Glucocorticoid receptor agonists and conjugates thereof

Abstract

The invention relates to the field of medicine, in particular to an antibody-drug conjugate of a glucocorticoid receptor stimulant. Specifically, the invention provides a glucocorticoid receptor agonist, and the glucocorticoid receptor agonist has excellent agonist activity. The present application also provides a drug-linker compound for conjugation, the drug being a glucocorticoid receptor agonist. The invention further provides an antibody-drug conjugate prepared by coupling the drug-connexon compound and an antibody, and the antibody-drug conjugate has good drug coupling uniformity and has an excellent treatment effect on inflammatory diseases such as rheumatoid arthritis. The invention further provides a preparation method of the glucocorticoid receptor agonist and the antibody-drug conjugate thereof and application of the glucocorticoid receptor agonist and the antibody-drug conjugate thereof in the field of medicine.

Description

Glucocorticoid receptor agonists and their conjugates Technical Field

[0001] The present application relates to the field of medicine, and specifically to an antibody-drug conjugate of a glucocorticoid receptor agonist. Specifically, the present application provides a class of glucocorticoid receptor agonists having excellent agonist activity. The present application also provides a drug-linker compound for coupling, wherein the drug is a glucocorticoid receptor agonist. The present application also provides an antibody-drug conjugate prepared by coupling the drug-linker compound to an antibody, which has good drug coupling uniformity and has excellent therapeutic effects on inflammatory diseases such as rheumatoid arthritis. The present application further provides a method for preparing the glucocorticoid receptor agonist and its antibody-drug conjugate and its application in the field of medicine. Background Art

[0002] Steroidal glucocorticoid receptor agonists are a class of drugs widely used to treat inflammatory and immune diseases. These drugs activate glucocorticoid receptors in cells, interfere with the recruitment of leukocytes to sites of inflammation, and inhibit the formation and release of inflammatory mediators from leukocytes and tissue cells, thereby exerting their anti-inflammatory effects. Long-term and excessive use of steroidal glucocorticoid receptor agonists may cause side effects such as infection, gastrointestinal reactions, endocrine disorders (such as moon face, buffalo hump, weight gain, hypokalemia, and abnormal blood sugar and blood pressure), and osteoporosis. To mitigate these side effects, inhalation formulations or topical formulations for skin application have been developed. Furthermore, the introduction of metabolically labile groups into the steroid structure to accelerate inactivation and reduce systemic exposure is another approach to reduce adverse reactions.

[0003] In recent years, in order to improve the efficacy and safety of steroid glucocorticoid receptor agonists, coupling them with antibodies has become a new direction in the research and development of this type of drugs.

[0004] SUMMARY OF THE INVENTION

[0005] The present application relates to steroidal glucocorticoid receptor agonists and linker conjugates and antibody conjugates thereof. The glucocorticoid receptor agonists and conjugates thereof have excellent agonistic activity and good drugability, and are expected to be used in the treatment of inflammatory and immune diseases.

[0006] In one aspect, the present application provides a steroid glucocorticoid receptor agonist, which is a compound represented by Formula I, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof:

[0007] in,

[0008] R1 is independently selected from hydrogen, halogen, -NR a R b , hydroxyl, cyano, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkyl, C substituted by one or more hydroxyl groups 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 alkyl halide;

[0009] R2 is selected from hydrogen, C 1-6 Alkyl, hydroxyl and C 1-6 alkoxy;

[0010] Ring A is selected from a single bond, C 6-10 Aromatic rings and 5-6 membered heteroaromatic rings;

[0011] X is selected from a single bond, C 1-6 Alkylene, C 3-6 Cycloalkylene, C 2-6 Alkenylene, C 2-6 Alkynylidene and -NR a -;

[0012] Y is selected from -O- and -S-;

[0013] Z is selected from hydroxy, halogen and cyano;

[0014] Q1 and Q2 are each independently selected from hydrogen and halogen;

[0015] m is selected from 1, 2, 3, 4, 5;

[0016] n is selected from 1 and 2;

[0017] R a 、R b are each independently selected from hydrogen and C 1-6 alkyl;

[0018] provided that: when R1 is each independently selected from 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, Ring A is not phenyl;

[0019] When ring A is a single bond, R1 is not hydrogen or C 1-6 alkyl; and

[0020] When Ring A is a 5-6 membered heteroaromatic ring, R1 is hydrogen and R2 is C 1-6 When it is an alkyl group, Q1 is not a halogen.

[0021] In another aspect, the present application provides an antibody-drug conjugate having the formula Ab-[MLED]x The structure shown, where:

[0022] Ab is an antibody or antigen-binding fragment thereof that specifically binds to an antigen;

[0023] M is a linker site with an antibody or antigen-binding fragment thereof;

[0024] L is a linker between the linkers M and E;

[0025] E is a structural fragment connecting L and D;

[0026] D is a glucocorticoid drug fragment, wherein the glucocorticoid drug fragment is a monovalent structure obtained by losing one H from -OH, -NH2 or a secondary amino group on the steroid glucocorticoid receptor agonist of the present invention;

[0027] x is an integer selected from 1 to 10.

[0028] In another aspect, the present application provides a drug-linker compound having the formula GM-[LED] x The structure shown, where:

[0029] G is a functional group or leaving group that reacts with specific amino acids or sugar groups and their derivatives in the antibody or antigen-binding fragment;

[0030] M is a linker site with an antibody or antigen-binding fragment thereof;

[0031] L is a linker between the linkers M and E;

[0032] E is a structural fragment connecting L and D;

[0033] D is a glucocorticoid drug fragment, wherein the glucocorticoid drug fragment is a monovalent structure obtained by losing one H from -OH, -NH2 or a secondary amino group on the steroid glucocorticoid receptor agonist of the present invention;

[0034] x is an integer selected from 1 to 10.

[0035] On the other hand, the present application provides a pharmaceutical composition comprising the steroid glucocorticoid receptor agonist of the present invention, the antibody-drug conjugate of the present invention or the drug-linker compound of the present invention, and one or more pharmaceutically acceptable carriers.

[0036] On the other hand, the present application provides the use of the steroid glucocorticoid receptor agonist of the present invention, the antibody-drug conjugate of the present invention, the drug-linker compound of the present invention or the pharmaceutical composition of the present invention in the preparation of a drug for treating inflammation or immune diseases.

[0037] On the other hand, 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, which is used to treat inflammation or immune diseases.

[0038] On the other hand, the present application provides a method for treating inflammatory or immune diseases, which comprises administering to an individual in need thereof a therapeutically effective amount of a steroidal 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.

[0039] In another aspect, the present application provides a method for preparing the compound of the present invention, comprising the following steps:

[0040] wherein Ring A, R1, R2, X, Y, Z, Q1, Q2, m and n are as defined above, and LG is a leaving group.

[0041] On the other hand, the present application provides a method for preparing the drug-linker compound of the present invention, comprising the following steps:

[0042] in,

[0043] Ring A, R1, R2, X, Y, Z, Q1, G, M, L, D, m and n are as described above;

[0044] x=1;

[0045] E is a single bond;

[0046] LG is a leaving group such as, but not limited to, halogen, methanesulfonyloxy (-OMs) and trifluoromethanesulfonyloxy (-OTf), preferably iodine; and

[0047] PG is an amino protecting group, such as but not limited to 9-fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), p-methoxytrityl (MMt) and allyloxycarbonyl (Alloc), preferably Fmoc.

[0048] On the other hand, the present application provides a method for preparing the antibody-drug conjugate of the present invention, which is selected from:

[0049] Coupling method A:

[0050] The drug-linker compound of the present invention is coupled to an antibody at a molar ratio of (8-10):1 to obtain an antibody-drug conjugate;

[0051] Coupling method B:

[0052] The drug-linker compound of the present invention is coupled to an antibody at a molar ratio of (4-6):1 to obtain an antibody-drug conjugate;

[0053] Coupling method C:

[0054] The drug-linker compound of the present invention is coupled to the antibody at a molar ratio of (4.0-4.5):1 to obtain an antibody-drug conjugate. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] FIG1 shows the results of the compounds of the present invention inhibiting the proliferation of mouse B cells in vitro.

[0056] FIG2 shows the results of the in vitro activation of glucocorticoid receptor activity in cells by the compounds of the present invention.

[0057] FIG3 shows the effect of the compounds of the present invention on the changes in arthritis scores in the CIA model of mice.

[0058] FIG4 shows the effects of the compounds of the present invention on body weight changes in CIA models of mice.

[0059] Detailed Description of the Invention

[0060] Steroid glucocorticoid receptor agonists

[0061] In one aspect, the present application provides a steroid glucocorticoid receptor agonist, which is a compound represented by Formula I, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof:

[0062] in,

[0063] R1 is independently selected from hydrogen, halogen, -NR a R b , hydroxyl, cyano, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkyl, C substituted by one or more hydroxyl groups 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 alkyl halide;

[0064] R2 is selected from hydrogen, C 1-6 Alkyl, hydroxyl and C 1-6 alkoxy;

[0065] Ring A is selected from a single bond, C 6-10 Aromatic rings and 5-6 membered heteroaromatic rings;

[0066] X is selected from a single bond, C 1-6 Alkylene, C 3-6 Cycloalkylene, C2-6 Alkenylene, C 2-6 Alkynylidene and -NR a -;

[0067] Y is selected from -O- and -S-;

[0068] Z is selected from hydroxy, halogen and cyano;

[0069] Q1 and Q2 are each independently selected from hydrogen and halogen;

[0070] m is selected from 1, 2, 3, 4, 5;

[0071] n is selected from 1 and 2;

[0072] R a 、R b are each independently selected from hydrogen and C 1-6 alkyl;

[0073] provided that: when R1 is each independently selected from 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, Ring A is not phenyl;

[0074] When ring A is a single bond, R1 is not hydrogen or C 1-6 alkyl; and

[0075] When Ring A is a 5-6 membered heteroaromatic ring, R1 is hydrogen and R2 is C 1-6 When it is an alkyl group, Q1 is not a halogen.

[0076] In some embodiments, each R1 is independently selected from hydrogen, halogen, -NR a R b , hydroxyl, C 1-6 Alkyl, -C 1-6 Alkyl-OH and C 1-6 Alkoxy.

[0077] In some embodiments, each R1 is independently selected from hydrogen, fluoro, chloro, amino, hydroxy, methyl, ethyl, and hydroxymethyl.

[0078] In some embodiments, each R1 is independently selected from hydrogen, fluoro, amino, hydroxy, methyl, ethyl, and hydroxymethyl.

[0079] In some embodiments, R2 is selected from hydrogen, methyl, hydroxy, and methoxy.

[0080] In some embodiments, R2 is selected from hydrogen and methyl.

[0081] In some embodiments, Ring A is selected from a single bond, a benzene ring, and a 5-6 membered heteroaryl ring.

[0082] In some embodiments, Ring A is selected from a single bond, a benzene ring, a pyridine ring, a furan ring, and a thiophene ring.

[0083] In some embodiments, X is selected from a single bond, methylene, ethylene, propylene, isopropylene, cyclopropylene, butylene, isobutylene, cyclobutylene, cyclohexylene, vinylene, and imino.

[0084] In some embodiments, X is selected from a single bond, methylene, ethylene, propylene, butylene, cyclopropylene, cyclohexylene, vinylene, and imino.

[0085] In some embodiments, X is selected from a single bond, methylene, ethylene, propylene, butylene, 1,1-cyclopropylene, 1,2-cyclopropylene, 1,4-cyclohexylene, and vinylene.

[0086] In some embodiments, Z is selected from the group consisting of hydroxy, fluoro, chloro, and cyano.

[0087] In some embodiments, Q1 and Q2 are each independently selected from hydrogen, fluorine, and chlorine.

[0088] In some embodiments, Q1 is selected from hydrogen and fluorine, and Q2 is fluorine.

[0089] In some embodiments, m is selected from 1 and 2.

[0090] In some embodiments, n is 1.

[0091] In some embodiments, the steroid glucocorticoid receptor agonist is a compound represented by Formula II, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof:

[0092] in,

[0093] R1, R2, Ring A, Y, Z, Q1, m and n are as defined above for the compound of formula I.

[0094] In some embodiments, the steroidal glucocorticoid receptor agonist is selected from a compound represented by Formula III, a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof:

[0095] in,

[0096] R1, Ring A, Y, Z, Q1, m and n are as defined above for the compound of formula I.

[0097] In some embodiments, the steroidal glucocorticoid receptor agonist is selected from a compound of Formula IV, a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof:

[0098] in,

[0099] R1, R2, Ring A, Y, Z, Q1, m and n are as defined above for the compound of formula I.

[0100] In some embodiments, the steroidal glucocorticoid receptor agonist is selected from a compound represented by Formula V, a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof:

[0101] in,

[0102] R1, R2, Ring A, Y, Z, Q1, m and n are as defined above for the compound of formula I.

[0103] In some embodiments, the steroidal glucocorticoid receptor agonist is selected from a compound represented by Formula VI, a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof:

[0104] in,

[0105] Ring B is C 3-6 a cycloalkane, preferably cyclopropane; and

[0106] R1, R2, Ring A, Y, Z, Q1, m and n are as defined above for the compound of formula I.

[0107] In some embodiments, the steroidal glucocorticoid receptor agonist is selected from the following compounds, their pharmaceutically acceptable salts, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites, or prodrugs:

[0108] In some embodiments, the steroidal glucocorticoid receptor agonist is selected from the following compounds, their pharmaceutically acceptable salts, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites, or prodrugs:

[0109] In some embodiments, the steroidal glucocorticoid receptor agonist is selected from the following compounds, their pharmaceutically acceptable salts, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites, or prodrugs:

[0110] Antibody Drug Conjugates

[0111] In one aspect, the present application provides an antibody-drug conjugate having the formula Ab-[MLED] x The structure shown, where:

[0112] Ab is an antibody or antigen-binding fragment thereof that specifically binds to an antigen;

[0113] M is a linker site with an antibody or antigen-binding fragment thereof;

[0114] L is a linker between the linkers M and E;

[0115] E is a structural fragment connecting L and D;

[0116] D is a glucocorticoid drug fragment, wherein the glucocorticoid drug is a glucocorticoid receptor agonist;

[0117] X is an integer selected from 1 to 10, preferably an integer from 3 to 8.

[0118] In the antibody-drug conjugate, the glucocorticoid receptor agonist can be linked to the antibody or antigen-binding fragment thereof via a linker (such as the "MLE" fragment shown in this application).

[0119] In some embodiments, in the antibody-drug conjugate, the antigen bound to the antibody includes but is not limited to TNFα, IL6R, BDCA2, NR3C1, MSR1, PRLR, CD25, CD40, CD70, CD74, CD163, etc.

[0120] In some embodiments, the antibodies or antigen-binding fragments thereof include, but are not limited to, adalimumab, tocilizumab, and IgG1 antibodies.

[0121] In some embodiments, M is wherein ring C is a single bond, halogen, 5-6 membered alicyclic or 5-20 membered aromatic ring system, wherein the alicyclic or aromatic ring system is optionally substituted by one or more independently selected from oxo (=O), halogen, cyano, amino, carboxyl, thiol and C 1-6 and M1 is selected from 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 heteroaryl, C 1-20 Alkylene, C 2-20 Alkenylene and C 2-20 Alkynylidene.

[0122] In some embodiments, M is wherein ring C is a single bond, a halogen, a 5-membered alicyclic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting 2-5 (preferably 3) units selected from 6-membered heteroaromatic rings and benzene rings through single bonds, wherein the alicyclic heterocycle, heteroaromatic ring or polycyclic ring is optionally substituted with one or more oxo (=O), halogen and C 1-4 and M1 is selected from a single bond or a fragment consisting of one or more groups selected from the following: -NH-, -C(=O)-, -NH-S(=O)2-NH-, C 1-10 Alkylene, C 2-10 Alkenylene and C 2-10 Alkynylidene.

[0123] In some embodiments, M is wherein ring C is selected from and M1 is selected from a single bond, -NH-, -NH-S(=O)2-NH-, C 1-6 Alkylene, C 2-6 Alkenylene and C 2-6 Alkynylidene.

[0124] In some embodiments, M is selected from

[0125] In some embodiments, M is selected from

[0126] In some embodiments, M is selected from

[0127] In some embodiments, M is selected from

[0128] 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 amino acids or non-natural amino acids and their analogs or derivatives (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys (COCH2CH2 (OCH2CH2) r OCH3)), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu- Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly),

[0129] Where R' represents hydrogen, C 1-6 Alkyl or containing -(CH2CH2O) r -C 4-30 alkyl; r is an integer selected from 1 to 10; s is an integer selected from 1 to 20. Preferably, r is an integer selected from 1 to 6; preferably, s is an integer selected from 1 to 10.

[0130] 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, Val-C it,Ala-Ala-Ala,Ala-Ala-Asn,Leu-Ala-Glu,Gly-Gly-Arg,Gly-Glu-Gly,Gly-Gly-Gly,Gly-Se r-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,

[0131] wherein s is an integer selected from 1-20.

[0132] In some embodiments, L is a structure consisting of one or more moieties selected from the group consisting of: 1-6 Alkylene, Carbonyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Lys, Val-C it,Ala-Ala-Ala,Ala-Ala-Asn,Leu-Ala-Glu,Gly-Gly-Arg,Gly-Glu-Gly,Gly-Gly-Gly,Gly-Se r-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, wherein s is an integer selected from 1-20, preferably an integer from 1-10.

[0133] In some embodiments, L is a structure consisting of one or more moieties selected from:

[0134] wherein s is an integer selected from 1-20, preferably an integer from 1-10.

[0135] In some embodiments, L is a structure consisting of one or more moieties selected from:

[0136] wherein s is an integer selected from 1-20, preferably an integer from 1-10.

[0137] In some embodiments, L is a structure consisting of one or more moieties selected from:

[0138] wherein s is an integer selected from 1-20, preferably an integer from 1-10.

[0139] In some embodiments, L is a structure consisting of one or more moieties selected from:

[0140]

[0141] wherein s is an integer selected from 1-20, preferably an integer from 1-10.

[0142] In some embodiments, L is selected from the following structures:

[0143] wherein s is an integer selected from 1-20, preferably an integer from 1-10.

[0144] In some embodiments, L is selected from the following structures:

[0145] wherein s is an integer selected from 1-20, preferably an integer from 1-10.

[0146] In some embodiments, L is selected from the following structures:

[0147] wherein s is an integer selected from 1-20, preferably an integer from 1-10.

[0148] In some embodiments, L is selected from the following structures:

[0149] wherein s is an integer selected from 1-20, preferably an integer from 1-10.

[0150] In some embodiments, L is selected from the following structures:

[0151] wherein s is an integer selected from 1-20, preferably an integer from 1-10.

[0152] In some embodiments, L is selected from the following structures:

[0153] In some embodiments, L is selected from the following structures:

[0154] In some embodiments, E is a single bond, a carbonyl group, -NHCH2-, or a structure selected from the group consisting of:

[0155] wherein s is an integer selected from 1-20, preferably an integer from 1-10.

[0156] In some embodiments, E is a single bond or -NHCH2-.

[0157] In some embodiments, E is a single bond.

[0158] In some embodiments, Selected from the following structures:

[0159] wherein s is an integer selected from 1-20, preferably an integer of 1-10.

[0160] In some embodiments, Selected from the following structures:

[0161] In some embodiments, Selected from the following structures:

[0162] In some embodiments, D is a corresponding fragment of the steroid glucocorticoid receptor agonist of the present invention obtained by linking the agonist to a linker.

[0163] In some embodiments, D is a monovalent structure obtained by losing one H from -OH, -NH2, or a secondary amino group on the steroidal glucocorticoid receptor agonist of the present invention.

[0164] In some embodiments, D is selected from the following structures:

[0165] In some embodiments, D is selected from the following structures:

[0166] In some embodiments, D is selected from the following structures:

[0167] In some embodiments, the antibody-drug conjugate is selected from ADC-A-01 to ADC-A-145 and ADC-B-01 to ADC-B-146 shown below: Ab in the following diagram is defined as above, wherein the sulfhydryl group on the antibody and the drug-linker compound form a thioether bond through an addition reaction or a substitution reaction to obtain a complete antibody-drug conjugate, x represents the amount of drug loading, and s represents an integer from 1 to 20, preferably an integer from 1 to 10:

[0168] in, Indicates the specific connection method between the thiol group in the antibody or its antigen-binding fragment and the linker

[0169] s is an integer selected from 1-20, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; and

[0170] n is selected from an integer of 0-20, for example 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0171] In some embodiments, Ab in the antibody-drug conjugate represents an antibody or antigen-binding fragment thereof comprising VH as shown in SEQ ID NO.1 and VL as shown in SEQ ID NO.2, for example, an antibody or antigen-binding fragment thereof comprising VH as shown in SEQ ID NO.1 and CH (heavy chain constant region) as shown in SEQ ID NO.3, and VL as shown in SEQ ID NO.2 and CL (light chain constant region) as shown in SEQ ID NO.4.

[0172] In some embodiments, Ab in the antibody-drug conjugate represents an antibody or antigen-binding fragment thereof comprising VH as shown in SEQ ID NO.7 and VL as shown in SEQ ID NO.8, for example, an antibody or antigen-binding fragment thereof comprising VH as shown in SEQ ID NO.7 and CH (heavy chain constant region) as shown in SEQ ID NO.9 or CH (mutant heavy chain constant region) as shown in SEQ ID NO.11, and VL as shown in SEQ ID NO.8 and CL (light chain constant region) as shown in SEQ ID NO.10.

[0173] In some embodiments, the DAR value (drug-antibody conjugate ratio) of the antibody-drug conjugate is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4 ~10, 5~6, 5~7, 5~8, 5~9, 5~10, 6~7, 6~8, 6~9, 6~10, 7~8, 7~9, 7~10, 8~9, 8~10, or 9~10, preferably 3~9, for example, 3.0~3.5, 3.0~4.0, 3.0~4.5, 3.0~5.0, 3.0~5.5, 3.0~6.0, 3.5~4.0, 3.5~4.5, 3.5~5.0, 3.5~5.5, 3.5 ~6.0, 3.5~6.5, 3.5~7.0, 3.5~7.5, 3.5~8.0, 4.0~4.5, 4.0~5.0, 4.0~5.5, 4.0~6.0, 4.0~6.5, 4.0~7.0, 4.0~7.5, 4.0~8.0, 4.5~5.0, 4.5~5.5, 4.5~6.0, 4.5~6.5, 4.5~7.0, 4.5~7.5, 4.5~8.0, 5.0~5 .5, 5.0-6.0, 5.0-6.5, 5.0-7.0, 5.0-7.5, 5.0-8.0, 5.5-6.0, 5.5-6.5, 5.5-7.0, 5.5-7.5, 5.5-8.0, 6.0-6.5, 6.0-7.0, 6.0-7.5, 6.0-8.5, 6.5-7.0, 6.5-7.5, 6.5-8.5, 7.0-7.5, 7.0-9.0 or 7.5-9.0.

[0174] In all of the above embodiments, the wavy line represents the point of attachment of the group to the rest of the molecule.

[0175] Drug-linker compounds

[0176] It will be understood by those skilled in the art that the antibody-drug conjugates described herein can be prepared modularly. For example, first obtain the free form of the "drug-linker" (which can be understood as GM-[LED] x, wherein GM is the structural form before covalently linking to the antibody or antigen-binding fragment thereof, and x is an integer selected from 1 to 10), which is then covalently linked to the antibody or antigen-binding fragment thereof to obtain the antibody-drug conjugate described herein. Accordingly, the GM in the free form of the "drug-linker" is linked to one or more sulfhydryl (-SH), amino (-NH2), or carboxyl (-COOH) groups on the antibody or antigen-binding fragment thereof via a substitution reaction (e.g., removal of structures such as -SO2Me or -Br) or an addition reaction.

[0177] In another aspect, the present invention provides a drug-linker compound having the formula GM-[LED] x The structure shown, wherein:

[0178] G is a functional group or leaving group that reacts with specific amino acids or sugar groups and their derivatives in the antibody or antigen-binding fragment;

[0179] M is a linker site with an antibody or antigen-binding fragment thereof;

[0180] L is a linker between the linkers M and E;

[0181] E is a structural fragment connecting L and D;

[0182] D is a glucocorticoid drug fragment, wherein the glucocorticoid drug fragment is a monovalent structure obtained by losing one H from -OH, -NH2 or a secondary amino group on the steroid glucocorticoid receptor agonist of the present invention;

[0183] x is an integer selected from 1 to 10, preferably 1 or 2.

[0184] In some embodiments, M, L, E, D, and x are as defined above in the "Antibody-Drug Conjugate" section.

[0185] In some embodiments, G is selected from halogen, halophenoxy, C 1-6 Haloalkyl, sulfonic acid (HOS (= O) 2-), C 1-6 Alkylsulfonyl, C 1-6 Halogenated alkylsulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, C 1-6 Halogenated alkyl sulfonate group, C 1-6 Alkyl sulfinate group, halosulfonate group, C 1-6 Alkyl sulfoxide, methylsulfonylmethacryloyl, bismethylsulfonylisobutyryl Haloformyl, haloacetyl, formyl, acetyl, nitro, azido, cyano, cyanovinyl, N-methylvinylsulfonylamino Tetrazine, methyl tetrazine, trans-cyclooctene carbonate, C 2-6 Alkenyl, C 2-6 alkynyl, benzazacyclooctinyl, (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy.

[0186] In some embodiments, GM is in,

[0187] G is a leaving group for nucleophilic substitution reaction, for example, halogen, methanesulfonyl, haloacetyl, fluorophenoxy or ); or G is a group that undergoes addition reaction or cyclization reaction, such as methylsulfonylmethacryloyl, cyanovinyl, N-methyl-vinylsulfonylamino, azido, tetrazine, methyltetrazine, trans-cyclooctene carbonate, C 2-6 alkynyl, benzazacyclooctinyl, (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy.

[0188] Ring C is a 5-6 membered alicyclic heterocyclic ring or a 5-20 membered aromatic ring system, wherein the alicyclic heterocyclic ring or aromatic ring system is optionally substituted by one or more independently selected from oxo (=O), halogen, cyano, amino, carboxyl, thiol and C 1-6 alkyl radicals; and

[0189] M1 is selected from 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 heteroarylene, C 1-20 Alkylene, C 2-20 Alkenylene and C 2-20 Alkynylidene.

[0190] In some embodiments, GM is in,

[0191] G is a methylsulfonyl group or a sulfonic acid group;

[0192] Ring C is a 5-membered alicyclic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting 2-5 (preferably 3) units selected from 6-membered heteroaromatic rings and benzene rings through single bonds, wherein the alicyclic heterocycle, heteroaromatic ring or polycyclic ring is optionally substituted by one or more selected from oxo (=O), halogen and C 1-4 alkyl radicals; and

[0193] M1 is selected from a single bond, -O-, -NH-, -NH-S(=O)2-NH-, C 1-10 Alkylene, C 2-10 Alkenylene and C 2-10 Alkynylidene.

[0194] In some embodiments, GM is in,

[0195] Selected from and

[0196] M1 is selected from a single bond, -O-, -NH-, -NH-S(=O)2-NH-, C 1-6 Alkylene, C 2-6 Alkenylene or C 2-6 Alkynylidene.

[0197] In some embodiments, GM is

[0198] In some embodiments, GM is

[0199] In some embodiments, GM is

[0200] In some embodiments, GM is

[0201] In some embodiments, the free form of the "drug-linker" compound is selected from:

[0202] wherein s is an integer selected from 1-20, preferably an integer of 1-10.

