Cleavable linker compound for prodrug delivery system, and preparation and use thereof

Abstract

Disclosed in present invention are a novel cleavable linker compound as shown in formula (1) for a prodrug delivery system, and a pharmaceutically acceptable salt, solvate, polymorph or isomer thereof. Further disclosed are a prodrug compound as shown in formula (J) and formula (L) formed by covalently bonding an albumin or antibody to a camptothecin derivative by means of a novel linker compound, and a pharmaceutically acceptable salt, solvate, polymorph or isomer thereof. Further disclosed are a synthesis method for the compound, and a preparation method for the compound in an enzyme release prodrug system. Further disclosed are a use of the compound in preparation of an anti-tumor drug, and a use of the compound in preparation of an albumin-bound anti-tumor drug or antibody-drug conjugate. The present invention innovatively proposes a structure in which a trigger fragment in the compound is linked to the novel linker by means of p-hydroxyl or para-amino, an active drug is linked to the novel linker by means of the hydroxyl part of the active drug, and a modified fragment is linked to the novel linker by means of an amino group.

Description

A cleavable linker compound for a prodrug delivery system and its preparation and application Technical Field

[0001] The present invention belongs to the field of biomedical technology and pharmaceutical preparation prodrug design, and relates to the design, synthesis and synthetic products of a novel linker of a cleavable prodrug delivery system, and its application in the prodrug delivery system. Background Art

[0002] As people's understanding of tumors deepens, different treatment methods such as small molecule cytotoxic drugs, nucleic acids, proteins or peptides, and immunomodulators are also developing. Among them, chemotherapy is still one of the important means of treating cancer. However, while chemotherapy uses cytotoxic drugs to kill tumor cells, it will inevitably affect normal tissues and cause various side effects.

[0003] Tumor cells are characterized by abnormal growth and rapid proliferation. Therefore, most chemotherapy drugs target these characteristics, using compounds with strong cytotoxicity. However, due to this lack of selectivity, these drugs can damage normal tissue while killing tumor cells, adding an additional burden to already vulnerable cancer patients. While some drugs, such as camptothecin derivatives, have a place among anticancer drugs due to their promising effects, their physical properties, such as poor water solubility, make administration difficult and patient compliance is poor.

[0004] Therefore, modifying these drugs to increase their targeting, solubility, stability, and clinical suitability has become a major challenge in the development of chemotherapy drugs. The novel linker of the present invention, which simultaneously provides a trigger, a site for modifying the side chain, and the ability to self-cleave under specific conditions to release the active drug, offers a solution to this problem.

[0005] Glucuronide prodrugs are selectively activated by β-glucuronidase, an enzyme highly expressed in the microenvironment of many solid tumors, including lung, breast, and gastrointestinal cancers. Over the past two decades, they have been used to deliver drugs around tumors. While prodrugs can reduce the toxicity of active drugs, their rapid metabolism through renal clearance has limited their development.

[0006] Human serum albumin (HSA) is an endogenous protein abundant in plasma. As a nutrient, it is preferentially taken up by tumors. Furthermore, when linked to albumin, its large molecular properties can impart an EPR effect to the prodrug. Albumin's stable structure and long half-life of 19 days stabilize and enhance the prodrug's half-life.

[0007] Antibody-Drug Conjugates (ADCs) are produced by coupling a bioactive small molecule drug to a monoclonal antibody (mAb) via a linker. Currently, the vast majority of ADCs are composed of tumor-targeting antibodies coupled to highly cytotoxic small molecule drugs via linkers. Leveraging the specific binding properties of antibodies to target antigens, ADCs deliver small molecule drugs to tumor cells, thereby exerting their tumor-killing effects. There are also cases where proteins and peptides with similar specific binding properties as antibodies, such as specific ligands, antigens, and peptides, are conjugated to linker compounds.

[0008] Summary of the Invention

[0009] In order to address the deficiencies in the prior art, the purpose of the present invention is to propose a new general formula compound of a cleavable linker / branched linker for use in a prodrug delivery system, and further synthetic products thereof, and preparation methods and applications thereof, including applications in a prodrug delivery system. The present invention innovatively proposes for the first time a new approach to trigger drug release by hydrolyzing peptide bonds or glycosidic bonds by cathepsin or β-glucuronidase. Specifically, the linker compound that can be used in a prodrug delivery system of the present invention hydrolyzes peptide bonds into amino groups by cathepsin, and glycosidic bonds into hydroxyl groups by β-glucuronidase or galactosidase; then 1.6 self-cleavage occurs and the corresponding active drug is released through a self-closed ring process.

[0010] The present invention proposes a novel cleavable linker compound for use in a prodrug delivery system, which has one or more independently modifiable sites, allowing different triggers, modified side chains, and / or active drugs to be combined together to form a new compound. The structure of the cleavable linker is shown in the following formula (1):

[0011] Wherein, T represents the linking site for connecting R-TR;

[0012] In the R-TR, TR is selected from one or more of a cathepsin substrate, a cathepsin substrate amino acid or short peptide chain, a β-glucuronidase substrate, a galactosidase substrate, a metalloprotease substrate, an aromatic boronic acid or boronate ester oxidizable to a hydroxyl group, a nitro group, an azide group, an azo group reducible to an amino group, a phosphate ester of a hydroxyl group, and a sulfate ester of a hydroxyl group; R is a substituent group selected from one or more of -H, PEG, maleimide-modified PEG, sugar, folic acid, polypeptides, antibodies, and protecting groups;

[0013] Wherein, A represents the attachment site for ligating LR;

[0014] The LR is selected from H, CH3(CH2) n, nitro group, methoxy group, PEG chain, maleimide-modified PEG, maleimide-modified fatty chain, maleimide-modified polyamino acid chain, fatty chain, polyamino acid chain, sugar chain, fatty acid, sugar, folic acid, polypeptide or antibody;

[0015] Wherein, D represents the attachment site for connecting to DR;

[0016] The DR contains naked -OH, -NH2, -NHR or -SH; the DR is a chemical drug, preferably a camptothecin compound and its derivatives SN-38, DXd, isitecan, belotecan, anti-tumor drugs paclitaxel, docetaxel, podophyllotoxin, CA4;

[0017] Wherein, X is O, N or S;

[0018] Wherein, R2 is H or CH3(CH2) n ; n = 0-20;

[0019] Wherein, R1 is an electron-withdrawing group, including halogen (eg, fluorine, chlorine, bromine), cyano, nitro, carboxylic acid, sulfonic acid, carboxylate, sulfonate, amide, and groups connected by amide bonds.

[0020] The specific structures of the linker compounds include those shown in the following formulas (1-1) and (1-2):

[0021] When the linking site T of formula (1) is connected by an amide bond, its structure is as shown in the following formula (1-1):

[0022] Wherein, TR is selected from single amino acids, dipeptides, and tetrapeptides in cathepsin substrates and metalloprotease substrates; wherein single amino acids are selected from single amino acids without protection or with protection groups, PEG chains, polyamino acid chains, and maleimide-modified amino acids, including -Lys-, -Cit-, -Arg-, -Leu-, -His-, -Asp-, -Glu-, -Ala-, -Val-, -Ile-, -Pro-, -Phe-, -Trp-, -Met-, -Ser-, -Cys-, or -Asn-; dipeptides are selected from single amino acids without protection or with protection groups, Protective groups, PEG chains, polyamino acid chains and maleimide-modified dipeptides include -Phe-Lys-, -Val-Lys-, -Ala-Lys-, -Val-Cit-, -Phe-Cit-, -Leu-Cit-, -Ile-Cit, -Trp-Cit-, -Phe-Gly-, -Ala-Ala- or -Phe-Arg-; tetrapeptides are selected from tetrapeptides without protection or with protective groups, PEG chains, polyamino acid chains and maleimide-modified tetrapeptides, including -Gly-Gly-Phe-Gly-.

[0023] When the linking site T of formula (1) is connected by an ether bond or an ester bond, its structure is as shown in the following formula (1-2):

[0024] Among them, TR includes β-glucoside, β-galactoside, phosphate, sulfate, and pyrophosphate.

[0025] Wherein, A represents the attachment site for ligating LR;

[0026] The LR is selected from H, CH3(CH2) n , nitro group, methoxy group, PEG chain, maleimide-modified PEG, maleimide-modified fatty chain, maleimide-modified polyamino acid chain, fatty chain, polyamino acid chain, sugar chain, fatty acid, sugar, folic acid, polypeptide or antibody;

[0027] Wherein, when the linking site A of formula (1) is preferably an amide bond, the structure of the linker compound is shown in the following formula (1-5):

[0028] Among them, LLR is a linker modification fragment, including one or more of PEG, sugar, alkyl chain, maleimide-modified PEG, modified alkyl chain, and fatty acid.

[0029] The group connected to the connection point T is preferably β-glucoside, β-galactoside, phosphate, sulfate, or pyrophosphate;

[0030] Wherein, when the group connected to the linking site A of formula (1) is preferably H, the structure of the linker compound is shown in the following formula (1-6):

[0031] T is selected from a single amino acid, a dipeptide, or a tetrapeptide selected from a cathepsin substrate or a metalloprotease substrate;

[0032] The single amino acid is selected from the group consisting of unprotected or protected, PEG chains, polyamino acid chains, and maleimide-modified single amino acids, including -Lys-, -Cit-, -Arg-, -Leu-, -His-, -Asp-, -Glu-, -Ala-, -Val-, -Ile-, -Pro-, -Phe-, -Trp-, -Met-, -Ser-, -Cys-, or -Asn-; the dipeptide is selected from the group consisting of unprotected or protected, PEG chains, polyamino acid chains, and maleimide-modified single amino acids. Imidyl-modified dipeptides include -Phe-Lys-, -Val-Lys-, -Ala-Lys-, -Val-Cit-, -Phe-Cit-, -Leu-Cit-, -Ile-Cit, -Trp-Cit-, -Phe-Gly-, -Ala-Ala- or -Phe-Arg-; tetrapeptides are selected from tetrapeptides without protection or with protecting groups, PEG chains, polyamino acid chains and maleimide-modified tetrapeptides, including -Gly-Gly-Phe-Gly-.

[0033] Wherein, in the structural formula (1), when R1 is a nitro group, the structure of the linker compound is shown in the following formula (J):

[0034] Wherein, in the structural formula (1), when the R1 is an amide bond-linked group, the linker compound further includes a linking site L1, and the structure of the linker compound is shown in the following formula (L):

[0035] The group connected to the linking site T is selected from: cathepsin substrate amino acids or short peptide chains; β-glucuronidase substrates; galactosidase substrates; metalloprotease substrates; aromatic boronic acids or boronic esters that can be oxidized to hydroxyl groups; nitro, azide, azo and other groups that can be reduced to amino groups; phosphate esters of hydroxyl groups; sulfate esters of hydroxyl groups, etc.

[0036] The group connected to the linking site A is a linker group LR used to adjust the physical and chemical properties of the compound or to connect antibodies, peptides, albumin, etc.; LR is selected from: H, a fatty chain, a PEG chain, a polyamino acid chain, a sugar chain, and a maleimide-modified PEG chain, a maleimide-modified fatty chain, a maleimide-modified polyamino acid chain, a maleimide-modified sugar chain, etc.;

[0037] D is a pharmaceutically active drug, wherein the active drug D has exposed -OH, -NH2, -NHR, -SH, including but not limited to anti-tumor drugs, anti-inflammatory drugs, anti-aging drugs and drugs for treating fibrosis, etc.;

[0038] X represents the linking site in the active drug D; it is O, N or S;

[0039] Wherein, R2 is H or CH3(CH2) n ; n = 0-20;

[0040] R3 is selected from H, C1-C6 alkyl or cycloalkyl;

[0041] L1 is selected from H, an amino protecting group, a C1-C6 alkyl or cycloalkyl group, a fatty chain, a linear or branched PEG chain, a sugar chain, a polyamino acid chain, a maleimide-modified PEG chain, a maleimide-modified fatty chain, a maleimide-modified polyamino acid chain, or a maleimide-modified sugar chain;

[0042] R3 and L1 can be optionally linked together to form 3 to 6 rings, and the rings formed by R3 and L1 can be selected from

[0043] R4 is selected from one or more of H, C1-C6 alkyl or cycloalkyl;

[0044] L2 is selected from H, an amino protecting group, a C1-C6 alkyl or cycloalkyl group, a fatty chain, a linear or branched PEG chain, a sugar chain, a polyamino acid chain, a maleimide-modified PEG chain, a maleimide-modified fatty chain, a maleimide-modified polyamino acid chain, or a maleimide-modified sugar chain;

[0045] The linker compound (L) includes (L-5) and (L-6), which are respectively represented by the following formulas:

[0046] in,

[0047] In formula (L-5), AA represents an amino acid, m represents the number of amino acids, and m is selected from 1-6; L3 is selected from one or more of H, an amino protecting group, a fatty chain, a PEG chain, a polyamino acid chain, a sugar chain, and a maleimide-modified PEG chain, a maleimide-modified fatty chain, a maleimide-modified polyamino acid chain, and a maleimide-modified sugar chain; R6 and R7 are selected from H or a C1-C6 alkyl or cycloalkyl group, L1 is selected from a C1-C6 alkyl or cycloalkyl group, or R6 and L1 can form a cycloalkyl group; L2 is selected from H, an amino protecting group, a fatty chain, a PEG chain, One or more of a polyamino acid chain, a sugar chain, a maleimide-modified PEG chain, a maleimide-modified fatty chain, a maleimide-modified polyamino acid chain, and a maleimide-modified sugar chain; L2 and L3 are simultaneously or independently selected from H, an amino protecting group, a fatty chain, a PEG chain, a polyamino acid chain, a sugar chain, and one or more of a maleimide-modified PEG chain, a maleimide-modified fatty chain, a maleimide-modified polyamino acid chain, and a maleimide-modified sugar chain; L2 and L3 are not simultaneously selected from a maleimide-modified PEG chain, a fatty chain, a polyamino acid chain, and a sugar chain;

[0048] In formula (L-6), T is selected from one or more of β-glucoside, β-galactoside, phosphate, pyrophosphate or sulfate; L4 is selected from hydrogen, C1-C6 alkyl or cycloalkyl, nitro, amino, methoxy, carboxyl, ester, fatty chain, linear or branched PEG chain, sugar chain, polyamino acid chain R 10 is selected from hydrogen, C1-C3 alkyl or cycloalkyl; L5 is selected from hydrogen, an amino protecting group, a C1-C6 alkyl or cycloalkyl, a fatty chain, a linear or branched PEG chain, a sugar chain, a polyamino acid chain, a maleimide-modified PEG chain, a maleimide-modified fatty chain, a maleimide-modified polyamino acid chain, and a maleimide-modified sugar chain; R6 and R7 are selected together or independently from H or a C1-C6 alkyl or cycloalkyl, L1 is selected from a C1-C6 alkyl or cycloalkyl, or R6 and L1 can form a cycloalkyl; L2 is selected from H, an amino protecting group, a fatty chain, a PEG chain, a polyamino acid chain, a sugar chain, and one or more of a maleimide-modified PEG chain, a maleimide-modified fatty chain, a maleimide-modified polyamino acid chain, and a maleimide-modified sugar chain.

[0049] The present invention also provides a covalently bound prodrug or antibody drug conjugate, which is formed by covalently linking the maleimide of the linker compound represented by Formula (L) and Formula (J) to albumin, an antibody, or an antigen-binding fragment thereof (formed by covalently binding the albumin or antibody fragment), and its structure is shown in the following Formula (L1) to Formula (L8) and Formula (J1) to Formula (J4):

[0050] Wherein, in the above formulas (L1) to (L8), and (J1) to (J4), n, o, m, p, and q represent the number of repeating units, and n, p, and q are selected from 1 to 24; o is selected from 2 to 6; and m is selected from 0 to 12;

[0051] In formula (L1) to formula (L4) and formula (L8), L' is selected from: One or more of;

[0052] In formula (L2), R' is selected from hydrogen, acetyl, amino protecting groups including Boc, Cbz, Fmoc, Alloc, and PEG chains, polyamino acid chains, sugar chains, and fatty chains; in formula (L4), R" is selected from hydrogen, nitro, and methoxy;

[0053] D1 represents camptothecin or other hydroxyl-containing drugs, including but not limited to:

[0054] The present invention also provides the covalently bound prodrug or antibody drug conjugates of formula (L1) to formula (L8) and formula (J1) to formula (J4) including the following formulas ((J1-1)-(J1-3), (J2-1)-(J2-2), (J3-1), (J4-1)-(J4-2), (L1-1)-(L1-4), (L2-1), (L3-1), (L4-1), (L5-1)-(L5-3), (L6-1), (L7-1)-(L7-3), and (L8-1)-(L8-3):

[0055] The antibody or antigen-binding fragment thereof is selected from rabbit antibodies, mouse antibodies, chimeric antibodies, humanized antibodies or fully human antibodies or protein fragments with similar functions to antibodies.

