LINOLEIC ACID DERIVATIVES, PHARMACEUTICAL COMPOSITION OR FOOD COMPOSITION COMPRISING SAID LINOLEIC ACID DERIVATIVES, AND THEIR USES

Abstract

Disclosed is a linoleic acid derivative of Formula (I) below including a hydrophobic part C.sub.17H.sub.31 linked to a polar head part “A”:

##STR00001## wherein the polar head part A is selected from A.sup.1 to A.sup.4 below:

##STR00002## or a pharmaceutically/food quality acceptable salt thereof.

Claims

1. Linoleic acid derivative of Formula (I) below comprising a hydrophobic part C.sub.17H.sub.31 linked to a polar head part “A”: ##STR00054## wherein said polar head part A is selected from A.sup.1 to A.sup.4 below: ##STR00055## wherein R.sup.1 and R.sup.2 are independently selected from the group composed of H or a saturated or unsaturated, straight or branched alkyl group containing 1 to 8 carbon atoms, or R.sup.1 and R.sup.2 are linked together to form a divalent radical of formula —R.sup.1-R.sup.2; R.sup.3 is independently selected from the group composed of: ##STR00056## wherein R.sup.4 is independently selected from the group composed of H or a saturated or unsaturated, straight or branched alkyl group containing 1 to 4 carbon atoms, n is an integer selected from 1 to 4, and n′ is an integer that is equal to 0 or 1, provided that R.sup.4 is different from H when n=4 and n′=0 or when n=1 and n′=0, “*” is the carbon atom which is attached to the oxygen atom of the A.sup.2 polar head; R.sup.12 is selected from H or a saturated or unsaturated, straight or branched alkyl group containing 1 to 4 carbon atoms, or an aromatic group or —CO.sub.2R.sup.13 in which R.sup.13 is a saturated alkyl group containing 1 to 4 carbon atoms, and R.sup.14, R.sup.15, R.sup.16 is independently selected from H or CH.sub.3; R.sup.5, R.sup.6 and R.sup.7 are independently selected from the group composed of H or a saturated or unsaturated, straight or branched alkyl group containing 1 to 4 carbon atoms, or —CO.sub.2R.sup.8 in which R.sup.8 is a saturated or unsaturated, straight or branched alkyl group containing 1 to 4 carbon atoms; with the proviso that when R.sup.7═H or CH.sub.3, R.sup.5 and R.sup.6 are different from each other; R.sup.9 to R.sup.11 are independently selected from the group composed of H or a saturated or unsaturated, straight or branched alkyl group containing 1 to 4 carbon atoms, provided that when R.sup.10 and R.sup.11═H, R.sup.9 is different from an ethyl-group or H; or a pharmaceutically/food quality acceptable salt thereof.

2. The linoleic acid derivative according to claim 1, having a molecular weight ranging from about 300 to about 600 g/mol.

3. The linoleic acid derivative according to claim 1, wherein in A.sup.1, R.sup.1 and R.sup.2 are a saturated or unsaturated, straight or branched alkyl group containing 1 to 2 carbon atoms, or a pharmaceutically/food quality acceptable salt thereof.

4. The linoleic acid derivative according to claim 1, wherein in A.sup.2, R.sup.3 is ##STR00057## wherein R.sup.4 is CH.sub.3, when n=1, and n′=0, or n=1 and n′=1, or still n=4 and n′=0. or a pharmaceutically/food quality acceptable salt thereof.

5. The linoleic acid derivative according to claim 1, wherein in A.sup.2, R.sup.3 is ##STR00058## wherein R.sup.12 is H, or a pharmaceutically/food quality acceptable salt thereof.

6. The linoleic acid derivative according to claim 1, wherein in A.sup.2, R.sup.3 is ##STR00059## or a pharmaceutically/food quality acceptable salt thereof.

7. The linoleic acid derivative according to claim 1, wherein in A.sup.3, R.sup.6 is H, R.sup.6 is —CO.sub.2CH.sub.3 and R.sup.7 is H, or a pharmaceutically/food quality acceptable salt thereof.

8. The linoleic acid derivative according to claim 1, wherein in A.sup.4, R.sup.9 is H, R.sup.10 is CH.sub.3, and R.sup.11 is H, or a pharmaceutically/food quality acceptable salt thereof.

9. A medicament comprising: a linoleic acid derivative of Formula (I) below comprising a hydrophobic part C.sub.17H.sub.31 linked to a polar head part “A”: ##STR00060## wherein said polar head part A is selected from A.sup.1 to A.sup.4 below: ##STR00061## wherein R.sup.1 and R.sup.2 are independently selected from the group composed of H or a saturated or unsaturated, straight or branched alkyl group containing 1 to 8 carbon atoms, or R.sup.1 and R.sup.2 are linked together to form a divalent radical of formula —R.sup.1-R.sup.2—; R.sup.3 is independently selected from the group composed of: ##STR00062## wherein R.sup.4 is independently selected from the group composed of H or a saturated or unsaturated, straight or branched alkyl group containing 1 to 4 carbon atoms, n is an integer selected from 1 to 4, and n′ is an integer that is equal to 0 or 1; “*” is the carbon atom which is attached to the oxygen atom of the A.sup.2 polar head; R.sup.12 is selected from H or a saturated or unsaturated, straight or branched alkyl group containing 1 to 4 carbon atoms, or an aromatic group or —CO.sub.2R.sup.13 in which R.sup.13 is a saturated alkyl group containing 1 to 4 carbon atoms, and R.sup.14, R.sup.15, R.sup.16 is independently selected from H or CH.sub.3; R.sup.5, R.sup.6 and R.sup.7 are independently selected from the group composed of H or a saturated or unsaturated, straight or branched alkyl group containing 1 to 4 carbon atoms, or —CO.sub.2R in which R.sup.8 is a saturated or unsaturated, straight or branched alkyl group containing 1 to 4 carbon atoms; R.sup.9 to R.sup.11 are independently selected from the group composed of H or a saturated or unsaturated, straight or branched alkyl group containing 1 to 4 carbon atoms; or a pharmaceutically/food quality acceptable salt thereof.

10. A pharmaceutical composition or food composition comprising, respectively, at least one pharmaceutically acceptable carrier and at least one linoleic acid derivative according to claim 9, or at least one food ingredient and/or at least one food additive and at least one linoleic acid derivative according to claim 9.

11. A method for treatment of a disorder modulated by the GPR120 receptor and/or the CD36 receptor, comprising administering to a subject in need thereof a therapeutically effective amount of said linoleic acid derivative of claim 1.

12. The method according to claim 11, wherein the disorder modulated by the GPR120 receptor and/or the CD36 receptor is selected from the group consisting of diabetes, hyperglycemia, impaired glucose tolerance, gestational diabetes, insulin resistance, hyperinsulinemia, retinopathy, neuropathy, nephropathy, diabetic kidney disease, acute kidney injury, cardiorenal syndrome, delayed wound healing, atherosclerosis and its sequelae, abnormal heart function, congestive heart failure, myocardial ischemia, stroke, Metabolic Syndrome, hypertension, obesity, body weight disorder, fatty liver disease, dyslipidemia, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, low high-density lipoprotein (HDL), high low-density lipoprotein (LDL), lipid disorders and liver diseases.

13. An appetite suppressant comprising the linoleic acid derivative according to claim 1.

14. A food supplement or dietary supplement for animal or human food, or ready-cooked dish, or animal feedstuff, comprising the food composition of claim 10.

15. A method for enhancing taste, modulating taste, suppressing appetite, or improving the physical appearance of individuals, comprising applying an effective amount of the linoleic acid derivative according to claim 1.

16. The linoleic acid derivative of claim 1, wherein: R.sup.1-R.sup.2— is —CH.sub.2—CH.sub.2— or —(CH.sub.2).sub.3—; R.sup.14═R.sup.15═R.sup.16═CH.sub.3 or R.sup.14═H or CH.sub.3 and R.sup.15═R.sup.16═H; and R.sup.5 and R.sup.6 are different from each other when they correspond to CH.sub.3 or to H.

17. The linoleic acid derivative according to claim 1, wherein in A.sup.1, R.sup.1 and R.sup.2 are a saturated or unsaturated, straight or branched alkyl group containing 1 to 2 carbon atoms, or a pharmaceutically/food quality acceptable salt thereof, and R.sup.1 and R.sup.2 are ethyl group.

18. The linoleic acid derivative of claim 9, wherein: —R.sup.1-R.sup.2— is —CH.sub.2—CH.sub.2— or —(CH.sub.2).sub.3—; R.sup.4 is different from H when n=4 and n′=0 or when n=1 and n′=0; R.sup.14═R.sup.15═R.sup.16═CH.sub.3 or R.sup.14═H or CH.sub.3 and R.sup.15═R.sup.16═H; when R.sup.7═H or CH.sub.3, R.sup.5 and R.sup.6 are different from each other, when they correspond to CH.sub.3 or to H; and when R.sup.10 and R.sup.11═H, R.sup.9 is different from an ethyl-group or H;

19. The linoleic acid derivative according to claim 2, wherein in A.sup.1, R.sup.1 and R.sup.2 are a saturated or unsaturated, straight or branched alkyl group containing 1 to 2 carbon atoms, or a pharmaceutically/food quality acceptable salt thereof.

