220] As can be seen from the data in the table, in the case of tripeptides with different variables under conditions where S and A are designed for high activation, the choice of amino acids and the difference in conformation affect the activation efficiency, and in particular D-Asn leads to a decrease in activation ability, while the other two positions have activation activity even when the amino acids are adjusted to the D-form.

[0221] Example 12: Comparison of Kinetic Rates of Enzymatic Cleavage of Preferred Compounds C3, QHL-087-DOX, QHL-090-DOX, QHL-093-DOX, QHL-094-DOX, QHL-093-DOX, and QHL-096-DOX samples were weighed out at 10 mg each, and an appropriate amount of water was added to make a sample stock solution of 4 umol / mL. Water was added to gradually dilute the sample to the concentration in Table 3 below, and then the sample was measured. Measure 20 ul of sample solutions with different concentrations, add 80 ul Legumain, and place in a 37°C water bath; take out after 2 h water bath, inject 10 ul, and detect by HPLC; read the area of ​​each corresponding product, calculate the concentration of the product according to the linear equation of the product, and substitute into the formula to obtain the corresponding V.

[0222] V(umoL / mL / min)=C(umoL / mL) / 120min

[0223] The intercept Km / Vmax is calculated by plotting V against [C]. At the intersection of the x-axis and the line, -Km, [C] is the concentration of each substrate, i.e., the concentration of the sample solution, expressed in umoL / mL.

[0224] [Table 29]

[0225] The experimental results under the same conditions as for other structures are shown in Figure 3. The 2PEG group of QHL-087 significantly increases the activation efficiency, but the efficiency decreases as the number of PEGs increases. This is compared to the same conditions with 6PEG groups attached: H2PABC-NH2H2 is replaced by leu, and the activation efficiency increases significantly.

[0226] <Example 13: Isolation and culture of mouse spleen and CD8+ T cells, isolation and culture of mouse bone marrow mononuclear cells, and induction of differentiation of M2 macrophages> 1. Mouse Spleen Cell Isolation 1) Collect the spleen from a C57BL / 6 mouse, place it on a petri dish with a 40uM sieve (ice bath), add approximately 10mL of saline, and gently crush with the tip of a sterile syringe. 2) Transfer the pulverized cell suspension to a 50 mL centrifuge tube, add approximately 5 mL of saline to wash the culture dish, transfer to the 50 mL centrifuge tube, and combine the suspension. 3) Centrifuge the cell suspension at 1000r / min for 10min, discard the supernatant, resuspend in an appropriate volume of saline, add 3 times the volume of ammonium chloride red blood cell lysis solution, and blow to homogenize. After about 10min of lysis on ice, add 10mL of saline to complete the lysis, and centrifuge at 1000r / min for 5min. 4) After centrifugation, discard the supernatant, add 10 mL of saline, shake well, and centrifuge at 1000 r / min for 5 minutes. Repeat this procedure once, and then resuspend the cells in 10% RMPI1640 medium for subsequent culture and 0.5% BSA for subsequent T cell sorting.

[0227] 2.CD8+ T cell sorting Mouse spleen cells were isolated as above, resuspended at 1E8 / mL, 100ul Miltenyibiotec CD8a(Ly-2) microBeads were added to each 1E8 cells, mixed well, incubated at 4℃ for 15 min in the dark, washed with 5-10 volumes of PBS, mixed well, centrifuged at 300g for 5 min, removed the supernatant, washed repeatedly, and resuspended. Cells were resuspended at 2E8 / mL for isolation on the column, and the cell suspension was loaded onto the LS column on the magnet plate (the LS column was pre-equilibrated with buffer (pH 7.2 PBS + 0.5% BSA + 2mM EDTA)). The cell suspension slowly flowed through the LS column, and after the CD8+ T cells bound to the magnetic particles in the LS column, the LS column was washed with 3 volumes of wash buffer, and after washing, the LS column was removed from under the magnet plate and placed into a 15mL centrifuge tube. All cells that passed through the column were collected, centrifuged to remove the supernatant, washed once more with buffer, and resuspended in an appropriate amount of 10% RMPI1640 medium, and the cells were counted and stored.

[0228] 3. Activation and expansion of CD8+ T cells A. Washing of CD3 / CD28 magnetic beads: a. Shake and suspend the immunomagnetic beads in the tube (vortex for more than 30 seconds or tilt spin for 5 minutes). b. Take the required amount of immunomagnetic beads into a 1.5 ml tube, add 1 ml of 1640 with serum, mix well, and vortex for more than 30 seconds or roll for more than 5 minutes.

