COMPOUND HAVING ANTI-ANDROGEN RECEPTOR ACTIVITY, AND USE THEREOF

Abstract

A compound represented by formula I, and a racemate, stereoisomer, tautomer, solvate, polymorph, or pharmaceutically acceptable salts thereof are provided. The compound has good prostate tumor cell proliferation inhibitory activity and anti-AR activity. The compound has good in-vivo metabolic properties, has high AUC and C.sub.max, is good in druggability, and can be used for preventing and/or treating androgen-related disorders. The compound also has a good hair growth promoting effect, and can effectively increase the number of hair follicles and the growth length of hair.

##STR00001##

Claims

1. A compound represented by formula I, a racemate thereof, a stereoisomer thereof, a tautomer thereof, a solvate thereof, a polymorph thereof or a pharmaceutically acceptable salt thereof, ##STR00054## wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are identical or different and are each independently selected from the following groups that are unsubstituted or optionally substituted with one, two or more Ra: C.sub.1-12 alkyl or deuterated C.sub.1-12 alkyl; ##STR00055## represents the following ring systems that are unsubstituted or optionally substituted with one, two or more Rb: C.sub.3-20 cycloalkyl, C.sub.4-20 cycloalkenyl, 3- to 20-membered heterocyclyl, a spiro ring formed from C.sub.3-20 cycloalkyl and C.sub.3-20 cycloalkyl, a spiro ring formed from C.sub.3-20 cycloalkyl and 3- to 20-membered heterocyclyl, and a spiro ring formed from 3- to 20-membered heterocyclyl and 3- to 20-membered heterocyclyl; Ra are identical or different and are each independently selected from halogen, O, hydroxyl, amino, C.sub.1-12 alkyl, and C.sub.1-12 alkoxy; Rb are identical or different and are each independently selected from deuterium, halogen, O, hydroxyl, amino, and the following groups that are unsubstituted or optionally substituted with one, two or more Rc: C.sub.1-12 alkyl, C.sub.1-12 alkoxy, C.sub.3-20 cycloalkyl, 3- to 20-membered heterocyclyl, NHC.sub.1-12 alkyl, N(C.sub.1-12 alkyl).sub.2, NHCOC.sub.1-12 alkyl, CONHC.sub.1-12 alkyl, NHC.sub.3-20 cycloalkyl, NHCOC.sub.3-20 cycloalkyl, CONHC.sub.3-20 cycloalkyl, S(O).sub.2C.sub.1-12 alkyl, and COOC.sub.1-12 alkyl; or two Rb attached to the same carbon atom, together with the carbon atom attached thereto, form the following ring systems that are unsubstituted or optionally substituted with one, two or more Rc: C.sub.3-20 cycloalkyl and 3- to 20-membered heterocyclyl; Rc is selected from halogen, hydroxyl, amino, C.sub.1-12 alkyl, C.sub.1-12 alkoxy, C.sub.3-20 cycloalkyl, 3- to 20-membered heterocyclyl, and 5- to 20-membered heteroaryl.

2. The compound according to claim 1, wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are identical or different and are each independently selected from C.sub.1-6 alkyl and deuterated C.sub.1-6 alkyl; ##STR00056## represents the following ring systems that are unsubstituted or optionally substituted with one, two or more Rb: C.sub.3-12 cycloalkyl, C.sub.4-12 cycloalkenyl, 3- to 12-membered heterocyclyl, a spiro ring formed from C.sub.3-12 cycloalkyl and C.sub.3-12 cycloalkyl, a spiro ring formed from C.sub.3-12 cycloalkyl and 3- to 12-membered heterocyclyl, and a spiro ring formed from 3- to 12-membered heterocyclyl and 3- to 12-membered heterocyclyl; Rb are identical or different and are each independently selected from deuterium, halogen, O, hydroxyl, amino, and the following groups that are unsubstituted or optionally substituted with one, two or more Rc: C.sub.3-12 cycloalkyl, 3- to 12-membered heterocyclyl, NHC.sub.1-6 alkyl, N(C.sub.1-6 alkyl).sub.2, NHCOC.sub.1-6 alkyl, NHC.sub.3-12 cycloalkyl, S(O).sub.2C.sub.1-6 alkyl, and COOC.sub.1-6 alkyl; Rc is selected from halogen, hydroxyl, C.sub.3-12 cycloalkyl, 3- to 12-membered heterocyclyl, and 5-to 12-membered heteroaryl.

3. The compound according to claim 1, wherein ##STR00057## is selected from the following ring systems that are unsubstituted or optionally substituted with one, two or more Rb: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, spiro[2.3]hexanyl, 1,3-dioxanyl, and 1,4-dioxaspiro[4,5]decanyl; Rb is selected from deuterium, F, Cl, Br, I, O, hydroxyl, amino, tert-butoxycarbonyl, S(O).sub.2CH.sub.3, COOC(CH.sub.3).sub.3, ##STR00058## N(C.sub.2H.sub.5).sub.2, N(CH.sub.3).sub.2, ##STR00059## R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are identical or different and are each independently selected from C.sub.1-3 alkyl and deuterated C.sub.1-3 alkyl.

4. The compound according to claim 1, wherein the compound represented by formula I is selected from the following: ##STR00060## ##STR00061## ##STR00062## ##STR00063## ##STR00064##

5. The compound according to claim 1, wherein the compound represented by formula I is selected from the following. ##STR00065##

6. The compound according to claim 1, wherein the pharmaceutically acceptable salt of the compound represented by formula I is hydrochloride thereof.

7. A pharmaceutical composition, comprising at least one of the compound represented by formula I, the racemate thereof, the stereoisomer thereof, the tautomer thereof, the solvate thereof, the polymorph thereof or the pharmaceutically acceptable salt thereof according to claim 1.

8. The pharmaceutical composition according to claim 7, further comprising one or more pharmaceutically acceptable auxiliary materials; preferably, suitable routes of administration for the pharmaceutical composition include: oral, rectal, topical, buccal, parenteral, intramuscular, intradermal, intravenous, and transdermal administration; preferably, the pharmaceutical composition is for topical administration, and the pharmaceutical composition can be an ointment, a cream, a paste, a tincture, a plaster, a gel, a film, a paint, an aerosol, a spray, a foam, or a microsponge.

9. Use of the compound represented by formula I, the racemate thereof, the stereoisomer thereof, the tautomer thereof, the solvate thereof, the polymorph thereof or the pharmaceutically acceptable salt thereof according to claim 1 for manufacturing a medicament for the treatment, prevention, or amelioration of symptoms or diseases of androgen-related disorders.

10. The use according to claim 9, wherein the symptoms or diseases of androgen-related disorders are selected from: androgen-related inflammation, including wounds, acne, atopic dermatitis, rheumatoid arthritis, psoriasis, and rosacea; polyglutamine-mediated degeneration of motor neurons, and Kennedy's disease (spinal and bulbar muscular atrophy or SBMA); androgen-related cancers such as prostate cancer, bladder cancer, breast cancer, ovarian cancer, endometrial cancer, hepatocellular carcinoma, hepatocellular carcinoma, central nervous system cancer, skin cancer, lymphoma, leukemia, esophageal cancer, gastric cancer, colon cancer and pancreatic cancer; alopecia, including androgenetic alopecia; comedone; and hirsutism.

11. Use of the pharmaceutical composition according to claim 7 for manufacturing a medicament for the treatment, prevention, or amelioration of symptoms or diseases of androgen-related disorders.

12. The use according to claim 11, wherein the symptoms or diseases of androgen-related disorders are selected from: androgen-related inflammation, including wounds, acne, atopic dermatitis, rheumatoid arthritis, psoriasis, and rosacea; polyglutamine-mediated degeneration of motor neurons, and Kennedy's disease (spinal and bulbar muscular atrophy or SBMA); androgen-related cancers such as prostate cancer, bladder cancer, breast cancer, ovarian cancer, endometrial cancer, hepatocellular carcinoma, hepatocellular carcinoma, central nervous system cancer, skin cancer, lymphoma, leukemia, esophageal cancer, gastric cancer, colon cancer and pancreatic cancer; alopecia, including androgenetic alopecia; comedone; and hirsutism.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0092] FIG. 1 shows Western blot images of reduced AR protein expression in LNCaP cells with different concentrations of the compounds of the present disclosure;

[0093] FIG. 2 shows pharmacokinetic curves of the compounds in the plasma of male rats after oral administration of the compounds of the present disclosure;

[0094] FIG. 3 shows photographs of hair growth and representative HE staining pathological images of skin tissues on day 18 of the test in Test Example 4 for various groups of mice in the hair growth promotion test.

DETAILED DESCRIPTION

[0095] The general-formula compounds of the present disclosure, the preparation method therefor, and use thereof are described in detail with reference to the following specific examples. It should be understood that the following examples are merely exemplary illustration and explanation of the present disclosure and should not be construed as limiting the protection scope of the present disclosure. All techniques implemented based on the content described above of the present disclosure are encompassed within the protection scope of the present disclosure.

