WO2024212922A1 - Camptothecin compound and conjugates thereof, preparation method therefor and use thereof - Google Patents

Abstract

The present invention relates to a camptothecin compound and conjugates thereof, or tautomers, enantiomers and diastereomers thereof, or a mixture of same, or pharmaceutically acceptable salts or solvates thereof, and a pharmaceutical composition thereof, a preparation method therefor and the use thereof. The compound and the conjugates can be used for treating proliferative diseases related to abnormal cell activity.

Description

Camptothecin compounds and conjugates thereof, preparation methods and uses thereof Technical Field

The present invention belongs to the field of medical technology, and relates to camptothecin compounds and conjugates thereof, preparation methods thereof and uses thereof in preventing and/or treating proliferative diseases (including but not limited to tumor diseases) associated with abnormal cell activity.

Background Art

Camptothecin (CPT) is a cytotoxic alkaloid isolated from the plant Camptotheca acuminata of the Davidiaceae family. It can form a ternary complex with cellular DNA topoisomerase I, thereby inhibiting DNA unwinding, leading to blocked DNA replication and further causing cell death (Cancer Res.1989,49,6365). It has a broad-spectrum antiproliferative activity. However, due to its low solubility, instability, acquired tumor cell resistance and significant toxicity, it is not suitable for clinical development. Camptothecin derivatives can increase water solubility and improve drugability by introducing water-soluble groups or preparing prodrugs. Several camptothecin derivatives with greatly improved solubility have been approved for marketing (Med.Res.Rev.2015,35,753), such as topotecan, irinotecan and belototecan, for the treatment of various types of cancer.

Camptothecin derivatives are also used to couple with antibodies as small molecule toxins contained in antibody-drug conjugates (ADCs), also known as payloads. ADC drugs combine the dual advantages of the high efficiency of cytotoxic small molecules and the high selectivity of antibodies for specific tumor cells. Compared with traditional chemotherapy drugs, ADCs can kill tumor cells more accurately and reduce the impact on normal cells. In recent years, ADCs with camptothecin derivatives as small molecule toxins have made great progress. Two camptothecin-based ADCs have been approved for the treatment of cancer, namely DS-8101a, in which the camptothecin analog dxd is coupled to the anti-HER2 antibody trastuzumab through a cleavable tetrapeptide-based linker, and Immu-132, in which the camptothecin analog SN-38 is coupled to the anti-Trop-2 antibody certolizumab through a hydrolyzable pH-sensitive linker.

However, ADCs with camptothecin drugs or derivatives as toxins generally have a large drug/antibody ratio (DAR), and the production process is difficult, which can easily cause ADC instability. In addition, camptothecin compounds often have blood toxicity caused by bone marrow suppression, such as neutropenia, leukopenia, thrombocytopenia, anemia, etc., as well as gastrointestinal side effects, such as nausea, vomiting, diarrhea, etc.

Therefore, there is still a high clinical demand and application value in developing camptothecin compounds and their conjugates with novel structures that can enhance effectiveness and improve safety issues.

Summary of the invention

The present application provides a camptothecin compound or its tautomer, mesomer, racemate, enantiomer, diastereoisomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, which may have one or more effects selected from the following groups: (1) having inhibitory activity on the proliferation of tumor cells in vitro; (2) having plasma stability; (3) having an in vivo tumor-suppressing effect; (4) having a bystander killing effect; (5) having anti-transporter transport ability; (6) having in vivo tumor targeting ability; and (7) having good in vivo safety.

In one aspect, the present application provides a compound represented by formula (Ia), or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof:

in,

R ^1a is selected from hydrogen, halogen, amino, hydroxy, -C _1-6 alkyl and -C _1-6 alkoxy;

R ^2a and R ^3a are each independently selected from hydrogen, -(C(R ^2a-1 ) ₂ ) _p -H, -(C(R ^2a-1 ) ₂ ) _p -OR ^2a-2 , -(C(R ^2a-1 ) ₂ ) _p -SR ^2a-2 , -(C(R ^2a-1 ) ₂ ) _p -S(O) ₂ R ^2a-2 , -(C(R ^2a-1 ) ₂ ) _p -N(R ^2a-2 )-S(O) ₂ R ^2a-2 and -C(＝O)R ^2a-2 ; or R ^2a and R ^3a and the atoms to which they are attached together form a 3-10 membered heterocyclic ring; the heterocyclic ring is optionally substituted by one or more selected from halogen, hydroxy, amino, -C _1-6 alkyl, -OC _1-6 alkyl, -NHC _1-6 alkyl, -N(C _{-C 1-6} alkyl) ₂ and -C _1-6 alkylene-OH substituents;

p is an integer selected from 0 to 10;

R ^2a-1 are each independently selected from hydrogen, halogen, hydroxy, amino, cyano, nitro, -C _1-6 alkyl and -halogenated C _1-6 alkyl;

Alternatively, any two R ^2a-1 together with the atoms to which they are attached form an oxo group, a 3-10 membered carbocyclic ring or a 3-10 membered heterocyclic ring, wherein the 3-10 membered carbocyclic ring or the 3-10 membered heterocyclic ring is optionally substituted with -C _1-6 alkyl;

R ^2a-2 is selected from hydrogen, hydroxy, amino, cyano, -C _1-6 alkyl, -C _3-6 cycloalkyl and 3-6 membered heterocycloalkyl; each of the alkyl, cycloalkyl and heterocycloalkyl groups is optionally substituted by one or more substituents selected from hydrogen, halogen, hydroxy, amino and -C _1-6 alkyl.

In some embodiments, a compound of the structure represented by formula (Ia) is provided, wherein R ^1a is selected from hydrogen, halogen, and -C _1-6 alkyl; preferably, R ^1a is selected from hydrogen and halogen; further preferably, R ^1a is selected from hydrogen, F, and Cl.

In some embodiments, a compound of the structure represented by formula (Ia) is provided, wherein R ^2a and R ^3a are each independently selected from hydrogen, -(C(R ^2a-1 ) ₂ ) _p -H, -(C(R ^2a-1 ) ₂ ) _p -OR ^2a-2 , -(C(R ^2a-1 ) ₂ ) _p -S(O) ₂ R ^2a-2 , -(C(R ^2a-1 ) ₂ ) _p -N(R ^2a-2 )-S(O) ₂ R ^2a-2 and -C(＝O)R ^2a-2 ;

p is an integer selected from 0-10; preferably, p is an integer selected from 0-5;

R ^2a-1 is independently selected from hydrogen, halogen, hydroxyl, amino or -C _1-6 alkyl; preferably, R ^2a-1 is selected from hydrogen or -C _1-6 alkyl; further preferably, R ^2a-1 is selected from hydrogen or methyl;

Alternatively, any two R ^2a-1 and the atoms to which they are connected together form an oxo group, a 3-6-membered carbocyclic ring or a heterocyclic ring; preferably, any two R ^2a-1 and the atoms to which they are connected together form an oxo group, a 3-6-membered carbocyclic ring;

R ^2a-2 is selected from hydrogen, -C _1-6 alkyl, -C _3-6 cycloalkyl, 3 to 6 membered heterocycloalkyl; preferably, R ^2a-2 is selected from hydrogen or -C _1-6 alkyl; further preferably, R ^2a-2 is selected from hydrogen or methyl.

In some embodiments, a compound of the structure represented by formula (Ia) is provided, wherein R ^2a and R ^3a together with the atoms to which they are connected form a 4-6 membered heterocyclic ring; the heterocyclic ring is optionally substituted with one or more substituents selected from halogen, hydroxyl, amino, -C _1-6 alkyl, -C _1-6 alkylene-OH.

In some embodiments, a compound of the structure shown in formula (Ia) is provided, wherein R ^2a and R ^3a are each independently selected from hydrogen, -C _1-6 alkyl, -C _1-6 alkylene-OH, -haloC _1-6 alkyl and -C _1-6 alkyl-OC _1-6 alkyl; preferably, R ^2a and R ^3a are each independently selected from hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl, -(CH ₂ ) ₂ OH, -(CH ₂ ) ₃ OH, -(CH ₂ ) ₂ OCH ₃ and -(CH ₂ ) ₃ OCH ₃

In some particularly preferred embodiments, a compound of the structure represented by formula (Ia) is provided, wherein R ^1a is selected from hydrogen, F and Cl; R ^2a and R ^3a are each independently selected from hydrogen, -C _1-6 alkyl, -C _1-6 alkylene-OH, -haloC _1-6 alkyl and -C _1-6 alkyl-OC _1-6 alkyl.

In some embodiments, the compound of the structure represented by formula (Ia) is selected from:

Preferably, the compound of the structure shown in formula (Ia) is selected from:

On the other hand, the present application provides a compound represented by formula (Ib), or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof:

in,

R ^1b , R ^2b are each independently selected from hydrogen, halogen, hydroxy, nitro, -C _1-6 alkyl and -C _1-6 alkoxy;

or R ^1b and R ^2b together with the atoms to which they are attached form a 4-6 membered carbocyclic ring or a 4-6 membered heterocyclic ring;

R ^3b is selected from -(C(R ^3b-1 ) ₂ ) _t -H, -(C(R ^3b-1 ) ₂ ) _t -OR ^3b-2 , -(C(R ^3b-1 ) ₂ ) _t - N(R ^3b-2 ) ₂ , -(C(R ^3b-1 ) ₂ ) _t -SR ^3b-2 and -(C(R ^3b-1 ) ₂ ) _t -S(O) ₂ R ^3b-2 ;

t is an integer selected from 0 to 10;

R ^3b-1 are each independently selected from hydrogen, halogen, hydroxy, amino, cyano, nitro, -C _1-6 alkyl and -halogenated C _1-6 alkyl;

Or, any two R ^3b-1 together with the atoms to which they are attached form an oxo group, a 3-6 membered carbocyclic ring or a 3-6 membered heterocyclic ring;

R ^3b-2 is selected from hydrogen, hydroxy, -C _1-6 alkyl, -C _3-6 cycloalkyl, 3 to 6 membered heterocycloalkyl; each of the alkyl, cycloalkyl, heterocycloalkyl is optionally substituted with one or more substituents selected from hydrogen, halogen, hydroxy, amino and -C _1-6 alkyl;

R ^4b is selected from hydrogen, hydroxyl, -C _1-6 alkyl, -C _3-6 cycloalkyl, -halogenated C _1-6 alkyl. In some embodiments, a compound of the structure shown in formula (Ib) is provided, wherein R ^1b is selected from hydrogen, halogen, -C _1-6 alkyl, -C _1-6 alkoxy; preferably, R ^1b is selected from hydrogen, F, Cl, methyl and methoxy.

In some embodiments, a compound of the structure represented by formula (Ib) is provided, wherein R ^2b is selected from hydrogen, halogen, -C _1-6 alkyl, -C _1-6 alkoxy; preferably, R ^2b is selected from hydrogen and halogen; further preferably, R ^2b is selected from F.

In some embodiments, a compound of the structure shown in formula (Ib) is provided, wherein R ^1b and R ^2b together with the atoms connected thereto form a 5-membered heterocyclic ring; preferably, the 5-membered heterocyclic ring is

In some embodiments, a compound of the structure shown in formula (Ib) is provided, wherein R ^3b is selected from hydrogen, -(C(R ^3b-1 ) ₂ ) _t -H, -(C(R ^3b-1 ) ₂ ) _t -OR ^3b-2 , -(C(R ^3b-1 ) ₂ ) _t -NHR ^3b-2 ;

t is selected from an integer of 0-10; preferably, t is selected from an integer of 3-5;

R ^3b-1 is independently selected from hydrogen, halogen, hydroxyl, amino or -C _1-6 alkyl; preferably, R ^3b-1 is selected from hydrogen or -C _1-6 alkyl; further preferably, R ^3b-1 is selected from hydrogen or methyl;

Alternatively, any two R ^3b-1 and the atoms to which they are connected together form an oxo group, a 3-6-membered carbocyclic ring or a 3-6-membered heterocyclic ring; preferably, any two R ^3b-1 and the atoms to which they are connected together form an oxo group or a 3-6-membered carbocyclic ring;

R ^3b-2 is selected from hydrogen, -C _1-6 alkyl, -C _3-6 cycloalkyl, 3 to 6 membered heterocycloalkyl; preferably, R ^3b-2 is selected from hydrogen or -C _1-6 alkyl; further preferably, R ^3b-2 is selected from hydrogen. In some embodiments, a compound of the structure shown in formula (Ib) is provided, wherein R ^3b is selected from hydrogen, -C _1-6 alkyl, -halogenated C _1-6 alkyl and -C _1-6 alkylene-OH.

In some embodiments, a compound of the structure represented by formula (Ib) is provided, wherein R ^4b is selected from hydrogen, methyl, and ethyl; preferably, R ^4b is selected from hydrogen and methyl; preferably, R ^4b is selected from hydrogen.

In some embodiments, a compound of the structure represented by formula (Ib) is provided, wherein R ^3b and R ^4b are not hydrogen at the same time.

In some embodiments, the compound of the structure shown in formula (Ib) is selected from:

Preferably, the compound of the structure shown in formula (Ib) is selected from:

In another aspect, the present application provides a compound represented by formula (IIa), or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof:

in,

R ^1a , R ^2a and R ^3a are as defined for the compound of formula (Ia);

L ¹ is the connection unit;

^L2 is -(C( ^RL21 ) ₂ ) _n- ,

Where n is a natural number from 0 to 50.

Any C(R ^L21 ) ₂ unit in L ² may be independently replaced by the following structural units: -Cy-, -C(O)-, -NR ^L22 -, -O-, -S-, -SO-, -SO ₂ -, -P(R ^L22 )-, -P(＝O)(R ^L22 )-, -C(＝S)-, -C(＝NR ^L22 )-, -N＝N-, -C＝N-, -N＝C-,

-Cy- is selected from phenylene, 5- to 8-membered heteroarylene, 3- to 10-membered heterocyclylene, or 3- to 10-membered cycloalkylene, wherein said -Cy- is unsubstituted or independently substituted with 1 or more R ^cx ,

R ^L21 , R ^L22 , and R ^cx are each independently selected from hydrogen, deuterium, halogen, -NO ₂ , -CN, -OR ^L2a , -SR ^L2a , -N(R ^L2a ) ₂ , -N ⁺ (R ^L2a ) ₃ , -C(O)R ^L2a , -CO ₂ R ^L2a , -C(O)C(O)R ^L2a , -C(O)CH ₂ C(O)R ^L2a , -S(O)R ^L2a , -S(O) ₂ R ^L2a , -C(O)N(R ^L2a ) ₂ , -SO ₂ N(R ^L2a ) ₂ , -OC(O)R ^L2a , -N(R ^L2a )SO ₂ R ^L2b , -N(R ^L2a )COR ^L2b , -(CH ₂ ) _y -CO-(N(Me)CH ₂ C(O)) _m -OR ^L2a , -(CH ₂ ) _y -CO-(N(Me)CH ₂ C(O)) _m -NHR ^L2a , -(CH ₂ ) _y -CO-(N(Me)CH ₂ C(O)) _m -N ⁺ (R ^L2a ) ₃ , -(CH ₂ ) _y -NHCOCH ₂ (OCH ₂ CH ₂ )OR ^L2a , -(CH ₂ ) _y -NH(COCH ₂ (N(Me)) _m -R ^L2a , -(CH ₂ ) _y -CONH-(CH ₂ CH ₂ O) _m -R ^L2a , -(CH ₂ ) _y -NHCO-(CH ₂ CH ₂ O) _m -R ^L2a , -(CH ₂ CH ₂ O) _m -R ^L2a , -(COCH _{2 -R} ^L2a , -COCH ₂ (OCH ₂ CH ₂ ) _m -OR ^L2a , -CO-(CH ₂ CH ₂ O) _m -R ^L2a , -CO-(CH ₂ ) _y -CONH-(CH ₂ CH ₂ O) _m -R ^L2a , -CO-(CH ₂ ) _y -NHCO-(CH ₂ CH ₂ O) _m -R ^L2a , or -C _1-6 ^alkyl , -C _1-6 alkenyl, -C _1-6 alkynyl, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 3-10 membered heteroaryl optionally substituted by R L2a,

m, y are natural numbers from 0 to 50,

^RL2a , ^RL2b are each independently selected from hydrogen, deuterium, halogen, _-NO2 , -CN, -OH, -SH, _-NH2 , -N(Me) ₂ , _-CO2H , -S(O) _2Me , _-S (O) _2OH , -C(O) _NH2 , _-SO2NH2 , _-C1-6alkyl , _-C1-6alkenyl , -C1-6alkynyl, 3-8-membered cycloalkyl, 4-10-membered heterocycloalkyl, _6-10 -membered aryl, or 3-10-membered heteroaryl;

L ³ does not exist or is an amino acid residue, a short peptide consisting of 2-10 amino acid residues, or any combination of the above groups, wherein the amino acid residue is a natural amino acid residue or a non-natural amino acid residue;

Tr does not exist or is or any combination of the above groups;

R ^Tr , R ^Tr1 and R ^Tr2 are each independently selected from hydrogen, deuterium, halogen, -NO ₂ , -CN, -OH, -SH, -NH ₂ , -CO ₂ H, _{-S(O)2OH, -C(O)NH2, -SO2NH2 , -OC (} O) _{NH2 , -CH2CO-} (N(Me) _CH2C (O)) _z- ^ORTra _, -CH2CO-(N(Me) _CH2C (O)) _z - ^NHRTra , -(CH2CH2O) _z - ^RTra , -CONH-( _CH2CH2O ) _z - ^RTra , or _-C1-6 alkyl, _-C1-6 alkenyl, _-C1-6 alkynyl, 3-8 membered cycloalkyl, _4-10 membered heterocycloalkyl, 6-10 membered _aryl or 3-10 membered heteroaryl optionally substituted by ^RTra ,

R ^Tra is hydrogen, deuterium, halogen, -NO ₂ , -CN, -OH, -SH, -NH ₂ , -N(Me) ₂ , -S(O) ₂ Me, -CO ₂ H, -S(O) ₂ OH, -C(O)NH ₂ , -SO ₂ NH ₂ , -C _1-6 alkyl, -C _1-6 alkenyl, -C _1-6 alkynyl, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 3-10 membered heteroaryl,

z is a natural number of 0 or greater.