[0229] Activation of T cells: a. Seed the appropriate number of cells into a culture plate (e.g. 1E6 / ml in a 6-well plate, 2 ml of culture medium, T cell density about 2E6 / m l b. Add washed magnetic beads so that the ratio of beads to cells is 1:1. c. Place in an incubator and culture for 3 days.

[0230] C. Growth: a. No medium change is required within 3 days. 30 U / ml on the 3rd day. lb. After 4-5 days of stimulation (to avoid overactivation), remove the magnetic beads and move up and down 5-10 times to avoid air bubbles and turbulence to isolate the cells and magnetic beads. Collect the cells in a 1.5 ml tube, place them on the magnetic beads for 1 minute until the magnetic beads stick to the tube wall, transfer the cells to another tube, remove the magnetic beads as completely as possible, and incubate the cells removed from the beads with 30 U / m l( Continue culturing in medium supplemented with additional medium (increased as appropriate) and monitor status, growth and viability.

[0231] 4. Isolation of Single Nucleated Cells from Mouse Bone Marrow and Induction of M2 Macrophage Differentiation Both femurs and tibias were removed from two C57BL / 6 mice under aseptic conditions, the bones were cut open on an ultraclean table, the bone marrow cavity was gently washed four times with 5mL of serum-free MEM culture medium, and all cell suspensions were collected. After centrifugation at 1000r / min for 10 minutes, the supernatant was discarded to obtain cell precipitates, and then the cells were resuspended in an appropriate amount of serum-free MEM medium, blown and homogenized repeatedly, filtered through a 40uM filter, and dissolved on ice for 10 minutes with three times the amount of red blood cell lysis solution; centrifuged at 1000r / min for 5 minutes, the supernatant was discarded to obtain cell precipitates, and washed twice with serum-free MEM medium to recover the cell precipitates. The cells were resuspended in an appropriate amount of MEM complete medium containing 10% volume fraction of FBS and 1% PS, and then counted after waiting for differentiation; 20,000 cells / well were seeded in 100uL into a 96-well cell culture plate, and 100ng M-CSF was added for M2 macrophage differentiation, and the cells were cultured at 37℃, 5% CO2 volume fraction incubator for 7 days to induce differentiation, and the cell morphology was observed. The morphology of the induced cells was confirmed as M2 macrophages with reference to Figure 4, and it can be seen that M2 macrophages are different from monocytes, DCs, and GM-macrophages.

[0232] <Example 14: Measurement of the cell proliferation inhibitory effect of drugs by MTT assay> The cells of Example 13 were counted, the cell concentration was adjusted with culture medium, and 100 μl of the cell suspension was inoculated per well into a 96-well culture plate to a concentration of 100,000 cells / well for CD8+ T cells and 20,000 cells / well for M2 macrophages. The 96-well culture plate was incubated overnight for 24 hours in a 37°C, CO2 (5%) incubator. After 24 hours, 100 ul of cell culture medium containing different concentrations of drugs was added to the 96-well culture plate, and control wells containing no drugs and the corresponding drug solvent (0.1% DMSO) only, and blank wells containing only medium and no cells were set up. Three parallel wells were set up for each group, and the plate was then incubated for 48 hours at 37°C in a CO2 (5%) incubator. After 48 hours, 20 μl of MTT (concentration 5 mg / ml) was added to each well, and the incubation was continued for 4 hours. Then, the culture medium was gently aspirated, and 150 μl of DMSO was added to each well as a solvent to dissolve the cells. After dissolution, the absorbance at 490 nm was measured using an enzyme marker.

[0233] The cell viability and the half-life inhibitory concentration of the drug for the cells were calculated. Cell viability = (ODtest-ODblankcontrol) / (ODtestcontrol-ODblankcontrol)*100%. The viability (%) was calculated using Excel software, and the dose-response curve of the drug for the cells was plotted using Prism5 software. Each index was expressed as an average value, and the coefficient of variation (CV) was displayed to evaluate the consistency of the data.

[0234] The maximum starting concentration of the test drug was 14uM, and it was diluted at a ratio of 1:3 to create 9 treatment groups (3 replicates per group). The drug solvent (DMSO) concentration in all treatment wells was controlled at 0.1%, and only the drug solvent (0.1% DMSO) was added as a control group (Control). Next, the tumor cell survival rate (%) of each treatment group relative to the control group (Control) was calculated according to the following method.

[0235] Cell viability (%) for each treatment group = (OD treatment group - OD blank group) / (OD 0.1% DMSO-OD blank group) * 100%.

[0236] The experimental results are shown in Figures 5 and 6. Under otherwise structurally compatible conditions, QHL-087-DOX exhibited significantly increased cytotoxicity against M2 macrophages, decreased toxicity against CD8+ T cells, and selectivity against immunosuppressive cells compared to C3 (i.e., EMC-AANL-DOX).