20. (canceled)

21. A method for treatment of a disorder modulated by the GPR120 receptor and/or the CD36 receptor, comprising: administering to a subject in need thereof a therapeutically effective amount of a pharmaceutically/food quality acceptable salts thereof or pharmaceutical composition according to claim 10.

Description

DESCRIPTION OF THE FIGURES

[0205] The present invention will be hereafter illustrated without being limited thereto by means of the following examples. It will be referred to the following figures in the examples:

[0206] FIG. 1: Action of the CD36 inhibitor (20 μM SSO, Sulfo-N-succinimidyl Oleate) on the increase in intracellular free calcium concentrations, [Ca.sup.2+]i, by linoleic acid (LA) as control test (FIG. 1A) and different linoleic acid derivatives according to the invention: NKS-3 (FIG. 1B) and NKS-5 (FIG. 1C) in mouse taste bud cells. The SSO completely blocked the responses triggered by LA and NKS-3, whereas the response of NKS-5 was partially inhibited by this blocker.

[0207] FIG. 2: Effect of GPR120 inhibitor (AH7614 that is a commercially available known GPR120 antagonist) at 20 μM on the increase in [Ca.sup.2+]i by the linoleic acid derivative NKS-5 according to the invention (at 50 μM) in mouse taste bud cells.

[0208] FIG. 3: Measurements of intracellular Ca.sup.2+ in freshly isolated taste bud cells. FIG. 3A shows the increases in [Ca.sup.2+]i, expressed as changes in pseudocolour images, as function of time in seconds in freshly isolated taste bud cells where 50 μM of the linoleic acid derivative NKS-3, according to the invention, was added, and FIG. 3B is a graphic which shows the dose-dependent increases in [Ca.sup.2+]i expressed as A ratio (ratio of F.sub.340/F.sub.390) in accordance with the increase of the NKS-3 concentration from 0 to 200 μM expressed in Log (the values are means±SD of n=6 assays).

[0209] FIG. 4: Measurements of intracellular Ca.sup.2+ in freshly isolated taste bud cells; especially FIG. 4A shows the increases in [Ca.sup.2+]i expressed as pseudocolour images, as function of time in seconds in freshly isolated taste bud cells where 50 μM of the linoleic acid derivative NKS-5 according to the invention was added, and FIG. 4B is a graphic which also shows the [Ca.sup.2+]i variation expressed as A ratio (ratio of F.sub.340/F.sub.380 in accordance with the increase of the NKS-5 concentration from 0 to 200 μM expressed in Log (the values are means±SD of n=6 assays);

[0210] FIG. 5: is a histogram showing the results of a two-bottle preference test: the tested mice were subjected to two bottles, one containing xanthan gum (white column) and the other one containing xanthan gum and linoleic acid (0.2%, v/v) (black column)—the asterisk shows the significant intake difference (g/12 h/mouse) as compared to the control solution (p<0.001) as per statistical students t-test of significance;

[0211] FIG. 6: Gustatory preference for the linoleic acid derivative NKS-3 according to the invention in a two bottle-preference test. FIG. 6A: shows the dose-dependent effect of NKS-3. n=18 per group; FIG. 6B: shows the preference for a solution-containing sugar (4%, w/v) and NKS-3 plus sugar (4%, w/v). The results are mean±SD. n=6 mice per group. The asterisk shows the significant difference as compared to the control solution (p<0.001) as per statistical students t-test of significance. NS=insignificant differences as compared to the respective control (sucrose) solution;

[0212] FIG. 7: Gustatory preference for the linoleic acid derivative NKS-5 according to the invention in a two bottle-preference test. Preferential consumption over a period of 12 hours (in g). The n represents the number of mice used in each experimental condition. The asterisk shows the highly significant difference (p<0.001) between the respective control solution and test molecule (NKS-5) according to the student t-test of significance.

[0213] FIG. 8: Effect of NKS-5 according to the invention on sweet preference. The mice were subjected to two bottles, one containing 4% (w/v) sugar (control) and the other one containing a test solution (sugar, 4% and NKS-5). Preferential consumption over a period of 12 hours (in g) was recorded as described here-above. The results are mean±SD. n=6 mice per group. The asterisk shows the highly significant difference (p<0.001) between the control solution and the test molecule (chemical compound) according to the student t-test of significance.

[0214] FIG. 9: 3D structure of the linoleic acid derivatives NKS-3 (FIG. 9A) and NKS-5 (FIG. 9B) according to the invention;

[0215] FIG. 10: Interaction of NKS-3, NKS-5 of the invention and Linoleic acid (control) with different amino acids (GLU.sup.418, LYS.sup.385, Phe.sup.266 and Ala.sup.251) of CD36 inside the 3D pocket of the receptor;

[0216] FIG. 11: Interaction of NKS-3, NKS-5 of the invention and Linoleic acid (control) with the LYS.sup.164 (lysine 164) residue of CD36 within the 3D pocket of the receptor;

[0217] FIG. 12: Interaction of NKS-5 of the invention and Linoleic acid (control) in the 3D structure complex of the GPR120.

[0218] FIG. 13: Effect of NKS-3 (FIG. 13A) and NKS-5 (FIG. 13B) on diet-induced obesity. The animals were fed a high-fat diet (HFD) for 28 weeks; however, from 10th week onwards, the baby-bottles/feeders contained either control solution (control group) or analogues (NKS-3 or NKS-5). The arrows indicate when the lipid analogues were added. The results are expressed as the means±SEM (n=10). p values represent the significant differences between the group of mice given either water and analogues in the feeder bottles.

[0219] FIG. 14: CCK concentrations in the peripheral circulation. The results are expressed as the means±SEM (n=10). p values represent the significant differences between the group of mice given either water and analogues in the feeder bottles. Abbreviations: ND=normal or standard diet; HFD=high-fat diet.

[0220] FIG. 15: GLP-1 concentrations in the peripheral blood circulation. The results are expressed as the means±SEM (n=10). p values represent the significant differences between the group of mice given either water and analogues in the feeder bottles. Abbreviations: ND=normal or standard diet; HFD=high-fat diet.

[0221] FIG. 16: PYY concentrations in the peripheral blood circulation. The results are expressed as the means±SEM (n=10). p values represent the significant differences between the group of mice given either water and analogues in the feeder bottles.

[0222] FIG. 17: IL-6 concentrations in the peripheral blood circulation. The results are expressed as the means±SEM (n=10). p values represent the significant differences between the group of mice given either water and analogues in the feeder bottles.

[0223] FIG. 18: Insulin concentrations in the peripheral blood circulation. The results are expressed as the means±SEM (n=10). p values represent the significant differences between the group of mice given either water and analogues in the feeder bottles.

[0224] FIG. 19: ALAT concentrations in the peripheral blood circulation. The results are expressed as the means±SEM (n=10). p values represent the significant differences between the group of mice given either water and analogues in the feeder bottles.

[0225] FIG. 20: Liver weight in normal diet fed and HFD-fed animals. The results are expressed as the means±SEM (n=10). p values represent the significant differences between the group of mice given either water and analogues in the feeder bottles.

[0226] FIG. 21: Liver histological examination of mice fed either normal diet or a HFD. The histological examinations were performed after the sacrifice of the animals. The liver tissues were embedded in the paraffin sections and 5-micron thick sections were cut by microtomes and the slides were stained in hematoxylin and eosin and the slides were observed under a light microscope. The circles with arrow show the liver steatosis in obese mice, fed a HFD only.

EXAMPLES

1. Synthesis of Different Linoleic Acid Derivatives of the Invention

1.1 Synthesis of methyl (S)-3-hydroxy-2-((9Z,12Z)-octadeca-9,12-dienamido)propanoate, Called Hereafter “NKS-2”

[0227] ##STR00041##

[0228] In a Schlenk were introduced linoleic acid (0.93 mL, 3.0 mmol), tetrahydrofyran (THF) (45 mL), L-serine methyl ester hydrochloride (0.47 g, 3.0 mmol) and (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) (1.75 g, 3.3 mmol). The suspension was treated with iPr.sub.2NEt (1.150 mL, 6.6 mmol) and stirred for 4 h at room temperature. Solvent was removed under vacuum and the residue was dissolved in ethyl acetate (EtOAc). The organic phase was successively treated by HCl 1M, water, saturated aqueous NaHC.sub.3, water and brine. The organic layer was concentrated and the product purified by column chromatography on SiO.sub.2 (petroleum ether/ethyl acetate (1:3)). m.sub.pure=0.87 g. Aspect: colorless oil. Yield: 76%.

