DRUG CONJUGATE OF GLUCOCORTICOID RECEPTOR AGONIST, AND APPLICATION THEREOF IN MEDICINE

Abstract

A drug conjugate of a glucocorticoid receptor agonist, and an application thereof in medicine. Specifically, the present invention relates to an antibody-drug conjugate as represented by formula (I): Ab-(L-D)k (I), wherein Ab is an antibody or an antigen-binding fragment thereof, L is a linker covalently linking Ab to D, k is 1 to 20, and D is as represented by formula (II-A) or (II-B). The groups in the formulas are as defined in the description. The antibody-drug conjugate can effectively treat autoimmune diseases.

##STR00001##

Claims

1. An antibody-drug conjugate of formula (I),
Ab-(L-D).sub.k (I) wherein Ab is an antibody or an antigen-binding fragment thereof; L is a linker covalently linking Ab to D, and k is 1 to 20; D is represented by formula (II-A) or (II-B): ##STR00109## wherein, represents a single bond or a double bond; each R.sub.1a is independently selected from the group consisting of hydrogen, alkyl and alkoxy, and the alkyl and alkoxy are each independently optionally substituted with one or more substituents selected from the group consisting of alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxy and hydroxyalkyl; ring A is aryl or heteroaryl optionally substituted with one or more Q.sub.1 substituents; ring B is aryl or heteroaryl optionally substituted with one or more Q.sub.1 substituents; X.sub.1 is (CR.sub.5aR.sub.5b)m- or is aryl or heteroaryl optionally substituted with one or more Q.sub.1 substituents; R.sub.5a and R.sub.5b are each independently selected from the group consisting of hydrogen, halogen, hydroxy, sulfhydryl, deuterium, nitro, cyano, and the following groups optionally substituted with one or more Q.sub.1 substituents: alkyl, NR.sub.iR.sub.j, C(O)R.sub.k, C(O)OR.sub.k, S(O)R.sub.k, S(O)OR.sub.k, S(O)(O)R.sub.k, S(O)(O)OR.sub.k, C(S)R.sub.k, alkoxy, alkylthio, alkenyl and alkynyl, or together R.sub.5a and R.sub.5b form oxo or thio; ring C and ring D are each independently selected from the group consisting of aryl and heteroaryl optionally substituted with one or more Q.sub.1 substituents, and at least one of ring C and ring D is fused cycloaryl or fused heteroaryl optionally substituted with one or more Q.sub.1 substituents; X.sub.2 is selected from the group consisting of (CR.sub.6aR.sub.6b)n-, aryl or heteroaryl optionally substituted with one or more Q.sub.1 substituents, O, S, S(O), S(O)(O), NR.sub.6c, CH.sub.2S, CH.sub.2O, NHCR.sub.6dR.sub.6e, CR.sub.6fCR.sub.6g and C?C, or X.sub.2 is absent; R.sub.6a and R.sub.6b are each independently selected from the group consisting of hydrogen, halogen, hydroxy, sulfhydryl, deuterium, nitro, cyano, and the following groups optionally substituted with one or more Q.sub.1 substituents: alkyl, NR.sub.iR.sub.j, C(O)R.sub.k, C(O)OR.sub.k, S(O)R.sub.k, S(O)OR.sub.k, S(O)(O)R.sub.k, S(O)(O)OR.sub.k, C(S)R.sub.k, alkoxy, alkylthio, alkenyl and alkynyl, or R.sub.6a and R.sub.6b, together with the carbon atom to which they are attached, form 3- to 10-membered cycloalkyl, or together R.sub.6a and R.sub.6b form oxo or thio; R.sub.6c, R.sub.6a, R.sub.6e, R.sub.6f and R.sub.6g are each independently selected from the group consisting of hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl and C.sub.1-C.sub.6 alkoxy; each R.sub.1 is independently selected from the group consisting of hydrogen, alkyl and alkoxy, wherein the alkyl and alkoxy are each independently optionally substituted with one or more substituents selected from the group consisting of alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxy and hydroxyalkyl; each R.sub.2 is independently selected from the group consisting of CH.sub.2OH, CH.sub.2SH, CH.sub.2Cl, SCH.sub.2Cl, SCH.sub.2F, SCH.sub.2CF.sub.3, OH, OCH.sub.2CN, OCH.sub.2Cl, OCH.sub.2F, OCH.sub.3, OCH.sub.2CH.sub.3, SCH.sub.2CN, ##STR00110## each R.sub.2a is independently hydrogen or C.sub.1-C.sub.6 alkyl; each R.sub.2b is independently C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkoxy; each R.sub.2c is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.6 alkyl, CH.sub.2OH and C.sub.1-C.sub.6 alkoxy; R.sub.2d and R.sub.2e are each independently hydrogen or C.sub.1-C.sub.6 alkyl; each R.sub.3 is independently hydrogen or a halogen; each R.sub.4 is independently selected from the group consisting of hydrogen, halogen and hydroxy; m and n are each independently an integer of 1 to 6; the Q.sub.1 substituents are each independently selected from the group consisting of C.sub.1-C.sub.6 alkyl, halogen, deuterium, hydroxy, sulfhydryl, NR.sub.iR.sub.j, oxo, thio, C(O)R.sub.k, C(O)OR.sub.k, S(O)R.sub.k, S(O)OR.sub.k, S(O)(O)R.sub.k, S(O)(O)OR.sub.k, C(S)R.sub.k, nitro, cyano, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 alkylthio, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, 3- to 10-membered cycloalkyl, 3- to 10-membered heterocyclyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, 8- to 12-membered fused cycloaryl, and 5- to 12-membered fused heteroaryl; each R.sub.k is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl, C.sub.1-C.sub.6 alkoxy, hydroxy and NR.sub.iR.sub.j, wherein the alkyl, alkoxy and haloalkyl are each independently optionally substituted with one or more substituents selected from the group consisting of C.sub.1-C.sub.6 alkyl, halogen, hydroxy, sulfhydryl, NR.sub.iR.sub.j, oxo, thio, carboxyl, nitro, cyano, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 alkylthio, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, 3- to 10-membered cycloalkyl, 3- to 10-membered heterocyclyl, 6- to 10-membered aryl, and 5- to 10-membered heteroaryl; provided that when R.sub.5a is hydrogen or alkyl, R.sub.5b is not hydrogen or alkyl.

2. The antibody-drug conjugate according to claim 1, wherein ring A is 6- to 10-membered aryl or 5- to 10-membered heteroaryl optionally substituted with one or more Q.sub.1 substituents, and the heteroaryl comprises at least one nitrogen atom; ring B is 6- to 10-membered aryl or 5- to 10-membered heteroaryl optionally substituted with one or more Q.sub.1 substituents, and the heteroaryl comprises at least one nitrogen atom.

3. (canceled)

4. The antibody-drug conjugate according to claim 1, wherein X.sub.1 is (CR.sub.5aR.sub.5b)m- or is 6- to 10-membered aryl or 5- to 10-membered heteroaryl optionally substituted with one or more Q.sub.1 substituents, and the heteroaryl comprises at least one nitrogen atom; R.sub.5a and R.sub.5b are each independently selected from the group consisting of hydrogen, halogen, hydroxy, sulfhydryl, deuterium, cyano, and the following groups optionally substituted with one or more Q.sub.1 substituents: C.sub.1-C.sub.6 alkyl, NR.sub.iR.sub.j, C(O)R.sub.k, C(O)OR.sub.k, S(O)R.sub.k, S(O)OR.sub.k, S(O)(O)R.sub.k, S(O)(O)OR.sub.k, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 alkylthio, C.sub.2-C.sub.6 alkenyl and C.sub.2-C.sub.6 alkynyl, or together R.sub.5a and R.sub.5b form oxo or thio.

5. The antibody-drug conjugate according to claim 1, wherein ring C and ring D are each independently selected from the group consisting of 6- to 10-membered aryl, 5- to 10-membered heteroaryl, 8- to 12-membered fused cycloaryl and 5- to 12-membered fused heteroaryl optionally substituted with one or more Q.sub.1 substituents, and the heteroaryl or fused heteroaryl comprises at least one nitrogen atom ##STR00111##

6. The antibody-drug conjugate according to claim 1, wherein X.sub.2 is selected from the group consisting of (CR.sub.6aR.sub.6b)n-, O, S, NR.sub.6c, CH.sub.2S, CH.sub.2O, NHCR.sub.6dR.sub.6e, and 6- to 10-membered aryl or 5- to 10-membered heteroaryl optionally substituted with one or more Q.sub.1 substituents, and the heteroaryl comprises at least one nitrogen atom; R.sub.6a and R.sub.6b are each independently selected from the group consisting of hydrogen, halogen, hydroxy, sulfhydryl, deuterium, cyano, and the following groups optionally substituted with one or more Q.sub.1 substituents: C.sub.1-C.sub.6 alkyl, NR.sub.iR.sub.j, C(O)R.sub.k, C(O)OR.sub.k, S(O)R.sub.k, S(O)(O)R.sub.k, C.sub.1-C.sub.6 alkoxy, C.sub.2-C.sub.6 alkenyl and C.sub.2-C.sub.6 alkynyl, or R.sub.6a and R.sub.6b, together with the carbon atom to which they are attached, form 3- to 10-membered cycloalkyl.