[0237] <Example 15: Cytotoxicity screening of multiple agents of the present invention against M2 macrophages> A cytotoxicity screening assay for M2 macrophage inhibition of several compounds was carried out according to the method of Example 14. Three wells were tested for each drug, and the following drugs were added to each well at 10 uM, and the inhibition rate was tested relative to the drug-free group. The experimental results are shown in Table 4.

[0238] Example 16: Comparison of water solubility of water-soluble, highly efficient, target-activated doxorubicin derivatives prepared according to embodiments of the present invention with that of a control compound The compounds prepared according to the embodiment of the present invention were freeze-dried (-70°C) from the compounds prepared above and reference compounds C1, C2, C3 and C4. The compounds were dissolved in water at different concentrations, and the water solubility was confirmed (95% or more) by observation and HPLC test. The results are shown in Table 4.

[0239] [Table 30]

[0240] [Table 31]

[0241] [Table 32]

[0242] [Table 33]

[0243] [Table 34]

[0244] [Table 35]

[0245] [Table 36]

[0246] [Table 37]

[0247] [Table 38]

[0248] The results showed that the 2PEG group significantly increased the solubility from insoluble to soluble in water under otherwise structurally compatible conditions, and the solubility increased with increasing PEG amount. Under the same conditions of PEG binding, the addition of Glu and Asp improved the water solubility. The change of group changed the water solubility of the drug conjugate, which could dramatically affect both the exit of the drug from the vascular membrane and the permeability of the tumor cell membrane, thus affecting the therapeutic effect. The improved water solubility provided the necessary conditions for drug formation and allowed the drug conjugate to be tailored.

[0249] <Example 17: Efficacy evaluation of injection administration of C3, QHL-085-DOX, QHL-087-DOX, QHL-091-DOX, and QHL-094-DOX in HT1080 nude mouse model> Objective: To investigate the antitumor effects of C3, QHL-085-DOX, QHL-087s-DOX, QHL-091-DOX, and QHL-94-DOX in mouse models undergoing tumor treatment. Test drugs: C3, QHL-085-DOX, QHL-087-DOX, QHL-091-DOX, and QHL-094-DOX were used as injection drugs and were diluted with saline to concentrations appropriate for the test.

[0250] Methods and Results: 1. Animals: Nude mice, 6-8 weeks old, all female.

[0251] 2. Preparation of Tumor Models 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 and 5% CO2. Subculture was performed every 3 days, and cells up to 15 generations were used.

[0252] 2) Tumor development: HT1080 cells 5 x 10 6 The tumors were injected subcutaneously into the backs of nude mice. 3 Once this threshold was reached, patients were randomly assigned to a group, after which treatment was initiated and the day treatment began was recorded as day 1.

[0253] 3) Treatment procedure C3: QHL-085-DOX, QHL-087-DOX, QHL-091-DOX, and QHL-094-DOX, drugs for intravenous infusion for clinical applications. C3, QHL-085-DOX, QHL-087-DOX, QHL-091-DOX, and QHL-094-DOX were administered at a low dose and the same dose of 18umol / kg, respectively. The control group was administered saline. The administration was once a week for 3 weeks.

[0254] 4) Results and Discussion: The grouping and trial results are shown in Figure 7. Compared to the equimolar low-dose group, the tumor-suppressing effect was sequentially enhanced in the 4peg group and the 2peg group.

[0255] <Example 18: Efficacy evaluation of injection administration of C1, C2, C3, QHL-086-DOX, QHL-092-DOX, QHL-095-DOX, QHL-087-DOX, QHL-010-DOX, and QHL-117-DOX in HT1080 nude mouse model> Objective: To investigate the antitumor effects of the above compounds in mouse models during tumor treatment. Test drugs: C1, C2, C3 and corresponding compound injections diluted to appropriate concentrations in test saline.

[0256] Methods and Results: 1. Animals: 6-8 week old nude mice (all female).

[0257] 2. Preparation of Tumor Models 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 and 5% CO2. Subculture was performed every 3 days, and cells up to 15 generations were used.

[0258] 2) Tumor development: HT1080 cells 5 x 10 6 The tumors were injected subcutaneously into the backs of nude mice. 3 Once this threshold was reached, patients were randomly assigned to a group, after which treatment was initiated and the day treatment began was recorded as day 1.

[0259] 3) Treatment procedure Intravenous (IV) administration of drugs according to the clinical application of the corresponding compound. The compounds shown in the table were administered at low doses and at the same dose of 36umol / kg. The control group received saline. The administration was done once a week for 3 weeks.