[0203] In some embodiments, the free form of the "drug-linker" compound is selected from:

[0204] in,

[0205] s is an integer selected from 1-20, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; and

[0206] n is selected from an integer of 0-20, for example 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0207] Pharmaceutical composition

[0208] 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.

[0209] The selection of a pharmaceutically acceptable carrier depends on the dosage form of the pharmaceutical composition, first on the route of administration of the dosage form, and secondly on the formulation of the dosage form. For example, the pharmaceutically acceptable carrier may include water (such as water for injection), a buffer, an isotonic saline 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, a polyalkylene glycol such as polyethylene glycol (e.g., polyethylene glycol 4000) or polypropylene glycol, triglycerides, etc.

[0210] The glucocorticoid receptor agonist, antibody-drug conjugate or drug-linker compound described herein is usually formulated in a unit injectable form together with a pharmaceutically acceptable parenteral vehicle for parenteral use, such as subcutaneous injection, intramuscular injection, intravenous push injection, intravenous drip, local injection into diseased tissue, intratumor 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 agent or solution (Remington's Pharmaceutical Sciences (1980) 16 th Administration of the glucocorticoid receptor agonist, antibody-drug conjugate, drug-linker compound, or pharmaceutical composition described herein can be by any route appropriate to the subject to be treated.

[0211] Treatment methods and uses

[0212] In another aspect, the present application provides use of the steroid glucocorticoid receptor agonist of the present invention, the antibody-drug conjugate of the present invention, the drug-linker compound of the present invention, or the pharmaceutical composition of the present invention in the preparation of a medicament for treating inflammatory or immune diseases.

[0213] 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 inflammation or immune diseases.

[0214] In another aspect, the present application provides a method for treating inflammatory or immune diseases, comprising administering to an individual in need thereof a therapeutically effective amount of a steroidal 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.

[0215] In one embodiment, the inflammatory or immune disease described herein is a disease with high expression of TNFα or IL-6R, including but not limited to rheumatoid arthritis, idiopathic arthritis, asthma, ulcerative colitis, neuromyelitis optica, and autoimmune liver disease.

[0216] definition

[0217] Unless otherwise defined below, the meanings of all technical and scientific terms used herein are intended to be the same as those generally understood by those skilled in the art. Reference to the techniques used herein is intended to refer to techniques generally understood in the art, including variations of those techniques that are obvious to those skilled in the art or replacements with equivalent techniques. Furthermore, laboratory procedures such as genomics, nucleic acid chemistry, and molecular biology used herein are conventional procedures widely used in the corresponding fields. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present invention.

[0218] The term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, with heavy chains also containing a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain, CL. The constant domains are not directly involved in the binding of antibodies to antigens, but exhibit a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen binding site, respectively. The allocation of amino acids in each region or domain can follow various numbering systems known in the art.

[0219] 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. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, as defined in the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), or the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268–9272). For a given antibody, a person skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0220] 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 those defined by the Kabat, Chothia, IMGT, or AbM numbering systems. In certain embodiments, the CDRs contained in an antibody or antigen-binding fragment thereof are defined by the Chothia numbering system.

[0221] The term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0222] The term "antigen-binding fragment" of an antibody refers to polypeptides that are fragments of an antibody, such as polypeptides that are fragments of a full-length antibody, which retain the ability to specifically bind to the same antigen bound by the full-length antibody and / or compete with the full-length antibody for specific binding to the antigen, and are also referred to as "antigen-binding portions." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be 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, di-scFv, (scFv)2, disulfide-stabilized Fv proteins ("dsFv"), single domain antibodies (sdAbs, nanobodies), and polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability on the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0223] The term "Fd" means an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means a fragment obtained after reducing the disulfide bonds linking the two heavy chain fragments in the F(ab')2 fragment, consisting of one complete light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain.

[0224] The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of the complete binding site.

[0225] The term "Fc" refers to an antibody fragment formed by disulfide bonds between the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0226] The term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by 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, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments, the VH and VL domains may be positioned relative to each other in any suitable arrangement. For example, containing NH2-VH-VH-COOH, NH 2- VL-VL-COOH scFv.

[0227] The term "single-domain antibody (sdAb)" has the meaning generally understood by those skilled in the art, and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as the full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also called nanobodies.

[0228] Each of the above antibody fragments 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 the antigen.

[0229] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0230] Antigen-binding fragments of antibodies (e.g., those described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as for intact antibodies.

[0231] The term "murine antibody" refers to antibodies obtained by fusing B cells from immunized mice with myeloma cells, screening for murine hybrid fusion cells that can both proliferate indefinitely and secrete antibodies, followed by screening, antibody preparation, and antibody purification; or refers to antibodies secreted by plasma cells formed by the differentiation and proliferation of B cells in mice after antigen invasion.

[0232] The term "humanized antibody" refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc. The donor antibody can be a mouse, rat, rabbit or non-human primate (e.g., cynomolgus monkey) antibody with the expected properties (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and / or ability to enhance immune response).

[0233] The term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 total positions match). Typically, two sequences are compared when aligned for maximum identity. Such alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent 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)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0234] The term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative 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 substitutions in which amino acid residues are substituted with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the 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. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0235] The twenty conventional amino acids referred to herein are denoted 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)), which is incorporated herein by reference. In the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0236] The terms "comprises," "comprising," "having," "containing," or "involving," and other variations thereof herein, are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

[0237] The term "alkyl" refers to a radical obtained by removing one hydrogen atom from a straight-chain or branched alkane, for example, "C 1-20Alkyl", "C 1-10 Alkyl", "C 1-6 Alkyl", "C 1-4 Alkyl", "C 1-3 alkyl”, etc., specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc.

[0238] The term "alkenyl" refers to a straight-chain or branched aliphatic hydrocarbon group having one or more unsaturated double bonds, for example, "C 2-20 Alkenyl", "C 2-10 Alkenyl", "C 2-6 Alkenyl", "C 2-4 Specific examples include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl, 2-buten-1-yl, 3-buten-1-yl, 2-penten-1-yl, 3-penten-1-yl, 4-penten-1-yl, 5-hexen-1-yl, 4-hexen-1-yl, 3-hexen-1-yl, 2-hexen-1-yl, 3-methyl-2-buten-1-yl, 3-methyl-3-penten-1-yl, 3-methyl-2-penten-1-yl, 4-methyl-3-penten-1-yl, 4-methyl-2-penten-1-yl, and 2-methyl-2-penten-1-yl.

[0239] The term "alkynyl" refers to a straight-chain or branched aliphatic hydrocarbon group having one or more unsaturated triple bonds, such as "C 2-20 Alkynyl", "C 2-10 Alkynyl", "C 2-6 Alkynyl", "C 2-4 Specific examples include, but are not limited to, ethynyl, 1-propyn-1-yl, 2-propyn-1-yl, 2-butyn-1-yl, 3-butyn-1-yl, 2-pentyn-1-yl, 3-pentyn-1-yl, 4-pentyn-1-yl, 5-hexyn-1-yl, 4-hexyn-1-yl, 3-hexyn-1-yl and 2-hexyn-1-yl.

[0240] The term "alkylene" refers to a radical obtained by removing two hydrogen atoms from a straight or branched chain alkane, for example, "C 1-20 Alkylene", "C 1- 10 Alkylene", "C 3-10 Alkylene", "C5-8 Alkylene", "C 1-6 Alkylene", "C 1-4 Alkylene", "C 1-3 Specific examples include, but are not limited to, methylene, ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene or 1,6-hexylene.

[0241] The term "alkenylene" refers to a divalent group derived from a linear or branched alkene containing at least one carbon-carbon double bond losing two hydrogen atoms, including, for example, "C 2-20 Alkenylene", "C 3-10 Alkenylene", "C 5-8 Examples include, but are not limited to, vinylene, 1-propenylene, 2-propenylene, 1-butenylene, 2-butenylene, 1,3-butadienylene, 1-pentenylene, 2-pentenylene, 3-pentenylene, 1,3-pentadienylene, 1,4-pentadienylene, 1-hexenylene, 2-hexenylene, 3-hexenylene, 1,4-hexadienylene, and the like.

[0242] The term "alkynylene" refers to a divalent group derived from a straight or branched alkyne containing at least one carbon-carbon triple bond losing two hydrogen atoms. 2-20 Alkynylidene", "C 3-10 Alkynylidene", "C 5-8 Examples include, but are not limited to, ethynylene, 1-propynylene, 2-propynylene, 1-butynylene, 2-butynylene, 1,3-butadiynylene, 1-pentynylene, 2-pentynylene, 3-pentynylene, 1,3-pentadiynylene, 1,4-pentadiynylene, 1-hexynylene, 2-hexynylene, 3-hexynylene, 1,4-hexadiynylene, and the like.

[0243] The term "cycloalkylene" refers to a radical obtained by removing two hydrogen atoms from a cycloalkane, for example, "C 3-20 Cycloalkylene", "C 3-10 Cycloalkylene", "C 3-6 Specific examples include but are not limited to cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, etc.

[0244] The term "alkoxy" means an "alkyl" as defined above attached to the parent molecular moiety through an oxygen atom, for example, C1-C6 alkoxy, C1-C3 alkoxy, specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, pentyloxy, isopentyloxy, hexyloxy, and the like.

[0245] The term "halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0246] The term "haloalkyl" refers to an alkyl group substituted by one or more (such as 1 to 3) identical or different halogen atoms. For example, the term "C1-C6 haloalkyl" as used herein refers to a haloalkyl group having 1 to 6 carbon atoms, including but not limited to -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, -CH2CH2CF3, -CH2Cl, etc.

[0247] The term "aromatic ring" refers to an all-carbon monocyclic or condensed polycyclic aromatic hydrocarbon with a conjugated π electron system. Common aromatic rings include, but are not limited to, benzene, naphthalene, anthracene, phenanthrene, acenaphthene, azulene, fluorene, indene, pyrene, etc. For example, the term "C6-C 10 By "aromatic ring" is meant an aromatic ring containing 6 to 10 carbon atoms, such as benzene or naphthalene.

[0248] The term "aliphatic heterocycle" refers to a saturated or partially saturated cyclic structure containing at least one ring member selected from N, O and S, including but not limited to 5-10 membered aliphatic heterocycle, 5-6 membered aliphatic heterocycle, etc., such as 5-6 membered nitrogen-containing aliphatic heterocycle, 5-6 membered oxygen-containing aliphatic heterocycle, etc. Specific examples include but are not limited to: tetrahydrofuran, pyrrolidine, piperidine, tetrahydropyran, etc.

[0249] The term "heteroaromatic ring" refers to an aromatic ring structure containing at least one ring member selected from N, O and S, including but not limited to 5-10 membered aromatic heterocycles, 5-6 membered aromatic heterocycles, and the like, such as 5-6 membered nitrogen-containing aromatic heterocycles, 5-6 membered oxygen-containing aromatic heterocycles, and the like. 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, and the like.

[0250] The term "aromatic ring system" refers to a monocyclic or polycyclic ring system comprising at least one aromatic ring (e.g., a benzene ring, etc.) or heteroaromatic ring (e.g., a pyrimidine ring, etc.), two or more aromatic rings and / or heteroaromatic rings may form a fused ring or be connected by a single bond (e.g., dipyrimidinylphenyl, etc.), and the aromatic ring system may be divalent or higher valent (e.g., trivalent or tetravalent), for example, a 5-20 membered aromatic ring system.

[0251] If a substituent is described as being "optionally substituted with," the substituent may be (1) unsubstituted or (2) substituted. If a carbon of a substituent is described as being optionally substituted with one or more of the substituents listed, one or more hydrogens on the carbon (to the extent of any hydrogens present) may be replaced, individually and / or collectively, with independently selected substituents or unsubstituted. If a nitrogen of a substituent is described as being optionally substituted with one or more of the substituents listed, one or more hydrogens on the nitrogen (to the extent of any hydrogens present) may each be replaced with an independently selected substituent or unsubstituted.

[0252] If a substituent is described as being "independently selected" from a group of groups, each substituent is selected independently of the other. Thus, each substituent may be the same as or different from another (other) substituent.

[0253] Unless otherwise indicated, as used herein, the point of attachment of a substituent can be from any suitable position of the substituent.

[0254] When a bond to a substituent is shown to pass through a bond connecting two atoms in a ring, then such substituent may be bonded to any ring atom in the substitutable ring.

[0255] 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 from acids that form pharmaceutically acceptable salts. Suitable base addition salts are formed from bases that form pharmaceutically acceptable salts. For a review of suitable salts, see Stahl and Wermuth, "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

[0256] The term "stereoisomer" (or "optical isomer") refers to a stable isomer that has a perpendicular asymmetric plane due to at least one chiral factor (including a chiral center, chiral axis, chiral plane, etc.), thereby being able to rotate plane-polarized light. Because the compounds of the present invention contain asymmetric centers and other chemical structures that may lead to stereoisomerism, the present invention also includes these stereoisomers and mixtures thereof. Because the compounds of the present invention (or pharmaceutically acceptable salts thereof) contain asymmetric carbon atoms, they can exist as single stereoisomers, racemates, enantiomers, and mixtures of diastereomers. Generally, these compounds can be prepared as racemates. However, if desired, such compounds can be prepared or isolated to obtain pure stereoisomers, i.e., single enantiomers or diastereomers, or mixtures enriched in a single stereoisomer (purity ≥98%, ≥95%, ≥93%, ≥90%, ≥88%, ≥85%, or ≥80%). As described below, a single stereoisomer of a compound is synthesized from an optically active starting material containing the desired chiral center, or by preparing a mixture of enantiomeric products followed by separation or resolution, for example, by conversion to a mixture of diastereoisomers followed by separation or recrystallization, chromatography, use of a chiral resolving agent, or direct separation of the enantiomers on a chiral chromatographic column. Starting compounds with a specific stereochemistry are either commercially available or prepared as described below and resolved by methods well known in the art. The term "enantiomer" refers to a pair of stereoisomers that are nonsuperimposable mirror images of each other. The term "diastereomer" or "diastereomers" refers to optical isomers that are not mirror images of each other. The term "racemic mixture" or "racemate" refers to a mixture containing equal parts of a single enantiomer (i.e., an equimolar mixture of two R and S enantiomers). The term "non-racemic mixture" refers to a mixture containing unequal parts of a single enantiomer. Unless otherwise indicated, all stereoisomeric forms of the compounds of the present invention are within the scope of the invention.

[0257] The term "tautomer" (or "tautomeric form") refers to structural isomers with different energies that can be interconverted via a low energy barrier. If tautomerism is possible (such as in solution), a chemical equilibrium of the tautomers can be achieved. For example, proton tautomers (or prototropic tautomers) include, but are not limited to, interconversions via proton migration, such as keto-enol isomerization, imine-enamine isomerization, amide-iminoalcohol isomerization, and the like. Unless otherwise indicated, all tautomeric forms of the compounds of the present invention are within the scope of the present invention.

[0258] The term "polymorph" (or "polymorphic form") refers to a solid crystalline form of a compound or complex. Polymorphs can be detected, classified, and identified using well-known techniques, including, but not limited to, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), X-ray powder diffraction (XRPD), single crystal X-ray diffraction (SCXRD), solid-state nuclear magnetic resonance (NMR), infrared spectroscopy (IR), Raman spectroscopy, and scanning electron microscopy (SEM).

[0259] The term "solvate" refers to a substance formed by the compound of the present invention (or a pharmaceutically acceptable salt thereof) and at least one solvent molecule bound by non-covalent intermolecular forces.

[0260] The term "nitrogen oxide" refers to a compound formed by oxidation of a nitrogen atom in a tertiary amine or nitrogen-containing (aromatic) heterocyclic compound structure.

[0261] The present invention also includes all pharmaceutically acceptable isotopically labeled compounds, which are identical to the compounds of the present invention except that one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number prevalent in nature. Examples of isotopes suitable for inclusion in the compounds of the present invention include, but are not limited to, isotopes of hydrogen (e.g., 2H, 3H, deuterium D, tritium T); isotopes of carbon (e.g., 11C, 13C, and 14C); isotopes of chlorine (e.g., 37Cl); isotopes of fluorine (e.g., 18F); isotopes of iodine (e.g., 123I and 125I); isotopes of nitrogen (e.g., 13N and 15N); isotopes of oxygen (e.g., 15O, 17O, and 18O); and isotopes of sulfur (e.g., 35S).

[0262] 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 the compounds of the invention. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc. of the administered compound. Thus, the present invention includes metabolites of the compounds of the invention, including compounds produced by contacting a compound of the invention with a mammal for a period of time sufficient to produce a metabolic product thereof.

[0263] The present invention further includes within its scope prodrugs of the compounds of the present invention, which are certain derivatives of the compounds of the present invention that may themselves have little or no pharmacological activity, and when administered to the body or thereon, can be converted into the compounds of the present invention having the desired activity by, for example, hydrolytic cleavage. Typically, such prodrugs will be functional group derivatives of the compounds that are readily converted into the desired therapeutically active compounds in vivo. Further information on the use of prodrugs can be found in "Pro-drugs as Novel Delivery Systems," Volume 14, ACS Symposium Series (T. Higuchi and V. Stella) and "Bioreversible Carriers in Drug Design," Pergamon Press, 1987 (E.B. Roche, ed., American Pharmaceutical Association). The prodrugs of the present invention can be prepared, for example, by replacing appropriate functional groups present in the compounds of the present invention with certain moieties known to those skilled in the art as "pro-moieties" (e.g., as described in "Design of Prodrugs," H. Bundgaard (Elsevier, 1985)).

[0264] Preparation of compounds

[0265] In another aspect, the present invention provides a method for preparing the compound represented by formula I.

[0266] In some embodiments, the compound of formula I of the present invention can be synthesized by the following synthetic route:

[0267] wherein Ring A, R1, R2, X, Y, Z, Q1, Q2, m and n are as defined above; and

[0268] LG is a leaving group, such as but not limited to halogen, methanesulfonyloxy (-OMs) or trifluoromethanesulfonyloxy (-OTf), preferably iodine.

[0269] The method comprises the following steps: an esterification reaction is carried out between the compounds of formula I-SM-1 and formula I-SM-2, and then a substitution reaction is carried out with the compound of formula I-SM-3 to obtain a compound of formula I.

[0270] In some embodiments, the above steps are carried out in the presence of a suitable condensation reagent, which can be selected from HATU, HBTU, EDCI, DCC, T3P, POCl3 and HOBT, preferably HATU.

[0271] In some embodiments, the above steps are carried out at a suitable temperature of 0-140°C, such as 0°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 0-35°C;

[0272] In some embodiments, the above steps are carried out in a suitable organic solvent, which can be selected from halogenated hydrocarbons (such as dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane (1,2-DCE), etc.), nitriles (such as acetonitrile (AN), etc.), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), tetrahydrofuran (THF), 1,4-dioxane (Dioxane), dimethyl sulfoxide (DMSO) and any combination thereof, preferably N,N-dimethylformamide (DMF).

[0273] In some embodiments, the above steps are carried out in the presence of a suitable base, which includes an organic base or an inorganic base. The organic base can be selected from N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK) and pyridine (Py); the inorganic base can be selected from 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).

[0274] Preparation of drug-linker compounds

[0275] In another aspect, the present invention provides a method for synthesizing a drug-linker compound of formula GM-[LED]x:

[0276] in,

[0277] Ring A, R1, R2, X, Y, Z, Q1, G, M, L, D, m and n have the same meanings as above;

[0278] x=1;

[0279] E is a single bond;

[0280] LG is a leaving group such as, but not limited to, halogen, -OMs, and -OTf, preferably iodine; and

[0281] PG is an amino protecting group, such as but not limited to Fmoc, Boc, MMt and Alloc, preferably Fmoc.

[0282] Step 1: Compound DL-SM-1 and DL-SM-2 undergo condensation reaction to obtain compound DL-IM-1

[0283] In some embodiments, this step is carried out in the presence of a suitable condensation reagent, which can be selected from HATU, HBTU, EDCI, DCC, T3P, POCl3 and HOBT, preferably HATU and T3P;

[0284] In some embodiments, this step is carried out at a suitable temperature, which is 0°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 0-35°C;

[0285] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from halogenated hydrocarbons such as dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane (1,2-DCE), etc.), nitriles (such as acetonitrile (AN), etc.), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), tetrahydrofuran (THF), 1,4-dioxane (Dioxane), dimethyl sulfoxide (DMSO) and any combination thereof, preferably dichloromethane (DCM) and N,N-dimethylformamide (DMF);

[0286] In some embodiments, this step is carried out in the presence of a suitable base, the base comprising an organic base or an inorganic base, the organic base can be selected from N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK) and pyridine (Py), the inorganic base can be selected from 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);

[0287] Step 2: Compound DL-IM-1 undergoes hydrolysis to obtain compound DL-IM-2

[0288] In some embodiments, this step is carried out at a suitable temperature, which is 0°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 0-35°C;

[0289] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from halogenated hydrocarbons such as dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane (1,2-DCE), etc.), nitriles (such as acetonitrile (AN), etc.), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), tetrahydrofuran (THF), 1,4-dioxane (Dioxane), dimethyl sulfoxide (DMSO) and any combination thereof, preferably tetrahydrofuran (THF);

[0290] In some embodiments, this step is carried out in the presence of a suitable Pd reagent, which can be selected from Pd(PPh3)4 and PdCl2(PPh3)2, preferably Pd(PPh3)4;

[0291] In some embodiments, this step is carried out in the presence of a suitable base, which can be selected from diethylamine, pyrrolidine, morpholine, piperidine and piperazine, preferably morpholine;

[0292] Step 3: After the compound DL-IM-2 undergoes esterification reaction with formula II-SM-1, it undergoes substitution reaction with formula II-SM-3 to obtain compound DL-IM-3

[0293] In some embodiments, this step is carried out in the presence of a suitable condensation reagent, which can be selected from HATU, HBTU, EDCI, DCC, T3P, POCl3 and HOBT, preferably HATU;

[0294] In some embodiments, this step is carried out at a suitable temperature, which is 0°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 0-35°C;

[0295] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from halogenated hydrocarbons (such as dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane (1,2-DCE), etc.), nitriles (such as acetonitrile (AN), etc.), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), tetrahydrofuran (THF), 1,4-dioxane (Dioxane), dimethyl sulfoxide (DMSO) and any combination thereof, preferably N,N-dimethylformamide (DMF);

[0296] In some embodiments, this step is carried out in the presence of a suitable base, the base including an organic base or an inorganic base, the organic base can be selected from N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK) and pyridine (Py), the inorganic base can be selected from 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);

[0297] Step 4: After removing the amino protecting group of compound DL-IM-3, compound DL-IM-4 is obtained.

[0298] In some embodiments, this step is carried out at a suitable temperature, which is 0°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 0-35°C;

[0299] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from halogenated hydrocarbons (such as dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane (1,2-DCE), etc.), nitriles (such as acetonitrile (AN), etc.), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), tetrahydrofuran (THF), 1,4-dioxane (Dioxane), dimethyl sulfoxide (DMSO) and any combination thereof, preferably acetonitrile (AN);

[0300] In some embodiments, this step is carried out in the presence of a suitable base, which can be selected from diethylamine, pyrrolidine, morpholine, piperidine and piperazine, preferably morpholine and diethylamine;

[0301] Step 5: Compound DL-SM-4 and GM-OH undergo condensation reaction to obtain a compound of formula GM-[LED]x

[0302] In some embodiments, this step is carried out in the presence of a suitable condensation reagent, which can be selected from HATU, HBTU, EDCI, DCC, T3P, POCl3 and HOBT, preferably HATU;

[0303] In some embodiments, this step is carried out at a suitable temperature, which is 0°C, 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 140°C, preferably 0-35°C;

[0304] In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from halogenated hydrocarbons such as dichloromethane (DCM), chloroform (TCM), 1,2-dichloroethane (1,2-DCE), etc.), nitriles (such as acetonitrile (AN), etc.), N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), tetrahydrofuran (THF), 1,4-dioxane (Dioxane), dimethyl sulfoxide (DMSO) and any combination thereof, preferably N,N-dimethylformamide (DMF);

[0305] In some embodiments, this step is carried out in the presence of a suitable base, which includes an organic base or an inorganic base. The organic base can be selected from N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), potassium tert-butoxide (t-BuOK) and pyridine (Py); the inorganic base can be selected from 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).

[0306] Preparation of Antibody-Drug Conjugates

[0307] On the other hand, the present application provides a method for preparing the antibody-drug conjugate of the present invention, which is selected from:

[0308] Coupling method A:

[0309] In a buffer solution at pH 7.0 to 9.0, the antibody interchain disulfide bonds are reduced using a reducing agent such as tris(2-carboxyethyl)phosphine, and the drug-linker compound of the present invention is coupled to the antibody at a molar ratio of 8 to 10:1 to obtain an antibody-drug conjugate.

[0310] Coupling method B:

[0311] In a buffer solution at pH 7.0-9.0, the antibody interchain disulfide bonds are reduced using a reducing agent such as tris(2-carboxyethyl)phosphine, and the drug-linker compound of the present invention is coupled to the antibody at a molar ratio of 4-6:1 to obtain an antibody-drug conjugate.

[0312] Coupling method C:

[0313] In a buffer solution at pH 7.0-9.0, the antibody interchain disulfide bonds are reduced using a reducing agent such as tris(2-carboxyethyl)phosphine, and the drug-linker compound of the present invention is coupled to the antibody at a molar ratio of 4.0-4.5:1 to obtain an antibody-drug conjugate.

[0314] In some embodiments, the method for preparing the antibody-drug conjugate of the present invention is selected from the group consisting of:

[0315] Coupling method A: (applicable to the monopyrimidine linker series, target DAR8)

[0316] Approximately 0.5 mL of antibody (3-10 mg / mL) was diluted with 0.1 M disodium edetate solution (pH 7.60). The pH was then adjusted to 7.60 with 1 M Na₂HPO₄ solution. 5.5 equivalents of 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution (pH 7.60) was added, mixed, and allowed to stand at room temperature for 1.5 hours. To this solution was added a drug-linker compound dissolved in DMSO at an amount 8-10 times the amount of the antibody. The solution was then allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the ADC product.

[0317] Coupling method B: (applicable to the bipyrimidine linker series, target DAR4)

[0318] Approximately 0.5 mL of antibody (3-10 mg / mL) was diluted with a 0.1 M disodium edetate solution (pH 7.60), then adjusted to pH 8.0 with a 1 M Na2HPO4 solution. 5.5 equivalents of a 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution (pH 7.60) were added, mixed, 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) was added to the solution at a 4-6-fold amount relative to the antibody. After addition, the solution was allowed to stand at room temperature for 18 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the ADC product.

[0319] Coupling method C: (applicable to bromoacetyl linker series (comparative compounds), target DAR4)

[0320] Approximately 0.5 mL of antibody (3-10 mg / mL) was diluted with a 0.1 M disodium edetate solution (pH 7.60). The pH was then adjusted to 8.0 with a 1 M Na2HPO4 solution. 2.4 equivalents of a 10 mM tris(2-carboxyethyl)phosphine (TCEP) solution (pH 7.60) were added, mixed, and allowed to stand at room temperature for 2 hours. A drug-linker compound dissolved in DMSO (4.0-4.5 times the amount of the antibody) was added to the solution. The mixture was allowed to stand at room temperature for 18 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the ADC product. DETAILED DESCRIPTION

[0321] The present invention will be further described below by describing specific embodiments, but this is not intended to limit the present invention. Those skilled in the art can make various modifications or improvements based on the teachings of the present invention without departing from the basic idea and scope of the present invention.

