Azo compound, azo prodrug compound, pharmaceutical composition, high-energy ray-responsive lipid molecule and high-energy ray-responsive liposome

Abstract

The present invention discloses an azo compound, an azo prodrug compound, a pharmaceutical composition, a high-energy ray-responsive lipid molecule, and a high-energy ray-responsive liposome. The hydroxyl end of the azo compound of the present invention can be covalently bonded to a drug to prepare an azo prodrug compound. When the prepared azo prodrug compound is used in combination with radiotherapy, under the irradiation of high-energy rays (e.g., α-rays, β-rays, γ-rays, X-rays), the azo group undergoes chemical transformation, thereby releasing the drug, thereby achieving drug chemotherapy while achieving radiotherapy. Compared with traditional chemotherapeutic drugs, the azo prodrug compound prepared by the azo compound of the present invention has a novel structure, good safety, and can achieve a stable activated radiotherapy response. In addition, the azo prodrug compound prepared by the azo compound of the present invention has a stable activation efficiency, a wide range of application of the original drug, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an azo compound, an azo prodrug compound, a pharmaceutical composition, a high-energy ray-responsive lipid molecule and a high-energy ray-responsive liposome. Background Art

[0002] In clinical treatment, combined radiotherapy and chemotherapy is the first-line treatment for more than 50% of cancer patients. While traditional chemotherapy drugs kill tumor cells, they also cause damage to normal cells and tissues, resulting in common toxic side effects such as hair loss, cardiotoxicity, and nephrotoxicity. How to reduce or eliminate the serious adverse reactions caused by chemotherapy drugs is one of the problems that urgently need to be solved in the current clinical treatment of cancer.

[0003] Anti-tumor drugs can be made into exogenous responsive prodrugs and activated at the tumor site after administration to effectively reduce the systemic toxicity of the drugs. Compared with other activation methods, X-rays can accurately penetrate deep tissues and have the advantage of high temporal and spatial resolution. Currently, X-ray technology used in medical radiotherapy is developing rapidly and can accurately locate tumors at the submillimeter level. Therefore, the strategy of X-ray activation is expected to achieve the targeted release of anti-cancer drugs at the tumor site, greatly reduce the killing of normal cells, and achieve precise tumor treatment on the basis of combined radiotherapy and chemotherapy.

[0004] According to existing research, the activation of prodrugs by X-rays mainly depends on the upregulated enzymes (such as caspase-3) expressed in the tumor site after X-ray irradiation and the ionization of water molecules to generate hydroxyl radicals (·OH), hydrogen radicals (·H), hydrated electrons (e - aq ) etc. Although the above-mentioned prodrug activation system has achieved certain results in in vitro activation and in vivo efficacy experiments, it is still in the early stage of exploration and faces challenges such as unstable activation efficiency and limited application scope of the original drug.

[0005] Therefore, it is necessary to further develop radiotherapy-responsive prodrug activation systems with novel structures, good safety, and stable activation for a variety of chemotherapeutic drugs with broad-spectrum anti-tumor activity. Summary of the invention

[0006] Based on this, it is necessary to provide an azo compound that can solve the above problems.

[0007] In addition, it is also necessary to provide an azo prodrug compound, a pharmaceutical composition, a high-energy ray-responsive lipid molecule, and a high-energy ray-responsive liposome.

[0008] An azo compound having the following general formula I:

[0009]

[0010] in, It is a single bond, which means that A and B are distributed on both sides or the same side of the azo double bond;

[0011] -A- and -B- are independently selected from: aryl or heteroaryl;

[0012] The term "aryl group" means an aromatic hydrocarbon group having 6 to 20 carbon atoms obtained by removing two hydrogen atoms from two carbon atoms in the aromatic nucleus of an aromatic hydrocarbon molecule;

[0013] The term "heteroaryl" refers to aromatic groups as 5-membered or 6-membered rings and fused ring systems comprising 5 to 20 atoms, wherein at least one ring is aromatic and contains one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, and if the ring contains multiple oxygen atoms, these oxygen atoms are not directly adjacent;

[0014] -C- is -X, -O-, -OC(=O)- or X is Cl, Br or I; when -C- is -X, R2 is an empty bond; when -C- is -O-, R2 is H; when -C- is -OC(=O)-, -R2 is -Cl, When -C- is When -R2 is -Cl;

[0015] -R1 is

[0016] -R3 and -R4 are independently selected from: -CN, -COOR, -CONR'R", -H, halogen, C1-C 12 Alkyl, C1~C 12 Heteroalkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The heteroatom in the aryl group is O or N;

[0017] -R5 and -R6 are independently selected from: -H, C1-C 12 Alkyl, C1~C 12 Heteroalkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The heteroatom in the aryl group is O or N;

[0018] -R7 is H, C1~C 12 Alkyl, C1~C 12 Heteroalkyl, C3~C 12Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The heteroatom in the aryl group is O or N;

[0019] -R8 is 1 to 4 substituted, and -R8 is selected from -NO2, -F, -CN, -CF3, -COOR, -CONR'R".

[0020] In one embodiment, the aryl or heteroaryl is substituted with one or more substituents, the substituents comprising halogen, -R9, -NR9R 10 , -CN, -NO2, -N3, -OR9, -SR9, -NHCOR9, -O-COR9, -CH=CR9R 10 , -C(=O)-R9, -C(=O)-OR9, -C(=O)-Cl, -C(=O)-NH2, -C(=CO)-NH-R9 and -C(=O)-NR9R 10 , where -R9 and -R 10 are independently selected from -H, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C3-C 10 Cycloalkyl, C6~C 20 The alkyl, alkenyl, cycloalkyl, aryl and heteroaryl groups described for the substituents are optionally replaced by one or more halogen, hydroxyl, mercapto, -NH2, -CN, -NO2, -N3, -NHCOH, -OC(=O)H, -C(=O)H, -C(=O)-OH, -C(=O)-Cl, -C(=O)-NH2, -C(=O)-NH-CH3, -C(=O)-CH3, -C(=O)-OCH3, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C2-C6 alkenyl, C3-C 10 Cycloalkyl, C6~C 20 substituted with an aryl group or a heteroaryl group having 5 to 20 ring atoms;

[0021] -R, -R' and -R" are independently selected from -H, Cl~C 12 Alkyl, C1~C 12 Heteroalkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The heteroatom in the aryl group or the heteroalkyl group is O or N.

[0022] In one embodiment, the structural formula of the azo compound is:

[0023]

[0024]

[0025] An azo prodrug compound, wherein the azo prodrug compound has the following general formula II:

[0026]

[0027] in, It is a single bond, which means that A and B are distributed on both sides or the same side of the azo double bond;

[0028] -A- and -B- are independently selected from: aryl or heteroaryl;

[0029] The term "aryl group" means an aromatic hydrocarbon group having 6 to 20 carbon atoms obtained by removing two hydrogen atoms from two carbon atoms in the aromatic nucleus of an aromatic hydrocarbon molecule;

[0030] The term "heteroaryl" refers to aromatic groups as 5-membered or 6-membered rings and fused ring systems comprising 5 to 20 atoms, wherein at least one ring is aromatic and contains one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, and if the ring contains multiple oxygen atoms, these oxygen atoms are not directly adjacent;

[0031] -C- is -O-, -N + (R a R b )-、-OC(=O)- or

[0032] -G1 is a drug group;

[0033] -G2 is a targeting group,

[0034] -R3 and -R4 are independently selected from: -CN, -COOR, -CONR'R", -H, halogen, C1-C 12 Alkyl, C1~C 12 Heteroalkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The heteroatom in the aryl group is O or N;

[0035] -R5 and -R6 are independently selected from: -H, C1-C 12 Alkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C12 Cycloalkyl or C6~C 20 The aromatic group;

[0036] -R7 is -H, C1~C 12 Alkyl, C1~C 12 Heteroalkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The heteroatom in the aryl group or the heteroalkyl group is O or N.

[0037] In one embodiment, the aryl or heteroaryl is substituted with one or more substituents, the substituents comprising halogen, -R9, -NR9R 10 , -CN, -NO2, -N3, -OR9, -SR9, -NHCOR9, -O-COR9, -CH=CR9R 10 , -C(=O)-R9, -C(=O)-OR9, -C(=O)-Cl, -C(=O)-NH2, -C(=CO)-NH-R9 and -C(=O)-NR9R 10 , where -R9 and -R 10 are independently selected from -H, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C3-C 10 Cycloalkyl, C6~C 20 The alkyl, alkenyl, cycloalkyl, aryl and heteroaryl groups described for the substituents are optionally replaced by one or more halogen, hydroxyl, mercapto, -NH2, -CN, -NO2, -N3, -NHCOH, -OC(=O)H, -C(=O)H, -C(=O)-OH, -C(=O)-Cl, -C(=O)-NH2, -C(=O)-NH-CH3, -C(=O)-CH3, -C(=O)-OCH3, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C2-C6 alkenyl, C3-C 10 Cycloalkyl, C6~C 20 substituted with an aryl group or a heteroaryl group having 5 to 20 ring atoms;

[0038] -R a and -R b Each independently selected from C1 to C 12 Alkyl, C1~C 12 Heteroalkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C20 The heteroatom in the aryl group is O or N;

[0039] -R, -R' and -R" are independently selected from -H, Cl~C 12 Alkyl, C1~C 12 Heteroalkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The heteroatom in the aryl group or the heteroalkyl group is O or N.

[0040] In one embodiment, the targeting group is covalently linked to other structures via N, O or S;

[0041] The targeting group is an antibody, a polypeptide, a sugar group, a small molecule ligand or a nucleic acid aptamer;

[0042] The drug group is covalently linked to other structures via N, O or S;

[0043] The drug group is

[0044] A pharmaceutical composition comprises the above-mentioned azo prodrug compound or a pharmaceutically acceptable salt thereof.

[0045] A high-energy ray-responsive lipid molecule has the following general formula III:

[0046]

[0047] in, It is a single bond, which means that A and B are distributed on both sides or the same side of the azo double bond;

[0048] -A- and -B- are independently selected from: aryl or heteroaryl;

[0049] The term "aryl" means an aromatic hydrocarbon group having 6 to 20 carbon atoms obtained by removing one hydrogen atom from each of two carbon atoms in the aromatic nucleus of an aromatic hydrocarbon molecule;

[0050] The term "heteroaryl" refers to aromatic groups as 5-membered or 6-membered rings and fused ring systems comprising 5 to 20 atoms, wherein at least one ring is aromatic and contains one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, and if the ring contains multiple oxygen atoms, these oxygen atoms are not directly adjacent;

[0051] -C- is -O-, -N + (R a R b )-、-OC(=O)- or

[0052] -G3 and -G4 are respectively one of a hydrophilic group and a hydrophobic group, and the high-energy ray-responsive lipid molecule is an amphiphilic molecule;

[0053] -R3 and -R4 are independently selected from: -CN, -COOR, -CONR'R", -H, halogen, C1-C 12 Alkyl, C1~C 12 Heteroalkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The heteroatom in the aryl group or the heteroalkyl group is O or N.

[0054] In one embodiment, the lipid molecule is a fatty acid ester or amide derivative, a sterol ester or amide derivative, a glyceride, a glycerophospholipid or a glycolipid containing an azo structural unit;

[0055] The aryl or heteroaryl is substituted by one or more substituents, and the substituents include halogen, -R9, -NR9R 10 , -CN, -NO2, -N3, -OR9, -SR9, -NHCOR9, -O-COR9, -CH=CR9R 10 , -C(=O)-R9, -C(=O)-OR9, -C(=O)-Cl, -C(=O)-NH2, -C(=CO)-NH-R9 and -C(=O)-NR9R 10 , where -R9 and -R 10 are independently selected from -H, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C3-C 10 Cycloalkyl, C6~C 20 The alkyl, alkenyl, cycloalkyl, aryl and heteroaryl groups described for the substituents are optionally replaced by one or more halogen, hydroxyl, mercapto, -NH2, -CN, -NO2, -N3, -NHCOH, -OC(=O)H, -C(=O)H, -C(=O)-OH, -C(=O)-Cl, -C(=O)-NH2, -C(=O)-NH-CH3, -C(=O)-CH3, -C(=O)-OCH3, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C2-C6 alkenyl, C3-C 10 Cycloalkyl, C6~C 20 substituted with an aryl group or a heteroaryl group having 5 to 20 ring atoms;

[0056] -Ra and -R b Each independently selected from C1 to C 12 Alkyl, C1~C 12 Heteroalkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The heteroatom in the aryl group is O or N;

[0057] -R, -R' and -R" are independently selected from -H, Cl~C 12 Alkyl, C1~C 12 Heteroalkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The heteroatom in the aryl group or the heteroalkyl group is O or N.

[0058] A high-energy ray-responsive liposome is formed by self-assembly of the above-mentioned high-energy ray-responsive lipid molecules.

[0059] The hydroxyl end of the azo compound of the present invention can be connected to a drug through a substitution reaction to prepare an azo prodrug compound. When the prepared azo prodrug compound is used in combination with radiotherapy, under the irradiation of high-energy rays (for example, α rays, β rays, γ rays, and X-rays), the azo group undergoes chemical transformation, thereby releasing the drug, thereby achieving drug chemotherapy while achieving radiotherapy.

[0060] Compared with traditional chemotherapy drugs, the azo prodrug compound prepared by using the azo compound of the present invention has a novel structure, good safety, and can achieve a stable activated radiotherapy response. In addition, the azo prodrug compound prepared by using the azo compound of the present invention has a stable activation efficiency, a wide range of application of the original drug, and has good application prospects.

[0061] In addition, the hydroxyl end of the azo compound can be connected to a drug through a substitution reaction to prepare an azo prodrug compound, and the carboxyl end of the azo compound can also be connected to a substance with a targeting function (such as an antibody) through a condensation reaction, so that the prepared azo prodrug compound has a targeting function.

