Dendritic polymer conjugate and preparation method therefor

Abstract

The present disclosure relates to a dendritic polymer conjugate and a preparation method therefor. Specifically, the present disclosure relates to a dendritic polymer conjugate or a pharmaceutically acceptable salt thereof, and use thereof in medicine. The conjugate comprises a dendritic polymer loaded with a drug and a pharmacokinetic modifier, and an antibody conjugated to the dendritic polymer.

Description

Dendritic polymer couplings and their preparation methods Technical Field

[0001] This disclosure pertains to the pharmaceutical field and relates to a dendritic polymer coupling compound or its pharmaceutically acceptable salt, as well as its preparation method and uses. Background Technology

[0002] Many drugs face challenges in clinical trials, some due to difficulties in preparation caused by their physical properties (especially solubility), or by producing high drug concentrations after administration that trigger toxic effects, resulting in a low therapeutic index. Other drawbacks include poor absorption, low bioavailability, instability, and systemic side effects, because targeted drug delivery cannot be achieved, and the biodistribution, metabolism, and renal and hepatic clearance of the drug cannot be controlled.

[0003] Recent studies have shown that dendritic polymers, due to their multi-branched structure, high tunability, and drug-loading capacity, are considered potential drug carriers. Their branched structure can effectively carry multiple drugs and achieve precise targeted delivery through their tunable functionalization properties. These characteristics help improve drug stability, loading capacity, and targeting, thereby enhancing the efficacy of antibody-drug conjugates (ADCs) and reducing adverse effects on non-target tissues. Therefore, dendritic polymers are considered a potentially effective drug delivery platform for ADCs, with the potential to optimize drug delivery and improve therapeutic efficacy.

[0004] For example, patent publication WO2012167309A1 discloses a macromolecule that uses dendritic polylysine as a carrier to deliver antitumor drugs. This type of macromolecule has high drug loading and controlled release, can passively target specific tissues and improve solubility, which allows for the reduction of side effects associated with the administration of oncology drugs, drug formulations without solubilizing excipients, and preoperative medications with or without reduced amounts of these drugs. Summary of the Invention

[0005] This disclosure provides a dendritic polymer coupling or a pharmaceutically acceptable salt thereof, comprising:

[0006] i) A dendritic polymer D having at least one surface amino group and at least one surface hydroxyl group or surface thiol group;

[0007] ii) A first end group, which is a residue A of a pharmaceutically active agent, its derivative or precursor containing a carboxyl, hydroxyl, amino or thiol group, optionally connected to the surface amino group of the dendritic polymer D via a linker portion;

[0008] iii) A second end group, which is a pharmacokinetic modifier M, optionally connected to the surface hydroxyl or surface thiol group of the dendritic polymer D via a linker portion;

[0009] iv) The third end group, which is the targeting portion Ab, is optionally connected to the core of the dendritic polymer D via the linker portion;

[0010] The targeted portion contains a targeting reagent.

[0011] In some embodiments, the dendritic polymer D is a polylysine analogue comprising i) a core; ii) a subsurface layer comprising building units selected from lysine or lysine analogues; and iii) a surface layer comprising structures selected from those shown in formula Ia or formula Ib.

[0012] in:

[0013] X is independently selected from O and S;

[0014] R 1 R 2 Each is independently selected from hydrogen, halogen, hydroxyl, mercapto, carboxyl, amino, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, the C 1-6 Alkyl, C 1-6 Alkyl groups are optionally replaced by halogen, hydroxyl, mercapto, carboxyl, amino, cyano, C 1-6 Alkyl, C 1-6 The alkoxy group is replaced by one or more substituents.

[0015] T 1 Each is an independent connection end to the first end base;

[0016] T 2 Each is an independent connection end to the second end base;

[0017] T 3 Each is an independent connection end that is linked to the building unit via an amide bond;

[0018] r is selected from 1, 2, 3, and 4;

[0019] s is selected from 0, 1, 2, 3, 4;

[0020] t is selected from 0, 1, 2, 3, 4.

[0021] In some embodiments, the subsurface layer comprises first-generation building units selected from lysine or lysine analogues.

[0022] In some embodiments, the subsurface layer comprises 2-7 generations of building units selected from lysine or lysine analogues. In some embodiments, the subsurface layer comprises 2 generations of building units selected from lysine or lysine analogues. In some embodiments, the subsurface layer comprises 3 generations of building units selected from lysine or lysine analogues. In some embodiments, the subsurface layer comprises 4 generations of building units selected from lysine or lysine analogues. In some embodiments, the subsurface layer comprises 5 generations of building units selected from lysine or lysine analogues. In some embodiments, the subsurface layer comprises 6 generations of building units selected from lysine or lysine analogues. In some embodiments, the subsurface layer comprises 7 generations of building units selected from lysine or lysine analogues.

[0023] In some embodiments, the dendritic polymer D comprises:

[0024] i) The core has a carbonyl carbon atom for connection with the target part Ab, and at least two amino nitrogen atoms for connection with lysine or lysine analog building units.

[0025] ii) Subsurface layer, which contains first-generation building blocks selected from lysine or lysine analogues;

[0026] iii) A surface layer comprising a structure selected from formula Ia or formula Ib.

[0027] in:

[0028] The targeted portion includes a targeting reagent;

[0029] X, R 1 R 2 T 1 T 2 T 3 r, s, and t are defined as described above.

[0030] In some embodiments, the dendritic polymer D comprises:

[0031] i) The core has a carbonyl carbon atom for connection with the target part Ab, and at least two amino nitrogen atoms for connection with lysine or lysine analog building units.

[0032] ii) Subsurface layer, which contains at least two generations of building units selected from lysine or lysine analogues;

[0033] iii) A surface layer comprising a structure selected from formula Ia or formula Ib.

[0034] in:

[0035] The targeted portion includes a targeting reagent;

[0036] X, R 1 R 2 T 1 T 2 T 3 r, s, and t are defined as described above.

[0037] In some embodiments, the building block is lysine, which has the following structure:

[0038] In some embodiments, the building block is a lysine analogue, which is selected from:

[0039] in:

[0040] a1 is selected from 1 and 2;

[0041] b1 and c1 are each independently selected from 1, 2, 3, and 4;

[0042] a2 is selected from 0, 1, and 2;

[0043] b2 and c2 are each independently selected from 2, 3, 4, 5, and 6;

[0044] a3 is selected from 0, 1, 2, 3, 4, and 5;

[0045] b3 and c3 are each independently selected from 1, 2, 3, 4, and 5;

[0046] a4 is selected from 0, 1, 2, 3, 4, and 5;

[0047] b4 and c4 are each independently selected from 0, 1, 2, 3, 4, and 5;

[0048] a5 is selected from 0, 1, 2, 3, 4, and 5;

[0049] b5 and c5 are each independently selected from 1, 2, 3, 4, and 5;

[0050] a6 is selected from 0, 1, 2, 3, 4, and 5;

[0051] b6, c6, and d6 are each independently selected from 1, 2, 3, 4, and 5;

[0052] a7 is selected from 0, 1, 2, 3, 4, and 5;

[0053] Phases b7 and c7 are each independently selected from 1, 2, 3, 4, and 5.

[0054] In some implementations, the core is selected from...

[0055] in,

[0056] a8 and b8 are each independently selected from 0, 1, 2, 3, 4, and 5;

[0057] Z 1 Each is an independently selected interval unit;

[0058] T 4 Each is an independent connection point to the target portion Ab;

[0059] T 5 Each is an independent connection end that is connected to the building unit via an amide bond.

[0060] In some implementations, the target portion Ab can be directly or preferably via the linking group Z. 1 It binds to the dendritic polymer core. Linking group Z 1 It can be any divalent group that can bind to the functional group on the core and the functional group on the target reagent.

[0061] In some implementations, the Z mentioned therein 1 -L P’ -M P -,

[0062] M P It is an extension unit covalently linked to the carbonyl group on the core, L P’ It is to connect the target portion Ab to M P The divalent linker portion, and its corresponding monovalent portion L P Contains a functional group W capable of connecting to the target portion Ab. P ;

[0063] The W mentioned P Selected from:

[0064] in,

[0065] R A R C Each is independently selected from hydrogen, halogen, hydroxyl, mercapto, nitro, carboxyl, amino, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, the C 1-6 Alkyl, C 1-6 Alkyl groups are optionally selected from halogens, hydroxyl groups, mercapto groups, nitro groups, carboxyl groups, amino groups, cyano groups, and C6 groups. 1-6 Alkyl, C 1-6 It is substituted by one or more substituents of alkoxy, 3 to 10-membered cycloalkyl groups;

[0066] R B Each group is independently selected from halogen, hydroxyl, mercapto, azide, hydrazine, carboxyl, amino, and cyano groups;

[0067] L 1 Selected from alkylene or heteroalkylene, wherein the alkylene or heteroalkylene is optionally interrupted by one or more groups selected from cycloalkylene, heterocycloalkylene, arylene, and heteroarylene, and wherein the alkylene or heteroalkylene is optionally selected from hydroxyl, C 1-6 Alkyl, 3- to 10-membered cycloalkyl, C 1-6 Alkoxy, halogen, nitro, cyano, oxo, thio, mercapto, sulfinyl, sulfonyl, -NR D R E The C is substituted by one or more substituents of aryl, heteroaryl, and heterocyclic groups. 1-6 Alkyl, C 1-6 Alkyl groups are optionally selected from halogens, hydroxyl groups, mercapto groups, nitro groups, carboxyl groups, amino groups, cyano groups, and C6 groups. 1-6 Alkyl, C 1-6 It is substituted by one or more substituents of alkoxy, 3 to 10-membered cycloalkyl groups;

[0068] Each of the numbers 'a' is independently selected from 1, 2, 3, 4, and 5.

[0069] b are each independently selected from 1, 2, 3, and 4;

[0070] c are each independently selected from 1 and 2;

[0071] d are each independently selected from 1, 2, and 3;

[0072] e is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13;

[0073] f are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0074] g is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12;

[0075] R D R E Whether the two are the same or different, each is independently selected from hydrogen, hydroxyl, and C. 1-6 Alkyl, cycloalkyl, C 1-6 Alkoxy, the C 1-6 Alkyl, C 1-6 Alkoxy and cycloalkyl groups are optionally selected from halogen, hydroxyl, mercapto, nitro, carboxyl, amino, cyano, C 1-6 Alkyl, C 1-6It is substituted by one or more substituents of alkoxy or 3- to 10-membered cycloalkyl groups.

[0076] In some implementations, the M P Selected from alkylene or heteroalkylene, wherein the alkylene or heteroalkylene is optionally interrupted by one or more groups selected from cycloalkylene, heterocycloalkylene, arylene, and heteroarylene, and wherein the alkylene or heteroalkylene is optionally selected from hydroxyl, C 1-6 Alkyl, 3- to 10-membered cycloalkyl, C 1-6 Alkoxy, halogen, nitro, cyano, oxo, thio, mercapto, sulfinyl, sulfonyl, -NR D R E The C is substituted by one or more substituents of aryl, heteroaryl, and heterocyclic groups. 1-6 Alkyl, C 1-6 Alkyl groups are optionally selected from halogens, hydroxyl groups, mercapto groups, nitro groups, carboxyl groups, amino groups, cyano groups, and C6 groups. 1-6 Alkyl, C 1-6 It is substituted by one or more substituents of alkoxy, 3 to 10-membered cycloalkyl groups;

[0077] Where R D R E As defined above.

[0078] In some implementations, the M mentioned therein P Selected from:

[0079] in,

[0080] j are each independently selected from integers from 1 to 50;

[0081] k is independently selected from integers from 1 to 50;

[0082] m are each independently selected from integers from 1 to 50;

[0083] m1 is selected from integers from 1 to 50;

[0084] m2 is selected from integers from 1 to 50;

[0085] h is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0086] i is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0087] l is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0088] n is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0089] In some implementations, the Z mentioned therein 1 Selected from Equations II-1, II-2, and II-3,

[0090] in:

[0091] * indicates the connection end with the target portion Ab;

[0092] j are each independently selected from integers from 1 to 50;

[0093] k is independently selected from integers from 1 to 50;

[0094] m are each independently selected from integers from 1 to 50;

[0095] m1 is selected from integers from 1 to 50;

[0096] m2 is selected from integers from 1 to 50;

[0097] h is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0098] i is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0099] l is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0100] n is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0101] In some implementations, h is independently selected from 0, 1, 2, 3, 4. In some implementations, h is independently selected from 0, 1, 2. In some implementations, h is independently selected from 0 and 1. In some implementations, h is independently selected from 1.

[0102] In some implementations, i is independently selected from 0, 1, 2, 3, 4. In some implementations, i is independently selected from 0, 1, 2. In some implementations, i is independently selected from 0 and 1. In some implementations, i is independently selected from 1.

[0103] In some implementations, j is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50. In some implementations, j is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. In some implementations, j is independently selected from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. In some implementations, j is independently selected from 11, 12, 13, 14, 15. In some implementations, j is independently selected from 12.

[0104] In some implementations, k is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50. In some implementations, k is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. In some implementations, k is independently selected from 2, 3, 4, 5, 6, 7, 8. In some implementations, k is independently selected from 3, 4, 5. In some implementations, k is independently selected from 4.

[0105] In some implementations, l is independently selected from 0, 1, 2, 3, 4. In some implementations, l is independently selected from 0, 1, 2. In some implementations, l is independently selected from 0 and 1. In some implementations, l is independently selected from 1.

[0106] In some implementations, m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50. In some implementations, m is independently selected from 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30. In some implementations, m is independently selected from 20, 21, 22, 23, 24, 25. In some implementations, m is independently selected from 22, 23, 24. In some implementations, m is independently selected from 23.

[0107] In some implementations, m1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50.

[0108] In some implementations, m2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50.

[0109] In some implementations, n is independently selected from 0, 1, 2, 3, and 4. In some implementations, n is independently selected from 0, 1, and 2. In some implementations, n is independently selected from 0 and 1. In some implementations, n is independently selected from 1.

[0110] In some implementations, the Z mentioned therein 1 for:

[0111] Wherein, * represents the connection end with the target portion Ab.

[0112] In some implementations, the first end base is connected via connector L A The L is covalently linked to the surface amino groups of the dendritic polymer D. A For -Y 1-L 2 -Y 2 -, Y 1 Selected from -C(O)- or -O-, optionally via a cleavable group Z 3 Linked to residue A of a pharmaceutically active agent, its derivative or its precursor, Y 2 It is -C(O)-, linked to the dendritic polymer D via an amide bond, wherein:

[0113] L 2 Selected from alkylene or heteroalkylene groups, wherein the alkylene or heteroalkylene group is optionally interrupted by one or more groups selected from cycloalkylene, heterocycloalkylene, aryl, and heteroaryl, and wherein the alkylene or heteroalkylene group is optionally selected from 3- to 10-membered cycloalkyl, C 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, halogen, nitro, cyano, acyl, amino, mercapto, sulfinyl, sulfonyl, -NR D R E The 3- to 10-membered cycloalkyl group is substituted by one or more substituents of aryl, heteroaryl, and heterocyclic groups. 1-6 Alkyl, C 1-6 Alkyl groups are optionally selected from hydroxyl, halogen, nitro, cyano, C 1-6 Alkyl, C 1-6 The alkoxy group is substituted by one or more substituents;

[0114] R D R E As defined above.

[0115] In some implementations, where L A Selected from the following structure:

[0116] in:

[0117] y3 is selected from integers from 1 to 50;

[0118] u is independently selected from 1, 2, 3, 4, 5, and 6;

[0119] v are each independently selected from 1, 2, 3, 4, 5, and 6;

[0120] w are each independently selected from 1, 2, 3, 4, 5, 6.

[0121] In some implementations, the connector L of this disclosure is selected. A It can provide the desired drug release rate, such as rapid release or slow release.

[0122] In some embodiments, the release rate of the pharmaceutically active agent of the conjugate is faster than that of the conjugate-independent agent, possibly at least twice as fast. In some embodiments, the release rate of the pharmaceutically active agent of the conjugate is slower than that of the conjugate-independent agent, possibly two, three, four, five, six, seven, eight, nine, ten, or more than ten, fifteen, twenty, or thirty times slower. Conjugates with low release rates are suitable for formulation into drugs that release slowly over long periods, such as 1 week to 3 months, 1 month to 6 months, or more than 6 months. Rapid release is preferably within 0-8 hours, especially within 0-4 hours, particularly within 0-2 hours, and even more particularly within 5-60 minutes, releasing more than 50% of the pharmaceutically active agent. Moderate release is preferably within 1-72 hours, especially within 2-48 hours, releasing more than 50% of the pharmaceutically active agent. The release rate of the pharmaceutically active agent can be determined by selecting a suitable linker L. A The release rate is controlled and also depends on the properties of the pharmaceutically active agent. In some embodiments, the pharmaceutically active agent is released via the same linker L. A Linked to a dendritic polymer. In other embodiments, the pharmaceutical active agent is connected via two or more linkers L. A It is linked with dendritic polymer D so that the pharmaceutical active agent can be released from the conjugate at different release rates.

[0123] In some embodiments, the first end group and the second end group exist in a ratio of 1:2 to 2:1, especially 1:2, 1:1, and 2:1. In some embodiments, the first end group and the second end group exist in a ratio of 1:1.

[0124] In some embodiments, the ratio of the first, second, and third terminal groups is 1:1:1 to 1:2:2, particularly 1:2:1. In some embodiments, the pharmaceutically active agent is bound to greater than 14%, 25%, 27%, 30%, 39%, 44%, or 48% of the surface amino groups. In some embodiments, the pharmacokinetic modifier is bound to greater than 15%, 25%, 30%, 33%, or 46% of the surface hydroxyl or surface thiol groups.

[0125] In some embodiments, the second end base is connected via connector L B The amino groups covalently bonded to the surface of the dendritic polymer D, wherein:

[0126] L B It is absent, or selected from alkylene or heteroalkylene groups, wherein the alkylene or heteroalkylene groups are optionally interrupted by one or more groups selected from cycloalkylene, heterocycloalkylene, arylene, and heteroarylene, and wherein the alkylene or heteroalkylene groups are optionally selected from 3- to 10-membered cycloalkyl, C 1-6 Alkyl, C 1-6Alkoxy, hydroxy, halogen, nitro, cyano, acyl, amino, mercapto, sulfinyl, sulfonyl, -NR D R E The 3- to 10-membered cycloalkyl group is substituted by one or more substituents of aryl, heteroaryl, and heterocyclic groups. 1-6 Alkyl, C 1-6 Alkyl groups are optionally selected from hydroxyl, halogen, nitro, cyano, C 1-6 Alkyl, C 1-6 The alkoxy group is replaced by one or more substituents.

[0127] R D R E As defined above.

[0128] In some implementations, L B It does not exist.

[0129] In some embodiments, the cleavable group Z is described as follows: 3 It contains a cleavable peptide portion.

[0130] In some embodiments, the cleavable group Z is described as follows: 3 It can be cleaved by an enzyme. In some embodiments, the cleavable group Z is described as... 3 It can be cleaved by cathepsins. In some embodiments, the cathepsin is cathepsin B.

[0131] In some embodiments, the cleavable group Z 3 The amino acid unit comprises 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, aspartic acid, homolysine, n-methylvaline, etc. The peptide residues are composed of amino acids, where x is an integer from 1 to 6. In some embodiments, the cleavable group Z is... 3 Selected from valine-citrulline (Val-Cit), valine-alanine (Val-Ala), alanine-phenylalanine (Ala-Phe); phenylalanine-lysine (Phe-Lys), phenylalanine-homolysine (Phe-Homolys), n-methyl-valine-citrulline (Me-Val-Cit), alanine-alanine (Ala-Ala), glycine-glutamic acid (Gly-Glu), glutamic acid-alanine-alanine (Glu-Ala-Ala), glycine-lysine (Gly-Lys), glycine-valine-citrulline (Gly-Val-Cit), glycine-glycine-glycine (Gly-Gly-Gly), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly) and...

[0132] In some embodiments, the cleavable group Z 3 Selected from valine-citrulline (Val-Cit), valine-alanine (Val-Ala), n-methyl-valine-citrulline (Me-Val-Cit), and glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

[0133] In some embodiments, the cleavable group Z 3 It contains a cleavable sulfonamide moiety.

[0134] In some embodiments, the cleavable group Z 3 It contains a pyrolytic disulfide component.

[0135] In some embodiments, the cleavable group Z 3 It can cleave under reducing conditions.

[0136] In some embodiments, the cleavable group Z 3 Selected from:

[0137] Among them, T 6 Each is an independent linker to residue A of the pharmaceutically active agent, its derivative or its precursor.

[0138] The pharmaceutically active agents described in this disclosure are slightly soluble or insoluble in aqueous solutions.

[0139] In some embodiments, residue A of the pharmaceutically active agent, its derivative, or its precursor is selected from cytotoxic compounds. In some embodiments, residue A of the pharmaceutically active agent, its derivative, or its precursor is selected from eribulin, trabectedin, MMAE, MMAF, alkylating agents, platinum-based drugs, antibiotics (e.g., mitomycin, bleomycin), tyrosine kinase inhibitors, protein degrading agents (e.g., protein degradation-targeting chimeras (PROTAC) or molecular glues), KRAS inhibitors (e.g., KRAS G12C inhibitors, KRAS G12D inhibitors, pan-KRAS inhibitors), anthracyclines (e.g., doxorubicin, PNU159682), taxanes (e.g., paclitaxel, docetaxel, cabazitaxel), vinca alkaloids (e.g., vincristine, vinorelbine, vinorelbine), camptothecin analogs (e.g., SN38, 9106-IM-2), and derivatives of the above drugs and their pharmaceutically acceptable salts. In some embodiments, residue A of the pharmaceutically active agent, its derivative, or its precursor is cabazitaxel or its derivative and a pharmaceutically acceptable salt thereof. In some embodiments, residue A of the pharmaceutically active agent, its derivative, or its precursor is docetaxel or its derivative and a pharmaceutically acceptable salt thereof. In some embodiments, residue A of the pharmaceutically active agent, its derivative, or its precursor is a camptothecin analog (e.g., SN38, 9106-IM-2) and a pharmaceutically acceptable salt thereof.

[0140] In some embodiments, the residue A of the pharmaceutically active agent, its derivative, or its precursor is of formula III-1, III-2, III-3, or III-4.

[0141] in:

[0142] Z 2 Each independently does not exist or is T 7 -C(R a R b )N(R c )-;

[0143] R a R b Each is independently selected from hydrogen, halogen, hydroxyl, mercapto, carboxyl, -NR D R E , cyano, C 1-6 Alkyl, C 1-6 Alkoxy, the C 1-6 Alkyl, C 1-6 Alkyl groups are optionally selected from halogens, hydroxyl groups, mercapto groups, carboxyl groups, and -NR groups. D R E , cyano, C 1-6 Alkyl, C1-6 The alkoxy group is substituted by one or more substituents;

[0144] R c Selected from hydrogen, C 1-6 Alkyl, C 1-6 Alkoxy, the C 1-6 Alkyl, C 1-6 Alkyl groups are optionally selected from halogen, hydroxyl, mercapto, carboxyl, amino, cyano, C 1-6 Alkyl, C 1-6 The alkoxy group is substituted by one or more substituents;

[0145] T 7 For connection to the oxygen atom in Formula III-1, Formula III-2 or Formula III-4;

[0146] R D R E As defined above.

[0147] The second end group is a pharmacokinetic modifier that can modify or modulate the pharmacokinetic characteristics of a pharmaceutical active agent or compound, including absorption, distribution, metabolism, and / or excretion. In some embodiments, the pharmacokinetic modifier prolongs the plasma half-life of the pharmaceutical active agent, such that the half-life of the pharmaceutical active agent linked to the compound is longer than that of the pharmaceutical active agent alone or on a non-dendritic polymer carrier. Preferably, the half-life of the compound or composition is at least 2 times longer and more preferably at least 10 times longer than that of the pharmaceutical active agent alone or on a non-dendritic polymer carrier.

[0148] The pharmacokinetic modifier may be selected from polyethylene glycol, polyethyloxazoline, polyvinylpyrrolidone, polypropylene glycol, folate, or folate derivatives relating to ligands of cell surface receptors. In some embodiments, the pharmacokinetic modifier is polyethylene glycol. In some embodiments, the polyethylene glycol has a molecular weight in the range of 220 to 5500 Da, for example, it may be 220-2500 Da, 570-2500 Da, 220-1100 Da, 570-1100 Da, 1000-5500 Da, 1000-2500 Da, or 1000-2300 Da. In some embodiments, the pharmacokinetic modifier is covalently linked to hydroxyl or thiol groups on the surface layer of the dendritic polymer.

[0149] In some embodiments, the number of residues A grafted onto the pharmaceutically active agent, its derivatives, or its precursors is selected from 1 to 4.

[0150] In some embodiments, the number of residues A grafted onto the pharmaceutically active agent, its derivative, or its precursor is selected from 4 to 32. In some embodiments, the number of residues A grafted onto the pharmaceutically active agent, its derivative, or its precursor is selected from 4 to 8. In some embodiments, the number of residues A grafted onto the pharmaceutically active agent, its derivative, or its precursor is selected from 10 to 16. In some embodiments, the number of residues A grafted onto the pharmaceutically active agent, its derivative, or its precursor is selected from 20 to 32.

[0151] The targeting fraction Abs described in this disclosure are agents that bind to selective biological target cells, organs, or tissues, thereby facilitating the delivery of pharmaceutically active agents to specific targets in vivo and their accumulation in those target cells, organs, or tissues. Additionally, the targeting fraction Abs can provide a mechanism for compounds to actively enter cells or tissues via receptor-mediated endocytosis. Specific examples include lectins and antibodies, as well as other ligands (including small molecules) targeting cell surface receptors. This interaction can occur through any type of bond or association (including covalent bonds, ionic bonds, hydrogen bonds, and van der Waals forces).

[0152] In some embodiments, the targeting portion Ab is selected from peptides, proteins, antibodies, or antigen-binding fragments. In some embodiments, the targeting portion Ab is selected from antibodies or antigen-binding fragments. In some embodiments, the targeting portion Ab is selected from monoclonal antibodies. In some embodiments, the targeting portion Ab is selected from adalimumab, alimazumab, atezolizumab, emecizumab, abavirin, oftadalafil, oxaijutuzumab, omalizumab, oxotuzumab, oxotuzumab, oxotuzumab, baliximab, bevacizumab, belimumab, brolimumab, brosuumab, daratumumab, daratumumab, daratumumab beta, etc. Trastuzumab, Denosumab, Iodine [131I] Metuximab, Duvarium, Duprenumab, Trastuzumab emtansine, Envorimab, Golimumab, Gosatuzumab, Gussageumab, Cantunilumab, Camrelizumab, Anti-human T-cell CD3 mouse monoclonal antibody, Anti-human interleukin-8 mouse monoclonal antibody, Lanalimumab, Ramosinumab, Ranibizumab, Rituximab, Romisvir, Mepolizumab, Mogliflozin, Nacetuzumab, Nivolumab, Nimotuzumab, Pembrolizumab, Pertuzumab, Pesolizumab, Pesolizumab, Putelizumab, Trastuzumab, Repertuzumab, Saturolizumab, Separibumab, Sugelizumab, Secukinumab, Secuximab, Slulizumab, Toripalimab Anti-CD25 humanized monoclonal antibody, tislelizumab, tocilizumab, brentuximab, vedelizumab, vedictizumab, vepotocilizumab, ustekinumab, cetuximab, sintilimab, inellizumab, inetuximab, ipilimumab, edasciizumab, ikuzumab, evolocumab, imalicumab, ixazolizumab, infliximab, recombinant anti-CD25 humanized monoclonal antibody.

[0153] In some embodiments, when the subsurface layer comprises second-generation building units selected from lysine or lysine analogues, the compounds of this disclosure or their pharmaceutically acceptable salts are as shown in Formula IV-A.

[0154] in,

[0155] p is 1 to 5 (including 1, 2, 3, 4, 5 or any value between any two values);

[0156] Ab、L A L B Z 1 Z 3 A, M, X, r, R 1 R 2 As defined above.

[0157] In some embodiments, the compounds of formula IV-A of this disclosure can also be simplified to formula IV-a.

[0158] p, Ab, L A L B Z 1 Z 3 A, M, X, r, R 1 R 2 As defined above.

[0159] In some embodiments, when the subsurface layer comprises second-generation building units selected from lysine or lysine analogues, the compounds of this disclosure or pharmaceutically acceptable salts thereof are as shown in Formula IV-B.

[0160] Among them, p, Ab, L A L B Z 1 Z 3 A, M, X, s, t, R 1 R 2 As defined above.

[0161] In some embodiments, the compound of formula IV-B of this disclosure can also be simplified to formula IV-b.

[0162] p, Ab, L A L B Z 1 Z 3 A, M, X, s, t, R 1 R 2 As defined above.

[0163] In some embodiments, when the subsurface layer comprises second-generation building units selected from lysine or lysine analogues, the compounds of this disclosure or their pharmaceutically acceptable salts are as shown in formula IV-C.

[0164] Where: q is 1 to 5 (including 1, 2, 3, 4, 5 or any value between any two values);

[0165] Ab、L A L B Z 1 Z 3 A, M, X, r, R 1 R 2 As defined above.

[0166] In some embodiments, the compounds of formula IV-c disclosed herein can also be represented by the simplified formula IV-c.

