Patent Description:
Receptors of immune system cells recognize a large variety of microbial pathogens, such as viruses, bacteria, fungi and parasites, via recognition of pathogen-associated molecular patterns (PAMPs) present on pathogens' surface.

Such receptors are known as pattern recognition receptors (PRRs) and may be of various typologies, depending on their localization in-cell, in cytosol or on the membrane, and on their function.

Toll-like receptors (TLRs) constitute the most well-known and studied family of innate immunity receptors, and have the role of promoting a rapid response to pathological menaces and aiding the development of adaptive immune response, which is the most appropriate and specific defense for such menaces.

In fact, innate immunity response to pathogens can be decisive in determining both the nature and the intensity of adaptive immunity response.

It is for that reason that TLR activators (agonists) have been studied and developed for the treatment of cancer, allergies and infective diseases, comprising adjuvants in prophylactic and therapeutic vaccines.

To date, there are various small molecules able to bind and activate TLR receptors, and some of those are in use as adjuvants: imidazoquinoline TLR7/<NUM> agonists, like imiquimod and resiquimod, as well as Pam2CS-type TLR2/TLR6 agonist and TLR4 agonists such as monophosphoryl lipid A (MPL) and aminoalkyl glucosaminide-<NUM>-phosphates (AGPs, also referred to as Corixa compounds, CRX).

TLR4 receptor is a highly interesting target for the development of immunostimulants and vaccine adjuvants, as TLR4 stimulation by agonists is the most effective way to activate innate and adaptive immunities.

The natural agonist of TLR4 is lipopolysaccharide (LPS), the main component of the outer membrane of Gram-negative bacteria. Lipid A is the immunologically active portion of LPS.

Lipid A agonistic activity is based on its binding affinity (ability to bind) to the TLR4 co-receptor, Myeloid Differentiation factor <NUM>, MD-<NUM>, with the entailed formation of the (TLR4/MD-<NUM>/LPS)<NUM> complex on the surface of innate immunity cells, i.e. macrophages and dendritic cells.

The activation process of the TLR4 receptor by LPS begins with the interaction of individual LPS molecules or aggregates in solution with Lipid Binding Protein (LBP), forming a complex with an LPS molecule. Thereafter, the LPS molecule is transferred from LBP to co-receptor CD14, which in turn transfers it from MD-<NUM>.

Natural endotoxins of bacteria, like LPS, lipooligosaccharides (LOS) and lipid A, are excessively toxic to be used as adjuvants.

Therefore, synthetic and natural proteins with a structure similar to lipid A, but with attenuated endotoxicity, are interesting candidates as vaccine adjuvants in the perspective of maintaining immunostimulatory activity while eliminating the toxic effects. Monophosphoryl lipid A (MPL) is a molecule identical to lipid A, but with the C<NUM> position stripped of the phosphate group through chemical modification. MPL has ~<NUM>% of the inflammatory toxicity of the parent molecule, LPS, and is used as adjuvant in a series of vaccines.

However, the MPL adjuvant used nowadays is chemically heterogeneous, as produced directly from natural LPS. The synthetic compounds named AGPs (also known as CRX adjuvants, Corixa) are comprised of a monosaccharide unit linked by glycosidation to a unit of an aminoalkyl aglycone N-acylate.

AGPs are potent agonists of TLR4 and are chemically homogeneous, as produced by chemical synthesis.

A further simplification of the structure of Lipid A still able to activate TLR4 is comprised of the monophosphorylated monosaccharide derivatives mimicking the reducing portion or the non-reducing portion of Lipid A (scheme below).

Compounds GLA <NUM> and GLA60 (scheme above) are comprised of a glucopyranoside skeleton, phosphorylated in position C<NUM> and with a <NUM>-Carbon linear chain in C<NUM> and a branched chain in C<NUM> (<NUM> + <NUM> in GLA <NUM> or <NUM> + <NUM> Carbons in GLA <NUM>) (<NPL>). These monosaccharides that partially mimic lipid A and mimic the monosaccharide lipid X, biosynthetic precursor of lipid A, are active in stimulating TLR4-dependent production of cytokines TNF-α and IL-<NUM>, in both murine and human cells.

Also compound SDZ MRL <NUM> demonstrated a potent activity in stimulating the release of inflammatory cytokines like interleukin-<NUM> (IL-<NUM>), interleukin-<NUM> (IL-<NUM>) and TNF-α factor in murine macrophages and neutrophil granulocytes, concomitantly exhibiting a toxicity reduced of a factor of at least <NUM><NUM> in galactosamine-sensitized mice compared to the parent endotoxin (Salmonella abortus equi).

In experimental microbial infection models, the compound proved to have a highly protective effect when administered prophylactically either once or thrice in myelo-suppressed or immunocompetent mice.

Doses effective to reach <NUM>% of response with SDZ MRL <NUM> vary depending on the infective agent and administration route. In all cases, however, EC<NUM> obtained are about <NUM><NUM> times greater than those obtained with endotoxin Salmonella abortus equi.

