Antibody-drug conjugate, preparation method therefor, and use thereof

Abstract

An antibody-drug conjugate, a preparation method therefor, and use thereof. Specifically, provided is a drug-linker compound for conjugation, the drug being a glucocorticoid receptor agonist. Also provided is an antibody-drug conjugate prepared by conjugating the drug-linker compound with an antibody. Further provided are a linker of a glucocorticoid receptor agonist, a preparation method for an antibody-drug conjugate thereof, and use thereof in the field of pharmaceutics.

Description

Antibody drug conjugates, methods of making and uses thereof

[0001] This application is based on Chinese Patent Application No. 202510304355.8, filed on March 14, 2025, and Chinese Patent Application No. 202511090300.8, filed on August 5, 2025, for which priority is claimed, and the disclosures of which are incorporated by reference herein in their entireties.

[0002] SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted in XML format via EFS-Web and is hereby incorporated by reference in its entirety. Said XML file, named “IEC260034PCT”, created on March 4, 2026, is 58 kKB in size. TECHNICAL FIELD

[0004] The present application relates to the field of medicine, in particular to an antibody drug conjugate, a drug-linker and intermediate compounds thereof, and methods of making and uses thereof. BACKGROUND

[0005] Steroidal glucocorticoid receptor agonists are a class of therapeutic drugs widely used for the treatment of inflammatory and immune diseases. This class of drugs exerts anti-inflammatory effects by activating glucocorticoid receptors in cells, interfering with the recruitment of leukocytes to the inflammatory site, and inhibiting the formation and release of inflammatory mediators from leukocytes and tissue cells. Long-term and large-dose use of steroidal glucocorticoid receptor agonist drugs can cause side effects such as infection, gastrointestinal reactions, endocrine disorders (such as moon face, buffalo hump, weight gain, hypokalemia, abnormal blood sugar and blood pressure, etc.), osteoporosis, etc. In order to reduce the above-mentioned side effects of steroidal glucocorticoid receptor agonist drugs, inhalation preparations or skin topical application preparations have made progress; in addition, introducing metabolically unstable groups into the steroidal structure to accelerate inactivation and reduce systemic exposure is also one of the measures to reduce adverse reactions.

[0006] In recent years, in order to improve the effectiveness and safety of steroidal glucocorticoid receptor agonist drugs, coupling them with antibodies is a new direction for the development of this class of drugs.

[0007] SUMMARY

[0008] The present application relates to steroidal glucocorticoid receptor agonists and linkers and antibody drug conjugates thereof, which have excellent agonistic activity and good drugability, and are expected to be used for the treatment of inflammatory and immune diseases.

[0009] Specifically, this application provides a drug linker compound for conjugation, wherein the drug is a glucocorticoid receptor agonist. This application also provides antibody-drug conjugates prepared by conjugating the drug-linker compound with an antibody, which exhibit good drug-like properties, including good pharmacodynamic activity, safety, and favorable physicochemical and pharmacokinetic properties. The conjugates of this application have excellent therapeutic effects on inflammatory diseases such as rheumatoid arthritis. This application further provides methods for preparing the glucocorticoid receptor agonist drug linker and its antibody-drug conjugate, as well as their applications in the pharmaceutical field.

[0010] Antibody-drug conjugates

[0011] On the one hand, this application provides an antibody-drug conjugate having the structure shown in formula HA-[MLED]x, wherein:

[0012] HA is an antibody or its antigen-binding fragment that specifically binds to an antigen;

[0013] M is the linker site that connects to the antibody or its antigen-binding fragment;

[0014] L is the connection structure that connects the joint portions M and E;

[0015] E is a structure that connects L and D;

[0016] D is a bioactive molecule or a fragment thereof; preferably, the bioactive molecule is a glucocorticoid drug; more preferably, the glucocorticoid drug is a glucocorticoid receptor agonist;

[0017] x is an integer selected from 1 to 10, preferably an integer from 3 to 8.

[0018] In the antibody-drug conjugate, the glucocorticoid receptor agonist can be linked to the antibody or its antigen-binding fragment via a linker (such as the “MLE” fragment shown in this application).

[0019] In some embodiments, the antigen bound to the antibody or its antigen-binding fragment in the antibody-drug conjugate is selected from one or more of TNFα, IL6R, BDCA2, NR3C1, MSR1, PRLR, CD19, CD25, CD40, D70, CD74, and CD163.

[0020] In some embodiments, the antigen that binds to the antibody or its antigen-binding fragment includes one or more of TNFα, IL6R, and CD19.

[0021] In some embodiments, the antibody or its antigen-binding fragment includes, but is not limited to, adalimumab with TNFα as the target antigen, tocilizumab with IL6R as the target antigen, and antibodies 1D3 or 21D4 with CD19 as the target antigen.

[0022] In some implementations, M is The C structure is a single bond, sulfonamide group, phosphoramidyl group, 4-6 membered aliphatic heterocycle or 5-20 membered aromatic ring system, wherein the aliphatic heterocycle or aromatic ring system is optionally selected by one or more groups, each independently selected from oxo (=O), halogen, cyano, nitro, amino, carboxyl, mercapto and C. 1-6 Alkyl group substitution; and M1 is selected from a single bond or a fragment consisting of one or more groups selected from the following: -O-, -NH-, -NH2, -C(=O)-, HO2C(=O)-, -S(=O)2-, HOS(=O)2-, -C=NO-, -NH-S(=O)2-NH-, 3-6 membered heterocyclic group, phenylene, 5-10 membered heteroaryl group, C 1-20 Alkylene, C 2-20 imide and C 2-20 Alynyl group.

[0023] In some implementations, M is Wherein, C is a single bond, sulfonamide group, phosphoramidyl group, 4-6 membered aliphatic heterocycle, 5-6 membered heteroaromatic ring, or a polycyclic ring formed by two or three units selected from 6 membered aromatic heterocycles and benzene rings linked by single bonds, wherein the aliphatic heterocycle, heteroaromatic ring, or polycyclic ring is optionally substituted by one or more groups selected from oxo (=O), halogen, cyano, and nitro; and M1 is selected from a single bond or a fragment composed of one or more groups selected from the following groups: -NH-, -C(=O)-, -NH-S(=O)2-NH-, C 1-10 Alkylene, C 2-10 imide and C 2- 10 Alynyl group.

[0024] In some implementations, M is The C structure is a single bond, halogen, sulfonamide group, phosphoramidyl group, 4-6 membered aliphatic heterocycle, or 5-20 membered aromatic ring system, wherein the aliphatic heterocycle or aromatic ring system is optionally selected by one or more groups, each independently selected from oxo (=O), halogen, cyano, nitro, amino, carboxyl, mercapto, and C. 1-6Alkyl group substitution; and M1 is selected from a single bond or a fragment consisting of one or more groups selected from the following: -O-, -NH-, -NH2, -C(=O)-, HO2C(=O)-, -S(=O)2-, HOS(=O)2-, -C=NO-, -NH-S(=O)2-NH-, 3-6 membered heterocyclic group, phenylene, 5-10 membered heteroaryl group, C 1-20 Alkylene, C 2-20 imide and C 2-20 Alynyl group.

[0025] In some implementations, M is Wherein, C is a single bond, sulfonamide group, phosphoramidyl group, 4-6 membered aliphatic heterocycle, 5-6 membered heteroaromatic ring, or a polycyclic ring formed by 2-5 (preferably 3) units selected from 6 membered aromatic heterocycles and benzene rings linked by single bonds, wherein the aliphatic heterocycle, heteroaromatic ring, or polycyclic ring is optionally substituted by one or more groups selected from oxo (=O), halogen, cyano, and nitro; and M1 is selected from a single bond or a fragment composed of one or more groups selected from the following groups: -NH-, -C(=O)-, -NH-S(=O)2-NH-, C 1-10 Alkylene, C 2- 10 imide and C 2-10 Alynyl group.

[0026] In some implementations, M is Where the C structure is M1 is selected from single bonds, carbonyl groups, sulfonyl groups, -NH-, -NH-S(=O)2-NH-, C 1-6 Alkylene, C 2-6 imide and C 2-6 Alynyl group.

[0027] In some implementations, M is Where the C structure is M1 is selected from single bonds, carbonyl groups, sulfonyl groups, -NH-, -NH-S(=O)2-NH-, C 1-6 Alkylene, C 2-6 imide and C 2-6 Alynyl group.

[0028] In some implementations, M is The C structure is selected from single bonds, M1 is selected from single bonds, carbonyl groups, sulfonyl groups, -NH-, -NH-S(=O)2-NH-, C 1-6 Alkylene, C 2-6 imide and C 2-6 Alynyl group.

[0029] In some implementations, M is selected from

[0030] In some implementations, M is selected from

[0031] In some implementations, M is

[0032] In some implementations, M is

[0033] In some implementations, M is

[0034] In some implementations, L is selected from a structure consisting of one or more of the following parts: C 1-6 Alkyl groups, -N(R')-, carbonyl groups, glycosyl groups, -O-, natural or non-natural amino acids and their analogs or derivatives (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH2CH2(OCH2CH2)) r OCH3)), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe -Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gl y-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Ala-Ala, Glu-Val-Ala, Glu-Val-Cit, L ys-Ala-Ala, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG, SEQ ID NO:22), Gly-Gly-Val-Ala (GGVA, SEQ ID NO:23), Gly-Phe-Leu-Gly (GFLG, SEQ ID NO:24), Glu-Ala-Ala-Ala (EAAA, SEQ ID NO:25), Gly-Gly-Gly-Gly-Gly (GGGGG, SEQ ID NO:26),

[0035] Where R' represents hydrogen, C 1-6 Alkyl groups or containing -(CH2CH2O) r -ofC 4-30 Alkyl; r is an integer selected from 1 to 10; s is an integer selected from 1 to 20. Preferably, r is an integer selected from 1 to 6; preferably, s is an integer selected from 1 to 10.

[0036] In some implementations, L is selected from a structure consisting of one or more of the following parts: C 1-6 Alkyl groups, -N(R')-, carbonyl groups, glycosyl groups, -O-, natural or non-natural amino acids and their analogs or derivatives (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH2CH2(OCH2CH2)) r OCH3), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG(SEQ ID))). NO:22)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO:23)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO:24)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO:25)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO:26)),

[0037] Where R' represents hydrogen, C 1-6 Alkyl groups or containing -(CH2CH2O) r -ofC 4-30Alkyl; r is an integer selected from 1 to 10; s is an integer selected from 1 to 20. Preferably, r is an integer selected from 1 to 6; preferably, s is an integer selected from 1 to 10.

[0038] In some implementations, L is a structure consisting of one or more parts selected from the following: Where s is an integer selected from 1 to 20, preferably an integer from 1 to 10.

[0039] In some implementations, L is selected from the following structures:

[0040] Each of n is independently selected from an integer from 1 to 20. In some implementations, each of n is independently selected from an integer from 1 to 15, such as an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or even 1, 2, 3, 4 or 5.

[0041] In some implementations, L is selected from the following structures:

[0042] Each of n is independently selected from an integer from 1 to 20. In some implementations, each of n is independently selected from an integer from 1 to 15, such as an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or even 1, 2, 3, 4 or 5.

[0043] In some implementations, L is selected from the following structures:

[0044] Each of n is independently selected from an integer from 1 to 20. In some implementations, each of n is independently selected from an integer from 1 to 15, such as an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or even 1, 2, 3, 4 or 5.

[0045] In some implementations, L is selected from the following structures:

[0046] Each of n is independently selected from an integer from 1 to 20. In some implementations, each of n is independently selected from an integer from 1 to 15, such as an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or even 1, 2, 3, 4 or 5.

[0047] In some implementations, E is a single bond or -NHCH2-.

[0048] In some implementations, E is a single bond.

[0049] In some implementation schemes, A structure composed of one or more of the following fragments: In some implementations, n is independently selected from integers from 1 to 20. In some implementations, n is independently selected from integers from 1 to 15, such as integers from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and again, such as 1, 2, 3, 4 or 5.

[0050] In some implementation schemes, Selected from the following structures:

[0051] In some implementation schemes, Selected from the following structures:

[0052] In some implementation schemes, Selected from the following structures:

[0053] In some implementation schemes, Selected from the following structures:

[0054] In some implementation schemes, Selected from the following structures:

[0055] In some implementation schemes, Selected from the following structures:

[0056] In some implementation schemes, Selected from the following structures:

[0057] In some implementation schemes, Selected from the following structures:

[0058] In some implementation schemes, Selected from the following structures:

[0059] In some implementation schemes, Selected from the following structures:

[0060] In some implementation schemes, Selected from the following structures:

[0061] In some implementation schemes, Selected from the following structures:

[0062] In some implementation schemes, Selected from the following structures:

[0063] In some implementation schemes, Selected from the following structures:

[0064] In some implementation schemes, Selected from the following structures:

[0065] In some implementations, -MLE- is selected from the following structures:

[0066] Where n is selected from an integer from 1 to 20; for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0067] In some implementations, -MLE- is selected from the following structures:

[0068] Where n is selected from an integer from 1 to 20; for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0069] In some implementations, n is preferably an integer from 1 to 10, for example, n is 1, 2, 3, 4, or 5.

[0070] In some implementations, the structure of D is as follows:

[0071] in,

[0072] R1 is independently selected from hydrogen, halogen, and -NR.a R b hydroxyl, cyano, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Alkyl groups, C groups substituted with one or more hydroxyl groups 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 Halogenated alkyl groups;

[0073] R2 is selected from hydrogen, C 1-6 Alkyl, hydroxyl and C 1-6 Alkoxy;

[0074] Ring A does not exist or is selected from C. 6-10 Aromatic rings and 5-6 quinary heterocyclic aromatic rings;

[0075] X1 is selected from single bond, C 1-6 Alkylene, C 3-6 Cycloalkylene, C 2-6 imidene group, C 2-6 Ethyne and -NR a -;

[0076] Y1 is selected from -O- and -S-;

[0077] Z1 is selected from hydroxyl, halogen, and cyano groups;

[0078] Q1 and Q2 are each independently selected from hydrogen and halogen;

[0079] Q3 is selected from -O- and -C. 1-6 Alkylene -O-, -C 1-6 alkoxides -O-, -C 1-6 Halogenated alkylene-O- and -NR a -;

[0080] a1 is 1 or 2;

[0081] a2 is selected from 0, 1, 2, 3, 4, and 5;

[0082] R a R b Each is independently selected from hydrogen and C. 1-6 alkyl;

[0083] The conditions are: when R1 is independently selected from halogen, amino and methyl, a2 is 2, R2 is methyl, X1 is a single bond, Y1 is -S-, Z1 is fluorine, a1 is 1, Q1 is hydrogen or fluorine, and Q2 is fluorine, ring A is not phenyl.

[0084] When ring A is absent, R1 is neither hydrogen nor C. 1-6 Alkyl; and

[0085] When ring A is a 5-6 membered heteroaromatic ring, R1 is hydrogen and R2 is C 1-6 When alkyl, Q1 is not halogen.

[0086] In some implementations, R1 is independently selected from hydrogen and halogen.

[0087] In some implementations, R2 is selected from hydrogen and C. 1-6 alkyl.

[0088] In some implementations, ring A is absent or selected from benzene rings and 5-6 membered heteroaromatic rings.

[0089] In some implementations, X1 is a single bond.

[0090] In some implementations, a2 is selected from 0, 1, and 2.

[0091] In some implementations, the structure of D is as follows:

[0092] in,

[0093] Q4 and Q5 are each independently selected from hydrogen or fluorine;

[0094] X2 is selected from HOCH2-, (HO)2P(=O)OCH2- and FCH2S-;

[0095] Z2 is oxygen or nitrogen;

[0096] R3 is selected from -O-(CR c R d ) n1 -、-HN-(CR c R b ) n1 -、-O-(CR c R d ) n1 -C(=O)-、-HN-(CR c R d ) n1 -C(=O)-、-O-(CR c R d ) n1 -OC(=O)-、-HN-(CR c R d ) n1 -OC(=O)-、-O-(CR c R d ) n1 -NR c -C(=O)- and -HN-(CR) c R d ) n1 -NR c-C(=O)-;

[0097] When Z2 is oxygen, R4 does not exist;

[0098] When Z2 is nitrogen, R4 is selected from hydrogen and C. 1-6 Alkyl group; or R3 and R4 are linked to an adjacent nitrogen atom to form a ring, said ring optionally being connected by one or more atoms selected from hydrogen, hydroxyl, amino, hydroxyalkylene, aminoalkylene, C 1-6 Alkyl, C 1-6 Substituted by alkoxy, halogen, and carbonyl groups;

[0099] R c and R b Each is independently selected from hydrogen and C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, halogen, and cycloalkyl, or R c and R b It connects with adjacent carbon atoms to form a ring;

[0100] Each time n1 appears, it is independently 1, 2, 3, 4, or 5.

[0101] In some implementations, X2 is FCH2S-.

[0102] In some implementations, Z2 is nitrogen.

[0103] In some implementations, R4 is hydrogen.

[0104] In some implementations, R3 is selected from -O-(CR c R d ) n1 -C(=O)-、-O-(CR c R d ) n1 -OC(=O)- and -O-(CR) c R d ) n1 -NR c -C(=O)-.

[0105] In some implementation schemes, R c and R b Each is independently selected from hydrogen and C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, halogen and C 3-6 cycloalkyl, or R c and R b It connects with adjacent carbon atoms to form C 3-6 Cycloalkyl or 3-6 membered heterocyclic groups.

[0106] In some implementations, D is selected from the following structures:

[0107] In some implementations, D is selected from the following structures:

[0108] In some implementations, D is selected from the following structures:

[0109] In some embodiments, the positions where the linker and bioactive molecule can be substituted can be replaced by suitable substituents.

[0110] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0111] (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system:

[0112] Heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 with sequence of SEQ ID NO:6 or a variant thereof, CDR-H2 with sequence of SEQ ID NO:7 or a variant thereof, and CDR-H3 with sequence of SEQ ID NO:8 or a variant thereof; and / or, light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with sequence of SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence of SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence of SEQ ID NO:15 or a variant thereof;

[0113] or,

[0114] (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

[0115] Heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 with sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with sequence of SEQ ID NO:8 or a variant thereof; and / or, light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with sequence of SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence of SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence of SEQ ID NO:15 or a variant thereof;

[0116] or,

[0117] (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system:

[0118] Heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 with sequence of SEQ ID NO:9 or a variant thereof, CDR-H2 with sequence of SEQ ID NO:10 or a variant thereof, and CDR-H3 with sequence of SEQ ID NO:8 or a variant thereof; and / or, light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with sequence of SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence of SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence of SEQ ID NO:15 or a variant thereof;

[0119] or,

[0120] (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

[0121] Heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 with sequence of SEQ ID NO:3 or a variant thereof, CDR-H2 with sequence of SEQ ID NO:4 or a variant thereof, and CDR-H3 with sequence of SEQ ID NO:5 or a variant thereof; and / or, light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with sequence of SEQ ID NO:13 or a variant thereof, CDR-L2 with sequence of SEQ ID NO:14 or a variant thereof, and CDR-L3 with sequence of SEQ ID NO:15 or a variant thereof;

[0122] Wherein, the variant described in any one of (1) to (4) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution.

[0123] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0124] (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system:

[0125] Heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 with sequence SEQ ID NO:6, CDR-H2 with sequence SEQ ID NO:7, and CDR-H3 with sequence SEQ ID NO:8; and / or, light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with sequence SEQ ID NO:16, CDR-L2 with sequence SEQ ID NO:17, and CDR-L3 with sequence SEQ ID NO:15;

[0126] or,

[0127] (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

[0128] Heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 with sequence SEQ ID NO:11, CDR-H2 with sequence SEQ ID NO:12, and CDR-H3 with sequence SEQ ID NO:8; and / or, light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with sequence SEQ ID NO:16, CDR-L2 with sequence SEQ ID NO:17, and CDR-L3 with sequence SEQ ID NO:15;

[0129] or,

[0130] (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system:

[0131] Heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 with sequence SEQ ID NO:9, CDR-H2 with sequence SEQ ID NO:10, and CDR-H3 with sequence SEQ ID NO:8; and / or, light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with sequence SEQ ID NO:16, CDR-L2 with sequence SEQ ID NO:17, and CDR-L3 with sequence SEQ ID NO:15;

[0132] or,

[0133] (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

[0134] Heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 with sequence SEQ ID NO:3, CDR-H2 with sequence SEQ ID NO:4, and CDR-H3 with sequence SEQ ID NO:5; and / or, light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with sequence SEQ ID NO:13, CDR-L2 with sequence SEQ ID NO:14, and CDR-L3 with sequence SEQ ID NO:15;

[0135] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0136] VH or a variant thereof shown in SEQ ID NO: 1, and / or VL or a variant thereof shown in SEQ ID NO: 2;

[0137] The variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0138] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0139] VH as shown in SEQ ID NO: 1, and VL as shown in SEQ ID NO: 2.

[0140] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 18 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 18.

[0141] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain constant region (CL) as shown in SEQ ID NO: 19 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 19.

[0142] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 18 and a light chain constant region (CL) as shown in SEQ ID NO: 19.

[0143] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0144] The heavy chain including the VH of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 18, and the light chain including the VL of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 19.

[0145] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0146] Includes the heavy chain of the sequence shown in SEQ ID NO: 20, and the light chain of the sequence shown in SEQ ID NO: 21.

[0147] In some embodiments, the antibody or its antigen-binding fragment in the antibody-drug conjugate of the present invention comprises:

[0148] (a) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:29; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO:30; or

[0149] (b) The heavy chain variable region (VH) containing CDR-H1, CDR-H2 and CDR-H3, and / or the light chain variable region (VL) containing CDR-L1, CDR-L2 and CDR-L3, wherein, compared with the heavy chain variable region (VH) and / or the light chain variable region (VL) described in (a), at least one CDR contains a mutation, said mutation being a substitution, deletion or addition of one or more amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids).

[0150] In some implementations, the permutation is a conservative permutation.

[0151] In some implementations, the CDR is defined according to the IMGT, Kabat, Chothia, or AbM numbering system.

[0152] In some embodiments, the antibody or its antigen-binding fragment in the antibody-drug conjugate of the present invention comprises the following CDR:

[0153] The heavy chain variable region (VH) shown in SEQ ID NO:29 contains CDR-H1, CDR-H2 and CDR-H3; and / or the light chain variable region (VL) shown in SEQ ID NO:30 contains CDR-L1, CDR-L2 and CDR-L3.

[0154] In some embodiments, the antibody or its antigen-binding fragment in the antibody-drug conjugate of the present invention comprises:

[0155] (1) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence SEQ ID NO:31 or a variant thereof; CDR-H2 with the sequence SEQ ID NO:32 or a variant thereof; CDR-H3 with the sequence SEQ ID NO:33 or a variant thereof; and / or,

[0156] The light chain variable region (VL) contains the following three CDRs: CDR-L1 with sequence SEQ ID NO:34 or a variant thereof; CDR-L2 with sequence SEQ ID NO:35 or a variant thereof; and CDR-L3 with sequence SEQ ID NO:36 or a variant thereof.

[0157] (2) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence SEQ ID NO:37 or a variant thereof; CDR-H2 with the sequence SEQ ID NO:38 or a variant thereof; CDR-H3 with the sequence SEQ ID NO:33 or a variant thereof; and / or,

[0158] The light chain variable region (VL) contains the following three CDRs: CDR-L1 with sequence SEQ ID NO:34 or a variant thereof; CDR-L2 with sequence SEQ ID NO:35 or a variant thereof; and CDR-L3 with sequence SEQ ID NO:36 or a variant thereof.

[0159] (3) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:39 or a variant thereof; CDR-H2 with the sequence of SEQ ID NO:40 or a variant thereof; CDR-H3 with the sequence of SEQ ID NO:33 or a variant thereof; and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:34 or a variant thereof; CDR-L2 with the sequence of SEQ ID NO:35 or a variant thereof; CDR-L3 with the sequence of SEQ ID NO:36 or a variant thereof;

[0160] or,

[0161] (4) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence SEQ ID NO:41 or a variant thereof; CDR-H2 with the sequence SEQ ID NO:42 or a variant thereof; CDR-H3 with the sequence SEQ ID NO:43 or a variant thereof; and / or,

[0162] The light chain variable region (VL) contains the following three CDRs: CDR-L1 with sequence SEQ ID NO:44 or a variant thereof; CDR-L2 with sequence SEQ ID NO:45 or a variant thereof; and CDR-L3 with sequence SEQ ID NO:36 or a variant thereof.

[0163] The variant described in any one of (1)-(4) has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution.

[0164] In some embodiments, the antibody or its antigen-binding fragment in the antibody-drug conjugate of the present invention comprises:

[0165] (1) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO:31; CDR-H2 with sequence SEQ ID NO:32; CDR-H3 with sequence SEQ ID NO:33; and / or,

[0166] The light chain variable region (VL) contains the following three CDRs: CDR-L1 with sequence SEQ ID NO:34; CDR-L2 with sequence SEQ ID NO:35; and CDR-L3 with sequence SEQ ID NO:36.

[0167] (2) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO:37; CDR-H2 with sequence SEQ ID NO:38; CDR-H3 with sequence SEQ ID NO:33; and / or,

[0168] The light chain variable region (VL) contains the following three CDRs: CDR-L1 with sequence SEQ ID NO:34; CDR-L2 with sequence SEQ ID NO:35; and CDR-L3 with sequence SEQ ID NO:36.

[0169] (3) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO:39; CDR-H2 with sequence SEQ ID NO:40; CDR-H3 with sequence SEQ ID NO:33; and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO:34; CDR-L2 with sequence SEQ ID NO:35; CDR-L3 with sequence SEQ ID NO:36;

[0170] or,

[0171] (4) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO:41; CDR-H2 with sequence SEQ ID NO:42; CDR-H3 with sequence SEQ ID NO:43; and / or,

[0172] The light chain variable region (VL) contains the following three CDRs: CDR-L1 with sequence SEQ ID NO:44; CDR-L2 with sequence SEQ ID NO:45; and CDR-L3 with sequence SEQ ID NO:36.

[0173] In some embodiments, the antibody or its antigen-binding fragment in the antibody-drug conjugate of the present invention comprises:

[0174] (1) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO:31; CDR-H2 with sequence SEQ ID NO:32; CDR-H3 with sequence SEQ ID NO:33; and,

[0175] The light chain variable region (VL) contains the following three CDRs: CDR-L1 with sequence SEQ ID NO:34; CDR-L2 with sequence SEQ ID NO:35; and CDR-L3 with sequence SEQ ID NO:36.

[0176] (2) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO:37; CDR-H2 with sequence SEQ ID NO:38; CDR-H3 with sequence SEQ ID NO:33; and,

[0177] The light chain variable region (VL) contains the following three CDRs: CDR-L1 with sequence SEQ ID NO:34; CDR-L2 with sequence SEQ ID NO:35; and CDR-L3 with sequence SEQ ID NO:36.

[0178] (3) Heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 with sequence SEQ ID NO:39; CDR-H2 with sequence SEQ ID NO:40; CDR-H3 with sequence SEQ ID NO:33; and light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with sequence SEQ ID NO:34; CDR-L2 with sequence SEQ ID NO:35; CDR-L3 with sequence SEQ ID NO:36;

[0179] or,

[0180] (4) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO:41; CDR-H2 with sequence SEQ ID NO:42; CDR-H3 with sequence SEQ ID NO:43; and,

[0181] The light chain variable region (VL) contains the following three CDRs: CDR-L1 with sequence SEQ ID NO:44; CDR-L2 with sequence SEQ ID NO:45; and CDR-L3 with sequence SEQ ID NO:36.

[0182] In some embodiments, the antibody or its antigen-binding fragment in the antibody-drug conjugate of the present invention comprises:

[0183] (1) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO:31; CDR-H2 with sequence SEQ ID NO:32; CDR-H3 with sequence SEQ ID NO:33; and,

[0184] The light chain variable region (VL) contains the following three CDRs: CDR-L1 with sequence SEQ ID NO:34; CDR-L2 with sequence SEQ ID NO:35; and CDR-L3 with sequence SEQ ID NO:36.

[0185] The CDR is defined according to the Kabat numbering system;

[0186] (2) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO:37; CDR-H2 with sequence SEQ ID NO:38; CDR-H3 with sequence SEQ ID NO:33; and,

[0187] The light chain variable region (VL) contains the following three CDRs: CDR-L1 with sequence SEQ ID NO:34; CDR-L2 with sequence SEQ ID NO:35; and CDR-L3 with sequence SEQ ID NO:36.

[0188] The CDR is defined according to the AbM numbering system;

[0189] (3) Heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 with sequence SEQ ID NO:39; CDR-H2 with sequence SEQ ID NO:40; CDR-H3 with sequence SEQ ID NO:33; and light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with sequence SEQ ID NO:34; CDR-L2 with sequence SEQ ID NO:35; CDR-L3 with sequence SEQ ID NO:36;

[0190] The CDR is defined according to the Chothia numbering system;

[0191] or,

[0192] (4) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO:41; CDR-H2 with sequence SEQ ID NO:42; CDR-H3 with sequence SEQ ID NO:43; and,

[0193] The light chain variable region (VL) contains the following three CDRs: CDR-L1 with sequence SEQ ID NO:44; CDR-L2 with sequence SEQ ID NO:45 (DAS); and CDR-L3 with sequence SEQ ID NO:36.

[0194] The CDR is defined according to the IMGT numbering system.

[0195] In some embodiments, the antibody or its antigen-binding fragment in the antibody-drug conjugate of the present invention comprises:

[0196] VH containing a sequence as shown in SEQ ID NO:29 or a variant thereof and / or VL containing a sequence as shown in SEQ ID NO:30 or a variant thereof;

[0197] The variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0198] In some embodiments, the antibody or its antigen-binding fragment in the antibody-drug conjugate of the present invention comprises:

[0199] VH containing the sequence shown in SEQ ID NO:29 and / or VL containing the sequence shown in SEQ ID NO:30.

[0200] In some embodiments, the antibody or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to antigens and may include post-translational modifications thereof (e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate conversion to pyroglutamic acid or pyroglutamate salt in the heavy or light chain), which may occur during recombinant expression in host cells (e.g., CHO cells) or during purification / storage.

