2-METHOXYESTRADIOL DERIVATIVES AND MEDICAL USES THEREOF

Abstract

The present invention relates to novel 2-Methoxyestradiol derivatives and their medical use. In particular, the novel derivatives of the present invention are useful for the treatment or prevention of liver or lung fibrosis. Accordingly, the present invention also provides medical uses of the 2-Methoxyestradiol derivatives of the present invention. The present invention also provides a method of treating or preventing liver or lung fibrosis comprising administering an effective amount of the 2-Methoxyestradiol derivatives of the present invention.

Claims

1. A compound of Chemical Formula 1: ##STR00080## or a pharmaceutically acceptable salt thereof, in Chemical Formula 1, n is 0, 1, 2, or 3 A is a moiety selected from the group consisting of: phenyl, naphthyl, pyridyl, and pyrimidyl, wherein A is not substituted or optionally substituted with at least one substituent selected from the group consisting of halogen, NO.sub.2, —O—C.sub.1-4 alkyl, OH, CN, NH.sub.2, COOH, —C.sub.1-4 alkyl, -halo-C.sub.1-4 alkyl, —C.sub.1-4 haloalkoxy, phenyl, pyrrolyl, and pyrrolidinyl, B is H, —S(O).sub.2—R.sup.1, —C(O)—R.sup.1, or —OC(O)—R.sup.1, and R.sup.1 is phenyl, —C.sub.1-4 alkyl, naphthyl, NH.sub.2, OH, or CN, wherein the phenyl, alkyl, and naphthyl of R.sup.1 is not substituted or optionally substituted with at least one substituent selected from the group consisting of halogen, NO.sub.2, —O—C.sub.1-4 alkyl, OH, CN, NH.sub.2, COOH, phenyl, —C.sub.1-4 alkyl, -halo-C.sub.1-4 alkyl, and —C.sub.1-4 haloalkoxy.

2. The compound or pharmaceutically acceptable salt of claim 1, wherein n is 0, 1, or 2, A is a moiety selected from the group consisting of: phenyl, naphthyl, and pyridyl, wherein A is not substituted or optionally substituted with at least one substituent selected from the group consisting of halogen, NO.sub.2, —O—C.sub.1-4 alkyl, OH, CN, NH.sub.2, COOH, —C.sub.1-4 alkyl, -halo-C.sup.1-4 alkyl, phenyl, pyrrolyl, and pyrrolidinyl, B is H, —S(O).sub.2—R.sup.1, or —C(O)—R.sup.1, and R.sup.1 is phenyl, —C.sub.1-4alkyl, naphthyl, NH.sub.2, OH, or CN, wherein the phenyl, alkyl, and naphthyl of R.sup.1 is not substituted or optionally substituted with at least one substituent selected from the group consisting of halogen, NO.sub.2, —O-C.sub.1-4 alkyl, OH, CN, NH.sub.2, COOH, —C.sup.1-4 alkyl, and -halo-C.sub.1-4 alkyl.

3. The compound or pharmaceutically acceptable salt of claim 1, wherein the compound is (13S, 17S)-17-hydroxy-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-yl benzenesulfonate, (13S,17S)-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diyldibenzenesulfonate, 13S,17S)-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diyl bis(4-fluorobenzenesulfonate), (13S,17S)-2-methoxy-13-methyl-3-((phenylsulfonyl)oxy)-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-yl acetate, (13S,17S)-2-methoxy-13-methyl-17-((methylsulfonyl)oxy)-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-yl benzenesulfonate, (13S,17S)-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diyl bis(3,4-difluorobenzenesulfonate), (13S,17S)-17-hydroxy-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-yl4-nitrobenzenesulfonate, (13S,17S)-17-hydroxy-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-yl4-methoxybenzenesulfonate, (13S,17S)-17-hydroxy-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-yl naphthalene-2-sulfonate, (13S,17S)-17-hydroxy-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-yl naphthalene-1-sulfonate, (13S,17S)-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diyl bis(naphthalene-2-sulfonate), (13S,17S)-2-methoxy-13-methyl-3-((naphthalen-2-ylsulfonyl)oxy)-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-yl acetate, (13S,17S)-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diyl bis(4-fluorobenzenesulfonate), (13S,17S)-2-methoxy-13-methyl-3-(((4-nitrophenyl)sulfonyl)oxy)-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-yl acetate, or (13S,17S)-2-methoxy-3-(((4-methoxyphenyl)sulfonyl)oxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-yl acetate.

4. The compound or pharmaceutically acceptable salt of claim 3, wherein the compound is (13S, 17S)-17-hydroxy-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-yl benzenesulfonate, (13S,17S)-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diyldibenzenesulfonate, (13S, 17S)-2-methoxy-13-methyl-17-((methylsulfonyl)oxy)-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-yl benzenesulfonate, or (13S,17S)-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diyl bis(naphthalene-2-sulfonate).

5. A composition comprising a compound or pharmaceutically acceptable salt of claim 1 and a pharmaceutically acceptable carrier.

6. A method for treating or preventing fibrosis, comprising: administering to a subject in need of treatment or prevention of fibrosis a compound or pharmaceutically acceptable salt of claim 1.

7. The method of claim 6, wherein the fibrosis is liver fibrosis or pulmonary fibrosis.

8. The method of claim 7, wherein the liver fibrosis is a fibrosis caused by chronic hepatitis virus infection, alcohol abuse, drug-induced liver injury (DILI), cholestasis, or NASH (Non-Alcoholic Steath Hepatitis).