However, thanks to the very low toxicity, the therapeutic indexes of this molecule, expressed, e.g., as LD<NUM>/ED<NUM> were significantly improved compared to the endotoxin and range from about <NUM> to ><NUM>, depending on the infective agent and the administration route.

The compound also proved efficient in inducing tolerance to endotoxins: repeated dosages of the compound induce a transient resistance (≥<NUM> week) to endotoxin-related lethal risks.

All these positive results were also confirmed in a model of advanced sepsis caused by Escherichia coli, in which antibiotic therapy had already proved inefficient: pre-treatment with one dose of SDZ MRL <NUM> one day prior to microbial inoculation dramatically increased the curative effects of antibiotics administered. For this reason, long-term survival was significantly increased with incremental doses of the immunostimulant in combined therapy.

Precisely for the tolerability demonstrated by SDZ MRL <NUM> in animals and in vitro, this compound was subsequently tested in human models.

On the basis of the known anti-tumour activity of Salmonella abortus equi endotoxin linked to its immunostimulating properties, Kiani et al. (<NPL>) conducted a randomized double-blind phase I trial with control medium, administering SDZ MRL <NUM> in tumour-affected patients in order to assess firstly its biological effects and its safety of administration in humans and, secondly, its influence on the reaction to a subsequent endotoxin (LPS) addition.

SDZ MRL <NUM> administration proved safe and of excellent tolerability. The same SDZ MRL <NUM> increases granulocyte counts and serum levels of G-CSF and interleukin-<NUM> (IL-<NUM>), but not of pro-inflammatory cytokines TNF-α, IL-1b, and IL-<NUM>.

Therefore, SDZ MRL <NUM> has three relevant features, i.e., <NUM>) a high tolerability and low toxicity, <NUM>) the ability to induce G-CSF production, and, as a result, <NUM>) the ability to stimulate an aspecific immune resistance expressed by an increased group of primary defenses in cells.

In spite of these positive results encouraging in the clinical use of SDZ MRL <NUM>, the action mechanism of this molecule has not yet been studied in molecular detail.

In vitro studies suggest that the compound acts independently of co-receptor CD14, as by using monoclonal antibodies for CD-<NUM> the compound-induced TNF-α release in human peripheral blood cells is not blocked.

This in turn seems to be indicative of a direct interaction with compound MD-<NUM>/TLR4, and might explain its different cytokine induction profile.

The synthesis of compound SDZ MRL <NUM> is made complex by the fact that the glucosamine core binds, in positions C<NUM>, C<NUM> and C<NUM>, chains of <NUM>(R)-hydroxymyristic acid as pure enantiomer. <NUM>-hydroxymyristic acid is in fact commercially available as racemate, whereas the pure enantiomer <NUM>(R)- hydroxymyristic acid needs to be first isolated, to then be used in the synthesis.

<CIT> discloses compounds having formula I
<CHM>
wherein R1, R2 and R3 independently are optionally substituted acyl, and describes a process for the preparation of the compound of formula I, and its use as pharmaceuticals, in particular for use as immunomodulators, as antiviral agents and as anti-inflammatory agents, and for use in the treatment of allergies.

The Authors of the present invention have surprisingly found that compounds of formula <NUM>
<CHM>.

said compounds are particularly effective in activating the TLR4 receptor and require synthesis processes that are less complex compared to compounds of the known art. In fact, the present invention enabled to identify compounds requiring simpler synthetic pathways, that are equally effective, or even more effective compared to those present in the known art.

Therefore, object of the invention are compounds of formula <NUM> as TLR4 receptor agonists, and their use as active principle or as adjuvant in the treatment of diseases requiring a TLR4 receptor activation, as well as drug or vaccine compositions comprising said compound.

HEK-Blue™ hTLR4 (A) and HEK-Blue™ Null2 (B) cells were treated with the indicated concentrations of compounds FP11, FP111, and with LPS (<NUM> ng/mL) and incubated for <NUM>-<NUM> hours. Results were normalized to stimulation with LPS alone, and are expressed as mean of percentage ± ES. of at least three independent experiments.

HEK-Blue™ hTLR4 cells were treated with increasing concentrations of compound FP11 and of LPS and incubated for <NUM>-<NUM> hours. Results were normalized to maximum activation of the reporter produced by LPS (A) and to maximum activation of the reporter produced by the compound itself (B). Data were fitted to a sigmoidal four-parameter logistic equation in order to determine IC<NUM> values, and expressed as mean of percentage ± ES of at least three independent experiments.

HEK-Blue™ hTLR4 cells were treated with the higher concentrations used in the preceding assays and incubated for <NUM>-<NUM> hours. Results were normalized to PBS addition and are expressed as mean of percentage ± ES. of at least three independent experiments.

