Uses of lipophenolic compounds

Abstract

The present invention relates to compound of formula (I): ##STR00001##
wherein
R is O—R.sub.3 or ##STR00002##
R.sub.1 and R.sub.2 are identical or different and are each independently H, (C.sub.1-C.sub.6)alkyl, —CO—(C.sub.1-C.sub.21)alkyl or —CO—(C.sub.11-C.sub.21)alkenyl group, provided that at least one of R.sub.1 or R.sub.2 is H or (C.sub.1-C.sub.6)alkyl,
R.sub.3 is a —CO—(C.sub.11-C.sub.21)alkyl or —CO—(C.sub.11-C.sub.21)alkenyl group,
or its pharmaceutically acceptable salts, racemates, diastereoisomers, enantiomers, or mixtures thereof,
for use in prevention and/or treatment of a disease or disorder linked to an exacerbated vascular, lymphatic or mucosal permeability.

Claims

1. A method for the treatment of encephalitis comprising administering, to a person in need thereof, a compound of formula (IIb): ##STR00064## with R.sub.3 selected from the group consisting of: ##STR00065## or its pharmaceutically acceptable salts, racemates, diastereoisomers, enantiomers, or mixtures thereof.

2. The method of claim 1, wherein the compound is administered for 1 to 4 days, and just before and/or during the acute phase response associated with factors inducing the increased vascular, lymphatic or mucosal permeability.

3. The method of claim 1, wherein the compound of formula (IIb) is administered in association with at least one additional active compound selected from the group consisting of antibiotic, antiviral, antifungal, antiparasitic, anti-inflammatory active compound and mixtures thereof.

Description

FIGURES

(1) FIG. 1: Chemical and enzymatic synthesis of 4′-Resv-C.sub.22 or 4′-Resv-BE, 4′-Resv-DHA, 4′-Resv-LA and 4′-Resv-ALA.

(2) FIG. 2: Chemical and enzymatic synthesis of 3-Resv-DHA.

(3) FIG. 3: Assessment of anti-MMP-9 activity of resveratrol lipophenolic derivatives.

(4) FIG. 4: Zymogram and Curve of the dose-response effect of Resv-LA (FIG. 4A) and Resv-C22 or Resv-BE (FIG. 4B) respectively on the inhibitory MMP-9 activity.

(5) FIG. 5: Assessment of in vitro vascular permeability assay of resveratrol lipophenolic derivatives.

(6) FIG. 6: Assessment of cell viability assay of Resv-LA (FIG. 6A) and Resv-C22 or Resv-BE (FIG. 6B) respectively.

(7) FIG. 7: Assessment of the inhibitory effect of Resv-LA (REV-LA) on TNF-α release by LPS-activated microglia.

EXAMPLES

(8) Materials and Methods

(9) THP-1 cells (human monocytic THP-1 cell line) were cultured in 10% heated inactivated FBS RPMI 1640 medium supplemented with penicillin G 100 units/mL and streptomycin 100 μg/mL purchased from Fisher Scientific™ (Illkirch-Graffenstaden, France).

(10) Human umbilical vascular endothelial cells (HUVECs) were cultured in low serum (1%) EndoGro™ medium kit purchased from Merck Millipore™ (Paris, France). TNF-α was purchased from PeproTech™ (Neuilly-Sur-Seine, France).

(11) Resveratrol has been isolated and purified from stalks of Vitis vinifera, Vitaceae, according to the process described by (Delaunay et al. 2002), SB-3CT #BML-E1325 (specific thiirane gelatinase inhibitor used as a reference: it blocks laminin degradation by MMP-9 so that prohibiting neuron apoptosis) was purchased from Enzo Life Sciences™ (Villeurbanne, France) and dissolved in Dimethyl sulfoxide (DMSO) Sigma-Aldrich™.

(12) Human CD31/PECAM-1 antibody (BBA7) and Streptavidin-Fluorescein (4800-30-14) were purchased from Bio-Techne™ (Abingdon, United Kingdom), and all other chemicals used in this study are highly purified molecular grade reagents.

Example 1: Synthesis of Resveratrol Derived Lipophenols

(13) To evaluate the activity of different lipid chains at the 4′ position, Resv-C22 or Resv-BE (5a), Resv-LA (5b), Resv-DHA (5c) and Resv-ALA (5d) were synthesized using enzymatic and chemical synthesis starting from resveratrol (FIG. 1). In the first step, the supported lipase Candida antartica (CALB, Novozyme 435, selective of the 4′ position) was used to introduce acetyl group regio-selectively at the resveratrol C4OH position. The reaction was performed in good yield (85%) without any acetyl derivatives at the 3 or 5 positions. Hydroxyl groups at 3 and 5 positions of compound 1 were then protected by triisopropylsilyl (TIPS) protecting groups using triflate reagent (TIPS-OTf) and diisopropylethylamine (DIPEA) as a base to obtain the protected derivative 2. The acetyl group of compound 2 was deprotected with a solution of sodium methanolate (MeONa) in anhydrous methanol and resulted resveratrol-diTIPS (3) in an excellent yield of 95%. The coupling reactions between compound 3 and the difference fatty acid, docosanoic acid or behenic acid (C22), linoleic acid (LA), docosahexaenoic acid (DHA) and linolenic acid (ALA) were initiated using dicyclohexylcardodiimide and dimethylaminopyridine (DCC/DMAP) as coupling reagents to access 4a-d. Final deprotection of TIPS protecting groups by Et.sub.3N-3HF in dry tetrahydrofuran (THF) yielded final lipophenols 5a-d.

(14) In order to study the importance of the position of the lipidic part on the resveratrol structure, the synthesis of a lipidic resveratrol having the fatty acid at the 3 position was developed. Starting from the fully protected resveratrol 2, one TIPS group at the 5 position was removed using mild Et.sub.3N-3HF carefully monitored by thin layer chromatography (48%, FIG. 2). Then, the mono deprotected derivative 6 was linked to the fatty acid (DHA) using DCC/DMAP. In order to preserve the ester linkage of the compound 7, the acetate group was deprotected using enzymatic lipase CALB in presence of butanol (89%) instead of MeONa solution. The final TIPS deprotection using Et.sub.3N-3HF afforded the desired 3-Resv-DHA compound (9).