[0229] .sup.1H NMR (500 MHz, CDCl.sub.3) δ (ppm): 6.41 (broad d, 1H, J=6.6 Hz), 5.40-5.34 (m, 4H), 4.72-4.69 (dt, 1H, J=3.6 Hz, J=3.7 Hz), 4.01-3.98 (ABX system, 1H, J=4.0 Hz, J=11.1 Hz), 3.96-3.93 (ABX system, 1H, J=3.4 Hz, J=11.2 Hz), 3.81 (s, 3H), 2.79 (t, 2H, J=7.0 Hz), 2.59 (broad s, 1H), 2.29 (m, 2H), 2.07 (m, 4H), 1.67 (m, 2H), 1.38-1.31 (m, 14H), 0.91 (t, 3H, J=6.9 Hz).

[0230] .sup.13C {.sup.1H} NMR (126 MHz, CDCl.sub.3) δ (ppm): 173.68, 171.01, 130.23, 130.03, 128.06, 127.91, 63.71, 54.69, 52.75, 36.50, 31.51, 29.60, 29.33, 29.22, 29.18, 29.12, 27.19, 25.63, 25.52, 22.56, 14.04. One C is missing.

[0231] IR (neat cm.sup.−1): v: 3363 (very broad), 3013, 2929, 2860, 1746, 1649, 1535, 1451, 1369, 1214, 1078, 982, 911, 718 (broad).

[0232] HRMS calcd for C.sub.22H.sub.39NO.sub.4Na [M+Na].sup.+ 404.2771, found 404.2761. [α].sub.D=+21.7 (c 0.76, CHCl.sub.3).

1.2 Synthesis of diethyl (9Z,12Z)-octadeca-9,12-dien-1-ylphosphonate, Called Hereafter “NKS-3”

[0233] ##STR00042##

[0234] This compound was prepared from linoleic acid via the alcohol (15mll-015) and the bromide (15 mll-016) described below. Both precursors are known in literature.

Step 1 (15 mll-015):

##STR00043##

[0235] In a round bottom flask was suspended LiAlH.sub.4 (0.18 g, 4.8 mmol) in THE (25 mL). A solution of linoleic acid (1.24 mL, 4.0 mmol) in THE (15 mL) was added dropwise at 0° C. The mixture was slowly warmed up to room temperature (20-25° C.) and stirred overnight. The mixture was quenched by addition of distilled water (2 mL) and 15% aqueous NaOH (5 mL). The mixture was diluted in EtOAc, transferred in a separating funnel then washed with brine. The organic phase was dried over MgSO.sub.4 and concentrated. The product was purified by a column chromatography on SiO.sub.2 (petroleum ether/ethyl acetate (5:1), R.sub.f=0.80). m.sub.pure=0.96 g. Aspect: colorless oil. Yield: 91%.

[0236] .sup.1H NMR (500 MHz, CDCl.sub.3) δ (ppm): 5.42-5.33 (m, 4H), 3.66 (t, 2H, J=6.6 Hz), 2.80 (m, 2H), 2.07 (m, 4H), 1.59 (m, 2H), 1.39-1.31 (m, 16H), 1.26 (broad s, 1H), 0.91 (t, 3H, J=7.0 Hz).

[0237] .sup.13C {.sup.1H} NMR (126 MHz, CDCl.sub.3) δ (ppm): 130.20, 130.10, 128.01, 127.93, 63.07, 32.81, 31.52, 29.64, 29.48, 29.39, 29.34, 29.22, 27.20, 25.73, 25.63, 22.55, 14.03. One C is missing HRMS calcd for C.sub.18H.sub.34ONa [M+Na].sup.+ 289.2501, found 289.2503.

[0238] Step 2 (15 mll-016):

##STR00044##

[0239] In a round bottom flask was introduced triphenylphosphine (1.90 g, 7.3 mmol), carbon tetrabromide (2.16 g, 6.5 mmol), and dichloromethane (DCM) (15 mL) was added at 0° C., the mixture is let stirred for 10 minutes (orange solution), then (9Z, 12Z)-octadecadien-1-ol (965.9 mg, 3.6 mmol) in DCM (10 mL) was transferred on the mixture via cannula at 0° C. A white precipitate was formed. The reaction mixture was let warm up overnight; then it was filtered over Celite® and concentrated under reduced pressure. The product was purified by a column chromatography on SiO.sub.2 (petroleum ether/ethyl acetate (9:1), R.sub.f=0.90). m.sub.pure=0.96 g. Aspect: colorless oil. Yield: 80%.

[0240] .sup.1H NMR (500 MHz, CDCl.sub.3) δ (ppm): 5.42-5.34 (m, 4H), 3.43 (t, 2H, J=6.9 Hz), 2.79 (m, 2H), 2.07 (m, 4H), 1.88 (m, 2H), 1.45 (m, 2H), 1.39-1.31 (m, 14H), 0.92 (t, 3H, J=7.0 Hz)

[0241] .sup.13C {.sup.1H} NMR (126 MHz, CDCl.sub.3) δ (ppm): 130.20, 130.10, 128.01, 63.07, 32.81, 31.52, 29.64, 29.48, 29.39, 29.34, 29.22, 27.20, 25.73, 25.63, 22.55, 14.03. One C is missing.

[0242] HRMS calcd for C.sub.18H.sub.32Br [M−H].sup.+ 327.1681, found 327.1682.

[0243] Step 3:

[0244] In a Schlenk were introduced diethyl phosphite (0.34 mL, 2.6 mmol) and THE (2 mL). The mixture was cooled to 0° C. and sodium bis(trimethylsilyl)amide (NaHMDS) 2M (1.5 mL, 3.0 mmol) was added. The mixture was stirred at room temperature (20-25° C.) for half an hour. Then the reaction was cooled to 0° C. and (6Z, 9Z)-18-bromooctadeca-6,9-diene (0.35 g, 1.07 mmol) in THE (2 mL) was transferred on the reaction mixture via cannula. After 30 minutes, the mixture was stirred overnight at 25° C. The reaction medium was concentrated under reduced pressure, and purified by column chromatography on SiO.sub.2 (petroleum ether/ethyl acetate (1:4), R.sub.f=0.50). m.sub.pure=0.35 g. Aspect slightly yellow oil. Yield: 85%.

[0245] .sup.1H NMR (500 MHz, CD.sub.2Cl.sub.2) δ (ppm): 5.44-5.34 (m, 4H), 4.12-4.03 (m, 4H), 2.82 (m, 2H), 2.09 (m, 4H), 1.74-1.68 (m, 2H), 1.62-1.57 (m, 2H), 1.39-1.32 (m, 22H), 0.93 (t, 3H, J=7.0 Hz)

[0246] .sup.13C {.sup.1H} NMR (126 MHz, CD.sub.2C.sub.2) δ (ppm): 130.08, 130.02, 127.92, 127.87, 61.19 (d, J=6.3 Hz), 31.51, 30.62, 30.49, 29.63, 29.33, 29.24 (d, J=6.3 Hz), 29.05, 27.15 (d, J=2.8 Hz), 26.11, 25.55, 25.00, 22.53, 22.40 (d, J=5.3 Hz), 16.26 (d, J=5.3 Hz), 13.79.

[0247] .sup.31P NMR (203 MHz, CDCl.sub.3) δ (ppm): +32.05 (s).

[0248] IR (neat cm.sup.−1): v: 3009, 2926, 2855, 1463, 1392, 1245, 1164, 1097, 1056, 1028, 954, 785, 722.

[0249] HRMS calcd for C.sub.22H.sub.44O.sub.3P [M+H].sup.+ 387.3023, found 387.3019.

1.3 Synthesis of (9Z,12Z)-(3-methyloxetan-3-yl)methyl octadeca-9,12-dienoate, Called Hereafter “NKS-5”

[0250] ##STR00045##

[0251] In a round bottom flask were introduced linoleic acid (1.24 mL, 4.0 mmol) and dichloromethane (8 mL) under inert atmosphere. The mixture was cooled to 0° C. and oxalyl chloride (0.38 mL, 4.4 mmol) was introduced dropwise. Then the catalytic quantity of dimethylformamide (DMF) (0.03 mL) was added. The reaction mixture was stirred for 1 h at 0° C. and for 2 h at room temperature (20-25° C.). Solvents and oxalyl chloride were removed under reduced pressure and the product was directly engaged in the following step (aspect: colorless oil with slight precipitate).

[0252] A solution of 3-methyl-3-oxetane methanol (0.52 mL, 5.2 mmol), triethylamine (0.73 mL, 5.2 mmol) in THE (20 mL) was prepared in a round bottom flask under inert atmosphere. The mixture was cooled to 0° C. and freshly prepared linoleoyl chloride (1.16 g, 4.0 mmol) in THE (20 mL) was added via cannula. After stirring for 1 h at RT, the mixture was worked up by addition of distilled water (6 mL). The product was extracted with ethyl acetate (3×15 mL), dried over MgSO.sub.4 and purified by column chromatography on SiO.sub.2 (petroleum ether/ethyl acetate (5:1), R.sub.f=0.70). m.sub.pure=0.85 g. Aspect: colorless oil. Yield: 80%.

