Patent ID: 12195437

The invention will be illustrated in the following non-limitative examples.

EXAMPLES

1—Synthesis of Lipophenolic Derivatives

a) Material and Methods

All solvents were anhydrous reagents from commercial sources. Unless otherwise noted, all chemicals and reagents were obtained commercially and used without purification. Commercial quercetin, rutin or (+)-catechin were dried at 100° C. for 48 h under vacuum before use. The reactions were monitored by using TLC on plates that were pre-coated with silica gel 60 (Merck). The reaction components were visualized by using a 254 nm UV lamp, staining with acidic p-anisaldehyde solution followed by gentle heating. Purifications of synthesized compounds by column chromatography were performed on silica gel 40-63 mm. Melting points (Mp) were determined on a Stuart capillary apparatus and are uncorrected. NMR spectra were recorded at 300 or 500 MHz (1H) and 75 or 126 MHz (13C) on Bruker spectrometers. The chemical shifts are reported in parts per million (ppm, δ) relative to residual deuterated solvent peaks. The NMR spectra were assigned with the help of 2D NMR analysis (COSY, HSQC, and HMBC). The multiplicities are reported as follows: bs=broad signal, m=multiplet, s=singlet, d=doublet, t=triplet, q=quadruplet, qt=quintuplet, or combinations thereof. For the peak assignation, the following abbreviations were used: Ar=aromatic, Cq=quaternary carbon, iP=O-isopropyl, TIPS=triisopropylsilyl, CH═CH=aliphatic alkene, tBu=tert-butyl. Proton numbering was assigned according to IUPAC nomenclature. The possible inversion of two values in the NMR spectra is expressed by an asterisk.

The compounds are numbered as follows in their NMR spectra interpretation:

b) Synthesis of Quercetin Derivatives
Synthesis of Q-3FA-7OiP Series

Three quercetin derivatives carrying an isopropyl group on their position 7 and a fatty acid on the position 3, have been synthesized. The synthesis is described on schema 1 hereunder.

From quercetin as starting material, after protection of hydroxyl group of the catechol with a diphenyldioxol (compound 1), followed by an acetylation, compound 2 was obtained. A selective deprotection of the position 7 was performed to lead to compound 3 which was then alkylated on said position 7 with an isopropyl group (compound 4). After diphenyldioxol deprotection, silylated protection of catechol hydroxyls and deacetylation of remaining OH at 3 and 5 positions, compounds 7 was obtained. A coupling step with three different fatty acids (LA, DHA and ALA) and a final deprotection of silylated protecting groups lead to the three desired quercetin derivatives: Q-3LA-70iP, Q-3DHA-7OiP and Q-3ALA-7OiP.

EXPERIMENTAL PART

(9Z,12Z)-5-hydroxy-2-(3,4-dihydroxyphenyl)-7-isopropoxy-4-oxo-4H-chromen-3-yl octadeca-9,12-dienoate (Q-3LA-70iP)

Rf(pentane/EtOAc, 50:50)=0.67. Mp 123.4° C.1H NMR (300 MHz, CDCl3) δ 7.37-7.28 (m, 2H, H2′, H6′), 6.90 (d, J=8.9 Hz, 1H, H5′), 6.36 (d, J=2.1 Hz, 1H, H8), 6.33 (d, J=2.1 Hz, 1H, H6), 5.46-5.24 (m, 4H, 2×CH═CH), 4.59 (qt, J=6.1 Hz, 1H, CHiP), 2.77 (t, J=5.7 Hz, 2H, H11″), 2.56 (t, J=7.6 Hz, 2H, H2″), 2.12-1.96 (m, 4H, H8″, H14″), 1.77-1.59 (m, 2H, H3″), 1.37 (d, J=6.1 Hz, 6H, 2×CH3iP), 1.34-1.18 (m, 14H, H4″, H5″, H6″, H7″, H15″, H16″, H17″), 0.88 (t, J=6.8 Hz, 3H, H18″).13C NMR (75 MHz, CDCl3) δ 175.89 (C4), 171.72 (C1″), 164.48 (C7), 161.71 (C5), 157.01 (C8a), 156.75 (C2), 147.45 (C3′), 143.72 (C4′), 130.53 (C3), 130.26 (CH═CH), 130.04 (CH═CH), 128.07 (CH═CH), 127.89 (CH═CH), 122.01 (C6′), 121.60 (C1′), 115.19 (C5′*), 114.96 (C2′*), 105.04 (C4a), 99.42 (C6), 93.95 (C8), 70.94 (CHiP), 33.91 (C2″), 31.50 (C16″), 29.56 (C15″*), 29.32 (C4″*), 29.13 (C5″*), 29.06 (C6″*), 28.98 (C7″*), 27.17 (2C, C8″, C14″), 25.61 (C11″), 24.66 (C3″), 22.55 (C17″), 21.84 (2C, CH3iP), 14.05 (C18″).

(4Z,7Z,10Z,13Z,16Z,19Z)-5-hydroxy-2-(3,4-dihydroxyphenyl)-7-isopropoxy-4-oxo-4H-chromen-3-yl docosa-4,7,10,13,16,19-hexaenoate (Q-3DHA-7OiP)

Rf(CH2Cl2/MeOH, 90:10)=0.65. Mp 116.8° C.1H NMR (300 MHz, MeOD) δ 7.35 (d, J=2.1 Hz, 1H, H2′), 7.30 (dd, J=8.4, 2.1 Hz, 1H, H6′), 6.89 (d, J=8.4 Hz, 1H, H5′), 6.58 (d, J=2.1 Hz, 1H, H8), 6.33 (d, J=2.1 Hz, 1H, H6), 5.47-5.20 (m, 12H, 6×CH═CH), 4.73 (qt, J=6.3 Hz, 1H, CHiP), 2.95-2.73 (m, 10H, H6″, H9″, H12″, H15″, H18″), 2.69 (t, J=7.3 Hz, 2H, H2″), 2.48 (dd, J=12.7, 7.3 Hz, 2H, H3″), 2.04 (qt, J=7.2 Hz, 2H, H21″), 1.36 (d, J=6.3 Hz, 6H, 2×CH3iP), 0.93 (t, J=7.2 Hz, 3H, H22″).13C NMR (75 MHz, MeOD) δ 177.22 (C4), 171.93 (C1″), 165.95 (C7), 162.93 (C5), 158.68 (C8a*), 158.61 (C2*), 150.44 (C3′), 146.68 (C4′), 132.76 (CH═CH), 131.62 (C3), 130.63 (2C,CH═CH), 129.40 (CH═CH), 129.15 (2C,CH═CH), 129.08 (2C,CH═CH), 128.92 (CH═CH), 128.69 (2C,CH═CH), 128.19 (CH═CH), 122.30 (C6′), 121.97 (C1′), 116.51 (C5′*), 116.34 (C2′*), 105.95 (C4a), 100.32 (C6), 94.83 (C8), 72.18 (CHiP), 34.71 (C2″), 30.69 (C6″*), 26.57 (2C, C9″*, C12″*), 26.40 (2C, C15″*, C18″*), 23.69 (C3″), 22.14 (2C, CH3iP), 21.45 (C21″), 14.59 (C22″).