[0260] 4) Subgroups and test results are shown in Table 5.

[0261] [Table 39]

[0262] 5) Results and Discussion: As shown in Table 5, compared with the equimolar AANL-DOX group, the high-dose treatment groups of QHL-086-DOX, QHL-092-DOX, QHL-095-DOX, QHL-087-DOX, QHL-010-DOX, and QHL-117-DOX significantly improved tumor growth inhibition and tumor disappearance, and also achieved a curative effect.

[0263] Example 19: Tissue distribution study of QHL-087-DOX and EMC-AANL-DOX in liver in situ transplanted CT26 tumors Aim of the experiment: To investigate the tissue distribution of the activator in liver tumors. Test animals: 6-8 week old BALB / c mice (all female).

[0264] Tumor model generation 1) CT26 cells were purchased from ATCC and cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. Subculture was performed every 3 days, and cells up to 15 generations were used.

[0265] 2) Tumor development: 5 x 10 CT26 cells 6 The tumors were injected subcutaneously into the backs of nude mice. 3 After reaching 100 mm, the tumor tissue was extracted and divided into random groups. 3 The tissue was cut into blocks and transplanted in situ into the liver of BALB / c mice.

[0266] 3) Drug administration: After 14 days, when the in situ transplanted tumors had grown, the drug was administered to 36 mice with in situ transplanted tumors. Then, after 1, 6, 12, 24, 36, and 72 hours, different tissues were collected to detect the concentration of doxorubicin released in different tissues. The AUClasth*nmol / g was calculated, and the average value and SEM are shown in Figure 8 and Figure 9.

[0267] 4) Results and Discussion: As shown in Figures 8 and 9, active doxorubicin in QHL-087-DOX and EMC-AANL-DOX was distributed predominantly in the liver and in situ tumors in the liver. The previously published use of EMC-AANL-DOX was for breast cancer treatment, but further research revealed that QHL-087-DOX has the property of delivering drugs to the liver, where adriamycin exposure is increased due to in situ activation of tumors. Compared with EMC-AANL-DOX, QHL-087-DOX promoted the delivery and activation of doxorubicin exposure in in situ hepatocellular carcinoma.

[0268] Example 20: Efficacy evaluation of QHL-087-DOX in CT26 tumors by in situ liver transplantation Study objective: To investigate the efficacy of QHL-087-DOX, PD-1 and their combination therapy against in situ implanted CT26 tumors. Test drugs: QHL-087-DOX 18 micromol / kg, mouse PD-1 5 mg / kg. Animals: 6-8 week old BALB / c mice, all female.

[0269] 1) Tumor model creation: CT26 tumor cells were obtained from ATCC. The cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. The cells were passaged every 3 days, and cells within the 15th generation were used. 5x10 5 CT26 cancer cells were injected subcutaneously into the backs of nude mice. Tumors were 800-1000 mm 3 After reaching 100 mm , the mice were randomly divided into groups. Tumor tissues were then extracted and 3 The tumors were cut into 100x blocks and implanted in situ into the livers of BALB / c mice. After one week, when the in situ tumors had grown, the mice bearing the in situ tumors were randomly assigned to groups.

[0270] 2) Treatment procedure: Six mice per group were administered the drug. The treatment date was the first day. In line with the clinical application of QHL-087-DOX, intravenous infusion was performed once a week for three weeks. Mouse PD-1 antibody was administered intravenously twice a week for three weeks. The subgroups and test results are shown in the attached Figure 10.

[0271] 3) Results and Discussion: The combination of QHL-087-DOX and PD-1 demonstrated immune synergistic effects against liver in situ tumors for the first time, and was found to have superior efficacy and immunotherapeutic properties compared to QHL-087-DOX alone.

[0272] <Example 21: Effect of mutual combination therapy of lenvatinib and PD-1 on in situ liver cancer using EMC-AANL-DOX (legubicin)> Study objective: To evaluate the efficacy of EMC-AANL-DOX, lenvatinib and PD-1 combination therapy against in situ hepatocellular carcinoma. Investigational drugs: EMC-AANL-DOX 18 μmol / kg and PD-1 5 mg / kg were administered to mice. Animals: 6-8 week old BALB / c mice, all female.

[0273] Tumor model preparation: CT26 tumor cells were obtained from ATCC. The cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. The cells were passaged every 3 days, and cells within the 15th generation were used. 5x10 5 CT26 cancer cells were injected subcutaneously into the backs of nude mice. Tumors were 800-1000 mm 3 After reaching 100 mm , the mice were randomly divided into groups. Tumor tissues were then extracted and 3 The tumors were cut into 100x blocks and implanted in situ into the livers of BALB / c mice. After one week, when the in situ tumors had grown, the mice bearing the in situ tumors were randomly assigned to groups.

