Camptothecin drug conjugate, preparation method and application thereof

Abstract

A camptothecin conjugate and a preparation method thereof. The camptothecin conjugate has excellent drug activity and kinetic characteristics, and is suitable for preparing a drug for treating tumor-related diseases.

Description

Camptothecin drug conjugates and preparation methods and applications thereof

[0001] This application claims the Chinese patent application filed with the China Patent Office on October 23, 2023, with application number 202311382346.8 and invention name “Camptothecin drug conjugates and preparation methods and applications thereof”, the Chinese patent application filed with the China Patent Office on January 26, 2024, with application number 202410118218.0 and invention name “Camptothecin drug conjugates and preparation methods and applications thereof”, and the Chinese patent application filed with the China Patent Office on May 30, 2024, with application number 202410693604.2 and invention name “ The present invention relates to a Chinese patent application entitled “Camptothecin-drug conjugates and their preparation methods and applications”, a Chinese patent application filed with the Patent Office of China on August 23, 2024, with application number 202411176314.7 and invention name “Camptothecin-drug conjugates and their preparation methods and applications”, and a Chinese patent application filed with the Patent Office of China on October 12, 2024, with application number 202411427303.1 and invention name “Camptothecin-drug conjugates and their preparation methods and applications”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of pharmaceutical chemistry technology, and specifically relates to camptothecin-type conjugated compounds and preparation methods thereof. Such camptothecin compounds have excellent pharmaceutical activity and kinetic properties and are suitable for preparing drugs for treating tumor-related diseases. Background Art

[0003] Camptothecin (CPT), a pyrroloquinoline cytotoxic alkaloid, is one of the most studied natural antitumor drugs besides paclitaxel. It is primarily found in the fruit or root bark of the Davidia involucrata plant, a species endemic to my country. In 1985, Hsiang et al. discovered that camptothecin and its derivatives exert their anticancer effects by inhibiting DNA synthesis through topoisomerase I (Topo I). This discovery led to the development of numerous derivatives, making them a new hotspot in anticancer research. Since then, a new generation of camptothecins, including 10-hydroxycamptothecin (HCPT), irinotecan, topotecan, SN-38, and belotecan, have been approved for the treatment of various tumors, including colorectal cancer, small cell lung cancer, and ovarian cancer. Research into their indications and dosage forms has also yielded promising results.

[0004] With the development of drug delivery systems and antibody-drug conjugate (ADC) technology, a series of camptothecins, which were previously undruggable and had significant side effects, have regained their applicability. ADCs are composed of monoclonal antibodies targeting specific antigens and small molecule cytotoxic drugs linked via a linker. They combine the powerful killing effects of traditional small molecule chemotherapy drugs with the tumor-targeting properties of antibody drugs. ADC technology can effectively overcome the side effects of camptothecins, such as their poor water solubility and insufficient tissue distribution. The powerful targeting ability of the antibody portion enables the camptothecin drug it carries to be effectively delivered to the target tissue, thereby enhancing the therapeutic efficacy of this compound.

[0005] Summary of the Invention

[0006] The first aspect of the present invention provides an antibody-drug conjugate represented by Formula I or a pharmaceutically acceptable salt thereof:

[0007] Ab-[-L1-L2-L3-L4-D] q Formula I

[0008] wherein Ab is an antibody portion or antigen-binding fragment; D is a drug molecule, -L1-L2-L3-L4- is a fragment connecting the antibody Ab and the drug molecule D, and q is selected from 2-9;

[0009] L1 is a linker portion connected to the antibody, and its structure is: -La-Lb-; wherein La is selected from the following structures: wherein R1 is independently selected from H, halogen atoms, -CN, -OH, -NH2, C 1-4 Alkyl, C 1-4 Halogenated alkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy, hydroxy-C 1-4 Alkyl, m is selected from 0, 1, 2, 3; La marked with an asterisk * indicates that the position is connected to the antibody Ab; Lb is selected from Or Lb is a chemical bond, wherein n is selected from 0 or 1; h, i are each independently selected from 0, 1, 2, 3, 4, 5, X1 is selected from CH2, N-Ra, O, Ra is selected from H, C 1-4 Alkyl, C 1-4 Halogenated alkyl, C 1-4 Alkyl-carbonyl; Lb marked with an asterisk * indicates that the position is connected to La;

[0010] L2 is a chemical bond or L2 is selected from wherein k is selected from 0-8, j is selected from 0-20 or j is selected from 0-10, and p is selected from 0, 1, 2, 3, or 4; and the -NH- terminus of L2 is connected to L1;

[0011] L3 is selected from a peptide group consisting of 2-10 amino acid residues; the amino acids are selected from natural amino acids or unnatural amino acids;

[0012] L4 is a chemical bond or L4 is selected from the following groups: -NH-CH2-, Wherein, R3 is selected from Or R3 is H, and r is selected from 0-10; the -NH- terminal of L4 is connected to L3.

[0013] In one technical solution of Formula I of the present invention, Ab is an antibody portion or antigen-binding fragment; D is a drug molecule, -L1-L2-L3-L4- is a fragment connecting the antibody Ab and the drug molecule D, and q is selected from 2-9; L1 is a linker portion connected to the antibody, and its structure is: -La-Lb-; wherein La is selected from the following structures:

[0014] wherein R1 is independently selected from H, halogen atoms, -CN, -OH, -NH2, C 1-4 Alkyl, C 1-4 Halogenated alkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy, hydroxy-C 1-4 Alkyl, m is selected from 0, 1, 2, 3; La marked with an asterisk * indicates that the position is connected to the antibody Ab; Lb is selected from Or Lb is a chemical bond, wherein n is selected from 0 or 1; h, i are each independently selected from 0, 1, 2, 3, 4, 5, X1 is selected from CH2, N-Ra, O, Ra is selected from H, C 1-4 Alkyl, C 1-4 Halogenated alkyl, C 1-4 Alkyl-carbonyl; Lb marked with an asterisk * indicates that the position is connected to La; L2 is a chemical bond or L2 is selected from wherein k is selected from 0-8, j is selected from 0-10, and p is selected from 0, 1, 2, 3, or 4; and the -NH-terminus of L2 is connected to L1; L3 is selected from a peptidyl group consisting of 2-10 amino acid residues; the amino acids are selected from natural amino acids or non-natural amino acids; L4 is a chemical bond or L4 is selected from the following groups: -NH-CH2-, Wherein, R3 is selected from Or R3 is H, and r is selected from 0-10; the -NH- terminal of L4 is connected to L3.

[0015] In one technical solution of Formula I of the present invention, Ab is an antibody portion; D is a drug molecule, -L1-L2-L3-L4- is a fragment connecting the antibody Ab and the drug molecule D, and q is selected from 2-6; L1 is a linker portion connected to the antibody, and its structure is: -La-Lb-; wherein La is selected from the following structures: wherein R1 is independently selected from H, halogen atoms, -CN, -OH, -NH2, C 1-4 Alkyl, C 1-4 Halogenated alkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy, hydroxy-C 1-4 Alkyl, m is selected from 0, 1, 2, 3; La marked with an asterisk * indicates that the position is connected to the antibody Ab; Lb is selected from Or Lb is a chemical bond, wherein n is selected from 0 or 1; h, i are each independently selected from 0, 1, 2, 3, 4, 5, X1 is selected from CH2, N-Ra, O, Ra is selected from H, C 1- 4 alkyl, C 1-4 Halogenated alkyl, C 1-4 Alkyl-carbonyl; Lb marked with an asterisk * indicates that the position is connected to La; L2 is a chemical bond or L2 is selected from wherein k is selected from 0-8, j is selected from 0-10; and the -NH- end of L2 is connected to L1; L3 is selected from a peptide group consisting of 2-10 amino acid residues; the amino acids are selected from natural amino acids or non-natural amino acids; L4 is a chemical bond or L4 is selected from the following groups: -NH-CH2-, Wherein, R3 is selected from Or R3 is H, and r is selected from 0-10; the -NH- terminal of L4 is connected to L3.

[0016] In any one of the technical solutions of formula I of the present invention, q is selected from 2-6, preferably 3-5, and more preferably 3.5-4.5.

[0017] In any one of the technical solutions of formula I of the present invention, q is selected from 7-9, preferably 7.5-8.5, and more preferably 7.5-8.0.

[0018] In any one of the technical solutions of formula I of the present invention, R1 is selected from H.

[0019] In any one of the technical solutions of formula I of the present invention, n is selected from 1.

[0020] In any technical solution of formula I of the present invention, h and i are each independently selected from 0, 1, and 2.

[0021] In any technical solution of formula I of the present invention, h is 0 and i is 1.

[0022] In any one of the technical solutions of formula I of the present invention, X1 is selected from CH2.

[0023] In any one of the technical solutions of formula I of the present invention, X1 is selected from O.

[0024] In any one of the technical solutions of formula I of the present invention, X1 is selected from N-Ra.

[0025] In any one of the technical solutions of formula I of the present invention, Ra is selected from methyl, ethyl, and isopropyl.

[0026] In any one of the technical solutions of formula I of the present invention, Ra is selected from methyl.

[0027] In any technical solution of formula I of the present invention, k is selected from 2-6, preferably, k is selected from 4.

[0028] In any one of the technical solutions of formula I of the present invention, j is selected from 4-18, or j is selected from 6-16, or j is selected from 7-15.

[0029] In any technical solution of formula I of the present invention, j is selected from 7, 11, and 15.

[0030] In any technical solution of formula I of the present invention, j is selected from 15, or j is selected from 11.

[0031] In any technical solution of formula I of the present invention, j is selected from 7, or j is selected from 8.

[0032] In any one of the technical solutions of formula I of the present invention, L2 is a chemical bond or L2 is selected from

[0033] wherein k is selected from 0-8, j is selected from 0-10; and the -NH- terminal of L2 is connected to L1.

[0034] In any one of the technical solutions of formula I of the present invention, L2 is

[0035] In any one of the technical solutions of formula I of the present invention, L2 is

[0036] In any one of the technical solutions of formula I of the present invention, L2 is

[0037] In any one of the technical solutions of formula I of the present invention, L2 is

[0038] In any one of the technical solutions of formula I of the present invention, L2 is

[0039] In any one of the technical solutions of formula I of the present invention, L2 is

[0040] In any one of the technical solutions of formula I of the present invention, L2 is

[0041] In any one of the technical solutions of formula I of the present invention, L2 is

[0042] In any technical solution of formula I of the present invention, L2 is a chemical bond.

[0043] In any one of the technical solutions of formula I of the present invention, the amino acid residue in L3 is an α-amino acid residue selected from the following amino acid residues: glycine, D-phenylalanine, D-valine, D-alanine, D-asparagine, citrulline, lysine, serine, glutamic acid, and aspartic acid.

[0044] In any one of the technical solutions of formula I of the present invention, the amino acid residue in L3 is an α-amino acid residue selected from the following amino acid residues: glycine, D-phenylalanine, D-valine, D-alanine, D-asparagine, and citrulline.

[0045] In any one of the technical solutions of formula I of the present invention, L3 is selected from the following polypeptide fragments: -Gly-Gly-Phe-Gly-, -Val-Ala-, -Ala-Ala-, -Ala-Ala-Asn-, -Val-Cit-; and the -NH-terminus of L3 is connected to L2.

[0046] In any one of the technical solutions of formula I of the present invention, r is selected from 6-10, or r is selected from 9.

[0047] In any technical solution of formula I of the present invention, L4 is a chemical bond.

[0048] In any one of the technical solutions of formula I of the present invention, L4 is selected from R3 has the same meaning as in formula I.

[0049] In any one of the technical solutions of formula I of the present invention, L4 is selected from

[0050] In any one of the technical solutions of formula I of the present invention, L4 is selected from

[0051] In any one of the technical solutions of formula I of the present invention, L4 is selected from -NH-CH2-.

[0052] In any one of the technical solutions of formula I of the present invention, La is selected from the following structures:

[0053] In any one of the technical solutions of formula I of the present invention, Lb is selected from the following structures:

[0054] In any one of the technical solutions of formula I of the present invention, Lb is selected from the following structures:

[0055] In any one of the technical solutions of formula I of the present invention, L1 is selected from the following fragments:

[0056] Wherein, X1 has the same meaning as in any technical solution of Formula I.

[0057] In any one of the technical solutions of formula I of the present invention, L1 is selected from the following fragments:

[0058] In any one of the technical solutions of formula I of the present invention, -L1-L2-L3-L4- is selected from the following structural fragments:

[0059] In any technical solution of the present invention, Ab can be selected from anti-Her2 antibody, anti-Trop2 antibody, anti-Claudin18.2 antibody, anti-LIV-1 antibody, anti-5T4 antibody, anti-CLDN6 antibody, anti-CDH6 antibody, and anti-folate receptor antibody.

[0060] In any technical solution of formula I of the present invention, the anti-Her2 antibody is selected from trastuzumab and pertuzumab.

[0061] In any technical solution of Formula I of the present invention, the anti-LIV-1 antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0062] In any technical solution of Formula I of the present invention, the anti-LIV-1 antibody comprises a heavy chain variable region as shown in SEQ ID NO: 4.

[0063] In any technical solution of Formula I of the present invention, the anti-LIV-1 antibody comprises a heavy chain as shown in SEQ ID NO: 5.

[0064] In any technical solution of Formula I of the present invention, the anti-LIV-1 antibody comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0065] In any technical solution of Formula I of the present invention, the anti-LIV-1 antibody comprises a light chain variable region as shown in SEQ ID NO:9.

[0066] In any technical solution of Formula I of the present invention, the anti-LIV-1 antibody comprises a light chain as shown in SEQ ID NO: 10.

[0067] In any technical solution of formula I of the present invention, the anti-LIV-1 antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0068] In any technical solution of Formula I of the present invention, the anti-LIV-1 antibody comprises a heavy chain variable region as shown in SEQ ID NO: 4, and a light chain variable region as shown in SEQ ID NO: 9.

[0069] In any technical solution of Formula I of the present invention, the anti-LIV-1 antibody comprises a heavy chain as shown in SEQ ID NO: 5 and a light chain as shown in SEQ ID NO: 10.

[0070] In any technical solution of Formula I of the present invention, the anti-LIV-1 antibody is Ladiratuzumab.

[0071] In any technical solution of formula I of the present invention, the anti-Her2 antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13.

[0072] In any technical solution of Formula I of the present invention, the anti-Her2 antibody comprises a heavy chain variable region as shown in SEQ ID NO: 14.

[0073] In any technical solution of Formula I of the present invention, the anti-Her2 antibody comprises a heavy chain as shown in SEQ ID NO: 15.

[0074] In any technical solution of formula I of the present invention, the anti-Her2 antibody comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.

[0075] In any technical solution of Formula I of the present invention, the anti-Her2 antibody comprises a light chain variable region as shown in SEQ ID NO: 19.

[0076] In any technical solution of Formula I of the present invention, the anti-Her2 antibody comprises a light chain as shown in SEQ ID NO: 20.

[0077] In any technical solution of formula I of the present invention, the anti-Her2 antibody comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and LCDR1, LCDR2, LCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18.

[0078] In any technical solution of Formula I of the present invention, the anti-Her2 antibody comprises a heavy chain variable region as shown in SEQ ID NO: 14, and a light chain variable region as shown in SEQ ID NO: 19.

[0079] In any technical solution of Formula I of the present invention, the anti-Her2 antibody comprises a heavy chain as shown in SEQ ID NO: 15 and a light chain as shown in SEQ ID NO: 20.

[0080] In any technical solution of Formula I of the present invention, the anti-Her2 antibody is Trastuzumab.

[0081] In any technical solution of formula I of the present invention, the drug molecule D is selected from camptothecin compounds, auristatin compounds, eribulin compounds, maytansine compounds, calicheamicin compounds, and anthramycin compounds.

[0082] In any technical solution of formula I of the present invention, the drug molecule D is selected from a fragment having a structure of formula IIa or a pharmaceutically acceptable salt thereof:

[0083] Wherein, R is selected from F, C 1-4 Alkoxy, C 1-4 Halogenated alkyl, C 1-4Haloalkoxy; X2 is selected from -(CH2)s-, s is selected from 0, 1, 2, 3, 4; Y is selected from -O-, -N(Rc)-, the Rc is selected from H, C 1-4 alkyl, 3-5 membered cycloalkyl, 3-5 membered heterocycloalkyl; and when s=1, Rc is not H.

[0084] In any technical solution of formula IIa of the present invention, R is F, or R is methoxy.

[0085] In any technical solution of Formula IIa of the present invention, s is 1, or s is 2, or s is 3.

[0086] In any technical solution of formula IIa of the present invention, Rc is selected from H, methyl, and cyclopropyl.

[0087] In any technical solution of formula IIa of the present invention, Y is selected from -O-.

[0088] In any technical solution of formula IIa of the present invention, Y is -N(Rc)-, and Rc is methyl or cyclopropyl.

[0089] In any one of the technical solutions of Formula I of the present invention, the drug molecule D is selected from the following structural fragments or pharmaceutically acceptable salts thereof:

[0090] In any one of the technical solutions of Formula I of the present invention, -L1-L2-L3-L4-D can be selected from the following structural fragments or pharmaceutically acceptable salts thereof:

[0091] In one technical solution of the present invention, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof represented by Formula I is an antibody-drug conjugate or a pharmaceutically acceptable salt thereof represented by the following Formula III:

[0092] Wherein, Ab, La, X1, L2, L3, and q have the same meanings as in Formula I or any other technical solution.

[0093] In any technical solution of formula III of the present invention, La can be selected from R1 and m have the same meanings as in Formula I or any other technical solution.

[0094] In any technical solution of formula III of the present invention, La can be selected from

[0095] In any technical solution of formula III of the present invention, X1 is selected from O or CH2.

[0096] In any technical solution of formula III of the present invention, L2 is selected from a chemical bond,

[0097] In any technical solution of Formula III of the present invention, L3 is a polypeptide fragment consisting of 2-4 amino acids selected from the following: glycine, D-phenylalanine, D-valine, D-alanine, D-asparagine, and citrulline, and the -NH-terminus of L3 is connected to L2.

[0098] In any technical solution of Formula III of the present invention, L3 is selected from -Val-Ala-, -Val-Cit-, and -Gly-Gly-Phe-Gly-, wherein the -NH-terminus of L3 is connected to L2.

[0099] In any technical solution of formula III of the present invention, q is selected from 7.5-8.5, preferably 7.5-8.0.

[0100] In any technical solution of formula III of the present invention, q is selected from 4.0-5.0, preferably 4.0-4.5.

[0101] In any technical solution of formula III of the present invention, La can be selected from X1 is selected from O or CH2; L2 is selected from chemical bonds, L3 is selected from -Val-Ala-, -Val-Cit-, and -Gly-Gly-Phe-Gly-, wherein the -NH-terminus of L3 is connected to L2; and q is selected from 7.5-8.5.

[0102] In one technical solution of the present invention, the antibody-drug conjugate represented by Formula I or Formula III or a pharmaceutically acceptable salt thereof is an antibody-drug conjugate represented by the following Formula IV or a pharmaceutically acceptable salt thereof:

[0103] Wherein, Ab, La, X1, k, p, j, and q have the same meanings as in Formula I, Formula III, or any other technical solutions.

[0104] In any technical solution of formula IV of the present invention, La is selected from R1 and m have the same meanings as in Formula I or any other technical solution.

[0105] In any technical solution of formula IV of the present invention, La is selected from

[0106] In any technical solution of formula IV of the present invention, X1 is CH2 or O.

[0107] In any technical solution of formula IV of the present invention, k is selected from 3, 4, 5, preferably 4; p is selected from 2, 3, 4, preferably 3; j is selected from 6, 7, 8, preferably 7.

[0108] In any technical solution of formula IV of the present invention, q is selected from 7-9, preferably 7.5-8.5, more preferably 7.5-8.0.

[0109] In any technical solution of formula IV of the present invention, q is selected from 4.5-5.5, preferably 4.0-4.5.

[0110] In any technical solution of formula IV of the present invention, La is selected from X1 is CH2, O; k is 4, p is 3, j is 7, and q is 7.5-8.0.

[0111] In any technical solution of Formula III or Formula IV of the present invention, Ab is selected from anti-Her2 antibody, anti-Trop2 antibody, anti-Claudin18.2 antibody, anti-LIV-1 antibody, anti-5T4 antibody, anti-CLDN6 antibody, anti-CDH6 antibody, and anti-folate receptor antibody.

[0112] In any one of the technical solutions of Formula III or Formula IV of the present invention, the anti-Her2 antibody is selected from Trastuzumab and Pertuzumab.

[0113] In any one of the technical solutions of Formula III or Formula IV of the present invention, the anti-LIV-1 antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0114] In any one of the technical solutions of Formula III or Formula IV of the present invention, the anti-LIV-1 antibody comprises a heavy chain variable region as shown in SEQ ID NO: 4, and a light chain variable region as shown in SEQ ID NO: 9.

[0115] In any one of the technical solutions of Formula III or Formula IV of the present invention, the anti-LIV-1 antibody comprises a heavy chain as shown in SEQ ID NO: 5 and a light chain as shown in SEQ ID NO: 10.

[0116] In one technical solution of the present invention, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof represented by Formula I is selected from the following antibody-drug conjugates or a pharmaceutically acceptable salt thereof:

[0117] Among them, q1, q2, q4, q5, q6, q7, q8, and q9 are each independently selected from 7.5-8.0, preferably 7.6-7.7; q3 is selected from 4.0-5.0, preferably 4.2-4.6; and Ab is each independently selected from Ladiratuzumab and Trastuzumab.

[0118] The second aspect of the present invention provides a compound represented by formula II or a pharmaceutically acceptable salt thereof:

[0119] Wherein, R is selected from F, C 1-4 Alkoxy, C 1-4 Halogenated alkyl, C 1-4 Haloalkoxy; X2 is selected from -(CH2)s-, s is selected from 0, 1, 2, 3, 4; Y is selected from -OH, -NH-Rc, the Rc is selected from H, C 1-4 alkyl, 3-5 membered cycloalkyl, 3-5 membered heterocycloalkyl; and when s=1, Rc is not H.

[0120] In any one of the technical solutions of formula II of the present invention, R is F, or R is methoxy.

[0121] In any one of the technical solutions of Formula II of the present invention, s is 1, or s is 2, or s is 3.

[0122] In any one of the technical solutions of formula II of the present invention, Rc is selected from H, methyl, and cyclopropyl.

[0123] In any one of the technical solutions of formula II of the present invention, Y is selected from -OH.

[0124] In any one of the technical solutions of formula II of the present invention, Y is -NH-Rc, and Rc is methyl or cyclopropyl.

[0125] In any one of the technical solutions of Formula II of the present invention, the compound represented by Formula II is selected from the following compounds:

[0126] The third aspect of the present invention provides the following compounds, or pharmaceutically acceptable salts thereof:

[0127] In one technical solution of the present invention, the present invention provides a compound represented by the following formula IIIa or a pharmaceutically acceptable salt thereof:

[0128] Wherein, X1, L2, and L3 have the same meanings as in Formula I, Formula III, or any other technical solution, and La' is selected from the following groups:

[0129] wherein R1 is independently selected from H, halogen atoms, -CN, -OH, -NH2, C 1- 4 alkyl, C 1-4 Halogenated alkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy, hydroxy-C 1-4 Alkyl, m is selected from 0, 1, 2, 3.

[0130] In any technical solution of formula IIIa of the present invention, La' can be selected from R1 and m have the same meanings as in Formula I or any other technical solution.

[0131] In any technical solution of formula IIIa of the present invention, La' can be selected from

[0132] In any technical solution of formula IIIa of the present invention, X1 is selected from O or CH2.

[0133] In any technical solution of formula IIIa of the present invention, L2 is selected from a chemical bond,

[0134] In any technical solution of formula IIIa of the present invention, L3 is a polypeptide fragment consisting of 2-4 amino acids selected from the following: glycine, D-phenylalanine, D-valine, D-alanine, D-asparagine, citrulline, and the -NH-terminus of L3 is connected to L2.

[0135] In any technical solution of formula IIIa of the present invention, L3 is selected from -Val-Ala-, -Val-Cit-, and -Gly-Gly-Phe-Gly-, wherein the -NH-terminus of L3 is connected to L2.

[0136] In any technical solution of formula IIIa of the present invention, La' can be selected from X1 is selected from O or CH2; L2 is selected from chemical bonds, L3 is selected from -Val-Ala-, -Val-Cit-, -Gly-Gly-Phe-Gly-, wherein the -NH-terminus of L3 is connected to L2.

[0137] In one technical solution of the present invention, the present invention provides a compound represented by the following formula IVa or a pharmaceutically acceptable salt thereof:

[0138] Wherein, X1, k, p, and j have the same meanings as in Formula I, Formula III, Formula IIIa, Formula IV or any other technical solutions, and La' has the same meaning as in Formula IIIa.

[0139] In any technical solution of formula IVa of the present invention, La' is selected from R1 and m have the same meanings as in Formula I or any other technical solution.

[0140] In any technical solution of formula IVa of the present invention, La' is selected from

[0141] In any technical solution of formula IVa of the present invention, X1 is CH2 or O.

[0142] In any technical solution of formula IVa of the present invention, k is selected from 3, 4, 5, preferably 4; p is selected from 2, 3, 4, preferably 3; j is selected from 6, 7, 8, preferably 7.

[0143] In any technical solution of formula IVa of the present invention, q is selected from 7-9, preferably 7.5-8.5, more preferably 7.5-8.0.