[0201] In some embodiments of the antibody or antigen-binding fragment disclosed herein, the heavy chain constant domain may contain a C-terminal lysine residue or lack a C-terminal lysine residue or a C-terminal glycine-lysine dipeptide. In some embodiments of the antibody or antigen-binding fragment thereof, the N-terminal amino acid of the antibody or antigen-binding fragment thereof may be cyclized to pyroglutamic acid. In some embodiments of the antibody or antigen-binding fragment thereof, the N-terminal amino acid of the antibody or antigen-binding fragment thereof may be cyclized to pyroglutamic acid salt.

[0202] As is known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0203] In some embodiments, compositions comprising antibody or antigen-binding fragments disclosed herein are provided, wherein the various antibody or antigen-binding fragments may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and / or comprise an N-terminal glutamine or glutamic acid, an N-terminal amino acid cyclized to pyroglutamic acid or an N-terminal amino acid cyclized to pyroglutamate salt.

[0204] In some embodiments, the N-terminal glutamate of the VH containing the sequence shown in SEQ ID NO:29 or a variant thereof undergoes cyclization to form pyroglutamic acid or a pyroglutamate salt.

[0205] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises a heavy chain constant region derived from or originating from human immunoglobulins (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises a wild-type Fc region, or comprises a mutated or chemically modified Fc region having altered effector functions (e.g., reduced ADCC activity) compared to the wild-type Fc region. In some embodiments, the antibody or its antigen-binding fragment comprises a variant of the human IgG1 heavy chain constant region as shown in SEQ ID NO:46. In some embodiments, the heavy chain constant region (CH) as shown in SEQ ID NO:46 or its variant lacks a C-terminal lysine.

[0206] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises a sequence as shown in SEQ ID NO:49 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, 10, or 5 conserved substitutions; e.g., 1, 2, 3, 4, or 5 conserved substitutions) compared to the antibody.

[0207] In some embodiments, the light chain of the antibody or its antigen-binding fragment comprises a light chain constant region derived from or originating from human immunoglobulins (e.g., κ or λ). In some embodiments, the light chain of the antibody or its antigen-binding fragment comprises a sequence as shown in SEQ ID NO:47 or a variant thereof, the variant having up to 20 conserved substitutions compared to it (e.g., up to 15, 10, or 5 conserved substitutions; e.g., 1, 2, 3, 4, or 5 conserved substitutions). In some embodiments, the light chain of the antibody or its antigen-binding fragment comprises a sequence as shown in SEQ ID NO:48 or a variant thereof, the variant having up to 20 conserved substitutions compared to it (e.g., up to 15, 10, or 5 conserved substitutions; e.g., 1, 2, 3, 4, or 5 conserved substitutions).

[0208] In some embodiments, the antibody or its antigen-binding fragment in the antibody-drug conjugate of the present invention comprises:

[0209] The heavy chain includes the VH shown in SEQ ID NO:29 and the heavy chain constant region (CH) shown in SEQ ID NO:46, and / or the light chain includes the VL shown in SEQ ID NO:30 and the light chain constant region (CL) shown in SEQ ID NO:47.

[0210] In some embodiments, the antibody or its antigen-binding fragment in the antibody-drug conjugate of the present invention comprises:

[0211] The heavy chain having the sequence shown in SEQ ID NO:49 and the light chain having the sequence shown in SEQ ID NO:50.

[0212] In some embodiments, the N-terminal glutamic acid of the heavy chain having the sequence shown in SEQ ID NO:49 undergoes cyclization to form pyroglutamic acid or pyroglutamate salt.

[0213] In some embodiments, the heavy chain having the sequence shown in SEQ ID NO:49 lacks a C-terminal lysine.

[0214] In some embodiments, the N-terminal glutamic acid of the heavy chain having the sequence shown in SEQ ID NO:49 undergoes cyclization to form pyroglutamic acid or pyroglutamate, and it lacks C-terminal lysine.

[0215] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises:

[0216] (a) A heavy chain having the sequence shown in SEQ ID NO:51 and / or a light chain having the sequence shown in SEQ ID NO:50;

[0217] (b) A heavy chain having the sequence shown in SEQ ID NO:52 and / or a light chain having the sequence shown in SEQ ID NO:50; or

[0218] (c) A heavy chain having the sequence shown in SEQ ID NO:53 and / or a light chain having the sequence shown in SEQ ID NO:50.

[0219] In some implementations, the formula HA-[MLED]x has the following structure:

[0220] ADC-A-1:

[0221] ADC-A-2:

[0222] ADC-A-3:

[0223] ADC-A-4:

[0224] ADC-A-5:

[0225] ADC-A-6:

[0226] ADC-B-1:

[0227] ADC-B-2:

[0228] ADC-B-3:

[0229] ADC-B-4:

[0230] ADC-C-1:

[0231] ADC-E-1:

[0232] ADC-E-2:

[0233] ADC-E-3:

[0234] ADC-E-4:

[0235] ADC-E-5:

[0236] ADC-E-6:

[0237] ADC-E-7:

[0238] ADC-E-8:

[0239] ADC-E-9:

[0240] ADC-E-10:

[0241] ADC-F-1:

[0242] ADC-F-2:

[0243] ADC-F-3:

[0244] ADC-F-4:

[0245] ADC-F-5:

[0246] ADC-F-6:

[0247] ADC-F-7:

[0248] ADC-F-8:

[0249] ADC-F-9:

[0250] ADC-F-10:

[0251] ADC-F-11:

[0252] ADC-F-12:

[0253] ADC-G-1:

[0254] ADC-G-2:

[0255] ADC-G-3:

[0256] ADC-G-4:

[0257] ADC-G-5:

[0258] ADC-G-6:

[0259] ADC-G-7:

[0260] ADC-G-8:

[0261] ADC-G-9:

[0262] ADC-G-10:

[0263] ADC-G-11:

[0264] ADC-G-12:

[0265] ADC-G-13:

[0266] ADC-G-14:

[0267] ADC-G-15:

[0268] ADC-G-17:

[0269] ADC-G-18:

[0270] ADC-G-19:

[0271] ADC-G-20:

[0272] ADC-G-21:

[0273] ADC-G-22:

[0274] ADC-H-1:

[0275] ADC-H-2:

[0276] ADC-H-3:

[0277] ADC-H-4:

[0278] ADC-H-5:

[0279] ADC-H-6:

[0280] ADC-N-1:

[0281] ADC-N-2:

[0282] ADC-N-3:

[0283] ADC-N-4:

[0284] ADC-N-5:

[0285] ADC-S-1:

[0286] ADC-S-2:

[0287] ADC-S-3:

[0288] ADC-S-4:

[0289] ADC-V-1:

[0290] ADC-V-2:

[0291] ADC-V-3:

[0292] ADC-V-4:

[0293] ADC-K-1:

[0294] ADC-K-2:

[0295] ADC-K-3:

[0296] ADC-K-4:

[0297] ADC-T-1:

[0298] ADC-T-1:

[0299] ADC-T-2:

[0300] ADC-T-3:

[0301] ADC-T-4:

[0302] ADC-T-5:

[0303] ADC-T-6:

[0304] ADC-T-7:

[0305] ADC-T-8:

[0306] ADC-T-9:

[0307] ADC-T-10:

[0308] ADC-T-11:

[0309] ADC-T-12:

[0310] ADC-T-13:

[0311] ADC-T-14:

[0312] ADC-T-15:

[0313] ADC-T-16:

[0314] ADC-T-17:

[0315] ADC-T-18:

[0316] ADC-T-19:

[0317] ADC-T-20:

[0318] ADC-T-21:

[0319] ADC-T-22:

[0320] ADC-T-23:

[0321] ADC-T-24:

[0322] ADC-T-25:

[0323] ADC-T-26:

[0324] ADC-T-27:

[0325] ADC-T-28:

[0326] ADC-T-29:

[0327] Where x is selected from integers from 1 to 10;

[0328] Among them, -S) x -Ab is any of the antibodies or antigen-binding fragments described above; This indicates the specific linkage between the thiol group and the pyrimidin group or methylene group in the antibody or its antigen-binding fragment.

[0329] In some embodiments, the antibody-drug conjugate (ADC) has an x ​​of 1 to 10, such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 7 to 8, 7 to 9, 7 to 10, 8 to 9, 8 to 10, or 9 to 10.

[0330] Composition

[0331] On the other hand, this application provides compositions of antibody-drug conjugates (ADCs) as described herein. Such compositions may comprise a plurality of ADCs as described herein, wherein each ADC contains a drug-linker as described herein, wherein x independently is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In other words, each antibody molecule in the composition may be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drug-linkers. Therefore, the compositions are characterized by a drug-antibody ratio (DAR) in the range of about 1 to about 10. Methods for determining DAR are well known to those skilled in the art, including methods using reversed-phase chromatography or HPLC-MS.

[0332] For example, in any embodiment, the ADC composition described herein has a DAR of about 1 to about 10 or any subrange therebetween, such as: about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, about 1 to 10, about 2 to 3, about 2 to 4, about 2 to 5, about 2 to 6, about 2 to 7, about 2 to 8, about 2 To 9, about 2 to 10, about 3 to 4, about 3 to 5, about 3 to 6, about 3 to 7, about 3 to 8, about 3 to 9, about 3 to 10, about 4 to 5, about 4 to 6, about 4 to 7, about 4 to 8, about 4 to 9, about 4 to 10, about 5 to 6, about 5 to 7, about 5 to 8, about 5 to 9, about 5 to 10, about 6 to 7, about 6 to 8, about 6 to 9, about 6 to 10, about 7 to 8, about 7 to 9, about 7 to 10, about 8 to 9, about 8 to 10 or about 9 to 10.

[0333] In some embodiments, the DAR of the ADC compositions described herein is about 3 to 9, for example, about 3.0 to 3.5, about 3.0 to 4.0, about 3.0 to 4.5, about 3.0 to 5.0, about 3.0 to 5.5, about 3.0 to 6.0, about 3.5 to 4.0, about 3.5 to 4.5, about 3.5 to 5.0, about 3.5 to 5.5, about 3.5 to 6.0, about 3.5 to 6.5, about 4.0 to 4.5, about 4.0 to 5.0, about 4.0 to 5.5, about 4.0 to 6.0, about 4.0 to 6.5, about 4.0 to 7.0, about 4 0 to 8.0, approximately 4.5 to 5.0, approximately 4.5 to 5.5, approximately 4.5 to 6.0, approximately 4.5 to 6.5, approximately 4.5 to 7.0, approximately 4.5 to 7.5, approximately 5.0 to 8.0, approximately 5.5 to 6.0, approximately 5.5 to 6.5, approximately 5.5 to 7.0, approximately 5.5 to 7.5, approximately 5.5 to 8.0, approximately 6.0 to 6.5, approximately 6.0 to 7.0, approximately 6.0 to 7.5, approximately 6.0 to 8.5, approximately 6.5 to 7.0, approximately 6.5 to 7.5, approximately 6.5 to 7.5, approximately 6.5 to 8.5 or approximately 7.0 to 7.5.

[0334] In some embodiments, the DAR of the ADC composition described herein is about 3 to 8, for example, about 3.0 to 3.5, about 3.0 to 4.0, about 3.0 to 4.5, about 3.0 to 5.0, about 6.0 to 6.5, about 6.0 to 7.0, about 6.0 to 7.5, about 6.0 to 8.0, about 6.0 to 8.5, about 6.5 to 7.0, about 6.5 to 7.5, about 6.5 to 8.0, about 6.5 to 8.5, about 7.0 to 7.5, about 7.0 to 8.0, about 7.5 to 8.0.

[0335] In some embodiments, the composition is composed of, for example, Ab-[MLED]. x The antibody-drug conjugate composition is shown. In some embodiments, the DAR of the composition is 1-10, for example 3-8, such as about 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5. In some embodiments, the antibody-drug conjugate with x=8 constitutes 60%, 70%, 80%, or 90% or more. In some embodiments, the antibody-drug conjugate with x=4 constitutes 60%, 70%, 80%, or 90% or more.

[0336] In some embodiments, the composition contains a plurality of ADC-A-1:

[0337] Where x is selected from integers from 1 to 8;

[0338] Ab contains:

[0339] (a) A heavy chain having the sequence shown in SEQ ID NO:20 and / or a light chain having the sequence shown in SEQ ID NO:21;

[0340] (b) A heavy chain having the sequence shown in SEQ ID NO:51 and / or a light chain having the sequence shown in SEQ ID NO:50;

[0341] (c) A heavy chain having the sequence shown in SEQ ID NO:52 and / or a light chain having the sequence shown in SEQ ID NO:50; or

[0342] (d) A heavy chain having the sequence shown in SEQ ID NO:53 and / or a light chain having the sequence shown in SEQ ID NO:50;

[0343] -S) x -Ab is any of the antibodies or antigen-binding fragments described above; This indicates the specific linkage between the thiol group and the pyrimidin group or methylene group in the antibody or its antigen-binding fragment. In some embodiments, the DAR value of the composition is selected from 1-8, for example 2-8, 3-8, 3-7, 3-6, 3-5; and for example 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0.

[0344] In some embodiments, the composition contains a plurality of ADC-A-3:

[0345] Where x is selected from integers from 1 to 8;

[0346] Ab contains:

[0347] (a) A heavy chain having the sequence shown in SEQ ID NO:20 and / or a light chain having the sequence shown in SEQ ID NO:21;

[0348] (b) A heavy chain having the sequence shown in SEQ ID NO:51 and / or a light chain having the sequence shown in SEQ ID NO:50;

[0349] (c) A heavy chain having the sequence shown in SEQ ID NO:52 and / or a light chain having the sequence shown in SEQ ID NO:50; or

[0350] (d) A heavy chain having the sequence shown in SEQ ID NO:53 and / or a light chain having the sequence shown in SEQ ID NO:50;

[0351] -S) x -Ab is any of the antibodies or antigen-binding fragments described above; This indicates the specific linkage between the thiol group and the pyrimidin group or methylene group in the antibody or its antigen-binding fragment. In some embodiments, the DAR value of the composition is selected from 1-8, for example 2-8, 3-8, 3-7, 3-6, 3-5; and for example 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0.

[0352] In some embodiments, the composition contains a plurality of ADC-E-5:

[0353] Where x is selected from integers from 1 to 8;

[0354] Ab contains:

[0355] (a) A heavy chain having the sequence shown in SEQ ID NO:20 and / or a light chain having the sequence shown in SEQ ID NO:21;

[0356] (b) A heavy chain having the sequence shown in SEQ ID NO:51 and / or a light chain having the sequence shown in SEQ ID NO:50;

[0357] (c) A heavy chain having the sequence shown in SEQ ID NO:52 and / or a light chain having the sequence shown in SEQ ID NO:50; or

[0358] (d) A heavy chain having the sequence shown in SEQ ID NO:53 and / or a light chain having the sequence shown in SEQ ID NO:50;

[0359] -S) x -Ab is any of the antibodies or antigen-binding fragments described above; This indicates the specific linkage between the thiol group and the pyrimidin group or methylene group in the antibody or its antigen-binding fragment. In some embodiments, the DAR value of the composition is selected from 1-8, for example 2-8, 3-8, 3-7, 3-6, 3-5; and for example 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0.

[0360] In some embodiments, the composition contains a plurality of ADC-N-4:

[0361] Where x is selected from integers from 1 to 8;

[0362] Ab contains:

[0363] (a) A heavy chain having the sequence shown in SEQ ID NO:20 and / or a light chain having the sequence shown in SEQ ID NO:21;

[0364] (b) A heavy chain having the sequence shown in SEQ ID NO:51 and / or a light chain having the sequence shown in SEQ ID NO:50;

[0365] (c) A heavy chain having the sequence shown in SEQ ID NO:52 and / or a light chain having the sequence shown in SEQ ID NO:50; or

[0366] (d) A heavy chain having the sequence shown in SEQ ID NO:53 and / or a light chain having the sequence shown in SEQ ID NO:50;

[0367] -S) x -Ab is any of the antibodies or antigen-binding fragments described above; This indicates the specific linkage between the thiol group and the pyrimidin group or methylene group in the antibody or its antigen-binding fragment. In some embodiments, the DAR value of the composition is selected from 1-8, for example 2-8, 3-8, 3-7, 3-6, 3-5; and for example 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0.

[0368] In some embodiments, the composition contains a plurality of ADC-S-3:

[0369] Where x is selected from integers from 1 to 8;

[0370] Ab contains:

[0371] (a) A heavy chain having the sequence shown in SEQ ID NO:20 and / or a light chain having the sequence shown in SEQ ID NO:21;

[0372] (b) A heavy chain having the sequence shown in SEQ ID NO:51 and / or a light chain having the sequence shown in SEQ ID NO:50;

[0373] (c) A heavy chain having the sequence shown in SEQ ID NO:52 and / or a light chain having the sequence shown in SEQ ID NO:50; or

[0374] (d) A heavy chain having the sequence shown in SEQ ID NO:53 and / or a light chain having the sequence shown in SEQ ID NO:50;

[0375] -S) x -Ab is any of the antibodies or antigen-binding fragments described above; This indicates the specific linkage between the thiol group and the pyrimidin group or methylene group in the antibody or its antigen-binding fragment. In some embodiments, the DAR value of the composition is selected from 1-8, for example 2-8, 3-8, 3-7, 3-6, 3-5; and for example 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0.

[0376] In some embodiments, the composition contains a plurality of ADC-T-1:

[0377] Where x is selected from integers from 1 to 8;

[0378] Ab contains:

[0379] (a) A heavy chain having the sequence shown in SEQ ID NO:20 and / or a light chain having the sequence shown in SEQ ID NO:21;

[0380] (b) A heavy chain having the sequence shown in SEQ ID NO:51 and / or a light chain having the sequence shown in SEQ ID NO:50;

[0381] (c) A heavy chain having the sequence shown in SEQ ID NO:52 and / or a light chain having the sequence shown in SEQ ID NO:50; or

[0382] (d) A heavy chain having the sequence shown in SEQ ID NO:53 and / or a light chain having the sequence shown in SEQ ID NO:50;

[0383] -S) x -Ab is any of the antibodies or antigen-binding fragments described above; This indicates the specific linkage between the thiol group and the pyrimidin group or methylene group in the antibody or its antigen-binding fragment. In some embodiments, the DAR value of the composition is selected from 1-8, for example 2-8, 3-8, 3-7, 3-6, 3-5; and for example 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0.

[0384] In some embodiments, the composition contains a plurality of ADC-V-3:

[0385] Where x is selected from integers from 1 to 8;

[0386] Ab contains:

[0387] (a) A heavy chain having the sequence shown in SEQ ID NO:20 and / or a light chain having the sequence shown in SEQ ID NO:21;

[0388] (b) A heavy chain having the sequence shown in SEQ ID NO:51 and / or a light chain having the sequence shown in SEQ ID NO:50;

[0389] (c) A heavy chain having the sequence shown in SEQ ID NO:52 and / or a light chain having the sequence shown in SEQ ID NO:50; or

[0390] (d) A heavy chain having the sequence shown in SEQ ID NO:53 and / or a light chain having the sequence shown in SEQ ID NO:50; -S) x -Ab is any of the antibodies or antigen-binding fragments described above; This indicates the specific linkage between the thiol group and the pyrimidin or methylene group in the antibody or its antigen-binding fragment. In some embodiments, the DAR value of the composition is selected from 1-8, for example 2-8, 3-8, 3-7, 3-6, 3-5; and further examples 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0. Drug-linker

[0391] On the other hand, this application provides a drug-linker and a pharmaceutically acceptable salt thereof, having the formula M a -The structure shown by the LED, wherein:

[0392] M a It is the structure before it is linked to an antibody or its antigen-binding fragment;

[0393] L, E, and D are defined as any one of the above terms.

[0394] In some implementation schemes, M a for The C structure is a single bond, sulfonamide group, phosphoramidyl group, 4-6 membered aliphatic heterocycle or 5-20 membered aromatic ring system, wherein the aliphatic heterocycle or aromatic ring system is optionally selected by one or more groups, each independently selected from oxo (=O), halogen, cyano, nitro, amino, carboxyl, mercapto and C. 1-6 Alkyl group substitution; and M1 is selected from a single bond or a fragment consisting of one or more groups selected from the following: -O-, -NH-, -NH2, -C(=O)-, HO2C(=O)-, -S(=O)2-, HOS(=O)2-, -C=NO-, -NH-S(=O)2-NH-, 3-6 membered heterocyclic group, phenylene, 5-10 membered heteroaryl group, C 1-20 Alkylene, C 2-20 imide and C 2-20 Alynyl group.

[0395] In some implementation schemes, M a for Wherein, C is a single bond, sulfonamide group, phosphoramidyl group, 4-6 membered aliphatic heterocycle, 5-6 membered heteroaromatic ring, or a polycyclic ring formed by two or three units selected from 6 membered aromatic heterocycles and benzene rings linked by single bonds, wherein the aliphatic heterocycle, heteroaromatic ring, or polycyclic ring is optionally substituted by one or more groups selected from oxo (=O), halogen, cyano, and nitro; and M1 is selected from a single bond or a fragment composed of one or more groups selected from the following groups: -NH-, -C(=O)-, -NH-S(=O)2-NH-, C 1-10 Alkylene, C 2-10 imide and C 2- 10 Alynyl group.

[0396] In some implementation schemes, M a for Where Q represents hydrogen, halogen, halomethylene, haloacetyl, alkenyl, alkynyl, cyano, isothiocyanate, or C. 1-6 Alkyl sulfonyl, fluorosulfonyl, fluorosulfonate, fluorophenol ester; C-structure is a single bond, halogen, sulfonamide, phosphoramidyl, 4-6 membered aliphatic heterocycle or 5-20 membered aromatic ring system, wherein the aliphatic heterocycle or aromatic ring system is optionally selected by one or more groups, each independently selected from oxo (=O), halogen, cyano, nitro, amino, carboxyl, mercapto, and C. 1-6Alkyl group substitution; and M1 is selected from a single bond or a fragment consisting of one or more groups selected from the following: -O-, -NH-, -NH2, -C(=O)-, HO2C(=O)-, -S(=O)2-, HOS(=O)2-, -C=NO-, -NH-S(=O)2-NH-, 3-6 membered heterocyclic group, phenylene, 5-10 membered heteroaryl group, C 1-20 Alkylene, C 2-20 imide and C 2-20 Alynyl group.

[0397] In some implementation schemes, M a for Where Q represents hydrogen, halogen, halomethylene, haloacetyl, alkenyl, alkynyl, cyano, isothiocyanate, or C. 1-6 Alkyl sulfonyl, fluorosulfonyl, fluorosulfonate, fluorophenol ester; C structure is a single bond, sulfonamide, phosphoramidyl, 4-6 membered aliphatic heterocycle, 5-6 membered heteroaromatic ring, or a polycyclic ring formed by 2-5 (preferably 3) units selected from 6 membered aromatic heterocycles and benzene rings linked by single bonds, wherein the aliphatic heterocycle, heteroaromatic ring, or polycyclic ring is optionally substituted by one or more groups selected from oxo (=O), halogen, cyano, and nitro; and M1 is selected from a single bond or a segment composed of one or more groups selected from the following groups: -NH-, -C(=O)-, -NH-S(=O)2-NH-, C 1-10 Alkylene, C 2-10 imide and C 2-10 Alynyl group.

[0398] In some implementation schemes, M a for The C structure is selected from single bonds, M1 is selected from single bonds, carbonyl groups, sulfonyl groups, -NH-, -NH-S(=O)2-NH-, C 1-6 Alkylene, C 2-6 imide and C 2-6 Alynyl group.

[0399] In some implementation schemes, M a for The C structure is selected from single bonds, M1 is selected from single bonds, carbonyl groups, sulfonyl groups, -NH-, -NH-S(=O)2-NH-, C 1-6 Alkylene, C 2-6 imide and C 2-6 Alynyl group.

[0400] In some implementation schemes, M a Selected from

[0401] In some implementation schemes, Ma for

[0402] In some implementation schemes, M a for

[0403] In some implementation schemes, M a for

[0404] In some implementation schemes, M a for

[0405] In some implementation schemes, M a for

[0406] In some implementation schemes, M a -LEDs have the following structure:

[0407] A-1:

[0408] A-2:

[0409] A-3:

[0410] A-4:

[0411] A-5:

[0412] A-6:

[0413] B-1:

[0414] B-2:

[0415] B-3:

[0416] B-4:

[0417] C-1:

[0418] E-1:

[0419] E-2:

[0420] E-3:

[0421] E-4:

[0422] E-5:

[0423] E-6:

[0424] E-7:

[0425] E-8:

[0426] E-9:

[0427] E-10:

[0428] F-1:

[0429] F-2:

[0430] F-3:

[0431] F-4:

[0432] F-5:

[0433] F-6:

[0434] F-7:

[0435] F-8:

[0436] F-9:

[0437] F-10:

[0438] F-11:

[0439] F-12:

[0440] G-1:

[0441] G-2:

[0442] G-3:

[0443] G-4:

[0444] G-5:

[0445] G-6:

[0446] G-7:

[0447] G-8:

[0448] G-9:

[0449] G-10:

[0450] G-11:

[0451] G-12:

[0452] G-13:

[0453] G-14:

[0454] G-15:

[0455] G-17:

[0456] G-18:

[0457] G-19:

[0458] G-20:

[0459] G-21:

[0460] G-22:

[0461] H-1:

[0462] H-2:

[0463] H-3:

[0464] H-4:

[0465] H-5:

[0466] H-6:

[0467] K-1:

[0468] K-2:

[0469] K-3:

[0470] K-4:

[0471] N-1:

[0472] N-2:

[0473] N-3:

[0474] N-4:

[0475] N-5:

[0476] S-1:

[0477] S-2:

[0478] S-3:

[0479] S-4:

[0480] V-1:

[0481] V-2:

[0482] V-3:

[0483] V-4:

[0484] T-1:

[0485] T-2:

[0486] T-3:

[0487] T-4:

[0488] T-5:

[0489] T-6:

[0490] T-7:

[0491] T-8:

[0492] T-9:

[0493] T-10:

[0494] T-11:

[0495] T-12:

[0496] T-13:

[0497] T-14:

[0498] T-15:

[0499] T-16:

[0500] T-17:

[0501] T-18:

[0502] T-19:

[0503] T-20:

[0504] T-21:

[0505] T-22:

[0506] T-23:

[0507] T-24:

[0508] T-25:

[0509] T-26:

[0510] T-27:

[0511] T-28:

[0512] T-29:

[0513] Connector

[0514] In some implementations, this application provides a connector with the following structure: -MLE-;

[0515] M, L, and E are each described independently as described in any of the above items.

[0516] In some embodiments, the M portion of the linker is coupled to the antibody or its antigen-binding fragment as described in any of the preceding claims; the E portion of the linker is coupled to the D portion as described in any of the preceding claims, forming the antibody-drug conjugate.

[0517] In some implementations, this application provides a connector with the following structure: M a -LE-;

[0518] Where M a L and E are each described independently as described in any of the above items.

[0519] In some embodiments, the E portion of the linker is connected to the D portion described in any of the preceding embodiments to form the drug-linker compound.

[0520] Synthetic intermediates

[0521] In some embodiments, this application provides synthetic intermediates with the following structures and pharmaceutically acceptable salts thereof:

[0522] Q1, Q2, Q3, Y1, Z1, and R1 are as described in any of the above descriptions; PG1 is an amino protecting group.

[0523] In some embodiments, PG1 is independently selected from Boc (tert-butyloxycarbonyl), Fmoc (9-fluorenyloxycarbonyl), benzyloxycarbonyl (Cbz), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), phthaloyl (Pht), allyloxycarbonyl (Alloc), triethoxycarbonyl (Teoc), 2,4-dimethoxybenzyl (Dmb), p-methoxybenzyl (PMB), and benzyl (Bn); preferably Boc (tert-butyloxycarbonyl) or Fmoc (9-fluorenyloxycarbonyl).

[0524] In some implementations, this application provides synthetic intermediates with the following structures:

[0525] Pharmaceutical Composition

[0526] In another aspect, this application provides a pharmaceutical composition comprising the glucocorticoid receptor agonist of the present invention, the antibody-drug conjugate of the present invention, or the drug-linker compound of the present invention, and one or more pharmaceutically acceptable carriers.

[0527] The choice of a pharmaceutically acceptable carrier depends on the dosage form of the pharmaceutical composition, primarily on the route of administration and secondarily on the formulation. For example, a pharmaceutically acceptable carrier may include water (e.g., water for injection), buffer solutions, isotonic saline solutions such as PBS (phosphate-buffered saline), glucose, mannitol, dextran glucose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ascorbic acid, lactic acid, ethanol, polyalkylene glycols such as polyethylene glycol (e.g., polyethylene glycol 4000) or polypropylene glycol, triglycerides, etc.

[0528] The glucocorticoid receptor agonists, antibody-drug conjugates, or drug-linked compounds described herein are typically formulated in a single injectable form with a pharmaceutically acceptable parenteral medium for parenteral use, such as subcutaneous injection, intramuscular injection, intravenous bolus, intravenous infusion, local injection into diseased tissue, or intratumoral injection. Optionally, glucocorticoid receptor agonists, antibody-drug conjugates, or drug-linked compounds of desired purity are mixed with a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer in the form of a lyophilized or solution form (Remington's Pharmaceutical Sciences (1980) 16). th (ed., Osol, A.Ed.). The glucocorticoid receptor agonists, antibody-drug conjugates, drug-linked compounds, or pharmaceutical compositions described herein can be administered via any route appropriate for the individual to be treated.

[0529] Treatment methods and uses

[0530] In another aspect, this application provides the use of the steroid glucocorticoid receptor agonists of the present invention, the antibody-drug conjugates of the present invention, the drug-linker compounds of the present invention, or the pharmaceutical compositions of the present invention in the preparation of medicaments for treating inflammatory or immune diseases.

[0531] In another aspect, this application provides the steroid glucocorticoid receptor agonist of the present invention, the antibody-drug conjugate of the present invention, the drug-linker compound of the present invention, or the pharmaceutical composition of the present invention for the treatment of inflammatory or immune diseases.

[0532] On the other hand, this application provides a method for treating inflammatory or immune diseases, comprising administering to an individual in need a therapeutically effective amount of the steroid glucocorticoid receptor agonist of the present invention, the antibody-drug conjugate of the present invention, the drug-linker compound of the present invention, or the pharmaceutical composition of the present invention.

[0533] In some implementations, the immune diseases described herein are autoimmune diseases.

