Compounds for the treatment or prevention of breast cancer

Abstract

It discloses compounds for the treatment and prevention of breast cancer, which are specifically 2-phenyl benzoselenazole compounds, pharmaceutically acceptable salts thereof and prodrugs thereof. The present invention further discloses pharmaceutical compositions containing the compounds and applications of the compounds in preparing medicines for the treatment and prevention of breast cancer in mammals. The compounds of the present invention can effectively inhibit or reduce the growth or proliferation of breast cancer cells in mammals, with no inhibition effect on the growth of part of the tested cell lines except for the breast cancer cell lines, and are highly selective.

Claims

1. A compound having general formula (II) shown as following, or its pharmaceutically acceptable salts thereof or prodrugs thereof: ##STR00024## wherein: R.sup.1 and R.sup.2 are independently selected from a group consisting of H, D, halogen, CN, C.sub.1-3 alkyl, substituted C.sub.1-3 alkyl, C.sub.1-3 alkoxy or substituted C.sub.1-3 alkoxy, and the substituent group is selected from D, halogen and C.sub.1-3 alkoxy; R.sup.3 is selected from a group consisting of halogen, OH, CN, NH.sub.2, substituted NH.sub.2, C.sub.1-3 alkyl, substituted C.sub.1-3 alkyl, C.sub.1-3 alkoxy or substituted C.sub.1-3 alkoxy, and the substituent group is selected from D, halogen, C.sub.1-3 alkyl and C.sub.1-3 alkoxy; and R.sup.4 is selected from a group consisting of H, D, halogen, OH, CN, NH.sub.2, substituted NH.sub.2, C.sub.1-3 alkyl, substituted C.sub.1-3 alkyl, C.sub.1-3 alkoxy or substituted C.sub.1-3 alkoxy, and the substituent group is selected from D, halogen, C.sub.1-3 alkyl and C.sub.1-3 alkoxy.

2. The compound according to claim 1, wherein: R.sup.1 and R.sup.2 are independently selected from a group consisting of H, D, halogen, CN, C.sub.1-3 alkyl, substituted C.sub.1-3 alkyl, C.sub.1-3 alkoxy or substituted C.sub.1-3 alkoxy, and the substituent group is selected from D, F and C.sub.1-3 alkoxy; R.sup.3 is selected from a group consisting of halogen, OH, CN, NH.sub.2, substituted NH.sub.2, C.sub.1-3 alkyl, substituted C.sub.1-3 alkyl, C.sub.1-3 alkoxy or substituted C.sub.1-3 alkoxy, and the substituent group is selected from D, F, C.sub.1-3 alkyl and C.sub.1-3 alkoxy; and R.sup.4 is selected from a group consisting of H, D, halogen, OH, CN, NH.sub.2, substituted NH.sub.2, C.sub.1-3 alkyl, substituted C.sub.1-3 alkyl, C.sub.1-3 alkoxy or substituted C.sub.1-3 alkoxy, and the substituent group is selected from D, F, C.sub.1-3 alkyl and C.sub.1-3 alkoxy.

3. The compound according to claim 1, wherein R.sup.1 and R.sup.2 are independently selected from a group consisting of H, D, F, CI, CN, CH.sub.3, CF.sub.3, OCH.sub.3, OCH.sub.2CH.sub.3, OCF.sub.3 and OCHF.sub.2.

4. The compound according to claim 1, wherein R.sup.3 is selected from a group consisting of halogen, OH, CN, NH.sub.2, CH.sub.3, CH.sub.2CH.sub.3, CF.sub.3, OCH.sub.3, OCH.sub.2CH.sub.3, OCHF.sub.2 or OCF.sub.3, and R.sup.4 is selected from H, D, halogen, OH, CN, NH.sub.2, CH.sub.3, CH.sub.2CH.sub.3CF.sub.3, OCH.sub.3, OCH.sub.2CH.sub.3,OCHF.sub.2 and OCF.sub.3.

5. The compound according to claim 1, wherein R.sup.1 and R.sup.2 are independently selected from a group consisting of H, D, F, CI, CN, CH.sub.3, CF.sub.3 or CHF.sub.2; R.sup.3 is selected from F, CI, Br, I, CN, CH.sub.3, CF.sub.3, OCH.sub.3, OCH.sub.2CH.sub.3, OCHF.sub.2 or OCF.sub.3; and, R.sup.4 is selected from H, D, F, CI, Br, I, CN, CH.sub.3, CF.sub.3, OCH.sub.3, OCH.sub.2CH.sub.3, OCHF.sub.2 and OCF.sub.3.

6. The compound according to claim 1, wherein the compounds are selected from a group consisting of 4-(benzoselenazole-2-yl)-2-bromoaniline, 2-bromo-4-(5-fluorobenzoselenazole-2-yl)aniline, 4-(5-fluorobenzoselenazole-2-yl)-2-methylaniline, 2-bromo-4-(5-fluorobenzoselenazole-2-yl)-6-methylaniline, 4-(5-bromobenzoselenazole-2-yl)-2-chloroaniline, 2-methyl-4-(5-methylbenzoselenazole-2-yl)aniline, 2-methyl-4[5-(trifluoromethyl)benzoselenazole-2-yl]aniline, 4-(6-ethoxybenzoselenazole-2-yl)-2-methylaniline, 4-(6-ethoxy-5-fluorobenzoselenazole-2-yl)-2-methylaniline, 2-fluoro-4-(5-fluorobenzoselenazole-2-yl)aniline, 2-bromo-6-fluoro-4-(5-fluorobenzoselenazole-2-yl)aniline, 5-(5-fluorobenzoselenazole-2-yl)-2-methylaniline, 4-(5-deuterobenzoselenazole-2-yl)-2-methylaniline, 2,6-difluoro-4-(5-fluorobenzoselenazole-2-yl)aniline, and 2-fluoro-4-(5-fluorobenzoselenazole-2-yl)-6-methylaniline.

7. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 1, or its pharmaceutically acceptable salts thereof or prodrugs thereof, and pharmaceutically acceptable adjuvants.

8. A process for treating breast cancer comprising administering to a subject in need a therapeutically effective amount of the compound of claim 1, or its pharmaceutically acceptable salts thereof or prodrugs thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) To make the objectives, technical schemes and advantages of the present invention clearer, the present invention will be further described below in details by specific implementations. It should be understood that the descriptions are merely exemplary and not intended to limit the scope of the present invention.

(2) Synthesis Embodiments

(3) Embodiment 1:

Synthesis of 4-(benzoselenazole-2-yl)-2-bromoaniline (7)

(4) ##STR00006##

(5) Step A: A solution of 2-nitroaniline(15.0 g, 108 mmol) in dichloromethane (150 mL) was added dropwise into boron trifluoride diethyl etherate (23.1 g, 163 mmol) at 10 to 15 and stirred for 15 min, and a solution of isoamyl nitrite (15.26 g, 130 mmol) in dichloromethane (75 mL) was added dropwise at this temperature. At the end of addition, the reaction mixture was continuously stirred for 30 minutes, and then stirred for 30 minutes at 10 to 0. The reaction system was added with cold petroleum ether (250 mL) dropwise and then filtered. The filter cake was washed with cold methyl tert-butyl ether (MTBE) (40 mL) to obtain 2-nitro-phenyl-diazonium tetrafluoroborate (1) (18.7 g). The yield was 73.1%.

(6) Step B: A solution of potassium selenocyanate (8.0 g, 55.5 mmol) in water (80 mL) was added into a mixture of the compound 1 (13.0 g, 54. 9 mmol) and water (300 mL) in an ice-water bath, and the reaction mixture was continuously stirred for 30 min at the end of addition. The reaction mixture was filtered, and the filter cake was washed with a small amount of water and then dried in vacuum at 60 to obtain 1-nitro-2-phenyl selenocyanate (2) (11.2 g). The yield was 89.8%.

(7) Step C: Sodium (6.0 g, 261 mmol) was added into a mixture of the compound 2 (10.5 g, 46.2 mol) and absolute ethyl alcohol (520 mL) at the room temperature, and the mixture was stirred for 1 h in a water bath. The reaction mixture was cooled to 0 to 5 and filtered, the filter cake was washed with a small amount of cold ethanol, and the collected solid was suspended in methylbenzene (100 mL), heated to reflux to dissolve the product, and filtered immediately. The filtrate was cooled to 0 to 5 to separate out solid and then filtered, and the filter cake was collected to obtain 1,2-di(2-nitrophenyl) diselenide (3) (4.5 g). The yield was 48.4%.

(8) Step D: Zinc powder (13.5 g, 206 mmol) was added into a suspension solution of the compound 3 (4.5 g, 11.2 mmol) in acetic acid (90 mL) at 40, and the reaction mixture was heated to 100 and continuously stirred for 2 h. The reaction mixture was cooled below 50, slowly added with 6M hydrochloric acid (40 mL) dropwise and filtered to remove insoluble substances. The filtrate was adjusted with 20% sodium acetate aqueous solution until the pH value was 2 to 3, and the solid was separated out. The reaction mixture was filtered, and the filter cake was dried to obtain di[(2-aminophenyl)seleno]zinc (4) (3.0 g). The yield was 77.8%.

(9) Step E: A mixture of the compound 4 (3.6 g, 17.4 mmol) and 4-nitro-benzoyl chloride (4.77 g, 25.7 mmol) was stirred for 2 h at 110. The mixture was cooled to the room temperature, added with saturated sodium bicarbonate aqueous solution (60 mL) and extracted with ethyl acetate (50 mL3), and the combined organic phase was washed with saturated saline solution (20 mL) and dried with anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by chromatography (200 to 300 meshes of silica gel, elution with ethyl acetate: petroleum ether=1:5 to 3:1) to obtain 2-(4-nitrophenyl)benzoselenazole (5) (500 mg). The yield was 6.42%. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) 8.40 (dd, J=2.0, 6.8 Hz, 2H), 8.21 (d, J=8.8 Hz, 2H), 7.76-7.74 (m, 1H), 7.36-7.33 (m, 2H), 7.27-7.23 (m, 1H).

(10) Step F: The compound 5 (450 mg, 1.48 mmol) was dissolved into ethanol (10 mL) and then added with 2M hydrochloric acid (15 mL) and tin powder (2.25 g, 6.74 mmol), and the mixture was refluxed and stirred for 2 h. Most of the solvent was evaporated under reduced pressure, then added with water (15 mL), adjusted with dilute sodium hydroxide solution until the pH value was 9 to 10, extracted with dichloromethane (20 mL3), and dried with anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by chromatography (200 to 300 meshes of silica gel, elution with ethyl acetate: petroleum ether=1:15 to 1:4) to obtain 4-(benzoselenazole-2-yl)aniline (6) (300 mg). The yield was 74.2%. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) 8.07 (dd, J=0.8, 8.0 Hz, 1H), 7.89 (dd, J=0.8, 8.0 Hz, 1H), 7.72-7.69 (m, 2H), 7.46-7.42 (m, 1H), 7.28-7.23 (m, 1H), 6.66-6.63 (m, 2H), 5.92 (s, 2H).