(A-B) Fluorescent measurements demonstrate that FP11, but not FP111, inhibits the binding of bis-ANS to MD-<NUM> in a dose-dependent manner; (C-D) FP11, but not FP111, displaces biotinylated LPS from MD-<NUM> hydrophobic pocket in a dose-dependent manner; (E-F) FP11, but not FP111, prevents the binding of monoclonal antibody to MD-<NUM> in a dose-dependent manner; (G-H) SPR experiments indicate that FP11, but not FP111, directly binds MD-<NUM> with a KD value of <NUM>.

To the ends of the present description, the term "TLR4 receptor agonist" denotes a compound that selectively binds to the TLR4 receptor inducing a conformational change of said receptor, in turn generating an intracellular stimulation by triggering a response similar to that induced by the natural ligand of said receptor. In the case of TLR4, the substances described as agonists bind to co-receptor MD-<NUM>, in turn non-covalently bound to TLR4, thereby generating the receptorial complex (TLR4/MD-<NUM>/agonist)<NUM>, which from the cell surface initiates a signal cascade leading to activation of nuclear transcription factors and synthesis of pro-inflammatory cytokines (mainly TNF-α and various interleukin types).

The present invention relates to a compound of formula <NUM>
<CHM>.

Therefore, according to the present description, each aliphatic chain, R<NUM>, R<NUM>, or R<NUM>, can have a length of C<NUM>-C<NUM>.

According to the description, R<NUM>, R<NUM>, and R<NUM> can be different from each other or alike to each other.

In one embodiment of the invention, R<NUM>, R<NUM>, and R<NUM> are alike to each other.

According to some possible non-limiting embodiments, the compound of formula <NUM> could be selected from the following ones:.

In one preferred embodiment, the compound of the invention is the compound having formula
<CHM>.

In the present description, such a compound is also referred to as compound FP11.

Data reported in the Examples section show the peculiar advantageous features of the above-indicated compounds FP.

According to the present description, and on the basis of experimental data obtained, it is evident that the compounds as described and claimed are effective agonists of TLR4 receptor. By "agonist of a receptor" (receptor agonist) it is meant as is commonly defined in the literature, i.e., a substance able to bind a specific receptor in the binding site for the endogenous ligand. Therefore, as the name suggests, the former competes with the latter for the binding with said site.

Following binding with the natural ligand, the receptor encounters conformational changes that mediate its biological activity at cell level. Agonists are molecules having inherent activity able to mimic ligand effects. When binding to the receptor, they cause conformational changes of an extent similar to those caused by binding with the endogenous ligand.

In the case of the present description, the agonist is an agonist selective for the TLR4 receptor.

Preliminary data (not shown) obtained by the Authors of the invention show that the compounds of formula <NUM> are more effective than analogous compounds having-OH groups bound on C<NUM> known in the literature, like, e.g., the compound described in the state of the art, similar to the compound FP11 but having -OH groups on the C<NUM> of each R chain.

Given the technical features observed for compounds of formula (<NUM>) as defined in the present description and in the claims, said compounds are useful as active principles or as adjuvants in diseases benefiting from a TLR4 receptor activation, i.e., in diseases in which an activation of the immune system, particularly of the innate activity, is therapeutic or prophylactic.

Therefore, by diseases requiring or benefiting from a TLR4 receptor activation, diseases are meant whose treatment or whose prevention are improved by a TLR4 receptor activation and by the related innate immune response triggered by the activation of said receptor. A non-limiting example of such diseases is represented by tumours.

An additional particularly advantageous use of the compound of formula <NUM> in any one of the embodiments provided in the description or in the claims is that as vaccine adjuvant. In fact, the relevance of immune response adjuvants during vaccine administration is known. Such adjuvants, in fact, substantially increase vaccine effectiveness and development of immunity, in the treated subject, toward antigens present in the vaccine.

Therefore, object of the present invention is also a vaccine composition comprising the compound of formula <NUM> in any one of the embodiments provided in the description or in the claims.

The vaccine composition according to the invention could therefore comprise the compound of formula <NUM> as described herein, in any one of the above-listed embodiments, at least one pharmaceutically acceptable carrier and at least one antigenic compound able to induce an immune response to a given pathology.

The composition could be prepared for a single administration of adjuvant and antigen, or for a concomitant administration thereof.

According to another embodiment, the present invention relates to a pharmaceutical composition for use in the treatment of diseases that require or benefit from a TLR4 receptor activation, comprising the compound of formula <NUM> in any one of the embodiments provided in the description or in the claims and at least one pharmaceutically acceptable excipient.

Said pharmaceutical composition can also be formulated as association between plural active principles.

According to the present description, said diseases may be cancer, allergies or infective diseases.

The pharmaceutical composition of the invention could comprise as sole active principle one or more compounds of formula <NUM> in any one of the embodiments provided in the description or in the claims, or could also comprise further active principles, such as anti-tumour active principles, kinase inhibitors, cytotoxic compounds and at least one pharmaceutically acceptable carrier or excipient.