Experimental Part

(E)-4-(3,5-dihydroxystyryl)phenyl acetate (Compound 1)

(15) resveratrol (2.88 g, 12.61 mmol) was dissolved in 2-methylbutan-2-ol (280 mL) and vinyl acetate (72.40 mL, 756.70 mmol) in presence of the supported lipase Candida Antarctica (Novozyme 435, CaIB, 14.40 g). The mixture was stirred with a rotary evaporator at 40° C. during 4 days, protected from sunlight by aluminium foil. The lipase was then filtered off and washed with AcOEt (10×50 mL) and diethyl ether (2×50 mL). The filtrate obtained was concentrated under reduced pressure and the residue obtained was purified by chromatography on silica gel using solid deposit (CH.sub.2Cl.sub.2/MeOH 99/1 to 98/2) to give the 4′-O-acetyl resveratrol 1 (2.89 mg, 85%) as white solid. R.sub.f (CH.sub.2Cl.sub.2/MeOH 95/5) 0.3; .sup.1H NMR (500 MHz; CD.sub.3OD) δ.sub.H 7.54 (d, J=7.6 Hz, 2H, H.sub.2′ and H.sub.6′), 7.07 (d, J=7.6 Hz, 2H, H.sub.3′ and H.sub.5′), 7.04 (d, J=16.2 Hz, 1H, H.sub.8), 6.97 (d, J=16.2 Hz, 1H, H.sub.7), 6.49 (s, 2H, H.sub.2, H.sub.6), 6.21-6.19 (m, 1H, Ha), 2.27 (s, 3H, CH.sub.3(OAc)); .sup.13C NMR (125 MHz; MeOD) δ.sub.C 171.1, 159.7 (2C), 151.5, 140.6, 136.6, 130.2, 128.3, 128.3, 122.9 (2C), 122.9 106.1 (2C), 103.2, 20.9.

(E)-4-(3,5-bis((triisopropylsilyl)oxy)styryl)phenyl acetate (Compound 2)

(16) 4′-O-acetyl resveratrol 1 (3.18 g, 11.78 mmol) was dissolved in dry THF (160 mL). DIPEA (4.20 mL, 24.70 mmol) and TIPS-OTf (6.70 mL, 24.70 mmol) were added dropwise to the solution and the reaction mixture was stirred at room temperature during 4.5 h. Additional amount of DIPEA (1.0 mL, 5.90 mmol) and TIPS-OTf (1.6 mL, 5.90 mmol) were added to reach completion of the reaction. After 2.5 additional hours of reaction, the solvent was evaporated under reduced pressure. The residue obtained was dissolved in 200 mL of AcOEt and washed with water ((2×100 mL) and brine (100 mL). The organic phase was dried (MgSO.sub.4) and concentrated under vacuum. The residue obtained was purified by chromatography on silica gel (pentane/AcOEt 99/1 to 70/30) to give the protected resveratrol 2 (6.85 g, 90%) as a colorless oil. R.sub.f (pentane/AcOEt 95/5) 0.5; .sup.1H NMR (500 MHz; CDCl.sub.3) δ.sub.H 7.51 (d, J=8.0 Hz, 2H, H.sub.2′ and H.sub.6′), 7.09 (d, J=8.0 Hz, 2H, H.sub.3′ and H.sub.5′), 6.98 (d, J=16.3 Hz, 1H, H.sub.8), 6.92 (d, J=16.3 Hz, 1H, H.sub.7), 6.65 (s, 2H, H.sub.2, H.sub.6), 6.37-6.36 (m, 1H, Ha), 2.31 (s, 3H, CH.sub.3(OAc)), 1.26 (m, 6H, CH—Si), 1.12 (d, J=7.6 Hz, 36H, (CH.sub.3—CH); .sup.13C NMR (125 MHz; CDCl.sub.3) δ.sub.C 169.7, 157.3 (2C), 150.2, 139.0, 135.3, 129.3, 127.8, 127.7 (2C), 122.0 (2C), 111.6 (2C), 111.5, 21.4, 18.2 (6C), 12.9 (12C).

(E)-4-(3,5-bis((triisopropylsilyl)oxy)styryl)phenol (Compound 3)

(17) The protected resveratrol 2 (6.85 g, 10.59 mmol) was dissolved in dry MeOH (58 mL) and CH.sub.2Cl.sub.2 (28 mL). Sodium methoxide (191 mg, 3.53 mmol) was added to the solution and the reaction mixture was stirred at room temperature during 4.5 h. Further, 0.3 eq of NaOMe (191 mg, 3.53 mmol) was added to drive the reaction to completion. After additional 2 h, the solvent was evaporated under reduced pressure. The residue obtained was purified by chromatography on silica gel (pentane/AcOEt 96/4 to 90/10) to give the 4′-deprotected resveratrol 3 (6.04 g, 95%) as an colorless oil. R.sub.f (hexane/AcOEt 90/10) 0.41; .sup.1H NMR (500 MHz; CD.sub.3OD) δ.sub.H 7.38 (d, J=8.5 Hz, 2H, H.sub.2′ and H.sub.6), 6.95 (d, J=16.2 Hz, 1H, H.sub.8), 6.84 (d, J=16.2 Hz, 1H, H.sub.7), 6.77 (d, J=8.5 Hz, 2H, H.sub.3′ and H.sub.5), 6.64-6.63 (m, 2H, H.sub.2, H.sub.6), 6.30-6.29 (m, 1H, H.sub.4), 1.30-1.22 (m, 6H, CH—Si), 1.14 (d, J=7.5 Hz, 36H, CH.sub.3—CH); .sup.13C NMR (125 MHz; MeOD) δ.sub.C 158.5, 158.3 (2C), 141.3, 130.1, 129.9, 129.0 (2C), 126.5, 116.5 (2C), 112.1 (2C), 111.4, 18.4 (6C), 13.9 (12C).

Description of 4′-Resv-C22 (4-Resv-BE) Series

(E)-4-(3,5-bis((triisopropylsilyl)oxy)styryl)phenyl docosanoate (Compound 4a)

(18) Compound 3 (3.00 g, 5.55 mmol) and docosanoic acid also named behenic acid (2.27 g, 6.67 mmol) were partially dissolved in dry DCM (180 mL) and the required amount of dry DMF (55 mL) was added to solubilize the acid entirely. Afterwards, DCC (1.70 g, 8.33 mmol) and DMAP (339 mg, 2.78 mmol) were added and the reaction was stirred at room temperature under argon until the conversion was completed according to TLC. After 6 h, the reaction was stored into the fridge (4° C.) to allow the formation of DCU precipitate, which was then filtered on frit. DCM (65 mL) was added to the filtrate and it was washed with water (2×150 mL) and brine (150 mL). The organic layer was dried over MgSO.sub.4 and evaporated to gain 8.00 g of crude product. Purification was performed by chromatography on silica gel (pentane/EtOAc 99:1) to yield 4a (6.63 g, 76%) as a white solid.