9. The method of claim 6, wherein the compound is (13S,17S)-17-hydroxy-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-ylbenzenesulfonate, (13S,17S)-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diyldibenzenesulfonate, (13S,17S)-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diyl bis(4-fluorobenzenesulfonate), (13S,17S)-2-methoxy-13-methyl-3-((phenylsulfonyl)oxy)-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-yl acetate, (13S,17S)-2-methoxy-13-methyl-17-((methylsulfonyl)oxy)-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-yl benzenesulfonate, (13S,17S)-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diyl bis(3,4-difluorobenzenesulfonate), (13S,17S)-17-hydroxy-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-yl4-nitrobenzenesulfonate, (13S,17S)-17-hydroxy-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-yl4-methoxybenzenesulfonate, (13S,17S)-17-hydroxy-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-ylnaphthalene-2-sulfonate, (13S,17S)-17-hydroxy-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-ylnaphthalene-1-sulfonate, (13S,17S)-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diyl bis(naphthalene-2-sulfonate), (13S,17S)-2-methoxy-13-methyl-3-((naphthalen-2-ylsulfonyl)oxy)-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-ylacetate, (13S,17S)-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diyl bis(4-fluorobenzenesulfonate), (13S,17S)-2-methoxy-13-methyl-3-(((4-nitrophenyl)sulfonyl)oxy)-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-ylacetate, or (13S,17S)-2-methoxy-3-(((4-methoxyphenyl)sulfonyl)oxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-ylacetate.

10. The method of claim 9, wherein the compound is (13S,17S)-17-hydroxy-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-ylbenzenesulfonate, (13S,17S)-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diyldibenzenesulfonate, (13S, 17S)-2-methoxy-13-methyl-17-((methylsulfonyl)oxy)-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-yl benzenesulfonate, or (13S,17S)-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diyl bis(naphthalene-2-sulfonate).

Description

BRIEF DESCRIPTION OF DRAWINGS

[0090] FIGS. 1 and 2 show validation results of in vivo efficacy of 2-ME derivatives on the RIPF. Representative images of hematoxylin and eosin stained lung sections from mice are shown at 2 weeks after irradiation. Lung sections were stained with Masson's Trichrome stain to visualize blue-colored collagen deposition and quantitative assessments of the degree of collagen deposition were determined (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns not significant vs vehicle).

[0091] FIGS. 3 to 6 show validation results of in vivo efficacy of 2-ME derivatives on the liver fibrosis.

[0092] In the FIGS. 1 to 6, No. means number of compounds listed in the Tables 3 and 4.

MODE FOR THE INVENTION

[0093] Hereinafter, the present disclosure is described in considerable detail with examples to help those skilled in the art understand the present disclosure. However, the following examples are offered by way of illustration and are not intended to limit the scope of the invention. It is apparent that various changes may be made without departing from the spirit and scope of the invention or sacrificing all of its material advantages.

[0094] Abbreviations and symbols utilized herein are in accordance with the common usage of such abbreviations and symbols by those skilled in the chemical and biological arts. Specifically, the following abbreviations may be used in the examples and throughout the specification. [0095] RIPF (Radiation-Induced Pulmonary Fibrosis) [0096] NASH (Non-Alcoholic Steath Hepatitis) [0097] ECM (ExtraCellular Matrix) [0098] 2-ME (2-Methoxyestradiol) [0099] EndMT (Endothelial-to-mesenchymal transition) [0100] HUVECs (Human Umbilical Vein Endothelial Cells) [0101] g (grams) mg (milligrams)

[0102] kg (kilograms) μg (micrograms) [0103] L (liters) mL (milliliters) [0104] μL (microliters) rt (room temperature) [0105] M (molar) mM (millimolar) [0106] μM (micromolar) nM (nanomolar) [0107] mol (moles) mmol (millimoles) [0108] min (minutes) h (hours) [0109] MeO H (methanol) EtOH (ethanol) [0110] DCM (dichloromethane) THF (tetrahydrofuran) [0111] EtOAc (ethyl acetate) Ac.sub.2O (acetic anhydride) [0112] TEA (triethlamine) K.sub.2CO.sub.3 (potassium carbonate) [0113] Pd/C (Palladium on Carbon) BnBr (benzyl bromide) [0114] DMAP (4-dimethylaminopyridine) KI (potassium iodide) [0115] Na.sub.2C.sub.3O (sodium carbonate) Na.sub.2SO.sub.4 (sodium sulfate)

[0116]

Preparation of Compounds of the Present Disclosure

[0117] Below, the illustrating synthetic examples of some compounds of the present disclosure are described, and other compounds can be prepared by the similar method to one described below with different starting or reacting materials.

##STR00018##

General Procedure for the Synthesis of A

[0118] To a stirred solution of 2-Methoxyestradiol (0.99 mmol) in pyridine (2 mL) were added benzenesulfonyl chloride (4.96 mmol) and the mixture was stirred for 6 hours at room temperature. After the reaction was completed, the reaction mixture was poured to the water and then extracted with EtOAc (15 mL) several times. The combined organic layer was washed with brine (10 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The resulting crude residue was purified by flash column chromatography to give A.

##STR00019##

General Procedure for the Synthesis of B

[0119] To a stirred solution of 2-Methoxyestradiol (0.99 mmol) in anhydrous DCM (2 mL) were added TEA (9.92 mmol), benzenesulfonyl chloride (4.96 mmol) and the mixture was stirred for 16 hours at room temperature. After the reaction was completed, the reaction mixture was poured to the water and then extracted with DCM (15 mL) several times. The combined organic layer was washed with brine (10 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The resulting crude residue was purified by flash column chromatography to give B.

##STR00020##

General Procedure for the Synthesis of C

[0120] To a stirred solution of B (0.45 mmol) in anhydrous DCM (5 mL) were added trimethylamine (1.36 mmol), Methanesulfonyl chloride (0.90 mmol) and the mixture was stirred for 16 hours at room temperature. After the reaction was completed, the reaction mixture was poured to the water and then extracted with DCM (15 mL) several times. The combined organic layer was washed with brine (10 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The resulting crude residue was purified by flash column chromatography to give C.

