Glucocorticoid receptor agonists and conjugates thereof

Abstract

The present application relates to the field of medicine, particularly to antibody-drug conjugates of glucocorticoid receptor agonists. Specifically, the present application provides glucocorticoid receptor agonists with good agonist activity. The present application also provides drug-linker compounds for coupling, where the drug is a glucocorticoid receptor agonist. The present application also provides antibody-drug conjugates prepared by coupling a drug with a linker compound, where the antibody-drug conjugates have good drug coupling uniformity and excellent therapeutic effects on inflammatory diseases such as rheumatoid arthritis. The present application also provides methods for preparing glucocorticoid receptor agonists and antibody-drug conjugates thereof, as well as uses of glucocorticoid receptor agonists and antibody-drug conjugates thereof in the field of medicine.

Description

[Technical Field]

[0001] The present application relates to the field of medicine, and in particular to antibody-drug conjugates of glucocorticoid receptor agonists. Specifically, the present application provides glucocorticoid receptor agonists with good agonist activity. The present application also provides drug-linker compounds for conjugation, in which the drug is a glucocorticoid receptor agonist. The present application also provides antibody-drug conjugates prepared by conjugating the drug-linker compounds to antibodies, which have good drug binding uniformity and excellent therapeutic effects against inflammatory diseases such as rheumatoid arthritis. The present application also provides methods for preparing glucocorticoid receptor agonists and their antibody-drug conjugates, as well as uses of glucocorticoid receptor agonists and their antibody-drug conjugates in the field of medicine. [Background technology]

[0002] Steroid glucocorticoid receptor agonists are a class of therapeutic agents widely used for inflammatory and immune disorders. These drugs exert their anti-inflammatory effects by activating intracellular glucocorticoid receptors, interfering with leukocyte recruitment to inflammatory sites while simultaneously inhibiting the production and release of inflammatory mediators from leukocytes and tissue cells. Long-term use of large doses of steroid glucocorticoid receptor agonists can cause side effects such as infections, gastrointestinal reactions, endocrine disorders (e.g., moon face, buffalo hump, weight gain, hypokalemia, and abnormal blood glucose and blood pressure levels), and osteoporosis. To mitigate the side effects of steroid glucocorticoid receptor agonists, inhaled formulations and topical formulations for dermal application are being developed; introducing metabolically labile functional groups into the steroid structure to promote inactivation and reduce systemic exposure is also a viable approach.

[0003] Recently, in order to improve the efficacy and safety of steroid glucocorticoid receptor agonists, conjugation of steroid glucocorticoid receptor agonists with antibodies is a new direction for the development of such drugs. Summary of the Invention

[0004] The present application relates to a steroid glucocorticoid receptor agonist, and a linker conjugate and antibody conjugate thereof. The steroid glucocorticoid receptor agonist and conjugate thereof of the present invention have excellent agonist activity and good druggability, and are therefore expected to be used as therapeutic agents for inflammatory diseases and immune diseases.

[0005] In one aspect, the present application provides a steroid glucocorticoid receptor agonist that is a compound of Formula I, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, or a prodrug thereof: [ka] During the ceremony, R1 is independently hydrogen, halogen, or -NR a R b , hydroxy, cyano, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkyl, C substituted with one or more hydroxy 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 haloalkyl; R2 is hydrogen, C 1-6 Alkyl, hydroxy and C 1-6 selected from the group consisting of alkoxy; Ring A is a single bond, C 6-10 selected from the group consisting of aromatic rings and 5-6 membered heteroaromatic rings; X is a single bond, C 1-6 Alkylene, C 3-6 Cycloalkylene, C 2-6Alkenylidene, C 2-6 Alkynylidene and -NR a - selected from the group consisting of; Y is selected from the group consisting of -O- and -S-; Z is selected from the group consisting of hydroxy, halogen, and cyano; Q1 and Q2 are each independently selected from the group consisting of hydrogen and halogen; m is selected from the group consisting of 1, 2, 3, 4 and 5; n is selected from the group consisting of 1 and 2; R a and R b are each independently hydrogen and C 1-6 selected from the group consisting of alkyl; With the proviso that: when each R1 is independently selected from the group consisting of halogen, amino, and methyl, m is 2, R2 is methyl, X is a single bond, Y is -S-, Z is fluorine, n is 1, Q1 is hydrogen or fluorine, and Q2 is fluorine, and ring A is not phenyl; When ring A is a single bond, R1 is hydrogen or C 1-6 is not alkyl; and When ring A is a 5- or 6-membered heteroaromatic ring, R1 is hydrogen and R2 is C 1-6 alkyl and Q1 is not a halogen.

[0006] In another embodiment, the present application provides a compound of formula Ab-[MLED] x and an antibody-drug conjugate having a structure represented by the formula: Ab is an antibody that specifically binds to an antigen or an antigen-binding fragment thereof; M is a linking site to an antibody or antigen-binding fragment thereof; L is a linker between linking sites M and E; E is a structural fragment connecting L with D; D is a glucocorticoid drug fragment that is a monovalent structure obtained by removing one H from the -OH, -NH, or secondary amino of a steroid glucocorticoid receptor agonist of the present invention; and x is an integer selected from 1 to 10.

[0007] In another embodiment, the present application provides a compound of formula GM-[LED] x and a drug-linker compound having a structure represented by the formula: G is a functional group or leaving group that reacts with specific amino acids or glycosyl groups and their derivatives in the antibody or antigen-binding fragment; M is a linking site to an antibody or antigen-binding fragment thereof; L is a linker between linking sites M and E; E is a structural fragment connecting L with D; D is a glucocorticoid drug fragment that is a monovalent structure obtained by removing one H from the -OH, -NH, or secondary amino of a steroid glucocorticoid receptor agonist of the present invention; and x is an integer selected from 1 to 10.

[0008] In another aspect, the present application provides a pharmaceutical composition comprising a steroid glucocorticoid receptor agonist of the present invention, an antibody-drug conjugate of the present invention, or a drug-linker compound of the present invention, and one or more pharmaceutically acceptable carriers.

[0009] In another aspect, the present application provides use of a steroid glucocorticoid receptor agonist of the present invention, an antibody-drug conjugate of the present invention, a drug-linker compound of the present invention, or a pharmaceutical composition of the present invention in the manufacture of a medicament for the treatment of an inflammatory disease or an immune disease.

[0010] In another aspect, the present application provides a steroid glucocorticoid receptor agonist of the present invention, an antibody-drug conjugate of the present invention, a drug-linker compound of the present invention, or a pharmaceutical composition of the present invention for use in treating an inflammatory or immune disorder.

[0011] In another aspect, the present application provides a method for treating an inflammatory or immune disease, comprising administering to a subject in need thereof a therapeutically effective amount of a steroid glucocorticoid receptor agonist of the present invention, an antibody-drug conjugate of the present invention, a drug-linker compound of the present invention, or a pharmaceutical composition of the present invention.

[0012] In another aspect, the present application provides a method for preparing a compound of the present invention, comprising the steps of: [ka] wherein ring A, R1, R2, X, Y, Z, Q1, Q2, m, and n are as defined above, and LG is a leaving group.

[0013] In another aspect, the present application provides a method for preparing a Drug-Linker Compound of the present invention, comprising the steps of: [ka] During the ceremony, Ring A, R1, R2, X, Y, Z, Q1, G, M, L, D, m and n are as defined above; X is 1; E is a single bond; LG is a leaving group, such as, but not limited to, halogen, methylsulfonyloxy (-OMs), and trifluoromethylsulfonyloxy (-OTf), preferably iodine; and PG is an amino protecting group, such as, but not limited to, 9-fluorenylmethoxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc), p-methoxytriphenylmethyl (MMt), and allyloxycarbonyl (Alloc), preferably Fmoc.

[0014] In another aspect, the present application provides a method for preparing an antibody-drug conjugate of the present invention, selected from the group consisting of: Conjugation Method A: conjugating the drug-linker compound of the present invention with an antibody at a molar ratio of (8 to 0):1 to obtain an antibody-drug conjugate; Conjugation Method B: conjugating the drug-linker compound of the present invention with an antibody at a molar ratio of (4-6):1 to obtain an antibody-drug conjugate; Conjugation Method C: The drug-linker compound of the present invention is conjugated to an antibody at a molar ratio of (4.0-4.5):1 to obtain an antibody-drug conjugate. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows the in vitro inhibitory results of the compounds of the present invention on B cell proliferation in mice. [Figure 2] FIG. 2 shows the in vitro activation results of the compounds of the present invention on the activity of glucocorticoid receptors in cells. [Figure 3] FIG. 3 shows the change in arthritis score in CIA model mice by the compound of the present invention. [Figure 4] FIG. 4 shows the changes in body weight in a mouse CIA model by the compounds of the present invention. Detailed Description of the Invention

[0016] Steroid glucocorticoid receptor agonists In one aspect, the present application provides a steroid glucocorticoid receptor agonist that is a compound of Formula I, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, or a prodrug thereof: [ka] During the ceremony, R1 is independently hydrogen, halogen, or -NR a R b , hydroxy, cyano, C 2-6 Alkenyl, C 2-6Alkynyl, C 1-6 Alkyl, C substituted with one or more hydroxy 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 haloalkyl; R2 is hydrogen, C 1-6 Alkyl, hydroxy and C 1-6 selected from the group consisting of alkoxy; Ring A is a single bond, C 6-10 selected from the group consisting of aromatic rings and 5-6 membered heteroaromatic rings; X is a single bond, C 1-6 Alkylene, C 3-6 Cycloalkylene, C 2-6 Alkenylidene, C 2-6 Alkynylidene and -NR a - selected from the group consisting of; Y is selected from the group consisting of -O- and -S-; Z is selected from the group consisting of hydroxy, halogen, and cyano; Q1 and Q2 are each independently selected from the group consisting of hydrogen and halogen; m is selected from the group consisting of 1, 2, 3, 4 and 5; n is selected from the group consisting of 1 and 2; R a and R b are each independently hydrogen and C 1-6 selected from the group consisting of alkyl; With the proviso that: when each R1 is independently selected from the group consisting of halogen, amino, and methyl, m is 2, R2 is methyl, X is a single bond, Y is -S-, Z is fluorine, n is 1, Q1 is hydrogen or fluorine, and Q2 is fluorine, and ring A is not phenyl; When ring A is a single bond, R1 is hydrogen or C 1-6 is not alkyl; and When ring A is a 5- or 6-membered heteroaromatic ring, R1 is hydrogen and R2 is C 1-6 alkyl and Q1 is not a halogen.

[0017] In some embodiments, each R is independently hydrogen, halogen, or —NR a R b , hydroxy, C 1-6 Alkyl, C 1-6 Alkyl, C 1-6 Alkyl-OH and C 1-6 alkoxy is selected from the group consisting of:

[0018] In some embodiments, each R 1 is independently selected from the group consisting of hydrogen, fluorine, chlorine, amino, hydroxy, methyl, ethyl, and hydroxymethyl.

[0019] In some embodiments, each R 1 is independently selected from the group consisting of hydrogen, fluorine, amino, hydroxy, methyl, ethyl, and hydroxymethyl.

[0020] In some embodiments, R2 is selected from the group consisting of hydrogen, methyl, hydroxy, and methoxy.

[0021] In some embodiments, R2 is selected from the group consisting of hydrogen and methyl.

[0022] In some embodiments, ring A is selected from the group consisting of a single bond, a benzene ring, and a 5-6 membered heteroaromatic ring.

[0023] In some embodiments, ring A is selected from the group consisting of a single bond, a benzene ring, a pyridine ring, a furan ring, and a thiophene ring.

[0024] In some embodiments, X is selected from the group consisting of a single bond, methylene, ethylene, propylene, isopropylene, cyclopropylene, butylene, isobutylene, cyclobutylene, cyclohexylene, ethenylidene, and imino.

[0025] In some embodiments, X is selected from the group consisting of a single bond, methylene, ethylene, propylene, butylene, cyclopropylene, cyclohexylene, ethenylidene, and imino.

[0026] In some embodiments, X is selected from the group consisting of a single bond, methylene, ethylene, propylene, butylene, 1,1-cyclopropylene, 1,2-cyclopropylene, 1,4-cyclohexylene, and ethenylidene.

[0027] In some embodiments, Z is selected from the group consisting of hydroxy, fluorine, chlorine, and cyano.

[0028] In some embodiments, Q1 and Q2 are each independently selected from the group consisting of hydrogen, fluorine, and chlorine.

[0029] In some embodiments, Q1 is selected from the group consisting of hydrogen and fluorine, and Q2 is fluorine.

[0030] In some embodiments, m is selected from the group consisting of 1 and 2.

[0031] In some embodiments, n is 1.

[0032] In some embodiments, the steroid glucocorticoid receptor agonist is a compound of Formula II, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, or a prodrug thereof: [ka] During the ceremony, R1, R2, Ring A, Y, Z, Q1, m, and n are as defined above for compounds of formula I.

[0033] In some embodiments, the steroid glucocorticoid receptor agonist is selected from a compound of Formula III, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, or a prodrug thereof: [ka] During the ceremony, R1, ring A, Y, Z, Q1, m, and n are as defined above for compounds of formula I.

[0034] In some embodiments, the steroid glucocorticoid receptor agonist is selected from a compound of Formula IV, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, or a prodrug thereof: [ka] During the ceremony, R1, ring A, Y, Z, Q1, m, and n are as defined above for compounds of formula I.

[0035] In some embodiments, the steroid glucocorticoid receptor agonist is selected from a compound of Formula V, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, or a prodrug thereof: [ka] During the ceremony, R1, R2, Ring A, Y, Z, Q1, m, and n are as defined above for compounds of formula I.

[0036] In some embodiments, the steroid glucocorticoid receptor agonist is selected from a compound of Formula VI, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, or a prodrug thereof: [ka] During the ceremony, Ring B is C 3-6 a cycloalkane, preferably cyclopropane; and R1, R2, Ring A, Y, Z, Q1, m, and n are as defined above for compounds of formula I.

[0037] In some embodiments, the steroid glucocorticoid receptor agonist is selected from the group consisting of the following compounds, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, or a prodrug thereof: [ka] TIFF2026501523000011.tif213170TIFF2026501523000012.tif244170TIFF2026501523000013.tif214170TIFF202 6501523000014.tif213170TIFF2026501523000015.tif230170TIFF2026501523000016.tif230170TIFF2026501523 000017.tif223170TIFF2026501523000018.tif250170TIFF2026501523000019.tif217170TIFF2026501523000020. tif217170TIFF2026501523000021.tif217170TIFF2026501523000022.tif217170TIFF2026501523000023.tif83170

[0038] In some embodiments, the steroid glucocorticoid receptor agonist is selected from the group consisting of the following compounds, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, or a prodrug thereof: [ka]

[0039] In some embodiments, the steroid glucocorticoid receptor agonist is selected from the group consisting of the following compounds, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, or a prodrug thereof: [ka] TIFF2026501523000026.tif213170TIFF2026501523000027.tif215170

[0040] antibody-drug conjugates In another embodiment, the present application provides a compound of formula Ab-[MLED] x and an antibody-drug conjugate having a structure represented by the formula: Ab is an antibody that specifically binds to an antigen or an antigen-binding fragment thereof; M is a linking site to an antibody or antigen-binding fragment thereof; L is a linker between linking sites M and E; E is a structural fragment connecting L with D; D is a glucocorticoid drug fragment, and the glucocorticoid drug is a glucocorticoid receptor agonist; X is an integer selected from 1 to 10, and preferably an integer selected from 3 to 8.

[0041] In the antibody-drug conjugate, the glucocorticoid receptor agonist can be linked to the antibody or antigen-binding fragment thereof via a linking moiety (e.g., an "MLE" fragment as set forth herein).

[0042] In some embodiments, the antigen bound to the antibody in the antibody-drug conjugate includes, but is not limited to, TNFα, IL6R, BDCA2, NR3C1, MSR1, PRLR, CD25, CD40, CD70, CD74, CD163, etc.

[0043] In some embodiments, the antibody or antigen-binding fragment thereof includes, but is not limited to, adalimumab, tocilizumab, and IgG1 antibodies.

[0044] In some embodiments, M is [ka] and ring C is a single bond, halogen, a 5-6 membered aliphatic heterocyclic ring system or a 5-20 membered aromatic ring system, and the aliphatic heterocyclic ring system or aromatic ring system is selected from the group consisting of oxo (=O), halogen, cyano, amino, carboxyl, mercapto, and C 1-6 alkyl; and M1 is selected from the group consisting of a single bond or a fragment consisting of one or more groups selected from the group consisting of: -O-, -NH-, -C(=O)-, -S(=O)2-, -C=NO-, -NH-S(=O)2-NH-, phenylene, 5-10 membered heteroaryl, C 1-20 Alkylene, C 2-20 Alkenylidene and C 2-20 Alkynylidene.

[0045] In some embodiments, M is [ka] wherein ring C is a polycyclic ring formed by 1 to 5 (preferably 3) units selected from a single bond, a halogen, a 5-membered aliphatic heterocycle, a 6-membered heteroaromatic ring, or a 6-membered heteroaromatic ring and a benzene ring connected via a single bond, and the aliphatic heterocycle, heteroaromatic ring, or polycyclic ring is selected from oxo (=O), halogen, and C 1-4 alkyl; and M1 is a single bond, or -NH-, -C(=O)-, -NH-S(=O)2-NH-, C 1-10 Alkylene, C 2-10 Alkenylidene and C 2-10 Alkynylidene is selected from the group consisting of fragments consisting of one or more groups selected from the group consisting of:

[0046] In some embodiments, M is [ka] and ring C is [ka] and M1 is selected from the group consisting of a single bond, -NH-, -C(=O)-, -NH-S(=O)2-NH-, C 1-6 Alkylene, C 2-6 Alkenylidene and C 2-6 Alkynylidene is selected from the group consisting of:

[0047] In some embodiments, M is [ka] is selected from the group consisting of:

[0048] In some embodiments, M is [ka] is selected from.

[0049] In some embodiments, M is [ka] is selected from the group consisting of:

[0050] In some embodiments, M is [ka] is selected from the group consisting of:

[0051] In some embodiments, L is a structure consisting of one or more moieties selected from the group consisting of: 1-6 Alkylene, -N(R')carbonyl-O-, natural or unnatural amino acids and analogs or derivatives thereof (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) as well as multiple amino acids (e.g., 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, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), [ka] a short polypeptide chain consisting of: R' is hydrogen, C 1-6 Alkyl or -(CH2CH2O) r -Contains C4-30 represents alkyl; r is an integer of 1 to 10; and s is an integer of 1 to 20. Preferably, r is an integer of 1 to 6; and preferably, s is an integer of 1 to 10.

[0052] In some embodiments, L is C 1-6 Alkylene, carbonyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Lys, V al-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, G ly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, [ka] a structure comprising one or more moieties selected from the group consisting of: s is an integer from 1 to 20.

[0053] In some embodiments, L is a structure consisting of one or more moieties selected from the group consisting of: 1-6Alkylene, carbonyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Lys, V al-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, G ly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, [ka] s is an integer of 1 to 20, preferably an integer of 1 to 10.

[0054] In some embodiments, L is a structure consisting of one or more moieties selected from the group consisting of: [ka] s is an integer of 1 to 20, and preferably an integer of 1 to 10.

[0055] In some embodiments, L is a structure consisting of one or more moieties selected from the group consisting of: [ka] s is an integer of 1 to 20, and preferably an integer of 1 to 10.

[0056] In some embodiments, L is a structure consisting of one or more moieties selected from the group consisting of: [ka] s is an integer of 1 to 20, and preferably an integer of 1 to 10.

[0057] In some embodiments, L is a structure consisting of one or more moieties selected from the group consisting of: [ka] TIFF2026501523000043.tif176170s is an integer from 1 to 20, and preferably an integer from 1 to 10.

[0058] In some embodiments, L is selected from the following structures: [ka] s is an integer of 1 to 20, and preferably an integer of 1 to 10.

[0059] In some embodiments, L is selected from the following structures: [ka] s is an integer of 1 to 20, and preferably an integer of 1 to 10.

[0060] In some embodiments, L is selected from the following structures: [ka] s is an integer of 1 to 20, and preferably an integer of 1 to 10.

[0061] In some embodiments, L is selected from the following structures: [ka] s is an integer of 1 to 20, and preferably an integer of 1 to 10.

[0062] In some embodiments, L is selected from the following structures: [ka] s is an integer of 1 to 20, and preferably an integer of 1 to 10.

[0063] In some embodiments, L is selected from the following structures: [ka]

[0064] In some embodiments, L is selected from the following structures: [ka]

[0065] In some embodiments, E is a single bond, a carbonyl, -NHCH2-, or a structure selected from the group consisting of: [ka] s is an integer of 1 to 20, and preferably an integer of 1 to 10.

[0066] In some embodiments, E is a single bond or -NHCH2-.

[0067] In some embodiments, E is a single bond.

[0068] In some embodiments, [ka] is selected from the following structures: [ka] TIFF2026501523000054.tif248170TIFF2026501523000055.tif237170TIFF2026501523000056.tif220170TIFF2026501523000057.tif219170TIFF2026501523000058.tif207170TIFF2026501523000059.tif103170s is an integer from 1 to 20, preferably an integer from 1 to 10.

[0069] In some embodiments, [ka] is selected from the following structures: [ka] TIFF2026501523000062.tif251170TIFF2026501523000063.tif233170TIFF2026501523000064.tif228170 TIFF2026501523000065.tif238170TIFF2026501523000066.tif227170TIFF2026501523000067.tif212170

[0070] In some embodiments, [ka] teeth, The structure is selected from the following: [ka] TIFF2026501523000070.tif215170TIFF2026501523000071.tif236170TIFF2026501523000072.tif238170TIFF2026501523000073.tif38170

[0071] In some embodiments, D is a corresponding agonist fragment obtained by linking a steroid glucocorticoid receptor agonist of the present invention to a linking moiety.

[0072] In some embodiments, D is a monovalent structure obtained by removing one H from the OH, -NH2, or secondary amino of a steroid glucocorticoid receptor agonist of the invention.

[0073] In some embodiments, D is selected from the following structures: [ka] TIFF2026501523000075.tif226170TIFF2026501523000076.tif244170TIFF2026501523000077.tif24117 0TIFF2026501523000078.tif223170TIFF2026501523000079.tif230170TIFF2026501523000080.tif22717 0TIFF2026501523000081.tif208170TIFF2026501523000082.tif246170TIFF2026501523000083.tif24917 0TIFF2026501523000084.tif249170TIFF2026501523000085.tif245170TIFF2026501523000086.tif45170

[0074] In some embodiments, D is selected from the following structures: [ka] JPEG2026501523000088.jpg231170JPEG2026501523000089.jpg229170JPEG2026501523000090.jpg139170

[0075] In some embodiments, D is selected from the following structures: [ka] JPEG2026501523000092.jpg172170

[0076] In some embodiments, the antibody-drug conjugate is selected from the group consisting of the following formulae: ADC-A-01 to ADC-A-145, ADC-B-01 to ADC-B-146, wherein Ab in the following formulae is as defined above, a mercapto on the antibody and a drug-linker compound form a thioether bond via an addition reaction or a substitution reaction to obtain a complete antibody-drug conjugate, x represents the drug loading capacity, and s represents an integer of 1 to 20, preferably an integer of 1 to 10. [ka] TIFF2026501523000094.tif199170TIFF2026501523000095.tif205170TIFF2026501523000096.tif185170TIFF2026501523000097.tif194170TIFF2026501523000098.tif206170TIFF2026501523000099.tif225170TIFF2026501523000100.tif201170TIFF2026501523000101.tif228170TIFF2026501523000102.tif187170TIFF2026501523000103.tif222170TIFF2026501523000104.tif206170TIFF2026501523000105.tif197170TIFF2026501523000106.tif211170TIFF2026501523000107.tif211170TIFF2026501523000108.tif218170TIFF2026501523000109.tif207170TIFF2026501523000110.tif213170TIFF2026501523000111.tif235170TIFF2026501523000112.tif171170TIFF2026501523000113.tif178170TIFF2026501523000114.tif229170TIFF2026501523000115.tif213170TIFF2026501523000116.tif222170TIFF2026501523000117.tif225170TIFF2026501523000118.tif202170TIFF2026501523000119.tif200170TIFF2026501523000120.tif192170TIFF2026501523000121.tif192170TIFF2026501523000122.tif189170TIFF2026501523000123.tif226170TIFF2026501523000124.tif220170TIFF2026501523000125.tif216170TIFF2026501523000126.tif224170TIFF2026501523000127.tif205170TIFF2026501523000128.tif215170TIFF2026501523000129.tif182170TIFF2026501523000130.tif208170TIFF2026501523000131.tif175170TIFF2026501523000132.tif229170TIFF2026501523000133.tif181170TIFF2026501523000134.tif230170TIFF2026501523000135.tif208170TIFF2026501523000136.tif216170TIFF2026501523000137.tif216170TIFF2026501523000138.tif218170TIFF2026501523000139.tif207170TIFF2026501523000140.tif229170TIFF2026501523000141.tif203170TIFF2026501523000142.tif177170TIFF2026501523000143.tif184170TIFF2026501523000144.tif222170TIFF2026501523000145.tif209170TIFF2026501523000146.tif204170TIFF2026501523000147.tif221170TIFF2026501523000148.tif229170TIFF2026501523000149.tif214170TIFF2026501523000150.tif221170TIFF2026501523000151.tif187170TIFF2026501523000152.tif211170TIFF2026501523000153.tif217170TIFF2026501523000154.tif166170TIFF2026501523000155.tif186170TIFF2026501523000156.tif221170TIFF2026501523000157.tif221170TIFF2026501523000158.tif206170TIFF2026501523000159.tif202170TIFF2026501523000160.tif199170TIFF2026501523000161.tif209170TIFF2026501523000162.tif224170TIFF2026501523000163.tif227 170TIFF2026501523000164.tif206170TIFF2026501523000165.tif226170TIFF2026501523000166.tif189170TIFF2026501523000167.tif187170In the expression. [ka] represents the specific linkage between a sulfhydryl group in an antibody or antigen-binding fragment thereof and a linking site. s is an integer selected from 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; and n is an integer selected from 0 to 20, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0077] In some embodiments, Ab in the antibody-drug conjugate refers to an antibody or antigen-binding fragment thereof comprising a VH set forth in SEQ ID NO: 1 and a VL set forth in SEQ ID NO: 2, for example, an antibody or antigen-binding fragment thereof comprising a VH set forth in SEQ ID NO: 1, a CH (heavy chain constant region) set forth in SEQ ID NO: 3, a VL set forth in SEQ ID NO: 2, and a CL (light chain constant region) set forth in SEQ ID NO: 4.

[0078] SEQ ID NO: 1 EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS

[0079] SEQ ID NO: 2 DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK

[0080] SEQ ID NO: 3 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0081] SEQ ID NO:4 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0082] In some embodiments, Ab in the antibody-drug conjugate represents an antibody or antigen-binding fragment thereof comprising a VH set forth in SEQ ID NO: 7 and a VL set forth in SEQ ID NO: 8, e.g., an antibody or antigen-binding fragment thereof comprising a VH set forth in SEQ ID NO: 7 and a CH (heavy chain constant region) set forth in SEQ ID NO: 9 or a CH (mutated heavy chain constant region) set forth in SEQ ID NO: 11, and a VL set forth in SEQ ID NO: 8 and a CL (light chain constant region) set forth in SEQ ID NO: 10.

[0083] SEQ ID NO:7 QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSS

[0084] SEQ ID NO:8 DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIK

[0085] SEQ ID NO:9 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0086] SEQ ID NO: 10 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0087] SEQ ID NO: 11 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0088] In some embodiments, the DAR value (drug to antibody conjugate ratio) of the antibody drug conjugate is 1 to 10, e.g., 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, preferably 3 to 9, for example, 3.0 to 3.5, 3.0 to 4.0, 3.0 to 4.5, 3.0 to 5.0, 3.0 to 5.5, 3.0 to 6.0, 3.5 to 4.0, 3.5 to 4.5, 3.5 to 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.

[0089] In some embodiments, the wavy line [ka] indicates the point of attachment between that moiety and the rest of the molecule.

[0090] Drug-Linker Compounds Those skilled in the art will appreciate that the antibody-drug conjugates of the present application can be prepared in a modular manner. For example, the free form of the "drug-linker" (GM-[LED] xwhere GM is in a structural form prior to covalent binding to an antibody or antigen-binding fragment thereof, and x is an integer selected from 1 to 10), which is then covalently bound to an antibody or antigen-binding fragment thereof to obtain the antibody-drug conjugate of the present application. Correspondingly, the free form of the "drug-linker," GM, is linked to one or more mercapto (-SH), amino (-NH), or carboxyl (-COOH) groups on the antibody or antigen-binding fragment thereof via a substitution reaction (e.g., removal of structures such as -SOMe or -Br), an addition reaction, or the like.

[0091] G is a functional group or leaving group that reacts with specific amino acids or glycosyl groups and their derivatives in the antibody or antigen-binding fragment; M is a linking site to an antibody or antigen-binding fragment thereof; L is a linker between linking sites M and E; E is a structural fragment connecting L with D; D is a glucocorticoid drug fragment that is a monovalent structure obtained by removing one H from the -OH, -NH, or secondary amino of a steroid glucocorticoid receptor agonist of the present invention; and x is an integer selected from 1 to 10, and is preferably 1 or 2.

[0092] In some embodiments, M, L, E, D, and x are as described above in the "Drug-Antibody Conjugate" section.

[0093] In some embodiments, G is halogen, halphenoxy, C 1-6 Haloalkyl, sulfonate (HOS(=O)2-), C 1-6 Alkyl sulfonyl, C 1-6 Haloalkylsulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate, C 1-6 Haloalkylsulfonates, C 1-6 Alkyl sulfite, halosulfonate, C 1-6Alkyl sulfoxide, methylsulfonyl methacryloyl, dimethylsulfonyl methacryloyl ( [ka] ), haloformyl, haloacetyl, formyl, acetyl, nitro, azido, cyano, cyanovinyl, N-methyl-vinylsulfonamide ( [ka] ), tetrazinyl, methyltetrazinyl, trans-cyclooctenyl carbonate group, C 2-6 Alkenyl, C 2-6 It is selected from the group consisting of alkynyl, benzazacyloctynyl, (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy.

[0094] Ring C is a 5-6 membered aliphatic heterocyclic ring system or a 5-20 membered aromatic ring system, and the aliphatic heterocyclic ring system or aromatic ring system is oxo (=O), halogen, cyano, amino, carboxyl, mercapto, and C 1-6 optionally substituted with one or more groups each independently selected from the group consisting of alkyl; and M1 is selected from the group consisting of a single bond or a fragment consisting of one or more groups selected from the following: -O-, -NH-, -C(=O)-, -S(=O)2-, -C=NO-, -NH-S(=O)2-NH-, phenylene, 5-10 membered heteroarylidene, C 1-20 Alkylene, C 2-20 Alkenylidene and C 2-20 Alkynylidene.

[0095] In some embodiments, GM is [ka] where: [ka] teeth, [ka] selected from the group consisting of: M1 is a single bond, -O-, -NH-, -NH-S(=O)2-NH-, C 1-10 Alkylene, C 2-6 Alkenylidene and C 2-6 Alkynylidene is selected from the group consisting of:

[0096] In some embodiments, GM is [ka] is.

[0097] In some embodiments, GM is [ka] is.

[0098] In some embodiments, GM is [ka] is.

[0099] In some embodiments, GM is [ka] is.