[0062] In addition, the hydroxyl end and carboxyl end of the azo compound of the present invention can also be connected to the hydrophilic group and the hydrophobic group through substitution reaction and condensation reaction respectively to form an amphiphilic lipid molecule, and the lipid molecule can achieve high-energy ray response through the structural transformation of the azo group before and after irradiation. The amphiphilic property of the lipid molecule enables it to self-assemble to form a high-energy ray-responsive liposome, and the interior of the high-energy ray-responsive liposome can be coated with drugs. Under the irradiation of high-energy rays (for example: α rays, β rays, γ rays, X-rays), the azo group undergoes structural transformation, the hydrophilic group and the hydrophobic group are separated, and the liposome structure is decomposed, thereby releasing the internally coated drugs, thereby achieving radiotherapy and drug chemotherapy at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0064] in:

[0065] Figures 1 to 18 This is a graph showing the toxicity comparison test results of various azo prodrug compounds and original drugs in various cells in Test Example 2.

[0066] Fig.19 This is a diagram showing the hydrophobic interaction chromatography (HIC-HPLC) test results of the antibody-drug conjugate XPADC-106-011 prepared in Example 24.

[0067] Fig. 20 This is a graph showing the LCMS chromatographic detection results of the 107-008 liposomes prepared in Example 25 before irradiation with 60 Gy and after the nanoparticles were demulsified with ethanol.

[0068] Fig.21 This is a graph showing the LCMS chromatographic detection results of the 107-008 liposomes prepared in Example 25 after irradiation with 60 Gy and the nanoparticles were demulsified with ethanol.

[0069] Fig. 22 This is a graph showing the particle size distribution test results of the 115-001 liposome prepared in Example 26 before irradiation with 60 Gy.

[0070] Fig.23 This is a graph showing the particle size distribution test results of the 115-001 liposome prepared in Example 26 after irradiation with 60 Gy. DETAILED DESCRIPTION

[0071] The following will be combined with the specific embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0072] It should be noted that all directional indications in the embodiments of the present invention (such as up, down, left, right, front, back, etc.) are only used to explain the relative position relationship, movement status, etc. between the various parts in a certain specific posture. If the specific posture changes, the directional indication will also change accordingly.

[0073] In addition, the descriptions of "first", "second", etc. in the present invention are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in the field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0074] The present invention discloses an azo compound according to an embodiment of the present invention, which has the following general formula I:

[0075]

[0076] in, It is a single bond, which means that A and B are distributed on both sides or the same side of the azo double bond;

[0077] -A- and -B- are independently selected from: aryl or heteroaryl;

[0078] The term "aryl group" means an aromatic hydrocarbon group having 6 to 20 carbon atoms obtained by removing two hydrogen atoms from two carbon atoms in the aromatic nucleus of an aromatic hydrocarbon molecule;

[0079] The term "heteroaryl" refers to aromatic groups as 5-membered or 6-membered rings and fused ring systems comprising 5 to 20 atoms, wherein at least one ring is aromatic and contains one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, and if the ring contains multiple oxygen atoms, these oxygen atoms are not directly adjacent;

[0080] -C- is -X, -O-, -OC(=O)- or X is Cl, Br or I; when -C- is -X, R2 is an empty bond; when -C- is -O-, R2 is H; when -C- is -OC(=O)-, -R2 is -Cl, When -C- is When -R2 is -Cl;

[0081] -R1 is

[0082] -R3 and -R4 are independently selected from: -CN, -COOR, -CONR'R", -H, halogen, C1-C 12 Alkyl, C1~C 12 Heteroalkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The heteroatom in the aryl group is O or N;

[0083] -R5 and -R6 are independently selected from: -H, C1-C 12 Alkyl, C1~C 12 Heteroalkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The heteroatom in the aryl group is O or N;

[0084] -R7 is H, C1~C 12 Alkyl, C1~C 12 Heteroalkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The heteroatom in the aryl group is O or N;

[0085] -R8 is 1 to 4 substituted, and -R8 is selected from -NO2, -F, -CN, -CF3, -COOR, -CONR'R".

[0086] in, etc. all represent the connection site between the drug group and other structures, that is, half of a single bond “-”.

[0087] The hydroxyl end of the azo compound of the present invention can be connected to a drug through a substitution reaction to prepare an azo prodrug compound. When the prepared azo prodrug compound is used in combination with radiotherapy, under the irradiation of high-energy rays (for example, α rays, β rays, γ rays, and X-rays), the azo group undergoes chemical transformation, thereby releasing the drug, thereby achieving drug chemotherapy while achieving radiotherapy.

[0088] Compared with traditional chemotherapy drugs, the azo prodrug compound prepared by using the azo compound of the present invention has a novel structure, good safety, and can achieve a stable activated radiotherapy response. In addition, the azo prodrug compound prepared by using the azo compound of the present invention has a stable activation efficiency, a wide range of application of the original drug, and has good application prospects.

[0089] In addition, the hydroxyl end of the azo compound can be connected to a drug through a substitution reaction to prepare an azo prodrug compound, and the carboxyl end of the azo compound can also be connected to a substance with a targeting function (such as an antibody) through a condensation reaction, so that the prepared azo prodrug compound has a targeting function.

[0090] In addition, the hydroxyl end and carboxyl end of the azo compound of the present invention can also be connected to the hydrophilic group and the hydrophobic group through substitution reaction and condensation reaction respectively to form an amphiphilic lipid molecule, and the lipid molecule can achieve high-energy ray response through the structural transformation of the azo group before and after irradiation. The amphiphilic property of the lipid molecule enables it to self-assemble to form a high-energy ray-responsive liposome, and the interior of the high-energy ray-responsive liposome can be coated with drugs. Under the irradiation of high-energy rays (for example: α rays, β rays, γ rays, X-rays), the azo group undergoes structural transformation, the hydrophilic group and the hydrophobic group are separated, and the liposome structure is decomposed, thereby releasing the internally coated drugs, thereby achieving radiotherapy and drug chemotherapy at the same time.

[0091] The heteroaryl group may be a monocyclic heteroaryl group, a bicyclic heteroaryl group, and a tricyclic heteroaryl group.

[0092] Exemplary monocyclic heteroaryl groups include pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, oxatotriazolyl, furazanyl, thiazolyl, isothiazolyl, and the like.

[0093] Exemplary bicyclic heteroaryl groups include indolyl, benzothiazolyl, benzodioxolyl, benzoxazolyl, benzothiophenyl, quinolyl, tetrahydroisoquinolyl, isoquinolyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, chromonyl, coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridinyl, furopyridinyl, dihydroisoindolyl, tetrahydroquinolyl, and the like.

[0094] Exemplary tricyclic heteroaryl groups include carbazolyl, dibenzofuranyl, dibenzothiophenyl, benzindolyl, phenanthrolinyl, acridinyl, phenanthridinyl, xanthene, and the like.

[0095] It should be noted that when -C- is -X and R2 is a null bond, -C-R2 is -X.

[0096] In one embodiment, the aryl or heteroaryl group has no substituents other than the linking azo and A / B groups.

[0097] Preferably, in this embodiment, the aryl or heteroaryl group is substituted by one or more substituents, and the substituents include halogen, -R9, -NR9R 10 , -CN, -NO2, -N3, -OR9, -SR9, -NHCOR9, -O-COR9, -CH=CR9R 10 , -C(=O)-R9, -C(=O)-OR9, -C(=O)-Cl, -C(=O)-NH2, -C(=CO)-NH-R9 and -C(=O)-NR9R 10 , where -R9 and -R 10 are independently selected from -H, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C3-C 10 Cycloalkyl, C6~C 20 The alkyl, alkenyl, cycloalkyl, aryl and heteroaryl groups described for the above substituents are optionally replaced by one or more halogen, hydroxyl, mercapto, -NH2, -CN, -NO2, -N3, -NHCOH, -OC(=O)H, -C(=O)H, -C(=O)-OH, -C(=O)-Cl, -C(=O)-NH2, -C(=O)-NH-CH3, -C(=O)-CH3, -C(=O)-OCH3, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C2-C6 alkenyl, C3-C 10 Cycloalkyl, C6~C 20 or a heteroaryl group having 5 to 20 ring atoms.

[0098] Preferably, in this embodiment, -R, -R' and -R" are independently selected from -H, C1-C 12 Alkyl, C1~C 12 Heteroalkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20The heteroatom in the aryl group or the heteroalkyl group is O or N.

[0099] More preferably, in one embodiment, the aryl or heteroaryl group is substituted with one or more substituents, and the substituents include alkoxy (eg, methoxy, ethoxy, propoxy, butoxy).

[0100] More preferably, in another embodiment, the aryl or heteroaryl group is substituted with one or more substituents, and the substituents include halogen, such as fluorine, chlorine, bromine, iodine, and more preferably include fluorine.

[0101] In a particular embodiment, -A- is -B- is

[0102] Specifically, in this embodiment, the structural formula of the azo compound is:

[0103]

[0104] The azo compound with the general formula I disclosed in the present invention can be prepared and synthesized through existing processes.

[0105] Specifically, the azo compound ON-3 (tert-butyl (E)-N-(4-((4-(hydroxymethyl)phenyl)diazenyl)benzoyl)-N-methylglycinate), the azo compound ON-4 (tert-butyl (E)-N-methyl-N-(4-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)diazenyl)benzoyl)glycinate) and the azo compound ON-5 ((E)-N-methyl-N-(4-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)diazenyl)benzoyl)glycine) can be prepared by the following synthetic route:

[0106]

[0107] Specifically, the azo compound YJY-2-67

[0108] ((E)-4-((4-((2-(dimethylamino)ethyl)carbamoyl)phenyl)diazenyl)benzyl(4-nitrophenyl)

[0109] carbonate) can be prepared by the following synthetic route:

[0110]

[0111] Specifically, the azo compound 2-MZL-Cl-2(tert-butyl(E)-N-(3-methoxy-4-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)diazenyl)benzoyl)-N-methylglycinate) can be prepared by the following synthetic route:

[0112]

[0113] Specifically, the azo compound ON-2F-4 (tert-butyl(E)-N-(3,5-difluoro-4-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)diazenyl)benzoyl)-N-methylglycinate) can be prepared by the following synthetic route:

[0114]

[0115] Specifically, the azo compound HJA-ON-4 (tert-butyl (E) -N- (4- ((2-methoxy-4- ((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)diazenyl)benzoyl) -N-methylglycinate) can be prepared by the following synthetic route:

[0116]

[0117] Specifically, the azo compound ON-3-Cl (tert-butyl (E) -N- (4- ((4- (((chlorocarbonyl)oxy)methyl)phenyl)diazenyl)benzoyl) -N-methylglycinate) can be prepared by the following synthetic route:

[0118]

[0119] Specifically, the azo compound ON-3-IMZ((E)-4-((4-((2-(tert-butoxy)-2-oxoethyl)(methyl)carbamoyl)phenyl)diazenyl)benzyl

[0120] 1H-imidazole-1-carboxylate) can be prepared by the following synthetic route:

[0121]

[0122] Specifically, the azo compound 12 (tert-butyl(E)-N-methyl-N-(5-((6-((((4-nitrophenoxy)carbonyl)oxy)methyl)pyridin-3-yl)diazenyl)picolinoyl)glycinate) can be prepared by the following synthetic route:

[0123]

[0124] Specifically, the azo compound 17 (tert-butyl(E)-N-methyl-N-(4-((2-((((4-nitrophenoxy)carbonyl)oxy)methyl)furan-3-yl)diazenyl)furan-2-carbonyl)glycinate) can be prepared by the following synthetic route:

[0125]

[0126] Specifically, the azo compound 19 (tert-butyl(E)-N-(4-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)diazenyl)benzoyl)-N-(tetrahydrofuran-2-yl)glycinate) can be prepared by the following synthetic route:

[0127]

[0128] Specifically, the azo compound 21 (1-(2-(tert-butoxy)-2-oxoethyl)piperidin-4-yl(E)-4-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)diazenyl)benzoate) can be prepared by the following synthetic route:

[0129]

[0130] The present invention also discloses an azo prodrug compound according to an embodiment of the present invention, which has the following general formula II:

[0131]

[0132] in, It is a single bond, which means that A and B are distributed on both sides or the same side of the azo double bond;

[0133] -A- and -B- are independently selected from: aryl or heteroaryl;

[0134] The term "aryl group" means an aromatic hydrocarbon group having 6 to 20 carbon atoms obtained by removing two hydrogen atoms from two carbon atoms in the aromatic nucleus of an aromatic hydrocarbon molecule;

[0135] The term "heteroaryl" refers to aromatic groups as 5-membered or 6-membered rings and fused ring systems comprising 5 to 20 atoms, wherein at least one ring is aromatic and contains one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, and if the ring contains multiple oxygen atoms, these oxygen atoms are not directly adjacent;

[0136] -C- is -O-, -N + (R a R b )-、-OC(=O)- or

[0137] -G1 is a drug group;

[0138] -G2 is a targeting group,

[0139] -R3 and -R4 are independently selected from: -CN, -COOR, -CONR'R", -H, halogen, C1-C 12 Alkyl, C1~C 12 Heteroalkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The heteroatom in the aryl group is O or N;

[0140] -R5 and -R6 are independently selected from: -H, C1-C 12 Alkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The aromatic group;

[0141] -R7 is -H, C1~C 12 Alkyl, C1~C 12 Heteroalkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The heteroatom in the aryl group or the heteroalkyl group is O or N.

[0142] in, etc. all represent the connection site between the drug group and other structures, that is, half of a single bond “-”.

[0143] When the azo prodrug compound of the present invention is used in combination with radiotherapy, under the irradiation of high-energy rays (such as α rays, β rays, γ rays, X-rays), the azo group undergoes chemical transformation, thereby releasing the drug, thereby achieving drug chemotherapy while achieving radiotherapy.