7. The antibody-drug conjugate according to claim 1, wherein D is represented by formula (II-A) or (II-B): ##STR00112## wherein, each R.sub.1a is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.6 alkyl and C.sub.1-C.sub.6 alkoxy; ring A is ##STR00113## and the ring A is optionally substituted with one or more Q.sub.1 substituents; ring B is ##STR00114## and the ring B is optionally substituted with one or more Q.sub.1 substituents; X.sub.1 is (CR.sub.5aR.sub.5b)m- or is 6- to 10-membered aryl or 5- to 10-membered heteroaryl optionally substituted with one or more Q.sub.1 substituents, and the heteroaryl comprises at least one nitrogen atom; R.sub.5a and R.sub.5b are each independently selected from the group consisting of hydrogen, halogen, hydroxy, sulfhydryl, deuterium, cyano, and the following groups optionally substituted with one or more Q.sub.1 substituents: C.sub.1-C.sub.6 alkyl, NR.sub.iR.sub.j, C(O)R.sub.k, C(O)OR.sub.k and C.sub.1-C.sub.6 alkoxy, or together R.sub.5a and R.sub.5b form oxo or thio; ring C is selected from the group consisting of ##STR00115## and the ring C is optionally substituted with one or more Q.sub.1 substituents; ring D is ##STR00116## and the ring D is optionally substituted with one or more Q.sub.1 substituents; X.sub.2 is selected from the group consisting of (CR.sub.6aR.sub.6b)n-, O, S, NR.sub.6c, CH.sub.2S, CH.sub.2O, NHCR.sub.6dR.sub.6e, and 6- to 10-membered aryl or 5- to 10-membered heteroaryl optionally substituted with one or more Q.sub.1 substituents; R.sub.6a and R.sub.6b are each independently selected from the group consisting of hydrogen, halogen, hydroxy, sulfhydryl, deuterium, cyano, and the following groups optionally substituted with one or more Q.sub.1 substituents: C.sub.1-C.sub.6 alkyl, NR.sub.iR.sub.j, C(O)R.sub.k, C(O)OR.sub.k and C.sub.1-C.sub.6 alkoxy; R.sub.6c, R.sub.6d and R.sub.6e are each independently selected from the group consisting of hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl and C.sub.1-C.sub.6 alkoxy; each R.sub.2 is independently selected from the group consisting of CH.sub.2OH, CH.sub.2SH, CH.sub.2Cl, SCH.sub.2Cl, SCH.sub.2F, SCH.sub.2CF.sub.3, OH, OCH.sub.2CN, OCH.sub.2Cl, OCH.sub.2F, OCH.sub.3, OCH.sub.2CH.sub.3, SCH.sub.2CN, ##STR00117## each R.sub.2a is independently hydrogen or C.sub.1-C.sub.6 alkyl; each R.sub.2b is independently C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkoxy; each R.sub.2c is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.6 alkyl, CH.sub.2OH and C.sub.1-C.sub.6 alkoxy; R.sub.2d and R.sub.2e are each independently hydrogen or C.sub.1-C.sub.6 alkyl; each R.sub.3 is independently hydrogen or a halogen; m and n are each independently an integer of 1 to 6; the Q.sub.1 substituents are each independently selected from the group consisting of halogen, hydroxy, sulfhydryl, deuterium, oxo, thio, cyano, amino, carboxyl, C.sub.1-C.sub.6 alkyl and C.sub.1-C.sub.6 alkoxy; R.sub.i and R.sub.j are each independently selected from the group consisting of hydrogen, hydroxy, C.sub.1-C.sub.6 alkyl and C.sub.1-C.sub.6 alkoxy; R.sub.k is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl, C.sub.1-C.sub.6 alkoxy, hydroxy and NR.sub.iR.sub.j; provided that when R.sub.5a is hydrogen or alkyl, R.sub.5b is not hydrogen or alkyl.

8. (canceled)

9. (canceled)

10. The antibody-drug conjugate according to claim 1, wherein k is 1-10.

11. The antibody-drug conjugate according to claim 1, wherein the linker comprises amino acid unit L.sub.1, and the amino acid unit L.sub.1 comprises a peptide residue consisting of 2 to 7 amino acids selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, aspartic acid, homolysine, n-methyl-valine and ##STR00118## wherein q is an integer of 1-6.

12. The antibody-drug conjugate according to claim 1, wherein the linker comprises a stretcher unit, the stretcher unit is selected from the group consisting of ##STR00119## wherein each p is independently 1, 2, 3, 4, 5 or 6.

13. The antibody-drug conjugate according to claim 1, wherein the linker is selected from the group consisting of ##STR00120## ##STR00121## ##STR00122##

14. The antibody-drug conjugate according to claim 1, being selected from the group consisting of: ##STR00123## ##STR00124## ##STR00125## wherein each p is independently 1, 2, 3, 4, 5 or 6; Ab, D and k are as defined in claim 1.

15. The antibody-drug conjugate according to claim 1, being selected from the group consisting of ##STR00126## ##STR00127## ##STR00128## wherein k is 1 to 10; Ab is as defined in claim 1.

16. The antibody-drug conjugate according to claim 1, wherein the antibody is selected from the group consisting of a murine antibody, a chimeric antibody, a humanized antibody and a fully human-derived antibody.

17. The antibody-drug conjugate according to claim 1, wherein the antibody or the antigen-binding fragment thereof is selected from the group consisting of an anti-TNF? antibody, an anti-IL-4R antibody, an anti-IL-6/IL-6R antibody, an anti-IL-13R antibody, an anti-IL-17/IL-17R antibody, an anti-IL-23/IL23R antibody, an anti-IL-36R antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD28 antibody, an anti-CD40 antibody and an anti-TSLP antibody or antigen-binding fragments thereof.

18. A compound of formula (III-A) or (III-B) or a pharmaceutically acceptable salt thereof, ##STR00129## wherein, represents a single bond or a double bond; ring A is aryl or heteroaryl optionally substituted with one or more Q.sub.1 substituents; ring B is aryl or heteroaryl optionally substituted with one or more Q.sub.1 substituents; X.sub.1 is (CR.sub.5aR.sub.5b)m- or is aryl or heteroaryl optionally substituted with one or more Q.sub.1 substituents; R.sub.5a and R.sub.5b are each independently selected from the group consisting of hydrogen, halogen, hydroxy, sulfhydryl, deuterium, nitro, cyano, and the following groups optionally substituted with one or more Q.sub.1 substituents: alkyl, NR.sub.iR.sub.j, C(O)R.sub.k, C(O)OR.sub.k, S(O)R.sub.k, S(O)OR.sub.k, S(O)(O)R.sub.k, S(O)(O)OR.sub.k, C(S)R.sub.k, alkoxy, alkylthio, alkenyl and alkynyl, or together R.sub.5a and R.sub.5b form oxo or thio; ring C and ring D are each independently selected from the group consisting of aryl and heteroaryl or fused heteroaryl optionally substituted with one or more Q.sub.1 substituents, and at least one of ring C and ring D is fused cycloaryl or fused heteroaryl optionally substituted with one or more Q.sub.1 substituents; X.sub.2 is selected from the group consisting of (CR.sub.6aR.sub.6b)n-, aryl or heteroaryl optionally substituted with one or more Q.sub.1 substituents, O, S, S(O), S(O)(O), NR.sub.6c, CH.sub.2S, CH.sub.2O, NHCR.sub.6dR.sub.6e, CR.sub.6f?CR.sub.6g and C?C, or X.sub.2 is absent; R.sub.6a and R.sub.6b are each independently selected from the group consisting of hydrogen, halogen, hydroxy, sulfhydryl, deuterium, nitro, cyano, and the following groups optionally substituted with one or more Q.sub.1 substituents: alkyl, NR.sub.iR.sub.j, C(O)R.sub.k, C(O)OR.sub.k, S(O)R.sub.k, S(O)OR.sub.k, S(O)(O)R.sub.k, S(O)(O)OR.sub.k, C(S)R.sub.k, alkoxy, alkylthio, alkenyl and alkynyl, or R.sub.6a and R.sub.6b, together with the carbon atom to which they are attached, form 3- to 10-membered cycloalkyl; R.sub.6c, R.sub.6d, R.sub.6e, R.sub.6f and R.sub.6g are each independently selected from the group consisting of hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl and C.sub.1-C.sub.6 alkoxy; each R.sub.1 is independently selected from the group consisting of hydrogen, alkyl and alkoxy, wherein the alkyl and alkoxy are each independently optionally substituted with one or more substituents selected from the group consisting of alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxy and hydroxyalkyl; each R.sub.2 is independently selected from the group consisting of CH.sub.2OH, CH.sub.2SH, CH.sub.2Cl, SCH.sub.2Cl, SCH.sub.2F, SCH.sub.2CF.sub.3, OH, OCH.sub.2CN, OCH.sub.2Cl, OCH.sub.2F, OCH.sub.3, OCH.sub.2CH.sub.3, SCH.sub.2CN, ##STR00130## each R.sub.2a is independently hydrogen or C.sub.1-C.sub.6 alkyl; each R.sub.2b is independently C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkoxy; each R.sub.2c is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.6 alkyl, CH.sub.2OH and C.sub.1-C.sub.6 alkoxy; R.sub.2d and R.sub.2e are each independently hydrogen or C.sub.1-C.sub.6 alkyl; each R.sub.3 is independently hydrogen or a halogen; each R.sub.4 is independently selected from the group consisting of hydrogen, halogen and hydroxy; m and n are each independently selected from an integer of 1 to 6; the Q.sub.1 substituents are each independently selected from the group consisting of C.sub.1-C.sub.6 alkyl, halogen, deuterium, hydroxy, sulfhydryl, NR.sub.iR.sub.j, oxo, thio, C(O)R.sub.k, C(O)OR.sub.k, S(O)R.sub.k, S(O)OR.sub.k, S(O)(O)R.sub.k, S(O)(O)OR.sub.k, C(S)R.sub.k, nitro, cyano, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 alkylthio, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, 3- to 10-membered cycloalkyl, 3- to 10-membered heterocyclyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, 8- to 12-membered fused cycloaryl, and 5- to 12-membered fused heteroaryl; R.sub.i and R.sub.j are each independently selected from the group consisting of hydrogen, hydroxy, C.sub.1-C.sub.6 alkyl and C.sub.1-C.sub.6 alkoxy; R.sub.k is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl, C.sub.1-C.sub.6 alkoxy, hydroxy and NR.sub.iR.sub.j, wherein the alkyl, alkoxy and haloalkyl are each independently optionally substituted with one or more substituents selected from the group consisting of C.sub.1-C.sub.6 alkyl, halogen, hydroxy, sulfhydryl, NR.sub.iR.sub.j, oxo, thio, carboxyl, nitro, cyano, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 alkylthio, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, 3- to 10-membered cycloalkyl, 3- to 10-membered heterocyclyl, 6- to 10-membered aryl, and 5- to 10-membered heteroaryl; each R.sub.1a is independently selected from the group consisting of hydrogen, alkyl and alkoxy, and the alkyl and alkoxy are each independently optionally substituted with one or more substituents selected from the group consisting of alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxy and hydroxyalkyl; each R.sub.1b is independently selected from the group consisting of hydrogen, PG-, H-L.sub.1-, PG-L.sub.1-, ##STR00131## or R.sub.1a and R.sub.1b, together with the nitrogen atom to which they are attached, form: ##STR00132## or R.sub.1a and R.sub.1b, together with the nitrogen atom to which they are attached, form a nitro group; each p is independently 1, 2, 3, 4, 5 or 6; L.sub.1 is an amino acid unit; X is a halogen; PG is an amino protecting group; provided that when R.sub.5a is hydrogen or alkyl, R.sub.5b is not hydrogen or alkyl.