In another aspect, the present application provides a compound represented by formula (IIb), or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof:

in,

X is selected from: -O-, -N(R ^3b-2 )-

L ¹ , L ² , L ³ , and Tr are as defined in the compound of formula (IIa);

R ^1b , R ^2b , R ^3b-1 , R ^3b-2 and R ^4b are as defined for the compound of formula (Ib), and t is an integer selected from 1-10.

In some embodiments, compounds of the structures shown in formula (IIa) and formula (IIb) are provided, wherein L ¹ is selected from:

In some embodiments, compounds of the structures shown in formula (IIa) and formula (IIb) are provided, wherein L ¹ is selected from: Preferably, ^L1 is selected from

In some embodiments, compounds of the structures shown in Formula (IIa) and Formula (IIb) are provided, wherein L ² is -(CHR ^L21 ) _n -;

Wherein, n is a natural integer from 0 to 50;

Any CHR ^L21 unit in ^L2 can be independently replaced by the following structural units: -Cy-, -C(O)-, -NR ^L22 -, -O-,

-Cy- is phenylene, 5- to 6-membered heteroarylene, 4- to 10-membered heterocyclylene, or 3- to 6-membered cycloalkylene, wherein said -Cy- is unsubstituted or independently substituted with 1 to 3 R ^cx ;

Each ^RL21 , ^RL22 , and ^Rcx is independently selected from hydrogen, halogen, -OR ^L2a , -N( ^RL2a ) ₂ , -C(O) ^RL2a , -S(O) ^2RL2a , -C(O)N( ^RL2a _{)2 , -SO2N} ( ^RL2a ) ₂ , -N( ^RL2a ) ^SO2RL2b , -N( ^RL2a )COR ^L2b , -( _CH2 ) _y -CO-(N(Me) _CH2C (O)) _m -OR ^L2a , _- ( _CH2 ) _y - _CO- (N(Me) _CH2C (O)) _m -NHR ^L2a , -( _CH2 ) _y -CONH-( _CH2CH2O ) _m - ^RL2a , -( _CH2 ) _y -NHCO-(CH ₂ CH ₂ O) _m ^-RL2a , -(CH ₂ ) _y -NHCOCH ₂ (OCH ₂ CH ₂ )OR ^L2a , -(CH ₂ ) _y -NH(COCH ₂ (N(Me)) _m ^-RL2a , -(CH ₂ ) _y -NHCO-(CH ₂ CH ₂ O) _m ^-RL2a , -(CH ₂ CH ₂ O) _m ^-RL2a , -(COCH ₂ N(Me)) _m ^-RL2a , -COCH ₂ (OCH ₂ CH ₂ ) _m -OR ^L2a , -CO-(CH ₂ CH ₂ O) _m ^-RL2a , or -C _1-6 alkyl, -C 1-6 alkenyl, -C _1-6 alkyl optionally substituted by ^RL2a , -C _1-6 membered alkynyl, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 3-10 membered heteroaryl;

m is a natural integer from 0 to 8;

y is 0, 1, 2, 3, or 4;

Each ^RL2a and ^RL2b is independently selected from hydrogen, halogen, -CN, -OH, _-NH2 , -N(Me) ₂ , _-CO2H , -C(O) _NH2 , and _-C1-6 alkyl.

In some embodiments, L ² is -(CH ₂ ) _n -;

Wherein, n is a natural integer from 0 to 50;

Any methylene unit in ^L2 can be independently replaced by the following structural units: 4- to 6-membered heterocyclylene, 3- to 6-membered cycloalkylene, -C(O)-, -NR ^L22 -, -O-,

Each R ^L22 is independently selected from hydrogen, -OR ^L2a , -C(O) ^RL2a , -S(O) ₂ R ^L2a , -C(O)N(R ^L2a ) ₂ , -SO ₂ N(R ^L2a ) ₂ , -(CH ₂ ) _y -CO-(N(Me)CH ₂ C(O)) _m -OR ^L2a , -(CH ₂ ) _y -CO-(N(Me)CH ₂ C(O) ) _m -NHR ^L2a , -(CH ₂ ) _y -CONH-(CH ₂ CH ₂ O) _m -R ^L2a , -(CH ₂ ) _y -NHCOCH ₂ (OCH ₂ CH ₂ )OR ^L2a , -(CH ₂ CH ₂ O) _m -R ^L2a , -(COCH ₂ N(Me)) _m -R ^L2a , -COCH ₂ (OCH ₂ CH ₂ ) _m -OR ^L2a , -CO-(CH ₂ CH ₂ O) _m -R ^L2a , or -C _1-6 alkyl optionally substituted by R ^L2a ;

m is a natural integer from 0 to 8;

y is 0, 1, 2, 3, or 4;

Each ^RL2a is independently selected from hydrogen, halogen, -CN, -OH, _-NH2 , -N(Me) ₂ , _-CO2H , -C(O) _NH2 , _-C1-6alkyl .

In some embodiments, compounds of the structures shown in formula (IIa) and formula (IIb) are provided, wherein L ² is selected from:

in,

n1, n2, n3, n4, m are each independently selected from natural numbers from 0 to 8;

n5 and n6 are each independently selected from 0 or 1;

-Cy- is a 4- to 6-membered heterocyclylene group or a 3- to 6-membered cycloalkylene group; preferably, -Cy- is Further preferably, -Cy- is

More preferably,

for

More preferably,

for Among them, c is connected to ^L1 and d is connected to ^L3 .

In some embodiments, compounds of the structures shown in formula (IIa) and formula (IIb) are provided, wherein: Selected from:

n1, n2, n3, n4, m are each independently selected from natural numbers from 0 to 8;

n5 and n6 are each independently selected from 0 or 1;

-Cy- is a 4- to 6-membered heterocyclylene or a 3- to 6-membered cycloalkylene; preferably, -Cy- is selected from: Further preferably, -Cy- is selected from

Preferably, Selected from: More preferably, Selected from:

In some embodiments, compounds of the structures shown in formula (IIa) and formula (IIb) are provided, wherein L ³ is selected from Val, D-Val, Phe, Lys, Leu, Ile, Gly, Ala, D-Ala, Cit, Asp, Asn, Glu, Gln, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), Leu-Lys, Leu-Lys(Ac), Ala-Ala, Ala-Lys, D-Ala-Ala, G ly-Glu, Gly-Asp, Gly-Asn, Val-Glu, Val-Asp, Asn-Asn, Asp-Glu, Gly-Gly-Glu, Gly-Gly-Asp, Gly-Gly-Asn, Gly-Ala-Ala, Gly-Val-Ala, Gly-Val-Cit, Glu-Val-Ci t, Ala-Ala-Ala, Ala-(D-Ala)-Ala, Ala-Ala-Asn, Ala-(D-Ala)-Asn, Ala-Ala-Asp, Val-Lys-Gly, D-Val-Leu-Lys, Gly-Gly-Arg, Gly-Gly-Gly, Lys-Ala-Asn, Gly-Phe-Gly, Gly-Gly-Phe, Asn-Pro -Val, Ala-Lys-Gly, Gly-Lys-Gly, Gly-Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Glu-Gly, Lys-Ala-Ala-Asn, Lys-Ala-Ala-Asp, Ala-Ala-Pro-Val, Ala-Ala-Pro-Nva, Or any combination of the above fragments.

In some embodiments, ^L3 is selected from Lys, Gly, Asp, Asn, Glu, Gln, Val-Cit, Val-Ala, Ala-Ala, Gly-Glu, Gly-Asp, Gly-Asn, Asn-Asn, Asp-Glu, Gly-Glu-Gly, Gly-Gly-Phe-Gly, or any combination of the above fragments; preferably, ^L3 is selected from Lys, Gly, Val-Ala, Ala-Ala, Gly-Glu, Gly-Asp, Gly-Asn, Asn-Asn, Asp-Glu, Gly-Gly-Phe-Gly, or any combination of the above fragments; More preferably, ^L3 is selected from Val-Ala, Ala-Ala, Gly-Glu, Gly-Asp, Gly-Asn, Asp-Asp, Asp-Glu, Gly-Gly-Phe-Gly or

In some embodiments, compounds of the structures shown in formula (IIa) and formula (IIb) are provided, wherein: Selected from:

In some embodiments, compounds of the structures shown in formula (IIa) and formula (IIb) are provided, wherein Tr is absent or is in

R ^Tr , R ^Tr1 and R ^Tr2 are independently selected from hydrogen, halogen, -NO ₂ , -CN, -OH, -NH ₂ , -CO ₂ H, -S(O) ₂ H, -C(O)NH ₂ , -SO ₂ NH ₂ , -OC(O)NH ₂ , -CH ₂ CO-(N(Me)CH ₂ C(O)) _z -NHMe, -(CH ₂ CH ₂ O) _z -H, -CONH-(CH ₂ CH ₂ O) _z -H;

z is a natural number from 0 to 8.

In some embodiments, compounds of the structures shown in formula (IIa) and formula (IIb) are provided, wherein Tr is absent or is

In some embodiments, the compound of the structure represented by formula (IIa) is selected from:

In some embodiments, the compound of the structure represented by formula (IIb) is selected from:

In another aspect, the present application provides a ligand-drug conjugate, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein the ligand-drug conjugate comprises a ligand and

-Structure represented by formula (IVa):

in,

R ^1a , R ^2a and R ^3a are as defined in the compound of formula (Ia), and the wavy line indicates that they are directly or indirectly connected to the ligand via the nitrogen atom; or

The structure shown in formula (IVb):

in,

X is selected from: -O-, -N(R ^3b-2 )-;

R ^1b , R ^2b , R ^3b-1 , R ^3b-2 and R ^4b are as defined in the compound of formula (Ib), t is an integer selected from 1-10, and the wavy line indicates direct or indirect connection with the ligand through X.

In another aspect, the present application provides a ligand-drug conjugate, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein the ligand-drug conjugate comprises a ligand and

-Structure represented by formula (Va):

in,

The wavy line indicates that the ligand is connected through the nitrogen or carbon atom on the L ^1a group;

L ^1a is selected from:

L ² , L ³ , and Tr are as defined in the compound of formula (IIa);

R ^1a , R ^2a and R ^3a are as defined for the compound of formula (Ia); or

-Structure represented by formula (Vb):

in,

The wavy line indicates that the ligand is connected through the nitrogen or carbon atom on the L ^1a group;

L ^1a is as defined for the compound of formula (Va);

L ² , L ³ , and Tr are as defined in the compound of formula (IIa);

X is selected from: -O-, -N(R ^3b-2 )-;

R ^1b , R ^2b , R ^3b-1 , R ^3b-2 and R ^4b are as defined for the compound of formula (Ib), and t is an integer selected from 1-10.

In some embodiments, a ligand-drug conjugate comprising a structure represented by formula (Va) and formula (Vb) is provided, wherein L ^1a is selected from: Preferably, L ^1a is selected from

In some embodiments, a ligand-drug conjugate comprising a structure represented by formula (Va) and formula (Vb) is provided, wherein Selected from:

n1, n2, n3, n4, m are each independently selected from natural numbers from 0 to 8;

n5 and n6 are each independently selected from 0 or 1;

-Cy- is a 4- to 6-membered heterocyclylene or a 3- to 6-membered cycloalkylene; preferably, -Cy- is selected from: Further preferably, -Cy- is selected from

In some embodiments, a ligand-drug conjugate comprising a structure represented by formula (Va) and formula (Vb) is provided, wherein: Selected from: More preferably, Selected from:

In some embodiments, a ligand-drug conjugate comprising a structure represented by formula (Va) and formula (Vb) is provided, wherein: Selected from:

In another aspect, the present application provides a ligand-drug conjugate represented by formula (IIIa), or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate:

in,

L ² , L ³ , and Tr are as defined in the compound of formula (IIa);

R ^1a , R ^2a , and R ^3a are as defined for the compound of formula (Ia).

Ab is the ligand that binds to the target;

q is the drug loading, which is an integer or decimal from 1 to 16;

L ^1a is as defined for the compound of formula (Va).

In another aspect, the present application provides a ligand-drug conjugate represented by formula (IIIb), or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate:

in,

Ab, q, L ^1a are as defined in the compound of formula (IIIa);

L ² , L ³ , and Tr are as defined in the compound of formula (IIa);

X is selected from: -O-, -N(R ^3b2 )-;

R ^1b , R ^2b , R ^3b-1 , R ^3b-2 and R ^4b are as defined for the compound of formula (Ib), and t is an integer selected from 1-10.

In some embodiments, a ligand-drug conjugate of formula (IIIa) and formula (IIIb) is provided, wherein Ab is a target-binding polypeptide, antibody, or antigen-binding fragment thereof.

In some embodiments, Ab is an antibody or an antigen-binding fragment thereof.

In some embodiments, the antibody is selected from the group consisting of: a fully human antibody, a humanized antibody, a chimeric antibody, a probody, a bispecific antibody, a multispecific antibody, a monoclonal antibody, and a polyclonal antibody.

In some embodiments, the antigen binding fragment is selected from the group consisting of: Fab, Fab', F(ab')2, Fv, scFv, diabody, Fd, dAb, VHH, big antibody and complementarity determining region (CDR) fragment.

In some embodiments, Ab is a monoclonal antibody.

In some embodiments, the Ab specifically binds to an antigen selected from the group consisting of 5T4, AGS-16, ANGPTL4, ApoE, CD19, CTGF, CXCR5, FGF2, MCPT8, MFI2, MS4A7, NCA, Sema5b, SLITRK6, STC2, TGF, 0772P, 5T4, ACTA2, ADGRE1, AG-7, AIF1, AKR1C1, AKR1C2, ASLG659, Axl, B7H3, BAFF-R, BCMA, BMPR1B, BNIP3, C1QA, C1QB, CA6, CADM1, CCD79b, CCL5, CCR5, CCR7, CD11c, CD123, CD138, CD142, CD147, CD166, CD19, CD19, CD22, CD21, CD2 0, CD205, CD22, CD223, CD228, CD25, CD30, CD33, CD37, CD38, CD40, CD45, CD45(PTPRC), CD46, CD47, CD49D(ITGA4), CD56, CD66e, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CDCP1, CDH 11, CDllb, CEA, CEACAM5, c-Met, COL6A3, COL7A1, CRIPTO, CSF1R, CTSD, CTSS, CXCL11, CXCL10, DDIT4, DLL3, DLL4, DR5, E16, EFNA4, EGFR, EGFRvIII, EGLN, EGLN3, EMR2, ENPP3, EpCAM, Ep hA2, EphB2R, ETBR, FcRH2, FcRHl, FGFR2, FGFR3, FLT3, FOLR-α, GD2, GEDA, GPC-1, GPNMB, GPR20, GZMB, HER2, HER3, HLA-DOB, HMOX1, IFI6, IFNG, IGF-1R, IGFBP3, IL10RA1, IL-13R, IL-2, IL20Ra, IL-3 , IL-4, IL-6, IRTA2, KISS1R, KRT33A, LIV-1, LOX, LRP-1, LRRC15, LUM, LY64, LY6E, Ly86, LYPD3, MDP, MMP10, MMP14, MMP16, MPF, MSG783, MSLN, MUC-1, NaPi2b, Napi3b, Nect in-4, Nectin-4, NOG, P2X5, pCAD, P-Cadherin, PDGFRA, PDK1, PD-L1, PFKFB3, PGF, PGK1, PIK3AP1, PIK3CD, PLOD2, PSCA, PSCAhlg, PSMA, PSMA, PTK7, P-Cadherin, RNF43, NaPi2b, ROR1, ROR2, SERPINE1, SLC39A6, SLTRK6, STAT1, STEAP1, STEAP2, TCF4, TENB2, TGFB1, TGFB2, TGFBR1, TNFRSF21, TNFSF9, Trop-2, TrpM4, Tyro7, UPK1B, VEGFA, WNT5A, ADAM9, epidermal growth factor, brevican, mesothelin, phosphodiesterase Sodium cotransporter 2B, Claudin 18.2, endothelin receptor, mucins (such as mucin 1 and mucin 16), guanylate cyclase C, integrin α4β7, integrin α4β6, trophoblast glycoprotein, or tissue factor.

In some preferred embodiments, the Ab specifically binds to an antigen selected from the group consisting of HER2, HER3, B7H3, TROP2, Claudin18.2, CD30, CD33, CD70, or EGFR.

In some embodiments, Ab is an antibody or antigen binding fragment that specifically binds to HER2, HER3, B7H3, TROP2, Claudin18.2, CD30, CD33, CD70, or EGFR. In some embodiments, the antibody or antigen binding fragment that specifically binds to HER2 is an anti-HER2 antibody or antigen binding fragment, for example, trastuzumab (Trastuzumab) or Pertuzumab (Pertuzumab) or its variant.

In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each of which has one, two, three, or four amino acid changes (e.g., amino acid substitutions or deletions) with one or more CDRs in HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the following molecules, respectively: trastuzumab or pertuzumab.

In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody, wherein the heavy chain variable region and the light chain variable region have at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity with the heavy chain variable region and the light chain variable region of the following molecules, respectively: Trastuzumab or Pertuzumab.

