Gem difluorocompounds as depigmenting or lightening agents

Abstract

The present invention relates to a compound having the formula (I), as well as a method for preparing such a compound, a cosmetic or pharmaceutic composition containing such a compound, and the use thereof as a depigmenting, lightening, bleaching or whitening agent and for treating pigmentation disorders, notably by topical application on the skin. ##STR00001##

Claims

1. A compound having the following formula (I): ##STR00036## or a cosmetically or a pharmaceutically acceptable salt thereof, a stereoisomer or a mixture of stereoisomers in any proportion, wherein: R.sub.1 and R.sub.2 represent, independently from each other, a hydrogen atom or OR.sub.6 or R.sub.1 and R.sub.2 form together an oxo group (O), or R.sub.1 and R.sub.2 are linked together by a chain of formula O(CH.sub.2).sub.nO, with n representing 2 or 3, X.sub.1, X.sub.2, X.sub.4, X.sub.5 represent, independently from one another, a hydrogen atom or OR.sub.16 X.sub.3 represents a hydrogen atom, OR.sub.16 or OC(O)R.sub.17 with: R.sub.6 and R.sub.16 representing, independently from one another, a hydrogen atom; or a (C.sub.1-C.sub.6)alkyl, (C.sub.2-C.sub.6)alkenyl, (C.sub.2-C.sub.6)alkynyl, (C.sub.3-C.sub.7)cycloalkyl, 5- to 7-membered heterocycloalkyl, aryl, heteroaryl, (C.sub.3-C.sub.7)cycloalkyl-(C.sub.1-C.sub.6)alkyl, (5- to 7-membered heterocycloalkyl)-(C.sub.1-C.sub.6)alkyl, aryl-(C.sub.1-C.sub.6)alkyl or heteroaryl-(C.sub.1-C.sub.6)alkyl group, said group being optionally substituted by one or several groups selected from a halogen atom, a (C.sub.1-C.sub.6)alkyl group and a (C.sub.1-C.sub.6)alkoxy group, and R.sub.17 representing a (C.sub.1-C.sub.6)alkyl, (C.sub.2-C.sub.6)alkenyl, (C.sub.2-C.sub.6)alkynyl, (C.sub.3-C.sub.7)cycloalkyl, 5- to 7-membered heterocycloalkyl, aryl, heteroaryl, (C.sub.3-C.sub.7)cycloalkyl-(C.sub.1-C.sub.6)alkyl, (5- to 7-membered heterocycloalkyl)-(C.sub.1-C.sub.6)alkyl, aryl-(C.sub.1-C.sub.6)alkyl or heteroaryl-(C.sub.1-C.sub.6)alkyl group, said group being optionally substituted by one or several groups selected from a halogen atom, a (C.sub.1-C.sub.6)alkyl group and a (C.sub.1-C.sub.6)alkoxy group, wherein an aryl is an aromatic hydrocarbon group comprising 6 to 10 carbon atoms and comprising one or more fused rings; wherein a heteroaryl is an aromatic group, having 5 to 10 members comprising one or more fused rings, in which the atoms of the ring(s) consist of one or more heteroatoms selected from nitrogen, oxygen and sulphur atoms, the remainder being carbon atoms; wherein a 5- to 7-membered heterocycloalkyl is a saturated hydrocarbon cycle having 5 to 7 members and in which one or several carbon atoms are each replaced with a nitrogen, oxygen or sulphur atom.

2. The compound according to claim 1, wherein X.sub.1, X.sub.2, X.sub.4 and X.sub.5 each represents a hydrogen atom and X.sub.3 does not represent a hydrogen atom.

3. The compound according to claim 1, wherein: R.sub.6 and R.sub.16 represent, independently from one another, a hydrogen atom; or a (C.sub.1-C.sub.6)alkyl, aryl, or aryl-(C.sub.1-C.sub.6)alkyl group, said group being optionally substituted by one or several groups selected from a halogen atom, a (C.sub.1-C.sub.6)alkyl group and a (C.sub.1-C.sub.6)alkoxy group, and R.sub.17 represents a (C.sub.1-C.sub.6)alkyl, aryl, or aryl-(C.sub.1-C.sub.6)alkyl group, said group being optionally substituted by one or several groups selected from a halogen atom, a (C.sub.1-C.sub.6)alkyl group and a (C.sub.1-C.sub.6)alkoxy group.

4. The compound according to claim 1, wherein it is chosen from the following compounds: ##STR00037## and cosmetically and pharmaceutically salts thereof.

5. A cosmetic or pharmaceutical composition comprising at least one compound of formula (I) as defined in claim 1 and at least one cosmetically or pharmaceutically acceptable excipient.

6. The compound according to claim 3, wherein: R.sub.6 and R.sub.16 represent, independently from one another, a hydrogen atom; or a (C.sub.1-C.sub.6)alkyl, aryl, or aryl-(C.sub.1-C.sub.6)alkyl group, said group being optionally substituted by one or several groups selected from a (C.sub.1-C.sub.6)alkyl group and a (C.sub.1-C.sub.6)alkoxy group, and R.sub.17 represents a (C.sub.1-C.sub.6)alkyl, aryl, or aryl-(C.sub.1-C.sub.6)alkyl group, said group being optionally substituted by one or several groups selected from a (C.sub.1-C.sub.6)alkyl group and a (C.sub.1-C.sub.6)alkoxy group.

7. A method for depigmenting, lightening, bleaching or whitening a skin comprising applying on said skin an efficient amount of a compound of formula (I) according to claim 1.

8. A method for treating a pigmentation disorder comprising applying on a skin of a person in need thereof an efficient amount of a compound of formula (I) according to claim 1.

9. The method according to claim 8, wherein the pigmentation disorder is a hyperpigmentation.

10. The method according to claim 9, wherein the hyperpigmentation is lentigo, melasma, ephelides, postinflammatory hyperpigmentation, or a hyperpigmentation caused by drugs, chemicals or sun.

11. A method for inhibiting or reducing oxidative stress comprising administering to a person in need thereof an efficient amount of a compound of formula (I) according to claim 1.