(9Z,12Z,15Z)-5-hydroxy-2-(3,4-dihydroxyphenyl)-7-isopropoxy-4-oxo-4H-chromen-3-yl octadeca-9,12,15-trienoate (Q-3ALA-70iP)

Rf(pentane/EtOAc, 50:50)=0.69. Mp 129.8° C.1H NMR (300 MHz, MeOD) δ 7.35 (d, J=2.2 Hz, 1H, H2′), 7.30 (dd, J=8.4, 2.2 Hz, 1H, H6′), 6.89 (d, J=8.4 Hz, 1H, H5′), 6.59 (d, J=2.2 Hz, 1H, H8), 6.33 (d, J=2.2 Hz, 1H, H6), 5.47-5.20 (m, 6H, 3×CH═CH), 4.73 (qt, J=6.1 Hz, 1H, CHiP), 2.89-2.73 (m, 4H, H11″, H14″), 2.63 (t, J=7.3 Hz, 2H, H2″), 2.15-1.99 (m, 4H, H8″, H17″), 1.70 (qt, J=7.3 Hz, 2H, H3″), 1.37 (d, J=6.1 Hz, 6H, 2×CH3iP), 1.41-1.25 (m, 8H, H4″, H5″, H6″, H7″), 0.95 (t, J=7.5 Hz, 3H, H18″).13C NMR (75 MHz, MeOD) δ 177.23 (C4), 172.51 (C1″), 165.92 (C7), 162.91 (C5), 158.69 (C8a*), 158.60 (C2*), 150.47 (C3′), 146.66 (C4′), 132.71 (CH═CH), 131.59 (C3), 131.13 (CH═CH), 129.24 (CH═CH), 129.17 (CH═CH), 128.83 (CH═CH), 128.24 (CH═CH), 122.23 (C6′), 121.90 (C1′), 116.47 (C5′*), 116.26 (C2′*), 105.88 (C4a), 100.28 (C6), 94.73 (C8), 72.10 (CHiP), 34.63 (C2″), 30.63 (C4″*), 30.20 (C5″*), 30.14 (C6″*), 29.99 (C7″*), 28.15 (C8″), 26.52 (C11″**), 26.40 (C14″**), 25.81 (C3″), 22.14 (2C, CH3iP), 21.48 (C17″), 14.66 (C18″).

Synthesis of Q-3FA-5OiP Series

Three quercetin derivatives carrying an isopropyl group on their position 5 and a fatty acid on the position 3, have been synthesized. The synthesis is described on schema 2 hereunder.

The synthesis starts from rutin, a natural quercetin diglycoside. Hydroxyl groups on position 7 and on the catechol moiety are first benzylated and the compound 9 is obtained. The free hydroxyl group at the 5 position is then etherified with an isopropyl group to lead to compound 10. Then, the diglycoside is removed in acidic conditions and the resulted compound 11 is acetylated to afford compound 12. A hydrogenation step allows the removal of benzyl group (compound 13) replaced by silylated protective groups to give compound 14 which is deacetylated with methanolic ammonia. The resulted compound 15 is then engaged in a coupling step with three fatty acids (LA, DHA and ALA) to give compounds 16-LA, 16-DHA and 16-ALA respectively. A final step of deprotection affords the three compounds of interest: Q-3LA-50iP, Q-3DHA-5OiP and Q-3ALA-5OiP.

EXPERIMENTAL PART

(9Z,12Z)-7-hydroxy-2-(3,4-dihydroxyphenyl)-5-isopropoxy-4-oxo-4H-chromen-3-yl octadeca-9,12-dienoate (Q-3LA-50iP)

Rf(CH2Cl2/MeOH, 90:10)=0.53. Mp 84.2° C.1H NMR (300 MHz, MeOD) δ 7.32 (d, J=2.2 Hz, 1H, H2′), 7.26 (dd, J=8.4, 2.2 Hz, 1H, H6′), 6.88 (d, J=8.4 Hz, 1H, H5′), 6.49 (d, J=2.0 Hz, 1H, H8), 6.42 (d, J=2.0 Hz, 1H, H6), 5.42-5.24 (m 4H, 2×CH═CH), 4.65 (qt, J=6.0 Hz, 1H, CHiP), 2.76 (t, J=5.8 Hz, 2H, H11″), 2.63 (t, J=7.3 Hz, 2H, H2″), 2.12-1.98 (m, 4H, H8″, H14″), 1.69 (qt, J=7.0 Hz, 2H, H3″), 1.41 (d, J=6.0 Hz, 6H, CH3iP), 1.38-1.23 (m, 14H, H4″, H5″, H6″, H7″, H15″, H16″, H17″), 0.88 (t, J=6.8 Hz, 3H, H18″).13C NMR (75 MHz, MeOD) δ 173.05 (C4), 172.76 (C1″), 164.94 (C7), 161.12 (C5), 160.66 (C8a), 155.76 (C2), 149.89 (C3′), 146.56 (C4′), 133.54 (C3), 130.92 (2C,CH═CH), 129.06 (2C, CH═CH), 122.21 (C1′), 121.80 (C6′), 116.39 (C5′), 116.04 (C2′), 108.90 (C4a), 99.83 (C6), 96.18 (C8), 73.17 (CHiP), 34.73 (C2″), 32.66 (C16″), 30.67 (C4″*), 30.47 (C5″*), 30.27 (C6″*), 30.17 (C7″*), 30.11 (C15″*), 28.15 (2C, C8″, C14″), 26.53 (C11″), 25.79 (C3″), 23.64 (C17″), 21.95 (2C, CH3iP), 14.46 (C18″).