(11) Step G: A solution of NBS (117 mg, 0.657 mmol) in dichloromethane (15 mL) was added dropwise into a solution of the compound 6 (200 mg, 0.732 mmol) in dichloromethane (5 mL) at 10, and the mixture was continuously stirred for 0.5 h at this temperature at the end of addition. The reaction mixture was washed with water (10 mL) and dried with anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by chromatography (200 to 300 meshes of silica gel, elution with ethyl acetate: petroleum ether=1:10) to obtain 4-(benzoselenazole-2-yl)-2-bromoaniline (7) (179 mg). The yield was 69.5%. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) 8.10 (dd, J=1.0, 8.0 Hz, 1H), 8.02 (d, J=1.0 Hz, 1H), 7.94 (dd, J=1.0, 8.0 Hz, 1H), 7.72 (dd, J=1.0, 8.0 Hz, 1H), 7.49-7.45 (m, 1H), 7.31-7.27 (m, 1H), 6.88 (d, J=8.4 Hz, 1H), 6.11 (s, 2H). MS (El, m/z):350.9 [MH].sup..

(12) Embodiment 2:

Synthesis of 4-(5-fluorobenzoselenazole-2-yl)aniline (13) and 2-bromo-4-(5-fluorobenzoselenazole-2-yl)aniline (14)

(13) ##STR00007##

(14) Step A: A solution of 2-nitro-4-fluoroaniline (5.0 g, 32.0 mmol) in dichloromethane (50 mL) was added dropwise into boron trifluoride diethyl etherate (6.8 g, 47.9 mmol) at 10 to 15 and stirred for 15 min, and a solution of isoamyl nitrite (4.5 g, 38.4 mmol) in dichloromethane (25 mL) was added dropwise at this temperature. At the end of addition, the mixture was continuously stirred for 30 minutes, and then stirred for 30 minutes at 10 to 0. The reaction mixture was added with cooled petroleum ether (80 mL) dropwise and then filtered. The filter cake was washed with cold MTBE (10 mL) to obtain 2-nitro-4-fluorophenyl-diazonium tetrafluoroborate (8) (13.0 g). The compound was directly used in the next reaction without purification.

(15) Step B: A solution of potassium selenocyanate (4.84 g, 33.6 mmol) in water (30 mL) was added into a mixture of the crude compound 8 (13.0 g) and water (170 mL) in an ice-water bath, and the reaction mixture was continuously stirred for 20 min at the end of addition. The reaction mixture was filtered, and the filter cake was washed with a small amount of water and then dried in vacuum at 60 to obtain 4-fluoro-2-nitro-1-phenyl selenocyanate (9) (9.1 g). The compound was directly used in the next reaction without purification.

(16) Step C: Sodium (4.1 g, 178 mmol) was added into a mixture of the crude compound 9 (9.1 g) and absolute ethyl alcohol (300 mL) at the room temperature, and the mixture was stirred for 1 h in a water bath. The mixture was cooled to 0 to 5 and filtered, and the filter cake was washed with a small amount of cold ethanol. The collected solid was suspended in methylbenzene (80 mL), heated to reflux to dissolve the product, and filtered immediately. The filtrate was cooled to 0 to 5 to separate out solid and then filtered, and the filter cake was collected to obtain 1,2-di(4-fluoro-2-nitrophenyl)diselenide (10) (2.0 g). The total yield of the reactions in the steps A, B and C was 21.9%.

(17) Step D: The compound 10 (1.49 g, 3.4 mmol) and raney nickel (1.2 g) were suspended in isopropanol (30 mL), 85% hydrazine hydrate (1.8 mL) was added, and the mixture was heated to reflux and continuously stirred for 2.5 h. The mixture was immediately filtered through a celite pad, the filter cake was washed with a small amount of isopropanol, and the filtrate was collected. The solvent was evaporated under reduced pressure, and the product was purified by chromatography (200 to 300 meshes of silica gel, elution with ethyl acetate: petroleum ether=1:10 to 1:5) to obtain 6,6-diselenodi(3-fluoroaniline) (11) (370 mg). The yield was 28.8%.

(18) Step E: A mixture containing the compound 11 (370 mg, 0.978 mmol), 4-nitrobenzaldehyde (296 mg, 1.958 mmol), sodium metabisulfite (372 mg, 1.956 mmol) and anhydrous DMSO (15 mL) was stirred for 72 h under nitrogen at 120. The mixture was cooled to the room temperature, added with saturated ammonium chloride aqueous solution (60 mL), extracted with ethyl acetate (25 mL3), and dried with anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified under reduced pressure by chromatography (200 to 300 meshes of silica gel, elution with ethyl acetate: petroleum ether=1:50) to obtain 5-fluoro-2-(4-nitrophenyl)benzoselenazole (12) (76 mg). The yield was 12.1%. .sup.1H NMR (DMSO-d.sub.6, 300 MHz) 8.39-8.28 (m, 5H), 8.01 (dd, J=2.4, 9.9 Hz, 1H), 7.41-7.34 (m, 1H).

(19) Step F: The compound 12 (70 mg, 0.218 mmol) was dissolved into ethanol (5 mL) and then added with stannous chloride hydrate (246 mg, 1.09 mmol), and the mixture was refluxed and stirred for 5 h. The mixture was cooled to the room temperature, added with saturated saline solution (20 mL), extracted with ethyl acetate (25 mL3), and dried with anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by chromatography (200 to 300 meshes of silica gel, elution with ethyl acetate: petroleum ether=1:15 to 1:5) to obtain 4-(5-fluorobenzoselenazole-2-yl)aniline (13) (50 mg). The yield was 78.8%, MS (El, m/z):291.0 [MH].sup..

(20) Step G: A solution of NBS (29.6 mg, 0.166 mmol) in dichloromethane (40 mL) was added dropwise into a solution of the compound 13 (44 mg, 0.151 mmol) in dichloromethane (40 mL) in an ice-salt bath, and the mixture was continuously stirred for 10 min at this temperature at the end of addition. The reaction mixture was washed with water (20 mL2) and dried with anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by chromatography (200 to 300 meshes of silica gel, elution with ethyl acetate: petroleum ether=1:20) to obtain 2-bromo-4-(5-fluorobenzoselenazole-2-yl)aniline (14). 1H NMR (DMSO-d6, 300 MHz) 8.14-8.09 (m, 1H), 8.03 (d, J=1.8 Hz, 1H), 7.78-7.70 (m, 2H), 7.23-7.16 (m, 1H), 6.87 (d, J=8.4 Hz, 1H), 6.14 (s, 2H). MS (El, m/z):369.0 [MH].sup..