12. The method according to claim 11, wherein the reducing oxidative stress is due to UV.

13. The method according to claim 11, wherein the administrating is performed via a topical administration on the skin.

Description

BRIEF DESCRIPTION OF THE FIGURE

(1) FIG. 1 represents the inhibition of human tyrosinase kinetics of compounds 11 and 16 at 1.12 mM.

EXAMPLES

(2) The following abbreviates have been used in the examples. Ac: Acetyl (COCH.sub.3) ACN: Acetonitrile ADDP: 1,1-(Azodicarbonyl)dipiperidine AP: Affinity-purification aq.: aqueous DAST: Diethylaminosulphurtrifluoride DCM: Dichloromethane DIPEA: N,N-Diisopropylethylamine eq: Equivalent GC/MS: Gas chromatography-mass spectrometry HPLC: High Performance Liquid Chromatography LC-MS/MS: Liquid chromatography coupled to tandem mass spectrometry LLOQ: Lower limit of quantification MOM: Methoxymethyl NMR: Nuclear Magnetic Resonance OD: Optical density RT: Room temperature sat.: saturated TLC: Thin Layer Chromatography UV/DAD: Ultraviolet diode array detector

1. Preparation of the Compounds According to the Invention

Synthesis of Intermediate Compound 1

(3) ##STR00017##

(4) Route 1:

(5) Under inert atmosphere, K.sub.2CO.sub.3 (0.42 g, 3.02 mmol, 2 eq) was added to a mixture of o-chlorocyclohexanone (0.20 g, 1.5 mmol, 1 eq) and p-benzyloxyphenol (0.45 g, 2.26 mmol, 1.5 eq) in acetone (3 mL) and the reaction mixture was refluxed for 1 h. The reaction can also be performed in acetonitrile at a temperature of 60 C. The reaction was monitored by TLC (cyclohexane/ethyl acetate 8:2stain: vanillin). Water (5 mL) and diethyl ether (10 mL) were then added at room temperature and the aqueous layer was extracted with diethyl ether (320 mL). The combined organic layer was then washed with 1N NaOH (420 mL), dried over sodium sulfate, filtered and concentrated. The crude brown oil was purified by flash chromatography (Biotage SNAP 25 g, cyclohexane/ethyl acetate 98:2 to 80:20) to afford intermediate compound 1 (0.28 g, 62%) as a white solid. Compound 1 can also be obtained by recrystallization in a mixture of heptane/isopropanol (5/1).

(6) Route 2:

(7) Under inert atmosphere, Dess-Martin periodinane (42.6 mg, 0.101 mmol, 1.5 eq) was added to a solution of intermediate compound 19 (20 mg, 0.067 mmol, 1 eq) in dry DCM (200 L). The mixture was stirred at 25 C. for 2.5 hours before aq. NaOH 1N was added. The mixture was then extracted with DCM (310 mL). The organic layers were combined, dried over sodium sulfate and concentrated to give intermediate compound 1 (16 mg, 81%) as a white solid.

(8) Mass (AP+): 297.1 [M+H].sup.+; 314.1 [M+NH.sub.4].sup.+; 319.1 [M+Na].sup.+; 335.1 [M+K].sup.+; 360.1 [M+Na+CH.sub.3CN].sup.+; 615.2 [2M+Na].sup.+.

Synthesis of Intermediate Compound 2

(9) ##STR00018##

(10) Under inert atmosphere, K.sub.2CO.sub.3 (2.08 g, 15.1 mmol, 2 eq) was added to a mixture of o-chlorocyclohexanone (1.00 g, 7.54 mmol, 1 eq) and p-methoxyphenol (1.12 g, 9.05 mmol, 1.2 eq) in toluene (12 mL). The reaction mixture was stirred at 90 C. for 1 h. The reaction was monitored by TLC (cyclohexane/ethyl acetate 8:2stain:vanillin). Water (30 mL) and ethyl acetate (50 mL) were then added at room temperature and the aqueous layer was extracted with ethyl acetate (350 mL). The combined organic layer was then washed with 1N NaOH (430 mL), dried over sodium sulfate, filtered and concentrated. The crude brown oil was purified by flash chromatography (Macherey Nagel CHROMABOND Flash RS 40 SiOH, cyclohexane/ethyl acetate 98:2 to 80:20) to afford intermediate compound 2 (1.08 g, 65%) as a white solid.

(11) Mass (GC/MS): 220 [M].sup.+, 202, 174, 124, 109, 95, 81, 69, 55.

Synthesis of Intermediate Compound 3

(12) ##STR00019##

(13) Hydroquinone (1.00 g, 9.08 mmol, 1 eq) was dissolved in acetic acid (2.27 mL) and the solution was heated to 110 C. Acetic anhydride (0.425 mL, 4.54 mmol, 0.5 eq) was then added at this temperature and the mixture was stirred at 110 C. for 2 h. The mixture was then allowed to reach room temperature and acetic acid was removed by evaporation. Toluene (4.5 mL) was then added to the white solid and the suspension was filtered to remove the excess of hydroquinone. The filtrate was then concentrated to afford intermediate compound 3 (651 mg, 94%) as a yellowish oil.

(14) Mass (GC/MS): 152 [M].sup.+, 143, 110, 81, 73, 55, 43.

Synthesis of Intermediate Compound 4

(15) ##STR00020##

(16) Under inert atmosphere, K.sub.2CO.sub.3 (0.21 g, 1.51 mmol, 2 eq) was added to a mixture of o-chlorocyclohexanone (0.100 g, 0.75 mmol, 1 eq) and intermediate compound 3 (0.172 g, 1.13 mmol, 1.5 eq) in acetone (1.5 mL). The reaction mixture was refluxed overnight. Water (5 mL) and diethyl ether (10 mL) were then added at room temperature and the aqueous layer was extracted with diethyl ether (320 mL). The combined organic layer was then washed with 1N NaOH (420 mL), dried over sodium sulfate, filtered and concentrated. The crude brown oil was purified by flash chromatography (Biotage SNAP 10 g, cyclohexane/ethyl acetate 95:5 to 75:25) to afford intermediate compound 4 (29 mg, 15%) as a white solid.

(17) Mass (GC/MS): 248 [M].sup.+, 206, 162, 110, 98, 91, 69, 55, 43.