(4Z,7Z,10Z,13Z,16Z,19Z)-7-hydroxy-2-(3,4-dihydroxyphenyl)-5-isopropoxy-4-oxo-4H-chromen-3-yl docosa-4,7,10,13,16,19-hexaenoate (Q-3DHA-5OiP)

Rf(CH2Cl2/MeOH, 90:10)=0.50. Mp 112.4° C.1H NMR (300 MHz, MeOD) δ 7.32 (d, J=2.2 Hz, 1H, H2′), 7.26 (dd, J=8.4, 2.2 Hz, 1H, H6′), 6.88 (d, J=8.4 Hz, 1H, H5′), 6.50 (d, J=2.1 Hz, 1H, H8), 6.42 (d, J=2.1 Hz, 1H, H6), 5.50-5.19 (m, 12H, 6×CH═CH), 4.66 (qt, J=6.0 Hz, 1H, CHiP), 2.92-2.65 (m, 12H, H2″, H6″, H9″, H12″, H15″, H18″), 2.52-2.45 (m, 2H, H3″), 2.14-1.97 (m, 2H, H21″), 1.41 (d, J=6.0 Hz, 6H, 2×CH3iP), 0.94 (t, J=7.5 Hz, 3H, H22″).13C NMR (75 MHz, MeOD) δ 172.97 (C4), 172.18 (C1″), 164.98 (C7), 161.15 (C5), 160.68 (C8a), 155.73 (C2), 149.91 (C3′), 146.60 (C4′), 133.58 (C3), 132.75 (CH═CH), 130.52 (2C,CH═CH), 129.39 (CH═CH), 129.14 (2C,CH═CH), 129.09 (2C,CH═CH), 128.91 (CH═CH), 128.84 (2C,CH═CH), 128.20 (CH═CH), 122.22 (C1′), 121.86 (C6′), 116.42 (C5′), 116.06 (C2′), 108.94 (C4a), 99.89 (C6), 96.21 (C8), 73.21 (CHiP), 34.79 (C2″), 26.56 (4C, C6″, C9″, C12″, C15″), 26.41 (C18″), 23.66 (C3″), 21.96 (2C, CH3iP), 21.48 (C21″), 14.66 (C22″).

(9Z,12Z,15Z)-7-hydroxy-2-(3,4-dihydroxyphenyl)-5-isopropoxy-4-oxo-4H-chromen-3-yl octadeca-9,12,15-trienoate (Q-3ALA-50iP)

Rf(CH2Cl2/MeOH, 90:10)=0.43. Mp 77.0° C.1H NMR (300 MHz, MeOD) δ 7.32 (d, J=2.1 Hz, 1H, H2′), 7.25 (dd, J=8.4, 2.1 Hz, 1H, H6′), 6.88 (d, J=8.4 Hz, 1H, H5′), 6.49 (d, J=2.0 Hz, 1H, H8), 6.42 (d, J=2.0 Hz, 1H, H6), 5.44-5.20 (m, 6H, 6×CH═CH), 4.65 (qt, J=6.0 Hz, 1H, CHiP), 2.80 (t, J=5.8 Hz, 4H, H11″, H14″), 2.63 (t, J=7.3 Hz, 2H, H2″), 2.13-1.99 (m, 4H, H8″, H17″), 1.70 (qt, J=7.3 Hz, 2H, H3″), 1.41 (d, J=6.0 Hz, 6H, 2×CH3iP), 1.38-1.23 (m, 8H, H4″, H5″, H6″, H7″), 0.95 (t, J=7.5 Hz, 3H, H18″).13C NMR (75 MHz, MeOD) δ 173.04 (C4), 172.79 (C1″), 164.93 (C7), 161.12 (C5), 160.66 (C8a), 155.77 (C2), 149.87 (C3′), 146.56 (C4′), 133.55 (C3), 132.70 (CH═CH), 131.13 (CH═CH), 129.22 (CH═CH), 129.17 (CH═CH), 128.80 (CH═CH), 128.23 (CH═CH), 122.24 (C1′), 121.80 (C6′), 116.40 (C5′), 116.06 (C2′), 108.94 (C4a), 99.90 (C6), 96.21 (C8), 73.20 (CHiP), 34.73 (C2″), 30.65 (C4″*), 30.26 (C5″*), 30.16 (C6″*), 30.10 (C7″*), 28.15 (C8″), 26.51 (C11″**), 26.39 (C14″**), 25.78 (C3″), 21.95 (2C, CH3iP), 21.47 (C17″), 14.65 (C18″).

Synthesis of Q-3FA Series

Compounds Q-3LA and Q-3DHA, without isopropyl group, are synthesized from quercetin, first engaged in a coupling step with free fatty acid (LA and DHA) to give compounds 17-LA and 17-DHA wherein all hydroxyl groups are acylated by fatty acids. Then, an enzymatic selective deprotection is performed to give desired compounds wherein only position 3 is carrying a fatty acid.

(9Z,12Z)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl octadeca-9,12-dienoate (Q-3LA)

Rf(pentane/EtOAc, 50:50)=0.25. Mp 193.8° C.1H NMR (500 MHz, MeOD) δ 7.33 (d, J=2.2 Hz, 1H, H2′), 7.28 (dd, J=8.4, 2.2 Hz, 1H, H6′), 6.89 (d, J=8.4 Hz, 1H, H5′), 6.43 (d, J=2.1 Hz, 1H, H8), 6.24 (d, J=2.1 Hz, 1H, H6), 5.41-5.27 (m, 4H, 2×CH═CH), 2.77 (t, J=6.5 Hz, 2H, H11″), 2.63 (t, J=7.3 Hz, 2H, H2″), 2.12-2.00 (m, 4H, H8″, H14″), 1.70 (qt, J=7.3 Hz, 2H, H3″), 1.43-1.24 (m, 14H, H4″, H5″, H6″, H7″, H15″, H16″, H17″), 0.89 (t, J=6.8 Hz, 3H, H18″).13C NMR (126 MHz, MeOD) δ 177.22 (C4), 172.55 (C1″), 166.30 (C7), 163.13 (C5), 158.68 (C8a), 158.45 (C2), 150.37 (C3′), 146.64 (C4′), 131.43 (C3), 130.91 (CH═CH), 130.90 (CH═CH), 129.07 (CH═CH), 129.06 (CH═CH), 122.13 (C6′), 121.97 (C1′), 116.45 (C5′), 116.18 (C2′), 105.27 (C4a), 100.14 (C6), 95.03 (C8), 34.61 (C2″), 32.65 (C16″), 30.63 (C4″*), 30.48 (C5″*), 30.19 (C6″*), 30.13 (C7″*), 29.98 (C15″*), 28.15 (C8″**), 28.12 (C14″**), 26.53 (C11″), 25.80 (C3″), 23.63 (C17″), 14.42 (C18″).