The composition could be for oral or injectable use, and suitable conventional carriers and/or excipients for liquid, semiliquid, solid formulations, granules or others can be selected by the technician in the field.

According to the invention, the composition could comprise <NUM> to <NUM> of compound of the invention per daily dosage: <NUM> to <NUM> of substance per Kg (test on animals).

The compounds of formula <NUM>, in particular FP11, can be synthesized in a simpler and industrially scalable way compared to compound SDZ MRL953. The latter requires the insertion of three acyl chains of (R)-<NUM>-hydroxymyristic acid. The optically pure compound (R-enantiomer) is not commercially available, as only the racemic mixture is marketed. Moreover, (R)-<NUM>-hydroxymyristic acid requires a reaction of protection of the hydroxyl group in <NUM> position prior to the condensation reaction with the sugar. Compound FP11 synthesis is therefore remarkably simplified by the use of non-hydroxylated myristic acid chains.

The compounds provided in the present invention exhibit activities comparable to, if not even better than the above-reported compound SDZ MRL953, despite differences, and can be synthesized in a much simpler and industrially scalable way. Therefore, the present invention also relates to a method of synthesis for the preparation of compounds of formula <NUM>
<CHM>.

According to some possible non-limiting embodiments, the compound of formula <NUM> could be selected from the following:.

The synthetic method enabling to obtain the compounds having formula <NUM>, like, e.g., FP11 and the variants indicated herein as FP111-<NUM>, comprises the following steps (also reported in Scheme <NUM>):.

The above-described method enables the synthesis/preparation of monophosphorylated derivatives of the compounds of formula <NUM>, like FP11, FP112 and FP114.

Alternatively, to have the compounds with two phosphate groups in positions C<NUM> and C<NUM>, after phosphorylation of the position described in <NUM>), the following steps are carried out: <NUM>) deblocking the para-methoxybenzyl ether in C<NUM> by catalytic hydrogenation with a Pd/C catalyst.

According to the present description, the protection of hydroxyls at <NUM>) can be carried out according to techniques commonly used by the technician in the field, like, e.g., reaction with paramethoxy acetaldheyde or dimethyl acetal thereof in the presence of a catalyst acid like camphorsulfonic acid (CSA).

The protection of the anomeric carbon at <NUM>) can be carried out by any suitable technique known to a technique in the field, like, e.g., sylilation of the hydroxyl on C<NUM> by reaction with tetrabutylammonium fluoride (TBAF).

Acylation by condensation with various linear-chain carboxylic acids at <NUM>) and/or <NUM>) enables the synthesis of variants with Carbon chains of different length on C<NUM> and C<NUM> positions, depending on the acids used.

The condensing agents at <NUM>) or <NUM>) can be any suitable condensing agent known to the technician in the field, like, e.g., dicyclohexylcarbodiimide (DCC) or <NUM>-Ethyl-<NUM>-(<NUM>-dimethylaminopropyl) carbodiimide (EDC).

According to the present description, it is possible to use C<NUM>-C<NUM> linear chain carboxylic acids; in one embodiment, leading to the synthesis of compound FP11, myristic acid is used in both steps.

The regioselective opening in reducing conditions according to <NUM>) can be carried out according to any suitable technique commonly used by the technician in the field, like, e.g., sodium cyanoborohydride and hydrochloric acid.

The process of synthesis according to the invention enables to make in an easy and industrially scalable way the compounds of formula <NUM>.

Object of the invention is also a process for the preparation of pharmaceutical formulations or of vaccine compositions comprising the steps of the above process, and at least one step wherein the product obtained at <NUM>) or <NUM>) in a pharmaceutically acceptable form is mixed with at least one pharmaceutically acceptable adjuvant or excipient.

Compound FP11, agonist of TLR4 (scheme below) having three myristic acid chains (C<NUM>) linked at positions C<NUM>, C<NUM> and C<NUM> of the sugar and a phosphate group in C<NUM> in α-anomeric configuration.

The compound FP111, having a second phosphate group in C<NUM>, was also synthesized.

Compounds FP11 and FP111 were synthesized according to the reaction scheme reported below.

Commercially available D-glucosamine was first of all transformed, through <NUM> steps, into intermediate <NUM>, which has two myristic acid chains in C<NUM> and C<NUM>, and is protected as p-methoxybenzylidene in positions C<NUM> and C<NUM>.

Regioselective opening of the benzyldene ring with sodium cyanoborohydride and HCl enables to obtain the compound <NUM> with a free hydroxyl in position <NUM>.

The third esterification in this position, followed by C<NUM> deprotection with TBAF provides intermediate <NUM>.

From this common intermediate, phosphorylation produces intermediate <NUM>, which in turn, subjected to hydrogenation, is deblocked of all benzyl groups, on the phosphate, and paramethoxybenzyl groups, on C<NUM>, to yield compound FP11; otherwise, hydrogenation before phosphorylation frees also position <NUM>, enabling the insertion of two phosphate groups, whose deprotection yields compound FP111.