Synthesis of Intermediate Compound 5

(18) ##STR00021##

(19) Hydroquinone (0.50 g, 4.54 mmol, 1 eq) was dissolved in acetonitrile (15 mL) under an inert atmosphere, and the solution was cooled to 0 C. MOMCl (517 L, 6.81 mmol, 1.5 eq) followed by diisopropylethylamine (1.5 mL, 9.08 mmol, 2 eq) were successively added and the mixture was stirred overnight at room temperature. Methanol (200 L) was added and the mixture was stirred at room temperature for 30 min. Acetonitrile was then removed under vacuum and 1M HCl (aq.) was added to the residue. The aqueous mixture was extracted twice with ethyl acetate and the combined organic extract was dried over sodium sulfate, filtered and concentrated to afford an orange oil (623 mg). This crude oil was purified by silica gel chromatography (Biotage SNAP 50 g, cyclohexane/ethyl acetate 98:2 to 70:30) to afford intermediate compound 5 (228 mg, 33%) as a yellowish oil.

(20) Mass (GC/MS):154 [M].sup.+, 124; 109; 93; 81; 65; 53.

Synthesis of Intermediate Compound 6

(21) ##STR00022##

(22) Under inert atmosphere intermediate compound 5 (1.0 g, 6.52 mmol, 1.2 eq.) followed by K.sub.2CO.sub.3 (1.50 g, 10.9 mmol, 2 eq) were added to a solution of 2-chlorocyclohexanone (720 mg; 5.43 mmol, 1 eq.) in dry toluene (11 mL). The mixture was heated at 90 C. for 1 h. At room temperature, water was added to the mixture, which was then extracted twice with ethyl acetate. The combined organic layer was washed twice with 1N NaOH, dried over sodium sulfate, filtered and concentrated to afford 1.02 g of crude oil. 720 mg of this crude material was purified by silica gel chromatography (Biotage SNAP 50 g, cyclohexane/ethyl acetate 96:4 to 78:22) to afford intermediate compound 6 (582 mg, 61% extrapolated yield) as a colorless oil.

(23) Mass (GC/MS): 250 [M].sup.+, 220; 154; 124; 110; 97; 81; 69; 55; 45.

Synthesis of Compound 7

(24) ##STR00023##

(25) Route 1:

(26) At room temperature, diethylaminosulfur trifluoride (3.33 mL, 27.3 mmol, 2.8 eq) was added to a solution of intermediate compound 1 (2.7 g, 9.11 mmol, 1 eq) in dry dichloromethane (55 mL) under inert atmosphere. The mixture was stirred overnight at room temperature before being poured on a mixture of ice and solid NaHCO.sub.3. The cold mixture was stirred 15 min and dichloromethane was added. The aqueous layer was then extracted with dichloromethane (250 mL), dried over sodium sulfate, filtered and concentrated. The crude brown oil was purified on silica gel chromatography (Biotage SNAP 100 g, cyclohexane/toluene 93:7 to 40:60) to afford compound 7 (1.87 g, 65%, 88% purity.sup.19F NMR) as colorless oil.

(27) Route 2:

(28) Under inert atmosphere, triethylamine trihydrofluoride (0.1 mL, 0.58 mmol, 2.8 eq.) was added at room temperature to a solution of XtalFluor-E (135 mg, 0.59 mmol, 2.8 eq) in dry dichloromethane (0.5 mL). Intermediate compound 1 (61.3 mg, 0.207 mmol, 1 eq.) was then added and the reaction was stirred at the same temperature for 3 h. Dichloromethane was then added followed by sat. aq. NaHCO.sub.3. The aqueous layer was extracted twice with dichloromethane and the combined organic layer was dried over sodium sulfate, filtered and concentrated. The crude material was purified by flash chromatography on silica gel (Biotage ZIP 10, cyclohexane/toluene 98:2 to 50:50) to afford compound 7 (31 mg, 47%) as a colorless oil with an estimated purity (.sup.19F NMR) of 98%.

(29) .sup.19F NMR (CDCl.sub.3, 282.5 MHz): 105.3 (d, J=244 Hz, 1 F); 107.8 (brs, 1 F).

(30) Mass (GC/MS): 318 [M].sup.+, 55, 77, 91, 109, 227.

Synthesis of Compound 8

(31) ##STR00024##

(32) Diethylaminosulfur trifluoride (35 L, 0.26 mmol, 2.7 eq) was added to a solution of intermediate compound 4 (24 mg, 0.1 mmol, 1 eq) in dry dichloromethane (537 L) under inert atmosphere. The mixture was stirred overnight at room temperature before being poured on a mixture of ice and solid NaHCO.sub.3. The cold mixture was stirred 15 min and dichloromethane was added. The aqueous layer was then extracted with dichloromethane (210 mL), dried over sodium sulfate, filtered and concentrated. The crude oil was purified on silica gel chromatography (Biotage SNAP 10 g, cyclohexane/Et.sub.2O 95:5 to 75:25) to afford compound 8 (16 mg, 61%, 84% purity-.sup.19F NMR) as a colorless oil.

(33) .sup.19F NMR (CDCl.sub.3, 282.5 MHz): 105.8 (d, J=243 Hz, 1 F); 107.8 (brs, 1 F).

(34) Mass (GC/MS): 270 [M].sup.+, 228, 110, 99, 77, 55, 43.

Synthesis of Compound 9

(35) ##STR00025##

(36) XtalFluor-E (0.208 g, 0.91 mmol, 2 eq) was suspended in dry DCM (2 mL), under inert atmosphere. Triethylamine trihydrofluoride (110 L, 0.68 mmol, 1.5 eq) was then added at room temperature followed by a solution of intermediate compound 2 (0.100 g, 0.45 mmol, 1 eq) in dry DCM (0.5 mL). The reaction was stirred under reflux for 2 h before being poured on a mixture of ice and solid NaHCO.sub.3. The cold mixture was stirred 15 min and dichloromethane was added. The aqueous layer was then extracted with dichloromethane (210 mL) and the combined organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated. The crude oil was purified on silica gel chromatography (Biotage SNAP 10 g, cyclohexane/toluene 93:7 to 40:60) to afford compound 9 (58 mg, 53%, 98% purity.sup.19F NMR) as a yellowish oil.

(37) .sup.19F NMR (CDCl.sub.3, 282.5 MHz): 105.3 (d, J=240 Hz, 1 F); 107.8 (brs, 1 F).