[0144] The azo prodrug compound of the present invention can be prepared by connecting a drug through a substitution reaction at the hydroxyl end of the above-mentioned azo compound.

[0145] It should be noted that the specific structure of the aryl or heteroaryl group in the azo prodrug compound is as described above and will not be repeated here.

[0146] Preferably, in this embodiment, -R a and -R b Each independently selected from C1 to C 12 Alkyl, C1~C 12 Heteroalkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The heteroatom in the aryl group or the heteroalkyl group is O or N.

[0147] Preferably, in this embodiment, -R, -R' and -R" are independently selected from -H, C1-C 12 Alkyl, C1~C 12 Heteroalkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The heteroatom in the aryl group or the heteroalkyl group is O or N.

[0148] It should be noted that when -G2 is a targeting group, the azo prodrug compound has a targeting function. At this time, the carboxyl end of the azo compound can also be connected to a substance with a targeting function (e.g., an antibody) through a condensation reaction to prepare an azo prodrug compound with a targeting function.

[0149] Preferably, the targeting group is covalently linked to other structures via N, O or S.

[0150] Generally speaking, the targeting group can be any group having a targeting effect.

[0151] Specifically, the targeting group is an antibody, a peptide, a sugar group, a small molecule ligand or a nucleic acid aptamer.

[0152] When -G2 is not a targeting group, the azo prodrug compound does not have a targeting function.

[0153] In general, the drug group can be any therapeutic agent that can be coupled to the azo structure, as long as it can be detached from the azo structure and release a therapeutically active molecule under the action of radiation.

[0154] In the present invention, the released therapeutically active molecule is generally the same structure as the therapeutic agent G1 in Formula II. In some embodiments, the released therapeutically active molecule is slightly different in structure from the therapeutic agent G1 in Formula II, i.e., the therapeutic agent molecule undergoes a chemical reaction under the action of radiation but does not significantly affect its therapeutic activity.

[0155] Active molecules that can be used as therapeutic agents include, but are not limited to, polypeptides, oligopeptides, peptidomimetics, amino acids, enzyme inhibitors, hormones, toxins, antibiotics, anti-inflammatory substances, and the like.

[0156] Preferably, the active molecule is a therapeutic agent for treating cancer.

[0157] When the active molecule is a therapeutic agent for treating cancer, the combination of radiotherapy and chemotherapy can be achieved by means of this radiotherapy-activated prodrug, that is, while radiation is treating cancer, irradiation also activates the release of chemical anti-tumor drugs to achieve chemotherapy.

[0158] More preferably, the present invention uses a drug containing a primary amine or a secondary amine as a therapeutic agent. Specifically, the present invention uses an anticancer drug containing a primary amine or a secondary amine as a therapeutic agent. Drugs containing primary amines or secondary amines include, for example, ibrutinib, acalabrutinib, zanubrutinib, doxorubicin, mitomycin-C, mitomycin-A, daunorubicin, aminopterin, actinomycin, bleomycin, 9-aminocamptothecin, N8-acetyl spermidine, bis(2-chloroethyl)amine, yunnanmycin, gemcitabine, cytarabine, dolastatin, dacarbazine, 5-fluorouracil and their derivatives.

[0159] Drugs containing primary or secondary amines also include amino derivatives of drugs that do not naturally contain amino groups. In other words, drugs that do not originally contain amino groups can be chemically modified to have amino groups, and then coupled to radiation-responsive groups or to radiation-responsive groups and linkers through the primary or secondary amine coupling method described in the present disclosure.

[0160] In particular, the therapeutic agent is monomethyl auristatin E.

[0161] In another embodiment, the present invention uses a hydroxyl-containing anticancer drug as a therapeutic agent. Hydroxyl-containing therapeutic agents include, for example, paclitaxel, docetaxel, gemcitabine, cytarabine, etc. Hydroxyl-containing therapeutic agents also include hydroxyl derivatives of drugs that do not naturally contain hydroxyl groups. In other words, drugs that do not originally contain hydroxyl groups can be chemically modified to have hydroxyl groups, and then coupled to the radiation-responsive group or to the radiation-responsive group and the linking group by the hydroxyl coupling method described in the present disclosure.

[0162] In another embodiment, the present invention uses a thiol-containing drug as a therapeutic agent. Preferably, the present disclosure uses a thiol-containing anticancer drug as a therapeutic agent. Examples of thiol-containing therapeutic agents include 6-mercaptopurine, etc. The thiol-containing therapeutic agent also includes thiol derivatives of drugs that do not naturally contain thiol groups. In other words, a drug that does not originally contain a thiol group can be chemically modified to have a thiol group, and then coupled to a radiation-responsive group or to a radiation-responsive group and a linker through the thiol coupling method described in the present disclosure.

[0163] Preferably, the drug group is covalently linked to other structures via N or O.

[0164] Specifically, in this embodiment, the drug group is

[0165] in, Represents the connection site between the drug group and other structures, that is, half of a single bond "-".

[0166] The present invention also discloses a pharmaceutical composition according to an embodiment, comprising the above-mentioned azo prodrug compound or a pharmaceutically acceptable salt thereof.

[0167] Specifically, pharmaceutically acceptable salts of azo prodrug compounds include inorganic salts or organic salts.

[0168] Among them, inorganic salts include hydrochlorides, hydrobromides, hydroiodides, perchlorates, sulfates, bisulfates, nitrates, phosphates, acid phosphates, sodium salts, calcium salts, potassium salts, magnesium salts, silver salts, and lithium salts; organic salts include formates, acetates, trifluoroacetates, propionates, pyruvates, glycolates, oxalates, malonates, succinates, glutarates, fumarates, maleates, lactates, malates, citrates, tartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, salicylates, p-toluenesulfonates, ascorbic acid salts, meglumine salts, tromethamine salts, diethylamine salts, lysine salts, choline salts, arginine salts, terbutylamine salts, and N,N-dibenzylethylenediamine salts.

[0169] Preferably, the above-mentioned pharmaceutical composition further comprises pharmaceutical excipients.

[0170] The pharmaceutical excipients include: at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, flocculants and deflocculating agents, filter aids and release retardants.

[0171] The present invention also discloses a high-energy ray-responsive lipid molecule according to an embodiment of the present invention, which has the following general formula III:

[0172]

[0173] in, It is a single bond, which means that A and B are distributed on both sides or the same side of the azo double bond;

[0174] -A- and -B- are independently selected from: aryl or heteroaryl;

[0175] The term "aryl" means an aromatic hydrocarbon group having 6 to 20 carbon atoms obtained by removing one hydrogen atom from each of two carbon atoms in the aromatic nucleus of an aromatic hydrocarbon molecule;

[0176] The term "heteroaryl" refers to aromatic groups as 5-membered or 6-membered rings and fused ring systems comprising 5 to 20 atoms, wherein at least one ring is aromatic and contains one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, and if the ring contains multiple oxygen atoms, these oxygen atoms are not directly adjacent;

[0177] -C- is -O-, -N + (R a R b )-、-OC(=O)- or

[0178] -G3 and -G4 are respectively one of a hydrophilic group and a hydrophobic group, and the high-energy ray-responsive lipid molecule is an amphiphilic molecule; -R3 and -R4 are respectively independently selected from: -CN, -COOR, -CONR'R", -H, halogen, C1~C 12 Alkyl, C1~C 12 Heteroalkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The heteroatom in the aryl group or the heteroalkyl group is O or N.

[0179] in, etc. all represent the connection site between the drug group and other structures, that is, half of a single bond “-”.

[0180] The high-energy ray-responsive lipid molecules of the present invention have lipid functions, so that the high-energy ray-responsive lipid molecules can self-assemble to form high-energy ray-responsive liposomes, and the interior of the high-energy ray-responsive liposomes can be coated with drugs. Under the irradiation of high-energy rays (for example, α rays, β rays, γ rays, and X-rays), the azo group undergoes structural transformation, the hydrophilic group and the hydrophobic group separate, and the high-energy ray-responsive liposome structure decomposes, thereby releasing the drugs coated inside, thereby achieving radiotherapy and drug chemotherapy at the same time.

[0181] The high-energy ray-responsive lipid molecule of the present invention can be prepared by connecting a hydrophilic group and a hydrophobic group through a substitution reaction and a condensation reaction of the hydroxyl end and the carboxyl end of an azo compound, respectively.

[0182] It should be pointed out that the specific structure of the aromatic or heteroaromatic group in the high-energy radiation-responsive lipid molecule is as described above and will not be repeated here.

[0183] Preferably, in this embodiment, -R a and -R b Each independently selected from C1 to C 12 Alkyl, C1~C 12 Heteroalkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The heteroatom in the aryl group or the heteroalkyl group is O or N.

[0184] Preferably, in this embodiment, -R, -R' and -R" are independently selected from C1 to C 12 Alkyl, C1~C 12 Heteroalkyl, C3~C12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The heteroatom in the aryl group or the heteroalkyl group is O or N.

[0185] Preferably, the hydrophilic group includes cationic groups, such as tertiary amine groups, quaternary amine groups, etc.; anionic groups, such as carboxylic acid groups, sulfonic acid groups, phosphoric acid groups, sulfate groups, etc.; polar nonionic groups, such as hydroxyl groups, ether groups, amine groups, amide groups, etc.

[0186] Preferably, the hydrophobic group comprises C 10 ~C 20 The hydrocarbon group may include a hydrocarbon group containing aromatic, ester, ether, amine, amide and the like; a hydrocarbon group containing a double bond; it may also be a lipophilic polymer such as polyoxypropylene, long-chain perfluoroalkyl, polysiloxane, etc.; it may also be a chemotherapeutic agent with hydrophobic effect such as bufalin, camptothecin, etc.

[0187] Specifically, the lipid molecule is a fatty acid ester or amide derivative, a sterol ester or amide derivative, a glyceride, a glycerophospholipid or a glycolipid containing an azo structural unit.

[0188] The present invention also discloses a high-energy ray-responsive liposome according to an embodiment of the present invention, which is formed by self-assembly of the high-energy ray-responsive lipid molecules mentioned above.

[0189] Specifically, the high-energy radiation-responsive liposomes can be prepared by mixing an aqueous phase with an organic phase containing high-energy radiation-responsive lipid molecules using an LNP intelligent synthesizer to prepare liposome nanoparticles (LNP).

[0190] The high-energy ray-responsive liposomes of the present invention can be coated with drugs. Under the irradiation of high-energy rays (for example, α rays, β rays, γ rays, and X-rays), the azo group undergoes structural transformation, the hydrophilic group and the hydrophobic group separate, and the high-energy ray-responsive liposome structure decomposes, thereby releasing the drugs coated inside, thereby achieving radiotherapy and drug chemotherapy at the same time.

[0191] The following are specific embodiments.

[0192] In a specific embodiment, the chromatographic conditions involved in the HPLC test process are shown in Table 1 below.

[0193] Table 1

[0194]

[0195] The irradiation parameters involved in the specific embodiment are as follows: the instrument is a small animal X-ray irradiator, model RS2000-PRO-225, the irradiation parameters are 225KV, 17.7mA, the position is placed in the fifth layer, 60Gy, and the required time is 9-10min. The sample is first dissolved in DMSO, an appropriate amount of surfactant Triton X-100 is added, and then diluted with pure water to a concentration of 5-10uM. The prepared liquid is placed in a glass bottle and ultrasonicated for 5-10min, 1mL is taken for sample retention, and the remaining liquid is passed through argon for 20min, then sealed and irradiated at room temperature.

[0196] Example 1

[0197] Preparation of azo prodrug XP-106-MMAE-010:

[0198] 1) Preparation of azo compound ON-2, azo compound ON-3 and azo compound ON-4:

[0199] 1.1)

[0200] Procedure: p-Aminobenzoic acid (2.0 g) was dissolved in dry DCM (40 mL), and aqueous potassium persulfate solution (8.64 g, 1.57 eq) was added thereto and stirred at room temperature for 2 hours.

[0201] Detection: The reaction changes from white suspension to yellow suspension.

[0202] Post-treatment: The reaction solution was directly filtered, the filter cake was rinsed with water 3-5 times, and the filter cake was dried to obtain the product ON-1 (2 g, 90% yield) as a yellow solid. (No purification required and used directly in the next step)

[0203] 1.2)

[0204] Procedure: Dissolve ON-1 (2 g, 1.0 eq) in dry DMF (200 mL), add p-aminobenzyl alcohol (2 g, 1.1 eq) and glacial acetic acid (50 mL) to the stirred reaction solution, and stir at room temperature for 24 hours.

[0205] Detection: LCMS detection.

[0206] Post-treatment: The reaction solution was spin-dried to obtain the product ON-2 (4.6 g, crude) as a brown solid. (It was used directly in the next step without purification)

[0207] 1.3)

[0208] Procedure: ON-2 (2.3 g, 1.0 eq) was dissolved in dry DCM (50 mL), and sarcosine tert-butyl ester hydrochloride (1.95 g, 1.2 eq), DCC (2.32 g, 1.2 eq), HOBt (1.52 g, 1.2 eq) and triethylamine (2.6 mL, 2.0 eq) were added to the reaction. The reaction was stirred at room temperature for 24 h.

[0209] Detection: LCMS showed that the starting material was completely reacted.

[0210] Post-treatment: the reaction was quenched with water, extracted with DCM (80 mL x 3), the combined organic phases were washed once with saturated brine and spin-dried.

[0211] Purification: The product ON-3 (2.3 g, 66% yield) was isolated by column purification (EA:PE=0:1, 1:10, 1:5, 1:2 to produce the product, 1:1) as an orange-yellow solid.

