ANALOGUES OF N-ACYL-HOMOSERINE LACTONES AND PHARMACEUTICAL COMPOSITION COMPRISING SAME

Abstract

The present invention relates to analogues of N-acyl homoserine lactones (AHLs) and pharmaceutical compositions comprising same. The invention also relates to their use in the treatment of inflammatory diseases of the epithelium.

Claims

1. A compound having the following general formula I: ##STR00049## wherein: X, Y, Z and W are independently of each other a carbon atom or a heteroatom selected from S, N and O, provided that X is different from O, X, Y, Z and W are independently of each other optionally substituted with a halogen selected from Cl, F, Br, and I, or a linear or branched C.sub.1 to C.sub.4 alkyl group, x, y, z, and w, independently of each other, are 0 or 1, provided that 3≤x+y+z+w≤4, R represents H or a linear or branched C.sub.1 to C.sub.4 alkyl group, or a hydroxyl group (OH) or an azido group (N.sub.3), represents a single or double bond (cis or trans), R′ represents H or a linear or branched C.sub.1 to C.sub.4 alkyl group for use in the treatment of an inflammatory disease of the epithelium.

2. The compound according to claim 1 selected from the group consisting of: the (D/L)-3-oxo-C.sub.12 aminothiolactone ((D/L)-3-oxo-C.sub.12-HTL) of the following formula I-1: ##STR00050## the (S,S)-3-oxo-C.sub.12 aminocyclohexanol ((S,S)-3-oxo C.sub.12-ACH) of the following formula I-2: ##STR00051## the (S)-3-oxo-C.sub.12 aminothiolactone (S)-3-oxo-C.sub.12-HTL) of the following formula I-3: ##STR00052## the (R,S)-3-oxo-C12-aminocyclohexanol of the following formula I-4: ##STR00053## the 3-oxo-C12-aminocyclohexanol of the following formula I-5: ##STR00054## the 3-oxo-C12-aminochlorophenol of the following formula I-6: ##STR00055##

3. A method for the treatment of an inflammatory disease of the intestine comprising administering an effective amount of the compound of claim 1.

4. A method for the treatment of psoriasis comprising administering an effective amount of the compound of claim 1.

5. A pharmaceutical composition comprising at least one compound having the following general formula I: ##STR00056## wherein: X, Y, Z and W are independently of each other a carbon atom or a heteroatom selected from S, N and O, provided that X is different from O, X, Y, Z and W are independently of each other optionally substituted with a halogen selected from Cl, F, Br, and I, or a linear or branched C.sub.1 to C.sub.4 alkyl group, x, y, z, and w, independently of each other, are 0 or 1, provided that 3≤x+y+z+w≤4, R represents H or a linear or branched C.sub.1 to C.sub.4 alkyl group, or a hydroxyl group (OH) or an azido group (N.sub.3), R′ represents H or a linear or branched C.sub.1 to C.sub.4 alkyl group, represents a single or double bond (cis or trans), and at least one pharmaceutically acceptable excipient.

6. The pharmaceutical composition of claim 5 wherein the at least one compound of formula I is selected from the group consisting of: the (D/L)-3-oxo-C12-aminothiolactone ((D/L)-3oxoC12-HTL) of the following formula I-1: ##STR00057## the (S,S)-3-oxo-C12-aminocyclohexanol (S,S) 3-oxo-C12-ACH) of the following formula I-2: ##STR00058## the (S)-3-oxo-C12-aminothiolactone ((S)-3-oxo-C12-HTL) of the following formula I-3: ##STR00059## the (R,S)-3-oxo-C12-aminocyclohexanol of the following formula I-4: ##STR00060## the 3-oxo-C12-aminocyclohexanol of the following formula I-5: ##STR00061## 3-oxo C12-aminochlorophenol of the following formula I-6: ##STR00062##

7. A method for the treatment of an inflammatory disease of the epithelium comprising administering an effective amount of the pharmaceutical composition of claim 5.

8. A method for the treatment of an inflammatory disease of the intestine comprising administering an effective amount of the pharmaceutical composition of claim 5.

9. A method for the treatment of psoriasis comprising administering an effective amount of the pharmaceutical composition of claim 5.

Description

FIGURES

[0077] FIG. 1 shows in bar graph form the results of stimulation of Caco-2/TC7 cells by the IL-1β in the presence of (D,L)-3oxo C.sub.12 aminothiolactone of formula I-1 used in the invention,

[0078] FIG. 2 shows the activation curves of the LasR receptor on bacterial reporter strain in relation to the natural molecules C4-HSL and 3-oxo-C.sub.12-HSL and to the molecule of formula I-1 used in the invention,

[0079] FIG. 3 shows in bar graph form the results of stimulation of Caco-2/TC7 cells by the IL-1β in the presence of (S,S)-3-oxo C.sub.12 aminocyclohexanol (S,S)-3-oxo C.sub.12-ACH) of formula I-2 used in the invention,

[0080] FIG. 4 represents the inhibition curve of the IL-8 secretion by human keratinocytes stimulated by IL-17 and TNF-α in the presence of increasing doses of (S,S) 3-oxo C.sub.12 aminocyclohexanol (S,S) 3-oxo C.sub.12-ACH) of formula I-2 used in the invention,

[0081] FIG. 5 shows the inhibition curve of the IL-2 secretion by human lymphocytes stimulated by CD2, CD3 and CD28, in the presence of increasing doses of (S,S)-3-oxo C.sub.12-aminocyclohexanol (S,S)-3-oxo C.sub.12-ACH) of formula I-2 used in the invention,

[0082] FIG. 6 shows the activation curves of the LasR receptor on the bacterial reporter strain in relation to the natural 3-oxo-C.sub.12HSL molecule and to the molecule of formula I-2 used in the invention,

[0083] FIG. 7 shows in bar graph form the results of stimulation of Caco-2/TC7 cells by the IL-1β in the presence of (S)-3-oxo C.sub.12 aminothiolactone ((S)-3oxoC.sub.12-HTL) of formula I-3 used in the invention,

[0084] FIG. 8 represents in bar graph form the results of stimulation of Raw 264.7 murine cells by the IFN-γ/LPS combination in the presence of (S,S)-3-oxo C.sub.12 aminocyclohexanol ((S,S)-3 oxo C.sub.12-ACH) of formula I-2 used in the invention,

[0085] FIG. 9 shows in bar graph form the results of stimulation of Caco-2/TC7 cells by the IL-1β in the presence of (R,S)-3-oxo C.sub.12 aminocyclohexanol (R,S)-3-oxo C.sub.12-ACH) of formula I-4 used in the invention,

[0086] FIG. 10 represents in bar graph form the results of stimulation of Raw 264.7 murine cells by the IFN-γ/LPS combination in the presence of the (R,S) 3-oxo C.sub.12-ACH) of formula I-4 used in the invention,

[0087] FIG. 11 shows the activation curves of the LasR receptor on the bacterial reporter strain in relation to the natural molecule 3-oxo-C.sub.12-HSL and to the (R,S)-3-oxo C.sub.12-aminocyclohexanol of formula I-4 used in the invention,

[0088] FIG. 12 shows in bar graph form the results of stimulation of Caco-2/TC7 cells by the IL-1β in the presence of the 3-oxo C.sub.12-aminochlorophenol of formula I-6 used in the invention,

[0089] FIG. 13 represents in bar graph form the results of stimulation of Raw 264.7 murine cells by the IFN-γ/LPS combination in the presence of the 3-oxo C.sub.12-aminochlorophenol of formula I-6 used in the invention, and

[0090] FIG. 14 shows the activation curves of the LasR receptor on the bacterial reporter strain in relation to the natural molecule 3-oxo-C.sub.12-HSL and to the 3-oxo C.sub.12-aminochlorophenol of formula I-6 used in the invention.

[0091] FIG. 15 represents as heat map form the secretion of 23 cytokines by Raw264.7 murine cells under stimulated conditions (LPS 10 ng/mL; IFN-γ 20 U/mL) (normalized to control),

[0092] FIGS. 16A and 16B show in histogram form:

[0093] At the top, the amount of TNF∝ secreted by Raw264.7.7 murine cells stimulated by LPS and interferonγ in the presence of 3-oxo-C.sub.12:2-HSL

[0094] At the bottom, the amount of TNF∝ secreted by Raw264.7 murine cells stimulated by LPS and interferonγ in the presence of PCA,

[0095] FIGS. 17A-17C represent in diagram form the gene expression results obtained by measuring the messenger RNA by quantitative PCR for 3 cytokines of interest: Rantes, TNF alpha, IL1-beta,

[0096] FIGS. 18A and 18B show in histogram form the cytotoxicity of the AHL 3oxoC.sub.12-HSL(A) and 3oxoC.sub.12:2-HSL(B) treatments on stimulated Caco-2/TC7 cells. Mean values of different replicates (n≥3)±SEM,

[0097] FIGS. 19A-19D show in histogram form the cytotoxicity of the AHL 3-oxo-C.sub.12-HSL(A,C) and 3-oxo-C12:2-HSL(B,D) treatments on Raw264.7 murine cells in the basal (A,B) or stimulated (C,D) state. Mean values of different replicates (n=3)±SEM. stimulated,

[0098] FIGS. 20A and 20B show in histogram form the LDH secretion under stimulated conditions in the Caco-2/TC7 (left) and Raw264.7 (right) cell lines. Mean values of multiple replicates (n≥6)±SEM,

[0099] FIGS. 21A and 21B show in histogram form the LDH secretion under stimulated conditions in the Caco-2/TC7 (A) and Raw264.7 (B) cell lines. Mean values of multiple replicates (n≥6)±SEM,

[0100] FIGS. 22A and 22B show in histogram form LDH secretion under stimulated conditions in the Caco-2/TC7 (A) and Raw264.7 (B) cell lines. Mean values of multiple replicates (n≥8)±SEM,

[0101] FIGS. 23A and 23B show in histogram form the comparative survival of the E. coli K12 strain after 18 h of incubation in the presence of control molecules or increasing doses of the 3oxoC12-HSL and 3oxoC.sub.12:2-HSL AHL. Mean values of different replicates (n=6)±SEM, and

[0102] FIG. 24 represents in histogram form the comparative survival of the E. coli K12 strain after 18 h of incubation in the presence of control molecules or 100 μM of molecule. Mean values of different replicates (n=6)±SEM.

[0103] The invention is based on the discovery of synthetic bio-inspired analogues of N-acyl-homoserine lactones (AHLs) with an anti-inflammatory activity and an action on the tight junctions at least equal to those of natural AHLs produced in the intestine while having a better stability, a delayed bacterial recognition and a lower toxicity.

[0104] The natural AHLs, produced in the intestine, of which the compounds used in the invention are analogs, are the 3-oxo-C.sub.12-HSL and the -oxo-C.sub.12:2-HSL.

[0105] The inventors have segmented these natural AHLs into three areas of interest: [0106] Lactone homoserine head [0107] Oxo substitution to form a ketone at the 3.sup.rd carbon of the carbon chain [0108] Acyl chain of 10 to 16 carbons.