(38) Mass (GC/MS): 242 [M].sup.+, 221, 124, 109, 95, 73, 55.

Synthesis of Compound 10

(39) ##STR00026##

(40) XtalFluor-E (174 mg, 0.76 mmol, 3.8 eq) was suspended in dry DCM (0.4 mL), under inert atmosphere. Triethylamine trihydrofluoride (91 L, 0.56 mmol, 2.8 eq) was then added at room temperature followed by a solution of intermediate compound 6 (50.0 mg, 0.20 mmol, 1 eq) in dry DCM (0.1 mL). The reaction was stirred at room temperature for 1 h30 before being poured on a saturated solution of NaHCO.sub.3. The mixture was stirred 5 min and dichloromethane was added. The aqueous layer was then extracted with dichloromethane (2) and the combined organic layer was dried over sodium sulfate, filtered and concentrated. The crude oil was purified on silica gel chromatography (Biotage SNAP 10 g, cyclohexane/toluene 100:0 to 90:10) to afford 10 (27 mg, 50%, 93% purity.sup.19F NMR) as a colorless oil.

(41) .sup.19F NMR (CDCl.sub.3, 282.5 MHz): 105.8 (brd, J=242 Hz, 1 F), 108.0 (brs, 1 F).

(42) Mass (GC/MS): 272[M].sup.+, 242, 216, 124, 73.

Synthesis of Compound 11

(43) ##STR00027##

(44) Pd/C 10% (6.42 g, 6.04 mmol, 0.1 eq) was added to a solution of compound 7 (19.2 g, 60.4 mmol, 1 eq) in ethyl acetate (275 mL). The mixture was stirred 16 h under hydrogen atmosphere at room temperature and was then filtered on millipore 0.45 M and concentrated to afford a colorless oil (13.9 g) which was purified on silica gel chromatography (Biotage SNAP 750 g, cyclohexane/diethyl ether 90:10 to 63:37 liquid injection) to afford compound 11 (9.8 g, 71%, racemate mixture) as an oil which slowly cristallizes in a white solid.

(45) HPLC analysis: compound 11 was analysed using a ThermoFisher P1000XR HPLC system with an elution solvent ratio of 95:5 v/v heptane/isopropyl alcohol and a 4.6250 mm, 5 m Chiralpak IA column, running 1 mL/min at 22 C. The detection system is a UV lamp at 225 nm. The enantiomers were eluted at tr=15.24 min and tr=16.80 min with relative amounts of 49.07% and 48.91% respectively.

(46) .sup.19F NMR (CDCl.sub.3, 282.5 MHz): 105.7 (d, J=241 Hz, 1 F); 108.4 (brs, 1 F).

(47) Mass (AP.sup.): 227.1 [MH].sup..

Synthesis of Compound 12

(48) ##STR00028##

(49) Sodium hydride (7.4 mg, 0.31 mmol, 1 eq) was added under inert atmosphere to a solution of compound 11 (70.0 mg, 0.31 mmol, 1 eq) in dry diethyl ether (0.7 mL). The reaction mixture was stirred overnight at room temperature. The resultant suspension was filtered, washed with diethyl ether and dried to afford compound 12 (43 mg, 61%) as white solid.

(50) .sup.19F NMR (MeOD, 282.5 MHz): 104.7 (d, J=244 Hz, 1 F); 108.6 (brs, 1 F).

Synthesis of Intermediate Compound 13

(51) ##STR00029##

(52) Under inert atmosphere, a solution of T15 (XBr) (prepared as disclosed in Chem. Eur. J. 2007, 13, 3739-3756) (4.78 g, 20.3 mmol, 1.2 eq) in dry DMF (40 mL) was added to a solution of intermediate compound 5 (2.61 g, 16.9 mmol, 1 eq) and K.sub.2CO.sub.3 (2.34 g, 16.9 mmol, 1 eq) in dry DMF (16 mL) at room temperature. The reaction was then stirred at 80 C. for 5 h. The reaction was then allowed to reach room temperature and water was added followed by 1N NaOH. The mixture was then extracted with diethyl ether (3) and the combined organic layer was washed with water, brine and was dried over sodium sulfate, filtered and concentrated to afford intermediate compound 13 (2.07 g, 40%) as a yellow solid. The crude intermediate compound 13 was engaged in the next step without further purification.

(53) Mass (GC/MS): 308 [M].sup.+, 278, 207, 155, 124, 111, 99, 86, 65, 55, 45.

Synthesis of compound 14

(54) ##STR00030##

(55) Route 1:

(56) Under inert atmosphere, diethylaminosulfur trifluoride (79 L, 0.59 mmol, 2.8 eq) was added dropwise to a solution of intermediate compound 13 (66.0 mg, 0.21 mmol, 1 eq) in dry dichloromethane (1.2 mL) and the reaction was stirred overnight at room temperature. The reaction mixture was then poured on a mixture of ice, water and solid NaHCO.sub.3. The agitation was maintained 15 min and the aqueous layer was then extracted twice with dichloromethane. The combined organic layer was dried over sodium sulfate, filtered and concentrated. The crude material was purified by flash chromatography on silica gel (Biotage SNAP 10 g, cyclohexane/ethyl acetate 97:3 to 72:28) to afford compound 14 (59 mg, 83%) with an estimated purity (.sup.19F NMR) of 87%.

(57) Route 2:

(58) Under inert atmosphere, XtalFluor-E (4.59 g, 20.0 mmol, 3 eq) and triethylamine trihydrofluoride (2.2 mL, 13.4 mmol, 2 eq.) were successively added at room temperature to a solution of intermediate compound 13 (2.06 g, 6.68 mmol, 1 eq.) in dry dichloromethane (13.4 mL). The reaction was stirred at the same temperature for 2 h. Dichloromethane was then added followed by sat. aq. NaHCO.sub.3. The aqueous layer was extracted twice with dichloromethane and the combined organic layer was dried over sodium sulfate, filtered and concentrated. The crude material was purified by flash chromatography on silica gel (Biotage SNAP 100 g, cyclohexane/ethyl acetate 98:2 to 75:25) to afford compound 14 (2.06 g, 76%) with an estimated purity (.sup.19F NMR) of 95%.