[0167] Where c8 is selected from 0, 1, 2, 3, 4;

[0168] q, Ab, L A L B Z 1 Z 3 A, M, X, r, R 1 R 2 As defined above.

[0169] In some embodiments, when the subsurface layer comprises second-generation building units selected from lysine or lysine analogues, the compounds of this disclosure or pharmaceutically acceptable salts thereof are as shown in Formula IV-D.

[0170] Among them, q, Ab, L A L B Z 1 Z 3 A, M, X, s, t, R 1 R 2 As defined above.

[0171] In some embodiments, the compounds of formula IV-D disclosed herein can also be simplified to formula IV-d.

[0172] q, c8, Ab, L A L B Z 1 Z 3 A, M, X, s, t, R 1 R 2 As defined above.

[0173] In some embodiments, when the subsurface layer comprises third-generation building blocks selected from lysine or lysine analogues, the compounds of this disclosure or their pharmaceutically acceptable salts are represented as Va, Vb, Vc, or Vd.

[0174] p, q, c8, Ab, L A L B Z 1 Z 3 A, M, X, r, s, t, R 1 R 2 As defined above.

[0175] In some embodiments, when the subsurface layer comprises third-generation building blocks selected from lysine or lysine analogues, the compounds of this disclosure or their pharmaceutically acceptable salts are as shown in formulas VI-a, VI-b, VI-c, or VI-d.

[0176] p, q, c8, Ab, L A L B Z 1 Z 3 A, M, X, r, s, t, R 1 R 2 As defined above.

[0177] In some embodiments, when the subsurface layer comprises first-generation building units selected from lysine or lysine analogues, the compounds of this disclosure or their pharmaceutically acceptable salts are represented as Aa, Ab, Ac, or Ad.

[0178] p, q, c8, Ab, L A L B Z 1 Z 3 A, M, X, r, s, t, R 1 R 2 As defined above.

[0179] The dendritic polymer conjugates or pharmaceutically acceptable salts thereof described in this disclosure are selected from formula VII:

[0180] in:

[0181] y1 is selected from integers from 1 to 100;

[0182] y2 is selected from 2, 4, 8, 16, and 32;

[0183] q, Ab, L A Z 1 Z 3 A is as defined above.

[0184] In some embodiments, when the subsurface layer comprises a first-generation building block selected from lysine or lysine analogues, the number of residues A grafted from the pharmaceutically active agent, its derivative, or its precursor is selected from 1 to 4. In some embodiments, when the subsurface layer comprises a first-generation building block selected from lysine or lysine analogues, the number of residues A grafted from the pharmaceutically active agent, its derivative, or its precursor is selected from 2 to 4. In some embodiments, when the subsurface layer comprises a first-generation building block selected from lysine or lysine analogues, the number of residues A grafted from the pharmaceutically active agent, its derivative, or its precursor is selected from 3 to 4.

[0185] In some embodiments, when the subsurface layer comprises two generations of building units selected from lysine or lysine analogues, the number of residues A grafted from the pharmaceutically active agent, its derivative, or its precursor is selected from 4 to 8. In some embodiments, when the subsurface layer comprises two generations of building units selected from lysine or lysine analogues, the number of residues A grafted from the pharmaceutically active agent, its derivative, or its precursor is selected from 5 to 8. In some embodiments, when the subsurface layer comprises two generations of building units selected from lysine or lysine analogues, the number of residues A grafted from the pharmaceutically active agent, its derivative, or its precursor is selected from 6 to 7.

[0186] In some embodiments, when the subsurface layer comprises third-generation building blocks selected from lysine or lysine analogs, the number of residues A grafted from the pharmaceutically active agent, its derivative, or its precursor is selected from 10 to 16. In some embodiments, when the subsurface layer comprises third-generation building blocks selected from lysine or lysine analogs, the number of residues A grafted from the pharmaceutically active agent, its derivative, or its precursor is selected from 13 to 16. In some embodiments, when the subsurface layer comprises third-generation building blocks selected from lysine or lysine analogs, the number of residues A grafted from the pharmaceutically active agent, its derivative, or its precursor is selected from 14 to 15.

[0187] In some embodiments, when the subsurface layer comprises four generations of building units selected from lysine or lysine analogues, the number of residues A grafted from the pharmaceutically active agent, its derivative, or its precursor is selected from 20 to 32. In some embodiments, when the subsurface layer comprises four generations of building units selected from lysine or lysine analogues, the number of residues A grafted from the pharmaceutically active agent, its derivative, or its precursor is selected from 27 to 32. In some embodiments, when the subsurface layer comprises four generations of building units selected from lysine or lysine analogues, the number of residues A grafted from the pharmaceutically active agent, its derivative, or its precursor is selected from 28 to 30.

[0188] In some embodiments, when the subsurface layer comprises 5 generations of building units selected from lysine or lysine analogs, the number of residues A grafted from the pharmaceutically active agent, its derivative, or its precursor is selected from 40 to 64. In some embodiments, when the subsurface layer comprises 4 generations of building units selected from lysine or lysine analogs, the number of residues A grafted from the pharmaceutically active agent, its derivative, or its precursor is selected from 54 to 64. In some embodiments, when the subsurface layer comprises 4 generations of building units selected from lysine or lysine analogs, the number of residues A grafted from the pharmaceutically active agent, its derivative, or its precursor is selected from 60 to 64.

[0189] In some embodiments, the dendritic polymer conjugates or pharmaceutically acceptable salts thereof described in this disclosure are as shown in Formula VIII:

[0190] Among them, L A y1, y2, q, Ab, Z 3 h, i, j, k, l, m, n, A are as defined above.

[0191] In some embodiments, the dendritic polymer conjugates described herein, or pharmaceutically acceptable salts thereof, are shown as in formula VIII-A or VIII-B:

[0192] in,

[0193] Z 4 Selected from O, N, S or C(R) a R b ),

[0194] y1, y2, y3, v, w, q, Ab, Z 3 h, i, j, k, l, m, n, A are as defined above.

[0195] The dendritic polymer conjugates or pharmaceutically acceptable salts thereof described in this disclosure are shown as those of formulas VIII-A-1, VIII-A-1', VIII-A-2, VIII-A-3, VIII-B-1, VIII-B-1', VIII-B-2, or VIII-B-3:

[0196] in,

[0197] Z 4 Selected from O, N, S or C(R) a R b ),

[0198] y1, y2, y3, v, w, q, Ab, Z 3 ,h,i,j,k,l,m,n,R a R b R c As defined above.

[0199] The dendritic polymer conjugates or pharmaceutically acceptable salts thereof described in this disclosure are shown in formulas IX-1, IX-1', IX-2, IX-3, IX-4, IX-5, IX-6, IX-6', IX-7, IX-8, or IX-9:

[0200] Wherein, y1, y2, y3, q, Ab, j, k, and m are as defined above. The dendritic polymer conjugates or their pharmaceutically acceptable salts described in this disclosure are selected from:

[0201] Where q is as defined above.

[0202] On the other hand, this disclosure also provides a dendritic polymer as shown in Formula X or a pharmaceutically acceptable salt thereof.

[0203] in,

[0204] R 3 Selected from hydrogen, C 1-6 Alkyl-L C -C(O), aryl-L C -C(O), heteroaryl-L C-C(O), wherein the alkyl, aryl, or heteroaryl group is optionally selected from halogen, hydroxyl, mercapto, carboxyl, -NR D R E , cyano, C 1-6 Alkyl, C 1-6 It is substituted by one or more substituents of alkoxy or phenyl groups;

[0205] L C The alkylene or heteroalkylene group is absent or selected from the group consisting of one or more groups selected from cycloalkylene, heterocycloalkylene, arylene, and heteroarylene, and the alkylene or heteroalkylene group is optionally selected from hydroxyl, C 1-6 Alkyl, 3- to 10-membered cycloalkyl, C 1-6 Alkoxy, halogen, nitro, cyano, acyl, amino, mercapto, sulfinyl, sulfonyl, -NR D R E The 3- to 10-membered cycloalkyl group is substituted by one or more substituents of aryl, heteroaryl, and heterocyclic groups. 1-6 Alkyl, C 1-6 Alkyl groups are optionally selected from hydroxyl, halogen, nitro, cyano, C 1-6 Alkyl, C 1-6 The alkoxy group is replaced by one or more substituents;

[0206] x1 is selected from 0 or 1;

[0207] y1 is selected from integers from 1 to 100;

[0208] y2 is selected from 2, 4, 8, 16, and 32;

[0209] A, R D R E L A Z 3 As defined above.

[0210] In some implementations, y1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 5 0, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100.

[0211] In some embodiments, the dendritic polymer of formula X or its pharmaceutically acceptable salt, such as the dendritic polymer of formula XI-1 or formula XI-2, is used.

[0212] in,

[0213] Z 4 ,y1,y2,y3,A,k,l,m,n,Z 3 v and w are as defined above.

[0214] In some embodiments, the dendritic polymer of formula X or its pharmaceutically acceptable salt, such as the dendritic polymer of formula XII or its pharmaceutically acceptable salt,

[0215] in,

[0216] Z 4 y1, y2, A, Z 3 v, w, R a R b x1 is as defined above.

[0217] In some embodiments, the dendritic polymer of formula X or its pharmaceutically acceptable salt, such as the dendritic polymer of formula XIII or its pharmaceutically acceptable salt,

[0218] Wherein, y1 and y2 are as defined above.

[0219] On the other hand, this disclosure also provides a dendritic polymer as shown in Formula XIV or a pharmaceutically acceptable salt thereof.

[0220] in,

[0221] R3’ Selected from hydrogen, C 1-6 Alkyl-L C -C(O), aryl-L C -C(O), heteroaryl-L C -C(O), N3-L C -C(O), wherein the alkyl, aryl, or heteroaryl group is optionally selected from halogen, hydroxyl, mercapto, carboxyl, -NR D R E , cyano, C 1-6 Alkyl, C 1-6 It is substituted by one or more substituents of alkoxy or phenyl groups;

[0222] R M R N Each is independently selected from either a hydrogen or amino protecting group;

[0223] y1 is selected from integers from 1 to 100;

[0224] y2 is selected from 2, 4, 8, 16, and 32;

[0225] R D R E L C As defined above.

[0226] In some embodiments, the amino protecting group is selected from benzyloxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, allyloxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, p-toluenesulfonylmethoxycarbonyl, ethoxycarbonyl, phthaloyl, trifluoroacetyl, triphenylmethyl, 2,4-dimethoxybenzyl, p-methoxybenzyl, and benzyl. In some embodiments, the amino protecting group is tert-butoxycarbonyl.

[0227] In some implementation schemes, R 3’ Selected from hydrogen,

[0228] Where k, l, m, and n are as defined above.

[0229] In some embodiments, the dendritic polymer of formula XIV or its pharmaceutically acceptable salt, such as the dendritic polymer of formula XIV-1, XIV-2, XIV-3 or its pharmaceutically acceptable salt,

[0230] Among them, y1, y2, R M R N k, l, m, n are as defined above.

[0231] In some embodiments, the dendritic polymer of formula XIV or its pharmaceutically acceptable salt, such as the dendritic polymers of formulas XV-1-A, XV-1-B, XIV-2-A, XIV-2-B, XIV-3-A, and XIV-3-B or their pharmaceutically acceptable salts,

[0232] Among them, y1, y2, R N As defined above.

[0233] On the other hand, this disclosure also provides a dendritic polymer coupling or a pharmaceutically acceptable salt thereof, selected from the dendritic polymer couplings of formula XV or pharmaceutically acceptable salts thereof.

[0234] in,

[0235] Ab represents the target region;

[0236] A is a residue A of a pharmaceutically active agent, its derivative or precursor containing a carboxyl, hydroxyl, amino or thiol group;

[0237] L A For optional connectors;

[0238] Z 1 The spacer group is optional;

[0239] y1 is selected from integers from 1 to 100;

[0240] y2 is selected from 2, 4, 8, 16, and 32;

[0241] Each q is independently 1 to 5 (including 1, 2, 3, 4, 5 or any value between any two values).

[0242] On the other hand, this disclosure also provides a dendritic polymer coupling or a pharmaceutically acceptable salt thereof, selected from the dendritic polymer couplings of formula XVI or pharmaceutically acceptable salts thereof.

[0243] in,

[0244] Ab represents the target region;

[0245] A is a residue A of a pharmaceutically active agent, its derivative or precursor containing a carboxyl, hydroxyl, amino or thiol group;

[0246] L A For optional connectors;

[0247] M P For optional extension units;

[0248] L P’For optional bivalent connectors;

[0249] y1 is selected from integers from 1 to 100;

[0250] y2 is selected from 2, 4, 8, 16, and 32;

[0251] Each q is independently 1 to 5 (including 1, 2, 3, 4, 5 or any value between any two values).

[0252] In some implementations, L P’ It is to connect the target portion Ab to M P The optional bivalent connector, its corresponding unit price portion L P Contains a functional group W capable of connecting to the target portion Ab. P W P As defined above.

[0253] On the other hand, this disclosure also provides a dendritic polymer coupling as defined above, or an isotopic substitute for a pharmaceutically acceptable salt thereof. In some embodiments, the isotopic substitute is a deuterated derivative.

[0254] This disclosure also includes compounds identical to those described herein, but in which one or more atoms are labeled with isotopes whose atomic weights or mass numbers differ from those commonly found in nature. Examples of isotopes that can be incorporated into compounds of this disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as... 2 H, 3 H, 11 C 13 C 14 C 13 N、 15 N、 15 O、 17 O、 18 O、 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.

[0255] Unless otherwise specified, when a position is specifically designated as deuterium (D), that position should be understood as having a deuterium abundance of at least 1000 times greater than the natural abundance of deuterium (which is 0.015%) (i.e., at least 10% deuterium incorporation). The natural abundance of deuterium in the example compounds can be at least 1000 times, at least 2000 times, at least 3000 times, at least 4000 times, at least 5000 times, at least 6000 times, or even higher. This disclosure also includes various deuterated forms of compounds of formula (I). Each available hydrogen atom bonded to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize the deuterated forms of compounds of formula (I) with reference to relevant literature. Commercially available deuterated starting materials can be used to prepare the deuterated form of the compound of formula (I), or they can be synthesized using conventional techniques with deuterated reagents, including but not limited to deuterated boranes, trideuterated borane tetrahydrofuran solutions, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane.

[0256] On the other hand, this disclosure also provides a pharmaceutical composition comprising a dendritic polymer conjugate as defined above or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient, or an isotopic substitute of a dendritic polymer conjugate as defined above or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is administered via transdermal, oral, or injectable routes.

[0257] The dendritic polymer conjugates disclosed herein, or compositions formulated with pharmaceutically acceptable salts thereof, include those suitable for oral, rectal, topical, nasal, inhalation, aerosol, ocular, or parenteral (including intraperitoneal, intravenous, subcutaneous, or intramuscular) administration. The compositions are readily available in unit dosage forms and can be prepared by any method well known in the pharmaceutical industry. All methods include the step of dispersing the compound in a carrier constituting one or more excipients. Typically, the compositions are prepared by dispersing the compound in a liquid carrier to form a solution or suspension, or optionally by dispersing the compound with a formulation component suitable for forming a solid, optionally particulate product, and then, if necessary, shaping the product into the desired delivery form. Solid dosage forms of the present disclosure, when in particulate form, will typically comprise a particle size range of about 1 nanometer to about 500 micrometers. Typically, for solid dosage forms intended for intravenous administration, the particle diameter range will typically be from about 1 nm to about 10 micrometers. The composition may contain the dendritic polymer conjugate or its pharmaceutically acceptable salt of the present disclosure, wherein the dendritic polymer conjugate or its pharmaceutically acceptable salt is nanoparticle with a particle diameter of less than 1000 nm, for example, 5 to 1000 nm, particularly 5 to 500 nm, particularly 5 to 400 nm (e.g., 5 to 50 nm and particularly 5 to 20 nm). In a particular embodiment, the composition contains a dendritic polymer conjugate or its pharmaceutically acceptable salt having an average size of 5 to 20 nm. In some embodiments, the dendritic polymer conjugate or its pharmaceutically acceptable salt is polydisperse in the composition, with a PDI (Polydispersity Index) between 1.01 and 1.8, particularly between 1.01 and 1.5, and particularly between 1.01 and 1.2. In a particular embodiment, the dendritic polymer conjugate or its pharmaceutically acceptable salt is monodisperse in the composition. Particularly preferred are sterile, lyophilized compositions reconstituted in an aqueous medium prior to injection.

[0258] In some embodiments, the composition contains a dendritic polymer conjugate or a pharmaceutically acceptable salt thereof having an average size of 5 to 20 nm. In some embodiments, the particle size D of the dendritic polymer conjugate or the pharmaceutically acceptable salt thereof is... 90 Or D 50 Less than 1000 nm, for example, 5 to 1000 nm, particularly 5 to 500 nm, especially 5 to 400 nm (e.g., 5 to 50 nm, particularly 5 to 20 nm). In some embodiments, the composition contains D 50 It is a dendritic polymer conjugate or its pharmaceutically acceptable salt with a wavelength of 5 to 20 nm.

[0259] The dendritic polymer conjugates or their pharmaceutically acceptable salts disclosed herein can also be used to provide controlled-release and / or sustained-release formulations of pharmaceutically active agents. In sustained-release formulations, the formulation component is selected to release the macromolecule from the formulation over an extended period (e.g., days, weeks, or months). Such formulations include transdermal patches or implantable devices capable of subcutaneous deposition, or via intravenous, subcutaneous, intramuscular, intradural, or intracranial injection. In controlled-release formulations, a diacid linker is selected to release the majority of its pharmaceutically active agent within a given time window. For example, when the time it takes for the majority of the macromolecule to accumulate in a target organ, tissue, or tumor is known, the linker can be selected to release the majority of its pharmaceutically active agent after the accumulation time has elapsed. This allows for the delivery of a high drug load at the site where its action is required at a given time point. Optionally, the linker is selected to release the pharmaceutically active agent at a therapeutic level over an extended period. In some embodiments, the formulation may have multiple controlled-release properties. For example, a formulation may contain compounds in which the drug is linked by different linkers, allowing for a burst release of the drug followed by a slower release at a lower but constant therapeutic level over an extended period. In some embodiments, the formulation may have sustained-release and controlled-release properties. For example, the formulation components may be selected to release a large molecule over an extended period, and the linkers may be selected to deliver a constant, low therapeutic level of the pharmaceutically active agent. In some embodiments, the pharmaceutically active agent is linked to the same molecule by different linkers.

[0260] In some embodiments, the dendritic polymer conjugate or its pharmaceutically acceptable salt in the pharmaceutical composition is formulated to release more than 50% of the pharmaceutically active agent between 5 minutes and 60 minutes. In some embodiments, the dendritic polymer conjugate or its pharmaceutically acceptable salt in the pharmaceutical composition is formulated to release more than 50% of the pharmaceutically active agent between 2 hours and 48 hours. In some embodiments, the dendritic polymer conjugate or its pharmaceutically acceptable salt in the pharmaceutical composition is formulated to release more than 50% of the pharmaceutically active agent between 5 days and 30 days.

[0261] On the other hand, this disclosure also provides the use of the dendritic polymer conjugate as defined above or its pharmaceutically acceptable salt or its isotopic substitute, or a pharmaceutical composition comprising the dendritic polymer conjugate as defined above or its pharmaceutically acceptable salt or its isotopic substitute, in the preparation of a medicament for treating cancer.

[0262] In some implementations, the cancer is selected from lung cancer (e.g., non-small cell lung cancer, non-squamous non-small cell lung cancer), gastric cancer, adenocarcinoma of the stomach or gastroesophageal junction, esophageal squamous cell carcinoma, colorectal cancer, esophageal cancer, non-epithelial malignant pleural mesothelioma, melanoma, nasopharyngeal carcinoma, urothelial carcinoma, Hodgkin's lymphoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, breast cancer, and ovarian cancer.

[0263] On the other hand, this disclosure also provides a method for delivering a cytotoxic compound to tumor target cells, including the step of contacting the tumor target cells with the dendritic polymer conjugate or its pharmaceutically acceptable salt as described in this disclosure.

[0264] This disclosure further provides a kit comprising the dendritic polymer conjugate described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0265] Methods for preparing dendritic polymers are known in the art. For example, dendritic polymers of compounds can be prepared by divergent or convergent methods or a combination thereof.

[0266] In the divergent method, each generation of building units is sequentially added to the core or the previous generation. The two surface amino groups on the outermost building unit are simultaneously capped with serine, threonine, and cysteine ​​to construct the outermost (or final) surface layer. Surface units with one surface amino group and one surface hydroxyl or thiol group are protected. The surface amino groups are then removed and bonded to L-type structures via amide bond formation methods known in the art. A -L A -A or -Z 3 -L A -A linkage. Surface hydroxyl or surface thiol groups with the hydroxyl or thiol protecting groups removed are linked to the pharmacokinetic modifier M via ester or thioester bond formation methods known in the art.

[0267] In the convergence method, each generation of building units is built upon the previous generation to form a dendritic motif. Before or after the dendritic motif is attached to the core, a first end group may be attached to a surface amino group as described above, and a second end group may be attached to a surface hydroxyl group or a surface thiol group as described above.

[0268] In the hybrid approach, each generation of building units is added to the core or the previous generation of the building unit. However, before the final generation is added to the dendrite, surface amino groups are functionalized with a first end group, and surface hydroxyl or thiol groups are functionalized with a second end group. The functionalized final generation is then added to the subsurface layer of the building unit, connecting the dendritic motif to the core.

[0269] Pharmaceutical active agents react with one of the carboxylic acids of the linker via ester formation, as is known in the art. For example, an activated carboxylic acid is formed, such as using an acyl chloride or an anhydride, and reacts with the hydroxyl group of the pharmaceutical active agent. If the pharmaceutical active agent has more than one hydroxyl group, then the other hydroxyl groups can be protected.

[0270] When the targeting reagent is attached to the core, the functional groups on the core can be protected during dendrite formation and then deprotected, and react with the targeting reagent, the linking group, or the targeting reagent-linking group. Optionally, the core can react with the linking group or the targeting reagent-linking group before dendrite formation.

[0271] Suitable protecting groups, their introduction and removal methods are described in Greene & Wuts, Protecting Groups in Organic Synthesis, 3rd Edition, 1999.

[0272] The dendritic polymer conjugates disclosed herein may be in the form of pharmaceutically acceptable salts. However, it should be understood that non-pharmaceutically acceptable salts also fall within the scope of this disclosure, as these may be useful as intermediates in the preparation of pharmaceutically acceptable salts or during storage or transport. Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids (such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, boric acid, aminosulfonic acid, and hydrobromic acid) or salts of pharmaceutically acceptable organic acids (such as acetic acid, propionic acid, butyric acid, tartaric acid, maleic acid, hydroxymaleic acid, fumaric acid, citric acid, lactic acid, mucoic acid, gluconic acid, benzoic acid, succinic acid, oxalic acid, phenylacetic acid, methanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, salicylic acid, sulfanilic acid, aspartic acid, glutamic acid, ethylenediaminetetraacetic acid, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid, and valeric acid). Basic salts include, but are not limited to, those that form with pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, ammonium, and alkylammonium.

[0273] The compounds disclosed herein can exist in specific geometric or stereoisomeric forms. This disclosure envisions all such compounds, including cis and trans isomers, (-)- and (+)- enantiomers, (R)- and (S)- enantiomers, diastereomers, (D)- isomers, (L)- isomers, and racemic mixtures thereof, as well as other mixtures, such as mixtures enriched with enantiomers or diastereomers, all of which are within the scope of this disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of this disclosure. The compounds containing asymmetric carbon atoms of this disclosure can be isolated in optically active pure form or in racemic form. Optically active pure forms can be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0274] Optically active (R)- and (S)- isomers, as well as D- and L- isomers, can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. To obtain an enantiomer of a compound disclosed herein, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the desired enantiomer in pure form. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a salt of the diastereomeric isomer is formed with a suitable optically active acid or base, followed by diastereomeric resolution using conventional methods known in the art, and then the pure enantiomer is recovered. Furthermore, the separation of enantiomers and diastereomeric isomers is typically accomplished by using chromatography employing a chiral stationary phase and optionally combined with chemical derivatization (e.g., from amines to carbamates).

[0275] In the chemical structure of the compounds described in this disclosure, the bonds... This indicates that the configuration is not specified; that is, if chiral isomers exist in the chemical structure, the bond... It can be or Or simultaneously include and Two configurations.

[0276] In the chemical structure of the compounds described in this disclosure, the bonds... No configuration is specified, meaning it can be Z configuration, E configuration, or both configurations.

[0277] The compounds and intermediates disclosed herein may also exist in different tautomer forms, and all such forms are included within the scope of this disclosure. The terms "tautomer" or "tautomer form" refer to structural isomers of different energies that can interconvert via low energy barriers. For example, proton tautomers (also known as proton transfer tautomers) include interconversions via proton migration, such as the compounds disclosed herein comprising tautomer changes between formulas M1, M2, and M3 as shown below.

[0278] All tautomers are within the scope of this disclosure. The nomenclature of compounds does not exclude any tautomers.

[0279] Unless otherwise stated, the terms used in the specification and claims shall have the following meanings.

[0280] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0281] The term "hydroxyl group" refers to -OH.

[0282] The term "amino" refers to -NH2.

[0283] The term "cyano" refers to -CN.

[0284] The term "nitro" refers to -NO2.

[0285] The term "oxo" or "oxo" refers to "=O".

[0286] The term "thio-" or "thio" means "=S".

[0287] The term "carbonyl" refers to C=O.

[0288] The term "carboxyl group" refers to -C(O)OH.

[0289] The term "azide" refers to -N = N + =N - Or -N3.

[0290] The terms "hydrazine" or "hydrazine group" refer to -NH-NH2.

[0291] The term "halogenation" refers to the substitution of one or more atoms selected from fluorine, chlorine, bromine, and iodine.

[0292] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight-chain and branched groups with 1 to 6 carbon atoms. The alkyl group includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, and their various branched isomers. The alkyl group can be substituted or unsubstituted; when substituted, the substituent can be replaced at any usable connection point, preferably one or more of the following groups, including, but not limited to, halogen, hydroxyl, mercapto, nitro, carboxyl, amino, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, 3 to 10-membered cycloalkyl.

[0293] The term "alkoxy" refers to -O- (alkyl), where alkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexyloxy. Alkoxy groups can be substituted or unsubstituted; when substituted, the substituent can be replaced at any usable connection point, preferably one or more of the following groups, including but not limited to halogens, hydroxyl, mercapto, nitro, carboxyl, amino, cyano, and C. 1-6 Alkyl, C 1-6 Alkoxy, 3 to 10-membered cycloalkyl.

[0294] The term "alkylene" refers to the portion of an alkane molecule remaining after the removal of two hydrogen atoms, including straight-chain and branched subgroups with 1 to 20 carbon atoms. Alkylenes containing 1 to 6 carbon atoms, non-limiting examples include, but are not limited to, methylene (-CH2-), ethylene (such as -CH2CH2- or -CH(CH3)-). Unless otherwise specified, alkylenes can be substituted or unsubstituted.

[0295] The term "heteroalkylene" refers to an alkylene group in which one or more -CH2- atoms are replaced by heteroatoms selected from N, O, and S; wherein the alkylene group is as defined above; the heteroalkylene group may be substituted or unsubstituted. Unless otherwise specified, the heteroalkylene group may be substituted or unsubstituted.

[0296] The term "cycloalkyl" or "alicyclic" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the alicyclic ring contains 3 to 10 carbon atoms. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups. Cycloalkyl or alicyclic groups can be substituted or unsubstituted. When substituted, the substituent can be replaced at any usable connection point, preferably one or more of the following groups, including but not limited to halogens, hydroxyl, mercapto, nitro, carboxyl, amino, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, 3 to 10-membered cycloalkyl.

[0297] The term "cycloalkylene" refers to a divalent cyclic hydrocarbon group derived from a cycloalkyl group. Examples of cycloalkylene groups include, but are not limited to, those derived from cycloalkyl groups. etc. Among them, cycloalkyl is defined as described above.

[0298] The terms "heterocyclic alkyl," "heterocyclic group," or "heterocycle" refer to a cyclic hydrocarbon in which one to four carbon atoms have been independently replaced by a heteroatom selected from N, S, S(O), S(O)₂, and O. The heterocycle can be saturated or unsaturated. Examples of suitable heterocyclic groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothiopheneyl, pyrrolyl, pyrrololinyl, pyrazolinyl, pyranyl, piperidinyl, pyrazolinyl, dithiohexacyclopentadienyl, oxathiohexacyclopentadienyl, dioxacyclohexyl, dioxacyclohexenyl, morpholino, and oxazinyl. Heterocyclic alkyl or heterocycles can be substituted or unsubstituted; when substituted, the substituent can be substituted at any usable junction, preferably one or more of the following groups, including but not limited to halogens, hydroxyl, mercapto, nitro, carboxyl, amino, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, 3 to 10-membered cycloalkyl.

[0299] The term "subheterocyclic group" refers to a heterocyclic group having two monovalent group centers, which are derived by removing two hydrogen atoms from the same or two different carbon atoms of the parent heterocycle, by removing two hydrogen atoms from two nitrogen atoms of the parent heterocycle, or by removing hydrogen atoms from both nitrogen and carbon atoms of the parent heterocycle. Subheterocyclic groups include, but are not limited to, piperidine-1,4-diyl, piperazine-1,4-diyl, tetrahydrofuran-2,4-diyl, tetrahydrofuran-3,4-diyl, aziridine-1,3-diyl, and pyrrolidine-1,3-diyl. The definition of a heterocyclic group is as described above.