##STR00021##

General Procedure for the Synthesis of D1

[0121] To a stirred solution of 2-Methoxyestradiol (16.53 mmol) in EtOH (100 mL) were potassium carbonate (49.60 mmol), BnBr (49.60 mmol) and the mixture was stirred for 16 hours at room temperature. After the reaction was completed, the reaction mixture was concentrated and then dissolved in EtOAc. The organic solution was washed with aqueous Na.sub.2CO.sub.3 (sat. 100 mL) and brine (10 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The resulting crude residue was purified by flash column chromatography to give D1.

[0122] Pale yellow solid; 1H NMR (400 MHz, Acetone-d.sub.6) δ 7.48 (d, J=7.6 Hz, 2H), 7.39 (t, J=2.8 Hz, 2H), 7.33-7.29 (m, 1H), 6.90 (s, 1H), 5.06 (s, 2H), 3.79 (s, 1H), 3.75-3.65 (m, 1H), 3.56 (d, J=5.2 Hz, 1H); LCMS (ESI) m/z 583 (M+H).sup.+.

General Procedure for the Synthesis of D2

[0123] To a stirred solution of D1 (1.27 mmol) in pyridine (5 ml) were added Ac.sub.2O (6.37 mmol) and the mixture was stirred for 16 hours at room temperature. After the reaction was completed, the reaction mixture was poured to the water and then extracted with EtOAc (15 mL) several times. The combined organic layer was washed with brine (10 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The resulting crude residue was purified by flash column chromatography to give D2.

[0124] White solid; 1H NMR (400 MHz, Acetone-d.sub.6) δ 7.48 (d, J=7.2 Hz, 2H), 7.38 (t, J=7.2 Hz, 2H), 7.37-7.29 (m, 1H), 6.89 (s, 1H), 6.73 (s, 1H), 5.06 (s, 2H), 4.67 (t, J=7.6 Hz, 1H), 3.80 (s, 3H), 2.89-2.73 (m, 3H), 2.38-2.30 (m, 1H), 2.27-2.12 (m. 2H), 1.97 (s, 3H), 1.91-1.83 (m, 2H), 1.80-1.71 (m, 1H), 1.60-1.51 (m, 1H), 1.50-1.39 (m, 5H), 1.38-1.28 (m, 3H), 0.86 (s, 3H); LCMS (ESI) m/z 435 (M+H.sup.+.

General Procedure for the Synthesis of D3

[0125] To a stirred solution of D2 (0.83 mmol) in a mixture of EtOAc and MeOH (3:1 ratio, 4 mL) were added 10% Pd/C (30 mg) and the resulting solution under H.sub.2 for 4 hours at room temperature. After the reaction was completed, the palladium was filtered off by silica and the filtrate was concentrated in vacuo. The resulting crude residue was purified by flash column chromatography to give D3.

[0126] Pale yellow solid; 1H NMR (400 MHz, Acetone-d.sub.6) δ 7.19 (brs, 1H), 6.85 (s, 1H), 6.52 (s, 1H), 4.66 (t, J=8.4 Hz, 1H), 3.80 (s, 3H), 2.77-2.60 (m, 2H), 2.47-2.28 (m, 1H), 2.26-2.12 (m. 2H), 2.00 (s, 3H), 1.92-1.83 (m, 2H), 1.81-1.73 (m, 1H), 1.60-1.52 (m, 1H), 1.50-1.40 (m, 4H), 1.38-1.26 (m, 3H), 0.86 (s, 3H); LCMS (ESI) m/z 345 (M+H).sup.+.

General Procedure for the Synthesis of D4

[0127] To a stirred solution of D3 (0.17 mmol) in anhydrous DCM (2 mL) were added TEA (0.52 mmol), benzenesulfonyl chloride (0.348 mmol) and the mixture was stirred for 16 hours at room temperature. After the reaction was completed, the reaction mixture was poured to the water and then extracted with DCM (15 mL) several times. The combined organic layer was washed with brine (10 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The resulting crude residue was purified by flash column chromatography to give D4.

[0128] White solid; 1H NMR (400 MHz, Acetone-d.sub.6) δ 7.87 (d, J=7.6 Hz, 2H), 7.79 (t, J=7.2 Hz, 1H), 7.66 (t, J=7.6 Hz, 2H), 6.90 (s, 1H), 6.83 (s, 1H), 4.66 (t, J=8.4 Hz, 1H), 3.50 (s, 3H), 2.76-2.70 (m, 2H), 2.60-2.30 (m, 1H), 2.20-2.15 (m, 2H), 1.99 (s, 3H), 1.87-1.83 (m, 2H), 1.77-1.73 (m, 1H), 1.55-1.30 (m 9H), 0.86 (s, 3H); LCMS (ESI) m/z 485 (M+H).sup.+.

[0129]

[0130] Structures and characteristics of the compounds according to the present disclosure and some comparative examples are written in Table 2 below.

