New eribulin-related active molecule, linker-drug toxin molecule compound containing same, and use thereof in antibody conjugate

Abstract

Disclosed in the present invention are a new eribulin-related active molecule, a linker-drug toxin molecule compound containing same, and the use thereof in an antibody conjugate. Provided in the present invention is a drug conjugate as represented by formula I. The drug conjugate, upon cleavage, first releases a new eribulin-related active molecule with low toxicity, and thus can effectively improve the safe therapeutic window. Further, the new eribulin-related active molecule of the present invention, the produced linker-payload compound, and the drug conjugate produced therefrom also have one or more of the advantages of blood stability, low toxicity and relatively rapid clearance.

Description

Novel active molecules related to eribulin, linker-drug toxin molecule compounds containing the same and their use in antibody conjugates

[0001] This application claims priority to Chinese patent application 2024111225360, filed on August 15, 2024. This application incorporates the entirety of the aforementioned Chinese patent application. TECHNICAL FIELD

[0002] The present application relates to a novel active molecule related to eribulin, linker-drug toxin molecule compounds containing the same and their use in antibody conjugates. BACKGROUND

[0003] Antibody-drug conjugates (ADCs) can selectively deliver drugs to target cells and produce pharmacologically active effects, but have less impact on normal cells, opening a new era of disease treatment. Currently, several ADC drugs have been approved for marketing by the FDA, such as DS-8201, which is a HER2 antibody connected with a camptothecin derivative Dxd, is used for the treatment of HER2-positive breast cancer patients, and Sacituzumab govitecan, which targets Trop-2 antigen (also known as epithelial glycoprotein 1, EGP-I). Among the ADCs approved for marketing by the FDA so far, the drugs contained mainly target DNA or tubulin as anti-tumor drugs. As small molecule compounds against tumors, camptothecin derivatives, such as SN-38, Dxd, and Dx-8951, which inhibit DNA topoisomerase I to achieve anti-tumor effects, have been confirmed to have killing effects on a variety of cancer cells in vivo and in vitro, showing strong anti-tumor effects. As compounds that inhibit tubulin to achieve anti-tumor effects, such as Eribulin, MMAE, MMAF, and maytansine, which have been confirmed to have killing effects on a variety of cancer cells in vivo and in vitro, show strong anti-tumor effects. By connecting small molecules to antibodies through appropriate linking methods, the stability of ADC drugs can be improved and the therapeutic window can be improved. An appropriate linking method can ensure the integrity of the ADC drug during blood circulation and avoid premature release, which can cause off-target toxicity. After entering the target cells, the toxin can be effectively released to exert a killing effect.

[0004] Current eribulin ADCs are connected to antibodies and drugs through a cleavable linker, which can directly release the drug during blood circulation. Because eribulin is highly toxic, the safe therapeutic window of ADC drugs is narrow. Moreover, the linkers in eribulin ADCs are all coupled to Cys on the antibody in a non-site-specific manner, which has problems of uniformity and stability. SUMMARY

[0005] The technical problem to be solved by the present application is that the existing art has strong toxicity of irinotecan ADC drugs and narrow safety treatment window; therefore, the present application provides a new active molecule related to irinotecan, a linker-drug toxin molecule containing the same, and their application in antibody conjugates. The drug conjugate provided by the present application, as shown in formula I, when broken, first releases the low-toxicity new active molecule related to irinotecan, which can effectively improve the safety treatment window. Further, the new active molecule related to irinotecan of the present application, as well as the generated linker-payload compound, and the drug conjugate generated therefrom, also have one or more of the advantages of stability in blood, low toxicity and fast clearance rate.

[0006] The present application provides a drug conjugate as shown in formula I,

[0007] wherein T is a ligand or targeting moiety that binds to a target,

[0008] n is a drug ligand coupling ratio;

[0009] L is a linker fragment connected to T;

[0010] L 1 is a connection unit;

[0011] L 2 is a single amino acid residue or a short peptide residue composed of 2-10 amino acid residues;

[0012] L 3 is a spacer;

[0013] L 4 is a single bond or Y is connected to L 3 ;

[0014] Y is O, S or NH;

[0015] s is 1, 2, 3, 4, 5 or 6;

[0016] R 3a and R 3b are independently hydrogen, deuterium, -COOH, C 1-6 alkyl, C 1-6 alkoxy or C 3a-1 alkyl substituted with one or more R 1-6 ;

[0017] R 3a-1 is independently halogen, -OH, -SH, -NH2, -COOH or C 6-10 aryl;

[0018] D is

[0019] R 1 and R 2 independently H, deuterium or C 1-6 alkyl;

[0020] and the drug conjugate of Formula I satisfies any one of the following conditions:

[0021] Condition (1): L 4 is

[0022] Condition (2): wherein Z 1 is m is 1, 2, 3, 4 or 5; and ring A is

[0023] ring A1 is C 5-10 alkene ring or 5-10 membered heteroalkene ring;

[0024] each ring A2 is independently 5-10 membered heteroaromatic ring;

[0025] ring A3 and ring A4 are independently C 5-10 alkane ring, 5-10 membered heteroalkane ring, C 5-10 alkene ring, 5-10 membered heteroalkene ring, C 6-10 aromatic ring or 5-10 membered heteroaromatic ring;

[0026] p is 0, 1, 2 or 3;

[0027] X 1 is CH, CH2, N or NH;

[0028] is single bond or double bond;

[0029] the heteroatom species of each 5-10 membered heteroalkane ring, 5-10 membered heteroalkene ring and 5-10 membered heteroaromatic ring is independently selected from one, two or three of N, O or S, and the number of heteroatoms is independently 1, 2 or 3;

[0030] the amino acid residue is natural amino acid residue or unnatural amino acid residue.

[0031] In the present application, the linker is a degradable or non-degradable linker fragment, one end of which is connected to the antibody.

[0032] In the present application, T is connected to by a compound coupling method understood by those skilled in the art; for example wherein Z 1with a thiol group contained in T (e.g., an antibody) itself after opening a disulfide bond at one end (e.g., a disulfide bond can be opened by reduction of the disulfide bond with a reducing agent TCEP to generate a thiol group -SH), that is, Z 1 -S- between T is not a sulfur atom externally attached again; or Z in 1 with an acyl acceptor of T (e.g., a peptide bond is formed between a glutamine side chain in an antibody and a linker precursor having a primary amine group catalyzed by a microbial transglutaminase (MTGase) enzyme), thus, Z 1 -S- between T is not a sulfur atom externally attached again; or is not a group externally attached again.

[0033] In a certain aspect, in the drug conjugate of Formula I, certain groups are defined as follows, and the definition of a group not mentioned is as described in any aspect of the application (hereinafter in this paragraph, referred to as "in a certain aspect").

[0034] In a certain aspect, in the drug conjugate of Formula I,

[0035] L 4 is a single bond or a single amino acid residue;

[0036] R 1 and R 2 are independently H or deuterium;

[0037] the amino acid residue is a natural amino acid residue or a non-natural amino acid residue;

[0038] in condition (1), L 4 is a single amino acid residue.

[0039] In a certain aspect, in the drug conjugate of Formula I,

[0040] T is an antibody,

[0041] n is a natural number or a decimal number from 1 to 16;

[0042] Z 1 is a single bond or

[0043] m is 1, 2, 3, 4, or 5;

[0044] ring A is a C 5-10 alkane ring, a 5-10 membered heteroalkane ring, substituted with one or more R a substituted with one or more R 5- 10 alkane ring, or a 5-10 membered heteroalkane ring substituted with one or more R a substituted with one or more R

[0045] Each R a It is oxygenated (=O);

[0046] Ring A1 is C 5-10 Olefin rings or 5-10 membered heteroolefin rings;

[0047] Each ring A2 is an independent 5-10 quintile heteroaryl ring;

[0048] Rings A3 and A4 are independently C 5-10 Alkane rings, 5-10 membered heteroalkane rings, C 5-10 olefin rings, 5-10 membered heteroolefin rings, C 6-10 Aromatic rings or 5-10 heterocyclic aromatic rings;

[0049] p is 0, 1, 2 or 3;

[0050] X 1 It can be CH, CH2, N or NH;

[0051] It can be a single bond or a double bond;

[0052] L 1 for

[0053] Each R b and R c Independently, it can be H, deuterium, or a halogen;

[0054] Each q1 is independently 1, 2, 3, 4, 5, or 6;

[0055] Each q2 is an independent integer from 1 to 16;

[0056] M is NH, S, O or

[0057] L 2 It can be a single amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue;

[0058] L 3 for R d It can be H, deuterium, or halogen;

[0059] The heteroatom types of each of the 5-10 member heteroalkane rings, 5-10 member heteroolefin rings, and 5-10 member heteroaromatic rings are independently selected from one, two, or three of N, O, or S; the number of heteroatoms is independently one, two, or three.

[0060] In one scheme, in rings A, A3, and A4, the respective C... 5-10 The alkane ring is independently C 5-6 Alkane ring.

[0061] In one aspect, in each of ring A3 and ring A4, the heteroatom identity of each 5-10 membered heteroalkane ring is N, and the number of heteroatoms can be 1.

[0062] In one aspect, in each of ring A3 and ring A4, the C 5-10 In one aspect, in each of ring A3 and ring A4, the C 5-10 In one aspect, in each of ring A3 and ring A4, the C 7-10 In one aspect, in each of ring A3 and ring A4, the C

[0063] In one aspect, in each of ring A3 and ring A4, the 5-10 membered heteroaromatic ring is independently a 5-6 membered heteroaromatic ring.

[0064] In one aspect, in ring A, the heteroatom identity of each 5-10 membered heteroalkane ring is N, and the number of heteroatoms can be 1.

[0065] In one aspect, in ring A, each 5-10 membered heteroalkane ring is independently a 5-6 membered heteroalkane ring, for example, aziridine; and further for example In one aspect, in ring A, each 5-10 membered heteroalkane ring is independently a 5-6 membered heteroalkane ring, for example, aziridine; and further for example In one aspect, in ring A, each 5-10 membered heteroalkane ring is independently a 5-6 membered heteroalkane ring, for example, aziridine; and further for example 1 In one aspect, in ring A, each 5-10 membered heteroalkane ring is independently a 5-6 membered heteroalkane ring, for example, aziridine; and further for example

[0066] In one aspect, in ring A1, the C 5-10 In one aspect, in ring A1, the C

[0067] In one aspect, in ring A1, the C 5-10 In one aspect, in ring A1, the C 7-10 In one aspect, in ring A1, the C 5-10 In one aspect, in ring A1, the C

[0068] In one aspect, in ring A1, the 5-10 membered heteroalkene ring is a 7-10 membered heteroalkene ring.

