Dual-toxin antibody-drug conjugate and pharmaceutical use thereof

Abstract

A dual-toxin antibody-drug conjugate and the pharmaceutical use thereof. The dual-toxin antibody-drug conjugate containing an eribulin compound and a camptothecin compound has a good tumor inhibitory activity and a synergistic tumor inhibitory effect between toxins.

Description

A dual-toxin antibody-drug conjugate and its pharmaceutical uses

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. CN202411961922.9, filed on December 27, 2024, entitled “A Bitoxic Antibody-Drug Conjugate and Its Pharmaceutical Use Thereof,” the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This application relates to the field of pharmaceutical technology, specifically to a bitoxic antibody-drug conjugate and its pharmaceutical uses. Background Technology

[0004] Antibody-drug conjugates (ADCs) combine the selectivity of antibodies with the cytotoxic effects of bioactive molecules. As a concrete manifestation of the "magic bullet" concept, they have experienced rapid development in recent years, providing new treatment options for cancer patients and prolonging their lives. Currently marketed ADC drugs all conjugate a single bioactive molecule; however, during the use of these ADC drugs, it has been observed that some patients show low or no response at the start of treatment, and some patients no longer show significant treatment response after a period of use.

[0005] Altered responses of tumor cells to the bioactive molecules in ADC drugs are a significant cause of ADC drug resistance. For example, upregulation of ABC-binding box drug transporters increases the efflux of the microtubule inhibitor MMAE, leading to insufficient effective concentrations of the active molecules of MMAE-conjugated ADC drugs within tumor cells, thus reducing therapeutic efficacy. Furthermore, changes in cell signaling pathways, such as decreased levels of the tumor suppressor PTEN found in T-DM1-resistant cells, result in abnormal activation of the PI3K / AKT signaling pathway, making it impossible to effectively inhibit rapid tumor cell growth. Target mutations of bioactive molecules can also induce ADC drug resistance. Studies have found that with the use of DS8201, topoisomerase I underwent point mutations, altering the enzyme's binding affinity to DNA, preventing the binding of drug active molecules to the enzyme-DNA complex, and reducing cell-killing activity. Tumor heterogeneity is also a crucial reason for poor or unsustainable responses to antitumor drugs. Heterogeneity is a typical characteristic of tumors, representing changes at the molecular or genetic level resulting from tumor cell proliferation and development. Tumor cells within a single tumor tissue typically differ in gene expression, growth rate, and response to treatment, making it impossible for a single molecule to effectively kill all tumor cells in the tumor tissue.

[0006] The combined use of two or more molecules can produce a wider killing effect on heterogeneous tumor cells, and can also produce synergistic mechanisms to further kill drug-resistant tumor cells, thereby improving the efficacy of anti-tumor therapy or reducing toxic side effects. Robles et al. found that when the microtubule inhibitor eribulin and the topoisomerase I inhibitor irinotecan were used in combination, the microtubule dynamics inhibition induced by eribulin enhanced the nuclear TP53 aggregation induced by irinotecan, thereby achieving rapid killing of tumor cells and demonstrating a strong anti-tumor effect in multiple mouse tumor models. Therefore, conjugating two or more molecules with different mechanisms of action or different structures to the same antibody molecule may produce synergistic effects, enhance anti-tumor efficacy, and has the potential to solve the drug resistance problem of current single-active molecule ADC drugs, expand more indications, and benefit a wider range of cancer patients. Summary of the Invention

[0007] To achieve the inventive objective of this application, in a first aspect, this application provides a bitoxin antibody-drug conjugate, which is a compound of formula (I), or an isotopically labeled compound thereof, or an optical isomer, geometric isomer, tautomer, or mixture of isomers thereof, and a pharmaceutically acceptable salt thereof.

[0008] in:

[0009] D1 is a fragment of the camptothecin-like toxin molecule;

[0010] D2 is a fragment of an eribulin toxin;

[0011] Ab represents an antibody or antigen-binding fragment;

[0012] Q is the linker group that connects to Ab;

[0013] L5 is a direct bond or a linking group connecting Ab and Q;

[0014] L1 and L2 are each an independent linking group;

[0015] L3 is a direct bond or a linking group;

[0016] L4 is a direct bond or a linking group;

[0017] n is an integer or decimal between 0 and 20; such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 0 and decimals between the above integers; preferably n is an integer or decimal between 0 and 8.

[0018] In one set of implementations, L1 and L2 are each independently -L9-L8-L7-L6-, where L6 is connected to D1 or D2;

[0019] L6 is selected from direct bond, in For the connection site connected to D1 or D2, This is the connection site that connects to L7;

[0020] L7 is a short peptide chain (e.g., 2-10 amino acid residues, which can be 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues) formed by two or more amino acid residues, wherein the amino acid residues are selected from natural or non-natural amino acid residues, the N-terminus of the short peptide chain is connected to L8, the C-terminus is connected to L6, and L7 optionally includes one or more structures selected from formulas (II-1) to (II-9):

[0021] s is selected from 1, 2, 3, 4, 5, 6, 7, 8; t is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; R1 is selected from H or C. 1-8 Alkyl group; preferably, s is selected from 2, 3, 4, 5, 6, t is selected from 4, 5, 6, 7, 8, and R1 is selected from H or C. 1-6 alkyl;

[0022] L9 is selected from r1 and r6 are each independently selected from 0, 1, 2, 3, 4, 5, 6; r2, r3, r4, r5 are each independently selected from 1, 2, 3, 4, 5, 6. The connection sites represent those connected to L8; preferably, r1 and r6 are each independently selected from 0, 1, 2, 3, and 4, and r2, r3, r4, and r5 are each independently selected from 1, 2, 3, and 4.

[0023] L8 is selected from direct key, r7 and r12 are each independently selected from 0, 1, 2, 3, 4, 5, 6; r9 and r10 are each independently selected from 1, 2, 3, 4, 5, 6; r8 and r11 are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; and R2 is selected from H or C. 1-8 Alkyl, wherein The r7 and r12 are each independently selected from 0, 1, 2, 3, and 4; the r9 and r10 are each independently selected from 1, 2, 3, and 4; the r8 and r11 are each independently selected from 4, 5, 6, 7, and 8; and the r2 is selected from H or C. 1-6 alkyl;

[0024] L3 is -L 11-L 10 -, where L 11 Connect to L4;

[0025] L 10 Selected from direct keys, r13 and r18 are each independently selected from 0, 1, 2, 3, 4, 5, 6; r15 and r16 are each independently selected from 1, 2, 3, 4, 5, 6; r14 and r17 are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; and R3 is selected from H or C. 1-8 Alkyl, wherein Representative and L 11 The connection sites are as follows: preferably, r13 and r18 are each independently selected from 0, 1, 2, 3, and 4; r15 and r16 are each independently selected from 1, 2, 3, and 4; r14 and r17 are each independently selected from 4, 5, 6, 7, and 8; and R3 is selected from H or C. 1-6 alkyl;

[0026] L 11 Selected from direct bonds, and one or more structures from formulas (II-1) to (II-9);

[0027] L4 is selected from -CO- and -(CH2). p1 -CO-, -(CH2CH2O) p2 -(CH2) p3 -CO-, -(CH2) p4 -O-(CH2) p5 -C(O)-, -NR4-(CH2) p1 -CO-, -NR4-(CH2CH2O) p2 -(CH2) p3 -CO-, -NR4-(CH2) p4 -O-(CH2) p5 -CO-, -O-(CH2) p1 -CO-, -O-(CH2CH2O) p2 -(CH2) p3 -CO-, -O-(CH2) p4 -O-(CH2) p5 -CO-, p1, p2, p3, p4, p5 are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, and R4 is selected from H or C. 1-8 Alkyl groups, wherein -CO- and L 10Connection; preferably, p1 is selected from 1, 2, 3, 4, 5, 6; p2 is selected from 1, 2, 3, 4; p3 is selected from 1, 2, 3, 4; p4 is selected from 1, 2, 3, 4; p5 is selected from 1, 2, 3, 4; R4 is selected from H or C. 1-6 alkyl.

[0028] In one set of implementations, L6 is selected from direct bonds,

[0029] L7 is a short peptide chain composed of 2-8 amino acid residues;

[0030] L8 is selected from direct key, r7 is selected from 0, 1, 2, 3; r9 is independently selected from 1, 2, 3; r8 is selected from 6, 7, 8; and R2 is selected from H or C. 1-6 alkyl;

[0031] L9 is selected from r1 is selected from 0, 1, 2, 3, and r2 and r3 are each independently selected from 1, 2, 3;

[0032] L 10 Selected from direct keys, r13 is selected from 0, 1, 2, 3; r15 is selected from 1, 2, 3; r14 is selected from 6, 7, 8; and R3 is selected from H or C. 1-6 alkyl;

[0033] L 11 Selected from direct keys, t = 8;

[0034] L4 is selected from direct bonds, -CO-, and -(CH2). p1 -CO-, -(CH2CH2O) p2 -(CH2) p3 -CO-, -NR4-(CH2) p1 -CO-, -NR4-(CH2CH2O) p2 -(CH2) p3 -CO-, p1 is selected from 1, 2, 3, 4, p2 is selected from 1, 2, 3, p3 is selected from 1, 2, 3, and R4 is selected from H or C. 1-6 alkyl.

[0035] In one set of implementations, L8, L 10 One of them is a direct key, and the other is not a direct key.

[0036] In one embodiment, L7 is a short peptide chain formed by 2-8 amino acid residues; the amino acid residues are amino acid residues selected from phenylalanine, alanine, glycine, valine, citrulline, leucine, isoleucine, tryptophan, tyrosine, histidine, lysine, serine, threonine, cysteine, glutamic acid, glutamine, aspartic acid, asparagine, methionine, and arginine; preferably, the amino acid residues are amino acid residues selected from alanine, valine, and citrulline.

[0037] In one set of implementations, L7 is - NH Val-Ala CO -、- NH Val-Cit CO -

[0038] In one set of implementations, L8 is selected from direct keys,

[0039] In one set of implementations, L9 is selected from...

[0040] In one set of embodiments, L1 and L2 are each independently -(CH2CH2O)2-(CH2)2-CO- NH Val-Ala CO -L6-, L6 is the direct key. or -(CH2CH2O)2-(CH2)2-CO- NH Val-Cit CO -L6-, L6 is the direct key. or -(CH2CH2O)2-(CH2)2-CO-NH-(CH2CH2O)8-(CH2)2-CO- NH Val-Ala CO -L6-, L6 is the direct key. or -(CH2CH2O)2-(CH2)2-CO-NH-(CH2CH2O)8-(CH2)2-CO- NH Val-Cit CO -L6-, L6 is the direct key.

[0041] In one set of implementations, L 10 Selected from direct keys, In one set of implementations, L 11 Selected from direct keys, t = 8.

[0042] In one set of implementations, L3 is selected from direct bonds; -L11 -NH-(CH2CH2O)8-(CH2)2-CO-,L 11 for t = 8; This represents the connection site with L4.

[0043] In one set of embodiments, L4 is selected from -CO-, -(CH2)3-CO-, -(CH2CH2O)-(CH2)2-CO-, -(CH2CH2O)2-(CH2)2-CO-, -NH-(CH2CH2O)-(CH2)2-CO-, and -NH-(CH2CH2O)2-(CH2)2-CO-.

[0044] In one set of embodiments, Q is selected from groups comprising a maleimide linker, a group comprising a methanesulfonylpyrimidine linker, a group comprising a methanesulfonylpyridinylthiazole linker, a group comprising a methanesulfonylpyrimidinethiazole linker, a group comprising a cycloeneyne linker, or a group comprising an oxime linker.

[0045] In one set of implementations, Q is selected from: The connection site is for L5. This represents the connection site with L4.

[0046] In one set of implementations, Q-L4 is selected from...

[0047] In one set of implementations, Q-L4 is selected from... p1 is selected from 2, 3, and 4; p2 is selected from 1 and 2; p3 is selected from 1 and 2; and R4 is selected from H. Preferably, p1 is selected from 3; p2 is selected from 1 and 2; and p3 is selected from 2. More preferably, L5 is a direct bond.

[0048] In one set of implementations, L5 is a direct key or Represents the connection site that connects to Ab. Z3 represents the connection between Z3 and Q; Z3 is a click probe or thiol group or its precursor that can be connected to Q after a reaction, such as a metal-free click reaction, preferably Z3 is an azide group;

[0049] Z1 and Z2 are each independently selected from -C(O)NH-, -C(O)O-, -C(O)-, -OC(O)-, -NHC(O)-, -NH-, -O-, -OC(O)NH- or -NHC(O)O-;

[0050] q1 is 0, 1, 2, 3, 4, 5, 6, 7, 8;

[0051] q2 is 1, 2, 3, 4, 5, 6, 7, 8;

[0052] q3 is 1, 2, 3, 4, 5, 6, 7, 8;

[0053] q4 is 1, 2, 3, 4, 5, 6, 7, 8.

[0054] In one set of implementations, L5 is a direct key or Preferably, L5 is a direct bond or Preferably, Z1 and Z2 are each independently selected from -C(O)NH-.

[0055] In one set of implementations, D1 is selected from the following structures:

[0056] L 12 Selected from -O-*, -NR a -*、-(CH2) m1 -O-*、-(CH2) m1 -NR a -*、-OC(=O)NR b -(CH2) m1 -O-*、-OC(=O)NR b -(CH2) m1 -NR a -*、-(CH2) m1 -C(=O)O-*、-(CH2) m1 -C(=O)NR a -*、-NR b -(CH2) m1 -O-*、-NR b -(CH2) m1 -NR a -*、-O-(CH2) m1 -O-*、-O-(CH2) m1 -NR a -*、-NR b C(=O)O-(CH2) m1 -O-*、-NR b C(=O)O-(CH2) m1 -NR a -*、-(CH2) m1 -NR b C(=O)-(CH2) m2 -NR a -*、-(CH2) m1 -NR b C(=O)-(CH2) m2-O-*, where * is the connection site with L1;

[0057] R a Selected from hydrogen and C1-C8 alkyl groups;

[0058] R b Selected from hydrogen, C1-C8 alkyl, -C(O)R c -S(O)R c -S(O)2R c R c Selected from hydrogen, hydroxyl, and C1-C8 alkyl groups;

[0059] m1 can be 1, 2, 3, 4, 5, or 6;

[0060] m2 is 1, 2, 3, 4, 5 or 6;

[0061] R 5 Selected from hydrogen, halogen, hydroxyl, amino, cyano, C1-C8 alkyl, C1-C8 alkoxy, C2-C8 alkenyl, C2-C8 alkynyl, C3-C6 cycloalkyl, -NR d R e -(CH2) m1 -OH, -(CH2) m -NR d R e -OC(=O)NR f -(CH2) m1 -OH, -OC(=O)NR f -(CH2) m1 -NR d R e -(CH2) m1 -C(=O)OH, -(CH2) m1 -C(=O)-NR d R e -(CH2) m1 -C(=O)NR f -(CH2) m2 -OH, -(CH2) m1 -C(=O)NR f -(CH2) m2 -NR d R e -NR f -(CH2) m1 -OH, -NR f -(CH2) m1 -NR d R e -O-(CH2) m1 -OH, -O-(CH2) m1-NR d R e -NR f C(=O)O-(CH2) m1 -OH, -NR b C(=O)O-(CH2) m1 -NR d R e -(CH2) m1 -NR f C(=O)O-(CH2) m2 -OH-, -(CH2) m1 -NR f C(=O)O-(CH2) m2 -NR d R e -(CH2) m1 -OC(=O)NR f -(CH2) m2 -NR d R e -(CH2) m1 -OC(=O)NR f -(CH2) m2 -OH, -(CH2) m1 -NR f C(=O)-(CH2) m2 -NR d R e -(CH2) m1 -NR f C(=O)-(CH2) m2 -OH, wherein the C1-C8 alkyl, C1-C8 alkoxy, C2-C8 alkenyl, C2-C8 alkynyl, or C3-C6 cycloalkyl may optionally be further substituted with one or more groups selected from halogens, deuterium, amino, C1-C8 alkyl, or hydroxyl; the above-mentioned -(CH2) m1 -、-(CH2) m2 -Optionally replaced by one or more deuterium or halogens;

[0062] R 6 The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, C1-C8 alkyl, C1-C8 alkoxy, C2-C8 alkenyl, and C2-C8 alkynyl, wherein the C1-C8 alkyl, C1-C8 alkoxy, C2-C8 alkenyl, and C2-C8 alkynyl are optionally further substituted by one or more groups selected from halogen.

[0063] R d and R e Each is independently selected from hydrogen and C1-C8 alkyl groups;

[0064] Rf Selected from hydrogen, C1-C8 alkyl, -C(O)R c -S(O)R c -S(O)2R c R c Selected from hydrogen, hydroxyl, and C1-C8 alkyl.

[0065] Preferably, L6 in L1 is selected from This represents the connection site with D1.

[0066] In one set of implementations, L 12 Selected from -NR b C(=O)O-(CH2) m1 -O-*、-NR b C(=O)O-(CH2) m1 -NR a -*,R a Selected from hydrogen, C1-C6 alkyl, R b Selected from hydrogen, C1-C6 alkyl, where m1 is 1, 2, 3, 4, 5, or 6. Preferably, R a Selected from hydrogen, R b Selected from hydrogen, m1 is 1, 2, 3, 4, 5 or 6.

[0067] In one set of implementations, R 5 Selected from C 1-6 Alkyl, C 1-6 alkoxy- or hydroxy-substituted C 1-6 Alkyl, preferably, R 5 It is a methyl group.

[0068] In one set of implementations, R 6 Selected from halogens, preferably, R 6 For F and Cl.

[0069] In one set of implementations, L 12 Selected from -NR b C(=O)O-(CH2) m1 -O-*,R b Selected from hydrogen, m1 is an integer of 1, 2, 3, 4, 5, or 6; R 5 Selected from methyl; R 6 Selected from F.

[0070] In one set of implementations, D1 is selected from the following structures:

[0071] In one set of implementations, D2 is selected from the following structures:

[0072] *、** Each is independently a single bond, double bond, or oxygen bond (=O); preferably, the * and ** positions are... All are double bonds;

[0073] L 13 Groups selected from those that are attached to L2 after the removal of the H from -NH2, -OH, -NH- on the chain, or -NH- on the ring:

[0074] X1 is selected from -CO-, -CH2, -S(O)- or -S(O)2-;

[0075] X2 is selected from direct key, -O-, or -NR7-;

[0076] X3 is selected from -(CR8R9) m3 -NH2, (CR8R9) m3 -OH, -R 10 -(CR8R9) m4 -NH2, -R 10 -(CR8R9) m4 -OH or

[0077] Y1 is selected from -(CR8R9) m3 -NH2, -(CR8R9) m3 -OH, -R 10 -(CR8R9) m4 -NH2, -R 10 -(CR8R9) m4 -OH or

[0078] R7 is selected from H, alkyl, hydroxy-substituted alkyl, alkyloxy-substituted alkyl, amino-substituted alkyl, haloalkyl or cyano-substituted alkyl;

[0079] R8 and R9 are each independently selected from H, alkyl, hydroxy, alkyloxy, amino, halogen, cyano, hydroxy-substituted alkyl, alkyloxy-substituted alkyl, amino-substituted alkyl, haloalkyl, cyano-substituted alkyl, substituted or unsubstituted carbocyclic substituted alkyl, substituted or unsubstituted heterocyclic substituted alkyl, substituted or unsubstituted carbocyclic or substituted or unsubstituted heterocyclic; the substituted or unsubstituted carbocyclic or substituted or unsubstituted heterocyclic refers to one or more substituents selected from oxo, alkyl, hydroxy, alkyloxy, amino, halogen, cyano, hydroxy-substituted alkyl, alkyloxy-substituted alkyl, amino-substituted alkyl, haloalkyl, cyano-substituted alkyl;

[0080] R 10Selected from substituted or unsubstituted carbocyclic groups or substituted or unsubstituted heterocyclic groups; the substituted or unsubstituted carbocyclic group or substituted or unsubstituted heterocyclic group refers to one or more substituents selected from oxo, alkyl, hydroxy, alkyloxy, amino, halogen, cyano, hydroxy-substituted alkyl, alkyloxy-substituted alkyl, amino-substituted alkyl, haloalkyl, cyano-substituted alkyl.

[0081] The ring is a substituted or unsubstituted heterocyclic group, wherein the ring of the heterocyclic group contains at least one -NH-, and the substitution refers to being substituted by one or more substituents selected from oxo, alkyl, alkyloxy, halogen, cyano, alkyloxy-substituted alkyl, haloalkyl, cyano-substituted alkyl;

[0082] The ring is a heterocyclic group, the ring of which contains at least one -NH- and / or the heterocyclic group is substituted with an amino group, a hydroxyl group, an amino-substituted alkyl group, or a hydroxyl-substituted alkyl group, wherein the heterocyclic group is optionally substituted with one or more substituents selected from oxo, alkyl, alkyloxy, halogen, cyano, alkyloxy-substituted alkyl, haloalkyl, cyano-substituted alkyl.

[0083] m3 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0084] m4 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0085] Specifically, the alkyl group is C 1-12 Straight-chain or branched alkyl groups (e.g., with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 carbon atoms). The carbocyclic group can be saturated, partially unsaturated, or completely unsaturated, such as cycloalkyl, aryl, cycloalkenyl, cycloynyl, cycloalkenyl, and cycloalkenylyl groups, with 3-20 carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). The heterocyclic group can be a saturated, partially unsaturated, or completely unsaturated heterocyclic group, such as a heterocyclic alkyl group or a heteroaryl group; the number of atoms can be 3-20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20), and can contain 1, 2, 3, or 4 heteroatoms selected from N, O, and S.

[0086] In one set of implementations, L 13 When L2 is connected after removing the H from -NH2, -NH- on the chain, or -NH- on the ring, L6 in L2 is a direct bond; 13 When the H group after removing the -OH group is connected to L2, L6 in L2 is...

[0087] In one set of implementations, L 13 Groups selected from those whose L2 is attached after removing the H from -NH2 or -OH: X1 is selected from -CO-; X2 is selected from -O-; X3 is selected from -(CR8R9) m3 -NH2, -(CR8R9) m3 -OH; Y1 is selected from -(CR8R9) m3 -NH2, -(CR8R9) m3 -OH; R7 is selected from H.

[0088] In one set of implementations, m3 is selected from 1, 2, 3, 4, 5, and 6.

[0089] In one set of embodiments, R8 and R9 are each independently selected from H and C. 1-6 Alkyl, C 3-6 Cycloalkyl groups, 3-6 membered heterocyclic alkyl groups containing one or two heteroatoms N or O.

[0090] In one set of embodiments, X3 is selected from the following groups:

[0091] Preferably, X3 is selected from the following groups:

[0092] In one set of embodiments, Y1 is selected from the following groups:

[0093] In one set of implementations, The ring is a 3-12 membered monocyclic, fused, spirocyclic, or bridged heterocyclic alkyl group containing 1, 2, or 3 heteroatoms N or O, wherein the heterocyclic alkyl group contains at least one -NH- and / or the heterocyclic alkyl group is substituted with an amino, hydroxyl, or amino group. 1-6 alkyl or hydroxy substituted C 1-6 Alkyl substitution.

[0094] Preferably, The ring is a 3-6 membered monocyclic heterocyclic alkyl group containing one nitrogen atom, wherein the C atom of the heterocyclic alkyl group is replaced by an amino, hydroxyl, or amino group. 1-6 alkyl or hydroxy substituted C 1-6 Alkyl substitution.

[0095] More preferably, The ring is selected from the following groups: After removing the H from the terminal -OH group, it reacts with L6. Connection at L6

[0096] In one set of implementations, L 13 The following groups are selected from those that have had the terminal -NH2 or -OH removed before being attached to L2:

[0097] Preferably, L 13 The following groups are selected from those that have had the terminal -NH2 or -OH removed before being attached to L2:

[0098] In one set of embodiments, the Ab is selected from murine antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, or antigen-binding fragments thereof; preferably, the Ab is selected from anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-ROR1 antibody, anti-CLDN6 antibody, anti-CLDN9 antibody, anti-CLDN18.2 antibody, anti-NaPi-2b antibody, anti-TNF-α antibody, etc. α antibody, anti-ENPP3 antibody, anti-DLL3 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD28 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD38 antibody, anti-CD44 antibody, anti-CD45 antibody, anti-CD47 antibody, anti-CD48 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD98 antibody, anti-CD105 antibody, anti-CEA antibody, anti-EphA2 antibody, anti-MUCI antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-CD79 antibody, anti-TROP-2 antibody, anti-CD79B antibody, anti-Mesothelin antibody, anti-Nectin-4 antibody, anti-TPBG antibody, or their antigen-binding fragments.

[0099] Preferably, Ab is an anti-HER2 (ErbB2) antibody or its antigen-binding fragment.

[0100] Preferably, Ab is selected from monoclonal antibodies or their antigen-binding fragments, and more preferably from trastuzumab, cetuximab, pertuzumab, nimotuzumab, enoblituzumab, emibetuzumab, Inotuzumab, vitine-pinatuzumab, and brentuximab. (Brentuximab), gemtuzumab, bivatuzumab, morovatuzumab, mutants of the above-mentioned antibodies that can bind to antigens, or antigen-binding fragments thereof; more preferably, wherein the Ab is selected from trastuzumab, trastuzumab mutants that can bind to antigens (such as Trastuzumab-A141C, Trastuzumab-A141C-C220S), or antigen-binding fragments thereof.

[0101] In one set of embodiments, equation (I) is one of the following:

[0102] Preferably, Ab is an anti-HER2 (ErbB2) antibody or its antigen-binding fragment, more preferably trastuzumab, a trastuzumab mutant that can bind to the antigen (such as Trastuzumab-A141C, Trastuzumab-A141C-C220S), or its antigen-binding fragment.

[0103] In a second aspect, this application provides a bitoxic compound, which is a compound of formula (III), or an isotopically labeled compound thereof, or an optical isomer, geometric isomer, tautomer, or mixture of isomers thereof, and a pharmaceutically acceptable salt thereof.

[0104] in:

[0105] Q' is a linker group that can be linked to an antibody or antigen-binding fragment; the group formed after Q' is linked to Ab is the Q described in the first aspect;

[0106] D1, D2, L1, L2, L3, and L4 are as described in the first aspect.

[0107] In one set of embodiments, Q' is selected from groups containing maleimide connectors, groups containing methanesulfonylpyrimidine connectors, groups containing methanesulfonylpyridinylthiazole connectors, groups containing methanesulfonylpyrimidinethiazole connectors, groups containing cycloeneyne connectors, or groups containing oxime connectors.

[0108] In one set of implementations, Q' is selected from...

[0109] In one set of embodiments, Q'-L4 is selected from... p1 is selected from 1, 2, 3, 4, 5, 6; p2 is selected from 1, 2, 3, 4; p3 is selected from 1, 2, 3, 4; p4 is selected from 1, 2, 3, 4; p5 is selected from 1, 2, 3, 4; R4 is selected from H or C. 1-6 alkyl.

[0110] In one set of embodiments, Q'-L4 is selected from... p1 is selected from 2, 3, and 4; p2 is selected from 1 and 2; p3 is selected from 1 and 2; and R4 is selected from H. Preferably, p1 is selected from 3; p2 is selected from 1 and 2; and p3 is selected from 2.

[0111] Specifically, equation (III) is one of the following:

[0112] In a third aspect, this application provides a pharmaceutical composition comprising the bitoxin antibody-drug conjugate described in the first aspect, or the bitoxin compound described in the second aspect, and one or more pharmaceutically acceptable carriers or excipients.

[0113] In a fourth aspect, this application provides the use of the bitoxic compound described in the second aspect in the preparation of antibody-drug conjugates.

[0114] In a fifth aspect, this application provides the use of the bitoxin antibody-drug conjugate described in the first aspect, or the bitoxin compound described in the second aspect, or the pharmaceutical composition described in the third aspect in the preparation of a medicament for treating tumors or cancer.

[0115] In a sixth aspect, this application provides a method for treating tumors or cancer, comprising administering to a subject in need an effective amount of the bitoxin antibody-drug conjugate of the first aspect, or the bitoxin compound of the second aspect, or the pharmaceutical composition of the third aspect.

[0116] In one set of embodiments, the tumor or cancer is selected from solid tumors or hematologic malignancies; preferably, the tumor or cancer is selected from metastatic or recurrent tumors or cancers; preferably, the tumor or cancer is selected from breast cancer, gastric cancer, colon cancer, pancreatic cancer, kidney cancer, ovarian cancer, lung cancer, soft tissue sarcoma, liposarcoma, lung cancer, non-small cell lung cancer, melanoma, head and neck cancer, cervical cancer, and prostate cancer; specifically, it may be HER-2 positive breast cancer, triple-negative breast cancer, HER-2 positive triple-negative breast cancer, renal clear gland adenocarcinoma, small cell lung cancer, non-small cell lung cancer, and large cell lung cancer.

[0117] In one set of embodiments, the tumor or cancer is selected from cancers or tumors that highly express HER2 (ErbB2) antibodies.

[0118] The dual-toxin ADC containing eribulin-like compounds and camptothecin-like compounds provided in this application has good tumor-suppressive activity and synergistic tumor-suppressive effects between the toxins. Attached Figure Description

[0119] Figure 1 shows the in vitro proliferative inhibitory activity of the dual-toxin ADC of this application on tumor cells overexpressing the ABCG2 transporter.

[0120] Figure 2A shows the inhibitory activity of the dual-toxin ADC T2-LEC22CD34BVC2 on the proliferation of the JIMT-1 tumor-bearing mouse model.

[0121] Figure 2B shows the inhibitory activity of the dual-toxin ADC T2-LEC22CD36BVC2 on the proliferation of the JIMT-1 tumor-bearing mouse model.

[0122] Figure 3 shows the inhibitory activity of the dual-toxin ADC T2-LEC2236P on the proliferation of the JIMT-1 xenograft mouse model. Detailed Implementation

[0123] Unless otherwise stated, all terms used in disclosing this application (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, and the following definitions are provided for a better understanding of the teachings of this application. The explanations of the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting of this application.

[0124] Features described or illustrated as part of one set of embodiments in this application may be used in another set of embodiments to produce further embodiments.

[0125] The terms "and / or," "or / and," and "and / or" as used in this application encompass any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0126] The terms “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, and do not exclude additional, uncited members, elements, or method steps.

[0127] The range of values ​​represented by endpoints in this application includes all values ​​and fractions contained within that range, as well as the endpoints referenced.

[0128] In this application, concentration values ​​are defined as those within a certain range of fluctuation. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% may fluctuate within ±0.1%. For larger values ​​or values ​​that do not require extremely fine control, even greater fluctuations are permitted. For example, 100mM may fluctuate within ranges of ±1%, ±2%, ±5%, etc. Regarding molecular weight, fluctuations of ±10% are allowed.

[0129] In this application, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0130] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0131] In this application, terms such as "preferred," "better," "more suitable," "specific," and "preferably" are merely descriptions of more effective implementation methods or embodiments, and should be understood not to limit the scope of protection of this application. In this application, terms such as "optional," "optionally," "optional," and "optional" mean that something is optional, that is, selected from either "with" or "without" parallel solutions. If multiple "optional" options appear in a technical solution, unless otherwise specified and without contradiction or mutual constraint, each "optional" option is independent.

[0132] The eribulin-like compounds described in this application include eribulin and derivatives containing the eribulin core skeleton structure; the camptothecin-like compounds described in this application refer to compounds containing the camptothecin core skeleton. Eeribulin-like toxin molecular fragments include molecular fragments of eribulin linked to L1, and molecular fragments of derivatives containing the eribulin core skeleton structure with tumor-suppressive activity linked to L1. Camptothecin-like toxin molecular fragments refer to molecular fragments of compounds containing the camptothecin core skeleton structure with tumor-suppressive activity linked to L1.

[0133] The term "isotope-labeled compound" refers to an isotope-labeled compound obtained by replacing any one atom in a compound with its isotopic atom. All pharmaceutically acceptable isotope-labeled compounds described in this application include those in which one or more atoms are replaced by atoms having the same atomic number as atoms commonly found in nature but with different atomic masses or mass numbers. Examples of isotopes suitable for inclusion in the compounds of this application include isotopes of hydrogen, such as... 2 H(D) and 3 H(T), isotopes of carbon, such as 11 C 13 C and 14 C, isotopes of chlorine, such as 37 Cl, an isotope of fluorine, such as 18 F, an isotope of iodine, such as 123 I and 125 I, isotopes of nitrogen, such as 13 N and 15 N, an isotope of oxygen, such as 15 O、 17 O and 18 O, and isotopes of sulfur, such as 35 S. Isotope-labeled compounds can generally be prepared using conventional techniques known to those skilled in the art, or by using a suitable isotope-labeling reagent instead of the previously used unlabeled reagent, in a manner similar to that described in the examples and preparations appended herein.

[0134] The term "optical isomer" refers to the various isomers formed when a compound has one or more chiral centers, each of which can exist in either an R or S configuration. Optical isomers include all diastereomers, enantiomers, meso compounds, racemates, or mixtures thereof. For example, optical isomers can be separated by chiral chromatography or by chiral synthesis.

[0135] The term "geometric isomer" refers to the fact that when a compound contains a double bond, it can exist as a cis isomer, a trans isomer, an E-isomer, or a Z-isomer. Geometric isomers include cis isomers, trans isomers, E-isomers, Z-isomers, or mixtures thereof.

[0136] The term "tautomer" refers to an isomer that results from the rapid movement of an atom in a molecule to two different positions. Those skilled in the art will understand that tautomers can interconvert and may coexist in an equilibrium state under certain conditions.

[0137] The compounds of this application may exist in the form of pharmaceutically acceptable salts, such as acid addition salts and / or base addition salts of compounds of formula (I) and (III). Unless otherwise specified, "pharmaceutically acceptable salt" as used herein includes acid addition salts or base addition salts that may appear in the compounds of this application. Suitable acid addition salts are formed from acids that form non-toxic salts. Examples include, but are not limited to: acetates, adipates, aspartates, benzoates, benzenesulfonates, bicarbonates / carbonates, hydrogen sulfates / sulfates, borates, camphor sulfonates, citrates, cyclohexylamine sulfonates, ethanedisulfonates, formates, fumarates, gluconate, glucuronide, glucuronide, hexafluorophosphate, 2-(4-hydroxybenzyl)benzoate, hydrochlorides / chlorides, hydrobromides / bromines, hydroiodides / iodides, 2-hydroxyethanesulfonates, lactates, malates, maleates, malonates, methanesulfonates, sulfates, methyl sulfates, naphthalates, 2-naphthalenesulfonates, nicotinates, nitrates, orotates, oxalates, hexadecates, phosphates / hydrogen phosphates / dihydrogen phosphates, pyroglutamates, gluconate, stearates, salicylates, tannins, tartrates, toluenesulfonates, and trifluoroacetates. Suitable base addition salts are formed by bases that form non-toxic salts. Examples include, but are not limited to: aluminum, arginine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tromethamine, and zinc salts. They can also form hemisalts of acids and bases, such as hemisulfates and hemicalcium salts. For a review of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection and Use by Stahl and Wermuth (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds described in this application are known to those skilled in the art.

[0138] Some compounds of this application may exist in different crystal forms or amorphous forms, and all such compounds are included within the scope of this application, regardless of their form.

[0139] In this application, the term "antibody" refers to immunoglobulin, which is 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 immunoglobulin heavy chain 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, based on differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in the heavy chain, different subclasses can be further divided; 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 or λ chain. The sequence of approximately 110 amino acids near the N-terminus of the antibody heavy and light chains varies considerably, forming the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable, forming the constant region. The variable region comprises three hypervariable regions (HVRs) and four relatively conserved backbone regions (FRs). The three hypervariable regions determine the antibody's specificity and are also known as 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. The CDR amino acid residues in the LCVR and HCVR regions of the antibody or antigen-binding fragment described in this application conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3) in number and position, or conform to the Kabat and Chothia numbering rules (HCDR1).