[0144] In any technical solution of formula IVa of the present invention, La' is selected from X1 is CH2, O; k is 4, p is 3, j is 7, and q is 7.5-8.0.

[0145] In any technical solution of formula IVa of the present invention, La' is selected from X1 is CH2, O; k is 4, p is 3, j is 7, and q is 4.5-5.0.

[0146] A fourth aspect of the present invention provides the following structural fragment:

[0147] In a fifth aspect, the present invention provides use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof (including the antibody-drug conjugates represented by Formula I, Formula III, and Formula IV or a pharmaceutically acceptable salt thereof) according to the first aspect of the present invention in the preparation of a drug for treating cancer.

[0148] The sixth aspect of the present invention provides use of the compound or a pharmaceutically acceptable salt thereof (including the compound represented by Formula II or a pharmaceutically acceptable salt thereof) according to the second aspect of the present invention in the preparation of a drug for treating cancer.

[0149] The seventh aspect of the present invention provides use of the compound according to the second aspect of the present invention or a pharmaceutically acceptable salt thereof in the preparation of the antibody-drug conjugate according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof.

[0150] The eighth aspect of the present invention provides use of the compound according to the second aspect of the present invention or a pharmaceutically acceptable salt thereof in the preparation of the compound according to the third aspect of the present invention or a pharmaceutically acceptable salt thereof.

[0151] The ninth aspect of the present invention provides use of the compound according to the third aspect of the present invention or a pharmaceutically acceptable salt thereof in preparing the antibody-drug conjugate according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof.

[0152] The tenth aspect of the present invention provides use of the structural fragment described in the fourth aspect of the present invention in preparing the antibody-drug conjugate or a pharmaceutically acceptable salt thereof described in the first aspect of the present invention.

[0153] In the eleventh aspect of the present invention, there is provided a method for treating cancer, comprising administering to a patient in need an effective amount of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to the first aspect of the present invention, or administering to a patient in need an effective amount of the compound or a pharmaceutically acceptable salt thereof according to the second aspect of the present invention.

[0154] The twelfth aspect of the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to the first aspect of the present invention.

[0155] The thirteenth aspect of the present invention provides use of the pharmaceutical composition according to the twelfth aspect of the present invention in the preparation of a drug for treating cancer.

[0156] In a fourteenth aspect, the present invention provides the antibody-drug conjugate or a pharmaceutically acceptable salt thereof (including the antibody-drug conjugate or a pharmaceutically acceptable salt thereof represented by Formula I, Formula III, or Formula IV) according to the first aspect of the present invention, the compound or a pharmaceutically acceptable salt thereof (including the compound represented by Formula II or a pharmaceutically acceptable salt thereof) according to the second aspect of the present invention, and the pharmaceutical composition according to the twelfth aspect of the present invention for use as a medicine (i.e., for use in treatment).

[0157] In any technical solution of the present invention, the cancer includes breast cancer.

[0158] It should be noted that for Ab-[-L1-L2-L3-L4-D] q In the present application, the S atom marked with an asterisk (*) at the end of L1 or La should be understood by those skilled in the art to be the S atom generated by the connection between L1 and the sulfhydryl group contained in the Ab itself. That is, the S atom between L1 and Ab is not an additional external sulfur atom. Even if the asterisked S atom is not shown or written in L1 or La in this application, it is generated when the Ab is coupled with L1-L2-L3-L4-D, and the resulting ADC molecule also contains this S atom.

[0159] In the present invention, the antibody hLIV22 is Ladiratuzumab, which has a sequence encoded by the Kabat system as shown in Table S1 below.

[0160] Sequence of the S1 antibody hLIV22

[0161] More specifically, the antibody hLIV22 has a heavy chain as shown in SEQ ID NO: 5 and a light chain as shown in SEQ ID NO: 10:

[0162] In the present invention, the anti-Her2 antibody may be Trastuzumab, which has a sequence encoded by the Kabat system as shown in Table S2 below.

[0163] Sequence of the S2 antibody Trastuzumab

[0164] More specifically, the antibody Trastuzumab has a heavy chain as shown in SEQ ID NO: 15 and a light chain as shown in SEQ ID NO: 20:

[0165] The present invention has the following outstanding technical effects: The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to the first aspect of the present invention, and the compound or a pharmaceutically acceptable salt thereof according to the second aspect of the present invention, exhibit excellent cancer inhibition and acceptable toxicity. The preparation method of the present invention also offers significant technical advantages. When preparing ADCs, the proportion of the DAR4 component in the ADCs obtained by the present invention is significantly superior to that of other existing technologies.

[0166] Definitions and Explanations of Terms

[0167] Unless otherwise indicated, the terms used herein have their ordinary meanings in the art. A particular term or phrase should not be construed as ambiguous or unclear unless specifically defined, but rather should be understood according to its ordinary meaning in the art. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[0168] The term "drug molecule," also known as "load," "drug payload," or "payload," refers to a substance that has the potential to prevent or treat a disease. The drug in an antibody-drug conjugate is typically a cytotoxic drug, a chemical molecule that has the potential to disrupt the normal growth of tumor cells.

[0169] The term "linker" refers to a chemical structure fragment or bond that is connected to a ligand at one end and to a drug at the other end. It can also be connected to other linkers before being connected to the drug.

[0170] The term "drug-linker conjugate" is also referred to as payload-linker conjugate, linker-payload conjugate, and has the same meaning in the present invention.

[0171] In the present invention, the sulfur atom (S) at one end of La marked with * in Formula I or any of its technical solutions used to connect to Ab is actually derived from Ab. The sulfur atom S can be reflected in the structural formula of La or can be omitted. For example, La is With La The same interpretation shall apply to the S atoms at corresponding positions in the structures of other ADC compounds described in the present invention.

[0172] In the present invention, H refers to hydrogen, i.e., an atom with a nucleus having one proton, including three isotopes: protium (P), deuterium (D), and tritium (T). H also represents an atom of the aforementioned hydrogen element.

[0173] In the present invention, the term "independently selected" means that when multiple substituents exist simultaneously between different variable groups and between the same substituent, each variable group can select the same or different selected ranges or options.

[0174] In the present invention, the "halogen" or "halogen atom" refers to fluorine, chlorine, bromine, and iodine; the "halo" refers to a group formed when one or more hydrogen atoms in a substituent are replaced by a halogen atom.

[0175] The term "alkyl" refers to a straight or branched hydrocarbon group in which carbon atoms and carbon atoms are connected to hydrogen atoms by single bonds. 1-4 or C 1-6 Alkyl; C 1-4 "Alkyl" means a straight or branched chain alkyl group having 1 to 4 carbon atoms; C 1-4 Specific examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl, preferably methyl, ethyl, n-propyl, and isopropyl; "C 1-6 "Alkyl" means a straight or branched chain alkyl group having 1 to 6 carbon atoms. 1-6 Specific examples of alkyl groups include but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl.

[0176] The term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by a halogen atom. 1-4 Halogenated alkyl, C 1-6 Halogenated alkyl. C 1-4 Specific examples of haloalkyl include, but are not limited to, monofluoromethyl, monochloromethane, difluoromethyl, dichloromethane, trifluoromethyl, trichloromethyl, and tribromomethyl.

[0177] The term "alkoxy" refers to an alkyl-O-, i.e., a substituent formed by replacing the hydrogen atom in -OH with an alkyl group. Alkyl groups are as described above, and alkoxy groups include C 1-4 Alkoxy, C 1-6 Alkoxy; C 1-4 Specific examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, preferably methoxy, ethoxy, isooxy; C 1-6 Specific examples of alkoxy groups include C 1-4 In addition to the specific examples of alkoxy, n-pentyloxy, neopentyloxy and n-hexyloxy are also included.

[0178] The term "haloalkoxy" refers to a substituent obtained by replacing one or more hydrogen atoms of an alkoxy group with a halogen atom. 1-4 Examples of haloalkoxy include, but are not limited to, trifluoromethoxy, trichloromethoxy, 2,2,2-trifluoroethoxy, 2,2,2-trichloroethoxy.

[0179] The term "hydroxy-C 1-4 "Alkyl" refers to C 1-4 A substituent obtained by replacing one or more hydrogen atoms of an alkyl group with a hydroxyl group. 1-4 Specific examples of the alkyl group include hydroxymethyl and hydroxyethyl.

[0180] The term "cycloalkyl" refers to a cyclic, saturated monocyclic structure formed by single bonds between carbon atoms. Specific examples of 3-6 membered cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl; specific examples of 5-6 membered cycloalkyl groups include, but are not limited to, cyclopentyl and cyclohexyl.

[0181] The term "heterocycloalkyl" refers to a substituent group formed by replacing one or more carbon atoms in the annular backbone of a cycloalkyl group with one or more heteroatoms or heteroatoms; the heteroatoms or heteroatoms are generally selected from N, N(O), O, S, S(O), and S(O). Specific examples of the 5- to 6-membered heterocycloalkyl group include, but are not limited to, tetrahydropyrrolyl, tetrahydrofuranyl, piperidinyl, and piperazinyl.

[0182] In the present invention, the "natural amino acids" refer to the following amino acids with α-configuration: glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile I), proline (Pro), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), serine (Ser), threonine (Thr), cysteine ​​(Cys), methionine (Met), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg), and histidine (His).

[0183] The term "unnatural amino acid" refers to amino acids other than the natural amino acids, such as citrulline.

[0184] The term "optionally" means that the substituent may be substituted by other substituents or may not be substituted by other substituents.

[0185] As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product resulting, directly or indirectly, from combination of the specified ingredients in the specified amounts. Those skilled in the art can modify the actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention so as to achieve the desired therapeutic response in an amount effective for a particular patient, composition, and route of administration.

[0186] The term "pharmaceutically acceptable carrier" refers to a medium generally accepted in the art for delivering biologically active agents to animals, especially mammals.

[0187] The term "excipient" generally refers to a carrier, diluent and / or vehicle required to formulate an effective pharmaceutical composition.

[0188] The term "effective amount" refers to a compound of the present invention or a pharmaceutically acceptable salt thereof, which is a sufficient amount of the compound to treat the disorder at a reasonable benefit / risk ratio applicable to any medical treatment and / or prevention.

[0189] The term "pharmaceutically acceptable salt" refers to salts that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, etc., and are commensurate with a reasonable benefit / risk ratio.

[0190] In the present invention, a chemical bond in a substituent is marked with When , it indicates that the substituent is connected to the adjacent group or structural fragment at this position. The presence of a dash "-" in the substituent structure indicates the point of attachment for the substituent, for example, -CH3 is connected through a C atom. and Indicates the absolute configuration of a stereocenter, i.e., R or S configuration.

[0191] When a substituent's bond crosses two atoms in a ring, the substituent may be bonded to any atom in the ring. The substituent R can be substituted at any position on the benzene ring.

[0192] The "isomers" described in the present invention include geometric isomers and stereoisomers, such as atropisomers, cis-trans isomers, enantiomers, diastereomers, tautomers, and racemic mixtures and other mixtures thereof, all of which fall within the scope of the present invention. The term "enantiomer" refers to stereoisomers that are mirror images of each other. The term "tautomer" refers to a type of functional group isomer that has different hydrogen attachment points due to the displacement of one or more double bonds, for example, a ketone and its enol form are keto-enol tautomers. The term "diastereomer" refers to stereoisomers in which the molecule has two or more chiral centers and is not a mirror image of the other molecules. The term "cis-trans isomers" refers to different spatial configurations in which double bonds or single bonds of ring carbon atoms in a molecule cannot rotate freely. The term "atropisomers" refers to stereoisomers that can be separated because single bond rotation is hindered or rotates very slowly.

[0193] Stereoisomers of the compounds of the present invention can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. For example, one enantiomer of a compound of the present invention can be prepared by asymmetric catalysis or chiral auxiliary derivatization. Alternatively, a compound of a single stereoconfiguration can be obtained from a mixture by chiral resolution techniques. Alternatively, they can be prepared directly using chiral starting materials. Separation of optically pure compounds in the present invention is typically accomplished by preparative chromatography using chiral chromatographic columns to achieve the purpose of separating chiral compounds.

[0194] In the present invention, the unit of solution concentration M represents mol / L, and nM represents mmol / L.

[0195] In the present invention, (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)2-oxoethyl)-3-phenylpropanamide refers to the compound with the following structure:

[0196] Compound A refers to the compound with the following structure, which can be prepared with reference to WO2022228493:

[0197] The compound Deruxtecan refers to the compound with the following structure:

[0198] SN-38 refers to the compound with the following structure:

[0199] Dxd refers to the compound with the following structure:

[0200] The chemical abbreviations used in the present invention and the chemical names they refer to are as follows: BRIEF DESCRIPTION OF THE DRAWINGS

[0201] FIG1 is an evaluation of the efficacy of ADC1-ADC6 of the present invention in a mouse transplanted HCC1806 breast cancer model;

[0202] Figure 2 shows the changes in body weight of ADC1-ADC6 of the present invention in a mouse transplanted HCC1806 breast cancer model;

[0203] FIG3 is an evaluation of the efficacy of ADC7-ADC9 of the present invention in a mouse transplanted HCC1806 breast cancer model;

[0204] FIG4 shows the changes in body weight of ADC7-ADC9 of the present invention in a mouse transplanted HCC1806 breast cancer model;

[0205] FIG5 shows the drug concentration-time curves of ADC10-ADC11 of the present invention. DETAILED DESCRIPTION

[0206] The present invention is further described in detail below through specific preparation examples and biological experiments. However, it should be understood that these examples and biological experiments are only for specific illustration purposes and should not be construed as limiting the present invention in any form. It will be clear to those skilled in the art that, unless otherwise specified, the materials used are well known in the art and can be purchased commercially or obtained by those skilled in the art according to published literature or conventional methods. Unless otherwise specified, all reactions of the present invention are carried out under continuous magnetic stirring in a dry nitrogen or argon atmosphere, with dry solvents, wherein: (i) temperatures are expressed in degrees Celsius (°C), and operations are carried out at room temperature, which generally refers to 15-35°C, preferably 20-30°C, and more preferably 20-25°C; (ii) solvent removal is performed by evaporation under reduced pressure on a rotary evaporator with a bath temperature not exceeding 60°C; (iii) the reaction process is monitored by thin layer chromatography (TLC); and (iv) the final product has satisfactory hydrogen nuclear magnetic resonance spectroscopy (1H-NMR) and / or mass spectrometry (MS) data.

[0207] Test equipment:

[0208] The structures of the compounds of the present invention are determined by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS). NMR chemical shifts (δ) are given in parts per million (ppm). NMR measurements are performed using a Bruker Neo 400M or Bruker Ascend 400 NMR instrument, using deuterated dimethyl sulfoxide (DMSO-d6), deuterated methanol (CD3OD), and / or deuterated chloroform (CDCl3) as the solvent, and tetramethylsilane (TMS) as the internal standard.

[0209] Liquid chromatography-mass spectrometry (LC-MS) was performed using a Shimadzu 2030Plus-LCMS2020 mass spectrometer, an Agilent 1260-6125B single quadrupole mass spectrometer, or a Shimadzu LCMS-2020 mass spectrometer. HPLC was performed using a Shimadzu LCMS-2020 or Agilent 1260 high-performance liquid chromatograph.

[0210] Preparative high-performance liquid chromatography was performed using a Shimadzu FRC-40 equipped with LC-20AP and PDA-20A (column: Synergi Max-RP, 150×30 mm, 4 m) or a GILSON GX-281 LC (columns: Boston Prime C18 150*30 mm*5 μm; YMC-Actus Triart C18 150*30 mm*5 μm; YMC-Triart PFP 150*30 mm*5 μm; YMC-Triart Phenyl 150*30 mm*5 μm).

[0211] The preparation and separation conditions of the payload-linker coupling compounds in the examples of this application are as follows: (Chromatographic column: Boston Prime C18 150*30mm*5μm; A: 0.225% FA in Water; B: MeCN). Those skilled in the art can appropriately adjust the elution gradient according to different example compounds.

[0212] Example A1: Preparation of Compound A1

[0213] Step 1: Synthesis of 6-iodo-2-(methylthio)benzothiazole (A1-2)

[0214] In a 100 mL single-necked flask, add 6-iodo-2-mercaptobenzothiazole A1-1 (2.8 g, 9.55 mmol) and dissolve it in tetrahydrofuran (20 mL). Add iodomethane (1.8 g, 12.42 mmol) and potassium carbonate (2.6 g, 19.10 mmol) to the resulting solution at 0°C. Stir the reaction mixture at room temperature for 4 hours. Add water (30 mL) and extract with ethyl acetate (30 mL x 3). The combined organic phases are washed with saturated brine (50 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue is purified on a silica gel column (PE:EA = 90:10) to afford A1-2 (2.9 g, 99%).

[0215] LC-MS: 308.0 [M+H] + .

[0216] Step 2: Synthesis of 6-(2-(methylthio)benzo[d]thiazol-6-yl)hex-5-ynoic acid (A1-3)

[0217] A1-2 (500.0 mg, 1.63 mmol) was added to a 10 mL single-necked vial and dissolved in diisopropylamine (3 mL). Bisacetonitrile palladium chloride (12.7 mg, 0.05 mmol), triphenylphosphine (42.7 mg, 0.16 mmol), and cuprous iodide (10.3 mg, 0.03 mmol) were added to the resulting solution. The reaction mixture was stirred at room temperature for 5 minutes, and 5-hexynoic acid (273.8 mg, 2.44 mmol) was added dropwise. The reaction mixture was stirred at 50°C overnight. The reaction mixture was concentrated under reduced pressure, and the residue was purified on a silica gel column (dichloromethane:methanol = 93:7) to obtain A1-3 (500.0 mg, 95%).

[0218] LC-MS: 292.0 [M+H] + .

[0219] Step 3: Synthesis of 6-(2-(methylsulfonyl)benzo[d]thiazol-6-yl)hexyl-5-ynoic acid (A1-4)

[0220] A1-3 (500.0 mg, 1.72 mmol) was added to a 10 mL single-necked vial and dissolved in dichloromethane (20 mL). Meta-chloroperbenzoic acid (888.3 mg, 5.15 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated to half its volume under reduced pressure and filtered. The organic phase was then concentrated under reduced pressure, and the residue was purified by preparative HPLC (column: Boston Prime C18 150*30 mm*5 μm; A: 0.225% FA in Water; B: MeCN) to afford A1-4 (80.0 mg, 14%).

[0221] LC-MS: 324.0 [M+H] + .

[0222] Step 4: Synthesis of 1,1-dimethylethyl (2S)-4-(2-benzothiazolylsulfonyl)-2-[[(1,1-dimethoxy)carbonyl]amino]butyrate (A1-5)

[0223] A1-4 (500.0 mg, 1.55 mmol) was added to a 10 mL single-necked vial and dissolved in dichloromethane (10 mL). N(e)-Boc-L-lysine tert-butyl ester hydrochloride (524.0 mg, 1.55 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (444.6 mg, 2.32 mmol), and 4-dimethylaminopyridine (566.7 mg, 4.64 mmol) were added to the resulting solution. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified on a silica gel column (dichloromethane:methanol = 90:10) to afford A1-5 (180.0 mg, 19%).

[0224] LC-MS: 552.2[M+H] + .

[0225] Step 5: Synthesis of 6-[[[2-(methylsulfonyl)-6-benzothiazolyl]carbonyl]amino]hexanoic acid (A1-6)

[0226] A1-5 (180.0 mg, 0.30 mmol) was added to a 50 mL single-necked flask and dissolved in trifluoroacetic acid (2.5 mL) and dichloromethane (5 mL). The reaction mixture was stirred at room temperature for 1 hour, then heated to 30°C and stirred for 2 hours. The reaction mixture was concentrated under reduced pressure, dissolved in 10 mL of toluene, and concentrated again under reduced pressure to yield A1-6 (167.4 mg, 100%).

[0227] LC-MS 452.2[M+H] + .

[0228] Step 6: Synthesis of (S)-31-(6-(2-(methylsulfonyl)benzo[d]thiazol-6-yl)hex-5-ynylamino)-25-oxo-2,5,8,11,14,17,20,23-octaoxa-26-azatricon-32-oic acid (A1-7)

[0229] A1-6 (133.7 mg, 0.30 mmol) was added to a 100 mL single-necked flask and dissolved in dichloromethane (10 mL). A solution of methyl-heptapeptide glycol-acetyl chloride (123.4 mg, 0.30 mmol) in dichloromethane (1 mL) and diisopropylethylamine (0.10 mL, 0.59 mmol) were added to the resulting solution. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure, and the residue was purified on a silica gel column (dichloromethane:methanol = 82:18) to afford A1-7 (110.0 mg, 45%).

[0230] LC-MS:832.4[M+H] + .

[0231] Step 7: Synthesis of N-((10S,19S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]nazo[1,2-b]quinolin-1-yl)amino)-19-(6-(2-(methylsulfonyl)benzo[d]thiazol-6-yl)hex-5-ynylamino)-1,6,9,12,15,18-hexaoxo-3-oxa-5,8,11,14,17-pentaazatriazol-23-yl)-2,5,8,11,11,14,17,20,23-octacosane-25amide (A1)

[0232] In a 100 mL single-necked bottle, A1-7 (80.0 mg, 0.10 mmol), diisopropylethylamine (0.05 mL, 0.3 mmol) and (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3' A solution of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (28.3 mg, 0.10 mmol) (4,4':6,7] indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)2-oxoethyl)-3-phenylpropanamide (80.9 mg, 0.10 mmol) was dissolved in N,N-dimethylformamide (2 mL). The resulting solution was added with 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (28.3 mg, 0.10 mmol) under ice-cooling. The reaction solution was stirred under ice-cooling for 1 hour. The reaction solution was filtered and analyzed by preparative high-performance liquid chromatography (column: Boston Prime C18 150*30 mm*5 μm; A: 0.225% FA in Water; B: MeCN) to obtain compound A1.

[0233] LC-MS: 1654.6[M+H] + .

[0234] Example A2: Preparation of Compound A2

[0235] Step 1: Synthesis of 5-(4-iodophenyl)-1,3,4-oxadiazole-2-thiol (A2-2)

[0236] To a 500 mL single-necked flask, add 4-iodobenzohydrazide (A2-1) (5.0 g, 19.08 mmol) and ethanol (200 mL). Add potassium hydroxide (1.1 g, 19.08 mmol) and carbon disulfide (4.4 g, 57.24 mmol) at room temperature and reflux overnight. Cool to room temperature, spin dry the reaction mixture, then dilute with water (30 mL), adjust the pH to 6 with 2 M HCl, and filter to yield A2-2 (5.0 g, 86%).

[0237] LC-MS: 305.0 [M+H] + .

[0238] Step 2: Synthesis of 2-(4-iodophenyl)-5-(methylthio)-1,3,4-oxadiazole (A2-3)

[0239] A2-2 (10.0 g, 32.88 mmol) and tetrahydrofuran (200 mL) were added to a 500 mL three-necked flask. Triethylamine (10.0 g, 98.65 mmol), iodomethane (5.1 g, 36.17 mmol), and 4-dimethylaminopyridine (0.4 g, 3.29 mmol) were then added at room temperature. After replacing the nitrogen atmosphere, the mixture was stirred at 25°C overnight. The reaction mixture was poured into water (100 mL) and extracted with dichloromethane (50 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether:ethyl acetate = 1:1) to afford A2-3 (10.0 g, 96%).

[0240] LC-MS: 319.0 [M+H] + .

[0241] Step 3: Synthesis of 6-(4-(5-(methylthio)-1,3,4-oxadiazol-2-yl)phenyl)hexyl-5-ynoic acid (A2-4)

[0242] A2-3 (10.0 g, 31.43 mmol) and toluene (100 mL) were added to a 250 mL three-necked flask. Hex-5-ynoic acid (7.1 g, 62.87 mmol), cuprous iodide (0.9 g, 4.72 mmol), triphenylphosphine (0.8 g, 3.14 mmol), bis(acetonitrile)palladium dichloride (0.4 g, 1.57 mmol), and diisopropylamine (19.1 g, 188.60 mmol) were then added at room temperature. The atmosphere was purged with nitrogen three times and stirred at room temperature overnight. The reaction mixture was filtered, concentrated, and purified by column chromatography (dichloromethane:methanol = 10:1) to afford A2-4 (5.0 g, 32%).

[0243] LC-MS: 303.0 [M+H] + .

[0244] Step 4: Synthesis of 6-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)hexyl-5-ynoic acid (A2-5)

[0245] A2-4 (1.4 g, 4.63 mmol) and dichloromethane (30 mL) were added to a 100 mL three-necked flask. At room temperature, m-chloroperbenzoic acid (4.0 g, 23.18 mmol) was added. The atmosphere was purged with nitrogen three times and stirred overnight at room temperature. The reaction mixture was filtered, and the filtrate was concentrated and separated by preparative HPLC to afford A2-5 (250.0 mg, 16%).

[0246] LC-MS: 335.0 [M+H] + .

[0247] Step 5: Synthesis of N-(((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)hexyl-5-ynamide (A2)

[0248] A2-5 (40.0 mg, 0.12 mmol) and N,N-dimethylformamide (3 mL) were added to a 50 mL single-necked bottle. (7-Azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (91.0 mg, 0.24 mmol) and N,N-diisopropylethylamine (46.4 mg, 0.36 mmol) were added at 0°C and stirred for 5 minutes. Then, (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-((((1 A solution of (5-(S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)2-oxoethyl)-3-phenylpropanamide 7 (100.6 mg, 0.12 mmol) in N,N-dimethylformamide (2 mL) was stirred at room temperature for 1 hour. The reaction solution was purified by preparative HPLC to give Compound A2 (20.2 mg, 15%).