19. The compound or the pharmaceutically acceptable salt thereof according to claim 18, being a compound of formula (III-A) or (III-B) or a pharmaceutically acceptable salt thereof: ##STR00133## wherein, ring A is ##STR00134## and the ring A is optionally substituted with one or more Q.sub.1 substituents; ring B is ##STR00135## and the ring B is optionally substituted with one or more Q.sub.1 substituents; X.sub.1 is (CR.sub.5aR.sub.5b)m- or is 6- to 10-membered aryl or 5- to 10-membered heteroaryl optionally substituted with one or more Q.sub.1 substituents, and the heteroaryl comprises at least one nitrogen atom; R.sub.5a and R.sub.5b are each independently selected from the group consisting of hydrogen, halogen, hydroxy, sulfhydryl, deuterium, cyano, and the following groups optionally substituted with one or more Q.sub.1 substituents: C.sub.1-C.sub.6 alkyl, NR.sub.iR.sub.j, C(O)R.sub.k, C(O)OR.sub.k and C.sub.1-C.sub.6 alkoxy, or together R.sub.5a and R.sub.5b form oxo or thio; ring C is selected from the group consisting of ##STR00136## and N and the ring C is optionally substituted with one or more Q.sub.1 substituents; ring D is ##STR00137## and the ring D is optionally substituted with one or more Q.sub.1 substituents; X.sub.2 is selected from the group consisting of (CR.sub.6aR.sub.6b)n-, O, S, NR.sub.6c, CH.sub.2S, CH.sub.2O, NHCR.sub.6dR.sub.6e, and 6- to 10-membered aryl or 5- to 10-membered heteroaryl optionally substituted with one or more Q.sub.1 substituents; R.sub.6a and R.sub.6b are each independently selected from the group consisting of hydrogen, halogen, hydroxy, sulfhydryl, deuterium, cyano, and the following groups optionally substituted with one or more Q.sub.1 substituents: C.sub.1-C.sub.6 alkyl, NR.sub.iR.sub.j, C(O)R.sub.k, C(O)OR.sub.k and C.sub.1-C.sub.6 alkoxy; R.sub.6c, R.sub.6d and R.sub.6e are each independently selected from the group consisting of hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl and C.sub.1-C.sub.6 alkoxy; each R.sub.2 is independently selected from the group consisting of CH.sub.2OH, CH.sub.2SH, CH.sub.2Cl, SCH.sub.2Cl, SCH.sub.2F, SCH.sub.2CF.sub.3, OH, OCH.sub.2CN, OCH.sub.2Cl, OCH.sub.2F, OCH.sub.3, OCH.sub.2CH.sub.3, SCH.sub.2CN, ##STR00138## each R.sub.2a is independently hydrogen or C.sub.1-C.sub.6 alkyl; each R.sub.2b is independently C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkoxy; each R.sub.2c is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.6 alkyl, CH.sub.2OH and C.sub.1-C.sub.6 alkoxy; R.sub.2d and R.sub.2e are each independently hydrogen or C.sub.1-C.sub.6 alkyl; each R.sub.3 is independently hydrogen or a halogen; m and n are each independently an integer of 1 to 6; the Q.sub.1 substituents are each independently selected from the group consisting of halogen, hydroxy, sulfhydryl, deuterium, oxo, thio, cyano, amino, carboxyl, C.sub.1-C.sub.6 alkyl and C.sub.1-C.sub.6 alkoxy; R.sub.i and R.sub.j are each independently selected from the group consisting of hydrogen, hydroxy, C.sub.1-C.sub.6 alkyl and C.sub.1-C.sub.6 alkoxy; R.sub.k is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl, C.sub.1-C.sub.6 alkoxy, hydroxy and NR.sub.iR.sub.j; each R.sub.1a is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.6 alkyl and C.sub.1-C.sub.6 alkoxy; each R.sub.1b is independently selected from the group consisting of hydrogen, PG-, H-L.sub.1-, PG-L.sub.1-, each p is independently 1, 2, 3, 4, 5 or 6; ##STR00139## L.sub.1 is an amino acid unit, preferably and L.sub.1 is -glycine-glutamic acid- or ##STR00140## X is a halogen; PG is an amino protecting group; provided that when R.sub.5a is hydrogen or alkyl, R.sub.5b is not hydrogen or alkyl.

20. The compound or the pharmaceutically acceptable salt thereof according to claim 18, being selected from the group consisting of ##STR00141## or pharmaceutically acceptable salts thereof.

21. The compound or the pharmaceutically acceptable salt thereof according to claim 18, being selected from the group consisting of ##STR00142## ##STR00143## ##STR00144## ##STR00145## or pharmaceutically acceptable salts thereof, wherein X is a halogen.

22. A pharmaceutical composition comprising the antibody-drug conjugate according to claim 1, and a pharmaceutically acceptable excipient.

23. A method of treating an immune disease in a subject in need thereof, the method comprising administering to the subject the antibody-drug conjugate according to claim 1, wherein the immune disease is selected from the group consisting of rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, adult Crohn's disease, pediatric Crohn's disease, ulcerative colitis, hidradenitis suppurativa, uveitis, Beh?et's disease, spondyloarthropathy and psoriasis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0311] FIG. 1: the anti-inflammatory activity of small-molecule steroids in the lipopolysaccharide's stimulation of human PBMCs' cytokine secretion.

[0312] FIG. 2: the activity of anti-TNF-ADCs in a membrane-bound TNF?-mediated GRE reporter gene system.

[0313] FIG. 3: the activity of anti-TNF-ADCs in lipopolysaccharide's stimulation of human monocytes' cytokine secretion.

[0314] FIG. 4: the activity of anti-TNF-ADCs (4 nM) in LPS-induced human monocytes' cytokine release.

[0315] FIG. 5: the experimental process of Test Example 5.

[0316] FIG. 6: the activity of anti-TNF-ADCs in a mouse collagen antibody-induced arthritis (CAIA) model.

[0317] FIG. 7: the activity of anti-TNF-ADCs in a mouse collagen antibody-induced arthritis (CAIA) model at different time points.

[0318] FIG. 8: the experimental process of Test Example 6.

[0319] FIG. 9: the activity of anti-TNF-ADCs in a delayed-type hypersensitivity (DTH) model.

DETAILED DESCRIPTION

[0320] The preparation of the compound and the pharmaceutically acceptable salt thereof of the present disclosure is further described below in conjunction with examples, which are not intended to limit the scope of the present disclosure.

[0321] Experimental methods without conditions specified in the examples of the present disclosure generally followed conventional conditions, or conditions recommended by the manufacturers of the starting materials or commercial products. Reagents without specific origins indicated were commercially available conventional reagents.

[0322] NMR shifts (?) were given in 10.sup.?6 (ppm). NMR analysis was performed on a Bruker AVANCE-400 nuclear magnetic resonance instrument, with deuterated dimethyl sulfoxide (DMSO-d.sup.6), deuterated chloroform (CDCl.sub.3) and deuterated methanol (CD.sub.3OD) as solvents and tetramethylsilane (TMS) as an internal standard.

[0323] MS analysis was performed on a Shimadzu 2010 Mass Spectrometer or Agilent 6110A MSD Mass Spectrometer.

[0324] HPLC analysis was performed on Shimadzu LC-20A systems, Shimadzu LC-2010HT series, or an Agilent 1200 LC high-performance liquid chromatograph (Ultimate XB-C18 3.0?150 mm chromatography column or Xtimate C18 2.1?30 mm chromatography column).

[0325] Chiral HPLC analysis used Chiralpak IC-3 100?4.6 mm I.D., 3 ?m, Chiralpak AD-3 150?4.6 mm I.D., 3 ?m, Chiralpak AD-3 50?4.6 mm I.D., 3 ?m, Chiralpak AS-3 150?4.6 mm I.D., 3 ?m, Chiralpak AS-3 100?4.6 mm I.D., 3 ?m, ChiralCel OD-3 150?4.6 mm I.D., 3 ?m, Chiralcel OD-3 100?4.6 mm I.D., 3 ?m, ChiralCel OJ-H 150?4.6 mm I.D., 5 m, and Chiralcel OJ-3 150?4.6 mm I.D., 3 ?m chromatography columns. Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates, 0.15-0.2 mm layer thickness, were adopted for thin-layer chromatography (TLC) analysis and 0.4-0.5 mm layer thickness for TLC separation and purification.

[0326] Yantai Huanghai silica gel of 100-200 mesh, 200-300 mesh or 300-400 mesh was generally used as a carrier in column chromatography.

[0327] Preparative chiral chromatography used a DAICEL CHIRALPAK IC (250 mm?30 mm, 10 ?m) or Phenomenex-Amylose-1 (250 mm?30 mm, 5 ?m) column.

[0328] The CombiFlash preparative flash chromatograph used was CombiFlash Rf150 (TELEDYNE ISCO).

[0329] The mean inhibition of kinase and the IC.sub.50 value were determined on a NovoStar microplate reader (BMG, Germany).

[0330] Known starting materials described herein may be synthesized using or according to methods known in the art, or may be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., Chembee Chemicals, and other companies.

[0331] In the examples, the reactions can all be performed in an argon atmosphere or a nitrogen atmosphere unless otherwise specified.

[0332] The argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to a balloon containing about 1 L of argon or nitrogen.

[0333] The hydrogen atmosphere means that the reaction flask is connected to a balloon containing about 1 L of hydrogen.

[0334] Pressurized hydrogenation reactions were conducted using a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogenator, or an HC2-SS hydrogenator.

[0335] Hydrogenation reactions generally involved 3 cycles of vacuumization and hydrogen purging.

[0336] Microwave reactions were conducted on a CEM Discover-S 908860 microwave reactor. In the examples, a solution was an aqueous solution unless otherwise specified.

[0337] In the examples, the reaction temperature was room temperature, i.e., 20? C. to 30? C., unless otherwise specified.

[0338] The monitoring of the reaction progress in the examples was conducted by thin-layer chromatography (TLC). The developing solvent for reactions, the eluent system of column chromatography for compound purification and the developing solvent system of thin-layer chromatography include: A: dichloromethane/methanol system, B: n-hexane/ethyl acetate system, C: petroleum ether/ethyl acetate system, and D: petroleum ether/ethyl acetate/methanol system. The volume ratio of the solvents was adjusted according to the polarity of the compound, or by adding a small amount of basic or acidic reagents such as triethylamine and acetic acid.