[0300] The term "aryl" or "aromatic ring" refers to any stable, monocyclic or bicyclic carbon ring with up to seven atoms in each ring, wherein at least one ring is aromatic. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indenyl, biphenyl, or binaphthyl. Aryl groups or aromatic rings can be substituted or unsubstituted; when substituted, the substituent can be replaced at any usable junction, preferably one or more of the following groups, including, but not limited to, one or more halogens, hydroxyl, mercapto, carboxyl, amino, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, phenyl.

[0301] The term "arylene" refers to a centrally located monovalent group derived from the same or two different carbon atoms of a parent aryl group by removing two hydrogen atoms. Examples of arylene groups include, but are not limited to, phenylene. The definition of aryl is as described above.

[0302] The term “heteroaryl” or “heteroary ring” refers to a stable monocyclic or bicyclic ring with up to seven atoms in each ring, wherein at least one ring is aromatic and at least one ring contains one to four heteroatoms selected from O, N and S. Examples of heteroaryl groups include, but are not limited to, acridinel, carbazolyl, cyclophosphinl, quinoxalinl, quinazolinl, pyrazolyl, indolyl, benzotriazolyl, furanyl, thiophene, phenylthio, 3,4-propylenedioxythiophenyl, benzothiophene, benzofuranyl, benzodioxane, benzodioxane, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, imidazolyl, pyrazinyl, pyridinyl, pyrimidinyl, pyrroleyl, tetrahydroquinoline, thiazolyl, isothiazolyl, 1,2,4-triazolyl, 1,2,3-triazolyl, 1,2,4-oxadiazolyl, 1,2,4-thiadiazolyl, 1,3,5-triazinyl, 1,2,4-triazinyl, 1,2,4,5-tetraazinyl, and tetrazolyl. The heteroaryl group or heterocyclic ring can be substituted or unsubstituted. When substituted, the substituent can be replaced at any usable connection point, preferably one or more of the following groups, including but not limited to one or more halogens, hydroxyl groups, mercapto groups, carboxyl groups, amino groups, cyano groups, C6 groups, etc.1-6 Alkyl, C 1-6 Alkoxy, phenyl.

[0303] The term "hybrid aryl" refers to a heteroaryl system having two connection sites attached to the rest of the molecule. Examples of hybrid aryl systems include, but are not limited to, pyridylene, pyrroleylene, thiazolyl, and imidazolyl. etc. Among them, the definition of heteroaryl is as described above.

[0304] The term "dendritic polymer" refers to a molecule containing a core and at least one dendritic unit attached to the core. Each dendritic unit consists of at least one layer or one generation of branching building units, resulting in an increasing number of branched structures with branches in each generation of building units. The maximum number of dendritic units attached to the core is limited by the number of functional groups on the core.

[0305] The term "building unit" refers to a molecule with at least three functional groups, one of which is used to attach to the core or the previous generation of the building unit and at least two functional groups are used to attach to the next generation of the building unit or to form the surface of a dendritic polymer.

[0306] The term "generation" refers to the number of layers of building units constituting a dendritic motif or dendritic polymer. For example, a first-generation dendritic polymer will have one layer of building units connected to the core, such as core-[[building unit]]u, where u is the number of dendritic motifs connected to the core. A second-generation dendritic polymer has two layers of building units in each dendritic motif connected to the core. When the building unit has a branch point, the dendritic polymer can be: core[[building unit][building unit]2]u. A third-generation dendritic polymer has three layers of building units in each dendritic motif connected to the core, such as core-[[building unit][building unit]2[building unit]4]u. A sixth-generation dendritic polymer has six layers of building units connected to the core, such as core-[[building unit][building unit]2[building unit]4[building unit]8[building unit]16[building unit]32]u, etc. The last generation (outermost generation) of building units provides the surface functionalization of the dendritic polymer and the number of functionalized groups that can be used to bind end groups. For example, in a dendritic polymer with two dendritic units connected to the core (u=2), if each building unit has a branch point and there are six generations, then the outermost generation has 64 building units and 128 functional groups that can be used to bind end groups.

[0307] The term "slightly soluble" refers to a drug or pharmaceutically active agent that has a solubility of 1 mg / mL to 10 mg / mL in water. Drugs with a solubility of less than 1 mg / mL in water are considered insoluble.

[0308] The term "solubilizing excipient" refers to a formulation additive used to dissolve insoluble or slightly soluble pharmaceutical active agents in aqueous formulations. Examples include surfactants such as polyethoxylated castor oil (including Cremophor EL, Cremophor RH40, and Cremophor RH60), D-α-tocopherol-polyethylene glycol 1000 succinate, polysorbate 20, polysorbate 80, polyethylene glycol 12-hydroxystearate (solutol HS15), sorbitan monooleate, poloxamer 407, and PEG-3-caprylate (Labrasol), etc.

[0309] In this disclosure, residue A of the pharmaceutically active agent, its derivative or its precursor refers to a molecule or group with pharmaceutical activity.

[0310] The term "pharmaceutical active agent" as used in this disclosure refers to a compound that has biological activity and provides the desired physiological effect when administered to a subject in need (e.g., an active pharmaceutical ingredient and a pharmaceutically acceptable salt thereof, a derivative of an active pharmaceutical ingredient and a pharmaceutically acceptable salt thereof, a prodrug modification of an active pharmaceutical ingredient and a pharmaceutically acceptable salt thereof), including but not limited to: cytotoxic compounds, cell growth inhibitory compounds, immunosuppressive compounds, anti-inflammatory compounds, or anti-infective compounds. The cytotoxic compounds mentioned include, but are not limited to, eribulin, trabectedin, MMAE, MMAF, alkylating agents (e.g., nitrogen mustard, cyclophosphamide, thiotepa, temozolomide, busulfan, semustine), platinum compounds (e.g., cisplatin, carboplatin, oxaliplatin, nedaplatin), antibiotics (e.g., mitomycin, bleomycin), tyrosine kinase inhibitors (e.g., axitinib, gefitinib, erlotinib, osimertinib, icotinib, imatinib, sunitinib), protein degrading agents (e.g., protein degradation-targeting chimeras (PROTAC) or molecular gels), and KRAS inhibitors (e.g., KRAS G12C inhibitors, KRAS...). G12D inhibitors, pan-KRAS inhibitors), anthracyclines (e.g., doxorubicin or PNU159682), vinblastines (e.g., vincristine, vinorelbine, vinorelbine), camptothecin analogs (e.g., SN38, 9106-IM-2), calicimycin, uncialamycin, orlistatine, tubulolysin analogs, maytansine, cryptophytin, benzodiazepine dimers (including pyrrole [2,1-c] known as PBDs). [1,4] Benzodiazepines), indobenzobenzodiazepine pseudodimer (IGN), ducamycin, Bcl2 and Bcl-xl inhibitors, Thailanstatins, amatoxins (including α-phalloidin), spindle kinin (KSP) inhibitors, cyclin-dependent kinase (CDK) inhibitors, actinomycins or radionuclides and their chelating agents (such as DOTA / 177Lu), and derivatives of the above drugs and their pharmaceutically acceptable salts. Anti-inflammatory compounds, such as corticosteroids, such as dexamethasone, fluticasone, fluocinolone, and triamcinolone. Anti-infectives, such as antibiotics, such as rifampin or vancomycin.

[0311] The terms “pharmaceutical active agent”, “pharmaceutical active agent or its derivative”, “residue of pharmaceutical active agent or its precursor”, or “residue A of pharmaceutical active agent, its derivative or its precursor” are used interchangeably and refer to molecules or groups that have pharmaceutical activity.

[0312] The term "spacer unit" refers to a divalent linear arm capable of covalently binding two components of a dendritic polymer conjugate, for example, it can be used to connect a pharmaceutically active agent to a dendritic polymer, or to connect a pharmacokinetic modifier to a dendritic polymer, or to connect a targeting moiety to a dendritic polymer. Examples of the spacer unit include, but are not limited to: carbonyl, alkylene, heteroalkylene, alkoxy, polyether (such as polyalkylene glycol, typically polyethylene glycol), one or more natural or non-natural amino acids (such as glycine, alanine, proline, valine, N-methylglycine), 3- to 8-membered heterocyclic alkyl, C3-8 carbon cyclic, aryl, and any combination thereof.

[0313] The term "cleavable group," also known as a "releaseable assembly unit," refers to a cleavable bond capable of forming a drug unit for release, functioning to release the drug at a ligand-targeted site. In some embodiments, the cleavable group is a cleavable peptide moiety. In some embodiments, dendritic polymer conjugates containing cleavable peptide moieties exhibit lower aggregation levels and improved antibody-to-drug ratios compared to dendritic polymer conjugates containing other cleavable groups. In some embodiments, the addition of a cleavable group increases cytotoxicity and / or potency compared to a non-cleavable linker. In some embodiments, the cleavable peptide moiety is enzymatically cleavable, and the linker is an enzyme-cleavable linker. In some embodiments, the enzyme is a cathepsin, and the linker is a cathepsin-cleavable linker. In some embodiments, an enzyme-cleavable linker (e.g., a cathepsin-cleavable linker) exhibits one or more of the aforementioned improved properties compared to other cleavage mechanisms.

[0314] The term "cleavable peptide moiety" refers to the portion containing amino acid units, wherein the amino acid units preferably comprise 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, aspartic acid, homolysine, n-methylvaline, ... The peptide residues are composed of amino acids, where x is an integer from 1 to 6. In some embodiments, the cleavable group Z is... 3Selected from valine-citrulline (Val-Cit), valine-alanine (Val-Ala), alanine-phenylalanine (Ala-Phe); phenylalanine-lysine (Phe-Lys), phenylalanine-homolysine (Phe-Homolys), n-methyl-valine-citrulline (Me-Val-Cit), alanine-alanine (Ala-Ala), glycine-glutamic acid (Gly-Glu), glutamic acid-alanine-alanine (Glu-Ala-Ala), glycine-lysine (Gly-Lys), glycine-valine-citrulline (Gly-Val-Cit), glycine-glycine-glycine (Gly-Gly-Gly), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly) and...

[0315] The terms “targeting fraction” and “targeting agent” are used interchangeably and refer to any macromolecule (peptide, protein, peptide, typically antibody) commonly used in antibody-drug conjugate (ADC) technology, or a small molecule (such as folic acid or aptamers) that can be covalently conjugated to a synthetic linker or drug linker using bioconjugation techniques (Greg T. Hermanson, Bioconjugate Techniques, 3). rdEdition, 2013, Academic Press. Ligands are traditionally selected compounds based on their targeting ability. A non-exhaustive list of ligands includes: proteins, polypeptides, peptides, antibodies, full-length antibodies and their antigen-binding fragments, interferons, lymphokines, hormones, growth factors, vitamins, transferrin, or any other cell-binding molecules or substances. The primary class of ligands used to prepare conjugates is antibodies. Targets of the antibodies include, but are not limited to, PD-1, Her-2, Her-3, CD20, EGFR, VEGF, VEGFR, VEGFR1, VEGFR2, BLCAM, BLyS, BSG, C5F, cADPR1, CCR4, CD19, CD20, CD25, CD3, CD30, TUBR, CD79B, COVID19 spike glycoprotein, CTLA4, and dabigatran inhibitors. Inhibitors), FGF23, FIX, FX, GD2, HSP, IL-6R, IFNG, IgE, IL-12R, IL-23, IL-13R, IL-4R, IL-17, IL-17A, IL-17RA, IL-1RL2, IL-23A, IL-2RA, IL-5R, IL-6R, IL-8R, ITGA4, ITGB7, MAdCAM, KLKB1, ODF, PCSK9, CTLA4, RANKL, TNFα. An example protein is human serum albumin. In some embodiments, the ligands are chimeric antibodies, humanized antibodies, or human antibodies.

[0316] As used herein, the term "antibody" is used in the broadest sense and encompasses monoclonal antibodies, polyclonal antibodies, modified monoclonal and polyclonal antibodies, monospecific antibodies, multispecific antibodies (such as bispecific antibodies), antibody fragments, and antibody mimics (Affibody, Affilin, Affimer, Nanofitin, Cell Penetrating Alphabody, Antiticalin, Avimer, Fynomer, Monobodies, or nanoCLAMP). Examples of monoclonal antibodies include, but are not limited to, adalimumab, alexizumab, atezolizumab, emecizumab, abavirin, oftadalafil, oxalitumumab, omalizumab, oxotuzumab, oxotuzumab, oxotuzumab, baliximab, bevacizumab, belimumab, bromolimumab, brosuumab, daratumumab, daratumumab beta, and detrastuzumab. Monoclonal antibodies, denosumab, iodine [131I] metuximab, durvalumab, duprexaumab, trastuzumab emtansine, envorimab, golimumab, gosatuzumab, gusetinumab, candunimarab, camrelizumab, anti-human T-cell CD3 mouse monoclonal antibody, anti-human interleukin-8 mouse monoclonal antibody, lanalimumab, ramoximumab, ranibizumab, rituximab, romi Severtumab, Meplezumab, Mogliflozin, Nacetuzumab, Nivolumab, Nimotuzumab, Pembrolizumab, Pertuzumab, Pesolizumab, Pesolizumab, Putelizumab, Trastuzumab, Repertuzumab, Saturolizumab, Separibumab, Sugelizumab, Secukinumab, Secuximab, Slulizumab, Toripalimab Tislelizumab, Tocilizumab, Brentuximab, Verdelizumab, Verdicituzumab, Verpotuzumab, Ustenozumab, Cetuximab, Sintilimab, Inellizumab, Inetuximab, Ipilimumab, Idacilizumab, Ikucilizumab, Ivolomumab, Imalimumab, Ikkizumab, Infliximab, Recombinant anti-CD25 humanized monoclonal antibody.

[0317] The term "amino acid" refers to an organic compound whose molecular structure contains both an amino group and a carboxyl group, with both groups directly attached to the -CH- structure. The general formula is H₂NCHRCOOH, where R is H, a substituted or unsubstituted alkyl group, etc. Based on the position of the amino group attached to the carbon atom in the carboxylic acid, amino acids can be classified into α, β, γ, δ, ε…-amino acids. In the biological world, the amino acids that constitute natural proteins have specific structural characteristics, namely, their amino groups are directly attached to the α-carbon atom, i.e., α-amino acids, including glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, and proline. Non-natural amino acids, such as citrulline, are also present. As is known to those skilled in the art, non-natural amino acids do not constitute natural proteins and therefore do not participate in the synthesis of the antibodies disclosed herein. The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0318] The term "dendritic polymer conjugate" refers to any conjugate that combines a dendritic polymer (in some embodiments, the surface layer of the dendritic polymer is covalently linked to a pharmaceutically active agent or a pharmacokinetic modifier) ​​as defined above with a targeting moiety and involves any of the means described above. In some embodiments, the targeting moiety is an antibody, in which case the dendritic polymer conjugate may refer to an antibody-drug conjugate (ADC).

[0319] The term "grafting number of residue A in the pharmaceutically active agent, its derivative, or its precursor" (hereinafter referred to as "grafting number of residue A") refers to the number of surface ammonia sites occupied by residue A in the dendritic polymer D. For example, a grafting number of 28 residues A indicates that 28 of the bindable surface ammonia sites in the dendritic polymer D are occupied by residue A. The grafting number of residue A is determined by... 1 Calculated using 1H NMR detection method.

[0320] The terms "grafting number of cabazitaxel," "grafting number of cabazitaxel derivatives," and "grafting number of cabazitaxel or its derivatives" are used interchangeably in this disclosure and all refer to the number of surface amine sites occupied by cabazitaxel in dendritic polymer D. For example, a grafting number of 28 for cabazitaxel or its derivatives indicates that 28 of the bindable surface amine sites in dendritic polymer D are occupied by cabazitaxel or its derivatives. The grafting number of cabazitaxel is determined by... 1 Calculated using 1H NMR detection method.

[0321] The term "drug loading" refers to the average amount of drug carried by each antibody-drug conjugate molecule in a population of antibody-drug conjugates, and can also be expressed as the ratio of drug amount to antibody amount. For example, for formula IV-A of this disclosure, the drug loading = 8 × p. For formula IV-a of this disclosure, the drug loading = 2 × 4 × p. For formula IV-C of this disclosure, the drug loading = 8 × q. For formula IV-c of this disclosure, the drug loading = 4 × 2 × p. The range of drug loading can be 1 to 64 pharmaceutically active agents linked to each antibody. In embodiments of this disclosure, the drug loading can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or the average of any two values. The average amount of drug in each ADC molecule after the coupling reaction can be identified using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA, monoclonal antibody size variant assay (CE-SDS), and HPLC. Furthermore, the p-value or q-value of the dendritic polymer conjugate molecule in this disclosure can be determined by CE-SDS.

[0322] The drug loading of dendritic polymer conjugates can be controlled using the following non-limiting methods, including:

[0323] (1) Control the molar ratio of the ligation reagent and the monoclonal antibody.

[0324] (2) Control the reaction time and temperature.

[0325] (3) Choose different reaction reagents.

[0326] The values ​​in this disclosure are instrument measurements and are subject to a certain degree of error. Generally, ±10% is within the reasonable error range. Of course, the context in which the value is used must be considered. For example, the number of grafts, which is calculated after measurement, is acceptable if the error variation does not exceed ±10%, and can be ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%, preferably ±5%.

[0327] The term "antibody" refers to immunoglobulin, a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain can further divide them into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ or λ chains based on differences in the constant region. Each of the five classes of Ig can have either a κ chain or a λ chain.

[0328] The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is known as the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are called the constant region. The variable region includes three hypervariable regions (HVRs) and four relatively conserved backbone regions (FRs). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0329] The antibodies disclosed herein include murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies, with humanized antibodies and fully human antibodies being preferred.

[0330] The term "mouse antibody" in this disclosure refers to antibodies prepared using mice in accordance with the knowledge and skills in the art. Preparation involves injecting a test subject with a specific antigen, followed by isolating a hybridoma expressing an antibody with the desired sequence or functional characteristics.

[0331] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody. It can reduce the immune response induced by murine antibodies. To create a chimeric antibody, a hybridoma that secretes murine-specific monoclonal antibodies must first be established. Then, the variable region gene is cloned from the murine hybridoma cells. Next, the constant region gene of the human antibody is cloned as needed. The murine variable region gene and the human constant region gene are then linked to form a chimeric gene, which is inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system.

[0332] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody generated by grafting a mouse CDR sequence into a human antibody variable region framework, i.e., a human germline antibody framework sequence of different types. This overcomes the heterologous response induced by chimeric antibodies carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or publicly available references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available at www.mrccpe.com.ac.uk / vbase) and in Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity along with a decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse or reversion mutations to maintain activity. The humanized antibodies disclosed herein also include humanized antibodies further matured by phage display with affinity for the CDR.

[0333] The term "fully human antibody," also known as a "fully human monoclonal antibody," refers to an antibody whose variable and constant regions are both human-derived, eliminating immunogenicity and toxicity. The development of monoclonal antibodies has gone through four stages: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies. This disclosure pertains to fully human monoclonal antibodies. Related technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology, and single B cell antibody preparation technology.

[0334] The term “antigen-binding fragment” refers to one or more fragments of an antibody that maintain the ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in “antigen-binding fragments” include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bridges on hinge regions; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VH and VL domains of a single arm of the antibody; (v) single-domain or dAb fragments (Ward et al., (1989) Nature 341: 544-546) consisting of a VH domain; and (vi) separate complementarity-determining regions (CDRs) or (vii) combinations of two or more separate CDRs optionally linked by synthetic linkers. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked by synthetic linkers using recombinant methods, thereby enabling the production of a single protein chain in which the VL and VH regions pair to form a monovalent molecule (referred to as a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding moieties can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0335] Fab is an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity obtained by treating IgG antibody molecules with the protease papain (which cleaves the amino acid residue at position 224 of the H chain). Approximately half of the N-terminal side of the H chain and the entire L chain are linked together by disulfide bonds.

[0336] F(ab')2 is an antibody fragment with a molecular weight of approximately 100,000, possessing antigen-binding activity, and containing two Fab regions connected at the hinge position, obtained by digesting the portion below the two disulfide bonds in the hinge region of IgG with the enzyme pepsin.

[0337] Fab' is an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity obtained by cleaving the disulfide bonds in the hinge region of the aforementioned F(ab')2.

[0338] In addition, the Fab' can be produced by inserting DNA encoding the Fab' fragment of an antibody into a prokaryotic or eukaryotic expression vector and then introducing the vector into a prokaryote or eukaryote to express the Fab'.

[0339] The terms “single-chain antibody,” “single-chain Fv,” or “scFv” refer to molecules containing a variable domain (or region; VH) of the antibody heavy chain and a variable domain (or region; VL) of the antibody light chain linked by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof, for example, using variants with 1–4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444–6448). Other connectors that may be used in this disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

[0340] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242. As used herein, the Kabat definition of CDR applies only to CDR1, CDR2, and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3) of the heavy chain variable domain. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable domain and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable domain. The amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the “Kabat” numbering rule (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the “Chothia” numbering rule (see Al-Lazikani et al., (1997) JMB 273: 927-948), and the ImMunoGenTics (IMGT) numbering rule (see Lefranc MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003)), etc. For example, in the classic format, following Kabat rules, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following Chothia rules, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).Combining the CDR definitions from Kabat and Chothia, the CDR is composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) from human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) from human VL. Following IMGT rules, the CDR amino acid residues in VH are approximately numbered 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), while those in VL are approximately numbered 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3). Following IMGT rules, the CDR region of an antibody can be determined using the IMGT / DomainGap Align procedure.

[0341] The term "antibody framework" refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain. Essentially, it is a variable domain without a CDR.

[0342] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to a pre-defined epitope on an antigen. Typically, antibodies bind at a concentration of approximately less than 10... -7 M, for example: approximately less than 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (KD) binding.

[0343] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop to which an additional DNA segment can be linked. In another embodiment, the vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. The vectors disclosed herein are capable of autonomous replication in host cells that have been introduced into them (e.g., bacterial vectors with bacterial origins of replication and episodic mammalian vectors) or can be integrated into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome (e.g., non-episodic mammalian vectors).

[0344] Methods for producing and purifying antibodies and antigen-binding fragments are well-known in the prior art, such as those described in Cold Spring Harbor's Guide to Antibody Laboratory Techniques, Chapters 5-8 and 15. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in this invention utilize genetic engineering methods to add one or more human FR regions to a non-human CDR region. Human FR germline sequences can be obtained by comparing with the IMGT Human Antibody Variable Region Germline Gene Database and MOE software, from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr, or from the journal Immunoglobulins, 2001 ISBN012441351.

[0345] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; members of the Bacillaceae family, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells.

[0346] The engineered antibody or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more preferred prior art, mammalian expression systems lead to glycosylation of the antibody, particularly at the highly conserved N-terminal site in the Fc region. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. The culture medium secreting the antibody can be purified using conventional techniques, such as using an A or G Sepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody is then eluted using a pH gradient, and the antibody fragments are detected by SDS-PAGE and collected. The antibody can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, e.g., at -70°C, or lyophilized.

[0347] Amino acid sequence “identity” refers to the percentage of amino acid residues in a first sequence that are identical to those in a second sequence, after aligning the amino acid sequences and, where necessary, introducing gaps to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. For the purpose of determining the percentage of amino acid sequence identity, alignment can be performed in a variety of ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0348] The term "Solid-Phase Peptide Synthesis (SPPS) Method" refers to the carrier commonly used in SPPS, in which peptides anchored to a carrier (insoluble polymer) are assembled via repeated deprotection-wash-coupling-wash cycles by the successive addition of Fmoc- or Boc-protected amino acids. Each amino acid addition refers to the following cycle: (i) cleavage of the Nα-protecting group, (ii) washing step, (iii) coupling of the fluorenylmethoxycarbonyl- (Fmoc-) or tert-butyloxycarbonyl- (Boc-)-protected amino acid with a coupling agent and a nonnucleophilic base, and (iv) washing step. Since the growth chain binds to the carrier, excess reagents and soluble byproducts can be removed by simple filtration. Because repeated coupling reactions with N-methylated amino acids protected with hindered Fmoc- or Boc- are difficult and generally suboptimal, this technique is expected to yield low crude purity, poor purification, and low yields. Examples of the carriers are Wang resin, Rink amide resin, triphenylmethyl and 2-chlorotriphenylmethyl resin, PAM resin, PAL resin, Sieber amide resin, MBHA resin, HMPB resin, and HMBA resin, which are commercially available and to which peptides are directly or indirectly bound.

[0349] The term "peptide" refers to a compound fragment that lies between amino acids and proteins. It is composed of two or more amino acid molecules linked together by peptide bonds. It is a structural and functional fragment of proteins, such as hormones and enzymes, which are essentially peptides.

[0350] The term "sugar" refers to a biological macromolecule composed of three elements: C, H, and O. It can be classified into monosaccharides, disaccharides, and polysaccharides.

[0351] "Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically pharmaceutically acceptable salts or prodrugs, along with other chemical components, such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and its biological activity.

[0352] "Pharmaceutical excipients" include, but are not limited to, any adjuvants, carriers, excipients, flow aids, sweeteners, diluents, preservatives, dyes / colorants, flavorings, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that have been approved by the U.S. Food and Drug Administration for use in humans or livestock.

[0353] The term "effective amount" or "effective therapeutic amount" as used in this disclosure includes an amount sufficient to improve or prevent symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0354] The terms "optional" or "optional" mean that the event or environment subsequently described may, but does not have to, occur. This description includes the possibility that the event or environment may or may not occur. For example, "the alkylene or heteroalkylene is optionally interrupted by one or more groups selected from cycloalkylene, heterocycloalkylene, arylene, and heteroarylene" means that the alkylene or heteroalkylene may be interrupted by cycloalkylene, heterocycloalkylene, arylene, or heteroarylene but is not necessarily interrupted. This description includes the case where the alkylene or heteroalkylene is interrupted by cycloalkylene, heterocycloalkylene, arylene, or heteroarylene and the case where the alkylene or heteroalkylene is not interrupted by cycloalkylene, heterocycloalkylene, arylene, or heteroarylene.

[0355] The term "substituted" refers to one or more hydrogen atoms in a group, preferably up to five, more preferably one to three hydrogen atoms, which are independently substituted by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort.

[0356] For pharmaceuticals or pharmacologically active agents, the term "effective amount" refers to a sufficient quantity of a drug or agent that is non-toxic but achieves the desired effect. The determination of the effective amount varies from person to person, depending on the recipient's age and general condition, as well as the specific active substance. The appropriate effective amount in a given case can be determined by a person skilled in the art based on routine testing.

[0357] The term "pharmaceutically acceptable" means that these compounds, materials, compositions, and / or dosage forms are suitable for contact with patient tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, within reasonable medical judgment, have a reasonable benefit / risk ratio, and are effective for their intended use.

[0358] The term "DMF" refers to N,N-dimethylformamide.

[0359] The term "DIPEA" refers to N,N-diisopropylethylamine.

[0360] The term "DMAP" refers to 4-dimethylaminopyridine.

[0361] The term "EDCI" refers to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0362] The term "PyBOP" refers to 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate.

[0363] As used herein, the singular forms of “a,” “an,” and “the” include plural references, and vice versa, unless the context clearly indicates otherwise. Attached Figure Description

[0364] Figure 1 shows the concentration of free docetaxel released at different time points after stable incubation of the test compound in human plasma.

[0365] Figure 2 shows the absolute white blood cell count index of the test compound. Detailed Implementation

[0366] The present disclosure is further described and explained below with reference to embodiments, but these embodiments are not intended to limit the scope of the present disclosure.

[0367] Experimental methods not specifying specific conditions in the embodiments of this disclosure are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. Reagents not specifying their source are commercially available, conventional reagents.

[0368] The structure of the compound was determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS). Chemical shifts were expressed in 10⁻¹⁰ increments. - 6 (ppm) is given as the unit.

[0369] MS measurements were performed using a Finnigan LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).

[0370] The determination by high performance liquid chromatography (HPLC) was performed using an Agilent 1200DAD high performance liquid chromatograph (Sunfire C18 150×4.6mm column) and a Waters 2695-2996 high performance liquid chromatograph (Gimini C18 150×4.6mm column).

[0371] Thin-layer chromatography (TLC) uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The silica gel plates used in TLC are 0.15mm to 0.2mm in diameter, while those used for TLC separation and purification are 0.4mm to 0.5mm in diameter.

[0372] Column chromatography typically uses 200-300 mesh silica gel from Yantai Huanghai as the carrier.

[0373] The known starting materials disclosed herein can be synthesized using or in accordance with methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organnics, Aldrich Chemical Company, Accela ChemBio Inc, and Darui Chemicals.

[0374] Unless otherwise specified in the examples, the reactions were carried out under an argon or nitrogen atmosphere.

[0375] Argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen gas balloon with a volume of approximately 1L.

[0376] A hydrogen atmosphere refers to a reaction flask connected to a hydrogen balloon with a volume of approximately 1L.

[0377] The pressurized hydrogenation reaction was performed using a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or an HC2-SS hydrogenator.