[0069] In one aspect, in ring A2, the heteroatom identity of the 5-10 membered heteroaromatic ring is N, and the number of heteroatoms is independently 1, 2, or 3, for example, 3.

[0070] In one aspect, in ring A2, the 5-10 membered heteroaromatic ring is a 5-6 membered heteroaromatic ring, for example, a triazole ring, and further for example

[0071] In one aspect, in each of ring A3 and ring A4, the C 6-10 In one aspect, in each of ring A3 and ring A4, the C

[0072] In a certain embodiment, R b , R c , and R d , the halogen is independently fluorine, chlorine, bromine, or iodine.

[0073] In a certain embodiment, T is an anti-HER2 antibody or a variant thereof that maintains at least equivalent antigen-binding function to the antibody; for example, an anti-HER2 antibody or a variant thereof; for example further, Trastuzumab or a mutant thereof having a difference in the amino acid residue at position 297 from the amino acid sequence of Trastuzumab; preferably, the amino acid residue at position 297 from the amino acid sequence of Trastuzumab is replaced by A or Q.

[0074] In a certain embodiment, n is a natural number or a decimal number from 1 to 8; for example, 1.31, 1.56, 1.91, 3.03, 3.22, 3.63, 3.67, 3.75, 3.78, 3.82, 3.83, 3.91, 4, or 8.

[0075] In a certain embodiment, m is 3.

[0076] In a certain embodiment, ring A is a 5-10 membered heteroalkane ring substituted with one or more R a ; preferably, the 5-10 membered heteroalkane ring is a 5-6 membered heteroalkane ring having one N as the heteroatom species and one N as the number of heteroatoms. In a certain embodiment, ring A is a 5-6 membered heteroalkane ring substituted with one or more R a ; preferably, the 5-6 membered heteroalkane ring has one N as the heteroatom species and one N as the number of heteroatoms.

[0077] In a certain embodiment, ring A is a 5-6 membered heteroalkane ring substituted with one or more R

[0078] In a certain embodiment, ring A1 is a C 5-10 alkene ring; preferably, ring A1 is a C 7-10 alkene ring.

[0079] In a certain embodiment, ring A2 is a 5-6 membered heteroaromatic ring; the 5-6 membered heteroaromatic ring has one N as the heteroatom species and one, two, or three N as the number of heteroatoms.

[0080] In a certain embodiment, ring A3 and ring A4 are independently a C 6-10 aromatic ring; for example, a benzene ring.

[0081] In a certain embodiment, is a double bond.

[0082] ​In one particular scheme, p is 1.

[0083] In one of the plans, X 1 It can be CH2 or NH, for example, NH.

[0084] In one possible solution, ring A is... At that time, Z 1 for -NH- is connected to T.

[0085] In one possible solution, ring A is C. 5-10 Alkane rings, 5-10 membered heteroalkane rings, and rings with one or more R groups a Replacement C 5-10 Alkyl ring or alkane ring with one or more R a When a 5-10 membered heteroalkane ring is substituted; Z 1 It is a single key.

[0086] In one scheme, rings A2 and Z 1 connect.

[0087] In one possible solution, ring A is... "1" bit connection Z 1 "2" bits connect L 1 V is CH2 or C(O), t1 is 1 or 2, t2 is 0, 1, 2 or 3; ring A3 and ring A4 are independently C 6-10 Aromatic ring, p is 0 or 1; X 1 It is CH2 or NH, for example, ring A is CH2 or NH2.

[0088] In a certain scheme, each R b and R c Independently, it can be H or deuterium, for example, H.

[0089] In a certain scheme, each q1 is independently either 1 or 2.

[0090] In one scheme, each q2 is an independent integer from 1 to 14; for example, 2, 4, or 12.

[0091] In a certain scheme, M is O or

[0092] In this invention, L 2 The amino terminus (-NH terminus) of individual amino acid residues and short peptide residues with L 1 Connection, carbonyl end (C=O end) and L 3 connect.

[0093] In one of the schemes, L 2 It is a dipeptide residue, and the amino terminus of the dipeptide residue is connected to L. 1 Connection, carbonyl end with L3 is connected to L NH -Val-Cit- C＝O , NH -Cit-Val- C＝O ; wherein the amino end is connected to L 1 and the carbonyl end is connected to L 3 .

[0094] In one aspect, L 2 is a dipeptide residue, a tripeptide residue, or a tetrapeptide residue, each amino acid residue in L 2 is independently a natural amino acid residue, for example Val, Leu, Lle, Gly, Ala, Cit, or Arg.

[0095] In one aspect, L 2 is L 2a is L 2b is

[0096] In one aspect, R d is H or deuterium, for example H.

[0097] In one aspect, L 4 is a single amino acid residue, the amino end of the amino acid residue is connected to L 3 and the carbonyl end is connected to D.

[0098] In one aspect, L 4 is a single bond.

[0099] In one aspect, L 4 is Y is O or S.

[0100] In one embodiment, R 3a is hydrogen or C 1-6 alkyl, for example hydrogen or methyl, and further for example hydrogen.

[0101] In one embodiment, R 3b is hydrogen, -COOH, C 1-6 alkyl, or C 3a-1 alkyl substituted with one or more R 1-6 , for example hydrogen, -COOH, methyl, ethyl, or n-butyl, and further for example H.

[0102] In one embodiment, R 2 is methyl.

[0103] In one aspect, R 1 and R 2For H.

[0104] In one implementation scheme, L 4 for Y is NH; preferably, R 3a For hydrogen, R 3b It is hydrogen or is formed by one or more R 3a-1 Replacement C 1-6 Alkyl, R 3a-1 Independently -OH or -COOH, m is 1, 2, 3, 4, 5 or 6.

[0105] In one scheme, -Z 1 - For single key or -NH- is connected to T.

[0106] In one possible solution, ring A is... for Ideally, one of them is related to Z. 1 Connect, 2 bits and L 1 Connect; better yet, for One of them is related to Z. 1 Connect, 2 bits and L 1 connect.

[0107] In one of the schemes, L 1 for Preferably, bit "1" is connected to ring A, and bit "2" is connected to L. 2 Connection; better yet, L 1 for Bit "1" is connected to ring A, and bit "2" is connected to L. 2 connect.

[0108] In one of the schemes, L 2 for The "1" position is related to L. 1 Connect, bit "2" and L 3 connect.

[0109] In one of the schemes, L 3 for The "1" position is related to L. 2 Connect, bit "2" and L 4 connect.

[0110] In one of the schemes, L 4 For single bond, L 4 The amino terminus of L 3L is connected to the amino end of D; preferably, L 4 is a single bond, L 4 is connected to the amino end of D; more preferably, L 3 is connected to the carbonyl end of D; 4 L 4

[0111] In one aspect, D is For example,

[0112] In one aspect, T is trastuzumab or a mutant thereof;

[0113] n is a natural number or a fraction from 1 to 8;

[0114] Z 1 is a single bond or -NH- is connected to T;

[0115] m is 1, 2, 3, 4, or 5;

[0116] Ring A is or a 5-6 membered heteroalkane ring substituted with one or more R a ; the heteroatom species of said 5-6 membered heteroalkane ring is N, and the number of heteroatoms is 1; Ring A2 is connected to Z 1 ;

[0117] Ring A1 is a C 7-10 alkene ring;

[0118] Ring A2 is independently a 5-6 membered heteroaromatic ring; the heteroatom species of said 5-6 membered heteroaromatic ring is N, and the number of heteroatoms is independently 1, 2, or 3;

[0119] Ring A3 and Ring A4 are independently a C 6-10 aromatic ring;

[0120] Each R a is oxo;

[0121] X 1 is CH2 or NH;

[0122] L 1 is connected to the amino end of D; preferably, L L 1 is connected to the carbonyl end of D; 2

[0123] Each R b and R c is independently H or deuterium;

[0124] ​​​each q1 is independently 1, 2, 3, 4, 5, or 6;

[0125] q2 is an integer from 1 to 16;

[0126] M is O or

[0127] L 2 is a dipeptide residue, the amino-terminal end of the dipeptide residue is attached to L 1 , the carbonyl end is attached to L 3 ;

[0128] L 3 is R d is H or deuterium;

[0129] L 4 is a single amino acid residue, the amino-terminal end of the amino acid residue is attached to L 3 , the carbonyl end is attached to D;

[0130] D is

[0131] In one aspect, T is trastuzumab or a mutant thereof;

[0132] n is a natural number or a fraction from 1 to 8;

[0133] Z 1 is -NH- is attached to T;

[0134] m is 1, 2, 3, 4, or 5;

[0135] Ring A is

[0136] Ring A1 is C 7-10 alkene ring;

[0137] Ring A2 is independently a 5-6 membered heteroaromatic ring; the heteroatom species of the 5-6 membered heteroaromatic ring is N, and the number of heteroatoms is independently 1, 2, or 3;

[0138] Ring A3 and Ring A4 are independently C 6-10 aromatic ring;

[0139] X 1 is CH2 or NH;

[0140] L 1 is L 1 , the carbonyl end is attached to L 2 ;

[0141] each R b and R cIndependently, it can be either H or deuterium;

[0142] Each q1 is independently 1, 2, 3, 4, 5, or 6;

[0143] Each q2 is an independent integer from 1 to 16;

[0144] M is O or

[0145] L 2 It is a dipeptide residue, and the amino terminus of the dipeptide residue is connected to L. 1 Connection, carbonyl end with L 3 connect;

[0146] L 3 for R d For H or deuterium;

[0147] L 4 It is a single bond or a single amino acid residue, with the amino terminus of the amino acid residue connected to L. 3 The carbonyl end is connected to D;

[0148] D is

[0149] In one scheme, the drug conjugate shown in Formula I is any of the compounds shown in Table A;

[0150] Preferably, the drug conjugate represented by Formula I is any of the compounds shown in Table B.

[0151] This invention provides a compound as shown in Formula II or a pharmaceutically acceptable salt thereof.

[0152] Where D is

[0153] For the connector precursor;

[0154] And the compound of formula II satisfies any of the following conditions:

[0155] Condition (1): L 4 for

[0156] Condition (2): Ring G is C 5-10 Alkyne rings, 5-10 heteroyne rings or

[0157] The heteroatoms in the 5-10 membered heteroyne ring are selected from one, two, or three of N, O, or S; the number of heteroatoms is one, two, or three.

[0158] X 1, p, ring A3, ring A4, L 1 , L 2 , L 3 , L 4 , R 1 , and R 2 are as described in any of the embodiments of the drug conjugate of Formula I.

[0159] In the present application, the linker precursor refers to a group whose one end has been connected to L 1 in the linker, but the other end has not been connected to T; after the other end of the linker precursor is connected to T, the linker precursor forms the linker segment in the drug conjugate. In the present application, G can be directly connected to T through a coupling reaction, or G can be first connected to an extension unit (e.g., NH2-PEG3-N3) and then connected to T; or T can be first connected to the extension unit and then connected to G.