(21) Embodiment 3:

Synthesis of 4-(5-fluorobenzoselenazole-2-yl)-2-methylaniline (17)

(22) ##STR00008##

(23) Step A: Zinc powder (5.5 g, 84.1 mmol) was added into a suspension of the compound 10 (2.0 g, 4.57 mmol) in acetic acid (40 mL) at 40, then heated to 100 and continuously stirred for 3 h. The reaction mixture was cooled below 50, slowly added with 6M hydrochloric acid (40 mL) and filtered to remove insoluble substances. The filtrate was adjusted with 20% sodium acetate until the pH value was 2 to 3, and the solid was collected by filtration and dried to obtain di[(2-amino-4-fluorophenyl)seleno]zinc (15) (1.2 g). The yield was 61.9%.

(24) Step B: A mixture of the compound 15 (590 mg, 3.10 mmol) and 3-methyl-4-nitrobenzoyl chloride (663 mg, 3.32 mmol) was stirred for 4 h at 100. The mixture was cooled to the room temperature, added with saturated sodium bicarbonate aqueous solution (15 mL) and extracted with ethyl acetate (30 mL2). The combined organic phase was washed with saturated saline solution (10 mL) and dried with anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by chromatography (200 to 300 meshes of silica gel, elution with ethyl acetate: petroleum ether=1:15 to 1:1) to obtain 5-fluoro-2-(3-methyl-4-nitrophenyl)benzoselenazole (16) (750 mg). The yield was 72.2%. .sup.1H NMR (DMSO-d.sub.6, 300 MHz) 8.32-8.27 (m, 1H), 8.19-8.13 (m, 3H), 7.98 (dd, J=2.4, 9.9 Hz, 1H), 7.40-7.33 (m, 1H), 2.64 (s, 3H).

(25) Step C: The compound 16 (200 mg, 0.597 mmol) was dissolved into ethanol (5 mL) and then added with 3M hydrochloric acid (4 mL) and tin powder (800 mg, 6.74 mmol), and the mixture was refluxed and stirred for 1.5 h. Most of the solvent was evaporated under reduced pressure, then added with water (15 mL), adjusted with dilute sodium hydroxide solution until the pH value was 9 to 10, extracted with ethyl acetate (20 mL2), and dried with anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by chromatography (200 to 300 meshes of silica gel, elution with ethyl acetate: petroleum ether=1:15 to 1:4) to obtain 4-(5-fluorobenzoselenazole-2-yl)-2-methylaniline (17) (103 mg). The yield was 56.4%. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) 8.08-8.05 (m, 1H), 7.72-7.69 (m, 1H), 7.62-7.57 (m, 2H), 7.18-7.13 (m, 1H), 6.68 (d, J=8.4 Hz, 1H), 5.75 (s, 2H), 2.14 (s, 3H). MS (El, m/z):305.0 [MH].sup..

(26) Embodiment 4:

Synthesis of 2-bromo-4-(5-fluorobenzoselenazole-2-yl)-6-methylaniline (18)

(27) ##STR00009##

(28) NBS (57 mg, 0.320 mmol) was added into a solution of the compound 17 (88 mg, 0.229 mmol) in DMF (5 mL) at the room temperature, and the mixture was continuously stirred for 20 min at this temperature at the end of addition. The mixture was added with water (25 mL) and extracted with ethyl acetate (20 mL2), and the combined organic phase was washed with saturated sodium bicarbonate aqueous solution (10 mL) and dried with anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by chromatography (200 to 300 meshes of silica gel, elution with ethyl acetate: petroleum ether=1:5) to obtain 2-bromo-4-(5-fluorobenzoselenazole-2-yl)-6-methylaniline (18) (73 mg). The yield was 83.0%. .sup.1H NMR (DMSO-d.sub.6, 500 MHz) 8.13-8.10 (m, 1H), 7.92 (d, J=2.0 Hz, 1H), 7.77-7.74 (m, 1H), 7.64 (s, 1H), 7.21-7.17 (m, 1H), 5.76 (s, 2H), 2.26 (s, 3H). MS (El, m/z):384.9 [M+H].sup.+.

(29) Embodiment 5:

Synthesis of 4-(5-bromobenzoselenazole-2-yl)-2-chloroaniline (25)

(30) ##STR00010##

(31) The steps A, B and C referred to the steps A, B and C in Embodiment 2.

(32) Step D: The compound 21 (1.9 g, 3.393 mmol) was dissolved into ethanol (40 mL) and then added with stannous chloride hydrate (3.8 g, 16.84 mmol), and the mixture was refluxed and stirred for 3.5 h under nitrogen. The mixture was cooled to the room temperature, added with saturated saline solution (20 mL), extracted with ethyl acetate (25 mL3), and dried with anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by chromatography (200 to 300 meshes of silica gel, elution with ethyl acetate: petroleum ether=1:10) to obtain 6,6-diselenodi(3-bromoaniline) (22) (450 mg). The yield was 26.5%.

(33) Step E: Referring to the step E in Embodiment 2, to get 5-bromo-2-(4-nitrophenyl)benzoselenazole (23). .sup.1H NMR (DMSO-d.sub.6, 300 MHz) 8.39-8.30 (m, 5H), 8.23 (d, J=9.0 Hz, 1H), 7.61 (d, J=9.0 Hz, 1H).