(4Z,7Z,10Z,13Z,16Z,19Z)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl docosa-4,7,10,13,16,19-hexaenoate (Q-3DHA)

Rf(CH2Cl2/MeOH, 95:5)=0.20. Mp 191.3° C.1H NMR (300 MHz, MeOD) δ 7.34 (d, J=2.2 Hz, 1H, H2′), 7.28 (dd, J=8.4, 2.2 Hz, 1H, H6′), 6.89 (d, J=8.4 Hz, 1H, H5′), 6.43 (d, J=2.1 Hz, 1H, H8), 6.24 (d, J=2.1 Hz, 1H, H6), 5.51-5.16 (m, 12H, 6×CH═CH), 2.89-2.74 (m, 10H, H6″, H9″, H12″, H15″, H18″), 2.70 (t, J=7.3 Hz, 2H, H2″), 2.48 (q, J=7.3 Hz, 2H, H3″), 2.04 (qt, J=7.5 Hz, 2H, H21″), 0.94 (t, J=7.5 Hz, 3H, H22″).13C NMR (75 MHz, MeOD) δ 177.15 (C4), 171.95 (C1″), 166.27 (C7), 163.13 (C5), 158.67 (C8a), 158.38 (C2), 150.38 (C3′), 146.67 (C4′), 132.73 (CH═CH), 131.46 (C3), 130.61 (2C,CH═CH), 129.38 (3C,CH═CH), 129.12 (2C,CH═CH), 129.06 (CH═CH), 128.89 (CH═CH), 128.68 (CH═CH), 128.18 (CH═CH), 122.19 (C6′), 121.96 (C1′), 116.47 (C5′), 116.20 (C2′), 105.30 (C4a), 100.14 (C6), 95.04 (C8), 34.68 (C2″), 26.55 (4C, C6″*, C9″*, C12″*, C15″*), 26.39 (C18″*), 23.67 (C3″), 21.46 (C21″), 14.65 (C22″).

c) Synthesis of Catechin Derivatives

Synthesis of C-3LA-7OiP and C-3LA-5OiP

The synthesis of compound C-3LA-50iP and C-3LA-70iP, carrying a linoleic acid on position 3 and an isopropyl group on position 7 or 5 is described on schema 4 above. The catechol moiety of the (+)-catechin is protected with a diphenyldioxol to give compound 18. An isopropyl group is then introduced on position 7 to afford compound 19 or in position 5 to afford compound 20. Both compounds are obtained in one step, separated on silicagel chromatography and then used separately. A hydrogenation step is then performed to remove the diphenyldioxol group on the catechol moiety (compounds 21 and 22 from compounds 19 and 20 respectively). A last coupling is then realized using linoleoyl chloride give the compounds C-3LA-7OiP and C-3LA-50iP. Final compounds are obtained in small amount (mg), sufficiently for identification and evaluation.

Synthesis of C-3LA

C-3LA was obtained starting from catechin by acylation using linoleoyl chloride. Mono acylated C-3LA was isolated in small amount due to degradation issue.

EXPERIMENTAL PART

(9Z,12Z)-(2R,3S)-2-(3,4-dihydroxyphenyl)-5-hydroxy-7-isopropoxy-chroman-3-yl octadeca-9,12-dienoate (C-3LA-70iP)

Rf(pentane/EtOAc, 50:50)=0.64.1H NMR (300 MHz, MeOD) δ 6.79 (d, J=1.9 Hz, 1H, H2′), 6.76-6.65 (m, 2H, H5′, H6′), 6.02-5.96 (m, 2H, H6, H8), 5.43-5.26 (m, 4H, 2×CH═CH), 5.21 (dd, J=12.2, 6.8 Hz, 1H, H3), 4.89-4.85 (m, 1H, H2), 4.46 (qt, J=6.0 Hz, 1H, CHiP), 2.89-2.73 (m, 3H, H4β, H11″), 2.62 (dd, J=16.3, 7.0 Hz, 1H, H4α), 2.21 (t, J=7.2 Hz, 2H, H2″), 2.06 (q, J=6.5 Hz, 4H, H8″, H14″), 1.45 (qt, J=7.0 Hz, 2H, H3″), 1.39-1.14 (m, 20H, 2×CH3iP, H4″, H5″, H6″, H7″, H15″, H16″, H17″), 0.90 (t, J=6.9 Hz, 3H, H18″).13C NMR (75 MHz, MeOD) δ 174.61 (C1″), 159.13 (C7), 156.64 (2C, C8a, C5), 146.34 (2C, C3′, C4′), 131.08 (C′), 130.99 (2C,CH═CH), 129.05 (2C,CH═CH), 119.47 (C6′), 116.11 (C5′), 114.73 (C2′), 100.80 (C4a), 97.17 (C6*), 95.86 (C8*), 79.69 (C2), 70.87 (2C, C3, CHiP), 35.23 (C2″), 32.68 (C16″), 30.71 (C4″*), 30.49 (C5″*), 30.19 (C6″*), 30.12 (C7″*), 29.90 (C15″*), 28.16 (2C, C8″, C14″), 26.56 (C11″), 26.03 (C3″), 25.03 (C4), 23.65 (C17″), 22.43 (2C, CH3iP), 14.45 (C18″).