[0249] LC-MS: 1157.2[M+H] + .

[0250] Example A3: Preparation of Compound A3

[0251] Step 1: Synthesis of (S)-tert-butyl 31-(6-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)hexyl-5-ynamido)-25-oxo-2,5,8,11,14,17,20,23-octaoxa-26-azatrioxane-32-oate (A3-1)

[0252] In a 100 mL three-necked flask, 6-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)hexyl-5-ynoic acid (i.e., compound A2-5) (70.0 mg, 0.21 mmol) and acetonitrile (3 mL) were added, and N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (88.1 mg, 0.31 mmol) and N-methylimidazole (34.4 mg, 0.42 mmol) were added at room temperature and stirred for 10 minutes. Then, a solution of 2-methylpropan-2-yl (2S)-2-amino-6-[(25-oxo-2,5,8,11,14,17,20,23-octaoxypentacosanyloxy)amino]hexanoate (122.0 mg, 0.21 mmol) in acetonitrile (2 mL) was added and stirred for 50 minutes. The reaction solution was concentrated and purified on a silica gel column (DCM:MeOH=10:1) to give compound A3-1 (188.0 mg, 100%).

[0253] LC-MS:899.4[M+H] + .

[0254] Step 2: Synthesis of (S)-31-(6-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)hexyl-5-ynamido)-25-oxo-2,5,8,11,14,17,20,23-octyloxy-26-azatrioxane-32-oic acid (A3-2)

[0255] In a 50 mL three-necked flask, A3-1 (100.0 mg, 0.11 mmol) was added to dichloromethane (1 mL) and methyltrifluoroacetic acid (0.5 mL) and stirred at room temperature for 1 hour. The solution was concentrated and separated by preparative HPLC to afford A3-2 (25.0 mg, 27%).

[0256] LC-MS:843.2[M+H] + .

[0257] Step 3: N-((10S,19S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-19-(6-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)hexyl-5-ynamido)-1,6,9,12,15,18-hexaoxo-3-oxo-5,8,11,14,17-pentaazatriazin-23-yl)-2,5,8,14,17,20,20 Synthesis of 3-octaoxapentacosane-25-amide A3

[0258] A3-2 (25.0 mg, 0.03 mmol) and acetonitrile (4 mL) were added to a 50 mL single-necked bottle, and (S)-2-(2-(2-(2-aminoacetamido-acetamido)-acetylamino)-N-(2-ethyl)-(1S,9S)-9-ethyl-5-fluorofluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,13-dioxo-1,2,3,10,10 The mixture was stirred at room temperature for 1 hour. The reaction mixture was purified by preparative high performance liquid chromatography to obtain compound A3 (11.4 mg, 23%).

[0259] LC-MS: 1665.6[M+H] + .

[0260] Example A4: Preparation of Compound A4

[0261] Step 1: Synthesis of tert-butyl (S)-31-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-25-oxo-2,5,8,11,14,17,20,23-octaoxa-26-azatriaza-32-oic acid ester (A4-2)

[0262] To a 10 mL single-necked vial was added tert-butyl ((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysine ester (176.9 mg, 0.38 mmol) and dissolved in dichloromethane (5 mL). A solution of methyl-heptapeptylethylene glycol-acetyl chloride (160.0 mg, 0.38 mmol) in dichloromethane (1 mL) and diisopropylethylamine (0.1 mL, 0.77 mmol) were added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was purified on a silica gel column (dichloromethane:methanol = 95:5) to afford A4-2 (251.0 mg, 87%).

[0263] LC-MS: 805.6[M+H] + .

[0264] Step 2: Synthesis of (S)-31-amino-25-oxo-2,5,8,11,14,17,20,23-octaoxa-26-azatricontan-32-oic acid tert-butyl ester (A4-3)

[0265] A4-2 (250.0 mg, 0.31 mmol) was added to a 10 mL single-necked vial and dissolved in dimethylformamide (5 mL). Diethylamine (0.3 mL, 3.11 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure to afford A4-3 (181.2 mg, 100%).

[0266] LC-MS: 583.4[M+H] + .

[0267] Step 3: Synthesis of tert-butyl (S)-31-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-25-oxo-2,5,8,11,14,17,20,23-octaoxa-26-azatriaza-32-oic acid ester (A4-4)

[0268] To a 10 mL single-necked vial, add benzoylacrylic acid (61.3 mg, 0.35 mmol), N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (146.5 mg, 0.52 mmol), and N-methylimidazole (57.2 mg, 0.70 mmol). Dissolve the mixture in acetonitrile (2 mL). Stir the resulting solution at room temperature for 10 minutes. Add A4-3 (202.8 mg, 0.35 mmol). Stir the reaction mixture at room temperature for 1 hour. The reaction mixture is concentrated under reduced pressure, and the residue is purified on a silica gel column (dichloromethane:methanol = 89:11) to afford A4-4 (150.0 mg, 58%).

[0269] LC-MS:741.6[M+H] + .

[0270] Step 4: Synthesis of (S,E)-25-oxo-31-(4-oxo-4-phenylbut-2-enamide)-2,5,8,11,14,17,20,23-octaoxa-26-azatriaza-32-enoic acid (A4-5)

[0271] A4-4 (150.0 mg, 0.20 mmol) was added to a 10 mL single-necked vial and dissolved in dichloromethane (2 mL). Trifluoroacetic acid (1 mL) was added to the resulting reaction mixture. The reaction mixture was stirred at 30°C for 1 hour. The reaction mixture was concentrated under reduced pressure to obtain A4-5 (138.3 mg, 100%).

[0272] LC-MS: 684.8[M] + .

[0273] Step 5: Synthesis of N-((10S,19S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-19-((E)-4-oxo-4-phenylbut-2-enamido)-3-oxa-5,8,11,14,17-pentaazatriazin-23-yl)-2,5,8,11,1,14,17,20,23-octaoxapentacan-25-amide A4

[0274] In a 100 mL single-necked bottle, A4-5 (138.3 mg, 0.20 mmol) and (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)2-oxoethyl)-3-phenylpropanamide (169.9 mg, 0.20 mmol) were added and dissolved with N,N-dimethylformamide (1 mL). To the resulting solution, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (59.54 mg, 0.202 mmol) was added under ice-cooling. The reaction mixture was stirred under ice-cooling for 1 hour. The reaction mixture was filtered and purified by preparative HPLC to afford A4 (23.0 mg, 8%).

[0275] LC-MS: 1507.6[M+H] + .

[0276] Example A5: Preparation of Compound A5

[0277] Step 1: Synthesis of N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyylidene-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]-2-{[(7S)-13-amino-7-benzyl-3,6,9,12-tetraoxyylidene-2,5,8,11-tetraazatridec-1-yl]oxy}acetamide (A5-2)

[0278] A5-1 (200.0 mg, 0.19 mmol) was added to a 50 mL single-necked flask and dissolved in N,N-dimethylformamide (2 mL). Diethylamine (0.19 mL, 1.9 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was slurried with petroleum ether to obtain A5-2 (120 mg, 75.1%).

[0279] LC-MS:841.3[M+H] + .

[0280] Step 2: Synthesis of N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyylidene-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]-2-{[(14S)-14-benzyl-1-{3,5-bis[5-(methyldioxyylidene-λ6-thio)-1,3,4-oxadiazacyclopentyl]phenyl}-6,9,12,15,18-pentaoxyylidene-4-oxa-7,10,13,16,19-pentaazaicos-1-yn-20-yl]oxy}acetamide A5

[0281] In a 10 mL single-necked vial, [(3-{3,5-bis[5-(methyldioxyylidene-λ6-thio)-1,3,4-oxadiazacyclopentan-2-yl]phenyl}prop-2-ynyl)oxy]acetic acid (B3-6) (19.3 mg, 0.04 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (24.5 mg, 0.06 mmol) were added and dissolved in N,N-dimethylformamide (1 mL). Diisopropylethylamine (0.01 mL, 0.09 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. A5-2 (33.7 mg, 0.04 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to yield A5 (22.5 mg, 43.1%).

[0282] LC-MS: 1305.3[M+H] + .

[0283] Example B1: Preparation of Compound B1

[0284] Step 1: Synthesis of 2,5,8,11,14,17,20,23,26-nonoxyoctadecane-28-yl methanesulfonate (B1-2)

[0285] Nonanediol monomethyl ether (22.0 g, 51.34 mmol) was added to a 100 mL single-necked flask and dissolved in dichloromethane (50 mL). Triethylamine (14.3 mL, 102.68 mmol) and methanesulfonyl chloride (8.8 g, 77.01 mmol) were added to the resulting solution at 0°C. The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified on a silica gel column (dichloromethane:methanol = 92:8) to obtain B1-2 (23.5 g, 90%).

[0286] LC-MS: 507.4[M+H] + .

[0287] Step 2: Synthesis of methyl-nonaethylene glycol-bromo (B1-3)

[0288] B1-2 (23.5 g, 46.39 mmol) was added to a 100 mL single-necked flask and dissolved in acetonitrile (200 mL). Tetrabutylammonium bromide (2.7 g, 8.29 mmol) was added to the resulting solution. The reaction mixture was stirred at 50°C overnight. The reaction mixture was concentrated under reduced pressure, and the residue was purified on a silica gel column (dichloromethane:methanol = 96:4) to obtain B1-3 (17.5 g, 77%).

[0289] LC-MS:491.2[M+H] + .

[0290] Step 3: Synthesis of 2-((2,5,8,11,14,17,20,23,26-nonanoxaoctan-28-yl)oxy)-4-nitrobenzaldehyde (B1-4)

[0291] B1-3 (5.5 g, 11.19 mmol) and 2-hydroxy-4-nitrobenzaldehyde (1.9 g, 11.19 mmol) were added to a 100 mL single-necked bottle and dissolved in N,N-dimethylformamide (10 mL). Potassium carbonate (7.7 g, 55.96 mmol) and potassium iodide (40.0 mg, 0.22 mmol) were added to the resulting solution. The reaction mixture was stirred at 100°C for 3 hours. The reaction mixture was cooled to room temperature, concentrated under reduced pressure, diluted with water (50 mL), and extracted with ethyl acetate (30 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified on a silica gel column (dichloromethane:methanol = 96:4) to yield B1-4 (14.3 g, 55%).

[0292] LC-MS:578.4[M+H] + .

[0293] Step 4: Synthesis of (2-((2,5,8,11,14,17,20,23,26-nonyloxyoctadecane-28-yl)oxy)-4-aminophenyl)methanol (B1-5)

[0294] B1-4 (500.0 mg, 0.87 mmol) was added to a 100 mL single-necked flask and dissolved in ethyl acetate (50 mL). Triethylamine (0.1 mL) was added to the resulting solution. After replacing the nitrogen atmosphere, 10% palladium on carbon (100.0 mg, 10%) was added to the reaction solution. The hydrogen atmosphere was replaced again, and the reaction solution was stirred at room temperature under a hydrogen atmosphere (15 psi) overnight. The palladium on carbon was filtered and concentrated under reduced pressure. The residue was purified on a silica gel column (dichloromethane:methanol = 92:8) to obtain B1-5 (500.0 mg, 53%).

[0295] LC-MS: 550.4 [M+H] + .

[0296] Step 5: Synthesis of (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-(3-((2,5,8,11,14,17,20,23,26-nonyloxyoctan-28-yl)oxy)-4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)carbamate (B1-6)

[0297] To a 100 mL single-necked bottle, add B1-5 (300.0 mg, 0.55 mmol), N-[Fluorenylmethyloxycarbonyl]-L-valyl-L-alanine (224.0 mg, 0.55 mmol), and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (160.9 mg, 0.55 mmol). Dissolve the mixture in dichloromethane (10 mL) and methanol (5 mL). Stir the reaction mixture at room temperature overnight. Concentrate the reaction mixture under reduced pressure, and purify the residue on a silica gel column (dichloromethane:methanol = 96:4) to obtain B1-6 (250.0 mg, 49%).

[0298] LC-MS: 964.6[M+Na] + .

[0299] Step 6: Synthesis of 9H-fluoren-9-ylmethyl (S)-1-((S)-1-((3-(2,5,8,11,14,17,20,23,26-nonaoxaoctadec-28-yl)oxy)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutane-2-carbamate (B1-7)

[0300] To a 100 mL single-necked flask, add B1-6 (600.0 mg, 0.64 mmol) and di(p-nitrobenzene) carbonate (581.2 mg, 1.91 mmol) and dissolve in N,N-dimethylformamide (3 mL). Diisopropylethylamine (0.4 mL, 2.55 mmol) and 4-dimethylaminopyridine (0.8 mg, 0.01 mmol) were added to the resulting solution. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure, and the residue was purified on a silica gel column (dichloromethane:methanol = 96:4) to obtain B1-7 (300.0 mg, 43%).

[0301] LC-MS: 1129.6[M+Na] + .

[0302] Step 7: Synthesis of 9H-fluoren-9-ylmethyl {[(2S)-1-{[(2S)-1-[(4-{[({[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyylidene-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}carbonyl)oxy]methyl}-3-(2,5,8,11,14,17,20,23,26-nonaoxaoctacosan-28-yloxy)phenyl)amino]-1-oxyylideneprop-2-yl]amino}-3-methyl-1-oxyylidenebut-2-yl]amino}methane

[0303] To a 10 mL single-necked vial, B1-7 (100.0 mg, 0.09 mmol), exitecan mesylate (48.0 mg, 0.09 mmol), and 1-hydroxybenzotriazole (12.2 mg, 0.09 mmol) were added and dissolved in N,N-dimethylformamide (1 mL). The resulting solution was stirred at room temperature for 30 minutes, followed by the addition of diisopropylethylamine (0.03 mL, 0.18 mmol). The reaction mixture was stirred at room temperature for 3 hours, during which time the turbid solution became clear. The reaction mixture was concentrated under reduced pressure, and the residue was purified on a silica gel column (dichloromethane:methanol = 94:6) to afford B1-8 (110.0 mg, 61%).

[0304] LC-MS: 1403.6[M+H] + .

[0305] Step 8: Synthesis of 2-((2,5,8,11,14,17,20,23,26-nonyloxyoctadecane-28-yl)oxy)-4-((S)-2-(S)2-amino-3-methylbutanamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,9,10,13,5-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)carbamate (B1-9)

[0306] B1-8 (100.0 mg, 0.07 mmol) was added to a 10 mL single-necked vial and dissolved in N,N-dimethylformamide (2 mL). Diethylamine (0.07 mL, 0.71 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was slurried with petroleum ether to obtain B1-9 (83.9 mg, 100%).

[0307] LC-MS: 1181.6[M+H] + .

[0308] Step 9: Synthesis of 2-((2,5,8,11,14,17,20,23,26-nonyloxyoctadecane-28-yl)oxy)-4-((S)-2-(S)2-amino-3-methylbutanamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,9,10,13,5-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)carbamate B1

[0309] In a 10 mL single-necked vial, 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (1.1 mg, 0.01 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.9 mg, 0.01 mmol) were added and dissolved in N,N-dimethylformamide (1 mL). Diisopropylethylamine (0.01 mL, 0.03 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 10 minutes. B1-9 (8.4 mg, 0.01 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was filtered and purified by preparative HPLC to yield B1 (13.2 mg, 13%).

[0310] LC-MS: 1374.6[M+H] + .

[0311] Example B2: Preparation of Compound B2

[0312] Step 1: Synthesis of 5-(4-iodophenyl)-1,3,4-oxadiazole-2-thiol (B2-2)

[0313] To a 500 mL single-necked flask, add 4-iodobenzohydrazide B2-1 (5.0 g, 19.08 mmol) and ethanol (200 mL). Potassium hydroxide (1.1 g, 19.08 mmol) and carbon disulfide (4.4 g, 57.24 mmol) were added at room temperature and stirred under reflux overnight. The reaction mixture was concentrated, diluted with water (30 mL), and the pH was adjusted to approximately 6 with 2 M hydrochloric acid. Filter the mixture to obtain B2-2 (5.0 g, 86%).

[0314] LC-MS: 305.0 [M+H] + .

[0315] Step 2: Synthesis of 2-(4-iodophenyl)-5-(methylthio)-1,3,4-oxadiazole (B2-3)

[0316] To a 500 mL three-necked flask, add B2-2 (10.0 g, 32.88 mmol) and tetrahydrofuran (200 mL). Triethylamine (10.0 g, 98.65 mmol), iodomethane (5.1 g, 36.17 mmol), and 4-dimethylaminopyridine (0.4 g, 3.29 mmol) were added at room temperature and stirred overnight. The reaction mixture was directly spin-dried, slurried with n-hexane, and filtered to obtain B2-3 (10.0 g, 96%).

[0317] LC-MS: 319.0 [M+H] + .

[0318] Step 3: Synthesis of 2-((3-(4-(5-(methylthio)-1,3,4-oxadiazol-2-yl)phenyl)prop-2-yn-1-yl)oxy)acetic acid (B2-4)

[0319] B2-3 (4.0 g, 12.57 mmol) and N,N-dimethylformamide (40 mL) were added to a 250 mL three-necked flask, and 2-(prop-2-yn-1-yloxy)acetic acid (1.9 g, 16.35 mmol), cuprous iodide (0.4 g, 1.89 mmol), triphenylphosphine (0.3 g, 1.26 mmol), bisacetonitrile palladium dichloride (0.2 g, 0.63 mmol), and diisopropylamine (7.6 g, 75.44 mmol) were added at room temperature and stirred at 50 degrees for 4 hours. The reaction solution was cooled to room temperature, poured into water (400 mL), adjusted to pH 6, extracted with ethyl acetate (100 mL*3), and the organic phases were combined and washed with saturated brine (150 mL*3), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (dichloromethane: methanol = 10:1) to obtain B2-4 (3.2 g, 84%).

[0320] LC-MS: 305.2[M+H] + .

[0321] Step 4: Synthesis of 2-((3-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)prop-2-yn-1-yl)oxy)acetic acid (B2-5)

[0322] To a 100 mL three-necked flask, add B2-4 (2.0 g, 6.57 mmol) and dichloromethane (30 mL). Add m-chloroperbenzoic acid (5.7 g, 32.86 mmol) at room temperature and stir overnight. The reaction mixture was filtered and directly purified by column chromatography (DCM:MeOH = 10:1) to afford 0.6 g of the crude product. This was then separated by preparative HPLC to afford B2-5 (0.1 g, 5%).

[0323] LC-MS: 337.0 [M+H] + .

[0324] Step 5: Synthesis of 2-(2,5,8,11,14,17,20,23,26-nonaoxaoctadec-28-yl)oxy)-4-(S)-2-(S)-3-methyl-2-(3-(4-(5-methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)propyl-2-yn-1-oxy)acetamido)butyramide)propionamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)carbamate B2

[0325] To a 10 mL single-necked vial, B2-5 (14.5 mg, 0.04 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (24.5 mg, 0.06 mmol) were added and dissolved in N,N-dimethylformamide (1 mL). Diisopropylethylamine (0.01 mL, 0.09 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. B1-9 (50.8 mg, 0.04 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to yield B2 (23.3 mg, 36%).

[0326] LC-MS: 1499.6[M+H] + .

[0327] Example B3: Preparation of Compound B3

[0328] Step 1: Synthesis of 5-iodoisophthalic acid hydrazide (B3-2)

[0329] To a 100 mL three-necked flask, add dimethyl 5-iodoisophthalate B3-1 (1.0 g, 3.12 mmol) and methanol (10 mL). Add hydrazine hydrate (0.8 g, 15.62 mmol) at room temperature and stir overnight at 70°C. The reaction mixture was concentrated and slurried with methanol to obtain B3-2 (1.0 g, 100%).

[0330] LC-MS: 321.0 [M+H] + .

[0331] Step 2: Synthesis of 5,5'-(5-iodo-1,3-phenylene)bis(1,3,4-oxadiazole-2-thiol) (B3-3)

[0332] To a 100 mL single-necked flask, add B3-2 (1.0 g, 3.12 mmol) and ethanol (30 mL). Potassium hydroxide (0.4 g, 6.25 mmol) and carbon disulfide (1.4 g, 18.75 mmol) were added at room temperature and stirred under reflux overnight. The reaction mixture was concentrated, diluted with water (10 mL), and the pH was adjusted to approximately 6 with 2 M hydrochloric acid. Filtering afforded B3-3 (1.3 g, 100%).

[0333] LC-MS: 404.9[M+H] + .

[0334] Step 3: Synthesis of 5,5'-(5-iodo-1,3-phenylene)bis(2-(methylthio)-1,3,4-oxadiazole) (B3-4)

[0335] To a 50 mL three-necked flask, add B3-3 (1.3 g, 3.22 mmol) and tetrahydrofuran (30 mL). Triethylamine (2.0 g, 19.30 mmol), iodomethane (1.8 g, 12.86 mmol), and 4-dimethylaminopyridine (80.0 mg, 0.64 mmol) were added at room temperature and stirred overnight. The reaction mixture was directly spin-dried, slurried with n-hexane, and filtered to obtain B3-4 (1.4 g, 100%).

[0336] LC-MS:433.0[M+H] + .

[0337] Step 4: Synthesis of 2-((3-(3,5-bis(5-(methylthio)-1,3,4-oxadiazol-2-yl)phenyl)prop-2-yn-1-yl)oxy)acetic acid (B3-5)

[0338] B3-4 (900.0 mg, 2.08 mmol) and N,N-dimethylformamide (10 mL) were added to a 100 mL three-necked flask, and 2-(prop-2-yn-1-yloxy)acetic acid (356.4 mg, 3.12 mmol), cuprous iodide (99.1 mg, 0.31 mmol), triphenylphosphine (54.6 mg, 0.21 mmol), bisacetonitrile palladium dichloride (27.0 mg, 0.10 mmol), and diisopropylamine (1.3 g, 12.49 mmol) were added at room temperature and stirred at 50 degrees for 3 hours. The reaction solution was cooled to room temperature, poured into water (50 mL), adjusted to pH 6, extracted with ethyl acetate (30 mL*3), and the organic phases were combined and washed with saturated brine (50 mL*3), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (dichloromethane: methanol = 10:1) to obtain B3-5 (0.6 g, 69%).

[0339] LC-MS:419.0[M+H] + .

[0340] Step 5: Synthesis of 2-((3-(3,5-bis(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)prop-2-yn-1-yl)oxy)acetic acid (B3-6)

[0341] To a 100 mL three-necked flask, add B3-5 (0.6 g, 1.43 mmol) and dichloromethane (20 mL). Add m-chloroperbenzoic acid (1.2 g, 7.17 mmol) at room temperature and stir overnight. The reaction mixture was filtered and directly purified by column chromatography (DCM:MeOH = 10:1) to afford 300 mg of the crude product. This was then separated by preparative HPLC to afford B3-6 (70.0 mg, 10%).

[0342] LC-MS:483.0[M+H] + .

[0343] Step 6: Synthesis of 4-((S)-2-(S)-3-methyl-2-(2-((3-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)prop-2-yn-1-yl)oxy)acetamido)propionamido)benzyl((1S,9S)-9-fluoro-9-hydroxy-4-methyl-10,13,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate B3

[0344] In a 10 mL single-necked bottle, B3-6 (63.9 mg, 0.13 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (75.6 mg, 0.20 mmol) were added, dissolved in N,N-dimethylformamide (2 mL), and diisopropylethylamine (34.3 mg, 0.26 mmol) was added. The resulting solution was stirred at room temperature for 10 minutes. 4-((S)-2-(S)2-amino-3-methylbutanamido)propionamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (100.0 mg, 0.13 mmol) was added to the mixture and stirred at room temperature for 2 hours. The reaction mixture was filtered and purified by preparative high-performance liquid chromatography to obtain B3 (22.1 mg, 14%).

[0345] LC-MS: 1219.2[M+H] + .

[0346] Example B4: Preparation of Compound B4

[0347] In a 10 mL single-necked bottle, B2-5 (44.6 mg, 0.13 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (75.6 mg, 0.20 mmol) were added. The mixture was dissolved in N,N-dimethylformamide (2 mL) and diisopropylethylamine (34.3 mg, 0.26 mmol) was added. The resulting solution was stirred at room temperature for 10 minutes. 4-((S)-2-(S)2-amino-3-methylbutanamido)propionamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (100.0 mg, 0.13 mmol) was added to the mixture and stirred at room temperature for 2 hours. The reaction mixture was filtered and purified by preparative HPLC to obtain B4 (21.3 mg, 15%).

[0348] LC-MS: 1073.4[M+H] + .

[0349] Example B5: Preparation of Compound B5

[0350] To a 10 mL single-necked vial, B3-6 (20.0 mg, 0.04 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (23.6 mg, 0.06 mmol) were added and dissolved in N,N-dimethylformamide (1 mL). Diisopropylethylamine (0.01 mL, 0.08 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. B1-9 (48.4 mg, 0.04 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to yield B5 (20.5 mg, 29%).

[0351] LC-MS: 1645.6[M+H] + .