[0212] 1.4)

[0213] Procedure: ON-3 (1.3 g, 1.0 eq) was dissolved in dry DCM (26 mL), p-nitrophenylcarbonyl chloride (820 mg, 1.2 eq), DMAP (41 mg, 0.1 eq) and triethylamine (565 μL, 1.2 eq) were added to the reaction, and the reaction was stirred at room temperature for 3 h.

[0214] Detection: LCMS showed that the starting material was completely reacted.

[0215] Post-treatment: the reaction was quenched with water, extracted with DCM (80 mL x 3), the combined organic phases were washed once with saturated brine and spin-dried.

[0216] Purification: The product ON-4 (1.3 g, 69% yield) was isolated by column purification (E:P = 0:1, 1:10, 1:5, 1:3, 1:2 to start producing the product, 1:1) in an orange-yellow semi-oil and semi-solid state.

[0217] 2) Preparation of azo prodrug XP-106-MMAE-010

[0218] Synthesis method:

[0219]

[0220] Procedure: MMAE (350 mg, 1.0 eq) was dissolved in dry DMF (10 mL), ON-4 (580 mg, 1.8 eq), HOBt (188 mg, 2 eq) and triethylamine (100 μL, 2 eq) were added to the reaction, and the reaction was stirred at room temperature for 24 h.

[0221] Detection: LCMS showed the reaction was complete.

[0222] Post-treatment and purification: DMF was spin-dried, dissolved in DCM and purified by column (E:P=0:1, 1:10, 1:5, 1:3, 1:1, 1:0) to obtain the product XP-106-MMAE-010 (350 mg, 63% yield) as a yellow solid.

[0223] 1 H NMR (400MHz, CDCl3) δ7.95(dd,J=25.6,13.2Hz,3H),7.62(d,J=8.0Hz,1H),7.52(dd,J=19.6,7.6Hz,2H),7.43-7.29(m,4H),7.24(t ,J=7.2Hz,1H),6.85(t,J=10.8Hz,1H),6.58(dd,J=24.0,8.4Hz,1H),5.38-5.06(m,2H),4.94(d,J=2.4Hz,1H),4.87-4.60(m,2H),4. 31-4.00(m,5H),3.86(dd,J=8.8,7.2Hz,1H),3.48(d,J=8.8Hz,3H),3.39(t,J=4.8Hz,4H),3.32(d,J=10.0Hz,3H),3.19-2.84(m,8H) ,2.52-2.15(m,4H),2.13-1.89(m,7H),1.82(dd,J=10.4,6.8Hz,2H),1.58-1.41(m,9H),1.26(t,J=10.4Hz,5H),1.08-0.65(m,21H).

[0224] LCMS(ESI):m / z Calcd for C 61 H 91 N8O 12 ,[M+H] + :1127.7,found:1128.1.

[0225] HPLC detection: The HPLC peak time of the original drug molecule MMAE is about 17.0min. The peak area (HPLC 315nm) of the prodrug before and after irradiation is reduced by 96.4% (60Gy, 10uM). After irradiation, the original drug MMAE (t = 17.02min) and the dropped linker 1_a (t = 20.491min) are produced. The HPLC detection results are shown in Table 2 below.

[0226]

[0227] Table 2 HPLC test results of XP-106-MMAE-010 before and after irradiation

[0228]

[0229] Conversion rate involved = (peak area of ​​azo prodrug before irradiation - peak area of ​​azo prodrug after irradiation) / peak area of ​​azo prodrug before irradiation.

[0230] Example 2

[0231] Preparation of azo prodrug XP-106-MMAE-011:

[0232]

[0233] Procedure: MMAE-010 (330 mg) was dissolved in dry DCM (8 mL), 0.5 mL of TMSOTf was added to the reaction and stirred at room temperature for 30 min.

[0234] Detection: TLC detection (PE:EA=1:1).

[0235] Post-treatment: the reaction was quenched with NaHCO3, extracted with EA, washed with water, washed with salt, filtered, and spin-dried to obtain a crude product (242 mg, 84% yield).

[0236] Purification: 20 mg was purified by Pre-HPLC to obtain XP-106-MMAE-011 (13 mg, 98.7% purity) as a yellow solid.

[0237] LCMS(ESI):m / z Calcd for C 57 H 83 O 12 N8,[M+H] + :1071.6,found:1072.0.

[0238] HPLC detection: The HPLC peak time of the original drug molecule MMAE is about 17.0min. The peak area (HPLC 315nm) of prodrug before and after irradiation is reduced by 66.4% (60Gy, 10uM), producing the original drug MMAE and the linker peak 2_a (HPLC254nm peak time is 15.793min). The HPLC detection results are shown in Table 3 below.

[0239]

[0240] Table 3 HPLC test results of XP-106-MMAE-011 before and after irradiation

[0241]

[0242] Example 3

[0243] Preparation of azo prodrug XP-107-BUF-009:

[0244] 1) Preparation of azo compound ON-5:

[0245]

[0246] Operation: 200 mg of compound ON-4 was dissolved in 4 mL of DCM / TFA (1 / 1) under ice bath, and then reacted at room temperature for two hours. TLC (DCM:MeOH=10:1) showed that the raw material reacted completely and a highly polar spot was generated.

[0247] Detection: TLC detection (DCM:MeOH=10:1).

[0248] Post-treatment: The reaction solution was evaporated at 40°C to remove dichloromethane, and then pumped dry with an oil pump to obtain 140 mg of yellow solid ON-5 (used directly in the next step without purification).

[0249] 2) Preparation of azo compound ON-6:

[0250]

[0251] Procedure: Dissolve 120 mg of compound ON-5 in 5 mL of anhydrous DCM, add 51 mg of Piperazine and 72 u of LTEA, and react at room temperature for 2 h.

[0252] Detection: TLC detection (PE:EA=1:1)

[0253] Post-treatment and purification: spin-dry at 37°C, and re-separate with dichloromethane-assisted wet column chromatography (PE:EA=1:0,0:1), (EA:MeOH=20:1,10:1,5:1) and add 1 / 1000 ammonia water. Finally, the product 6 (92.2 mg) was obtained as a yellow solid ON-6.

[0254] 3) Preparation of compound 7:

[0255]

[0256] Procedure: Dissolve 100 mg of bufalin in 1 mL of anhydrous DCM, add 120 mg of p-nitrophenyl chloroformate, add 38 mg of DMAP and 60 uL of TEA at 0°C, and react at room temperature for 24 h.

[0257] Detection: TLC detection (PE:EA=1:1)

[0258] Post-treatment and purification: spin-dry at 37°C, dissolve again in dichloromethane and separate by wet column chromatography (PE:EA=1:0, 5:1, 3:1, 1:1). Finally, the product compound 7 (110 mg, 77.1% yield) was obtained as a white solid.

[0259] 2) Preparation of azo prodrug XP-107-BUF-009:

[0260]

[0261] Procedure: 100 mg of compound 7 was dissolved in 3 mL of DCM at room temperature, and then 92 mg of compound 2 was added. The reaction was allowed to proceed overnight at room temperature.

[0262] Detection: LCMS showed that the reaction of compound 2 was complete and product MS was generated.

[0263] Post-treatment and purification: After the reaction is completed, add 10 mL of water and 20 mL of dichloromethane to precipitate solids. Filter and collect the filter cake. The filtrate is extracted with dichloromethane. The organic phase is dried with anhydrous sodium sulfate. Dichloromethane is removed by rotary evaporation at 40°C to obtain a yellow solid. The filter cake and solid are combined. Use TLC (DCM / MeOH=10:1, 1 / 1000 formic acid) and dry with an oil pump. XP-107-BUF-009 64.0 mg (99% purity) yellow solid is obtained.

[0264] LCMS(ESI):m / z Calcd for C 47 H 58 O 10 N5,[M+H] + :852.4,found:852.2.

[0265] HPLC detection: The HPLC peak time of the original drug molecule 3-2-3 is about 17.2min. The peak area (HPLC 315nm) of prodrug before and after irradiation is reduced by 88.4% (60Gy, 10uM), producing the original drug 3-2-3 (315nm) and the linker peak 2_a (HPLC254nm peak time is 15.796min). The HPLC detection results are shown in Table 4 below.

[0266] Table 4 HPLC test results of XP-107-BUF-009 before and after irradiation

[0267]

[0268] Example 4

[0269] Preparation of azo prodrug XP-101-DAU-002:

[0270]

[0271] Procedure: Dissolve 50 mg DAU in 2 mL anhydrous DMF, add 58 mg ON-4, 14.3 mg HOBt and 37 u LTEA, and react at room temperature for 24 h.

[0272] Detection: TLC detection (DCM: MeOH = 10: 1)

[0273] Post-treatment and purification: spin-dry at 37°C, redissolve in dichloromethane and separate on a silica gel plate (DCM:MeOH=10:1). Finally, the product XP-101-DAU-002 (22.7 mg) was obtained as a red solid.

[0274] LCMS(ESI):m / z Calcd for C 49 H 53 O4N 15 ,[M+H] + :937.3,found:937.1.

[0275] HPLC detection: The HPLC peak time of the original drug molecule DAU is 17.2min. The peak area (HPLC 315nm) of prodrug before and after irradiation is reduced by 50.1% (60Gy, 10uM), and the original drug DAU and linker 1_a are produced. The HPLC detection results are shown in Table 5 below.

[0276] Table 5 HPLC test results of XP-101-DAU-002 before and after irradiation

[0277]

[0278]

[0279] Example 5

[0280] Preparation of azo prodrug XP-104-CPT-010:

[0281]

[0282] Procedure: 200 mg CPT was dissolved in 5 mL DCM, 200 mg compound 3, 20 mg DMAP and 40 uL TEA were added, and the mixture was reacted at room temperature for 24 h.

[0283] Detection: TLC detection (PE:EA=1:1)

[0284] Post-treatment and purification: spin-dry at 37°C, redissolve in dichloromethane and separate on a silica gel plate (DCM:MeOH=30:1). Finally, the product CPT_1 (55.6 mg) was obtained as a yellow solid.

[0285] Synthesis method:

[0286]

[0287] Procedure: Dissolve 55.6 mg CPT_1 in 2 mL DCM, add 50 mg ON-3 and 30 uL TEA, and react at room temperature for 24 h.

[0288] Detection: TLC detection (EA)

[0289] Post-treatment and purification: spin-dry at 37°C, redissolve in MeOH and separate by preparative HPLC to obtain the product XP-104-CPT-010 (10 mg) as a white solid.

[0290] LCMS(ESI):m / z Calcd for C 42 H 40 O9N5,[M+H] + :758.3,found:758.1.

[0291] HPLC detection: The HPLC peak time of the original drug molecule is 18.5min. The peak area (HPLC315nm) of prodrug before and after irradiation is reduced by 61.1% (60Gy, 10uM), producing the original drug camptothecin and the linker compound 1_a. The HPLC detection results are shown in Table 6 below.

[0292] Table 6 HPLC test results of XP-104-CPT-010 before and after irradiation

[0293]

[0294] Example 6

[0295] Azo prodrug XP-109-EXA-002:

[0296]

[0297] Procedure: Dissolve 15 mg of isoproterenol in 0.5 mL of DMF and 0.5 mL of DCM, add ON-4 (23 mg) and 5 mg of DMAP, and triethylamine (9 uL) to the reaction solution. React at room temperature for 48 hours.

[0298] Detection: LCMS detection showed that the reaction of the starting material was incomplete (about 40% remained).

[0299] Post-treatment and purification: Preparative HPLC separation gave 2 mg of yellow solid XP-109-EXA-002.

[0300] 1 H NMR(400MHz,d6-DMSO)δ8.48(s,1H),8.22(d,J=9.2Hz,1H),8.16-7.92(m,4H),7.87-7.47(m,4H),7 .35(d,J=20.0Hz,1H),6.53(s,1H),5.45(s,1H),5.37-5.19(m,4H),4.20(d,J=26.4Hz,1H),3.00(d, J=16.7Hz,2H),2.39(s,2H),2.31-2.15(m,1H),2.00(dd,J=15.2,7.2Hz,2H),1.86(tt,J=14.0,7.2 Hz,1H),1.75(s,1H),1.47(s,3H),1.37(d,J=12.8Hz,3H),1.24(s,9H),0.85(dd,J=7.2,3.2Hz,3H).

[0301] LCMS(ESI):m / z Calcd for C 46 H 46 O9N6F1,[M+H] + :845.3,found:845.1.

[0302] HPLC detection: The HPLC peak time of the original drug molecule is about 15.2min. The peak area (HPLC 254nm) of prodrug before and after irradiation decreases by -% (60Gy, 10uM), but there is no obvious new peak. The HPLC detection results are shown in Table 7 below.

[0303] Table 7 HPLC test results of XP-109-EXA-002 before and after irradiation

[0304]

[0305] Example 7

[0306] Preparation of azo prodrug XP-111-RES-004:

[0307]

[0308] Procedure: RES (50 mg) was dissolved in dry DMF (40 mL) and stirred. To the stirred solution were added ON-4 (96 mg), HOBt (30 mg) and triethylamine (66 μL), and the mixture was stirred at room temperature for 24 hours.

[0309] Detection: LCMS detection, the raw material has not reacted completely.

[0310] Work-up and purification: The product (4 mg, 3% yield) was directly prepared as a yellow solid.

[0311] 1 H NMR (400MHz, CDCl3) δ8.46(s,1H),8.23(s,1H),8.17(d,J=8.4Hz,1H),7.98(d,J=8.4Hz,3H),7.70-7.63(m,3H),7.58(d,J=7.6Hz,1H),7.50( d,J=8.0Hz,1H),7.43-7.38(m,1H),5.43(s,2H),4.93(s,2H),4.81(s, 2H),4.22(s,1H),3.90(s,1H),3.68(q,J=6.8Hz,2H),3.12(d,J=36.8Hz 3H),1.55(s,5H),1.49(s,4H),1.35(s,5H),1.30-1.24(m,4H).