[0056] Furthermore, the antibody or antigen-binding fragment thereof is selected from the group consisting of anti-CD3 antibody, anti-FOLR1 antibody, anti-ROR1 antibody, anti-TNFα antibody, anti-TF antibody, anti-EpCAM antibody, anti-EGFRvIII antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-MUC16 antibody, anti-ENPP3 antibody, anti-TDGF1 antibody, anti-ETBR antibody, anti-MSLN antibody, anti-TIM-1 antibody, anti-LRRC15 antibody, anti-LIV-1 antibody, anti-CanAg / AFP antibody, anti-Claudin 6 antibody, anti-Claudin 9 antibody, anti-Claudin 18.2 antibody, anti-Mesothelin antibody, anti-HER2 antibody, anti-EGFR antibody, anti-c-MET antibody, anti-SLITRK6 antibody, anti-KIT / CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7 / CS1 antibody, anti-NaPi2B / SLC34A2 antibody, anti-GPNMB antibody, anti-HER3 antibody, anti-MUC1 / CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7-H3 (CD276) antibody, anti-PTK7 / CCK4 antibody, anti-PRLR antibody, anti-EFNA4 antibody, anti-5T4 antibody, anti-NOTCH3 antibody, anti-Nectin 4 antibody, anti-TROP-2 antibody, anti-CD142 antibody, anti-CA6 antibody, anti-GPR20 antibody, anti-CD174 antibody, anti-CD70 antibody, anti-CD71 antibody, anti-EphA2 antibody, anti-LYPD3 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FRα antibody, anti-CEACAMs antibody, anti-GCC antibody, anti-IntegrinAv antibody, anti-CAIX antibody, anti-P-cadherin antibody, anti-GD3 antibody, anti-Cadherin 6 antibody, anti-LAMP1 antibody, anti-FLT3 antibody, anti-BCMA antibody, anti-CD79b antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD74 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD37 antibody, anti-CD47 antibody, anti-CD138 antibody, anti-CD352 antibody, anti-CD25 antibody, and anti-CD123 antibody;

[0057] And / or, the antibody or antigen-binding fragment thereof is selected from:

[0058] i) Trastuzumab antibody;

[0059] ii) ILB-3101 antibody;

[0060] iii) 59H4 antibody.

[0061] The coupling ratio of the antibody or antigen-binding fragment thereof to the linker compound is 3-8.

[0062] The coupling ratio is optimized to be 6-8 or 4-6.

[0063] The present invention also provides a method for preparing a linker of formula (L), the preparation reaction process of which is shown in the following formula:

[0064] The reaction steps include:

[0065] a) Substitution reaction: Under alkaline conditions, compound 1 undergoes a nucleophilic substitution reaction with a conventional hydroxyl protecting group (P) such as benzyl bromide or p-methoxybenzyl bromide (MOMCl). The base is potassium carbonate, cesium carbonate, sodium carbonate, etc., and the solvent is acetone, acetonitrile, or NN-dimethylformamide.

[0066] b) Reduction reaction: Compound 2 is reduced with zinc powder / acid or iron powder / acid system to obtain compound 3, and the solvent is a conventional organic solvent / acid.

[0067] c) Condensation reaction: Under nitrogen conditions, compound 3 reacts with formic acid and acetic anhydride to produce compound 4;

[0068] d) Reduction reaction: Under nitrogen conditions, compound 4 and borane dimethyl sulfide are dissolved in a solvent and undergo a reduction reaction to produce compound 5;

[0069] e) Substitution reaction: Under alkaline conditions, compound 5 and BOC anhydride undergo substitution reaction to obtain compound 6, where the base is DIPEA, DMAP, etc.

[0070] f) Hydrolysis reaction: Under alkaline conditions, compound 6 undergoes hydrolysis reaction to obtain compound 7, and the base is LiOH, NaOH, KOH, etc.

[0071] g) Condensation reaction: Compound 7 undergoes a condensation reaction with an amine condensing agent to produce compound 8. The condensing agent can be a commonly used condensing agent such as DCC, EEDQ, HATU, EDC / HOBt, PyBOP, etc.

[0072] h) Decomposition reaction: Compound 8 is subjected to a deprotection reaction such as Pd / C to generate compound 9, and the solvent is methanol or a mixed solvent of methanol, tetrahydrofuran and ethyl acetate.

[0073] i) Etherification reaction: Under nitrogen protection, benzyl bromide substrates with different trigger fragments are reacted with silver oxide or base to generate compound 10 in anhydrous tetrahydrofuran, anhydrous dichloromethane or anhydrous acetonitrile as solvent.

[0074] j) Deprotection reaction: Compound 10 is deprotected by N-Boc under acidic conditions to obtain compound 11. The acid is hydrochloric acid, trifluoroacetic acid, etc., and the solvent is tetrahydrofuran or dichloromethane, etc.

[0075] k) Condensation reaction: Under nitrogen protection, compound 11 is dissolved in an organic solvent, and bis(trichloromethyl) carbonate and an anhydrous organic base such as triethylamine, DIPEA, pyridine, etc. are added. After reacting for 1 hour, drug (D) and DMAP are added. The reaction is carried out at 20-60°C overnight to obtain compound 12. The drug (D) must have exposed -OH, -NH2, -NHR, or -SH. The solvent is anhydrous tetrahydrofuran or dichloromethane.

[0076] In a specific embodiment, the method comprises the following steps:

[0077] a) Substitution reaction: Under alkaline conditions, compound 1 and a conventional hydroxyl protecting group (P) such as benzyl bromide, p-methoxybenzyl bromide (MOMCl), etc., undergo a nucleophilic substitution reaction to produce compound 2. The base is potassium carbonate, cesium carbonate, sodium carbonate, etc., and the solvent is acetone, acetonitrile, or NN-dimethylformamide. The reaction temperature is 20-80°C, wherein the molar ratio of compound 1 to the base is 1:1.5-2; and the molar ratio of compound 1 to the protecting group is 1:1.5-2.

[0078] b) Reduction reaction: Compound 2 is reduced with zinc powder / acid or iron powder / acid to obtain compound 3. The solvent is a conventional organic solvent / acid. The reaction temperature is 20-80°C. The molar ratio of compound 2 to iron powder or zinc powder is 1:5.

[0079] c) Condensation reaction: Under nitrogen conditions, compound 3 reacts with formic acid and acetic anhydride to produce compound 4. The molar ratio of compound 3 to formic acid is 1:5-10, and the molar ratio of compound 3 to acetic anhydride is 1:5-10. The reaction temperature is 0-80°C and the reaction time is 2-3 hours. The solvent is anhydrous tetrahydrofuran.

[0080] d) Reduction reaction: Under nitrogen conditions, compound 4 and borane dimethyl sulfide are dissolved in a solvent and subjected to a reduction reaction to produce compound 5. The molar ratio of compound 4 to borane dimethyl sulfide is 1:3. The reaction temperature is 0-5°C and the reaction time is 2-3 hours. The solvent is anhydrous tetrahydrofuran.

[0081] e) Substitution reaction: Under alkaline conditions, compound 5 and BOC anhydride undergo substitution reaction to obtain compound 6. The base is DIPEA, DMAP, etc. The reaction temperature is 20-80°C and the solvent is BOC anhydride.

[0082] f) Hydrolysis reaction: Under alkaline conditions, compound 6 undergoes hydrolysis reaction to obtain compound 7. The base is LiOH, NaOH, KOH, etc., the molar ratio of compound 4 to borane dimethyl sulfide is 1:2-5, the reaction temperature is 0-30°C, and the solvent is tetrahydrofuran / methanol / acetonitrile / dioxane and water.

[0083] g) Condensation reaction: Compound 7 reacts with an amine in the presence of a condensing agent to produce compound 8. The condensing agent may be a commonly used condensing agent such as DCC, EEDQ, HATU, EDC / HOBt, or PyBOP. The molar ratio of compound 7 to amine is 1:1-2, and the molar ratio of compound 7 to condensing agent is 1:2-5. The reaction temperature is 0-30°C, and the solvent is a conventional organic solvent such as dichloromethane, NN-dimethylformamide, or acetonitrile.

[0084] h) Decomposition reaction: Compound 8 undergoes a deprotection reaction such as Pd / C to generate compound 9. The molar ratio of compound 7 to Pd / C is 1:0.1-0.2. The reaction temperature is 0-60°C. The solvent is methanol or a mixed solvent of methanol and other organic solvents including tetrahydrofuran and / or ethyl acetate.

[0085] i) Etherification: Under nitrogen protection, benzyl bromide, a triggering substrate, reacts with silver oxide or a base to produce compound 10. Compound 9 can also react with a benzyl alcohol, a triggering substrate, in the presence of a phosphine reagent and an azo compound to produce compound 10. The molar ratio of compound 9 to various benzyl bromides or benzyl alcohols is 1:1-1.2, and the molar ratio of compound 9 to silver oxide / phosphine reagent / azo compound is 1:1.2-2. The reaction temperature is 0-60°C, and the solvent is anhydrous tetrahydrofuran, anhydrous dichloromethane, or anhydrous acetonitrile.

[0086] j) Deprotection reaction: Compound 10 is deprotected by N-Boc under acidic conditions to obtain compound 11. The acid is hydrochloric acid, trifluoroacetic acid, etc. The reaction temperature is 0-60°C. The solvent is tetrahydrofuran / water or dichloromethane, etc.

[0087] l) Condensation reaction: Under nitrogen protection, compound 11 is dissolved in an organic solvent, and bis(trichloromethyl) carbonate and an anhydrous organic base are added. The molar ratio of compound 11 to bis(trichloromethyl) carbonate is 1:0.5, and the molar ratio of compound 11 to anhydrous organic base is 1:3-5. After reacting for 1 hour, drug (D) and DMAP are added, and the reaction is carried out at 20-60°C overnight to obtain compound 12. The molar ratio of compound 11 to the drug (D) derivative is 1:1-1.2. The drug (D) must contain exposed -OH, -NH2, -NHR, or -SH. The solvent is anhydrous tetrahydrofuran or dichloromethane.

[0088] The present invention also provides a method for preparing a covalently bound prodrug or antibody-drug conjugate (J1). The reaction process of the preparation method is shown in the following formula:

[0089] The reaction steps include:

[0090] 1) Bromination reaction: Compound 1 is brominated to obtain compound 2; the bromination reagent is phosphorus tribromide, triphenylphosphine, carbon tetrabromide, etc., and the solvent is anhydrous dichloromethane;

[0091] 2) Etherification reaction: Compound 2 is subjected to an etherification reaction to generate compound 3, wherein the organic solvent is anhydrous acetonitrile;

[0092] 3) Reduction reaction: Under nitrogen conditions, compound 3 and borane dimethyl sulfide are dissolved in a solvent to undergo a reduction reaction to produce compound 4, and the solvent is anhydrous tetrahydrofuran;

[0093] 4) Condensation reaction: Under nitrogen protection, compound 4 was dissolved in an organic solvent, and bis(trichloromethyl) carbonate and triethylamine were added. After reacting for 1 hour, a hydroxyl-containing drug was added and the reaction was carried out at room temperature overnight to obtain compound 5;

[0094] 5) Deprotection: Under nitrogen protection, compound 5 is dissolved in an organic solvent, and a 0.2 M aqueous base solution is added under ice bath condition to deprotect to obtain compound 6, wherein the base is one or a mixture of lithium hydroxide, sodium hydroxide, and potassium hydroxide; the organic solvent is one or a mixture of methanol, acetonitrile, tetrahydrofuran and water;

[0095] 6) Condensation reaction: Compound 6 and Compound 9 are dissolved in an organic solvent and stirred at room temperature for 6-8 hours to react to form the prodrug having the general formula (J1). The organic solvent is one or a mixture of methanol, acetonitrile, tetrahydrofuran, N,N-dimethylformamide and water.

[0096] In a specific embodiment, the method comprises the following steps:

[0097] 1) Bromination reaction: Compound 1, triphenylphosphine, and carbon tetrabromide are dissolved in a solvent and reacted at room temperature overnight to obtain Compound 2. The molar ratio of Compound 1 to triphenylphosphine is 1:3, and the molar ratio of Compound 1 to carbon tetrabromide is 1:3. The solvent is anhydrous dichloromethane.

[0098] 2) Substitution reaction: Compound 2, N-(2-hydroxy-5-nitrophenyl)formamide, and silver oxide react in an organic solvent to produce Compound 3. The molar ratio of Compound 2 to N-(2-hydroxy-5-nitrophenyl)formamide is 1:1, and the molar ratio of Compound 2 to silver oxide is 1:1.4. The reaction time is 12-14 hours. The organic solvent is anhydrous acetonitrile.

[0099] 3) Reduction reaction: Under nitrogen conditions, compound 3 and borane dimethyl sulfide are dissolved in a solvent and subjected to a reduction reaction to produce compound 4. The molar ratio of compound 3 to borane dimethyl sulfide is 1:3. The reaction temperature is 0-5°C and the reaction time is 2-3 hours. The solvent is anhydrous tetrahydrofuran.

[0100] 4) Condensation reaction: Under nitrogen protection, compound 4 is dissolved in an organic solvent, and bis(trichloromethyl) carbonate and triethylamine are added. The molar ratio of compound 4 to bis(trichloromethyl) carbonate is 1:0.5, and the molar ratio of compound 4 to triethylamine is 1:4. After reacting for 1 hour, a camptothecin derivative is added and reacted at room temperature overnight to obtain compound 5. The molar ratio of compound 4 to the camptothecin derivative is 1:1. The camptothecin derivative is SN38.

[0101] 5) Deprotection: Under nitrogen protection, compound 5 is dissolved in an organic solvent, and a 0.2 M aqueous base solution is added under ice bath conditions to deprotect to obtain compound 6. The reaction time is 0.2-1 hour, and the molar ratio of compound 5 to the base is 1:8. The base is one or a mixture of lithium hydroxide, sodium hydroxide, and potassium hydroxide. The organic solvent is one or a mixture of methanol, acetonitrile, tetrahydrofuran, and water.

[0102] 6) Condensation reaction: Compound 6 and Compound 9 are dissolved in an organic solvent and stirred at room temperature for 6-8 hours to react to form the prodrug having the general formula (J-1). The molar ratio of Compound 6 to Compound 9 is 1:1. The organic solvent is selected from methanol, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, and water, or a mixture thereof.

[0103] Another object of the present invention is to provide a possible drug release mechanism under different conditions by connecting a ligand, a trigger and a drug to a linker to form a targeted drug linker.

[0104] The linker hydrolyzes the peptide bond into an amino group by tissue protease, or the glycosidic bond into a hydroxyl group by β-glucuronidase or galactosidase; then 1.6 self-cleavage occurs and the corresponding active drug is released through a self-closing ring process.

[0105] The present invention also provides a drug or pharmaceutical composition comprising the linker compound, its pharmaceutically acceptable salt, solvate, polymorph, isomer, derivative, or the covalently bound prodrug or antibody conjugate, and a pharmaceutically acceptable excipient, diluent or carrier.

[0106] The present invention also proposes an application, wherein the linker compound (1) includes but is not limited to compounds of formula (J1-1), formula (J4-1), formula (L4-1), formula (L7-1), formula (L1-1), formula (L7-2), formula (L7-3), formula (L7-4), etc., and / or the covalently bound prodrugs or antibody-drug conjugates, as well as drugs and / or pharmaceutical compositions containing the aforementioned compounds, are used in the preparation of anti-tumor drugs and in the preparation of albumin-bound anti-tumor drugs; and also includes the application in the preparation of tumor treatment for albumin-bound β-glucuronidase prodrugs and antibody-drug conjugates for tumor treatment.

[0107] The tumor is a solid tumor or blood tumor such as breast cancer, ovarian cancer, ovarian teratoma, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethra cancer, testicular cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, head and neck cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, neural tumor, glioma, neuroblastoma, glioblastoma multiforme, squamous cell carcinoma, myeloma, sarcoma, lymphoma and leukemia.

[0108] The present invention has advantages and beneficial effects including, but not limited to, a novel cleavable linker (Formula (I)) for use as a prodrug, proposed herein. The linker has multiple independently modifiable sites, allowing different triggers, modified side chains, and / or active drugs to be combined to form a new compound. This makes the active drug more stable, less toxic, and more targeted while maintaining activity. The present invention provides albumin-binding camptothecin derivative prodrugs that are cleaved by β-glucuronidase, triggering the self-cleavage of the linker, as well as methods for their preparation and use.

[0109] In a specific embodiment, experiments have shown that the compound formula (J) composed of the present linker, including but not limited to formula (J1-1) and formula (J4-1), has excellent in vitro and in vivo stability and can rapidly release active drugs under conditions simulating a tumor microenvironment. After binding to albumin, the in vivo metabolic time is effectively improved. Among them, compound (J1-1) significantly prolonged the survival period of mice and inhibited tumor growth in an anti-tumor experiment in an HCT116 xenograft tumor mouse (NCG or equivalent) model. This shows that the new linker and the compound constructed based on it can achieve the purpose of rapid drug release at the tumor site, improve the drugability of the drug, and increase the anti-cancer effect of the drug.

[0110] In specific embodiments, experiments have shown that compounds of Formula (J) composed of the present linker, including but not limited to Formula (J1-2), Formula (J1-3), Formula (J2-1), Formula (L1-3), Formula (L1-4), Formula (L5-1), and Formula (L5-3), after being conjugated to multiple antibodies, produce ADC drugs with different DAR values, exhibiting anti-tumor activity in various cell lines in vitro and strong anti-tumor effects in various mouse CDX models, some of which were superior to the positive control ADC. This indicates that the novel linker and the compounds constructed based on it can exhibit excellent anti-tumor activity after conjugation with antibodies and can be used to treat various cancers such as lung cancer, breast cancer, and colon cancer.

[0111] The prodrug compound, linker compound and linker-payload of the present invention have the same meaning.

[0112] The antibodies are selected from the invention patent document CN 2023108539993, including:

[0113] Antibody 3101 is clone 7B7, an anti-human B7-H3 antibody, with the following heavy and light chain sequences:

[0114] 3101 heavy chain sequence:

[0115] 3101 light chain sequence:

[0116] Trastuzumab, an anti-human HER2 antibody, has the following heavy and light chains:

[0117] Trastuzumab heavy chain sequence:

[0118] Trastuzumab light chain sequence:

[0119] Clone 59H4 is an anti-human DLL3 antibody, selected from invention patent document CN 2023108539993, and the sequence is as follows:

[0120] 59H4 heavy chain sequence:

[0121] 59H4 light chain sequence:

[0122] The antibody of DS-7300 is derived from Daiichi Sankyo's invention patent US9808537.

[0123] Antiviral antibodies were used as negative control antibodies and did not bind to the target antigen.

[0124] The isotype antibody sequences are as follows:

[0125] Isotype heavy chain sequence:

[0126] Isotype light chain sequence:

[0127] All of the above antibodies can be expressed, purified and used for conjugate preparation according to the method of invention patent document CN 2023108539993.