(9Z,12Z)-(2R,3S)-2-(3,4-dihydroxyphenyl)-7-hydroxy-5-isopropoxy-chroman-3-yl octadeca-9,12-dienoate (C-3LA-50iP)

Rf(pentane/EtOAc, 50:50)=0.63.1H NMR (300 MHz, MeOD) δ 6.78 (d, J=1.9 Hz, 1H, H2′), 6.77-6.63 (m, 2H, H5′, H6′), 6.04 (d, J=2.1 Hz, 1H, H8), 5.95 (d, J=2.1 Hz, 1H, H6), 5.44-5.25 (m, 4H, 2×CH═CH), 5.18 (dd, J=12.3, 6.9 Hz, 1H, H3), 4.86 (d, J=6.9 Hz, 1H, H2), 4.49 (qt, J=6.0 Hz, 1H, CHiP), 2.86-2.70 (m, 3H, H4β, H11″), 2.58 (dd, J=16.5, 7.1 Hz, 1H, H4α), 2.21 (t, J=7.2 Hz, 2H, H2″), 2.06 (q, J=6.5 Hz, 4H, H8″, H14″), 1.45 (qt, J=7.3 Hz, 2H, H3″), 1.40-1.13 (m, 20H, 2×CH3iP, H4″, H5″, H6″, H7″, H15″, H16″, H17″), 0.90 (t, J=6.8 Hz, 3H, H18″).13C NMR (75 MHz, MeOD) δ 174.61 (C1″), 158.41 (C5*), 158.24 (C7*), 156.54 (C8a), 146.47 (C3′**), 146.33 (C4′**), 131.06 (C1′), 130.97 (2C,CH═CH), 129.06 (2C,CH═CH), 119.48 (C6′), 116.10 (C5′), 114.73 (C2′), 101.57 (C4a), 96.32 (C6*), 95.13 (C8*), 79.62 (C2), 71.23 (C3**), 70.93 (CHiP**), 35.22 (C2″), 32.68 (C16″), 30.68 (C4″*), 30.48 (C5″*), 30.17 (C6″*), 30.10 (C7″*), 29.88 (C15″*), 28.15 (2C, C8″, C14″), 26.55 (C11″), 26.02 (C3″), 25.31 (C4), 23.64 (C17″), 22.51 (CH3iP), 22.46 (CH3iP), 14.45 (C18″).

(9Z,12Z)-(2R,3S)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-chroman-3-yl octadeca-9,12-dienoate (C-3LA)

Rf(CH2Cl2/MeOH, 80:20)=0.80.1H NMR (500 MHz, MeOD) δ 6.80 (d, J=2.0 Hz, 1H, H2′), 6.73 (d, J=8.1 Hz, 1H, H5′), 6.68 (dd, J=8.1, 2.0 Hz, 1H, H6′), 5.95 (d, J=2.2 Hz, 1H, H8), 5.89 (d, J=2.2 Hz, 1H, H6), 5.42-5.27 (m, 4H, 2×CH═CH), 5.20 (dd, J=12.4, 7.0 Hz, 1H, H3), 4.85 (d, J=7.0 Hz, 1H, H2), 2.84-2.74 (m. 3H, H4β, H11″), 2.60 (dd, J=16.3, 7.0 Hz, 1H, H4α), 2.20 (td, J=7.3, 1.8 Hz, 2H, H2″), 2.06 (q, J=6.9 Hz, 4H, H8″, H14″), 1.50-1.40 (m, 2H, H3″), 1.40-1.11 (m, 14H, H4″, H5″, H6″, H7″, H15″, H16″, H17″), 0.90 (t, J=6.9 Hz, 3H, H18″).13C NMR (126 MHz, MeOD) δ 174.61 (C1″), 158.17 (C7), 157.61 (C5), 156.60 (C8a), 146.45 (C3′*), 146.31 (C4′*), 131.15 (C1′), 130.98 (CH═CH), 130.93 (CH═CH), 129.09 (CH═CH), 129.04 (CH═CH), 119.53 (C6′), 116.10 (C5′), 114.79 (C2′), 99.72 (C4a), 96.49 (C8), 95.56 (C6), 79.68 (C2), 70.97 (C3), 35.22 (C2″), 32.65 (C16″), 30.68 (C4″*), 30.47 (C5″*), 30.17 (C6″*), 30.10 (C7″*), 29.90 (C15″*), 28.16 (C8″**), 28.14 (C14″**), 26.54 (C11″), 26.00 (C3″), 25.13 (C4), 23.61 (C17″), 14.41 (C18″).

2—Evaluation of Biological Activity of Lipophenolic Derivatives

a) Material and Methods

Chemicals

All lipophenols were dissolved in dimethylsulfoxide (DMSO) to prepare a stock solution at 80 mM. Hydrogen peroxide solution (H2O2, 30 wt. % in H2O), α-tocopherol and all-trans-retinal were purchased from Sigma-Aldrich. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was obtained from Sigma-Aldrich. N-retinylidene-N-retinylethanolamine (A2E) was synthesized as previously described by Parish et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 14609-613. 2′,7′-dichlorofluorescin diacetate (DCFDA) was purchased from Abcam and dissolved in DMSO to prepare stock solution at 20 mM. All stock solutions of lipophenols, α-tocopherol, all-trans-retinal, A2E and probe were stored at −20° C. in the dark.

Cell Culture

ARPE-19 cells (cells from the retinal pigment epithelium) were obtained from ATCC, and were grown following the instructions in Dulbecco's Modified Eagle's Medium (DMEM)/Ham F12 (GIBCO) containing 10% v/v fetal bovine serum (FBS) and 1% v/v penicillin/streptomycin under a humidified air (95%)/CO2(5%) atmosphere at 37° C. For experimental cell seeding and sub-culturing, cells were dissociated with 0.25% trypsin-EDTA, re-suspended in the culture medium and then plated at 1-3×105cells/mL.

Cell Viability

Cell viability was determined by MTT colorimetric assay. Cells were incubated for 2 h with MTT reagent (0.5 mg/mL). During this incubation time, dehydrogenases of living cells reduced MTT to insoluble purple formazan, which was then dissolved in DMSO. The absorbance at 570 nm and 655 nm of individual wells was measured using a microplate reader (BioRad 550). The percentage of viable cells was calculated as [(OD570 sample−OD655 sample)/(OD570 control−OD655 control)]×100%.

Statistical Analyses for Biological Tests

The data are presented as means±SEM determined from at least three independent experiments. In each experiment, all conditions were done at least in quadruplicate.

Cytotoxicity of Lipophenols

ARPE-19 cells were plated into 96-well plates (4×104cells/well) and cultured for 24 h to reach confluence before lipophenol treatment. The cell cultures were treated with serum free medium containing the lipophenols at different concentrations (0-160 μM) for 24 h. Control cells were incubated with DMSO (0.2%). The viability of the cells was determined using a MTT colorimetric assay. All samples are expressed as percentage of viability compared to non-treated control cells (normalized at 100% survival).