[0322] The information of the adalimumab sequence involved in the present invention is described as follows:

[0323] The sequence information of Tocilizumab involved in the present invention is described as follows:

[0324] The abbreviations used in this document have the following meanings:

[0325] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0326] Nuclear magnetic resonance (NMR) 1 H NMR measurements were performed using a Bruker 400 MHz nuclear magnetic resonance instrument; the deuterated reagent was hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0327] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the examples are shown below.

[0328] 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.

[0329] Mass spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0330] Preparation Example 1: (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-

[0331] 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)

[0332] Step 1:

[0333] 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 system was stirred at 25 ° C for 2 hours and then filtered. The filter cake was washed with water (30 ml x 3) and then dried in vacuo to obtain the crude product A-1-2 (900 mg, 2.35 mmol). The crude product was used directly in the next step without purification.

[0334] ESI-MS (m / z): 383.1 (M+H) + .

[0335] Step 2:

[0336] A-1-2 (0.9 g, 2.35 mmol) was dissolved in DMF (3 mL), and CDI (763 mg, 4.71 mmol) was added, followed by stirring at 25°C for 2 hours. DMF (7 mL) was added to the reaction solution, which was then cooled to -20°C. Hydrogen sulfide gas (15 PSI) was continuously introduced over 30 minutes, and then the temperature was raised to 25°C and stirred for 2 hours. Water (100 mL) was added to the reaction solution, and a solid precipitated. The solid was filtered and the filter cake was dried under vacuum to obtain crude A-1-3 (1.3 g, 2.11 mmol), which was used directly in the next step without purification.

[0337] ESI-MS (m / z): 399.2 (M+H) + .

[0338] Step 3:

[0339] 3-(tert-Butyloxycarbonylamino)-4-fluoro-benzoic acid (289 mg, 1.13 mmol) was dissolved in DMF (7 mL). HATU (517 mg, 1.36 mmol) and DIEPA (440 mg, 3.40 mmol) were added and stirred at 25°C for 1.5 hours. A-1-3 (700 mg, 1.13 mmol) was then added and the reaction continued for 1.5 hours. Fluoroiodomethane (272 mg, 1.70 mmol) was then added to the reaction solution and the reaction continued for 1 hour. Water (50 mL) was added to the reaction solution, and a solid precipitated. The solid was filtered and the filter cake was dried under vacuum to obtain crude A-1-4 (950 mg, 754 mmol), which was used directly in the next step without purification.

[0340] ESI-MS (m / z): 612.5 (M+H) + .

[0341] Step 4:

[0342] A-1-4 (900 mg, 715 μmol) was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (1.47 g, 12.9 mmol) was added, followed by stirring at 25°C for 1 hour. The reaction solution was directly concentrated to obtain a crude product, which was then purified by HPLC to obtain A-1 (310.39 mg, 532 mmol).

[0343] Chromatographic column: Phenomenex C18 150mm×25mm×10mm

[0344] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate)

[0345] The structural characterization of A-1 is as follows:

[0346] ESI-MS (m / z): 568.5 [M+H] + .

[0347] 1 H NMR (400MHz, DMSO) δ7.24-7.37(m,2H),7.15(dd,J=11.2,8.4Hz,1H),7.05(ddd,J=8.4,4.4,2.1H z,1H),6.33(dd,J=10.0,1.8Hz,1H),6.12(s,1H),5.90-6.02(m,1H),5.78-5.88(m,1H),5.57-5.7 4(m,2H),5.53(s,1H),4.32(s,1H),2.86-2.94(m,1H),2.53-2.75(m,2H),2.25-2.38(m,2H),2.01 -2.17(m,2H),1.94(d,J=13.6Hz,1.2H),1.69-1.82(m,1H),1.43-1.59(m,5H),0.88-1.00(m,3H).

[0348] 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)

[0349] Step 1:

[0350] P-1-1 (10 g, 24.3 mmol) was dissolved in THF (135 mL) and water (75 mL), followed by the addition of periodic acid (16.6 g, 73.0 mmol, 16.6 mL). The reaction mixture was stirred at 25°C for 2 hours, and the THF was concentrated under reduced pressure. The filter cake was washed with water (100 mL x 3) and then dried under vacuum to afford crude P-1-2 (9.00 g, 22.7 mmol, 93.18% yield). The crude product was used in the next step without further purification.

[0351] ESI-MS (m / z): 397.0 (M+H) + .

[0352] Step 2:

[0353] P-1-2 (9.00 g, 22.7 mmol) was dissolved in DMF (100 mL), and CDI (7.36 g, 45.41 mmol) was added. The mixture was stirred at 25°C for 4 hours. Hydrogen sulfide gas (15 psi) was continuously introduced into the reaction mixture for 30 minutes, followed by stirring for another 3 hours. The pH of the reaction mixture was adjusted to 2-3 with 1N aqueous hydrochloric acid. A solid precipitated, which was filtered and the filter cake was dried under vacuum to yield P-1-3 (8.5 g, 20.61 mmol).

[0354] ESI-MS (m / z): 413.2 (M+H) + .

[0355] Step 3:

[0356] 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 DIEPA (141 mg, 1.09 mmol) were added, and the mixture was stirred at 25°C for 0.5 h. Fluoroiodomethane (174 mg, 1.09 mmol) and DIEPA (141 mg, 1.09 mmol) were then added to the reaction mixture, and stirring was continued for 2 h. The reaction mixture was filtered and concentrated to afford crude P-1-4 (350 mg), which was used in the next step without purification.

[0357] ESI-MS (m / z): 595.5 (M+H) + .

[0358] Step 4:

[0359] P-1-4 (350 mg, 588 μmol) was added to EtOH (5.00 mL) and water (5.00 mL) at 25°C, followed by NH4Cl (251 mg, 4.71 mmol) and iron powder (263 mg, 4.71 mmol). After the addition, the reaction mixture was heated to 80°C for 6 hours. The reaction mixture was filtered and concentrated to obtain the crude product, which was purified by HPLC to afford P-1 (0.169 g, 284 μmol).

[0360] Chromatographic column: Phenomenex C18 250mm×50mm×10μm

[0361] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate)

[0362] The structural characterization of P-1 is as follows:

[0363] ESI-MS (m / z): 565.5 [M+H] + .

[0364] 1 H NMR (400MHz, DMSO) δ8.1-8.3(d,J=5.2Hz,1H),7.1-7.2(d,J=10Hz,1H),7.0-7.1(d d,J=5.2Hz,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=7.2Hz,3H).

[0365] 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)

[0366] Step 1:

[0367] Methyl 4-bromofuran-2-carboxylate (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). 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) were then added sequentially. The reaction mixture was heated to 130°C and stirred for 18 hours. Water (200 mL) and dichloromethane (100 mL x 3) were added to the reaction mixture. The organic phases were combined, dried, filtered, and concentrated to obtain the crude product, which was purified by flash column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 5 / 1) to afford F-2-2 (700 mg, 2.90 mmol).

[0368] ESI-MS (m / z): 242.0 (M+H) + .

[0369] Step 2:

[0370] 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 reaction was allowed to react at 25°C for 1 hour. Water and 1N aqueous hydrochloric acid (10.0 mL) were added to the reaction solution, which was then extracted with dichloromethane (20.0 mL x 3). The combined organic phases were dried, filtered, and concentrated to afford F-2-3 (350 mg, 1.54 mmol).

[0371] ESI-MS (m / z): 228.1 (M+H) + .

[0372] Step 3:

[0373] F-2-3 (100 mg, 440 μmol) and HATU (200 mg, 528 μmol) were added to DMF (10.0 mL), followed by DIPEA (284 mg, 2.20 mmol, 383 μL). The reaction mixture was allowed to react at 25°C for 2 hours, followed by the addition of P-1-3 (181 mg, 440 μmol). Stirring was continued for 2 hours, followed by the addition of fluoroiodomethane (105 mg, 660 μmol), and the reaction was continued at 25°C for 1 hour. Water (100 mL) and ethyl acetate (100 mL x 3) were added to the reaction mixture for extraction. The combined organic phases were dried, filtered, and concentrated to afford crude F-2-4 (220 mg, 336 μmol), which was used directly in the next step without further purification.

[0374] ESI-MS (m / z): 654.2 (M+H)+ .

[0375] Step 4:

[0376] 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 solution was stirred at 25 °C for 1 hour and then concentrated to obtain a crude product. The crude product was purified by HPLC to obtain F-2 (27.9 mg, 45.54 μmol).

[0377] Chromatographic column: Phenomenex C18 150mm×25mm×10μm

[0378] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate)

[0379] The structural characterization of F-2 is as follows:

[0380] ESI-MS (m / z): 554.2 [M+H] + .

[0381] 1 H NMR (400MHz, DMSO) δ7.34 (dd, J=1.2, 10.0Hz, 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=9.6Hz,1H),3.46(dt, J=3.2,6.97Hz,1H),2.54-2.72(m,1H),2.36(dd,J=3.2,11.2Hz,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).

[0382] 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)

[0383] Step 1:

[0384] 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-dioxaborolan-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 atmosphere was purged with nitrogen three times, then heated to 100°C and reacted for 3 hours. The reaction system was filtered through a silica gel pad and extracted with water (80.0 mL) and ethyl acetate (50.0 mL x 3). The organic phases were combined, dried, filtered, and concentrated to obtain a crude product, which was purified by flash column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 3 / 1) and concentrated to obtain C-1-2 (4.20 g, 12.9 mmol).

[0385] ESI-MS (m / z): 292.1 (M+H) + .

[0386] Step 2:

[0387] C-1-2 (3.60 g, 12.3 mmol) was added to DMSO (40.0 mL), followed by trimethylsulfoxide iodide (3.26 g, 14.8 mmol) and potassium tert-butoxide (1.53 g, 13.5 mmol). The reaction system was stirred at 25°C for 2 hours, then extracted with water (50.0 mL) and ethyl acetate (50.0 mL x 3). The organic phases were combined, dried, filtered, and concentrated to obtain the crude product, which was purified by flash column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 3 / 1) and concentrated to obtain C-1-3 (400 mg, 1.31 mmol).

[0388] ESI-MS (m / z): 306.0 (M+H) + .

[0389] Step 3:

[0390] 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 lithium hydroxide (62.7 mg, 2.62 mmol). The reaction system was reacted at 25°C for 1 hour. The pH of the reaction solution was adjusted to 2-3 with 1N aqueous hydrochloric acid. A solid precipitated and was filtered. The filter cake was vacuum dried to obtain C-1-4 (300 mg, 1.08 mmol), which was used directly in the next step without purification.

[0391] ESI-MS (m / z): 278.1 (M+H) +.

[0392] 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)

[0393] 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 system was stirred at 25°C for 1 hour before P-1-3 (356 mg, 865 μmol) was added and stirring continued for 2 hours. Fluoroiodomethane (173 mg, 1.08 mmol) was then added and the reaction continued for 1 hour. The reaction system was extracted with water (10.0 mL) and dichloromethane (10.0 mL x 3). The combined organic phases were dried, filtered, and concentrated to afford C-1-5 (150 mg, 213 μmol), which was used directly in the next step without purification.

[0394] ESI-MS (m / z): 704.3 (M+H) + .

[0395] Step 5:

[0396] 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 system was stirred at 25°C for 1 hour, then concentrated to obtain the crude product, which was purified by HPLC to afford C-1 (11.89 mg, 18.4 μmol).

[0397] Chromatographic column: Phenomenex C18 150mm×25mm×10μm

[0398] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate)

[0399] The structural characterization of C-1 is as follows:

[0400] ESI-MS (m / z): 604.2 [M+H] + .

[0401] 1H NMR (400MHz, DMSO) δ7.32 (dd, J=6.0, 9.57Hz, 1H), 6.78-6.96 (m, 2H), 6.65 (dd, J=1.6, 8.4Hz, 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=10.0Hz,1H),3.41(d,J=4.0 0Hz,2H),2.52-2.66(m,1H),2.19-2.41(m,4H),1.93-2.06(m,2H),1.78(ddd,J=4.8,9.0,13.6Hz,1H),1.5 5-1.69(m,4H),1.41-1.48(m,1H),1.35(tt,J=3.74,7.52Hz,2H),1.12(d,J=1.8Hz,3H),0.92-1.10(m,3H).

[0402] Preparation Example 5: (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-carboxylic acid fluoromethyl ester (O-1)

[0403] Step 1:

[0404] The 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 reacted at 25°C for 15 minutes. DIPEA (146.71 mg, 1.14 mmol) was added to the reaction system, followed by 3-(tert-butoxycarbonylamino)-4-fluoro-benzoic acid (300 mg, 756.77 μmol). After the addition was complete, the reaction was continued at 25°C for 16 hours. The reaction was monitored by LC-MS, and iodomethane (604.49 mg, 3.78 mmol) was added to the reaction system. The reaction was carried out at 25°C for 4 h and monitored by LC-MS. Water was added to the reaction system to precipitate a light yellow solid. The solid was collected by filtration to obtain a crude product, which was purified by column chromatography (ethyl acetate / petroleum ether = 0-35%) to obtain O-1-1 (390 mg).

[0405] ESI-MS (m / z): 610.1 [M+1-56] + .

[0406] Step 2:

[0407] Compound O-1-2 (70 mg, 105.16 μmol) was added to DCM (3 mL) and TFA (1 mL) and reacted at 25°C for 1.5 hours. The reaction was monitored by LC-MS until completion. The reaction system was concentrated to dryness to obtain the crude product, which was purified by preparative HPLC to afford O-1 (23.8 mg).

[0408] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0409] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0410] The structure of O-1 is characterized as follows:

[0411] ESI-MS (m / z): 566.3 [M+1] + .

[0412] 1 H NMR (400MHz, DMSO-d6): δ7.34-7.24(m,2H),7.16(dd,J=11.2Hz,8.4Hz,1H),7.07–7.01(m,1H) ,6.33(dd,J=10.0Hz,2.0Hz,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=7.2Hz,3H).

[0413] Preparation Example 6: (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-carboxylic acid chloromethyl ester (O-3)

[0414] Step 1:

[0415] 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 (7.5 mL) and reacted at 25°C for 15 minutes. DIPEA (73.35 mg, 567.57 μmol) and 3-(tert-butoxycarbonylamino)-4-fluorobenzoic acid (150 mg, 378.38 μmol) were then added. The mixture was allowed to react at 25°C for 3 hours. The reaction was monitored by LC-MS. Chloroiodomethane (668.05 mg, 3.78 mmol) was then added to the reaction system. The reaction was allowed to react at 45°C for 20 hours, also monitored by LC-MS. The reaction system was extracted with water and dichloromethane. The organic phase was dried and concentrated to yield 230 mg of crude O-3-1, which was used directly in the next step.

[0416] Step 2:

[0417] Compound O-3-1 (150 mg, 105.16 μmol) was added to DCM (4 mL) and TFA (2 mL) and reacted at 25°C for 1.5 hours. The reaction was monitored by LC-MS. The reaction system was concentrated to dryness to obtain a crude product, which was purified by HPLC to obtain O-3 (20 mg).

[0418] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0419] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0420] The O-3 structure is characterized as follows:

[0421] ESI-MS (m / z): 582.2 [M+1] + .

[0422] 1H NMR (400MHz, DMSO-d6): δ7.33-7.26(m,2H),7.16(dd,J=11.2Hz,8.8Hz,1H),7.05–6.99(m,1H),6.33( dd,J=10.0Hz,2.0Hz,1H),6.14(s,1H),5.96(d,J=6.0Hz,1H),5.86(d,J=6.0Hz,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),1.7 8–1.70(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).

[0423] 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)

[0424] Step 1:

[0425] 4-Bromo-1-fluoro-2-nitrobenzene (1.00 g, 4.55 mmol), ethyl (E)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-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). The atmosphere was purged with nitrogen three times, then the temperature was raised to 100°C and the reaction was allowed to react for 2 hours. Water (100 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate three times (60 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. After purification by flash column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 5 / 1), the residue was concentrated again to give ethyl (E)-3-(4-fluoro-3-nitrophenyl)acrylate (450 mg, 1.76 mmol).

[0426] ESI-MS (m / z): 240.1 (M+H) + .

[0427] Step 2:

[0428] 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 added and stirred at room temperature for 2 hours. The reaction solution was adjusted to a pH of 2-3 with 1N dilute hydrochloric acid. A solid precipitated and was filtered. The filter cake was vacuum dried to yield (E)-3-(4-fluoro-3-nitrophenyl)acrylic acid (305 mg, 895 μmol).

[0429] ESI-MS (m / z): 212.0 (M+H) + .

[0430] Step 3:

[0431] (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, 50% purity) were added. The 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-carboxylic acid (527.46 mg, 1.28 mmol) was added to the above reaction solution and stirring was continued for 2 hours. Then, iodomethane (224 mg, 1.41 mmol) was added and stirring was continued for 0.5 hour. Water (100 mL) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (60 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product, which was purified by flash column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 1 / 1) and concentrated again to obtain (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).

[0432] ESI-MS (m / z): 638.2 (M+H) + .

[0433] Step 4:

[0434] (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). Iron powder (43.79 mg, 784 μmol) and ammonium chloride (20.9 mg, 392 μmol) were added, the temperature was raised to 80°C, and the mixture was stirred for 2 hours. Water (50 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (40 mL x 3). The organic phases were combined and dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product, which was purified by HPLC 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).

[0435] Chromatographic column: Phenomenex luna C18 150mm×25mm×10μm

[0436] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0437] Its structural characterization is as follows:

[0438] ESI-MS (m / z): 608.2 [M+H] + .

[0439] 1H NMR (400MHz, DMSO) δ7.50(d,J=16.0Hz,1H),7.35(dd,J=10.4,1.2Hz,1H),7.07(dd,J=8.4,2.4Hz,1H),6.9 7(dd,J=10.8,8.4Hz,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=7.2Hz,3H).

[0440] 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-6,7,8,9,1 0,11,12,13,14,15,16,17-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-triazaheptatriacontane-37-ynoic acid (DL-B-11')

[0441] Step 1:

[0442] The raw 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 system was stirred at 90°C under a nitrogen atmosphere for 3 hours. The reaction was monitored by LC-MS, filtered through celite, and water and ethyl acetate were added to the filtrate. The mixture was extracted and concentrated to give a crude product, which was purified by column chromatography (EA / PE = 0-25%) to give 710 mg of methyl 3,5-di(2-(methylthio)pyrimidin-5-yl)benzoate.

[0443] Its structural characterization data are as follows:

[0444] ESI-MS (m / z): 385.1 [M+H] + .

[0445] Step 2:

[0446] 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 H₂O (2 mL). The reaction was stirred at 25°C for 2 hours and monitored by LC-MS. The pH of the system was adjusted to approximately 2 with 1N HCl, resulting in the precipitation of a large amount of solid. The filter cake was collected by filtration and dried to yield 560 mg of 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoic acid.

[0447] Its structural characterization data are as follows:

[0448] ESI-MS (m / z): 371.1 [M+H] + .

[0449] Step 3:

[0450] 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-ate (4.03 g, 8.10 mmol) were added to DMF (40 mL), and 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) were added in sequence, and the reaction system was stirred at 60 ° C for 2 hours. Water (100 mL) and ethyl acetate (60 mL x 3) were added to the reaction solution, and 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-oate (4.20 g, 4.14 mmol), which was used in the next step without purification.

[0451] Step 4:

[0452] Dissolve tert-butyl 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosadecane-29-oate (3.60 g, 4.24 mmol) in dichloromethane (30 mL). Add TFA (15.3 g, 134 mmol, 10 mL). Stir the reaction system at 25°C for 6 hours. Add water (60 mL) and extract with ethyl acetate (40 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by preparative high performance liquid chromatography and freeze-dried 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-oic acid (2.93 g, 3.63 mmol).

[0453] Its structural characterization data are as follows:

[0454] ESI-MS (m / z): 794.3 [M+H] + .

[0455] The purification method is as follows:

[0456] Chromatographic column: Phenomenex luna C18 (250mm*70mm*10mm)

[0457] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0458] Step 5:

[0459] 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosanetan-29-oic 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 were added. The hydrate (15.47 mg, 74.56 μmol) was added to the reaction system, stirred at 25 ° C for 30 minutes, and the reaction system 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-oic acid (155 mg).

[0460] Its structural characterization data are as follows:

[0461] ESI-MS (m / z): 858.3 [M+H] +.

[0462] Step 6:

[0463] To DCM (1 mL) were added (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-(tert-butoxy)-5-oxopentanoic acid (764 mg, 1.58 mmol), allyl 3-amino-4-fluorobenzoate (324.50 mg, 1.66 mmol), pyridine (375.73 mg, 4.75 mmol), and a 50% solution of 1-propylphosphonic anhydride in DMF (5.57 g) in sequence. After addition, the mixture was stirred at 25°C for 3 h. Completion of the reaction was monitored by TLC (PE:EA = 0:1). Water (12 mL) was added to the reaction solution, which was then extracted with DCM (15 mL x 2). The organic phase 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 (SiO2, CHCl2 / MeOH = 100 / 1 to 10 / 1). To obtain (S)-3-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5-oxopentamide)-4-fluorobenzoic acid allyl ester (814 mg, 1.14 mmol).

[0464] Its structural characterization data are as follows:

[0465] ESI-MS (m / z): 659.70 (M+H) + .

[0466] Step 7:

[0467] To THF (7 mL) were added (S)-3-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5-oxopentamide)-4-fluorobenzoic acid allyl ester (734 mg, 1.11 mmol), morpholine (193.86 mg, 2.23 mmol), and Pd(PPh3)4 (64.29 mg, 55.63 μmol) in sequence. After the addition was complete, the reaction was stirred at 25°C for 2 h. Water (26 mL) was added to the reaction solution, and the 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 dryness. PE (8 mL) was added and the mixture was slurried at room temperature for 1 h. The mixture was filtered and the filter cake was dried under reduced pressure to give (S)-3-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5-oxopentamide)-4-fluorobenzoic acid (450 mg, 697.18 μmol).

[0468] Its structural characterization data are as follows:

[0469] ESI-MS (m / z): 620 (M+H) + .

[0470] Step 8:

[0471] To DMF (4 mL) were added (S)-3-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)-5-(tert-butoxy)-5-oxopentanamide)-4-fluorobenzoic acid (200 mg, 322.77 μmol), DIPEA (83.43 mg, 645.54 μmol), and HATU (147.73 mg, 387.32 μmol) in sequence. After addition, the mixture was stirred at 16°C for 35 min. To the reaction solution was added A-1-3 (128.86 mg, 320.28 μmol). After addition, the mixture was stirred at 20°C for 1 h. To the above reaction solution were added DIPEA (31.02 mg, 239.98 μmol) and iodomethane (211.09 mg, 1.32 mmol). After addition, the mixture was stirred at 20°C for 1 h. Water (12 mL) was added to the reaction solution, and EA (15 mL*2) was added for extraction. The organic phase was washed with water (8 mL) and saturated brine (8 mL), respectively. The liquids were separated, and the organic phase was concentrated to dryness under reduced pressure. It was purified by column chromatography (SiO2, PE / EA=12 / 1 to 1 / 1) to obtain (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-1 1-Hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl-3-((S)-2-((((9H-fluoro-9-yl)methoxy)carbonylamino)acetamido)-5-(tert-butoxy)-5-oxopentanamido)-4-fluorobenzoate (88 mg, 80.15 μmol).

[0472] Its structural characterization data are as follows:

[0473] ESI-MS (m / z): 1032 (M+H) + .

[0474] Step 9:

[0475] To MeCN (4 mL) 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-((((9H-fluoro-9-yl)methoxy)carbonylamino)acetamido)-5-(tert-butoxy)-5-oxopentanamido)-4-fluorobenzoate (88 mg, 80.15 μmol) and diethylamine (31.18 mg, 426.32 μmol) in sequence. After the addition was complete, the reaction was stirred at 25°C for 2 h. The reaction solution 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]phenanthrene-17-yl-3-(((S)-2-(2-aminoacetamide)-5-(tert-butoxy)-5-oxopentanamide)-4-fluorobenzoate (69 mg, 80.94 μmol), which was used in the next step without purification.

[0476] Its structural characterization data are as follows:

[0477] ESI-MS (m / z): 810 (M+H) + .

[0478] Step 10:

[0479] To DCM (3 mL) was added crude (69 mg, 80.94 μmol) 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-(((S)-2-(2-aminoacetamide)-5-(tert-butoxy)-5-oxopentanamide)-4-fluorobenzoate, and TFA (0.5 mL) was added. After the addition was complete, the mixture was stirred at 25°C for 2 h. The reaction mixture was stirred for 3 h. DCM and TFA were removed from the reaction mixture under reduced pressure to give (S)-4-(2-aminoacetamide)-5-(5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-5-oxopentanoic acid (88 mg, 83.15 μmol), which was used in the next step without purification.

[0480] Its structural characterization data are as follows:

[0481] ESI-MS (m / z): 754 (M+H) + .

[0482] Step 11:

[0483] To DMF (2 mL) were added (S)-4-(2-aminoacetamide)-5-(5-((((6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)oxy)carbonyl)-2-fluorophenyl)amino The crude product of 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid (23.73 mg, 27.66 μmol) was added, and the mixture was stirred at 15°C for 1 h. The reaction solution was directly purified by HPLC to obtain the title compound (4.55 mg, 2.77 μmol).

[0484] The purification method is as follows:

[0485] Chromatographic column: SunFire Prep C18 OBD 19*150mm*5μm

[0486] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0487] Its structural characterization data are as follows:

[0488] ESI-MS (m / z): 1594 (M+H) + .

[0489] 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]phenanthrene-17-yl)oxy)carbonyl)-2-fluorophenyl)amino)-4-(2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)acetamido)-5-oxopentanoic acid (DL-A-05)

[0490] Step 1:

[0491] To DMF (2 mL) 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 -(2-aminoacetamide)-5-(tert-butoxy)-5-oxopentylamide)-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) and DIPEA (21.38 mg, 165.46 μmol) were added and the reaction was stirred at 15 °C for 1 h. Purified water (10 mL) and ethyl acetate (6 mL*2) were added to the reaction solution for extraction. The mixture was separated, dried over anhydrous sodium sulfate (3 g), filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (SiO2, PE / EA=100 / 1 to 1 / 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)-5-(tert-butoxy)-2-(2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)acetamido)-5-oxopentanamido)-4-fluorobenzoate (64 mg, 54.33 μmol).

[0492] Step 2:

[0493] To DCM (2 mL) 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)-5-(tert-Butoxy)-2-(2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)acetamido)-5-oxopentanamido)-4-fluorobenzoate (64 mg, 54.33 μmol) and TFA (1 mL) were added and reacted at 16°C for 1.5 h. The reaction solution was purified by HPLC to obtain the title compound (42.00 mg, 41.41 μmol).

[0494] The purification method is as follows:

[0495] Chromatographic column: SunFire Prep C18 OBD 19*150mm*5μm

[0496] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% TFA)

[0497] Its structural characterization data are as follows:

[0498] ESI-MS (m / z): 1004.3 (M+H) + .