[0109] In addition, the L-enantiomer of the AHL is the active form (no activity of the D-enantiomer).

[0110] The inventors then studied the influence of a modification with various chemical groups of each of these three areas of interest on the biological activity on human and murine cells, on the stability and the bacterial recognition of the analogues thus obtained.

[0111] They then discovered that the length of the carbon chain can be 10 and up to 16 carbon atoms, with an optimal length of 14 carbon atoms, and only tolerates the addition of chemical groups of low steric hindrance. The presence of the lactone group at the C3 position is necessary, because its removal or its transformation into an acetal inhibits the anti-inflammatory activity of the AHLs. The head group can only tolerate minor modifications that do not excessively increase its size and retain chemical groups capable of providing hydrogen bonds.

[0112] Thus, the compounds used in the invention for the treatment of inflammatory diseases of the epithelium, and in particular inflammatory diseases of the intestine and the psoriasis, have the following general formula I:

##STR00017##

wherein: [0113] X, Y, Z and W are independently of each other a carbon atom or a heteroatom selected from S, N and O, provided that X is different from O, [0114] X, Y, Z and W are independently of each other optionally substituted with a halogen selected from Cl, F, Br, and I, or a linear or branched C.sub.1 to C.sub.4 alkyl group, [0115] x, y, z, and w, independently of each other, are 0 or 1, provided that 3≤x+y+z+w≤4, [0116] R represents H or a linear or branched C.sub.1 to C.sub.4 alkyl group, or a hydroxyl group (OH) or an azido group (N.sub.3), [0117] R′ represents H or a linear or branched C.sub.1 to C.sub.4 alkyl group [0118] represents a single or double bond (-cis or trans).

[0119] These compounds of formula I can be used in combination with a pharmaceutically acceptable excipient to form a pharmaceutical composition.

[0120] The pharmaceutical composition can be formulated for any form of administration, in a solid or liquid or semi-solid form, such as a gel, cream, balm and can be administered orally, rectally, intravenously, or topically.

[0121] This pharmaceutical composition is in particular intended for the treatment of an inflammatory disease of the epithelium, and in particular of an inflammatory disease of the intestine and the psoriasis.

[0122] The preferred compounds for use in the treatment of an inflammatory disease of the epithelium are the following compounds.

[0123] The first of these compounds is the (D/L)-3oxoC12 aminothiolactone (D/L)-3oxoC.sub.12-HTL) of the following formula I-1:

##STR00018##

[0124] The compound of formula I-1 corresponds to the natural 3-oxo-C.sub.12 HSL in which the lactone head has been replaced by a thiolactone group.

[0125] As shown in FIG. 1, which represents in bar graph form the results of stimulation of Caco-2/TC7 cells by the IL-1β in the presence of the (D/L)-3oxo C.sub.12 aminothiolactone of formula I-1 used in the invention, in comparison to DMSO, this compound shows in the human Caco-2/TC7 enterocyte line (cells of the intestinal epithelium) an activity equivalent to that of the natural 3-oxo-C.sub.12 HSL, and is more active than the latter at the 100 μM dose.

[0126] FIG. 2 shows the activation curves of the LasR receptor on bacterial reporter strain in relation to the natural molecules C4-HSL and 3-oxo-C.sub.12-HSL and to the molecule of formula I-1 used in the invention.

[0127] It can be seen from this FIG. 2 that the racemate of formula I-1 shows a delayed recognition capacity with an EC.sub.50 of 125 nM vs. 0.9 nM for the natural molecule 3-oxo-C.sub.12-HSL and vs. no recognition (EC.sub.50>1000 μM) for the natural molecule C.sub.4-HSL.

[0128] The second of these compounds is the (S,S) 3-oxo C.sub.12 aminocyclohexanol ((S,S) 3 oxo C.sub.12-ACH) of formula I-2 below:

##STR00019##

[0129] The compound of formula I-2 corresponds to the 3-oxo-C.sub.12 HSL in which the lactone head has been replaced by a (S,S)-aminocyclohexanol group.

[0130] It can be seen from FIG. 3, which represents in bar graph form the results of stimulation of Caco-2/TC7 cells by the IL-1B in presence of the (S,S)-3-oxo C.sub.12 aminocyclohexanol ((S,S)-3-oxo C.sub.12-ACH) of formula I-2 used in the invention, that this compound exhibits in the human Caco-2/TC7 enterocyte line an activity equivalent to that of the natural 3-oxo-C.sub.12 HSL in the 1-50 μM range. As seen in FIG. 4, which represents the inhibition curve of the IL-8 secretion by human keratinocytes stimulated by IL-17 and TNF-α in the presence of increasing doses of the (S,S) 3-oxo C.sub.12 aminocyclohexanol ((S,S) 3-oxo C.sub.12-ACH) of formula I-2 used in the invention, this compound presents a relative EC.sub.50 of 111 nM and a maximum inhibition corresponding to 39% of the inhibition obtained in the presence of the reference compound betamethasone, which allows to show the generalization of the anti-inflammatory effects of this molecule to several cell lines, resulting from various organs of the human body.

[0131] It can be seen from FIG. 5, which represents the inhibition curve of the IL-2 secretion by the human lymphocytes stimulated by CD2, CD3 and CD28, in the presence of increasing doses of the (S,S)-3-oxo C.sub.12-aminocyclohexanol ((S,S)-3-oxo C.sub.12-ACH) of formula I-2 used in the invention, that this compound presents a relative EC.sub.50 of 89.9 nM and a maximum inhibition corresponding to 46% of the inhibition obtained in the presence of the reference compound betamethasone, which confirms the generalization of the anti-inflammatory effects of this molecule to several cell lines, resulting from different organs of the human body.

[0132] It can also be seen from FIGS. 4 and 5 that the natural AHL of Formulae A and B are inactive in the scope of the tests, the results of which are shown in FIGS. 4 and 5, which demonstrates the superiority of the molecules of Formula I used in the invention.

[0133] From FIG. 6, which shows the activation curves of the LasR receptor on bacterial reporter strain in relation to the natural 3-oxo-C.sub.12HSL molecule and the molecule of formula I-2 used in the invention, the compound of formula I-2 has an EC.sub.50 of 165 nM. It therefore has an even more delayed recognition ability than the compound of formula I-1.

[0134] Finally, this molecule of formula I-2 is also resistant to the hydrolysis giving an open form (both enzymatic and spontaneous).

[0135] FIG. 8 shows in bar graph form the results of stimulation of Raw 264.7 murine cells by the IFN-γ/LPS combination in the presence of (S,S)-3-oxo C.sub.12 aminothiolactone ((S,S)-3 oxo C.sub.12-ACH) of formula I-2. This figure shows that the compound of formula I-2 is more active than the reference molecule 3-oxo-C.sub.12-HSL, at all doses in the concentration range of 1 to 50 μM.

[0136] The third compound is (S)-3-oxo C.sub.12 aminothiolactone ((S)-3oxoC.sub.12-HTL) of the following formula I-3:

##STR00020##

[0137] The compound of formula I-3 corresponds to the 3-oxo-C.sub.12 HSL in which the lactone head has been replaced by a thiolactone group.

[0138] As shown in FIG. 7, which represents in bar graph form the results of stimulation of Caco-2/TC7 cells by the IL-1β in the presence of (S)-3-oxo C.sub.12 aminothiolactone ((S)-3oxoC.sub.12-HTL) of formula I-3, the effects observed with the molecule of formula I-3 used in the invention are similar to those observed with the natural 3-oxo-C.sub.12-HSL molecule, in particular a 27% decrease in the inflammatory secretion at 5 μM.

[0139] The fourth compound is (R,S)-3-oxo C12-aminocyclohexanol of the following formula I-4:

##STR00021##

[0140] FIG. 9 shows in bar graph form the results of stimulation of Caco-2/TC7 cells by the IL-1β in the presence of the (R,S)-3-oxo C.sub.12 aminocyclohexanol ((R,S)-3-oxo C.sub.12-ACH) of formula I-4 used in the invention.

[0141] From FIG. 9, we see in the human enterocyte line Caco-2/TC7 an activity equivalent to that of the natural 3-oxo-C.sub.12 HSL in the 1-50 μM range.

[0142] FIG. 10 represents in bar graph form the results of stimulation of Raw 264.7 murine cells by the IFN-γ/LPS combination in the presence of the (R,S) 3-oxo C.sub.12-ACH of formula I-4 used in the invention.

[0143] It can be seen from FIG. 10 that in the Raw 264.7 murine macrophage line the molecule shows a higher activity than the natural 3-oxo-C.sub.12 HSL in the 1-50 μM range.

[0144] FIG. 11 shows the activation curves of the LasR receptor on bacterial reporter strain in relation to the natural molecule 3-oxo-C.sub.12-HSL and to the (R,S) 3-oxo C.sub.12-aminocyclohexanol of formula I-4 used in the invention. As can be seen from FIG. 11, the (R,S)-3-oxo C.sub.12-aminocyclohexanol of formula I-4 has an EC.sub.50 of 200 μM. It therefore has a much-delayed recognition capacity compared to the natural AHLs. [0145] The fifth compound is the 3-oxo C.sub.12-aminocyclohexanol of the following formula I-5:

##STR00022##

[0146] The compound of formula I-5 corresponds to the 3-oxo-C.sub.12 HSL in which the lactone head has been replaced by an aminocyclohexanol group. [0147] The sixth compound is the 3-oxo C.sub.12-aminochlorophenol of the following formula I-6:

##STR00023##

[0148] The compound of formula I-6 corresponds to the 3-oxo-C.sub.12 HSL in which the lactone head has been replaced by an aminochlorophenol group.

[0149] FIG. 12 shows in bar graph form the results of stimulation of Caco-2/TC7 cells by the IL-1β in the presence of the 3-oxo C.sub.12-aminochlorophenol of formula I-6 used in the invention.

[0150] It can be seen from FIG. 12 that in the human enterocyte line Caco-2/TC7 the molecule shows a significantly higher activity than that of the natural 3-oxo-C.sub.12 HSL in the 10-100 μM range.

[0151] FIG. 13 represents in bar graph form the results of stimulation of Raw 264.7 murine cells by the IFN-γ/LPS combination in the presence of the 3-oxo C.sub.12-aminochlorophenol of formula I-6 used in the invention.

[0152] As can be seen, the molecule of formula I-6 shows in the Raw 264.7 murine macrophage line a significantly higher activity than that of the natural 3-oxo-C.sub.12 HSL in the 10-50 μM range.

[0153] FIG. 14 shows the activation curves of the LasR receptor on bacterial reporter strain in relation to the natural molecule 3-oxo-C.sub.12-HSL and to the 3-oxo C.sub.12-aminochlorophenol of formula I-6 used in the invention.

[0154] As can be seen, the molecule of formula I-6 has an EC.sub.50 greater than 1000 μM. It has the most delayed recognition ability compared to the natural AHLs and to the molecules of formula I-1 to 1-5 used in the invention.