[0339] Preparation of 1.0 M Tris buffer, pH=8.30?0.1:

[0340] 6.0 g of tris was weighed into a 50 mL volumetric flask and dissolved by shaking in 40 mL of purified water, 1.2 mL of concentrated hydrochloric acid was added dropwise to adjust the pH to 8.30, and the solution was brought to volume with purified water.

[0341] Preparation of Buffer Solution A:

[0342] KH.sub.2PO.sub.4 (8.50 g), K.sub.2HIPO.sub.4 (8.56 g), NaCl (5.86 g) and EDTA (1.50 g) were added to a 2.0 L vessel and completely dissolved in 1.6 L of water for injection by half an hour's stirring, and the solution was then brought to volume (2.0 L) with water for injection. Measurement showed the pH was 6.30?0.1.

[0343] The abbreviations used in the following experiments have the following meanings:

[0344] DAST: diethylaminosulfur trifluoride; THF: tetrahydrofuran; NMP: N-methylpyrrolidone; DCM: dichloromethane; m-CPBA: m-chloroperoxybenzoic acid; DIEA: N,N-diisopropylethylamine; TEA: triethylamine; Boc: tert-butyloxycarbonyl; MeOH: methanol; Et.sub.2O: diethyl ether.

Example 1

[0345] ##STR00089##

[0346] Step 1

[0347] Compound 1-1 (500 mg, 1.9 mmol, 1.0 eq), compound 1-2 (730 mg, 2.3 mmol, 1.2 eq), Pd(PPh.sub.3).sub.4 (660 mg, 0.57 mmol, 0.3 eq), K.sub.2CO.sub.3 (960 mg, 6.9 mmol, 3.6 eq) and DMF (35 mL, 70 V) were added to a 100 mL single-necked flask, purged with nitrogen three times and stirred at 80? C. until the reaction was complete. EA (50 mL) and H.sub.2O (130 mL) were added to the reaction mixture. The aqueous phase was separated and extracted with EA (30 mL?2). The organic phases were combined, washed with H.sub.2O (50 mL?3), a saturated solution of LiCl (50 mL) and then a saturated solution of NaCl (30 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and then concentrated by rotary evaporation to remove the solvent. The residue was purified by column chromatography to give a crude product. The crude product was triturated with PE and EA (2:1), and the triturate was filtered to give compound 1-3 (220 mg, 31% yield).

[0348] Ms (ESI): m/z 318 [M-55].sup.+.

[0349] Step 2

[0350] Compound 1-3 (100 mg, 0.268 mmol, 1.1 eq), compound 1-4 (92 mg, 0.243 mmol, 1.0 eq), anhydrous MgSO.sub.4 (146 mg, 1.215 mmol, 5.0 eq) and anhydrous CH.sub.3CN (5 mL) were added to a 50 mL three-necked flask and stirred in a nitrogen atmosphere at room temperature for 0.7 h. The mixture was cooled to 0? C. in an ice bath, and CF.sub.3SO.sub.3H (182 mg, 1.215 mmol, 5.0 eq) was slowly added dropwise. After the addition, the reaction was naturally warmed to room temperature. After the reaction was complete, it was quenched with EA and a saturated solution of sodium bicarbonate in an ice bath, and the pH was adjusted to be greater than 8. Then the aqueous phase was extracted with EA, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by prep-HPLC to give compound 1-A (64.5 mg, 42% yield).

[0351] Ms (ESI): m/z 632.3 [M+H].sup.+.

[0352] .sup.1H NMR (400 MHz, DMSO) ? 7.73 (dd, J=8.3, 2.5 Hz, 4H), 7.64 (d, J=8.3 Hz, 2H), 7.57 (d, J=8.2 Hz, 2H), 7.33 (d, J=10.1 Hz, 1H), 7.11 (t, J=7.8 Hz, 1H), 6.89 (s, 1H), 6.83 (d, J=7.8 Hz, 1H), 6.57 (d, J=7.9 Hz, 1H), 6.17 (d, J=10.1 Hz, 1H), 5.95 (s, 1H), 5.52 (s, 1H), 5.17 (s, 2H), 5.12 (t, J=5.9 Hz, 1H), 4.97 (d, J=5.1 Hz, 1H), 4.82 (d, J=2.8 Hz, 1H), 4.56 (dd, J=19.6, 6.4 Hz, 1H), 4.32 (s, 1H), 4.22 (dd, J=19.6, 5.5 Hz, 1H), 2.61-2.53 (m, 1H), 2.34 (d, J=10.4 Hz, 1H), 2.20-2.01 (m, 2H), 1.90-1.60 (m, 5H), 1.41 (s, 3H), 1.10-1.01 (m, 2H), 0.89 (s, 3H).

Example 2

[0353] ##STR00090##

[0354] In a nitrogen atmosphere, compound 1-4 (176 mg, 0.47 mmol, 1.0 eq) and anhydrous magnesium sulfate (282 mg, 2.34 mmol, 5.0 eq) were added to a 25 mL reaction flask, and anhydrous acetonitrile (5 mL) was added. The reaction was stirred at room temperature for 90 min. Compound 2-1 (160 mg, 0.49 mmol, 1.05 eq) was then added to the above mixture. The mixture was cooled to 0-5? C. in an ice bath, and trifluoromethanesulfonic acid (351 mg, 2.34 mmol, 5.0 eq) was added using a syringe. The reaction was stirred in the ice bath until completion. The reaction mixture was filtered with celite, and the filter cake was rinsed with ethyl acetate. Ethyl acetate and water were added to the filtrate. The organic phase was separated, washed once with saturated brine, dried, and concentrated under reduced pressure to give a crude product. The crude product was purified by prep-HPLC to give compound 2-A (88 mg, 32.2% yield).

[0355] Ms (ESI): m/z 584.42 [M+1].sup.+.

Example 3

[0356] ##STR00091##

[0357] Step 1

[0358] In a nitrogen atmosphere, compound 3-1 (10.0 g, 45.03 mmol), (Bpin).sub.2 (bis(pinacolato)diboron, 18.3 g, 72.05 mmol), X-Phos (1.36 g, 2.70 mmol) and KOAc (8.84 g, 90.06 mmol) were dissolved in 1,4-dioxane (150 mL). Pd.sub.2(dba).sub.3 (1.65 g, 1.80 mmol) was added, and the reaction was heated to 90? C. The reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated and separated by column chromatography (PE/EA) to give compound 3-2 (13 g).

[0359] MS-ESI: m/z 270.2 [M+H].sup.+.

[0360] Step 2

[0361] Compound 3-2 (13 g, 45.03 mmol) was dissolved in toluene (100 mL), and Boc.sub.2O (13.4 mL, 58.54 mmol) was added. The reaction was heated to 100? C. The reaction mixture was cooled to room temperature, concentrated, and separated by column chromatography (PE/EA) to give compound 3-3 (16 g, 96.2% yield over two steps).

[0362] MS-ESI: m/z 396.1 [M+Na].sup.+.

[0363] Step 3

[0364] In a nitrogen atmosphere, compound 3-3 (2.0 g, 5.42 mmol), compound 3-4 (2.16 g, 10.84 mmol) and K.sub.2CO.sub.3 (3.75 g, 27.10 mmol) were dissolved in tetrahydrofuran (50 mL). Pd(dppf)C.sub.12 DCM (441 mg, 0.54 mmol) was added, and the reaction was heated to 80? C. The reaction mixture was cooled to room temperature, quenched with water, and extracted with ethyl acetate. After concentration, the residue was separated by column chromatography (PE/EA) to give compound 3-5 (1.33 g, 67.9% yield).

[0365] MS-ESI: m/z 384.1 [M+Na].sup.+.

[0366] Step 4

[0367] In a nitrogen atmosphere, compound 1-4 (123 mg, 0.327 mmol) and MgSO.sub.4 (197 mg, 1.635 mmol) were dissolved in acetonitrile (10 mL). The mixture was allowed to react at room temperature for 1 h. A solution of compound 3-5 (130 mg, 0.360 mmol) in acetonitrile (10 mL) was added. The mixture was cooled to 0? C., and trifluoromethanesulfonic acid (145 ?L, 1.635 mmol) was slowly added dropwise. After the addition, the reaction was naturally warmed to room temperature. After filtration, the filtrate was concentrated and separated by prep-HPLC (CH.sub.3CN/H.sub.2O) to give compound 3-A (90 mg, 44.4% yield).

[0368] MS-ESI: m/z 620.3 [M+H].sup.+.

[0369] .sup.1H NMR (400 MHz, DMSO) ? 7.96 (dd, J=7.0, 2.2 Hz, 1H), 7.38-7.26 (m, 5H), 7.20 (d, J=8.2 Hz, 3H), 7.17-7.09 (m, 1H), 6.67 (d, J=7.2 Hz, 1H), 6.14 (d, J=10.1 Hz, 1H), 5.91 (s, 1H), 5.36 (s, 1H), 4.90 (d, J=4.9 Hz, 1H), 4.75 (brs, 1H), 4.47 (d, J=19.4 Hz, 1H), 4.32 (s, 2H), 4.27 (brs, 1H), 4.15 (d, J=19.4 Hz, 1H), 2.58-2.50 (m, 3H), 2.34-2.23 (m, 1H), 2.08 (dd, J=16.2, 6.0 Hz, 1H), 2.03-1.93 (m, 1H), 1.81-1.53 (m, 5H), 1.38 (s, 3H), 1.23 (s, 1H), 1.03 (ddd, J=27.9, 11.3, 2.6 Hz, 2H), 0.84 (s, 3H).

Example 4

[0370] ##STR00092## ##STR00093##

[0371] Step 1

[0372] To a 1000 mL single-necked flask, compound 4-1 (48 g, 176.4 mmol, 1.0 eq), bis(pinacolato)diboron (71.7 g, 282.2 mmol, 1.6 eq), potassium acetate (34.6 g, 352.8 mmol, 2.0 eq), PdCl.sub.2(dppf) (6.4 g, 8.82 mmol, 0.05 eq) and dioxane (500 mL) were added. The mixture was heated to 95? C. and stirred in a nitrogen atmosphere until the reaction was complete. The reaction was stopped, and the reaction mixture was cooled. Compound 4-2 (80.8 g, 352.8 mmol, 2.0 eq), potassium carbonate (48.8 g, 352.8 mmol, 2.0 eq) and PdCl.sub.2(dppf) (6.4 g, 8.82 mmol, 0.05 eq) were added, and water (100 mL) was then added. The mixture was stirred, heated to 80? C. and stirred in a nitrogen atmosphere until the reaction was complete. After the reaction mixture was cooled, ethyl acetate and water were added, and the mixture was stirred and separated. After drying over anhydrous sodium sulfate and concentration, the crude product was purified by column chromatography to give compound 4-3 (about 54 g, 90% yield).