HEK-BlueTM hTLR4 cells are HEK cells transfected so as to stably express human receptors hTLR4, hMD-<NUM>, and hCD14 for endotoxin recognition. In addition, said cells have a reporter gene encoding for a secreted alkaline phosphatase (SEAP) placed under the control of specific transcription factors (NF-κB and AP-<NUM>) activated by the TLR4 signaling pathway. Therefore, the presence of LPS or of an agonist of TLR4 causes receptor dimerization, activation of the signaling pathway and of transcription factors NF-κB and AP-<NUM>, and finally SEAP production and secretion into the culture medium. SEAP levels can be subsequently quantitated by incubating the medium with compound p-nitrophenylphosphate (pNPP) and monitoring with a spectrophotometer the formation of p-nitrophenol chromogenic product at <NUM>.

The test conducted on HEK-BlueTM hTLR4 cells shows that compound FP11 is able to induce activation of NF-κB e AP-<NUM> at concentrations of ><NUM>. Conversely, compound FP111 proved completely inactive, demonstrating how the number of phosphate groups plays a crucial role in determining the agonist activity of the compound (<FIG>). In order to be certain that the agonist effect of compound FP11 be due to the interaction with the TLR4 receptor, the compounds were tested on the HEK-Blue™ Null2 cell line. Said line expresses the same reporter gene of HEK-BlueTM hTLR4 cells (SEAP), but has none of the receptors involved in endotoxin recognition (TLR4, MD-<NUM>, CD14). Both compounds tested proved unable to activate transcription factors NF-κB and AP-<NUM>, demonstrating how FP11 effect is due to interaction with the LPS receptor complex (<FIG>).

To determine the extent of the agonist activity of compound FP11, two dose-response curves were constructed (<FIG>): the first curve shows FP11 activity in connection with LPS (<FIG>), whereas the second curve indicates the compound activity normalized on its maximum TLR4 activation power (<FIG>). EC<NUM> value calculated for FP11 (<NUM>) is remarkably greater than that of LPS (<NUM>), causing a more moderate activation of TLR4 signaling pathway.

Finally, compound FP11 cytotoxicity was assessed by MTT (<NUM>-(<NUM>,<NUM>-dimethylthiazol-<NUM>-yl)-<NUM>,<NUM>-diphenyltetrazolium bromide) viability assay. HEK-BlueTM hTLR4 cells were treated with the highest compound concentrations used in the preceding assay (<NUM> and <NUM>). The MTT assay reveals that compound FP11 is not toxic at the concentrations assayed.

Interaction studies of synthetic molecules FP11 and FP111 with purified human MD-<NUM> receptor were carried out by using four different techniques: two ELISA method-based assays with blocked MD-<NUM>, a fluorescent molecule displacement assay and SPR measurements.

It has been demonstrated that compound <NUM>,<NUM>'-Bis(anilino)-<NUM>,<NUM>'-bis (naphtalene)-<NUM>,<NUM>' disulfonate (bis-ANS) binds MD-<NUM> and is displaced by LPS (<NPL>). bis-ANS presumably binds the same hydrophobic pocket of MD-<NUM> accountable for the binding of lipid A lipophilic chains; therefore, TLR4 modulators that interact with MD-<NUM> compete with compound bis-ANS, and are able to displace it from MD-<NUM>. FP11 causes a concentration-dependent decrease of compound bis-ANS fluorescence, indicating a competitive-type binding of FP11 to MD-<NUM> (Figure 3A). FP111 is not able to induce a decrease of compound bis-ANS fluorescence at the assayed concentrations (Figure 3B).

The ability to displace LPS from MD-<NUM> hydrophobic pocket was assessed by using an ELISA assay. Molecules FP11 and FP111 were added at increasing concentrations to MD-<NUM> which had previously been incubated with biotinylated LPS. FP11 exhibits the ability to displace biotinylated LPS from MD-<NUM> hydrophobic pocket in a dose-dependent manner, with a <NUM>% displacement obtained at a concentration of <NUM> (Figure 3C). FP111 was unable to displace biotinylated LPS at the highest concentration assayed, of <NUM> (Figure 3D).

The direct binding of molecules FP11 and FP111 to MD-<NUM> was analyzed by using a monoclonal antibody binding MD-<NUM>, but not LPS-bound MD-<NUM> (<NPL>).

Anti-MD-<NUM> monoclonal antibody (9B4) specifically binds an epitope near the MD-<NUM> hydrophobic pocket, which is available for 9B4 antibody binding only when the MD-<NUM> hydrophobic pocket is empty. Compound FP11 is accountable for a decrease of 9B4 antibody binding to MD-<NUM> equal to <NUM>% at a concentration of <NUM> (Figure 3E). FP111 was unable to cause a decrease of 9B4 antibody binding at the concentration of <NUM> (Figure 3F).