[0253] .sup.1H NMR (500 MHz, CDCl.sub.3) δ (ppm): 5.39-5.36 (m, 4H), 4.54 (d, 2H, J=6.0 Hz), 4.40 (d, 2H, J=6.0 Hz), 4.18 (s, 2H), 2.79 (t, 2H, J=6.9 Hz), 2.37 (t, 2H, J=7.5 Hz), 2.07 (q, 4H, J=6.8 Hz), 1.66 (m, 2H, J=7.2 Hz), 1.38-1.31 (m, 17H), 0.91 (t, 3H, J=6.9 Hz).

[0254] .sup.13C {.sup.1H} NMR (126 MHz, CDCl.sub.3) δ (ppm): 174.00, 130.36, 130.15, 128.22, 128.05, 79.74, 68.60, 39.26, 34.36, 31.67, 29.73, 29.48, 29.29, 29.26, 29.24, 27.34, 27.32, 25.78, 25.12, 22.70, 21.34, 14.19. One C is missing. IR (neat cm.sup.−1): v: 3010 (small), 2928 (broad), 2857 (broad), 1739, 1460, 1378, 1351, 1240, 1163 (broad), 1099, 984, 942, 834, 723 (broad). HRMS calcd for C.sub.23H.sub.41O.sub.3 [M+H].sup.+ 365.3050, found 365.3043.

1.4 Synthesis of methyl (S)-2-((8Z,11Z)-heptadeca-8,11-dien-1-yl)-4,5-dihydrooxazole-4-carboxylate, Called Hereafter “NKS-6”

[0255] ##STR00046##

[0256] In a Schlenk was introduced methyl (S)-3-hydroxy-2-((9Z,12Z)-octadeca-9,12-dienamido)propanoate (0.19 g, 0.5 mmol), DCM (1 mL) and then (iPr).sub.2NEt (0.19 mL, 1.1 mmol). After 30 min, the mixture was cooled to 0° C. and Deoxofluor (0.24 mL, 1.1 mmol) was added dropwise. After 16 h, the crude mixture was directly engaged on SiO.sub.2 gel chromatography for purification (petroleum ether/ethyl acetate (1:1), R.sub.f=0.75). m.sub.pure=0.17 g. Aspect: colorless oil. Yield: 92%.

[0257] .sup.1H NMR (600 MHz, CDCl.sub.3) δ (ppm):

[0258] Step-1 (15mll-052): 5.42-5.34 (m, 4H), 4.76-4.73 (m, 1H), 4.50 (1H, J=7.8 Hz), 4.41 (m, 1H), 3.81 (s, 3H), 2.80 (m, 2H), 2.35 (m, 2H), 2.08 (m, 4H), 1.67 (m, 2H), 1.39-1.31 (m, 14H), 0.92 (t, 3H, J=6.7 Hz).

[0259] .sup.13C {.sup.1H} NMR (126 MHz, CDCl.sub.3) δ (ppm):

[0260] Step-2 (15mll-030): 171.67, 171.12, 130.20, 130.04, 128.04, 127.91, 69.32, 67.92, 52.56, 31.51, 29.57, 29.32, 29.08, 27.95, 27.19, 25.87, 25.62, 22.54, 14.02. 3 C are missing

[0261] IR (neat cm.sup.−1): v: 3009, 2925, 2854, 1743, 1660, 1459, 1436, 1362, 1270, 1204, 1177, 1060, 1037, 986, 955, 915, 723.

[0262] HRMS calcd for C.sub.22H.sub.38NO.sub.3 [M+H].sup.+ 364.2846, found 364.2842. [α].sub.D=+33.5 (c 1.7, EtOAc).

1.5 Synthesis of (9Z,12Z)-(tetrahydrofuran-2-yl)methyl octadeca-9,12-dienoate, Called Hereafter “1x”

[0263] ##STR00047##

[0264] 17FM024: In a round bottom flask were introduced linoleic acid (200 mg, 0.7 mmol) and dichloromethane (2 mL) under inert atmosphere. The mixture was cooled to 0° C. and oxalyl chloride (0.07 mL, 0.78 mmol) was introduced dropwise. Then the catalytic quantity of DMF (5 μL) was added. The reaction mixture was stirred for 1 h at 0° C. and for 2 h at room temperature. Solvents and oxalyl chloride were removed under reduced pressure and the product was directly engaged in the following step. Aspect: yellow oil with slight precipitate.

[0265] A solution of tetrahydrofurfuryl alcohol (0.14 mL, 1.42 mmol), triethylamine (0.2 mL, 1.42 mmol) in THE (4 mL) was prepared in a round bottom flask under inert atmosphere. The mixture was cooled to 0° C. and freshly prepared linoleoyl chloride (212 mg, 0.7 mmol) in THE (4 mL) was added via cannula. After stirring for overnight at RT, the mixture was worked up by addition of distilled water (6 mL). The product was extracted with ethyl acetate (3×15 mL), dried over MgSO.sub.4 and purified by column chromatography on SiO.sub.2 (petroleum ether/ethyl acetate (5:1), R.sub.f=0.70). m.sub.pure=0.23 g. Aspect: colorless oil. Yield: 88%.

[0266] .sup.1H NMR (500 MHz, CDCl.sub.3) δ (ppm): 5.41-5.30 (m, 4H), 4.16 (dd, 1H, J=3.6 Hz, J=11.2 Hz), 4.11 (ddd, 1H, J=3.6 Hz, J=6.9 Hz, J=14.0 Hz), 3.99 (dd, 1H, J=6.7 Hz, J=11.2 Hz), 3.89 (dd, 1H, J=6.7 Hz, J=8.2 Hz), 3.82-3.77 (m, 1H), 2.77 (t, 2H, J=6.6 Hz), 2.34 (t, 2H, J=7.6 Hz), 2.07-1.86 (m, 6H), 1.65-1.56 (m, 4H), 1.38-1.25 (m, 14H), 0.89 (t, 3H, J=6.9 Hz).

[0267] .sup.13C {.sup.1H} NMR (126 MHz, CDCl.sub.3) δ (ppm): 173.80, 130.20, 130.04, 128.02, 127.90, 76.55, 68.43, 66.31, 34.20, 31.51, 29.59, 29.33, 29.15, 29.10 (2×), 27.99, 27.19 (2×), 25.65, 25.62, 24.91, 22.56, 14.06.

[0268] IR (neat cm.sup.−1): v: 3008, 2924, 2854, 1737, 1458, 1362, 1239, 1169, 1082, 1023, 991, 916, 722.

1.6 Synthesis of (9Z,12Z)-(tetrahydrofuran-3-yl)methyl octadeca-9,12-dienoate, Called Hereafter “1a”

[0269] ##STR00048##

[0270] 17FM090: In a round bottom flask were introduced linoleic acid (220 mg, 0.78 mmol) and dichloromethane (2 mL) under inert atmosphere. The mixture was cooled to 0° C. and oxalyl chloride (0.08 mL, 0.86 mmol) was introduced dropwise. Then the catalytic quantity of DMF (5.5 μL) was added. The reaction mixture was stirred for 1 h at 0° C. and for 2 h at room temperature. Solvents and oxalyl chloride were removed under reduced pressure and the product was directly engaged in the following step. Aspect: yellow oil with slight precipitate.

[0271] A solution of tetrahydro-3-furanmethanol (0.14 mL, 1.42 mmol), triethylamine (0.2 mL, 1.42 mmol) in THE (4 mL) was prepared in a round bottom flask under inert atmosphere. The mixture was cooled to 0° C. and freshly prepared oleoyl chloride (233 mg, 0.78 mmol) in THE (4 mL) was added via cannula. After stirring for overnight at RT, the mixture was worked up by addition of distilled water (6 mL). The product was extracted with ethyl acetate (3×15 mL), dried over MgSO.sub.4 and purified by column chromatography on SiO.sub.2 (petroleum ether/ethyl acetate (5:1), R.sub.f=0.70). m.sub.pure=234.1 mg. Aspect: colorless oil. Yield: 82%.

[0272] .sup.1H NMR (500 MHz, CDCl.sub.3) δ (ppm): 5.46-5.25 (m, 4H), 4.08 (dd, 1H, J=6.5 Hz, J=10.9 Hz), 3.97 (dd, 1H, J=8.0 Hz, J=10.9 Hz), 3.88-3.81 (m, 2H), 3.75 (dt, 1H, J=7.2 Hz, J=8.5 Hz), 3.56 (dd, 1H, J=5.6 Hz, J=8.8 Hz), 2.80-2.72 (m, 2H), 2.80-2.52 (m, 1H), 2.30 (t, 2H, J=7.5 Hz), 2.09-1.98 (m, 5H), 1.67-1.57 (m, 3H), 1.41-1.24 (m, 14H), 0.88 (t, 3H, J=6.9 Hz).

[0273] .sup.13C {.sup.1H} NMR (126 MHz, CDCl.sub.3) δ (ppm): 173.74, 130.20, 130.00, 128.04, 127.88, 70.53, 67.70, 65.65, 38.26, 34.24, 31.50, 29.57, 29.32, 29.13, 29.09, 29.08, 28.94, 27.18, 27.16, 25.61, 24.94, 22.55, 14.04.

[0274] IR (neat cm.sup.−1): v: 3009, 2925, 2854, 1738, 1456, 1394, 1357, 1240, 1167, 1079, 1011, 914, 723, 600.