[0128] The linker-payload of the positive control conjugate can be Deruxtecan (CAS 1599440-13-7), which is described in US Pat. No. 9,808,537 and is commercially available or synthesized according to the patented method.

[0129] The linker-payload of the positive control conjugate can be AZ0133, which is the linker-payload of compound SG3932 described in patent WO2022 / 053685, that is, AZD8205, which is commercially available or synthesized according to its patented method.

[0130] The antibody was conjugated with the linker compound of the present invention, Deruxtecan and AZ0133 respectively to prepare conjugates for in vitro and in vivo activity evaluation.

[0131] The present invention innovatively proposes a new cleavable linker compound that can be used in a prodrug delivery system, wherein the trigger fragment is connected to the new linker through a para-hydroxyl group or a para-amino group, the active drug is connected to the new linker through its own hydroxyl group, and the modified fragment is connected to the new linker through the amino group. According to its abundant modification sites, different modification methods are selected to adapt to different drug delivery needs, so as to achieve the purpose of improving targeting, reducing the toxicity of active drugs, and improving drugability. After administration of the present invention, the trigger fragment is released at a specific target site in the body, the modified fragment is stably circulated in the body for a long time, and the active drug portion provides a therapeutic effect, ultimately achieving accumulation and directional drug release at the target site, reducing the toxic and side effects of the active drug, and improving the drugability of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0133] FIG1 shows the stability of the prodrug compound (J1-1) according to an example of the present invention in PBS and mouse plasma.

[0134] FIG2 shows the release rate of the prodrug compound (J1-1) according to an example of the present invention under the action of β-glucuronidase.

[0135] FIG3 shows the inhibition of cell growth in the presence of a prodrug compound (J1-1) according to an example of the present invention and β-glucuronidase.

[0136] FIG4 is a graph showing the time and tumor volume changes of the in vivo tumor inhibition experiment of the prodrug compound (J1-1) of the example of the present invention.

[0137] Figures 5A, B, and C are graphs showing the time and tumor volume changes of the in vivo tumor inhibition experiment of the B7-H3 ADC according to an embodiment of the present invention.

[0138] Figures 6A and 6B are graphs showing the time and tumor volume changes of the in vivo tumor inhibition experiment of the B7-H3 ADC according to an embodiment of the present invention.

[0139] FIG7 is a graph showing the time and tumor volume changes of an in vivo tumor inhibition experiment of a HER2 ADC according to an embodiment of the present invention.

[0140] FIG8 is a graph showing the time and tumor volume changes of an in vivo tumor inhibition experiment of a DLL3 ADC according to an embodiment of the present invention.

[0141] FIG9 shows the inhibitory activity of the payloads according to the examples of the present invention on tumor cell growth in vitro.

[0142] FIG10 shows the inhibitory activity of the conjugates of the examples of the present invention on tumor cell growth in vitro. DETAILED DESCRIPTION

[0143] The invention is further described in detail with reference to the following specific examples and accompanying drawings. The processes, conditions, experimental methods, etc. for implementing the present invention, except for those specifically mentioned below, are common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0144] Example 1 Synthesis of Compound (J1-1)

[0145] The synthesis of J1-1 refers to Route A.

[0146] Synthesis of compound 2

[0147] Carbon tetrabromide (1.77 g, 5.36 mmol) was weighed into a three-necked flask, the atmosphere was replaced with nitrogen, and 20 mL of anhydrous dichloromethane was injected. A solution of triphenylphosphine (1.4 g, 5.36 mmol) in anhydrous dichloromethane was added dropwise under an ice bath. After stirring for 30 minutes, a solution of compound 1 (1 g, 1.34 mmol) in anhydrous dichloromethane was added dropwise. The reaction was allowed to react at room temperature overnight. TLC confirmed the reaction was complete, and the product was concentrated under reduced pressure to yield a yellow oil. Column chromatography (dichloromethane:methanol = 100:1) was performed to obtain 750 mg of a light yellow, fluffy solid, with a yield of 83.9%. NMR (400MHz, CDCl3) δ8.45(d,J=20.7Hz,1H),8.03(d,J=21.8Hz,1H),7.73(dd,J=21.2,7.0Hz,2H) ,7.57(d,J=13.9Hz,2H),7.41–7.28(m,3H),7.21(s,2H),7.06(dd,J=21.1,8.2Hz,1H),6.89(dd,J= 21.4,8.3Hz,1H),5.60(s,1H),5.41–5.21(m,3H),5.03(dd,J=21.1,6.9Hz,1H),4.44(d,J=21.7Hz ,2H),4.39–4.16(m,3H),3.71(d,J=21.5Hz,3H),3.55(s,2H),2.68(s,2H),2.02(d,J=18.8Hz,9H).

[0148] Synthesis of compound 3

[0149] Silver oxide (300 mg, 1.29 mmol), 2-hydroxy-5-nitroformamidobenzene (186 mg, 1.02 mmol), and compound 2 (750 mg, 0.92 mmol) were placed in a three-necked flask. The atmosphere was purged with nitrogen and 10 mL of anhydrous dichloromethane was injected. The mixture was allowed to react overnight at room temperature. TLC confirmed the reaction was complete, and the filtrate was filtered through celite. The filtrate was collected and concentrated under reduced pressure to obtain the crude product. Column chromatography (dichloromethane:methanol = 100:1 to 50:1) afforded 500 mg of a white, fluffy solid in a yield of 52.2%. NMR (400MHz, CDCl3) δ9.27(d,J=17.6Hz,1H),8.47(t,J=21.4Hz,2H),8.12(d,J=19.8Hz,1 H),8.02–7.91(m,2H),7.52(s,3H),7.45(s,3H),7.34(d,J=11.2Hz,1H),7.22(s,2H),7.06 –6.88(m,3H),5.43–5.35(m,1H),5.29(d,J=7.9Hz,2H),5.12(d,J=20.3Hz,3H),4.34(s,2 H), 4.17 (s, 2H), 3.71 (d, J = 14.6Hz, 3H), 3.56 (s, 2H), 2.70 (s, 2H), 2.03 (d, J = 17.1Hz, 9H).

[0150] Synthesis of compound 4

[0151] Under strict nitrogen protection, compound 3 (500 mg, 0.55 mmol) was weighed into a three-necked flask. The nitrogen atmosphere was replaced, and 10 mL of anhydrous tetrahydrofuran was injected. Borane dimethyl sulfide (0.875 mL, 1.64 mmol) was slowly added dropwise under an ice-salt bath. The reaction was allowed to react at room temperature for three hours. TLC confirmed the reaction was complete, and the mixture was concentrated under reduced pressure to yield 420 mg of a yellow solid, in an 85.4% yield.

[0152] Synthesis of compound 5

[0153] Compound 4 (100 mg, 0.11 mmol) and triphosgene (12.56 mg, 0.04 mmol) were weighed in a three-necked flask, the atmosphere was replaced with nitrogen, and 10 mL of anhydrous tetrahydrofuran was injected. DIEA (51.7 mg, 0.42 mmol) was slowly added dropwise under an ice bath and allowed to react at room temperature for one hour. SN38 (48 mg, 0.12 mmol) was added to the reaction system and stirred at 40°C overnight. The reaction was monitored for completion by TLC. 10 mL of dichloromethane was added, and the mixture was washed twice with 1M dilute hydrochloric acid and once with saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. Purification by column chromatography (dichloromethane:methanol = 100:1 to 50:1) afforded 118 mg of a yellow solid, with a two-step yield of 81.0%.1H NMR (400MHz, CDCl3) δ8.56(s,1H),8.33(s,1H),8.28–8.22(m,1H),8.19–8.06(m,2H),7.84(s,1H),7.64(t,J=22.0Hz,5H),7.47(d ,J=6.8Hz,2H),7.30(d,J=8.0Hz,2H),7.21–7.06(m,4H),7.01(s,1H),5.97(s,1H),5.73(d,J=16.2Hz,1H),5.50–5.36(m,1H),5.35 –5.26(m,4H),5.22(d,J=14.3Hz,3H),5.14(d,J=7.0Hz,3H),4.33–4.18(m,3H),3.61(s,2H),3.40(s,3H),3.15(dd,J=15.2,7.5Hz ,1H),2.97(s,2H),2.75(s,2H),2.06(s,9H),1.86(dd,J=14.0,7.0Hz,2H),1.40(t,J=7.7Hz,3H),1.01(dd,J=15.6,7.5Hz,3H).13C NMR (101MHz, CDCl3) δ173.89,173.80,170.47,170.44,169.82,169.45,166.70,159.20,157.63,157.51,156.50,150.19,146.9 3,146.84,146.56,143.88,141.45,141.11,131.86,131.61,129.51,127.53,127.37,127.30,127.19,126.94,125.06,124.98,1 19.74,118.62,114.36,114.16,112.63,112.61,98.16,98.14,72.87,72.44,71.25,71.01,70.84,69.17,66.64,66.30,66.25,53.48,53.25,49.43,49.33,47.06,37.90,37.08,36.96,31.59,31.50,23.18,23.03,20.85,20.58,20.49,14.00,13.93,7.86.

[0154] Synthesis of compound 6

[0155] Under ice, compound 5 (118 mg, 0.09 mmol) was weighed into a vial, 1 mL of water and 1 mL of acetonitrile were added, and 0.5 mL of 1 M aqueous lithium hydroxide solution was slowly added dropwise. The reaction was allowed to react at room temperature for one hour. LC-MS monitored the reaction completion. The solution was concentrated under reduced pressure to afford 66 mg of a yellow solid (92.1% yield).

[0156] Synthesis of compound (J1-1)

[0157] Compound 6 (66 mg, 0.07 mmol) and succinimidyl 6-(maleimido)hexanoate (53 mg, 0.17 mmol) were weighed into a vial, 2 mL of acetonitrile and 1 mL of methanol were added, and the pH of the system was adjusted to 8 with DIEA. The reaction was allowed to react at room temperature for five hours. LC-MS monitored the reaction for completion, and the product was concentrated under reduced pressure. The product was purified by reverse-phase column chromatography (0.1% aqueous trifluoroacetic acid:acetonitrile = 15:1 to 30:1), concentrated under reduced pressure, and lyophilized to obtain 45 mg of an off-white solid (59.2% yield). 1H NMR(400MHz,DMSO)δ12.81(s,1H),9.12(s,1H),8.53(s,1H),8.34(s,1H) ,8.28(s,1H),8.12(d,J=8.6Hz,1H),7.87(s,1H),7.80(s,1H),7.49(s,2H ),7.32(s,1H),7.22(d,J=7.6Hz,1H),7.15(d,J=7.2Hz,1H),6.98(s,1H), 6.53(s,1H),5.87(s,1H),5.44(s,2H),5.36(d,J=31.7Hz,4H),4.93(d,J= 6.1Hz,1H),3.93(d,J=9.1Hz,1H),3.42(d,J=7.3Hz,10H),3.30(s,3H),3.06(s,2 H),2.00(d,J=5.0Hz,2H),1.93–1.80(m,2H),1.44(d,J=6.6Hz,4H),1.22(d,J=9.0 Hz,3H),1.15(d,J=7.3Hz,2H),0.88(s,3H).13CNMR(101MHz,DMSO)δ172.96,172.5 9,171.53,170.50,170.14,159.64,157.28,153.73,152.28,150.50,150.13,146. 79,146.54,146.39,145.76,145.68,141.14,134.89,131.86,131.61,130.40,129.35,128.99,127.42,127.36,125.18,123.41,120.90,119.41,116.70,115.35,1 14.37,102.04,97.09,75.93,75.44,73.33,72.86,71.81,71.11,65.74,50.03,37.88,37.40,36.98,35.56,35.46,30.74,28.22,26.25,25.19,22.57,14.24,8.25.

[0158] Example 2 Stability Experiment

[0159] A PBS solution (1 mM, pH 7.4) of the compound (J1-1) prepared in Example 1 of the present invention was incubated at 37° C. Corresponding samples (n=3) were collected on day 1, day 2, day 3, day 5, and day 7, and the contents of (J1-1) and SN38 were determined by HPLC.

[0160] Mouse plasma stability: 40 μL of a PBS solution (1 mM, pH 7.4) of the compound (J1-1) prepared in Example 1 of the present invention was incubated with 360 μL of mouse plasma at 37°C on a shaker. Corresponding samples (n=3) were taken on day 1, day 2, day 3, day 5, and day 7, precipitated with cold acetonitrile, centrifuged at 4°C for 30 minutes, and the supernatant was collected and the SN38 content was determined by HPLC. The experimental results are shown in Figure 1, which shows that within seven days, the compound (J1-1) prepared in Example 1 of the present invention released less than 2% of SN38 in mouse plasma containing PBS at pH 7.4, indicating that the compound (J1-1) prepared in Example 1 of the present invention has excellent stability, indicating that the phenolic hydroxyl prodrug design of the present invention is sufficiently stable.

[0161] Example 3 Enzyme release experiment

[0162] A solution of compound (J1-1) prepared in Example 1 of the present invention (100 μM, pH 7.4 or pH 5.5) was mixed with β-glucuronidase (300 U) and incubated on a shaker at 37°C. Corresponding samples (n=3) were collected at 1, 2, 5, 10, 20, 45, 60, and 90 minutes, precipitated with cold acetonitrile, and centrifuged at 4°C for 15 minutes. The supernatant was collected and the SN38 content was determined by HPLC.

[0163] The experimental results are shown in Figure 2. Within 45 minutes, SN38 was released 100% under the triggering of β-glucuronidase at either pH 7.4 or pH 5.5, demonstrating that compound (J1-1) prepared in Example 1 of the present invention can rapidly release the prodrug, converting it to SN38 with antitumor activity. This demonstrates the unexpectedly complete release of the phenolic hydroxyl group of SN38.

[0164] Example 4 In vitro drug cytotoxicity experiment

[0165] The MTT method was used to determine the in vitro cytotoxicity of the compound (J1-1) prepared in Example 1 of the present invention to HCT116, A549, MCF-7, HepG2, 4T1 cells, and in the presence of β-glucuronidase. 200 μL of culture fluid containing 4000 cells was spread in the wells of a 96-well plate. After adhering to the wall in an incubator (37° C., 5% CO2) for 24 hours, the culture fluid was aspirated, and 200 μL of drug solution containing different concentrations or with the addition of β-glucuronidase was added, and the cells were cultured in an incubator for 72 hours. The culture fluid in the plate was aspirated, 100 μL of MTT solution (0.5 mg / mL) was added, and after being placed in an incubator for 3 hours, 100 μL of triple solution was added to each well and placed in an incubator for 12 hours. The absorbance (OD) at 570 nm was measured using a microplate reader. Cell viability was calculated according to the following formula:

[0166] OD sample is the absorbance value of the drug-treated group, OD blank OD is the absorbance value of the complete culture medium only. control The absorbance value of the inoculated cells without drug.

[0167] The experimental results are shown in FIG3 , which show that, at the cellular level, the compound (J1-1) prepared in Example 1 of the present invention has low toxicity and can release active drugs under the triggering of β-glucuronidase to kill tumor cells.

[0168] Example 5 Antitumor Effect on HCT116 Xenograft Tumor in Nude Mice

[0169] Female BALB / c nude mice (4 weeks old) were selected and adapted to the center of the barrier for three days. HCT116 cells (5×10 6 ) was subcutaneously inoculated into the right flank of nude mice. When the tumor grew to 80-100 mm 3 Afterwards, tumor-bearing nude mice were randomly divided into four groups of six mice each. Each group was injected with physiological saline (blank group), 30 mg / kg of (J1-1), and 60 mg / kg of the compound prepared in Example 1 of the present invention (J1-1) (supplementary treatment group) via the tail vein. The administration was repeated twice a week for a total of five times. The body weight and tumor volume of the nude mice were then recorded.

[0170] The experimental results are shown in Figure 4, which show that the compound (J1-1) prepared in Example 1 of the present invention exhibited excellent therapeutic effects in the HCT116 cell xenograft tumor model. After five doses, the tumor volume was significantly reduced or even disappeared, indicating that the compound (J1-1) prepared in Example 1 of the present invention has the ability to inhibit tumor growth.

[0171] Example 6 Synthesis of Compound (L1-1)

[0172] Key intermediate A1 was synthesized using route 1

[0173] To a solution of methyl 3-nitro-4-hydroxybenzoate (20 g, 100 mmol) in N,N-dimethylformamide (100 mL) were added potassium carbonate (27.6 g, 200 mmol) and benzyl bromide (17.8 mL, 150 mmol). After stirring at 60°C for 6 hours, the mixture was poured into a mixture of 400 mL of water and 150 mL of ethyl acetate. The organic phase was washed sequentially with water (50 mL x 2) and saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was washed with petroleum ether and purified to yield the desired product A1-1 (27.5 g, 96% yield). MS: 288 (M+H). + . 1H NMR (400MHz, CDCl3) δ8.52(d,J=2.0Hz,1H),8.18(dd,J=8.8,2.0Hz,1H),7.58–7.30(m,5H),7.18(d,J=8.8Hz,1H),5.31(s,2H),3.94(s,3H).

[0174] To a solution of A1-1 (27.5 g, 96 mmol) in a mixed solvent of acetic acid and tetrahydrofuran (80 / 80 ml) was added activated zinc powder (31.2 g, 480 mol). The mixture was stirred at 70°C for 2 hours and filtered. The filtrate was slowly poured into a saturated aqueous sodium bicarbonate solution to precipitate a solid. The solid was filtered and the filter cake was dried to obtain the desired product A1-2 (21.9 g, 89% yield). MS: 258 (M+H). + . 1H NMR (400MHz, CDCl3) δ7.49–7.33 (m, 7H), 6.87 (d, J = 8.3Hz, 1H), 5.14 (s, 2H), 3.86 (s, 3H).