In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain of an antibody, wherein the heavy chain and the light chain have at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity with the heavy chain and light chain of the following molecules, respectively: Trastuzumab or Pertuzumab.

Sequences of exemplary anti-HER2 antibodies or antigen-binding fragments thereof are provided in Table 1.

Table 1: Trastuzumab amino acid sequence

In some embodiments, the antibody or antigen-binding fragment that specifically binds to TROP2 is an anti-Trop-2 antibody or antigen-binding fragment, for example, Sacituzumab or a variant thereof.

In some embodiments, the anti-Trop-2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each of which has one, two, three, or four amino acid changes (e.g., amino acid substitutions or deletions) with one or more CDRs in HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the following molecule: Sacituzumab.

In some embodiments, the anti-Trop-2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody, wherein the heavy chain variable region and the light chain variable region have at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity with the heavy chain variable region and the light chain variable region of the following molecule, respectively: Sacituzumab.

In some embodiments, the anti-Trop-2 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain of an antibody, wherein the heavy chain and the light chain have at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity to the heavy chain and the light chain of the following molecule, respectively: Sacituzumab.

Sequences of exemplary anti-Trop-2 antibodies or antigen-binding fragments thereof are provided in Table 2.

Table 2: Certolizumab amino acid sequence

In some embodiments, the ligand-drug conjugate of formula (IIIa) is selected from:

wherein Ab and q are as defined in the compound of formula (IIIa).

In some preferred embodiments, the ligand-drug conjugate represented by formula (IIIa) is selected from:

wherein q is as defined for the compound of formula (IIIa).

In some embodiments, the ligand-drug conjugate of formula (IIIb) is selected from:

wherein Ab and q are as defined in the compound of formula (IIIa).

In some preferred embodiments, the ligand-drug conjugate represented by formula (IIIb) is selected from:

wherein q is as defined for the compound of formula (IIIa).

In some embodiments, q is an integer or decimal selected from 2 to 8.

The present invention also provides a pharmaceutical composition, which comprises the above-mentioned compounds with structures shown in formula (Ia) or (IIa), ligand-drug conjugates shown in formula (IIIa), or ligand-drug conjugates with structures shown in formula (IVa) or (Va), compounds with structures shown in formula (Ib) or (IIb), ligand-drug conjugates shown in formula (IIIb), or ligand-drug conjugates with structures shown in formula (IVb) or (Vb), or tautomers, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof, and a pharmaceutically acceptable carrier.

The present invention also provides a pharmaceutical preparation, which comprises the above-mentioned compounds with structures shown in formula (Ia) or (IIa), ligand-drug conjugates shown in formula (IIIa), or ligand-drug conjugates with structures shown in formula (IVa) or (Va), compounds with structures shown in formula (Ib) or (IIb), ligand-drug conjugates shown in formula (IIIb), or ligand-drug conjugates with structures shown in formula (IVb) or (Vb), or tautomers, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof.

The present invention also provides a use of a substance S in the preparation of a drug for preventing or treating cancer; the substance S is the compound of the structure shown in the above-mentioned formula (Ia) or formula (IIa), the ligand-drug conjugate shown in formula (IIIa), or the ligand-drug conjugate containing the structure shown in formula (IVa) or formula (Va), the compound of the structure shown in formula (Ib) or formula (IIb), the ligand-drug conjugate shown in formula (IIIb), or the ligand-drug conjugate containing the structure shown in formula (IVb) or formula (Vb), or its tautomer, enantiomer, diastereoisomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, and the above-mentioned drug The cancer disease is a solid tumor or a non-solid tumor. For example, it is selected from esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, non-Hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer or thyroid cancer.

The present invention also provides a use of a substance S in the preparation of a drug for preventing or treating a disease associated with abnormal cell activity; the substance S is the compound of the structure shown in the above-mentioned formula (Ia), formula (IIa), a ligand-drug conjugate shown in formula (IIIa), or a ligand-drug conjugate containing a structure shown in formula (IVa) or formula (Va), a compound of the structure shown in formula (Ib), formula (IIb), a ligand-drug conjugate shown in formula (IIIb), or a ligand-drug conjugate containing a structure shown in formula (IVb) or formula (Vb), or its tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, the above-mentioned pharmaceutical composition, or the above-mentioned pharmaceutical preparation. The disease associated with abnormal cell activity may be cancer. The definition of cancer is as described above.

Definition of terms:

In this application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory operation procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

In this application, the term "pharmaceutical excipients" refers to excipients and additives used in the production of drugs and the preparation of prescriptions. It refers to substances that have been reasonably evaluated in terms of safety and are included in drug preparations in addition to active ingredients. In addition to excipients, acting as carriers, and improving stability, pharmaceutical excipients also have important functions such as solubilization, solubilization, and sustained and controlled release. They are important ingredients that may affect the quality, safety, and effectiveness of drugs. According to their sources, they can be divided into natural products, semi-synthetic products, and fully synthetic products. According to their functions and uses, they can be divided into: 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, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, etc. According to their administration routes, they can be divided into oral, injection, mucosal, transdermal or topical administration, nasal or oral inhalation administration and ocular administration, etc. The same pharmaceutical excipients can be used in drug preparations with different administration routes and have different functions and uses.

In the present application, the term "pharmaceutical composition" refers to various suitable dosage forms that can be prepared according to the route of administration. For example, tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder sprays, sprays, etc. Among them, the pharmaceutical composition or suitable dosage form can contain 0.01 mg to 1000 mg of the compound of the present disclosure or its pharmaceutically acceptable salt or conjugate, preferably 0.1 mg to 800 mg, preferably 0.5-500 mg, preferably 0.5 to 350 mg, and particularly preferably 1-250 mg.

The pharmaceutical composition can be used in the form of injection, including injection, sterile powder for injection and concentrated solution for injection. Among them, the carriers and solvents that can be used include water, Ringer's solution and isotonic sodium chloride solution. In addition, sterilized non-volatile oils can also be used as solvents or suspension media, such as monoglycerides or diglycerides.

In the present application, the term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" generally refers to a salt of the compound or ligand-drug conjugate of the present application, or a salt of the compound described in the present application. Such salts may be safe and/or effective when used in mammals, and may have the desired biological activity. The antibody-antibody drug conjugate compound of the present application may form a salt with an acid. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

In the present application, the term "solvate" or "solvate compound" generally refers to a pharmaceutically acceptable solvate formed by a compound or ligand-drug conjugate compound of the present application and one or more solvent molecules, and non-limiting examples of solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.

The term "drug loading" generally refers to the average number of cytotoxic drugs loaded on each ligand, and can also be expressed as the ratio of the cytotoxic drug to the amount of antibody, for example, drug/antibody ratio (DAR), and the range of the cytotoxic drug loading can be an integer or decimal of 0-20. In an embodiment of the present application, the drug loading is expressed as q, which can be exemplarily an integer or decimal of 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10. For example, the drug loading q is 7.8 or 7.9. The drug loading of each ADC molecule after the coupling reaction can be identified by conventional methods such as UV/visible light spectroscopy, mass spectrometry, HIC, ELISA test and HPLC characteristics.

In the present application, the term "ligand-drug conjugate" generally refers to a ligand connected to a biologically active cytotoxic drug via a stable linker. In some embodiments of the present application, the "ligand-drug conjugate" may be an antibody-drug conjugate (ADC), and the ADC may refer to a monoclonal antibody or antibody fragment connected to a biologically active cytotoxic drug via a stable linker.

In the present application, the term "ligand" generally refers to small molecules, polypeptides, RNA, DNA, carbohydrates and macromolecular compounds that can recognize and bind to antigens or receptors associated with target cells. The role of the ligand can be to present the drug to the target cell population bound to the ligand. These ligands include but are not limited to protein hormones, lectins, growth factors, antibodies or other molecules that can bind to cells, receptors and/or antigens. In the present application, the ligand can be expressed as Ab, and the ligand antigen forms a connection bond with the connecting unit (also called "linker" or "linker") through the heteroatom on the ligand. The ligand can be an antibody or an antigen-binding fragment thereof, and the antibody can be selected from a chimeric antibody, a humanized antibody, a fully human antibody or a mouse antibody; the antibody can be a monoclonal antibody. For example, the antibody can be an antibody targeting the following targets: HER2, HER3, B7H3, TROP2, Claudin18.2, CD30, CD33, CD70 or EGFR. For example, the antibody can be an antibody targeting the following targets: 5T4, AGS-16, ANGPTL4, ApoE, CD19, CTGF, CXCR5, FGF2, MCPT8, MFI2, MS4A7, NCA, Sema5b, SLITRK6, STC2, TGF, 0772P, 5T4, ACTA2, ADGRE1, AG-7, AIF1, AKR1C1, AKR1C2, ASLG65 9. Axl, B7H3, BAFF-R, BCMA, BMPR1B, BNIP3, C1QA, C1QB, CA6, CADM1, CCD79b, CCL5, CCR5, CCR7, CD1lc, CD123, CD138, CD142, CD147, CD166, CD19, CD19, CD22, CD21, CD20, CD205, CD22, CD 223, CD228, CD25，CD30，CD33，CD37，CD38，CD40，CD45，CD45(PTPRC)，CD46，CD47，CD49D(ITGA4)，CD56，CD66e，CD70，CD71，CD72，CD74，CD79a，CD79b，CD80，CDCP1，CDH11，CDllb，CEA，CEACAM5，c-Met，COL6A3，COL7A1，CRIPTO，CSF1R，CTSD，CTSS，CXCL11，CXCL10，DDIT4，DLL3，DLL4，DR5，E16，EFNA4，EGFR，EGFRvIII，EGLN，EGLN3，EMR2，ENPP3，EpCAM，EphA2，EphB2R，ETBR，FcRH2，FcRHl，FGFR2，FGFR3，FLT3， FOLR-α, GD2, GEDA, GPC-1, GPNMB, GPR20, GZMB, HER2, HER3, HLA-DOB, HMOX1, IFI6, IFNG, IGF-1R, IGFBP3, IL10RA1, IL-13R, IL-2, IL20Ra, IL-3, IL-4, IL-6, IRTA2, KISS1R, KRT33A, LIV-1, LOX, LRP -1, LRRC15, LUM, LY64, LY6E, Ly86, LYPD3, MDP, MMP10, MMP14, MMP16, MPF, MSG783, MSLN, MUC-1, NaPi2b, Napi3b, Nectin-4, Nectin-4, NOG, P2X5, pCAD, P-Cadherin, PDGFRA, PDK1, PD-L1, PFKFB3, P GF, PGK1, PIK3AP1, PIK3CD, PLOD2, PSCA, PSCAhlg, PSMA, PSMA, PTK7, P-cadherin, RNF43, NaPi2b, ROR1, ROR2, SERPINE1, SLC39A6, SLTRK6, STAT1, STEAP1, STEAP2, TCF4, TENB2, TGFB1, TGFB2, TGFBR1, TNFRSF21, TNFSF9, Trop-2, TrpM4, Tyro7, UPK1B, VEGFA, WNT5A, ADAM9, epidermal growth factor, brevican, mesothelin, sodium phosphate cotransporter 2B, Claudin18.2, endotenin receptor, mucins (such as mucin 1 and mucin 16), guanylate cyclase C, integrin α4β7, integrin α4β6, trophoblast glycoprotein, or tissue factor.

In the present application, the term "antibody or its antigen-binding fragment" generally refers to an immunological binder, which extends to all antibodies from all species, including dimers, trimers and multimers; bispecific antibodies; chimeric antibodies; fully human antibodies; humanized antibodies; recombinant and remodeled antibodies and their fragments. The term "antibody or its fragment" may refer to any antibody-like molecule with an antigen-binding region, and the term includes small molecule fragments such as Fab', Fab, F(ab') ₂ , single domain antibodies (DABs), Fv, scFv (single chain Fv), linear antibodies, diabodies, etc. The term "antigen-binding fragment" may refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. For example, a fragment of a full-length antibody can be used to implement the antigen-binding function of an antibody. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. The antibodies may include: anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-TROP2 antibody, anti-Claudin One or more of the antibodies selected from 18.2 antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-ADAM9 antibody or anti-Mesothelin antibody, for example, may be Trastuzumab or Pertuzumab.

In this application, the term "chimeric antibody" generally refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody. The method for establishing a chimeric antibody can, for example, establish a hybridoma that secretes mouse-specific monoclonal antibodies, then clone the variable region gene from the mouse hybridoma cells, clone the constant region gene of the human antibody as needed, and then connect the mouse variable region gene with the human constant region gene to form a chimeric gene and insert it into an expression vector, and the chimeric antibody molecule can be expressed in a eukaryotic system or a prokaryotic system.

In this application, the term "humanized antibody", also known as CDR-grafted antibody, generally refers to an antibody produced by transplanting mouse CDR sequences into the human antibody variable region framework, that is, transplanting into different types of human germline antibody framework sequences. Humanized antibodies can overcome the problem that chimeric antibodies carry a large amount of mouse protein components, thereby inducing a strong heterologous response. Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references. For example, the germline DNA sequences of human heavy chain variable region and light chain variable region genes can be found in the "VBase" human germline sequence database.

In the present application, the terms "fully human antibody", "fully human antibody" or "completely human antibody" are used interchangeably, and both the variable region and the constant region of the antibody may be of human origin, eliminating immunogenicity and toxic side effects.

The development of monoclonal antibodies has gone through four stages, namely: mouse monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies. The antibody or ligand described in this application can be a fully human monoclonal antibody. The relevant technologies for the preparation of fully human antibodies can be: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology and single B cell antibody preparation technology, etc.

In this application, the term "CDR" generally refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding. The most commonly used definitions of the six CDRs are provided, for example, by Kabat E.A. et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242), Chothia et al., "Canonical Structures For the Hypervariable Regions of Immunoglobulins," J. Mol. Biol. 196:901 (1987); and MacCallum et al., "Antibody-Antigen Interactions: Contact Analysis and Binding Site Topography," J. Mol. Biol. 262:732 (1996)). As used in this application, the Kabat definition of CDR can be applied to CDR1, CDR2 and CDR3 of the light chain variable domain (CDRL1, CDRL2, CDRL3 or L1, L2, L3), and CDR1, CDR2 and CDR3 of the heavy chain variable domain (CDR H1, CDRH2, CDRH3 or H1, H2, H3).

The term "one or more" or the similar expression "at least one" may mean, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more;

When the lower and upper limits of a numerical range are disclosed, any value and any included range falling within the range is specifically disclosed. In particular, each range of values disclosed herein (in the form "about a to b", or equivalently, "approximately a to b", or equivalently, "about a-b") should be understood to mean each value and range encompassed in the broader range;

For example, the expression "C _1-6 " should be understood to cover any sub-ranges and each point value therein, such as C _2-5 , C _3-4 , C _1-2 , C _1-3 , C _1-4 , C _1-5 , etc., as well as C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , etc. For example, the expression "C _3-10 " should also be understood in a similar manner, for example, it can cover any sub-ranges and point values contained therein, such as C _3-9 , C _6-9 , C _6-8 , C _6-7 , C _7-10 , C _7-9 , C _7-8 , C _8-9 , etc., as well as C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , etc. For another example, the expression "3-10 yuan" should be understood to include any sub-ranges and each point value therein, such as 3-4 yuan, 3-5 yuan, 3-6 yuan, 3-7 yuan, 3-8 yuan, 3-9 yuan, 4-5 yuan, 4-6 yuan, 4-7 yuan, 4-8 yuan, 5-7 yuan, 5-8 yuan, 6-7 yuan, etc., as well as 3, 4, 5, 6, 7, 8, 9, 10 yuan, etc. For another example, the expression "5-10 yuan" should also be understood in a similar manner, for example, it can include any sub-ranges and point values contained therein, such as 5-6 yuan, 5-7 yuan, 5-8 yuan, 5-9 yuan, 5-10 yuan, 6-7 yuan, 6-8 yuan, 6-9 yuan, 6-10 yuan, 7-8 yuan, etc., as well as 5, 6, 7, 8, 9, 10 yuan, etc.

In the present application, the term "alkyl" refers to a saturated straight or branched hydrocarbon group. As used herein, the term "C _1-6 alkyl" refers to a saturated straight or branched hydrocarbon group having 1 to 6 carbon atoms (e.g., 1, 2, 3, 4, 5 or 6 carbon atoms). "C _1-6 alkyl" For example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl or n-hexyl, etc. The alkyl group in the present invention is optionally substituted with one or more substituents described in the present invention.

In the present application, the term "alkylene" refers to a saturated straight or branched divalent hydrocarbon group. As used herein, the term "C _1-6 alkylene" refers to a saturated straight or branched divalent hydrocarbon group having 1 to 6 carbon atoms. "C _1-6 alkylene" includes, but is not limited to, methylene, ethylene, propylene or butylene. The alkylene in the present invention is optionally substituted with one or more substituents described in the present invention.

In the present application, the term "alkenyl" refers to a straight or branched aliphatic hydrocarbon group with one or more carbon-carbon double bonds. For example, the term " _C2-6 alkenyl" as used herein refers to an alkenyl group (such as vinyl, 1-propenyl, 2-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 2-methyl-2-propenyl, 4-methyl-3-pentenyl, etc.) with 2-6 carbon atoms and one, two or three (preferably one) carbon-carbon double bonds, which is optionally substituted with one or more (such as 1-3) substituents described herein.

In the present application, the term "alkenylene" refers to a straight or branched divalent aliphatic hydrocarbon group having one or more carbon-carbon double bonds, and the two groups (or fragments) connected thereto may be connected to the same carbon atom or to different carbon atoms. For example, the term " _C2-6 alkenylene" as used herein refers to an alkenylene group having 2 to 6 carbon atoms (e.g. , etc.), which are optionally substituted with one or more (e.g., 1-3) substituents described herein.

