Drug conjugate, pharmaceutical composition and preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a medicine conjugate, a medicine composition and a preparation method and application of the medicine conjugate. The invention particularly relates to a linker drug conjugate which has a structure as shown in a formula (I), an antibody drug conjugate prepared from a compound as shown in the formula (I), a drug composition and a preparation method and application of the antibody drug conjugate and the drug composition. The definition of each group in the compound in the formula (I) is shown in the specification. The antibody drug conjugate provided by the invention has a good inhibition effect on tumor cells.

Description

Technical Field

[0001] This disclosure belongs to the field of biomedical technology, specifically relating to drug conjugates, pharmaceutical compositions, their preparation methods and uses. Background Technology

[0002] Antibody-drug conjugates (ADCs) are an innovative biologic drug therapy approach, consisting of a small molecule drug (i.e., a cytotoxic payload) linked to a biological component (i.e., an antibody) via a specially designed linker. Traditional ADCs utilize the binding of antibodies to tumor-specific antigens to deliver the payload to target cancer cells, followed by the release of the payload leading to cancer cell death. ADCs combine target-selective antibodies with highly active cytotoxic drugs and have shown the potential to significantly improve the therapeutic window (providing a safe and effective therapeutic drug dosage range) compared to current standard of care therapies.

[0003] Therefore, it is essential to develop antibody-drug conjugates that have a good inhibitory effect on tumor cells. Summary of the Invention

[0004] This disclosure provides drug conjugates, pharmaceutical compositions, methods for their preparation, and uses. The antibody-drug conjugates disclosed herein exhibit good inhibitory effects on tumor cells.

[0005] To achieve the above-mentioned technical objectives, the technical solution adopted in this disclosure is as follows:

[0006] On the one hand, this disclosure provides a linker drug conjugate having the structure shown in formula (I):

[0007]

[0008] In the formula,

[0009] C' is a group that can be coupled to an antibody or its antigen-binding fragment;

[0010] H is a group having the structure shown in formula (IA);

[0011] P is a peptide containing 1 to 10 amino acids;

[0012] V represents either the absence of a group or a self-degrading group;

[0013] The structure shown in equation (IA) is as follows:

[0014]

[0015] In the formula,

[0016] n x It can be any integer from 1 to 10;

[0017] A is *-NH-C(=O)-C 1-8 Alkylene-(O-CH2) m1 -C(=O)-** or *-NH-C(=O)-C 1-8 Alkylene-**, where * indicates the group with -(CH2)n x - indicates connection with a Hy group; ** indicates connection with a Hy group.

[0018] m1 is any integer from 0 to 8;

[0019] Hy is a hydrophilic group;

[0020] D represents a biologically active molecule or its fragment.

[0021] In some preferred embodiments of this disclosure, D has the structure shown in formulas (II), (II2), (II3), (II4), (II5), and (II6):

[0022]

[0023]

[0024]

[0025] In formula (II),

[0026] BM is -CH- or -(CH2)n y -;

[0027] n y It can be any integer from 1 to 8;

[0028] BZ does not exist, -C (=O)-C 0-8 Alkylene -O-, -C(=O)-C 0-8 Alkylene-CH(CH3)-C 0-8 Alkylene -O- or -C(=O)-C 0-8 Alkylene-C(CH3)2-C 0-8 Alkylene-O-;

[0029] R 1 R 2 Each is independently hydrogen, halogen, C 1-8 Alkyl or C 3-8 cycloalkyl, wherein the C 1-8 Alkyl and C 3-8 One or more hydrogen atoms in the cycloalkyl group may optionally be replaced by halogens;

[0030] R 3 For hydrogen, C 1-8 Alkyl or C 3-8 cycloalkyl;

[0031] R 4 For hydrogen, C 1-8 Alkylene, C 1-8 Alkyl or C 3-8 cycloalkyl;

[0032] When BM is -CH- and R 4 C 1-8 When alkylene forms, BM and R 4 Connected to form a ring;

[0033] In formula (Ⅱ2),

[0034] Each R 5 Each is independently a halogen or C 1-3 alkyl;

[0035] R 6 C 2-3 olefin group or C 1-3 Alkoxy;

[0036] na and nb are each independent integers from 0 to 4;

[0037] In formula (Ⅱ3),

[0038] R 12 For oxygen or C 1-3 alkyl;

[0039] nd is any integer from 0 to 2;

[0040] In formula (Ⅱ4),

[0041] R 13 It is a hydroxyl group or C 1-3 alkyl;

[0042] R 14 Halogen or C 1-3 alkyl;

[0043] In formula (Ⅱ5),

[0044] R 16 It is hydrogen or C 1-3 alkyl;

[0045] R 17 C 1-3 Alkyl or C 1-3 Alkoxy;

[0046] In formula (Ⅱ6),

[0047] R 10 R 11 Each independently is C 1-3 alkyl.

[0048] In some preferred embodiments of this disclosure, it has the structure shown in formula (VII):

[0049]

[0050] In the formula,

[0051] C', P, V, A, Hy, n x Each custom definition is as previously defined;

[0052] na, nb, R 5 R 6 Each custom expression is defined in formula (Ⅱ2).

[0053] In some preferred embodiments of this disclosure, each R 5 Each is independently fluorine, chlorine, bromine or C 1-3 Alkyl groups. In some preferred embodiments of this disclosure, each R 5 Each can be fluorine, chlorine, or bromine independently.

[0054] In some preferred embodiments of this disclosure, each R 5 Each is fluorine, independent of the others.

[0055] In some preferred embodiments of this disclosure, R 6 It is -CH=CH- or -O-CH2-.

[0056] In some preferred embodiments of this disclosure, na and nb are each independently any integer from 0 to 3.

[0057] In some preferred embodiments of this disclosure, na and nb are each independently any integer from 0 to 2.

[0058] In some preferred embodiments of this disclosure, na and nb are each independently any integer from 0 to 1.

[0059] In some preferred embodiments of this disclosure, na is 0 or 1; nb is 1.

[0060] In some preferred embodiments of this disclosure, it has the structure shown in formula (VIII):

[0061]

[0062] In the formula,

[0063] C', P, V, A, Hy, n x Each custom definition is as previously defined;

[0064] R 10 R 11Each custom expression is defined in formula (Ⅱ6).

[0065] In some preferred embodiments of this disclosure, R 10 It is a methyl group.

[0066] In some preferred embodiments of this disclosure, R 11 for

[0067] In some preferred embodiments of this disclosure, it has the structure shown in formula (IX):

[0068]

[0069] In the formula,

[0070] C', H, P, V, n x Each custom definition is as previously defined;

[0071] nd、R 12 Each custom expression is defined in formula (Ⅱ3).

[0072] In some preferred embodiments of this disclosure, R 12 For oxygen or C 1-2 alkyl.

[0073] In some preferred embodiments of this disclosure, R 12 It can be oxo or methyl.

[0074] In some preferred embodiments of this disclosure, R 12 It is an oxygen-based process.

[0075] In some preferred embodiments of this disclosure, n d It can be 1 or 2.

[0076] In some preferred embodiments of this disclosure, it has the structure shown in formula (X):

[0077]

[0078] In the formula,

[0079] C', P, V, A, Hy, n x Each custom definition is as previously defined;

[0080] R 13 R 14 Each custom expression is defined in formula (Ⅱ4).

[0081] In some preferred embodiments of this disclosure, R 13 It is a hydroxyl group.

[0082] In some preferred embodiments of this disclosure, R14 It is a halogen.

[0083] In some preferred embodiments of this disclosure, R 14 It can be fluorine, chlorine, or bromine.

[0084] In some preferred embodiments of this disclosure, R 14 It is chlorine.

[0085] In some preferred embodiments of this disclosure, it has the structure shown in formula (XI):

[0086]

[0087] In the formula,

[0088] C', P, V, A, Hy, n x Each custom definition is as previously defined;

[0089] R 16 R 17 Each custom expression is defined in formula (Ⅱ5).

[0090] In some preferred embodiments of this disclosure, R 16 It can be hydrogen or methyl.

[0091] In some preferred embodiments of this disclosure, R 16 It is hydrogen.

[0092] In some preferred embodiments of this disclosure, R 17 C 1-3 Alkyl group.

[0093] In some preferred embodiments of this disclosure, R 17 C 1-2 Alkyl group.

[0094] In some preferred embodiments of this disclosure, R 17 It is a methoxy group.

[0095] Another aspect of this disclosure provides a linker drug conjugate having the structure shown in formula (I):

[0096]

[0097] In the formula,

[0098] C' is a group that can be coupled to an antibody or its antigen-binding fragment;

[0099] H is a group having the structure shown in formula (IA);

[0100] P is a peptide containing 1 to 10 amino acids;

[0101] V represents either the absence of a group or a self-degrading group;

[0102] The structure shown in equation (IA) is as follows:

[0103]

[0104] In the formula,

[0105] n x n is any integer from 1 to 10. x The numbers are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0106] A is *-NH-C(=O)-C 1-8 Alkylene-(O-CH2) m1 -C(=O)-** or *-NH-C(=O)-C 1-8 Alkylene-**, where * indicates the group with -(CH2)n x - indicates connection with a Hy group; ** indicates connection with a Hy group.

[0107] m1 is any integer from 0 to 8, that is, m1 is 0, 1, 2, 3, 4, 5, 6, 7 or 8;

[0108] Hy is a hydrophilic group;

[0109] D represents a biologically active molecule or its fragment.

[0110] In some preferred embodiments of this disclosure, D is a biologically active molecule or fragment thereof having the structure shown in formula (II):

[0111]

[0112] In the formula,

[0113] BM is -CH- or -(CH2)n y -;

[0114] n y n is any integer from 1 to 8. y It can be 1, 2, 3, 4, 5, 6, 7, or 8;

[0115] BZ does not exist, -C (=O)-C 0-8 Alkylene -O-, -C(=O)-C 0-8 Alkylene-CH(CH3)-C 0-8 Alkylene -O- or -C(=O)-C 0-8 Alkylene-C(CH3)2-C 0-8 Alkylene-O-;

[0116] R 1 R2 Each is independently hydrogen, halogen, C 1-8 Alkyl or C 3-8 cycloalkyl, wherein the C 1-8 Alkyl and C 3-8 One or more hydrogen atoms in the cycloalkyl group may optionally be replaced by halogens;

[0117] R 3 For hydrogen, C 1-8 Alkyl or C 3-8 cycloalkyl;

[0118] R 4 For hydrogen, C 1-8 Alkylene, C 1-8 Alkyl or C 3-8 cycloalkyl;

[0119] When BM is CH and R 4 C 1-8 When alkylene forms, BM and R 4 They are connected to form a ring.

[0120] In some preferred embodiments of this disclosure, BM is -(CH2)n y -;n y n is any integer from 1 to 8. y The digits are 1, 2, 3, 4, 5, 6, 7, or 8.

[0121] In some preferred embodiments of this disclosure, BM is -(CH2)n y -;n y n is any integer from 1 to 6. y It can be 1, 2, 3, 4, 5, or 6.

[0122] In some preferred embodiments of this disclosure, BM is -(CH2)n y -;n y n is any integer from 1 to 4. y It can be 1, 2, 3 or 4.

[0123] In some preferred embodiments of this disclosure, BM is -(CH2)n y -;n y n is any integer from 1 to 3. y It can be 1, 2, or 3.

[0124] In some preferred embodiments of this disclosure, BM is -(CH2)n y -;n y It can be 1 or 2.

[0125] In some preferred embodiments of this disclosure, BM is -(CH2)ny -;n y The value is 1.

[0126] In some preferred embodiments of this disclosure, BM is -CH-; R 4 C 1-8 Alkylenes; BM and R 4 They are connected to form a ring.

[0127] In some preferred embodiments of this disclosure, BM is -CH-; R 4 C 1-6 Alkylenes; BM and R 4 They are connected to form a ring.

[0128] In some preferred embodiments of this disclosure, BM is -CH-; R 4 C 1-3 Alkylenes; BM and R 4 They are connected to form a ring.

[0129] In some preferred embodiments of this disclosure, BM is -CH-; R 4 -CH2CH2-; BM and R 4 They are connected to form a ring.

[0130] In some preferred embodiments of this disclosure, the linker drug conjugate has the structure shown in formula (Ⅳ):

[0131]

[0132] In the formula,

[0133] C', A, Hy, P, V, BZ, R 1 R 2 R 3 R 4 n x n y Each custom expression is defined in formula (I).

[0134] In some preferred embodiments of this disclosure, n y It can be any integer from 1 to 8.

[0135] In some preferred embodiments of this disclosure, n y It can be any integer from 1 to 6.

[0136] In some preferred embodiments of this disclosure, n y It can be any integer from 1 to 4.

[0137] In some preferred embodiments of this disclosure, n y It can be any integer from 1 to 3.

[0138] In some preferred embodiments of this disclosure, n y It can be 1 or 2.

[0139] In some preferred embodiments of this disclosure, n y The value is 2.

[0140] In some preferred embodiments of this disclosure, the linker drug conjugate has the structure shown in formula (V):

[0141]

[0142] In the formula,

[0143] C', A, Hy, P, V, BZ, R 1 R 2 R 3 R 4 n x Each custom expression is defined in formula (I).

[0144] In some preferred embodiments of this disclosure, R 4 C 1-8 Alkylene.

[0145] In some preferred embodiments of this disclosure, R 4 C 1-6 Alkylene.

[0146] In some preferred embodiments of this disclosure, R 4 C 1-3 Alkylene.

[0147] In some preferred embodiments of this disclosure, R 4 C 1-2 Alkylene.

[0148] In some preferred embodiments of this disclosure, R 4 It is -CH2CH2-.

[0149] In some preferred embodiments of this disclosure, R 4 For hydrogen, C 1-8 Alkyl or C 3-8 Cycloalkyl.

[0150] In some preferred embodiments of this disclosure, R 4 For hydrogen, C 1-6 Alkyl or C 3-6 Cycloalkyl.

[0151] In some preferred embodiments of this disclosure, R 4 For hydrogen, C 1-4 Alkyl or C 3-4Cycloalkyl.

[0152] In some preferred embodiments of this disclosure, R 4 For hydrogen, C 1-3 Alkyl or C 3-4 Cycloalkyl.

[0153] In some preferred embodiments of this disclosure, R 4 It is hydrogen or C 1-3 alkyl.

[0154] In some preferred embodiments of this disclosure, R 4 It is hydrogen or C 1-2 alkyl.

[0155] In some preferred embodiments of this disclosure, R 4 It can be hydrogen or methyl.

[0156] In some preferred embodiments of this disclosure, R 4 It is hydrogen.

[0157] In some preferred embodiments of this disclosure, BZ is non-existent, -C (=O) -C 0-6 Alkylene -O-, -C(=O)-C 0-6 Alkylene-CH(CH3)-C 0-6 Alkylene -O- or -C(=O)-C 0-6 Alkylene-C(CH3)2-C 0-6 Alkylene-O-.

[0158] In some preferred embodiments of this disclosure, BZ is non-existent, -C (=O) -C 0-4 Alkylene -O-, -C(=O)-C 0-4 Alkylene-CH(CH3)-C 0-4 Alkylene -O- or -C(=O)-C 0-4 Alkylene-C(CH3)2-C 0-4 Alkylene-O-.

[0159] In some preferred embodiments of this disclosure, BZ is non-existent, -C (=O) -C 0-3 Alkylene -O-, -C(=O)-C 0-3 Alkylene-CH(CH3)-C 0-3 Alkylene -O- or -C(=O)-C 0-3 Alkylene-C(CH3)2-C 0-3 Alkylene-O-.

[0160] In some preferred embodiments of this disclosure, BZ is non-existent, -C (=O) -C 1-3Alkylene -O-, -C(=O)-C 0-3 Alkylene-CH(CH3)-C 0-3 Alkylene -O- or -C(=O)-C 0-3 Alkylene-C(CH3)2-C 1-3 Alkylene-O-.

[0161] In some preferred embodiments of this disclosure, BZ is non-existent, -C (=O) -C 1-3 Alkylene -O-, -C(=O)-CH(CH3)-O-, -C(=O)-C 1-3 Alkylenes -CH(CH3)-O-, -C(=O)-CH(CH3)-C 1-3 Alkylene -O- or -C(=O)-C(CH3)2-C 1-3 Alkylene-O-.

[0162] In some preferred embodiments of this disclosure, BZ is absent, -C(=O)-CH2-O-, -C(=O)-CH(CH3)-O-, -C(=O)-CH2-CH(CH3)-O-, -C(=O)-CH(CH3)-CH2-O-, or -C(=O)-C(CH3)2-CH2-O-.

[0163] In some preferred embodiments of this disclosure, BZ is absent, -C(=O)-CH2-O-, -C(=O)-CH(CH3)-O-, -C(=O)-CH2-CH(CH3)-O-, -C(=O)-CH(CH3)-CH2-O-, or -C(=O)-C(CH3)2-CH2-O-.

[0164] In some preferred embodiments of this disclosure, BZ is absent.

[0165] In some preferred embodiments of this disclosure, BZ is -C (=O)-C 0-8 Alkylene-O-.

[0166] In some preferred embodiments of this disclosure, BZ is -C (=O)-C 0-8 Alkylene-CH(CH3)-C 0-8 Alkylene-O-.

[0167] In some preferred embodiments of this disclosure, BZ is -C (=O)-C 0-8 Alkylene-C(CH3)2-C 0-8 Alkylene-O-.

[0168] In some preferred embodiments of this disclosure, BM is -CH-; R 4-CH2CH2-; BM and R 4 Connected to form a ring; BZ does not exist.

[0169] In some preferred embodiments of this disclosure, BM is -CH-; R 4 -CH2CH2-; BM and R 4 They are connected to form a ring; BZ is -C(=O)-CH2-O-.

[0170] In some preferred embodiments of this disclosure, BM is -CH-; R 4 -CH2CH2-; BM and R 4 They are connected to form a ring; BZ is -C(=O)-CH(CH3)-O-.

[0171] In some preferred embodiments of this disclosure, BM is -CH-; R 4 -CH2CH2-; BM and R 4 They are connected to form a ring; BZ is -C(=O)-CH2-CH(CH3)-O-.

[0172] In some preferred embodiments of this disclosure, BM is -CH-; R 4 -CH2CH2-; BM and R 4 They are connected to form a ring; BZ is -C(=O)-CH(CH3)-CH2-O-.

[0173] In some preferred embodiments of this disclosure, BM is -CH-; R 4 -CH2CH2-; BM and R 4 They are connected to form a ring; BZ is -C(=O)-C(CH3)2-CH2-O-.

[0174] In some preferred embodiments of this disclosure, R 1 R 2 Each is independently hydrogen, halogen, C 1-6 Alkyl or C 3-6 cycloalkyl, wherein the C 1-6 Alkyl and C 3-6 One or more hydrogen atoms in a cycloalkyl group may optionally be replaced by halogens.

[0175] In some preferred embodiments of this disclosure, R 1 R 2 Each is independently hydrogen, halogen, C 1-3 Alkyl or C 3-4 cycloalkyl, wherein the C 1-3 Alkyl and C 3-4 One or more hydrogen atoms in a cycloalkyl group may optionally be replaced by halogens.

[0176] In some preferred embodiments of this disclosure, R 1 R 2 Each is independently hydrogen, halogen, or C 1-3 Alkyl, wherein the C 1-3 One or more hydrogen atoms of the alkyl group may optionally be replaced by halogens.

[0177] In some preferred embodiments of this disclosure, R 1 R 2 Each is independently hydrogen, halogen, or C 1-2 Alkyl, wherein the C 1-2 One or more hydrogen atoms of the alkyl group may optionally be replaced by halogens.

[0178] In some preferred embodiments of this disclosure, R 1 R 2 Each can be independently hydrogen, halogen, or methyl.

[0179] In some preferred embodiments of this disclosure, R 1 R is hydrogen or halogen. 2 It can be hydrogen, halogen, or methyl.

[0180] In some preferred embodiments of this disclosure, R 1 For halogens, R 2 It is a methyl group.

[0181] In some preferred embodiments of this disclosure, R 1 For halogens, R 2 It is a halogen.

[0182] In some preferred embodiments of this disclosure, R 1 R 2 Both are hydrogen.

[0183] In some preferred embodiments of this disclosure, R 1 For hydrogen, halogen, C 1-6 Alkyl or C 3-6 cycloalkyl, wherein the C 1-6 Alkyl and C 3-6 One or more hydrogen atoms in a cycloalkyl group may optionally be replaced by halogens.

[0184] In some preferred embodiments of this disclosure, R 1 For hydrogen, halogen, C 1-3 Alkyl or C 3-4 cycloalkyl, wherein the C 1-3 Alkyl and C 3-4 One or more hydrogen atoms in a cycloalkyl group may optionally be replaced by halogens.

[0185] In some preferred embodiments of this disclosure, R 1 It is hydrogen, halogen or C 1-3 Alkyl, wherein the C 1-3 One or more hydrogen atoms of the alkyl group may optionally be replaced by halogens.

[0186] In some preferred embodiments of this disclosure, R 1 It is hydrogen, halogen or C 1-2 Alkyl, wherein the C 1-2 One or more hydrogen atoms of the alkyl group may optionally be replaced by halogens.

[0187] In some preferred embodiments of this disclosure, R 1 It can be hydrogen, halogen, or methyl.

[0188] In some preferred embodiments of this disclosure, R 1 It can be hydrogen or halogen.

[0189] In some preferred embodiments of this disclosure, R 1 It is a halogen.

[0190] In some preferred embodiments of this disclosure, R 1 It is hydrogen.

[0191] In some preferred embodiments of this disclosure, R 2 For hydrogen, halogen, C 1-6 Alkyl or C 3-6 cycloalkyl, wherein the C 1-6 Alkyl and C 3-6 One or more hydrogen atoms in a cycloalkyl group may optionally be replaced by halogens.

[0192] In some preferred embodiments of this disclosure, R 2 For hydrogen, halogen, C 1-3 Alkyl or C 3-4 cycloalkyl, wherein the C 1-3 Alkyl and C 3-4 One or more hydrogen atoms in a cycloalkyl group may optionally be replaced by halogens.

[0193] In some preferred embodiments of this disclosure, R 2 It is hydrogen, halogen or C 1-3 Alkyl, wherein the C 1-3 One or more hydrogen atoms of the alkyl group may optionally be replaced by halogens.

[0194] In some preferred embodiments of this disclosure, R 2 It is hydrogen, halogen or C 1-2 Alkyl, wherein the C 1-2One or more hydrogen atoms of the alkyl group may optionally be replaced by halogens.

[0195] In some preferred embodiments of this disclosure, R 2 It can be hydrogen, halogen, or methyl.

[0196] In some preferred embodiments of this disclosure, R 2 It can be hydrogen, halogen, or methyl.

[0197] In some preferred embodiments of this disclosure, R 2 It is a methyl group.

[0198] In some preferred embodiments of this disclosure, R 2 It is a halogen.

[0199] In some preferred embodiments of this disclosure, R 2 It is hydrogen.

[0200] In some preferred embodiments of this disclosure, R 3 For hydrogen, C 1-6 Alkyl or C 3-6 Cycloalkyl.

[0201] In some preferred embodiments of this disclosure, R 3 For hydrogen, C 1-3 Alkyl or C 3-4 Cycloalkyl.

[0202] In some preferred embodiments of this disclosure, R 3 It is hydrogen or C 1-3 alkyl;

[0203] Preferably, R 3 It is hydrogen or C 1-2 alkyl;

[0204] Preferably, R 3 It can be hydrogen or methyl.

[0205] In some preferred embodiments of this disclosure, D is selected from the following compound fragments:

[0206]

[0207]

[0208]

[0209] In some preferred embodiments of this disclosure, D is selected from the following compound fragments:

[0210]

[0211] In some preferred embodiments of this disclosure, D is selected from the following compound fragments:

[0212]

[0213] In some preferred embodiments of this disclosure, D is selected from fragments of the following compounds:

[0214]

[0215] In some preferred embodiments of this disclosure, D is selected from fragments of the following compounds:

[0216]

[0217] In some preferred embodiments of this disclosure, D is selected from fragments of the following compounds:

[0218]

[0219] In some preferred embodiments of this disclosure, A is *-NH-C(=O)-C 1-6 Alkylene-(O-CH2) m1 -C(=O)-** or *-NH-C(=O)-C 1-6 Alkylene-**, where * indicates the group with -(CH2)n x - indicates connection to a group, ** indicates connection to a Hy group; m1 is any integer from 0 to 6.

[0220] In some preferred embodiments of this disclosure, A is *-NH-C(=O)-C 1-4 Alkylene-(O-CH2) m1 -C(=O)-** or *-NH-C(=O)-C 1-4 Alkylene-**, where * indicates the group with -(CH2)n x - indicates connection to a group, ** indicates connection to a Hy group; m1 is any integer from 0 to 4.

[0221] In some preferred embodiments of this disclosure, A is *-NH-C(=O)-C 1-3 Alkylene-(O-CH2) m1 -C(=O)-** or *-NH-C(=O)-C 1-3 Alkylene-**, where * indicates the group with -(CH2)n x - indicates connection to a group, ** indicates connection to a Hy group; m1 is any integer from 0 to 3.

[0222] In some preferred embodiments of this disclosure, A is or in With -(CH2)n x - Groups are linked together, It is attached to the Hy group.

[0223] In some preferred embodiments of this disclosure, Hy is or

[0224]

[0225] In some preferred embodiments of this disclosure, C' is selected from:

[0226] or

[0227] Among them, R C1 It is hydrogen or C 1-8 Alkyl; R C2 It is a halogen;

[0228] n1, n2, n3, n4, n5, n6, n7, n8, and n9 are each an independent integer from 0 to 10.

[0229] In some preferred embodiments of this disclosure, C' is selected from: or

[0230] Where n1 and n2 are each an independent integer from 0 to 10.

[0231] In some preferred embodiments of this disclosure, C' is or

[0232] In some preferred embodiments of this disclosure, V is selected from the absence of, or Where *L1 indicates a connection to part P, and **L1 indicates a connection to part D.

[0233] In some preferred embodiments of this disclosure, V is selected from the absence of, or Where *L1 indicates a connection to part P, and **L1 indicates a connection to part D.

[0234] In some preferred embodiments of this disclosure, the amino acid is selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit), arginine (Arg), and asparagine (Asn), and the side chain of the amino acid is optionally substituted with an alkyl or hydrophilic group.

[0235] In some preferred embodiments of this disclosure, P is a peptide containing 2 amino acids, a peptide containing 3 amino acids, or a peptide containing 4 amino acids.

[0236] In some preferred embodiments of this disclosure, P is a peptide containing two amino acids, selected from... *L2 -Phe-Lys- **L2 , *L2 -Val-Ala- **L2 , *L2 -Val-Lys- **L2 、* L2 -Ala-Lys- **L2 , *L2 -Val-Cit- **L2 , *L2 -Phe-Cit- **L2 , *L2 -Leu-Cit- **L2 , *L2 -Phe-Arg- **L2 and *L2 -Gly-Val- **L2 , where *L2 indicates connection to part H, and **L2 indicates connection to part V.

[0237] In some preferred embodiments of this disclosure, P is a peptide containing three amino acids, selected from... *L2 -Glu-Val-Ala- **L2 , *L2 -Glu-Val-Cit- **L2 , *L2 -Ala-Ala-Ala- **L2 , *L2 -Ala-Ala-Asn- **L2 and Where *L2 indicates connection to part H, and **L2 indicates connection to part V.

[0238] In some preferred embodiments of this disclosure, P is a peptide containing four amino acids, selected from... *L2 -Gly-Gly-Phe-Gly-** L2 , *L2-Gly-Gly-Val-Ala- **L2 and *L2 -Gly-Phe-Gly-Gly- **L2 Preferred *L2 -Gly-Gly-Phe-Gly- **L2 and *L2 -Gly-Gly-Val-Ala- **L2 , where *L2 indicates connection to part H, and **L2 indicates connection to part V.

[0239] In some preferred embodiments of this disclosure, P is selected from: or Where *L2 indicates connection to part H, and **L2 indicates connection to part V.

[0240] In some preferred embodiments of this disclosure, the compound of formula (I) is selected from the following compounds:

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262] On the other hand, this disclosure provides antibody-drug conjugates obtained by conjugating a linker drug conjugate as described above with an antibody or its antigen-binding fragment.

[0263] In some preferred embodiments of this disclosure, the antibody-drug conjugate has the structure shown in formula (Ⅲ):

[0264]

[0265] Wherein, C is the group formed by coupling C' with the antibody or its antigen-binding fragment;

[0266] Ab represents an antibody or its antigen-binding fragment;

[0267] n xy It can be any integer from 1 to 10;

[0268] H, P, V, and D are each defined in the same formula (I).

[0269] According to formula (Ⅲ) as described above, C'-HPVD is a linker drug conjugate;

[0270] The CHPVD is a group formed by coupling C'-HPVD with an antibody or its antigen-binding fragment.

[0271] In some preferred embodiments of this disclosure, the antibody-drug conjugate according to this disclosure has a structure as shown in formula (VII):

[0272]

[0273] In the formula,

[0274] C', P, V, A, Hy, n x Each custom definition is as previously defined;

[0275] na, nb, R 5 R 6 Each custom expression is defined in formula (Ⅱ2).

[0276] na, nb, R 5R 6 The customizations are as described above and will not be repeated here.

[0277] In some preferred embodiments of this disclosure, the antibody-drug conjugate according to this disclosure has the structure shown in formula (VIII):

[0278]

[0279] In the formula,

[0280] C', P, V, A, Hy, n x Each custom definition is as previously defined;

[0281] R 10 R 11 Each custom expression is defined in formula (Ⅱ6).

[0282] R 10 R 11 The customizations are as described above and will not be repeated here.

[0283] In some preferred embodiments of this disclosure, the antibody-drug conjugate according to this disclosure has a structure as shown in formula (IX):

[0284]

[0285] In the formula,

[0286] C', H, P, V, n x Each custom definition is as previously defined;

[0287] nd、R 12 Each custom expression is defined in formula (Ⅱ3).

[0288] nd、R 12 The customizations are as described above and will not be repeated here.

[0289] In some preferred embodiments of this disclosure, the antibody-drug conjugate according to this disclosure has a structure as shown in formula (X):

[0290]

[0291] In the formula,

[0292] C', P, V, A, Hy, n x Each custom definition is as previously defined;

[0293] R 13 R 14 Each custom expression is defined in formula (Ⅱ4).

[0294] R 13 R 14 The customizations are as described above and will not be repeated here.

[0295] In some preferred embodiments of this disclosure, the antibody-drug conjugate according to this disclosure has the structure shown in formula (XI):

[0296]

[0297] In the formula,

[0298] C', P, V, A, Hy, n x Each custom definition is as previously defined;

[0299] R 16 R 17 Each custom expression is defined in formula (Ⅱ5).

[0300] R 16 R 17 The customizations are as described above and will not be repeated here.

[0301] In some preferred embodiments of this disclosure, the linker drug conjugate has the structure shown in formula (Ⅳ):

[0302]

[0303] In the formula,

[0304] C', A, Hy, P, V, BZ, R 1 R 2 R 3 R 4 n x n y Each custom expression is defined in formula (I).

[0305] In some preferred embodiments of this disclosure, n y It can be any integer from 1 to 8.

[0306] In some preferred embodiments of this disclosure, n y It can be any integer from 1 to 6.

[0307] In some preferred embodiments of this disclosure, n y It can be any integer from 1 to 4.

[0308] In some preferred embodiments of this disclosure, n y It can be any integer from 1 to 3.

[0309] In some preferred embodiments of this disclosure, n yIt can be 1 or 2.

[0310] In some preferred embodiments of this disclosure, R 4 For hydrogen, C 1-8 Alkyl or C 3-8 Cycloalkyl.

[0311] In some preferred embodiments of this disclosure, R 4 For hydrogen, C 1-6 Alkyl or C 3-6 Cycloalkyl.

[0312] In some preferred embodiments of this disclosure, R 4 For hydrogen, C 1-4 Alkyl or C 3-4 Cycloalkyl.

[0313] In some preferred embodiments of this disclosure, R 4 For hydrogen, C 1-3 Alkyl or C 3-4 Cycloalkyl.

[0314] In some preferred embodiments of this disclosure, R 4 It is hydrogen or C 1-3 alkyl.

[0315] In some preferred embodiments of this disclosure, R 4 It is hydrogen or C 1-2 alkyl.

[0316] In some preferred embodiments of this disclosure, R 4 It can be hydrogen or methyl.

[0317] In some preferred embodiments of this disclosure, R 4 It is hydrogen.

[0318] In some preferred embodiments of this disclosure, the linker drug conjugate has the structure shown in formula (V):

[0319]

[0320] In the formula,

[0321] C', A, Hy, P, V, BZ, R 1 R 2 R 3 R 4 n x Each custom expression is defined in formula (I).

[0322] In some preferred embodiments of this disclosure, R 4 C 1-8 Alkylene.

[0323] In some preferred embodiments of this disclosure, R 4 C 1-6 Alkylene.

[0324] In some preferred embodiments of this disclosure, R 4 C 1-3 Alkylene.

[0325] In some preferred embodiments of this disclosure, R 4 C 1-2 Alkylene.

[0326] In some preferred embodiments of this disclosure, BZ is non-existent, -C (=O) -C 0-6 Alkylene -O-, -C(=O)-C 0-6 Alkylene-CH(CH3)-C 0-6 Alkylene -O- or -C(=O)-C 0-6 Alkylene-C(CH3)2-C 0-6 Alkylene-O-.

[0327] In some preferred embodiments of this disclosure, BZ is non-existent, -C (=O) -C 0-4 Alkylene -O-, -C(=O)-C 0-4 Alkylene-CH(CH3)-C 0-4 Alkylene -O- or -C(=O)-C 0-4 Alkylene-C(CH3)2-C 0-4 Alkylene-O-.

[0328] In some preferred embodiments of this disclosure, BZ is non-existent, -C (=O) -C 0-3 Alkylene -O-, -C(=O)-C 0-3 Alkylene-CH(CH3)-C 0-3 Alkylene -O- or -C(=O)-C 0-3 Alkylene-C(CH3)2-C 0-3 Alkylene-O-.

[0329] In some preferred embodiments of this disclosure, BZ is non-existent, -C (=O) -C 1-3 Alkylene -O-, -C(=O)-C 0-3 Alkylene-CH(CH3)-C 0-3 Alkylene -O- or -C(=O)-C 0-3 Alkylene-C(CH3)2-C 1-3 Alkylene-O-.

[0330] In some preferred embodiments of this disclosure, BZ is non-existent, -C (=O) -C1-3 Alkylene -O-, -C(=O)-CH(CH3)-O-, -C(=O)-C 1-3 Alkylenes -CH(CH3)-O-, -C(=O)-CH(CH3)-C 1-3 Alkylene -O- or -C(=O)-C(CH3)2-C 1-3 Alkylene-O-.

[0331] In some preferred embodiments of this disclosure, BZ is absent, -C(=O)-CH2-O-, -C(=O)-CH(CH3)-O-, -C(=O)-CH2-CH(CH3)-O-, -C(=O)-CH(CH3)-CH2-O-, or -C(=O)-C(CH3)2-CH2-O-.

[0332] In some preferred embodiments of this disclosure, R 1 R 2 Each is independently hydrogen, halogen, C 1-6 Alkyl or C 3-6 cycloalkyl, wherein the C 1-6 Alkyl and C 3-6 One or more hydrogen atoms in a cycloalkyl group may optionally be replaced by halogens.

[0333] In some preferred embodiments of this disclosure, R 1 R 2 Each is independently hydrogen, halogen, C 1-3 Alkyl or C 3-4 cycloalkyl, wherein the C 1-3 Alkyl and C 3-4 One or more hydrogen atoms in a cycloalkyl group may optionally be replaced by halogens.