[0499] 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-((34 S,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-tetraazatriacont-38-amino)-4-fluorobenzoate (DL-B-07')

[0500] Step 1:

[0501] Allyl 3-amino-4-fluorobenzoate (5.00 g, 25.6 mmol) and N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(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, 50.0% purity) and DIPEA (9.93 g, 76.8 mmol, 13.3 mL) were added and stirred at 25 °C for 2 hours. Water (200 mL) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (100 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product, which was purified on a silica gel column (SiO2, CH2Cl2 / MeOH = 100 / 1 to 10 / 1) and concentrated again to obtain DL-B-07'-2 (10.5 g, 16.2 mmol).

[0502] Its structural characterization data are as follows:

[0503] ESI-MS (m / z): 668.4 [M+Na] + .

[0504] Step 2:

[0505] DL-B-07'-2 (10.0 g, 15.4 mmol) was dissolved in tetrahydrofuran (100 mL). Morpholine (2.70 g, 30.9 mmol, 2.73 mL) and Pd(PPh3)4 (894 mg, 774 μmol) were added and stirred at 25°C for 1 hour. The reaction mixture was adjusted to a pH of 2-3 with 1N dilute hydrochloric acid, diluted with water (500 mL), and extracted three times with dichloromethane (500 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The mixture was stirred at 25°C for 2 hours with petroleum ether / ethyl acetate (5 / 1), then filtered and dried to obtain DL-B-07'-3 (8.00 g, 13.2 mmol).

[0506] Its structural characterization data are as follows:

[0507] ESI-MS (m / z): 623.2 [M+H] + .

[0508] Step 3:

[0509] DL-B-07'-3 (5.00 g, 8.26 mmol) was dissolved in DMF (100 mL), and HATU (3.14 g, 8.26 mmol) and DIPEA (3.20 g, 24.7 mmol, 4.31 mL) were added. After stirring at 25°C for 2 hours, (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-11,17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,11,12,14,15,16-octahydrocyclopenta[a]phenanthrene-17-carboxylic acid (3.29 g, 8.26 mmol) was added to the reaction mixture, and stirring was continued for 2 hours. Then, iodomethane (1.32 g, 8.26 mmol) was added to the reaction mixture, and the reaction was continued for 2 hours. Water (200 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (100 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product, which was purified on a silica gel column (SiO2, CH2Cl2 / MeOH = 100 / 1 to 10 / 1) and concentrated again to obtain DL-B-07'-4 (2.60 g, 2.55 mmol).

[0510] Its structural characterization data are as follows:

[0511] ESI-MS (m / z): 1018.1 [M+H] + .

[0512] Step 4:

[0513] 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, followed by stirring at 25°C for 1 hour. The reaction solution was used directly in the next step without further treatment.

[0514] Step 5:

[0515] To the reaction mixture from the previous step, (((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 sequentially, and the mixture was stirred at 25°C for 1 hour. Water (200 mL) was then added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (100 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified on a silica gel column (SiO2, CH2Cl2 / MeOH = 100 / 1 to 10 / 1) and concentrated again to obtain DL-B-07'-6 (2.00 g, 1.81 mmol).

[0516] Its structural characterization data are as follows:

[0517] ESI-MS (m / z): 1105.5 [M+H] + .

[0518] Step 6:

[0519] 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, followed by stirring at 25°C for 1 hour. The reaction solution was used directly in the next step without further treatment.

[0520] Step 7:

[0521] To the reaction mixture from the previous step were added (((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), followed by stirring at 25°C for 1 hour. Water (200 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (50.0 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified on a silica gel column (SiO2, CH2Cl2 / MeOH = 100 / 1 to 10 / 1) and concentrated again to afford DL-B-07'-8 (903 mg, 700 μmol).

[0522] Its structural characterization data are as follows:

[0523] ESI-MS (m / z): 1162.5 [M+H] + .

[0524] Step 8:

[0525] DL-B-07'-8 (500 mg, 0.43 mmol) was dissolved in DMF (5 mL), diethylamine (125.8 mg, 1.72 mmol) was added, and the reaction was carried out at 25 ° C for 1 hour. Most of the DMF was concentrated, and the crude product was purified by C18 reverse column (H2O / ACN = 10-60%, 0.1% formic acid) and lyophilized to obtain the formate salt of DL-B-07'-9 (325 mg).

[0526] Its structural characterization data are as follows:

[0527] ESI-MS (m / z): 940.4 [M+H] + .

[0528] Step 9:

[0529] 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 DL-B-07'-9 formate (30 mg, 0.030 mmol) were added in sequence. The reaction was carried out at 25 ° C for 1 hour. The reaction solution was directly purified by Pre-HPLC to obtain DL-B-07'-10 (21 mg).

[0530] The purification method is as follows:

[0531] Chromatographic column: SunFire Prep C18 OBD 19*150mm*5μm

[0532] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0533] Retention time: 7.00~9.00min.

[0534] Its structural characterization data are as follows:

[0535] ESI-MS (m / z): 1779.5 (M+H) + .

[0536] Step 10:

[0537] 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 mixture was reacted at 25°C for 2 hours, and concentrated to obtain a crude product. The crude product was purified by Pre-HPLC to obtain the trifluoroacetate salt of DL-B-07' (14.0 mg).

[0538] The purification method is as follows:

[0539] Chromatographic column: SunFire Prep C18 OBD 19*150mm*5μm

[0540] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0541] Retention time: 10.5~12.5min.

[0542] Its structural characterization data are as follows:

[0543] ESI-MS (m / z): 1679.6 (M+H) + .

[0544] 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-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)acetamido)propionamido)-4-fluorobenzoate (DL-A-01)

[0545] Step 1:

[0546] The formate salt of 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 reacted at 25°C for 2 hours. The reaction solution was directly purified by Pre-HPLC and lyophilized to obtain DL-A-01-1 (22.0 mg).

[0547] The purification method is as follows:

[0548] Chromatographic column: SunFire Prep C18 OBD 19*150mm*5μm

[0549] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0550] Retention time: 7.00~9.00min.

[0551] Its structural characterization data are as follows:

[0552] ESI-MS (m / z): 1190.5 (M+H) + .

[0553] Step 2:

[0554] DL-A-01-1 (22.0 mg, 0.018 mmol) was dissolved in DCM (1 mL), and TFA (105.4 mg, 0.924 mmol) was added. The mixture was allowed to react at 25°C for 1 hour. The reaction solution was directly concentrated to obtain the crude product, which was purified by Pre-HPLC to obtain the trifluoroacetic acid salt of DL-A-01 (14.6 mg).

[0555] The purification method is as follows:

[0556] Chromatographic column: SunFire Prep C18 OBD 19*150mm*5μm

[0557] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0558] Retention time: 10.50~12.50min.

[0559] Its structural characterization data are as follows:

[0560] ESI-MS (m / z): 1090.4 (M+H) + .

[0561] 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]phenanthren-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')

[0562] Step 1:

[0563] Allyl 3-amino-4-fluorobenzoate (3.30 g, 16.91 mmol) and (tert-butoxycarbonyl)-L-alanyl-L-alanine (4.40 g, 16.91 mmol) were dissolved in DMF (40.00 mL), followed by the addition of T3P (23.67 g, 37.19 mmol, 50.0% purity) and pyridine (5.35 g, 67.63 mmol). The reaction mixture was stirred at 25°C for 16 hours. After completion, the reaction was monitored by LC-MS. Water and ethyl acetate were added for extraction, and the crude product was concentrated to afford the product. Purification by column chromatography (MeOH / DCM = 0-10%) afforded DL-B-09'-1 (3.40 g, 7.77 mmol).

[0564] Its structural characterization is as follows:

[0565] ESI-MS (m / z): 382.2 (M+H-56) + .

[0566] Step 2:

[0567] Under nitrogen, DL-B-09'-1 (3.39 g, 7.75 mmol) was dissolved in THF (100.00 mL), followed by the addition of tetrakistriphenylphosphine palladium (895.47 mg, 774.93 μmol) and morpholine (1.35 g, 15.50 mmol). The reaction mixture was stirred at 25°C for 16 hours. LC-MS monitored the reaction for completion. The pH was adjusted to approximately 8 with sodium bicarbonate and extracted with ethyl acetate to remove impurities. The aqueous phase was then adjusted to approximately pH 3 with dilute hydrochloric acid. The product was extracted with ethyl acetate, dried, and concentrated to yield DL-B-09'-2 (2.40 g, 6.04 mmol) as a white solid.

[0568] Its structural characterization is as follows:

[0569] ESI-MS (m / z): 342.2 (M+H-56) + .

[0570] Step 3:

[0571] 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). The reaction solution was stirred at 25 °C for 30 min, and then DIPEA (146.35 mg, 1.13 mmol) and (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-11,17-dihydroxy-10,13-dimethyl-3- -Oxo-6,7,8,11,12,14,15,16-octahydrocyclopenta[a]phenanthrene-17-carboxylic acid (300.80 mg, 754.91 μmol) was stirred for 1 hour, and then iodomethane (482.93 mg, 3.02 mmol) was added to the reaction system. The reaction solution was stirred at 25 ° C for 2 hours. The reaction was monitored by LC-MS. Water and ethyl acetate were added for extraction, and the crude product was concentrated to obtain the crude product, which was purified by column chromatography (ACN / H2O = 0-60%, 0.05% formic acid) and lyophilized to obtain the crude product DL-B-09'-3 (199 mg).

[0572] Its structural characterization is as follows:

[0573] ESI-MS (m / z): 810.3 (M+H) + .

[0574] Step 4:

[0575] DL-B-09'-3 (255 mg, 316.42 mmol) was added to DCM (8.00 mL), followed by TFA (4.00 mL). The reaction solution was stirred at 25°C for 1 hour. The reaction was completed as monitored by LC-MS. The reaction system was concentrated to obtain the crude product, which was purified by HPLC to obtain DL-B-09'-4 (100 mg, 121.39 μmol) as a white solid.

[0576] The purification method is as follows:

[0577] Chromatographic column: Phenomenex C18 250mm×50mm×10μm

[0578] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% TFA)

[0579] Its structural characterization is as follows:

[0580] ESI-MS (m / z): 710.3 (M+H) + .

[0581] Step 5:

[0582] 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 reacted at 25°C for 1 hour. The reaction mixture was filtered to obtain a crude product, which was purified by HPLC to obtain DL-B-09' (4 mg, 2.56 μmol).

[0583] The purification method is as follows:

[0584] Chromatographic column: Phenomenex C18 250mm×50mm×10μm

[0585] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0586] Its structural characterization is as follows:

[0587] ESI-MS (m / z): 1549.5 (M+H) + .

[0588] 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 Preparation of phenanthren-17-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-triazapentatriacontanoate-35-amino)isonicotinate (DL-B-17′)

[0589] Step 1:

[0590] 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-carboxylic acid (1.23 g, 2.97 mmol) were dissolved in DMF (10.00 mL), and T3P (10.00 g, 15.7 mmol, 50.0% purity) and DIPEA (1.15 g, 8.92 mmol) were added. The reaction solution was stirred at 25°C for 1 hour, and then iodomethane (475 mg, 2.97 mmol) was added to the reaction system. The reaction solution was stirred at 25°C for 1 hour. The reaction was monitored by LC-MS. Water and ethyl acetate were added for extraction, and the mixture was dried over sodium sulfate to obtain a crude yellow oil (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 2-nitroisonicotinate (2.1 g). The crude product was used in the next step without purification.

[0591] Its structural characterization is as follows:

[0592] ESI-MS (m / z): 595.2 (M+H) + .

[0593] Step 2:

[0594] (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 system was heated to 80° C. and stirred for 2 hours. The reaction solution was filtered through a pad of celite, and the filtrate was concentrated to obtain a crude product. The crude product 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).

[0595] Its structural characterization is as follows:

[0596] ESI-MS (m / z): 565.1 (M+H) + .

[0597] 1 H NMR: (400MHz, CD3OD) δ8.04 (d, J=5.6Hz, 1H), 7.29-7.39 (m, 1H), 7.00 (s, 1H), 6.92-6.96 (m, 1H) ),6.36(dd,J=10.0,1.6Hz,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=7.2Hz,3H).

[0598] Step 3:

[0599] (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), and phosphorus oxychloride (2.17 g, 14.1 mmol) was added. After maintaining the system at 0 ° C. and stirring for 1 hour, water and DCM were added for extraction. The organic phase was dried over sodium sulfate, filtered 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]phenanthrene-17-yl-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionylamino)isonicotinate crude product (1.5 g) which was used in the next step without purification.

[0600] Its structural characterization is as follows:

[0601] ESI-MS (m / z): 858.3 (M+H) + .

[0602] Step 4:

[0603] (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)propionylamino)isonicotinate (1.50 g, 1.75 mmol) was added to acetonitrile (10.00 mL) followed by diethylamine (2.13 g, 29.1 mmol). The reaction solution was stirred at 25°C for 1 hour and then concentrated to give a crude product (1.5 g) 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]phenanthrene-17-yl-2-((S)-2-aminopropionylamino)isonicotinate, which was used in the next step without purification.

[0604] Its structural characterization is as follows:

[0605] ESI-MS (m / z): 636.1 (M+H) + .

[0606] Step 5:

[0607] (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-Aminopropionylamino)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 DIPEA (792 mg, 6.14 mmol, 1.07 mL) and T3P (6.84 g, 10.7 mmol, 6.40 mL, 50% purity). After stirring at 25°C for 1 hour, water and ethyl acetate were added to the reaction solution for extraction. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by column chromatography (petroleum ether / ethyl acetate = 0-40%) 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-9-yl)methoxy)carbonylamino)propionamido)propionamide)isonicotinate (264 mg, 252 μmol).

[0608] Its structural characterization is as follows:

[0609] ESI-MS (m / z): 929.5 (M+H) + .

[0610] Step 6:

[0611] (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-9-yl)methoxy)carbonylamino)propionamido)propionamide)isonicotinate (50 mg, 53.82 μmol) was dissolved in DMF (2.00 mL) and diethylamine (19.68 mg, 269.10 μmol) was added. The reaction mixture was stirred at 25°C for 2 hours, and then DMF and diethylamine were concentrated under reduced pressure to give crude (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-aminopropionylamino)propionylamino isonicotinate (38 mg, 53.77 μmol), which was used in the next step without purification.

[0612] Its structural characterization is as follows:

[0613] ESI-MS (m / z): 707.3 (M+H) + .

[0614] Step 7:

[0615] 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-aminopropionylamino)propionylamino isonicotinate (38 mg, 53.77 μmol), 1-(3,5-bis(2-(methylsulfonyl)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-aminopropionylamino)propionylamino isonicotinate (38 mg, 53.77 μmol), To DMF (2.00 mL) was added 1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid (46.13 mg, 53.77 μmol), HATU (40.89 mg, 107.53 μmol), and DIPEA (34.74 mg, 268.83 μmol). 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 HPLC to obtain the title compound (28 mg, 17.74 μmol).

[0616] The preparation and purification conditions are as follows:

[0617] Chromatographic column: Phenomenex C18 250mm×50mm×10μm

[0618] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0619] Its structural characterization is as follows:

[0620] ESI-MS (m / z): 1547.6 [M+H] + .

[0621] 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)propionylamino)isonicotinate (DL-A-08)

[0622] 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-aminopropionylamino)propionylamino isonicotinate (38 To the mixture of 1,4-dioxopyrrolidin-1-yl (5,6-dioxopyrrolidin-1-yl)-6-(2-methylsulfonylpyrimidin-5-yl)hex-5-ynoic acid (19.64 mg, 53.77 μmol), and DIPEA (13.90 mg, 107.53 μmol) were added to DMF (3.00 mL), 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 HPLC to obtain the title compound (21 mg, 21.72 μmol).

[0623] The preparation and purification conditions are as follows:

[0624] Chromatographic column: Phenomenex C18 250mm×50mm×10μm

[0625] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0626] Its structural characterization is as follows:

[0627] ESI-MS (m / z): 957.3 [M+H] + .

[0628] 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)

[0629] Step 1:

[0630] Methyl 3-hydroxybenzoate (1.00 g, 6.57 mmol) was dissolved in tetrahydrofuran (10.0 mL), cooled to 0°C, and NaH (526 mg, 13.1 mmol) was added, followed by stirring for 30 minutes. Chloromethyl methyl ether (1.26 g, 15.7 mmol) was then added to the reaction mixture, warmed to 25°C, and stirred for 2 hours. Saturated aqueous ammonium chloride (10.0 mL) was added to the reaction system, and the mixture was 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 afford crude methyl 3-(methoxymethoxy)benzoate (1.40 g), which was used directly in the next step without purification.

[0631] Its structural characterization is as follows:

[0632] ESI-MS (m / z): 197.1 (M+H) + .

[0633] Step 2:

[0634] Methyl 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 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 layers were separated. The aqueous phase was adjusted to pH 3 with 10% aqueous citric acid and 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 afford crude 3-(methoxymethoxy)benzoic acid (351 mg), which was used directly in the next step without purification.

[0635] Its structural characterization is as follows:

[0636] ESI-MS (m / z): 183.1 (M+H) + .

[0637] Step 3:

[0638] 3-(Methoxymethoxy)benzoic acid (200 mg, 1.10 mmol) and HATU (417 mg, 1.10 mmol) were dissolved in DMF (4.00 mL). DIPEA (568 mg, 4.39 mmol) was added, and the mixture was stirred at 25°C for 1 hour. To the reaction solution was added (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), and stirring was continued for 2 hours. Then, fluoroiodomethane (351 mg, 2.20 mmol) was added to the reaction solution and stirred for 1 hour. DCM (30.0 mL) and water (30.0 mL) were added to the reaction solution, and the mixture was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product (1.00 g) 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]phenanthrene-17-yl-3-(methoxymethoxy)benzoate, which was used in the next step without purification.

[0639] Its structural characterization is as follows:

[0640] ESI-MS (m / z): 609.2 (M+H) + .

[0641] Step 4:

[0642] (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 mixture was stirred at 25°C for 1 hour. The reaction solution was directly concentrated and purified by HPLC to give the title compound (134 mg, 230 μmol).

[0643] The purification conditions are as follows:

[0644] Chromatographic column: Phenomenex luna C18 150mm×25mm×10μm

[0645] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0646] Its structural characterization is as follows:

[0647] ESI-MS (m / z): 565.3 [M+H] + .

[0648] 1 H NMR(400MHz,DMSO)δ9.97(s,1H),7.23-7.26(m,1H),7.21-7.38(m,3H),7.02-7.09(m ,1H),6.32(dd,J=10,1.8Hz,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=7.2Hz,3H).

[0649] 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)

[0650] Step 1:

[0651] 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 stirred 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-S-carboxylic acid (300 mg, 727 μmol) was added and stirring continued for another 6 hours. Iodofluoromethane (232 mg, 1.45 mmol) was then added to the reaction system and stirring continued for 1 hour. Dichloromethane (30.0 mL) and water (30.0 mL) were added to the reaction solution. After separation, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product 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]phenanthrene-17-yl-4-(methoxymethoxy)benzoate (1.02 g), which was used in the next step without purification.

[0652] Its structural characterization is as follows:

[0653] ESI-MS (m / z): 609.3 (M+H) + .

[0654] Step 2:

[0655] (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 mixture was stirred at 25°C for 30 minutes. The reaction mixture was concentrated to obtain the crude product, which was purified by preparative HPLC to afford the title compound (116 mg, 205 μmol).

[0656] The purification conditions are as follows:

[0657] Chromatographic column: Phenomenex luna C18 250mm×70mm×15μm

[0658] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0659] Its structural characterization is as follows:

[0660] ESI-MS (m / z): 565.3 [M+H] + .

[0661] 1 H NMR (400MHz, DMSO) δ10.50(s,1H),7.72(d,J=8.8Hz,2H),7.28(d,J=10Hz,1H),6.82-6.9 4(m,2H),6.33(dd,J=10.2,1.8Hz,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),1.8 4-2.01(m,2H),1.49-1.62(m,4H),1.26-1.37(m,1H),1.05(s,3H),0.91(d,J=7.0Hz,3H).

[0662] 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)

[0663] Step 1:

[0664] Methyl 3-hydroxybenzoate (3.00 g, 19.7 mmol) was dissolved in tetrahydrofuran (30.0 mL), cooled to 0°C, and NaH (1.58 g, 39.4 mmol, 60.0% purity) was added and stirred for half an hour. Chloromethyl methyl ether (3.18 g, 39.4 mmol) was then added to the reaction mixture, and the temperature was raised to 25°C and stirred for 2.5 hours. Water (20.0 mL) was added to the reaction mixture to quench the reaction, and the mixture was extracted three times with ethyl acetate (20.0 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to afford crude methyl 2-(methoxymethoxy)benzoate (4.00 g), which was used directly in the next step without purification.

[0665] Its structural characterization is as follows:

[0666] ESI-MS (m / z): 197.1 (M+H) + .

[0667] Step 2:

[0668] 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, followed by stirring at 25°C for 2 hours. Water (20.0 mL) and ethyl acetate (20.0 mL) were added to the reaction system. After separation, the aqueous phase was adjusted to pH 3 with 1N dilute hydrochloric acid and extracted three times with ethyl acetate (25.0 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, adjusted to pH 9 with triethylamine, and filtered and concentrated to afford crude 2-(methoxymethoxy)benzoic acid (2.00 g), which was used directly in the next step without purification.

[0669] Its structural characterization is as follows:

[0670] ESI-MS (m / z): 183.1 (M+H) + .

[0671] Step 3:

[0672] 2-(Methoxymethoxy)benzoic acid (350 mg, 1.92 mmol) was dissolved in DMF (1.00 mL), and DIPEA (993 mg, 7.69 mmol) and HATU (657 mg, 1.73 mmol) were added, followed by stirring 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-S-carboxylic acid (713 mg, 1.73 mmol) was then added to the reaction mixture, and the mixture was heated to 30°C and stirred for 12 hours. Finally, iodomethane (614 mg, 3.84 mmol) was added to the reaction solution and stirring was continued for 1 hour. After cooling to 20°C, water (20.0 mL) was added to the reaction solution to quench the reaction, and the mixture was extracted with dichloromethane three times (20.0 mL x 3). The organic phases were combined and dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product 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]phenanthrene-17-yl-2-(methoxymethoxy)benzoate (1.30 g), which was used in the next step without purification.

[0673] Its structural characterization is as follows:

[0674] ESI-MS (m / z): 609.2 (M+H) + .

[0675] Step 4:

[0676] (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 mL). Trifluoroacetic acid (3.07 g, 26.92 mmol) was added and the mixture was stirred at 25°C for 2 hours. The reaction solution was directly concentrated to obtain the crude product, which was purified by preparative HPLC to obtain the title compound (168 mg, 297 μmol).

[0677] The purification conditions are as follows:

[0678] Chromatographic column: Phenomenex luna C18 150mm×25mm×10μm

[0679] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0680] Its structural characterization is as follows:

[0681] ESI-MS (m / z): 565.3 [M+H] + .

[0682] 1 H NMR (400MHz, DMSO) δ10.23(s,1H),7.70-7.72(m,1H),7.48-7.49(m,1H),7.12-7.19(m,1H),6.97(d,J=8.5Hz,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=7.0Hz,3H).

[0683] 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)

[0684] 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 in sequence. The mixture was stirred at 15 °C for 1 hour, and 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-thioxoS-carboxylic acid (84.44 mg, 204.71 μmol) was added and stirred at 15°C for 4 hours. Then, iodomethane (130.96 mg, 818.86 μmol) was added and stirred at 15°C for 1 hour. The reaction solution was directly prepared by high performance liquid chromatography and freeze-dried, and then purified by preparative thin layer chromatography (developing solvent: petroleum ether / ethyl acetate = 1 / 1, R f =0.4) to obtain the title compound (10 mg, 16.41 μmol).

[0685] The purification conditions are as follows:

[0686] Chromatographic column: Waters Sunfire Prep C18 OBD 150mm×19mm×5μm

[0687] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0688] Its structural characterization is as follows:

[0689] ESI-MS (m / z): 549.3 [M+H] + ,1197.4[2M+H] + .

[0690] 1H NMR (400MHz, CDCl3) δ7.95 (dd, J=8.4, 1.2Hz, 2H), 7.60 (tt, J=7.6, 1.2Hz, 1H), 7.46 (t, J=8.0Hz, 2H), 7.14 (dd, J= 10.4,1.2Hz,1H),6.48(s,1H),6.42(dd,J=10.0,1.6Hz,1H),5.91(ddd,J=50.0,44.8,9.2Hz,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=14.8,3.2Hz,1H),2.51–2.42(m,2H),2.31-2.35(m, 1H),1.94-2.01(m,2H),1.83(t,J=9.6Hz,1H),1.55(s,3H),1.38-1.44(m,1H),1.18(s,3H),1.04(d,J=7.2Hz,3H).

[0691] 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)

[0692] 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 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-thioxo S-carboxylic acid (58.88 mg, 142.74 μmol) was added, and the mixture was stirred at 15°C for 16 hours. Then, fluoroiodomethane (91.32 mg, 570.97 μmol) was added to the reaction system and stirred at 15°C for 1 hour. The reaction solution was directly prepared by HPLC and freeze-dried, and then purified 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).

[0693] The purification conditions are as follows:

[0694] Chromatographic column: Waters Sunfire Prep C18 OBD 150mm×19mm×5μm

[0695] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0696] Its structural characterization is as follows:

[0697] ESI-MS (m / z): 567.3 [M+H] + ,1133.4[2M+H] + .

[0698] 1 H NMR(400MHz, CDCl3)δ7.90-8.00(m,2H),7.09–7.17(m,3H),6.48(s,1H),6.43(dd,J=10.0 ,1.6Hz,1H),5.91(ddd,J=50.0,36.4,9.2Hz,2H),5.32–5.52(m,1H),4.50(ddd,J=6.4,4. 0,2.8Hz,1H),3.51(ddd,J=10.4,6.8,3.2Hz,1H),2.27–2.61(m,4H),1.92–2.05(m,2H),1 .82(t,J=12.0Hz,1H),1.55(s,3H),1.36–1.45(m,1H),1.18(s,3H),1.04(d,J=7.2Hz,3H).

[0699] 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)

[0700] Step 1: Synthesis of methyl 2-(triphenylmethoxy)acetate (D-1-2)

[0701] Methyl 2-hydroxyacetate (323 mg, 3.59 mmol, 276.88 μL) and triphenylmethane (1.00 g, 3.59 mmol) were dissolved in pyridine (4.00 mL) and heated to 80°C for 15 hours. 1N dilute hydrochloric acid (50.0 mL x 3) was added to the reaction solution, followed by extraction with dichloromethane (50.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to afford the crude title compound (1.10 g, 3.31 mmol), which was used directly in the next step without purification.

[0702] Its structural characterization is as follows:

[0703] 1 H NMR (400MHz, DMSO) δ7.33-7.41(m,12H),7.27-7.31(m,3H),3.69-3.75(m,2H),3.53-3.58(m,3H).

[0704] Step 2: Synthesis of 2-(triphenylmethoxy)acetic acid (D-1-3)

[0705] 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 mixture was heated to 40°C for 3 hours. Water (50.0 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (50.0 mL). The aqueous phase was adjusted to pH 1 with 1N dilute hydrochloric acid and filtered. The filtrate was extracted with dichloromethane three times (50.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (700 mg, 2.20 mmol), which was used directly in the next step without purification.

[0706] Its structural characterization is as follows:

[0707] 1 H NMR (400MHz, DMSO) δ7.42 (s, 6H), 7.33-7.38 (m, 6H), 7.27-7.30 (m, 3H), 3.54-3.59 (m, 2H).