In the present application, the term "alkynyl" refers to a straight or branched aliphatic hydrocarbon group with one or more carbon-carbon triple bonds. For example, the term " _C2-6alkynyl " as used herein refers to an alkynyl group (such as ethynyl, 1-propynyl, 2-propynyl, 2-butynyl, 3-butynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, etc.) with 2-6 carbon atoms and one, two or three (preferably one) carbon-carbon triple bonds, which is optionally substituted with one or more (such as 1-3) substituents described herein.

In the present application, the term "alkynylene" refers to a straight or branched divalent aliphatic hydrocarbon group having one or more carbon-carbon triple bonds, wherein the two groups (or fragments) connected thereto are respectively connected to different carbon atoms. For example, the term " _C2-6 alkynylene" as used herein refers to an alkynylene group having 2 to 6 carbon atoms (e.g. , etc.), which are optionally substituted with one or more (e.g., 1-3) substituents described herein.

In the present application, the term "aryl" refers to a monocyclic or condensed aromatic hydrocarbon group having a conjugated π electron system. For example, the term "6-10 membered aryl" as used herein refers to an aryl group having 6-10 carbon atoms (such as phenyl, naphthyl, etc.), which is optionally substituted with one or more substituents described herein (such as halogen substitution, etc.).

In the present application, the term "arylene" refers to a monocyclic or condensed divalent aromatic hydrocarbon group having a conjugated π electron system. For example, the term "C _6-10 arylene" used herein refers to an arylene group having 6 to 10 carbon atoms, which is optionally substituted by one or more substituents described herein (such as substituted by halogen, etc.).

In the present application, the term "heteroaryl" or "heteroaromatic ring" refers to a monocyclic and fused heterocyclic ring system having one or more conjugated π-electron systems, wherein one or more (e.g., 1, 2, or 3) ring atoms are heteroatoms selected from N, O, P, and S, and the remaining ring atoms are C. A heteroaryl or heteroaromatic ring can be characterized by the number of ring atoms. For example, a 5-12 membered heteroaryl can contain 5-12 (e.g., 5, 6, 7, 8, 9, 10, 11, or 12) ring atoms, in particular 5, 6, 9, or 10 ring atoms. Examples of heteroaryl include thienyl, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, indolyl, and the like; which are optionally substituted with one or more substituents described herein.

In the present application, the term "heteroarylene" refers to a monocyclic or condensed divalent aromatic group with a conjugated π electron system, which has one or more carbon atoms (such as 1, 2, 3, 4, 5, 6, 9 or 10 carbon atoms) and one or more (such as 1, 2, 3 or 4) heteroatoms independently selected from N, O, P and S in the ring, for example, a total of 5-12 (preferably 5-10, more preferably 5, 6, 9 or 10) ring atoms. For example, the term "5-12 membered heteroarylene" as used herein refers to a heteroarylene having 5-12 ring atoms, which is optionally substituted with one or more substituents described herein (such as substituted with C _1-6 alkyl). Halogen substituted wait).

In the present application, the term "cycloalkyl" or "carbocycle" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic) non-aromatic hydrocarbon group. For example, "C _3-12 cycloalkyl" or "3-12 membered cycloalkyl" refers to a cycloalkyl group having 3-12 ring carbon atoms (such as 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12). Common cycloalkyl groups include (but are not limited to) monocyclic cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclobutene, cyclopentene, cyclohexene, etc.; or bicyclic cycloalkyl groups, including fused rings, bridged rings or spiro rings, such as bicyclo[1.1.1]pentyl, bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, bicyclo[5.2.0]nonyl, decalinyl, etc., which are optionally substituted with one or more substituents described herein.

In the present application, the term "cycloalkylene" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic) non-aromatic divalent cyclic group. For example, "C _3-12 cycloalkylene" or "3-12 membered cycloalkylene" refers to a cycloalkylene having 3-12 ring carbon atoms (such as 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12). Common cycloalkylenes include (but are not limited to) monocyclic cycloalkylenes, such as cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclobutene, cyclopentene, cyclohexene, etc.; or bicyclic cycloalkylenes, including fused rings, bridged rings or spiro rings, such as bicyclo[1.1.1]pentylene, bicyclo[2.2.1] heptyl, bicyclo[3.2.1]octylene, bicyclo[5.2.0]nonylene, decalinylene, and the like, optionally substituted with one or more substituents described herein.

The term "heterocycloalkyl" or "heterocycle" refers to a saturated or partially saturated non-aromatic cyclic group containing at least one ring member selected from N, O, P and S heteroatom, preferably, the number of the heteroatoms is 1, 2, 3 or 4. For example, 3-8-membered, 3-6-membered heterocycloalkyl. Specific examples include, but are not limited to, oxirane, oxocyclobutane, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, homopiperazinyl, etc., which are optionally substituted with one or more oxo groups or substituents described herein.

The term "heterocycloalkylene" refers to a saturated or partially saturated non-aromatic divalent cyclic group containing at least one ring member being a heteroatom selected from N, O, P and S, preferably, the number of the heteroatoms is 1, 2, 3 or 4. For example, 3-8-membered, 3-6-membered heterocycloalkylene. Specific examples include, but are not limited to, oxiranediylene, oxocyclobutanediylene, pyrrolidinylene, tetrahydrofuranylene, piperidinylene, piperazinylene, tetrahydropyranylene, homopiperazinylene, etc., which are optionally substituted with one or more oxo groups or substituents described herein.

The term "condensed ring (condensed ring system)" refers to a polycyclic structure formed by two or more (e.g., 3, 4, or 5) carbocyclic rings or heterocyclic rings with a common ring edge, wherein the carbocyclic ring includes a cycloalkyl group and an aryl group, and the heterocyclic ring includes a heteroaromatic ring and a heterocycloalkyl group. The condensed ring system includes, but is not limited to, a condensed ring system formed by a cycloalkyl group and a cycloalkyl group, a condensed ring system formed by a cycloalkyl group and a heterocycloalkyl group, a condensed ring system formed by a cycloalkyl group and an aromatic ring, a condensed ring system formed by a cycloalkyl group and a heteroaromatic ring, a condensed ring system formed by a heterocycloalkyl group and a heteroaromatic ring, a condensed ring system formed by a heterocycloalkyl group and an aromatic ring, a condensed ring system formed by a heteroaromatic ring and a heteroaromatic ring, a condensed ring system formed by a heteroaromatic ring and an aromatic ring, and the like.

In the present application, the term "halogen" generally refers to fluorine, chlorine, bromine, iodine, for example, it can be fluorine, chlorine.

In the present application, the term "each independently" means that at least two groups (or fragments) with the same or similar value ranges in the structure may have the same or different meanings in specific circumstances. For example, substituent X and substituent Y are each independently hydrogen, halogen, hydroxyl, cyano, alkyl or aryl. When substituent X is hydrogen, substituent Y can be either hydrogen or halogen, hydroxyl, cyano, alkyl or aryl; similarly, when substituent Y is hydrogen, substituent X can be either hydrogen or halogen, hydroxyl, cyano, alkyl or aryl.

In this application, the term "optional" or "optionally" generally means that the subsequently described event or circumstance may but need not occur, and the description includes occasions where the event or circumstance occurs or does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that the alkyl group may but need not be present, and the description may include situations where the heterocyclic group is substituted with an alkyl group and situations where the heterocyclic group is not substituted with an alkyl group.

In the present application, the term "replacement" and its other variant forms in this article refer to that one or more (such as 1,2,3 or 4) atoms or atomic groups (such as hydrogen atoms) on the specified atom are replaced by other equivalents, provided that the normal valence of the specified atom or atomic group in the current situation is not exceeded, and stable compounds can be formed. If a certain atom or atomic group is described as "optionally replaced by...", it can be substituted or unsubstituted. Unless otherwise indicated, the attachment site of the substituent herein can be from any suitable position of the substituent. When the connecting bond in the substituent is shown as a chemical bond between two atoms connected to each other through the ring system, it means that the substituent can be connected to any ring-forming atom in the ring system.

In the present application, the term 0 or more (e.g., 0 or more than 1, 0 or 1, 0) methylene units are "replaced" generally means that when the structure contains 1 or more methylene units, the one or more methylene units may be unsubstituted or replaced with one or more groups described herein (e.g., -NHC(O)-, -C(O)NH-, -C(O)-, -OC(O)-, -C(O)O-, -NH-, -O-, -S-, -SO-, -SO ₂ -, -PH-, -P(＝O)H-, -NHSO ₂ -, -SO ₂ NH-, -C(＝S)-, -C(＝NH)-, -N＝N-, -C＝N-, -N＝C-, or -C(＝N ₂ )-, etc.).

This article uses wavy lines The bonds in the structural formula represented are intended to indicate that the structure represents either a cis or trans isomer, or a mixture of cis and trans isomers in any ratio.

The term "oxo," as used herein alone or in combination with other groups, refers to =0.

In the present application, one or more hydrogen atoms, for example up to 5, for example 1 to 3 hydrogen atoms, in a group are replaced independently of one another by a corresponding number of substituents. The substituents are only in their possible chemical positions, and a person skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g. olefinic) bond.

In the present application, the term "amino acid" includes natural amino acids and non-natural amino acids, and the writing of conventional amino acids follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this article, the terms "polypeptide" and "protein" have the same meaning and can be used interchangeably. And in the present application, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala; arginine can be represented by R or Arg; glycine can be represented by G or Gly; glutamine can be represented by Q or Gln;

In this application, the term "unnatural amino acid" has the following structure: wherein r is selected from 0, ₁ , ₂ , 3, 4, ₅ ; wherein ^Ra and ^Rb are each independently selected from -C1-6alkyl-NH2, _-C1-6alkyl -NH- _C1-6alkyl , _-C1-6alkyl -N( _C1-6alkyl ) ₂ , _-C1-6alkyl -NH-C3-10cycloalkyl, _-C1-6alkyl -N(3-10 membered cycloalkyl)( _C1-6alkyl ), _-C1-6alkyl- -C _1-6 alkyl-S(O) 2 -C _1-6 alkyl, -C _1-6 alkyl-S(O) _{2 -C 3-10} cycloalkyl, -C _1-6 alkyl-S(O) _{2 -NH 2} , -C _1-6 alkyl _{-COOH, -C 1-6 alkyl-CONH 2 , -C 1-6 alkyl-CONHC 1-6 alkyl , -C 1-6 alkyl - CO ( 3-10 membered heterocycloalkyl )} , The alkyl, cycloalkyl and heterocycloalkyl groups are each optionally substituted by one or more substituents selected from H, halogen, -OH, -NH ₂ , -SH, -NO ₂ , CN, -COOH and oxo groups; or any of ^Ra and ^Rb together with the atoms to which they are connected form a 3-10 membered heterocycloalkyl or 3-10 membered cycloalkyl group; the cycloalkyl and heterocycloalkyl groups are each optionally substituted by one or more substituents selected from H, halogen, -OH, -NH ₂ , -SH, -NO ₂ , CN, -COOH and oxo groups;

In the present application, the term "compound" generally refers to a substance having two or more different elements. For example, the compound of the present application may be an organic compound, for example, the compound of the present application may be a compound with a molecular weight of less than 500 Daltons, a compound with a molecular weight of less than 1000 Daltons, a compound with a molecular weight of more than 1000 Daltons, or a compound with a molecular weight of more than 10000 Daltons or more than 100000 Daltons. In the present application, a compound may also refer to a compound connected by chemical bonds, for example, a compound in which one or more molecules with a molecular weight of less than 1000 Daltons are connected to a biomacromolecule by chemical bonds, and the biomacromolecule may be a high polysaccharide, protein, nucleic acid, polypeptide, etc. For example, the compound of the present application may include a compound in which a protein is connected to one or more molecules with a molecular weight of less than 1000 Daltons, a compound in which a protein is connected to one or more molecules with a molecular weight of less than 10000 Daltons, or a compound in which a protein is connected to one or more molecules with a molecular weight of less than 10000 Daltons.

In the present application, the term "stereoisomer" means an isomer formed by at least one asymmetric center. In compounds with one or more (e.g., one, two, three, or four) asymmetric centers, it can produce racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. Specific individual molecules can also exist as geometric isomers (cis/trans). Similarly, the compounds of the present invention can exist as mixtures (commonly referred to as tautomers) of two or more structurally different forms in rapid equilibrium. Representative examples of tautomers include keto-enol tautomers, phenol-ketone tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is to be understood that the scope of the present application covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%).

In this article, solid lines can be used Solid wedge Virtual wedge Carbon-carbon bonds of the compounds of the invention are depicted. The use of solid lines to depict bonds to asymmetric carbon atoms is intended to indicate that all possible stereoisomers at that carbon atom are included (e.g., specific enantiomers, racemic mixtures, etc.). The use of solid or dashed wedges to depict bonds to asymmetric carbon atoms is intended to indicate that the stereoisomer shown is present. When present in a racemic mixture, the solid and dashed wedges are used to define relative stereochemistry, not absolute stereochemistry. Unless otherwise indicated, the compounds of the invention are intended to exist in the form of stereoisomers, which include cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, atropisomers, and mixtures thereof. The compounds of the invention may exhibit more than one type of isomerism and consist of mixtures thereof (e.g., racemic mixtures and diastereomeric pairs).

In the present application, the term "comprising" generally refers to including the features explicitly specified, but not excluding other elements. The terms "above" and "below" generally refer to the case where the number is inclusive.

In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

Unless otherwise indicated, the structures described herein may also include compounds that differ only in the presence or absence of one or more isotopically enriched atoms. For example, compounds that are consistent with the structures of the present application except that a hydrogen atom is replaced by deuterium or tritium, or a carbon atom is replaced by carbon 13 or carbon 14, are within the scope of the present application.

The terms "active ingredient," "therapeutic agent," "active substance," or "active agent" refer to a chemical entity that is effective in treating one or more symptoms of a target disorder or condition.

As used herein, the term "effective amount" (e.g., "therapeutically effective amount" or "prophylactically effective amount") refers to the amount of active ingredient that, after administration, will achieve the desired effect to some extent, such as alleviating one or more symptoms of the condition being treated or preventing the occurrence of the condition or its symptoms.

As used herein, unless otherwise indicated, the terms "treat," ...

As used herein, "individual" includes humans or non-human animals. Exemplary human individuals include human individuals (referred to as patients) suffering from diseases (e.g., diseases described herein) or normal individuals. "Non-human animals" in the present invention include all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock and/or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

Those skilled in the art will appreciate that, since nitrogen requires available lone pairs of electrons to be oxidized to oxides, not all nitrogen-containing heterocycles can form nitrogen oxides. Those skilled in the art will recognize nitrogen-containing heterocycles that can form nitrogen oxides. Those skilled in the art will also recognize that tertiary amines can form nitrogen oxides. The synthetic method for preparing the nitrogen oxides of heterocycles and tertiary amines is well known to those skilled in the art, including oxidizing heterocycles and tertiary amines with peroxyacids such as peracetic acid and metachloroperbenzoic acid (mCPBA), hydrogen peroxide, alkyl hydroperoxides such as tert-butyl hydroperoxide, sodium perborate and dioxirane such as dimethyl dioxirane. These methods for preparing nitrogen oxides have been widely described and reviewed in the literature, see, for example: T.L.Gilchrist, Comprehensive Organic Synthesis, vol.7, pp.748-750 (A.R.Katritzky and A.J.Boulton, Eds., Academic Press); and G.W.H.Cheeseman and E.S.G.Werstiuk, Advances in Heterocyclic Chemistry, vol.22, pp.390-392 (A.R.Katritzky and A.J.Boulton, Eds., Academic Press).

The present invention also encompasses compounds of the present invention containing protecting groups. In any process for preparing the compounds of the present invention, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules involved, thereby forming a chemically protected form of the compounds of the present invention. This can be achieved by conventional protecting groups, for example, those described in T.W.Greene & P.G.M.Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 2006, which references are incorporated herein by reference. Protecting groups may be removed at an appropriate subsequent stage using methods known in the art.

The present invention also encompasses methods for preparing the compounds described herein. It should be understood that the compounds of the present invention can be synthesized using the methods described below and synthetic methods known in the field of synthetic organic chemistry or variations thereof known to those skilled in the art. Preferred methods include (but are not limited to) those described below. The reaction can be carried out in a solvent or solvent mixture suitable for the reagents and materials used and suitable for achieving the transformation.

Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

The reagents and raw materials used in the present invention are commercially available.

The beneficial technical effects of the present invention are that the compounds of the present invention:

(1) It has inhibitory activity on the proliferation of tumor cells in vitro;

(2) Having plasma stability;

(3) Has tumor-suppressing effect in vivo;

(4) Has a bystander effect;

(5) Possessing the ability to resist transporter transport;

(6) Possessing the ability to target tumors in vivo;

(7) Has good in vivo safety.

In addition, the coupling method disclosed in the present disclosure has a wide range of applications and can be widely used for coupling with bioactive molecules such as antibodies or targeted small molecule ligands. In summary, the cytotoxic agent, linker, antibody and ADC of the present invention have great clinical value.

Preparation method:

In order to achieve the synthesis purpose of the present disclosure, the present application adopts the following synthesis technology scheme:

Scheme 1: A method for preparing a compound represented by formula (Ia), formula (IIa) and a ligand-drug conjugate represented by formula (IIIa) or a pharmaceutically acceptable salt or solvate thereof, the method comprising:

in,

Ab, q, L ^1a are as defined in formula (IIIa);

L ¹ , L ² , L ³ , and Tr are as defined in formula (IIa);

R ^1a , R ^2a , and R ^3a are as defined in formula (Ia);

X' is a halogen; preferably, X' is selected from Cl, Br;

Lg ^Tr and Lg ¹ are leaving groups selected from halogen, hydroxyl, -OAc, etc.