1.7 Synthesis of (9Z,12Z)-(oxetan-3-yl)methyl octadeca-9,12-dienoate, Called Hereafter “1b”

[0275] ##STR00049##

[0276] In a round bottom flask were introduced linoleic acid (200 mg, 0.7 mmol) and dichloromethane (2 mL) under inert atmosphere. The mixture was cooled to 0° C. and oxalyl chloride (0.07 mL, 0.78 mmol) was introduced dropwise. Then the catalytic quantity of DMF (5 μL) was added. The reaction mixture was stirred for 1 h at 0° C. and for 2 h at room temperature. Solvents and oxalyl chloride were removed under reduced pressure and the product was directly engaged in the following step. Aspect: yellow oil with slight precipitate.

[0277] A solution of 3-oxetane alcohol (0.12 mL, 1.42 mmol), triethylamine (0.2 mL, 1.42 mmol) in THE (4 mL) was prepared in a round bottom flask under inert atmosphere. The mixture was cooled to 0° C. and freshly prepared linoleoyl chloride (212 mg, 0.7 mmol) in THE (4 mL) was added via cannula. After stirring for overnight at RT, the mixture was worked up by addition of distilled water (6 mL). The product was extracted with ethyl acetate (3×15 mL), dried over MgSO.sub.4 and purified by column chromatography on SiO.sub.2 (petroleum ether/ethyl acetate (5:1), R.sub.f=0.73). m.sub.pure=0.23 g. Aspect: colorless oil. Yield: 94%.

[0278] .sup.1H NMR (500 MHz, CDCl.sub.3) δ (ppm): 5.41-5.30 (m, 4H), 4.81-4.78 (m, 2H), 4.47 (dt, 2H, J=2.1 Hz, J=6.2 Hz), 4.30 (dd, 2H, J=1.9 Hz, J=6.7 Hz), 3.33-3.24 (m, 1H), 2.77 (t, 2H, J=6.6 Hz), 2.32 (dt, 2H, J=1.8 Hz, J=7.7 Hz), 2.04 (q, 4H, J=6.9 Hz), 1.65-1.59 (m, 2H), 1.38-1.27 (m, 14H), 0.89 (dt, 3H, J=1.8 Hz, J=6.9 Hz).

[0279] .sup.13C {.sup.1H} NMR (126 MHz, CDCl.sub.3) δ (ppm): 173.80, 130.21, 129.99, 128.05, 127.87, 74.10 (2×), 64.99, 34.16, 34.09, 31.51, 29.57, 29.33, 29.13, 29.08 (2×), 27.19, 27.17, 25.62, 24.92, 22.53, 14.06.

[0280] IR (neat cm.sup.−1): v: 3008, 2925, 2855, 1737, 1461, 1362, 1239, 1163, 1051, 982, 853, 723.

1.8 Synthesis of (9Z,12Z)-(oxyran-3-yl)methyl octadeca-9,12-dienoate, Called Hereafter “1c”

[0281] ##STR00050##

[0282] 17FM33: In a round bottom flask were introduced linoleic acid (200 mg, 0.7 mmol) and dichloromethane (2 mL) under inert atmosphere. The mixture was cooled to 0° C. and oxalyl chloride (0.07 mL, 0.78 mmol) was introduced dropwise. Then the catalytic quantity of DMF (5 μL) was added. The reaction mixture was stirred for 1 h at 0° C. and for 2 h at room temperature. Solvents and oxalyl chloride were removed under reduced pressure and the product was directly engaged in the following step. Aspect: yellow oil with slight precipitate.

[0283] A solution of glycidol (105.2 mg, 1.42 mmol), triethylamine (0.2 mL, 1.42 mmol) in THE (4 mL) was prepared in a round bottom flask under inert atmosphere. The mixture was cooled to 0° C. and freshly prepared linoleoyl chloride (212 mg, 0.7 mmol) in THE (4 mL) was added via cannula. After stirring for overnight at RT, the mixture was worked up by addition of distilled water (6 mL). The product was extracted with ethyl acetate (3×15 mL), dried over MgSO.sub.4 and purified by column chromatography on SiO.sub.2 (petroleum ether/ethyl acetate (5:1), R.sub.f=0.73). m.sub.pure=0.21 g. Aspect: colorless oil. Yield: 91%.

[0284] .sup.1H NMR (500 MHz, CDCl.sub.3) δ (ppm): 5.41-5.30 (m, 4H), 4.41 (dd, 1H, J=3.1 Hz, J=12.3 Hz), 3.91 (dd, 1H, J=6.3 Hz, J=12.3 Hz), 3.20 (ddd, 1H, J=2.9 Hz, J=4.1 Hz, J=9.4 Hz), 2.84 (t, 1H, J=4.5 Hz), 2.77 (t, 2H, J=6.6 Hz), 2.64 (dd, 1H, J=2.6 Hz, J=4.9 Hz), 2.35 (t, 2H, J=7.6 Hz), 2.04 (q, 4H, J=6.8 Hz), 1.66-1.60 (m, 2H), 1.38-1.25 (m, 14H), 0.89 (t, 3H, J=6.9 Hz).

[0285] .sup.13C {.sup.1H} NMR (126 MHz, CDCl.sub.3) δ (ppm): 173.50, 130.21, 130.02, 128.04, 127.89, 64.74, 49.38, 44.66, 34.05, 31.52, 29.58, 29.34, 29.14, 29.08 (2×), 27.19, 27.18, 25.62, 24.85, 22.56, 14.06.

[0286] IR (neat cm.sup.−1): v: 3008, 2924, 2854, 1739, 1458, 1374, 1245, 1171, 1013, 910, 853, 722.

1.9 Synthesis of (9Z,12Z)-(2,2,5-trimethyl-1,3-dioxan-5-yl)methyl octadeca-9,12-dienoate, Called Hereafter “1d”

[0287] ##STR00051##

[0288] This compound was prepared by reaction of linoleic acid and 2,2,5-trimethyl-1,3-dioxane-5-methanol (17FM025), known in literature and prepared as described below.

##STR00052##

[0289] 17FM025: In a round bottom flask were introduced tris(hydroxymethyl)ethane (2 g, 16.7 mmol) and a catalytic quantity of PTSA (2 mg) in acetone (20 mL) under inert atmosphere. The mixture was stirred at room temperature for 2 days. The reaction was neutralized with 50 mg of K.sub.2CO.sub.3, filtrated and evaporated to give the desired product with 96% of purity. m.sub.pure=2.64 g. Aspect: colorless oil. Yield: 98%.

[0290] .sup.1H NMR (500 MHz, DMSO) (ppm): 4.57 (t, 1H, J=5.4 Hz), 3.57 (AB system, 2H, J=11.7 Hz), 3.44 (AB system, 2H, J=11.7 Hz), 3.35 (d, 1H, J=5.3 Hz), 1.33 (s, 3H), 1.27 (s, 3H), 0.75 (s, 3H).

[0291] .sup.13C {.sup.1H} NMR (126 MHz, DMSO) δ (ppm): 96.92, 65.34, 34.22, 25.80, 21.57, 17.61.

[0292] Compound 1d: In a round bottom flask were introduced linoleic acid (200 mg, 0.7 mmol) and dichloromethane (2 mL) under inert atmosphere. The mixture was cooled to 0° C. and oxalyl chloride (0.07 mL, 0.78 mmol) was introduced dropwise. Then the catalytic quantity of DMF (5 μL) was added. The reaction mixture was stirred for 1 h at 0° C. and for 2 h at room temperature. Solvents and oxalyl chloride were removed under reduced pressure and the product was directly engaged in the following step. Aspect: yellow oil with slight precipitate.

[0293] A solution of 2,2,5-trimethyl-1,3-dioxane-5-methanol (228 mg, 1.42 mmol), triethylamine (0.2 mL, 1.42 mmol) in THE (4 mL) was prepared in a round bottom flask under inert atmosphere. The mixture was cooled to 0° C. and freshly prepared linoleoyl chloride (212 mg, 0.7 mmol) in THE (4 mL) was added via cannula. After stirring for overnight at RT, the mixture was worked up by addition of distilled water (6 mL). The product was extracted with ethyl acetate (3×15 mL), dried over MgSO.sub.4 and purified by column chromatography on SiO.sub.2 (petroleum ether/ethyl acetate (5:1), R.sub.f=0.73). m.sub.pure=213.6 mg. Aspect: colorless oil. Yield: 91%.

[0294] .sup.1H NMR (500 MHz, CDCl.sub.3) δ (ppm): 5.41-5.30 (m, 4H), 4.15 (s, 2H), 3.60 (AB system, 2H, J=12.0 Hz), 3.53 (AB system, 2H, J=12.0 Hz), 2.77 (t, 2H, J=6.6 Hz), 2.34 (t, 2H, J=7.6 Hz), 2.05 (q, 4H, J=7.0 Hz), 1.66-1.60 (m, 2H), 1.44 (s, 3H), 1.40 (s, 3H), 1.37-1.25 (m, 14H), 0.89 (t, 3H, J=6.9 Hz), 0.85 (s, 3H).