[0334] In some preferred embodiments of this disclosure, R 1 R 2 Each is independently hydrogen, halogen, or C 1-3 Alkyl, wherein the C 1-3 One or more hydrogen atoms of the alkyl group may optionally be replaced by halogens.

[0335] In some preferred embodiments of this disclosure, R 1 R 2 Each is independently hydrogen, halogen, or C 1-2 Alkyl, wherein the C 1-2 One or more hydrogen atoms of the alkyl group may optionally be replaced by halogens.

[0336] In some preferred embodiments of this disclosure, R 1 R 2 Each can be independently hydrogen, halogen, or methyl.

[0337] In some preferred embodiments of this disclosure, R 3 For hydrogen, C 1-6 Alkyl or C 3-6 Cycloalkyl.

[0338] In some preferred embodiments of this disclosure, R 3 For hydrogen, C 1-3 Alkyl or C 3-4 Cycloalkyl.

[0339] In some preferred embodiments of this disclosure, R 3 It is hydrogen or C 1-3 alkyl.

[0340] In some preferred embodiments of this disclosure, R 3 It is hydrogen or C 1-2 alkyl.

[0341] In some preferred embodiments of this disclosure, R 3 It can be hydrogen or methyl.

[0342] In some preferred embodiments of this disclosure, D is selected from the following compound fragments:

[0343]

[0344]

[0345]

[0346] In some preferred embodiments of this disclosure, A is *-NH-C(=O)-C 1-6 Alkylene-(O-CH2) m1 -C(=O)-** or *-NH-C(=O)-C 1-6 Alkylene-**, where * indicates the group with -(CH2)n x - indicates connection to a group, ** indicates connection to a Hy group; m1 is any integer from 0 to 6.

[0347] In some preferred embodiments of this disclosure, A is *-NH-C(=O)-C 1-4 Alkylene-(O-CH2) m1 -C(=O)-** or *-NH-C(=O)-C 1-4 Alkylene-**, where * indicates the group with -(CH2)n x - indicates connection to a group, ** indicates connection to a Hy group; m1 is any integer from 0 to 4.

[0348] In some preferred embodiments of this disclosure, A is *-NH-C(=O)-C 1-3 Alkylene-(O-CH2) m1 -C(=O)-** or *-NH-C(=O)-C 1-3 Alkylene-**, where * indicates the group with -(CH2)n x - indicates connection to a group, ** indicates connection to a Hy group; m1 is any integer from 0 to 3.

[0349] In some preferred embodiments of this disclosure, A is or in With -(CH2)n x - Groups are linked together, It is attached to the Hy group.

[0350] In some preferred embodiments of this disclosure, Hy is or

[0351] In some preferred embodiments of this disclosure, C' is selected from:

[0352] or

[0353] Among them, R C1 It is hydrogen or C 1-8 Alkyl; R C2 It is a halogen;

[0354] n1, n2, n3, n4, n5, n6, n7, n8, and n9 are each an independent integer from 0 to 10.

[0355] In some preferred embodiments of this disclosure, C' is selected from: or

[0356] Where n1 and n2 are each an independent integer from 0 to 10.

[0357] In some preferred embodiments of this disclosure, C' is or

[0358] In some preferred embodiments of this disclosure, V is selected from the absence of, or Where *L1 indicates a connection to part P, and **L1 indicates a connection to part D.

[0359] In some preferred embodiments of this disclosure, V is selected from non-existent, or Where *L1 indicates a connection to part P, and **L1 indicates a connection to part D.

[0360] In some preferred embodiments of this disclosure, the amino acid is selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit), arginine (Arg), and asparagine (Asn), and the side chain of the amino acid is optionally substituted with an alkyl or hydrophilic group.

[0361] In some preferred embodiments of this disclosure, P is a peptide containing 2 amino acids, a peptide containing 3 amino acids, or a peptide containing 4 amino acids.

[0362] In some preferred embodiments of this disclosure, P is a peptide containing two amino acids, selected from... *L2 -Phe-Lys- **L2 , *L2 -Val-Ala- **L2 , *L2 -Val-Lys- **L2 、* L2 -Ala-Lys- **L2 , *L2 -Val-Cit- **L2 , *L2 -Phe-Cit- **L2 , *L2 -Leu-Cit- **L2 , *L2 -Phe-Arg- **L2 and *L2 -Gly-Val- **L2 , where *L2 indicates connection to part H, and **L2 indicates connection to part V.

[0363] In some preferred embodiments of this disclosure, P is a peptide containing three amino acids, selected from... *L2 -Glu-Val-Ala- **L2 , *L2 -Glu-Val-Cit- **L2 , *L2 -Ala-Ala-Ala- **L2 , *L2 -Ala-Ala-Asn- **L2 and Where *L2 indicates connection to part H, and **L2 indicates connection to part V.

[0364] In some preferred embodiments of this disclosure, P is a peptide containing four amino acids, selected from... *L2 -Gly-Gly-Phe-Gly-** L2 , *L2 -Gly-Gly-Val-Ala- **L2 and *L2 -Gly-Phe-Gly-Gly- **L2 Preferred *L2 -Gly-Gly-Phe-Gly- **L2 and *L2 -Gly-Gly-Val-Ala- **L2 , where *L2 indicates connection to part H, and **L2 indicates connection to part V.

[0365] In some preferred embodiments of this disclosure, P is selected from: or Where *L2 indicates connection to part H, and **L2 indicates connection to part V.

[0366] In some preferred embodiments of this disclosure, the antibody-drug conjugate is selected from the following compounds:

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387] In some preferred embodiments of this disclosure, the Ab is selected from any one of mouse antibodies or their antigen-binding fragments, chimeric antibodies or their antigen-binding fragments, nanodomain antibodies, human antibodies or their antigen-binding fragments, or humanized antibodies or their antigen-binding fragments.

[0388] In some preferred embodiments of this disclosure, the Ab is selected from the following targets: HER2, TROP2, Claudin18.2, ROR1, PD-L1, HER1, HER3, HER4, VEGFR, EpCAM, Mesothelin, CD19, CD20, GPRC5D, SLAMF7, BCMA, CD38, CD56, CD44v6, FLT3, CD33, CD123, CD37, TCRβ-chain, AnnexinA2, B7-H3, CD171, Chlorotoxin, Claudin-6, EIIIB, Glypican-1, IntegrinαVβ3, Mucl, CD22, CD30, SLAM (Signaling lymphocytic activation) molecule), GD2, GPC3, CEA, EGFRvIII, PSMA, EGFR, PIK3CA, KRAS, BRAF, PD-1, CTLA-4, BRCA1 / 2, PARP, FSHR, LILRB4, CD33, CDH17.

[0389] In some preferred embodiments of this disclosure, the Ab is selected from the following targets: HER2, TROP2, Claudin18.2, ROR1, PD-L1, HER1, HER3, or HER4.

[0390] In some preferred embodiments of this disclosure, the Ab is selected from the following targets: HER2, TROP2, Claudin18.2, ROR1, or PD-L1.

[0391] In some preferred embodiments of this disclosure, the Ab is selected from the target HER2.

[0392] In some preferred embodiments of this disclosure, the Ab is trastuzumab.

[0393] On the other hand, this disclosure provides pharmaceutical compositions comprising one or more of the linker drug conjugates or antibody drug conjugates as described above, and optionally a pharmaceutically acceptable carrier.

[0394] On the other hand, this disclosure provides the use of the linker drug conjugate, antibody drug conjugate, or pharmaceutical composition as described above in the preparation of a medicament for treating cancer.

[0395] On the other hand, this disclosure provides a method of treating cancer, comprising administering to a patient in need a therapeutically effective amount of the linker drug conjugate, the antibody drug conjugate, or the pharmaceutical composition as described above.

[0396] In some preferred embodiments of this disclosure, the cancer is breast cancer, gastric cancer, leukemia, bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, or thyroid cancer.

[0397] In some preferred embodiments of this disclosure, the cancer is breast cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, ovarian cancer, bile duct cancer, or endometrial cancer.

[0398] In some preferred embodiments of this disclosure, the cancer is breast cancer or gastric cancer.

[0399] On the other hand, this disclosure provides a method for preparing the antibody-drug conjugate as described above, comprising the following steps: reacting an antibody or its antigen-binding fragment with the linker drug conjugate as described above to obtain the antibody-drug conjugate.

[0400] On the other hand, this disclosure provides a method for preparing a drug, wherein the method includes the step of conjugating an antibody or an antigen-binding fragment thereof with a linker drug conjugate as described above.

[0401] On the other hand, this disclosure provides a method for preparing the antibody-drug conjugate as described above, which includes the following steps: adding TCEP reducing agent to the antibody, mixing and reacting; after the reaction is completed, adding the linker drug conjugate as described above, mixing and reacting to obtain the antibody-drug conjugate.

[0402] This disclosure has the following advantages:

[0403] (1) The antibody-drug conjugates provided in this disclosure have a good inhibitory effect on tumor cells. Attached Figure Description

[0404] To more clearly illustrate the technical solutions in the specific embodiments of this disclosure or the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. The accompanying drawings are incorporated in and constitute a part of this specification, illustrating embodiments consistent with this specification, and are used together with the specification to explain the principles of this specification.

[0405] Figure 1 , Figure 2 The study shows changes in tumor volume in female C57 patients with MC38-HER2 tumors at different days after administration of different antibody-drug conjugates.

[0406] Figure 3 The changes in tumor volume in female nude mice with A549-Claudin18.2 tumors at different days after drug administration are shown.

[0407] Figure 4 The effects of antibody-drug conjugates T-mab-SJ-LP15 and T-mab-SJ-LP18 on the survival rate of Her2-positive SK-BR-3 cells are shown; where T-mab-SJ-LP15 represents Tmab+SJ-LP15 and T-mab-SJ-LP18 represents Tmab+SJ-LP18.

[0408] Figure 5 The effects of antibody-drug conjugates T-mab-SJ-LP15 and T-mab-SJ-LP18 on the survival rate of Her2-negative MDA-MB-468 cells are shown; where T-mab-SJ-LP15 represents Tmab+SJ-LP15 and T-mab-SJ-LP18 represents Tmab+SJ-LP18.

[0409] Figure 6 The effects of antibody-drug conjugates T-mab-SJ-LP10, T-mab-SJ-LP11, and T-mab-SJ-LP12 on the survival rate of Her2-positive SK-BR-3 cells are shown; where T-mab-SJ-LP10 represents Tmab+SJ-LP10, T-mab-SJ-LP11 represents Tmab+SJ-LP11, and T-mab-SJ-LP12 represents Tmab+SJ-LP12.

[0410] Figure 7 The effects of antibody-drug conjugates T-mab-SJ-LP10, T-mab-SJ-LP11, and T-mab-SJ-LP12 on the survival rate of Her2-negative MDA-MB-468 cells are shown; where T-mab-SJ-LP10 represents Tmab+SJ-LP10, T-mab-SJ-LP11 represents Tmab+SJ-LP11, and T-mab-SJ-LP12 represents Tmab+SJ-LP12.

[0411] Figure 8 The effects of antibody-drug conjugates T-mab-SJ-LP17 and T-mab-SJ-LP20 on the survival rate of Her2-positive SK-BR-3 cells are shown; where T-mab-SJ-LP17 represents Tmab+SJ-LP17 and T-mab-SJ-LP20 represents Tmab+SJ-LP20.

[0412] Figure 9 The effects of antibody-drug conjugates T-mab-SJ-LP17 and T-mab-SJ-LP20 on the survival rate of Her2-negative MDA-MB-468 cells are shown; where T-mab-SJ-LP17 represents Tmab+SJ-LP17 and T-mab-SJ-LP20 represents Tmab+SJ-LP20.

[0413] Figure 10 The effects of antibody-drug conjugates T-mab-SJ-LP13 and T-mab-SJ-LP19 on the survival rate of Her2-positive SK-BR-3 cells are shown; where T-mab-SJ-LP13 represents Tmab+SJ-LP13 and T-mab-SJ-LP19 represents Tmab+SJ-LP19.

[0414] Figure 11 The effects of antibody-drug conjugates T-mab-SJ-LP13 and T-mab-SJ-LP19 on the survival rate of Her2-negative MDA-MB-468 cells are shown; where T-mab-SJ-LP13 represents Tmab+SJ-LP13 and T-mab-SJ-LP19 represents Tmab+SJ-LP19.

[0415] Figure 12The effects of antibody-drug conjugates T-mab-SJ-LP30 and T-mab-SJ-LP31 on the survival rate of Her2-positive SK-BR-3 cells were shown; where T-mab-SJ-LP30 represents Tmab+SJ-LP30 and T-mab-SJ-LP31 represents Tmab+SJ-LP31.

[0416] Figure 13 The effects of antibody-drug conjugates T-mab-SJ-LP30 and T-mab-SJ-LP31 on the survival rate of Her2-negative MDA-MB-468 cells are shown; where T-mab-SJ-LP30 represents Tmab+SJ-LP30 and T-mab-SJ-LP31 represents Tmab+SJ-LP31.

[0417] Figure 14 The effect of antibody-drug conjugate T-mab-SJ-LP33 on the survival rate of Her2-positive SK-BR-3 cells was shown; where T-mab-SJ-LP33 represents Tmab+SJ-LP33.

[0418] Figure 15 The effect of antibody-drug conjugate T-mab-SJ-LP33 on the survival rate of Her2-negative MDA-MB-468 cells was shown; where T-mab-SJ-LP33 represents Tmab+SJ-LP33.

[0419] Figure 16 The effects of antibody-drug conjugates T-mab-SJ-LP34 and T-mab-SJ-LP35 on the survival rate of Her2-positive SK-BR-3 cells are shown; where T-mab-SJ-LP34 represents Tmab+SJ-LP34 and T-mab-SJ-LP35 represents Tmab+SJ-LP35.

[0420] Figure 17 The effects of antibody-drug conjugates T-mab-SJ-LP34 and T-mab-SJ-LP35 on the survival rate of Her2-negative MDA-MB-468 cells are shown; where T-mab-SJ-LP34 represents Tmab+SJ-LP34 and T-mab-SJ-LP35 represents Tmab+SJ-LP35. Detailed Implementation

[0421] Definitions and Explanations

[0422] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the related terms and laboratory procedures used herein are those widely used and standard practices in their respective fields. To better understand this disclosure, definitions and explanations of related terms are provided below.

[0423] In the description herein, references to “some embodiments,” “some implementations,” or “some implementation schemes” describe a subset of all possible embodiments. However, it is understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0424] As used herein and unless otherwise stated, the terms “comprising,” “including,” “having,” “containing,” and their grammatical equivalents, including their grammatical equivalents, should generally be understood as open-ended and non-restrictive, e.g., not excluding other unlisted elements or steps.

[0425] When listing a range of values, it is assumed that each value and the subranges within that range are included. For example, "C 1-6 Alkyl groups include C1, C2, C3, C4, C5, C6, and C6. 1-6 C 1-5 C 1-4 C 1-3 C 1-2 C 2-6 C 2-5 C 2-4 C 2-3 C 3-6 C 3-5 C 3-4 C 4-6 C 4-5 and C 5-6 alkyl.

[0426] As used herein, the term "substitution" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include deuterium and hydrogen variants, provided that the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is an oxo group (i.e., =O), it means that two hydrogen atoms are replaced. The terms "optional substitution" or "optionally substituted" mean that substitution is optional, and unless otherwise specified, the type and number of substituents can be arbitrary on a chemically feasible basis.

[0427] When any variable (e.g., R) appears more than once in the composition or structure of a compound, its definition is independent in each case. Thus, for example, if a group is substituted by 0-2 Rs, the group can optionally be substituted by at most two Rs, and the Rs in each case have independent options. Furthermore, combinations of substituents and / or their variants are only permitted if such combinations produce a stable compound.

[0428] In any embodiment, any or all hydrogen atoms present in the compound, or hydrogen atoms in a specific group or portion of the compound, may be replaced by deuterium or tritium. One to a maximum number of hydrogen atoms present in the compound may be replaced by deuterium. One to a maximum number of hydrogen atoms present in any group of the general formula compound or a specific compound may be replaced by deuterium. For example, when a group is described as ethyl, the ethyl group may be C2H5 or a C2H5 in which x (1 to 5) hydrogen atoms are replaced by deuterium, such as C2D. x H 5-x When a group is described as a deuterated ethyl group, the deuterated ethyl group can be a C2H5 with x (1 to 5) hydrogen atoms replaced by deuterium, such as C2D. x H 5-x The stable deuterated derivatives described in this disclosure are preferably stable deuterated isotope derivatives obtained by replacing any deuterated hydrogen atom in each formula with 1 to a maximum number (e.g., 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, etc.) of deuterium atoms.

[0429] This disclosure refers to compounds of formula (I), which include the compound of formula (I) itself, as well as its tautomers, stereoisomers, mixtures of stereoisomers, solvates or derivatives, etc.

[0430] This disclosure of "compounds" also includes tautomer forms. A tautomer form arises from the exchange of a single bond with an adjacent double bond, accompanied by the migration of a proton. The terms "tautomer" or "tautomer form" refer to isomers of different functional groups in dynamic equilibrium at room temperature that can rapidly interconvert. It refers to one of two or more structural isomers that exist in equilibrium and readily transform from one isomer form to another. This transformation results in the formal migration of a hydrogen atom, accompanied by the conversion of adjacent conjugated double bonds. Tautomers exist as a mixture of tautomer groups in solution. In solutions where tautomerization is possible, chemical equilibrium of the tautomers will be reached. The exact proportions of the tautomers depend on several factors, including temperature, solvent, and pH conditions. The concept of tautomers that can interconvert through tautomerization is called tautomerism.

[0431] When this specification describes a compound that is readily tautomerizable, but only one of its tautomers is described, it should be understood that all tautomers are included as part of the chemical meaning described. It should be understood that when a compound has tautomeric forms, it is intended to include all tautomeric forms, and the naming of the compound does not exclude any tautomeric form.

[0432] Of the various possible types of tautomerism, two are typically observed. In keto-enol tautomerism, both electrons and hydrogen atoms move simultaneously.

[0433] Common tautomer pairs are: keto-enol, amide-nitrile, lactam-lactam, amide-imine tautomer in heterocycles, imine-enamine, and enamine-enamine.

[0434] The term "isomer" refers to different compounds having the same molecular formula but different atomic arrangements and configurations. Depending on their structure, the compounds of this disclosure can exist in different stereoisomeric forms. These forms include configurational isomers or optical conformational isomers (enantiomers and / or diastereomers, including those that are blocked from rotation). Therefore, this disclosure includes enantiomers, diastereomers, and mixtures thereof. This disclosure further includes all mixtures of the above-described stereoisomers, regardless of proportions, including racemic mixtures.

[0435] Depending on their structure, the compounds disclosed herein can exist in various stable isotopic forms. These forms include those in which one or more hydrogen atoms are replaced by deuterium atoms, those in which one or more nitrogen atoms are replaced by 15N atoms, or those in which one or more carbon, fluorine, chlorine, bromine, sulfur, or oxygen are replaced by stable isotopes of their respective original atoms.

[0436] According to this disclosure, some compounds and salts can exist in different crystalline forms (polymorphs) within the scope of this disclosure.

[0437] In this disclosure, the term is used. It represents the absolute configuration of the center of a solid. In It refers to the junction of chemical bonds.

[0438] When the ring appears Furthermore, if the connection location is uncertain, it indicates that the connection site is located at... Any atom on the monocyclic ring, as long as its valence allows.

[0439] The term "prodrug" refers to a precursor or derivative of a pharmaceutically active substance that exhibits lower cytotoxicity to tumor cells compared to the parent drug and can be activated by enzymes or converted into a more active parent form. Prodrugs disclosed herein include, but are not limited to, phosphate (ester)-containing prodrugs, thiophosphate (ester)-containing prodrugs, sulfate (ester)-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, prodrugs containing optionally substituted phenoxyacetamide or phenylacetamide, 5-fluorocytosine, and other 5-fluorouridine prodrugs capable of being converted into more active cytotoxic free drugs. Examples of cytotoxic agents that can be derived into prodrug forms for use in the cytotoxic drugs of this disclosure include, but are not limited to, the aforementioned chemotherapeutic agents.

[0440] The term "alkyl" refers to a chain-like (straight-chain or branched) saturated aliphatic hydrocarbon group. The term "alkyl" can refer to a straight-chain or branched alkyl group containing 1 to 10 carbon atoms. 1-10 Alkyl groups, preferably alkyl groups containing 1 to 6 carbon atoms (C 1-6 Alkyl groups. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and various branched isomers thereof. More preferably are lower alkyl groups containing 1 to 3 carbon atoms (C... 1-3 Alkyl groups, including methyl, ethyl, n-propyl, isopropyl, etc., are used in non-limiting embodiments. Alkyl groups may be substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups described in this application.

[0441] In one embodiment, the substituent is independently selected from oxo, halogen, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, alkyl (including straight-chain, branched and / or unsaturated alkyl), substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, fluoroalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, fluoroalkoxy, -S-alkyl, S(=O)2alkyl, -C(=O)NH (substituted or unsubstituted alkyl, or substituted or unsubstituted). -Phenyl), -C(=O)N(H or alkyl)2, -OC(=O)N(substituted or unsubstituted alkyl)2, NHC(=O)NH(substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl), -NHC(=O)alkyl, -N(substituted or unsubstituted alkyl)C(=O)(substituted or unsubstituted alkyl), -NHC(=O)(substituted or unsubstituted alkyl), -C(OH)(substituted or unsubstituted alkyl)2 and -C(NH2)(substituted or unsubstituted alkyl)2. In another embodiment, for example, the optional substituents are selected from oxo, fluorine, chlorine, bromine, iodine, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, -CH3, -CH2CH3, -CH(CH3)2, -CF3, -CH2CF3, -OCH3, -OCH2CH3, -OCH(CH3)2, -OCF3, -OCH2CF3, -S(=O)2-CH3, -C(=O)NH2, -C(=O)-NHCH3, -NHC(=O)NHCH3, -C(=O)CH3, -ON(O)2, and C(=O)OH. In yet another embodiment, the substituents are independently selected from C 1-6 Alkyl, -OH, C 1-6 Alkoxy, halogen, amino, acetamino, oxo, and nitro groups. In yet another embodiment, the substituents are independently selected from C10. 1-6 Alkyl, C 1-6 Alkoxy, halogen, acetamino, and nitro groups. As used herein, when the substituent is alkyl or alkoxy, the carbon chain can be branched, linear, or cyclic.

[0442] The term "alkylene" refers to a divalent group formed by removing one hydrogen atom from an alkyl group, which may be substituted or unsubstituted. The "alkylene" is preferably a divalent group of a straight-chain or branched saturated aliphatic hydrocarbon containing 1 to 8 carbon atoms, more preferably a divalent alkyl group containing 1 to 6 carbon atoms (C1 to C2). 1-6 Alkylenes, examples of which include, but are not limited to, -CH2-, -CH(CH3)-, -CH2CH2-, -CH(CH3)CH2-, -CH2CH2CH2- or -(CH2)4- and their stereoisomers.

[0443] The terms "heterocyclic" and "heterocyclic group" are used interchangeably to refer to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon group, wherein one or more (preferably 1 to 4, 1 to 3, or 1 to 2) ring atoms are selected from nitrogen, oxygen, or S(O). t3 The heteroatom (where t3 is an integer from 0 to 2) is a heteroatom, excluding the ring portion of -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. The term "heterocyclic group" preferably refers to a 3- to 6-membered heterocyclic alkyl group; wherein one or more (preferably 1 to 4) ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O)t3 (where t3 is an integer from 0 to 2), excluding the ring portion of -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. The nitrogen atom may be substituted or unsubstituted (i.e., N or NR, R being hydrogen or any substituent already defined herein). The ring carbon atoms of the heterocyclic group may optionally be substituted with 1, 2, or 3 oxo groups to form a cyclic ketone, cyclic lactone, or cyclic lactam structure.

[0444] The term "heteroatom" is selected from nitrogen, oxygen, or sulfur. Nitrogen may optionally be substituted; sulfur may also optionally be substituted, for example, by oxidation, thus forming S(O). t3 (where t3 is an integer from 0 to 2).

[0445] The term "aryl" refers to phenyl or naphthyl, or phenyl or naphthyl substituted with the following groups: halogen, C 1-8 Alkyl, hydroxyl, nitro, trifluoromethyl, etc. Phenyl or monosubstituted phenyl groups are preferred; phenyl is the most preferred.

[0446] The term "solvent" as used in this disclosure refers to a complex formed by the compound of this disclosure with a solvent. These complexes either react in the solvent or precipitate or crystallize out of the solvent. For example, a complex formed with water is called a "hydrate". Solvents of the compounds represented by formula (I) of this disclosure are within the scope of this disclosure.

[0447] This disclosure includes prodrugs of the aforementioned compounds. The prodrugs include known amino and carboxyl protecting groups, which are released under physiological conditions by hydrolysis or via enzymatic reactions to yield the parent compound. Specific methods for preparing the prodrugs can be found in (Saulnier, MG; Frenneson, DB; Deshpande, MS; Hansel, SB and Vysa, DMB Bioorg. Med. Chem Lett. 1994, 4, 1985-1990; and Greenwald, RB; Choe, YH; Conover, CD; Shum, K.; Wu, D.; Royzen, MJ Med. Chem. 2000, 43, 475.).

[0448] As used in this article, the term "hydroxyl group" refers to -OH.

[0449] As used in this article, the term "cyano" refers to -CN.

[0450] As used in this article, the term "nitro" refers to -NO2.

[0451] As used in this article, the term "oxo" refers to =O.

[0452] As used in this article, the term "carboxyl group" refers to -C(=O)OH.

[0453] As used in this article, the term "acetyl (Ac)" refers to -COCH3.

[0454] As used in this article, the term "aldehyde group" refers to -C(=O)H.

[0455] As used in this article, the term "ethynyl" refers to -CH≡C.

[0456] As used in this article, the term "vinyl" refers to -CH=CH2.

[0457] As used herein, the term "propylene" refers to CH2=C=CH2, whose structure or representation is as follows:

[0458] As used in this article, the term "azido group" refers to -N3.

[0459] As used in this article, the term "isocyanate group" refers to -N=C=O.

[0460] The term "hydrophilic group" refers to chemical structures that enhance the solubility of a molecule in water. By forming hydrogen bonds or other interactions with water molecules, it increases the overall hydrophilicity of the molecule, thereby improving its solubility in water. In the design of ADCs (Advanced Drug Acquisition Factor) drugs, the introduction of hydrophilic groups helps to improve the overall hydrophilicity of the ADC molecule, thus improving the solubility and stability of the ADC. The introduction of hydrophilic groups also helps to improve the pharmacokinetic properties of ADC drugs, such as increasing stability in blood circulation and reducing aggregate formation.

[0461] The types of hydrophilic groups can be diverse, such as polyethylene glycol (PEG) chains, sugar chains, and polysarcosine. They improve the physicochemical properties and biological behavior of ADCs by increasing the hydrophilicity of the molecule. For example, the hydrophilicity of linkers can be improved by introducing PEG groups, or by increasing PEG chains or sugar chains (glucosamine), thereby improving the physicochemical properties of the drug.

[0462] The term "polyhydroxy group" is one type of hydrophilic group, including monosaccharides, disaccharides, and polysaccharides, and their stereoisomers or mixtures thereof. Exemplary monosaccharides may be C5 or C6 sugars, and further may include:

[0463] Glucose, ribose, galactose, mannose, arabinose, 2-deoxyglucose, glyceraldehyde, erythrose, threose, xylose, lysose, allose, adroose, gulose, idoleose, deoxyribose, fructose, amino sugars, uronic acid, nucleotide sugars, dihydroxyacetone, ribulose, xylulose, allulose, sorbose, and tarose, etc.

[0464] Glucuronic acid, aldonic acid, uronic acid, fructonic acid, galactonic acid, mannitol, arabinic acid, and ketonic acid, etc.;

[0465] Or glucosamine, N-acetylglucosamine, galactosamine, N-glucosamine sulfate, N-acetymannosamine, fucose, rhamnosamine, fructosamine, N-acetyhexosamine, N-methylglucosamine, chitosan oligosamine, and N-acetylglucosamine, etc.

[0466] The amino acid sequences disclosed herein contain conventional single-letter or three-letter codes for naturally occurring amino acids, as well as generally accepted three-letter codes for other amino acids, such as Gly (α-aminoacetic acid) or G (glycine). The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

[0467] An amino acid is a molecule that contains both amino and carboxyl functional groups. In α-amino acids, the amino and carboxyl groups are attached to the same carbon atom (α-carbon). The α-carbon may also have one or two additional organic substituents. Amino acids include L and D isomers and racemic mixtures.

[0468] The term "antibody-drug conjugate (ADC)" refers to a small molecule drug with biological activity linked to an antibody via a chemical link. The antibody then acts as a carrier to target and deliver the small molecule drug to the target cell.

[0469] The term "antibody" as used in this disclosure refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in its heavy chain, it can be further divided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ chains or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ chain or a λ chain.

[0470] Typically, the compounds disclosed herein, or their pharmaceutically acceptable salts, solvates, stereoisomers, prodrugs, or antibody-drug conjugates, can be formulated with one or more pharmaceutical carriers to form suitable dosage forms for administration. These dosage forms are suitable for oral, rectal, topical, intraoral, and other non-gastrointestinal administration (e.g., subcutaneous, intramuscular, intravenous, etc.). For example, suitable dosage forms for oral administration include capsules, tablets, granules, and syrups. The compounds of this disclosure contained in these formulations can be solid powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; water-in-oil or oil-in-water emulsions, etc. The above dosage forms can be prepared from the active compound and one or more carriers or excipients using conventional pharmaceutical methods. The carriers described above need to be compatible with the active compound or other excipients. For solid dosage forms, commonly used non-toxic carriers include, but are not limited to, mannitol, lactose, starch, magnesium stearate, cellulose, glucose, and sucrose. Carriers used for liquid dosage forms include water, physiological saline, glucose aqueous solution, ethylene glycol, and polyethylene glycol. The active compound can form solutions or suspensions with the above carriers.

[0471] The compositions disclosed herein are formulated, quantified, and administered in accordance with medical practice guidelines. The "therapeutic effective amount" of the compound administered is determined by factors such as the specific condition to be treated, the individual being treated, the cause of the condition, the target of the drug, and the route of administration.

[0472] As used herein, "therapeutic effective amount" means the amount of the disclosed compound that will elicit a biological or medical response in an individual, such as reducing or inhibiting enzyme or protein activity or improving symptoms, alleviating symptoms, slowing or delaying disease progression, or preventing disease.

[0473] As used herein, “pharmaceuticalally acceptable carrier” means a non-toxic, inert, solid, or semi-solid substance or liquid filling machine, diluent, encapsulation material, or excipient or any type of excipient that is compatible with patients, preferably mammalian, more preferably human, and suitable for delivering an active agent to a target site without terminating the agent’s activity.

[0474] As used in this article, "patient" or "subject" refers to an animal, preferably a mammal, and more preferably a human. The term "mammal" refers to warm-blooded vertebrate mammals, including animals such as cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, rats, pigs, and humans.

[0475] As used in this article, the term "disease" is a state of health in an animal in which the animal is unable to maintain homeostasis and the animal's health continues to deteriorate if the disease does not improve.

[0476] As used herein, “treatment” means to reduce, slow the progression of, attenuate, prevent, or maintain an existing disease or condition (e.g., cancer). Treatment also includes curing, preventing the development of, or reducing to some extent one or more symptoms of a disease or condition. As used herein, the term “treatment” or “treatment” is defined as the administration or application of a therapeutic agent, namely a compound of this disclosure (alone or in combination with another pharmaceutical agent), or the administration or application of a therapeutic agent to an isolated tissue or cell from a patient who suffers from a disease or condition considered herein, a sign or symptom of a disease or condition considered herein, or has the potential to develop a disease or condition considered herein, with the aim of curing, resolving, alleviating, reducing, altering, remedying, improving, ameliorating, or influencing the disease or condition considered herein, a sign or symptom of a disease or condition considered herein, or the possibility of developing a disease or condition considered herein. Such treatments can be specifically tailored or modified based on knowledge obtained from the field of pharmacogenomics. As used herein, the term “treatment” for a disease refers to reducing the frequency or severity of at least one sign or symptom of a disease or condition experienced by a subject.

[0477] As used in this article, the term "cancer" refers to any of the various types of malignant tumors, most of which invade surrounding tissues, can metastasize to several sites, and may recur after attempted resection, leading to the patient's death unless adequately treated. As used in this article, tumor formation includes cancer. Representative cancers include squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, hepatocellular carcinoma and renal cell carcinoma, bladder cancer, colorectal cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, head cancer, kidney cancer, liver cancer, lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, and stomach cancer; leukemia, including non-acute and acute leukemia, such as acute myeloid leukemia, acute lymphoblastic leukemia, acute promyelocytic leukemia (APL), acute T-cell lymphoblastic leukemia, T-cell acute lymphoblastic leukemia (T-ALL), adult T-cell leukemia, basophilic leukemia, eosinophilic leukemia, granulocytic leukemia, hairy cell leukemia, leukopenic leukemia, lymphocytic leukemia, lymphocytic leukemia, megakaryocytic leukemia, microcytic leukemia, monocytic leukemia, neutrophilic leukemia, and stem cell leukemia; Benign and malignant lymphomas, particularly Burkitt lymphoma and non-Hodgkin's lymphoma; benign and malignant melanomas; myeloproliferative disorders; sarcomas, including Ewing sarcoma, angiosarcoma, Kaposi's sarcoma, liposarcoma, sarcoma, myoma, peripheral neuroepithelial tumor, synovial sarcoma, glioma, astrocytoma, oligodendroglioma, ependymoma, glioblastoma, neuroblastoma, ganglioneuroma, ganglioglioma, medulloblastoma, pineal cell carcinoma, hemangioma, meningeal sarcoma, neurofibroma, and schwannoma; colorectal cancer, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, esophageal cancer, pancreatic cancer, gastric cancer, liver cancer, colon cancer, melanoma; carcinosarcoma, Hodgkin's disease, nephroblastoma, and teratoma, etc., can be treated with one or more of the compounds disclosed herein.

[0478] The disease or condition is "reduced" if the severity of the signs or symptoms of the disease or condition, the frequency with which the patient experiences such signs or symptoms, or both are reduced.

[0479] As used herein, the term "inhibition" means the inhibition or blocking of activity or function by at least about 10 percent relative to a control. Preferably, the activity is inhibited or blocked by 50%, more preferably 75%, and even more preferably 95% or more compared to a control.

[0480] The term "pharmaceutical composition" refers to a mixture of at least one compound that can be used in this disclosure with a pharmaceutically acceptable carrier. This pharmaceutical composition facilitates the administration of the compound to a patient or subject. Various techniques for administering compounds exist in the art, including but not limited to intravenous, oral, aerosol, parenteral, ocular, pulmonary, and topical administration. The terms "pharmaceutical composition," "therapeutic composition," "therapeutic formulation," or "pharmaceuticalally acceptable formulation" can refer to, but are by no means limited to, compositions or formulations that allow for the effective distribution of the pharmaceutical agents provided in this disclosure, in a form suitable for administration to the physical site most optimal for their desired activity, such as systemic administration.

[0481] "Therapeutic" treatment refers to treatment applied to a subject who exhibits signs or symptoms of a pathological disease or condition, with the aim of reducing or eliminating those signs or symptoms.

[0482] The abbreviation mpk stands for milligrams per kilogram, which refers to the number of milligrams of drug administered per kilogram of animal body weight.