[0378] The hydrogenation reaction is usually carried out under vacuum, filled with hydrogen gas, and repeated 3 times.

[0379] The microwave reaction was performed using a CEM Discover-S 908860 microwave reactor.

[0380] Unless otherwise specified in the examples, the solution in the reaction refers to an aqueous solution.

[0381] Unless otherwise specified in the examples, the reaction temperature is room temperature.

[0382] Room temperature is the optimal reaction temperature, with a range of 20℃ to 30℃.

[0383] Preparation of PBS buffer solution with pH=6.5 in the example: Take 8.5g of KH2PO4, 8.56g of K2HPO4·3H2O, 5.85g of NaCl and 1.5g of EDTA and put them into a bottle, make up to 2L, sonicate to dissolve completely, and shake well to obtain the solution.

[0384] The eluent systems for column chromatography and the developing solvent systems for thin-layer chromatography used to purify the compounds include: A: dichloromethane and isopropanol system, B: dichloromethane and methanol system, and C: petroleum ether and ethyl acetate system. The volume ratio of the solvents is adjusted according to the polarity of the compounds, and small amounts of triethylamine and acidic or basic reagents can also be added for adjustment.

[0385] Some of the compounds disclosed herein were characterized by Q-TOF LC / MS. The Q-TOF LC / MS was performed using an Agilent 6530 Precision Mass Number Quadrupole-Time-of-Flight Mass Spectrometer and an Agilent 1290-Infinity Ultra-High Performance Liquid Chromatography System (Agilent Poroshell 300SB-C8 5 μm, 2.1 × 75 mm column).

[0386] Preparation of sodium acetate buffer:

[0387] Mobile phase A: In a 1L container, add sodium acetate (4.10g), add 900mL of distilled water, sonicate to dissolve for 15min, adjust the pH to 5.0 with acetic acid, continue stirring for 10min, and then bring the volume to 1L.

[0388] Mobile phase B: In a 1L container, add sodium acetate (4.10g) and sodium chloride (58.44g), add 900mL of distilled water, sonicate for 15min, adjust the pH to 5.0 with acetic acid, continue stirring for 10min, and then bring the volume to 1L.

[0389] 1×PBS 7.2 (pH 7.2): purchased from Shanghai Yuanpei Biotechnology Co., Ltd.

[0390] Preparation of 1×PBS 6.3 (pH 6.3): Adjust the pH to 6.3 with phosphate based on 1×PBS 7.2.

[0391] The following antibodies were prepared using standard antibody methods, such as vector construction, transfection into eukaryotic cells like HEK293 cells (Life Technologies Cat. No. 11625019), purification, and expression.

[0392] The sequence of Trastuzumab is as follows:

[0393] Light chain SEQ ID NO.1

[0394] Heavy chain SEQ ID NO.2

[0395] The sequence of Bevacizumab is as follows:

[0396] Light chain: SEQ ID NO.3

[0397] Heavy chain: SEQ ID NO.4

[0398] The sequence of Cetuximab is as follows:

[0399] Light chain: SEQ ID NO.5

[0400] Heavy chain: SEQ ID NO.6

[0401] The sequence of Telisotuzumab is as follows:

[0402] Light chain: SEQ ID NO.7

[0403] Heavy chain: SEQ ID NO.8

[0404] Preparation of compound A

[0405] Step 1:

[0406] Compound A1 (25.00 g, 72.80 mmol, purchased from Shaoyuan) was dissolved in dichloromethane (150 mL), and trifluoroacetic acid (150 mL) was added dropwise at 0 °C. The reaction was continued at 0 °C until complete. The reaction solution was concentrated under reduced pressure and dissolved in 300 mL of water. The solution was washed with ethyl acetate (400 mL × 2). The separated aqueous phase was added with tetrahydrofuran (300 mL), sodium carbonate (15.47 g, 145.60 mmol), and di-tert-butyl dicarbonate (31.78 g, 145.60 mmol). The reaction was stirred at room temperature under a nitrogen atmosphere until complete. The solution was extracted with ethyl acetate (200 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was purified by column chromatography (petroleum ether: ethyl acetate = 9:1) to give 8.9 g of compound A2 (yield 58.5%).

[0407] MS(ESI): m / z 102.2[M-Boc+1] + .

[0408] 1H NMR (400MHz, CDCl3) δ3.78(s,3H),3.04(dd,1H),2.53(dd,1H),2.42(dd,1H),1.46(s,9H).

[0409] Step 2:

[0410] Compound mPEG (molecular weight 1000 Da, 20.00 g, 19.13 mmol, purchased from Bio-Pharmaceutical) was dissolved in dichloromethane (200 mL), and compound A2 (7.70 g, 38.27 mmol) was added. After mixing and dissolving, boron trifluoride diethyl ether (271.57 mg, 1.91 mmol) was added dropwise, and the reaction was continued under a nitrogen atmosphere until complete. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (acetonitrile:water = 35%-40%) to give 12.5 g of compound A3 (yield 55%).

[0411] 1 H NMR (400MHz, DMSO-d6): δ7.07(d,1H),4.23-4.18(m,1H),3.65-3.62(m,5H),3.54-3.48(m,92H),3.24(s,3H),1.38(s,9H).

[0412] Step 3:

[0413] Compound A3 (6.60 g, 5.29 mmol) was dissolved in tetrahydrofuran (70 mL) and water (70 mL). Lithium hydroxide monohydrate (444.35 mg, 10.59 mmol) was added at 0 °C, and the reaction was allowed to proceed to completion at room temperature. The reaction solution was concentrated at low temperature, and the pH was adjusted to 4-5 with 2N HCl aqueous solution. The solution was purified by column chromatography (acetonitrile:water = 35%-40%), and lyophilized to give 4.2 g of compound A (yield 63.6%).

[0414] 1 H NMR (400MHz, DMSO-d6): δ12.69(brs,1H),6.83(d,1H),4.12-4.07(m,1H),3.70-3.66(m,2H),3.54-3.49(m,92H),3.24(s,3H),1.38(s,9H).

[0415] Preparation of compound B

[0416] Compound B1 (4.41 g, 17.9 mmol, purchased from Bioderm) and compound B2 (20 g, 16.3 mmol, purchased from Xiamen Sinobond) were added to a 500 mL three-necked reaction flask, purged three times with nitrogen, and anhydrous N,N-dimethylformamide (200 mL) was added. The mixture was stirred until the starting materials were completely dissolved and cooled in an ice-water bath. Then, N,N-diisopropylethylamine (4.63 g, 35.86 mmol) was added, and the reaction was stirred at room temperature until complete. The mixture was concentrated under reduced pressure and purified by column chromatography (acetonitrile:water = 40%). After concentration, the mixture was freeze-dried to give 20.2 g of compound B (yield 86%).

[0417] 1 H NMR (400MHz, CD3OD): δ4.06-4.03(m,1H),3.73-3.70(m,2H),3.63-3.61(m,90H),3.54-3.52 (m,3H),3.35(s,3H),3.20-3.17(m,2H),2.44-2.41(m,2H),1.83-1.50(m,6H),1.45(s,9H).

[0418] Preparation of compound C

[0419] Step 1:

[0420] Compound C1 (20 g, 85.8 mmol, purchased from Bidet) was added to a 1 L three-necked flask, followed by the addition of water (40 mL) and stirring. Tetrahydrofuran (200 mL) was then added and stirred until the mixture separated into layers. Compound C2 (84.2 g, 180.2 mmol, purchased from Bidet) was added, and the mixture was purged twice with nitrogen. The mixture was stirred until it separated into layers. The reaction mixture was cooled to 0 °C, and N,N-diisopropylethylamine was added and stirred for 10 minutes. The reaction was allowed to proceed at room temperature until complete. The mixture was concentrated under reduced pressure and purified by column chromatography (dichloromethane:methanol = 10:1) to give 42 g of compound C3 (60% yield).

[0421] MS(ESI): m / z 817[M+1] + .

[0422] 1 H NMR (400MHz, CD3OD): δ8.32-8.30(m,1H),7.99-7.10(m,1H),4.45-4.38(m,1H),4.10 -3.96(m,2H),3.66(s,3H),3.21-3.20(m,2H),3.06-3.03(m,4H),1.91-1.30(m,54H).

[0423] Step 2:

[0424] Compound C3 (42 g, 51.5 mmol) and tetrahydrofuran (200 mL) were added to a reaction flask, followed by an aqueous solution (80 mL) of lithium hydroxide (6.5 g, 154.8 mmol). The mixture was stirred at room temperature until complete. The solution was concentrated, and the residue was adjusted to pH 5-6 with hydrochloric acid, extracted with dichloromethane, concentrated, and purified by column chromatography (acetonitrile:water = 0-50%) to give 40 g of compound C (60% yield).

[0425] MS(ESI): m / z 802[M+1] + .

[0426] 1 H NMR (400MHz, CD3OD): δ8.12-8.10(m,1H),7.99-7.10(m,1H),4.41-4.38(m,1H) ,4.09-3.97(m,2H),3.21-3.20(m,2H),3.06-3.03(m,4H),1.91-1.30(m,54H).

[0427] Preparation of compound D

[0428] Step 1:

[0429] Compound D1 (10.0 g, 7.73 mmol, purchased from Xiamen Sainuobangge), compound D2 (3.3 g, 10.05 mmol, purchased from Leyan), and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (4.40 g, 11.6 mmol) were dissolved in N,N-dimethylformamide (100 mL) and stirred. N,N-diisopropylethylamine (4.99 g, 38.65 mmol) was added, and the reaction was allowed to proceed to completion at room temperature. The reaction mixture was concentrated to dryness and purified by column chromatography (dichloromethane:methanol = 100:0-20:1) to give 11.0 g of compound D3 (yield 68%).

[0430] 1 H NMR (400MHz, CD3OD): δ7.88-7.80(m,4H),3.89-3.86(m,2H),3.77-3.72(m,4H),3.63-3.53(m,90H ),3.40-3.34(m,4H),3.07-2.97(m,4H),2.65-2.62(m,2H),1.82-1.67(m,4H),1.44-1.43(m,18H).

[0431] Step 2:

[0432] Compound D3 (10.0 g, 6.23 mmol) was dissolved in ethanol (200 mL), and hydrazine hydrate (10 mL) was added. The reaction was heated until complete. The mixture was filtered at room temperature, the filter cake was washed with ethanol, and the filtrate was concentrated to dryness. The filtrate was purified by column chromatography (dichloromethane:methanol = 60:1-10:1) to give 5.3 g of compound D4 (yield 52%).

[0433] 1 H NMR (400MHz, CD3OD): δ3.77-3.74(m,2H),3.64-3.56(m,96H),3.40-3.36(m,4H),3.09-2 .98(m,4H),2.89-2.86(m,2H),2.64-2.61(m,2H),1.80-1.67(m,4H),1.44-1.43(m,18H).

[0434] Step 3:

[0435] Compound D4 (2.00 g, 1.37 mmol), compound D5 (0.72 g, 1.64 mmol, purchased from Leyan), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (1.07 g, 2.06 mmol) were dissolved in N,N-dimethylformamide (20 mL) and stirred. N-methylmorpholine (0.416 g, 4.11 mmol) was added, and the reaction was allowed to proceed to completion at room temperature. The reaction mixture was concentrated to dryness and purified by column chromatography (acetonitrile:water = 0%–50%) to give 1.80 g of compound D6 (80% yield).

[0436] MS(ESI): m / z 1878[M+1] + .

[0437] 1 H NMR (400MHz, CD3OD): δ8.50(d,2H),7.18(d,2H),4.30-4.28(m,2H),3.71-3.65(m,108H),3.40-3.36(m,2H),3.10-3.0 6(m,2H),3.02(s,3H),3.01-2.98(m,1H),2.73-2.70(m,2H),2.48-2.45(m,2H),2.09-1.94(m,4H),1.46-1.45(m,18H).

[0438] Step 4:

[0439] Compound D6 (2.50 g, 1.33 mmol) was dissolved in methanol (25 mL) and stirred. 2.0 M hydrochloric acid in methanol (20 mL) was added, and the reaction was allowed to proceed at room temperature until complete. The reaction mixture was concentrated to dryness and purified by column chromatography (acetonitrile:water = 0%–40%) to give 1.00 g of compound D7 (yield 63%).

[0440] 1 H NMR (400MHz, CD3OD): δ8.50(d,2H),7.18(d,2H),4.28-4.26(m,2H),3.91-3.52(m,108H),3.37-3.35(m,2H), 3.09-3.04(m,2H),3.00(s,3H),2.97-2.95(m,1H),2.70-2.68(m,2H),2.46-2.42(m,2H),2.07-1.91(m,4H).

[0441] Step 5:

[0442] Compound D7 (0.70 g, 0.42 mmol), compound C (0.77 g, 0.96 mmol), and 1H-benzotriazol-1-yloxytripyrrolyl hexafluorophosphate (0.60 g, 1.05 mmol) were dissolved in N,N-dimethylformamide (10 mL) and stirred. N-methylmorpholine (0.21 g, 2.11 mmol) was added, and the reaction was allowed to proceed to completion at room temperature. The reaction mixture was concentrated to dryness and purified by column chromatography (acetonitrile:water = 0%–100%) to give 0.90 g of compound D8 (70% yield).

[0443] 1 H NMR (400MHz, CD3OD): δ8.50(d,2H),7.18(d,2H),4.28-4.26(m,4H),4.06-3.51(m,100H),3.38-3.34 (m,4H),3.18-2.98(m,16H),3.00(s,3H),2.65-2.62(m,2H),2.46-2.42(m,2H),1.75-1.28(m,138H).

[0444] Step 6:

[0445] Compound D8 (3.00 g, 0.92 mmol) was dissolved in methanol (60 mL) and stirred. 4.0 M dioxane hydrochloride solution (30 mL) was added, and the reaction was allowed to proceed at room temperature until complete. The reaction mixture was concentrated to dryness and purified by column chromatography (acetonitrile:water = 0%–50%) to give 2.10 g of compound D (yield 65%).

[0446] 1 H NMR (400MHz, CD3OD): δ8.50(d,2H),7.19(d,2H),4.28-4.26(m,4H),4.12-3.54(m,112H),3.37-3.34 (m,18H),3.00(s,3H),2.98-2.80(m,6H),2.78-2.75(m,2H),2.45-2.40(m,2H),1.95-1.51(m,40H).

[0447] Preparation of compound E

[0448] Step 1:

[0449] Compound E1 (1 g, 1.62 mmol, purchased from Hanlai Biochemical), compound E2 (518 mg, 1.78 mmol, purchased from Bio-Tech), and N,N-diisopropylethylamine (737 mg, 5.67 mmol) were dissolved in N,N-dimethylformamide (15 mL) and reacted at room temperature until complete. The mixture was purified by column chromatography (water:acetonitrile = 100:0-45:55) to give 750 mg of compound E3 (yield 60.17%).

[0450] MS(ESI): m / z 787.6 [M+18] + .

[0451] Step 2:

[0452] Compound E3 (360 mg, 0.47 mmol), compound E4 (129 mg, 0.47 mmol, purchased from Leyan), N,N-diisopropylethylamine (151 mg, 1.17 mmol), and benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (365 mg, 0.71 mmol) were dissolved in dry N,N-dimethylformamide (5 mL) and reacted at room temperature until complete. The solution was purified by column chromatography (acetonitrile:water = 0-40%) to give 281 mg of compound E (yield 58.55%).

[0453] MS(ESI): m / z 1028.8 [M+1] + .

[0454] 1H NMR (400MHz, CD3OD-d4): δ7.68-7.66(m,1H),7.40-7.32(m,2H),7.51-7.45(m,4 H),7.28-7.25(m,1H),5.63-5.43(m,2H),5.15(d,J=13.2Hz,1H),4.31-4.28(m,1 H),3.71(d,J=14.0Hz,1H),3.62-3.44(m,50H),3.26-3.21(m,2H),3.18-3.11(m, 1H),2.55-2.48(m,1H),2.35-2.26(m,4H),2.08-1.95(m,5H),1.78-1.56(m,3H).

[0455] Preparation of compound F

[0456] Step 1:

[0457] Compound F1 (15.1 g, 41.0 mmol, purchased from Bidet) was dissolved in anhydrous dichloromethane (500 mL). Under an argon atmosphere, trimethylchlorosilane (137 mL, purchased from Energie) was added, and the reaction was allowed to proceed to completion at room temperature. The solution was concentrated under reduced pressure to give 18 g of compound F2, which was used directly in the next step.

[0458] MS(ESI): m / z 368.1 [M+23] + .

[0459] Step 2:

[0460] Compound F3 (cabatamethasone, 17.05 g, 20.4 mmol, purchased from Anegy) was dissolved in anhydrous tetrahydrofuran (700 mL, purchased from Anegy) and stirred under an argon atmosphere for 1–2 hours. The solution was cooled to approximately -60°C with dry ice and ethanol, and hexamethyldisilamide lithium (31 mL, 1.0 M tetrahydrofuran solution) was added dropwise. The reaction mixture was stirred until complete and the solution was reserved. Compound F2 (18.0 g, 41.0 mmol) was dissolved in anhydrous tetrahydrofuran (300 mL) and added dropwise to the above reaction solution. The mixture was stirred overnight at room temperature. The reaction solution was cooled to approximately 0°C, and a saturated ammonium chloride solution (1 L) was added. The mixture was then extracted with ethyl acetate (500 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography (ethyl acetate: petroleum ether = 20%–100%) to give 18 g of compound F4 (yield 77.1%).

[0461] MS(ESI): m / z 1144.3 [M+1] + .

[0462] 1 H NMR (400MHz, DMSO-d6): δ8.76(brs,1H),7.98(d,J=7.6Hz,2H),7.90(d,J=7.2Hz,2H),7.73-7.69(m,3H),7.63-7.53(m,3H),7. 44-7.32(m,9H),7.23-7.19(m,1H),5.97-5.93(m,1H),5.38(d,J=6.8Hz,1H),5.00-4.90(m,1H),4.87(d,J=9.6Hz,1H),4.72-4 .69(m,2H),4.59-4.53(m,2H),4.46(d,J=6.0Hz,1H),4.33-4.21(m,3H),4.01(s,2H),3.75-3.60(m,4H),3.28(s,3H),3.15(s, 3H),2.57-2.55(m,1H),2.25(s,3H),1.91-1.77(m,5H),1.51(s,3H),1.46-1.43(m,1H),1.34(s,9H),1.02(s,3H),0.98(s,3H).

[0463] Step 3:

[0464] Compound F4 (18.0 g, 15.7 mmol) was dissolved in dichloromethane (500 mL). A solution of 1,8-diazacyclo[5,4,0]undecene-7 (18.0 g, 47.2 mmol, purchased from Anegy) in dichloromethane (100 mL) was added dropwise with stirring at 0 °C. The reaction was continued at 0 °C until complete. The solution was diluted with 1000 mL of water, and the organic phase was separated. The aqueous phase was then extracted with dichloromethane (500 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography (methanol:dichloromethane = 0%–10%) to give 13 g of compound F5 (yield 89.6%).

[0465] MS(ESI): m / z 922.4 [M+1] + .

[0466] 1H NMR (400MHz, DMSO-d6): δ8.68(brs,1H),7.73-7.69(m,1H),7.64-7.60(m,2H),7.64-7.60(m,2H),7.44 -7.21(m,6H),5.94-5.90(m,1H),5.40-5.38(m,1H),4.98-4.94(m,2H),4.71-4.58(m,4H),4.45-4.44(m ,1H),4.03(m,2H),3.79-3.74(m,1H),3.65-3.63(m,1H),3.31(s,6H),3.22(s,3H),3.10(s,3H),2.70- 2.63(m,1H),2.29(s,3H),1.99-1.74(m,5H),1.52-1.48(m,4H),1.36(s,9H),1.02(s,3H),0.98(s,3H).

[0467] Step 4:

[0468] Compound F5 (12.0 g, 13.01 mmol) was dissolved in N,N-dimethylformamide (200 mL). Compound F6 (6.53 g, 13.0 mmol, purchased from Chengdu Yunda), (7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (5.44 g, 14.3 mmol, purchased from Bailingwei), and N,N-diisopropylethylamine (2.69 g, 20.8 mmol, purchased from Anaiji) were added under stirring in an ice-water bath. The reaction was carried out at 0°C until complete. The mixture was diluted with 1000 mL of water, extracted with ethyl acetate (500 mL × 3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography (methanol:dichloromethane = 0%–10%) to give 17.2 g of compound F7 (yield 94.0%).

[0469] MS(ESI): m / z 1427.3 [M+23] + .

[0470] 1H NMR (400MHz, DMSO-d6): δ8.66(brs,1H),8.26(brs,1H),8.14(d,J=8.0Hz,1H),8.03-7.97(m,3H),7.89(d,J=7.2Hz,2H),7.72-7.68(m,3H), 7.63-7.57(m,3H),7.46-7.37(m,5H),7.34-7.31(m,4H),7.27-7.16(m,6H),5.95(brs,1H),5.38(d,J=7.2Hz,1H),5.01-4.91(m,2H),4.69- 4.65(m,2H),4.61-4.51(m,3H),4.45(d,J=6.4Hz,1H),4.30-4.21(m, 3H),3.81-3.58(m,8H),3.28(s,3H),3.13-3.08(m,1H),2.83-2.78(m, 1H),2.60-2.53(m,1H),2.27(s,3H),1.97-1.77(m,5H),1.51(s,3H),1 .46-1.43(m,1H),1.34(s,9H),1.26(m,2H),1.02(s,3H),0.98(s,3H).

[0471] Step 5:

[0472] Compound F7 (17.2 g, 12.2 mmol) was dissolved in dichloromethane (500 mL) and stirred while cooling in an ice-water bath. A solution of 1,8-diazacyclo[5,4,0]undecene-7 (5.59 g, 36.7 mmol, purchased from Anage) in dichloromethane (100 mL) was added dropwise. The reaction was carried out at 0 °C until complete. The mixture was diluted with 1000 mL of water, and the organic phase was separated. The aqueous phase was then extracted with dichloromethane (500 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (methanol:dichloromethane = 0%–20%) to give 13.1 g of compound F8 (yield 90.5%).

[0473] MS(ESI): m / z 1183.4 [M+1] + .

[0474] 1H NMR (400MHz, DMSO-d6): δ8.72(brs,1H),8.33-8.27(m,2H),8.20(brs,1H),7.98(d,J=7.6Hz,2H),7.73-7.69(m,1H),7.61(t,J=7.6Hz,2H ),7.49-7.41(m,1H),7.42-7.33(m,4H),7.28-7.16(m,6H),5.94(brs,1H),5.38(d,J=7.2Hz,1H),5.00-4.92(m,2H),4.70-4.53(m,5H),4 .46(d,J=6.4Hz,1H),4.12(brs,1H),3.82-3.63(m,6H),3.57-3.46(m,2H),3.29(s,3H),3.26-3.21(m,3H),3.14-3.09(m,1H),2.85-2.79 (m,1H),2.69-2.59(m,2H),2.28(s,3H),1.95-1.88(m,4H),1.81-1.61(m,3H),1.55-1.44(m,4H),1.35(s,9H),1.02(s,3H),0.98(s,3H).

[0475] Step 6:

[0476] Compound F8 (13.1 g, 11.1 mmol) was dissolved in N,N-dimethylformamide (200 mL). Compound F9 (1.93 g, 16.6 mmol, purchased from Anaiji) and triethylamine (3.36 g, 33.2 mmol, purchased from Sinopharm) were added to the solution under stirring at 0 °C. The reaction was stirred at room temperature until complete. The solution was concentrated under reduced pressure, and the residue was purified by column chromatography (acetonitrile:water = 0-40%) to give 12.8 g of compound F (yield 89.0%).

[0477] MS(ESI): m / z 1299.3 [M+1] + .

[0478] 1H NMR (400MHz, CD3OD): δ8.42-8.34(m,1H),8.16-8.05(m,3H),7.66-7.60(m,1H),7.56-7.52(m,2H),7.40(d,J=4.4Hz,4H), 7.30-7.18(m,6H),6.24-6.19(m,1H),5.60(d,J=6.8Hz,1H),5.22(s,1H),4.99(d,J=8.8Hz,1H),4.88(s,2H),4.65-4.52(m ,3H),4.22(s,2H),4.19-4.12(m,4H),3.93(s,2H),3.90-3.67(m,6H),3.40(s,3H),3.29-3.23(m,4H),3.04-2.98(m,1H), 2.68-2.60(m,1H),2.43(s,3H),2.32-2.06(m,2H),1.65(s,3H),1.62-1.58(m,4H),1.39(s,9H),1.18(s,3H),1.12(s,3H).

[0479] Preparation of compound G

[0480] Step 1:

[0481] Compound G1 (1.0 g, 1.94 mmol, prepared according to the method disclosed in patent JP3359955B2) was dissolved in N,N-dimethylformamide (32 mL). Under an argon atmosphere, N,N-diisopropylethylamine (0.50 g, 3.88 mmol, purchased from Anage) and Fmoc-Val-Cit-PAB-PNP (1.60 g, 2.14 mmol, purchased from Shaoyuan) were added, and the reaction was carried out at room temperature until complete. The mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (acetonitrile / water = 0-40%) to give 1.2 g of compound G2 (yield 72%).

[0482] MS(ESI): m / z 1063.3 [M+1] + .

[0483] Step 2:

[0484] Compound G2 (1.0 g, 0.94 mmol) was dissolved in N,N-dimethylformamide (20 mL), purged with nitrogen three times, and piperidine (240 mg, 2.82 mmol, purchased from Sinopharm) was added at -10 °C. After the reaction was complete, methyl tert-butyl ether (50 mL) was added to the reaction solution, filtered, and dichloromethane (2 mL) was added to the filter cake. The solution was purified by column chromatography (acetonitrile / water = 0-50%) and lyophilized to give 0.56 g of compound G3 (yield 72%).

[0485] MS(ESI): m / z 841.3 [M+1] + .

[0486] Step 3:

[0487] Compound G3 (50 mg, 0.059 mmol) was dissolved in N,N-dimethylformamide (5 mL), and compound G4 (9 mg, 0.059 mmol, purchased from Sinopharm) and triethylamine (12 mg, 0.119 mmol, purchased from Sinopharm) were added separately. The mixture was purged with nitrogen three times, and the reaction was stirred at 60 °C until complete. The mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (acetonitrile:water = 0-40%) to give 38 mg of compound G (yield 70%).

[0488] MS(ESI): m / z 927.3 [M+1] + .

[0489] Preparation of compound H

[0490] Step 1:

[0491] Compound H1 (500 mg, 1.80 mmol) and polyethylene glycol monomethyl ether 500 (751 mg, 1.50 mmol, purchased from Aladdin, batch number A2010094) were dissolved in dichloromethane (15 mL). The temperature was lowered to 0 °C, and boron trifluoride diethyl ether (43 mg, 300 μmol) was added under a nitrogen atmosphere. The mixture was stirred until the reaction was complete, and purified by column chromatography (methanol / dichloromethane = 0-3%) to give 550 mg of compound H2 (yield 46.1%).

[0492] 1 H NMR(400MHz,CD3OD-d4)δ7.38-7.31(m,5H),5.23-5.11(m,2H),4.36-4.34 (m,1H),3.91-3.87(m,1H),3.73-3.35(m,45H),3.31(s,3H),1.45(s,9H).

[0493] Step 2:

[0494] Compound H2 (570 mg, 718 μmol) was dissolved in methanol (15 mL), and Pd / C (38 mg) was added. After the addition was complete, the mixture was purged with hydrogen three times, and the reaction was stirred at room temperature until complete. The mixture was filtered, and the filtrate was collected and concentrated under reduced pressure to give 500 mg of compound H (yield 98.9%).

[0495] 1 H NMR (400MHz, CD3OD-d4) δ4.30-4.26(m,1H),3.88-3.85(m,1H),3.71-3.52(m,46H),3.35(s,3H),1.45(s,9H).

[0496] Preparation of Compound I

[0497] Compound G3 (150 mg, 0.18 mmol) was dissolved in N,N-dimethylformamide (8 mL), and glutaric anhydride (31 mg, 0.27 mmol) and triethylamine (54 mg, 0.53 mmol) were added. The mixture was heated to 60 °C until complete. The reaction solution was cooled to room temperature, purified by column chromatography (acetonitrile / water = 0-45%), concentrated, and lyophilized to give 150 mg of compound I (yield 88%).

[0498] MS(ESI): m / z 955.6 [M+1] + .

[0499] Preparation of compound J

[0500] Step 1:

[0501] Compound J1 (3.0 g, 5.39 mmol, purchased from Leyan) was dissolved in tetrahydrofuran (40 mL), and potassium tert-butoxide (181 mg, 1.62 mmol) was added under ice bath conditions. The mixture was stirred at room temperature until the reaction was complete. Then, compound J2 (3.45 g, 26.94 mmol, purchased from Adamas) was added, and the mixture was stirred at room temperature until the reaction was complete. Ethyl acetate (100 mL) and water (80 mL) were added to the reaction solution, and the organic phase was separated. The aqueous phase was extracted twice with ethyl acetate (80 mL). The organic phases were combined, dried, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (methanol / dichloromethane = 0-10%), concentrated, and lyophilized to give 2.5 g of compound J3 (yield 57%).

[0502] MS(ESI): m / z 820.7 [M+18] + .