(19) R.sub.f (pentane/EtOAc 99:1) 0.4; .sup.1H NMR (500 MHz, CDCl.sub.3): δ.sub.H 7.50 (d, J=8.5 Hz, 2H, H.sub.2′ and H.sub.6′), 7.07 (d, J=8.5 Hz, 2H, H.sub.3′ and H.sub.5), 6.99 (d, J=16.0 Hz, 1H, H.sub.8), 6.92 (d, J=16.0 Hz, 1H, H.sub.7), 6.65 (d, J=2.0 Hz, 2H, H.sub.2 and H.sub.6), 6.36 (t, J=2.5 Hz, 1H, Ha), 2.56 (t, J=7.5 Hz, 2H, CH.sub.2—C═O), 1.76 (quint, J=7.5 Hz, 2H, CH.sub.2—CH.sub.2—C═O), 1.43-1.22 (m, 42H, CH.sub.2—CH.sub.2 and CH—Si), 1.11 (d, J=7.5 Hz, 36H, CH.sub.3—CH), 0.89 (t, J=7.1 Hz; 3H, CH.sub.3—CH.sub.2). .sup.13C NMR (126 MHz, CDCl.sub.3) δ.sub.C 172.4, 157.2, 157.2, 150.2, 138.9, 135.1, 129.1, 127.7, 127.5 (2C), 121.9 (2C), 111.5 (2C), 111.4, 34.6, 32.1, 29.9 (8C), 29.8, 29.8, 29.8, 29.7, 29.6, 29.5, 29.4, 29.2, 25.1, 22.8, 18.1 (6C), 14.3, 12.8 (12C).

(E)-4-(3,5-dihydroxystyryl)phenyl docosanoate (Compound 5a)

(20) Compound 4a (3.63 g, 4.20 mmol) was dissolved in dry THF (220 mL) under argon atmosphere. Addition of Et.sub.3N-3HF (4.11 mL, 25.21 mmol) was arranged via plastic syringe and the reaction was allowed to stir at room temperature and monitored by TLC (pentane/EtOAc 7:3 and 9:1). Additional equivalents of Et.sub.3N-3HF were added after 3.5 h (2.06 mL, 12.61 mmol) and 6 h (2.06 mL, 12.61 mmol) of reaction. After 8 hours, the THF was evaporated under reduced pressure and the residue was dissolved in 400 mL of EtOAc, and then washed with H.sub.2O (3×200 mL) and brine (200 mL). The organic layer was dried over MgSO.sub.4 and evaporated to gain 2.70 g of crude product. Purification was performed by chromatography on silica gel using solid deposit (pentane/EtOAc 7:3 to 0:1) and resulted in 503 mg (17%) of mono protected derivative and 1.82 g (79%) of compound 5a as a white solid.

(21) R.sub.f (pentane EtOAc 7:3) 0.3; .sup.1H NMR (500 MHz, CDCl.sub.3/MeOD 10:1) δ.sub.H 7.31 (d, J=8.5 Hz, 2H, H.sub.2′ and H.sub.6′), 6.87 (d, J=8.5 Hz, 2H, H.sub.3′ and H.sub.5′), 6.84 (d, J=16.5 Hz, 1H, H.sub.8), 6.74 (d, J=16.5 Hz, 1H, H.sub.7), 6.34 (d, J=2.0 Hz, 2H, H.sub.2 and H.sub.6), 6.08 (t, J=2.0 Hz, 1H, H.sub.4), 2.39 (t, J=7.0 Hz, 2H, CH.sub.2—C═O), 1.57 (quint, J=7.0 Hz, 2H, CH.sub.2—CH.sub.2—C═O), 1.23-1.07 (m, 36H, CH.sub.2—CH.sub.2), 0.69 (t, J=6.6 Hz, 3H, CH.sub.3—CH.sub.2); .sup.13C NMR (126 MHz, CDCl.sub.3/MeOD 10:1) δ.sub.C 172.8, 157.9 (2C), 149.8, 139.1, 135.0, 128.9, 127.3, 127.2 (2C), 121.6 (2C), 105.1 (2C), 102.1, 34.2, 31.7, 29.5 (9C), 29.4, 29.4, 29.4, 29.3, 29.2, 29.0, 28.9, 24.7, 22.5, 13.8.

Description of 4′-Resv-LA Series

(9,12Z)-4-((E)-3,5-bis((triisopropylsilyl)oxy)styryl)phenyl octadeca-9,12-dienoate (Compound 4b)

(22) Compound 3 (1.00 g, 1.67 mmol) and linoleic acid (LA; 623 mg, 2.22 mmol) were dissolved in dry DCM (40 mL). Next, DCC (573 mg, 2.78 mmol) and DMAP (113 mg, 0.93 mmol) were added to the reaction mixture which was stir at room temperature under inert atmosphere (monitored by TLC pentane/EtOAc 95:5). The reaction was terminated after 3 hours. Flask was put into the fridge (4° C.) for 1 h min to maximize the amount of DCU crystals. White DCU precipitate was then removed by filtration on frit, rinsed by a few drops of cold DCM. Filtrate was diluted by 40 mL of DCM and washed twice with water (30 mL) and once with brine (30 mL). Aqueous phases were re-extracted with 100 mL of DCM. Organic layers were collected, dried over MgSO.sub.4 and evaporated. Purification by silica gel column chromatography (pentane/EtOAc 99.5/5 to 99/1) resulted in 1.10 g (74%) of compound 4b (colorless oil).