[0100] In some embodiments, the free form of the "drug-linker" is selected from the group consisting of: [ka] TIFF2026501523000180.tif193170TIFF2026501523000181.tif198170TIFF2026501523000182.tif184170TIFF2026501523000183.tif199170TIFF2026501523000184.tif201170TIFF2026501523000185.tif215170TIFF2026501523000186.tif186170TIFF2026501523000187.tif214170TIFF2026501523000188.tif216170TIFF2026501523000189.tif186170TIFF2026501523000190.tif220170TIFF2026501523000191.tif188170TIFF2026501523000192.tif197170TIFF2026501523000193.tif197170TIFF2026501523000194.tif211170TIFF2026501523000195.tif207170TIFF2026501523000196.tif214170TIFF2026501523000197.tif199170TIFF2026501523000198.tif189170TIFF2026501523000199.tif206170TIFF2026501523000200.tif227170TIFF2026501523000201.tif203170TIFF2026501523000202.tif223170TIFF2026501523000203.tif227170TIFF2026501523000204.tif229170TIFF2026501523000205.tif228170TIFF2026501523000206.tif184170TIFF2026501523000207.tif230170TIFF2026501523000208.tif230170TIFF2026501523000209.tif211170TIFF2026501523000210.tif220170TIFF2026501523000211.tif194170TIFF2026501523000212.tif200170TIFF2026501523000213.tif179170TIFF2026501523000214.tif209170TIFF2026501523000215.tif194170TIFF2026501523000216.tif190170TIFF2026501523000217.tif170170TIFF2026501523000218.tif188170TIFF2026501523000219.tif170170TIFF2026501523000220.tif170170TIFF2026501523000221.tif186170TIFF2026501523000222.tif175170TIFF2026501523000223.tif175170TIFF2026501523000224.tif182170TIFF2026501523000225.tif216170TIFF2026501523000226.tif202170TIFF2026501523000227.tif177170TIFF2026501523000228.tif180170TIFF2026501523000229.tif193170TIFF2026501523000230.tif191170TIFF2026501523000231.tif176170TIFF2026501523000232.tif180170TIFF2026501523000233.tif174170TIFF2026501523000234.tif174170TIFF2026501523000235.tif185170TIFF2026501523000236.tif221170TIFF2026501523000237.tif221170TIFF2026501523000238.tif203170TIFF2026501523000239.tif220170TIFF2026501523000240.tif167170TIFF2026501523000241.tif170170TIFF2026501523000242.tif170170TIFF2026501523000243.tif197170TIFF2026501523000244.tif212170TIFF2026501523000245.tif193170TIFF2026501523000246.tif193170TIFF2026501523000247.tif185170TIFF2026501523000248.tif170170TIFF202650152300024 9.tif175170TIFF2026501523000250.tif175170TIFF2026501523000251.tif229170TIFF2026501523000252.tif194170TIFF20265015230002 53.tif189170TIFF2026501523000254.tif196170TIFF2026501523000255.tif205170TIFF2026501523000256.tif179170TIFF2026501523000257.tif182170TIFF2026501523000258.tif193170TIFF2026501523000259.tif190170In the formula, s is an integer selected from 1 to 20, preferably an integer selected from 1 to 10.

[0101] In some embodiments, the free form of the "drug-linker" is selected from the group consisting of: [ka] TIFF2026501523000261.tif191170TIFF2026501523000262.tif198170TIFF2026501523000263.tif183170TIFF2026501523000264.tif193170TIFF2026501523000265.tif201170TIFF2026501523000266.tif217170TIFF2026501523000267.tif186170TIFF2026501523000268.tif214170TIFF2026501523000269.tif216170TIFF2026501523000270.tif186170TIFF2026501523000271.tif186170TIFF2026501523000272.tif192170TIFF2026501523000273.tif189170TIFF2026501523000274.tif194170TIFF2026501523000275.tif222170TIFF2026501523000276.tif223170TIFF2026501523000277.tif203170TIFF2026501523000278.tif186170TIFF2026501523000279.tif189170TIFF2026501523000280.tif219170TIFF2026501523000281.tif231170TIFF2026501523000282.tif200170TIFF2026501523000283.tif224170TIFF2026501523000284.tif197170TIFF2026501523000285.tif214170TIFF2026501523000286.tif212170TIFF2026501523000287.tif190170TIFF2026501523000288.tif197170TIFF2026501523000289.tif209170TIFF2026501523000290.tif209170TIFF2026501523000291.tif221170TIFF2026501523000292.tif187170TIFF2026501523000293.tif194170TIFF2026501523000294.tif215170TIFF2026501523000295.tif228170TIFF2026501523000296.tif216170TIFF2026501523000297.tif183170TIFF2026501523000298.tif224170TIFF2026501523000299.tif183170TIFF2026501523000300.tif171170TIFF2026501523000301.tif223170TIFF2026501523000302.tif219170TIFF2026501523000303.tif229170TIFF2026501523000304.tif228170TIFF2026501523000305.tif216170TIFF2026501523000306.tif188170TIFF2026501523000307.tif212170TIFF2026501523000308.tif169170TIFF2026501523000309.tif192170TIFF2026501523000310.tif181170TIFF2026501523000311.tif218170TIFF2026501523000312.tif218170TIFF2026501523000313.tif221170TIFF2026501523000314.tif211170TIFF2026501523000315.tif202170TIFF2026501523000316.tif186170TIFF2026501523000317.tif222170TIFF2026501523000318.tif196170TIFF2026501523000319.tif170170TIFF2026501523000320.tif170170TIFF2026501523000321.tif182170TIFF2026501523000322.tif202170TIFF2026501523000323.tif177170TIFF2026501523000324.tif177170TIFF2026501523000325.tif226170TIFF2026501523000326.tif216170TIFF2026501523000327.tif216170TIFF2026501523000328.tif222170TIFF20265 01523000329.tif192170TIFF2026501523000330.tif191170TIFF2026501523000331.tif196 170TIFF2026501523000332.tif210170TIFF2026501523000333.tif176170TIFF20265015230 00334.tif182170TIFF2026501523000335.tif193170TIFF2026501523000336.tif192170In the expression. s is selected from the group consisting of integers ranging from 1 to 20, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; and n is selected from the group consisting of integers ranging from 1 to 20, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0102] Pharmaceutical Composition In another aspect, the present application provides a pharmaceutical composition comprising a glucocorticoid receptor agonist of the present invention, an antibody-drug conjugate of the present invention, or a drug-linker compound of the present invention, and one or more pharmaceutically acceptable carriers.

[0103] The selection of pharmaceutically acceptable carriers depends on the dosage form of the pharmaceutical composition, firstly on the route of administration of the dosage form, and secondly on the formulation of the dosage form.For example, pharmaceutically acceptable carriers can include water (e.g., water for injection), buffer, isotonic salt solution such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ascorbic acid, lactic acid, ethanol, polyalkylene glycols (e.g., polyethylene glycol 4000), such as polyethylene glycol, polypropylene glycol, triglycerides, etc.

[0104] The glucocorticoid receptor agonist, antibody-drug conjugate or drug-linker compound according to the present invention is usually formulated together with a pharmaceutically acceptable parenteral vehicle as a unit injectable form for parenteral use, such as subcutaneous injection, intramuscular injection, intravenous injection, drip injection, local injection into diseased tissue, intratumoral injection, etc. Optionally, the glucocorticoid receptor agonist, antibody-drug conjugate or drug-linker compound having a desired purity is mixed with a pharmaceutically acceptable diluent, carrier, excipient or stabilizer in the form of a lyophilized preparation or solution (Remington's Pharmaceutical Sciences (1980) 16 th (Osol, A. Ed.) The glucocorticoid receptor agonist, antibody drug conjugate or drug-linker compound according to the present invention or the pharmaceutical composition according to the present invention can be administered by any route appropriate to the subject to be treated.

[0105] Treatment Methods and Uses In another aspect, the present application provides use of a steroid glucocorticoid receptor agonist of the present invention, an antibody-drug conjugate of the present invention, a drug-linker compound of the present invention, or a pharmaceutical composition of the present invention in the manufacture of a medicament for treating an inflammatory disease or an immune disease.

[0106] In another aspect, the present application provides a steroid glucocorticoid receptor agonist of the present invention, an antibody-drug conjugate of the present invention, a drug-linker compound of the present invention, or a pharmaceutical composition of the present invention for use in the treatment of an inflammatory disease or an immune disease.

[0107] In another aspect, the present application provides a method for treating an inflammatory disease or an immune disease, comprising administering a therapeutically effective amount of a steroid glucocorticoid receptor agonist of the present invention, an antibody-drug conjugate of the present invention, a drug-linker compound of the present invention, or a pharmaceutical composition of the present invention to a subject in need thereof.

[0108] In one embodiment, the inflammatory or immune disease is a TNFα or IL-6R overexpressing disease, including but not limited to rheumatoid arthritis, idiopathic arthritis, asthma, ulcerative colitis, neuromyelitis optica, and autoimmune liver disease.

[0109] definition Unless otherwise specified below, the meanings of all technical and scientific terms used in the present invention are intended to be the same as those commonly understood by those skilled in the art. The techniques used in the present invention are intended to refer to techniques commonly understood in the art, including modifications or equivalent substitutions that are obvious to those skilled in the art. Furthermore, the experimental procedures used in the present invention, such as genomics, nucleic acid chemistry, and molecular biology, are all conventional procedures widely used in their respective fields. Although the following terms are believed to be easily understood by those skilled in the art, the following definitions are provided as examples to better explain the present invention.

[0110] The term "antibody" refers to an immunoglobulin molecule consisting of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). Antibody light chains can be classified as kappa (κ) light chains and lambda (λ) 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. In light and heavy chains, the variable and constant regions are connected via a "J" region of about 12 or more amino acids, and heavy chains also contain a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain, CL. The constant regions are not directly involved in antibody binding to antigens but exhibit multiple effector functions, such as mediating immunoglobulin interactions with host tissues and factors, including binding to various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). The VH and VL regions can be further subdivided into highly variable regions (called complementarity-determining regions (CDRs)) interspersed with conserved regions called framework regions (FRs). VH and VL each consist of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of each heavy / light chain pair form the respective antigen-binding site. The distribution of amino acids in each region or domain follows various numbering systems known in the art.

[0111] 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. The variable regions of the heavy and light chains each contain three CDRs, designated CDR1, CDR2, and CDR3, respectively. The exact boundaries of these CDRs can be defined according to 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) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272). For a given antibody, one skilled in the art would readily identify the CDRs defined according to each numbering system. Furthermore, the correspondence between different numbering systems is known to those skilled in the art (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0112] In the present invention, the CDRs contained in an antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art, such as the Kabat numbering system, the Chothia numbering system, the IMGT numbering system, or the AbM numbering system. In some embodiments, the CDRs contained in an antibody or antigen-binding fragment thereof are determined according to the Chothia numbering system.

[0113] The terms "framework region" or "FR" residues refer to amino acid residues in antibody variable regions other than the CDR residues defined above.

[0114] The term "antigen-binding fragment" of an antibody refers to a polypeptide fragment of an antibody, e.g., a polypeptide fragment of a full-length antibody, that retains the ability to specifically bind to the same antigen as the full-length antibody and / or competes with the full-length antibody for specific binding to an antigen, referred to as an "antigen-binding portion." See generally Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition), Raven Press, NY (1989), the disclosure of which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques 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, d-scFv, (scFv)2, disulfide bond stabilized Fv proteins ("dsFv"), single domain antibodies (sdAb, nanobodies), and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. A review of artificial antibody variants is summarized in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0115] The term "Fd" refers to an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" refers to an antibody fragment consisting of the VH domain (Ward et al, Nature 341:544 546 (1989)); the term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; and the term "Fab' fragment" refers to a fragment obtained after reducing the disulfide bond linking the two heavy chain fragments of the F(ab')2 fragment, and consists of an intact light chain and an Fd fragment of the heavy chain (composed of the VH and CH1 domains).

[0116] The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single antibody arm. An Fv fragment is generally considered the minimum antibody fragment capable of forming an intact antigen-binding site. It is generally recognized that the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable region (e.g., an Fd fragment containing only three antigen-specific CDRs) can recognize and bind to an antigen, although perhaps with lower affinity than the intact binding site.

[0117] The term "Fc" refers to an antibody fragment formed by disulfide bonding between the second and third constant regions of a first antibody heavy chain and the second and third constant regions of a second antibody heavy chain. The Fc fragment of an antibody has a variety of different functions, but is not involved in antigen binding.

[0118] The term "scFv" refers to a single polypeptide chain comprising VL and VH domains, where the VL and VH are linked via a linker (see, e.g., 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, Volume 113, edited by Roseburg and Moore, Springer-Verlag, New York, pages 269-315 (1994)). Such scFv molecules can have the general structure NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable existing linkers consist of GGGGS amino acid repeats or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, although 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 the present invention are described in 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 instances, there may be a disulfide bond between the VH and VL of the scFv. In some instances, the VH and VL domains are linked together, e.g., NH2-VH-VH-COOH and NH 2- They can be arranged relative to each other in any suitable configuration, such as an scFv comprising VL-VL-COOH.

[0119] The term "single domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art and refers to an antibody fragment consisting of a single monomeric variable antibody domain (such as a single heavy chain variable region) and which retains the ability to specifically bind to the same antigen as a full-length antibody (Holt, L. et al, Trends in Biotechnology, 21(11):484-490, 2003). Single domain antibodies are also called nanobodies.

[0120] All of the above antibody fragments retain the ability to specifically bind to the same antigen as the full-length antibody and / or compete with the full-length antibody for specific binding to an antigen.

[0121] Herein, unless the context clearly indicates otherwise, reference to the term "antibody" includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0122] Antigen-binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided in the present invention) via conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies are screened for specificity in the same manner as are intact antibodies.

[0123] The term "mouse-derived antibody" refers to an antibody obtained by the following method: fusing B cells from an immunized mouse with myeloma cells, screening for mouse hybrid fusion cells that can achieve both infinite proliferation and antibody secretion, followed by screening, antibody preparation, and antibody purification; or it refers to an antibody secreted by plasma cells formed by the differentiation and proliferation of B cells after antigen infiltration into a mouse.

[0124] The term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been altered to improve homology with that of a human antibody. Generally, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (e.g., variable region FRs and / or constant regions) are derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain the desired properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activation, and ability to enhance immune responses. The donor antibody can be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody with the desired properties (e.g., antigen specificity, affinity, reactivity, ability to enhance immune cell activation, and / or ability to enhance immune responses).

[0125] The term "identity" refers to the sequence match between two polypeptides or two nucleic acids. If a position in two compared sequences is occupied by the same base or amino acid monomer subunit (e.g., if a position in each of two DNA molecules is occupied by adenine, or if a position in each of two polypeptides is occupied by lysine), the molecules are identical at that position. The "percentage identity" between two sequences refers to a function calculated by dividing the number of matching positions shared by the two sequences by the total number of compared positions and multiplying by 100. For example, if 6 out of 10 positions in two sequences are identical, the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (3 out of 6 positions are identical). Generally, comparisons are performed when the two sequences are aligned to maximize identity. Such alignment can be achieved, for example, using a method that can be easily performed using a computer program such as the Align program (DNAstar, Inc.) (Needleman et al. (1970) J. Mol. Biol. 48: 443-453). The percentage of 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)). This algorithm has been integrated into the ALIGN program (version 2.0), and uses a PAM120 weight residue table with a gap length penalty score of 12 and a gap length penalty score of 4. The percentage of identity between two amino acid sequences can also be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)).This algorithm has been integrated into the GAP program in the GCC software package (available at www.gcg.com) and uses the Blossum 62 matrix or the PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0126] The term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the expected properties of a protein / polypeptide containing the amino acid sequence. For example, conservative amino acid substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include replacing an amino acid residue with an amino acid residue having a similar side chain, e.g., using a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include, but are not limited to, 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), beta-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., 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. Sci. USA 94:412-417 (1997), the disclosures of which are incorporated herein by reference).

[0127] The 20 common amino acids included in the present invention are designated according to conventional usage. See, for example, "Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991))," the disclosure of which is incorporated herein by reference. In the present invention, amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented as A or Ala.

[0128] In the present invention, the words "include," "comprise," "have," "contain," or "involve," and variations thereof, are intended to be inclusive or open-ended and do not exclude other elements or method steps not listed.

[0129] The term "alkyl" refers to a group obtained by removing one hydrogen atom from a straight or branched chain alkane, e.g., "C 1-20 Alkyl”, “C 1-10 Alkyl”, “C 1-6 Alkyl”, “C 1-4 Alkyl”, “C 1-3 Illustrative 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, 2,2-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, and 1,2-dimethylpropyl.

[0130] The term "alkenyl" refers to a straight or branched chain aliphatic hydrocarbon group having one or more unsaturated double bonds, such as C 2-20Alkenyl, C 2-10 Alkenyl, C 2-6 Alkenyl, C 2-4 Alkenyl, etc. 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, 2-methyl-2-penten-1-yl, etc.

[0131] The term "alkynyl" refers to a straight or branched chain aliphatic hydrocarbon group having one or more unsaturated triple bonds, e.g., "C 2-20 Alkynyl”, “C 2-10 Alkynyl”, “C 2-6 Alkynyl”, “C 2-4 alkynyl", 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, 2-hexyn-1-yl, etc.

[0132] The term "alkylene" refers to a group obtained by removing two hydrogen atoms from a straight or branched chain alkane, e.g., "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 1-3Specific examples include, but are not limited to, methylene, ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene, and 1,6-hexylene.

[0133] The term "alkenylidene" refers to a divalent group obtained by removing two hydrogen atoms from a straight or branched chain alkene, e.g., "C 2-20 Alkenylidene”, “C 3-10 Alkenylidene”, “C 5-8 Specific examples include, but are not limited to, ethenylidene, 1-propenylidene, 2-propenylidene, 1-butenylidene, 2-butenylidene, 1,3-butadienylidene, 1-pentenylidene, 2-pentenylidene, 3-pentenylidene, 1,3-pentadienylidene, 1,4-pentadienylidene, 1-hexenylidene, 2-hexenylidene, 3-hexenylidene, 1,4-hexadienylidene, and the like.

[0134] The term "alkynylidene" refers to a divalent group obtained by removing two hydrogen atoms from a straight- or branched-chain alkene, and includes "C 2-20 Alkynylidene”, “C 3-10 Alkynylidene”, “C 5-8 Specific examples include, but are not limited to, ethynylidene, 1-propynylidene, 2-propynylidene, 1-butynylidene, 2-butynylidene, 1,3-butadiynylidene, 1-pentynylidene, 2-pentynylidene, 3-pentynylidene, 1,3-pentadiynylidene, 1,4-pentadiynylidene, 1-hexynylidene, 2-hexynylidene, 3-hexynylidene, 1,4-hexadiynylidene, and the like.

[0135] The term "cycloalkylene" refers to a group obtained by removing two hydrogen atoms from a cycloalkane, e.g., "C 3-20 Cycloalkylene”, “C 3-10 Cycloalkylene”, “C 3-6Specific examples include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and the like.

[0136] The term "alkoxy" means an "alkyl," as defined above, attached to the parent molecular moiety through an oxygen atom, e.g., C1-C6 alkoxy and C1-C3 alkoxy. Specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutyloxy, pentoxy, isopentyloxy, hexyloxy, and the like.

[0137] The term "halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0138] The term "haloalkyl" refers to an alkyl substituted with one or more (e.g., 1 to 3) of the same or different halogen atoms. For example, the term "C1-C6 haloalkyl" refers to a haloalkyl having 1 to 6 carbon atoms, including, but not limited to, -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, -CH2CH2CF3, -CH2Cl, and the like.

[0139] The term "aromatic ring" refers to an all-carbon, monocyclic or polycyclic aromatic hydrocarbon. Common aromatic rings include, but are not limited to, benzene, naphthalene, anthracene, phenanthrene, acenaphthene, azulene, fluorene, indene, and pyrene. For example, "C6-C 10 The term "aromatic ring" refers to an aromatic ring containing 6 to 10 carbon atoms, such as benzene or naphthalene.

[0140] The term "aliphatic heterocycle" refers to a saturated or partially saturated cyclic structure containing at least one ring member selected from the group consisting of N, O, and S, including, but not limited to, 5- to 10-membered aliphatic heterocycles, 5- to 6-membered aliphatic heterocycles, etc., such as, for example, 5- to 6-membered nitrogen-containing aliphatic heterocycles, 5- to 6-membered oxygen-containing aliphatic heterocycles, etc. Specific examples include, but are not limited to, tetrahydrofuran, pyrrolidine, piperidine, tetrahydropyran, etc.

[0141] The term "heteroaromatic ring" refers to an aromatic ring structure containing at least one ring member selected from the group consisting of N, O, and S, including, but not limited to, 5- to 10-membered heteroaromatic rings, 5- to 6-membered heteroaromatic rings, etc., such as a 5- to 6-membered nitrogen-containing heteroaromatic ring, a 5- to 6-membered oxygen-containing heteroaromatic ring, 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-tetrazine, etc.

[0142] The term "aromatic ring system" refers to a monocyclic or polycyclic system containing at least one aromatic ring (e.g., a benzene ring) or heteroaromatic ring (e.g., a pyrimidine ring). Two or more aromatic and / or heteroaromatic rings can form fused rings or can be linked via a single bond (e.g., dipyrimidylphenyl). The aromatic ring system can be divalent or higher (e.g., trivalent or tetravalent), for example, a 5- to 20-membered aromatic ring system.

[0143] When a substituent is described as "optionally substituted with...," the substituent can be (1) unsubstituted or (2) substituted. When a carbon in a substituent is described as optionally substituted with one or more substituents in a substitution list, one or more hydrogens on the carbon (to the extent there are hydrogens) can be, singly and / or together, substituted with independently selected substituents or can be unsubstituted. When a nitrogen in a substituent is described as optionally substituted with one or more substituents in a substitution list, one or more hydrogens on the nitrogen (to the extent there are hydrogens) can be, singly and / or together, substituted with independently selected substituents, respectively, or can be unsubstituted.

[0144] When substituents are described as being "independently selected from the group consisting of," each substituent is selected independently from the other substituents. Thus, each substituent can be the same as or different from the other (other) substituents.

[0145] Unless otherwise specified, as used herein, the point of attachment of a substituent may be from any suitable position on the substituent.

[0146] When a bond of a substituent is depicted as passing through a bond connecting two atoms in a ring, then such substituent may be bonded to any ring-forming atom in the substitutable ring.

[0147] Pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts and base addition salts thereof. Suitable acid addition salts are formed with acids that form pharmaceutically acceptable salts. Suitable base addition salts are formed with bases that form pharmaceutically acceptable salts. A review of suitable salts is summarized in "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002) by Stahl and Wermuth. Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

[0148] The term "stereoisomer" (also referred to as "enantiomer") refers to a stable isomer that possesses a perpendicular plane of asymmetry due to the presence of at least one chiral factor (including a chiral center, a chiral axis, a chiral plane, etc.) and is capable of rotating plane-polarized light. Because the compounds of the present invention have asymmetric centers and chemical structures that can undergo stereoisomerization, the present invention also encompasses these stereoisomers and mixtures thereof. Because the compounds of the present invention (or pharmaceutically acceptable salts thereof) contain asymmetric carbon atoms, they can exist in the form of single stereoisomers, racemates, enantiomeric and diastereomeric mixtures. Generally, these compounds can be prepared in the form of racemates. However, if necessary, such compounds can be prepared or separated to obtain pure stereoisomers, i.e., single enantiomers or asteromers, or mixtures enriched in a single stereoisomer (having a purity of ≥98%, ≥95%, ≥93%, ≥90%, ≥88%, ≥85%, or ≥80%). As described below, single stereoisomers of a compound can be obtained by synthesis from optically active starting materials containing the desired chiral center, or by preparing and then separating or resolving a mixture of enantiomers, for example, by conversion to a mixture of diastereomers followed by separation or recrystallization, chromatographic procedures, the use of chiral resolving reagents, or by directly separating the enantiomers on a chiral HPLC column. Starting compounds of a particular stereochemistry are not only commercially available, but can also be prepared using the methods described below and then separated using methods known in the art. The term "enantiomer" refers to a pair of stereoisomers that are mirror images of each other and are non-superimposable. The term "diastereoisomer" refers to optical isomers that are not mirror images of each other. The terms "racemic mixture" or "racemate" refer to a mixture containing equal amounts 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 amounts of a single enantiomer. Unless otherwise specified, all stereoisomeric forms of the compounds of the present invention are within the scope of the present invention.

[0149] The term tautomer (or "tautomeric form") refers to structural isomers that have different energies and are interconvertible via a low energy barrier. Where tautomerism is possible (such as in solution), a chemical equilibrium of the tautomers can be achieved. For example, proton tautomers (also known as proton transfer tautomers) include, but are not limited to, interconversions via proton transfer, such as ketone-enol isomerization, imine-enamine isomerization, and amide-imine alcohol isomerization. Unless otherwise specified, all tautomers of the compounds of the invention are within the scope of the invention.

[0150] The term "polymorph" (or "polymorphic form") refers to a solid crystalline form of a compound or composition. Polymorphs can be detected, classified, and identified by known techniques, including, but not limited to, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), powder X-ray diffraction (XRPD), single crystal X-ray diffraction (SCXRD), solid state nuclear magnetic resonance (NMR), infrared spectroscopy (IR), Raman spectroscopy, scanning electron microscopy (SEM), and the like.

[0151] The term "solvate" refers to a substance formed by binding a compound of the invention (or a pharmaceutically acceptable salt) to at least one solvent molecule via non-covalent intermolecular forces.

[0152] The term "N-oxide" refers to compounds formed by oxidation of a nitrogen atom in the structure of a tertiary amine or a nitrogen-containing (aromatic) heterocyclic compound.

[0153] The present invention also includes all pharmaceutically acceptable isotope-labeled compounds identical to the compounds of the present invention, except that one or more atoms are replaced with atoms that have the same atomic number as the predominant atom in nature but different atomic mass or mass number.The examples of isotopes suitable for inclusion in the compounds of the present invention include but are not limited to hydrogen isotopes (H, H, deuterium D, tritium T, etc.); carbon isotopes (C, C, and C, etc.); chlorine isotopes (Cl, etc.); fluorine isotopes (F, etc.); iodine isotopes (I, I, etc.); nitrogen isotopes (N, N, etc.); oxygen isotopes (O, O, O, etc.); and sulfur isotopes (S, etc.).

[0154] Also included within the scope of the present invention are metabolites of the compounds of the invention, i.e., substances formed in vivo upon administration of a compound of the invention. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc. of the administered compound. Accordingly, the present invention includes metabolites of compounds of the invention, including compounds produced by a process comprising contacting a compound of the invention with a mammal for a period of time sufficient to produce the metabolite.

[0155] The scope of the present invention also includes prodrugs of the compounds of the present invention, which are derivatives of the compounds of the present invention and have relatively little or no pharmacological activity by themselves, but which can be converted into the compounds of the present invention having the desired activity when administered to or on the body, for example, by hydrolytic cleavage. Generally, such prodrugs are functional derivatives of the compounds and can be easily converted into compounds having the desired therapeutic activity in vivo. For further information on the use of prodrugs, see "Prodrugs as Novel Delivery Systems," volume 14, ACS Symposium Series (T. Higuchi and V. Stella) and "Bioreversible Carriers in Drug Design," Pergamon Press, 1987 (EB Roche edition, American Pharmaceutical Association). The prodrugs of the present invention can be prepared by replacing appropriate functional groups in the compounds of the present invention with several moieties known to those skilled in the art as "promoieties" (for example, as described in "Design of Prodrugs," H. Bundgaard (Elsevier, 1985)).

[0156] Preparation of compounds In another aspect, the present invention provides a method for preparing a compound of formula I.

[0157] In some embodiments, compounds of formula I of the present invention can be prepared according to the following scheme: [ka] wherein ring A, R1, R2, X, Y, Z, Q1, Q2, m, and n are as defined above; and LG is a leaving group, for example, but not limited to, halogen, methylsulfonyloxy (-Oms) or trifluoromethylsulfonyloxy (-OTf), preferably iodine.

[0158] The method comprises the following steps: subjecting the compounds of formula I-SM-1 and I-SM-2 to an esterification reaction, followed by a substitution reaction with a compound of formula I-SM-3 to obtain a compound of formula I.

[0159] In some embodiments, the above step is carried out in the presence of a suitable condensing reagent, which can be selected from the group consisting of HATU, HBTU, EDCI, DCC, T3P, POCl3 and HOBT, preferably HATU.

[0160] In some embodiments, the above steps are carried out at a suitable temperature between 0 and 140°C, such as 0°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably between 0 and 35°C.

[0161] In some embodiments, the above steps are carried out in a suitable organic solvent, which may be selected from the group consisting of halogenated hydrocarbons (e.g., dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane (e.g., 1,2-DCE), etc.), nitriles (e.g., acetonitrile (AN)), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), tetrahydrofuran (THF), 1,4-dioxane, dimethyl sulfoxide (DMSO), and any combination thereof, preferably N,N-dimethylformamide (DMF).

[0162] In some embodiments, the above step is carried out in the presence of a suitable base, including an organic base or an inorganic base. The organic base can be selected from the group consisting of N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK), and pyridine (Py). The inorganic base can be selected from the group consisting of potassium phosphate (K3PO4), sodium hydride (NaH), potassium carbonate (K2CO3), sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), cesium carbonate (Cs2CO3), and NaOH, preferably N,N-diisopropylethylamine (DIPEA).

[0163] Preparation of drug-linker compounds In another aspect, the present invention provides a compound of formula GM-[LED] x The present invention provides a method for synthesizing the drug-linker compounds of formula (I). [ka] During the ceremony, Ring A, R1, R2, X, Y, Z, Q1, G, M, L, D, m and n are as defined above. x is 1; LG is a leaving group, such as, but not limited to, halogen, -OMs, and -OTf, preferably iodine; and PG is a protecting group, such as, but not limited to, Fmoc, Boc, MMt and Alloc, preferably Fmoc.

[0164] Step 1: performing a condensation reaction on the compounds of formula DL-SM-1 and DL-SM-2 to obtain a compound of formula DL-IM-1.

[0165] In some embodiments, this step is carried out in the presence of a suitable condensing reagent, which may be selected from the group consisting of HATU, HBTU, EDCI, DCC, T3P, POCl3 and HOBT, preferably HATU and T3P.

[0166] In some embodiments, this step is carried out at a suitable temperature, such as 0°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably 0-35°C.

[0167] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from the group consisting of halogenated hydrocarbons (e.g., dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane (e.g., 1,2-DCE), etc.), nitriles (e.g., acetonitrile (AN)), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), tetrahydrofuran (THF), 1,4-dioxane, dimethyl sulfoxide (DMSO), and any combination thereof, preferably dichloromethane (DCM) and N,N-dimethylformamide (DMF).

[0168] In some embodiments, this step is carried out in the presence of a suitable base, including an organic base or an inorganic base. The organic base can be selected from the group consisting of N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK), and pyridine (Py). The inorganic base can be selected from the group consisting of potassium phosphate (K3PO4), sodium hydride (NaH), potassium carbonate (K2CO3), sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), cesium carbonate (Cs2CO3), and NaOH, preferably pyridine (Py) and N,N-diisopropylethylamine (DIPEA).

[0169] Step 2: Hydrolysis of the compound of formula DL-IM-1 to obtain the compound of formula DL-IM-2

[0170] In some embodiments, this step is carried out at a suitable temperature, such as 0°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably 0-35°C.

[0171] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from the group consisting of halogenated hydrocarbons (e.g., dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane (e.g., 1,2-DCE), etc.), nitriles (e.g., acetonitrile (AN)), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), tetrahydrofuran (THF), 1,4-dioxane, dimethyl sulfoxide (DMSO), and any combination thereof, preferably tetrahydrofuran (THF).

[0172] In some embodiments, this step is carried out in the presence of a suitable Pd reagent, which may be selected from the group consisting of Pd(PPh3)4 and PdCl2(PPh3)2, preferably Pd(PPh3)4.

[0173] In some embodiments, this step is carried out in the presence of a suitable base, which may be selected from the group consisting of diethylamine, pyrrolidine, morpholine, piperidine, preferably morpholine.

[0174] Step 3: carrying out an esterification reaction on the compounds of formula DL-IM-2 and II-SM-1, followed by a substitution reaction with a compound of formula DL-IM-3

[0175] In some embodiments, this step is carried out in the presence of a suitable condensing reagent, which may be selected from the group consisting of HATU, HBTU, EDCI, DCC, T3P, POCl3 and HOBT, preferably HATU.

[0176] In some embodiments, this step is carried out at a suitable temperature, such as 0°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably 0-35°C.

[0177] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from the group consisting of halogenated hydrocarbons (e.g., dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane (e.g., 1,2-DCE), etc.), nitriles (e.g., acetonitrile (AN)), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), tetrahydrofuran (THF), 1,4-dioxane, dimethyl sulfoxide (DMSO), and any combination thereof, preferably N,N-dimethylformamide (DMF).

[0178] In some embodiments, this step is carried out in the presence of a suitable base, including an organic base or an inorganic base. The organic base can be selected from the group consisting of N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK), and pyridine (Py). The inorganic base can be selected from the group consisting of potassium phosphate (K3PO4), sodium hydride (NaH), potassium carbonate (K2CO3), sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), cesium carbonate (Cs2CO3), and NaOH, preferably N,N-diisopropylethylamine (DIPEA).

[0179] Step 4: Removal of the amino protecting group from the compound of formula DL-IM-3 to obtain the compound of formula DL-IM-4

[0180] In some embodiments, this step is carried out at a suitable temperature, such as 0°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably 0-35°C.

[0181] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from the group consisting of halogenated hydrocarbons (e.g., dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane (e.g., 1,2-DCE), etc.), nitriles (e.g., acetonitrile (AN)), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), tetrahydrofuran (THF), 1,4-dioxane, dimethyl sulfoxide (DMSO), and any combination thereof, preferably acetonitrile (AN).

[0182] In some embodiments, this step is carried out in the presence of a suitable base, which may be selected from the group consisting of diethylamine, pyrrolidine, morpholine, piperidine and piperazine, preferably morpholine and diethylamine.

[0183] Step 5: Condensation reaction of the compound of formula DL-SM-4 and GM-OH to obtain a compound of formula GM-[LED]x

[0184] In some embodiments, this step is carried out in the presence of a suitable condensing reagent, which may be selected from the group consisting of HATU, HBTU, EDCI, DCC, T3P, POCl3 and HOBT, preferably HATU.