[0274] Treatment procedure: 6 mice per group were administered the drug. The treatment date was the first day. According to the clinical application of EMC-AANL-DOX, it was administered intravenously once a week for 3 weeks. Mouse PD-1 antibody was administered intravenously twice a week for 3 weeks. The subgroups and test results are shown in the attached Figure 11.

[0275] Results and Discussion: For the first time, we found that the effect of combination therapy of EMC-AANL-DOX and PD-1 was superior to that of combination therapy of lenvatinib and PD-1, and that the effect of combination therapy of EMC-AANL-DOX and lenvatinib was enhanced compared to that of each monotherapy.

[0276] Example 22: Study of the efficacy of injection of some compounds of the present invention against human hepatocellular carcinoma HepG2 cells in nude mice Experimental objective: To study the antitumor effects of some compounds of the present invention in mouse tumor treatment models. Test drug: Injection of the corresponding compound in the table and control injection diluted with saline to the appropriate concentration at the time of testing.

[0277] Methods and Results: 1. Test animals: 6-8 week old nude mice (all female).

[0278] 2. Preparation of Tumor Models 1) Human hepatocellular carcinoma HepG2 cells were purchased from ATCC and identified according to the instructions provided by ATCC. The cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. Subculture was performed every 3 days, and cells up to 15 generations were used.

[0279] 2) Tumor development: 5 x 10 HepG2 cells 6 The tumors were injected subcutaneously into the backs of nude mice. 3 Once the age limit was reached, randomization to a group was performed, after which treatment was initiated and the day treatment began was recorded as day 1.

[0280] 3) Treatment procedure Intravenous (IV) administration according to the clinical application of the corresponding compounds. Compounds and controls were administered at a dose of 54umol / kg, DOX was only available at a dose of 18umol / kg due to toxicity restrictions. The control group received saline. Treatment was once a week for 4 weeks.

[0281] 4) Subgroups and test results are shown in Table 6.

[0282] [Table 40]

[0283] 5) Results and Discussion: As shown in Table 6, EMC-AANL-DOX showed superior efficacy against hepatocellular carcinoma, and the preferred compounds of the present invention showed increased therapeutic effect against tumor growth compared to the use of the same molar amount of EMC-AANL-DOX.

[0284] Example 23: Efficacy evaluation 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 Purpose of the study: To examine the antitumor effect of the above compounds in immunotherapy using a CT26 tumor model. Test drugs: QHL-096-DOX, QHL-087-DOX, QHL-090-DOX, QHL-093-DOX, QHL-117-DOX and control, dose 36umol / k, mouse PD-1 antibody, 5mg / kg. Test animals: 6-8 week old BALB / c mice (all female).

[0285] Tumor model generation 1) CT26 cells were purchased from ATCC and cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. Subculture was performed every 3 days, and cells up to 15 generations were used.

[0286] 2) Tumor development: 5x10 corresponding cells 6The mice were subcutaneously injected with 100 μg of 100 μg of 100% ethanol in the back of the nude mice. 3 After reaching 1 day, the mice were randomly assigned to groups, after which treatment began, marking the first day.

[0287] 3) Treatment course: The drug was administered at an equimolar dose of 36umol / kg. The control group was administered saline. The drug was administered once a week for three weeks.

[0288] 4) Tumor CD8 + T cell analysis. Tumor tissue was homogenized, and individual cells in the tumor were isolated by filtration, washed twice with buffer, and then incubated with antibodies labeled with leukocyte common antigens CD45-PE and CD8-FITC for 1 h at room temperature. Cells were washed twice with phosphate buffer containing 1% fetal bovine serum and analyzed by flow cytometry for the percentage of T lymphocyte antigen (CD8) positive cells among leukocyte common antigen (CD45) positive cells. An increase in this ratio indicated an increase in T lymphocytes, and thus an improvement in the animal's immunity against the tumor.

[0289] 5 ) Groupings and test results are shown in Table 7.

[0290] [Table 41]

[0291] 6 ) Results and Discussion: It was found that the combination of C3, QHL-096-DOX, QHL-087-DOX, QHL-090-DOX, QHL-093-DOX, and QHL-117-DOX with PD-1 enhanced the therapeutic effect and cured tumors compared to single agents. QHL-090-Darafenib and QHL-090-Darafenib also showed a certain degree of synergistic effect when combined with PD-1.

[0292] Example 24: Efficacy test of QHL-087-DOX injection in various tumor models Study Objective: To investigate the antitumor spectrum of QHL-087 in various tumor models in mice. Test drug: QHL-087-DOX was injected and diluted in saline to the appropriate concentration for the test.