[0160] In an embodiment, in the compound of Formula II or a pharmaceutically acceptable salt thereof, certain groups are defined as follows, and the definitions of the groups not mentioned are as described in any of the embodiments of the present application (hereinafter this paragraph is referred to as “in an embodiment”).

[0161] In an embodiment, ring G, L 4 , R 1 , and R 2 are defined as follows:

[0162] Scheme I:

[0163] ring G is a C 5-10 alkene ring, a 5-10 membered heteroalkene ring, a C 5-10 aldehyde ring, a 5-10 membered heteroaldehyde ring, C a alkene ring substituted with one or more R 5-10 or a 5-10 membered heteroalkene ring substituted with one or more R a ;

[0164] L 4 is a single amino acid residue;

[0165] R 1 and R 2 are independently H or deuterium;

[0166] Scheme II:

[0167] ring G is a C 5-10 aldehyde ring, a 5-10 membered heteroaldehyde ring, or

[0168] L 4 is a single bond or a single amino acid residue;

[0169] R 1 and R 2 Independently, it can be either H or deuterium;

[0170] The heteroatom types of each of the 5-10 member heteroolefin rings and 5-10 member heteroyne rings are independently selected from one, two, or three of N, O, or S; the number of heteroatoms is independently one, two, or three.

[0171] L 1 L 2 L 3 L 4 A3, A4 p, X 1 R 1 and R 2 The definition is as described in any of the drug conjugates shown in Formula I.

[0172] In one scheme, in ring G, the C 5-10 The number of olefin bonds in olefin rings and 5-10 member heteroolefin rings can be 1, 2 or 3 independently.

[0173] In one scheme, in ring G, the C 5-10 The olefin ring is C 5-6 Olefin ring.

[0174] In one embodiment, in ring G, the 5-10 membered heteroolefin ring is a 5-6 membered heteroolefin ring, and the heteroatom type of the 5-6 membered heteroolefin ring can be N, and the number of heteroatoms can be 1; for example For example for

[0175] In one scheme, in ring G, the C 5-10 The number of alkyne bonds in alkyne rings and 5-10 membered heteroyne rings can be independently 1, 2, or 3, for example, 1.

[0176] In one scheme, in ring G, the C 5-10 The alkyne ring is C 7-10 Alkyne ring, the C 5-10 Alkyne rings can be monocyclic or fused rings, for example...

[0177] In one embodiment, in ring G, the 5-10 member heteroyne ring is a 7-10 member heteroyne ring, and the heteroatom type of the 7-10 member heteroyne ring is N, with one or two heteroatoms.

[0178] In a certain scheme, scheme I, ring G is C. 5-10 Alkyne rings, 5-10 heteroyne rings, Or by one or more R a Substituted 5-10 membered heteroolefin rings; preferably, ring G is C. 7-10 Alkyne rings, 7-10 heteroyne rings, Or by one or more R a The substituted 5-6 membered heteroolefin ring; the heteroatom type of the 5-6 membered heteroolefin ring is N, and the number of heteroatoms is 1; the heteroatom type of the 7-10 membered heteroyne ring is N, and the number of heteroatoms is 1 or 2.

[0179] In one scheme, scheme II, ring A is C. 5-10 Alkyne rings, 5-10 heteroyne rings or The preferred location is C. 5-10 Alkyne ring or For example, C 5-10 The alkyne ring is C 7-10 Alkyne ring.

[0180] In one scheme, ring G is V is CH2 or C(O), t1 is 1 or 2, t2 is 0, 1, 2, or 3; ring A3 and ring A4 are independently C 6-10 Aromatic ring, p is 0 or 1; X 1 It can be CH2 or NH.

[0181] In one of the solutions, for

[0182] In one scheme, ring G is C. 7-10 Alkyne rings, 7-10 heteroyne rings, Or by one or more R a The substituted 5-6 membered heteroolefin ring; the heteroatom type of the 5-6 membered heteroolefin ring is N, and the number of heteroatoms is 1; the heteroatom type of the 7-10 membered heteroyne ring is N, and the number of heteroatoms is 1 or 2;

[0183] Each R a For oxygenation;

[0184] Rings A3 and A4 are independently C 6-10 Aromatic rings;

[0185] X 1 It is CH2 or NH;

[0186] L 1 for L 1 carbonyl end and L 2 connect;

[0187] Each R b and Rc independently H or deuterium;

[0188] each q1 is independently 1, 2, 3, 4, 5, or 6;

[0189] each q2 is independently an integer from 1 to 16;

[0190] M is O or

[0191] L 2 is a dipeptide residue, the amino terminus of which is attached to L 1 , the carbonyl terminus is attached to L 3 ;

[0192] L 3 is R d is H or deuterium;

[0193] L 4 is a single amino acid residue, the amino terminus of which is attached to L 3 , the carbonyl terminus is attached to D;

[0194] R 1 and R 2 are independently H or deuterium.

[0195] In one aspect,

[0196] Ring G is a C 7-10 alkyne ring or

[0197] Ring A3 and Ring A4 are independently a C 6-10 aromatic ring;

[0198] X 1 is CH2 or NH;

[0199] L 1 is L 1 , the carbonyl terminus is attached to L 2 ;

[0200] M is O or

[0201] each R b and R c are independently H or deuterium;

[0202] each q1 is independently 1, 2, 3, 4, 5, or 6;

[0203] each q2 is independently an integer from 1 to 16;

[0204] L 2is a dipeptide residue, the amino terminus of which is attached to L 1 is a dipeptide residue, the amino terminus of which is attached to L 3 ;

[0205] L 3 is a single bond or a single amino acid residue, the amino terminus of which is attached to L R d is H or deuterium;

[0206] L 4 is a single bond or a single amino acid residue, the amino terminus of which is attached to L 3 , the carbonyl terminus of which is attached to D;

[0207] R 1 and R 2 are independently H or deuterium.

[0208] In one embodiment, the compound of Formula II is any one of the compounds listed in Table C.

[0209] The present application provides a compound of Formula III, or a pharmaceutically acceptable salt thereof:

[0210] R 1 and R 2 are independently H, deuterium or C 1-6 alkyl;

[0211] R 3 is

[0212] Y 1 is -OH, -SH or -NH2;

[0213] s is 1, 2, 3, 4, 5 or 6;

[0214] R 3a and R 3b are independently hydrogen, deuterium, -COOH, C 1-6 alkyl, C 1-6 alkoxy or C 3a-1 alkyl substituted with one or more R 1-6 ;

[0215] R 3a-1 is independently halogen, -OH, -SH, -NH2, -COOH or C 6-10 aryl.

[0216] In one embodiment, R 3 is a single amino acid residue that has lost a hydroxyl group; the amino acid residue is a natural amino acid residue or a non-natural amino acid residue.

[0217] In one embodiment, R 3 is Y 1 is -OH or -SH.

[0218] In one embodiment, R 3a is hydrogen or C 1-6 alkyl, for example hydrogen or methyl, and for example hydrogen.

[0219] In one embodiment, R 3b is hydrogen, -COOH, C 1-6 alkyl or C 3a-1 alkyl substituted by one or more R 1-6 , for example hydrogen, -COOH, methyl, ethyl or n-butyl, and for example H.

[0220] In one embodiment, R 1 and R 2 are H.

[0221] In one embodiment, R 2 is methyl.

[0222] In one embodiment, R 3 is Y 1 is -NH2; preferably, R 3a is hydrogen, R 3b is hydrogen or C 3a-1 alkyl substituted by one or more R 1-6 , R 3a-1 is independently -OH or -COOH, and m is 1, 2, 3, 4, 5 or 6.

[0223] In one embodiment, R 3 is for example for example for example

[0224] In one embodiment, R 3 is

[0225] In one embodiment, the compound of Formula III is any one of the following:

[0226] The present application provides a method for preparing a drug conjugate of Formula I, which is Method 1 or Method 2 as follows:

[0227] Method 1 comprises the following steps: coupling reaction of an antibody with a compound shown in formula II or a pharmaceutically acceptable salt thereof in a solvent in the presence of a reducing agent to obtain a drug conjugate shown in formula I,

[0228] wherein ring G is C 5-10 alkane ring, 5-10 membered heteroalkane ring or 5-10 membered heteroalkane ring substituted by one or more R a substituted 5-10 membered heteroalkane ring; ring A is C 5-10 alkane ring, 5-10 membered heteroalkane ring or 5-10 membered heteroalkane ring substituted by one or more R a substituted 5-10 membered heteroalkane ring; Z 1 is a single bond; T, D, n, ring A, ring G, L 1 , L 2 , L 3 and L 4 The definitions of T, D, n, ring A, ring G, L

[0229] Method 2 comprises the following steps: reaction of an antibody shown in formula IV with a compound shown in formula II or a pharmaceutically acceptable salt thereof in a solvent to obtain a drug conjugate shown in formula I,

[0230] ring A and ring G are ring A2 is ring A1, ring A3, ring A4, L 1 , L 2 , L 3 , L 4 , D, T, m, n, p, X 1 The definitions of T, D, n, ring A, ring G, L

[0231] The amount of the solvent, the reducing agent and each raw material is conventional in the art and the amount is conventional in such coupling reaction, for example, the solvent is PBS buffer or sodium borate buffer; the reducing agent can be a thiol reducing agent (TCEP).

[0232] The present application provides a pharmaceutical composition comprising (a therapeutically effective amount of) the drug conjugate shown in formula I according to any one of the present application, the compound shown in formula II or III or a pharmaceutically acceptable salt thereof; and, a pharmaceutical excipient.

[0233] The present application provides a use of the drug conjugate shown in formula I according to any one of the present application, the compound shown in formula II or III or a pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition in the preparation of a drug for preventing or treating cancer; the cancer can be breast adenocarcinoma, breast cancer, lung cancer or gastric cancer.

[0234] The application provides a use of the drug conjugate shown in the formula I, the compound shown in the formula II or III or a pharmaceutically acceptable salt thereof or the above-mentioned pharmaceutical composition in any aspect of the application in the preparation of a drug for preventing or treating a cancer related to a HER2 or Trop2 target point; the cancer related to the HER2 or Trop2 target point can be breast adenocarcinoma, breast cancer, lung cancer or gastric cancer.

[0235] The application provides a method for treating a cancer, which comprises administering (a therapeutically effective amount of) the drug conjugate shown in the formula I, the compound shown in the formula II or III or a pharmaceutically acceptable salt thereof or the above-mentioned pharmaceutical composition in any aspect of the application to a subject; the cancer can be breast adenocarcinoma, breast cancer, lung cancer or gastric cancer.

[0236] In an aspect, the cancer is gastric cancer.