[0295] .sup.13C {.sup.1H} NMR (126 MHz, CDCl.sub.3) δ (ppm): 173.80, 130.24, 130.06, 128.07, 127.92, 98.07, 66.65, 66.33 (2×), 34.33, 33.55, 31.55, 29.62, 29.37, 29.20, 29.17, 29.14, 27.23 (2×), 26.82, 25.66, 24.98, 22.60, 20.56, 17.83, 14.10.

[0296] IR (neat cm.sup.−1): v: 2992, 2925, 2855, 1737, 1456, 1395, 1373, 1343, 1264, 1246, 1226, 1205, 1186, 1155, 1089, 1042, 1024, 933, 913, 829, 728.

1.10 Synthesis of (9Z,12Z)-(2-oxopropyl)-octadeca-9,12-dienoate, Called Hereafter “1e”

[0297] ##STR00053##

[0298] In a round bottom flask were introduced linoleic acid (200 mg, 0.7 mmol) and dichloromethane (2 mL) under inert atmosphere. The mixture was cooled to 0° C. and oxalyl chloride (0.07 mL, 0.78 mmol) was introduced dropwise. Then the catalytic quantity of DMF (5 μL) was added. The reaction mixture was stirred for 1 h at 0° C. and for 2 h at room temperature. Solvents and oxalyl chloride were removed under reduced pressure and the product was directly engaged in the following step. Aspect: yellow oil with slight precipitate.

[0299] A solution of hydoxyacetone (0.2 mL, 1.42 mmol), triethylamine (0.2 mL, 1.42 mmol) in THE (4 mL) was prepared in a round bottom flask under inert atmosphere. The mixture was cooled to 0° C. and freshly prepared linoleoyl chloride (212 mg, 0.7 mmol) in THE (4 mL) was added via cannula. After stirring for overnight at RT, the mixture was worked up by addition of distilled water (6 mL). The product was extracted with ethyl acetate (3×15 mL), dried over MgSO.sub.4 and purified by column chromatography on SiO.sub.2 (petroleum ether/ethyl acetate (5:1.5), R.sub.f=0.6). m.sub.pure=151.4 mg. Aspect: colorless oil. Yield: 64%.

[0300] .sup.1H NMR (500 MHz, CDCl.sub.3) δ (ppm): 5.41-5.30 (m, 4H), 4.64 (s, 2H), 2.77 (t, 2H, J=6.7 Hz), 2.42 (t, 2H, J=7.6 Hz), 2.16 (s, 3H), 2.05 (q, 4H, J=6.8 Hz), 1.70-1.64 (m, 2H), 1.38-1.25 (m, 14H), 0.89 (t, 3H, J=6.9 Hz).

[0301] .sup.13C {.sup.1H} NMR (126 MHz, CDCl.sub.3) δ (ppm): 201.69, 173.03, 130.21, 130.03, 128.03, 127.89, 68.14, 33.80, 31.52, 29.59, 29.34, 29.15, 29.09, 29.04, 27.20, 27.18, 26.08, 25.62, 24.82, 22.57, 14.06.

[0302] IR (neat cm.sup.−1): v: 3008, 2924, 2854, 1734, 1460, 1417, 1372, 1270, 1154, 1099, 966, 909, 722.

2. Biological Properties

2.1 Materials and Methods

[0303] a—Animals

[0304] Eight weeks old, male mice (C57Bl/6 black) were purchased from the Janvier Elevage (Le Mans) and used for the experiments.

[0305] b—Isolation of taste bud cells

[0306] The experimental protocol for isolation/purification of taste cells from fungiform papillae was approved by the Regional Ethical Committee (protocol number: A0508).

[0307] The technique to isolate taste bud cells from mouse fungiform papillae has been described in the publication of El-Yassimi A, Hichami A, Besnard P, Khan N A. “Linoleic acid induces calcium signaling, Src kinase phosphorylation, and neurotransmitter release in mouse CD36-positive gustatory cells”—J Biol Chem. 2008 May 9; 283(19):12949-59.

[0308] Briefly, lingual epithelium was separated by enzymatic dissociation which contained the following: elastase and dispase mixture, 2 mg/ml each, in Tyrode's buffer (120 mM NaCl; 5 mM KCl; 10 mM HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); 1 mM CaCl.sub.2; 1 mM MgCl.sub.2; 10 mM glucose; 10 mM Na pyruvate, pH 7.4). The taste bud cells were isolated by incubating lingual epithelium in RPMI 1640 medium containing 2 mM EDTA, 1.2 mg/ml elastase, 0.6 mg/ml collagenase (type 1), and 0.6 mg/ml trypsin inhibitor at 37° C. for 10 minutes, followed by centrifugation (600 g, 10 minutes). The cell populations, after separation, were suspended in fresh RPMI 1640 medium containing 10% fetal calf serum, 200 U/ml penicillin, and 0.2 mg/ml streptomycin, seeded onto a Poly-D-Lysine-coated dishes, and cultured for 24 hours. At the end of this period, the cells were used for the experiments or stained with trypan blue to assess their viability.

[0309] c—“Ca.sup.2+ Signaling” for Showing the Interaction with CD36 and/or GPR120 Fat Taste Receptors Ca.sup.2+ is the key second messenger in mammalian cells. It regulates a variety of cellular functions. Phospholipase C (PLC) is responsible for the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP.sub.2), generating two second messengers, i.e., inositol-1,4,5-triphosphate (hereafter IP.sub.3) and diacylglycerol (DAG). The PLCβ subfamily is of particular interest, given its prominent role in neuronal and Taste Bud Cells (TBC) signaling, and its regulation by G Protein-Coupled Receptors (hereafter GPCR). IP.sub.3 is freely diffusible and binds to IP.sub.3-specific receptors, leading to the release of Ca.sup.2+ from the endoplasmic reticulum (ER) which represents the intracellular Ca.sup.2+ pool.

[0310] As regards taste perception, an increase in [Ca.sup.2+]i has been considered as one of the earliest mechanisms, involved in the transfer of taste message from the tongue to the brain. The publication of El-Yassimi et al. mentioned above shows that CD36 and GPR120 (Ozdener M H et al. “CD36- and GPR120-mediated Ca.sup.2+ signaling in human taste bud cells mediates differential responses to fatty acids and is altered in obese mice”—Gastroenterology 2014, April, 146(4), 995-1005) in human and taste bud cells are coupled to an increase in free [Ca.sup.2+]i during their activation by fatty acids like linoleic acid. In mouse taste bud cells, the linoleic acid via CD36 induced the phosphorylation of src-kinases, (Fyn.sup.59 and Yes.sup.62) and regulated the increases in [Ca.sup.2+]i by opening of calcium channels whose opening was controlled by stromal interaction molecule-1, STIM-1 (publication Dramane G et al. “STIM1 regulates calcium signaling in taste bud cells and preference for fat in mice”, J. Clin. Invest. 2012, June, 122(6), 2267-82).

[0311] Thus, it can be stated that Ca.sup.2+ signaling (under the control of STIM1), plays a key role in the signaling of fat taste transduction in mice.

[0312] Therefore, the mobilization of intracellular calcium in response to the linoleic acid derivatives of the invention was studied by the Applicant.

[0313] d—Method of Measurement of the Ca.sup.2+ Signaling in Mouse Bud Cells

[0314] The mouse taste bud cells were seeded in 24-well plates (with glass bottom), containing poly-D-lysine for better adhesion to the surface. After 24 h, taste cells were incubated for 45 minutes with a fluorescent probe, Fura-2/AM (1 μM) in calcium buffer (110 mM NaCl, 5.4 mM, KCl, 25 mM, NaHCO.sub.3, 0.8 mM, MgCl.sub.2, 0.4 mM, KH.sub.2PO.sub.4, 20 mM, Hepes-Na, 0.33 mM, NaHPO.sub.4, 1.2 mM, CaCl.sub.2, pH 7.4). Then, the cells are washed with phosphate buffer saline (PBS) and taken up in calcium buffer.

[0315] The changes in intracellular Ca.sup.2+ (F.sub.340/F.sub.380) were monitored under a Nikon microscope (TiU) by using an S Fluor 40× oil immersion objective.

[0316] Especially, the increases in [Ca.sup.2+ ]i were measured as follows: the mice taste bud cells were cultured on Willico-Dish wells with a glass bottom and loaded with a fluorescent probe, Fura-2/AM. The changes in intracellular Ca.sup.2+ (F.sub.340/F.sub.380) were monitored under the Nikon microscope (TiU) by using S-fluor 40× oil immersion objective. The planes were taken at Z intervals of 0.3 μm, and NIS-Elements software was used to deconvolve the images. The microscope was equipped with EM-CCD (Lucas) camera for real time recording of 16-bit digital images. The dual excitation fluorescence imaging system was used for studies of individual cells. The changes in intracellular Ca.sup.2 were expressed as ΔRatio, which was calculated as the difference between the peak F.sub.340/F.sub.380 ratio. The data were summarized from the large number of individual cells (20-40 cells in a single run, with 3-9 identical experiments done in at least three cell preparations). The results were analyzed with the NIS-elements software, the variation in [Ca.sup.2+]i is expressed as Δ ratio (F.sub.340/F.sub.380 which was calculated with the difference of spectra at two wavelengths, i.e., 340 nm and 380 nm.