EXAMPLES

[0155] 1. Experimental Procedures and Protocols Used in Biology [0156] The penicillin-streptomycin antibiotics, the nonessential amino acids (NEAA), and the L-glutamine were from Invitrogen (Thermo Fisher Scientific, Waltham, USA). The saline solution buffered with Dulbecco's phosphate (DPBS 10×), the high glucose cell culture medium (DMEM GlutaMAX 4.5 g/L glucose), the DMEM and the DMEM without phenol red were from Gibco (Thermo Fisher Scientific, Waltham, Mass., USA). The foetal calf serum was from GE Healthcare (Life Sciences, South Logan, Utah, USA). [0157] The 2-Hydroxyquinoline (CAS [59-31-4]), the 3oxo C.sub.12-HSL molecule, and the sterile DMSO were purchased from Sigma. The molecule 3oxoC.sub.12: 2-HSL was synthesized on demand by Diverchim (Roissy-en-France, France) [0158] All the absorbance and luminescence tests were read on the SpectraMax M5 spectrometers from Molecular Devices®.

[0159] 1.1. Cell Culture

[0160] 1.1.1. The Caco-2/TC7 Cell Line [0161] The Caco-2 cell line is derived from a human colon adenocarcinoma and represents a cell culture model of enterocytes lining the epithelium of the small intestinal. The Caco-2/TC7 cell line is a clonal population of Caco-2 cells that reproduces to a large extent and homogeneously most of the morphological and functional characteristics of the normal human enterocytes. [0162] The Caco-2/TC7 cells exhibit a contact inhibition property leading to a growth arrest when the cells reach the confluence, which allows the establishment of a cell monolayer. During the exponential growth phase (from seeding to confluence), the cells remain undifferentiated. At confluence, they can spontaneously (in the absence of differentiation inducers) differentiate and progressively polarize. The differentiation process, which is a growth-related mechanism, is maximal at the end of confluence (stationary phase of the growth curve). [0163] The cells develop a brush border at the apical pole containing microvilli and with characteristic enterocytic enzymes, such as hydrolases. [0164] In accordance with the published literature, the Caco-2/TC7 cells in our experiments were seeded at 10.sup.5 cells/well (equivalent to 10-12×10.sup.3 cells/cm.sup.2) in 6-well plastic culture plates. The cells were maintained in a glucose-rich medium (high glucose in the DMEM GlutaMAX medium 4.5 g/I glucose) supplemented with 20% heat-inactivated foetal calf serum, 1% nonessential amino acids NEAA, and 1% penicillin streptomycin. The cells were grown at 37° C. in a 10% CO.sub.2/air atmosphere. The medium was changed every day. Under these conditions, the cells were confluent on day 6. On day 17, the cells were serum-starved, and were used on day 18.

[0165] 1.1.2. The Raw 264.7 Cell Line [0166] The Raw 264.7 cell line consists of murine cells of macrophage type derived from cell line transformed by the virus of the Abelson leukaemia and providing from BALB/c mice. This cell line is very often used as a model of macrophages in vitro. The Raw 264.7 cells are capable of a phagocytosis and a pinocytosis, can kill the target cells by antibody-dependent cytotoxicity, and secrete a wide range of inflammatory cytokines as well as nitric oxide (NO). In addition, the Raw 264.7 cells are a very convenient cell line: the cells grow rapidly, appreciate the small diameter wells, and should be used before confluence. These conditions make it an advantageous line for the compound screening.

[0167] The cells were used between the passages 13 and 26. [0168] The Raw 264.7 cells used are from the ATCC bank. They were grown in DMEM supplemented with 10% heat-inactivated foetal calf serum and 1% L-glutamine to 200 mM, and maintained at 37° C. with a 5% CO2/air atmosphere. The medium was changed every two days.

[0169] 1.1.3. The Bacterial Reporter Strain E. coli pSB1075 [0170] A bacterial reporter strain of the Quorum Sensing was used to study the ability of the molecules to be recognized by the Pseudomonas aeruginosa AHL receptor (LasR) and induce an activation. Thus, the strain pSB1075 of Escherichia coli was used. [0171] The enterobacterium Escherichia coli does not naturally produce AHL, nor does it have an orphan receptor capable of recognizing AHL. This bioluminescent strain was modified by addition of a plasmid pSB1075. This plasmid contains genes encoding both tetracycline resistance and the expression of the LasR AHL receptor from the Pseudomonas aeruginosa bacteria, as well as a fusion gene derived from the LasR promoter and the luxCDABE gene from Photorhabdus luminescence. The bacterial strain was grown in the LB medium supplemented with 5 μg/ml tetracycline to exert a selection pressure and ensure that only the desired strain was amplified. The fusion gene included in the plasmid gives the bacteria the ability to emit a bioluminescence when the LasR receptor is activated, and provides the user with a robust test for the molecular screening. The LasR receptor of P. aeruginosa is well suited for the recognition of the long-chain AHL, particularly its natural partner, the 3oxoC.sub.12-HSL, which causes the highest bioluminescent response. In contrast, short-chain AHL such as the C.sub.4-HSL do not induce the bioluminescence emission. [0172] Briefly, the bacterial culture was started on day 0 in 10 ml LB medium supplemented with 5 μg/ml tetracycline and maintained for 24 hours at 37° C. and under stirring at 70 rpm. On day 1, the culture was diluted 1:100 (P1) in 10 ml of LB medium supplemented with 5 μg/ml tetracycline and maintained for 24 hours at 37° C. under stirring at 70 rpm. On day 2, the experiment took place: a bacterial suspension extemporaneously diluted 1:10 in LB medium supplemented with 5 μg/ml tetracycline (P2) was dispensed into a black opaque 96-well plate and incubated for 4 hours with a range of AHL concentrations or controls until the resulting luminescence was read at the end point. All experiments were performed in triplicates.

[0173] 1.1.4. Bactericidal Dosage [0174] The K12 strain of E. coli was grown on agar on day 0 and a colony was transferred to the LYBHI liquid bacterial culture medium on day 1. On day 2, the colony was diluted 1:100 in LYBHI medium and amplified for 18 hours before transfer to an opaque 96-well plate. In each well, LYBHI, controls or increasing doses of tested molecules and bacteria were distributed. The absorbance at 600 nm was read at start-up (t=0) and after an 18-hour incubation. The raw absorbance values were corrected using the absorbance of solutions without bacteria. All experiments were performed twice, each with 4 replicates.

[0175] 1.2. Evaluation of the Biological Activity of the Molecules in the Mammalian Cells

[0176] 1.2.1. Stimulation of Caco-2/TC7 with Cytokines [0177] The Caco-2/TC7 cells were seeded in 6-well plates at 100,000 cells/well and grown for 18 days. On day 17, the cells were serum-starved, which means that the cell medium was replaced with a foetal calf serum-free medium, and used on day 18. [0178] The stimulation medium was composed of a medium referred to as “starvation medium”, i.e. without foetal calf serum (DMEM GlutaMAX, 1% NEAA, 1% penicillin-streptomycin) with 100 μM of 2-HQ. The cells were incubated for 18 h at 37° C. with 2 ml of stimulation medium containing 0.1% DMSO (negative control) or stimulation medium containing the compounds tested at desired concentrations, with or without proinflammatory cytokines to induce an inflammation. To induce the inflammation, either the IL-1β at 25 ng/ml or the combination of TNF-α and IFN-γ at 50 ng/ml each were used. After 18 h, the supernatants were collected and stored at −80° C. before analysis by ELISA test. The cells were washed with 1 ml of PBS 1×/well, and lysed in 100 μl of PBS 1× containing 1% Triton X-100. The cells were harvested by scraping and stored at −80° C. before the quantification of the protein. The dosing of the LDH was performed immediately before freezing. [0179] All the experiments on the cells were performed in triplicates.

[0180] 1.2.2. Stimulation of Raw 264.7 with LIPS and IFN-γ [0181] Raw 264.7 cells were seeded in 12-well plates at 75,000 cells/well, or 24-well plates at 40,000 cells/well, to reach 80-90% confluence after 3 days of culture. For the stimulation, the cells were incubated for 6 h at 37° C. with 750 μL (respectively 500 μL) of 100 μM 2-HQ-enriched cell medium containing 0.1% DMSO (negative control) or compounds tested at desired concentrations, with or without LPS (10 ng/ml) and IFN-γ (20 U/ml) to establish an inflammation. After 6 h, the supernatants were collected and stored at −80° C. before analysis by ELISA test. The cells were harvested by scraping in 100 μL of PBS 1×/well and stored at −80° C. before the quantification of the total protein. The dosing of the LDH was performed immediately before freezing. [0182] All experiments were performed in triplicates.

[0183] 1.2.3. Measurement of Protein Concentration in Cell Lysate [0184] The total protein concentrations were determined in cell lysates using the assay reagents of the protein which are bicinchoninic acid (BCA) and bovine serum albumin (BSA) according to the instructions of the manufacturer (Uptima-Interchim, Montlugon, France).

[0185] 1.2.4. Quantification of the Human Cytokines by ELISA [0186] The levels of the proinflammatory cytokine IL-8 produced by the cells were determined in the cell supernatants and/or cell lysates using the commercially available IL-8 ELISA detection kit (Duoset Human C×CL8/IL-8, ref. DY208) provided by R & D Systems (Minneapolis, Minn., USA) according to the instructions of the manufacturer. [0187] All the cytokine levels were first normalized to the protein content determined in the corresponding cell lysates. Then, to compare the experiments, they could be further normalized using the activated control condition (DMSO+cytokines) as the 100% response.

[0188] 1.2.5. Quantification of the Murine Cytokines [0189] The levels of IL-6 murine cytokines produced by cells were determined in cell supernatants or lysates using the commercially available “BD OptEIA Mouse IL-6 ELISA Set” from BD Biosciences (San Jose, Calif., USA, ref. 555240). All the ELISA kits were used according to the instructions of the manufacturer. [0190] The cytokine rates were first normalized to the protein content determined in the corresponding cell lysates. To compare the experiments, they could be further normalized by using the activated control condition (DMSO+cytokines) as the 100% response.