[0534] In some embodiments, the inflammatory or immune diseases described herein are diseases with high expression of TNFα or IL-6R. In some embodiments, the inflammatory or immune diseases are selected from rheumatoid arthritis, idiopathic arthritis, asthma, ulcerative colitis, neuromyelitis optica, autoimmune liver disease, systemic lupus erythematosus, psoriasis, systemic sclerosis, and atopic dermatitis. In some embodiments, the inflammatory or immune diseases are selected from rheumatoid arthritis, idiopathic arthritis, asthma, ulcerative colitis, neuromyelitis optica, and autoimmune liver disease.

[0535] definition

[0536] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art. References to technical terms herein refer to techniques commonly understood in the art, including variations or equivalent substitutions of techniques obvious to those skilled in the art. Furthermore, laboratory procedures used herein, such as those related to genomics, nucleic acid chemistry, and molecular biology, are standard procedures widely used in their respective fields. While it is believed that the following terms will be readily understood by those skilled in the art, the following definitions are set forth to better explain the invention.

[0537] The term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form antigen-binding sites. The amino acid distribution in each region or domain can follow various numbering systems known in the art.

[0538] The term "complementarity-determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs, named CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003), or the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268-9272). For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0539] In this invention, the CDR contained in the antibody or its antigen-binding fragment can be determined according to various numbering systems known in the art, such as the Kabat, Chothia, IMGT, or AbM numbering systems. In some embodiments, the CDR contained in the antibody or its antigen-binding fragment is defined using the Chothia numbering system.

[0540] The term "framework region" or "FR" residues refers to the amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0541] The term "antigen-binding fragment" in antibody refers to a fragment of the antibody polypeptide, such as a fragment of the full-length antibody polypeptide, which retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the "antigen-binding moiety". See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fd, Fv, scFv, di-scFv, (scFv)2, disulfide-stabilized Fv proteins (“dsFv”), single-domain antibodies (sdAb, nanobodies), and peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0542] The term "Fd" refers to an antibody fragment composed of VH and CH1 domains; the term "dAb fragment" refers to an antibody fragment composed of VH domain (Ward et al., Nature 341:544 546 (1989)); the term "Fab fragment" refers to an antibody fragment composed of VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments connected by disulfide bridges on the hinge region; the term "Fab' fragment" refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, which consists of a complete light chain and heavy chain Fd fragment (composed of VH and CH1 domains).

[0543] The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0544] The term "Fc" refers to an antibody fragment formed by disulfide bonds connecting the second and third constant regions of the first heavy chain to the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0545] The term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS (SEQ ID NO:27) amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO:28) can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between VH and VL of scFv. In some implementations, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, domains containing NH2-VH-VH-COOH, NH 2- VL-VL-COOH of scFv.

[0546] The term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art as an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that maintains the ability to specifically bind to the same antigen bound by a full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also known as nanobodies.

[0547] Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0548] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0549] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0550] The term "mouse antibody" refers to antibodies obtained by fusing B cells from immunized mice with myeloma cells, screening for mouse hybrid fusion cells that can proliferate indefinitely and secrete antibodies, and then screening, preparing and purifying the antibodies; or it refers to antibodies secreted by plasma cells formed by the differentiation and proliferation of B cells after the antigen enters the mouse body.

[0551] The term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase its homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and ability to enhance the immune response. Donor antibodies can be mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibodies with the intended properties (e.g., antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and / or ability to enhance the immune response).

[0552] The term "identity" is used to refer to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are considered identical at that position when a position is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods conveniently performed, for example, by computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0553] The term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0554] The twenty common amino acids mentioned herein are written in accordance with conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ESGolob and DRGren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine may be represented by A or Ala. In this application, unless otherwise expressly stated, all amino acids mentioned are naturally occurring L-configured amino acids (except glycine).

[0555] The terms “including,” “comprising,” “having,” “containing,” or “involving,” and their other variations herein, are inclusive or open-ended and do not exclude other unlisted elements or method steps.

[0556] The term "alkyl" refers to a group obtained by removing one hydrogen atom from a straight-chain or branched alkane, such as "C". 1-20 Alkyl", C 1-10 Alkyl", C 1-6 Alkyl", C 1-4 Alkyl", C 1-3 Alkyl groups, etc., specific examples include but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc.

[0557] The term "alkenyl" refers to a straight-chain or branched aliphatic hydrocarbon group having one or more unsaturated double bonds, such as "C". 2-20 "alkenyl", "C" 2- 10 "alkenyl", "C" 2-6 "alkenyl", "C" 2-4 "Alkenyl" and other specific examples include, but are not limited to: vinyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl, 2-buten-1-yl, 3-buten-1-yl, 2-penten-1-yl, 3-penten-1-yl, 4-penten-1-yl, 5-hexen-1-yl, 4-hexen-1-yl, 3-hexen-1-yl, 2-hexen-1-yl, 3-methyl-2-buten-1-yl, 3-methyl-3-penten-1-yl, 3-methyl-2-penten-1-yl, 4-methyl-3-penten-1-yl, 4-methyl-2-penten-1-yl, and 2-methyl-2-penten-1-yl, etc.

[0558] The term "alkynyl" refers to a straight-chain or branched aliphatic hydrocarbon group having one or more unsaturated triple bonds, such as "C". 2-20 "Alkyne", "C" 2- 10 "Alkyne", "C" 2-6 "Alkyne", "C" 2-4 "Alynyl", etc., specific examples include but are not limited to: ethynyl, 1-propyn-1-yl, 2-propyn-1-yl, 2-butyn-1-yl, 3-butyn-1-yl, 2-pentyn-1-yl, 3-pentyn-1-yl, 4-pentyn-1-yl, 5-hexyn-1-yl, 4-hexyn-1-yl, 3-hexyn-1-yl and 2-hexyn-1-yl, etc.

[0559] The term "alkylene" refers to a group obtained by removing two hydrogen atoms from a straight-chain or branched alkane, such as "C".1-20 Alkylene, C 1-10 Alkylene, C 3-10 Alkylene, C 5-8 Alkylene, C 1-6 Alkylene, C 1-4 Alkylene, C 1-3 "alkylene", etc., specific examples include but are not limited to: methylene, ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene or 1,6-hexylene, etc.

[0560] The term "alkenyl" refers to a divalent group formed by the loss of two hydrogen atoms from a straight-chain or branched alkene containing at least one carbon-carbon double bond, including, for example, "C". 2-20 "Ideinyl", "C" 3-10 "Ideinyl", "C" 5-8 Examples of these include, but are not limited to: vinylidene, 1-propenyne, 2-propenyne, 1-butenyne, 2-butenyne, 1,3-butadiene, 1-pentenyne, 2-pentenyne, 3-pentenyne, 1,3-pentadiene, 1,4-pentadiene, 1-hexenyne, 2-hexenyne, 3-hexenyne, 1,4-hexadiene, etc.

[0561] The term "acetylenic" refers to a divalent group formed by the loss of two hydrogen atoms from a straight-chain or branched alkyne containing at least one carbon-carbon triple bond. This includes, for example, "C..." 2-20 "Immyne", "C" 3-10 "Immyne", "C" 5-8 Examples of "ethynyl" include, but are not limited to: ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1,3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1,3-pentynyl, 1,4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,4-hexynyl, etc.

[0562] The term "cycloalkylene" refers to the group obtained by removing two hydrogen atoms from a cycloalkane, such as "C". 3-20 "Cycloalkylene", "C" 3-10 "Cycloalkylene", "C" 3-6 "Cycloalkylene", etc., specific examples include but are not limited to: cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, etc.

[0563] The term "alkoxy" refers to an alkyl group as defined above, which is attached to a part of the parent molecule by an oxygen atom. Examples include C1-C6 alkoxy and C1-C3 alkoxy, and specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, pentoxy, isopentoxy, hexoxy, etc.

[0564] The term "halogenated" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0565] The term "haloalkyl" refers to an alkyl group substituted with one or more (such as 1 to 3) identical or different halogen atoms. For example, the term "C1-C6 haloalkyl" as used herein refers to a haloalkyl group having 1 to 6 carbon atoms, including but not limited to -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, -CH2CH2CF3, -CH2Cl, etc.

[0566] The term "aromatic ring" refers to all-carbon monocyclic or fused-ring polycyclic aromatic hydrocarbons with a conjugated π-electron system. Common aromatic rings include, but are not limited to, benzene, naphthalene, anthracene, phenanthrene, acenaphthene, azulene, fluorene, indene, and pyrene. For example, the term "C6-C..." 10 An aromatic ring is a ring containing 6 to 10 carbon atoms, such as benzene or naphthalene.

[0567] The term "aliphatic heterocycle" refers to a saturated or partially saturated cyclic structure containing at least one ring member selected from N, O and S, including but not limited to 5-10 member aliphatic heterocycles, 5-6 member aliphatic heterocycles, such as 5-6 member nitrogen-containing aliphatic heterocycles, 5-6 member oxygen-containing aliphatic heterocycles, etc. Specific examples include but are not limited to: tetrahydrofuran, pyrrolidine, piperidine, tetrahydropyran, etc.

[0568] The term "heteroaromatic ring" refers to an aromatic ring structure containing at least one ring member selected from N, O, and S, including but not limited to 5-10 membered aromatic heterocycles, 5-6 membered aromatic heterocycles, such as 5-6 membered nitrogen-containing aromatic heterocycles, 5-6 membered oxygen-containing aromatic heterocycles, etc. Specific examples include but are not limited to: furan, thiophene, pyrrole, thiazole, isothiazole, thiadiazole, oxazole, isoxazole, oxadiazole, imidazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, pyridine, pyrimidine, pyridazine, pyrazine, 1,2,3-triazine, 1,3,5-triazine, 1,2,4,5-tetraazine, etc.

[0569] The term "aromatic ring system" refers to a monocyclic or polycyclic system containing at least one aromatic ring (e.g., a benzene ring) or a heteroaromatic ring (e.g., a pyrimidine ring). Two or more aromatic rings and / or heteroaromatic rings may form a fused ring or be linked by a single bond (e.g., a diamyrimidinylphenyl ring). The aromatic ring system may be divalent or higher valence (e.g., trivalent or tetravalent), such as 5-20 member aromatic ring systems.

[0570] If a substituent is described as “optionally substituted with…”, then the substituent may be (1) unsubstituted or (2) substituted. If the carbon of the substituent is described as being optionally substituted with one or more of the substituents in the list, then one or more hydrogens on the carbon (to the extent that any hydrogens are present) may be substituted individually and / or together with independently selected substituents or not substituted. If the nitrogen of the substituent is described as being optionally substituted with one or more of the substituents in the list, then one or more hydrogens on the nitrogen (to the extent that any hydrogens are present) may each be substituted with independently selected substituents or not substituted.

[0571] If a substituent is described as being "independently selected" from a group of groups, then each substituent is selected independently of the others. Therefore, each substituent may be the same as or different from another (other) substituent.

[0572] Unless otherwise specified, as used herein, the connection point of a substituent may be derived from any suitable location of the substituent.

[0573] When the bond of a substituent is such that it passes through the ring and connects two atoms, then such a substituent can be bonded to any cyclic atom in the substituted ring.

[0574] Pharmaceutically acceptable salts of the compounds of the present invention include their acid addition salts and base addition salts. Suitable acid addition salts are formed by acids that form pharmaceutically acceptable salts. Suitable base addition salts are formed by bases that form pharmaceutically acceptable salts. A review of suitable salts can be found in Stahl and Wermuth's "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

[0575] The term "stereoisomer" (or "optical isomer") refers to a stable isomer that has a perpendicular asymmetric plane due to having at least one chiral element (including a chiral center, chiral axis, chiral plane, etc.), thereby enabling the rotation of plane-polarized light. Since the compounds of this invention contain asymmetric centers and other chemical structures that may lead to stereoisomerism, this invention also includes these stereoisomers and mixtures thereof. Because the compounds of this invention (or pharmaceutically acceptable salts thereof) comprise asymmetric carbon atoms, they can exist as single stereoisomers, racemates, or mixtures of enantiomers and diastereomers. Typically, these compounds can be prepared as racemates. However, if desired, such compounds can be prepared or isolated to obtain pure stereoisomers, i.e., single enantiomers or diastereomers, or mixtures enriched with single stereoisomers (purity ≥98%, ≥95%, ≥93%, ≥90%, ≥88%, ≥85%, or ≥80%). As described below, a single stereoisomer of a compound is synthesized from an optically active starting material containing the desired chiral center, or prepared by separating or resolving a mixture of enantiomers, for example, by converting it into a mixture of diastereomers and then separating or recrystallizing, chromatographically processing, using chiral resolving reagents, or by directly separating the enantiomers on a chiral chromatographic column. Starting compounds with specific stereochemistry are commercially available or prepared according to the methods described below and then resolved by methods well known in the art. The term "enantiomer" refers to a pair of stereoisomers that are mirror images of each other. The term "diastereomer" or "diastereomer" refers to an optically active isomer that is not mirror images of each other. The term "racemic mixture" or "racemate" refers to a mixture containing equal parts of a single enantiomer (i.e., an equimolar mixture of two R and S enantiomers). The term "non-racemic mixture" refers to a mixture containing unequal parts of a single enantiomer. Unless otherwise stated, all stereoisomers of the compounds of this invention are within the scope of this invention.

[0576] The term "tautomer" (or "tautomer form") refers to structural isomers with different energies that can interconvert through a low energy barrier. If tautomerism is possible (e.g., in solution), chemical equilibrium can be achieved in the tautomer. For example, proton tautomers (or proton transfer tautomers) include (but are not limited to) interconversions via proton transfer, such as keto-enol isomerization, imine-enamine isomerization, amide-imine alcohol isomerization, etc. Unless otherwise stated, all tautomer forms of the compounds of this invention are within the scope of this invention.

[0577] The term "polymorph" (or "polymorphic form") refers to the solid crystalline form of a compound or complex. Polymorphs can be detected, classified, and identified using well-known techniques, including (but not limited to) differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), X-ray powder diffraction (XRPD), single-crystal X-ray diffraction (SCXRD), solid-state nuclear magnetic resonance (NMR), infrared spectroscopy (IR), Raman spectroscopy, and scanning electron microscopy (SEM).

[0578] The term "solvent" refers to a substance formed by the combination of the compound of the present invention (or a pharmaceutically acceptable salt thereof) with at least one solvent molecule through non-covalent intermolecular forces.

[0579] The term "nitrogen oxides" refers to compounds formed by the oxidation of nitrogen atoms in the structure of tertiary amines or nitrogen-containing (aromatic) heterocyclic compounds.

[0580] This invention also includes all pharmaceutically acceptable isotopically labeled compounds that are identical to the compounds of this invention, except that one or more atoms are replaced by atoms having the same atomic number but with a different atomic mass or mass number than the dominant atomic mass or mass number found in nature. Examples of isotopes suitable for inclusion in the compounds of this invention include, but are not limited to, isotopes of hydrogen (e.g., 2H, 3H, deuterium D, tritium T); isotopes of carbon (e.g., 11C, 13C, and 14C); isotopes of chlorine (e.g., 37Cl); isotopes of fluorine (e.g., 18F); isotopes of iodine (e.g., 123I and 125I); isotopes of nitrogen (e.g., 13N and 15N); isotopes of oxygen (e.g., 15O, 17O, and 18O); and isotopes of sulfur (e.g., 35S).

[0581] The scope of this invention also includes metabolites of the compounds of this invention, i.e., substances formed in the body when the compounds of this invention are administered. Such products can be generated, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc., of the administered compound. Therefore, this invention includes metabolites of the compounds of this invention, including compounds obtained by methods that expose the compounds of this invention to mammals for a time sufficient to produce their metabolites.

[0582] This invention further includes, within its scope, prodrugs of the compounds of the invention, which are certain derivatives of the compounds of the invention that may themselves have little or no pharmacological activity, and which, when administered to or onto the body, can be converted, for example, by hydrolysis and cleavage into the compounds of the invention having the desired activity. Typically, such prodrugs are functional group derivatives of the compounds that are readily converted in vivo into the compounds with the desired therapeutic activity. Further information regarding the use of prodrugs can be found in “Pro-drugs as Novel Delivery Systems,” Vol. 14, ACS Symposium Series (T. Higuchi and V. Stella) and “Bioreversible Carriers in Drug Design,” Pergamon Press, 1987 (EB Roche, editor, American Pharmaceutical Association). The prodrugs of the invention can be prepared, for example, by replacing suitable functional groups present in the compounds of the invention with certain portions known to those skilled in the art as “pro-moiety” (e.g., as described in “Design of Prodrugs,” H. Bundgaard (Elsevier, 1985)).

[0583] Preparation of antibody-drug conjugates

[0584] On the other hand, this application provides a method for preparing the antibody-drug conjugate of the present invention, comprising the following steps:

[0585] a) Provide a solution containing antibodies;

[0586] b) Contact the solution from a) with a reducing agent;

[0587] c) The solution of b) is contacted with a solution containing the drug-linker compound or its salt described herein to prepare an ADC.

[0588] In one embodiment, the reducing agent is tris(2-carboxyethyl)phosphine (TCEP).

[0589] In some embodiments, the method for preparing the antibody-drug conjugate of the present invention is selected from:

[0590] Take approximately 0.5 mL of antibody (3-10 mg / mL), dilute with 0.1 M disodium edetate solution (pH 7.60), then adjust the pH to 7.60 with 1 M Na₂HPO₄ solution. Add 2.0-5.5 equivalents of 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution (pH 7.60), mix well, and incubate at room temperature for 1.5 h. Add 4-10 times the amount of the antibody dissolved in DMSO to the above solution system. After the addition is complete, incubate at room temperature for 2 h. Finally, use a NAP-5 gel column (Cytiva) to replace the buffer solution with 20 mM histidine buffer solution at pH 6.0 to obtain the ADC product. Attached image description:

[0591] Figure 1A: Results of endocytic activity assay of antibody-drug conjugates on Raji cells.

[0592] Figure 1B: Results of endocytic activity assay of antibody-drug conjugate on SU-DHL-6 cells.

[0593] Figure 2A: Detection results of the activation activity of antibody-drug conjugates on glucocorticoid receptors in CD19-overexpressing luciferin reporter system cells.

[0594] Figure 2B: Detection results of the activation activity of antibody-drug conjugates on glucocorticoid receptors in CD19-negative blank fluorescein reporter system cells.

[0595] Figure 3A: Results of urine protein detection at the endpoint (15 weeks) of administration of the antibody-drug conjugate of the present invention (10 mg / kg, iv, QW) to NZBWF1 / J spontaneous SLE model mice.

[0596] Figure 3B: Urine protein score data of NZBWF1 / J spontaneous SLE model mice after administration of the antibody-drug conjugate of the present invention (10 mg / kg, iv, QW).

[0597] Figure 3C: Kidney injury score at the endpoint (15w) of the NZBWF1 / J spontaneous SLE model mice after administration of the antibody-drug conjugate of the present invention (10mg / kg, iv, QW).

[0598] Figure 3D: Results of renal immune complex deposition detection at the endpoint (15w) after administration of the antibody-drug conjugate of the present invention (10mg / kg, iv, QW) to NZBWF1 / J spontaneous SLE model mice.

[0599] Figure 4A: Total score of skin desquamation and erythema in the model area of ​​mice after administration of the antibody-drug conjugate of the present invention (10 mg / kg, IV).

[0600] Figure 4B: Skin erythema score in the modeling area of ​​mice after administration of the antibody-drug conjugate of the present invention (10 mg / kg, IV).

[0601] Figure 4C: Skin desquamation scores in the modeling area of ​​mice after administration of the antibody-drug conjugate of the present invention (10 mg / kg, IV).

[0602] Figure 4D: Evaluation results of skin thickening in the model area after administration of the antibody-drug conjugate of the present invention (10 mg / kg, IV) to mice with psoriasis.

[0603] Figure 4E: Evaluation results of spleen / body weight ratio in mice after administration of the antibody-drug conjugate of the present invention (10 mg / kg, iv).

[0604] Figure 4F: Pathological scoring of skin tissue in the modeling area of ​​mice after administration of the antibody-drug conjugate of this invention (10 mg / kg, IV). Detailed Implementation

[0605] The present invention will be further described below through specific embodiments, but this is not intended to limit the invention. Those skilled in the art can make various modifications or improvements based on the teachings of the present invention without departing from the basic ideas and scope of the invention.

[0606] The sequence information involved in this invention is described as follows:

[0607] The abbreviations used in this article have the following meanings:

[0608] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance (NMR). 1 It can be determined by 1H NMR or mass spectrometry (MS).

[0609] Nuclear magnetic resonance (NMR) 1 The H NMR measurements were performed using a Bruker 400MHz NMR spectrometer; the deuterated reagent was hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0610] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the embodiments are shown below.

[0611] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: dimethyl sulfoxide deuterated. δ values ​​are expressed in ppm.

[0612] Mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0613] Synthesis Examples

[0614] Example 1 of intermediate preparation: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-3-amino-4-fluorobenzoate (INT-1)

[0615] Step 1:

[0616] Periodic acid (1.72 g, 7.57 mmol) was added to a solution of INT-1-1 (1 g, 2.52 mmol) in THF (13.5 mL) and water (7.5 mL). The reaction system was stirred at 25 °C for 2 hours and then filtered. The filter cake was washed with water (30 mL x 3) and then dried under vacuum to obtain crude INT-1-2 (900 mg, 2.35 mmol). The crude product was used directly in the next step without purification.

[0617] Its structural characterization is as follows:

[0618] ESI-MS (m / z): 383.1 (M+H) + .

[0619] Step Two:

[0620] INT-1-2 (0.9 g, 2.35 mmol) was dissolved in DMF (3 mL), and CDI (763 mg, 4.71 mmol) was added. The mixture was stirred at 25 °C for 2 hours. DMF (7 mL) was added to the reaction mixture, which was then cooled to -20 °C. Hydrogen sulfide gas (15 PSI) was continuously bubbled through the mixture for 30 minutes, and then the temperature was raised to 25 °C and stirring continued for 2 hours. Water (100 mL) was added to the reaction mixture, resulting in the precipitation of a solid. This solid was filtered, and the filter cake was dried under vacuum to obtain crude INT-1-3 (1.3 g, 2.11 mmol). The crude product was used directly in the next step without purification.

[0621] Its structural characterization is as follows:

[0622] ESI-MS (m / z): 399.2 (M+H) + .

[0623] Step 3:

[0624] 3-(tert-Butoxycarbonylamino)-4-fluorobenzoic acid (289 mg, 1.13 mmol) was dissolved in DMF (7 mL), and HATU (517 mg, 1.36 mmol) and DIEPA (440 mg, 3.40 mmol) were added. The mixture was stirred at 25 °C for 1.5 hours. Then, INT-1-3 (700 mg, 1.13 mmol) was added, and the reaction was continued for another 1.5 hours. Fluoroiodomethane (272 mg, 1.70 mmol) was then added to the above reaction solution, and the reaction was continued for another hour. Water (50 mL) was added to the reaction solution, and a solid precipitated. The solid was filtered, and the filter cake was dried under vacuum to obtain crude INT-1-4 (950 mg, 754 μmol). The crude product was used directly in the next step without purification.

[0625] Its structural characterization is as follows:

[0626] ESI-MS (m / z): 612.5 (M+H) + .

[0627] Step Four:

[0628] INT-1-4 (900 mg, 715 μmol) was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (1.47 g, 12.9 mmol) was added. The mixture was stirred at 25 °C for 1 hour. The reaction solution was directly concentrated to obtain the crude product, which was then purified by high performance liquid chromatography to obtain INT-1 (310.39 mg, 532 μmol).

[0629] Column: Phenomenex C18 150mm×25mm×10μm

[0630] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0631] Its structural characterization is as follows:

[0632] ESI-MS (m / z): 568.5 [M+H] + .

[0633] 1H NMR (400MHz, DMSO) δ7.24-7.37(m,2H),7.15(dd,J=11.2,8.4Hz,1H),7.05(ddd,J=8.4,4.4,2.1H z,1H),6.33(dd,J=10.0,1.8Hz,1H),6.12(s,1H),5.90-6.02(m,1H),5.78-5.88(m,1H),5.57-5.7 4(m,2H),5.53(s,1H),4.32(s,1H),2.86-2.94(m,1H),2.53-2.75(m,2H),2.25-2.38(m,2H),2.01 -2.17(m,2H),1.94(d,J=13.6Hz,1.2H),1.69-1.82(m,1H),1.43-1.59(m,5H),0.88-1.00(m,3H).

[0634] Example 2 of intermediate preparation: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentadien[a]phenanthrene-17-yl-3-((S)-2-((S)-2-aminopropionylamino)propionylamino)-4-fluorobenzoate (INT-2)

[0635] Step 1:

[0636] Allyl 3-amino-4-fluorobenzoate (3.30 g, 16.91 mmol) and (tert-butoxycarbonyl)-L-alanyl-L-alanine (4.40 g, 16.91 mmol) were dissolved in DMF (40.00 mL), followed by the addition of T3P (23.67 g, 37.19 mmol, 50.0% purity) and pyridine (5.35 g, 67.63 mmol). The reaction mixture was stirred at 25 °C for 16 hours. The reaction was monitored by LC-MS until complete. The mixture was extracted with water and ethyl acetate, concentrated to obtain a crude product, and purified by column chromatography (MeOH / DCM = 0-10%) to give INT-2-2 (3.40 g, 7.77 mmol).

[0637] Its structural characterization is as follows:

[0638] ESI-MS (m / z): 382.2 (M+H-56) + .

[0639] Step Two:

[0640] Under nitrogen protection, INT-2-2 (3.39 g, 7.75 mmol) was dissolved in THF (100.00 mL), followed by the addition of tetrakis(triphenylphosphine)palladium (895.47 mg, 774.93 μmol) and morpholine (1.35 g, 15.50 mmol). The reaction mixture was stirred at 25 °C for 16 hours. The reaction was monitored for completeness by LC-MS. The pH of the system was adjusted to approximately 8 with sodium bicarbonate, and the product was extracted with ethyl acetate (to remove impurities). The aqueous phase was then adjusted to approximately 3 with dilute hydrochloric acid, and the product was extracted with ethyl acetate, dried, and concentrated to obtain INT-2-3 (2.40 g, 6.04 mmol).

[0641] Its structural characterization is as follows:

[0642] ESI-MS (m / z): 342.2 (M+H-56) + .

[0643] Step 3:

[0644] INT-2-3 (300 mg, 754.91 μmol), HTUA (287.05 mg, 754.91 μmol), and DIPEA (146.35 mg, 1.13 mmol) were added to DMF (9.00 mL). The reaction solution was stirred at 25 °C for 30 min. Then, DIPEA (146.35 mg, 1.13 mmol) and (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-11,17-dihydroxy-10,13-dimethyl-3- -O-6,7,8,11,12,14,15,16-octahydrocyclopentadieno[a]phenanthrene-17-carboxylic acid (300.80 mg, 754.91 μmol) was stirred for 1 h, and then fluoroiodomethane (482.93 mg, 3.02 mmol) was added to the reaction system. The reaction solution was stirred at 25 °C for 2 h. The reaction was monitored by LC-MS until complete. Water and ethyl acetate were added for extraction, and the crude product was concentrated. The crude product was purified by column chromatography (ACN / H2O = 0-60%, 0.05% formic acid) and lyophilized to obtain crude product INT-2-4 (199 mg).

[0645] Its structural characterization is as follows:

[0646] ESI-MS (m / z): 810.3 (M+H) + .

[0647] Step Four:

[0648] INT-2-4 (255 mg, 316.42 mmol) was added to DCM (8.00 mL), followed by TFA (4.00 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction was monitored by LC-MS until it was complete. The reaction system was concentrated to obtain the crude product, which was then purified by high performance liquid chromatography to obtain INT-2 (100 mg, 121.39 μmol).

[0649] The purification method is as follows:

[0650] Column: Phenomenex C18 250mm×50mm×10μm

[0651] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% TFA)

[0652] Its structural characterization is as follows:

[0653] ESI-MS (m / z): 710.3 (M+H) + .

[0654] Example 3 of intermediate preparation: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-3-((S)-2-((S)-2-(2-aminoacetamido)-3-hydroxypropionylamino)-6-((tert-butoxycarbonyl)amino)hexanoylamino)-4-fluorobenzoate (INT-3)

[0655] Step 1:

[0656] Allyl 3-amino-4-fluorobenzoate (5.00 g, 25.6 mmol), N 2 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 6-(tert-butoxycarbonyl)-L-lysine (10.8 g, 23.0 mmol) was dissolved in dichloromethane (100 mL), and T3P (24.6 g, 38.7 mmol, 23.0 mL, 50.0% purity) and DIPEA (9.93 g, 76.8 mmol, 13.3 mL) were added. The mixture was stirred at 25 °C for 2 hours. Water (200 mL) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (100 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (SiO2, CH2Cl2 / MeOH = 100 / 1-10 / 1) and concentrated again to obtain INT-3-1 (10.5 g, 16.2 mmol).

[0657] Its structural characterization data are as follows:

[0658] ESI-MS (m / z): 668.4 [M+Na] + .

[0659] Step Two:

[0660] INT-3-1 (10.0 g, 15.4 mmol) was dissolved in tetrahydrofuran (100 mL), and morpholine (2.70 g, 30.9 mmol, 2.73 mL) and Pd(PPh3)4 (894 mg, 774 μmol) were added. The mixture was stirred at 25 °C for 1 hour. The pH of the reaction solution was adjusted to 2-3 with 1 N dilute hydrochloric acid, then diluted with water (500 mL) and extracted three times with dichloromethane (500 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was stirred at 25 °C for 2 hours with petroleum ether / ethyl acetate (5 / 1), filtered, and dried to obtain INT-3-2 (8.00 g, 13.2 mmol).

[0661] Its structural characterization data are as follows:

[0662] ESI-MS (m / z): 623.2 [M+H] + .

[0663] Step 3:

[0664] INT-3-2 (5.00 g, 8.26 mmol) was dissolved in DMF (100 mL), and HATU (3.14 g, 8.26 mmol) and DIPEA (3.20 g, 24.7 mmol, 4.31 mL) were added. After stirring at 25 °C for 2 hours, (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-11,17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,11,12,14,15,16-octahydrocyclopentadien[a]phenanthrene-17-carboxylic acid (3.29 g, 8.26 mmol) was added to the above reaction solution, and stirring was continued for 2 hours. Then, fluoroiodomethane (1.32 g, 8.26 mmol) was added to the reaction solution, and the reaction was continued for another 2 hours. Add water (200 mL) to the reaction solution, extract three times with ethyl acetate (100 mL x 3), combine the organic phases, dry with anhydrous sodium sulfate, filter and concentrate to obtain crude product, purify by silica gel column chromatography (SiO2, CH2Cl2 / MeOH = 100 / 1 to 10 / 1) and concentrate again to obtain INT-3-3 (2.60 g, 2.55 mmol).

