TERPENE COUPLING CONJUGATE

Abstract

The subject matter of the present invention relates to the use of an optionally branched linear terpene having at most one C═C unsaturation for the production of conjugates provided with self-assembly properties, as well as a self-assembly agent of formula (I):

X(-Spacer-Y-Terpene).sub.p   (I)

in which: “Terpene” is linear, optionally branched, having at most one C═C unsaturation; “Y” is a bond or a molecular fragment with a biodegradable bond; “Spacer” is a bond or a fragment comprising at least one carbon atom; “X” is a molecular fragment comprising at least one biodegradable bond; “p” ranges between 0.1 and 4; and the “-Spacer-Y-” group optionally can be a bond,
as well as the conjugate resulting from the combination of the self-assembly agent of formula (I) with an active molecule MA.

Claims

1. A method of producing conjugates with self-assembly properties comprising a linear terpene, optionally branched, having at most one C═C unsaturation.

2. The method according to claim 1, wherein the terpene comprises between 15 and 25 carbon atoms.

3. The method according to claim 1, wherein the terpene is bio-sourced.

4. The method according to any one of claim 1, wherein the terpene is phytol or a phytol derivative, such as isophytol.

5. A self-assembly agent of formula (I):
X(-Spacer-Y-Terpene).sub.p   (I) wherein: “Terpene” is as defined in claim 1; “Y” is a bond or a molecular fragment with a biodegradable bond; “Spacer” is a bond or a fragment comprising at least one carbon atom; “X” is a molecular fragment comprising at least one biodegradable bond; “p” is comprised between 0.1 and 4; and the “-Spacer-Y-” group optionally can be a bond.

6. The self-assembly agent according to claim 5, wherein the spacer comprises any one of the following fragments: ##STR00033## wherein “n” are independently whole numbers ranging between 0 and 6, preferably ranging between 1 and 4.

7. The self-assembly agent according to claim 5, wherein “Y” and/or “X” comprise any one of the following fragments: ##STR00034## wherein: “u” are independently whole numbers comprised between 0 and 6, preferably between 0 and 1; “R” is a hydrogen atom, a C1-C6 alkyl group, a C4-C8 aromatic group or a monocyclic or polycyclic (C1-C6)-aryl (C4-C8) alkyl group, for example, R can represent a hydrogen atom, a methyl, ethyl, propyl, butyl, phenyl, or even a benzyl group.

8. The self-assembly agent according to claim 5, wherein said at least one biodegradable bond of “X” comprises an ionic bond and/or the biodegradable bond of “Y” is a covalent bond.

9. A conjugate with self-assembly properties of formula (II):
MA(-AA).sub.k   (II) wherein “AA” is a self-assembly agent as defined in claim 5; “MA” is a biologically active molecule; and “k” is comprised between 0.1 and 6, as well as to the pharmaceutically acceptable salts and/or solvates thereof.

10. The conjugate according to claim 9, wherein the MA is selected from ibuprofen, paracetamol, 4-nBu-resorcinol, 6-nHex-resorcinol, azelaic acid, caffeic acid, ferulic acid, glycyrrhizic acid, hyaluronic acid, kojic acid, linoleic acid, lipoic acid, adenosine di-phosphate, adenosine mono-phosphate, adenosine tri-phosphate, aescin, arbutin, bakuchiol, bis-(Et)-Hexyl-dihydroxymethoxybenzyl-malonate, bisabolole, boldine, caffeine, canabidiole, carotenoids, coenzyme A, coenzyme Q10, dihydroxy acetone, dihydroxymethylchromonyl-palmitate, D-panthenol, ectoin, glabridin, idebenone, L-carnitine, licochalchone A, menthol, N-acetyl-tetrapeptide-2, N-acetyl-tetrapeptide-9, niaccinamide, oleuropein, phycocyanin, pro-xylan, resorcinol, resveratrol, superoxide dismutase, tripeptide-29, tyamine pyrrophosphate, vanillin, vitamin A, vitamin B3, vitamin B8, vitamin C and vitamin E.

Description

FIGURES

[0148] FIG. 1 is a graph showing the stability of the nanoparticulate suspensions over time for conjugates 3 (dashed line) and conjugates 4 (solid line).

[0149] FIG. 2 is a graph showing the stability of the nanoparticulate suspensions over time for conjugates 7 (large spaced dashed line), conjugates 8 (solid line) and conjugates 9 (closely spaced dashed line).

[0150] FIG. 1 is a histogram showing the variation of areas as a function of time for conjugate 9 (black column) and retinol (gray column), at 0, 1 and 2 days.

EXAMPLES

[0151] The following abbreviations are provided for the following examples:

[0152] CAS: “Chemical Abstracts Service” English international standard.

[0153] Dm: Dermis.

[0154] Ø: Diameter.

[0155] Ep: Epidermis.

[0156] e: Thickness.

[0157] FZ: Franz type diffusion cell.

[0158] h: Hour.

[0159] INCI: International Nomenclature of Cosmetic Ingredients.

[0160] LOD: Limit of detection threshold.

[0161] LOQ: Limit of quantification threshold.

[0162] LR: Liquid Receiver.

[0163] OECD: Organization for Economic Cooperation and Development.

[0164] PBS: Phosphate buffered saline solution (pH 7.4).

[0165] IWL: Insensible Water Loss.

[0166] SC: Stratum Corneum.

[0167] sem: Standard error of the mean.

[0168] SD: Standard deviation.

[0169] V: Volume.

[0170] rpm: revolution(s) per minute.

[0171] The .sup.1H and .sup.13C NMR spectra were measured on a Brucker Advance 300 MHz spectrometer in CDCl.sub.3 using tetramethylsilane (TMS) as a reference. Chemical shifts are expressed in ppm.

[0172] The mean particle sizes were measured using the “Dynamic Light Scattering” (DLS) method on a Malvern-Panalytical Nano-Sizer ZS® at 25° C. with a detection angle of 173° and a wavelength of 633 nm. The reported sizes are determined by the mean of three measurements. The measurements were carried out in polystyrene cuvettes.

[0173] The HPLC analyzes were carried out with an “Ultimate 3000 system” chain, Dionex®, France on a C18 “Vintage series KR C18—5 μm-150×4.6 mm (Interchim®) column. The samples were detected by UV absorption at λ=325 nm.