[0140] 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 CH domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bridges on the chain region; (iii) F fragments consisting of VH and CH domains; (iv) Fv fragments consisting of VH and VL domains of a single arm of the antibody; (v) single-domain or dAb fragments 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 (called a single-chain Fv (scFv)) in which the VL and VH regions pair to form a monovalent molecule. Such single-chain antibodies are also included in the term "antigen-binding fragment." 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 technology or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be different types of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0141] The term "linker group" refers to a chemical structural fragment or bond that is linked to an antibody at one end and to a toxin drug at the other end. It can also be linked to other linker groups before being linked to an antibody or toxin drug.

[0142] The term "antibody drug conjugate" (ADC) refers to an antibody or antigen-binding fragment linked to a biologically active toxic drug via a stable linker unit.

[0143] The term "bitoxic antibody-drug conjugate" refers to an antibody or antigen-binding fragment linked to two different biologically active toxic drugs via a stable linker unit.

[0144] The term "short peptide," in its common sense within the art, refers to a compound covalently linked together by the condensation and dehydration of an amino acid molecule's carboxyl group with the amino group of another amino acid molecule, forming an amide bond (peptide bond). The naming conventions used to define peptides are commonly used in the art, where the N-terminal amino group appears on the left and the C-terminal carboxyl group appears on the right. The short peptide chain described in this application refers to a peptide chain obtained by the condensation and dehydration of two or more amino acids (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12), with the N-terminal amino group forming -NH- on the left and the C-terminal carboxyl group forming -CO- on the right. For example, "- NH Gly-Gly-Phe-Gly CO -” represents a tetrapeptide chain formed by the sequential condensation of Gly, Gly, Phe, and Gly. The left side is -NH- formed by the N-terminal Gly amino group, and the right side is -CO- formed by the C-terminal Gly carboxyl group.

[0145] The term "amino acid residue" has its common meaning in the art, referring to a group formed after an amino acid (as a single amino acid or as part of a peptide) reacts with a peptide, another amino acid, or another amino acid residue. Generally, when an amino acid combines with another amino acid or amino acid residue, water is removed, and the remaining amino acid is called an amino acid residue.

[0146] The term "amino acid" has its common meaning in the art and can include both natural and non-natural amino acids. The abbreviations for amino acid residues used in this invention are standard 3-letter and / or 1-letter codes used in the art to refer to one of 20 commonly used amino acids.

[0147] Amino acid residues or amino acids have isomer forms, including L- and D-forms.

[0148] The following table shows examples of the three-letter and single-letter codes for amino acids and their structures used in this application:

[0149] In this application, some of the substituents are... This indicates the connection point. Indicates the connection site. For example, L6 is... For the connection site connected to D1 or D2, This is the connection site with L7. For example, X3 is... hour, This indicates the connection site with X2.

[0150] Unless otherwise stated, "optional substitution" means that the hydrogen atom on the substituted group is not substituted or that one or more substituted sites of the substituted group are independently substituted by a substituent. The substituent is independently selected from one or more of the following: deuterium, oxo, halogen, amino, hydroxyl, nitro, cyano, mercapto, optionally substituted alkyl, optionally substituted alkyloxy, optionally substituted alkylthio, optionally substituted alkylamino, optionally substituted carbocyclic, optionally substituted heterocyclic; the substituent on the alkyl, carbocyclic, or heterocyclic group is selected from one or more of the following: oxo, deuterium, halogen, amino, hydroxyl, nitro, cyano, mercapto, alkyl, haloalkyl, alkyloxy, hydroxy-substituted alkyl, alkyloxyalkyl, etc. When the substituent is selected as "oxo," it means that two hydrogen atoms on the carbon atom at the same substitution position are replaced by an oxygen atom, or a heteroatom is bonded to oxygen, such as nitrogen oxides bonded to oxygen, or sulfinyl or sulfonyl groups bonded to oxygen. "Optional oxo" means that the hydrogen atoms on the substituted group are not substituted, or that two hydrogen atoms on the carbon at the same substitution position are replaced by oxygen atoms, or that a heteroatom is bonded to oxygen, such as nitrogen oxides bonded to oxygen, or sulfinyl or sulfonyl groups bonded to oxygen. The term "independently" means that when there are more than one substituent, these substituents can be the same or different.

[0151] The term "oxygen bond" (=O) refers to the situation where two hydrogen atoms on a carbon atom bonded to it are replaced by oxygen atoms to form a carbonyl group.

[0152] In this application This refers to the formation of N with other groups attached to it. ring; This means that X3 or Y1 is The ring contains at least one -NH-.

[0153] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms (the number of carbon atoms is between 1 and 12, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12), more preferably an alkyl group containing 1 to 10 carbon atoms, most preferably an alkyl group containing 1 to 8 carbon atoms (i.e. C1-8 alkyl, the number of carbon atoms is between 1 and 8, specifically 1, 2, 3, 4, 5, 6, 7 or 8), and an alkyl group containing 1 to 6 carbon atoms (i.e. C1-6 alkyl, the number of carbon atoms is between 1 and 6, specifically 1, 2, 3, 4, 5 or 6). Examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, n-heptyl, n-octyl, 3-hexyl, 2-pentyl, etc.

[0154] The term "alkyloxy" refers to -O-alkyl, and the definition of alkyl is the same as above. Examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 1-methylpropoxy, 2-methylpropoxy, tert-butoxy, etc.

[0155] The term "halogen" refers to F, Cl, Br, and I.

[0156] The terms “hydroxyl-substituted alkyl,” “alkyloxy-substituted alkyl,” “amino-substituted alkyl,” “halogenated alkyl,” “cyano-substituted alkyl,” “carbocyclic-substituted alkyl,” and “heterocyclic-substituted alkyl” refer to alkyl groups that are substituted with one or more hydroxyl groups, alkyl groups that are substituted with one or more alkyloxy groups, alkyl groups that are substituted with one or more amino groups, alkyl groups that are substituted with one or more halogens, alkyl groups that are substituted with one or more cyano groups, alkyl groups that are substituted with one or more carbocyclic groups, and alkyl groups that are substituted with one or more heterocyclic groups.

[0157] The term "carbocyclic group" refers to a saturated, partially unsaturated, or fully unsaturated carbocyclic group, such as cycloalkyl, aryl, cycloalkenyl, cycloynyl, and cycloenynyl. These can be monocyclic or polycyclic (including bicyclic, tricyclic, etc.), and include monocyclic, fused, spirocyclic, and bridged rings. The number of carbon atoms can range from 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20).

[0158] The term "cycloalkyl" refers to a saturated monocyclic or polycyclic (including bicyclic, tricyclic, etc.) cyclic hydrocarbon substituent, including monocyclic, fused, spirocyclic, and bridged rings. Cycloalkyl groups may contain 3 to 20 carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20), preferably 3 to 15 carbon atoms, and more preferably 3 to 12 carbon atoms. Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0159] The term "aryl" refers to a monocyclic or bicyclic aromatic carbocyclic system containing 6-10 carbon atoms. Examples of aryl groups include phenyl and naphthyl groups, such as 1-naphthyl, 2-naphthyl, 3-naphthyl, and 4-naphthyl.

[0160] The term "heterocyclic group" refers to a saturated, partially unsaturated, or fully unsaturated heterocyclic group, such as heterocyclic alkyl groups and heteroaryl groups. These can be monocyclic or polycyclic (including bicyclic, tricyclic, etc.), including monocyclic, fused, spirocyclic, and bridged rings. The number of atoms can range from 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20), and may contain 1, 2, 3, or 4 heteroatoms selected from N, O, and S, such as 1, 2, or 3 heteroatoms selected from N or O.

[0161] The term "heterocyclic alkyl" refers to a saturated monocyclic or polycyclic (including bicyclic, tricyclic, etc.) hydrocarbon substituent, including monocyclic, fused, combined, spirocyclic, and bridged rings, containing 3 to 20 ring atoms (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20), wherein one or more ring atoms (e.g., 1, 2, 3, 4) are heteroatoms selected from N, O, or S. Preferably, it contains 3 to 12 ring atoms (including 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 ring atoms), wherein 1 to 3 are heteroatoms (1, 2, or 3 heteroatoms), and the heteroatoms are selected from N, O, or S. Heterocyclic alkyl groups can be selected from 3-11 member monocyclic, fused, bicyclic, spirocyclic, and bridged heterocyclic alkyl groups with one or two nitrogen atoms, such as 3-6 member monocyclic heterocyclic alkyl groups, 6-12 member fused heterocyclic alkyl groups, 6-12 member bicyclic heterocyclic alkyl groups, 6-12 member spirocyclic heterocyclic alkyl groups, and 6-12 member bridged heterocyclic alkyl groups. Non-limiting examples include pyrrolidinyl, imidazoalkyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, azacyclopropane, and oxacyclopropane. Thionecyclopropane, azironecyclobutane, oxetanecyclobutane, thiohexane, morpholinyl, thiomorpholinyl, dioxane, dithiohexyl, oxazolyl, thiazolyl, pyrazolyl, imidazolinidine, tetrahydrofuran-1-yl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydropyran-1-yl, tetrahydropyran-2-yl, tetrahydropyran-3-yl, tetrahydropyran-4-yl, piperidin-1-yl, piperidin-4-yl wait.

[0162] The term "heteroaryl" refers to an aromatic monocyclic or polycyclic system containing a 5-10 member structure, or preferably a 5-8 member structure or a 6-10 member structure, more preferably a 5-6 member structure, wherein one, two, three, four or more ring atoms are heteroatoms and the remaining atoms are carbon atoms, the heteroatoms being independently selected from O, N or S, and the number of heteroatoms is preferably one, two, three or four. A heteroaryl group can be an aromatic monocyclic ring containing one or two 5-6 member structures selected from N, S, or O, or an aromatic fused ring containing one, two, three or four 8-10 member structures selected from N, S, or O.

[0163] The term "ring-linked ring" refers to two rings sharing two connected ring atoms.

[0164] The term "link" refers to two rings connected by a single bond.

[0165] The term "spiro ring" refers to two rings that share a single ring atom.

[0166] The term "bridged ring" refers to two rings that share two ring atoms that are not directly connected.

[0167] In this application, the ranges related to the number of substituents, carbon atoms, ring atoms, heteroatoms, and groups represent a list of all integers within that range, and the range is merely a simplified representation. For example, "1-4 substituents" means 1, 2, 3, or 4 substituents; "3-8 ring atoms" means 3, 4, 5, 6, 7, or 8 ring atoms. Therefore, the ranges related to the number of substituents, carbon atoms, ring atoms, heteroatoms, and groups also encompass any of their subranges, and each subrange is also considered to be disclosed in this application.

[0168] The term "pharmaceutical composition" refers to a mixture containing one or more compounds described in this application and one or more pharmaceutically acceptable carriers or excipients. The pharmaceutical composition may not contain other active ingredients, or may further contain one or more other active ingredients. The purpose of the pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredients and enabling them to exert their biological activity.

[0169] The terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" include, but are not limited to, any flow aids, sweeteners, diluents, preservatives, dyes / colorants, flavoring agents, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, emulsifiers, disintegrants, binders, fillers, antioxidants, fragrances, solubilizers, osmotic pressure regulators, and the base portion of semi-solid preparations such as ointments and creams.

[0170] The pharmaceutical compositions described in this application can be prepared into liquid, solid, or semi-solid dosage forms. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o / w, w / o, and double emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments, etc.; solid dosage forms can be tablets (including regular tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, pills, suppositories, films, patches, aerosols, and sprays, etc.; semi-solid dosage forms can be ointments, gels, pastes, etc. The pharmaceutical compositions of this application can be formulated into ordinary preparations, as well as sustained-release preparations, controlled-release preparations, targeted preparations, and various microparticle delivery systems.

[0171] The term "subject" refers to any human or non-human organism that may potentially benefit from treatment with the compounds described in this application. Exemplary subjects include humans or mammals of any age. Preferably, the subject is a human.

[0172] The term “treatment” includes treating a disease or symptom in mammals, particularly humans, and includes: (a) suppressing an infection, disease, or symptom, i.e., halting or delaying the development of an infection, disease, or symptom; (b) alleviating an infection, disease, or symptom, i.e. causing the remission of a disease or symptom; and / or (c) curing an infection, disease, or symptom.

[0173] The term "prevention" includes preventative therapy in mammals, particularly humans, aimed at reducing the likelihood of infection, disease, or symptoms occurring. Patients receiving preventative therapy may be selected based on an increased risk of infection or disease or symptoms compared to the general population. "Prevention" can include the management of subjects who have not yet presented with an infection or clinical condition, and the prevention of a second occurrence of the same or similar infection or clinical condition.

[0174] "Effective amount" refers to the amount of the compound of this application that, when administered alone or in combination, is effective in treating or preventing cancer, inhibiting cancer cell proliferation, and / or reducing tumor volume.

[0175] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art can make various modifications and changes to this application without departing from the scope or spirit of this application. For experimental methods in the following embodiments where specific conditions are not specified, the guidance given in this application should be used first, or experimental manuals or conventional conditions in the art can be followed, or other experimental methods known in the art can be referred to, or the conditions recommended by the manufacturer can be followed.

[0176] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0177] In addition, this application may use protecting groups to protect certain functional groups from unwanted reactions. Protecting groups suitable for various functional groups and their protection or deprotection conditions are well known to those skilled in the art. For example, TW Greene and GMWuts' "Protecting Groups in Organic Preparations" (3rd edition, Wiley, New York, 1999 and cited references in the book) describes in detail a large number of protecting or deprotecting groups.

[0178] The separation and purification of compounds and intermediates employ appropriate methods and procedures depending on specific needs, such as filtration, extraction, distillation, crystallization, column chromatography, preparative thin-layer chromatography, preparative high-performance liquid chromatography, or a combination of the above methods. Specific methods can be found in the examples described in this application. Of course, other similar separation and purification techniques can also be used. Conventional methods (including physical constants and spectroscopic data) can be used for characterization.

[0179] Preparation Example 1: Preparation of compound LA-25-b

[0180] Step 1: Preparation of compound ((9H-fluorene-9-yl)methyl((13S,16S)-2,2,3,3,13,17-hexamethyl-12,15-dioxo-4,9-dioxa-11,14-diaza-3-silicatadecane-16-yl)carbamate

[0181] Compound (5S,8S)-1-(9H-fluorene-9-yl)-5-isopropyl-8-methyl-3,6,9-trioxo-2-oxo-4,7,10-triazaundecane-11-yl acetate (commercially available from Xingzhi Biotechnology) (500 mg, 1.04 mmol, 1 eq) was dissolved in THF (10 mL), followed by the addition of compound 4-((tert-butyldimethylsilyl)oxy)but-1-ol (424.42 mg, 2.08 mmol, 2 eq) and TsOH (16.46 mg, 15.38 μL, 95.59 μmol, 1 eq). The reaction mixture was stirred at 30 °C for 4 hours. LC-MS showed the presence of the target product. The reaction mixture was used directly in the next step according to the theoretical yield.

[0182] LCMS(ESI): m / z, 648.4 [M+Na] + .

[0183] Step 2: Preparation of compound (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-(4-hydroxybutoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)carbamate

[0184] The compound ((9H-fluorene-9-yl)methyl((13S,16S)-2,2,3,3,13,17-hexamethyl-12,15-dioxo-4,9-dioxa-11,14-diaza-3-silicatadecane-16-yl)carbamate (500 mg, 798.88 μmol, 1 eq) was dissolved in THF (10 mL). HCl (29.13 mg, 199.72 μL, 798.88 μmol, 1 eq, 1,4-Dioxane solution, 4 M) was added to this solution, and the mixture was stirred at 15 °C for 3 hours. LC-MS showed the reaction was complete. Most of the solvent was removed by vortexing, and the residue was purified by high-performance liquid chromatography to give a white solid product LY-22CC-b (70 mg, yield 17.13%).

[0185] The high-performance liquid chromatography (HPLC) conditions are as follows:

[0186] The preparative chromatography system used was manufactured by Shimadzu, model LC-20AP. The column was XT-15*30-10. The mobile phase was water (0.225% HCOOH)-acetonitrile, with acetonitrile elution ratios ranging from 40% to 70%.

[0187] LCMS(ESI): m / z, 534.3 [M+Na] + .

[0188] Step 3: Preparation of compound (9H-fluorene-9-yl)methyl((11S,14S)-11,15-dimethyl-1-(4-nitrophenoxy)-1,10,13-trioxy-2,7-dioxy-9,12-diazahexadecane-14-yl)carbamate

[0189] The compound (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-(4-hydroxybutoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate (70 mg, 136.82 μmol, 1 eq) was dissolved in a solution of DMF (5 mL), and bis(4-nitrophenyl) carbonate (83.24 mg, 55.50 μL, 273.64 μmol, 2 eq) and DIPEA (53.05 mg, 67.84 μL, 410.46 μmol, 3 eq) were added. The reaction mixture was stirred at 15 °C for 12 hours. The reaction solution was concentrated to obtain most of the DMF, DCM (30 mL) was added, and the mixture was washed with water (30 mL). The organic phase was dried and filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: DCM:MeOH = 0:1 to 5:1). A yellow solid product LY-22CC-c (70 mg, yield 75.60%) was obtained.

[0190] LCMS(ESI): m / z, 699.3 [M+Na] + .

[0191] Step 4: Preparation of compound LA-25-a

[0192] At room temperature, LY-22CC-c (12 mg, 18 μmol, 1.5 eq), HOBT (0.8 mg, 6 μmol, 0.5 eq), and pyridine (5 mg, 60 μmol, 5 eq) were added to a solution of eribulin methanesulfonate (10 mg, 12.11 μmol, 1 eq) and DIPEA (2 μL, 12 μmol, 1 eq) in dimethylacetamide (2 mL). The reaction mixture was stirred at 25 °C for 12 hours. LC-MS showed that the reaction was complete. The mixture was prepared by high performance liquid chromatography (preparative chromatograph manufacturer: Oriendo, model BRIX-2860-R1; column: Phenomenex Luna C18 250×30mm, 10μm; mobile phase: water (0.225% HCOOH)-acetonitrile, elution ratio of acetonitrile from 50% to 80%) to give a white solid LA-25-a (15 mg, yield 97.75%).

[0193] Step 5: Preparation of compound LA-25-b

[0194] Compound LA-25-a (2.40 g, 4.65 mmol, 1 eq) was dissolved in DMF (2 mL), followed by the addition of DBU (15 mg, 12 μmol, 1 eq). The reaction was stirred at 25 °C for half an hour. LC-MS showed the reaction was complete. The reaction was quenched by adding 15 mg of TsOH to the reaction solution, and the reaction solution was then used directly for the next step (12.37 mg of crude product in 2 mL DMF).

[0195] Preparation Example 2: Preparation of compound LA-27-d

[0196] Step 1: Preparation of compound LA-27-a

[0197] At room temperature, 5-((tert-butyldimethylsilyl)oxy)pentan-1-ol (181.42 mg, 830.65 μmol, 2 eq) and TsOH (21.46 mg, 20.05 μL, 124.60 μmol, 0.3 eq) were added to a solution of compound LY-27a (commercially purchased from Xingzhi Biotechnology) in THF (8 mL). After addition, the reaction was carried out at 33 °C for 3 hours. LC-MS showed that the reaction was complete. The reaction was concentrated to obtain the crude product, which was then prepared by high-performance liquid chromatography (HPLC) (Shimadzu LC-20AP, Phenomenex Luna C18 column). 150×25mm, 10μm. Purified by mobile phase water (0.225% FA)-acetonitrile (elution ratio of acetonitrile from 30% to 60%), and lyophilized to give a yellow solid compound LA-27-a (102 mg, yield 46.72%).

[0198] LCMS(ESI): m / z, 548.4 [M+Na] + Rt = 2.009 min.

[0199] Step 2: Preparation of compound LA-27-b

[0200] At 0 °C, di(p-nitrobenzene) carbonate (118.06 mg, 388.09 μmol, 2 eq) and DIPEA (75.24 mg, 96.21 μL, 582.14 μmol, 3 eq) were added to a DMF (5 mL) solution of compound LA-27-a (102 mg, 194.05 μmol, 1 eq). After the addition was complete, the reaction solution was reacted at 33-35 °C for 3 hours. LCMS showed that the reaction was complete. The reaction solution was poured into 50 mL of ice water and extracted with ethyl acetate (40 mL × 3). The organic phases were combined and washed successively with water (60 mL × 2) and brine (50 mL). The organic phases were dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent: methanol / dichloromethane = 1:20 to 1:10) to obtain the yellow solid compound LA-27-b (95 mg, crude product yield 56.70%, purity 80%).

[0201] LCMS(ESI): m / z, 713.4 [M+Na] + Rt = 2.783 min.

[0202] Step 3: Preparation of compound LA-27-c

[0203] DIPEA (1.56 mg, 2 μL, 12.11 μmol, 1 eq) was added to a DMAC (2 mL) solution of eribulin methanesulfonate (10 mg, 12.11 μmol, 1 eq) at 0 °C, and the mixture was stirred at 25 °C for 10 minutes. Then, LA-27-b (10.45 mg, 80%, 12.11 μmol, 1 eq), HOBt (817.91 μg, 6.05 μmol, 0.5 eq) and pyridine (4.79 mg, 4.90 μL, 60.53 μmol, 5 eq) were added to the reaction solution. After adding the reagent, the reaction was carried out at 25°C for 16 hours. LCMS showed that the reaction was complete. The reaction solution was purified by high-performance liquid chromatography (preparative chromatograph manufacturer: Oriendo, model BRIX-2860; column: Welch Xtimate C18 250×30mm, 10μm; mobile phase: water (0.225% HCOOH)-acetonitrile; acetonitrile elution ratio: 50% to 80%). The purified solution was lyophilized to give a yellow solid compound LA-27-c (11 mg, yield 70.90%).

[0204] LCMS(ESI): m / z, 1303.8 [M+Na] + Rt = 3.103 min.

[0205] Step 4: Preparation of compound LA-27-d

[0206] DBU (3.92 mg, 3.85 μL, 25.75 μmol, 3 eq) was added to a DMF (2 mL) solution of compound LA-27-c (11 mg, 8.58 μmol, 1 eq) at 0 °C. After the addition was complete, the reaction solution was reacted at 25 °C for half an hour. LCMS showed that the reaction was complete. TsOH (4.43 mg, 25.75 μmol, 3 eq) was added to the reaction solution, and then the reaction solution was used directly for the next step.

[0207] LCMS(ESI): m / z, 1059.8 [M+H] + Rt = 1.700 min.

[0208] Preparation Example 3: Preparation of compound LA-28-d

[0209] Step 1: Preparation of compound LA-28-a

[0210] TsOH (21 mg, 124.6 μmol, 0.3 eq) was added to a THF (6 mL) solution of 6-((tert-butyldimethylsilyl)oxy)hexane-1-ol (193 mg, 830.65 μmol, 2 eq) and LY-27a (200 mg, 415.33 μmol, 1 eq), and the mixture was stirred at 33 °C for 3 h. LC-MS showed that the reaction was complete. HCl (4 M, 0.5 mL) was added to the mixture, and the solution was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (eluent: methanol / dichloromethane = 0:1 to 1:10) to give a white solid LA-28-a (277 mg, crude product yield not calculated).

[0211] Step 2: Preparation of compound LA-28-b

[0212] DIPEA (254.49 μL) was added to a DMF (6 mL) solution of compound LA-28-a (277 mg) and NPC (312.29 mg, 1.03 mmol, 2 eq), and the mixture was stirred overnight at room temperature. LCMS showed that the reaction was complete. The mixture was prepared by high performance liquid chromatography (preparative chromatograph manufacturer: Oriendo, model BRIX-2860; column: Welch Xtimate C18 250 × 30 mm, 10 μm; mobile phase: water (0.225% HCOOH)-acetonitrile, elution ratio of acetonitrile from 50% to 80%), to give a white solid LA-28-b (16.90 mg, yield 4.67%).

[0213] Step 3: Preparation of compound LA-28-c

[0214] DIPEA (1.56 mg, 12.11 μmol, 1 eq) was added to a 2 mL solution of eribulin methanesulfonate (10 mg, 12.11 μmol, 1 eq) in DMF at 0 °C. Then, LA-28-b (11 mg, 15.74 μmol, 1.3 eq), HOBT (818 μg, 6.05 μmol, 0.5 eq), and pyridine (5 mg, 60.53 μmol, 5 eq) were added to the mixture, and the mixture was stirred overnight at room temperature. LC-MS showed that the reaction was complete. The mixture was prepared by high performance liquid chromatography (preparative chromatograph manufacturer: Oriendo, model BRIX-2860; column: Welch Xtimate C18 250×30mm, 10μm; mobile phase: water (0.225% HCOOH)-acetonitrile; elution ratio of water from 30% to 60%) to give a white solid LA-28-c (14 mg, yield 89.26%).

[0215] Step 4: Preparation of compound LA-28-d

[0216] Compound LA-28-c (14 mg, 10.81 μmol, 1 eq) was dissolved in DMF (2 mL), and then DBU (4.94 mg, 32.42 μmol, 3 eq) was added. The reaction was stirred at room temperature for half an hour. LCMS showed that the reaction was complete. p-Toluenesulfonic acid (5 mg, 32.42 μmol, 3 eq) was added to the reaction solution to give product LA-28-d (11.60 mg, crude product in 2 mL DMF).

[0217] Preparation Example 4: Preparation of compound LA-29-f

[0218] Step 1: Preparation of compound LA-29-c

[0219] Compound LY-27a (250 mg, 519 μmol, 1 eq) was dissolved in THF (3 mL), cooled to 0 °C, and then TsOH (89.4 mg, 83.5 μL, 519 μmol, 1 eq) and (S)-butane-1,3-diol (140 mg, 1.56 mmol, 3 eq) were added. The reaction mixture was reacted at 25 °C for 3 hours. LC-MS showed that the reaction was complete. The reaction mixture was concentrated to dryness and then prepared by high performance liquid chromatography (preparative chromatograph manufacturer: Oriendo, model BRIX-2860; column: Phenomenex Luna C18 250 × 50 mm, 10 μm; mobile phase: water (0.225% HCOOH)-acetonitrile, elution ratio of acetonitrile from 30% to 60%), to give a white solid compound LA-29-c (100 mg, yield 37.65%).

[0220] LCMS(ESI): m / z, 534.3 [M+Na] + Rt = 1.971 min.

[0221] Step 2: Preparation of compound LA-29-d

[0222] To a DMF (5 mL) solution of compound LA-29-c (100 mg, 195.46 μmol, 1 eq), bis(4-nitrophenyl) carbonate (237.84 mg, 781.83 μmol, 4 eq) and DIPEA (151.57 mg, 193.82 μL, 1.17 mmol, 6 eq) were added, and the reaction was carried out at 33 °C for 1 h. LC-MS showed that the reaction was complete. The compound was purified by high-performance liquid chromatography (HPLC) (ISCO manufacturer, model ISCO; Welch Xtimate C18 250 × 30 mm, 10 μm column; mobile phase: water (0.225% HCOOH)-acetonitrile; acetonitrile elution ratio from 60% to 90%) to give a white solid compound LA-29-d (30 mg, yield 22.68%).

[0223] LCMS(ESI): m / z, 699 [M+Na] + .

[0224] Step 3: Preparation of compound LA-29-e

[0225] DIPEA (4.69 mg, 6 μL, 36.32 μmol, 3 eq) and HOBT (1.64 mg, 12.11 μmol, 1 eq) were added to a DMF (1 mL) solution of compound LA-29-d (9.01 mg, 13.32 μmol, 1.1 eq) and eribulin mesylate (10 mg, 12.11 μmol, 1 eq). After reacting at room temperature for 2 hours, the reaction was complete as shown by LC-MS. The product was purified by high-performance liquid chromatography (ISCO, ISCO model; Welch Xtimate C18 150 × 30 mm, 10 μm column; mobile phase: water (0.225% HCOOH)-acetonitrile; elution ratio: water from 50% to 80%) to give a white solid product LA-29-e (5.80 mg, yield 37.80%).

[0226] LCMS(ESI): m / z, 1289 [M+Na] + .

[0227] Step 4: Preparation of compound LA-29-f

[0228] DBU (2.09 mg, 2.05 μL, 13.73 μmol, 3 eq) was added to a DMF (1 mL) solution of compound LA-29-e (5.80 mg, 4.58 μmol, 1 eq). After reacting at room temperature for 40 minutes, LCMS showed that the starting material disappeared. TsOH (2.36 mg, 13.73 μmol, 3 eq) was added to give a yellow oily compound LA-29-f (4.78 mg, crude product yield not considered).

[0229] LCMS(ESI): m / z, 1067 [M+Na] + .

[0230] Preparation Example 5: Preparation of compound LA-31A-d

[0231] Step 1: Preparation of compound 2-(benzyloxy)-1-cyclopropyl-1-ethanol

[0232] Cyclopropylmagnesium bromide (47.95 mL, 23.97 mmol, 1.2 eq, 0.5 M) was added to a THF (25 mL) solution of benzyloxyacetaldehyde (3 g, 19.98 mmol, 1 eq) at room temperature and stirred at 70 °C for 2 hours. LC-MS showed that the starting material reacted completely. A saturated ammonium chloride solution (10 mL) and water (20 mL) were slowly added to the reaction solution under ice bath conditions. The aqueous phase was extracted three times with ethyl acetate (20 mL). The organic phases were combined, washed once with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1:5-1:3) to give a colorless oily 2-(benzyloxy)-1-cyclopropyl-1-ethanol (1.2 g, yield 31.22%).

[0233] LCMS(ESI): m / z, 216[M+Na] + Rt = 1.442. 1 H NMR(400MHz,Chloroform-d)δ7.39-7.28(m,5H),4.58(s,2H),3.64(dd,J=9.6,3.0Hz,1H),3.48(dd,J=9.6,8.2Hz,1H),3.12(td,J= 8.2,3.0Hz,1H),2.36(s,1H),0.87(qt,J=8.2,4.8Hz,1H),0.60-0.43(m,2H),0.38(dtd,J=9.2,5.2,4.2Hz,1H),0.25-0.18(m,1H).

[0234] Step 2: Preparation of compound 1-cyclopropylethane-1,2-diol

[0235] Palladium on carbon (60 mg) was added to a solution of 2-(benzyloxy)-1-cyclopropyl-1-ethanol (600 mg, 3.12 mmol, 1 eq) in MeOH (6 mL) and concentrated hydrochloric acid (56 μL), and hydrogen gas was purged three times. The mixture was stirred at room temperature for 2 hours, and TLC showed that the reaction proceeded completely. The palladium on carbon was removed by diatomaceous earth filtration, and the filter cake was washed with methanol (3 mL). The solution was concentrated to give a colorless oily 1-cyclopropylethane-1,2-diol (320 mg, 99% yield).

[0236] 1 H NMR(400MHz,Chloroform-d)δ3.77(d,J=10.8Hz,1H),3.61(dd,J=11.2,7.2Hz,1H),3.02(t,J=8.0H z,1H),1.01–0.84(m,1H),0.55(d,J=7.8Hz,2H),0.36(d,J=9.6Hz,1H),0.26(dd,J=9.6,4.6Hz,1H).

[0237] Step 3: Preparation of compound LA-31A-a

[0238] To a THF (20 mL) solution of 1-cyclopropylethane-1,2-diol (320 mg, 3.13 mmol, 1.5 eq) and LY-27a (1.01 g, 2.09 mmol, 1 eq), p-toluenesulfonic acid (359.70 mg, 2.09 mmol, 1 eq) was added, and the mixture was stirred at 30 °C for 2 hours. After concentration, the solution was dissolved in DMF and filtered. The solution was then prepared by high performance liquid chromatography (HPLC) (Oriendo BRIX-2860 chromatographic column, Welch Xtimate C18 150 × 30 mm, 10 μm; mobile phase: water (0.225% HCOOH)-acetonitrile, elution ratio of acetonitrile from 30% to 60%), yielding a white solid LA-31A-a (515.70 mg, yield 47.15%).

[0239] LCMS(ESI): m / z, 546 [M+Na] + Rt = 2.058 min.

[0240] Step 4: Preparation of compound LA-31A-b

[0241] DIPEA (1.10 g, 1.41 mL, 8.50 mmol, 8 eq) and NPC (1.94 g, 6.38 mmol, 6 eq) were added to an 8 mL DMF solution of compound LA-31A-a (556.50 mg, 1.06 mmol, 1 eq) and stirred at 35 °C for 2 hours. The solution was concentrated by an oil pump, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 0-50%, methanol / dichloromethane = 0-8%) to give LA-31A-b (161.40 mg, 50% purity) as a yellow oil.

[0242] LCMS(ESI): m / z, 711[M+Na] + Rt = 1.953 min.

[0243] Step 5: Preparation of compound LA-31A-c

[0244] DIPEA (6.26 mg, 48.43 μmol, 4 eq) and HOBT (1.64 mg, 12.11 μmol, 1 eq) were added to a DMF (1 mL) solution of compound LA-31A-b (20.01 mg, 50% purity, 14.53 μmol, 1.2 eq) and iribulin methanesulfonate (10 mg, 12.11 μmol, 1 eq). The mixture was stirred at room temperature for 1 hour, and LC-MS showed complete reaction of the starting material. The solution was prepared by high-performance liquid chromatography (Preparative chromatograph manufacturer: Oriendo, model BRIX-2860; column: Welch Xtimate C18 150 × 30 mm, 10 μm; mobile phase: water (0.225% HCOOH)-acetonitrile; acetonitrile elution ratio: 50-80%), yielding a white solid LA-31A-c (6.40 mg, yield 41.32%).

[0245] LCMS(ESI): m / z, 1301[M+Na] + Rt = 3.168 min.

[0246] Step 6: Preparation of compound LA-31A-d

[0247] DBU (2.28 mg, 15.01 μmol, 3 eq) was added to a 1 mL DMF solution of compound LA-31A-c (6.40 mg, 5 μmol, 1 eq) under ice bath conditions and stirred at room temperature for 1 hour. LC-MS showed that the starting material reacted completely. Then, p-toluenesulfonic acid (2.58 mg, 15.01 μmol, 3 eq) was added under ice bath conditions and directly added to the next step. The theoretical mass of LA-31A-d is 5.29 mg.

[0248] LCMS(ESI): m / z, 1057 [M+H] + , Rt=1.799min.

[0249] Preparation Example 6: Preparation of compound LA-27C-b

[0250] Step 1: Preparation of compound LA-27C-a

[0251] At 0°C, 10.60 mg (8.27 μmol, 1 eq) of compound LA-27-c (for the synthesis of this compound, please refer to the preparation example of LA-27-d) (3 mL) of DCM was added to a solution of LA-27-c (15.77 μL, 49.63 μmol, 6 eq) containing 21.48 mg (98%). After addition, the reaction was carried out at 25°C for 16 hours. LC-MS showed that the reaction was complete. The reaction solution was combined with another small batch of the reaction solution and poured into ice water / saturated sodium bicarbonate / saturated water. The mixture was stirred for 5 minutes in a mixed solution of sodium bicarbonate and sodium sulfite (1V / 1V / 1V, total volume 60mL), extracted three times with DCM, 40mL each time, and the organic phases were combined. The organic phases were then washed with a mixed solution of saturated sodium bicarbonate / saturated sodium sulfite (1V / 1V, total volume 30mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give a yellow solid compound LA-27C-a (14mg, crude product, 1.28 batch; another small-scale product of 0.28 scale was combined with this product).

[0252] LCMS(ESI): m / z, 1279.9 [M+H] + Rt = 3.257 min.

[0253] Step 2: Preparation of compound LA-27C-b

[0254] Diethylamine (4 mg, 54.71 μmol, 5 eq) was added to a DMF (1 mL) solution of compound LA-27C-a (14 mg, 10.94 μmol, 1 eq) at 0 °C. After the addition was complete, the reaction solution was reacted at 25 °C for 1 hour. LCMS showed that the reaction was complete. The reaction solution was directly evaporated to dryness to obtain a yellow solid compound LA-27C-b (12 mg, crude product).