[0237] The application provides a method for treating the above-mentioned cancer related to a HER2 or Trop2 target point, which comprises administering (a therapeutically effective amount of) the drug conjugate shown in the formula I, the compound shown in the formula II or III or a pharmaceutically acceptable salt thereof or the above-mentioned pharmaceutical composition in any aspect of the application to a subject.

[0238] Definitions of terms

[0239] As will be understood by those skilled in the art, the use of a name or description of a group in the structural formulae described herein is used according to convention in the art. means that the corresponding group is attached to the rest of the compound through this site.

[0240] In this specification, groups and substituents thereof can be chosen by one skilled in the art to provide stable moieties and compounds. When a substituent is described using a conventional chemical formula written from left to right, the substituent is also meant to include an enantiomeric form written from right to left.

[0241] If a linking group is denoted as "absence or single bond", the structure on both sides of the linking group is directly connected by a single bond, for example, -A-B-C-, when B is a single bond, -A-B-C- is -A-C-.

[0242] In the application, any combination of variables is only allowed if the combination results in a stable compound.

[0243] In the application, any variable, when present more than once in the composition or structure of a compound, is defined independently at each occurrence. For example, when R is substituted with one or more groups, each substituent is an independent substituent and can be the same or different.

[0244] The term "plurality" means 2, 3, 4, or 5, preferably 2 or 3.

[0245] The term "pharmaceutically acceptable" means that the salt, solvent, adjuvant, etc. is generally non-toxic, safe, and neither biologically nor otherwise undesirable, and includes that the subject is preferably a mammal, and more preferably a human.

[0246] The term "pharmaceutically acceptable salt" means a salt of a compound of the present application prepared from a relatively non-toxic, pharmaceutically acceptable acid or base.

[0247] The term "ligand" generally refers to small molecules, polypeptides, RNA, DNA, carbohydrates, and macromolecular compounds that recognize and bind to antigens or receptors associated with a target cell. The role of ligands includes the delivery of a drug to a target cell population with which the ligand binds, and these ligands include antibodies. For example, the antibody is an antibody that targets a HER2 target or a Trop2 target.

[0248] The term "halogen" means fluorine, chlorine, bromine, or iodine.

[0249] The term "alkyl" means a straight or branched chain alkyl group having the specified number of carbon atoms (e.g., C1-C6). Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl, sec-butyl, n-pentyl, n-hexyl.

[0250] The term "C 5-10 Alkanecycle" means a saturated ring system consisting solely of carbon atoms having the specified number of carbon atoms (e.g., C 5-6 ) atoms; preferably a monocyclic ring. C 5-10 Alkanecycles include, but are not limited to, cyclopentane, cyclohexane, and the like. C 5-10 Alkanecycles are attached to the remainder of the molecule by a single bond; or two atoms and a single bond to the remainder of the molecule.

[0251] The term "5-10 membered heteroalkanecycle" means a saturated ring system having the specified number of ring atoms (e.g., 5-6 membered), the specified number of heteroatoms (e.g., 1, 2, or 3), and the specified type of heteroatoms (one or more of N, O, and S), preferably a monocyclic ring. For example, a pyrrolidine ring. The 5-10 membered heteroalkanecycle is attached to the remainder of the molecule by a single bond; or two atoms and a single bond to the remainder of the molecule.

[0252] The term "C 5-10 Alkenecycle" means a ring system consisting solely of carbon atoms having the specified number of carbon atoms (e.g., C 5-6 , C 7-10 ) atoms; containing one or more double bonds and no triple bonds; and having no aromaticity. C 5-10 Alkenecycles include, but are not limited to, cyclopentene, cyclohexene. The C5-10 Olefinic ring is connected to the rest of the molecule by a single bond or shares two atoms and one bond with the rest of the molecule.

[0253] The term "C 5-10 Alkynyl ring" means a cyclic system consisting only of carbon atoms, having the specified number of carbon atoms (e.g., C 7-10 ) containing one or more triple bonds and no double bonds, and no aromaticity. C 5-10 Alkynyl rings include, but are not limited to, cycloheptatriene. The C 5-10 Alkynyl ring is connected to the rest of the molecule by a single bond or shares two atoms and one bond with the rest of the molecule.

[0254] The term "5-10 membered heteroolefinic ring" means a cyclic system having the specified number of ring atoms (e.g., 7-10 membered), the specified number of heteroatoms (e.g., 1, 2, or 3), the specified type of heteroatoms (one or more of N, O, and S), containing one or more double bonds and no triple bonds, and no aromaticity, preferably monocyclic. For example, azacycloheptene ring. The 5-10 membered heteroolefinic ring is connected to the rest of the molecule by a single bond or shares two atoms and one bond with the rest of the molecule.

[0255] The term "aromatic ring" means a cyclic system consisting only of carbon atoms, having the specified number of carbon atoms (e.g., C 6-10 ) which is monocyclic or polycyclic, and each ring is aromatic (satisfies Huckel's rule). Aromatic rings include, but are not limited to, benzene and naphthalene rings, and the like. The aromatic ring is connected to the rest of the molecule by a single bond or shares two atoms and one bond with the rest of the molecule.

[0256] The term "5-10 membered heteroaromatic ring" means a heteroaromatic cyclic system having the specified number of ring atoms (e.g., 5-6 membered, 8-10 membered), the specified number of heteroatoms (e.g., 1, 2, 3), the specified type of heteroatoms (one or more of N, O, and S), preferably monocyclic; for example, triazole ring. The heteroaromatic ring is connected to the rest of the molecule by a single bond or shares two atoms and one bond with the rest of the molecule.

[0257] The term "pharmaceutically acceptable excipient" means an excipient and additional agent used in the manufacture of pharmaceutical products and dispensing of prescriptions, which is all material contained in a drug formulation other than an active ingredient.

[0258] The term "treatment" refers to therapeutic treatment. With respect to a particular condition, treatment refers to: (1) relieving the disease or condition, or one or more of the biological manifestations thereof, (2) interfering with (a) one or more points in the biological cascade leading to or causing the condition or (b) one or more of the biological manifestations of the condition, (3) ameliorating one or more symptoms, effects, or side effects associated with the condition, or one or more symptoms, effects, or side effects associated with the condition or treatment thereof, or (4) slowing the development of the condition or one or more of the biological manifestations thereof.

[0259] The term "prevention" refers to a reduction in the risk of acquiring or developing a disease or disorder.

[0260] The term "therapeutically effective amount" refers to the amount of a compound that, when administered to a patient, is sufficient to effect treatment of a disease or condition as described herein. The "therapeutically effective amount" will vary depending on the compound, the condition and its severity, and the age of the patient to be treated, but can be adjusted by those skilled in the art as needed.

[0261] The term "patient" refers to any animal, preferably a mammal, and most preferably a human, who is to receive or has received administration of the compound or composition according to embodiments of the present application.

[0262] The above-mentioned preferred conditions can be combined in any way, to obtain preferred embodiments of the present application.

[0263] The reagents and materials used in the present application are commercially available.

[0264] The positive progress effect of the present application is that the ADCs of the novel active molecules (irinotecan prodrugs) related to irinotecan have lower toxicity and higher therapeutic activity than the ADCs of irinotecan toxins, effectively improving the safe treatment window of the ADCs. Further, the novel active molecules related to irinotecan of the present application, as well as the linker-payload compounds produced thereby, and the drug conjugates produced therefrom, also have the advantages of being stable in blood, having low toxicity, and having a faster clearance rate. DETAILED DESCRIPTION

[0265] The present application is further illustrated by the following examples, but the present application is not limited to the scope of the examples. The experimental methods in the following examples, for which no specific conditions are indicated, are selected according to conventional methods and conditions, or according to the instructions of the commercial products.

[0266] General synthesis method of irinotecan derivatives (novel active molecules):

[0267] Synthesis route 1:

[0268] Step one: Eribulin (1.0 equiv.) and N-Fmoc-amino acid (P#-1) (1.1 equiv.) were mixed and dissolved in N,N-dimethylformamide (1 mL per 10 mg of eribulin) at room temperature. To the solution, HATU (1.5 equiv.) and N,N-diisopropylethylamine (2.0 equiv.) were added successively. The resulting reaction solution was stirred at room temperature for 2 hours. The reaction was monitored by LCMS until eribulin was completely converted. The reaction solution was directly purified by reverse phase flash chromatography (C18 column, eluted with 0-60% acetonitrile in water containing 0.01% trifluoroacetic acid) to give compound P#-2.

[0269] Step two: Compound P#-2 (1.0 equiv.) obtained in step one was dissolved in acetonitrile or N,N-dimethylformamide (1 mL per 10 mg of compound P#-2) at room temperature. Diethylamine (10 equiv.) was added to the solution. The resulting mixture was stirred at room temperature for 2 hours. The reaction was monitored by LCMS until the Fmoc protecting group was completely removed. The reaction solution was directly purified by reverse phase flash chromatography (C18 column, eluted with acetonitrile in water containing 0.01% formic acid or trifluoroacetic acid) to give compound P# or compound P#-3 with other protecting groups. P#-3 was further deprotected to give compound P#.

[0270] Example 1. Synthesis of a novel active molecule related to eribulin (glycine eribulin) P1

[0271] According to the general synthetic method of eribulin derivatives, starting from eribulin (40 mg, 51 μmol) and N-Fmoc-glycine (P1-1), glycine eribulin P1 was obtained as a white solid (32 mg, 80% yield over two steps) via step one and step two in synthetic route 1. ESI m / z: 787.5 (M+H) + .

[0272] The synthesis of eribulin derivatives in the following table is the same as Example 1:

[0273] Example 2: Synthesis of a novel active molecule related to eribulin (serine eribulin) P3

[0274] Following the general synthetic procedure for Eribulin derivatives, white solid P3-3 (16 mg, 45% yield over two steps, trifluoroacetate salt) was obtained from Eribulin (25 mg, 34 μmol) and O-[(tert-butyl)dimethylsilyl]-N-[fluorenylmethoxycarbonyl]-L-serine (P3-1, N-Fmoc-Ser(TBDMS)-OH) starting from step one and step two in Synthetic Route 1. ESI m / z: 931.5 (M+H) + .

[0275] Step three: The resulting white solid P3-3 (6 mg, 5.7 μmol) was dissolved in dichloromethane (2 mL) and trifluoroacetic acid (0.4 mL) was added. The resulting reaction was stirred at room temperature for 4 hours. The reaction was monitored by LCMS until the complete removal of the protecting group and the reaction was concentrated under reduced pressure. The crude product was purified by reverse phase flash chromatography (C18 column eluted with 0-60% acetonitrile in water containing 0.01% trifluoroacetic acid) to give white solid P3 (2 mg, trifluoroacetate salt, 37% yield). ESI m / z: 817.6 (M+H) + .