[0665] Its structural characterization data are as follows:

[0666] ESI-MS (m / z): 1018.1 [M+H] + .

[0667] Step Four:

[0668] INT-3-3 (2.50 g, 2.46 mmol) was dissolved in DMF (25.0 mL), and DBU (373 mg, 2.46 mmol, 370 μL) was added. The mixture was stirred at 25 °C for 1 hour to obtain INT-3-4. The reaction solution was used directly in the next step without any further treatment.

[0669] Step 5:

[0670] To the reaction solution from the previous step, (((9H-fluorene-9-yl)methoxy)carbonyl)-L-serine (781 mg, 2.39 mmol), EDCI (686 mg, 3.58 mmol), and HOBt (483 mg, 3.58 mmol) were added sequentially, and the mixture was stirred at 25°C for 1 hour. Then, water (200 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (100 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was then purified by silica gel column chromatography (SiO2, CH2Cl2 / MeOH = 100 / 1 to 10 / 1) and concentrated again to obtain INT-3-5 (2.00 g, 1.81 mmol).

[0671] Its structural characterization data are as follows:

[0672] ESI-MS (m / z): 1105.5 [M+H] + .

[0673] Step Six:

[0674] INT-3-5 (2.00 g, 1.81 mmol) was dissolved in DMF (25.0 mL), and DBU (275 mg, 1.81 mmol, 272 μL) was added. The mixture was then stirred at 25 °C for 1 hour to obtain INT-3-6. The reaction solution was used directly in the next step without any further treatment.

[0675] Step Seven:

[0676] Add (((9H-fluorene-9-yl)methoxy)carbonyl)glycine (538 mg, 1.81 mmol), EDCI (521 mg, 2.72 mmol), and HOBt (367 mg, 2.72 mmol) sequentially to the reaction solution from the previous step, and then stir at 25 °C for 1 hour. Add water (200 mL) to the reaction solution and extract three times with ethyl acetate (50.0 mL x 3). Combine the organic phases, dry them over anhydrous sodium sulfate, filter and concentrate to obtain crude product, which is then purified by silica gel column chromatography (SiO2, CH2Cl2 / MeOH = 100 / 1 to 10 / 1) and concentrated again to obtain INT-3-7 (903 mg, 700 μmol).

[0677] Its structural characterization data are as follows:

[0678] ESI-MS (m / z): 1162.5 [M+H] + .

[0679] Step 8:

[0680] INT-3-7 (500 mg, 0.43 mmol) was dissolved in DMF (5 mL), and diethylamine (125.8 mg, 1.72 mmol) was added. The mixture was reacted at 25 °C for 1 hour, and most of the DMF was concentrated. The crude product was purified by C18 reverse column chromatography (H2O / ACN = 10-60%, 0.1% formic acid) and lyophilized to obtain INT-3 formate (325 mg).

[0681] Its structural characterization data are as follows:

[0682] ESI-MS (m / z): 940.4 [M+H] + .

[0683] Example 4 of intermediate preparation: Preparation of (S)-4-(2-aminoacetamide)-5-(5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-oxopentanoic acid (INT-4)

[0684] Step 1:

[0685] Add (S)-2-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)-5-(tert-butoxy)-5-oxopentanoic acid (764 mg, 1.58 mmol), allyl 3-amino-4-fluorobenzoate (324.50 mg, 1.66 mmol), pyridine (375.73 mg, 4.75 mmol), and 50% 1-propylphosphonic anhydride-DMF (5.57 g) solution sequentially to DCM (1 mL). After addition, stir the mixture at 25 °C for 3 h. Monitor the reaction for completeness by TLC (PE:EA = 0:1). Add 12 mL of water to the reaction mixture, extract with DCM (15 mL * 2), wash the organic phase with water (8 mL), dry with anhydrous sodium sulfate (3 g), filter, concentrate the filtrate under reduced pressure, and purify by column chromatography (SiO2, CH2Cl2 / MeOH = 100 / 1-10 / 1). (S)-3-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5-oxopentanamide)-4-fluorobenzoic acid allyl ester (814 mg, 1.14 mmol).

[0686] Its structural characterization data are as follows:

[0687] ESI-MS (m / z): 659.70 (M+H) + .

[0688] Step Two:

[0689] Add (S)-3-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5-oxopentanamide)-4-fluorobenzoate (734 mg, 1.11 mmol), morpholine (193.86 mg, 2.23 mmol), and Pd(PPh3)4 (64.29 mg, 55.63 μmol) to THF (7 mL) in sequence. After the addition is complete, stir the mixture at 25 °C for 2 h. Add water (26 mL) to the reaction solution, extract with DCM (26 mL * 2), wash the organic phase with water (8 mL) and saturated brine (8 mL) respectively, dry with anhydrous sodium sulfate (4 g), concentrate under reduced pressure until almost dry, add PE (8 mL), slurry at room temperature for 1 h, filter, dry the filter cake under reduced pressure to obtain (S)-3-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5-oxopentanamide)-4-fluorobenzoic acid (450 mg, 697.18 μmol).

[0690] Its structural characterization data are as follows:

[0691] ESI-MS (m / z): 620 (M+H) + .

[0692] Step 3:

[0693] Add (S)-3-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5-oxopentanamide)-4-fluorobenzoic acid (200 mg, 322.77 μmol), DIPEA (83.43 mg, 645.54 μmol), and HATU (147.73 mg, 387.32 μmol) sequentially to DMF (4 mL). After addition, stir at 16 °C for 35 min. Add A-1-3 (128.86 mg, 320.28 μmol) to the reaction solution. After addition, stir at 20 °C for 1 h. Add DIPEA (31.02 mg, 239.98 μmol) and fluoroiodomethane (211.09 mg, 1.32 mmol) to the above reaction solution. After addition, stir at 20 °C for 1 h. Add water (12 mL) to the reaction solution, extract with EA (15 mL * 2), wash the organic phase with water (8 mL) and saturated brine (8 mL) respectively, separate the layers, concentrate the organic phase under reduced pressure to dryness, and purify by column chromatography (SiO2, PE / EA = 12 / 1-1 / 1) to obtain (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-1 1-Hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-3-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonylamino)acetamido)-5-(tert-butoxy)-5-oxopentanoylamino)-4-fluorobenzoate (88 mg, 80.15 μmol).

[0694] Its structural characterization data are as follows:

[0695] ESI-MS (m / z): 1032 (M+H) + .

[0696] Step Four:

[0697] (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-3-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonylamino)acetamido)-5-(tert-butoxy)-5-oxopentanoylamino)-4-fluorobenzoate (88 mg, 80.15 μmol) and diethylamine (31.18 mg, 426.32 μmol) were added sequentially to MeCN (4 mL). After the addition was complete, the mixture was stirred at 25 °C for 2 h. The reaction solution was concentrated under reduced pressure to obtain crude (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-3-(((S)-2-(2-aminoacetamido)-5-(tert-butoxy)-5-oxopentamido)-4-fluorobenzoate (69 mg, 80.94 μmol), which was used directly in the next step without purification.

[0698] Its structural characterization data are as follows:

[0699] ESI-MS (m / z): 810 (M+H) + .

[0700] Step 5:

[0701] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-3-(((S)-2-(2-aminoacetamido)-5-(tert-butoxy)-5-oxopentamido)-4-fluorobenzoate crude product (69 mg, 80.94 μmol) to DCM (3 mL), add TFA (0.5 mL), stir at 25 °C after the addition is complete. The reaction was carried out for 3 hours. The reaction solution was then subjected to reduced pressure to remove DCM and TFA, yielding crude (S)-4-(2-aminoacetamide)-5-(5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-oxopentanoic acid (88 mg, 83.15 μmol), which was used directly in the next step without purification.

[0702] Its structural characterization data are as follows:

[0703] ESI-MS (m / z): 754 (M+H) + .

[0704] Example 5 of intermediate preparation: Preparation of S-(fluoromethyl)(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxacyclopentene-8b-thioester (INT-5)

[0705] Step 1: Preparation of (3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecylhydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxacyclopenten-10-yl)benzyl)phenyl)tert-butyl carbamate (INT-5-2)

[0706] (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecylhydro-4H-naphtho[ [2',1':4,5]indeno[1,2-d][1,3]dioxane-4-one (1.5 g, 2.48 mmol) was dissolved in tetrahydrofuran (29.29 mL) and water (5.86 mL), and then di-tert-butyl dicarbonate (810.77 mg, 3.71 mmol, 853.44 μL) and sodium bicarbonate (624.15 mg, 7.43 mmol) were added. The mixture was reacted overnight at room temperature. The mixture was then extracted with water (60 mL) and ethyl acetate (100 mL). The organic phase was collected, dried, and concentrated under vacuum to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–60%) and concentrated to obtain the title compound (1.270 g, 1.80 mmol).

[0707] Its structural characterization is as follows:

[0708] ESI-MS(m / z): 706.3 [M+H]+.

[0709] Step 2: Preparation of (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((tert-butoxycarbonyl)amino)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxacyclopentene-8b-carboxylic acid (INT-5-3)

[0710] (3-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecylhydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxacyclopenten-10-yl)benzyl)phenyl)tert-butyl carbamate (1.270 g, 1.80 mmol) was dissolved in acetonitrile (22 mL) and water (22 mL), and then NaIO4 (1.54 g, 7.20 mmol) was added and stirred overnight at room temperature. Add water (100 mL) and stir for 20 min. Filter and dry to obtain crude product (0.95 g, 1.37 mmol).

[0711] Its structural characterization is as follows:

[0712] ESI-MS(m / z): 692.3[M+H]+.

[0713] Step 3: Preparation of (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((tert-butoxycarbonyl)amino)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxacyclopentene-8b-carboxylic acid dimethylthioanhydride (INT-5-4)

[0714] (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((tert-butoxycarbonyl)amino)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2',1] [4,5]indeno[1,2-d][1,3]dioxane-8β-carboxylic acid (0.95 g, 1.37 mmol) was dissolved in acetonitrile (40 mL), followed by the addition of dimethylthiocarbamoyl chloride (424.37 mg, 3.43 mmol), DIPEA (532.46 mg, 4.12 mmol), and potassium iodide (136.78 mg, 823.99 μmol) at room temperature overnight. After slowly adding 200 mL of water, a large amount of solid precipitated. The solid was filtered, washed with water, and then washed with petroleum ether to obtain the crude product (1.0 g, 1.28 mmol).

[0715] Its structural characterization is as follows:

[0716] ESI-MS(m / z): 779.3 [M+H]+.

[0717] Step 4: Preparation of S-(fluoromethyl)(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((tert-butoxycarbonyl)amino)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxacyclopentene-8b-thioester (INT-5-5)

[0718] (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((tert-butoxycarbonyl)amino)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxacyclopentene-8b-carboxylic acid dimethylthioic anhydride (1.0 g, 1.28 mmol) was dissolved in N,N-dimethylacetamide (12 mL), and then NaSH (215.92 mg, 3.85 mmol) was added. The reaction was carried out at room temperature for two hours. Fluoroiodomethane (1.02 g, 6.36 mmol) was added to the system and the reaction was carried out at room temperature for two hours. Water and ethyl acetate were added for extraction, and the organic phase was collected, dried and concentrated to give crude product (0.94 g, 1.27 mmol).

[0719] Its structural characterization is as follows:

[0720] ESI-MS(m / z): 740.3[M+H]+.

[0721] Step 5: Preparation of S-(fluoromethyl)(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxacyclopentene-8b-thioester (INT-5)

[0722] S-(fluoromethyl)(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-((tert-butoxycarbonyl)amino)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxacyclopentene-8b-thioester (0.94 g, 1.27 mmol) was dissolved in dichloromethane (4 mL), and then trifluoroacetic acid (1 mL) was added. The reaction was carried out at room temperature for eight hours. The reaction solution was concentrated to obtain a crude product, which was then purified by high performance liquid chromatography and freeze-dried to obtain the title compound (720 mg, 955.23 μmol).

[0723] Column: Waters SunFire Prep C18 OBD 19mm×150mm×5μm

[0724] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0725] Its structural characterization is as follows:

[0726] ESI-MS (m / z): 694.1 [M+H] + .

[0727] 1 H NMR (400MHz, DMSO) δ7.33 (t, J = 8.7Hz, 2H), 7.29-7.18 (m, 3H), 6.92-6.86 (m, 1H), 6.42-

[0728] 6.32(m,3H),6.30(dd,J=10.2,1.8Hz,1H),6.13(s,1H),6.06-5.93(m,1 H),5.87(d,J=27.6Hz,1H),5.59(m,2H),5.51(s,1H),4.94(m,3H),4.21 (d,J=6.4Hz,1H),3.75(s,2H),2.72-2.59(m,1H),2.32-2.16(m,2H),2. 02(d,J=13.7Hz,1H),1.82-1.69(m,3H),1.60-1.46(m,4H),0.97(s,3H).

[0729] Example 6 of intermediate preparation: Preparation of 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (INT-6)

[0730] Step 1: Preparation of 3,3'-((2-((3-(benzyloxy)-3-oxopropoxy)methyl)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxo))dipropionic acid (INT-6-2)

[0731] 3,3'-((2,2-bis((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (0.94 g, 4.71 mmol) and 2-benzyloxy-1-methylpyridine trifluoromethanesulfonate (1.65 g, 4.71 mmol) were reacted at 80-85 °C for 16 hours. The reaction solution was directly purified by C18 reverse-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 10-70%) and lyophilized to give the title compound (0.78 g, 1.51 mmol).

[0732] Its structural characterization data are as follows:

[0733] ESI-MS (m / z): 515.2 (M+H) +

[0734] Step 2: Preparation of 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)benzyl propionate (INT-6-3)

[0735] 3,3'-((2-((3-(benzyloxy)-3-oxopropoxy)methyl)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (0.78 g, 1.51 mmol) and 2-amino-2-(hydroxymethyl)propane-1,3-diol (0.82 g, 6.80 mmol) were dissolved in DMF (15 mL), DIPEA (1.17 g, 9.07 mmol) was added, and HATU (2.59 g, 6.80 mmol) was added in portions. The reaction was carried out at 25 °C for 2 hours. The reaction solution was directly purified by C18 reverse-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 10-70%) and lyophilized to give the title compound (0.74 g, 0.90 mmol).

[0736] Its structural characterization data are as follows:

[0737] ESI-MS (m / z): 824.4 (M+H) +

[0738] Step 3: Preparation of 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (INT-6)

[0739] Benzyl 3-(3-(((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-(((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propoxy)propionate (0.74 g, 0.90 mmol) was dissolved in ethanol (30 mL), 10% palladium on carbon (0.15 g) and acetic acid (0.14 g, 2.42 mmol) were added, the air was removed, hydrogen gas (balloon) was introduced, the temperature was raised to 40 °C and reacted for 4 hours, palladium on carbon was filtered off, the filtrate was concentrated, the residue was dissolved in water (15 mL) and acetonitrile (15 mL) and clarified, and lyophilized to give the title compound (0.59 g, 0.80 mmol).

[0740] Its structural characterization data are as follows:

[0741] ESI-MS (m / z): 734.3 (M+H) +

[0742] Example 7 of intermediate preparation: Preparation of 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (INT-7)

[0743] Step 1: Preparation of 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)benzyl propionate (INT-7-1)

[0744] 3,3'-((2-((3-(benzyloxy)-3-oxopropoxy)methyl)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (1.07 g, 2.08 mmol) and (2R,3R,4R,5S)-6-(methylamino)hexane-1,2,3,4,5-pentaol (1.62 g, 8.32 mmol) were dissolved in DMF (15 mL), DIPEA (1.34 g, 10.40 mmol) was added, and HATU (3.56 g, 9.36 mmol) was added in portions. The reaction was carried out at 25 °C for 1 hour. The reaction solution was directly purified by C18 reverse-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 10-70%) and lyophilized to give the title compound (0.66 g, 0.63 mmol).

[0745] Its structural characterization data are as follows:

[0746] ESI-MS (m / z): 1046.5 (M+H) +

[0747] Step 2: Preparation of 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (INT-7)

[0748] Benzyl 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propoxy)propionate (0.66 g, 0.63 mmol) was dissolved in ethanol (14 mL) and water (7 mL). 10% palladium on carbon (0.13 g) and acetic acid (0.10 g, 1.70 mmol) were added. The air was removed, and hydrogen gas (in a balloon) was introduced. The mixture was heated to 40 °C and reacted for 4 hours. The palladium on carbon was filtered off, the filtrate was concentrated, and the residue was dissolved and clarified in water (15 mL) and acetonitrile (15 mL). The solution was lyophilized to give the title compound (0.54 g, 0.56 mmol).

[0749] Its structural characterization data are as follows:

[0750] ESI-MS (m / z): 956.4 (M+H) +

[0751] Example 8 of intermediate preparation: N 6 -(diphenyl(p-tolyl)methyl)-N 2Preparation of -(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-lysine (INT-8)

[0752] Step 1: N 6 Preparation of 1-(diphenyl(p-tolyl)methyl)-L-lysine (INT-8-2)

[0753] N 2 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 6 -(diphenyl(p-tolyl)methyl)-L-lysine (150.0 mg, 0.24 mmol) was dissolved in DMF (2 mL), and diethylamine (182.3 mg, 2.5 mmol) was added. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, 10 mL of a mixed solvent (EA / PE = 1 / 3) was added for recrystallization, and the solution was filtered to give the title compound (90.1 mg, 0.22 mmol).

[0754] Step Two: N 6 -(diphenyl(p-tolyl)methyl)-N 2 Preparation of -(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-lysine (INT-8)

[0755] N 6 -(diphenyl(p-tolyl)methyl)-L-lysine (90.1 mg, 0.22 mmol) was dissolved in DMF (3 mL), DIPEA (86.3 mg, 0.66 mmol) was added, followed by 2,5-dioxopyrrolidone-1-yl-6-(2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-acetylacetate (160.6 mg, 0.44 mmol). After the reaction was complete, the mixture was purified by rapid column chromatography (C18, water / acetonitrile = 0.5) and then freeze-dried to give the title compound (75.2 mg, 0.11 mmol).

[0756] Its structural characterization data are as follows:

[0757] MS m / z (ESI): 653.2 [M+H] +

[0758] Example 9 of intermediate preparation: N 6 -(tert-Butoxycarbonyl)-N 2 Preparation of -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (INT-9):

[0759] Step 1: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine-L-lysine (INT-9-1)

[0760] Add N to DCM (3 mL) and TFA (1 mL) 6 -(diphenyl(p-tolyl)methyl)-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (800 mg, 1.06 mmol), after addition, the mixture was stirred at 20 °C for 3 hours. After the reaction solution was evaporated to dryness, the residue was slurried with DCM (10 mL) and THF (3 mL) for 1 hour, filtered, and the filter cake was dried to give crude trifluoroacetate of the title compound (550 mg).

[0761] Its structural characterization data are as follows:

[0762] ESI-MS (m / z): 496.2 (M+H) + .

[0763] Step Two: N 6 -(tert-Butoxycarbonyl)-N 2 Preparation of -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (INT-9)

[0764] To 1,4-dioxane (5 mL), (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine-L-lysine (250 mg, 410.10 μmol), triethylamine (207.49 mg, 2.05 mmol), and di-tert-butyl dicarbonate (134.25 mg, 615.14 μmol) were added. After the addition was complete, the mixture was stirred at 20 °C for 16 hours. The reaction solution was purified by rapid column chromatography (C18, 0.05% formic acid aqueous solution / acetonitrile = 0%-80%) and then freeze-dried to give the title compound (198 mg, 305.79 μmol).

[0765] Its structural characterization data are as follows:

[0766] ESI-MS (m / z): 595.2 (M+H) + .

[0767] Example 10: Preparation of (S)-3-(6-((diphenyl(p-tolyl)methyl)amino)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-yneamide)hexamide)propionic acid (INT-10)

[0768] N 6-(diphenyl(p-tolyl)methyl)-N 2 -(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-lysine (1.15 g, 1.76 mmol), DIPEA (683.02 mg, 5.28 mmol), and HATU (736.83 mg, 1.94 mmol) were added to DMF (20 mL), and the mixture was stirred at 25 °C for 30 minutes. Then, 3-aminopropionic acid (235.42 mg, 2.64 mmol, FR) was added to the reaction mixture, and the mixture was stirred at 25 °C for 2.5 hours. The reaction mixture was concentrated to dryness, and the final product was purified by high-performance liquid chromatography to obtain the title compound (170 mg, 234.85 μmol).

[0769] Its structural characterization data are as follows:

[0770] MS m / z(ESI): 724.1 [M+H] +

[0771] Its preparation method is as follows:

[0772] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0773] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0774] Example 11: Preparation of intermediate (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl 3-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyramide)propionamide)-4-fluorobenzoate (INT-11):

[0775] Step 1: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentan[a]phenanthrene-17-yl 3-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)propionamido)-4-fluorobenzoate (INT-11-1)

[0776] (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-amino-4-fluorobenzoate (10.0) (17.6 mmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanine (4.39 g, 14.0 mmol) were dissolved in DMF (100 mL). T3P (59.9 g, 94.1 mmol, 56.0 mL, 50.0% purity) and pyridine (4.18 g, 52.8 mmol, 4.27 mL) were added, and the mixture was stirred at 25 °C for 1 hour. The reaction mixture was poured into water until a white solid precipitated. This solid was dissolved in ethyl acetate, dried, and concentrated to obtain the crude product. After purification by silica gel column chromatography (dichloromethane / methanol = 100 / 1 -3 / 97), the product was concentrated again to obtain the title compound (7.46 g, 7.97 mmol, 92.0% purity).

[0777] Its structural characterization data are as follows:

[0778] ESI-MS (m / z): 861.2 (M+H) + .

[0779] Step 2: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl 3-((S)-2-aminopropionamido)-4-fluorobenzoate (INT-11-2)

[0780] (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentan[a]phenanthrene-17-yl 3-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)propionamido)-4-fluorobenzoate (6.96 g, 8.08 mmol) was dissolved in DMF (70.0 mL), and DBU (984 mg, 6.47 mmol, 974 μL) was added. The mixture was stirred at 25 °C for 1 hour. The reaction solution was used directly in the next step without further treatment.

[0781] Its structural characterization data are as follows:

[0782] ESI-MS (m / z): 639.1 (M+H) + .

[0783] Step 3: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl 3-((S)-2-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-methylbutyramide)propionamide)-4-fluorobenzoate (INT-11)

[0784] To the reaction solution from the previous step, (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine (2.74 g, 8.08 mmol), HOBt (1.64 g, 12.1 mmol), and EDCI (2.32 g, 12.1 mmol) were added sequentially, and the mixture was stirred at 25 °C for 2 hours. The reaction solution was then poured into water, resulting in the precipitation of a solid. After drying, the crude product was obtained. Purification was performed by silica gel column chromatography (dichloromethane / methanol = 100 / 1 - 97 / 3), followed by further concentration to obtain the title compound (6.12 g, 6.03 mmol).

[0785] Its structural characterization data are as follows:

[0786] ESI-MS (m / z): 960.0 (M+H) + .

[0787] Example 12: Preparation of (S)-5-(allyloxy)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)-5-oxovaleric acid (INT-12)

[0788] Step 1: Preparation of 5-(tert-butyl)-(((9H-fluorene-9-yl)methoxy)carbonyl)-L-glutamic acid allyl ester (INT-12-2)

[0789] (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxovaleric acid (6.00 g, 14.10 mmol) and lithium hydroxide monohydrate (1.18 g, 28.20 mmol) were added sequentially to DMF (30 mL). After the addition was complete, the mixture was stirred at 25 °C for 10 min, and then allyl bromide (5.12 g, 42.31 mmol) was added. The reaction mixture was reacted at 25 °C for 1 h. The reaction solution was poured into dilute hydrochloric acid (100 mL of 0.5 N HCl), extracted with EA (40 mL * 2), the organic phase was washed with water (20 mL), dried over anhydrous sodium sulfate (3 g), filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (SiO2, EA / PE = 0-45%). The title compound (4.67 g, 10.03 mmol) was obtained.

[0790] Its structural characterization data are as follows:

[0791] ESI-MS (m / z): 410.3 (M+H-56) + .

[0792] Step 2: Preparation of 5-(tert-butyl)-L-glutamic acid allyl ester (INT-12-3)

[0793] 5-(tert-butyl)-(((9H-fluorene-9-yl)methoxy)carbonyl)-L-glutamic acid allyl ester (4.40 g, 9.45 mmol) and DBU (4.32 g, 28.35 mmol) were added sequentially to DMF (22 mL). After the addition was complete, the mixture was stirred at 25 °C for 1 h. The reaction solution was concentrated under reduced pressure to obtain the crude title compound (2.29 g, 9.41 mmol), which was used directly in the next step without purification.

[0794] Its structural characterization data are as follows:

[0795] ESI-MS (m / z): 244.2 (M+H) + .

[0796] Step 3: Preparation of 5-(tert-butyl)(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamic acid allyl ester (INT-12-4)

[0797] Crude 5-(tert-butyl)-L-glutamic acid allyl ester (2.29 g, 9.41 mmol), 6-(2-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ethynic acid (3.03 g, 11.29 mmol), DIPEA (4.87 g, 37.65 mmol), and HATU (5.37 g, 14.12 mmol) were added sequentially to DMF (22 mL). After addition, the mixture was stirred at 25 °C for 1 h. The reaction was monitored by LC-MS until complete. The crude product was concentrated and purified by column chromatography (ACN / H2O = 0.75%, 0.05% formic acid). The product was then lyophilized to obtain the title compound (1.42 g, 2.87 mmol).

[0798] Its structural characterization data are as follows:

[0799] ESI-MS (m / z): 511.2 (M+H2O) + .

[0800] Step 4: Preparation of (S)-5-(allyloxy)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-5-oxovalerate (INT-12)

[0801] 5-(tert-butyl)(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamic acid allyl ester (830.00 mg, 1.48 mmol) was dissolved in DCM (10 mL), and TFA (5 mL) was added. After the addition was complete, the mixture was stirred at 25 °C for 3 h. The reaction solution was subjected to reduced pressure to remove DCM and TFA to obtain the crude product, which was purified by high performance liquid chromatography to obtain the title compound (361.00 mg, 825.21 μmol).

[0802] The purification method is as follows:

[0803] Column: Phenomenex C18 250mm×50mm×10μm

[0804] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[0805] Its structural characterization is as follows:

[0806] ESI-MS (m / z): 438.1 (M+H) + .

[0807] Example 13: Preparation of (S)-5-(tert-butoxy)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)-5-oxovaleric acid (INT-13)

[0808] 5-(tert-butyl)(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamic acid allyl ester (450.00 mg, 911.72 μmol), 1,3-dimethyl barbituric acid (711.78 mg, 4.56 mmol), and Pd(PPh3)4 (347.67 mg, 300.87 μmol) were added sequentially to DMF (25 mL). After the addition was complete, the mixture was stirred at 25 °C for 2 h under nitrogen protection. The reaction was monitored by LC-MS until complete. The reaction system was concentrated to obtain a crude product, which was purified by high performance liquid chromatography to obtain (S)-5-(tert-butoxy)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)-5-oxovalerate (INT-13) (387.00 mg, 853.35 μmol).

[0809] The purification method is as follows:

[0810] Column: Phenomenex C18 250mm×50mm×10μm

[0811] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[0812] Its structural characterization is as follows:

[0813] ESI-MS (m / z): 471.2 (M+H2O) + .

[0814] Example 14: Preparation of (S)-9-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-23-(2-methylsulfonyl)pyrimidin-5-yl)-8,11,18-trioxo-4,14-dioxa-7,10,17-triazatridecane-22-alkynic acid (INT-14)

[0815] Step 1: Preparation of (S)-5-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-1-(9H-fluorene-9-yl)-3,6-dioxo-2,10-dioxa-4,7-diazatriadecane-13-acid (INT-14-1)

[0816] N 2 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 6-(diphenyl(p-tolyl)methyl)-L-lysine (8.5 g, 13.61 mmol), 3-(2-aminoethoxy)propionic acid (5.95 g, 15.65 mmol), HATU (6.21 g, 16.33 mmol), and DIPEA (3.52 g, 27.21 mmol) were dissolved in DMF (60 mL), and the reaction was stirred at room temperature for 1 hour. After quenching the reaction with water, the pH was adjusted to 5-6 with formic acid. The organic phase was extracted with dichloromethane (200 mL), and then concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography. Further purification by high performance liquid chromatography yielded the title compound (10.07 g, 2.44 mmol).

[0817] Its structural characterization is as follows:

[0818] ESI-MS (m / z): 740.3 (M+H) + .

[0819] Step 2: Preparation of (S)-3-(2-(2-amino-6-((diphenyl(p-tolyl)methyl)amino)hexamethylene)ethoxy)propionic acid (INT-14-2)

[0820] (S)-5-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-1-(9H-fluorene-9-yl)-3,6-dioxo-2,10-dioxa-4,7-diazatriadecane-13-acid (1.90 g, 2.57 mmol, FR) was dissolved in DMF (12 mL), and diethylamine (563.15 mg, 7.70 mmol) was added. The mixture was stirred at 25 °C for 2 hours. After the reaction solvent was dried, the crude product was purified by reversed-phase column chromatography to obtain the title compound (1.34 g, 2.02 mmol).

[0821] Its structural characterization is as follows:

[0822] ESI-MS (m / z): 518.04 (M+H) + .

[0823] Step 3: Preparation of tert-butyl propionate (INT-14-4)

[0824] 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-alkynic acid (9.92 g, 36.99 mmol) and tert-butyl 3-(2-aminoethoxy)propionate (7.0 g, 36.99 mmol) were dissolved in DMF (20 mL), followed by the addition of HATU (21.09 g, 55.48 mmol) and DIEPA (14.34 g, 110.96 mmol), and the mixture was stirred at 25 °C for 1.5 hours. The reaction solution was purified by reversed-phase column chromatography to obtain a crude brown solid (15 g, 34.13 mmol) of the title compound. The product was used directly in the next reaction step.