[0483] Tmab (Trastuzumab) is a monoclonal antibody targeting the HER2 receptor. In ADC drugs, it acts as a "navigation system," specifically recognizing and binding to the HER2 receptor on the surface of tumor cells. This targeted delivery is achieved through antigen-antibody binding, reducing damage to healthy tissues and mediating the entry of the ADC into tumor cells for the treatment of HER2-positive cancers. In this disclosure, Tmab may also be referred to as T-mab; both Tmab and T-mab refer to trastuzumab.

[0484] GGFG is a cleavable tetrapeptide linker (Gly-Gly-Phe-Gly) composed of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly). Its key characteristic is its stability in the bloodstream, reducing the risk of premature toxin release and thus minimizing side effects. This GGFG linker is cleaved by proteases (such as phospholipids) within lysosomes, releasing the toxin and exerting its anti-tumor effect.

[0485] Tmab+GGFG-Dxd refers to an antibody-drug conjugate formed by Tmab, GGFG, and Dxd.

[0486] Dxd is a cytotoxic drug, belonging to the class of topoisomerase I inhibitors. Dxd induces DNA damage by inhibiting key enzymes in the DNA replication and transcription process, leading to cancer cell death, and also has a "bystander effect"—it can spread to nearby antigen-negative cells, expanding the killing range.

[0487] +GGFG-Dxd: This trade name refers to a novel antibody-drug conjugate (ADC) composed of a humanized anti-monoclonal antibody conjugated to a topoisomerase I inhibitor (camptothecin derivative) via a cleavable linker. The antibody (Ab) targets the HER2 receptor, the linker optimizes drug stability and homogeneity, and the payload (D) is rapidly released intracellularly, thereby enhancing anti-antibody activity and reducing systemic toxicity.

[0488] Claudin-18 isoform 2 (Claudin 18.2 or CLDN18.2) is a highly selective cell lineage marker. Its expression in normal tissues is strictly limited to epithelial cells differentiated from the gastric mucosa, but not to gastric stem cell regions. CLDN18.2 is expressed in a significant proportion of primary gastric cancers and retains its expression levels in metastatic gastric cancer tissues. In addition to gastric cancer, CLDN18.2 expression has also been found in pancreatic cancer, making it an ideal target molecule for the treatment of these cancers.

[0489] In this disclosure, there is no substantial difference in the different symbolic representations of the same antibody-drug conjugate. For example, the product obtained by conjugating SJ-LP20 with T-mab can be arbitrarily written as T-mab-SJ-LP20, Tmab+SJ-LP20, or Tmab-SJ-LP20, all referring to the same molecule.

[0490] Example

[0491] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions of the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. The following is merely a further description of this disclosure, and the scope of protection of this disclosure is not limited thereto. Unless otherwise stated, the methods and materials of the embodiments, experimental examples, and other examples described below are all conventional products that can be purchased from the market.

[0492] The experimental conditions for Examples 1-15 of this application are as follows:

[0493] Reagent instructions:

[0494] The antibody T-mab was prepared in-house based on the published sequence.

[0495] Both VC-MMAE and GGFG-DXD were obtained through market purchases.

[0496] Hydrophobic interaction chromatography-high performance liquid chromatography (SEC-HPLC) conditions

[0497] Instrument model: Agilent 1260HPLC

[0498] Chromatographic column: Advance Bio SEC 300A 2.7μm 4.6×150mm

[0499] Detector: VWD 280nm

[0500] Mobile phase: 100 mM PB, pH 7.0

[0501] Elution gradient: isocratic elution

[0502]

[0503] Sample loading amount: 15 μg

[0504] Size exclusion chromatography-high performance liquid chromatography (HIC-HPLC) conditions

[0505] Instrument model: Agilent 1260HPLC

[0506] Chromatographic column: TSKgel Butyl-NPR 4.6*3.5mm 2.5μm

[0507] Detector: VWD 214nm

[0508] Mobile phase: Buffer A: 1.5M (NH4)2SO4 in 50mM PB, pH 7.0

[0509] Buffer B: 50mM PB+20% IPA, pH7.0

[0510] Elution gradient:

[0511]

[0512] Sample loading amount: 8 μg

[0513] Liquid chromatography-mass spectrometry conditions for LC-MS molecular weight analysis:

[0514] Sample preparation: Dilute the sample to 1 mg / mL with ultrapure water, take 50 μL and add 2 U PF enzyme (glycosidase F derived from Elizabethan bacillus meningoseptica) and incubate overnight in a metal bath at 37°C for 16 h.

[0515] The ADC sample was subjected to LC-MS molecular weight analysis under the following chromatographic conditions:

[0516] Instrument model: Agilent 1290U HPLC

[0517] Column: AdvanceBio SEC 200A 1.9μm, 2.1×150mm

[0518] Mobile phase A: Ultrasound Water

[0519] Mobile phase B: 250mM ammonium acetate

[0520] Column temperature: Uncontrolled column temperature

[0521] Elution gradient: 80% A + 20% B, isocratic elution at 0.06 mL / min

[0522] The mass spectrometry conditions are as follows:

[0523] Mass spectrometer model: 6545XT

[0524] Mass spectrometry conditions:

[0525]

[0526] Example 1: Synthesis of SJ-LP1

[0527] Synthesis route:

[0528]

[0529] Synthesis of (9H-fluorene-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-(((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)1-oxobutan-2-yl)carboxylate:

[0530] Synthesis of (9H-fluorene-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-(((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)1-oxobutan-2-yl)carboxylate:

[0531]

[0532] (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate 1 (5.00 g, 8.31 mmol) and 4-nitrophenyl[(4-nitrophenyl)oxy]carbamate (7.58 g, 24.93 mmol) were dissolved in N,N-dimethylformamide (100 mL), and N,N-diisopropylethylamine (3.22 g, 24.93 mmol) was added. The reaction was stirred at room temperature for 3 hours. The reaction was confirmed by TLC. The reaction solution was filtered, concentrated, and subjected to column chromatography (DCM:MeOH = 10:1) to give a white solid (9H-fluorene-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-(((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)1-oxobutan-2-yl)carbamate 2 (4.00 g, 5.22 mmol, 63% yield). This was used directly in the next step.

[0533] Synthesis of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)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] indo[1,2-b]quinoline-1-yl)carbamate:

[0534]

[0535] (9H-fluorene-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-(((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)1-oxobut-2-yl)carbamate 2 (600.00 mg, 0.78 mmol), eczemabium methanesulfonate (340.73 mg, 0.78 mmol), HOBt (126.88 mg, 0.93 mmol), and DIPEA (201.88 mg, 1.57 mmol) were dissolved in DMF (10 mL), and the reaction was stirred at room temperature for 12 hours. The reaction was detected by LCMS. The reaction solution was filtered, concentrated, and subjected to column chromatography (DCM:MeOH = 20:1) to give a white solid 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)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] indo[1,2-b]quinoline-1-yl)carbamate 3 (500.00 mg, 0.47 mmol, 60% yield).

[0536] LC-MS: 1 / 2[M+2H]+: 532.4

[0537] Synthesis of 4-((S)-2-((S)-2-amino-3-methylbutamido)-5-ureidopentamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indo[1,2-b]quinoline-1-yl)carbamate:

[0538]

[0539] 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)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] indo[1,2-b]quinoline-1-yl)carbamate 3 (400.00 mg, 0.38 mmol) and diethylamine (0.10 mL, 0.94 mmol) were dissolved in N,N-dimethylformamide (10 mL), and the reaction was stirred at room temperature for 2 hours. The reaction was detected by LCMS. The reaction solution was filtered, concentrated, and subjected to column chromatography (DCM:MeOH = 20:1) to give a white solid 4-((S)-2-((S)-2-amino-3-methylbutamido)-5-ureidopentamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indo[1,2-b]quinoline-1-yl)carbamate 4 (240.00 mg, 0.29 mmol, 76% yield).

[0540] LC-MS:[M+H]+:841.2

[0541] Synthesis of 8-(2-((tert-Butoxycarbonyl)amino)ethyl)-2,2-dimethyl-4,9-dioxo-3,11-dioxo-5,8-diazatriadecane-13-acid:

[0542]

[0543] Di-tert-butyl(azadiylbis(ethane-2,1-diyl)) dicarboxylate 5-1 (25.00 g, 82.40 mmol) and 1,4-dioxane-2,6-dione 5-2 (9.56 g, 82.40 mmol) were dissolved in THF (100 mL), and the reaction was stirred at 80 °C for 12 hours. The reaction was confirmed by LCMS. The reaction solution was filtered, concentrated, and subjected to column chromatography (DCM:MeOH = 10:1) to give a colorless, transparent oily substance 8-(2-((tert-butoxycarbonyl)amino)ethyl)-2,2-dimethyl-4,9-dioxo-3,11-dioxo-5,8-diazatridecane-13-acid 5-3 (25.00 g, 59.60 mmol, 72% yield).

[0544] LC-MS: [M+Na]+: 442.2

[0545] Synthesis of tert-butyl(S)-19-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-8-(2-((tert-butoxycarbonyl)amino)ethyl)-2,2-dimethyl-4,9,13-trioxo-3,11-dioxa-5,8,14-triazolidine-20-ester:

[0546]

[0547] 8-(2-((tert-Butoxycarbonyl)amino)ethyl)-2,2-dimethyl-4,9-dioxo-3,11-dioxo-5,8-diazatridecane-13-acid 5-3 (5.00 g, 11.92 mmol), tert-butyl(((9H-fluorene-9-yl)methoxy)carbonyl)-L-lysine hydrochloride 5-4 (5.50 g, 11.92 mmol), HATU (4.99 g, 13.11 mmol), and DIPEA (3.24 g, 25.03 mmol) were dissolved in N,N-dimethylformamide (50 mL), and the reaction was stirred at room temperature for 3 hours. The reaction was detected by LCMS. The reaction solution was filtered, concentrated, and subjected to column chromatography (DCM:MeOH = 20:1) to give a white solid tert-butyl(S)-19-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-8-(2-((tert-butoxycarbonyl)amino)ethyl)-2,2-dimethyl-4,9,13-trioxo-3,11-dioxa-5,8,14-triazolidine-20-ester 5-5 (7.00 g, 8.48 mmol, 71% yield).

[0548] LC-MS:[M+H]+:826.6

[0549] Synthesis of N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(2-(2-(bis(2-aminoethyl)amino)-2-oxoethoxy)acetyl)-L-lysine:

[0550]

[0551] 7.00 g (8.48 mmol) of tert-butyl(S)-19-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-8-(2-(((tert-butoxycarbonyl)amino)ethyl)-2,2-dimethyl-4,9,13-trioxo-3,11-dioxa-5,8,14-triazolidine-20-ester 5-5 was dissolved in DCM (100 mL), and TFA (25 mL) was added. The reaction was stirred at room temperature for 2 hours. The reaction was confirmed by LCMS. After concentration, the reaction solution yielded a white solid N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(2-(2-(bis(2-aminoethyl)amino)-2-oxoethoxy)acetyl)-L-lysine 5-6 (4.00 g, 7.02 mmol, 83% yield).

[0552] LC-MS:[M+H]+:570.4

[0553] Synthesis of (2R, 3R, 4R, 5S, 21S)-21-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-10-(2-(bis((2S, 3R, 4R, 5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)-1,2,3,4,5-pentahydroxy-11,15-dioxo-7-((2S, 3R, 4R, 5R)-23,4,5,6-pentahydroxyhexyl)-13-oxa-7,10,16-triazacosanodecane-22-acid:

[0554]

[0555] N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(2-(2-(bis(2-aminoethyl)amino)-2-oxoethoxy)acetyl)-L-lysine 5-6 (2.00 g, 3.51 mmol), (2R,3S,4R,5R)-2,3,4-5,6-pentahydroxyhexanal 5-7 (6.33 g, 35.11 mmol), NaBH3CN (1.76 g, 28.09 mmol), and AcOH (1.04 g, 17.55 mmol) were dissolved in MeOH (20 mL), and the reaction was stirred at 90 °C for 12 hours. The reaction was confirmed by LCMS. The reaction solution was filtered and subjected to column chromatography / C18 (0.1% trifluoroacetic acid: acetonitrile = 3:1). After concentration, a white solid (2R,3R,4R,5S,21S)-21-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-10-(2-(bis((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)-1,2,3,4,5-pentahydroxy-11,15-dioxo-7-((2S,3R,4R,5R)-23,4,5,6-pentahydroxyhexyl)-13-oxa-7,10,16-triazacosano-22-acid 5 (600.00 mg, 0.49 mmol, 14% yield) was obtained.

[0556] LC-MS: 1 / 2[M+2H]+: 614.0

[0557] 4-((2S,5S,8S,24S,25R,26R,27R)-8-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2, Synthesis of 3,4,5,6-pentahydroxyhexyl)-2-(3-ureopropyl)-16-oxa-3,6,13,19,22-pentazaoctacosanamino)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indo[1,2-b]quinoline-1-yl)carbamate:

[0558]

[0559] 4-((S)-2-((S)-2-amino-3-methylbutamido)-5-ureidopentamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indene[1,2-b]quinoline-1-yl)carbamate 4 (137.15 mg, 0.16 mmol), (2R,3R,4R,5S,21S)-21-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-10-(2-(bis(((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl) -1,2,3,4,5-pentahydroxy-11,15-dioxo-7-((2S,3R,4R,5R)-23,4,5,6-pentahydroxyhexyl)-13-oxa-7,10,16-triazacosico ... The reaction was confirmed by LCMS. The reaction solution was filtered, and subjected to column chromatography / C18 (0.1% trifluoroacetic acid: acetonitrile = 3:1). After lyophilization, a white solid was obtained: 4-((2S,5S,8S,24S,25R,26R,27R)-8-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-4,7,14,18-tetraoxo-22-((2S... ,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-2-(3-ureopropyl)-16-oxa-3,6,13,19,22-pentazaoctacosanamino)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indene[1,2-b]quinoline-1-yl)carbamate 6 (90.00 mg, 0.044 mmol, 27% yield).

[0560] LC-MS: [M+2H]+ / 2: 1024.2

[0561] 4-((2S,5S,8S,24S,25R,26R,27R)-8-amino-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxy Synthesis of (hexyl)-2-(3-ureopropyl)-16-oxa-3,6,13,19,22-pentazazole octamido)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] indo[1,2-b]quinoline-1-yl)carbamate:

[0562]

[0563] 4-((2S,5S,8S,24S,25R,26R,27R)-8-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-2-(3-ureopropyl)-16-oxa-3,6, 13,19,22-pentazaoctacosanamino)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indo[1,2-b]quinoline-1-yl)carbamate 6 (90.00 mg, 0.044 mmol) was dissolved in N,N-dimethylformamide (1 mL), and diethylamine (32.06 mg, 0.44 mmol) was slowly added. The reaction was stirred at room temperature for 1 hour. LCMS analysis confirmed the reaction was complete. The reaction solution was concentrated to obtain the crude product 4-((2S,5S,8S,24S,25R,26R,27R)-8-amino-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl) 2-(3-Uripropyl)-16-oxa-3,6,13,19,22-pentazazole octamido)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]indo[1,2-b]quinoline-1-yl)carbamate 7 (80.00 mg, 0.044 mmol, 99% yield). Used directly in the next step.

[0564] LC-MS: 1 / 3 [M+3H]+: 609.7

[0565] 4-((2S,5S,8S,24S,25R,26R,27R)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-8-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-24,25,26,27,28-pentahydroxy-5-isopropyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6 ... Synthesis of R)-2,3,4,5,6-pentahydroxyhexyl)-2-(3-ureidopropyl)-16-oxa-3,6,13,19,22-pentazaoctacosanamino)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] indo[1,2-b]quinoline-1-yl)carbamate:

[0566]

[0567] 4-((2S,5S,8S,24S,25R,26R,27R)-8-amino-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-2-(3-ureopropyl)-16-oxa-3,6,13,19,22-pentazazole octamido)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] indo[1,2-b]quinoline-1-yl)carbamate 7 (80.00 mg, 0.044 mmol) and 2,5-dioxopyrrolidine-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoate 8 (13.56 mg, 0.044 mmol) were dissolved in N,N-dimethylformamide (1 mL), and N,N-diisopropylethylamine (12.09 mg, 0.090 mmol) was added. The reaction was stirred at room temperature for 2 hours. LCMS analysis confirmed the reaction was complete. The reaction solution was adjusted to weakly acidic with formic acid, filtered, and separated (formic acid system). After lyophilization, a white solid was obtained: 4-((2S,5S,8S,24S,25R,26R,27R)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-8-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-24,25,26,27,28-pentahydroxy-5-isopropyl-4,7,14,18-tetraoxo-22-((2 S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-2-(3-ureopropyl)-16-oxa-3,6,13,19,22-pentazaoctacosanamino)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]indo[1,2-b]quinoline-1-yl)carbamate SJ-LP1 (2.10 mg, 0.0010 mmol, 2% yield).

[0568] LC-MS: 1 / 3[M+3H]+: 674.2;

[0569] The retention time Rt of SJ-LP1 by LC-MS was 1.13 min.

[0570] Example 2: Synthesis of SJ-LP3

[0571] Synthesis route:

[0572]

[0573] 4-((2S,5S,8S,24S,25R,26R,27R)-8-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18-tetraoxo-22-((2S,3R,4R)... Synthesis of (5R)-2,3,4,5,6-pentahydroxyhexyl)-16-oxa-3,6,13,19,22-pentazaoctacosanamido)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]indolo[1,2-b]quinoline-1-yl)carbamate:

[0574]

[0575] 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)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]indolo[1,2-b]quinoline-1-yl)carbamate 6 (100.00 mg, 0.13 mmol), (2R,3R,4R,5S,21S)-21-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-10-(2-(bis(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-1, 2,3,4,5-Pentahydroxy-11,15-dioxo-7-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-13-oxa-7,10,16-triazapolysaccharide-22-acid 7 (178.71 mg, 0.15 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (75.51 mg, 0.20 mmol), and benzo[d][1,2,3]triazole-1-ol (26.83 mg, 0.20 mmol) were dissolved in N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (34.18 mg, 0.27 mmol) was added. The reaction was stirred at room temperature for 2 hours. The reaction was confirmed by LCMS. The reaction solution was filtered and subjected to column chromatography / C18 (0.1% trifluoroacetic acid: acetonitrile = 3:1). After concentration, a white solid was obtained: 4-((2S,5S,8S,24S,25R,26R,27R)-8-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18-tetraoxo- 22-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-16-oxa-3,6,13,19,22-pentazaoctacosanamido)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]indolo[1,2-b]quinoline-1-yl)carbamate 8 (10.00 mg, 0.0050 mmol, 99% yield).

[0576] LC-MS: 1 / 2[M+2H]+: 982.6

[0577] 4-((2S,5S,8S,24S,25R,26R,27R)-8-amino-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3,4,5 Synthesis of (6-pentahydroxyhexyl)-16-oxo-3,6,13,19,22-pentazaoctacosanamino)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indo[1,2-b]quinoline-1-yl)carbamate:

[0578]

[0579] 4-((2S,5S,8S,24S,25R,26R,27R)-8-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-16-oxa-3,6,13,1 9,22-pentazaoctadecanoamide)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]indolo[1,2-b]quinoline-1-yl)carbamate 8 (10.00 g, 0.0050 mmol) was dissolved in N,N-dimethylformamide (1 mL), and diethylamine (3.72 mg, 0.050 mmol) was slowly added. The reaction was stirred at room temperature for 1 hour. LCMS analysis confirmed the reaction was complete. The reaction solution was concentrated to obtain the crude product 4-((2S,5S,8S,24S,25R,26R,27R)-8-amino-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3,4,5,6) -Pentahydroxyhexyl)-16-oxo-3,6,13,19,22-pentazaoctacosanamino)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indo[1,2-b]quinoline-1-yl)carbamate 9 (8.00 mg, 0.0050 mmol, 90% yield).

[0580] LC-MS: 1 / 2[M+2H]+: 871.2

[0581] 4-((2S,5S,8S,24S,25R,26R,27R)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-8-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexylamino)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18-tetraoxo-22-((2S,3R) Synthesis of (4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-16-oxo-3,6,13,19,22-pentazaoctacosamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7] indo[1,2-b]quinoline-1-yl)carbamate:

[0582]

[0583] 4-((2S,5S,8S,24S,25R,26R,27R)-8-amino-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-16-oxo-3,6,13,19,22-pentazaoctacosanamino)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10 ,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7] indo[1,2-b]quinoline-1-yl)carbamate 9 (8.00 mg, 0.0050 mmol) and 2,5-dioxopyrrolidone-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoate 10 (1.42 mg, 0.0050 mmol) were dissolved in N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (1.19 mg, 0.010 mmol) was added. The reaction was stirred at room temperature for 2 hours. LCMS analysis confirmed the reaction was complete. The reaction solution was adjusted to weakly acidic with formic acid, filtered, and preparatively separated (formic acid system). After lyophilization, a white solid was obtained: 4-((2S,5S,8S,24S,25R,26R,27R)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-8-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexylamino)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18-tetraoxo-2 2-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-16-oxo-3,6,13,19,22-pentazaoctacosamide)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indo[1,2-b]quinoline-1-yl)carbamate SJ-LP3 (0.95 mg, 0.0010 mmol, 11% yield).

[0584] LC-MS: 1 / 2[M+2H]+: 967.4;

[0585] The retention time Rt of SJ-LP3 by LC-MS was 1.14 min.

[0586] Example 3: Synthesis of SJ-LP5

[0587] Synthesis route:

[0588]

[0589] Synthesis of 3-(bis(2-((tert-butoxycarbonyl)amino)ethyl)amino)propionate benzyl ester):

[0590]

[0591] 1,7-bis-BOC-1,4,7-triazaheptane 1 (5.00 g, 16.48 mmol) and benzyl propionate 2 (4.01 g, 24.72 mmol) were dissolved in methanol (50 mL), and the reaction was stirred at room temperature for 16 hours. The reaction was confirmed by LCMS. The reaction solution was concentrated, and then extracted with ethyl acetate (50 mL) and saturated brine (50 mL). The aqueous phase was back-extracted once with ethyl acetate (30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a yellow oily crude product, 3-(bis(2-((tert-butoxycarbonyl)amino)ethyl)amino)propionate 3 (7.50 g, 16.11 mmol, 98% yield).

[0592] LC-MS:[M+H]+:466.3

[0593] Synthesis of 3-(bis(2-((tert-butoxycarbonyl)amino)ethyl)amino)propionic acid:

[0594]

[0595] 3-(bis(2-((tert-Butoxycarbonyl)amino)ethyl)amino)propionate 3 (3.50 g, 7.52 mmol) was dissolved in methanol (35 mL), and palladium on carbon (0.70 g) was added. The reaction was stirred at room temperature for 2 hours under a hydrogen system. The reaction was confirmed by LCMS. The reaction solution was filtered with diatomaceous earth, and the filter cake was washed three times with methanol (5 mL), filtered, and concentrated to obtain a colorless oily crude product 3-(bis(2-((tert-Butoxycarbonyl)amino)ethyl)amino)propionate 4 (2.50 g, 6.66 mmol, 89% yield).

[0596] LC-MS:[M+H]+:376.2

[0597] Synthesis of (S)-17-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-8-(2-((tert-butoxycarbonyl)amino)ethyl)-2,2-dimethyl-4,11-dioxo-3-oxo-5,8,12-triazaoctadecane-18-ester:

[0598]

[0599] 3-(bis(2-((tert-butoxycarbonyl)amino)ethyl)amino)propionic acid 4 (1.00 g, 2.66 mmol), (((9H-fluorene-9-yl)methoxy)carbonyl)-L-lysine tert-butyl hydrochloride 5 (1.20 g, 2.66 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.20 g, 3.20 mmol), and N,N-diisopropylethylamine (1.00 g, 7.99 mmol) were dissolved in N,N-dimethylformamide (10 mL), and the reaction was stirred at room temperature for 2 hours. The reaction was detected by LC-MS. Water (50 mL) and ethyl acetate (30 mL) were added to the reaction solution. The organic layer was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (dichloromethane:methanol = 8:1). After concentration, a colorless oily (S)-17-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-8-(2-((tert-butoxycarbonyl)amino)ethyl)-2,2-dimethyl-4,11-dioxo-3-oxo-5,8,12-triazaoctadecane-18-ester 6 (1.00 g, 1.28 mmol, 48% yield) was obtained. LC-MS: [M+H]+: 782.4

[0600] Synthesis of N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(3-(bis(2-aminoethyl)amino)propionyl)-L-lysine:

[0601]

[0602] (S)-17-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-8-((2-((tert-butoxycarbonyl)amino)ethyl)-2,2-dimethyl-4,11-dioxo-3-oxo-5,8,12-triazaoctadecane-18-ester 6 (200.00 mg, 0.26 mmol) was dissolved in dichloromethane (4 mL), and trifluoroacetic acid (1 mL) was slowly added. The reaction was stirred at room temperature for 1 hour. The reaction was confirmed to be complete by LCMS. The reaction solution was concentrated to obtain a colorless oily crude product N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(3-(bis(2-aminoethyl)amino)propionyl)-L-lysine 7 (130.00 mg, 0.25 mmol, 97% yield).

[0603] LC-MS:[M+H]+:526.3

[0604] Synthesis of N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(3-(bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)amino)propionyl)-L-lysine:

[0605]

[0606] N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(3-(bis(2-aminoethyl)amino)propionyl)-L-lysine 7 (130.00 mg, 0.25 mmol), (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal 8 (801.00 mg, 4.45 mmol), and sodium cyanoborohydride (189.00 mg, 3.00 mmol) were dissolved in methanol (5 mL), and the reaction was heated to 90 °C and stirred for 16 hours under a nitrogen system. The reaction was detected by LCMS. The reaction solution was cooled, concentrated, and water (2 mL) was added. The solution was then subjected to column chromatography on a C18 column (0.1% trifluoroacetic acid: acetonitrile = 5:1). After lyophilization, a white solid N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(3-(bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)amino)propionyl)-L-lysine 9 (200.00 mg, 0.17 mmol, 70% yield) was obtained.

[0607] LC-MS: [M+2H]+ / 2: 591.6

[0608] 4-((2S,5S,8S,22S,23R,24R,25R)-8-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-17-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-22,23,24,25,26-pentahydroxy-5-isopropyl-4,7,14-trioxy-20-((2S,3R,4R,5R)) Synthesis of 2,3,4,5,6-pentahydroxyhexyl)-2-(3-ureidopropyl)-3,6,13,17,20-pentazahexaamino)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]indolo[1,2-b]quinoline-1-yl)carbamate:

[0609]

[0610] N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(3-(bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)amino)propionyl)-L-lysine 9 (180.00 mg, 0.15 mmol), 4-((S)-2-((S)-2-amino-3-methylbutamido)-5-ureidopentamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12 H-benzopyran[3',4':6,7]indolo[1,2-b]quinoline-1-yl)carbamate 10 (128.00 mg, 0.15 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (86.80 mg, 0.23 mmol), benzo[d][1,2,3]triazol-1-ol (30.90 mg, 0.23 mmol), and N,N-diisopropylethylamine (39.30 mg, 0.30 mmol) were dissolved in N,N-dimethylformamide (5 mL), and the reaction was stirred at room temperature for 2 hours. The reaction was confirmed by LCMS. The reaction solution was filtered, and subjected to column chromatography / C18 (0.1% trifluoroacetic acid: acetonitrile = 4:1). After lyophilization, a white solid was obtained: 4-((2S,5S,8S,22S,23R,24R,25R)-8-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-17-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-22,23,24,25,26-pentahydroxy-5-isopropyl-4,7,14-trioxy-20-((2 S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-2-(3-ureopropyl)-3,6,13,17,20-pentazahexaamino)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]indolo[1,2-b]quinoline-1-yl)carbamate 11 (120.00 mg, 0.06 mmol, 39% yield).

[0611] LC-MS: [M+2H]+ / 2:1003.6

[0612] 4-((2S,5S,8S,22S,23R,24R,25R)-8-amino-17-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-22,23,24,25,26-pentahydroxy-5-isopropyl-4,7,14-trioxy-20-((2S,3R,4R,5R)-2,3,4,5,6) Synthesis of (-pentahydroxyhexyl)-2-(3-ureapropyl)-3,6,13,17,20-pentazahexaamino)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]indolo[1,2-b]quinoline-1-yl)carbamate:

[0613]

[0614] 4-((2S,5S,8S,22S,23R,24R,25R)-8-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-17-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-22,23,24,25,26-pentahydroxy-5-isopropyl-4,7,14-trioxy-20-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-2-(3-ureopropyl)-3,6,13,1 7,20-pentazahexaamino)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]indolo[1,2-b]quinoline-1-yl)carbamate 11 (100.00 mg, 0.05 mmol) was dissolved in N,N-dimethylformamide (2 mL), and diethylamine (36.40 mg, 0.50 mmol) was slowly added. The reaction was stirred at room temperature for 1 hour. LCMS analysis confirmed the reaction was complete. After concentration, ethyl acetate was added and the mixture was slurried to obtain a gray powder: 4-((2S,5S,8S,22S,23R,24R,25R)-8-amino-17-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-22,23,24,25,26-pentahydroxy-5-isopropyl-4,7,14-trioxy-20-((2S,3R,4R,5R)-2,3,4, 5,6-pentahydroxyhexyl)-2-(3-ureapropyl)-3,6,13,17,20-pentazahexaamino)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]indolo[1,2-b]quinoline-1-yl)carbamate 12 (85.00 mg, 0.05 mmol, 96% yield).

[0615] LC-MS:[M+3H]+ / 3:595.0

[0616] 4-((2S,5S,8S,22S,23R,24R,25R)-17-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-8-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexylamino)-22,23,24,25,26-pentahydroxy-5-isopropyl-4,7,14-trioxy-20-((2S,3R, Synthesis of (4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-2-(3-ureapropyl)-3,6,13,17,20-pentazahexaamino)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7]indolo[1,2-b]quinoline-1-yl)carbamate:

[0617]

[0618] 4-((2S,5S,8S,22S,23R,24R,25R)-8-amino-17-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-22,23,24,25,26-pentahydroxy-5-isopropyl-4,7,14-trioxy-20-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-2-(3-ureapropyl)-3,6,13,17,20-pentazahexaamino)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]indolo[1,2-b]quinoline-1-yl)carbamate 12 (85.00 mg, 0.05 mmol) and 2,5-dioxopyrrolidone-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoate 13 (14.70 mg, 0.05 mmol) were dissolved in N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (12.30 mg, 0.10 mmol) was added. The reaction was stirred at room temperature for 2 hours. LCMS analysis confirmed the reaction was complete. The pH of the reaction solution was adjusted to weakly acidic with formic acid, and the mixture was filtered. The residue was purified (using formic acid) to obtain a white solid: 4-((2S,5S,8S,22S,23R,24R,25R)-17-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-8-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexylamino)-22,23,24,25,26-pentahydroxy-5-isopropyl-4,7,14-trioxy-20- ((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-2-(3-ureapropyl)-3,6,13,17,20-pentazahexaamino)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7]indolo[1,2-b]quinoline-1-yl)carbamate SJ-LP5 (12.00 mg, 0.006 mmol, 13% yield).

[0619] LC-MS:[M+3H]+ / 3:659.4.

[0620] The retention time Rt of SJ-LP5 by LC-MS was 0.99 min.

[0621] Example 4: Synthesis of SJ-LP6

[0622] Synthesis route:

[0623]

[0624] 4-((2S,5S,8S,24S,25R,26R,27R)-8-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)) Synthesis of 2,3,4,5,6-pentahydroxyhexyl)-2-(3-ureapropyl)-3,6,13,19,22-pentazaoctacosanamino)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7]indolo[1,2-b]quinoline-1-yl)carbamate:

[0625]

[0626] N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(5-(bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)amino)-5-oxopentanoyl)-L-lysine 1 (160.00 mg, 0.13 mmol), 4-((S)-2-((S)-2-amino-3-methylbutamido)-5-ureidopentanoyl)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H 12H-benzopyran[3',4':6,7]indolo[1,2-b]quinoline-1-yl)carbamate 2 (109.89 mg, 0.13 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (74.49 mg, 0.20 mmol), benzo[d][1,2,3]triazol-1-ol (26.49 mg, 0.20 mmol), and N,N-diisopropylethylamine (33.72 mg, 0.26 mmol) were dissolved in N,N-dimethylformamide (5 mL), and the reaction was stirred at room temperature for 2 hours. The reaction was confirmed by LCMS. The reaction solution was filtered, and subjected to column chromatography / C18 (0.1% trifluoroacetic acid: acetonitrile = 4:1). After lyophilization, a white solid was obtained: 4-((2S,5S,8S,24S,25R,26R,27R)-8-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-4,7,14,18-tetraoxo-22-(( 2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-2-(3-ureapropyl)-3,6,13,19,22-pentazaoctacosanamino)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7]indolo[1,2-b]quinoline-1-yl)carbamate 3 (140.00 mg, 0.070 mmol, 52% yield).

[0627] LC-MS:[M+3H]+ / 3:683.2

[0628] 4-((2S,5S,8S,24S,25R,26R,27R)-8-amino-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3,4,5,6) Synthesis of (-pentahydroxyhexyl)-2-(3-ureapropyl)-3,6,13,19,22-pentazaoctacosanamino)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]indolo[1,2-b]quinoline-1-yl)carbamate:

[0629]

[0630] 4-((2S,5S,8S,24S,25R,26R,27R)-8-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-2-(3-ureapropyl)-3,6,13, 19,22-pentazaoctacosanamino)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7]indolo[1,2-b]quinoline-1-yl)carbamate 3 (120.00 mg, 0.06 mmol) was dissolved in N,N-dimethylformamide (2 mL), and diethylamine (42.79 mg, 0.59 mmol) was slowly added. The reaction was stirred at room temperature for 1 hour. LCMS analysis confirmed the reaction was complete. After concentration, the reaction solution was slurried with ethyl acetate to obtain a gray powder: 4-((2S,5S,8S,24S,25R,26R,27R)-8-amino-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3,4,5, 6-Pentahydroxyhexyl)-2-(3-Urepropyl)-3,6,13,19,22-pentazaoctacosanamino)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]indolo[1,2-b]quinoline-1-yl)carbamate 4 (100.00 mg, 0.06 mmol, 93% yield).

[0631] LC-MS:[M+3H]+ / 3:609.0.

[0632] 4-((2S,5S,8S,24S,25R,26R,27R)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-8-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamamide)-24,25,26,27,28-pentahydroxy-5-isopropyl-4,7,14,18-tetraoxo-22-((2S,3R,4R) Synthesis of (5R)-2,3,4,5,6-pentahydroxyhexyl)-2-(3-ureapropyl)-3,6,13,19,22-pentazaoctacosanamido)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]indolazino[1,2-b]quinoline-1-yl)carbamate:

[0633]

[0634] 4-((2S,5S,8S,24S,25R,26R,27R)-8-amino-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-2-(3-ureapropyl)-3,6,13,19,22-pentazaoctacosanamino)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)carbamate (100 mg, 0.054 mmol) was dissolved in N,N-dimethylformamide (2 mL), and 2,5-dioxopyrrolidine-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoate 4 (16.89 mg, 0.054 mmol) and N,N-diisopropylethylamine (14.14 mg, 0.11 mmol) were added. The reaction was stirred at room temperature for 2 hours. LCMS analysis confirmed the reaction was complete. The reaction solution was adjusted to weakly acidic with formic acid, filtered, and the residue was purified (in formic acid system) to give a white solid 4-((2S,5S,8S,24S,25R,26R,27R)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-8-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamethylene)-24,25,26,27,28-pentahydroxy-5-isopropyl-4,7,14,18-tetraoxo-22-((2 S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-2-(3-ureapropyl)-3,6,13,19,22-pentazaoctacosamide)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]indolazino[1,2-b]quinoline-1-yl)carbamate SJ-LP6 (13.00 mg, 0.006 mmol, 12% yield).