[0352] Example B6: Preparation of Compound B6

[0353] Step 1: Synthesis of 2-methylpropan-2-yl (2S)-2-({[(9H-fluoren-9-ylmethyl)oxy]carbonyl}amino)-6-[(25-oxyylidene-2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)amino]hexanoate (B6-3)

[0354] To a 10 mL single-necked vial, (2,5,8,11,14,17,20-heptaoxadocosan-22-yloxy)acetic acid (B6-1) (400.0 mg, 1.00 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (380.0 mg, 1.00 mmol) were added and dissolved in N,N-dimethylformamide (5 mL). Diisopropylethylamine (0.25 mL, 2.00 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. B6-2 (460.2 mg, 1.00 mmol, prepared according to WO 2019208820) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to obtain B6-3 (620 mg, 77.1%).

[0355] LC-MS: 805.4[M+H] + .

[0356] Step 2: Synthesis of (2S)-2-({[(9H-fluoren-9-ylmethyl)oxy]carbonyl}amino)-6-[(25-oxyylidene-2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)amino]hexanoic acid (B6-4)

[0357] B6-3 (580.0 mg, 0.72 mmol) was added to a 50 mL single-necked flask and dissolved in trifluoroacetic acid (2 mL) and dichloromethane (4 mL). The reaction mixture was stirred at room temperature for 1 hour, then heated to 30°C and stirred for 2 hours. The reaction mixture was concentrated under reduced pressure, dissolved in 10 mL of toluene, and concentrated again under reduced pressure to yield B6-4 (540.0 mg, 100%).

[0358] LC-MS:749.4[M+H] + .

[0359] Step 3: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S, Synthesis of 37S)-31-({[(9H-fluoren-9-ylmethyl)oxy]carbonyl}amino)-37-methyl-25,32,35,38-tetraoxylidene-34-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,33,36-triazatriacont-38-yl]amino}phenyl)methyl ester (B6-6)

[0360] To a 100 mL single-necked bottle, B6-5 (75.4 mg, 0.10 mmol), diisopropylethylamine (0.05 mL, 0.3 mmol), and B6-4 (74.9 mg, 0.10 mmol) were added and dissolved in N,N-dimethylformamide (2 mL). 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (28.3 mg, 0.10 mmol) was added to the resulting solution under ice-cooling. The reaction mixture was stirred under ice-cooling for 1 hour. The reaction mixture was filtered and purified by preparative HPLC to obtain B6-6 (100 mg, 67.3%).

[0361] LC-MS: 1485.7[M+H] + .

[0362] Step 4: Synthesis of {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyidene-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S)-31-amino-37-methyl-25,32,35,38-tetraoxyidene-34-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,33,36-triazatriacont-38-yl]amino}phenyl)methyl ester (B6-7)

[0363] B6-6 (100.0 mg, 0.07 mmol) was added to a 10 mL single-necked vial and dissolved in N,N-dimethylformamide (2 mL). Diethylamine (0.07 mL, 0.71 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was slurried with petroleum ether to obtain B6-7 (80 mg, 90.5%).

[0364] LC-MS: 1263.6[M+H] + .

[0365] Step 5: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S)-37-methyl-31-({2-[( Synthesis of 3-{4-[5-(methyldioxyylidene-λ6-thio)-1,3,4-oxadiazacyclopentan-2-yl]phenyl}prop-2-ynyl)oxy]acetyl}amino)-2,5,32,35,38-tetraoxyylidene-34-(prop-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,33,36-triazatriacont-38-yl]amino}phenyl)methyl ester B6

[0366] To a 10 mL single-necked vial, B6-7 (80.0 mg, 0.06 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (22.8 mg, 0.06 mmol) were added and dissolved in N,N-dimethylformamide (2 mL). Diisopropylethylamine (0.15 mL, 1.2 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. B2-5 (20.2 mg, 0.06 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to yield B6 (25 mg, 26.3%).

[0367] LC-MS: 1581.6[M+H] + .

[0368] Example B7: Preparation of Compound B7

[0369] To a 10 mL single-necked vial, add B6-7 (80.0 mg, 0.06 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (22.8 mg, 0.06 mmol) and dissolve in N,N-dimethylformamide (2 mL). Add diisopropylethylamine (0.15 mL, 1.2 mmol) to the resulting solution, and stir the mixture at room temperature for 15 minutes. Then, add B3-6 (28.9 mg, 0.06 mmol). Stir the reaction mixture at room temperature for 3 hours. Filter the reaction mixture and purify it by preparative HPLC to obtain B7 (30 mg, 28.9%).

[0370] LC-MS: 1727.6[M+H] + .

[0371] Example B8: Preparation of Compound B8

[0372] Step 1: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S)-31-{[1-(9H-fluorene Synthesis of 2,5-(1,2-dioxo-3,18-dioxy-4-aza-2,7,10,13,16-pentaoxaoctadec-18-yl)amino}-37-methyl-25,32,35,38-tetraoxo-34-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,33,36-triazatriacont-38-yl)amino}phenyl)methyl ester (B8-1)

[0373] B6-7 (80.0 mg, 0.06 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (22.8 mg, 0.06 mmol) were added to a 10 mL single-necked vial and dissolved in N,N-dimethylformamide (2 mL). Diisopropylethylamine (0.15 mL, 1.2 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. {[1-(9H-fluoren-9-yl)-3-oxyylidene-4-aza-2,7,10,13-tetraoxapentadecan-15-yl]oxy}acetic acid (28.4 mg, 0.06 mmol) was then added to the mixture. The reaction mixture was spin-dried and eluted onto a silica gel column with 5% methanol in dichloromethane to afford B8-1 (50 mg, 48.2%).

[0374] LC-MS: 1718.9[M+H] + .

[0375] Step 2: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S)-31 Synthesis of 1,4-[(14-amino-1-oxyylidene-3,6,9,12-tetraoxatetradec-1-yl)amino]-37-methyl-25,32,35,38-tetraoxatetradec-34-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,33,36-triazatriacont-38-yl]amino}phenyl)methyl ester (B8-2)

[0376] B8-1 (50.0 mg, 0.03 mmol) was added to a 10 mL single-necked vial and dissolved in N,N-dimethylformamide (1 mL). Diethylamine (0.45 mL, 0.3 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was slurried with petroleum ether to obtain B8-2 (40 mg, 89.1%).

[0377] LC-MS: 1496.8[M+H] + .

[0378] Step 3: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyylidene-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S)-37-methyl-31-[(21-{4-[5-(methyldioxyylidene-λ 6 Synthesis of [(1,3,4-oxadiazacyclopentan-2-yl)phenyl]-1,16-dioxy-15-aza-3,6,9,12,18-pentaoxahistriazolyl-20-yn-1-yl)amino]-25,32,35,38-tetraoxy-34-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,33,36-triazatriazodec-38-yl)amino]phenyl]methyl ester B8

[0379] In a 10 mL single-necked vial, B8-2 (40.0 mg, 0.03 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (11.4 mg, 0.03 mmol) were added and dissolved in N,N-dimethylformamide (1 mL). Diisopropylethylamine (0.03 mL, 0.06 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. B2-5 (10.1 mg, 0.03 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to yield compound B8 (15 mg, 27.6%).

[0380] LC-MS: 1814.8[M+H] + .

[0381] 1H NMR (400MHz, DMSO) δ9.99 (s, 1H), 8.17 (d, J = 6.7Hz, 1H), 8.10 (d, J = 8.5Hz, 2H), 8.05 (d, J = 8. 8Hz,1H),7.88(d,J=8.7Hz,1H),7.82(t,J=5.7Hz,1H),7.77(d,J=10.9Hz,1H),7.72(d,J=8. 5Hz,2H),7.64(dd,J=10.5,5.3Hz,2H),7.59(d,J=8.4Hz,2H),7.36(d,J=8.5Hz,2H),7.31(s ,1H),6.51(s,1H),5.44(s,2H),5.34–5.21(m,3H),5.07(s,2H),4.53(s,2H),4.38(dd,J=13 .5,6.5Hz,2H),4.23–4.16(m,1H),4.02(s,2H),3.91(s,2H),3.84(s,2H),3.71(s,3H),3.60 –3.49(m,37H),3.42(dt,J=6.7,5.2Hz,4H),3.29–3.25(m,4H),3.23(s,3H),3.13(s,1H),3. 07–3.02(m,2H),2.37(s,3H),2.19(s,2H),2.00–1.93(m,1H),1.92–1.81(m,2H),1.65(s,1H ),1.54(d,J=8.8Hz,1H),1.39(s,2H),1.29(d,J=7.0Hz,3H),1.24(s,2H),0.90–0.81(m,9H).

[0382] Example B9: Preparation of Compound B9

[0383] Step 1: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S)-31-[(21-{3,5-bis[5-(methyldioxy-1-yl)-1-yl]amino}methane- 6 Synthesis of [(1,3,4-oxadiazacyclopentan-2-yl)phenyl]-1,16-dioxy-15-aza-3,6,9,12,18-pentaoxahistriazolylidene-20-yn-1-yl)amino]-37-methyl-25,32,35,38-tetraoxy-34-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,33,36-triazatriazodec-38-yl)amino]phenyl]methyl ester B9

[0384] To a 10 mL single-necked vial, add B8-2 (40.0 mg, 0.03 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (11.4 mg, 0.03 mmol) and dissolve in N,N-dimethylformamide (1 mL). Add diisopropylethylamine (0.03 mL, 0.06 mmol) to the resulting solution, and stir the mixture at room temperature for 15 minutes. Add B3-6 (14.5 mg, 0.03 mmol) to the mixture. Stir the reaction mixture at room temperature for 3 hours. Filter the reaction mixture and purify it by preparative HPLC to obtain compound B9 (15 mg, 25.5%).

[0385] LC-MS: 1960.8[M+H] + .

[0386] Example B10: Preparation of Compound B10

[0387] Step 1: Synthesis of 9H-fluoren-9-ylmethyl {[(2S,5S,8S)-2-(formamidomethyl)-5-methyl-1-({4-[({[(4-nitrophenyl)oxy]carbonyl}oxy)methyl]phenyl}amino)-1,4,7-trioxydeoxy-3,6-diazanonan-8-yl]amino}methane

[0388] To a 100 mL single-necked bottle, 9H-fluoren-9-ylmethyl {[(2S,5S,8S)-2-(formamidomethyl)-1-{[4-(hydroxymethyl)phenyl]amino}-5-methyl-1,4,7-trioxyde-3,6-diazanonan-8-yl]amino}methane was added and dissolved in N,N-dimethylformamide (6 mL). Diisopropylethylamine (0.4 mL, 2.55 mmol) and 4-dimethylaminopyridine (0.8 mg, 0.01 mmol) were added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure and slurried with methanol to obtain B10-2 (600.0 mg, 78.3%).

[0389] LC-MS:767.3[M+H] + .

[0390] Step 2: Synthesis of 9H-fluoren-9-ylmethyl {[(2S,5S,8S)-2-(formamidomethyl)-1-[(4-{[({[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyylidene-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}carbonyl)oxy]methyl}phenyl)amino]-5-methyl-1,4,7-trioxyylidene-3,6-diazanonan-8-yl]amino}methane

[0391] To a 100 mL single-necked bottle, add B10-2 (100.0 mg, 0.13 mmol), exitecan mesylate (69.3 mg, 0.13 mmol), and 1-hydroxybenzotriazole (17.6 mg, 0.13 mmol). Dissolve the mixture in N,N-dimethylformamide (2 mL). The resulting solution was stirred at room temperature for 30 minutes, followed by the addition of diisopropylethylamine (0.06 mL, 0.39 mmol). The reaction mixture was stirred at room temperature for 3 hours, during which time the turbid solution became clear. The reaction mixture was concentrated under reduced pressure, and the residue was purified on a silica gel column (dichloromethane:methanol = 94:6) to afford B10-3 (110.0 mg, 79.6%).

[0392] LC-MS: 1063.4[M+H] + .

[0393] Step 3: Synthesis of {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyidene-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(2S,5S,8S)-8-amino-2-(formamidomethyl)-5-methyl-1,4,7-trioxyidene-3,6-diazanonan-1-yl]amino}phenyl)methyl ester (B10-4)

[0394] B10-3 (110.0 mg, 0.10 mmol) was added to a 10 mL single-necked vial and dissolved in N,N-dimethylformamide (1 mL). Diethylamine (73 mg, 1.0 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was slurried with petroleum ether to obtain B10-4 (75 mg, 89.2%).

[0395] LC-MS:841.3[M+H] + .

[0396] Step 4: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S,40S) Synthesis of 40-(formamidomethyl)-31-({[(9H-fluoren-9-ylmethyl)oxy]carbonyl}amino)-34,37-dimethyl-25,32,35,38,41-pentaoxadiene-2,5,8,11,14,17,20,23-octaoxa-26,33,36,39-tetraazaheteracontan-41-yl]amino}phenyl)methyl ester (B10-5)

[0397] To a 10 mL single-necked vial, add B10-4 (40.0 mg, 0.03 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (11.4 mg, 0.03 mmol) and dissolve in N,N-dimethylformamide (1 mL). Add diisopropylethylamine (0.03 mL, 0.06 mmol) to the resulting solution, and stir the mixture at room temperature for 15 minutes. Then, add B6-4 (22.5 mg, 0.03 mmol). Stir the reaction mixture at room temperature for 3 hours. Filter the reaction mixture and purify it by preparative HPLC to obtain B10-5 (35 mg, 74.2%).

[0398] LC-MS:1571.8[M+H] + .

[0399] Step 5: Synthesis of {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyylidene-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S,40S)-31-amino-40-(formamidomethyl)-34,37-dimethyl-25,32,35,38,41-pentaoxyylidene-2,5,8,11,14,17,20,23-octaoxa-26,33,36,39-tetraazatetradecane-41-yl]amino}phenyl)methyl ester (B10-6)

[0400] B10-5 (35.0 mg, 0.02 mmol) was added to a 10 mL single-necked vial and dissolved in N,N-dimethylformamide (1 mL). Diethylamine (14.6 mg, 0.2 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was slurried with petroleum ether to obtain B10-6 (20 mg, 74.1%).

[0401] LC-MS: 1349.6[M+H] + .

[0402] Step 6: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S,40S)-31-({2-[(3-{3,5-bis[5-(methyldioxy-1-yl)-1-yl]amino}methane-1-yl)- 6 Synthesis of (4,5-dimethyl-2,5-dimethyl-3-[(1,3,4-oxadiazacyclopenta-2-yl]phenyl}prop-2-ynyl)oxy]acetyl}amino)-40-(formamidomethyl)-34,37-dimethyl-25,32,35,38,41-pentaoxydeoxy-2,5,8,11,14,17,20,23-octaoxa-26,33,36,39-tetraazaheteradecane-41-yl)amino}phenyl)methyl ester B10

[0403] To a 10 mL single-necked vial, add B10-6 (20.0 mg, 0.015 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.7 mg, 0.015 mmol) and dissolve in N,N-dimethylformamide (0.5 mL). Add diisopropylethylamine (0.1 mL, 0.03 mmol) to the resulting solution, and stir the mixture at room temperature for 15 minutes. Add B3-6 (7.3 mg, 0.015 mmol) to the mixture. Stir the reaction mixture at room temperature for 3 hours. Filter the reaction mixture and purify it by preparative HPLC to obtain B10 (8 mg, 29.4%).

[0404] LC-MS: 1813.6[M+H] + .

[0405] Example B11: Preparation of Compound B11

[0406] Step 1: Synthesis of {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyidene-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(5S,8S)-8-{3-[(aminocarbonyl)amino]propyl}-1-(9H-fluoren-9-yl)-3,6,9-trioxyidene-5-(propan-2-yl)-4,7-diaza-2-oxanonan-9-yl]amino}phenyl)methyl ester B11-2

[0407] To a 50 mL single-necked bottle, add B11-1 (70.0 mg, 0.09 mmol, CAS: 863971-53-3, purchased from Leyan), exitecan mesylate (48.0 mg, 0.09 mmol), and 1-hydroxybenzotriazole (12.2 mg, 0.09 mmol). Dissolve the mixture in N,N-dimethylformamide (1 mL). The resulting solution was stirred at room temperature for 30 minutes, followed by the addition of diisopropylethylamine (0.03 mL, 0.18 mmol). The reaction mixture was stirred at room temperature for 3 hours, during which time the turbid solution became clear. The reaction mixture was concentrated under reduced pressure, and the residue was purified on a silica gel column (dichloromethane:methanol = 94:6) to obtain B11-2 (85.0 mg, 88.9%).

[0408] LC-MS: 1063.1[M+H] + .

[0409] Step 2: Synthesis of {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyidene-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(2S)-5-[(aminocarbonyl)amino]-2-{[(2S)-2-amino-3-methyl-1-oxyidenebutyl]amino}-1-oxyidenepentyl]amino}phenyl)methyl ester B11-3

[0410] B11-2 (85.0 mg, 0.08 mmol) was added to a 25 mL single-necked vial and dissolved in N,N-dimethylformamide (2 mL). Diethylamine (0.07 mL, 0.71 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was slurried with petroleum ether to obtain B11-3 (60 mg, 89.5%).

[0411] LC-MS:841.2[M+H] + .

[0412] Step 3: {[(1S,9S)-5-fluoro-9-hydroxy-4,9-dimethyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S)-37- Synthesis of {3-[(aminocarbonyl)amino]propyl}-31-({[(9H-fluoren-9-ylmethyl)oxy]carbonyl}amino)-25,32,35,38-tetraoxydeoxy-34-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,33,36-triazatriacont-38-yl]amino}phenyl)methyl ester B11-4

[0413] To a 100 mL single-necked bottle, B11-3 (60 mg, 0.07 mmol), diisopropylethylamine (0.05 mL, 0.3 mmol), and B6-4 (74.9 mg, 0.10 mmol) were added and dissolved in N,N-dimethylformamide (2 mL). HOBT (2 mg, 0.01 mmol) and HATU (38 mg, 0.10 mmol) were added to the resulting solution under ice-cooling. The reaction solution was stirred under ice-cooling for 1 hour. The reaction solution was filtered and purified by preparative HPLC to obtain B11-4 (86 mg, 78.9%).

[0414] LC-MS: 1558.1[M+H] + .

[0415] Step 4: Synthesis of {[(1S,9S)-5-fluoro-9-hydroxy-4,9-dimethyl-10,13-dioxyylidene-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S)-31-amino-37-{3-[(aminocarbonyl)amino]propyl}-25,32,35,38-tetraoxyylidene-34-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,33,36-triazatriacont-38-yl]amino}phenyl)methyl ester B11-5

[0416] B11-4 (86.0 mg, 0.055 mmol) was added to a 25 mL single-necked vial and dissolved in N,N-dimethylformamide (2 mL). Diethylamine (0.05 mL, 0.55 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was slurried with petroleum ether to obtain B11-5 (60 mg, 81.6%).

[0417] LC-MS: 1335.8[M+H] + .

[0418] Step 5: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S)-37-{3-[(aminocarbonyl)amino]propyl}-31-({2 Synthesis of 1-[(1-{3,5-bis[5-(methyldioxyylidene-λ6-thio)-1,3,4-oxadiazacyclopentan-2-yl]phenyl}prop-1-yn-3-yl)oxy]acetyl}amino)-25,32,35,38-tetraoxyylidene-34-(prop-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,33,36-triazatriacont-38-yl]amino}phenyl)methyl ester B11

[0419] To a 10 mL single-necked vial, B11-5 (20.0 mg, 0.015 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.7 mg, 0.015 mmol) were added and dissolved in N,N-dimethylformamide (0.5 mL). Diisopropylethylamine (0.1 mL, 0.03 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. B3-6 (7.3 mg, 0.015 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to obtain B11 (8 mg, 29.4%).

[0420] LC-MS: 1813.7 [M+H] + .

[0421] Example B12: Preparation of Compound B12

[0422] Step 1: {[(31S,34S,37S)-38-[(4-{[({[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyidene-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}carbonyl)oxy]methyl}-3-(2 Synthesis of 9H-fluoren-9-ylmethyl-2,5,8,11,14,17,20,23-octaoxa-26,33,36-triazatriacont-31-yl]amino]-37-methyl-25,32,35,38-tetraoxydeoxy-34-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,33,36-triazatriacont-31-yl]amino}methane

[0423] To a 100 mL single-necked bottle, B1-9 (100 mg, 0.08 mmol), diisopropylethylamine (0.03 mL, 0.18 mmol), and B6-4 (60.0 mg, 0.08 mmol) were added and dissolved in N,N-dimethylformamide (2 mL). 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (28.3 mg, 0.10 mmol) was added to the resulting solution under ice-cooling. The reaction mixture was stirred under ice-cooling for 1 hour. The reaction mixture was filtered and purified by preparative HPLC to obtain B12-2 (90 mg, 58.8%).

[0424] LC-MS: 1913.0 [M+H] + .

[0425] Step 2: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S)-31-amino- Synthesis of 37-methyl-25,32,35,38-tetraoxylidene-34-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,33,36-triazatriacont-38-yl]amino}-2-(2,5,8,11,14,17,20,23,26-nonaoxaoctacocant-28-yloxy)phenyl)methyl ester (B12-3)

[0426] B12-2 (90.0 mg, 0.05 mmol) was added to a 10 mL single-necked vial and dissolved in N,N-dimethylformamide (1 mL). Diethylamine (36.5 mg, 0.5 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was slurried with petroleum ether to afford B12-3 (70 mg, 82.8%).

[0427] LC-MS: 1690.1[M+H] + .

[0428] Step 3: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S)-37-methyl-31-({2-[(3-{4-[5-(methyldioxy-1-yl) ... 6 Synthesis of (2,5,8,11,14,17,20,23,26-nonaoxaoctacosan-28-yloxy)phenyl)methyl)- ...32,35,38-tetrahydro-34-(prop-2-yl)-2,5

[0429] To a 10 mL single-necked vial, B12-3 (25.0 mg, 0.015 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.7 mg, 0.015 mmol) were added and dissolved in N,N-dimethylformamide (0.5 mL). Diisopropylethylamine (0.1 mL, 0.03 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. B2-5 (5.0 mg, 0.015 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to yield B12 (5.2 mg, 17.3%).

[0430] LC-MS: 2008.9[M+H] + .

[0431] Example B13: Preparation of Compound B13

[0432] Step 1: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S)-31-({2-[(3-{3,5-bis[5-(methyldioxy-1-yl)-1-yl]amino}methane-1-yl]amino}methane-(4-{[(31S,34S,37S)-31-({2-[(3-{3,5-bis[5-(methyldioxy-1-yl)-1-yl]amino}methane-1-yl]amino}methane-1-yl]amino}methane- 6 Synthesis of (2,5,8,11,14,17,20,23,26-nonaoxaoctacosan-28-yloxy)phenyl)methyl)-3,7-methyl-2,5,32,35,38-tetrahydroylidene-34-(prop-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,33,36-triazaoctatriacont-38-yl)amino)-2-(2,5,8,11,14,17,20,23,26-nonaoxaoctacosan-28-yloxy)phenyl)methyl ester B13

[0433] To a 10 mL single-necked vial, B12-3 (25.0 mg, 0.015 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.7 mg, 0.015 mmol) were added and dissolved in N,N-dimethylformamide (0.5 mL). Diisopropylethylamine (0.1 mL, 0.03 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. B3-6 (7.2 mg, 0.015 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to obtain B13 (8.5 mg, 26.3%).

[0434] LC-MS: 2154.9 [M+H] + .

[0435] Example B14: Preparation of Compound B14

[0436] Step 1: Synthesis of 2-methylpropan-2-yl {[(7S)-7-benzyl-11-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyylidene-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2,5,8,11-tetraoxyylidene-3,6,9-triazaundec-1-yl]amino}methane (B14-2)

[0437] In a 100 mL single-necked bottle, add N-[(12S)-12-benzyl-2,2-dimethyl-4,7,10,13-tetraoxylidene-3-oxa-5,8,11-triazatridec-13-yl]glycine (B14-1) (100.0 mg, 0.23 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (87.5 mg, 0.23 mmol). Dissolve the mixture in N,N-dimethylformamide (2 mL). Add diisopropylethylamine (89 mg, 0.69 mmol) to the resulting solution, and stir the mixture at room temperature for 15 minutes. Then, add exitecan mesylate (122.2 mg, 0.23 mmol). The reaction mixture is stirred at room temperature for 3 hours. The reaction solution was evaporated to dryness and purified on a silica gel column (dichloromethane:methanol=94:6) to obtain B14-2 (120 mg, 61.2%).

[0438] LC-MS:854.3[M+H] + .

[0439] Step 2: Synthesis of N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]-2-{[(2S)-8-amino-2-benzyl-1,4,7-trioxy-3,6-diazaoctan-1-yl]amino}acetamide (B14-3)

[0440] B14-2 (120.0 mg, 0.14 mmol) was added to a 50 mL single-necked flask and dissolved in trifluoroacetic acid (1 mL) and dichloromethane (2 mL). The reaction mixture was stirred at room temperature for 1 hour, then heated to 30°C and stirred for 2 hours. The reaction mixture was concentrated under reduced pressure, dissolved in 10 mL of toluene, and concentrated again under reduced pressure to yield B14-3 (106.0 mg, 100%).

[0441] LC-MS:754.3[M+H] + .