[0503] Step 2:

[0504] Compound J3 (2.1 g, 2.62 mmol) was dissolved in dichloromethane (40 mL), and trifluoroacetic acid (3 mL) was added. The mixture was stirred at room temperature until the reaction was complete. The solution was concentrated, purified by column chromatography (acetonitrile / water = 0-35%), and lyophilized to give 620 mg of compound J4 (yield 31.7%).

[0505] MS(ESI): m / z 764.6 [M+18] + .

[0506] 1 H NMR (400MHz, CD3OD-d4) δ3.74-3.63(m,4H), 3.62-3.58(m,48H), 2.56-2.46(m,4H), 1.45(s,9H).

[0507] Step 3:

[0508] Compound G3 (100 mg, 0.12 mmol), compound J4 (98 mg, 0.13 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (106 mg, 0.15 mmol), N,N-diisopropylethylamine (46 mg, 0.36 mmol), and N,N-dimethylformamide (3 mL) were added to a reaction flask. The mixture was purged with nitrogen three times, and the reaction was stirred at room temperature until complete. The mixture was purified by column chromatography (acetonitrile / water = 0-35%), concentrated, and lyophilized to give 0.12 g of compound J5 (70% yield).

[0509] MS(ESI): m / z 1570.0[M+1]+.

[0510] Step 4:

[0511] Compound J5 (0.12 g, 0.076 mmol) and dichloromethane (11 mL) were added to a reaction flask. Trifluoroacetic acid (1 mL) was added under ice-water bath conditions, and the reaction mixture was allowed to rise naturally to room temperature while stirring until the reaction was complete. The mixture was concentrated and purified by column chromatography (acetonitrile / water = 40%), and then lyophilized to give 80 mg of compound J (yield: 65%).

[0512] MS(ESI): m / z 1515.0 [M+1] + .

[0513] Preparation of compound K

[0514] Step 1:

[0515] Compound G3 (500 mg, 0.60 mmol), compound K1 (180 mg, 0.75 mmol, purchased from Bide), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (335 mg, 0.9 mmol), N,N-diisopropylethylamine (285 mg, 2.25 mmol), and N,N-dimethylformamide (3 mL) were added to a reaction flask. The mixture was purged with nitrogen three times, and the mixture was stirred at room temperature until the reaction was complete. The mixture was purified by column chromatography (acetonitrile / water = 70%), concentrated, and lyophilized to give 0.30 g of compound K (yield 50%).

[0516] MS(ESI): m / z 1001.3[M+1] + .

[0517] Preparation of compound L

[0518] Step 1:

[0519] Compound L2 (600 mg, 1.41 mmol, prepared by the method disclosed in patent US2023 / 54458) and compound L1 (662 mg, 1.25 mmol, purchased from Wuyan) were placed in a 100 mL single-necked flask. Under argon protection, dry dichloromethane (10 mL) and methanol (10 mL) were added. The mixture was stirred in an ice-water bath, and then 4-(4,6-dimethoxy-1,3,5,-triazine-2-yl)-4-methylmorpholine hydrochloride (534 mg, 1.94 mmol, purchased from Shaoyuan) and triethylamine (391 mg, 3.87 mmol, purchased from Sinopharm) were added. The reaction was carried out in an ice-water bath until complete. Concentration was recommended, and the residue was purified by column chromatography (acetonitrile / water = 0-60%) to give 0.92 g of compound L3 (yield 86%).

[0520] MS(ESI): m / z 842.3 [M+1] + .

[0521] Step 2:

[0522] Compound L3 (0.76 g, 0.90 mmol) was dissolved in tetrahydrofuran (10 mL), purged three times with nitrogen, and then 1,8-diazobisspirocyclic [5.4.0]undec-7-ene (412 mg, 2.71 mmol, purchased from Sinopharm) was added. The reaction solution was allowed to react completely under ice-water bath conditions. Methyl tert-butyl ether (30 mL) was added, and a solid precipitated. The solid was filtered to give 0.56 g of compound L4 (yield 72%).

[0523] MS(ESI): m / z 620.3 [M+1] + .

[0524] Step 3:

[0525] Compound L4 (559 mg, 0.90 mmol) and compound L5 (498 mg, 0.99 mmol, purchased from Bidex) were added to a 50 mL single-necked flask. Under argon protection, dry tetrahydrofuran (10 mL) and methanol (10 mL) were added, and the mixture was stirred and dissolved in an ice-water bath. 4-(4,6-dimethoxy-1,3,5,-triazine-2-yl)-4-methylmorpholine hydrochloride (375 mg, 1.35 mmol, purchased from Shaoyuan) was added, and the reaction was carried out in an ice-water bath until complete. The mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (acetonitrile / water = 0-60%) to give 0.46 g of compound L6 (yield 65%).

[0526] MS(ESI): m / z 1103.4 [M+1] + .

[0527] Step 4:

[0528] Compound L6 (0.26 g, 0.24 mmol) was dissolved in tetrahydrofuran (5.2 mL), purged three times with nitrogen, and then 1,8-diazobisspirocyclic [5.4.0]undec-7-ene (108 mg, 0.71 mmol, purchased from Sinopharm) was added. The reaction was carried out at -10 °C until complete. Methyl tert-butyl ether (20 mL) was added to the reaction solution, and a solid precipitated. The solid was filtered to give 0.16 g of compound L7 (70% yield).

[0529] MS(ESI): m / z 881.3 [M+1] + .

[0530] Step 5:

[0531] Compound L7 (160 mg, 0.18 mmol) was dissolved in N,N-dimethylformamide (5 mL), and compound L8 (25.3 mg, 0.22 mmol, purchased from Sinopharm) and N,N-isopropylethylamine (70 mg, 0.54 mmol, purchased from Sinopharm) were added separately. The mixture was purged with nitrogen three times and reacted at room temperature until complete. The solution was concentrated under reduced pressure and purified by column chromatography (acetonitrile:water = 0-50%) to give 80 mg of compound L (yield 50%).

[0532] MS(ESI): m / z 997.3 [M+1] + .

[0533] 1H NMR (400MHz, CD3OD): δ7.51(s,1H),7.52(d,1H),7.24-7.11(m,5H),5.72 -5.70(d,1H),5.55-5.31(m,3H),4.97(d,1H),4.82(d,1H),4.68(d,1H),4 .37-4.35(m,1H),3.98-3.53(m,15H),3.36-2.37(m,9H),2.36(s,3H),2.3 5-1.72(m,12H),1.32-1.29(m,5H),1.00-0.97(m,3H),0.67-0.61(m,4H).

[0534] Preparation of compound M

[0535] Step 1:

[0536] Compound D7 (0.35 g, 0.21 mmol) and N,N-dimethylformamide (3.5 mL) were added to a reaction flask, followed by N-methylmorpholine (0.23 g, 2.30 mmol) and compound C2 (0.312 g, 0.67 mmol). The mixture was purged with nitrogen three times and stirred at room temperature until complete. The reaction solution was concentrated to dryness and purified by column chromatography (acetonitrile / water = 0-60%) to give 0.42 g of compound M1 (yield 80%).

[0537] 1 H NMR(400MHz,CD3OD):8.50(d,2H),7.19(d,2H),4.28-4.25(m,2H),3.94-3.89(m,4H),3.78-3.66(m,107H),3 .40-3.34(m,7H),3.20-3.10(m,3H),3.04-3.00(m,8H),2.65-2.62(m,2H),2.44(t,2H),1.83-1.52(m,53H).

[0538] Step 2:

[0539] With stirring, compound M1 (0.4 g, 0.17 mmol) was dissolved in methanol (12 mL), and 2.0 M hydrochloric acid methanol solution (5 mL) was added. The reaction was allowed to proceed at room temperature until complete. The reaction solution was concentrated to dryness and purified by column chromatography (acetonitrile:water = 0%-40%) to give 0.3 g of compound M2 (yield 85%).

[0540] 1H NMR(400MHz,CD3OD):8.50(d,2H),7.19(d,2H),4.28-4.26(m,2H),3.92-3.89(m,4H),3.78-3.58(m ,107H),3.55-3.34(m,12H),3.03-2.98(m,7H),2.67-2.66(m,2H),2.44(t,2H),1.93-1.52(m,17H).

[0541] Step 3:

[0542] Under stirring, compound M2 (0.30 g, 0.14 mmol), compound A (1.00 g, 0.83 mmol), and 1H-benzotriazol-1-yloxytripyrrolyl hexafluorophosphate (0.75 g, 1.44 mmol) were dissolved in N,N-dimethylformamide (10 mL), and N-methylmorpholine (0.73 g, 7.2 mmol) was added. The mixture was purged with nitrogen and reacted at room temperature until complete. The reaction solution was concentrated to dryness and purified by column chromatography (acetonitrile:water = 0%–40%) to give 0.43 g of compound M3 (60% yield).

[0543] 1 H NMR (400MHz, CD3OD): δ8.50(d,2H),7.18(d,2H),3.91-3.44(m,502H),3.35(s,12H), 3.28-3.18(m,7H),3.00(s,3H),2.65-2.60(m,2H),2.44(t,2H),1.89-1.26(m,55H).

[0544] Step 4:

[0545] Compound M3 (0.33 g, 0.048 mmol) was dissolved in methanol (5 mL) with stirring, and 4.0 M dioxane hydrochloride solution (2 mL) was added. The reaction was allowed to proceed at room temperature until complete. The reaction solution was concentrated to dryness and purified by column chromatography (acetonitrile:water = 0%–40%) to give 0.3 g of compound M (yield 80%).

[0546] 1 H NMR (400MHz, CD3OD): δ8.50(d,2H),7.18(d,2H),3.91-3.44(m,502H),3.35(s,12H), 3.28-3.18(m,7H),3.00(s,3H),2.71-2.67(m,2H),2.44(t,2H),1.92-1.34(m,19H).

[0547] Preparation of compound N

[0548] Compound N1 (350 mg, 0.46 mmol, prepared using the method disclosed in patent WO2022 / 58395) was dissolved in N,N-dimethylformamide (3.5 mL). Compound L8 (64.5 mg, 0.56 mmol, purchased from Sinopharm) and N,N-diisopropylethylamine (179 mg, 1.39 mmol, purchased from Sinopharm) were added separately. The mixture was purged with nitrogen three times, and the reaction was allowed to proceed completely at room temperature. The mixture was concentrated under reduced pressure and purified by column chromatography (acetonitrile:water = 0-60%) to give 242 mg of compound N (yield 60%).

[0549] MS(ESI): m / z 871.3 [M+1] + .

[0550] 1 H NMR (400MHz, CD3OD): δ7.78(d,1H),7.59-7.52(m,4H),7.39(d,2H),5.59-5.55(m,1H),5.39-5.05(m,6H),4.49-4.47(m,1H),4.28(d,1H) ),4.22(s,2H),4.18-4.09(m,2H),2.36(s,3H),2.34-2.31(m,1H),2.20-2.13(m,2H),2.03-1.92(m,3H),1.44(d,3H),1.01-0.89(m,9H).

[0551] Preparation of compound O

[0552] Step 1:

[0553] Compound O1 (345 mg, 0.78 mmol, purchased from Leyan), compound E2, and N,N-diisopropylethylamine (352 mg, 2.73 mmol) were dissolved in N,N-dimethylformamide (5 mL) and reacted at room temperature until complete. The mixture was purified by column chromatography (water:acetonitrile = 100:0-45:55) to give 278 mg of compound O2 (60% yield).

[0554] MS(ESI): m / z = 594.34 [M+1] + .

[0555] Step 2:

[0556] Compound O2 (260 mg, 0.44 mmol), compound E4 (110 mg, 0.40 mmol), N,N-diisopropylethylamine (155 mg, 1.20 mmol), and (7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (198 mg, 0.52 mmol) were dissolved in dry N,N-dimethylformamide (5 mL) and reacted at room temperature until complete. The mixture was purified by column chromatography (acetonitrile:water = 0-40%) to give 200 mg of compound O (50% yield).

[0557] MS(ESI): m / z 852.5 [M+1] + .

[0558] 1 H NMR (400MHz, CD3OD-d4): δ7.67-7.65(m,1H),7.51-7.26(m,7H),5.63-5.43(m,2H),5.15(d,1H),4.31-4.28(m,1H),3.70( d,1H),3.87-3.42(m,34H),3.26-3.11(m,4H),2.55-2.48(m,1H),2.35-2.26(m,5H),2.07-1.87(m,5H),1.77-1.53(m,3H).

[0559] Synthesis of compound P

[0560] Compound E1 (250 mg, 404.7 μmol) was dissolved in a mixed solvent of dichloromethane (3 mL) and N,N-dimethylformamide (10 mL). N,N-diisopropylethylamine (184 mg, 1.42 mmol) and succinimide 6-(maleimide)hexanoate (135 mg, 437.1 μmol, purchased from Bide) were added, and the mixture was stirred at room temperature until complete. 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (316 mg, 607 μmol) and azadibenzocyclooctylenylamine (123 mg, 445.2 μmol, purchased from Hanlai Biochemical) were added to the reaction solution, and the mixture was stirred until complete. The reaction solution was concentrated, purified by HPLC (0-40% acetonitrile / 0.3% FA aqueous solution), and lyophilized to give 180 mg of compound P (yield: 41.6%).

[0561] MS(ESI): m / z = 1069.5(M+1) + .

[0562] 1H NMR(400MHz,CD3OD-d4)δ7.69(d,J=6.8Hz,1H),7.53-7.47(m,4H),7.43-7.35(m,2H) ,7.30-7.28(m,1H),6.83(s,2H),5.16(d,J=14.0Hz,1H),3.73(d,J=13.6Hz,1H),3.6 7-3.61(m,46H),3.60-3.47(m,8H),3.38-3.35(m,1H),3.30-3.27(m,1H),3.20-3.13 (m,1H),2.57-2.50(m,1H),2.32-2.29(m,2H),2.23-2.19(m,2H),1.68-1.56(m,4H).

[0563] Synthesis of compound Q

[0564] Step 1:

[0565] Compound Q1 (11 g, 51.83 mmol) and compound D2 (17.18 g, 51.83 mmol) were dissolved in anhydrous dichloromethane (220 mL). N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (29.72 g, 77.74 mmol) and 4-dimethylaminopyridine (9.50 g, 77.74 mmol) were added with stirring in an ice bath. The reaction mixture was stirred at room temperature until complete. The reaction solution was concentrated under reduced pressure and purified by column chromatography (petroleum ether:ethyl acetate = 2:1) to give 21.9 g of compound Q2 (yield: 80%).

[0566] MS(ESI): m / z = 526.5 [M+1] + .

[0567] 1 H NMR (400MHz, CDCl3): δ7.35-7.28(m,4H),7.28-7.21(m,6H),5.43-5.33(m,1H),5.17(s,1H),4.55-4.4 3(s,1H),3.44(t,2H),3.33–3.25(m,2H),3.07(dq,4H),1.75(q,2H),1.70-1.60(m,2H),1.43(d,18H).

[0568] Step 2:

[0569] Compound Q2 (21.86 g, 41.58 mmol) was dissolved in tetrahydrofuran (44 mL), and 4.0 M HCl / 1,4-dioxane (110 mL) was added. The mixture was then stirred at room temperature for 2–4 hours. The reaction solution was concentrated under reduced pressure to give 19.1 g of compound Q3, which could be used directly in the next step without purification.

[0570] MS(ESI): m / z = 326.3[M+1] + .

[0571] 1 H NMR (400MHz, CD3OD-d4): δ7.38-7.22(m,10H),5.48(s,1H),3.63-3.50(m,4H),2.89(q,J=7.6Hz,4H),2.01-1.91(m,4H).

[0572] Step 3:

[0573] Compound Q3 (16.0 g, 40.16 mmol) and compound C (70.95 g, 88.36 mmol) were dissolved in dichloromethane (870 mL). 4-Dimethylaminopyridine (24.53 g, 200.82 mmol) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (46.06 g, 120.49 mmol) were added with stirring in an ice bath. The reaction mixture was stirred at room temperature until complete. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (water:acetonitrile 0-80%) to give 57.3 g of compound Q4 (yield: 75.3%).

[0574] MS(ESI): m / z = 948.7[(M+2) / 2] + .

[0575] 1 H NMR (400MHz, CD3OD-d4): δ8.15-7.83(m,3H),7.38-7.19(m,10H),6.93-6.48(m,3H),5.42-5.35(m,1H),4.36-4.20(m,2H) ),4.10-3.91(m,4H),3.52-3.31(m,4H),3.29-3.08(m,8H),3.07-2.93(m,8H),1.99-1.55(m,16H),1.55-1.30(m,101H).

[0576] Step 4:

[0577] Compound Q4 (29.18 g, 15.40 mmol) was dissolved in tetrahydrofuran (150 mL), and 4.0 M HCl / 1,4-dioxane (150 mL) was added. The mixture was then stirred at room temperature until complete. The reaction solution was concentrated under reduced pressure to give 22.80 g of compound Q, which could be used directly in the next step without purification.

[0578] MS(ESI): m / z = 1095.0 [M+1] + .

[0579] 1 H NMR (400MHz, D2O): δ7.37-7.25(m,6H),7.19-7.10(m,4H),5.36(s,1H),4.29-4.17(m,2H),4.05-3.97(m,2H), 3.95-3.85(m,2H),3.54(q,2H),3.49-3.32(m,4H),3.25-3.08(m,6H),3.03-2.86(m,8H),1.96-1.25(m,40H).

[0580] Example 1: Preparation of Compound 1

[0581] Step 1:

[0582] Compound D (0.70 g, 0.26 mmol), compound B (3.78 g, 2.81 mmol), and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (1.60 g, 3.07 mmol) were dissolved in N,N-dimethylformamide (100 mL). N-methylmorpholine (1.88 g, 18.7 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness, dissolved in 10 mL of water, purified by centrifugation (H₂O), and lyophilized to give 2.2 g of compound 1a (72% yield).

[0583] 1 H NMR (400MHz, CD3OD): δ8.50(d,2H),7.19(d,2H),4.07-3.44(m,835H),3.35(s,2 4H),3.19-3.12(m,30H),3.00(s,3H),2.64-2.43(m,17H),1.83-1.41(m,166H).

[0584] Step 2:

[0585] Compound 1a (2.20 g, 0.16 mmol) was dissolved in methanol (20 mL), and 4.0 M dioxane hydrochloride solution (20 mL) was added with stirring. The reaction was allowed to proceed at room temperature until complete. The reaction solution was concentrated to dryness, then dissolved in 5 mL of water, centrifuged, filtered (H2O), purified, and lyophilized to give 2.00 g of compound 1b (yield 86%).

[0586] 1 H NMR (400MHz, CD3OD): δ8.50(d,2H),7.19(d,2H),4.07-3.44(m,835H),3.35(s, 24H)3.29-3.12(m,30H),3.00(s,3H),2.50-2.43(m,17H),1.88-1.31(m,94H).

[0587] Step 3:

[0588] Compound 1b (0.83 g, 0.064 mmol), compound F (0.83 g, 0.64 mmol), and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.37 g, 0.70 mmol) were dissolved in N,N-dimethylformamide (20 mL). N-methylmorpholine (0.41 g, 4.03 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness, then dissolved in 6 mL of water, purified by centrifugation (H₂O), lyophilized, and purified by SEC (acetonitrile) to give 1.1 g of compound 1 (75% yield, NMR characterization showed 8 grafts of cabazitaxel).

[0589] 1 H NMR (400MHz, CD3OD): δ8.50(d,2H),8.11-8.09(m,16H),7.64-7.19(m,104H),7.19(d,2H),6.23-6.23(m,8H),5.60-5.49(m,8H),5.3 0-5.20(m,8H),4.98-4.96(m,8H),4.65-4.15(m,91H),3.94-3.66(m,910H),3.38-3.12(m,126H),3.00(s,3H),2.90-1.12(m,344H).

[0590] Example 2 Preparation of Compound 2

[0591] Step 1:

[0592] Compound D (0.50 g, 0.183 mmol), compound A (2.19 g, 1.83 mmol), and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (1.05 g, 2.01 mmol) were dissolved in N,N-dimethylformamide (30 mL). N-methylmorpholine (1.35 g, 1.90 mmol) was added with stirring, and the reaction was allowed to proceed at room temperature until complete. The reaction solution was concentrated to dryness and purified by centrifugation (H₂O) to give 1.8 g of compound 2a (76% yield).

[0593] 1 H NMR (400MHz, CD3OD): δ8.52(d,2H),77.19(d,2H),4.42-4.18(m,14H),4.04-3.8 6(m,15H),3.83-3.43(m,844H),3.35(s,24H),3.00(s,3H),1.90-1.29(m,117H).

[0594] Step 2:

[0595] Compound 2a (0.70 g, 0.059 mmol) was dissolved in methanol (7 mL), and 4.0 M dioxane hydrochloride solution (10 mL) was added with stirring. The reaction was allowed to proceed at room temperature until complete. The reaction solution was concentrated to dryness, then dissolved in 3 mL of water, purified by centrifugation (H2O), and lyophilized to give 0.60 g of compound 2b (yield 86%).

[0596] 1 H NMR (400MHz, CD3OD): δ8.53(d,2H),7.19(d,2H),4.44-4.18(m,14H),4.04-3.86(m,15H),3.83-3. 43(m,840H),3.35(s,24H),3.00(s,3H),2.71-2.63(m,2H),2.46-2.42(t,2H),1.73-1.21(m,40H).

[0597] Step 3:

[0598] Compound 2b (0.50 g, 0.044 mmol), compound F (0.57 g, 0.44 mmol), and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.25 g, 0.48 mmol) were dissolved in N,N-dimethylformamide (10 mL). N-methylmorpholine (0.28 g, 2.77 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness, dissolved in 3 mL of water, purified by centrifugation (H2O), and lyophilized. The product was then purified by SEC (acetonitrile) to give 0.72 g of compound 2 (65% yield, NMR characterization showed 8 grafts of cabazitaxel).

[0599] 1 H NMR (400MHz, CD3OD): δ8.50(d,2H),8.11-8.09(d,16H),7.67-7.59(t,8H),7.58-7.49(m,16H),7.44-7.35(d,30H),7.3 1-7.15(m,47H),7.19(d,2H),6.30-6.13(t,8H),5.67-5.53(d,8H),5.29-5.15(d,8H),4.73-4.49(m,33H),4.40-4.05(m ,54),4.0-3.75(m,78H),3.74-3.48(m,830H),3.35(s,24H),3.21(s,24H),3.0-2.90(m,10H),2.60(brs,10H),2.43(s,2 4H),2.35-2.22(m,10H),2.20-2.06(m,9H),2.01(s,24H),1.88-1.55(m,47H),1.53-1.24(m,103H),1.24-1.06(d,48H).

[0600] Example 3 Preparation of Compound 3

[0601] Step 1:

[0602] Compound 3a (0.80 g, 0.20 mmol, prepared by the method disclosed in patent WO2021 / 243415), compound A (5.68 g, 4.78 mmol), and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (2.60 g, 5.00 mmol) were dissolved in N,N-dimethylformamide (30 mL). N-methylmorpholine (1.68 g, 16.6 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness and purified by centrifugation (H2O) to give 3.2 g of compound 3b (73% yield).

[0603] 1 H NMR(400MHz,CD3OD):8.52(d,2H),7.19(d,2H),4.53-4.17(m,28H),4.06-3.86(m,27H),3.84-3.40(m,1584H ),3.36(s,48H),3.27-3.11(m,35H),3.00(s,3H),2.68-2.62(t,2H),2.46-2.42(t,2H),1.84-1.29(m,237H).

[0604] Step 2:

[0605] Compound 3b (1.60 g, 0.071 mmol) was dissolved in methanol (16 mL), and 4.0 M dioxane hydrochloride solution (20 mL) was added with stirring. The reaction was allowed to proceed at room temperature until complete. The reaction solution was concentrated to dryness, then dissolved in 3 mL of water, and purified by centrifugation (H2O) to give 1.3 g of compound 3c (yield 80%).

[0606] 1H NMR (400MHz, CD3OD): δ8.52(d,2H),7.19(d,2H),4.53-4.17(m,28H),4.06-3.86(m,27H),3.84-3.40(m,1584 H),3.36(s,48H),3.27-3.11(m,35H),3.01(s,3H),2.48-2.42(t,2H),2.30-2.24(t,2H),1.98-1.24(m,90H).

[0607] Step 3:

[0608] Compound 3c (1.00 g, 0.047 mmol), compound F (1.22 g, 0.94 mmol), and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.54 g, 1.03 mmol) were dissolved in N,N-dimethylformamide (20 mL). N-methylmorpholine (0.77 g, 7.66 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness, dissolved in 3 mL of water, purified by centrifugation (H2O), and lyophilized. The product was purified by SEC (acetonitrile) to give 1.3 g of compound 3 (60% yield, NMR characterization showed 16 grafts of cabazitaxel).

[0609] 1 H NMR (400MHz, CD3OD): δ8.49(d,2H),8.11-8.09(d,32H),7.67-7.59(t,16H),7.59-7.49(t,32H),7.49-7.34(d,62H),7.37-7.13 (m,94H),7.06-6.90(m,8H),6.36-6.06(t,16H),5.69-5.48(d,16H),5.31-5.15(brs,16H),5.03-4.92(d,16H),4.76-4.47(m,63 H),4.41-4.04(m,112H),4.01-3.77(m,118H),3.73-3.41(m,1616H),3.73(s,48H),3.35(s,48H),3.24-3.07(m,78H),3.06-2.92 (m,24H),2.60(brs,20H),2.52-2.23(m,65H),2.20-1.90(m,69H),1.89-1.54(m,99H),1.52-1.24(m,212H),1.24-1.03(d,96H).

[0610] Example 4: Preparation of Compound 4

[0611] Step 1:

[0612] Compound 4a (0.30 g, 0.045 mmol, prepared using the method disclosed in patent WO2021 / 243415), compound A (2.24 g, 1.86 mmol), and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.98 g, 1.89 mmol) were dissolved in N,N-dimethylformamide (10 mL). N-methylmorpholine (0.27 g, 2.66 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness and purified by centrifugation (H2O) to give 1.4 g of compound 4b (66% yield).

[0613] 1 H NMR (400MHz, CD3OD): δ8.53(d,2H),7.24(d,2H),4.52-4.18(m,58H),3.84-3.43(m,3145H ),3.38(s,96H),3.04(s,3H),2.50-2.45(t,2H),2.25-2.18(t,2H),2-05-1.26(m,506H).

[0614] Step 2:

[0615] Compound 4b (1.60 g, 0.071 mmol) was dissolved in methanol (16 mL), and 4.0 M dioxane hydrochloride solution (20 mL) was added with stirring. The reaction was allowed to proceed at room temperature until complete. The reaction solution was concentrated to dryness, then dissolved in 3 mL of water, purified by centrifugation (H2O), and lyophilized to give 1.3 g of compound 4c (80% yield).

[0616] 1 H NMR (400MHz, CD3OD): δ8.53(d,2H),7.24(d,2H),4.50-4.18(m,58H),3.88-3.43(m,3150H ),3.38(s,96H),3.04(s,3H),2.50-2.45(t,2H),2.25-2.18(t,2H),2-00-1.23(m,218H).

[0617] Step 3:

[0618] Compound 4c (0.80 g, 0.02 mmol), compound F (1.05 g, 0.81 mmol), and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.43 g, 0.82 mmol) were dissolved in N,N-dimethylformamide (16 mL). N-methylmorpholine (0.32 g, 3.20 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness, dissolved in 3 mL of water, purified by centrifugation (H₂O), and lyophilized. The product was purified by SEC (acetonitrile) to give 1.05 g of compound 4 (yield 58%, NMR characterization showed 30 grafts of cabazitaxel).

[0619] 1 H NMR (400MHz, CD3OD): δ8.49(d,2H),8.10-8.08(d,64H),7.67-7.59(d,35H),7.59-7.49(t,65H),7.46-7.34(d,126H),7.3 2-7.14(m,187H),7.08-6.89(m,22H),6.36-6.09(s,30H),5.65-5.49(s,31H),5.32-5.15(brs,31H),5.07-4.92(s,39H), 4.74-4.43(m,128H),4.42-4.07(m,207H),4.02-3.43(m,3121H),3.37(s,86H),3.35(s,96H),3.24-3.07(m,158H),3.07- 2.88(m,66H),2.74-2.22(m,183H),2.21-1.86(m,148H),1.85-1.51(m,200H),1.51-1.25(m,412H),1.24-1.00(d,202H).

[0620] Example 5: Preparation of 1-ADC

[0621] Step 1:

[0622] Add 30 mg of Ab (antibody Trastuzuma, 0.21 μmol, 14.14 mg / mL, 1×PBS 6.3) to a 15 mL EP tube, then add EndoS enzyme (483.9 μL at a dose of 0.01 mg / mL, concentration 0.62 mg / mL). Vortex to mix and incubate at 37°C with shaking for 4 h. After incubation, Ab-1 (deglycosylated) is obtained and can be used directly in the next step.

[0623] Step 2:

[0624] 1×PBS 7.2 (200 μL) solution of compound 5a (3.6 mg, 6.3 μmol, prepared according to the method disclosed in patent WO2023051814) was directly added to the obtained Ab-1. After vortexing and mixing, the mixture was incubated at 37°C for 2 h. The mixture was then removed and passed through a desalting column (Zeba). TM Thermo) yielded 27.0 mg of Ab-2 (90% yield).