Pg ² is a protecting group selected from: Boc, Fmoc, Cbz, Sem, etc.

Step 1 : Compound Ia-1 and compound E are cyclized under acidic conditions and heated in a solvent (eg, 80-130° C.) to obtain compound Ia-2.

In some embodiments, the solvent is selected from methanol, tetrahydrofuran, DMF, NMP, DMSO, toluene, n-hexane, n-heptane, etc.; preferably toluene.

In some embodiments, the reagent providing acidic conditions is selected from PTSA, acetic acid, hydrochloric acid, sulfuric acid, InCl ₃ , p-toluenesulfonic acid, formic acid, trifluoroacetic acid; preferably acetic acid, hydrochloric acid, InCl ₃ .

Step 2 : Compound Ia-2 is reacted with an acetylating agent (such as acetyl chloride or acetic anhydride) in acetic acid to obtain compound Ia-3.

Step 3 : Compound Ia-3 is oxidized in the presence of an oxidant in a polar solvent (such as dichloromethane, chloroform, ethyl acetate, etc.) to obtain compound Ia-4.

In some embodiments, the oxidant in step 3 is m-CPBA, oxone, urea peroxide, hydrogen peroxide, etc., preferably m-CPBA.

Step 4 : Compound Ia-4 is reacted in a suitable organic solvent (eg, toluene, DMF, NMP, DMSO, chloroform, etc.) in the presence of a halogenating agent to obtain compound Ia-5.

In some embodiments, the halogenating agent in step 4 is selected from oxalyl chloride, oxalyl bromide, thionyl chloride, phosphorus oxychloride, phosphorus oxybromide, etc., preferably oxalyl chloride.

Step 5 : Compound Ia-5 undergoes substitution reaction in a suitable organic solvent (e.g., acetonitrile, DMF, tetrahydrofuran, dioxane, dichloromethane, NMP, DMSO) in the presence of an organic base (e.g., DIPEA, triethylamine, DBU, pyridine, N-methylmorpholine, etc.) or an inorganic base (e.g., potassium carbonate, sodium carbonate, sodium bicarbonate, cesium carbonate, potassium phosphate, etc.) to obtain compound Ib-6.

Step 6 : Compound Ia-6 is subjected to reduction of the nitro group in the presence of a reducing agent (e.g., palladium carbon, iron powder/acetic acid, zinc powder/ammonium chloride, ammonium sulfide, _SnCl2 , etc.) in a suitable solvent (e.g., methanol, ethanol, ethyl acetate, dichloromethane, tetrahydrofuran, dioxane, etc.) to obtain a compound of formula (Ia-7).

Step 7 : Compound Ia-7 is deacetylated in the presence of an inorganic base (e.g., sodium carbonate, potassium carbonate, cesium carbonate, etc.) or an organic base (e.g., DBU, N-methylmorpholine, etc.) in a suitable solvent (e.g., methanol, ethanol, DMF, tetrahydrofuran, dioxane, etc.), and then acidified with an acid (e.g., hydrochloric acid, trifluoroacetic acid, etc.) to obtain a compound of formula (Ia).

Step 8 : The compound of formula (Ia) and compound F are reacted in the presence of a condensation agent through a condensation reaction or a substitution reaction to obtain a compound of formula (IIa).

Step 8a : The compound of formula (Ia) and compound Fa are reacted by condensation reaction or substitution reaction in the presence of a condensation reagent; then the protecting group is removed to obtain compound Ia-8.

Step 8b : Compound Ia-8 and compound L ¹ -Lg ¹ are reacted in the presence of a condensation agent through a condensation reaction or a substitution reaction to obtain a compound of formula (IIa).

In some embodiments, the condensing agent is selected from 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, O-benzotriazole- N,N,N',N'-tetramethyluronium hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate or benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, preferably 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride or 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

Step 9 : The compound of formula (IIa) reacts with Ab after disulfide bond reduction to obtain a ligand-drug conjugate of formula (IIIa).

In some embodiments, the reducing agent for reducing Ab includes but is not limited to tris(2-carboxyethyl)phosphine, mercaptoethanol, dithiothreitol, cysteine, reduced glutathione, etc.; in particular, it is preferred to reduce the disulfide bonds on the antibody;

Scheme 2: A method for preparing a compound represented by formula (Ib), formula (IIb), a ligand-drug conjugate represented by formula (IIIb), or a pharmaceutically acceptable salt or solvate thereof, the method comprising:

in,

Ab, q, L ^1a are as defined in formula (IIIa);

X' is a halogen; preferably, X' is selected from Cl, Br;

X is selected from: -O-, -N(R ^3b-2 )-;

L ¹ , L ² , L ³ , and Tr are as defined in formula (IIa);

R ^1b , R ^2b , R ^3b-1 , R ^3b-2 and R ^4d are as defined in formula (Ib), and t is an integer selected from 1-10.

The specific preparation method of formula (Ib) is described in Scheme 1.

The specific preparation methods of formula (IIb) and (IIIb) are carried out by referring to the methods described in step 8, step 8a, step 8b and step 9 of scheme 1 respectively.

Those skilled in the art will appreciate that, depending on the desired product structure, one or more steps in the above-described preparation method may be omitted, and the order of the reaction steps may be appropriately adjusted as well as protection/deprotection reaction steps may be added or omitted as needed.

DETAILED DESCRIPTION

The present invention includes all combinations of the described specific embodiments. Further embodiments of the present invention and the full scope of applicability will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific examples indicate preferred embodiments of the present invention, these descriptions and examples are provided only by way of illustration, because various changes and modifications within the spirit and scope of the present invention will become apparent to those familiar with the art from this detailed description. For all purposes, all publications, patents and patent applications cited herein, including citations, will be incorporated herein by reference in their entirety. The present invention is further described below by way of example, but the present invention is not therefore limited to the scope of the described embodiments. The experimental methods for which specific conditions are not indicated in the following examples are selected according to conventional methods and conditions, or according to the product specifications.

Mass spectrometry (MS) was measured using an Agilent (ESI) mass spectrometer, manufacturer: Agilent, model: Agilent 6120B.

Preparative high performance liquid chromatography (HPLC) was performed using a Shimadzu LC-8A preparative liquid chromatograph (YMC, ODS, 250×20 mm column).

Thin layer chromatography purification was performed using GF 254 (0.4-0.5 nm) silica gel plates produced in Yantai.

The reaction is monitored by thin layer chromatography (TLC) or liquid chromatography-mass spectrometry (LC-MS), and the developing solvent system used includes but is not limited to: dichloromethane and methanol system, n-hexane and ethyl acetate system, and petroleum ether and ethyl acetate system. The volume ratio of the solvent is adjusted according to the polarity of the compound, or adjusted by adding triethylamine or the like.

Column chromatography generally uses Qingdao Ocean 200-300 mesh silica gel as the stationary phase. The eluent system includes but is not limited to the dichloromethane and methanol system and the n-hexane and ethyl acetate system. The volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of triethylamine can also be added for adjustment.

If not otherwise specified in the examples, the reaction temperature is room temperature (20°C to 30°C).

Unless otherwise specified, the reagents used in the examples were purchased from Acros Organics, Aldrich Chemical Company, Nanjing Yao Shi Technology, or Shanghai Shuya Pharmaceutical Technology.

The above-described embodiments do not limit the scheme of the present application in any way. Except those described herein, according to the foregoing description, multiple modifications of the present invention will be apparent to those skilled in the art. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in the present application (including all patents, patent applications, journal articles, books and any other disclosures) is incorporated by reference in its entirety.

In conventional synthesis methods, preparation examples, embodiments and intermediate synthesis examples, the meanings of the abbreviations are shown in the following table.

Example 1: Preparation of Camptothecin Compounds

Example 1.1: Preparation of compound A1.

Step 1: Preparation of compound A1-2.

Compound A1-1 (6 g, 36.13 mmol) and compound E (6 g, 22.81 mmol) were placed in a 2L three-necked flask, protected by nitrogen, 600 mL of glacial acetic acid was added to dissolve, and then 120 mL of concentrated hydrochloric acid was added, and refluxed at 125°C for 16 h. The reaction solution was then dried and purified by column chromatography to obtain compound A1-2 (8.6 g, yield 95.9%).

Step 2: Preparation of compound A1-3.

Compound A1-2 (8.60 g, 21.87 mmol) was placed in a 500 mL three-necked flask, 123 mL of glacial acetic acid was added to dissolve, acetyl chloride (20.60 g, 262.52 mmol) was added under nitrogen protection, and the mixture was heated at 75°C for 1 h. The reaction solution was dried by rotary evaporation, and the residue was purified by column chromatography to obtain compound A1-3 (9.4 g, 98.7%).

Step 3: Preparation of compound A1-4.

Compound A1-3 (9.4 g, 21.6 mmol) was placed in a 250 mL single-mouth bottle, 108 mL DCM was added to dissolve, and m-CPBA (13.15 g, 64.81 mmol) was added after cooling to 0 °C. After the addition, the mixture was returned to room temperature for 1 h. Saturated sodium bicarbonate aqueous solution was then added dropwise to the reaction solution to adjust the pH to about 8, the layers were separated, the organic phase was collected, the aqueous phase was extracted with DCM (80 mL × 3), the organic phases were combined, washed once with saturated brine and dried over anhydrous sodium sulfate, the organic phase was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain compound A1-4 (7.5 g, yield 78.9%).

Step 4: Preparation of compound A1-5.

Compound A1-4 (3 g, 6.65 mmol) was placed in a 100 mL single-mouth bottle, 38 mL DMF was added to dissolve, the temperature was lowered to 0°C, and 8.3 mL oxalyl chloride (2 M DCM solution) was slowly added dropwise, and the temperature was restored to room temperature for 2 h. The reaction solution was poured into 400 mL water, extracted with DCM (100 mL × 4), the organic phase was washed once with 150 mL, and dried by spin drying. The residue was purified by column chromatography to obtain compound A1-5 (2.4 g, yield 77.1%).

Step 5: Preparation of compound A1-6.

Compound A1-5 (1.2 g, 2.55 mmol), 3-methylbutan-1-amine (267 mg, 3.06 mmol), and K ₂ CO ₃ (1.06 mg, 7.67 mmol) were placed in a 100 mL single-mouth bottle, 1,4-dioxane was added to dissolve, and stirred at 80° C. for 16 h. The reaction solution was spin-dried, and the residue was purified by column chromatography to obtain compound A1-6 (0.42 g, yield 34.7%).

Step 6: Preparation of compound A1-7.

Compound A1-6 (360 mg, 0.69 mmol) and 10% Pd/C (360 mg) were placed in a 25 mL single-necked bottle, and 4 mL of DCM/MeOH (10:1) mixed solvent was added to dissolve, and the mixture was reacted at 25 °C in a hydrogen atmosphere for 16 h. The reaction solution was filtered to remove Pd/C, and the organic phase was concentrated under reduced pressure to obtain compound A1-7 (317 mg crude product).

Step 7: Preparation of compound A1.

Compound A1-7 (310 mg, 0.63 mmol) was placed in a 50 mL single-mouth bottle, 15 mL of methanol was added to dissolve, and 3 mL of K ₂ CO ₃ (0.63 M) aqueous solution was added, stirred at room temperature for 2 h, and then adjusted to pH 4 with 5 M dilute hydrochloric acid, and stirred for 10 min. The reaction solution was spin-dried, and the crude product was subjected to HPLC to obtain compound A1 (22 mg, yield 7.7%).

MS m/z(ESI):449.2[M+H] ⁺

¹ H-NMR (400MHz, DMSO-d ₆ ): δ7.72 (d, J = 9.0Hz, 1H), 7.59 (s, 1H), 7.39–7.27 (m, 2H), 6.66 (s, 1H), 5.51(s,2H),5.44(s,2H),3.74(d,J＝7.4Hz,2H),1.92–1.72(m,3H),1.65(dd,J＝14.8,6.9Hz,2H),0.99 (d, J＝6.6Hz, 6H), 0.88 (dd, J＝9.7, 5.1Hz, 3H).

Example 1.2: Preparation of compound A13.

Compound A13 was synthesized by a similar method as described in Steps 5 to 6 of Example 1.1.

MS m/z(ESI):423.2[M+H] ⁺

Example 1.3: Preparation of Compound A14

Compound A14 was synthesized by a similar method as described in the fifth to sixth steps of Example 1.1.

MS m/z(ESI):437.2[M+H] ⁺

¹ H-NMR (400MHz, DMSO-d ₆ ): δ14.38 (s, 1H), 8.62 (s, 1H), 7.72 (d, J = 9.2Hz, 1H), 7.61 (brs, 1H), 7.38– 7.35(m,1H),7.38–7.35(m,1H),7.29(d,J＝1.6Hz,1H),6.67(s,1H),5.56(s,2H),5.43(d,J＝1.6Hz ,2H),3.85(q,J＝6.4Hz,2H),3.58(t,J＝8.0Hz,2H),1.98–1.80(m,4H),0.87(t,J＝7.2Hz,3H).

According to the method of Example 1.1, the following compounds were synthesized using appropriate starting materials.

Example 1.2: Preparation of compound B1.

Steps 1 to 5: Preparation of compound B1-6.

Compound B1-6 was synthesized by a similar method as described in the first to fifth steps of Example 1.1.

Step 6: Preparation of compound B1.

Compound B1-6 (305 mg, 0.5 mmol) was placed in a 50 mL single-mouth bottle, 15 mL of methanol was added to dissolve, and then 3 mL of K ₂ CO ₃ (0.63 M) aqueous solution was added, stirred at room temperature for 2 h, and then the pH was adjusted to about 4 with 5 M dilute hydrochloric acid, and stirred for 20 min. The reaction solution was spin-dried, and the crude product was purified by reverse phase HPLC to obtain compound B1 (4 mg, yield 17.6%).

MS m/z(ESI):454.2[M+H] ⁺

Referring to the method of Example 1.4, the following compounds were synthesized using appropriate starting materials.

Example 2: Preparation of linker intermediate

Example 2.1: Preparation of intermediate Int1

Compound Int9-1 (500 mg, 1.42 mmol) was dissolved in DMF (5 ml), HATU (810 mg, 2.13 mmol) and DIEA (549 mg, 4.26 mmol) were added, and then stirred at room temperature for 30 minutes, and compound Int9-2 (369 mg, 1.42 mmol) was added, and the reaction was continued for 2 hours. After the reaction was completed, ethyl acetate was added, and the pH was adjusted to about 6 with 2M citric acid under stirring, and the liquid was separated, and the organic phase was washed twice with saturated brine, and the organic phase was dried, concentrated, and prepared and purified by reverse phase HPLC to obtain compound Int9 (422 mg, yield 50%).

The following intermediates were synthesized by referring to the method of Example 2.1:

Example 3: Preparation of linker-payload for ligand drug conjugates

Example 3.1: Preparation of Compound AA1

Compound Int2 (100 mg, 0.28 mmol), compound A1 (125 mg, 0.28 mmol), HATU (159 mg, 0.42 mmol), and DIPEA (108 mg, 0.84 mmol) were dissolved in DMF (5 mL) and stirred at room temperature for 12 h. The residue was added into water and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The crude product was purified by reverse phase HPLC to obtain compound AA1 (110 mg, yield 50%).

MS: [M+H] ⁺ , 784.4.

Example 3.2: Preparation of Compound AA15

Step 1: Preparation of compound AA15-1

Compound A1 (125 mg, 0.28 mmol), compound Int9 (105 mg, 0.28 mmol), HATU (159 mg, 0.42 mmol), and DIPEA (108 mg, 0.84 mmol) were dissolved in DMF (5 mL) and stirred at room temperature for 12 h. After the reaction was completed, water was added, extracted with ethyl acetate, separated, the organic phase was dried over anhydrous sodium sulfate and concentrated to dryness, and the crude product was purified by column chromatography to obtain compound AA15-1 (200 mg, yield 74%).

Step 2: Preparation of compound AA15-2

Compound AA15-1 (200 mg, 0.21 mmol) was dissolved in piperidine/dichloromethane (5 mL, 1/5) and stirred at room temperature for 1 h. After the reaction was completed, ethyl acetate was added and the liquids were separated. The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain a crude compound AA15-2 (168 mg).

Step 3: Preparation of compound AA15-3

Compound AA15-2 (168 mg, 0.21 mmol), 2-(methylsulfonyl)pyrimidine-5-carboxylic acid (42 mg, 0.21 mmol), and HATU (121 mg, 0.32 mmol) were dissolved in anhydrous DMF (5 mL) and stirred at room temperature for 12 h. After the reaction was completed, water was added, extracted with ethyl acetate, separated, the organic phase was dried over anhydrous sodium sulfate and concentrated to dryness, and the residue was purified by reverse phase HPLC to obtain compound AA15-3 (50 mg, yield 24%).

Step 4: Preparation of Compound AA15

Compound AA15-3 (50 mg, 0.05 mmol) was dissolved in TFA/dichloromethane (3 mL, 1/5) and stirred at room temperature for 1 h. After the reaction was completed, the mixture was concentrated to dryness and the residue was purified by reverse phase HPLC to obtain compound AA15 (5 mg, yield 10%). MS: [M+H] ⁺ , 932.3.

The following compounds were synthesized according to the methods of Examples 3.1 and 3.2.

Example 3.3: Preparation of Compound BB1

Step 1: Preparation of compound B1-1

Compound B1 (500 mg, 1.150 mmol), [[2-(FMOC-amino)acetylamino]methyl]acetate (2.12 g, 5.75 mmol), p-toluenesulfonic acid (20 mg, 0.115 mmol) were dissolved in DMAc (10 mL), heated to 50° C. and reacted for 17 h. After the reaction was complete as detected by LCMS, it was cooled to room temperature, dichloromethane and water were added, stirred and separated, the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was prepared and purified by reverse phase HPLC to obtain compound B1-1 (600 mg, yield 70%).