Surface plasmon resonance (SPR) experiments enable the study of direct interactions between the molecules to be characterized and MD-<NUM>. SPR data show binding interactions between FP11 and MD-<NUM>, and the results indicate that FP11, but not FP111, directly binds MD-<NUM> with a KD value of <NUM> (Figure <NUM>-H).

These results obtained from the <NUM> in vitro assays on purified MD-<NUM> receptor clearly demonstrate that FP11 directly binds the MD-<NUM> hydrophobic pocket, whereas the molecule FP111 does not bind MD-<NUM>.

THE CLAIMS: FP112, FP113, FP114, FP115
<CHM>.

All reagents were available on the market and used without further purifications, unless noted otherwise.

When dry solvents were used, the reactions were carried out in stove-dried glassware under light argon pressure.

The reactions were magnetically stirred.

The reactions were monitored by thin-layer chromatography (TLC) on silica gel. TLC was performed on <NUM> F254 plates of silica gel (Merck). Spot revealing was carried out using UV light (<NUM>) or a molibdate development solution [aqueous H<NUM>SO<NUM> (<NUM>%) with (NH<NUM>)<NUM>Mo<NUM>O<NUM>·<NUM><NUM>O (<NUM>%) and <NUM>% Ce(SO<NUM>)<NUM>] or a H<NUM>SO<NUM> solution [H<NUM>O (<NUM>%) <NUM> EtOH (<NUM>%) with aqueous H<NUM>SO<NUM> (<NUM>%)], followed by heating to <NUM>.

A flash column chromatography was performed on <NUM>-<NUM> mesh (Merck) silica gel. The petroleum ether used as eluent in the chromatography has a boiling range of <NUM>-<NUM>.

<NUM>H and <NUM>C NMR spectra were recorded with a Varian <NUM> Mercury instrument at <NUM>. Chemical shifts are reported in ppm downfield from TMS as internal standard. Mass spectra were recorded with an ESI-MS triple quadrupole instrument (API2000 QTrap model, Applied Biosystems).

Myristic acid (<NUM>, <NUM> mmol) was dissolved in anhydrous CH<NUM>Cl<NUM> (<NUM>) and DCC (<NUM>, <NUM> mmol) was added. After <NUM>, compound <NUM> (scheme <NUM> above) (<NUM>, <NUM> mmol) dissolved in anhydrous CH<NUM>Cl<NUM> (<NUM>) and DMAP (<NUM>, <NUM> mmol) were added and the mixture was mixed at room temperature for <NUM> hour. Precipitate was removed by filtration and solvents evaporated under vacuum. The raw product was purified by flash chromatography (petroleum ether-AE <NUM>:<NUM>) to yield compound <NUM> (scheme <NUM> above) as a white solid (<NUM>, <NUM>%).

<NUM>H-NMR: (<NUM>, CDCl<NUM>, <NUM>, TMS): δ= <NUM> (d, <NUM>J(H,H) = <NUM>, <NUM>, 2x H-ortho), <NUM> (d, 3J(H,H) = <NUM>, <NUM>, 2x H-meta), <NUM> (d, <NUM>, NH), <NUM> (t, <NUM>J(H,H) = <NUM>, <NUM>, H-<NUM>), <NUM> (t, 3J(H,H) = <NUM>, <NUM>, H-<NUM>), <NUM> (d, <NUM>J(H,H) = <NUM>, <NUM>, H-<NUM>), <NUM> (s, <NUM>, CH<NUM>-PMP), <NUM> - <NUM> (m, <NUM>, H-<NUM>), <NUM> (s, <NUM>, OCH<NUM>), <NUM> - <NUM> (m, <NUM>, H-<NUM>), <NUM> (m, <NUM>, H-6a, H-6b), <NUM> (t, <NUM>J(H,H) = <NUM>, <NUM>, CH<NUM>α-chain1), <NUM> (t, <NUM>J(H,H) = <NUM>, <NUM>, CH<NUM>α-chain2), <NUM> - <NUM> (m, <NUM>, CH<NUM>α-chain3),<NUM> - <NUM> (m, <NUM>, CH<NUM>β-chains1,<NUM>,<NUM>), <NUM> (m, <NUM>, 30xCH2), <NUM> - <NUM> (m, <NUM>, 2xCH<NUM>- chains1,<NUM> ,t-Bu-Si), <NUM> (s, <NUM>; CH<NUM>-Si), <NUM> (s, <NUM>; CH<NUM>-Si).

<NUM>C NMR (<NUM>, CDCl<NUM>, <NUM>, TMS) δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, -<NUM>, -<NUM>.