[0312] LCMS(ESI):m / z Calcd for C 39 H 46 O7N7,[M+H] + :724.3,found:724.2.

[0313] HPLC detection: The HPLC (254nm) peak time of the original drug molecule is about 15.1min. The peak area (HPLC 315nm) of prodrug before and after irradiation is reduced by 27.1% (60Gy, 10uM), producing resiquimod original drug and linker 1_a. The HPLC detection results are shown in Table 8 below.

[0314] Table 8 HPLC test results of XP-111-RES-004 before and after irradiation

[0315]

[0316] Example 8

[0317] Preparation of azo prodrug XP-113-PTX-002:

[0318]

[0319] Procedure: Dissolve 25 mg of paclitaxel in 2 mL of DCM, add 20 mg of ON-4, then add 3 mg of DMAP and 7 uL of TEA, and react at room temperature for 24 h.

[0320] Detection: LCMS detection, the raw material has not reacted completely.

[0321] Post-treatment and purification: After a portion of the solvent was removed at 37°C, separation was performed on a silica gel plate (DCM:MeOH=20:1). The obtained solid contained the raw material, which was then separated by HPLC to obtain the product XP-113-PTX-002 as a yellow solid (7 mg).

[0322] 1 H NMR(400MHz, CDCl3) δ8.15(d,J=7.6Hz,2H),7.94(dd,J=12.4,8.4Hz,4H),7.73( d,J=7.6Hz,2H),7.67-7.46(m,7H),7.46-7.32(m,6H),6.88(dd,J=33.2,8.4Hz,2 H),6.38-6.23(m,2H),6.01(dd,J=9.2,2.4Hz,1H),5.70(d,J=7.2Hz,1H),5.48(d ,J=2.4Hz,1H),5.36-5.18(m,2H),4.98(d,J=8.4Hz,1H),4.45(s,1H),4.32(d,J= 8.4Hz,1H),4.21(d,J=8.4Hz,2H),3.88(s,1H),3.83(d,J=7.2Hz,1H),3.72(dd, J=14.0,6.8Hz,1H),3.15(s,1H),3.05(s,1H),2.63-2.52(m,1H),2.51-2.43(m,3 H),2.43-2.35(m,1H),2.26-2.18(m,3H),2.06-1.98(m,1H),1.96-1.87(m,3H),1 .87-1.79(m,1H),1.69(s,3H),1.59(s,9H),1.50(d,J=22.0Hz,6H),1.14(s,3H).

[0323] LCMS(ESI):m / z Calcd for C 69 H 75 N4O 19 ,[M+H] + :1263.5,found:1263.1.

[0324] HPLC detection: The HPLC 254nm peak time of the original drug molecule is about 24.006min. The peak area (HPLC 315nm) of prodrug before and after irradiation is reduced by 39.2% (60Gy, 10uM), producing paclitaxel original drug and linker 1_a. The HPLC detection results are shown in Table 9 below.

[0325] Table 9 HPLC test results of XP-113-PTX-002 before and after irradiation

[0326]

[0327] Example 9

[0328] Preparation of azo prodrug XP-102-DOX-011:

[0329]

[0330] Procedure: Dissolve 50 mg DOX in 2 mL anhydrous DMF, add 55 mg ON-4, 15 mg HOBt and 37 uL TEA, and react at room temperature for 24 h.

[0331] Detection: TLC detection (DCM: MeOH = 10: 1)

[0332] Post-treatment and purification: spin-dry at 37°C, redissolve in dichloromethane and separate on a silica gel plate (DCM:MeOH=10:1). Finally, the product XP-102-DOX-011 (11 mg) was obtained as a red solid.

[0333] LCMS(ESI):m / z Calcd for C 49 H 53 O 16 N4,[M+H] + :953.3,found:952.9.

[0334] 1H NMR (400MHz, CDCl3) δ13.99(d,J=9.1Hz,1H),13.24(s,1H),8.03(d,J=7.6Hz,1H),7.96-7.69(m,5H),7.58(dd,J=30.0,8.0Hz,2H ),7.42(dd,J=26.1,8.2Hz,3H),5.51(d,J=3.5Hz,1H),5.36-5.26(m,1H),4.77(d,J=11.8Hz,2H),4.16(dd,J=17.8,11.3Hz,2H),4 .07(s,3H),3.87(s,2H),3.68(s,1H),3.28(d,J=18.2Hz,1H),3.14(s,1H),3.08-2.92(m,3H),2.34(d,J=14.8Hz,1H),2.23-2.10( m,1H),2.06-1.98(m,1H),1.96-1.86(m,1H),1.84-1.74(m,2H),1.68(s,3H),1.47(dd,J=24.4,14.2Hz,9H),1.29(d,J=6.5Hz,3H)

[0335] HPLC detection: The HPLC peak time of the original drug molecule DOX is about 16.2min. The peak area (HPLC 315nm) of prodrug before and after irradiation is reduced by 95.2% (60Gy, 5uM), and the original drug DOX and linker 1_a are produced. The HPLC detection results are shown in Table 10 below.

[0336] Table 10 HPLC test results of XP-102-DOX-011 before and after irradiation

[0337]

[0338] Example 10

[0339] Preparation of azo prodrug XP-107-BUF-008

[0340] 1) Preparation of azo compound YJY-2-67:

[0341]

[0342] 1.1)

[0343] Procedure: ON-2 (200 mg) was dissolved in DCM (5 mL), 200 mg DCC, 120 mg HOBt and 200 u LTEA were added, and then 80 mg of compound 2 was added, and stirred at room temperature for 24 hours.

[0344] Detection: TLC detection (DCM:MeOH=10:1).

[0345] Post-treatment: spin-dry, dissolve with DCM:MeOH=5:1 solution, apply to a large plate, and then use DCM:MeOH=10:1 to scrape the yellow band in the middle to obtain the product YJY-2-61 (180 mg) as a yellow solid.

[0346] 1.2)

[0347] Procedure: 120 mg of YJY-2-61 was dissolved in DCM (2 mL), 10 mg of DMAP and 130 uL of TEA were added, and then 60 mg of compound 3 was added, and the mixture was stirred at room temperature for 24 hours.

[0348] Detection: TLC detection (DCM:MeOH=10:1).

[0349] Post-treatment: The reaction solution was used directly in the next step without purification, and the product mixture was YJY-2-67.

[0350] 2) Preparation of azo prodrug XP-107-BUF-008:

[0351] Synthesis method:

[0352]

[0353] Operation: Add 32 mg of 3-2-3 to the reaction mixture YJY-2-67 in the previous step and react at room temperature for 24 hours.

[0354] Detection: TLC detection (DCM: MeOH = 10: 1)

[0355] Post-treatment and purification: Concentrate at 37°C and separate using a silica gel plate (DCM:MeOH=10:1). Finally, the product YJY-2-69 (40 mg) was obtained as a yellow solid.

[0356] LCMS(ESI):m / z Calcd for C 48 H 63 O8N6,[M+H] + :851.5,found:851.6

[0357]

[0358] Procedure: Dissolve 40 mg of YJY-2-69 in 2 mL of DCM, add 0.5 mL of iodomethane, and react at room temperature for 16 h.

[0359] Detection: LCMS detection.

[0360] Post-treatment and purification: A yellow solid was produced during the reaction. The precipitate was collected by centrifugation and washed with DCM and PE in sequence to obtain the product XP-107-BUF-008 (40 mg) as a yellow solid.

[0361] LCMS(ESI):m / z Calcd for C 49 H 65 O8N6 + ,[M] + :865.5,found:865.1.

[0362] 1 H NMR(400MHz,d6-DMSO)δ8.98(t,J=5.6Hz,1H),8.13-7.79(m,6H),7.66-7.47(m,3H),6.29(d,J=9.8Hz,1H),5.75(s, 1H),5.22(s,2H),4.90(s,1H),4.15(s,1H),3.73(d,J=6.0Hz,1H),3.53(t,J=6.5Hz,1H),3.39(d,J=14.6Hz,8H),3.1 5(d,J=7.6Hz,9H),2.86(d,J=8.3Hz,2H),2.67(s,1H),2.33(s,1H),2.13-1.89(m,4H),1.77(d,J=10.2Hz,2H),1.58 (t,J=21.8Hz,9H),1.34(t,J=12.0Hz,4H),1.19(ddd,J=36.9,18.4,11.7Hz,5H),0.91(d,J=4.2Hz,3H),0.60(s,3H).

[0363] HPLC detection: The HPLC peak time of the original drug molecule 3-2-3 is about 17.142min. The peak area (HPLC 315nm) of prodrug before and after irradiation is reduced by 88.4% (60Gy, 10uM), and the original drug 3-2-3 is produced. The HPLC detection results are shown in Table 11 below.

[0364] Table 11 HPLC test results of XP-107-BUF-008 before and after irradiation

[0365]

[0366] Embodiment 11

[0367] Preparation of azo prodrug XP-102-DOX-012:

[0368]

[0369] Procedure: Dissolve 50 mg DOX in 2 mL anhydrous DMF, add 76 mg ON-5, 30 mg HOBt and 30 uL TEA, and react at room temperature for 24 h.

[0370] Detection: TLC detection (DCM: MeOH = 10: 1)

[0371] Post-treatment and purification: spin-dry at 37°C, redissolve in dichloromethane and separate on a silica gel plate (DCM:MeOH=10:1). Finally, the product XP-102-DOX-012 (73 mg) was obtained as a red solid.

[0372] LCMS(ESI):m / z Calcd for C 45 H 45 O 16 N4,[M+H] + :897.3,found:896.9.

[0373] 1 H NMR (400MHz, d6-DMSO) δ13.97(d,J=38.1Hz,1H),13.26(s,1H),8.07-7.76(m,5H),7.76-7.34(m,5H),7.01(t,J= 19.3Hz,1H),5.47(s,1H),5.28(d,J=34.4Hz,1H),5.08(q,J=13.7Hz,2H),4.98-4.69(m,3H),4.58(s,2H),4.18( s,2H),3.92(d,J=33.3Hz,3H),3.78(d,J=24.8Hz,1H),3.45(d,J=21.8Hz,2H),3.10-2.83(m,5H),2.73(s,1H),2 .28-2.06(m,2H),1.88(dd,J=12.6,9.4Hz,1H),1.50(d,J=8.1Hz,1H),1.22(d,J=10.0Hz,1H),1.21-1.06(m,3H).

[0374] Example 12

[0375] Preparation of azo prodrug XP-102-DOX-013:

[0376]

[0377] Procedure: Dissolve 73 mg XP-102-DOX-012 in 2 mL anhydrous DMF, add 40 mg compound 5 ((2R,3R,4R,5R,6S)-2-(2-aminoethoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol), add 20 mg HATU and 20 uL DIPEA, and react at room temperature for 2 h.

[0378] Detection: LCMS detection

[0379] Post-treatment and purification: Filter through a filter membrane (0.22uM) and prepare and purify. Finally, the product XP-102-DOX-013 (26 mg) was obtained as a red solid.

[0380] LCMS(ESI):m / z Calcd for C 53 H 59 O 21 N5Na,[M+Na] + :1124.4,found:1123.8.

[0381] 1 H NMR (400MHz, d6-DMSO) δ14.20-13.92(m,1H),13.32-13.07(m,1H),8.05(s,1H),7.91(dd,J=16.4,5.3Hz,4H),7.58(dd,J=31.7,21. 4Hz,5H),7.03(d,J=8.1Hz,1H),5.76(s,1H),5.47(s,1H),5.24(s,1H),5.08(s,2H),4.88(dd,J=28.4,22.3Hz,3H),4.80-4.46(m,6H ),4.24-4.07(m,3H),4.01(d,J=20.5Hz,3H),3.84(s,1H),3.74(s,1H),3.60(s,3H),3.17(d,J=5.2Hz,2H),2.97(d,J=6.3Hz,5H),2 .33(s,1H),2.26-2.07(m,2H),1.99(s,1H),1.92-1.82(m,1H),1.76(s,1H),1.47(s,2H),1.23(s,2H),1.16(dd,J=20.6,6.7Hz,3H).

[0382] HPLC detection: The HPLC peak time of the original drug molecule DOX is about 16.2min. The peak area (HPLC 254nm) of prodrug before and after irradiation is reduced by 22.1% (24Gy, 5uM), and the original drug DOX is produced. The HPLC detection results are shown in Table 12 below.

[0383] Table 12 HPLC test results of XP-102-DOX-013 before and after irradiation

[0384]

[0385]

[0386] Embodiment 13

[0387] Preparation of azo prodrug XP-106-MMAE-019

[0388] 1) Preparation of azo compounds:

[0389] 1.1)

[0390] Procedure: 100 mg of 4-amino-3-methoxybenzoic acid was dissolved in dry DCM (2 mL), and aqueous potassium hydrogen persulfate solution (307.38 mg, 1.6 eq) was added thereto and stirred at room temperature for 2 hours.

[0391] Detection: LC-MS detection.

[0392] Post-treatment: water was added to the reaction and the product was filtered to obtain 74.5 mg of a yellow solid with a yield of 68.8%.

[0393] 1.2)

[0394] Procedure: 2-MZL-4 (74.5 mg, 1.0 eq) was dissolved in dry DMF (3 mL), p-aminobenzyl alcohol (55.4 mg, 1.1 eq) and formic acid (3 mL) were added to the stirred reaction solution, and stirred at room temperature for 24 hours.

[0395] Detection: LC-MS detection.

[0396] Work-up: extract with EA and H2O three times to remove DMF to give the product 2-MZL-5 (114.8 mg, crude) as a brown solid.

[0397] 1.3)

[0398] Procedure: 2-MZL-5 (114.8 mg, 1.0 eq) was dissolved in dry DCM (5 mL), sarcosine tert-butyl ester hydrochloride (87.2 mg, 1.2 eq), DCC (99.04 mg, 1.2 eq), HOBt (64.9 mg, 1.2 eq) and triethylamine (167 μL, 3.0 eq) were added to the reaction. The reaction was stirred at room temperature for 24 h.