[0255] LCMS(ESI): m / z, 1057.9 [M+H] + .

[0256] Preparation Example 7: Preparation of compound LA-27AA-2-e

[0257] Step 1: Preparation of compound LA-27AA-2-a

[0258] To a DMF (8 mL) solution of 5-amino-1-pentanol (55.29 mg, 535.98 μmol, 1.1 eq) and N-[fluorenylmethoxycarbonyl]-L-valine-L-alanine (200 mg, 487.26 μmol, 1 eq), DIEA (157.43 mg, 1.22 mmol, 2.5 eq) and HATU (222.33 mg, 584.71 μmol, 1.2 eq) was added and the mixture was stirred at room temperature for 2 hours. LC-MS showed that the reaction proceeded completely. The residue was purified by silica gel column chromatography (C18, ISCO, R-330 g SepaFlash Silica Flash Column, eluted with 10-50% water / CH3CN, 100 mL / min) to give a white solid LA-27AA-2-a (227.4 mg, yield 94.16%).

[0259] LCMS(ESI): m / z, 496 [M+H] + Rt = 2.049 min.

[0260] Step 2: Preparation of compound LA-27AA-2-e

[0261] DIEA (178 mg, 1.38 mmol, 3 eq) and NPC (279 mg, 0.92 mmol, 2 eq) were added to a DMF (10 mL) solution of compound LA-27AA-2-a (227 mg, 0.46 mmol, 1 eq) and stirred at 35 °C for 1.5 h. The solution was concentrated by an oil pump, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 0-50%, methanol / dichloromethane = 0-8%) to give a yellow oily LA-27AA-2-e (197 mg, yield 65%).

[0262] LCMS (ESI): m / z, 683.2 [M+Na] + .

[0263] Preparation Example 8: Preparation of compound LA-27AAVc-d

[0264] Step 1: Preparation of compound LA-27AAVC-a

[0265] DIEA (2.34 g, 18.125 mmol, 5 eq), 5-amino-1-pentanol (0.81 g, 7.854 mmol, 1.3 eq), and HATU (2.99 g, 7.854 mmol, 1.3 eq) were added to a solution of Fmoc-Val-Cit-OH (3 g, 6.042 mmol, 1 eq) in DMF (10 mL) at room temperature. The mixture was then stirred at 25 °C for 2 h. LCMS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was then slurried with ethyl acetate (30 mL) to give LA-27AAVC-a (1.1 g, yield 31.30%) as a yellow solid. LCMS (ESI): m / z, 582.2 [M+1] + Rt = 1.336 min.

[0266] Step 2: Preparation of compound LA-27AAVC-b

[0267] At 25 °C, DIEA (0.781 mL, 4.727 mmol, 2.5 eq) and di(p-nitrobenzene) carbonate (1.73 g, 5.673 mmol, 3 eq) were added to a DMF (15 mL) solution of compound LA-27AAVC-a (1.1 g, 1.891 mmol, 1 eq). The reaction mixture was stirred at 25 °C for 1 hour, and LC-MS showed that the starting material reacted completely. The reaction mixture was passed through a reverse-phase C18 reactor (54% H2O (0.025% TFA) / ACN) to give LA-27AAVC-b (770 mg, yield 54.52%) as a white solid. LC-MS (ESI): m / z, 746.6 [M]. + Rt = 1.598 min.

[0268] Step 3: Preparation of compound LA-27AAVC-c

[0269] At room temperature, DIEA (47 mg, 0.363 mmol, 3 eq), LA-27AAVC-b (108.50 mg, 0.145 mmol, 1.2 eq), and HOBt (16.36 mg, 0.121 mmol, 1 eq) were added to 3 mL of DMF containing eribulin methanesulfonate (100 mg, 0.121 mmol, 1 eq). The mixture was then stirred at 25 °C for 2 h. LCMS showed that the reaction was complete. The reaction solution was purified by high performance liquid chromatography (HPLC) using C18 (82% H2O (0.025% TFA) / ACN, 82%) to give LA-27AAVC-c (67 mg, yield 41.37%) as a white solid. LCMS (ESI): m / z, 1337.4 [M]. + Rt = 1.692 min.

[0270] Step 4: Preparation of compound LA-27AAVC-d

[0271] Diethylamine (0.022 mL, 0.217 mmol, 10 eq) was added to a DMF (1 mL) solution of compound LA-27AAVC-c (29 mg, 0.022 mmol, 1 eq) at 25 °C. The reaction solution was stirred at 25 °C for 1.5 hours, and LCMS showed that the starting material reacted completely. The reaction solution was concentrated under vacuum at low temperature to obtain crude LA-27AAVC-d (24 mg, crude product), which was a pale yellow oil. LCMS (ESI): m / z, 1114.1 [M-1] + , Rt=1.310min.

[0272] Preparation Example 9: Preparation of compound LA-34B-b

[0273] Step 1: Preparation of compound PE-AM1-b

[0274] Sodium periodate (11.76 mg, 99.5%, 3.04 μL, 54.73 μmol, 2 eq) was added to a tetrahydrofuran (1 mL) and water (0.5 mL) solution of compound PE-AM1-a (20 mg, 27.36 μmol, 1 eq, purchased from Haoyuan Pharmaceutical). The mixture was stirred at 25 °C for 4 hours. LC-MS showed that the starting material reacted completely. The reaction solution was extracted with water (15 mL) and ethyl acetate (10 mL × 3). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure to give a white solid compound PE-AM1-b (27 mg, crude product). LC-MS (ESI): m / z, 721.5 [M + Na] + .

[0275] Step 2: Preparation of compound PE-AM1-c

[0276] At 0 °C, a solution of sodium chlorite (18.44 mg, 80%, 7.38 μL, 163.13 μmol, 6 eq) and sodium dihydrogen phosphate (6.59 mg, 99%, 54.38 μmol, 2 eq) in water (1 mL) was added to a tert-butanol (1 mL) solution of compound PE-AM1-b (19 mg, 27.19 μmol, 1 eq) and 2-methyl-2-butene (21.29 mg, 90%, 32.10 μL, 271.88 μmol, 10 eq). After the addition was complete, the reaction mixture was stirred at 25 °C for 1 hour. LC-MS showed that the starting material reacted completely. The reaction mixture was then added to water (15 mL) and extracted with ethyl acetate (10 mL × 3). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain a white solid compound PE-AM1-c (24 mg, crude product).

[0277] LCMS(ESI): m / z, 715.4 [M+H] + 737.4 [M+Na] + .

[0278] Step 3: Preparation of compound LA-34A-a

[0279] To a THF (10 mL) solution of tert-butyl 3-hydroxymethylazetane-1-carboxylic acid (238.06 mg, 1.25 mmol, 2 eq) and LY-27a (300 mg, 622.99 μmol, 1 eq), p-toluenesulfonic acid (108.36 mg, 622.99 μmol, 1 eq) was added, and the mixture was reacted at 30 °C for 2 h. LC-MS showed that the reaction was complete. The residue was purified by silica gel column chromatography (C18, ISCO, R-330 g SepaFlash Silica Flash Column, elution of 10-60% H2O / CH3CN, 100 mL / min) under medium pressure to give a white solid compound LA-34A-a (252 mg, yield 66.4%).

[0280] LCMS(ESI): m / z, 631[M+Na] + Rt = 2.589 min.

[0281] Step 4: Preparation of compound LA-34A-b

[0282] TFA (3.0 mL, 41.40 mmol, 100 eq) was added to a DCM (12 mL) solution of compound LA-34A-a (252 mg, 413.97 μmol, 1 eq), and the reaction was carried out at 0 °C for 30 min. The reaction was completed by LCMS. After evaporation to dryness, the residue was purified by silica gel column chromatography (C18, ISCO, R-330 g SepaFlash Silica Flash Column, elution of 5-40% H2O / CH3CN, 100 mL / min) under medium pressure to give a white solid LA-34A-b (95.40 mg, yield 45.31%).

[0283] Step 5: Preparation of compound LA-34B-a

[0284] DIPEA (7.01 μL, 41.97 μmol, 3 eq) and HATU (6.38 mg, 16.79 μmol, 1.2 eq) were added to a DMF (2 mL) solution of compound LA-34A-b (8.54 mg, 16.79 μmol, 1.2 eq) and compound PE-AM1-c (10 mg, 13.99 μmol, 1 eq). The reaction was carried out at room temperature for 1 hour. The reaction was confirmed to be complete by LC-MS. After filtration, the solution was purified by high performance liquid chromatography (Preparative chromatograph manufacturer: Oriendo, model BRIX-2860. Column: Welch Xtimate C18 150 × 30 mm, 10 μm. Mobile phase: water (0.225% HCOOH)-acetonitrile, elution ratio of acetonitrile from 50% to 80%) to give a white solid compound LA-34B-a (9 mg, yield 53.37%).

[0285] LCMS(ESI): m / z, 1205 [M+H] + Rt = 2.983 min.

[0286] Step 6: Preparation of compound LA-34B-b

[0287] Et₂NH₃ (3.88 μL, 37.33 μmol, 5 eq) was added to a DMF (1 mL) solution of compound LA-34B-a (9 mg, 7.47 μmol, 1 eq). The reaction was carried out at room temperature for 1 hour. LC-MS showed that the reaction was complete. The solution was evaporated to dryness to obtain a white solid compound LA-34B-b (7.34 mg, crude product not considered in yield), which was used directly for the next step. LC-MS (ESI): m / z, 983 [M+H] + Rt = 1.677 min.

[0288] Preparation Example 10: Preparation of compound LA-34BVC-d

[0289] Step 1: Preparation of compound LA-34BVC-b

[0290] Compounds (5S,8S)-1-(9H-fluorene-9-yl)-5-isopropyl-3,6,9-trioxo-8-(3-ureopropyl)-2-oxo-4,7,10-triazaundecane-11-yl acetate (purchased from autogenous organisms, CAS: 3028612-25-8) (200 mg, 0.352 mmol, 1 eq) and 3-(hydroxymethyl)azacyclobutane-1-carboxylic acid 2-methylpropyl-2-yl ester (…) were also mentioned. TsOH (60.67 mg, 0.352 mmol, 1 eq) was added to a solution of DMF (5 mL) and THF (5 mL) containing 263.89 mg, 1.409 mmol, 4 eq, and the mixture was reacted at 20 °C for 18 hours. The reaction was completed by LCMS. The product LA-34BVC-b (150 mg, 61.27%) was obtained by evaporation and purification by reverse-phase C18 (40% aqueous solution of ACN).

[0291] LCMS(ESI): m / z, 695.3 [M+H] + Rt = 1.676 min.

[0292] Step 2: Preparation of compound LA-34BVC-c

[0293] At 0 °C, TFA (0.5 mL) was added to a DCM (2.5 mL) solution of compound LA-34BVC-b (150 mg, 0.054 mmol, 1 eq) and the mixture was reacted at 0 °C for 1 hour. LCMS showed that the reaction was complete. The mixture was then purified by reverse-phase C18 (40% ACN aqueous solution) to obtain LA-34BVC-c (102.71 mg, 80%).

[0294] LCMS(ESI): m / z, 594.7 [M+H] + Rt = 1.229 min.

[0295] Step 3: Preparation of compound LA-34BVC-d

[0296] At 0 °C, DIPEA (54.24 mg, 0.420 mmol, 3 eq) and HATU (63.83 mg, 0.168 mmol, 1.2 eq) were added to a DMF (5 mL) solution of compound LA-34AVC-c (91.59 mg, 0.154 mmol, 1.1 eq) and compound PE-AM1-c (100 mg, 0.140 mmol, 1 eq). After the addition was complete, the reaction was carried out at 20 °C for 1 hour. LCMS showed that the reaction was complete. The reaction solution was filtered and purified by reverse-phase C18 (mobile phase: acetonitrile and water; acetonitrile elution ratio from 50% to 80%). The solution was lyophilized to give a white solid compound LA-34BVC-d (144.54 mg, yield 80.03%).

[0297] LCMS(ESI): m / z, 1291.9 [M+H] + Rt = 1.657 min.

[0298] Preparation Example 11: Preparation of compound LA-35A-d

[0299] Step 1: Preparation of compound LA-35A-a

[0300] To a solution of compound LY-27a (300 mg, 622.99 μmol, 1 eq) and 5-(N-tert-butoxycarbonylamino)-1-pentanol (258.45 mg, 1.25 mmol, 2 eq) in tetrahydrofuran (10 mL), p-toluenesulfonic acid (108.36 mg, 622.99 μmol, 1 eq) was added. After the addition was complete, the reaction mixture was stirred at 30 °C for 1 hour. LC-MS showed that the starting material reacted completely. The reaction mixture was concentrated and purified by silica gel column chromatography (C18, ISCO, R-120 g SepaFlash Silica Flash Column, elution of 10-65% H2O (0.225% FA) / CH3CN, 60 mL / min). The purified compound was lyophilized to give a white solid compound LA-35A-a (222 mg, yield 57.04%, purity 97.76%).

[0301] LCMS(ESI): m / z, 647.3 [M+Na] + .

[0302] Step 2: Preparation of compound LA-35A-b

[0303] Trifluoroacetic acid (3.27 g, 99%, 2.13 mL, 28.43 mmol, 80 eq) was added to a dichloromethane (10 mL) solution of compound LA-35A-a (222 mg, 355.33 μmol, 1 eq) at 0 °C. After the addition was complete, the reaction solution was stirred at 0 °C for 1 hour. LCMS showed that the starting material reacted completely. The reaction solution was concentrated and purified by silica gel column chromatography (C18, ISCO, R-40 g SepaFlash Silica Flash Column, 5-40% H2O (0.225% FA) / CH3CN elution, 40 mL / min). The purified compound LA-35A-b (132 mg, yield 70.81%, purity 93.11%) was obtained by lyophilization.

[0304] LCMS(ESI): m / z, 525.3 [M+H] + 547.3 [M+Na] + .

[0305] Step 3: Preparation of compound LA-35A-c

[0306] DIPEA (13.70 mg, 99%, 17.51 ​​μL, 104.92 μmol, 3eq) and HATU (16.45 mg, 97%, 41.97 μmol, 1.2eq) were added to a DMF (0.5 mL) solution of compounds LA-35A-b (18.35 mg, 34.97 μmol, 1eq) and PE-AM1-c (25 mg, 34.97 μmol, 1eq). The mixture was stirred at 25 °C for 1 hour. LC-MS showed that the reaction was complete. The reaction solution was filtered and then prepared by high-performance liquid chromatography (HPLC) (Oriendo BRIX-2860 chromatographic column, Synergi Max-RP). 200×30mm, 10μm. Purification was carried out using water (0.225% HCOOH)-acetonitrile as the mobile phase (elution ratio of acetonitrile from 50% to 80%), followed by lyophilization to give a white solid compound LA-35A-c (27 mg, yield 63.20%, purity 90.13%).

[0307] LCMS(ESI): m / z, 1221.6 [M+H] + 1243.5 [M+Na] + .

[0308] Step 4: Preparation of compound LA-35A-d

[0309] Diethylamine (8.17 mg, 110.52 μmol, 5 eq) was added to a DMF (0.5 mL) solution of compound LA-35A-c (27 mg, 22.10 μmol, 1 eq), and the mixture was stirred at 25 °C for 1 hour. LCMS showed that the reaction was complete. After concentration, the reaction solution yielded a white oily compound LA-35A-d (22 mg, yield 99.60%).

[0310] LCMS(ESI): m / z, 999.7 [M+H] + 1021.8 [M+Na] + .

[0311] Preparation Example 12: Preparation of compound LA-36dPAB-b

[0312] Step 1: Preparation of compound PE-E2K-a

[0313] NaHCO3 (6.13 mg, 72.64 μmol, 2 eq) and Fmoc-Osu (13.75 mg, 39.95 μmol, 1.1 eq) were added to a THF (2 mL) solution of eribulin methanesulfonate (30 mg, 36.32 μmol, 1 eq) at 0 °C. The reaction mixture was stirred at 25 °C for 2 hours. LCMS showed that the reaction was complete. The residue of the mixture was purified by silica gel column chromatography (C18, ISCO, R-40 g SepaFlash Silica Flash Column, elution of 10-75% H2O / CH3CN, 100 mL / min) to give a colorless oily product PE-E2K-a (30 mg, yield 86.75%). LCMS (ESI): m / z, 952.6 [M+H] + .

[0314] Step 2: Preparation of compound PE-E2K-b

[0315] Potassium osmium tetroxide dihydrate (490.97 μg, 1.58 μmol, 0.1 eq) and NaIO4 (67.73 mg, 315.08 μmol, 20 eq) were added to a THF (3 mL) and water (1 mL) solution of compound PE-E2K-a (15 mg, 15.75 μmol, 1 eq) at 0 °C. The reaction solution was stirred at 25 °C for 2 hours. NaIO4 (67.73 mg, 315.08 μmol, 20 eq) was then added to the reaction solution, and the reaction solution was stirred overnight at room temperature. LC-MS showed that the reaction was complete. The mixture (and the previous batch, 5 mg, small-scale) was prepared by high-performance liquid chromatography (Preparative chromatograph manufacturer: Oriendo, model BRIX-2860. Column: Synergi Max-RP 200×30 mm, 10 μm. Mobile phase: water (0.1% TFA)-acetonitrile, elution ratio of acetonitrile from 40% to 70%) to give a white solid product PE-E2K-b (16.60 mg, yield 82.68%), LCMS (ESI): m / z, 956.4 [M+H). + .

[0316] Step 3: Preparation of compound PE-E2K

[0317] Et2NH (6.41 mg, 86.81 μmol, 5 eq) was added to a DMF (2 mL) solution of compound PE-E2K-b (16.6 mg, 17.36 μmol, 1 eq). The reaction mixture was stirred at 25 °C for 1 hour, and LC-MS showed that the starting material reacted completely. The mixture was then prepared by high-performance liquid chromatography (Preparative chromatograph manufacturer: Oriendo, model BRIX-2860-R6; column: Welch Xtimate C18 250 × 30 mm, 10 μm; mobile phase: water (0.1% TFA)-acetonitrile, elution ratio of acetonitrile from 10% to 40%) to give a white solid PE-E2K (7.50 mg, yield 58.86%).

[0318] LCMS(ESI): m / z, 734.3 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ7.67(s,2H),5.23(s,1H),4.70–4.53(m,2H),4.46–4.28(m,2H),4.19–4.05(m,3H),3.94–3.67(m,5H),3.61–3.47(m ,1H),3.33–3.21(m,4H),3.01–2.70(m,4H),2.65–2.52(m,2H),2.41–2 .34(m,2H),2.30–1.87(m,8H),1.85–1.10(m,14H),0.97–0.90(m,2H).

[0319] Step 4: Preparation of compound LA-36dPAB-a

[0320] At 0 °C, DIEA (0.042 mL, 0.253 mmol, 3 eq), N-[(5S)-1-(9H-fluorene-9-yl)-3,6-dioxo-5-(propanoyl-2-yl)-4-aza-2-oxahexyl-6-yl]-L-alanine (41.61 mg, 0.101 mmol, 1.2 eq), and HATU (38.55 mg, 0.101 mmol, 1.2 eq) were added to a 2 mL DMF solution of compound PE-2K (62 mg, 0.084 mmol, 1 eq). The mixture was then stirred at 25 °C for 1 h.

[0321] LCMS indicated the reaction was complete. The reaction solution was purified by silica gel column chromatography (C18, H2O (0.25% TFA):ACN = 36%:64%) to give LA-36dPAB-a (43 mg, yield 45.19%) as a white solid. LCMS (ESI): m / z, 1126.1 [m⁻¹] + Rt = 1.611 min.

[0322] Step 5: Preparation of compound LA-36dPAB-b

[0323] Diethylamine (9 mg, 0.120 mmol, 10 eq) was added to a 1 mL DMF solution of compound LA-36dPAB-a (13.0 mg, 82% purity, 0.012 mmol, 1 eq) at room temperature and stirred at 25 °C for 15 minutes. LC-MS showed that the reaction proceeds were completely reacted. The reaction solution was concentrated under reduced pressure to obtain a yellow oily crude product, LA-36dPAB-b. This crude product was used directly in the next step without purification.

[0324] LCMS(ESI): m / z, 903.9 [(M+H)] + .

[0325] Preparation Example 13: Preparation of compound LA-36VCdPAB-b

[0326] Step 1: Preparation of compound LA-36VCdPAB-a

[0327] At 0 °C, DIEA (40 mg, 0.307 mmol, 3 eq), compound CAS: 159858-21-6 (60.90 mg, 0.123 mmol, 1.2 eq), and HATU (46.63 mg, 0.123 mmol, 1.3 eq) were added to a DMF (3 mL) solution of compound PE-E2K (Reference Preparation Example 12) (75 mg, 0.102 mmol, 1 eq). The mixture was then stirred at 25 °C for 2 h. LCMS showed that the reaction was complete. The reactants were purified by silica gel column chromatography (C18, H2O (0.25% TFA): ACN = 36%: 64%) to give LA-36VCdPAB-a (60 mg, yield 48.42%) as a white solid. LCMS (ESI): m / z, 1212.8 [M]. + , Rt = 1.508 min.

[0328] Step 2: Preparation of compound LA-36VCdPAB-b

[0329] Diethylamine (5 mg, 0.070 mmol, 5 eq) was added to a DMF (1 mL) solution of compound LA-36VCdPAB-a (17 mg, 0.014 mmol, 1 eq). The reaction mixture was stirred at 25 °C for 1 hour, and LCMS showed that the starting material reacted completely. The reaction mixture was concentrated under vacuum at low temperature to obtain crude LA-36VCdPAB-b (13.88 mg, crude product), which was a pale yellow oil. LCMS (ESI): m / z, 989.7 [M-1] + , Rt=1.151min.

[0330] Preparation Example 14: Preparation of compound LA-36OVCdPAB-b

[0331] Step 1: Preparation of compound LA-36OVCdPAB-a1

[0332] At 0°C, TsOH (106 mg, 0.62 mmol) was added to a DMF / THF (5 mL / 5 mL) solution of compound PE-AM1 (purchased from Haoyuan Pharmaceutical) (300 mg, 0.41 mmol) and (5S, 8S)-1-(9H-fluoro-9-yl)-5-isopropyl-3,6,9-trioxo-8-(3-ureopropyl)-2-oxo-4,7,10-triazaundecane-11-ylacetate (purchased from Zizhi Biotechnology, CAS: 3028612-25-8) (350 mg, 0.62 mmol). The mixture was then stirred at 30°C for 1 hour. The reaction solution was purified by C18 reversed-phase column chromatography with water (0.1% TFA) and acetonitrile as the mobile phase. The acetonitrile elution ratio ranged from 0% to 60%, and the purified product was a yellow solid compound LA-36OVCdPAB-a1 (350 mg, crude product).

[0333] LCMS (ESI): m / z, 1238.3 [M] + Rt = 2.62 min.

[0334] Step 2: Preparation of compound LA-36OVCdPAB-a

[0335] At 0 °C, potassium osmium dihydrate (11 mg, 0.028 mmol) and sodium periodate (604 mg, 2.8 mmol) were added to a THF / H₂O (5 mL / 5 mL) solution of compound LA-36OVCdPAB-a1 (350 mg, 0.28 mmol). The mixture was then stirred at 30 °C for 1 hour. The reaction solution was extracted with EtOAc (50 mL × 3), the organic phases were combined and washed with brine (50 mL), the organic phase was dried, filtered and evaporated to dryness to obtain the crude product. The crude product was purified by C18 reversed-phase column chromatography with water (0.1% TFA)-acetonitrile as the mobile phase, and the acetonitrile elution ratio was from 0% to 65%. The purified product was a yellow oily compound LA-36OVCdPAB-a (200 mg, crude product).

[0336] LCMS (ESI): m / z, 1242.2 [M+1] + Rt = 2.304 min.

[0337] Step 3: Preparation of compound LA-36OVCdPAB-b

[0338] At 0 °C, diethylamine (0.08 mL, 0.805 mmol) was added to a DMF (5 mL) solution of compound LA-36OVCdPAB-a (100 mg, 0.08 mmol). The mixture was then stirred at 25 °C for 2 hours. The reaction solution was concentrated under vacuum to obtain a brown solid compound LA-36OVCdPAB-b (50 mg, 60% yield).

[0339] LCMS (ESI): m / z, 1019.7 [M+1] + Rt = 1.08 min.

[0340] Example 1: Preparation of compound LEC-01

[0341] Step 1: Preparation of di-tert-butyl 10-(((9H-fluorene-9-yl)methoxy)carbonyl)-4,7,13,16-tetraoxa-10-azadecanoate

[0342] At 0°C, 9-fluorenemethyl-N-succinimide carbonate (412.67 mg, 294.76 μL, 1.22 mmol, 1.1 eq) was added to a solution of compound 4,7,13,16-tetraoxa-10-docosahexadecanoic acid di-tert-butyl ester (500 mg, 1.11 mmol, 1 eq) and sodium bicarbonate (280.29 mg, 3.34 mmol, 3 eq) in tetrahydrofuran (8 mL) and water (3 mL). After addition, 18 mL was added. The reaction was carried out at -20℃ for 16 hours. LCMS showed that the reaction was complete. Ethyl acetate (40 mL) and water (40 mL) were added to the reaction solution. The mixture was then separated. The aqueous layer was extracted twice with ethyl acetate (50 mL × 2). The organic phases were combined, washed once with brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness to give 10-(((9H-fluorene-9-yl)methoxy)carbonyl)-4,7,13,16-tetraoxa-10-azadecanoic acid di-tert-butyl ester (750 mg, crude product, yield negligible).

[0343] LCMS(ESI): m / z, 694.4 [M+Na] + Rt = 3.55 min.

[0344] Step 2: Preparation of compound 10-(((9H-fluorene-9-yl)methoxy)carbonyl)-4,7,13,16-tetraoxa-10-docosahexaic acid

[0345] TFA (8 mL) was added to a DCM (40 mL) solution of compound 10-(((9H-fluorene-9-yl)methoxy)carbonyl)-4,7,13,16-tetraoxa-10-azadecanoic acid di-tert-butyl ester (750 mg, 1.12 mmol, 1 eq, crude product). The reaction mixture was incubated at 18-20 °C for 2 hours. LC-MS showed the reaction was complete. The reaction mixture was evaporated to dryness to give crude compound 10-(((9H-fluorene-9-yl)methoxy)carbonyl)-4,7,13,16-tetraoxa-10-docosahexaic acid (625 mg, crude product yield negligible).

[0346] LCMS(ESI): m / z, 560.3 [M+H] + , Rt=1.899min.

[0347] Step 3: Preparation of compound LC-CL-03-a

[0348] At 0 °C, NHS (637.55 mg, 398.47 μL, 5.54 mmol, 5 eq) and DIC (489.37 mg, 604.16 μL, 3.88 mmol, 3.5 eq) were added to a DMF (10 mL) solution of compound 10-(((9H-fluorene-9-yl)methoxy)carbonyl)-4,7,13,16-tetraoxa-10-docosahexadic acid (620 mg, 1.11 mmol, 1 eq, crude product). After addition, the reaction mixture was reacted at 25 °C for 16 hours. LC-MS showed approximately 3% product, mostly of the starting material. NHS (637.55 mg, 398.47 μL, 5.54 mmol, 5 eq) and DIC (489.37 mg, 604.16 μL, 3.88 mmol, 3.5 eq) were added to the reaction solution at 25 °C. After the addition was complete, the reaction solution was reacted at 50 °C for 3 hours. LCMS showed that the reaction was complete. The reaction solution was prepared by high performance liquid chromatography (preparative chromatograph manufacturer: Oriendo, model BRIX-2860. Column: YMC-Triart Prep C18 150×30 mm, 10 μm. Mobile phase: water (0.225% HCOOH)-acetonitrile, elution ratio of acetonitrile from 30% to 60%). After lyophilization, a yellow oily compound LC-CL-03-a (653 mg, yield 78.19%) was obtained.

[0349] LCMS(ESI): m / z, 754.2 [M+H] + Rt = 2.382 min.

[0350] Step 4: Preparation of compound LEC-01-a

[0351] DIEA (5.14 mg, 6.58 μL, 39.80 μmol, 1 eq) was added to a DMF (2 mL) solution of compounds LC-CL-03-a (30 mg, 39.80 μmol, 1 eq) and LY-22CD-b (11.95 mg, 15.92 μmol, 0.4 eq) at 0 °C. After adding the reagent, the reaction was carried out at 25°C for 3 hours. LCMS showed that the reaction was complete. The reaction solution was purified by high performance liquid chromatography (Shimadzu LC-20AP, Synergi Max-RP 200×30mm, 10μm column; mobile phase: water (0.225% FA)-acetonitrile, elution ratio of acetonitrile from 40% to 70%). The purified solution was lyophilized to give a white solid compound LEC-01-a (9 mg, crude product, yield 13.02%, purity 80%).

[0352] LCMS(ESI): m / z, 1411.8 [M+Na] + Rt = 2.548 min.

[0353] Step 5: Preparation of compound LEC-01-b

[0354] LA-28-d (5.55 mg, 5.17 μmol, 0.95 eq) and DIEA (1.41 mg, 1.80 μL, 10.88 μmol, 2 eq) were added to a 2 mL DMF solution of compound LEC-01-a (9.45 mg, 80%, 5.44 μmol, 1 eq) at 0 °C. After the addition was complete, the reaction solution was allowed to react at 25 °C for 1 hour. DBU (2.48 mg, 3 eq) was added to the reaction solution, and stirring was continued for 30 minutes. LCMS showed that the reaction was complete. The reaction solution and another batch (0.88 times the volume of this batch) were combined and purified by high performance liquid chromatography (preparative chromatograph manufacturer: Oriendo, model BRIX-2860; column: Welch Xtimate C18 150×21.2 mm, 5 μm; mobile phase: water (0.225% HCOOH)-acetonitrile; acetonitrile elution ratio: 30% to 60%). The purified compound was lyophilized to give a white solid compound LEC-01-b (7 mg, yield 32.20%).

[0355] LCMS(ESI):m / z,1063.6[1 / 2M+H] + Rt = 2.333 min.

[0356] Step 6: Preparation of compound LEC-01

[0357] At 0 °C, DIEA (708.37 μg, 5.48 μmol, 2.5 eq) and HATU (1 mg, 2.63 μmol, 1.2 eq) were added to a 2 mL DMF solution of compounds LEC-01-b (4.66 mg, 2.19 μmol, 1 eq) and LY-22CD-3-d (for the synthesis of this compound, please refer to WO2024175069A1) (2.45 mg, 3.07 μmol, 1.4 eq). After the addition was complete, the reaction solution was reacted at 25 °C for 1 hour. LC-MS showed that the reaction was complete. The reaction solution and another batch of small-scale test were combined and purified by high-performance liquid chromatography (HPLC) (preparative chromatograph manufacturer: Oriendo, model BRIX-2860; column: Welch Xtimate C18 150×21.2mm, 5μm; mobile phase: water (0.225% HCOOH)-acetonitrile; elution ratio of acetonitrile from 30% to 60%). The purified compound was lyophilized to give a white solid compound LEC-01 (1.14 mg, yield 17.90%, purity 100%).

[0358] LCMS(ESI):m / z,1475.8[1 / 2M+Na] + , Rt=2.513min. HPLC: Rt=2.897min, 100.0%.

[0359] Example 2: Preparation of compound LEC-01B

[0360] Step 1: Preparation of compound LEC-01B

[0361] Compound LEC-01-b (5.44 mg, 2.56 μmol, 1 eq; for the synthesis of this compound, please refer to the example of LEC-01) was dissolved in DMF (500 μL, cooled to 0 °C), and then LEC-03-d (4.29 mg, 2.56 μmol, 1 eq; for the synthesis of this compound, please refer to the example of LEC-03), DIEA (992.56 μg, 7.68 μmol, 3 eq), and HATU (1.46 mg, 3.84 μmol, 1.5 eq) were added. The reaction mixture was reacted at 25 °C for 30 min. LC-MS showed that the reaction was complete. The reaction mixture was prepared by high performance liquid chromatography (preparative chromatograph manufacturer: Oriendo, model BRIX-2860(R1). The chromatographic column was Phenomenex Luna C18). 250×30mm, 10μm. Mobile phase: water (0.225% HCOOH)-acetonitrile (elution ratio of acetonitrile from 30% to 60%), yielding a white solid LEC-01B (130μg, yield 1.34%, purity 92.54%).

[0362] LCMS(ESI):m / z,1284.6[M / 3+Na] + Rt = 2.164 min.

[0363] Example 3: Preparation of compound LEC-02

[0364] Step 1: Preparation of compound LEC-02-a

[0365] At 0°C, LA-25-d (9.30 mg, 8.90 μmol, 1 eq) and DIEA (3.45 mg, 26.69 μmol, 3 eq) were added to a 2 mL DMF solution of compound LEC-01-a (for the synthesis of this compound, please refer to the example of LEC-01) (12.36 mg, 8.90 μmol, 1 eq). After the addition was complete, the reaction solution was reacted at 35°C for 6 hours. DBU (4.06 mg, 3.99 μL, 26.69 μmol, 3 eq) was added to the reaction solution, and stirring was continued for 20 minutes. LC-MS showed that the reaction was complete. The reaction solution was purified by high-performance liquid chromatography (preparative chromatograph by Oriendo, model BRIX-2860; column: Welch Xtimate C18 150 × 21.2 mm, 5 μm; mobile phase: water (0.225% HCOOH)-acetonitrile; acetonitrile elution ratio from 30% to 60%). The purified solution was lyophilized to give a white solid compound LEC-02-a (840 μg, yield 4.50%).

[0366] LCMS(ESI):m / z,1049.4[1 / 2M+H] + Rt = 2.251 min.

[0367] Step 2: Preparation of compound LEC-02

[0368] At 0 °C, DIEA (155.28 μg, 1.20 μmol, 3 eq) and HATU (182.74 μg, 480.59 nmol, 1.2 eq) were added to a 2 mL DMF solution of compounds LEC-02-a (840 μg, 400.49 nmol, 1 eq) and LY-22CD-3-d (synthesis of which can be found in WO2024175069A1) (382.98 μg, 480.59 nmol, 1.2 eq). After the addition was complete, the reaction mixture was allowed to react at 25 °C for 0.5 h. LC-MS showed that the reaction was complete. The reaction solution was prepared by high performance liquid chromatography (Shimadzu LC-20AP, column: Welch Xtimate C18 150×21.2 mm, 5 μm; mobile phase: water (0.225% FA)-acetonitrile, elution ratio of acetonitrile from 40% to 70%) and purified by lyophilization to obtain a white solid compound LEC-02 (420 μg, yield 36.46%, purity 95.84%).

[0369] LCMS(ESI): m / z, 1461.7[1 / 2M+Na] + , Rt=2.423min. HPLC: Rt=2.817min, 95.84%.

[0370] Example 4: Preparation of compound LEC-02B

[0371] Step 1: Preparation of compound LEC-02B

[0372] At 0°C, DIEA (5.11 mg, 5.64 μmol, 1 eq) and HATU (3.86 mg, 10.16 μmol, 1.8 eq) were added to a 2 mL DMF solution of compounds LEC-02-a (synthesis of which can be found in the example of LEC-02) (11.84 mg, 5.64 μmol, 1 eq) and LEC-03-d (synthesis of which can be found in the example of LEC-03) (14.20 mg, 8.47 μmol, 1.5 eq). After the addition was complete, the reaction mixture was allowed to react at 25°C for 1 hour. LCMS showed that the reaction was complete. The reaction solution was purified by high-performance liquid chromatography (using Oriendo BRIX-2860 chromatographic column, YMC-Triart Prep C18 150×30mm, 10μm; mobile phase: water (0.225% HCOOH)-acetonitrile, elution ratio from 30% to 60%), and then lyophilized to give a white solid compound LEC-02B (2.05 mg, yield 9.67%).

[0373] LCMS(ESI): m / z, 1274[1 / 2M+Na] + , Rt=2.071min. HPLC: Rt=2.457min, 97.7%.