(23) Rf (pentane/EtOAc 95:5) 0.7; .sup.1H NMR (500 MHz, CDCl.sub.3): δ.sub.H 7.79 (d, J=8.5 Hz, 2H, H.sub.2′ and H.sub.6′), 7.06 (d, J=8.5 Hz, 2H, H.sub.3′ and H.sub.5′), 6.97 (d, J=16 Hz, 1H, H.sub.8), 6.91 (d, J=16 Hz, 1H, H.sub.7), 6.64 (d, J=1.5 Hz, 2H, H.sub.2 and H.sub.6), 6.3 (t, J=1.5 Hz, 1H, H.sub.4), 5.41-5.33 (m, 4H, CH═CH), 2.78 (t, J=6.5 Hz, 2H, CH.sub.2 bis-allylic), 2.55 (t, J=7.5 Hz, 2H, CH.sub.2—C═O), 2.07-2.03 (m, 4H, CH.sub.2 allylic), 1.75 (quint, J=7.5 Hz, 2H, CH.sub.2—CH.sub.2—C═O), 1.42-1.22 (m, 20H, CH.sub.2—CH.sub.2 and CH—Si), 1.11 (d, J=7.5 Hz, 36H, CH.sub.3—CH), 0.89 (t, J=7 Hz, 3H, CH.sub.3—CH.sub.2); .sup.13C NMR (126 MHz, CDCl.sub.3) δ.sub.C 172.6, 157.4 (2C), 150.4, 139.1, 135.3, 130.6, 130.3, 129.3, 128.4, 128.2, 127.9, 127.7 (2C), 122.1 (2C), 111.7 (2C), 111.6, 34.8, 31.8, 29.9, 29.7, 29.5, 29.4, 29.4, 27.5, 27.5, 26.0, 25.3, 22.9, 18.2 (6C), 14.4, 13.0 (12C).

(9,12Z)-4-((E)-3,5-dihydroxystyryl)phenyl octadeca-9,12-dienoate (Compound 5b)

(24) Et.sub.3N-3HF (1.32 mL, 8.08 mmol) was added via plastic syringe to a solution of Compound 4b (1.08 g, 1.35 mmol) dissolved in dry THF (60 mL) The reaction was stirred at room temperature under argon. Further equivalents of Et.sub.3N-3HF (2×0.66 mL, 2×4.04 mmol) were added at four and six hours of reaction time. Reaction was terminated after another two hours (reaction time 8 h, monitored by TLC pentane/EtOAc 7:3 and 9:1). Reaction media was evaporated and the residue was dissolved in EtOAc (120 mL). Organic phases was washed with H.sub.2O (3×60 mL) and brine (60 mL), dried over MgSO.sub.4, filtered and evaporated under reduced pressure. Crude product was purified by column chromatography on silica gel (pentane/EtOAc 7:3 to 6:4) to obtain 5b (546 mg, 83%) as a white solid.

(25) R.sub.f (pentane/EtOAc 7:3) 0.3; .sup.1H NMR (500 MHz, CDCl.sub.3) δ.sub.H 7.34 (d, J=8.5 Hz, 2H, H.sub.2′ and H.sub.6′), 7.00 (d, J=8.5 Hz, 2H, H.sub.3′ and H.sub.5′), 6.79 (d, J=16.5 Hz, 1H, H.sub.8), 6.70 (d, J=16.5 Hz, 1H, H.sub.7), 6.40 (d, J=2.0 Hz, 2H, H.sub.2 and H.sub.6), 6.23 (t, J=2.0 Hz, 1H, H.sub.4), 5.99 (Br, 2H, OH), 5.41-5.32 (m, 4H, CH═CH), 2.77 (t, J=6.5 Hz, 2H, CH.sub.2 bis-allylic), 2.56 (t, J=7.5 Hz, 2H, CH.sub.2—C═O), 2.06-2.03 (m, 4H, CH.sub.2 allylic), 1.75 (quint, J=7.5 Hz, 2H, CH.sub.2—CH.sub.2—C═O), 1.42-1.25 (m, 14H, CH.sub.2—CH.sub.2), 0.88 (t, J=7 Hz, 3H, CH.sub.3—CH.sub.2); .sup.13C NMR (126 MHz, CDCl.sub.3) δ.sub.C 173.5, 157.0 (2C), 150.1, 139.7, 135.1, 130.4, 130.2, 128.4, 128.2, 128.2, 128.0, 127.7 (2C), 121.8 (2C), 106.3 (2C), 102.6, 34.6, 31.7, 29.7, 29.5, 29.3, 29.3, 29.2, 27.3, 27.3, 25.7, 25.0, 22.7, 14.2.

Description of 4′-Resv-DHA Series (4c and 5c)

(26) The synthesis of Resv-DHA is described in the publication Crauste et al. (2014).

(4,7,10,13,16,19 Z)-4-((E)-3,5-bis(triisopropylsilyloxy)styryl)phenyl docosa-4,7,10,13,16,19-hexaenoate (Compound 4c)

(27) Coupling of the di-protected resveratrol 3 (103 mg, 0.18 mmol) and DHA (67 mg, 0.20 mmol) was performed with the general procedure and afforded 4c (130 mg, 80%) as an uncolored oil after purification on silicagel chromatography (hexane/AcOEt 99/1).

(28) R.sub.f (hexane/AcOEt 95/5) 0.73; .sup.1H NMR (500 MHz; CDCl.sub.3) δ.sub.H 7.50 (d, J=8.5 Hz, 2H, H.sub.2′ and H.sub.6′), 7.07 (d, J=8.4 Hz, 2H, H.sub.3′ and H.sub.5), 6.98 (d, J=16.5 Hz, 1H, H.sub.8), 6.92 (d, J=16.5 Hz, 1H, H.sub.7), 6.64 (d, J=2.3 Hz, 2H, H.sub.2, H.sub.6), 6.36 (t, J=2.3 Hz 1H, H.sub.4), 5.50-5.29 (m, 12H, CH═CH), 2.90-2.80 (m, 10H, CH.sub.2 bis-allylic), 2.64 (t, J=7.0 Hz, 2H, CH.sub.2—C═O), 2.55-2.51 (m, 2H, CH.sub.2 allylic), 2.08 (quint, J=7.0 Hz, 2H, CH.sub.2 allylic), 1.29-1.22 (m, 6H, CH—Si), 1.12 (d, J=7.5 Hz, 36H, (CH.sub.3).sub.2C); 0.98 (t, J=7.5 Hz, 3H, CH.sub.3); .sup.13C NMR (125 MHz; CDCl.sub.3) δ.sub.C 171.8, 157.4, 150.4, 139.1, 135.3, 132.3, 130.0, 129.3, 128.9, 128.7, 128.6, 128.6, 128.4, 128.4, 128.3, 128.2, 127.9, 127.8, 127.7, 127.3, 122.0, 111.7, 111.6, 34.6, 25.9, 25.9, 25.8, 23.1, 20.9, 18.2, 14.6, 13.0

(4,7,10,13,16,19 Z)-4-((E)-3,5-dihydroxyphenylstyryl)phenyl docosa-4,7,10,13,16,19-hexaenoate (Compound 5c)

(29) deprotection of the protected DHA-resveratrol 4c (142 mg, 0.17 mmol) was performed with the general procedure and afforded 5c (55 mg, 61%) as white solid after 7 h of reaction and purification on silicagel chromatography (hexane/AcOEt 95/5 to 70/30).