[0191] 1.2.6. Cytotoxicity Test [0192] The cytotoxicity of the tested compounds and of the controls, with or without proinflammatory cytokines, was assessed using a release test of the lactate dehydrogenase (LDH), which tracks the release of this enzyme in the cell supernatants, a good indicator of the damages of the membrane and of the cell death. A compound was considered cytotoxic when its secreted LDH rate was greater than 10%. [0193] Two methods can be used to perform the test: measurement with a pyruvate/NADH solution (Sigma), or using the Cytotoxicity detection kit.sup.PLUS (LDH) of Roche (Sigma-Aldrich). [0194] For the Pyruvate/NADH method, a pyruvate/NADH solution was prepared with 4.1 mg pyruvic acid (0.62 mM) and 7.7 mg NADH (0.18 mM) in 60 ml of 0.1 M PBS (pH 7.4). [0195] To measure the concentration of LDH in the supernatant, 800 μL of NADH was added to 200 μL of supernatant in a plastic cuvette and the decrease in the absorbance at 340 nm was monitored for 1 min. To measure the concentration of LDH in the cell lysate, 800 μL of NADH was added to 10 μL of supernatant and 190 μL of 0.1 M PBS in a plastic cuvette, and the decrease in the absorbance at 340 nm was monitored for 1 min. The percentage of LDH released in the supernatant was calculated as the ratio of the corrected slopes in the supernatant and the cell lysate. [0196] Using the Cytotoxicity Detection Kit.sup.PLUS (LDH-Roche-Ref. 04744934001), the LDH levels were determined in the cell supernatants and the lysates by means of the absorption-based and colorimetric test, and performed according to the instructions of the manufacturer. The percentage of cytotoxicity could be established with the formula:

[00001]$\begin{array}{cc}{\%}_{\mathrm{LDH}}=\frac{{\mathrm{DO}}_{\mathrm{sample}}-{\mathrm{DO}}_{\mathrm{low}\mathrm{control}}}{{\mathrm{DO}}_{\mathrm{high}\mathrm{control}}-{\mathrm{DO}}_{\mathrm{low}\mathrm{control}}}\times 100& \left[\mathrm{Math}1\right]\end{array}$

[0197] 1.3. Biological Activity of the Molecules on the Bacterial Reporter Strain E. coli pSB1075 [0198] Escherichia coli does not naturally produce AHL. This bioluminescent strain was developed by the addition of a plasmid pSB1075 containing genes encoding the tetracycline resistance and the expression of the LasR AHL receptor, as well as a fusion gene of the LasR promoter and the luxCDABE gene of Photorhabdus luminescence. [0199] On day 1, a 1/100 dilution of the bacterial strain (P1) was grown for 24 hours at 37° C. under stirring (70 rpm) in a LB medium containing 5 μg/ml tetracycline (pressure selective). On day 2, P1 was diluted 1:10 in the same medium to obtain P2. In a black 96-well plate were placed 200 μL of P2 and 10 μL of the short chain compound (medium, water and DMSO for the negative controls, C4-HSL as positive control, and the test sample at the desired concentration). The plate was incubated for 4 hours at 37° C. under stirring at 70 rpm. The luminescence was then read at all wavelengths (integration time 200 ms) on a microplate reader. [0200] For the competition assays the culture protocol was similar but the bacteria were first pre-incubated with 1, 10 or 100 nM of 3oxoC.sub.12-HSL for different durations (1, 2, 6 or 16 h) before dilution to obtain P2. After that, the incubation was continued according to the conventional protocol, with the test compound at the desired concentrations.

[0201] 1.4. Statistical Analysis [0202] All the data are represented as the mean plus or minus SEM of n independent experiments, and were tested for the Gaussian distribution. The statistical significance was examined by a Student test t, a one-way ANOVA, a two-way ANOVA, or a Kruskal-Wallis test, depending on the data set, combined with a post-test (Tukey or Dunn multiple comparison test). The differences were considered significant when p<0.05. All the statistical analyses were performed using the Prism 6.0, GraphPad software.

[0203] 2. Materials and Methods in Chemistry [0204] Unless otherwise stated, all the reactions were performed under argon atmosphere in dry glassware. The reagents were purchased from commercial suppliers Sigma-Aldrich and TCI Chemicals and were used without further purification. [0205] The flash chromatography was performed on pre-packaged silica gel columns (40-63 μm irregular SiO.sub.2 silica gel) of CHROMABOND® Flash (Macherey-Nagel, Duren, Germany), mounted on an automated SPOT platform from ArMen. [0206] The thin layer chromatography was performed on aluminium sheets coated with silica gel 60 F.sub.254 (Millipore, Merck) and revealed with potassium permanganate (KMnO4), iodine on silica, bromocresol green or under an UV light (254 nm or 365 nm).

[0207] 2.1. Convention for the Numbering of the Atoms in N-Acyl Homoserine Lactones and their Analogues [0208] the numbering of the atoms adopted in the AHL molecules and their analogues is as follows:

##STR00024##

[0209] 2.2. Experimental Procedures for the Synthesis and Physicochemical Characterization of the Natural AHL, Intermediates and Non-Natural Analogues

[0210] General Procedure for the Preparation of Meldrum 3a-b Acid Derivatives (GP1) [0211] The appropriate carboxylic acid (1.0 equiv.) was dissolved in dichloromethane (denoted DCM) (1.5 ml/mmol acid) at room temperature. DCC (1.1 equiv.), DMAP (1.05 eq.), and Meldrum acid (1.0 eq.) were added to the mixture sequentially. The reaction mixture was stirred overnight at room temperature under argon atmosphere. The reaction was monitored by TLC in a 1:1 EtOAc/cyclohexane mixture and revealed with iodine. [0212] After completion of the reaction, the reaction mixture was filtered to remove the precipitated DCU and the filtration residue thoroughly washed with dichloromethane. The filtrate was collected and the solvent removed under vacuum. The resulting oil was diluted in EtOAc and the organic phase was extracted with HCl 1 M (×2), while the aqueous phase was washed with EtOAc (×2). The combined organic phases are dried over MgSO.sub.4, before removing the solvent under vacuum. The raw product, obtained in the form of oil, was directly engaged in the next step.

2,2-dimethyl-5-(1-oxodecyl)-1,3-dioxane-4,6-dione 3a [182359-65-5]

[0213] ##STR00025## [0214] Prepared by GP1 with a yield of 94%. .sup.1H RMN (300 MHz, CDCl.sub.3): δ:12-3.03 (m, 2H, C (4)H.sub.2), 1.74 (s, 6H, OC(CH.sub.3).sub.2O), 1.45-1:24 (m, 14H C (5) H.sub.2 to C (11) H.sub.2), 0.91 to 0.86 (m, 3H, C (12) H.sub.3). .sup.13C RMN (75 MHz, CDCl.sub.3): δ 198.32 (C(3)), 170.57 (ester), 160.18 (ester), 104.74 (OC(CH.sub.3).sub.2O), 91.23, 35.74, 31.83, 29.37, 26.76, 26.15, 22.65, 14.08 (C (12)). R.sub.f (1:3 EtOAc/Cychex): 0.22.

2,2-dimethyl-5-(1-oxodecyl)-1,3-dioxane-4,6-dione 3b azide

[0215] ##STR00026##

[0216] Prepared by GP1 with a yield of 87%. .sup.1H RMN (300 MHz, chloroform-d): 3.24 (t, J=6.9 Hz, 2H, C (12)H), 3:08-3:02 (m, 2H, C (4) H.sub.2), 2.12 (s, 1H, C (2) H), 1.72 (s, 6H, C(CH.sub.3).sub.2), 1.62-1.54 (m, 4H, C (5) H.sub.2 and C(6)H.sub.2, 1.31-1.28 (m, 10H, C (7) H.sub.2 to C (11) H.sub.2). .sup.13C RMN (75 MHz, CDCl.sub.3): δ198.33 (ketone), 170.68 (ester), 160.30 (ester), 104.88 (C.sub.2), 91.36 (OC(CH3).sub.2O), 51.56 (C.sub.12, 43.87 (C.sub.4), 35.82 (C.sub.5), 29.37, 29.17, 28.92, 26.91 ((CH.sub.3).sub.2), 26.77, 26.20, 23.91. R.sub.f (1:3 EtOAc/Cychex): 0,25.

[0217] General Procedure for the Methanolysis of the Meldrum 4a-b Acid Derivative (GP2) [0218] The meldrum 3a-c acid derivative (1.0 equiv.) was dissolved in excess methanol and the reaction flask was equipped with a reflux apparatus under an argon atmosphere. The reaction was heated at reflux for 2 hours, then the heating was stopped and the mixture allowed to cool spontaneously to room temperature and stirred overnight. The progress of the reaction was followed by TLC in an EtOAc/cyclohexane mixture at 1:1 and revealed in iodine. On completion, the solvent was removed under vacuum and the oily product was used raw in the following step.

3-oxododecanoate of methyl 4a [76835-64-8]

[0219] ##STR00027## [0220] Prepared by GP2 with a yield of 96%. .sup.1H RMN (300 MHz, CDCl.sub.3) 3.75 (s, 3H, OCH.sub.3), 3.46 (s, 2H, C (2) H.sub.2), 2.54 (t, J=7.4 Hz, 2H, C (4) H.sub.2), 1.59 (d, J=7.4 Hz, 2H, C (5) H.sub.2), 1.28 (s, 12H, C (6) H.sub.2 to C (11) H.sub.2), 0.92 to 0.87 (m, 3H, C (12) H.sub.3). .sup.13C RMN (75 MHz, CDCl.sub.3): δ202.86 (C.sub.3), 167.70 (C.sub.1), 52.31 (OCH.sub.3), 49.01, 43.09, 31.85, 29.39, 29.34, 29.24, 29.00, 23.47, 22.66, 14:09 (C.sub.12). R.sub.f (1:3 EtOAc/Cychex): 0.55.

Methyl ester of the acid 12-Azido-3-oxododecanoic 4b [1421598-01-7]

[0221] ##STR00028## [0222] Prepared by GP2 with a yield of 81%. .sup.1H RMN (300 MHz, CDCl.sub.3): δ3.71 (s, 3H, OCH.sub.3), 3.42 (s, 2H, C (2) H.sub.2), 3.22 (t, J=6.9 Hz, 2H, C (12) H.sub.2), 2.50 (t, J=7.3 Hz, 2H, C (4) H.sub.2), 01.55 (q, J=6.8 Hz, 2H, C (5) H.sub.2), 1.27 (t, J=4.7 Hz, 12H, C (6) H.sub.2 to C (11) H.sub.2). .sup.13C RMN (75 MHz, CDCl.sub.3): 202.85, 167.77, 52.40, 51.56, 49.11, 43.12, 29.36, 29.30, 29.14, 29.02, 28.91, 26.77, 23.50. R.sub.f (1:3 EtOAc/cyclohexane): 0.6.

[0223] General Procedure for Installing an Acetal in Position C.sub.3 5a-b (GP3) [0224] The ester 3-ketomethyl 4a-b (1.0 equiv.) was diluted in toluene (about 1 ml/mmol ester) and camphorsulfonic acid (0.2 eq.), trimethylorthoformate (5.0 eq.), and ethylene glycol (8.9 eq.) were successively added. The reaction mixture was heated to 80° C. for 3 hours, allowed to cool spontaneously and stirred overnight at room temperature. The reaction was followed by TLC in an EtOAc/cyclohexane 1:6 mixture and revealed with iodine. On completion, the toluene was removed under vacuum and the resulting oil was dissolved in DCM. The organic phase was extracted with a saturated Na—HCO3 solution (×3) while the aqueous phase was washed with DCM. The combined organic phases were dried over MgSO.sub.4 and the solvent removed under vacuum. [0225] If necessary, the oily product was purified by Flash Chromatography on a silica column using an elution gradient of 1:8 to 1:2 EtOAc/cyclohexane.