[0096] The intermediate compounds of the present disclosure can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other chemical synthetic methods, and equivalent substitutions thereof known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present disclosure.

[0097] The chemical reactions of the specific embodiments of the present disclosure are carried out in a suitable solvent that must be suitable for the chemical changes in the present disclosure and the reagents and materials required. In order to obtain the compounds of the present disclosure, it is sometimes necessary for those skilled in the art to modify or select a synthesis procedure or a reaction scheme based on the existing embodiments.

[0098] The present disclosure is described in detail below through examples; however, these examples are not intended to limit the present disclosure in any way.

[0099] Experimental methods without specified conditions in the following examples are generally conducted under conventional conditions, or conditions recommended by the manufacturer. Unless otherwise indicated, the percentages and the number of parts are calculated by weight, and the starting materials and reagents used in the following examples are commercially available or may be manufactured by known methods.

[0100] The following abbreviations are used in the present disclosure: DHT for dihydrotestosterone; clascoterone (purchased from Nanjing Chemlin Chemical Industry Co., Ltd.), which is a chemical drug on the market, the target indications including androgenetic alopecia, acne, and the like, and the mechanism of action thereof being as follows: competitively inhibiting the binding of DHT to AR, thereby achieving an anti-androgen effect; dimethylcurcumin (purchased from Nanjing Chemlin Chemical Industry Co., Ltd.); starting material A having a structural formula of

##STR00024##

(purchased from Nanjing Chemlin Chemical Industry Co., Ltd.); starting material B having a structural formula of

##STR00025##

starting material C having a structural formula of

##STR00026##

and starting material D having a structural formula of

##STR00027##

(starting materials B to D are all purchased from Nanjing Jiming Biotechnology Co., Ltd.).

Example 1. Synthesis of Compound 1

##STR00028##

[0101] Starting material A (408 mg, 1.00 mmol, 1.0 eq), 1,2-dibromoethane (225 mg, 1.20 mmol), Cs.sub.2CO.sub.3 (814 mg, 2.5 mmol), and DMF (15 mL) were added to a reaction flask, and the resulting mixture was stirred at 50 C. for 20 h. After completion of the reaction, water (50 mL) and ethyl acetate (20 mL) were added. Liquid separation was performed. The aqueous phase was extracted with ethyl acetate (20 mL). The organic phases were combined and washed with saturated brine to remove DMF. The resulting solution was dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (petroleum ether/ethyl acetate 10:1-2:1) to give a light yellow solid (39 mg).

[0102] .sup.1H NMR (400 MHz, DMSO-d.sub.6) 7.53 (d, J=15.8 Hz, 2H), 7.30 (d, J=2.0 Hz, 2H), 7.24 (dd, J=8.4, 2.1 Hz, 2H), 6.97 (d, J=8.3 Hz, 2H), 6.94 (d, J=15.7 Hz, 2H), 1.54 (brs, 4H). MS m/z: 435.14 [M+H].sup.+.

Example 2. Synthesis of Compound 2

##STR00029##

[0103] Starting material A (408 mg, 1.00 mmol, 1.0 eq), 1,3-dibromopropane (242 mg, 1.20 mmol), Cs.sub.2CO.sub.3 (814 mg, 2.5 mmol), and DMF (15 mL) were added to a reaction flask, and the resulting mixture was stirred at 50 C. for 20 h. After completion of the reaction, water (50 mL) and ethyl acetate (20 mL) were added. Liquid separation was performed. The aqueous phase was extracted with ethyl acetate (20 mL). The organic phases were combined and washed with saturated brine to remove DMF. The resulting solution was dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (petroleum ether/ethyl acetate 10:1-2:1) to give a light yellow solid (90 mg).

[0104] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.57 (d, J=15.6 Hz, 1H), 7.33 (d, J=15.8 Hz, 1H), 7.20-6.91 (m, 6H), 6.84 (d, J=8.2 Hz, 1H), 6.79 (d, J=8.2 Hz, 1H), 4.36-4.06 (m, 2H), 2.76-2.46 (m, 2H), 2.12-1.83 (m, 2H). MS m/z: 449.22 [M+H].sup.+.

Example 3. Synthesis of Compound 3

##STR00030##

[0105] Starting material A (408 mg, 1.00 mmol, 1.0 eq), 1,4-dibromobutane (260 mg, 1.20 mmol), Cs.sub.2CO.sub.3 (814 mg, 2.5 mmol), and DMF (15 mL) were added to a reaction flask, and the resulting mixture was stirred at 50 C. for 20 h. After completion of the reaction, water (50 mL) and ethyl acetate (20 mL) were added. Liquid separation was performed. The aqueous phase was extracted with ethyl acetate (20 mL). The organic phases were combined and washed with saturated brine to remove DMF. The resulting solution was dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (petroleum ether/ethyl acetate 10:1-2:1) to give a light yellow solid (87 mg).

[0106] .sup.1H NMR (400 MHz, DMSO-d.sub.6) 7.55 (d, J=15.6 Hz, 2H), 7.32-7.18 (m, 4H), 6.96 (d, J=8.3 Hz, 2H), 6.81 (d, J=15.7 Hz, 2H), 2.31-2.15 (m, 4H), 1.67-1.50 (m, 4H). MS m/z: 463.20 [M+H].sup.+.

Example 4. Synthesis of Compound 4

##STR00031##

[0107] Starting material A (408 mg, 1.00 mmol, 1.0 eq), 1,4-dibromopentane (277 mg, 1.20 mmol), Cs.sub.2CO.sub.3 (814 mg, 2.5 mmol), and DMF (15 mL) were added to a reaction flask, and the resulting mixture was stirred at 50 C. for 20 h. After completion of the reaction, water (50 mL) and ethyl acetate (20 mL) were added. Liquid separation was performed. The aqueous phase was extracted with ethyl acetate (20 mL). The organic phases were combined and washed with saturated brine to remove DMF. The resulting solution was dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (petroleum ether/ethyl acetate 10:1-2:1) to give a light yellow solid (92 mg).

[0108] .sup.1H NMR (400 MHz, DMSO-d.sub.6) 7.56 (d, J=15.5 Hz, 2H), 7.37-7.18 (m, 4H), 7.03 (d, J=15.5 Hz, 2H), 6.96 (d, J=8.3 Hz, 2H), 2.21-1.96 (m, 4H), 1.60-1.28 (m, 6H). MS m/z: 477.23 [M+H].sup.+.

Example 5. Synthesis of Compound 5

##STR00032##

[0109] Dimethylcurcumin (500 mg, 1.26 mmol, 1.0 eq), 1,1-bis-(bromomethyl)cyclopropane (340 mg, 1.50 mmol), Cs.sub.2CO.sub.3 (1.00 g, 3.11 mmol), and DMF (15 mL) were added to a reaction flask, and the resulting mixture was stirred at 50 C. for 20 h. After completion of the reaction, water (50 mL) and ethyl acetate (20 mL) were added. Liquid separation was performed. The aqueous phase was extracted with ethyl acetate (20 mL). The organic phases were combined and washed with saturated brine to remove DMF. The resulting solution was dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (petroleum ether/ethyl acetate 10:1-2:1) to give a light yellow solid (20 mg).

[0110] .sup.1H NMR (400 MHz, CDCl.sub.3) 8.20 (d, J=15.8 Hz, 1H), 7.56 (d, J=15.6 Hz, 1H), 7.33 (d, J=16.1 Hz, 1H), 7.24-6.79 (m, 6H), 6.72 (d, J=15.5 Hz, 1H), 3.92 (s, 12H), 1.63 (brs, 4H), 0.70 (brs, 4H). MS m/z: 463.16 [M+H].sup.+.

Example 6. Synthesis of Compound 6

##STR00033##

[0111] Starting material A (408 mg, 1.00 mmol, 1.0 eq), deuterated dibromoethane (230 mg, 1.20 mmol), Cs.sub.2CO.sub.3 (814 mg, 2.5 mmol), and DMF (15 mL) were added to a reaction flask, and the resulting mixture was stirred at 50 C. for 20 h. After completion of the reaction, water (50 mL) and ethyl acetate (20 mL) were added. Liquid separation was performed. The aqueous phase was extracted with ethyl acetate (20 mL). The organic phases were combined and washed with saturated brine to remove DMF. The resulting solution was dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (petroleum ether/ethyl acetate 10:1-2:1) to give a light yellow solid (30 mg).

[0112] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.62 (d, J=15.8 Hz, 2H), 7.12 (dd, J=8.3, 2.1 Hz, 2H), 7.00 (d, J=2.0 Hz, 2H), 6.83 (d, J=8.3 Hz, 2H), 6.79 (d, J=15.7 Hz, 2H). MS m/z: 439.18 [M+H].sup.+.