[0293] Methods and Results: 1. Animals: 6-8 week old nude mice (all female).

[0294] 2. Preparation of Tumor Models 1) The corresponding tumor cells were purchased from the American Typical Culture Collection (ATCC) and identified according to the specifications provided by ATCC. The cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. The cells were passaged every 3 days, and cells within the 15th generation were used.

[0295] 2) Tumor development: 5x10 corresponding cells 6 The mice were subcutaneously injected with 100 μg of 100 μg of 100% ethanol in the back of the nude mice. 3 After reaching 1 day, the mice were randomly assigned to groups, after which treatment began, marking the first day.

[0296] 3) Treatment course: QHL-087-DOX was administered at a dose of 36umol / kg in accordance with clinical application. Saline was administered to the control group. The treatment was administered once a week for three weeks.

[0297] 4) The groupings and test results are as shown in Table 8.

[0298] [Table 42]

[0299] 5) Results and Discussion: QHL-087-DOX showed excellent efficacy in various tumor models, demonstrating that this drug has a broad antitumor spectrum.

[0300] Example 25: Solubility of HSA-EMC-AANL-DOX, HSA-QHL-087-DOX, and HSA-QHL-087-N-CBP compared to control compound Lyophilized EMC-AANL-DOX, HSA-EMC-AANL-DOX, HSA-QHL-087-DOX and HSA-QHL-087-N-CBP prepared according to this embodiment of the present invention were dispensed in a sterile room and reconstituted with water for injection. HSA-EMC-AANL-DOX, HSA-QHL-087-DOX and HSA-QHL-087-N-CBP could all be completely dissolved, as shown in Table 9.

[0301] [Table 43]

[0302] As can be seen from Table 9, binding the compounds to human albumin further improves their solubility, making HSA-EMC-AANL-DOX, HSA-QHL-087-DOX and HSA-QHL-087-N-CBP large protein drugs that can be dissolved in high concentrations directly in water for injection or saline without the need for harsh organic solvents required to dissolve EMC-AANL-DOX. Unlike water-insoluble small molecule compounds such as EMC-AANL-DOX, changes in solubility properties have dramatic effects on both the distribution and metabolism of the drug and the mechanism of action of the drug.

[0303] Example 26: Solution stability of water-soluble, highly efficient, target-activated doxorubicin derivatives prepared according to embodiments of the present invention compared to a control compound 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 were accurately weighed and dispensed in 5.0 mg portions in a sterile room, and 0.5 ml of sterile water was added to make a master mix of 10 mg / ml. 50% ethanol was required to dissolve EMC-AANL-DOX. 30 ul of the mother liquor was taken and 570 ul of different pH 5.5 buffers were added to make a sample solution of 0.5 mg / ml. After clarification, the mixture was left to stand in a water bath at 25°C / 37°C for 8 h, and the relative 0h content was measured by HPLC and electrophoresis to obtain the stability data of each compound. The results are shown in Table 10.

[0304] [Table 44]

[0305] As can be seen from the data in the above table, the stability of the albumin-bound compounds increased at 25°C and pH=5.5, which was more pronounced for QHL-087-N-CBP and HSA-QHL-087-N-CBP.

[0306] Example 27: Activation efficiency test of HSA-EMC-AANL-DOX, HSA-QHL-087-DOX, and HSA-QHL-087-N-CBP EMC-AANL-DOX was dissolved using a solvent (50% water for injection + 50% alcohol), and HSA-EMC-AANL-DOX, HSA-QHL-087-DOX and HSA-QHL-087-N-CBP were dissolved uniformly using water for injection and diluted 10 times with water to 1 mg / ml. In the present experiment, 1 mg / ml of the sample compound was added to 100 μg of tumor tissue homogenate acidified at 37° C. (pH 6.0), doxorubicin was released by the enzyme in the tumor tissue homogenate, and the drug activation efficiency in the tumor tissue was compared by HPLC, which can detect the decrease of the compound and the increase of doxorubicin. It was found that EMC-AANL-DOX, HSA-EMC-AANL-DOX, HSA-QHL-087-DOX and HSA-QHL-087-N-CBP combinations had the highest activation efficiency among the compounds screened. The results are shown in Table 11.

[0307] [Table 45]

[0308] Example 28: Toxicity assessment 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 Purpose of the study: To understand the acute toxicity of the drug substance of the present invention by measuring the intravenous MTD test using mice. Test drugs: EMC-AANL-DOX was dissolved in the solvent (50% water for injection, 50% alcohol), and 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 then diluted appropriately with physiological saline for use. Animals: Grade 1 BALB / C mice (purchased from Shanghai Slake Laboratory Animal Co., Ltd.), weighing 19–21 g, all female.