(30) R.sub.f (hexane/AcOEt 70/30)=0.22; .sup.1H NMR (500 MHz; CDCl.sub.3) δ.sub.H 7.45 (d, J=8.6 Hz, 2H, H.sub.2′ and H.sub.6′), 7.07 (d, J=8.5 Hz, 2H, H.sub.3′ and HO, 6.95 (d, J=16.2 Hz, 1H, H.sub.8), 6.85 (d, J=16.2 Hz, 1H, H.sub.7), 6.51 (d, J=2.1 Hz, 2H, H.sub.2, H.sub.6), 6.26 (t, J=2.1 Hz, 1H, H.sub.4), 5.52-5.29 (m, 12H, CH.sub.2 bis-allylic), 5.13 (br, 2H, OH), 2.90-2.80 (m, 10H, CH.sub.2 allylic), 2.66 (t, J=7.4 Hz, 2H, CH.sub.2—C═O), 2.52-2.56 (m, 2H, CH.sub.2 allylic), 2.08 (quint, J=7.8 Hz, 2H, CH.sub.2 allylic), 0.98 (t, J=7.3 Hz, 3H, CH.sub.3); .sup.13C NMR (125 MHz; CDCl.sub.3) δ.sub.C 172.3, 157.3, 150.4, 140.0, 135.2, 132.4, 130.1, 128.9, 128.7, 128.6, 128.6, 128.6, 128.5, 128.4, 128.4, 128.3, 128.2, 127.8, 127.7, 127.3, 122.1, 106.4, 102.7, 34.6, 25.9, 25.8, 23.1, 20.9, 14.6

Description 4′-Resv-ALA Series

(9,12, 15Z)-4-((E)-3,5-bis((triisopropylsilyl)oxy)styryl)phenyl octadeca-9,12,15-trienoate (Compound 4d)

(31) A solution of linolenic acid (ALA; 56 mg, 0.20 mmol) in dry DCM (2.50 mL) was added to the protected resveratrol 3 (100 mg, 0.18 mmol) in solution in DCM (2.50 mL). DCC (42 mg, 0.20 mmol) and DMAP (6 mg, 0.05 mmol) were added to the reaction mixture and the solution was left to stir at room temperature under inert atmosphere during 2 h (monitored by TLC pentane/EtOAc 95:5). Flask was put into the fridge (4° C.) for 1 h to maximize the amount of DCU crystals. White DCU precipitate was then removed by filtration on frit, rinsed by a few drops of cold DCM. Filtrate was diluted by 10 mL of DCM and washed twice with water (10 mL) and once with brine (10 mL). The organic layer was dried over MgSO.sub.4 and evaporated. Purification by silica gel column chromatography (pentane/EtOAc 99.5/5 to 99/1) resulted in 115 mg (76%) of compound 4d as a colorless oil.

(32) Rf (pentane/EtOAc 95:5) 0.5; .sup.1H NMR (500 MHz, CDCl.sub.3): δ.sub.H 7.50 (d, J=8.5 Hz, 2H, H.sub.2′ and HO, 7.07 (d, J=8.5 Hz, 2H, H.sub.3′ and HO, 6.98 (d, J=16.5 Hz, 1H, H.sub.8), 6.92 (d, J=16.5 Hz, 1H, H.sub.7), 6.65 (d, J=2.5 Hz, 2H, H.sub.2 and H.sub.6), 6.36 (t, J=2.5 Hz, 1H, Ha), 5.39-5.36 (m, 6H, CH═CH), 2.82 (t, J=6.5 Hz, 4H, CH.sub.2 bis-allylic), 2.56 (t, J=7.5 Hz, 2H, CH.sub.2—C═O), 2.10-2.06 (m, 4H, CH.sub.2 allylic), 1.75 (quint, J=7.5 Hz, 2H, CH.sub.2—CH.sub.2—C═O), 1.43-1.21 (m, 14H, CH.sub.2—CH.sub.2 and CH—Si), 1.11 (d, J=7.5 Hz, 36H, CH.sub.3—CH), 0.89 (t, J=7 Hz, 3H, CH.sub.3—CH.sub.2); .sup.13C NMR (126 MHz, CDCl.sub.3) δ.sub.C 172.3, 157.2, 150.2, 138.9, 135.1, 132.1, 132.1, 130.4, 129.1, 128.4, 128.4, 127.9, 127.7, 127.5 (2C), 127.2, 121.9 (2C), 111.5 (2C), 111.4, 34.5, 29.7, 29.3, 29.2, 29.2, 27.3, 25.7, 25.6, 25.0, 20.7, 18.1 (6C), 14.4, 12.8 (12C).

(9, 12, 15Z)-4-((E)-3,5-dihydroxystyryl)phenyl octadeca-9,12-dienoate (Compound 5d)

(33) Et.sub.3N-3HF (134 μl, 0.82 mmol) was added via plastic syringe to a solution of ALA-resveratrol 4d (110 mg, 0.14 mmol) dissolved in dry THF (6 mL). Further equivalents of Et.sub.3N-3HF (2×70 μL, 2×0.41 mmol) were added at four and six hours of reaction time. Reaction was terminated after another two hours (reaction time 8 h, monitored by TLC pentane/EtOAc 7:3 and 9:1). Reaction media was evaporated and the residue was dissolved in EtOAc (20 mL) and then washed with H.sub.2O (3×20 mL) and brine (20 mL). Organic phase was dried over MgSO.sub.4, filtered and evaporated under reduced pressure. Crude product was purified by column chromatography on silica gel (cyclohexane/AcOEt 80/20) and afforded Resv-ALA 5d (57 mg, 84%) as white solid.