Example 7. Synthesis of Compound 7

##STR00034##

[0113] Dimethylcurcumin (10.0 g, 25.22 mmol, 1.0 eq), 1,5-dichloropentanone (3.91 g, 25.22 mmol), KBr (12.01 g, 100.9 mmol), K.sub.2CO.sub.3 (10.46 g, 75.67 mmol), and DMF (300 mL) were added to a reaction flask, and the resulting mixture was stirred at 50 C. for 20 h. After completion of the reaction, water (700 mL) and ethyl acetate (400 mL) were added. Liquid separation was performed. The aqueous phase was extracted with ethyl acetate (400 mL). The organic phases were combined and washed with saturated brine to remove DMF. The resulting solution was dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (petroleum ether/ethyl acetate 10:1-2:1) to give a yellow foamy solid (3.6 g).

[0114] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.75 (d, J=15.5 Hz, 2H), 7.15 (dd, J=8.4, 2.0 Hz, 2H), 7.02 (d, J=2.0 Hz, 2H), 6.85 (d, J=8.4 Hz, 2H), 6.74 (d, J=15.4 Hz, 2H), 3.91 (s, 12H), 2.56-2.40 (m, 8H). MS m/z: 479.29 [M+H].sup.+.

Example 8. Synthesis of Compound 8

##STR00035##

[0115] Under nitrogen atmosphere, compound 7 (2.0 g, 4.18 mmol), dichloromethane (20 mL), and 1 drop of pyridine hydrofluoride were added to a reaction flask. The resulting mixture was cooled to 0 C. under stirring, and 4-tert-butyl-2,6-dimethylphenylsulfur trifluoride (2.1 g, 8.36 mmol) was added in one portion. The mixture was reacted at 10-15 C. for 6 h. The reaction liquid was washed with water, dried over anhydrous sodium sulfate, and subjected to column chromatography (petroleum ether/ethyl acetate 10:1-2:1) to give a crude product, which was subjected to preparative HPLC (acetonitrile/water 10:90-80:20) to give a light yellow solid (150 mg).

[0116] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.71 (d, J=15.4 Hz, 2H), 7.14 (d, J=8.3 Hz, 2H), 7.09-6.96 (m, 2H), 6.85 (d, J=8.4 Hz, 2H), 6.72 (d, J=15.4 Hz, 2H), 3.91 (s, 12H), 2.45-2.18 (m, 4H), 2.12-1.91 (m, 4H). .sup.19F NMR (376 MHz, CDCl.sub.3) 97.28. MS m/z: 501.27 [M+H].sup.+.

Example 9. Synthesis of Compound 9

##STR00036##

Step (1): Synthesis of Intermediate 9-1

[0117] Dimethylcurcumin (2.00 g, 5.0 mmol) and tetrahydrofuran (16 mL) were successively added to a reaction flask. The resulting mixture was stirred and dissolved. The reaction temperature of the mixture was controlled to be 1 C. to 0 C., and an aqueous formaldehyde solution (37-40%, 860 mg, 10.6 mmol) and a catalytic amount of 1,8-diazabicyclo[5.4.0]undec-7-ene (93 mg) were successively added into the mixture. The mixture was then reacted at 0-10 C. for 1-2 h, concentrated by rotary evaporation at room temperature, and subjected to column chromatography (MeOH/CH.sub.2Cl.sub.2 1:100-1:50) to give a yellow solid (900 mg).

[0118] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.73 (d, J=15.4 Hz, 2H), 7.15 (dd, J=8.3, 2.1 Hz, 2H), 7.01 (d, J=2.1 Hz, 2H), 6.84 (d, J=8.4 Hz, 2H), 6.73 (d, J=15.4 Hz, 2H), 4.34 (d, J=8.0 Hz, 4H), 3.90 (s, 12H), 2.97 (t, J=8.0 Hz, 2H).

Step (2): Synthesis of Compound 9

[0119] Intermediate 9-1 (300 mg, 0.65 mmol), dichloromethane (7 mL), and pyridine (156 mg, 1.97 mmol) were added to a reaction flask under nitrogen atmosphere, and the resulting mixture was cooled to 70 C. under stirring. A solution of bis(trichloromethyl)carbonate (97 mg, 0.33 mmol) in dichloromethane (1 mL) was added dropwise into the mixture, and the mixture was reacted at 70 C. for 2 h. The reaction liquid was washed with water, dried over anhydrous sodium sulfate, and subjected to column chromatography (petroleum ether/ethyl acetate 10:1-2:1) to give a light yellow solid (210 mg). MS m/z: 483.35 [M+H].sup.+.

Example 10. Synthesis of Compound 10

##STR00037##

[0120] Dimethylcurcumin (500 mg, 1.26 mmol, 1.0 eq), 1,5-dibromopentane (435 mg, 1.89 mmol), K.sub.2CO.sub.3 (523 mg, 3.78 mmol), and DMF (15 mL) were added to a reaction flask, and the resulting mixture was stirred at 50 C. for 20 h. After completion of the reaction, water (50 mL) and ethyl acetate (20 mL) were added into the mixture. Liquid separation was performed. The aqueous phase was extracted with ethyl acetate (20 mL). The organic phases were combined and washed with saturated brine to remove DMF. The resulting solution was dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (petroleum ether/ethyl acetate 10:1-2:1) to give a yellow foamy solid (150 mg).

[0121] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.66 (d, J=15.5 Hz, 2H), 7.12 (dd, J=8.3, 1.9 Hz, 2H), 7.01 (d, J=2.0 Hz, 2H), 6.83 (d, J=8.3 Hz, 2H), 6.75 (d, J=15.4 Hz, 2H), 3.90 (s, 12H), 2.17-2.02 (m, 4H), 1.66-1.53 (m, 4H), 1.50-1.37 (m, 2H). MS m/z: 465.31 [M+H].sup.+.

Example 11. Synthesis of Compound 11

##STR00038##

[0122] Compound 7 (500 mg, 1.04 mmol), ethylene glycol (324 mg, 5.22 mmol), acetonitrile (5 mL), and oxalic acid (94 mg, 1.04 mmol) were added to a reaction flask, and the resulting mixture was stirred at 25 C. for 20 h. After completion of the reaction, the reaction liquid was diluted with ethyl acetate (50 mL), washed with a saturated aqueous sodium bicarbonate solution, dried over sodium sulfate, concentrated, and subjected to column chromatography (petroleum ether/ethyl acetate 10:1-2:1) to give a light yellow solid (420 mg).

[0123] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.66 (d, J=15.5 Hz, 2H), 7.12 (dd, J=8.3, 2.0 Hz, 2H), 7.00 (d, J=1.9 Hz, 2H), 6.82 (d, J=8.4 Hz, 2H), 6.75 (d, J=15.4 Hz, 2H), 3.93 (s, 4H), 3.89 (s, 12H), 2.36-2.22 (m, 4H), 1.82-1.68 (m, 4H). MS m/z: 523.33 [M+H].sup.+.

Example 12. Synthesis of Compound 12 and Hydrochloride Thereof

##STR00039##

[0124] Compound 7 (200 mg, 0.4 mmol), acetic acid (25 mg, 0.4 mmol), 1,2-dichloroethane (1.5 mL), and morpholine (36 g, 0.4 mmol) were added to a reaction flask. Sodium triacetoxyborohydride (106 mg, 0.5 mmol) was added in one portion into the mixture, and the resulting mixture was stirred at 25 C. for 7 h. The reaction liquid was washed with a saturated aqueous sodium bicarbonate solution, dried over sodium sulfate, concentrated, and subjected to preparative TLC (petroleum ether/ethyl acetate 1:1) to give a light yellow solid (30 mg).

[0125] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.68 (d, J=15.5 Hz, 1H), 7.66 (d, J=15.5 Hz, 1H), 7.16-7.09 (m, 2H), 7.03-6.98 (m, 2H), 6.83 (dd, J=8.4, 3.1 Hz, 2H), 6.75 (d, J=15.5 Hz, 1H), 6.69 (d, J=15.5 Hz, 1H), 3.90 (s, 12H), 3.88-3.72 (m, 4H), 2.87-2.40 (m, 7H), 2.10-1.45 (m, 6H). MS m/z: 550.48 [M+H].sup.+.

Synthesis of Compound 12 Hydrochloride:

[0126] Compound 12 (200 mg), methanol (0.5 mL) and ethyl acetate (5 mL) were added to a reaction flask. The resulting mixture was stirred and dissolved. Hydrochloric acid-ethyl acetate (1 mol/L, 0.5 mL) was added into the mixture. The mixture was filtered, and the filtrate was dried under vacuum to give a light yellow solid (120 mg).