[0276] Example 3: Synthesis of a novel active molecule related to Eribulin (lysine eribulin) P4

[0277] Step one: N 6 -Fmoc-N 2 -Boc-lysine (P4-1) (16 mg, 34 μmol) was dissolved in N,N-dimethylformamide (1 mL / 10 mg of Eribulin) and to this solution was added HATU (15 mg, 41 μmol), N,N-diisopropylethylamine (5 mg, 41 μmol) and Eribulin (25 mg, 34 μmol) sequentially. The resulting reaction was stirred at room temperature for 2 hours. The reaction was monitored by LCMS until the complete conversion of Eribulin and the reaction was directly purified by reverse phase flash chromatography (C8 column eluted with 0-85% acetonitrile in water containing 0.01% trifluoroacetic acid) to give compound P4-2 (30 mg, 75% yield). ESI m / z: 1180.7 (M+H) + .

[0278] Step two: To a solution of compound P4-2 (30 mg, 25 μmol) in dichloromethane (2 mL) was added trifluoroacetic acid (0.2 mL) at room temperature. The resulting mixture was stirred at room temperature for 2 hours. The reaction was monitored by LCMS until the Boc protecting group was completely removed. The reaction mixture was concentrated and the resulting crude product was purified by reverse phase flash chromatography (C8 column, eluted with 0-56% acetonitrile in water containing 0.01% trifluoroacetic acid) to give compound P4-3 (23 mg, 85% yield) as a white solid. ESI m / z: 1080.4 (M+H) + .

[0279] Step three: To a solution of compound P4-3 (8 mg, 7 μmol) in N,N- dimethylformamide (1 mL) was added diethylamine (1 mL) at room temperature. The resulting mixture was stirred at room temperature for 2 hours. The reaction was monitored by LCMS until the Fmoc protecting group was completely removed. The reaction mixture was directly purified by reverse phase flash chromatography (C8 column, eluted with 0-51% acetonitrile in water containing 0.01% trifluoroacetic acid) to give P4 (5.2 mg, 82% yield) as a white solid. ESI m / z: 859.7 (M+H) + .

[0280] Example 4: Synthesis of a novel active molecule related to eribulin (glutamic acid eribulin) P5

[0281] Step one: According to the general procedure for synthesis of eribulin derivatives, step one, starting from eribulin (25 mg, 34 μmol) and N-Fmoc-L- glutamic acid-γ-methyl ester (CAS: 145038-50-2) gave P5-1 (27 mg, 72% yield) as a white solid. ESI m / z: 1095.7 (M+H) + .

[0282] Step two: To a solution of compound P5-1 (27 mg, 25 μmol) in acetonitrile / water (1 mL / 1 mL) was added solid lithium hydroxide (0.6 mg, 25 μmol) at room temperature. The resulting mixture was stirred at room temperature for 1 hour. The reaction was monitored by LCMS until the methyl ester and Fmoc were completely removed. The reaction mixture was directly purified by reverse phase flash chromatography (C18 column, eluted with 0-50% acetonitrile in water containing 0.01% trifluoroacetic acid) to give P5 (12.9 mg, 60% yield) as a white solid. ESI m / z: 859.1 (M+H) + .

[0283] Example 5: Synthesis of a novel active molecule related to eribulin (eribulin derivative) P5a

[0284] Following the procedure of Example 4, N-Fmoc-L-glutamic acid- methyl ester (CAS: 145038-49-9) was used to replace N-Fmoc-L-glutamic acid-γ-methyl ester to give eribulin derivative P5a.

[0285] Structural confirmation data of eribulin derivatives

[0286] Synthesis of eribulin and its derivative linkers

[0287] General synthetic procedure of eribulin and derivative-maleimide linkers (L0, L2 or L3-P#) (Scheme 2):

[0288] Scheme 2:

[0289] Step one: To a stirred solution of compound A (Fmoc-vcPAB-PNP) (1 equiv.) in N,N-dimethylformamide (1 mL per 40 mg of compound A) was added eribulin mesylate or eribulin derivative P# or eribulin derivative P#-3 with protecting group (1 equiv.), HOBt (0.5 equiv.) and N,N-diisopropylethylamine (2.0 equiv.) sequentially at room temperature under nitrogen. The resulting mixture was stirred at room temperature for 2 hours and monitored by LCMS until the starting material was completely consumed. The reaction mixture was directly purified by reverse phase flash chromatography (C18 column eluted with 5-65% acetonitrile in water containing 0.1% trifluoroacetic acid) to give Fmoc-protected L0-P#-1, which was lyophilized.

[0290] Step two: The compound obtained above was dissolved in N,N-dimethylformamide (1 mL per 50-60 mg of compound) and diethylamine (5% in N,N-dimethylformamide) was added. The reaction mixture was stirred at room temperature for 2 hours and monitored by LCMS until the Fmoc group was completely removed. The resulting mixture was concentrated under reduced pressure and the residue was purified by reverse phase flash chromatography (C18 column eluted with 5-40% acetonitrile in water containing 0.1% trifluoroacetic acid) to give L0-P#-1 (trifluoroacetate salt), which was lyophilized.

[0291] Step 3: The above obtained compound L0-P#-1 (1 equiv.) was dissolved in N,N- dimethylformamide (1 mL per 50-70 mg of L0-P#-1). To the solution was added N,N- diisopropylethylamine (2.0 equiv.) and the corresponding compound B (1.1 equiv.) sequentially with stirring at room temperature. The reaction was stirred at room temperature for 1-2 hours, monitored by LCMS until the starting material was consumed completely. The resulting mixture was directly purified by reverse phase flash chromatography (C18 column, eluted with 5-75% acetonitrile in water containing 0.1% trifluoroacetic acid) and lyophilized to give eribulin or its derivative-maleimide linker L0, L2 or L3-P#.

[0292] Example 6: Eribulin-maleimide linker L0-P0

[0293] Following the general synthetic procedure for eribulin and its derivative-maleimide linker (L0, L2 or L3-P#) (Scheme 2), compound L0-P0 (30 mg, 27% overall yield for three steps) was obtained from eribulin mesylate (76 mg, 0.10 mmol) via steps one, two and three. ESI m / z: 1375.6 (M+H) + .

[0294] The starting materials in the above table were prepared as eribulin linkers in the following table, all of which were synthesized following the general synthetic procedure for eribulin and its derivative-maleimide linker (L0, L2 or L3-P#) (Scheme 2), as in Example 6:

[0295] General synthetic procedure for eribulin and its derivative-linker (L1, L4 or L5-P#), Scheme 3:

[0296] To a stirred solution of eribulin (mesylate) or eribulin derivative P# or eribulin derivative P#-3 with protecting group (1 equiv.) in N,N-dimethylformamide (1 mL per 40 mg of reactant) was added compound C or D or E (1 equiv.), HOBt (0.5 equiv.) and N,N- diisopropylethylamine (2.0 equiv.) sequentially at room temperature under nitrogen. The resulting mixture was stirred at room temperature for 2 hours, monitored by LCMS until the starting material was consumed completely. The reaction was directly purified by reverse phase flash chromatography (C18 column, eluted with 5-75% acetonitrile in water containing 0.01% trifluoroacetic acid) and lyophilized to give the corresponding eribulin or eribulin derivative linker.

[0297] Synthesis route 4 of Eribulin derivative linker

[0298] For example, L4-P1, the general procedure of synthesis route 4 is as follows:

[0299] Step 1: To a stirred solution of glycine (1 equiv.) in N,N-dimethylformamide (1 mL per 40 mg of reactant) was added active ester D (1.1 equiv.), HOBt (0.5 equiv.) and N,N-diisopropylethylamine (2.0 equiv.) successively at room temperature under nitrogen. The resulting mixture was stirred at room temperature for 2 hours and monitored by LCMS until the starting material was completely converted. The reaction mixture was directly purified by reverse phase flash chromatography (C18 column, eluted with 0.01% trifluoroacetic acid in 5-95% acetonitrile in water) and the corresponding intermediate Fl was obtained after lyophilization.

[0300] Step 2: Eribulin (1.0 equiv.) and intermediate Fl from step 1 (1.1 equiv.) were mixed and dissolved in N,N-dimethylformamide (1 mL per 10 mg of Eribulin) at room temperature. To the solution was added HATU (1.5 equiv.) and N,N-diisopropylethylamine (2.0 equiv.) successively and the resulting reaction mixture was stirred at room temperature for 2 hours. The reaction was monitored by LCMS until Eribulin was completely converted. The reaction mixture was directly purified by reverse phase flash chromatography (C18 column, eluted with 0.01% trifluoroacetic acid in 0-60% acetonitrile in water) to give compound L4-P1 (3.3 mg, 40% yield (calculated based on Eribulin mesylate)). ESI m / z: 1604.7 (M+H) + .

[0301] Example 7: Glycine Eribulin-linker L1-P1

[0302] Following the general procedure for the synthesis of Eribulin and derivative-linker (L1, L4 or L5-P#) (synthesis route 3), compound L1-P1 (4.3 mg, 28% yield) was obtained starting from glycine Eribulin (8.5 mg, 11 μmol) and active ester C. ESI m / z: 1386.6 (M+H) + .

[0303] The synthesis of Eribulin and derivative-linkers in the following table was carried out following the general procedure for the synthesis of Eribulin and derivative-linker (L1, L4 or L5-P#) (synthesis route 3), as in Example 7:

[0304] The synthetic route, yield, ESI data of the above compounds are shown in the following table.

[0305] Example 8: Lysine eribulin-linker L4-P4

[0306] Step one: according to the general synthetic procedure of eribulin and its derivatives-linker (L1, L4 or L5-P#) (synthetic route 3), the reaction of P4-3 (10 mg, 9.2 μmol) and compound D gave Fmoc-protected L4-P4 (13 mg, yield 72%) as a white solid. ESI m / z: 1897.8 (M+H) + .

[0307] Step two: the compound (13 mg) from step one was dissolved in N,N- dimethylformamide (1 mL) at room temperature, diethylamine (0.2 mL) was added to the reaction solution. The reaction solution was stirred at room temperature for 1 hour, the reaction was monitored by LCMS until Fmoc was completely removed. The reaction solution was directly purified by reverse phase flash chromatography (C8 column, eluted with 0-50% acetonitrile in water containing 0.01% trifluoroacetic acid) and lyophilized to give L4-P4 (7.9 mg, yield 72%) as a white solid. ESI m / z: 838.3 (M / 2+H) + .

[0308] Example 9: Serine eribulin-linker L4-P3

[0309] Step one: according to the general synthetic procedure of eribulin and its derivatives-linker (L1, L4 or L5-P#) (synthetic route 3), the reaction of P3-3 (6.5 mg, 7 μmol) and compound D gave TBDMS-protected L4-P3 (9.2 mg, yield 76%) as a white solid. ESI m / z: 874.5 (M / 2+H) + .