(34) Steps F and G: Referring to the step F in Embodiment 2 to get compound 24. The compound 23 was reduced to obtain a compound 24. The compound 24 (80 mg, 0.227 mmol) was dissolved into DMF (5 mL) and added with NCS (34 mg, 0.255 mmol), and the mixture was stirred overnight at the room temperature. The mixture was added with water (25 mL) and extracted with ethyl acetate (15 mL3), and the combined organic phase was washed with saturated sodium bicarbonate aqueous solution (10 mL) and dried with anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by chromatography (200 to 300 meshes of silica gel, elution with ethyl acetate: petroleum ether=1:15) to obtain 4-(5-bromobenzoselenazole-2-yl)-2-chloroaniline (25). .sup.1H NMR (DMSO-d.sub.6, 300 MHz) 8.10-8.04 (m, 2H), 7.87 (s, 1H), 7.68 (d, J=9.0 Hz, 1H), 7.43 (d, J=9.0 Hz, 1H), 6.88 (d, J=9.0 Hz, 1H), 6.16 (s, 2H). MS (El, m/z):384.9 [MH].sup..

(35) Embodiment 6:

Synthesis of 2-methyl-4-(5-methylbenzoselenazole-2-yl)aniline (26)

(36) ##STR00011##

(37) The method for preparing the compound 26 refers to the steps A, B, C and D in Embodiment 2 and the steps E and F in Embodiment 5. 2-Nitro-4-fluoroaniline in the step A of Embodiment 2 was replaced with 2-nitro-4-methylaniline, and 4-nitrobenzaldehyde in the step E of Embodiment 5 was replaced with 3-methyl-4-nitrobenzaldehyde. .sup.1H NMR (DMSO-d.sub.6, 500 MHz) 7.89 (d, J=8.0 Hz, 1H), 7.71 (s, 1H), 7.59-7.54 (m, 2H), 7.08 (d, J=8.0 Hz, 1H), 6.67 (d, J=8.0 Hz, 1H), 5.62 (s, 2H), 2.41 (s, 3H), 2.14 (s, 3H). MS (El, m/z):301.0 [MH].sup..

(38) Embodiment 7:

Synthesis of 2-methyl-4-[5-(trifluoromethyl)benzoselenazole-2-yl]aniline (27)

(39) ##STR00012##

(40) The method for preparing the compound 27 refers to the steps A, B, C and D in Embodiment 2 and the steps E and F in Embodiment 5. 2-nitro-4-fluoroaniline in the step A of Embodiment 2 was replaced with 2-nitro-4-trifluoromethylaniline, and 4-nitrobenzaldehyde in the step E of Embodiment 5 was replaced with 3-methyl-4-nitrobenzaldehyde. .sup.1H NMR (DMSO-d.sub.6, 500 MHz) 8.31 (d, J=8.0 Hz, 1H), 8.16 (s, 1H), 7.65-7.62 (m, 2H), 7.55 (d, J=8.5 Hz, 1H), 6.70 (d, J=8.5 Hz, 1H), 5.77 (s, 2H), 2.15 (s, 3H). MS (El, m/z):355.0 [MH].sup..

(41) Embodiment 8:

Synthesis of 2-(3,4-dimethoxy-phenyl)-5-fluoro-benzoselenazole (28)

(42) ##STR00013##

(43) The method for preparing the compound 28 refers to the step B in Embodiment 3, wherein acyl chloride in the reaction equation was prepared by reacting a corresponding acid with thionyl chloride. MS (El, m/z):338.1 [M+H].sup.+.

(44) Embodiment 9:

Synthesis of 4-(6-ethoxybenzoselenazole-2-yl)-2-methylaniline (29)

(45) ##STR00014##

(46) The method for preparing the compound 29 refers to the steps A, B, C and D in Embodiment 2 and the steps E and F in Embodiment 5. 2-nitro-4-fluoroaniline in the step A was replaced with 2-nitro-5-fluoroaniline, and 3-methyl-4-nitrobenzaldehyde in the step E of Embodiment 5 was replaced with 3-methyl-4-nitrobenzaldehyde. In the reduction reactions in the steps C and F, the solvent was ethanol so that F at the sixth site of benzaldehyde was substituted with ethoxy. The final resulting product was 4-(6-ethoxybenzoselenazole-2-yl)-2-methylaniline (29). .sup.1H NMR (DMSO-d.sub.6, 300 MHz) 7.76 (d, J=8.7 Hz, 1H), 7.63 (s, 1H), 7.55-7.50 (m, 2H), 6.99 (d, J=8.1 Hz, 1H), 6.66 (d, J=8.1 Hz, 1H), 5.58 (s, 2H), 4.09 (q, J=6.6 Hz, 2H), 2.13 (s, 3H), 1.35 (t, J=6.6 Hz, 3H). MS (El, m/z):333.0 [M+H].sup.+.

(47) Embodiment 10:

Synthesis of 4-(6-ethoxy-5-fluorobenzoselenazole-2-yl)-2-methylaniline (30)

(48) ##STR00015##

(49) The method for preparing the compound 30 refers to the steps A, B, C and D in Embodiment 2 and the steps E and F in Embodiment 5. 2-nitro-4-fluoroaniline in the step A of Embodiment 2 was replaced with 2-nitro-4,5-difluoroaniline, and 4-nitrobenzaldehyde in the step E of Embodiment 5 was replaced with 3-methyl-4-nitrobenzaldehyde. In the reduction reactions in the steps C and F, the solvent was ethanol so that F at the sixth site of benzaldehyde was substituted with ethoxy. The final resulting product was 4-(6-ethoxy-5-fluorobenzoselenazole-2-yl)-2-methylaniline (30). .sup.1H NMR (DMSO-d.sub.6, 300 MHz) 7.85 (d, J=8.4 Hz, 1H), 7.72 (d, J=12.0 Hz, 1H), 7.55-7.50 (m, 2H), 6.66 (d, J=8.1 Hz, 1H), 5.61 (s, 2H), 4.16 (q, J=6.9 Hz, 2H), 2.13 (s, 3H), 1.38 (t, J=6.9 Hz, 3H). MS (El, m/z):351.0 [M+H].sup.+.