[0127] .sup.1H NMR (400 MHz, CDCl.sub.3) 12.96 (brs, 1H), 7.72 (d, J=15.2 Hz, 1H), 7.69 (d, J=15.3 Hz, 1H), 7.17-7.10 (m, 2H), 7.03-6.96 (m, 2H), 6.84 (dd, J=8.4, 3.5 Hz, 2H), 6.71 (d, J=15.4 Hz, 1H), 6.62 (d, J=15.4 Hz, 1H), 4.56-4.29 (m, 2H), 4.05-3.80 (m, 2H), 3.91 (s, 12H), 3.34-2.88 (m, 5H), 2.83-2.68 (m, 2H), 2.43-2.27 (m, 2H), 1.91-1.69 (m, 4H). MS m/z: 550.48 [M+H].sup.+.

Example 13. Synthesis of Compound 13

##STR00040##

[0128] Compound 7 (200 mg, 0.4 mmol), acetic acid (25 mg, 0.4 mmol) and 1,2-dichloroethane (5 mL) were added to a reaction flask. Sodium triacetoxyborohydride (106 mg, 0.5 mmol) was added in one portion into the mixture, and the resulting mixture was stirred at 25 C. for 5 h. The reaction liquid was washed with a saturated aqueous sodium bicarbonate solution, dried over sodium sulfate, concentrated, and subjected to preparative TLC (petroleum ether/ethyl acetate 2:1) to give a light yellow solid (20 mg).

[0129] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.68 (d, J=15.5 Hz, 1H), 7.66 (d, J=15.5 Hz, 1H), 7.17-7.10 (m, 2H), 7.03-6.99 (m, 2H), 6.83 (dd, J=8.3, 2.6 Hz, 2H), 6.76 (d, J=15.4 Hz, 1H), 6.73 (d, J=15.5 Hz, 1H), 3.90 (s, 12H), 3.79-3.69 (m, 1H), 2.00-1.86 (m, 4H), 1.72-1.50 (m, 4H). MS m/z: 481.24 [M+H].sup.+.

Example 14. Synthesis of Compound 14 and Hydrochloride Thereof

##STR00041##

[0130] Under nitrogen atmosphere, compound 7 (300 mg, 0.63 mmol), ammonium trifluoroacetate (160 mg, 1.25 mmol), and tetrahydrofuran (10 mL) were added to a reaction flask. The resulting mixture was stirred at 25 C. for 30 mins, and sodium triacetoxyborohydride (270 mg, 1.25 mmol) was then added. The mixture was reacted at room temperature for 5 h, and ethyl acetate (30 mL) and diluted hydrochloric acid (0.2 N/30 mL) were added. Liquid separation was performed. The organic phase was washed with purified water, dried over sodium sulfate, and concentrated to give a yellow oil, which was subjected to preparative HPLC (acetonitrile/water 10:90-80:20) to give compound 14 (MS m/z: 480.26. [M+H].sup.+) as a yellow solid.

[0131] Compound 14, ethyl acetate (7 mL) and methanol (0.1 mL) were added to a reaction flask. The resulting mixture was stirred and dissolved at room temperature, and hydrochloric acid-ethyl acetate (0.5 N/1.2 mL) was added dropwise into the mixture. The resulting mixture was filtered, and the filtrate was dried under vacuum at 40 C. to give compound 14 hydrochloride (20 mg) as a yellow solid.

[0132] .sup.1H NMR (400 MHz, CDCl.sub.3) 8.39 (brs, 3H), 7.68 (d, J=15.4 Hz, 1H), 7.66 (d, J=15.4 Hz, 1H), 7.18-7.06 (m, 2H), 7.05-6.95 (m, 2H), 6.83 (d, J=8.4 Hz, 1H), 6.79 (d, J=8.4 Hz, 1H), 6.73 (d, J=15.5 Hz, 1H), 6.68 (d, J=15.5 Hz, 1H), 3.89 (s, 6H), 3.88 (s, 3H), 3.87 (s, 3H), 3.32-3.10 (m, 1H), 2.77-2.52 (m, 2H), 2.31-2.06 (m, 2H), 1.99-1.53 (m, 4H). MS m/z: 480.26. [M+H].sup.+.

Example 15. Synthesis of Compound 16 and Hydrochloride Thereof

##STR00042##

[0133] Under nitrogen atmosphere, compound 7 (300 mg, 0.63 mmol), diethylamine (9 mg, 1.25 mmol) and tetrahydrofuran (10 mL) were added to a reaction flask. The resulting mixture was stirred at 25 C. for 30 mins, and sodium triacetoxyborohydride (270 mg, 1.25 mmol) was then added. The mixture was reacted at room temperature for 5 h, and ethyl acetate (30 mL) and diluted hydrochloric acid (0.2 N/30 mL) were added. Liquid separation was performed. The organic phase was washed with purified water, dried over sodium sulfate, and concentrated to give a yellow oily compound, which was subjected to preparative TLC (petroleum ether/ethyl acetate 1:1) to give compound 16 (MS m/z: 536.44 [M+H].sup.+) as a light yellow solid.

[0134] Compound 16, ethyl acetate (12 mL) and methanol (0.2 mL) were added to a reaction flask. The resulting mixture was stirred and dissolved at room temperature, and hydrochloric acid-ethyl acetate (0.5 N/1.2 mL) was added dropwise into the mixture. The resulting mixture was filtered, and the filtrate was dried under vacuum at 40 C. to give a yellow solid (160 mg), which was hydrochloride of compound 16.

[0135] .sup.1H NMR (400 MHz, CDCl.sub.3) 11.95 (brs, 1H), 7.72 (d, J=15.1 Hz, 1H), 7.69 (d, J=14.8 Hz, 1H), 7.14 (dd, J=8.3, 1.9 Hz, 2H), 7.04-6.96 (m, 2H), 6.85 (dd, J=8.4, 2.7 Hz, 2H), 6.71 (d, J=15.4 Hz, 1H), 6.63 (d, J=15.4 Hz, 1H), 3.91 (s, 12H), 3.42-3.22 (m, 1H), 3.18-2.96 (m, 4H), 2.82-2.66 (m, 2H), 2.37-2.22 (m, 2H), 1.92-1.67 (m, 4H), 1.51 (t, J=7.2 Hz, 6H). MS m/z: 536.44 [M+H].sup.+.

Example 16. Synthesis of Compound 18

##STR00043##

[0136] Under nitrogen atmosphere, compound 7 (300 mg, 0.63 mmol), 3,3-difluorocyclobutanamine hydrochloride (200 mg, 1.25 mmol), triethylamine (130 mg, 1.25 mmol), and tetrahydrofuran (10 mL) were added to a reaction flask. The resulting mixture was stirred at 25 C. for 30 min, and sodium triacetoxyborohydride (270 mg, 1.25 mmol) was then added. The mixture was reacted at room temperature for 2 h, and ethyl acetate (30 mL) and diluted hydrochloric acid (0.2 N/30 mL) were added. Liquid separation was performed. The organic phase was washed with purified water, dried over anhydrous sodium sulfate, concentrated, and subjected to preparative TLC (petroleum ether/ethyl acetate 1:1) to give compound 18 (6 mg) as a light yellow solid. MS m/z: 584.50 [M+H].sup.+.

[0137] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.68 (d, J=15.5 Hz, 1H), 7.65 (d, J=15.5 Hz, 1H), 7.17-7.06 (m, 2H), 7.01 (d, J=2.0 Hz, 1H), 7.00 (d, J=2.0 Hz, 1H), 6.83 (dd, J=8.4, 3.4 Hz, 2H), 6.76 (d, J=15.5 Hz, 1H), 6.70 (d, J=15.5 Hz, 1H), 3.90 (s, 12H), 2.72-2.56 (m, 2H), 2.50-2.36 (m, 1H), 2.06-1.88 (m, 4H), 1.80-1.41 (m, 1OH). .sup.19F NMR (376 MHz, CDCl.sub.3) 97.80.

Example 17. Synthesis of Compound 19 and Hydrochloride Thereof

##STR00044##

[0138] Under nitrogen atmosphere, compound 7 (300 mg, 0.63 mmol), piperidine (110 mg, 1.25 mmol), and tetrahydrofuran (10 mL) were added to a reaction flask. The resulting mixture was stirred at 25 C. for 30 mins, and sodium triacetoxyborohydride (270 mg, 1.25 mmol) was then added. The mixture was reacted at room temperature for 3 h, and ethyl acetate (30 mL) and diluted hydrochloric acid (0.2 N/30 mL) were added. Liquid separation was performed. The organic phase was washed with purified water, dried over sodium sulfate, and concentrated to give a yellow oily compound, which was subjected to preparative TLC (petroleum ether/ethyl acetate 1:1) to give compound 19 (MS m/z: 548.45 [M+H].sup.+) as a light yellow solid.

[0139] Compound 19, ethyl acetate (12 mL) and methanol (0.2 mL) were added to a reaction flask. The resulting mixture was stirred and dissolved at room temperature, and hydrochloric acid-ethyl acetate (0.5 N/1.2 mL) was added dropwise. The resulting mixture was filtered, and the filtrate was dried under vacuum at 40 C. to give compound 19 hydrochloride (120 mg) as a yellow solid.