(59) .sup.19F NMR (CDCl.sub.3, 282.5 MHz): 107.8 (dm, J=238 Hz, 1 F); 120.9 (brd, J=236 Hz, 1 F).

(60) Mass (GC/MS): 330 [M].sup.+, 177, 157, 133, 113, 99, 85, 77, 65, 55, 45.

Synthesis of Compound 15

(61) ##STR00031##

(62) Under inert atmosphere, trifluoroacetic acid (8.1 mL, 109 mmol, 25 eq) was added at room temperature to a solution of compound 14 (1.44 g, 4.36 mmol, 1 eq) in dry dichloromethane (44 mL). The reaction was stirred overnight at room temperature. The mixture was diluted with dichloromethane and was poured onto sat. aq. NaHCO.sub.3. The aqueous layer was extracted twice with dichloromethane and the combined organic layer was dried over sodium sulfate, filtered and concentrated. The crude material was purified by flash chromatography on silica gel (Biotage ZIP 30 g, cyclohexane/ethyl acetate 98:2 to 60:40) to afford compound 15 (245 mg, 23%) as a white solid.

(63) .sup.19F NMR (CDCl.sub.3, 282.5 MHz): 108.9 (dm, J=251 Hz, 1 F); 110.4 (dm, J=251 Hz, 1 F).

(64) Mass (GC/MS): 242 [M].sup.+, 143, 133, 110, 104, 91, 85, 81, 77, 68, 63, 59, 55, 43.

Synthesis of Compound 16

(65) ##STR00032##

(66) Under inert atmosphere, sodium borohydride (75.0 mg, 1.97 mmol, 2 eq) was added at 0 C. to a solution of compound 15 (239 mg, 0.99 mmol, 1 eq) in dry methanol (9.9 mL). The mixture was stirred at this temperature for 2 h. Sat. aq. NH.sub.4Cl followed by brine were then added at 0 C. and the mixture was stirred for 15 min before being extracted twice with ethyl acetate. The combined organic layer was washed with water and brine, dried over sodium sulfate, filtered and concentrated. The crude material was purified by flash chromatography on silica gel (Biotage SNAP 25 g, cyclohexane/ethyl acetate 90:10 to 65:35) to afford compound 16 (183 mg, 76%) as a white solid.

(67) .sup.19F NMR (MeOD, 282.5 MHz): 110.6 (dd, J=239 Hz, J=4 Hz, 1 F); 122.0 (brd, J=234 Hz, 1 F).

(68) Mass (ESI): 223.1 [MHFH].sup., 243.1 [MH].sup., 285.1.

Synthesis of Compound 17

(69) ##STR00033##

(70) Under inert atmosphere, 4N HCl in dioxane (380 L, 1.52 mmol, 10 eq) was added to a solution of compound 14 (50.0 mg, 0.15 mmol, 1 eq) in dry dichloromethane (1.5 mL). The mixture was stirred at room temperature for 4 h30 and at 40 C. overnight. The reaction was monitored by TLC (cyclohexane/ethyl acetate 6:4stain: vanillin). An aliquot of the reaction was treated with sat. aq. NaHCO.sub.3 and extracted with CDCl.sub.3. The organic layer was filtered through Na.sub.2SO.sub.4 and analysed by .sup.19F NMR. The analysis showed that the reaction was completed and that compound 17 was obtained with 61% conversion.

(71) .sup.19F-decoupled .sup.1H NMR (CDCl.sub.3, 282.5 MHz): 107.9 (d, J=255 Hz, 1 F); 120.9 (brd, J=240 Hz, 1 F).

(72) Mass (GC/MS): 286 [M].sup.+, 221, 177, 110, 73, 85.

Synthesis of Intermediate Compound 18

(73) ##STR00034##

(74) Benzyloxyphenol (245 mg, 1.2 mmol, 1.2 eq) and 1,1-(azodicarbonyl)dipiperidine (303 mg, 1.2 mmol, 1.2 eq) were added under inert atmopshere to a solution of cyclohex-2-en-1-ol (0.1 mL, 1 mmol, 1 eq) in dry toluene (3.2 mL). The orange mixture was cooled to 0 C. and tri-n-butylphosphine (0.316 mL, 1.2 mmol, 1.2 eq) was added. The mixture was stirred at 25 C. for 5 min until the mixture jellified. Dichloromethane was added to the mixture, which was then concentrated to give the desired crude product as a white paste. The latter was purified by flash chromatography (Biotage SNAP 25 g, cyclohexane/ethyl acetate 100:0 to 75:25) to afford intermediate compound 18 (40 mg, 92%) as a white solid.

(75) Mass (GC/MS): 280 [M].sup.+, 200, 131, 91, 79, 65, 51, 44.

Synthesis of Intermediate Compound 19

(76) ##STR00035##

(77) A solution of borane dimethyl sulfide complex (2M in THF, 2.27 mL, 4.55 mmol, 5 eq) was slowly added to a cold solution (0 C.) of intermediate compound 18 (255 mg, 0.91 mmol, 1 eq) in THF (4.6 mL) under an inert atmosphere. The mixture was stirred at 25 C. for 20 hours before being cooled to 0 C. Hydrogen peroxide 30% w/v (2.79 mL, 27.3 mmol, 30 eq), water (1.15 mL, 63.7 mmol, 70 eq) and 2M aq. NaOH (3.64 mL, 7.28 mmol, 8 eq) were then successively added. The mixture was stirred at 25 C. for an additional 3 h. Water was added to the mixture which was then extracted with ethyl acetate (330 mL). The organic layers were combined, dried over Na.sub.2SO.sub.4 and concentrated. The crude mixture was purified by flash chromatography (Biotage ZIP 30 g, cyclohexane/ethyl acetate 100:0 to 35:65) to afford intermediate compound 19 (153 g, 56%) as a yellowish liquid which slowly crystallized.

(78) Mass (API+): 321.1 [M+Na].sup.+.

2. Biological Activity of the Compounds According to the Invention

2.1. In Vitro Stability of Compound 11

(79) The stability of compound 11 was evaluated by the released of hydroquinone, which is a potentially toxic compound, in different chemical conditions (extreme conditions, simulating the application of these molecules on the skin) and it was compared to the deoxyarbutin.