[0708] 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)

[0709] 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, followed by stirring at 25°C for 1 hour. To the reaction mixture was added (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 thioate (453 mg, 1.10 mmol), and stirring was continued for 2 hours. Fluoroiodomethane (175 mg, 1.10 mmol) was then added to the reaction mixture and stirring continued for 0.4 hour. Water (50.0 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (50.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (600 mg) as a yellow oil.

[0710] 3-(Triphenylmethoxy)propionic acid (300 mg, 902 μmol) was dissolved in DMF (2.00 mL), and HATU (343 mg, 903 μmol) and DIPEA (349 mg, 2.71 mmol, 472 μL) were added, followed by stirring 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 thioate (372 mg, 902 μmol) was added to the reaction mixture, and stirring was continued for 12 hours. Fluoroiodomethane (144 mg, 903 μmol) was then added to the reaction mixture, and stirring was continued for 1 hour. Water (100 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (80.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the title compound (450 mg), which was used directly in the next step without purification.

[0711] Its structural characterization is as follows:

[0712] ESI-MS (m / z): 745.3 (M+H) + .

[0713] 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]phenanthrene-17-yl-2-hydroxyacetate (D-1)

[0714] (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). 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 solution was directly concentrated to obtain a crude product, which was purified by HPLC to obtain the title compound (141 mg, 259 μmol).

[0715] Chromatographic column: Phenomenex luna C18 200mm×40mm×10μm

[0716] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0717] Its structural characterization is as follows:

[0718] ESI-MS (m / z): 503.3 [M+H] + .

[0719] 1H NMR (400MHz, CD3OD) δ7.27-7.39(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).

[0720] 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-hydroxypropanoate (D-9)

[0721] Step 1: Synthesis of methyl 2-(triphenylmethoxy)propionate (D-9-2)

[0722] Methyl 2-hydroxypropionate (373 mg, 3.59 mmol, 341 μL) and triphenylmethane (1.00 g, 3.59 mmol) were dissolved in pyridine (4.00 mL), heated to 80°C, and reacted for 15 hours. 1N dilute hydrochloric acid (50.0 mL) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (50.0 mL x 3). The organic phases were combined and washed twice with 1N dilute hydrochloric acid (50.0 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (1.00 g, 2.89 mmol), which was used directly in the next step without purification.

[0723] Its structural characterization is as follows:

[0724] 1 H NMR (400MHz, DMSO) δ7.37-7.40(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).

[0725] Step 2: Synthesis of 2-(triphenylmethoxy)propionic acid (D-9-3)

[0726] Methyl 2-(triphenylmethoxy)propionate (1.00 g, 2.89 mmol) was dissolved in a mixture 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 mixture was heated to 40°C and allowed to react for 3 hours. Water (50.0 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (50.0 mL). The aqueous phase was adjusted to pH 1 with 1N dilute hydrochloric acid, filtered, and the filtrate was extracted three times with dichloromethane (50.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the title compound (250 mg, 752 μmol), which was used directly in the next step without purification.

[0727] Its structural characterization is as follows:

[0728] 1 H NMR (400MHz, DMSO) δ7.38-7.48(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).

[0729] 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]phenanthrene-17-yl-2-(triphenylmethoxy)propionate (D-9-4)

[0730] 2-(Triphenylmethoxy)propionic acid (200 mg, 601 μmol) was dissolved in DMF (5.00 mL), and HATU (228 mg, 601 μmol) and DIPEA (233 mg, 1.81 mmol, 314 μL) were added, followed by stirring 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 thioate (248 mg, 601 μmol) was added to the reaction mixture, and stirring was continued for 2 hours. Fluoroiodomethane (96.2 mg, 601 μmol) was then added and stirring continued for 0.5 hour. Water (100 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (50.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to afford the title compound (500 mg, crude product), which was used directly in the next step without purification.

[0731] Its structural characterization is as follows:

[0732] ESI-MS (m / z): 759.3 (M+H) + .

[0733] 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]phenanthrene-17-yl-2-hydroxypropionate (D-9)

[0734] (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)propanoate (300 mg, 395 μmol) was dissolved in a mixed solvent of dichloromethane (4.00 mL) and methanol (1.00 mL). 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 solution was directly concentrated to obtain a crude product, which was purified by HPLC to obtain the title compound (102 mg, 189 μmol).

[0735] Chromatographic column: Phenomenex luna C18 200mm×40mm×10μm

[0736] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0737] Its structural characterization is as follows:

[0738] ESI-MS (m / z): 517.1 [M+H] + .

[0739] 1 H NMR (400MHz, CD3OD) δ7.28-7.37(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).

[0740] 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]phenanthrene-17-yl-3-hydroxypropanoate (D-5)

[0741] Step 1: Synthesis of methyl 3-(triphenylmethoxy)propionate (D-5-2)

[0742] Dissolve methyl 3-hydroxypropionate (375 mg, 3.60 mmol) in pyridine (4.00 mL), add triphenylmethane (1.00 g, 3.60 mmol), and heat to 80°C. Stir for 12 hours. Add 1N dilute hydrochloric acid (60.0 mL) to the reaction mixture, and extract three times with dichloromethane (50.0 mL x 3). Combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate to obtain the title compound (1.10 g, 3.18 mmol).

[0743] Its structural characterization is as follows:

[0744] 1 H NMR (400MHz, DMSO) δ7.21-7.47 (m, 19H), 3.60 (s, 3H), 3.21 (t, J = 6.4Hz, 2H), 2.58 (t, J = 6.4Hz, 2H).

[0745] Step 2: Synthesis of 3-(triphenylmethoxy)propionic acid (D-5-3)

[0746] 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 stirred at 25°C for 2 hours. Water (60.0 mL) was added to the reaction solution, and the pH was adjusted to 3-4 with 1N dilute hydrochloric acid. The mixture was extracted three times with dichloromethane (80.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the title compound (980 mg, 2.95 mmol).

[0747] Its structural characterization is as follows:

[0748] 1 H NMR (400MHz, DMSO) δ7.19-7.42 (m, 17H), 3.19 (t, J = 6.4Hz, 2H), 2.44-2.49 (m, 2H).

[0749] 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)

[0750] 3-(Triphenylmethoxy)propionic acid (300 mg, 902 μmol) was dissolved in DMF (2.00 mL), and HATU (343 mg, 903 μmol) and DIPEA (349 mg, 2.71 mmol, 472 μL) were added, followed by stirring 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 thioate (372 mg, 902 μmol) was added to the reaction mixture, and stirring was continued for 12 hours. Then, add iodomethane (144 mg, 903 μmol) to the reaction mixture and continue stirring for 1 hour. Add water (100 mL) to the reaction mixture and extract three times with ethyl acetate (80.0 mL x 3). The combined organic phases are dried over anhydrous sodium sulfate, filtered, and concentrated to yield the title compound (450 mg, crude product).

[0751] Its structural characterization is as follows:

[0752] ESI-MS (m / z): 781.3 (M+Na) + .

[0753] 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]phenanthrene-17-yl-3-hydroxypropionate (D-5)

[0754] (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)propanoate (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 mixture was stirred at 25°C for 2 hours. The reaction mixture was concentrated to give the crude product, which was purified by preparative HPLC to give the title compound (152.45 mg, 295.12 μm).

[0755] Chromatographic column: Phenomenex luna C18 150mm×25mm×5μm

[0756] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0757] Its structural characterization is as follows:

[0758] ESI-MS (m / z): 517.2 [M+H] + .

[0759] 1 H NMR (400MHz, DMSO) δ7.25 (d, J=10.0Hz, 1H), 6.30 (dd, J=10.0, 1.6Hz, 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=6.4Hz,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=7.2Hz,3H).

[0760] 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]phenanthrene-17-yl-3-hydroxybutyrate (D-11)

[0761] Step 1: Synthesis of methyl 3-(triphenylmethoxy)butyrate (D-11-2)

[0762] Dissolve methyl 3-hydroxybutyrate (211 mg, 1.79 mmol) and triphenylmethane (500 mg, 1.79 mmol) in pyridine (3.00 mL) and heat to 80°C for 10 hours. Add 1N dilute hydrochloric acid (50.0 mL x 3) to the reaction solution, and extract with dichloromethane three times (50.0 mL x 3). Combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate to obtain the title compound (520 mg, 1.44 mmol).

[0763] Its structural characterization is as follows:

[0764] 1 H NMR (400MHz, DMSO) δ7.41 (d, J=7.2Hz, 6H), 7.33 (t, J=7.2Hz, 6H), 7.26-7.28 (m, 3H), 3.76-3.8 4(m,1H),3.48(s,3H),2.21(dd,J=15.2,7.6Hz,1H),2.02-2.11(m,1H),0.88(d,J=6.0Hz,3H).

[0765] Step 2: Synthesis of 3-(triphenylmethoxy)butyric acid (D-11-3)

[0766] Methyl 3-(triphenylmethoxy)butanoate (500 mg, 1.39 mmol) was dissolved in a mixture 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 mixture was heated to 40°C for 2 hours. Water (50.0 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (50.0 mL). The aqueous phase was adjusted to pH 1 with 1N dilute hydrochloric acid and filtered. The filtrate was extracted with dichloromethane three times (50.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the title compound (250 mg, 721 μmol), which was used directly in the next step without purification.

[0767] Its structural characterization is as follows:

[0768] 1 H NMR(400MHz,DMSO)δ7.39-7.45(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).

[0769] 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)butyrate (D-11-4)

[0770] 3-(Triphenylmethoxy)butanoic 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, followed by stirring at 25° C. for 1 hour. To the reaction mixture, (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 (226 mg, 548 μmol) was added and stirring continued for 2 hours. Iodofluoromethane (87.7 mg, 548 μmol) was then added and stirring continued for 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 x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to afford the crude title compound (600 mg, crude product), which was used directly in the next step without purification.

[0771] Its structural characterization is as follows:

[0772] ESI-MS (m / z): 773.3 (M+H) + .

[0773] 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]phenanthrene-17-yl-3-hydroxybutyrate (D-11)

[0774] (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)butyrate (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 mixture was stirred at 25°C for 0.5 hour. The reaction solution was directly concentrated to obtain a crude product, which was purified by HPLC to obtain the title compound (76.0 mg, 142 μmol).

[0775] Chromatographic column: Welch Xtimate C18 150mm×25mm×5μm

[0776] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0777] Its structural characterization is as follows:

[0778] ESI-MS (m / z): 531.2 [M+H] + .

[0779] 1 H NMR (400MHz, CD3OD) δ7.26-7.37(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=7.2Hz,3H).

[0780] 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]phenanthrene-17-yl-(S)-3-hydroxybutyrate (D-15)

[0781] Step 1: Synthesis of (S)-3-(trityloxy)butyric acid methyl ester (D-15-2)

[0782] (S)-Methyl 3-hydroxybutyrate (300 mg, 2.54 mmol) was added to a single-necked flask, along with pyridine (3 mL) and triphenylmethane (849 mg, 3.05 mmol). The temperature was raised to 80°C and stirred for 15 hours. The mixture was then cooled to room temperature, and water (15 mL) was added to the reaction solution. The mixture was extracted twice with ethyl acetate (8 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by flash column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 3 / 1) to obtain the title compound (460 mg, 1.15 mmol).

[0783] Its structural characterization is as follows:

[0784] 1 H NMR (400MHz, DMSO) δ7.48–7.41(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=14.8,7.6Hz,1H),2.06(dd,J=14.8,4.8Hz,1H),0.89(d,J=6.0Hz,3H).

[0785] Step 2: Synthesis of (S)-3-(trityloxy)butyric acid (D-15-3)

[0786] Methyl (S)-3-(trityloxy)butanoate (460 mg, 1.15 mmol) was added to a mixture of methanol (1 mL), tetrahydrofuran (1 mL), and water (0.5 mL). Lithium hydroxide (36.9 mg, 1.54 mmol) was added and stirred at 25°C for 2 hours. Water and 1N dilute hydrochloric acid (2 mL) were added to the reaction solution, and the mixture was extracted three times with dichloromethane (10 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the title compound (142 mg, 0.41 mmol).

[0787] 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]phenanthrene-17-yl-(S)-3-(triphenylmethoxy)butyrate (D-15-4)

[0788] (S)-3-(Trityloxy)butanoic 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 reacted at 25°C for 2 hours, and 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-carboxylic acid thioate (63.2 mg, 0.16 mmol) was added and stirring was continued for 2 hours. Then, iodomethane (34.7 mg, 0.22 mmol) was added and the reaction was continued at 25°C for 1 hour. Water (10 mL) was added to the reaction solution, 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 obtain the crude title compound (60 mg), which was used directly in the next step without purification.

[0789] Its structural characterization is as follows:

[0790] ESI-MS (m / z): 773.3 (M+H) + .

[0791] 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]phenanthrene-17-yl-(S)-3-hydroxybutyrate (D-15)

[0792] (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), followed by acetic acid (0.5 mL). The reaction mixture was stirred at 40°C for 3 hours and then concentrated to obtain the crude product, which was purified by HPLC to obtain the title compound (4.2 mg, 0.008 mmol).

[0793] Chromatographic column: SunFire prep C18 OBD 150mm×19mm×5μm

[0794] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0795] Its structural characterization is as follows:

[0796] ESI-MS (m / z): 574.1 [M+Na] + .

[0797] 1 H NMR (400MHz, DMSO) δ7.25(d,J=10.2Hz,1H),6.30(d,J=10.2,1H),6.11(s,1H),5.99(s,1H),5.87(s,1 H),5.74–5.54(m,1H),5.58(d,J=4.0Hz,1H),4.74(d,J=5.2Hz,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=6.2Hz,3H),0.99(s,2H),0.91(d,J=7.1Hz,3H).

[0798] 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]phenanthrene-17-yl-(R)-3-hydroxybutyrate (D-19)

[0799] Step 1: Synthesis of (R)-3-(trityloxy)butyric acid methyl ester (D-19-2)

[0800] Methyl (R)-3-hydroxybutyrate (300 mg, 2.54 mmol) was added to a single-necked flask, along with pyridine (3 mL) and triphenylmethane (849 mg, 3.05 mmol). The temperature was raised to 80°C and stirred for 15 hours. The mixture was then cooled to room temperature, and water (15 mL) was added to the reaction solution. The mixture was extracted twice with ethyl acetate (8 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by flash column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 5 / 1) to obtain the title compound (278 mg, 0.77 mmol).

[0801] Its structural characterization is as follows:

[0802] 1 H NMR (400MHz, DMSO) δ7.47–7.41(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=14.8,7.6Hz,1H),2.07(dd,J=14.8,4.8Hz,1H),0.89(d,J=6.0Hz,3H).

[0803] Step 2: Synthesis of (R)-3-(trityloxy)butyric acid (D-19-3)

[0804] Methyl (R)-3-(trityloxy)butanoate (278 mg, 0.77 mmol) was added to a mixture of methanol (1 mL), tetrahydrofuran (1 mL), and water (0.5 mL). Lithium hydroxide (36.9 mg, 1.54 mmol) was added and stirred at 25°C for 2 hours. Water and 1N dilute hydrochloric acid (2 mL) were added to the reaction solution, and the mixture was extracted three times with dichloromethane (10 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the title compound (142 mg, 0.41 mmol).

[0805] 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]phenanthrene-17-yl-(R)-3-(triphenylmethoxy)butanoate (D-19-4)

[0806] (R)-3-(Trityloxy)butanoic 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 reacted at 25°C for 2 hours, and 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-carboxylic acid thioate (63.2 mg, 0.16 mmol) was added and stirring was continued for 2 hours. Then, iodomethane (34.7 mg, 0.22 mmol) was added and the reaction continued at 25°C for 1 hour. Water (10 mL) was added to the reaction solution, 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 obtain the crude title compound. Flash column chromatography (C18, water / acetonitrile = 0.8) afforded the title compound (32 mg, 0.04 mmol).

[0807] Its structural characterization is as follows:

[0808] ESI-MS (m / z): 773.3 (M+H) + .

[0809] 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]phenanthrene-17-yl-(R)-3-hydroxybutyrate (D-19)

[0810] (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), followed by acetic acid (0.5 mL). The reaction mixture was stirred at 40°C for 3 hours and then concentrated to obtain the crude product, which was purified by HPLC to obtain the title compound (5.2 mg, 0.01 mmol).

[0811] Chromatographic column: SunFire prep C18 OBD 150mm×19mm×5μm

[0812] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0813] Its structural characterization is as follows:

[0814] ESI-MS (m / z): 531.2 [M+H] +. .

[0815] 1 H NMR (400MHz, CD3OD) δ7.33 (d, J=10.0Hz, 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=9.6Hz,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=6.4Hz,3H),1.13(s,3H),1.02(d,J=7.2Hz,3H).

[0816] 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]phenanthrene-17-yl-4-hydroxybutyrate (D-23)

[0817] Step 1: Synthesis of 4-(triphenylmethoxy)benzyl butyrate (D-23-2)

[0818] Dissolve benzyl 4-hydroxybutyrate (500 mg, 2.57 mmol) in pyridine (3.00 mL), add triphenylmethane (718 mg, 2.57 mmol), and heat to 90°C for 10 hours. Add 1N dilute hydrochloric acid (20.0 mL) to the reaction solution, extract three times with dichloromethane (30.0 mL x 3). Combine the organic phases, wash twice with 1N dilute hydrochloric acid (20.0 mL x 2), dry over anhydrous sodium sulfate, filter, and concentrate to obtain the crude title compound (900 mg, crude product), which is used directly in the next step without purification.

[0819] Its structural characterization is as follows:

[0820] ESI-MS (m / z): 459.3 (M+Na) + .

[0821] Step 2: Synthesis of 4-(triphenylmethoxy)butyric acid (D-23-3)

[0822] Benzyl 4-(triphenylmethoxy)butyrate (850 mg, 1.95 mmol) was dissolved in a mixture of methanol (0.50 mL), tetrahydrofuran (0.50 mL), and sodium hydroxide (0.50 mL). Lithium hydroxide (140 mg, 5.84 mmol) was added and the mixture was heated to 40°C for 1 hour. The pH of the reaction mixture was adjusted to 2-3 with 1N dilute hydrochloric acid. Water (10.00 mL) was added and the mixture was extracted three times with ethyl acetate (20.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (230 mg, crude product), which was used directly in the next step without purification.

[0823] Its structural characterization is as follows:

[0824] ESI-MS (m / z): 369.2 (M+H) + .

[0825] 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)butyrate (D-23-4)

[0826] 4-(Triphenylmethoxy)butanoic acid (180 mg, 519 μmol) was dissolved in DMF (3.00 mL), and HATU (198 mg, 520 μmol) and DIPEA (201 mg, 1.56 mmol, 272 μL) were added, followed by reaction at 25° C. for 1 hour. To the reaction mixture, (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 (193 mg, 468 μmol) was added and stirring continued for 0.5 hour. Iodofluoromethane (83.1 mg, 520 μmol) was then added and stirring continued for 0.5 hour. Water (10.0 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (20.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to afford the crude title compound (480 mg, crude product), which was used directly in the next step without purification.

[0827] Its structural characterization is as follows:

[0828] ESI-MS (m / z): 796.3 (M+H) + .

[0829] 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]phenanthrene-17-yl-4-hydroxybutyrate (D-23)

[0830] (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)butyrate (430 mg, 556 μmol) was dissolved in a mixed solvent of dichloromethane (4.00 mL) and methanol (1.00 mL). Trifluoroacetic acid (1.54 g, 13.5 mmol, 1.00 mL) was added, and the mixture was stirred at 25°C for 1 hour. The reaction solution was directly concentrated to obtain a crude product, which was purified by HPLC to obtain the title compound (49.0 mg, 92.2 μmol).

[0831] Chromatographic column: Phenomenex Luna C18 200mm×40mm×10μm

[0832] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0833] Its structural characterization is as follows:

[0834] ESI-MS (m / z): 531.1 [M+H] + .

[0835] 1 H NMR(400MHz,CD3OD)δ7.32(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=6.4Hz,2H),3.41(m,1H),2.51-2.6 5(m,1H),2.46(t,J=7.6Hz,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=7.2Hz,3H).

[0836] 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]phenanthrene-17-yl-5-hydroxyvalerate (D-24)

[0837] Step 1: Synthesis of methyl 5-(triphenylmethoxy)valerate (D-24-2)

[0838] Methyl 5-hydroxyvalerate (450 mg, 3.41 mmol) was dissolved in pyridine (15.0 mL). Triphenylmethane (949 mg, 3.41 mmol) was added and the mixture was heated to 80°C for 10 hours. 1N dilute hydrochloric acid (20.0 mL) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (30.0 mL x 3). The organic phases were combined and washed twice with 1N dilute hydrochloric acid (20.0 mL x 2). After drying over anhydrous sodium sulfate, the mixture was filtered and concentrated to obtain the crude title compound (937 mg, crude product).

[0839] Its structural characterization is as follows:

[0840] 1 H NMR (400MHz, DMSO) δ7.35-7.37(m,6H),7.27-7.30(m,6H),7.21(d,J=1.6Hz,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).

[0841] Step 2: Synthesis of 5-(triphenylmethoxy)valeric acid (D-24-3)

[0842] Methyl 5-(triphenylmethoxy)pentanoate (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 mixture was heated to 40°C and stirred for 1 hour. The reaction mixture was adjusted to a pH of 2-3 with 1N dilute hydrochloric acid. Water (10.0 mL) was added, and the mixture was extracted three times with ethyl acetate (20.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (285 mg, crude product), which was used directly in the next step without purification.

[0843] Its structural characterization is as follows:

[0844] 1 H NMR (400MHz, DMSO) δ7.34-7.39(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).

[0845] 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)pentanoate (D-24-4)

[0846] 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 mixture was stirred at 25°C for 1 hour. To the reaction mixture was added (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 thioate (237 mg, 574 μmol), and the reaction was continued for 0.5 hour. Then, iodomethane (102 mg, 638 μmol) was added and stirring continued for 0.5 hour. Water (10.0 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (20.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (600 mg, crude product), which was used directly in the next step without purification.

[0847] Its structural characterization is as follows:

[0848] ESI-MS (m / z): 809.3 (M+Na) + .

[0849] 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]phenanthrene-17-yl-5-hydroxyvalerate (D-24)

[0850] (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)pentanoate (600 mg, 762 μmol) was dissolved in a mixed solvent of dichloromethane (4.00 mL) and methanol (1.00 mL). Trifluoroacetic acid (1.54 g, 13.5 mmol, 1.00 mL) was added, and the mixture was stirred at 25°C for 1 hour. The reaction solution was directly concentrated to obtain a crude product, which was purified by HPLC to obtain the title compound (72.0 mg, 132 μmol).

[0851] Chromatographic column: Phenomenex Luna C18 200mm×40mm×10μm

[0852] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0853] Its structural characterization is as follows:

[0854] ESI-MS (m / z): 545.2 [M+H] + .

[0855] 1 H NMR (400MHz, CD3OD) δ7.32(m,1H),6.34(m,1H),6.30(s,1H),5.91-5.97(m,1H),5.79-5.85(m,1 H),5.44-5.64(m,1H),4.27-4.32(m,1H),3.54(t,J=6.4Hz,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=7.2Hz,3H).

[0856] 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-carboxylate (D-25)

[0857] Step 1: Synthesis of methyl (1S,4S)-4-hydroxycyclohexane-1-carboxylate (D-25-2)

[0858] (1S,4S)-4-Hydroxycyclohexane-1-carboxylic acid (300 mg, 2.1 mmol) was added to DMF (2 mL). Anhydrous potassium carbonate (345 mg, 2.5 mmol) and iodomethane (591.6 mg, 4.17 mmol) were then added and stirred at 25°C for 2 hours. Water (20 mL) was added to the reaction solution, which was then extracted with ethyl acetate three times (10 mL x 3) and washed twice with saturated sodium chloride solution (10 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (310 mg, 1.96 mmol), which was used directly in the next step without purification.

[0859] Step 2: Synthesis of methyl (1S,4S)-4-(trityloxy)cyclohexane-1-carboxylate (D-25-3)

[0860] Methyl (1S,4S)-4-hydroxycyclohexane-1-carboxylate (310 mg, 1.96 mmol) was added to a single-necked flask, along with pyridine (3 mL) and triphenylmethane chloride (820.5 mg, 2.94 mmol). The temperature was raised to 80°C and stirred for 15 hours. After cooling to room temperature, water (15 mL) was added to the reaction solution, 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 obtain the crude product. Purification by flash column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 5 / 1) afforded the title compound (320 mg, 0.8 mmol).

[0861] Its structural characterization is as follows:

[0862] 1 H NMR (400MHz, DMSO) δ7.46–7.41(m,6H),7.36–7.29(m,6H),7.28–7.22(m,3H),3.50(s,3H),1.68(d,J=10.4Hz,2H),1.26–1.01(m,8H).

[0863] Step 3: Preparation of (1S,4S)-4-(trityloxy)cyclohexane-1-carboxylic acid (D-25-4)

[0864] Methyl (1S,4S)-4-(trityloxy)cyclohexane-1-carboxylate (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 stirred at 25°C for 2 hours. Water and 1N dilute hydrochloric acid (3 mL) were added to the reaction solution, and the mixture was extracted three times with dichloromethane (10 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification by flash column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 3 / 1) afforded the title compound (280 mg, 0.72 mmol).

[0865] 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-(triphenoxy)cyclohexane-1-carboxylate (D-25-5)

[0866] (1S,4S)-4-(Trityloxy)cyclohexane-1-carboxylic acid (50 mg, 0.13 mmol) and HATU (59.0 mg, 0.16 mmol) were added to DMF (2.0 mL), followed by the addition of DIPEA (50.1 mg, 0.39 mmol) and the mixture was allowed to react 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-carboxylic acid thioate (63.2 mg, 0.16 mmol) was added to the reaction mixture, and stirring was continued for 2 hours. Then, iodomethane (31.0 mg, 0.19 mmol) was added and the reaction continued at 25°C for 1 hour. Water (10 mL) was added to the reaction solution, 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 obtain the crude title compound. Flash column chromatography (C18, water / acetonitrile = 0.8) afforded the title compound (16 mg, 0.02 mmol).

[0867] 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]phenanthrene-17-yl-(1S,4S)-4-hydroxycyclohexane-1-carboxylate (D-25)

[0868] (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-(triphenoxy)cyclohexane-1-carboxylate (60 mg, 0.073 mmol) was dissolved in dichloromethane (1 mL). Acetic acid (0.5 mL) was added and the mixture was stirred at 40°C for 3 hours. The reaction mixture was concentrated to obtain the crude product, which was purified by HPLC to obtain the title compound (3.1 mg, 0.005 mmol).

[0869] Chromatographic column: SunFire prep C18 OBD 150mm×19mm×5μm

[0870] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0871] Its structural characterization is as follows:

[0872] ESI-MS (m / z): 571.1 [M+H] + .

[0873] 1 H NMR(400MHz,CD3OD)δ7.33(dd,J=10.0,1.6Hz,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,3 H), 1.54–1.41 (m, 2H), 1.36–1.21 (m, 4H), 1.11 (s, 3H), 0.96 (d, J = 7.2Hz, 3H).

[0874] 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]phenanthrene-17-yl-(1R,4R)-4-hydroxycyclohexane-1-carboxylate (D-26)

[0875] Step 1: Synthesis of (1R,4R)-4-hydroxycyclohexane-1-carboxylic acid methyl ester (D-26-2)

[0876] (1R,4R)-4-Hydroxycyclohexane-1-carboxylic acid (300 mg, 2.1 mmol) was added to DMF (2 mL). Anhydrous potassium carbonate (345 mg, 2.5 mmol) and iodomethane (591.6 mg, 4.17 mmol) were then added and stirred at 25°C for 2 hours. Water (20 mL) was added to the reaction solution, which was then extracted with ethyl acetate three times (10 mL x 3) and washed twice with saturated sodium chloride solution (10 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (310 mg, 1.96 mmol), which was used directly in the next step without purification.