[0140] .sup.1H NMR (400 MHz, CDCl.sub.3) 11.95 (brs, 1H), 7.71 (d, J=15.4 Hz, 1H), 7.68 (d, J=15.4 Hz, 1H), 7.14 (dd, J=8.4, 2.0 Hz, 2H), 7.05-6.96 (m, 2H), 6.84 (dd, J=8.4, 3.3 Hz, 2H), 6.71 (d, J=15.4 Hz, 1H), 6.63 (d, J=15.4 Hz, 1H), 3.91 (s, 12H), 3.45-3.28 (m, 2H), 3.17-3.03 (m, 1H), 2.86-2.62 (m, 4H), 2.56-2.29 (m, 4H), 2.00-1.52 (m, 8H).MS m/z: 548.45 [M+H].sup.+.

Example 18. Synthesis of Compound 22

##STR00045##

[0141] Under nitrogen atmosphere, compound 7 (1.00 g, 2.09 mmol) and dichloromethane (20 mL) were added to a reaction flask. The resulting mixture was stirred at 0 C. for 10 mins, and diethylaminosulfur trifluoride (0.40 g, 2.51 mmol) was added dropwise. The mixture was then reacted at room temperature overnight, and a saturated aqueous sodium bicarbonate solution was added. Liquid separation was performed. The aqueous phase was extracted with dichloromethane, concentrated, and then subjected to column chromatography (petroleum ether/ethyl acetate 10:1-2:1) to give a crude product (320 mg), which was subjected to preparative HPLC (acetonitrile/water 10:90-80:20) to give a light yellow solid (0.10 g).

[0142] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.704 (d, J=15.4 Hz, 2H), 7.138 (dd, J=8.3, 1.9 Hz, 2H), 7.013 (d, J=1.9 Hz, 2H), 6.842 (d, J=8.3 Hz, 2H), 6.727 (d, J=15.4 Hz, 2H), 5.326-5.169 (m, 1H), 3.910 (s, 6H), 3.904 (s, 6H), 2.822-2.606 (m, 2H), 2.469-2.352 (m, 2H), 2.317-2.198 (m, 2H). .sup.19F NMR (377 MHz, CDCl.sub.3) 102.82. MS m/z: 481.99 [M+H].sup.+.

Example 19. Synthesis of Compound 23 and Hydrochloride Thereof

##STR00046##

[0143] Under nitrogen atmosphere, compound 7 (300 mg, 0.63 mmol), 3,3-difluorocyclobutanamine hydrochloride (180 mg, 1.25 mmol), triethylamine (130 mg, 1.25 mmol), and tetrahydrofuran (10 mL) were added to a 50 mL reaction flask. The resulting mixture was stirred at 25 C. for 30 mins, and sodium triacetoxyborohydride (270 mg, 1.25 mmol) was then added. The mixture was reacted at room temperature for 2 h. Ethyl acetate (30 mL) and diluted hydrochloric acid (0.2 N/30 mL) were added. Liquid separation was performed. The organic phase was washed with purified water, dried over anhydrous sodium sulfate, and concentrated to give compound 23 (MS m/z: 570.43 [M+H].sup.+) as a yellow oil.

[0144] Compound 23, ethyl acetate (15 mL) and methanol (0.3 mL) were added. The mixture was stirred and dissolved at room temperature, and hydrochloric acid-ethyl acetate (0.5 N/1.2 mL) was added dropwise. The resulting mixture was filtered, and the filtrate was dried under vacuum at 40 C. to give compound 23 hydrochloride (120 mg) as a yellow solid.

[0145] .sup.1H NMR (400 MHz, CDCl.sub.3) 10.26 (brs, 2H), 7.71 (d, J=15.4 Hz, 1H), 7.67 (d, J=15.4 Hz, 1H), 7.18-7.06 (m, 2H), 7.03-6.97 (m, 2H), 6.82 (d, J=8.4 Hz, 1H), 6.79 (d, J=8.4 Hz, 1H), 6.74 (d, J=15.4 Hz, 1H), 6.69 (d, J=15.5 Hz, 1H), 3.88 (s, 12H), 3.66-3.50 (m, 1H), 3.41-3.22 (m, 2H), 3.13-2.88 (m, 3H), 2.81-2.60 (m, 2H), 2.28-2.13 (m, 2H), 1.94-1.73 (m, 4H).MS m/z: 570.43 [M+H].sup.+.

Example 20. Synthesis of Compound 24

##STR00047##

[0146] Dimethylcurcumin (500 mg, 1.26 mmol), dibromoethyl methanesulfonamide (460 mg, 1.50 mmol), K.sub.2CO.sub.3 (520 mg, 3.76 mmol), and DMF (15 mL) were added to a reaction flask, and the resulting mixture was stirred at 50 C. for 20 h. After completion of the reaction, water (50 mL) and ethyl acetate (20 mL) were added. Liquid separation was performed. The aqueous phase was extracted with ethyl acetate (20 mL). The organic phase was washed with saturated brine to remove DMF, dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (petroleum ether/ethyl acetate 10:1-2:1) to give a light yellow solid (20 mg). MS m/z: 544.42 [M+H].sup.+.

[0147] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.72 (d, J=15.4 Hz, 2H), 7.15 (dd, J=8.4, 2.0 Hz, 2H), 7.01 (d, J=2.0 Hz, 2H), 6.85 (d, J=8.3 Hz, 2H), 6.69 (d, J=15.4 Hz, 2H), 3.91 (s, 12H), 3.36-3.27 (m, 4H), 2.76 (s, 3H), 2.42-2.28 (m, 4H).

Example 21. Synthesis of Compound 25

##STR00048##

[0148] Dimethylcurcumin (500 mg, 1.26 mmol), tert-butyl bis(2-bromoethyl)carbamate (500 mg, 1.50 mmol), K.sub.2CO.sub.3 (520 mg, 3.76 mmol), and DMF (15 mL) were added to a reaction flask, and the resulting mixture was stirred at 50 C. for 20 h. After completion of the reaction, water (50 mL) and ethyl acetate (20 mL) were added. Liquid separation was performed. The aqueous phase was extracted with ethyl acetate (20 mL). The organic phase was washed with saturated brine to remove DMF, dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (petroleum ether/ethyl acetate 10:1-2:1) to give a light yellow solid (90 mg).

[0149] MS m/z: 566.47 [M+H].sup.+.

[0150] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.69 (d, J=15.4 Hz, 2H), 7.13 (dd, J=8.4, 2.0 Hz, 2H), 7.00 (d, J=2.0 Hz, 2H), 6.83 (d, J=8.4 Hz, 2H), 6.70 (d, J=15.5 Hz, 2H), 3.90 (s, 12H), 3.54-3.26 (m, 4H), 2.30-2.10 (m, 4H), 1.44 (s, 9H).

Example 22. Synthesis of Compound 26

##STR00049##

[0151] Dimethylcurcumin (500 mg, 1.26 mmol), (dibromoethyl)cyclopropane (390 mg, 1.52 mmol), K.sub.2CO.sub.3 (520 mg, 3.76 mmol), and DMF (15 mL) were added to a reaction flask, and the resulting mixture was stirred at 50 C. for 20 h. After completion of the reaction, water (50 mL) and ethyl acetate (20 mL) were added. Liquid separation was performed. The aqueous phase was extracted with ethyl acetate (20 mL). The organic phase was washed with saturated brine to remove DMF, dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (petroleum ether/ethyl acetate 10:1-2:1) to give a light yellow solid (50 mg).

[0152] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.69 (s, 2H), 7.13 (dd, J=8.3, 1.9 Hz, 2H), 7.01 (d, J=2.0 Hz, 2H), 6.83 (d, J=8.3 Hz, 2H), 6.77 (d, J=15.5 Hz, 2H), 3.90 (s, 12H), 2.26-2.13 (m, 4H), 1.45-1.37 (m, 4H), 0.25 (s, 4H). MS m/z: 491.42 [M+H].sup.+.

Example 23. Synthesis of Compound 27

##STR00050##

[0153] Starting material B (500 mg, 1.25 mmol), 1,5-dibromo-3,3-difluoropentane (317 mg, 1.2 mmol), K.sub.2CO.sub.3 (520 mg, 3.76 mmol), and DMF (15 mL) were added to a reaction flask, and the resulting mixture was stirred at 50 C. for 20 h. After completion of the reaction, water (50 mL) and ethyl acetate (20 mL) were added. Liquid separation was performed. The aqueous phase was extracted with ethyl acetate (20 mL). The organic phase was washed with saturated brine to remove DMF, dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (petroleum ether/ethyl acetate 10:1-2:1) to give a light yellow solid (30 mg).