[0175] 40 ml of formic acid and 100 ml of acetic anhydride were stirred at 70°C for 2 h and then cooled to room temperature. A solution of A1-2 (21.9 g, 85 mmol) in tetrahydrofuran (150 ml) was slowly added to the reaction system. After stirring at room temperature for 2 hours, most of the solvent was removed by distillation under reduced pressure. The remaining solution was slowly poured into saturated aqueous sodium bicarbonate solution to precipitate a white solid. The solid was filtered and the filter cake was dried to give the desired product A1-3 (21 g, 87% yield). MS: 286 (M+H). +. 1H NMR (400MHz, CDCl3) δ9.89 (S, J=7.8Hz, 2H), δ7.54 (d, J=7.8Hz, 2H), 7.42 (d, J=8 .3Hz,1H), δ7.44-7.24(m,5H),7.08(d,J=8.4Hz,1H),5.08(s,2H),3.88(s,3H).

[0176] To a solution of A1-3 (21 g, 74 mmol) in anhydrous tetrahydrofuran (150 mL) was slowly added dropwise 2M borane dimethyl sulfide to obtain a tetrahydrofuran solution (74 mL, 148 mmol) under nitrogen at 0°C. After stirring at 0°C for 4 hours, the reaction was quenched with cold methanol. Most of the solvent was removed by distillation under reduced pressure, and the remaining solution was slowly poured into saturated aqueous sodium bicarbonate solution to precipitate a white solid. The solid was filtered and the filter cake was dried to give the desired product A1-4 (16.4 g, 82% yield). MS: 272 (M+H). + . 1H NMR (400MHz, CDCl3) δ7.49–7.33(m,7H),6.87(d,J=8.3Hz,1H),5.14(s,2H),3.86(s,3H),2.91(s,3H).

[0177] To a solution of A1-4 (16.4 g, 60.7 mmol) in tetrahydrofuran / methanol / water (30 / 30 / 10 ml) was added sodium hydroxide (4.8 g, 120 mmol). The mixture was stirred at room temperature under a hydrogen atmosphere for 4 hours. The pH was adjusted to 3-4 with 1M hydrochloric acid to precipitate a white solid. The solid was filtered and the filter cake was dried to give the desired product A1-7 (13.8 g, 89% yield). MS: 258 (M+H) + . .MS:372(M+H)+. 1H NMR (400MHz, CDCl3) δ7.51–7.33 (m, 7H), 6.88 (d, J = 8.3Hz, 1H), 5.13 (s, 2H), 2.90 (s, 3H).

[0178] To a solution of A1-5 (13.8 g, 53.4 mmol) in tert-butanol (50 mL) were added Boc anhydride (58 g, 270 mmol), DMAP (1.2 g, 10 mmol) and DIPEA (4.7 mL, 270 mmol). The mixture was stirred at 60°C for 48 hours and then poured into saturated aqueous ammonium chloride solution. The mixture was extracted with EA, dried over anhydrous magnesium sulfate, filtered, and the filtrate was collected and subjected to column chromatography to obtain the target product A1-6 (16.3 g, 74% yield). MS: 414 (M+H) +. MS:372(M+H)+. 1H NMR (400MHz, CDCl3) δ7.51–7.33 (m, 7H), 6.88 (d, J = 8.3Hz, 1H), 5.13 (s, 2H), 2.89 (s, 3H), 1.45 (s, 9H), 1.20 (s, 9H).

[0179] To a solution of A1-6 (16.3 g, 39 mmol) in methanol (100 ml) was added palladium carbon (1.6 g) and stirred under hydrogen atmosphere at room temperature for 4 hours. The mixture was filtered and the filtrate was dried to give the target product A1-7 (12.6 g, yield 89%). MS: 324 (M+H) + .

[0180] A1-8 was synthesized according to the general method.

[0181] General method 1:

[0182] To a dry two-necked flask, add A1-7 (1 equivalent), benzyl bromide intermediates of different trigger fragments (1 equivalent), and silver oxide (1.5 equivalents). Add anhydrous tetrahydrofuran or anhydrous acetonitrile solution under nitrogen and light-shielding conditions. Stir at room temperature for 12-24 hours, filter, concentrate the filtrate, and purify the crude product by column chromatography.

[0183] A1-8 yield 78%, MS: 683 (M+H) + . 1H NMR(400MHz, CDCl3)δ8.99(s,1H),7.84–7.65(m,2H),7.62–7.41(m,3H),7.19(dd,J=25.8,11.3Hz ,2H),6.85(d,J=8.1Hz,1H),5.95–5.71(m,2H),5.25–5.15(m,2H),5.10(d,J=10.4Hz,1H),5.01(s ,2H),4.67(p,J=6.9Hz,1H),4.55–4.43(m,2H),4.13(t,J=7.3Hz,1H),3.05(s,3H),2.09–1.91(m, 1H), 1.47 (d, J = 22.7Hz, 9H), 1.36 (d, J = 6.9Hz, 4H), 1.20 (d, J = 13.9Hz, 9H), 0.86 (t, J = 5.7Hz, 6H).

[0184] To a solution of A1-8 (1.3 g, 1.9 mmol) in dichloromethane (10 ml) was added trifluoroacetic acid (2 ml). After stirring at room temperature for 4 hours, dichloromethane and trifluoroacetic acid were removed by distillation under reduced pressure to obtain the target crude product A1-10 (0.92 g, yield 92%). MS: 527 (M+H) +.

[0185] To a solution of A1-9 (0.92 g, 1.75 mmol) in chloromethane (10 mL) were added EDCI-HCl (668 mg, 3.5 mmol), Hobt (270 mg, 2 mmol), N-Boc-piperazine (651 mg, 3.5 mmol), and triethylamine (478 μL, 3.5 mmol). After stirring at room temperature for 6 hours, the mixture was poured into saturated aqueous ammonium chloride solution and extracted with dichloromethane. The organic phase was collected, dried, filtered, concentrated, and column chromatography to give the desired product A1 (1.08 g, 89% yield). MS: 695 (M+H). + . 1H NMR (400MHz, CDCl3) δ8.69(s,1H),7.56(d,J=8.1Hz,2H),7.33(d,J=8.3Hz,2H),6.77(dd,J=13.0,7.8Hz ,2H),6.69–6.56(m,2H),5.99–5.82(m,1H),5.34(dd,J=18.5,7.7Hz,2H),5.22(d,J=10.2Hz,1H),5.01( s,2H),4.65(p,J=7.0Hz,1H),4.56(t,J=9.7Hz,2H),4.35(s,1H),4.04(t,J=6.6Hz,1H),3.76–3.31(m,9 H),2.83(s,3H),2.16(dq,J=13.3,6.7Hz,1H),1.71(s,6H),1.46(s,9H),0.94(dt,J=25.8,12.9Hz,6H).

[0186] The second connection route between the key intermediate and the drug DXD is carried out

[0187] Route 2:

[0188] Synthesis of intermediate A1-a1

[0189] To a dry two-necked flask was added A1 (300 mg, 0.432 mmol) and dissolved in 10 mL of anhydrous dichloromethane. Anhydrous DIPEA (300 μL, 1.73 mmol) was added under nitrogen. A dichloromethane solution (2 mL) of triphosgene (64 mg, 0.216 mmol) was added at 0°C. After stirring at room temperature for 1 hour, 2-(trimethylsilyl)ethyl 2-hydroxyacetate (176 mg, 1.29 mmol) and DMAP (157 mg, 1.29 mmol) were added. The mixture was stirred at 50°C for 16 hours and poured into saturated aqueous ammonium chloride. The mixture was extracted with dichloromethane and the organic phase was collected, dried, filtered, concentrated, and subjected to column chromatography to obtain the desired product A1 (279 mg, 72% yield). MS: 897 (M+H). +. 1H NMR(400MHz, CDCl3)δ8.92(s,1H),7.52(t,J=12.0Hz,2H),7.42–7.19(m,5H),6.96(d,J=8.4Hz ,1H),5.87(ddt,J=16.1,10.6,5.4Hz,1H),5.68(d,J=7.9Hz,1H),5.34–5.22(m,2H),5.17(d,J= 10.4Hz,1H),5.06(s,2H),4.73–4.48(m,4H),4.31–4.06(m,3H),3.70–3.35(m,7H),3.17(s,2H) ,2.28–2.04(m,2H),1.44(s,9H),1.38(d,J=6.8Hz,3H),0.95–0.83(m,6H),0.04–-0.02(m,9H).

[0190] Synthesis of intermediate A1-a2

[0191] To a solution of A1-a1 (279 mg, 0.311 mmol) in tetrahydrofuran (15 mL) was added tetrabutylammonium fluoride (162 mg, 0.622 mmol). After stirring at room temperature for 8 hours, the mixture was poured into saturated aqueous ammonium chloride solution, extracted with EA, dried over anhydrous magnesium sulfate, filtered, and the filtrate was collected and dried to give the desired product A1-a2 (220 mg, 89% yield). The crude product was directly used in the next step without purification. MS: 797 (M+H) + .

[0192] Synthesis of intermediate A1-a3

[0193] A1-a2 (277 mg, 0.277 mmol), isotecan mesylate (153 mg, 0.277 mmol), and DIPEA (97 μl, 0.554 mmol) were dissolved in 15 mL of DMF. A solution of DMTMM (150 mg, 0.554 mmol) in DMF (2 mL) was added dropwise under ice-cooling. After stirring under ice-cooling for 4 hours, the mixture was poured into saturated aqueous ammonium chloride, extracted with EA, dried over anhydrous magnesium sulfate, filtered, and the filtrate was collected and subjected to column chromatography to obtain the desired product A1-a3 (238 mg, 71% yield). MS: 1213 (M+H). +. 1H NMR (400MHz, CDCl3) δ8.24-7.94(m,2H),7.64-7.47(m,3H),7.43-7.41(m,3H),7.31(s,1H),7.19(d,J=12.0Hz,1H),7.00(d, J=12.4Hz,2H),5.97-5.93(m,1H),5.53-5.49(m,4H),5.40(s,1H),5.35-5.31(m,1H),5.30-5.21(m,2H),5.15(q,J=9.7Hz,1H ),5.03(s,2H),4.95(s,2H),4.49(dt,J=9.7,1.5Hz,2H),4.23(s,1H),3.59-3.54(m,4H),3.43-3.28(m,5H),3.24(s,3H),3.0 1-2.52(m,4H),2.41-1.95(m,6H),1.44(d,J=6.8Hz,2H),1.42-1.39(m,12H),1.02(d,J=10.2Hz,6H),0.96(t,J=10.8Hz,3H).

[0194] Synthesis of intermediate A1-a4

[0195] To a solution of A1-a3 (238 mg, 0.197 mmol) in DMF (10 ml) were added tetrakistriphenylphosphine palladium (23 mg) and piperidine (46 μl, 0.394 mmol). The mixture was stirred at room temperature under nitrogen for 12 hours, then filtered, and the filtrate was collected and dried by column chromatography to give the desired product A1-a4 (160 mg, 72% yield). MS: 1130 (M+H) +. 1H NMR (400MHz, CDCl3) δ8.23-7.96(m,2H),7.62-7.47(m,3H),7.43-7.39(m,3H),7.32(s,1H),7.20(d,J=11.6Hz,1H ),7.01(d,J=9.9Hz,2H),5.52-5.49(m,2H),5.40(s,1H),5.29-5.21(m,2H),5.15-5.09(m,1H),5.05(s,2H),4.95( s,2H),4.48–4.42(m,2H),4.23-4.19(m,2H),3.56-3.51(m,3H),3.41-3.28(m,5H),3.24(s,3H),3.010-2.53(m,3 H),2.41-1.95(m,7H),1.42(d,J=6.7Hz,2H),1.40-1.397(m,12H),1.04(d,J=10.1Hz,6H),0.96(t,J=10.6Hz,3H).

[0196] Synthesis of intermediate A1-a5

[0197] To a solution of A1-a4 (160 mg, 0.142 mmol) in DMF (5 mL) were added 3-(2-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)ethoxy)ethoxy)propanoic acid (50 mg, 0.142 mmol) and DIPEA (50 μL, 0.284 mmol). A solution of DMTMM (78 mg, 0.284 mmol) in DMF (2 mL) was added dropwise at 0°C. After stirring for 12 hours, DMF was evaporated under reduced pressure. The crude product was purified by C18 flash chromatography to afford the desired product A1-a5 (142 mg, 68% yield). MS: 1468 (M+H). + . 1H NMR (400MHz, CDCl3) δ8.28(s,1H),8.17(d,J=2.9Hz,1H),7.77(dd,J=15.0,3.1Hz,1H),7.62–7.51(m,3H),7.44(dt,J=4.1,2.6H z,3H),7.20(d,J=15.0Hz,1H),7.10(d,J=4.8Hz,3H),7.03(d,J=14.1Hz,2H),5.58(d,J=8.1Hz,3H),5.45–5.20(m,4H),5.15–4.9 0(m,5H),4.00(t,J=10.0Hz,2H),3.75–3.53(m,8H),3.49(d,J=28.9Hz,7H),3.37–3.21(m,9H),2.99–2.61(m,4H),2.46(t,J=8.4 Hz,2H),2.38–2.07(m,7H),1.97(q,J=13.6Hz,1H),1.68–1.52(m,2H),1.48–1.39(m,12H),1.38–1.11(m,4H),1.02–0.83(m,9H).

[0198] Synthesis of intermediate A1-a6

[0199] To a solution of A1-a5 (142 mg, 0.096 mmol) in dichloromethane (10 mL) was added trifluoroacetic acid (2 mL). After stirring at room temperature for 4 hours, the dichloromethane and trifluoroacetic acid were removed by distillation under reduced pressure to obtain the target crude product A1-a6 (121 mg, 92% yield). The crude product was directly used for the next step without purification. MS: 1368 (M+H) + .

[0200] Synthesis of L1-1

[0201] To a solution of A1-a6 (121 mg, 0.088 mmol) in DMF (5 mL) were added 2,5,8,11,14,17,20,23-octaoxadocosano-25-oic acid (35 mg, 0.088 mmol) and DIPEA (30 μL, 0.176 mmol). A solution of DMTMM (49 mg, 0.176 mmol) in DMF (2 mL) was added dropwise at 0°C. After stirring for 12 hours, DMF was evaporated under reduced pressure. The crude product was separated and purified by HPLC to give the desired product L1-1 (62 mg, 68% yield). MS: 1748 (M+H). + . 11H NMR (400 MHz, DMSO-d6) δ 8.29 (s, 1H), 8.16 (d, J = 3.0 Hz, 1H), 7.76 (dd, J = 15.0, 3.1 Hz, 1H), 7.61–7.51 (m, 3H), 7.43 (dt, J = 4.0, 2.6 Hz, 3H), 7.20 - 7.16 (m, 1H), 7.11–7.06 (m, 3H), 7.03 - 7.68 (m, 2H), 5.58 (d, J = 8.0 Hz, 3H), 5.43–5.20 (m, 5H), 5.14–4.90 (m, 4H), 4.00 - 3.89 (m, 2H), 3.75–3.53 (m, 12H), 3.49 - 3.40 (m, 56H), 3.37–3.21 (m, 16H), 2.98–2.63 (m, 6H), 2.46–2.39 (m, 6H), 2.38–2.07 (m, 7H), 1.97 (q, J = 13.6 Hz, 1H), 1.69–1.54 (m, H), 1.47–1.36 (m, 16H), 1.34–1.20 (m, 4H), 1.04–0.82 (m, 9H).

[0202] Synthesis of L8-3

[0203] Intermediate A1-a4 is condensed with maleimide PEG chain acid (refer to the synthesis of A1-a5 of the present invention), deprotected with Boc (refer to the synthesis of A1-a6 of the present invention), and condensed with polysarcosine chain (refer to the synthesis of L1-1 of the present invention) to obtain the target product L8-3. NMR (400MHz, DMSO-d6) δ8.28 (s, 1H), 8.17 (d, J = 1.4Hz, 1H), 7.93 (s, 1H), 7.77 (dd,J=6,1.1Hz,1H),7.56(d,J=6Hz,2H),7.51-7.37(m,4H),7.20(d,J=6Hz,1H ),7.13(s,2H),7.02(s,1H),6.40(s,1H),5.56(s,2H),5.53(d,J=2.0Hz,1H),5 .42(t,J=6.2Hz,1H),5.36(s,1H),5.14(s,1H),5.04(s,2H),4.99-4.92(m,3H) ,4.88(s,1H),4.75(t,J=6.4Hz,2H),3.91(s,16H),3.74-3.56(m,12H),3.52(s ,12H),3.39(s,1H),3.31-3.19(m,5H),2.96(s,24H),2.85(dt,J=7.8,3.8Hz,1 H),2.80-2.69(m,3H),2.63–2.58(m,4H),2.50–2.43(m,1H),2.37-2.25(m,4H) ,2.11(s,3H),1.91(q,J=5.4Hz,1H),1.44(d,J=4.8Hz,3H),1.00-0.85(m,9H).