[0825] Step 4: Preparation of 3-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)ethoxy)propionic acid (INT-14-5)

[0826] 3-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)ethoxy)tert-butyl propionate (6.2 g, 14.11 mmol) was dissolved in dichloromethane (30 mL), and trifluoroacetic acid (15 mL) was added. The mixture was stirred at 25 °C for 1 hour. The reaction solution was directly concentrated to obtain a crude product, which was purified by high performance liquid chromatography to obtain the title compound (2.4 g, 6.26 mmol).

[0827] Its structural characterization is as follows:

[0828] ESI-MS (m / z): 880.1 (2M+H) + .

[0829] Step 5: Preparation of 2,5-dioxopyrrolidone-1-yl 3-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)ethoxy)propionate (INT-14-6)

[0830] 3-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)ethoxy)propionic acid (2.6 g, 6.78 mmol) and 1-hydroxypyrrolidine-2,5-dione (936.50 mg, 8.14 mmol) were dissolved in DMF (25 mL), and EDCI (1.95 g, 10.17 mmol) was added. The mixture was stirred at 25 °C for 6 hours. The reaction was quenched with water and extracted with DCM. The combined organic phases were concentrated to remove the solvent, yielding the crude title compound (4.85 g, 6.56 mmol). The crude compound was used directly in the next reaction.

[0831] Its structural characterization is as follows:

[0832] ESI-MS (m / z): 481.2 (M+H) + .

[0833] Step Six: Preparation of (S)-9-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-23-(2-methylsulfonyl)pyrimidin-5-yl)-8,11,18-trioxo-4,14-dioxa-7,10,17-triazatridecane-22-alkynic acid (INT-14)

[0834] (S)-3-(2-(2-amino-6-((diphenyl(p-tolyl)methyl)amino)hexamethylene)ethoxy)propionic acid (1.34 g, 2.02 mmol) and 2,5-dioxopyrrolidone-1-yl 3-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)ethoxy)propionate (1.49 g, 2.02 mmol) were dissolved in DMF (25 mL), and DIPEA (520.90 mg, 4.03 mmol) was added. The mixture was stirred at 25 °C for 2 hours. The reaction solution was purified by reversed-phase column chromatography to give the title compound (1.35 g, 1.45 mmol).

[0835] Its structural characterization is as follows:

[0836] ESI-MS (m / z): 883.4 (M+H) + .

[0837] Example 15: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadiene[a]phenanthrene-17-yl-3-((S)-2-((S)-2-((S)-2-aminopropamido)propamido)propamido)-4-fluorobenzoate (INT-15)

[0838] Step 1: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentadiene[a]phenanthrene-17-yl-3-((5S,8S,11S)-1-(9H-fluorene-9-yl)-5,8-11-trimethyl-3,6,9-trioxo-2-oxa-4,7,10-triazadodecane-12-amino)-4-fluorobenzoate (INT-15-1)

[0839] The (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadiene[a]phenanthrene-17-yl-3-((S)-2-((S)-2- (Aminopropylamino)propylamino)-4-fluorobenzoate (30 mg, 36.42 μmol), (2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)propionic acid (17.01 mg, 54.63 μmol), and DIPEA (18.83 mg, 145.67 μmol) were added sequentially to DMF (2 mL), followed by DMTMM (16.10 mg, 54.63 mg). The reaction mixture was stirred at 25 °C for 2 hours. The reaction solution was purified by reverse-phase column chromatography (acetonitrile / 0.5% formic acid aqueous solution = 20-25%), and then freeze-dried to give the title compound (15 mg, 14.95 μmol).

[0840] Step 2: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadiene[a]phenanthrene-17-yl-3-((S)-2-((S)-2-((S)-2-aminopropylamino)propylamino)propylamino)-4-fluorobenzoate (INT-15)

[0841] (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadiene[a]phenanthrene-17-yl-3-((5S,8S,11) S)-1-(9H-fluorene-9-yl)-5,8-11-trimethyl-3,6,9-trioxo-2-oxa-4,7,10-triazadodecane-12-amino)-4-fluorobenzoate (15 mg, 14.95 μmol) was dissolved in N,N-dimethylformamide (2 mL), and diethylamine (1.09 mg, 14.95 μmol) was added. The mixture was stirred at 25 °C for 1 hour. The reaction solution was directly placed on a lyophilizer to remove the solvent and residual diethylamine, yielding the crude title compound (12 mg), which was used directly in the next step without purification.

[0842] Small molecule hormone example: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-3-amino-4-fluorobenzoate (compound P1)

[0843] Step 1:

[0844] Periodic acid (1.72 g, 7.57 mmol) was added to a solution of P-1-1 (1 g, 2.52 mmol) in THF (13.5 mL) and water (7.5 mL). The reaction system was stirred at 25 °C for 2 hours and then filtered. The filter cake was washed with water (30 mL x 3) and then dried under vacuum to obtain crude P-1-2 (900 mg, 2.35 mmol). The crude product was used directly in the next step without purification.

[0845] ESI-MS (m / z): 383.1 (M+H) + .

[0846] Step Two:

[0847] P-1-2 (0.9 g, 2.35 mmol) was dissolved in DMF (3 mL), and CDI (763 mg, 4.71 mmol) was added. The mixture was stirred at 25 °C for 2 hours. DMF (7 mL) was added to the reaction mixture, and the mixture was cooled to -20 °C. Hydrogen sulfide gas (15 PSI) was continuously bubbled through the mixture for 30 minutes, then the temperature was raised to 25 °C and stirring continued for 2 hours. Water (100 mL) was added to the reaction mixture, resulting in the precipitation of a solid. The solid was filtered, and the filter cake was dried under vacuum to obtain crude P-1-3 (1.3 g, 2.11 mmol). This crude product was used directly in the next step without purification.

[0848] ESI-MS (m / z): 399.2 (M+H) + .

[0849] Step 3:

[0850] 3-(tert-Butoxycarbonylamino)-4-fluorobenzoic acid (289 mg, 1.13 mmol) was dissolved in DMF (7 mL), and HATU (517 mg, 1.36 mmol) and DIEPA (440 mg, 3.40 mmol) were added. The mixture was stirred at 25 °C for 1.5 hours. Then, P-1-3 (700 mg, 1.13 mmol) was added, and the reaction was continued for another 1.5 hours. Fluoroiodomethane (272 mg, 1.70 mmol) was then added to the above reaction solution, and the reaction was continued for another hour. Water (50 mL) was added to the reaction solution, and a solid precipitated. The solid was filtered, and the filter cake was dried under vacuum to obtain crude P-1-4 (950 mg, 754 mmol). The crude product was used directly in the next step without purification.

[0851] ESI-MS (m / z): 612.5 (M+H) + .

[0852] Step Four:

[0853] P-1-4 (900 mg, 715 μmol) was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (1.47 g, 12.9 mmol) was added. The mixture was stirred at 25 °C for 1 hour. The reaction solution was directly concentrated to obtain the crude product, which was then purified by high performance liquid chromatography to obtain P1 (310.39 mg, 532 mmol).

[0854] Column: Phenomenex C18 150mm×25mm×10mm

[0855] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0856] The structural characterization data for P1 are as follows:

[0857] ESI-MS (m / z): 568.5 [M+H] + .

[0858] 1H NMR (400MHz, DMSO) δ7.24-7.37(m,2H),7.15(dd,J=11.2,8.4Hz,1H),7.05(ddd,J=8.4,4.4,2.1H z,1H),6.33(dd,J=10.0,1.8Hz,1H),6.12(s,1H),5.90-6.02(m,1H),5.78-5.88(m,1H),5.57-5.7 4(m,2H),5.53(s,1H),4.32(s,1H),2.86-2.94(m,1H),2.53-2.75(m,2H),2.25-2.38(m,2H),2.01 -2.17(m,2H),1.94(d,J=13.6Hz,1.2H),1.69-1.82(m,1H),1.43-1.59(m,5H),0.88-1.00(m,3H).

[0859] Example 1: 2,2',2”-(10-((2S,5S,12S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro Preparation of -3H-cyclopentadien[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-2,5-dimethyl-12-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamino)-1,4,7,11,18-pentoxa-3,6,10,17-tetraazanonadecan-19-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-1)

[0860] Step 1: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((2S,5S,12S)-12-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-2,5-dimethyl-19-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,11,14-tetraoxo-3,6,10,13-tetraazanonadecan-18-acetylamidamide)-4-fluorobenzoate (A-1-1)

[0861] (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-3-((S)-2-((S)-2-aminopropionylamino)propionylamino)-4-fluorobenzoate (180mg, 2 53.61 μmol of (S)-3-(6-((diphenyl(p-tolyl)methyl)amino)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)hexamide)propionic acid (212 mg, 278.97 μmol) was dissolved in DMF (10 mL), followed by the addition of 2,6-dimethylpyridine (108.70 mg, 1.01 mmol) and HATU (150.00 mg, 394.51 mmol), and the mixture was stirred at 25 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (165.00 mg, 116.56 μmol).

[0862] Its structural characterization data are as follows:

[0863] ESI-MS (m / z): 1416.6 (M+H) + .

[0864] Step 2: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((2S,5S,12S)-12-(4-aminobutyl)-2,5-dimethyl-19-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,11,14-tetraoxo-3,6,10,13-tetraazanonadecan-18-acetylamidamide)-4-fluorobenzoate (A-1-2)

[0865] The (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl3-((2S,5S,12S)-1 2-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-2,5-dimethyl-19-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,11,14-tetraoxo-3,6,10,13-tetraazanonadecan-18-acetylamidamide)-4-fluorobenzoate (165.00 mg, 116.56 μmol) was dissolved in DCM (3 mL), and formic acid (3 mL) was added. The reaction mixture was stirred at 25 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to give the title compound (95.00 mg, 81.95 μmol).

[0866] Its structural characterization data are as follows:

[0867] ESI-MS (m / z): 1160.3 (M+H) + .

[0868] Step 3 2,2',2”-(10-((2S,5S,12S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro- Preparation of 3H-cyclopentadien[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-2,5-dimethyl-12-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamino)-1,4,7,11,18-pentaoxo-3,6,10,17-tetraazanonadecan-19-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-1)

[0869] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-3-((2S,5S,12S)-12-(4-aminobutyl)-2,5-dimethyl-19-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,11,14-tetraoxo-3,6,10,13 -Tetraazanonadecano-18-acetylamido-4-fluorobenzoate (36.00 mg, 31.05 μmol), 2,2',2”-(10-(2-((2,5-dioxocyclopentyl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (31.08 mg, 62.11 μmol), and DIPEA (16.05 mg, 124.22 μmol) were added, and the mixture was stirred at 25 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (33.26 mg, 21.52 μmol).

[0870] Its structural characterization data are as follows:

[0871] ESI-MS (m / z): 1546.6 (M+H) + .

[0872] The purification method is as follows:

[0873] Column: Agilent Prep C18 OBD 19*150mm*5μm

[0874] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[0875] Example 2: (2S,5S,12S)-23,23-bis((2-carboxyethoxy)methyl)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14, Preparation of 15,16,17-dodecano-3H-cyclopentadien[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-2,5-dimethyl-12-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)-1,4,7,11,18-pentoxo-21,25-dioxa-3,6,10,17-tetraazaoctacosane-28-acid (A-2)

[0876] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((2S,5S,12S)-12-(4-aminobutyl)-2,5-dimethyl-19-(2-(methylsulfonyl)pyrimidin-5-yl) 4,7,11,14-tetraoxo-3,6,10,13-tetraazanonadecan-18-acetylamido)-4-fluorobenzoate (20.00 mg, 17.25 μmol), 3,3'-((2,2-bis((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxo))dipropionic acid (7.32 mg, 17.25 μmol), DIPEA (11.15 mg, 86.26 μmol), and HATU (13.12 mg, 34.50 μmol) were added, and the mixture was stirred at 25 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (5.98 mg, 3.67 μmol).

[0877] The purification method is as follows:

[0878] Column: Agilent Prep C18 OBD 19*150mm*5μm

[0879] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[0880] Its structural characterization data are as follows:

[0881] ESI-MS (m / z): 1566.6 (M+H) + .

[0882] Example 3: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((2S,5S,12S)-23,23-bis((3-((1, Preparation of 3-dihydroxy-2-(hydroxymethyl)propane-2-yl)amino)-3-oxopropoxy)methyl)-31-hydroxy-30,30-bis(hydroxymethyl)-2,5-dimethyl-12-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-4,7,11,18,28-pentoxo-21,25-dioxa-3,6,10,17,29-pentazatriacontivacanamide)-4-fluorobenzoate (A-3).

[0883] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-3-((2S,5S,12S)-12-(4-aminobutyl)-2,5-dimethyl-19-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,11,14-tetraoxo-3,6,10,13 -Tetraazanonadecano-18-acetylamido-4-fluorobenzoate (11.06 mg, 9.54 μmol), 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-(((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (7.00 mg, 9.54 μmol), DIPEA (3.72 mg, 28.62 μmol), HATU (5.44 mg, 14.31 μmol) were added, and the mixture was stirred at 25 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then freeze-dried to obtain the title compound (4.05 mg, 2.05 μmol).

[0884] The purification method is as follows:

[0885] Column: Agilent Prep C18 OBD 19*150mm*5μm

[0886] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[0887] Its structural characterization data are as follows:

[0888] ESI-MS (m / z): 1876.2 (M+H) + .

[0889] Example 4: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-4-fluoro-3-((2S,5S,12S,31S,32R,33R,34R)-31,32,33, Preparation of 34,35-pentahydroxy-2,5,29-trimethyl-23,23-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-12-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-4,7,11,18,28-pentoxo-21,25-dioxa-3,6,10,17,29-pentazapentacarbonamide)benzoate (A-4).

[0890] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentan[a]phenanthrene-17-yl-3-((2S,5S,12S)-12-(4-aminobutyl)-2,5-dimethyl-19-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,11,14-tetraoxo-3,6,10,13-tetraazanonadecan-18-yne sequentially to DMF (2 mL). The following compounds were added: 4-fluorobenzoate (12.00 mg, 10.35 μmol), 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (9.90 mg, 10.35 μmol), DIPEA (6.69 mg, 51.76 μmol), and HATU (7.87 mg, 20.71 μmol). The mixture was stirred at 25 °C for 2 hours. The reaction solution was directly purified by high-performance liquid chromatography and then lyophilized to obtain the title compound (12.60 mg, 5.71 μmol).

[0891] The purification method is as follows:

[0892] Column: Agilent Prep C18 OBD 19*150mm*5μm

[0893] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[0894] Its structural characterization data are as follows:

[0895] ESI-MS (m / z): 1049.2 (M / 2+1) + .

[0896] Example 5: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-3-((48S,55S,58S)-55,58-dimethyl-48-(6-(2-(methyl)) Preparation of sulfonyl(pyrimidin-5-yl)hex-5-yneamide)-15,22,38,42,49,53,56-heptaoxo-20,20-bis(15-oxo-2,5,8,11,18-pentaoxa-14-azanonadecan-19-yl)-2,5,8,11,18,25,28,31,34-nonaoxa-14,21,37,43,50,54,57-heptaazanonadecan-59-amide)-4-fluorobenzoate (A-5).

[0897] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-3-((2S,5S,12S)-12-(4-aminobutyl)-2,5-dimethyl-19-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,11,14-tetraoxo-3,6,10,13 -tetraazanonadecano-18-acetylamido-4-fluorobenzoate (16.38 mg, 14.13 μmol), 2,5-dioxocyclopentyl-15,22,38-trioxo-20,20-bis(15-oxo-2,5,8,11,18-pentaoxa-14-azanonadecano-19-yl)-2,5,8,11,18,25,28,31,34-nonaoxa-14,21,37-triazatetracosano-42-ester (28.88 mg, 21.19 μmol), and DIPEA (7.30 mg, 56.51 μmol) were added, and the reaction mixture was stirred at 25 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (16.20 mg, 6.39 μmol).

[0898] The purification method is as follows:

[0899] Column: Agilent Prep C18 OBD 19*150mm*5μm

[0900] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[0901] Its structural characterization data are as follows:

[0902] ESI-MS (m / z): 1204.2 (M / 2+1) + .

[0903] Example 6: S-(fluoromethyl)(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-(((9S,12S,15S)-9,12-dimethyl-15-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-8,11,14,21,25,41,48-heptaoxo-43,43-bis(15-oxo-2,5,8,11,18-pentaoxa-14-azanonadecan-19-yl)-2,5,29,32,35,38,4 Preparation of 5,52,55,58,61-Undecano-7,10,13,20,26,42,49-Heptaazahexadecanoyl)amino)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecano-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxacyclopentene-8b-thioester (A-6)

[0904] Step 1: Preparation of (9H-fluorene-9-yl)methyl(S)-(1-(((2-hydroxyethoxy)methyl)amino)-1-oxopropane-2-yl)carbamate (A-6-2)

[0905] Ethylene glycol (3.9 g, 62.76 mmol) was dissolved in a mixed solvent of tetrahydrofuran (10 mL), followed by (S)-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)propionamido)methyl ester (4.0 g, 10.46 mmol) and p-toluenesulfonic acid monohydrate (298.1 mg, 1.57 mmol) in sequence, and stirred at 35 °C for 3 hours. The crude product was concentrated under reduced pressure, purified by C18 column chromatography (0.05% formic acid aqueous solution-acetonitrile = 0%-95%), and freeze-dried to give the title compound (3.4 g, 8.84 mmol).

[0906] Its structural characterization data are as follows:

[0907] ESI-MS (m / z): 385.2 (M+H) + .

[0908] Step 2: Preparation of (9H-fluorene-9-yl)methyl(S)-(1-(((2-(((4-nitrophenoxy)carbonyl)oxy)ethoxy)methyl)amino)-1-oxopropane-2-yl)carbamate (A-6-3)

[0909] In a mixture of (9H-fluorene-9-yl)methyl(S)-(1-(((2-hydroxyethoxy)methyl)amino)-1-oxopropane-2-yl)carbamate (3.4 g, 8.84 mmol / L) and DMF (15 mL), DIPEA (2.29 g, 17.69 mmol) and bis(4-nitrophenyl) carbonate (3.5 g, 11.50 mmol) were added. After the addition was complete, the mixture was stirred at 25 °C for 2 hours. The reaction solution was purified by C18 column chromatography (0.05% formic acid aqueous solution-acetonitrile = 0%-95%) and lyophilized to give the title compound (4.06 g, 7.39 mmol).

[0910] Its structural characterization data are as follows:

[0911] ESI-MS (m / z): 567.2 (M+H2O) + .

[0912] Step 3: Preparation of S-(fluoromethyl)(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-(((S)-1-(9H-fluorene-9-yl)-5-methyl-3,6-dioxo-2,9,12-trioxa-4,7-diazatridecan-13-acyl)amino)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxacyclopentene-8b-thioester (A-6-4)

[0913] (9H-fluorene-9-yl)methyl(S)-(1-(((2-(((4-nitrophenoxy)carbonyl)oxy)ethoxy)methyl)amino)-1-oxopropane-2-yl)carbamate (434.7 mg, 0.79 mmol l), S-(fluoromethyl)(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-aminobenzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH -Naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxane-8b-thioester (460 mg, 0.72 mmol) was dissolved in DMF (8 mL), and 2,6-dimethylpyridine (385.2 mg, 3.6 mmol) and HOBT (194.3 mg, 1.50 mmol) were added. After the addition was complete, the mixture was heated to 45 °C and stirred for 16 hours. The reaction solution was purified by C18 column chromatography (0.05% formic acid aqueous solution-acetonitrile = 0%-95%) and lyophilized to give the title compound (450 mg, 0.43 mmol).

[0914] Its structural characterization data are as follows:

[0915] ESI-MS (m / z): 1050.3 (M+H) + .

[0916] Step 4: Preparation of S-(fluoromethyl)(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-(((2-((((S)-2-aminopropamido)methoxy)ethoxy)carbonyl)amino)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxacyclopentene-8b-thioester (A-6-5)

[0917] S-(fluoromethyl)(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-(((S)-1-(9H-fluoren-9-yl)-5-methyl-3,6-dioxo-2,9,12-trioxa-4,7-diazatridecan-13-acyl)amino)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a- Dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxacyclopentene-8b-thioester (450 mg, 0.43 mmol) was dissolved in DMF (3 mL), and diethylamine (0.3 mL) was added. After the addition was complete, the mixture was stirred at 25 °C for 0.5 h. The solvent was removed by lyophilization, and 50 mL of petroleum ether / ethyl acetate = 20% was added for recrystallization for 1 h. The mixture was then filtered and dried to give the title compound (350 mg, 0.42 mmol).

[0918] Its structural characterization data are as follows:

[0919] ESI-MS (m / z): 828.1 (M+H) + .

[0920] Step 5: S-(fluoromethyl)(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-(((5S,8S)-1-(9H-fluorene-9-yl)-5,8-dimethyl-3,6,9-trioxo-2,12,15-trioxa-4,7,10-triazahexadecane-16- Preparation of acyl)amino)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxanepentene-8b-thioester (A-6-6)

[0921] S-(fluoromethyl)(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-(((2-(((S)-2-aminopropamido)methoxy)ethoxy)carbonyl)amino)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxane-8b-thioester (175.1 mg, 0.21 mmol) (l), (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanine (79 mg, 0.25 mmol) was dissolved in DMF (2 mL), and HATU (104.4 mg, 0.27 mmol) and DIPEA (82.3 mg, 0.63 mmol) were added. After the addition was complete, the mixture was stirred at 25 °C for 2 hours. The reaction solution was purified by C18 column chromatography (0.05% formic acid aqueous solution-acetonitrile = 0%-95%) and lyophilized to give the title compound (170 mg, 0.15 mmol).

[0922] Its structural characterization data are as follows:

[0923] ESI-MS (m / z): 1121.3 (M+H) + .

[0924] Step Six: Preparation of S-(fluoromethyl)(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-(((9S,12S)-12-amino-9-methyl-8,11-dioxo-2,5-dioxa-7,10-diazatridecyl)amino)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxacyclopentene-8b-thioester (A-6-7)

[0925] S-(fluoromethyl)(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-(((5S,8S)-1-(9H-fluorene-9-yl)-5,8-dimethyl-3,6,9-trioxo-2,12,15-trioxa-4,7,10-triazahexadecane-16-acyl)amino)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6 α,8a-Dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxacyclopentene-8b-thioester (170 mg, 0.15 mmol) was dissolved in DMF (1.5 mL), and diethylamine (0.15 mL) was added. After the addition was complete, the mixture was stirred at 25 °C for 0.5 h. The solvent was removed by lyophilization, and 20 mL (petroleum ether / ethyl acetate = 20%) was added for recrystallization for 1 h. The mixture was then filtered and dried to give the title compound (121.3 mg, 0.13 mmol).

[0926] Its structural characterization data are as follows:

[0927] ESI-MS (m / z): 899.3 (M+H) + .

[0928] Step 7: S-(fluoromethyl)(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-(((9S,12S,15S)-15-(4-((diphenyl(m-tolyl)methyl)amino)butyl)-9,12-dimethyl-22-(2-(methylsulfonyl)pyrimidin-5-yl)-8,11,14,17-tetraoxo-2,5-dioxane Preparation of -7,10,13,16-tetraaza-22-carbon-21-acetylacetyl)amino)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxacyclopentene-8b-thioester (A-6-8)

[0929] S-(fluoromethyl)(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-(((9S,12S)-12-amino-9-methyl-8,11-dioxo-2,5-dioxa-7,10-diazatridecoyl)amino)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecano-8bH-naphtho[2 [1,1:4,5]Indo[1,2-d][1,3]dioxacyclopenten-8b-thioester (121.3 mg, 0.13 mmol), N6-(diphenyl(m-tolyl)methyl)-N2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-lysine (96 mg, 0.15 mmol) were dissolved in DMF (2 mL), and HATU (74.1 mg, 0.2 mmol) and DIPEA (57.7 mg, 0.4 mmol) were added. After the addition was complete, the mixture was stirred at 25 °C for 2 hours. The reaction solution was purified by C18 column chromatography (0.05% formic acid aqueous solution-acetonitrile = 0%-95%) and lyophilized to give the title compound (92 mg, 0.06 mmol).

[0930] Its structural characterization data are as follows:

[0931] ESI-MS (m / z): 1533.3 (M+H) + .

[0932] Step 8: S-(fluoromethyl)(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-(((9S,12S,15S)-15-(4-aminobutyl)-9,12-dimethyl-22-(2-(methanesulfonyl)pyrimidin-5-yl)-8,11,14,17-tetraoxo-2,5-dioxa-7,10,1 Preparation of 3,16-tetraazacos-21-ynyl)amino)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxacyclopentene-8b-thioester (A-6-9)

[0933] S-(fluoromethyl)(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-(((9S,12S,15S)-15-(4-((diphenyl(m-tolyl)methyl)amino)butyl)-9,12-dimethyl-22-(2-(methylsulfonyl)pyrimidin-5-yl)-8,11,14,17-tetraoxo-2,5-dioxa-7,10,13,16-tetraaza-tetracoco-21-ynyl) (92 mg, 0.06 mmol)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxacyclopentene-8b-thioester (3 mL) was dissolved in DCM, and formic acid (1.5 mL) was added. After the addition was complete, the mixture was heated to 50 °C and stirred for 6 hours. The crude product was concentrated under reduced pressure, purified by C18 column chromatography (0.05% formic acid aqueous solution-acetonitrile = 0%-95%), and freeze-dried to give the title compound (61 mg, 0.05 mmol).

[0934] Its structural characterization data are as follows:

[0935] ESI-MS (m / z): 1277.3 (M+H) + .

[0936] Step Nine: S-(fluoromethyl)(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-(((9S,12S,15S)-9,12-dimethyl-15-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-8,11,14,21,25,41,48-heptaoxo-43,43-bis(15-oxo-2,5,8,11,18-pentaoxa-14-azanonadecan-19-yl)-2,5,29,32,35,38,4 Preparation of 5,52,55,58,61-Undecano-7,10,13,20,26,42,49-Heptaazahexadecanoyl)amino)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecano-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxacyclopentene-8b-thioester (A-6)

[0937] S-(fluoromethyl)(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-(((9S,12S,15S)-15-(4-aminobutyl)-9,12-dimethyl-22-(2-(methylsulfonyl)pyrimidin-5-yl)-8,11,14,17-tetraoxo-2,5-dioxa-7,10,13,16-tetraazacos-21-ynyl)amino)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2 [4,5]Indo[1,2-d][1,3]dioxacyclopenten-8b-thioester (22 mg, 0.017 mmol) was dissolved in DMF (1.5 mL), and DIPEA (6.7 mg, 51.7 μmol) and 2,5-dioxopyrrolidine-1-yl15,22,38-trioxo-20,20-bis(15-oxo-2,5,8,11,18-pentaoxa-14-aza-napto-19-yl)-2,5,8,11,18,25,28,31,34-nonaoxa-14,21,37-triazatetraconcanane-42-oate (35.3 mg, 25.7 μmol) were added. After the addition was complete, the mixture was stirred at 25 °C for 1 hour. The reaction solution was directly purified by high performance liquid chromatography and then freeze-dried to obtain the title compound (10.97 mg, 4.32 μmol).

[0938] The purification method is as follows:

[0939] Column: Agilent Prep C18 OBD 19*150mm*5μm

[0940] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[0941] Its structural characterization data are as follows:

[0942] ESI-MS (m / z): 1263.1 (M+H) + / 2.

[0943] Example 7: Preparation of (S)-5-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-4-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamino)acetamido)-5-oxopentanoic acid (E-1)

[0944] Step 1: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentanyl[a]phenanthrene-17-yl 3-((S)-5-(tert-butoxy)-2-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)acetamide)-5-oxopentanamide)-4-fluorobenzoate (E-1-1)

[0945] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-3-(((S)-2-(2-aminoacetamide)-5-(tert-butoxy)-5-oxopentanamide)-4-fluorobenzoate (67) to DMF (2 mL) in sequence. (mg, 82.73μmol), (2,5-dioxopyrrolidone-1-yl)6-(2-methylsulfonylpyrimidin-5-yl)hex-5-acetylacetate (33.25mg, 91.00μmol), and DIPEA (21.38mg, 165.46μmol) were added. After the addition was complete, the mixture was stirred at 15℃ for 1h. Purified water (10mL) and ethyl acetate (6mL*2) were added to the reaction solution for extraction. The mixture was separated, dried over anhydrous sodium sulfate (3g), filtered, and the filtrate was concentrated under reduced pressure and then subjected to column chromatography (SiO2, PE / EA = 100 / 1). -1 / 4) Purification. (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadienyl[a]phenanthrene-17-yl-3-(((S)-5-(tert-butoxy)-2-(2-(6-(2-((methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)acetamido)-5-oxopentanylamido)-4-fluorobenzoate (64 mg, 54.33 μmol).

[0946] Step 2: Preparation of (S)-5-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-4-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)acetamido)-5-oxopentanoic acid (E-1)

[0947] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-3-((( S)-5-(tert-butoxy)-2-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)acetamido)-5-oxopentanoylamido)-4-fluorobenzoate (64 mg, 54.33 μmol) and TFA (1 mL) were added, and the mixture was reacted at 16 °C for 1.5 h. The reaction solution was purified by high performance liquid chromatography to obtain the title compound (42.00 mg, 41.41 μmol).

[0948] The purification method is as follows:

[0949] Column: SunFire Prep C18 OBD 19*150mm*5μm

[0950] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% TFA)

[0951] Its structural characterization data are as follows:

[0952] ESI-MS (m / z): 1004.3 (M+H) + .

[0953] Example 8: (2S,5S,8S)-19,19-bis((2-carboxyethoxy)methyl)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,1 Preparation of 3,14,15,16,17-dodecanehydro-3H-cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-2,5-dimethyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-1,4,7,14-tetraoxo-17,21-dioxa-3,6,13-triazatetracosane-24-acid (N-1)

[0954] Step 1: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl 3-((S)-2-((S)-2-((S)-6-((diphenyl(p-tolyl)methyl)amino)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)hexamide)propionamide)propionamide)-4-fluorobenzoate (N-1-1)

[0955] (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-3-((S)-2-((S)-2-aminopropionylamino)propionylamino)-4-fluorobenzoate (3 50 mg (493.13 μmol) and N6-(diphenyl(p-tolyl)methyl)-N2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-lysine (354.11 mg, 542.45 μmol) were dissolved in DMF (10 mL), followed by the addition of HATU (375.02 mg, 986.27 μmol) and DIPEA (318.66 mg, 2.47 mmol). The mixture was stirred at 25 °C for 1 hour. The solution was purified by rapid column chromatography (ACN / H2O = 0.65%, 0.05% formic acid) and lyophilized to give the title compound (420 mg, 249.90 μmol, purity 80%).