[0399] Detection: LC-MS detection.

[0400] Post-treatment: The reaction solution was purified by chromatography on a silica gel plate to obtain 37.7 mg of an orange-yellow solid, with an eluent ratio of (EA:PE=1:5) and a yield of 22.8%. 1.4)

[0402]

[0403] Procedure: 2-MZL-O-1 (37.7 mg, 1.0 eq) was dissolved in dry DCM (2 mL), p-nitrophenylcarbonyl chloride (27.2 mg, 1.5 eq), DMAP (22 mg, 2 eq) and triethylamine (38 μL, 3 eq) were added to the reaction, and the reaction was stirred at room temperature for 16 h.

[0404] Detection: LC-MS detection.

[0405] Post-treatment: The reaction solution was purified by chromatography on a silica gel plate to obtain 41.3 mg of an orange-yellow solid, with an eluent ratio of (EA:PE=1:5) and a yield of 80.5%.

[0406] 2) Preparation of azo prodrug XP-106-MMAE-019:

[0407]

[0408] Procedure: MMAE (57 mg, 1.1 eq) was dissolved in dry DCM (2 mL), 2-MZL-Cl-2 (41.3 mg, 1 eq), HOBt (18.9 mg, 2 eq) and triethylamine (20 μL, 2 eq) were added to the reaction, and the reaction was stirred at room temperature for 24 h.

[0409] Detection: LC-MS detection.

[0410] Post-treatment: DCM was spin-dried with a water pump, dissolved with DMF and MeOH, and semi-preparative purification was performed to obtain 21 mg of an orange-yellow solid with a yield of 24.7%.

[0411] LCMS(ESI):m / z Calcd for C 62 H 93 N8O13 ,[M+H] + :1157.7,found:1157.1.

[0412] HPLC detection: The HPLC peak time of the original drug molecule MMAE is about 17.0min. The peak area (HPLC 315nm) of the prodrug before and after irradiation decreased by 45.2% (18Gy, 5uM). After irradiation, the original drug MMAE (t=16.957min) and the dropped linker 4_a (t=21.125min) were produced. The HPLC detection results are shown in Table 13 below.

[0413] Table 13 HPLC test results of XP-106-MMAE-019 before and after irradiation

[0414]

[0415] Embodiment 14

[0416] Preparation of azo prodrug XP-106-MMAE-020:

[0417] 1) Preparation of azo compound ON-2F-2, azo compound ON-2F-3 and azo compound ON-2F-4:

[0418] 1.1)

[0419] Procedure: The starting material 6 (500 mg) was dissolved in dry DCM (10 mL), and aqueous potassium persulfate solution (600 mg, 1.5 eq) was added thereto and stirred at room temperature for 2 hours.

[0420] Detection: LC-MS detection.

[0421] Post-treatment: Filtration gave the solid product ON-2F-1 (520 mg, 96% yield) as a white solid.

[0422] 1.2)

[0423] ON-2F-1 (260 mg, 1.0 eq) was dissolved in dry DMF (10 mL), p-aminobenzyl alcohol (256 mg, 1.5 eq) and glacial ethyl (15 mL) were added to the stirred reaction solution, and the mixture was stirred at room temperature for 24 hours.

[0424] Detection: LC-MS detection.

[0425] Post-treatment: the reaction was quenched with water, extracted with EA, and dried by spin drying to obtain the product ON-2F-2 (380 mg, crude) as a brown oil. 1.3)

[0427]

[0428] Procedure: ON-2F-2 (200 mg, 1.0 eq) was dissolved in dry DCM (4 mL), sarcosine tert-butyl ester hydrochloride (149 mg, 1.2 eq), HATU (312 mg, 1.2 eq) and DIPEA (177 mg, 2 eq) were added to the reaction. The reaction was stirred at room temperature for 4 h.

[0429] Detection: LC-MS detection.

[0430] Post-treatment: The reaction solution was separated by prep-TLC (E:P=1:2.5) to obtain the product ON-2F-3 (47 mg, 17% yield) as an orange oily solid. 1.4)

[0432]

[0433] Procedure: ON-2F-3 (47 mg, 1.0 eq) was dissolved in dry DCM (2 mL), p-nitrophenylcarbonyl chloride (26 mg, 1.2 eq), DMAP (4 mg, 0.1 eq) and triethylamine (30 μL, 2 eq) were added to the reaction, and the reaction was stirred at room temperature for 24 h.

[0434] Detection: LC-MS detection.

[0435] Post-treatment: The reaction solution was separated by prep-TLC (EA:PE=1:2) to obtain the product ON-2F-4 (60 mg, 90% yield) as a yellow solid.

[0436] 2) Preparation of azo prodrug XP-106-MMAE-020:

[0437]

[0438] Procedure: MMAE (50 mg, 1.0 eq) was dissolved in dry DMF (2 mL), ON-4 (60 mg, 1.5 eq), HOBt (19 mg, 2 eq) and triethylamine (19 μL, 2 eq) were added to the reaction, and the reaction was stirred at room temperature for 24 h.

[0439] Detection: LC-MS detection.

[0440] Post-treatment: DMF was spin-dried, dissolved in MeOH and separated by prep-TLC (M:D=1:10) to obtain the product MMAE-020 (17 mg, 20% yield) as a yellow solid.

[0441] LCMS(ESI):m / z Calcd for C 61 H 89 N8O 12 F2,[M+H] + :1163.6,found:1163.0.

[0442] 1 H NMR (400MHz, CDCl3) δ7.94(d,J=8.1Hz,1H),7.54(t,J=11.2Hz,1H),7.44-7.30(m,4H),7.27-7.09(m,2H),6.96(dd,J=15.9,7.7H z,1H),6.57(s,1H),5.24(ddt,J=44.5,30.9,8.7Hz,2H),4.97(s,1H),4.80-4.62(m,1H),4.27(d,J=6.7Hz,1H),4.13(dd,J=27.5 ,16.5Hz,4H),3.94-3.70(m,2H),3.50(dd,J=12.5,5.9Hz,1H),3.46-3.30(m,6H),3.16-2.88(m,9H),2.54-2.17(m,4H),2.03(dd ,J=48.6,16.9Hz,4H),1.91-1.60(m,7H),1.49(dd,J=23.5,12.8Hz,8H),1.33(d,J=13.4Hz,2H),1.27(s,6H),1.07-0.71(m,20H).

[0443] HPLC detection: The HPLC peak time of the original drug molecule MMAE is about 17.0min. The peak area (HPLC 315nm) of the prodrug before and after irradiation decreased by 47.5% (18Gy, 5uM), and the original drug MMAE (t=16.942min) was produced after irradiation. The HPLC detection results are shown in Table 14 below.

[0444] Table 14 HPLC test results of XP-106-MMAE-020 before and after irradiation

[0445]

[0446] Embodiment 15

[0447] Preparation of azo prodrug XP-106-MMAE-023:

[0448] 1) Preparation of azo compound HJA-ON-2, azo compound HJA-ON-3 and azo compound HJA-ON-4:

[0449] 1.1)

[0450] Procedure: ON-1 (39.0 mg, 2.0 eq) was dissolved in dry DMF (1 mL), 4-amino-3-methoxybenzyl alcohol (20 mg, 1.0 eq) and glacial acetic acid (1 mL) were added to the stirred reaction solution, and stirred at room temperature for 24 hours.

[0451] Detection: LC-MS detection.

[0452] Post-treatment: The reaction solution was spin-dried to obtain the product HJA-ON-2 (37.3 mg, crude) as a yellow solid.

[0453] 1.2)

[0454] Procedure: HJA-ON-2 (37.3 mg, 1.0 eq) was dissolved in dry DCM (2 mL), and sarcosine tert-butyl ester hydrochloride (22.0 mg, 1.2 eq), DCC (25.0 mg, 1.2 eq), HOBT (16.2 mg, 1.2 eq) and triethylamine (17 uL, 2.0 eq) were added to the reaction. The reaction was stirred at room temperature for 24 h.

[0455] Detection: LC-MS detection.

[0456] Post-treatment: spin-drying, p-TLC (PE:EA = 1:1) purification and isolation of the product HJA-ON-3 (13.8 mg) as a yellow solid.

[0457] 1.3)

[0458] Procedure: HJA-ON-3 (13.8 mg, 1.0 eq) was dissolved in dry DCM (2 mL), p-nitrophenylcarbonyl chloride (8.04 mg, 1.2 eq), DMAP (1.00 mg, 0.1 eq) and triethylamine (5.6 μL, 1.2 eq) were added to the reaction, and the reaction was stirred at room temperature for 3 h.

[0459] Detection: LC-MS detection.

[0460] Post-treatment: spin-drying, prep-TLC (PE:EA=1:1) purification to isolate the product ON-4 (10.0 mg, 50% yield) as a yellow solid.

[0461] 2) Preparation of azo prodrug XP-106-MMAE-023

[0462]

[0463] Procedure: MMAE (9.00 mg, 1.0 eq) was dissolved in dry DMF (1 mL), HJA-ON-4 (10.0 mg, 2.0 eq), HOBt (3.39 mg, 2 eq) and triethylamine (3.5 μL, 2 eq) were added to the reaction, and the reaction was stirred at room temperature for 24 h.

[0464] Detection: LC-MS detection.

[0465] Post-treatment: DMF was spin-dried, dissolved in DCM, and purified by prep-TLC (EA=100%) to isolate the product XP-106-MMAE-023 (4.20 mg, 28% yield, 95% purity) as a yellow solid.

[0466] LCMS(ESI):m / z Calcd for C 62 H 93 N8O 13 ,[M+H] + :1157.7,found:1157.1.

[0467] HPLC detection: The HPLC peak time of the original drug molecule MMAE is about 17.0min. The peak area (HPLC 315nm) of the prodrug before and after irradiation decreased by 36.6% (18Gy, 5uM), and the original drug MMAE (t=16.954min) was produced after irradiation. The HPLC detection results are shown in Table 15 below.

[0468] Table 15 HPLC test results of XP-106-MMAE-023 before and after irradiation

[0469]

[0470] Example 16

[0471] 1) Preparation of azo compound ON-3-Cl:

[0472]

[0473] Procedure: To a solution of trichloromethyl chloroformate (158 mg, 0.8 mmol) in dry tetrahydrofuran (15 mL) at 0°C was added about ON-3 (383 mg) dissolved in dry THF (40 mL) and TEA (102 mg) and stirred at room temperature for 2 hours.

[0474] Detection: TLC (PE:EA=1:1).

[0475] Work-up: the resulting suspension was filtered, the filter cake was washed with a little tetrahydrofuran, and the combined organic phases were concentrated in vacuo to give ON-3-Cl (200 mg) as a crude yellow oil.

[0476] 2) Preparation of azo compound XP-106-MMAE-010:

[0477]

[0478] Procedure: MMAE (50 mg, 1.0 eq) was dissolved in DCM (1 mL), ON-3-Cl (50 mg) was added to the reaction, and the reaction was stirred at room temperature for 3 h.

[0479] Detection: LCMS detection.

[0480] Post-treatment and purification: DCM was partially concentrated and then purified by prep-TLC (EA=100%) to isolate the product XP-106-MMAE-010 (10 mg) as a yellow solid.

[0481] LCMS(ESI):m / z Calcd for C 61 H 91 N8O 12 ,[M+H] + :1127.7,found:1128.1.

[0482] Embodiment 17

[0483] 1) Preparation of azo compound ON-3-IMZ:

[0484]

[0485] Workup: ON-3 (383 mg) was dissolved in DCM (40 mL) at room temperature, CDI (162 mg) was added and stirred at room temperature for 2 hours.

[0486] Detection: TLC (PE:EA=1:1).

[0487] Post-treatment: spin-drying and purification by prep-TLC gave ON-3-IMZ (300 mg) as a yellow solid.

[0488] 2) Preparation of azo compound XP-106-MMAE-010:

[0489]

[0490] Procedure: MMAE (50 mg, 1.0 eq) was dissolved in DCM (1 mL), ON-3-IMZ (50 mg) was added to the reaction, and the reaction was stirred at room temperature for 3 h.

[0491] Detection: LCMS detection.

[0492] Post-treatment and purification: DCM was partially concentrated and then purified by prep-TLC (EA=100%) to isolate the product XP-106-MMAE-010 (10 mg) as a yellow solid.

[0493] LCMS(ESI):m / z Calcd for C 61 H 91 N8O 12 ,[M+H] + :1127.7,found:1128.1.

[0494] Embodiment 18

[0495] 1) Preparation of azo compound 10, azo compound 11 and azo compound 12:

[0496] 1.1)

[0497] Procedure: 5-Amino-2-pyridinecarboxylic acid (100 mg) was dissolved in dry DCM (2 mL), aqueous potassium hydrogen persulfate solution (450 mg) was added thereto and stirred at room temperature for 2 hours.

[0498] Detection: The reaction changes from white suspension to yellow suspension.

[0499] Post-treatment: Filter and wash with water 3-5 times to obtain product 9 (80 mg) as a yellow solid. (Used directly in the next step without purification)

[0500] 1.2)

[0501] Procedure: 9 (80 mg, 1.0 eq) was dissolved in dry DMF (2 mL), 3-amino-6-pyridinemethanol (70 mg) and glacial ethyl (2 mL) were added to the stirred reaction solution, and the mixture was stirred at room temperature for 24 hours.

[0502] Detection: LCMS detection.