Step 2: Preparation of compound B1-2

Compound B1-1 (600 mg, 0.80 mmol) was dissolved in piperidine/dichloromethane (10 mL, 1/5) and stirred at room temperature for 1 h. After the reaction was completed, ethyl acetate and water were added, the liquids were separated, and the organic phase was dried over anhydrous sodium sulfate and concentrated to obtain a crude compound B1-2 (420 mg).

Step 3: Preparation of compound BB1

Compound BB1 was synthesized by referring to the method of Example 3.1. MS: [M+H] ⁺ , 980.4.

Example 3.4: Preparation of Compound BB17

Step 1: Preparation of compound BB17-1

Compound Int15 (200 mg, 0.41 mmol), B1-2 (215 mg, 0.41 mmol), HATU (233 mg, 0.62 mmol), and DIPEA (158 mg, 1.23 mmol) were dissolved in DMF (8 mL) and stirred at room temperature for 12 h. After the reaction was completed, water was added, extracted with ethyl acetate, separated, the organic phase was dried over anhydrous sodium sulfate and concentrated to dryness, and the crude product was purified by column chromatography to obtain compound BB17-1 (300 mg, yield 73%).

Step 2: Preparation of compound BB17

Compound BB17 was synthesized by referring to the method described in Example 3.2, Steps 2 to 3. MS: [M+H] ⁺ , 1084.4.

The following compounds were synthesized by referring to the methods described in Examples 3.3 and 3.4.

Example 4: Preparation of ligand drug conjugates

The antibody used as the ligand is prepared according to conventional methods, for example, after constructing the vector, it can be transfected into eukaryotic cells such as HEK293 cells and CHO cells for purification and expression. Trastuzumab and Sacituzumab are used as examples to prepare ligand-drug conjugates.

Amino acid sequence of trastuzumab:

Light chain (SEQ ID NO: 10)

Heavy chain (SEQ ID NO:9)

Amino acid sequence of certolizumab pegol:

Light chain (SEQ ID NO: 20)

Heavy chain (SEQ ID NO: 19)

Example 4.1: Preparation of ADC-1A

At 37°C, add prepared tri(2-carbonylethyl)phosphine hydrochloride (10mM, 0.135mL, 1.35μmol) to the buffer of antibody trastuzumab (14.0mM succinic acid-sodium hydroxide + 108mM NaCl pH 6.0; 20mg, 10.0mg/mL, 0.135μmol), place in a water bath oscillator, oscillate at 37°C for 3 hours, stop the reaction, and use 14.0mM succinic acid-sodium hydroxide + 108mM NaCl pH 6.0 buffer to ultrafilter and replace to remove excess TCEP;

Compound AA1 (1.29 mg, 1.65 μmol) was dissolved in 0.2 mL DMSO, added to the above solution, placed in a water bath oscillator, oscillated at 22°C for 2 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 20 mM histidine-hydrochloric acid pH 5.5) to obtain a solution of the exemplary product ADC-1A (20 mM histidine-hydrochloric acid pH 5.5; 18.4 mg, 4.96 mg/mL, yield: 92%), which was stored at 4°C.

The DAR value q was 7.82 as detected and calculated by LC-MS analysis.

Referring to the method of Example 4.1, the following compounds were synthesized using appropriate linker-payloads.

Example 5: In vitro tumor cell proliferation inhibition test of compounds

Purpose of the test

In order to detect the inhibitory activity of drug compounds on the proliferation of NCI-N87 cells, JIMT-1 and MBA-MB-231 tumor cells in vitro, cells were treated with different concentrations of compounds in vitro and cultured for 6 days. The cell proliferation was detected by using Luminescent Cell Viability Assay (Promega, Catalog No.: G7558), and the in vitro activity of the compound was evaluated based on the IC ₅₀ value.

Experimental methods

The following is an example of the in vitro proliferation inhibition test method for NCI-N87 cells to illustrate the method for testing the in vitro proliferation inhibition activity of the compounds of the present application on tumor cells in the present application. The present method is also applicable to, but not limited to, in vitro proliferation inhibition activity tests on other tumor cells.

1. Cell culture: NCI-N87 was cultured in 10% FBS RPMI-1640 medium.

2. Cell preparation: Take NCI-N87 cells in the logarithmic growth phase, wash once with PBS, add 2-3 ml of trypsin to digest for 2-3 minutes, after the cells are completely digested, add 10-15 ml of cell culture medium to wash out the digested cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and then add 10-20 ml of cell culture medium to resuspend the cells to make a single cell suspension.

3. Cell plating: Mix the NCI-N87 single cell suspension, adjust the live cell density to 6x10 ⁴ cells/ml with cell culture medium, mix the cell suspension after density adjustment, and add 50ul/well to a 96-well cell culture plate. Incubate the culture plate in an incubator for 18 hours (37°C, 5% CO ₂ ).

4. Compound preparation: Dissolve the compound in DMSO and prepare a storage solution with an initial concentration of 10 mM.

There are 8 concentrations of small molecule compounds, namely: 300, 100, 30, 10, 3, 1, 0.3, and 0.1 nM.

5. Sample addition operation: Add the samples to be tested of different concentrations to the culture plate, with two replicate wells for each sample. Incubate the culture plate in an incubator for 6 days (37° C., 5% CO ₂ ).

6. Color development operation: Take out the 96-well cell culture plate, add 50ul CTG reagent to each well, and incubate at room temperature for 10 minutes.

7. Plate reading operation: Take out the 96-well cell culture plate, place it in an ELISA reader, and measure the chemiluminescence using the ELISA reader.

Data analysis

The data were processed and analyzed using Microsoft Excel and Graphpad Prism 5.

Table 3. IC ₅₀ values of the small molecule fragments in the present application for inhibiting proliferation of NCI-N87 cells in vitro.

Conclusion: According to the results in Table 3, the small molecule fragments in the present application have obvious proliferation inhibitory activity on NCI-N87 cells, JIMT-1 cells, and MDA-MB-231 cells.

Example 6: In vitro cell proliferation inhibition activity test of antibody-drug conjugates

Implementation: 6.1: NCI-N87/JIMT-1 cell in vitro proliferation inhibition activity test

use The chemiluminescent cell viability assay (CTG method) was used to evaluate the inhibitory effect of the anti-Her2 antibody trastuzumab (Trastuzumab) coupled to a camptothecin compound ADC drug on cell proliferation in Her2-positive human gastric cancer cells NCI-N87 and human breast cancer cells JIMT-1 after incubation for 6 days.

Cells in logarithmic growth phase were collected and plated in a 96-well cell culture plate at a density of 6000 cells/well. The cell plate was placed in a 37°C, 5% CO ₂ incubator for overnight culture. On the second day of the experiment, the ADC drug of camptothecin compounds was diluted 3 times with complete culture medium to obtain 9 concentration gradients (starting with the highest concentration of 300nM). After the drug, 100μL/well was added to the cell culture plate, and the complete culture medium was used as a blank control. Three replicates were set up; the incubation continued in a 37°C, 5% CO ₂ incubator for 6 days. After the incubation, the cell culture plate was removed, equilibrated to room temperature, and 50μL CTG detection reagent (Promega, Cat#: G7573) was added to each well. After shaking and mixing, it was placed in the dark for 10 minutes, and the signal value was read using an enzyme marker. GraphPad Prism software was used to draw an S-type dose-response curve using a nonlinear regression model and calculate the IC ₅₀ value. The cell viability calculation formula = (Lum _{test drug} - Lum _{blank control} ) / (Lum _{solvent blank control -} Lum _{blank control} ) × 100%.

Experimental conclusion: The antibody-drug conjugate of the present application has significant proliferation inhibitory activity on Her2-positive human gastric cancer cell NCI-N87 and human breast cancer JIMT-1 cells.

Example 7: In vivo tumor inhibition test of antibody drug conjugates

In order to evaluate the inhibitory effect of the ADC drug of the present invention on tumor formation in vivo, after forming transplanted tumors in mice using Her2-positive human breast cancer cells JIMT-1, the anti-tumor effect of the ADC drug of the present invention was evaluated.

Evaluation of the efficacy of antibody-drug conjugates in JIMT-1 human breast cancer cell-bearing mice

1. Test drugs and materials

Blank control group (control group): normal saline

ADC (treatment group): 5 mg/kg, single dose

2. Preparation method: All samples were diluted with physiological saline.

3. Experimental animals: 8-week-old female BALB/c-nude mice were purchased from Jicui Pharmaceutical Biotechnology Co., Ltd.

4. Test methods:

1×10 ⁷ JIMT-1 cells were inoculated subcutaneously at the right anterior shoulder of 8-week-old female BALB/c-nude mice. When the tumor grew to about 125 mm ³ , the tumor-bearing mice were randomly divided into groups using StudyDirectorTM and injected with the ADC drug of the present invention intravenously (iv) starting on the same day (day 0), once every 7 days for a total of 2 injections, with a dose of 5 mg/kg. The tumor volume and body weight were measured twice a week and the data were recorded.

There were 5 mice in each vehicle control group or treatment group. The tumor inhibition rate was calculated by measuring the tumor volume. Tumor inhibition rate (TGI%) = 100% - (tumor volume of the treatment group on the day of measurement - tumor volume of the treatment group on day 0) / (tumor volume of the control group on the day of measurement - tumor volume of the control group on day 0).

The experimental results show that the antibody-drug conjugate of the present invention exhibits significant tumor-suppressing activity after a single administration.

Example 8: Antibody Drug Conjugate Plasma Stability Test

To evaluate the plasma stability of the antibody drug conjugate of the present invention, the antibody drug conjugate of the present invention was incubated in human, rat and monkey plasma for 21 days, and samples were taken at 0 hours, 8 hours, 1 day, 4 days, 7 days, 14 days and 21 days to detect the drug ligand coupling rate (DAR value) and free drug.

Incubation of Antibody Drug Conjugates in Plasma

The antibody drug conjugate of the present invention was diluted with plasma to a final concentration of 150 μg/mL and incubated at 37°C in the dark for 21 days. Samples were collected at T0 (the sample was immediately frozen to -70°C after dilution within 30 minutes), 2 hours, 8 hours, day 1 (24 hours), day 4, day 7, day 14, and day 21. All samples were stored in a -70°C refrigerator before analysis. Only the DAR values were analyzed for samples collected at 2 hours and 8 hours.

Analysis of free drugs by LC-MS/MS

Protein precipitation

Plasma samples were thawed and 291.9 μL was taken for free drug analysis. 400 μL of precipitant (acetonitrile solution containing 0.1% formic acid, 200 ng/mL tolbutamide, and 200 ng/mL labetalol) was added to each sample and mixed thoroughly. The mixture was shaken for at least 20 minutes to precipitate the protein in the plasma. The samples were centrifuged at 4°C and 4000 rpm for 20 min, and 150 μL of the supernatant was taken for LC-MS/MS analysis.

The equipment used for free drug analysis is shown in the following table:

The LC and MS parameters are as follows:

LC-MS determination of drug-ligand coupling rate

Immobilized magnetic beads capture antibody drug conjugates

Add 25 μL of streptavidin magnetic beads to each well of a 96-well plate and discard the stock buffer. Elute with 200 μL of PBS buffer and treat with 80 μL of biotin-labeled antigen protein and another 100 μL of PBS buffer at room temperature for 120 minutes. After the antigen protein is completely immobilized, elute twice with 200 μL of PBS buffer. Add 20 μL of antibody drug conjugate plasma sample and 180 μL of PBS buffer to each well, then shake at room temperature for 120 minutes to ensure that the antibody drug conjugate in the plasma is completely captured, and then remove the supernatant. Wash twice with PBS buffer, then add 50 μL of elution buffer (1% formic acid in water) and treat at room temperature for 20 minutes. Then add 5 μL of neutralization buffer (1M NH ₄ HCO ₃ , pH 8.5) and 5 μL of DTT (1M) to reduce the captured antibody drug conjugate at room temperature for 60 minutes, and then analyze by LC-MS.

Drug-ligand coupling rate analysis

The LC-MS parameters are as follows:

The results showed that after the antibody-drug conjugate of the present invention was incubated in plasma for 21 days, no drug or only a very small amount of drug was shed, and the drug-ligand coupling rate did not change significantly. The antibody-drug conjugate of the present invention has extremely high plasma stability, indicating that the antibody-drug conjugate of the present invention has better safety.

Example 9: Stability of Antibody Drug Conjugate Stock Solution (Liquid Preparation)

In order to evaluate the stock solution stability of the antibody drug conjugate of the present invention, the ADC stock solution of the present invention was concentrated to 20 mg/mL, dispensed into 2 mL cryovials, and incubated in a constant temperature box at 40° C. Samples were taken at 0 hour, 1 week, 2 weeks, 1 month, and 2 months for concentration, drug-ligand coupling rate (DAR value), SEC purity and aggregation, CE-SDS (non-reduced) purity, CE-SDS (reduced) purity, charge heterogeneity and free drug detection, and the appearance of the liquid was observed with the naked eye to see whether it was clear and whether there was precipitation.

The sample concentration was detected by UV method.

The drug-ligand coupling rate (DAR value) was detected by hydrophobic HPLC (HIC) or reverse phase HPLC (RP-HPLC).

The drug-ligand coupling rate (DAR value) was detected by LC-MS.

Reverse phase HPLC (RP-HPLC) was used to detect free drugs.

The SEC purity and aggregation of the samples were determined by SEC-HPLC (TOSOH G3000 SW SEC column).

The purity was determined by CE-SDS (non-reduced) and CE-SDS (reduced) detection using Maurice.

The charge heterogeneity was detected by imaging capillary isoelectric focusing (iCIEF).

The results show that after incubation at 40°C for 1 week to 2 months, the concentration, drug ligand coupling rate (DAR), SEC purity and aggregation, CE-SDS (non-reduced) purity, CE-SDS (reduced) purity, charge heterogeneity and free drug of the antibody drug conjugate stock solution of the present invention have no significant changes. The antibody drug conjugate stock solution of the present invention has extremely high stability.

Example 10: Pharmacokinetic and toxicological studies of single or multiple doses in monkeys

After a single or multiple intravenous injections of the antibody-drug conjugate of the present invention are given to monkeys, the pharmacokinetic properties of the drug in the monkeys are investigated, and the toxicity of the animals is observed.

Test methods

Pharmacokinetics: After a single intravenous infusion of different doses of the antibody-drug conjugate of the present invention to monkeys, blood samples were collected at multiple consecutive time points, and the concentration of the drug in the blood was detected by an appropriate specific detection method.

Toxicology study: After single or multiple intravenous infusions of different doses of the antibody-drug conjugate of the present invention to monkeys, the tolerance of the animals to the antibody-drug conjugate of the present invention, as well as drug-related toxicity manifestations, were investigated through clinical observation, body weight and food intake, hematology, blood biochemistry, urine, gross anatomy, histopathology, etc.

The results showed that after single or multiple intravenous infusions of the antibody-drug conjugate of the present invention in monkeys, the pharmacokinetic properties of the total antibody and ADC were similar, and the concentration of free toxin was very low, indicating that the antibody-drug conjugate of the present invention has high stability and good pharmacokinetic properties in vivo. The animals were well tolerated and did not show severe or intolerable drug-related toxicity, indicating that the safety of the antibody-drug conjugate of the present invention is controllable.