Protection of Lipophenols Against all-Trans-Retinal

ARPE-19 cells were plated into 96-well plates (4×104cells/well) and cultured for 24 h to reach confluence before lipophenol treatment. The cell cultures were treated with serum free medium containing lipophenols at different concentrations (0-80 μM) for 1 h. Then all-trans-retinal was added to a final concentration of 15 μM for 4 h, before rinsing with medium. Control cells were incubated with DMSO (0.2%)±all-trans-retinal. The cell viability was determined 16-20 h later using a MTT colorimetric assay. All samples are expressed as percentage of viability compared to non-treated and exposed to all-trans-retinal control cells. Non-treated and non-exposed to all-trans-retinal control cells are normalized at 100% survival.

Protection of Lipophenols Against ROS Production

Reactive oxygen species (ROS) were measured in ARPE-19 cells with the probe DCFDA. The cell permeant reagent DCFDA is deacetylated by cellular esterases to dichlorofluorescein (DCFH), which can be oxidized by ROS into the fluorophore 2′,7′-dichlorofluorescein (DCF). ARPE-19 cells were plated into black, opaque bottom 96-well plates (4×104cells/well) and cultured for 24 h to reach confluence before drug treatment. The cell cultures were incubated with 2 μM of DCFDA for 45 min in DMEM/F12 medium without phenol red+1% FBS. The cells were rinsed and incubated with medium containing lipophenols at different concentrations (0-80 μM) for 1 h. Then H2O2was added to a final concentration of 600 μM for 4 h, followed by the measurement of DCF production by fluorescence spectroscopy with excitation wavelength at 485 nm and emission wavelength at 535 nm. Control cells were incubated with DMSO (0.2%)±DCFDA±H2O2. All samples are expressed as percentage of ROS production compared to non-oxidized control cells (normalized at 100% of ROS production).

Protection of Lipophenols Against A2E

ARPE-19 cells were plated into 96-well plates (4×104cells/well) and cultured for 24 h to reach confluence before lipophenol treatment. The cell cultures were treated with serum free DMEM/F12 medium without phenol red containing lipophenols at different concentrations (0-80 μM) for 1 h. Then A2E was added to a final concentration of 20 μM for 6 h, before rinsing with medium. Control cells were incubated with DMSO (0.2%)+A2E. Cells were exposed to intense blue light (4600 LUX) for 30 min to induce phototoxicity of A2E and incubated at 37° C. The cell viability was determined 16-20 h later using a MTT colorimetric assay. All samples are expressed as percentage of viability compared to non-treated and exposed to A2E control cells. Non-treated and non-exposed to A2E control cells are normalized at 100% survival.

b) Results

Compounds according to the invention, from quercetin and catechin series, are compared to phloroglucinol compounds already known in the art.

The discussion below is based on the results illustrated onFIGS.1to5.

Cytotoxicity on ARPE-19 Cells

Results on the measurement of lipophenol toxicity will be discussed in % cell survival at a given lipophenol concentration compared to untreated cells. Any toxicity observed will give an indication of the cytotoxic concentration 50 (CC50) of the derivatives. Any concentration of lipophenol resulting in cell survival of less than 5% (zero survival) will be considered a highly toxic concentration.

Phloroglucinol Series (Comparative)

A slight toxicity is observed on ARPE-19 cells regardless of the phloroglucinol derivative. This toxicity does not appear to increase with phloroglucinol concentration (between 10 and 160 μM) and allows survival between 61% (commercial phloroglucinol) and 86% (P-OiP-ODHA) after incubation of a maximum concentration of 160 μM.

Catechin Series

Commercial catechin and both C-50iP and C-70iP derivative (which do not carry a fatty acid portion) show no toxicity for concentration until 160 μM. However, for compound carrying a linoleic acid C-3LA (without isopropyl group), the toxicity increases with a CC50<160 μM. Similar toxicity is observed for compound C-3LA-50iP, suggesting that the introduction of an isopropyl group in position 5 does not allow to decrease the toxicity due to the linoleic acid. C-3LA-7OiP derivative ended in a toxicity much more important than C-3LA-5OiP with a cell survival equal to 0 starting from 80 μM.

Quercetin Series

Commercial quercetin induces cytotoxicity at a concentration below 160 μM with a CC50of approximately 120 μM. When substituted with isopropyl at position 5 (Q-50iP), this toxicity decreases to 72% survival at 160 μM. On the other hand, if isopropyl is introduced in position 7 (Q-70iP), a high toxicity is observed with zero survival as early as 40 μM of alkyl-phenol, suggesting an importance of isopropyl position in the toxicity of the alkylated-quercetin without a fatty acid moiety.

The introduction of a lipid chains on commercial quercetin lead to different toxicity profiles according to the type of fatty acid (LA or DHA). Thus, Q-3LA show a protective ability regardless cells since it increases survival up to 156% at 80 μM and remains protective even at 160 μM (124%). However, Q-3DHA is protective at low concentrations but is toxic at higher concentrations with a CC50<160 μM.

For doubly substituted derivatives, the series having isopropyl in position 5 (Q-3FA-5OiP wherein FA=LA or DHA or ALA) induces cytotoxicity from 40-80 μM. Since Q-50iP shows almost no toxicity, the induced toxicity seems to be due to the introduction of the lipid chain in position 3 when an isopropyl is at the position 5. The observed toxicity profile appears to be in the same direction as for Q-3FA (where FA=LA or DHA since the toxicity observed for the DHA derivative (Q-3DHA-5OiP, CC50>40 μM) is higher than for the LA derivative (Q-3LA-50iP, CC50<80 μM). However, for the series having an isopropyl in position 7 (Q-3FA-7OiP where FA=LA or DHA or ALA) no toxicity is observed up to 160 μM, whatever the lipid introduced. These derivatives even seem to have a more or less important protective effect on the cells, going up to 153% survival for Q-3LA-7OiP at 160 μM. These protections are all the more surprising as the Q-70iP derivative (without fatty acid) shows a very important cytotoxicity even at low concentration, suggesting that the ester bond is stable in cellular medium during the duration of the test (24 h).

Conclusion on toxicity: This study shows that the toxicity profile of quercetin and catechin series differs from one derivative to another and seems to be related to the position of the substituents. Concerning the quercetin series, compounds with an isopropyl group in position 7 and a fatty acid in position 3 allow avoiding any toxicity until 160 μM.

Anti-Carbonyl Stress Activity: Protection Against Trans-Retinal Toxicity in ARPE-19 Cells.