[0310] Step two: the compound (9.2 mg) from step one was dissolved in THF (1.5 mL) at room temperature, TBAF (1.5 mg, 6 μmol) was added to the reaction solution. The reaction solution was stirred at room temperature for 2 hours, the reaction was monitored by LCMS until TBDMS was completely removed. The reaction solution was directly purified by reverse phase flash chromatography (C8 column, eluted with 0-60% acetonitrile in water containing 0.01% trifluoroacetic acid) and lyophilized to give L4-P3 (5.5 mg, yield 67%) as a white solid. ESI m / z: 817.8 (M / 2+H) + .

[0311] Example 10: Lysine eribulin-N 6 Linker L4-P4’

[0312] Using a similar synthetic procedure as P4-3, except that N 2 -Fmoc-N 6 -Boc-Lysine (P4’-1) instead of N 6 -Fmoc-N 2 -Boc-Lysine (P4-1), by the procedure of Step One and Step Two, to give P4’-3 as a white solid (15.8 mg, 44% yield). ESI m / z: 1080.9 (M+H) + .

[0313] Using P4’-3 instead of P4-3, by the procedure of Step One and Step Two of Example 8, to give L4-P4’ as a white solid (2.2 mg, 10% yield). ESI m / z: 838.4 (M / 2+H) + .

[0314] Drug linker analysis data

[0315] Synthesis of antibody-drug conjugates of eribulin and its derivatives

[0316] Example 11: Glutamic acid-maleimide conjugation to synthesize antibody-drug conjugates

[0317] General procedure for glutamic acid-maleimide conjugation

[0318] Take 2 mg of Trastuzumab (ChemPartner, CP202307101801-MA), dilute to 5 mg per milliliter with PBS buffer, and add TCEP (8 equivalents of TCEP when the DAR of the target ADC is 8; 2.7 equivalents of TCEP when the DAR of the target ADC is 4), and reduce for 2 hours at 37 degrees Celsius. After cooling to room temperature, add the DMSO solution of the corresponding drug linker (15 equivalents of drug linker when the DAR of the target ADC is 8; 9 equivalents of drug linker when the DAR of the target ADC is 4), with a DMSO ratio of 10% of the total volume, and react for 2 hours at 25 degrees Celsius. Purify through a desalting column, and filter through a 0.22 μm sterilization filter to obtain the antibody-drug conjugate.

[0319] The drug conjugates obtained by the method of Example 11, and their DAR values, purity, are shown in the following table:

[0320] Example 12: Synthesis of antibody-drug conjugate by two-step conjugation method of transglutaminase (MTG)

[0321] General operation procedure of two-step conjugation method of transglutaminase (MTG)

[0322] Step 1: Mutated Trastuzumab antibody (N297A, ChemPartner, CP202305311801-MA) or N297Q mutated Trastuzumab antibody (ChemPartner, CP202309011801-MA) was dialyzed against sodium borate buffer (25 mM, pH 7.4) for 24 hours with two buffer changes. The collected antibody was quantified by UV absorbance method. If the concentration was too low, it was concentrated to a concentration of >1.0 mg / ml by ultrafiltration. The mixture was mixed with sodium borate buffer solution (0.98 mg / mL, final concentration 1 U / mg Ab) of MTG enzyme (Jiangsu Yiming Biological, purified by gel filtration, 4495.5 U / g) and DMSO solution (50 mM) containing a linker with primary amino groups and azido groups (such as NH2-PEG3-N3, CAS: 134179-38-7). The mixture was reacted at 37°C for 24 hours. The reaction was tracked by SDS gel electrophoresis. The reaction product was desalted column chromatography to remove uncoupled small molecules and DMSO, analyzed by LC-MS for drug-antibody ratio (DAR), and analyzed by SEC-HPLC for aggregation to obtain an antibody with an azido group.

[0323] Step 2: The antibody with azido group obtained in step 1 was mixed with a linking unit with alkyne (such as COT, DBCO, etc.) in a molar ratio of 1:10 to 1:30 in sodium borate buffer (25 mM, pH 7.4). The mixture was reacted at 25 or 37°C for 24-96 hours, and the reaction was tracked by SDS gel electrophoresis, the conjugate was purified by desalting column chromatography, the drug-antibody ratio (DAR) was analyzed by LC-MS, and the aggregation was analyzed by SEC-HPLC.

[0324] The drug conjugates obtained by the method of Example 12 and their DAR values, purity are shown in the following table:

[0325] The structure of the ADC is shown in the following table:

[0326] Cell activity test of antibody conjugate of eribulin and its derivatives

[0327] Example 13: Inhibition effect of antibody-drug conjugate on cells

[0328] The cell lines used for the inhibition of proliferation test were SK-BR-3 (ATCC), JIMT-1 (ATCC) and MDA-MB-231 (ATCC); the cells were cultured in McCoy's 5a Medium + 10% FBS. In a 96-well plate, each well was added with cells (80 μL, 1000 cells) and incubated in a 37°C carbon dioxide incubator for 24 hours. Then, different concentrations (ADC concentrations were 150, 30, 6, 1.2, 0.24, 0.048, 0.0096, 0.0019, 0.0004 nM respectively; eribulin derivative concentrations were 1000, 200, 40, 8, 1.6, 0.32, 0.064, 0.0128, 0.0026 nM respectively) of test compounds (20 μL) were placed in the appropriate cell culture solution (total amount 0.1 mL) for treatment. The control wells contained cells and culture medium but no test compounds. Incubation was carried out in a 37°C carbon dioxide incubator for 144 hours. The 96-well plate was taken out, and the cell state was observed under a microscope. White bottom membrane was pasted, CTG reagent (100 μL) was added to the wells, the plate was shaken at room temperature for 10 minutes, and then was allowed to stand for 10 minutes. The luminescence was recorded by a Microplate Reader.

[0329] The calculation formula of inhibition rate was: Survival% = ((T sample -T blank ) / (T DMSO -T blank )) x 100%. Here, the IC 50 The calculated values were based on the concentration of eribulin corresponding to the conjugate, i.e. Conc. = Conc. (ADC) x DAR, and the results are shown in the following table. The tested ADCs had strong inhibition rates in Her2-positive cells SKBR3; the inhibition rates of the tested ADCs were weak in Her2-negative cells MDA-MB-231; and in JIMT-1 cells, the ADC with DXd as the drug had no inhibition rate, while the antibody conjugates of eribulin and its derivatives had strong inhibition rates. In the SKBR3 cell line, the inhibition rate of the eribulin derivative was 85 times higher than that of eribulin, and the inhibition rates of the antibody conjugates of the eribulin derivative and eribulin were comparable.

[0330] Example 14: Pharmacodynamic experiment of antibody conjugate of eribulin-related novel active molecule on NCI-N87 & MDA-MB-468-Luc xenograft tumor model in mice.

[0331] HER2 positive NCI-N87 cells and HER2 negative MDA-MB-468-Luc cells were mixed at a ratio of 1:3. After mixing the cell suspension (containing 3x10 NCI-N87 cells and 9x106 MDA-MB-468-Luc cells) with Matrigel matrix, the mixture was subcutaneously inoculated into the right side of CB17 SCID mice. Luc fluorescence intensity represents the growth status of MDA-MB-468-Luc cells in vivo.

[0332] After 7 days of inoculation, the tumor volume and luciferase signal obtained by in vivo imaging were randomly divided into 9 groups. Except for the control group, different antibody drug conjugates were injected into each group through the tail vein. A single dose of 1 mg / kg or 5 mg / kg was observed for 28 days. The experimental results are as follows:

[0333] The anti-HER2 antibody drug conjugate delivers chemotherapeutic drugs to HER2 positive NCI-N87 cells due to its antibody targeting effect, showing strong cell killing activity; for HER2 negative cells MDA-MB-468-Luc, it generally does not show killing activity. If NCI-N87 and MDA-MB-468-Luc cells are mixed and transplanted into animals, i.e. in the mouse NCI-N87 & MDA-MB-468-Luc xenograft tumor model, the observation of the inhibitory or killing effect of antibody conjugates on NCI-N87 & MDA-MB-468-Luc co-growth tumors, i.e. the bystander effect of MDA-MB-468-Luc killing.

[0334] T-DM1 is a representative antibody conjugate without bystander effect, and the tumor inhibition rate TGI% and Luc fluorescence intensity inhibition rate of the mouse NCI-N87 & MDA-MB-468-Luc xenograft tumor model are low. The antibody conjugates ADC13, ADC31, ADC51 of the new active molecules related to eribulin provided in this patent have strong tumor inhibition rate TGI% and Luc fluorescence intensity inhibition rate in the mouse NCI-N87 & MDA-MB-468-Luc xenograft tumor model, showing obvious tumor growth inhibition and bystander killing effect.

Claims

1. A drug conjugate of formula I, ###0001### I wherein T is a ligand or targeting moiety that binds to a target, n is a drug ligand coupling ratio; is a linker fragment of a linker connected to T; L 1 for connecting the units; L 2 a single amino acid residue or a short peptide residue consisting of 2-10 amino acid residues; L 3 spacer; L 4 is a single bond or Y connects L 3 ; Y is O, S or NH; s is 1, 2, 3, 4, 5 or 6; R 3a and R 3b are independently hydrogen, deuterium, -COOH, C 1-6 alkyl, C 1-6 alkoxy or C 3a-1 alkyl substituted by one or more R 1-6 ; R 3a-1 independently halogen, -OH, -SH, -NH2, -COOH, or C 6-10 aryl; D is R 1 and R 2 independently H, deuterium, or C 1-6 alkyl; and the drug conjugate of Formula I satisfies any one of the following conditions: Condition (1): L 4 To Condition (2): In some embodiments, Z is -CH2-. 1 For m is 1, 2, 3, 4, or 5; ring A is Ring A1is C 5-10 an olefinic ring or a 5-10 membered heteroolefinic ring; each ring A2 is independently a 5-10 membered heteroaromatic ring; Rings A3and A4are independently C 5-10 alkane ring, 5-10 membered heteroalkane ring, C 5-10 alkene ring, 5-10 membered heteroalkene ring, C 6-10 aromatic ring or 5-10 membered heteroaromatic ring; p is 0, 1, 2 or 3; X 1 is CH, CH2, N or NH; is a single bond or a double bond; the heteroatom species of each 5-10 membered heteroalkane ring, 5-10 membered heteroalkene ring and 5-10 membered heteroaromatic ring is independently selected from one, two or three of N, O or S, and the number of heteroatoms is independently 1, 2 or 3; the amino acid residue is a natural amino acid residue or a non-natural amino acid residue.