[0373] Ms (ESI): m/z 342.1 [M+1].sup.+.

[0374] Step 2

[0375] To a 500 mL three-necked flask, compound 4-3 (7.0 g, 20.5 mmol, 1.0 eq), potassium permanganate (9.7 g, 61.6 mmol, 3.0 eq) and tetra-tert-butylammonium bromide (20.0 g, 61.6 mmol, 3.0 eq) were added, and dichloroethane (140 mL) was then added. The mixture was stirred at room temperature in a nitrogen atmosphere until the reaction was complete. The reaction was stopped, and the reaction mixture was cooled in iced water. 10% sodium bisulfite and acetic acid were added. The organic layer was separated, dried over anhydrous sodium sulfate, filtered under reduced pressure, and concentrated. The crude product was purified by column chromatography to give compound 4-4 (about 6.2 g, 85% yield).

[0376] Ms (ESI): m/z 378.1 [M+23].sup.+.

[0377] Step 3:

[0378] To a 500 mL three-necked flask, compound 4-4 (20.0 g, 56.0 mmol, 1.0 eq), propanedithiol (12.1 g, 112.0 mmol, 2.0 eq) and boron trifluoride diethyl etherate (23.8 g, 168.0 mmol, 3.0 eq) were added, and chloroform (100 mL) was then added. The mixture was heated at reflux and stirred in a nitrogen atmosphere until the reaction was complete. The reaction was stopped, and the reaction mixture was cooled in iced water. Water was added, and the mixture was stirred to completely dissolve the solid. The organic layer was separated, dried over anhydrous sodium sulfate, filtered under reduced pressure, and concentrated. Petroleum ether was added to the crude product, and the mixture was stirred and filtered under reduced pressure to give compound 4-5 (about 22 g). The product was directly used in the next step.

[0379] Ms (ESI): m/z 346.0 [M+1].sup.+.

[0380] Step 4:

[0381] To a 100 mL three-necked flask, compound 4-5 (22.0 g, 56.0 mmol, 1.0 eq) and di-tert-butyl dicarbonate (24.4 g, 112.0 mmol, 2.0 eq) were added, and ethanol (60 mL) was then added. The mixture was heated to 50? C. and stirred in a nitrogen atmosphere until the reaction was complete. The reaction was stopped, and the reaction mixture was concentrated and purified by column chromatography to give compound 4-6 (about 18.5 g, 71% yield).

[0382] Ms (ESI): m/z 468.1 [M+23].sup.+.

[0383] Step 5:

[0384] To a 100 mL three-necked flask, compound 4-6 (4.7 g, 13.6 mmol, 1.0 eq) and DAST (6.6 g, 40.9 mmol, 3.0 eq) were added, and dichloromethane (50 mL) was then added. The mixture was heated to 50? C. and stirred in a nitrogen atmosphere until the reaction was complete. The reaction was stopped. The reaction was quenched with water under cooling in iced water. The organic layer was separated, dried over anhydrous sodium sulfate, filtered under reduced pressure, concentrated, and purified by column chromatography to give compound 4-7 (about 3.9 g, 76% yield).

[0385] Ms (ESI): m/z 400.1 [M+23].sup.+.

[0386] Step 6:

[0387] To a 100 mL three-necked flask, compound 4-7 (2.0 g, 5.3 mmol, 1.0 eq) was added and then dissolved in tetrahydrofuran (25 mL). In a nitrogen atmosphere, the solution was cooled to about 0? C., and a 1.0 M solution of lithium aluminum hydride in tetrahydrofuran (8.0 mL, 8.0 mmol, 1.5 eq) was slowly added dropwise. The mixture was stirred at about 0? C. until the reaction was complete. The reaction was stopped. The reaction was quenched with water (0.8 mL) under cooling in iced water, and a 3.0 M aqueous solution of potassium hydroxide (0.8 mL) was added. Then water (1.6 mL) was added, and the mixture was stirred for 15 min and filtered under reduced pressure. The filtrate was dried over anhydrous sodium sulfate, filtered under reduced pressure, and concentrated to give compound 4-8 (about 2.2 g). The product was directly used in the next step.

[0388] Ms (ESI): m/z 372.1 [M+23].sup.+.

[0389] Step 7:

[0390] To a 100 mL three-necked flask, compound 4-8 (2.2 g, 5.3 mmol, 1.0 eq) was added and then dissolved in ethyl acetate (25 mL). In a nitrogen atmosphere, the solution was cooled to about 5? C., and the Dess-Martin oxidant (6.7 g, 15.9 mmol, 3.0 eq) was added. The mixture was stirred at about 10? C. until the reaction was complete. The reaction was stopped, and the reaction mixture was filtered under reduced pressure. The filtrate was concentrated, and the crude product was purified by column chromatography to give compound 4-9 (about 1.5 g, 82% yield).

[0391] Ms (ESI): m/z 370.1 [M+23].sup.+.

[0392] Step 8:

[0393] To a 100 mL three-necked flask, compound 1-4 (1.2 g, 3.0 mmol, 1.0 eq) and compound 4-9 (1.1 g, 3.17 mmol, 1.05 eq) were added, anhydrous magnesium sulfate (1.8 g, 15.0 mmol, 5.0 eq) was added, and acetonitrile (25 mL) was then added. The mixture was stirred. In a nitrogen atmosphere, the mixture was cooled to below 0? C., and trifluoromethanesulfonic acid (2.3 g, 15.0 mmol, 5.0 eq) was added. The mixture was stirred at about 0? C. until the reaction was complete. The reaction was stopped, and the reaction mixture was filtered under reduced pressure. The filtrate was directly purified by a preparative method to give compound 4-A (about 1.5 g, 70% yield).

[0394] Ms (ESI): m/z 606.3 [M+1].sup.+.

[0395] .sup.1H-NMR (400 MHz, MeOD) ? 7.53 (m, 4H), 7.44 (m, 2H), 7.28 (m, 3H), 6.23 (dd, 1H), 5.99 (t, 1H), 5.52 (s, 1H), 5.08 (d, 1H), 4.63 (d, 1H), 4.37 (m, 2H), 2.65 (td, 1H), 2.36 (d, 1H), 2.25 (m, 1H), 2.13 (m, 1H), 1.95 (dd, 1H), 1.80 (m, 4H), 1.49 (s, 3H), 1.11 (dt, 1H), 1.10 (m, 4H).

Example 5

[0396] ##STR00094##

[0397] Compound 5-1 (85.3 mg, 0.262 mmol, 1.1 eq), compound 5-2 (94 mg, 0.238 mmol, 1.0 eq, Macklin/C.sub.10492138/P>98%), anhydrous MgSO.sub.4 (143 mg, 1.19 mmol, 5.0 eq) and anhydrous CH.sub.3CN (4 mL) were added to a 25 mL Schlenk reaction flask and stirred in a nitrogen atmosphere at room temperature for 0.7 h. The mixture was cooled to 0? C. in an ice bath, and CF.sub.3SO.sub.3H (179 mg, 1.19 mmol, 5.0 eq) was slowly added dropwise. After the dropwise addition, the reaction was naturally warmed and completed. The reaction was quenched with EA and a saturated solution of sodium bicarbonate in an ice bath. Then the aqueous phase was extracted with EA, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give a crude product. The crude product was purified by prep-HPLC to give compound 5-A (67.1 mg, 42.6% yield).

[0398] Ms (ESI): m/z 602.3 [M+H].sup.+.

[0399] .sup.1H NMR (400 MHz, DMSO) ? 7.71 (d, J=8.1 Hz, 2H), 7.59 (d, J=8.2 Hz, 2H), 7.29 (d, J=10.2 Hz, 1H), 7.16 (t, J=7.7 Hz, 1H), 6.92 (s, 1H), 6.87-6.76 (m, 2H), 6.22 (d, J=10.2 Hz, 1H), 6.02 (s, 1H), 5.59 (s, 1H), 5.47 (d, J=2.9 Hz, 1H), 5.39 (s, 2H), 5.13 (t, J=5.9 Hz, 1H), 5.00 (d, J=4.3 Hz, 1H), 4.57 (dd, J=19.6, 6.5 Hz, 1H), 4.30-4.15 (m, 2H), 2.71-2.56 (m, 2H), 2.38-2.31 (m, 1H), 2.20-2.12 (m, 1H), 2.09-2.01 (m, 1H), 1.90-1.80 (m, 1H), 1.78-1.64 (m, 3H), 1.50 (s, 3H), 1.45-1.34 (m, 1H), 0.89 (s, 3H).

Example 6

[0400] ##STR00095##

[0401] In a nitrogen atmosphere, compound 3-A (90.0 mg, 0.145 mmol, 1.0 eq) was added to 2 mL of toluene, and BOC anhydride (63.3 mg, 0.290 mmol, 2.0 eq) was then added. The system was heated at 100? C. until the reaction was complete. The reaction mixture was directly concentrated to dryness under reduced pressure and purified by column chromatography to give compound 3-A-1 (48.0 mg).

[0402] MS-ESI: m/z 742.3 [M+Na].sup.+.

[0403] Step 2

[0404] In a nitrogen atmosphere, compound 3-A-1 (45.0 mg, 0.063 mmol) and tetrazole (66.0 mg, 0.945 mmol) were dissolved in N,N-dimethylacetamide (1.5 mL), and di-tert-butyl N,N-diethylphosphoramidite (187.0 mg, 0.756 mmol) was added. The mixture was allowed to react at room temperature for 2 h. The reaction was cooled to 0? C., and H.sub.2O.sub.2 (50.0 mg, 0.82 mmol) was slowly added. After the addition, the mixture was stirred at room temperature until the reaction was complete. 2 mL of water was added, and the mixture was filtered. The filter cake was dried to give compound 3-A-2 (about 40.0 mg). The product was directly used in the next step.

[0405] MS-ESI: m/z 943.3 [M+Na].sup.+.