Methyl ester of the acid 2-Nonyl-1,3-dioxolane-2-acetic 5a [109873-29-2]

[0226] ##STR00029##

[0227] Prepared by GP3 with a yield of 95%. .sup.1H RMN (300 MHz, CDCl.sub.3): δ04:01-3.95 (m, 4H, OCH.sub.2CH.sub.2O), 3.70 (s, 3H, OCH.sub.3), 2.67 (s, 2H, C (2) H.sub.2), 1.83-1.76 (m, 2H, C (4) H.sub.2), 1.41-1:36 (m, 2H, C (5) H.sub.2), 1.28 (d, J=5.4 Hz, 12H, C (6) H.sub.2 to C (11) H.sub.2), 0.91-0.85 (m, 3H, C (12) H.sub.3). .sup.13C RMN (75 MHz, CDCl.sub.3): δ170.05 (C.sub.1), 109.41 (C.sub.3), 65.10 (ketal), 51.72 (OCH.sub.3), 42.42, 37.75, 31.88, 29.68, 29.56, 29.51, 29.29, 23.51, 22.67, 14.10 (C.sub.12). R.sub.f (1:8 EtOAc/Cychex): 0.31.

Methyl ester of the acid 2-(9-azido) nonyl-1,3-dioxolane-2-acetic 5b

[0228] ##STR00030## [0229] Prepared by GP3 with a yield of 93%. .sup.1H RMN (300 MHz, CDCl.sub.3): δ04:00-3.93 (m, 4H, OCH.sub.2CH.sub.2O), 3.67 (d, J=0.9 Hz, 3H, OCH.sub.3), 3.23 (t, J=6.9 Hz, 2H, C (12) H.sub.2), 2.64 (s, 2H, C (2) H.sub.2), 1.80-1.74 (m, 2H, C (4) H.sub.2), 1.61-1.55 (m, 2H C (5) H.sub.2), 1.28 (d, J=5.3 Hz, 12H, C(6) H.sub.2 to C(11) H.sub.2). .sup.13C RMN (75 MHz, CDCl.sub.3): δ170.15 (C.sub.1), 109.49 (C.sub.3), 65.22 (acetal), 51.87 (OCH.sub.3), 51.59 (C.sub.12), 42.52, 37.81, 29.71, 29.51, 29.45, 29.20, 28.93, 26.79, 23.56. R.sub.f (1:4 EtOAc/Cychex): 0.45.

[0230] General Procedure for the Basic Hydrolysis of the Methyl Ester 6a-b (GP4) [0231] The ketal-protected methyl ester 5a-b (1.0 eq.) was dissolved in THF (1.25 ml/mmol ester) and a NaOH 1 M solution (about 2.5 eq.) was added. The reaction mixture was refluxed for 3 hours. [0232] The progress of the reaction was followed by TLC in EtOAc/cyclohexane at 1:8 and revealed with iodine. [0233] Once completed and after the reaction mixture was cooled to room temperature, the pH was fixed at 4-5 with HCl IM. The organic phase was extracted with DCM (×3). The combined organic phases were dried over MgSO.sub.4 and the solvent removed under vacuum to give the desired product as an oil.

Acid 2-Nonyl-1,3-dioxolane-2-acetic 6a [596104-60-8]

[0234] ##STR00031## [0235] Prepared by GP4 with a yield of 95%. .sup.1H RMN (300 MHz, CDCl.sub.3): δ10.93 (s, 1H, OH), 4:06-3.99 (m, 4H, OCH.sub.2CH.sub.2O), 2.72 (s, 2H, C (2) H.sub.2), 1.85-1.78 (m, 2H, C (4) H.sub.2), 1.43-1.37 (m, 2H, C (5) H.sub.2) 1.29 (d, J=5.2 Hz, 12H, C (6) H.sub.2 to C (11) H.sub.2), 0.92-0.87 (m, 3H, C (12) H.sub.3). .sup.13C RMN (75 MHz, CDCl.sub.3): δ174.61 (C.sub.1), 109.32 (C.sub.3), 65.10 (OCH.sub.2CH.sub.2O), 42.36 (C.sub.2), 37.61 (C.sub.4), 31.88, 29.65, 29.51, 29.30, 26.91, 23.51, 22.67, 14.10 (C.sub.12). Does not migrate under normal TLC conditions.

Acid 2-(9-azido) nonyl-1,3-dioxolane-2-acetic 6b [1421598-02-8]

[0236] ##STR00032## [0237] Prepared by GP4 with a yield of 97%. .sup.1H RMN (300 MHz, CDCl.sub.3): δ8.77 (s, 1H, OH), 4.07 to 3.93 (m, 4H, OCH.sub.2CH.sub.2O), 3.24 (t, J=6.9 Hz, 2H, C (12) H.sub.2), 2.68 (s, 2H, C (2) H.sub.2), 1.95 to 1.82 (m, 2H, C (4) H.sub.2), 1.61 to 1.55 (m, 2H, C (5) H.sub.2), 1.29 (d, J=6.5 Hz, 12H, C (6) H.sub.2 to C (11) H.sub.2). .sup.13C RMN (75 MHz, CDCl.sub.3): δ174.32 (C.sub.1), 109.42 (C.sub.3), 65.21 (acetal), 51.59 (C.sub.12), 42.50, 37.66, 29.68, 29.51, 29.45, 29.20, 28.93, 26.19, 23.56. Does not migrate under normal TLC conditions.

[0238] General Preparation of Ketoamides Using EDC and DMAP with Various Head Group Facilities (GP5) [0239] For the general head groups, the ketal-protected carboxylic acid 6a (1.0 equiv.) was dissolved in DCM (about 7 ml/mmol acid) and were added sequentially: EDC (1.2 eq.), DMAP (1.7 eq.) and the appropriate amino head group (1.3 eq.). [0240] For the (S)-(−)-(α)-amino-(γ) butyrolactone, the ketal-protected acids 6a-b (1.0 equiv.) and the EDC (1.1 equiv.) were dissolved in DCM (about 2 ml/mmol substrate) under argon atmosphere and left under stirring at room temperature for 20 min. Then hydrochloride (S)-(−)-(α)-amino-(γ) butyrolactone (1.3 eq.) and DMAP (1.7 eq.) in DCM (about 2 ml/mmol substrate) were added. The reaction was stirred for 12-22 hours under argon atmosphere at room temperature. [0241] The progress of the reaction was followed by TLC in an EtOAc/cyclohexane mixture and the revelation was obtained with iodine, an UV light, potassium permanganate or bromocresol green, depending on the nature of the head group. Upon completion of the reaction, an additional DCM was added (13 ml/mmol substrate) and the organic phase was washed with HCl 1 M (×3). The phases were separated, the combined organic phases were dried over MgSO.sub.4 and the solvent removed in vacuo to give the desired compound as an oil. If necessary, the product was purified by flash chromatography on a silica column.

[0242] Alternative Procedure Using ECD and Et.sub.3N for the Preparation of Ketoamides from (S)-(−)-(α)-Amino-(γ) Butyrolactone (GP5)

[0243] A solution of (S)-(−)-(α)-amino-(γ) hydrochloride butyrolactone (0.91 eq.) in DCM (approx. 6 ml/mmol substrate) was stirred. Et.sub.3N (1.0 eq.), the protected acid 6a (1.0 eq.) and the EDC (1.37 eq.) were added successively. The reaction mixture was stirred at room temperature for 40 hours. [0244] The reaction was followed by TLC in EtOAc/Cychex 1:2 and revealed with iodine, potassium permanganate and UV light. Upon completion, the mixture was evaporated to dryness under vacuum. The residue was partitioned between water (8 ml/mmol substrate) and EtOAc (17 ml/mmol substrate), and the organic phase was washed successively with a saturated solution of NaHCO3 (×2) and brine (×2). The organic layer was dried over MgSO.sub.4 and evaporated to dryness to give the desired compound as an oil. If necessary, the product could be purified by flash chromatography on a silica column.

(S)-2-(2-nonyl-1,3-dioxolan-2-yl)-N-(2-oxotetrahydrofuran-3-yl) acetamide 7a [182359-61-1]

[0245] ##STR00033##

[0246] Prepared by GP5 and GP5bis with yields of 88% and 91% respectively [0247] .sup.1H RMN (300 MHz, CDCl.sub.3): δ6.99 (d, J=6.3 Hz, 1H, NH), 04.58 (ddd, J=11.6, 8.6, 6.3 Hz, 1H, C.sub.αH), 4.46 (td, J=9.1, 1.3 Hz, 1H, H.sub.A), 4.27 (ddd, J=11.2, 9.1, 5.9 Hz, 1H, H.sub.B), 4.11 to 3.96 (m, 4H, OCH.sub.2CH.sub.2O), 2.80 (dddd, J=12.5, 8.7, 5.9, 1.3 Hz, 1H, H.sub.D), 2.64 (s, 2H, C (2)H.sub.2), 2.13 (dtd, J=12.5, 11.3, 8.8 Hz, 1H, H.sub.C), 1.71-1.65 (m, 2H, C (4)H.sub.2), 1.39-1.33 (m, 2H, C (5) H.sub.2), 1.27 (d, J=7.0 Hz, 12H, C (6) H.sub.2 to C (11) H.sub.2), 0.91-0.84 (m, 3H, C (12) H.sub.3). .sup.13C RMN (75 MHz, CDCl.sub.3): δ175.35 (C.sub.1), 169.95 (ester), 109.76 (C.sub.3), 66.06 (CH.sub.AH.sub.B), 65.25 (CH.sub.2 acetal), 65.12 (CH.sub.2 acetal), 49.11 (CH), 44.33, 37.66, 32.02, 30.53 (CH.sub.C H.sub.D), 29.82, 29.67, 29.65, 29.45, 29.85, 22.82, 14.26 (C.sub.12). R.sub.f (4:1 EtOAc/Cychex): 0,35. HRMS (ESI): exact mass calculated for C.sub.18H.sub.31NO.sub.5Na ([M+Na].sup.+): 362.2094. Found 364.2095.

12-azido-3-(1,3-dioxolane)-N-((3S)-tetrahydro-2-oxo-3-furanyl) dodecanamide 7b

[0248] ##STR00034## [0249] Prepared by GP5 with a yield of 69%. .sup.1H RMN (300 MHz, CDCl.sub.3: δ.99 (d, J=6.4 Hz, 1H, NH), 4.57 (ddd, J=11.6, 8.7, 6.4 Hz, 1H, C.sub.αH), 4.45 (td, J=9.1, 1.3 Hz, 1H, H.sub.A), 4.26 (ddd, J=11.1, 9.1, 5.9 Hz, 1H, H.sub.B), 4.08-3.95 (m, 4H, OCH.sub.2CH.sub.2O), 3.24 (t, J=6.9 Hz, 2H, C (12)H.sub.2), 2.84 to 2.73 (m, 1H, H.sub.D), 2.63 (s, 2H, C (2)H.sub.2), 2.13 (dtd, J=12.5, 11.4, 8.9 Hz, 1H, H.sub.C), 1.71 to 1.65 (m, 2H, C (4)H.sub.2), 1.63 to 1.53 (m, 2H, C (5)H.sub.2), 1:38-1:28 (m, 12H, C (6)H.sub.2 to C (11)H.sub.2). .sup.13C RMN (75 MHz, CDCl.sub.3): δ175.33 (C.sub.1), 169.90 (ester), 109.71 (C.sub.3), 66.03 (CH.sub.AH.sub.B), 65.23 (CH.sub.2 acetal), 65.11 (CH2 acetal), 51.60 (C.sub.12), 49.07 (CH), 44.31, 37.60, 30.41 (CH.sub.CH.sub.D), 29.72, 29.48, 29.45, 29.21, 28.94, 26.80, 23.77. R.sub.f (2:1 EtOAc/cyclohexane): 0.20. HRMS (ESI): exact mass calculated for C.sub.18H.sub.30N.sub.4O.sub.4Na ([M+Na].sup.+): 405.2108. Found: 405.2110.