Example 1: Synthesized Products

Preparation of Phytyl Mono-Succinate

[0174] ##STR00005##

[0175] The following are added to a solution of phytol (10.00 g, 33.78 mmol, 1.0 equiv) and succinic anydride (3.54 g, 35.47 mmol, 1.05 equiv) in PhMe (135 mL): Et.sub.3N (5.40 mL, 38.85 mmol, 1.05 equiv), then DMAP (204 mg, 1.69 mmol, 0.05 equiv), and the reaction is heated to 50° C. with stirring for 7 h.

[0176] The TLC analysis (EtOAC/CyH—60:40, revealed by CAM) shows complete conversion of the starting material. The formation of the desired compound is confirmed by a comparison with an authentic sample.

[0177] The reaction medium is hydrolyzed with aq. saturated NH.sub.4Cl, then transferred to a separating funnel and the organic phase is separated. The aqueous phase is extracted with EtOAc (3×50 mL). The organic extracts are combined, washed with aq. HCl (0.1 N), dried over MgSO.sub.4, filtered and concentrated under reduced pressure.

[0178] The concentrate is then purified by silica gel chromatography (EtOAc/CyH—30:70 to 50:50) to provide the expected compound (12.84 g, 32.42 mmol, 96%) in the form of a yellow oil.

[0179] .sup.1H NMR (300 MHz, CDCl.sub.3): δ 5.33 (td, J=7.1, 1.3 Hz, 1H), 4.62 (d, J=7.1 Hz, 2H), 2.91-2.47 (m, 4H), 1.97 (t, J=7.6 Hz, 2H), 1.72 (brs, 3H), 1.55-1.00 (m, 19H), 0.92-0.73 (m, 12H) ppm.

[0180] .sup.13C NMR (75 MHz, CDCl.sub.3): δ 178.3, 172.2, 142.8, 117.9, 61.7, 39.8, 39.4, 37.4, 37.4, 37.3, 36.6, 32.8, 32.7, 29.0, 28.9, 27.8, 25.0, 24.8, 24.5, 22.7, 22.6, 19.7, 19.7, 16.3 ppm.

Preparation of Phytyl Mono-Dithioglycolate

[0181] ##STR00006##

[0182] Dithioglycolic acid (0.5 g, 2.74 mmol, 2.95 equiv) and acetic anhydride (2 ml) are stirred under an inert atmosphere for 2 h at 21° C. Then the mixture is azeotropically distilled under reduced pressure with PhMe (3×20 mL), while controlling the temperature of the bath (<30° C.). The residue that is obtained is then used in the next step without further purification. The anhydride that is obtained is dissolved in CH.sub.2Cl.sub.2 (20 mL), then phytol (275 mg, 0.928 mmol, 1.0 equiv) and DMAP (11 mg, 0.092 mmol, 0.1 equiv) are added. The reaction is stirred at 21° C. for 1 h and the end of the reaction is checked by TLC (EtOAc/CyH=1:1). The crude compound is then isolated by filtration and dried under reduced pressure (T<30° C.) to yield a yellow semi-solid (0.627 g). The residue that is obtained is then purified by silica gel chromatography (EtOAc/CyH=20:80+1% AcOH) to yield the expected compound in the form of a yellow solid (338 mg, 0.734 mmol, 79%).

[0183] .sup.1H NMR (300 MHz, CDCl.sub.3): δ 10.84 (s, 1H), 5.37 (t, J=7.2 Hz, 1H), 4.69 (dd, J=7.0, 3.7 Hz, 2H), 3.63 (dd, J=11.6, 3.9 Hz, 5H), 2.18-1.90 (m, 2H), 1.72 (d, J=3.6 Hz, 4H), 1.62-0.98 (m, 20H), 0.87 (td, J=6.3, 4.0 Hz, 12H) ppm.

[0184] General Procedure A for Molecules Containing Carboxylic Acids:

[0185] The following is added to a solution of carboxylic acid (1.05 equiv) in CH.sub.2Cl.sub.2 (0.2 M): EDC-HCl (1.1 equiv) and the reaction medium is stirred for 10 min. Phytol (1.0 equiv) followed by DMAP (0.1 equiv) are added and the reaction medium is stirred at 21° C. for 12 h.

[0186] The reaction medium is hydrolyzed with aq. saturated NH.sub.4Cl, then transferred to a separating funnel and the organic phase is separated. The aqueous phase is extracted with EtOAc (3×30 mL). The organic extracts are combined, washed with aq. saturated NaCl (2×30 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure.

Phytyl Nicotinate

[0187] ##STR00007##

[0188] Prepared from nicotinic acid (200 mg, 1.625 mmol).

[0189] The residue that is obtained is purified by silica gel chromatography (EtOAc/CyH—0:100 at 20:80) to provide the expected compound (474 mg, 1.182 mmol, 73%) in the form of a yellow oil.

[0190] .sup.1H NMR (300 MHz, CDCl.sub.3): δ 9.24 (s, 1H), 8.78 (d, J=3.8 Hz, 1H), 8.36 (dt, J=7.8, 1.9 Hz, 1H), 7.44 (dd, J=7.8, 5.0 Hz, 1H), 5.46 (tq, J=7.2, 1.2 Hz, 1H), 4.88 (d, J=7.2 Hz, 2H), 2.04 (t, J=7.6 Hz, 2H), 1.76 (d, J=1.2 Hz, 3H), 1.57-1.00 (m, 19H), 0.88-0.80 (m, 12H) ppm.

[0191] .sup.13C NMR (75 MHz, CDCl.sub.3): δ 165.0, 153.1, 150.9, 143.2, 136.8, 126.3, 123.0, 117.7, 62.1, 39.8, 39.3, 37.3, 37.3, 37.2, 36.5, 32.7, 32.6, 27.9, 24.9, 24.7, 24.4, 22.6, 22.5, 19.7, 19.6, 16.4 ppm.

Phytyl Sinapinate

[0192] ##STR00008##

[0193] Prepared from sinapic acid (113 mg, 0.530 mmol).