(80) This study involved different tests as degradation test and chemical stability (various chemical solutions, pH and temperatures).

(81) Methods

(82) Preparation of Biological Solutions

(83) Human Skin Solution

(84) Eight pieces of skin of about 1 cm.sup.2 were scratched and 7.2 mL of purified water were added. The solution was placed in ultrasonic bath.

(85) Cell Extract (Fibroblasts or Keratinocytes)

(86) The cultures of cells were performed in two steps.

(87) In the first step, cells were pre-cultured: when the confluence was obtained, the culture medium was removed, replaced by trypsin in order to take off the cells, centrifuged and the sediment was taken back in suspension in a growing medium containing 10% of Fetal Bovine Serum. This cells suspension was divided into two flasks and put back in culture.

(88) In the second step: when the confluence was obtained, the culture medium was removed, replaced by trypsin in order to take off the cells, centrifuged and the sediment was taken back in suspension in water. The cells in this suspension were counted and a solution containing 1.Math.10.sup.5 cells/mL was prepared. An extraction was performed using ultrasounds to lyse the cells in order to obtain a keratinocyte or fibroblast extract solution.

(89) Analytical Method (HPLC-UV/DAD Method)

(90) Column Atlantis dC18 150 mm*4.6 mm*3 m Waters, 30 C. Injection 50 l, 25 C. : 220 nm, 265 nm, 285 nm. A-Acetonitrile B-Water; 0.8 ml/min; elution gradient:

(91) TABLE-US-00001 Time (min) % phase A % phase B 0.0 20 80 5.0 25 75 6.0 90 10 9.0 90 10 9.5 20 80 12.0 20 80
retention time 11=9.55 min; retention time Deoxyarbutin=9.19 min; retention time Hydroquinone=3.61 min.

(92) This analytical method gave linear response for compound 11, Deoxyarbutin and Hydroquinone from 25 ng/mL to 1000 ng/mL. Dilution of samples had to be taken into account for each stability study to calculate the LLOQ of each compound.

(93) Assay

(94) The tested compounds are incubated in different solutions, at different times (see table 1). Then the analytical method was used to quantify remaining compounds and potential hydroquinone released (as possible degradation of compounds).

(95) Hydroquinone (HQ) apparition is expressed as a percentage of initial compound 11 or Deoxyarbutin. The calculation was done in molar units and the limit of quantification fluctuates with the initial concentration.

(96) $\mathrm{Percentage}\mathrm{released}\mathrm{HQ}=\frac{\mathrm{Concentration}\mathrm{HQ}}{\mathrm{Concentration}\mathrm{material}}100$
Results

(97) TABLE-US-00002 TABLE 1 Mean percentage of Hydroquinone released from Deoxyarbutin or compound 11 in various conditions % Hydroquinone released From From Conditions Time Deoxyarbutin 11 Water at room temperature .sup.14 Days 4.20 0.00 Ringer's solution pH 5.5 at 24 H 11.83 0.00 70 C. Ringer's solution pH 8.5 at 24 H 88.76 0.00 70 C. Synthetic perspiration at RT 48 H 128.61 0.00

(98) As shown in the table 1 above, hydroquinone was never released whatever the tested conditions from compound 11 unlike Deoxyarbutin.

2.2. In Vitro Efficacy of Compound 11 as Human Tyrosinase Inhibitor

(99) The efficacy of compound 11 was evaluated by the inhibition of human tyrosinase in-tubo and compared to the deoxyarbutin and both -arbutin and P-arbutin.

(100) Methods

(101) Preparation of Sample Solutions

(102) Bis-Tris Buffer 100 mM pH=6.5 (Bis Tris free Base 2.09 g/Purified water up to 100 mL/HCl up to pH=6.5).

(103) Substrate solution: L-DOPA (1 mg/mL) Solution B (L-DOPA 20 mg/Purified water up to 20 mL)

(104) Enzyme solution: Tyrosinase (384.6 U/mL) Solution A (R-Human Like active Tyrosinase (5000 U/mL) 100 L/Purified water 1200 L)

(105) Preparation of Test Solutions (for IC50 TestExamples for Deoxyarbutin)

(106) Inhibitors solutions deoxyarbutin (1 mg/mL): Deoxyarbutin 20 mg in purified water up to 20 mL

(107) TABLE-US-00003 TABLE 2 Example of test solutions Test Test Test Test Deoxyarbutin Deoxyarbutin Deoxyarbutin Deoxyarbutin Positive Negative 1 2 3 4 control control Solution B (substrate) 25 25 25 25 25 25 (L) 95 L of Deoxyarbutin 0.2 0.1 0.01 0.004 0 0.2 solutions at (mg/mL) Bis Tris buffer (L) 20 20 20 20 20 20 Solution A (enz) (L) 20 20 20 20 20 0
Assay

(108) This assay used a 96-well plate. Test solutions and controls were made several times. The absorbance (OD at 477 nm) was measured during all the experiment (kinetic), i.e. during 1 h, for each sample.

(109) The kinetic profiles were determined for each concentration of enzyme inhibitors and the IC50 values, i.e. the concentration of inhibitor giving 50% of enzyme inhibition, were calculated and the results at time 20 min was used for the IC50 calculations.

(110) In the conditions of the experiment the rate of conversion was stable between 5 and 30 minutes. The results were calculated from the OD measured at time point 20 min.

(111) The determination of half maximal inhibitory concentration (IC50) was performed using the following formula.

(112) $\%\mathrm{inhibition}=100-\frac{{\mathrm{OD}}_{\mathrm{sample}T20\min }100}{{\mathrm{OD}}_{\mathrm{positive}\mathrm{control}T20\min }}$
Results

(113) Arbutins, Deoxyarbutin and compound 11 were tested as inhibitors of the human tyrosinase activity at different concentrations.

(114) The IC50 values of both Deoxyarbutin and compound 11 were determined and compared in the following table 3. The IC50 values of both arbutins are reported in table 4.