[0625] Step 3:

[0626] Compound E (2.12 mg, 1.90 μmol, prepared as a 0.835 mg / mL solution with 1×PBS 7.2:DMSO = 80:20) was added to Ab-2 (27.0 mg, 0.19 μmol), vortexed, and reacted overnight at room temperature. The mixture was then desalted using a Zeba column. TM Thermo yielded 24.57 mg of Ab-5b (91% yield).

[0627] Step 4:

[0628] Ab-5b (24.57 mg, 0.17 μmol) and compound 1 (23.3 mg, 0.85 μmol, dissolved in 1×PBS 7.2 to a concentration of 10 mg / mL) were mixed and incubated at room temperature for 16 h. The crude product was adjusted to pH 5.0 with acetic acid, and then an equal volume of sodium acetate buffer (50 mM NaOAc / HOAc, pH 5.0, 2.33 mL) was added and mixed. The mixture was then passed through a HiTrap Capto S cation exchange chromatography column (mobile phase A: 50 mM NaOAc / HOAc, pH 5.0; mobile phase B: 50 mM NaOAc / HOAc, 1 M NaCl, pH 5.0, elution with 0%-60% B phase solution) and concentrated using a 30 kDa ultrafiltration tube. Finally, the concentrate was passed through a HiLoad Superdex 200 pg preparative grade SEC chromatography column (mobile phase: 1×PBS 7.2), and the collected solution was concentrated to obtain 58.8 mg of 1-ADC (yield 65.3%, q = 2 and drug loading 16 as determined by CE-SDS method), and stored at 4°C.

[0629] Example 6: Preparation of 2-ADC

[0630] The same method as in step 4 of Example 5 was used to prepare the 2-ADC, except that compound 1 was replaced with compound 2 (18.30 mg, 0.85 μmol, dissolved in 1×PBS 7.2 to make a 10 mg / mL solution), and 7.91 mg of 2-ADC was finally obtained (q was 1.9 and drug loading was 15.2 as determined by CE-SDS method), and it was stored at 4°C.

[0631] Example 7: Preparation of 3-ADC

[0632] The same method as in step 4 of Example 5 was used to prepare the 3-ADC, except that compound 1 was replaced with compound 3 (56.67 mg, 1.36 μmol, dissolved in 1×PBS 7.2 to a concentration of 10 mg / mL). A total of 20.64 mg of 3-ADC was obtained (yield 84.0%, q = 1.8 and drug loading 28.8 as determined by CE-SDS), which was stored at 4°C.

[0633] Example 8: Preparation of 4-ADC

[0634] The same method as in step 4 of Example 5 was used to prepare the 4-ADC, except that compound 1 was replaced with compound 4 (124.08 mg, 1.69 μmol, dissolved in 1×PBS 7.2 to a concentration of 10 mg / mL). A total of 10.88 mg of 4-ADC was obtained (yield 44.3%, q = 1.8, drug loading 57.6 g as determined by CE-SDS), which was stored at 4°C.

[0635] Example 9 Preparation of Compound 5

[0636] Step 1:

[0637] Compound 3a (0.30 g, 0.074 mmol), compound B (1.99 g, 1.48 mmol), and benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.85 g, 1.63 mmol, purchased from Bailingwei) were dissolved in N,N-dimethylformamide (20 mL). N-methylmorpholine (0.62 g, 6.14 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness and purified by centrifugation (H2O) to give 1.3 g of compound 5a (yield 85%).

[0638] 1 H NMR(400MHz,CD3OD):8.52(d,2H),7.19(d,2H),4.56(s,16H),4.30-4.26(m,17H),4.01-3.3.99(m,16H),3.9 1-3.44(m,1563H),3.35(s,48H),3.18-3.15(m,60H),3.00(s,3H),2.45-2.40(m,32H),1.84-1.29(m,327H).

[0639] Step 2:

[0640] Compound 5A (1.3 g, 0.053 mmol) was dissolved in methanol (13 mL), and 4.0 M dioxane hydrochloride solution (6 mL) was added with stirring. The reaction was allowed to proceed at room temperature until complete. The reaction solution was concentrated to dryness, then dissolved in 10 mL of water, and purified by centrifugation (H2O) to give 0.9 g of compound 5b (yield 85%).

[0641] 1 H NMR (400MHz, CD3OD): δ8.52(d,2H),7.19(d,2H),4.44-4.26(m,16H),3.95-3.43(m,1601H) ,3.36(s,48H),3.22-3.18(m,60H),3.01(s,3H),2.45-2.41(m,32H),1.98-1.24(m,190H).

[0642] Step 3:

[0643] Compound 5B (0.20 g, 0.0084 mmol), compound F (0.219 g, 0.17 mmol), and benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.097 g, 0.19 mmol) were dissolved in N,N-dimethylformamide (2 mL). N-methylmorpholine (0.138 g, 1.37 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness, dissolved in 3 mL of water, purified by centrifugation (H₂O), and lyophilized. The product was purified by SEC (acetonitrile) to give 0.38 g of compound 5 (90% yield, NMR characterization showed 15 grafted drug molecules).

[0644] 1 H NMR (400MHz, CD3OD): δ8.50(d,2H),8.11-8.09(m,32H),7.69-7.52(m,48H),7.41-7.39(m,64H),7.26-7.1 7(m,93H),7.06-6.94(m,9H),6.28-6.15(m,15H),5.60-5.49(m,15H),5.30-5.20(m,15H),4.98-4.96(m,16 H),4.65-4.15(m,178H),3.94-3.66(m,1654H),3.38(s,43H),3.34(s,48H),3.29-3.10(m,120H),3.00(s, 3H),2.70-2.63(m,20H),2.52-2.48(m,81H),2.35-1.89(m,88H),1.88-1.29(m,380H),1.18-1.02(d,90H).

[0645] Example 10 Preparation of Compound 6

[0646] Compound 4c (0.080 g, 0.0020 mmol), compound I (0.071 g, 0.067 mmol), and benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.037 g, 0.072 mmol) were dissolved in N,N-dimethylformamide (2 mL). N-methylmorpholine (0.032 g, 0.33 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness, dissolved in 3 mL of water, purified by centrifugation (H2O), and lyophilized. The product was purified by SEC (acetonitrile) to give 0.1 g of compound 6 (60% yield, NMR characterization showed 28 grafted drug molecules).

[0647] 1 H NMR (400MHz, CD3OD): δ8.49(d,2H),8.23-6.64(m,168H),6.17-6.19(m,15H),5.35-4.89(m,133H),4 .57-3.44(m,3019H),3.34(s,96H),3.26-2.79(m,129H),2.57-1.17(m,610H),0.96-0.88(m,208H).

[0648] Example 11 Preparation of Compound 7

[0649] Compound 4c (0.080 g, 0.0020 mmol), compound K (0.067 g, 0.066 mmol), and benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.037 g, 0.072 mmol) were dissolved in N,N-dimethylformamide (2 mL). N-methylmorpholine (0.032 g, 0.33 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness, dissolved in 3 mL of water, purified by centrifugation (H2O), and lyophilized. The product was purified by SEC (acetonitrile) to give 0.09 g of compound 7 (70% yield, NMR characterization showed 30 grafted drug molecules).

[0650] 1H NMR (400MHz, CD3OD): δ8.49(d,2H),8.05-6.94(m,182H),5.71-4.96(m,32H),4.75-4.05(m,120H),4.02-3 .43(m,2952H),3.34(s,96H),3.24-3.07(m,93H),2.92(s,58H),2.18-1.93(m,298H),0.94-0.88(m,240H).

[0651] Example 12 Preparation of Compound 8

[0652] Compound 3c (0.10 g, 0.0047 mmol), compound K (0.089 g, 0.094 mmol), and benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.054 g, 0.103 mmol) were dissolved in N,N-dimethylformamide (1 mL). N-methylmorpholine (0.077 g, 0.766 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness, dissolved in 3 mL of water, purified by centrifugation (H2O), and lyophilized. The product was purified by SEC (acetonitrile) to give 0.09 g of compound 8 (80% yield, NMR characterization showed 15 grafted drug molecules).

[0653] 1 H NMR (400MHz, CD3OD): δ8.49(d,2H),7.62-7.05(m,92H),5.84-4.96(m,56H),4.74-3.96(m,142H),3.99-3 .45(m,1518H),3.36(s,48H),3.24-3.12(m,36H),3.00(s,3H),2.43-1.20(m,314H),0.95-0.82(m,160H).

[0654] Example 13 Preparation of Compound 9

[0655] Compound 3c (0.15 g, 0.0070 mmol), compound J (0.213 g, 0.14 mmol), and benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.080 g, 0.15 mmol) were dissolved in N,N-dimethylformamide (3 mL). N-methylmorpholine (0.116 g, 1.14 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness, dissolved in 3 mL of water, purified by centrifugation (H₂O), and lyophilized. The product was purified by SEC (acetonitrile) to give 0.26 g of compound 9 (yield 85%, NMR characterization showed 15 grafted drug molecules).

[0656] 1 H NMR (400MHz, CD3OD): δ8.49(d,2H),7.62-7.14(m,92H),7.06(d,2H),5.54-5.06(m,44H),4.47-4.16(m,60H),3.99-3.45(m,1 611H),3.25(s,48H),3.36(s,48H),3.10-3.01(m,45H),2.86(s,3H),2.45(s,35H),2.13-1.32(m,160H),0.87-0.78(m,90H).

[0657] Example 14 Preparation of Compound 10

[0658] Compound 4c (0.10 g, 0.0025 mmol), compound G (0.080 g, 0.086 mmol), and benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.054 g, 0.103 mmol) were dissolved in N,N-dimethylformamide (2 mL). N-methylmorpholine (0.040 g, 0.40 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness, dissolved in 3 mL of water, purified by centrifugation (H2O), and lyophilized. The product was purified by SEC (acetonitrile) to give 0.13 g of compound 10 (60% yield, NMR characterization showed 27 grafted drug molecules).

[0659] 1H NMR (400MHz, CD3OD): δ8.49(d,2H),7.80-7.15(m,166H),5.83-4.99(m,108H),4.74-4.13(m,103H), 4.00-3.39(m,3019H),3.35(s,96H),3.24-2.90(m,83H),2.57-1.15(m,522H),0.96-0.50(m,287H).

[0660] Example 15 Preparation of Compound 11

[0661] Compound 4c (0.05 g, 0.0012 mmol), compound J (0.080 g, 0.043 mmol), and benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.065 g, 0.103 mmol) were dissolved in N,N-dimethylformamide (1 mL). N-methylmorpholine (0.020 g, 0.20 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness, dissolved in 3 mL of water, purified by centrifugation (H2O), and lyophilized. The product was purified by SEC (acetonitrile) to give 0.07 g of compound 11 (80% yield, NMR characterization showed 30 grafted drug molecules).

[0662] 1 H NMR (400MHz, CD3OD): δ8.49(d,2H),7.62-7.26(m,180H),7.06(d,2H),5.76-5.03(m,137H),4.64-4.11(m,99H),3.73-3.4 3(m,3100H),3.37(s,96H),,3.19-3.11(m,90H),2.95(s,3H),2.55(br,111H),2.21-1.29(m,563H),1.09-0.88(m,282H).

[0663] Example 16 Preparation of Compound 12

[0664] Compound 3c (0.05 g, 0.0023 mmol), compound G (0.039 g, 0.042 mmol), and benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.024 g, 0.046 mmol) were dissolved in N,N-dimethylformamide (1 mL). N-methylmorpholine (0.037 g, 1.14 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness, dissolved in 3 mL of water, purified by centrifugation (H2O), and lyophilized. The product was purified by SEC (acetonitrile) to give 0.07 g of compound 12 (80% yield, NMR characterization showed 15 grafted drug molecules).

[0665] 1 H NMR (400MHz, CD3OD): δ8.49(d,2H),8.01-7.13(m,92H),7.06(d,2H),5.72-4.92(m,56H),4.84-4.08(m,58H),4.00-3.43(m,1563H) ,3.35(s,48H),3.30-2.90(m,43H),2.95(s,3H),2.49-1.98(m,116H),1.87-1.49(m,35H),1.40-1.28(m,63H),1.09-0.88(m,154H).

[0666] Example 17 Preparation of Compound 13

[0667] Step 1:

[0668] Compound 3a (0.10 g, 0.025 mmol), compound L (1.10 g, 0.49 mmol, prepared according to the method for compound H), and benzotriazol-1-yl-oxytripyrrolylphosphine hexafluorophosphate (0.282 g, 0.55 mmol) were dissolved in N,N-dimethylformamide (10 mL). N-methylmorpholine (0.207 g, 2.08 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness and purified by centrifugation (H₂O) to give 1.1 g of compound 13a (90% yield).

[0669] 1H NMR(400MHz,CD3OD):8.52(d,2H),7.19(d,2H),4.56(br,178H),4.06-4.00(m,32H),3.99-3.40(m,3020H), 3.38(s,48H),3.27-3.11(m,35H),3.00(s,3H),2.68-2.62(t,2H),2.46-2.42(t,2H),1.84-1.29(m,283H).

[0670] Step 2:

[0671] Compound 13a (0.9 g, 0.023 mmol) was dissolved in methanol (10 mL) and stirred. 4.0 M dioxane hydrochloride solution (5 mL) was added, and the reaction was allowed to proceed at room temperature until complete. The reaction solution was concentrated to dryness, then dissolved in 50 mL of water. The solution was purified by ultrafiltration (10 kD, Hydrosart) using water (1 L), and lyophilized to give 0.6 g of compound 13b (85% yield).

[0672] 1 H NMR (400MHz, CD3OD): δ8.52(d,2H),7.19(d,2H),4.56(br,178H),4.06-4.00(m,32H),3.99-3.40(m,3020H) ,3.38(s,48H),3.27-3.11(m,35H),3.00(s,3H),2.68-2.62(t,2H),2.46-2.42(t,2H),1.98-1.24(m,90H).

[0673] Step 3:

[0674] Compound 13b (0.20 g, 0.0053 mmol), compound F (0.136 g, 0.105 mmol), and benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.061 g, 0.116 mmol) were dissolved in N,N-dimethylformamide (2 mL). N-methylmorpholine (0.087 g, 0.42 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness, dissolved in 3 mL of water, purified by centrifugation (H₂O), and lyophilized. The product was purified by SEC (acetonitrile) to give 0.23 g of compound 13 (70% yield, NMR characterization showed 15 grafted drug molecules).

[0675] 1H NMR (400MHz, CD3OD): δ8.49(d,2H),8.11-8.09(d,32H),7.67-7.59(t,16H),7.59-7.49(t,32H),7.49-7.34 (d,62H),7.37-7.13(m,35H),6.36-6.06(m,15H),5.69-5.48(d,15H),5.31-5.15(brs,15H),5.03-4.92(d, 15H),4.76-4.47(m,115H),4.41-4.04(m,98H),3.73-3.41(m,3140H),3.73(s,43H),3.35(s,48H),3.24-3. 07(m,42H),3.06-2.92(m,76H),2.60(m,75H),2.52-2.23(m,57H),1.52-1.24(m,285H),1.24-1.03(d,96H).

[0676] Example 18 Preparation of Compound 14

[0677] Step 1:

[0678] Compound 3a (0.15 g, 0.037 mmol), compound H (0.50 g, 0.74 mmol), and benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.42 g, 0.81 mmol) were dissolved in N,N-dimethylformamide (10 mL). N-methylmorpholine (0.31 g, 3.07 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness and purified by centrifugation (H₂O) to give 0.43 g of compound 14a (80% yield).

[0679] 1 H NMR(400MHz,CD3OD):8.50(d,2H),7.19(d,2H),7.19(d,2H),4.54(s,10H),4.30-4.20(m,30H),3.84-3.40(m,83 9H),3.36(s,48H),3.27-3.11(m,32H),3.00(s,3H),2.68-2.62(t,2H),2.46-2.42(t,2H),1.84-1.29(m,225H).

[0680] Step 2:

[0681] Compound 14a (0.43 g, 0.028 mmol) was dissolved in methanol (5 mL) and stirred. 2 mL of 4.0 M dioxane hydrochloride solution was added, and the reaction was allowed to proceed at room temperature until complete. The reaction solution was concentrated to dryness, then dissolved in 50 mL of water. The solution was purified by ultrafiltration (10 kD, Hydrosart) using water (1 L), and lyophilized to give 0.3 g of compound 14b (85% yield).

[0682] 1H NMR (400MHz, CD3OD): δ8.50(d,2H),7.19(d,2H),4.53-4.17(m,33H),4.00-3.40(m,839H),3.36(s ,48H),3.27-3.11(m,30H),3.01(s,3H),2.48-2.42(t,2H),2.30-2.24(t,2H),1.98-1.29(m,90H).

[0683] Step 3:

[0684] Compound 14b (0.10 g, 0.0073 mmol), compound F (0.19 g, 0.15 mmol), and benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.083 g, 0.16 mmol) were dissolved in N,N-dimethylformamide (1 mL). N-methylmorpholine (0.12 g, 1.19 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness, and the remaining solution was diluted with acetonitrile (10 mL) and purified by ultrafiltration (10 kD, Hydrosart) using acetonitrile (1 L) to give 0.12 g of compound 14 (yield 85%, NMR characterization showed 15 grafted drug molecules).

[0685] 1H NMR (400MHz, CD3OD): δ8.49(d,2H),8.11-8.09(d,32H),7.67-7.59(t,16H),7.59-7.49(t,32H),7.49-7.34(d,6 2H),7.37-7.13(m,94H),7.06-6.90(m,8H),6.36-6.06(t,15H),5.69-5.48(d,15H),5.31-5.15(brs,15H),5.03- 4.92(d,15H),4.76-4.57(m,63H),4.41-4.06(m,112H),4.01-3.45(m,947H),3.37(s,43H),3.35(s,48H),3.24- 3.07(m,78H),3.06-2.92(m,24H),2.60(brs,20H),2.52-2.23(m,65H),1.90-1.24(m,298H),1.24-1.03(d,96H).

[0686] Example 19 Preparation of Compound 15

[0687] Step 1:

[0688] Compound 3a (1.60 g, 0.39 mmol) and compound C2 (3.70 g, 7.89 mmol) were dissolved in N,N-dimethylformamide (36 mL). N-methylmorpholine (6.40 g, 63.57 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness and purified by column chromatography (acetonitrile / water = 20%) to give 3.2 g of compound 15a (80% yield).

[0689] 1 H NMR(400MHz,CD3OD):8.51(d,2H),7.18(d,2H),4.31-4.25(m,16H),4.06-4.02(m,16H),3.94-3.52(m,112H),3.50 -3.34(m,16H),3.30-3.09(m,32H),3.04-2.99(m,35H),2.68-2.62(t,2H),2.46-2.42(t,2H),1.84-1.29(m,470H).

[0690] Step 2:

[0691] In an ice-water bath, compound 15a (1.8 g, 0.20 mmol) and methanol (18 mL) were added to a reaction flask, followed by 10 mL of 4.0 M dioxane hydrochloride solution. The mixture was stirred at room temperature until complete. The solution was concentrated and purified by column chromatography (acetonitrile / water = 40%), and then lyophilized to give 1.1 g of compound 15b (yield 85%).

[0692] 1 H NMR(400MHz,CD3OD):8.51(d,2H),7.18(d,2H),4.51-4.25(m,16H),4.16-4.00(m,18H),3.94-3.52(m,15 4H),3.30-3.09(m,26H),3.04-2.99(m,35H),2.68-2.62(t,2H),2.46-2.42(t,2H),1.84-1.29(m,187H).

[0693] Step 3:

[0694] Compound 15b (0.30 g, 0.035 mmol), compound B (1.88 g, 1.40 mmol), and benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.76 g, 1.47 mmol) were dissolved in N,N-dimethylformamide (20 mL). N-methylmorpholine (0.58 g, 5.71 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness and purified by centrifugation (acetonitrile) to give 1.0 g of compound 15c (70% yield).

[0695] 1 H NMR(400MHz,CD3OD):8.52(d,2H),7.19(d,2H),4.31-4.02(m,77H),4.02-3.41(m,3062H), 3.35(s,96H),3.27-3.11(m,112H),3.01(s,3H),2.60-2.45(m,70H),1.84-1.29(m,660H).

[0696] Step 4:

[0697] Compound 15c (1.5 g, 0.0029 mmol) was dissolved in methanol (15 mL), and 4.0 M dioxane hydrochloride solution (10 mL) was added with stirring. The reaction was allowed to proceed at room temperature until complete. The reaction solution was concentrated to dryness, then dissolved in 10 mL of water, and purified by centrifugation (H2O) to give 1.1 g of compound 15d (yield 85%).

[0698] 1H NMR (400MHz, CD3OD): δ8.52(d,2H),7.19(d,2H),4.53-4.17(m,33H),3.95-3.45(m,3117H) ,3.36(s,96H),3.18-3.11(m,110H),3.01(s,3H),2.60-2.45(m,66H),1.98-1.24(m,388H).

[0699] Step 5:

[0700] Compound 15d (0.30 g, 0.0066 mmol), compound F (0.343 g, 0.26 mmol), and benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (0.144 g, 0.28 mmol) were dissolved in N,N-dimethylformamide (3 mL). N-methylmorpholine (0.109 g, 1.08 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness, and the remaining solution was diluted with acetonitrile (100 mL). The solution was then purified by ultrafiltration (10 kD, Hydrosart) using acetonitrile (0.3 L), followed by lyophilization to obtain 0.5 g of compound 15 (90% yield, NMR characterization showed 28 grafted drug molecules).

[0701] 1 H NMR (400MHz, CD3OD): δ8.49(d,2H),8.10-8.08(d,64H),7.67-7.59(d,35H),7.59-7.49(t,65H),7.46-7.34(d,126H),7. 32-7.14(m,187H),7.08-6.89(m,22H),6.36-6.09(s,28H),5.65-5.49(s,29H),5.32-5.15(brs,27H),5.07-4.92(s,39H) ,4.74-4.43(m,90H),4.42-4.07(m,207H),4.02-3.43(m,3140H),3.37(s,86H),3.35(s,96H),3.24-3.07(m,158H),3.07- 2.88(m,66H),2.74-2.22(m,183H),2.21-1.86(m,148H),1.85-1.51(m,200H),1.51-1.25(m,412H),1.24-1.00(d,202H).

[0702] Example 20: Preparation of 5-ADC

[0703] Prepared using the same method as in step 4 of Example 5, except that compound 1 was replaced with compound 13 (45.55 mg, 0.82 μmol, dissolved in 1×PBS 7.2 to obtain a 10 mg / mL solution), yielding 11 mg of 5-ADC (yield 27.5%, q was 1.81 and drug loading was 29.0 as determined by non-reducing RP method), and stored at 4°C.

[0704] Example 21: Preparation of 6-ADC

[0705] The same method as in step 4 of Example 5 was used to prepare the 6-ADC, except that compound 1 was replaced with compound 8 (16.3 mg, 0.82 μmol, dissolved in 1×PBS 7.2 to form a 10 mg / mL solution), and 13 mg of 6-ADC was finally obtained (yield 32.5%, q was 1.9 and drug loading was 30.4 as determined by non-reducing RP method), and it was stored at 4°C.

[0706] Example 22 Preparation of 7-ADC

[0707] The same method as in step 4 of Example 5 was used to prepare the 7-ADC, except that compound 1 was replaced with compound 7 (56.1 mg, 0.82 μmol, dissolved in 1×PBS 7.2 to form a 10 mg / mL solution). The final result was 37 mg of 7-ADC (yield 92.5%, q was 1.86 and drug loading was 59.5 as determined by non-reducing RP method), which was stored at 4 °C.

[0708] Example 23 Preparation of 8-ADC

[0709] The same method as in step 4 of Example 5 was used to prepare the ADC, except that compound 1 was replaced with compound 6 (63.6 mg, 0.82 μmol, dissolved in 1×PBS 7.2 to form a 10 mg / mL solution), and 33 mg of 8-ADC was finally obtained (yield 82.5%, q was determined to be 2 by non-reducing RP method, and drug loading was 64.0), which was stored at 4°C.

[0710] Example 24 Preparation of 9-ADC

[0711] The same method as in step 4 of Example 5 was used to prepare the 9-ADC, except that compound 1 was replaced with compound 5 (36 mg, 0.82 μmol, dissolved in 1×PBS 7.2 to form a 10 mg / mL solution), and 15 mg of 9-ADC was finally obtained (yield 37.5%, q was 1.82 and drug loading was 29.1 as determined by non-reducing RP method), and it was stored at 4 °C.

[0712] Example 25 Preparation of 10-ADC

[0713] The same method as in step 4 of Example 5 was used to prepare the 10-ADC, except that compound 1 was replaced with compound 15 (57 mg, 0.81 μmol, dissolved in 1×PBS 7.2 to prepare a 10 mg / mL solution). The final result was 10 mg of 10-ADC (yield 62%, q = 1.82 determined by non-reducing RP, drug loading 58.2), which was stored at 4°C.

[0714] Example 26 Preparation of 11-ADC

[0715] Dissolve Ab-5b (37 mg, 0.25 μmol) in 1×PBS 7.2 to a concentration of 5 mg / mL, add compound 12 (44 mg, 1.26 μmol) in DMSO (0.37 mL), mix well, and incubate at room temperature for 16 h. The crude product was adjusted to pH 5.0 with acetic acid, and then an equal volume of sodium acetate buffer (50 mM NaOAc / HOAc, pH 5.0, 2.33 mL) was added and mixed. The mixture was then passed through a HiTrap Capto S cation exchange chromatography column (mobile phase A: 150 mM NaOAc / HOAc, pH 5.0; mobile phase B: 150 mM NaOAc / HOAc, 0.5 M NaCl, pH 5.0, elution with 0%-30% B phase solution) and concentrated using a 30 kDa ultrafiltration tube to obtain 9.5 mg of 11-ADC (yield 17.6%, q = 1.9 and drug loading 30.4 g as determined by RP-HPLC). The ADC was stored at 4 °C.

[0716] Example 27 Preparation of 12-ADC

[0717] Prepared using the same method as in Example 26, except that compound 12 was replaced with compound 10 (22 mg, 0.335 μmol), yielding 11 mg of 12-ADC (yield 59.8%, q = 1.9 and drug loading 60.8 as determined by RP-HPLC), and stored at 4°C.

[0718] Example 28 Preparation of 13-ADC

[0719] Prepared using the same method as in Example 26, except that compound 12 was replaced with compound 9 (14.6 mg, 0.335 μmol), yielding 10 mg of 13-ADC (yield 64%, q = 1.89 and drug loading 30.2 as determined by RP-HPLC), which was stored at 4°C.

[0720] Example 29 Preparation of 14-ADC

[0721] Prepared using the same method as in Example 26, except that compound 12 was replaced with compound 14 (9.19 mg, 0.335 μmol), yielding 14 mg of 14-ADC (yield 70%, q = 1.90 and drug loading = 30.4 as determined by RP-HPLC), and stored at 4°C.

[0722] Example 30 Preparation of 15-ADC

[0723] Prepared using the same method as in Example 26, except that compound 12 was replaced with compound 11 (43 mg, 0.50 μmol), yielding 7 mg of 15-ADC (yield 22%, q = 1.94 and drug loading 62.1 as determined by RP-HPLC), which was stored at 4°C.

[0724] Example 31 Preparation of Compound 16

[0725] Compound M (0.065 g, 0.01 mmol), compound L (0.051 g, 0.052 mmol), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (0.032 g, 0.06 mmol) were dissolved in N,N-dimethylformamide (20 mL). N-methylmorpholine (0.082 g, 0.82 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness, then dissolved in 2 mL of acetonitrile. The solution was purified by gel column chromatography (acetonitrile), concentrated, dissolved in water (6 mL), filtered through a 0.2 μm filter, and lyophilized to give 0.1 g of compound 16 (yield 85%, NMR characterization showed a grafting number of 4 drug molecules).

[0726] 1 H NMR (400MHz, CD3OD): δ8.41(d,2H),7.44-7.11(m,30H),5.75-5.63(m,4H),5.50-5 .46(m,4H),5.33-5.22(m,4H),5.18-5.07(m,4H),4.64-4.04(m,38H),3.93-3.43(m ,493H),3.34(s,3H),3.24-2.96(m,20H),2.95(s,3H),2.83-2.78(m,4H),2.71-2.6 7(m,2H),2.43(t,2H),2.35-1.22(m,42H),0.97-0.94(m,10H),0.75-0.52(m,15H).

[0727] Example 32 Synthesis of Compound 17

[0728] Step 1:

[0729] Compound M (50 mg, 0.0078 mmol), compound N (35 mg, 0.041 mmol), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (24 mg, 0.047 mmol) were dissolved in N,N-dimethylformamide (2 mL). N-methylmorpholine (0.39 mg, 0.39 mmol) was added with stirring. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness, then dissolved in 2 mL of acetonitrile. The solution was purified by gel column chromatography (acetonitrile), concentrated, dissolved in water (6 mL), and lyophilized to give 0.065 g of compound 17 (yield 85%, NMR characterization showed a grafting number of 4).

[0730] 1 H NMR (400MHz, CD3OD): δ8.41(d,2H),7.44-7.11(m,30H),5.48-5.03(m,16H),4.71-4.02(m,38H) ,3.93-3.43(m,493H),3.20-3.12(m,10H),2.95(s,3H),2.60-1.41(m,60H),0.97-0.88(m,36H).