[0442] Step 3: N-[(7S)-7-benzyl-11-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyylidene-2,3,9,10,12,15-hexahydro-1H-cyclohexyl[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2,5,8,11-tetraoxyylidene-3,6,9-triazaundec-1-yl]-2-[(3-{4-[2-(methyldioxyylidene-λ 6 Synthesis of [(1,3,4-oxadiazacyclopentyl-5-yl)phenyl]prop-2-ynyl)oxy]acetamide B14]

[0443] To a 10 mL single-necked vial, add B14-3 (11.3 mg, 0.015 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.7 mg, 0.015 mmol) and dissolve in N,N-dimethylformamide (0.5 mL). Add diisopropylethylamine (0.1 mL, 0.03 mmol) to the resulting solution, and stir the mixture at room temperature for 15 minutes. Add B2-5 (5.0 mg, 0.015 mmol) to the mixture. Stir the reaction mixture at room temperature for 3 hours. Filter the reaction mixture and purify it by preparative HPLC to obtain compound B14 (5.5 mg, 34.2%).

[0444] LC-MS: 1072.3[M+H] + .

[0445] Example B15: Preparation of Compound B15

[0446] Step 1: N-[(7S)-7-benzyl-11-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyylidene-2,3,9,10,12,15-hexahydro-1H-cyclohexyl[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2,5,8,11-tetraoxyylidene-3,6,9-triazaundec-1-yl]-6-{4-[2-(methyldioxyylidene-λ 6 Synthesis of [(1,3,4-oxadiazacyclopentyl)-1,3,4-oxadiazacyclopentyl]phenyl]hex-5-ynamide B15]

[0447] To a 10 mL single-necked vial, add B14-3 (11.3 mg, 0.015 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.7 mg, 0.015 mmol) and dissolve in N,N-dimethylformamide (0.5 mL). Add diisopropylethylamine (0.1 mL, 0.03 mmol) to the resulting solution, and stir the mixture at room temperature for 15 minutes. Add A2-5 (5.0 mg, 0.015 mmol) to the mixture. Stir the reaction mixture at room temperature for 3 hours. Filter the reaction mixture and purify it by preparative HPLC to obtain compound B15 (4.8 mg, 30.0%).

[0448] LC-MS: 1070.4[M+H] + .

[0449] Example B16: Preparation of Compound B16

[0450] Step 1: Synthesis of N-methyl-N-(3-{4-[2-(methylthio)-1,3,4-oxadiazacyclopentyl-5-yl]phenyl}prop-2-ynyl)glycine (B16-1)

[0451] A2-3 (500.0 mg, 1.58 mmol) was added to a 10 mL single-necked vial and dissolved in diisopropylamine (3 mL). Bisacetonitrile palladium chloride (12.7 mg, 0.05 mmol), triphenylphosphine (42.7 mg, 0.16 mmol), and cuprous iodide (10.3 mg, 0.03 mmol) were added to the resulting solution. The reaction mixture was stirred at room temperature for 5 minutes, and N-methyl-N-(prop-2-ynyl)glycine (301.0 mg, 2.37 mmol) was added dropwise. The reaction mixture was stirred at 50°C overnight. The reaction mixture was concentrated under reduced pressure, and the residue was purified on a silica gel column (dichloromethane:methanol = 93:7) to obtain B16-1 (450.0 mg, 89.5%).

[0452] LC-MS: 318.1[M+H] + .

[0453] Step 2: N-methyl-N-(3-{4-[2-(methyldioxy- 6 Synthesis of [(1,3,4-oxadiazacyclopentyl-5-yl)phenyl]prop-2-ynyl)glycine (B16-2)

[0454] B16-1 (450.0 mg, 1.42 mmol) was added to a 50 mL single-necked flask and dissolved in dichloromethane (20 mL). Meta-chloroperbenzoic acid (888.3 mg, 5.15 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated to half its volume under reduced pressure and filtered. The organic phase was then concentrated under reduced pressure, and the residue was purified by preparative HPLC to afford B16-2 (80.0 mg, 16.1%).

[0455] LC-MS: 350.1[M+H] + .

[0456] Step 3: N-[(7S)-7-benzyl-11-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyylidene-2,3,9,10,12,15-hexahydro-1H-cyclohexyl[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2,5,8,11-tetraoxyylidene-3,6,9-triazaundec-1-yl]-2-[methyl(3-{4-[2-(methyldioxyylidene-λ 6 Synthesis of [(1,3,4-oxadiazacyclopent-5-yl)phenyl]prop-2-ynyl)amino]acetamide B16

[0457] In a 10 mL single-necked vial, B14-3 (11.3 mg, 0.015 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.7 mg, 0.015 mmol) were added and dissolved in N,N-dimethylformamide (0.5 mL). Diisopropylethylamine (0.1 mL, 0.03 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. B16-2 (5.2 mg, 0.015 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to yield B16 (4.5 mg, 27.6%).

[0458] LC-MS: 1085.4[M+H] + .

[0459] Example B17: Preparation of Compound B17

[0460] Step 1: Synthesis of 6-{3,5-bis[5-(methylthio)-1,3,4-oxadiazacyclopentan-2-yl]phenyl}hex-5-ynoic acid B17-1

[0461] B3-4 (683.0 mg, 1.58 mmol) was added to a 10 mL single-necked vial and dissolved in diisopropylamine (3 mL). Bisacetonitrile palladium chloride (12.7 mg, 0.05 mmol), triphenylphosphine (42.7 mg, 0.16 mmol), and cuprous iodide (10.3 mg, 0.03 mmol) were added to the resulting solution. The reaction mixture was stirred at room temperature for 5 minutes, and hex-5-ynoic acid (265.4 mg, 2.37 mmol) was added dropwise. The reaction mixture was stirred at 50°C overnight. The reaction mixture was concentrated under reduced pressure, and the residue was purified on a silica gel column (dichloromethane:methanol = 93:7) to obtain B17-1 (550.0 mg, 83.5%).

[0462] LC-MS:417.1[M+H] + .

[0463] Step 2: 6-{3,5-bis[5-(methyldioxy- 6 Synthesis of [(1,3,4-oxadiazacyclopentyl)-1,3,4-oxadiazacyclopentyl)-2-yl]phenyl]hex-5-yne B17-2

[0464] B17-1 (417.0 mg, 1.0 mmol) was added to a 50 mL single-necked flask and dissolved in dichloromethane (20 mL). Meta-chloroperbenzoic acid (888.3 mg, 5.15 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated to half its volume under reduced pressure and filtered. The organic phase was then concentrated under reduced pressure, and the residue was purified by preparative HPLC to afford B17-2 (90.0 mg, 18.8%).

[0465] LC-MS:481.1[M+H] + .

[0466] Step 3: N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]-2-{[(2S)-2-benzyl-15-{3,5-bis[5-(methyldioxy-λ 6Synthesis of [(1,3,4-oxadiazacyclopentadien-2-yl)phenyl]-1,4,7,10-tetrahydroylidene-3,6,9-triazapentadeca-14-yn-1-yl)amino]acetamide B17

[0467] To a 10 mL single-necked vial, add B14-3 (11.3 mg, 0.015 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.7 mg, 0.015 mmol) and dissolve in N,N-dimethylformamide (0.5 mL). Add diisopropylethylamine (0.1 mL, 0.03 mmol) to the resulting solution, and stir the mixture at room temperature for 15 minutes. Add B17-2 (7.2 mg, 0.015 mmol) to the mixture. Stir the reaction mixture at room temperature for 3 hours. Filter the reaction mixture and purify it by preparative HPLC to obtain compound B17 (6.4 mg, 35.1%).

[0468] LC-MS: 1216.4[M+H] + .

[0469] Example B18: Preparation of Compound B18

[0470] Step 1: N-[(7S)-7-benzyl-11-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyylidene-2,3,9,10,12,15-hexahydro-1H-cyclohexyl[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2,5,8,11-tetraoxyylidene-3,6,9-triazaundec-1-yl]-2-[(3-{3,5-bis[5-(methyldioxyylidene-λ 6 Synthesis of [(1,3,4-oxadiazacyclopentyl-2-yl)phenyl]prop-2-ynyl)oxy]acetamide B18

[0471] To a 10 mL single-necked vial, add B14-3 (11.3 mg, 0.015 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.7 mg, 0.015 mmol) and dissolve in N,N-dimethylformamide (0.5 mL). Add diisopropylethylamine (0.1 mL, 0.03 mmol) to the resulting solution, and stir the mixture at room temperature for 15 minutes. Add B3-6 (7.2 mg, 0.015 mmol) to the mixture. Stir the reaction mixture at room temperature for 3 hours. Filter the reaction mixture and purify it by preparative HPLC to obtain compound B18 (6.2 mg, 33.9%).

[0472] LC-MS: 1218.3[M+H] + .

[0473] Example B19: Preparation of Compound B19

[0474] Step 1: Synthesis of N-(3-{3,5-bis[5-(methylthio)-1,3,4-oxadiazacyclopentyl-2-yl]phenyl}prop-2-ynyl)-N-methylglycine (B19-1)

[0475] B3-4 (683.0 mg, 1.58 mmol) was added to a 100 mL single-necked flask and dissolved in diisopropylamine (3 mL). Bisacetonitrile palladium chloride (12.7 mg, 0.05 mmol), triphenylphosphine (42.7 mg, 0.16 mmol), and cuprous iodide (10.3 mg, 0.03 mmol) were added to the resulting solution. The reaction solution was stirred at room temperature for 5 minutes, and N-methyl-N-(prop-2-ynyl)glycine (301.8 mg, 2.37 mmol) was added dropwise. The reaction solution was stirred at 50°C overnight. The reaction solution was concentrated under reduced pressure, and the residue was purified on a silica gel column (dichloromethane:methanol = 93:7) to obtain B19-1 (600.0 mg, 87.8%).

[0476] LC-MS:432.1[M+H] + .

[0477] Step 2: N-(3-{3,5-bis[5-(methyldioxy- 6 Synthesis of [(1,3,4-oxadiazacyclopentyl-2-yl]phenyl}prop-2-ynyl)-N-methylglycine (B19-2)]

[0478] B19-1 (432.0 mg, 1.0 mmol) was added to a 50 mL single-necked flask and dissolved in dichloromethane (20 mL). Meta-chloroperbenzoic acid (888.3 mg, 5.15 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated to half its volume under reduced pressure and filtered. The organic phase was then concentrated under reduced pressure, and the residue was purified by reverse-phase preparative HPLC to yield B19-2 (80.0 mg, 16.2%).

[0479] LC-MS:496.1[M+H] + .

[0480] Step 3: N-[(7S)-7-benzyl-11-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyylidene-2,3,9,10,12,15-hexahydro-1H-cyclohexyl[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2,5,8,11-tetraoxyylidene-3,6,9-triazaundec-1-yl]-2-[(3-{3,5-bis[5-(methyldioxyylidene-λ 6 Synthesis of [(1,3,4-oxadiazacyclopentyl-2-yl)phenyl]prop-2-ynyl](methyl)amino]acetamide B19

[0481] In a 10 mL single-necked vial, B14-3 (11.3 mg, 0.015 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.7 mg, 0.015 mmol) were added and dissolved in N,N-dimethylformamide (0.5 mL). Diisopropylethylamine (0.1 mL, 0.03 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. B19-2 (7.4 mg, 0.015 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to obtain B19 (5.2 mg, 28.2%).

[0482] LC-MS: 1231.3[M+H] + .

[0483] Example B20: Preparation of Compound B20

[0484] Step 1: Synthesis of 9H-fluoren-9-ylmethyl {[(7S)-7-benzyl-11-{[4-(hydroxymethyl)phenyl]amino}-2,5,8,11-tetrahydroylidene-3,6,9-triazaundec-1-yl]amino}methane

[0485] To a 100 mL single-necked bottle, add B20-1 (559.0 mg, 1.0 mmol, prepared according to WO 2015155976) and (4-aminophenyl)methanol (123 mg, 1.0 mmol). Dissolve the mixture in dichloromethane (5 mL) and methanol (5 mL). Add 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (296.7 mg, 1.2 mmol) dropwise to the solution. Stir the reaction mixture overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and the residue was purified on a silica gel column (dichloromethane:methanol = 93:7) to obtain B20-2 (550.0 mg, 82.8%).

[0486] LC-MS: 664.3[M+H] + .

[0487] Step 2 to Step 5: Synthesis of B20-3 to B20

[0488] The synthesis of B20-3 to B20 was carried out by referring to the synthesis method of compounds B10-2 to B10-5 to obtain 6.2 mg of compound B20.

[0489] LC-MS: 1367.4[M+H] + .

[0490] Example B21: Preparation of Compound B21

[0491] Referring to the synthesis method of compound B6, 5 mg of compound B21 was obtained by liquid preparative chromatography.

[0492] LC-MS: 1667.7[M+H] + .

[0493] Example B22: Preparation of Compound B22

[0494] Step 1: Synthesis of 2-methylpropan-2-yl {[(22S)-22-benzyl-26-[(4-{[({[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyylidene-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}carbonyl)oxy]methyl}phenyl)amino]-14,17,20,23,26-pentaoxyylidene-15,18,21,24-tetraaza-3,6,9,12-tetraoxahexacosa-1-yl]amino}methanoate (B22-1)

[0495] B20-5 (90.3 mg, 0.1 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (57 mg, 0.15 mmol) were added to a 50 mL single-necked bottle and dissolved in N,N-dimethylformamide (1 mL). Diisopropylethylamine (25.8 mg, 0.2 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. [(2,2-dimethyl-4-oxyylidene-5-aza-3,8,11,14-tetraoxahexadecan-16-yl)oxy]acetic acid (52.6 mg, 0.15 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was dried by vortexing and purified by preparative HPLC to obtain B22-1 (60 mg, 48.5%).

[0496] LC-MS: 1236.5[M+H] + .

[0497] Step 2: Synthesis of {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyylidene-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(22S)-1-amino-22-benzyl-14,17,20,23,26-pentaoxyylidene-15,18,21,24-tetraaza-3,6,9,12-tetraoxacosaccharin-26-yl]amino}phenyl)methyl ester (B22-2)

[0498] B22-1 (60 mg, 0.049 mmol) was added to a 50 mL single-necked bottle and dissolved in dichloromethane (1 mL). Trifluoroacetic acid (0.2 mL) was added to the solution and the mixture was stirred at room temperature for 30 minutes. The reaction solution was spin-dried to dryness to obtain the crude product B22-2 (70 mg, 100%).

[0499] LC-MS: 1136.5[M+H] + .

[0500] Step 3: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,55S)-55-benzyl-31 Synthesis of 2-({[(9H-fluoren-9-ylmethyl)oxy]carbonyl}amino)-25,32,47,50,53,56,59-heptaoxylidene-2,5,8,11,14,17,20,23,36,39,42,45-dodecano-26,33,48,51,54,57-hexaazanonac-59-yl]amino}phenyl)methyl ester (B22-3)

[0501] To a 50 mL single-necked bottle, add B22-2 (70 mg, 0.06 mmol) and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (26.5 mg, 0.09 mmol) and dissolve in N,N-dimethylformamide (1 mL). Add diisopropylethylamine (25.8 mg, 0.2 mmol) to the resulting solution, and stir the mixture at room temperature for 15 minutes. Add B6-4 (67 mg, 0.09 mmol) to the mixture. Stir the reaction mixture at room temperature for 3 hours. The reaction mixture is then dried and purified by preparative HPLC to obtain B22-3 (55 mg, 49.1%).

[0502] LC-MS: 1867.9[M+H] + .

[0503] Step 4: Synthesis of {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyylidene-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,55S)-31-amino-55-benzyl-25,32,47,50,53,56,59-heptaoxyylidene-2,5,8,11,14,17,20,23,36,39,42,45-dodeca-26,33,48,51,54,57-hexaazanonacontan-59-yl]amino}phenyl)methyl ester (B22-4)

[0504] B22-3 (55.0 mg, 0.03 mmol) was added to a 50 mL single-necked flask and dissolved in N,N-dimethylformamide (1 mL). Diethylamine (22.2 mg, 0.3 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was slurried with petroleum ether to obtain B22-4 (55 mg, 100%).

[0505] LC-MS:1844.8[M+H] + .

[0506] Step 5: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,55S)-55-benzyl-31-({2-[(3-{4-[5-(methyldioxy)- Synthesis of [(1,3,4-oxadiazacyclopentadecyl-2-yl)phenyl]([(1,6-(6-thio)-1,3,4-oxadiazacyclopentadecyl-2-yl)phenyl]([(1,3,4-prop-2-ynyl)oxy]acetyl)amino]-2,5,32,47,50,53,56,59-heptaoxydeoxy-2,5,8,11,14,17,20,23,36,39,42,45-dodecano-2,6,33,48,51,54,57-hexaazanonadecyl-59-yl)amino]phenyl]methyl]ester B22

[0507] To a 10 mL single-necked vial, B22-4 (20 mg, 0.01 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.7 mg, 0.015 mmol) were added and dissolved in N,N-dimethylformamide (0.5 mL). Diisopropylethylamine (0.1 mL, 0.03 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. B2-5 (5.0 mg, 0.015 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to yield B22 (7.2 mg, 36.7%).

[0508] LC-MS: 1963.8[M+H] + .

[0509] Example B23: Preparation of Compound B23

[0510] Step 1: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,55S)-55-benzyl-31-({2-[(3-{3,5-bis[5-(methyl) Synthesis of (1,3,4-oxadiazacyclopentadecyl-2-yl)phenyl}prop-2-ynyl)oxy]acetyl}amino)-2,5,32,47,50,53,56,59-heptaoxylidene-2,5,8,11,14,17,20,23,36,39,42,45-dodecano-2,6,33,48,51,54,57-hexaazanonadec-59-yl)amino}phenyl)methyl ester B23

[0511] In a 10 mL single-necked vial, B22-4 (20 mg, 0.01 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.7 mg, 0.015 mmol) were added and dissolved in N,N-dimethylformamide (0.5 mL). Diisopropylethylamine (0.1 mL, 0.03 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. B3-6 (7.2 mg, 0.015 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to yield B23 (8.3 mg, 39.3%).

[0512] LC-MS: 2109.8[M+H] + .

[0513] Example B24: Preparation of Compound B24

[0514] Step 1: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S)-37-{3-[(aminocarbonyl)amino] Synthesis of 2-(4-(propyl)-31-[(2,2-dimethyl-4,19-dioxyylidene-5-aza-3,8,11,14,17-pentaoxanonadecan-19-yl)amino]-25,32,35,38-tetraoxyylidene-34-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,33,36-triazatriacont-38-yl]amino}phenyl)methyl ester B24-1

[0515] B11-1 (80.0 mg, 0.06 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (22.8 mg, 0.06 mmol) were added to a 50 mL single-necked bottle and dissolved in N,N-dimethylformamide (2 mL). Diisopropylethylamine (0.15 mL, 1.2 mmol) was added to the resulting solution, followed by [(2,2-dimethyl-4-oxyylidene-5-aza-3,8,11,14-tetraoxahexadecan-16-yl)oxy]acetic acid (21.0 mg, 0.06 mmol). The mixture was stirred at room temperature for 1 hour. The reaction mixture was dried and purified on a silica gel column (DCM:MeOH = 10:1) to afford B24-1 (50 mg, 49.5%).

[0516] LC-MS: 1682.9[M+H] + .

[0517] Step 2: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S)-37-{3-[( Synthesis of methyl 2-[(1,4-amino-1-oxyylidene-3,6,9,12-tetraoxatetradec-1-yl)amino]-2,5,32,35,38-tetraoxyylidene-34-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,33,36-triazatriacont-38-yl)amino]phenyl]methyl ester B24-2

[0518] B24-1 (50 mg, 0.03 mmol) was added to a 50 mL single-necked flask and dissolved in dichloromethane (1 mL). Trifluoroacetic acid (0.2 mL) was added to the solution, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was spin-dried to dryness to obtain the crude product B24-2 (60 mg, 100%). The crude product was directly carried on to the next reaction without purification.

[0519] LC-MS: 1582.9[M+H] + .

[0520] Step 3: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S)-37-{3-[(aminocarbonyl)amino]propyl}-31-[(21-{3,5-bis[5-( Synthesis of [(1,6-(methyldioxy)-[1,3,4-oxadiazacyclopentan-2-yl]phenyl]-1,16-dioxy-15-aza-3,6,9,12,18-pentaoxahistriacont-20-yn-1-yl)amino]-2,5,32,35,38-tetraoxy-34-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,33,36-triazatriacont-38-yl]amino]phenyl]methyl ester B24

[0521] To a 50 mL single-necked vial, add B24-2 (32 mg, 0.02 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (11.4 mg, 0.03 mmol) and dissolve in N,N-dimethylformamide (1 mL). Diisopropylethylamine (0.2 mL, 0.06 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. B3-6 (14.5 mg, 0.03 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to yield B24 (12 mg, 29.3%).

[0522] LC-MS: 2046.8[M+H] + .

[0523] Example B25: Preparation of Compound B25

[0524] Step 1: Synthesis of 1-[(25-oxyylidene-2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)oxy]tetrahydropyrrole-2,5-dione B25-2

[0525] (2,5,8,11,14,17,20-Heptadioxacosadocosan-22-yloxy)acetic acid (B25-1) (1 g, 2.5 mmol) was dissolved in anhydrous dichloromethane (10 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (575 mg, 3 mmol) and N-hydroxysuccinimide (345 mg, 3 mmol) were added to the reaction system, and the reaction solution was stirred for 3 h. The reaction solution was spin-dried and purified on a silica gel column to obtain B25-2 (1 g, 80.6%).

[0526] LC-MS:496.2[M+H] + .

[0527] Step 2: Synthesis of (2S)-2-({[(9H-fluoren-9-ylmethyl)oxy]carbonyl}amino)-4-[(25-oxyylidene-2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)amino]butanoic acid B25-3

[0528] Compound B25-2 (500 mg, 1.0 mmol) and (2S)-4-amino-2-({[(9H-fluoren-9-ylmethyl)oxy]carbonyl}amino)butyric acid (374 mg, 1.1 mmol) were dissolved in N,N-dimethylformamide (8 mL), triethylamine (202 mg, 2 mmol) was added to the reaction system, the reaction solution was stirred for 3 h, the reaction solution was spin-dried, and liquid preparative chromatography was used to obtain B25-3 (550 mg, 76.3%).

[0529] LC-MS:721.5[M+H] + .

[0530] Step 3: {[(29S,32S,35S)-36-[(4-{[({[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyidene-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline Synthesis of 9H-fluoren-9-ylmethyl]-3-[(1-yl]amino}carbonyl]oxy]methyl]phenyl]amino]-35-methyl-25,30,33,36-tetraoxyde-32-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,31,34-triazahexatriacont-29-yl]amino]methane

[0531] To a 100 mL single-necked bottle, B6-5 (75.4 mg, 0.10 mmol), diisopropylethylamine (0.05 mL, 0.3 mmol), and B25-3 (72 mg, 0.10 mmol) were added and dissolved in N,N-dimethylformamide (2 mL). 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (28.3 mg, 0.10 mmol) was added to the resulting solution under ice-cooling. The reaction mixture was stirred under ice-cooling for 1 hour. The reaction mixture was filtered and purified by preparative HPLC to obtain B25-4 (80 mg, 54.9%).

[0532] LC-MS:1457.8[M+H] + .

[0533] Step 4: Synthesis of {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyidene-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(29S,32S,35S)-29-amino-35-methyl-25,30,33,36-tetraoxyidene-32-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,31,34-triazahexatriacont-36-yl]amino}phenyl)methyl ester B25-5

[0534] B25-4 (80.0 mg, 0.055 mmol) was added to a 50 mL single-necked flask and dissolved in N,N-dimethylformamide (2 mL). Diethylamine (40 mg, 0.55 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was slurried with petroleum ether to obtain B25-5 (60 mg, 88.3%).

[0535] LC-MS: 1235.6[M+H] + .

[0536] Step 5: {[(29S)-29-[(3S,6S)-7-[(4-{[({[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyidene-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}carbonyl] Synthesis of 2-methylprop-2-yl]-6-methyl-1,4,7-trioxy-3-(prop-2-yl)-2,5-diazahept-1-yl]-2,5,31-dioxy-2,6,30-diaza-2,5,8,11,14,17,20,23,33,36,39,42-dodecaoxatetradec-44-yl]amino]methane

[0537] B25-5 (60.0 mg, 0.048 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (18 mg, 0.048 mmol) were added to a 50 mL single-necked bottle and dissolved in N,N-dimethylformamide (2 mL). Diisopropylethylamine (0.15 mL, 1.2 mmol) was added to the resulting solution, followed by [(2,2-dimethyl-4-oxyylidene-5-aza-3,8,11,14-tetraoxahexadecan-16-yl)oxy]acetic acid (21.0 mg, 0.06 mmol). The mixture was stirred at room temperature for 1 hour. The reaction mixture was dried and purified on a silica gel column (DCM:MeOH = 10:1) to afford B25-6 (40 mg, 53.1%).

[0538] LC-MS: 1568.8[M+H] + .

[0539] Step 6: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(29S,32S,35S)-2 Synthesis of 9-[(14-amino-1-oxyylidene-3,6,9,12-tetraoxatetradec-1-yl)amino]-35-methyl-25,30,33,36-tetraoxyylidene-32-(prop-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,31,34-triazahexatriacont-36-yl]amino}phenyl)methyl ester B25-7

[0540] B25-6 (40 mg, 0.025 mmol) was added to a 50 mL single-necked flask and dissolved in dichloromethane (1 mL). Trifluoroacetic acid (0.2 mL) was added to the solution, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was spin-dried to dryness to obtain the crude product B25-7 (45 mg, 100%). The crude product was directly carried on to the next reaction without purification.