(50) Embodiment 11:

Synthesis of 5-(benzoselenazole-2-yl)-2-methoxyphenol (32)

(51) ##STR00016##

(52) According to the step D in Embodiment 5 and the step E in Embodiment 2, compound 32 was synthesized as the aimed product while using compound 3 and 4-nitrobenzaldehyde as the starting materials. The compound 3 was used as raw material, and the method for preparing the compound 32 refers to the step D in Embodiment 5 and the step E in Embodiment 2. 4-nitrobenzaldehyde in the step E of Embodiment 2 was replaced with 3-hydroxyl-4-methoxybenzaldehyde. .sup.1H NMR (DMSO-d.sub.6, 300 MHz) 9.55 (s, 1H), 8.13 (d, J=7.5 Hz, 1H), 7.99 (d, J=7.5 Hz, 1H), 7.51-7.44 (m, 3H), 7.36-7.30 (m, 1H), 7.06 (d, J=8.4 Hz, 1H), 3.85 (s, 3H). MS (El, m/z):306.0 [M+H].sup.+.

(53) Embodiment 12:

Synthesis of 2-(3,4-dimethoxyphenyl)benzoselenazole (33)

(54) ##STR00017##

(55) A mixture of the compound 32 (90 mg, 0.296 mmol), potassium carbonate (61 mg, 0.441 mmol), iodomethane (126 mg, 0.888 mmol) and DMF (8 mL) was stirred overnight at 50. The mixture was added with water (40 mL) and extracted with ethyl acetate (20 mL3), and the combined organic phase was washed with saturated saline solution (15 mL2) and dried with anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by chromatography (200 to 300 meshes of silica gel, elution with ethyl acetate: petroleum ether=1:5) to obtain 2-(3,4-dimethoxy- phenyl)benzoselenazole (33). .sup.1H NMR (DMSO-d.sub.6, 300 MHz) 8.13 (d, J=7.8 Hz, 1H), 8.02 (d, J=7.8 Hz, 1H), 7.60-7.55 (m, 2H), 7.52-7.47 (m, 1H), 7.36-7.31 (m, 1H), 7.10 (d, J=8.1 Hz, 1H), 3.89 (s, 3H), 3.85 (s, 3H). MS (El, m/z):320.0 [M+H].sup.+.

(56) Embodiment 13:

Synthesis of 2-fluoro-4-(5-fluorobenzoselenazole-2-yl)aniline (34) and 2-bromo-6-fluoro-4-(5-fluorobenzoselenazole-2-yl)aniline (35)

(57) ##STR00018##

(58) Step A: Tributylphosphine (1.28 g, 6.33 mmol) was added into a solution of the compound 11 (800 mg, 2.12 mmol) in methylbenzene (15 mL), then stirred for 5 min under nitrogen. To the mixture was added 3-fluoro-4-nitrobenzoic acid (392 mg, 2.12 mmol), and the resulting mixture was refluxed for 48 h under nitrogen. The mixture was cooled to the room temperature, added with water (25 mL) and adjusted with saturated sodium carbonate solution until the pH value was 9 to 10. The mixture was extracted with ethyl acetate (20 mL3), and the combined organic phase was washed with saturated saline solution (15 mL) and dried with anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by chromatography (200 to 300 meshes of silica gel, elution with ethyl acetate: petroleum ether=1:15) to obtain 2-fluoro-4-(5-fluoro- benzoselenazole-2-yl)aniline (34). .sup.1H NMR (DMSO-d.sub.6, 300 MHz) 8.13-8.09 (m, 1H), 7.77-7.73 (m, 1H), 7.69-7.64 (m, 1H), 7.59-7.56 (m, 1H), 7.22-7.16 (m, 1H), 6.88-6.83 (m, 1H), 6.00 (s, 2H). MS (El, m/z):309.0 [MH].sup..

(59) Step B: 2-bromo-6-fluoro-(5-fluorobenzoselenazole-2-yl)aniline (35) was prepared by using the compound 34 as raw material, and the method refers to the step G in Embodiment 2. .sup.1H NMR (DMSO-d.sub.6, 500 MHz) 8.16-8.13 (m, 1H), 7.90 (s, 1H), 7.79 (dd, J=2.0, 10.0 Hz, 1H), 7.73 (dd, J=2.0, 10.0 Hz, 1H), 7.24-7.20 (m, 1H), 6.12 (s, 2H). MS (El, m/z):388.9 [M+H].sup.+.

(60) Embodiment 14:

Synthesis of 5-(5-fluorobenzoselenazole-2-yl)-2-methylaniline (37)

(61) ##STR00019##

(62) Step A: A solution of the compound 11 (800 mg, 2.12 mmol) and tributylphosphine (1.28 g, 6.32 mmol) in methylbenzene (15 mL) was stirred for 5 min under nitrogen and added with 3-nitro-4-methylbenzoic acid (380 mg, 2.09 mmol), and the mixture was refluxed and stirred for 48 h under nitrogen. The mixture was cooled to the room temperature, added with water (30 mL) and adjusted with 2M sodium hydroxide solution until the pH value was 9 to 10. The mixture was extracted with ethyl acetate (20 mL3) and dried with anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by chromatography (200 to 300 meshes of silica gel, elution with ethyl acetate: petroleum ether=1:20 to 1:2) to obtain 5-fluoro-2-(4-methyl-3-nitrophenyl)benzoselenazole (36) (107 mg). The yield was 15.3%.

(63) Step B: 5-(5-fluorobenzoselenazole-2-yl)-2-methylaniline (37) was prepared by using the compound 36 as raw material, and the experimental operation refers to the step F in Embodiment 2. .sup.1H NMR (DMSO-d.sub.6, 500 MHz) 8.17-8.14 (m, 1H), 7.82 (dd, J=2.5, 10.0 Hz, 1H), 7.38 (s, 1H), 7.27-7.23 (m, 1H), 7.18 (d, J=8.0 Hz, 1H), 7.10 (d, J=8.0 Hz, 1H), 2.13 (s, 3H). MS (El, m/z):305.1 [MH].sup..