[0194] The residue that is obtained is purified by silica gel chromatography (EtOAc/CyH—0:100 to 30:70) to provide the expected compound (205 mg, 0.341 mmol, 67%) in the form of a waxy white solid.

[0195] .sup.1H NMR (300 MHz, CDCl.sub.3): δ 7.75 (d, J=15.9 Hz, 1H), 6.78 (s, 2H), 6.39 (d, J=15.9 Hz, 1H), 5.34 (tq, J=7.2, 1.2 Hz, 1H), 4.64 (d, J=7.2 Hz, 2H), 3.85 (s, 6H), 2.98 (t, J=7.2 Hz, 2H), 2.77 (t, J=7.2 Hz, 2H), 2.00 (t, J=7.5 Hz, 2H), 1.69 (s, 3H), 1.58-0.98 (m, 19H), 0.89-0.82 (m, 12H) ppm.

[0196] .sup.13C NMR (75 MHz, CDCl.sub.3): δ 172.1, 172.0, 170.0, 152.5, 146.7, 142.9, 132.4, 130.8, 118.0, 117.7, 105.0, 61.8, 56.2 (2C), 39.9, 39.4, 37.5, 37.4, 37.3, 36.7, 32.8, 32.7, 29.8, 29.4, 28.9, 28.0, 25.1, 24.8, 24.5, 22.8, 22.7, 19.8, 19.8, 16.4 ppm.

Phytyl Ibuprofenate

[0197] ##STR00009##

[0198] Prepared from ibuprofen (206 mg, 1.00 mmol).

[0199] The residue that is obtained is purified by silica gel chromatography (EtOAc/CyH—30:70) to provide the expected compound (426 mg, 0.880 mmol, 88%) in the form of a pale yellow oil.

[0200] .sup.1H NMR (300 MHz, CDCl.sub.3): δ 7.49-7.35 (d, 2H), 7.28-7.13 (d, 2H), 5.49 (t, 1H), 4.79-4.66 (d, 2H), 3.46 (q, 1H), 2.62-2.45 (d, 2H), 2.25 (t, 2H), 2.04-2.00 (s, 3H), 1.77-1.73 (m, 1H), 1.66 (m, 1H), 1.59 (d, 3H), 1.57-1.22 (m, 14H), 1.07-1.06 (2d, 6H), 1.11 (s, 25H), 1.02-0.93 (m, 12H) ppm.

[0201] .sup.13C NMR (75 MHz, CDCl.sub.3): δ 177.1, 142.0, 140.1, 140.0, 131.1, 131.0, 126.5, 126.5, 121.2, 61.5, 45.7, 45.3, 39.4, 39.3, 36.81 (3C), 35.8, 34.82 (2C), 28.3, 27.6, 25.1, 23.9, 23.7, 22.73 (2C), 22.2 (2C), 20.40 (2C), 20.3, 16.5 ppm.

Phytyl Diclofenate

[0202] ##STR00010##

[0203] Prepared from diclofenac (296 mg, 1.00 mmol).

[0204] The residue that is obtained is purified by silica gel chromatography (EtOAc/CyH—15:85) to provide the expected compound (473 mg, 0.83 mmol, 83%) in the form of a pale yellow oil.

[0205] .sup.1H NMR (300 MHz, CDCl.sub.3): δ 7.36 (d, J=7.5 Hz, 1H), 7.23 (t, J=7.5 Hz, 1H), 7.13 (d, J=7.5 Hz, 2H), 7.03 (d, J=7.5 Hz, 1H), 6.93 (t, J=7.5 Hz, 1H), 6.80 (t, J=7.5 Hz, 1H), 5.48 (t, J=6.2 Hz, 1H), 4.80 (s, 1H), 4.73 (d, J=6.2 Hz, 2H), 3.61 (s, 2H), 2.08 (t, J=5.5 Hz, 2H), 1.66 (t, J=2.9 Hz, 4H), 1.65-1.60 (m, 2H), 1.54 (dq, J=14.6, 7.2 Hz, 1H), 1.43-1.15 (m, 16H), 1.01 (d, J=6.4 Hz, 12H) ppm.

[0206] .sup.13C NMR (75 MHz, CDCl.sub.3): δ 172.4, 145.7, 140.1, 137.9, 132.4, 130.62, 130.6, 130.3, 129.8 (2C), 123.5, 122.2, 121.2, 120.5, 118.7, 60.8, 39.3, 39.3, 36.8 (3C), 36.3, 35.8, 34.8 (2C), 28.3, 25.1, 23.9, 23.7, 22.7 (2C), 20.4, 20.4, 16.5 ppm.

[0207] General Procedure B for Molecules Containing Alcohols:

[0208] The following is added to a solution of phytyl mono-succinate (1.05 equiv) in CH.sub.2Cl.sub.2 (0.2 M): EDC-HCl (1.1 equiv) and the reaction medium is stirred for 10 min. The corresponding alcohol (1.0 equiv) followed by DMAP (0.1 equiv) are added and the reaction medium is stirred at 21° C. for 12 h.

[0209] The reaction medium is hydrolyzed with aq. NH.sub.4Cl, then transferred to a separating funnel and the organic phase is separated. The aqueous phase is extracted with EtOAc (3×30 mL). The organic extracts are combined, washed with aq. saturated NaCl (2×30 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure.

Phytyl (4-tert-butyl cyclohexyl) succinate

[0210] ##STR00011##

[0211] Prepared from 4-tert-butyl cyclohexanol (79 mg, 0.530 mmol, as an 80:20 mixture of cis and trans isomers).

[0212] The residue that is obtained is purified by silica gel filtration (10 cm) and eluted with EtOAc/CyH (10:90) to provide the expected compound (260 mg, 0.488 mmol, 97%, isolated as an 80:20 mixture of cis and trans isomers) as a colorless oil.

[0213] .sup.1H NMR (300 MHz, CDCl.sub.3): δ 5.33 (m, 1H), 4.66 (m, 3H), 2.60 (m, 4H), 1.98 (t, J=7.5 Hz, 4H), 1.80 (m, 2H), 1.66 (brs, 3H), 1.59-0.98 (m, 28H), 0.93-0.75 (m, 21H) ppm.