[0503] Post-treatment: The reaction solution was spun dry and pumped dry with an oil pump to obtain product 10 (160 mg, crude) as a brown solid. (Used directly in the next step without purification)

[0504] 1.3)

[0505] Procedure: 10 (130 mg, 1.0 eq) was dissolved in dry DCM (5 mL), sarcosine tert-butyl ester hydrochloride (80 mg), DCC (85 mg), HOBt (65 mg) and triethylamine (40 uL) were added to the reaction. The reaction was stirred at room temperature for 24 h.

[0506] Detection: LCMS showed that the reaction of the starting material was almost complete.

[0507] Post-treatment: the reaction was quenched with water, extracted with DCM (8 mL x 3), the combined organic phases were washed once with saturated brine and spin-dried.

[0508] Purification: pre-TLC (EA:PE=2:1) ​​separated the product 11 (60 mg) as a yellow solid. 1.4)

[0510]

[0511] Procedure: 11 (60 mg, 1.0 eq) was dissolved in dry DCM (2 mL), p-nitrophenylcarbonyl chloride (38 mg, 1.2 eq), DMAP (6 mg) and triethylamine (30 μL) were added to the reaction, and the reaction was stirred at room temperature for 3 h.

[0512] Detection: TLC (EA:PE=2:1) ​​detection.

[0513] Post-treatment: the reaction was quenched with water, extracted with DCM (8 mL x 3), the combined organic phases were washed once with saturated brine and spin-dried.

[0514] Purification: prep-TLC (EA:PE=1:1) separated the product 12 (30 mg) as an orange-yellow oily solid.

[0515] 2) Preparation of azo compound XP-106-MMAE-024:

[0516]

[0517] Procedure: MMAE (50 mg, 1.0 eq) was dissolved in DMF (1 mL), 12 (50 mg), HOBt (33 mg) and triethylamine (30 μL) were added to the reaction, and the reaction was stirred at room temperature for 3 h.

[0518] Detection: LCMS detection.

[0519] Post-treatment and purification: DMF was concentrated and then purified by prep-TLC (EA=100%) to isolate the product XP-106-MMAE-024 (5 mg) as a yellow solid.

[0520] LCMS(ESI):m / z Calcd for C 59 H 89 N 10 O 12 ,[M+H] + :1129.7,found:1129.2.

[0521] Embodiment 19

[0522] 1) Preparation of azo compound 15, azo compound 16 and azo compound 17:

[0523] 1.1)

[0524] Procedure: 4-Amino-2-furancarboxylic acid (100 mg) was dissolved in dry DCM (2 mL), and aqueous potassium hydrogen persulfate solution (400 mg) was added thereto and stirred at room temperature for 2 hours.

[0525] Detection: The reaction changes from white suspension to brown suspension.

[0526] Post-treatment: Filter and wash with water 3-5 times to obtain product 14 (60 mg) as a yellow solid. (Used directly in the next step without purification)

[0527] 1.2)

[0528] Procedure: 14 (60 mg, 1.0 eq) was dissolved in dry DMF (2 mL), 3-aminofuran-2-methanol (50 mg) and glacial ethyl (2 mL) were added to the stirred reaction solution, and the mixture was stirred at room temperature for 24 hours.

[0529] Detection: LCMS detection.

[0530] Post-treatment: The reaction solution was spun dry and pumped dry with an oil pump to obtain product 15 (120 mg, crude) as a brown solid. (Used directly in the next step without purification)

[0531] 1.3)

[0532] Procedure: 15 (100 mg, 1.0 eq) was dissolved in dry DCM (5 mL), sarcosine tert-butyl ester hydrochloride (70 mg), DCC (75 mg), HOBt (60 mg) and triethylamine (30 uL) were added to the reaction. The reaction was stirred at room temperature for 24 h.

[0533] Detection: LCMS showed that the reaction of the starting material was almost complete.

[0534] Post-treatment: the reaction was quenched with water, extracted with DCM (8 mL x 3), the combined organic phases were washed once with saturated brine and spin-dried.

[0535] Purification: pre-TLC (EA:PE=2:1) ​​isolated the product 16 (40 mg) as a yellow solid.

[0536] 1.4)

[0537] Procedure: 16 (40 mg, 1.0 eq) was dissolved in dry DCM (2 mL), p-nitrophenylcarbonyl chloride (28 mg, 1.2 eq), DMAP (4 mg) and triethylamine (15 μL) were added to the reaction, and the reaction was stirred at room temperature for 3 h.

[0538] Detection: TLC (EA:PE=2:1) ​​detection.

[0539] Post-treatment: the reaction was quenched with water, extracted with DCM (8 mL x 3), the combined organic phases were washed once with saturated brine and spin-dried.

[0540] Purification: prep-TLC (EA:PE=1:1) separated the product 17 (15 mg) as an orange-yellow oily solid.

[0541] 2) Preparation of azo compound XP-106-MMAE-025:

[0542]

[0543] Procedure: MMAE (50 mg, 1.0 eq) was dissolved in DMF (1 mL), 12 (50 mg), HOBt (30 mg) and triethylamine (25 μL) were added to the reaction, and the reaction was stirred at room temperature for 3 h.

[0544] Detection: LCMS detection.

[0545] Post-treatment and purification: DMF was concentrated and purified by prep-TLC (EA=100%), and then separated by prep-HPLC to obtain the product XP-106-MMAE-025 (6 mg) as a yellow solid.

[0546] LCMS(ESI):m / z Calcd for C 57 H 87 N8O 14 ,[M+H] + :1107.7,found:1107.1.

[0547] Embodiment 20

[0548] 1) Preparation of azo compound 18 and azo compound 19:

[0549] 1.1)

[0550] Procedure: ON-2 (50 mg) was dissolved in dry DCM (2 mL), and tert-butyl (tetrahydrofuran-2-yl) glycinate (40 mg), DCC (35 mg), HOBt (30 mg) and triethylamine (10 uL) were added thereto, and stirred at room temperature for 24 hours.

[0551] Detection: LCMS detection.

[0552] Post-treatment: prep-TLC (EA:PE=1:1) separated the product 18 (20 mg) as a yellow solid. 1.2)

[0554]

[0555] Procedure: 18 (20 mg, 1.0 eq) was dissolved in dry DCM (2 mL), p-nitrophenylcarbonyl chloride (15 mg, 1.2 eq), DMAP (2 mg) and triethylamine (8 μL) were added to the reaction, and the reaction was stirred at room temperature for 3 h.

[0556] Detection: TLC (EA:PE=1:1) detection.

[0557] Post-treatment: the reaction was quenched with water, extracted with DCM (8 mL x 3), the combined organic phases were washed once with saturated brine and spin-dried.

[0558] Purification: prep-TLC (EA:PE=1:1) separated the product 19 (10 mg) as an orange-yellow oily solid.

[0559] 2) Preparation of azo compound XP-106-MMAE-026:

[0560]

[0561] Procedure: MMAE (15 mg, 1.0 eq) was dissolved in DMF (1 mL), 19 (10 mg), HOBt (8 mg) and triethylamine (6 μL) were added to the reaction, and the reaction was stirred at room temperature for 3 h.

[0562] Detection: LCMS detection.

[0563] Post-treatment and purification: The product XP-106-MMAE-026 (2 mg) was isolated as a yellow solid by prep-HPLC.

[0564] LCMS(ESI):m / z Calcd for C 64 H 95 N8O 13 ,[M+H] + :1183.7,found:1183.2.

[0565] Embodiment 21

[0566] 1) Preparation of azo compound 20 and azo compound 21: 1.1)

[0568]

[0569] Procedure: ON-2 (50 mg) was dissolved in dry DCM (2 mL), tert-butyl peroxy 2-(4-hydroxypiperidin-1-yl)acetate (40 mg), DCC (35 mg), DMAP (5 mg) were added thereto, and the mixture was stirred at room temperature for 24 hours.

[0570] Detection: LCMS detection.

[0571] Post-treatment: prep-TLC (EA:PE=1:1) separated the product 17 (15 mg) as a yellow solid. 1.2)

[0573]

[0574] Procedure: 20 (15 mg, 1.0 eq) was dissolved in dry DCM (2 mL), p-nitrophenylcarbonyl chloride (10 mg, 1.2 eq), DMAP (2 mg) and triethylamine (5 μL) were added to the reaction, and the reaction was stirred at room temperature for 3 h.

[0575] Detection: TLC (EA:PE=1:1) detection.

[0576] Post-treatment: quench the reaction with water, extract with DCM (5 mL x 2), combine the organic phases, wash once with saturated brine, and spin dry.

[0577] Purification: prep-TLC (EA:PE=1:1) separated the product 17 (15 mg) as an orange-yellow oily solid.

[0578] 2) Preparation of azo compound XP-106-MMAE-027:

[0579]

[0580] Procedure: MMAE (20 mg, 1.0 eq) was dissolved in DMF (1 mL), 21 (15 mg), HOBt (8 mg) and triethylamine (6 μL) were added to the reaction, and the reaction was stirred at room temperature for 3 h.

[0581] Detection: LCMS detection.

[0582] Post-treatment and purification: DMF was concentrated and purified by prep-TLC (EA=100%), and then separated by prep-HPLC to obtain the product XP-106-MMAE-027 (3 mg) as a yellow solid.

[0583] LCMS(ESI):m / z Calcd for C 65 H 97 N8O 13 ,[M+H] + :1197.7,found:1197.1.

[0584] Embodiment 22

[0585] 1) Preparation of azo compound 22:

[0586]

[0587] Procedure: Dissolve ON-2 (50 mg) in dry DCM (2 mL), add tert-butyl 2-(4-hydroxypiperidin-1-yl)acetate (40 mg), DCC (35 mg), DMAP (5 mg). Stir at room temperature for 24 hours. Dissolve ON-3 (1040 mg, 1.0 eq) in dry DCM (10 mL), add pyridine (110 μL, 0.5 eq), stir in a 45 ° C oil bath, slowly add phosphorus tribromide (50 μL x 3) dropwise, and reflux for 3 hours. TLC detects that the raw material reacts completely, cool the reaction solution to room temperature, add isopropanol, and stir for ten minutes. Add saturated NaHCO3 to the reaction solution to quench, extract with EA (10 mL x 3), combine the organic phases and spin dry to obtain the product (1126 mg, crude).

[0588] 2) Preparation of azo compound XP-103-SN38-028:

[0589]

[0590] Procedure: SN-38 (50 mg, 1.0 eq) was dissolved in dry DCM (2 mL) and DMF (2 mL), 73 mg of compound 22 was added, K2CO3 (18 mg) was added to the stirred reaction solution, and stirred at room temperature for 24 hours.

[0591] Detection: TLC detection (DCM:MeOH=10:1).

[0592] Post-treatment and purification: DMSO was added to the reaction solution to completely dissolve it, and the filtrate was filtered to obtain the product (4 mg, 5% Yield) as a yellow solid.

[0593] LCMS(ESI):m / z Calcd for C 43 H 44 N5O8,[M+H] + :758.3,found:758.2.

[0594] Embodiment 23

[0595] 1) Preparation of azo compound XP-114-NM-029:

[0596]

[0597] Procedure: NM (119 mg, 1.0 eq) was dissolved in dry THF (2 mL), 490 mg of compound 22 (1.1 eq) was added, and the mixture was refluxed and stirred for 24 hours.

[0598] Detection: TLC detection (DCM:MeOH=10:1).

[0599] Post-treatment and purification: Silica gel column purification gave 411 mg NM-1 with a yield of 85% as a yellow solid.

[0600] Procedure: NM-1 (242 mg, 0.5 mmol) was added dropwise into a dichloromethane solution of thionyl chloride (10 eq.) and stirred at room temperature for 12 hours.

[0601] Post-treatment and purification: The reaction mixture was filtered to obtain a yellow solid, which was the target product XP-114-NM-029 (247 mg, yield 95%).

[0602] LCMS(ESI):m / z Calcd for C 26 H 36 C l2 N4O3,[M] + :521.2,found:521.1.

[0603] Test Example 1

[0604] The azo prodrug compounds prepared in Examples 1 to 9 (irradiation dose of 60 Gy), 12 (irradiation dose of 24 Gy) and 13-15 (irradiation dose of 18 Gy) were irradiated and released, and the test results are shown in Table 16 below:

[0605] Table 16

[0606]

[0607]

[0608] It can be seen from Table 12 that after irradiation, the prodrug peak areas of the azo prodrug compounds prepared in Examples 1 to 15 were significantly reduced, and obvious prodrugs were detected.

[0609] Test Example 2

[0610] The cytotoxicity comparison test of the azo prodrug compound prepared above and the original drug was performed respectively. The corresponding cells (such as HeLa, HT29, LOVO, HepG2, NPC / HK-1, SCC-090, 143B, purchased from ATCC) were inoculated in a 96-well cell culture plate at a density of 4000 per well and incubated at 37°C, 4% carbon dioxide for 18 hours to ensure cell adhesion; then the cells were treated with different concentrations of the corresponding drugs and continued to be incubated at a constant temperature for 72 hours; after 72 hours, the original culture medium was aspirated and the cells were washed 3 times with PBS, 100 μL of diluted CCK-8 solution (with a ratio of 1:10 to the culture medium) was added to each well, and after incubation at a constant temperature for 2 hours, the ultraviolet absorption at 450nm in each well was detected with an ELISA instrument, and the absorbance of the treated cells was compared with that of the untreated cells to obtain the cell activity value.

[0611] The test results are shown in Tables 17 to 23 and Figures 1 to 18 As shown in the figure, the higher the prodrug / original drug IC50 ratio is, the lower the cytotoxicity of the prodrug is.

[0612] Table 17

[0613]

[0614] Table 18

[0615]

[0616] Table 19

[0617]

[0618]

[0619] Table 20

[0620]

[0621] Note: “-” means that the XP-104-CPT-010 drug did not reach the half-lethal concentration of HT29 cells at a concentration of 10000 nM.