[0877] Step 2: Synthesis of methyl (1R, 4R)-4-(trityloxy)cyclohexane-1-carboxylate (D-26-3)

[0878] Methyl (1R,4R)-4-hydroxycyclohexane-1-carboxylate (310 mg, 1.96 mmol) was added to a single-necked flask, along with pyridine (3 mL) and triphenylmethane chloride (820.5 mg, 2.94 mmol). The temperature was raised to 80°C and stirred for 15 hours. After cooling to room temperature, water (15 mL) was added to the reaction solution, 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 obtain the crude product. Purification by flash column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 5 / 1) afforded the title compound (691 mg, 1.73 mmol).

[0879] Its structural characterization is as follows:

[0880] 1H NMR (400MHz, DMSO) δ7.44–7.40(m,6H),7.38–7.30(m,6H),7.28–7.20(m,3H),3.48(s,3H),1.67(d,J=10.4Hz,2H),1.24–1.01(m,8H).

[0881] Step 3: Preparation of (1R, 4R)-4-(trityloxy)cyclohexane-1-carboxylic acid (D-26-4)

[0882] Methyl (1R,4R)-4-(trityloxy)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 1N dilute hydrochloric acid (3 mL) were added to the reaction solution, and the mixture was extracted three times with dichloromethane (10 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The title compound (410 mg, 1.06 mmol) was obtained by flash column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 3 / 1).

[0883] 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]phenanthrene-17-yl-(1r,4R)-4-(triphenoxy)cyclohexane-1-carboxylate (D-26-5)

[0884] (1R,4R)-4-(Trityloxy)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, and the mixture was reacted at 25°C for 2 hours. To the reaction mixture was added (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 thioate (63.2 mg, 0.16 mmol), and stirring was continued for 2 hours. Then, iodomethane (31.0 mg, 0.19 mmol) was added and the reaction continued at 25°C for 1 hour. Water (10 mL) was added to the reaction solution, 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 obtain the crude title compound (60 mg, 0.073 mmol), which was used directly in the next step without purification.

[0885] 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)

[0886] (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-(triphenoxy)cyclohexane-1-carboxylate (60 mg, 0.073 mmol) was dissolved in dichloromethane (1 mL). Acetic acid (0.5 mL) was added and the mixture was stirred at 40°C for 3 hours. The reaction mixture was concentrated to obtain a crude product, which was purified by HPLC to obtain the title compound (3.5 mg, 0.006 mmol).

[0887] Chromatographic column: SunFire prep C18 OBD 150mm×19mm×5μm

[0888] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0889] Its structural characterization is as follows:

[0890] ESI-MS (m / z): 594.2 [M+Na] + .

[0891] 1 H NMR(400MHz,DMSO)δ7.26(d,J=10.1Hz,1H),6.30(dd,J=10.2,1.8Hz,1H),6.11(s,1H),5.98(s,1H ),5.86(s,1H),5.73–5.54(m,1H),5.58(d,J=4.0Hz,1H),4.59(d,J=4.3Hz,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=7.2Hz,3H).

[0892] 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)

[0893] 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]phenanthrene-3-one (A-2-2)

[0894] Ethyl 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-oxoacetate (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 was adjusted to 2-3 with 1N dilute hydrochloric acid, water (30.0 mL) was added, and the mixture was extracted three times with dichloromethane (20.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to give the title compound (110 mg, 290 μmol).

[0895] Its structural characterization is as follows:

[0896] ESI-MS (m / z): 378.9 [M+H] + .

[0897] 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)

[0898] (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]phenanthrene-3-one (200 mg, 528 μmol) was dissolved in a mixture of tetrahydrofuran (1.30 mL) and water (1.00 mL). Periodic acid (361 mg, 1.59 mmol, 361 μL) was added and stirred at 25°C for 1 hour. The pH was adjusted to 2-3 with 1N dilute hydrochloric acid, filtered, and the filter cake was dried to obtain the title compound (100 mg, 274 μmol).

[0899] Its structural characterization is as follows:

[0900] ESI-MS (m / z): 365.0 [M+H] + .

[0901] 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)

[0902] (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 stirred at 25°C for 2 hours. H2S gas (9.35 mg, 274 μmol, 4.52 μL) (15 PSI) was introduced into the reaction system and stirring continued for 2.5 hours. The pH was adjusted to 2-3 with 1N dilute hydrochloric acid, the mixture was filtered, and the filter cake was dried to obtain the title compound (70.0 mg, 183 μmol).

[0903] Its structural characterization is as follows:

[0904] ESI-MS (m / z): 380.9 [M+Na] + .

[0905] 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)

[0906] 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, and the mixture was reacted at 25°C for 2 hours. To the reaction mixture was added (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 thioate (60.0 mg, 157 μmol), and stirring was continued for 1 hour. Then, iodomethane (25.2 mg, 157 μmol) was added, and the reaction was continued at 25°C for 1 hour. Water (10 mL) was added to the reaction solution, 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 obtain the crude title compound. Purification by p-TLC (SiO2, CH2Cl2 / MeOH = 100 / 1 to 10 / 1) and further concentration were performed to obtain the title compound (12.0 mg, 18.4 μmol).

[0907] Its structural characterization is as follows:

[0908] ESI-MS (m / z): 650.2 [M+H] + .

[0909] 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)

[0910] (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 mixture was stirred at 25°C for 1 hour. The reaction mixture was concentrated to obtain the crude product, which was purified by HPLC to obtain the title compound (2.36 mg, 4.07 μmol).

[0911] Chromatographic column: Phenomenex luna C18 150mm×25mm×10mm

[0912] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate)

[0913] The structural characterization of A-2 is as follows:

[0914] ESI-MS (m / z): 550.2 [M+H] + .

[0915] 1 H NMR (400MHz, CD3OD) δ7.36-7.49(m,2H),7.22-7.32(m,1H),7.06(dd,J=10.8,8.4Hz,1H),6.33(dd ,J=10.0,1.8Hz,1H),6.11(s,1H),5.68-6.00(m,2H),4.40(d,J=8.4Hz,1H),3.03(dd,J=14.4,11.2 Hz,1H),2.76(td,J=13.6,5.6Hz,1H),2.53-2.67(m,1H),2.38-2.51(m,2H),2.18-2.27(m,1H),2.0 2-2.14(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).

[0916] 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)

[0917] Step 1: Synthesis of (5-bromofuran-3-yl)methanol (F-9-2)

[0918] Dissolve 5-bromofuran-3-carboxylic acid (1.00 g, 5.24 mmol) in tetrahydrofuran (20.0 mL), cool to 0°C, add borane in tetrahydrofuran (2.5 M, 4.19 mL) under a nitrogen atmosphere, and heat to 50°C, stirring for 3 hours. Cool to 25°C, quench the reaction with methanol (10.0 mL), concentrate, add 2 M aqueous sodium hydroxide (30.0 mL), and extract twice with ethyl acetate (30.0 mL x 2). The organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated to afford the crude title compound (1.06 g), which is used directly in the next step without purification.

[0919] Its structural characterization is as follows:

[0920] ESI-MS (m / z): 177.1 [M+H] + .

[0921] Step 2: Synthesis of ((5-bromofuran-3-yl)methoxy)(tert-butyl)diphenylsilane (F-9-3)

[0922] Dissolve (5-bromofuran-3-yl)methanol (1.00 g, 5.65 mmol) in dichloromethane (10.0 mL), add imidazole (1.15 g, 16.9 mmol) and tert-butyldiphenylsilyl chloride (2.33 g, 8.47 mmol), and stir at 25°C for 3 hours. Add water (20.0 mL) to the reaction solution, and extract with dichloromethane (20.0 mL). The organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purify by silica gel column chromatography (petroleum ether:ethyl acetate = 100 / 0 to 100 / 5) and reconcentrate to obtain the crude title compound (1.20 g, crude product).

[0923] Its structural characterization is as follows:

[0924] ESI-MS (m / z): 415.1 [M+H] + .

[0925] Step 3: Synthesis of methyl 4-(((tert-butyldiphenylsilyl)oxy)methyl)furan-2-carboxylate (F-9-4)

[0926] ((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) and Pd(dppf)Cl2 (158 mg, 216 μmol) were 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 obtain a crude product, which was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:0 to 10:2) and concentrated again to obtain the title compound (746 mg, 1.89 mmol).

[0927] Its structural characterization is as follows:

[0928] ESI-MS (m / z): 395.3 [M+H] + .

[0929] Step 4: Synthesis of methyl 4-(hydroxymethyl)furan-2-carboxylate (F-9-5)

[0930] Methyl 4-(((tert-Butyldiphenylsilyl)oxy)methyl)furan-2-carboxylate (643 mg, 1.63 mmol) was dissolved in tetrahydrofuran (6.00 mL). Tetrabutylammonium fluoride solution (1 M, 1.63 mL) was added and stirred at 20°C for 1 hour. The reaction mixture was concentrated to obtain a crude product, which was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10 / 1 to 1 / 1) and concentrated again to obtain the crude title compound (191 mg, crude product).

[0931] Its structural characterization is as follows:

[0932] ESI-MS (m / z): 157.0 [M+H] + .

[0933] Step 5: Synthesis of methyl 4-((triphenylmethoxy)methyl)furan-2-carboxylate (F-9-6)

[0934] Methyl 4-(hydroxymethyl)furan-2-carboxylate (191 mg, 1.22 mmol) was dissolved in pyridine (2.00 mL), triphenylmethane (341 mg, 1.22 mmol) was added, and the mixture was heated to 80°C for 12 hours. 0.5 M dilute hydrochloric acid (20.0 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (20.0 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (517 mg, crude product), which was used directly in the next step without purification.

[0935] Step 6: Synthesis of 4-((triphenylmethoxy)methyl)furan-2-carboxylic acid (F-9-7)

[0936] Methyl 4-((triphenylmethoxy)methyl)furan-2-carboxylate (517 mg, 1.30 mmol) was dissolved in a mixture of 4.00 mL, methanol (4.00 mL), and water (4.00 mL). Lithium hydroxide monohydrate (163 mg, 3.89 mmol) was added, and the mixture was heated to 40°C and stirred for 1 hour. Water (30.0 mL) was added to the reaction solution, 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 three times with dichloromethane (10.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (98.0 mg, crude product), which was used directly in the next step without purification.

[0937] 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)

[0938] 4-((Triphenylmethoxy)methyl)furan-2-carboxylic acid (65.0 mg, 169 μmol) (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 thioate (69.8 mg, 169 μmol) was dissolved in DMF (2.00 mL). HATU (64.3 mg, 169 μmol) and DIPEA (65.6 mg, 507 μmol) were added, and the mixture was stirred at 25°C for 2 hours. Then, fluoroiodomethane (27.1 mg, 169 μmol) was added, and stirring was continued for 3 hours. After the reaction mixture was concentrated, saturated aqueous sodium chloride solution (30.0 mL) was added and extracted twice with ethyl acetate (30.0 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude title compound (234 mg, crude product), which was used directly in the next step without purification.

[0939] 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)

[0940] (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 (315 mg, 388 μmol) was dissolved in a mixed solvent of dichloromethane (3.00 mL) and methanol (3.00 mL). Trifluoroacetic acid (4.61 g, 40.4 mmol) was added, and the mixture was stirred at 20°C for 1 hour. The reaction solution was directly concentrated to obtain a crude product, which was purified by HPLC to obtain the title compound (18.0 mg, 0.031 mmol).

[0941] Chromatographic column: Phenomenex luna C18 150mm×25mm×10μm

[0942] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0943] Its structural characterization is as follows:

[0944] ESI-MS (m / z): 569.3 [M+H] + .

[0945] 1H NMR(400MHz,CD3OD)δ7.72(d,J=0.8Hz,1H),7.34(dd,J=1.4,10.2Hz,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=3.6,10.4Hz,1H),2.74-2.53(m,1H),2.37(dd,J=3.4,10.8Hz,3H),2.05-1.96(m,2H),1.74 -1.63(m,1H),1.59(s,3H),1.37(dd,J=7.8,11.8Hz,1H),1.17(s,3H),1.02(d,J=7.4Hz,3H).

[0946] 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)

[0947] 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)

[0948] Under a nitrogen atmosphere, 4-((triphenylmethoxy)methyl)furan-2-carboxylic acid (850 mg, 2.21 mmol) was dissolved in DMF (30.0 mL), and DIPEA (857 mg, 6.63 mmol) and HATU (841 mg, 2.21 mmol) were added, followed by stirring 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-carboxylic acid thioate (881 mg, 2.21 mmol) was added to the reaction system, and stirring was continued for 2 hours. Then, fluoroiodomethane (354 mg, 2.21 mmol) was added and stirring continued for 1 hour. The reaction solution was poured into water (100 mL) and stirred for 30 minutes. The mixture was filtered and the filter cake was washed with water three times (10.0 mL x 3) and dried under vacuum to obtain the title compound (1.40 g, 1.76 mmol), which was used in the next step without purification.

[0949] Its structural characterization is as follows:

[0950] ESI-MS (m / z): 796.3 [M+H] + .

[0951] 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)

[0952] (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 (900 mg, 1.13 mmol) was dissolved in dichloromethane (6.00 mL) and methanol (2.00 mL). Trifluoroacetic acid (1.54 g, 13.5 mmol) was added, and the mixture was stirred at 25°C for 2 hours. The reaction solution was directly concentrated to obtain a crude product, which was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 1 to 10 / 1) and concentrated again to obtain the title compound (461 mg, 803.34 μmol).

[0953] Its structural characterization is as follows:

[0954] ESI-MS (m / z): 555.0 [M+H] + .

[0955] 1 H NMR (400MHz, CD3OD) δ7.68 (s, 1H), 7.31 (d, J = 10.2Hz, 1H), 7.20 (s, 1H), 6.2 5-6.34(m,2H),5.66-5.95(m,2H),5.44-5.61(m,1H),4.44(s,2H),4.32(br d,J=8.4Hz,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),2.06-2.1 3(m,1H),1.97(d,J=14.2Hz,1H),1.72-1.80(m,1H),1.64(d,J=12.8Hz,1H),1.55(s,3H),1.51(s,1H),1.01(s,3H).

[0956] 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)

[0957] Step 1: Synthesis of methyl 5-((triphenylmethoxy)methyl)furan-2-carboxylate (F-13-2)

[0958] Dissolve methyl 5-(hydroxymethyl)furan-2-carboxylate (200 mg, 1.28 mmol) in pyridine (2.00 mL). Add triphenylmethane (357 mg, 1.28 mmol). Heat to 90°C and stir for 12 hours. Add 0.5M dilute hydrochloric acid (20.0 mL) to the reaction solution, and extract with dichloromethane (20.0 mL). Dry the organic phase over anhydrous sodium sulfate, filter, and concentrate to obtain the crude title compound (621 mg, crude product), which is used directly in the next step without purification.

[0959] Step 2: Synthesis of 5-((triphenylmethoxy)methyl)furan-2-carboxylic acid (F-13-3)

[0960] 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 stirred at 25°C for 3 hours. Water (30.0 mL) was added to the reaction solution, 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 three times with dichloromethane (10.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (246 mg, crude product), which was used directly in the next step without purification.

[0961] 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]phenanthrene-17-5-((triphenylmethoxy)methyl)furan-2-carboxylate (F-13-4)

[0962] 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-carboxylic acid (196 mg, 476 μmol) were dissolved in DMF (10.0 mL). DIPEA (184 mg, 1.43 mmol) and HATU (181 mg, 476 μmol) were added, and the mixture was stirred at 25°C for 2 hours. Then, fluoroiodomethane (76.1 mg, 476 μmol) was added, and stirring was continued for 3 hours. After the reaction mixture was concentrated, saturated aqueous sodium chloride solution (30.0 mL) was added and extracted twice with ethyl acetate (30.0 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude title compound (488 mg, crude product), which was used directly in the next step without purification.

[0963] 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)

[0964] (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). Trifluoroacetic acid (6.14 g, 53.9 mmol) was added, and the mixture was stirred at 25°C for 1 hour. The reaction solution was directly concentrated to obtain a crude product, which was purified by HPLC to obtain the title compound (53.8 mg, 93.9 μmol).

[0965] Chromatographic column: Welch Xtimate C18 150mm×25mm×5μm

[0966] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0967] Its structural characterization is as follows:

[0968] ESI-MS (m / z): 569.3 [M+H] + .

[0969] 1H NMR (400MHz, CD3OD) δ7.34(d,J=10.0Hz,1H),7.13(d,J=3.4Hz,1H),6.50(d,J=3.4Hz,1 H),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=13.0Hz,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=7.2Hz,3H).

[0970] 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-yl-furan-2-carboxylate (F-21)

[0971] 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-6,7,8,9,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl-furan-2-carboxylate (F-21-2)

[0972] 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, followed by stirring 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 (400 mg, 969 μmol) was added to the reaction mixture and stirred for 1 hour. Then, 1-iodo-2-(methoxymethoxy)ethane (209 mg, 969 μmol) was added and stirred for another 1 hour. Water was added to the reaction mixture to precipitate a solid, which was filtered and the filter cake dried under vacuum to obtain the crude title compound (458 mg, crude product) which was used directly in the next step without purification.

[0973] Its structural characterization is as follows:

[0974] ESI-MS (m / z): 595.2 [M+H] + .

[0975] 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-yl-furan-2-carboxylate (F-21)

[0976] (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-6,7,8,9,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-yl-furan-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 stirred at 25°C for 1 hour. The reaction mixture was concentrated to obtain the crude product, which was purified by preparative HPLC to obtain the title compound (86.9 mg, 154 μmol).

[0977] Chromatographic column: Welch Xtimate C18 200mm×40mm×10μm

[0978] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0979] Its structural characterization is as follows:

[0980] ESI-MS (m / z): 551.1 [M+H] + .

[0981] 1 H NMR (400MHz, DMSO-d6) δ8.00(d,J=1.2Hz,1H),7.27(d,J=10.8Hz,1H),7.18(d,J=3.2Hz,1 H),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,1 H),3.47-3.51(m,3H),3.00(t,J=6.8Hz,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).

[0982] 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)

[0983] 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]-17-yl(E)-3-(3-nitro-4-fluorophenyl)acrylate (E-4-1)

[0984] To dichloromethane (10 mL) were added (E)-3-(4-fluoro-3-nitrophenyl)prop-2-enoic acid (263 mg, 1.25 mmol), pyridine (344.84 mg, 4.36 mmol), and a 50% DMF solution of 1-propylphosphonic anhydride (2.30 g, 7.22 mmol). The 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-carboxylic acid thioate (71 mg, 181.76 mmol) was added. The mixture was reacted at room temperature for 3 hours. Fluoroiodomethane (400 mg, 2.50 mmol) was added and the reaction was allowed to proceed at room temperature. After the reaction was complete, water (30 mL) and dichloromethane (20 mL x 2) were added for extraction. The mixture was dried and 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 crude title compound (94 mg).

[0985] Its structural characterization data are as follows:

[0986] MS m / z(ESI):624.1[M+H] + .

[0987] 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)

[0988] To ethanol (1 mL) and water (0.3 mL) 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]-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). After the addition was complete, the mixture was heated to 80°C for reaction. After the reaction was completed, water (8 mL) was added, extracted with ethyl acetate (6 mL x 2), filtered through celite, and the organic phase was dried. After concentration to remove the solvent, it was directly purified by preparative HPLC and freeze-dried to obtain the title compound (2.46 mg, 0.004 mmol).

[0989] Its structural characterization is as follows:

[0990] MS m / z(ESI):594.2[M+H] + .

[0991] The purification method is as follows:

[0992] Column: Waters SunFire Prep C18 OBD (5 μm x 19 mm x 150 mm)

[0993] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0994] 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)

[0995] 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)

[0996] 4-(((tert-Butyldimethylsilyl)oxy)methyl)benzoic acid (70 mg, 262.76 μmol) was added to DMF (8.00 mL), followed by HATU (99.91 mg, 262.76 μmol) and DIPEA (50.94 mg, 394.15 μmol), and the mixture was 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 system. EA (50.94 mg, 394.15 μmol), the reaction solution was stirred at 45 ° C for 1.5 hours, fluoroiodomethane (210.11 mg, 1.31 mmol) was added dropwise to the reaction system, and then DIPEA (102 mg, 788.28 μmol) was added. After the reaction solution was stirred at 45 ° C for 45 minutes, water and EA were added for extraction, and the crude product was concentrated to obtain the title compound (14 mg, 20.20 μmol).

[0997] Chromatographic column: Phenomenex C18 250mm×50mm×10μm

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

[0999] Its structural characterization is as follows:

[1000] ESI-MS (m / z): 693.4 (M+H) + .

[1001] 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)

[1002] (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). The reaction solution was stirred at 25°C for 1 hour and then concentrated to give the crude product, which was purified by preparative HPLC to give the title compound (7 mg, 11.98 μmol).

[1003] Chromatographic column: Phenomenex C18 250mm×50mm×10μm

[1004] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1005] Its structural characterization is as follows:

[1006] ESI-MS (m / z): 579.3 (M+H) + .

[1007] 1H NMR (400MHz, DMSO) δ7.83(d,J=8.4Hz,2H),7.49(d,J=8.4Hz,2H),7.29(d,J=10.0Hz,1H),6.33(dd,J= 1.6Hz,10.0Hz,1H),6.14(s,1H),6.03(s,1H),5.90(s,1H),5.80-5.56(m,2H),5.38(t,J=6.0Hz,1H),4 .56(d,J=5.6Hz,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),2.0 2-1.88(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=6.8Hz,3H).

[1008] 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)

[1009] 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)

[1010] 3-(((tert-Butyldimethylsilyl)oxy)methyl)benzoic acid (280 mg, 1.05 mmol) was added to DMF (15.00 mL), followed by HATU (399.64 mg, 1.05 mmol) and DIPEA (203.75 mg, 1.58 mmol), and the mixture was 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 system, and DIPEA (203.75 mg, 1.58 mmol) was added. After the reaction solution was stirred at 45°C for 1.5 hours, fluoroiodomethane (672.36 mg, 4.20 mmol) was added dropwise to the reaction system, and DIPEA (407.50 mg, 3.16 mmol) was added. The reaction mixture was stirred at 45°C for 2 hours. The reaction was complete as monitored by LC-MS. Water and EA were added for extraction and concentrated to obtain a crude product. The crude product was purified by HPLC to obtain the title compound (35 mg, 50.51 μmol).

[1011] Chromatographic column: Phenomenex C18 250mm×50mm×10μm

[1012] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1013] Its structural characterization is as follows:

[1014] ESI-MS (m / z): 693.4 (M+H) + .

[1015] 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)

[1016] (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). The reaction solution was stirred at 25°C for 1.5 hours. The reaction was completed as monitored by LC-MS. The crude product was concentrated to give the title compound (11 mg, 18.82 μmol). The crude product was purified by preparative HPLC to give the title compound (11 mg, 18.82 μmol).

[1017] Chromatographic column: Phenomenex C18 250mm×50mm×10μm

[1018] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1019] Its structural characterization is as follows:

[1020] ESI-MS (m / z): 579.3 (M+H) + .

[1021] 1 H NMR (400MHz, DMSO) δ7.87(s,1H),7.74(d,J=7.6Hz,1H),7.61(d,J=8.0Hz,1H),7.52(d,J=7.6Hz,1H),7.29(dd,J =1.2Hz,10.4Hz,1H),6.33(dd,J=2.0Hz,10.0Hz,1H),6.14(s,1H),6.03(s,1H),5.90(s,1H),5.80-5.55(m,2H),5 .34(t,J=6.0Hz,1H),4.52(d,J=5.6Hz,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=6.8Hz,3H).

[1022] 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)

[1023] Step 1: Synthesis of (5-(methoxycarbonyl)furan-3-yl)boronic acid (F-17-2)

[1024] Methyl 4-bromofuran-2-carboxylate (2 g, 9.76 mmol), bis-pinacol boronate (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 atmosphere was purged with nitrogen, followed by the addition of Pd(dppf)Cl2 (722.38 mg, 975.58 μmol). The atmosphere was purged with nitrogen again, and the reaction was repeated three times. The mixture was then placed in an 80°C oil bath to react for 24 hours. After completion of the reaction, the reaction mixture was cooled to room temperature, filtered through a pad of Celite, and the filter cake was washed with ethyl acetate. The filtrate was then directly spin-dried to dryness. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0% to 40%) and concentrated again to yield the title compound (4.6 g, 9.12 mmol) (overweight, residual boronate).

[1025] ESI-MS (m / z): 253.2 (M+H) + .

[1026] Step 2: Synthesis of methyl 4-hydroxyfuran-2-carboxylate (F-17-3)

[1027] (5-(Methoxycarbonyl)furan-3-yl)boronic acid (4.7 g, 27.66 mmol) was dissolved in THF (40 mL). H₂O₂ (40.00 g, 352.79 mmol, 40 mL, 30% purity) was slowly added dropwise. Excessive heat was released, and the reaction was allowed to proceed for 5 hours. After completion, 20 mL of water was added to the reaction solution, which was then extracted twice with ethyl acetate (10 mL x 2). The organic phase was washed alternately with saturated aqueous sodium sulfite and saturated aqueous sodium chloride until the separated aqueous layer no longer showed a blue color using potassium iodide (PI) paper. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to yield the crude title compound (1.54 g, 10.84 mmol), which was used directly in the next step without purification.

[1028] ESI-MS (m / z): 143.0 (M+H) + .

[1029] Step 3: Synthesis of methyl 4-(methoxymethoxy)furan-2-carboxylate (F-17-4)

[1030] Methyl 4-hydroxyfuran-2-carboxylate (1.64 g, 11.54 mmol) was dissolved in DMF (50 mL) and cooled to 0°C under nitrogen. NaH (553.89 mg, 13.85 mmol, 60% purity) was then added portionwise, causing the mixture to change from orange-yellow to brick-red. The mixture was stirred at 0°C for 30 minutes. A solution of bromomethyl methyl ether (2.88 g, 23.08 mmol) in DMF (1 mL) was slowly added dropwise at 0°C, causing the mixture to gradually turn yellow. The mixture was allowed to react for 30 minutes. After the addition, water (100 mL) was slowly added to the reaction mixture to quench the reaction. The mixture was extracted three times with ethyl acetate (50 mL x 3). The organic phases were combined, washed with saturated brine, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0% to 20%) and concentrated again to yield the title compound (400 mg, 2.15 mmol).

[1031] ESI-MS (m / z): 187.1 (M+H) + .

[1032] Step 4: Synthesis of 4-(methoxymethoxy)furan-2-carboxylic acid (F-17-5)

[1033] 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, the reaction mixture was directly dried, water (5 mL) was added, and the pH was adjusted to 4-5 with 2N dilute hydrochloric acid. Extraction with ethyl acetate was ineffective. The organic phase was concentrated under reduced pressure and combined with the aqueous phase. The title compound (180 mg, 1.05 mmol) was obtained by preparative purification via HPLC.

[1034] The purification conditions are as follows:

[1035] Column: Waters Sunfire Prep C18 OBD (5μmx19mmx150mm)

[1036] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1037] Its structural characterization is as follows:

[1038] ESI-MS (m / z): 173.1 [M+H] + .