[0214] .sup.13C NMR (75 MHz, CDCl.sub.3): δ 172.3, 171.8, 142.7, 118.1, 47.2, 39.9, 39.5, 37.5, 37.5, 37.4, 36.7, 32.88, 32.8, 32.3, 32.1, 29.8, 29.6, 29.4, 28.1, 27.7, 27.5, 25.5, 25.1, 24.9, 24.6, 22.8, 22.7, 19.8, 19.8, 16.4 ppm.

(Vanillyl) Phytyl Succinate

[0215] ##STR00012##

[0216] Prepared from vanillin (900 mg, 1.316 mmol).

[0217] The residue that is obtained is purified by silica gel chromatography (EtOAc/CyH—5:95 to 15:85) to provide the expected compound (521 mg, 0.489 mmol, 57%) in the form of a pale yellow oil.

[0218] .sup.1H NMR (300 MHz, CDCl.sub.3): δ 9.94 (s, 1H), 7.49 (s, 1H), 7.46 (dd, J=7.8, 1.8 Hz, 1H), 7.23 (d, J=7.8 Hz, 1H), 5.34 (td, J=7.1, 1.1 Hz, 1H), 4.64 (d, J=7.1 Hz, 2H), 3.89 (s, 3H), 2.95 (t, J=6.8 Hz, 2H), 2.76 (t, J=6.9 Hz, 2H), 2.08-1.92 (m, 2H), 1.69 (s, 3H), 1.56-1.01 (m, 19H), 0.84 (dd, J=9.3, 3.7 Hz, 12H) ppm.

[0219] .sup.13C NMR (75 MHz, CDCl.sub.3): δ 190.9, 171.9, 169.9, 151.9, 144.9, 142.9, 135.3, 124.6, 123.4, 117.9, 110.9, 61.8, 56.0, 39.9, 39.4, 37.4, 37.4, 37.3, 36.6, 32.8, 32.7, 29.2, 29.0, 28.0, 25.0, 24.8, 24.5, 22.7, 22.6, 19.8, 19.7, 16.4 ppm.

Phytyl Retinyl Succinate

[0220] ##STR00013##

[0221] Prepared from retinol (200 mg, 0.699 mmol).

[0222] The residue that is obtained is purified by silica gel chromatography (MTBE/CyH—10:90) to provide the expected compound (115 mg, 0.173 mmol, 25%) in the form of a yellow oil.

[0223] .sup.1H NMR (300 MHz, CDCl.sub.3): δ 6.64 (dd, J=15.0, 11.3 Hz, 1H), 6.27 (d, J=15.1 Hz, 1H), 6.18 (d, J=16.2 Hz, 1H), 6.13 (d, J=14.5 Hz, 1H), 6.10 (d, J=16.5 Hz, 1H), 5.60 (t, J=7.1 Hz, 1H), 5.32 (t, J=7.0 Hz, 1H), 4.75 (d, J=7.2 Hz, 2H), 4.61 (d, J=7.1 Hz, 2H), 2.64 (s, 4H), 2.05-1.98 (m, 4H), 1.95 (s, 3H), 1.88 (s, 3H), 1.71 (s, 3H), 1.68 (s, 3H), 1.63-1.05 (m, 23H), 1.02 (s, 6H), 0.85 (t, J=6.3 Hz, 12H).

[0224] .sup.13C NMR (75 MHz, CDCl.sub.3): δ 172.4, 172.3, 143.0, 139.3, 137.9, 137.7, 136.7, 135.9, 130.1, 129.4, 127.1, 125.9, 124.4, 118.0, 61.8, 61.6, 40.0, 39.7, 39.5, 37.5, 37.5, 37.4, 36.8, 34.4, 33.2, 32.9, 32.8, 29.3 (2C), 29.1 (2C), 28.1, 27.1, 25.2, 24.9, 24.6, 22.8, 22.7, 21.8, 19.9, 19.8, 19.4, 16.5, 12.9 ppm.

(1,3-dimethyl acetonidyl)penthenoyl-(phytyl)-dithiodiglycolate

[0225] ##STR00014##

[0226] Prepared from panthenol acetonide (338 mg, 0.734 mmol).

[0227] The residue that is obtained is purified by silica gel chromatography (EtOAc/CyH—20:80) to provide the expected compound (369 mg, 0.536 mmol, 73%) in the form of a colorless oil.

[0228] .sup.1H NMR (300 MHz, CDCl.sub.3): δ 6.74 (s, 1H), 5.34 (t, J=6.6 Hz, 1H), 4.66 (d, J=7.2 Hz, 2H), 4.20 (t, J=6.2 Hz, 2H), 4.08 (s, 1H), 3.68 (d, J=11.8 Hz, 1H), 3.51-3.16 (m, 3H), 3.32-3.17 (m, 1H), 1.99 (t, J=7.6 Hz, 2H), 1.95-1.84 (m, 2H), 1.69 (s, 2H), 1.46 (s, 3H), 1.42 (s, 2H), 1.58-0.92 (m, 29H), 1.04 (s, 3H), 0.98 (s, 3H), 0.84 (d, J=6.5 Hz, 8H) ppm.

[0229] General Procedure C:

[0230] The following are successively added to a solution of carboxylic acid (1.0 equiv) and 2-hydroxyethyl disulfide (5.0 equiv) in THE (0.2 M): DCC (1.3 equiv), DMAP (0.1 equiv), then Et.sub.3N (2 equiv) and the reaction medium is then stirred at 21° C. for 12 h. The reaction medium is then filtered, concentrated under reduced pressure and purified by silica gel chromatography.

[0231] Compound “11”:

##STR00015##

[0232] Prepared from ibuprofen (206 mg, 1 mmol).

[0233] The residue that is obtained is purified by silica gel chromatography (EtOAc/CyH—30:70 to 50:50) to provide the expected compound (250 mg, 0.762 mmol, 76%) in the form of a colorless oil.

[0234] .sup.1H NMR (300 MHz, CDCl.sub.3): δ 7.22 (d, J=8.1 Hz, 2H), 7.11 (d, J=8.1 Hz, 2H), 4.44-4.22 (m, 2H), 3.83 (t, J=5.9 Hz, 2H), 3.73 (q, J=7.2 Hz, 1H), 2.88 (t, J=6.7 Hz, 2H), 2.83 (t, J=5.9 Hz, 2H), 2.47 (d, J=7.2 Hz, 2H), 1.97-1.75 (m, 1H), 1.51 (d, J=7.2 Hz, 3H), 0.92 (d, J=6.6 Hz, 6H) ppm.