[0185] In some embodiments, this step is carried out at a suitable temperature, such as 0°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, or 140°C, preferably 0-35°C.

[0186] In some embodiments, this step is carried out in a suitable organic solvent, which may be selected from the group consisting of halogenated hydrocarbons (e.g., dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane (e.g., 1,2-DCE), etc.), nitriles (e.g., acetonitrile (AN)), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), tetrahydrofuran (THF), 1,4-dioxane, dimethyl sulfoxide (DMSO), and any combination thereof, preferably N,N-dimethylformamide (DMF).

[0187] In some embodiments, this step is carried out in the presence of a suitable base, including an organic base or an inorganic base. The organic base can be selected from the group consisting of N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK), and pyridine (Py). The inorganic base can be selected from the group consisting of potassium phosphate (K3PO4), sodium hydride (NaH), potassium carbonate (K2CO3), sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), cesium carbonate (Cs2CO3), and NaOH, preferably N,N-diisopropylethylamine (DIPEA).

[0188] Preparation of drug-antibody conjugates In another aspect, the present application provides a method for preparing an antibody-drug conjugate of the present invention, selected from the group consisting of: Conjugation Method A: reducing the antibody interchain disulfide bond using a reducing agent such as tris(2-carboxyethyl)phosphine in a buffer solution of pH 7.0 to 9.0, and then binding the drug-linker compound of the present invention to the antibody at a molar ratio of (8 to 10:1) to obtain an antibody-drug conjugate; Conjugation Method B: reducing the antibody interchain disulfide bond using a reducing agent such as tris(2-carboxyethyl)phosphine in a buffer solution of pH 7.0 to 9.0, and conjugating the drug-linker compound of the present invention with the antibody at a molar ratio of (4 to 6):1 to obtain an antibody-drug conjugate; Conjugation Method C: The antibody-drug conjugate is obtained by reducing the interchain disulfide bond of the antibody using a reducing agent such as tris(2-carboxyethyl)phosphine in a buffer solution of pH 7.0 to 9.0, and then binding the drug-linker compound of the present invention to the antibody at a molar ratio of (4.0 to 4.5):1.

[0189] In some embodiments, the present application provides a method for preparing an antibody-drug conjugate of the present invention, selected from the group consisting of: Conjugation Method A: (Suitable for single pyrimidine linker series, targeting DAR8) Approximately 0.5 mL of antibody (3-10 mg / mL) was diluted with 0.1 M edetate disodium solution (pH 7.60), the pH was adjusted to 8.0 with 1 M NaHPO solution, 5.5 equivalents of 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution (pH 7.60) was added, mixed uniformly, and allowed to stand at room temperature for 1.5 hours. A drug-linker compound dissolved in DMSO in an amount 8-10 times the molar amount of antibody was added to the above solution, and after addition, the solution was allowed to stand at room temperature for 2 hours. The buffer was then replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the ADC product. Conjugation Method B: (Suitable for dipyrimidine linker series, targeting DAR4) Approximately 0.5 mL of antibody (3–10 mg / mL) was diluted with 0.1 M edetate disodium solution (pH 7.60), the pH adjusted to 7.60 with 1 M NaHPO solution, 5.5 equivalents of 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution (pH 7.60) was added, mixed uniformly, and allowed to stand at room temperature for 2 hours. A drug-linker compound dissolved in DMSO (10 mM, 1 equivalent added every 30 minutes) in an amount 4–6 times the molar amount of antibody was added to the above solution, and after addition, the solution was allowed to stand at room temperature for 18 hours. The buffer was then replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the ADC product. Conjugation Method C: (Suitable for Bromoacetyl Linker Series (Comparative Compounds), Targeting DAR4) Approximately 0.5 mL of antibody (3–10 mg / mL) was diluted with 0.1 M edetate disodium solution (pH 7.60), the pH was adjusted to 7.60 with 1 M NaHPO solution, 2.4 equivalents of 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution (pH 7.60) was added, mixed uniformly, and allowed to stand at room temperature for 2 hours. A drug-linker compound dissolved in DMSO in an amount 4.0–4.5 times the molar amount of antibody was added to the above solution, and after addition, the mixture was allowed to stand at room temperature for 18 hours. The buffer was then replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the ADC product. [Example]

[0190] The present invention will be further described with reference to specific examples, but these examples are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and improvements in accordance with the teachings of the present invention without departing from the basic spirit and scope of the present invention.

[0191] The sequence information of adalimumab relevant to the present invention is set forth below: Full length heavy chain (SEQ ID NO: 5) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0192] Full length light chain (SEQ ID NO: 6) DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0193] The information regarding the sequence of tocilizumab involved in the present invention is set forth below: Full length heavy chain (SEQ ID NO: 12) QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQG SLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0194] Full length light chain (SEQ ID NO: 13) DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0195] Full length mutant heavy chain (SEQ ID NO: 14) QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQG SLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0196] The abbreviations used in this invention have the following meanings: [Table 1]

[0197] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 The molecular weight is determined by 1 H NMR or mass spectrometry (MS).

[0198] Nuclear magnetic resonance ( 1 H NMR measurements were performed using a Bruker 400 MHz nuclear magnetic resonance spectrometer; the deuteration reagent was hexadeuterated dimethyl sulfoxide (DMSO-d6); the internal standard was tetramethylsilane (TMS).

[0199] Nuclear magnetic resonance (NMR) spectrum abbreviations used in the examples are as follows: s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: deuterated dimethyl sulfoxide. δ values ​​are expressed in ppm.

[0200] MS determinations are performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0201] Preparation Example 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-amino-4-fluorobenzoate (A-1) [ka]

[0202] Step 1: To a solution of A-1-1 (1 g, 2.52 mmol) in THF (13.5 mL) and water (7.5 mL) was added periodic acid (1.72 g, 7.57 mmol). The reaction was stirred at 25 °C for 2 h and then filtered. The filter cake was washed with water (30 ml x 3) and dried under vacuum to give crude product A-1-2 (900 mg, 2.35 mmol). This crude product was used directly in the next step without further purification. ESI-MS (m / z): 383.1 (M+H) + .

[0203] Step 2: A-1-2 (0.9 g, 2.35 mmol) was dissolved in DMF (3 mL), CDI (763 mg, 4.71 mmol) was added, and 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 introduced within 30 minutes, and then the temperature was raised to 25 °C and stirring was continued for 2 hours. Water (100 mL) was added to the reaction mixture to precipitate a solid, which was then filtered. The filter cake was dried under vacuum to obtain crude product A-1-3 (1.3 g, 2.11 mmol). ESI-MS (m / z): 399.2 (M+H) + .

[0204] Step 3: 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 DIPEA (440 mg, 3.40 mmol) were added. The reaction mixture was stirred at 25 °C for 1.5 h. A-1-3 (700 mg, 1.13 mmol) was then added, and the reaction was continued for 1.5 h. Fluoroiodomethane (272 mg, 1.70 mmol) was then added to the reaction mixture, and the reaction was continued for 1 h. Water (50 mL) was added to the reaction mixture to precipitate a solid, which was then filtered. The filter cake was dried under vacuum to give crude product A-1-4 (950 mg, 754 μmol), which was used directly in the next step without further purification.

[0205] Step 4: A-1-4 (900 mg, 715 μmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (1.47 g, 12.9 mmol) was added, and the reaction mixture was stirred at 25° C. for 1 hour. The reaction mixture was concentrated to give a crude product, which was purified by high-performance liquid chromatography to give A-1 (310.39 mg, 532 μmol).

[0206] Chromatography column: Phenomenex C18 150mm x 25mm x 10μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate) [ka] The structure of A-1 is: ESI-MS (m / z): 568.5 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 7.24-7.37 (m, 2H), 7.15 (dd, J =11.2, 8.4 Hz, 1H), 7.05 (ddd, J = 8.4, 4.4, 2.1 Hz, 1H), 6.33 (dd, J = 10.0, 1.8 Hz, 1H), 6.12 (s, 1H), 5.90-6.02 (m, 1H), 5.78-5.88 (m, 1H), 5.57-5.74 (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 = 13.6 Hz, 1.2 H), 1.69-1.82 (m, 1H), 1.43-1.59 (m, 5H), 0.88-1.00 (m, 3H).

[0207] Preparation Example 2: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-aminoisonicotinate (P-1) [ka]

[0208] Step 1: P-1-1 (10 g, 24.3 mmol) was dissolved in THF (135 mL) and water (75 mL), and periodic acid (16.6 g, 73.0 mmol, 16.6 mL) was added. The reaction mixture was stirred at 25 °C for 2 h and then concentrated under reduced pressure to remove THF. The filter cake was washed with water (100 mL × 3) and then dried under vacuum to give crude product P-1-2 (9.00 g, 22.7 mmol, 93.18% yield), which was used directly in the next step without further purification. ESI-MS (m / z): 397.0 (M+H) + .

[0209] Step 2: P-1-2 (9.00 g, 22.7 mmol) was dissolved in DMF (100 mL), followed by the addition of CDI (7.36 mg, 45.41 mmol), and the reaction mixture was stirred at 25 °C for 4 h. Hydrogen sulfide gas (15 PSI) was continuously introduced into the reaction mixture for 30 min, followed by stirring for 3 h. The reaction mixture was adjusted to pH 2-3 using 1 N aqueous hydrochloric acid to precipitate a solid. After filtration, the filter cake was vacuum dried to obtain P-1-3 (8.5 g, 20.61 mmol). ESI-MS (m / z): 413.2 (M+H) + .

[0210] Step 3: P-1-3 (300 mg, 727 μmol) and 2-nitropyridine-4-carboxylic acid (146 mg, 872 μmol) were dissolved in DMF (10.0 mL). T3P (694 mg, 1.09 mmol) and DIPEA (141 mg, 1.09 mmol) were added, and the reaction mixture was stirred at 25 °C for 0.5 h. Fluoroiodomethane (174 mg, 1.09 mmol) and DIPEA were then added to the reaction mixture, and stirring was continued for 2 h. The reaction mixture was filtered and concentrated to give the crude product P-1-4 (350 mg), which was used directly in the next step without further purification. ESI-MS (m / z): 595.5 (M+H) + .

[0211] Step 4: At 25 °C, P-1-4 (350 mg, 588 μmol) was added to EtOH (5.00 mL) and water (5.00 mL), followed by the addition of NHCl (251 mg, 4.71 mmol) and iron powder (263 mg, 4.71 mmol). After the addition was complete, the reaction mixture was heated to 80 °C and reacted for 6 hours. The reaction mixture was filtered and concentrated to obtain the crude product, which was purified by high-performance liquid chromatography to obtain P-1 (0.169 g, 284 μmol).

[0212] Chromatography column: Phenomenex C18 250mm x 50mm x 10μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate) [ka] The structure of P-1 is: ESI-MS (m / z): 565.5 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 8.1-8.3 (d, J = 5.2 Hz, 1H), 7.1-7.2 (d, J = 10 Hz, 1H), 7.0-7.1 (dd, J = 5.2 Hz, 1H), 6.94 (s, 1H), 6.3-6.5 (m, 2H), 5.7-6.1 (m, 2H), 5.3-5.5 (m, 1H), 4.6-4.8 (s, 2H), 4.4-4.6 (m, 1H), 3.4-3.6 (m, 1H), 2.3-2.6 (m, 4H), 1.9-2.0 (m, 3H), 1.8-1.9 (m, 1H), 1.56 (s, 3H), 1.4-1.5 (m, 1H), 1.18 (s, 3H), 1.0-1.1 (d, J = 7.2 Hz, 3H).

[0213] Preparation Example 3: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-aminofuran-2-carboxylate (F-2) [ka]

[0214] Step 1: Methyl 4-bromofuran-2-formate (F-2-1, 1.5 g, 7.32 mmol) and tert-butyl carbamate (4.29 g, 36.5 mmol) were added to toluene (20.0 mL), followed by the addition of N,N'-dimethylethylenediamine (322 mg, 3.66 mmol, 393 μL), cuprous iodide (836 mg, 4.39 mmol), and potassium carbonate (3.03 g, 21.9 mmol). The reaction mixture was heated to 130 °C and stirred for 18 h. The mixture was extracted with water (200 mL) and dichloromethane (100 mL × 3). The combined organic phases were dried, filtered, and concentrated to give the crude product, which was purified by flash column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 5 / 1) to give F-2-2 (700 mg, 2.90 mmol). ESI-MS (m / z): 242.0 (M+H) + .

[0215] Step 2: F-2-2 (700 mg, 2.90 mmol) was added to methanol (4.00 mL), tetrahydrofuran (4.00 mL), and water (2.00 mL), followed by the addition of lithium hydroxide (138 mg, 5.80 mmol). The mixture was allowed to react at 25 °C for 1 hour. Water and 1 N aqueous hydrochloric acid (10.0 mL) were added to the reaction mixture, which was then extracted with dichloromethane (20.0 mL x 3). The organic phases were combined, dried, filtered, and concentrated to give F-2-3 (350 mg, 1.54 mmol). ESI-MS (m / z): 228.1 (M+H) + .

[0216] Step 3: F-2-3 (100 mg, 440 μmol) and HATU (200 mg, 528 μmol) were added to DMF (10.0 mL), followed by the addition of DIPEA (284 mg, 2.20 mmol, 383 μL). The reaction mixture was stirred at 25 °C for 2 h, followed by the addition of P-1-3 (181 mg, 440 μmol). After stirring for 2 h, fluoroiodomethane (105 mg, 660 μmol) was added, and the mixture was stirred at 25 °C for 1 h. The reaction mixture was extracted with water (100 mL) and ethyl acetate (100 mL x 3). The organic phases were combined, dried, filtered, and concentrated to give crude product F-2-4 (220 mg, 336 μmol), which was used directly in the next step without further purification. ESI-MS (m / z): 654.2 (M+H) + .

[0217] Step 4: F-2-4 (150 mg, 229 μmol) was added to dichloromethane (3 mL) and trifluoroacetic acid (1.54 g, 13.5 mmol) was added. The reaction mixture was stirred at 25°C for 1 hour, then concentrated to give the crude product directly. This was purified by high-performance liquid chromatography to give F-2 (27.9 mg, 45.54 μmol). Chromatography column: Phenomenex 150mm x 25mm x 10μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate) [ka] The structure of F-2 is: ESI-MS (m / z): 554.2 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 7.34 (dd, J=1.2, 10.0 Hz, 1H), 6.87-7.31 (m, 1H), 6.27-6.38 (m, 2H), 5.88-6.03 (m, 1H), 5.74-5.88 (m, 1H), 5.47-5.65 (m, 1H), 4.33 (d, J=9.6 Hz, 1H), 3.46 (dt, J=3.2, 6.97 Hz, 1H), 2.54-2.72 (m, 1H), 2.36 (dd, J=3.2, 11.2 Hz, 3H), 1.86-2.08 (m, 2H), 1.62-1.71 (m, 1H), 1.56-1.60 (m, 3H), 1.32-1.42 (m, 1H), 1.16 (s, 3H), 0.98-1.06 (m, 3H).

[0218] Preparation Example 4: 6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl (1S)-2-(4-aminophenyl)cyclopropane-1-carboxylate (C-1) [ka]

[0219] Step 1: Tert-butyl N-(4-bromophenyl)carbamate (C-1-1, 5.00 g, 18.3 mmol), ethyl (E)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)prop-2-enoate (4.57 g, 20.2 mmol), XPhos Pd G3 (1.24 g, 1.47 mmol), and potassium phosphate (11.70 g, 55.1 mmol) were added to 1,4-dioxane (50.0 mL) and water (17.0 mL). The reaction mixture was purged with nitrogen three times, then heated to 100 °C and reacted for 3 h. The reaction mixture was filtered through a silica gel pad and extracted with water (80.0 mL) and ethyl acetate (50.0 mL × 3). The organic phases were combined, dried, filtered, and concentrated to give the crude product, which was purified by flash column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 3 / 1) and concentrated to give C-1-2 (4.20 g, 12.9 mmol). ESI-MS (m / z): 292.1 (M+H) + .

[0220] Step 2: C-1-2 (3.60 g, 12.3 mmol) was added to DMSO (40.0 mL), and trimethylsulfoxonium iodide (3.26 g, 14.8 mmol) and potassium t-butoxide (1.53 g, 13.5 mmol) were added. The reaction mixture was stirred at 25 °C for 2 h and then extracted with water (50.0 mL) and ethyl acetate (50.0 mL × 3). The combined organic phases were dried, filtered, and concentrated to give the crude product, which was purified by flash column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 3 / 1) and concentrated to give C-1-3 (400 mg, 1.31 mmol). ESI-MS (m / z): 306.0 (M+H) + .

[0221] Step 3: C-1-3 (400 mg, 1.31 mmol) was added to methanol (2.00 mL), water (2.00 mL), and THF (2.00 mL), followed by the addition of lithium hydroxide (62.7 mg, 2.62 mmol), and the reaction mixture was allowed to react at 25 °C for 1 h. The reaction mixture was adjusted to pH 2-3 with 1N aqueous hydrochloric acid to precipitate a solid, which was then filtered. The filter cake was dried under vacuum to give C-1-4 (300 mg, 1.08 mmol), which was used directly in the next step without further purification. ESI-MS (m / z): 278.1 (M+H) + .

[0222] Step 4: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl(1S,2S)-2-(4-(tert-butoxycarbonyl)amino)phenyl)cyclopropane-1-carboxylate (C-1-5) C-1-4 (200 mg, 721 μmol), DIPEA (466 mg, 3.61 mmol, 628 μL), and HATU (548 mg, 1.44 mmol) were added to DMF (15.0 mL). The reaction mixture was stirred at 25 °C for 1 h, and then P-1-3 (356 mg, 865 μmol) was added and stirred for an additional 2 h. Fluoroiodomethane (173 mg, 1.08 mmol) was then added, and the reaction mixture was allowed to react for 1 h. The reaction mixture was extracted with water (10.0 mL) and dichloromethane (10.0 mL × 3). The organic phases were combined, dried, filtered, and concentrated to give C-1-5 (150 mg, 213 μmol), which was used directly in the next step without further purification. ESI-MS (m / z): 704.3 (M+H) + .

[0223] Step 5: C-1-5 (50.0 mg, 71.0 μmol) was added to dichloromethane (1.00 mL), followed by TFA (462 mg, 4.05 mmol, 0.30 mL). The reaction mixture was incubated at 25°C for 1 hour, then directly concentrated to give the crude product, which was purified by high-performance liquid chromatography to give C-1 (11.89 mg, 18.4 μmol).

[0224] Chromatography column: Phenomenex 150mm x 25mm x 10μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate) [ka] The structure of C-1 is: ESI-MS (m / z): 604.2 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 7.32 (dd, J = 6.0, 9.57 Hz, 1H), 6.78-6.96 (m, 2H), 6.65 (dd, J = 1.6, 8.4 Hz, 2H), 6.25-6.41 (m, 2H), 5.91-6.00 (m, 1H), 5.78-5.87 (m, 1H), 5.33-5.66 (m, 2H), 4.30 (t, J = 10.0 Hz, 1H), 3.41 (d, J = 4.00 Hz, 2H), 2.52-2.66 (m, 1H), 2.19-2.41 (m, 4H), 1.93-2.06 (m, 2H), 1.78 (ddd, J = 4.8, 9.0, 13.6 Hz, 1H), 1.55-1.69 (m, 4H), 1.41-1.48 (m, 1H), 1.35 (tt, J = 3.74, 7.52 Hz, 2H), 1.12 (d, J = 1.8 Hz, 3H), 0.92-1.10 (m, 3H).

[0225] Preparation Example 5: Fluoromethyl (6S,8S,9R,10S,11S,13S,14S,16R,17R)-17-((3-amino-4-fluorobenzoyl)oxy)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylate (O-1) [ka]

[0226] Step 1: Starting material P-1-2 (193.16 mg, 756.77 μmol), DIPEA (146.71 mg, 1.14 mmol), and HATU (345.30 mg, 908.12 μmol) were added to DMF (15 mL) and the reaction mixture was incubated at 25°C for 15 minutes. DIPEA (146.71 mg, 1.14 mmol) was added to the reaction mixture, followed by 3-(tert-butoxycarbonylamino)-4-fluorobenzoic acid (300 mg, 756.77 μmol). After the addition was complete, the reaction mixture was allowed to react at 25°C for 16 hours. The reaction was monitored by LC-MS, and fluoroiodomethane (604.49 mg, 3.78 mmol) was added to the reaction mixture and the reaction mixture was allowed to react at 25°C for 4 hours. The reaction was monitored by LC-MS, and water was added to the reaction system to precipitate a pale yellow solid, which was collected by filtration to obtain a crude product. The crude product was purified by column chromatography (ethyl acetate / petroleum ether = 0 to 35%) to obtain O-1-1 (390 mg). ESI-MS (m / z): 610.1 [M+1-56] + .

[0227] Step 2: Compound O-1-2 (70 mg, 105.16 μmol) was added to DCM (3 mL) and TFA (1 mL), and the reaction mixture was reacted at 25° C. for 1.5 hours. The reaction was monitored by LC-ML until completion. The reaction mixture was concentrated to dryness to obtain the crude product, which was purified by high-performance liquid chromatography to obtain O-1 (23.8 mg). Chromatography column: Waters XBridge Prep C18OBD (5 μm*19 mm*150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [ka] The structure of O-1 is: ESI-MS (m / z): 566.3 [M+1] + . 1 H NMR (400 MHz, DMSO-d6): δ 7.34-7.24 (m, 2H), 7.16 (dd, J =11.2 Hz, 8.4 Hz, 1H), 7.07-7.01 (m, 1H), 6.33 (dd, J =10.0 Hz, 2.0 Hz, 1H), 6.14 (s, 1H), 5.97-5.55 (m, 4H), 4.30-4.20 (m, 1H), 3.36-3.25 (m, 1H), 2.68-2.54 (m, 1H), 2.33-2.21 (m, 2H), 2.20-2.12 (m, 1H), 1.95-1.83 (m, 1H), 1.81-1.73 (m, 1H), 1.67-1.54 (m, 1H), 1.51 (s, 3H), 1.34-1.25 (m, 1H), 1.06 (s, 3H), 0.87 (d, J = 7.2 Hz, 3H).

[0228] Preparation Example 6: Chloromethyl (6S,8S,9R,10S,11S,13S,14S,16R,17R)-17-((3-amino-4-fluorobenzoyl)oxy)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylate (O-3) [ka]

[0229] Step 1: P-1-2 (96.58 mg, 378.33 μmol), DIPEA (73.35 mg, 567.57 μmol), and HATU (172.65 mg, 454.06 μmol) were added to DMF (172.65 mg, 454.06 μmol), and the reaction mixture was reacted at 25 °C for 15 min. Then, DIPEA (73.35 mg, 567.57 μmol) and 3-(tert-butoxycarbonylamino)-4-fluorobenzoic acid (150 mg, 378.38 μmol) were added. After the addition was complete, the reaction mixture was reacted at 25 °C for 3 h. The reaction was monitored by LC-MS. Then, chloroiodomethane (668.05 mg, 3.78 mmol) was added to the reaction mixture, and the reaction mixture was reacted at 45 °C for 20 h. The reaction was monitored by LC-MS. The reaction mixture was extracted with water and dichloromethane, and the organic phase was dried and concentrated to give 230 mg of crude product O-3-1, which was used directly in the next step.

[0230] Step 2: Compound O-3-1 (150 mg, 105.16 μmol) was added to DCM (4 mL) and TFA (2 mL), and the reaction mixture was reacted at 25° C. for 1.5 hours. The reaction was monitored by LC-MS. The reaction mixture was concentrated under reduced pressure to give the crude product, which was purified by high-performance liquid chromatography to give O-3 (20 mg). Chromatography column: Waters XBridge Prep C18OBD (5 μm*19 mm*150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [ka] The structure of O-3 is: ESI-MS (m / z): 582.2 [M+1] + . 1H NMR (400 MHz, DMSO-d6): δ 7.33-7.26 (m, 2H), 7.16 (dd, J =11.2 Hz, 8.8 Hz, 1H), 7.05-6.99 (m, 1H), 6.33 (dd, J =10.0 Hz, 2.0 Hz, 1H), 6.14 (s, 1H), 5.96 (d, J = 6.0 Hz, 1H), 5.86 (d, J = 6.0 Hz, 1H), 5.76-5.56 (m, 2H), 4.28-4.20 (m, 1H), 3.48-3.25 (m, 1H), 2.65-2.55 (m, 1H), 2.31-2.11 (m, 3H), 1.95-1.82 (m, 1H), 1.78-1.70 (m, 1H), 1.64-1.54 (m, 1H), 1.51 (s, 3H), 1.34-1.24 (m, 1H), 1.07 (s, 3H), 0.87 (d, J = 7.2Hz, 3H).

[0231] Preparation Example 7: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl(E)-3-(3-amino-4-fluorophenyl)acrylate (E-3) [ka]

[0232] Step 1: 4-Bromo-1-fluoro-2-nitrobenzene (1.00 g, 4.55 mmol), ethyl (E)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)acrylate (1.25 g, 5.45 mmol), Xphos-Pd-G3 (384.76 mg, 455 μmol), and potassium phosphate (2.89 g, 13.6 mmol) were added to 1,4-dioxane (12 mL) and water (4 mL). After purging with nitrogen three times, the reaction mixture was heated to 100 °C and reacted for 2 hours. Water (100 mL) was added to the reaction mixture, which was then extracted three times with ethyl acetate (60 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product, which was purified by flash column chromatography (petroleum ether / ethyl acetate=100 / 1 to 5 / 1) and concentrated again to give ethyl (E)-3-(4-fluoro-3-nitrophenyl)acrylate (450 mg, 1.76 mmol). ESI-MS (m / z): 240.1 (M+H) + .

[0233] Step 2: Ethyl (E)-3-(4-fluoro-3-nitrophenyl)acrylate (300 mg, 1.25 mmol) was dissolved in tetrahydrofuran (3 mL) and water (3 mL). Lithium hydroxide monohydrate (210 mg, 5.02 mmol) was then added and stirred at room temperature for 2 hours. The reaction mixture was adjusted to pH 2-3 using 1 N dilute hydrochloric acid, and the solid precipitated and filtered. The filter cake was dried under vacuum to give (E)-3-(4-fluoro-3-nitrophenyl)acrylic acid (305 mg, 895 μmol). ESI-MS (m / z): 212.0 (M+H) + .

[0234] Step 3: (E)-3-(4-Fluoro-3-nitrophenyl)acrylic acid (270 mg, 1.28 mmol) was dissolved in dichloromethane (10 mL), and DIPEA (826 mg, 6.39 mmol) and T3P (2.44 g, 3.84 mmol, 2.28 mL, purity 50%) were added, and the reaction mixture was then stirred at 25°C for 1 hour. (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylic acid (527.46 mg, 1.28 mmol) was added to the reaction mixture, and the mixture was stirred for an additional 2 hours. Fluoroiodomethane (224 mg, 1.41 mmol) was added, and the mixture was stirred for an additional 0.5 hours. Water (100 mL) was added, and the mixture was extracted three times with dichloromethane (60 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product, which was purified by flash column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 1 / 1) and concentrated to give (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl(E)-3-(4-fluoro-3-nitrophenyl)acrylate (52.0 mg, 28.6 μmol). ESI-MS (m / z): 638.2 (M+H) + .

[0235] Step 4: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl(E)-3-(4-fluoro-3-nitrophenyl)acrylate (50.0 mg, 78.4 μmol) was dissolved in ethanol (1.5 mL) and water (0.5 mL), followed by the addition of iron powder (43.79 mg, 784 μmol) and ammonium chloride (20.9 mg, 392 μmol). The reaction mixture was heated to 80°C and stirred for 2 hours. Water (50 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (40 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product, which was purified by high-performance liquid chromatography to give (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl(E)-3-(3-amino-4-fluorophenyl)acrylate (4.49 mg, 7.15 μmol). Chromatography column: Henomenex Luna C18 150mm x 25mm x 10μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) TIFF2026501523000353.tif20170Its structure is as follows: ESI-MS (m / z): 608.2 [M+H] + . 1H NMR (400 MHz, DMSO) δ 7.50 (d, J = 16.0 Hz, 1H), 7.35 (dd, J = 10.4, 1.2 Hz, 1H), 7.07 (dd, J = 8.4, 2.4 Hz, 1H), 6.97 (dd, J = 10.8, 8.4 Hz, 1H), 6.82-6.90 (m, 1H), 6.34-6.42 (m, 2H), 6.32 (s, 1H), 5.91-6.03 (m, 1H), 5.79-5.90 (m, 1H), 5.46-5.67 (m, 1H), 4.34 (d, J = 9.6Hz, 1H), 3.43-3.50 (m, 1H), 2.52-2.72 (m, 1H), 2.28-2.40 (m, 3H), 1.95-2.08 (m, 2H), 1.63-1.73 (m, 1H), 1.59 (s, 3H), 1.33-1.40 (m, 1H), 1.16 (s, 3H), 1.01 (d, J = 7.2 Hz, 3H).

[0236] Preparation Example 8: (S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-34-(5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,1 2,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)oxy)carbonyl)-2-fluorophenyl)carbamoyl)-1,29,32-trioxo-5,8,11,14,17,20,23,26-octaoxa-2,30,33-triazaheptatriacontan-37-ynoic acid (DL-B-11') [ka]

[0237] Step 1: The starting materials, methyl 3,5-dibromobenzoate (720 mg, 2.45 mmol), 2-methylthiopyrimidine-5-boronic acid (874 mg, 5.14 mmol), XPhosPd G3 (207 mg, 245 μmol), and K3PO4 (1.56 g, 7.35 mmol), were added to dioxane (12 mL) and water (4 mL). The reaction mixture was stirred under a nitrogen atmosphere at 90 °C for 3 h. The reaction was monitored by LC-MS. The reaction mixture was filtered through a pad of diatomaceous earth, and the filtrate was extracted with water and ethyl acetate and concentrated to give the crude product, which was purified by column chromatography (EA / PE = 0-25%) to give 710 mg of methyl 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoate. The structural property data are as follows: ESI-MS (m / z): 385.1 [M+H] + .

[0238] Step 2: The compound methyl 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoate (650 mg, 1.69 mol) and lithium hydroxide (121 mg, 5.07 mmol) were dissolved in THF (2 mL), MeOH (2 mL), and HO (2 mL). The reaction mixture was stirred at 25 °C for 2 hours. The reaction was monitored by LC-MS. The system was adjusted to pH 2 with 1 N hydrochloric acid, causing a large amount of solid to precipitate. This was filtered, and the filter cake was collected and dried to obtain 560 mg of 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoic acid. ESI-MS (m / z): 371.1 [M+H] + .

[0239] Step 3: 3,5-Bis(2-(methylthio)pyrimidin-5-yl)benzoic acid (3.00 g, 8.10 mmol) and tert-butyl 1-amino-3,6,9,12,15,18,21,24-octaoxaheptane-27-carboxylate (4.03 g, 8.10 mmol) were added to DMF (40 mL), followed by HOBt (3.28 g, 24.3 mmol), EDCI (4.66 g, 24.3 mmol), and DIPEA (4.19 g, 32.4 mmol, 5.64 mL), and the reaction mixture was stirred at 60 °C for 2 h. The reaction mixture was extracted with water (100 mL) and ethyl acetate (60 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give tert-butyl 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-carboxylate (4.20 g, 4.14 mmol), which was used directly in the next step.

[0240] Step 4: Tert-butyl 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-carboxylate (3.60 g, 4.24 mmol) was dissolved in dichloromethane (30 mL), TFA (15.3 g, 134 mmol, 10 mL) was added, and the reaction mixture was stirred at 25° C. for 6 hours. The reaction mixture was extracted with water (60 mL) and ethyl acetate (40 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product, which was purified by high-performance liquid chromatography and lyophilized to give 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-carboxylic acid (2.93 g, 3.63 mmol). The structural property data are as follows: ESI-MS (m / z): 794.3 [M+H] + . The purification method is as follows: Chromatography column: Phenomenex luna C18 (250mm*70mm*10μm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 2]

[0241] Step 5: 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-carboxylic acid (148 mg, 0.186 mmol) was added to acetonitrile (15 mL) and water (7.5 mL), and sodium periodate (398.71 mg, 1.86 mmol) and ruthenium trichloride hydrate (15.47 mg, 74.56 μmol) were added. The reaction mixture was stirred at 25°C for 30 minutes. The reaction mixture was extracted with water and ethyl acetate and concentrated to give 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-carboxylic acid (155 mg). The structural property data are as follows: ESI-MS (m / z): 858.3 [M+H] + .