[0541] LC-MS: 1468.8[M+H] + .

[0542] Step 7: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(29S,32S,35S)-35-methyl-29-[(21-{4-[5-(methyldioxy-λ 6 Synthesis of [(1,3,4-oxadiazacyclopentan-2-yl)phenyl]-1,16-dioxyylidene-15-aza-3,6,9,12,18-pentaoxahistriacont-20-yn-1-yl)amino]-2,5,30,33,36-tetraoxyylidene-32-(prop-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,31,34-triazahexatriacont-36-yl)amino]phenyl]methyl ester B25

[0543] To a 50 mL single-necked vial, add B25-7 (37 mg, 0.03 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (11.4 mg, 0.03 mmol) and dissolve in N,N-dimethylformamide (1 mL). Add diisopropylethylamine (0.03 mL, 0.06 mmol) to the resulting solution, and stir the mixture at room temperature for 15 minutes. Then, add B2-5 (10.1 mg, 0.03 mmol). Stir the reaction mixture at room temperature for 3 hours. Filter the reaction mixture and purify it by preparative HPLC to yield compound B25 (18 mg, 33.6%).

[0544] LC-MS:1786.8[M+H] + .

[0545] Example B26: Preparation of Compound B26

[0546] Step 1: Synthesis of (2S)-2-({[(9H-fluoren-9-ylmethyl)oxy]carbonyl}amino)-3-[(25-oxyylidene-2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)amino]propanoic acid B26-1

[0547] Compound B25-2 (500 mg, 1.0 mmol) and (2S)-3-amino-2-({[(9H-fluoren-9-ylmethyl)oxy]carbonyl}amino)propionic acid (359 mg, 1.1 mmol) were dissolved in N,N-dimethylformamide (8 mL), triethylamine (202 mg, 2 mmol) was added to the reaction system, the reaction solution was stirred for 3 h, the reaction solution was spin-dried, and liquid preparative chromatography was used to obtain B26-1 (520 mg, 73.6%).

[0548] LC-MS: 707.3[M+H] + .

[0549] Step 2: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(28S,31S, Synthesis of 34S)-28-({[(9H-fluoren-9-ylmethyl)oxy]carbonyl}amino)-34-methyl-25,29,32,35-tetraoxydeoxy-31-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,30,33-triazapentatriacont-35-yl]amino}phenyl)methyl ester B26-2

[0550] To a 100 mL single-necked bottle, B6-5 (75.4 mg, 0.10 mmol), diisopropylethylamine (0.05 mL, 0.3 mmol), and B26-1 (70 mg, 0.10 mmol) were added and dissolved in N,N-dimethylformamide (2 mL). 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (28.3 mg, 0.10 mmol) was added to the resulting solution under ice-cooling. The reaction mixture was stirred under ice-cooling for 1 hour. The reaction mixture was filtered and purified by preparative HPLC to obtain B26-2 (75 mg, 51.9%).

[0551] LC-MS: 1443.8[M+H] + .

[0552] Step 3: Synthesis of {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyylidene-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(28S,31S,34S)-28-amino-34-methyl-25,29,32,35-tetraoxyylidene-31-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,30,33-triazapentatriacont-35-yl]amino}phenyl)methyl ester B26-3

[0553] B26-2 (80.0 mg, 0.052 mmol) was added to a 50 mL single-necked flask and dissolved in N,N-dimethylformamide (2 mL). Diethylamine (40 mg, 0.55 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was slurried with petroleum ether to obtain B26-3 (55 mg, 87.3%).

[0554] LC-MS: 1221.6[M+H] + .

[0555] Step 4: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(28S,31S,34S)-28-[(2,2- Synthesis of dimethyl-4,19-dioxy-5-aza-3,8,11,14,17-pentaoxa-19-yl)amino-34-methyl-25,29,32,35-tetraoxy-31-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,30,33-triazapentatriacont-35-yl)aminophenyl)methyl ester B26-4

[0556] B26-3 (55.0 mg, 0.045 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (18 mg, 0.048 mmol) were added to a 50 mL single-necked bottle and dissolved in N,N-dimethylformamide (2 mL). Diisopropylethylamine (0.15 mL, 1.2 mmol) was added to the resulting solution, followed by [(2,2-dimethyl-4-oxyylidene-5-aza-3,8,11,14-tetraoxahexadecan-16-yl)oxy]acetic acid (21.0 mg, 0.06 mmol). The mixture was stirred at room temperature for 1 hour. The reaction mixture was evaporated to dryness and purified on a silica gel column (DCM:MeOH = 10:1) to afford B26-4 (40 mg, 57.2%).

[0557] LC-MS: 1554.8[M+H] + .

[0558] Step 5: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(28S,31S,34S)-2 Synthesis of 8-[(14-amino-1-oxyylidene-3,6,9,12-tetraoxatetradec-1-yl)amino]-34-methyl-25,29,32,35-tetraoxatetradec-31-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,30,33-triazapentatriacont-35-yl]amino}phenyl)methyl ester B26-5

[0559] B26-4 (40 mg, 0.025 mmol) was added to a 50 mL single-necked vial and dissolved in dichloromethane (1 mL). Trifluoroacetic acid (0.2 mL) was added to the solution, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was spin-dried to dryness to obtain the crude product B26-5 (45 mg, 100%). The crude product was directly carried on to the next reaction without purification.

[0560] LC-MS: 1454.9 [M+H] + .

[0561] Step 6: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(28S,31S,34S)-34-methyl-28-[(21-{4-[5-(methyldioxy-6 Synthesis of [(1,3,4-oxadiazacyclopentacyclopentan-2-yl)phenyl]-1,16-dioxyylidene-15-aza-3,6,9,12,18-pentaoxahistriacont-20-yn-1-yl)amino]-25,29,32,35-tetraoxyylidene-31-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,30,33-triazapentatriacont-35-yl)amino]phenyl]methyl ester B26

[0562] To a 50 mL single-necked vial, add B26-5 (45 mg, 0.03 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (11.4 mg, 0.03 mmol) and dissolve in N,N-dimethylformamide (1 mL). Diisopropylethylamine (0.045 mL, 0.09 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. B2-5 (10.1 mg, 0.03 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to yield compound B26 (15 mg, 28.2%).

[0563] LC-MS:1772.8[M+H] + .

[0564] Example B27: Preparation of Compound B27

[0565] Step 1: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxyylidene-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S)-31-{[6-(2,5-dioxyylidene-2,5-dihydro-1 Synthesis of methyl 2-(2,5,8,11,14,17,20,23,26-nonaoxaoctacosan-28-yloxy)phenyl)-1-(2-H-pyrrol-1-yl)-1-oxyylidenehexyl]amino}-37-methyl-25,32,35,38-tetraoxyylidene-34-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,33,36-triazatriacont-38-yl)amino}-2-(2,5,8,11,14,17,20,23,26-nonaoxaoctacosan-28-yloxy)phenyl)methyl ester B27

[0566] B12-3 (25.0 mg, 0.015 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.7 mg, 0.015 mmol) were added to a 10 mL single-necked vial and dissolved in N,N-dimethylformamide (0.5 mL). Diisopropylethylamine (0.1 mL, 0.03 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. 6-(2,5-dioxyylidene-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (3.0 mg, 0.015 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours, filtered, and purified by preparative HPLC to obtain B27 (8.3 mg, 29.4%).

[0567] LC-MS:1883.1[M+H] + .

[0568] Example B28: Preparation of Compound B28

[0569] Step 1: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S)-37-methyl-31-({21-[2-(methyl)- Synthesis of [(1,16-dioxyylidene-λ6-thio)pyrimidin-5-yl]-1,16-dioxyylidene-15-aza-3,6,9,12-tetraoxahistriacont-20-yn-1-yl)amino]-25,32,35,38-tetraoxyylidene-34-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,33,36-triazatriacont-38-yl]amino]phenyl]methyl ester B28

[0570] B8-2 (40.0 mg, 0.03 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (11.4 mg, 0.03 mmol) were added to a 10 mL single-necked bottle and dissolved in N,N-dimethylformamide (1 mL). Diisopropylethylamine (0.03 mL, 0.06 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. 6-[2-(methyldioxy- 6 [-(2-Methyl-1,3-thio)pyrimidin-5-yl]hex-5-ynoic acid (8 mg, 0.03 mmol) was added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to obtain compound B28 (14.8 mg, 28.2%).

[0571] LC-MS: 1746.8[M+H] + .

[0572] 1H NMR (400MHz, DMSO) δ9.99(s,1H),9.11(s,2H),8.17(d,J=6.7Hz,1H),8.05(d,J=8.1H z,1H),7.95–7.85(m,2H),7.78(d,J=11.1Hz,1H),7.64(d,J=7.8Hz,2H),7.58(d,J=8. 4Hz,2H),7.36(d,J=8.3Hz,2H),7.31(s,1H),6.51(s,1H),5.45(s,2H),5.29(s,3H),5 .07(s,2H),4.38(d,J=6.6Hz,2H),4.24–4.15(m,1H),3.91(s,2H),3.84(s,2H),3.60– 3.48(m,40H),3.41(d,J=3.6Hz,6H),3.23(s,3H),3.20(d,J=5.7Hz,2H),3.14(s,1H) ,3.05(d,J=6.1Hz,2H),2.55(d,J=7.1Hz,2H),2.38(s,3H),2.26(t,J=7.3Hz,2H),2.1 9(s,2H),1.98(dd,J=13.5,6.4Hz,1H),1.91–1.83(m,2H),1.83–1.76(m,2H),1.65(s, 1H),1.55(s,1H),1.40(s,2H),1.29(d,J=7.1Hz,3H),1.23(s,2H),0.89–0.81(m,9H).

[0573] Example B29: Preparation of Compound B29

[0574] Step 1: Synthesis of 2-methylpropan-2-yl ({3-[2-(methylthio)pyrimidin-5-yl]prop-2-ynyl}oxy)acetate (B29-2)

[0575] To a 250 mL three-necked flask, add B29-1 (2.05 g, 10 mmol) and toluene (30 mL). Add 2-methylprop-2-yl (prop-2-ynyloxy)acetate (2.04 g, 12 mmol), cuprous iodide (38 mg, 0.2 mmol), triphenylphosphine (262 mg, 1 mmol), bis(acetonitrile)palladium dichloride (77.7 mg, 0.3 mmol), and diisopropylamine (2.02 g, 20 mmol) at room temperature. The atmosphere was purged with nitrogen three times and stirred at room temperature overnight. The reaction mixture was filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate) to afford B29-2 (2.0 g, 67.8%).

[0576] LC-MS: 295.1[M+H] + .

[0577] Step 2: ({3-[2-(methyldioxy- 6 Synthesis of 2-methylprop-2-yl]-1-[[(-thio)pyrimidin-5-yl]prop-2-ynyl]oxy]acetate (B29-3)]

[0578] To a 100 mL three-necked flask, add B29-2 (2.0 g, 6.79 mmol) and dichloromethane (30 mL). At room temperature, add m-chloroperbenzoic acid (5.86 g, 33.95 mmol). The atmosphere was replaced with nitrogen three times and stirred overnight at room temperature. The reaction mixture was filtered, and the filtrate was concentrated and purified by column chromatography (eluent: petroleum ether / ethyl acetate) to afford B29-3 (1.5 g, 67.6%).

[0579] LC-MS: 327.1[M+H] + .

[0580] Step 3: ({3-[2-(methyldioxy- 6 Synthesis of [5-(2-(2-thio)pyrimidin-5-yl]prop-2-ynyl}oxy)acetic acid (B29-4)

[0581] B29-3 (1.5 g, 4.58 mmol) was added to a 100 mL single-necked bottle and dissolved in trifluoroacetic acid (4 mL) and dichloromethane (20 mL). The reaction mixture was stirred at room temperature for 1 hour, then concentrated under reduced pressure. The crude product was purified by preparative HPLC to afford B29-4 (900 mg, 72.5%).

[0582] LC-MS 271.0 [M+H] + .

[0583] Step 4: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S)-37-methyl-31-({21-[2-(methyl)- Synthesis of [1,16-dioxyylidene-15-aza-3,6,9,12,18-pentaoxahistriacont-20-yn-1-yl]amino]-25,32,35,38-tetraoxyylidene-34-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,33,36-triazatriacont-38-yl]amino]phenyl]methyl ester B29

[0584] In a 10 mL single-necked vial, B8-2 (40.0 mg, 0.03 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (11.4 mg, 0.03 mmol) were added and dissolved in N,N-dimethylformamide (1 mL). Diisopropylethylamine (0.03 mL, 0.06 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. B29-4 (8 mg, 0.03 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to yield compound B29 (13.6 mg, 25.9%).

[0585] LC-MS:1748.8[M+H] + .

[0586] 1 H NMR (400MHz, DMSO) δ9.99 (s, 1H), 9.20 (s, 2H), 8.17 (d, J = 6.6Hz, 1H), 8.05 (d, J = 8.5Hz, 1H),7.89(d,J=8.8Hz,1H),7.83(t,J=5.8Hz,1H),7.78(d,J=10.9Hz,1H),7.64(t,J=7. 0Hz,2H),7.59(d,J=8.4Hz,2H),7.36(d,J=8.5Hz,2H),7.31(s,1H),6.51(s,1H),5.45( s,2H),5.28(s,3H),5.07(s,2H),4.59(s,2H),4.38(dd,J=13.6,6.7Hz,2H),4.23–4.15( m,1H),4.04(s,2H),3.91(s,2H),3.84(s,2H),3.59–3.47(m,39H),3.45–3.40(m,7H),3 .28–3.24(m,2H),3.23(s,3H),3.14(s,1H),3.05(dd,J=13.5,7.0Hz,2H),2.38(s,3H),2 .19(s,2H),1.98(dd,J=13.7,6.9Hz,1H),1.87(tt,J=14.1,7.1Hz,2H),1.65(s,1H),1. 54(d,J=9.6Hz,1H),1.40(s,2H),1.29(d,J=7.1Hz,3H),1.23(s,2H),0.91–0.79(m,9H).

[0587] Example B30: Preparation of Compound B30

[0588] Step 1: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,55S)-55-benzyl-31-{[2-({3-[ Synthesis of 2-((2-(methyldioxyylidene-λ6-thio)pyrimidin-5-yl)prop-2-ynyl)oxy)acetyl)amino)-25,32,47,50,53,56,59-heptaoxyylidene-2,5,8,11,14,17,20,23,36,39,42,45-dodecano-26,33,48,51,54,57-hexaazanonacontan-59-yl)amino)phenyl)methyl ester B30

[0589] To a 10 mL single-necked vial, B22-4 (49.0 mg, 0.03 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (11.4 mg, 0.03 mmol) were added and dissolved in N,N-dimethylformamide (1 mL). Diisopropylethylamine (0.03 mL, 0.06 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. B29-4 (8 mg, 0.03 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to yield compound B30 (14.8 mg, 26.0%).

[0590] LC-MS: 1896.8[M+H] + .

[0591] Example B31: Preparation of Compound B31

[0592] Step 1: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,55S)-55-benzyl-31-({6-[2- Synthesis of (( ...

[0593] B22-4 (49.0 mg, 0.03 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (11.4 mg, 0.03 mmol) were added to a 10 mL single-necked bottle and dissolved in N,N-dimethylformamide (1 mL). Diisopropylethylamine (0.03 mL, 0.06 mmol) was added to the resulting solution and the mixture was stirred at room temperature for 15 minutes. 6-[2-(methyldioxy-λ 6 [-(2-Methyl-1,3-thio)pyrimidin-5-yl]hex-5-ynoic acid (8 mg, 0.03 mmol) was added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to obtain compound B31 (16.2 mg, 29.5%).

[0594] LC-MS: 1894.8[M+H] + .

[0595] Example B32: Preparation of Compound B32

[0596] Step 1: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S)-37-{3-[(aminocarbonyl)amino]propyl}-31-[(21-{4-[5-(methyl) Synthesis of [(1,6-dioxy-1,3,4-oxadiazacyclopentan-2-yl)phenyl]-1,16-dioxy-15-aza-3,6,9,12,18-pentaoxahistriacont-20-yn-1-yl)amino]-2,5,32,35,38-tetraoxy-34-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,33,36-triazatriacont-38-yl)amino]phenyl]methyl ester B32

[0597] To a 50 mL single-necked vial, B24-2 (32 mg, 0.02 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (11.4 mg, 0.03 mmol) were added and dissolved in N,N-dimethylformamide (1 mL). Diisopropylethylamine (0.2 mL, 0.06 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. B2-5 (10 mg, 0.03 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to obtain B32 (10 mg, 26.3%).

[0598] LC-MS: 1900.8[M+H] + .

[0599] Example B33: Preparation of Compound B33

[0600] Step 1: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S)-37-{3-[(aminocarbonyl)amino]propyl}-31-({2 Synthesis of 1-[2-(methyldioxyylidene-λ6-thio)pyrimidin-5-yl]-1,16-dioxyylidene-15-aza-3,6,9,12-tetraoxahistriacont-20-yn-1-yl]amino)-25,32,35,38-tetraoxyylidene-34-(propan-2-yl)-2,5,8,11,14,17,20,23-octaoxa-26,33,36-triazatriacont-38-yl]amino}phenyl)methyl ester B33

[0601] B24-2 (32 mg, 0.02 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (11.4 mg, 0.03 mmol) were added to a 50 mL single-necked bottle and dissolved in N,N-dimethylformamide (1 mL). Diisopropylethylamine (0.2 mL, 0.06 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. 6-[2-(methyldioxy- 6 [-(2-Methyl-1,3-thio)pyrimidin-5-yl]hex-5-ynoic acid (8 mg, 0.03 mmol) was added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to obtain B33 (9.8 mg, 26.8%).

[0602] LC-MS:1832.8[M+H] + .

[0603] Example B34: Preparation of Compound B34

[0604] Step 1: {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,12,15-hexahydro-1H-cyclohexane[1,2,3-de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}methane-(4-{[(31S,34S,37S)-37-{3-[(aminocarbonyl)amino]propyl}-31-({21 Synthesis of methyl ester B34

[0605] To a 50 mL single-necked vial, B24-2 (32 mg, 0.02 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (11.4 mg, 0.03 mmol) were added and dissolved in N,N-dimethylformamide (1 mL). Diisopropylethylamine (0.2 mL, 0.06 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. B29-4 (8 mg, 0.03 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to yield B34 (11.5 mg, 31.3%).

[0606] LC-MS:1834.8[M+H] + .

[0607] Example B35: Preparation of Compound B35

[0608] Compound B35 was obtained by referring to the preparation methods of compounds B20 and B24.

[0609] LC-MS:1834.8[M+H] + .

[0610] Example B36: Preparation of Compound B36

[0611] The synthesis of compound B36-3 was based on the synthesis method of compound B6-4. The overall synthesis method of compound B36 was based on the synthesis method of compound B8. Liquid preparative chromatography purification finally gave 20 mg of compound B36.

[0612] LC-MS: 1094.1[M / 2+H]+.

[0613] 1 H NMR (400MHz, DMSO-d6) δ10.04(s,1H),9.20(s,2H),8.12(d,J=7.3Hz,1H),8.05(d,J=8.8H z,1H),7.90(d,J=8.7Hz,1H),7.83(t,J=5.7Hz,1H),7.78(d,J=10.9Hz,1H),7.70–7.63(m ,2H),7.60(d,J=8.3Hz,2H),7.36(d,J=8.4Hz,2H),7.31(s,1H),6.52(s,1H),5.98(t,J=5 .9Hz,1H),5.45(s,2H),5.41(s,2H),5.29(s,3H),5.07(s,2H),4.59(s,2H),4.38(d,J=6.4 Hz,2H),4.27–4.15(m,1H),4.04(s,2H),3.91(s,2H),3.84(s,2H),3.62–3.54(m,8H),3.5 3–3.45(m,57H),3.43(d,J=5.0Hz,8H),3.27(t,J=6.1Hz,5H),3.23(s,5H),3.05(p,J=7.4 Hz,4H),2.97–2.89(m,1H),2.38(d,J=1.7Hz,3H),2.17(d,J=13.1Hz,2H),2.03–1.77(m,3 H),1.73–1.50(m,4H),1.40(dt,J=12.6,8.1Hz,4H),1.29–1.14(m,2H),0.94–0.73(m,9H).

[0614] Example B37: Preparation of Compound B37

[0615] The overall synthesis method of compound B37 refers to the synthesis method of compound B36, and liquid preparative chromatography purification is finally performed to obtain 30 mg of compound B37.

[0616] LC-MS: 1006.6 [M / 2+H] + .

[0617] 1H NMR (400MHz, DMSO-d6) δ10.04(s,1H),9.20(s,2H),8.12(d,J=7.2Hz,1H),8.05(d,J=8.8Hz,1H),7.90(d,J=8.6Hz,1H),7.83( t,J=5.8Hz,1H),7.77(d,J=10.9Hz,1H),7.65(dd,J=7.3,3.8Hz,2H),7.60(d,J=8.3Hz,2H),7.36(d,J=8.3Hz,2H),7.31(s,1H ),6.51(s,1H),5.97(t,J=5.9Hz,1H),5.45(s,2H),5.41(s,2H),5.29(d,J=4.1Hz,3H),5.07(s,2H),4.59(s,2H),4.38(q,J=7 .5,7.1Hz,2H),4.21(dd,J=8.7,6.8Hz,1H),4.04(s,2H),3.91(s,2H),3.84(s,2H),3.60–3.49(m,52H),3.43(d,J=4.2Hz,8H), 3.27(q,J=5.8Hz,3H),3.23(s,3H),3.18–2.86(m,6H),2.42–2.34(m,3H),2.27–2.08(m,2H),1.98(q,J=6.8Hz ,1H),1.87(dt,J=16.7,7.0Hz,2H),1.74–1.49(m,4H),1.40(s,4H),1.23(d,J=8.1Hz,2H),0.91–0.77(m,9H).

[0618] Example B38: Preparation of Compound B38

[0619] The overall synthesis method of compound B38 refers to the synthesis method of compound B36, and liquid preparative chromatography purification is finally performed to obtain 12.5 mg of compound B38.

[0620] LC-MS: 963.0 [M / 2+H] + .

[0621] 1H NMR (400MHz, DMSO-d6) δ9.99(s,1H),9.20(s,2H),8.17(d,J=6.8Hz,1H),8.05(d,J=8.8Hz,1H),7.89(d,J=8.7Hz,1H),7.82(t, J=5.7Hz,1H),7.78(d,J=10.9Hz,1H),7.65(d,J=7.9Hz,2H),7.59(d,J=8.2Hz,2H),7.36(d,J=8.3Hz,2H),7.31(s,1H),6.51(s ,1H),5.45(s,2H),5.29(s,3H),5.07(s,2H),4.59(s,2H),4.44–4.31(m,2H),4.25–4.15(m,1H),4.04(s,2H),3.91(s,2H),3.8 4(s,2H),3.59–3.54(m,7H),3.53–3.49(m,42H),3.46–3.37(m,8H),3.27(t,J=5.9Hz,4H),3.23(s,3H),3.05(q,J=6.8Hz,2H), 2.38(s,3H),2.19(s,2H),1.98(q,J=7.1Hz,2H),1.86(dq,J=14.1,6.9Hz,2H),1.65(s,1 H),1.55(d,J=9.6Hz,1H),1.40(s,2H),1.33–1.11(m,8H),0.86(dt,J=15.7,7.1Hz,9H).

[0622] Example C1: Preparation of Compound C1

[0623] Step 1: Synthesis of (2-amino-4,5-difluorophenyl)methanol (C1-2)

[0624] C1-1 (3 g, 17.3 mmol) was added to a 100 mL three-necked flask and dissolved in anhydrous tetrahydrofuran (30 mL). The solution was stirred at 0°C for 15 minutes. Lithium aluminum tetrahydride (10.38 mL, 2.5 mol / L in THF) was then added dropwise to the mixture. The reaction was stirred at 0°C for 30 minutes and allowed to react at room temperature overnight. The reaction was quenched by adding sodium sulfate decahydrate at 0°C, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the organic phase was evaporated to dryness. Purification on a silica gel column (petroleum ether:ethyl acetate = 5:1) afforded C1-2 (2.5 g, 90.9%).

[0625] LC-MS: 160.1[M+H] + .

[0626] Step 2: Synthesis of 2-amino-4,5-difluorobenzene-1-carbaldehyde (C1-3)

[0627] C1-2 (2.5 g, 15.6 mmol) was added to a 100 mL three-necked flask and dissolved in chloroform (30 mL). 2 g of active manganese dioxide was added to the reaction solution, and the reaction solution was reacted at 50°C overnight. The solution was filtered, dried, and purified on a silica gel column (petroleum ether: ethyl acetate = 8:1) to obtain C1-3 (2.2 g, 89.4%).

[0628] LC-MS: 158.0 [M+H] + .

[0629] Step 3: Synthesis of (4S)-4-ethyl-8,9-difluoro-4-hydroxy-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14-dione (C1-4)

[0630] In a 250 mL single-necked bottle, (4S)-4-ethyl-4-hydroxy-3,4,6,7,8,10-hexahydro-1H-pyrano[3,4-f]indolizine-3,6,10-trione (3 g, 11.4 mmol), C1-3 (2.2 g, 13.7 mmol) and p-toluenesulfonic acid (189.4 g, 1.1 mmol) were added and dissolved with anhydrous toluene (50 mL). The reaction solution was reacted at 120 ° C overnight, the solvent was dried, slurried with methanol, and filtered to obtain C1-4 (2.2 g, 50.3%).