[0374] Example 5: Preparation of compound LEC-03

[0375] Step 1: Preparation of compound LY-22CD-a

[0376] At 0℃, DIEA (142.94 mg, 1.11 mmol, 3 eq) and HOBt (49.81 mg, 33.21 μL, 368.66 μmol, 1 eq) were added to a DMF (5 mL) solution of compound LY-22CC-c (297 mg, 84%, 368.66 μmol, 1 eq) (for the synthesis of this compound, please refer to WO2024175069A1), eczema mesylate (195.6 mg, 368.66 μmol, 1 eq). After addition, the reaction was carried out at 25℃ (outer temperature) for 1.5 hours. LC-MS showed that the reaction was complete. The reaction solution was purified by silica gel column chromatography (C18, ISCO, R-80g SepaFlash Silica Flash Column, 5-30%). Eluting with H2O / CH3CN (100 mL / min), the purified compound was lyophilized to give a white solid compound LY-22CD-a (230 mg, crude product, yield 56.42%, purity 88%).

[0377] LCMS (ESI): m / z, 973.6 [M+H] + Rt = 2.674 min.

[0378] Step 2: Preparation of compound LY-22CD-b

[0379] Diethylamine (91.28 mg, 1.25 mmol, 6 eq) was added to a DMF (4 mL) solution of compound LY-22CD-a (230 mg, 88%, 208 μmol, 1 eq) at 0 °C. The reaction mixture was stirred at 25 °C for 1 hour. LC-MS showed the reaction was complete. The reaction mixture was purified by silica gel column chromatography (C18, ISCO, R-80 g SepaFlash Silica Flash Column, 5-55% H2O / CH3CN elution, 100 mL / min) to obtain compound LY-22CD-b (93 mg, yield 57.76%, purity 97%) and another batch of LY-22CD-b with slightly lower purity (70 mg, yield 40.34%, purity 90%).

[0380] LCMS(ESI): m / z, 751.5 [M+H] + Rt = 1.412 min.

[0381] Step 3: Preparation of compound LEC-03-a

[0382] At 0°C, DIEA (5.32 mg, 41.13 μmol, 1 eq) was added to a DMF (3 mL) solution of compounds LA-27-d (31 mg, 41.13 μmol, 1 eq) and LC-CL-03-a (17.43 mg, 16.45 μmol, 0.4 eq; for the synthesis of this compound, please refer to the example of LEC-01). After the addition, the reaction solution was stirred at 25°C for 1 hour, and the LCMS showed that the reaction was complete. The reaction solution was prepared by high performance liquid chromatography (preparative chromatograph manufacturer: Oriendo, model BRIX-2860; column: Welch Xtimate C18 250×30 mm, 10 μm; mobile phase: water (0.225% FA)-acetonitrile, elution ratio of acetonitrile from 50% to 80%). After lyophilization, a white solid product LEC-03-a (13 mg, yield 46.5%) was obtained.

[0383] LCMS(ESI): m / z, 1720.9[M+Na] + Rt = 2.951 min.

[0384] Step 4: Preparation of compound LEC-03-b

[0385] DIEA (8.91 mg, 68.91 μmol, 9 eq) was added to a DMF (3 mL) solution of compounds LEC-03-a (13 mg, 7.66 μmol, 1 eq) and LY-22CD-b (8.19 mg, 84.2%, 9.19 μmol, 1.2 eq) at 0 °C. The reaction mixture was stirred at 25 °C for 2 hours. LC-MS showed the reaction was complete. The reaction mixture was purified by high-performance liquid chromatography (HPLC) (Oriendo BRIX-2860 chromatographic column: Phenomenex Luna C18 150 × 25 mm, 10 μm; mobile phase: water (0.225% HCOOH)-acetonitrile; acetonitrile elution ratio: 50% to 80%). The purified compound was lyophilized to give a white solid, LEC-03-b (8 mg, yield 44.77%).

[0386] LCMS(ESI): m / z, 1189.6[(M+2Na) / 2] + Rt = 3.053 min.

[0387] Step 5: Preparation of compound LEC-03-c

[0388] Diethylamine (12.54 mg, 171.40 μmol, 50 eq) was added to a DMF (3 mL) solution of compound LEC-03-b (8 mg, 3.43 μmol, 1 eq) at 0 °C. After the addition was complete, the reaction was carried out at 25 °C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was evaporated to dryness to obtain a white solid compound LEC-03-c (7.30 mg, crude product).

[0389] LCMS(ESI): m / z, 1078.3[(M+2Na) / 2] + Rt = 2.158 min.

[0390] Step 6: Preparation of compound LEC-03-d1

[0391] Compound (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-aminohexanoate tert-butyl hydrochloride (119.77 mg, 259.80 μmol, 1 eq) was dissolved in DMF (5 mL), followed by the addition of DIEA (67.15 mg, 519.60 μmol, 2 eq) and HOBt (52.66 mg, 35.10 μL, 389.70 μmol, 1.5 eq). Finally, LY-10b (synthesis of which can be found in WO2024175069A1) (200 mg, 259.80 μmol, 1 eq) and EDCI (74.70 mg, 389.70 μmol, 1.5 eq) was added. The mixture was stirred at 25 °C for 1 hour, then at 40 °C for 10 hours. The product was detected by LCMS. The reaction solution was prepared by high performance liquid chromatography to obtain a yellow oily product (296 mg, yield 96.85%).

[0392] The preparation conditions for high performance liquid chromatography are as follows:

[0393] The preparative chromatography system used Oriendo, model BRIX-2860. The column was a Phenomenex Luna C18, 250×50mm, 10μm. The mobile phase was water (0.225% HCOOH)-acetonitrile, with acetonitrile elution ratios ranging from 5% to 45%.

[0394] LCMS(ESI): m / z, 1198.7 [M+Na] + .

[0395] Step 7: Preparation of compound LEC-03-d2

[0396] Compound LEC-03-d1 (270 mg, 229.52 μmol, 1 eq) was dissolved in DCM (3 mL), and TFA (6.14 g, 4 mL, 53.85 mmol, 234.6189 eq) was added at 0 °C. The reaction was stirred at 25 °C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was directly evaporated to dryness to give a yellow oily liquid product (250 mg, yield 97.23%).

[0397] LCMS(ESI): m / z, 1120.9 [M+H] + .

[0398] Step 8: Preparation of compound LEC-03-d3

[0399] Compound LEC-03-d2 (250 mg, 223.16 μmol, 1 eq) was dissolved in DMF (4 mL), followed by the addition of DIEA (230.73 mg, 1.79 mmol, 8 eq) and amino-octaethylene glycol-tert-butyl propionate (166.58 mg, 334.75 μmol, 1.5 eq). Finally, HATU (127.28 mg, 334.75 μmol, 1.5 eq) was added at 0 °C, and the reaction mixture was stirred at 25 °C for 2 hours. LCMS showed that the starting material reacted completely. The reaction mixture was further analyzed by high performance liquid chromatography to obtain a yellow oily product (279 mg, yield 78.14%).

[0400] The preparation conditions for high performance liquid chromatography are as follows:

[0401] The preparative chromatography system used Oriendo, model BRIX-2860. The column was a Welch Xtimate C18 150×30mm, 10μm. The mobile phase was water (0.225% HCOOH)-acetonitrile, with acetonitrile elution ratios ranging from 20% to 50%.

[0402] LCMS(ESI): m / z, 1622.2 [M+Na] + .

[0403] Step 9: Preparation of compound LEC-03-d4

[0404] Compound LEC-03-d3 (260 mg, 162.51 μmol, 1 eq) was dissolved in DMF (3 mL), and compound Et2NH (59.43 mg, 812.57 μmol, 5 eq) was added. The reaction mixture was stirred at 25 °C for 1 hour. LCMS showed the target product and the product after removing the tert-butyl group. The reaction solution was evaporated to dryness and used directly in the next step.

[0405] Step 10: Preparation of compound LEC-03-d5

[0406] Compound LEC-03-d4 (180 mg, 130.66 μmol, 1 eq) was dissolved in DMF (3 mL), and compound LY-22CD-3-c (92.21 mg, 195.99 μmol, 1.5 eq; for the synthesis of this compound, please refer to WO2024175069A1) and DIEA (67.55 mg, 522.64 μmol, 4 eq) were added. Then, HATU (49.68 mg, 130.66 μmol, 1 eq) was added, and the reaction was stirred at 25 °C for 1 hour. The target product was observed by LCMS. The reaction solution was purified by high performance liquid chromatography to obtain a colorless oily product (170 mg, yield 75.08%).

[0407] Because the active ester LEC-03-d4 is unstable and contains 40% of the corresponding acid, condensation conditions were used here, and the corresponding DIEA was also added to 4 eq to promote the reaction of the active ester, acid and amine.

[0408] The preparation conditions for high performance liquid chromatography are as follows:

[0409] The preparative chromatogram was manufactured by Shimadzu, model LC-20AP. The column was a Welch Xtimate C18 250×30mm, 10μm. The mobile phase was water (0.225% HCOOH)-acetonitrile, with acetonitrile elution ratios ranging from 20% to 50%.

[0410] LCMS(ESI):m / z,839.0[[(M-56)+2H] / 2] + .

[0411] Step 11: Preparation of compound LEC-03-d

[0412] Compound LEC-03-d5 (170 mg, 98.10 μmol, 1 eq) was dissolved in DCM (5 mL), and TFA (1.64 g, 1.07 mL) was added at 0 °C. The reaction mixture was stirred at 25 °C for 1 hour. LCMS showed the target product. The reaction mixture was evaporated to dryness using an oil pump and then purified by high performance liquid chromatography to obtain a yellow oily product (100 mg, yield 60%).

[0413] LCMS(ESI): m / z, 839.7 [1 / 2M+H] + .

[0414] Step 12: Preparation of compound LEC-03

[0415] At 0℃, DIEA (2.08 mg, 2.66 μL, 16.11 μmol, 7 eq) and HATU (1.58 mg, 4.14 μmol, 1.8 eq) were added to a DMF (3 mL) solution of compounds LEC-03-d (5.40 mg, 3.22 μmol, 1.4 eq) and LEC-03-c (4.86 mg, 2.30 μmol, 1 eq). After addition, the reaction was carried out at 25℃ for 1 hour. LC-MS showed that the reaction was complete. The reaction solution and another batch of small-scale test were combined and prepared by high-performance liquid chromatography (HPLC) (Oriendo BRIX-2860 chromatographic column, Welch Xtimate C18). 150×21.2mm, 5μm. Purified by mobile phase water (0.225% HCOOH)-acetonitrile (elution ratio of acetonitrile from 30% to 60%), and lyophilized to give white solid compound LEC-03 (4.70 mg, yield 36.11%, purity 97%).

[0416] LCMS(ESI): m / z, 1279.6[(M+3Na) / 3] + ,1908.4[(M+2Na) / 2] + Rt = 2.112 min.

[0417] 1 H NMR (400MHz, DMSO-d) 6)δ9.08 (s, 1H), 8.86 (d, J = 8.6Hz, 1H), 8.58–8.48 (m, 2H), 8.37 (d, J = 8.6Hz, 1H), 8.04 (d, J=7.1Hz, 2H), 7.96–7.81 (m, 4H), 7.77 (d, J=10.6Hz, 2H), 7.31 (s, 1H), 6.91 (s, 1H ), 6.51(s, 1H), 5.42(s, 2H), 5.24(s, 2H), 5.05(s, 1H), 4.99(s, 1H), 4.82(s, 1H), 4.7 5(s, 1H), 4.62(d, J=4.9Hz, 1H), 4.58–4.42(m, 7H), 4.32(q, J=8.3, 5.6Hz, 3H), 4.20(d q, J=9.3, 7.2Hz, 6H), 4.12–3.96 (m, 5H), 3.93–3.69 (m, 5H), 3.69–3.53 (m, 13H), 3.61 –3.43(m, 52H), 3.43–3.33(m, 69H), 3.25(s, 7H), 3.22–2.88(m, 11H), 2.87–2.71(m, 4 H), 2.63–2.54(m, 4H), 2.45–2.35(m, 6H), 2.31–2.07(m, 9H), 2.04–1.80(m, 9H), 1.75 –1.39 (m, 15H), 1.38–1.08 (m, 15H), 1.00 (dd, J=22.8, 9.2Hz, 4H), 0.90–0.77 (m, 10H).

[0418] Example 6: Preparation of compound LEC-04

[0419] Step 1: Preparation of compound LEC-04-a

[0420] At 0°C, LA-29-f (16.64 mg, 15.92 μmol, 1 eq) was added to a DMF (2 mL) solution of compound LC-CL-03-a (30 mg, 39.80 μmol, 2.5 eq) (for the synthesis of this compound, please refer to the example of LEC-01) and DIEA (4.12 mg, 31.84 μmol, 2 eq). After addition, the reaction was carried out at 25°C (outer temperature) for 1.5 hours. LCMS showed that the reaction was complete. The reaction solution was purified by high performance liquid chromatography (preparative chromatograph manufacturer: Oriendo, model BRIX-2860; column: Welch Xtimate C18 150×21.2 mm, 5 μm; mobile phase: water (0.225% HCOOH)-acetonitrile; acetonitrile elution ratio: 50% to 80%). The purified solution was lyophilized to give a white solid compound LEC-04-a (20 mg, yield 74.60%).

[0421] LCMS(ESI): m / z, 1707.1 [M+Na] + Rt = 2.925 min.

[0422] Step 2: Preparation of compound LEC-04-b

[0423] At 0 °C, LY-22CD-b (8.92 mg, 11.88 μmol, 1 eq; for the synthesis of this compound, please refer to WO2024175069A1) and DIEA (13.81 mg, 106.89 μmol, 9 eq) were added to a DMF (1 mL) solution of compound LEC-04-a (20 mg, 11.88 μmol, 1 eq) at 0 °C. The reaction mixture was stirred at 25°C for 1 hour. LCMS showed approximately 20% product, indicating the presence of some acid corresponding to LEC-04-a. Therefore, HATU (1.81 mg, 4.75 μmol, 0.4 eq) was added, and stirring continued for another 2 hours. LCMS indicated the reaction was complete. The reaction mixture was purified by high-performance liquid chromatography (preparative chromatograph manufacturer: Shimadzu, model LC-20AP; column: Welch Xtimate C18 150 × 21.2 mm, 5 μm; mobile phase: water (0.225% FA)-acetonitrile; acetonitrile elution ratio: 30% to 60%). After lyophilization, compound LEC-04-b (12 mg, yield 43.56%) was obtained.

[0424] LCMS(ESI):m / z,1182.4[1 / 2M+Na] + Rt = 3.02 min.

[0425] Step 3: Preparation of compound LEC-04-c

[0426] Diethylamine (0.2 mL) was added to a 2 mL DMF solution of compound LEC-04-b (8 mg, 3.45 μmol, 1 eq) at 0 °C. The reaction mixture was stirred at 25 °C for 30 minutes, and LC-MS showed the reaction was complete. The reaction mixture was directly evaporated to dryness to give a white solid product, LEC-04-c (7.50 mg, crude product).

[0427] LCMS(ESI):m / z,1071.5[1 / 2M+Na] + Rt = 2.138 min.

[0428] Step 4: Preparation of compound LEC-04

[0429] At 0°C, DIEA (1.85 mg, 14.30 μmol, 4 eq) and HATU (1.77 mg, 4.65 μmol, 1.3 eq) were added to a DMF (2 mL) solution of compounds LEC-03-d (6 mg, 3.58 μmol, 1 eq; for the synthesis of this compound, please refer to the example of LEC-03) and LEC-04-c (7.50 mg, 3.58 μmol, 1 eq). After addition, the reaction was carried out at 25°C for 1 hour. LC-MS showed that the reaction was complete. The reaction solution was prepared by high-performance liquid chromatography (HPLC) (Shimadzu LC-20AP, column: Welch Xtimate C18). 150×21.2mm, 5μm. Purified by mobile phase water (0.225% FA)-acetonitrile (elution ratio of acetonitrile from 30% to 60%), and lyophilized to give white solid compound LEC-04 (2.04 mg, yield 15.19%, purity 100%).

[0430] LCMS(ESI): m / z, 1274.8[(M+3Na) / 3] + Rt = 2.109 min.

[0431] Example 7: Preparation of compound LEC-05

[0432] Step 1: Preparation of compound LEC-05-a

[0433] DIEA (2.71 mg, 21.01 μmol, 1 eq) was added to a DMF (3 mL) solution of compounds LC-CL-03-a (synthesis of which can be found in the example of LEC-01) (39.58 mg, 52.52 μmol, 2.5 eq) and LA-31A-d (22.21 mg, 21.01 μmol, 1 eq) at 0 °C. After the addition was complete, the reaction mixture was allowed to react at 25 °C for 1 hour. LCMS showed that the reaction was complete. The reaction solution was purified by high performance liquid chromatography (preparative chromatograph manufacturer: Oriendo, model BRIX-2860; column: Welch Xtimate C18 150×30mm, 10μm; mobile phase: water (0.225% HCOOH)-acetonitrile; acetonitrile elution ratio: 50% to 80%), and then lyophilized to give a white solid compound LEC-05-a (20.50 mg, yield 57.54%).

[0434] LCMS(ESI): m / z, 1719 [M+Na] + Rt = 2.993 min.

[0435] Step 2: Preparation of compound LEC-05-b

[0436] At 0°C, DIEA (14.06 mg, 108.79 μmol, 9 eq) was added to a 2 mL DMF solution of compounds LEC-05-a (20.50 mg, 12.09 μmol, 1 eq) and LY-22CD-b (synthesis of which can be found in WO2024175069A1) (11.23 mg, 14.51 μmol, 1.2 eq). After the addition, the reaction solution was reacted at 25°C for 2 hours, and then HATU (4.60 mg, 12.09 μmol, 1 eq) was added (a condensing agent was added because the acid corresponding to LEC-05-a was found). LCMS showed that the reaction was complete. The reaction solution was purified by high performance liquid chromatography (preparative chromatograph manufacturer: Oriendo, model BRIX-2860; column: YMC-Triart Prep C18 150×30mm, 10μm; mobile phase: water (0.225% HCOOH)-acetonitrile; elution ratio of acetonitrile from 50% to 80%), and then lyophilized to give a white solid compound LEC-05-b (18 mg, yield 63.86%).

[0437] LCMS(ESI): m / z, 1188 [1 / 2M+Na] + Rt = 3.076 min.

[0438] Step 3: Preparation of compound LEC-05-c

[0439] Et₂NH (2.82 mg, 38.60 μmol, 5 eq) was added to a DMF (1.5 mL) solution of compound LEC-05-b (18 mg, 7.72 μmol, 1 eq), and the mixture was stirred at 25 °C for 1 hour. LCMS showed that the reactants reacted completely, and the mixture was directly added to the next step after the oil pump was drained. The theoretical mass of LEC-05-c is 16.28 mg.

[0440] LCMS(ESI): m / z, 1055 [1 / 2M+H] + Rt = 2.301 min.

[0441] Step 4: Preparation of compound LEC-05

[0442] At 0°C, DIEA (2.99 mg, 23.15 μmol, 3 eq) and HATU (3.52 mg, 9.26 μmol, 1.2 eq) were added to a 2 mL DMF solution of compounds LEC-05-c (16.28 mg, 7.72 μmol, 1 eq) and LEC-03-d (see the example for the synthesis of LEC-03) (15.53 mg, 9.26 μmol, 1.2 eq). After the addition was complete, the reaction mixture was allowed to react at 25°C for 1 hour. LCMS showed that the reaction was complete. The reaction solution was purified by high performance liquid chromatography (preparative chromatograph manufacturer: Oriendo, model BRIX-2860; column: Welch Xtimate C18 250×30mm, 10μm; mobile phase: water (0.225% HCOOH)-acetonitrile; elution ratio of acetonitrile from 30% to 60%), and then lyophilized to give a white solid compound LEC-05 (3.50 mg, yield 12.03%).

[0443] LCMS(ESI): m / z, 1278 [1 / 3M+Na] + , Rt=2.140min; HPLC: Rt=2.457min, 90.03%.

[0444] Example 8: Preparation of compound LEC-06

[0445] Step 1: Preparation of compound LEC-06-a

[0446] DIEA (3.13 mg, 4 μL, 24.19 μmol, 1 eq) was added to a 2 mL DMF solution of compounds LC-CL-03-a (synthesis of which can be found in the example of LEC-01) (45.58 mg, 60.46 μmol, 2.5 eq) and LA-32AS-d (synthesis of which can be found in the example of LEC-2932AS) (25.62 mg, 24.19 μmol, 1 eq) at 0 °C. After the addition was complete, the reaction mixture was allowed to react at 25 °C for 1 hour. LCMS showed that the reaction was complete. The reaction solution was purified by high performance liquid chromatography (preparative chromatograph manufacturer: Oriendo, model BRIX-2860; column: Synergi Max-RP 200×30mm, 10μm; mobile phase: water (0.225% HCOOH)-acetonitrile; elution ratio of acetonitrile from 50% to 80%), and then lyophilized to give a white solid compound LEC-06-a (31.30 mg, yield 76.22%).

[0447] LCMS(ESI): m / z, 1720 [M+Na] + Rt = 2.963 min.

[0448] Step 2: Preparation of compound LEC-06-b

[0449] At 0°C, DIEA (21.44 mg, 165.90 μmol, 9 eq) was added to a 2 mL DMF solution of compounds LEC-06-a (31.30 mg, 18.43 μmol, 1 eq) and LY-22CD-b (synthesis of which can be found in WO2024175069A1) (17.12 mg, 22.12 μmol, 1.2 eq). After the addition, the reaction solution was reacted at 25°C for 2 hours, and then HATU (7.01 mg, 18.43 μmol, 1 eq) was added (a condensing agent was added because the acid corresponding to LEC-06-a was found). LCMS showed that the reaction was complete. The reaction solution was purified by high performance liquid chromatography (preparative chromatograph manufacturer: Oriendo, model BRIX-2860; column: YMC-Triart Prep C18 150×30mm, 10μm; mobile phase: water (0.225% HCOOH)-acetonitrile; elution ratio of acetonitrile from 50% to 80%), and then lyophilized to give a white solid compound LEC-06-b (29.30 mg, yield 68.11%).

[0450] LCMS(ESI): m / z, 1189 [1 / 2M+Na] + Rt = 3.046 min.

[0451] Step 3: Preparation of compound LEC-06-c

[0452] Et₂NH (4.59 mg, 62.78 μmol, 5 eq) was added to a DMF (2 mL) solution of compound LEC-06-b (29.30 mg, 12.56 μmol, 1 eq), and the mixture was stirred at 25 °C for 1 hour. LCMS showed that the reactants reacted completely, and the mixture was directly added to the next step after the oil pump was drained. The theoretical mass of LEC-06-c is 26.51 mg.

[0453] LCMS(ESI): m / z, 1078 [1 / 2M+Na] + Rt = 2.309 min.

[0454] Step 4: Preparation of compound LEC-06

[0455] At 0°C, DIEA (3.25 mg, 25.11 μmol, 2 eq) and HATU (5.73 mg, 15.07 μmol, 1.2 eq) were added to a 2 mL DMF solution of compounds LEC-06-c (26.51 mg, 12.56 μmol, 1 eq) and LEC-03-d (for the synthesis of this compound, please refer to the preparation example of LEC-03) (25.26 mg, 15.07 μmol, 1.2 eq). After the addition was complete, the reaction solution was reacted at 25°C for 1 hour. LCMS showed that the reaction was complete. The reaction solution was purified by high performance liquid chromatography (preparative chromatograph manufacturer: Oriendo, model BRIX-2860; column: Welch Xtimate C18 250×30mm, 10μm; mobile phase: water (0.225% HCOOH)-acetonitrile, elution ratio of acetonitrile from 30% to 60%), and then lyophilized to give a white solid compound LEC-06 (5.49 mg, yield 11.60%).

[0456] LCMS(ESI): m / z, 1279 [1 / 3M+Na] + , Rt=2.140min; HPLC: Rt=2.500min, 96.07%. 1H NMR (400MHz, DMSO-d6) δ9.09 (s, 1H), 8.86 (d, J = 8.6Hz, 1H), 8.52 (d, J = 21.8Hz, 3H) , 8.37 (d, J = 8.6Hz, 1H), 8.06 (d, J = 7.2Hz, 2H), 7.99–7.82 (m, 5H), 7.81–7.74 (m, 2H) , 7.31 (s, 1H), 6.85 (s, 1H), 6.52 (s, 1H), 5.43 (s, 2H), 5.24 (s, 3H), 5.02 (d, J=22.8 Hz, 3H), 4.79 (d, J=31.4Hz, 3H), 4.66–4.41 (m, 11H), 4.40–4.15 (m, 11H), 4.08 (d, J= 11.8Hz, 6H), 3.95–3.71(m, 4H), 3.70–3.43(m, 81H), 3.40(d, J=6.2Hz, 12H), 3.25( s, 15H), 2.97 (d, J=7.2Hz, 5H), 2.88–2.81 (m, 2H), 2.72 (d, J=12.6Hz, 3H), 2.66–2.5 3(m, 7H), 2.47–2.35(m, 10H), 2.31–2.06(m, 11H), 1.89(d, J=23.2Hz, 11H), 1.80–1 .39 (m, 19H), 1.38–1.07 (m, 19H), 1.00 (dd, J=23.2, 9.0Hz, 5H), 0.93–0.76 (m, 14H).

[0457] Example 9 Preparation of compound LEC-22CD27AA-2

[0458] Step 1: Preparation of compound LA-27AA-2-f

[0459] At 25°C, HOBt (9.86 mg, 0.073 mmol, 1.0 eq) and DIEA (0.036 mL, 0.219 mmol, 3.0 eq) were added to a DMF (2.0 mL) solution of compound LA-27AA-2-e (57.88 mg, 0.088 mmol, 1.2 eq) and eribulin methanesulfonate (60.0 mg, 0.073 mmol, 1.0 eq), and the mixture was stirred at 25°C for 2 hours. LC-MS showed that the reaction was complete. The reaction solution was quenched with H2O (10 mL) and then extracted with ethyl acetate (10 mL × 2). The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The crude product was obtained by concentration under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 10:1) to obtain a yellow solid LA-27AA-2-f (114.0 mg, purity 85%, yield 87.99%).

[0460] LCMS(ESI): m / z, 1273.3[(M+Na)] + .

[0461] Step 2: Preparation of compound LA-27AA-2-g

[0462] Diethylamine (0.018 mL, 0.170 mmol, 10 eq) was added to a 1 mL DMF solution of compound LA-27AA-2-f (25.0 mg, 85% purity, 0.017 mmol, 1 eq) at 25 °C and stirred at 25 °C for 1 hour. LC-MS showed that the starting material reacted completely. The reaction solution was concentrated under reduced pressure to obtain a yellow solid crude product LA-27AA-2-g. This product was used directly in the next step without purification.

[0463] LCMS(ESI): m / z, 1029.3 [(M+H)] + .

[0464] Step 3: Preparation of compound LEC-22CD27AA-2-a

[0465] At 0°C, DIEA (0.008 mL, 0.051 mmol, 3.0 eq) was added to a DMF (2.0 mL) solution of compound LEC-22CD34B-a (see the example of LEC-22CD34B for its synthesis) (23.6 mg, 0.017 mmol, 1.0 eq) and the crude LA-27AA-2-g (0.017 mmol, 1.0 eq) described above. The mixture was stirred at 25°C for 1 hour, and LC-MS showed that the reaction proceeds were completely reacted. The reaction solution was purified by silica gel column chromatography (C18) using water (0.25% TFA)-acetonitrile (0.25% TFA) as the mobile phase, with acetonitrile elution ratios ranging from 0% to 60%. The purified product was a yellow solid, LEC-22CD27AA-2-a (14.0 mg, yield 35.75%).

[0466] LCMS(ESI): m / z, 1173.7[(M / 2+Na)] + .

[0467] Step 4: Preparation of compound LEC-22CD27AA-2-b

[0468] Diethylamine (0.006 mL, 0.061 mmol, 10 eq) was added to a 1 mL DMF solution of compound LEC-22CD27AA-2-a (14.0 mg, 0.006 mmol, 1 eq) at 25 °C and stirred at 25 °C for 1 hour. LC-MS showed that the reaction proceeds were completely reacted. The reaction solution was concentrated under reduced pressure to obtain a yellow solid crude product, LEC-22CD27AA-2-b. This product was used directly in the next step without purification.

[0469] LCMS(ESI): m / z, 1040.7 [(M / 2)] + .

[0470] Step 5: Preparation of compound LEC-22CD27AA-2

[0471] At 0°C, DIEA (0.002 mL, 0.012 mmol, 2.0 eq), LEC-03-d (12.07 mg, 0.007 mmol, 1.2 eq) and HATU (2.74 mg, 0.007 mmol, 1.2 eq) were added to a DMF (1 mL) solution of crude LEC-22CD27AA-2-b (0.006 mmol, 1.0 eq) and stirred at 25°C for 1 hour. LCMS showed that the starting material reacted completely. The reaction solution was purified by high performance liquid chromatography (JB-C235-04 column, Bonnasil-BS C18, 20×250mm, 8μm, mobile phase: water (0.1% TFA)-acetonitrile, elution ratio of acetonitrile from 20% to 60%) to obtain a yellow solid product LEC-22CD27AA-2 (3.64 mg, yield 16.01%).

[0472] LCMS(ESI): m / z, 1869.5[(M / 2+H)] + .

[0473] 1 H NMR (400MHz, DMSO) δ9.08 (t, J=5.4Hz, 1H), 8.86 (d, J=8.6Hz, 1H), 8.54 (d, J=6.6Hz, 1H), 8.38 (d, J=8.8Hz, 1H), 8.13 (s , 1H), 8.04 (d, J=6.8Hz, 1H), 7.96–7.72 (m, 10H), 7.32 (s, 1H), 7.22 (s, 1H), 6.91 (s, 1H), 6.66 (s, 1H), 6.52 (s, 1H), 5.43 (s, 3H), 5.33 (t, J = 4.8Hz, 1H), 5.24 (s, 3H), 5.02 (d, J = 24.0Hz, 3H), 4.79 (d, J = 30.0Hz, 5H), 4.67–4.37 (m, 14H), 4.31– 3.95 (m, 23H), 3.93–3.50 (m, 83H), 2.40–1.83 (m, 37H), 1.76–1.13 (m, 61H), 1.02 (t, J=10.4Hz, 4H), 0.91–0.77 (m, 19H).

[0474] Example 10 Compound LEC-22CD27AAVC

[0475] Step 1: Preparation of compound LEC-22CD27AAVC-a

[0476] At 0 °C, DIEA (0.017 mL, 0.105 mmol, 5 eq) and LA-27AAVC-d (23 mg, 0.021 mmol, 1 eq) were added to a solution of compound LEC-22CD34B-a (30.09 mg, 0.022 mmol, 1.05 eq; for the synthesis of this compound, please refer to the example of LEC-22CD34B) in DMF (2 mL). The mixture was then stirred at 25 °C for 1 h. LCMS showed that the reaction was complete. The reactants were purified by silica gel column chromatography (C18, 71% H2O (0.025% TFA) / ACN) to give LEC-22CD27AAVC-a (24 mg, yield 48.70%) as a white solid, LCMS (ESI): m / z, 1194.8 [M / 2]. + Rt = 1.666 min.

[0477] Step 2: Preparation of compound LEC-22CD27AAVC-b

[0478] Diethylamine (0.010 mL, 0.100 mmol, 10 eq) was added to a 2 mL DMF solution of compound LEC-22CD27AAVC-a (24 mg, 0.010 mmol, 1 eq) at 25 °C. The reaction mixture was stirred at 25 °C for 1.5 hours, and LCMS showed that the starting material reacted completely. The reaction mixture was concentrated under vacuum at low temperature to obtain crude LEC-22CD27AAVC-b (21 mg, crude product), which was a pale yellow oil. LCMS (ESI): m / z, 1083.7 [M / 2] + Rt = 1.448 min.

[0479] Step 3: Preparation of compound LEC-22CD27AAVC

[0480] At 0 °C, DIEA (0.005 mL, 0.029 mmol, 3 eq), LY-22CD-3-d (8.49 mg, 0.011 mmol, 1.1 eq) and HATU (4.79 mg, 0.013 mmol, 1.3 eq) were added to 2 mL of DMF containing compound LEC-22CD27AAVC-b (21 mg, 0.010 mmol, 1 eq). The mixture was then stirred at 25 °C for 1 h. LC-MS showed the reaction was complete.

[0481] The reaction solution was purified by high-performance liquid chromatography (Bonnasil-BS C18, 20×250 mm, 8 μm, 38-68% H2O (0.1% FA) / ACN) to obtain LEC-22CD27AAVC (11.34 mg, yield 39.72%), as a white solid. LCMS (ESI): m / z, 1472.9 [M / 2-1] + Rt = 1.468 min.

[0482] 1 H NMR (400MHz, DMSO) δ9.05 (t, J=5.6Hz, 1H), 8.86 (d, J=8.6Hz, 1H), 8.54 (t, J=6.5Hz, 1H), 8.37 (d, J=8.6Hz, 1H), 8.04 ( d, J=7.2Hz, 1H), 7.94–7.74 (m, 7H), 7.31 (s, 1H), 6.91 (d, J=5.3Hz, 1H), 6.58 (d, J=58.4Hz, 1H), 5.92 (s, 1H), 5.42 (s, 2 H), 5.34–5.30 (m, 1H), 5.26 (d, J=19.3Hz, 3H), 5.02 (d, J=22.7Hz, 2H), 4.83 (s, 1H), 4.75 (s, 1H), 4.63 (s, 1H), 4.59–4 .52 (m, 2H), 4.47 (dd, J=10.1, 6.6Hz, 1H), 4.29–4.13 (m, 6H), 4.12–3.97 (m, 6H), 3.88 (t, J=6.5Hz, 2H), 3.79 (dd, J=20. 4, 9.2Hz, 2H), 3.64 (d, J=3.3Hz, 3H), 3.63–3.52 (m, 11H), 3.50–3.45 (m, 38H), 3.25 (s, 4H), 3.20–3.16 (m, 3H), 3.13 (s , 3H), 3.05–2.99 (m, 3H), 2.93 (d, J=5.4Hz, 4H), 2.84 (d, J=9.7Hz, 1H), 2.79–2.65 (m, 3H), 2.57 (dd, J=12.2, 5.4Hz, 3H) , 2.47–2.42(m, 2H), 2.34(dd, J=17.0, 10.9Hz, 9H), 2.29–2.12(m, 7H), 2.06–1.78(m, 13H), 1.77–1.58(m, 8H), 1.57–1 .43 (m, 9H), 1.30 (ddd, J=32.4, 14.7, 7.4Hz, 22H), 1.00 (dd, J=22.9, 9.1Hz, 4H), 0.84 (ddd, J=16.2, 13.6, 7.2Hz, 16H).

[0483] Example 11 Preparation of compound LEC-22CD34B

[0484] Step 1: Preparation of compound LEC-22CD34B-a

[0485] DIEA (4 mg, 31.17 μmol, 1 eq) was added to a DMF (3 mL) solution of compounds LC-CL-03-a (synthesis of which can be found in Example LEC-01) (58.73 mg, 77.91 μmol, 2.5 eq) and LY-22CD-b (synthesis of which can be found in WO2024175069A1) (23.40 mg, 31.17 μmol, 1 eq) at 0 °C. After the addition was complete, the reaction mixture was allowed to react at 25 °C for 1 hour. LCMS showed that the reaction was complete. The reaction solution was purified by high-performance liquid chromatography (preparative chromatograph manufacturer: Oriendo, model BRIX-2860; column: YMC-Triart Prep C18 150×30mm, 10μm; mobile phase: water (0.225% HCOOH)-acetonitrile; elution ratio of acetonitrile from 35% to 70%), and then lyophilized to give a yellow solid compound LEC-22CD34B-a (33 mg, yield 46.42%).