[0731] Example 33 Preparation of 16-ADC

[0732] The same method as in step 4 of Example 5 was used to prepare the 16-ADC, except that compound 1 was replaced with compound 16 (6.93 mg, 0.67 μmol, dissolved in 1×PBS 7.2 to prepare a 10 mg / mL solution), and 12 mg of 16-ADC was obtained (yield 48%, q was 1.90 and drug loading was 7.60 as determined by RP-HPLC). The ADC was stored at 4 °C.

[0733] Example 34 Preparation of 17-ADC

[0734] The same method as in step 4 of Example 5 was used to prepare 17-ADC, except that compound 1 was replaced with compound 17 (7.93 mg, 0.82 μmol, dissolved in 1×PBS 7.2 to prepare a 10 mg / mL solution), and 20 mg of 17-ADC was obtained (yield 81%, q was 1.92 and drug loading was 7.68 as determined by RP-HPLC). The ADC was stored at 4 °C.

[0735] Preparation of Compound 18 in Example 35

[0736] Step 1:

[0737] Compound 18b (100 mg, 0.23 mmol, purchased from Leyan) and compound 18a (132 mg, 0.25 mmol, purchased from Shanghai Bide Pharmaceutical) were dissolved in N,N-dimethylformamide (2 mL), and N-methylmorpholine (114 mg, 1.13 mmol) was added. The reaction was carried out at room temperature until complete. The mixture was purified by column chromatography (acetonitrile:water = 0%-60%) to give 120 mg of compound 18c (yield 73%).

[0738] MS(ESI): m / z = 860.3[M+1] + .

[0739] Step 2:

[0740] Compound 18c (0.40 g, 0.47 mmol), compound D2 (170 mg, 0.51 mmol), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (293 mg, 0.56 mmol) were dissolved in N,N-dimethylformamide (5 mL), and N-methylmorpholine (142 mg, 1.41 mmol) was added. The reaction was carried out at room temperature until complete. The solution was purified by column chromatography (acetonitrile:water = 0%–60%) to give 436 mg of compound 18d (yield 80%).

[0741] 1 H NMR (400MHz, CD3OD): δ8.49(d,2H),7.18(d,2H),4.27-4.25(m,2H),3.91-3.88(m,4H),3.76-3.57(m,44H),3.54-3.51(m,2H),3 .39-3.30(m,6H),3.08-3.02(m,4H),3.00(s,3H),2.64-2.61(m,2H),2.45-2.42(m,2H),1.86-1.65(m,4H),1.44-1.43(m,18H).

[0742] Step 3:

[0743] Compound 18d (0.5 g, 0.43 mmol) was dissolved in methanol (5 mL), and 2.0 M hydrochloric acid-methanol solution (5 mL) was added. The reaction was allowed to proceed at room temperature until complete. The reaction solution was concentrated to dryness and purified by column chromatography (acetonitrile:water = 0%-40%) to give 0.43 g of compound 18e (100% yield).

[0744] 1H NMR (400MHz, CD3OD): δ8.50(d,2H),7.18(d,2H),4.28-4.26(m,2H),3.92-3.85(m,4H),3.78-3.59(m,44H),3.54-3.5 1(m,2H),3.40-3.30(m,6H),3.08-3.02(m,4H),2.99(s,3H),2.64-2.61(m,2H),2.47-2.42(m,2H),1.96-1.68(m,4H).

[0745] Step 4:

[0746] Compound 18f (1.85 g, 1.08 mmol, prepared using the method disclosed in patent WO1990011778), compound 18e (0.50 g, 0.51 mmol), and 1H-benzotriazol-1-yloxytripyrrolyl hexafluorophosphate (0.70 g, 1.17 mmol) were dissolved in N,N-dimethylformamide (15 mL), and N-methylmorpholine (0.50 g, 5.11 mmol) was added. The reaction was carried out at room temperature until complete. The reaction solution was concentrated to dryness and purified by column chromatography (acetonitrile:water = 0%-100%) to give 1.30 g of compound 18 (yield 80%).

[0747] 1 H NMR (400MHz, CD3OD): δ8.50(d,2H),7.18(d,2H),4.28-3.51(m,67H),3.38-3.34(m,15H),3 .18-2.98(m,16H),3.00(s,3H),2.68-2.64(m,2H),2.46-2.42(m,2H),1.75-1.28(m,242H).

[0748] Step 5:

[0749] 18 g (1.30 g, 2.97 mmol) of the compound was dissolved in methanol (13 mL), and 8 mL of 2.0 M hydrochloric acid-methanol solution was added. The reaction was allowed to proceed at room temperature until complete. The reaction solution was concentrated to dryness, and the residue was not purified but directly added to water (10 mL) and lyophilized to give 1.00 g of the compound for 18 h (100% yield).

[0750] 1H NMR (400MHz, CD3OD): δ8.50(d,2H),7.18(d,2H),4.35-3.57(m,80H),3.38-3.21(m,13H),3 .18-3.01(m,21H),3.00(s,3H),2.68-2.63(m,2H),2.45-2.42(m,2H),1.75-1.28(m,98H).

[0751] Step 6:

[0752] Compound 18i (0.1 g, 0.035 mmol), compound B (0.83 g, 0.69 mmol), and 1H-benzotriazol-1-yloxytripyrrolyl hexafluorophosphate (0.40 g, 0.77 mmol) were dissolved in N,N-dimethylformamide (8 mL), and N-methylmorpholine (0.175 g, 2.91 mmol) was added. The reaction was carried out under a nitrogen atmosphere at room temperature until complete. The reaction solution was concentrated to dryness, centrifuged, filtered, and purified (acetonitrile) to give 0.6 g of compound 18i (yield 80%).

[0753] 1 H NMR(400MHz,CD3OD):8.52(d,2H),7.19(d,2H),4.53-4.21(m,30H),3.84-3.44(m,1473H),3.37(s, 48H),3.27-3.13(m,33H),3.02(s,3H),2.68-2.62(t,2H),2.46-2.42(t,2H),1.84-1.29(m,237H).

[0754] Step 7:

[0755] Compound 18i (0.60 g, 0.028 mmol) was dissolved in methanol (10 mL), and 4.0 M dioxane hydrochloride solution (6 mL) was added with stirring. The reaction was allowed to proceed at room temperature until complete. The reaction solution was concentrated to dryness, then dissolved in 5 mL of water, centrifuged, filtered (H2O), purified, and lyophilized to give 0.45 g of compound 18j (70% yield).

[0756] 1 H NMR(400MHz,CD3OD):8.52(d,2H),7.19(d,2H),4.53-4.21(m,30H),3.80-3.44(m,1473H),3.37(s ,48H),3.28-3.13(m,33H),3.00(s,3H),2.68-2.62(t,2H),2.46-2.42(t,2H),1.88-1.31(m,94H).

[0757] Step 8:

[0758] Compound 18j (0.13 g, 0.0064 mmol), compound F (0.167 g, 0.13 mmol), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (0.073 g, 0.14 mmol) were dissolved in N,N-dimethylformamide (2.6 mL), and N-methylmorpholine (0.105 g, 1.04 mmol) was added. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness, then dissolved in 3 mL of water, purified by centrifugation (H2O), and lyophilized. The product was purified by SEC (acetonitrile) to give 0.18 g of compound 18 (yield 80%, NMR characterization showed 15 grafts of cabazitaxel).

[0759] 1 H NMR (400MHz, CD3OD): δ8.49(d,2H),8.11-8.09(d,32H),7.67-7.59(t,16H),7.59-7.49(t,32H),7.49-7.34(d,62H),7.37-7.13 (m,94H),7.06-6.90(m,8H),6.36-6.06(t,15H),5.69-5.48(d,15H),5.31-5.15(brs,16H),5.03-4.92(d,16H),4.76-4.47(m,63 H),4.41-4.04(m,112H),4.01-3.77(m,118H),3.73-3.41(m,1616H),3.73(s,48H),3.35(s,48H),3.24-3.07(m,78H),3.06-2.92 (m,24H),2.60(brs,20H),2.52-2.23(m,65H),2.20-1.90(m,69H),1.89-1.54(m,99H),1.52-1.24(m,212H),1.24-1.03(d,96H).

[0760] Example 36 Preparation of Compound 19

[0761] Step 1:

[0762] Compound D3 (1.00 g, 0.60 mmol), compound C (1.10 g, 1.37 mmol), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (0.78 g, 1.50 mmol) were dissolved in N,N-dimethylformamide (10 mL), and N-methylmorpholine (0.303 g, 3.00 mmol) was added. The reaction was carried out at room temperature until complete. The reaction solution was concentrated to dryness and purified by column chromatography (acetonitrile:water = 0%–100%) to give 1.8 g of compound 19a (yield 80%).

[0763] 1 H NMR (400MHz, CD3OD): δ7.86-7.82(m,4H),4.30-4.20(m,2H),4.06-3.73(m,12H),3.62-3.5 2(m,94H),3.45-3.30(m,4H),3.19-3.02(m,18H),2.68-2.58(m,2H),1.97-1.28(m,120H).

[0764] Step 2:

[0765] Compound 19a (1.80 g, 0.57 mmol) was dissolved in ethanol (20 mL), and hydrazine hydrate (2 mL) was added. The mixture was heated to 60–65 °C until the reaction was complete. The mixture was filtered at room temperature, the filter cake was washed with ethanol, and the filtrate was concentrated to dryness. The filtrate was purified by column chromatography (dichloromethane:methanol = 60:1–10:1) to give 1.0 g of compound 19b (yield 52%).

[0766] 1 H NMR (400MHz, CD3OD): δ4.30-4.22(m,2H),4.05-3.95(m,4H),3.82-3.52(m,94H),3.37-3. 30(m,5H),3.19-3.02(m,16H),2.80-2.77(m,2H),2.63-2.58(m,2H),1.83-1.28(m,120H).

[0767] Step 3:

[0768] N3-(CH2CH2O)4-CH2CH2COOH (56 mg, 0.19 mmol, purchased from Leyan), compound 19b (0.5 g, 0.18 mmol), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (118 mg, 0.23 mmol) were dissolved in N,N-dimethylformamide (5 mL), and N-methylmorpholine (106 mg, 1.05 mmol) was added. The reaction was carried out at room temperature until complete. The solution was purified by column chromatography (acetonitrile:water = 0%-100%) to give 0.38 g of compound 19c (yield 82%).

[0769] 1 H NMR (400MHz, CD3OD): δ4.27-4.22(m,2H),4.05-3.81(m,4H),3.82-3.51(m,120H),3.35-3. 31(m,8H),3.27-3.00(m,16H),2.80-2.75(m,2H),2.63-2.56(m,2H),1.93-1.28(m,120H).

[0770] Step 4:

[0771] Compound 19c (0.38 g, 0.12 mmol) was dissolved in methanol (5 mL), and 2.0 M hydrochloric acid methanol solution (3 mL) was added. The reaction was allowed to proceed at room temperature until complete. The reaction solution was concentrated to dryness, and then lyophilized with water (10 mL) to obtain 280 mg of crude compound 19d, which was used directly in the next step.

[0772] 1 H NMR (400MHz, CD3OD): δ4.32-4.12(m,2H),4.05-3.81(m,4H),3.82-3.51(m,120H),3.36-3 .31(m,8H),3.28-3.00(m,16H),2.80-2.75(m,2H),2.64-2.56(m,2H),1.93-1.48(m,48H).

[0773] Step 5:

[0774] Compound 19d (0.15 g, 0.057 mmol), compound B (0.687 g, 0.57 mmol), and 1H-benzotriazol-1-yloxytripyrrolyl hexafluorophosphate (0.356 g, 0.68 mmol) were dissolved in N,N-dimethylformamide (8 mL), and N-methylmorpholine (0.288 g, 2.85 mmol) was added. The reaction was carried out under a nitrogen atmosphere at room temperature until complete. The reaction solution was concentrated to dryness, centrifuged, filtered, and purified (acetonitrile) to give 0.5 g of compound 19e (yield 85%).

[0775] 1 H NMR (400MHz, CD3OD): δ4.33-4.14(m,14H),3.81-3.61(m,850H),3.54-3.36(m,57H ),3.26-3.07(m,16H),2.80-2.75(m,2H),2.64-2.56(m,2H),1.93-1.48(m,120H).

[0776] Step 6:

[0777] Compound 19e (0.38 g, 0.033 mmol) was dissolved in methanol (5 mL), and 4.0 M dioxane hydrochloride solution (3 mL) was added with stirring. The reaction was allowed to proceed at room temperature until complete. The reaction solution was concentrated to dryness, then dissolved in 5 mL of water, centrifuged, filtered (H2O), and lyophilized to give 0.30 g of compound 19f (90% yield).

[0778] 1 H NMR (400MHz, CD3OD): δ4.35-4.15(m,12H),3.81-3.62(m,850H),3.54-3.37(m,57 H),3.28-3.07(m,16H),2.80-2.73(m,2H),2.65-2.57(m,2H),1.83-1.48(m,48H).

[0779] Step 7:

[0780] Compound 19f (0.14 g, 0.012 mmol), compound F (0.161 g, 0.12 mmol), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (0.077 g, 0.15 mmol) were dissolved in N,N-dimethylformamide (2 mL), and N-methylmorpholine (61 mg, 0.60 mmol) was added. The reaction was carried out at room temperature under a nitrogen atmosphere until complete. The reaction solution was concentrated to dryness, then dissolved in 3 mL of water, purified by centrifugation (H2O), and lyophilized. The product was purified by SEC (acetonitrile) to give 0.07 g of compound 19 (yield 50%, NMR characterization showed 8 grafts of cabazitaxel).

[0781] 1H NMR (400MHz, CD3OD): δ8.61-8.39(m,8H),8.17-8.04(m,16H),7.64-7.22(m,103H),6.30-6.13(t,8 H),5.67-5.53(d,8H),5.34-5.23(m,16H),4.66-4.58(m,30H),4.20-4.16(m,48H),3.74-3.48(m,83 0H),3.38(s,24H),3.24(s,24H),3.0-2.90(m,10H),2.60(brs,10H),2.43(s,24H),2.35-2.22(m,1 0H),2.20-2.06(m,9H),2.01(s,24H),1.88-1.55(m,47H),1.53-1.24(m,103H),1.24-1.06(d,48H).

[0782] Example 37 Preparation of Compound 20

[0783] Step 1:

[0784] Compound Q (450 mg, 0.32 mmol) was dissolved in dry N,N-dimethylformamide (12 mL), and compound H (2.5 g, 3.55 mmol), 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (2.5 g, 4.81 mmol), and N,N-diisopropylethylamine (1.26 g, 9.69 mmol) were added. The mixture was stirred at room temperature until complete. The reaction solution was purified by column chromatography (acetonitrile / water = 45-53%) and lyophilized to give 2.0 g of compound 20a (yield: 93.2%).

[0785] 1 H NMR (400MHz, CD3OD-d4): δ7.32-7.25(m,10H),5.41(s,1H),4.57-4.50(m,13H),4.30-4.19(m,14H ),3.81-3.52(m,367H),3.40-3.38(m,6H),3.35(s,24H),3.23-3.19(m,18H),1.91-1.26(m,133H).

[0786] Step 2:

[0787] Compound 20a (2.0 g, 0.30 mmol) was dissolved in ethyl acetate hydrochloride (4 M / L, 20 mL) and stirred at room temperature until the reaction was complete. The reaction solution was concentrated, and the residue was purified by column chromatography (acetonitrile / water = 45-53%) and lyophilized to give 1.8 g of compound 20b (yield: 97.0%).

[0788] Step 3:

[0789] Compound 20b (600 mg, 0.10 mmol) was dissolved in dry N,N-dimethylformamide (8 mL), and compound R (950 mg, 0.98 mmol, prepared according to Example 4 on page 29 of the specification of patent WO2022042583A1), 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (1 g, 1.92 mmol) and N,N-diisopropylethylamine (1 g, 7.69 mmol) were added sequentially. The mixture was stirred at room temperature until complete. The reaction solution was purified by SEC (eluent methanol), concentrated and lyophilized to give 760 mg of compound 20 (yield: 62.2%, NMR characterization with 7 docetaxel).

[0790] 1 H NMR(400MHz,CD3OD-d4)δ8.12(d,14H),7.71-7.63(m,7H),7.61-7.53(m,14H),7.47-7.35(m,27H),7.3 3-7.20(m,20H),6.16-6.14(m,7H),5.66-5.63(m,7H),5.42-5.28(m,21H),5.01-4.99(m,7H),4.57-4. 50(m,13H),4.30-4.20(m,27H),3.89-3.71(m,383H),3.34(s,24H),3.28-3.07(m,25H),2.52-2.39(m, 61H),2.35-2.18(m,61H),2.05-1.77(m,70H),1.69(s,21H),1.58-1.29(m,105H),1.16-1.13(m,42H).

[0791] Example 38 Preparation of Compound 21

[0792] Step 1:

[0793] Compound 21a (5.0 g, 10.0 mmol, purchased from Amex) was dissolved in dichloromethane (25 mL), and tert-butyl acrylate (25.6 g, 200.0 mmol, purchased from Adamas) and potassium tert-butoxide tetrahydrofuran solution (1 M) (1.0 mL, 1 mmol, purchased from Adamas) were added sequentially. The reaction was allowed to proceed at room temperature until complete. The reaction mixture was poured into water (50 mL) and extracted with dichloromethane (50 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The residue was purified by column chromatography (petroleum ether: ethyl acetate = 2:1) to give 5.77 g of compound 21b (yield: 89.0%).

[0794] MS(ESI): m / z = 645[M+1] + .

[0795] 1 H-NMR (400MHz, CDCl3): δ3.71(t,2H),3.62-3.61(m,42H),3.59-3.55(m,1H),3.38(s,3H),2.50(t,2H),1.44(s,9H).

[0796] Step 2:

[0797] Compound 21b (5.0 g, 7.75 mmol) was dissolved in dichloromethane (60 mL), and trifluoroacetic acid (8.84 g, 77.5 mmol) was added. The reaction was carried out at room temperature until complete. The reaction solution was concentrated, and the residue was purified by column chromatography (acetonitrile:water = 1:3), lyophilized, and yielded 3.05 g of compound 21c (yield: 67.0%).

[0798] MS(ESI): m / z = 589[M+1] + .

[0799] 1 H-NMR (400MHz, CDCl3): δ3.18(t,2H), 3.67-3.62(m,42H), 3.55(t,2H), 3.38(s,3H), 2.60(t,2H).

[0800] Step 3:

[0801] Compound 21c (4.80 g, 8.15 mmol) was dissolved in dichloromethane (48 mL), and N-hydroxysuccinimide (1.13 g, 9.78 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.87 g, 9.78 mmol) were added. The reaction was allowed to proceed at room temperature until complete. The reaction mixture was poured into water (50 mL) and extracted with dichloromethane (50 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The residue was purified by column chromatography (acetonitrile:water = 1:3) and lyophilized to give 4.50 g of compound 21d (yield: 80.0%).

[0802] MS(ESI): m / z = 686[M+1] + .

[0803] 1 H-NMR (400MHz, CDCl3): δ3.85(t,2H),3.65-3.63(m,42H),3.56-3.53(m,2H),3.38(s,3H),2.90(t,2H),2.83(s,4H).

[0804] Step 4:

[0805] Compound 21d (8.40 g, 12.24 mmol) was dissolved in N,N-dimethylformamide (40 mL), and compound 21e (3.16 g, 12.81 mmol, purchased from Anegig) was added. The reaction was carried out at room temperature until complete. The reaction solution was poured into water (50 mL), extracted with ethyl acetate (50 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography (acetonitrile:water = 1:3). The purified residue was lyophilized to give 7.0 g of compound 21f (yield: 70.0%).

[0806] MS(ESI): m / z = 817[M+1] + .

[0807] 1 H-NMR (400MHz, CDCl3): δ6.94(s,1H),5.32(d,1H),4.26(d,1H),3.73(t,2H),3.68-3.65(m,42H),3.59-3. 55(m,2H),3.38(s,3H),3.27-3.22(m,2H),2.49(t,2H),1.79-1.75(m,2H),1.54-1.51(m,2H),1.45(s,9H).

[0808] Step 5:

[0809] Compound 21f (7.0 g, 8.57 mmol) was dissolved in N,N-dimethylformamide (100 mL), and compound Q (1.19 g, 0.86 mmol), N,N-diisopropylethylamine (2.12 g, 16.45 mmol), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (8.55 g, 16.45 mmol) were added. The reaction was carried out at room temperature for 15–17 hours until complete. The reaction solution was poured into water (200 mL), extracted with ethyl acetate (100 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography (acetonitrile:water = 1:3). The residue was lyophilized to give 2.10 g of compound 21f (yield: 32.6%).

[0810] 1 H-NMR (400MHz, CD3OD): δ7.31-7.25(m,10H),5.40(s,1H),4.31-4.02(m,6H),4.03-3.95(m,6H), 3.79-3.44(m,352H),3.35(s,24H),3.18-3.13(m,78H),2.41-2.36(m,16H),1.76-1.24(m,144H).

[0811] Step 6:

[0812] 21 g (2.10 g, 0.28 mmol) of compound was dissolved in methanol (5 mL), and HCl / EA solution (45 mL, 89.76 mmol) was added. The mixture was stirred at room temperature until the reaction was complete. The reaction solution was concentrated, and the residue was purified by column chromatography (acetonitrile:water = 1:3). The residue was lyophilized to give 1.4 g of compound 21h (yield: 71.0%).

[0813] MS m / z(ESI): 7051 [M+1] + .

[0814] 1 H-NMR (400MHz, CD3OD): δ7.32-7.26(m,10H),5.41(s,1H),4.39-4.25(m,6H),4.01-3.91(m,6H), 3.73-3.49(m,352H),3.35(s,24H),3.27-3.14(m,27H),2.46-2.38(m,16H),1.87-1.30(m,72H).

[0815] Step 7:

[0816] Compound 21h (700 mg, 0.10 mmol) was dissolved in N,N-dimethylformamide (10 mL), and compound R (958 mg, 0.99 mmol), N,N-diisopropylethylamine (2.12 g, 16.45 mmol), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (8.55 g, 16.45 mmol) were added. The reaction was carried out at room temperature until complete. The reaction solution was purified by SEC (eluent methanol), concentrated, and lyophilized to give 1.15 g of compound 21 (yield: 81.0%, NMR characterization with 7 docetaxel).

[0817] 1 H-NMR (400MHz, CD3OD): δ8.12(d,14H),7.62-7.69(m,7H),7.61-7.52(s,14H),7.37-7.50(m,27H),7.35-7.2 0(m,20H),6.17(s,7H),5.64(d,7H),5.48(s,14H),5.39(s,1H),5.28(s,7H),5.01(d,7H),4.37-4.14(m,35H) ,3.88(d,7H),3.82-3.47(m,361H),3.34(s,24H),3.17(s,33H),2.61(s,40H),2.50-2.37(m,45H),2.27(s,2 2H),2.07(s,14H),2.34-2.04(m,34H),1.92(s,25H),2.03-1.61(m,98H),1.59-1.25(m,134H),1.13(d,42H).

[0818] Example 39 Preparation of 18-ADC

[0819] Following the preparation method of Example 5, antibody Ab (Trastuzuma) was replaced with antibody mAb1 (Bevacizumab), and compound 1 was replaced with compound 3 (56.67 mg, 1.36 μmol, dissolved in 1×PBS 7.2 to a 10 mg / mL solution), yielding 22.11 mg of 18-ADC (yield 90.0%, q was 1.85 and drug loading was 29.6 as determined by MS), which was stored at 4°C.

[0820] Example 40 Preparation of 19-ADC

[0821] Using the same preparation method as in Example 39, antibody mAb1 (Bevacizumab) was replaced with antibody mAb2 (Cetuximab) to obtain 20.00 mg of 19-ADC (yield 81.4%, q was 1.90 and drug loading was 30.4 as determined by MS), which was stored at 4°C.

[0822] Example 41: Preparation of 20-ADC

[0823] Using the same preparation method as in Example 39, antibody mAb1 (Bevacizumab) was replaced with antibody mAb3 (Telisotuzumab) to obtain 20.50 mg of 20-ADC (yield 83.4%, q was 1.80 and drug loading was 28.8 as determined by MS), which was stored at 4°C.

[0824] Biological evaluation

[0825] The following test examples further describe and explain this disclosure, but these test examples are not intended to limit the scope of this disclosure.

[0826] Test Example 1: In vitro cell-killing activity of the dendritic polymer conjugate disclosed herein

[0827] 1) Sample to be tested

[0828] 1-ADC (control), 2-ADC, 3-ADC, 4-ADC.

[0829] 2) Testing Methods

[0830] JIMT-1 cells (purchased from ATCC) suspension were prepared and seeded into 96-well plates. The cells were incubated overnight at 37°C. The analyte was added at a maximum concentration of 500 nM, serially diluted 5-fold, with two parallel test wells for each condition. Cells were incubated at 37°C for 120 h. Cell viability was assessed using the CTG assay (CellTiter-Glo). The IC50 (IC50) of the analyte on the cell lines was calculated. 50 .

[0831] 3) Results

[0832] The disclosed compound exhibits cytotoxic activity (IC50) against JIMT-1 cells. 50 The values ​​are shown in Table 1 below.

[0833] Table 1

[0834] As shown in the table above, the in vitro activity of 2-ADC is comparable to that of 1-ADC, the in vitro activity of 3-ADC is about twice that of 2-ADC, and the in vitro activity of 4-ADC is about twice that of 3-ADC.

[0835] Test Example 2: In vivo efficacy of the dendritic polymer conjugate of this disclosure

[0836] 1) Sample to be tested

[0837] 1-ADC (control), 2-ADC, 3-ADC, 4-ADC.

[0838] 2) Testing Methods

[0839] The JIMT-1 cell line used in this experiment (purchased from ATCC) was cultured in DMEM medium (with 10% FBS) at 37°C in an incubator containing 5% CO2. Before ten consecutive passages of cell culture, approximately 5 × 10⁶ cells were cultured... 6 One JIMT-1 cell was suspended in 100 μL of PBS and mixed with an equal volume of Matrigel (Corning Cellgro, catalog number: 354234). The mixture was then subcutaneously injected into the right back near the axilla of mice (CB17 SCID strain, from Shanghai Jihui Laboratory Animal Breeding Co., Ltd.), with an injection volume of approximately 200 μL. When the tumor grew to an average size of approximately 100-150 mm³, mice were randomly assigned to groups of 7 mice each using E-WorkBook software. Mouse weight was measured and recorded twice weekly during the treatment period. Twenty-one days after treatment, tumor volume was recorded, and the tumor inhibition ratio (TGI) was calculated.

[0840] TGI = (1-T / C) × 100%, where T and C are the relative tumor volumes (RTV) of the experimental group and the negative control group (administered with normal saline) at a specific time point, respectively.

[0841] 3) Results

[0842] The in vivo efficacy of 2-ADC and 1-ADC is basically equivalent (TGI is about 60%), and the efficacy of 3-ADC and 4-ADC is basically equivalent (tumor near regression, TGI is about 90%).

[0843] Test Example 3: In vitro cell-killing activity of the dendritic polymer conjugate disclosed herein

[0844] 1) Sample to be tested

[0845] 1-ADC (control), 2-ADC, 3-ADC, 4-ADC.

[0846] 2) Testing Methods

[0847] NCI-N87 (purchased from ATCC) cell suspension was prepared and seeded into 96-well plates. The cells were incubated overnight at 37°C. The analyte was added at a maximum concentration of 500 nM, serially diluted 5-fold, with two parallel test wells for each condition. Cells were incubated at 37°C for 144 h. Cell viability was assessed using the CTG (CellTiter-Glo) assay, and the IC50 (intrinsic value) of the analyte on the cell lines was calculated. 50 .

[0848] 3) Results

[0849] The disclosed dendritic polymer conjugate exhibits cytotoxic activity (IC50) against NCI-N87 cells. 50 The values ​​are shown in Table 2 below.

[0850] Table 2

[0851] As shown in Table 2 above, the in vitro activity of 2-ADC is 3 to 4 times that of 1-ADC, and the activity of 4-ADC and 3-ADC is twice that of 2-ADC.

[0852] Test Example 4: Rat PK of the Dendritic Polymer Conjugate of the Presently Disclosed

[0853] 1) Sample to be tested

[0854] 2-ADC, 3-ADC, 4-ADC

[0855] 2) Testing Methods

[0856] Three groups of SD rats (SBF / Sberger) were established, with three animals (single sex) in each group. 2-ADC, 3-ADC, and 4-ADC were administered intravenously over 10 minutes. Blood samples were collected from the animals before administration and at 0.5h, 6h, 24h, 72h, and 168h after administration. Serum was separated, and the concentrations of total antibodies (total Her2) and ADC in the serum were measured using ELISA. Pharmacokinetic parameters were calculated.

[0857] 3) Results

[0858] The pharmacokinetic parameters of the dendritic polymer conjugates disclosed herein in rats are shown in Table 3 below.

[0859] Table 3

[0860] Note: Total resistance refers to total Her2.

[0861] As shown in the table above, the half-life of 2-ADCs is shorter than that of 3-ADCs and 4-ADCs.

[0862] Test Example 5: In vitro stability of the dendritic polymer conjugates disclosed herein

[0863] 1) Sample to be tested

[0864] 2-ADC, 3-ADC, 4-ADC

[0865] 2) Testing Methods

[0866] Each ADC molecule was concentrated into a PBS pH=7.2 buffer system to a final concentration of approximately 15 mg / ml. The solutions were incubated at 4°C and 25°C for 14 days. Size exclusion chromatography (SEC) was used to detect the purity of the ADCs in the solution at 0, 7, and 14 days of incubation.