[0406] Step 3

[0407] In a nitrogen atmosphere, compound 3-A-2 (40.0 mg, 0.043 mmol) was dissolved in dichloromethane (1.0 mL). The solution was cooled to 0? C., and trifluoroacetic acid (0.3 mL) was added. The mixture was allowed to react at room temperature for 3 h. After concentration, the residue was separated by prep-HPLC (CH.sub.3CN/H.sub.2O+0.1% trifluoroacetic acid) to give compound 3-B (about 12.0 mg).

[0408] ESI: m/z 700.3 [M+H].sup.+.

Example 7

[0409] ##STR00096##

[0410] Step 1

[0411] In a nitrogen atmosphere, compound 4-A (45.0 mg, 0.074 mmol, 1.0 eq) was added to 2 mL of toluene, and BOC anhydride (32.0 mg, 0.158 mmol, 2.0 eq) was then added. The system was heated at 100? C. until the reaction was complete. The reaction mixture was directly concentrated under reduced pressure and purified by column chromatography to give compound 4-A-1 (45.2 mg).

[0412] MS-ESI: m/z 728.2 [M+Na].sup.+.

[0413] Step 2

[0414] In a nitrogen atmosphere, compound 4-A-1 (45.0 mg, 0.063 mmol) and tetrazole (66.0 mg, 0.945 mmol) were dissolved in N,N-dimethylacetamide (1.0 mL), and di-tert-butyl N,N-diethylphosphoramidite (249.0 mg, 0.756 mmol) was added. The mixture was allowed to react at room temperature for 2 h. The reaction was cooled to 0? C., and H.sub.2O.sub.2 (50.0 mg, 0.82 mmol) was slowly added. After the addition, the mixture was stirred at room temperature until the reaction was complete. 2 mL of water was added, and the mixture was filtered. The filter cake was dried to give compound 4-A-2 (about 43.0 mg). The product was directly used in the next step.

[0415] MS-ESI: m/z 920.3 [M+Na].sup.+.

[0416] Step 3

[0417] In a nitrogen atmosphere, compound 4-A-2 (40.0 mg, 0.042 mmol) was dissolved in dichloromethane (1.0 mL). The solution was cooled to 0? C., and trifluoroacetic acid (0.3 mL) was added. The mixture was allowed to react at room temperature for 3 h. After concentration, the residue was separated by prep-HPLC (CH.sub.3CN/H.sub.2O+0.1% trifluoroacetic acid) to give compound 4-B (about 15.0 mg).

[0418] ESI: m/z 686.2 [M+H].sup.+.

Example 8

[0419] ##STR00097## ##STR00098##

[0420] Step 1

[0421] In a nitrogen atmosphere, compounds 3-A (2.60 g, 4.20 mmol) and 3-6 (2.03 g, 4.20 mmol) were dissolved in N,N-dimethylacetamide (30 mL), and triethylamine (1.27 g, 12.60 mmol) was added. The mixture was cooled to 0? C., and T.sub.3P (5.35 g, 8.40 mmol, 50% in DMF) was slowly added. The reaction was completed at room temperature. The reaction mixture was directly purified by prep-HPLC to give the product 3-7 (965 mg, 21.2% yield).

[0422] MS-ESI: m/z 1106.5 [M+Na].sup.+.

[0423] Step 2

[0424] In a nitrogen atmosphere, compound 3-7 (805 mg, 0.778 mmol) and tetrazole (818 mg, 11.670 mmol) were dissolved in N,N-dimethylacetamide (10 mL), and di-tert-butyl N,N-diethylphosphoramidite (2.6 mL, 9.336 mmol) was added. The mixture was allowed to react at room temperature for 2 h. The reaction was cooled to 0? C., and H.sub.2O.sub.2 (437 ?L, 4.279 mmol, 30% in water) was slowly added. After the addition, the mixture was stirred at room temperature for 1 h. After concentration, the residue was separated by column chromatography (CH.sub.3CN/H.sub.2O) to give compound 3-8 (692 mg, 73.1% yield).

[0425] Step 3

[0426] In a nitrogen atmosphere, compound 3-8 (830 mg, 0.650 mmol) was dissolved in acetonitrile (20 mL), and piperidine (302 ?L, 3.250 mmol) was added. The mixture was allowed to react at room temperature. After concentration, the residue was triturated with 15 mL of petroleum ether three times to give compound 3-9 (645 mg). The product was directly used in the next step.

[0427] MS-ESI: m/z 1054.5 [M+H].sup.+.

[0428] Step 4

[0429] In a nitrogen atmosphere, 2-bromoacetic acid (170 mg, 1.224 mmol) and EEDQ (305 mg, 1.224 mmol) were dissolved in N,N-dimethylacetamide (5 mL). The mixture was allowed to react at room temperature for 1 h. A solution of compound 3-9 (645 mg, 0.612 mmol) in N,N-dimethylacetamide (5 mL) was added, and the mixture was allowed to react at room temperature. The reaction mixture was diluted with dichloromethane (200 mL) and washed with a 1 M aqueous solution of hydrobromic acid, a saturated solution of sodium bicarbonate and then saturated brine. The organic phase was concentrated to give compound 3-10 (830 mg). The product was directly used in the next step.

[0430] MS-ESI: m/z 1196.4 [M+Na].sup.+.

[0431] Step 5

[0432] In a nitrogen atmosphere, compound 3-10 (830 mg, 0.706 mmol) was dissolved in dichloromethane (8 mL). The solution was cooled to 0? C., and trifluoroacetic acid (4 mL) was added. The mixture was allowed to react at room temperature. After concentration, the residue was separated by prep-HPLC (CH.sub.3CN/H.sub.2O+0.1% trifluoroacetic acid) to give compound 3-B00 (220 mg, 33.6% yield over three steps).

[0433] MS-ESI: m/z 1028.2 [M+Na].sup.+.

[0434] .sup.1H NMR (400 MHz, DMSO) ? 9.98 (s, 1H), 8.55 (t, J=5.4 Hz, 1H), 8.36 (d, J=7.4 Hz, 1H), 7.97-7.81 (m, 2H), 7.55-7.46 (m, 2H), 7.45-7.38 (m, 2H), 7.36-7.26 (m, 3H), 7.23-7.16 (m, 2H), 6.14 (dd, J=10.0, 1.1 Hz, 1H), 5.90 (s, 1H), 5.44 (s, 1H), 4.97-4.79 (m, 3H), 4.65-4.50 (m, 2H), 4.42 (s, 2H), 4.27 (brs, 1H), 3.94 (s, 2H), 3.86 (d, J=5.5 Hz, 2H), 2.41-2.22 (m, 4H), 2.17-1.88 (m, 6H), 1.81-1.56 (m, 6H), 1.37 (s, 3H), 1.06-0.92 (m, 2H), 0.85 (s, 3H).

Example 9

[0435] ##STR00099## ##STR00100## ##STR00101##

[0436] Step 1

[0437] To a 25 mL three-necked flask, compound 4-A (0.329 g, 0.544 mmol, 1.05 eq) was added, and compound 4-10 (0.25 g, 0.52 mmol, 1.0 eq), triethylamine (0.25 g, 1.56 mmol, 3.0 eq) and DMF (2 mL) were added. After the addition, the flask was cooled in an ice bath for 5-10 min to reduce the internal temperature to ?5? C., and then T3P (50% DMF) (0.9 mL, 1.82 mmol, 3.5 eq) was slowly added. After the addition, the mixture was naturally warmed and stirred until the reaction was complete. The reaction mixture was directly purified by pre-HPLC to give compound 4-11 (223 mg, 40% yield).

[0438] Ms (ESI): m/z 1092.4 [M+Na].sup.+.

[0439] Step 2

[0440] To a 50 mL three-necked flask, compound 4-11 (0.22 g, 0.206 mmol, 1.0 eq) was added, and the starting material tetrazole (0.20 g, 2.87 mmol, 14.0 eq), di-tert-butyl N,N-diethylphosphoramidite (0.616 g, 2.47 mmol, 12.0 eq) and DMF (2.6 mL) were added. After the addition, the mixture was allowed to react at room temperature for 2 h, cooled to 0? C. in an ice bath, and then H.sub.2O.sub.2 (30%) (0.13 g, 0.57 mmol, 5.5 eq) was slowly added.

[0441] After the addition, the mixture was stirred at room temperature until the reaction was complete. The reaction mixture was directly purified by pre-HPLC to give compound 4-12 (184.1 mg, 70.8% yield).

[0442] Ms (ESI): m/z 1284.6 [M+Na].sup.+.

[0443] Step 3

[0444] To a 25 mL single-necked flask, compound 4-12 (0.285 g, 0.233 mmol, 1.0 eq) was added, and the starting material piperidine (0.17 g, 1.96 mmol, 8.5 eq) and acetonitrile (5 mL) were added. After the addition, the mixture was stirred at room temperature. The reaction mixture was concentrated under reduced pressure and triturated with 5 mL of petroleum ether. The triturate was stirred at room temperature and then filtered. The filter cake was washed with 2 mL of petroleum ether twice to give compound 4-13 (209 mg, 91% yield).

[0445] Ms (ESI): m/z 1004.4 [M+H].sup.+.

[0446] Step 4

[0447] To a 25 mL single-necked flask, the starting material 2-bromoacetic acid (0.074 g, 0.523 mmol, 2.6 eq) was added, and the starting material EEDQ (0.13 g, 0.523 mmol, 2.6 eq) and DMF (1 mL) were added. After the addition, the mixture was stirred at room temperature for 1 h, and a solution of compound 4-13 (0.21 g, 0.201 mmol, 1.0 eq) in DMF (0.5 mL) was added. After the addition, the mixture was stirred at room temperature until the reaction was complete. The reaction mixture was first diluted with dichloromethane (40 mL), then washed with 1 M HBr (10 mL?2), then washed with saturated sodium bicarbonate (20 mL?2), and finally washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure using an oil pump to give compound 4-14 (230 mg, crude).

[0448] Ms (ESI): m/z 1182.4 [M+Na].sup.+.

[0449] Step 5

[0450] To a 25 mL single-necked flask, compound 4-14 (0.240 g, 0.201 mmol, 1.0 eq) and DCM (2 mL) were added. After the addition, the mixture was cooled to 0? C. in an ice bath, and trifluoroacetic acid (1 mL) was slowly added. After the addition, the mixture was stirred at room temperature until the reaction was complete. The reaction mixture was concentrated under reduced pressure in an ice bath, then purified by pre-HPLC, and lyophilized to give compound 4-B00 (78 mg, 39.2% yield).

[0451] Ms (ESI): m/z 1014.2 [M+Na].sup.+.