(115) TABLE-US-00004 TABLE 3 Calculated IC50 of Deoxyarbutin and compound 11 Tyrosinase Molecular Concentration Test Test Test Test Mean weight IC50 (300 U/mL) #1 #2 #3 #4 (mg/mL) (g/mol) (mM) Compound 11 0.008 0.008 0.008 0.008 0.008 228.24 0.035 Deoxyarbutin 0.026 0.045 0.080 0.060 0.053 194.23 0.272

(116) TABLE-US-00005 TABLE 4 Calculated IC50 of -arbutin and -arbutin Tyrosinase Molecular Concentration Test Test Mean weight IC50 (170 U/mL) #1 #2 (mg/mL) (g/mol) (mM) -arbutin 0.068 0.065 0.067 272.5 0.244 -arbutin 0.116 0.132 0.124 272.5 0.454

(117) The comparison of IC50 has shown that the compound 11 inhibits the human recombinant tyrosinase 7-8 times more than the Deoxyarbutin with IC50 of 0.035 mM and 0.272 mM respectively. Moreover compound 11 inhibits the human recombinant tyrosinase better than -arbutin, and -arbutin.

2.3. In Vitro Efficacy of Compound 16 as Human Tyrosinase Inhibitor

(118) The efficacy of compound 16 was evaluated by the inhibition of human tyrosinase in tubo and compared to the compound 11.

(119) Methods

(120) The assay was performed with a ready-to-use kit from Feldan Inc (Canada): the HumanLike Tyrosinase Assay kit (ref A021-a-001Kit).

(121) The protocol was performed as described in the instructions for use of the manufacturer. Briefly, this kit is intended for the determination of human tyrosinase activity in presence of different inhibitors. The kit measures the conversion of L-Tyrosine into a dopachrome complex absorbing at 490 nm. The time course of the assay is 20 minutes, after which the results are analyzed and compared.

(122) Results

(123) The compounds 11 and 16 have been tested at a final concentration of 1.12 mM.

(124) The measured absorbances at 490 nm are reported in the table 5 and plotted in function of time in the FIG. 1.

(125) TABLE-US-00006 TABLE 5 measured OD at 490 nm for 20 minutes with compound 11 and 16 OD at 490 nm Negative Positive compound 11 compound 16 control control 1.12 mM 1.12 mM time 0 0.0000 0.0000 0.0000 0.0000 (min) 1 0.0030 0.0100 0.0095 0.0105 2 0.0060 0.0210 0.0190 0.0185 3 0.0080 0.0320 0.0295 0.0285 4 0.0110 0.0430 0.0400 0.0385 5 0.0140 0.0550 0.0505 0.0505 6 0.0180 0.0670 0.0610 0.0605 7 0.0210 0.0820 0.0715 0.0720 8 0.0230 0.0940 0.0825 0.0815 9 0.0270 0.1080 0.0925 0.0925 10 0.0310 0.1210 0.1035 0.1025 11 0.0350 0.1340 0.1135 0.1125 12 0.0390 0.1470 0.1245 0.1225 13 0.0430 0.1590 0.1355 0.1330 14 0.0460 0.1720 0.1465 0.1435 15 0.0500 0.1850 0.1575 0.1545 16 0.0550 0.1960 0.1685 0.1650 17 0.0580 0.2080 0.1790 0.1765 18 0.0630 0.2200 0.1900 0.1885 19 0.0660 0.2320 0.2010 0.2000 20 0.0700 0.2450 0.2120 0.2115

(126) Moreover for each compound the efficacy as tyrosinase inhibitor was calculated using the following formula:

(127) $\begin{array}{c}\mathrm{Inhibitor}\mathrm{efficacy}\frac{{\mathrm{OD}}_{490\mathrm{nm}}\mathrm{assay}}{{\mathrm{OD}}_{490\mathrm{nm}}\mathrm{positive}\mathrm{control}\left(\mathrm{no}\mathrm{inhibitor}\right)}100\end{array}$

(128) The results are reported in the table 6.

(129) TABLE-US-00007 TABLE 6 Inhibitor efficacy of compounds 11 and 16 at T = 20 min Compound at 1.12 mM Inhibitor efficacy (%) Compound 11 13.5 Compound 16 13.7

(130) In these tested conditions, compounds 16 and 11 have shown a similar efficacy as human tyrosinase inhibitor in vitro.

2.4. Evaluation of the Antioxidant Activity of Compound 11

(131) The aim of the study was to evaluate the antioxidant activity of compound 11 by the spectrophotometric method of the free radical DPPH (2,2-diphenyl-1-picrylhydrazyl). Indeed, antioxidants react with DPPH.sup. (purple), a stable free radical which is reduced to DPPH-H (yellow), and in consequence, the absorbance is decreased from the DPPH.sup. radical to the DPPH-H form. The degree of discoloration indicates the scavenging potential of the antioxidant compounds in terms of hydrogen donating ability (Popovici et al. Revue de gnie industriel 2009, 4, 25-39).

(132) The tested compounds were: Trolox (()-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid, Sigma Aldrich), as reference, and compound 11.

(133) Methods

(134) Preparation of Solutions

(135) A stock solution of DPPH was prepared at 200 mol/mL in methanol and diluted in methanol in order to have a final concentration of around 150 mol/mL to obtain a DO close to 0.9 in a plate of 96 wells. The antioxidant stock solutions were prepared at 1 mg/mL in methanol and the tested solutions were prepared from these antioxidant stock solutions as described in Table 7 below.

(136) TABLE-US-00008 TABLE 7 Four working solutions for each antioxidant tested were prepared in methanol at the following concentrations: Concentration (mg/mL) Solution Solution Solution Solution Antioxidant 1 2 3 4 Trolox 0.01 0.02 0.03 0.05 Compound 11 0.05 0.10 0.15 0.2
Assay

(137) 50 L of each solution of antioxidant was added to the wells. Then, 200 L of DPPH.sup. was added to each well.

(138) The blank was prepared with only 250 l of methanol and the negative control was prepared with 200 L DPPH and 50 L of methanol.

(139) The analysis was started immediately after the addition of DPPH lasting for 2 hours (readings every 20 seconds). The absorbance was read at 515 nm. Each experiment was performed three times and had shown that the absorbance decreases while the concentration of antioxidant increased.

(140) Results

(141) In order to evaluate the antioxidant activity of compound 11, the absorbance at the time T=30 min was selected for the further calculation of EC50.