[0242] Step 6: (S)-2-(2-(((9H-Fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)-5-(tert-butoxy)-5-oxovaleric 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 a 50% solution of 1-propylphosphoric anhydride in DMF (5.57 g) were added sequentially to DCM (1 mL). After the addition was complete, the reaction mixture was stirred at 25 °C for 3 h. The reaction was monitored by TLC (PE:EA = 0:1). After the reaction was complete, 12 mL of water was added to the reaction mixture, followed by extraction with DCM (15 mL). The organic layer was washed with water (8 mL), dried over anhydrous sodium sulfate (3 g), filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (SiO, CHCl / MeOH = 100 / 1 to 10 / 1) to give allyl (S)-3-(2-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)-5-(tert-butoxy)-5-oxopentanamido)-4-fluorobenzoate (814 mg, 1.14 mmol). The structural property data are as follows: ESI-MS (m / z): 659.70(M+H) + .

[0243] Step 7: Allyl (S)-3-(2-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)-5-(tert-butoxy)-5-oxopentanamido)-4-fluorobenzoate (734 mg, 1.11 mmol), morpholine (193.86 mg, 2.23 mmol), and Pd(PPh3)4 (64.29 mg, 55.63 μmol) were added sequentially to THF (7 mL). After the addition was complete, the reaction mixture was reacted at 25 °C for 2 hours. Water (26 mL) was added, and the reaction mixture was extracted with DCM (26 mL × 2). The organic phase was washed with water (8 mL) and saturated brine (8 mL), dried over anhydrous sodium sulfate (4 g), and concentrated under reduced pressure to near dryness. PE (8 mL) was added, stirred at room temperature for 1 hour, and then filtered. The filter cake was dried under reduced pressure to give (S)-3-(2-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)-5-(tert-butoxy)-5-oxopentanamido)-4-fluorobenzoic acid. The structural property data are as follows: ESI-MS (m / z): 620 (M+H) + .

[0244] Step 8: (S)-3-(2-(2-(((9H-Fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)-5-(tert-butoxy)-5-oxopentanamido)-4-fluorobenzoic acid (200 mg, 322.77 μmol), DIPEA (83.43 mg, 645.54 μmol), and HATU (147.73 mg, 387.32 μmol) were added sequentially to DMF (4 mL). After the addition was complete, the reaction mixture was stirred at 16°C for 35 minutes. A-1-3 (128.86 mg, 320.28 μmol) was added, and after the addition was complete, the reaction mixture was stirred at 20°C for 1 hour. To the reaction mixture, DIPEA (31.02 mg, 239.98 μmol) was added, followed by fluoroiodomethane (211.09 mg, 1.32 mmol). After the addition was complete, the reaction mixture was stirred at 20°C for 1 hour. Water (12 mL) was added to the reaction mixture, followed by extraction with EA (15 mL*2). The organic phase was washed with water (8 mL) and saturated brine (8 mL) and separated. The organic phase was concentrated to dryness under reduced pressure and purified by column chromatography (SiO, PE / EA = 12 / 1 to 1 / 1) to give (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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonylamino)acetamino)-5-(tert-butoxy)-5-oxopentanamido)-4-fluorobenzoate (88 mg, 80.15 μmol). The structural property data are as follows: ESI-MS (m / z): 1032 (M+H) + ..

[0245] Step 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonylamino)acetamino)-5-(tert-butoxy)-5-oxopentanamido)-4-fluorobenzoate (88 mg, 80.15 μmol) and diethylamine (31.18 mg, 426.32 μmol) were dissolved in MeCN (4 mL), and after the addition was completed, the reaction mixture was stirred at 25° C. for 2 hours. The reaction mixture was concentrated under reduced pressure to give 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-dodecahydro-3H-cyclopenta[a]phenanthren-17yl 3-((S)-2-(2-aminoacetamido)-5-(tert-butoxy)-5-oxopentanamido)-4-fluorobenzoate (69 mg, 80.94 μmol), which was used directly in the next step without purification. The structural property data are as follows: ESI-MS (m / z): 810 (M+H) + .

[0246] Step 10: The 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-((S)-2-(2-aminoacetamido)-5-(tert-butoxy)-5-oxopentanamido)-4-fluorobenzoate (69 mg, 80.94 μmol) was added to DCM (3 mL), followed by TFA (0.5 mL). After the addition was complete, the reaction mixture was stirred at 25° C. for 3 hours. The reaction mixture was concentrated under reduced pressure to remove DCM and TFA, and the crude product (S)-4-(2-aminoacetamido)-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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-oxovaleric acid (88 mg, 83.15 μmol) was obtained, which was used directly in the next step without purification. The structural property data are as follows: ESI-MS (m / z): 754 (M+H) + .

[0247] Step 11: (S)-4-(2-aminoacetamido)-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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-oxovaleric acid crude product (24 mg, To DMF (2 mL), 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-carboxylic acid (23.73 mg, 27.66 μmol), DIPEA (17.87 mg, 138.28 μmol), and HATU (20 mg, 52.60 μmol) were added sequentially. After the addition was complete, the reaction mixture was stirred at 15°C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography to give the title compound (4.55 mg, 2.77 μmol). The purification method is as follows: Chromatography column: SunFire Preparative C18 OBD 19*150mm*5μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) TIFF2026501523000356.tif26170Structural characteristic data are as follows: ESI-MS (m / z): 1594 (M+H) + .

[0248] Preparation Example 9: (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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-4-(2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamino)acetamino)-5-oxovaleric acid (DL-A-05) [ka]

[0249] 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-((S)-2-(2-aminoacetamido)-5-(tert-butoxy)-5-oxopentanamido)-4-fluorobenzoate (67 mg, 82.73 μmol), (2,5-dioxopyrrolidin-1-yl) 6-(2-methylsulfonylpyrimidin-5-yl)hex-5-ynoate (33.25 mg, 91.00 μmol) After the addition was completed, the reaction mixture was stirred at 15°C for 1 hour. The reaction mixture was extracted with purified water (10 mL) and ethyl acetate (6 mL*2). After separation, the mixture was dried over anhydrous sodium sulfate (3 g), filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (SiO2, Purification by PE / EA = 100 / 1 to 1 / 4 gave (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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-((S)-5-(tert-butoxy)-2-(2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamino)acetamino)-5-oxopentanamido)-4-fluorobenzoate (64 mg, 54.33 μmol).

[0250] 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-((S)-5-(tert-butoxy)-2-(2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamino)acetamino)-5-oxopentanamido)-4-fluorobenzoate (64 mg, 54.33 μmol) and TFA (1 mL) were added sequentially to DCM (2 mL). After the addition was complete, the reaction mixture was allowed to react at 16° C. for 1.5 hours. The reaction mixture was purified by high performance liquid chromatography to obtain the title compound (42.00 mg, 41.41 μmol). The purification method is as follows: Chromatography column: SunFire Prep C18 OBD 19*150mm*5μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% TFA) [Table 4] The structural property data are as follows: ESI-MS (m / z): 1004.3 (M+H) + .

[0251] Preparation 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-(34S,37S) -37-(4-aminobutyl)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-34-(hydroxymethyl)-1,29,32,35-tetraoxo-5,8,11,14,17,20,23,26-octaoxa-2,30,33,36-tetraazaoctatriacontane-38-amino-4-fluorobenzoate (DL-B-07') [ka]

[0252] Step 1: Allyl 3-amino-4-fluorobenzoate: Allyl 3-amino-4-fluorobenzoate (5.00 g, 25.6 mmol) and N-(((9H-fluoren-9-yl))methoxy)carbonyl)-N-(tert-butoxycarbonyl)-L-lysine (10.8 g, 23.0 mmol) were dissolved in dichloromethane (100 mL). T3P (24.6 g, 38.7 mmol, 23.0 mL, purity 50.0%) and DIPEA (9.93 g, 76.8 mmol, 13.3 mL) were then added, and the reaction mixture was stirred at 25 °C for 2 hours. Water was added to the reaction mixture, and the mixture was extracted three times with dichloromethane (100 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product, which was then purified by silica gel column (SiO 2, The mixture was purified with CH2Cl2 / MeOH (100 / 1 to 10 / 1) and concentrated again to give DL-B-07'-2 (10.5 g, 16.2 mmol). The structural property data are as follows: ESI-MS (m / z): 668.4 [M+Na] + .

[0253] Step 2: DL-B-07'-2 (10.0 g, 15.4 mmol) was dissolved in THF (100 mL), morpholine (2.70 g, 30.9 mmol, 2.73 mL) and Pd(PPh3)4 (894 mg, 774 μmol) were added, and the reaction mixture was stirred at 25 °C for 1 h. The reaction mixture was adjusted to pH 2-3 with 1 N dilute hydrochloric acid, diluted with water (500 mL), and extracted three times with dichloromethane (500 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product, which was stirred in petroleum ether / ethyl acetate = 5 / 1 at 25 °C for 2 h, filtered, and dried to give DL-B-07'-3 (8.00 g, 13.2 mmol). The structural property data are as follows: ESI-MS (m / z): 623.2 [M+H] + .

[0254] Step 3: DL-B-07'-3 (5.00 g, 8.26 mmol) was dissolved in DMF (100 mL), HATU (3.14 g, 8.26 mmol) and DIPEA (3.20 g, 24.7 mmol, 4.31 mL) were added, and the reaction mixture was stirred for 2 hours at 25 °C. Next, (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-octahydrocyclopenta[a]phenanthrene-17-carboxylic acid (3.29 g, 8.26 mmol) was added to the reaction mixture, and the reaction mixture was stirred for an additional 2 hours. Fluoroiodomethane (1.32 g, 8.26 mmol) was then added to the reaction solution, and the reaction solution was stirred for an additional 2 hours. Water (200 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (100 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product. The crude product was purified on a silica gel column (SiO2, CHCl2 / MeOH = 100 / 1 to 10 / 1) and concentrated again to give DL-B-07'-4 (2.60 g, 2.55 mmol). The structural property data are as follows: ESI-MS (m / z): 1018.1 [M+H] + .

[0255] Step 4: DL-B-07'-4 (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 reaction mixture was stirred at 25° C. for 1 hour. The reaction mixture was used directly in the next step without further treatment.

[0256] Step 5: (((9H-Fluoren-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 to the reaction mixture obtained in the previous step, and the reaction mixture was stirred at 25 °C for 1 h. Water (200 mL) was then added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (100 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product. The crude product was purified on a silica gel column (SiO2, CHCl2 / MeOH = 100 / 1 to 10 / 1) and concentrated again to give DL-B-07'-6 (2.00 g, 1.81 mmol). The structural property data are as follows: ESI-MS (m / z): 1105.5 [M+H] + .

[0257] Step 6: DL-B-07'-6 (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 reaction mixture was stirred at 25° C. for 1 h. The reaction mixture was used directly in the next step without further treatment.

[0258] Step 7: ((9H-Fluoren-9-yl)methoxy)carbonyl)glycine (538 mg, 1.81 mmol), EDCI (521 mg, 2.72 mmol), and HOBt (367 mg, 2.72 mmol) were added to the reaction mixture obtained in the previous step, and the reaction mixture was stirred at 25 °C for 1 h. Water (200 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (50.0 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product. The crude product was purified on a silica gel column (SiO2, CHCl2 / MeOH = 100 / 1 to 10 / 1) and concentrated again to give DL-B-07'-8 (903 mg, 700 μmol). The structural property data are as follows: ESI-MS (m / z): 1162.5 [M+H] + .

[0259] Step 8: DL-B-07'-8 (500 mg, 0.43 mmol) was dissolved in DMF (5 mL), and diethylamine (125.8 mg, 1.72 mmol) was added. The mixture was allowed to react for 1 hour at 25 °C. The reaction mixture was concentrated to remove most of the DMF, yielding a crude product. The crude product was purified on a C18 reverse column (HO / CAN = 10-60%, 0.1% formic acid) and lyophilized to give the formate salt of DL-B-07'-9 (325 mg). The structural property data are as follows: ESI-MS (m / z): 940.4 [M+H] + .

[0260] Step 9: DL-B-11'-6 (27.4 mg, 0.032 mmol) was dissolved in DMF (0.5 mL), and HATU (23.14 mg, 0.061 mmol), DIPEA (11.8 mg, 0.091 mmol), and the formate salt of DL-B-07'-9 (30 mg, 0.030 mmol) were added sequentially. The reaction mixture was reacted at 25 °C for 1 hour. The reaction mixture was directly purified by pre-HPLC to give DL-B-07'-10 (21 mg). The structural property data are as follows: Chromatography column: SunFire Prep C18 OBD 19*150mm*5μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 5] Holding time: 7.00-9.00 min The structural property data are as follows: ESI-MS (m / z): 1779.5 (M+H) + .

[0261] Step 10: Compound DL-B-07'-10 (21 mg, 0.012 mmol) was dissolved in dichloromethane (1 mL), and TFA (67.3 mg, 0.59 mmol) was added. The reaction mixture was reacted at 25°C for 2 hours, then concentrated to give the crude product, which was purified by pre-HPLC to give the trifluoroacetate salt of DL-B-07' (14.0 mg). Holding time: 7.00-9.00 min Chromatography column: SunFire Prep C18 OBD 19*150mm*5μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 6] Holding time: 10.5-12.5 min The structural property data are as follows: ESI-MS (m / z): 1679.6 (M+H) + .

[0262] Preparation Example 11: (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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-((S)-6-amino-2-(S)-3-hydroxy-2-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamino)acetamidopropionamido)-4-fluorobenzoate (DL-A-01) [ka]

[0263] Step 1: DL-B-07'-9 (30.0 mg, 0.030 mmol), (2,5-dioxopyrrolidin-1-yl) 6-(2-methylsulfonylpyrimidin-5-yl)hex-5-ynoate (11.7 mg, 0.032 mmol), and DIPEA (11.8 mg, 0.091 mmol) were added to DMF (0.5 mL), and the reaction mixture was reacted at 25°C for 2 hours. The reaction mixture was directly purified by pre-HPLC and lyophilized to give DL-A-01-1 (22.0 mg). The purification method is as follows: Chromatography column: SunFire Prep C18 OBD 19*150mm*5μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 7] Holding time: 7.00-9.00 min The structural property data are as follows: ESI-MS (m / z): 1190.5 (M+H) + .

[0264] Step 2: DL-A-01-1 (22.0 mg, 0.018 mmol) was dissolved in DCM (1 mL), TFA (105.4 mg, 0.924 mmol) was added, and the reaction mixture was reacted at 25° C. for 1 hour. The reaction mixture was directly concentrated to give the crude product, which was purified by pre-HPLC to give the trifluoroacetate salt of DL-A-01 (14.6 mg). The purification method is as follows: Chromatography column: SunFire Prep C18 OBD 19*150mm*5μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 8] Holding time: 10.50-12.50 min The structural property data are as follows: ESI-MS (m / z): 1090.4 (M+H) + .

[0265] Preparation Example 12: (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-dodecahydro-3H-cyclopenta[a]phenanthrene-17-yl 3-((31S,34S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-31,34-dimethyl-1,2,32-trioxo-5,8,11,14,17,20,23,26-octaoxa-2,30,33-triazapentatriacontane-35-amino)-4-fluorobenzoate (DL-B-09') [ka]

[0266] Step 1: 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), and T3P (23.67 g, 37.19 mmol, 50.0% purity) and pyridine (5.35 g, 67.63 mmol) were added. The reaction mixture was stirred at 25 °C for 16 h and monitored by LC-MS until completion. The reaction mixture was then extracted with water and ethyl acetate, concentrated to give the crude product, which was purified by column chromatography (MeOH / DCM = 0-10%) to give DL-B-09'-1 (3.40 g, 7.77 mmol). The structure is characterized as follows: ESI-MS (m / z): 382.2 (M+H-56) + .

[0267] Step 2: Under a nitrogen atmosphere, DL-B-09'-1 (3.39 g, 7.75 mmol) was dissolved in THF (100.00 mL), and tetra(triphenylphosphine)palladium (895.47 mg, 774.93 μmol) and morpholine (1.35 g, 15.50 mmol) were added. The reaction mixture was stirred at 25 °C for 16 h, and the reaction was monitored by LC-MS until completion. The pH of the system was adjusted to approximately 8 using sodium bicarbonate, and the mixture was extracted with ethyl acetate (to remove impurities). The pH of the aqueous phase was adjusted to approximately 3 using dilute hydrochloric acid, and the product was extracted with ethyl acetate, dried, and concentrated to give DL-B-09'-2 (2.40 g, 6.04 mmol) as a white solid. The structure is characterized as follows: ESI-MS (m / z): 342.2 (M+H-56) + .

[0268] Step 3: DL-B-09'-2 (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), and the reaction mixture was stirred at 25 °C for 30 min. 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-oxo-6,7,8,11,12,14,15,16-octahydrocyclopenta[a]phenanthrene-17-carboxylic acid (300.80 mg, 754.91 μmol) were added, and the reaction mixture was stirred for 1 h. Fluoroiodomethane (482.93 mg, 3.02 mmol) was added, and the reaction mixture was stirred at 25°C for 2 hours. The reaction was monitored by LC-MS until completion. The reaction mixture was extracted with water and ethyl acetate and concentrated to give the crude product, which was purified by column chromatography (CAN / HO = 0-60%, 0.05% formic acid) and lyophilized to give the crude product DL-B-09'-3 (199 mg). The structure is characterized as follows: ESI-MS (m / z): 810.3 (M+H) + .

[0269] Step 4: DL-B-09'-3 (255 mg, 316.42 mmol) was added to DCM (8.00 mL), and TFA (4.00 mL) was added and stirred at 25 °C for 1 h. The reaction was monitored by LC-MS until completion. The reaction was concentrated to give the crude product, which was purified by high-performance liquid chromatography to give DL-B-09'-4 (100 mg, 121.39 μmol) as a white solid. The purification method is as follows: Chromatography column: Phenomenex C18 250mm x 50mm x 10μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% TFA) [Table 9] The structure is characterized as follows: ESI-MS (m / z): 710.3 (M+H) + .

[0270] Step 5: DL-B-09'-4 (30 mg, 36.42 μmol), DL-B-11'-6 (31.24 mg, 36.42 μmol), HATU (27.70 mg, 72.84 μmol), and DIPEA (23.53 mg, 182.09 μmol) were added to DMF (2.00 mL), and the reaction mixture was incubated at 25 °C for 1 h. The reaction mixture was filtered to obtain the crude product, which was purified by high-performance liquid chromatography to give DL-B-09' (4 mg, 2.56 μmol). The purification method is as follows: Chromatography column: Phenomenex C18 250mm x 50mm x 10μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 10] The structure is characterized as follows: ESI-MS (m / z): 1549.5 (M+H) + .

[0271] Preparation Example 13: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene Preparation of 1,317-yl 2-((31S,34R)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-31,34-dimethyl-1,2,32-trioxo-5,8,11,14,17,20,23,26-octaoxa-2,30,33-triazapentatriacontane-35-amino) isonicotinate (DL-B-17') [ka]

[0272] Step 1: 2-Nitroisonicotinic acid (500 mg, 2.97 mmol) and (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-thiocarboxylic acid (1.23 g, 2.97 mmol) were dissolved in DMF (10.00 mL), followed by the addition of T3P (10.00 g, 15.7 mmol, 50.0% purity) and DIPEA (1.15 g, 8.92 mmol). After stirring the reaction mixture at 25° C. for 1 hour, fluoroiodomethane (475 mg, 2.97 mmol) was added and the mixture was stirred at 25° C. for 1 hour. The reaction was monitored by LC-MS until completion. The reaction mixture was then extracted with water and ethyl acetate and dried over sodium sulfate to give the crude product (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-nitroisonicotinate (2.1 g) as a yellow oil, which was used directly in the next step without purification. The structure is characterized as follows: ESI-MS (m / z): 595.2 (M+H) + .

[0273] Step 2: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-nitroisonicotinate (1.50 g, 2.40 mmol) was dissolved in ethanol (10.00 mL) and water (3.00 mL), and iron powder (1.34 g, 23.9 mmol) and ammonium chloride (641 mg, 11.9 mmol) were added. The mixture was heated to 80°C and stirred for 2 hours. The reaction mixture was then filtered through a pad of diatomaceous earth, and the filtrate was concentrated to give the crude product, which was purified by column chromatography (MeOH / MeOH = 0-5%) to give (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-aminoisonicotinate (1.02 g, 1.69 mmol). The structure is characterized as follows: ESI-MS (m / z): 565.1 (M+H) + . 1H NMR: (400 MHz, CD3OD)δ 8.04 (d, J =5.6 Hz, 1H), 7.29-7.39 (m, 1H), 7.00 (s, 1H), 6.92-6.96 (m, 1H), 6.36 (dd, J= 10.0, 1.6 Hz, 1H), 6.32 (s, 1H), 5.94-6.02 (m, 1H), 5.80-5.89 (m, 1H), 5.49-5.66 (m, 1H), 4.33-4.41 (m, 1H), 3.46-3.57 (m, 1H), 2.57-2.72 (m, 1H), 2.33-2.42 (m, 3H), 1.99-2.08 (m, 2H), 1.65-1.77 (m, 1H), 1.59 (s, 3H), 1.36-1.44 (m, 1H), 1.19 (s, 3H), 1.00 (d, J = 7.2 Hz, 3H).

[0274] ステップ3: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-aminoisonicotinate (700 mg, 1.24 mmol), N-fluorenylmethoxycarbonyl-L-alanine (385 mg, 1.24 mmol), and pyridine (980 mg, 12.4 mmol) were added to dichloromethane (15.00 mL), followed by the addition of phosphorus oxychloride (2.17 g, 14.1 mmol). The mixture was stirred at 0° C. for 1 hour and then extracted with water and DCM. The organic phase was dried over sodium sulfate, filtered, and concentrated to give the crude product (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido) isonicotinate (1.5 g), which was used directly in the next step without purification. The structure is characterized as follows: ESI-MS (m / z): 858.3 (M+H) + .

[0275] Step 4: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido) isonicotinate (1.50 g, 1.75 mmol) was added to acetonitrile (10.00 mL), followed by the addition of diethylamine (2.13 g, 29.1 mmol). After stirring at 25° C. for 1 hour, the reaction mixture was directly concentrated to give the crude product (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-((S)-2-aminopropionamido)isonicotinate (1.5 g), which was used directly in the next step without purification. The structure is characterized as follows: ESI-MS (m / z): 636.1 (M+H) + .

[0276] Step 5: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-((S)-2-aminopropionamido)isonicotinate (1.30 g, 2.05 mmol) and N-fluorenylmethoxycarbonyl-L-alanine (636 mg, 2.05 mmol) were added to DMF (10.00 mL), followed by addition of DIPEA (792 mg, 6.14 mmol, 1.07 mL) and T3P (6.84 g, To the mixture was added 10.7 mmol, 6.40 mL, 50% purity), and the mixture was stirred at 25° C. for 1 hour, and then the reaction mixture was extracted with water and ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude product, which was purified by column chromatography (petroleum ether / ethyl acetate = 0 to 40%) to give (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-(((S)-2-(((9H-fluoro-yl)methoxy)carbonylamino)propionamido)propanamido) isonicotinate (264 mg, 252 μmol). The structure is characterized as follows: ESI-MS (m / z): 929.5 (M+H) + .

[0277] Step 6: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-(((S)-2-(((9H-fluoro-yl)methoxy)carbonylamino)propionamido)propanamido) isonicotinate (50 mg, 53.82 μmol) was dissolved in DMF (2.00 mL) and diethylamine (19.68 mg, 269.10 μmol) was added. After stirring at 25°C for 2 hours, the reaction mixture was concentrated under reduced pressure to remove DMF and diethylamine to give the crude product (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-(((S)-2-aminopropionamido)propanamido)isonicotinate (38 mg, 53.77 µmol), which was used directly in the next step without purification. The structure is characterized as follows: ESI-MS (m / z): 707.3 (M+H) + .

[0278] Step 7: At 25°C, (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-(((S)-2-aminopropionamido)propanamido) isonicotinate (38 mg, 53.77 To a mixture of 1,2-dimethyl-2,4-diphenyl-1,2,4-triazano-2-propanediol (46.13 mg, 53.77 μmol), 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-carboxylic acid (46.13 mg, 53.77 μmol), HATU (40.89 mg, 107.53 μmol), and DIPEA (34.74 mg, 268.83 μmol) was added to DMF (2.00 mL), and the reaction mixture was reacted at 25°C for 1 hour. The reaction mixture was filtered to obtain the crude product, which was purified by high-performance liquid chromatography to obtain the title compound (28 mg, 17.74 μmol). The preparation and purification conditions are as follows: Chromatography column: Phenomenex C18 250mm x 50mm x 10μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 11] The structure is characterized as follows: ESI-MS (m / z): 1547.6 [M+H] + .

[0279] Preparation Example 14: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-(((R)-2-(6-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamino)propionamido) isonicotinate (DL-A-08) [ka]

[0280] At 25°C, (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-(((S)-2-aminopropionamido)propionamido isonicotinate (38 mg, 53.77 μmol), (2,5-dioxopyrrolidin-1-yl) 6-(2-methylsulfonylpyrimidin-5-yl)hex-5-ynoic acid (19.64 mg, 53.77 μmol), To DMF (3.00 mL), DIPEA (13.90 mg, 107.53 μmol) and DIPEA (13.90 mg, 107.53 μmol) were added, and the reaction mixture was reacted at 25° C. for 1 hour. The reaction mixture was filtered to obtain a crude product, which was purified by high-performance liquid chromatography to obtain the title compound (21 mg, 21.72 μmol). The preparation and purification conditions are as follows: Chromatography column: Phenomenex C18 250mm x 50mm x 10μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 12] The structure is characterized as follows: ESI-MS (m / z): 957.3[M+H] + .

[0281] Preparation Example 15: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-hydroxybenzoate (B-1) [Table 13]

[0282] Step 1: Methyl 3-hydroxybenzoate (1.00 g, 6.57 mmol) was dissolved in tetrahydrofuran (10.0 mL) and cooled to 0 °C. NaH (526 mg, 13.1 mmol) was added and the reaction mixture was stirred for 30 min. Chloromethyl methyl ether (1.26 g, 15.7 mmol) was then added, and the reaction mixture was warmed to 25 °C and stirred for 2 h. Saturated aqueous ammonium chloride solution (10.0 mL) was added to the reaction mixture, which was then extracted three times with ethyl acetate (10.0 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product, methyl 3-(methoxymethoxy)benzoate (1.40 g), which was used directly in the next step without further purification. The structure is characterized as follows: ESI-MS (m / z): 197.1 (M+H) + .

[0283] Step 2: 3-(Methoxymethoxy)benzoate (700 mg, 3.57 mmol) was dissolved in tetrahydrofuran (3.00 mL), methanol (2.00 mL), and water (1.00 mL). Lithium hydroxide monohydrate (449 mg, 10.7 mmol) was added, and the reaction mixture was stirred at 25°C for 2 hours. Ethyl acetate (10.0 mL) and water (10.0 mL) were added to the reaction mixture, and the mixture was separated. The aqueous phase was adjusted to pH 3 with 10% aqueous citric acid and then extracted three times with ethyl acetate (10.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product, 3-(methoxymethoxy)benzoic acid (351 mg), which was used directly in the next step without further purification. The structure is characterized as follows: ESI-MS (m / z): 183.1 (M+H) + .

[0284] Step 3: 3-(Methoxymethoxy)benzoic acid (200 mg, 1.10 mmol) and HATU (417 mg, 1.10 mmol) were added to DMF (4.00 mL), and DIPEA (568 mg, 4.39 mmol) was added. The reaction mixture was stirred for 1 hour at 25° C. (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-S-carboxylic acid (435 mg, 1.10 mmol) was added, and the reaction mixture was stirred for an additional 2 hours. Next, fluoroiodomethane (351 mg, 2.20 mmol) was added, and the reaction mixture was stirred for an additional 1 hour. DCM (30.0 mL) and water (30.0 mL) were added to the reaction mixture, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-(methoxymethoxy)benzoate (1.00 g), which was used directly in the next step without further purification. The structure is characterized as follows: ESI-MS (m / z): 609.2 (M+H) + .

[0285] Step 4: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-(methoxymethoxy)benzoate (1.00 g, 1.64 mmol) was dissolved in dichloromethane (15.0 mL), trifluoroacetic acid (7.68 g, 67.3 mmol) was added, and the reaction mixture was stirred at 25° C. for 1 hour. The reaction mixture was directly concentrated and purified by high-performance liquid chromatography to obtain the title compound (134 mg, 230 μmol). The purification conditions were as follows: Chromatography column: Phenomenex luna C18 150mm x 25mm x 10μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 14] The structure is characterized as follows: ESI-MS (m / z): 565.3 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 9.97 (s, 1H), 7.23-7.26 (m, 1H), 7.21-7.38 (m, 3H), 7.02-7.09 (m, 1H), 6.32 (dd, J = 10, 1.8 Hz, 1H), 6.14 (s, 1H), 6.02 (s, 1H), 5.90 (s, 1H), 5.74 (m, 1H), 4.22-4.35 (m, 1H), 3.36-3.46 (m, 1H), 2.56-2.64 (m, 1H), 2.20-2.31 (m, 3H), 1.87-2.00 (m, 2H), 1.49-1.62 (m, 4H), 1.29-1.38 (m, 1H), 1.07 (s, 3H), 0.92 (d, J = 7.2 Hz, 3H).

[0286] Preparation Example 16: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-hydroxybenzoate (B-13) [ka]

[0287] Step 1: 4-(Methoxymethoxy)benzoic acid (146 mg, 800 μmol) and HATU (277 mg, 727 μmol) were dissolved in DMF (3.00 mL) at 25°C. DIPEA (376 mg, 2.91 mmol) was added and the reaction was stirred for 1 h. (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-S-carboxylic acid (300 mg, 727 μmol) was added and the reaction was stirred for an additional 6 h. Fluoroiodomethane (232 mg, 1.45 mmol) was then added and the mixture was stirred for an additional hour. Dichloromethane (30.0 mL) and water (30.0 mL) were added to the reaction mixture, and the mixture was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-(methoxymethoxy)benzoate (1.02 g), which was used directly in the next step without further purification. The structure is characterized as follows: ESI-MS (m / z): 609.3 (M+H) + .

[0288] Step 2: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-(methoxymethoxy)benzoate (1.02 g, 1.68 mmol) was dissolved in dichloromethane (30.0 mL), trifluoroacetic acid (15.7 g, 137 mmol) was added, and the reaction mixture was stirred at 25° C. for 30 minutes. The reaction mixture was directly concentrated to give the crude product, which was purified by high-performance liquid chromatography to give the title compound (116 mg, 205 μmol). The purification conditions were as follows: Chromatography column: Phenomenex luna C18 250mm x 70mm x 15μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 15] The structure is characterized as follows: ESI-MS (m / z): 565.3 [M+H] + . 1H NMR (400 MHz, DMSO) δ 10.50 (s, 1H), 7.72 (d, J = 8.8 Hz, 2H), 7.28 (d, J = 10 Hz, 1H), 6.82-6.94 (m, 2H), 6.33 (dd, J = 10.2, 1.8 Hz, 1H), 6.14 (s, 1H), 6.01 (s, 1H), 5.88 (s, 1H), 5.57-5.74 (m, 2H), 4.27-4.28 (m, 1H), 3.40-3.37 (m, 1H), 2.59-2.70 (m, 1H), 2.19-2.31 (m, 3H), 1.84-2.01 (m, 2H), 1.49-1.62 (m, 4H), 1.26-1.37 (m, 1H), 1.05 (s, 3H), 0.91 (d, J = 7.0 Hz, 3H).

[0289] Preparation Example 17: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-hydroxybenzoate (B-25) [ka]

[0290] Step 1: Methyl 3-hydroxybenzoate (3.00 g, 19.7 mmol) was dissolved in tetrahydrofuran (30.0 mL) and cooled to 0 °C. NaH (1.58 g, 39.4 mmol, 60.0% purity) was added, and the reaction mixture was stirred for 30 min. Chloromethyl methyl ether (3.18 g, 39.4 mmol) was then added, and the reaction mixture was warmed to 25 °C and stirred for 2.5 h. The reaction mixture was quenched by the addition of water (20.0 mL), and the reaction mixture was extracted three times with ethyl acetate (20.0 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product, methyl 2-(methoxymethoxy)benzoate (4.00 g), which was used directly in the next step without further purification. The structure is characterized as follows: ESI-MS (m / z): 197.1 (M+H) + .

[0291] Step 2: Methyl 2-(methoxymethoxy)benzoate (2.00 g, 10.2 mmol) was dissolved in ethanol (5.00 mL), and lithium hydroxide monohydrate (1.28 g, 30.6 mmol) was added. The reaction mixture was stirred at 25 °C for 2 h. Water (20.0 mL) and ethyl acetate (20.0 mL) were added, and the reaction mixture was separated. The aqueous phase was adjusted to pH 3 with 1N dilute hydrochloric acid and then extracted three times with ethyl acetate (25.0 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, adjusted to pH 9 with triethylamine, filtered, and concentrated to give the crude product, 2-(methoxymethoxy)benzoic acid (2.00 g), which was used directly in the next step without further purification. The structure is characterized as follows: ESI-MS (m / z): 183.1 (M+H) + .