Compound <NUM>, scheme <NUM> above (<NUM>, <NUM> mmol) was dissolved in anhydrous THF (<NUM>), cooled to -<NUM>, and a solution of TBAF (<NUM>, <NUM> mmol) and AcOH (<NUM>µl, <NUM> mmol) in THF (<NUM>µL) was added. The reaction was mixed at -<NUM>° C for <NUM>, then left warming to room temperature and left mixing at room temperature for <NUM>. The solution was diluted in water and extracted with CH<NUM>Cl<NUM>. Organic layer was dried with Na<NUM>SO<NUM>, filtered, and solvents were evaporated under vacuum. The raw product was purified by flash chromatography (petroleum ether-AE <NUM>:<NUM>) to yield compound <NUM> as a red oil (<NUM>, <NUM>%).

Compound <NUM> (<NUM>, <NUM> mmol) was dissolved in anhydrous CH<NUM>Cl<NUM> (<NUM>), then imidazolium triflate (<NUM>, <NUM> mmol) and dibenzyl N,N-diisopropyl phosphoramidite (<NUM>µl, <NUM> mmol) were added and the reaction was mixed at room temperature for <NUM>. The solution was then cooled in ice bath, and mCPBA (<NUM>, <NUM> mmol) was added. The reaction was mixed at room temperature overnight, then the mixture was diluted with CH<NUM>Cl<NUM>, washed with a saturated NaHCO<NUM> solution and saline. Organic layer was dried on anhydrous Na<NUM>SO<NUM>, filtered, and solvents were evaporated under vacuum. The raw product was purified by flash chromatography (petroleum ether-AE <NUM>:<NUM>) yielding compound <NUM> as a brown solid (<NUM>, <NUM>%).

Compound <NUM> (<NUM>, <NUM> mmol) was dissolved in anhydrous CH<NUM>Cl<NUM> / MeOH <NUM>:<NUM> (<NUM>), and added in catalytic amount Pd on activated coal. The reaction mixture was mixed at room temperature under H<NUM> atmosphere overnight. Triethylamine (<NUM>µL) was added to the reaction mixture and the suspension was filtered with a syringe filter. The triethylammonium salt was dissolved in CH<NUM>Cl<NUM> /MeOH1:<NUM> (<NUM>) and treated first with an Amberlite IRA <NUM>+ exchange resin and then with an IR <NUM> Na+ exchange resin to remove triethylamine and form the sodium salt, yielding compound FP11 as a white solid (<NUM>, <NUM>%).

Compound <NUM> (<NUM>, <NUM> mmol) was dissolved in anhydrous CH<NUM>Cl<NUM> /MeOH <NUM>:<NUM> (<NUM>) and was added in catalytic amount Pd on activated coal. The reaction mixture was mixed at room temperature under H<NUM> atmosphere overnight. Solvents were evaporated under vacuum, yielding compound <NUM> as a white solid (<NUM>, <NUM>%).

Compound <NUM> (<NUM>, <NUM> mmol) was dissolved in anhydrous CH<NUM>Cl<NUM> (<NUM>), then imidazolium triflate (<NUM>, <NUM> mmol) and dibenzyl N,N-diisopropil fosforamidite (<NUM>µl, <NUM> mmol) were added and the reaction was mixed at room temperature for <NUM> hours. The solution was then cooled in an ice bath and mCPBA (<NUM>, <NUM> mmol). The reaction was mixed at room temperature overnight, then the mixture was diluted with CH<NUM>Cl<NUM>, washed with a saturated NaHCO<NUM> solution and saline. Organic layer was dried on anhydrous Na<NUM>SO<NUM>, filtered, and solvents were evaporated under vacuum. The raw product was purified by flash chromatography (petroleum ether-AE <NUM>:<NUM>), yielding compound <NUM> as a brown compound (<NUM>, <NUM>%).

Compound <NUM> (<NUM>, <NUM> mmol) was dissolved in anhydrous CH<NUM>Cl<NUM> / MeOH <NUM>:<NUM> (<NUM>), and was added in catalytic amount Pd on activated coal. The reaction mixture was mixed at room temperature under H<NUM> atmosphere overnight. Then, triethylamine (<NUM>µL) was added to the reaction mixture, and the suspension was filtered with a syringe filter. The triethylammonium salt was dissolved in CH<NUM>Cl<NUM>/MeOH <NUM>:<NUM> (<NUM>) and treated first with an Amberlite IRA <NUM>+ exchange resin and then with an IR <NUM> Na+ exchange resin to remove triethylamine and form the sodium salt, yielding FP111 as a white solid (<NUM>, <NUM>%).

In order to obtain a <NUM> stock solution of the concentrated compounds, a milligram of each compound was resuspended in the solvent indicated in the table below:.

The solution was stirred to complete dissolution of the compound.

HEK-Blue ™ hTLR4 cells (InvivoGen) were cultured according to manufacturer's instruction.

Briefly, cells were cultured in DMEM high glucose medium supplemented with <NUM>% fetal bovine serum (FBS), <NUM> glutamine, antibiotics and 1XHEK-Blue ™ Selection (InvivoGen).

Cells were detached using a cell scraper, counted and seeded in a <NUM>-well multiwell plate at a density of <NUM>×<NUM><NUM> cells per well.