[0154] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.707 (d, J=15.5 Hz, 2H), 7.139 (dd, J=8.3, 2.0 Hz, 2H), 7.009 (d, J=2.0 Hz, 2H), 6.840 (dd, J=8.4, 2.4 Hz, 2H), 6.722 (d, J=15.4 Hz, 2H), 3.907 (s, 6H), 3.903 (s, 3H), 2.38-2.27 (m, 4H), 2.12-1.85 (m, 4H). MS m/z: 504.44 [M+H].sup.+.

Example 24. Synthesis of Compound 28

##STR00051##

[0155] Starting material C (500 mg, 1.24 mmol), 1,5-dibromo-3,3-difluoropentane (317 mg, 1.2 mmol), K.sub.2CO.sub.3 (520 mg, 3.76 mmol), and DMF (15 mL) were added to a reaction flask, and the resulting mixture was stirred at 50 C. for 20 h. After completion of the reaction, water (50 mL) and ethyl acetate (20 mL) were added. Liquid separation was performed. The aqueous phase was extracted with ethyl acetate (20 mL). The organic phase was washed with saturated brine to remove DMF, dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (petroleum ether/ethyl acetate 10:1-2:1) to give a light yellow solid (35 mg).

[0156] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.709 (d, J=15.4 Hz, 2H), 7.140 (dd, J=8.3, 2.0 Hz, 2H), 7.027-6.982 (m, 2H), 6.843 (dd, J=8.4, 2.5 Hz, 2H), 6.722 (d, J=15.4 Hz, 2H), 3.910 (s, 3H), 3.907 (s, 3H), 2.372-2.227 (m, 4H), 2.116-1.949 (m, 4H). MS m/z: 507.46 [M+H].sup.+.

Example 25. Synthesis of Compound 29

##STR00052##

[0157] Starting material D (500 mg, 1.24 mmol), 1,5-dibromo-3,3-difluoropentane (317 mg, 1.2 mmol), K.sub.2CO.sub.3 (520 mg, 3.76 mmol), and DMF (15 mL) were added to a reaction flask, and the resulting mixture was stirred at 50 C. for 20 h. After completion of the reaction, water (50 mL) and ethyl acetate (20 mL) were added. Liquid separation was performed. The aqueous phase was extracted with ethyl acetate (20 mL). The organic phase was washed with saturated brine to remove DMF, dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (petroleum ether/ethyl acetate 10:1-2:1) to give a light yellow solid (25 mg).

[0158] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.704 (d, J=15.4 Hz, 2H), 7.136 (dd, J=8.4, 2.0 Hz, 2H), 7.007 (d, J=2.0 Hz, 2H), 6.834 (d, J=8.3 Hz, 2H), 6.720 (d, J=15.4 Hz, 2H), 3.904 (s, 6H), 2.366-2.227 (m, 4H), 2.117-1.939 (m, 4H). MS m/z: 507.51 [M+H].sup.+.

Example 26. Synthesis of Compound 30 and Hydrochloride Thereof

##STR00053##

Step 1: Synthesis of Intermediate 30-1

[0159] Starting material D (1.0 g, 2.50 mmol, 1.0 eq), 1,5-dichloropentanone (0.391 g, 2.52 mmol), KBr (1.20 g, 10.1 mmol), K.sub.2CO.sub.3 (1.05 g, 7.57 mmol), and DMF (30 mL) were added to a reaction flask, and the resulting mixture was stirred at 50 C. for 20 h. After completion of the reaction, water (70 mL) and ethyl acetate (40 mL) were added. Liquid separation was performed. The aqueous phase was extracted with ethyl acetate (400 mL). The organic phase was washed with saturated brine to remove DMF, dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (petroleum ether/ethyl acetate 10:1-2:1) to give a yellow solid (600 mg).

Step 2: Synthesis of Compound 30 and Hydrochloride Thereof

[0160] Under nitrogen atmosphere, intermediate 30-1 (300 mg, 0.62 mmol), diethylamine (9 mg, 1.25 mmol), and tetrahydrofuran (10 mL) were added to a reaction flask. The resulting mixture was stirred at 25 C. for 30 mins, and sodium triacetoxyborohydride (270 mg, 1.25 mmol) was then added. The mixture was reacted at room temperature for 5 h. Ethyl acetate (30 mL) and diluted hydrochloric acid (0.2 N/30 mL) were added. Liquid separation was performed. The organic phase was washed with purified water, dried over sodium sulfate, and concentrated to give a yellow oily compound, which was subjected to preparative TLC (petroleum ether/ethyl acetate 1:1) to give compound 30 (MS m/z: 542.57 [M+H].sup.+) as a light yellow solid.

[0161] Compound 30, ethyl acetate (12 mL) and methanol (0.2 mL) were added to a reaction flask. The resulting mixture was stirred and dissolved at room temperature, and hydrochloric acid-ethyl acetate (0.5 N/1.2 mL) was added dropwise. The resulting mixture was filtered, and the filtrate was dried under vacuum at 40 C. to give hydrochloride of compound 30 (60 mg) as a yellow solid.

[0162] .sup.1H NMR (400 MHz, CDCl.sub.3) 11.83 (brs, 1H), 7.77-7.60 (m, 2H), 7.14 (d, J=8.2 Hz, 2H), 7.01 (dd, J=4.2, 1.8 Hz, 2H), 6.84 (dd, J=8.3, 2.7 Hz, 2H), 6.72 (d, J=15.4 Hz, 1H), 6.63 (d, J=15.4 Hz, 1H), 3.91 (s, 6H), 3.37-3.20 (m, 1H), 3.17-2.88 (m, 4H), 2.82-2.63 (m, 2H), 2.37-2.19 (m, 2H), 1.91-1.63 (m, 4H), 1.56-1.37 (m, 6H).MS m/z: 542.57 [M+H].sup.+.

Test Example 1. Cell Viability Inhibition Assay of Compounds of the Present Disclosure on Human Prostate Cancer Cells

[0163] Human prostate cancer cells LNCaP and human prostate cancer cells 22Rvl are present in patients with prostate cancer. An androgen, DHT, is able to promote the growth of human prostate cancer cells LNCaP. Using the cell model described above was intended to study the inhibitory effects of the compounds of the present disclosure on the growth of human prostate cancer cells (human prostate cancer cells LNCaP and human prostate cancer cells 22Rvl) in the presence or absence of DHT.

Assay Materials:

[0164] Test compounds (prepared by the method of the present disclosure), human prostate cancer cells LNCaP (American Type Culture Collection (ATCC), Cat No: CRL-1740), human prostate cancer cells 22Rvl (American Type Culture Collection (ATCC), Cat No: CRL-2505), RPMI 1640 medium (Invitrogen; Cat. No. 11875119), fetal bovine serum (Certified FBS Charcoal Stripped, Biolohivsl Industries, Cat. No. 04-204-1A), penicillin-streptomycin mixed solution (Solarbio; Cat No: P1400), and CellTiter-Glo (CTG) reagent (Promega, Cat #G7573).

Assay Method:

[0165] Human prostate cancer cells LNCaP and human prostate cancer cells 22Rvl in the exponential growth phase were placed under a microscope, and the growth state of the cells was observed to be good. The medium in the culture dish was discarded, and 5 mL of trypsin was added for digestion for about 3 mins. The digestion was stopped by adding 10 mL of a fresh medium. The cells were fully pipetted and transferred to a 15 mL centrifuge tube, and the cells were collected by centrifugation at 1000 rpm for 5 mins. The cells collected by centrifugation were resuspended in 11 mL of a fresh medium. 1 mL of the cells was taken and counted, and the cell viability was detected. The cell density was adjusted to 110.sup.4 cells/mL by adding an appropriate amount of the medium. The cells were seeded in a 96-well plate at 200 L/well, and the plate was incubated overnight in a cell incubator.

[0166] After the cells were attached to the well, for the human prostate cancer cells LNCaP group, test compounds (0.1 M, 0.3 M, 1 M, 3 M, 10 M, and 30 M) and DHT (1 nM, inducing the expression of AR) were added to the 96-well plate, and the plate was incubated for another 5 days; for the human prostate cancer cells 22Rvl group, test compounds (0.1 M, 0.3 M, 1 M, 3 M, 10 M, and 30 M) were added to the 96-well plate, and the plate was incubated for another 5 days. After 5 days, the 96-well plate was taken out from the incubator and observed under the microscope for no abnormality in the cell state. The 96-well plate was equilibrated at room temperature for 30 mins. The cell viability was detected by using a CTG method. Before detection, CellTiter-Glo Buffer and a substrate thereof (collectively called CTG reagent) were thawed and equilibrated to room temperature. Then, 100 mL of Buffer was gently mixed with the substrate to form a homogeneous solution. The homogeneous solution was added to the 96-well plate at 100 L/well, and the 96-well plate was incubated on a shaker for 15 mins. The fluorescence value in each well was then detected. The detection method for the fluorescence value in each well was as follows: after adding the CTG reagent, luminescence detection (integration time: 500 ms) was performed by using a correspondingly equipped multi-mode microplate reader (M200Pro, TECAN, Switzerland) according to the supplier's manual to quantify the effect of the inhibitor on the cell viability. For data analysis, an assay background value was subtracted from all data points, which was determined in wells containing the medium but no cells. Data were processed using the XLfit software (XLfit 5.2., IDBS, UK), and IC.sub.50 values of the test compounds were calculated. The IC.sub.50 values of the typical compounds of the present disclosure on human prostate cancer cells LNCaP and human prostate cancer cells 22Rvl are shown in Table 1 below.