[0452] .sup.1H NMR (400 MHz, DMSO) ? 10.15 (s, 1H), 8.53 (t, J=5.2 Hz, 1H), 8.29 (br, d, J=7.5 Hz, 1H), 7.83 (s, 1H), 7.73 (d, J=7.8 Hz, 1H), 7.68-7.58 (m, 2H), 7.57-7.47 (m, 2H), 7.43 (t, J=7.9 Hz, 1H), 7.32 (d, J=9.9 Hz, 1H), 7.20 (d, J=8.2 Hz, 1H), 6.17 (d, J=10.1 Hz, 1H), 5.92 (s, 1H), 5.58 (s, 1H), 4.95-4.80 (m, 3H), 4.58 (br, dd, J=18.4, 8.2 Hz, 1H), 4.40-4.37 (m, 1H), 4.33-4.27 (m, 1H), 3.93 (s, 2H), 3.82-3.78 (m, 2H), 2.30-2.25 (m, 3H), 2.16-2.07 (m, 1H), 2.10-1.90 (m, 2H), 1.88-1.59 (m, 6H), 1.39 (s, 3H), 1.18-1.05 (m, 2H), 0.89 (s, 3H).

Example 10

[0453] ##STR00102## ##STR00103##

[0454] Step 1

[0455] To a 25 mL three-necked flask, compound 4-A (0.260 g, 0.430 mmol, 1.05 eq), compound 4-C-1 (0.193 g, 0.551 mmol, 1.32 eq), triethylamine (0.125 g, 1.23 mmol, 3.0 eq) and DMF (2 mL) were added. After the addition, the mixture was cooled to ?5? C. in an ice bath, and then T3P (50% DMF) (0.5 mL, 1.024 mmol, 2.5 eq) was slowly added. The mixture was naturally warmed and stirred until the reaction was complete. The reaction mixture was directly purified by pre-HPLC (TFA (0.05%) water-acetonitrile) to give compound 4-C-2 (140 mg, 35.1% yield).

[0456] Ms (ESI): m/z 949.3 [M+Na].sup.+.

[0457] Step 2

[0458] To a 50 mL three-necked flask, compound 4-C-2 (0.34 g, 0.366 mmol, 1.0 eq) was added, and the starting material tetrazole (0.360 g, 5.13 mmol, 14.0 eq), di-tert-butyl N,N-diethylphosphoramidite (1.099 g, 4.40 mmol, 12.0 eq) and DMF (5 mL) were added. After the addition, the mixture was allowed to react at room temperature for 2 h, cooled to 0? C. in an ice bath, and then H.sub.2O.sub.2 (30%) (0.234 g, 2.02 mmol, 5.5 eq) was slowly added. After the addition, the mixture was stirred at room temperature until the reaction was complete. The reaction mixture was directly purified using a medium-pressure boston reversed-phase column to give compound 4-C-3 (304 mg, 64.1% yield).

[0459] Ms (ESI): m/z 1141.3 [M+Na].sup.+.

[0460] Step 3

[0461] To a 25 mL single-necked flask, compound 4-C-3 (0.304 g, 0.272 mmol, 1.0 eq) was added, and the starting material piperidine (0.255 g, 1.63 mmol, 9.0 eq) and acetonitrile (8 mL) were added. After the addition, the mixture was stirred at room temperature until the reaction was complete. The reaction mixture was concentrated under reduced pressure and triturated with 4 mL of petroleum ether. The triturate was stirred at 35? C. for 2 h and then filtered. The filter cake was washed with 2 mL of petroleum ether twice to give compound 4-C-4 (243 mg, 99% yield).

[0462] Ms (ESI): m/z 897.6 [M+H].sup.+.

[0463] Step 4

[0464] To a 25 mL single-necked flask, the starting material 2-bromoacetic acid (0.112 g, 0.813 mmol, 3.0 eq) was added, and the starting material EEDQ (0.201 g, 0.813 mmol, 3.0 eq) and DMF (2 mL) were added. After the addition, the mixture was stirred at room temperature for 0.6 h, and a solution of compound 4-C-4 (0.243 g, 0.271 mmol, 1.0 eq) in DMF (1 mL) was added. After the addition, the mixture was stirred at room temperature until the reaction was complete. The reaction mixture was first diluted with dichloromethane (50 mL), then washed with 1 M HBr (15 mL?2), then washed with saturated sodium bicarbonate (15 mL?2), and finally washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated to dryness by rotary evaporation under reduced pressure using an oil pump, and dried by nitrogen blowing for 10 min to give compound 4-C-5 (275 mg, crude).

[0465] Ms (ESI): m/z 1039.1 [M+Na].sup.+ and 1041.1 [M+Na+2].sup.+.

[0466] Step 5

[0467] To a 25 mL single-necked flask, the starting material compound 4-C-5 (0.275 g, 0.271 mmol, 1.0 eq) and DCM (2.5 mL) were added. After the addition, the mixture was cooled to 0? C. in an ice bath, and trifluoroacetic acid (1 mL) was slowly added. After the addition, the mixture was stirred at room temperature until the reaction was complete. The reaction mixture was concentrated under reduced pressure in an ice bath, then purified by pre-HPLC, and lyophilized to give compound 4-C.sub.00 (120 mg, 48.3% yield).

[0468] Ms (ESI): m/z 905.2 [M+H].sup.+.

Example 11

[0469] ##STR00104##

[0470] To adalimumab in buffer A (a 0.05 M aqueous buffer solution having a pH of 6.3; 10.0 mg/mL, 6.0 mL, 405.11 nmol), a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (2.5 mM, 356.8 ?L, 891.24 nmol) was added at 37? C. The mixture was allowed to react in a shaking water bath at 37? C. for 3 h, and the reaction was stopped. The reaction mixture was cooled to 25? C. in a water bath. 1.0 M Tris buffer (840 ?L) was added to the above reaction mixture, and then a solution of compound 3-B00 (4.08 mg, 4051.10 nmol) in 300 ?L of DMSO was added to the above reaction mixture. The mixture was allowed to react in a shaking water bath at 25? C. for 3 h, and the reaction was stopped. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: buffer A) and concentrated using an ultrafiltration tube to give antibody-drug conjugate Humira-3-B00 in buffer A (2.55 mg/mL, 23.5 mL). The product was stored frozen at 4? C.

Example 12

[0471] ##STR00105##

[0472] To adalimumab in buffer A (a 0.05 M aqueous buffer solution having a pH of 6.3; 10.0 mg/mL, 6.0 mL, 405.11 nmol), a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (2.5 mM, 405.4 ?L, 1012.77 nmol) was added at 37? C. The mixture was allowed to react in a shaking water bath at 37? C. for 3 h, and the reaction was stopped. The reaction mixture was cooled to 25? C. in a water bath. 1.0 M Tris buffer (840 ?L) was added to the above reaction mixture, and then a solution of compound 4-B00 (4.02 mg, 4051.10 nmol) in 300 ?L of DMSO was added to the above reaction mixture. The mixture was allowed to react in a shaking water bath at 25? C. for 3 h, and the reaction was stopped. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: buffer A) and concentrated using an ultrafiltration tube to give the title product antibody-drug conjugate Humira-4-B00 in buffer A (2.58 mg/mL, 23.25 mL). The product was stored frozen at 4? C.

Biological Evaluation

[0473] The present disclosure is further described and explained below with reference to test examples, which are not intended to limit the scope of the present disclosure.

Test Example 1: In Vitro Activity of Small-Molecule Steroids

[0474] 1. Test Samples

[0475] Compounds 1-A to 5-A, and compound A-A (prepared according to Example 2 of WO2017210471).

##STR00106##

[0476] 2. Glucocorticoid Receptor Binding Assay

[0477] The binding activity of small-molecule steroids for glucocorticoid receptor (GR) was tested using a human glucocorticoid NHR (radiolabeled agonist) binding assay (#232020, eurofins). The assays were based on the following principle: Different concentrations of a small-molecule steroid and 5 nM [3H]dexamethasone are incubated with human recombinant GR at 4? C. for 24 h. The small-molecule steroid competes with [3H]dexamethasone for binding to human GR. The binding activity of the small-molecule steroid for GR can be calculated by counting the number of [3H]dexamethasone molecules that bind specifically to the receptor. The results are detailed in Table 1.

[0478] 3. Mineralocorticoid Receptor Agonist Activity Assay

[0479] The mineralocorticoid receptor (MR) agonist activity of small-molecule steroids was tested using a PathHunter? NHR nuclear translocation assay. PathHunter NHR CHO-K.sub.1 cells were plated onto a 384-well white-wall microplate and co-incubated with test small-molecule steroids at 37? C. in 5% CO.sub.2 to induce reactions. Assay signals were generated using the PathHunter assay reagent mix. After one hour of incubation at room temperature, chemiluminescent signals were detected. The data were analyzed using four-parameter curve fitting to generate EC.sub.50 values. The results are detailed in Table 1.

[0480] 4. GRE reporter gene assay A549 cells were plated (40,000 cells/96-well plate), transfected with pGL4.36 (MMTV-Luc, 100 ng/well) using lipo3000, cultured overnight and then incubated with different concentrations of small-molecule steroids. After 24 h, the expression of luciferase in the reporter gene system was detected using the Bright-Glo (promega) assay reagent.

TABLE-US-00002 TABLE 1 In vitro activity of small-molecule steroids GRE GRE reporter reporter MR Compound GR binding gene gene agonist No. IC.sub.50 (nM) EC.sub.50 (nM) E.sub.max (RLU) EC.sub.50 (?M) Compound 2.52 0.60 1.45E6 0.06 A-A Compound 4.37 22.65 7.67E5 >200 1-A Compound 6.69 / / 0.22 2-A Compound 2.24 1.47 1.53E6 0.19 3-A Compound 2.12 2.16 1.69E6 0.26 4-A Compound 5.23 4.3 2.3E7 / 5-A

Test Example 2: Anti-Inflammatory Activity of Small-Molecule Steroids in Lipopolysaccharide (LPS)'s Stimulation of Human PBMCs' Cytokine Secretion

[0481] 1. Test Samples

[0482] Compounds 1-A to 5-A, and compound A-A.

[0483] 2. Test Method

[0484] Frozen primary human peripheral blood mononuclear cells (PBMCs) were resuspended in RPMI (2% FBS, 1% penicillin-streptomycin) and plated onto a 96-well plate. After PBMCs were co-incubated with different concentrations of small-molecule steroids at 37? C. in 5% CO.sub.2 for 4 h, 0.01 ng/mL LPS was added for overnight stimulation. The following day, the medium supernatants were collected and assayed for IL-6 concentration using alpha LISA (Cisbio).