[0486] LCMS(ESI): m / z, 1411[M+Na] + Rt = 2.558 min.

[0487] Step 2: Preparation of compound LEC-22CD34B-b

[0488] DIEA (3.48 μL, 21.03 μmol, 2 eq) was added to a DMF (3 mL) solution of compounds LEC-22CD34B-a (27 mg, 19.43 μmol, 1 eq) and LA-34B-b (22.93 mg, 23.32 μmol, 1.2 eq) at 0 °C. After addition, the reaction solution was reacted at 25 °C for 3 hours, and LC-MS showed that the reaction was essentially complete. The reaction solution was purified by high-performance liquid chromatography (preparative chromatograph manufacturer: Oriendo, model BRIX-286; column: Welch Xtimate C18 150×30 mm, 10 μm; mobile phase: water (0.225% HCOOH)-acetonitrile; acetonitrile elution ratio: 50% to 80%), and lyophilized to obtain a white solid compound LEC-22CD34B-b (19.30 mg, yield 36%).

[0489] LCMS(ESI): m / z, 1151[1 / 2M+Na] +Rt = 2.984 min.

[0490] Step 3: Preparation of compound LEC-22CD34B-c

[0491] Et₂NH (4.45 μL, 42.74 μmol, 5 eq) was added to a 2 mL DMF solution of compound LEC-22CD34B-b (19.30 mg, 8.55 μmol, 1 eq) at 0 °C and stirred at 25 °C for 1 hour. LCMS showed that the reaction proceeded completely. After concentration by an oil pump, the solution was directly added to the next step. The theoretical mass of LEC-22CD34B-c is 17.40 mg.

[0492] LCMS(ESI): m / z, 1018 [1 / 2M+H] + Rt = 2.046 min.

[0493] Step 4: Preparation of compound LEC-22CD34B

[0494] At 0°C, DIEA (2.85 μL, 17.10 μmol, 2 eq) and HATU (3.90 mg, 10.26 μmol, 1.2 eq) were added to a 2 mL DMF solution of compounds LEC-22CD34B-c (17.40 mg, 8.55 μmol, 1 eq) and LEC-03-d (see the example for LEC-03 for synthesis) (17.20 mg, 10.26 μmol, 1.2 eq). After the addition was complete, the reaction mixture was allowed to react at 25°C for 1 hour. LC-MS showed that the reaction was complete. The reaction solution was prepared by high performance liquid chromatography (preparative chromatograph manufacturer: Oriendo, model BRIX-2860; column: YMC-Triart Prep C18 150×30mm, 10μm; mobile phase: water (0.225% HCOOH)-acetonitrile, elution ratio of acetonitrile from 30% to 60%), and purified by lyophilization to obtain a white solid compound LEC-22CD34B (8.22 mg, yield 26.03%, purity 100%).

[0495] LCMS(ESI): m / z, 1253 [1 / 3M+Na] + , Rt=2.062min; HPLC: Rt=5.180min, 100%.

[0496] 1H NMR (400MHz, DMSO-d6) δ9.09 (t, J=5.8Hz, 1H), 8.86 (d, J=8.6Hz, 1H), 8.58 (dt, J=24.2, 7.0Hz, 2H), 8 .37 (d, J=8.6Hz, 1H), 8.31 (s, 1H), 8.06 (dd, J=14.8, 7.2Hz, 2H), 7.94 (d, J=8.4Hz, 1H), 7.89 (d, J=5.4 Hz, 1H), 7.87–7.81 (m, 2H), 7.81–7.74 (m, 2H), 7.31 (s, 1H), 6.51 (s, 1H), 5.42 (s, 2H), 5.24 (d, J=2.8 Hz, 3H), 5.03 (d, J=11.4Hz, 2H), 4.82 (s, 1H), 4.75 (s, 1H), 4.67–4.43 (m, 9H), 4.36–4.15 (m, 10H), 4.1 4–3.95 (m, 8H), 3.89 (d, J=5.0Hz, 1H), 3.78 (dd, J=16.4, 7.4Hz, 4H), 3.71–3.35 (m, 100H), 3.28–3.02 (m, 13H), 2.97 (q, J=7.2Hz, 2H), 2.86–2.81 (m, 1H), 2.80–2.69 (m, 3H), 2.57 (q, J=12.8, 9.8Hz, 5H), 2. 47–2.32 (m, 11H), 2.27 (d, J=6.6Hz, 4H), 2.23–2.07 (m, 5H), 2.07–1.76 (m, 11H), 1.74–1.40 (m, 12H), 1.32 (d, J=8.4Hz, 5H), 1.21 (dd, J=7.2, 2.4Hz, 8H), 1.00 (dd, J=22.6, 9.2Hz, 5H), 0.90–0.78 (m, 12H).

[0497] Example 12 Preparation of compound LEC-22CD34BVA2

[0498] Step 1: Preparation of compound LEC-22CD34BVA2-f

[0499] At 25°C, HATU (11.55 mg, 0.030 mmol, 1.3 eq) and DIEA (0.012 mL, 0.070 mmol, 3.0 eq) were added to a DMF (1.5 mL) solution of compounds LEC-22CD34BVC2-c (see the example of LEC-22CD34BVC2 for its synthesis) (50.0 mg, 0.023 mmol, 1.0 eq) and LA-34B-b (33 mg, 0.027 mmol, 1.15 eq), and the mixture was stirred at 25°C for one hour. LCMS showed that the reaction was complete. The reaction solution was purified by C18 reversed-phase column chromatography with water (0.25% TFA) and acetonitrile (0.25% TFA) as the mobile phase. The acetonitrile elution ratio ranged from 0% to 60%, and the yellow solid product LEC-22CD34BVA2-f (40.0 mg, yield 55.13%) was obtained.

[0500] LCMS(ESI): m / z, 1573.7[(M / 2+Na)] + .

[0501] Step 2: Preparation of compound LEC-22CD34BVA2-g

[0502] Diethylamine (0.013 mL, 0.129 mmol, 10.0 eq) was added to a 1.5 mL DMF solution of compound LEC-22CD34BVA2-f (40 mg, 0.013 mmol, 1.0 eq) at 25 °C and stirred at 25 °C for 1 hour. LC-MS showed that the reaction proceeds were completely reacted. The reaction solution was concentrated under reduced pressure to obtain a yellow oily crude product of LEC-22CD34BVA2-g (the yield of the crude product was not considered). The crude product was used directly in the next step without purification.

[0503] LCMS(ESI): m / z, 1440.8 [(M / 2)] + .

[0504] Step 3: Preparation of compound LEC-22CD34BVA2

[0505] At 25°C, HATU (6.37 mg, 0.017 mmol, 1.3 eq) and DIEA (0.006 mL, 0.039 mmol, 3.0 eq) were added to a DMF (1.5 mL) solution of the above crude LEC-22CD34BVA2-g (37.14 mg, 0.013 mmol, 1.0 eq) and LY-22CD-3-b (for the synthesis of this compound, please refer to WO2024175069A1) (6.25 mg, 0.017 mmol, 1.3 eq) and stirred at 25°C for 1 hour. LCMS showed that the starting material reacted completely. The reaction solution was evaporated to dryness and purified by high performance liquid chromatography (JB-C235-04 column, Bonnasil-BS C18, 20×250mm, 8μm), with water (0.1% TFA) and acetonitrile as the mobile phase, and acetonitrile elution ratio from 20% to 60%, to obtain a yellow solid product LEC-22CD34BVA2 (14.62 mg, yield 35.04%).

[0506] LCMS(ESI): m / z, 1618.4[(M / 2+H)] + .

[0507] 1H NMR (400MHz, DMSO-d6) δ9.05 (t, J=5.6Hz, 1H), 8.86 (d, J=8.6Hz, 1H), 8.62–8.57 (m, 1H), 8.53 (t, J=6.5Hz, 1H), 8.37 (d, J=8.6Hz, 1H), 8.04 (dd, J=15.8, 7.1Hz, 2H), 7.93–7.82 (m, 5H), 7.77 (d, J= 10.9Hz, 1H), 7.31 (s, 1H), 5.42 (s, 2H), 5.32 (t, J=4.7Hz, 1H), 5.24 (s, 3H), 5.03 (d, J=11.7Hz, 2H ), 4.82(s, 1H), 4.75(s, 1H), 4.65–4.62(m, 1H), 4.58–4.52(m, 4H), 4.49–4.45(m, 1H), 4.27–3.96( m, 17H), 3.84–3.75 (m, 4H), 3.65 (s, 3H), 3.60–3.53 (m, 20H), 3.47–3.36 (m, 55H), 3.33–3.30 (m, 2 H), 3.26 (d, J=2.6Hz, 3H), 3.21–3.15 (m, 5H), 2.83 (d, J=10.6Hz, 2H), 2.75 (d, J=11.0Hz, 3H), 2.6 8–2.66 (m, 1H), 2.60 (t, J=6.6Hz, 3H), 2.46–2.40 (m, 2H), 2.38 (s, 3H), 2.36–2.11 (m, 17H), 2.04– 1.83 (m, 13H), 1.74–1.42 (m, 12H), 1.37–1.18 (m, 21H), 1.03 (d, J=6.4Hz, 3H), 0.89–0.78 (m, 17H).

[0508] Example 13 Preparation of compound LEC-22CD34BVC

[0509] Step 1: Preparation of compound LA-34BVC-e

[0510] Diethylamine (0.032 mL, 0.310 mmol, 10 eq) was added to a DMF (1.5 mL) solution of compound LA-34BVC-d (40.0 mg, 0.031 mmol, 1 eq) at 25 °C and stirred at 25 °C for 1 hour. LC-MS showed that the starting material reacted completely. The reaction solution was concentrated under reduced pressure to obtain crude LA-34BVC-e as a yellow solid. It was used directly in the next step without purification.

[0511] LCMS(ESI): m / z, 1068.8 [(M+H)] +.

[0512] Step 2: Preparation of compound LEC-22CD34BVC-a

[0513] DIEA (0.014 mL, 0.087 mmol, 3.0 eq) was added to a DMF (2.0 mL) solution of compound LEC-22CD34B-a (41.0 mg, 0.031 mmol, 1.0 eq) (synthetic method as described in the LEC-22CD34B example) and the crude LA-34BVC-b (0.031 mmol, 1.0 eq) at 0 °C. The mixture was stirred at 25 °C for 1 hour, and LC-MS showed complete reaction of the starting material. The reaction solution was purified by C18 reversed-phase column chromatography with water (0.25% TFA) and acetonitrile (0.25% TFA) as the mobile phase, with acetonitrile elution ratios ranging from 0% to 70%, yielding a yellow solid product LEC-22CD34BVC-a (33.0 mg, yield 48.91%).

[0514] LCMS(ESI): m / z, 1171.7[(M / 2+H)] + .

[0515] Step 3: Preparation of compound LEC-22CD34BVC-b

[0516] Diethylamine (0.015 mL, 0.141 mmol, 10.0 eq) was added to a 2.0 mL DMF solution of compound LEC-22CD34BVC-a (33.0 mg, 0.014 mmol, 1.0 eq) at 0 °C and stirred at 25 °C for 1 hour. LC-MS showed that the reaction proceeds were completely reacted. The reaction solution was purified by C18 reversed-phase column chromatography using water (0.25% TFA)-acetonitrile (0.25% TFA) as the mobile phase. The reaction solution was then concentrated under reduced pressure to give crude yellow solid LEC-22CD34BVC-b. This crude product was used directly in the next step without further purification.

[0517] LCMS(ESI): m / z, 1060.7 [(M / 2)] + .

[0518] Step 4: Preparation of compound LEC-22CD34BVC

[0519] At 0°C, DIEA (0.007 mL, 0.042 mmol, 3.0 eq), LY-22CD-3-d (12.34 mg, 0.015 mmol, 1.1 eq) and HATU (6.42 mg, 0.017 mmol, 1.2 eq) were added to a DMF (1.5 mL) solution of crude LEC-22CD34BVC-b (0.014 mmol, 1.0 eq) at WO2024175069A1 for synthesis of this compound, and the mixture was stirred at 25°C for 1 hour. LCMS showed that the starting material reacted completely. The reaction solution was purified by high performance liquid chromatography (HPLC) using a Bonnasil-BS C18 column (model JB-C235-04, 20×250mm, 8μm) with water (0.1% TFA) and acetonitrile as the mobile phase, with acetonitrile elution ratios ranging from 20% to 60%, to obtain a yellow solid product LEC-22CD34BVC (14.24 mg, yield 34.87%).

[0520] LCMS(ESI): m / z, 1449.8 [(M / 2)] + .

[0521] 1H NMR (400MHz, DMSO-d6) δ9.04 (t, J=5.6Hz, 1H), 8.88–8.83 (m, 1H), 8.65 (t, J=6.4Hz, 1 H), 8.54 (t, J=6.6Hz, 1H), 8.39–8.35 (m, 1H), 8.03 (t, J=7.7Hz, 2H), 7.92–7.82 (m, 4H) , 7.77(d, J=10.9Hz, 1H), 7.30(s, 1H), 5.94(s, 1H), 5.75(s, 2H), 5.42(s, 2H), 5.23(d , J=3.5Hz, 3H), 5.03 (d, J=12.1Hz, 2H), 4.78 (d, J=27.5Hz, 2H), 4.63 (d, J=2.0Hz, 1H), 4.59–4.53(m, 4H), 4.49–4.44(m, 1H), 4.30–3.96(m, 17H), 3.87–3.75(m, 4H), 3.65(s , 3H), 3.63–3.48 (m, 43H), 3.33–3.05 (m, 15H), 2.94 (d, J=5.8Hz, 3H), 2.83 (d, J=10.4H z, 1H), 2.79–2.63 (m, 4H), 2.58 (t, J=6.7Hz, 3H), 2.47–2.42 (m, 2H), 2.40–2.08 (m, 20H ), 2.04–1.79 (m, 12H), 1.75–1.11 (m, 26H), 1.02 (d, J=6.3Hz, 3H), 0.90–0.77 (m, 17H).

[0522] Example 14 Preparation of compound LEC-22CD34BVC2

[0523] Step 1: Preparation of compound LEC-22CD34BVC2-a

[0524] At 0°C, DIEA (0.789 mL, 4.776 mmol, 3.0 eq) was added to a DMF (30.0 mL) solution of compound LC-CL-03-a (synthesis of which can be found in the example of LEC-01) (1200 mg, 1.592 mmol, 1.0 eq) and amino-octavalent polyethylene glycol tert-butyl ester (1900.0 mg, 3.821 mmol, 2.4 eq), and the mixture was stirred at 0°C for 20 minutes. LC-MS showed that the reaction was complete. The reaction solution was purified by C18 reversed-phase column chromatography with water (0.25% TFA) and acetonitrile (0.25% TFA) as the mobile phase. The acetonitrile elution ratio ranged from 0% to 60%, yielding a yellow oily product LEC-22CD34BVC2-a (1980.0 mg, yield 81.89%).

[0525] LCMS(ESI): m / z, 1517.5 [(M)] + .

[0526] Step 2: Preparation of compound LEC-22CD34BVC2-b

[0527] TFA (8.0 mL) was added to a DCM (40 mL) solution of compound LEC-22CD34BVC2-a (1980 mg, 1.304 mmol, 1 eq) at 25 °C and stirred at 25 °C for 1 hour. LC-MS showed that the reaction proceeds were completely reacted. After concentration under reduced pressure, the crude product was purified by C18 reversed-phase column chromatography using water (0.25% TFA) and acetonitrile (0.25% TFA) as the mobile phase. The acetonitrile elution ratio ranged from 0% to 50%, yielding a yellow oily product LEC-22CD34BVC2-b (1640.0 mg, yield 89.44%).

[0528] LCMS(ESI): m / z, 1405.5 [(M)] + .

[0529] Step 3: Preparation of compound LEC-22CD34BVC2-c

[0530] HATU (50.64 mg, 0.133 mmol, 1.0 eq) and DIEA (0.044 mL, 0.266 mmol, 2.0 eq) were added to a DMF (6.0 mL) solution of compounds LEC-22CD34BVC2-b (374.68 mg, 0.266 mmol, 2.0 eq) and LY-22CD-b (for the synthesis of this compound, please refer to WO2024175069A1) (100 mg, 0.133 mmol, 1.0 eq) at 0 °C, and the mixture was stirred at 25 °C for 1 hour. LCMS showed that the starting materials reacted completely. The reaction solution was purified by C18 reversed-phase column chromatography with water (0.25% TFA) and acetonitrile (0.25% TFA) as the mobile phase. The acetonitrile elution ratio ranged from 0% to 60%, and the purified product was a yellow solid, LEC-22CD34BVC2-c (210.0 mg, yield 73.70%).

[0531] LCMS(ESI): m / z, 1069.5 [(M / 2)] + .

[0532] Step 4: Preparation of compound LEC-22CD34BVC2-e

[0533] Compounds LEC-22CD34BVC2-c (50 mg, 0.023 mmol, 1 eq), DIEA (0.012 mL, 0.070 mmol, 3.0 eq), and HATU (10.66 mg, 0.028 mmol, 1.3 eq) were added to a DMF (2 mL) solution of compound LA-34BVC-b (27.49 mg, 0.026 mmol, 1.1 eq) at 0 °C and stirred at 25 °C for 1 hour. LC-MS showed that the reaction proceeds were completely reacted. The reaction solution was purified by C18 reversed-phase column chromatography with a mobile phase of water (0.25% TFA)-acetonitrile (0.25% TFA), eluting at acetonitrile ratios from 0% to 41%, yielding a yellow solid product LEC-22CD34BVC2-e (28 mg, yield 37.55%).

[0534] LCMS(ESI): m / z, 1594.8 [M / 2] + , Rt=1.551min.

[0535] Step 5: Preparation of compound LEC-22CD34BVC2-f

[0536] Diethylamine (0.009 mL, 0.088 mmol, 10 eq) was added to 2 mL of DMF containing LEC-22CD34BVC2-e (28 mg, 0.009 mmol, 1 eq) at 0 °C. The mixture was then stirred at 25 °C for 1 h. LCMS showed the reaction was complete. The reaction solution was concentrated under vacuum to obtain LEC-22CD34BVC2-f (26.0 mg, crude product), a yellow oil. The crude product was used directly in the next step without any purification. LCMS (ESI): m / z, 1483.9 [M / 2] + Rt = 1.400 min.

[0537] Step 6: Preparation of compound LEC-22CD34BVC2

[0538] At 0 °C, compound DIEA (0.004 mL, 0.026 mmol, 3.0 eq), compound LY-22CD-3-b (4.91 mg, 0.013 mmol, 1.5 eq) and HATU (4.33 mg, 0.011 mmol, 1.3 eq) were added to a DMF (2 mL) solution of compound LEC-22CD34BVC2-f (26 mg, 0.009 mmol, 1.0 eq) at WO2024175069A1 at 2 mL. The mixture was stirred at 25 °C for 1 hour. LCMS showed that the starting material reacted completely. The reaction solution was purified by BSC 18, 20×250 mm, 8 μm (37-57% H2O (0.1% TFA) / ACN) to obtain LEC-22CD34BVC2 (7.18 mg, yield 24.67%) as a yellow solid. LCMS (ESI): m / z, 1681.6 [M-1] + Rt = 1.327 min.

[0539] 1H NMR (400MHz, DMSO) δ9.06 (t, J=5.5Hz, 1H), 8.86 (d, J=8.6Hz, 1H), 8.64 (t, J=6.3Hz, 1H), 8.53 (t, J=6.3Hz, 1H) , 8.37 (d, J=8.6Hz, 1H), 8.02 (t, J=6.6Hz, 2H), 7.85 (ddd, J=34.6, 21.9, 10.4Hz, 6H), 7.31 (s, 1H), 7.20 (s, 1H) , 6.65 (s, 1H), 6.51 (s, 1H), 5.92 (s, 1H), 5.42 (s, 2H), 5.33 (dd, J=13.1, 8.3Hz, 5H), 5.24 (s, 3H), 5.03 (d, J=11 .5Hz, 2H), 4.82 (s, 1H), 4.75 (s, 1H), 4.63 (s, 1H), 4.54 (dd, J=8.5, 4.8Hz, 4H), 4.47 (dd, J=10.3, 6.4Hz, 1H), 4 .19 (dd, J=15.1, 8.0Hz, 4H), 4.12–3.96 (m, 7H), 3.79 (dt, J=13.4, 6.8Hz, 2H), 3.65 (d, J=4.9Hz, 4H), 3.56 (dt, J=10.7, 5.5Hz, 10H), 3.49 (d, J=1.6Hz, 46H), 3.43 (s, 5H), 3.26 (d, J=3.0Hz, 5H), 3.20–3.16 (m, 7H), 2.96–2.9 2(m, 4H), 2.83(d, J=10.3Hz, 2H), 2.80–2.73(m, 2H), 2.38(s, 5H), 2.31–2.27(m, 8H), 2.04–1.91(m, 19H), 1.66 –1.59 (m, 6H), 1.46 (d, J=6.8Hz, 4H), 1.23 (s, 41H), 1.03 (d, J=6.3Hz, 3H), 0.83 (ddd, J=9.5, 9.0, 4.8Hz, 20H).

[0540] Example 15 Preparation of compound LEC-22CD35A

[0541] Step 1: Preparation of compound LEC-22CD35A-a

[0542] At 0°C, DIEA (81.36 mg, 623.25 μmol, 20 eq) was added to a DMF (2 mL) solution of compound LEC-22CD34B-a (see the example for the synthesis of this compound LEC-22CD34B) (43.30 mg, 31.16 μmol, 1 eq) and compound LA-35A-d (31.14 mg, 31.16 μmol, 1 eq). After addition, the reaction mixture was stirred at 25°C for 2 hours. LC-MS showed that the starting materials reacted completely. The reaction mixture was filtered and then prepared by high-performance liquid chromatography (HPLC) (Oriendo BRIX-2860 chromatographic column, Welch Xtimate C18). 250×30mm, 10μm. The mobile phase was H2O (0.225% HCOOH)-acetonitrile (elution ratio of acetonitrile from 50% to 80%). After purification, the compound was lyophilized to give a white solid compound LEC-22CD35A-a (32.10 mg, yield 45.31%).

[0543] LCMS(ESI): m / z, 1137.2[(M+2) / 2] + .

[0544] Step 2: Preparation of compound LEC-22CD35A-b

[0545] Diethylamine (5.22 mg, 7.35 μL, 70.59 μmol, 5 eq) was added to a DMF (1 mL) solution of compound LEC-22CD35A-a (32.10 mg, 14.12 μmol, 1 eq) at 0 °C and stirred at 25 °C for 1 hour. LCMS showed that the reaction was complete. After concentration, the reaction solution yielded a yellow oily compound LEC-22CD35A-b (28.96 mg, crude product).

[0546] LCMS(ESI):m / z,1026.8[(M+2) / 2] + .

[0547] Step 3: Preparation of compound LEC-22CD35A

[0548] At 0°C, DIEA (5.53 mg, 42.35 μmol, 3 eq) and HATU (6.64 mg, 16.94 μmol, 1.2 eq) were added to a DMF (1 mL) solution of compound LEC-22CD35A-b (28.96 mg, 14.12 μmol, 1 eq) and compound LEC-03-d (for the synthesis of this compound, please refer to the example of LEC-03) (28.41 mg, 16.94 μmol, 1.2 eq). The mixture was stirred at 25°C for 1 hour. LC-MS showed that the reaction was complete. The reaction solution was filtered and then prepared by high-performance liquid chromatography (HPLC) (Oriendo BRIX-2860 chromatographic column, Welch Xtimate C18). 150×30mm, 10μm. Purification was carried out using water (0.225% HCOOH)-acetonitrile as the mobile phase (elution ratio of acetonitrile from 30% to 60%), followed by lyophilization to obtain a white solid compound LEC-22CD35A (17.02 mg, yield 32.49%, purity 96.31%).

[0549] LCMS(ESI): m / z, 1237.2[(M+3) / 3] + .

[0550] 1H NMR (400MHz, DMSO-d6) δ9.10 (t, J=5.6Hz, 1H), 8.86 (d, J=8.6Hz, 1H), 8.54 (q, J=6.9Hz, 2H), 8.37 (d, J=8.6Hz, 1H), 8.06 (d, J=7.2Hz, 2H), 7.97–7.76(m, 8H), 7.30(s, 1H), 6.53(s, 1H), 5.42(s, 2H), 5.23(s, 3H), 5.03(d, J=14.5Hz, 2H), 4.81(s, 1H), 4.74 (s, 1H), 4.63 (s, 1H), 4.58–4.44 (m, 8H), 4.32 (s, 2H), 4.20 (dt, J=14.8, 7.3Hz, 8H), 4.11–3.98 (m, 7H), 3.79 (t, J=10.7 Hz, 2H), 3.65 (s, 6H), 3.61 (d, J=8.2Hz, 3H), 3.57 (q, J=6.3Hz, 12H), 3.53 (d, J=2.9Hz, 3H), 3.48 (dtd, J=8.1, 5.2, 3.1Hz, 73 H), 3.38 (dd, J=12.6, 7.4Hz, 13H), 3.30 (dd, J=7.1, 3.1Hz, 4H), 3.25 (s, 4H), 3.18 (s, 2H), 3.00 (dd, J=23.5, 6.8Hz, 4H), 2.8 4–2.71 (m, 4H), 2.68–2.66 (m, 1H), 2.58 (t, J=6.4Hz, 4H), 2.43 (t, J=7.0Hz, 3H), 2.36 (d, J=12.7Hz, 7H), 2.34–2.30 (m, 3H), 2.26 (q, J=8.5, 7.6Hz, 5H), 2.15 (d, J=28.1Hz, 4H), 2.03–1.80 (m, 11H), 1.63 (td, J=31.9, 27.8, 17.5Hz, 10H), 1.43 (t, J=7. 1Hz, 4H), 1.34 (d, J=7.5Hz, 6H), 1.20 (d, J=7.1Hz, 13H), 1.02 (d, J=6.4Hz, 3H), 0.97 (d, J=11.6Hz, 1H), 0.90–0.74 (m, 16H).

[0551] Example 16 Preparation of compound LEC-22CD36BVA2

[0552] Step 1: Preparation of compound LEC-22CD36BVA2-a

[0553] At 25°C, HATU (15.31 mg, 0.040 mmol, 1.3 eq) and DIEA (0.015 mL, 0.093 mmol, 3.0 eq) were added to a DMF (1.5 mL) solution of compounds LEC-22CD34BVC2-c (for the synthesis of this compound, please refer to the example of LEC-22CD34BVC2) (72.89 mg, 0.034 mmol, 1.1 eq) and LA-36dPAB-b (28 mg, 0.031 mmol, 1.0 eq), and the mixture was stirred at 25°C for one hour. LCMS showed that the reaction was complete. The reaction solution was purified by C18 reversed-phase column chromatography with water (0.25% TFA) and acetonitrile (0.25% TFA) as the mobile phase. The acetonitrile elution ratio ranged from 0% to 60%, and the purified product was a yellow solid, LEC-22CD36BVA2-a (30.0 mg, yield 32.02%).

[0554] LCMS(ESI): m / z, 1512.2[(M / 2+H)] + .

[0555] Step 2: Preparation of compound LEC-22CD36BVA2-b

[0556] Diethylamine (0.013 mL, 0.129 mmol, 10.0 eq) was added to a 1.5 mL DMF solution of compound LEC-22CD36BVA2-a (30.0 mg, 0.010 mmol, 1.0 eq) at 25 °C and stirred at 25 °C for 1 hour. LC-MS showed that the reaction proceeds were completely reacted. The reaction solution was concentrated under reduced pressure to obtain a yellow oily crude product of LEC-22CD36BVA2-b (the yield of the crude product was not considered). The crude product was used directly in the next step without purification.

[0557] LCMS(ESI): m / z, 1401.2[(M / 2+H)] + .

[0558] Step 3: Preparation of compound LEC-22CD36BVA2

[0559] HATU (5.66 mg, 0.015 mmol, 1.5 eq) and DIEA (0.005 mL, 0.030 mmol, 3.0 eq) were added to a DMF (1.5 mL) solution of crude LEC-22CD36BVA2-b (27.8 mg, 0.010 mmol, 1.0 eq) and LY-22CD-3-b (for the synthesis of this compound, please refer to WO2024175069A1) (5.55 mg, 0.015 mmol, 1.5 eq) at 25 °C and stirred at 25 °C for 1 hour. LCMS showed that the starting materials reacted completely. The reaction solution was evaporated to dryness and purified by high performance liquid chromatography (HPLC) using a Bonnasil-BS C18 column (model JB-C235-04, 20×250mm, 8μm) with water (0.1% TFA) and acetonitrile as the mobile phase, with acetonitrile elution ratios ranging from 20% to 60%, to obtain a yellow solid product LEC-22CD36BVA2 (10.26 mg, yield 32.76%).

[0560] LCMS(ESI): m / z, 1578.7 [(M / 2)] + .

[0561] 1H NMR (400MHz, DMSO-d6) δ9.05 (s, 1H), 8.86 (d, J=8.6Hz, 1H), 8.53 (s, 1H), 8.37 (d, J=8.6Hz, 1H), 8.03 (d, J=7.2Hz, 1H), 7.95 (d, J=7.4Hz, 1H), 7.92–7.82 (m, 5H), 7. 77 (d, J=10.9Hz, 1H), 7.72 (s, 1H), 7.31 (s, 1H), 5.42 (s, 2H), 5.32 (t, J=4.6Hz, 1H ), 5.24(s, 3H), 4.64(t, J=4.5Hz, 1H), 4.54(dd, J=9.2, 5.6Hz, 2H), 4.50–4.46(m, 1 H), 4.41–4.36 (m, 1H), 4.30–4.02 (m, 13H), 3.88–3.72 (m, 7H), 3.65 (s, 3H), 3.61– 3.51(m, 28H), 3.45–3.35(m, 35H), 3.23(s, 3H), 3.20–3.15(m, 5H), 3.14–3.05(m, 3 H), 3.03–2.96(m, 2H), 2.90–2.55(m, 10H), 2.45–2.10(m, 21H), 2.07–1.77(m, 14H ), 1.72–1.35(m, 17H), 1.31–1.17(m, 20H), 0.95–0.93(m, 2H), 0.90–0.77(m, 18H).

[0562] Example 17 Preparation of compound LEC-22CD36BVC2

[0563] Step 1: Preparation of compound LEC-22CD36BVC2-a

[0564] At 25°C, HATU (11.55 mg, 0.030 mmol, 1.3 eq) and DIEA (0.012 mL, 0.070 mmol, 3.0 eq) were added to a DMF (1.5 mL) solution of compounds LEC-22CD34BVC2-c (see the example of LEC-22CD34BVC2 for its synthesis) (50.0 mg, 0.023 mmol, 1.0 eq) and LA-36VCdPAB-b (25.45 mg, 0.026 mmol, 1.1 eq), and the mixture was stirred at 25°C for one hour. LCMS showed that the reaction was complete. The reaction solution was purified by C18 reversed-phase column chromatography with water (0.25% TFA) and acetonitrile (0.25% TFA) as the mobile phase. The acetonitrile elution ratio ranged from 0% to 60%, and the purified product was a yellow solid, LEC-22CD36BVC2-a (36.0 mg, yield 49.50%).

[0565] LCMS(ESI): m / z, 1555.3[(M / 2+H)] + .

[0566] Step 2: Preparation of compound LEC-22CD36BVC2-b

[0567] Diethylamine (0.010 mL, 0.096 mmol, 10.0 eq) was added to a 1.5 mL DMF solution of compound LEC-22CD36BVC2-a (30 mg, 0.013 mmol, 1.0 eq) at 25 °C and stirred at 25 °C for 1 hour. LC-MS showed that the reaction proceeds were completely reacted. The reaction solution was concentrated under reduced pressure to obtain a yellow oily crude product of LEC-22CD36BVC2-b (the yield of the crude product was not considered). The crude product was used directly in the next step without purification.

[0568] LCMS(ESI): m / z, 1444.3[(M / 2+H)] + .

[0569] Step 3: Preparation of compound LEC-22CD36BVC2

[0570] HATU (4.76 mg, 0.013 mmol, 1.3 eq) and DIEA (0.005 mL, 0.029 mmol, 3.0 eq) were added to a DMF (1.5 mL) solution of crude LEC-22CD36BVC2-b (27.8 mg, 0.010 mmol, 1.0 eq) and LY-22CD-3-b (for the synthesis of this compound, please refer to WO2024175069A1) (4.67 mg, 0.013 mmol, 1.3 eq) at 25 °C and stirred at 25 °C for 1 hour. LCMS showed that the starting material reacted completely. The reaction solution was evaporated to dryness and purified by high performance liquid chromatography (HPLC) using a Bonnasil-BS C18 column (model JB-C235-04, 20×250mm, 8μm) with water (0.1% TFA) and acetonitrile as the mobile phase, with acetonitrile elution ratios ranging from 20% to 60%, to obtain a yellow solid product LEC-22CD36BVC2 (8.83 mg, yield 28.28%).

[0571] LCMS(ESI): m / z, 1621.8 [(M / 2)] + .

[0572] 1H NMR (400MHz, DMSO-d6) δ9.05 (t, J=5.6Hz, 1H), 8.86 (d, J=8.6Hz, 1H), 8.53 (t, J=6.5Hz, 1H ), 8.37 (d, J=8.6Hz, 1H), 8.03 (d, J=7.1Hz, 1H), 7.87 (dt, J=12.9, 8.3Hz, 6H), 7.75 (dd, J= 14.6, 8.1Hz, 2H), 7.31 (s, 1H), 5.90 (s, 1H), 5.42 (s, 2H), 5.32 (t, J=4.7Hz, 1H), 5.24 (s, 3 H), 4.64 (t, J=4.4Hz, 1H), 4.55 (dd, J=10.1, 6.6Hz, 2H), 4.50–4.46 (m, 1H), 4.39 (s, 1H), 4. 27–4.01 (m, 13H), 3.65 (s, 3H), 3.61–3.55 (m, 48H), 3.43 (dd, J=8.8, 4.1Hz, 13H), 3.38 (t, J=5.7Hz, 6H), 3.27–3.23(m, 5H), 3.18(dd, J=10.8, 5.2Hz, 5H), 3.04(s, 3H), 2.93(s, 3H), 2 .87–2.73(m, 4H), 2.69–2.57(m, 5H), 2.46–2.25(m, 18H), 2.24–2.09(m, 6H), 2.06–1.74(m , 15H), 1.72–1.41 (m, 19H), 1.37–1.18 (m, 19H), 0.94 (d, J=6.5Hz, 3H), 0.90–0.78 (m, 18H).

[0573] Example 18: Preparation of LEC-2236M

[0574] Step 1: Preparation of compound LEC-2236M

[0575] At room temperature, HATU (16 mg, 0.065 mmol, 1.5 eq) and DIEA (17 mg, 0.129 mmol, 3.0 eq) were added to a DMF (3 mL) solution of compound LEC-22CD36BVC2-b (for the synthesis of this compound, please refer to the example of LEC-22CD36BVC2) (125 mg, 0.043 mmol, 1.0 eq) and 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethoxy)ethoxy)propionic acid (17 mg, 0.065 mmol, 1.5 eq). The mixture was stirred at room temperature for one hour, and LCMS showed that the reaction was complete. The reaction solution was purified by high performance liquid chromatography (JB-C235-04 column, Bonnasil-BS C18, 20×250mm, 8μm) with water (0.1% TFA) and acetonitrile as the mobile phase, with acetonitrile elution ratios ranging from 20% to 60%, to obtain a yellow solid product LEC-2236M (25.71 mg, yield 19%).

[0576] LCMS(ESI):m / z[M / 2] + =1563.9.