[0235] .sup.13C NMR (75 MHz, CDCl.sub.3): δ 172.05, 143.14, 133.71, 130.29, 130.29, 130.06, 130.06, 62.69, 61.13, 45.74, 40.95, 40.81, 38.51, 27.63, 22.18, 22.18 ppm.

[0236] Compound “12”:

##STR00016##

[0237] Prepared from diclofenac (296 mg, 1 mmol).

[0238] The residue that is obtained is purified by silica gel chromatography (EtOAc/CyH—40:60) to provide the expected compound (203 mg, 0.469 mmol, 47%) in the form of a yellow solid.

[0239] .sup.1H NMR (300 MHz, CDCl.sub.3): δ 7.35 (d, J=7.5 Hz, 1H), 7.22 (t, J=7.5 Hz, 1H), 7.16 (d, J=7.5 Hz, 2H), 7.01 (d, J=7.5 Hz, 1H), 6.90 (t, J=7.5 Hz, 1H), 6.84 (q, J=7.4 Hz, 1H), 4.47 (s, 1H), 4.44 (t, J=5.0 Hz, 2H), 3.79 (t, J=7.7 Hz, 2H), 3.42 (s, 2H), 2.81 (t, J=5.0 Hz, 2H), 2.74 (t, J=7.7 Hz, 2H) ppm.

[0240] .sup.13C NMR (75 MHz, CDCl.sub.3): δ 172.13, 145.82, 143.69, 132.41, 130.31, 130.18, 130.18, 129.80, 129.80, 123.51, 122.23, 120.13, 118.69, 62.69, 61.13, 40.81, 38.51, 37.01 ppm.

[0241] General Procedure D:

[0242] The following is added to a cooled solution (0° C.) of diphosgene (2.5 equiv) in CH.sub.2Cl.sub.2 (5 mL): a solution of the corresponding alcohol (1.0 equiv) and DIPEA (5.0 equiv) in CH.sub.2Cl.sub.2 (5 mL). After 45 min stirring at 0° C., the reaction medium is concentrated. The residue is then re-dissolved in CH.sub.2Cl.sub.2 (5 mL), and a solution composed of phytol (1.2 equiv), Et.sub.3N (1.2 equiv) and DMAP (0.1 equiv) in CH.sub.2Cl.sub.2 (5 mL) is then added at 0° C. After 1.5 h with stirring, the reaction medium is hydrolyzed with aq. HCl (1 N, 10 mL), then extracted with CH.sub.2Cl.sub.2 (3×20 mL). The organic phases are combined, washed with aq. saturated NaCl (2×20 mL), then dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure.

[0243] Compound “5”:

##STR00017##

[0244] Prepared from ibuprofen 11 derivative (420 mg, 1.28 mmol).

[0245] The residue that is obtained is purified by silica gel chromatography (EtOAc/CyH—3:97) to provide the expected compound (570 mg, 0.857 mmol, 67%) in the form of a colorless oil.

[0246] M=665.05 g/mole

[0247] SM: 665.6 [M+H]

[0248] Compound “6”:

##STR00018##

[0249] Prepared from diclofenac derivative 12 (127 mg, 0.295 mmol).

[0250] The residue that is obtained is purified by silica gel chromatography (EtOAc/CyH—3:97) to provide the expected compound (138 mg, 0.182 mmol, 62%) in the form of a yellow oil.

[0251] M=754.91 g/mole

[0252] SM: 754.5 [M+H]

TABLE-US-00001 TABLE 1 Examples of synthesized products Products Structures 1 [00019] 2 [00020] 3 [00021] 4 [00022] 5 [00023] 6 [00024] 7 [00025] 8 [00026] 9 [00027] 10 [00028] 11 [00029] 12 [00030] 13 [00031] 14 [00032]

[0253] With the exception of compounds 11 and 12, these structures all comprise a phytol fragment.

Example 2: Examples of Self-Assembly

[0254] The self-assembly properties were confirmed for all these bio-conjugates. Indeed, nanoobjects could be formed using the nanoprecipitation/solvent evaporation method.

[0255] The formation stages are:

(1) Dissolution of the phytolized conjugate in an organic solvent miscible with water;
(2) Nanoprecipitation in water;
(3) Evaporation of the solvent under reduced pressure.

[0256] For all the conjugates, the prepared nanoobjects were characterized and generally have the following characteristics:

[0257] Observed size ranging between 150 and 170 nm;

[0258] Polydispersity index (PDI) ranging between 0.070 and 0.270;

[0259] Zeta potential ranging between −19.0 and −35 mV.

[0260] The stability of these suspensions was studied over time and shows that the suspensions are stable. In certain borderline cases, the nanoobjects tend to aggregate and lead to precipitation of the conjugates in the medium. However, it has been shown that the stability of these conjugate suspensions can be improved by adding surfactants such as, for example, Pluronic F68. Thus, certain conjugates have had a tendency to aggregate and have been able to lead to stable suspensions for up to 10 days by the addition of 0.5 to 5% (m/m) of Pluronic F68. Other surfactants, like coconut amidopropyl betaine, sodium laureth sulfate, sorbitan palmitate, lauryl glucoside, fatty alcohols, acids and the mixture thereof, phospholipids, phosphatidyl choline have also been used and are currently being studied for their stabilizing effect on conjugate suspensions.

Example 3: Physico-Chemical Study

[0261] Nanoprecipitation:

[0262] A solution of conjugate in EtOH (2 mg for 0.5 mL) is added dropwise to vigorously stirred MiliQ water (1 mL). The formation of nanoparticles is observed in that the solution becomes partially cloudy. The suspension that is thus obtained is transferred to a flask and the EtOH is evaporated on a rotary evaporator (200 mbar for 5 min, then 130 mbar for 1 min at 40° C. and at 50 revolutions per minute).

[0263] The residual suspension is transferred to a vial and stored at 23° C.