[0317] CD36: in order to assess if the linoleic acid derivatives according to the invention exert their actions via CD36, sulfo-N-succinimidyl oleate (SSO) was used. SSO is a CD36 inhibitor/blocker/antagonist.

[0318] Experiments were performed in the presence or absence of SSO with respect to calcium signaling to verify whether our analogs act well via this lipid receptor. Especially, for this assay, NKS-3, NKS-5 and linoleic acid (control) were tested.

[0319] GPR120: in order to assess if the linoleic acid derivatives according to the invention exert their actions via GPR120, AH7614 was used. This commercially available compound is a GPR120 antagonist.

[0320] Experiments were performed in the presence or absence of SSO and/or AH7614 with respect to calcium signaling to verify whether our analogs act well via this lipid receptor.

[0321] Especially, for this assay, NKS-3, NKS-5 and linoleic acid (control) were tested.

2.1 Results

[0322] a—Action on CD36 Fat Taste Receptor (FIG. 1)

[0323] FIG. 1A shows that LA, the control molecule, in mice taste bud cells, induces a huge increase in [Ca.sup.2+]i via CD36. This phenomenon is suppressed by the presence of SSO.

[0324] FIG. 1B proves that NKS-3 is a complete CD36 agonist as its action was completely suppressed by SSO. The NKS-5 appears to be a partial agonist because its action on the increases in [Ca.sup.2+]i was curtailed, but not completely suppressed, by SSO (FIG. 1C).

[0325] In addition, FIG. 3A shows that NKS-3 induced in mouse taste bud cells an increase in [Ca.sup.2+]i which is very rapid. Especially, this compound induces the increases in [Ca.sup.2+]i in a dose-dependent manner with an EC.sub.50 value of 25±0.01 μM (FIG. 3B).

b—Action on GPR120 Fat Taste Receptor (FIG. 2)

[0326] As it is shown by FIG. 2, NKS-5 according to the invention also acts via GPR120 since AH7614 abolished partially its action on the increase in [Ca.sup.2+]i in mouse gustatory cells. Indeed, FIG. 2 shows that the response of NKS-5 was significantly curtailed, but not suppressed, by AH7614. The results at 25 μM and 50 μM of NKS-3 are identical.

[0327] Similar to NKS-3, the NKS-5-induced increases in [Ca.sup.2]i is also very rapid (FIG. 4A) in mouse taste bud cells. This compound according to the invention induces in particular the increases in a dose-dependent manner with an EC.sub.50 value of 30±0.01 μM (FIG. 4B).

3. Fat-Like Taste Perception

3.1 Materials and Methods

[0328] a—Animals

[0329] Eight weeks old, male mice (C57Bl/6 black) were purchased from the Janvier Elevage (Le Mans) and used for these experiments.

[0330] b—Methods of Two-Bottle Preference Test

[0331] The mice are placed individually in cages in a controlled environment (humidity and constant temperature) and have free access to a standard diet. 6 hours before the tests, the mice are deprived of water.

[0332] During the behavioral experiments, the mice were offered two bottles simultaneously for 12 hours (night period).

[0333] The mice are subjected to a choice between the “control” solution and the experimental solution.

[0334] Experiment 1 (FIG. 5, FIG. 6A, FIG. 7) [0335] control solutions contain 0.3% xanthan gum, w/v, (Sigma Life Science, USA) in water to reproduce lipid texture and minimize bias due to differences in appearance between the two bottles; [0336] the experimental solution contains, in addition to xanthan gum, 0.3% (w/v), either the “test compound” according to the invention at a concentration varying from 10 to 200 μM (NKS-3, FIG. 6A or NKS-5, FIG. 7) or linoleic acid at 0.2% (v/v) (control test, FIG. 5). To avoid the preference for one side, the position of each bottle is changed during each test.

[0337] Experiment 2 (FIG. 6B and FIG. 8): gustatory properties [0338] control solutions contain 2% of sucrose (w/v); [0339] the experimental solution contains, in addition to sucrose (2% w/v), the “test compound” according to the invention (NKS-3, FIG. 6B or NKS-5, FIG. 8) at a concentration of 50 μM.

[0340] To estimate the consumption of control and experimental solutions, the bottles are weighed before and after each experiment. The mice are allowed to take rest for 48 hours between each experiment.

3.2 Results (FIG. 5 to FIG. 8)

[0341] a—NKS-3

[0342] As shown in FIG. 5, the mice exhibit a strong preference for the natural fat linoleic acid (LA) as compared to xanthan gum in the two-bottle preference test. As it is similarly shown by FIG. 6A, mice exhibit spontaneous preference for a solution containing the linoleic acid according to the invention, i.e. NKS-3 as compared to the control solution containing xanthan gum. The preference is optimal at a concentration of 50 μM of NKS-3.

[0343] In addition, in order to check whether the NKS-3 might increase the gustatory properties of a palatable solution, comparative tests have been performed between a bottle of sucrose and a bottle comprising, in addition to sucrose, NKS-3 (FIG. 6B) (experiment 2 described above). It was observed that the NKS-3 failed to enhance the palatability of a sucrose-containing solution. The mice exhibited a spontaneous preference for a sweet solution as it is an innate taste modality (FIG. 8).

b—NKS-5

[0344] Similarly, mice exhibit spontaneous preference for a solution containing NKS-5. It can be observed from FIG. 7 that mice prefer NKS-5 at 75 μM. This is much lesser (93 times) than a fatty acid solution employed for two-bottle preference test.

[0345] In addition, once dissolved in a palatable solution, the NKS-5 also conserves its gustatory properties (FIG. 8). Alternatively, it can be also alluded that NKS-5 increases the palatability of a sweet solution.

4. Binding Properties

[0346] The interactions/binding properties of NKS-3 and NKS-5 with CD36 and GPR120 were studied by simulation.

3.1 Materials and Methods

[0347] We used the “Docking” properties by employing Autodock software. The structure box was determined by Autodock grid box, and Autodock Vina was used to calculate the preferred orientation of the analogous molecules towards the protein CD36 and GPR120 to calculate the affinity score of binding of these FAA with CD36 and GPR120.

[0348] We used the Docking Discovery Studio to visualize the types of interactions between these molecules and the proteins.

3.2 Results (FIG. 9 to FIG. 12)

[0349] a—CD36

[0350] It was observed from FIG. 9A and FIG. 9B that NKS-3 and NKS-5 retain a 3D structure with their free ends for binding with lipid receptors.

[0351] Their kinetic interactions with CD36 have been explored by the Applicant (FIG. 10). It can be seen from this figure that NKS-3 and NKS-5 bound, like linoleic acid, to different amino acid residues with an affinity score of −7.3 to −7.9 Kcal/mol (Table 1 below).

TABLE-US-00001 TABLE 1 Affinity score of agonists with the 3D structure of CD36 Affinity score Ligands Target (Kcal/mol) Amino acids Linoleic acid CD36 −7.3 Glu.sup.418, IIe.sup.275 NKS-3 CD36 −7.4 Glu.sup.418, Lys.sup.385, Phe.sup.266, Asp.sup.256, Asp.sup.206 NKS-5 CD36 −7.9 Glu.sup.418, Lys.sup.385, Phe.sup.266, Ala.sup.251

[0352] Moreover, in the literature, it has been demonstrated that the lead molecule (linoleic acid) binds particularly to the Lys.sup.164 residue in the CD36 receptor pocket (Kuda O et al, “Sulfo-N-succinimidyl oleate (SSO) inhibits fatty acid uptake and signaling for intracellular calcium via binding CD36 lysine 164: SSO also inhibits oxidized low-density lipoprotein uptake by macrophages”. J. Biol. Chem. 2013, May 31, 288(22), 15547-55). Thus, this interaction with Lys.sup.164 of CD36 has also been studied by the Applicant (FIG. 11).

[0353] On this figure, it can be observed that the two FAA according to the invention (NKS-3 and NKS-5) behave as linoleic acid with respect to their binding with CD36 Lys.sup.164. The affinity of this interaction between the three molecules (NKS-3, NKS-5 and LA) is from −3 to −3.2 Kcal/mol (Table 2 below).

TABLE-US-00002 TABLE 2 Affinity score of agonists with the 3D structure of CD36 with Lysine.sup.164. Ligand Target/ Affinity (Kcal/mol) Lys.sup.164 score Amino acids Linoleic acid CD36 −3.2 Lys.sup.164 NKS-3 CD36 −3.0 Lys.sup.164 NKS-5 CD36 −2.8 Lys.sup.164, Leu.sup.161 SSO CD36 −3.1 Lys.sup.164

[0354] All these observations demonstrate that the two FAA according to the invention have a very important potential in terms of their modulating properties of lingual CD36.

b—GPR120

[0355] As shown by FIG. 12, the FAA according to the invention, especially NKS-5, also bind to GPR120 receptor.