2. The drug conjugate of claim 1, represented by Formula I: ###0001### I L 4 is a single bond or a single amino acid residue; R 1 and R 2 is independently H or deuterium; the amino acid residue is a natural amino acid residue or a non-natural amino acid residue; In the condition (1), L 4 is a single amino acid residue; Preferably, in the drug conjugate of Formula I, T is an antibody, n is a natural number or a decimal number from 1 to 16; Z 1 is a single bond or m is 1, 2, 3, 4 or 5; Ring A is C 5-10 alkane ring, 5-10 membered heteroalkane ring, substituted with one or more R a substituted with one or more R 5- 10 substituted with one or more R a substituted with one or more R each R a is oxo; Ring A1is C 5-10 an olefinic ring or a 5-10 membered heteroolefinic ring; each ring A2 is independently a 5-10 membered heteroaromatic ring; Rings A3and A4are independently C 5-10 alkane ring, 5-10 membered heteroalkane ring, C 5-10 alkene ring, 5-10 membered heteroalkene ring, C 6-10 aromatic ring or 5-10 membered heteroaromatic ring; p is 0, 1, 2 or 3; X 1 is CH, CH2, N or NH; is a single bond or a double bond; L 1 For each R b and R c is independently H, deuterium, or halogen; each q1 is independently 1, 2, 3, 4, 5 or 6; each q2 is independently an integer from 1 to 16; M is NH, S, O or L 2 is a single amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue; L 3 For R d H, deuterium or halogen; the heteroatom species of each 5-10 membered heteroalkane ring, 5-10 membered heteroalkene ring and 5-10 membered heteroaromatic ring is independently selected from one, two or three of N, O and S, and the number of heteroatoms is independently 1, 2 or 3.

3. The drug conjugate of Formula I as described in claim 2, characterized in that, satisfies one or more of the following conditions: (1) in ring A, the heteroatom species of each 5-10 membered heteroalkane ring is N, and the number of heteroatoms can be 1; (2) in ring A, each 5-10 membered heteroalkane ring is independently a 5-6 membered heteroalkane ring; (3) in ring A1, the C 5-10 the number of olefinic bonds in the olefinic and 5-10 membered heteroolefinic rings is independently 1, 2, or 3, e.g., 1; (4) in ring A1, the C 5-10 the olefinic ring is C 7-10 the olefinic ring; the C 5-10 the olefinic ring can be monocyclic or fused; (5) in ring A1, the 5-10 membered heteroalkene ring is a 7-10 membered heteroalkene ring; (6) in ring A2, the heteroatom species of the 5-10 membered heteroaromatic ring is N, and the number of heteroatoms is independently 1, 2 or 3, for example, 3; (7) in ring A2, the 5-10 membered heteroaromatic ring is a 5-6 membered heteroaromatic ring; (8) in ring A3and ring A4, the C 6-10 the aromatic ring is a benzene ring or a naphthalene ring, for example a benzene ring; (9) R b , R c , and R d , wherein the halogen is independently fluorine, chlorine, bromine, or iodine; (10) in each of ring A, ring A3and ring A4, each C 5-10 alkane ring is independently C 5-6 alkane ring; (11) in ring A3 and ring A4, the heteroatom species of each 5-10 membered heteroalkane ring is N, and the number of heteroatoms can be 1; (12) in rings A3and A4, said C 5-10 the number of olefinic bonds in the olefinic ring and the 5-10 membered heteroolefinic ring is independently 1, 2, or 3, said C 5-10 the olefinic ring can be a C 7-10 olefinic ring; the 5-10 membered heteroolefinic ring can be a 7-10 membered heteroolefinic ring; (13) in ring A3 and ring A4, the 5-10 membered heteroaromatic ring is independently a 5-6 membered heteroaromatic ring; Preferably, the drug conjugate of Formula I satisfies one or more of the following conditions: (1) at each occurrence, each 5-10 membered heteroalkylene ring within ring A is independently aziridine; for example For For example, N atom and L 1 Connection; (2) in ring A1, the C 5-10 the olefinic ring is (3) in ring A2, the 5-10 membered heteroaromatic ring is a triazole ring, for example 4. The drug conjugate of Formula I as described in claim 1 or 2, characterized in that, satisfies one or more of the following conditions: (1) T is an anti-HER2 antibody or a variant thereof or an anti-Trop2 antibody or a variant thereof, which maintains at least equivalent antigen binding function to the antibody; for example, an anti-HER2 antibody or a variant thereof; for example, Trastuzumab or a mutant thereof, which has a difference in the amino acid residue at position 297 in the amino acid sequence of Trastuzumab; preferably, the amino acid residue at position 297 in the amino acid sequence of Trastuzumab is replaced by A or Q; (2) n is a natural number or a decimal number from 1 to 8; for example, 1.31, 1.56, 1.91, 3.03, 3.22, 3.63, 3.67, 3.75, 3.78, 3.82, 3.83, 3.91, 4, or 8; (3) m is 3; (4) Ring A is or substituted with one or more R a a substituted 5-10 membered heteroalkane ring; preferably, the 5-10 membered heteroalkane ring is a 5-6 membered heteroalkane ring, the heteroatom species of which is N, the number of heteroatoms of which is 1; more preferably, ring A is or substituted with one or more R a substituted 5-6 membered heteroalkyl ring having one heteroatom Alternatively, ring A is "1" position is connected to Z 1 , the "2" position is connected to L 1 , V is CH2or C(O), t1 is 1 or 2, t2 is 0, 1, 2 or 3; ring A3 and ring A4 are independently C 6-10 aromatic ring, p is 0 or 1; X 1 is CH2or NH, for example ring A is (5) Ring A1is C 5-10 an olefinic ring; preferably, Ring A1is C 7-10 an olefinic ring; (6) ring A2 is a 5-6 membered heteroaromatic ring; the heteroatom species of the 5-6 membered heteroaromatic ring is N, and the number of heteroatoms is 1, 2, or 3; (7) Ring A3and Ring A4are independently C 6-10 aromatic ring; for example, a benzene ring; (8) is a double bond; (9) p is 1; (10) X 1 is CH2or NH, for example NH; (11) Ring A is Time, Z 1 To -NH- is connected to T; (12) Ring A is C 5-10 alkane ring, 5-10 membered heteroalkane ring, substituted C a substituted C 5-10 alkane ring, or 5-10 membered heteroalkane ring substituted with one or more R a substituted C 1 is a single bond; (13) ring A2and Z 1 connection; (14) each R b and R c is independently H or deuterium, e.g., H; (15) each q1 is independently 1 or 2; (16) each q2 is independently an integer from 1 to 14; for example, 2, 4, or 12; (17) M is O or (18) L 2 is a dipeptide residue, the amino terminus of the dipeptide residue is attached to L 1 , the carbonyl terminus is attached to L 3 ; preferably, the dipeptide residue is NH -Val-Cit- C＝O , NH -Cit-Val- C＝O ; wherein the amino end is attached to L 1 and the carbonyl end is attached to L 3 and the carbonyl end is attached to L or L is a dipeptide residue, a tripeptide residue or a tetrapeptide residue, wherein each amino acid residue in L is independently a natural amino acid residue, e.g. Val, Leu, Lle, Gly, Ala, Cit or Arg; and 2 or L is a dipeptide residue, a tripeptide residue or a tetrapeptide residue, wherein each amino acid residue in L is independently a natural amino acid residue, e.g. Val, Leu, Lle, Gly, Ala, Cit or Arg; and 2 or L is a dipeptide residue, a tripeptide residue or a tetrapeptide residue, wherein each amino acid residue in L Preferably, L 2 is L 2a For L 2b for (19) R d H or deuterium, for example H; (20) L 4 is either of the following, Scheme 1 : L 4 is a single amino acid residue, the amino terminus of which is attached to L 3 , the carbonyl terminus is attached to D; Scheme 2: L 4 is a single bond; Scheme 3: L 4 is Y is O or S; Scheme 4: L 4 is Y is NH; (21) R 3a is hydrogen or C 1-6 alkyl, for example hydrogen or methyl, and furthermore, for example, hydrogen; (22)R 3b For hydrogen, -COOH, C 1-6 Alkyl or with one or more R 3a-1 Replacement C 1-6 Alkyl groups, such as hydrogen, -COOH, methyl, ethyl, or n-butyl, and for example, H; (23) R 1 is H; (24) R 2 is H or R 2 is methyl.

5. The drug conjugate of Formula I as described in claim 1 or 2, characterized in that, which satisfies one or more of the following conditions: (1) -Z 1 - is a single bond or -NH- is connected to T; (2) Ring A is For Preferably, the group at position 1 is connected to Z 1 and the group at position 2 is connected to L 1 ; more preferably, For wherein 1 is connected to Z 1 and 2 is connected to L 1 and 1 is connected to L (3) L 1 To Preferably, the "1 " position is attached to ring A and the "2" position is attached to L 2 is attached to ring A; more preferably, L 1 is the "1 " position is attached to ring A and the "2" position is attached to L 2 attached; (4) L 2 To wherein the "1" position is attached to L 1 and the "2" position is attached to L 3 and the "2" position is attached to L (5) L 3 To wherein the "1" position is attached to L 2 and the "2" position is attached to L 4 and the "2" position is attached to L (6) L 4 is a single bond, L 4 the amino terminus and L 3 the carbonyl terminus; preferably, L 4 is a single bond, L 4 the amino terminus and L 3 the carbonyl terminus; more preferably, L 4 is (7) D is 6. The drug conjugate of Formula I as described in claim 1 or 2, characterized in that, which is the following Scheme 1 or Scheme 2: Scheme 1: T is trastuzumab or a mutant thereof; n is a natural number or a decimal number from 1 to 8; Z 1 is a single bond or -NH- is connected to T; m is 1, 2, 3, 4, or 5; Ring A is or one or more R a substituted 5-6 membered heteroalkyl ring; the 5-6 membered heteroalkyl ring has 1 heteroatom 1 linking; Ring A1is C 7-10 Olefinic ring; ring A2 is independently a 5-6 membered heteroaromatic ring; the heteroatom species of the 5-6 membered heteroaromatic ring is N, and the number of heteroatoms is independently 1, 2, or 3; Rings A3and A4are independently C 6-10 aromatic ring; each R a is oxo; X 1 is CH2or NH; L 1 for L 1 the carbonyl end of L 2 ; each R b and R c is independently H or deuterium; each q1 is independently 1, 2, 3, 4, 5, or 6; q2 is an integer from 1 to 16; M is O or S, and L 2 is a dipeptide residue, the amino terminus of the dipeptide residue is attached to L 1 , the carbonyl terminus is attached to L 3 ; L 3 for R d is H or deuterium; L 4 is a single amino acid residue, the amino terminus of the amino acid residue is attached to L 3 and the carbonyl terminus is attached to D; D is Scheme 2: T is trastuzumab or a mutant thereof; n is a natural number or a decimal number from 1 to 8; Z 1 for -NH- is connected to T; m is 1, 2, 3, 4, or 5; Ring A is Ring A1is C 7-10 Olefinic ring; ring A2 is independently a 5-6 membered heteroaromatic ring; the heteroatom species of the 5-6 membered heteroaromatic ring is N, and the number of heteroatoms is independently 1, 2, or 3; Ring A3 and Ring A4 are independently C 6-10 aromatic ring; X 1 is CH2or NH; L 1 for L 1 the carbonyl end of L 2 is connected to L each R b and R c is independently H or deuterium; each q1 is independently 1, 2, 3, 4, 5, or 6; each q2 is independently an integer from 1 to 16; M is O or S, and L 2 is a dipeptide residue, the amino terminus of the dipeptide residue is attached to L 1 , the carbonyl terminus is attached to L 3 ; L 3 for R d is H or deuterium; L 4 is a single bond or a single amino acid residue, the amino terminus of the amino acid residue is connected to L 3 and the carbonyl terminus is connected to D; D is 7. The drug conjugate of Formula I as described in claim 1, characterized in that, the drug conjugate of the formula I is any one of the following compounds shown in Table A: Table A n is a natural number or a decimal number from 1 to 8; preferably, the drug conjugate of the formula I is any one of the following compounds shown in Table B: Table B 8. A compound of Formula II or a pharmaceutically acceptable salt thereof, wherein, D is is a linker precursor; and the compound of the formula II satisfies any one of the following conditions: Condition (1): L 4 To Condition (2): ring G is C 5-10 alkyne ring, 5-10 heteroalkyne ring, or the heteroatom species of the 5-10 membered heteroalkyne ring is selected from one, two, or three of N, O, or S; the number of heteroatoms is 1, 2, or 3; X 1 , p, ring A3, ring A4, L 1 , L 2 , L 3 , L 4 , R 1 and R 2 are as defined in any one of claims 2-7.