[0577] 1H NMR (400MHz, DMSO) δ8.54 (t, J=6.6Hz, 1H), 8.03 (d, J=7.2Hz, 1H), 7.96–7.81 (m, 6H), 7.79–7.72(m, 2H), 7.31(s, 1H), 7.01(s, 2H), 6.51(s, 1H), 5.88(s, 1H), 5.42 (s, 2H), 5.35–5.30 (m, 1H), 5.24 (s, 3H), 4.64 (t, J=4.5Hz, 1H), 4.58–4.52 (m, 2H) , 4.48 (d, J=6.5Hz, 1H), 4.43–4.36 (m, 1H), 4.25–4.05 (m, 10H), 3.89–3.72 (m, 5H) , 3.56 (dt, J=6.7, 5.7Hz, 12H), 3.52–3.45 (m, 78H), 3.27–3.22 (m, 5H), 3.21–3.1 6(m, 5H), 3.04(s, 2H), 2.93(s, 2H), 2.88–2.81(m, 2H), 2.79–2.72(m, 1H), 2.64(d , J=6.9Hz, 1H), 2.56(t, J=6.7Hz, 2H), 2.45–2.11(m, 20H), 2.05–1.77(m, 12H), 1. 71–1.42 (m, 17H), 1.40–1.12 (m, 14H), 0.94 (d, J=6.5Hz, 2H), 0.90–0.76 (m, 17H).

[0578] Example 19: Preparation of compound LEC-2236P

[0579] Step 1: Preparation of compound LEC-2236P

[0580] At room temperature, HATU (16 mg, 0.065 mmol, 1.5 eq) and DIEA (17 mg, 0.129 mmol, 3.0 eq) were added to a DMF (3.0 mL) solution of compound LEC-22CD36BVC2-b (for the synthesis of this compound, please refer to the example of LEC-22CD36BVC2) (125 mg, 0.043 mmol, 1.0 eq) and 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-alkynic acid (17 mg, 0.065 mmol, 1.5 eq). The mixture was stirred at room temperature for one hour, and LCMS showed that the reaction was complete. The reaction solution was prepared by high performance liquid chromatography (JB-C235-04 column, Bonnasil-BS C18, 20×250mm, 8μm, mobile phase water (0.1% TFA)-acetonitrile, elution ratio of acetonitrile from 20% to 60%), and purified to give a white solid product LEC-2236P (35.57mg, yield 26.19%).

[0581] LCMS(ESI): m / z, [M / 2+H] + =1569.4.

[0582] 1 H NMR (400MHz, DMSO-d6) δ9.11 (s, 2H), 8.53 (t, J=6.7Hz, 1H), 8.03 (d, J=7.3Hz , 1H), 7.94–7.82(m, 6H), 7.80–7.73(m, 2H), 7.31(s, 1H), 6.50(s, 1H), 5.89(s , 1H), 5.42 (s, 2H), 5.32 (t, J = 4.6Hz, 2H), 5.24 (s, 3H), 4.64 (t, J = 4.5Hz, 1H), 4.58–4.52(m, 2H), 4.47(dd, J=10.2, 6.6Hz, 1H), 4.39(s, 1H), 4.26–4.03(m, 1 1H), 3.88–3.72 (m, 5H), 3.56 (d, J=6.9Hz, 6H), 3.51–3.45 (m, 69H), 3.27–3.2 3(m, 5H), 3.18(dd, J=10.8, 5.7Hz, 5H), 3.04(s, 3H), 2.96–2.69(m, 7H), 2.43– 2.26(m, 15H), 2.24–2.08(m, 6H), 2.03–1.89(m, 9H), 1.87–1.75(m, 6H), 1.71– 1.42 (m, 18H), 1.37–1.19 (m, 18H), 0.94 (d, J=6.5Hz, 2H), 0.89–0.79 (m, 17H).

[0583] Example 20 Preparation of compound LEC-22CD36dPAB

[0584] Step 1: Preparation of compound PE-E2K-a

[0585] NaHCO3 (91.54 mg, 1.090 mmol, 3 eq) and Fmoc-Osu (367.55 mg, 1.090 mmol, 3 eq) were added to a THF (6 mL) solution of eribulin methanesulfonate (330 mg, 0.363 mmol, 1 eq) at 0 °C. The reaction mixture was stirred at 25 °C for 18 hours. LCMS showed that the reaction was complete. The reaction mixture was concentrated under vacuum, and the crude product was purified by silica gel column chromatography (5% dichloromethane / methanol) to give a white solid product PE-E2K-a (330 mg, yield 95.43%), LCMS (ESI): m / z, 951.7 [M]. + Rt = 1.872 min.

[0586] Step 2: Preparation of compound PE-E2K-b

[0587] Potassium osmium tetroxide dihydrate (10.79 mg, 0.035 mmol, 0.1 eq) and NaIO4 (1482.62 mg, 6.932 mmol, 20 eq) were added to a THF (10 mL) and water (3 mL) solution of compound PE-E2K-a (330 mg, 0.347 mmol, 1 eq) at 0 °C. The reaction solution was stirred at 25 °C for 2 hours. NaIO4 (1482.62 mg, 6.932 mmol, 20 eq) was then added to the reaction solution, and the reaction solution was stirred at 25 °C overnight. LC-MS showed that the reaction was complete. The reaction solution was concentrated under vacuum at low temperature and extracted with dichloromethane. The separated organic phase was concentrated under vacuum at low temperature, and the crude product was purified by silica gel column chromatography (10 g, DCM:MeOH:THF = 10:1:1) to obtain a white solid product PE-E2K-b (95 mg, yield 28.67%), LCMS (ESI): m / z, 955.8 [M-1]. + Rt = 1.641 min.

[0588] Step 3: Preparation of compound PE-2K

[0589] Diethylamine (0.049 mL, 0.476 mmol, 5 eq) was added to a DMF (1.5 mL) solution of compound PE-E2K-b (91 mg, 0.095 mmol, 1 eq). The reaction solution was stirred at 25 °C for 1 hour, and LCMS showed that the starting material reacted completely. The reaction solution was concentrated under vacuum at low temperature to obtain crude PE-E2K (69 mg, crude product), which was a pale yellow oil. LCMS (ESI): m / z, 734.1 [M] + Rt = 1.152 min.

[0590] Step 4: Preparation of compound LEC-22CD36dPAB-b1

[0591] At 0 °C, DIEA (0.042 mL, 0.253 mmol, 3 eq), N-[(5S)-1-(9H-fluorene-9-yl)-3,6-dioxo-5-(propanoyl-2-yl)-4-aza-2-oxahexyl-6-yl]-L-alanine (41.61 mg, 0.101 mmol, 1.2 eq), and HATU (38.55 mg, 0.101 mmol, 1.2 eq) were added to a 2 mL DMF solution of compound PE-2K (62 mg, 0.084 mmol, 1 eq). The mixture was then stirred at 25 °C for 1 h. LC-MS showed that the reaction was complete. The reaction solution was purified by silica gel column chromatography (C18, H2O (0.25% TFA); ACN = 36%:64%) to obtain LEC-22CD36dPAB-b1 (43 mg, yield 45.19%), a white solid, LCMS (ESI): m / z, 1126.1 [M-1]. + Rt = 1.611 min.

[0592] Step 5: Preparation of compound LEC-22CD36dPAB-b

[0593] Diethylamine (0.023 mL, 0.220 mmol, 10 eq) was added to a 2 mL DMF solution of LEC-22CD36dPAB-b1 (30 mg, 0.022 mmol, 1 eq) at 0 °C. The mixture was then stirred at 25 °C for 1 h. LCMS showed the reaction was complete. The reaction solution was concentrated under vacuum to obtain LEC-22CD36dPAB-b (19.75 mg, crude product), a yellow oil. The crude product was used directly in the next step without any purification. LCMS (ESI): m / z, 904.2 [M+1] + Rt = 1.144 min.

[0594] Step 6: Preparation of compound LEC-22CD34B-a

[0595] At 0°C, LY-22CD-b (50 mg, 0.067 mmol, 1 eq) and DIEA (0.028 mL, 0.166 mmol, 2.5 eq) were added to a DMF (3 mL) solution of compound LC-CL-03-a (synthesis of which can be found in Example LEC-01) (125.49 mg, 0.166 mmol, 2.5 eq) (synthesis of which can be found in WO2024175069A1 ... The crude product was purified by silica gel column chromatography (10 g, DCM:MeOH:THF = 10:1:1) to give LEC-22CD34B-a (22 mg, yield 23.78%) as a white solid. LCMS (ESI): m / z, 1389.8 [M] + Rt = 1.566 min.

[0596] Step 7: Preparation of compound LEC-22CD36dPAB-a

[0597] DIEA (0.018 mL, 0.109 mmol, 5 eq) and LEC-22CD34B-a (30.35 mg, 0.022 mmol, 1 eq) were added to a 2 mL DMF solution of LEC-22CD36dPAB-b (19.75 mg, 0.022 mmol). The mixture was then stirred at 35 °C for 1 h. LCMS showed that the reaction was complete.

[0598] The reactants were purified by silica gel column chromatography (C18, 47% H2O (0.25% TFA) / ACN) to give LEC-22CD36dPAB-a (24 mg, yield 50.43%) as a white solid. LCMS (ESI): m / z, 1089.0 [1 / 2 M]. + Rt = 1.592 min.

[0599] Step 8: Preparation of compound LEC-22CD36dPAB-b

[0600] Diethylamine (0.011 mL + 0.110 mmol, 10 eq) was added to a 2 mL solution of LEC-22CD36dPAB-a (24 mg, 0.011 mmol, 1 eq) in DMF at 0 °C. The mixture was then stirred at 25 °C for 1 h. LCMS showed that the reaction was complete. The reaction solution was purified by silica gel column chromatography (C18, 47% H2O (0.25% TFA) / ACN) to give LEC-22CD36dPAB-b (20 mg, yield 92.81%) as a white solid. LCMS (ESI): m / z, 1955.7 [M] + Rt = 1.349 min.

[0601] Step 9: Preparation of compound LEC-22CD36dPAB

[0602] At 0 °C, DIEA (0.005 mL, 0.030 mmol, 3 eq), LEC-03-d (18.45 mg, 0.011 mmol, 1.1 eq) and HATU (4.94 mg, 0.013 mmol, 1.3 eq) were added to a DMF (1.5 mL) solution of compound LEC-22CD36dPAB-b (20 mg, 0.010 mmol, 1 eq) at 0 °C. The mixture was then stirred at 25 °C for 1 h. LCMS showed that the reaction was complete.

[0603] The reaction solution was purified by high performance liquid chromatography (SunFire Prep C18 OBD, 50×250 mm, 10 μm (25-55% H2O (0.1% TFA) / ACN) to obtain LEC-22CD36dPAB (4.73 mg, yield 12.80%) as a white solid. LCMS (ESI): m / z, 1807.0 [M / 2] + Rt = 1.254 min.

[0604] 1H NMR (400MHz, DMSO-d6) δ9.08 (s, 1H), 8.86 (d, J = 8.6Hz, 1H), 8.54 (s, 1H), 8.38 ( d,J=8.6Hz, 1H), 8.12(s, 1H), 8.04(d,J=7.2Hz, 1H), 7.99–7.66(m, 10H), 7.32(s , 1H), 6.53 (s, 1H), 5.43 (s, 4H), 5.24 (s, 3H), 4.79 (s, 2H), 4.53 (ddd, J=42.7, 3 4.6, 15.2Hz, 11H), 4.28–3.97 (m, 15H), 3.80 (t, J=29.0Hz, 9H), 3.65 (s, 4H), 3.5 9–3.55(m, 12H), 3.53–3.46(m, 74H), 3.24(s, 6H), 3.17(s, 2H), 3.09(d,J=6.1H z, 4H), 2.98 (s, 5H), 2.89–2.81 (m, 3H), 2.77 (s, 2H), 2.58 (t, J = 6.7Hz, 4H), 2.37 (dd,J=20.3, 8.4Hz, 9H), 2.27(d,J=6.4Hz, 3H), 2.24–2.02(m, 7H), 2.00–1.78( m, 10H), 1.71–1.39 (m, 18H), 1.25 (dt,J=15.0, 7.8Hz, 16H), 1.01–0.78 (m, 20H).

[0605] Example 21 Preparation of compound LEC-22CD36dPABVA

[0606] Step 1: Preparation of compound LEC-22CD36dPABVA-a

[0607] At 25°C, HATU (16.83 mg, 0.044 mmol, 1.3 eq) and DIEA (0.017 mL, 0.102 mmol, 3.0 eq) were added to a DMF (2.0 mL) solution of compounds LEC-22CD34B-a (see the example of LEC-22CD34B for its synthesis) (44.0 mg, 0.034 mmol, 1.0 eq) and LA-36dPAB-b (30.78 mg, 0.034 mmol, 1.0 eq), and the mixture was stirred at 25°C for one hour. LCMS showed that the reaction was complete. The reaction solution was purified by C18 reversed-phase column chromatography with water (0.25% TFA) and acetonitrile (0.25% TFA) as the mobile phase. The acetonitrile elution ratio ranged from 0% to 60%, and the purified product was a yellow solid, LEC-22CD36dPABVA-a (20.89 mg, yield 28.17%).

[0608] LCMS(ESI): m / z, 1098.9 [fragment peak] + .

[0609] Step 2: Preparation of compound LEC-22CD36dPABVA-b

[0610] Diethylamine (0.010 mL, 0.096 mmol, 10.0 eq) was added to a 2.0 mL DMF solution of compound LEC-22CD36dPABVA-a (20.89 mg, 0.010 mmol, 1.0 eq) at 25 °C and stirred at 25 °C for 1 hour. LC-MS showed that the reaction proceeds were completely reacted. The reaction solution was concentrated under reduced pressure to obtain a yellow oily crude product of LEC-22CD36dPABVA-b (the yield of the crude product was not considered). The crude product was used directly in the next step without purification.

[0611] LCMS (ESI): m / z, 1954.6 [M] + .

[0612] Step 3: Preparation of compound LEC-22CD36dPABVA

[0613] At 25°C, HATU (5.47 mg, 0.010 mmol, 1.5 eq) and DIEA (0.010 mL, 0.058 mmol, 6.0 eq) were added to a DMF (2.0 mL) solution of crude LEC-22CD36dPABVA-b (18.76 mg, 0.010 mmol, 1.0 eq) and LY-22CD-3-b (for the synthesis of this compound, please refer to WO2024175069A1) (7.64 mg, 0.010 mmol, 1.0 eq). The mixture was stirred at 25°C for 1 hour. LCMS showed that the reaction of the starting materials was complete. The reaction solution was evaporated to dryness and purified by high performance liquid chromatography (JB-C235-04 column, Bonnasil-BS C18, 20×250mm, 8μm, mobile phase: water (0.1% TFA)-acetonitrile, elution ratio of acetonitrile from 20% to 60%) to obtain a yellow solid product LEC-22CD36dPABVA (2.40 mg, yield 9.15%).

[0614] LCMS(ESI): m / z, 1367.1 [(M / 2)] + .

[0615] Example 22 Preparation of compound LEC-22CD36VCdPAB

[0616] Step 1: Preparation of compound LEC-22CD36VCdPAB-a

[0617] At 0 °C, DIEA (0.043 mL, 0.260 mmol, 5 eq) and compound LA-36VCdPAB-b (51 mg, 0.052 mmol, 1 eq) were added to a 2 mL solution of DMF containing compound LEC-22CD34B-a (see the example for the synthesis of LEC-22CD34B) (72.25 mg, 0.052 mmol, 1 eq). The mixture was then stirred at 35 °C for 1 h. LCMS showed that the reaction was complete. The reactants were purified by silica gel column chromatography (C18, 41% H2O (0.025% TFA) / ACN) to give LEC-22CD36VCdPAB-a (67 mg, yield 56.90%) as a white solid. LCMS (ESI): m / z, 1132.1 [M+1] + Rt = 1.542 min.

[0618] Step 2: Preparation of compound LEC-22CD36VCdPAB-b

[0619] Diethylamine (0.021 mL, 0.207 mmol, 10 eq) was added to a 1.5 mL DMF solution of compound LEC-22CD36VCdPAB-a (67 mg, 0.021 mmol, 1 eq) at 25 °C. The reaction mixture was stirred at 25 °C for 1 hour, and LCMS showed that the starting material reacted completely. The reaction mixture was concentrated under vacuum at low temperature to obtain crude LEC-22CD36VCdPAB-b (40 mg, crude product), which was a pale yellow oil. LCMS (ESI): m / z, 1021.1 [M / 2] + Rt = 1.285 min.

[0620] Step 3: Preparation of compound LEC-22CD36VCdPAB

[0621] At 0 °C, DIEA (0.010 mL, 0.059 mmol, 3 eq), LY-22CD-3-d (18.73 mg, 0.024 mmol, 1.3 eq) and HATU (9.89 mg, 0.026 mmol, 1.3 eq) were added to a 2 mL DMF solution of compound LEC-22CD36VCdPAB-b (40 mg, 0.020 mmol, 1 eq) at 25 °C. The mixture was then stirred at 25 °C for 1 h. LC-MS showed that the reaction was complete.

[0622] The reaction solution was purified by high-performance liquid chromatography (Bonnasil-BS C18, 20×250 mm, 8 μm, 33-63% H2O (0.1% TFA) / ACN) to obtain LEC-22CD36VCdPAB (18.69 mg, yield 33.82%), a white solid, LCMS (ESI): m / z, 1410.3 [M / 2]. + Rt = 1.352 min.

[0623] 1H NMR (400MHz, DMSO) δ9.05 (t, J=5.6Hz, 1H), 8.86 (d, J=8.6Hz, 1H), 8.54 (t, J=6.4Hz, 1H), 8.37 (d, J=8.6Hz, 1H), 8.04 (d, J=7.2Hz, 1H), 7.93–7.82 (m, 4H), 7.77 (d, J=11.0Hz, 2H), 7.31 (s, 1H), 5.92 (s, 1H), 5.42 (s, 2H), 5.21 (t, J=11.8 Hz, 3H), 4.64 (t, J=4.4Hz, 1H), 4.54 (d, J=10.2Hz, 2H), 4.47 (dd, J=10.2, 6.4Hz, 1H), 4.38 (s, 1H), 4.28–3.99 (m, 10H ), 3.90–3.71(m, 5H), 3.65(s, 3H), 3.62(d, J=6.4Hz, 3H), 3.58(d, J=6.4Hz, 4H), 3.56–3.52(m, 5H), 3.50–3.45(m, 42 H), 3.36–3.30(m, 12H), 3.25(dd, J=8.0, 4.4Hz, 6H), 3.18(dd, J=11.8, 6.0Hz, 3H), 3.13(s, 1H), 3.04(s, 2H), 2.93(s , 2H), 2.88–2.81 (m, 2H), 2.76 (dd, J=13.0, 6.4Hz, 1H), 2.69–2.61 (m, 1H), 2.58 (t, J=6.8Hz, 2H), 2.47–2.41 (m, 2H), 2.34 (dd, J=15.8, 9.2Hz, 8H), 2.24 (d, J=17.8Hz, 2H), 2.17–2.08 (m, 3H), 2.07–2.00 (m, 1H), 1.98–1.76 (m, 9H), 1.73 –1.41 (m, 16H), 1.33 (dd, J=26.0, 14.2Hz, 3H), 1.25–1.19 (m, 5H), 0.97 (dd, J=21.0, 6.6Hz, 3H), 0.90–0.77 (m, 15H).

[0624] Example 23 Preparation of compound LEC-22CD36OdPAB

[0625] Step 1: Preparation of compound LA-36OdPAB-a1

[0626] Compound PE-AM1-a (200 mg, 0.274 mmol, 1 eq) (commercially purchased from Haoyuan Pharmaceutical) was dissolved in DMF / THF (4 mL / 4 mL), and LY-27a (197.66 mg, 0.410 mmol, 1.5 eq) (commercially purchased from Shandong Xingzhi) was added. TsOH (70.68 mg, 0.410 mmol, 1.5 eq) was added at room temperature, and the reaction mixture was stirred at 25 °C for 1 hour. LC-MS showed the target product. The reaction mixture was purified by C18 reversed-phase column chromatography with a mobile phase of water (0.25% TFA)-acetonitrile (0.25% TFA), eluting at acetonitrile ratios from 0% to 70%, yielding a yellow solid product LA-36OdPAB-a1 (125 mg, yield 39.64%).

[0627] LCMS(ESI): m / z, 1151.7 [M] + .

[0628] Step 2: Synthesis of compound LA-36OdPAB-a

[0629] K₂O₅O₄·2H₂O (3.24 mg, 0.010 mmol, 0.1 eq) and NaIO₄ (446.0 mg, 2.082 mmol, 20 eq) were added to compound LA-36OdPAB-a1 (125.0 mg, 0.108 mmol, 1.0 eq) at 0 °C, and the mixture was stirred at 25 °C for 18 hours. LC-MS showed that the reaction proceeds were completely reacted. The reaction solution was purified by C18 reversed-phase column chromatography with water (0.25% TFA) and acetonitrile (0.25% TFA) as the mobile phase (elution ratio of acetonitrile from 0% to 70%), yielding a yellow solid product LA-36OdPAB-a (32.5 mg, yield 26.02%).

[0630] LCMS(ESI): m / z, 1155.7 [M] + .

[0631] Step 3: Preparation of compound LA-36OdPAB-b

[0632] Compound LA-36OdPAB-a (32.5 mg, 0.028 mmol, 1 eq) was dissolved in DMF (2.0 mL), and diethylamine (0.029 mL, 0.281 mmol, 10 eq) was added. The reaction mixture was stirred at 25 °C for 1 hour. LC-MS showed that the starting material reacted completely. The reaction mixture was concentrated under reduced pressure to obtain crude LA-36OdPAB-b (26.25 mg, yield not calculated).

[0633] LCMS(ESI): m / z, 933.7 [M] + .

[0634] Step 4: Preparation of compound LEC-22CD36OdPAB-a

[0635] At 0°C, N,N-diisopropylethylamine (0.058 mL, 0.35 mmol, 3.0 eq) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (66 mg, 0.18 mmol, 1.5 eq) were added to a DMF (30.0 mL) solution of compound LEC-22CD34BVC2-c (for the synthesis of this compound, please refer to the example of LEC-22CD34BVC2) (250 mg, 0.12 mmol, 1.0 eq) and compound LA-36OdPAB-b (110 mg, 0.12 mmol, 1.0 eq), and stirred at 25°C for 30 minutes. The reaction solution was purified by C18 reversed-phase column chromatography with water (0.25% TFA) and acetonitrile (0.25% TFA) as the mobile phase. The acetonitrile elution ratio ranged from 0% to 60%, and the purified product was a yellow oil LEC-22CD36OdPAB-a (300 mg, yield 80%).

[0636] LCMS(ESI): m / z, 1527.4 [M / 2+1] + Rt = 1.35 min.

[0637] Step 5: Preparation of compound LEC-22CD36OdPAB-b

[0638] Diethylamine (0.09 mL, 0.88 mmol, 10 eq) was added to a 2 mL DMF solution of LEC-22CD36OdPAB-a (270 mg, 0.088 mmol, 1 eq) at 0 °C. The mixture was then stirred at 25 °C for 1 h. The reaction solution was concentrated under vacuum to obtain LEC-22CD36OdPAB-b (100 mg, crude product), which was a yellow oil. The crude product was used directly in the next step without any purification.

[0639] LCMS(ESI): m / z, 1416.4 [M / 2+1] + Rt = 1.98 min.

[0640] Step 6: Preparation of compound LEC-22CD36OdPAB

[0641] At 0 °C, N,N-diisopropylethylamine (0.018 mL, 0.106 mmol, 3.0 eq) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (20 mg, 0.053 mmol, 1.5 eq) were added to a DMF (3.0 mL) solution of compound LEC-22CD36OdPAB-b (100 mg, 0.035 mmol, 1.0 eq) and compound LY-22CD-3-b (for the synthesis of this compound, please refer to WO2024175069A1) (13 mg, 0.035 mmol, 1.0 eq) and stirred at 25 °C for 30 minutes. The reaction solution was purified by BSC18 (20×250mm, 8μm, 35-57% H2O (0.1% TFA) / ACN) to obtain LEC-22CD36OdPAB (6.2 mg, yield 5.5%), as a yellow solid.

[0642] LCMS(ESI):m / z,1615.6[M / 2+11] + , Rt=1.253min.

[0643] Example 24 Preparation of compound LEC-22CD36OVCdPAB

[0644] Step 1: Preparation of compound LEC-22CD36OVCdPAB-a

[0645] At 0°C, compound LA-36OVCdPAB-b (38 mg, 0.037 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (28.0 mg, 0.076 mmol), and N,N-diisopropylethylamine (14.5 mg, 0.12 mmol) were added to a solution of compound LEC-22CD34BVC2-c (for the synthesis of this compound, please refer to the example of LEC-22CD34BVC2) (80 mg, 0.037 mmol) in N,N-dimethylformamide (5 mL). The mixture was then stirred at 30°C for 1 hour. The organic phase was dried, filtered, and evaporated to obtain a crude product. The crude product was purified by C18 reversed-phase column chromatography with water (0.1% TFA) and acetonitrile as the mobile phase. The acetonitrile elution ratio ranged from 0% to 65%. The purified product yielded a yellow oily compound LEC-22CD36OVCdPAB-a (100 mg, yield 85%).

[0646] LCMS(ESI): m / z, 1570.4[M / 2+1] + Rt = 2.24 min.

[0647] Step 2: Preparation of compound LEC-22CD36OVCdPAB-b

[0648] At 0°C, diethylamine (0.03 mL, 0.318 mmol) was added to a 5 mL DMF solution of compound LEC-22CD36OVCdPAB-a (100 mg, 0.032 mmol). The mixture was then stirred at 25°C for 2 hours. The reaction solution was concentrated under vacuum to obtain a brown solid compound LEC-22CD36OVCdPAB-b (90 mg, crude product).

[0649] LCMS (ESI): m / z, 1459.3 [M+1] + , Rt = 1.19 min.

[0650] Step 3: Preparation of compound LEC-22CD36OVCdPAB

[0651] At 0 °C, compound LY-22CD-3-b (synthesis of which is referenced in WO2024175069A1) (11.5 mg, 0.031 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (17.5 mg, 0.046 mmol), and N,N-diisopropylethylamine (12 mg, 0.092 mmol) were added to a solution of compound LEC-22CD36OVCdPAB-b (90 mg, 0.031 mmol) in N,N-dimethylformamide (5 mL). The mixture was then stirred at 30 °C for 1 hour. The organic phase was concentrated and separated under vacuum to obtain the crude product. The crude product was prepared by high performance liquid chromatography (preparative chromatograph manufacturer: Oriendo, model BRIX-2860; column: Phenomenex Luna C18 250×50mm, 10μm; mobile phase: water (0.225% CF3COOH)-acetonitrile, elution ratio of acetonitrile from 12% to 42%). The eluent was lyophilized to give a white solid product LEC-22CD36OVCdPAB (10.5 mg, yield 10.4%).

[0652] 1H NMR (400MHz, DMSO) δ9.06 (t, J=5.8Hz, 1H), 8.86 (d, J=8.6Hz, 1H), 8.56 (dt, J=13.3, 6.4Hz, 2H), 8.37 (d, J=8.6Hz, 1H), 8.03 (d, J=7.2Hz, 1H), 7 .96(d, J=7.2Hz, 1H), 7.85(s, 5H), 7.77(d, J=11.0Hz, 1H), 7.31(s, 1H), 6.51(s, 1H), 5.92(s, 1H), 5.33(d, J=75.0Hz, 8H), 4.65–4.44(m, 7H), 4. 23–4.09 (m, 9H), 3.89–3.73 (m, 5H), 3.65 (s, 6H), 3.57 (dt, J=10.7, 6.4 Hz, 14H), 3.49 (t, J=2.9Hz, 58H), 3.43 (d, J=4.8Hz, 8H), 3.25 (s, 6H), 3. 21–3.13(m, 6H), 2.98–2.59(m, 10H), 2.38(s, 19H), 2.01–1.80(m, 11H), 1.69–1.39 (m, 18H), 1.23 (s, 12H), 0.94 (d, J=6.4Hz, 3H), 0.83 (m, 18H).

[0653] LCMS(ESI): m / z, 1636.9 [M / 2+H] + Rt = 1.24 min.

[0654] Example 25 Preparation of compound LEC-27C27C

[0655] Step 1: Preparation of compound LEC-27C27C-a

[0656] At 0°C, LA-27C-b (12.16 mg, 85%, 13.72 μmol, 1 eq) and DIEA (10.74 mg, 82.31 μmol, 6 eq) were added to a DMF (3 mL) solution of compound LC-CL-03-a (synthesis of which can be found in the example of LEC-01) (28.86 mg, 27.30 μmol, 1.99 eq), and the mixture was stirred at 25°C for 16 hours. LCMS showed that the starting material reacted completely. The reaction solution was then purified by high performance liquid chromatography to obtain a white solid product LEC-27C27C-a (25 mg, yield 69.08%).

[0657] The preparation conditions for high performance liquid chromatography are as follows:

[0658] The preparative chromatography system used Oriendo, model BRIX-2860. The column was a Welch Xtimate C18, 150×21.2 mm, 5 μm. The mobile phase was H2O (0.225% HCOOH)-acetonitrile, with acetonitrile elution ratios ranging from 60% to 90%.

[0659] LCMS(ESI): m / z, 1342.2[(1 / 2M+Na)] + .

[0660] Step 2: Preparation of compound LEC-27C27C-b

[0661] Diethylamine (0.2 mL) was added to a 2 mL DMF solution of compound LEC-27C27C-a (25 mg, 9.48 μmol, 1 eq) at 0 °C and stirred at 25 °C for 30 minutes. LC-MS showed that the reaction proceeds were completely reacted. The reaction solution was directly evaporated to dryness to give a yellow solid product LEC-27C27C-b (23 mg, crude product).

[0662] LCMS(ESI): m / z, 1230.8[(M+2Na) / 2] + .

[0663] Step 3: Preparation of compound LEC-27C27C

[0664] At 0°C, DIEA (4.97 mg, 38.08 μmol, 4 eq) was added to a DMF (2 mL) solution of compound LEC-03-d (synthesis of which is described in the example of LEC-03) (15.96 mg, 9.52 μmol, 1 eq) and compound LEC-27C27C-b (23 mg, 9.52 μmol, 1 eq), followed by HATU (5.43 mg, 14.28 μmol, 1.5 eq). The mixture was stirred at 25°C for 1 hour, and LCMS showed that the reaction proceeds were completely reacted. The reaction solution was then purified by high performance liquid chromatography to obtain a white solid product, LEC-27C27C (2.12 mg, yield 5.46%, purity 95%).

[0665] The high-performance liquid chromatography (HPLC) conditions are as follows:

[0666] The preparative chromatography system used Oriendo, model BRIX-2860. The column was a Welch Xtimate C18 250×30mm, 10μm. The mobile phase was water (0.225% HCOOH)-acetonitrile, with acetonitrile elution ratios ranging from 40% to 70%.

[0667] LCMS(ESI): m / z, 1180.4 (fragment peak).

[0668] Example 26: Preparation of compound LEC-27C22CD

[0669] Step 1: Preparation of compound LEC-2927C-a

[0670] At 0°C, LC-CL-03-a (52.42 mg, 85%, 59.11 μmol, 2.5 eq) and DIEA (7.72 mg, 59.11 μmol, 2.5 eq) were added to a DMF (1 mL) solution of compound LA-27C-b (25 mg, 23.65 μmol, 1 eq) (see example LEC-01 for the synthesis of this compound), and the mixture was stirred at 25°C for 2 hours. LCMS showed that the starting material reacted completely. The reaction solution was prepared by high performance liquid chromatography to give a white solid product (30 mg, yield 74.81%).

[0671] The preparation conditions for high performance liquid chromatography are as follows:

[0672] The preparative chromatogram was manufactured by Shimadzu, model LC-20AP. The column was a Welch Xtimate C18 150×30mm, 10μm. The mobile phase was water (0.225% HCOOH)-acetonitrile, with acetonitrile elution ratios ranging from 50% to 80%.

[0673] LCMS(ESI): m / z, 1696 [(M+H)] + .

[0674] Step 2: Preparation of compound LEC-27C22CD-a

[0675] Compound LY-22CD-b (14.61 mg, 19.46 μmol, 1 eq) and DIEA (16.16 mg, 123.82 μmol, 7 eq) were added to a DMF (2 mL) solution of compound LEC-2927C-a (30 mg, 17.69 μmol, 1 eq) and stirred at 25 °C for 2 hours. LCMS showed the presence of the acid corresponding to the active ester of LEC-2927C, so HATU (3.36 mg, 8.84 μmol, 0.5 eq) was added. After the addition was complete, the reaction was continued at 25 °C for 2 hours. LCMS showed that the starting material reacted completely. The reaction solution was prepared by high performance liquid chromatography to obtain a white solid product LEC-27C22CD-a (22 mg, yield 46.40%, purity 87%).

[0676] The high-performance liquid chromatography (HPLC) conditions are as follows:

[0677] The preparative chromatography system used Oriendo, model BRIX-2860. The column was a Welch Xtimate C18 250×30mm, 10μm. The mobile phase was water (0.225% HCOOH)-acetonitrile, with acetonitrile elution ratios ranging from 60% to 90%.

[0678] LCMS(ESI): m / z, 1188.4[(M+2Na) / 2] + .

[0679] Step 3: Preparation of compound LEC-27C22CD-b

[0680] Et2NH (143.43 mg, 99%, 202.02 μL) was added to a DMF (2 mL) solution of compound LEC-27C22CD-a (22 mg, 87%, 8.21 μmol, 1 eq) at 0 °C and stirred at 25 °C for 1.5 h. LC-MS showed that the starting material reacted completely. The reaction solution was directly evaporated to dryness to give a yellow oily product LEC-27C22CD-b (17 mg, crude product).

[0681] LCMS(ESI): m / z, 1055.2[(M+2H) / 2] + .

[0682] Step 4: Preparation of compound LEC-27C22CD

[0683] At 0°C, a DMF (1 mL) solution of compound LEC-27C27CD-b (17 mg, 8.06 μmol, 1 eq) was mixed with LEC-03-d (17.49 mg, 85%, 8.86 μmol, 1.1 eq) and DIEA (4.21 mg, 32.24 μmol, 4 eq), followed by HATU (3.68 mg, 9.67 μmol, 1.2 eq). The mixture was stirred at 25°C for 40 minutes, and LC-MS showed complete reaction of the starting material. The reaction solution was then purified by high-performance liquid chromatography to obtain a white solid product, LEC-27C22CD (9.67 mg, yield 31.84%).

[0684] The high-performance liquid chromatography (HPLC) conditions are as follows:

[0685] The preparative chromatography system used Oriendo, model BRIX-2860. The column was a Welch Xtimate C18 250×30mm, 10μm. The mobile phase was water (0.225% HCOOH)-acetonitrile, with acetonitrile elution ratios ranging from 30% to 60%.

[0686] LCMS(ESI): m / z, 942.7[(M+4H) / 4] + .

[0687] 1 H NMR (400MHz, DMSO-d6) δ9.09 (t, J=5.6Hz, 1H), 8.86 (d, J=8.6Hz, 1H), 8.55 (t, J=4.2Hz, 2H), 8.41 (s, 1H), 8. 37 (d, J=8.6Hz, 1H), 8.05 (d, J=7.1Hz, 2H), 7.97–7.82 (m, 5H), 7.80–7.73 (m, 2H), 7.29 (d, J=8.4Hz, 2H), 6.52 (s, 1H), 5.42 (s, 2H), 5.28–5.19 (m, 3H), 5.06–4.99 (m, 2H), 4.82 (s, 1H), 4.74 (s, 1H), 4.65–4.61 (m, 1H), 4.5 8–4.41 (m, 8H), 4.32 (q, J=8.6, 5.7Hz, 4H), 4.20 (ddd, J=19.1, 9.1, 5.2Hz, 8H), 4.11–3.97 (m, 7H), 3.91 (t, J= 6.6Hz, 2H), 3.85–3.74(m, 4H), 3.71(s, 1H), 3.65(s, 4H), 3.63–3.52(m, 15H), 3.51–3.43(m, 61H), 3.43–3.3 5 (m, 12H), 3.22 (s, 3H), 3.20–3.07 (m, 4H), 2.97 (d, J=6.7Hz, 3H), 2.86–2.69 (m, 5H), 2.70–2.63 (m, 4H), 2.62 –2.51 (m, 10H), 2.43 (dt, J=13.4, 6.9Hz, 7H), 2.39–2.24 (m, 12H), 2.23–2.08 (m, 5H), 2.04–1.80 (m, 11H), 1.7 5–1.39 (m, 17H), 1.38–1.23 (m, 8H), 1.20 (d, J=7.1Hz, 8H), 1.00 (dd, J=22.7, 9.2Hz, 5H), 0.91–0.73 (m, 14H).