[1039] 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)

[1040] 4-(Methoxymethoxy)furan-2-carboxylic acid (85 mg, 493.80 μmol) was dissolved in DMF (10 mL), and DIPEA (191.46 mg, 1.48 mmol) and HATU (187.76 mg, 493.80 μmol) were added. The temperature was raised to 45° C. and the mixture was stirred 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-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylic acid (217.50 mg, 493.80 μmol) in DMF (2 mL) was added and stirred at room temperature for 16 hours. After completion of the reaction, water (60 mL) was added to the reaction solution, and the pH was adjusted to 4-5 with 0.5 M aqueous citric acid. A large amount of white flocculent material precipitated. The solid was filtered, the filter cake was collected, and vacuum dried to obtain the crude title compound (220 mg, 399.60 μmol), which was used directly in the next reaction without purification.

[1041] ESI-MS (m / z): 551.2 [M+H] + .

[1042] 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-carboxylic acid dimethylaminothiocarboxylic anhydride (F-17-7)

[1043] (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). Dimethylaminothiocarbonyl chloride (56.13 mg, 454.10 μmol) was added in portions under nitrogen protection. After the addition was complete, The reaction was stirred at room temperature for 4 hours. After completion of the reaction, water (10 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (5 mL x 3). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain the crude title compound (110 mg, 172.50 μmol), which was used directly in the next step without purification.

[1044] ESI-MS (m / z): 639.3 (M+H) + .

[1045] 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)

[1046] (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 dimethylaminothiocarboxylic anhydride (110 mg, 172.50 μmol) was dissolved in DMAc (4.0 mL). NaSH (29.01 mg, 517.50 μmol) was added portionwise. The system instantly turned dark green. After addition, the reaction was stirred at room temperature for 3 hours. LCMS monitored the remaining starting material. NaSH (29.01 mg, 517.50 μmol) was added and stirred at room temperature for another 0.5 hour. LCMS monitored the reaction, indicating that the starting material had reacted completely and the thiocarboxylic acid intermediate was evident. Fluoroiodomethane (275.87 mg, 1.72 mmol) was added dropwise, and the system instantly turned khaki. Stir at room temperature for 0.5 hour. After the reaction was complete, water (10 mL) was added to the reaction solution. Ethyl acetate (10 mL x 2) was added for extraction. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by thin-layer chromatography (ethyl acetate / petroleum ether = 1:1) to obtain the title compound (86 mg, 143.66 μmol).

[1047] ESI-MS (m / z): 599.2 (M+H) + .

[1048] 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)

[1049] 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) was added a hydrogen chloride-1,4-dioxane solution (4 mL). The mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction solution was directly concentrated under reduced pressure. The crude product was purified by preparative HPLC to obtain the title compound (180 mg, 1.05 mmol).

[1050] The preparation conditions are as follows:

[1051] Column: Waters Sunfire Prep C18 OBD (5μm x19mm x150mm)

[1052] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1053] Its structural characterization is as follows:

[1054] ESI-MS (m / z): 555.3 [M+H] + .

[1055] 1 H NMR (400MHz, DMSO) δ9.13(s,1H),7.50(d,J=1.2Hz,1H),7.28(dd,J=10.2,1.6Hz,1H),6.86(d,J=1.1Hz,1H),6.3 0(dd,J=10.4,1.6Hz,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=7.2Hz,1H),3.37(ddd,J=10.6,7.2,3.6Hz,1H),2.61(ddd,J=21.6,16.4,3.2Hz,1H),2.35–2.19(m,3H),1.95( dd,J=22.4,11.6Hz,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=7.2Hz,3H).

[1056] 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)hexyl-5-ynylamino)propionylamino)propionylamino)benzoate (DL-A-03)

[1057] (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-amino)-3-((S ... To DMF (2.00 mL) was added 4-fluorobenzoate (25 mg, 30.35 μmol), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoate (11.09 mg, 30.35 μmol), and DIPEA (11.77 mg, 91.04 μmol). 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 preparative HPLC to obtain the title compound (18 mg, 18.56 μmol).

[1058] The purification method is as follows:

[1059] Chromatographic column: Phenomenex C18 250mm×50mm×10μm

[1060] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1061] Its structural characterization is as follows:

[1062] ESI-MS (m / z): 960.3 (M+H) + .

[1063] 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-trioxy-12,15,18,21-tetraoxy-5,8,24-triazatriacontacosa-nonacosynoic acid (DL-A-47)

[1064] Step 1: Preparation of tert-butyl 22-(2-(methylsulfonyl)pyrimidin-5-yl)-17-oxo-4,7,10,13-tetraoxo-16-azadocosa-21-ynoate (DL-A-47-2)

[1065] 1-Amino-3,6,9,12-tetraoxopentadecane-15-oic acid tert-butyl ester (117 mg, 364.02 μmol), 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoate (146.30 mg, 400.43 μmol), and DIPEA (94.09 mg, 728.05 μmol) were added sequentially to DMF (2 mL) and stirred at 25°C for 3 hours. Water (8 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (8 mL x 3). The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and filtered, concentrated, and used directly in the next step without purification.

[1066] Step 2: Preparation of 22-(2-(methylsulfonyl)pyrimidin-5-yl)-17-oxo-4,7,10,13-tetraoxo-16-azadocosa-21-ynoic acid (DL-A-47-3)

[1067] Tert-butyl 22-(2-(methylsulfonyl)pyrimidin-5-yl)-17-oxo-4,7,10,13-tetraoxo-16-azadocosa-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 solution was directly concentrated under reduced pressure to remove dichloromethane and placed on a lyophilizer to remove trifluoroacetic acid to obtain the crude title compound (86 mg), which was used directly in the next step without purification.

[1068] Step 3: 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]phenanthrene-17-yl)oxy)carbonyl-2-fluorophenyl)carbamoyl)-30-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,25-trioxy-12,15,18,21-tetraoxy-5,8,24-triazatriacontacosa-nonacosynoic acid tert-butyl ester (DL-A-47-4)

[1069] To DMF (2 mL) 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-(2-aminoacetamido) The reaction mixture was stirred at 16°C for 1.5 h. Purified water (8 mL) was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate (6 mL x 2). The organic phase was washed once with saturated brine (4 mL). The solvent was removed under reduced pressure to obtain the crude title compound (130 mg), which was used in the next step without purification.

[1070] 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-trioxy-12,15,18,21-tetraoxy-5,8,24-triazatriacontacosa-nonacosynoic acid (DL-A-47)

[1071] (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)- Crude tert-butyl 30-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,25-trioxy-12,15,18,21-tetraoxy-5,8,24-triazatriacontacosa-nonacosynoate (51 mg) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (0.5 mL) was added. After addition, the mixture was stirred at 16°C for 3 h. The reaction solution was directly purified by HPLC to obtain the title compound (17.51 ​​mg, 13.85 μmol).

[1072] The purification method is as follows:

[1073] Chromatographic column: SunFire Prep C18 OBD 19*150mm*5μm

[1074] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% TFA)

[1075] Its structural characterization data are as follows:

[1076] ESI-MS (m / z): 1251.4 (M+H) + .

[1077] 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-decafluoro- dihydro-3H-cyclopenta[a]phenanthren-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-octaoxo-5,8,36-triazatetradecane-41-ynoic acid (DL-A-48)

[1078] Step 1: Preparation of tert-butyl 34-(2-(methylsulfonyl)pyrimidin-5-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatetratriacontanoate-ynoate (DL-A-48-1)

[1079] 2,5-Dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoate (100 mg, 0.274 mmol), tert-butyl 1-amino-3,6,9,12,15,18,21,24-octa-oxaheptacosane-27-oate (136.2 mg, 0.274 mmol), and DIPEA (70.8 mg, 0.547 mmol) were added to THF (2 mL) and reacted at 25°C for 2 hours. Ethyl acetate (20 mL) and water (6 mL) were added to the reaction solution and stirred for 5 minutes. The organic phase was separated, washed with saturated aqueous sodium chloride (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the title compound (174 mg), which was used directly in the next step without purification.

[1080] Its structural characterization data are as follows:

[1081] ESI-MS (m / z): 748.4 (M+H) + .

[1082] Step 2: Preparation of 34-(2-(methylsulfonyl)pyrimidin-5-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatetratriacontanoic acid (DL-A-48-2)

[1083] 34-(2-(Methylsulfonyl)pyrimidin-5-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatetratriacontanoic acid tert-butyl ester (174 mg, 232.65 μmol, FR) and trifluoroacetic acid (530.54 mg, 4.65 mmol) were added to dichloromethane (3 mL), the temperature was raised to 35 ° C, and the reaction was carried out for 2 hours. The reaction solution was directly concentrated to obtain the title compound (185 mg), which was used directly in the next step without purification.

[1084] Its structural characterization data are as follows:

[1085] ESI-MS (m / z): 692.4 (M+H) + .

[1086] Step 3: (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] Preparation of tert-butyl phenanthren-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-octaoxo-5,8,36-triazatetradecane-41-ynoate (DL-A-48-1)

[1087] To DMF (2 mL) 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-(2-aminoacetamido)-5-(tert-butyloxy)-5-oxopentanamido To the reaction mixture were added 4-(2-(methylsulfonyl)pyrimidin-5-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxo-28-azatetratriacontane-33-ynoic acid (36.73 mg, 53.10 μmol), DIPEA (20 mg, 154.7 μmol), and HATU (24.23 mg, 63.71 μmol). The mixture was stirred at 16°C for 1.5 h. Water (8 mL) was added to the reaction solution, and the mixture was extracted twice with ethyl acetate (6 mL x 2). The organic phase was washed once with saturated brine (6 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (40 mg), which was used directly in the next step without purification.

[1088] Its structural characterization data are as follows:

[1089] ESI-MS (m / z): 1483.6 (M+H) + .

[1090] 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- Preparation of 3H-cyclopenta[a]phenanthren-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-octaoxo-5,8,36-triazatetradecane-41-ynoic acid (DL-A-48)

[1091] (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 Tert-butyl 4-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,37-trioxo-12,15,18,21,24,27,30,33-octaoxo-5,8,36-triazatetradecane-41-ynoate (40 mg) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (1 mL) was added. After the addition was complete, the mixture was stirred at 15°C for 2 h. The reaction solution was directly purified by HPLC to obtain the title compound (16.54 mg, 11.01 μmol).

[1092] The purification method is as follows:

[1093] Chromatographic column: SunFire Prep C18 OBD 19*150mm*5μm

[1094] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% TFA)

[1095] Its structural characterization data are as follows:

[1096] ESI-MS (m / z): 1427.6 (M+H) + .

[1097] 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-tetraazaoctacosane-27-ynamide)-4-fluorobenzoate (DL-A-49)

[1098] Step 1: Preparation of tert-butyl 19-(2-(methylsulfonyl)pyrimidin-5-yl)-14-oxo-4,7,10-trioxo-13-azacyclononadecan-18-ynoate (DL-A-49-2)

[1099] 2,5-Dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoate (200 mg, 0.547 mmol), tert-butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate (151.8 mg, 0.547 mmol) and DIPEA (141.5 mg, 1.09 mmol) were added to DMF (3 mL) and reacted at 25 ° C for 2 hours. EA (30 ml) and water (10 ml) were added and stirred for 5 minutes. The organic phase was separated and concentrated to give a crude product, which was purified by column chromatography (MeOH / DCM = 5-20%) to give the title compound (260 mg, 443.49 μmol) and used directly in the next step without purification.

[1100] Its structural characterization data are as follows:

[1101] ESI-MS (m / z): 528.2 [M+H] + .

[1102] Step 2: Preparation of 19-(2-(methylsulfonyl)pyrimidin-5-yl)-14-oxo-4,7,10-trioxo-13-azacyclononadecan-18-ynoic acid (DL-A-49-3)

[1103] Tert-butyl 19-(2-(methylsulfonyl)pyrimidin-5-yl)-14-oxo-4,7,10-trioxo-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 temperature was raised to 35-40°C for 3 hours. The mixture was concentrated, and ACN (10 ml) and deionized water (5 ml) were added to the residue for dispersion. The title compound (290 mg, 492.02 μmol) was obtained by lyophilization and used directly in the next step without purification.

[1104] Its structural characterization data are as follows:

[1105] ESI-MS (m / z): 472.2 [M+H] + .

[1106] 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- Preparation of ((tert-Butyloxycarbonyl)amino)butyl)-5-(hydroxymethyl)-28-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,23-tetraoxo-13,16,19-trioxo-3,6,9,22-tetraazaoctacosane-27-ynamide)-4-fluorobenzoate (DL-A-49-4)

[1107] 19-(2-(methylsulfonyl)pyrimidin-5-yl)-14-oxo-4,7,10-trioxo-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), (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-hydroxypropionamido)-6-((tert-butoxycarbonyl)amino)hexanoylamino)-4-fluorobenzoate (50.0 mg, 0.051 mmol, formate) was reacted at 25°C for 1 h. EA (10 ml) / H2O (3 ml) was added and stirred for 5 minutes. The organic phase was separated and concentrated to obtain the crude product, which was purified by thin layer chromatography (MeOH / DCM = 10%) to give the title compound (41 mg, 27.95 μmol).

[1108] Its structural characterization data are as follows:

[1109] ESI-MS (m / z): 1393.4 [M+H] + .

[1110] 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-tetraazaoctacosane-27-ynamide)-4-fluorobenzoate (DL-A-49)

[1111] (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-butyloxycarbonyl)amino)butyl)-5-(hydroxymethyl)-28-(2-(methylsulfonyl)amino)-1-hydroxy-1-hydroxy-1-hydroxy-1-hydroxy-1-hydroxy-1-hydroxy-1-hydroxy-1-hydroxy-1-hydroxy-1-hydroxy-1-hydroxy-1-hydroxy-1-hydroxy-1-hydroxy-1-hydroxy-1-hydroxy-1-hydroxy-1-hydroxy-1-hydroxy-1-hydroxy-1-hydroxy-1-hydroxy-1-hydroxy-1-hydroxy-1-hydroxy-1 To the mixture of 4-fluorobenzoic acid ester (41.0 mg, 0.029 mmol) and 5-(4-yl)pyrimidin-5-yl)-4,7,10,23-tetraoxo-13,16,19-trioxo-3,6,9,22-tetraazaoctacosane-27-ynamide) was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (167.7 mg, 1.47 mmol) was added. The mixture was reacted at 25°C for 1 h, and the crude product was concentrated to give the crude product, which was purified by Pre-HPLC to give the trifluoroacetate salt of the title compound (22.9 mg, 15.78 μmol).

[1112] The purification method is as follows:

[1113] Chromatographic column: SunFire Prep C18 OBD 19*150mm*5μm

[1114] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1115] Its structural characterization data are as follows:

[1116] ESI-MS (m / z): 1293.5 (M+H) + .

[1117] Preparation Example 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]phenanthrene-17-yl- 3-((2S,5S)-2-(4-aminobutyl)-5-(hydroxymethyl)-4,3-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,10,38-tetraoxo-13,16,19,22,25,28,31,34-octaoxo-3,6,9,37-tetraazatricarboxane-42-ynamide)-4-fluorobenzoate (DL-A-50)

[1118] 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 Preparation of 4-(tert-butyloxycarbonyl)amino)butyl)-5-(hydroxymethyl)-4,3-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,38-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,6,9,37-tetraazatricarboxane-42-ynamide)-4-fluorobenzoate (DL-A-50-1)

[1119] 34-(2-(methylsulfonyl)pyrimidin-5-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxo-28-azatetratriacontane-33-ynoic acid (28.9 mg, 0.033 mmol) was dissolved in DMF (1 mL), and HATU (23.1 mg, 0.061 mmol), DIPEA (11.8 mg, 0.091 mmol), (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-hydroxypropionamido)-6-((tert-butoxycarbonyl)amino)hexanoylamino)-4-fluorobenzoate (30.0 mg, 0.030 mmol, formate) was reacted at 25°C for 1 hour. Ethyl acetate (10 ml) and water (3 ml) were added and stirred for 5 minutes. The organic phase was separated and concentrated to obtain the 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).

[1120] Its structural characterization data are as follows:

[1121] ESI-MS (m / z): 1613.7 (M+H) + .

[1122] 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-( Preparation of (2S,5S)-2-(4-aminobutyl)-5-(hydroxymethyl)-4,3-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,10,38-tetraoxo-13,16,19,22,25,28,31,34-octaoxo-3,6,9,37-tetraazatricarboxane-4,2-ynamide)-4-fluorobenzoate (DL-A-50)

[1123] (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-((2 S,5S)-2-(4-(tert-Butyloxycarbonyl)amino)butyl)-5-(hydroxymethyl)-4,3-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,10,38-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,6,9,37-tetraazatricarboxane-42-ynamide)-4-fluorobenzoate (17.0 mg, 0.010 mmol) was dissolved in dichloromethane (0.5 mL), and trifluoroacetic acid (60.0 mg, 0.527 mmol) was added. The reaction was carried out 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).

[1124] The purification method is as follows:

[1125] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1126] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1127] Its structural characterization data are as follows:

[1128] ESI-MS (m / z): 1513.6 (M+H) + .

[1129] 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]phenanthrene-17-yl)oxy)-4-oxobutan-2-yl)oxy)methyl)amino)-4-(2-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynylamino)acetamido)-5-oxopentanoic acid (DL-A-139)

[1130] 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)

[1131] To tetrahydrofuran (40 mL) was added N,N'-dicyclohexylcarbodiimide (2.22 g, 10.75 mmol), (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(allyloxy)-5-oxopentanoic acid (4.0 g, 9.77 mmol), and N-hydroxysuccinimide (1.19 g, 10.34 mmol). After addition, the mixture was stirred at 16°C for 2 hours. The mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain the crude title compound (4.9 g, 9.67 mmol), which was used in the next step without purification.

[1132] Its structural characterization data are as follows:

[1133] ESI-MS (m / z): 507.2 (M+H) + .

[1134] Step 2: Preparation of (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(allyloxy)-5-oxopentanoyl)glycine (DL-A-139-3)

[1135] To acetone (50 mL) and water (50 mL) was added 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). The reaction was allowed to proceed at 15°C for 1 hour. Purified water (120 mL) was added, and the pH was adjusted to 3-4 with 1N dilute hydrochloric acid. The mixture was extracted three times with ethyl acetate (50 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (5.6 g), which was used directly in the next step without purification.

[1136] Its structural characterization data are as follows:

[1137] ESI-MS (m / z): 467.2 (M+H) + .

[1138] Step 3: Preparation of (S)-(5-(allyloxy)-2-amino-5-oxopentanoyl)glycine (DL-A-139-4)

[1139] Crude (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(allyloxy)-5-oxopentanoyl)glycine (5.6 g) was added to DMF (12 mL), followed by diethylamine (2.5 mL). After addition, the mixture was stirred at 16°C for 1 h. The reaction mixture was concentrated under reduced pressure to remove residual diethylamine, affording the crude title compound (2.3 g), which was used in the next step without purification.

[1140] Its structural characterization data are as follows:

[1141] ESI-MS (m / z): 245.1 (M+H) + .

[1142] Step 4: Preparation of (S)-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)-5-(allyloxy)-5-oxopentanoyl)glycine (DL-A-139-5)

[1143] To DMF (24 mL) was added crude (S)-(5-(allyloxy)-2-amino-5-oxopentanoyl)glycine (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). After addition, the mixture was stirred at 17°C for 3 hours. Water (110 mL) was added to the reaction solution, and impurities were extracted with ethyl acetate (15 mL x 2). The aqueous phase was adjusted to pH 3-4 with 1N dilute hydrochloric acid and extracted three times with ethyl acetate (50 mL x 3). The organic phase was dried and concentrated to dryness under reduced pressure to obtain the title compound (3.8 g, 7.25 mmol).

[1144] Its structural characterization data are as follows:

[1145] ESI-MS (m / z): 524.2 (M+H) + .

[1146] Step 5: Preparation of (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)-5-((acetoxymethyl)amino)-5-oxopentanoic acid allyl ester (DL-A-139-6)

[1147] To tetrahydrofuran (9 mL) and toluene (3 mL) was added (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). After addition, the atmosphere was replaced with nitrogen three times and the temperature was raised to 84°C for 3 hours. The reaction solution was filtered, purified by column chromatography (petroleum ether / ethyl acetate = 4 / 1 to 1 / 4), and concentrated again to obtain the title compound (177 mg, 296.34 μmol).

[1148] Its structural characterization data are as follows:

[1149] ESI-MS (m / z): 560.3 (M+H) + .

[1150] Step 6: Preparation of (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-((chloromethyl)amino)-5-oxopentanoic acid allyl ester (DL-A-139-7)

[1151] To DCM (2 mL) was added (S)-allyl 4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-((acetoxymethyl)amino)-5-oxopentanoate (80 mg, 148.82 μmol) and trimethylsilyl chloride (162 mg, 1.49 mmol). The reaction was allowed to proceed at 17°C for 0.5 h. The reaction solution was evaporated to dryness under reduced pressure to obtain the crude title compound (49 mg), which was used in the next step without purification.

[1152] 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-3,69-trioxo-2,12-dioxo-4,7,10-triazapentadecane-15-propionate (DL-A-139-8)

[1153] To DCM (4 mL) were added crude (S)-allyl 4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-((chloromethyl)amino)-5-oxopentanoate (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]phenanthrene-17-yl-3-hydroxybutyrate (30 mg, 56.54 μmol). After the addition was complete, the temperature was raised to reflux and the reaction was carried out for 5 hours. The reaction solution was quenched with methanol and purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 4 / 1 to 1 / 4) to give the crude title compound (39 mg), which was used in the next step without purification.

[1154] Its structural characterization data are as follows:

[1155] ESI-MS (m / z): 1008.4 (M+H) + .

[1156] Step 8: Preparation of (4S)-4-(2-aminoacetamide)-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-oxobutan-2-yl)oxy)methyl)amino)-5-oxopentanoic acid (DL-A-139-9)

[1157] To DMF (3 mL) was added (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-1 Crude 7-methyl(8S)-8-(3-(allyloxy)-3-oxopropyl)-1-(9H-fluoren-9-yl)-13-methyl-3,69-trioxo-2,12-dioxo-4,7,10-triazapentadecane-15-propionate (39 mg), diethylamine (16 mg, 218.7 μmol), and tetrakistriphenylphosphine palladium (13 mg, 11.25 μm) were added and reacted at 17°C for 1 hour. Residual diethylamine was removed from the reaction mixture under reduced pressure to obtain the crude title compound (28 mg), which was used directly in the next step without purification.

[1158] Its structural characterization data are as follows:

[1159] ESI-MS (m / z): 746.2 (M+H) + .

[1160] 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]phenanthrene-17-yl)oxy)-4-oxobutan-2-yl)oxy)methyl)amino)-4-(2-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynylamino)acetamido)-5-oxopentanoic acid (DL-A-139)

[1161] To DMF (3 mL) was added (4S)-4-(2-aminoacetamide)-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 Crude product of 5-oxopentadecanoic acid (28 mg), 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, 37.54-37.54 μmol) were added, and the mixture was allowed to react at 17°C for 1 hour. The reaction solution was purified by HPLC to obtain the title compound (9.49 mg, 9.24 μmol).

[1162] The purification method is as follows:

[1163] Chromatographic column: SunFire Prep C18 OBD 19*150mm*5μm

[1164] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% TFA)

[1165] Its structural characterization data are as follows:

[1166] ESI-MS (m / z): 996.3 (M+H) + .

[1167] 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-tetraazaoctatriacont-38-amino)furan-2-carboxylate (DL-B-22')

[1168] 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)hexanoylamino)furan-2-carboxylate (DL-B-22'-1)

[1169] 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, followed by stirring 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 stirring was continued at 25°C for 5 hours. Water (150 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (150 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude title compound (450 mg), which was used directly in the next step without purification.

[1170] Its structural characterization data are as follows:

[1171] ESI-MS (m / z): 904.0 (M+H-Boc) + .

[1172] 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)hexanoylamino)furan-2-carboxylate (DL-B-22'-2)

[1173] (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)hexanoylamino)furan-2-carboxylate (387 mg, 385 μmol) was dissolved in DMF (6.00 mL). DBU (58.6 mg, 385 μmol, 58.0 μL) was added and the mixture was stirred at 25°C for 1 hour. The reaction solution was used in the next step without treatment.

[1174] Its structural characterization data are as follows:

[1175] ESI-MS (m / z): 782.0 (M+H) + .

[1176] 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-trioxy-2-oxo-4,7,10-triazadodec-12-amino)furan-2-carboxylate (DL-B-22'-3)

[1177] The next step is (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- To a solution of ((tert-butoxycarbonyl)amino)hexanoylamino)furan-2-carboxylate (301 mg, 384 μmol) in DMF (6.00 mL) were added (((9H-fluoren-9-yl)methoxy)carbonyl)glycyl-L-serine (147 mg, 384 μmol) and HOBt (78.0 mg, 577 μmol), followed by EDCI (110 mg, 577 μmol), and the mixture was stirred at 25°C for 1 hour. Water (100 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (100 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound, which was purified by preparative HPLC and lyophilized to afford the title compound (33.4 mg, 28.5 μmol).

[1178] The purification method is as follows:

[1179] Chromatographic column: Phenomenex C18 (10μm*25mm*150mm)

[1180] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% ammonium bicarbonate)

[1181] Its structural characterization data are as follows:

[1182] ESI-MS (m / z): 1148.5 (M+H) + .

[1183] 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)hexanoylamino)furan-2-carboxylate (DL-B-22'-4)

[1184] (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) 1-(4-(tert-Butyloxycarbonyl)amino)butyl)-1-(9H-fluoren-9-yl)-8-(hydroxymethyl)-3,6,9-trioxy-2-oxo-4,7,10-triazadodec-12-amino)furan-2-carboxylate (30 mg, 26.13 μmol, FR) was dissolved in DMF (1 mL), and diethylamine (9.55 mg, 130.63 μmol) was added. The mixture was reacted at 25°C for 1 hour. The reaction solution was directly purified by HPLC and lyophilized to obtain the formate salt of the title compound (23.0 mg, 22.48 μmol).

[1185] The purification method is as follows:

[1186] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1187] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1188] Its structural characterization data are as follows:

[1189] ESI-MS (m / z): 926.4 (M+H) + .

[1190] 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,3 Preparation of 7S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-37-(4-(tert-butyloxycarbonyl)amino)butyl)-34-(hydroxymethyl)-1,29,32,35-tetraoxo-5,8,11,14,17,20,23,26-octaoxa-2,30,33,36-tetraazatriacont-38-amino)furan-2-carboxylate (DL-B-22'-5)

[1191] 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oic acid (22.4 mg, 0.026 mmol) was dissolved in DMF (1 mL), and HATU (18.9 mg, 0.050 mmol), DIPEA (9.6 mg, 0.074 mmol) and (6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11 4-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)hexanoylamino)furan-2-carboxylate (23.0 mg, 0.025 mmol, formate) was reacted at 25°C for 1 hour; the reaction solution was directly purified by HPLC and lyophilized to obtain the title compound (20 mg, 10.76 μmol).

[1192] The purification method is as follows:

[1193] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1194] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1195] Its structural characterization data are as follows:

[1196] ESI-MS (m / z): 1765.8 (M+H) + .