[0204] The key intermediates and the connection route of all phenolic hydroxy drugs are carried out in three

[0205] Route 3 (taking camptothecin phenolic hydroxyl as an example):

[0206] Synthesis of A1-b1

[0207] To a dry two-necked flask was added A1 (300 mg, 0.432 mmol) and dissolved in 10 mL of anhydrous dichloromethane. Anhydrous DIPEA (300 μL, 1.73 mmol) was added under nitrogen. A dichloromethane solution (2 mL) of triphosgene (64 mg, 0.216 mmol) was added at 0°C. After stirring at room temperature for 1 hour, camptothecin phenolic hydroxy compound (218 mg, 0.52 mmol) and DMAP (157 mg, 1.29 mmol) were added. The mixture was stirred at 50°C for 16 hours and poured into saturated aqueous ammonium chloride solution. The mixture was extracted with dichloromethane and the organic phase was collected, dried, filtered, concentrated, and subjected to column chromatography to obtain the desired product A1-b1 (279 mg, 72% yield). MS: 1143 (M+H). + . 1H NMR (400MHz, CDCl3) δ8.22(s,1H),8.17(d,J=1.1Hz,1H),7.77-7.67(m,1H),7.56(d,J=6Hz,2H),7.44(d,J=6Hz,2H),7.38-7.32(m,2H ),7.20-7.15(m,1H),7.05(s,1H),5.98-5.92(m,1H),5.78-5.60(m,2H),5.52(d,J=8.8Hz,1H),5.27-5.19(m,3H),5.04(s,2H),4.96(t ,J=3.4Hz,2H),4.50(d,J=4.9Hz,2H),4.26(s,1H),3.75(t,J=4.6Hz,2H),3.59(t,J=4.1Hz,4H),3.45(s,1H),3.34–3.29(m,4H),3.25 (s,3H),2.97(t,J=4.7Hz,2H),2.78-2.62(m,1H),2.28-2.19(m,3H),1.50-1.34(m,12H),0.96(d,J=5.1Hz,6H),0.89(t,J=5.4Hz,3H).

[0208] Synthesis of A1-b2

[0209] To a solution of A1-b1 (0.2 mmol) in DCM (10 ml) were added TMS-DMA (1.2 mmol) and TMS-TFA (1.2 mmol). The mixture was stirred at room temperature under nitrogen for 1 hour. Tetrakistriphenylphosphine palladium (0.04 mmol) was then added and stirred at room temperature under nitrogen for 12 hours. The mixture was poured into saturated aqueous ammonium chloride solution and extracted with dichloromethane. The organic phase was collected, dried, filtered, concentrated, and column chromatography to obtain the desired product A1-b2 (yield 69%). MS: 1058 (M+H). +. 1H NMR (400 MHz, CDCl3) δ 8.17 (d, J = 2.3 Hz, 1H), 7.77 (dd, J = 12.0, 2.5 Hz, 1H), 7.60 - 7.51 (m, 2H), 7.47 - 7.41 (m, 2H), 7.37 (d, J = 12.7 Hz, 1H), 7.20 (d, J = 15.0 Hz, 1H), 7.05 (s, 1H), 6.14 (s, 1H), 5.76 (d, J = 16.5 Hz, 2H), 5.12 - 5.00 (m, 3H), 4.98 (s, 2H), 3.82 - 3.69 (m, 3H), 3.60 (dd, J = 12.2, 4.1 Hz, 4H), 3.42 (s, 1H), 3.33 (dd, J = 12.2, 4.1 Hz, 4H), 3.25 (s, 3H), 2.97 (t, J = 9.4 Hz, 2H), 2.32 - 1.96 (m, 6H), 1.74 (s, 1H), 1.49 - 1.36 (m, 12H), 0.96 (d, J = 10.2 Hz, 6H), 0.89 (t, J = 10.8 Hz, 3H).

[0210] Synthesis of L1-3

[0211] Intermediate A1-b2 is condensed with maleimide PEG chain acid (refer to the synthesis of A1-a5 of the present invention), deprotected with Boc (refer to the synthesis of A1-a6 of the present invention), and condensed with 1,2-polyethylene glycol propionic acid (refer to the synthesis of L1-1 of the present invention) to obtain the target product L1-3. NMR (400MHz, DMSO-d6) δ9.83 (s, 1H), 8.16 (d, J = 2.3Hz, 1H), 7.79-7.70 (m, 2H ),7.61-7.50(m,2H),7.48-7.40(m,2H),7.19(d,J=12.0Hz,1H),7.14(s,2H), 6.92(s,1H),5.68(s,1H),5.61(s,1H),5.54(s,1H),5.21(s,1H),5.16(q,J= 9.9Hz,1H),5.03(s,2H),4.95(s,2H),4.74(t,J=12.8Hz,2H),4.61(d,J=17.3 Hz,1H),3.74(t,J=9.2Hz,2H),3.69-3.58(m,13H),3.57-3.52(m,4H),3.51(s,52 H),3.40(s,3H),3.27(t,J=6.2Hz,2H),3.25(s,3H),3.11(s,1H),2.97(t,J=9.2H z,2H),2.79-2.60(m,5H),2.53(t,J=6.8Hz,2H),2.31-2.17(m,3H),1.93(q,J=10 .8Hz,1H),1.44(d,J=9.9Hz,3H),0.96(d,J=10.1Hz,6H),0.89(t,J=10.8Hz,3H).

[0212] Synthesis of L1-4

[0213] Intermediate A1-b2 was condensed with maleimidocaproic acid (refer to the synthesis of A1-a5 of the present invention), deprotected with Boc (refer to the synthesis of A1-a6 of the present invention), and condensed with 1,2-polyethylene glycol propionic acid (refer to the synthesis of L1-1 of the present invention) to obtain the target product L1-4. 1H NMR (400 MHz, DMSO-d6) δ 8.64 (s, 1H), 8.17 (d, J = 2.3 Hz, 1H), 7.96 (s, 1H), 7.77 (dd, J = 12.0, 2.3 Hz, 1H), 7.61-7.51 (m, 2H), 7.48-7.40 (m, 2H), 7.30 (d, J = 12.7 Hz, 1H), 7.20 (d,J=11.8Hz,1H),7.12(s,2H),7.00(s,1H),5.74(d,J=12.6Hz,2H),5.39(s,1H),5. 20(d,J=14.6Hz,1H),5.04(s,2H),4.97(s,2H),4.95-4.86(m,1H),4.00(t,J=11.2Hz ,2H),3.75(t,J=9.3Hz,2H),3.67-3.49(m,54H),3.40(s,3H),3.40(s,3H),3.33(s,1 H),3.33(s,1H),3.25(s,3H),3.25(s,3H),2.97(t,J=9.4Hz,2H),2.81-2.62(m,1H), 2.54(t,J=12.4Hz,2H),2.31-2.15(m,3H),2.13-1.93(m,3H),1.72-1.55(m,2H),1.4 4(d,J=9.7Hz,3H),1.40-1.11(m,4H),0.96(d,J=10.2Hz,6H),0.89(t,J=10.8Hz,3H).

[0214] Synthesis of L8-1

[0215] Intermediate A1-b2 is condensed with maleimide PEG chain acid (refer to the synthesis of A1-a5 of the present invention), deprotected with Boc (refer to the synthesis of A1-a6 of the present invention), and condensed with 10-polysarcosine (refer to the synthesis of L1-1 of the present invention) to obtain the target product L8-1. NMR(400MHz,DMSO-d6)δ8.17(d,J=1.4Hz,1H),7.77(dd,J=7.5,1.4Hz,1H),7.56 (d,J=7.5Hz,2H),7.44(d,J=7.5Hz,2H),7.33(d,J=8.1Hz,1H),7.20(d,J=7.5Hz, 1H),7.14(s,1H),7.00(s,1H),6.78(s,1H),6.74(s,2H),5.63–5.55(m,1H),5.53 (s,1H),5.39(s,2H),5.04(s,2H),4.96(t,J=6.8Hz,2H),4.75(t,J=7.9Hz,2H),4 .59(d,J=2.5Hz,1H),3.91(s,20H),3.75(t,J=5.8Hz,2H),3.71–3.55(m,12H),3 .51(d,J=8.4Hz,13H),3.28(q,J=4.5Hz,2H),3.25(s,3H),3.00–2.90(m,32H),2. 81–2.69(m,1H),2.66(t,J=7.9Hz,2H),2.35–2.15(m,5H),2.11(s,3H),1.99(q,J =6.9Hz,1H),1.44(d,J=6.0Hz,3H),0.96(d,J=6.4Hz,6H),0.89(t,J=6.8Hz,3H).

[0216] Synthesis of L1-2

[0217] Using A1 and different camptothecin phenolic hydroxy compounds as raw materials, the target product L1-2 was obtained by referring to the synthesis of L1-3 of the present invention via route 3. MS: 1999 (M+H) +. 1H NMR (400MHz, DMSO-d6) δ8.20 (s, 1H), 8.16 (d, J = 2.3Hz, 1H), 7.76 (dd, J = 12.0, 2.5Hz ,1H),7.59-7.52(m,2H),7.46-7.39(m,3H),7.19(d,J=12.0Hz,1H),7.13(s,2H),7. 01(d,J=4.5Hz,2H),6.38(s,1H),6.27(s,1H),5.56(d,J=5.4Hz,3H),5.41(t,J=3.8 Hz,1H),5.39(s,1H),5.37-5.23(m,3H),5.04(s,2H),4.93(s,2H),4.75(t,J=12.8H z,2H),3.71-3.56(m,15H),3.56-3.53(m,4H),3.52(s,52H),3.46(s,1H),3.40(s,3 H),3.30(t,J=3.5Hz,1H),3.27(t,J=3.3Hz,1H),3.25(s,3H),2.95-2.67(m,5H),2. 66-2.52(m,4H),2.51-2.37(m,2H),2.36-2.29(m,4H),2.32-2.14(m,2H),1.96(q,J =10.8Hz,1H),1.44(d,J=9.8Hz,3H),0.96(d,J=10.2Hz,6H),0.89(t,J=10.8Hz,3H).

[0218] Synthesis of L8-2

[0219] Using A1 and different camptothecin phenolic hydroxy compounds as raw materials, the target product L8-2 was obtained by referring completely to the synthesis of L8-1 of the present invention via route three. 1H NMR (400MHz, DMSO-d6) δ8.31(s,1H),8.17(d,J=2.3Hz,1H),7.77(dd,J=12.0,2.3Hz,1H),7.64-7.38(m,6H),7.24–7.18(m,2H),7.13(d,J= 2.5Hz,3H),7.01(s,1H),5.74(d,J=6.2Hz,2H),5.31(d,J=3.0Hz,1H),5.25(s,1H),5.08-4.98(m,3H),4.97(s,2H),4.75(t,J=12.8Hz,2H) ,3.91(s,20H),3.81-3.55(m,14H),3.52(s,12H),3.47(s,1H),3.34-3.18(m,5H),3.01-2.85(m,32H),2.79-2.59(m,3H),2.53(t,J=12.5Hz,2H),2.31-2.16(m,3H),2.11(s,3H),1.98(q,J=10.8Hz,1H),1.44(d,J=9.8Hz,3H),0.96(d,J=10.2Hz,6H),0.89(t,J=10.8Hz,3H). Example 7 1 Synthesis of Compounds L3-1 and L4-1

[0220] Intermediates C1 and D1 were synthesized using Route 3

[0221] Route 3

[0222] C1-1 and D1-1 were synthesized according to General Method 1.

[0223] C1-1 yield 81%, MS: 791 (M+H) + . 1 H NMR (400MHz, CDCl3) δ8.21(dd,J=4.0,2.4Hz,1H),7.79(dd,J=12.0,2.3Hz,1H),7.38(dd,J =11.9,2.4Hz,1H),7.17–7.00(m,1H),6.55(d,J=12.7Hz,1H),5.53(dd,J=13.4,12.3Hz,1H ),5.16(s,1H),5.01-4.74(m,1H),4.61(dd,J=15.4,12.2Hz,1H),4.30(d,J=15.4Hz,1H),3 .70(s,1H),3.25(s,2H),2.04(s,3H),2.02(s,3H),1.99(s,3H),1.42(s,12H),1.40(s,6H).

[0224] D1-1 yield 79%, MS: 746 (M+H) + . 1 H NMR (400MHz, CDCl3) δ8.21(d,J=1.1Hz,1H),7.79(dd,J=6,1.1Hz,1H),7.13(d,J=6Hz,1H),6. 99(d,J=6Hz,1H),6.89(d,J=6Hz,1H),5.91(dd,J=7.9,6.6Hz,1H),5.61–5.50(m,1H),5.46(d , J = 6.2 Hz, 1H), 5.28–4.98 (m, 1H), 3.85–3.55 (m, 1H), 3.25 (s, 1H), 2.06 (s, 3H), 2.03 (s, 3H), 2.01 (s, 3H), 1.43 (s, 12H), 1.41 (s, 6H). C1-2 and D1-2 were synthesized according to the synthesis method of A1-9. The yield of C1-1 was 91%, MS: 691 (M+H). + D1-1 yield 93%, MS: 646 (M+H) + C1-2 and D1-2 were used directly in the next step without purification.

[0225] The synthesis of C1 and D1 was based on the synthesis method of A1. The yield of C1 was 84%, MS: 805 (M+H). + . 1 H NMR (400MHz, CDCl3) δ8.22(d,J=2.4Hz,1H),7.37(td,J=12.0,2.3Hz,2H),7.20(dd,J=11.4,7 .2Hz,2H),7.00(d,J=12.0Hz,1H),6.67(d,J=12.8Hz,1H),5.69(dd,J=13.4,12.8Hz,1H),5.1 6 (s, 2H), 4.92-4.65 (m, 3H), 4.29 (d, J = 16 Hz, 1H), 3.70 (s, 3H), 3.67-3.27 (m, 4H), 3.23 (s, 6H), 2.68 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H), 1.99 (s, 3H), 1.40 (s, 9H). Yield of D1-1: 82%, MS: 760 (M+H) + . 1H NMR (400MHz, CDCl3) δ7.35 (dd, J=6, 1.1Hz, 1H), 7.19 (d, J=1.1Hz, 1H), 7.00 (dd, J=6, 3.7Hz,3H),6.89(d,J=6Hz,2H),6.60(d,J=6.3Hz,1H),5.78(t,J=6.5Hz,1H),5.16(s, 2H),4.78-4.67(m,2H),4.58(s,1H),4.50(d,J=7.7Hz,1H),3.70(s,3H),3.59(t,J=3 .8Hz,4H),3.34(t,J=3.8Hz,4H),2.68(s,3H),2.02(s,6H),2.00(s,3H),1.41(s,9H).

[0226] Synthesis reference route 4 of L3-1

[0227] Route 4:

[0228] Synthesis of intermediate C1-a1

[0229] The synthesis of C1-a1 was carried out according to the synthesis method of A1-b1. The yield of C1-a1 was 74%, and the MS was 1486 (M+H). +. 1H NMR (400MHz, CDCl3) δ8.25 (s, 1H), 8.17 (d, J = 2.5Hz, 1H), 7.91 (d, J = 2.6Hz, 1H), 7.88(d,J=2.5Hz,1H),7.84(d,J=2.4Hz,1H),7.77(dd,J=12.0,2.4Hz,1H),7.65 (d,J=11.9Hz,1H),7.47-7.10(m,10H),6.87(s,1H),6.83(dd,J=12.0,2.3Hz,1H ),6.18(d,J=5.3Hz,1H),5.47(dd,J=3.2,0.9Hz,1H),5.31-5.23(m,2H),5.19-5 .11(m,3H),4.99-4.92(m,4H),4.72(d,J=14.0Hz,2H),4.49(d,J=3.9Hz,1H),3. 70(s,3H),3.64-3.43(m,8H),3.37-3.27(m,1H),3.24(d,J=8.1Hz,9H),3.00(t, J=8.8Hz,1H),2.71(t,J=12.8Hz,2H),2.24(q,J=10.8Hz,1H),2.02(s,9H),1.92 (q,J=10.8Hz,1H),1.42(s,9H),1.18(t,J=10.5Hz,3H),0.89(t,J=10.8Hz,3H).

[0230] Synthesis of intermediate C1-a2

[0231] To a solution of C1-a1 (1 equivalent) in tetrahydrofuran / methanol / water (3:3:1) was added 1 M aqueous lithium hydroxide solution (5 equivalents). After stirring at room temperature for 4 hours, the product C1-a2 was purified by C18 flash chromatography (yield 69%). MS: 1124 (M+H) +. 1H NMR (400MHz, CDCl3) δ8.27-8.12(m,2H),7.87-7.72(m,2H),7.65(d,J=11.8Hz,1H),7.46-7.29(m,2H),7.19(dd,J=12.0,7.6 Hz,2H),7.00(s,1H),6.83(dd,J=12.0,2.3Hz,1H),5.80(s,1H),5.60(s,1H),5.22-5.11(m,3H),4.97(s,2H),4.49(dd,J=5.4 ,0.9Hz,1H),4.18-3.98(m,2H),3.66-3.42(m,6H),3.39-3.30(m,2H),3.24(d,J=8.1Hz,9H),3.11(td,J=12.5,0.9Hz,2H),2. 81-2.63(m,2H),2.24(q,J=10.8Hz,1H),1.98(q,J=10.8Hz,1H),1.42(s,9H),1.18(t,J=10.5Hz,3H),0.89(t,J=10.8Hz,3H).

[0232] Synthesis of L3-1

[0233] Intermediate C1-a2 was condensed with maleimidocaproic acid (refer to the synthesis of A1-a5), deprotected with Boc (refer to the synthesis of A1-a6), and condensed with 8-polyethylene glycol propionic acid (refer to the synthesis of L1-1) to obtain the target product L3-1 with a multi-step yield of 12%. MS: 1611 (M+H) +. 1H NMR (400MHz, DMSO-d6) δ8.12(d,J=2.3Hz,1H),7.79(d,J=2.4Hz,1H),7.72(dd,J=11.9,2.4Hz ,1H),7.60(d,J=11.8Hz,1H),7.38-7.25(m,2H),7.23(s,1H),7.14(dd,J=11.9,7.5Hz,2H),7. 06(s,2H),6.95(s,1H),6.79(dd,J=12.0,2.3Hz,1H),6.27(d,J=2.3Hz,1H),5.80(s,1H),5.33 -5.26(m,2H),5.13(s,2H),4.93(s,2H),4.40(s,2H),4.34(s,1H),4.29(dd,J=5.2,0.6Hz,1H) ,4.17(dd,J=4.7,2.3Hz,1H),4.10(t,J=4.9Hz,1H),3.97(t,J=7.9Hz,2H),3.85(d,J=0.5Hz,1 H),3.77(d,J=12.8Hz,1H),3.71-3.50(m,4H),3.50(s,32H),3.49-3.41(m,4H),3.37(s,1H),3 .23(s,3H),3.21(s,3H),2.92(s,3H),2.69(t,J=12.7Hz,2H),2.38(s,1H),2.24-2.03(m,4H), 1.70-1.52(m,2H),1.44(s,1H),1.41-1.23(m,4H),1.21-1.03(m,6H),0.88(t,J=10.8Hz,3H).