[0635] LC-MS: [M+3H]+ / 3: 673.4;

[0636] The retention time Rt of SJ-LP6 by LC-MS was 0.99 min.

[0637] Example 5: Synthesis of SJ-LP11

[0638] Synthesis route:

[0639]

[0640] Synthesis of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7] indo[1,2-b]quinoline-11-yl)ethyl)(isopropyl)carbamate:

[0641]

[0642] Dissolve (9H-fluorene-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-(((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropane-2-yl)amino)-1-oxobutane-2-yl)carbamate 1 (500.00 mg, 0.74 mmol) in N,N-dimethylformamide (20 mL), and add (S)-4-ethyl-4-hydroxy-11-(2- (Isopropylamino)ethyl)-1,12-dihydro-14H-pyrano[3',4':6,7] indo[1,2-b]quinoline-3,14(4H)-dione 2 (318.00 mg, 0.74 mmol), 1-hydroxybenzotriazole (123.00 mg, 0.89 mmol), and finally N,N-diisopropylethylamine (0.40 mL, 2.24 mmol) were added. The mixture was purged with nitrogen three times and reacted at room temperature for 2 hours. LCMS was used to monitor complete consumption of the starting materials and detection of the product. The reaction solution was concentrated under reduced pressure and purified by column chromatography (dichloromethane:methanol = 10:1) to give a pale yellow solid product 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(2-(((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7] indo[1,2-b]quinoline-11-yl)ethyl)(isopropyl)carbamate 3 (600.00 mg, 0.62 mmol, 84% yield).

[0643] LC-MS:[M+H]+:975.4

[0644] Synthesis of 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7] indo[1,2-b]quinoline-11-yl)ethyl)(isopropyl)carbamate:

[0645]

[0646] 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7] indo[1,2-b]quinoline-11-yl)ethyl)(isopropyl)carbamate 3 (600.00 mg, 0.62 mmol) was dissolved in N,N-dimethylformamide (10 mL), and diethylamine (452.00 mg, 6.20 mmol) was added. The mixture was purged with nitrogen three times and reacted at room temperature for 2 hours. LCMS was used to monitor complete consumption of the starting material and detection of the product. The reaction solution was concentrated under reduced pressure, slurried with n-hexane, filtered, and the filter cake was concentrated under reduced pressure to finally obtain a pale yellow solid product 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indo[1,2-b]quinoline-11-yl)ethyl)(isopropyl)carbamate 4 (400.00 mg, 0.53 mmol, 86% yield).

[0647] LC-MS:[M+H]+:753.2

[0648] 4-((2S,5S,8S,24S,25R,26R,27R)-8-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-19-(2-(bis((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18-tetraoxo-22 Synthesis of -((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctacosanamino)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3':4,7] indo[1,2-b]quinoline-11-yl)ethyl)(isopropyl)carbamate:

[0649]

[0650] 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7] indene[1,2-b]quinoline-11-yl)ethyl)(isopropyl)carbamate 4 (400.00 mg, 0.53 mmol), N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(5-(bis(2-(bis( (2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)amino)-5-oxopentanoyl)-L-lysine 5 (716.00 mg, 0.58 mmol) was dissolved in N,N-dimethylformamide (20 mL), HATU (243.00 mg, 0.64 mmol), 1-hydroxybenzotriazole (88.00 mg, 0.64 mmol) and N,N-diisopropylethylamine (0.3 mL, 1.59 mmol) were added, and the mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS until completion. The direct reaction solution was purified by reverse-phase reaction with 30% ACN, 70% H₂O, and 0.1% TFA to give a white solid 4-((2S,5S,8S,24S,25R,26R,27R)-8-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-19-(2-(bis((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7 ,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctacosanamino)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3':4,7]indo[1,2-b]quinoline-11-yl)ethyl)(isopropyl)carbamate 6 (600.00 mg, 0.31 mmol, 58% yield).

[0651] LC-MS: 1 / 2 [M+2H]+: 980.2

[0652] 4-((2S,5S,8S,24S,25R,26R,27R)-8-amino-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18-tetraoxo-22-((2S,3R,4R, Synthesis of 5R)-2,3,4,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctacosanamino)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7] indo[1,2-b]quinoline-11-yl)ethyl)(isopropyl)carbamate:

[0653]

[0654] 4-((2S,5S,8S,24S,25R,26R,27R)-8-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-19-(2-(bis(((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3,4,5,6- Pentahydroxyhexyl)-3,6,13,19,22-pentazaoctadecanoamino)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3':4,7]indo[1,2-b]quinoline-11-yl)ethyl)(isopropyl)carbamate 6 (600.00 mg, 0.31 mmol) was added to DMF (10 mL), followed by diethylamine (226.00 mg, 3.10 mmol). The mixture was stirred at room temperature for one hour. LC-MS showed the reaction was complete. The mixture was then evaporated to dryness using an oil pump. 5 mL of dichloromethane was added, and the mixture was ultrasonically slurried and filtered to obtain a gray solid: 4-((2S,5S,8S,24S,25R,26R,27R)-8-amino-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18-tetraoxo-2 2-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctacosanamino)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indo[1,2-b]quinoline-11-yl)ethyl)(isopropyl)carbamate 7 (300.00 mg, 0.18 mmol, 56% yield).

[0655] LC-MS: 1 / 2[M+2H]+: 869.2

[0656] 4-((2S,5S,8S,24S,25R,26R,27R)-19-(2-(bis((2S,3R,4R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-8-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18-tetraoxo Synthesis of -22-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctacosamide)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7] indo[1,2-b]quinoline-11-yl)ethyl)(isopropyl)carbamate:

[0657]

[0658] 4-((2S,5S,8S,24S,25R,26R,27R)-8-amino-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctacosanamino)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14- Dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indo[1,2-b]quinoline-11-yl)ethyl)(isopropyl)carbamate 7 (200.00 mg, 0.17 mmol), 2,5-dioxopyrrolidone-1-yl-6-(2,5-diketone-2,5-dihydro-1H-pyrrolo-1-yl)hexanoate 8 (43.00 mg, 0.14 mmol) were dissolved in N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (0.09 mL, 0.51 mmol) were added. The mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS until completion. The pH was adjusted to weakly acidic with formic acid, and the formic acid was purified to give a white solid 4-((2S,5S,8S,24S,25R,26R,27R)-19-(2-(bis((2S,3R,4R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-8-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18- Tetraoxo-22-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctacosamide)benzyl(2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indo[1,2-b]quinoline-11-yl)ethyl)(isopropyl)carbamate SJ-LP11 (17.00 mg, 0.0080 mmol, 5% yield).

[0659] LC-MS: [M+2H]+ / 2: 965.8;

[0660] The retention time Rt of SJ-LP11 by LC-MS was 1.66 min.

[0661] Example 6: Synthesis of SJ-LP10 and SJ-LP12

[0662] Synthesis route:

[0663]

[0664]

[0665] Synthesis of (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)carbamate:

[0666]

[0667] (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine-L-alanine 1 (2.00 g, 4.87 mmol) was dissolved in methanol (10 mL) and dichloromethane (50 mL). p-Aminobenzyl alcohol (1.80 g, 14.62 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (3.61 g, 14.62 mmol) were added. The mixture was purged three times with nitrogen and reacted at room temperature for 18 hours in the dark. The reaction was confirmed by LCMS. The reaction solution was concentrated under reduced pressure, slurried with petroleum ether:ethyl acetate in a ratio of 5:1, filtered, and the filter cake was concentrated to obtain a white solid (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)carbamate 2 (2.4 g, 4.66 mmol, 96% yield).

[0668] LC-MS:[M+H]+:516.4

[0669] Synthesis of (9H-fluorene-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-(((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropane-2-yl)amino)-1-oxobutane-2-yl)carbamate:

[0670]

[0671] (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)carbamate 2 (1.90 g, 3.69 mmol) was dissolved in N,N-dimethylformamide (50 mL), followed by the addition of bis(4-nitrobenzene) carbonate (1.12 g, 11.06 mmol). After the reaction was completely dissolved, N,N-diisopropylethylamine (1.83 mL, 11.06 mmol) was added. The mixture was purged three times with nitrogen and reacted at room temperature for 3 hours. LC-MS monitoring showed complete consumption of the starting material and detection of the product. The reaction solution was concentrated under reduced pressure, slurried with petroleum ether:ethyl acetate (5:1), filtered, and the filter cake was concentrated to obtain a white solid (9H-fluorene-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-(((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropane-2-yl)amino)-1-oxobutane-2-yl)carbamate 3 (2.10 g, 3.08 mmol, 83% yield).

[0672] LC-MS: [M+Na]+: 703.4

[0673] Synthesis of (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-(((((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]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxoisobutane-2-yl)carbamate:

[0674]

[0675] (9H-fluorene-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-(((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropane-2-yl)amino)-1-oxobutane-2-yl)carbamate 3 (2.50 g, 3.67 mmol), eczetidine methanesulfonate 4 (1.95 g, 3.67 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (2.09 g, 5.51 mmol), 1-hydroxybenzotriazole (0.74 g, 5.51 mmol), and N,N-diisopropylethylamine (0.95 g, 7.35 mmol) were dissolved in N,N-dimethylformamide (5 mL), and the reaction was stirred at room temperature for 2 hours. The reaction was detected by LCMS. The reaction solution was slowly added dropwise to 100 mL of water, filtered, and the filter cake was washed with 30 mL of water, dried, and subjected to column chromatography (dichloromethane:methanol = 20:1). After concentration, a pale yellow solid (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-)) was obtained. Dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxoisobutane-2-yl)carbamate 5 (2.30 g, 2.35 mmol, 64% yield).

[0676] LC-MS:[M+H]+:977.4

[0677] Synthesis of 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)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]indolo[1,2-b]quinoline-1-yl)carbamate:

[0678]

[0679] (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-((((((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]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxoisobutane-2-yl)carbamate 5 (600.00 mg, 0.61 mmol) was dissolved in N,N-dimethylformamide (6 mL), and diethylamine (448.28 mg, 6.14 mmol) was slowly added. The reaction was stirred at room temperature for 1 hour. The reaction was confirmed by LCMS. The reaction solution was concentrated, and N,N-dimethylformamide (6 mL) in ethyl acetate was added and the mixture was stirred. The filter cake was dried to obtain crude product 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)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]indolo[1,2-b]quinoline-1-yl)carbamate 6 (300.00 mg, 0.40 mmol, 66% yield).

[0680] LC-MS:[M+H]+:755.2

[0681] 4-((2S,5S,8S,24S,25R,26R,27R)-8-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18-tetraoxo-22-((2S,3R) Synthesis of (4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctacosamide)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]indolo[1,2-b]quinoline-1-yl)carbamate:

[0682]

[0683] 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)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]indolo[1,2-b]quinoline-1-yl)carbamate 6 (300.00 mg, 0.40 mmol), N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(5-(bis(2-(bis((2S,3R,4R,5) R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)amino)-5-oxopentanoyl)-L-lysine 7 (486.60 mg, 0.40 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (226.54 mg, 0.60 mmol), and 1-hydroxybenzotriazole (80.48 mg, 0.60 mmol) were dissolved in N,N-dimethylformamide (5 mL), and N,N-diisopropylethylamine (102.54 mg, 0.80 mmol) was added. The reaction was stirred at room temperature for 2 hours. The reaction was detected by LCMS. The reaction solution was filtered and purified by reverse-phase column chromatography (acetonitrile:water = 50:50) to give a white solid 4-((2S,5S,8S,24S,25R,26R,27R)-8-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18-tetraoxo-2 2-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctacosamide)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]indolo[1,2-b]quinoline-1-yl)carbamate 8 (400.00 mg, 0.20 mmol, 50% yield).

[0684] LC-MS:[M+2H]+ / 2:981.6

[0685] 4-((2S,5S,8S,24S,25R,26R,27R)-8-amino-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)- Synthesis of 2,3,4,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctaamino)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]indolo[1,2-b]quinoline-1-yl)carbamate:

[0686]

[0687] 4-((2S,5S,8S,24S,25R,26R,27R)-8-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3,6,13,19,2 2-Pentazaoctacosamido)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]indolo[1,2-b]quinoline-1-yl)carbamate 8 (400.00 mg, 0.20 mmol) was dissolved in N,N-dimethylformamide (5 mL), and diethylamine (148.89 mg, 2.04 mmol) was slowly added. The reaction was stirred at room temperature for 1 hour. LCMS analysis confirmed the reaction was complete. The reaction solution was slurried with ethyl acetate (5 mL), filtered, and concentrated to obtain a creamy white solid: 4-((2S,5S,8S,24S,25R,26R,27R)-8-amino-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18-tetraoxo-22-((2S,3R) ,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctaamino)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]indolo[1,2-b]quinoline-1-yl)carbamate 9 (230.00 mg, 0.13 mmol, 65% yield).

[0688] LC-MS: [M+2H]+ / 2: 870.2

[0689] 4-((2S,5S,8S,24S,25R,26R,27R)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-8-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexylamino)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18-tetraoxo-22-((S,3R,4R,5R)-2,3,4,5 Synthesis of 5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctacosamide)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]indolo[1,2-b]quinoline-1-yl)carbamate:

[0690]

[0691] 4-((2S,5S,8S,24S,25R,26R,27R)-8-amino-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctaamino)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,1 3-Dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)carbamate 9 (230.00 mg, 0.13 mmol) and 2,5-dioxopyrrolidone-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoate 10 (40.78 mg, 0.13 mmol) were dissolved in N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (34.13 mg, 0.26 mmol) was added. The reaction was stirred at room temperature for 2 hours. LCMS analysis confirmed the reaction was complete. The reaction solution was adjusted to weakly acidic with formic acid, filtered, and prepared by separation (ammonium acetate system). After lyophilization, a white solid was obtained: 4-((2S,5S,8S,24S,25R,26R,27R)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-8-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexylamino)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18-tetraoxo-22-((S,3R,4R,5R)-2,3,4,5 5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctacosamide)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]indolo[1,2-b]quinoline-1-yl)carbamate SJ-LP12 (26.80 mg, 0.014 mmol, 11% yield).

[0692] LC-MS: [M+2H]+ / 2: 966.7;

[0693] The retention time Rt of SJ-LP12 by LC-MS was 1.04 min.

[0694] 4-((2S,5S,8S,24S,25R,26R,27R)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamine)-4,7,14,18-tetraoxo-22-((2S... Synthesis of (3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctacosanamino)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]indolo[1,2-b]quinoline-1-yl)carbamate:

[0695]

[0696] 4-((2S,5S,8S,24S,25R,26R,27R)-8-amino-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctaamino)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]indo[1,2-b]quinoline-1-yl)carbamate 9 (90.00 mg, 0.050 mmol), 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl acid 11 (13.89 mg, 0.050 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (29.50 mg, 0.080 mmol), benzo[d][1,2,3]triazole-1-ol (10.49 mg, 0.080 mmol) were dissolved in N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (13.35 mg, 0.10 mmol) was added. The reaction was stirred at room temperature for 2 hours. LCMS analysis confirmed the reaction was complete. The reaction solution was adjusted to weakly acidic with formic acid, filtered, and prepared by separation (ammonium acetate system). After lyophilization, a white solid was obtained: 4-((2S,5S,8S,24S,25R,26R,27R)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-5-isopropyl-2-methyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamino)-4,7,14,18-tetraoxonyl 22-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctacosanamino)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]indolo[1,2-b]quinoline-1-yl)carbamate SJ-LP10 (20.71 mg, 0.010 mmol, 20% yield).

[0697] LC-MS: [M+2H]+ / 2: 994.6;

[0698] The retention time Rt of SJ-LP10 by LC-MS was 2.704 min.

[0699] Example 7: Synthesis of SJ-LP13 and 19

[0700] Synthesis route:

[0701]

[0702]

[0703] Synthesis of (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)carboxylate:

[0704]

[0705] (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine 1 (2.00 g, 5.23 mmol) was dissolved in methanol (10 mL) and dichloromethane (50 mL). Then, p-aminobenzyl alcohol (0.64 g, 5.23 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (1.29 g, 5.23 mmol) were added. The mixture was purged with nitrogen three times and reacted at room temperature in the dark for 16 hours. LC-MS monitoring showed complete consumption of the starting materials and detection of the product. The reaction solution was concentrated under reduced pressure, slurried with petroleum ether:ethyl acetate = 5:1, filtered, and the filter cake was concentrated to give a white solid product (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)carbamate 2 (2.30 g, 4.72 mmol, 90% yield).

[0706] LC-MS: [M+Na]+: 510.2

[0707] Synthesis of (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-(((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)carboxylate:

[0708]

[0709] (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)carboxylate 2 (1.80 g, 3.69 mmol) was dissolved in N,N-dimethylformamide (40 mL), followed by the addition of bis(4-nitrobenzene) carbonate (3.37 g, 11.08 mmol). After the reaction was completely dissolved, N,N-diisopropylethylamine (1.83 mL, 11.08 mmol) was added. The mixture was purged three times with nitrogen and reacted at room temperature for 3 hours. LC-MS monitoring showed complete consumption of the starting material and detection of the product. The reaction solution was concentrated under reduced pressure, slurried with petroleum ether:ethyl acetate (5:1), filtered, and the filter cake was concentrated to give a white solid product (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-(((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)carbamate 3 (1.40 g, 2.15 mmol, 58% yield).

[0710] LC-MS: [M+Na]+: 675.3

[0711] Synthesis of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)propamido)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] indo[1,2-b]quinoline-1-yl)carbamate:

[0712]

[0713] (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-(((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)carbamate 3 (800.00 mg, 1.23 mmol) was dissolved in N,N-dimethylformamide (10 mL), followed by the addition of eczemab mesylate 4 (533.77 mg, 1.23 mmol) and 1-hydroxybenzotriazole (182.20 mg, 1.35 mmol), and finally N,N-diisopropylethylamine (0.41 mL, 2.45 mmol). The mixture was purged with nitrogen three times and reacted at room temperature for 18 hours. LCMS was used to monitor complete consumption of the starting material and detection of the product. The reaction solution was concentrated under reduced pressure and purified by column chromatography (dichloromethane:methanol = 10:1) to give a pale yellow solid product 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)propamido)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] indo[1,2-b]quinoline-1-yl)carbamate 5 (500.00 mg, 0.53 mmol, 43% yield).

[0714] LC-MS:[M+H]+:949.4

[0715] Synthesis of 4-((S)-2-((S)-2-aminopropylamino)propamido)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] indo[1,2-b]quinoline-1-yl)carbamate:

[0716]

[0717] 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)propamido)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] indo[1,2-b]quinoline-1-yl)carbamate 5 (500.00 mg, 0.53 mmol) was dissolved in N,N-dimethylformamide (8 mL), and diethylamine (0.55 mL, 5.27 mmol) was added. The mixture was purged with nitrogen three times and reacted at room temperature for 2 hours. LCMS was used to monitor complete consumption of the starting material and detection of the product. The reaction solution was concentrated under reduced pressure, slurried with n-hexane, filtered, and the filter cake was concentrated under reduced pressure to finally obtain the pale yellow solid product 4-((S)-2-((S)-2-aminopropylamino)propamido)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]indo[1,2-b]quinoline-1-yl)carbamate 6 (265.00 mg, 0.37 mmol, 69% yield).

[0718] LC-MS:[M+H]+:727.4.

[0719] 4-((2S,5S,8S,24S,25R,26R,27R)-8-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-2,5-dimethyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-2,5-dimethyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl) Synthesis of (R,4R,5R)-2,3,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctadecanoamide)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] indo[1,2-b]quinoline-1-yl)carbamate:

[0720]

[0721] 4-((S)-2-((S)-2-aminopropylamino)propamido)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] indene[1,2-b]quinoline-1-yl)carbamate 6 (50.00 mg, 0.069 mmol), N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(5-(bis(2-( Bis((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)amino)-5-oxopentanoyl)-L-lysine 7 (92.65 mg, 0.076 mmol) was dissolved in N,N-dimethylformamide (2 mL), HATU (39.24 mg, 0.103 mmol), 1-hydroxybenzotriazole (13.92 mg, 0.103 mmol) and N,N-diisopropylethylamine (0.034 mL, 0.206 mmol) were added, and the mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS until completion. The solution was directly purified by reverse-phase reaction (ACN:H₂O = 30:70), and lyophilized to obtain a white solid: 4-((2S,5S,8S,24S,25R,26R,27R)-8-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-2,5-dimethyl-4,7,14,18-tetraoxy 22-((2S,3R,4R,5R)-2,3,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctadecanoamide)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]indo[1,2-b]quinoline-1-yl)carbamate 8 (100.00 mg, 0.052 mmol, 75% yield).

[0722] LC-MS:[M+H]+:1932.0

[0723] 4-((2S,5S,8S,24S,25R,26R,27R)-8-amino-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-2,5-dimethyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3, Synthesis of 4,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctacosanamino)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] indo[1,2-b]quinoline-1-yl)carbamate:

[0724]

[0725] 4-((2S,5S,8S,24S,25R,26R,27R)-8-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-2,5-dimethyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3,5,6-pentahydroxyhexyl)-3, 6,13,19,22-pentazaoctadecanoamide)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] indo[1,2-b]quinoline-1-yl)carbamate 8 (100.00 mg, 0.047 mmol) was added to DMF (2 mL), followed by diethylamine (0.048 mL, 0.47 mmol). The mixture was stirred at room temperature for one hour. LC-MS showed the reaction was complete. The mixture was then evaporated to dryness using an oil pump. 2 mL of dichloromethane was added, and the mixture was ultrasonically slurried and filtered to obtain a gray solid: 4-((2S,5S,8S,24S,25R,26R,27R)-8-amino-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-2,5-dimethyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-22-((2S,3R)-22-dimethyl)-24,25,26,27,28-pentahydroxy-2,5-dimethyl-4,7,14,18-tetraoxo-22-((2S,3R)-22-dimethyl ...22-dimethyl)-24,25,26,27,28-dimethyl)-24,25,26,27,28-dimethyl)-24,25,26,27,28-dimethyl)-24,25,26,27,28-dimethyl)-24,25,26,27,28-dimethyl)-24,25,26,27,28-dimethyl)-24,25,26 R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctacosanamino)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]indo[1,2-b]quinoline-1-yl)carbamate 9 (90.00 mg, 0.053 mmol, equivalent yield).

[0726] LC-MS:[M+H]+:1710.1

[0727] 4-((2S,5S,8S,24S,25R,26R,27R)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-8-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-24,25,26,27,28-pentahydroxy-2,5-dimethyl-4,7,14,18-tetraoxo-22-((2 Synthesis of (1S,9S)-2,3,4,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctacosanamino)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] indo[1,2-b]quinoline-1-yl)carbamate:

[0728]

[0729] 4-((2S,5S,8S,24S,25R,26R,27R)-8-amino-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-2,5-dimethyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctacosanamino)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,1 3-Dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7] indo[1,2-b]quinoline-1-yl)carbamate 9 (90.00 mg, 0.053 mmol) and 2,5-dioxopyrrolidine-1-yl-6-(2,5-diketone-2,5-dihydro-1H-pyrrolo-1-yl)hexanoate 10 (16.22 mg, 0.053 mmol) were dissolved in N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (0.026 mL, 0.16 mmol) was added. The mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS until completion. The pH was adjusted to weakly acidic with formic acid, and the mixture was purified by reverse-phase reaction to obtain a white solid: 4-((2S,5S,8S,24S,25R,26R,27R)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-8-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-24,25,26,27,28-pentahydroxy-2,5-dimethyl-4,7,14,18-tetraoxo-22 -((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctacosanamino)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]indo[1,2-b]quinoline-1-yl)carbamate SJ-LP13 (25.00 mg, 0.013 mmol, 25% yield).

[0730] LC-MS: [M+2H]+ / 2: 952.8;

[0731] The retention time Rt of SJ-LP13 by LC-MS was 1.29 min.

[0732] Synthesis of 5-(bis(2-((tert-butoxycarbonyl)amino)ethyl)amino)-5-oxopentanoic acid:

[0733]

[0734] Di-tert-butyl(azadiylbis(ethane-2,1-diyl)) dicarboxylate 7a (5.00 g, 16.48 mmol), dihydro-2H-pyran-2,6(3H)-dione 7b (1.79 g, 15.66 mmol), and triethylamine (2.50 g, 24.72 mmol) were dissolved in dichloromethane (50 mL), and the reaction was stirred at room temperature for 16 hours. The reaction was detected by LCMS. The reaction solution was concentrated and dissolved in water (50 mL). The pH was adjusted to about 3 with 1 N hydrochloric acid. The aqueous phase was extracted with dichloromethane (50 mL * 3). The organic phases were combined and washed with saturated brine (100 mL). The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to give a white solid 5-(bis(2-((tert-butoxycarbonyl)amino)ethyl)amino)-5-oxovaleric acid 7c (5.50 g, 13.17 mmol, 80% yield).

[0735] LC-MS: [M+Na]+: 440.2

[0736] Synthesis of tert-butyl(S)-19-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-8-(2-((tert-butoxycarbonyl)amino)ethyl)-2,2-dimethyl-4,9,13-trioxo-3-oxa-5,8,14-triazolidine-20-ester:

[0737]

[0738] 5-(bis(2-((tert-butyloxycarbonyl)amino)ethyl)amino)-5-oxovaleric acid 7c (500.00 mg, 1.20 mmol), tert-butyl(((9H-fluorene-9-yl)methoxy)carbonyl)-L-lysine hydrochloride 7d (552.10 mg, 1.20 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (546.11 mg, 1.44 mmol), and N,N-diisopropylethylamine (463.47 mg, 3.59 mmol) were dissolved in N,N-dimethylformamide (5 mL), and the reaction was stirred at room temperature for 2 hours. The reaction was detected by LCMS. The reaction solution was diluted with water (50 mL) and ethyl acetate (30 mL). The organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (dichloromethane:methanol = 5:1). After concentration, a white solid tert-butyl(S)-19-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-8-(2-((tert-butoxycarbonyl)amino)ethyl)-2,2-dimethyl-4,9,13-trioxo-3-oxa-5,8,14-triazolidine-20-ester 7e (850.00 mg, 1.03 mmol, 86% yield) was obtained.

[0739] LC-MS:[M+H]+:824.4

[0740] Synthesis of N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(5-(bis(2-aminoethyl)amino)-5-oxopentanoyl)-L-lysine:

[0741]

[0742] 200.00 mg (0.24 mmol) of tert-butyl(S)-19-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-8-(2-(((tert-butoxycarbonyl)amino)ethyl)-2,2-dimethyl-4,9,13-trioxo-3-oxa-5,8,14-triazolidine-20-ester 7e was dissolved in dichloromethane (4 mL), and trifluoroacetic acid (1 mL) was slowly added. The reaction was stirred at room temperature for 1 hour. The reaction was confirmed by LCMS. The reaction solution was concentrated to give a pale yellow oil N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(5-(bis(2-aminoethyl)amino)-5-oxopentanoyl)-L-lysine 7f (130.00 mg, 0.23 mmol, 94% yield).

[0743] LC-MS:[M+H]+:568.3.

[0744] Synthesis of N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(5-(bis(2-(bis((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)amino)-5-oxopentanoyl)-L-lysine:

[0745]

[0746] N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(5-(bis(2-aminoethyl)amino)-5-oxopentanoyl)-L-lysine 7f (130.00 mg, 0.23 mmol), (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal 7g (742.62 mg, 4.12 mmol), and sodium cyanoborohydride (173.12 mg, 2.75 mmol) were dissolved in methanol (5 mL). The reaction was heated to 90 °C and stirred for 16 hours under a nitrogen system. The reaction was detected by LCMS. The reaction solution was cooled, concentrated, and water (2 mL) was added. The solution was purified by reverse-phase column chromatography (0.1% trifluoroacetic acid: acetonitrile = 85:15). After lyophilization, a white solid N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(5-(bis(2-(bis((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)amino)-5-oxopentanoyl)-L-lysine 7 (180.00 mg, 0.15 mmol, 64% yield) was obtained.

[0747] LC-MS:[M+2H]+ / 2:613.0.

[0748] 4-((2S,5S,8S,24S,25R,26R,27R)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-2,5-dimethyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-enamido)-4,7,14,18-tetraoxo-22-((2S, Synthesis of (3R, 4R, 5R)-2,3,4,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctacosanamino)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] indo[1,2-b]quinoline-1-yl)carbamate:

[0749]

[0750] 4-((2S,5S,8S,24S,25R,26R,27R)-8-amino-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-2,5-dimethyl-4,7,14,18-tetraoxo-22-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctadecanoamino)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] indo[1,2-b]quinoline-1-yl)carbamate 9 (100.00 mg, 0.058 mmol), 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-alkynic acid 11 (18.16 mg, 0.059 mmol) were dissolved in N,N-dimethylformamide (3 mL), HATU (33.34 mg, 0.088 mmol), HOBt (11.85 mg, 0.088 mmol), and N,N-diisopropylethylamine (22.66 mg, 0.18 mmol) were added, and the mixture was stirred at room temperature for 1 h. After the reaction was monitored by LCMS, the mixture was purified by HPLC and given a white solid in a 0.05% formic acid system: 4-((2S,5S,8S,24S,25R,26R,27R)-19-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-24,25,26,27,28-pentahydroxy-2,5-dimethyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-enamido)-4,7,14,18-tetraoxo-22-(( 2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3,6,13,19,22-pentazaoctacosanamino)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]indo[1,2-b]quinoline-1-yl)carbamate SJ-LP19 (9.50 mg, 0.0050 mmol, 8% yield).

[0751] LC-MS:[M+H]+:1960.2;

[0752] The retention time Rt of SJ-LP19 by LC-MS was 2.654 min.

[0753] Example 8: Synthesis of SJ-LP15 and 18

[0754] Synthesis route:

[0755]

[0756]

[0757] Synthesis of (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine-L-alanylglycine tert-butyl ester:

[0758]

[0759] (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine-L-alanine 1 (12.50 g, 30.45 mmol) was dissolved in dichloromethane (300 mL) and tetrahydrofuran (300 mL). Glycine tert-butyl ester 2 (4.80 g, 131.18 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (22.60 g, 91.36 mmol) were added. The mixture was purged with nitrogen three times and reacted at room temperature for 18 hours. LC-MS monitoring showed complete consumption of the starting material and detection of the product. The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane:methanol = 10:1) to give a white solid product (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine-L-alanylglycine tert-butyl ester 3 (12.60 g, 24.06 mmol, 79% yield).

[0760] LC-MS: [M+Na]+: 546.4

[0761] Synthesis of (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine-L-alanylglycine:

[0762]

[0763] (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine-L-alanylglycine tert-butyl ester 3 (12.60 g, 24.06 mmol) was dissolved in dichloromethane (160 mL), and trifluoroacetic acid (40 mL) was added dropwise at 0 °C. The reaction was carried out overnight at room temperature, and the starting material was completely consumed and the product was detected by LCMS. The reaction solution was concentrated under reduced pressure, slurried with diethyl ether, filtered, and the filter cake was concentrated under reduced pressure to give a white solid product (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine-L-alanylglycine 4 (10.00 g, 21.39 mmol, 89% yield).

[0764] LC-MS:[M+H]+:468.4

[0765] Synthesis of (5S,8S)-1-(9H-fluorene-9-yl)-5-isopropyl-8-methyl-3,6,9-trioxo-2-oxo-4,7,10-triazaundecane-11-ylacetate:

[0766]

[0767] (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine-L-alanylglycine 4 (10.00 g, 21.39 mmol), lead tetraacetate (11.40 g, 25.67 mmol), and pyridine (2.10 mL, 25.67 mmol) were dissolved in tetrahydrofuran (240 mL) and toluene (60 mL). The mixture was purged with nitrogen three times and the temperature was slowly increased to 80 °C for 12 hours. The reaction solution was concentrated, and the residue was purified by column chromatography (dichloromethane:methanol = 10:1) to give a white solid product (5S,8S)-1-(9H-fluorene-9-yl)-5-isopropyl-8-methyl-3,6,9-trioxo-2-oxo-4,7,10-triazaundecane-11-ylacetate 5 (6.00 g, 12.46 mmol, 58% yield).

[0768] LC-MS: [M+Na]+: 504.4

[0769] Synthesis of (5S,8S)-1-(9H-fluorene-9-yl)-5-isopropyl-8-methyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazatetradecane-14-acid benzyl ester:

[0770]

[0771] (5S,8S)-1-(9H-fluorene-9-yl)-5-isopropyl-8-methyl-3,6,9-trioxo-2-oxo-4,7,10-triazaundecane-11-yl acetate 5 (4.00 g, 8.31 mmol) and benzyl glycolate 6 (6.90 g, 41.53 mmol) were dissolved in tetrahydrofuran (40 mL), and 4A molecular sieves were added. After stirring at room temperature for 0.5 hours, scandium trifluoromethanesulfonate (1.20 g, 2.49 mmol) was added, and the reaction was carried out at room temperature for 3 hours. The starting materials were completely consumed and the product was detected by LCMS. The reaction solution was filtered, concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether: ethyl acetate = 1:2). The product was concentrated to give a white solid product (5S,8S)-1-(9H-fluorene-9-yl)-5-isopropyl-8-methyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazatetradecane-14-acid benzyl ester 7 (1.10 g, 1.87 mmol, 23% yield).

[0772] LC-MS: [M+Na]+: 609.7

[0773] Synthesis of (5S,8S)-1-(9H-fluorene-9-yl)-5-isopropyl-8-methyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazatetradecane-14-acid:

[0774]

[0775] (5S,8S)-1-(9H-fluorene-9-yl)-5-isopropyl-8-methyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazatetradecane-14-acid benzyl ester 7 (800.00 mg, 1.23 mmol) was dissolved in THF (20 mL), and Pd / C 10% (80.00 mg) was added. The mixture was purged with hydrogen three times and reacted at room temperature for 16 hours. LC-MS showed that the reaction was complete. The mixture was filtered through diatomaceous earth and evaporated to dryness to give a white solid (5S,8S)-1-(9H-fluorene-9-yl)-5-isopropyl-8-methyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazatetradecane-14-acid 8 (500.00 mg, 1.01 mmol, 82% yield).

[0776] LC-MS: [M+Na]+: 519.5

[0777] Synthesis of (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-(((2-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxoisobutane-2-yl)carbamate:

[0778]

[0779] (5S,8S)-1-(9H-fluorene-9-yl)-5-isopropyl-8-methyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazatetradecane-14-acid 8 (400.00 mg, 0.48 mmol) was dissolved in N,N-dimethylformamide (5 mL), and HATU (275.12 mg, 0.72 mmol) and HOBt (97.77 mg) were added. 0.72 mmol (mg), N,N-diisopropylethylamine (0.24 mL, 1.45 mmol), stirred at room temperature for 10 minutes, then (1S,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzopyrano[3',4':6,7] indo[1,2-b]quinoline-10,13-dione was added.

[0780] 9 (219.90 mg, 0.48 mmol). The reaction was stirred at room temperature for 2 hours, and the reaction was monitored by LCMS to indicate completion. The reaction solution was directly purified by reverse phase (acetonitrile / water = 50 / 50, TFA system) to give a white solid (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-(((2-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxoisobutane-2-yl)carbamate 10 (240.00 mg, 0.26 mmol, 54% yield).

[0781] LC-MS:[M+H]+:934.8

[0782] Synthesis of (S)-2-amino-N-((S)-1-(((2-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropane-2-yl)-3-methylbutyramide:

[0783]

[0784] Add (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-(((2-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxoisobutane-2-yl)carbamate 10 (240.00 mg, 0.26 mmol) to DMF (5 mL), and add diethylamine (0.27 mL, 2.57 mmol). The mixture was stirred at room temperature for 1 hour. LCMS showed that the reaction was complete. The mixture was evaporated to dryness using an oil pump, and 3 mL of ethyl acetate was added. The mixture was ultrasonically slurried and filtered to obtain a gray solid (S)-2-amino-N-((S)-1-(((2-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropane-2-yl)-3-methylbutyramide 11 (130.00 mg, 0.18 mmol, 69% yield).