TABLE-US-00002 TABLE 2 No. structure Name NMR Characterization 1 [00022] (13S,17S)-2- methoxy-13-methyl- 7,8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthrene-3,17-diyl diacetate White solid; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 6.98 (s, 1H), 6.71 (s, 1H), 4.67 (t, J = 8.8 Hz, 1H), 3.78 (s, 3H), 2.76-2.70 (m, 2H), 2.38-2.32 (m, 1H), 2.28-2.23 (m, 1H), 2.22 (s, 3H), 2.18- 2.11 (m, 1H), 2.00 (s, 3H), 1.93-1.83 (m, 2H), 1.82-1.75 (m, 1H), 1.59-1.28 (m 9H), 0.87 (s, 3H); LCMS (ESI) m/z 387 (M + H).sup.+. 2 [00023] (13S,17S)-2- methoxy-13-methyl- 7,8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthrene-3,17-diyl dipropionate White solid; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 6.98 (s, 1H), 6.71 (s, 1H), 4.67 (t, J = 8.8 Hz, 1H), 3.77 (s, 3H), 2.80-2.73 (m, 2H), 2.54 (q, J = 7.2 Hz, 2H), 2.50-2.15 (m, 6H), 1.95-1.85 (m, 2H), 1.83-1.74 (m, 1H), 1.60-1.30 (m, 9H), 1.19 (t, J = 7.6 Hz, 3H), 1.10 (t, J = 7.6 Hz, 3H), 0.88 (s, 3H); LCMS (ESI) m/z 415 (M + H).sup.+. 3 [00024] (13S,17S)-17- hydroxy-2-methoxy- 13-methyl- 7,8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthren-3-yl dimethylcarbamate White solid; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 6.95 (s, 1H), 6.71 (s, 1H), 3.78 (s, 3H), 3.68 (t, J = 8.4 Hz, 1H), 3.07 (s, 3H), 2.93 (s, 3H), 2.38-2.32 (m, 1H), 2.26-2.15 (m, 1H), 2.03-1.83 (m, 3H), 1.72-1.64 (m, 1H), 1.55-1.16 (m, 9H), 0.80 (s, 3H); LCMS (ESI) m/z 374 (M + H).sup.+. 4 [00025] (13S,17S)-17- hydroxy-2-methoxy- 13-methyl- 7,8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthren-3-yl benzenesulfonate White solid; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 7.87 (d, J = 7.6 Hz, 2H), 7.78 (t, J = 7.6 Hz, 1H), 7.65 (t, J = 8.0 Hz, 2H), 6.89 (s, 1H), 6.81 (s, 1H), 3.66 (t, J = 7.6 Hz, 1H), 3.60 (brs, 1H), 3.50 (s, 3H), 2.76-2.31 (m, 2H), 2.31-2.21 (m, 1H), 2.28-2.15 (m, 1H), 2.06-1.85 (m, 3H), 1.70-1.62 (m, 1H), 1.54-1.15 (m 9H), 0.78 (s, 3H); LCMS (ESI) m/z 443 (M + H).sup.+. 5 [00026] (13S,17S)-2- methoxy-13-methyl- 7,8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthrene-3,17-diyl dibenzenesulfonate White solid; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 7.94-7.87 (m, 2H), 7.86- 7.80 (m, 2H), 7.79-7.76 (m, 2H), 7.70- 7.62 (m, 4H), 6.86 (s, 1H), 6.80 (s, 1H), 4.42 (t, J = 8.8 Hz, 1H), 3.49 (s, 3H), 2.79-2.73 (m, 2H), 2.71-2.19 (m, 2H), 2.01-1.96 (m, 1H), 1.83-1.73 (m, 1H), 1.72-1.63 (m, 3H), 1.45-1.34 (m 3H), 1.33-1.21 (m, 3H), 1.15-1.11(m, 1H), 0.84 (s, 3H); LCMS (ESI) m/z 583 (M + H).sup.+. 6 [00027] (13S,17S)-17- hydroxy-2-methoxy- 13-methyl- 7,8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthren-3-yl acetate White solid; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 6.98 (s, 1H), 6.70 (s, 1H), 3.82 (s, 3H), 3.72-3.65 (m, 1H), 3.59 (d, J = 4.8 Hz, 1H), 2.77-2.72 (m, 2H), 2.38- 2.32 (m, 1H), 2.21 (s, 3H), 2.03-1.92 (m, 2H), 1.91-1.83 (m, 1H), 1.73-1.64 (m, 1H), 1.55-1.15 (m, 9H), 0.80 (s, 3H); LCMS (ESI) m/z 345 (M + H).sup.+. 7 [00028] (13S,17S)-17- hydroxy-2-methoxy- 13-methyl- 7,8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthren-3-yl propionate White solid; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 6.98 (s, 1H), 6.70 (s, 1H), 3.77 (s, 3H), 3.68 (t, J = 8.4 Hz, 1H), 3.60 (brs, 1H), 2.78-2.72 (m, 2H), 2.54 (t, J = 7.2 Hz, 2H), 2.38-2.31 (m, 1H), 2.25- 2.16 (m, 1H), 2.07-1.92 (m, 2H), 1.91- 1.84 (m, 1H), 1.72-1.64 (m, 1H), 1.55- 1.23 (m, 8H), 1.17 (t, J = 8.0 Hz, 1H), 0.80 (s, 3H); LCMS (ESI) m/z 359 (M + H).sup.+. 8 [00029] (13S,17S)-3- hydroxy-2-methoxy- 13-methyl- 7,8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthren-17-yl acetate Pale yellow solid; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 7.19 (brs, 1H), 6.85 (s, 1H), 6.52 (s, 1H), 4.66 (t, J = 8.4 Hz, 1H), 3.80 (s, 3H), 2.77-2.60 (m, 2H), 2.47- 2.28 (m, 1H), 2.26-2.12 (m, 2H), 2.00 (s, 3H), 1.92-1.83 (m, 2H), 1.81-1.73 (m, 1H), 1.60-1.52 (m, 1H), 1.50-1.40 (m, 4H), 1.38-1.26 (m, 3H), 0.86 (s, 3H); LCMS (ESI) m/z 345 (M + H).sup.+. 9 [00030] (13S,17S)-2- methoxy-13-methyl- 7,8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthrene-3,17-diyl dimethanesulfonate White solid; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 7.08 (s, 1H), 6.97 (s, 1H), 4.56 (t, J = 8.4 Hz, 1H), 3.90 (s, 3H), 3.22 (s, 3H), 3.10 (s, 3H), 2.85-2.81 (m, 3H), 2.48-2.41 (m, 1H), 2.37-2.25 (m, 2H), 2.05-2.01 (m, 1H), 1.96-1.87 (m, 1H), 1.85-1.76 (m, 2H), 1.54-1.45 (m 4H), 1.41-1.33 (m, 2H), 0.90 (s, 3H); LCMS (ESI) m/z 459 (M + H).sup.