[0234] Synthesis of L4-1 Reference Route 5:

[0235] Route 5

[0236] The synthesis of D1-a1 was carried out according to the synthesis method of A1-b1. The yield of D1-a1 was 72%, MS: 1486 (M+H) +. 1H NMR (400MHz, CDCl3) δ8.22(d,J=1.2Hz,1H),8.17(d,J=1.1Hz,1H),7.77(dd,J=6.0,1.1Hz,1H),7.65(d,J=6.0Hz,1H),7.43-7.30(m,3H),7.21(dd,J=7 .9,6.0Hz,2H),7.03(s,1H),6.05(s,1H),5.67(s,1H),5.42(d,J=6.2Hz,1H ),5.19(dd,J=6.9,4.7Hz,1H),5.16(s,2H),5.03(td,J=8.0,3.1Hz,1H),4.9 9(d,J=1.1Hz,2H),4.70(d,J=7.9Hz,1H),3.97(dd,J=6.1,2.5Hz,1H),3.70 (s,3H),3.59-3.55(m,2H),3.52-3.44(m,4H),3.41-3.35(m,1H),3.24(d,J= 4.2Hz,3H),3.23(s,6H),2.21(q,J=5.4Hz,1H),2.15-2.06(m,1H),2.04(s, 6H), 2.01 (s, 3H), 1.40 (s, 9H), 1.18 (t, J = 5.3Hz, 3H), 0.89 (t, J = 5.4Hz, 3H).

[0237] Synthesis of L4-1:

[0238] D1-a1 was subjected to hydrolysis (refer to the synthesis of C1-a2), deprotection of Boc (refer to the synthesis of A1-a6), and condensation with C3 maleimide 8 polyethylene glycol propionic acid (refer to the synthesis of L1-1) to obtain the target product L4-1 with a multi-step yield of 11% and MS: 1557 (M+H). + . 1H NMR (400MHz, CDCl3) δ8.22(d,J=2.3Hz,1H),8.17(d,J=2.4Hz,1H),7.77(dd,J=12. 0,2.3Hz,1H),7.65(d,J=11.8Hz,1H),7.37(ddd,J=11.4,6.4,2.3Hz,3H),7.26(s,2 H),7.21(dd,J=12.0,7.5Hz,2H),6.86(s,1H),5.92(d,J=7.4Hz,1H),5.81(s,1H),5 .77(s,1H),5.65(s,1H),5.16(s,2H),4.96(s,2H),4.78(s,1H),4.65(dd,J=9.6,1. 2Hz,1H),4.60(s,2H),4.44(dd,J=7.36,4.6Hz,1H),4.21(dd,J=9.6,4.6Hz,1H),3. 70-3.53(m,10H),3.52(s,30H),3.51-3.45(m,4H),3.31-3.26(m,3H),3.25(s,3H), 3.23(s,3H),3.11(s,1H),2.94(s,3H),2.80(s,1H),2.69(t,J=12.4Hz,2H),2.40(q ,J=10.8Hz,1H),2.20-2.08(m,2H),1.18(t,J=10.5Hz,3H),0.89(t,J=10.8Hz,3H).

[0239] Example: Synthesis of L5-L7 Reference Route 6

[0240] Route 6:

[0241] Camptothecin compounds with different substrates can be synthesized into key intermediate E1-a3 according to Route 6 by referring to General Method 1 and known synthetic conditions (reduction, condensation, de-Alloc protection, etc.). Intermediate E1-a3 is then condensed with different side chains (referring to the synthesis of L1-1 of the present invention) to obtain L5, L6, L7 and J4;

[0242] Synthesis of L5 side chains

[0243] L5-1 multi-step yield 8%, MS: 1103 (M / 2+H) +. 1H NMR (400 MHz, DMSO-d6) δ 8.59 (d, J = 3.1 Hz, 1H), 8.33 (s, 1H), 7.86 (s, 2H), 7.80 - 7.73 (m, 2H), 7.69 (s, 1H), 7.61 - 7.52 (m, 2H), 7.43 - 7.39 (m, 3H), 7.20 (d, J = 15.0 Hz, 1H), 7.05 (d, J = 17.8 Hz, 2H), δ 6.37 (s, 1H), 5.76 (s, 1H), 5.56 (s, 1H), 5.41 - 5.34 (m, 2H), 5.21 (s, 2H), 5.16 - 5.05 (m, 1H), 5.04 (s, 2H), 4.94 (s, 2H), 4.50 (tt, J = 12.3, 5.3 Hz, 1H), 4.35 (s, 4H), 4.14 (t, J = 13.8 Hz, 1H), 3.98 (t, J = 11.1 Hz, 2H), 3.72 - 3.67 (m, 9H), 3.55 - 3.52 (m, 6H), 3.52 (s, 52H), 3.47 - 3.43 (m, 8H), 3.25 - 3.19 (m, 8H), 3.12 - 3.08 (m, 4H), 2.95 - 2.67 (m, 2H), 2.66 - 2.47 (m, 1H), 2.34 (s, 3H), 2.30 - 1.90 (m, 10H), 1.70 - 1.47 (m, 5H), 1.44 - 1.17 (m, 8H), 1.02 - 0.80 (m, 9H).

[0244] The multi-step yield of L5-2 is 11%, MS:​。1H NMR (400 MHz, DMSO-d6) δ 8.57 - 8.54 (m, 1H), 8.32 (s, 1H), 7.88 (s, 2H), 7.80 - 7.72 (m, 2H), 7.68 (s, 1H), 7.61 - 7.54 (m, 2H), 7.42 - 7.39 (m, 3H), 7.20 - 7.18 (m, 1H), 7.05–7.00 (m, 2H), δ 6.37 (s, 1H), 5.78 (s, 1H), 5.54 (s, 1H), 5.41 - 5.34 (m, 3H), 5.21 (s, 1H), 5.16 - 5.02 (m, 1H), 5.04 - 4.94 (m, 2H), 4.50 - 4.35 (m, 4H), 4.14 - 3.97 (m, 3H), 3.72 - 3.67 (m, 8H), 3.55 - 3.52 (m, 5H), 3.52 (s, 54H), 3.47 - 3.40 (m, 9H), 3.26 - 3.19 (m, 7H), 3.13 - 3.08 (m, 5H), 2.96 - 2.67 (m, 4H), 2.65 - 2.47 (m, 2H), 2.30 - 1.89 (m, 10H), 1.69 - 1.47 (m, 4H), 1.40 - 1.19 (m, 6H), 1.02 - 0.80 (m, 9H).

[0245] The multi-step yield of L5-3 is 14%, MS: 1058 (M / 2 + H) + 。1H NMR (400 MHz, DMSO-d6) δ 8.56 - 8.50 (m, 1H), 8.33 (s, 1H), 7.86 (s, 2H), 7.80 - 7.72 (m, 2H), 7.68 (s, 2H), 7.60 - 7.56 (m, 2H), 7.41 - 7.39 (m, 3H), 7.22 - 7.18 (m, 1H), 7.06 - 7.00 (m, 2H), 5.78 - 5.70 (m, 2H), 5.56 (s, 2H), 5.40 - 5.32 (m, 2H), 5.21 - 5.02 (m, 2H), 5.04 - 4.96 (m, 2H), 4.51 - 4.37 (m, 3H), 4.14 - 3.99 (m, 3H), 3.74 - 3.66 (m, 10H), 3.55 - 3.52 (m, 8H), 3.52 (s, 50H), 3.50 - 3.40 (m, 8H), 3.25 - 3.19 (m, 6H), 3.15 - 3.08 (m, 4H), 2.95 - 2.67 (m, 4H), 2.65 - 2.47 (m, 4H), 2.30 - 1.90 (m, 8H), 1.64 - 1.45 (m, 3H), 1.40 - 1.18 (m, 6H), 1.02 - 0.80 (m, 9H).

[0246] Synthesis of L6 side chains

[0247] L6-1 multi-step yield 7%, MS: 1785 (M+H) + . 1H NMR (400MHz, DMSO-d6) δ8.59(d,J=1.4Hz,1H),8.18(s,1H),7.83(dd,J=7.5,1.4Hz,1H),7.56(d ,J=7.5Hz,2H),7.44(d,J=7.5Hz,2H),7.34(d,J=8.1Hz,1H),7.20(d,J=7.5Hz,1H),7.14(s,2H), 6.99(s,1H),6.53(d,J=9.5Hz,2H),6.33(s,1H),5.73(d,J=25.5Hz,2H),5.44(s,1H),5.38(q,J= 6.2Hz,1H),5.32(s,1H),5.21(t,J=6.4Hz,1H),5.18(d,J=1.4Hz,2H),5.04(s,2H),4.99(d,J=11 .0Hz,1H),4.75(t,J=7.9Hz,2H),3.75(t,J=6.0Hz,2H),3.70-3.65(m,4H),3.62(t,J=7.8Hz,2H) ,3.55(t,J=3.0Hz,2H),3.52(s,38H),3.44-3.37(m,6H),3.28(t,J=4.0Hz,2H),3.25(s,3H),2.9 7(t,J=5.8Hz,2H),2.79-2.62(m,5H),2.57(q,J=6.9Hz,1H),2.33-2.12(m,4H),2.05(t,J=5.5Hz ,2H),1.93(q,J=6.8Hz,1H),1.44(d,J=6.1Hz,3H),0.96(d,J=6.4Hz,6H),0.89(t,J=6.8Hz,3H).

[0248] Synthesis of L7 side chains

[0249] The multi-step yield of L7-1 is 10%. 1H NMR (400 MHz, DMSO-d6) δ 8.90 (s, 1H), 8.59 (d, J = 2.3 Hz, 1H), 8.38 (s, 1H), 7.83 (dd, J = 12.0, 2.5 Hz, 1H), 7.66–7.11 (m, 8H), 7.00 (d, J = 3.9 Hz, 3H), 6.35 (s, 1H), 5.81–5.72 (m, 2H), 5.66 (q, J = 6.7 Hz, 1H), 5.49 (s, 1H), 5.33 (d, J = 17.6 Hz, 1H), 5.03 (d, J = 10.8 Hz, 4H), 4.75 (t, J = 8.3 Hz, 2H), 4.22 (t, J = 12.0 Hz, 1H), 3.91 (s, 20H), 3.81 - 3.58 (m, 10H), 3.52 (s, 16H), 3.41 (td, J = 11.4, 1.1 Hz, 2H), 3.34 - 3.16 (m, 8H), 3.01 - 2.90 (m, 32H), 2.82 - 2.57 (m, 5H), 2.37 - 2.00 (m, 11H), 1.44 (d, J = 9.8 Hz, 3H), 0.96 (d, J = 10.2 Hz, 6H), 0.89 (t, J = 10.8 Hz, 3H).

[0250] The multi-step yield of L7-2 is 9%. 1H NMR (400 MHz, DMSO-d6) δ 8.58 (d, J = 2.3 Hz, 1H), 8.23 (s, 1H), 8.08 (s, 1H), 7.82 (dd, J = 11.9, 3.4 Hz, 1H), 7.62 - 7.37 (m, 5H), 7.32 - 7.10 (m, 4H), 7.05 (s, 1H), 6.58 (s, 1H), 5.81 (s, 1H), 5.73 (s, 1H), 5.49 (s, 1H), 5.38 (d, J = 9.2 Hz, 2H), 5.17 (d, J = 17.6 Hz, 1H), 5.06 - 4.90 (m, 6H), 4.75 (t, J = 8.5 Hz, 2H), 4.59 - 4.46 (m, 1H), 3.91 (s, 20H), 3.68 - 3.56 (m, 10H), 3.52 (s, 16H), 3.41 (dd, J = 13.6, 6.9 Hz, 3H), 3.33 - 3.17 (m, 7H), 3.08 - 2.89 (m, 33H), 2.82 - 2.52 (m, 5H), 2.32 - 1.94 (m, 11H), 1.44 (d, J = 9.8 Hz, 3H), 0.96 (d, J = 10.2 Hz, 6H), 0.89 (t, J = 10.8 Hz, 3H).

[0251] The multi-step yield of L7-3 was 6%. 1H NMR (400 MHz, DMSO-d6) δ8.59 (d, J = 2.3 Hz, 1H), 8.07 (s, 1H), 7.83 (dd, J = 12.0, 2.3 Hz, 1H), 7.59-7.27 (m, 9H), 7.20 (d, J = 12.0 Hz, 1H), 7.00 (s, 1H), 6.92 (s, 1H), 6.45 (s, 1H), 6.34 (s, 1H), 5.63 (t, J = 11.0Hz,1H),5.54(d,J=13.9Hz,2H),5.27(d,J=6.3Hz,3H),5.05(d,J=4.2Hz,4H),5.01-4.93(m,1 H),4.75(t,J=8.3Hz,2H),4.52-4.41(m,1H),3.91(s,2H),3.72-3.57(m,8H),3.52(s,16H),3.50- 3.36(m,3H),3.33-3.23(m,7H),2.96(s,3H),2.93-2.49(m,8H),2.34(s,3H),2.30-2.12(m,2H),2.10(d,J=5.1Hz ,3H),2.09-1.92(m,4H),1.84–1.79(m,1H),1.44(d,J=9.8Hz,3H),0.96(d,J=10.3Hz,6H),0.89(t,J=10.8Hz,3H).

[0252] J4 side chains were purchased from various reagent companies.

[0253] The multi-step yield of J4-1 is 13%. 1H NMR (400 MHz, DMSO-d6) δ 8.59 (d, J = 2.3 Hz, 1H), 7.83 - 7.78 (m, 1H), 7.65 - 7.60 (m, 1H), 7.58 - 7.53 (m, 2H), 7.41 - 7.36 (m, 4H), 7.23 - 7.17 (m, 2H), 7.12 (s, 2H), 7.00 (s, 1H), 6.25 (s, 1H), 5.60 (d, J = 6.9 Hz, 2H), 5.42 (s, 1H), 5.22 - 5.01 (m, 4H), 4.98 (s, 2H), 4.75 (t, J = 11.0 Hz, 2H), 3.65 - 3.60 (m, 4H), 3.54 - 3.49 (m, 29H), 3.48 (s, 1H), 3.41 (s, 1H), 3.28 (t, J = 8.0 Hz, 2H), 3.25 (s, 3H), 2.84 - 2.69 (m, 1H), 2.66 (t, J = 11.0 Hz, 2H), 2.37 (t, J = 13.2 Hz, 2H), 2.18 - 2.01 (m, 2H), 1.44 (d, J = 10.5 Hz, 3H), 1.18 (t, J = 10.7 Hz, 3H), 0.96 (d, J = 10.2 Hz, 6H), 0.89 (t, J = 10.8 Hz, 3H).

[0254] The multi-step yield of J4-2 is 13%. 1H NMR (400 MHz, DMSO-d6) δ 9.43 (s, 1H), 8.59 (d, J = 1.4 Hz, 1H), 7.83 (dd, J = 7.5, 1.4 Hz, 1H), 7.56 (d, J = 7.5 Hz, 2H), 7.44 (d, J = 7.5 Hz, 2H), 7.29 (d, J = 8.1 Hz, 1H), 7.20 (d, J = 7.5 Hz, 1H), 7.11 (s, 2H), 6.99 (s, 1H), 6.81 (s, 1H), 5.64 (d, J = 10.6 Hz, 2H), 5.46 (s, 1H), 5.30 - 5.13 (m, 2H), 5.04 (s, 2H), 5.00 - 4.90 (m, 2H), 4.75 (t, J = 7.7 Hz, 2H), 3.84 - 3.56 (m, 6H), 3.52 (s, 28H), 3.44 (s, 1H), 3.34 - 3.20 (m, 5H), 2.97 (t, J = 5.8 Hz, 2H), 2.80 - 2.53 (m, 3H), 2.35 (t, J = 4.3 Hz, 2H), 2.31 - 2.02 (m, 4H), 1.44 (d, J = 6.1 Hz, 3H), 0.96 (d, J = 6.4 Hz, 6H), 0.89 (t, J = 6.8 Hz, 3H).

[0255] The synthesis of J1-2 was carried out by referring to J1-1 and Route A using different camptothecins as starting materials. The multi-step yield was 11%. MS: 1558 (M+H) + . 1HNMR (500MHz, DMSO-d6) δ8.56(d,J=2.9Hz,1H),8.33(s,1H),7.86-7.71(m,2H),7.40(s,2H),7.16(dd,J=14.9,9.4Hz,2H),7.01(d,J=15. 9Hz,1H),6.92-6.75(m,2H),6.19(s,1H),5.84(s,1H),5.57(dd,J=18.0,10.4Hz,3H),5.14(s,2H),4.96(s,2H),4.73(t,J=10.2Hz,2H),4. 64-4.48(m,2H),4.22-4.03(m,2H),3.86-3.55(m,8H),3.51(s,28H),3.48(s,2H),3.38(s,1H),3.27(t,J=7.6Hz,2H),3.24(s,3H),2.96(t ,J=11.8Hz,2H),2.67(dt,J=19.4,13.0Hz,4H),2.50(t,J=8.4Hz,2H),2.35(s,1H),2.30-2.10(m,4H),2.07(s,1H),0.89(t,J=10.8Hz,3H).