TABLE-US-00001 TABLE 1 Inhibitory activities of compounds of the present disclosure against human prostate cancer cells LNCaP and human prostate cancer cells 22Rvl Inhibitory activity Inhibitory activity against human against human prostate cancer cell prostate cancer cells Compound LNCaP IC.sub.50 (M) 22Rvl IC.sub.50 (M) 1 1.71 2 1.82 3 1.75 4 1.77 5 5.48 6 2.56 7 0.83 8 0.61 0.35 9 3.31 10 1.71 11 0.98 12 1.29 12 hydrochloride 0.45 13 0.95 16 hydrochloride 0.37 18 hydrochloride 0.61 19 hydrochloride 0.35 22 3.14 23 hydrochloride 0.62 24 0.61 25 1.37 26 0.62
indicates that the relevant assay was not performed.
As can be seen from the assay data in the table above, the compounds of the present disclosure had good inhibitory activities against the growth of prostate cancer cells, and were able to effectively inhibit the growth of prostate cancer cells.

Test Example 2. Test of Effects of Compounds of the Present Disclosure on AR Protein Expression in Human Prostate Cancer Cells LNCaP

[0167] AR is a key factor for regulating the response of prostate cancer cells to androgens. AR is also a key factor for the response of hair follicles or tissues around the hair follicles to androgens, and the reduction of the AR content not only can down-regulate the growth of the prostate cancer cells, but also can inhibit androgenetic alopecia. This test example is to test the effects of the compounds of the present disclosure on the reduction of AR protein expression in human prostate cancer cells LNCaP in the presence of DHT. The compounds of the present disclosure were analyzed for anti-AR activity in human prostate cancer cells LNCaP by measuring the reduction of AR protein using the Western blot method.

Assay Materials:

[0168] Test compounds (prepared by the method of the present disclosure), human prostate cancer cells LNCaP (American Type Culture Collection (ATCC), Cat No: CRL-1740), RPMI 1640 medium (Invitrogen; Cat. No. 11875119), fetal bovine serum (Certified FBS Charcoal Stripped, Biolohivsl Industries, Cat. No. 04-204-1A), BCA protein quantification kit (Thermo; Cat. No. 23225), color prestained protein marker (Beyotime; Cat. No. P0069), GAPDH antibody (Millipore; Cat. No. MAB374), and AR antibody (CST; Cat. No. 5153).

Assay Method:

[0169] Human prostate cancer cells LNCaP were seeded in a 6-well plate at 410.sup.5 cells/mL and at a volume of 2 mL per well. The plate was incubated overnight in a cell incubator. DMSO and dihydrotestosterone (DHT, 1 nM) were used as control groups. Assays were performed in the presence of dihydrotestosterone (DHT, 1 nM). After incubating with the test compounds for a specified time, the cells were collected and dissolved according to the Western blot technique known in the biochemical art. A DMSO group, a DHT group (1 nM) and test compound groups (containing 1 nM DHT) were set during the assay. DMSO and DHT (1 nM DHT) were added to 6-well plates corresponding to the DMSO group and the DHT group, respectively. DHT (1 nM) and the test compounds (0.6 M, 1.2 M, 2.5 M, 5 M, and 10 M) were added to 6-well plates corresponding to the test compound groups. After cells in the control groups and the test compound groups were incubated for 48 h, the cells were collected for lysis. The protein concentration was quantified using the BCA kit, and then the samples were stored for later use.

[0170] The content of the AR protein in each group was determined by using Western Blot, and the loading amount of the sample in each group was 30 ag. Western blot analysis has been disclosed in the prior art. Specifically, the cells were collected in 2 sodium dodecyl sulfate/polyacrylamide gel electrophoresis loading buffer or in radioimmunoprecipitation assay (RIPA) buffer strengthened with 10 g/mL benzamidine, 10 g/mL trypsin inhibitor, and 1 mM phenylmethylsulfonyl fluoride. A sample of total protein (about 40 g) from each cell lysate was separated by electrophoresis on an SDS/PAGE gel. After separation by electrophoresis, the proteins were transferred from the gel to a nitrocellulose membrane following the standard procedures. The membrane was then incubated with 10% non-fat milk in phosphate-buffered saline supplemented with 0.1% Tween 20 (PBST) for 1 h, followed by incubation with a primary human AR-specific antibody (purchased from BD-Harlingen) at 4 C. overnight. After incubation, the membrane was washed 3 times with PBST buffer, each for 10 mins. An alkaline phosphatase-conjugated secondary antibody was then added, and the membrane was incubated at room temperature for 1 h. After the secondary incubation, the membrane was washed again with PBST, and the AR protein signal in the membrane was visualized by adding an alkaline phosphatase medium, bromochloroindolyl phosphate, and nitrotetrazole to the membrane. To ensure that an equal amount of protein from each sample was analyzed, a portion of the membrane was kept with a specific antibody for the regulatory protein GAPDH (Santa Cruz Biotechnology), and the GAPDH signal was visualized with the secondary antibody described above. The protein signal intensity (as indicated by color bands on the membrane) was measured using a densitometer and analyzed by the NIH Image J software (NIH1.33). The AR protein signal in each sample was normalized (relative to GAPDH), and the data were presented as the ratio of AR grey values to GAPDH grey values, as shown in FIG. 1.

[0171] The above experiments tested the ability of some compounds of the present disclosure to reduce AR expression (i.e., anti-AR activity) in human prostate cancer cells LNCaP, and the anti-AR activity of each compound was compared to the DHT and DMSO control groups at various concentrations. The relative potency of the anti-AR activity of the compounds of the present disclosure was expressed as the AR reduction percentage after the compounds were incubated at concentrations of 2.5 M, 5 M, and 10 M for 48 h (with 1 nM DHT). The evaluation index is shown in Table 2 below:

TABLE-US-00002 TABLE 2 Evaluation of relative potency Relative potency AR reduction percentage (%) + 12.5 < * 25 ++ 25 < * 37.5 +++ 37.5 < * 50 ++++ 50 < * 62.5 +++++ 62.5 < * 75 ++++++ 75 < * 100

[0172] The AR reduction percentage (%) was calculated as follows: AR reduction percentage (%)=(AR in DHT group/GAPDH valueAR in test compound group/GAPDH value)/AR in DHT group/GAPDH value100%.

[0173] The ability of the compounds of the present disclosure to reduce AR expression (i.e., anti-AR activity) in the human prostate cancer cells LNCaP is shown in Table 3. The values in Table 3 refer to AR reduction percentage (%), the concentrations in Table 3 refer to the concentrations of the test compounds, and a greater number of + in the relative potency column in Table 3 indicates better anti-AR activity of the compound.

TABLE-US-00003 TABLE 3 Ability of compounds of the present disclosure to reduce AR expression in human prostate cancer cells LNCaP Concentration Relative Compound 2.5 mol/L 5 mol/L 10 mol/L potency 7 0 67.3% 95.6% +++++ 8 57.5% 87.3% 92.2% ++++++ 10 0 35.6% 92.5% ++ 12 hydrochloride 86.2% ++++++ 16 hydrochloride 82.1% ++++++ 19 hydrochloride 81.4% ++++++
indicates the assay was not performed. Since the hydrochloride salts of compounds 12, 16 and 19 already showed excellent relative potency at a concentration of 2.5 mol/L, the relevant assay was not performed at higher concentrations.

Conclusion:

[0174] DHT could up-regulate the expression of AR protein in human prostate cancer cells LNCaP, and the compounds of the present disclosure could inhibit the expression of AR protein in human prostate cancer cells LNCaP in a dose-dependent manner, that is, the compounds of the present disclosure have anti-AR activities. In combination with the relative potency, the compounds of the present disclosure, particularly compound 7, compound 8, compound 12 hydrochloride, compound 16 hydrochloride, and compound 19 hydrochloride, have significant anti-AR activities.

Test Example 3. Pharmacokinetic Study

[0175] This test example was intended to study single oral administration of solutions of the compounds of the present disclosure to SD rats, to detect the concentration of the active ingredient in the plasma, and to evaluate their pharmacokinetic (PK) profiles in SD rats.

[0176] Experimental materials: male SD rats (weighing 180-220 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., production license No. SCXK(Beijing)2016-0006), experimental compounds (prepared according to the methods of the examples of the present disclosure), and purified water (made in-house).