[0485] 3. Test Results

[0486] The test results are shown in FIG. 1: the small-molecule steroid compounds of the present disclosure can significantly inhibit LPS-induced IL-6 release.

Test Example 3: Activity of Anti-TNF-ADCs in Membrane-Bound TNF?-Mediated GRE Reporter Gene System

[0487] 1. Test Samples

[0488] Humira-3-B00, Humira-4-B00, and Humira-A-B00 (prepared according to Example 7 of WO2019106609).

##STR00107##

[0489] 2. Test Method

[0490] A reporter gene cell line stably transfected with MMLV-Luc was established using lentiviruses in Hela cells. The stably transfected Hela-MMTV-Luc cell line was plated onto a 96-well plate (30000 cells/well), and cells in each well were transfected with an empty plasmid or a human TNF? mutant plasmid (TNF??12, with the restriction enzyme cutting site for TACE removed, 50 ng/well) using lipo3000. After being left overnight, the cells were incubated with different concentrations of anti-TNF-ADCs. After 24 h, the expression of luciferase in the reporter gene system was detected using the Bright-Glo (promega) assay reagent.

[0491] 3. Test Results

[0492] The test results are shown in Table 2 and FIG. 2.

TABLE-US-00003 TABLE 2 The activity of anti-TNF-ADCs in a membrane- bound TNF?-mediated GRE reporter gene system hTNF?12 Humira-A-B00 Humira-3-B00 Humira-4-B00 EC.sub.50 (nM) 0.84 7.4 0.55

Test Example 4: Activity of Anti-TNF-ADCs in Lipopolysaccharide (LPS)'s Stimulation of Human Monocytes' Cytokine Secretion

[0493] Monocytes were selected and enriched from frozen primary human peripheral blood PBMCs by sorting using an EsaySep? human CD14 sorting kit and plated onto a 96-well plate. After monocytes were co-incubated with different concentrations of anti-TNF-ADCs at 37? C. in 5% CO.sub.2 for 4 h, 0.01 ng/mL LPS was used for overnight stimulation. The following day, the medium supernatants were collected and assayed for IL-6 concentration using alpha LISA (cisbio).

[0494] Compared to adalimumab (Humira), the ADCs of the present disclosure are effective in inhibiting LPS-induced IL-6 secretion in high concentrations (4-100 nM) (as shown in FIG. 3). In addition, 4 nM Humira-3-B00 and Humira-4-B00 have significantly higher anti-inflammatory activity than the ADC molecule Humira-A-B00 in that concentration (as shown in FIG. 4).

Test Example 5: Mouse Collagen Antibody-Induced Arthritis (CAIA) Model

[0495] Test Animals:

[0496] Male balb/c mice, 6 w, purchased from the Laboratory Animal Management Department, Shanghai Institute of Planned Parenthood Research. Housing environment: SPF; production license SCXK (Shanghai) 2018-0006; Balb/c mouse certification number: 20180006023393.

[0497] Test Samples:

[0498] Humira-3-B00, Humira-4-B00, and Humira-A-B00.

[0499] Test Method:

[0500] Upon arrival, the test animals were acclimatized for 7 days and randomly grouped. On day 0, each group of mice was intraperitoneally injected with 1.5 mg/mouse of type II collagen antibody cocktail (purchased from Chondrex Inc.) for modeling except for the control group. On day 3, each group of mice was intraperitoneally injected with 50 g/mouse of LPS (100 ?L) to boost the immune response except for the control group. Administration to each group of mice began on day 5, and limb arthritis was scored every 1-3 days. The specific experimental process is shown in FIG. 5. Mice were dosed according to the following administration regimens. The severity of arthritis in each group of mice was semi-quantitatively scored every 1-3 days and further, the anti-inflammatory activity of anti-TNF-ADCs was evaluated.

TABLE-US-00004 TABLE 3 Administration regimens Group CII Ab Dose Route of Frequency of No. Group Number (mg/mouse) (mg/kg) administration administration 1 Control group 4 NA Intraperitoneally Twice a week 2 Model group 8 1.5 10 Intraperitoneally Twice a week 3 Humira 8 1.5 10 Intraperitoneally Twice a week 4 Humira-A-B00 8 1.5 10 Intraperitoneally Twice a week 5 Humira-3-B00 8 1.5 10 Intraperitoneally Twice a week 6 Humira-4-B00 8 1.5 10 Intraperitoneally Twice a week

[0501] Test Results:

[0502] Humira has weak anti-inflammatory activity in the CAIA model. Humira-4-B00 has a rapid onset of action (began to take effect on day 5) and can cause a sustained reduction in arthritic inflammation. The control ADC molecule Humira-A-B00 showed an alleviating effect on arthritis only late in the course of arthritis (from day 11 onwards) (as shown in FIG. 6). From day 8 to day 14 after arthritis modeling, Humira-4-B00 significantly reduced the mouse arthritis score (*, p<0.05) compared to the model group, whereas the control ADC molecule Humira-A-B00 showed no significant difference from the model group in anti-inflammatory activity (as shown in FIG. 7).

Test Example 6: Delayed-Type Hypersensitivity (DTH) Model

[0503] Test Animals:

[0504] Male ICR mice, 6 w, purchased from the Laboratory Animal Management Department, Shanghai Institute of Planned Parenthood Research. Housing environment: SPF; production license SCXK (Shanghai) 2018-0006; certification number: 20180006023622.

[0505] Test Samples:

[0506] Humira-3-B00, Humira-4-B00, Humira-A-B00, and Humira-A-A00 (prepared according to Example 2 of WO2017210471 and Example 7 of WO2019106609).

##STR00108##

[0507] Test Method:

[0508] Upon arrival, the animals were acclimatized for 7 days and randomly grouped. On day 0, mice were immunized by applying 50 ?L of a 1% solution of DNFB (2,4-dinitrofluorobenzene) to the abdomen where the hair had been removed. On day 5, 10 L of a 0.5% solution of DNFB was applied to each of the inner and outer sides of the right ears of the mice as a booster (20 ?L in total). On day 6 (after 24 h), the mice were sacrificed, and ear samples 8 mm in diameter were taken from both sides using a punch and weighed. Each group of mice was dosed on day 0 and day 4. The specific experimental process is shown in FIG. 8. Mice were dosed according to the following administration regimens. The ear samples from the control sides and the model sides of the mice were weighed, and the weights (indicating degrees of swelling) were used to evaluate the anti-inflammatory activity of the anti-TNF-ADCs.

TABLE-US-00005 TABLE 4 Administration regimens Group Dose Route of Frequency of No. Group Number (mg/kg) administration administration 1 Model group 8 10 Intraperitoneally Twice a week 2 Humira 8 10 Intraperitoneally Twice a week 3 Humira-A-A00 8 10 Intraperitoneally Twice a week 4 Humira-A-B00 8 10 Intraperitoneally Twice a week 5 Humira-3-B00 8 10 Intraperitoneally Twice a week 6 Humira-4-B00 8 10 Intraperitoneally Twice a week

[0509] Test Results:

[0510] There was no difference in weight between the untreated left ears of these groups of mice. Humira-4-B00 showed comparable anti-inflammatory activity to the positive control Humira-A-B00 in the model. Humira-3-B00 showed relatively low anti-inflammatory activity but is still significantly better than Humira mAb (as shown in FIG. 9).

Test Example 7: Stability of FADC Samples in Plasma

[0511] Test Samples:

[0512] Humira-3-BOO, Humira-4-B00, Humira-A-B00, and Humira-A-A00.

[0513] Test Plasma:

[0514] Human, cynomolgus monkey, rat, mouse and 100 BSA

[0515] Test Method:

[0516] 1 mg/mL solutions of test molecules were prepared in PBS and sterilized by filtration through a 0.22 ?m filter membrane. 15 ?L of a 1 mg/mL sample was added to 135 ?L of a reaction medium to make a final concentration of 100 ?g/mL. The mixture was incubated at 37? C. in a dark place for 0 days, 7 days, 14 days and 21 days, and samples were tested for free toxins.

[0517] The data are shown in Table 5.

TABLE-US-00006 TABLE 5 Plasma stability data Free toxins (%) Humira-A-B00 Humira-4-B00 Humira-3-B00 Humira-A-A00 Toxin Toxin Toxin Toxin Toxin Toxin 100 ?g/mL, Toxin compound compound compound compound compound compound plasma, 37? C. compound A-A A-A A-B 4-A 4-B 3-A 3-B Human D 0 0.000 0.000 0.000 0.000 0.000 0.000 0.000 D 7 0.000 0.000 0.000 0.000 0.000 0.000 0.000 D 14 0.000 0.000 0.000 0.000 0.000 0.000 0.000 D 21 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Cynomolgus D 0 0.000 0.000 0.000 0.000 0.000 0.000 0.000 monkey D 7 0.000 0.000 0.000 0.000 0.000 0.000 0.000 D 14 0.000 0.000 0.000 0.000 0.000 0.000 0.000 D 21 0.000 0.000 0.000 0.000 0.000 0.000 0.000 BSA (1%) D 0 0.000 0.000 0.000 0.000 0.000 0.000 0.000 D 7 0.000 0.000 0.000 0.000 0.000 0.000 0.000 D 14 0.000 0.000 0.000 0.000 0.000 0.000 0.000 D 21 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Rat D 0 0.000 0.000 0.000 0.000 0.000 0.000 0.000 D 7 0.000 0.000 0.000 0.000 0.000 0.000 0.000 D 14 0.232 0.232 0.168 0.000 0.000 0.000 0.000 D 21 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Mouse D 0 0.000 0.000 0.145 0.000 0.000 0.000 0.000 D 7 0.000 0.000 0.540 0.000 0.000 0.000 0.000 D 14 0.000 0.000 0.818 0.000 0.000 0.000 0.000 D 21 0.000 0.000 0.975 0.000 0.000 0.000 0.000

[0518] In the concentration of 100 ?g/mL, Humira-3-B00 and Humira-4-B00 showed excellent stability in human, cynomolgus monkey, mouse and rat plasma and 1% BSA, with free toxin levels below the lower limit of detection; Humira-A-A00 released a small amount of a free toxin in rat plasma; Humira-A-B00 released small amounts of free toxins in rat and mouse plasma.