[0622] Table 21

[0623]

[0624] Table 22

[0625]

[0626] Table 23

[0627]

[0628] Embodiment 24

[0629] Preparation and analysis of antibody-drug conjugate XPADC-106-011:

[0630]

[0631] The synthetic route of XPADC-106-011 is as follows:

[0632]

[0633] first step:

[0634]

[0635] Procedure: 106-011 (100 mg) was dissolved in dry DCM (4 mL), and DCC (23.4 mg), TEA (16.7 ul) and HOBt (16.7 mg) were added thereto. After stirring evenly, piperazine (16.7 mg) was added and stirred at room temperature for 7 hours.

[0636] Detection: LCMS detection.

[0637] Post-treatment: DCM was dried and purified by PLC (DCM: MeOH = 10:1) to obtain the product 106-011-piperazine (90.1 mg, 86% yield) as a yellow solid. (No purification required and used directly in the next step)

[0638] Step 2

[0639]

[0640] Operation: 106-011-piperazine (45 mg) was dissolved in dry DCM (10 mL), TEA (11 ul, 2 eq.) and HOBt (6.4 mg, 1.2 eq.) were added, and then SMCC (39.7 mg, 3 eq.) dissolved in 1.5 ml DCM was added under an ice-water bath, and then stirred at room temperature for 24 hours. LC-MS showed that about 40% of 106-011-piperazine was not completely reacted, and then 1.5 ml of SMCC (39.7 mg, 3 eq.) dissolved in DCM was added to continue the reaction for 2 hours.

[0641] Detection: LCMS detection.

[0642] Post-treatment: the reaction solution was spin-dried and purified by Pre-HPLC to obtain 106-011-SMCC (12.3 mg) as a yellow solid.

[0643] Step 3:

[0644]

[0645] TCEP (20 mM His) solution was added to the antibody solution and reacted at room temperature for 3 hours. Then, 10% v / v DMSO-dissolved 106-011-SMCC solution was added and reacted for 1 hour. Finally, samples were taken for characterization. The characterization results are shown in Table 24 below.

[0646] Antibody name: Cetuximab injection, antibody concentration: 5 mg / mL, antibody purchase source: Merck Pharmaceuticals (Jiangsu) Co., Ltd.

[0647] UV-Vis test sample pretreatment: The sample needs to remove unreacted Linker-Drug and DMSO, which can be removed by filtration, ultrafiltration or desalting.

[0648] HIC test sample pretreatment: The sample can be injected after filtering.

[0649] Table 24

[0650]

[0651] The results of the hydrophobic interaction chromatography (HIC-HPLC) test are as follows: Fig.19 shown.

[0652] The DAR value is calculated based on the above test results, and the calculation results are shown in Table 25 below.

[0653] Table 25

[0654] Detection Methods DAR value Ultraviolet spectrophotometry (UV-Vis) 3.98 Hydrophobic Interaction Chromatography (HIC-HPLC) 3.71

[0655] The antibody-conjugated azo prodrug and the original drug prepared above were subjected to cytotoxicity comparison tests. The corresponding cells (such as LOVO, HepG2, NPC / HK-1, Panc-1, purchased from ATCC) were inoculated in a 96-well cell culture plate at a density of 4000 per well and incubated at 37°C, 4% carbon dioxide for 18 hours to ensure cell adhesion; then the cells were treated with different concentrations of the corresponding drugs and continued to be incubated at a constant temperature for 72 hours; after 72 hours, the original culture medium was aspirated and the cells were washed 3 times with PBS, 100 μL of diluted CCK-8 solution (1:10 ratio with culture medium) was added to each well, and after incubation at a constant temperature for 2 hours, the ultraviolet absorption at 450nm in each well was detected with an enzyme marker, and the absorbance of the treated cells was compared with that of the untreated cells to obtain the cell activity value. The test results are shown in Table 26 below:

[0656] Table 26: Tumor cell inhibitory activity

[0657]

[0658] Embodiment 25

[0659] Synthesis and analysis of lipid component XP-107-BUF-008:

[0660] Synthesis method:

[0661]

[0662] Operation: Add 32 mg of 3-2-3 to the reaction mixture YJY-2-67 in the previous step and react at room temperature for 24 hours.

[0663] Detection: TLC detection (DCM: MeOH = 10: 1)

[0664] Post-treatment and purification: Concentrate at 37°C and separate using a silica gel plate (DCM:MeOH=10:1). Finally, the product YJY-2-69 (40 mg) was obtained as a yellow solid.

[0665] LCMS(ESI):m / z Calcd for C 48 H 63 O8N6,[M+H] + :851.5,found:851.6

[0666]

[0667] Procedure: Dissolve 40 mg of YJY-2-69 in 2 mL of DCM, add 0.5 mL of iodomethane, and react at room temperature for 16 h.

[0668] Detection: LCMS detection.

[0669] Post-treatment and purification: A yellow solid was produced during the reaction. The precipitate was collected by centrifugation and washed with DCM and PE in sequence to obtain the product XP-107-BUF-008 (40 mg) as a yellow solid.

[0670] LCMS(ESI):m / z Calcd for C49H65O8N6 + ,[M] + :865.5,found:865.1.

[0671] 1 H NMR(400MHz,d6-DMSO)δ8.98(t,J=5.6Hz,1H),8.13-7.79(m,6H),7.66-7.47(m,3H),6.29(d,J=9.8Hz,1H),5.75(s, 1H),5.22(s,2H),4.90(s,1H),4.15(s,1H),3.73(d,J=6.0Hz,1H),3.53(t,J=6.5Hz,1H),3.39(d,J=14.6Hz,8H),3.1 5(d,J=7.6Hz,9H),2.86(d,J=8.3Hz,2H),2.67(s,1H),2.33(s,1H),2.13-1.89(m,4H),1.77(d,J=10.2Hz,2H),1.58 (t,J=21.8Hz,9H),1.34(t,J=12.0Hz,4H),1.19(ddd,J=36.9,18.4,11.7Hz,5H),0.91(d,J=4.2Hz,3H),0.60(s,3H).

[0672] XP-107-BUF-008 liposome preparation (107-008-LNPs):

[0673] Method: According to the molar ratio, 69.3% cholesterol, 18% DSPC, 2.7% PEG-2000-DMG and 10% XP-107-BUF-008 (hereinafter referred to as 107-008) were prepared into a 12 mM solution with ethanol, and then filtered with a 0.22 micron filter membrane to obtain an organic phase. The aqueous phase was PBS.

[0674] Liposome nanoparticles (LNP) were prepared by LNP intelligent synthesizer at a ratio of organic phase: aqueous phase = 1:3. The prepared LNP was passed through a 30 kD ultrafiltration tube for solution replacement. The ethanol concentration was diluted to less than 0.5% with PBS and concentrated to the target concentration, and then stored at 4°C.

[0675] Radiological Detection:

[0676] The particle size distribution of the XP-107-BUF-008 liposomes prepared above was tested before and after 60 Gy irradiation. The test results are shown in Table 27 below.

[0677] Table 27: Particle size intensity distribution before and after irradiation

[0678]

[0679] Combined with Table 27, it can be seen that the particle size distribution of the XP-107-BUF-008 liposome mixture with a content of 10% changes significantly after irradiation with 60Gy. This is because the XP-107-BUF-008 liposomes rupture after 60Gy irradiation, and the ruptured mixture reorganizes to form new aggregates.

[0680] The XP-107-BUF-008 liposomes prepared above were respectively demulsified with ethanol before and after 60 Gy irradiation, and then detected by LCMS. The detection results are as follows: Fig. 20 and Fig.21 shown.

[0681] Combination Fig. 20 and Fig.21 , it can be seen that after 60Gy irradiation, the prodrug basically disappeared.

[0682] Embodiment 26

[0683] Synthesis of lipid component 115-001:

[0684] The specific steps include:

[0685] first step

[0686]

[0687] Procedure: DSPE (50 mg) was dissolved in dry chloroform (3 mL), and ON-4 (50 mg) and TEA (40 uL) were added thereto, followed by stirring at 50°C for 3 hours.

[0688] Detection: TLC (DCM:MeOH=10:1).

[0689] Post-treatment: After concentrating to 1 mL, directly scrape on a large plate (DCM:MeOH=10:1) to obtain YJY-2-87 as a yellow solid 25 mg (yield 32.3%).

[0690] Step 2

[0691]

[0692] Procedure: Dissolve YJY-2-87 (25 mg, 1.0 eq) in DCM (2 mL), add TFA (0.5 mL) to the stirred reaction solution at 0°C, and stir at room temperature for 1 hour.

[0693] Detection: TLC (DCM:MeOH=10:1).

[0694] Post-treatment: The reaction solution was concentrated to obtain the product YJY-2-98 (22 mg, crude) as a yellow-brown solid. (It was used directly in the next step without purification)

[0695] Step 3

[0696]

[0697] Procedure: Dissolve YJY-2-98 (30 mg, 1.0 eq) in DCM (2 mL), add compound 4 (10 mg), HATU (12 mg), and DIPEA (60 uL) to the reaction. Stir the reaction at room temperature for 24 h.

[0698] Detection: TLC (DCM:MeOH=10:1).

[0699] Post-treatment: After concentrating to 1 mL, directly scrape on a large plate (DCM:MeOH=10:1) to obtain 26 mg of the product as a yellow solid (yield 81.5%).

[0700] HRMS(ESI):m / z Calcd for C63H108O12N6P,[M+H]+:1171.7685,found:1171.7790.

[0701]

[0702] Procedure: YJY-2-98 (38 mg, 1.0 eq) was dissolved in DCM (5 mL), 1 mL of iodomethane was added to the reaction, and the reaction was stirred at 50 °C overnight.

[0703] Detection: HRMS showed that the reaction of the starting material was complete.

[0704] Post-treatment: spin-drying, washing the solid with ethyl acetate and centrifuging three times, dissolving it with a small amount of DCM, adding a large amount of petroleum ether to precipitate a yellow solid, and centrifuging to obtain 30 mg of product 115-001.

[0705] HRMS(ESI):m / z Calcd for C 64 H 111 O 12 N6P,[M+H] +:1185.7914,found:1185.7917.

[0706] 1 H NMR (400MHz, CDCl3) δ8.32 (s, 1H), 7.83 (d, J = 12.5Hz, 3H), 7.71-7.35 (m, 4H), 6. 45(s,1H),5.19(d,J=41.1Hz,2H),4.37(s,1H),4.14(dd,J=12.9,5.7Hz,2H),4.0 6(s,4H),3.87(s,3H),3.69(s,4H),3.42(s,9H),3.37(s,6H),3.07(s,13H),2.96 (s,1H),2.31(d,J=7.6Hz,3H),1.59(s,4H),1.26(s,46H),0.89(t,J=6.6Hz,6H).

[0707] 115-001 Liposome Preparation:

[0708] Methods: According to the molar ratio, 40% cholesterol, 50% Dlin-MC3-DMA, 1.5% PEG-2000-DMG and 10% 115-001 were prepared into a 12mM solution with ethanol, and then filtered with a 0.22 micron filter to obtain an organic phase. The aqueous phase was PBS. According to the ratio of organic phase: aqueous phase = 1:3, liposome nanoparticles (LNP) were prepared by LNP intelligent synthesizer, and the prepared LNP was replaced by solution through a 30kD ultrafiltration tube, and the ethanol concentration was diluted to less than 0.5% with PBS and concentrated to the target concentration, and then stored at 4°C.

[0709] The particle size distribution of the 115-001 liposomes prepared above was tested before and after 60 Gy irradiation. The test results are as follows: Fig. 22 and Fig.23 shown.

[0710] Combination Fig. 22 and Fig.23 It can be seen that the particle size distribution of the 115-001 liposomes prepared above changes significantly after irradiation with 60 Gy. This is because the 115-001 liposomes rupture after irradiation with 60 Gy, and the ruptured mixture reorganizes to form new aggregates.

[0711] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An azo compound, characterized in that It has the following general formula I: The structural formula of the azo compound shown in the general formula I is:

2. An azo compound, characterized in that The structural formula of the azo compound is:

3. An azo prodrug compound, characterized in that The azo prodrug compound has the following general formula II: in, It is a single bond, which means that A and B are distributed on both sides or the same side of the azo double bond; -A- and -B- are independently selected from: aryl or heteroaryl; The term "aryl group" means an aromatic hydrocarbon group having 6 to 20 carbon atoms obtained by removing two hydrogen atoms from two carbon atoms in the aromatic nucleus of an aromatic hydrocarbon molecule; The term "heteroaryl" refers to aromatic groups as 5-membered or 6-membered rings and fused ring systems comprising 5 to 20 atoms, wherein at least one ring is aromatic and contains one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, and if the ring contains multiple oxygen atoms, these oxygen atoms are not directly adjacent; -C- is -O-, -OC(=O)- or -G1 is a drug group; the drug group is -G2 is -R3 and -R4 are independently selected from: -CN, -COOR, -CONR'R", -H, halogen, C1-C 12 Alkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The aromatic group; -R5 and -R6 are independently selected from: -H, C1-C 12 Alkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The aromatic group; -R7 is -H, C1~C 12 Alkyl, C3~C 12 Cycloalkyl, C2~C 12 Alkenyl, C3~C 12 Cycloalkyl or C6~C 20 The aromatic group.

4. An azo prodrug compound, characterized in that The specific structure is as follows:

5. An antibody-drug conjugate, characterized in that: The specific structure is as follows: in, It is cetuximab.

6. A pharmaceutical composition, characterized in that The invention comprises the azo prodrug compound or a pharmaceutically acceptable salt thereof as claimed in any one of claims 3 to 4.

7. A high-energy ray-responsive lipid molecule, characterized in that: It has the following general formula III: The structural formula of the high-energy ray-responsive lipid molecule shown in the general formula III is:

8. A high-energy radiation-responsive liposome, characterized in that: It is formed by self-assembly of the high-energy ray-responsive lipid molecules described in claim 7.

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