[0688] Example 27: Preparation of compound LEC-2927

[0689] Step 1: Preparation of compound LEC-2927-a

[0690] At 0°C, LC-CL-03-a (46.25 mg, 61.36 μmol, 2.5 eq) and DIEA (6.34 mg, 49.09 μmol, 2 eq) were added to a DMF (3 mL) solution of compound LA-27-d (26 mg, 24.54 μmol, 1 eq) (the synthesis of this compound is described in the example of LEC-01), and the mixture was stirred at 25°C for 1 hour. LCMS showed that the starting material reacted completely. The reaction solution was prepared by high performance liquid chromatography to give a white solid product (25 mg, yield 59.99%).

[0691] The preparation conditions for high performance liquid chromatography are as follows:

[0692] The preparative chromatogram was manufactured by Shimadzu, model LC-20AP. The column was a Welch Xtimate C18 150×30mm, 10μm. The mobile phase was water (0.225% HCOOH)-acetonitrile, with water elution ratios ranging from 30% to 60%.

[0693] LCMS(ESI): m / z, 1719.8[(M+Na)] + .

[0694] Step 2: Preparation of compound LEC-2927-b

[0695] LY-29b (8.92 mg, 14.72 μmol, 1 eq) and DIEA (3.81 mg, 29.45 μmol, 2 eq) were added to a 2 mL DMF solution of compound LEC-2927-a (25 mg, 14.72 μmol, 1 eq) and stirred at 25 °C for 1 hour. LC-MS showed that the starting material reacted completely. The reaction solution was then purified by high performance liquid chromatography to obtain a white solid product (12 mg, yield 37.24%).

[0696] The high-performance liquid chromatography (HPLC) conditions are as follows:

[0697] The preparative chromatography system used Oriendo, model BRIX-2860. The column was a Welch Xtimate C18 250×30mm, 10μm. The mobile phase was water (0.225% HCOOH)-acetonitrile, with acetonitrile elution ratios ranging from 30% to 60%.

[0698] LCMS(ESI):m / z,1094.5[(M+2H) / 2] + .

[0699] Step 3: Preparation of compound LEC-2927-c

[0700] Et₂NH (2.01 mg, 27.42 μmol, 5 eq) was added to a DMF (1 mL) solution of compound LEC-2927-b (12 mg, 5.48 μmol, 1 eq), and the mixture was stirred at 25 °C for 1 hour. LC-MS showed that the reaction proceeds were completely reacted. The reaction solution was directly evaporated to dryness to give a yellow oily product (10 mg, yield 92.75%).

[0701] LCMS(ESI): m / z, 1967.3 [(M+H)] + .

[0702] Step 4: Preparation of compound LEC-2927

[0703] To a DMF (0.5 mL) solution of compound LEC-2927-c (8 mg, 4.07 μmol, 1 eq), LEC-03-d (6.82 mg, 4.07 μmol, 1 eq) and DIEA (1.06 mg, 8.14 μmol, 2 eq) were added, followed by HATU (2.32 mg, 6.10 μmol, 1.5 eq). The mixture was stirred at 25 °C for 0.5 h, and LCMS showed complete reaction of the starting material. The reaction solution was prepared by high performance liquid chromatography to give a white solid product (740 μg, yield 5.02%, purity 95%).

[0704] The high-performance liquid chromatography (HPLC) conditions are as follows:

[0705] The preparative chromatography system used Oriendo, model BRIX-2860. The column was a Welch Xtimate C18 250×30mm, 10μm. The mobile phase was water (0.225% HCOOH)-acetonitrile, with acetonitrile elution ratios ranging from 30% to 60%.

[0706] LCMS(ESI): m / z, 824.4, 860.5 (fragment peaks).

[0707] Example 28 Preparation of compound LEC-2927AA-2

[0708] Step 1: Preparation of compound LEC-2934A-a

[0709] At 0°C, DIEA (0.082 mL, 0.495 mmol, 3.0 eq) was added to a DMF (5.0 mL) solution of compound LY-29b (synthesis reference WO2024175069A1) (100.0 mg, 0.165 mmol, 1.0 eq) and LC-CL-03-a (synthesis reference LEC-01) (310.9 mg, 0.412 mmol, 2.5 eq) and stirred at 25°C for 2 hours. LC-MS showed that the reaction was complete. The reaction solution was quenched with H2O (10 mL) and then extracted with ethyl acetate (15 mL × 2). The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The crude product was obtained by concentration under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane:(methanol / THF=1:1)=10:1) to obtain a white solid LEC-2934A-a (32.81 mg, yield 15.97%).

[0710] LCMS(ESI): m / z, 1243.6 [(M)] + .

[0711] Step 2: Preparation of compound LEC-2927AA-2-a

[0712] DIEA (0.014 mL, 0.078 mmol, 3.0 eq) was added to a DMF (1.5 mL) solution of compound LEC-2934A-a (32.81 mg, 0.026 mmol, 1 eq) and crude LA-27AA-2-g (26.8 mg, 0.026 mmol, 1 eq) (for the synthesis of this compound, please refer to the example of LEC-22CD27AA-2). The mixture was stirred at 25 °C for 1 hour. LCMS showed that the starting materials reacted completely. The reaction solution was purified by high performance liquid chromatography (model JB-C235-04, column Bonnasil-BS C18, 20×250mm, 8μm, mobile phase water (0.1% TFA)-acetonitrile, elution ratio of acetonitrile from 20% to 60%) to obtain a yellow solid product LEC-2927AA-2-a (14.0 mg, yield 22.25%).

[0713] LCMS(ESI): m / z, 1079.3[(M / 2+H)] + .

[0714] Step 3: Preparation of compound LEC-2927AA-2-b

[0715] Diethylamine (0.007 mL, 0.065 mmol, 10.0 eq) was added to a 2.0 mL DMF solution of compound LEC-2927AA-2-a (14.0 mg, 0.006 mmol, 1.0 eq) at 25 °C and stirred at 25 °C for 1 hour. LC-MS showed that the starting material reacted completely. The reaction solution was concentrated under reduced pressure to give a yellow crude solid, LEC-2927AA-2-b (12.56 mg, 100% yield).

[0716] LCMS(ESI): m / z, 1936.2 [(M)] + .

[0717] Step 4: Preparation of compound LEC-2927AA-2

[0718] HATU (2.96 mg, 0.006 mmol, 1 eq) and DIEA (0.003 mL, 0.019 mmol, 3.0 eq) were added to a DMF (1.5 mL) solution of crude compound LEC-2927AA-2-b (12.56 mg, 0.006 mmol, 1 eq) and LEC-03-d (see the example of LEC-03 for the synthesis of this compound) (11.97 mg, 0.007 mol, 1.1 eq) at 25 °C. The mixture was stirred at 25 °C for 1 hour. LCMS showed that the reaction of the starting materials was complete. The reaction solution was purified by high performance liquid chromatography (model JB-C235-04, column Bonnasil-BS C18, 20×250mm, 8μm, mobile phase water (0.1% TFA)-acetonitrile, elution ratio of acetonitrile from 20% to 60%) to obtain a yellow solid product LEC-2927AA-2 (7.04 mg, yield 30.19%).

[0719] LCMS(ESI): m / z, 1797.0[(M / 2+H)] + .

[0720] 1H NMR (400MHz, DMSO-d6) δ9.08 (t, J=5.6Hz, 1H), 8.86 (d, J=8.6Hz, 1H), 8.37 (d, J =8.6Hz, 1H), 8.16 (t, J = 5.6Hz, 1H), 8.03–7.99 (m, 1H), 7.95–7.72 (m, 9H), 7.31 (s, 1H), 6.90 (t, J=5.6Hz, 1H), 6.51 (s, 1H), 5.43 (s, 3H), 5.26 (s, 2H), 5.02 (d, J=22.4Hz, 2H), 4.78 (d, J=31.0Hz, 3H), 4.54 (dt, J=37.8, 28.8Hz, 10H), 4.29–4. 08(m, 11H), 4.03–3.95(m, 2H), 3.88(t, J=6.6Hz, 3H), 3.84–3.71(m, 3H), 3.65( s, 3H), 3.57–3.44 (m, 91H), 3.25 (s, 3H), 3.05–2.92 (m, 14H), 2.69–2.66 (m, 3H), 2.36–2.21 (m, 16H), 2.07–1.82 (m, 18H), 1.62 (dd, J=42.8, 32.0Hz, 18H), 1.39– 1.21 (m, 16H), 1.17 (d, J=6.8Hz, 6H), 1.03 (d, J=6.4Hz, 3H), 0.90–0.73 (m, 19H).

[0721] Example 29: Preparation of compound LEC-2927C

[0722] Step 1: Preparation of compound LEC-2927C-a

[0723] At 0°C, LC-CL-03-a (25.16 mg, 85%, 28.37 μmol, 2.5 eq; for the synthesis of this compound, please refer to the example of LEC-01) and DIEA (2.96 mg, 22.70 μmol, 2 eq) were added to a DMF (1 mL) solution of compound LA-27C-b (12 mg, 11.35 μmol, 1 eq), and the mixture was stirred at 25°C for 2 hours. LC-MS showed that the starting material reacted completely. The reaction solution was prepared by high-performance liquid chromatography to give a white solid product (6 mg, yield 31.17%).

[0724] The preparation conditions for high performance liquid chromatography are as follows:

[0725] The preparative chromatogram was manufactured by Shimadzu, model LC-20AP. The column was a Welch Xtimate C18 150×30mm, 10μm. The mobile phase was water (0.225% HCOOH)-acetonitrile, with acetonitrile elution ratios ranging from 50% to 80%.

[0726] LCMS(ESI): m / z, 1718.9[(M+Na)] + .

[0727] Step 2: Preparation of compound LEC-2927C-b

[0728] Compounds LY-29b (2.14 mg, 3.54 μmol, 1 eq) and DIEA (923.69 μg, 7.08 μmol, 2 eq) were added to a DMF (1 mL) solution of compound LEC-2927C-a (6 mg, 3.54 μmol, 1 eq) and stirred at 25 °C for 2 hours. LC-MS showed that the starting material reacted completely. The reaction solution was then purified by high-performance liquid chromatography to obtain a white solid product (5 mg, yield 64.64%).

[0729] The high-performance liquid chromatography (HPLC) conditions are as follows:

[0730] The preparative chromatography system used Oriendo, model BRIX-2860. The column was a Welch Xtimate C18 250×30mm, 10μm. The mobile phase was water (0.225% HCOOH)-acetonitrile, with water elution ranging from 50% to 80%.

[0731] LCMS(ESI):m / z,1093.53[(M+2H) / 2] + .

[0732] Step 3: Preparation of compound LEC-2927C-c

[0733] Et₂NH (844.70 μg, 11.43 μmol, 5 eq) was added to a DMF (1 mL) solution of compound LEC-2927C-b (5 mg, 2.29 μmol, 1 eq), and the mixture was stirred at 25 °C for 2 hours. LC-MS showed that the starting material reacted completely. The reaction solution was directly evaporated to dryness to give a yellow oily product (10 mg, yield 92.75%).

[0734] LCMS(ESI): m / z, 1967.3 [(M+H)] + .

[0735] Step 4: Preparation of compound LEC-2927C

[0736] To a DMF (1 mL) solution of compound LEC-2927C-c (4 mg, 2.04 μmol, 1 eq), LEC-03-d (3.41 mg, 2.04 μmol, 1 eq) and DIEA (531.67 μg, 4.07 μmol, 2 eq) were added, followed by HATU (2.32 mg, 6.10 μmol, 1.5 eq). The mixture was stirred at 25 °C for 1 hour, and LC-MS showed complete reaction of the starting material. The reaction solution was prepared by high-performance liquid chromatography to obtain a white solid product, LEC-2927C (2.41 mg, yield 32.66%, purity 95%).

[0737] The high-performance liquid chromatography (HPLC) conditions are as follows:

[0738] The preparative chromatography system used Oriendo, model BRIX-2860. The column was a Welch Xtimate C18 250×30mm, 10μm. The mobile phase was water (0.225% HCOOH)-acetonitrile, with water elution ratios ranging from 30% to 60%.

[0739] LCMS(ESI): m / z, 824.4, 840.5 (fragment peaks).

[0740] Example 30 Preparation of compound LEC-2929

[0741] Step 1: Preparation of compounds LEC-2929-a1 and LEC-2929-a2

[0742] At 0°C, LC-CL-03-a (synthesis of which is based on the example of LEC-01) (117.13 mg, 85%, 132.09 μmol, 1 eq) and DIEA (120.70 mg, 924.60 μmol, 7 eq) were added to a DMF (1 mL) solution of compound LY-29b (synthesis of which is based on WO2024175069A1) (80 mg, 132.09 μmol, 1 eq) and stirred at 25°C for 1 hour. LCMS showed that the starting material reacted completely. The reaction solution was prepared by high performance liquid chromatography to give white solid products LEC-2929-a1 (36 mg, yield 21.90%) and LEC-2929-a2 (20 mg, yield 12.16%).

[0743] The preparation conditions for high performance liquid chromatography are as follows:

[0744] The preparative chromatogram was manufactured by Shimadzu, model LC-20AP. The column was a Welch Xtimate C18 150×30mm, 10μm. The mobile phase was water (0.225% HCOOH)-acetonitrile, with acetonitrile elution ratios ranging from 50% to 80%.

[0745] LEC-2929-a2:LCMS(ESI):m / z, 1148.4[(M+H)] + ;LEC-2929-a1:LCMS(ESI):m / z, 1244.5[(M+H)] + .

[0746] Step 2: Preparation of compound LEC-2929-b

[0747] Compounds LY-29b (synthesis reference WO2024175069A1) (13.20 mg, 21.79 μmol, 1 eq), DIEA (17.07 mg, 130.75 μmol, 6 eq), and HATU (9.94 mg, 26.15 μmol, 1.2 eq) were added to a DMF (1 mL) solution of compound LEC-2929-a2 (25.25 mg, 21.79 μmol, 1 eq) at 0 °C and stirred at 25 °C for 1 hour. LC-MS showed that the starting material reacted completely. The reaction solution was prepared by high-performance liquid chromatography to give a white solid product (9 mg, yield 23.81%).

[0748] The high-performance liquid chromatography (HPLC) conditions are as follows:

[0749] The preparative chromatography system used Oriendo, model BRIX-2860. The column was a YMC-Triart Prep C18, 150×30mm, 10μm. The mobile phase was H2O (0.225% HCOOH)-acetonitrile, with acetonitrile elution ratios ranging from 40% to 70%.

[0750] LCMS(ESI): m / z, 868[(M+2H) / 2] + 1757.7[(M+Na)] + .

[0751] Step 3: Preparation of compound LEC-2929-c

[0752] Et₂NH (200 μL) was added to a DMF (2 mL) solution of compound LEC-2929-b (9 mg, 5.19 μmol, 1 eq) at 0 °C, and the mixture was stirred at 25 °C for 2 hours. LC-MS showed that the starting material reacted completely. The reaction solution was directly evaporated to dryness to give a yellow oily product (8 mg, crude product).

[0753] LCMS(ESI): m / z, 1513.6 [M+H] + .

[0754] Step 4: Preparation of compound LEC-2929

[0755] At 0 °C, LEC-03-d (8.87 mg, 5.29 μmol, 1 eq) and DIEA (3.45 mg, 26.44 μmol, 5 eq) were added to a 2 mL DMF solution of compound LEC-2929-c (8 mg, 5.29 μmol, 1 eq), followed by HATU (2.41 mg, 6.35 μmol, 1.2 eq). The mixture was stirred at 25 °C for 40 min, and LC-MS showed complete reaction of the starting material. The reaction solution was then purified by high-performance liquid chromatography to obtain a white solid product, LEC-2929 (760 μg, yield 4.53%).

[0756] The high-performance liquid chromatography (HPLC) conditions are as follows:

[0757] The preparative chromatography system used Oriendo, model BRIX-2860. The column was a YMC-Triart Prep C18, 150×30mm, 10μm. The mobile phase was H2O (0.225% HCOOH)-acetonitrile, with acetonitrile elution ratios ranging from 20% to 50%.

[0758] LCMS(ESI): m / z, 770.8, 1044.4 (fragment peak).

[0759] Example 31 Preparation of compound LEC-2932AS

[0760] Step 1: Preparation of compound LA-32AS-c

[0761] At 0 °C, a solution of (9H-fluorene-9-yl)methyl ((3S,11S,14S)-3,11,15-trimethyl-1-(4-nitrophenoxy)-1,0,13-trioxy-2,7-dioxy-9,12-diazahexadecane-14-yl)carbamate (35.12 mg, 50.85 μmol, 1.2 eq) in DMF (3 mL) was added, along with eribulin methanesulfonate (35 mg, 42.37 μmol, 1 eq), DIEA (16.59 mg, 127.12 μmol, 3 eq), and HOBt (5.73 mg, 42.37 μmol, 1 eq). The reaction mixture was stirred at 25 °C for 2 hours, and LC-MS showed complete reaction of the starting material. The reaction mixture was further analyzed by high-performance liquid chromatography to obtain a white solid product (30 mg, yield 55.25%).

[0762] The preparation conditions for high performance liquid chromatography are as follows:

[0763] The preparative chromatography system used Oriendo, model BRIX-2860. The column was a Welch Xtimate C18 250×30mm, 10μm. The mobile phase was water (0.225% HCOOH)-acetonitrile, with acetonitrile elution ratios ranging from 50% to 80%.

[0764] LCMS(ESI): m / z, 1304.8[(M+Na)] + .

[0765] Step 2: Preparation of compound LA-32AS-d

[0766] Et₂NH (8.65 mg, 117.05 μmol, 5 eq) was added to a DMF (2 mL) solution of compound LA-32AS-c (30 mg, 23.41 μmol, 1 eq) at 0 °C. The reaction solution was stirred at 25 °C for 1 hour, and LCMS showed that the starting material reacted completely. The reaction solution was directly concentrated and used in the next step to give a yellow oily product (22 mg, yield 88.72%).

[0767] LCMS(ESI): m / z, 1059.8 [(M+H)] + .

[0768] Step 3: Preparation of compound LA-32AS-e

[0769] At 0 °C, DIEA (5.42 mg, 41.54 μmol, 2 eq) and bis(2,5-dioxopyrrolidone-1-yl)10-((9H-fluorene-9-yl)methoxy)carbonyl)-4,7,13,16-tetraoxa-10-azapentadecanedioate (the synthesis of this compound is described in Example LEC-01) (39.14 mg, 51.92 μmol, 2.5 eq) were added to a DMF (2 mL) solution of compound LA-32AS-d (22 mg, 20.77 μmol, 1 eq) at 0 mL. The reaction mixture was stirred at 25 °C for 1 hour, and LC-MS showed that the starting material reacted completely. The reaction mixture was then purified by high-performance liquid chromatography to obtain a white solid product (16 mg, yield 45.37%).

[0770] The preparation conditions for high performance liquid chromatography are as follows:

[0771] The preparative chromatogram was manufactured by Shimadzu, model LC-20AP. The column was a Welch Xtimate C18 250×30mm, 10μm. The mobile phase was water (0.225% HCOOH)-acetonitrile, with acetonitrile elution ratios ranging from 50% to 80%.

[0772] LCMS(ESI): m / z, 1720.9 [M+Na)] + .

[0773] Step 4: Preparation of compound LA-32AS-f

[0774] At 0 °C, LY-29b (5.71 mg, 9.42 μmol, 1 eq) and DIEA (2.46 mg, 18.85 μmol, 2 eq) were added to a DMF (2 mL) solution of LA-32AS-e (16 mg, 9.42 μmol, 1 eq) (synthesis of this compound is referenced in WO2024175069A1). The reaction solution was stirred at 25 °C for 1 hour, and LC-MS showed that the starting material reacted completely. The reaction solution was then purified by high-performance liquid chromatography to obtain a white solid product (2.41 mg, yield 32.66%).

[0775] The high-performance liquid chromatography (HPLC) conditions are as follows:

[0776] The preparative chromatography system used Oriendo, model BRIX-2860. The column was a Welch Xtimate C18 250×30mm, 10μm. The mobile phase was water (0.225% HCOOH)-acetonitrile, with acetonitrile elution ratios ranging from 50% to 80%.

[0777] Step 5: Preparation of compound LA-32AS-g

[0778] Compound LA-32AS-f (11 mg, 5.03 μmol, 1 eq) was dissolved in DMF (2 mL) at 0 °C, and Et2NH (1.86 mg, 25.13 μmol, 5 eq) was added. The reaction solution was stirred at 25 °C for 1 hour, and LC-MS showed that the starting material reacted completely. The reaction solution was concentrated to give a yellow oily product LA-32AS-g (30 mg, yield 55.25%).

[0779] The preparation conditions for high performance liquid chromatography are as follows:

[0780] The preparative chromatography system used Oriendo, model BRIX-2860. The column was a Welch Xtimate C18 250×30mm, 10μm. The mobile phase was water (0.225% HCOOH)-acetonitrile, with acetonitrile elution ratios ranging from 50% to 80%.

[0781] LCMS(ESI): m / z, 1988.1[(M+Na)] + .

[0782] Step 6: Preparation of compound LEC-2932AS

[0783] Compound LA-32AS-g (7 mg, 3.56 μmol, 1 eq) was dissolved in DMF (0.5 mL) at 0 °C. LEC-03-d (synthesis of which is described in the LEC-03 example) (5.97 mg, 3.56 μmol, 1 eq) and DIEA (929.48 μg, 7.12 μmol, 2 eq) were added, followed by HATU (2.03 mg, 5.34 μmol, 1.5 eq). The reaction mixture was stirred at 25 °C for 1 hour, and LC-MS showed complete reaction of the starting material. The reaction mixture was further analyzed by high-performance liquid chromatography to obtain a white solid product (6.62 mg, yield 51.30%, purity 98%).

[0784] The preparation conditions for high performance liquid chromatography are as follows:

[0785] The preparative chromatography column was Oriendo, model BRIX-2860. The column was a Synergi Max-RP 200×30mm, 10μm. The mobile phase was water (0.225% HCOOH)-acetonitrile, with acetonitrile elution ratios ranging from 30% to 60%.

[0786] LCMS(ESI): m / z, 860.4 [fragment peak] + .

[0787] 1H NMR (400MHz, DMSO-d6) δ9.10 (s, 1H), 8.86 (d, J = 8.6Hz, 1H), 8.55 (s, 1H), 8.37 (d, J = 8.5Hz, 1H), 8.1 8 (s, 1H), 8.04 (d, J = 8.5Hz, 2H), 7.95 (d, J = 8.2Hz, 1H), 7.85 (s, 2H), 7.75 (d, J = 11.1Hz, 3H), 7.32 (s , 1H), 6.86(s, 1H), 6.53(s, 1H), 5.44(s, 2H), 5.27(s, 2H), 5.05(s, 1H), 4.99(s, 1H), 4.82(s, 1H), 4 .75(s, 1H), 4.70(s, 2H), 4.63(s, 3H), 4.57–4.49(m, 5H), 4.46(s, 2H), 4.34(s, 2H), 4.32–4.11(m, 9H ), 4.10(s, 2H), 4.02(s, 2H), 3.89(s, 3H), 3.77(d, J=20.5Hz, 4H), 3.70–3.40(m, 61H), 3.25(s, 15H) , 3.17 (s, 8H), 2.97 (d, J = 9.0Hz, 8H), 2.84 (d, J = 9.5Hz, 2H), 2.73 (s, 4H), 2.58 (d, J = 6.6Hz, 5H), 2.40 (dt, J=13.7, 7.0Hz, 6H), 2.32–2.17 (m, 8H), 2.08 (s, 5H), 2.06–1.83 (m, 12H), 1.67 (dd, J=24.8, 12. 0Hz, 8H), 1.46 (s, 7H), 1.25–1.08 (m, 11H), 1.06–0.95 (m, 5H), 0.82 (ddd, J=28.0, 13.1, 7.4Hz, 12H).

[0788] ADC fabrication:

[0789] Procedure 1A, trastuzumab (A030, T), Thiomab Random DAR 4 or Site-specific DAR 8;

[0790] Operation 1B, TJ106A028(T2), Site-specific DAR 4;

[0791] Operation 1C, TJ106A014(T1), Site-specific DAR 2.

[0792] Operation 1D, A030-H114C(T4), Thiomab Random DAR 1.

[0793] The light and heavy chain encoding genes for TJ106A014 and TJ106A028 were designed using codons optimized for mammalian / human expression, respectively, and cloned into the expression vector pcDNA3.4 (Invitrogen) to obtain light and heavy chain plasmids for expressing TJ106A014 and TJ106A028. Gene synthesis and plasmid construction were performed by Genwiz Suzhou Co., Ltd.

[0794] The light and heavy chain plasmids were mixed separately at a ratio of 1:1.5, and then 1 / 10 of the transfection volume of OPM-CD Trans293 medium (OPM, P82019) was added. FectoPRO transfection reagent (PolyPlus, PT-116-010) was added to the medium at a ratio of 1 μl / 1.2 ml cells. The medium containing the plasmids and transfection reagent was mixed and incubated for 10 min. The mixture was then added to a shake flask containing Expi293F cells (Invitrogen, A14635) and cultured with shaking at 37°C and 5% CO2. Sixteen h after transfection, 10% of the transfection volume of feed (OPM, F081918-001) was added, and the cells were cultured for another 6 days before harvesting.

[0795] A 1 ml Protein A Diamond chromatography column (Borglon, AA0273) was equilibrated with 5 column volumes of equilibration buffer (20 mM sodium phosphate, pH 7.4). Clear cell culture medium was loaded onto the column at a flow rate of 0.5 column volumes / min. The packing material was then washed sequentially with 5 column volumes of equilibration buffer (20 mM sodium phosphate, pH 7.4) and pre-elution buffer (20 mM acetate-sodium acetate, pH 5.2). Finally, elution was performed with 10 column volumes of eluent (20 mM acetate-sodium acetate, pH 3.2), and the collected eluent was adjusted to pH 6.0 with 1.5 M Tris base. Trastuzumab mutants TJ106A014 (T1) and TJ106A028 (T2) were obtained.

[0796] Example 32: Preparation of T-LEC-2927 (Procedure 1A)

[0797] At 25°C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (5 mM, 8 μL, 40 nmol) was added to a PBS buffer solution of trastuzumab (Herceptin, Genentech, 10.0 mg / mL, 0.2 mL, 13.3 nmol) (pH = 7.4, 0.01 M), and the mixture was placed in a water bath and reacted at 25°C for 2 hours, after which the reaction was stopped.

[0798] Compound LEC-2927 (4.83 mg, 1330 nmol) was dissolved in 83.3 μL of DMSO. 8.3 μL of this solution was added to the above reaction mixture, and the mixture was placed in a water bath and reacted at 25 °C for 60 minutes. The reaction mixture was purified using an ultrafiltration centrifuge tube (BIOFIL, 30 K MWCO) to obtain a histidine buffer of T-LEC-2927 (1.68 mg / mL, 0.89 mL, 20 mM Histidine-HCl, pH 5.5), which was stored at 4 °C and kept at -80 °C for long-term preservation.

[0799] The average drug loading calculated by RP: n = 3.58.

[0800] Example 33: Preparation of T2-LEC-22CD36BVC2 (Procedure 1B)

[0801] At 25°C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (40 mM, 11.7 μL, 467 nmol) was added to a PBS buffer solution (pH = 7.4, 0.01 M) containing antibody T2 (US9527927B2, Trastuzumab-A141C-C220S, 10.0 mg / mL, 1.0 mL, 66.7 nmol). The mixture was placed in a water bath and reacted at 25°C for 2 hours, after which the reaction was stopped.

[0802] The compound LEC-22CD36BVC2 (1.73 mg, 533 nmol) was dissolved in 33.3 μL of DMSO and added to the above reaction solution. The mixture was placed in a water bath and reacted at 25 °C for 90 minutes. The reaction solution was purified using an ultrafiltration centrifuge tube (BIOFIL, 30 K MWCO) to obtain a histidine buffer of T2-LEC-22CD36BVC2 (11.48 mg / mL, 0.78 mL, 20 mM Histidine-HCl, pH 5.5), which was stored at 4 °C and kept at -80 °C for long-term storage.

[0803] The average drug loading calculated by RP is n = 3.78.

[0804] Example 34: Preparation of T1-LEC-2927C (Operation 1C)

[0805] At 25°C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (40 mM, 11.7 μL, 467 nmol) was added to a PBS buffer solution (pH = 7.4, 0.01 M) containing antibody T1 (US9527927B2, Trastuzumab-A141C, 10.0 mg / mL, 1.0 mL, 66.7 nmol). The mixture was placed in a water bath and reacted at 25°C for 2 hours, after which the reaction was stopped.

[0806] Compound LEC-2927C (1.45 mg, 400 nmol) was dissolved in 25 μL DMSO and added to the above reaction solution. The mixture was placed in a water bath and reacted at 25 °C for 120 minutes. The reaction solution was purified using an ultrafiltration centrifuge tube (BIOFIL, 30 KMWCO) to obtain T1-LEC-2927C histidine buffer (11.20 mg / mL, 0.75 mL, 20 mM Histidine-HCl, pH 5.5), which was stored at 4 °C and kept at -80 °C for long-term storage.

[0807] The average drug loading calculated by RP: n = 1.86.

[0808] Example 35T4-BA1 (Procedure 1D)

[0809] At 25°C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (40 mM, 24.3 μL, 966 nmol) was added to a PBS buffer solution (10 mM, pH 7.4) containing antibody T4 (12 mg / mL, 0.583 mL, 48 nmol). The mixture was placed in a water bath and reacted at 25°C for 2 hours, after which the reaction was stopped.

[0810] The reaction solution was replaced with an ultrafiltration centrifuge tube (Cobetter, 30K MWCO) to an EDTA-PBS buffer solution (2mM EDTA, 10mM PBS, pH 7.4). A DMSO solution of dehydroascorbic acid (dHAA) (10mM, 243μL, 2,400nmol) was added to the above reaction solution. The mixture was then placed in a water bath and reacted at 25°C for 2 hours.

[0811] The reaction solution was replaced with an ultrafiltration centrifuge tube (Cobetter, 30K MWCO) to an EDTA-PBS buffer solution. DMSO (135 μL) and BA1 (16 mM, 3 μL, 48 nmol) DMSO solution were added sequentially. The mixture was placed in a water bath and reacted at 25°C for 1 hour.

[0812] The reaction solution was replaced with His-HCl buffer solution (20 mM Histidine-HCl, pH 5.5) using an ultrafiltration centrifuge tube (Cobetter, 30 K MWCO) to remove small molecules and organic solvents, yielding T4-BA1 (3.6 mg, 52%), which was stored at 4 °C and kept at -80 °C for long-term preservation.

[0813] The average drug loading calculated by HIC-HPLC: n = 1.17.

[0814] Preparation of A030-H114C(T4) antibody

[0815] The trastuzumab light chain variable region sequence was fused with the human Kappa light chain constant region sequence to form the full-length light chain sequence of the anti-Her2 antibody; the trastuzumab heavy chain variable region sequence was fused with the human IgG1 heavy chain constant region sequence (CH1 with the A114C mutation) to form the full-length heavy chain sequence of the anti-Her2 antibody. The synthesized antibody heavy chain and light chain genes were inserted into the vector pcDNA3.4 (Invitrogen) to obtain the plasmid for expressing the anti-Her2 monoclonal antibody.

[0816] Plasmid transfection was performed according to the instructions of the ExpiCHO expression system kit (Gibco, A29133). The antibody light and heavy chain plasmids and ExpiFectamine CHO transfection reagent were mixed separately in OptiPRO SFM medium (Gibco, 12309019). After incubation, the mixture was added to ExpiCHO cells (Invitrogen, A14635), and the supernatant was harvested after 8 days. The supernatant was captured using a Protein ADiamond chromatography column (Borglon, AA0273) to obtain the anti-Her2 antibody T4.

[0817] Test Example 1: Inhibitory Activity of the ADC of the Present Invention on Cancer Cells

[0818] 1) Experimental Procedure

[0819] Day 0: After digesting and sieving cells in good growth condition, count them. Use 2000 cells / 100μL of culture medium (N87 cells) per well in a 96-well plate, and 1000 cells / 100μL of culture medium (MDA-MB-468 cells or JIMT-1) per well in a 96-well plate. Add 200μL of sterile water or PBS to the outer ring and incubate for 24 hours.

[0820] Day 1: Gradual dilution of samples in culture medium (9 gradients + 1 negative control), N87 plate with corresponding ADC added, two parallel controls for each sample, starting at 100 nM, the subsequent 8 wells were each 4-fold diluted, and 100 μL of the corresponding sample was added to each well of the Day 0 plate. Culture medium was added to column 11 as a control. Two parallel controls for each sample, starting at 1000 nM, 4-fold gradient dilution, added sequentially to the subsequent 8 wells, 100 μL of the corresponding sample was added to each well of the Day 0 plate, and culture medium was added to column 11 as a control.

[0821] Day 7: Dissolve the chromogenic agent 30 minutes before cell treatment 144 hours later. Add 100 μL to each well and develop the color in the dark for 2-5 minutes. Take two readings and calculate the average.

[0822] 2) Experimental Results

[0823] The results show that the dual-toxin ADC of eribulin and camptothecin derivative of this application has good anticancer activity.

[0824] Table 1. Inhibitory IC50 of the ADC in this application against N87 gastric cancer cells, MDA-MB-468 and JIMT-1 breast cancer cells. 50 value

[0825] Test Example 2: In vitro inhibitory activity of the ADC of the present invention against N87 cancer cells overexpressing the hABCG2 transporter.

[0826] 1) Experimental methods

[0827] Cell (from WuXi AppTec) inoculation

[0828] 1. Suspend cells in culture medium (RPMI 1640 + 10% FBS) and count the trypan blue-stained cells using a hematology counter.

[0829] 2. Adjust cell density.

[0830] 3. Add 135 μL of cell suspension to the detection plate.

[0831] 4. Add 135 μL of culture medium to plate 1B-1G, and add 200 μL of PBS to columns A, H and 12 in the horizontal direction of the plate.

[0832] 5. Incubate the cells overnight at 37°C, 5% CO2, 95% air and 100% relative humidity.

[0833] Compound treatment

[0834] 6. Prepare compound plates by diluting the stock solution of the compound to be tested from the highest concentration of 100 nmol to the lowest concentration in a 5-fold gradient.

[0835] 7. Add 7.5 μL or 15 μL of the compound to each well.

[0836] 8. Incubate the test plate in an incubator for 7 days.

[0837] Plate reading

[0838] 9. Equilibrate the test plate and CellTiter Glo reagent to room temperature for approximately 30 minutes.

[0839] 10. Add 75 μL of CellTiter Glo reagent to each well.

[0840] 11. Shake for 2 minutes and incubate the plate at room temperature for 10 minutes to stabilize the luminescence signal.

[0841] 12. Detected using the EnVision (luminescence) detection board.

[0842] As shown in Table 2 and Figure 1, camptothecin T-LY22CD3 loses its activity in ABCG2-overexpressing N87 cells, while both toxin molecules maintain good activity, which may solve the drug resistance problem caused by the overexpression of ABCG2 transporter in existing camptothecin-based ADC drugs.

[0843] Table 2. In vitro proliferative inhibitory activity of the ADC of this application against tumor cells overexpressing ABCG2 transporter.