[0309] Methods and Results: BALB / C mice weighing 19-21g 70 All females were weighed according to their weight.10 The mice were randomly divided into 7 groups, each consisting of 100 mice. 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 administered intravenously in a single dose as shown in Table 12. Control experiments were also conducted for saline and paclitaxel injections (commercially available, Beijing Yuekang), and each mouse was administered 0.2 ml of drug. The mice were observed for 17 consecutive days, and were observed daily for piloerection, disheveledness, lack of shine, lethargy, stooping, hyperresponsiveness, etc., and their body weight and mortality were recorded. Blood was collected on the 3rd, 5th, and 14th days for complete blood count, and the animals were dissected on the 14th day to collect the heart, liver, kidney, lung, spleen, and pancreas, which were stained with HE and observed.

[0310] [Table 46]

[0311] Results and Discussion: When injected with HSA-EMC-AANL-DOX, HSA-QHL-087-DOX and HSA-QHL-087-N-CBP of the present invention, the animals did not show piloerection, messy and dull appearance, lethargy, crouching, hyper-reactivity and death, indicating that the toxicity of the albumin conjugates is significantly reduced compared to the unconjugated drugs.

[0312] <Example 29: Effect of combination therapy of HSA-EMC-AANL-DOX, HSA-QHL-087-DOX and anti-PD-1 antibody> Purpose of the study: To compare the effects of combination therapy of EMC-AANL-DOX, HSA-QHL-087-DOX and anti-PD-1 antibody. Test drugs: HSA-EMC-AANL-DOX and HSA-QHL-087-DOX, each at 18 μmol / kg; mouse PD-1 antibody, 5 mg / kg. Test animals: 6-8 week old BALB / c mice (all female).

[0313] Tumor model preparation: CT26 cells were purchased from ATCC and cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. Subculture was performed every 3 days, and cells up to 15 generations were used. 5x10 6 CT26 cancer cells were injected subcutaneously into mice, who were then injected with the drug three times a week and the anti-PD-1 antibody twice a week for a total of eight injections.

[0314] Results and Discussion: The results of this study are shown in Figure 12. HSA-QHL-087-DOX combined with anti-PD-1 antibody demonstrated improved therapeutic efficacy and a higher cure rate compared to HSA-EMC-AANL-DOX.

[0315] <Example 30. Examination of tumor cell proliferation inhibitory effect of N-CBP and HSA-QHL-095-N-CBP by MTT method> The isolated and cultured cells were counted and the cell concentration was adjusted with culture medium. 100 μl of cell suspension was seeded per well in a 96-well culture plate, with CD8+ T cells at 100,000 cells / well and CT26 tumor cells at 20,000 cells / well. The 96-well culture plate was incubated overnight for 24 hours in a 37°C, CO2 (5%) incubator. After 24 hours, 100 ul of cell culture medium containing different concentrations of drugs was added to the 96-well plate, control wells (0.1% DMSO) were set up with the corresponding drug solvent without drugs, and blank wells (blank) were set up with only medium without cells. Three parallel wells were set up for each group, and the plate was then incubated for 48 hours at 37°C in a CO2 (5%) incubator. After 48 hours, 20 μl of MTT (concentration 5 mg / ml) was added to each well, and the incubation continued for 4 hours. Then, the culture medium was gently aspirated, and 150 μl of DMSO was added to each well as a solvent to dissolve the cells. After dissolution, the absorbance at 490 nm was measured using an enzyme marker.

[0316] Results and Discussion: The results of the trial are shown in Figures 13 and 14. The in vitro cytotoxicity of N-CBP was stronger than that of carboplatin and oxaliplatin, but weaker than that of cisplatin (Figure 13). HSA-QHL-095-N-CBP had reduced in vitro cytotoxicity compared to N-CBP and carboplatin (Figure 14).

[0317] Example 31: Effect of HSA-QHL-095-N-CBP alone and in combination with anti-PD-1 antibody Study objective: To compare the efficacy of combination therapy with carboplatin, HSA-QHL-095-N-CBP, and anti-PD-1 antibody. Investigational drugs: Carboplatin, HSA-QHL-095-N-CBP (18 μmol / kg), and mouse-derived PD-1 antibody (5 mg / kg) were administered. Test animals: 6-8 week old BALB / c mice (all female).

[0318] Tumor model preparation: CT26 cells were purchased from ATCC and cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. Subculture was performed every 3 days, and cells up to 15 generations were used. 5x10 6 CT26 cancer cells were subcutaneously injected into mice.