[0956] Its structural characterization data are as follows:

[0957] ESI-MS (m / z): 1344.5 (M+H) + .

[0958] Step 2: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl 3-((S)-2-((S)-2-((S)-6-amino-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)hexamide)propionamide)propionamide)-4-fluorobenzoate (N-1-2)

[0959] (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl 3-((S) 2-((S)-2-((S)-6-((diphenyl(p-tolyl)methyl)amino)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)hexamide)propionamide)propionamide)-4-fluorobenzoate (220 mg, 130.90 μmol, purity 80%) was added to a mixed solvent of DCM (4 mL) and TFA (4 mL). The mixture was stirred at 25 °C for 1 hour. The crude product was concentrated, purified by high performance liquid chromatography, and lyophilized to obtain the title compound (90 mg, 74.86 μmol).

[0960] The purification method is as follows:

[0961] Column: Agilent Prep C18 OBD 19*150mm*5μm

[0962] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% TFA)

[0963] Its structural characterization data are as follows:

[0964] ESI-MS (m / z): 1088.3 (M+H) + .

[0965] Step 3: (2S,5S,8S)-19,19-bis((2-carboxyethoxy)methyl)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,1 Preparation of 3,14,15,16,17-dodecanehydro-3H-cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-2,5-dimethyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-1,4,7,14-tetraoxo-17,21-dioxa-3,6,13-triazatetracosane-24-acid (N-1)

[0966] (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl 3-((S)-2-((S)-2-((S)-6-amino-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5 4-fluorobenzoate (45 mg, 37.43 μmol) and 3,3'-((2,2-bis((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (15.89 mg, 37.43 μmol) were dissolved in DMF (2 mL), followed by the addition of HATU (14.23 mg, 37.43 μmol) and DIPEA (24.19 mg, 187.15 μmol). The mixture was stirred at 25 °C for 1.5 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (12 mg, 7.95 μmol).

[0967] The purification method is as follows:

[0968] Column: Agilent Prep C18 OBD 19*150mm*5μm

[0969] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% TFA)

[0970] Its structural characterization data are as follows:

[0971] ESI-MS (m / z): 1494.4 (M+H) + .

[0972] Example 9: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((2S,5S,8S)-19,19-bis((3- Preparation of ((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)-27-hydroxy-26,26-bis(hydroxymethyl)-2,5-dimethyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-4,7,14,24-tetraoxo-17,21-dioxa-3,6,13,25-tetraazaheptacapamide)-4-fluorobenzoate (N-2)

[0973] (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadienyl[a]phenanthrene-17-yl-3-((S)-2-((S)-2-((S)-6-amino-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)hexamide)propionamide)propionamide)-4-fluorobenzoate (20 20.34 mg (16.64 μmol) and 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-(((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (20.34 mg, 24.95 μmol, purity 90%) were dissolved in DMF (3 mL), followed by the addition of HATU (12.65 mg, 33.27 μmol) and DIPEA (10.75 mg, 83.18 μmol). The mixture was stirred at 25 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (11 mg, 6.04 μmol).

[0974] The purification method is as follows:

[0975] Column: Agilent Prep C18 OBD 19*150mm*5μm

[0976] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[0977] Its structural characterization data are as follows:

[0978] ESI-MS (m / z): 1803.6 (M+H) + .

[0979] Example 10: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-4-fluoro-3-((2S,5S,8S,27S,28R,29R,30R)-27,28, Preparation of 29,30,31-pentahydroxy-2,5,25-trimethyl-19,19-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-4,7,14,24-tetraoxo-17,21-dioxa-3,6,13,25-tetraazatriacontivacanamide)benzoate (N-3)

[0980] (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadienyl[a]phenanthrene-17-yl-3-((S)-2-((S)-2-((S)-6-amino-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)hexamide)propionamide)propionamide)-4-fluorobenzoate (15 mg, 12.48) 14.31 mg (14.97 μmol) and 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (14.97 μmol) were dissolved in DMF (3 mL), and then HATU (9.49 mg, 24.95 μmol) and DIPEA (8.06 mg, 62.38 μmol) were added. The mixture was stirred at 25 °C for 1.5 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (19 mg, 9.28 μmol).

[0981] The purification method is as follows:

[0982] Column: Agilent Prep C18 OBD 19*150mm*5μm

[0983] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[0984] Its structural characterization data are as follows:

[0985] ESI-MS (m / z): 1013.6 (0.5M+H) + .

[0986] Example 11: S-(fluoromethyl)(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-10-(4-(3-(((9S,12S,15S,34S,35R,36R,37R)-34,35,36,37,38-pentahydroxy-9,12,32-trimethyl-26,26-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-1 Preparation of 5-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide)-8,11,14,21,31-pentoxa-2,5,24,28-tetraoxa-7,10,13,20,32-pentazaoctadecanoyl)amino)benzyl)phenyl)-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxacyclopentene-8b-thiocarbamate (N-4)

[0987] S-(fluoromethyl)(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-(((9S,12S,15S)-15-(4-aminobutyl)-9,12-dimethyl-22-(2-(methanesulfonyl)pyrimidin-5-yl)-8,11,14,17-tetraoxo-2,5-dioxa-7,10,13,16-tetraazacos-21-ynyl)amino)benzyl)phenyl)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2',1':4, 5] Indo[1,2-d][1,3]dioxacyclopentene-8b-thioester (25 mg, 0.02 mmol), 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (37.4 mg, 0.04 mmol) were dissolved in DMF (1.5 mL), HATU (11.2 mg, 0.03 mmol) and DIPEA (8.7 mg, 0.06 mmol) were added. After the addition was complete, the mixture was stirred at 25 °C for 1 hour. The reaction solution was directly purified by high performance liquid chromatography and then freeze-dried to obtain the title compound (10.76 mg, 4.62 μmol).

[0988] The purification method is as follows:

[0989] Column: Agilent Prep C18 OBD 19*150mm*5μm

[0990] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[0991] Its structural characterization data are as follows:

[0992] ESI-MS (m / z): 1256.4 (M+H) + .

[0993] Example 12: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentadien[a]phenanthrene-17-yl-3-((S)-6-amino-2-((S)-3-hydroxy-2-(2-(2-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamino)propionylamino)-4-fluorobenzoate (S-1)

[0994] Step 1: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((S)-6-((tert-butyloxycarbonyl)amino)-2-((S)-3-hydroxy-2-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)acetamido)propionamide)hexamide)-4-fluorobenzoate (S-1-1)

[0995] The (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((S)-2-((S)-2-(2-aminoacetamido)-3-hydroxypropyl Formate of (-amido)-6-((tert-butoxycarbonyl)amino)hexamido)-4-fluorobenzoate (30.0 mg, 0.030 mmol), (2,5-dioxopyrrolidone-1-yl)6-(2-methylsulfonylpyrimidin-5-yl)hex-5-acetylacetate (11.7 mg, 0.032 mmol), and DIPEA (11.8 mg, 0.091 mmol) were added to DMF (0.5 mL) and reacted at 25 °C for 2 hours. The reaction solution was directly purified by Pre-HPLC and then lyophilized to obtain S-1-1 (22.0 mg).

[0996] The purification method is as follows:

[0997] Column: SunFire Prep C18 OBD 19*150mm*5μm

[0998] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0999] Retention time: 7.00-9.00 min.

[1000] Its structural characterization data are as follows:

[1001] ESI-MS (m / z): 1190.5 (M+H) + .

[1002] Step 2: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-3-((S)-6-amino-2-((S)-3-hydroxy-2-(2-(2-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamino)propionylamino)-4-fluorobenzoate (S-1)

[1003] (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl3-((S)-6-((tert-)) Butyloxycarbonyl)amino)-2-((S)-3-hydroxy-2-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)acetamido)propionamide)hexamide)-4-fluorobenzoate (22.0 mg, 0.018 mmol) was dissolved in DCM (1 mL), and TFA (105.4 mg, 0.924 mmol) was added. The reaction was carried out at 25 °C for 1 hour. The reaction solution was directly concentrated to obtain the crude product, which was purified by Pre-HPLC to obtain trifluoroacetate of S-1 (14.6 mg).

[1004] The purification method is as follows:

[1005] Column: SunFire Prep C18 OBD 19*150mm*5μm

[1006] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1007] Retention time: 10.50-12.50 min.

[1008] Its structural characterization data are as follows:

[1009] ESI-MS (m / z): 1090.4 (M+H) + .

[1010] Example 13: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((S)-9,9-bis((3-((1,3-dihydroxy) Preparation of 2-(hydroxymethyl)prop-2-yl)amino)-3-oxopropoxy)methyl)-1-hydroxy-20-((S)-3-hydroxy-2-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)acetamide)propionamide)-2,2-bis(hydroxymethyl)-4,14-dioxo-7,11-dioxa-3,15-diazaeicosano-21-amide)-4-fluorobenzoate (S-3)

[1011] (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadienyl[a]phenanthrene-17-yl 3-((S)-6-amino-2-((S)-3-hydroxy-2-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)acetamide)propionamide)hexamide)-4-fluorobenzoate (15.0 mg, 0.012 mmol, trifluoroacetate) was dissolved in D In MF (1 mL), 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (13.6 mg, 0.018 mmol) and DIPEA (5.0 mg, 0.038 mmol) were added and stirred for 1 minute. Then, HATU (14.6 mg, 0.038 mmol) was added, and the mixture was reacted at 25 °C for 1 hour. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (7.51 mg, 0.004 mmol).

[1012] The purification method is as follows:

[1013] Column: Agilent Waters XBridge Prep C18 OBD (19*150mm*5μm)

[1014] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1015] Its structural characterization data are as follows:

[1016] ESI-MS (m / z): 1805.7 (M+H) +

[1017] Example 14: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-4-fluoro-3-((2S,21S,22R,23R,24R)-21,22,23,24,2 Preparation of 5-pentahydroxy-2-((S)-3-hydroxy-2-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)acetamide)propionamide)-19-methyl-13,13-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8,18-dioxo-11,15-dioxa-7,19-diazapentacarbamide)benzoate (S-4)

[1018] Dissolve (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl 3-((S)-6-amino-2-((S)-3-hydroxy-2-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)acetamide)propionamide)hexamide)-4-fluorobenzoate (15.0 mg, 0.012 mmol, trifluoroacetate) in DMF (1 mL), add 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (17.7 mg, 0.018 mmol) and DIPEA (5.0 mg, 0.038 mmol) were stirred for 1 minute, and then HATU (14.6 mg, 0.038 mmol) was added. The mixture was reacted at 25 °C for 1 hour. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (5.82 mg, 0.003 mmol).

[1019] The purification method is as follows:

[1020] Column: Agilent Waters SunFire Prep C18 OBD (19*150mm*5μm)

[1021] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1022] Its structural characterization data are as follows:

[1023] ESI-MS (m / z): 1014.5 (M / 2+H) +

[1024] Example 15: 2,2',2”-(10-((2S,5S,12S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3 Preparation of H-cyclopentadieno[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-12-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamino)-1,4,7,11,18-pentoxa-3,6,10,17-tetraazanonadecan-19-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (V-1)

[1025] Step 1: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-3-((S)-2-((S)-2-amino-3-methylbutamido)propamido)-4-fluorobenzoate (V-1-1)

[1026] (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl3-((S)- 2-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-methylbutyramide)propionamide)-4-fluorobenzoate (400 mg, 416.65 μmol) was dissolved in DMF (8 mL), and 1,8-diazacyclo[5,4,0]undecene-7 (190.29 mg, 1.25 mmol) was added. The mixture was stirred at 25 °C for 20 min. After purification by rapid column chromatography (ACN / H2O = 0-65%, 0.05% trifluoroacetic acid), the compound was lyophilized to give the title compound (334 mg, 388.18 μmol).

[1027] Its structural characterization data are as follows:

[1028] ESI-MS (m / z): 738.3 (M+H) + .

[1029] Step 2: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl Preparation of 3-((2S,5S,12S)-12-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-5-isopropyl-2-methyl-19-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,11,14-tetraoxo-3,6,10,13-tetraazanonadecan-18-acetylamidamide)-4-fluorobenzoate (V-1-2)

[1030] (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-3-((S)-2-((S)-2-amino-3-methylbutamido)propamido)-4-fluorobenzoate (90 mg, 121.98 μmol) and N6-(diphenyl(p-tolyl)methyl)-N2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-lysine (97.13 mg, 134.18 μmol) were dissolved in DMF (4 mL), followed by the addition of DMTMM (31.96 mg, 243.97 μmol) and DIPEA (78.83 mg, 609.92 μmol). The mixture was stirred at 25 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (95 mg, 65.80 μmol).

[1031] The purification method is as follows:

[1032] Column: Agilent Prep C18 OBD 19*150mm*5μm

[1033] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[1034] Its structural characterization data are as follows:

[1035] ESI-MS (m / z): 1443.5 (M+H) + .

[1036] Step 3: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-3-((2S,5S,12S)-12-(4-aminobutyl)-5-isopropyl-2-methyl-19-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,11,14-tetraoxo-3,6,10,13-tetraazanonadecan-18-acetylamidamide)-4-fluorobenzoate (V-1-3)

[1037] The (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-3-((2S,5S,12S)-12 -(4-((diphenyl(p-tolyl)methyl)amino)butyl)-5-isopropyl-2-methyl-19-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,11,14-tetraoxo-3,6,10,13-tetraazanonadecan-18-acetylamidamide)-4-fluorobenzoate (77 mg, 53.34 μmol) was added to a mixed solvent of DCM (2 mL) and TFA (2 mL). The reaction was stirred at 25 °C for 1.5 h. The crude product was concentrated, purified by high performance liquid chromatography, and lyophilized to give the title compound (60 mg, 46.11 μmol).

[1038] The purification method is as follows:

[1039] Column: Agilent Prep C18 OBD 19*150mm*5μm

[1040] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% TFA)

[1041] Its structural characterization data are as follows:

[1042] ESI-MS (m / z): 1187.4 (M+H) + .

[1043] Step 4: 2,2',2”-(10-((2S,5S,12S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H Preparation of cyclopentadieno[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-12-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamino)-1,4,7,11,18-pentoxa-3,6,10,17-tetraazanonadecan-19-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (V-1)

[1044] The (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentan[a]phenanthrene-17-yl-3-((2S,5S,12S)-12-(4-aminobutyl)-5-isopropyl-2-methyl-19-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,11,14-tetraoxo-3,6,10,13-tetraazanonadecane- 18-Acynylamide)-4-fluorobenzoate (38 mg, 30.81 μmol) and 2,2',2”-(10-(2-((2,5-dioxopyrrolidone-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (46.35 mg, 92.43 μmol) were dissolved in DMF (5 mL), and then DIPEA (39.82 mg, 308.10 μmol) was added. The mixture was stirred at 25 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (27 mg, 16.34 μmol).

[1045] The purification method is as follows:

[1046] Column: Agilent Prep C18 OBD 19*150mm*5μm

[1047] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% TFA)

[1048] Its structural characterization data are as follows:

[1049] ESI-MS (m / z): 1573.5 (M+H) + .

[1050] Example 16: (2S,5S,12S)-23,23-bis((2-carboxyethoxy)methyl)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,1 Preparation of 4,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-12-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-1,4,7,11,18-pentoxo-21,25-dioxa-3,6,10,17-tetraazaoctacosane-28-acid (V-2)

[1051] The (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-3-((2S,5S,12S)-12-(4-aminobutyl)-5-isopropyl-2-methyl-19-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7, 11,14-Tetraoxo-3,6,10,13-tetraazanonadecano-18-acetylamido-4-fluorobenzoate (35 mg, 26.90 μmol) and 3-[3-(2-carboxyethoxy)-2,2-bis(2-carboxyethoxymethyl)propoxy]propionic acid (11.41 mg, 26.90 μmol) were dissolved in DMF (3 mL), followed by the addition of HATU (10.23 mg, 26.90 μmol) and DIPEA (17.38 mg, 134.48 μmol). The mixture was stirred at 25 °C for 1.5 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (4 mg, 2.46 μmol).

[1052] The purification method is as follows:

[1053] Column: Agilent Prep C18 OBD 19*150mm*5μm

[1054] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% TFA)

[1055] Its structural characterization data are as follows:

[1056] ESI-MS (m / z): 1593.5 (M+H) + .

[1057] Example 17: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-3-((2S,5S,12S)-23,23-bis((3-((1, Preparation of 3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)-31-hydroxy-30,30-bis(hydroxymethyl)-5-isopropyl-2-methyl-12-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-4,7,11,18,28-pentaoxo-21,25-dioxa-3,6,10,17,29-pentazatriacontanamide)-4-fluorobenzoate (V-3)

[1058] (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-3-((2S,5S,12S)-12-(4-aminobutyl)-5-isopropyl-2-methyl-19-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,11,14-tetraoxo-3,6,10,13-tetraazanonadecan-18-yne 4-fluorobenzoate (12 mg, 9.22 μmol) and 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (11.28 mg, 13.83 μmol, 90% purity) were dissolved in DMF (3 mL), followed by the addition of HATU (7.01 mg, 18.44 μmol) and DIPEA (5.96 mg, 46.11 μmol). The mixture was stirred at 25 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (9 mg, 4.63 μmol).

[1059] The purification method is as follows:

[1060] Column: Agilent Prep C18 OBD 19*150mm*5μm

[1061] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[1062] Its structural characterization data are as follows:

[1063] ESI-MS (m / z): 1902.6 (M+H) + .

[1064] Example 18: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-4-fluoro-3-((2S,5S,12S,31S,32R,33R,34R)-31,32,33,34 Preparation of 3,5-pentahydroxy-5-isopropyl-2,29-dimethyl-23,23-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-12-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-4,7,11,18,28-pentoxo-21,25-dioxa-3,6,10,17,29-pentazatripentadecanoamide)benzoate (V-4)

[1065] (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-3-((2S,5S,12S)-12-(4-aminobutyl)-5-isopropyl-2-methyl-19-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,11,14-tetraoxo-3,6,10,13-tetraazanonadecan-18-acetylamidamide)-4-fluorobenzene Formate (12 mg, 9.22 μmol) and 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (10.58 mg, 11.07 μmol) were dissolved in DMF (3 mL), followed by the addition of HATU (7.01 mg, 18.44 μmol) and DIPEA (5.96 mg, 46.11 μmol). The mixture was stirred at 25 °C for 1.5 h. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (4 mg, 1.83 μmol).

[1066] The purification method is as follows:

[1067] Column: Agilent Prep C18 OBD 19*150mm*5μm

[1068] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[1069] Its structural characterization data are as follows:

[1070] ESI-MS (m / z): 1063.1 (0.5M+H) + .

[1071] Example 19: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-(2-((S)-6-amino-2-((S)-3-methyl-2-(6-(2-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)hexamide)acetamide)-4-fluorobenzoate (K-1)

[1072] Step 1: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentadien[a]phenanthrene-17-yl 3-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-4-fluorobenzoate (K-1-1)

[1073] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl- 3-Amino-4-fluorobenzoate (240 mg, 422.84 μmol), (((9H-fluorene-9-yl)methoxy)carbonyl)glycine (188.57 mg, 634.26 μmol), DIPEA (273.24 mg, 2.11 mmol), and HATU (321.53 mg, 845.68 μmol) were added, and the mixture was stirred at 20 °C for 16 hours. The reaction solution was purified by rapid column chromatography (C18, 0.05% formic acid aqueous solution / acetonitrile = 0%-80%) and then freeze-dried to give the title compound (200 mg, 188.93 μmol, 80% purity).

[1074] Its structural characterization data are as follows:

[1075] ESI-MS (m / z): 847.2 (M+H) + .

[1076] Step 2: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl 3-(2-aminoacetamide)-4-fluorobenzoate (K-1-2)

[1077] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl 3-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)-4-fluorobenzoate (200 mg, 188.93 μmol, 80% purity) and diethylamine (41.94 mg, 566.79 μmol) to DMF (5 mL). After the addition is complete, stir the mixture at 20 °C for 3 hours. The reaction solution was purified by rapid column chromatography (C18, 0.05% trifluoroacetic acid aqueous solution / acetonitrile = 0%-80%) and then freeze-dried to obtain the trifluoroacetate of the title compound (109 mg, 135.76 μmol).

[1078] Its structural characterization data are as follows:

[1079] ESI-MS (m / z): 625.2 (M+H) + .

[1080] Step 3: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-(2-((S)-6-((tert-butoxycarbonyl)amino)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)hexamide)acetamide)-4-fluorobenzoate (K-1-3)

[1081] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl 3-(2-aminoacetamide)-4-fluorobenzoate to DMF (3 mL). 109 mg (135.76 μmol), N6-(tert-butoxycarbonyl)-N2-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (105.49 mg (177.08 μmol), DIPEA (76.28 mg (590.25 μmol), and HATU (84.16 mg (221.35 μmol)) were added, and the mixture was stirred at 20 °C for 3 hours. The reaction mixture was then diluted with water (10 mL), extracted with ethyl acetate (15 mL * 3), washed with saturated brine, and dried to obtain the crude title compound (170 mg).

[1082] Its structural characterization data are as follows:

[1083] ESI-MS(m / z): 1203.4(M+H)+.

[1084] Step 4: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-(2-((S)-6-amino-2-((S)-3-methyl-2-(6-(2-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)hexamide)acetamide)-4-fluorobenzoate (K-1)

[1085] Add 170 mg of crude (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-(2-((S)-6-((tert-butoxycarbonyl)amino)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)hexamide)acetamide)-4-fluorobenzoate to DCM (5 mL) and TFA (1 mL). After the addition is complete, stir the mixture at 20 °C for 3 hours. The reaction solution was directly purified by high performance liquid chromatography and then freeze-dried to obtain the trifluoroacetate of the title compound (115 mg, 89.83 μmol).

[1086] The purification method is as follows:

[1087] Column: Agilent Prep C18 OBD 19*150mm*5μm

[1088] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% TFA)

[1089] Its structural characterization data are as follows:

[1090] ESI-MS (m / z): 1202.2 (M-100+H) + .

[1091] Example 20: (17S,20S)-6,6-bis((2-carboxyethoxy)methyl)-17-((2-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13 Preparation of 14,15,16,17-dodecanehydro-3H-cyclopentadieno[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-2-oxoethyl)carbamoyl)-20-isopropyl-27-(2-(methylsulfonyl)pyrimidin-5-yl)-11,19,22-trioxo-4,8-dioxa-12,18,21-triazahepta-26-acetylic acid (K-2)

[1092] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-(2-((S)-6-amino-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl))) to DMF (2 mL) in sequence. Trifluoroacetate of pyrimidin-5-ylhexyl-5-ynylamide (butyramide)hexylamide (acetamide)-4-fluorobenzoate (25 mg, 20.56 μmol), 3,3'-((2,2-bis((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (8.72 mg, 20.56 μmol), DIPEA (10.63 mg, 82.22 μmol), and HATU (7.82 mg, 20.56 μmol) were added, and the mixture was stirred at 20 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to give the title compound (3.92 mg, 2.47 μmol).

[1093] The purification method is as follows:

[1094] Column: Agilent Prep C18 OBD 19*150mm*5μm

[1095] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[1096] Its structural characterization data are as follows:

[1097] ESI-MS (m / z): 1509.5 (M+H) + .

[1098] Example 21: (17S,20S)-6,6-bis((2-carboxyethoxy)methyl)-17-((2-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13 Preparation of 14,15,16,17-dodecanehydro-3H-cyclopentadieno[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-2-oxoethyl)carbamoyl)-20-isopropyl-27-(2-(methylsulfonyl)pyrimidin-5-yl)-11,19,22-trioxo-4,8-dioxa-12,18,21-triazahepta-26-acetylic acid (K-3)

[1099] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-(2-((S)-6-amino-2-((S)-3-methyl-2-(6-(2-((methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)hexamide)acetamide)- The trifluoroacetate of 4-fluorobenzoate (25 mg, 20.56 μmol), 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-(((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (13.57 mg, 18.50 μmol), DIPEA (10.63 mg, 82.22 μmol), and HATU (7.82 mg, 20.56 μmol) were added, and the mixture was stirred at 20 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to give the title compound (7.52 mg, 3.93 μmol).

[1100] The purification method is as follows:

[1101] Column: Agilent Prep C18 OBD 19*150mm*5μm

[1102] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[1103] Its structural characterization data are as follows:

[1104] ESI-MS (m / z): 1819.6 (M+H) + .

[1105] Example 22: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-4-fluoro-3-((5S,24S,25R,26R,27R)-24,25,26,27, Preparation of 28-pentahydroxy-22-methyl-16,16-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-5-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)-4,11,21-trioxo-14,18-dioxa-3,10,22-triazaoctacosanamide)benzoate (K-4)

[1106] Add trifluoro(6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-(2-((S)-6-amino-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)hexamide)acetamide)-4-fluorobenzoate to DMF (2 mL) in sequence. Acetate (15 mg, 12.33 μmol), 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (14.15 mg, 14.80 μmol), DIPEA (4.78 mg, 37.00 μmol), and HATU (7.03 mg, 18.50 μmol) were added, and the mixture was stirred at 20 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (14.52 mg, 6.76 μmol).

[1107] The purification method is as follows:

[1108] Column: Agilent Prep C18 OBD 19*150mm*5μm

[1109] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[1110] Its structural characterization data are as follows:

[1111] ESI-MS (m / z): 1020.5 (1 / 2M+H) + .

[1112] Example 23: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((2S,5S,12S)-12-(2 Preparation of 1-bromoacetamyl)-23,23-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)-31-hydroxy-30,30-bis(hydroxymethyl)-2,5-dimethyl-4,7,11,18,28-pentoxo-21,25-dioxa-3,6,10,17,29-pentazatrionetamamido)-4-fluorobenzoate (B-3)

[1113] Step 1: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentan[a]phenanthrene-17-yl 3-((9S,12S)-1-(9H-fluorene-9-yl)-9,12-dimethyl-3,7,10-trioxo-2-oxa-4,8,11-triazatridecane-13-amide)-4-fluorobenzoate (B-3-1)

[1114] (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-3-((S)-2-((S)-2-aminopropionylamine) 3-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)propionic acid (987.00 mg, 3.17 mmol), HATU (1.61 g, 4.23 mmol), and DIPEA (820.00 mg, 6.34 mmol) were added sequentially, and the mixture was stirred at 25 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (1.35 g, 1.35 mmol).

[1115] Its structural characterization data are as follows:

[1116] ESI-MS (m / z): 1003.5 (M+H) + .

[1117] Step 2: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl 3-((S)-2-((S)-2-(3-aminopropionamide)propionamide)propionamide)-4-fluorobenzoate (B-3-2)

[1118] (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl3-((9S,12) S)-1-(9H-fluorene-9-yl)-9,12-dimethyl-3,7,10-trioxo-2-oxa-4,8,11-triazatridecane-13-amide)-4-fluorobenzoate (500.00 mg, 498.47 μmol) was dissolved in DMF (3 mL), and diethylamine (109.38 mg, 1.5 mmol) was added. The mixture was stirred at 25 °C for 2 hours. The organic phase was concentrated to obtain the crude title compound (315 mg), which was used directly in the next step without purification.

[1119] Step 3: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((5S,12S,15S)-5-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-1-(9H-fluorene-9-yl)-12,15-dimethyl-3,6,10,13-tetraoxo-2-oxa-4,7,11,14-tetraazahexadecane-16-amide)-4-fluorobenzoate (B-3-3)

[1120] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl 3-((S)-2-((S)-2-(3-amino) Crude 4-fluorobenzoate (315 mg), N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(diphenyl(p-tolyl)methyl)-L-lysine (378.06 mg, 605.13 μmol), DIPEA (156.41 mg, 1.21 mmol), and HATU (306.76 mg, 806.84 μmol) were added and the mixture was stirred at 20 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then freeze-dried to obtain the title compound (386.00 mg, 278.18 μmol).

[1121] The purification method is as follows:

[1122] Column: Agilent Prep C18 OBD 19*150mm*5μm

[1123] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[1124] Its structural characterization data are as follows:

[1125] ESI-MS (m / z): 1387.5 (M+H) + .

[1126] Step 4: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentan[a]phenanthrene-17-yl 3-((5S,12S,15S)-5-(4-aminobutyl)-1-(9H-fluorene-9-yl)-12,15-dimethyl-3,6,10,13-tetraoxo-2-oxa-4,7,11,14-tetraazahexadecane-16-amide)-4-fluorobenzoate (B-3-4)

[1127] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-3-((5S,12S) 15S)-5-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-1-(9H-fluorene-9-yl)-12,15-dimethyl-3,6,10,13-tetraoxo-2-oxa-4,7,11,14-tetraazahexadecane-16-amide)-4-fluorobenzoate (386.00 mg, 278.18 μmol), formic acid (2 mL), were added, and the mixture was stirred at 25 °C for 2 hours. The organic phase was concentrated to obtain the crude title compound (258 mg), which was used directly in the next step without purification.

[1128] Step 5: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((2S,5S,12S)-12-((((9H-fluorene-9- Preparation of (B-3-5)-23,23-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)-31-hydroxy-30,30-bis(hydroxymethyl)-2,5-dimethyl-4,7,11,18,28-pentoxo-21,25-dioxa-3,6,10,17,29-pentazatrionecanamide)-4-fluorobenzoate (B-3-5)

[1129] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-3-((5S,12S,15S)-5-(4-aminobutyl)-1-(9H-fluorene-9-yl)-12,15-dimethyl-3,6,10,13-tetraoxo-2-oxa-4,7,11, Crude 14-tetraazahexadecane-16-amide)-4-fluorobenzoate (60 mg), 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (51.89 mg, 63.65 μmol), DIPEA (16.18 mg, 159.12 μmol), and HATU (40.33 mg, 106.08 μmol) were added and the mixture was stirred at 20 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then freeze-dried to obtain the title compound (65.00 mg, 35.19 μmol).

[1130] The purification method is as follows:

[1131] Column: Agilent Prep C18 OBD 19*150mm*5μm

[1132] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[1133] Its structural characterization data are as follows:

[1134] ESI-MS (m / z): 1846.6 (M+H) + .