[0177] Experimental method: Male SD rats were randomly grouped (3 rats per group), given ad libitum access to water during the assay, fasted for 12 or more hours before administration, and given food 4 hours after administration. These groups of SD rats were intragastrically administered, by mouth, 0.5% aqueous suspensions of the test compounds (formula comprised: 0.5% test compound, 1% CMCNa, 0.5% Tween 80, and 98% purified water) at a dose of 50 mg/kg (calculated based on the test compound).

[0178] Blood samples were collected into K.sub.2EDTA anticoagulation tubes at 0 min before administration and at 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, and 12 h after administration, and then were temporarily placed on ice before centrifugation.

[0179] The blood was centrifuged within 60 min of being collected to isolate plasma (centrifuged at 8000 rpm at 2-8 C. for 5 min). After the centrifugation, the plasma was transferred to a 96-well plate or centrifuge tubes, transported in a box with ice, and stored at 15 C. before LC-MS/MS analysis. The drug concentration in the plasma of SD rats was measured by LC-MS/MS bioanalysis, and the plasma concentration-time data were analyzed using WinNonlin (Version 8.3, Certara, USA) and a non-compartmental model to evaluate the pharmacokinetic (PK) profiles of the compounds in SD rats. The data are shown in Table 4, and the pharmacokinetic curves are shown in FIG. 2.

TABLE-US-00004 TABLE 4 Pharmacokinetic parameters of compounds of the present disclosure in plasma of male rats after oral administration of compounds of the present disclosure Compound Parameter Compound 8 Compound 7 Compound 10 T.sub.1/2 (h) 1.57 2.95 3.80 T.sub.max (h) 0.33 0.19 0.25 C.sub.max (ng/ml) 282.20 297.87 44.01 AUC.sub.last (h * ng/ml) 1013.88 460.22 92.47

[0180] The plasma concentration-time curves in FIG. 2 show that compound 10 had the lowest C.sub.max and the lowest AUC.sub.last in rats; compound 7 and compound 8 had similar C.sub.max, but compound 8 had higher AUC.sub.last than that of compound 7. These results indicate that after the compounds were intragastrically administered to SD rats at the same dose, compound 10 likely exerted the worst efficacy due to the lowest exposure in the rats, followed by compound 7, and compound 8 likely exerted the best efficacy due to the significantly increased C.sub.max and AUC.sub.last.

[0181] The results described above indicate that the compounds of the present disclosure had significantly improved pharmacokinetic properties. Particularly, AUC and C.sub.max were both very significantly improved after administration of the compounds of the present disclosure. Therefore, the compounds of the present disclosure have good druggability and are able to obtain better efficacy at a lower administration dose.

Test Example 4. Hair Growth Promotion Test of Compounds of the Present Disclosure in Mice with Alopecia

Objective:

[0182] The compounds of the present disclosure were tested for their hair growth-promoting effects on an alopecia mouse model.

Test Materials:

[0183] C57BL/6 mice (purchased from SPF (Beijing) Biotechnology Co., Ltd., production license No.: SCXK (Beijing) 2019-0010), test compounds (prepared by the method of the present disclosure), rosin (Shanghai YuanYe Bio-Technology Co., Ltd., lot No. Y18M10C83144), liquid paraffin (Shanghai YuanYe Bio-Technology Co., Ltd., lot No.: Z22S11Y125555), chloral hydrate (Sinopharm Chemical Reagent Co., Ltd., lot No.: 20190823, a 2% chloral hydrate solution was prepared by using a 0.9% sodium chloride solution before use), testosterone propionate (Aladdin Reagent (Shanghai) Co., Ltd., lot No.: T101368, a 0.5% testosterone propionate solution was prepared by using an a soybean oil for injection before use), soybean oil for injection (Liaoning Shinsun Pharmaceutical Co., Ltd., lot No.: NMPA Approval No. H21024303), paraformaldehyde (Xilong Scientific Co., Ltd., lot No.: 1705022), a 0.9% sodium chloride solution (Jiangsu Huai'an Shuanghe Pharmaceutical Co., Ltd., lot No.: 210322-3C), an electronic balance (Beijing Sartorius Scientific Instruments Co., Ltd., model: BS224 S; GZX-9140MBE), and a digital display electric heating constant temperature air blast drying oven (Shanghai Boxun Medical Biological Instrument Corp.).

Test Method:

[0184] 8-week-old male C57BL/6 mice were selected and injected with 2% chloral hydrate (400 mg/kg) for anesthesia. Equal amounts of rosin and paraffin were weighed out, heated, and mixed well. The mixture was applied on the front back of the mice in an area of about 2 cm2.5 cm. After the mixture was cooled to be hard, the solidified hair was torn off by tweezers gently. Animals found to have a dark skin color after hair removal, indicating that they were in the anagen phase with active hair follicle growth, were excluded from the study.

[0185] Qualified C57BL/6 mice (3 mice per group) were randomly assigned to a blank group, a model group, a positive control drug group (7.5% clascoterone group), and test groups (including 0.3% compound 8 group and 0.3% compound 7 group). Mice in the blank group were coated with 0.1 mL of normal saline in the depilated area every morning (1 time a day for 17 consecutive days); mice in the model group were coated with 0.1 mL of 0.5% testosterone propionate solution in the depilated area every morning (1 time a day, starting on the first day, for 17 consecutive days), and with 0.5 mL of the control solvent in the depilated area every afternoon (1 time a day, starting on the second day, for 16 consecutive days); mice in the positive control drug group were coated with 0.1 mL of 0.5% testosterone propionate solution in the depilated area every morning (1 time a day, starting on the first day, for 17 consecutive days), and with 0.5 mL of 7.5% clascoterone solution in the depilated area every afternoon (1 time a day, starting on the second day, for 16 consecutive days); and mice in the test groups were coated with 0.1 mL of 0.5% testosterone propionate solution in the depilated area every morning (1 time a day, starting on the first day, for 17 consecutive days), and with 0.5 mL of 0.3% test compound solution in the depilated area every afternoon (1 time a day, starting on the second day, for 16 consecutive days). The formula of the test compound solution was 0.3% test compound, 40% DMSO, 30% ethanol, and the balance of water; the formula of the control solvent was 40% DMSO, 30% ethanol, and the balance of water; the formula of the positive control drug group was 7.5% clascoterone, 40% DMSO, 30% ethanol, and the balance of water. The hair regrowth in the depilated area of the mice in each group was observed and photographed 16 days after the start of topical administration of the test compound solution.

[0186] On day 18 of the test, 5 new hairs were pulled out from the back depilated area of each mouse in each group, the lengths of the hairs were measured using a vernier caliper, and the lengths of the hairs of the mice in each group were counted. The skin of the depilated area of the mice in each group was cut out, and hematoxylin-eosin staining (HE staining) was performed. The morphology of the hair follicles was observed under a microscope. 6 fields (100) were randomly selected for each sample to be observed, and the average number of hair follicles in each field was calculated. The software used for data statistics was Graph Pad 8.0, and the data of the groups were expressed as meanstandard deviation (XSD), as shown in Table 5 below. Photographs of hair growth and representative HE-stained sections of the skin tissues from representative mice in each group on day 18 are shown in FIG. 3.

TABLE-US-00005 TABLE 5 Hair length and number of hair follicles for different groups (expressed as X SD) Number of hair Daily dose of Hair length follicles Group administration (X SD, mm) (X SD) Blank group 0 6.035 0.038 110.00 41.867 Model group 0 0.948 0.090 18.20 2.615 Positive control 37.5 mg of 5.649 0.059 53.533 24.929 drug group clascoterone Compound 7 1.5 mg of 3.850 0.009 95.600 26.091 group compound 7 Compound 8 1.5 mg of 4.493 0.024 116.733 35.809 group compound 8

[0187] FIG. 3 shows that compared to the blank group, the mice in the model group had poor hair growth and significantly fewer hair follicles than the blank group, indicating that coating 0.5% testosterone propionate solution may result in an alopecia mouse model; compared to the model group, the mice in the positive control drug group had significantly grown hairs and the number of hair follicles was significantly increased, indicating that the alopecia mouse model is reliable; under the condition that the daily dose of administration of compound 7 and compound 8 was 1.5 mg, the growth of hairs and hair follicles was better than that of the positive control drug group, indicating that the compounds of the present disclosure at a low dose have better alopecia treatment effects than the positive control drug at a high dose.

[0188] As can be seen from the above test data, the compounds of the present disclosure can significantly increase the hair length and the number of hair follicles of mice in the test groups at a relatively small dose of administration, can promote the generation of the hair follicles and the growth of the hair in the mice with alopecia, and have good hair growth-promoting effects.

[0189] The embodiments of the present disclosure have been described above. However, the present disclosure is not limited to the embodiments described above. Any modification, equivalent replacement, improvement, and the like made without departing from the spirit and principle of the present disclosure shall fall within the protection scope of the present disclosure.