[0785] LC-MS:[M+H]+:712.5

[0786] (9H-fluorene-9-yl)methyl((7S,10S,13S,29S,30R,31R,32R)-24-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3] Synthesis of [6,7]indo[1,2-b]quinoline-1-yl)amino)-29,30,31,32,33-pentahydroxy-10-isopropyl-7-methyl-1,6,9,12,19,23-hexaoxo-27-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3-oxo-5,8,11,18,24,27-hexaazatriazacyclopentane-13-yl)carbamate:

[0787]

[0788] (S)-2-amino-N-((S)-1-(((2-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropane-2-yl)-3-methylbutyramide 11 (240.00 mg, 0.34 mmol), N2-(((9H-fluorene-9-yl)methoxy) Carbonyl)-N6-(5-(bis(2-(bis((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)amino)-5-oxopentanoyl)-L-lysine 12 (412.02 mg, 0.34 mmol) was dissolved in N,N-dimethylformamide (5 mL), HATU (191.94 mg, 0.51 mmol), HOBt (68.21 mg, 0.51 mmol) and N,N-diisopropylethylamine (0.17 mL, 1.01 mmol) were added, and the mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS until completion. The solution was directly purified by reverse-phase reaction (acetonitrile / water = 30 / 70, TFA system) to obtain a white solid (9H-fluorene-9-yl)methyl((7S, 10S, 13S, 29S, 30R, 31R, 32R)-24-(2-(bis((2S, 3R, 4R, 5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-1-(((1S, 9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-Benzopyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-29,30,31,32,33-pentahydroxy-10-isopropyl-7-methyl-1,6,9,12,19,23-hexaoxo-27-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3-oxo-5,8,11,18,24,27-hexaazatriazacyclopentane-13-yl)carbamate 13 (150.00 mg, 0.078 mmol, 23% yield).

[0789] LC-MS:[M+H]+:1917.4

[0790] Synthesis of N1-((7S, 10S, 13S)-13-amino-1-(((S, 9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3', 4': 6,7]indolo[1,2-b]quinoline-1-yl)amino)-10-isopropyl-7-methyl-1,6,9,12-tetraoxo-3-oxo-5,8,11-triazacycloheptane-17-acyl)-N5,N5-bis(2-(bis((2S, 3R, 4R, 5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)pentadiamide:

[0791]

[0792] (9H-fluorene-9-yl)methyl((7S,10S,13S,29S,30R,31R,32R)-24-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7)) Indo[1,2-b]quinoline-1-yl)amino)-29,30,31,32,33-pentahydroxy-10-isopropyl-7-methyl-1,6,9,12,19,23-hexaoxo-27-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3-oxo-5,8,11,18,24,27-hexaazatriazacyclopentane-13-yl)carbamate 13 (150.00 mg, 0.078 mmol) Add to DMF (5 mL), add diethylamine (0.081 mL, 0.78 mmol), stir at room temperature for 1 hour, LCMS shows complete reaction, evaporate to dryness with an oil pump, add 2 mL of ethyl acetate, sonicate and filter to obtain gray solid N1-((7S,10S,13S)-13-amino-1-(((S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- Benzopyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-10-isopropyl-7-methyl-1,6,9,12-tetraoxo-3-oxo-5,8,11-triazacycloheptane-17-acyl)-N5,N5-bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)glutaramide 14 (100.00 mg, 0.059 mmol, 76% yield).

[0793] LC-MS:[M+H]+:1695.9

[0794] N1,N1-bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-N5-((7S,10S,13S)-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H) Synthesis of benzopyran[3',4':6,7] indo[1,2-b]quinoline-1-yl)amino)-13-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-10 isopropyl-7-methyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazaheptadecane-17-yl)pentadiamide:

[0795]

[0796] N1-((7S, 10S, 13S)-13-amino-1-(((S, 9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3', 4': 6,7]indolo[1,2-b]quinoline-1-yl)amino)-10-isopropyl-7-methyl-1,6,9,12-tetraoxo-3-oxo-5,8,11-triazacycloheptane-17-acyl)-N5,N5-bis(2-(bis) ((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)pentadiamide 14 (100.00 mg, 0.059 mmol) and 2,5-dioxopyrrolidone-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoate 15 (18.16 mg, 0.059 mmol) were dissolved in N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (0.029 mL, 0.18 mmol) was added. The mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS until completion. Formic acid was added to adjust the pH to weakly acidic. HPLC purification was then performed using a 0.05% formic acid system to obtain a white solid, N1,N1-bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-N5-((7S,10S,13S)-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15) -Hexahydro-1H,12H-benzopyran[3',4':6,7]indox[1,2-b]quinoline-1-yl)amino)-13-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-10isopropyl-7-methyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazaheptadecane-17-yl)glutaramide SJ-LP15 (4.00 mg, 0.0020 mmol, 3% yield).

[0797] LC-MS:[M+H]+:1889.4;

[0798] The retention time Rt of SJ-LP15 by LC-MS was 2.533 min.

[0799] Synthesis of 5-bromo-4-chloro-3-fluoroaniline:

[0800]

[0801] 5-Bromo-3-fluoroaniline 9a (200.00 g, 1052.52 mmol) was dissolved in N,N-dimethylformamide (2000 mL), and N-chlorosuccinimide (168.65 g, 1263.02 mmol) was slowly added. After the addition was complete, the reaction was carried out at 25 °C for 48 hours. The reaction was confirmed by LCMS. The reaction solution was slowly poured into water and then extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was distilled under reduced pressure to obtain the crude product. The crude product was purified by rapid silica gel column chromatography to give a colorless oily liquid 5-bromo-4-chloro-3-fluoroaniline 9b (80.00 g, 356.41 mmol, 34% yield).

[0802] LC-MS:[M+H]+:225.3

[0803] 1H NMR (400MHz, DMSO-d6) δ6.76 (dd, J=2.4, 1.6Hz, 1H), 6.51 (dd, J=12.0, 2.4Hz, 1H), 5.83 (s, 2H).

[0804] Synthesis of N-(5-bromo-4-chloro-3-fluorophenyl)acetamide:

[0805]

[0806] 5-Bromo-4-chloro-3-fluoroaniline 9b (80.00 g, 356.41 mmol) was dissolved in acetic acid (800 mL), and acetic anhydride (145.54 g, 1425.64 mmol) was added. The reaction was stirred at 25 °C for 16 hours. LCMS analysis showed the reaction was complete. The reaction solution was slowly poured into ice water under stirring, and a white solid product precipitated. The product was filtered, and the filter cake was dried using an oil pump to obtain a white solid N-(5-bromo-4-chloro-3-fluorophenyl)acetamide 9c (83.00 g, 311.45 mmol, 87% yield).

[0807] LC-MS:[M+H]+:267.5

[0808] Synthesis of (Z / E)-4-(5-acetamido-2-chloro-3-fluorophenyl)but-3-enoic acid:

[0809]

[0810] N-(5-bromo-4-chloro-3-fluorophenyl)acetamide 9c (83.00 g, 311.45 mmol) and 3-butenoic acid 9d (32.18 g, 373.75 mmol) were dissolved in a mixed solvent of 1,4-dioxane (830 mL) and water (83 mL). Then, N,N-diisopropylethylamine (120.77 g, 934.37 mmol), tris(o-methylphenyl)phosphine (9.48 g, 31.14 mmol), and palladium acetate (3.53 g, 15.57 mmol) were added. After the addition was complete, the reaction system was purged three times with nitrogen, and the reaction was carried out at 100 °C for 16 hours under a nitrogen atmosphere. The reaction was confirmed by LCMS. After the reaction solution was cooled to room temperature, 1N sodium hydroxide aqueous solution and ethyl acetate were added and the mixture was shaken to separate the layers. After separating the lower aqueous phase, the pH was adjusted to about 3 with 4 mol / L hydrochloric acid aqueous solution, and then extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain crude grayish-white solid (Z / E)-4-(5-acetamido-2-chloro-3-fluorophenyl)but-3-enoic acid 9e (84.61 g, 311.45 mmol, equivalent yield).

[0811] LC-MS:[M+H]+:271.6

[0812] Synthesis of 4-(5-acetamido-2-chloro-3-fluorophenyl)butyric acid:

[0813]

[0814] (Z / E)-4-(5-acetamido-2-chloro-3-fluorophenyl)but-3-enoic acid 9e (84.61 g, 311.45 mmol) was dissolved in tetrahydrofuran (850 mL), and then 10% palladium on carbon (8.50 g) was added. After the addition was complete, the reaction system was purged with hydrogen three times, and the reaction was carried out at 25 °C for 48 hours under a hydrogen atmosphere. The reaction was confirmed by LCMS. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to give crude grayish-white solid 4-(5-acetamido-2-chloro-3-fluorophenyl)butyric acid 9f (80.00 g, 292.30 mmol, 94% yield).

[0815] LC-MS:[M+K]+:312.5

[0816] Synthesis of N-(4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide:

[0817]

[0818] 9g (80.00g, 292.30mmol) of 4-(5-acetamido-2-chloro-3-fluorophenyl)butyric acid was dissolved in 400mL of trifluoroacetic acid. The solution was cooled to 5°C, and 306.96g (1461.50mmol) of trifluoroacetic anhydride was slowly added. After the addition was complete, the reaction was maintained at 5°C for 16 hours. The reaction system was purged with nitrogen three times, and the reaction was carried out under a nitrogen atmosphere for 48 hours. The reaction was confirmed by LCMS. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 9g (50.00g, 195.56mmol, 67% yield) of crude grayish-white solid N-(4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide.

[0819] LC-MS:[M+H]+:255.4

[0820] 1H NMR (400MHz, DMSO-d6) δ12.16(s,1H),8.48(d,J=12.8Hz,1H),3.02(t,J=6.0Hz,2H),2.72–2.65(m,2H),2.18(s,3H),2.09–1.99(m,2H).

[0821] Synthesis of (Z)-N-(4-chloro-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide:

[0822]

[0823] 9 g (45.00 g, 176.01 mmol) of N-(4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide was dissolved in tetrahydrofuran (450 mL). The solution was cooled to 0 °C, and potassium tert-butoxide (43.45 g, 387.20 mmol) was slowly added. After 15 minutes, tert-butyl nitrite (29.04 g, 281.61 mmol) was added. After the addition was complete, the reaction was maintained at 0 °C for 1 hour. The reaction was detected by LCMS to indicate completion. The reaction solution was quenched with saturated ammonium chloride aqueous solution, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product (Z)-N-(4-chloro-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide was obtained in 9 h (50.10 g, 176.01 mmol, equivalent yield).

[0824] LC-MS:[M+H]+:284.5

[0825] Synthesis of N,N'-(4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide:

[0826]

[0827] Crude (Z)-N-(4-chloro-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (9h, 50.10 g, 176.01 mmol) was dissolved in acetic acid (250 mL) and acetic anhydride (250 mL). The solution was cooled to 0 °C, and after 30 minutes, zinc powder (22.97 g, 351.20 mmol) was slowly added. After the addition was complete, the reaction was maintained at 0 °C for 1 hour. The reaction was confirmed by LCMS. The reaction solution was filtered, and the pH of the filtrate was adjusted to 8-9 with saturated sodium bicarbonate aqueous solution. The filtrate was then extracted with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by rapid silica gel column chromatography to give a yellow solid N,N'-(4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide 9i (24.00 g, 76.74 mmol, 44% yield).

[0828] LC-MS:[M+H]+:312.6

[0829] Synthesis of N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)acetamide:

[0830]

[0831] N,N'-(4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthyl-1,7-diyl)diacetamide 9i (24.00 g, 76.74 mmol) was dissolved in methanol (120 mL) and 2N hydrochloric acid (120 mL), and the mixture was heated to 50 °C and reacted for 7 hours. The reaction was confirmed by LCMS. The reaction solution was concentrated under pressure, dissolved in dichloromethane, and the pH was adjusted to neutral with saturated sodium bicarbonate aqueous solution. The solution was extracted with dichloromethane, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by rapid silica gel column chromatography to give N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthyl-2-yl)acetamide 9j (14.60 g, 53.97 mmol, 70% yield) as a brown solid.

[0832] LC-MS:[M+H]+:270.6

[0833] Synthesis of N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7] indo[1,2-b]quinoline-1-yl)acetamide:

[0834]

[0835] N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)acetamide 9j (14.60 g, 53.97 mmol) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]-indene-3,6,10(4H)-trione 9k (17.10 g, 64.77 mmol) were added to ultra-dry toluene (150 mL), followed by p-toluenesulfonic acid (9.29 g, 53.90 mmol). After the addition was complete, the mixture was heated to 140 °C and reacted for 16 hours. The reaction was confirmed by LCMS. The filter cake was filtered, washed with methyl tert-butyl ether, and concentrated under reduced pressure to give a brown solid N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7] indene[1,2-b]quinoline-1-yl)acetamide 9l (26.00 g, 52.22 mmol, 97% yield).

[0836] LC-MS:[M+H]+:497.6.

[0837] Synthesis of (1S,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzopyrano[3',4':6,7] indo[1,2-b]quinoline-10,13-dione:

[0838]

[0839] 9l (26.00 g, 52.22 mmol) of N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7] indo[1,2-b]quinoline-1-yl)acetamide was added to concentrated hydrochloric acid (260 mL), and the mixture was heated to 70 °C and reacted for 72 hours. The reaction was confirmed by LCMS. The filtrate was filtered and subjected to reversed-phase column chromatography to obtain a brown solid (1S,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzopyrano[3',4':6,7] indo[1,2-b]quinoline-10,13-dione 9 (5.30 g, 11.63 mmol, 22% yield).

[0840] LC-MS:[M+H]+:455.4

[0841] 1H NMR (400MHz, DMSO-d6) δ8.74(s,2H),8.15(d,J=10.4Hz,1H),7.38(s,1H),6.57(s,1H),5.91(d,J=19.6Hz,1H),5.52–5.41(m,3H), 5.13(s,1H),3.43-3.36(m,1H),3.29–3.15(m,1H),2.62-2.58(m,1H),2.26-2.21(m,1H),1.97–1.81(m,2H),0.89(t,J=7.6Hz,3H).

[0842] N1,N1-bis(2-(bis((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)-N5-((7S,10S,13S)-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12) Synthesis of H-benzopyrano[3',4':6,7] indo[1,2-b]quinoline-1-yl)amino)-10-isopropyl-7-methyl-13-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-enamido)-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazaheptadecane-17-yl)pentadiamide:

[0843]

[0844] N1-((7S, 10S, 13S)-13-amino-1-(((S, 9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3', 4': 6,7]indolo[1,2-b]quinoline-1-yl)amino)-10-isopropyl-7-methyl-1,6,9,12-tetraoxo-3-oxo-5,8,11-triazacycloheptane-17-acyl)-N5,N5-bis(2-(bis((2S, 3R, 4R, 5R) 2,3,4,5,6-Pentahydroxyhexyl)amino)ethyl)glutaramide 14 (40.00 mg, 0.024 mmol), 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-alkynic acid 16 (18.16 mg, 0.059 mmol) were dissolved in N,N-dimethylformamide (2 mL), HATU (13.44 mg, 0.036 mmol), HOBt (4.78 mg, 0.036 mmol) and N,N-diisopropylethylamine (9.14 mg, 0.071 mmol) were added, and the mixture was stirred at room temperature for 1 h. After the reaction was monitored by LCMS, the reaction solution was prepared and purified by HPLC. A white solid N1,N1-bis(2-(bis((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)-N5-((7S,10S,13S)-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro- 1H,12H-Benzopyrano[3',4':6,7]indo[1,2-b]quinoline-1-yl)amino)-10-isopropyl-7-methyl-13-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-enamido)-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazaheptadecane-17-yl)glutaramide SJ-LP18 (4.00 mg, 0.0020 mmol, 8% yield).

[0845] LC-MS:[M+H]+:1946.7;

[0846] The retention time Rt of SJ-LP18 by LC-MS was 2.267 min.

[0847] Example 9: Synthesis of SJ-LP17 and SJ-LP20

[0848] Synthesis route:

[0849]

[0850]

[0851] Synthesis of (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanyl-L-propylaminoglycine tert-butyl ester:

[0852]

[0853] (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine 1 (15.00 g, 39.22 mmol) and glycine tert-butyl ester 2 (15.40 g, 117.67 mmol) were dissolved in dichloromethane (300 mL) and tetrahydrofuran (300 mL). 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (29.10 g, 117.67 mmol) was added, and the mixture was purged with nitrogen three times. The reaction was carried out at room temperature for 18 hours. LCMS was used to monitor complete consumption of the starting materials and detection of the product. The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane:methanol = 10:1) to give a white solid (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanyl-L-propylaminoglycine tert-butyl ester 3 (16.00 g, 32.29 mmol, 82% yield).

[0854] LC-MS: [M+Na]+: 518.4.

[0855] Synthesis of (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanyl-L-alanylglycine:

[0856]

[0857] (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanyl-L-propylaminoglycine tert-butyl ester 3 (16.00 g, 32.29 mmol) was dissolved in dichloromethane (180 mL), and trifluoroacetic acid (60 mL) was added dropwise at 0 °C. The mixture was purged with nitrogen three times, and the reaction was carried out at room temperature for 18 hours. LC-MS monitoring showed complete consumption of the starting material and detection of the product. The reaction solution was concentrated under reduced pressure, and the residue was slurried with diethyl ether, filtered, and the filter cake was concentrated to give a pink solid (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanyl-L-propylaminoglycine 4 (13.60 g, 30.95 mmol, 96% yield).

[0858] LC-MS:[M+H]+:440.2.

[0859] Synthesis of (5S,8S)-1-(9H-fluorene-9-yl)-5,8-dimethyl-3,6,9-trioxo-2-oxo-4,7,10-triazaundecane-11-ylacetate:

[0860]

[0861] (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanyl-L-propylaminoglycine 4 (5.00 g, 11.38 mmol), lead tetraacetate (6.10 g, 13.65 mmol), and pyridine (1.10 mL, 13.65 mmol) were dissolved in tetrahydrofuran (120 mL) and toluene (30 mL), purged three times with nitrogen, and the reaction was slowly heated to 80 °C for 18 hours. The reaction solution was concentrated, and the residue was purified by column chromatography (dichloromethane:methanol = 10:1) to give a white solid, (5S,8S)-1-(9H-fluorene-9-yl)-5,8-dimethyl-3,6,9-trioxo-2-oxo-4,7,10-triazaundecane-11-ylacetate 5 (2.0 g, 4.41 mmol, 39% yield).

[0862] LC-MS: [M+Na]+: 476.2.

[0863] Synthesis of (5S,8S)-1-(9H-fluoren-9-yl)-5,8-dimethyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazatetradecane-14-acid benzyl ester:

[0864]

[0865] (5S,8S)-1-(9H-fluorene-9-yl)-5,8-dimethyl-3,6,9-trioxo-2-oxo-4,7,10-triazaundecane-11-yl acetate 5 (2.00 g, 4.41 mmol) and 2-hydroxyacetic acid benzyl ester 6 (0.73 g, 4.41 mmol) were dissolved in tetrahydrofuran (20 mL), and 4A molecular sieves were added. After stirring at room temperature for 0.5 hours, scandium trifluoromethanesulfonate (2.20 g, 4.41 mmol) was added, and the reaction was carried out at room temperature for 2 hours. LCMS monitoring showed complete consumption of the starting materials and detection of the product. The reaction solution was filtered, concentrated under reduced pressure, and purified by column chromatography (dichloromethane: ethyl acetate = 2:3). The purified solution was concentrated to give a white solid, (5S,8S)-1-(9H-fluorene-9-yl)-5,8-dimethyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazatetradecane-14-acid benzyl ester 7 (500.00 mg, 0.89 mmol, 20% yield).

[0866] LC-MS: [M+Na]+: 582.4.

[0867] Synthesis of (5S,8S)-1-(9H-fluorene-9-yl)-5,8-dimethyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazatetradecane-14-acid:

[0868]

[0869] (5S,8S)-1-(9H-fluorene-9-yl)-5,8-dimethyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazatetradecane-14-acid benzyl ester 7 (500.00 mg, 0.89 mmol) was dissolved in tetrahydrofuran (5 mL), palladium on carbon (50.00 mg), 10% Pd / C was added, and the mixture was purged three times with hydrogen. The reaction was carried out at room temperature for 2 hours. LC-MS monitoring showed complete consumption of the starting material and detection of the product. The reaction solution was filtered and concentrated under reduced pressure to give a white solid (5S,8S)-1-(9H-fluorene-9-yl)-5,8-dimethyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazatetradecane-14-acid 8 (417.85 mg, 0.89 mmol, equivalence yield).

[0870] LC-MS: [M+Na]+: 492.2

[0871] Synthesis of (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-(((2-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)carbamate:

[0872]

[0873] (5S,8S)-1-(9H-fluorene-9-yl)-5,8-dimethyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazatetradecane-14-acid 8 (417.85 mg, 0.89 mmol) was dissolved in N,N-dimethylformamide (5 mL), and 1-hydroxybenzotriazole (185.60 mg, 1.37 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (520.93 mg) were added. (mg, 1.37mmol) and N,N-diisopropylethylamine (0.35g, 2.67mmol), stirred at room temperature for 10 minutes, then (1S,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzopyrano[3',4':6,7] indo[1,2-b]quinoline-10,13-dione 9 (417.50mg, 0.92mmol) were added, and the reaction was carried out at room temperature for 2 hours. The reaction solution was filtered and purified by reverse-phase column chromatography (water: acetonitrile = 50:50). After lyophilization, a white solid (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-(((2-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indene[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)carbamate 10 (100.00 mg, 0.11 mmol, 12% yield) was obtained.

[0874] LCMS:[MH]+:906.5

[0875] Synthesis of (S)-2-amino-N-((S)-1-(((2-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)1-oxopropane-2-yl)propionamide:

[0876]

[0877] Add 100.00 mg (0.11 mmol) of (9H-fluorene-9-yl)methyl((S)-1-((S,-1-(2-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indene[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)carbamate 10 (100.00 mg, 0.11 mmol) to DMF (2 mL), add diethylamine (0.11 mL, 1.10 mmol), and stir at room temperature for one hour. At that time, LCMS showed that the reaction was complete. The oil pump was used to evaporate the solution, 2 mL of ethyl acetate was added, and the mixture was ultrasonically slurried and filtered to obtain a gray solid (S)-2-amino-N-((S)-1-(((2-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropane-2-yl)propionamide 11 (80.00 mg, 0.11 mmol, equivalent yield).

[0878] LCMS:[MH]+:684.5

[0879] (9H-fluorene-9-yl)methyl((7S,10S,13S,29S,30R,31R,32R)-24-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano) Synthesis of [3', 4': 6,7]indo[1,2-b]quinoline-1-yl)amino)-29,30,31,32,33-pentahydroxy-7,10-dimethyl-1,6,9,12,19,23-hexaoxo-27-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3-oxo-5,8,11,18,24,27-hexaazatriazacyclopentane-13-yl)carbamate:

[0880]

[0881] (S)-2-amino-N-((S)-1-(((2-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropane-2-yl)propionamide 11 (80.00 mg, 0.11 mmol), N2-(((9H-fluorene-9-yl)methoxy)carbonyl 12 lysine (142.96 mg, 0.12 mmol) was dissolved in N,N-dimethylformamide (2 mL), and HATU (66.60 mg, 0.18 mmol), HOBt (23.67 mg, 0.18 mmol) and N,N-diisopropylethylamine (0.058 mL, 0.35 mmol) were added. The mixture was stirred at room temperature for 2 hours. The reaction was monitored by LCMS until completion. The solution was directly purified by reverse-phase reaction (acetonitrile / water = 30 / 70) to give a white solid (9H-fluorene-9-yl)methyl((7S,10S,13S,29S,30R,31R,32R)-24-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,1) 2H-Benzopyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-29,30,31,32,33-pentahydroxy-7,10-dimethyl-1,6,9,12,19,23-hexaoxo-27-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3-oxo-5,8,11,18,24,27-hexaazatriazacyclopentane-13-yl)carbamate 13 (100.00 mg, 0.053 mmol, 48% yield).

[0882] LCMS:[MH]+:1890.7

[0883] Synthesis of N1-((7S, 10S, 13S)-13-amino-1-(((1S, 9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3', 4': 6,7]indolo[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazacycloheptane-17-yl)-N5-bis(2-(bis((2S, 3R, 4R, 5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)pentadiamide:

[0884]

[0885] (9H-fluorene-9-yl)methyl((7S,10S,13S,29S,30R,31R,32R)-24-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1) H,12H-Benzopyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-29,30,31,32,33-pentahydroxy-7,10-dimethyl-1,6,9,12,19,23-hexoxo-27-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3-oxo-5,8,11,18,24,27-hexaza Triazacyclopentan-13-yl)carbamate 13 (100.00 mg, 0.053 mmol) was added to DMF (2 mL), followed by diethylamine (0.055 mL, 0.53 mmol). The mixture was stirred at room temperature for one hour. LC-MS showed that the reaction was complete. The mixture was evaporated to dryness using an oil pump, and 2 mL of ethyl acetate was added. The mixture was then sonicated and filtered to obtain a gray solid, N1-((7S, 10S, 13S)-13-amino-1-yl)carbamate 13. (((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazacycloheptane-17-yl)-N5N5-bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)glutaramide 14 (100 mg, 0.053 mmol, equivalent yield).

[0886] LCMS:[MH]+:1667.4

[0887] N1,N1-bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-N5-((7S,10S,13S)-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,1) Synthesis of 2H-benzopyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-13-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoamide)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxo-5,8,11-triazaheptane-17-yl)pentanediamide:

[0888]

[0889] N1-((7S, 10S, 13S)-13-amino-1-(((1S, 9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazacycloheptane-17-yl)-N5 N5-bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)pentadiamide 14 (55.00 mg, 0.033 mmol) and 2,5-dioxopyrrolidone-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoate 15 (8.84 mg, 0.033 mmol) were dissolved in N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (0.016 mL, 0.099 mmol) was added. The mixture was stirred at room temperature for 2 hours. The reaction was monitored by LCMS until completion. Formic acid was added to adjust the pH to weakly acidic. HPLC purification was then performed using a 0.05% formic acid system to obtain a white solid, N1,N1-bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-N5-((7S,10S,13S)-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13, 15-Hexahydro-1H,12H-Benzopyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-13-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexamamide)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxo-5,8,11-triazaheptane-17-yl)glutaramide SJ-LP17 (5.00 mg, 0.0027 mmol, 8% yield).

[0890] LCMS:[MH]+:1861.8;

[0891] The retention time Rt of SJ-LP17 by LC-MS was 2.66 min.

[0892] N1,N1-bis(2-(bis((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)-N5-((7S,10S,13S)-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, Synthesis of 12H-benzopyrano[3',4':6,7]-indo[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-13-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-enamido)-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazaheptadecane-17-yl)pentadiamide:

[0893]

[0894] N1-((7S, 10S, 13S)-13-amino-1-(((1S, 9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazacycloheptane-17-yl)-N5 N5-bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)glutaramide 14 (55.00 mg, 0.033 mmol) and 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-alkynyl acid 16 (8.84 mg, 0.033 mmol) were dissolved in N,N-dimethylformamide (2 mL), and HATU (18.82 mg, 0.050 mmol), HOBt (6.69 mg, 0.050 mmol), and N,N-diisopropylethylamine (0.016 mL, 0.099 mmol) were added. The mixture was stirred at room temperature for 1 hour. After the reaction was monitored by LCMS, the mixture was purified by HPLC in a 0.05% formic acid system to give a white solid N1,N1-bis(2-(bis((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)-N5-((7S,10S,13S)-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro- 1H,12H-Benzopyrano[3',4':6,7]indo[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-13-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-enamido)-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazaheptadecane-17-yl)glutaramide SJ-LP20 (8.00 mg, 0.0040 mmol, 12% yield).

[0895] LCMS:[MH]+:1918.6;

[0896] The retention time Rt of SJ-LP20 by LC-MS was 2.387 min.

[0897] Example 10: Synthesis of SJ-LP30

[0898] Synthesis route:

[0899]

[0900] Synthesis of (5S,8S,13S)-1-(9H-fluorene-9-yl)-5,8,13-trimethyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazatetradecane-14-acid benzyl ester:

[0901]

[0902] (5S,8S)-1-(9H-fluorene-9-yl)-5,8-dimethyl-3,6,9-trioxo-2-oxo-4,7,10-triazaundecane-11-yl acetate 5 (1.00 g, 2.21 mmol) and (2S)-2-hydroxypropanoate benzyl ester 6 (1.99 g, 11.03 mmol) were dissolved in tetrahydrofuran (30 mL), and 4A molecular sieves were added. The mixture was stirred at room temperature for 0.5 h. Scandium trifluoromethanesulfonate (1.30 g, 2.65 mmol) was added at room temperature, and the reaction was carried out at room temperature for 2 h. The starting materials were completely consumed and the product was detected by LCMS. The reaction solution was filtered, water (50 mL) was added, and the mixture was extracted with ethyl acetate (50 mL * 3). The organic phases were combined, dried over anhydrous sodium sulfate, and the residue was concentrated under reduced pressure and purified by column chromatography (dichloromethane: ethyl acetate = 1:1) to give a white solid product (5S, 8S, 13S)-1-(9H-fluorene-9-yl)-5,8,13-trimethyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazatetradecane-14-acid benzyl ester 7 (0.36 g, 0.63 mmol, 28% yield).

[0903] LC-MS: [M+Na]+: 595.6

[0904] Synthesis of (5S, 8S, 13S)-1-(9H-fluorene-9-yl)-5,8,13-trimethyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazatetradecane-14-acid:

[0905]

[0906] (5S,8S,13S)-1-(9H-fluorene-9-yl)-5,8,13-trimethyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazatetradecane-14-acid benzyl ester 7 (360.00 mg, 0.63 mmol) was dissolved in tetrahydrofuran (5 mL), and palladium on carbon (Pd / C, 10%) (72.00 mg) was added. The mixture was stirred at room temperature for 3 h. LCMS monitoring showed complete consumption of the starting material and detection of the product. The reaction solution was filtered and concentrated under reduced pressure to give a white solid (5S,8S,13S)-1-(9H-fluorene-9-yl)-5,8,13-trimethyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazatetradecane-14-acid 8 (300.00 mg, 0.62 mmol, 99% yield).

[0907] LC-MS: [M+Na]+: 505.6

[0908] Synthesis of (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-10,13-dione:

[0909]

[0910] (5S,8S,13S)-1-(9H-fluorene-9-yl)-5,8,13-trimethyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazatetradecane-14-acid 8 (300.00 mg, 0.62 mmol) was dissolved in N,N-dimethylformamide (10 mL), and 1-hydroxybenzotriazole (125.80 mg, 0.93 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (353 mg, 10 mL, 10 mL, 10 mL, 10 mL, 125.80 mg, 0.93 mmol) were added. 0.90 mg (0.93 mmol) and N,N-diisopropylethylamine (0.31 mL, 1.86 mmol) were stirred for 10 minutes, and then (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7] indo[1,2-b]quinoline-10,13-dione 9 (270.20 mg, 0.62 mmol) were added. The reaction was carried out at room temperature for 2 hours. The starting material was completely consumed and the product was detected by LCMS. The reaction solution was filtered, purified by reverse-phase purification (pure water: acetonitrile = 1:1), and lyophilized to give a yellow solid (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-10,13-dione (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10H,13H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-10,13-dione 10 (300.00 mg, 0.33 mmol, 54% yield).

[0911] LCMS:[M+H]+:900.8

[0912] Synthesis of (S)-2-amino-N-((S)-1-(((((S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-1-oxopropane-2-yl)oxy)methyl)amino)1-oxopropane-2-yl)propionamide:

[0913]

[0914] (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-10,13-dione 10 (300.00 mg, 0.33 mmol) was dissolved in N,N-dimethylformamide (5 mL), and diethylamine (0.34 mL, 3.33 mmol) was added. After stirring at room temperature for 1 hour, LCMS showed that the starting material was completely consumed and the product was detected. The reaction solution was concentrated, slurried with ethyl acetate, filtered, and the filter cake was concentrated under reduced pressure to obtain a yellow solid (S)-2-amino-N-((S)-1-(((((S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-1-oxopropane-2-yl)oxy)methyl)amino)-1-oxopropane-2-yl)propionamide 11 (210.00 mg, 0.31 mmol, 93% yield).

[0915] LCMS:[M+H]+:678.8

[0916] (9H-fluorene-9-yl)methyl((2S,7S,10S,13S,29S,30R,31R,32R)-24-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]) Synthesis of pyran[3',4':6,7]indole[1,2-b]quinoline-1-yl)amino)-29,30,31,32,33-pentahydroxy-2,7,10-trimethyl-1,6,9,12,19,23-hexaoxo-27-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3-oxo-5,8,11,18,24,27-hexaazatriazacyclopentane-13-yl)carbamate:

[0917]

[0918] N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(5-(bis(2-(bis((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)amino)-5-oxopentanoyl)-L-lysine 12 (378.80 mg, 0.31 mmol), (S)-2-amino-N-((S)-1-((((S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4'] [6,7]Indo[1,2-b]quinoline-1-yl)amino)-1-oxopropane-2-yl)oxy)methyl)amino)-1-oxopropane-2-yl)propionamide 11 (210.00 mg, 0.31 mmol) was dissolved in N,N-dimethylformamide (2 mL), and 1-hydroxybenzotriazole (62.70 mg, 0.46 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (176.50 mg, 0.46 mmol) and N,N-diisopropylethylamine (0.15 mL, 0.93 mmol) were added. The reaction was carried out at room temperature for 2 hours. LCMS showed that the starting material was completely consumed and the product was detected. The reaction solution was filtered, purified by reverse-phase purification (pure water: acetonitrile = 1:1), and lyophilized to obtain a yellow solid (9H-fluorene-9-yl)methyl((2S,7S,10S,13S,29S,30R,31R,32R)-24-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H) -Benz[de]pyran[3',4':6,7]indol[1,2-b]quinoline-1-yl)amino)-29,30,31,32,33-pentahydroxy-2,7,10-trimethyl-1,6,9,12,19,23-hexaoxo-27-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3-oxo-5,8,11,18,24,27-hexaazatriazacyclopentane-13-yl)carbamate 13 (240.00 mg, 0.13 mmol, 41% yield).

[0919] LCMS:1 / 2[M+2H]+:942.4

[0920] Synthesis of N1-((2S,7S,10S,13S)-13-amino-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7]indo[1,2-b]quinoline-1-yl)amino)-2,7,10-trimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazaheptadecane-17-yl)-N5,N5-bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)pentadiamide:

[0921]

[0922] (9H-fluorene-9-yl)methyl((2S,7S,10S,13S,29S,30R,31R,32R)-24-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indol[1,2-b]quinoline-1-yl)amino)-29 30,31,32,33-Pentahydroxy-2,7,10-trimethyl-1,6,9,12,19,23-hexano-27-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3-oxo-5,8,11,18,24,27-hexaazatriazacyclopentan-13-yl)carbamate 13 (240.00 mg, 0.13 mmol) was dissolved in N,N-dimethylformamide (5 mL), and diethylamine (0.13 mL, 1.27 mmol) was added. The reaction was carried out at room temperature for 0.5 h. LC-MS showed that the starting material was completely consumed and the product was detected. The reaction solution was concentrated, ethyl acetate was added and the mixture was stirred, filtered, and the filter cake was concentrated under reduced pressure to obtain a yellow solid product N1-((2S,7S,10S,13S)-13-amino-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran [3',4':6,7] indene[ [1,2-b]quinoline-1-yl)amino)-2,7,10-trimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazaheptadecane-17-yl)-N5,N5bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)pentadiamide 14 (200.00 mg, 0.12 mmol, 94% yield).