(34) Rf (pentane/EtOAc 7/3) 0.3; .sup.1H NMR (500 MHz, CDCl.sub.3): δ.sub.H 7.38 (d, J=8.5 Hz, 2H, H.sub.2′ and HO, 7.02 (d, J=8.5 Hz, 2H, H.sub.3′ and HO, 6.84 (d, J=16.5 Hz, 1H, H.sub.8), 6.75 (d, J=16.5 Hz, 1H, H.sub.7), 6.43 (d, J=2.0 Hz, 2H, H.sub.2 and H.sub.6), 6.23 (t, J=2.0 Hz, 1H, Ha), 5.41-5.32 (m, 6H, CH═CH), 2.81 (t, J=6.0 Hz, 4H, CH.sub.2 bis-allylic), 2.57 (t, J=7.5 Hz, 2H, CH.sub.2—C═O), 2.11-2.04 (m, 4H, CH.sub.2 allylic), 1.75 (quint, J=7.5 Hz, 2H, CH.sub.2—CH.sub.2—C═O), 1.42-1.30 (m, 8H, CH.sub.2—CH.sub.2), 0.97 (t, J=7.5 Hz, 3H, CH.sub.3—CH.sub.2); .sup.13C NMR (126 MHz, CDCl.sub.3) δ.sub.C 173.3, 157.0 (2C), 150.1, 139.7, 135.0, 132.1, 130.4, 128.4, 128.4, 128.4, 128.2, 127.9 (2C), 127.7, 127.2, 121.9 (2C), 106.3 (2C), 102.6, 34.6, 29.7, 29.3, 29.2, 26.2, 27.3, 25.7, 25.6, 25.0, 20.7, 14.4.

Description 3-Resv-DHA Series

(E)-4-(3-hydroxy-5-((triisopropylsilyl)oxy)styryl)phenyl acetate (Compound 6)

(35) Et.sub.3N-3HF (554 μL, 3.40 mmol) was added dropwise via plastic syringe to a solution protected resveratrol 2 (1.00 g, 1.70 mmol) dissolved in dry THF (60 mL). The reaction was stirred at room temperature during 3 h. AcOEt (60 mL) was added to the mixture and the organic layer was washed with water (20 mL) and brine (20 mL). The organic phase was dried on MgSO.sub.4 and concentrated under reduced pressure. The residue obtained was purified by chromatography on silica gel (cyclohexane/AcOEt 95/5 to 80/20) to give the mono-protected resveratrol 6 (350 mg, 48%) as a white solid. The di-deprotected resveratrol was isolated in 26% as a white solid (118 mg).

(36) R.sub.f (Hexane/AcOEt 70/30) 0.6; 1H NMR (500 MHz, CDCl.sub.3) δ.sub.H 7.46 (d, J=8.5 Hz, 2H, H.sub.2′ and H.sub.6′), 7.07 (d, J=8.5 Hz, 2H, H.sub.3′ and H.sub.5′), 6.93 (d, J=16.5 Hz, 1H, H.sub.8), 6.86 (d, J=16.5 Hz, 1H, H.sub.7), 6.59 (t, J=1.5 Hz, 1H, H.sub.2), 6.51 (s, 1H, H.sub.4), 6.32 (t, J=2.0 Hz, 1H, H.sub.6), 5.55 (Br, 1H, OH), 2.32 (s, 3H, CH.sub.3—CO), 1.32-1.23 (m, 3H, CH—Si), 1.12 (d, J=7.0 Hz, 18H, CH.sub.3—CH); .sup.13C NMR (126 MHz, CDCl.sub.3) δ.sub.C 170.2, 157.5, 156.9, 150.1, 139.2, 135.2, 128.9, 127.9, 127.6 (2C), 121.8 (2C), 111.3, 107.0, 106.3, 21.3, 18.1 (3C), 12.8 (6C).

(4,7,10,13,16,19Z)-3-((E)-4-acetoxystyryl)-5-((triisopropylsilyl)oxy)phenyl docosa-4,7,10,13,16,19-hexaenoate (Compound 7)

(37) Compound 6 (470 mg, 1.1 mmol) and DHA (397 mg, 1.2 mmol) were dissolved in dry DCM (20 mL) under argon. Then, DCC (250 mg, 1.21 mmol) and DMAP (13 mg, 0.1 mmol) were added to the reaction mixture and the solution was stirred at room temperature under inert atmosphere during 2 h (monitored by TLC pentane/EtOAc 90:10). The reaction was put into the fridge (4° C.) for 1 h to maximize the amount of DCU crystals. White DCU precipitate was then removed by filtration on frit, rinsed by a few drops of cold DCM. Filtrate was diluted by 20 mL of DCM and washed twice with water (15 mL) and once with brine (15 mL). Aqueous phase was re-extracted with 50 mL of DCM. Organic layers were collected, dried over MgSO.sub.4 and evaporated. Purification on silica gel chromatography (cyclohexane/AcOEt 98/2) afforded compound 7 (391 mg, 49%) as a white solid.

(38) R.sub.f (Hexane/AcOEt 90/10) 0.5; .sup.1H NMR (500 MHz, CDCl.sub.3) δ.sub.H 7.49 (d, J=8.7 Hz, 2H, H.sub.2′ and H.sub.6), 7.08 (d, J=8.7 Hz, 2H, H.sub.3′ and H.sub.5′), 7.00 (d, J=16.2 Hz, 1H, H.sub.8), 6.93 (d, J=16.2 Hz, 1H, H.sub.7), 6.84 (t, J=2.0 Hz, 2H, H.sub.2 and H.sub.6), 6.53 (t, J=2.0 Hz, 1H, H.sub.4), 5.50-5.28 (m, 12H, CH═CH), 2.89-2.79 (m, 10H, CH.sub.2 bis-allylic), 2.63-2.60 (m, 2H, CH.sub.2—C═O), 2.56-2.50 (m, 2H, CH.sub.2 allylic), 2.30 (s, 3H, CH.sub.3—C═O), 2.10-2.04 (m, 2H, CH.sub.2 allylic), 1.30-1.23 (m, 3H, CH—Si), 1.12 (d, J=7.3, 18H, CH.sub.3—CH), 0.97 (t, J=7.5, 3H, CH.sub.3—CH.sub.2); .sup.13C NMR (126 MHz, CDCl.sub.3) δ.sub.C 171.4, 169.5, 157.1, 151.8, 150.3, 139.2, 134.9, 132.1, 129.8, 128.7, 128.7, 128.5, 128.4, 128.4, 128.3, 128.2, 128.2, 128.1, 128.0, 127.7, 127.6 (2C), 127.1, 121.9 (2C), 115.8, 112.9, 112.3, 34.4, 25.8 (2C), 25.7, 25.7, 25.6, 22.9, 21.3, 20.7, 18.0 (3C), 14.4, 12.7 (6C).