+. 10 [00031] (13S,17S)-2- methoxy-13-methyl- 17- ((phenylsulfonyl) oxy)- 7.8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthren-3-yl acetate White solid; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 7.97 (d, J = 7.6 Hz, 2H), 7.79 (t, J = 7.6 Hz, 1H), 7.70 (t, J = 8.0 Hz, 2H), 6.94 (s, 1H), 6.69 (s, 1H), 4.43 (t, J = 7.6 Hz, 1H), 3.77 (s, 3H), 2.75-2.70 (m, 2H), 2.31-2.25 (m, 1H), 2.20 (s, 3H), 2.18-2.12 (m, 1H), 2.05-1.95 (m, 1H), 1.89-1.81 (m, 1H), 1.77-1.64 (m, 3H), 1.49-1.38 (m, 3H), 1.36-1.21 (m, 2H), 1.18-1.10 (m, 1H), 0.83 (s, 3H); LCMS (ESI) m/z 485 (M + H).sup.+. 11 [00032] (13S,17S)-3- hydroxy-2-methoxy- 13-methyl- 7,8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthren-17-yl benzenesulfonate Pale yellow solid; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 7.97 (d, J = 7.6 Hz, 2H), 7.79 (t, J = 7.6 Hz, 1H), 7.70 (t, J = 8.0 Hz, 2H), 7.18 (s, 1H), 6.81 (s, 1H), 6.50 (s, 1H), 4.43 (t, J = 8.0 Hz, 1H), 3.79 (s, 3H), 2.72-2.68 (m, 2H), 2.27-2.18 (m, 1H), 2.17-2.10 (m, 1H), 2.00-1.95 (m, 1H), 1.86-1.77 (m, 1H), 1.75-1.63 (m, 4H), 1.45-1.35 (m, 3H), 1.30-1.18 (m, 3H), 1.18-1.08 (m 1H), 0.85 (s, 3H); LCMS (ESI) m/z 443 (M + H).sup.+. 12 [00033] diethyl ((13S,17S)-3- hydroxy-2-methoxy- 13-methyl- 7,8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthren-17-yl) phosphate Pale yellow solid; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 7.22 (s, 1H), 6.85 (s, 1H), 6.52 (s, 1H), 4.28 (q, J = 7.6 Hz, 1H), 4.12-4.02 (m, 4H), 3.80 (s, 3H), 2.78- 2.69 (m, 2H), 2.38-2.32 (m, 1H), 2.28- 2.13 (m, 2H), 2.03-1.98 (m, 1H), 1.90- 1.82 (m, 1H), 1.78-1.67 (m, 2H), 1.51- 1.38 (m, 4H), 1.36-1.26 (m, 8H), 0.83 (s, 3H); LCMS (ESI) m/z 439 (M + H).sup.+. 13 [00034] (13S,17S)-2- methoxy-13-methyl- 7,8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthrene-3,17-diyl bis(4-fluoro- benzenesulfonate) White solid; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 8.10-8.00 (m, 2H), 7.93- 7.83 (m, 2H), 7.44 (q, J = 9.6, 8.8 Hz, 4H), 6.86 (d, J = 10.4 Hz, 2H), 4.43 (t, J = 7.6 Hz, 1H), 3.53 (s, 3H), 2.74-2.68 (m, 2H), 2.28-2.15 (m, 2H), 1.89-1.83 (m, 1H), 1.74-1.63 (m, 3H), 1.45-1.40 (m, 3H), 1.35-1.27 (m, 3H), 1.17-1.10 (m, 1H), 0.85 (s, 3H); LCMS (ESI) m/z 619 (M + H).sup.+. 14 [00035] (13S,17S)-2- methoxy-13-methyl- 3-((phenylsulfonyl) oxy)- 7,8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthren-17-yl acetate White solid; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 7.87 (d, J = 7.6 Hz, 2H), 7.79 (t, J = 7.2 Hz, 1H), 7.66 (t, J = 7.6 Hz, 2H), 6.90 (s, 1H), 6.83 (s, 1H), 4.66 (t, J = 8.4 Hz, 1H), 3.50 (s, 3H), 2.76- 2.70 (m, 2H), 2.60-2.30 (m, 1H), 2.20- 2.15 (m, 2H), 1.99 (s, 3H), 1.87-1.83 (m, 2H), 1.77-1.73 (m, 1H), 1.55-1.30 (m 9H), 0.86 (s, 3H); LCMS (ESI) m/z 485 (M + H).sup.+. 15 [00036] (13S,17S)-17- hydroxy-2-methoxy- 13-methyl- 7,8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthren-3-yl 4- methoxybenzene- sulfonate White solid; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 7.78 (d, J = 9.2 Hz, 2H), 7.13 (d, J = 8.8 Hz, 2H), 6.90 (s, 1H), 6.81 (s, 1H), 3.93 (s, 3H), 3.70-3.57 (m, 1H), 3.55 (s, 3H), 2.75-2.69 (m, 2H), 2.38-2.28 (m, 1H), 2.24-2.15 (m, 1H), 2.04-1.96 (m, 1H), 1.94-1.86 (m, 2H), 1.73-1.63 (m, 1H), 1.55-1.42 (m, 2H), 1.41-1.26 (m, 4H), 1.23-1.15 (m, 2H), 0.78 (s, 3H); LCMS (ESI) m/z 473 (M + H).sup.+. 16 [00037] (13S,17S)-2- methoxy-13-methyl- 3-(((4- nitrophenyl)sulfonyl) oxy)- 7,8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthren-17-yl acetate White solid; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 8.50 (d, J = 8.8 Hz, 2H), 8.16 (d, J = 8.8 Hz, 2H), 6.92 (s, 2H), 4.66 (t, J = 8.0 Hz, 1H), 3.50 (s, 3H), 2.78-2.73 (m, 1H), 2.45-2.25 (m, 2H), 2.24-2.12 (m, 1H), 2.00 (s, 3H), 1.95- 1.85 (m, 2H), 1.84-1.73 (m, 1H), 1.59- 1.40 (m, 5H), 1.39-1.30 (m, 4H), 0.86 (s, 3H); LCMS (ESI) m/z 530 (M + H).sup.+. 17 [00038] (13S,17S)-2- methoxy-3-(((4- methoxyphenyl) sulfonyl) oxy)-13-methyl- 7,8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthren-17-yl acetate White solid; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 7.78 (d, J = 8.8 Hz, 2H), 7.13 (d, J = 8.4 Hz, 2H), 6.90 (s, 1H), 6.82 (s, 1H), 4.66 (t, J = 7.6 Hz, 1H), 3.93 (s, 3H), 3.54 (s, 3H), 2.88-2.84 (m, 2H), 2.38-2.31 (m, 1H), 2.28-2.12 (m, 2H), 2.00 (s, 3H), 1.95-1.83 (m, 2H), 1.81- 1.75 (m, 1H), 1.58-1.38 (m, 5H), 1.37- 1.28 (m, 3H), 0.86 (s, 3H); LCMS (ESI) m/z 515 (M + H).sup.