[0292] Step 3: 2-(Methoxymethoxy)benzoic acid (350 mg, 1.92 mmol) was dissolved in DMF (1.00 mL), DIPEA (993 mg, 7.69 mmol) and HATU (657 mg, 1.73 mmol) were added, and the reaction mixture was stirred at 25° C. for 2 hours. Then, (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-S-carboxylic acid (713 mg, 1.73 mmol) was added, and the reaction mixture was warmed to 30° C. and stirred for 12 hours. Finally, fluoroiodomethane (614 mg, 3.84 mmol) was added, and the mixture was stirred for another hour. After cooling to 20°C, the reaction mixture was quenched by adding water (20.0 mL) and extracted three times with dichloromethane (20.0 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-(methoxymethoxy)benzoate (1.30 g), which was used directly in the next step without purification. The structure is characterized as follows: ESI-MS (m / z): 609.2 (M+H) + .

[0293] Step 4: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-(methoxymethoxy)benzoate (1.20 g, 1.97 mmol) was dissolved in dichloromethane (10.0 mmol), and trifluoroacetic acid (3.07 g, 26.92 mmol) was added. The reaction mixture was stirred at 25°C for 2 hours. The reaction mixture was directly concentrated to give the crude product, which was purified by high-performance liquid chromatography to give the title compound (168 mg, 297 µmol). The purification conditions were as follows: Chromatography column: Phenomenex luna C18 150mm x 25mm x 10μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 16] The structure is characterized as follows: ESI-MS (m / z): 565.3 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 10.23 (s, 1H), 7.70-7.72 (m, 1H), 7.48-7.49 (m, 1H), 7.12-7.19 (m, 1H), 6.97 (d, J = 8.5 Hz, 1H), 6.88-6.94 (m, 1H), 6.39-6.49 (m, 2H), 5.75-6.05 (m, 2H), 5.25-5.58 (m, 1H), 4.39-4.56 (m, 1H), 3.50-3.54 (m, 1H), 2.40-2.59 (m, 3H), 2.28-2.37 (m, 1H), 1.73-2.05 (m, 6H), 1.38-1.40 (m, 1H), 1.18 (s, 3H), 1.06 (d, J = 7.0 Hz, 3H).

[0294] Preparation Example 18: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-yl benzoate (H-1) [ka]

[0295] Benzoic acid (25 mg, 204.71 μmol) was dissolved in DMF (1 mL), and DIPEA (79.37 mg, 614.14 μmol) and HATU (65.13 mg, 171.29 μmol) were added sequentially. The reaction mixture was stirred at 15° C. for 1 hour. Then, (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-thiocarboxylic acid (84.44 mg, 204.71 μmol) was added. After the addition was complete, the reaction mixture was stirred at 15° C. for 4 hours. Then, fluoroiodomethane (130.96 mg, 818.86 μmol) was added, and the reaction mixture was further stirred at 15° C. for 1 hour. The reaction mixture was directly subjected to high-performance liquid chromatography, freeze-dried, and then purified by preparative thin-layer chromatography (developing solvent: petroleum ether / ethyl acetate=1 / 1, R f =0.4) to give the title compound (10 mg, 16.41 μmol). The purification conditions were as follows: Chromatography column: Waters Sunfire Prep C18 OBD 150mm x 19mm x 5μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 17] The structure is characterized as follows: ESI-MS (m / z): 549.3[M+H] + , 1197.4[2M+H] + . 1H NMR (400 MHz, CDCl3) δ 7.95 (dd, J = 8.4, 1.2 Hz, 2H), 7.60 (tt, J = 7.6, 1.2 Hz, 1H), 7.46 (t, J = 8.0 Hz, 2H), 7.14 (dd, J = 10.4, 1.2 Hz, 1H), 6.48 (s, 1H), 6.42 (dd, J = 10.0, 1.6 Hz, 1H), 5.91 (ddd, J = 50.0, 44.8, 9.2 Hz, 2H), 5.34-5.51 (m, 1H), 4.50 (dt, J =8.4, 2.4Hz, 1H), 3.48-3.55 (m, 1H), 2.59 (dt, J =14.8, 3.2 Hz, 1H), 2.51-2.42 (m, 2H), 2.31-2.35 (m, 1H), 1.94-2.01 (m, 2H), 1.83 (t, J = 9.6 Hz, 1H), 1.55 (s, 3H), 1.38-1.44 (m, 1H), 1.18 (s, 3H), 1.04 (d, J = 7.2 Hz, 3H).

[0296] Preparation Example 19: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-fluorobenzoate (H-5) [ka]

[0297] 4-Fluorobenzoic acid (20 mg, 142.74 μmol) was dissolved in DMF (1 mL), and DIPEA (55.34 mg, 428.23 μmol) and HATU (65.13 mg, 171.29 μmol) were added sequentially, and the reaction mixture was stirred at 15° C. for 1 hour. Then, (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-thiocarboxylic acid (58.88 mg, 142.74 μmol) was added, and after the addition was complete, the reaction mixture was stirred at 15°C for 16 hours. Fluoroiodomethane (91.32 mg, 570.97 μmol) was then added, and the reaction mixture was stirred at 15°C for an additional hour. The reaction mixture was directly subjected to high-performance liquid chromatography, lyophilized, and then analyzed by preparative thin-layer chromatography (developing solvent: petroleum ether / ethyl acetate = 1 / 1, R f =0.3) to give the title compound (13 mg, 21.80 μmol). The purification conditions were as follows: Waters Sunfire Prep C18 OBD Chromatography Column, 150mm x 19mm x 5μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 18] Its structure is characterized as follows: ESI-MS (m / z): 567.3[M+H] + , 1133.4[2M+H] + . 1H NMR (400 MHz, CDCl3) δ 7.90-8.00 (m, 2H), 7.09 - 7.17 (m, 3H), 6.48 (s, 1H), 6.43 (dd, J = 10.0, 1.6 Hz, 1H), 5.91 (ddd, J =50.0, 36.4, 9.2 Hz, 2H), 5.32-5.52 (m, 1H), 4.50 (ddd, J = 6.4, 4.0, 2.8 Hz, 1H), 3.51 (ddd, J = 10.4, 6.8, 3.2 Hz, 1H), 2.27-2.61(m, 4H), 1.92-2.05 (m, 2H), 1.82 (t, J = 12.0 Hz, 1H), 1.55 (s, 3H), 1.36-1.45 (m, 1H), 1.18 (s, 3H), 1.04 (d, J = 7.2 Hz, 3H).

[0298] Preparation Example 20: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-hydroxyacetate (D-1) [ka]

[0299] Step 1: Synthesis of methyl 2-(triphenylmethoxy)acetate (D-1-2) Methyl 2-hydroxyacetate (323 mg, 3.59 mmol, 276.88 μL) and triphenylmethyl chloride (1.00 g, 3.59 mmol) were dissolved in pyridine (4.00 mL), and the reaction mixture was heated to 80° C. and reacted for 15 hours. To the reaction mixture was added 1 N dilute hydrochloric acid (50.0 mL × 3), followed by extraction with dichloromethane three times (50.0 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product (1.10 g, 3.31 mmol), which was used directly in the next step without purification. The structure is characterized as follows: 1 H NMR (400 MHz, DMSO) δ 7.33-7.41 (m, 12H), 7.27-7.31 (m, 3H), 3.69-3.75 (m, 2H), 3.53-3.58 (m, 3H).

[0300] Step 2: Synthesis of 2-(triphenylmethoxy)acetic acid (D-1-3) Methyl 2-(triphenylmethoxy)acetate (1.10 g, 3.31 mmol) was dissolved in a mixture of tetrahydrofuran (3.00 mL), methanol (3.00 mL), and water (3.00 mL). Lithium hydroxide monohydrate (416 mg, 9.93 mmol) was added, and the reaction mixture was heated to 40°C and reacted for 3 hours. Water (50.0 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (50.0 mL). The aqueous phase was adjusted to pH 1 with 1 N dilute hydrochloric acid and filtered. The filtrate was extracted three times with dichloromethane (50.0 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product (700 mg, 2.20 mmol), which was used directly in the next step without purification. The structure is characterized as follows: 1 H NMR (400 MHz, DMSO) δ 7.42 (s, 6H), 7.33-7.38 (m, 6H), 7.27-7.30 (m, 3H), 3.54-3.59 (m, 2H).

[0301] Step 3: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-(triphenylmethoxy)acetate (D-1-4) 2-(Triphenylmethoxy)acetic acid (350 mg, 1.10 mmol) was dissolved in DMF (2.00 mL), and HATU (418 mg, 1.10 mmol) and DIEA (426 mg, 3.30 mmol, 574 μL) were added sequentially. The reaction mixture was stirred at 25° C. for 1 h. (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-thiocarboxylic acid (453 mg, 1.10 mmol) was added, and the reaction mixture was stirred for an additional 2 h. Next, fluoroiodomethane (175 mg, 1.10 mmol) was added, and the reaction mixture was stirred for an additional 0.4 hours. Water (50.0 mL) was added to the reaction mixture, which was then extracted three times with ethyl acetate (50.0 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude title compound (600 mg) as a yellow oil.

[0302] 3-(Triphenylmethoxy)propionic acid (300 mg, 902 μmol) was dissolved in DMF (2.00 mL), followed by the addition of HATU (343 mg, 903 μmol) and DIPEA (349 mg, 2.71 mmol, 472 μL), and the reaction mixture was stirred at 25° C. for 1 h. (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-thiocarboxylic acid (372 mg, 902 μmol) was added, and the mixture was stirred for an additional 12 h. Then, fluoroiodomethane (144 mg, 903 μmol) was added and stirred for another 1 hour. Water (100 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (80.0 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound (450 mg), which was used directly in the next step without purification. The structure is characterized as follows: ESI-MS (m / z): 745.3 (M+H) + .

[0303] Step 4: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-hydroxyacetate (D-1) (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-(triphenylmethoxy)acetate (500 mg, 671 μmol) was dissolved in a mixed solvent of dichloromethane (5.00 mL) and methanol (1.00 mL), and trifluoroacetic acid (1.54 g, 13.4 mmol, 1.00 mL) was added and the mixture was stirred at 25 ° C. for 1 hour. The reaction mixture was directly concentrated to give a crude product, which was purified by high performance liquid chromatography to give the title compound (141 mg, 259 μmol). Chromatography column: Phenomenex luna C18 200mm x 40mm x 10μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid) [Table 19] The structure is characterized as follows: ESI-MS (m / z): 503.3 [M+H] + . 1 H NMR (400 MHz, CD3OD) δ 7.27-7.39 (m, 1H), 6.31-6.40 (m, 1H), 6.22-6.31 (m, 1H), 5.90-5.99 (m, 1H), 5.78-5.89 (m, 1H), 5.44-5.63 (m, 1H), 4.25-4.32 (m, 1H), 4.10-4.23 (m, 2H), 3.38-3.46 (m, 1H), 2.49-2.69 (m, 1H), 2.29-2.37 (m, 1H), 2.17-2.27 (m, 2H), 1.90-2.03 (m, 2H), 1.59-1.69 (m, 1H), 1.57 (s, 3H), 1.31-1.40 (m, 1H), 1.10-1.18 (m, 3H), 0.97-1.05 (m, 3H).

[0304] Preparation Example 21: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-hydroxypropionate (D-9) [ka]

[0305] Step 1: Synthesis of methyl 2-(triphenylmethoxy)propionate (D-9-2) Methyl 2-hydroxypropionate (373 mg, 3.59 mmol, 341 μL) and triphenylmethyl chloride (1.00 g, 3.59 mmol) were dissolved in pyridine (4.00 mL), and the reaction mixture was heated to 80 °C and reacted for 15 hours. 1 N dilute hydrochloric acid (50.0 mL) was added to the reaction mixture, which was then extracted three times with dichloromethane (50.0 mL × 3). The combined organic phases were washed twice with 1 N dilute hydrochloric acid (50.0 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product (1.00 g, 2.89 mmol), which was used directly in the next step without further purification. The structure is characterized as follows: 1 H NMR (400 MHz, DMSO) δ 7.37-7.40 (m, 6H), 7.30-7.34 (m, 6H), 7.18-7.23 (m, 3H), 4.00-4.06 (m, 1H), 3.14-3.21 (m, 3H), 1.24-1.28 (m, 3H).

[0306] Step 2: Synthesis of 2-(triphenylmethoxy)propionic acid (D-9-3) Methyl 2-(triphenylmethoxy)propionate (1.00 g, 2.89 mmol) was dissolved in a mixed solvent of tetrahydrofuran (3.00 mL), methanol (3.00 mL), and water (3.00 mL). Lithium hydroxide monohydrate (363 mg, 8.66 mmol) was added, and the reaction mixture was heated to 40°C and reacted for 3 hours. Water (50 mL) was added to the reaction mixture, followed by extraction with dichloromethane (50 mL). The aqueous phase was adjusted to pH 1 with 1N dilute hydrochloric acid and filtered. The filtrate was extracted three times with dichloromethane (50 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound (250 mg, 752 μmol), which was used directly in the next step without purification. The structure is characterized as follows: 1 H NMR (400 MHz, DMSO) δ 7.38-7.48 (m, 6H), 7.30-7.35 (m, 6H), 7.25-7.29 (m, 3H), 3.89-4.00 (m, 1H), 0.92-1.04 (m, 3H).

[0307] Step 3: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-(triphenylmethoxy)propionate (D-9-4) 2-(Triphenylmethoxy)propionic acid (200 mg, 601 μmol) was dissolved in DMF (5.00 mL), HATU (228 mg, 601 μmol) and DIPEA (233 mg, 1.81 mmol, 314 μL) were added, and the reaction mixture was stirred for 1 h at 25° C. (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-thiocarboxylic acid (248 mg, 601 μmol) was added, and the reaction mixture was stirred for an additional 2 h. Next, fluoroiodomethane (96.2 mg, 601 μmol) was added, and the reaction mixture was stirred for an additional 0.5 hours. Water (100 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (50.0 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound (500 g, crude), which was used directly in the next step without purification. The structure is characterized as follows: ESI-MS (m / z): 759.3 (M+H) + .

[0308] Step 4: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-hydroxypropionate (D-9) (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 2-(triphenylmethoxy)propionate (300 mg, 395 μmol) was dissolved in a mixed solvent of dichloromethane (4.00 mL) and methanol (1.00 mL), and trifluoroacetic acid (1.54 g, 13.4 mmol, 1.00 mL) was added. The reaction mixture was then stirred at 25° C. for 1 hour. The reaction mixture was directly concentrated to give a crude product, which was purified by high performance liquid chromatography to give the title compound (102 mg, 189 μmol). Chromatography column: Phenomenex luna C18 200mm x 40mm x 10μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid) [Table 20] The structure is characterized as follows: ESI-MS (m / z): 517.1 [M+H] + . 1H NMR (400 MHz, CD3OD) δ 7.28-7.37 (m, 1H), 6.31-6.37 (m, 1H), 6.26-6.31 (m, 1H), 5.90-5.99 (m, 1H), 5.77-5.86 (m, 1H), 5.44-5.65 (m, 1H), 4.22-4.37 (m, 2H), 3.37-3.50 (m, 1H), 2.50-2.70 (m, 1H), 2.30-2.38 (m, 1H), 2.19-2.29 (m, 2H), 1.91-2.05 (m, 2H), 1.59-1.70 (m, 1H), 1.57 (s, 3H), 1.38-1.44 (m, 3H), 1.30-1.38 (m, 1H), 1.08-1.18 (m, 3H), 0.95-1.04 (m, 3H).

[0309] Preparation Example 22: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-hydroxypropionate (D-5) [ka]

[0310] Step 1: Synthesis of methyl 3-(triphenylmethoxy)propionate (D-5-2) Methyl 3-hydroxypropionate (375 mg, 3.60 mmol) was dissolved in pyridine (4.00 mL), triphenylmethyl chloride (1.00 g, 3.60 mmol) was added, and the reaction mixture was heated to 80°C and stirred for 12 hours. 1 N dilute hydrochloric acid (60.0 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (50.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound (1.10 g, 3.18 mmol). The structure is characterized as follows: 1 H NMR (400 MHz, DMSO) δ 7.21-7.47 (m, 19 H), 3.60 (s, 3H), 3.21 (t, J = 6.4 Hz, 2H), 2.58 (t, J = 6.4 Hz, 2H).

[0311] Step 2: Synthesis of 3-(triphenylmethoxy)propionic acid (D-5-3) Methyl 3-(triphenylmethoxy)propionate (1.10 g, 3.18 mmol) was added to a mixture of methanol (4.00 mL), water (4.00 mL), and tetrahydrofuran (4.00 mL). Lithium hydroxide monohydrate (399 mg, 9.53 mmol) was added, and the reaction mixture was stirred at 25°C for 2 hours. Water (60.0 mL) was added, and the reaction mixture was adjusted to pH 3-4 with 1 N dilute hydrochloric acid. The mixture was then extracted three times with dichloromethane (80.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound (980 mg, 2.95 mmol). The structure is characterized as follows: 1 H NMR (400 MHz, DMSO) δ 7.19-7.42 (m, 17H), 3.19 (t, J = 6.4 Hz, 2H), 2.44-2.49 (m, 2H).

[0312] Step 3: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-(triphenylmethoxy)propionate (D-5-4) 3-(Triphenylmethoxy)propionic acid (300 mg, 902 μmol) was dissolved in DMF (2.00 mL), followed by the addition of HATU (343 mg, 903 μmol) and DIPEA (349 mg, 2.71 mmol, 472 μL), and the reaction mixture was stirred at 25° C. for 1 h. (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-thiocarboxylic acid (372 mg, 902 μmol) was added, and the mixture was stirred for an additional 12 h. Then, fluoroiodomethane (144 mg, 903 μmol) was added and the mixture was stirred for another hour. Water (100 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (80.0 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound (450 mg, crude). The structure is characterized as follows: ESI-MS (m / z): 781.3 (M+Na) + .

[0313] Step 4: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-hydroxypropionate (D-5) (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-(triphenylmethoxy)propionate (450 mg, 592 μmol) was dissolved in dichloromethane (5.00 mL), TFA (1.68 g, 14.8 mmol, 1.10 mL) was added, and the reaction mixture was stirred at 25° C. for 2 hours. The reaction mixture was directly concentrated to give a crude product, which was purified by high performance liquid chromatography to give the title compound (152.45 mg, 295.12 μm). Chromatography column: Phenomenex luna C18 150mm x 25mm x 5μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid) [Table 21] The structure is characterized as follows: ESI-MS (m / z): 517.2 [M+H] + . 1H NMR (400 MHz, DMSO) δ 7.25 (d, J = 10.0 Hz, 1H), 6.30 (dd, J = 10.0, 1.6 Hz, 1H), 6.11 (s, 1H), 5.99 (s, 1H), 5.86 (s, 1H), 5.52-5.75 (m, 2H), 4.16-4.26 (m, 1H), 3.61 (t, J = 6.4 Hz, 2H), 3.22-3.32 (m, 1H), 2.51-2.67 (m, 1H), 2.45-2.48 (m, 2H), 2.23-2.26 (m, 1H), 2.05-2.19 (m, 2H), 1.79-1.93 (m, 2H), 1.50-1.54 (m, 1H), 1.48 (s, 3H), 1.20-1.30 (m, 1H), 0.99 (s, 3H), 0.91 (d, J = 7.2 Hz, 3H).

[0314] Preparation Example 23: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-hydroxybutyrate (D-11) [ka]

[0315] Step 1: Synthesis of methyl 3-(triphenylmethoxy)butyrate (D-11-2) Methyl 3-hydroxybutyrate (211 mg, 1.79 mmol) and triphenylmethyl chloride (500 mg, 1.79 mmol) were dissolved in pyridine (3.00 mL), and the reaction mixture was heated to 80°C and reacted for 10 hours. 1N dilute hydrochloric acid (50.0 mL x 3) was added to the reaction mixture, which was then extracted three times with dichloromethane (50.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound (520 mg, 1.44 mmol). The structure is characterized as follows: 1 H NMR (400 MHz, DMSO) δ 7.41 (d, J = 7.2 Hz, 6H), 7.33 (t, J = 7.2 Hz, 6H), 7.26-7.28 (m, 3H), 3.76-3.84 (m, 1H), 3.48 (s, 3H), 2.21 (dd, J = 15.2, 7.6 Hz, 1H), 2.02-2.11 (m, 1H), 0.88 (d, J = 6.0 Hz, 3H).

[0316] Step 2: Synthesis of 3-(triphenylmethoxy)butyric acid (D-11-3) Methyl 3-(triphenylmethoxy)butyrate (500 mg, 1.39 mmol) was dissolved in a mixed solvent of tetrahydrofuran (1.50 mL), methanol (1.50 mL), and water (1.50 mL). Lithium hydroxide monohydrate (174 mg, 4.16 mmol) was added, and the reaction mixture was heated to 40°C and reacted for 2 hours. Water (50 mL) was added to the reaction mixture, followed by extraction with dichloromethane (50 mL). The aqueous phase was adjusted to pH 1 with 1 N dilute hydrochloric acid and filtered. The filtrate was extracted three times with dichloromethane (50 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound (250 mg, 721 μmol), which was used directly in the next step without purification. The structure is characterized as follows: 1 H NMR (400 MHz, DMSO) δ 7.39-7.45 (m, 6H), 7.29-7.36 (m, 6H), 7.22-7.28 (m, 3H), 3.79-3.85 (m, 1H), 1.98-2.10 (m, 1H), 1.86-1.95 (m, 1H), 0.77-0.87 (m, 3H).

[0317] Step 3: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-(triphenylmethoxy)butanoate (D-11-4) 3-(Triphenylmethoxy)butyric acid (190 mg, 548 μmol) was dissolved in DMF (6.00 mL), and HATU (208 mg, 548 μmol) and DIPEA (212 mg, 1.65 mmol, 286 μL) were added. The mixture was stirred for 1 hour at 25° C. (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-thiocarboxylic acid (226 mg, 548 μmol) was added, and the reaction mixture was stirred for an additional 2 hours. Fluoroiodomethane (87.7 mg, 548 μmol) was then added, and the reaction was stirred for an additional 0.5 hours. Water (100 mL) was added to the reaction, and the mixture was extracted three times with ethyl acetate (50.0 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product (600 mg, crude), which was used directly in the next step without purification. The structure is characterized as follows: ESI-MS (m / z): 773.3 (M+H) + .

[0318] Step 4: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-hydroxybutyrate (D-11) (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-(triphenylmethyl) (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-(triphenylmethoxy)butanoate (550 mg, 711 μmol) was dissolved in dichloromethane (5.00 mL) and methanol (1.00 mL), trifluoroacetic acid (1.54 g, 13.4 mmol, 1.00 mL) was added, and the reaction mixture was stirred at 25 °C for 0.5 hours. The reaction mixture was directly concentrated to give a crude product, which was purified by high performance liquid chromatography to give the title compound (76.0 mg, 142 μmol). Chromatography column: Welch Xtimate C18 150mm x 25mm x 5μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid) [Table 22] The structure is characterized as follows: ESI-MS (m / z): 531.2 [M+H] + . 1 H NMR (400 MHz, CD3OD) δ 7.26-7.37 (m, 1H), 6.32-6.36 (m, 1H), 6.28-6.31 (m, 1H), 5.89-5.98 (m, 1H), 5.77-5.86 (m, 1H), 5.45-5.64 (m, 1H), 4.25-4.32 (m, 1H), 4.10-4.19 (m, 1H), 3.35-3.43 (m, 1H), 2.52-2.66 (m, 1H), 2.45 (s, 2H), 2.29-2.36 (m, 1H), 2.20-2.29 (m, 2H), 1.89-2.01 (m, 2H), 1.59-1.71 (m, 1H), 1.54-1.59 (m, 3H), 1.30-1.41 (m, 1H), 1.17-1.25 (m, 3H), 1.10-1.15 (m, 3H), 1.02 (d, J = 7.2 Hz, 3H).

[0319] Preparation Example 24: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl (S)-3-hydroxybutyrate (D-15) [ka]

[0320] Step 1: Synthesis of methyl (S)-3-(triphenylmethyloxy)butyrate (D-15-2) Methyl (S)-3-hydroxybutyrate (300 mg, 2.54 mmol) was added to a single-neck flask, followed by the addition of pyridine (3 mL) and triphenylmethyl chloride (849 mg, 3.05 mmol). The reaction mixture was heated to 80 °C and stirred for 15 hours. The mixture was then cooled to room temperature. Water (15 mL) was then added to the reaction mixture, which was then extracted twice with ethyl acetate (8 mL × 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product, which was purified by flash column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 3 / 1) to give the title compound (460 mg, 1.15 mmol). The structure is characterized as follows: 1 H NMR (400 MHz, DMSO) δ 7.48 - 7.41 (m, 6H), 7.36 - 7.26 (m, 6H), 7.26 - 7.21 (m, 3H), 3.86 - 3.80 (m, 1H), 3.46 (s, 3H), 2.20 (dd, J =14.8, 7.6 Hz, 1H), 2.06 (dd, J = 14.8, 4.8 Hz, 1H), 0.89 (d, J = 6.0 Hz, 3H).

[0321] Step 2: Synthesis of (S)-3-(triphenylmethyloxy)butyric acid (D-15-3) Methyl (S)-3-(triphenylmethyloxy)butyrate (460 mg, 1.15 mmol) was added to a mixture of methanol (1 mL), tetrahydrofuran (1 mL), and water (0.5 mL), and lithium hydroxide (36.9 mg, 1.54 mmol) was added. The reaction mixture was stirred at 25°C for 2 hours. Water and 1 N dilute hydrochloric acid (2 mL) were added to the reaction mixture, which was then extracted three times with dichloromethane (10 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound (142 mg, 0.41 mmol).

[0322] Step 3: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl (S)-3-(triphenylmethoxy)butanoate (D-15-4) (S)-3-(Triphenylmethyloxy)butyric acid (50 mg, 0.14 mmol) and HATU (60.4 mg, 0.16 mmol) were added to DMF (2.0 mL), followed by DIPEA (56.0 mg, 0.43 mmol), and the reaction mixture was stirred at 25° C. for 2 hours. (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-thiocarboxylic acid (63.2 mg, 0.16 mmol) was added, and the mixture was stirred for an additional 2 hours. Then, fluoroiodomethane (34.7 mg, 0.22 mmol) was added and the mixture was reacted at 25° C. for 1 hour. Water (10 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate three times (10 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product (60 mg), which was used directly in the next step without purification. The structure is characterized as follows: ESI-MS (m / z): 773.3 (M+H) + .

[0323] Step 4: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl (S)-3-hydroxybutyrate (D-15) (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl(S)-3-(triphenylmethoxy)butanoate (32 mg, 0.04 mmol) was added to dichloromethane (1 mL) and acetic acid (0.5 mL) was added. After stirring at 40 °C for 3 hours, the reaction mixture was concentrated to give the crude product, which was purified by high-performance liquid chromatography to give the title compound (4.2 mg, 0.008 mmol). Chromatography column: SunFire prep C18 OBD 150mm x 19mm x 5μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 23] The structure is characterized as follows: ESI-MS (m / z): 574.1[M+Na] + . 1H NMR (400 MHz, DMSO) δ 7.25 (d, J = 10.2 Hz, 1H), 6.30 (d, J = 10.2, 1H), 6.11 (s, 1H), 5.99 (s, 1H), 5.87 (s, 1H), 5.74 - 5.54 (m, 1H), 5.58 (d, J = 4.0 Hz, 1H), 4.74 (d, J = 5.2 Hz, 1H), 4.20 (s, 1H), 3.98 - 3.90 (m, 1H), 3.30 - 3.25 (m, 1H), 2.47 - 2.36 (m, 3H), 2.28 - 2.21 (m, 1H), 2.17 - 2.07 (m, 2H), 1.92 - 1.80 (m, 2H), 1.58 - 1.42 (m, 1H), 1.48 (s, 3H), 1.31 - 1.15 (m, 2H), 1.09 (d, J = 6.2 Hz, 3H), 0.99 (s, 2H), 0.91 (d, J = 7.1 Hz, 3H).

[0324] Preparation Example 25: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl (R)-3-hydroxybutyrate (D-19) [ka]

[0325] Step 1: Synthesis of methyl (R)-3-(triphenylmethoxy)butyrate (D-19-2) Methyl (R)-3-hydroxybutyrate (300 mg, 2.54 mmol) was added to a single-neck flask, followed by pyridine (3 mL) and triphenylmethyl chloride (849 mg, 3.05 mmol). The reaction mixture was heated to 80 °C and stirred for 15 h. The mixture was then cooled to room temperature. Water (15 mL) was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate (8 mL × 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product, which was purified by flash column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 5 / 1) to give the title compound (278 mg, 0.77 mmol). The structure is characterized as follows: 1 H NMR (400 MHz, DMSO) δ 7.47 - 7.41 (m, 6H), 7.37 - 7.28 (m, 6H), 7.28 - 7.23 (m, 3H), 3.88 - 3.80 (m, 1H), 3.48 (s, 3H), 2.22 (dd, J =14.8, 7.6 Hz, 1H), 2.07 (dd, J = 14.8, 4.8 Hz, 1H), 0.89 (d, J = 6.0 Hz, 3H).

[0326] Step 2: Synthesis of (R)-3-(triphenylmethoxy)butyric acid (D-19-3) Methyl (R)-3-(triphenylmethoxy)butyrate (278 mg, 0.77 mmol) was added to a mixture of methanol (1 mL), tetrahydrofuran (1 mL), and water (0.5 mL), and lithium hydroxide (36.9 mg, 1.54 mmol) was added. The reaction mixture was stirred at 25°C for 2 hours. Water and 1 N dilute hydrochloric acid (2 mL) were added to the reaction mixture, which was then extracted three times with dichloromethane (10 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound (142 mg, 0.41 mmol).

[0327] Step 3: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl (R)-3-(triphenylmethoxy)butanoate (D-19-4) (R)-3-(Triphenylmethoxy)butyric acid (50 mg, 0.14 mmol) and HATU (60.4 mg, 0.16 mmol) were added to DMF (2.0 mL), followed by DIPEA (56.0 mg, 0.43 mmol). The reaction mixture was stirred at 25 °C for 2 h, followed by (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-thiocarboxylic acid (63.2 mg, 0.16 mmol). The mixture was stirred for an additional 2 h. Then, fluoroiodomethane (34.7 mg, 0.22 mmol) was added and the mixture was allowed to react at 25°C for 1 hour. Water (10 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (10 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product. The crude product was purified by flash column chromatography (C18, water / acetonitrile = 0.8) to give the title compound (32 mg, 0.04 mmol). The structure is characterized as follows: ESI-MS (m / z): 773.3 (M+H) + .

[0328] Step 4: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl (R)-3-hydroxybutyrate (D-19) (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl(R)-3-(triphenylmethoxy)butanoate (32 mg, 0.04 mmol) was added to dichloromethane (1 mL) and acetic acid (0.5 mL) was added. After stirring at 40 °C for 3 hours, the reaction mixture was concentrated to give the crude product, which was purified by high-performance liquid chromatography to give the title compound (5.2 mg, 0.01 mmol). Chromatography column: SunFire prep C18 OBD 150mm x 19mm x 5μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 24] The structure is characterized as follows: ESI-MS (m / z): 531.2[M+H] + . 1H NMR (400 MHz, CD3OD) δ 7.33 (d, J = 10.0 Hz, 1H), 6.37 - 6.28 (m, 2H), 5.94 - 5.90 (m, 1H), 5.85 - 5.76 (m, 1H), 5.65 - 5.45 (m, 1H), 4.29 (d, J = 9.6 Hz, 1H), 4.20 - 4.10 (m, 1H), 3.45 - 3.35 (m, 1H), 2.68 - 2.52 (m, 1H), 2.49 - 2.45 (m, 2H), 2.37 - 2.20 (m, 3H), 2.02 - 1.90 (m, 2H), 1.70 - 1.60 (m, 1H), 1.57 (s, 3H), 1.39 - 1.32 (m, 1H), 1.21 (d, J = 6.4 Hz, 3H), 1.13 (s, 3H), 1.02 (d, J = 7.2 Hz, 3H).

[0329] Preparation Example 26: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-hydroxybutanoate (D-23) [ka]

[0330] Step 1: Synthesis of benzyl 4-(triphenylmethoxy)butyrate (D-23-2) Benzyl 4-hydroxybutyrate (500 mg, 2.57 mmol) was dissolved in pyridine (3.00 mL), triphenylmethyl chloride (718 mg, 2.57 mmol) was added, and the reaction mixture was heated to 90 °C and reacted for 10 hours. 1 N dilute hydrochloric acid (20.0 mL) was added to the reaction mixture, which was then extracted three times with dichloromethane (30.0 mL × 3). The organic phases were combined, washed with 1 N hydrochloric acid (20.0 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product (900 mg, crude), which was used directly in the next step without purification. The structure is characterized as follows: ESI-MS (m / z): 459.3 (M+Na) + .