(4,7,10,13,16,19Z)-3-((E)-4-hydroxystyryl)-5-((triisopropylsilyl)oxy)phenyl docosa-4,7,10,13,16,19-hexaenoate (Compound 8)

(39) The protected DHA-resveratrol 7 (345 mg, 0.47 mmol) was dissolved in t-buthylmethylether (55 mL) and n-BuOH (2 mL). The supported lipase Candida Antarctica (Novozyme 435, CaIB, 345 mg) was added to the solution and the mixture was stirred at 40° C. during 3 days. The lipase was filtered off and washed with 5×30 mL of AcOEt and 2×30 mL of diethyl ether. The filtrate was concentrated under reduced pressure and the residue obtained was purified by chromatography on silica gel (cyclohexane/AcOEt 95/5 to 90/10) to give the compound 8 (291 mg, 89%) as a yellow oil.

(40) R.sub.f (Hexane/AcOEt 90/10) 0.55; .sup.1H NMR (500 MHz, CDCl.sub.3) δ.sub.H 7.37 (d, J=8.6 Hz, 2H, H.sub.2′ and H.sub.6′), 6.96 (d, J=16.2 Hz, 1H, H.sub.8), 6.85-6.79 (m, 5H, H.sub.3′, H.sub.5′, H.sub.2, H.sub.6 and H.sub.7), 6.50 (t, J=2.1 Hz, 1H, H.sub.4), 5.48-5.28 (m, 12H, CH═CH), 2.89-2.79 (m, 10H, CH.sub.2 bis-allylic), 2.63-2.60 (m, 2H, CH.sub.2—C═O), 2.56-2.50 (m, 2H, CH.sub.2 allylic), 2.10-2.04 (m, 2H, CH.sub.2 allylic), 1.30-1.24 (m, 3H, CH—Si), 1.11 (d, J=7.4 Hz, 18H, CH.sub.3—CH), 0.97 (t, J=7.5 Hz, 3H, CH.sub.3—CH.sub.2); .sup.13C NMR (126 MHz, CDCl.sub.3) δ.sub.C 171.5, 157.0, 155.5, 151.8, 139.7, 132.2, 130.1, 129.8, 129.2, 128.7, 128.5, 128.4, 128.4, 128.2, 128.2, 128.2 (2C), 128.1, 128.0, 127.7, 127.2, 126.0, 115.7 (2C), 115.5, 112.5, 112.1, 34.5, 25.8 (2C), 25.8, 25.8, 25.7, 22.9, 20.7, 18.1 (3C), 14.4, 12.8 (6C).

(4,7,10,13,16,19Z)-3-hydroxy-5-((E)-4-hydroxystyryl)phenyl docosa-4,7,10,13,16,19-hexaenoate (Compound 9)

(41) Et.sub.3N-3HF (232 μl, 1.41 mmol) was added via plastic syringe to a solution of DHA-resveratrol 8 (330 mg, 0.47 mmol) dissolved in dry THF (20 mL) The reaction was stirred at room temperature under argon during 4 h30. TLC monitoring was carried out using pentane/EtOAc (7:3 and 9:1) to observe the desired product formation and the departure of starting material respectively. Reaction media was evaporated and the residue was dissolved in EtOAc (20 mL) and then washed with H.sub.2O (20 mL) and brine (20 mL). Organic phase was dried over MgSO.sub.4, filtered and evaporated. Crude product was purified by column chromatography on silica gel (Cyclohexane/AcOEt 80/20) and afforded 3-Resv-DHA 9 (191 mg, 75%) as a white solid

(42) R.sub.f (Hexane/AcOEt 70/30) 0.33; .sup.1H NMR (500 MHz, CDCl.sub.3) δ.sub.H 7.34 (d, J=8.6, 2H, H.sub.2′ and H.sub.6), 6.95 (d, J=16.2, 1H, H.sub.8), 6.83-6.73 (m, 5H, H.sub.3′, H.sub.5′, H.sub.2, H.sub.6 and H.sub.7), 6.46 (t, J=2.1, 1H, H.sub.4), 5.51-5.28 (m, 12H, CH═CH), 5.13 (br, 1H, OH), 5.09 (br, 1H, OH), 2.89-2.79 (m, 10H, CH.sub.2 bis-allylic), 2.65-2.62 (m, 2H, CH.sub.2—C═O), 2.58-2.51 (m, 2H, CH.sub.2 allylic), 2.10-2.03 (m, 2H, CH.sub.2 allylic), 0.97 (t, J=7.5, 3H, CH.sub.3—CH.sub.2); .sup.13C NMR (126 MHz, CDCl.sub.3) δ.sub.H 172.0, 156.6, 155.6, 151.8, 140.3, 132.2, 129.9, 129.9, 129.6, 128.7, 128.5, 128.4, 128.4, 128.2 (2C), 128.2, 128.2, 128.1, 128.0, 127.5, 127.1, 125.5, 115.8 (2C), 111.9, 110.9, 108.1, 34.5, 25.8 (2C), 25.8, 25.7, 25.6, 22.9, 20.7, 14.4.

Example 2: Effect of Lipophenolic Derivative of Resveratrol on MMP-9 Activity

(43) THP-1 cells were seeded (3×10.sup.5 cell/well) in 24-well plate with 10 ng/mL TNF-α with or without 30 μM of resveratrol derivatives dissolved in DMSO. After 24 hours incubation at 37° C., supernatant was collected and tested on zymography. Gel bands demonstrates MMP-9 activity of activated THP-1 cell line in presence of resveratrol lipophenolic derivatives.

(44) Assessment of MMP-9 Activity in THP-1 Cell Line

(45) Low-serum (1%) RPMI-1640 medium was used for the assessment of MMP-9 activity on TNF-alpha activated THP-1 cell line 3×10.sup.6 cell/mL. Firstly, resveratrol and its derived lipophenolic compounds were dissolve in DMSO, and incubated at a final concentration of 30 μM with 10 ng/mL TNF-alpha treated THP-1 in CO.sub.2 incubator chamber for 24 hours at 37° C. After incubation, cell suspension was centrifuged at 1200 r.p.m for five minutes, supernatant was recovered and stored at −80° C. for zymogram analysis.

(46) Zymogram

(47) The anti MMP-9 activity was assessed using gelatin zymography. In brief, the collected supernatants were loaded on 10% SDS-polyacrylamide gel electrophoresis (PAGE) supplemented with 1% gelatin without reducing agents. After separation, gels were washed three times with 2.5% Triton X-100 and incubated with gelatinase buffer (NaCl 200 mM, Tris Base 50 mM, CaCl2) 5 mM and ZnCl2 0.25 mM; pH 7.5), for 24 hr at 37° C. on an orbital shaker at 100 r.p.m/min. Gels were further stained for one hour with Commassie Blue-staining solution (0.025% Commassie Blue, 40% methanol and 10% acetic acid) followed by destaining with 20% methanol and 10% glacial acetic acid solution until the clear bands appearance. Gels were photographed and analyzed by GelAnalyzer 2010a™ software.