[0867] 3) Results

[0868] Table 4

[0869] As shown in Table 4 above, the in vitro stability of 3-ADC and 4-ADC is basically the same at 4℃ and 25℃, and both are better than 2-ADC.

[0870] Test Example 6: In vitro cell-killing activity of the dendritic polymer conjugates disclosed herein

[0871] 1) Analytical molecule

[0872] 3-ADC, 9-ADC.

[0873] 2) Testing Methods

[0874] Step 1) Preparation of cell suspension

[0875] Cell suspensions of NCI-H2170, HCC1954, SK-OV-3, and OE19 (purchased from ATCC) were prepared and seeded into 384-well plates, 40 μL per well, with cell numbers of 900 (NCI-H2170), 500 (HCC1954), 80 (SK-OV-3), and 900 (OE19) per well, respectively. The cells were incubated overnight at 37°C.

[0876] Step 2) IC50 of the analyte 50

[0877] The target molecule was added to the OE19 cell line at a maximum concentration of 500 nM, serially diluted 3-fold. Cells were cultured at 37°C for 144 h, and cell viability was assessed using the CTG (CellTiter-Glo) assay. The IC50 (intrinsic value) of the target molecule on the cell line was calculated. 50 .

[0878] The target molecule was added to NCI-H2170, HCC1954, and SK-OV-3 cell lines, with a maximum concentration of 100 nM and serial dilutions of 3-fold. Cells were cultured at 37°C for 144 h, and cell viability was assessed using the CTG (CellTiter-Glo) assay. The IC50 (intrinsic value) of the target molecule on the cell lines was calculated. 50 .

[0879] 3) Experimental Results

[0880] The dendritic polymer conjugate disclosed herein exhibits cytotoxic activity (IC50) against NCI-H2170, HCC1954, SK-OV-3, and OE19 cells. 50 The values ​​are shown in Table 5 below.

[0881] Table 5

[0882] As shown in the table above, the in vitro killing activity of 3-ADC in the four cell lines was superior to that of 9-ADC.

[0883] Test Example 7: Human Plasma Stability of the Dendritic Polymers of the Present Disclosure

[0884] 1. Analytical molecule

[0885] Compound 20, Compound 21.

[0886] 2. Testing Methods

[0887] The analyte (dissolved in physiological saline) at a final concentration of 100 μg / ml was mixed with human plasma (anticoagulated with sodium heparin, sourced from Weifang High-tech Industrial Development Zone People's Hospital) to prepare drug-containing plasma samples. Three aliquots of the resulting sample were immediately precipitated, centrifuged, and the supernatant was collected for immediate analysis or stored at -20℃ for later testing to determine the initial concentration (0h). The remaining drug-containing plasma samples were incubated at 37℃ for the corresponding times (0.5h, 6h, 24h, 72h). At each time point, three aliquots were taken from each plasma sample tube, immediately precipitated, centrifuged, and the supernatant was collected for immediate analysis or stored at -20℃ for later testing. The concentration of free toxin (i.e., free docetaxel) in the samples was determined using LC-MS / MS.

[0888] 3. Results

[0889] As shown in Table 6 below, the AUC of the free toxin of compound 20 was significantly lower than that of compound 21. The concentrations of free docetaxel in plasma at each time point are shown in Figure 1. The plasma stability of compound 20 was significantly better than that of compound 21.

[0890] Table 6

[0891] Test Example 8: Hematologic toxicity of the dendritic polymers disclosed herein in rats

[0892] 1. Analytical molecule

[0893] Compound 20, Compound 21.

[0894] 2. Testing Methods

[0895] Two groups of SD rats (Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) were used (each group corresponded to one test molecule), with three animals (single sex) in each group. Each animal was administered 10 mg / kg of the test molecule (dissolved and diluted in physiological saline) via intravenous infusion (10 min), with an administration volume of 20 ml / kg. Approximately 0.3 mL of blood was collected from the retroocular venous plexus or a suitable site before administration, 24 h after administration, and 72 h after administration (accurate measurement). The blood samples were placed in EDTA-K2 tubes, thoroughly mixed immediately (inverted at least 10 times), and stored at 2-8℃. The samples were sent for testing on the same day for hematological parameters (including absolute white blood cell count, absolute neutrophil count, absolute monocyte count, and absolute lymphocyte count).

[0896] 3. Results

[0897] Figure 2 shows the white blood cell count values ​​for compounds 20 and 21. This figure indicates that compound 20 exhibits lower hematological toxicity than compound 21 in rats. The other three hematological parameters also show a similar trend towards superiority.

[0898] Test Example 9: In vivo pharmacokinetic properties of the dendritic polymer disclosed herein in rats

[0899] Compound 20 was observed to exhibit superior hematologic toxicity, which is typically C in vivo. max Driven toxicity. To investigate whether better plasma stability leads to lower free toxin C in pharmacokinetic behavior. max This results in lower hematological toxicity. The pharmacokinetic behavior of the dendritic polymer molecules in rats was further tested.

[0900] 1. Sample to be tested

[0901] Compound 20, Compound 21.

[0902] 2. Testing Methods

[0903] Two groups of SD rats (Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) were used (each group corresponding to one test molecule), with three animals (single sex) in each group. Each animal was administered 10 mg / kg of the test molecule (dissolved and diluted in physiological saline) via intravenous infusion (10 min), with an administration volume of 20 ml / kg. At 10 min, 20 min, 40 min, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, and 72 h post-administration, approximately 0.15 mL of blood was collected from the retroocular venous plexus or a suitable site and placed in EDTA-K2 tubes. The plasma (at least 65 μL) was separated within 1 hour at 11000 rpm for 5 min (4℃) and stored frozen at -70℃. Blood collection and plasma separation were performed under ice bath conditions. The concentration of free toxin (i.e., free docetaxel) in each group was determined by LC-MS / MS.

[0904] 3. Results

[0905] Compound 20: Free PK toxin C in rats max The concentration was 907 ng / ml, significantly lower than the 1250 ng / ml of compound 21.

Claims

1. A dendritic polymer conjugate or a pharmaceutically acceptable salt thereof, comprising: i) A dendritic polymer D having at least one surface amino group and at least one surface hydroxyl group or surface thiol group; ii) A first end group, which is a residue A of a pharmaceutically active agent, its derivative or precursor containing a carboxyl, hydroxyl, amino or thiol group, optionally connected to the surface amino group of the dendritic polymer D via a linker portion; iii) A second end group, which is a pharmacokinetic modifier M, optionally connected to the surface hydroxyl or surface thiol group of the dendritic polymer D via a linker portion; iv) The third end group, which is the targeting portion Ab, is optionally connected to the core of the dendritic polymer D via the linker portion; The targeted portion Ab contains a targeting reagent.

2. The dendritic polymer conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the dendritic polymer D comprises: i) The core has a carbonyl carbon atom for connection with the target part Ab, and at least two amino nitrogen atoms for connection with lysine or lysine analog building units. ii) Subsurface layer, which contains at least two generations of building units selected from lysine or lysine analogues; iii) A surface layer comprising a structure selected from formula Ia or formula Ib. in: X is independently selected from O and S; R 1 R 2 Each is independently selected from hydrogen, halogen, hydroxyl, mercapto, carboxyl, amino, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, the C 1-6 Alkyl, C 1-6 Alkyl groups are optionally selected from halogen, hydroxyl, mercapto, carboxyl, amino, cyano, C 1-6 Alkyl, C 1-6 The alkoxy group is replaced by one or more substituents. T 1 Each is an independent connection end to the first end base; T 2 Each is an independent connection end to the second end base; T 3 Each is an independent connection end that is linked to the building unit via an amide bond; r is selected from 1, 2, 3, and 4; s is selected from 0, 1, 2, 3, 4; t is selected from 0, 1, 2, 3, 4.

3. The dendritic polymer conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the dendritic polymer D comprises: i) The core has a carbonyl carbon atom for connection with the target part Ab, and at least two amino nitrogen atoms for connection with lysine or lysine analog building units. ii) Subsurface layer, which contains first-generation building blocks selected from lysine or lysine analogues; iii) A surface layer comprising a structure selected from formula Ia or formula Ib. in: X, R 1 R 2 T 1 T 2 T 3 r, s, and t are defined as in claim 2.

4. The dendritic polymer conjugate or its pharmaceutically acceptable salt according to claim 2 or 3, wherein the subsurface layer comprises lysine building units having a structure of 5. The dendritic polymer conjugate according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein the core is selected from... in, a8 and b8 are each independently selected from 0, 1, 2, 3, 4, and 5; Z 1 Each is an independently selected interval unit; T 4 Each is an independent connection point to the target portion Ab; T 5 Each is an independent connection end that is connected to the building unit via an amide bond.

6. The dendritic polymer conjugate of claim 5 or its pharmaceutically acceptable salt, wherein the Z... 1 -L P’ -M P -, M P It is an extension unit covalently linked to the carbonyl group on the core, L P’ It is to connect the target portion Ab to M P The divalent linker portion, and its corresponding monovalent portion L P Contains a functional group W capable of connecting to the target portion Ab. P ; The W mentioned P Selected from: in, R A R C Each is independently selected from hydrogen, halogen, hydroxyl, mercapto, nitro, carboxyl, amino, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, the C 1-6 Alkyl, C 1-6 Alkyl groups are optionally selected from halogens, hydroxyl groups, mercapto groups, nitro groups, carboxyl groups, amino groups, cyano groups, and C6 groups. 1-6 Alkyl, C 1-6 It is substituted by one or more substituents of alkoxy, 3 to 10-membered cycloalkyl groups; R B Each group is independently selected from halogen, hydroxyl, mercapto, azide, hydrazine, carboxyl, amino, and cyano groups; L 1 Selected from alkylene or heteroalkylene, wherein the alkylene or heteroalkylene is optionally interrupted by one or more groups selected from cycloalkylene, heterocycloalkylene, arylene, and heteroarylene, and wherein the alkylene or heteroalkylene is optionally selected from hydroxyl, C 1-6 Alkyl, 3- to 10-membered cycloalkyl, C 1-6 Alkoxy, halogen, nitro, cyano, oxo, thio, mercapto, sulfinyl, sulfonyl, -NR D R E The C is substituted by one or more substituents of aryl, heteroaryl, and heterocyclic groups. 1-6 Alkyl, C 1-6 Alkyl groups are optionally selected from halogens, hydroxyl groups, mercapto groups, nitro groups, carboxyl groups, amino groups, cyano groups, and C6 groups. 1-6 Alkyl, C 1-6 It is substituted by one or more substituents of alkoxy, 3 to 10-membered cycloalkyl groups; Each of the numbers 'a' is independently selected from 1, 2, 3, 4, and 5. b are each independently selected from 1, 2, 3, and 4; c are each independently selected from 1 and 2; d are each independently selected from 1, 2, and 3; e is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13; f are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; g is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; R D R E Whether the two are the same or different, each is independently selected from hydrogen, hydroxyl, and C. 1-6 Alkyl, cycloalkyl, C 1-6 Alkoxy, the C 1-6 Alkyl, C 1-6 Alkoxy and cycloalkyl groups are optionally selected from halogen, hydroxyl, mercapto, nitro, carboxyl, amino, cyano, C 1-6 Alkyl, C 1-6 It is substituted by one or more substituents of alkoxy or 3- to 10-membered cycloalkyl groups.

7. The dendritic polymer conjugate of claim 6 or its pharmaceutically acceptable salt, wherein the M... P Selected from alkylene or heteroalkylene, wherein the alkylene or heteroalkylene is optionally interrupted by one or more groups selected from cycloalkylene, heterocycloalkylene, arylene, and heteroarylene, and wherein the alkylene or heteroalkylene is optionally selected from hydroxyl, C 1-6 Alkyl, 3- to 10-membered cycloalkyl, C 1-6 Alkoxy, halogen, nitro, cyano, oxo, thio, mercapto, sulfinyl, sulfonyl, -NR D R E The C is substituted by one or more substituents of aryl, heteroaryl, and heterocyclic groups. 1-6 Alkyl, C 1-6 Alkyl groups are optionally selected from halogens, hydroxyl groups, mercapto groups, nitro groups, carboxyl groups, amino groups, cyano groups, and C6 groups. 1-6 Alkyl, C 1-6 It is substituted by one or more substituents of alkoxy, 3 to 10-membered cycloalkyl groups; Where R D R E As defined in claim 6.

8. The dendritic polymer coupling according to any one of claims 5 to 7, or a pharmaceutically acceptable salt thereof, wherein the Z... 1 As shown in Equation II-1 in: * indicates the connection end with the target portion Ab; j is selected from integers from 1 to 50; k is selected from integers from 1 to 50; m is selected from integers from 1 to 50; h is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; i is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; l is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; n is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

9. The dendritic polymer coupling according to any one of claims 1 to 8, wherein the dendritic polymer coupling is selected from the dendritic polymer coupling of formula VIII or the pharmaceutically acceptable salt thereof. in, Ab represents the target region; A is a residue A of a pharmaceutically active agent, its derivative or precursor containing a carboxyl, hydroxyl, amino or thiol group; L A For optional connectors; Z 3 These are optional cleavable groups; y1 is selected from integers from 1 to 100; y2 is selected from 2, 4, 8, 16, and 32; Each q is independently 1 to 5 (including 1, 2, 3, 4, 5 or any value between any two values); h, i, j, k, l, m, n are as defined in claim 8.

10. The dendritic polymer coupling according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, wherein the first end group is connected by a linker L A The L is covalently linked to the surface amino groups of the dendritic polymer D. A For -Y 1 -L 2 -Y 2 -,in: Y 1 Selected from -C(O)- or -O-, optionally via a cleavable group Z 3 Linked to residue A of a pharmaceutically active agent, its derivative or its precursor; Y 2 It is -C(O)-, and is linked to the dendritic polymer D via an amide bond; L 2 Selected from alkylene or heteroalkylene groups, wherein the alkylene or heteroalkylene group is optionally interrupted by one or more groups selected from cycloalkylene, heterocycloalkylene, aryl, and heteroaryl, and wherein the alkylene or heteroalkylene group is optionally selected from 3- to 10-membered cycloalkyl, C 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, halogen, nitro, cyano, acyl, amino, mercapto, sulfinyl, sulfonyl, -NR D R E The 3- to 10-membered cycloalkyl group is substituted by one or more substituents of aryl, heteroaryl, and heterocyclic groups. 1-6 Alkyl, C 1-6 Alkyl groups are optionally selected from hydroxyl, halogen, nitro, cyano, C 1-6 Alkyl, C 1-6 The alkoxy group is substituted by one or more substituents; R D R E As defined in claim 6.

11. The dendritic polymer conjugate of claim 10 or a pharmaceutically acceptable salt thereof, wherein L A Selected from the following structure: in: y3 is selected from integers from 1 to 50; u is independently selected from 1, 2, 3, 4, 5, and 6; v are each independently selected from 1, 2, 3, 4, 5, and 6; w are each independently selected from 1, 2, 3, 4, 5, 6.

12. The dendritic polymer coupling according to any one of claims 9 to 11, or a pharmaceutically acceptable salt thereof, wherein the cleavable group Z... 3 It contains a cleavable peptide portion.

13. The dendritic polymer coupling according to any one of claims 9 to 12, or a pharmaceutically acceptable salt thereof, wherein the cleavable group Z... 3 It can be cleaved by an enzyme, preferably by a cathepsin, and the cathepsin is preferably cathepsin B.

14. The dendritic polymer coupling according to any one of claims 9 to 13, or a pharmaceutically acceptable salt thereof, wherein the cleavable group Z 3 The amino acid unit comprises 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, aspartic acid, homolysine, and n-methylvaline. The peptide residues composed of amino acids, where x is an integer from 1 to 6, preferably valine-citrulline (Val-Cit), valine-alanine (Val-Ala), alanine-phenylalanine (Ala-Phe), phenylalanine-lysine (Phe-Lys), phenylalanine-homolysine (Phe-Homolys), n-methyl-valine-citrulline (Me-Val-Cit), alanine-alanine (Ala-Ala), glycine-glutamic acid (Gly-Glu), glutamic acid-alanine-alanine (Glu-Ala-Ala), glycine-lysine (Gly-Lys), glycine-valine-citrulline (Gly-Val-Cit), glycine-glycine-glycine (Gly-Gly-Gly), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly) and The preferred choices are valine-citrulline (Val-Cit), valine-alanine (Val-Ala), n-methyl-valine-citrulline (Me-Val-Cit), and glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

15. The dendritic polymer coupling according to any one of claims 9 to 14, or a pharmaceutically acceptable salt thereof, wherein the cleavable group Z 3 It contains a cleavable sulfonamide moiety.

16. The dendritic polymer coupling according to any one of claims 9 to 15, or a pharmaceutically acceptable salt thereof, wherein the cleavable group Z 3 It contains a crackable disulfide portion.

17. The dendritic polymer coupling according to any one of claims 9 to 16, or a pharmaceutically acceptable salt thereof, wherein the cleavable group Z 3 It can cleave under reducing conditions.

18. The dendritic polymer coupling according to any one of claims 9 to 17, or a pharmaceutically acceptable salt thereof, wherein the cleavable group Z 3 Selected from: in, T 6 Each is an independent linker to residue A of the pharmaceutically active agent, its derivative or its precursor.

19. The dendritic polymer conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 18, wherein residue A of the pharmaceutically active agent, its derivative or precursor thereof is selected from cytotoxic compounds, preferably eribulin, trabectedin, MMAE, MMAF, alkylating agents, platinum compounds, antibiotics (e.g., mitomycin, bleomycin), tyrosine kinase inhibitors, protein degrading agents (e.g., protein degradation-targeting chimeras (PROTAC) or molecular glues), KRAS inhibitors (e.g., KRAS G12C inhibitors, KRAS G12D inhibitors, pan-KRAS inhibitors), anthracyclines (e.g., doxorubicin, PNU159682), taxanes (e.g., paclitaxel, docetaxel, cabazitaxel), vinblastines (e.g., vincristine, vinblastine, vinorelbine), camptothecin analogs (e.g., SN38, 9106-IM-2), and derivatives of the above-mentioned drugs and their pharmaceutically acceptable salts.

20. The dendritic polymer conjugate or its pharmaceutically acceptable salt according to any one of claims 1 to 19, wherein residue A of the pharmaceutically active agent, its derivative or its precursor is selected from formula III-1, formula III-2, formula III-3 or formula III-4. in: Z 2 Each independently does not exist or is T 7 -C(R a R b )N(R c )-; R a R b Each is independently selected from hydrogen, halogen, hydroxyl, mercapto, carboxyl, -NR D R E , cyano, C 1-6 Alkyl, C 1-6 Alkoxy, the C 1-6 Alkyl, C 1-6 Alkyl groups are optionally selected from halogens, hydroxyl groups, mercapto groups, carboxyl groups, and -NR groups. D R E , cyano, C 1-6 Alkyl, C 1-6 The alkoxy group is substituted by one or more substituents; R c Selected from hydrogen, C 1-6 Alkyl, C 1-6 Alkoxy, the C 1-6 Alkyl, C 1-6 Alkyl groups are optionally selected from halogen, hydroxyl, mercapto, carboxyl, amino, cyano, C 1-6 Alkyl, C 1-6 The alkoxy group is substituted by one or more substituents; T 7 For connection to the oxygen atom in Formula III-1, Formula III-2 or Formula III-4; R D R E As defined in claim 6.

21. The dendritic polymer conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 20, wherein the pharmacokinetic modifier M is selected from polyethylene glycol, polyethyloxazoline, polyvinylpyrrolidone, polypropylene glycol, folate, or folate derivatives relating to ligands of cell surface receptors, preferably polyethylene glycol.

22. The dendritic polymer coupling of claim 21 or a pharmaceutically acceptable salt thereof, wherein the polyethylene glycol has a molecular weight in the range of 220 to 5500 Da, preferably 1000-5500 Da, more preferably 1000-2500 Da, and most preferably 1000-2300 Da.

23. The dendritic polymer conjugate or its pharmaceutically acceptable salt according to any one of claims 1 to 22, wherein the conjugate is selected from: in, Each q is independently 1 to 5 (including 1, 2, 3, 4, 5 or any value between any two values), and the target portion Ab is as defined in claim 1.

24. The dendritic polymer conjugate or its pharmaceutically acceptable salt according to any one of claims 1 to 23, wherein the targeting portion Ab is selected from polypeptides, proteins, antibodies, or antigen-binding fragments, preferably antibodies or antigen-binding fragments, most preferably monoclonal antibodies, and more preferably adalimumab, alisimumab, atezolizumab, emecizumab, abavirin, oftatumumab, oxametuzumab, oxalitumumab, omalizumab, and oxotuzumab. Oxtuzumab, Baliximab, Bevacizumab, Belimumab, Brolimumab, Broshumab, Daratuzumab, Datoximab β, Detrastuzumab, Denosumab, Iodine [131I] Metuximab, Duvarium, Duprenumab, Trastuzumab Emmy, Envorimab, Golimumab, Gosatuzumab, Gussageumab, Candunimarab, Camrelizumab, Anti-human T-cell CD3 mouse monoclonal antibody, Anti- Human interleukin-8 mouse monoclonal antibody, lanazalimumab, ramoximumab, ranibizumab, rituximab, romisivir, mepolizumab, moglizumab, nalcetuzumab, nivolumab, nimotuzumab, pembrolizumab, pertuzumab, penprilisimumab, pesolizumab, pesolizumab, ptetalizumab, trastuzumab, reperatuzumab, saturolizumab, cepalimumab, sugemalumab, secukinumab Sestoximab, Slulimab, Toripalimab, Tislelizumab, Tocilizumab, Vebrutuximab, Vedollizumab, Vedicetumab, Vepotetumab, Ustenomab, Cetuximab, Sintilimab, Inellizumab, Inetetumab, Ipilimumab, Idacilizumab, Ikucilizumab, Ivolomab, Imalimumab, Ikkizumab, Infliximab, Recombinant anti-CD25 humanized monoclonal antibody.

25. A dendritic polymer as shown in Formula X, or a pharmaceutically acceptable salt thereof. in, R 3 Selected from hydrogen, C 1-6 Alkyl-L C -C(O), aryl-L C -C(O), heteroaryl-L C -C(O), wherein the alkyl, aryl, or heteroaryl group is optionally selected from halogen, hydroxyl, mercapto, carboxyl, -NR D R E , cyano, C 1-6 Alkyl, C 1-6 It is substituted by one or more substituents of alkoxy or phenyl groups; L C The alkylene or heteroalkylene group is absent or selected from the group consisting of one or more groups selected from cycloalkylene, heterocycloalkylene, arylene, and heteroarylene, and the alkylene or heteroalkylene group is optionally selected from hydroxyl, C 1-6 Alkyl, 3- to 10-membered cycloalkyl, C 1-6 Alkoxy, halogen, nitro, cyano, acyl, amino, mercapto, sulfinyl, sulfonyl, -NR D R E The 3- to 10-membered cycloalkyl group is substituted by one or more substituents of aryl, heteroaryl, and heterocyclic groups. 1-6 Alkyl, C 1- The 6-alkoxy group is optionally selected from halogen, hydroxyl, mercapto, nitro, carboxyl, amino, cyano, C 1-6 Alkyl, C 1-6 It is substituted by one or more substituents of alkoxy, 3 to 10-membered cycloalkyl groups; x1 is selected from 0 or 1; y1 is selected from integers from 1 to 100; y2 is selected from 2, 4, 8, 16, and 32; A as defined in claim 1, R D R E As defined in claim 6, L A Z 3 As defined in claim 9.

26. The dendritic polymer or pharmaceutically acceptable salt thereof according to claim 25, wherein it is a dendritic polymer or pharmaceutically acceptable salt thereof as shown in Formula XI. in, Z 4 Selected from O, N, S or C(R) a R b ); y1 is selected from integers from 1 to 100; y2 is selected from 2, 4, 8, 16, and 32; A is as defined in claim 1, k, l, m, n are as defined in claim 8, and Z is... 3 As defined in claim 9, v and w as defined in claim 11, R a R b As defined in claim 20.

27. The dendritic polymer or its pharmaceutically acceptable salt according to claim 25, wherein it is the dendritic polymer or its pharmaceutically acceptable salt represented by formula XII. in, Z 4 Selected from O, N, S or C(R) a R b ); y1 is selected from integers from 1 to 100; y2 is selected from 2, 4, 8, 16, and 32; A as defined in claim 1, Z 3 As defined in claim 9, v and w as defined in claim 11, R a R b As defined in claim 20, x1 is as defined in claim 25.

28. The dendritic polymer coupling according to any one of claims 1 to 24 or a pharmaceutically acceptable salt thereof, or an isotope-substituted derivative of the dendritic polymer or a pharmaceutically acceptable salt thereof according to any one of claims 25 to 27, preferably said isotope-substituted derivative being a deuterated derivative.

29. A pharmaceutical composition comprising a dendritic polymer conjugate according to any one of claims 1 to 24 or a pharmaceutically acceptable salt thereof, or a dendritic polymer according to any one of claims 25 to 27 or an isotopic substitute according to claim 28, and a pharmaceutically acceptable excipient.

30. Use of the dendritic polymer conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 24, or the dendritic polymer or pharmaceutically acceptable salt thereof according to any one of claims 25 to 27, or the isotope substitute according to claim 28, or the pharmaceutical composition according to claim 29, in the preparation of a medicament for treating cancer.

31. The use according to claim 30, wherein the cancer is selected from lung cancer (e.g., non-small cell lung cancer, non-squamous non-small cell lung cancer), gastric cancer, adenocarcinoma of the stomach or gastroesophageal junction, esophageal squamous cell carcinoma, colorectal cancer, esophageal cancer, non-epithelial malignant pleural mesothelioma, melanoma, nasopharyngeal carcinoma, urothelial carcinoma, Hodgkin's lymphoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, breast cancer, and ovarian cancer.

32. A method of treating cancer, comprising administering to a patient in need a therapeutically effective amount of the dendritic polymer conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 24, or the dendritic polymer or a pharmaceutically acceptable salt thereof according to any one of claims 25 to 27, or the isotope substitute according to claim 28, or the pharmaceutical composition according to claim 29.

33. The method of claim 32, wherein the cancer is selected from lung cancer (e.g., non-small cell lung cancer, non-squamous non-small cell lung cancer), gastric cancer, adenocarcinoma of the stomach or gastroesophageal junction, esophageal squamous cell carcinoma, colorectal cancer, esophageal cancer, non-epithelial malignant pleural mesothelioma, melanoma, nasopharyngeal carcinoma, urothelial carcinoma, Hodgkin's lymphoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, breast cancer, and ovarian cancer.

34. A dendritic polymer as shown in Formula XIV, or a pharmaceutically acceptable salt thereof. in, R 3’ Selected from hydrogen, C 1-6 Alkyl-L C -C(O), aryl-L C -C(O), heteroaryl-L C -C(O), N3-L C -C(O), wherein the alkyl, aryl, or heteroaryl group is optionally selected from halogen, hydroxyl, mercapto, carboxyl, -NR D R E , cyano, C 1-6 Alkyl, C 1-6 It is substituted by one or more substituents of alkoxy or phenyl groups; L C The alkylene or heteroalkylene group is absent or selected from the group consisting of one or more groups selected from cycloalkylene, heterocycloalkylene, arylene, and heteroarylene, and the alkylene or heteroalkylene group is optionally selected from hydroxyl, C 1-6 Alkyl, 3- to 10-membered cycloalkyl, C 1-6 Alkoxy, halogen, nitro, cyano, oxo, thio, mercapto, sulfinyl, sulfonyl, -NR D R E The alkyl or alkoxy group is substituted with one or more substituents of aryl, heteroaryl, and heterocyclic groups, wherein the alkyl or alkoxy group is optionally selected from halogen, hydroxyl, mercapto, nitro, carboxyl, amino, cyano, C 1-6 Alkyl, C 1-6 It is substituted by one or more substituents of alkoxy, 3 to 10-membered cycloalkyl groups; R M R N Each is independently selected from either a hydrogen or amino protecting group; y1 is selected from integers from 1 to 100; y2 is selected from 2, 4, 8, 16, and 32; R D R E As defined in claim 6.

35. The dendritic polymer or its pharmaceutically acceptable salt according to claim 34, wherein it is a dendritic polymer or its pharmaceutically acceptable salt shown in formula XIV-1, XIV-2, or XIV-3. in, y1 is selected from integers from 1 to 100; y2 is selected from 2, 4, 8, 16, and 32; k, l, m, n as defined in claim 8, R M R N As defined in claim 34.

36. The dendritic polymer or its pharmaceutically acceptable salt according to claim 34 or 35, wherein the dendritic polymer or its pharmaceutically acceptable salt represented by formula XIV is a dendritic polymer or its pharmaceutically acceptable salt represented by formula XV-1-A, XV-1-B, XIV-2-A, XIV-2-B, XIV-3-A, or XIV-3-B. in, y1 is selected from integers from 1 to 100; y2 is selected from 2, 4, 8, 16, and 32; R N As defined in claim 34.

37. Use of the dendritic polymer or pharmaceutically acceptable salt thereof according to any one of claims 34 to 36 in the preparation of antibody-drug conjugates.

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