[0256] The synthesis of J1-3 was carried out by referring to J1-1 and Route A using different camptothecins as starting materials. The multi-step yield was 10%. MS: 1541 (M+H) +. 1H NMR (500 MHz, DMSO-d6) δ 9.11 (s, 1H), 8.59 (d, J = 3.1 Hz, 1H), 8.29 (s, 1H), 7.92 - 7.74 (m, 2H), 7.50 (d, J = 15.0 Hz, 1H), 7.28 (d, J = 14.1 Hz, 3H), 7.19 (dd, J = 15.0, 9.5 Hz, 2H), 7.00 (s, 1H), 6.83 (dd, J = 15.0, 2.9 Hz, 1H), 5.92 (s, 1H), 5.75 (s, 1H), 5.63 (s, 1H), 5.25 - 5.07 (m, 3H), 4.98 (s, 2H), 4.75 (t, J = 10.2 Hz, 2H), 4.45 - 4.39 (m, 2H), 3.96 (s, 1H), 3.91 - 3.57 (m, 9H), 3.52 (s, 28H), 3.31 - 3.27 (m, 4H), 3.25 (s, 3H), 2.97 (t, J = 11.7 Hz, 2H), 2.81 - 2.52 (m, 6H), 2.34 - 2.02 (m, 5H), 1.75 (s, 1H), 0.89 (t, J = 10.8 Hz, 3H).

[0257] Synthesis of J2-1

[0258] The multi-step yield of J2-1 is 8%. 1H NMR (400 MHz, DMSO-d6) δ 8.59 (d, J = 1.4 Hz, 1H), 8.12 (s, 1H), 7.83 (dd, J = 8.8, 1.4 Hz, 2H), 7.19 (dd, J = 9.9, 7.5 Hz, 2H), 7.11 (s, 2H), 6.98 - 6.89 (m, 2H), 6.86 - 6.76 (m, 2H), 6.45 (s, 1H), 5.83 (s, 1H), 5.64 (s, 1H), 5.59 (s, 1H), 5.16 (s, 2H), 5.05 (d, J = 7.7 Hz, 1H), 4.96 (t, J = 6.8 Hz, 2H), 4.52 (t, J = 6.9 Hz, 1H), 4.35 (dd, J = 9.7, 8.3 Hz, 1H), 4.22 (t, J = 8.0 Hz, 1H), 4.00 (t, J = 5.1 Hz, 2H), 3.82 (t, J = 8.3 Hz, 1H), 3.75 (t, J = 5.8 Hz, 2H), 3.65 - 3.58 (m, 4H), 3.55 (ddd, J = 6.2, 5.0, 2.2 Hz, 3H), 3.52 - 3.50 (m, 44H), 3.41 (d, J = 5.5 Hz, 4H), 3.25 (s, 3H), 2.99 (dt, J = 11.8, 5.4 Hz, 4H), 2.76 - 2.64 (m, 3H), 2.57 (t, J = 4.2 Hz, 2H), 2.35 (t, J = 8.0 Hz, 2H), 2.27 - 2.07 (m, 4H), 1.98 - 1.79 (m, 4H), 1.63 - 1.59 (m, 5H), 1.35 - 1.21 (m, 6H), 0.89 (t, J = 6.8 Hz, 3H).

[0259] J2-2 had a multi-step yield of 7%. 1H NMR (400 MHz, DMSO-d6) δ8.59 (d, J = 2.9 Hz, 1H), 7.88-7.75 (m, 2H), 7.50 (d, J = 15.0 Hz, 1H), 7.32-7.16 (m, 3H), 7.13 (s, 2H), 7.03 (s, 1H), 6.83 (dd, J = 15.0, 2.9 Hz, 1H), 6.17 (d, J = 18.5 Hz, 2H), 6.07 (d, J = 15.6 Hz, 1H), 5.73 (d, J = 29.1 Hz, 2H), 5.51 (s, 1H), 5.16 (s, 2H), 5.05 (t, J = 13.0 Hz, 1H). z,1H),4.97(s,2H),4.26(ddd,J=17.2,15.8,4.5Hz,2H),4.05-3.71(m,7H),3.66-3.58(m,4H),3.57-3.47(m,49H),3.40(s,3H),3.25(s,3H) ),3.08-2.90(m,4H),2.77-2.45(m,6H),2.31-2.15(m,3H),2.09-1.80 (m,4H),1.71-1.55(m,3H),1.53-1.17(m,8H),0.89(t,J=13.6Hz,3H).

[0260] Synthesis of J3-1:

[0261] The key intermediate F1 and the side chain L7 were condensed to obtain the target product J3-1. The multi-step yield was 9%. NMR (400MHz, DMSO-d6) δ8.76 (s, 1H), 8.59 (d, J = 2.9Hz, 1H), 8.04 (s, 1H), 7.9 1-7.75(m,2H),7.19(dd,J=15.0,9.5Hz,2H),7.08(s,2H),6.91(s,1H),6.82 (dt,J=14.3,7.1Hz,1H),6.77(s,1H),6.20(s,1H),5.83(s,1H),5.70(s,1H) ,5.62(s,1H),5.43(d,J=15.6Hz,2H),5.16(s,2H),4.97(s,2H),4.75(t,J=10 .9Hz,2H),4.33-4.10(m,2H),3.94-3.89(m,20H),3.99-3.79(m,5H),3.89-3 .56(m,14H),3.51(d,J=5.7Hz,8H),3.49-3.35(m,3H),3.32-3.23(m,7H),3.2 1-3.11(m,1H),3.01-2.93(m,32H),2.74-2.58(m,9H),2.30-2.17(m,3H),2. 13(d,J=20.0Hz,6H),2.09-1.73(m,5H),1.57(s,2H),0.89(t,J=10.8Hz,3H).

[0262] Example 8 Antibody Preparation

[0263] The antibody light and heavy chains were constructed into expression cassettes in plasmid vectors, and the plasmids were extracted. Suspension-acclimated CHO cells were revived in OPM-CD medium (manufacturer: OPM, catalog number P83059) and cultured to a density of 2 million cells / ml with a viability of over 95% in a volume of 1000 ml. 0.5 mg of the light chain plasmid and 0.5 mg of the heavy chain plasmid were combined and dissolved in 10 ml of culture medium. 3 mg of PEI (manufacturer: Polyscience, catalog number: 24765-1) dissolved in culture medium was diluted in 10 ml of culture medium. The mixed plasmid and PEI solutions were incubated at room temperature for 10 minutes and then added dropwise to 1000 ml of cell culture medium. After incubation at 37°C for 5 days, the supernatant was harvested by centrifugation at 12,000 g for 15 minutes. The supernatant was purified by affinity chromatography and eluted with 50 mM acetic acid. The neutralized peak was replaced with PBS pH 7.4 using a 30KD ultrafiltration tube (Merck, Cat. No. UFC9030). The antibody was measured for absorbance at 280 nm, and the concentration was calculated by dividing the absorbance by the theoretical extinction coefficient.

[0264] Example 9 Preparation of Antibody Conjugates

[0265] Take 30 mg of the antibody and dilute it to 3 mg / ml with 10 mM sodium phosphate buffer, pH 7.4. Add tris(2-hydroxyethyl)phosphine hydrochloride (TCEP) stock solution (prepared from 10 mM sodium phosphate buffer, pH 7.4, TCEP manufacturer: Sigma, Cat. No. C4706-2G, CAS. 51805-45-9) to ensure that the final molar concentration of TCEP is X:1 to the final molar concentration of the antibody. Incubate the antibody at 37°C for 45 minutes. Add the linker compound stock solution (prepared from DMSO to 5 mM) to ensure that the final molar concentration of the linker compound is Y:1 to the final molar concentration of the antibody. Incubate the antibody at 2-8°C for 2 hours. The reaction was terminated with cysteine ​​at a final concentration of 20 mM, and the solution was transferred to a 30KD ultrafiltration tube (Merck, product number UFC9030), placed in a centrifugal force of 3000 g at 2-8°C for liquid exchange, and the residual small molecules were reduced to one thousandth of the reaction concentration. The solution was then sterile filtered, the concentration was determined, and the solution was aliquoted and frozen.

[0266] To obtain products with different coupling ratios, adjust the molar ratio of antibody: reducing agent: linker compound between 1:X:Y, while keeping all other conditions unchanged. The DAR value of the final coupling product is determined by the test results. Generally, X=3, Y=6 can produce a conjugate with a coupling ratio of 4, while X=5, Y=15 can produce a conjugate with a coupling ratio of 8.

[0267] Antibodies and conjugates were analyzed by SEC-HPLC molecular sieve analysis using a TSKgel G3000SWXL, 7.8 mm x 30 cm, 5 μm, catalog number 08541 analytical column. Approximately 0.3 mg of sample was centrifuged in 300 μl at 10,000 g for 5 minutes, and the supernatant was collected. The mobile phase consisted of 50 mM sodium phosphate + 0.1 M sodium chloride, pH 6.8. The analytical column was connected to an Agilent 1260 HPLC at a flow rate of 0.8 ml / min. The mobile phase was flushed for at least 30 minutes until the UV baseline at 280 nm stabilized. 100 μg of sample was injected and the mobile phase was flushed for 20 minutes. The percentages of the high-molecular-weight, monomer-weight, and low-molecular-weight peaks were calculated based on the peak areas. The monomer-weight peak was used as the sample purity value.

[0268] The average number of linker payloads conjugated per antibody molecule is the coupling ratio (DAR), which is determined by hydrophobic chromatography (HIC)-HPLC using a TSKgel Butyl-NPR column, 4.6 mm × 10 cm, 2.5 μm, Catalog No. 042168. Approximately 0.3 mg of sample was added to 150 μl of mobile phase A, centrifuged at 10,000 g for 5 minutes, and the supernatant was collected. Mobile phase A consisted of 20 mM sodium phosphate + 1.5 M ammonium sulfate, pH 7.0. Mobile phase B consisted of 20 mM sodium phosphate + 20% (v / v) acetonitrile, pH 7.0. The analytical column was connected to an Agilent 1260 HPLC at a flow rate of 0.6 ml / min. Mobile phase A was rinsed for over 30 minutes until a stable UV baseline at 280 nm was achieved. A 50 μg sample was injected, and after a 2-minute rinse with mobile phase A, an 18-minute gradient elution (0% B–100% B) was performed. The elution was continued with 100% mobile phase B for 5 minutes before terminating. The percentage of each DAR value was calculated based on the peak area. The product of the percentage of each peak and the DAR value was added and then divided by the total percentage to obtain the average DAR value. The conjugates of the present invention are not limited to a DAR of 8 and can be extended to 2–8.

[0269] The conjugates are named as Target-Compound-DAR, as shown in Table 1.

[0270] Table 1 Antibody conjugates

[0271] Example 10 In vivo activity of conjugates

[0272] Human tumor cell lines were revived to the logarithmic growth phase with a viability greater than 90%. Immunocompromised mice (NCG or equivalent) were inoculated with varying numbers of cells, ranging from 10,000 to 1,000,000. When tumors grew to approximately 120 square millimeters, mice were divided into groups of 4 to 5 mice per group. Drugs were administered once intravenously, with the first dose occurring on the day of grouping. The inhibitory activity of each drug group was determined by comparing tumor size between the drug groups or with the saline group. The horizontal axis of the graph represents the treatment time / inoculation time (days after treatment or days after inoculation) for each drug group, and the vertical axis represents tumor volume.

[0273] Some linker compounds were conjugated to an antibody (3101) targeting B7-H3, and the activity of several conjugates in inhibiting tumor cell growth was evaluated in an immunodeficient mouse tumor transplantation model of the human lung cancer cell line Calu-6. The results are shown in Figure 5A. The tumor volumes are ranked from large to small in the order of G1>G6>G7>G8>G5>G4>G3>G2. The results showed that there was no significant difference in the activity of the conjugates of the candidate compounds and that they were better than the positive control.

[0274] Furthermore, the activity of the above-mentioned conjugates in inhibiting tumor cell growth was evaluated in an immunodeficient mouse tumor transplantation model of the human colorectal cancer cell line RKO. The results are shown in Figure 5B. The tumor volume is ranked from large to small in the order of G1 or G6>G4>G7>G2>G3>G5. The results show that the conjugate G3 of the candidate compound has better activity.

[0275] Furthermore, the activity of the above-mentioned conjugates in inhibiting tumor cell growth was evaluated in an immunodeficient mouse tumor transplantation model of the human lung cancer cell line SHP-77. The results are shown in Figure 5C. The tumor volume is ranked from large to small in the order of G1 or G6>G7>G4 or G5 or G2>G3. The results show that the conjugate G3 of the candidate compound has better activity.

[0276] Furthermore, the linker compound was optimized and synthesized, and the conjugates were conjugated. The tumor cell growth inhibition activity of the conjugates was re-evaluated in immunodeficient mouse tumor transplant models of human colorectal cancer cell line RKO and human lung cancer cell line SHP-77. The results are shown in Figure 6A. The tumor volume of the RKO model is ranked from large to small in the order of G1>G6>G2>G3>G4>G7>G5, indicating that the conjugate G5 of the candidate compound has the best activity. The results are shown in Figure 6B. The tumor volume of the SHP-77 model is ranked from large to small in the order of G1>G6>G2>G3>G4>G7>G5, indicating that the conjugate G5 of the candidate compound has the best activity.

[0277] In summary, it can be seen that the activity of the G5 conjugate in inhibiting tumor growth in vivo is slightly better than that of other groups. Further, the linker compound was conjugated to the HER2 target antibody Trastuzumab, and the activity of the conjugate in inhibiting tumor cell growth was evaluated in the immunodeficient mouse tumor transplantation model of the human breast cancer tumor cell line JIMT-1. The results are shown in Figure 7. The model tumor volume is G1>G6 or G8>G3>G2>G4>G6>G7 from large to small. The results show that the activity of the candidate compound conjugate G4 is better in the intermediate stage, or worse than or equal to the control conjugate.

[0278] Furthermore, the linker compound J2-1 was conjugated to the DLL3-targeting antibody 59H4, and the activity of the conjugate in inhibiting tumor cell growth was evaluated in an immunodeficient mouse tumor transplantation model of the human lung cancer tumor cell line NCI-H82. The results are shown in Figure 8. The tumor volumes of the model are G1>G2>G3>G2>G4>G5 from large to small. The results show that the activity of the conjugate of the candidate compound J2-1 is better than that of the control.

[0279] In summary, the antibody conjugates of linker compounds J1-2, J1-3, J2-1, and J2-2 have in vivo tumor inhibition activity that is better than or similar to that of the control conjugates.

[0280] Example 11 In vitro activity of conjugates

[0281] The in vitro activity of candidate linker compounds and their conjugates was determined. Human tumor cell lines were revived to the logarithmic growth phase. When viability was greater than 90%, 500 to 5,000 cells were seeded into 96-well plates in a 150 μl volume. The cells were incubated at 37°C in a 5% CO2 incubator. After approximately 20 hours, 50 μl of culture medium containing different antibody-drug conjugates was added to achieve a final drug concentration of 12 dilutions between 0 and 1,000 nM (1000 nM in the first well, followed by 10 5-fold dilutions, with the final drug concentration at zero, with each sample in duplicate). After 4 to 7 days of co-culture, the plates were removed and 50 μl of CTG detection reagent (CellTiterGlo, Promega, Cat. No. G7575) was added to each well. The cells were reacted for 2 minutes according to the kit's recommended protocol. Fluorescence intensity (MFI) was then measured using a Tecan Spark. The average MFI value of the replicate wells was taken as the Y-axis, and the log10 value (nM) of the dilution gradient was used as the X-axis to make a smooth curve graph, and the EC / IC50 value was calculated according to the four-parameter fitting.

[0282] First, the free payload of the linker compound, Deruxtecan payload Dxd (payload of Deruxtecan), J2-1 payload (payload of J2-1), J2-2 payload (payload of J2-2), and FL118 were subjected to in vitro activity assays in various tumor cells. The results are shown in FIG9 , which show that the payloads have different sensitivities to different cells.

[0283] Next, the in vitro activity of the conjugates was measured, as shown in Figure 10. There were differences in the in vitro and in vivo activity between the different antibody conjugates. Tumor inhibition activity was measured using the in vivo CDX model as a reference.

[0284] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. A linker compound that can be used in a prodrug delivery system and a pharmaceutically acceptable salt, solvate, polymorph or isomer thereof, characterized in that: The structure of the linker compound is shown in formula (1): Wherein, T represents the attachment site for connecting R-TR; In the R-TR, TR is selected from one or more of a cathepsin substrate, a cathepsin substrate amino acid or short peptide chain, a β-glucuronidase substrate, a β-galactosidase substrate, a metalloprotease substrate, an aromatic boronic acid or boric acid ester that can be oxidized to a hydroxyl group, a nitro group, an azide group, an azo group that can be reduced to an amino group, a phosphate ester of a hydroxyl group, and a sulfate ester of a hydroxyl group; R is a substituent group selected from one or more of -H, PEG, maleimide-modified PEG, sugar, folic acid, polypeptides, antibodies, and protective groups; Wherein, A represents the attachment site for ligating LR; The LR is selected from H, CH3(CH2) n , nitro, methoxy, PEG chain, maleimide-modified PEG, maleimide-modified fatty chain, maleimide-modified polyamino acid chain, fatty chain, polyamino acid chain, sugar chain, fatty acid, sugar, folic acid, polypeptide or antibody; Wherein, D represents the connection site for connecting DR; The DR contains exposed -OH, -NH2, -NHR or -SH; the DR is a chemical drug; Wherein, X is O, N or S; Wherein, R2 is H or CH3(CH2) n ; n = 0-20; Wherein, R1 is an electron-withdrawing group.

2. The linker compound according to claim 1, characterized in that The chemical drug is a camptothecin compound or a paclitaxel compound, or a derivative of the two compounds; And / or, the R1 electron-withdrawing group includes halogen, cyano, nitro, carboxylic acid, sulfonic acid, carboxylate, sulfonate, amide, and / or a group connected by an amide bond.

3. The linker compound according to claim 2, characterized in that The chemical drugs include SN-38, DXd, isitecan, belotecan, paclitaxel, docetaxel, podophyllotoxin, or CA4.