+. 18 [00039] (13S,17S)-2- methoxy-13-methyl- 17- ((methylsulfonyl) oxy)- 7,8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthren-3-yl benzenesulfonate White solid; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 7.87 (d, J = 7.6 Hz, 2H), 7.79 (t, J = 7.6 Hz, 1H), 7.66 (t, J = 8.0 Hz, 2H), 6.91 (s, 1H), 6.83 (s, 1H), 4.57 (t, J = 7.6 Hz, 1H), 3.50 (s, 3H), 3.09 (s, 3H), 2.75-2.74 (m, 2H), 2.37-2.31 (m, 1H), 2.30-2.17 (m, 2H), 2.08-1.95 (m, 1H), 1.93-1.85 (m, 1H), 1.84-1.77 (m, 2H), 1.54-1.43 (m, 5H), 1.39-1.28 (m 3H), 0.88 (s, 3H); LCMS (ESI) m/z 521 (M + H).sup.+. 19 [00040] (13S,17S)-2- methoxy-13-methyl- 7,8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthrene-3,17-diyl bis(3,4-difluoro- benzenesulfonate) White solid; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 8.03-7.91 (m, 1H), 7.90- 7.78 (m, 2H), 7.76-7.69 (m, 1H), 7.65 (q, J = 9.2 Hz, 2H), 6.88 (d, J = 14.4 Hz, 2H), 4.48 (t, J = 7.6 Hz, 1H), 3.57 (s, 3H), 2.74 (d, J = 4.4 Hz, 2H), 2.25 (d, J = 13.2 Hz, 1H), 2.23-2.06 (m, 1H), 1.83 (d, J = 10.8 Hz, 1H), 1.75-1.65 (m, 3H), 1.51-1.33 (m, 3H), 1.30-1.11 (m, 4H), 0.85 (s, 3H); LCMS (ESI) m/z 655 (M + H).sup.+. 20 [00041] (13S,17S)-17- hydroxy-2-methoxy- 13-methyl- 7,8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthren-3-yl 4- nitrobenzene- sulfonate Yellow solid; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 8.49 (d, J = 9.2 Hz, 2H), 8.16 (d, J = 8.8 Hz, 2H), 6.92 (s, 2H), 3.72-3.51 (m, 1H), 3.51 (s, 3H), 2.78- 2.68 (m, 1H), 2.37-2.27 (m, 1H), 2.25- 2.14 (m, 1H), 2.03-1.96 (m, 1H), 1.94- 1.85 (m, 2H), 1.73-1.62 (m, 1H), 1.54- 1.42 (m, 3H), 1.41-1.28 (m, 3H), 1.27- 1.15 (m, 2H), 0.78 (s, 3H); LCMS (ESI) m/z 488 (M + H).sup.+. 21 [00042] (13S,17S)-17- hydroxy-2-methoxy- 13-methyl- 7,8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthren-3-yl naphthalene-2- sulfonate White solid; .sup.1H NMR (400 MHz, 8.17 (d, J = 8.0 Hz, 2H), 8.09 (d, J = 8.0 Hz, 1H), 7.89 (dd, J = 8.4, 1.6 Hz, 1H), 7.80-7.69 (m, 2H), 6.85 (s, 2H), 3.68- 3.63 (m, 1H), 3.59 (d, J = 4.8 Hz, 1H), 3.37 (s, 3H), 2.74-2.69 (m, 2H), 2.29- 2.24 (m, 1H), 2.19-2.08 (m, 1H), 2.05- 1.98 (m, 1H), 1.96-1.84 (m, 2H), 1.69- 1.61 (m, 1H), 1.54-1.45 (m, 3H), 1.44- 1.29 (m, 3H), 1.28-1.12 (m, 1H), 0.77 (s, 3H); LCMS (ESI) m/z 493 (M + H).sup.+. 22 [00043] (13S,17S)-17- hydroxy-2-methoxy- 13-methyl- 7,8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthren-3-yl naphthalene-1- sulfonate White solid; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 8.83 (d, J = 8.8 Hz, 1H), 8.33 (d, J = 8.8 Hz, 1H), 8.13 (td, J = 7.2, 1.2 Hz, 2H), 7.86 (td, J = 7.2, 1.6 Hz, 1H), 7.74 (td, J = 7.2, 1.2 Hz, 1H), 7.63 (t, J = 7.6 Hz, 1H), 6.80 (s, 1H), 6.72 (s, 1H), 3.68-3.62 (m, 1H), 3.60 (d, J = 4.8 Hz, 1H), 3.19 (s, 3H), 2.70-2.62 (m, 2H), 2.28-2.22 (m, 1H), 2.17-2.11 (m, 1H), 2.02-1.88 (m, 2H), 1.85-1.81 (m, 1H), 1.68-1.61 (m, 1H), 1.53-1.42 (m, 2H), 1.39-1.32 (m, 2H), 1.30-1.21 (m, 2H), 1.19-1.11 (m, 1H), 0.76 (s, 3H); LCMS (ESI) m/z 493 (M + H).sup.+. 23 [00044] (13S,17S)-2- methoxy-13-methyl- 7,8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthrene-3,17-diyl bis(naphthalene-2- sulfonate) White solid; .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 8.60 (s, 1H), 8.44 (s, 1H), 8.23-8.14 (m, 4H), 8.10 (t, J = 6.8 Hz, 2H), 7.93-7.86 (m, 2H), 7.79-7.69 (m, 4H), 6.82 (s, 1H), 6.77 (s, 1H), 4.45 (t, J = 8.4 Hz, 1H), 3.34 (s, 3H), 2.71-2.66 (m, 2H), 2.20-2.14 (m, 1H), 2.04-1.94 (m, 1H), 1.82-1.74 (m, 1H), 1.72-1.64 (m, 3H), 1.46-1.34 (m, 3H), 1.31-1.18 (m, 3H), 1.16-1.02 (m, 1H), 0.85 (s, 3H); LCMS (ESI) m/z 683 (M + H).sup.+. 24 [00045] (13S,17S)-2- methoxy-13-methyl- 3-((naphthalen-2- ylsulfonyl)oxy)- 7,8,9,11,12,13,14,15, 16,17-decahydro-6H- cyclopenta[a]phe- nanthren-17-yl acetate White solid: .sup.1H NMR (400 MHz, Acetone-d.sub.6) δ 8.46 (t, J = 0.8 Hz, 1H), 8.17 (d, J = 8.0 Hz, 2H), 8.09 (d, J = 8.0 Hz, 1H), 7.89 (dd, J = 8.8, 1.6 Hz, 1H), 7.80-7.69 (m, 2H), 6.85 (d, J = 5.6 Hz, 2H), 4.64 (t, J = 9.2 Hz, 1H), 3.36 (s, 3H), 2.78-2.71 (m, 2H), 2.29-2.26 (m, 1H), 2.24-2.14 (m, 2H), 1.98 (s, 3H), 1.96- 1.84 (m, 2H), 1.78-1.73 (m, 1H), 1.55- 1.46(m, 3H), 1.45-1.19 (m, 4H), 0.85 (s, 3H); LCMS (ESI) m/z 535 (M + H).sup.+.