[0923] LCMS:[M+H]+:1662.1

[0924] N1,N1bis(2-(bis((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)-N5-((2S,7S,10S,13S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,1) Synthesis of 2H-benzo[de]pyran[3',4':6,7]indo[1,2-b]quinoline-1-yl)amino)-2,7,10-trimethyl-13-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-enamido)-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazaheptadecane-17-yl)pentadiamide:

[0925]

[0926] 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl acetic acid 15 (32.30 mg, 0.12 mmol) was dissolved in N,N-dimethylformamide (5 mL), followed by the addition of 1-hydroxybenzotriazole (24.40 mg, 0.18 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (68.60 mg, 0.18 mmol), and N,N-diisopropylethylamine (0.11 mL, 0.36 mmol). After stirring for 10 minutes, N1-((2S,7S,10S,13S)-13-amino-1-(((1S,9S)- 9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7]indo[1,2-b]quinoline-1-yl)amino)-2,7,10-trimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazaheptadecane-17-yl)-N5,N5bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)pentanediamide 14 (200.00 mg, 0.12 mmol), reacted at room temperature for 1 hour. The starting material was completely consumed and the product was detected by LCMS. The reaction solution was filtered, purified by Prep-HPLC reverse phase (formic acid system), and lyophilized to obtain a white solid product N1,N1bis(2-(bis((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)-N5-((2S,7S,10S,13S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- Hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indo[1,2-b]quinoline-1-yl)amino)-2,7,10-trimethyl-13-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-enamido)-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazaheptadecane-17-yl)glutaramide SJ-LP30 (21.50 mg, 0.011 mmol, 9% yield).

[0927] LCMS: 1 / 2[M+2H]+: 956.6;

[0928] The retention time Rt of SJ-LP30 by LC-MS was 2.106 min.

[0929] Example 11: Synthesis of SJ-LP31

[0930] Synthesis route:

[0931]

[0932] Synthesis of (5S, 8S, 13S)-1-(9H-fluorene-9-yl)-5,8,13-trimethyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazapentadecan-15-acid:

[0933]

[0934] (5S,8S)-1-(9H-fluorene-9-yl)-5,8-dimethyl-3,6,9-trioxo-2-oxo-4,7,10-triazaundecane-11-ylacetate 1 (500.00 mg, 1.10 mmol) was dissolved in dichloromethane (5 mL), and (S)-3-hydroxybutyric acid 2 (459.00 mg, 4.41 mmol) and trifluoroacetic acid (251.00 mg, 2.20 mmol) were slowly added. After the addition was complete, the mixture was reacted at room temperature for 3 hours. The reaction was confirmed by LCMS. The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane:methanol = 10:1 system) to give a white solid product (5S,8S,13S)-1-(9H-fluorene-9-yl)-5,8,13-trimethyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazapentadecan-15-acid 3 (140.00 mg, 0.28 mmol, 26% yield).

[0935] LC-MS: [M+Na] + 519.6

[0936] Synthesis of (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-(((((S)-4-(((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]indolazino[1,2-b]quinoline-1-yl)amino)-4-oxobutane-2-yl)oxy)methyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)carbamate:

[0937]

[0938] (5S,8S,13S)-1-(9H-fluorene-9-yl)-5,8,13-trimethyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazapentadecan-15-acid 3 (140.00 mg, 0.28 mmol) was dissolved in N,N-dimethylformamide (3 mL), and then (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzopyrene The reaction mixture of nazo[3',4':6,7]-indo[1,2-b]quinoline-10,13-dione 4 (98.00 mg, 0.23 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (160.00 mg, 0.42 mmol), 1-hydroxybenzotriazole (57.00 mg, 0.42 mmol) and N,N-diisopropylethylamine (109.00 mg, 0.84 mmol) was stirred at room temperature for 1 hour. The reaction was confirmed by LCMS. The reaction solution was filtered, purified by reverse-phase purification (pure water: acetonitrile = 1:1), and lyophilized to give a white solid product (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-(((((S)-4-(((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]indolazino[1,2-b]quinoline-1-yl)amino)-4-oxobutane-2-yl)oxy)methyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)carbamate 5 (140.00 mg, 0.15 mmol, 54% yield).

[0939] LC-MS:[M+H]+:914.8.

[0940] Synthesis of (S)-3-(((S)-2-((S)-2-aminopropamido)propamido)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indo[1,2-b]quinoline-1-yl)butyramide:

[0941]

[0942] (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-(((((S)-4-(((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]indolazino[1,2-b]quinoline-1-yl)amino)-4-oxobutane-2-yl)oxy)methyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)carbamate 5 (140.00 mg, 0.15 mmol) was dissolved in N,N-dimethylformamide (5 mL), and then diethylamine (112.00 mg, 1.53 mmol) was added. After the addition was complete, the reaction was stirred at room temperature for 1 hour. The reaction was confirmed by LCMS. The reaction solution was concentrated, slurried with ethyl acetate, filtered, and the filter cake was concentrated under reduced pressure to obtain a yellow solid product (S)-3-(((S)-2-((S)-2-aminopropamido)propamido)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indene[1,2-b]quinoline-1-yl)butyramide 6 (110.00 mg, 0.14 mmol, 93% yield).

[0943] LC-MS:[M+H]+:692.7

[0944] (9H-fluorene-9-yl)methyl((3S,8S,11S,14S,30S,31R,32R,33R)-25-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran) Synthesis of [3', 4': 6, 7] indo[1,2-b]quinoline-1-yl)amino)-30,31,32,33,34-pentahydrohydroxy-3,8,11-trimethyl-1,7,10,13,20,24-hexaoxo-28-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-4-oxa-6,9,12,19,25,28-hexaazatetrazane-14-yl)carbamate:

[0945]

[0946] Dissolve (S)-3-(((S)-2-((S)-2-aminopropamido)propamido)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indene[1,2-b]quinoline-1-yl)butyramide 6 (110.00 mg, 0.14 mmol) in N,N-dimethylformamide (5 mL), then add N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6 -(5-(bis(2-(bis((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)amino)-5-oxopentanoyl)-L-lysine 7 (203.00 mg, 0.16 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (94.00 mg, 0.24 mmol), 1-hydroxybenzotriazole (34.00 mg, 0.24 mmol) and N,N-diisopropylethylamine (65.00 mg, 0.49 mmol), the reaction was stirred at room temperature for 1 hour. LCMS analysis confirmed the reaction was complete. The reaction solution was filtered, purified by reverse-phase purification (pure water: acetonitrile = 1:1), and lyophilized to obtain a white solid product: (9H-fluorene-9-yl)methyl((3S,8S,11S,14S,30S,31R,32R,33R)-25-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro) -1H,12H-benzopyran[3',4':6,7]indo[1,2-b]quinoline-1-yl)amino)-30,31,32,33,34-pentahydrohydroxy-3,8,11-trimethyl-1,7,10,13,20,24-hexaoxo-28-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-4-oxa-6,9,12,19,25,28-hexaazatetrazane-14yl)carbamate 8 (140.00 mg, 0.070 mmol, 44% yield).

[0947] LC-MS:[M+H]+:1898.2

[0948] Synthesis of N1-((3S,8S,11S,14S)-14-amino-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7]indo[1,2-b]quinoline-1-yl)amino)-3,8,11-trimethyl-1,7,10,13-tetraoxo-4-oxa-6,9,12-triazaoctadecane-18-yl)-N5,N5-bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)pentadiamide:

[0949]

[0950] The (9H-fluorene-9-yl)methyl((3S,8S,11S,14S,30S,31R,32R,33R)-25-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indene[1,2-b]quinoline-1-yl)amino)-30,31 32,33,34-Pentahydrohydroxy-3,8,11-trimethyl-1,7,10,13,20,24-hexaoxo-28-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-4-oxa-6,9,12,19,25,28-hexaazatetrazane-14yl)carbamate 8 (140.00 mg, 0.070 mmol) was dissolved in N,N-dimethylformamide (2 mL), and then diethylamine (57.00 mg, 0.77 mmol) was added. After the addition was complete, the reaction was stirred at room temperature for 1 hour. The reaction was detected by LCMS to indicate completion. The reaction solution was concentrated, slurried with ethyl acetate, filtered, and the filter cake was concentrated under reduced pressure to obtain a yellow solid product N1-((3S,8S,11S,14S)-14-amino-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indene[1, [2-b] Quinolin-1-yl)amino)-3,8,11-trimethyl-1,7,10,13-tetraoxo-4-oxa-6,9,12-triazaoctadecane-18-yl)-N5,N5-bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)pentadiamide 9 (120.00 mg, 0.060 mmol, 92% yield).

[0951] LC-MS:[M+H]+:1676.1

[0952] N1,N1bis(2-(bis((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)-N5-((3S,8S,11S,14S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, Synthesis of 12H-benzopyran[3',4':6,7] indo[1,2-b]quinoline-1-yl)amino)-3,8,11-trimethyl-14-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-enamido)-1,7,10,13-tetraoxo-4-oxa-6,9,12-triazaoctadecane-18-yl)pentadiamide:

[0953]

[0954] The N1-((3S,8S,11S,14S)-14-amino-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indene[1,2-b]quinoline-1-yl)amino)-3,8,11-trimethyl-1,7,10,13-tetraoxo-4-oxa-6,9,12-triazaoctadecane-18-yl)-N5,N5-bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino 9-(7-Azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (42.00 mg, 0.12 mmol) was dissolved in 2 mL of N,N-dimethylformamide. 1-hydroxybenzotriazole (15.00 mg, 0.12 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (42.00 mg, 0.12 mmol), and N,N-diisopropylethylamine (28.00 mg, 0.21 mmol) were added. After stirring for 10 minutes, 10-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ethynic acid 10 (19.00 mg, 0.060 mmol) was added, and the reaction was carried out at room temperature for 1 hour. LC-MS monitoring showed complete consumption of the starting material and detection of the product. The reaction solution was filtered, purified by Prep-HPLC reverse phase (formic acid system), and lyophilized to obtain a white solid product N1,N1bis(2-(bis((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)-N5-((3S,8S,11S,14S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- Hexahydro-1H,12H-benzopyran[3',4':6,7] indene[1,2-b]quinoline-1-yl)amino)-3,8,11-trimethyl-14-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-enamido)-1,7,10,13-tetraoxo-4-oxa-6,9,12-triazaoctadecane-18-yl)glutaramide SJ-LP31 (19.10 mg, 0.010 mmol, 13% yield).

[0955] LC-MS:[M+H]+:1926.3

[0956] The retention time Rt of SJ-LP31 by LC-MS was 2.125 min.

[0957] Example 12: Synthesis of SJ-LP33

[0958] Synthesis route:

[0959]

[0960] Synthesis of (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzopyran[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-acyl)carbamate:

[0961]

[0962] (5S,8S)-1-(9H-fluorene-9-yl)-5,8-dimethyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazatetradecane-14-acid 8 (500.00 mg, 1.07 mmol) was dissolved in N,N-dimethylformamide (20 mL), and 1-hydroxybenzotriazole (215.90 mg, 1.60 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (6) were added. 0.40 mg (1.60 mmol) and N,N-diisopropylethylamine (0.53 mL, 3.20 mmol) were stirred for 10 minutes, and then (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydrobenzopyrano[3',4':6,7]-aza[1,2-b]quinoline-10,13(1H,9H)-dione 9 (463.8 mg, 1.07 mmol) were added. The reaction was carried out at room temperature for 4 hours. The starting material was completely consumed and the product was detected by LCMS. The reaction solution was filtered, purified by reverse-phase purification (pure water: acetonitrile = 1:1), and lyophilized to give a yellow solid product (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzopyran[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-acyl)carbamate 10 (220.00 mg, 23% yield).

[0963] LCMS:[M+H]+:886.6

[0964] Synthesis of (S)-2-amino-N-((S)-1-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzopyran[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)1-oxopropane-2-yl)propionamide:

[0965]

[0966] (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzopyran[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-acyl)carbamate 10 (220.00 mg, 0.25 mmol) was dissolved in N,N-dimethylformamide (5 mL), and diethylamine (0.26 mL, 2.48 mmol) was added. After stirring at room temperature for 30 minutes, LCMS showed that the starting material was completely consumed and the product was detected. The reaction solution was concentrated, and the residue was pulped with ethyl acetate, filtered, and the filter cake was concentrated under reduced pressure to obtain a yellow solid product (S)-2-amino-N-((S)-1-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzopyran[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropane-2-yl)propionamide 11 (180.00 mg, 98% yield).

[0967] LCMS:[M+H]+:664.7

[0968] (9H-fluorene-9-yl)methyl((7S,10S,13S,29S,30R,31R,32R)-24-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo) [3', 4': 6,7] Indo[1,2-quinolin-1-yl)amino)-29,30,31,32,33-pentahydroxy-7,10-dimethyl-1,6,9,12,19,23-hexoxo-27-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3-oxo-5,8,11,18,24,27-hexaazatriazacyclopentane-13-yl)carbamate

[0969] Synthesis:

[0970]

[0971] The following ingredients were added: (S)-2-amino-N-((S)-1-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzopyran[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropane-2-yl)propionamide 11 (180.00 mg, 0.27 mmol), N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(5-(bis(2-(bis((2) S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)amino)-5-oxopentanoyl)-L-lysine 12 (331.60 mg, 0.27 mmol) was dissolved in N,N-dimethylformamide (5 mL), followed by the addition of 1-hydroxybenzotriazole (54.90 mg, 0.41 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (154.50 mg, 0.41 mmol), and N,N-diisopropylethylamine (0.13 mL, 0.81 mmol). The reaction mixture was incubated at room temperature for 2 hours. LC-MS analysis showed complete consumption of the starting material and detection of the product. The reaction solution was filtered, purified by reverse-phase purification (pure water: acetonitrile = 1:1), and lyophilized to obtain a yellow solid product (9H-fluorene-9-yl)methyl((7S, 10S, 13S, 29S, 30R, 31R, 32R)-24-(2-(bis((2S, 3R, 4R, 5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-1-(((1S, 9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,1) 3,15-Octahydrobenzo[3',4':6,7]indolo[1,2-quinolin-1-yl)amino)-29,30,31,32,33-pentahydroxy-7,10-dimethyl-1,6,9,12,19,23-hexaoxo-27-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3-oxo-5,8,11,18,24,27-hexaazatriazacyclopentane-13-yl)carbamate 13 (220.00 mg, 43% yield).

[0972] LCMS:[M+H]+:1870.2

[0973] Synthesis of N1-((7S, 10S, 13S)-13-amino-1-(((1S, 9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzopyrano[3', 4': 6,7]indolo[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazohepane-17-yl)-N5,N5-bis(2-(bis((2S, 3R, 4R, 5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)pentadiamide:

[0974]

[0975] (9H-fluorene-9-yl)methyl((7S,10S,13S,29S,30R,31R,32R)-24-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[3',4':6,7]indolo[1,2-quinoline-1-yl)amino)-29,30,3 1,32,33-pentahydroxy-7,10-dimethyl-1,6,9,12,19,23-hexano-27-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-3-oxo-5,8,11,18,24,27-hexaazatriazacyclopentan-13-yl)carbamate 13 (220.00 mg, 0.12 mmol) was dissolved in N,N-dimethylformamide (5 mL), and diethylamine (0.12 mL, 1.18 mmol) was added. The reaction was carried out at room temperature for 0.5 h. LC-MS showed that the starting material was completely consumed and the product was detected. The reaction solution was concentrated, ethyl acetate was added and the mixture was stirred, filtered, and the filter cake was concentrated under reduced pressure to obtain a yellow solid product N1-((7S, 10S, 13S)-13-amino-1-(((1S, 9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzopyrano[3', 4': 6,7]in Dodo[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazolidone-17-yl)-N5,N5-bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)pentadiamide 14 (180.00 mg, 93% yield).

[0976] LCMS:[M+H]+:1648.2

[0977] Synthesis of N1,N1 bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-N5-((7S,10S,13S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzopyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-13(6-(2-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamido)-1,6,9,12-tetraoxo-3-oxo-5,8,11-triazaheptadecane-17-yl)pentadiamide:

[0978] 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl acetic acid 15 (29.30 mg, 0.11 mmol) was dissolved in N,N-dimethylformamide (5 mL), followed by the addition of 1-hydroxybenzotriazole (22.10 mg, 0.16 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (62.30 mg, 0.16 mmol), and N,N-diisopropylethylamine (0.054 mL, 0.33 mmol). After stirring for 10 minutes, N1-((7S, 10S, 13S)-13-amino-1-(((1S, 9S) 9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzopyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazohepane-17-yl)-N5,N5-bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)pentanediamide 14 (180.00 mg, 0.11 mmol), reacted at room temperature for 1 hour. The starting material was completely consumed and the product was detected by LCMS. The reaction solution was filtered, purified by Prep-HPLC (formic acid system), and lyophilized to obtain a white solid product N1,N1bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-N5-((7S,10S,13S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9, 10,12,13,15-Octahydrobenzopyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-13(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamido)-1,6,9,12-tetraoxo-3-oxo-5,8,11-triazaheptadecane-17-yl)glutaramide SJ-LP33 (20.20 mg, 10% yield).

[0979] LCMS:[M+H]+:1898.4;

[0980] The retention time Rt of SJ-LP33 by LC-MS was 2.049 min.

[0981] Example 13: Synthesis of SJ-LP34

[0982] Synthesis route:

[0983]

[0984] Synthesis of (S)-N-((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] indo[1,2-b]quinoline-1-yl)-3-hydroxy-2-methylpropionamide:

[0985]

[0986] Dissolve (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzopyrano[3',4':6,7] indo[1,2-b]quinoline-10,13-dione 1 (300.00 mg, 0.69 mmol) in N,N-dimethylformamide (15.00 mL), and slowly add (S)3-hydroxy 2-Methylpropionic acid 2 (107.00 mg, 1.03 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (524.00 mg, 1.38 mmol), 1-hydroxybenzotriazole (186.00 mg, 1.38 mmol), and N,N-diisopropylethylamine (267.00 mg, 2.07 mmol) were added, and the reaction was carried out at room temperature for 1 hour. The reaction was confirmed by LCMS. The reaction solution was filtered, purified by reverse-phase purification (pure water: acetonitrile = 1:1), and lyophilized to give a yellow solid product (S).

[0987] -N-((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] indo[1,2-b]quinoline-1-yl)-3-hydroxy-2-methylpropionamide SJ-LP40 (350.00 mg, 0.67 mmol, 57% yield).

[0988] LC-MS:[M+H]+:521.6

[0989] Synthesis of (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((((S)-3-(((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]indolo[1,2-b]quinoline-1-yl)amino)-2-methyl-3-oxopropoxy)methyl)amino)-1-oxoprop-2-yl)amino)-1-oxopropane-2-yl)carbamate:

[0990]

[0991] Dissolve ((S)-N-((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] indo[1,2-b]quinoline-1-yl)-3-hydroxy-2-methylpropionamide SJ-LP40 (350.00 mg, 0.67 mmol) in tetrahydrofuran (5 mL) Add (5S,8S)-1-(9H-fluorene-9-yl)-5,8-dimethyl-3,6,9-trioxo-2-oxo-4,7,10-triazaundecane-11-ylacetate 3 (304.00 mg, 0.67 mmol) and boron trifluoride diethyl ether (190.00 mg, 1.34 mmol) to the solution. Stir the mixture at room temperature for 1 hour. The reaction was confirmed by LCMS. The reaction solution was filtered and purified by reverse-phase chromatography (using pure water).

[0992] After purification and lyophilization with acetonitrile (1:1), a white solid product (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((((S)-3-(((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]indolo[1,2-b]quinoline-1-yl)amino)-2-methyl-3-oxopropoxy)methyl)amino)-1-oxoprop-2-yl)amino)-1-oxopropane-2-yl)carbamate 4 (350.00 mg, 0.38 mmol, 57% yield) was obtained.

[0993] LC-MS:[M+H]+:914.9

[0994] Synthesis of (S)-3-(((S)-2-((S)-2-aminopropamido)propamido)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indo[1,2-b]quinoline-1-yl)-2-methylpropionamide:

[0995]

[0996] (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((((S)-3-(((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]indolo[1,2-b]quinoline-1-yl)amino)-2-methyl-3-oxopropoxy)methyl)amino)-1-oxoprop-2-yl)amino)-1-oxopropane-2-yl)carbamate 4 (350.00 mg, 0.38 mmol) was dissolved in N,N-dimethylformamide (5 mL), and then diethylamine (280.00 mg, 3.82 mmol) was added. After the addition was complete, the reaction was stirred at room temperature for 1 hour. The reaction was confirmed by LCMS. The reaction solution was concentrated, slurried with ethyl acetate, filtered, and the filter cake was concentrated under reduced pressure to obtain a yellow solid product (S)-3-(((S)-2-((S)-2-aminopropamido)propamido)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indene[1,2-b]quinoline-1-yl)-2-methylpropionamide 5 (228.00 mg, 0.33 mmol, 86% yield).

[0997] LC-MS:[M+H]+:692.8

[0998] (9H-fluorene-9-yl)methyl((2S,8S,11S,14S,30S,31R,32R,33R)-25-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrene) Synthesis of [3',4':6,7]indo[1,2-b]quinoline-1-yl)amino)-30,31,32,33,34-pentahydroxy-2,8,11-trimethyl-1,7,10,13,20,24-hexaoxo-28-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-4-oxo-6,9,12,19,25,28-hexaazatazanaphthalene-14-yl)carbamate:

[0999]

[1000] Dissolve (S)-3-(((S)-2-((S)-2-aminopropamido)propamido)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indene[1,2-b]quinoline-1-yl)-2-methylpropamid 5 (228.00 mg, 0.33 mmol) in N,N-dimethylformamide (5 mL), then add N2-(((9H-fluorene-9-yl)methoxy)carbonyl)- N6-(5-(bis(2-(bis((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)amino)-5-oxopentanoyl)-L-lysine 6 (403.00 mg, 0.33 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (187.00 mg, 0.49 mmol), 1-hydroxybenzotriazole (67.00 mg, 0.49 mmol) and N,N-diisopropylethylamine (127.00 mg, 0.98 mmol) were reacted and stirred at room temperature for 1 hour. LCMS analysis confirmed the reaction was complete. The reaction solution was filtered, purified by reverse-phase purification (pure water: acetonitrile = 1:1), and lyophilized to obtain a white solid product: (9H-fluorene-9-yl)methyl((2S,8S,11S,14S,30S,31R,32R,33R)-25-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-) Hexahydro-1H,12H-benzopyran[3',4':6,7]indole[1,2-b]quinoline-1-yl)amino)-30,31,32,33,34-pentahydroxy-2,8,11-trimethyl-1,7,10,13,20,24-hexaoxo-28-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-4-oxo-6,9,12,19,25,28-hexaazatetrazananaphthalene-14-yl)carbamate 7 (400.00 mg, 0.21 mmol, 64% yield).

[1001] LC-MS:[M+H]+:1898.4

[1002] Synthesis of N1-((2S,8S,11S,14S)-14-amino-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indo[1,2-b]quinoline-1-yl)amino)-2,8,11-trimethyl-1,7,10,13-tetraoxo-4-oxa-6,9,12-triazaoctadecane-18-yl)-N5,N5-bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)pentadiamide:

[1003]

[1004] The (9H-fluorene-9-yl)methyl((2S,8S,11S,14S,30S,31R,32R,33R)-25-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7]indol[1,2-b]quinoline-1-yl)amino)-30,31, 32,33,34-Pentahydroxy-2,8,11-trimethyl-1,7,10,13,20,24-hexaoxo-28-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-4-oxo-6,9,12,19,25,28-hexaazatetrazananaphthalene-14-yl)carbamate 7 (400.00 mg, 0.21 mmol) was dissolved in N,N-dimethylformamide (5 mL), followed by the addition of diethylamine (158.00 mg, 2.15 mmol). After the addition was complete, the reaction was stirred at room temperature for 1 hour. The reaction was confirmed by LCMS. The reaction solution was concentrated, slurried with ethyl acetate, filtered, and the filter cake was concentrated under reduced pressure to obtain a yellow solid product N1-((2S,8S,11S,14S)-14-amino-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indene[1] [2-b] Quinolin-1-yl)amino)-2,8,11-trimethyl-1,7,10,13-tetraoxo-4-oxa-6,9,12-triazaoctadecane-18-yl)-N5,N5bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)pentadiamide 8 (355.00 mg, 0.21 mmol, 98% yield).

[1005] LC-MS:[M+H]+:1676.3

[1006] N1,N1-bis(2-(bis((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)-N5-((2S,8S,11S,14S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H) Synthesis of indo[1,2-b]quinoline-1-yl)amino)-2,8,11-trimethyl-14-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-enamido)-1,7,10,13-tetraoxo-4-oxa-6,9,12-triazaoctadecane-18-yl)pentadiamide from 12H-benzopyran[3',4':6,7]:

[1007]

[1008] The N1-((2S,8S,11S,14S)-14-amino-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indene[1,2-b]quinoline-1-yl)amino)-2,8,11-trimethyl-1,7,10,13-tetraoxo-4-oxa-6,9,12-triazaoctadecane-18-yl)-N5,N5bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino 1,N-dimethylformamide (5 mL) was dissolved in 1-hydroxybenzotriazole (19.00 mg, 0.14 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (53.00 mg, 0.14 mmol), and N,N-diisopropylethylamine (36.00 mg, 0.28 mmol). After stirring for 10 minutes, 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl acetic acid (25.00 mg, 0.090 mmol) was added, and the reaction was carried out at room temperature for 1 hour. LCMS was used to monitor complete consumption of the starting materials and detection of the product. The reaction solution was filtered, purified by Prep-HPLC reverse phase (formic acid system), and lyophilized to obtain a white solid product N1,N1-bis(2-(bis((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)-N5-((2S,8S,11S,14S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15) -Hexahydro-1H,12H-benzopyran[3',4':6,7]indo[1,2-b]quinoline-1-yl)amino)-2,8,11-trimethyl-14-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-enamido)-1,7,10,13-tetraoxo-4-oxa-6,9,12-triazaoctadecane-18-yl)glutaramide SJ-LP34 (22.23 mg, 0.012 mmol, 12% yield).

[1009] LC-MS:[M+H]+:1926.2;

[1010] The retention time Rt of SJ-LP34 by LC-MS was 2.073 min.

[1011] Example 14: Synthesis of SJ-LP35

[1012] Synthesis route:

[1013]

[1014] Synthesis of (5S,8S)-1-(9H-fluorene-9-yl)-5,8,14,14-tetramethyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazapentadecan-15-acid:

[1015]

[1016] (5S,8S)-1-(9H-fluorene-9-yl)-5,8-dimethyl-3,6,9-trioxo-2-oxo-4,7,10-triazaundecane-11-yl acetate 5 (500.00 mg, 1.10 mmol) and 3-hydroxy-2,2-dimethylpropionic acid 6 (130.20 mg, 1.11 mmol) were dissolved in dichloromethane (6 mL), and trifluoroacetic acid (251.40 mg, 2.21 mmol) was added. The mixture was stirred at room temperature for 1.0 h. LCMS was used to monitor complete consumption of the starting materials and detection of the product. The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane:methanol = 20:1) to give a colorless oily product (5S,8S)-1-(9H-fluorene-9-yl)-5,8,14,14-tetramethyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazapentadecan-15-acid 8 (300.00 mg, 43% yield).

[1017] LCMS:[M+Na]+:533.7

[1018] Synthesis of (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-(((3-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indo[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-1-oxoprop-2-yl)amino)-1-oxoprop-2-yl)carbamate:

[1019]

[1020] (5S,8S)-1-(9H-fluorene-9-yl)-5,8,14,14-tetramethyl-3,6,9-trioxo-2,12-dioxo-4,7,10-triazapentadecan-15-acid 8 (300.00 mg, 0.59 mmol) was dissolved in N,N-dimethylformamide (5 mL), and 1-hydroxybenzotriazole (118.90 mg, 0.88 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (334 mg) were added. 0.50 mg (0.88 mmol) and N,N-diisopropylethylamine (0.29 mL, 1.76 mmol) were stirred for 10 minutes, and then (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzopyrano[3',4':6,7] indo[1,2-b]quinoline-10,13-dione 9 (255.40 mg, 0.59 mmol) were added. The reaction was carried out at room temperature for 0.5 hours. The starting material was completely consumed and the product was detected by LCMS. The reaction solution was filtered, purified by reverse-phase (formic acid system), and lyophilized to give a white solid product (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-(((3-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indene[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-1-oxoprop-2-yl)amino)-1-oxoprop-2-yl)carbamate 10 (120.00 mg, 22% yield).

[1021] LCMS:[M+H]+:929.0

[1022] Synthesis of 3-(((S)-2-((S)-2-aminopropamido)propamido)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indo[1,2-b]quinoline-1-yl)-2,2-dimethylpropionamide:

[1023]

[1024] (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-(((3-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indene[1,2-b]quinoline-1-yl)amino)-2,2-dimethyl-3-oxopropoxy)methyl)amino)-1-oxoprop-2-yl)amino)-1-oxoprop-2-yl)carbamate 10 (120.00 mg, 0.13 mmol) was dissolved in N,N-dimethylformamide (3 mL), and diethylamine (0.13 mL, 1.29 mmol) was added. The reaction was carried out at room temperature for 0.5 hours. LCMS monitoring showed complete consumption of the raw materials and detection of the product. The reaction solution was concentrated, and the residue was slurried with ethyl acetate to give a yellow solid product 3-(((S)-2-((S)-2-aminopropamido)propamido)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indo[1,2-b]quinoline-1-yl)-2,2-dimethylpropionamide 11 (97.00 mg, 99% yield).

[1025] LCMS:[M+H]+:706.8

[1026] (9H-fluorene-9-yl)methyl((8S,11S,14S,30S,31R,32R,33R)-25-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-1-(((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]indo[1,2-b]quinoline-1-yl)amino)-30,31,32,33,34-pentahydroxy-2,2 Synthesis of 8,11-tetramethyl-1,7,10,13,20,24-hexaoxo-28-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-4-oxa-6,9,12,19,25,28-hexaazatetrazane-14-yl)carbamate:

[1027]

[1028] N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(5-(bis(2-(bis((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)amino)-5-oxopentanoyl)-L-lysine 12 (150.70 mg, 0.12 mmol) was dissolved in N,N-dimethylformamide (3 mL), and 1-hydroxybenzotriazole (24.60 mg, 0.19 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (70.20 mg, 0.19 mmol) were added. After stirring with N,N-diisopropylethylamine (0.06 mL, 0.37 mmol) for 10 minutes, 3-(((S)-2-((S)-2-aminopropamido)propamido)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] indo[1,2-b]quinoline-1-yl)-2,2-dimethylpropionamide 11 (87.00 mg, 0.12 mmol) was added, and the reaction was carried out at room temperature for 0.5 hours. LCMS was used to monitor complete consumption of the starting material and detection of the product. The reaction solution was filtered, purified by reverse-phase purification (pure water: acetonitrile = 1:1), and lyophilized to obtain a yellow solid product (9H-fluorene-9-yl)methyl((8S, 11S, 14S, 30S, 31R, 32R, 33R)-25-(2-(bis((2S, 3R, 4R, 5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-1-(((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] indo[1,2-b]quinoline-1-yl)amino)-30,31,32,33,34-pentahydroxy-2,2 8,11-Tetramethyl-1,7,10,13,20,24-hexaoxo-28-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-4-oxa-6,9,12,19,25,28-hexaazatetrazane-14yl)carbamate 13 (110.00 mg, 47% yield).

[1029] LCMS:[M+H]+:1912.5

[1030] Synthesis of N1-((8S, 11S, 14S)-14-amino-1-(((1S, 9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3', 4':6,7]-aza[1,2-b]quinoline-1-yl)amino)-2,2,8,11-tetramethyl-1,7,10,13-tetraoxo-4-oxa-6,9,12-triazaoctadecane-18-yl)-N5,N5-bis(2-(bis((2S, 3R, 4R, 5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)pentadiamide:

[1031]

[1032] The (9H-fluorene-9-yl)methyl((8S,11S,14S,30S,31R,32R,33R)-25-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)-1-(((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] indenzo[1,2-b]quinoline-1-yl)amino)-30,31,32,33,34-pentahydroxy-2,2 8,11-Tetramethyl-1,7,10,13,20,24-hexaoxo-28-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-4-oxa-6,9,12,19,25,28-hexaazatetrazane-14yl)carbamate 13 (110.00 mg, 0.060 mmol) was dissolved in N,N-dimethylformamide (3 mL), and diethylamine (0.06 mL, 0.58 mmol) was added. The reaction was carried out at room temperature for 30 minutes. LCMS was used to monitor complete consumption of the starting material and detection of the product. The reaction solution was concentrated, pulped, and filtered to obtain a yellow solid product N1-((8S,11S,14S)-14-amino-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3',4':6,7] with aza[1,2-b] in its zirconium. Quinolino-1-yl)amino)-2,2,8,11-tetramethyl-1,7,10,13-tetraoxo-4-oxa-6,9,12-triazaoctadecane-18-yl)-N5,N5-bis(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)pentadiamide 14 (100.00 mg, 99% yield).

[1033] LCMS:[M+H]+:1690.4

[1034] N1,N1-bis(2-(bis((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)-N5-((8S,11S,14S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12) Synthesis of H-benzopyrano[3',4':6,7] indo[1,2-b]quinoline-1-yl)amino)-2,2,8,11-tetramethyl-14-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-enamide)-1,7,10,13-tetraoxo-4-oxa-6,9,12-triazaoctadecane-18-yl)pentadiamide:

[1035]

[1036] N1-((8S, 11S, 14S)-14-amino-1-(((1S, 9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyran[3', 4':6,7] in aza[1,2-b]quinoline-1-yl)amino)-2,2,8,11-tetramethyl-1,7,10,13-tetraoxo-4-oxa-6,9,12-triazaoctadecane-18-yl)-N5,N5-bis(2-(bis((2S, 3R, 4R, 5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)glutaryl Amine 14 (100.00 mg, 0.060 mmol) and 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-alkynyl acid 15 (15.90 mg, 0.059 mmol) were dissolved in N,N-dimethylformamide (5 mL), and 1-hydroxybenzotriazole (11.10 mg, 0.090 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (33.70 mg, 0.090 mmol) and N,N-diisopropylethylamine (0.03 mL, 0.18 mmol) were added. The reaction was carried out at room temperature for 1 hour. The starting materials were completely consumed and the product was detected by LCMS. The reaction solution was filtered, sent to Prep-HPLC for preparation (formic acid system), and lyophilized to obtain a white solid product N1,N1-bis(2-(bis((2S,3R,4R,5R)-2,3,4-5,6-pentahydroxyhexyl)amino)ethyl)-N5-((8S,11S,14S)-1-(((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]indo[1,2-b]quinoline-1-yl)amino)-2,2,8,11-tetramethyl-14-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-enamide)-1,7,10,13-tetraoxo-4-oxa-6,9,12-triazaoctadecane-18-yl)glutaramide SJ-LP35 (13.40 mg, 12% yield).

[1037] LCMS:[MH]+:1940.4

[1038] The retention time Rt of SJ-LP35 by LC-MS was 2.176 min.

[1039] Example 15: Synthesis of SJ-LP21

[1040] The chemical structural formula of SJ-LP21 is as follows:

[1041]

[1042] LP21 was purchased from Bid Pharmaceuticals, CAS: 1616493-44-7, as follows:

[1043]

[1044] SJ-LP21 was synthesized according to the synthesis method of Example 5 (SJ-LP11), with compound 2 in Example 5 replaced by LP21. The raw materials were completely consumed by LCMS and the product was detected. SJ-LP21 was obtained by freeze-drying using the Prep-HPLC method.