2-nonyl-N-(3-tetrahydro-2-oxo-3-thienyl)-1,3-dioxalane-2-acetamide 7f

[0250] ##STR00035## [0251] Prepared by modified GP5 with a yield of 53%. .sup.1H RMN (300 MHz, CDCl.sub.3): δ6.89 (d, J=6.6 Hz, 1H, NH), 4.56 (dt, J=13.1, 6.7 Hz, 1H, C.sub.αH), 4.10 to 3.93 (m, 4H, OCH.sub.2CH.sub.2O), 03:34 (td, J=11.7, 5.1 Hz, 1H, H.sub.B), 3.22 (ddd, J=11.4, 7.1, 1.3 Hz, 1H, H.sub.A), 2.86 (dddd, J=12.1, 6.7, 5.1, 1.4 Hz, 1H, H.sub.C), 2.61 (s, 2H, C (2) H.sub.2), 1.92 (dq, J=12.4, 7.0 Hz, 1H, H.sub.D), 1.70-1.62 (m, 2H, C (4) H.sub.2), 1:38-1:32 (m, 2H, C (5) H.sub.2), 1.23 (s, 12H, C (6) H.sub.2 to C (11) H.sub.2), from 0.90 to 0.81 (m, 3H, C (12) H.sub.3). .sup.13C RMN (75 MHz, CDC.sub.3): δ 205.32 (C═O), 169.79 (C.sub.1, 109.75 (C.sub.3), 65.17 (OCH.sub.2CH.sub.2O), 65.03 (OCH.sub.2CH.sub.2O), 59.27 (CH), 44.36 (C.sub.2), 37.59, 31.96, 31.92, 29.77, 29.60, 29.59, 29.39, 27.59, 23.79, 22.76, 14:21 (C.sub.12). R.sub.f (1:1 EtOAc/Cychex): 0.31. HRMS (ESI): exact mass calculated for C.sub.18H.sub.31NO.sub.4SH ([M+H].sup.+): 358.2047. Found: 358.2047.

2-Nonyl-N-(3(S)-tetrahydro-2-oxo-3-thienyl)-1,3-dioxalane-2-acetamide 7g[429675-24-1]

[0252] ##STR00036## [0253] Prepared by GP5bis with a yield of 64%. .sup.1H RMN (300 MHz, CDCl.sub.3): δ 6.89 (d, J=6.6 Hz, 1H, NH), 4.56 (dt, J=13.1, 6.7 Hz, 1H, H), 4.10 to 3.93 (m, 4H, OCH.sub.2CH.sub.2O), 03:34 (td, J=11.7, 5.1 Hz, 1H, H.sub.B), 3.22 (ddd, J=11.4, 7.1, 1.3 Hz, 1H, H.sub.A), 2.86 (dddd, J=12.1, 6.7, 5.1, 1.4 Hz, 1H, H.sub.C), 2.61 (s, 2H, C (2) H.sub.2), 1.92 (dq, J=12.4, 7.0 Hz, 1H, H.sub.D), 1.70 to 1.62 (m, 2H, C (4) H.sub.2), 1.38-1.32 (m, 2H, C (5) H.sub.2), 1.23 (s, 12H, C (6) H.sub.2 to C (11) H.sub.2), 0.90 to 0.81 (m, 3H, C (12) H.sub.3). .sup.13C RMN (75 MHz, CDCl.sub.3): δ205.32 (C═O), 169.79 (C), 109.75 (C.sub.3), 65.17 (OCH.sub.2CH.sub.2O), 65.03 (OCH.sub.2CH.sub.2O), 59.27 (CH), 44.36 (C.sub.2), 37.59, 31.96, 31.92, 29.77, 29.60, 29.59, 29.39, 27.59, 23.79, 22.76, 14:21 (C.sub.12). R.sub.f (1:1 EtOAc/Cychex): 0.31. HRMS (ESI): exact mass calculated for C.sub.18H.sub.31NO.sub.4SH ([M+HA].sup.+): 358.2047. Found: 358.2047.

N-((1S,2S)-2-hydroxycyclohexyl)-2-(2-nonyl-1,3-dioxalan-2-yl) acetamide 7h

[0254] ##STR00037## [0255] Prepared by GP5 with a yield of 54%. .sup.1H RMN (300 MHz, CDCl.sub.3): δ6.47 (d, J=7.5 Hz, 1H, N H), 3.96 (p, J=3.7, 3.1 Hz, 4H), 3.66-3.53 (m, 1H, NHCHC(OH) H), 3.28 (td, J=9.9, 4.3 Hz, 1H, NHC H), 2.63-2.49 (m, 2H, C (2) H.sub.2), 2.01 (ddd, J=11.9, 4.9, 2.5 Hz, 1H, NHCHC(H) H.sub.eq.), 1.89 (dq, J=14.3, 4.6, 3.3 Hz, 1H, NHCHC(H) H.sub.ax), 1.73-1.59 (m, 4H, C (4) H.sub.2 and NHCHCH(OH) CH.sub.2), 1.35-1.27 (m, 2H, C (5) H.sub.2), 1.21 (s, 16H, C (6) H.sub.2 to C (11) H.sub.2 and NHCHCH.sub.2CH.sub.2CH.sub.2CH.sub.2C(OH) H), 0.83 (t, J=6.7 Hz, 3H, C (12) H.sub.3). .sup.13C RMN (75 MHz, CDCl.sub.3): δ170.95 (C.sub.1), 109.89 (C.sub.3), 75.31 (CHOH), 65.04 (ketal), 65.02 (ketal), 55.45 (NHCH), 44.56 (C.sub.2), 37.51, 34.35, 31.92 (C.sub.4), 31.44 (C.sub.5), 29.73, 29.58, 29.55, 29.35, 24.62, 24.62, 24.06, 23.71, 22.72, 14.16 (C.sub.12). R.sub.f (2:1 EtOAc/Cychex)=0.15. MS (ESI): exact mass calculated for C.sub.20H.sub.37NO.sub.4Na ([M+Na].sup.+): 355.52. Found: 378.1.

N-((1S,2R)-2-hydroxycyclohexyl)-2-(2-nonyl-1,3-dioxolan-2-yl) acetamide 7i

[0256] ##STR00038## [0257] Prepared by GP5 with a yield of 51%. .sup.1H RMN (300 MHz, CDCl.sub.3): δ6.70 (d, J=8.1 Hz, 1H, N H), 4:01-3,96 (m, 4H, ketal), 3.95-3.87 (m, 2H, NHCHC(OH) H and NHC H), 2.56 (d, J=2.7 Hz, 2H, C (2) H.sub.2), 2:37 (s, 1H), 1.71-1.57 (m, 7H), 01:38 (ddd, J=14.3, 6.9, 4.0 Hz, 4H), 1.24 (d, J=2.2 Hz, 12H, C (6) H.sub.2 to C (11) H.sub.2), from 0.89 to 0.83 (m, 3H, C (12) H.sub.3). .sup.13C RMN (75 MHz, CDCl.sub.3): δ169.32 (C.sub.1), 110.03 (C.sub.3), 69.52 (CHOH), 65.09 (ketal), 50.78 (NHCH), 44.75 (C.sub.2), 37.52, 31.99, 31.53, 29.83, 29.64, 29.43, 27.37, 23.80, 23.48, 22.79, 20.45, 14.23 (C.sub.12). R.sub.f (3:1 EtOAc/Cychex)=0.26. HRMS (ESI): exact mass calculated for C.sub.20H.sub.37NO.sub.4Na ([M+Na].sup.+): 378.2615. Found: 378.2615.

N-(5-chloro-2-hydroxyphenyl)-2-(2-nonyl-1,3-dioxolan-2-yl) acetamide 7m

[0258] ##STR00039## [0259] Prepared by GP5 with a yield of 37%. .sup.1H RMN (300 MHz, CDCl.sub.3): δ 8.78 (s, 1H, OH), 8.67 (s, 1H, NH), 7.07 (dd, J=8.7, 12.5 Hz, 1H, aromatic), 6.98 (d, J=2.5 Hz, 1H, aromatic), 6.94 (d, J=8.7 Hz, 1H, aromatic), 4.07 (s, 4H, OCH.sub.2CH.sub.2O), 2.80 (s, 2H, C (2) H.sub.2), 1.75-1.69 (m, 2H, C (4) H.sub.2), 1.43-1.36 (m, 2H, C (5) H.sub.2), 1.28-1.25 (m, 12H, C (6) H.sub.2 to C (11) H.sub.2), 0.90-0.85 (m, 3H, C (12) H.sub.3). .sup.13C RMN (75 MHz, CDCl.sub.3): δ169.75 (C.sub.1), 147.68 (C OH), 127.04 (NH C), 126.73 (CCl), 124.94 (aromatic), 121.94 (aromatic), 121.17 (aromatic) 109.77 (C.sub.3), 65.29 (acetal), 44.67 (C.sub.2), 37.65, 32.01, 29.73, 29.62, 29.42, 23.80, 22.81, 14.25 (C.sub.12). R.sub.f(1:4 EtOAc/Cychex): 0.14. HRMS (ESI): exact mass calculated for C.sub.20H.sub.30ClNO.sub.4H ([M+H.sup.+]): 384.1936. Found: 384.1936.

[0260] Procedure for the Synthesis of the Terminal Azido-Carboxylic Acid [0261] The appropriate bromocarboxylic acid (1.0 equivalent) and the sodium azide (1.5 equivalents) were dissolved in DMF (5 ml/mmol carboxylic acid). The reaction mixture was heated to 60° C. and stirred overnight under argon atmosphere. After completion, the solvent was removed under reduced pressure. The resulting oil was dissolved in 1:1:1 (v/v/v) mixture of EtOAc, H.sub.2O and brine. The organic phases are extracted with EtOAc (×3) and washed with semi-saturated brine (×2) before drying over MgSO.sub.4. The solvents were removed under vacuum to give the product as an oil.