[0631] LC-MS: 385.1[M+H] + .

[0632] Step 4: Synthesis of (4S)-4-ethyl-8,9-difluoro-4-hydroxy-11-(hydroxymethyl)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14-dione (C1-5)

[0633] C1-4 (500 mg, 1.3 mmol) was added to a 100 mL single-necked flask and dissolved in 15 mL of methanol and 12.5 mL of water. 6.5 mL of concentrated sulfuric acid and 400 mg of ferrous sulfate heptahydrate were added dropwise at 0°C. Finally, 1.2 mL of 30% hydrogen peroxide was added dropwise. The reaction solution was stirred at room temperature overnight. 50 mL of ice water was added to the reaction system and filtered to obtain C1-5 (400 mg, 74.1%).

[0634] LC-MS:415.1[M+H] + .

[0635] Step 5: Synthesis of ({1-[(4S)-4-ethyl-8,9-difluoro-4-hydroxy-3,14-dioxy-1,3,4,12-tetrahydropyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl]-5-oxy-4-aza-2-oxahexan-6-yl}amino)methane-9H-fluoren-9-ylmethyl ester (C1-6)

[0636] In a 100 mL single-necked bottle, C1-5 (380 mg, 0.92 mmol) was added, and 1-(9H-fluoren-9-yl)-3,6-dioxy-4,7-diaza-2-oxoctan-8-yl acetate (169 mg, 0.46 mmol) was dissolved in a mixed solution of 1 mL of trifluoroacetic acid and 4 mL of dichloromethane. The reaction solution was stirred at room temperature overnight, the solvent was dried by rotary evaporation, and the mixture was purified by liquid preparative chromatography to give C1-6 (180 mg, 54.2%).

[0637] LC-MS:723.2[M+H] + .

[0638] Step 6: Synthesis of 2-amino-N-[({[(4S)-4-ethyl-8,9-difluoro-4-hydroxy-3,14-dioxyylidene-1,3,4,12-tetrahydropyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl]methyl}oxy)methyl]acetamide (C1-7)

[0639] C1-6 (180.0 mg, 0.25 mmol) was added to a 50 mL single-necked flask and dissolved in N,N-dimethylformamide (2 mL). Diethylamine (182 mg, 2.5 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was slurried with petroleum ether to afford C1-7 (110 mg, 88.0%).

[0640] LC-MS: 501.2[M+H] + .

[0641] Step 7: Synthesis of 9H-fluoren-9-ylmethyl {[(9S)-9-benzyl-1-[(4S)-4-ethyl-8,9-difluoro-4-hydroxy-3,14-dioxyylidene-1,3,4,12-tetrahydropyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl]-5,8,11,14-tetraoxyylidene-2-oxa-4,7,10,13-tetraazapentadecan-15-yl]amino}methane

[0642] C1-7 (110 mg, 0.22 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (91 mg, 0.24 mmol) were added to a 100 mL single-necked bottle and dissolved in N,N-dimethylformamide (2 mL). Diisopropylethylamine (57 mg, 0.44 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. N-[1-(9H-fluoren-9-yl)-3,6,9-trioxyde-4,7-diaza-2-oxanonan-9-yl]-L-phenylalanine (120 mg, 0.24 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The solution was then dried and purified on a silica gel column (methanol:dichloromethane = 1:15) to afford C1-8 (169 mg, 78.2%).

[0643] LC-MS:984.3[M+H] + .

[0644] Step 8: Synthesis of 2-amino-N-[(9S)-9-benzyl-1-[(4S)-4-ethyl-8,9-difluoro-4-hydroxy-3,14-dioxyylidene-1,3,4,12-tetrahydropyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl]-5,8,11-trioxyylidene-2-oxa-4,7,10-triazadodec-12-yl]acetamide (C1-9)

[0645] C1-8 (169.0 mg, 0.17 mmol) was added to a 50 mL single-necked flask and dissolved in N,N-dimethylformamide (2 mL). Diethylamine (124 mg, 1.7 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was slurried with petroleum ether to afford C1-9 (115 mg, 89.1%).

[0646] LC-MS:762.2[M+H] + .

[0647] Step 9: N-[(9S)-9-benzyl-1-[(4S)-4-ethyl-8,9-difluoro-4-hydroxy-3,14-dioxyylidene-1,3,4,12-tetrahydropyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl]-5,8,11-trioxyylidene-2-oxa-4,7,10-triazadodec-12-yl]-2-({2-[(3-{4-[5-(methyldioxyylidene-λ 6 Synthesis of [(1,3,4-oxadiazacyclopentyl-2-yl)phenyl]prop-2-ynyl)oxy]acetyl]amino]acetamide C1]

[0648] In a 10 mL single-necked vial, C1-9 (11.4 mg, 0.015 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.7 mg, 0.015 mmol) were added and dissolved in N,N-dimethylformamide (0.5 mL). Diisopropylethylamine (0.1 mL, 0.03 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. B2-5 (5.0 mg, 0.015 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to yield C1 (5.6 mg, 34.6%).

[0649] LC-MS: 1080.3[M+H] + .

[0650] Example C2: Preparation of Compound C2

[0651] Step 1: N-[(9S)-9-benzyl-1-[(4S)-4-ethyl-8,9-difluoro-4-hydroxy-3,14-dioxyylidene-1,3,4,12-tetrahydropyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl]-5,8,11-trioxyylidene-2-oxa-4,7,10-triazadodec-12-yl]-2-({2-[(3-{3,5-bis[5-(methyldioxyylidene-λ 6 Synthesis of [(1,3,4-oxadiazacyclopentyl-2-yl)phenyl]prop-2-ynyl)oxy]acetyl]amino]acetamide C2]

[0652] In a 10 mL single-necked vial, C1-9 (11.4 mg, 0.015 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.7 mg, 0.015 mmol) were added and dissolved in N,N-dimethylformamide (0.5 mL). Diisopropylethylamine (0.1 mL, 0.03 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. Compound B3-6 (7.2 mg, 0.015 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to yield C2 (4.5 mg, 24.5%).

[0653] LC-MS: 1226.3[M+H] + .

[0654] Example C3: Preparation of Compound C3

[0655] Step 1: N-[(9S)-9-benzyl-1-[(4S)-4-ethyl-8,9-difluoro-4-hydroxy-3,14-dioxyylidene-1,3,4,12-tetrahydropyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl]-5,8,11-trioxyylidene-2-oxa-4,7,10-triazadodec-12-yl]-2-{[(31S)-31-({6-[2-(methyldioxyylidene-λ 6 Synthesis of acetamide C3 (2,5,8,11,14,17,20,23-octaoxatriacont-32-yl)benzo[d][1,3]thiazolyl-6-yl]-1-oxyylidenehex-5-ynyl}amino)-2,5,32-dioxyylidene-2,6-aza-2,5,8,11,14,17,20,23-octaoxatriacont-32-yl]amino)

[0656] To a 10 mL single-necked vial, C1-9 (11.4 mg, 0.015 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.7 mg, 0.015 mmol) were added and dissolved in N,N-dimethylformamide (0.5 mL). Diisopropylethylamine (0.1 mL, 0.03 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. A1-7 (12.5 mg, 0.015 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to obtain compound C3 (7.6 mg, 32.2%).

[0657] LC-MS: 1575.6[M+H] + .

[0658] Example C4: Preparation of Compound C4

[0659] Step 1: Synthesis of ({3-[2-(methylthio)benzo[d][1,3]thiazolin-6-yl]prop-2-ynyl}oxy)acetic acid (C4-1)

[0660] A1-2 (485.0 mg, 1.58 mmol) was added to a 50 mL single-necked flask and dissolved in diisopropylamine (3 mL). Bisacetonitrile palladium chloride (12.7 mg, 0.05 mmol), triphenylphosphine (42.7 mg, 0.16 mmol), and cuprous iodide (10.3 mg, 0.03 mmol) were added to the resulting solution. The reaction mixture was stirred at room temperature for 5 minutes, and (prop-2-ynyloxy)acetic acid (270.0 mg, 2.37 mmol) was added dropwise to the solution. The reaction mixture was stirred at 50°C overnight. The reaction mixture was concentrated under reduced pressure, and the residue was purified on a silica gel column (dichloromethane:methanol = 93:7) to obtain C4-1 (430.0 mg, 92.9%).

[0661] LC-MS: 294.1[M+H] + .

[0662] Step 2: ({3-[2-(methyldioxy- 6 Synthesis of 2-(4-thio)benzo[d][1,3]thiazolyl-6-yl]prop-2-ynyl}oxy)acetic acid (C4-2)

[0663] C4-1 (416.0 mg, 1.42 mmol) was added to a 50 mL single-necked flask and dissolved in dichloromethane (20 mL). Meta-chloroperbenzoic acid (888.3 mg, 5.15 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated to half its volume under reduced pressure and filtered. The organic phase was then concentrated under reduced pressure, and the residue was purified by preparative liquid chromatography to afford C4-2 (70.0 mg, 15.2%).

[0664] LC-MS: 326.1[M+H] + .

[0665] Step 3: Synthesis of (2S)-2-({[(9H-fluoren-9-ylmethyl)oxy]carbonyl}amino)-6-[(25-oxyylidene-2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)amino]hexanoic acid (C4-3)

[0666] A4-2 (200.0 mg, 0.25 mmol) was added to a 50 mL single-necked flask and dissolved in trifluoroacetic acid (1 mL) and dichloromethane (2 mL). The reaction mixture was stirred at room temperature for 1 hour, then heated to 30°C and stirred for 2 hours. The reaction mixture was concentrated under reduced pressure, dissolved in 10 mL of toluene, and concentrated again under reduced pressure to afford C4-3 (106.0 mg, 100%).

[0667] LC-MS:749.4[M+H] + .

[0668] Step 4: Synthesis of 9H-fluoren-9-ylmethyl {[(9S,18S)-9-benzyl-1-[(4S)-4-ethyl-8,9-difluoro-4-hydroxy-3,14-dioxyylidene-1,3,4,12-tetrahydropyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl]-5,8,11,14,17,24-hexaoxyylidene-4,7,10,13,16,23-hexaaza-2,26,29,32,35,38,41,44,47-nonaoxatetraoctadec-18-yl]amino}methane

[0669] C1-9 (11.4 mg, 0.015 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.7 mg, 0.015 mmol) were added to a 10 mL single-necked vial and dissolved in N,N-dimethylformamide (0.5 mL). Diisopropylethylamine (0.1 mL, 0.03 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. C4-3 (11.2 mg, 0.015 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to obtain C4-4 (10 mg, 44.7%).

[0670] LC-MS: 1492.6[M+H] + .

[0671] Step 5: Synthesis of N-[(9S)-9-benzyl-1-[(4S)-4-ethyl-8,9-difluoro-4-hydroxy-3,14-dioxyylidene-1,3,4,12-tetrahydropyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl]-5,8,11-trioxyylidene-2-oxa-4,7,10-triazadodec-12-yl]-2-{[(31S)-31-amino-25,32-dioxyylidene-26-aza-2,5,8,11,14,17,20,23-octaoxatriadodec-32-yl]amino}acetamide (C4-5)

[0672] C4-4 (10.0 mg, 0.007 mmol) was added to a 50 mL single-necked flask and dissolved in N,N-dimethylformamide (2 mL). Diethylamine (5 mg, 0.07 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was slurried with petroleum ether to afford C4-5 (7 mg, 78.7%).

[0673] LC-MS: 1270.6[M+H] + .

[0674] Step 6: N-[(9S)-9-benzyl-1-[(4S)-4-ethyl-8,9-difluoro-4-hydroxy-3,14-dioxy-1,3,4,12-tetrahydropyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl]-5,8,11-trioxy-2-oxa-4,7,10-triazadodec-12-yl]-2-{[(31S)-31-{[2-({3-[2-(methyldioxy-λ 6 Synthesis of 2-(4-(2-(2-nitro-1,2-thio)benzo[d][1,3]thiazolyl-6-yl]prop-2-ynyl]oxy)acetyl]amino]-2,5,32-dioxyylidene-2,6-aza-2,5,8,11,14,17,20,23-octaoxatriacont-32-yl]amino]acetamide C4

[0675] C4-5 (19 mg, 0.015 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.7 mg, 0.015 mmol) were added to a 10 mL single-necked vial and dissolved in N,N-dimethylformamide (0.5 mL). Diisopropylethylamine (0.1 mL, 0.03 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. C4-2 (5.1 mg, 0.015 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to yield C4 (8.8 mg, 37.2%).

[0676] LC-MS: 1577.6[M+H] + .

[0677] Example C5: Preparation of Compound C5

[0678] Step 1: Synthesis of N-{[(2-methylpropan-2-yl)oxy]carbonyl}-N-{3-[2-(methylthio)benzo[d][1,3]thiazolin-6-yl]prop-2-ynyl}glycine (C5-1)

[0679] A1-2 (485.0 mg, 1.58 mmol) was added to a 50 mL single-necked flask and dissolved in diisopropylamine (3 mL). Bisacetonitrile palladium chloride (12.7 mg, 0.05 mmol), triphenylphosphine (42.7 mg, 0.16 mmol), and cuprous iodide (10.3 mg, 0.03 mmol) were added to the resulting solution. The reaction solution was stirred at room temperature for 5 minutes, and N-{[(2-methylprop-2-yl)oxy]carbonyl}-N-(prop-2-ynyl)glycine (505.0 mg, 2.37 mmol) was added dropwise to the solution. The reaction solution was stirred at 50°C overnight. The reaction solution was concentrated under reduced pressure, and the residue was purified on a silica gel column (dichloromethane:methanol = 93:7) to obtain C5-1 (550.0 mg, 88.8%).

[0680] LC-MS: 393.1[M+H] + .

[0681] Step 2: N-{3-[2-(methyldioxy- 6 Synthesis of 2-(2-methyl-1,2-thio)benzo[d][1,3]thiazolin-6-yl]prop-2-ynyl}-N-{[(2-methylprop-2-yl)oxy]carbonyl}glycine (C5-2)

[0682] C5-1 (550.0 mg, 1.42 mmol) was added to a 50 mL single-necked flask and dissolved in dichloromethane (20 mL). Meta-chloroperbenzoic acid (888.3 mg, 5.15 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated to half its volume under reduced pressure and filtered. The organic phase was then concentrated under reduced pressure, and the residue was purified by preparative liquid chromatography to afford C5-2 (90.0 mg, 14.9%).

[0683] LC-MS:425.1[M+H] + .

[0684] Step 3: [(31S)-31-[(9S)-9-benzyl-1-[(4S)-4-ethyl-8,9-difluoro-4-hydroxy-3,14-dioxy-1,3,4,12-tetrahydropyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl]-5,8,11,14,17-pentaoxy-2-oxa-4,7,10,13,16-pentaazaheptadecan-17-yl]-38-[2-(methyldioxy-λ 6Synthesis of 2-methylprop-2-yl]-2,5,8,11,14,17,20,23-octaoxa-26,32,35-triazatriacontria-37-yn-35-yl]methane (C5-3)

[0685] C4-5 (19 mg, 0.015 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.7 mg, 0.015 mmol) were added to a 50 mL single-necked vial and dissolved in N,N-dimethylformamide (0.5 mL). Diisopropylethylamine (0.1 mL, 0.03 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. C5-2 (6.3 mg, 0.015 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to yield C5-3 (10 mg, 39.8%).

[0686] LC-MS: 1676.6[M+H] + .

[0687] Step 4: N-[(9S)-9-benzyl-1-[(4S)-4-ethyl-8,9-difluoro-4-hydroxy-3,14-dioxyylidene-1,3,4,12-tetrahydropyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl]-5,8,11-trioxyylidene-2-oxa-4,7,10-triazadodec-12-yl]-2-{[(31S)-31-{[2-({3-[2-(methyldioxyylidene-λ 6 Synthesis of 2-(4-(2-(2-nitro-1,2-thio)benzo[d][1,3]thiazolyl-6-yl]prop-2-ynyl]amino)acetyl]amino]-2,5,32-dioxyylidene-2,6-aza-2,5,8,11,14,17,20,23-octaoxatriacont-32-yl]amino]acetamide C5

[0688] C5-3 (10.0 mg, 0.006 mmol) was added to a 50 mL single-necked bottle and dissolved in trifluoroacetic acid (0.2 mL) and dichloromethane (0.4 mL). The reaction mixture was stirred at room temperature for 1 hour, then heated to 30°C and stirred for 2 hours. The reaction mixture was concentrated under reduced pressure and purified by preparative HPLC to yield C5 (3 mg, 31.7%).

[0689] LC-MS: 1576.6[M+H] + .

[0690] Example C6: Preparation of Compound C6

[0691] Step 1: Synthesis of N-methyl-N-{3-[2-(methylthio)benzo[d][1,3]thiazolin-6-yl]prop-2-ynyl}glycine (C6-1)

[0692] A1-2 (485.0 mg, 1.58 mmol) was added to a 50 mL single-necked flask and dissolved in diisopropylamine (3 mL). Bisacetonitrile palladium chloride (12.7 mg, 0.05 mmol), triphenylphosphine (42.7 mg, 0.16 mmol), and cuprous iodide (10.3 mg, 0.03 mmol) were added to the resulting solution. The reaction solution was stirred at room temperature for 5 minutes, and N-methyl-N-(prop-2-ynyl)glycine (301.0 mg, 2.37 mmol) was added dropwise to the solution. The reaction solution was stirred at 50°C overnight. The reaction solution was concentrated under reduced pressure, and the residue was purified on a silica gel column (dichloromethane:methanol = 93:7) to obtain C6-1 (435.0 mg, 90.1%).

[0693] LC-MS: 307.1[M+H] + .

[0694] Step 2: N-methyl-N-{3-[2-(methyldioxy- 6 Synthesis of 6-(2-thio)benzo[d][1,3]thiazolyl-6-yl]prop-2-ynyl}glycine (C6-2)

[0695] C6-1 (435.0 mg, 1.42 mmol) was added to a 50 mL single-necked flask and dissolved in dichloromethane (20 mL). Meta-chloroperbenzoic acid (888.3 mg, 5.15 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated to half its volume under reduced pressure and filtered. The organic phase was then concentrated under reduced pressure, and the residue was purified by preparative liquid chromatography to afford C6-2 (85.0 mg, 17.7%).

[0696] LC-MS: 339.1[M+H] + .

[0697] Step 3: N-[(9S)-9-benzyl-1-[(4S)-4-ethyl-8,9-difluoro-4-hydroxy-3,14-dioxyylidene-1,3,4,12-tetrahydropyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl]-5,8,11-trioxyylidene-2-oxa-4,7,10-triazadodec-12-yl]-2-{[(31S)-31-{[2-(methyl{3-[2-(methyldioxyylidene-λ 6Synthesis of 2-(4-(2-(2-nitro-1,2-thio)benzo[d][1,3]thiazolyl-6-yl]prop-2-ynyl]amino)acetyl]amino]-2,5,32-dioxyylidene-2,6-aza-2,5,8,11,14,17,20,23-octaoxatriacont-32-yl]amino]acetamide C6

[0698] C4-5 (19 mg, 0.015 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.7 mg, 0.015 mmol) were added to a 50 mL single-necked bottle and dissolved in N,N-dimethylformamide (0.5 mL). Diisopropylethylamine (0.1 mL, 0.03 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. C6-2 (5.1 mg, 0.015 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to obtain C6 (7.8 mg, 32.7%).

[0699] LC-MS: 1590.6 [M+H] + .

[0700] Example C7: Preparation of Compound C7

[0701] Step 1: Synthesis of (2S)-N-[(9S)-9-benzyl-1-[(4S)-4-ethyl-8,9-difluoro-4-hydroxy-3,14-dioxyylidene-1,3,4,12-tetrahydropyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl]-5,8,11,14-tetraoxyylidene-2-oxa-4,7,10,13-tetraazapentadecan-15-yl]-2-{[(2E)-1,4-dioxyylidene-4-phenylbut-2-enyl]amino}-6-[(25-oxyylidene-2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)amino]hexanamide C7

[0702] C4-5 (19 mg, 0.015 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.7 mg, 0.015 mmol) were added to a 50 mL single-necked bottle and dissolved in N,N-dimethylformamide (0.5 mL). Diisopropylethylamine (0.1 mL, 0.03 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. (2E)-4-Oxylidene-4-phenylbut-2-enoic acid (2.7 mg, 0.015 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative HPLC to yield C7 (6.3 mg, 29.4%).

[0703] LC-MS: 1428.6[M+H] + .

[0704] Example D1: Preparation of Compound D1

[0705] Step 1: Synthesis of (4S)-11-(2-chloroethyl)-4-ethyl-8,9-difluoro-4-hydroxy-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14-dione (D1-1)

[0706] C1-4 (200 mg, 0.52 mmol) was added to a 100 mL single-necked bottle and dissolved in 5 mL of water. 2.6 mL of concentrated sulfuric acid, 3 mL of 3-chloro-1,1-diethoxypropane, and 160 mg of ferrous sulfate heptahydrate were added dropwise at 0°C. Finally, 0.48 mL of 30% hydrogen peroxide was added dropwise. The reaction solution was stirred at room temperature overnight. 20 mL of ice water was added to the reaction system, and the precipitate was filtered to obtain D1-1 (100 mg, 43.0%).

[0707] LC-MS:447.1[M+H] + .

[0708] Step 2: Synthesis of (4S)-4-ethyl-8,9-difluoro-4-hydroxy-11-(2-hydroxyethyl)-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14-dione (D1-2)

[0709] D1-1 (100 mg, 0.22 mmol) was added to a 50 mL single-necked bottle and dissolved in 5 mL of 10% sulfuric acid. The mixture was reacted at 100°C for 48 h. 10 mL of ice water was added to the reaction system to precipitate. D1-2 (40 mg, 42.5%) was obtained by filtration.

[0710] LC-MS:429.2[M+H] + .

[0711] Steps 3 to 7: Synthesis of compounds D1-3 to D1

[0712] The synthesis method of this compound refers to the synthesis method of compounds C1-5 to C1, and liquid preparative chromatography purification is performed to finally obtain 5.5 mg of compound D1.

[0713] LC-MS: 1240.3[M+H] + .

[0714] Example D2: Preparation of Compound D2

[0715] The synthesis method of this compound refers to the synthesis method of compound D1, and liquid preparative chromatography purification is performed to finally obtain 6.3 mg of compound D2.

[0716] LC-MS: 1254.3[M+H] + .

[0717] Example D3: Preparation of Compound D3

[0718] The synthesis method of this compound refers to the synthesis method of compound C1, and liquid preparative chromatography purification is finally performed to obtain 5.3 mg of compound D3.

[0719] LC-MS: 1252.3[M+H] + .

[0720] Example D4: Preparation of Compound D4

[0721] The synthesis method of this compound refers to the synthesis method of compound D1, and liquid preparative chromatography purification is performed to finally obtain 6.8 mg of compound D4.

[0722] LC-MS: 1236.3[M+H] + .

[0723] Example D5: Preparation of Compound D5

[0724] Step 1: Synthesis of (4S)-11-(2-azidoethyl)-4-ethyl-8,9-difluoro-4-hydroxy-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14-dione (D5-1)

[0725] D1-1 (100 mg, 0.22 mmol) was added to a 50 mL single-necked bottle and dissolved in 5 mL of N,N-dimethylformamide. Sodium azide (17 mg, 0.26 mmol) and potassium iodide (3.6 mg, 0.022 mmol) were added. The reaction solution was stirred at 50 ° C overnight. 20 mL of ice water was added to the reaction system, and the precipitate was precipitated and filtered to obtain D5-1 (80 mg, 80.3%).

[0726] LC-MS:454.1[M+H] + .

[0727] Step 2: Synthesis of (4S)-11-(2-aminoethyl)-4-ethyl-8,9-difluoro-4-hydroxy-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14-dione (D5-2)

[0728] D5-1 (80 mg, 0.18 mmol) was added to a 50 mL single-necked bottle, dissolved in 4 mL of tetrahydrofuran and 1 mL of water, and triphenylphosphine (57 mg, 0.22 mmol). The reaction solution was stirred at 50 ° C overnight and the reaction solution was dried to obtain D5-2 (60 mg, 77.9%).

[0729] LC-MS:428.1[M+H] + .

[0730] Step 3 to Step 5: Synthesis of Compounds D5-3 to D5

[0731] Referring to the synthesis method of compound C1, 7.2 mg of compound D5 was finally obtained by liquid preparative chromatography purification.

[0732] LC-MS: 1210.3[M+H] + .

[0733] Example D6: Preparation of Compound D6

[0734] Referring to the synthesis method of compound C1, 6.5 mg of compound D6 was finally obtained by liquid preparative chromatography purification.

[0735] LC-MS: 1238.3[M+H] + .

[0736] Example D7: Preparation of Compound D7

[0737] To a 50 mL single-necked flask, add D7-1 (22.5 mg, 0.02 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (7.6 mg, 0.02 mmol) and dissolve in N,N-dimethylformamide (0.5 mL). Add diisopropylethylamine (5 mg, 0.04 mmol) to the resulting solution, and stir the mixture at room temperature for 15 minutes. Add B3-6 (10.0 mg, 0.02 mmol) to the mixture. Stir the reaction mixture at room temperature for 3 hours. Filter the reaction mixture and purify it by preparative HPLC to obtain compound D7 (8.5 mg, 26.8%).