[1045] Example 16: Synthesis of SJ-LP22

[1046] The chemical structural formula of SJ-LP22 is as follows:

[1047]

[1048] LP21 was purchased from Bid Pharmaceuticals, CAS: 1616493-44-7, as follows:

[1049]

[1050] SJ-LP22 was synthesized according to the synthesis method of Example 6 (SJ-LP10), with compound 4 in Example 6 replaced by LP21. The raw materials were completely consumed by LCMS and the product was detected. SJ-LP22 was obtained by freeze-drying using the Prep-HPLC method.

[1051] LC-MS:[M+H]+:1943.3;

[1052] The retention time Rt of SJ-LP22 by LC-MS was 2.16 min.

[1053] Example 17: Synthesis of SJ-LP23

[1054] The chemical structural formula of SJ-LP23 is as follows:

[1055]

[1056] LP21 was purchased from Bid Pharmaceuticals, CAS: 1616493-44-7, as follows:

[1057]

[1058] SJ-LP23 was synthesized according to the synthetic method of Example 5 (SJ-LP11), except that compound 1 in Example 5 was replaced with Compound 2 was replaced with LP21. LCMS monitoring showed that the raw material was completely consumed and the product was detected. SJ-LP23 was obtained by freeze-drying using the Prep-HPLC method.

[1059] Example 18: Synthesis of SJ-LP24

[1060] The chemical structural formula of SJ-LP24 is as follows:

[1061]

[1062] LP21 was purchased from Bid Pharmaceuticals, CAS: 1616493-44-7, as follows:

[1063]

[1064] SJ-LP24 was synthesized using the same method as in Example 6 (SJ-LP10), except that compound 3 in Example 6 was replaced with... Compound 4 was replaced with LP21. LCMS monitoring showed that the raw material was completely consumed and the product was detected. SJ-LP24 was obtained by freeze-drying using the Prep-HPLC method.

[1065] LC-MS:[M+H]+:1794.1;

[1066] The retention time Rt of SJ-LP24 by LC-MS was 1.881 min.

[1067] Example 19: Synthesis of SJ-LP27

[1068] The chemical structural formula of SJ-LP27 is as follows:

[1069]

[1070] LP21 was purchased from Bid Pharmaceuticals, CAS: 1616493-44-7, as follows:

[1071]

[1072] SJ-LP27 was synthesized according to the synthesis method of Example 7 (SJ-LP19), except that compound 4 in Example 7 was replaced with LP21. The raw materials were completely consumed by LCMS and the product was detected. SJ-LP27 was obtained by freeze-drying using the Prep-HPLC method.

[1073] LC-MS: 1 / 2[M+2H]+: 958.1;

[1074] The retention time Rt of SJ-LP27 by LC-MS was 2.059 min.

[1075] Example 20: Synthesis of SJ-LP28

[1076] The chemical structural formula of SJ-LP28 is as follows:

[1077]

[1078] LP28 was purchased from Shanghai Titan Technology Co., Ltd., CAS: 209783-80-2, as follows:

[1079]

[1080] SJ-LP28 was synthesized according to the synthesis method of Example 7 (SJ-LP19), except that compound 4 in Example 7 was replaced with LP28. The raw materials were completely consumed by LCMS and the product was detected. SJ-LP28 was obtained by freeze-drying using the Prep-HPLC method.

[1081] LC-MS: 1 / 2[M+2H]+: 951.1;

[1082] The retention time Rt of SJ-LP28 by LC-MS was 1.962 min.

[1083] Example 21: Synthesis of SJ-LP29

[1084] The chemical structural formula of SJ-LP29 is as follows:

[1085]

[1086] LP28 was purchased from Shanghai Titan Technology Co., Ltd., CAS: 209783-80-2, as follows:

[1087]

[1088] SJ-LP29 was synthesized according to the synthetic method of Example 7 (SJ-LP19), except that compound 3 in Example 7 was replaced with Compound 4 was replaced with LP28. LCMS monitoring showed that the raw material was completely consumed and the product was detected. SJ-LP29 was obtained by freeze-drying using the Prep-HPLC method.

[1089] LC-MS:[M+H]+:1752.3;

[1090] The retention time Rt of SJ-LP29 by LC-MS was 1.606 min.

[1091] Example 22: Synthesis of SJ-LP32

[1092] The chemical structural formula of SJ-LP32 is as follows:

[1093]

[1094] LP21 was purchased from Bid Pharmaceuticals, CAS: 1616493-44-7, as follows:

[1095]

[1096] SJ-LP32 was synthesized according to the synthetic method of Example 7 (SJ-LP19), except that compound 3 in Example 7 was replaced with Compound 4 was replaced with LP21. LCMS monitoring showed that the raw material was completely consumed and the product was detected. SJ-LP32 was obtained by freeze-drying using the Prep-HPLC method.

[1097] Example 23: Synthesis of SJ-LP43

[1098] The chemical structural formula of SJ-LP43 is as follows:

[1099]

[1100] LP43 was purchased from Nanjing Yilan Biotechnology Co., Ltd., CAS: 1346574-57-9, as follows:

[1101]

[1102] SJ-LP43 was synthesized according to the synthesis method of Example 7 (SJ-LP19), except that compound 4 in Example 7 was replaced with LP43. The raw materials were completely consumed by LCMS and the product was detected. SJ-LP43 was obtained by freeze-drying using the Prep-HPLC method.

[1103] LC-MS: 1 / 2[M+2H]+: 1026.4;

[1104] The retention time Rt of SJ-LP43 by LC-MS was 2.36 min.

[1105] Example 24: Synthesis of SJ-LP44

[1106] The chemical structural formula of SJ-LP44 is as follows:

[1107]

[1108] LP44 was purchased from Leyan, CAS: 191732-72-6, as follows:

[1109]

[1110] SJ-LP44 was synthesized according to the synthetic method of Example 7 (SJ-LP19), except that compound 3 in Example 7 was replaced with Compound 4 was replaced with LP44. LCMS monitoring showed that the raw material was completely consumed and the product was detected. SJ-LP44 was obtained by freeze-drying using the Prep-HPLC method.

[1111] LC-MS:[M+H]+:1663.3;

[1112] The retention time Rt of SJ-LP44 by LC-MS was 1.591 min.

[1113] Example 25: Synthesis of SJ-LP45

[1114] The chemical structural formula of SJ-LP45 is as follows:

[1115]

[1116] LP45 was purchased from Leyan, CAS number: 19171-19-8 as follows:

[1117]

[1118] SJ-LP45 was synthesized according to the synthetic method of Example 7 (SJ-LP19), except that compound 3 in Example 7 was replaced with Compound 4 was replaced with LP45. LCMS monitoring showed that the raw material was completely consumed and the product was detected. SJ-LP45 was obtained by freeze-drying using the Prep-HPLC method.

[1119] LC-MS: 1 / 2[M+2H]+: 839.0;

[1120] The retention time Rt of SJ-LP45 by LC-MS was 1.809 min.

[1121] Example 26: Synthesis of SJ-LP48

[1122] The chemical structural formula of SJ-LP48 is as follows:

[1123]

[1124] LP48: Leyan CAS: 474645-27-7

[1125]

[1126] SJ-LP48 was synthesized according to the synthesis method of Example 7 (SJ-LP19), except that compound 4 in Example 7 was replaced with LP48. The raw materials were completely consumed by LCMS and the product was detected. SJ-LP48 was obtained by freeze-drying using the Prep-HPLC method.

[1127] LC-MS: 1 / 2[M+2H]+: 1122.8;

[1128] The retention time Rt of SJ-LP48 by LC-MS was 1.81 min.

[1129] Example 27: Synthesis of SJ-LP49

[1130] The chemical structural formula of SJ-LP49 is as follows:

[1131]

[1132] LP49 was purchased from Nantong Quanyi Biotechnology Co., Ltd., CAS: 1001264-89-6, as follows:

[1133]

[1134] SJ-LP49 was synthesized according to the synthesis method of Example 7 (SJ-LP19), except that compound 4 in Example 7 was replaced with LP49. The raw materials were completely consumed by LCMS and the product was detected. SJ-LP49 was obtained by freeze-drying using the Prep-HPLC method.

[1135] LC-MS: 1 / 2[M+2H]+: 992.2;

[1136] The retention time Rt of SJ-LP49 by LC-MS was 1.002 min.

[1137] Example 28: Preparation of antibody-drug conjugates 1

[1138] The purified antibody (T-mab) with a monomer content greater than 97% was transferred to a buffer solution containing 10 mM PBS and 2.5 mM EDTA (pH 6.8) using a dialysis bag with a molecular weight cutoff of 8000-14000. Ten molar equivalents (relative to the antibody) of 10 mM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) were added, mixed, and reacted at 37°C for 2 hours. Subsequently, 20 molar equivalents (relative to the antibody) of the linker drug conjugates (SJ-LP10, SJ-LP12, GGFG-Dxd) were added, mixed, and reacted at 4°C for 16 hours. After the reaction was complete, dialysis was performed using a dialysis bag with a molecular weight cutoff of 8000-14000, with the buffer changed to PBS buffer, and dialysis was performed for 48 hours, with three buffer changes during this period. After dialysis, the obtained antibody-drug conjugates (ADCs) (T-mab-SJ-LP10, T-mab-SJ-LP12, T-mab-GGFG-Dxd) were subsequently detected by high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS).

[1139] The chromatographic data of T-mab, T-mab-SJ-LP10, T-mab-SJ-LP12, and T-mab-GGFG-Dxd in this embodiment are as follows:

[1140] The retention time Rt of T-mab by HPLC-HIC was 7.682 min; the retention time Rt of T-mab by HPLC-SEC was 3.37 min.

[1141] The HPLC-HIC retention time Rt of T-mab-SJ-LP10 was 8.085 min; the HPLC-SEC retention time Rt of T-mab-SJ-LP10 was 3.34 min.

[1142] The HPLC-HIC retention time Rt of T-mab-SJ-LP12 was 7.937 min; the HPLC-SEC retention time Rt of T-mab-SJ-LP12 was 3.265 min.

[1143] The HPLC-HIC retention time Rt of T-mab-GGFG-Dxd was 9.664 min; the HPLC-SEC retention time Rt of T-mab-GGFG-Dxd was 3.35 min.

[1144] Example 29: Preparation of antibody-drug conjugates 2

[1145] The purified antibody (T-mab) with a monomer content greater than 97% was transferred to a buffer solution containing 10 mM PBS and 2.5 mM EDTA (pH 6.8) using a dialysis bag with a molecular weight cutoff of 8000-14000. Ten molar equivalents (relative to the antibody) of 10 mM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) were added, mixed, and reacted at 37°C for 2 hours. Subsequently, 20 molar equivalents (relative to the antibody) of the linker drug conjugate (SJ-LP11) were added, mixed, and reacted at 4°C for 16 hours. After the reaction was complete, dialysis was performed using a dialysis bag with a molecular weight cutoff of 8000-14000, with the buffer changed to PBS buffer, and dialysis was performed for 48 hours, with three buffer changes during this period. After dialysis, the resulting antibody-drug conjugate (ADC) (T-mab-SJ-LP11) sample was subsequently detected by high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS).

[1146] The chromatographic data of T-mab and T-mab-SJ-LP11 in this embodiment are as follows:

[1147] The retention time Rt of T-mab by HPLC-HIC was 7.468 min; the retention time Rt of T-mab by HPLC-SEC was 3.28 min.

[1148] The HPLC-HIC retention time Rt of T-mab-SJ-LP11 was 8.08 min; the HPLC-SEC retention time Rt of T-mab-SJ-LP11 was 3.34 min.

[1149] Example 30: Preparation of antibody-drug conjugates 3

[1150] The purified antibody (T-mab) with a monomer content greater than 97% was transferred to a buffer solution containing 10 mM PBS and 2.5 mM EDTA (pH 6.8) using a dialysis bag with a molecular weight cutoff of 8000-14000. Ten molar equivalents (relative to the antibody) of 10 mM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) were added, mixed, and reacted at 37°C for 2 hours. Subsequently, 20 molar equivalents (relative to the antibody) of the linker drug conjugate (SJ-LP13) were added, mixed, and reacted at 4°C for 16 hours. After the reaction was complete, dialysis was performed using a dialysis bag with a molecular weight cutoff of 8000-14000, with the buffer changed to PBS buffer, and dialysis was performed for 48 hours, with three buffer changes during this period. After dialysis, the resulting antibody-drug conjugate (ADC) (T-mab-SJ-LP13) sample was subsequently detected by high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS).

[1151] The chromatographic data of T-mab and T-mab-SJ-LP13 in this embodiment are as follows:

[1152] The retention time Rt of T-mab by HPLC-HIC was 7.468 min; the retention time Rt of T-mab by HPLC-SEC was 3.28 min.

[1153] The HPLC-HIC retention time Rt of T-mab-SJ-LP13 was 7.7 min; the HPLC-SEC retention time Rt of T-mab-SJ-LP13 was 3.24 min.

[1154] Example 31: Preparation of antibody-drug conjugates 4

[1155] The purified antibody (T-mab) with a monomer content greater than 97% was transferred to a buffer solution containing 10 mM PBS and 2.5 mM EDTA (pH 6.8) using a dialysis bag with a molecular weight cutoff of 8000-14000. Ten molar equivalents (relative to the antibody) of 10 mM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) were added, mixed, and reacted at 37°C for 2 hours. Subsequently, 20 molar equivalents (relative to the antibody) of the linker drug conjugate (SJ-LP15) were added, mixed, and reacted at 4°C for 16 hours. After the reaction was complete, dialysis was performed using a dialysis bag with a molecular weight cutoff of 8000-14000, with the buffer changed to PBS buffer, and dialysis was performed for 48 hours, with three buffer changes during this period. After dialysis, the resulting antibody-drug conjugate (ADC) (T-mab-SJ-LP15) sample was subsequently detected by high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS).

[1156] The chromatographic data of T-mab and T-mab-SJ-LP15 in this embodiment are as follows:

[1157] The retention time Rt of T-mab by HPLC-HIC was 7.287 min; the retention time Rt of T-mab by HPLC-SEC was 3.31 min.

[1158] The retention time Rt of T-mab-SJ-LP15 by HPLC-HIC was 7.412 min; the retention time Rt of T-mab-SJ-LP15 by HPLC-SEC was 3.24 min.

[1159] Example 32: Preparation of antibody-drug conjugates 5

[1160] The purified antibody (T-mab) with a monomer content greater than 97% was transferred to a buffer solution containing 10 mM PBS and 2.5 mM EDTA (pH 6.8) using a dialysis bag with a molecular weight cutoff of 8000-14000. Ten molar equivalents (relative to the antibody) of 10 mM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) were added, mixed, and reacted at 37°C for 2 hours. Subsequently, 20 molar equivalents (relative to the antibody) of the linker drug conjugates (SJ-LP17, SJ-LP20) were added, mixed, and reacted at 4°C for 16 hours. After the reaction was complete, dialysis was performed using a dialysis bag with a molecular weight cutoff of 8000-14000, with the buffer changed to PBS buffer, and dialysis was performed for 48 hours, with three buffer changes during this period. After dialysis, the obtained antibody-drug conjugates, namely ADCs (T-mab-SJ-LP17 and T-mab-SJ-LP20), were subsequently detected by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS).

[1161] The chromatographic data of T-mab, T-mab-SJ-LP17, and T-mab-SJ-LP20 in this embodiment are as follows:

[1162] The retention time Rt of T-mab by HPLC-HIC was 7.757 min; the retention time Rt of T-mab by HPLC-SEC was 3.41 min.

[1163] The HPLC-HIC retention time Rt of T-mab-SJ-LP17 was 7.855 min; the HPLC-SEC retention time Rt of T-mab-SJ-LP17 was 3.35 min.

[1164] The retention time Rt of T-mab-SJ-LP20 by HPLC-HIC was 7.749 min; the retention time Rt of T-mab-SJ-LP20 by HPLC-SEC was 3.34 min.

[1165] Example 33: Preparation of antibody-drug conjugates 6

[1166] The purified antibody (T-mab) with a monomer content greater than 97% was transferred to a buffer solution containing 10 mM PBS and 2.5 mM EDTA (pH 6.8) using a dialysis bag with a molecular weight cutoff of 8000-14000. Ten molar equivalents (relative to the antibody) of 10 mM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) were added, mixed, and reacted at 37°C for 2 hours. Subsequently, 20 molar equivalents (relative to the antibody) of the linker drug conjugate (SJ-LP18) were added, mixed, and reacted at 4°C for 16 hours. After the reaction was complete, dialysis was performed using a dialysis bag with a molecular weight cutoff of 8000-14000, with the buffer changed to PBS buffer, and dialysis was performed for 48 hours, with three buffer changes during this period. After dialysis, the resulting antibody-drug conjugate (ADC) (T-mab-SJ-LP18) sample was subsequently detected by high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS).

[1167] The chromatographic data of T-mab and T-mab-SJ-LP18 in this embodiment are as follows:

[1168] The retention time Rt of T-mab by HPLC-HIC was 7.256 min; the retention time Rt of T-mab by HPLC-SEC was 3.25 min.

[1169] The retention time Rt of T-mab-SJ-LP18 in HPLC-HIC was 7.252 min; the retention time Rt of T-mab-SJ-LP18 in HPLC-SEC was 3.2 min.

[1170] Example 34: Preparation of antibody-drug conjugates 7

[1171] The purified antibody (T-mab) with a monomer content greater than 97% was transferred to a buffer solution containing 10 mM PBS and 2.5 mM EDTA (pH 6.8) using a dialysis bag with a molecular weight cutoff of 8000-14000. Ten molar equivalents (relative to the antibody) of 10 mM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) were added, mixed, and reacted at 37°C for 2 hours. Subsequently, 20 molar equivalents (relative to the antibody) of the linker drug conjugate (SJ-LP19) were added, mixed, and reacted at 4°C for 16 hours. After the reaction was complete, dialysis was performed using a dialysis bag with a molecular weight cutoff of 8000-14000, with the buffer changed to PBS buffer, and dialysis was performed for 48 hours, with three buffer changes during this period. After dialysis, the resulting antibody-drug conjugate (ADC) (T-mab-SJ-LP19) sample was subsequently detected by high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS).

[1172] The chromatographic data of T-mab and T-mab-SJ-LP19 in this embodiment are as follows:

[1173] The retention time Rt of T-mab by HPLC-HIC was 7.363 min; the retention time Rt of T-mab by HPLC-SEC was 3.41 min.

[1174] The HPLC-HIC retention time Rt of T-mab-SJ-LP19 was 7.465 min; the HPLC-SEC retention time Rt of T-mab-SJ-LP19 was 3.38 min.

[1175] Example 35: Preparation of antibody-drug conjugates 8

[1176] The purified antibody (T-mab) with a monomer content greater than 97% was transferred to a buffer solution containing 10 mM PBS and 2.5 mM EDTA (pH 6.8) using a dialysis bag with a molecular weight cutoff of 8000-14000. Ten molar equivalents (relative to the antibody) of 10 mM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) were added, mixed, and reacted at 37°C for 2 hours. Subsequently, 20 molar equivalents (relative to the antibody) of the linker drug conjugate (SJ-LP30) were added, mixed, and reacted at 4°C for 16 hours. After the reaction was complete, dialysis was performed using a dialysis bag with a molecular weight cutoff of 8000-14000, with the buffer changed to PBS buffer, and dialysis was performed for 48 hours, with three buffer changes during this period. After dialysis, the resulting antibody-drug conjugate (ADC) (T-mab-SJ-LP30) sample was subsequently detected by high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS).

[1177] The chromatographic data of T-mab and T-mab-SJ-LP30 in this embodiment are as follows:

[1178] The retention time Rt of T-mab by HPLC-HIC was 7.37 min; the retention time Rt of T-mab by HPLC-SEC was 3.53 min.

[1179] The retention time Rt of T-mab-SJ-LP30 in HPLC-HIC was 7.3 min; the retention time Rt of T-mab-SJ-LP30 in HPLC-SEC was 3.45 min.

[1180] Example 36: Preparation of antibody-drug conjugates 9

[1181] The purified antibody (T-mab) with a monomer content greater than 97% was transferred to a buffer solution containing 10 mM PBS and 2.5 mM EDTA (pH 6.8) using a dialysis bag with a molecular weight cutoff of 8000-14000. Ten molar equivalents (relative to the antibody) of 10 mM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) were added, mixed, and reacted at 37°C for 2 hours. Subsequently, 20 molar equivalents (relative to the antibody) of the linker drug conjugates (SJ-LP31, SJ-LP33, SJ-LP34, SJ-LP35) were added, mixed, and reacted at 4°C for 16 hours. After the reaction was complete, dialysis was performed using a dialysis bag with a molecular weight cutoff of 8000-14000, with the buffer changed to PBS buffer, and dialysis was performed for 48 hours, with three buffer changes during this period. After dialysis, the obtained antibody-drug conjugates (ADCs) (T-mab-SJ-LP31, T-mab-SJ-LP33, T-mab-SJ-LP34, T-mab-SJ-LP35) were subsequently detected by high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS).

[1182] The chromatographic data of T-mab, T-mab-SJ-LP31, T-mab-SJ-LP33, T-mab-SJ-LP34, and T-mab-SJ-LP35 in this embodiment are as follows:

[1183] The retention time Rt of T-mab by HPLC-HIC was 7.48 min; the retention time Rt of T-mab by HPLC-SEC was 3.53 min.

[1184] The HPLC-HIC retention time Rt of T-mab-SJ-LP31 was 7.47 min; the HPLC-SEC retention time Rt of T-mab-SJ-LP31 was 3.46 min.

[1185] The retention time Rt of T-mab-SJ-LP33 by HPLC-HIC was 7.53 min; the retention time Rt of T-mab-SJ-LP33 by HPLC-SEC was 3.33 min.

[1186] The HPLC-HIC retention time Rt of T-mab-SJ-LP34 was 7.46 min; the HPLC-SEC retention time Rt of T-mab-SJ-LP34 was 3.46 min.

[1187] The retention time Rt of T-mab-SJ-LP35 by HPLC-HIC was 7.47 min; the retention time Rt of T-mab-SJ-LP35 by HPLC-SEC was 3.45 min.

[1188] Example 37: Preparation of antibody-drug conjugates 10

[1189] The purified antibody (T-mab) with a monomer content greater than 97% was transferred to a buffer solution containing 10 mM PBS and 2.5 mM EDTA (pH 6.8) using a dialysis bag with a molecular weight cutoff of 8000-14000. Ten molar equivalents (relative to the antibody) of 10 mM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) were added, mixed, and reacted at 37°C for 2 hours. Subsequently, 20 molar equivalents (relative to the antibody) of the linker drug conjugates (SJ-LP22, SJ-LP24, SJ-LP27, SJ-LP28, SJ-LP29, SJ-LP43, SJ-LP44, SJ-LP45, SJ-LP48, SJ-LP49, VC-MMAE) were added, mixed, and reacted at 4°C for 16 hours. After the reaction was complete, dialysis was performed using dialysis bags with a molecular weight cutoff of 8000-14000. The solution was changed to PBS buffer, and dialysis was continued for 48 hours, with the solution changed 3 times during this period. After dialysis, the obtained antibody-drug conjugates (ADCs) (T-mab-SJ-LP22, T-mab-SJ-LP24, T-mab-SJ-LP27, T-mab-SJ-LP28, T-mab-SJ-LP29, T-mab-SJ-LP43, T-mab-SJ-LP44, T-mab-SJ-LP45, T-mab-SJ-LP48, T-mab-SJ-LP49, T-mab-VC-MMAE) were subsequently detected by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS).

[1190] The chromatographic data for T-mab, T-mab-SJ-LP22, T-mab-SJ-LP24, T-mab-SJ-LP27, T-mab-SJ-LP28, T-mab-SJ-LP29, T-mab-SJ-LP43, T-mab-SJ-LP44, T-mab-SJ-LP45, T-mab-SJ-LP48, T-mab-SJ-LP49, and T-mab-VC-MMAE in this embodiment are as follows:

[1191] The retention time Rt of T-mab by HPLC-HIC was 7.71 min; the retention time Rt of T-mab by HPLC-SEC was 3.438 min.

[1192] The HPLC-HIC retention time Rt of T-mab-SJ-LP22 was 9.107 min; the HPLC-SEC retention time Rt of T-mab-SJ-LP22 was 3.378 min.

[1193] The HPLC-HIC retention time Rt of T-mab-SJ-LP24 was 8.142 min; the HPLC-SEC retention time Rt of T-mab-SJ-LP24 was 3.388 min.

[1194] The HPLC-HIC retention time Rt of T-mab-SJ-LP27 was 8.716 min; the HPLC-SEC retention time Rt of T-mab-SJ-LP27 was 3.394 min.

[1195] The HPLC-HIC retention time Rt of T-mab-SJ-LP28 was 8.571 min; the HPLC-SEC retention time Rt of T-mab-SJ-LP28 was 3.395 min.

[1196] The HPLC-HIC retention time Rt of T-mab-SJ-LP29 was 7.977 min; the HPLC-SEC retention time Rt of T-mab-SJ-LP29 was 3.38 min.

[1197] The HPLC-HIC retention time Rt of T-mab-SJ-LP43 was 8.803 min; the HPLC-SEC retention time Rt of T-mab-SJ-LP43 was 3.342 min.

[1198] The HPLC-HIC retention time Rt of T-mab-SJ-LP44 was 7.737 min; the HPLC-SEC retention time Rt of T-mab-SJ-LP44 was 3.374 min.

[1199] The HPLC-HIC retention time Rt of T-mab-SJ-LP45 was 7.709 min; the HPLC-SEC retention time Rt of T-mab-SJ-LP45 was 3.366 min.

[1200] The HPLC-HIC retention time Rt of T-mab-SJ-LP48 was 10.221 min; the HPLC-SEC retention time Rt of T-mab-SJ-LP48 was 3.384 min.

[1201] The HPLC-HIC retention time Rt of T-mab-SJ-LP49 was 8.803 min; the HPLC-SEC retention time Rt of T-mab-SJ-LP49 was 3.39 min.

[1202] The HPLC-HIC retention time Rt of T-mab-VC-MMAE was 13.705 min; the HPLC-SEC retention time Rt of T-mab-VC-MMAE was 3.253 min.

[1203] ADC activity verification

[1204] Experimental Example 1: Verification of Cell Viability

[1205] Cells in the logarithmic growth phase were digested with trypsin and counted. The cell density of SK-BR-3 and MDA-MB-468 cells was adjusted to 5×10^4 cells / mL using experimental culture medium. 100 μL of cell suspension was added to the corresponding position of each well in a 96-well cell culture plate and incubated overnight in a 37℃ CO2 incubator.

[1206] On the second day, the ADC drug was serially diluted using their respective experimental media. The initial concentration was 100 μg / mL, followed by 5× serial dilutions, for a total of 8 concentrations: 100 μg / mL, 20 μg / mL, 4 μg / mL, 0.8 μg / mL, 0.16 μg / mL, 0.032 μg / mL, 0.0064 μg / mL, and 0.00128 μg / mL. After dilution, the original culture medium was removed from the 96-well cell culture plates, and 100 μL of the drug dilution was added to each well. Two replicates were set up for each concentration. The negative control group received 100 μL of experimental culture medium per well, while the blank control group received only 100 μL of experimental culture medium. After addition, the 96-well cell culture plates were incubated at 37°C in a CO2 incubator for 120 hours.

[1207] After incubation, remove the cell culture plate and add 100 μL of CellTiter-Lumi™ Plus chemiluminescence assay reagent equilibrated to room temperature to each well. Incubate at room temperature for 10 min. After incubation, place the plate in a microplate reader for chemiluminescence detection. Analyze the obtained data, plot the dose-response curve, and calculate the IC50. 50 .

[1208] The IC50 of the compound was obtained using four-parameter nonlinear fitting analysis (XLfit, IDBS). 50 value.

[1209] IC50 of some compounds 50 The values ​​are shown in Table 1 and Table 2.

[1210] Table 1. IC50 of some compounds on Her2-positive SK-BR-3 cells 50 value

[1211]

[1212] For IC in Table 150 The value, "+" indicates IC 50 A value greater than or equal to 0.085 μg / mL, "++" indicates IC50 concentration. 50 Values ​​greater than 0.060 μg / mL and less than or equal to 0.085 μg / mL, with "+++" indicating IC50. 50 Values ​​greater than 0.035 μg / mL and less than or equal to 0.060 μg / mL, with "++++" indicating IC50 concentration. 50 Values ​​greater than 0.010 μg / mL and less than or equal to 0.035 μg / mL, with "+++++" indicating IC50 value. 50 The value is less than or equal to 0.010 μg / mL. T-mab-GGFG-Dxd means Tmab+GGFG-Dxd, T-mab-SJ-LP17 means Tmab+SJ-LP17, and T-mab-SJ-LP20 means Tmab+SJ-LP20.

[1213] Table 2. IC50 of some compounds on Her2-negative MDA-MB-468 cells 50 value

[1214]

[1215] For IC in Table 2 50 The value, "+" indicates IC 50 A value greater than or equal to 90 μg / mL, "++" indicates IC50. 50 Values ​​greater than 70 μg / mL and less than or equal to 90 μg / mL, "+++" indicates IC50. 50 Values ​​greater than 50 μg / mL and less than or equal to 70 μg / mL, "++++" indicates IC50 value. 50 Values ​​greater than 30 μg / mL and less than or equal to 50 μg / mL, with "+++++" indicating IC50. 50 The value is less than or equal to 30 μg / mL. T-mab-GGFG-Dxd means Tmab+GGFG-Dxd, T-mab-SJ-LP17 means Tmab+SJ-LP17, and T-mab-SJ-LP20 means Tmab+SJ-LP20.

[1216] The effects of antibody-drug conjugates T-mab-SJ-LP15 and T-mab-SJ-LP18 on the survival rate of Her2-positive SK-BR-3 cells are shown in the figure. Figure 4 ; where T-mab-SJ-LP15 means Tmab+SJ-LP15, and T-mab-SJ-LP18 means Tmab+SJ-LP18.

[1217] The effects of antibody-drug conjugates T-mab-SJ-LP15 and T-mab-SJ-LP18 on the survival of Her2-negative MDA-MB-468 cells are shown in the figure. Figure 5 ; where T-mab-SJ-LP15 means Tmab+SJ-LP15, and T-mab-SJ-LP18 means Tmab+SJ-LP18.

[1218] The effects of antibody-drug conjugates T-mab-SJ-LP10, T-mab-SJ-LP11, and T-mab-SJ-LP12 on the survival rate of Her2-positive SK-BR-3 cells are shown in the figure. Figure 6 Where T-mab-SJ-LP10 represents Tmab+SJ-LP10, T-mab-SJ-LP11 represents Tmab+SJ-LP11, and T-mab-SJ-LP12 represents Tmab+SJ-LP12.

[1219] The effects of antibody-drug conjugates T-mab-SJ-LP10, T-mab-SJ-LP11, and T-mab-SJ-LP12 on the survival of Her2-negative MDA-MB-468 cells are shown in the table below. Figure 7 Where T-mab-SJ-LP10 represents Tmab+SJ-LP10, T-mab-SJ-LP11 represents Tmab+SJ-LP11, and T-mab-SJ-LP12 represents Tmab+SJ-LP12.

[1220] The effects of antibody-drug conjugates T-mab-SJ-LP17 and T-mab-SJ-LP20 on the survival rate of Her2-positive SK-BR-3 cells are shown in the figure. Figure 8 ; where T-mab-SJ-LP17 means Tmab+SJ-LP17, and T-mab-SJ-LP20 means Tmab+SJ-LP20.

[1221] The effects of antibody-drug conjugates T-mab-SJ-LP17 and T-mab-SJ-LP20 on the survival of Her2-negative MDA-MB-468 cells are shown in the figure. Figure 9 ; where T-mab-SJ-LP17 means Tmab+SJ-LP17, and T-mab-SJ-LP20 means Tmab+SJ-LP20.

[1222] The effects of antibody-drug conjugates T-mab-SJ-LP13 and T-mab-SJ-LP19 on the survival rate of Her2-positive SK-BR-3 cells are shown in the figure. Figure 10; where T-mab-SJ-LP13 means Tmab+SJ-LP13, and T-mab-SJ-LP19 means Tmab+SJ-LP19.

[1223] The effects of antibody-drug conjugates T-mab-SJ-LP13 and T-mab-SJ-LP19 on the survival of Her2-negative MDA-MB-468 cells are shown in the figure. Figure 11 ; where T-mab-SJ-LP13 means Tmab+SJ-LP13, and T-mab-SJ-LP19 means Tmab+SJ-LP19.

[1224] The effects of antibody-drug conjugates T-mab-SJ-LP30 and T-mab-SJ-LP31 on the survival rate of Her2-positive SK-BR-3 cells are shown in the figure. Figure 12 Where T-mab-SJ-LP30 means Tmab+SJ-LP30, and T-mab-SJ-LP31 means Tmab+SJ-LP31.

[1225] The effects of antibody-drug conjugates T-mab-SJ-LP30 and T-mab-SJ-LP31 on the survival of Her2-negative MDA-MB-468 cells are shown in the figure. Figure 13 Where T-mab-SJ-LP30 means Tmab+SJ-LP30, and T-mab-SJ-LP31 means Tmab+SJ-LP31.

[1226] The effect of antibody-drug conjugate T-mab-SJ-LP33 on the survival rate of Her2-positive SK-BR-3 cells is shown in the figure. Figure 14 ; where T-mab-SJ-LP33 means Tmab+SJ-LP33.

[1227] The effect of antibody-drug conjugate T-mab-SJ-LP33 on the survival rate of Her2-negative MDA-MB-468 cells is shown in the figure. Figure 15 ; where T-mab-SJ-LP33 means Tmab+SJ-LP33.

[1228] The effects of antibody-drug conjugates T-mab-SJ-LP34 and T-mab-SJ-LP35 on the survival rate of Her2-positive SK-BR-3 cells are shown in the figure. Figure 16 Where T-mab-SJ-LP34 means Tmab+SJ-LP34, and T-mab-SJ-LP35 means Tmab+SJ-LP35.

[1229] The effects of antibody-drug conjugates T-mab-SJ-LP34 and T-mab-SJ-LP35 on the survival rate of Her2-negative MDA-MB-468 cells are shown in the figure. Figure 17 Where T-mab-SJ-LP34 means Tmab+SJ-LP34, and T-mab-SJ-LP35 means Tmab+SJ-LP35.

[1230] Experimental Example 2

[1231] MC38 hHER2 In vivo efficacy evaluation of cell-derived tumors in mouse xenograft models

[1232] (1) Efficacy evaluation of T-mab-GGFG-Dxd, T-mab-SJ-LP31, T-mab-SJ-LP34 and T-mab-SJ-LP35

[1233] MC38 hHER2 Cells (2×10) 6 (Number) were injected subcutaneously into the right rib area of ​​C57BL6J mice. Twelve days after inoculation, when the tumor volume approached 200 mm... 3 After removing animals with excessive weight or tumors that were too large or too small, the mice were randomly divided into 8 groups according to tumor volume, with each group consisting of 6 animals of similar weight.

[1234] The ADC and control samples were administered intraperitoneally on day 0 (D12) and day 7 (D19) after grouping. Each animal received 10 g / 0.1 mL of the drug based on its body weight. The dosages for T-mab-SJ-LP30 were 5 mg / kg, 2.5 mg / kg, and 1.25 mg / kg, respectively. The dosages for the other sample groups (T-mab-GGFG-Dxd, T-mab-SJ-LP31, T-mab-SJ-LP34, and T-mab-SJ-LP35) were all 5 mg / kg. Tumor volume and body weight were measured every 3 days and recorded for a total of 12 days (D24).