(64) Embodiment 15:

Synthesis of 2-[3-chloro-4-(trifluoromethoxy)phenyl)-5-fluorobenzoselenazole (38)

(65) ##STR00020##

(66) The method for preparing the compound 38 refers to the step E in Embodiment 2, wherein 4-nitrobenzaldehyde in the step E of Embodiment 2 was replaced with 3-chloro-4(trifluoromethoxy)benzaldehyde. .sup.1H NMR (DMSO-d.sub.6, 300 MHz) 8.35 (d, J=1.2 Hz, 1H), 8.30-8.27 (m, 1H), 8.17-8.15 (m, 1H), 7.98-7.96 (m, 1H), 7.77-7.75 (m, 1H), 7.38-7.34 (m, 1H). MS (El, m/z):394.0 [MH].sup..

(67) Embodiment 16:

Synthesis of 4-(5-deuterobenzoselenazole-2-yl)-2-methylaniline (40)

(68) ##STR00021##

(69) Step A: The compound 22 and 3-methyl-4-nitrobenzaldehyde were used as raw materials, and the experimental operation of preparing the compound 39 refers to the step E in Embodiment 2. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) 8.35 (s, 1H), 8.24 (d, J=8.4 Hz, 1H), 8.19 (s, 1H), 8.15-8.14 (m, 2H), 7.62 (dd, J=2.0, 8.4 Hz, 1H), 2.65 (s, 3H).

(70) Step B: The compound 39 (42 mg, 0.106 mmol) was suspended in DMF (5 mL) and then added with deuteroxide (0.5 mL) and 5% palladium carbon, and the mixture was stirred overnight under deuterium gas at the normal pressure. The mixture was filtered through a celite pad, added with water (20 mL) and extracted with ethyl acetate (20 mL2), and the combined organic phase was washed with saturated saline solution (10 mL2) and dried with anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by chromatography (200 to 300 meshes of silica gel, elution with ethyl acetate: petroleum ether=1:10) to obtain 4-(5-deuterobenzoselenazole-2-yl)-2-methylaniline (40). .sup.1H NMR (DMSO-d.sub.6, 400 MHz) 8.06-8.02 (m, 2H), 7.62-7.59 (m, 2H), 8.16 (dd, J=2.0, 8.8 Hz, 1H), 6.68 (d, J=8.0 Hz, 1H), 5.78 (s, 2H), 2.14 (s, 3H). MS (El, m/z):288.0 [MH].sup..

(71) Embodiment 17:

Synthesis of 2,6-difluoro-4-(5-fluorobenzoselenazole-2-yl)aniline (41)

(72) ##STR00022##

(73) The method for preparing the compound 41 refers to the step A in Embodiment 13, wherein 3-fluoro-4-nitrobenzaldehyde in the step A of Embodiment 13 was replaced with 3,5-difluoro-4-nitrobenzaldehyde. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) 8.18-8.14 (m, 1H), 7.79 (dd, J=2.4, 10.0 Hz, 1H), 7.62 (dd, J=2.4, 7.2 Hz, 2H), 7.26-7.21 (m, 1H), 6.11 (s, 2H). MS (El, m/z):327.0 [MH].sup..

(74) Embodiment 18:

Synthesis of 2-fluoro-4-(5-fluorobenzoselenazole-2-yl)-6-methylaniline (45)

(75) ##STR00023##

(76) Step A: A solution of NBS (5.0 g, 28.1 mmol) in DMF (20 mL) was added dropwise into a solution of 2-fluoro-6-methylaniline (3.5 g, 28.0 mmol) in DMF (10 mL) in an ice-water bath, and the mixture was continuously stirred for 5 min at the end of addition. The ice-water bath was removed, and the reaction mixture was stirred 0.5 h at the room temperature. The mixture was added with water (150 mL) and extracted with ethyl acetate (80 mL3), and the combined organic phase was successively washed with saturated sodium bicarbonate solution (40 mL2) and saturated saline solution (40 mL2), and dried with anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to obtain 4-bromo-2-fluoro-methylaniline (42) (5.3 g). The yield was 92.8%.

(77) Step B: A mixture of the compound 42 (5.3 g, 26.0 mmol), cuprous cyanide (3.0 g, 33.5 mmol) and N-methylpyrrolidone (15 mL) was stirred overnight under nitrogen at 180. The mixture was added with water (75 mL) and extracted with ethyl acetate (50 mL3), and the combined organic phase was successively washed with water (30 mL2) and saturated saline solution (30 mL) and dried with anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by chromatography (200 to 300 meshes of silica gel, elution with ethyl acetate: petroleum ether=1:30 to 1:5) to obtain 4-amino-3-fluoro-5-methylbenzonitrile (43) (2.96 g). The yield was 75.8%.

(78) Step C: A mixture of the compound 43 (2.95 g, 19.6 mmol), 1M sodium hydroxide solution (50 mL) and ethanol (5 mL) was refluxed and stirred overnight. The mixture was cooled to the room temperature, added with water (50 mL) and washed with MTBE (20 mL2), and the water phase is used as the product. The water phase was adjusted with 2M hydrochloric acid until the pH value was 3 to 4, and the solid was separated out. The mixture was filtered, and the filter cake was dried to obtain 4-amino-3-fluoro-5-methylbenzoic acid (44) (2.90 g). The yield was 87.5%.