[0131]

Evaluation of Compounds

Evaluation of 2-Methoxyestradiol (2-ME) Derivatives as Therapeutics for Radiation-Induced Pulmonary Fibrosis (RIPF)

[0132] RIPF triggers physiological abnormalities. EndMT is the phenotypic conversion of endothelial cells to fibroblast-like cells and is involved in RIPF.

[0133] We established a phenomic screening platform to measure radiation-induced stress fibers and optimized the conditions for high-throughput screening using HUVEC cells to develop compounds targeting RIPF. The results of screening indicated that 2-ME derivatives reduced radiation-induced fibrosis, as evidenced by an enlargement of cell size and increases in actin stress fibers and α-smooth muscle actin expression.

[0134] 2-ME derivatives were prepared and their anti-fibrotic activities were tested in vitro [Table 3] and in vivo mouse models [FIGS. 1 and 2]. Some compounds among the total synthesized compounds showed relatively high anti-fibrotic activities on RIPF. In this test, HUVECs were pre-treated with 2-ME derivatives at 1 hour before radiation and irradiated with 10 Gy. After 48 hours, HUVECs stained with Phalloidin and Hoechst to assess morphological changes

[0135] Table 3 shows validation results of in vitro efficacy of 2-ME derivatives on the RIPF

[0136]

TABLE-US-00003 TABLE 3 Stress Fiber NO. Structure Cell number Pre- Post- 1 [00046] O X X 2 [00047] X X X 3 [00048] O X X 4 [00049] O X X 5 [00050] O O X 6 [00051] O O X 7 [00052] O X X 8 [00053] O X X 9 [00054] O X X 10 [00055] O O O 11 [00056] O O O 12 [00057] O X X 13 [00058] O X X 14 [00059] O X X *Cell number: O (there were no change in the cell number compare to IR (control) treated group), X (there were more than 10% changes in the cell number compare to IR (control) treated group) *Stress fiber: O (there were stress fiber decreasing more than 20% compare to IR (control) treated group), X (there were no stress fiber decreasing compare to IR (control) treated group)

[0137]

Materials and Methods

Validation of 2-ME Derivatives as Regulators of Radiation-Induced EndMT Using the HCS System

[0138] Collagen type 1 (BD Collagen I, 354236, Thermo Fisher Scientific) was diluted in 70% ethanol, and then the collagen solution (400 μg/ml) was dispensed into each 384-well plate (6007550, Perkin Elmer, MA, US). After a 1-hour incubation at room temperature, the wells were rinsed twice with Dulbecco's phosphate-buffered saline. HUVECs were seeded at a density of 7×10.sup.2 cells per well onto collagen-coated, 384-well plates and allowed to attach in complete growth media. Cells were then exposed to 10 Gy radiation at room temperature. After irradiation, the cells were incubated for an additional 3 hours. The compounds were tested at a final concentration of 10 μM in 0.5% dimethyl sulfoxide (DMSO; v/v) using an automated liquid handling system (Hummingbird, Analytik Jena, Jena, DE). After the 48-hour treatment, cells were fixed with 4% paraformaldehyde (w/v). The expression of actin filament was determined by incubation with 488-Phalloidin (1:100, MOP-A7466, Thermo Fisher Scientific) using direct immunofluorescence staining. The nuclei were stained using Hoechst 33342 (1:1000, MOP-H3570, Thermo Fisher Scientific) at room temperature. For assay validation, a control run was performed. The low control consisted of three 384-well plates that contained irradiated cells, and the high control consisted of three 384-well plates that contained non-irradiated cells. For detecting and visualizing filamentous actin (F-actin) and nuclei, images were collected using an automated high-content imaging system with a 20× magnifying objective (Operetta, PerkinElmer). The acquired images were analyzed using an in-house software tool and Harmony 3.5.1® high-content imaging (Harmony, PerkinElmer) for segmentation of cells.