9. The compound of claim 8 having the formula II ###0002### or a pharmaceutically acceptable salt thereof. G, L 4 , R 1 , and R 2 are defined as either Scheme I or Scheme II below: Scheme I: Ring G is C 5-10 olefinic ring, 5-10 membered heteroolefinic ring, C 5-10 acetylenic ring, 5-10 membered heteroacetylenic ring, substituted C a substituted C 5-10 substituted C a substituted C L 4 is a single amino acid residue; R 1 and R 2 are independently H or deuterium; Scheme II: Ring G is C 5-10 alkyne ring, 5-10 heteroalkyne ring, or L 4 is a single bond or a single amino acid residue; R 1 and R 2 is independently H or deuterium; the heteroatom species of each of the 5-10 membered heteroalkane ring, 5-10 membered heteroalkene ring, 5-10 membered heteroalkyne ring, and 5-10 membered heteroaromatic ring is independently selected from one, two, or three of N, O, and S; the number of heteroatoms is independently 1, 2, or 3; preferably, the compound of the formula II satisfies one or more of the following conditions: (1) in ring G, said C 5-10 The number of olefinic bonds in the olefinic ring and the 5-10 membered heteroolefinic ring is independently 1, 2, or 3; said C 5-10 The olefinic ring can be a C 5-6 The olefinic ring; said 5-10 membered heteroolefinic ring can be a 5-6 membered heteroolefinic ring, the kind of heteroatom of said 5-6 membered heteroolefinic ring can be N, the number of heteroatoms can be 1; for example For example For (4) in ring G, said C 5-10 The number of alkynyl groups in an alkynyl ring and 5-10 membered heteroalkynyl ring is independently 1, 2, or 3, e.g., 1; said C 5-10 The alkynyl ring can be a C 7-10 The alkynyl ring can be a C 5-10 The alkynyl ring can be monocyclic or annelated, e.g. the 5-10 membered heteroalkyne ring can be a 7-10 membered heteroalkyne ring, the heteroatom species of the 7-10 membered heteroalkyne ring is N, and the number of heteroatoms is 1 or 2; (5) in Scheme I, ring G is C 5-10 alkyne ring, 5-10 heteroalkyne ring, or substituted with one or more R a substituted 5-10 membered heteroaromatic ring; preferably, ring G is C 7-10 alkyne ring, 7-10 membered heteroalkyne ring, or one or more R a substituted 5-6 membered heteroalkene ring; the heteroatom type of said 5-6 membered heteroalkene ring is N, the number of heteroatoms is 1; the heteroatom type of said 7-10 membered heteroalkyne ring is N, the number of heteroatoms is 1 or 2; More preferably, ring G is V is CH2or C(O), t1is 1 or 2, t2is 0, 1, 2, or 3; ring A3and ring A4are independently C 6-10 aromatic ring, p is 0 or 1; X 1 is CH2or NH; (6) in Scheme II, ring A is C 5-10 alkyne ring, 5-10 heteroalkyne ring, or Preferably, R is C 5-10 alkyne ring or For example, the C 5-10 The alkyne ring is C 7-10 The alkyne ring; more preferably, the compound of the formula II is the following Scheme I or Scheme II: Scheme I: Ring G is C 7-10 alkyne ring, 7-10 membered heteroalkyne ring, or one or more R a substituted 5-6 membered heteroalkene ring; the heteroatom type of said 5-6 membered heteroalkene ring is N, the number of heteroatoms is 1; the heteroatom type of said 7-10 membered heteroalkyne ring is N, the number of heteroatoms is 1 or 2; each R a is oxo; Ring A3 and Ring A4 are independently C 6-10 aromatic ring; X 1 CH2or NH; L 1 for L 1 the carbonyl end of L 2 is connected to L each R b and R c is independently H or deuterium; each q1 is independently 1, 2, 3, 4, 5, or 6; each q2 is independently an integer from 1 to 16; M is O or S, and L 2 is a dipeptide residue, the amino terminus of which is attached to L 1 and the carbonyl terminus is attached to L 3 ; L 3 for R d is H or deuterium; L 4 is a single amino acid residue, the amino terminus of the amino acid residue is attached to L 3 and the carbonyl terminus is attached to D; R 1 and R 2 is independently H or deuterium; Scheme II: Ring G is C 7-10 alkyne ring or Ring A3 and Ring A4 are independently C 6-10 aromatic ring; X 1 is CH2or NH; L 1 for L 1 the carbonyl end of L 2 is attached; M is O or S, and each R b and R c is independently H or deuterium; each q1 is independently 1, 2, 3, 4, 5, or 6; each q2 is independently an integer from 1 to 16; L 2 is a dipeptide residue, the amino terminus of which is attached to L 1 and the carbonyl terminus is attached to L 3 ; L 3 For R d is H or deuterium; L 4 is a single bond or a single amino acid residue, the amino terminus of the amino acid residue is connected to L 3 and the carbonyl terminus is connected to D; R 1 and R 2 are independently H or deuterium.

10. The compound of claim 8 having the formula II ###0002### or a pharmaceutically acceptable salt thereof. the compound of the formula II is any one of the following compounds shown in Table C: Table C 11. A compound of Formula III or a pharmaceutically acceptable salt thereof, R 1 and R 2 are independently H, deuterium, or C 1-6 alkyl; R 3 for Y 1 is -OH, -SH or -NH2; s is 1, 2, 3, 4, 5, or 6; R 3a and R 3b are independently hydrogen, deuterium, -COOH, C 1-6 alkyl, C 1-6 alkoxy or C 3a-1 alkyl substituted by one or more R 1-6 alkyl; R 3a-1 independently halogen, -OH, -SH, -NH2, -COOH, or C 6-10 aryl.

12. The compound of claim 11 having the formula III: ###0002### III or a pharmaceutically acceptable salt thereof. the compound of the formula III satisfies one or two of the following conditions: (1) R 3 is a single amino acid residue that has lost one hydroxyl group; the amino acid residue is a natural amino acid residue or an unnatural amino acid residue; (2) R 3 To Y 1 is -OH or -SH; or R 3 is Y 1 is -NH2; (3) R 3a is hydrogen or C 1-6 alkyl, for example hydrogen or methyl, and furthermore, for example, hydrogen; (4) R 3b is hydrogen, -COOH, C 1-6 alkyl or C 3a-1 substituted C 1-6 alkyl, for example hydrogen, -COOH, methyl, ethyl or n-butyl, and for example H; Preferably, the compound of Formula III satisfies one or more of the following conditions: (1) R 1 is H; (2) R 2 is H or R 2 is methyl; (3) R 3 To For example For example R 3 For 13. The compound of claim 11 having the formula III: ###0003### or a pharmaceutically acceptable salt thereof. The compound of Formula III is any one of the following:

14. A method for preparing a drug conjugate as shown in Formula I, characterized in that, which is the following Method 1 or Method 2: The method 1 comprises the following steps: coupling an antibody with a compound shown as formula II or a pharmaceutically acceptable salt thereof in a solvent in the presence of a reducing agent to obtain a drug conjugate shown as formula I, wherein ring G is C 5-10 alkane ring, 5-10 membered heteroalkane ring, or 5-10 membered heteroalkane ring substituted with one or more R a substituted 5-10 membered heteroalkane ring; ring A is C 5-10 alkane ring, 5-10 membered heteroalkane ring, or 5-10 membered heteroalkane ring substituted with one or more R a substituted 5-10 membered heteroalkane ring; Z 1 is a single bond; T, D, n, ring A, ring G, L 1 , L 2 , L 3 , and L 4 are as defined in any one of claims 1-7; Method 2 comprises the following steps: reacting an antibody shown in formula IV with a compound shown in formula II or a pharmaceutically acceptable salt thereof in a solvent to obtain a drug conjugate shown in formula I, Ring A and Ring G are Ring A2 is Ring A1, Ring A3, Ring A4, L 1 L 2 L 3 L 4 D, T, m, n, p, X 1 The definition can also be as described in any one of claims 1-7.

15. A pharmaceutical composition comprising, which comprises a drug conjugate of Formula I as described in any one of claims 1-7, or a compound of Formula II as described in any one of claims 8-10, or a pharmaceutically acceptable salt thereof, or a compound of Formula III as described in any one of claims 11-13, or a pharmaceutically acceptable salt thereof; and, a pharmaceutical excipient.

16. Use of a drug conjugate of Formula I as described in any one of claims 1-7, or a compound of Formula II as described in any one of claims 8-10, or a pharmaceutically acceptable salt thereof, or a compound of Formula III as described in any one of claims 11-13, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in claim 15, in the manufacture of a medicament for the prevention or treatment of a cancer or a cancer associated with a HER2 or Trop2 target, which cancer and cancer associated with a HER2 or Trop2 target can independently be breast adenocarcinoma, breast carcinoma, lung carcinoma, or gastric carcinoma.

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