(79) Step D: Tributylphosphine (2.15 g, 10.6 mmol) was added into a mixture of the compound 11 (1.74 g, 4.60 mmol), the compound 44 (600 mg, 3.55 mmol) and methylbenzene (25 mL), and the mixture was refluxed and stirred for 48 h under nitrogen. The mixture was cooled to the room temperature, added with water (40 mL) and adjusted with 2M sodium hydroxide solution until the pH value was 9 to 10. The mixture was extracted with ethyl acetate (40 mL3), and the combined organic phase was washed with saturated saline solution (25 mL) and dried with anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by chromatography (200 to 300 meshes of silica gel, elution with ethyl acetate: petroleum ether=1:100 to 1:30) to obtain 2-fluoro-4-(5-fluorobenzoselenazole-2-yl)-6-methylaniline (45). .sup.1H NMR (DMSO-d.sub.6, 400 MHz) 8.14-8.10 (m, 1H), 7.77-7.74 (m, 1H), 7.58-7.55 (m, 1H), 7.51 (s, 1H), 7.22-7.17 (m, 1H), 5.76 (s, 2H), 2.22 (s, 3H). MS (El, m/z):323.0 [MH].sup..

(80) Bioactivity Embodiments

(81) Embodiment 19: inhibition effects of the compounds on the growth of breast cancer cell Lines of MCF-7 and MDA-MB-468

(82) Experimental Methods and Results

(83) 1. Breast cancer cells MCF-7 (Luminal type cells) and MDA-MB-468 (basal-like cells, which are of the triple-negative type without epithelial-mesenchymal transition) were purchased from Cell Resource Center of Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences, and cultured with DMEM culture medium(containing 10% of fetal bovine serum, 10 U/mL of penicillin and 0.1 mg/mL of streptomycin) in a 5% CO2 incubator at 37 until the cell density was about 90%.

(84) 2. The cells were inoculated to a 96-well plate at a cell population of 310.sup.3/well and then cultured in the 5% CO.sub.2 incubator for 24 h at 37.

(85) 3. Tested compounds of different concentration gradients were prepared by using the DMEM culture medium, and added into wells at 100 L/well as tested compounds wells; and, the DMEM culture liquid was added into wells at 100 L/well as negative control wells. At 37 and in the 5% CO.sub.2 incubator, the MCF-7 cells were cultured for 120 h, and MDA-MB-468 cells were cultured for 72 h.

(86) 4. Resazurin (15 mg/50 mL), Methylene Blue (25 mg/10 mL), Potassium ferricyanide (0.329 g/100 mL) and Potassium ferrocyanide (0.422 g/100 mL) were dissolved into PBS (0.1 M, pH=7.4) to obtain Alamar Blue solution for standby.

(87) 5. The cells were washed with PBS (0.1 M, pH=7.4) for two times, and the Alamar Blue solution was added into wells at 100 L/well; and 100 L of Alamar Blue solution was added into wells without cells to serve as blank control cells. The 96-well plate was placed into the 5% CO.sub.2 incubator at 37 and cultured for 3 h.

(88) 6. The fluorescence value of the cells was detected at 530/590 nm by ELIASA Victor X4 (Perkin Elmer). The fluorescence value at each concentration was repetitively measured for 4 times to obtain an average value and a standard deviation. The cell viability was calculated by the following formula[User2]:

(89) $\mathrm{Cell}\mathrm{viability}\left(\%\right)=\frac{\mathrm{tested}\mathrm{compound}\mathrm{wells}-\mathrm{blank}\mathrm{control}\mathrm{wells}}{\mathrm{negative}\mathrm{control}\mathrm{wells}-\mathrm{blank}\mathrm{control}\mathrm{wells}}100\%$

(90) 7. The half inhibitory concentration (IC.sub.50) of the tested compounds againstt he cell lines was obtained according to the cell viability by Prism Graph software. The experimental results are shown in Table 1.

(91) TABLE-US-00001 TABLE 1 Half inhibitory concentration (IC.sub.50, nM) of the compounds againstcell lines ofMCF-7 and MDA-MB-468 Compound ID MCF-7 MDA-MB-468 7 56.69 74.07 14 187.71 137.94 17 72.81 58.45 18 91.86 70.93 26 133.8 34 52.38 29.06 35 41.59 39.67 37 449.56 113.63 41 56.77 20.1 Paclitaxel 4.6* 4.43 *Note: In Table 1, the positive control drugpaclitaxel has only 70% inhibition rate to MCF-7 cell line in itshighestconcentration at 500 nM. Therefore, the IC.sub.50 of the paclitaxel for the MCF-7 cell strains calculated by the software is lower.

(92) Embodiment 20: inhibition effects of the compounds on the growth of human lung cancer cells H1299, human colon cancer cells HT29, human liver cancer cells SK-HEP-1, human colon cancer cells HCT116, human normal liver cells L-02 and WRL-68

(93) Growth inhibition tests of the tested compounds 7, 17, 18, 34, 35 and 41 on the human lung cancer cells H1299, human colon cancer cells HT29, human liver cancer cells SK-HEP-1, human colon cancer cells HCT116, human normal liver cells L-02 and WRL-68 were conducted, and the method refers to Embodiment 19 Inhibition effects of the compounds on the growth of breast cancer cells MCF-7 and MDA-MB-468. The compounds 7, 17, 18, 34, 35 and 41 had a half inhibitory concentration (IC.sub.50) greater than 10 M for the cells, and had no obvious inhibition effects. The half inhibitory concentration (IC.sub.50) of the positive control drug paclitaxel for the six cell lines was within a range of 1.59 nM to 15.31 nM.

(94) The experimental results indicated that the compounds 7, 17, 18, 34, 35 and 41 had very good growth inhibition effects on the breast cancer cells MCF-7 and MDA-MB-468; and the half inhibitory concentration (IC.sub.50) for other tested cell strains such as H1299, HT29, SK-HEP-1, HCT116, L-02 and WRL-68 was greater than 10 M; and, the positive medicine paclitaxel still had strong inhibition toxicity on cancer cells in addition to the breast cancer cells and normal cells. Therefore, it was indicated that the compounds of the present application had remarkable selectivity for the inhibition of breast cancer cell lines.