[0319] Treatment course: The corresponding drug was injected once a week for 3 weeks, and anti-PD-1 antibody was administered once a week for 4 weeks.

[0320] Results and Discussion: The results of this study are shown in Figure 15. It can be seen that the equimolar amount of HSA-QHL-095-N-CBP is significantly superior to carboplatin, and the curative effect is improved by combining with anti-PD-1 antibody compared to single agent.

[0321] <Example 32: Effect of inflammatory response in nonalcoholic fatty liver disease (NAFLD) model mice> Experimental Method: C57 mice were randomly divided into normal group (standard feed), model group (high-fat feed), simvastatin group (positive control, 3mg / kg), and drug-treated group (50mg / kg), with 6 mice in each group. The mice in the normal group were fed standard feed, and the mice in the remaining groups were fed high-fat feed to induce the NAFLD model. At the same time, the mice in each group were intravenously administered an appropriate amount of 48umol / kg twice a week for a total of 8 weeks. Serum biochemical parameters: HDL-C and LDL-C were measured by an automatic biochemical analyzer 12 hours after the final administration. The results are shown in Table 13.

[0322] [Table 47]

[0323] Results: Compared with the normal group, serum HDL-C level in the model group was significantly decreased by 66.2%, and LDL-C level was significantly increased by 135.6% (P<0.05). HDL-C and LDL-C levels in the QHL-158-T3 and QHL-159-T3 treatment groups were better restored to normal levels.

Claims

1. A conjugate or a pharma- ceutically acceptable salt thereof, characterized in that it is selected from the following: 【Chemistry 1】 【Chemistry 2】 【Chemistry 3】 【Chemistry 4】 【Chemistry 5】 【Chemistry 6】 【Chemistry 7】 【Chemistry 8】 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 【Chemistry 18】 【Chemistry 19】 【Chemistry 20】 【Chemistry 21】 【Chemical Formula 22】 【Chemistry 23】 【Chemistry 24】 【Chemistry 25】 【Chemistry 26】 【Chemical 27】 【Chemistry 28】 【Chemical 29】 【Chemistry 30】 【Chemistry 31】 【Chemistry 32】 【Chemical 33】 【Chemical 34】 【Chemistry 35】 【Chemical 36】 【Chemical 37】 【Chemical Formula 38】 【Chemical 39】 【Chemistry 40】 【Chemistry 41】 【Chemistry 42】 【Chemistry 43】 【Chemistry 44】 【Chemistry 45】 【Chemistry 46】 【Chemistry 47】 【Chemistry 48】 【Chemistry 49】 【Chemistry 50】 【Chemistry 51】 【Chemistry 52】 【Chemistry 53】 【Chemical 54】 【Chemistry 55】 【Chemistry 56】 【Chemistry 57】 【Chemistry 58】 【Chemistry 59】

2. A complex formed by covalently bonding the complex according to claim 1 with albumin, or a pharma- ceutically acceptable salt thereof.

3. A pharmaceutical composition comprising the complex of claim 1 or a pharma- ceutically acceptable salt thereof, or the complex of claim 2 or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

4. Use of the complex according to claim 1 or a pharma- ceutically acceptable salt thereof, or the complex according to claim 2 or a pharma- ceutically acceptable salt thereof, in the preparation of a medicament for the treatment or prevention of the following steatotic phenomena: cancer, fatty liver (including alcoholic and non-alcoholic fatty liver), steatohepatitis, fatty liver disease, liver fibrosis, liver inflammation, and hepatocellular injury.

5. Use of the conjugate of claim 1 or a pharma- ceutically acceptable salt thereof in the preparation of a drug molecule for delivering a drug to the liver, or in the preparation of a drug having increased water solubility, reduced drug toxicity, improved drug efficacy, and / or improved drug selectivity for immune cells, or in the preparation of a drug molecule for delivering a drug to the liver, referring to enhanced water solubility of a compound drug, reduced drug toxicity, improved drug efficacy, and / or improved drug selectivity for immune cells.

6. Use of the complex according to claim 1 or a pharma- ceutically acceptable salt thereof, or the complex according to claim 2 or a pharma- ceutically acceptable salt thereof, in the preparation of an immunosuppressant cell inhibitor, a tumor-associated macrophage inhibitor, an MDSC cell inhibitor, an angiogenesis inhibitor, an antitumor immune promoter and / or a T lymphocyte proliferation promoter.

7. 13. Use of the conjugate of claim 1 or a pharma- ceutically acceptable salt thereof, or the conjugate of claim 2 or a pharma- ceutically acceptable salt thereof, and an anti-PD-1 antibody in the preparation of a combination therapeutic agent for a tumor.

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