Claims (40)

A ligand-drug conjugate, or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate comprises a ligand and: -Structure represented by formula (IVa):
in, The wavy lines indicate direct or indirect attachment to the ligand through the nitrogen atom; R ^1a is selected from hydrogen, halogen, amino, hydroxy, -C _1-6 alkyl and -C _1-6 alkoxy; R ^2a and R ^3a are each independently selected from hydrogen, -(C(R ^2a-1 ) ₂ ) _p -H, -(C(R ^2a-1 ) ₂ ) _p -OR ^2a-2 , -(C(R ^2a-1 ) ₂ ) _p -SR ^2a-2 , -(C(R ^2a-1 ) ₂ ) _p -S(O) ₂ R ^2a-2 , -(C(R ^2a-1 ) ₂ ) _p -N(R ^2a-2 )-S(O) ₂ R ^2a-2 and -C(＝O)R ^2a-2 ; or R ^2a and R ^3a and the atoms to which they are attached together form a 3-10 membered heterocyclic ring; the heterocyclic ring is optionally substituted by one or more selected from halogen, hydroxy, amino, -C _1-6 alkyl, -OC _1-6 alkyl, -NHC _1-6 alkyl, -N(C _{-C 1-6} alkyl) ₂ and -C _1-6 alkylene-OH substituents; p is an integer selected from 0 to 10; R ^2a-1 are each independently selected from hydrogen, halogen, hydroxy, amino, cyano, nitro, -C _1-6 alkyl and -halogenated C _1-6 alkyl; Alternatively, any two R ^2a-1 together with the atoms to which they are attached form an oxo group, a 3-10 membered carbocyclic ring or a 3-10 membered heterocyclic ring, wherein the 3-10 membered carbocyclic ring or the 3-10 membered heterocyclic ring is optionally substituted with -C _1-6 alkyl; R ^2a-2 is selected from hydrogen, hydroxy, amino, cyano, -C _1-6 alkyl, -C _3-6 cycloalkyl and 3-6 membered heterocycloalkyl; each of the alkyl, cycloalkyl and heterocycloalkyl is optionally substituted with one or more substituents selected from hydrogen, halogen, hydroxy, amino and -C _1-6 alkyl; or -Structure represented by formula (IVb):
in, The wavy line indicates direct or indirect connection to the ligand through X; X is selected from: -O-, -N(R ^3b-2 )-; R ^1b , R ^2b are each independently selected from hydrogen, halogen, hydroxy, nitro, -C _1-6 alkyl and -C _1-6 alkoxy; or R ^1b and R ^2b together with the atoms to which they are attached form a 4-6 membered carbocyclic ring or a 4-6 membered heterocyclic ring; t is an integer selected from 1 to 10; R ^3b-1 are each independently selected from hydrogen, halogen, hydroxy, amino, cyano, nitro, -C _1-6 alkyl and -halogenated C _1-6 alkyl; Or, any two R ^3b-1 together with the atoms to which they are attached form an oxo group, a 3-6 membered carbocyclic ring or a 3-6 membered heterocyclic ring; R ^3b-2 is selected from hydrogen, hydroxy, -C _1-6 alkyl, -C _3-6 cycloalkyl, 3 to 6 membered heterocycloalkyl; each of the alkyl, cycloalkyl, heterocycloalkyl is optionally substituted with one or more substituents selected from hydrogen, halogen, hydroxy, amino and -C _1-6 alkyl; R ^4b is selected from hydrogen, hydroxy, -C _1-6 alkyl, -C _3-6 cycloalkyl, -halogenated C _1-6 alkyl. The ligand-drug conjugate according to claim 1, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein the ligand-drug conjugate comprises a ligand and: -Structure represented by formula (Va):
or -Structure represented by formula (Vb):
in, The wavy line indicates the connection to the ligand through L ^1a ; L ^1a is selected from: ^L2 is -(C( ^RL21 ) ₂ ) _n- , Where n is a natural number from 0 to 50. Any -C(R ^L21 ) ₂ - unit in L ² may be independently replaced by the following structural units: -Cy-, -C(O)-, -NR ^L22 -, -O-, -S-, -SO-, -SO ₂ -, -P(R ^L22 )-, -P(＝O)(R ^L22 )-, -C(＝S)-, -C(＝NR ^L22 )-, -N＝N-, -C＝N-, -N＝C-, -Cy- is selected from phenylene, 5- to 8-membered heteroarylene, 3- to 10-membered heterocyclylene, or 3- to 10-membered cycloalkylene, wherein said -Cy- is unsubstituted or independently substituted with 1 or more R ^cx , R ^L21 , R ^L22 , and R ^cx are each independently selected from hydrogen, deuterium, halogen, -NO ₂ , -CN, -OR ^L2a , -SR ^L2a , -N(R ^L2a ) ₂ , -N ⁺ (R ^L2a ) ₃ , -C(O)R ^L2a , -CO ₂ R ^L2a , -C(O)C(O)R ^L2a , -C(O)CH ₂ C(O)R ^L2a , -S(O)R ^L2a , -S(O) ₂ R ^L2a , -C(O)N(R ^L2a ) ₂ , -SO ₂ N(R ^L2a ) ₂ , -OC(O)R ^L2a , -N(R ^L2a )SO ₂ R ^L2b , -N(R ^L2a )COR ^L2b , -(CH ₂ ) _y -CO-(N(Me)CH ₂ C(O)) _m -OR ^L2a , -(CH ₂ ) _y -CO-(N(Me)CH ₂ C(O)) _m -NHR ^L2a , -(CH ₂ ) _y -CO-(N(Me)CH ₂ C(O)) _m -N ⁺ (R ^L2a ) ₃ , -(CH ₂ ) _y -NHCOCH ₂ (OCH ₂ CH ₂ )OR ^L2a , -(CH ₂ ) _y -NH(COCH ₂ (N(Me)) _m -R ^L2a , -(CH ₂ ) _y -CONH-(CH ₂ CH ₂ O) _m -R ^L2a , -(CH ₂ ) _y -NHCO-(CH ₂ CH ₂ O) _m -R ^L2a , -(CH ₂ CH ₂ O) _m -R ^L2a , -(COCH _{2 -R} ^L2a , -COCH ₂ (OCH ₂ CH ₂ ) _m -OR ^L2a , -CO-(CH ₂ CH ₂ O) _m -R ^L2a , -CO-(CH ₂ ) _y -CONH-(CH ₂ CH ₂ O) _m -R ^L2a , -CO-(CH ₂ ) _y -NHCO-(CH ₂ CH ₂ O) _m -R ^L2a , or -C _1-6 ^alkyl , -C _1-6 alkenyl, -C _1-6 alkynyl, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 3-10 membered heteroaryl optionally substituted by R L2a, m, y are natural numbers from 0 to 50, ^RL2a , ^RL2b are each independently selected from hydrogen, deuterium, halogen, _-NO2 , -CN, -OH, -SH, _-NH2 , -N(Me) ₂ , _-CO2H , -S(O) _2Me , _-S (O) _2OH , -C(O) _NH2 , _-SO2NH2 , _-C1-6alkyl , _-C1-6alkenyl , -C1-6alkynyl, 3-8-membered cycloalkyl, 4-10-membered heterocycloalkyl, _6-10 -membered aryl, or 3-10-membered heteroaryl; L ³ does not exist or is an amino acid residue, a short peptide consisting of 2-10 amino acid residues, or any combination of the above groups, wherein the amino acid residue is a natural amino acid residue or a non-natural amino acid residue; Tr does not exist or is or any combination of the above groups; R ^Tr , R ^Tr1 and R ^Tr2 are each independently selected from hydrogen, deuterium, halogen, -NO ₂ , -CN, -OH, -SH, -NH ₂ , -CO ₂ H, -S(O) ₂ OH, -C(O)NH ₂ , -SO ₂ NH ₂ , -OC(O)NH ₂ , -CH ₂ CO-(N(Me)CH ₂ C(O) ⁾ _z -OR ^Tra , -CH ₂ CO-(N(Me)CH ₂ C(O)) _z -NHR ^Tra , -(CH ₂ CH ₂ O) _z -R ^Tra , -CONH-(CH ₂ CH ₂ O) _z -R ^Tra , or -C _1-6 alkyl, -C _1-6 alkenyl, -C _1-6 alkynyl, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 3-10 membered heteroaryl optionally substituted by R Tra , R ^Tra is hydrogen, deuterium, halogen, -NO ₂ , -CN, -OH, -SH, -NH ₂ , -N(Me) ₂ , -S(O) ₂ Me, -CO ₂ H, -S(O) ₂ OH, -C(O)NH ₂ , -SO ₂ NH ₂ , -C _1-6 alkyl, -C _1-6 alkenyl, -C _1-6 alkynyl, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 3-10 membered heteroaryl, z is a natural number 0 or greater; X is selected from: -O-, -N(R ^3b-2 )-; R ^1a , R ^2a , R ^3a , R ^1b , R ^2b , R ^3b-1 , R ^3b-2 , R ^4b and t are as defined in claim 1 . The ligand-drug conjugate according to claim 2, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein the ligand-drug conjugate has a structure shown in formula (IIIa) or formula (IIIb):
in: Ab is the ligand; q is the drug loading, which is an integer or decimal from 1 to 16; R ^1a , R ^2a , R ^3a , R ^1b , R ^2b , R ^3b-1 , R ^3b-2 , R ^4b and t are as defined in claim 1; and L ^1a , L ² , L ³ , Tr and X are as defined in claim 2 . The ligand-drug conjugate according to claim 3, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein Ab is a target-binding polypeptide, an antibody or an antigen-binding fragment thereof, in particular an antibody or an antigen-binding fragment thereof, for example, selected from fully human antibodies, humanized antibodies, chimeric antibodies, probodies, bispecific antibodies, multispecific antibodies, monoclonal antibodies and polyclonal antibodies; the antibody or antigen-binding fragment thereof can be selected from Fab, Fab', F(ab')2, Fv, scFv, diabodies, Fd, dAb, VHH and antigen-binding fragments of a complementarity determining region (CDR) fragment. The ligand-drug conjugate according to claim 3, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein Ab is a monoclonal antibody. The ligand-drug conjugate according to claim 3, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein Ab is a specific binding selected from HER2, Antibodies or antigen-binding fragments of HER3, B7H3, TROP2, Claudin18.2, CD30, CD33, CD70 and EGFR, for example, Trastuzumab or Pertuzumab or variants thereof. The ligand-drug conjugate according to any one of claims 3 to 6, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein q is an integer or decimal selected from 2 to 8, in particular an integer or decimal selected from 4 to 8. A compound represented by formula (Ia) or formula (Ib), or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof,
in: R ^1a is selected from hydrogen, halogen, amino, hydroxy, -C _1-6 alkyl and -C _1-6 alkoxy; R ^2a and R ^3a are each independently selected from hydrogen, -(C(R ^2a-1 ) ₂ ) _p -H, -(C(R ^2a-1 ) ₂ ) _p -OR ^2a-2 , -(C(R ^2a-1 ) ₂ ) _p -SR ^2a-2 , -(C(R ^2a-1 ) ₂ ) _p -S(O) ₂ R ^2a-2 , -(C(R ^2a-1 ) ₂ ) _p -N(R ^2a-2 )-S(O) ₂ R ^2a-2 and -C(＝O)R ^2a-2 ; or R ^2a and R ^3a and the atoms to which they are attached together form a 3-10 membered heterocyclic ring; the heterocyclic ring is optionally substituted by one or more selected from halogen, hydroxy, amino, -C _1-6 alkyl, -OC _1-6 alkyl, -NHC _1-6 alkyl, -N(C _{-C 1-6} alkyl) ₂ and -C _1-6 alkylene-OH substituents; p is an integer selected from 0 to 10; R ^2a-1 are each independently selected from hydrogen, halogen, hydroxy, amino, cyano, nitro, -C _1-6 alkyl and -halogenated C _1-6 alkyl; Alternatively, any two R ^2a-1 together with the atoms to which they are attached form an oxo group, a 3-10 membered carbocyclic ring or a 3-10 membered heterocyclic ring, wherein the 3-10 membered carbocyclic ring or the 3-10 membered heterocyclic ring is optionally substituted with -C _1-6 alkyl; R ^2a-2 is selected from hydrogen, hydroxy, amino, cyano, -C _1-6 alkyl, -C _3-6 cycloalkyl and 3-6 membered heterocycloalkyl; each of the alkyl, cycloalkyl and heterocycloalkyl groups is optionally substituted with one or more substituents selected from hydrogen, halogen, hydroxy, amino and -C _1-6 alkyl; R ^1b , R ^2b are each independently selected from hydrogen, halogen, hydroxy, nitro, -C _1-6 alkyl and -C _1-6 alkoxy; or R ^1b and R ^2b together with the atoms to which they are attached form a 4-6 membered carbocyclic ring or a 4-6 membered heterocyclic ring; R ^3b is selected from -(C(R ^3b-1 ) ₂ ) _t -H, -(C(R ^3b-1 ) ₂ ) _t -OR ^3b-2 , -(C(R ^3b-1 ) ₂ ) _t - N(R ^3b-2 ) ₂ , -(C(R ^3b-1 ) ₂ ) _t -SR ^3b-2 and -(C(R ^3b-1 ) ₂ ) _t -S(O) ₂ R ^3b-2 ; t is an integer selected from 0 to 10; R ^3b-1 are each independently selected from hydrogen, halogen, hydroxy, amino, cyano, nitro, -C _1-6 alkyl and -halogenated C _1-6 alkyl; Or, any two R ^3b-1 together with the atoms to which they are attached form an oxo group, a 3-6 membered carbocyclic ring or a 3-6 membered heterocyclic ring; R ^3b-2 is selected from hydrogen, hydroxy, -C _1-6 alkyl, -C _3-6 cycloalkyl, 3 to 6 membered heterocycloalkyl; each of the alkyl, cycloalkyl, heterocycloalkyl is optionally substituted with one or more substituents selected from hydrogen, halogen, hydroxy, amino and -C _1-6 alkyl; R ^4b is selected from hydrogen, hydroxy, -C _1-6 alkyl, -C _3-6 cycloalkyl, -halogenated C _1-6 alkyl. A compound represented by formula (IIa) or formula (IIb), or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof:
in: L ¹ is the connection unit; ^L2 is -(C( ^RL21 ) ₂ ) _n- , Where n is a natural number from 0 to 50. Any -C(R ^L21 ) ₂ - unit in L ² may be independently replaced by the following structural units: -Cy-, -C(O)-, -NR ^L22 -, -O-, -S-, -SO-, -SO ₂ -, -P(R ^L22 )-, -P(＝O)(R ^L22 )-, -C(＝S)-, -C(＝NR ^L22 )-, -N＝N-, -C＝N-, -N＝C-, -Cy- is selected from phenylene, 5- to 8-membered heteroarylene, 3- to 10-membered heterocyclylene, or 3- to 10-membered cycloalkylene, wherein said -Cy- is unsubstituted or independently substituted with 1 or more R ^cx , R ^L21 , R ^L22 , and R ^cx are each independently selected from hydrogen, deuterium, halogen, -NO ₂ , -CN, -OR ^L2a , -SR ^L2a , -N(R ^L2a ) ₂ , -N ⁺ (R ^L2a ) ₃ , -C(O)R ^L2a , -CO ₂ R ^L2a , -C(O)C(O)R ^L2a , -C(O)CH ₂ C(O)R ^L2a , -S(O)R ^L2a , -S(O) ₂ R ^L2a , -C(O)N(R ^L2a ) ₂ , -SO ₂ N(R ^L2a ) ₂ , -OC(O)R ^L2a , -N(R ^L2a )SO ₂ R ^L2b , -N(R ^L2a )COR ^L2b , -(CH ₂ ) _y -CO-(N(Me)CH ₂ C(O)) _m -OR ^L2a , -(CH ₂ ) _y -CO-(N(Me)CH ₂ C(O)) _m -NHR ^L2a , -(CH ₂ ) _y -CO-(N(Me)CH ₂ C(O)) _m -N ⁺ (R ^L2a ) ₃ , -(CH ₂ ) _y -NHCOCH ₂ (OCH ₂ CH ₂ )OR ^L2a , -(CH ₂ ) _y -NH(COCH ₂ (N(Me)) _m -R ^L2a , -(CH ₂ ) _y -CONH-(CH ₂ CH ₂ O) _m -R ^L2a , -(CH ₂ ) _y -NHCO-(CH ₂ CH ₂ O) _m -R ^L2a , -(CH ₂ CH ₂ O) _m -R ^L2a , -(COCH _{2 -R} ^L2a , -COCH ₂ (OCH ₂ CH ₂ ) _m -OR ^L2a , -CO-(CH ₂ CH ₂ O) _m -R ^L2a , -CO-(CH ₂ ) _y -CONH-(CH ₂ CH ₂ O) _m -R ^L2a , -CO-(CH ₂ ) _y -NHCO-(CH ₂ CH ₂ O) _m -R ^L2a , or -C _1-6 ^alkyl , -C _1-6 alkenyl, -C _1-6 alkynyl, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 3-10 membered heteroaryl optionally substituted by R L2a, m, y are natural numbers from 0 to 50, ^RL2a , ^RL2b are each independently selected from hydrogen, deuterium, halogen, _-NO2 , -CN, -OH, -SH, _-NH2 , -N(Me) ₂ , _-CO2H , -S(O) _2Me , _-S (O) _2OH , -C(O) _NH2 , _-SO2NH2 , _-C1-6alkyl , _-C1-6alkenyl , -C1-6alkynyl, 3-8-membered cycloalkyl, 4-10-membered heterocycloalkyl, _6-10 -membered aryl, or 3-10-membered heteroaryl; L ³ does not exist or is an amino acid residue, a short peptide consisting of 2-10 amino acid residues, or any combination of the above groups, wherein the amino acid residue is a natural amino acid residue or a non-natural amino acid residue; Tr does not exist or is or any combination of the above groups; R ^Tr , R ^Tr1 and R ^Tr2 are each independently selected from hydrogen, deuterium, halogen, -NO ₂ , -CN, -OH, -SH, -NH ₂ , -CO ₂ H, -S(O) ₂ OH, -C(O)NH ₂ , -SO ₂ NH ₂ , -OC(O)NH ₂ , -CH ₂ CO-(N(Me)CH ₂ C(O) ⁾ _z -OR ^Tra , -CH ₂ CO-(N(Me)CH ₂ C(O)) _z -NHR ^Tra , -(CH ₂ CH ₂ O) _z -R ^Tra , -CONH-(CH ₂ CH ₂ O) _z -R ^Tra , or -C _1-6 alkyl, -C _1-6 alkenyl, -C _1-6 alkynyl, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 3-10 membered heteroaryl optionally substituted by R Tra , R ^Tra is hydrogen, deuterium, halogen, -NO ₂ , -CN, -OH, -SH, -NH ₂ , -N(Me) ₂ , -S(O) ₂ Me, -CO ₂ H, -S(O) ₂ OH, -C(O)NH ₂ , -SO ₂ NH ₂ , -C _1-6 alkyl, -C _1-6 alkenyl, -C _1-6 alkynyl, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 3-10 membered heteroaryl, z is a natural number 0 or greater; X is selected from: -O-, -N(R ^3b-2 )-; R ^1a is selected from hydrogen, halogen, amino, hydroxy, -C _1-6 alkyl and -C _1-6 alkoxy; R ^2a and R ^3a are each independently selected from hydrogen, -(C(R ^2a-1 ) ₂ ) _p -H, -(C(R ^2a-1 ) ₂ ) _p -OR ^2a-2 , -(C(R ^2a-1 ) ₂ ) _p -SR ^2a-2 , -(C(R ^2a-1 ) ₂ ) _p -S(O) ₂ R ^2a-2 , -(C(R ^2a-1 ) ₂ ) _p -N(R ^2a-2 )-S(O) ₂ R ^2a-2 and -C(＝O)R ^2a-2 ; or R ^2a and R ^3a and the atoms to which they are attached together form a 3-10 membered heterocyclic ring; the heterocyclic ring is optionally substituted by one or more selected from halogen, hydroxy, amino, -C _1-6 alkyl, -OC _1-6 alkyl, -NHC _1-6 alkyl, -N(C _{-C 1-6} alkyl) ₂ and -C _1-6 alkylene-OH substituents; p is an integer selected from 0 to 10; R ^2a-1 are each independently selected from hydrogen, halogen, hydroxy, amino, cyano, nitro, -C _1-6 alkyl and -halogenated C _1-6 alkyl; Alternatively, any two R ^2a-1 together with the atoms to which they are attached form an oxo group, a 3-10 membered carbocyclic ring or a 3-10 membered heterocyclic ring, wherein the 3-10 membered carbocyclic ring or the 3-10 membered heterocyclic ring is optionally substituted by -C _1-6 alkyl; R ^2a-2 is selected from hydrogen, hydroxy, amino, cyano, -C _1-6 alkyl, -C _3-6 cycloalkyl and 3-6 membered heterocycloalkyl; each of the alkyl, cycloalkyl and heterocycloalkyl groups is optionally substituted with one or more substituents selected from hydrogen, halogen, hydroxy, amino and -C _1-6 alkyl; R ^1b , R ^2b are each independently selected from hydrogen, halogen, hydroxy, nitro, -C _1-6 alkyl and -C _1-6 alkoxy; or R ^1b and R ^2b together with the atoms to which they are attached form a 4-6 membered carbocyclic ring or a 4-6 membered heterocyclic ring; t is an integer selected from 1 to 10; R ^3b-1 are each independently selected from hydrogen, halogen, hydroxy, amino, cyano, nitro, -C _1-6 alkyl and -halogenated C _1-6 alkyl; Or, any two R ^3b-1 together with the atoms to which they are attached form an oxo group, a 3-6 membered carbocyclic ring or a 3-6 membered heterocyclic ring; R ^3b-2 is selected from hydrogen, hydroxy, -C _1-6 alkyl, -C _3-6 cycloalkyl, 3 to 6 membered heterocycloalkyl; each of the alkyl, cycloalkyl, heterocycloalkyl is optionally substituted with one or more substituents selected from hydrogen, halogen, hydroxy, amino and -C _1-6 alkyl; R ^4b is selected from hydrogen, hydroxy, -C _1-6 alkyl, -C _3-6 cycloalkyl, -halogenated C _1-6 alkyl. The compound according to claim 9, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein L ¹ is selected from:
Preferably, ^L1 is selected from More preferably, ^L1 is selected from The ligand-drug conjugate according to any one of claims 1 to 7 or the compound according to any one of claims 8 to 10, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein R ^1a is selected from hydrogen, halogen, -C _1-6 alkyl; preferably, R ^1a is selected from hydrogen and halogen; further preferably, R ^1a is selected from hydrogen, F, Cl. The ligand-drug conjugate according to any one of claims 1 to 7 or the compound according to any one of claims 8 to 10, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein: R ^2a and R ^3a are each independently selected from hydrogen, -(C(R ^2a-1 ) ₂ ) _p -H, -(C(R ^2a-1 ) ₂ ) _p -OR ^2a-2 , -(C(R ^2a-1 ) ₂ ) _p -S(O) ₂ R ^2a-2 , -(C(R ^2a-1 ) ₂ ) _p -N(R ^2a-2 )-S(O) ₂ R ^2a-2 and -C(＝O)R ^2a-2 ; p is an integer selected from 0-10; preferably, p is an integer selected from 0-5; R ^2a-1 is independently selected from hydrogen, halogen, hydroxyl, amino or -C _1-6 alkyl; preferably, R ^2a-1 is selected from hydrogen or -C _1-6 alkyl; further preferably, R ^2a-1 is selected from hydrogen or methyl; Alternatively, any two R ^2a-1 and the atoms to which they are connected together form an oxo group, a 3-6-membered carbocyclic ring or a heterocyclic ring; preferably, any two R ^2a-1 and the atoms to which they are connected together form an oxo group, a 3-6-membered carbocyclic ring; R ^2a-2 is selected from hydrogen, -C _1-6 alkyl, -C _3-6 cycloalkyl, 3 to 6 membered heterocycloalkyl; preferably, R ^2a-2 is selected from hydrogen or -C _1-6 alkyl; further preferably, R ^2a-2 is selected from hydrogen or methyl. The ligand-drug conjugate according to any one of claims 1 to 7 or the compound according to any one of claims 8 to 10, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein R ^2a and R ^3a together with the atoms to which they are attached form a 4-6 membered heterocyclic ring; the heterocyclic ring is optionally substituted with one or more substituents selected from halogen, hydroxyl, amino, -C _1-6 alkyl, -C _1-6 alkylene-OH. The ligand-drug conjugate according to any one of claims 1 to 7 or the compound according to any one of claims 8 to 10, or its tautomers, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof, wherein R ^2a and R ^3a are each independently selected from hydrogen, -C _1-6 alkyl, -C _1-6 alkylene-OH, -haloC _1-6 alkyl and -C _1-6 alkyl-OC _1-6 alkyl; preferably, R ^2a and R ^3a are each independently selected from hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl, -(CH ₂ ) ₂ OH, -(CH ₂ ) ₃ OH, -(CH ₂ ) ₂ OCH ₃ and -(CH ₂ ) ₃ OCH ₃ . The ligand-drug conjugate according to any one of claims 1 to 7 or the compound according to any one of claims 8 to 10, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein R ^1b is selected from hydrogen, halogen, -C _1-6 alkyl, -C _1-6 alkoxy; preferably, R ^1b is selected from hydrogen, F, Cl, methyl and methoxy. The ligand-drug conjugate according to any one of claims 1 to 7 or the compound according to any one of claims 8 to 10, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein R ^2b is selected from hydrogen, halogen, -C _1-6 alkyl, -C _1-6 alkoxy; preferably, R ^2b is selected from hydrogen, halogen; further preferably, R ^2b is selected from F. The ligand-drug conjugate according to any one of claims 1 to 7 or the compound according to any one of claims 8 to 10, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt wherein R ^1b and R ^2b together with the atoms to which they are attached form a 5-membered heterocyclic ring; preferably, the 5-membered heterocyclic ring is The ligand-drug conjugate according to any one of claims 1 to 7 or the compound according to any one of claims 8 to 10, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein R ^3b-1 is independently selected from hydrogen, halogen, hydroxyl, amino or -C _1-6 alkyl; preferably, R ^3b-1 is selected from hydrogen or -C _1-6 alkyl; further preferably, R ^3b-1 is selected from hydrogen or methyl; Alternatively, any two R ^3b-1 and the atoms to which they are connected together form an oxo group, a 3-6-membered carbocyclic ring or a 3-6-membered heterocyclic ring; preferably, any two R ^3b-1 and the atoms to which they are connected together form an oxo group or a 3-6-membered carbocyclic ring; R ^3b-2 is selected from hydrogen, -C _1-6 alkyl, -C _3-6 cycloalkyl, 3 to 6 membered heterocycloalkyl; preferably, R ^3b-2 is selected from hydrogen or -C _1-6 alkyl; further preferably, R ^3b-2 is selected from hydrogen. The ligand-drug conjugate according to any one of claims 1 to 7 or the compound according to any one of claims 8 to 10, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein R ^4b is selected from hydrogen, methyl, ethyl; preferably, R ^4b is selected from hydrogen and methyl. The ligand-drug conjugate or compound according to any one of claims 2-7 and 9-19, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein ^L2 is -(CHR ^L21 ) _n -; Where n is a natural number from 0 to 50; Any CHR ^L21 unit in ^L2 can be independently replaced by the following structural units: -Cy-, -C(O)-, -NR ^L22 -, -O-, -Cy- is phenylene, 5- to 6-membered heteroarylene, 4- to 10-membered heterocyclylene, or 3- to 6-membered cycloalkylene, wherein said -Cy- is unsubstituted or independently substituted with 1 to 3 R ^cx ; Each ^RL21 , ^RL22 , and ^Rcx is independently selected from hydrogen, halogen, -OR ^L2a , -N( ^RL2a ) ₂ , -C(O) ^RL2a , -S(O) ^2RL2a , -C(O)N( ^RL2a _{)2 , -SO2N} ( ^RL2a ) ₂ , -N( ^RL2a ) ^SO2RL2b , -N( ^RL2a )COR ^L2b , -( _CH2 ) _y -CO-(N(Me) _CH2C (O)) _m -OR ^L2a , _- ( _CH2 ) _y - _CO- (N(Me) _CH2C (O)) _m -NHR ^L2a , -( _CH2 ) _y -CONH-( _CH2CH2O ) _m - ^RL2a , -( _CH2 ) _y -NHCO-(CH ₂ CH ₂ O) _m -R ^L2a , -(CH ₂ ) _y -NHCOCH ₂ (OCH ₂ CH ₂ )OR ^L2a , -(CH ₂ ) _y -NH(COCH ₂ (N(Me)) _m -R ^L2a , -(CH ₂ ) _y -NHCO-(CH ₂ CH ₂ O) _m ^-RL2a , -(CH ₂ CH ₂ O) _m ^-RL2a , -(COCH ₂ N(Me)) _m ^-RL2a , ^-COCH ₂ (OCH ₂ CH ₂ ) _m -OR ^L2a , -CO-(CH ₂ CH ₂ O) _m ^-RL2a , or -C _1-6 alkyl, -C _1-6 alkenyl, -C _1-6 alkynyl, 3-8 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 3-10 membered heteroaryl optionally substituted by RL2a ; m is a natural number from 0 to 8; y is 0, 1, 2, 3, or 4; Each ^RL2a , ^RL2b is independently selected from hydrogen, halogen, -CN, -OH, _-NH2 , -N(Me) ₂ , _-CO2H , -C(O) _NH2 , _-C1-6 alkyl. The ligand-drug conjugate or compound according to any one of claims 2-7 and 9-19, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein ^L2 is -( _CH2 ) _n- ; Where n is a natural number from 0 to 50; Any methylene unit in ^L2 can be independently replaced by the following structural units: 4- to 6-membered heterocyclylene, 3- to 6-membered cycloalkylene, -C(O)-, -NR ^L22 -, -O-, Each R ^L22 is independently selected from hydrogen, -OR ^L2a , -C(O) ^RL2a , -S(O) ₂ R ^L2a , -C(O)N(R ^L2a ) ₂ , -SO ₂ N(R ^L2a ) ₂ , -(CH ₂ ) _y -CO-(N(Me)CH ₂ C(O)) _m -OR ^L2a , -(CH ₂ ) _y -CO-(N(Me)CH ₂ C(O) ) _m -NHR ^L2a , -(CH ₂ ) _y -CONH-(CH ₂ CH ₂ O) _m -R ^L2a , -(CH ₂ ) _y -NHCOCH ₂ (OCH ₂ CH ₂ )OR ^L2a , -(CH ₂ CH ₂ O) _m -R ^L2a , -(COCH ₂ N(Me)) _m -R ^L2a , -COCH ₂ (OCH ₂ CH ₂ ) _m -OR ^L2a , -CO-(CH ₂ CH ₂ O) _m -R ^L2a , or -C _1-6 alkyl optionally substituted by R ^L2a ; m is a natural number from 0 to 8; y is 0, 1, 2, 3, or 4; Each ^RL2a is independently selected from hydrogen, halogen, -CN, -OH, _-NH2 , -N(Me) ₂ , _-CO2H , -C(O) _NH2 , _-C1-6alkyl . The ligand-drug conjugate or compound according to any one of claims 2-7 and 9-19, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein ^L2 is selected from:
in, n1, n2, n3, n4, m are each independently selected from natural numbers from 0 to 8; n5 and n6 are each independently selected from 0 or 1; -Cy- is a 4- to 6-membered heterocyclylene group or a 3- to 6-membered cycloalkylene group; preferably, -Cy- is Further preferably, -Cy- is Preferably, for More preferably, for Among them, the c-terminal is connected to ^L1 or ^L1a , and the d-terminal is connected to ^L3 . A compound as defined in any one of claims 9 to 19, or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein: Selected from:
n1, n2, n3, n4, m are each independently selected from natural numbers from 0 to 8; n5 and n6 are each independently selected from 0 or 1; -Cy- is a 4- to 6-membered heterocyclylene or a 3- to 6-membered cycloalkylene; preferably, -Cy- is selected from: Further preferably, -Cy- is selected from Preferably, Selected from: More preferably, Selected from: The ligand-drug conjugate or compound according to any one of claims 2-7 and 9-23, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein L ³ is selected from Val, D-Val, Phe, Lys, Leu, Ile, Gly, Ala, D-Ala, Cit, Asp, Asn, Glu, Gln, Val-Cit, Val-Ala, Val-Lys, Val- Lys(Ac), Phe-Lys, Phe-Lys(Ac), Leu-Lys, Leu-Lys(Ac), Ala-Ala, Ala-Lys, D-Ala-Ala, Gly-Glu, Gly-Asp, Gly -Asn, Val-Glu, Val-Asp, Asn-Asn, Asp-Glu, Gly-Gly-Glu, Gly-Gly-Asp, Gly-Gly-Asn, Gly-Ala-Ala, Gly-Val-Ala, Gly -Val-Cit, Glu-Val-Ci t, Ala-Ala-Ala, Ala-(D-Ala)-Ala, Ala-Ala-Asn, Ala-(D-Ala)-Asn, Ala-Ala-Asp, Val-Lys-Gly, D-Val- Leu-Lys, Gly-Gly-Arg, Gly-Gly-Gly, Lys-Ala-Asn, Gly-Phe-Gly, Gly-Gly-Phe, Asn-Pro-Val, Ala-Lys-Gly, Gly-Lys- Gly, Gly-Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Glu-Gly, Lys-Ala-Ala-Asn, Lys-Ala-Ala-Asp, Ala-Ala-Pro-Val, Ala-Ala-Pro-Nva, or any combination of the above fragments; Preferably, L ³ is selected from Lys, Gly, Asp, Asn, Glu, Gln, Val-Cit, Val-Ala, Ala-Ala, Gly-Glu, Gly-Asp, Gly-Asn, Asn-Asn, Asp-Glu ,Gly-Glu-Gly, Gly-Gly-Phe-Gly, or any combination of the above fragments; preferably, ^L3 is selected from Lys, Gly, Val-Ala, Ala-Ala, Gly-Glu, Gly-Asp, Gly-Asn, Asn-Asn, Asp-Glu, Gly-Gly- Phe-Gly, or any combination of the above fragments; more preferably, ^L3 is selected from Val-Ala, Ala-Ala, Gly-Glu, Gly-Asp, Gly-Asn, Asp-Asp, Asp-Glu, Gly-Gly-Phe-Gly or The compound as defined in claim 23, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein: Selected from:


Preferably, Selected from: The ligand-drug conjugate or compound according to any one of claims 2-7 and 11-22, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein Selected from: More preferably, Selected from: The ligand-drug conjugate or compound according to any one of claims 2-7, 11-22 and 24, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein
Selected from: Preferably, Selected from The ligand-drug conjugate or compound according to any one of claims 2-7 and 9-27, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein Tr does not exist or is in R ^Tr , R ^Tr1 and R ^Tr2 are independently selected from hydrogen, halogen, -NO ₂ , -CN, -OH, -NH ₂ , -CO ₂ H, -S(O) ₂ H, -C(O)NH ₂ , -SO ₂ NH ₂ , -OC(O)NH ₂ , -CH ₂ CO-(N(Me)CH ₂ C(O)) _z -NHMe, -(CH ₂ CH ₂ O) _z -H, -CONH-(CH ₂ CH ₂ O) _z -H; z is a natural number from 0 to 8; Preferably, Tr does not exist or is The compound according to claim 8, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein R ^3b is selected from hydrogen, -(C(R ^3b-1 ) ₂ ) _t -H, -(C(R ^3b-1 ) ₂ ) _t -OR ^3b-2 and -(C(R ^3b-1 ) ₂ ) _t -NHR ^3b-2 ; t is selected from an integer of 0-10; preferably, t is selected from an integer of 3-5; and R ^3b-1 and R ^3b-2 are as defined in claim 8. The compound according to claim 8, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein R ^3b is selected from hydrogen, -C _1-6 alkyl, -haloC _1-6 alkyl and -C _1-6 alkylene-OH. The compound according to claim 8, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein the compound is selected from


The compound according to claim 8, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein the compound is selected from
The compound according to claim 9, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein the compound is selected from



The ligand-drug conjugate according to claim 3, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein the ligand-drug conjugate is selected from:






wherein Ab and q are as defined in claim 3. The ligand-drug conjugate according to claim 3, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, wherein the ligand-drug conjugate is selected from:







wherein q is as defined in claim 3. A pharmaceutical composition comprising the ligand-drug conjugate or compound as described in any one of claims 1 to 35, or its tautomer, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt or solvate, and a pharmaceutically acceptable carrier. A pharmaceutical preparation comprising the ligand-drug conjugate or compound as described in any one of claims 1 to 35 or its tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof. Use of a ligand-drug conjugate or compound as described in any one of claims 1 to 35, or a tautomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition as described in claim 35, or a pharmaceutical preparation as described in claim 36 in the preparation of a drug for preventing or treating a disease associated with abnormal cell activity. The use according to claim 38, wherein the disease associated with abnormal cell activity is cancer. The use according to claim 39, wherein the cancer is a solid tumor or a non-solid tumor, for example, selected from esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, non-Hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer or thyroid cancer.