4. The linker compound according to claim 1, characterized in that When the linking site T is connected by an amide bond, the structure of the linker compound is as shown in the following formula (1-1): wherein TR is selected from a single amino acid, a dipeptide, or a tetrapeptide selected from a tissue protease substrate or a metalloprotease substrate; The single amino acid is selected from the group consisting of unprotected or protected, PEG chains, polyamino acid chains, and maleimide-modified single amino acids, including -Lys-, -Cit-, -Arg-, -Leu-, -His-, -Asp-, -Glu-, -Ala-, -Val-, -Ile-, -Pro-, -Phe-, -Trp-, -Met-, -Ser-, -Cys- or -Asn-; the dipeptide is selected from the group consisting of unprotected or protected, PEG chains, polyamino acid chains, and maleimide-modified single amino acids. Imidyl modified dipeptides include -Phe-Lys-, -Val-Lys-, -Ala-Lys-, -Val-Cit-, -Phe-Cit-, -Leu-Cit-, -Ile-Cit, -Trp-Cit-, -Phe-Gly-, -Ala-Ala- or -Phe-Arg-; tetrapeptides selected from tetrapeptides without protection or with protection groups, PEG chains, polyamino acid chains and maleimide modified tetrapeptides include -Gly-Gly-Phe-Gly-; and / or, When the linking site T is connected by an ether bond or an ester bond, the structure of the linker compound is as shown in the following formula (1-2): Among them, TR includes β-glucoside, β-galactoside, phosphate, sulfate, and pyrophosphate. And / or, when the linking site A is connected by an amide bond, the structure of the linker compound is as shown in the following formula (1-5): Wherein, LLR is a linker modification fragment, including one or more of PEG, sugar, alkyl chain, maleimide-modified PEG, modified alkyl chain, and fatty acid; The groups connected to the connection point T include β-glucoside, β-galactoside, phosphate, sulfate, and pyrophosphate; And / or, when the group connected to the connection site A is H, the structure of the linker compound is as shown in the following formula (1-6): T is selected from a single amino acid, a dipeptide, or a tetrapeptide selected from a cathepsin substrate or a metalloprotease substrate; The single amino acid is selected from single amino acids without protection or with protection groups, PEG chains, polyamino acid chains and maleimide-modified single amino acids, including -Lys-, -Cit-, -Arg-, -Leu-, -His-, -Asp-, -Glu-, -Ala-, -Val-, -Ile-, -Pro-, -Phe-, -Trp-, -Met-, -Ser-, -Cys- or -Asn-; the dipeptide is selected from the dipeptide without protection or with a protecting group, a PEG chain, a polyamino acid chain and a maleimide-modified dipeptide, including -Phe-Lys-, -Val-Lys-, -Ala-Lys-, -Val-Cit-, -Phe-Cit-, -Leu-Cit-, -Ile-Cit, -Trp-Cit-, -Phe-Gly-, -Ala-Ala- or -Phe-Arg-; the tetrapeptide is selected from the tetrapeptide without protection or with a protecting group, a PEG chain, a polyamino acid chain and a maleimide-modified tetrapeptide, including -Gly-Gly-Phe-Gly-.

5. The linker compound according to claim 1, characterized in that When the R1 is an amide bond-connected group, the linker compound further includes a connection site L1, and the structure of the linker compound is shown in the following formula (L): Wherein, R3 is selected from one or more of H, C1-C6 alkyl or cycloalkyl; L1 is selected from one or more of H, amino protecting group, aliphatic chain, PEG chain, polyamino acid chain, sugar chain, and maleimide-modified PEG chain, maleimide-modified aliphatic chain, maleimide-modified polyamino acid chain, maleimide-modified sugar chain; R3 and L1 can be connected to form 3 to 6 rings, including The L2 is selected from one or more of H, an amino protecting group, a C1-C6 alkyl or cycloalkyl group, a fat chain, a linear or branched PEG chain, a sugar chain, a polyamino acid chain, a maleimide-modified PEG chain, a maleimide-modified fat chain, a maleimide-modified polyamino acid chain, and a maleimide-modified sugar chain; R4 is selected from one or more selected from H, C1-C6 alkyl or cycloalkyl; and / or, When the R1 is a nitro group, the structure of the linker compound is as shown in the following formula (J):

6. The linker compound according to claim 5, characterized in that The linker compound (L) includes (L-5) and (L-6), which are respectively shown in the following formulas: in, In formula (L-5), AA represents an amino acid, m represents the number of amino acids, and m is selected from 1-6; L3 is selected from H, an amino protecting group, a fatty chain, a PEG chain, a polyamino acid chain, a sugar chain, and one or more of a maleimide-modified PEG chain, a maleimide-modified fatty chain, a maleimide-modified polyamino acid chain, and a maleimide-modified sugar chain; R6 and R7 are selected from H or a C1-C6 alkyl or cycloalkyl group or independently, L1 is selected from a C1-C6 alkyl or cycloalkyl group, or R6 and L1 can form a cycloalkyl group; L2 is selected from H, an amino protecting group, a fatty chain, a PEG chain, One or more of a polyamino acid chain, a sugar chain, a maleimide-modified PEG chain, a maleimide-modified fatty chain, a maleimide-modified polyamino acid chain, and a maleimide-modified sugar chain; L2 and L3 are selected together or independently from H, an amino protecting group, a fatty chain, a PEG chain, a polyamino acid chain, a sugar chain, and one or more of a maleimide-modified PEG chain, a maleimide-modified fatty chain, a maleimide-modified polyamino acid chain, and a maleimide-modified sugar chain; L2 and L3 are not simultaneously selected from a maleimide-modified PEG chain, a fatty chain, a polyamino acid chain, and a sugar chain; In formula (L-6), T is selected from one or more of β-glucoside, β-galactoside, phosphate, pyrophosphate or sulfate; L4 is selected from hydrogen, C1-C6 alkyl or cycloalkyl, nitro, amino, methoxy, carboxyl, ester, fatty chain, linear or branched PEG chain, sugar chain, polyamino acid chain R 10 Selected from hydrogen, C1-C3 alkyl or cycloalkyl; L5 is selected from hydrogen, amino protecting group, C1-C6 alkyl or cycloalkyl, fatty chain, linear or branched PEG chain, sugar chain, polyamino acid chain, maleimide-modified PEG chain, maleimide-modified fatty chain, maleimide-modified polyamino acid chain, maleimide-modified sugar chain; R6 and R7 are selected from H or C1-C6 alkyl or cycloalkyl together or independently, L1 is selected from C1-C6 alkyl or cycloalkyl, or R6 and L1 can form a cycloalkyl; L2 is selected from H, amino protecting group, fatty chain, PEG chain, polyamino acid chain, sugar chain, and one or more of maleimide-modified PEG chain, maleimide-modified fatty chain, maleimide-modified polyamino acid chain, and maleimide-modified sugar chain.

7. A covalently bound prodrug or antibody drug conjugate, characterized in that: It is formed by covalently linking albumin, an antibody or an antigen-binding fragment thereof with the maleimide of the linker compound represented by formula (L) and formula (J) described in claim 5, and its structure is shown in the following formula (L1) to formula (L8) and formula (J1) to formula (J4): ， Wherein, in the above formula (L1) to formula (L8), formula (J1) and formula (J4), n, o, and m represent the number of repeating units, n is selected from 1-24; o is selected from 2-6; and m is selected from 0-12; In formula (L1) to formula (L4) and formula (L8), L' is selected from: One or more of; In formula (L2), R' is selected from hydrogen, acetyl, amino protecting groups including Boc, Cbz, Fmoc, Alloc, and PEG chains, polyamino acid chains, sugar chains, and fatty chains; in formula (L4), R" is selected from hydrogen, nitro, and methoxy; D1 represents hydroxyl-containing drugs including camptothecins. D1 includes:

8. The covalently bound prodrug or antibody drug conjugate according to claim 7, characterized in that: Formula (L1) to Formula (L8) and Formula (J1) to Formula (J4) include the following formulas (J1-1) to (J1-4), (J2-1) to (J2-2), (J4-1) to (J4-2), (L1-1) to (L1-4), (L4-1), (L5-1) to (L5-3), (L7-1) to (L7-4), and (L8-1) to (L8-3):

9. The covalently bound prodrug or antibody drug conjugate according to claim 7 or 8, characterized in that: The antibody or antigen-binding fragment thereof is selected from rabbit antibody, mouse antibody, chimeric antibody, humanized antibody or fully human antibody or a protein fragment having similar functions to an antibody.

10. The covalently bound prodrug or antibody drug conjugate according to claim 9, characterized in that: The antibody or antigen-binding fragment thereof is selected from the group consisting of anti-CD3 antibody, anti-FOLR1 antibody, anti-ROR1 antibody, anti-TNFα antibody, anti-TF antibody, anti-EpCAM antibody, anti-EGFRvIII antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-MUC16 antibody, anti-ENPP3 antibody, anti-TDGF1 antibody, anti-ETBR antibody, anti-MSLN antibody, anti-TIM-1 antibody, anti-LRRC15 antibody, anti-LIV-1 antibody, anti-CanAg / AFP antibody, anti-Claudin 6 antibody, anti-Claudin 9 antibody, anti-Claudin 18.2 antibody, anti-Mesothelin antibody, anti-HER2 antibody, anti-EGFR antibody, anti-c-MET antibody, anti-SLITRK6 antibody, anti-KIT / CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7 / CS1 antibody, anti-NaPi2B / SLC34A2 antibody, anti-GPNMB antibody, anti-HER3 antibody, anti-MUC1 / CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7-H3 (CD276) antibody, anti-PTK7 / CCK4 antibody, anti-PRLR antibody, anti-EFNA4 antibody, anti-5T4 antibody, anti-NOTCH3 antibody, anti-Nectin 4 antibody, anti-TROP-2 antibody, anti-CD142 antibody, anti-CA6 antibody, anti-GPR20 antibody, anti-CD174 antibody, anti-CD70 antibody, anti-CD71 antibody, anti-EphA2 antibody, anti-LYPD3 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FRα antibody, anti-CEACAMs antibody, anti-GCC antibody, anti-IntegrinAv antibody, anti-CAIX antibody, anti-P-cadherin antibody, anti-GD3 antibody, anti-Cadherin 6 antibody, anti-LAMP1 antibody, anti-FLT3 antibody, anti-BCMA antibody, anti-CD79b antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD74 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD37 antibody, anti-CD47 antibody, anti-CD138 antibody, anti-CD352 antibody, anti-CD25 antibody and anti-CD123 antibody; And / or, the antibody or antigen-binding fragment thereof is selected from: i) Trastuzumab antibody; ii) ILB-3101 antibody; iii) 59H4 antibody.

11. The covalently bound prodrug or antibody drug conjugate according to any one of claims 7 to 10, characterized in that: The coupling ratio of the antibody or antigen-binding fragment thereof to the linker compound is 3-8; preferably, the coupling ratio is optimized to 6-8 or 4-6.

12. A pharmaceutical composition comprising a linker compound of the prodrug delivery system according to any one of claims 1 to 6, a pharmaceutically acceptable salt, solvate, polymorph, isomer, derivative thereof, or a covalently bound prodrug or antibody drug conjugate according to claims 7 to 11, and a pharmaceutically acceptable excipient, diluent or carrier thereof.

13. Use of a linker compound of a prodrug drug delivery system according to any one of claims 1 to 6, a pharmaceutically acceptable salt, solvate, polymorph, isomer, derivative thereof, or a covalently bound prodrug or antibody drug conjugate according to claims 7 to 11, or a pharmaceutical composition according to claim 12 in the preparation of a drug for treating tumors.

14. The use according to claim 13, characterized in that The tumor is breast cancer, ovarian cancer, ovarian teratoma, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, testicular cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, head and neck cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, neural tumor, glioma, neuroblastoma, multiforme glioma, squamous cell carcinoma, myeloma, sarcoma, lymphoma and leukemia or blood tumor.

15. A method for preparing the linker compound (L) as claimed in claim 5, characterized in that: The preparation reaction process of this method is shown in the following formula: The reaction steps include: a) Substitution reaction: Under alkaline conditions, compound 1 and the hydroxyl protecting group undergo a nucleophilic substitution reaction in a solvent to generate compound 2; b) Reduction reaction: Compound 2 is reduced by zinc powder / acid or iron powder / acid system to obtain compound 3, and the solvent is a conventional organic solvent / acid; c) Condensation reaction: Under nitrogen conditions, compound 3 reacts with formic acid and acetic anhydride to generate compound 4; d) Reduction reaction: Under nitrogen conditions, compound 4 and borane dimethyl sulfide are dissolved in a solvent and undergo a reduction reaction to generate compound 5; e) Substitution reaction: Under alkaline conditions, compound 5 and BOC anhydride undergo substitution reaction to obtain compound 6; f) Hydrolysis reaction: Under alkaline conditions, compound 6 undergoes hydrolysis reaction to obtain compound 7; g) condensation reaction: compound 7 reacts with amine under the action of a condensing agent to generate compound 8; h) Decomposition reaction: Compound 8 is subjected to a Pd / C deprotection reaction to generate compound 9, and the solvent is methanol or a mixed solvent of methanol, tetrahydrofuran and / or ethyl acetate; i) Etherification reaction: under nitrogen protection, benzyl bromide substrates with different trigger fragments are reacted with silver oxide or base to generate compound 10, and the solvent is anhydrous tetrahydrofuran, anhydrous dichloromethane or anhydrous acetonitrile; j) Deprotection reaction: Compound 10 is deprotected from N-Boc under acidic conditions in a solvent to obtain compound 11; k) Condensation reaction: Under nitrogen protection, compound 11 was dissolved in an organic solvent, and di(trichloromethyl) carbonate and anhydrous organic base were added. After reacting for 1 hour, active drug and DMAP were added, and the reaction was carried out at 20-60°C overnight to obtain compound 12.

16. The preparation method according to claim 15, characterized in that: In step a), the hydroxyl protecting group includes benzyl bromide, p-methoxybenzyl bromide MOMCl; the base includes potassium carbonate, cesium carbonate, sodium carbonate; the solvent includes acetone, acetonitrile or NN-dimethylformamide; the molar ratio of compound 1 to the base is 1:(1.5-2); the molar ratio of compound 1 to the protecting group is 1:(1.5-2); In step b), the molar ratio of the compound 2 to the zinc powder or iron powder is 1:5; In step c), the molar ratio of compound 3 to formic acid is 1:(5-10), and the molar ratio of compound 3 to acetic anhydride is 1:(5-10); In step d), the molar ratio of compound 4 to borane dimethyl sulfide is 1:3; In step e), the base includes DIPEA and DMAP; In step f), the base comprises LiOH, NaOH or KOH; the molar ratio of compound 4 to borane dimethyl sulfide is 1:(2-5); In step g), the condensing agent includes DCC, EEDQ, HATU, EDC / HOBt, PyBOP; the molar ratio of compound 7 to amine is 1:(1-2), and the molar ratio of compound 7 to condensing agent is 1:(2-5); In step h), the molar ratio of compound 8 to Pd / C is 1:(0.1-0.2); In step i), the molar ratio of compound 9 to different benzyl bromides or benzyl alcohols is 1:(1-1.2), and the molar ratio of compound 9 to silver oxide / phosphorus reagent / azo is 1:(1.2-2); In step j), the acid includes hydrochloric acid or trifluoroacetic acid, and the solvent includes tetrahydrofuran or dichloromethane; In step k), the anhydrous organic base includes triethylamine, DIPEA, and pyridine; the drug contains exposed -OH, -NH2, -NHR, and -SH; the molar ratio of compound 11 to di(trichloromethyl)carbonate is 1:0.5, and the molar ratio of compound 11 to the anhydrous organic base is 1:(3-5); the molar ratio of compound 11 to the active drug is 1:(1-1.2).

17. A method for preparing a covalently bound prodrug or antibody drug conjugate (J1), characterized in that: The reaction process of the preparation method is shown in the following formula: The reaction steps include: 1) Bromination reaction: Compound 1 is subjected to bromination reaction to obtain compound 2; the bromination reagent includes phosphorus tribromide, triphenylphosphine and carbon tetrabromide, the The solvent was anhydrous dichloromethane; 2) Etherification reaction: Compound 2 is subjected to an etherification reaction to generate compound 3, wherein the organic solvent is anhydrous acetonitrile; 3) Reduction reaction: Under nitrogen conditions, compound 3 and borane dimethyl sulfide are dissolved in a solvent to undergo a reduction reaction to generate compound 4, and the solvent is anhydrous tetrahydrofuran; 4) Condensation reaction: Under nitrogen protection, compound 4 is dissolved in an organic solvent, di(trichloromethyl) carbonate and triethylamine are added, and after reacting for 1 hour, a hydroxyl-containing drug is added, and the reaction is carried out at room temperature overnight to obtain compound 5; 5) Deprotection: Under nitrogen protection, compound 5 was dissolved in an organic solvent, and a 0.2 M aqueous base solution was added under ice bath condition to deprotect to obtain compound 6; 6) Condensation reaction: Compound 6 and compound 9 are dissolved in an organic solvent and stirred at room temperature for 6-8 hours to react to generate the prodrug having the general structure of formula (J1).

18. The preparation method according to claim 17, characterized in that: In step 1), the molar ratio of compound 1 to the bromination reagent is 1:3; In step 3), the molar ratio of compound 3 to borane dimethyl sulfide is 1:3; In step 4), the molar ratio of compound 4 to bis(trichloromethyl) carbonate is 1:0.5, the molar ratio of compound 4 to triethylamine is 1:4; the molar ratio of compound 4 to the hydroxyl-containing drug is 1:1; In step 5), the base includes one or more of lithium hydroxide, sodium hydroxide, and potassium hydroxide; the organic solvent includes one or more of methanol, acetonitrile, tetrahydrofuran, and water; the molar ratio of the compound 5 to the base is 1:8; In step 6), the molar ratio of compound 6 to compound 9 is 1:1; the organic solvent is one or a mixture of methanol, acetonitrile, tetrahydrofuran, N,N-dimethylformamide and water.

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