[0738] LC-MS: 1587.7[M+H] + .

[0739] Example D8: Preparation of Compound D8

[0740] Step 1: {[(2S)-2-hydroxy-3-[(1S,4S,7S,10S,12R,14R,16S,18R,19R,20S,24R,27S,28S,29S,30R,31R,32S,33R)-19-methoxy-12-methyl-6,13-dimethylidene-22-oxyidene-10,14-oxo-4,7-oxo-30,33-oxo-24,28-oxo-17,35,36,37-tetraoxopentacyclo[27.5.1.1 1,32 .1 27,31 .0 16,20 Synthesis of 4-{[(5S,8S)-8-{3-[(aminocarbonyl)amino]propyl}-1-(9H-fluoren-9-yl)-3,6,9-trioxydeoxy-5-(propan-2-yl)-4,7-diaza-2-oxanon-9-yl]amino}phenyl]methyl ester (D8-2)

[0741] To a 50 mL single-necked bottle, D8-1 (100.0 mg, 0.13 mmol, prepared according to WO 2004010957) and eribulin (CAS No. 253128-41-5, 95 mg, 0.13 mmol) were added and dissolved in N,N-dimethylformamide (2 mL). The resulting solution was stirred at room temperature for 30 minutes, followed by the addition of diisopropylethylamine (0.06 mL, 0.39 mmol). The reaction mixture was stirred at room temperature for 3 hours, during which time the turbid solution became clear. The reaction mixture was concentrated under reduced pressure, and the residue was purified on a silica gel column (dichloromethane:methanol = 94:6) to yield D8-2 (110.0 mg, 79.6%).

[0742] LC-MS: 1357.7[M+H] + .

[0743] Step 2: {[(2S)-2-hydroxy-3-[(1S,4S,7S,10S,12R,14R,16S,18R,19R,20S,24R,27S,28S,29S,30R,31R,32S,33R)-19-methoxy-12-methyl-6,13-dimethylidene-22-oxyidene-10,14-oxo-4,7-oxo-30,33-oxo-24,28-oxo-17,35,36,37-tetraoxopentacyclo[27.5.1.1 1,32 .1 27,31 .0 16,20 Synthesis of 4-{[(2S)-5-[(aminocarbonyl)amino]-2-{[(2S)-2-amino-3-methyl-1-oxobutylidenebutyl]amino}-1-oxopentylidene]amino}phenyl]methyl ester (D8-3)

[0744] D8-2 (100.0 mg, 0.07 mmol) was added to a 50 mL single-necked flask and dissolved in N,N-dimethylformamide (2 mL). Diethylamine (51 mg, 0.7 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was slurried with petroleum ether to obtain D8-3 (70 mg, 88.6%).

[0745] LC-MS: 1135.6[M+H] + .

[0746] Step 3: {[(2S)-2-hydroxy-3-[(1S,4S,7S,10S,12R,14R,16S,18R,19R,20S,24R,27S,28S,29S,30R,31R,32S,33R)-19-methoxy-12-methyl-6,13-dimethylidene-22-oxyidene-10,14-oxo-4,7-oxo-30,33-oxo-24,28-oxo-17,35,36,37-tetraoxopentacyclo[27.5.1.1 1,32 .1 27,31 .0 16,20 ]heptatriacont-18-yl]propyl]amino}methane acid-(4-{[(8S,11S)-11-{3-[(aminocarbonyl)amino]propyl}-1-{3,5-bis[5-(methyldioxy- 6Synthesis of methyl ester D8

[0747] To a 50 mL single-necked bottle, add D8-2 (22.7 mg, 0.02 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (7.6 mg, 0.02 mmol) and dissolve in N,N-dimethylformamide (0.5 mL). Add diisopropylethylamine (5 mg, 0.04 mmol) to the resulting solution, and stir the mixture at room temperature for 15 minutes. Add B3-6 (10.0 mg, 0.02 mmol) to the mixture. Stir the reaction mixture at room temperature for 3 hours. Filter the reaction mixture and purify it by preparative HPLC to obtain D8 (7.5 mg, 23.4%).

[0748] LC-MS: 1599.6[M+H] + .

[0749] Example E Sequence Expression and Purification of Antibody hLIV22

[0750] Antibody hLIV22 can be prepared according to WO2012078668 or according to the following method: dilute cells to 6×10 6 viable cells / mL, with a viability greater than 95%. Prepare ExpiFectamine CHO Reagent:DNA complex at a 4:1 ratio and incubate at room temperature for 3 minutes. Add the complex to the cells to be transfected and mix thoroughly. Incubate at 37°C in an atmosphere of 80% humidified air containing 8% CO2. 18–22 hours after transfection, add ExpiFectamine CHO Enhancer and ExpiCHO Supplement. Transfer the culture flask to a 32°C, 80% humidified air atmosphere containing 5% CO2 and continue incubation. On day 10, collect the supernatant and incubate overnight at 4°C with Protein A MagBEADS (GenScript). Wash four times with PBS and elute with glycine, pH 3.2, to obtain hLIV22 antibody. The yield is then calculated.

[0751] Example F ADC conjugation preparation

[0752] 1. ADC coupling conditions:

[0753] DAR8 ADC: The present invention uses the LIV1-targeting antibody hLIV22 (having a heavy chain as shown in SEQ ID NO: 5 and a light chain as shown in SEQ ID NO: 10) or the Her2-targeting antibody trastuzumab (purchased from Sanyou Biopharmaceuticals (Shanghai) Co., Ltd., having a heavy chain as shown in SEQ ID NO: 15 and a light chain as shown in SEQ ID NO: 20) to reduce the reaction with 6-20 equivalents of tris(2-carboxyethyl)phosphine in 25 mM histidine buffer (pH 6.5) at a temperature of 4-40°C for 0.5-6 hours. After reduction, 5-30% DMSO (DMF or DMAC can also be used as an organic solvent) is added, and the reaction is carried out at 4-40°C with 6-30 equivalents of linker-load compound for 0.5-24 hours. The product is quenched with excess NAC. After purification, the product is exchanged for storage in 25 mM histidine buffer (pH 5.5).

[0754] DAR4 ADC: The present invention uses the LIV1-targeting antibody hLIV22, which is reduced with 2-6 equivalents of tris(2-carboxyethyl)phosphine in 25 mM histidine buffer (pH 6.5) at 4-40°C for 0.5-2 hours. Following reduction, 5-30% DMSO (DMF or DMAC can also be used as an organic solvent) is added, and the reaction is continued at 4-40°C with 4-12 equivalents of linker-to-payload compound for 0.5-12 hours. The product is quenched with excess N-acetylcysteine. The product is purified and stored in 25 mM histidine buffer (pH 5.5).

[0755] The ADC products coupled by this method were subjected to RP-UPLC and SEC-HPLC tests to obtain RP DAR values ​​and SEC purities, respectively. The DAR values ​​are shown in Table 1. The SEC purities of the ADC compounds obtained in the examples of the present invention were all ≥99.5%.

[0756] 2. ADC analysis method:

[0757] The PB used in the present invention refers to a sodium phosphate buffer solution with disodium hydrogen phosphate and sodium dihydrogen phosphate as the main components. Disodium hydrogen phosphate and sodium dihydrogen phosphate buffer solutions of different pH values ​​are usually prepared using sodium dihydrogen phosphate and disodium hydrogen phosphate solutions of the same concentration.

[0758] Determination of DAR value by conjugated drug hydrophobic chromatography RP-UPLC;

[0759] Sample treatment: Sample concentration is 1.0-5 mg / mL, filtered through a 0.22 μm filter membrane;

[0760] Common detection methods include:

[0761] (1) Size Exclusion Chromatography (SEC)-HPLC

[0762] Sample treatment: Sample concentration is 1.0-5 mg / mL, filtered through a 0.22 μm filter membrane;

[0763] Chromatographic column: TOSOH, TSKgel G3000SWxL, 5 μm, 7.8 mm × 300 mm;

[0764] Mobile phase: 0.2 M PB, 5-15% isopropanol pH 7.0;

[0765] Flow rate: 0.5-1 mL / min;

[0766] Detection wavelength: 280nm & 248nm (or 360nm);

[0767] Column temperature: RT;

[0768] Loading amount: 30 μg;

[0769] SEC chromatography elution method: isocratic elution.

[0770] (2) Reversed-Phase Chromatography (RP-UPLC)

[0771] Sample treatment: Samples were used at a concentration of 0.1–0.5 mg / mL and fully reduced with excess DTT;

[0772] Chromatographic column: Waters BioResolve TM RP mAb polyphenyl,2.7μm,4.6×100mm;

[0773] Mobile phase A: 0.1TFA% water;

[0774] Mobile phase B: 0.1TFA% acetonitrile;

[0775] Flow rate: 0.3 mL / min;

[0776] Column temperature: 60-90°C;

[0777] Sample volume: 5 μL;

[0778] RP chromatography elution method: Phase B at equilibrium increased from ~30% to ~50% within 30 minutes.

[0779] Table 1 ADC compounds obtained in the present invention and their properties

[0780] Biological activity experiments

[0781] 1. Evaluation of drug efficacy in HCC1806 tumor-bearing mice

[0782] BALB / c Nude mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., grouped into 6 mice per group) were used as test animals to evaluate the efficacy of hLIV1-ADC in nude mice bearing human breast cancer cell HCC1806 xenografts after tail vein injection.

[0783] Under sterile conditions, HCC1806 cells cultured in vitro (purchased from ATCC, passaged and preserved by the Non-clinical R&D Department of the Drug R&D Center of Qilu Pharmaceutical Co., Ltd., and expanded in vitro) were prepared into a cell suspension for inoculation. After centrifugation, the cell concentration was adjusted to 4 × 10 cells using 0.9% NaCl injection. 7 cells / mL, and Matrigel was added at a 1:1 ratio to obtain a final concentration of 2×10 7 The mice were inoculated subcutaneously in the right axilla (0.1 mL / mouse). A single dose was administered via tail vein injection, with the first dose designated as day 0 (D0). Tumor volume and body weight were measured twice weekly, with data recorded until day 19 after administration. Tumor inhibition rate (%) = (Crtv - Trtv) / Crtv (%), where Crtv and Trtv represent the relative tumor volumes of the blank control group (Vehicle, PBS) and the experimental group at the end of the experiment, respectively. The results of the tumor inhibition rate determination are shown in Table 2, Figures 1 and 3, and the results of the body weight determination are shown in Figures 2 and 4.

[0784] The above data show that the ADC conjugate of the present invention has a better tumor inhibition rate, and the ADC conjugate of the present invention has a small change in body weight during the experiment, indicating that it has excellent safety.

[0785] Table 2 Conjugates obtained in the present invention and their properties Note: * indicates that the data are calculated based on the tumor volume recorded until the 20th day after administration.

[0786] 2. In vivo PK study of ADC compounds in mice

[0787] PK protocol: Nine B-hFcRn mice (Biocytogen, product number: 110001), three per cage, were administered trastuzumab, ADC10, or ADC11 via tail vein injection (IV, n=3, with free access to food and water). The vehicle was normal saline at a dose of 10 mg / kg in a 10 mL / kg volume. Blood was collected from the fundus vein of the mice at 5 minutes, 8 hours, 1 day, 2 days, 4 days, 7 days, 10 days, 14 days, and 21 days after administration. The collected blood samples were allowed to stand at room temperature for approximately half an hour until coagulation occurred. Approximately 25 μL of serum was obtained by centrifugation at 1000 g for 15 minutes at 4°C. The serum samples were immediately stored at -80°C for subsequent LBA (HTFR) analysis to determine serum antibody concentrations.

[0788] ADC concentration detection method: To a 96-well HTRF low-capacity plate (Cisbio-66PL96100), 10 μL / well of a reagent mixture consisting of PAb Anti Human IgG-Tb cryptate (1:200, Cisbio-61HFCTAB) & Streptavidin-XL665 (1:200, cisbio-610SAXLB) & goat anti-human IgG (Fab')2 (Biotin) (2 μg / mL, Abcam-ab64666) was added at 1000 rpm for 1 min. Then, 10 μL of the diluted standard curve and the serum sample to be tested (MRD=50) were added and incubated at 1000 rpm for 1 min at room temperature for 1-2 h. The plate was read using the HTRF module of an EnVision microplate reader (laser excitation). The fluorescence signals at 620 nm and 665 nm were read, and the signal ratio at 665 nm / 620 nm was calculated. WinNonlin statistical software was used to calculate the pharmacokinetic parameters using the non-compartmental model method. The results are shown in Table 3 and Figure 5.

[0789] Table 3 Pharmacokinetic parameters of some ADC compounds determined in the present invention

[0790] The experimental data in Table 3 show that ADC10 using B29 exhibits better in vivo exposure and lower metabolic elimination rate.

[0791] 3. Cytostatic activity experiment of loaded compound C1-5

[0792] a) All cell lines were cultured in complete medium at 37°C and 5% CO2.

[0793] b) Harvest cells in the logarithmic growth phase and count the cells using a platelet counter. Detect cell viability using the trypan blue exclusion method to ensure that the cell viability is above 90%.

[0794] c) Complete medium was used to adjust the cell density, and then 80 μL of the cell suspension was inoculated into each well of a 96-well cell culture plate, with 3,000 cells per well.

[0795] d) The cells in the 96-well plate were cultured at 37°C and 5% CO2.

[0796] e) Prepare a 10x drug solution with the highest working concentration of the test compound being 10 μM, 8 concentrations, and 4-fold dilutions with a working concentration of 5 μM. For the test group, transfer 10 μL of the serially diluted test compound to the corresponding experimental wells of a 96-well cell plate, and add 10 μL of the diluent containing an equal amount of DMSO (1%). Set up three replicate wells for each drug concentration; add the corresponding volume of diluent to the control wells.

[0797] f) The cells in the drug-added 96-well plate were cultured at 37° C. and 5% CO 2 for 72 hours.

[0798] g) Melt the CTG reagent ( Luminescent Cell Viability Assay (purchased from Promega, catalog number G7571) was used and the cell plate was equilibrated to room temperature for 30 minutes.

[0799] h) Add 100 μL of CTG solution to each well.

[0800] i) Lyse the cells by shaking on an orbital shaker for 5 minutes.

[0801] j) Place the cell plate at room temperature for 20 minutes to stabilize the luminescence signal.

[0802] k) Read the luminescence value and collect the data.

[0803] l) GraphPad Prism 7.0 software was used to analyze the data, and nonlinear S-curve regression was used to fit the data to obtain the dose-effect curve, from which the IC was calculated. 50 The specific experimental results are shown in Table 4. Cell survival rate (%) = (Lum 待测药 -Lum 培养液对照 ) / (Lum 溶剂对照 -Lum 培养液对照 )×100%.

[0804] Table 4 Cytostatic activity of the loaded compounds of the present invention

Claims

1. The antibody drug conjugate of formula I or a pharmaceutically acceptable salt thereof: Ab-[-L1-L2-L3-L4-D] q Formula I in, Ab is an antibody portion or an antigen-binding fragment; D is a drug molecule, -L1-L2-L3-L4- is a fragment connecting the antibody Ab and the drug molecule D, and q is selected from 2-9; L1 is a linker portion connected to the antibody, and its structure is: -La-Lb-; wherein La is selected from the following structures: Wherein R1 is independently selected from H, halogen atoms, -CN, -OH, -NH2, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, hydroxy-C 1-4 Alkyl, m is selected from 0, 1, 2, 3; La is marked with an asterisk * at one end to indicate that the position is connected to the antibody Ab; Lb is selected from Or Lb is a chemical bond, wherein n is selected from 0 or 1; h and i are each independently selected from 0, 1, 2, 3, 4, 5, X1 is selected from CH2, N-Ra, O, Ra is selected from H, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkyl-carbonyl; Lb marked with an asterisk * indicates that the position is connected to La; L2 is a chemical bond or L2 is selected from wherein k is selected from 0-8, j is selected from 0-20, and p is selected from 0, 1, 2, 3, 4; and the -NH- terminal of L2 is connected to L1; L3 is selected from a peptide group consisting of 2-10 amino acid residues; the amino acids are selected from natural amino acids or unnatural amino acids; L4 is a chemical bond or L4 is selected from the following groups: -NH-CH2-, Wherein R3 is selected from Or R3 is H, and r is selected from 0-10; the -NH- terminal of L4 is connected to L3.

2. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: q is selected from 2-6, preferably 3-5, more preferably 3.5-4.

5.

3. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: q is selected from 7-9, preferably 7.5-8.5, more preferably 7.5-8.

0.

4. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: R1 is selected from H.

5. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: n is 1; h is 0, i is 1; k is selected from 2-6, preferably 4; j is selected from 4-18, or j is selected from 6-16, or j is selected from 7-15, or j is selected from 11, 15, or j is selected from 7, 8.

6. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: L2 is a chemical bond or L2 is selected from k, j are as defined in claim 1 or 5.

7. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1 or 6, characterized in that: L2 is selected from Chemical bond.

8. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: L3 is selected from the following polypeptide fragments: -Gly-Gly-Phe-Gly-, -Val-Ala-, -Ala-Ala-, -Ala-Ala-Asn-, -Val-Cit-, preferably -Val-Ala-, -Val-Cit-, -Gly-Gly-Phe-Gly-.

9. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: r is selected from 6-10, or r is selected from 9.

10. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: L4 is a chemical bond or L4 is selected from -NH-CH2-.

11. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: La is selected from the following structures:

12. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Lb is selected from the following fragments:

13. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Lb is selected from the following fragments:

14. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: L1 is selected from the following fragments:

15. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: L1 is selected from the following fragments:

16. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: -L1-L2-L3-L4- is selected from the following structural fragments:

17. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 16, characterized in that: Ab is selected from anti-Her2 antibody, anti-Trop2 antibody, anti-Claudin18.2 antibody, anti-LIV-1 antibody, anti-5T4 antibody, anti-CLDN6 antibody, anti-CDH6 antibody, anti-folate receptor antibody; preferably, the anti-Her2 antibody comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and LCDR1, LCDR2, LCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18; or, the anti-Her2 antibody comprises a heavy chain variable region as shown in SEQ ID NO: 14 and a light chain variable region as shown in SEQ ID NO: 19; or, the anti-Her2 antibody comprises a heavy chain as shown in SEQ ID NO: 15 and a light chain as shown in SEQ ID NO: 20; or, the anti-Her2 antibody is Trastuzumab.

18. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 17, characterized in that: Ab is anti-LIV-1 antibody.

19. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 18, characterized in that: The anti-LIV-1 antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO:

8.

20. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 19, characterized in that: The anti-LIV-1 antibody comprises a heavy chain variable region as shown in SEQ ID NO: 4 and a light chain variable region as shown in SEQ ID NO: 9; preferably, the anti-LIV-1 antibody comprises a heavy chain as shown in SEQ ID NO: 5 and a light chain as shown in SEQ ID NO:

10.

21. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 20, characterized in that: The drug molecule D is selected from camptothecin compounds, auristatin compounds, eribulin compounds, maytansine compounds, calicheamicin compounds, and anthramycin compounds; preferably, the drug molecule D is selected from a fragment having a structure of formula IIa or a pharmaceutically acceptable salt thereof: Wherein, R is selected from F, C 1-4 Alkoxy, C 1-4 Haloalkyl, C 1-4 Haloalkoxy; X2 is selected from -(CH2)s-, s is selected from 0, 1, 2, 3, 4; Y is selected from -O-, -N(Rc)-, Rc is selected from H, C 1-4 alkyl, 3-5 membered cycloalkyl, 3-5 membered heterocycloalkyl; and when s=1, Rc is not H.

22. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 21, characterized in that: R is F, or R is methoxy.

23. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 21, characterized in that: s is 1, or s is 2, or s is 3.

24. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 21, characterized in that: Rc is selected from H, methyl, cyclopropyl.

25. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 21, characterized in that: Y is selected from -O-.

26. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 21, characterized in that: Y is -N(Rc)-, and Rc is methyl or cyclopropyl.

27. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The drug molecule D is selected from the following structural fragments or pharmaceutically acceptable salts thereof:

28. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: -L1-L2-L3-L4-D can be selected from the following structural fragments or pharmaceutically acceptable salts thereof:

29. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The antibody-drug conjugate or a pharmaceutically acceptable salt thereof represented by Formula I is an antibody-drug conjugate or a pharmaceutically acceptable salt thereof represented by Formula III below: wherein Ab, La, X1, L2, L3, and q have the same meanings as in any one of claims 1-27.

30. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 29, characterized in that: La is selected from R1 and m have the same meanings as in claim 1.

31. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 30, characterized in that: La is selected from 32. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 29, characterized in that: X1 is selected from O or CH2.

33. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 29, characterized in that: L2 is selected from a chemical bond, 34. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 29, characterized in that: L3 is a polypeptide fragment consisting of 2-4 amino acids selected from the following: glycine, D-phenylalanine, D-valine, D-alanine, D-asparagine, citrulline, and the -NH-terminus of L3 is connected to L2.

35. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 34, characterized in that: L3 is selected from -Val-Ala-, -Val-Cit-, -Gly-Gly-Phe-Gly-, wherein the -NH-terminus of L3 is connected to L2.

36. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 29, characterized in that: q is selected from 7.5-8.

5.

37. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 29, characterized in that: La can be selected from X1 is selected from O or CH2; L2 is selected from chemical bonds, L3 is selected from -Val-Ala-, -Val-Cit-, -Gly-Gly-Phe-Gly-, and the -NH-terminus of L3 is connected to L2; q is selected from 7.5-8.5, preferably 7.5-8.

0.

38. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1 or 29, characterized in that: The antibody-drug conjugate or a pharmaceutically acceptable salt thereof represented by Formula I or Formula III is an antibody-drug conjugate or a pharmaceutically acceptable salt thereof represented by Formula IV below: wherein Ab, La, X1, k, p, j, q have the same meanings as in any one of claims 1 to 29.

39. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 38, characterized in that: k is selected from 3, 4, 5, preferably 4; p is selected from 2, 3, 4, preferably 3; j is selected from 6, 7, 8, preferably 7.

40. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 38, characterized in that: La is selected from X1 is CH2, O; k is 4, p is 3, j is 7, and q is 7.5-8.

0.

41. The antibody drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, which is selected from the following antibody drug conjugates or pharmaceutically acceptable salts thereof: in, q1, q2, q4, q5, q6, q7, q8, and q9 are each independently selected from 7.5-8.0, preferably 7.6-7.7; q3 is selected from 4.0-5.0, preferably 4.2-4.6; Ab is each independently selected from Ladiratuzumab and Trastuzumab.

42. A compound represented by formula II or a pharmaceutically acceptable salt thereof: in, R is selected from F, C 1-4 Alkoxy, C 1-4 Haloalkyl, C 1-4 Haloalkoxy; X2 is selected from -(CH2)s-, s is selected from 0, 1, 2, 3, 4; Y is selected from -OH, -NH-Rc, wherein Rc is selected from H, C 1-4 alkyl, 3-5 membered cycloalkyl, 3-5 membered heterocycloalkyl; and when s=1, Rc is not H.

43. The compound according to claim 42 or a pharmaceutically acceptable salt thereof, characterized in that R is F, or R is methoxy.

44. The compound according to claim 42 or a pharmaceutically acceptable salt thereof, characterized in that s is 1, or s is 2, or s is 3.

45. The compound according to claim 42 or a pharmaceutically acceptable salt thereof, characterized in that Rc is selected from H, methyl, cyclopropyl.

46. ​​The compound according to claim 42 or a pharmaceutically acceptable salt thereof, characterized in that Y is selected from -OH, -NH-Rc; Rc is methyl or cyclopropyl.

47. The following compounds or pharmaceutically acceptable salts thereof; 48. The following compound or a pharmaceutically acceptable salt thereof:

49. A compound represented by formula IIIa or a pharmaceutically acceptable salt thereof, in, X1, L2, L3 have the same meanings as in any one of claims 1 or 29, and La' is selected from the following groups: Wherein R1 is independently selected from H, halogen atoms, -CN, -OH, -NH2, C 1- 4 alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy, hydroxy-C 1-4 Alkyl, m is selected from 0, 1, 2, 3; preferably, La' is selected from the following groups:

50. A compound represented by formula IVa or a pharmaceutically acceptable salt thereof. in, X1, k, p, and j have the same meanings as in claim 1 and any one of claims 38 to 40, and La' has the same meaning as in claim 49.

51. The following structural fragment:

52. Use of the antibody drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 41 in the preparation of a drug for treating cancer.

53. Use of the compound according to any one of claims 42 to 47 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating cancer.

54. Use of the compound according to any one of claims 42 to 47 or a pharmaceutically acceptable salt thereof in the preparation of the antibody drug conjugate according to any one of claims 1 to 41 or a pharmaceutically acceptable salt thereof.

55. Use of the compound according to any one of claims 42 to 47 or a pharmaceutically acceptable salt thereof in the preparation of the compound according to any one of claims 48 to 50 or a pharmaceutically acceptable salt thereof.

56. Use of the compound according to any one of claims 48 to 50 or a pharmaceutically acceptable salt thereof in the preparation of the antibody-drug conjugate according to any one of claims 1 to 41 or a pharmaceutically acceptable salt thereof.

57. Use of the structural fragment of claim 51 in preparing the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 41.

58. A pharmaceutical composition comprising the antibody drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 41.

59. Use of the pharmaceutical composition according to claim 58 in the preparation of a drug for treating cancer.

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