[1235] Data were recorded using Excel statistical software: the mean was calculated as AVG; SEM values ​​were calculated as STDEV / SQRT (number of animals per group); graphs were created using Graphpad Prism software; and statistical analysis of the data was performed using two-way ANOVA or one-way ANOVA.

[1236] The formula for calculating tumor volume (V) is: V = 1 / 2 × L_length × L_short. 2 .

[1237] The relative tumor proliferation rate T / C (%) = T / C × 100, where T and C are the tumor volumes of the treatment group and the control group at the end of the time period.

[1238] Tumor inhibition rate TGI (%) = 1 - T / C (%).

[1239] The results are shown in Table 3 and Appendix below. Figure 1 This indicates that the ADC compound coupled with SJ-LP30, SJ-LP31, SJ-LP34, and SJ-LP35 is effective against MC38. hHER2 Transplanted tumors have a strong tumor-suppressing effect.

[1240] Table 3. Effects of ADC on tumor-bearing C57BL6J mice MC38 hHER2 The efficacy of transplanted tumors

[1241]

[1242] For the tumor inhibition rate TGI value, "+" indicates a TGI value greater than 1 and less than or equal to 10%, "++" indicates a TGI value greater than 10 and less than or equal to 50%, "+++" indicates a TGI value greater than 50% and less than or equal to 70%, "++++" indicates a TGI value greater than 70% and less than 90%, and "+++++" indicates a TGI value greater than or equal to 90%.

[1243] (2) Efficacy evaluation of T-mab-LP10, T-mab-LP12, T-mab-LP13, T-mab-LP15, T-mab-LP17, T-mab-LP18, T-mab-LP19 and T-mab-LP20

[1244] MC38 hHER2 Cells (2×10) 6 (Number) mice were injected subcutaneously into the right rib area of ​​C57BL6J mice. Seven days after inoculation, when the tumor volume was close to 165 mm3, animals with excessive weight or tumors that were too large or too small were removed. The mice were then randomly divided into 10 groups according to the tumor volume, with animals of similar weight in each group and 6 mice in each group.

[1245] The ADC and control samples were administered via intraperitoneal injection on day 0 (D7) after grouping, with a single dose of 10 g / 0.1 mL per animal based on body weight, at a dose of 5 mg / kg. Tumor volume and body weight were measured twice every 3 days, and data were recorded for a total of 18 days (D25).

[1246] Data were recorded using Excel statistical software: the mean was calculated as AVG; SEM values ​​were calculated as STDEV / SQRT (number of animals per group); graphs were created using Graphpad Prism software; and two-way ANOVA and one-way ANOVA were used for statistical analysis of the data.

[1247] The formula for calculating tumor volume (V) is: V = 1 / 2 × L_length × L_short 2.

[1248] The relative tumor proliferation rate T / C (%) = (T-T0) / (C-C0) × 100, where T and C are the tumor volumes of the treatment group and the control group at the end of the time period; T0 and C0 are the tumor volumes at the beginning of the experiment.

[1249] Tumor inhibition rate TGI (%) = 1 - T / C (%).

[1250] The results are shown in Table 4 and Appendix below. Figure 2 This indicates that at a dose of 5 mpk, the ADC compound conjugated with LP10, LP12, LP13, and LP19 has a strong tumor-suppressing effect on MC38hHER2 xenografts.

[1251] Table 4. Effects of ADC on tumor-bearing C57BL6J mice MC38 hHER2 The efficacy of transplanted tumors

[1252]

[1253]

[1254] For the tumor inhibition rate TGI value, "+" indicates a TGI value greater than 1 and less than or equal to 10%, "++" indicates a TGI value greater than 10 and less than or equal to 50%, "+++" indicates a TGI value greater than 50% and less than or equal to 70%, "++++" indicates a TGI value greater than 70% and less than 90%, and "+++++" indicates a TGI value greater than or equal to 90%.

[1255] Experimental Example 3

[1256] A549 hClaudin 18.2 In vivo efficacy evaluation of cell-derived drug in mouse xenograft tumor model

[1257] A549 hClaudin 18.2 Cells (1×10) 7 One tumor was injected subcutaneously into the right rib area of ​​Nude mice. Sixteen days after inoculation, when the tumor volume reached approximately 120 mm, the tumor was observed to be 16 days later. 3 After removing animals with excessive weight or tumors that were too large or too small, the mice were randomly divided into 7 groups according to tumor volume, with animals of similar weight in each group and 6 animals in each group.

[1258] The ADC and control samples were administered via intraperitoneal injection on days 0 (D16), 7 (D23), and 14 (D30) after grouping. Each sample received 10 g / 0.1 mL of the ADC based on body weight. The dosages for Hz69H9-SJ-LP30 were 10 mg / kg and 5 mg / kg, respectively. The dosages for the other sample groups (Hz69H9-GGFG-Dxd, Hz69H9-SJ-LP10, Hz69H9-SJ-LP19, and Hz69H9-SJ-LP31) were all 5 mg / kg. Tumor volume and body weight were measured every 3 days and recorded for a total of 18 days (D34).

[1259] Data were recorded using Excel statistical software: the mean was calculated as AVG; SEM values ​​were calculated as STDEV / SQRT (number of animals per group); graphs were created using Graphpad Prism software; and statistical analysis of the data was performed using two-way ANOVA or one-way ANOVA.

[1260] The formula for calculating tumor volume (V) is: V = 1 / 2 × L_length × L_short. 2 .

[1261] The relative tumor proliferation rate T / C (%) = T / C × 100, where T and C are the tumor volumes of the treatment group and the control group at the end of the time period.

[1262] Tumor inhibition rate TGI (%) = 1 - T / C (%).

[1263] The results are shown in Table 5 and Appendix below. Figure 3 This indicates that the ADC compounds coupled with SJ-LP10, SJ-LP19, SJ-LP30, and SJ-LP31 are effective against MC38. hHER2 Transplanted tumors have a strong tumor-suppressing effect.

[1264] The anti-Claudin 18.2 antibody used in this experiment is derived from patent application CN115943162A.

[1265] Table 5. Effects of ADC on tumor-bearing Nude mice A549 hClaudin 18.2 The efficacy of transplanted tumors

[1266]

[1267] For the tumor inhibition rate TGI value, "+" indicates a TGI value greater than 1 and less than or equal to 10%, "++" indicates a TGI value greater than 10 and less than or equal to 50%, "+++" indicates a TGI value greater than 50% and less than or equal to 70%, "++++" indicates a TGI value greater than 70% and less than 90%, and "+++++" indicates a TGI value greater than or equal to 90%.

[1268] The foregoing description of specific exemplary embodiments of this disclosure is for illustrative and explanatory purposes. These descriptions are not intended to limit this disclosure to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of this disclosure and their practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of this disclosure, as well as various different choices and variations. The scope of this disclosure is intended to be defined by the claims and their equivalents.

Claims

1. A linker drug conjugate having the structure shown in formula (I): In the formula, C' is a group that can be coupled to an antibody or its antigen-binding fragment; H is a group having the structure shown in formula (IA); P is a peptide containing 1 to 10 amino acids; V represents either the absence of a group or a self-degrading group; in, The structure shown in equation (IA) is as follows: In the formula, n x It can be any integer from 1 to 10; A is *-NH-C(=O)-C 1-8 Alkylene-(O-CH2) m1 -C(=O)-** or *-NH-C(=O)-C 1-8 Alkylene-**, where * indicates the group with -(CH2)n x - indicates connection with a Hy group; ** indicates connection with a Hy group; m1 is any integer from 0 to 8; Hy is a hydrophilic group; D represents a biologically active molecule or its fragment.

2. The linker drug conjugate according to claim 1, wherein, D has the structures shown in equations (Ⅱ), (Ⅱ2), (Ⅱ3), (Ⅱ4), (Ⅱ5), and (Ⅱ6): In formula (II), BM is -CH- or -(CH2)n y -; n y It can be any integer from 1 to 8; BZ does not exist, -C (=O)-C 0-8 Alkylene -O-, -C(=O)-C 0-8 Alkylene-CH(CH3)-C 0-8 Alkylene -O- or -C(=O)-C 0-8 Alkylene-C(CH3)2-C 0-8 Alkylene-O-; R 1 R 2 Each is independently hydrogen, halogen, C 1-8 Alkyl or C 3-8 cycloalkyl, wherein the C 1-8 Alkyl and C 3-8 One or more hydrogen atoms in the cycloalkyl group may optionally be replaced by halogens; R 3 For hydrogen, C 1-8 Alkyl or C 3-8 cycloalkyl; R 4 For hydrogen, C 1-8 Alkylene, C 1-8 Alkyl or C 3-8 cycloalkyl; When BM is -CH- and R 4 C 1-8 When alkylene forms, BM and R 4 Connected to form a ring; In formula (Ⅱ2), Each R 5 Each is independently a halogen or C 1-3 alkyl; R 6 C 2-3 olefin group or C 1-3 Alkoxy; na and nb are each independent integers from 0 to 4; In formula (Ⅱ3), R 12 For oxygen or C 1-3 alkyl; nd is any integer from 0 to 2; In formula (Ⅱ4), R 13 It is a hydroxyl group or C 1-3 alkyl; R 14 Halogen or C 1-3 alkyl; In formula (Ⅱ5), R 16 It is hydrogen or C 1-3 alkyl; R 17 C 1-3 Alkyl or C 1-3 Alkoxy; In formula (Ⅱ6), R 10 R 11 Each independently is C 1-3 alkyl; Preferably, the linker drug conjugate has the structure shown in formula (VII): In the formula, C', P, V, A, Hy, n x Each custom definition is the same as that defined in claim 1; na, nb, R 5 R 6 Each custom definition is the same as that in formula (Ⅱ2); Preferably, each R 5 Each is independently fluorine, chlorine, bromine or C 1-3 alkyl; Preferably, each R 5 Each can be independently fluorine, chlorine, or bromine; Preferably, each R 5 Each is independently fluorine; Preferably, R 6 It is -CH=CH- or -O-CH2-; Preferably, na and nb are each an independent integer from 0 to 3; Preferably, na and nb are each an independent integer from 0 to 2; Preferably, na and nb are each an independent integer from 0 to 1; Preferably, na is 0 or 1; nb is 1; Preferably, the linker drug conjugate has the structure shown in formula (VIII): In the formula, C', P, V, A, Hy, n x Each custom definition is the same as that defined in claim 1; R 10 R 11 Each custom definition is the same as that in formula (Ⅱ6); Preferably, R 10 It is methyl; Preferably, R 11 for Preferably, the linker drug conjugate has the structure shown in formula (IX): In the formula, C', H, P, V, n x Each custom definition is the same as that defined in claim 1; nd、R 12 Each custom definition is defined in formula (Ⅱ3); Preferably, R 12 For oxygen or C 1-2 alkyl; Preferably, R 12 It can be oxo or methyl; Preferably, R 12 For oxygenation; Preferably, n d 1 or 2 Preferably, the linker drug conjugate has the structure shown in formula (X): In the formula, C', P, V, A, Hy, n x Each custom definition is the same as that defined in claim 1; R 13 R 14 Each custom definition is the same as that in formula (Ⅱ4); Preferably, R 13 It is a hydroxyl group; Preferably, R 14 It is a halogen; Preferably, R 14 It is fluorine, chlorine, or bromine; Preferably, R 14 It is chlorine; Preferably, the linker drug conjugate has the structure shown in formula (XI): In the formula, C', P, V, A, Hy, n x Each custom definition is the same as that defined in claim 1; R 16 R 17 Each custom definition is the same as that in formula (Ⅱ5); Preferably, R 16 It can be hydrogen or methyl; Preferably, R 16 It is hydrogen; Preferably, R 17 C 1-3 Alkoxy; Preferably, R 17 C 1-2 Alkoxy; Preferably, R 17 It is a methoxy group.

3. A linker drug conjugate having the structure shown in formula (I): In the formula, C' is a group that can be coupled to an antibody or its antigen-binding fragment; H is a group having the structure shown in formula (IA); P is a peptide containing 1 to 10 amino acids; V represents either the absence of a group or a self-degrading group; in, The structure shown in equation (IA) is as follows: In the formula, n x It can be any integer from 1 to 10; A is *-NH-C(=O)-C 1-8 Alkylene-(O-CH2) m1 -C(=O)-** or *-NH-C(=O)-C 1-8 Alkylene-**, where * indicates the group with -(CH2)n x - indicates connection with a Hy group; ** indicates connection with a Hy group. m1 is any integer from 0 to 8; Hy is a hydrophilic group; D is a biologically active molecule or fragment thereof, having the structure shown in formula (II): In the formula, BM is -CH- or -(CH2)n y -; n y It can be any integer from 1 to 8; BZ does not exist, -C (=O)-C 0-8 Alkylene -O-, -C(=O)-C 0-8 Alkylene-CH(CH3)-C 0-8 Alkylene -O- or -C(=O)-C 0-8 Alkylene-C(CH3)2-C 0-8 Alkylene-O-; R 1 R 2 Each is independently hydrogen, halogen, C 1-8 Alkyl or C 3-8 cycloalkyl, wherein the C 1-8 Alkyl and C 3-8 One or more hydrogen atoms in the cycloalkyl group may optionally be replaced by halogens; R 3 For hydrogen, C 1-8 Alkyl or C 3-8 cycloalkyl; R 4 For hydrogen, C 1-8 Alkylene, C 1-8 Alkyl or C 3-8 cycloalkyl; When BM is -CH- and R 4 C 1-8 When alkylene forms, BM and R 4 They are connected to form a ring.

4. The linker drug conjugate according to claim 1 or 3, wherein, BM is -(CH2)n y -; n y It can be any integer from 1 to 8; Preferably, BM is -(CH2)n y -; n y It can be any integer from 1 to 6; Preferably, BM is -(CH2)n y -; n y It can be any integer from 1 to 4; Preferably, BM is -(CH2)n y -; n y It can be any integer from 1 to 3; Preferably, BM is -(CH2)n y -; n y It can be 1 or 2; Preferably, wherein, BM stands for -CH-; R 4 C 1-8 Alkylene; BM and R 4 Connected to form a ring; Preferably, BM is -CH-; R 4 C 1-6 Alkylene; BM and R 4 Connected to form a ring; Preferably, BM is -CH-; R 4 C 1-3 Alkylene; BM and R 4 Connected to form a ring; Preferably, it has the structure shown in formula (Ⅳ): In the formula, C', A, Hy, P, V, BZ, R 1 R 2 R 3 R 4 n x n y Each custom definition is the same as that defined in claim 3; Preferably, n y It can be any integer from 1 to 8; preferably, n y It can be any integer from 1 to 6; preferably, n y It can be any integer from 1 to 4; preferably, n y It can be any integer from 1 to 3; preferably, n y It can be 1 or 2; Preferably, it has the structure shown in formula (V): In the formula, C', A, Hy, P, V, BZ, R 1 R 2 R 3 R 4 n x Each custom definition is the same as that defined in claim 3; Preferably, R 4 C 1-8 Alkylene; preferably, R 4 C 1-6 Alkylene; preferably, R 4 C 1-3 Alkylene; preferably, R 4 C 1-2 Alkylene; Preferably, R 4 For hydrogen, C 1-8 Alkyl or C 3-8 cycloalkyl; preferably, R 4 For hydrogen, C 1-6 Alkyl or C 3-6 cycloalkyl; preferably, R 4 For hydrogen, C 1-4 Alkyl or C 3-4 cycloalkyl; preferably, R 4 For hydrogen, C 1-3 Alkyl or C 3-4 cycloalkyl; preferably, R 4 It is hydrogen or C 1-3 Alkyl; preferably, R 4 It is hydrogen or C 1-2 Alkyl; preferably, R 4 It is hydrogen or methyl; preferably, R 4 It is hydrogen; Preferably, BZ is non-existent, -C (=O) -C 0-6 Alkylene -O-, -C(=O)-C 0-6 Alkylene-CH(CH3)-C 0-6 Alkylene -O- or -C(=O)-C 0-6 Alkylene-C(CH3)2-C 0-6 Alkylene-O-; Preferably, BZ is non-existent, -C (=O) -C 0-4 Alkylene -O-, -C(=O)-C 0-4 Alkylene-CH(CH3)-C 0-4 Alkylene -O- or -C(=O)-C 0-4 Alkylene-C(CH3)2-C 0-4 Alkylene-O-; Preferably, BZ is non-existent, -C (=O) -C 0-3 Alkylene -O-, -C(=O)-C 0-3 Alkylene-CH(CH3)-C 0-3 Alkylene -O- or -C(=O)-C 0-3 Alkylene-C(CH3)2-C 0-3 Alkylene-O-; Preferably, BZ is non-existent, -C (=O) -C 1-3 Alkylene -O-, -C(=O)-C 0-3 Alkylene-CH(CH3)-C 0-3 Alkylene -O- or -C(=O)-C 0-3 Alkylene-C(CH3)2-C 1-3 Alkylene-O-; Preferably, BZ is non-existent, -C (=O) -C 1-3 Alkylene -O-, -C(=O)-CH(CH3)-O-, -C(=O)-C 1-3 Alkylenes -CH(CH3)-O-, -C(=O)-CH(CH3)-C 1-3 Alkylene -O- or -C(=O)-C(CH3)2-C 1-3 Alkylene-O-; Preferably, BZ is absent, -C(=O)-CH2-O-, -C(=O)-CH(CH3)-O-, -C(=O)-CH2-CH(CH3)-O-, -C(=O)-CH(CH3)-CH2-O-, or -C(=O)-C(CH3)2-CH2-O-; Preferably, R 1 R 2 Each is independently hydrogen, halogen, C 1-6 Alkyl or C 3-6 cycloalkyl, wherein the C 1-6 Alkyl and C 3-6 One or more hydrogen atoms in the cycloalkyl group may optionally be replaced by halogens; Preferably, R 1 R 2 Each is independently hydrogen, halogen, C 1-3 Alkyl or C 3-4 cycloalkyl, wherein the C 1-3 Alkyl and C 3-4 One or more hydrogen atoms in the cycloalkyl group may optionally be replaced by halogens; Preferably, R 1 R 2 Each is independently hydrogen, halogen, or C 1-3 Alkyl, wherein the C 1-3 One or more hydrogen atoms of the alkyl group may optionally be replaced by halogens; Preferably, R 1 R 2 Each is independently hydrogen, halogen, or C 1-2 Alkyl, wherein the C 1-2 One or more hydrogen atoms of the alkyl group may optionally be replaced by halogens; Preferably, R 1 R 2 Each can be independently hydrogen, halogen, or methyl; Preferably, R 3 For hydrogen, C 1-6 Alkyl or C 3-6 cycloalkyl; Preferably, R 3 For hydrogen, C 1-3 Alkyl or C 3-4 cycloalkyl; Preferably, R 3 It is hydrogen or C 1-3 alkyl; Preferably, R 3 It is hydrogen or C 1-2 alkyl; Preferably, R 3 It can be hydrogen or methyl; Preferably, D is selected from the following compound fragments: Preferably, A is *-NH-C(=O)-C 1-6 Alkylene-(O-CH2) m1 -C(=O)-** or *-NH-C(=O)-C 1-6 Alkylene-**, where * indicates the group with -(CH2)n x - indicates connection to a group, ** indicates connection to a Hy group; m1 is any integer from 0 to 6; Preferably, A is *-NH-C(=O)-C 1-4 Alkylene-(O-CH2) m1 -C(=O)-** or *-NH-C(=O)-C 1-4 Alkylene-**, where * indicates the group with -(CH2)n x - indicates connection to a group, ** indicates connection to a Hy group; m1 is any integer from 0 to 4; Preferably, A is *-NH-C(=O)-C 1-3 Alkylene-(O-CH2) m1 -C(=O)-** or *-NH-C(=O)-C 1-3 Alkylene-**, where * indicates the group with -(CH2)n x - indicates connection to a group, ** indicates connection to a Hy group; m1 is any integer from 0 to 3; Preferably, A is in With -(CH2)n x - Groups are linked together, Connected to a Hy group; Preferably, Hy is Preferably, C' is selected from: Among them, R C1 It is hydrogen or C 1-8 Alkyl; R C2 It is a halogen; n1, n2, n3, n4, n5, n6, n7, n8, and n9 are each an independent integer from 0 to 10; Preferably, C' is selected from: Where n1 and n2 are each an independent integer from 0 to 10; Preferably, C' is Preferably, V is selected from non-existent, Where *L1 indicates connection to part P, and **L1 indicates connection to part D; Preferably, V is selected from non-existent, Where *L1 indicates connection to part P, and **L1 indicates connection to part D; Preferably, the amino acid is selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit), arginine (Arg), and asparagine (Asn), and the side chain of the amino acid is optionally substituted with an alkyl or hydrophilic group. Preferably, P is a peptide containing 2 amino acids, a peptide containing 3 amino acids, or a peptide containing 4 amino acids; Preferably, P is a peptide containing two amino acids, selected from... *L2 -Phe-Lys- **L2 , *L2 -Val-Ala- **L2 , *L2 -Val-Lys- **L2 , *L2 -Ala-Lys- **L2 , *L2 -Val-Cit- **L2 , *L2 -Phe-Cit- **L2 , *L2 -Leu-Cit- **L2 , *L2 -Phe-Arg- **L2 and *L2 -Gly-Val- **L2 , where *L2 indicates connection to part H, and **L2 indicates connection to part V; Preferably, P is a peptide containing 3 amino acids, selected from... *L2 -Glu-Val-Ala- **L2 , *L2 -Glu-Val-Cit- **L2 , *L2 -Ala-Ala-Ala- **L2 , *L2 -Ala-Ala-Asn- **L2 and Where *L2 indicates connection to part H, and **L2 indicates connection to part V; Preferably, P is a peptide containing four amino acids, selected from... *L2 -Gly-Gly-Phe-Gly- **L2 , *L2 -Gly-Gly-Val-Ala-** L2 and *L2 -Gly-Phe-Gly-Gly- **L2 Preferred *L2 -Gly-Gly-Phe-Gly- **L2 and *L2 -Gly-Gly-Val-Ala- **L2 , where *L2 indicates connection to part H, and **L2 indicates connection to part V; Preferably, P is selected from: Where *L2 indicates connection to part H, and **L2 indicates connection to part V; Preferably, the compound of formula (I) is selected from the following compounds:

5. An antibody-drug conjugate, which is obtained by conjugating the linker drug conjugate of any one of claims 1-4 with an antibody or an antigen-binding fragment thereof; Preferably, the antibody-drug conjugate has the structure shown in formula (Ⅲ): in, C is the group formed by C' being coupled with an antibody or its antigen-binding fragment; Ab represents an antibody or its antigen-binding fragment; n xy It can be any integer from 1 to 10; H, P, V, and D are each defined as in claim 1 or 2; The C'-HPVD is a linker drug conjugate; The CHPVD is a group formed by coupling C'-HPVD with an antibody or its antigen-binding fragment; Preferably, the linker drug conjugate has the structure shown in formula (VII): In the formula, C', P, V, A, Hy, n x Each custom definition is the same as that defined in claim 1; na, nb, R 5 R 6 Each custom definition is the same as that in formula (Ⅱ2); Preferably, each R 5 Each is independently fluorine, chlorine, bromine or C 1-3 alkyl; Preferably, each R 5 Each can be independently fluorine, chlorine, or bromine; Preferably, each R 5 Each is independently fluorine; Preferably, R 6 It is -CH=CH- or -O-CH2-; Preferably, na and nb are each an independent integer from 0 to 3; Preferably, na and nb are each an independent integer from 0 to 2; Preferably, na and nb are each an independent integer from 0 to 1; Preferably, na is 0 or 1; nb is 1. Preferably, the linker drug conjugate has the structure shown in formula (VIII): In the formula, C', P, V, A, Hy, n x Each custom definition is the same as that defined in claim 1; R 10 R 11 Each custom definition is the same as that in formula (Ⅱ6); Preferably, R 10 It is methyl; Preferably, R 11 for Preferably, the linker drug conjugate has the structure shown in formula (IX): In the formula, C', H, P, V, n x Each custom definition is the same as that defined in claim 1; nd、R 12 Each custom definition is the same as that in formula (Ⅱ3); Preferably, R 12 For oxygen or C 1-2 alkyl; Preferably, R 12 It can be oxo or methyl; Preferably, R 12 For oxygenation; Preferably, n d It can be 1 or 2; Preferably, the linker drug conjugate has the structure shown in formula (X): In the formula, C', P, V, A, Hy, n x Each custom definition is the same as that defined in claim 1; R 13 R 14 Each custom definition is the same as that in formula (Ⅱ4); Preferably, R 13 It is a hydroxyl group; Preferably, R 14 It is a halogen; Preferably, R 14 It is fluorine, chlorine, or bromine; Preferably, R 14 It is chlorine; Preferably, the linker drug conjugate has the structure shown in formula (XI): In the formula, C', P, V, A, Hy, n x Each custom definition is the same as that defined in claim 1; R 16 R 17 Each custom definition is the same as that in formula (Ⅱ5); Preferably, R 16 It can be hydrogen or methyl; Preferably, R 16 It is hydrogen; Preferably, R 17 C 1-3 Alkoxy; Preferably, R 17 C 1-2 Alkoxy; Preferably, R 17 It is a methoxy group; Preferably, the linker drug conjugate has the structure shown in formula (Ⅳ): In the formula, C', A, Hy, P, V, BZ, R 1 R 2 R 3 R 4 n x n y Each custom expression is defined in expression (I); Preferably, n y It can be any integer from 1 to 8; preferably, n y It can be any integer from 1 to 6; preferably, n y It can be any integer from 1 to 4; preferably, n y It can be any integer from 1 to 3; preferably, n y It can be 1 or 2; Preferably, R 4 For hydrogen, C 1-8 Alkyl or C 3-8 cycloalkyl; preferably, R 4 For hydrogen, C 1-6 Alkyl or C 3-6 cycloalkyl; preferably, R 4 For hydrogen, C 1-4 Alkyl or C 3-4 cycloalkyl; preferably, R 4 For hydrogen, C 1-3 Alkyl or C 3-4 cycloalkyl; preferably, R 4 It is hydrogen or C 1-3 Alkyl; preferably, R 4 It is hydrogen or C 1-2 Alkyl; preferably, R 4 It is hydrogen or methyl; preferably, R 4 It is hydrogen; Preferably, the linker drug conjugate has the structure shown in formula (V): In the formula, C', A, Hy, P, V, BZ, R 1 R 2 R 3 R 4 n x Each custom expression is defined in expression (I); Preferably, R 4 C 1-8 Alkylene; preferably, R 4 C 1-6 Alkylene; preferably, R 4 C 1-3 Alkylene; preferably, R 4 C 1-2 Alkylene; Preferably, BZ is non-existent, -C (=O) -C 0-6 Alkylene -O-, -C(=O)-C 0-6 Alkylene-CH(CH3)-C 0-6 Alkylene -O- or -C(=O)-C 0-6 Alkylene-C(CH3)2-C 0-6 Alkylene-O-; Preferably, BZ is non-existent, -C (=O) -C 0-4 Alkylene -O-, -C(=O)-C 0-4 Alkylene-CH(CH3)-C 0-4 Alkylene -O- or -C(=O)-C 0-4 Alkylene-C(CH3)2-C 0-4 Alkylene-O-; Preferably, BZ is non-existent, -C (=O) -C 0-3 Alkylene -O-, -C(=O)-C 0-3 Alkylene-CH(CH3)-C 0-3 Alkylene -O- or -C(=O)-C 0-3 Alkylene-C(CH3)2-C 0-3 Alkylene-O-; Preferably, BZ is non-existent, -C (=O) -C 1-3 Alkylene -O-, -C(=O)-C 0-3 Alkylene-CH(CH3)-C 0-3 Alkylene -O- or -C(=O)-C 0-3 Alkylene-C(CH3)2-C 1-3 Alkylene-O-; Preferably, BZ is non-existent, -C (=O) -C 1-3 Alkylene -O-, -C(=O)-CH(CH3)-O-, -C(=O)-C 1-3 Alkylenes -CH(CH3)-O-, -C(=O)-CH(CH3)-C 1-3 Alkylene -O- or -C(=O)-C(CH3)2-C 1-3 Alkylene-O-; Preferably, BZ is absent, -C(=O)-CH2-O-, -C(=O)-CH(CH3)-O-, -C(=O)-CH2-CH(CH3)-O-, -C(=O)-CH(CH3)-CH2-O-, or -C(=O)-C(CH3)2-CH2-O-; Preferably, R 1 R 2 Each is independently hydrogen, halogen, C 1-6 Alkyl or C 3-6 cycloalkyl, wherein the C 1-6 Alkyl and C 3-6 One or more hydrogen atoms in the cycloalkyl group may optionally be replaced by halogens; Preferably, R 1 R 2 Each is independently hydrogen, halogen, C 1-3 Alkyl or C 3-4 cycloalkyl, wherein the C 1-3 Alkyl and C 3-4 One or more hydrogen atoms in the cycloalkyl group may optionally be replaced by halogens; Preferably, R 1 R 2 Each is independently hydrogen, halogen, or C 1-3 Alkyl, wherein the C 1-3 One or more hydrogen atoms of the alkyl group may optionally be replaced by halogens; Preferably, R 1 R 2 Each is independently hydrogen, halogen, or C 1-2 Alkyl, wherein the C 1-2 One or more hydrogen atoms of the alkyl group may optionally be replaced by halogens; Preferably, R 1 R 2 Each can be independently hydrogen, halogen, or methyl; Preferably, R 3 For hydrogen, C 1-6 Alkyl or C 3-6 cycloalkyl; Preferably, R 3 For hydrogen, C 1-3 Alkyl or C 3-4 cycloalkyl; Preferably, R 3 It is hydrogen or C 1-3 alkyl; Preferably, R 3 It is hydrogen or C 1-2 alkyl; Preferably, R 3 It can be hydrogen or methyl; Preferably, D is selected from the following compound fragments: Preferably, A is *-NH-C(=O)-C 1-6 Alkylene-(O-CH2) m1 -C(=O)-** or *-NH-C(=O)-C 1-6 Alkylene-**, where * indicates the group with -(CH2)n x - indicates connection to a group, ** indicates connection to a Hy group; m1 is any integer from 0 to 6; Preferably, A is *-NH-C(=O)-C 1-4 Alkylene-(O-CH2) m1 -C(=O)-** or *-NH-C(=O)-C 1-4 Alkylene-**, where * indicates the group with -(CH2)n x - indicates connection to a group, ** indicates connection to a Hy group; m1 is any integer from 0 to 4; Preferably, A is *-NH-C(=O)-C 1-3 Alkylene-(O-CH2) m1 -C(=O)-** or *-NH-C(=O)-C 1-3 Alkylene-**, where * indicates the group with -(CH2)n x - indicates connection to a group, ** indicates connection to a Hy group; m1 is any integer from 0 to 3; Preferably, A is in With -(CH2)n x - Groups are linked together, Connected to a Hy group; Preferably, Hy is Preferably, C' is selected from: Among them, R C1 It is hydrogen or C 1-8 Alkyl; R C2 It is a halogen; n1, n2, n3, n4, n5, n6, n7, n8, and n9 are each an independent integer from 0 to 10; Preferably, C' is selected from: Where n1 and n2 are each an independent integer from 0 to 10; Preferably, C' is Preferably, V is selected from non-existent, Where *L1 indicates connection to part P, and **L1 indicates connection to part D; Preferably, V is selected from non-existent, Where *L1 indicates connection to part P, and **L1 indicates connection to part D; Preferably, the amino acid is selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit), arginine (Arg), and asparagine (Asn), and the side chain of the amino acid is optionally substituted with an alkyl or hydrophilic group. Preferably, P is a peptide containing 2 amino acids, a peptide containing 3 amino acids, or a peptide containing 4 amino acids; Preferably, P is a peptide containing two amino acids, selected from... *L2 -Phe-Lys- **L2 , *L2 -Val-Ala- **L2 , *L2 -Val-Lys-** L2 、* L2 -Ala-Lys- **L2 , *L2 -Val-Cit- **L2 , *L2 -Phe-Cit- **L2 , *L2 -Leu-Cit- **L2 , *L2 -Phe-Arg- **L2 and *L2 -Gly-Val- **L2 , where *L2 indicates connection to part H, and **L2 indicates connection to part V; Preferably, P is a peptide containing 3 amino acids, selected from... *L2 -Glu-Val-Ala- **L2 , *L2 -Glu-Val-Cit- **L2 , *L2 -Ala-Ala-Ala- **L2 , *L2 -Ala-Ala-Asn- **L2 and Where *L2 indicates connection to part H, and **L2 indicates connection to part V; Preferably, P is a peptide containing four amino acids, selected from... *L2 -Gly-Gly-Phe-Gly- **L2 , *L2 -Gly-Gly-Val-Ala-** L2 and *L2 -Gly-Phe-Gly-Gly- **L2 Preferred *L2 -Gly-Gly-Phe-Gly- **L2 and *L2 -Gly-Gly-Val-Ala- **L2 , where *L2 indicates connection to part H, and **L2 indicates connection to part V; Preferably, P is selected from: Where *L2 indicates connection to part H, and **L2 indicates connection to part V; Preferably, the compounds are selected from the following: Preferably, the Ab is selected from any one of mouse antibodies or their antigen-binding fragments, chimeric antibodies or their antigen-binding fragments, nanodomain antibodies, human antibodies or their antigen-binding fragments, or humanized antibodies or their antigen-binding fragments; Preferably, the Ab is selected from the following targets: HER2, TROP2, Claudin18.2, ROR1, PD-L1, HER1, HER3, HER4, VEGFR, EpCAM, Mesothelin, CD19, CD20, GPRC5D, SLAMF7, BCMA, CD38, CD56, CD44v6, FLT3, CD33, CD123, CD37, TCRβ-chain, Annexin A2, B7-H3, CD171, Chlorotoxin, Claudin-6, EIIIB, Glypican-1, IntegrinαVβ3, Mucl, CD22, CD30, SLAM (Signaling lymphocytic activation). molecule), GD2, GPC3, CEA, EGFRvIII, PSMA, EGFR, PIK3CA, KRAS, BRAF, PD-1, CTLA-4, BRCA1 / 2, PARP, FSHR, LILRB4, CD33, CDH17; Preferably, the Ab is selected from the following targets: HER2, TROP2, Claudin18.2, ROR1, PD-L1, HER1, HER3, or HER4; Preferably, the Ab is selected from the following targets: HER2, TROP2, Claudin18.2, ROR1, or PD-L1; Preferably, the Ab is selected from the following target: HER2; Preferably, the Ab is trastuzumab.

6. A pharmaceutical composition comprising one or more of the linker drug conjugates of any one of claims 1-4 or the antibody drug conjugates of claim 5, and optionally a pharmaceutically acceptable carrier.

7. Use of the linker drug conjugate of any one of claims 1-4, the antibody drug conjugate of claim 5, or the pharmaceutical composition of claim 6 in the preparation of a medicament for treating cancer.

8. A method of treating cancer, comprising administering to a patient in need a therapeutically effective amount of the linker drug conjugate of any one of claims 1-4, the antibody drug conjugate of claim 5, or the pharmaceutical composition of claim 6; Preferably, the cancer is breast cancer, stomach cancer, leukemia, bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, or thyroid cancer. Preferably, the cancer is breast cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, ovarian cancer, bile duct cancer, or endometrial cancer. Preferably, the cancer is breast cancer or stomach cancer.

9. A method for preparing the antibody-drug conjugate as described in claim 5, comprising the following steps: reacting an antibody or its antigen-binding fragment with the linker drug conjugate as described in any one of claims 1-4 to obtain the antibody-drug conjugate.

10. A method for preparing a drug, wherein the method comprises the step of conjugating an antibody or an antigen-binding fragment thereof with a linker drug conjugate according to any one of claims 1-4.

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