[0331] Step 2: Synthesis of 4-(triphenylmethoxy)butyric acid (D-23-3) Benzyl 4-(triphenylmethoxy)butyrate (850 mg, 1.95 mmol) was dissolved in a mixture of methanol (0.50 mL), tetrahydrofuran (0.50 mL), and water (0.50 mL). Lithium hydroxide (140 mg, 5.84 mmol) was added, and the reaction mixture was heated to 40°C and reacted for 1 hour. The reaction mixture was adjusted to pH 2-3 with 1 N dilute hydrochloric acid and extracted with ethyl acetate (20.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product (230 mg, crude), which was used directly in the next step without purification. The structure is characterized as follows: ESI-MS (m / z): 369.2 (M+H) + .

[0332] Step 3: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-(triphenylmethoxy)butanoate (D-23-4) 4-(Triphenylmethoxy)butyric acid (180 mg, 519 μmol) was dissolved in DMF (3.00 mL), HATU (198 mg, 520 μmol) and DIPEA (201 mg, 1.56 mmol, 272 μL) were added, and the reaction mixture was incubated at 25° C. for 1 hour. (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-thiocarboxylic acid (193 mg, 468 μmol) was added, and the mixture was stirred for an additional 0.5 hours. Then, fluoroiodomethane (83.1 mg, 520 μmol) was added and stirred for an additional 0.5 hours. Water (10.0 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (20.0 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product (480 mg, crude), which was used directly in the next step without purification. The structure is characterized as follows: ESI-MS (m / z): 796.3 (M+H) + .

[0333] Step 4: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-hydroxybutanoate (D-23) (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-(triphenylmethoxy)butanoate (430 mg, 556 μmol) was dissolved in a mixed solvent of dichloromethane (4.00 mL) and methanol (1.00 mL), and trifluoroacetic acid (1.54 g, 13.5 mmol, 1.00 mL) was added. The reaction mixture was then stirred at 25° C. for 1 hour. The reaction mixture was directly concentrated to give a crude product, which was purified by high performance liquid chromatography to give the title compound (49.0 mg, 92.2 μmol). Chromatography column: Phenomenex Luna C18 200mm x 40mm x 10μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid) [Table 25] The structure is characterized as follows: ESI-MS (m / z): 531.1 [M+H] + . 1H NMR (400 MHz, CD3OD) δ 7.32 (m, 1H), 6.33 (m, 1H), 6.30 (s, 1H), 5.91-5.97 (m, 1H), 5.79-5.85 (m, 1H), 5.44-5.64 (m, 1H), 4.27-4.32 (m, 1H), 3.56 (t, J = 6.4 Hz, 2H), 3.41 (m, 1H), 2.51-2.65 (m, 1H), 2.46 (t, J = 7.6 Hz, 2H), 2.30-2.35 (m, 1H), 2.18-2.28 (m, 2H), 1.93-2.01 (m, 2H), 1.81 (m, 2H), 1.58-1.66 (m, 1H), 1.57 (s, 3H), 1.34 (m, 1H), 1.12 (s, 3H), 0.99 (d, J = 7.2 Hz, 3H).

[0334] Preparation Example 27: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 5-hydroxyvalerate (D-24) [ka]

[0335] Step 1: Synthesis of methyl 5-(triphenylmethoxy)valerate Methyl 5-hydroxyvalerate (450 mg, 3.41 mmol) was dissolved in pyridine (15.0 mL), triphenylmethyl chloride (949 mg, 3.41 mmol) was added, and the reaction mixture was heated to 80°C and reacted for 10 hours. 1 N dilute hydrochloric acid (20.0 mL) was added to the reaction mixture, which was then extracted three times with dichloromethane (30.0 mL x 3). The combined organic phase was washed twice with 1 N dilute hydrochloric acid (20.0 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product (937 mg, crude). The structure is characterized as follows: 1 H NMR (400 MHz, DMSO) δ 7.35-7.37 (m, 6H), 7.27-7.30 (m, 6H), 7.21 (d, J = 1.6 Hz, 3H), 3.55-3.59 (m, 3H), 2.93-2.98 (m, 2H), 2.22-2.29 (m, 2H), 1.53-1.63 (m, 4H).

[0336] Step 2: Synthesis of 5-(triphenylmethoxy)valeric acid (D-24-3) Methyl 5-(triphenylmethoxy)valerate (937 mg, 2.50 mmol) was dissolved in a mixture of tetrahydrofuran (2.00 mL), water (2.00 mL), and methanol (2.00 mL). Lithium hydroxide monohydrate (315 mg, 7.51 mmol) was added, and the reaction mixture was warmed to 40°C and stirred for 1 hour. The reaction mixture was adjusted to pH 2-3 with 1 N dilute hydrochloric acid, and water (10.0 mL) was added. The mixture was then extracted with ethyl acetate (20.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product (285 mg), which was used directly in the next step without further purification. The structure is characterized as follows: 1H NMR (400 MHz, DMSO) δ 7.34-7.39 (m, 8H), 7.31-7.34 (m, 4H), 7.26 (m, 3H), 2.92-2.99 (m, 2H), 2.17 (m, 2H), 1.53-1.60 (m, 4H).

[0337] Step 3: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 5-(triphenylmethoxy)valerate (D-24-4) 5-(Triphenylmethoxy)valeric acid (230 mg, 638 μmol) was dissolved in DMF (3.00 mL), HATU (243 mg, 638 μmol) and DIPEA (247 mg, 1.91 mmol, 333 μL) were added, and the reaction mixture was stirred at 25° C. for 1 h. (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-thiocarboxylic acid (237 mg, 574 μmol) was added, and the reaction mixture was allowed to continue for an additional 0.5 h. Then, fluoroiodomethane (102 mg, 638 μmol) was added and the reaction was continued for another 0.5 hours. Water (10.0 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (20.0 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product (600 mg, crude), which was used directly in the next step without purification. The structure is characterized as follows: ESI-MS (m / z): 809.3 (M+Na) + .

[0338] Step 4: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 5-hydroxyvalerate (D-24) (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 5-(triphenylmethoxy)valerate (600 mg, 762 μmol) was dissolved in a mixture of dichloromethane (4.00 mL) and methanol (1.00 mL), and trifluoroacetic acid (1.54 g, 13.5 mmol, 1.00 mL) was added. The reaction mixture was then stirred at 25° C. for 1 hour. The reaction mixture was directly concentrated to give a crude product, which was purified by high performance liquid chromatography to give the title compound (72.0 mg, 132 μmol). Chromatography column: Phenomenex Luna C18 200mm x 40mm x 10μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid) [Table 26] The structure is characterized as follows: ESI-MS (m / z): 545.2 [M+H] + . 1H NMR (400 MHz, CD3OD) δ 7.32 (m, 1H), 6.34 (m, 1H), 6.30 (s, 1H), 5.91-5.97 (m, 1H), 5.79-5.85 (m, 1H), 5.44-5.64 (m, 1H), 4.27-4.32 (m, 1H), 3.54 (t, J = 6.4 Hz, 2H), 3.40 (m, 1H), 2.51-2.63 (m, 1H), 2.39-2.44 (m, 2H), 2.30-2.37 (m, 1H), 2.19-2.28 (m, 2H), 1.93-2.01 (m, 2H), 1.64-1.70 (m, 2H), 1.59 (m, 2H), 1.57 (s, 3H), 1.53-1.56 (m, 1H), 1.34 (m, 1H), 1.12 (s, 3H), 0.99 (d, J = 7.2 Hz, 3H).

[0339] Preparation Example 28: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl (1S,4S)-4-hydroxycyclohexane-1-formate (D-25) [ka]

[0340] Step 1: Synthesis of methyl (1S,4S)-4-hydroxycyclohexane-1-formate (D-25-2) (1S,4S)-4-Hydroxycyclohexane-1-formic acid (345 mg, 2.5 mmol) was added to DMF (2 mL), and anhydrous potassium carbonate (345 mg, 2.5 mmol) and methyl iodide (591.6 mg, 4.17 mmol) were added. The reaction mixture was stirred at 25 °C for 2 hours. Water (20 mL) was added, and the mixture was extracted with ethyl acetate three times (10 mL × 3) and washed with saturated aqueous sodium chloride twice (10 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product (310 mg, 1.96 mmol), which was used directly in the next step without purification.

[0341] Step 2: Synthesis of methyl (1S,4S)-4-(triphenylmethoxy)cyclohexane-1-formate (D-25-3) Methyl (1S,4S)-4-hydroxycyclohexane-1-formate (310 mg, 1.96 mmol) was added to a single-neck flask, followed by pyridine (3 mL) and triphenylmethyl chloride (820.5 mg, 2.94 mmol). The reaction mixture was heated to 80 °C and stirred for 15 h. The mixture was then cooled to room temperature. Water (15 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (8 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product, which was purified by flash column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 5 / 1) to give the title compound (320 mg, 0.8 mmol). The structure is characterized as follows: 1 H NMR (400 MHz, DMSO) δ 7.46 - 7.41 (m, 6H), 7.36 -7.29 (m, 6H), 7.28 - 7.22 (m, 3H), 3.50 (s, 3H), 1.68 (d, J = 10.4 Hz, 2H), 1.26 - 1.01 (m, 8H).

[0342] Step 3: Synthesis of (1S,4S)-4-(triphenylmethoxy)cyclohexane-1-formic acid (D-25-4) Methyl (1S,4S)-4-(triphenylmethoxy)cyclohexane-1-formate (320 mg, 0.8 mmol) was added to tetrahydrofuran (2 mL), methanol (2 mL), and water (1 mL). Lithium hydroxide (67.2 mg, 1.6 mmol) was added, and the reaction mixture was stirred at 25 °C for 2 h. Water and 1 N dilute hydrochloric acid (3 mL) were added to the reaction mixture, which was then extracted three times with dichloromethane (67.2 mg, 1.6 mmol). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product, which was purified by flash column chromatography (SiO, petroleum ether / ethyl acetate = 100 / 1 to 3 / 1) to give the title compound (280 mg, 0.72 mmol).

[0343] Step 4: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl(1S,4S)-4-(triphenylmethoxy)cyclohexane-1-formate (D-25-5) (1S,4S)-4-(triphenylmethoxy)cyclohexane-1-formate (50 mg, 0.13 mmol) and HATU (59.0 mg, 0.16 mmol) were added to DMF (2.0 mL), and DIPEA (50.1 mg, 0.39 mmol) was added. The reaction mixture was then reacted for 2 hours at 25° C. (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-thiocarboxylic acid (63.2 mg, 0.16 mmol) was added to the reaction mixture, and the mixture was stirred for an additional 2 hours. Fluoroiodomethane (31.0 mg, 0.19 mmol) was then added, and the reaction mixture was allowed to react for an additional hour at 25°C. Water (10 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (8 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product, which was purified by flash column chromatography (C18, water / acetonitrile = 0.8) to give the title compound (16 mg, 0.02 mmol).

[0344] Step 5: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl(1S,4S)-4-hydroxycyclohexane-1-formate (D-25) (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl(1S,4S)-4-(triphenylmethoxy)cyclohexane-1-formate (60 mg, 0.073 mmol) was dissolved in dichloromethane (1 mL), acetic acid (0.5 mL) was added, and the reaction mixture was stirred at 40°C for 3 hours. The reaction mixture was directly concentrated to give the crude product, which was purified by high-performance liquid chromatography to give the title compound (3.1 mg, 0.005 mmol). Chromatography column: SunFire prep C18 OBD 150 mm x 19 mm x 5 μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 27] The structure is characterized as follows: ESI-MS (m / z): 571.1[M+H] + . 1 H NMR (400 MHz, CD3OD) δ 7.33 (dd, J = 10.0, 1.6 Hz, 1H), 6.38 - 6.28 (m, 2H), 5.98 - 5.88 (m, 1H), 5.86 - 5.76 (m, 1H), 5.65 - 5.45 (m, 1H), 4.35 - 4.26 (m, 1H), 3.51 - 3.45 (m, 1H), 2.69 - 2.50 (m, 1H), 2.39 - 2.18 (m, 4H), 2.08 - 1.90 (m, 6H), 1.57 (s, 3H), 1.54 -1.41 (m, 2H), 1.36 - 1.21 (m, 4H), 1.11 (s, 3H), 0.96 (d, J = 7.2 Hz, 3H).

[0345] Preparation Example 29: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl(1R,4R)-4-hydroxycyclohexane-1-formate (D-26) [ka]

[0346] Step 1: Synthesis of (1R,4R)-4-hydroxycyclohexane-1-carboxylate (D-26-2) (1R,4R)-4-Hydroxycyclohexane-1-formic acid (300 mg, 2.1 mmol) was dissolved in DMF (2 mL), and anhydrous potassium carbonate (345 mg, 2.5 mmol) and methyl iodide (591.6 mg, 4.17 mmol) were added. The mixture was stirred at 25°C for 2 hours. Water (20 mL) was added to the reaction mixture, which was extracted three times with ethyl acetate (10 mL x 3) and then washed twice with saturated aqueous sodium chloride (10 mL x 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound (310 mg, 1.96 mmol) as a crude product, which was used directly in the next step without further purification.

[0347] Step 2: Synthesis of methyl (1R,4R)-4-(triphenylmethoxy)cyclohexane-1-carboxylate (D-26-3) Methyl (1R,4R)-4-hydroxycyclohexane-1-carboxylate (310 mg, 1.96 mmol) was added to a single-neck flask, and pyridine (3 mL) and triphenylmethyl chloride (820.5 mg, 2.94 mmol) were added. The reaction mixture was heated to 80 °C and stirred for 15 hours. The reaction mixture was cooled to room temperature. Water (15 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (8 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product, which was purified by flash column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 5 / 1) to give the title compound (691 mg, 1.73 mmol). The structure is characterized as follows: 1 H NMR (400 MHz, DMSO) δ 7.44 - 7.40 (m, 6H), 7.38 -7.30 (m, 6H), 7.28 - 7.20 (m, 3H), 3.48 (s, 3H), 1.67 (d, J = 10.4 Hz, 2H), 1.24 - 1.01 (m, 8H).

[0348] Step 3: Synthesis of (1R,4R)-4-(triphenylmethoxy)cyclohexane-1-carboxylic acid (D-26-4) Methyl (1R,4R)-4-(triphenylmethoxy)cyclohexane-1-carboxylate (691 mg, 1.73 mmol) was added to tetrahydrofuran (6 mL), methanol (6 mL), and water (3 mL). Lithium hydroxide (123.96 mg, 5.18 mmol) was added and the mixture was stirred at 25°C for 2 hours. Water and 1 N dilute hydrochloric acid (3 mL) were added to the reaction mixture, which was then extracted three times with dichloromethane (10 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product, which was purified by flash column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 3 / 1) to give the title compound (410 mg, 1.06 mmol).

[0349] Step 4: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl(1R,4R)-4-(triphenylmethoxy)cyclohexane-1-carboxylate (D-26-5) (1R,4R)-4-(triphenylmethoxy)cyclohexane-1-carboxylic acid (50 mg, 0.13 mmol) and HATU (59.0 mg, 0.16 mmol) were added to DMF (2.0 mL), and DIPEA (50.1 mg, 0.39 mmol) was added. The reaction mixture was then reacted for 2 hours at 25° C. (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-thiocarboxylic acid (63.2 mg, 0.16 mmol) was added, and the reaction mixture was stirred for an additional 2 hours. Fluoroiodomethane (31.0 mg, 0.19 mmol) was then added, and the reaction mixture was allowed to react for an additional hour at 25° C. Water (10 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (10 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound (60 mg, 0.073 mmol) as a crude product, which was used directly in the next step without purification.

[0350] Step 5: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl(1R,4R)-4-hydroxycyclohexane-1-carboxylate (D-26) (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl (1R,4R)-4-(triphenylmethoxy)cyclohexane-1-carboxylate (60 mg, 0.073 mmol) was dissolved in dichloromethane (1 mL), acetic acid (0.5 mL) was added, and the reaction mixture was stirred at 40 ° C. for 3 hours. The reaction mixture was directly concentrated to give a crude product, which was purified by high performance liquid chromatography to give the title compound (3.5 mg, 0.006 mmol). Chromatography column: SunFire prep C18 OBD 150mm x 19mm x 5μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 28] The structure is characterized as follows: ESI-MS (m / z): 594.2[M+Na] + . 1H NMR (400 MHz, DMSO) δ 7.26 (d, J = 10.1 Hz, 1H), 6.30 (dd, J = 10.2, 1.8 Hz, 1H), 6.11 (s, 1H), 5.98 (s, 1H), 5.86 (s, 1H), 5.73 - 5.54 (m, 1H), 5.58 (d, J = 4.0 Hz, 1H), 4.59 (d, J = 4.3 Hz, 1H), 4.22 (s, 1H), 3.32 - 3.25 (m, 1H), 2.61 - 2.32 (m, 2H), 2.30 - 2.20 (m, 2H), 2.14 - 2.03 (m, 2H), 1.89 - 1.77 (m, 5H), 1.59 - 1.44 (m, 1H) 1.48 (s, 3H), 1.39 - 1.21 (m, 4H), 1.20 - 1.09 (m, 2H), 0.98 (s, 3H), 0.86 (d, J = 7.2 Hz, 3H).

[0351] Preparation Example 30: (8S,9R,10S,11S,13S,14S,17R)-9-fluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-amino-4-fluorobenzoate (A-2) [ka]

[0352] Step 1: Synthesis of (8S,9R,10S,11S,13S,14S,17R)-9-fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (A-2-2) 2-((8S,9R,10S,11S,13S,14S,17R)-9-fluoro-11,17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl acetate (150 mg, 356 μmol) was dissolved in a mixed solvent of water (1.00 mL), dichloromethane (1.00 mL), and methanol (1.00 mL). Sodium hydroxide (21.4 mg, 535 μmol) was added and the mixture was stirred at 25°C for 1 hour. The pH of the reaction solution was adjusted to 2-3 using 1 N diluted hydrochloric acid. The reaction mixture was added with water (30.0 mL) and extracted with dichloromethane three times (20.0 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound (110 mg, 290 μmol). The structure is characterized as follows: ESI-MS (m / z): 378.9 [M+H] + .

[0353] Step 2: Synthesis of (8S,9R,10S,11S,13S,14S,17R)-9-fluoro-11,17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylic acid (A-2-3) (8S,9R,10S,11S,13S,14S,17R)-9-fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (200 mg, 528 μmol) was dissolved in a mixture of tetrahydrofuran (1.30 mL) and water (1.00 mL), and hydrogen peroxide (361 mg, 1.59 mmol, 361 μL) was added and stirred at 25°C for 1 hour. The reaction mixture was adjusted to pH 2-3 with 1 N dilute hydrochloric acid, filtered, and the filter cake was dried to give the title compound (100 mg, 274 μmol). The structure is characterized as follows: ESI-MS (m / z): 365.0 [M+H] + .

[0354] Step 3: Synthesis of (8S,9R,10S,11S,13S,14S,17R)-9-fluoro-11,17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-thiocarboxylic acid (A-2-4) (8S,9R,10S,11S,13S,14S,17R)-9-Fluoro-11,17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylic acid (100 mg, 274 μmol) was dissolved in DMF (2.00 mL), CDI (88.9 mg, 548 μmol) was added, and the reaction mixture was stirred at 25 °C for 2 h. HS gas (9.35 mg, 274 μmol, 4.52 μL) was introduced, and the reaction mixture was stirred for an additional 2.5 h. The reaction mixture was adjusted to pH 2-3 with 1 N dilute hydrochloric acid, filtered, and the filter cake was dried to give the title compound (70.0 mg, 183 μmol). The structure is characterized as follows: ESI-MS (m / z): 380.9 [M+Na] + .

[0355] Step 4: Synthesis of (8S,9R,10S,11S,13S,14S,17R)-9-fluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-((tert-butoxycarbonyl)amino)-4-fluorobenzoate (A-2-5) 3-(tert-Butoxycarbonyl)amino)-4-fluorobenzoic acid (40.2 mg, 157 μmol) was dissolved in DMF (4.00 mL), and HATU (59.9 mg, 157 μmol) and DIPEA (20.3 mg, 157 μmol, 27.4 μL) were added. The mixture was allowed to react for 2 hours at 25° C. (8S,9R,10S,11S,13S,14S,17R)-9-fluoro-11,17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-thiocarboxylic acid (60.0 mg, 157 μmol) was added, and the reaction mixture was stirred for an additional hour. Fluoroiodomethane (25.2 mg, 157 μmol) was then added, and the reaction mixture was allowed to react at 25°C for 1 hour. Water (10 mL) was added to the reaction mixture, and the mixture was extracted three times with dichloromethane (10.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product. The crude product was purified by p-TLC (SiO2, CHCl2 / MeOH = 100 / 1 to 10 / 1), and concentrated again to give the title compound (12.0 mg, 18.4 μmol). The structure is characterized as follows: ESI-MS (m / z): 650.2 [M+H] + .

[0356] Step 5: Synthesis of (8S,9R,10S,11S,13S,14S,17R)-9-fluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-amino-4-fluorobenzoate (A-2) (8S,9R,10S,11S,13S,14S,17R)-9-fluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-((tert-butoxycarbonyl)amino)-4-fluorobenzoate (10.0 mg, 15.3 μmol) was dissolved in dichloromethane (1.00 mL), trifluoroacetic acid (460 mg, 4.04 mmol, 0.30 mL) was added, and the reaction mixture was stirred at 25° C. for 1 hour. The reaction mixture was directly concentrated to give the crude product, which was purified by high-performance liquid chromatography to give the title compound (2.36 mg, 4.07 μmol). Chromatography column: Phenomenex luna C18 150mm x 25mm x 10μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate) [Table 29] The structure is characterized as follows: ESI-MS (m / z): 550.2 [M+H] + . 1H NMR (400 MHz, CD3OD) δ 7.36-7.49 (m, 2H), 7.22-7.32 (m, 1H), 7.06 (dd, J = 10.8, 8.4 Hz, 1H), 6.33 (dd, J = 10.0, 1.8 Hz, 1H), 6.11 (s, 1H), 5.68-6.00 (m, 2H), 4.40 (d, J =8.4 Hz, 1H), 3.03 (dd, J = 14.4, 11.2 Hz, 1H), 2.76 (td, J = 13.6, 5.6 Hz, 1H), 2.53-2.67 (m, 1H), 2.38-2.51 (m, 2H), 2.18-2.27 (m, 1H), 2.02-2.14 (m, 2H), 1.94-2.01 (m, 1H), 1.73-1.83 (m, 1H), 1.62 (s, 3H), 1.50-1.60 (m, 2H), 1.07 (s, 3H).

[0357] Preparation Example 31: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-(hydroxymethyl)furan-2-carboxylate (F-9) [ka]

[0358] Step 1: Synthesis of (5-bromofuran-3-yl)methanol (F-9-2) 5-Bromofuran-3-formic acid (1.00 g, 5.24 mmol) was dissolved in tetrahydrofuran (20.0 mL), and the reaction mixture was cooled to 0 °C. Borane-tetrahydrofuran complex (2.5 M, 4.19 mL) was added under a nitrogen atmosphere, and the reaction mixture was heated to 50 °C and stirred for 3 h. After that, the reaction mixture was cooled to 25 °C. Methanol (10.0 mL) was added to the reaction mixture to quench the reaction, and the reaction mixture was concentrated. 2 M aqueous sodium hydroxide solution (30.0 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (30.0 mL × 2). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product (1.06 g, crude), which was used directly in the next step without further purification. The structure is characterized as follows: ESI-MS (m / z): 177.1 [M+H] + .

[0359] Step 2: Synthesis of (5-bromofuran-3-yl)methoxy)(tert-butyl)diphenylsilane (F-9-3) (5-Bromofuran-3-yl)methanol (1.00 g, 5.65 mmol) was dissolved in dichloromethane (10.0 mL), and imidazole (1.15 g, 16.9 mmol) and tert-butyldiphenylsilane chloride (2.33 g, 8.47 mmol) were added. The reaction mixture was stirred at 25°C for 3 hours. Water (20.0 mL) was added to the reaction mixture, followed by extraction with dichloromethane (20.0 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product. This was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 100 / 0 to 100 / 5) and concentrated again to give the title compound as a crude product (1.20 g, crude). The structure is characterized as follows: ESI-MS (m / z): 415.1 [M+H] + .

[0360] Step 3: Synthesis of methyl 4-((tert-butyldiphenylsilyl)oxy)methyl)furan-2-carboxylate (F-9-4) (5-Bromofuran-3-yl)methoxy)(tert-butyl)diphenylsilane (900 mg, 2.17 mmol) was dissolved in methanol (45.0 mL), triethylamine (438 mg, 4.33 mmol) was added, and Pd(dppf)Cl2 (158 mg, 216 μmol) was added under a nitrogen atmosphere. CO gas (40 psi) was introduced into the reaction system, and the reaction was carried out at 60 °C for 6 hours. The reaction solution was concentrated to give the crude product, which was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1 to 10:2) and concentrated again to give the title compound (746 mg, 1.89 mmol). The structure is characterized as follows: ESI-MS (m / z): 395.3 [M+H] + .

[0361] Step 4: Synthesis of methyl 4-(hydroxymethyl)furan-2-carboxylate (F-9-5) Methyl 4-((tert-butyldiphenylsilyl)oxy)methyl)furan-2-carboxylate (643 mg, 1.63 mmol) was dissolved in tetrahydrofuran (6.00 mL), and tetrabutylammonium fluoride solution (1 M, 1.63 mL) was added. The reaction mixture was stirred at 20° C. for 1 hour. The reaction mixture was concentrated to give a crude product, which was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10 / 1 to 1 / 1) and concentrated again to give the title compound as a crude product (190 mg, crude). The structure is characterized as follows: ESI-MS (m / z): 157.0 [M+H] + .

[0362] Step 5: Synthesis of methyl 4-((triphenylmethoxy)methyl)furan-2-carboxylate (F-9-6) Methyl 4-(hydroxymethyl)furan-2-carboxylate (191 mg, 1.22 mmol) was dissolved in pyridine (2.00 mL), triphenylmethyl chloride (341 mg, 1.22 mmol) was added, and the reaction mixture was heated to 80° C. and reacted for 12 hours. 0.5 M dilute hydrochloric acid (20.0 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (20.0 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product (517 mg, crude), which was used directly in the next step without purification.

[0363] Step 6: Synthesis of 4-((triphenylmethoxy)methyl)furan-2-carboxylic acid (F-9-7) Methyl 4-((triphenylmethoxy)methyl)furan-2-carboxylate (517 mg, 1.30 mmol) was dissolved in a mixture (4.00 mL) of methanol (4.00 mL) and water (4.00 mL). Lithium hydroxide monohydrate (163 mg, 3.89 mmol) was added, and the reaction mixture was heated to 40°C and stirred for 1 hour. Water (30.0 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (30.0 mL). The aqueous phase was adjusted to pH 2-3 with 0.5 M dilute hydrochloric acid and extracted with dichloromethane (10.0 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product (98.0 mg, crude), which was used directly in the next step without further purification.

[0364] Step 7: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-((triphenylmethoxy)methyl)furan-2-carboxylate (F-9-8) 4-((triphenylmethoxy)methyl)furan-2-carboxylic acid (65.0 mg, 169 μmol) and (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-thiocarboxylic acid (69.8 mg, 169 μmol) were dissolved in DMF (2.00 mL), and HATU (64.3 mg, 169 μmol) and DIPEA (65.6 mg, 507 μmol) were added. The reaction mixture was stirred at 25°C for 2 hours. Fluoroiodomethane (27.1 mg, 169 μmol) was then added and stirred for an additional 3 hours. The reaction mixture was concentrated, saturated aqueous sodium chloride solution (30.0 mL) was added, and the reaction mixture was extracted twice with ethyl acetate (30.0 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product (234 mg, crude), which was used directly in the next step without purification.

[0365] Step 8: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-(hydroxymethyl)furan-2-carboxylate (F-9) (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-yl 4-((triphenylmethoxy)methyl)furan-2-carboxylate (315 mg, 388 μmol) was dissolved in a mixed solvent of dichloromethane (3.00 mL) and methanol (3.00 mL), and trifluoroacetic acid (4.61 g, 40.4 mmol) was added. The reaction mixture was stirred at 20 ° C. for 1 hour. The reaction mixture was directly concentrated to give a crude product, which was purified by high performance liquid chromatography to give the title compound (18.0 mg, 0.031 mmol). Chromatography column: Phenomenex luna C18 150mm x 25mm x 10μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 30] The structure is characterized as follows: ESI-MS (m / z): 569.3 [M+H] + . 1H NMR (400 MHz, CD3OD) δ 7.72 (d, J = 0.8 Hz, 1H), 7.34 (dd, J = 1.4, 10.2 Hz, 1H), 7.20 (s, 1H), 6.41 - 6.28 (m, 2H), 6.03 - 5.78 (m, 2H), 5.68 - 5.47 (m, 1H), 4.47 (s, 2H), 4.38 - 4.29 (m, 1H), 3.49 (dd, J = 3.6, 10.4 Hz, 1H), 2.74 - 2.53 (m, 1H), 2.37 (dd, J = 3.4, 10.8 Hz, 3H), 2.05 - 1.96 (m, 2H), 1.74 - 1.63 (m, 1H), 1.59 (s, 3H), 1.37 (dd, J = 7.8, 11.8 Hz, 1H), 1.17 (s, 3H), 1.02 (d, J = 7.4 Hz, 3H).

[0366] Preparation 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-(hydroxymethyl)furan-2-carboxylate (F-10) [ka]

[0367] Step 1: Synthesis 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-((tributoxy)methyl)furan-2-carboxylate (F-10-1) 4-((triphenylmethoxy)methyl)furan-2-carboxylic acid (850 mg, 2.21 mmol) was dissolved in DMF (30.0 mL) under a nitrogen atmosphere. DIPEA (857 mg, 6.63 mmol) and HATU (841 mg, 2.21 mmol) were added, and the reaction mixture was stirred at 25° C. for 1 hour. (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-11,17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-thiocarboxylic acid (881 mg, 2.21 mmol) was added, and the mixture was stirred for an additional 2 hours. Fluoroiodomethane (354 mg, 2.21 mmol) was then added and stirred for an additional 1 hour. The reaction mixture was poured into water (100 mL), stirred for 30 minutes, and filtered. The filter cake was washed with water three times (10.0 mL × 3) and dried under vacuum to give the title compound (1.40 g, 1.76 mmol), which was used directly in the next step without purification. The structure is characterized as follows: ESI-MS (m / z): 796.3 [M+H] + .

[0368] Step 2: Synthesis 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-(hydroxymethyl)furan-2-carboxylate (F-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-dodecahydro-3H-cyclopenta[a]phenanthrene-17-yl 4-((tributoxy)methyl)furan-2-carboxylate (900 mg, 1.13 mmol) was dissolved in dichloromethane (6.00 mL) and methanol (2.00 mL), and trifluoroacetic acid (1.54 g, 13.5 mmol) was added. The reaction mixture was stirred at 25°C for 2 hours. The reaction mixture was directly concentrated to give a crude product, which was purified by silica gel column chromatography (dichloromethane / methanol=100 / 1 to 10 / 1) and concentrated again to give the title compound (461 mg, 803.34 μmol). The structure is characterized as follows: ESI-MS (m / z): 555.0 [M+H] + . 1 H NMR (400 MHz, CD3OD) δ 7.68 (s, 1H), 7.31 (d, J = 10.2 Hz, 1H), 7.20 (s, 1H), 6.25-6.34 (m, 2H), 5.66-5.95 (m, 2H), 5.44-5.61 (m, 1H), 4.44 (s, 2H), 4.32 (br d, J = 8.4 Hz, 1H), 3.27 (s, 1H), 2.94-3.03 (m, 1H), 2.56-2.69 (m, 1H), 2.32-2.43 (m, 2H), 2.19-2.27 (m, 1H), 2.06-2.13 (m, 1H), 1.97 (d, J = 14.2 Hz, 1H), 1.72-1.80 (m, 1H), 1.64 (d, J = 12.8 Hz, 1H), 1.55 (s, 3H), 1.51 (s, 1H), 1.01 (s, 3H).

[0369] Preparation Example 33: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 5-(hydroxymethyl)furan-2-carboxylate (F-13) [ka]

[0370] Step 1: Synthesis of methyl 5-((triphenylmethoxy)methyl)furan-2-carboxylate (F-13-2) Methyl 5-(hydroxymethyl)furan-2-carboxylate (200 mg, 1.28 mmol) was dissolved in pyridine (2.00 mL), triphenylmethyl chloride (357 mg, 1.28 mmol) was added, and the reaction mixture was heated to 90° C. and stirred for 12 hours. 0.5 M dilute hydrochloric acid (20.0 mL) was added to the reaction mixture, which was then extracted with dichloromethane (20.0 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product (621 mg, crude), which was used directly in the next step without purification.

[0371] Step 2: Synthesis of 5-((triphenylmethoxy)methyl)furan-2-carboxylic acid (F-13-3) Methyl 5-((triphenylmethoxy)methyl)furan-2-carboxylate (612 mg, 1.54 mmol) was dissolved in a mixture of methanol (5.00 mL), tetrahydrofuran (5.00 mL), and water (5.00 mL). Lithium hydroxide monohydrate (193 mg, 4.61 mmol) was added, and the reaction mixture was stirred at 25°C for 3 hours. Water (30.0 mL) was added to the reaction mixture, followed by extraction with dichloromethane (30.0 mL). The aqueous phase was adjusted to pH 2-3 with 0.5 M dilute hydrochloric acid and extracted three times with dichloromethane (10.0 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product (246 mg, crude), which was used directly in the next step without further purification.