The results on the measurement of anti-carbonyl stress activity will be discussed in % cell survival gain compared to cells stressed by trans-retinal (at 15 μM) and not treated with lipophenols. Any survival gain greater than +5% will be considered as a proven anti-carbonyl stress activity.

Phloroqlucinol Series (Comparative)

The need for double functionalization of phloroglucinol by isopropyl and lipid has already been demonstrated in order to obtain anti-carbonyl stress activity (patent WO2015162265A1, Brabet et al., New lipophenol compounds and uses thereof). The results reproduced here for the phloroglucinol series confirm the importance of the two groups. Indeed, commercial phloroglucinol, P-OiP as well as P-OLA and P-ODHA show no survival gain on cells in the presence of trans-retinal whatever the concentration used. On the other hand, the two derivatives P-OiP-OLA and P-OiP-ODHA increase cell survival in the presence of trans-retinal up to +31% and +25% respectively, at 80 μM.

Catechin Series

Like the phloroglucinol series, commercial catechin, C-50iP and C-70iP show no anti-carbonyl stress activity up to 80 μM. However, unlike the phloroglucinol series, C-3LA (isopropyl-free) shows slight cell protection up to +11% at 80 μM.

Concerning the double functionalization of catechins, C-3LA-5OiP shows moderate anti-carbonyl stress activity (+9% at 80 μM) while C-3LA-7OiP seems three times more protective at lower concentrations (+27% at 40 μM).

Compared to the lead of the phloroglucinol P-OiP-OLA series, C-3LA-7OiP has a higher anti-carbonyl stress activity at low doses (40 μM) increasing cell survival by +27% compared to +20% for phloroglucinol derivative.

Quercetin Derivatives

Like in the phloroglucinol and catechin series, the commercial quercetin and the isopropylated analogues (Q-50iP and Q-70iP, without fatty acid), did not show anti-carbonyl stress potency. Conversely, the two derivatives Q-3LA and Q-3DHA (without isopropyl group) show moderate cell protection against trans-retinal toxicity, with a maximum protection of +9% (obtained at 20 μM) and +21% (at 60 μM) respectively.

All quercetins doubly functionalized with isopropyl and fatty acid, Q-3FA-5OiP and Q-3FA-70iP (where FA=LA or DHA or ALA) show interesting anti-carbonyl stress activity in the tested concentration range.

The Q-3FA-5OiP series (where FA=LA or DHA or ALA) where isopropyl is in position 5, has a protective effect against the toxicity of trans-retinal despite significant toxicity of lipophenols above 40 μM. The Q-3LA-5OiP shows the best anti-carbonyl stress properties with a maximum survival gain of +49% to 80 μM. Even at lower doses (40 μM, non-cytotoxic concentration) the Q-3LA-5OiP allows to reach a cellular survival increased by +34% which is much higher than that observed by the lead P-OiP-OLA at the same concentration (+20% survival). The Q-3DHA-5OiP and Q-3ALA-5OiP derivatives have a similar activity profile, since at low concentrations (up to 40 μM) they are protective but their beneficial effect is counteracted by their toxicity (from 60 μM). Thus at 40 μM the Q-3DHA-5OiP and Q-3ALA-5OiP allow a survival gain of +27% and +24% respectively, which is higher than the gain observed for the P-OiP-ODHA lead (+20% at 40 μM).

Concerning the series Q-3FA-70iP (where FA=LA or DHA or ALA) having isopropyl in position 7, the derivative showing the best anti-carbonyl stress activity is also the derivative LA: Q-3LA-70iP, which makes it possible to obtain a survival gain of +38% at 80 μM. Its protective activity seems to reach a limit value as early as 20 μM suggesting that this concentration would be sufficient to obtain the maximum anti-carbonyl stress activity of this derivative. For Q-3DHA-70iP, the maximum protection is reached less rapidly and it is necessary to increase the concentrations to observe the protective effect: +29% at 80 μM. This anti-carbonyl stress activity is however slightly higher than that observed for P-OiP-ODHA lead (+25% at 80 μM). Finally, for the Q-3ALA-7OiP derivative, the maximum observed survival gain is similar to that of Q-3DHA-7OiP (+29% at 80 μM). The higher value observed for Q-3LA-7OiP giving maximum anti-carbonyl stress activity is not reached for the two derivatives Q-3DHA-7OiP and Q-3ALA-7OiP suggesting that they may confer better cell protection against trans-retinal at higher concentrations since they show no toxicity up to 160 μM.

Conclusion on anti-carbonyl stress activity: These results show that compounds substituted by isopropyl only, do not present any anti-carbonyl stress activity. In some cases, derivative with only one fatty acid can lead to a slight protective effect against carbonyl stress. Best protective results are obtained for di-substituted (by isopropyl and fatty acid) derivative in quercetin series>catechin series and with 7-OiP close to 5-OiP activities, however 7-OiP series would be preferred due to toxicity issue of the 5-OiP series.

Antioxidant Activity: Reduction of ROS

The results on the measurement of antioxidant activity linked to the trapping of ROS (Reactive Oxygen Species) of lipophenols, will be discussed by comparing the gain of % of ROS production compared to non-stressed cells (to be as comparable as possible given the high variability of the % of ROS produced by cells stressed by hydrogen peroxide and not treated by lipophenols). We therefore consider that above +300% ROS, lipophenol derivatives do not have antioxidant activity. The lowest percentages of increase will therefore be representative of the most protective derivatives.

Phloroqlucinol Series (Comparative)

Measurement of the antioxidant activity of the phloroglucinol series shows that only commercial phloroglucinol can significantly reduce the production of ROS in stressed cells (+50% ROS at 80 μM). The mono-substituted P-OiP derivative retains part of the antioxidant activity with +142% ROS at 80 μM. The lipidic mono-substituted P-OLA and P-ODHA derivatives lose almost all of the antioxidant properties with +238% and +231% ROS at 80 μM respectively. Concerning the two di-substituted leads (P-OiP-OLA and P-OiP-ODHA), no antioxidant property could be observed based on ROS trapping (+300% of ROS whatever the lipophenol concentration).

Catechin Series

Measurement of the antioxidant activity of the catechin series shows that the derivatives provide good cell protection with a relatively small increase in the % ROS. The commercial catechin, C-50iP and C-70iP derivatives make it possible to fully protect cells with 0% increase in ROS from 20 μM. For C-3LA, the increase in ROS is also small with only +24% at 80 μM. The ROS trapping activity of the two di-substituted derivatives is slightly reduced but remains nevertheless interesting with only +67% of ROS for C-3LA-50iP and +37% of ROS for C-3LA-7OiP at 80 μM.