[0139]

Validation of In Vivo Efficacy of 2-ME Derivatives on the RIPF

[0140] Radiation was delivered using an X-RAD 320 platform (Precision X-ray). The left main bronchi of 8-week-old male C57BL/6 mice were irradiated at 90 Gy using a 4-mm diameter field to mimic an ablative dose. 1-hour prior to irradiation, 2-ME and 2-ME derivatives were injected intraperitoneally (5 per group). 2-ME and 2-ME derivatives were administered at a concentration of 30 mg/kg for 2 weeks (3 times a week). After 2 weeks of irradiation, lung tissue was harvested and fixed in 10% (v/v) neutral buffered formalin before preparing paraffin sections. Paraffin-embedded sections were deparaffinized and stained with hematoxylin and eosin (H&E; Sigma-Aldrich), and using a Masson's trichrome stain kit (Sigma-Aldrich) to detect collagen. Images were obtained using a Zeiss microscope. At least five images per section were acquired for quantification, and positively stained areas were evaluated with ImageJ software. Lung fibrosis was scored on a scale of 0 to 8 according to the following criteria: grade 0, normal lung; grade 1, minimal fibrous thickening of alveolar or bronchiolar wall; grade 2-3, moderate thickening of walls without obvious damage to lung architecture; grade 4-5, increased fibrosis with definite damage to lung architecture and formation of fibrous bands or small fibrous mass; grade 6-7, severe distortion of structure and large fibrous areas; grade 8, total fibrous obliteration of the field.

[0141]

Evaluation of 2-Methoxyestradiol (2-ME) Derivatives as Therapeutics for Liver Fibrosis

[0142] Despite liver fibrosis are highly prevalent disease, there are still no approved therapies because liver fibrosis has numerous causes and complications. The main causes of liver fibrosis are chronic hepatitis virus infection, alcohol abuse, drug-induced liver injury (DILI), cholestasis and NASH etc.

[0143] We evaluated activity of 2-ME derivatives as inhibitors of liver fibrosis through multicellular hepatic spheroids (MCHSs) model-based phenomic screening [Table 4] and in vivo mouse models [FIGS. 3 to 6]. Some compounds among the total synthesized compounds showed relatively high anti-fibrotic activities in liver.

[0144] Table 4 shows validation results of in vitro efficacy of 2-ME derivatives on the liver fibrosis using MCHSs.

[0145]

TABLE-US-00004 TABLE 4 NO. Structure Anti-fibrotic activity 1 [00060] X 2 [00061] X 3 [00062] X 4 [00063] X 5 [00064] X 6 [00065] X 7 [00066] O 8 [00067] O 9 [00068] O 10 [00069] X 11 [00070] X 12 [00071] X 13 [00072] X 14 [00073] X 15 [00074] X 16 [00075] X 17 [00076] O 18 [00077] X 19 [00078] X 20 [00079] X *Anti-fibrotic activity: O (there were dimeter or size increasing compare to 2-ME (control)), X (there no dimeter or size change compare to 2-ME (control))

Materials and Methods

Validation of In Vitro Efficacy of 2-ME and it's Derivatives in 3D Fibrosis Model

[0146] Huh? (hepatocellular carcinoma cell), LX2 (hepatic stellate cell), WI38 (fibroblast cell), and HUVEC (endothelial cell) were seeded on 96-well ULA U-bottom plate (Corning, 7007) at density of 6,000cells/well with 5.5:1.5:1.5:1.5 ratio. Spheroid was cultivated for 3 days, and 2-ME or 2-ME derivatives were treated for further 2 days from 10 uM to 39 nM concentration (9-points, 2-fold dilution from 10 uM). After 2 days incubation, spheroids were captured with automated high-content imaging system with a 10× magnifying objective (Operetta, PerkinElmer). Size of spheroids were analyzed using an in-house software tool and Harmony 3.5.1® high-content imaging (Harmony, PerkinElmer) for segmentation of cells. Increased dimeter of spheroids compared to 0.5% DMSO were selected as hit compounds.

[0147]

CCl.SUB.4 .Model of Liver Fibrosis

[0148] 9 week old C57BL/6 male mice (CentralLab) were dosed 8 times with 1 mg/kg CCl.sub.4 (Sigma) diluted olive oil for a total of 4 weeks. 2-ME, MC-011, or MC-015 were injected intraperitoneally at a concentration of 30 mg/kg (three times) or 60 mg/kg (two times) for 2 weeks. After 2 weeks of injection, liver tissue was harvested and fixed in 10% (v/v) neutral buffered formalin before preparing paraffin sections. Paraffin-embedded sections were deparaffinized and stained with hematoxylin and eosin (H&E; Sigma-Aldrich), and using a Masson's trichrome stain kit (Sigma-aldrich) to detect collagen. Images were obtained using a Zeiss microscope. At least five images per section were acquired for quantification, and positively stained areas were evaluated with ImageJ software.

[0149]

[0150] All mentioned documents are incorporated by reference as if herein written. When introducing elements of the present invention or the exemplary embodiment(s) thereof, the articles “a,” “an,” “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising,” “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. Although this invention has been described with respect to specific embodiments, the details of these embodiments are not to be construed as limitations.