[0356] Table 3 below shows the affinity constants between NKS-5, LA and GPR120 receptor. It

TABLE-US-00003 TABLE 3 Affinity score of agonists with the 3D structure of the GPR120. Ligands Affinity (Kcal/mol) Target score Amino acids Linoleic GPR120 −5.3 Arg.sup.99, Val.sup.98, acid (LA) Phe.sup.320, Ser.sup.317 NKS-5 GPR120 −5.7 Arg.sup.99, Phe.sup.320, Val.sup.98, Phe.sup.319, Pro.sup.316

[0357] It can be observed that linoleic acid and NKS-5 possess almost similar affinity for GPR120.

5. Anti-Obesity Effects

5.1 Materials and Methods

[0358] a—Mice and Diet-Induced Obesity:

[0359] C57BL/6J male mice, aged between 6 to 10 weeks (16-20 g), were obtained from Janvier Labs (France). They were housed individually in a controlled environment with a 12 h light/dark cycle with food (SAFE, France) and water ad libitum. The palm oil (Huilerie Vigean, France) was the main fat component in high-fat diets. The high-fat diet was prepared weekly and stored at 4° C. until further use. The study was conducted as per Declaration of Helsinki and European ethical guidelines for the care and use of animals for experimentation. All the experimental protocols (protocol number: 16158) were approved by the Regional Ethical Committee of the University of Burgundy (Dijon, France).

[0360] The mice were grouped at random (n=10/group) and fed with either of the following diets: standard diet (STD) or high-fat diet (HFD). The body weight, food, and water intake were measured, weekly.

TABLE-US-00004 Composition of the diets Content (%) STD HFD Proteins 66.8 40.07 starch 16.10 14.6 Fats 3.10 35.3 Cholesterol 0.03 Cellulose 3.9 2.7 Vitamins 5 3.4 Minerals 5.1 3.9 Energy (Kcal/100g) 359.5 536.65 Fat energy (% of 8 60 total energy) Standard diet (STD); high-fat diet (HFD)

TABLE-US-00005 Fatty acid composition of the diets Fatty acids (%) STD HFD SFA 18.82 45.85 MUFA 26.38 40.02 PUFA 54.8 14.13 Standard diet (STD); high-fat diet (HFD) SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids.

[0361] After 10 weeks of feeding the diets, obese animals were divided into two groups: one groups continued to be fed on the same HFD; however, another group was fed on the same diet and received either NKS-3 or NKS-5) until 28.sup.th of weeks of experimentation. At the end, the animals were sacrificed, under a fasting condition, and used for different analysis in blood and different tissues.

b—Determination of Biochemical Parameters:

[0362] The rat/mouse insulin ELISA kit was obtained from EMD Millipore (USA). ELISA kits for CCK, PYY and GLP-1 were purchased from Cloud-Clone Corp. (USA). The ALAT (Alanine Amino Transferase) was determined by an automatic analyzer (Gilford Model 2000 system, a Beckman System T.R).

c—Liver Histology:

[0363] The liver of the mice at the time of sacrifice were fixed in formaldehyde and then dehydrated by passaging into ethanoic media. The liver tissues were embedded into the paraffin and 5μ thick section were cut by the microtome as per standard procedures. The slides, containing tissues, were stained by hematoxylin and eosin dyes as per standard methods. The stained slides were observed under Zeiss light microscope at 10× or 20× magnifications.

d—Statistical Analysis:

[0364] Results are expressed as mean±SEM (standard error of mean). Data were analyzed by using Statistica (4.1, Statsoft, Paris, France). The significance of difference between groups was determined by one-way analysis of variance (ANOVA), followed by least-significant-difference (LSD) test. For all the tests, the significance level chosen was p<0.05.

5.2 Fat Taste Analogues According to the Invention Exert Anti-Obesity Effects in Mice

[0365] In order to elucidate the beneficial effects and especially during obesity, experiments were conducted on obese C57BL/6J mice. The mice were maintained on a high-fat diet for 10 weeks and an increase in body weight in a progressive and linear manner was observed in these animals. In order to test the curative/interventional effects on obesity, the animals were divided into two groups: one group that consume only the high-fat diet (HFD) and the other consuming the same diet and also receiving NKS-3 or NKS-5 in adlibitum baby bottles/feeders. From the 10.sup.th week of the diet, lipid analogues were introduced into the bottles at concentrations that corresponded to their preference in the double choice test (NKS-5 at 75 μM; NKS-3 at 50 μM). From the first week of administration of analogues, there was a significant decrease in body weight in the obese mouse by these two lipid analogues (NKS-5 was more powerful than NKS-3). In addition, the decrease in obesity was maintained until the 28.sup.th week of feeding a HFD (FIGS. 13A and 13B). It should be noted that these lipid analogues exerted no effect on the body weight of the control animals, receiving the normal diet, suggesting that the lipid analogues act only in the case of a pathology.

[0366] The study was stopped after the 28.sup.th week of the diet because the animals reached the age of 34 weeks and beyond that age, the aging factor may interfere with the growth.

5.3 Lipid Analogues Reduce Obesity by Releasing Anorectic Peptides/Hormones

[0367] The blood/sera concentrations of various factors/hormones/peptides released by the gastrointestinal tract were quantified. The secretion of cholecystokinin (CCK) by the pancreas is considered to be one of the most anorexigen factors that plays a role in satiety, exerting its action on the vagus nerve (nerve X) that conveys the message from the duodenum/intestine to the central nervous system to stop food intake. Some agents that increase the secretion of CCK very significantly can trigger satiety and decrease food intake. Other peptides such as glucagon-like petide-1 (GLP-1), released by enterochromaffin cells of the intestine (jejunum) and a peptide of the NPY family, called PYY, released by the stomach, also exert strong satiety effects in the late phase of food intake. Lipid analogues according to the invention were observed to trigger high release of CCK (FIG. 14), GLP-1 (FIG. 15) and PYY (FIG. 16) in the blood circulation of animals in the obese group, receiving fat taste analogues.

[0368] These observations suggest that the anti-obesity effects of lipid analogues of the invention are exerted via their action on the release of anorectic/satiety agents that may decrease the food intake.

5.4 Fat Taste Analogues Decrease the Pro-Inflammatory Status in Obese Animals

[0369] It is now well established that obesity is associated with low-grade inflammation, characterized by the increase of pro-inflammatory factors in the peripheral blood. These factors are, principally, as follows: interleukin-1 (IL-1), inetroleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha). These factors are released mainly by adipocytes that differentiate into macrophage-like cells during obesity.

[0370] NKS-3 and NKS-4 lipid analogues according to the invention were observed to decrease the circulating concentrations of IL-6 (FIG. 17) and other mediators of inflammation in obese animals, who received both NKS-3 and NKS-5 in the bottle/feeders.

5.5 Fat Taste Analogues Decrease Insulinemia in Obese Animals

[0371] Insulin is a polypeptide hormone synthetized by the β-cells of islets of Langerhans in the pancreas. Its role is to maintain the level of sugar present in the blood at a stable level. When blood sugar levels increase a little because of the consumption of high-sugar products, insulin is responsible for transporting this excess sugar to the cells of the muscles, the liver and also adipose tissue.

[0372] The obese subjects suffer from pre-diabetic state that is marked by high concentrations of blood glucose. This state is called insulin-resistance as this hormone is not able to control blood glucose. In our study, we observed that obese animals were marked by insulin levels and addition of NKS-3 or NKS-5 decreases the insulin concentrations (FIG. 18).

5.6 Lipid Analogues Protect Liver Function

[0373] The liver is the mainly involved in lipid metabolism and elimination of toxic substances. In case of obesity (or high alcohol consumption), we observe a liver steatosis.

[0374] The ALAT (Alanine Amino Transferase, also called Pyruvic Glutamic Transaminase), is a detoxifying enzyme, present mainly in striated muscles, voluntary muscles, and especially in the liver. Its dosage is very useful for the diagnosis of certain diseases, in particular muscular and hepatic diseases. Thus, in case of destruction of the liver cells as in hepatitis or cirrhosis, the level of ALT in the blood is increased. The blood concentrations of ALAT was observed to increase significantly in the blood of obese animals compared to normal mice, and the fat taste analogues NKS-3 and NKS-5 according to the invention decreased its concentration in obese animals (FIG. 19).

[0375] It is also noteworthy that weight of the liver that was increased in obese mice was significantly curtailed only in obese animals (FIG. 20).

[0376] The liver of obese subjects suffers from metabolic steatohepatitis or NASH (non-alcoholic steatosis hepatitis) syndrome. The prevalence of NASH syndrome and severe steatosis (fibrosis/cirrhosis) is correlated with the degree of obesity. The histological changes in the liver were also studied. Very beneficial effects of fat taste analogues were surprisingly observed. It is to note that the analogues (NKS-3 and NKS-5) did not exert any effect in normal diet fed mice (FIG. 21). However, the liver of obese mice was affected with steatosis and the lipid analogues exerted curative effects in liver sections (FIG. 21).