[1197] 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-((3 Preparation of 4S,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-tetraazaoctatriacont-38-amino)furan-2-carboxylate (DL-B-22')

[1198] (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-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-37-(4-(tert-butyloxycarbonyl)amino)butyl) 3,4-(Hydroxymethyl)-1,29,32,35-tetraoxo-5,8,11,14,17,20,23,26-octaoxa-2,30,33,36-tetraazaoctatriacont-38-amino)furan-2-carboxylate (20.0 mg, 0.011 mmol) was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (64.6 mg, 0.566 mmol) was added. The mixture was reacted at 25°C for 2 hours. The crude product was concentrated to obtain a crude product, which was purified by HPLC and lyophilized to obtain the trifluoroacetate salt of the title compound (12.2 mg, 6.65 μmol).

[1199] The purification method is as follows:

[1200] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1201] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1202] Its structural characterization data are as follows:

[1203] ESI-MS (m / z): 1665.7 (M+H) + .

[1204] Preparation Example 46:

[1205] (S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-34-(((((R)-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-6,7,8,9,10,1 Preparation of 1,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)oxy)-4-oxobutan-2-yl)oxy)methyl)carbamoyl)-1,29,32-trioxy-5,8,11,14,17,20,23,26,26-octaoxy-2,30,33-triazaheptatriacontane-37-ynoic acid (DL-B-65')

[1206] 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(8S,13R)-8-(3-(allyloxy)-3-oxopropyl)-1-(9H-fluoren-9-yl)-13-methyl-3,69-trioxo-2,12-dioxo-4,7,10-triazapentadecan-15-propionate (DL-B-65'-1)

[1207] To DCM (8 mL) were added crude (S)-allyl 4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-((chloromethyl)amino)-5-oxopentanoate (65 mg, 126.47 μmol), (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17 1-(((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 (67.10 mg, 126.47 μmol) was added and the mixture was heated to reflux for 6 hours. The reaction mixture was quenched with water (40 mL) and extracted twice with DCM (4 mL x 2). The organic phase was dried and concentrated to obtain the crude product, which was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 4 / 1 to 1 / 4) and concentrated again to obtain the title compound (27 mg, 26.78 μmol).

[1208] Its structural characterization data are as follows:

[1209] ESI-MS (m / z): 1008.4 (M+H) + .

[1210] Step 2: Preparation of (S)-4-(2-aminoacetamido)-5-(R)-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]phenanthrene-17-yl)oxy)-4-oxobutan-2-yl)oxy)methyl)amino)-5-oxopentanoic acid (DL-B-65'-2)

[1211] To DMF (3 mL) was added (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 (8 S,13R)-8-(3-(allyloxy)-3-oxopropyl)-1-(9H-fluoren-9-yl)-13-methyl-3,69-trioxo-2,12-dioxo-4,7,10-triazapentadecane-15-propionate (27 mg, 26.78 μmol), diethylamine (16 mg, 219 μmol), and tetrakistriphenylphosphine palladium (7 mg, 6.06 μmol) were added and reacted at 18°C ​​for 3 hours. Residual diethylamine was removed from the reaction mixture under reduced pressure to obtain the crude title compound (19 mg), which was used directly in the next step without purification.

[1212] Step 3: (S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-34-(((((R)-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 Preparation of 1,29,32-trioxy-5,8,11,14,17,20,23,26,26-octaoxy-2,30,33-triazaheptatriacontane-37-ynoic acid (DL-B-65')

[1213] To DMF (3 mL) was added (S)-4-(2-aminoacetamido)-5-(R)-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]phenanthrene-17-yl)-17-ol After the addition of 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-propionic acid succinimidyl ester (24.33 mg, 25.48 μmol), and DIPEA (6.59 mg, 50.95 μmol), the mixture was reacted at 18°C ​​for 2 hours. The reaction solution was directly purified by high performance liquid chromatography to obtain the title compound (1.44 mg, 0.899 μmol).

[1214] The purification method is as follows:

[1215] Chromatographic column: SunFire Prep C18 OBD 19*150mm*5μm

[1216] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% TFA)

[1217] Its structural characterization data are as follows:

[1218] ESI-MS (m / z): 793.5 (M / 2+H) + .

[1219] Preparation Example 47: (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-hydroxybenzoate (B-5)

[1220] Step 1: Preparation of methyl 3-(triphenylmethoxy)benzoate (B-5-2)

[1221] Dissolve methyl 3-hydroxybenzoate (2.00 g, 13.2 mmol) in pyridine (20.0 mL), add triphenylmethane chloride (3.66 g, 13.2 mmol), and heat to 80°C for 12 hours. Cool the reaction mixture to 25°C, add 1 M dilute hydrochloric acid (40.0 mL), and extract with dichloromethane three times (50.0 mL x 3). Combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate to obtain the crude title compound (6.80 g), which is used directly in the next step without purification.

[1222] Its structural characterization is as follows:

[1223] ESI-MS (m / z): 417.0 (M+Na) + .

[1224] Step 2: Preparation of 3-(triphenylmethoxy)benzoic acid (B-5-3)

[1225] Methyl 3-(triphenylmethoxy)benzoate (6.80 g, 17.2 mmol) was dissolved in a mixture of tetrahydrofuran (22.0 mL), methanol (22.0 mL), and water (22.0 mL). Lithium hydroxide monohydrate (2.17 g, 51.7 mmol) was added and the mixture was heated to 40°C for 1 hour. Water (30.0 mL) and ethyl acetate (20.0 mL) were added to the reaction solution, and the pH was adjusted to 2-3 with 1N dilute hydrochloric acid. The layers were separated, and the aqueous phase was extracted three times with ethyl acetate (30.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to afford the crude title compound (554 mg, 646 μmol), which was used in the next step without purification.

[1226] Its structural characterization is as follows:

[1227] ESI-MS (m / z): 381.0 (M+H) + .

[1228] Step 3: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-yl-3-(triphenylmethoxy)benzoate (B-5-4)

[1229] 3-(Triphenylmethoxy)benzoic acid (334 mg, 878 μmol) was dissolved in DMF (3.00 mL), and HATU (334 mg, 878 μmol) and DIPEA (340 mg, 2.63 mmol, 459 μL) were added and stirred for 1 hour. Then, (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-carboxylic acid thioate (140 mg, 351 μmol) was added to the reaction mixture and stirring was continued for 1 hour. Fluoroiodomethane (140 mg, 878 μmol) was then added to the reaction mixture and stirring continued for 0.5 hour. The reaction mixture was poured into water (30.0 mL), stirred for 30 minutes, and filtered. The filter cake was washed with water three times (10.0 mL x 3) and dried under vacuum to obtain the crude product. The product was purified on a silica gel column (dichloromethane / methanol = 100 / 1 to 10 / 1) and concentrated again to obtain the title compound (200 mg, 22.5 μmol).

[1230] Its structural characterization is as follows:

[1231] ESI-MS (m / z): 815.3 (M+Na) + .

[1232] 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]phenanthrene-17-yl-3-hydroxybenzoate (B-5)

[1233] (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-(triphenylmethoxy)benzoate (170 mg, 214 μmol) was dissolved in dichloromethane (3.00 mL). A hydrogen chloride / 1,4-dioxane solution (2 M, 1.00 mL) was added, and the mixture was stirred at 25°C for 1 hour. The reaction mixture was concentrated to obtain the crude product, which was purified by preparative HPLC to afford the title compound (2.83 mg, 3.03 μmol).

[1234] The purification method is as follows:

[1235] Chromatographic column: Phenomenex Luna C18 100mm×30mm×5mm

[1236] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% hydrochloric acid)

[1237] Its structural characterization is as follows:

[1238] ESI-MS (m / z): 551.0 [M+H] + .

[1239] 1 H NMR (400MHz, CD3OD) δ7.39 (s, 1H), 7.34-7.37 (m, 2H), 7.33 (dd, J = 2.4Hz, 1H), 7.0 2-7.06(m,1H),6.34-6.40(m,2H),5.75-5.95(m,2H),5.49-5.56(m,1H),4.34(d,J =1.2Hz,1H),3.04(d,J=7.2Hz,1H),2.61-2.67(m,1H),2.52(d,J=3.6Hz,1H),2.29 -2.35(m,2H),1.99-2.07(m,2H),1.65(s,1H),1.61(d,J=3.6Hz,5H),1.07(s,3H).

[1240] Preparation Example 48: (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-hydroxybenzoate (B-17)

[1241] Step 1: Preparation of methyl 4-(triphenylmethoxy)benzoate (B-17-2)

[1242] Methyl 4-hydroxybenzoate (1.50 g, 9.86 mmol) was dissolved in pyridine (15.0 mL), and triphenylmethane (2.75 g, 9.86 mmol) was added. The temperature was raised to 80°C and the reaction was allowed to react for 12 hours. The reaction solution was cooled to 25°C, and ethyl acetate (100 mL) and 1M dilute hydrochloric acid (100 mL) were added. The layers were separated, and the organic phase was washed with saturated aqueous sodium bicarbonate (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (9.21 g), which was used directly in the next step without purification.

[1243] Its structural characterization is as follows:

[1244] ESI-MS (m / z): 417.0 (M+Na) + .

[1245] Step 2: Preparation of 4-(triphenylmethoxy)benzoic acid (B-17-3)

[1246] Methyl 4-(triphenylmethoxy)benzoate (9.00 g, 22.8 mmol) was dissolved in a mixture of tetrahydrofuran (30.0 mL), methanol (30.0 mL), and water (30.0 mL). Lithium hydroxide monohydrate (2.87 g, 68.4 mmol) was added, and the mixture was heated to 45°C for 2 hours. Dichloromethane (100 mL) and water (100 mL) were added to the reaction solution, and the layers were separated. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (1.30 g), which was used directly in the next step without purification.

[1247] Its structural characterization is as follows:

[1248] ESI-MS (m / z): 403.0 (M+Na) + .

[1249] Step 3: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-yl-4-(triphenylmethoxy)benzoate (B-17-4)

[1250] 4-(Triphenylmethoxy)benzoic acid (50.0 mg, 52.5 μmol) was dissolved in DMF (2.00 mL), and HATU (19.9 mg, 52.5 μmol) and DIPEA (20.3 mg, 157 μmol, 27.4 μL) were added, followed by stirring for 1 hour. Then, (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-carboxylic acid thioate (16.7 mg, 42.0 μmol) was added to the reaction mixture, and stirring was continued for 1 hour. Then, fluoroiodomethane (8.41 mg, 52.5 μmol) was added to the reaction solution and stirring was continued for 0.5 h. Water (20.0 mL) was added to the reaction solution and the mixture was freeze-dried to obtain the crude title compound (60 mg), which was used in the next step without purification.

[1251] Its structural characterization is as follows:

[1252] ESI-MS (m / z): 793.3 (M+H) + .

[1253] 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]phenanthrene-17-yl-4-hydroxybenzoate (B-17)

[1254] (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-(triphenylmethoxy)benzoate (130 mg, 163 μmol) was dissolved in a mixture of dichloromethane (1.00 mL) and methanol (1.00 mL). A hydrochloric acid / 1,4-dioxane solution (2 M, 1.00 mL) was added and stirred at 25°C for 1 hour. The reaction mixture was concentrated to obtain the crude product, which was purified by preparative HPLC to afford the title compound (6.30 mg, 11.2 μmol).

[1255] The purification method is as follows:

[1256] Chromatographic column: Welch Ultimate C18 150mm×25mm×5mm

[1257] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[1258] Its structural characterization is as follows:

[1259] ESI-MS (m / z): 551.0 [M+H] + .

[1260] 1 H NMR(400MHz,CD3OD)δ77.70-7.86(m,2H),7.37(dd,J=10.0,1.2Hz,1H),6.72-6.92(m,2 H),6.38(dd,J=10.0,2.0Hz,1H),6.32(s,1H),5.67-6.02(m,2H),5.47-5.66(m,1H),4. 34-4.45(m,1H),2.96-3.09(m,1H),2.60-2.78(m,1H),2.48(dt,J=14.0,3.2Hz,1H),2. 25-2.43(m,2H),2.02-2.18(m,2H),1.74-1.86(m,1H),1.51-1.71(m,5H),1.07(s,3H).

[1261] Preparation Example 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-4-hydroxymethylbenzoate (B-32)

[1262] Step 1: Preparation of methyl 4-((triphenylmethoxy)methyl)benzoate (B-32-2)

[1263] Methyl 4-hydroxymethylbenzoate (3.00 g, 18.1 mmol) was dissolved in pyridine (30.0 mL). Triphenylmethane (5.03 g, 18.1 mmol) was added and the mixture was heated to 80°C for 12 hours. The reaction mixture was cooled to 25°C, 1 M dilute hydrochloric acid (40.0 mL) was added, and the mixture was extracted three times with dichloromethane (50.0 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to afford the crude title compound (7.26 g), which was used directly in the next step without purification.

[1264] Step 2: Preparation of 4-((triphenylmethoxy)methyl)benzoic acid (B-32-3)

[1265] Methyl 4-((triphenylmethoxy)methyl)benzoate (7.26 g, 17.8 mmol) was dissolved in a mixture of tetrahydrofuran (24.0 mL), methanol (24.0 mL), and water (24.0 mL). Lithium hydroxide monohydrate (2.24 g, 53.3 mmol) was added and the mixture was heated to 40°C for 1 hour. Water (30.0 mL) and ethyl acetate (20.0 mL) were added to the reaction mixture. The pH was adjusted to 2-3 with 1N dilute hydrochloric acid. The layers were separated and the aqueous phase was extracted three times with ethyl acetate (30.0 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (6.41 g, crude product).

[1266] Its structural characterization is as follows:

[1267] ESI-MS (m / z): 417.0 (M+Na) + .

[1268] Step 3: Preparation of (6S,8S,9R,10S,11S,13S,14S,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-yl-4-((triphenylmethoxy)methyl)benzoate (B-32-4)

[1269] 4-((Triphenylmethoxy)methyl)benzoic acid (150 mg, 380 μmol) was dissolved in DMF (3.00 mL), and HATU (145 mg, 380 μmol) and DIPEA (148 mg, 1.14 mmol, 199 μL) were added, followed by stirring 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-carboxylic acid thioate (121 mg, 304 μmol) was then added to the reaction mixture, and stirring was continued for 1 hour. Fluoroiodomethane (60.8 mg, 380 μmol) was then added to the reaction mixture and stirring continued for 0.5 hour. Water (30.0 mL) was added to the reaction mixture, stirred for 30 minutes, and filtered. The filter cake was washed with water three times (10.0 mL x 3) to obtain the crude title compound (300 mg), which was used directly in the next step without purification.

[1270] Its structural characterization is as follows:

[1271] ESI-MS (m / z): 829.3 (M+Na) + .

[1272] 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]phenanthrene-17-yl-4-hydroxymethylbenzoate (B-32)

[1273] (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-((triphenylmethoxy)methyl)benzoate (300 mg, 372 μmol) was dissolved in a mixed solvent of dichloromethane (6.00 mL) and methanol (1.20 mL). Trifluoroacetic acid (1.84 g, 16.1 mmol, 1.20 mL) was added, and the mixture was stirred at 25°C for 0.5 hour. The reaction solution was directly concentrated to obtain the crude product, which was purified by preparative HPLC to obtain the title compound (62.0 mg, 108 μmol).

[1274] The purification method is as follows:

[1275] Chromatographic column: Phenomenex Luna C18 200mm×40mm×10mm

[1276] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[1277] Its structural characterization is as follows:

[1278] ESI-MS (m / z): 565.2 [M+H] + .

[1279] 1H NMR (400MHz, CD3OD) δ7.93(d,J=8.4Hz,2H),7.48(d,J=8.4Hz,2H),7.37(dd,J=10.4,1.2Hz, 1H),6.38(dd,J=10.4,2.0Hz,1H),6.32(s,1H),5.68-6.01(m,2H),5.47-5.67(m,1H),4.67( s,2H),4.37-4....

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof: in, R1 is independently selected from hydrogen, halogen, -NR a R b , hydroxyl, cyano, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkyl, C substituted with one or more hydroxyl groups 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 Preferably, R1 is independently selected from hydrogen, halogen, -NR a R b , hydroxyl, C 1-6 Alkyl, -C 1-6 Alkyl-OH and C 1-6 Alkoxy; preferably, R1 is each independently selected from hydrogen, fluorine, chlorine, amino, hydroxyl, methyl, ethyl and hydroxymethyl; preferably, R1 is each independently selected from hydrogen, fluorine, amino, hydroxyl, methyl, ethyl and hydroxymethyl; R2 is selected from hydrogen, C 1-6 Alkyl, hydroxyl and C 1-6 Alkoxy; preferably, R2 is selected from hydrogen, methyl, hydroxy and methoxy; preferably, R2 is selected from hydrogen and methyl; Ring A is selected from a single bond, C 6-10 Aromatic ring and 5-6 membered heteroaromatic ring; preferably, ring A is selected from a single bond, a benzene ring and a 5-6 membered heteroaromatic ring; preferably, ring A is selected from a single bond, a benzene ring, a pyridine ring, a furan ring and a thiophene ring; X is selected from a single bond, C 1-6 Alkylene, C 3-6 Cycloalkylene, C 2-6 Alkenylene, C 2-6 Alkynylidene and -NR a -; preferably, X is selected from a single bond, methylene, ethylene, propylene, butylene, cyclopropylene, cyclohexylene, vinylene and imino; preferably, X is selected from a single bond, methylene, ethylene, propylene, butylene, 1,1-cyclopropylene, 1,2-cyclopropylene, 1,4-cyclohexylene and vinylene; Y is selected from -O- and -S-; Z is selected from hydroxy, halogen and cyano; preferably, Z is selected from hydroxy, fluorine, chlorine and cyano; Q1 and Q2 are each independently selected from hydrogen and halogen; preferably, Q1 and Q2 are each independently selected from hydrogen, fluorine and chlorine; preferably, Q1 is selected from hydrogen and fluorine, and Q2 is fluorine; m is selected from 1, 2, 3, 4, 5; preferably, m is selected from 1 and 2; n is selected from 1 and 2; preferably, n is 1; R a , R b are each independently selected from hydrogen and C 1-6 alkyl; Provided that: when R1 is each independently selected from 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, Ring A is not phenyl; When ring A is a single bond, R1 is not hydrogen or C 1-6 Alkyl; and When Ring A is a 5-6 membered heteroaromatic ring, R1 is hydrogen and R2 is C 1-6 When it is an alkyl group, Q1 is not a halogen.

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

3. The compound of claim 2, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, wherein the compound is a compound of formula III:

4. The compound of claim 2, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, wherein the compound is a compound of formula IV:

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

6. The compound of claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, wherein the compound is a compound of formula VI: in, Ring B is C 3-6 Cycloalkane, preferably cyclopropane.

7. The compound of any one of claims 1-6, its pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug, wherein the compound is selected from: Preferably selected from:

8. A compound, a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof, wherein the compound is selected from: Preferably selected from:

9. An antibody-drug conjugate having the formula Ab-[MLED] x The structure shown, where: Ab is an antibody or antigen-binding fragment thereof that specifically binds to an antigen; M is a linker site with an antibody or antigen-binding fragment thereof; L is a linker between the linkers M and E; E is a structural fragment connecting L and D; D is a glucocorticoid drug fragment, which is a monovalent structure obtained by losing one H from -OH, -NH2 or secondary amino group on the compound according to any one of claims 1 to 8, its pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug; x is an integer selected from 1 to 10, preferably an integer from 3 to 8; Preferably, D is selected from the following structures: Preferably, D is selected from the following structures: More preferably, D is selected from the following structures: The wavy line It indicates the point of attachment of the group to the rest of the molecule.

10. The antibody-drug conjugate of claim 9, wherein the antigen is selected from 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 adalimumab and tocilizumab.

12. The antibody-drug conjugate according to any one of claims 9 to 11, wherein M is Wherein ring C is a single bond, halogen, 5-6 membered alicyclic ring or 5-20 membered aromatic ring system, wherein the alicyclic ring or aromatic ring system is optionally substituted by one or more independently selected from oxo, halogen, cyano, amino, carboxyl, thiol and C 1-6 and M1 is selected from a single bond or a fragment consisting of one or more groups selected from the following groups: -O-, -NH-, -C(=O)-, -S(=O)2-, -C=NO-, -NH-S(=O)2-NH-, phenylene, 5-10 membered heteroarylene, C 1-20 Alkylene, C 2-20 Alkenylene and C 2-20 Alkyne; Preferably, M is wherein ring C is a single bond, a halogen, a 5-membered alicyclic ring, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting 2 to 5 (preferably three) units selected from a 6-membered heteroaromatic ring and a benzene ring through a single bond, wherein the alicyclic ring, heteroaromatic ring or polycyclic ring is optionally substituted with one or more selected from oxo, halogen and C 1-4 and M1 is selected from a single bond or a fragment consisting of one or more groups selected from the following groups: -NH-, -C(=O)-, -NH-S(=O)2-NH-, C 1-10 Alkylene, C 2-10 Alkenylene and C 2-10 Alkyne; Preferably, M is wherein ring C is selected from and M1 is selected from a single bond, -NH-, -NH-S(=O)2-NH-, C 1-6 Alkylene, C 2-6 Alkenylene and C 2-6 Alkyne; Preferably, M is selected from Preferably, M is selected from Preferably, M is selected from Preferably, M is selected from The wavy line It indicates the point of attachment of the group to the rest of the molecule.

13. The antibody-drug conjugate of any one of claims 9 to 12, wherein L is a structure consisting of one or more moieties selected from the group consisting of: C 1-6 Alkylene, -N(R')-, carbonyl, -O-, natural amino acids or unnatural amino acids and their analogs or derivatives (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys (COCH2CH2 (OCH2CH2) r OCH3)), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys- Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), Where R' represents hydrogen, C 1-6 Alkyl or containing -(CH2CH2O) r -C 4-30 Alkyl; r is an integer selected from 1-10, preferably an integer selected from 1-6; s is an integer selected from 1-20, preferably an integer selected from 1-10; Preferably, L is a structure consisting of one or more moieties selected from the following: C 1-6Alkylene, Carbonyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Lys, Val-C it,Ala-Ala-Ala,Ala-Ala-Asn,Leu-Ala-Glu,Gly-Gly-Arg,Gly-Glu-Gly,Gly-Gly-Gly,Gly-Se r-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, wherein s is an integer selected from 1-20, preferably an integer from 1-10; Preferably, L is a structure consisting of one or more moieties selected from the following: wherein s is an integer selected from 1-20, preferably an integer from 1-10; Preferably, L is selected from the following structures: wherein s is an integer selected from 1-20, preferably an integer from 1-10; Preferably, L is selected from the following structures: wherein s is an integer selected from 1-20, preferably an integer from 1-10; The wavy line It indicates the point of attachment of the group to the rest of the molecule.

14. The antibody-drug conjugate of any one of claims 9 to 13, wherein E is a single bond, -NHCH2-, or a structure selected from the following: wherein s is an integer selected from 1-20, preferably an integer from 1-10; Preferably, E is a single bond or -NHCH2-; Preferably, E is a single bond; The wavy line It indicates the point of attachment of the group to the rest of the molecule.

15. The antibody-drug conjugate of any one of claims 9 to 14, wherein Select from the following structures: in, s is an integer selected from 1-20, preferably an integer from 1-10; Preferably, Select from the following structures:

16. The antibody-drug conjugate of any one of claims 9 to 14, wherein Select from the following structures: in, s is an integer selected from 1-20, preferably an integer from 1-10; Preferably, Select from the following structures:

17. The antibody-drug conjugate of any one of claims 9 to 16, selected from: in, s is selected from an integer from 1 to 20; and n is selected from an integer of 0-20.

18. The antibody-drug conjugate of any one of claims 9 to 17, wherein the drug-antibody conjugation ratio is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10, preferably 3-9, for example, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 3.0-5.5, 3.0-6.0, 3.5-4.0, 3.5-4.5, 3.5-5.0, 3.5-5.5, 3. 5~6.0、3.5~6.5、3.5~7.0、3.5~7.5、3.5~8.0、4.0~4.5、4.0~5.0、4.0~5.5、4.0~6.0、4.0~6.5、4.0~7.0、4.0~7.5、4.0~8.0、4.5~5.0、4.5~5.5、4.5~6.0、4.5~6.5、4.5~7.0、4.5~7.5、4.5~8.0、5.0~5 .5, 5.0-6.0, 5.0-6.5, 5.0-7.0, 5.0-7.5, 5.0-8.0, 5.5-6.0, 5.5-6.5, 5.5-7.0, 5.5-7.5, 5.5-8.0, 6.0-6.5, 6.0-7.0, 6.0-7.5, 6.0-8.5, 6.5-7.0, 6.5-7.5, 6.5-8.5, 7.0-7.5, 7.0-9.0 or 7.5-9.

0.

19. A drug-linker compound having the formula GM-[LED] x The structure shown, where: G is a functional group or leaving group that reacts with specific amino acids or sugar groups and their derivatives in the antibody or antigen-binding fragment; preferably, G is selected from halogen, halogenated phenoxy, C 1-6 Halogenated alkyl, sulfonic acid, C 1-6 Alkylsulfonyl, C 1-6 Haloalkylsulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, C 1-6 Halogenated alkyl sulfonate, C 1-6 Alkyl sulfinate group, halosulfonate group, C 1-6 Alkyl sulfoxide, methylsulfonyl isobutylene, bismethylsulfonyl isobutylene, haloformyl, haloacetyl, formyl, acetyl, nitro, azido, cyano, cyanovinyl, N-methyl-vinylsulfonylamino, tetrazine, methyltetrazine, trans-cyclooctene carbonate, C 2-6 Alkenyl, C 2-6 Preferably, G is selected from halogen, methanesulfonyl, haloacetyl, fluorophenoxy, Methanesulfonylmethacryloyl, cyanovinyl, N-methylvinylsulfonylamino, azido, tetrazine, methyltetrazine, trans-cyclooctene carbonate, C 2-6 alkynyl, benzazacyclooctinyl, (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy; and M, L, E, D and x are as defined in any one of claims 9-18.

20. The drug-linker compound of claim 19, selected from: in, s is selected from an integer from 1 to 20; and n is selected from an integer of 0-20.

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

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

23. A method for preparing the compound of any one of claims 1 to 8, comprising the following steps: wherein Ring A, R1, R2, X, Y, Z, Q1, Q2, m and n are as defined in any one of claims 1 to 8; and LG is a leaving group, such as halogen, methanesulfonyloxy or trifluoromethanesulfonyloxy, preferably iodine.

24. A method for preparing the drug-linker compound according to any one of claims 19 to 20, comprising the following steps: in, Ring A, R1, R2, X, Y, Z, Q1, G, M, L, D, m and n are as described in any one of claims 1 to 20; x＝1； E is a single bond; LG is a leaving group, such as halogen, methanesulfonyloxy or trifluoromethanesulfonyloxy, preferably iodine; and PG is an amino protecting group, for example, 9-fluorenylmethoxycarbonyl, tert-butyloxycarbonyl, p-methoxytrityl and allyloxycarbonyl, preferably 9-fluorenylmethoxycarbonyl.

25. The method for preparing the antibody-drug conjugate according to any one of claims 9 to 18, which is selected from: Coupling method A: Conjugating the drug-linker compound according to any one of claims 19 to 20 to an antibody at a molar ratio of (8-10):1 to obtain an antibody-drug conjugate; Coupling method B: Conjugating the drug-linker compound according to any one of claims 19 to 20 to an antibody at a molar ratio of (4-6):1 to obtain an antibody-drug conjugate; Coupling method C: The drug-linker compound according to any one of claims 19 to 20 is coupled to an antibody at a molar ratio of (4.0-4.5):1 to obtain an antibody-drug conjugate.

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