[1135] Step Six: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((2S,5S,12S)-12- Preparation of amino-23,23-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)-31-hydroxy-30,30-bis(hydroxymethyl)-2,5-dimethyl-4,7,11,18,28-pentoxo-21,25-dioxa-3,6,10,17,29-pentazatrionecanamide)-4-fluorobenzoate (B-3-6)

[1136] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl3-((2S,5S,12S)-12-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-23,23- Bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)-31-hydroxy-30,30-bis(hydroxymethyl)-2,5-dimethyl-4,7,11,18,28-pentoxo-21,25-dioxa-3,6,10,17,29-pentazahexamonadecanamide)-4-fluorobenzoate (45.00 mg, 26.39 μmol) and diethylamine (10.10 mg, 138.06 μmol) were added, and the mixture was stirred at 25 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to give the title compound (30.00 mg, 18.46 μmol).

[1137] The purification method is as follows:

[1138] Column: Agilent Prep C18 OBD 19*150mm*5μm

[1139] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% TFA)

[1140] Its structural characterization data are as follows:

[1141] ESI-MS (m / z): 1624.6 (M+H) +.

[1142] Step 7: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((2S,5S,12S)-12-(2- Preparation of bromoacetamido)-23,23-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)-31-hydroxy-30,30-bis(hydroxymethyl)-2,5-dimethyl-4,7,11,18,28-pentoxo-21,25-dioxa-3,6,10,17,29-pentazatrionetamamido)-4-fluorobenzoate (B-3)

[1143] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((2S,5S,12S)-12-amino-23,23-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3 (-Oxopropoxy)methyl)-31-hydroxy-30,30-bis(hydroxymethyl)-2,5-dimethyl-4,7,11,18,28-pentoxo-21,25-dioxa-3,6,10,17,29-pentazatetradecanoamide)-4-fluorobenzoate (30.00 mg, 18.46 μmol), 2,5-dioxopyrrolidone-1-yl-2-bromoacetate (26.15 mg, 110.79 μmol), DIPEA (16.18 mg, 159.12 μmol), were added, and the mixture was stirred at 20 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (18.40 mg, 10.07 μmol).

[1144] The purification method is as follows:

[1145] Column: Agilent Prep C18 OBD 19*150mm*5μm

[1146] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[1147] Its structural characterization data are as follows:

[1148] ESI-MS (m / z): 1745.6 (M+H) + .

[1149] Example 24: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((2S,5S,12S,31S,32R,33R,34R)-12-( Preparation of 2-bromoacetamyl)-31,32,33,34,35-pentahydroxy-2,5,29-trimethyl-23,23-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-4,7,11,18,28-pentoxo-21,25-dioxa-3,6,10,17,29-pentazapentadecanoamide)-4-fluorobenzoate (B-4)

[1150] Step 1: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((2S,5S,12S,31S,32R,33R,34R)-12-((((9H-fluorene-9 Preparation of 31,32,33,34,35-pentahydroxy-2,5,29-trimethyl-23,23-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-4,7,11,18,28-pentaoxo-21,25-dioxa-3,6,10,17,29-pentazatripentadecanoamide)-4-fluorobenzoate (B-4-1)

[1151] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentan[a]phenanthrene-17-yl-3-((5S,12S,15S)-5-(4-aminobutyl)-1-(9H-fluorene-9-yl)-12,15-dimethyl-3,6,10,13-tetraoxo-2-oxa-4,7,11,14-tetraazahexadecane-16-amide) sequentially to DMF (2 mL). 4-Fluorobenzoate (66 mg, 58.34 μmol), 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (55.78 mg, 58.34 μmol), DIPEA (16.18 mg, 159.12 μmol), and HATU (44.36 mg, 116.69 μmol) were added, and the mixture was stirred at 20 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (65.00 mg, 31.41 μmol).

[1152] The purification method is as follows:

[1153] Column: Agilent Prep C18 OBD 19*150mm*5μm

[1154] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[1155] Its structural characterization data are as follows:

[1156] ESI-MS (m / z): 1035.0 (M / 2+1) + .

[1157] Step 2: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-3-((2S,5S,12S,31S,32R,33R,34R)-12 Preparation of 3-amino-31,32,33,34,35-pentahydroxy-2,5,29-trimethyl-23,23-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-4,7,11,18,28-pentaoxo-21,25-dioxa-3,6,10,17,29-pentazapentadecanoamide)-4-fluorobenzoate (B-4-2)

[1158] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((2S,5S,12S,31S,32R,33R,34R)-12-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-31,3 2,33,34,35-pentahydroxy-2,5,29-trimethyl-23,23-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-4,7,11,18,28-pentoxo-21,25-dioxa-3,6,10,17,29-pentazapentadecanoamide)-4-fluorobenzoate (65.00 mg, 31.41 μmol) and diethylamine (6.89 mg, 94.24 μmol) were added, and the mixture was stirred at 25 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to give the title compound (40.00 mg, 21.66 μmol).

[1159] The purification method is as follows:

[1160] Column: Agilent Prep C18 OBD 19*150mm*5μm

[1161] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% TFA)

[1162] Its structural characterization data are as follows:

[1163] ESI-MS (m / z): 924.0 (M / 2+1) + .

[1164] Step 3: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((2S,5S,12S,31S,32R,33R,34R)-12-(2 Preparation of 3-bromoacetamyl)-31,32,33,34,35-pentahydroxy-2,5,29-trimethyl-23,23-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-4,7,11,18,28-pentoxo-21,25-dioxa-3,6,10,17,29-pentazapentadecanoamide)-4-fluorobenzoate (B-4)

[1165] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((2S,5S,12S,31S,32R,33R,34R)-12-amino-31,32,33,34,35-pentahydroxy-2,5,29-trimethyl-23,23-bis((3 -(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-4,7,11,18,28-pentoxo-21,25-dioxa-3,6,10,17,29-pentazapentadecanoamide)-4-fluorobenzoate (40.00 mg, 21.66 μmol), 2,5-dioxopyrrolidone-1-yl-2-bromoacetate (40.89 mg, 173.26 μmol), 2,6-dimethylpyridine (18.56 mg, 173.26 μmol), after addition, the reaction mixture was stirred at 20 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (20.30 mg, 9.80 μmol).

[1166] The purification method is as follows:

[1167] Column: Agilent Prep C18 OBD 19*150mm*5μm

[1168] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[1169] Its structural characterization data are as follows:

[1170] ESI-MS (m / z): 1967.8 (M+H) + .

[1171] Example 25: (2S,5S,15S)-26,26-bis((2-carboxyethoxy)methyl)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17S)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16, Preparation of 17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-2,5-dimethyl-15-(3-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)ethoxy)propionamide)-1,4,7,14,21-pentoxo-10,24,28-trioxa-3,6,13,20-tetraazatrione-31-acid (C-1)

[1172] Step 1: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((2S, Preparation of 5S,15S)-15-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-2,5-dimethyl-29-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,14,17,24-pentoxo-10,20-dioxa-3,6,13,16,23-pentazanonadecano-28-acetylamidamide)-4-fluorobenzoate (C-1-1)

[1173] INT-14 (89.58 mg, 101.44 μmol), INT2 (60 mg, 84.54 μmol), and HATU (48.22 mg, 126.81 μmol) were dissolved in DMF (5 mL). DIPEA (32.78 mg, 253.61 μmol) was added to the reaction system. The reaction was stirred at 25 °C for 1 hour. After the reaction was complete, the reaction solution was analyzed by high-performance liquid chromatography-mass spectrometry to obtain a white solid title compound (44 mg, 25.15 μmol).

[1174] Its structural characterization data are as follows:

[1175] ESI-MS (m / z): 1575.5 (M+H) + .

[1176] Step 2: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17- Preparation of 3-((2S,5S,15S)-15-(4-aminobutyl)-2,5-dimethyl-29-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,14,17,24-pentoxo-10,20-dioxa-3,6,13,16,23-pentazanonadecano-28-acetylamido)-4-fluorobenzoate (C-1-2)

[1177] The (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl3-((2S,5S,15S)-15-(4-(( Diphenyl(p-tolyl)methyl)amino)butyl)-2,5-dimethyl-29-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,14,17,24-pentoxo-10,20-dioxa-3,6,13,16,23-pentazanonadecano-28-acetylamido)-4-fluorobenzoate (44 mg, 27.94 μmol) was dissolved in DCM (3 mL), and formic acid (1 mL) was added. The reaction mixture was stirred at 25 °C for 1 hour. The reaction solution was purified by high performance liquid chromatography to give the title compound (30 mg, 20.48 μmol).

[1178] Its structural characterization data are as follows:

[1179] ESI-MS (m / z): 1318.4 (M+H) + .

[1180] Step 3 (2S,5S,15S)-26,26-bis((2-carboxyethoxy)methyl)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17S)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17) Preparation of -dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-2,5-dimethyl-15-(3-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)ethoxy)propionamide)-1,4,7,14,21-pentoxo-10,24,28-trioxa-3,6,13,20-tetraazatrionecan-31-acid (C-1)

[1181] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-3-((2S,5S,15S)-15-(4-aminobutyl)-2,5-dimethyl-29-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7 14,17,24-pentaoxo-10,20-dioxa-3,6,13,16,23-pentazanonadecano-28-acetylamido)-4-fluorobenzoate (30 mg, 22.75 μmol), 3-[3-(2-carboxyethoxy)-2,2-bis(2-carboxyethoxymethyl)propoxy]propionic acid (11.59 mg, 27.30 μmol), HATU (10.38 mg, 27.30 μmol), and DIPEA (6.87 mg, 53.19 μmol) were added, and the mixture was stirred at 20 °C for 1.5 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to give the title compound (5.71 mg, 3.18 μmol).

[1182] The purification method is as follows:

[1183] Column: Agilent Prep C18 OBD 19*150mm*5μm

[1184] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[1185] Its structural characterization data are as follows:

[1186] ESI-MS (m / z): 1724.7 (M+H) + .

[1187] Example 26: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((S)-9,9-bis((3-((1,3-dihydroxy) Preparation of 2-(hydroxymethyl)propane-2-yl)amino)-3-oxopropoxy)methyl)-1-hydroxy-2,2-bis(hydroxymethyl)-33-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)acetamide)-4,11,30-trioxo-7,14,17,20,23,26-hexaoxa-3,10,29-triazatetratetran-34-amide)-4-fluorobenzoate (E-5)

[1188] Step 1: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentanyl[a]phenanthrene-17-yl 3-((S)-5-(tert-butoxy)-2-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)acetamide)-5-oxopentanamide)-4-fluorobenzoate (E-5-1)

[1189] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-3-(((S)-2-(2-aminoacetamide)-5-(tert-butoxy)-5-oxopentanamide)-4-fluorobenzoate (400 mL) to DMF (4 mL). The following compounds were added: 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-alkynic acid (139.14 mg, 518.61 μmol), DIPEA (255.33 mg, 1.98 mmol), and HATU (281.68 mg, 740.87 μmol). After addition, the mixture was stirred at 22 °C for 3 h. The reaction solution was purified by C18 reverse-phase column chromatography (H2O / ACN = 10-80%, 0.1% formic acid) and lyophilized to give the title compound (427 mg, 370.56 μmol).

[1190] Its structural characterization is as follows:

[1191] ESI-MS (m / z): 1060.4 (M+H) + .

[1192] Step 2: Preparation of (S)-5-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-4-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)acetamide)-5-oxopentanoic acid (E-5-2)

[1193] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((S)-5-(tert-butoxy)-2-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)acetamide)-5-oxopentanamide)-4-fluorobenzoate (427 mg, 402.78 μmol) to DCM (6 nL) TFA (2 mL). After the addition is complete, stir the mixture at 22 °C for 3 h. After rotary evaporation, the compound was purified by C18 reverse column chromatography (H2O / ACN = 10-80%, 0.1% formic acid) and lyophilized to give the title compound (279 mg, 263.99 μmol).

[1194] Its structural characterization is as follows:

[1195] ESI-MS (m / z): 1004.2 (M+H) + .

[1196] Step 3: Preparation of (S)-23-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)carbamoyl)-33-(2-(methylsulfonyl)pyrimidin-5-yl)-20,25,28-trioxo-4,7,10,13,16-pentaoxa-19,24,27-triazatridodecane-32-acetylgynyl tert-butyl ester (E-5-3)

[1197] Add (S)-5-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-4-(2 -(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)acetamide)-5-oxovaleric acid (50 mg, 49.80 μmol), tert-butyl 1-amino-3,6,9,12,15-pentadecaoxoctadecane-18-oate (21.84 mg, 59.76 μmol), DIPEA (19.31 mg, 149.40 μmol), and HATU (28.40 mg, 78.40 μmol) were added, and the reaction mixture was stirred at 22 °C for 2 h. The reaction solution was purified by C18 reverse-phase column chromatography (H2O / ACN = 10-80%, 0.1% formic acid) and lyophilized to give the title compound (64 mg, 43.57 μmol).

[1198] Its structural characterization is as follows:

[1199] ESI-MS (m / z): 1352.5 (M+H) + .

[1200] Step 4: Preparation of (S)-23-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)carbamoyl)-33-(2-(methylsulfonyl)pyrimidin-5-yl)-20,25,28-trioxo-4,7,10,13,16-pentaoxa-19,24,27-triazatridodecane-32-alkynic acid (E-5-4)

[1201] Add tert-butyl(S)-23-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17 to DCM (3 mL) and TFA (1 mL). -Dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)carbamoyl)-33-(2-(methanesulfonyl)pyrimidin-5-yl)-20,25,28-trioxo-4,7,10,13,16-penta-19,24,27-triazatridodecane-32-acetylacetate (64 mg, 43.57 μmol), after addition, the reaction mixture was stirred at 22 °C for 2 h. The reaction solution was evaporated to dryness to give the crude product of the title compound (60 mg).

[1202] Its structural characterization is as follows:

[1203] ESI-MS (m / z): 1296.4 (M+H) + .

[1204] Step 5: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((S)-9,9-bis((3-((1,3-dihydroxy- Preparation of 2-(hydroxymethyl)propane-2-yl)amino)-3-oxopropoxy)methyl)-1-hydroxy-2,2-bis(hydroxymethyl)-33-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)acetamide)-4,11,30-trioxo-7,14,17,20,23,26-hexaoxa-3,10,29-triazatetradecane-34-amide)-4-fluorobenzoate (E-5)

[1205] Add (S)-23-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)carbamoyl)-33-(2-(methylsulfonyl)pyrimidin-5-yl)-20,25,28-trioxo-4,7,10,13,16 -Penta-19,24,27-triazatridodecane-32-alkynyl acid crude product (30 mg), 3,3'-((2-amino-2-((3-((1,3-dihydroxy-2-(hydroxymethyl)prop-2-yl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))bis(N-(1,3-dihydroxy-2-(hydroxymethyl)prop-2-yl)propionamide (29.95 mg, 46.32 μmol), DIPEA (8.98 mg, 69.48 μmol) and HATU (13.21 mg, 34.74 μmol) were added, and the reaction mixture was stirred at 22 °C for 2 h. The reaction solution was purified by high performance liquid chromatography to obtain the title compound (11.80 mg, 5.83 μmol).

[1206] Column: Phenomenex C18 150mm×25mm×10μm

[1207] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1208] Its structural characterization is as follows:

[1209] ESI-MS (m / z): 1924.8 (M+H) + .

[1210] Example 27: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-4-fluoro-3-((2S,34S,35R,36R,37R)-34,35,36,37,3 Preparation of 8-pentahydroxy-32-methyl-26,26-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-2-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)acetamide)-5,24,31-trioxo-9,12,15,18,21,28-hexaoxo-6,25,32-triazaoctadecanoamide)benzoate (E-6)

[1211] Add (S)-23-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)carbamoyl)-33-(2-(methylsulfonyl)pyrimidin-5-yl)-20,25,28-trioxo-4,7,10,13,16-pentaoxa-19,24,27 Crude triazatridodecane-32-alkynyl acid (30 mg), 3,3'-((2-amino-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))bis(N-methyl-N-(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide) (30.19 mg, 34.74 μmol), DIPEA (8.98 mg, 69.48 μmol), and HATU (13.21 mg, 34.74 μmol) were added, and the mixture was stirred at 22 °C for 2 h. The reaction solution was purified by high performance liquid chromatography to obtain the title compound (24.0 mg, 10.62 μmol).

[1212] Column: Phenomenex C18 150mm×25mm×10μm

[1213] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1214] Its structural characterization is as follows:

[1215] ESI-MS (m / z): 1074.2 (M1 / 2+H) + .

[1216] Example 28: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-4-fluoro-3-((2S,5S,10S,28S,29R,30R,31R)-28,29,30, Preparation of 31,32-pentahydroxy-5-isopropyl-2,26-dimethyl-20,20-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-4,7,11,18,25-pentoxo-15,22-dioxa-3,6,12,19,26-pentazatridodecanoamide)benzoate (F-1)

[1217] Step 1: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl 3-((S)-2-((S)-2-amino-3-methylbutamido)propamido)-4-fluorobenzoate (F-1-1)

[1218] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl 3-((S)-2-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-methylbutyramide)propionamide)-4-fluorobenzoate (3.00 g, 3.12 mmol) and diethylamine (685.66 mg, 9.37 mmol) to DMF (10 mL) in sequence. After the addition is complete, stir the mixture at 25 °C for 1 h. The reaction solution was concentrated under reduced pressure and then purified by column chromatography (ACN / H2O = 0-90%, 0.05% trifluoroacetic acid) to obtain the title compound (1.90 g, 2.23 mmol).

[1219] Its structural characterization is as follows:

[1220] ESI-MS (m / z): 738.3 (M+H) + .

[1221] Step 2: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl 3-((S)-2-((S)-2-((S)-5-(allyloxy)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-5-oxopentanamide)-3-methylbutyramide)propionamide)-4-fluorobenzoate (F-1-2)

[1222] (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-3-((S)-2-((S)-2-amino-3-methylbutamido)propamido)-4-fluorobenzoate (177.02 mg, 207.81 μmol), (S)-5-(allyloxy)- 4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-5-oxovaleric acid (100.00 mg, 228.59 μmol), DIPEA (107.43 mg, 831.23 μmol), and HTUA (157.93 mg, 415.62 μmol) were added to DMF (9.00 mL). The reaction solution was stirred at 25 °C for 1 hour. The reaction solution was concentrated under reduced pressure and purified by column chromatography (ACN / H2O = 0-90%, 0.05% formic acid) to give the title compound (180.00 mg, 155.54 μmol).

[1223] Its structural characterization is as follows:

[1224] ESI-MS (m / z): 1157.3 (M+H) + .

[1225] Step 3: N 5 -((S)-1-(((S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-N 2 Preparation of -(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (F-1-3)

[1226] Under nitrogen protection, (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-3-((S)-2-((S)-2-((S)-5-(ene) Propoxy)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)-5-oxopentanamide)-3-methylbutyramide)propionamide)-4-fluorobenzoate (180.00 mg, 155.54 μmol) was dissolved in DMF (1.00 mL), followed by the addition of tetrakis(triphenylphosphine)palladium (53.90 mg, 46.66 μmol) and 1,3-dimethylbarbituric acid (72.79 mg, 466.62 μmol). The reaction mixture was stirred at 25 °C for 1 hour, and the reaction was monitored by LC-MS until complete. The crude product was concentrated and purified by column chromatography (ACN / H₂O = 0-90%, 0.05% trifluoroacetic acid), and then lyophilized to give the title compound (170.00 mg, 138.08 μmol).

[1227] Its structural characterization data are as follows:

[1228] ESI-MS (m / z): 1117.3 (M+18) + .

[1229] Step 4: (2S,5S,10S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17- Preparation of dodecahydro-3H-cyclopentadieno[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-carboxylic acid tert-butyl ester (F-1-4)

[1230] N 5-((S)-1-(((S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-N 2 -(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (60.00 mg, 53.71 μmol), tert-butyl 3-(2-aminoethoxy)propionate (20.33 mg, 107.41 μmol), DIPEA (27.76 mg, 214.83 μmol), and HTUA (40.82 mg, 107.41 μmol) were added to DMF (3.00 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction was monitored by LC-MS until complete. The reaction system was concentrated to obtain a crude product, which was then purified by high performance liquid chromatography to obtain the title compound (65.00 mg, 50.45 μmol).

[1231] The purification method is as follows:

[1232] Column: Phenomenex C18 250mm×50mm×10μm

[1233] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% TFA)

[1234] Its structural characterization is as follows:

[1235] ESI-MS (m / z): 1288.4 (M+H) + .

[1236] Step 5: (2S,5S,10R)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,1) Preparation of 7-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-carboxylic acid (F-1-5)

[1237] Add (2S,5S,10S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadiene[a] to DCM (5 mL) 65.00 mg (50.45 μmol) of phenanthrene-17-yl(oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-carboxylic acid tert-butyl ester was added to TFA (2.00 mL). After the addition was complete, the mixture was stirred at 25 °C for 3 h. DCM and TFA were removed from the reaction solution under reduced pressure to obtain the crude title compound (65.00 mg, 48.28 μmol), which was used directly in the next step without purification.

[1238] Its structural characterization data are as follows:

[1239] ESI-MS (m / z): 1232.4 (M+H) + .

[1240] Step Six: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-4-fluoro-3-((2S,5S,10S,28S,29R,30R,31R)-28,29,30,3 Preparation of 1,32-pentahydroxy-5-isopropyl-2,26-dimethyl-20,20-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-4,7,11,18,25-pentoxo-15,22-dioxa-3,6,12,19,26-pentazatridodecanoamide)benzoate (F-1)

[1241] (2S,5S,10R)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadiene) Crude 1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-carboxylic acid (65.00 mg, 48.28 μmol), 3,3'-((2-(a)phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-carboxylic acid (65.00 mg, 48.28 μmol), 3,3'-((2-(a) ... -Amino-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))bis(N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide) (83.90 mg, 96.56 μmol), DIPEA (62.40 mg, 482.79 μmol), and HTUA (36.72 mg, 96.56 μmol) were added to DMF (3.00 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction was monitored by LC-MS until complete. The reaction system was concentrated to obtain a crude product, which was purified by high performance liquid chromatography to obtain the title compound (53.00 mg, 24.68 μmol, purity 97%).

[1242] The purification method is as follows:

[1243] Column: Phenomenex C18 250mm×50mm×10μm

[1244] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[1245] Its structural characterization is as follows:

[1246] ESI-MS (m / z): 1042.1 (M / 2+H) + .

[1247] Example 29: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-4-fluoro-3-((2S,5S,10S,34S,35R,36R,37R)-34,35,36,37, Preparation of 38-pentahydroxy-5-isopropyl-2,32-dimethyl-26,26-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-4,7,11,24,31-pentoxo-15,18,21,28-tetraoxa-3,6,12,25,32-pentazaoctadecanoamide)benzoate (F-2)

[1248] Step 1: (2S,5S,10S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro- Preparation of 3H-cyclopentadieno[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)-1,4,7,11-tetraoxo-15,18,21-trioxa-3,6,12-triazatetracosane-24-carboxylic acid tert-butyl ester (F-2-1)

[1249] N 5-((S)-1-(((S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-N 2 -(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (60.00 mg, 53.71 μmol), tert-butyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propionate (27.03 mg, 97.47 μmol), DIPEA (25.19 mg, 194.93 μmol), and HTUA (37.04 mg, 97.47 μmol) were added to DMF (1.00 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction was monitored by LC-MS until complete. The reaction system was concentrated to obtain a crude product, which was purified by high performance liquid chromatography to obtain the title compound (65.00 mg, 47.22 μmol).

[1250] The purification method is as follows:

[1251] Column: Phenomenex C18 250mm×50mm×10μm

[1252] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% TFA)

[1253] Its structural characterization is as follows:

[1254] ESI-MS (m / z): 1393.5 (M+18) + .

[1255] Step 2: (2S,5S,10S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecyl) Preparation of hydroxy-3H-cyclopentadieno[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamino)-1,4,7,11-tetraoxo-15,18,21-trioxa-3,6,12-triazatetracosane-24-carboxylic acid (F-2-2)

[1256] Add (2S,5S,10S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentadien[a]phenanthrene- 17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)-1,4,7,11-tetraoxo-15,18,21-trioxa-3,6,12-triazatetracosane-24-carboxylic acid tert-butyl ester (65.00 mg, 47.22 μmol) was added, followed by the addition of TFA (2 mL). The mixture was stirred at 25 °C for 3 h. DCM and TFA were removed from the reaction solution under reduced pressure to obtain the crude title compound (65.00 mg, 45.31 μmol), which was used directly in the next step without purification.

[1257] Its structural characterization data are as follows:

[1258] ESI-MS (m / z): 1342.4 (M+Na) + .

[1259] Step 3: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-4-fluoro-3-((2S,5S,10S,34S,35R,36R,37R)-34,35,36,37,3 Preparation of 8-pentahydroxy-5-isopropyl-2,32-dimethyl-26,26-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-4,7,11,24,31-pentoxo-15,18,21,28-tetraoxa-3,6,12,25,32-pentazaoctacosanamide)benzoate (F-2)

[1260] The (2S,5S,10S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadiene) [a]Phenanthroline-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamino)-1,4,7,11-tetraoxo-15,18,21-trioxa-3,6,12-triazatetracosane-24-acid crude product (65.00 mg, 45.31 μmol), 3,3'- ((2-amino-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))bis(N-methyl-N-(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide)(78.75 mg, 90.63 μmol), DIPEA (58.56 mg, 453.14 μmol), and HTUA (34.46 mg, 90.63 μmol) were added to DMF (3.00 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction was monitored by LC-MS until complete. The reaction system was concentrated to obtain a crude product, which was purified by high performance liquid chromatography to obtain the title compound (57.00 mg, 25.46 μmol, purity 97%).

[1261] The purification method is as follows:

[1262] Column: Phenomenex C18 250mm×50mm×10μm

[1263] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[1264] Its structural characterization is as follows:

[1265] ESI-MS (m / z): 1086.6 (M / 2+H) + .

[1266] Example 30: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-4-fluoro-3-((2S,5S,10S,40S,41R,42R,43R)-40,41,42,43,44- Preparation of pentahydroxy-5-isopropyl-2,38-dimethyl-32,32-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-4,7,11,30,37-pentoxo-15,18,21,24,27,34-hexaoxa-3,6,12,31,38-pentazatetratetradecanoamide)benzoate (F-3)

[1267] Step 1: (2S,5S,10S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H Preparation of cyclopentadieno[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-1,4,7,11-tetraoxo-15,18,21,24,27-pentaoxa-3,6,12-triazatriacontane-30-carboxylic acid tert-butyl ester (F-3-1)

[1268] N 5-((S)-1-(((S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-N 2 -(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (60.00 mg, 48.73 μmol), tert-butyl 1-amino-3,6,9,12,15-pentadecaoxaoctadecane-18-oate (35.62 mg, 97.47 μmol), DIPEA (25.19 mg, 194.93 μmol), and HTUA (37.04 mg, 97.47 μmol) were added to DMF (1.00 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction was monitored by LC-MS until complete. The reaction system was concentrated to obtain a crude product, which was purified by high performance liquid chromatography to obtain the title compound (65.00 mg, 44.38 μmol).

[1269] The purification method is as follows:

[1270] Column: Phenomenex C18 250mm×50mm×10μm

[1271] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% TFA)

[1272] Its structural characterization is as follows:

[1273] ESI-MS (m / z): 1481.5 (M+H2O) + .

[1274] Step 2: (2S,5S,10S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3 Preparation of H-cyclopentadieno[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-1,4,7,11-tetraoxo-15,18,21,24,27-pentaoxa-3,6,12-triazatriacontane-30-carboxylic acid (F-3-2)

[1275] Add (2S,5S,10S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentadien[a]phenanthrene-17 (-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-1,4,7,11-tetraoxo-15,18,21,24,27-pentaoxa-3,6,12-triazatriacontane-30-carboxylic acid tert-butyl ester (65.00 mg, 44.38 μmol) was added, followed by the addition of TFA (2 mL). The mixture was stirred at 25 °C for 3 h. DCM and TFA were removed from the reaction solution under reduced pressure to obtain the crude title compound (65.00 mg, 42.69 μmol), which was used directly in the next step without purification.

[1276] Its structural characterization data are as follows:

[1277] ESI-MS (m / z): 1408.5 (M+H) + .

[1278] Step 3:

[1279] (2S,5S,10S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadiene)[ [a]Phenanthroline-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-1,4,7,11-tetraoxo-15,18,21,24,27-pentaoxa-3,6,12-triazatriacontane-30-carboxylic acid crude product (65.00 mg, 42.69 μmol), 3, 3'-((2-amino-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))bis(N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide) (74.19 mg, 85.38 μmol), DIPEA (55.17 mg, 426.92 μmol), and HTUA (32.45 mg, 85.38 μmol) were added to DMF (3.00 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction was monitored by LC-MS until complete. The reaction system was concentrated to obtain a crude product, which was purified by high performance liquid chromatography to obtain the title compound (50.00 mg, 21.47 μmol, purity 97%).

[1280] The purification method is as follows:

[1281] Column: Phenomenex C18 250mm×50mm×10μm

[1282] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[1283] Its structural characterization is as follows:

[1284] ESI-MS (m / z): 1130.1 (M / 2+H) + .

[1285] Example 31: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-4-fluoro-3-((2S,5S,8S,28S,29R,30R,31R)-28,29,30,3 Preparation of 1,32-pentahydroxy-5-isopropyl-2,26-dimethyl-20,20-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-4,7,11,18,25-pentoxo-15,22-dioxa-3,6,12,19,26-pentazatridodecanoamide)benzoate (F-4)

[1286] Step 1: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-3-((S)-2-((S)-2-((S)-5-(tert-butoxy)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-5-oxopentanamide)-3-methylbutyramide)propionamide)-4-fluorobenzoate (F-4-1)

[1287] (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-3-((S)-2-((S)-2-amino-3-methylbutamido)propamido)-4-fluorobenzoate (169.05 mg, 198.45 μmol), (S)-5 -(tert-butoxy)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-5-oxovaleric acid (90.00 mg, 198.45 μmol), DIPEA (128.24 mg, 992.26 μmol), and HATU (150.92 mg, 396.91 μmol) were added to DMF (6.00 mL). The reaction solution was stirred at 25 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (180.00 mg, 153.41 μmol).

[1288] The purification method is as follows:

[1289] Column: Phenomenex C18 250mm×50mm×10μm

[1290] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1291] Its structural characterization is as follows:

[1292] ESI-MS (m / z): 1191.4 (M+H2O) + .