[0844] Test Example 3: Efficacy Experiment of the ADC of the Present Invention in the JIMT-1 Xenograft Model of Human Breast Cancer

[0845] Human breast cancer JIMT-1 cell line (Nanjing Kebai Biotechnology, CBP60378) was cultured in DMEM medium (Gibco, catalog number: 11995-065) + 10% FBS (Excell Bio, catalog number: FCS500) at 37°C in an incubator containing 5% CO2. Cells were digested with 0.25% trypsin-EDTA, washed twice with PBS, counted using a cell counter, and diluted with PBS to a cell concentration of 1×10⁻⁶. 8 / mL.

[0846] CB17 SCID mice, female, 4-6 weeks old, were purchased from Vital River Laboratory Animal Co., Ltd. After acclimatization to the laboratory environment for 2-5 days, CB17 SCID mice were subcutaneously inoculated with JIMT-1 cells at a dose of 5 × 10⁻⁶ cells per rib. 6 / animal, inoculated with 0.1 mL (containing 50% Matrigel), until the tumor grows to 150-200 mm 3 At that time, the medication was administered according to the regimen shown in Table 3, and the DAR value was adjusted to 8 for all doses. Tumor volume and body weight were measured twice a week, and the tumor proliferation rate and relative tumor inhibition rate were calculated.

[0847] Table 3. Dosing regimens of ADC in human breast cancer JIMT-1 xenograft mice

[0848] At the end of the experiment, mice were euthanized, and tumors were collected and weighed. Tumor volume and body weight were measured twice a week, and tumor proliferation rate and relative tumor inhibition rate were calculated. The calculation formula is: Relative tumor proliferation rate T / C (%) = TRTV / C RTV ×100%

[0849] (T RTV The average RTV in the treatment group; C RTV The average RTV of the control group; RTV = V t / V o V o V represents the tumor volume of the animal when grouped. t (Tumor volume in the animal after treatment); Relative tumor inhibition rate (TGI%) = (1 - T / C) × 100%

[0850] (T and C represent the relative tumor volume (RTV) of the treatment group and the control group at a specific time point, respectively).

[0851] The formula for calculating tumor volume (V) is: V = l / 2 × L 长 ×L 短 2 ,

[0852] Where L 长 L 短 These represent the long diameter and short diameter of the tumor, respectively.

[0853] The experimental results are shown in Table 4 and Figures 2A-2B.

[0854] Table 4 Tumor volume data of the JIMT-1 breast cancer-bearing mouse model

[0855] Results: On day 28, the mean tumor volume in the PBS group reached 855 mm. 3 The dual-toxin ADC T2-LEC-22CD36BVC2 showed better tumor inhibition rates than its corresponding single-toxin ADCs T2-LY22CD3 and T2-LA36VCdPAB, as well as the combination of the corresponding single toxins T2-LA36VCdPAB+T2-LY22CD3. The dual-toxin ADC T2-LEC-22CD34BVC2 also showed similar results. During administration, no significant weight loss or drug toxicity was observed in any of the groups.

[0856] Conclusion: The dual-toxin ADC of eribulin derivative and camptothecin derivative in this invention has good tumor inhibitory activity and synergistic tumor inhibitory effect between the toxins.

[0857] Test Example 4: Efficacy Experiment of the ADC of the Present Invention in the JIMT-1 Xenograft Model of Human Breast Cancer

[0858] Human breast cancer JIMT-1 cell line (Nanjing Kebai Biotechnology, CBP60378) was cultured in DMEM medium (Gibco, catalog number: 11995-065) + 10% FBS (Excell Bio, catalog number: FCS500) at 37°C in an incubator containing 5% CO2. Cells were digested with 0.25% trypsin-EDTA, washed twice with PBS, counted using a cell counter, and diluted with PBS to a cell concentration of 1×10⁻⁶. 8 / mL.

[0859] CB17 SCID mice, female, 6-8 weeks old, were purchased from Shanghai Lingchang Biotechnology Co., Ltd. After acclimatization to the laboratory environment for 2-5 days, CB17 SCID mice were subcutaneously inoculated with JIMT-1 cells at a dose of 5 × 10⁻⁶ cells per rib. 6 / animal, inoculated with 0.1 mL (containing 50% Matrigel), until the tumor grows to 200-250 mm 3 At that time, administer medication according to the protocol shown in Table 3 and adjust based on the measured DAR value. Measure tumor volume and body weight weekly, and calculate tumor proliferation rate and relative tumor inhibition rate.

[0860] Table 5. Dosing regimens of ADC in human breast cancer JIMT-1 xenograft mice

[0861] Dosage volume: The dosage volume for animals was adjusted to 10 μL / g body weight.

[0862] At the end of the experiment, mice were euthanized, and tumors were collected and weighed. Tumor volume and body weight were measured twice a week, and tumor proliferation rate and relative tumor inhibition rate were calculated. The calculation formula is: Relative tumor proliferation rate T / C (%) = T RTV / C RTV ×100%

[0863] (T RTV The average RTV in the treatment group; C RTV The average RTV of the control group; RTV = V t / V o V o V represents the tumor volume of the animal when grouped. t (Tumor volume in the animal after treatment); Relative tumor inhibition rate (TGI%) = (1 - T / C) × 100%

[0864] (T and C represent the relative tumor volume (RTV) of the treatment group and the control group at a specific time point, respectively).

[0865] The formula for calculating tumor volume (V) is: V = l / 2 × L 长 ×L 短 2L 长 L 短 These represent the long and short diameters of the tumor, respectively.

[0866] The experimental results are shown in Table 6 and Figure 3.

[0867] Table 6 Tumor volume data of the JIMT-1 breast cancer-bearing mouse model

[0868] Conclusions: First, at day 53, T2-LEC2236P exhibited a tumor inhibition rate of 73%, significantly superior to the corresponding single-toxin ADCs T2-LA36VCP (50% tumor inhibition rate) and T2-LY22CD3 (58% tumor inhibition rate). Meanwhile, the eryribulin-based ADC T4-BA1 showed a tumor inhibition rate of 42% in the randomized DAR1 setting, while the less cytotoxic PE-E2K-based ADC T2-LA36VCP showed a tumor inhibition rate of 50% in the DAR4 setting. Therefore, in the JIMT-1 tumor model, the PE-E2K-based ADC exhibits tumor inhibitory activity up to four times weaker than the eryribulin-based ADC, with activity similar to the TOPO1 inhibitor (T2-LY22CD3). These two toxins with different mechanisms of action are more suitable for use together in equal proportions as dual-toxin ADCs. In this case, the two toxins can be directly coupled via cysteine ​​in a 1:1 ratio, which is much cheaper than using Eribulin as the toxin and employing different DAR values, which requires enzyme-catalyzed coupling.

[0869] Test Example 5: PK experiment of the toxin of the present invention in rats

[0870] Experimental objective:

[0871] The pharmacokinetic behavior of the compounds of this invention in male SD rats was evaluated.

[0872] Experimental methods:

[0873] 1) Formulation

[0874] Dissolve PE-E2K and Eribulin mesylate (Haoyuan Pharmaceutical) in a solvent (Saline). The specific steps are as follows:

[0875] a. Weigh 1.441 mg of PE-E2K into a glass bottle;

[0876] b. Add 2525 μL of saline and stir for 2 minutes to obtain a clear solution;

[0877] c. Weigh 1.228 mg of Eribulin mesylate into a glass bottle;

[0878] d. Add 2140 μL of saline and stir for 2 minutes to obtain a clear solution;

[0879] d. After filtration through a 0.22 μm PVDF membrane, the solution was used for drug delivery.

[0880] 2) Animal administration

[0881] Six male SD rats were randomly divided into two groups of three. PE-E2K and Eribulin mesylate were administered via single intravenous bolus (iv) injection at a dose of 1 mg / kg, respectively. Whole blood samples (approximately 0.2 mL) were collected from the jugular vein at 0.083, 0.5, 1, 2, 4, 9, 24, 33, and 48 hours post-administration and placed on moist ice. The actual blood collection time was recorded. A deviation within ±1 minute was acceptable for collections within 1 hour of administration, and within 5% of the theoretical time was acceptable for other collection times.

[0882] 3) Sample collection and processing

[0883] Within 60 minutes of blood collection, centrifuge at 3200×g for 10 minutes at 4°C, then aspirate the supernatant plasma (approximately 0.1 mL) onto another 96-well plate, quickly place it on dry ice, and then store it at -60°C or lower for LC-MS / MS analysis.

[0884] 4) Sample pretreatment for analysis

[0885] Take 20 μL of plasma and add 400 μL of precipitant containing internal standard. Mix at 800 rpm for 10 minutes, then centrifuge at 3200 × g, 4 °C for 15 minutes. Transfer 50 μL of the supernatant to another 96-well plate, centrifuge at 3200 × g, 4 °C for 5 minutes, and inject directly. Analyze the drug concentration using LC-MS / MS (Triple Quad 6500plus).

[0886] 5) Data processing

[0887] The drug concentration data of PE-E2K and Eribulin mesylate were processed using WinNonlin Version 8.3.5 (Certara) pharmacokinetic software with a non-compartmental model. Relevant pharmacokinetic parameters were calculated using the linear logarithmic trapezoidal method.

[0888] Individual plasma concentrations for BQL were excluded when calculating PK parameters. All drug concentrations and pharmacokinetic parameters were reported to three significant figures. Pharmacokinetic parameters were calculated using the theoretical sampling time and theoretical dose concentration specified in the protocol.

[0889] Experimental results: The pharmacokinetic results of PE-E2K and Eribulin mesylate in rats are shown in Table 7.

[0890] Table 7. Pharmacokinetic results of PE-E2K and Eribulin mesylate in rats.

[0891] Conclusion: The plasma exposure (CO) of the compound PE-E2K of this invention is twice that of the reference compound Eribulin mesylate, and the vitamin D content is also significantly higher. ss It is 1 / 62 and AUC 0-last The comparable results suggest that PE-E2K has lower off-target toxicity. Simultaneously, PE-E2K has a shorter half-life, meaning that after completing its tumor-killing action and returning to the bloodstream, it will be cleared from the body more quickly, significantly reducing toxin accumulation and preventing untargeted damage to normal tissues. Based on the above comparison data, PE-E2K is safer and more suitable as an ADC toxin.

[0892] Test Example 6: Maximum tolerance test of the ADC of the present invention in SD rats after a single intravenous injection of TJ01-TJ04

[0893] Experimental Methods: SD rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and housed in an SPF-grade animal facility. Twenty-four SD rats, aged 6–7 weeks (males 240–260g, females 180–200g), were selected (half male and half female) and divided into four groups: a solvent control group, TJ01 group, TJ02 group, TJ03 group, and TJ04 group, with six rats in each group (half male and half female). The drug dosages were 0 mg / kg, 20 mg / kg, 200 mg / kg, and 200 mg / kg, with an administration volume of 10 mL / kg. The day of administration was designated as D1, and the recovery period was 21 days. On the day of administration, rats were observed at approximately 15 min, 0.5 h, 1 h, 2 h, and 4 h post-administration. During the recovery period, at least one cage-side observation was conducted daily. Weight checks were performed before administration on D1 and on D3, D5, D9, D12, D16, D21, and D22. Blood and biochemical tests were performed once each on D-1 (before administration), D7, and D22; a coagulation test was performed once on D22, and the animals were necropsy to examine the body surface, neck, thoracic cavity and thoracic organs, pelvic cavity and pelvic organs, abdominal cavity and abdominal organs, lymph nodes, head, mammary glands, sciatic nerve, and administration site.

[0894] Experimental results:

[0895] Clinical observation: No obvious abnormalities were observed in the animals during administration and recovery.

[0896] Weight: Male animals in groups TJ01 to TJ04 showed slow weight gain from day 9 to day 21.

[0897] Table 8. Hematological results of the ADC of the present invention in the maximal tolerance test of SD rats.

[0898] Conclusion: In terms of blood parameters, on the seventh day after administration, in both female and male animals, at a dose of 20 mg / kg (T2-BA1, DAR4) of Eribulin as an ADC, key indicators such as platelet (PLT), white blood cell lines (WBC, NEUT#, LYMP#, etc.), and reticulocytes (RET#) decreased less than those of the corresponding 200 mg / kg T-LA36VCP (DAR8). This indicates that Eribulin as a toxin ADC is more than 20 times more toxic than its derivative PE-E2K as a toxin ADC. However, the dual toxin T2-LEC2236P, at the same dose of 200 mg / kg, did not show stronger toxicity than the corresponding single toxin ADC T-LA36VCP. This suggests that this modified toxin is more suitable as an ADC.

Claims

1. A bi-toxin antibody drug conjugate, which is a compound of Formula (I), or an isotopically-labeled compound thereof, or an optical isomer, a geometric isomer, a tautomer, or an isomer mixture thereof, and a pharmaceutically acceptable salt thereof, Formula (I) ###0001### (I). wherein: D1 is a fragment of a duocarmycin toxin molecule; D2 is a fragment of an eribulin toxin molecule; Ab is an antibody or antigen binding fragment; Q is a linker group attached to Ab; L5 is a direct bond or a linker group connecting Ab to Q; L1, L2 are each independently a linker group; L3 is a direct bond or a linker group; L4 is a direct bond or a linker group; n is an integer or decimal number between 0 and 20; preferably n is an integer or decimal number between 0 and 8.

2. The diabody drug conjugate of claim 1, wherein, L1, L2 are each independently -L9-L8-L7-L6-, wherein L6 is attached to D1 or D2; L6is selected from a direct bond, wherein a linking site for linking to D1 or D2, represents the attachment site to L7; L7 is a short peptide chain formed from two or more amino acid residues selected from natural amino acid residues or non-natural amino acid residues, the N-terminus of which is connected to L8, the C-terminus of which is connected to L6, and L7 optionally comprises one or more structures selected from the group consisting of formula (II-1) to formula (II-9): s is selected from 1, 2, 3, 4, 5, 6, 7, 8, t is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, R1is selected from H or C 1-8 alkyl; L9 is selected from r1, r6are each independently selected from 0, 1, 2, 3, 4, 5, 6, r2, r3, r4, r5are each independently selected from 1, 2, 3, 4, 5, 6, wherein represents the attachment site to L8; L8is selected from a direct bond, r7, r12 are each independently selected from 0, 1, 2, 3, 4, 5, 6, r9, r10 are each independently selected from 1, 2, 3, 4, 5, 6, r8, r11 are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, R2 is selected from H or C 1-8 alkyl, wherein represents the attachment site to L7; L3 is -L 11 -L 10 - wherein L 11 is connected to L4; L 10 selected from a direct bond, r13, r18 are each independently selected from 0, 1, 2, 3, 4, 5, 6, r15, r16 are each independently selected from 1, 2, 3, 4, 5, 6, r16, r17 are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, R3 is selected from H or C 1-8 alkyl, wherein representing a L 11 a connection site to which a connection is made; L 11 one or more structures selected from a direct bond, one of formula (II-1) to formula (II-9); L4is selected from -CO-, -(CH2) p1 -CO-, -(CH2CH2O) p2 -(CH2) p3 -CO-, -(CH2) p4 -O-(CH2) p5 -C(O)-, -NR4-(CH2) p1 -CO-, -NR4-(CH2CH2O) p2 -(CH2) p3 -CO-, -NR4-(CH2) p4 -O-(CH2) p5 -CO-, -O-(CH2) p1 -CO-, -O-(CH2CH2O) p2 -(CH2) p3 -CO-, -O-(CH2) p4 -O-(CH2) p5 -CO-, p1, p2, p3, p4, p5 are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, R4is selected from H or C 1-8 alkyl, wherein -CO- is attached to L 10 .

3. The diabody drug conjugate of claim 2, wherein, L6is selected from a direct bond, L7 is a short peptide chain formed by 2-8 amino acid residues; L8is selected from a direct bond, r7 is selected from 0, 1, 2, 3, each r9 is independently selected from 1, 2, 3, r8 is selected from 6, 7, 8, R2 is selected from H or C 1-6 alkyl; L9 is selected from r1 is selected from 0, 1, 2, 3, r2, r3 are each independently selected from 1, 2, 3; L 10 selected from a direct bond, r13 is selected from 0, 1, 2, 3, r15 is selected from 1, 2, 3, r14 is selected from 6, 7, 8, R3 is selected from H or C 1-6 alkyl; L 11 selected from a direct bond, L4is selected from a direct bond, -CO-, -(CH2) p1 -CO-, -(CH2CH2O) p2 -(CH2) p3 -CO-, -NR4-(CH2) p1 -CO-, -NR4-(CH2CH2O) p2 -(CH2) p3 -CO-, p1is selected from 1, 2, 3, 4, p2is selected from 1, 2, 3, p3is selected from 1, 2, 3, R4is selected from H or C 1-6 alkyl.

4. The diabody drug conjugate of claim 2 or 3, wherein, L8, L 10 one is a direct bond and one is not a direct bond.

5. The diabody drug conjugate of any one of claims 3-4, wherein, L7 is a short peptide chain formed by 2-8 amino acid residues, which are amino acid residues selected from phenylalanine, alanine, glycine, valine, citrulline, leucine, isoleucine, tryptophan, tyrosine, histidine, lysine, serine, threonine, cysteine, glutamic acid, glutamine, aspartic acid, asparagine, methionine, arginine; preferably the amino acid residues are amino acid residues selected from alanine, valine, citrulline.

6. The diabody drug conjugate of claim 5, wherein, L7 is -CH2-6-, NH Val-Ala CO -, NH Val-Cit CO - 7. The diabody drug conjugate of any one of claims 3-6, wherein, L8is selected from a direct bond, L9is selected from 8. The diabody drug conjugate of claim 7, wherein, L1, L2are each independently -(CH2CH20)2-(CH2)2-CO- NH Val-Ala CO -L6-, L6is a direct bond, or -(CH2CH20)2-(CH2)2-CO- NH Val-Cit CO -L6-, L6is a direct bond, or -(CH2CH20)2-(CH2)2-CO-NH-(CH2CH20)8-(CH2)2-CO- NH Val-Ala CO -L6-, L6is a direct bond, or -(CH2CH20)2-(CH2)2-CO-NH-(CH2CH20)8-(CH2)2-CO- NH Val-Cit CO -L6-, L6is a direct bond, 9. The diabody drug conjugate of any one of claims 3-8, wherein, L 10 selected from a direct bond, L 11 selected from a direct bond, 10. The diabody drug conjugate of claim 9, wherein, L3is selected from a direct bond; L 11 is represents a connection site for L4.

11. The diabody drug conjugate of any one of claims 3-10, wherein, L4 is selected from -CO-, -(CH2)3-CO-, -(CH2CH2O)-(CH2)2-CO-, -(CH2CH2O)2-(CH2)2-CO-, -NH-(CH2CH2O)-(CH2)2-CO-, -NH-(CH2CH2O)2-(CH2)2-CO-.

12. The diabody drug conjugate of any one of claims 3-11, wherein, Q is selected from a group comprising a maleimide linker, a group comprising a methanesulfonylpyrimidine linker, a group comprising a methanesulfonylpyridothiazole linker, a group comprising a methanesulfonylpyrimidothiazole linker, a group comprising a cycloalkynene linker or a group comprising an oxime bond linker.

13. The diabody drug conjugate of claim 12, wherein, Q is selected from: for the attachment site to L5, represents the attachment site to L4.

14. The diabody drug conjugate of claim 13, wherein, Q-L4 is selected from 15. The diabody drug conjugate of claim 14, wherein, Q-L4 is selected from pi is selected from 2, 3, 4, p2 is selected from 1, 2, p3 is selected from 1, 2, R4 is selected from H; Preferably, p1 is selected from 3, p2 is selected from 1, 2, p3 is selected from 2.

16. The diabody drug conjugate of any one of claims 1-15, wherein, L5is a direct bond or represents a linking site to Ab, represents Z3linked to Q; Z3is a click probe or a thiol or a precursor thereof that is capable of being linked to Q after reaction. Z1 and Z2 are each independently selected from -C(O)NH-, -C(O)O-, -C(O)-, -OC(O)-, -NHC(O)-, -NH-, -O-, -OC(O)NH- or -NHC(O)O-; q1 is 0, 1, 2, 3, 4, 5, 6, 7, 8; q2 is 1, 2, 3, 4, 5, 6, 8; q3 is 1, 2, 3, 4, 5, 6,7, 8; q4 is 1, 2, 3, 4, 5, 6,8.

17. The diabody drug conjugate of claim 16, wherein, Z3 is a click probe or a thiol group or a precursor thereof, which is capable of being attached to Q after a metal-free click reaction, preferably Z3 is an azido group.

18. The diabody drug conjugate of claim 16 or 17, wherein, L5is a direct bond or Preferably, L5is a direct bond or 19. The diabody drug conjugate of any one of claims 16-18, wherein, Z1 and Z2 are each independently selected from -C(O)NH-.

20. The diabody drug conjugate of claim 15, wherein, L5 is a direct bond.

21. The diabody drug conjugate of any one of claims 1-20, wherein, D1 is selected from the following structures: L 12 selected from -O-*, -NR a *-, -(CH2) m1 -O-*, -(CH2) m1 -NR a *-, -OC(=O)NR b -(CH2) m1 -O-*, -OC(=O)NR b -(CH2) m1 -NR a *-, -(CH2) m1 -C(=O)O-*, -(CH2) m1 -C(=O)NR a *-, -NR b -(CH2) m1 -O-*, -NR b -(CH2) m1 -NR a *-, -O-(CH2) m1 -O-*, -O-(CH2) m1 -NR a *-, -NR b C(=O)O-(CH2) m1 -O-*, -NR b C(=O)O-(CH2) m1 -NR a *-, -(CH2) m1 -NR b C(=O)-(CH2) m2 -NR a *-, -(CH2) m1 -NR b C(=O)-(CH2) m2 -O-*, wherein * is the point of attachment to L1; R a selected from hydrogen, C1-C8alkyl; R b selected from the group consisting of hydrogen, C1-C8alkyl, -C(O)R c , -S(O)R c , -S(O)2R c , R c is selected from the group consisting of hydrogen, hydroxyl, C1-C8alkyl; m1 is 1, 2, 3, 4, 5 or 6; m2 is 1, 2, 3, 4, 5 or 6; R 5 selected from hydrogen, halogen, hydroxyl, amino, cyano, Ci-C8-alkyl, Ci-C8-alkoxy, C2-C8-alkenyl, C2-C8-alkynyl, C3-C6-cycloalkyl, -NR d R e -(CH2) m1 -OH, -(CH2) m -NR d R e -(CH2) f -OH, -OC(=O)NR m1 -(CH2) f -NR m1 R d -(CH2) e -C(=O)OH, -(CH2) m1 -C(=O)-NR m1 R d -(CH2) e -C(=O)NR m1 -(CH2) f -OH, -(CH2) m2 -C(=O)NR m1 -(CH2) f -NR m2 R d -(CH2) e -NR f -(CH2) m1 -OH, -NR f -(CH2) m1 -NR d R e -O-(CH2) m1 -OH, -O-(CH2) m1 -NR d R e -NR f C(=O)O-(CH2) m1 -OH, -NR b C(=O)O-(CH2) m1 -NR d R e -(CH2) m1 -NR f C(=O)O-(CH2) m2 -OH-, -(CH2) m1 -NR f C(=O)O-(CH2) m2 -NR d R e -(CH2) m1 -OC(=O)NR f -(CH2) m2 -NR d R e , -(CH2) m1 -OC(=O)NR f -(CH2) m2 -OH, -(CH2) m1 -NR f C(=O)-(CH2) m2 -NR d R e , -(CH2) m1 -NR f C(=O)-(CH2) m2 -OH, the Ci-C8alkyl, Ci-C8alkyloxy, C2-C8alkenyl, C2-C8alkynyl, C3-C6cycloalkyl groups are optionally further substituted by one or more groups selected from halogen, deuterium, amino, Ci-C8alkyl, hydroxyl; the above -(CH2) m1 -, -(CH2) m2 - optionally substituted by one or more deuterium or halogen; R 6 selected from the group consisting of hydrogen, halogen, hydroxyl, amino, cyano, Ci-C8-alkyl, Ci-C8-alkoxy, C2-C8-alkenyl, C2-C8-alkynyl, said Ci-C8-alkyl, Ci-C8-alkoxy, C2-C8-alkenyl, C2-C8-alkynyl being optionally further substituted by one or more radicals selected from halogen; R d and R e each independently is selected from the group consisting of hydrogen and Ci-C8alkyl; R f selected from the group consisting of hydrogen, C1-C8alkyl, -C(O)R c , -S(O)R c , -S(O)2R c , R c is selected from the group consisting of hydrogen, hydroxyl, C1-C8alkyl.

22. The diabody drug conjugate of claim 21, wherein, L6 in L1 is selected from represents the point of attachment to D1.

23. The diabody drug conjugate of claim 21 or 22, wherein, L 12 -NR b C(=O)O-(CH2) m1 -O-*, -NR b C(=O)O-(CH2) m1 -NR a -*, R a selected from hydrogen, C1-C6alkyl, R b selected from hydrogen, C1-C6alkyl, m1 is 1, 2, 3, 4, 5 or 6; Preferably, R a selected from hydrogen, R b selected from hydrogen.

24. The diabody drug conjugate of any one of claims 21-23, wherein, R 5 selected from C 1-6 alkyl, C 1-6 alkoxy, hydroxy-substituted C 1-6 alkyl; preferably, R 5 is methyl.

25. The diabody drug conjugate of any one of claims 21-24, wherein, R 6 selected from halogen, preferably R 6 is F, Cl.

26. The diabody drug conjugate of claim 21, wherein, D1 is selected from the following structures:

27. The diabody drug conjugate of any one of claims 1-26, wherein, D2 is selected from the following structures: at the * and ** places are each independently a single bond, a double bond, or an oxygen bond =0; preferably, * and ** are each independently a single bond or a double bond; more preferably, * and ** are each independently a single bond; and are each a double bond; L 13 a group selected from the following groups, after removal of the H of -NH2, -OH, -NH- in the chain or -NH- in the ring, followed by attachment to L2: X1 is selected from -CO-, -CH2, -S(O)- or -S(O)2-; X2 is selected from a direct bond, -O- or -NR7-; X3is selected from -(CR8R9) m3 -NH2, -(CR8R9) m3 -OH, -R 10 -(CR8R9) m4 -NH2, -R 10 -(CR8R9) m4 -OH or Y1is selected from -(CR8R9) m3 -NH2, -(CR8R9) m3 -OH, -R 10 -(CR8R9) m4 -NH2, -R 10 -(CR8R9) m4 -OH or R7is selected from H, alkyl, hydroxy-substituted alkyl, alkyloxy-substituted alkyl, amino-substituted alkyl, haloalkyl, or cyano-substituted alkyl; R8, R9are each independently selected from H, alkyl, hydroxy, alkyloxy, amino, halo, cyano, hydroxy-substituted alkyl, alkyloxy-substituted alkyl, amino-substituted alkyl, haloalkyl, cyano-substituted alkyl, substituted or unsubstituted carbocyclyl-substituted alkyl, substituted or unsubstituted heterocyclyl-substituted alkyl, substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl; said substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, means optionally substituted with one or more substituents selected from oxo, alkyl, hydroxy, alkyloxy, amino, halo, cyano, hydroxy-substituted alkyl, alkyloxy-substituted alkyl, amino-substituted alkyl, haloalkyl, cyano-substituted alkyl; R 10 substituted or unsubstituted heterocyclyl; said substituted or unsubstituted heterocyclyl means optionally substituted with one or more substituents selected from oxo, alkyl, hydroxy, alkyloxy, amino, halo, cyano, hydroxysubstituted alkyl, alkyloxy-substituted alkyl, aminosubstituted alkyl, haloalkyl, cyano-substituted alkyl; the ring is a substituted or unsubstituted heterocyclyl, said heterocyclyl containing at least one -NH- in the ring, said substitution means substitution with one or more substituents selected from oxo, alkyl, alkyloxy, halo, cyano, alkyloxy-substituted alkyl, haloalkyl, cyano-substituted alkyl; the ring is a heterocyclyl, said heterocyclyl containing at least one -NH- in the ring and / or said heterocyclyl is substituted with amino, hydroxy, amino-substituted alkyl, or hydroxy-substituted alkyl, said heterocyclyl being optionally substituted with one or more substituents selected from oxo, alkyl, alkyloxy, halo, cyano, alkyloxy-substituted alkyl, haloalkyl, cyano-substituted alkyl; m3is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m4is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

28. The diabody drug conjugate of claim 27, wherein, said alkyl is C 1-12 linear or branched alkyl; said carbocyclic group is a saturated, partially unsaturated or fully unsaturated carbocyclic group having 3 to 20 carbon atoms; said heterocyclic group is a saturated, partially unsaturated or fully unsaturated heterocyclic group having 3 to 20 atoms, containing 1, 2, 3, 4 heteroatoms selected from N, O, S.

29. The diabody drug conjugate of claim 27 or 28, wherein, L 13 L6is a direct bond when L2is attached by removal of the H of -NH2, -NH- on a chain, or -NH- on a ring; L 13 L6is a direct bond when L2is attached by removal of the H of -OH 30. The diabody drug conjugate of any one of claims 27-29, wherein, L 13 a group selected from the following groups, after removal of the H of -NH2, -OH, which is attached to L2: X1is selected from -CO-; X2is selected from -0-; X3is selected from -(CR8R9) m3 -NH2, -(CR8R9) m3 -OH; Y1is selected from -(CR8R9) m3 -NH2, -(CR8R9) m3 -OH; R7is selected from H.

31. The diabody drug conjugate of any one of claims 27-30, wherein, R8, R9are each independently selected from the group consisting of H, C 1-6 alkyl, C 3-6 cycloalkyl, 3-6 membered heterocycloalkyl containing 1 or 2 heteroatoms N or O.

32. The diabody drug conjugate of claim 31, wherein, X3is selected from the following groups: Preferably, X3is selected from the following groups:

33. The diabody drug conjugate of claim 31, wherein, Y1is selected from the following groups:

34. The diabody drug conjugate of claim 30, wherein, Ring is a 3-12 membered monocyclic, fused, spiro, or bridged heterocycloalkyl containing 1, 2, or 3 heteroatoms N or O, said heterocycloalkyl containing at least one -NH- in the ring and / or said heterocycloalkyl being substituted by amino, hydroxy, amino-substituted C 1-6 alkyl or hydroxy-substituted C 1-6 alkyl-substituted; Preferably, The ring is a 3-6 membered monocyclic heterocyclic alkyl group containing one nitrogen atom, wherein the C atom of the heterocyclic alkyl group is replaced by an amino, hydroxyl, or amino group. 1-6 alkyl or hydroxy substituted C 1-6 Alkyl substitution; More preferably, Ring is selected from the following groups: by removal of the H of the terminal -OH followed by coupling with L6 At the connection, L6 is 35. The diabody drug conjugate of claim 30, wherein, L 13 a group selected from the following groups, after removal of the H of -NH2, -OH, which is attached to L2: Preferably, L 13 a group selected from the following groups, after removal of the terminal -NH2, -OH H, which is attached to L2:

36. The diabody drug conjugate of any one of claims 1-35, wherein, Ab is selected from a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof; preferably, the Ab is selected from an anti-HER2 (ErbB2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-c-Met antibody, an anti-HER3 (ErbB3) antibody, an anti-HER4 (ErbB4) antibody, an anti-ROR1 antibody, an anti-CLDN6 antibody, an anti-CLDN9 antibody, an anti-CLDN18.2 antibody, an anti-NaPi-2b antibody, an anti-TNF-a antibody, an anti-ENPP3 antibody, an anti-DLL3 antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD28 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD37 antibody, an anti-CD38 antibody, an anti-CD44 antibody, an anti-CD45 antibody, an anti-CD47 antibody, an anti-CD48 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD73 antibody, an anti-CD98 antibody, an anti-CD105 antibody, an anti-CEA antibody, an anti-EphA2 antibody, an anti-MUC1 antibody, an anti-Lewis Y antibody, an anti-VEGFR antibody, an anti-GPNMB antibody, an anti-Integrin antibody, an anti-PSMA antibody, an anti-Tenascin-C antibody, an anti-SLC44A4 antibody, an anti-CD79 antibody, an anti-TROP-2 antibody, an anti-CD79B antibody, an anti-Mesothelin antibody, an anti-Nectin-4 antibody, an anti-TPBG antibody, or an antigen-binding fragment thereof.

37. The diabody drug conjugate of claim 36, wherein, Ab is an anti-HER2 (ErbB2) antibody or an antigen-binding fragment thereof. Preferably, wherein Ab is selected from a monoclonal antibody or an antigen-binding fragment thereof; More preferably, Ab is selected from Trastuzumab, Cetuximab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, a mutant of the above antibodies that can bind to an antigen, or an antigen-binding fragment thereof; Most preferably, Ab is selected from Trastuzumab, a mutant of Trastuzumab that can bind to an antigen (such as Trastuzumab-A141C, Trastuzumab-A141C-C220S), or an antigen-binding fragment thereof.

38. The diabody drug conjugate of any one of claims 1, 36-37, wherein, Formula (I) is one of the following:

39. A bi-toxin compound which is a compound of Formula (III), or an isotopically-labeled compound thereof, or an optical isomer, a geometric isomer, a tautomer, or an isomer mixture thereof, and pharmaceutically acceptable salts, wherein: Q’ is a linker group that can be linked to an antibody or an antigen-binding fragment, the group after Q’ is linked to Ab is Q as defined in any one of claims 1-35, 38; D1, D2, L1, L2, L3, L4 are as defined in any one of claims 1-35, 38.

40. The bi-toxin compound of claim 39, wherein, Q' is selected from 41. The bi-toxin compound of claim 40, wherein, Q' -L4 is selected from p1 is selected from 1, 2, 3, 4, 5, 6, p2 is selected from 1, 2, 3, 4, p3 is selected from 1, 2, 3, 4, p4 is selected from 1, 2, 3, 4, p5 is selected from 1, 2, 3, 4, R4 is selected from H or C 1-6 alkyl.

42. The bi-toxin compound of claim 41, wherein, Q' -L4 is selected from p1 is selected from 2, 3, 4, p2 is selected from 1, 2, p3 is selected from 1, 2, R4 is selected from H; Preferably, pi is selected from 3, p2 is selected from 1, 2, and p3 is selected from 2.

43. The bi-toxin compound of claim 42, wherein, Formula (III) is one of the following:

44. A pharmaceutical composition comprising the antibody drug conjugate of any one of claims 1-38, or the bi-toxin compound of any one of claims 39-43, and one or more pharmaceutically acceptable carriers or excipients.

45. Use of the bi-toxin compound of any one of claims 39-43 in the manufacture of an antibody drug conjugate.

46. Use of the antibody drug conjugate of any one of claims 1-38, or the bi-toxin compound of any one of claims 39-43, or the pharmaceutical composition of claim 44 in the manufacture of a medicament for treating a tumor or cancer.

47. A method of treating a tumor or cancer, comprising administering to a subject in need thereof an effective amount of the antibody drug conjugate of any one of claims 1-38, or the bi-toxin compound of any one of claims 39-43, or the pharmaceutical composition of claim 44.

48. The use according to claim 46, or the method according to claim 47, characterized in that, The tumor or cancer is selected from a solid tumor or a hematological tumor; preferably selected from breast cancer, gastric cancer, colon cancer, pancreatic cancer, renal cancer, ovarian cancer, lung cancer, soft tissue sarcoma, liposarcoma, lung cancer, non-small cell lung cancer, melanoma, head and neck cancer, cervical cancer, prostate cancer; more preferably is HER-2 positive breast cancer, triple negative breast cancer, HER-2 positive triple negative breast cancer, renal clear cell adenocarcinoma, small cell lung cancer, non-small cell lung cancer, large cell lung cancer.

49. The use or method of claim 48, wherein, The tumor or cancer is selected from a cancer or tumor with high expression of a HER2 (ErbB2) antibody. The tumor or cancer is selected from a cancer or tumor with high expression of a HER2 (ErbB2) antibody.

Images