[0372] Step 3: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 5-((triphenylmethoxy)methyl)furan-2-carboxylate (F-13-4) 5-((triphenylmethoxy)methyl)furan-2-carboxylic acid (183 mg, 476 μmol) and (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-thiocarboxylic acid (196 mg, 476 μmol) were dissolved in DMF (10.0 mL), and DIPEA (184 mg, 1.43 mmol) and HATU (181 mg, 476 μmol) were added. Fluoroiodomethane (76.1 mg, 476 μmol) was then added, and the reaction mixture was stirred for an additional 3 h. The reaction mixture was concentrated, saturated aqueous sodium chloride solution (30.0 mL) was added, and the reaction mixture was extracted twice with ethyl acetate (30.0 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product (488 mg, crude), which was used directly in the next step without purification.

[0373] Step 4: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 5-(hydroxymethyl)furan-2-carboxylate (F-13) (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 5-((triphenylmethoxy)methyl)furan-2-carboxylate (488 mg, 601 μmol) was dissolved in a mixed solvent of dichloromethane (4.00 mL) and methanol (4.00 mL), and trifluoroacetic acid (6.14 g, 53.9 mmol) was added. The reaction mixture was stirred at 25° C. for 1 hour. The reaction mixture was directly concentrated to give a crude product, which was purified by high performance liquid chromatography to give the title compound (53.8 mg, 93.9 μmol). Chromatography column: Welch Xtimate C18 150mm x 25mm x 5μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 31] The structure is characterized as follows: ESI-MS (m / z): 569.3 [M+H] + . 1H NMR (400 MHz, CD3OD) δ 7.34 (d, J = 10.0 Hz, 1H), 7.13 (d, J = 3.4 Hz, 1H), 6.50 (d, J = 3.4 Hz, 1H), 6.40 - 6.26 (m, 2H), 6.02 - 5.77 (m, 2H), 5.68 - 5.45 (m, 1H), 4.55 (s, 2H), 4.34 (d, J = 9.2 Hz, 1H), 3.55 - 3.42 (m, 1H), 2.74 - 2.51 (m, 1H), 2.44 - 2.26 (m, 3H), 2.01 (d, J = 13.0 Hz, 2H), 1.74 - 1.61 (m, 1H), 1.58 (s, 3H), 1.41 - 1.31 (m, 1H), 1.17 (s, 3H), 1.02 (d, J = 7.2 Hz, 3H).

[0374] Preparation Example 34: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-17-((2-hydroxyethyl)thio)carbonyl-10,13,16-trimethyl-3-oxo-6,7,8,9,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-ylfuran-2-carboxylate (F-21) [ka]

[0375] Step 1: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-17-((2-(methoxymethoxy)ethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-6,7,8,9,11,12,13,14,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-ylfuran-2-carboxylate (F-21-2) Furan-2-carboxylic acid (108 mg, 969 μmol) was dissolved in DMF (15.0 mL), and HATU (368 mg, 969 μmol) and DIPEA (375 mg, 2.91 mmol, 506 μL) were added. The reaction mixture was stirred at 25° C. for 1 hour. (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-thiocarboxylic acid (400 mg, 969 μmol) was added, followed by stirring for an additional hour. After that, 1-iodo-2-(methoxymethoxy)ethane (209 mg, 969 μmol) was added, and the reaction mixture was stirred for another 1 h. Water was added to the reaction mixture to precipitate a solid, which was then filtered. The filter cake was dried under vacuum to give the title compound (458 mg, crude) as a crude product, which was used directly in the next step without purification. The structure is characterized as follows: ESI-MS (m / z): 595.2 [M+H] + .

[0376] Step 2: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-17-((2-hydroxyethyl)thio)carbonyl-10,13,16-trimethyl-3-oxo-6,7,8,9,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-ylfuran-2-carboxylate (F-21) (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-17-((2-(methoxymethoxy)ethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-6,7,8,9,11,12,13,14,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-ylfuran-2-carboxylate (450 mg, 756 μmol) was dissolved in dichloromethane (13.5 mL), trifluoroacetic acid (4.14 g, 36.3 mmol, 2.70 mL) was added, and the reaction mixture was stirred at 25°C for 1 hour. The reaction mixture was directly concentrated to give the crude product, which was purified by high-performance liquid chromatography to give the title compound (86.9 mg, 154 μmol). Chromatography column: Welch Xtimate C18 200mm x 40mm x 10μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid) TIFF2026501523000410.tif21170The structure is characterized as follows: ESI-MS (m / z): 551.1 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 8.00 (d, J = 1.2 Hz, 1H), 7.27 (d, J = 10.8 Hz, 1H), 7.18 (d, J = 3.2 Hz, 1H), 6.68-6.71 (m, 1H), 6.29-6.34 (m, 1H), 6.12 (s, 1H), 5.54-5.63 (m, 2H), 4.22-4.27 (m, 1H), 3.47-3.51 (m, 3H), 3.00 (t, J = 6.8 Hz, 2H), 2.53-2.68 (m, 2H), 2.17-2.28 (m, 3H), 1.84-1.93 (m, 2H), 1.53-1.61 (m, 1H), 1.50 (s, 3H), 1.26-1.32 (m, 1H), 1.04 (s, 3H), 0.94 (m, 3H).

[0377] Preparation 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl(E)-3-(3-amino-4-fluorophenyl)acrylate (E-4) [ka]

[0378] Step 1: Synthesis 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl (E)-3-(3-nitro-4-fluorophenyl)acrylate (E-4-1) (E)-3-(4-Fluoro-3-nitro-phenyl)prop-2-enoic acid (263 mg, 1.25 mmol), pyridine (344.84 mg, 4.36 mmol), and a 50% solution of 1-propylphosphonic anhydride in DMF (2.30 g, 7.22 mmol) were added sequentially to dichloromethane (10 mL), and after the addition was complete, the reaction mixture was reacted at 25°C for 1 hour. (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-11,17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-thiocarboxylic acid (71 mg, 181.76 mmol) was added, and the reaction mixture was allowed to react at room temperature for 3 hours. Fluoroiodomethane (400 mg, 2.50 mmol) was added, and the reaction mixture was allowed to react at room temperature for 3 hours. After completion of the reaction, the reaction mixture was extracted with water (30 mL) and dichloromethane (20 mL × 2), dried, and then concentrated. The concentrate was purified on a silica gel column (petroleum ether / ethyl acetate = 100 / 1 to 50 / 1) and concentrated again to obtain the title compound (94 mg) as a crude product. The structure is characterized as follows: MS m / z (ESI): 624.1[M+H] + .

[0379] Step 2: Synthesis 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl(E)-3-(3-amino-4-fluorophenyl)acrylate (E-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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl(E)-3-(3-nitro-4-fluorophenyl)acrylate (94 mg, crude), iron powder (24.18 mg, 432.96 μmol), and ammonium chloride (11.58 mg, 216.48 μmol) were added to ethanol (1 mL) and water (0.3 mL). After the addition was complete, the reaction mixture was heated to 80°C and allowed to react. After the reaction was completed, water (8 mL) was added, and the reaction mixture was extracted with ethyl acetate (6 mL × 2) and filtered through diatomaceous earth. The organic layer was dried, concentrated by distillation, purified by preparative high-performance liquid chromatography, and lyophilized to give the title compound (2.46 mg, 0.004 mmol). The structure is characterized as follows: MS m / z (ESI): 594.2[M+H] + . The purification method is as follows: Chromatography column: Waters SunFire Prep C18 OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 32]

[0380] Preparation Example 36: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-(hydroxymethyl)benzoate (B-31) [ka]

[0381] Step 1: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-((tert-butyldimethylsilyl)oxy)methyl)benzoate (B-31-2) 4-((tert-Butyldimethylsilyl)oxy)methyl)benzoic acid (70 mg, 262.76 μmol) was added to DMF (8.00 mL), followed by the addition of HATU (99.91 mg, 262.76 μmol) and DIPEA (50.94 mg, 394.15 μmol). The reaction mixture was then stirred at 25° C. for 15 minutes. (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-S-carboxylic acid (127.51 mg, 262.76 μmol) was added to the reaction mixture, followed by DIPEA (50.94 mg, 394.15 μmol). The reaction mixture was stirred at 45°C for 1.5 hours. Fluoroiodomethane (210.11 mg, 1.31 mmol) was then added dropwise to the reaction mixture, followed by DIPEA (102 mg, 788.28 μmol). The reaction mixture was stirred at 45° C. for 45 minutes, extracted with water and EA, and then concentrated to give a crude product, which was purified by high performance liquid chromatography to give the title compound (14 mg, 20.20 μmol). Chromatography column: Phenomenex C18 250mm x 50mm x 10μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 33] The structure is characterized as follows: ESI-MS (m / z): 693.4 (M+H) + .

[0382] Step 2: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-(hydroxymethyl)benzoate (B-31) (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-((tert-butyldimethylsilyl)oxy)methyl)benzoate (16 mg, 23.09 μmol) was dissolved in DCM (3.00 mL) and TFA (1.5 mL), and the reaction mixture was stirred at 25° C. for 1 hour, then concentrated to give the crude product, which was purified by high-performance liquid chromatography to give the title compound (7 mg, 11.98 μmol). Chromatography column: Phenomenex C18 250mm x 50mm x 10μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 34] The structure is characterized as follows: ESI-MS (m / z): 579.3 (M+H) + . 1H NMR (400 MHz, DMSO) δ 7.83 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 10.0 Hz, 1H), 6.33 (dd, J = 1.6 Hz, 10.0 Hz, 1H), 6.14 (s, 1H), 6.03 (s, 1H), 5.90 (s, 1H), 5.80-5.56 (m, 2H), 5.38 (t, J =6.0 Hz, 1H), 4.56 (d, J = 5.6 Hz, 2H), 4.33-4.24 (m, 1H), 3.48-3.37 (m, 1H), 2.67-2.53 (m, 1H), 2.32-2.21 (m, 3H), 2.02-1.88 (m, 2H), 1.65-1.54 (m, 1H), 1.51 (s, 3H), 1.38-1.29 (m, 1H), 1.07 (s, 3H), 0.92 (d, J = 6.8 Hz, 3H).

[0383] Preparation Example 37: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-(hydroxymethyl)benzoate (B-35) [ka]

[0384] Step 1: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17- ((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-((tert-butyldimethylsilyl)oxy)methyl)benzoate (B-35-2) 3-((tert-Butyldimethylsilyl)oxy)methyl)benzoic acid (280 mg, 1.05 mmol) was added to DMF (15.00 mL), followed by the addition of HATU (399.64 mg, 1.05 mmol) and DIPEA (203.75 mg, 1.58 mmol). The reaction mixture was then stirred at 25° C. for 15 minutes. (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-S-carboxylic acid (510.04 mg, 1.05 mmol) was added to the reaction mixture, followed by DIPEA (203.75 mg, 1.58 mmol). The reaction mixture was stirred at 45°C for 1.5 hours. Fluoroiodomethane (672.36 mg, 4.20 mmol) was then added dropwise to the reaction mixture, followed by DIPEA (407.50 mg, 3.16 mmol). The reaction mixture was stirred at 45°C for 2 hours and then monitored by LC-MS until completion. The reaction mixture was extracted with water and EA and concentrated to give the crude product, which was purified by high-performance liquid chromatography to give the title compound (35 mg, 50.51 µmol). Chromatography column: Phenomenex C18 250mm x 50mm x 10μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 35] The structure is characterized as follows: ESI-MS (m / z): 693.4 (M+H) + .

[0385] Step 2: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-(hydroxymethyl)benzoate (B-35) (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-((tert-butyldimethylsilyl)oxy)methyl)benzoate (35 mg, 50.51 μmol) was dissolved in DCM (4.00 mL) and TFA (2.00 mL), and the reaction was stirred at 25° C. for 1.5 hours. The reaction was monitored by LC-MS until completion. The reaction mixture was concentrated to give a crude product, which was purified by high performance liquid chromatography to give the title compound (11 mg, 18.82 μmol). Chromatography column: Phenomenex C18 250mm x 50mm x 10μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 36] The structure is characterized as follows: ESI-MS (m / z): 579.3 (M+H) + . 1H NMR (400 MHz, DMSO) δ 7.87 (s, 1H), 7.74 (d, J = 7.6 Hz, 1H), 7.61 (d, J = 8.0 Hz, 1H), 7.52 (d, J = 7.6 Hz, 1H), 7.29 (dd, J = 1.2 Hz, 10.4 Hz, 1H), 6.33 (dd, J = 2.0 Hz, 10.0 Hz, 1H), 6.14 (s, 1H), 6.03 (s, 1H), 5.90 (s, 1H), 5.80-5.55 (m, 2H), 5.34 (t, J = 6.0 Hz, 1H), 4.52 (d, J = 5.6 Hz,2H), 4.33-4.25 (m, 1H), 3.48-3.36 (m, 1H), 2.69-2.54 (m, 1H), 2.33-2.21 (m, 3H), 2.02-1.88 (m, 2H), 1.67-1.52 (m, 1H), 1.51 (s, 3H), 1.38-1.30 (m, 1H), 1.08 (s, 3H), 0.93 (d, J = 6.8 Hz, 3H).

[0386] Preparation Example 38: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-hydroxyfuran-2-carboxylate (F-17) [ka]

[0387] Step 1: Synthesis of (5-(methoxycarbonyl)furan-3-yl)boronic acid (F-17-2) Methyl 4-bromofuran-2-carboxylate (2 g, 9.76 mmol), bis(pinacolato)diboron (4.95 g, 19.51 mmol), and KOAc (2.87 g, 29.27 mmol, 167.68 μL) were added to 1,4-dioxane (200 mL). The reaction mixture was purged with nitrogen, and then Pd(dppf)Cl2 (722.38 mg, 975.58 μmol) was added. The reaction mixture was purged with nitrogen three times, and then the mixture was heated in an 80 °C oil bath for 24 h. After completion of the reaction, the reaction mixture was cooled to room temperature and filtered through diatomaceous earth. The filter cake was washed with ethyl acetate, and the filtrate was directly dried to obtain the crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0% to 40%) and concentrated again to obtain the title compound (4.6 g, 9.12 mmol) (overweight, boric acid residue). ESI-MS (m / z): 253.2 (M+H) + .

[0388] Step 2: Synthesis of methyl 4-hydroxyfuran-2-carboxylate (F-17-3) (5-(Methoxycarbonyl)furan-3-yl)boronic acid (4.7 g, 27.66 mmol) was dissolved in THF (40 mL) and HO (40.00 g, 352.79 mmol, 40 mL, 30% purity) was slowly added dropwise. A large amount of heat was released from the system, and the reaction mixture was then allowed to react for 5 h while maintaining the temperature. After completion of the reaction, 20 mL of water was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate (10 mL × 2). The organic phase was washed alternately with aqueous sodium sulfite and aqueous sodium chloride solutions until the separated aqueous layer no longer showed a blue color when tested with potassium iodide-starch test paper. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the title compound as a crude product (1.54 g, 10.84 mmol), which was used directly in the next step without further purification. ESI-MS (m / z): 143.0 (M+H) + .

[0389] Step 3: Synthesis of methyl 4-(methoxymethoxy)furan-2-carboxylate (F-17-4) Methyl 4-hydroxyfuran-2-carboxylate (1.64 g, 11.54 mmol) was dissolved in DMF (50 mL), and the reaction mixture was cooled to 0 °C under nitrogen protection. NaH (553.89 mg, 13.85 mmol, 60% purity) was then added in several portions until the mixture changed color from orange-yellow to brick-colored. The mixture was stirred and reacted at 0 °C for 30 minutes while maintaining the temperature. A solution of bromomethyl methyl ether (2.88 g, 23.08 mmol) in DMF (1 mL) was slowly added dropwise at a controlled temperature of 0 °C, and the mixture gradually changed color to yellow. After the addition, the mixture was reacted for 30 minutes while maintaining the temperature. After the reaction was completed, water (100 mL) was added to the reaction mixture to quench the reaction. The reaction mixture was extracted three times with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated brine, and concentrated under reduced pressure to give a crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether=0% to 20%) and concentrated again to give the title compound (400 mg, 2.15 mmol). ESI-MS (m / z): 187.1 (M+H) + .

[0390] Step 4: Synthesis of 4-(methoxymethoxy)furan-2-carboxylic acid (F-17-5) Methyl 4-(methoxymethoxy)furan-2-carboxylate (400 mg, 2.15 mmol) was dissolved in methanol (10 mL), KOH (301.38 mg, 5.37 mmol) was added, and the reaction mixture was heated to 35°C and stirred for 6 hours. After completion of the reaction, the reaction mixture was directly evaporated to dryness. Water (5 mL) was then added, and the pH was adjusted to 4-5 with 2 N dilute hydrochloric acid. Extraction with ethyl acetate had undesirable effects. The organic phase was concentrated under reduced pressure, combined with the aqueous phase, and purified by high-performance liquid chromatography to obtain the title compound (180 mg, 1.05 mmol). The purification conditions were as follows: Chromatography column: Waters Sunfire Prep C18 OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 37] The structure is characterized as follows: ESI-MS (m / z): 173.1 [M+H] + .

[0391] Step 5: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-17-(4-(methoxymethoxy)furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylic acid (F-17-6) 4-(Methoxymethoxy)furan-2-carboxylic acid (85 mg, 493.80 μmol) was dissolved in DMF (10 mL), DIPEA (191.46 mg, 1.48 mmol) and HATU (187.76 mg, 493.80 μmol) were added, and the reaction mixture was heated to 45°C and reacted with stirring for 1 hour, and then cooled to room temperature. A solution of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylic acid (217.50 mg, 493.80 μmol) in DMF was added and stirred at room temperature for 16 hours. After completion of the reaction, water (60 mL) was added, and the reaction solution was adjusted to pH 4-5 with 0.5 M aqueous citric acid, resulting in the precipitation of a large amount of white cohesive solids. The solid was filtered, and the filter cake was collected and dried under vacuum to obtain the title compound (220 mg, 399.60 μmol) as a crude product, which was used directly in the next step without further purification. ESI-MS (m / z): c551.2 [M+H] + .

[0392] Step 6: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-17-(4-(methoxymethoxy)furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxydimethylaminothioformic anhydride (F-17-7) (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-17-(4-(methoxymethoxy)furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylic acid (100 mg, 181.64 μmol) was dissolved in acetone (2 mL), followed by the addition of DIPEA (35.21 mg, 272.46 μmol) and KI (18.09 mg, 108.98 μmol). Dimethylaminothioform chloride (56.13 mg, 454.10 μmol) was added in several portions under a nitrogen atmosphere. After the addition was complete, the mixture was stirred at room temperature for 4 hours. After the reaction was complete, water (10 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (5 mL x 3). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the title compound (110 mg, 172.50 μmol) as a crude product, which was used directly in the next step without further purification. ESI-MS (m / z): 639.3 (M+H) + .

[0393] Step 7: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-(methoxymethoxy)furan-2-carboxylate (F-17-8) (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-17-(4-(methoxymethoxy)furan-2-carbonyl)oxy)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxydimethylaminothioformic anhydride (110 mg, 172.50 μmol) was dissolved in DMAc (4.0 mL), and NaSH (29.01 mg, 517.50 μmol) was added in one portion so that the mixture instantly turned dark green. After the addition was complete, the mixture was stirred at room temperature for 3 hours. The remaining starting material, NaSH (29.01 mg, 517.50 μmol), was replenished, and the mixture was stirred at room temperature for an additional 0.5 h. The reaction was monitored by LCMS. The reaction was monitored by LCMS until no starting material remained and a significant amount of the thiocarboxylic acid intermediate was present. Fluoroiodomethane (275.87 mg, 1.72 mmol) was added dropwise, and the mixture instantly turned earth-yellow. The mixture was stirred at room temperature for 0.5 h. After completion of the reaction, water (10 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (10 mL × 2). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product, which was purified by thin-layer chromatography (ethyl acetate / petroleum ether = 1:1) to give the title compound (86 mg, 143.66 μmol). ESI-MS (m / z): 599.2 (M+H) + .

[0394] Step 8: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-hydroxyfuran-2-carboxylate (F-17) A solution of hydrogen chloride in 1,4-dioxane (4 mL) was added to (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-(methoxymethoxy)furan-2-carboxylate (35 mg, 58.47 μmol). After the addition was complete, the reaction mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give the crude product, which was purified by high-performance liquid chromatography to give the title compound (180 mg, 1.05 mmol). The preparation conditions are as follows: Chromatography column: Waters Sunfire Prep C18 OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 38] The structure is characterized as follows: ESI-MS (m / z): 555.3 [M+H] + . 1H NMR (400 MHz, DMSO) δ 9.13 (s, 1H), 7.50 (d, J = 1.2 Hz, 1H), 7.28 (dd, J = 10.2, 1.6 Hz, 1H), 6.86 (d, J = 1.1 Hz, 1H), 6.30 (dd, J = 10.4, 1.6 Hz, 1H), 6.16 (s, 1H), 6.00 (s, 1H), 5.88 (s, 1H), 5.72 - 5.53 (m, 1H), 5.46 - 5.42 (m, 1H), 4.29 (d, J = 7.2 Hz, 1H), 3.37 (ddd, J = 10.6, 7.2, 3.6 Hz, 1H), 2.61 (ddd, J =21.6, 16.4, 3.2 Hz, 1H), 2.35 - 2.19 (m, 3H), 1.95 (dd, J = 22.4, 11.6 Hz, 2H), 1.65 - 1.54 (m, 1H), 1.53 (s, 3H), 1.37 - 1.29 (m, 1H), 1.09 (s, 3H), 0.96 (d, J = 7.2 Hz, 3H).

[0395] Preparation Example 39: (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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-fluoro-3-((S)-2-((S)-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamino)propioamino)benzoate (DL-A-03) [ka]

[0396] (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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-((S)-2-((S)-2-aminopropionamido)propionamido)-4-fluorobenzoate (25 mg, 30.35 μmol), 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (11.09 mg, 30.35 μmol), and DIPEA (11.77 mg, The reaction mixture was added to DMF (2.00 mL) and reacted at 25° C. for 1 hour. The reaction mixture was filtered to obtain a crude product, which was purified by high-performance liquid chromatography to obtain the title compound (18 mg, 18.56 μmol). The purification method is as follows: Chromatography column: Phenomenex C18 250mm x 50mm x 10μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 39] The structure is characterized as follows: ESI-MS (m / z): 960.3 (M+H) + .

[0397] Preparation Example 40: (S)-4-((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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)oxy)carbonyl-2-fluorophenyl)carbamoyl)-30-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,25-trioxo-12,15,18,21-tetraoxa-5,8,24-triazatriacontan-29-ynoic acid (DL-A-47) [ka]

[0398] Step 1: Preparation of tert-butyl 22-(2-(methylsulfonyl)pyrimidin-5-yl)-17-oxo-4,7,10,13-tetraoxa-16-azadocosan-21-ynoate (DL-A-47-2) Tert-butyl 1-amino-3,6,9,12-tetraoxypentadecane-15-carboxylate (117 mg, 364.02 μmol), 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (146.30 mg, 400.43 μmol), and DIPEA (94.09 mg, 728.05 μmol) were added sequentially to DMF (2 mL), and the reaction mixture was reacted at 25° C. for 3 hours. Water (8 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (8 mL × 3). The organic phases were combined, washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound (259 mg), which was used directly in the next step without purification.

[0399] Step 2: Preparation of 22-(2-(methylsulfonyl)pyrimidin-5-yl)-17-oxo-4,7,10,13-tetraoxa-16-azadocosan-21-ynoic acid (DL-A-47-3) Tert-butyl 22-(2-(methylsulfonyl)pyrimidin-5-yl)-17-oxo-4,7,10,13-tetraoxa-16-azadocosan-21-ynoate (100 mg, 167.93 μmol, FR) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (0.5 mL) was added, and the mixture was stirred at 25° C. for 2 hours. The reaction mixture was directly concentrated under reduced pressure to remove dichloromethane, and then lyophilized to remove trifluoroacetic acid, affording the title compound as a crude product (86 mg), which was used directly in the next step without purification.

[0400] Step 3: Preparation of tert-butyl (S)-4-((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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)oxy)carbonyl-2-fluorophenyl)carbamoyl)-30-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,25-trioxo-12,15,18,21-tetraoxa-5,8,24-triazatriacontan-29-ynoate (DL-A-47-4) (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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-((S)-2-(2-aminoacetamido)-5-(tert-butoxy)-5-oxopentanamido)-4-fluorobenzoate (40 mg, 49.39 μmol), 22-(2-(methylsulfonyl)pyrimidin-5-yl)-17-oxo-4,7,10,13-tetraoxa-16-azadocosan-21-ynoic acid (40 mg, 49.39 μmol), 22-(2-(methylsulfonyl)pyrimidin-5-yl)-17-oxo-4,7,10,13-tetraoxa-16-azadocosan-21-ynoic acid (25.46 mg, 49.39 μmol), and DIPEA (19.15 mg, 148.17 μmol) were added sequentially to DMF (2 mL). After the addition was completed, the reaction mixture was reacted at 16°C for 1.5 hours. Purified water (8 mL) was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate (6 mL*2). The organic phase was washed once with saturated brine (4 mL). After the solvent was evaporated under reduced pressure, the title compound was obtained as a crude product (130 mg), which was used directly in the next step without further purification.

[0401] Step 4: Preparation of (S)-4-((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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)oxy)carbonyl-2-fluorophenyl)carbamoyl)-30-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,25-trioxo-12,15,18,21-tetraoxa-5,8,24-triazatriacontan-29-ynoic acid (DL-A-47) The crude (S)-4-((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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)oxy)carbonyl-2-fluorophenyl)carbamoyl)-30-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,25-trioxo-12,15,18,21-tetraoxa-5,8,24-triazatriacontan-29-ynoate (51 mg) was dissolved in dichloromethane (3 Trifluoroacetic acid (0.5 mL) was added, and after the addition was completed, the reaction was carried out for 3 hours at 16° C. The reaction solution was purified directly by high performance liquid chromatography to obtain the title compound (17.51 ​​mg, 13.85 μmol). The purification method is as follows: Chromatography column: SunFire Prep C18 OBD 19*150mm*5μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% TFA) [Table 40] The structure is characterized as follows: ESI-MS (m / z): 1251.4 (M+H) + .

[0402] Preparation Example 41: (S)-4-((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-dodecahydro-3H-cyclopene thia[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)carbamoyl)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,37-trioxo-12,15,18,21,24,27,30,33-octaoxa-5,8,36-triazatetratriacontan-41-ynoic acid (DL-A-48) [ka]

[0403] Step 1: Preparation of tert-butyl 34-(2-(methylsulfonyl)pyrimidin-5-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatetratriacontan-ynoate (DL-A-48-1) 2,5-Dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (100 mg, 0.274 mmol), tert-butyl 1-amino-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-carboxylate (136.2 mg, 0.274 mmol), and DIPEA (70.8 mg, 0.547 mmol) were added to THF (2 mL), and the reaction mixture was stirred at 25 °C for 2 hours. Ethyl acetate (20 mL) and water (6 mL) were added, and the reaction mixture was stirred for 5 minutes. The organic phase was separated, washed with saturated aqueous sodium chloride (5 mL), dried over anhydrous sodium sulfate, and concentrated to give the title compound (174 mg), which was used directly in the next step without purification. The structure is characterized as follows: ESI-MS (m / z): 748.4 (M+H) + .

[0404] Step 2: Preparation of 34-(2-(methylsulfonyl)pyrimidin-5-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatetratriacontan-ynoic acid (DL-A-48-2) Tert-butyl 34-(2-(methylsulfonyl)pyrimidin-5-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatetratriacontan-ynoate (530.54 mg, 4.65 mmol) and trifluoroacetic acid (530.54 mg, 4.65 mmol) were added to dichloromethane (3 mL), and the reaction mixture was heated to 35° C. and reacted for 2 hours. The reaction mixture was concentrated to give the title compound (185 mg), which was used directly in the next step without purification. The structure is characterized as follows: ESI-MS (m / z): 692.4 (M+H) + .

[0405] Step 3: tert-Butyl (S)-4-((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-dodecahydro-3H-cyclopenta[pi]phenyl]propanol Preparation of (penta[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)carbamoyl)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,37-trioxo-12,15,18,21,24,27,30,33-octaoxa-5,8,36-triazatetratriacontan-41-ynoate (DL-A-48-1) (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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-((S)-2-(2-aminoacetamido)-5-(tert-butoxy)-5-oxopentanamido)-4-fluorobenzoate (43 mg, 53.10 To a mixture of 2 mL of DMF (2 mL), 34-(2-(methylsulfonyl)pyrimidin-5-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatetratriacontan-ynoic acid (36.73 mg, 53.10 μmol), DIPEA (20 mg, 154.7 μmol), and HATU (24.23 mg, 63.71 μmol) was added. After the addition was complete, the reaction mixture was stirred at 16°C for 1.5 hours. Water (8 mL) was added to the mixture, and the mixture was extracted twice with ethyl acetate (6 mL × 2). The organic phase was washed once with saturated brine (6 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product (40 mg). The structure is characterized as follows: ESI-MS (m / z): 1483.6 (M+H) + .

[0406] Step 4: (S)-4-((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-dodecahydro-3H-cyclopenta[3-(hydroxymethyl)thio]carbonyl] Preparation of [a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)carbamoyl)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,37-trioxo-12,15,18,21,24,27,30,33-octaoxa-5,8,36-triazatetratriacontan-41-ynoic acid (DL-A-48) Tert-butyl (S)-4-((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-dodecahydro-3H-cyclohexyl Ropenta[a]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)carbamoyl)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,37-trioxo-12,15,18,21,24,27,30,33-octaoxa-5,8,36-triazatetratriacontan-41-ynoate (40 mg) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (1 mL) was added, and after the addition was complete, the reaction mixture was stirred at 15°C for 2 hours. The reaction mixture was purified directly by high-performance liquid chromatography to obtain the title compound (16.54 mg, 11.01 μmol). The purification method is as follows: Chromatography column: SunFire Prep C18 OBD 19*150mm*5μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% TFA) [Table 41] The structure is characterized as follows: ESI-MS (m / z): 1427.6 (M+H) + .

[0407] Preparation Example 42: (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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-((2S,5R)-2-(4-aminobutyl)-5-(hydroxymethyl)-28-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,10,16,19-trioxo-3,6,9,22-tetraazaoctacosan-27-ynamido)-4-fluorobenzoate (DL-A-49) [ka]

[0408] Step 1: Preparation of tert-butyl 19-(2-(methylsulfonyl)pyrimidin-5-yl)-14-oxo-4,7,10-trioxa-13-azacyclononadecan-18-ynoate (DL-A-49-2) 2,5-Dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (200 mg, 0.547 mmol), tert-butyl 3-(2-(2-aminoethoxy)ethoxy)propionate (151.8 mg, 0.547 mmol), and DIPEA (141.5 mg, 1.09 mmol) were added to DMF (3 mL), and the reaction mixture was reacted at 25 °C for 2 h. EA (30 mL) and water (10 mL) were then added, and the reaction mixture was stirred for 5 min. The organic phase was separated and concentrated to give the crude product, which was purified by column chromatography (MeOH / DCM = 5-20%) to give the title compound (260 mg, 443.49 μmol), which was used directly in the next step without further purification. The structure is characterized as follows: ESI-MS (m / z): 528.2 [M+H] + .

[0409] Step 2: Preparation of 9-(2-(methylsulfonyl)pyrimidin-5-yl)-14-oxo-4,7,10-trioxa-13-azacyclononadecan-18-ynoic acid (DL-A-49-2) Tert-butyl 19-(2-(methylsulfonyl)pyrimidin-5-yl)-14-oxo-4,7,10-trioxa-13-azacyclononadecan-18-ynoate (260 mg, 0.493 mmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (1.12 g, 9.86 mmol) was added, and the reaction mixture was heated to 35-40°C for 3 hours. The mixture was then concentrated. The residue was dispersed in ACN (10 mL) and deionized water (5 mL) and lyophilized to give the title compound (290 mg, 492.02 μmol), which was used directly in the next step without further purification. The structure is characterized as follows: ESI-MS (m / z): 472.2 [M+H] + .