Quercetin Derivatives

The measurement of the antioxidant activity of the quercetin series shows that all the derivatives have a more or less important antioxidant activity and allow reducing the production of ROS. Both Q-70iP and Q-50iP derivatives have good antioxidant properties with +31% ROS at 20 μM (non-cytotoxic concentration) and +46% ROS at 80 μM respectively.

The Q-3FA derivatives (where FA=LA or DHA) show a similar antioxidant profile ranging from +109 to +120% ROS (at 80 μM) suggesting that antioxidant activity does not depend on the type of lipid introduced.

Concerning the series of di-functionalized derivatives Q-3FA-5OiP (where FA=LA or DHA or ALA) having isopropyl in 5, the antioxidant activity profiles are similar whatever the lipid introduced in position 3 ranging from +88 to +114% of ROS at 80 μM. Similarly, for the series of di-functionalized derivatives Q-3FA-7OiP (where FA=LA or DHA or ALA) having isopropyl in 7, the profiles are similar whatever the fatty acid in position 3 with slightly weaker activities ranging from +141 to +192% of ROS at 80 μM. Although these antioxidant activities appear more moderate than those observed for commercial quercetin, they represent a maximum decrease in ROS production of 53% for the Q-3FA-70iP series (where FA=LA or DHA or ALA) and 71% for the Q-3FA-5OiP series (where FA=LA or DHA or ALA) compared to stressed and untreated cells.

Conclusion on antioxidant activity: It appears from this study that three of the catechin derivatives have dual anti-carbonyl stress and antioxidant activity: C-3LA, C-3LA-5OiP and C-3LA-7OiP. Concerning the quercetin series, two mono-substituted derivatives have a double anti-carbonyl stress and antioxidant activity and are potentially interesting for the development of anti-COS: Q-3LA and Q-3DHA. In addition, the six di-functionalized quercetin derivatives have shown strong dual anti-carbonyl stress and antioxidant activity and are of interest for future development and/or in vivo evaluations: Q-3LA-5OiP, Q-3DHA-5OiP, Q-3ALA-5OiP, Q-3LA-7OiP, Q-3DHA-7OiP and Q-3ALA-7OiP. Due to their low toxicity, Q-3FA-7OiP derivatives (where FA=LA or DHA or ALA) with isopropyl at the 7 position are preferred in this study.

Protection Against Toxicity of Photoactivated A2E in ARPE-19 Cells:

Photoactivation of A2E leads to its oxidation and can generate cytotoxic derivatives, in particular carbonyl derivatives responsible for carbonyl stress. Results on lipophenol protection against photoactivated A2E toxicity will be discussed in % cell survival gain compared to cells stressed by A2E and not treated by lipophenols. This additional evaluation was not performed for all synthesized lipophenolic derivatives but only a selection of them to discuss the results obtained.

Concerning the series of phloroglucinol derivatives, the two leads P-OiP-OLA and P-OiP-ODHA (anti-carbonyl stress activities but without effectiveness in ROS trapping) were selected to evaluate their protection against toxicity induced by photoactivated A2E. For comparison, commercial phloroglucinol (no anti-carbonyl stress activity but strong anti-oxidative stress activity) and P-OLA (no anti-carbonyl stress activity but anti-oxidative stress activity) were also evaluated.

For catechin derivatives, C-3LA-5OiP being the least toxic and having shown both anti-carbonyl stress and anti-oxidative stress properties, its protection against photoactivated A2E induced toxicity was also evaluated.

Finally, for the quercetin series, the three derivatives having obtained the best double anti-carbonyl stress and anti-oxidative stress activities, and an absence of toxicity up to 160 μM, are Q-3LA-70iP, Q-3DHA-7OiP and Q-3ALA-70iP. They were therefore selected to evaluate their protection against photoactivated A2E induced toxicity. In order to serve as a comparison, commercial quercetin (without anti-carbonyl stress activity but with antioxidant activity) as well as Q-3LA (with low anti-carbonyl stress activity and good antioxidant activity) were also evaluated.

Phloroqlucinol Series (Comparative)

For commercial phloroglucinol and P-OiP, no cell protection could be observed. In contrast, for the two derivatives with anti-carbonyl stress activity P-OiP-OLA and P-OiP-ODHA, a survival gain of +52% and +25% respectively was observed at 80 μM of lipophenol. As these two derivatives do not have ROS trapping activity in the tests performed (unlike commercial phloroglucinol) it would seem that their protection against A2E toxicity could be linked to their anti-carbonyl stress activity.

Catechin Derivative

For C-3LA-5OiP having shown a double activity anti-carbonyl stress and antioxidant, it was observed a gain of survival of +30%. Even with moderate anti-carbonyl stress activity (+9% at 80 μM), this lipophenol is protective against A2E.

Quercetin Derivatives

Commercial quercetin shows a cell protection with a +36% survival gain from 40 μM, which seems to reach a plateau and no longer increase up to 80 μM. This protection against A2E does not seem related to the anti-carbonyl stress activity of commercial quercetin because it shows no cellular protection in the presence of trans-retinal at the concentrations evaluated. All quercetin derivatives tested are protective whether they are antioxidant or active against carbonyl stressor. Q-3LA having shown an anti-carbonyl stress activity and moderate antioxidant properties, allows to reach a +48% survival gain from 60 μM. The Q-3LA-70iP derivative is the one allowing the best protection with a +86% survival gain from 40 μM. It seems to reach a maximum protection plateau from this concentration. The Q-3DHA-7OiP derivative also seems to reach a plateau as early as 20 μM with a survival gain of +62%. The Q-3ALA-7OiP derivative seems to require higher concentrations to confer good cell protection and allow a survival gain of +77% at 80 μM. Concerning the quercetin derivative series, part of the cells protection against photoactivated A2E is conferred by the skeleton (and potentially its antioxidant properties) but in order to exacerbate this protection, the activity against carbonyl stress seems essential, allowing to pass from a survival gain of +36% to +86%.

All these biological results demonstrate that the double substituted flavonoid derivatives, preferably the double substituted quercetin, and even more preferably quercetin carrying an isopropyl group on position 7 and a fatty acid DHA or LA on position 3, are the most efficient compounds against both carbonyl and oxidative stresses.