##STR00040##

Acid 10-Azidodecanoic 14 [186788-32-9]

[0262] ##STR00041## [0263] Prepared with a quantitative yield. .sup.1H RMN (300 MHz, CDCl.sub.3): δ 3.24 (t, J=6.9 Hz, 2H, C (10) H.sub.2), 2:33 (t, J=7.5 Hz, 2H, C (2) H.sub.2), 1.60 (dt, J=10.1, 6.9 Hz, 4H, C (3) H.sub.2 and C (9) H.sub.2), 1.30 (m, 10H, C (4) H.sub.2 to C (8) H.sub.2). .sup.13C RMN (75 MHz, CDCl.sub.3): δ180.31 (acid), 51.59 (C.sub.10), 34.18 (C.sub.2), 29.36, 29.23, 29.19, 29.11, 28.94, 26.80, 24.76.

[0264] General Procedure for Removing the Acetal Protecting Group (GP6) [0265] The AHL precursor or a protected analogue (1.0 equiv.) was dissolved in TFA (4 ml/mmol substrate) and water (1 ml/mmol substrate). DCM could be added if necessary to improve the solubility (up to 10 ml/mmol substrate). The reaction mixture was stirred at room temperature under argon atmosphere until a TLC analysis in EtOAc/CycHex indicate a complete consumption of the starting reagent (overnight). The reaction was stopped by adding a saturated NaHCO.sub.3 solution and NaHCO3.sub.(s) until the pH was stabilized at 4-5, and the organic phase was extracted with DCM (×3). The combined organic phases were dried over MgSO.sub.4 and the solvent removed under vacuum. If necessary, the product could be purified by flash chromatography on a silica column.

N-(3(S)-oxododecanoyl) homoserine lactone 1 (=15a) [168982-69-2]

[0266] ##STR00042## [0267] Prepared by GP6 with a yield of 98%. .sup.1H RMN (300 MHz, CDCl.sub.3): δ 7.68 (d, J=6.6 Hz, 1H, N H), 4.59 (ddd, J=11.5, 8.7, 6.6 Hz, 1H, C.sub.αH), 4.48 (dt, J=9.1, 1.4 Hz, 1H, H.sub.A), 4.27 (ddd, J=11.1, 9.1, 6.0 Hz, 1H, H.sub.B), 3.47 (s, 2H, C (2) H.sub.2), 2.76 (dddd, J=12.6, 8.8, 6.0, 1.4 Hz, 1H, H.sub.C), 2.52 (t, J=7.3 Hz, 2H, C (4) H.sub.2), 2:30-2:15 (m, 1H, HD), 01.57 (d, J=6.9 Hz, 2H, C (5) H.sub.2), 1.26 (t, J=3.1 Hz, 12H, C (6) H.sub.2 to C (11) H.sub.2), 0.90-0.85 (m, 3H, C (12) H.sub.3). .sup.13C RMN (75 MHz, CDCl.sub.3): δ206.77 (C.sub.3), 174.85 (ester), 166.44 (C.sub.1), 65.99 (CH.sub.AH.sub.B), 49.19 (C.sub.α), 48.12, 44.12, 31.99, 30.04, 29.52, 29.47, 29.38, 29.13, 23.51, 22.80, 14.24 (C.sub.12). R.sub.f(2:1 EtOAc/Cychex): 0.35. HRMS (ESI): exact mass calculated for C.sub.16H.sub.27NO.sub.4Na ([M+Na].sup.+): 320.1832. Found: 320.1834.

12-azido-3-oxo N-((3S)-tetrahydro-2-oxo-3-furanyl) dodecanamide 15b [1175052-13-7]

[0268] ##STR00043## [0269] Prepared by GP6 with a yield of 87%. .sup.1H RMN (300 MHz, CDCl.sub.3): δ 7.61 (d, J=4.9 Hz, 1H, N H), 4.58 (ddd, J=11.5, 8.7, 6.5 Hz, 1H, C.sub.αH), 4.47 (dt, J=9.1, 1.5 Hz, 1H, H.sub.A), 4.27 (ddd, J=11.0, 9.3, 6.1 Hz, 1H, H.sub.B), 3.46 (s, 2H, C (2) H.sub.2), 3.25 (t, J=6.9 Hz, 2H, C (12) H.sub.2), 2.76 (dddd, J=12.6, 8.7, 6.0, 1.5 Hz, 1H, H.sub.D), 2.52 (t, J=7.3 Hz, 2H, C (4) H.sub.2), 2.22 (dtd, J=12.5, 11.2, 8.9 Hz, 1H, H.sub.C), 1.59 (t, J=6.6 Hz, 2H, C (5) H.sub.2), 1:37-1:27 (m, 12H, C (6) H.sub.2 to C (11) H.sub.2). .sup.13C RMN (75 MHz, CDCl.sub.3): δ 210.56 (C.sub.3), 168.45 (ester), 165.97 (C.sub.1), 65.99 (CH.sub.AH.sub.B), 51.61 (C.sub.12), 49.21, 48.19, 44.06, 30.06 (CH.sub.CH.sub.D), 29.35, 29.33, 29.18, 29.06, 28.96, 26.81, 23.45. HRMS (ESI): exact mass calculated for C.sub.16H.sub.26N.sub.4O.sub.4Na ([M+Na].sup.+): 361.1846. Found: 361.1848.

3-oxo-N-(tetrahydro-2-oxo-3-thienyl)-dodecanamide 15f [663883-93-0]

[0270] ##STR00044## [0271] Prepared by GP6 with quantitative yield. .sup.1H RMN (300 MHz, CDCl.sub.3): δ7.47 (s, 1H, N H), 4.58 (dt, J=13.2, 6.8 Hz, 1H, C.sub.αH), 3.45 (s, 2H, C (2) H.sub.2), 3.40-3.22 (m, 2H, CH.sub.AH.sub.B), 2.86 (dddd, J=12.2, 6.7, 5.1, 1.5 Hz, 1H, H.sub.D), 2.52 (t, J=7.4 Hz, 2H, C (4) H.sub.2), 2.01 (dq, J=12.4, 7.1 Hz, 1H, H.sub.C) 1.62-1.55 (m, 2H, C (5) H.sub.2), 1.26 (t, J=2.8 Hz, 12H, C (6) H.sub.2 to C (11) H.sub.2), 0.91-0.84 (m, 3H, C (12) H.sub.3). .sup.13C RMN (75 MHz, CDCl.sub.3): δ 206.76 (C.sub.3), 204.67 (thioester), 166.39 (C.sub.1), 59.40 (C.sub.α), 48.34, 44.09, 31.99, 31.66, 29.52, 29.47, 29.38, 29.13, 27.62, 23.50, 22.80, 14.24 (C.sub.12). HRMS (ESI): exact mass calculated for C.sub.16H.sub.27NO.sub.3Na ([M+Na].sup.+): 336.1604. Found 336.1605.

3-oxo N-[(3S)-tetrahydro-2-oxo-3-thienyl]-dodecanamide 15g [177158-29-1]

[0272] ##STR00045## [0273] Prepared by GP6 with quantitative yield. .sup.1H RMN (300 MHz, CDCl.sub.3): δ7.48 (d, J=6.6 Hz, 1H, N H), 4.58 (dt, J=13.3, 6.6 Hz, 1H, C.sub.αH), 3.45 (s, 2H, C (2) H.sub.2), 3.35 (td, J=11.5, 5.1 Hz, 1H, CH.sub.A), 3.25 (ddd, J=11.5, 7.1, 1.5 Hz, 1H, CH.sub.B), 2.84 (dddd, J=12.4, 6.7, 5.1, 1.5 Hz, 1H, H.sub.D), 2.52 (t, J=7.3 Hz, 2H, C (4) H.sub.2), 2.01 (dq, J=12.4, 7.1 Hz, 1H, H.sub.C) 1.57 (t, J=7.3 Hz, 2H, C (5) H.sub.2), 1.25 (t, J=3.1 Hz, 12H, C (6) H.sub.2 to C (11) H.sub.2), 0.91-0.82 (m, 3H, C (12) H.sub.3). .sup.13C RMN (75 MHz, CDCl.sub.3): δ206.72 (C.sub.3), 204.71 (thioester), 166.36 (C.sub.1), 59.36 (C.sub.α), 48.41 (CS), 44.06 (C.sub.2), 31.98, 31.64, 29.51, 29.47, 29.37, 29.12, 27.61, 23.48, 22.79, 14.24 (C.sub.12). HRMS (ESI): exact mass calculated for C.sub.16H.sub.27NO.sub.3SH ([M+H].sup.+): 314.1784. Found: 314.1785.

N-[(1S,2S)-2-hydroxycyclohexyl]-3-oxo-dodecanamide 15h [886755-19-7]

[0274] ##STR00046## [0275] Prepared by GP6 with a yield of 54%. .sup.1H RMN (300 MHz, CDCl.sub.3): δ7.17 (d, J=7.4 Hz, 1H, N H), 3.65 (dddd, J=11.2, 9.1, 7.4, 4.3 Hz, 1H, NHCHC(OH) H.sub.ax), 3.41 (s, 2H, C (2) H.sub.2), 3:38-3:27 (m, 1H, NHC H), 2.51 (t, J=7.3 Hz, 2H, C (4) H.sub.2), 2.9 to 1.99 (m, 1H, NHCHC(H) H.sub.eq), 1.95 (tdd, J=7.4, 3.9, 2.3 Hz, 1H, NHCHC(H) H.sub.ax), 1.71 (ddt, J=8.8, 5.6, 2.7 Hz, 2H NHCHCH(OH) CH.sub.2), 1.56 (p, J=6.9 Hz, 2H, C (5) H.sub.2), 1.36-1.16 (m, 16H, C (6) H.sub.2 to C (11) H.sub.2 and NHCHCH.sub.2CH.sub.2CH.sub.2CH.sub.2C(OH) H, 0.90-0.81 (m, 3H, C (12) H.sub.3). .sup.13C RMN (75 MHz, CDCl.sub.3): δ207.55 (C.sub.3), 167.37 (C.sub.1), 75.24 (CHOH), 55.88 (NHCH), 48.60, 44.08 (C.sub.2), 34.39, 31.97, 31.38, 29.50, 29.46, 29.36, 29.11, 24.67, 24.10, 23.48, 22.78, 14.22 (C.sub.12). HRMS (ESI): exact mass calculated for C.sub.18H.sub.33NO.sub.3Na ([M+Na].sup.+): 334.2353. Found 334.2353.