[0410] 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-((2S,5S)-2-(4)-(tert-butoxycarbonyl)amino)butyl)-5-(hydroxymethyl)-28-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,23-tetraoxo-13,16,19-trioxa-3,6,9,22- Preparation of Tetraazaoctacosan-27-ynamido)-4-fluorobenzoate (DL-A-49-1) 9-(2-(methylsulfonyl)pyrimidin-5-yl)-14-oxo-4,7,10-trioxa-13-azacyclononadecan-18-ynoic acid (32.9 mg, 0.056 mmol) was dissolved in DMF (1 mL) and HATU (38.6 mg, 0.10 mmol), DIPEA (19.7 mg, 0.152 mmol), and HCl were added. (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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-((S)-2-((S)-2-(2-aminoacetamido)-3-hydroxypropioamino)-6-(tert-butoxycarbonyl)amino)hexanamido)-4-fluorobenzoate (50.0 mg, 0.051 mmol, formic acid) was added, and the reaction mixture was incubated at 25°C for 1 hour. EA (10 ml) / HO (3 ml) was added, and the reaction mixture was stirred for 5 minutes. The organic phase was separated and concentrated to give a crude product, which was purified by thin layer chromatography (MeOH / DCM=10%) to give the title compound (41 mg, 27.95 μmol). The structure is characterized as follows: ESI-MS (m / z): 1393.4 [M+H] + .

[0411] 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-((2S,5R)-2-(4-aminobutyl)-5-(hydroxymethyl)-28-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,10,16,19-trioxo-3,6,9,22-tetraazaoctacosan-27-ynamido)-4-fluorobenzoate (DL-A-49) (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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-(( 2S,5S)-2-(4)-(tert-butoxycarbonyl)amino)butyl)-5-(hydroxymethyl)-28-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,23-tetraoxo-13,16,19-trioxa-3,6,9,22-tetraazaoctacosan-27-ynamido)-4-fluorobenzoate (41.0 mg, 0.029 mmol) was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (167.7 mg, 1.47 mmol) was added. The reaction mixture was reacted at 25°C for several hours, concentrated to obtain a crude product, which was purified by pre-HPLC to obtain the trifluoroacetate salt of the title compound (22.9 mg, 15.78 µmol). The purification method is as follows: Chromatography column: SunFire Prep C18 OBD 19*150mm*5μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 42] The structure is characterized as follows: ESI-MS (m / z): 1293.5 (M+H) + .

[0412] Preparation 43: (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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-((2S,5S)-2-(4-aminobutyl)-5-(hydroxymethyl)-43-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,38-tetraoxo-13,16,19,22,25,28,31,34,34-octaoxa-3,6,9,37-tetraazatritetracontan-42-ynamido)-4-fluorobenzoate (DL-A-50) [ka]

[0413] 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-((2S,5S)-2-( Preparation of 4-(tert-butoxycarbonyl)amino)butyl)-5-(hydroxymethyl)-43-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,38-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,6,9,37-tetraazatritetracontan-42-ynamido)-4-fluorobenzoate (DL-A-50-1) 34-(2-(methylsulfonyl)pyrimidin-5-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatetratriacontan-33-ynoic acid (28.9 mg, 0.033 mmol) was dissolved in DMF (1 mL), followed by addition of HATU (23.1 mg, 0.061 mmol), DIPEA (11.8 mg, 0.091 mmol), and HCl (1 mL). To the reaction mixture was added (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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl-3-((S)-2-((S)-2-(2-aminoacetamido)-3-hydroxypropanamido)-6-((tert-butoxycarbonyl)amino)hexamido)-4-fluorobenzoate (30.0 mg, 0.030 mmol, formic acid), and the reaction mixture was incubated at 25°C for 1 hour. Ethyl acetate (10 ml) and water (3 ml) were added, and the mixture was stirred for 5 minutes. The organic phase was separated and concentrated to give a crude product, which was purified by thin layer chromatography (methanol / dichloromethane=10%) and concentrated again to give the title compound (17 mg, 10.01 μmol). The structure is characterized as follows: ESI-MS (m / z): 1613.7 (M+H) + .

[0414] 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-((2S, Preparation of 5S)-2-(4-aminobutyl)-5-(hydroxymethyl)-43-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,38-tetraoxo-13,16,19,22,25,28,31,34,34-octaoxa-3,6,9,37-tetraazatritetracontan-42-ynamido)-4-fluorobenzoate (DL-A-50) (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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-((2S,5S)-2- (4-(tert-butoxycarbonyl)amino)butyl)-5-(hydroxymethyl)-43-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,38-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,6,9,37-tetraazatritetracontan-42-ynamido)-4-fluorobenzoate (17.0 mg, 0.010 mmol) was dissolved in dichloromethane (0.5 mL), trifluoroacetic acid (60.0 mg, 0.527 mmol) was added, and the reaction mixture was reacted at 25 ° C. for 2 hours. The reaction mixture was concentrated to obtain a crude product, which was purified by pre-HPLC to obtain the trifluoroacetate salt of the title compound (8.4 mg, 5.01 μmol). The purification method is as follows: Chromatography column: Waters SunFire Prep C18 OBD (5 μm*19 mm*150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 43] The structure is characterized as follows: ESI-MS (m / z): 1513.6 (M+H) + .

[0415] Preparation Example 44: (4S)-5-(((4-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)oxy)-4-oxobut-2-yl)oxy)methyl)amino)-4-(2-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)acetamido)-5-oxovaleric acid (DL-A-139) [ka]

[0416] Step 1: Preparation of 5-allyl-1-(2,5-dioxopyrrolidin-1-yl)(((9H-fluoren-9-yl)methoxy)carbonyl)-L-glutamic acid (DL-A-139-2) N,N'-Dicyclohexylcarbodiimide (2.22 g, 10.75 mmol), (S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(allyloxy)-5-oxovaleric acid (4.0 g, 9.77 mmol), and N-hydroxysuccinimide (1.19 g, 10.34 mmol) were added to tetrahydrofuran (40 mL). After the addition was complete, the reaction mixture was stirred at 16°C for 2 hours. The reaction mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure to give the title compound as a crude product (4.9 g, 9.67 mmol). Its structural property data are as follows: ESI-MS (m / z): 507.2(M+H) + .

[0417] Step 2: Preparation of (S)-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(allyloxy)-5-oxopentanoyl)glycine (DL-A-139-3) 5-Allyl-1-(2,5-dioxopyrrolidin-1-yl)(((9H-fluoren-9-yl)methoxy)carbonyl)-L-glutamic acid (4.9 g, 9.67 mmol), glycine (1.45 g, 19.35 mmol), and sodium bicarbonate (1.63 g, 19.35 mmol) were added to acetone (50 mL) and water (50 mL). After the addition was complete, the reaction mixture was allowed to react at 15°C for several hours. Purified water (120 mL) was added, and the reaction mixture was adjusted to pH 3-4 with 1 N dilute hydrochloric acid and extracted three times with ethyl acetate (50 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product (5.6 g). Its structural property data are as follows: ESI-MS (m / z): 467.2(M+H) + .

[0418] Step 3: Preparation of (S)-(5-(allyloxy)-2-amino-5-oxopentanoyl)glycine (DL-A-139-4) The crude (S)-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(allyloxy)-5-oxopentanoyl)glycine product (5.6 g) was added to DMF (12 mL), followed by the addition of diethylamine (2.5 mL). After the addition was complete, the reaction mixture was stirred at 16°C for several hours. The reaction mixture was concentrated under reduced pressure to remove residual diethylamine, affording the title compound as a crude product (2.3 g). Its structural property data are as follows: ESI-MS (m / z): 245.1 (M+H) + .

[0419] Step 4: Preparation of (S)-(2-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-(allyloxy)-5-oxopentanoyl)glycine (S)-(5-(allyloxy)-2-amino-5-oxopentanoyl)glycine crude product (2.2 g), 2,5-dioxopyrrolidin-1-yl((9H-fluoren-9-yl)methoxy)carbonyl)glycine (4.26 g, 10.81 mmol), and DIPEA (2.33 g, 18.01 mmol) were added to DMF (24 mL). After the addition, the mixture was stirred at 17°C for 3 hours. Water (110 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (15 mL × 2) to remove impurities. The aqueous phase was adjusted to pH 3-4 with 1 N dilute hydrochloric acid and extracted three times with ethyl acetate (50 mL × 3). The organic phase was dried and concentrated to dryness under reduced pressure to give the title compound (3.8 g, 7.25 mmol). Its structural property data are as follows: ESI-MS (m / z): 524.2 (M+H) + .

[0420] Step 5: Preparation of allyl (S)-4-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-((acetoxymethyl)amino)-5-oxovalerate (DL-A-139-6) (S)-(2-(2-(((9H-Fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-(allyloxy)-5-oxopentanoyl)glycine (602 mg, 1.15 mmol), pyridine (272.89 mg, 3.45 mmol), and lead tetraacetate (1.02 g, 2.30 mmol) were added to tetrahydrofuran (9 mL) and toluene (3 mL). After the addition was complete, nitrogen was introduced into the system three times and replaced. The system was then heated to 84°C and reacted for 3 hours. The reaction mixture was filtered, purified by column chromatography (petroleum ether / ethyl acetate = 4 / 1 to 1 / 4), and concentrated again to give the title compound (177 mg, 296.34 μmol). Its structural property data are as follows: ESI-MS (m / z): 560.3(M+H) + .

[0421] Step 6: Preparation of allyl (S)-4-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-((chloromethyl)amino)-5-oxovalerate (DL-A-139-7) Allyl (S)-4-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-((acetoxymethyl)amino)-5-oxovalerate (80 mg, 148.82 μmol) and trimethylsilyl chloride (162 mg, 1.49 mmol) were added to DCM (2 mL), and after the addition was complete, the reaction mixture was reacted at 17° C. for 0.5 h. The reaction mixture was evaporated to dryness under reduced pressure to give the title compound as a crude product (49 mg), which was used directly in the next step without purification.

[0422] Step 7: Preparation of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl(8S)-8-(3-(allyloxy)-3-oxopropyl)-1-(9H-fluoren-9-yl)-13-methyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazapentadecane-15-propionate (DL-A-139-8) Crude allyl (S)-4-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-((chloromethyl)amino)-5-oxovalerate (49 mg) and (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-hydroxybutyrate (30 mg, 56.54 μmol) were added to DCM (4 mL). After the addition was complete, the reaction mixture was heated to reflux and then reacted for 5 hours. The reaction mixture was quenched with methanol and then purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 4 / 1 to 1 / 4) to give the title compound as a crude product (39 mg), which was used directly in the next step without purification. Its structural property data are as follows: ESI-MS (m / z): 1008.4 (M+H) + .

[0423] Step 8: Preparation of (4S)-4-(2-aminoacetamido)-5-((4(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)oxy)-4-oxobut-2-yl)oxy)methyl)amino)-5-oxovaleric acid (DL-A-139-9) (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl(8S)-8-(3-(allyloxy)-3-oxopropyl)-1-(9H-fluoren-9-yl)-13-methyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazapentadecane-15-propionate crude product (39 mg), diethylamine (16 mg, 218.7 To DMF (3 mL) were added tetra(triphenylphosphine)palladium (13 mg, 11.25 μmol) and tetra(triphenylphosphine)palladium (13 mg, 11.25 μm), and after the addition was complete, the reaction mixture was reacted at 17° C. for 1 hour. Residual diethylamine was removed from the reaction mixture under reduced pressure to give the title compound as a crude product (28 mg), which was used directly in the next step without purification. Its structural property data are as follows: ESI-MS (m / z): 746.2(M+H) + .

[0424] Step 9: Preparation of (4S)-5-(((4-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)oxy)-4-oxobut-2-yl)oxy)methyl)amino)-4-(2-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)acetamido)-5-oxovaleric acid (DL-A-139) (4S)-4-(2-aminoacetamido)-5-((4(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)oxy)-4-oxobut-2-yl)oxy)methyl)amino)-5-oxovaleric acid crude product (28 mg), 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate, 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (13.72 mg, 37.54 μmol) and DIPEA (4.85 mg, 37.54 μmol) were added to DMF (3 mL), and after the addition was complete, the reaction mixture was reacted at 17°C for 1 hour. The reaction mixture was purified by high-performance liquid chromatography to obtain the title compound (9.49 mg, 9.24 μmol). The purification method is as follows: Chromatography column: SunFire Prep C18 OBD 19*150mm*5μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% TFA) [Table 44] Its structural property data are as follows: ESI-MS (m / z): 996.3 (M+H) + .

[0425] Preparation Example 45: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-((34S, 37S)-37-(4-aminobutyl)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-34-(hydroxymethyl)-1,29,32,35-tetraoxo-5,8,11,14,17,20,23,26-octaoxa-2,30,33,36-tetraazaoctatriacontane-38-amino)furan-2-carboxylate (DL-B-22') [ka]

[0426] Step 1: Preparation of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(tert-butoxycarbonyl)amino)hexanamido)furan-2-carboxylate (DL-B-22'-1) N 2 -((9H-fluoren-9-yl)methoxy)carbonyl)-N 6-(tert-Butoxycarbonyl)-L-lysine (224 mg, 479 μmol) was dissolved in DMF (10 mL), and HATU (191 mg, 503 μmol) and DIPEA (185 mg, 1.44 mmol, 250 μL) were added thereto. The reaction mixture was stirred at 25°C for 1.5 hours. Then, (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-aminofuran-2-carboxylate trifluoroacetate (320 mg, 479 μmol) was added, and the reaction was stirred at 25 °C for an additional 5 hours. Water (150 mL) was added to the reaction, followed by extraction with ethyl acetate (150 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product (450 mg), which was used directly in the next step without purification. Its structural property data are as follows: ESI-MS (m / z): 904.0 (M+H-Boc) + .

[0427] Step 2: Preparation of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-((S)-2-amino-6-(tert-butoxycarbonyl)amino)hexanamido)furan-2-carboxylate (DL-B-22'-2) (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(tert-butoxycarbonyl)amino)hexanamido)furan-2-carboxylate (387 mg, 385 μmol) was dissolved in DMF (6.00 mL) and DBU (58.6 mg, 385 μmol, 58.0 mL) was added. 1 μL) was added and the reaction was stirred at 25 °C for 1 h and then used directly in the next step without purification. Its structural property data are as follows: ESI-MS (m / z): 782.0 (M+H) + .

[0428] Step 3: Preparation of (6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-((8S,11S)-11-(4-(tert-butoxycarbonyl)amino)butyl)-1-(9H-fluoren-9-yl)-8-(hydroxymethyl)-3,6,9-trioxo-2-oxa-4,7,10-triazadodecane-12-amino)furan-2-carboxylate (DL-B-22'-3) ((9H-Fluoren-9-yl)methoxy)carbonyl)glycyl-L-serine (147 mg, 384 μmol) and HOBt (78.0 mg, 577 μmol) were used to prepare (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-((S)-2-amino-6-(tert-butoxycarbonyl)amino)hexanamide)furan-2-carboxylate (301 mg, 384 μmol). To a solution of 110 mg (577 μmol) of EDCI in DMF (6.00 mL) was added, and EDCI (110 mg, 577 μmol) was added, and the reaction mixture was stirred at 25°C for 1 hour. Water (100 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (100 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a crude product, which was purified by high-performance liquid chromatography and lyophilized to give the title compound (33.4 mg, 28.5 μmol). The purification method is as follows: Chromatography column: Phenomenex C18 (10 μm*25 mm*150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate) [Table 45] Its structural property data are as follows: ESI-MS (m / z): 1148.5 (M+H) + .

[0429] Step 4: Preparation of 6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-((S)-2-((S)-2-(2-aminoacetamido)-3-hydroxypropionamido)-6-(tert-butoxycarbonyl)amino)hexanamido)furan-2-carboxylate (DL-B-22'-4) (6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-((8S,11S)-11-(4-(tert-butoxycarbonyl)amino)butyl)-1-(9H-fluoren-9-yl)-8-(hydroxymethyl)-3,6,9-trioxo-2-oxa-4,7,10-triazadodecane-12-amino)furan-2-carboxylate (30 mg, 26.13 μmol, FR) was dissolved in DMF (1 mL), diethylamine (9.55 mg, 130.63 μmol) was added, and the reaction mixture was reacted at 25° C. for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography and lyophilized to obtain the formate salt of the title compound (23.0 mg, 22.48 μmol). The purification method is as follows: Chromatography column: Waters SunFire Prep C18 OBD (5 μm*19 mm*150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 46] Its structural property data are as follows: ESI-MS (m / z): 926.4 (M+H)+ .

[0430] Step 5: (6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-((34S,37S)-1-(3,5 Preparation of -bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-37-(4-(tert-butoxycarbonyl)amino)butyl)-34-(hydroxymethyl)-1,29,32,35-tetraoxo-5,8,11,14,17,20,23,26-octaoxa-2,30,33,36-tetraazaoctatriacontane-38-amino)furan-2-carboxylate (DL-B-22'-5) 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-carboxylic acid (22.4 mg, 0.026 mmol) was dissolved in DMF (1 mL) and added with HATU (18.9 mg, 0.050 mmol), DIPEA (9.6 mg, 0.074 mmol), and HCl (1 mL). To the reaction mixture were added 6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-((S)-2-((S)-2-(2-aminoacetamido)-3-hydroxypropionamido)-6-(tert-butoxycarbonyl)amino)hexanamido)furan-2-carboxylate (23.0 mg, 0.025 mmol, formic acid), and the reaction mixture was allowed to react at 25°C for 1 hour. The reaction mixture was directly purified by high performance liquid chromatography and lyophilized to give the title compound (20 mg, 10.76 μmol). The purification method is as follows: Chromatography column: Waters SunFire Prep C18 OBD (5 μm*19 mm*150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [Table 47] Its structural property data are as follows: ESI-MS (m / z): 1765.8 (M+H) + .

[0431] Step 6: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4-((34S,37 Preparation of S)-37-(4-aminobutyl)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5...

Claims

1. A compound of Formula I, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, or a prodrug thereof: 【Chemistry 185】 During the ceremony, R 1 are each independently hydrogen, halogen, or -NR a R b , hydroxy, cyano, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkyl, C substituted with one or more hydroxy 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 haloalkyl; preferably, R 1 are each independently hydrogen, halogen, or -NR a R b , hydroxy, C 1-6 Alkyl, C 1-6 Alkyl-OH and C 1-6 alkoxy; preferably, R 1 are each independently selected from the group consisting of hydrogen, fluorine, chlorine, amino, hydroxy, methyl, ethyl, and hydroxymethyl; preferably, R 1 are each independently selected from the group consisting of hydrogen, fluorine, amino, hydroxy, methyl, ethyl, and hydroxymethyl; R 2 is hydrogen, C 1-6 Alkyl, hydroxy and C 1-6 alkoxy; preferably, R 2 is selected from the group consisting of hydrogen, methyl, hydroxy and methoxy; preferably, R 2 is selected from the group consisting of hydrogen and methyl; Ring A is a single bond, C 6-10 Preferably, ring A is selected from the group consisting of a single bond, a benzene ring, and a 5- to 6-membered heteroaromatic ring; preferably, ring A is selected from the group consisting of a single bond, a benzene ring, a pyridine ring, a furan ring, and a thiophene ring; X is a single bond, C 1-6 Alkylene, C 3-6 Cycloalkylene, C 2-6 Alkenylidene, C 2-6 Alkynylidene and -NR a -preferably, X is selected from the group consisting of a single bond, methylene, ethylene, propylene, butylene, cyclopropylene, cyclohexylene, ethenylidene, and imino; preferably, X is selected from the group consisting of a single bond, methylene, ethylene, propylene, butylene, 1,1-cyclopropylene, 1,2-cyclopropylene, 1,4-cyclohexylene, and ethenylidene; Y is selected from the group consisting of -O- and -S-; Z is selected from the group consisting of hydroxy, halogen, and cyano; preferably, Z is selected from the group consisting of hydroxy, fluorine, chlorine, and cyano; Q 1 and Q 2 are each independently selected from the group consisting of hydrogen and halogen; preferably, Q 1 and Q 2 are each independently selected from the group consisting of hydrogen, fluorine and chlorine; preferably, Q 1 is selected from the group consisting of hydrogen and fluorine, and Q 2 is fluorine; M is selected from the group consisting of 1, 2, 3, 4 and 5; preferably, m is selected from the group consisting of 1 and 2; n is selected from the group consisting of 1 and 2; preferably, n is 1; R a and R b are each independently hydrogen and C 1-6 selected from the group consisting of alkyl; However: R 1 are each independently selected from the group consisting of halogen, amino, and methyl, m is 2, and R 2 is methyl, X is a single bond, Y is -S-, Z is fluorine, n is 1, and Q 1 is hydrogen or fluorine, and Q 2 is fluorine and ring A is not phenyl; When ring A is a single bond, R 1 is hydrogen or C 1-6 is not alkyl; and When ring A is a 5- or 6-membered heteroaromatic ring, R 1 is hydrogen and R 2 is C 1-6 is alkyl, and Q 1 is not a halogen, A compound, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, or a prodrug thereof.

2. The compound of claim 1, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, or a prodrug thereof, wherein the compound is a compound of formula II: 【Chemistry 186】

3. The compound of claim 1, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, or a prodrug thereof, wherein the compound is a compound of Formula III: 【Chemistry 187】

4. The compound of claim 1, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, or a prodrug thereof, wherein the compound is a compound of formula IV: 【Chemical 188】

5. The compound of claim 1, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, or a prodrug thereof, wherein the compound is a compound of formula V: 【Chemical 189】

6. 10. The compound of claim 1, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, or a prodrug thereof, wherein the compound is a compound of formula VI: 【Chemistry 190】 During the ceremony, Ring B is C 3-6 a cycloalkane, preferably cyclopropane; A compound, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, or a prodrug thereof.

7. The compound is selected from the group consisting of: 【Chemistry 191】 【change】 【change】 【change】 Preferably, the compound according to any one of claims 1 to 6, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, or a prodrug thereof, is selected from the group consisting of: 【Chemistry 192】

8. The compound is selected from the group consisting of: 【Chemistry 193】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 Preferably, the compound according to any one of claims 1 to 6, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, or a prodrug thereof, is selected from the group consisting of: 【Chemistry 194】 【change】 【change】

9. Formula Ab-[MLED] x An antibody-drug conjugate having a structure represented by the formula: Ab is an antibody that specifically binds to an antigen or an antigen-binding fragment thereof; M is a linking site to an antibody or antigen-binding fragment thereof; L is a linker between linking sites M and E; E is a structural fragment connecting L with D; D is -OH, -NH of a steroid glucocorticoid receptor agonist that is a compound according to any one of claims 1 to 8, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, or a prodrug thereof. 2 or a glucocorticoid drug fragment that is a monovalent structure obtained by removing one H from a secondary amino; x is an integer selected from 1 to 10, preferably an integer selected from 3 to 8; Preferably, D is selected from the following structures: 【Chemistry 195】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 Preferably, D is selected from the following structures: 【Chemistry 196】 【change】 【change】 【change】 More preferably, D is selected from the following structures: 【Chemistry 197】 【change】 In the formula, wavy line 【Chemistry 198】 indicates the point of attachment between that moiety and the rest of the molecule, Antibody-drug conjugates.

10. The antibody-drug conjugate of claim 9, wherein the antigen is selected from the group consisting of TNFα, IL6R, BDCA2, NR3C1, MSR1, PRLR, CD25, CD40, CD70, CD74, and CD163.

11. The antibody-drug conjugate of claim 9 or 10, wherein the antibody is selected from the group consisting of adalimumab and tocilizumab.

12. The antibody-drug conjugate according to any one of claims 9 to 11, wherein: M, 【Chemistry 199】 wherein ring C is a single bond, halogen, a 5-6 membered aliphatic heterocyclic ring system or a 5-20 membered aromatic ring system, and the aliphatic heterocyclic ring system or aromatic ring system is selected from the group consisting of oxo, halogen, cyano, amino, carboxyl, mercapto, and C 1-6 optionally substituted with one or more substituents each independently selected from the group consisting of alkyl; and M 1 represents a single bond, or -O-, -NH-, -C(=O)-, -S(=O) 2 -, -C=NO-, -NH-S(=O) 2 -NH-, phenylene, 5-10 membered heteroarylidene, C 1-20 Alkylene, C 2-20 Alkenylidene and C 2-20 alkynylidene; Preferably, M is 【Chemistry 200】 wherein ring C is a polycyclic ring formed by 2 to 5 (preferably 3) units selected from a single bond, a halogen, a 5-membered aliphatic heterocycle, a 6-membered heteroaromatic ring, or a 6-membered heteroaromatic ring and a benzene ring connected via a single bond, and the aliphatic heterocycle, heteroaromatic ring, or polycyclic ring is selected from oxo (=O), halogen, and C 1-4 optionally substituted with one or more groups selected from the group consisting of alkyl; and M 1 is a single bond, or -NH-, -C(=O)-, -NH-S(=O) 2 -NH-, C 1-10 Alkylene, C 2-10 Alkenylidene and C 2-10 alkynylidene; Preferably, M is 【Chemical Engineering 201】 and ring C is 【Chemical Engineering 202】 and M 1 is a single bond, -NH-, -NH-S(=O) 2 -NH-, C 1-6 Alkylene, C 2-6 Alkenylidene and C 2-6 alkynylidene; Preferably, M is 【Chemical 203】 selected from the group consisting of: Preferably, M is 【Chemical 204】 Selected from: Preferably, M is 【Chemical 205】 selected from the group consisting of: Preferably, M is 【Chemical 206】 is selected from the group consisting of In the formula, wavy line 【Chemical 207】 indicates the point of attachment between that moiety and the rest of the molecule, Antibody-drug conjugates.

13. The antibody-drug conjugate according to any one of claims 9 to 12, wherein L is C 1-6 Alkylene, -N(R')carbonyl-O-, natural or unnatural amino acids and analogs or derivatives thereof (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH 2 CH 2 (OCH 2 CH 2 ) r OCH 3 )), as well as short polypeptides consisting of multiple amino acids (e.g., 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, Va l-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), 【Chemical 208】 a structure consisting of one or more moieties selected from the group consisting of R' is hydrogen, C 1-6 Alkyl or -(CH 2 CH 2 O) r -Contains C 4-30 represents alkyl; r is an integer selected from 1 to 10, preferably an integer selected from 1 to 6; s is an integer selected from 1 to 20, preferably an integer selected from 1 to 10; Preferably、Lが、C 1-6 Alkylene, carbonyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Lys, 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, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly, 【Chemical Engineering 209】 wherein s is an integer selected from 1 to 20, preferably an integer selected from 1 to 10; Preferably, L is selected from the following structures: 【Chemical 210】 wherein s is an integer selected from 1 to 20, preferably an integer selected from 1 to 10; Preferably, L is selected from the following structures: 【Chemistry 211】 wherein s is an integer selected from 1 to 20, preferably an integer selected from 1 to 10; In the formula, wavy line 【Chemical Engineering 212】 indicates the point of attachment between that moiety and the rest of the molecule, Antibody-drug conjugates.

14. The antibody-drug conjugate according to any one of claims 9 to 13, wherein E is a single bond, -NHCH 2 - or a structure selected from the group consisting of: 【Chemistry 213】 wherein s is an integer selected from 1 to 20, preferably an integer selected from 1 to 10; Preferably, E is a single bond or -NHCH 2 - and; Preferably, E is a single bond; In the formula, wavy line 【Chemical 214】 indicates the point of attachment between that moiety and the rest of the molecule, Antibody-drug conjugates.

15. The antibody-drug conjugate according to any one of claims 9 to 14, wherein: 【Chemical 215】 is selected from the following structures: 【Chemical 216】 【change】 【change】 【change】 【change】 【change】 【change】 wherein s is an integer selected from 1 to 20, preferably an integer selected from 1 to 10; Preferably, 【Chemical 217】 is selected from the following structures: 【Chemistry 218】 【change】 【change】

16. The antibody-drug conjugate according to any one of claims 9 to 14, wherein: 【Chemical 219】 is selected from the following structures: 【Chemical 220】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 wherein s is an integer selected from 1 to 20, preferably an integer selected from 1 to 10; Preferably, 【Chemistry 221】 is selected from the following structures: 【Chemistry 222】

17. 17. The antibody-drug conjugate of any one of claims 9 to 16, wherein the antibody-drug conjugate is selected from the following structures: 【Chemistry 223】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 During the ceremony, s is an integer selected from 1 to 20; and An antibody-drug conjugate wherein n is an integer selected from 1 to 20.

18. 1 to 10, for example, 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, preferably 3 to 9, for example, 3.0 to 3.5, 3.0 to 4.0, 3.0 to 4.5, 3.0 to 5.0, 3.0 to 5.5, 3.0 to 6.0, 3.5 to 4.0, 3.5 to 4.5, 3.5 to 5.0, 3.5 to 5.5, 3.5 to 6.0, 3.5 to 6.5, 3.5 to 7.0, 3.5 to 7.5, 3.5 to 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 to 6.0, 5.5 to 6.5, 5.5 to 7.0, 5.5 to 7.5, 5.5 to 8.0, 6.0 to 6.5, 6.0 to 7.0, 6.0 to 7.5, 6.0 to 8.5, 6.5 to 7.0, 6.5 to 7.5, 6.5 to 8.5, 7.0 to 7.5, 7.0 to 9.0, or 7.5 to 9.

0.

19. Formula GM-[LED] x A drug-linker compound having a structure represented by the formula: G is a functional group or leaving group that reacts with specific amino acids or glycosyl groups and their derivatives in the antibody or antigen-binding fragment; preferably, G is halogen, halofenoxy, C 1-6 Haloalkyl, sulfonate, C 1-6 Alkylsulfonyl, C 1-6 Haloalkylsulfonyl, halosulfonyl, C 1-6 Alkyl sulfonates, C 1-6 Haloalkylsulfonates, C 1-6 Alkyl sulfite, halosulfonate, C 1-6 Alkyl sulfoxide, methylsulfonyl methacryloyl, dimethylsulfonyl methacryloyl, haloformyl, haloacetyl, formyl, acetyl, nitro, azido, cyano, cyanovinyl, N-methyl-vinylsulfonamide, tetrazinyl, methyltetrazinyl, trans-cyclooctenyl carbonate, C 2-6 Alkenyl, C 2-6 alkynyl, benzazacylcyclooctynyl, and (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy, preferably G is selected from the group consisting of halogen, methylsulfonyl, haloacetyl, fluorophenoxy, 【Chemistry 224】 Methylsulfonyl methacryloyl, cyanovinyl, N-methyl-vinylsulfonamide, azide, tetrazinyl, methyltetrazinyl, trans-cyclooctenyl carbonate, C 2-6 selected from the group consisting of alkynyl, benzazacyclooctynyl, and (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy; M, L, E, D and x are as defined in any one of claims 9 to 18. Drug-linker compounds.

20. 20. The drug-linker compound of claim 19, selected from the group consisting of: 【Chemical 225】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 During the ceremony, s is an integer selected from 1 to 20; and A Drug-Linker compound wherein n is an integer selected from 1 to 20.

21. A pharmaceutical composition comprising the compound according to any one of claims 1 to 8, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, or a prodrug thereof, the antibody-drug conjugate according to any one of claims 9 to 18, or the drug-linker compound according to claim 19 or 20, and one or more pharmaceutically acceptable carriers.

22. Use of a compound according to any one of claims 1 to 8, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, or a prodrug thereof, an antibody-drug conjugate according to any one of claims 9 to 18, a drug-linker compound according to claim 19 or 20, or a pharmaceutical composition according to claim 21 in the manufacture of a medicament for treating an inflammatory disease or an immune disease, Preferably, the inflammatory or immune disease is selected from the group consisting of rheumatoid arthritis, idiopathic arthritis, asthma, ulcerative colitis, neuromyelitis optica and autoimmune liver disease.

23. A method for preparing a compound according to any one of claims 1 to 8, comprising the steps of: 【Chemistry 226】 In the formula, rings A, R 1 , R 2 , X, Y, Z, Q 1 , Q 2 , m and n are as defined in any one of claims 1 to 8; and The method wherein LG is a leaving group such as halogen, methylsulfonyloxy or trifluoromethylsulfonyloxy, preferably iodine.

24. A method for preparing the drug-linker compound of any one of claims 19 to 20, comprising the steps of: 【Chemistry 227】 During the ceremony, Ring A, R 1 , R 2 , X, Y, Z, Q 1 , G, M, L, D, m and n are as defined in any one of claims 1 to 20; x is 1; E is a single bond; LG is a leaving group such as halogen, methylsulfonyloxy, or trifluoromethylsulfonyloxy, preferably iodine; and The method wherein PG is a protecting group such as 9-fluorenylmethoxycarbonyl, tert-butoxycarbonyl, p-methoxytriphenylmethyl and allyloxycarbonyl, preferably 9-fluorenylmethoxycarbonyl.

25. A method for preparing the antibody-drug conjugate of any one of claims 9 to 18, selected from the group consisting of: Conjugation Method A: Conjugating the drug-linker compound of any one of claims 19 to 20 with an antibody at a molar ratio of (8-10):1 to obtain an antibody-drug conjugate; Conjugation Method B:

21. The drug-linker compound according to claim 19, wherein the compound is conjugated to an antibody at a molar ratio of (4-6):1 to obtain an antibody-drug conjugate; Conjugation Method C:

21. The drug-linker compound according to any one of claims 19 to 20, which is conjugated with an antibody at a molar ratio of (4.0-4.5):1 to obtain an antibody-drug conjugate.

Images