[0264] The samples are prepared as follows: 40 μL of the residual suspension is dissolved in 500 μL of MiliQ H.sub.2O.

[0265] 1. Stability of the Nanoparticulate Suspensions

[0266] The stability of the suspensions was measured over time and the results are summarized in Tables 2 and 3 and in the graph of FIG. 1 and in the graph of FIG. 2.

[0267] Table 2: Stability of the nanoparticulate suspensions over time (Conjugate 3 and 4)

TABLE-US-00002 TABLE 2 d (nm) Time (h) Conjugate 3 Conjugate 4 1 132.9 270.0 24 154.5 369.7 168 156.6 502.4 196 164.1 436.8

[0268] Table 3: Stability of the nanoparticulate suspensions over time (Conjugate 7, 8 and 9 and 4).

TABLE-US-00003 TABLE 3 d (nm) Time (h) Conjugate 7 Conjugate 8 Conjugate 9 1 256.2 208.9 167.4 24 269.7 227.8 165.2 168 271.2 214.0 151.8 196 269.6 233.3 131.3

[0269] 2. Influence of the Phytolization Process on the Stability of Retinol:

[0270] Vitamin A (retinol) is known to be sensitive to oxygen and UV radiation. The phytolization process allows retinol to be stabilized. This protection was demonstrated by HPLC monitoring of a nanoparticulate suspension of the conjugate 9 at 20° C. compared to a solution of retinol under the same conditions.

[0271] The solutions of retinol and conjugate 9 in nanoparticulate form (6 mg/L in an H.sub.2O/iPrOH 1:1 mixture) were stored at 21° C. in ambient light and analyzed by HPLC over time (24 and 48 h).

[0272] Table 4: HPLC condition.

TABLE-US-00004 TABLE 4 Column Interchim Vintage series KR C18-5 micometers -150 × 4.6 mm T Column 25° C. Elution iPrOH/H2O 85:15 isocratic Flow 1 mL/min Injection volume 20 microliters Tr Retinol = 3.41 min Tr conjugate 9 = 11.4 min

[0273] The variation in the area of the compounds over time is shown over time (average of two measurements)—see Table 5 and the graph of FIG. 3.

[0274] Table 5: Variation of areas as a function of time.

TABLE-US-00005 TABLE 5 t0 t24 h t48 h Conju- Conju- Conju- gate 9 Retinol gate 9 Retinol gate 9 Retinol A (mU) A (mU) A (mU) A (mU) A (mU) A (mU) 4.1604 0.0502 3.4609 0.0354 2.9536 0.0218 4.0495 0.0694 3.3380 0.0360 2.9385 0.0273 Average 4.1050 0.0598 3.3995 0.0357 2.9461 0.0246 Variation 0.0000 0.0000 0.1719 0.4030 0.2823 0.5895

[0275] CCL: Retinol breaks down twice as quickly when in it is free form.

[0276] 3. Inclusion of the Conjugates in Cosmetic Formulations:

[0277] Preparation of a solution of conjugate 9 at 1%.

[0278] 800 mg of conjugate 9 is dissolved in EtOH (40 mL), then poured dropwise into H.sub.2O (80 mL) with vigorous stirring (addition 1 mL/min). The suspension is then concentrated in a rotavapor (T=40° C., 50 rpm at 200, then 130 mbar). The volume is then adjusted to 80 mL by adding H.sub.2O.

[0279] Face cream:

[0280] The face cream was prepared as follows:

[0281] Operating mode: Homogenize a phase A (see Table 6), then introduce a phase B (see Table 6) and homogenize for 10 minutes with vigorous stirring (1500 revolutions/min). Produce the emulsion by pouring phase C (see Table 6) into the mixture and then homogenize with vigorous stirring for 10 min. Finally, introduce a phase D (see Table 6).

[0282] Table 6: Composition of a face cream.

TABLE-US-00006 TABLE 6 Phase Ingredients (INCI) % A Solution of Conjugate 9 at 1% 75.22 Glycerin 3 B Sodium Polyacrylate 0.8 C Coco-Caprylate/Caprate 6.66 Caprylic/Capric Triglyceride 6.66 Olus Oil 6.66 D Phenoxyethanol (and) Ethylhexylglycerin 1

[0283] A smooth, pale yellow cream is thus obtained.

[0284] Aqueous gel:

[0285] A conjugate 9 was included in a cosmetic gel as follows:

[0286] Operating mode: Homogenize a phase A (see Table 7) with vigorous stirring (1500 revolutions/min) for 20 minutes. Then, introduce a phase B (see Table 7) and homogenize until the powders have completely dissolved. Prepare the premix for phase C (see Table 7), then introduce it into the mixture and homogenize with vigorous stirring for 15 minutes. Introduce a phase D (see Table 7), then homogenize until the powders have completely dissolved. Finally, adjust to pH 5.0-5.5 with phase E.

[0287] Table 6: Composition of an aqueous gel.

TABLE-US-00007 TABLE 7 Phase Ingredients (INCI) % A Solution of Conjugate 9 at 1% 80.2 Triethyl Citrate 5 B Erythritol 5 C Xanthan Gum 1 Propylene glycol 3 Potassium Lactate 5 D Aqua (and) Citric Acid QSP

[0288] A bright yellow gel is thus obtained.

Example 4: Biological Application

[0289] 1. Aim

[0290] The aim of this study is the ex vivo evaluation, on skin explants of human origin, of the promoting effect of the transdermal passage of an innovative skin vectorization system according to the present invention. The tracer that is used to compare the 2 formulations is retinol.

[0291] Each formulation is applied to 3 explants from a single donor. At the end of the contact period (24 hours), the total concentration of retinol is measured in different skin layers (Stratum Corneum, Epidermis and Dermis) and diffusion kinetics are performed on 4 points.

[0292] In order to study the promoting effect of the concept of phytolization, two formulas were compared: [0293] F1: Retinol (0.9% retinol equivalent) vectorized with phytol in nanoparticulate form; [0294] F2: Retinol (0.9% retinol equivalent) in free form with a propenetrating agent (5% transcutol) included in the dosage form.

[0295] NB: The use of trancutol is regulated and limited to 2.6% for unrinsed body applications.