[1293] Step 2: Preparation of (S)-5-(((S)-1-(((S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)amino)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-5-oxopentanoic acid (F-4-2)

[1294] Add (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentadien[a]phenanthrene- 17-yl-3-((S)-2-((S)-2-((S)-5-(tert-butoxy)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)-5-oxopentanamide)-3-methylbutyramide)propionamide)-4-fluorobenzoate (180.00 mg, 153.41 μmol) was added to TFA (2.50 mL), and the mixture was stirred at 25 °C for 1 h. The reaction mixture was concentrated to dryness to obtain crude product (165.00 mg, 147.69 μmol), which was used directly in the next step without purification.

[1295] Its structural characterization data are as follows:

[1296] ESI-MS (m / z): 1134.3 (M+H2O) + .

[1297] Step 3: (2S,5S,8S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,1) Preparation of 7-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-carboxylic acid tert-butyl ester (F-4-3)

[1298] The (S)-5-(((S)-1-(((S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)amino)-4-(6 -(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-5-oxovaleric acid (35.00 mg, 31.33 μmol), tert-butyl 3-(2-aminoethoxy)propionate (11.86 mg, 62.66 μmol), DIPEA (20.24 mg, 156.64 μmol), and HTUA (23.82 mg, 62.66 μmol) were added to DMF (3.00 mL). The reaction solution was stirred at 25 °C for 1 hour. The reaction solution was directly purified by high performance liquid chromatography and then freeze-dried to obtain the title compound (27.00 mg, 20.96 μmol).

[1299] The purification method is as follows:

[1300] Column: Phenomenex C18 250mm×50mm×10μm

[1301] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1302] Its structural characterization is as follows:

[1303] ESI-MS (m / z): 1288.4 (M+H) + .

[1304] Step 4: Preparation of (2S,5S,8S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-carboxylic acid (F-4-4)

[1305] Add tert-butyl(2S,5S,8S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H- to DCM (3.00 mL). Cyclopentanyl[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-ester (27.00 mg, 20.96 μmol) was added to TFA (1.50 mL), and the mixture was stirred at 25 °C for 1 h. The reaction mixture was concentrated to dryness to obtain crude product (25.00 mg, 20.29 μmol), which was used directly in the next step without purification.

[1306] Its structural characterization data are as follows:

[1307] ESI-MS (m / z): 1232.3 (M+H) + .

[1308] Step 5: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-4-fluoro-3-((2S,5S,8S,28S,29R,30R,31R)-28,29,30,31, Preparation of 3,2-pentahydroxy-5-isopropyl-2,26-dimethyl-20,20-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-4,7,11,18,25-pentoxo-15,22-dioxa-3,6,12,19,26-pentazatridodecanoamide)benzoate (F-4)

[1309] The (2S,5S,8S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro- 3H-Cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-acid (25.00 mg, 20.29 μmol), 3,3 '-((2-amino-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))bis(N-methyl-N-(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide) (26.44 mg, 30.43 μmol), DIPEA (13.11 mg, 101.43 μmol), and HTUA (15.43 mg, 40.57 μmol) were added to DMF (3.00 mL). The reaction solution was stirred at 25 °C for 1 hour. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (6.00 mg, 2.85 μmol).

[1310] The purification method is as follows:

[1311] Column: Phenomenex C18 250mm×50mm×10μm

[1312] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1313] Its structural characterization is as follows:

[1314] ESI-MS (m / z): 1042.4 (M / 2+H) + .

[1315] Example 32: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-4-fluoro-3-((2S,5S,8S,34S,35R,36R,37R)-34,35,36,37,3 Preparation of 8-pentahydroxy-5-isopropyl-2,32-dimethyl-26,26-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-4,7,11,24,31-pentoxo-15,18,21,28-tetraoxa-3,6,12,25,32-pentazaoctadecanoamide)benzoate (F-5)

[1316] Step 1: (2S,5S,8S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H- Preparation of cyclopentane[a]benzo[a]pyridineheptane-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)-1,4,7,11-tetraoxo-15,18,21-trioxa-3,6,12-triazatetracosane-24-carboxylic acid tert-butyl ester (F-5-1)

[1317] The (S)-5-(((S)-1-(((S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)amino)-4-(6-(2-(methyl) Sulfonylpyrimidin-5-ylhex-5-acetylamide-5-oxovalerate (35.00 mg, 31.33 μmol), tert-butyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propionate (17.38 mg, 62.66 μmol), DIPEA (20.24 mg, 156.64 μmol), and HTUA (23.82 mg, 62.66 μmol) were added to DMF (3.00 mL). The reaction solution was stirred at 25 °C for 1 hour. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (25.00 mg, 18.16 μmol).

[1318] The purification method is as follows:

[1319] Column: Phenomenex C18 250mm×50mm×10μm

[1320] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1321] Its structural characterization is as follows:

[1322] ESI-MS (m / z): 1376.4 (M+H) + .

[1323] Step 2: (2S,5S,8S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro Preparation of -3H-cyclopentadienyl[a]p-hep-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-1,4,7,11-tetraoxo-15,18,21-trioxa-3,6,12-triazatetracosane-24-acid (F-5-2)

[1324] Add tert-butyl(2S,5S,8S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentane[a]benzo[a] to DCM (3.00 mL). [a]pyridine-heptanthane-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)-1,4,7,11-tetraoxo-15,18,21-trioxa-3,6,12-triazatetracosane-24-ester (24.00 mg, 17.44 μmol) was added, and the mixture was stirred at 25 °C for 1 h. The reaction mixture was concentrated to dryness to obtain crude product (23.00 mg, 17.42 μmol), which was used directly in the next step without purification.

[1325] Its structural characterization data are as follows:

[1326] ESI-MS (m / z): 1320.4 (M+H) + .

[1327] Step 3: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-4-fluoro-3-((2S,5S,8S,34S,35R,36R,37R)-34,35,36,37,38- Preparation of pentahydroxy-5-isopropyl-2,32-dimethyl-26,26-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-4,7,11,24,31-pentoxo-15,18,21,28-tetraoxa-3,6,12,25,32-pentazaoctadecanoamide)benzoate (F-5)

[1328] The (2S,5S,8S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclo Pentadienyl[a]p-heptyl-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)-1,4,7,11-tetraoxo-15,18,21-trioxa-3,6,12-triazatetracosane-24-acid (23.00 mg, 17.42 μmol) l), 3,3'-((2-amino-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))bis(N-methyl-N-(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide) (22.70 mg, 26.13 μmol), DIPEA (22.51 mg, 174.19 μmol), and HTUA (13.25 mg, 34.84 μmol) were added to DMF (3.00 mL). The reaction solution was stirred at 25 °C for 1 hour. The reaction solution was directly purified by high performance liquid chromatography and then freeze-dried to obtain the title compound (24.00 mg, 10.94 μmol).

[1329] The purification method is as follows:

[1330] Column: Phenomenex C18 250mm×50mm×10μm

[1331] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1332] Its structural characterization is as follows:

[1333] ESI-MS (m / z): 1086.1 (M / 2+H) + .

[1334] Example 33: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-4-fluoro-3-((2S,5S,8S,40S,41R,42R,43R)-40,41,42,43,44-pentanylhydro-3H-cyclopentanyl[a]phenanthrene-17-yl ... Preparation of hydroxy-5-isopropyl-2,38-dimethyl-32,32-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-4,7,11,30,37-pentoxo-15,18,21,24,27,34-hexaoxa-3,6,12,31,38-pentazatetratetradecanoamide)benzoate (F-6)

[1335] Step 1: tert-Butyl(2S,5S,8S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,1 7-Dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-isopropyl-2-methyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)-1,4,7,11-tetraoxo-15,18,21,24,27-pentaoxa-3,6,12-triazatriacontane-30-ester

[1336] The (S)-5-(((S)-1-(((S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)amino)-4-(6-(2-(methyl) Sulfonylpyrimidin-5-ylhex-5-acetylamide-5-oxovaleric acid (35.00 mg, 31.33 μmol), tert-butyl amino-3,6,9,12,15-pentadecaoxoctadecane-18-oate (22.90 mg, 62.66 μmol), DIPEA (20.24 mg, 156.64 μmol), and HTUA (23.82 mg, 62.66 μmol) were added to DMF (3.00 mL). The reaction solution was stirred at 25 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (40.00 mg, 27.31 μmol).

[1337] The purification method is as follows:

[1338] Column: Phenomenex C18 250mm×50mm×10μm

[1339] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1340] Its structural characterization is as follows:

[1341] ESI-MS (m / z): 1481.5 (M+18) + .

[1342] Step 2: (2S,5S,8S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]) 1,4,7,11-Tetraoxo-15,18,21,24,27-pentaoxa-3,6,12-triazatriacontane-30-acid was added to DCM (3.00 mL) with tert-butyl(2S,5S,8S)-1- ((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluoro) Phenyl(amino)-5-isopropyl-2-methyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-1,4,7,11-tetraoxo-15,18,21,24,27-pentaoxa-3,6,12-triazatridodecane-30-ester (40.00 mg, 27.31 μmol) was added to TFA (1.50 mL). After the addition was complete, the mixture was stirred at 25 °C for 1 h. The reaction mixture was concentrated to dryness to obtain crude product (38.00 mg, 26.98 μmol), which was used directly in the next step without purification.

[1343] Its structural characterization data are as follows:

[1344] ESI-MS (m / z): 1408.4 (M+H) + .

[1345] Step 3: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-4-fluoro-3-((2S,5S,8S,40S,41R,42R,43R)-40,41,42,43,44-pentahydroxy Preparation of 5-isopropyl-2,38-dimethyl-32,32-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-4,7,11,30,37-pentoxo-15,18,21,24,27,34-hexaoxa-3,6,12,31,38-pentazatetratetradecanoamide)benzoate (F-6)

[1346] The (2S,5S,8S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclo Pentadienyl[a]p-heptanone-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-2,5-dimethyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)-1,4,7,11-tetraoxo-15,18,21,24,27-pentaoxa-3,6,12-triazatriacontane-30-carboxylic acid (25.00 mg, 17.75 μg) 23.13 mg (26.62 μmol), 3,3'-((2-amino-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))bis(N-methyl-N-(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide) (23.13 mg, 26.62 μmol), DIPEA (22.94 mg, 177.49 μmol), and HTUA (13.50 mg, 35.50 μmol) were added to DMF (3.00 mL). The reaction solution was stirred at 25 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then freeze-dried to obtain the title compound (21.00 mg, 9.20 μmol).

[1347] The purification method is as follows:

[1348] Column: Phenomenex C18 250mm×50mm×10μm

[1349] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1350] Its structural characterization is as follows:

[1351] ESI-MS (m / z): 1130.4 (M / 2+H) + .

[1352] Example 34: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-4-fluoro-3-((2S,5S,10S,28S,29R,30R,31R)-28,29,30 Preparation of 31,32-pentahydroxy-2,5,26-trimethyl-20,20-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-4,7,11,18,25-pentoxo-15,22-dioxa-3,6,12,19,26-pentazatridodecanoamide)benzoate (G-1)

[1353] Step 1: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-3-((S)-2-((S)-2-((S)-5-(allyloxy)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-5-oxopentanamide)propionamide)propionamide)-4-fluorobenzoate (G-1-1)

[1354] (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-3-((S)-2-((S)-2-aminopropionylamino)propionylamino)-4-fluorobenzoate (112.98 mg, 137.15 μmol), (S) 5-(allyloxy)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-5-oxovaleric acid (60.00 mg, 137.15 μmol), DIPEA (88.63 mg, 685.77 μmol), and HTUA (104.30 mg, 274.31 μmol) were added to DMF (5.00 mL). The reaction solution was stirred at 25 °C for 4 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (112 mg, 99.19 μmol).

[1355] The purification method is as follows:

[1356] Column: Phenomenex C18 250mm×50mm×10μm

[1357] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1358] Its structural characterization is as follows:

[1359] ESI-MS (m / z): 1130.3 (M+H) + .

[1360] Step 2: Preparation of N5-((S)-1-(((S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)-N2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (G-1-2)

[1361] Under nitrogen protection, (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl-3-((S)-2-((S)-2-((S)-5) -(allyloxy)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)-5-oxopentanamide)propionamide)propionamide)-4-fluorobenzoate (87.00 mg, 77.05 μmol) was dissolved in DMF (6.00 mL), followed by the addition of tetrakis(triphenylphosphine)palladium (44.52 mg, 38.52 μmol) and 1,3-dimethylbarbituric acid (60.15 mg, 385.23 μmol). The reaction mixture was stirred at 25 °C for 1.5 hours. The resulting solution was then purified directly by high-performance liquid chromatography and lyophilized to obtain the title compound (112.00 mg, 99.19 μmol).

[1362] The purification method is as follows:

[1363] Column: Phenomenex C18 250mm×50mm×10μm

[1364] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1365] Its structural characterization is as follows:

[1366] ESI-MS (m / z): 1089.3 (M+H) + .

[1367] Step 3: tert-Butyl(2S,5S,10S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15, Preparation of 16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-2,5-dimethyl-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-ester (G-1-3)

[1368] The N5-((S)-1-(((S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)-N2-(6-(2-( Methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (20.00 mg, 18.36 μmol), tert-butyl 3-(2-aminoethoxy)propionate (6.59 mg, 36.73 μmol), DIPEA (11.87 mg, 91.82 μmol), and HTUA (13.96 mg, 36.73 μmol) were added to DMF (3.00 mL). The reaction solution was stirred at 25 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (17.00 mg, 13.49 μmol).

[1369] The purification method is as follows:

[1370] Column: Phenomenex C18 250mm×50mm×10μm

[1371] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1372] Its structural characterization is as follows:

[1373] ESI-MS (m / z): 1260.3 (M+H) + .

[1374] Step 4: Preparation of (2S,5S,10S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-2,5-dimethyl-10-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-acid (G-1-4)

[1375] Add tert-butyl(2S,5S,10S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3- H-cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-2,5-dimethyl-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-ester (17.00 mg, 13.49 μmol) was added, and the mixture was stirred at 25 °C for 1 h. The reaction mixture was concentrated to dryness to obtain crude product (16.00 mg, 13.29 μmol), which was used directly in the next step without purification.

[1376] Its structural characterization data are as follows:

[1377] ESI-MS (m / z): 1204.3 (M+H) + .

[1378] Step 5: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-4-fluoro-3-((2S,5S,10S,28S,29R,30R,31R)-28,29,30,3 Preparation of 1,32-pentahydroxy-2,5,26-trimethyl-20,20-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-4,7,11,18,25-pentoxo-15,22-dioxa-3,6,12,19,26-pentazatridodecanoamide)benzoate (G-1)

[1379] (2S,5S,10S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro) -3H-cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-2,5-dimethyl-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-acid (16.00 mg, 13.29 μmol), 3,3' -((2-amino-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))bis(N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide)(23.09 mg, 26.57 μmol), DIPEA (17.17 mg, 132.86 μmol), and HTUA (10.10 mg, 26.57 μmol) were added to DMF (3.00 mL). The reaction solution was stirred at 25 °C for 1 hour. The reaction solution was directly purified by high performance liquid chromatography and then freeze-dried to obtain the title compound (16.00 mg, 7.71 μmol).

[1380] The purification method is as follows:

[1381] Column: Phenomenex C18 250mm×50mm×10μm

[1382] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1383] Its structural characterization is as follows:

[1384] ESI-MS (m / z): 1028.0 (M / 2+H) + .

[1385] Example 35: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-4-fluoro-3-((2S,5S,10S,34S,35R,36R,37R)-34,35,36,37 Preparation of 3,8-pentahydroxy-2,5,32-trimethyl-26,26-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)-4,7,11,24,31-pentoxo-15,18,21,28-tetraoxa-3,6,12,25,32-pentazaoctadecanoamide)benzoate (G-2)

[1386] Step 1: (2S,5S,10S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecyl) Preparation of 3H-cyclopentadieno[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-2,5-dimethyl-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-1,4,7,11-tetraoxo-15,18,21-trioxa-3,6,12-triazatetracosane-2,4-carboxylic acid tert-butyl ester (G-2-1)

[1387] N 5-((S)-1-(((S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)-N2-(6-(2-(methylsulfonyl)pyrimidine- 5-yl)hexyl-5-ynyl)-L-glutamine (38.00 mg, 34.89 μmol), tert-butyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propionate (19.35 mg, 69.78 μmol), DIPEA (22.55 mg, 174.45 μmol), and HTUA (26.53 mg, 69.78 μmol) were added to DMF (4.00 mL). The reaction solution was stirred at 25 °C for 2 hours. The reaction solution was directly purified by high performance liquid chromatography and then lyophilized to obtain the title compound (31.00 mg, 22.99 μmol).

[1388] The purification method is as follows:

[1389] Column: Phenomenex C18 250mm×50mm×10μm

[1390] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1391] Its structural characterization is as follows:

[1392] ESI-MS (m / z): 1348.4 (M+H) + .

[1393] Step 2: (2S,5S,10S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17- Preparation of dodecahydro-3H-cyclopentadiene[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-2,5-dimethyl-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-1,4,7,11-tetraoxo-15,18,21-trioxa-3,6,12-triazatetracosane-24-carboxylic acid (G-2-2)

[1394] Add (2S,5S,10S)-1-((5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadiene to DCM (3.00 mL) and [ [a]Phenylenol-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-2,5-dimethyl-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-1,4,7,11-tetraoxo-15,18,21-trioxa-3,6,12-triazatetracosane-24-oic acid tert-butyl ester (21.00 mg, 15.57 μmol) was added to TFA (1.50 mL), and the mixture was stirred at 25 °C for 1 h. The reaction mixture was concentrated to dryness to obtain crude product (20.00 mg, 15.48 μmol), which was used directly in the next step without purification.

[1395] Its structural characterization data are as follows:

[1396] ESI-MS (m / z): 1292.4 (M+H) + .

[1397] Step 3: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentan[a]phenanthrene-17-yl-4-fluoro-3-((2S,5S,10S,34S,35R,36R,37R)-34,35,36,37,3 Preparation of 8-pentahydroxy-2,5,32-trimethyl-26,26-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)-10-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-4,7,11,24,31-pentoxo-15,18,21,28-tetraoxa-3,6,12,25,32-pentazaoctadecanoamide)benzoate (G-2)

[1398] (2S,5S,10S)-1-((5-((((6S,8S,9R,10S,11S,13S,1...

Claims

An antibody-drug conjugate having the structure shown in formula HA-[MLED]x, wherein: HA is an antibody or its antigen-binding fragment that specifically binds to an antigen; M is the linker site that is attached to the antibody or its antigen-binding fragment; L is the connection structure that connects the joint portions M and E; E is a structure that connects L and D; D is a bioactive molecule or a fragment thereof; preferably, the bioactive molecule is a glucocorticoid drug; more preferably, the glucocorticoid drug is a glucocorticoid receptor agonist; x is an integer selected from 1 to 10, preferably an integer from 3 to 8. The antibody-drug conjugate of claim 1, wherein the antigen is selected from one or more of TNFα, IL6R, BDCA2, NR3C1, MSR1, PRLR, CD19, CD25, CD40, D70, CD74, and CD163. The antibody-drug conjugate of claim 1 or 2, wherein the antibody is a 1D3 monoclonal antibody. The antibody-drug conjugate according to any one of claims 1-3, wherein, M is The C structure is a single bond, sulfonamide group, phosphoramidyl group, 4-6 membered aliphatic heterocycle or 5-20 membered aromatic ring system, wherein the aliphatic heterocycle or aromatic ring system is optionally selected by one or more groups, each independently selected from oxo (=O), halogen, cyano, nitro, amino, carboxyl, mercapto and C. 1-6 Alkyl group substitution; and M1 is selected from a single bond or a fragment consisting of one or more groups selected from the following: -O-, -NH-, -NH2, -C(=O)-, HO2C(=O)-, -S(=O)2-, HOS(=O)2-, -C=NO-, -NH-S(=O)2-NH-, 3-6 membered heterocyclic group, phenylene, 5-10 membered heteroaryl group, C 1-20 Alkylene, C 2-20 imide and C 2-20 Ethyne group; Preferably, M is Wherein, C is a single bond, sulfonamide group, phosphoramidyl group, 4-6 membered aliphatic heterocycle, 5-6 membered heteroaromatic ring, or a polycyclic ring formed by two or three (preferably three) units selected from 6 membered aromatic heterocycles and benzene rings linked by single bonds, wherein the aliphatic heterocycle, heteroaromatic ring, or polycyclic ring is optionally substituted by one or more groups selected from oxo (=O), halogen, cyano, and nitro; and M1 is selected from a single bond or a fragment composed of one or more groups selected from the following groups: -NH-, -C(=O)-, -NH-S(=O)2-NH-, C 1-10 Alkylene, C 2-10 imide and C 2-10 Ethyne group; Preferably, M is Where the C structure is Furthermore, M1 is selected from single bonds, carbonyl groups, sulfonyl groups, -NH-, -NH-S(=O)2-NH-, and C. 1-6 Alkylene, C 2-6 imide and C 2-6 Ethyne group; Preferably, M is selected from Preferably, M is Preferably, M is In any one of claims 1-4, the antibody-drug conjugate L is a structure composed of one or more fragments selected from the following: C 1-6 Alkyl groups, -N(R')-, carbonyl groups, glycosyl groups, -O-, natural or non-natural amino acids and their analogs or derivatives (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH2CH2(OCH2CH2)) r OCH3), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly- Gly, Gly-Ser-Lys, Glu-Ala-Ala, Glu-Val-Ala, Glu-Val-Cit, Lys-Ala-Ala, Ser-Ala-Pr0, Val-Leu-Lys, Val-Lys-Al a. Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), Where R' represents hydrogen, C 1-6 Alkyl groups or C groups containing -(CH2CH2O)r- 4-30 Alkyl; r is an integer selected from 1 to 10; s is an integer selected from 1 to 20; Preferably, L is a structure composed of one or more parts selected from the following: Where s is an integer selected from 1 to 20, preferably an integer from 1 to 10. The antibody-drug conjugate of any one of claims 1-5, wherein E is a single bond or -NHCH2-. The antibody-drug conjugate according to any one of claims 1-6, Selected from the following structures: in, n is an integer selected from 1 to 20. The antibody-drug conjugate of any one of claims 1-7 is selected from: ADC-A-1: ADC-A-2: ADC-A-3: ADC-A-4: ADC-A-5: ADC-A-6: ADC-B-1: ADC-B-2: ADC-B-3: ADC-B-4: ADC-C-1: ADC-E-1: ADC-E-2: ADC-E-3: ADC-E-4: ADC-E-5: ADC-E-6: ADC-E-7: ADC-E-8: ADC-E-9: ADC-E-10: ADC-F-1: ADC-F-2: ADC-F-3: ADC-F-4: ADC-F-5: ADC-F-6: ADC-F-7: ADC-F-8: ADC-F-9: ADC-F-10: ADC-F-11: ADC-F-12: ADC-G-1: ADC-G-2: ADC-G-3: ADC-G-4: ADC-G-5: ADC-G-6: ADC-G-7: ADC-G-8: ADC-G-9: ADC-G-10: ADC-G-11: ADC-G-12: ADC-G-13: ADC-G-14: ADC-G-15: ADC-G-17: ADC-G-18: ADC-G-19: ADC-G-20: ADC-G-21: ADC-G-22: ADC-H-1: ADC-H-2: ADC-H-3: ADC-H-4: ADC-H-5: ADC-H-6: ADC-N-1: ADC-N-2: ADC-N-3: ADC-N-4: ADC-N-5: ADC-S-1: ADC-S-2: ADC-S-3: ADC-S-4: ADC-V-1: ADC-V-2: ADC-V-3: ADC-V-4: ADC-K-1: ADC-K-2: ADC-K-3: ADC-K-4: ADC-T-1: ADC-T-1: ADC-T-2: ADC-T-3: ADC-T-4: ADC-T-5: ADC-T-6: ADC-T-7: ADC-T-8: ADC-T-9: ADC-T-10: ADC-T-11: ADC-T-12: ADC-T-13: ADC-T-14: ADC-T-15: ADC-T-16: ADC-T-17: ADC-T-18: ADC-T-19: ADC-T-20: ADC-T-21: ADC-T-22: ADC-T-23: ADC-T-24: ADC-T-25: ADC-T-26: ADC-T-27: ADC-T-28: in, x is an integer selected from 1 to 10; Wherein, -S)x-Ab is any of the antibodies or antigen-binding fragments described above; This indicates the specific linkage between the thiol group and the pyrimidin group or methylene group in the antibody or its antigen-binding fragment. The composition comprises the antibody-drug conjugate according to any one of claims 1-8, wherein the drug-antibody conjugate ratio (DAR) is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10, preferably 3-9, for example, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 3.0-5.5, 3.0-6.0, 3.5-4.0, 3.5-4.5, 3.5-5.0, 3.5-5.5 3.5-6.0, 3.5-6.5, 3.5-7.0, 3.5-7.5, 3.5-8.0, 4.0-4.5, 4.0-5.0, 4.0-5.5, 4.0-6.0, 4.0-6.5, 4.0-7.0, 4.0-7.5, 4.0-8.0, 4.5-5.0, 4.5-5.5, 4.5-6.0, 4.5-6.5, 4.5-7.0, 4.5-7.5, 4.5-8.0, 5.0 -5.5, 5.0-6.0, 5.0-6.5, 5.0-7.0, 5.0-7.5, 5.0-8.0, 5.5-6.0, 5.5-6.5, 5.5-7.0, 5.5-7.5, 5.5-8.0, 6.0-6.5, 6.0-7.0, 6.0-7.5, 6.0-8.5, 6.5-7.0, 6.5-7.5, 6.5-8.5, 7.0-7.5, 7.0-9.0 or 7.5-9.

0. A drug-linker compound having the formula M a -The structure shown by the LED, wherein: M a It is the linker site before the antibody or its antigen-binding fragment is attached; L, E, D, and x are as defined in any one of claims 1-9; Preferably, M a for Where Q represents hydrogen, halogen, halomethylene, haloacetyl, alkenyl, alkynyl, cyano, isothiocyanate, or C. 1-6 Alkyl sulfonyl, fluorosulfonyl, fluorosulfonate, fluorophenol ester; C-structure is a single bond, sulfonamide, phosphoramidyl, 4-6 membered aliphatic heterocycle or 5-20 membered aromatic ring system, wherein the aliphatic heterocycle or aromatic ring system is optionally selected by one or more groups, each independently selected from oxo (=O), halogen, cyano, nitro, amino, carboxyl, mercapto, and C. 1-6 Alkyl group substitution; and M1 is selected from a single bond or a fragment consisting of one or more groups selected from the following: -O-, -NH-, -NH2, -C(=O)-, HO2C(=O)-, -S(=O)2-, HOS(=O)2-, -C=NO-, -NH-S(=O)2-NH-, 3-6 membered heterocyclic group, phenylene, 5-10 membered heteroaryl group, C 1-20 Alkylene, C 2-20 imide and C 2-20 Ethyne group; Preferably, M a for Where Q represents hydrogen, halogen, halomethylene, haloacetyl, alkenyl, alkynyl, cyano, isothiocyanate, or C. 1-6 Alkyl sulfonyl, fluorosulfonyl, fluorosulfonate, fluorophenol ester; C structure is a single bond, sulfonamide, phosphoramidyl, 4-6 membered aliphatic heterocycle, 5-6 membered heteroaromatic ring, or a polycyclic ring formed by two or three (preferably three) units selected from 6 membered aromatic heterocycles and benzene rings linked by single bonds, wherein the aliphatic heterocycle, heteroaromatic ring, or polycyclic ring is optionally substituted by one or more groups selected from oxo (=O), halogen, cyano, and nitro; and M1 is selected from a single bond or a segment composed of one or more groups selected from the following groups: -NH-, -C(=O)-, -NH-S(=O)2-NH-, C 1-10 Alkylene, C 2-10 imide and C 2-10 Ethyne group; Preferably, M a for The C structure is selected from single bonds, M1 is selected from single bonds, carbonyl groups, sulfonyl groups, -NH-, -NH-S(=O)2-NH-, C 1-6 Alkylene, C 2-6 imide and C 2-6 Ethyne group; Preferably, M a Selected from Preferably, M a for Preferably, M a for Preferably, M a for Preferably, M a for Preferably, M a for The drug-linker compound of claim 10 is selected from: A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1-9, or the drug-linker compound of claim 10 or 11, and one or more pharmaceutically acceptable carriers. Use of the antibody-drug conjugate of any one of claims 1-8, the composition of claim 9, the drug-linker compound of claim 10 or 11, or the pharmaceutical composition of claim 12 in the preparation of a medicament for treating inflammatory or immune diseases; Preferably, the immune disease is an autoimmune disease; Preferably, the inflammatory or immune disease is selected from rheumatoid arthritis, idiopathic arthritis, asthma, ulcerative colitis, neuromyelitis optica, autoimmune liver disease, systemic lupus erythematosus, and psoriasis. The antibody-drug conjugate of any one of claims 1-8, the composition of claim 9, the drug-linker compound of claim 10 or 11, or the pharmaceutical composition of claim 12, for the treatment of inflammatory or immune diseases; Preferably, the immune disease is an autoimmune disease; Preferably, the inflammatory or immune disease is selected from rheumatoid arthritis, idiopathic arthritis, asthma, ulcerative colitis, neuromyelitis optica, autoimmune liver disease, systemic lupus erythematosus, and psoriasis. A method of treating an inflammatory or immune disease, comprising administering to an individual in need a therapeutically effective amount of the antibody-drug conjugate of any one of claims 1-8, the composition of claim 9, the drug-linker compound of claim 10 or 11, or the pharmaceutical composition of claim 12; Preferably, the immune disease is an autoimmune disease; Preferably, the inflammatory or immune disease is selected from rheumatoid arthritis, idiopathic arthritis, asthma, ulcerative colitis, neuromyelitis optica, autoimmune liver disease, systemic lupus erythematosus, psoriasis, systemic sclerosis, and atopic dermatitis. The method for preparing the antibody-drug conjugate according to any one of claims 1-8, the method comprising the following steps: a) Provide a solution containing antibodies; b) Contact the solution from a) with a reducing agent; c) Contacting the solution of b) with a solution containing any one of the drug-linker compounds or salts thereof according to claims 10-11 to prepare an ADC; Preferably, the reducing agent is tris(2-carboxyethyl)phosphine (TCEP); Preferably, the molar ratio of the drug-linker compound to the antibody in any one of claims 10-11 is (1-10):

1. A compound or a pharmaceutically acceptable salt thereof, said compound having the following structure: Wherein, Q1, Q2, Q3, Y1, Z1, and R1 are as described in any one of claims 1-11; PG1 is an amino protecting group.

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