N-[(1S,2R)-2-hydroxycyclohexyl]-3-oxo-dodecanamide 15i [897031-37-7]

[0276] ##STR00047## [0277] Prepared by GP6 with a yield of 67%. .sup.1H RMN (300 MHz, CDCl.sub.3): δ7.25-7.14 (m, 1H, N H), 3.98 (ddd, J=8.2, 6.2, 2.7 Hz, 1H, NHC H), 3.92 (dt, J=5.8, 2.6 Hz, 1H, NHCHC(OH) H.sub.eq), 3.40 (d, J=1.5 Hz, 2H, C (2) H.sub.2), 2.52 (t, J=7.3 Hz, 2H, C (4) H.sub.2), 2.20-2.08 (m, 2H), 1.68-1.53 (m, 6H), 1.42 (ttd, J=11.5, 5.5, 5.0, 2.8 Hz, 2H), 1.26 (d, J=3.5 Hz, 12H, C(6)H.sub.2 to C(11)H.sub.2), 0.91-0.83 (m, 3H, C (12) H.sub.3). .sup.13C RMN (75 MHz, CDCl.sub.3): δ 207.31 (C.sub.3), 165.84 (C.sub.1), 69.38 (COH), 51.23 (NHCH), 49.17, 44.07, 31.99, 31.51, 29.52, 29.48, 29.38, 29.15, 27.32, 23.52, 23.44, 22.79, 20.43, 14.23 (C.sub.12). R.sub.f (3:1 EtOAc/CycHex)=0.38. HRMS (ESI): exact mass calculated for C.sub.18H.sub.33NO.sub.3H ([M+H].sup.+): 312.2533. Found: 312.2534.

N-(5-chloro-2-hydroxyphenyl)-3-oxododecanamide 15 m [663883-68-9]

[0278] ##STR00048## [0279] Prepared by GP6 with a yield of 98%. .sup.1H RMN (300 MHz, MeOD-d.sub.4): δ8:01 (d, J=2.5 Hz, 1H, aromatic H), 6.94 (dd, J=8.6, 2.6 Hz, 1H, aromatic H), 6.84 to 6.76 (m, 1H, aromatic H), 3.64 (dd, J=5.5, 3.3 Hz, 1H, C (2) H-form-enol), 2.60 (t, J=7.3 Hz, 2H, C (4) H.sub.2), 1.59 (p, J=7.2 Hz, 2H, C (5) H.sub.2), 1.34-1.26 (m, 12H, C (6) H.sub.2 to C (11) H.sub.2), 0.92 to 0.87 (m, 3H, C (12) H.sub.3). .sup.13C RMN (75 MHz, MeOD-d.sub.4): δ 207.41 (C.sub.3), 167.64 (C.sub.1), 147.59 (COH), 128.30 (aromatic C), 125.51 (aromatic C H), 124.91 (aromatic C), 122.45 (aromatic C H), 116.95 (aromatic C H), 43.99 (C.sub.2), 33.03 (C.sub.4), 30.57, 30.53, 30.40, 30.13, 24.45, 23.72, 14.43 (C.sub.12). HRMS (ESI): exact mass calculated for C.sub.18H.sub.26ClNO.sub.3H ([M+H].sup.+): 340.1654. Found 340.1663.

[0280] 3. Anti-Inflammatory Effect of the Compounds of the Invention

[0281] In order to evaluate the properties of compounds of interest, a murine macrophage line, the RAW264.7, was used.

[0282] To assess the effect on the inflammation, the cells were treated or not with the addition of a proinflammatory cocktail (interferon-γ (IFN-γ, 20 U/mL) and lipopolysaccharide (LPS, 10 ng/mL)). The inflammatory state was assessed by Multiplex analysis by dosing the secretion of 23 cytokines in the supernatant of the cells. The heatmap results are shown in FIG. 15, which shows the secretion of the cytokines by RAW264.7 under stimulated conditions (LPS 10 ng/mL; IFN-γ 20 U/mL) (normalized in relation to the control).

[0283] These results allow to visualize globally a decrease of the production of the cytokines in the presence of 50 μM PCA. The cytokines whose production was modulated by the compounds of the invention are interleukin-13 (IL-1p), IL-2, IL-6, IL-12, RANTES, TNFα, pro-inflammatory cytokines. The decrease in these proteins was dose-dependent, and the largest effects were observed in the presence of 50 μM PCA.

[0284] To illustrate this, 2 histograms (the first with the 3oxoC.sub.12:2; the 2.sup.nd with the PCA) showing the results observed for the TNF alpha (results from the Multiplex analysis) are shown in FIGS. 16A and 16B (at the top: TNF∝ secreted by RAW264.7 stimulated by LPS and interferon-γ in the presence of 3oC.sub.12:2 and at the bottom: TNF∝ secreted by RAW264.7 stimulated by LPS and interferonγ—in the presence of PCA).

[0285] The confirmation of some gene expression results was performed by measuring the messenger RNA by quantitative PCR, in particular for 3 cytokines of interest: Rantes, TNF aplha, IL1-beta. The results are shown in the diagrams shown in FIGS. 17A-17C.

[0286] Thus, we observe an anti-inflammatory effect of the compounds on the cells of the macrophage type both at the protein level and in mRNA expression.

[0287] 4 Measurement of the Toxicity of the Compounds of the Invention

[0288] 4.1 Measurement of the Toxicity of the Compounds on Eukaryotic Cells

[0289] The cytotoxicity of the tested and control compounds was evaluated using a measurement test of the LDH (Lactate Dehydrogenase) secretion. This test is based on the measurement of the amount of LDH secreted by the cells in their supernatant, compared to the amount of LDH that will remain in the intracellular compartment. This ratio provides an indication of the membrane damage suffered by the cells, and thus of the cytotoxicity of the compound. A compound is considered toxic when the ratio is higher than 10%.

[0290] Two methods were used to perform this test: the measurement using an extemporaneously prepared pyruvate/NADH solution, or using the Cytotoxicity Detection Kit.sup.PLUS(LDH) from the manufacturer Roche (Sigma-Aldrich).

[0291] Pyruvate/NADH method: pyruvate/NADH solution prepared with 4.1 mg pyruvic acid (0.62 mM) and 7.7 mg NADH (0.18 mM) in 60 ml of 0.1M PBS (pH 7.4).

[0292] For the measurement of the LDH concentration in the supernatants, 800 μL of NADH was added to 200 μL of supernatant in plastic spectrometry cuvettes, and the decrease in absorbance of the final solution was read at 340 nm for 1 min. For the measurement of the LDH concentration in the cell lysates, 800 μL of NADH was added to 10 μL of cell lysates and 190 μL of 0.1M PBS in plastic spectrometry cuvettes, and the decrease in absorbance of the final solution was read at 340 nm for 1 min. The percentage of LDH secreted was then calculated by the ratio of the decay slopes of the absorbance of the supernatants and of the cell lysates (respectively).

[0293] Cytotoxicity Detection Kit.sup.PLUS (LDH) method: absorbance test performed according to the manufacturer's instructions. The percentage of LDH secreted was then calculated by the formula:

[00002]$\begin{array}{cc}{\%}_{\mathrm{LDH}}=\frac{{\mathrm{DO}}_{\mathrm{sample}}-{\mathrm{DO}}_{\mathrm{basal}\mathrm{control}}}{{\mathrm{DO}}_{\mathrm{control}\mathrm{activated}}-{\mathrm{DO}}_{\mathrm{basal}\mathrm{control}}}*100& \left[\mathrm{Math}2\right]\end{array}$

[0294] 4.2 Method for Measuring the Toxicity of the Compounds on Bacterial Line

[0295] The E. coli K12 strain was cultured at D0 on agar gel, and then a selected colony was transferred to a liquid bacterial culture LYBHI medium at D1. At day 2, this colony was diluted 1:100 in LYBHI medium, and maintained for 18 hours for expansion before distribution in an opaque 96-well plate. In each well were distributed: LYBHI medium, bacterial culture, and test or control compounds. The absorbance of the wells was read at 600 nm at t0 and t18h. The raw absorbance values were corrected against the absorbance of the wells containing only LYBHI medium without bacteria or compounds.

[0296] 4.3 Results on Eukaryotic Cells

[0297] a. Natural AHL 3oxoC.sub.12-HSL and 3oxoC.sub.12:2-HSL

[0298] FIGS. 18A-18B show that in the Caco-2/TC7 cell line, the 2 molecules are well tolerated in the concentration range 1-100 μM, in the presence and absence of pro-inflammatory cytokines:

[0299] the measured cytotoxicity does not exceed 2.5% for 3oxoC.sub.12-HSL and 1.5% for 3oxoC.sub.12:2-HSL. In comparison, the toxicity of the activated control (DMSO 0.1% and cytokines) is about 4%.

[0300] FIGS. 19A-19D show that in the Raw264.7 murine cell line, an increase in secreted LDH was observed as early as 50 μM for both AHL, with a cytotoxicity greater than 10% at the 100 μM concentration. This phenomenon is observed in basal and stimulated conditions, and is therefore attributable to a toxicity of the molecules. This justifies the use of the 1-50 μM doses in the rest of the study on the macrophage line.

[0301] b. (D/L)-3oxoC.sub.12-HTL (Formula I-1) and (S)-3oxoC.sub.12-HTL (Formula I-3)

[0302] As can be seen in FIGS. 20A-20B, neither of these two thiolactone-headed compounds showed toxicity on the two cell lines, with LDH secretions below 10% even at the highest concentrations.

[0303] c. (S,S)-3oxoC.sub.12-ACH (Formula (I-2) and (R,S)-3oxoC.sub.12-ACH (Formula (I-4)

[0304] Both compounds are not cytotoxic at low concentrations as shown in FIGS. 21A-21B, but the (R,S)-3oxoC.sub.12-ACH molecule shows an increase in LDH secretion at the higher concentrations (≥50 μM). On the Caco-2/TC7 epithelium cells, without exceeding the 10% mark, we observe a secretion of 9% at 100 μM. This effect is not as significant for the (S,S)-3oxoC.sub.12-ACH diastereomer.

[0305] This observation is found in the Raw264.7 macrophage line, where consistently, the LDH secreted in the presence of (R,S)-3oxoC.sub.12-ACH is greater than that secreted in the presence of (S,S)-3oxoC.sub.12-ACH, at all concentrations. The (R,S)-3oxoC.sub.12-ACH molecule is also toxic at 50 μM.

[0306] d. 3oxoC.sub.12-Aminochlorophenol (Formula I-6)

[0307] The molecule is not toxic to either line at any of the concentrations tested as shown in FIGS. 22A-22B.

[0308] 4.4 Results on Bacterial Strain

[0309] a. Natural AHL 3oxoC.sub.12-HSL and 3oxoC12:2-HSL

[0310] The 3oxoC.sub.12-HSL and 3oxoC.sub.12:2-HSL AHL were tested for bactericidal effects in the 1-100 μM range. No toxic effect was observed after 18 h of incubation: the absorbances recorded were identical to those for the LYBHI medium alone and in the presence of 0.1% DMOS: see FIGS. 23A-23B.

[0311] 4.5 Results on Bacterial Strain

[0312] a. (D/L)-3oxoC12-HTL (Formula I-1), (S)-3oxoC12-HTL (Formula I-3), (S,S)-3oxoC12-ACH (Formula I-2), (R,S)-3oxoC12-ACH (Formula I-4) and 3oxoC12-Aminochlorophenol (Formula I-6) Analogues

[0313] No significant differences were observed among all the concentrations and all the molecules tested, so only the maximum concentration (100 μM) is shown in FIG. 24.

[0314] In general, no compound, natural or synthetic, is bactericidal on the E. coli K12 strain.