[0296] 2. Materials and Methods

[0297] 2.1 Products Tested and Molecule Assayed

[0298] 2.1.1. Inclusion of Retinol in Cosmetic Formulations:

[0299] Preparation of a solution of conjugate 9 at 1%.

[0300] 800 mg of conjugate 9 is dissolved in EtOH (40 mL), then poured dropwise into H.sub.2O (80 mL) with vigorous stirring (addition 1 mL/min). The suspension is then concentrated in a rotavapor (T=40° C., 50 rpm at 200, then 130 mbar). The volume is then adjusted to 80 mL by adding H.sub.2O.

[0301] Formula F1:

[0302] Preparation of a solution of conjugate 9 at 3%.

[0303] 2.1 g of conjugate 9 is dissolved in EtOH (35 mL), then poured dropwise into H.sub.2O (70 mL) with vigorous stirring (addition 1 mL/min). The suspension is then concentrated in a rotavapor (T=40° C., 50 rpm at 200, then 130 mbar). The volume is then adjusted to 70 mL by adding H.sub.2O.

[0304] Formula F1 was prepared as below.

[0305] Table 8: formula F1.

TABLE-US-00008 TABLE 8 Phase Ingredients (INCI) % A Solution of Conjugate 9 at 3% 70 Water 7.22 Glycerin 3 B Sodium Polyacrylate 0.8 C Coco-Caprylate/Caprate 6.66 Caprylic/Capric Triglyceride 6.66 Olus Oil 4.66 D Phenoxyethanol (and) Ethylhexylglycerin 1

[0306] Operating mode: Homogenize phase A, then introduce phase B and homogenize for 10 minutes with vigorous stirring (1500 revolutions/min). Make the emulsion by pouring phase C into the mixture, then homogenize with vigorous stirring for 10 min. Finally, introduce phase D.

[0307] A smooth, pale yellow cream is thus obtained.

[0308] Formula F2:

[0309] Preparation of a 1.29% retinol solution containing 7.14% of 2-(2-ethoxyethoxy) ethanol (Transcutol).

[0310] 0.9 g of retinol is dissolved in EtOH (35 mL), then poured dropwise into an aqueous solution of 2-(2-ethoxyethoxy) ethanol (Transcutol) (5 g in 70 mL) with vigorous stirring (addition 1 mL/min). The suspension is then concentrated in a rotavapor (T=40° C., 50 rpm at 200, then 130 mbar). The volume is then adjusted to 70 mL by adding H.sub.2O.

[0311] Formula F2 was prepared as below.

[0312] Table 9: formula F2.

TABLE-US-00009 TABLE 9 Phase Ingredients (INCI) % A H.sub.2O Solution/Retinol transcultol at 1.28% 70 Water 7.22 Glycerin 3 B Sodium Polyacrylate 0.8 C Coco-Caprylate/Caprate 6.66 Caprylic/Capric Triglyceride 6.66 Olus Oil 4.66 D Phenoxyethanol (and) Ethylhexylglycerin 1

[0313] Operating mode: Homogenize phase A, then introduce phase B and homogenize for 10 minutes with vigorous stirring (1500 revolutions/min). Make the emulsion by pouring phase PGP-62T 1 C into the mixture, then homogenize with vigorous stirring for 10 min. Finally, introduce phase D.

[0314] A smooth, pale yellow cream is thus obtained.

[0315] 2.2. Materials and Equipment

[0316] 2.2.1. Biological Material

[0317] Human skin samples are obtained from a plastic surgery department of a clinic in Tours (France), after an abdominoplasty operation. Following the operation, the skin is placed in a chamber at a temperature of 4′C, then transferred to our establishment.

[0318] Upon receipt, the hypodermis is gently removed and the skin sample is recorded with an encrypted identification number and then stored at −20° C. According to the OECD guidelines (Test N.sup.0428), the skin can be preserved at this temperature for a maximum period of one year without modifying its permeability.

[0319] For this study, 10 skin explants from a single donor are used.

[0320] 2.3. Sequence of the Study

[0321] 2.3.1. Characterization of the Explants

[0322] A sample of human skin is divided into 10 skin explants measuring 3×3 cm. The explants are thawed at room temperature for 10 minutes, then cleaned with PBS.

[0323] The integrity of the skin barrier of each explant is controlled by measuring the insensible water loss (IWL). With the values of IWL measured for the 10 skin explants ranging between 5.2 and 7.6 gm.sup.−2.Math.h.sup.−1, the skin explants are considered suitable for experimentation. The thickness of each explant is measured at five different places.

[0324] The average values obtained per formula for these two parameters (IWL and thickness) are shown in Table 10.

[0325] Table 10: Average thickness and IWL of skin explants per condition (n=3; *n=1; average±wk).

TABLE-US-00010 TABLE 10 Condition Thickness (micrometers) IWL (gm.sup.−2 .Math. h.sup.−1) F1 964 ± 67 6.6 ± 1.1 F2 856 ± 62 6.3 ± 1.1 White* 881 ± 29 5.6 ± 1. 

[0326] 2.3.2. Transdermal Passage Test

[0327] All the skin explants are placed in Franz type diffusion cells with the stratum corneum placed facing the donor compartment. Clips are used to keep the two compartments together and thus provide the seal.

[0328] The receiving compartments are filled with receiving liquid. Special care is taken to avoid the formation of air bubbles under the skin explants.

[0329] For 1 hour, the diffusion cells are placed on a magnetic plate to keep stirring the receiving liquid, and the whole is placed in an oven enabling a temperature of 32° C. on the surface of the skin and a 50% moisture content to be obtained. The stirring speed of the receiving liquid during the experiment is set to 400 rpm.

[0330] After one hour, with the thermal equilibrium of each diffusion cell being reached, the formulations are carefully applied to the surface of the skin explants in accordance with the distribution described in Table 11.

[0331] The formulations are in the form of an emulsion, therefore the applications are carried out with a positive displacement pipette.

[0332] The amount deposited on the surface of the skin is 500 mg.

[0333] The diffusion cells are placed in an oven for 24 hours.