(142) TABLE-US-00009 TABLE 8 Measure of the absorbance (A) at 515 nm in the assays with compound 11 Compound 11 Solution Solution Solution Solution Negative 1 2 3 4 control A at 515 nm 0.505 0.373 0.234 0.233 (T = 30 min) A.sub.n at 515 nm 0.71 (T = 30 min)

(143) The EC50 corresponds to the concentration of antioxidant necessary to reduce by 50% the activity of DPPH. The activity of DPPH is a percentage calculated as the ratio of A/A.sub.n100%.

(144) The EC50 is expressed as a molar ratio of antioxidant (compound 11)/DPPH (Popovici et al. Revue de gnie industriel 2009, 4, 25-39). The EC50 was determined from the graph representing the percentage of DPPH in function of the above mentioned molar ratio (moles of compound 11/moles of DPPH).

(145) The antioxidant activity of compound 11 was compared to that of Trolox used as a reference (considered as 100%). The results are reported in table 9 below:

(146) TABLE-US-00010 TABLE 9 Comparison of EC50 of compound 11 and Trolox EC50 (Ratio antioxidant/DPPH) Standard Antioxidant Mean Deviation % Trolox 0.240 0.012 Compound 11 0.772 0.009

(147) The results in table 9 showed that compound 11 has an antioxidant activity, as a free radical scavenger activity, with an efficacy of 31.1% compared to that of Trolox.

2.5. In Vitro Human Skin Absorption of Compound 11

(148) The aim of this study was to evaluate the absorption of compound 11 applied to excised human skin.

(149) In vitro method using a Franz diffusion cell allows measuring the diffusion of chemicals into and across skin.

(150) The tested compound is applied to the surface of a human skin explant separating the two chambers of a Franz diffusion cell. The compound remains on the skin for a specified time under specified conditions. The receptor fluid is sampled at time points throughout the experiment and analyzed for the tested compound. The skin may also be fractioned for separate analysis in epidermis or dermis layers.

(151) Methods

(152) The transcutaneous absorption was measured on human skin collected from the abdominoplasty of a single donor. At reception, the skin was defatted and cut in several fragments in order to perform Franz experiment. After that, it was frozen until use in Franz experiment.

(153) The tested compounds were prepared at a defined concentration. After thawing, the skin was placed on the receptor side containing Ringer solution (6 mL).

(154) The tested solutions were applied to the outer surface of the skin (exchange surface: 2 cm.sup.2). The temperature was regulated at 35 C. in receptor side corresponding to a temperature of 32 C. at the surface of the skin during the experiment.

(155) The tested compounds were quantified, at T0 h and T24 h in the receptor solution bathing the inner surface of the skin (determination of flux), or at T24 h in the fractionated skin extracts (epidermis or dermis).

(156) To prepare the extracts, the skin explant is firstly divided in dermis and epidermis layers, then 500 L of methanol were added on each piece of skin and incubated in an ultrasonic bath for 4 hours. 10 l of this sample were collected, completed with 990 l of methanol and vigorously shaken (vortex). Secondly the quantification of compounds by LC-MS/MS method was realized on 100 l of the previous preparation added with 1000 l of ultrapure water and 10 l of an internal standard

(157) Conditions of LC-MS/MS Method

(158) Symmetri C18, 50 mm*2.1 mm, 3.5 m, Waters, 40 C.

(159) Gradient of elution: A-Purified water/B-Acetonitrile, flow 0.3 mL/min

(160) TABLE-US-00011 Time (min) % phase A % phase B 0.0 90 10 2.0 90 10 2.1 0 100 7 0 100 7.1 90 10 9 90 10 Q1 Q3 Mass Mass Dwell (amu) (amu) (msec) Parameters Parameters of 227 108 150 DP = 41; FP = 156; compound 11 EP = 5; CE = 22; CXP = 18 Parameters of 193 108 150 DP = 35; FP = 120; deoxyarbutin EP = 8; CE = 25; CXP = 5 Internal standard: 207 136 150 DP = 56; FP = 200; Pentylparaben EP = 10; CE = 26; CXP = 11

(161) Lower Limit of Quantification: 0.2 ng/ml for compound 11 and 0.5 ng/mL for deoxyarbutin

(162) Upper Limit of quantification: 100 ng/ml

(163) Assays and Results

(164) Quantification of Compound in the Receptor Fluid of Franz Cell

(165) 200 l of compound 11 or deoxyarbutin, at a final concentration of 2 mg/ml (in water), were deposited on the skin explant.

(166) Sampling: 500 L from the receptor fluid were removed at T0 h (replaced by Ringer solution) and at T24 h. These samples were analyzed by LC-MS/MS method for quantification of compound 11 and deoxyarbutin (results not shown). The quantity measured allowed to calculate a flux and to compare the absorption profile across the skin of compound 11 and deoxyarbutin. The results are reported in table 10 below.

(167) TABLE-US-00012 TABLE 10 skin absorption of compound 11 and deoxyarbutin Flux compound 11 (g/h/cm.sup.2) Flux deoxyarbutin (g/h/cm.sup.2) mean (n = 3) sd mean (n = 3) sd 2.71 0.63 3.98 0.37

(168) These results showed that compound 11 is able to go through the skin and that its flux in these conditions is lower than that of deoxyarbutin. There is more risk of skin resorption (risk to reach the bloodstream) for deoxyarbutin.

(169) Quantification of Compound in Skin Fractions

(170) An emulsion of compound 11 was prepared at a final concentration of 0.1 mg/mL (in 90% purified water/5% ethanol/5% DMSO) and was deposited on the skin explant (200 L).

(171) The skin extracts were prepared at T24 h and the quantification of compound 11 was performed. The experiment was repeated 3 times.

(172) The mean of compound 11 quantities measured for each fraction of dermis or epidermis is reported in the table 11 below.

(173) TABLE-US-00013 TABLE 11 quantification of compound 11 in epidermis and dermis Skin extract Compound 11 (g/g of tissue) Standard deviation (g/g) EPIDERMIS 22.3 1.3 DERMIS 5.1 0.8

(174) The results in table 11 showed that compound 11 is able to go into the different compartment of the skin and that the quantity measured in epidermis was drastically higher than the one measured in dermis. So compound 11 is able to reach the compartment of the skin where its activity is required (on melanocytes).