(48) The results are presented in FIG. 3, showing that Resv-LA, Resv-C22 (Resv-BE), and to a lesser extent 4′-Resv-DHA, demonstrated inhibition of MMP9-activity in TNF-alpha-activated THP-1 monocytes (FIG. 3). While 4′-Resv-DHA reduced MMP-9 activity at 30 μM, its regio-isomer, having the lipid chain linked with the hydroxyl group in position 3 of resveratrol, the 3-Resv-DHA (FIG. 2), was not active at the same concentration.

(49) Resv-LA and Resv-C22 (Resv-BE) have demonstrated interesting inhibition of activity of MMP-9 (FIG. 3).

(50) And the results in FIG. 4 showed a dose-response effect of Resv-LA (FIG. 4A) and respectively Resv-C22 (Resv-BE) (FIG. 4B) on the inhibitory activity of MMP9.

Example 3: Effect of Lipophenolic Derivative of Resveratrol on In Vitro Vascular Permeability Assay (FITC-Dextran Endothelial Permeability Assay)

(51) The permeability of HUVEC monolayer seeded on collagen coated semi-permeable inserts was examined using in vitro vascular permeability assay kit, Millipore™ (Paris, France) according to the manufacturer protocol.

(52) Endothelial cells are seeded into the inserts and cultured until complete monolayer formation occurs. After forming confluent monolayer by seeding HUVECs (4×10.sup.5 cells/insert) for 48 hours, medium was replaced with 100 ng/mL TNF-α in medium with and without MMP-9 inhibitors at 10 μM and incubated for 24 hours in CO.sub.2 chamber at 37° C. Lipophenolic derivatives of resveratrol were dissolved in DMSO, and SB-3CT (gelatinase inhibitor) was dissolved in DMSO.

(53) At the end of the permeability treatment, 150 μl FITC-dextran in media solution were incubated for 20 minutes at room temperature protected from light. FITC-dextran permeates the treated cell monolayer into the plate well. Permeation was stopped by removing the inserts and 100 μl were withdrawn from the receiving tray and added to 96-well opaque plate for fluorescence measurement. The resulting fluorescence in the plate well is measured and used as an indicator of the extent of monolayer permeability. Filters used are 485 nm and 535 nm for excitation and emission, respectively.

(54) The results are presented in FIG. 5, showing that Resv-LA demonstrated inhibition of TNF-alpha-enhanced permeability of the HUVEC monolayer.

Example 4: Effect of Lipophenolic Derivative of Resveratrol on Cell Viability Assay (Mtt Assay)

(55) The MTT assay is a test used to evaluate the cytotoxicity of compounds.

(56) The cytotoxicity assay was carried out as described by Mosmann (Mosmann 1983). HUVEC cells and THP-1 cell line (1×10.sup.4 cell/well) were seeded in 96-well plate and incubated CO.sub.2 incubator chamber at 37° C. for 24 h. Cells were treated with serial dilutions of lipophenolic derivative of resveratrol at a final concentration of 10, 20, 40 and 80 μM, and plate was incubated for 72 hours. Supernatant was discarded and MTT-serum-free medium was added to each plate and incubated for three additional hours. The formed formazan blue crystals were further dissolved by addition of 100 μL of 10% SDS in 0.1 N HCl to each plate for 2 hours. The optical density was measured at 570 nm (reference filter 690 nm) using a TECAN™ plate reader. Lethal Concentration 50% (LC50) was calculated using GraphPad Prism v.5 software.

(57) The results are presented in FIG. 6, showing that Resv-LA and Resv-C22 (Resv-BE) at concentration 10, 20, 40 μM does not have cytotoxic effect on viability.

(58) All these results demonstrate that the lipophenolic compounds of the invention, in particular Resv-LA (FIG. 6A) and Resv-C22 (Resv-BE) (FIG. 6B), have inhibition effect of activity of MMP-9 and are able to decrease the TNF-alpha induced endothelial permeability. Such lipophenolic compounds, as new MMP-9 inhibitors, are capable of protecting the endothelial integrity and decrease the exacerbated vascular permeability, so may be advantageously used to protect the endothelial barrier integrity in infections and other diseases.

Example 5: Effect of Resv-LA (REV-LA) on TNF-α Release by LPS-Activated Microglia

(59) Cell Culture

(60) BV-2 cells that are derived from raf/myc-immortalised murine neonatal microglia are the most frequently used substitute for primary microglia (Blasi et al., 1990).

(61) BV-2 cells were maintained in 75 cm2 culture flasks in Dulbecco's Modified Eagle's Medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1% Penicillin-Streptomycin solution (Sigma), and cultured at 37° C. in a humidified atmosphere of 5% CO2.

(62) Treatment of the BV-2 Microglial Cell Culture

(63) BV-2 cells were plated on 24-well plates at a density of 105 cells per well. On the following day cells were subjected to different treatments. To study the effect of Resv-LA on TNF-alpha production by LPS activated BV-2 cells, the cells were incubated in medium containing 1 mg/ml LPS (Sigma-Aldrich) for 24 h, with or without Resv-LA (REV-LA) 30 μM.

(64) TNF-Alpha Measurement

(65) Supernatants from untreated and treated cells were centrifuged at 5000 rpm for 5 minutes and assayed for their TNF-α contents using the TNF-α (mouse) AlphaLISA Detection Kit of Perkin-Elmer. TNF-α released was normalized by cell number and expressed as percentage of the maximal released obtained in the LPS-stimulated condition.

(66) Results

(67) REV-LA (30 μM) is efficient for reducing microglial activation and neurotoxic TNF-α secretion by LPS-activated BV2 microglia cells. Twenty-four hours after LPS treatment, TN-α expression was reduced to 66.1±4.2 in REV-LA-treated BV2.

(68) Data are expressed as mean±SEM. Statistical analysis of differences between groups was performed by using unpaired t-test/Mann-Whitney. The level of significance is set at p<0.05. *P<0.05 and ***P<0.001 versus control (Ctr., non LPS-challenged control), #P<0.05 ##P<0.001 versus LPS-stimulated condition.

(69) The results are presented in the FIG. 7. These data suggest that Resv-LA (REV-LA) inhibits TNF-α release, which is a key factor of inflammatory cascades as disclosed above, so Resv-LA may advantageously be used for preventing or treating central nervous system disorders.

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