[0334] Table 11: Breakdown of explants per condition

TABLE-US-00011 TABLE 11 Condition Explants White # T Formula 1 # 1; # 2; # 3 Formula 2 # 7; # 8; # 9

[0335] During the 24 hours of diffusion, 3 kinetic points are achieved at the following times: 1 h, 4 h and 8 h. To this end, a volume of 300 μL of receiving liquid is taken from each cell, then replaced by “new” receiving liquid. Each sample is kept frozen.

[0336] At the end of the diffusion time (24 hours), the following procedure is carried out for all the diffusion cells:

[0337] Cleaning the skin surface: [0338] Absorption of the unabsorbed fraction; [0339] Cleaning the skin surface with 2 cotton swabs impregnated with micellar water; [0340] Rinsing the skin surface with 2 cotton swabs impregnated with demineralized water; [0341] Drying the skin surface with 1 cotton swab; [0342] Application of D-Squame adhesive to remove product residue remaining on the skin.

[0343] Recovery of the Receiving Liquid: [0344] All the receiving liquid is placed in a 15 mL Falcon tube, then frozen.

[0345] Recovery of the Stratum Corneum: [0346] Successive application of 2 D-Squame adhesives on the treated area. The two adhesives are placed together in a 15 mL Falcon tube, then frozen, with each adhesive being folded back on itself.

[0347] Recovery of the Epidermis and Dermis: [0348] The epidermis and dermis are separated by lightly scraping the surface or, if necessary, by heating at 65° C. for 15 seconds. [0349] The epidermis and dermis are individually placed in a 15 mL Falcon tube, weighed and finally frozen.

[0350] 2.4. Analysis and Dosage of the Samples

[0351] All the samples were recovered.

[0352] The extraction and analytical assaying of retinol in the samples was carried out according to the following procedure:

[0353] 2.4.1. Retinol Assay Method: HPLC.

[0354] HPLC analysis procedure: [0355] For the receiving liquids: direct injection [0356] For SC, Ep, Dm: extraction by ethanol (with stirring) before injection. [0357] Tested extraction time=12 h and 24 h [0358] Ethanol volume=10 mL for SC, 1 mL for Ep and 2 mL for Dm [0359] The triplicates of SC, Ep and Dm are “pooled” before extraction [0360] After extraction, the tubes are centrifuged at 3000 g for 5 minutes [0361] 300 μL is taken for HPLC analyzes

[0362] HPCL analysis conditions: [0363] Column: (C18 Vintage series KR C18—5 μm-150×4.6 mm) [0364] Mobile phase: Isopropanol-water (85/15) [0365] Column temperature: 25° C. [0366] Injection volume: 20 μL [0367] Pump flow: 1 mL/min [0368] Detection: UV—325 nm [0369] Retinol retention time: 11.8 min [0370] Total time per injection: 15 mins

[0371] 3. Transdermal Passage of Retinol from the 2 Formulations

[0372] The results of the assaying of retinol in the different skin layers and in the receiving liquids are presented in the following chapters.

[0373] 3.1. Distribution of Retinol in the Skin Layers

[0374] The average amounts of retinol obtained in the skin layers are presented in Table 12 and Table 13.

[0375] Table 12: Average amount of retinol (μg/cm.sup.2) in the skin layers (12 hours of extraction).

TABLE-US-00012 TABLE 12 F1 F3 Control Stratum 10.76 ± 0.25 9.34 ± 0.08 0.22 Corneum Epidermis 12.13 ± 0.58 6.28 ± 0.10 0.22 ± 0.01 Dermis  0.24 ± 0.06 0.22 ± 0.04 0.20 ± 0.04

[0376] Table 13: Average amount of retinol (μg/cm.sup.2) in the skin layers (24 hours of extraction).

TABLE-US-00013 TABLE 13 F1 F3 Control Stratum 10.67 ± 0.27 9.27 ± 0.08 0.24 ± 0.03 Corneum Epidermis 12.46 ± 0.29 7.26 ± 0.22 0.21 ± 0.03 Dermis  0.21 ± 0.05 0.19 ± 0.02 0.17 ± 0.05

[0377] Two durations for extracting retinol from skin layers were applied: 12 h and 24 h. The results of the test for the transdermal passage of retinol in the skin layers do not show any difference between the values obtained after 12 h of extraction (Table 12) and those obtained after 24 h of extraction (Table 13). This result validates the extraction method.

[0378] In the remainder of the document, only the results obtained within 12 hours of extraction are retained and reviewed.

[0379] The retinol assay results for the control sample condition show very low values (less than 0.30 μg/cm.sup.2) in the 3 layers. This result confirms that the human skin explant used in this study does not contain endogenous retinol.

[0380] For the three formulations, the amount of retinol measured in the dermis is of the same order as that of the control sample (˜0.2 μg/cm.sup.2). The three formulas do not seem to allow retinol to diffuse into the dermis.

[0381] The lowest retinol transdermal diffusion results are obtained with the F2 formulation. In fact, with this formulation, the values obtained in the stratum corneum and in the epidermis are less than 10 μg/cm.sup.2.

[0382] The results of transdermal diffusion of retinol obtained with F1 are higher overall than those obtained with formulation F2. The amount of retinol in the stratum corneum is only slightly higher, with 10.76 μg/cm.sup.2 for F1 against 9.34 μg/cm.sup.2 for F2. This is twice as high in the epidermis with 12.13 μg/cm.sup.2 for F1 against 6.28 μg/cm.sup.2 for F2, showing the effectiveness of this formulation for transporting the retinol in this skin layer.

[0383] 3.2. Result of Diffusion of Retinol in the Receiving Fluids

[0384] No detection of the molecule of interest was observed in the receiving fluids. This result is consistent with the previous result where there was a very small amount of retinol in the dermis, irrespective of the condition.

[0385] 4. Conclusions

[0386] In conclusion, this study highlights the following elements: [0387] The transdermal absorption of retinol varies depending on the formulation applied to the skin. [0388] Irrespective of the formulation that is used, retinol was not found in the receiving fluid and in the dermis. [0389] Among the 2 formulations that were studied, formulation F2 is the formulation that is the least effective in allowing transdermal diffusion of retinol.

[0390] Formulation F1 has the most interesting results in terms of the amount of retinol transported to the stratum corneum and the epidermis, irrespective of the condition.