After overnight incubation (<NUM>, <NUM>% CO<NUM>, <NUM>% humidity), the culture medium was replaced by DMEM without Phenol Red, with addition of the compound to be tested. The cells were incubated overnight.

The SEAP-containing supernatants were collected and incubated with para-nitrophenylphosphate (pNPP) for <NUM>-<NUM> in the dark at room temperature.

The wells' optical density was determined using a microplate reader set at <NUM>. The results were normalized with positive control (LPS alone) and expressed as a mean of percentage ± SEM of at least three independent experiments.

HEK-Blue™ hTLR4 cells were grown in DMEM supplemented with <NUM>% FBS, <NUM> glutamine and antibiotics.

Cells were seeded in <NUM>µL of DMEM without Phenol Red at a density of <NUM>×<NUM><NUM> cells per well and incubated overnight (<NUM>, <NUM>% CO<NUM>, <NUM>% humidity).

Cells were treated with the higher dose of compound used in the previous experiments and incubated overnight. MTT solution (<NUM>/mL in PBS) was added to each well, and after <NUM> incubation, <NUM> N HCl in <NUM>-propanol solution was used to dissolve formazan crystals.

Formazan concetration was determined by measuring the absorbance at <NUM>.

The results were normalized with untreated control (PBS) and expressed as the mean of percentage ± SEM of three independent experiments.

The method of antibody-sandwich ELISA for the detection of the binding of compounds to MD-<NUM> was modified from a previous study [<NUM>].

A microtiter plate was coated overnight at <NUM> with <NUM>µL/well of <NUM>µg/mL of chicken polyclonal anti-MD-<NUM> antibodies, diluted in <NUM> Na<NUM>CO<NUM> buffer, pH <NUM>, and blocked with <NUM>% BSA in PBS. After washing, <NUM> MD-<NUM> with tested compounds was added and incubated for <NUM>.

Mouse anti-MD-<NUM> mAb (<NUM>µg/mL 9B4) and goat antimouse IgG conjugated with HRP (<NUM>µg/mL) in PBS were added, followed by detection at <NUM> after the addition of <NUM>µL of ABTS (Sigma).

Chicken anti-MD-<NUM> polyclonal antibodies were prepared against recombinant MD-<NUM> by GenTel (Madison, WI, USA), monoclonal mouse anti-MD-9B4 antibodies were from eBioscience (San Diego, CA, USA), and secondary goat antimouse IgG conjugated with horseradish peroxidase was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Fluorescence was measured on PerkinElmer fluorimeter LS <NUM> (PerkinElmer, UK) as previously described [<NUM>].

All measurements were done at <NUM> in a <NUM> × <NUM> quartz glass cuvette (Hellma Suprasil, Müllheim, Germany). MD-<NUM> protein (<NUM>) and <NUM>,<NUM>'-Bis(anilino)-<NUM>,<NUM>'-bis (naphtalene)-<NUM>,<NUM>' disulfonate (bis-ANS, <NUM>) were mixed and incubated until reaching stable relative fluorescence units (RFUs) emitted at <NUM>-<NUM> under excitation at <NUM>.

Compounds, at different concentrations, were then added, followed by relative fluorescence unit (RFU) measurement at <NUM>-<NUM>.

The ability of the compounds to displace LPS from MD-<NUM> hydrophobic pocket was determined by ELISA.

A microtiter plate was coated overnight at <NUM> with <NUM>µL/well of <NUM>µg/mL chicken polyclonal anti-MD-<NUM> antibodies, diluted in <NUM> Na<NUM>CO<NUM> buffer, pH <NUM>, and blocked with <NUM>% BSA in PBS. After washing, <NUM> MD-<NUM> with biotin-labeled LPS was added and incubated for <NUM>. After washing, the compounds were added at different concentrations and incubated for <NUM>.

After washing, <NUM>µg/mL HRP-conjugated streptavidin (Sigma) in PBS was added, followed by detection at <NUM> after the addition of <NUM>µL of ABTS (Sigma). Chicken anti-MD-<NUM> polyclonal antibodies were prepared against recombinant MD-<NUM> by GenTel (Madison, WI, USA),.

The binding affinity of the compounds to recombinant MD-<NUM> was determined using a Biacore X100 with an NTA sensor chip (Biacore, GE Healthcare, Uppsala, Sweden). Briefly, <NUM> MD-<NUM> (in <NUM> TRIS, <NUM> NaCl, <NUM>% Tween <NUM>, pH <NUM>) was immobilized onto the sensor chip previously activated with <NUM> pulse of <NUM> NiSO<NUM>. The first flow cell was used as a reference surface to control nonspecific binding.

Claim 1:
A compound of formula <NUM>
<CHM>
wherein R<NUM> is a saturated C<NUM>-C<NUM> aliphatic chain having a =O on C<NUM>, said chain being free from -OH substituents on C<NUM>,
wherein R<NUM> is a saturated C<NUM>-C<NUM> aliphatic chain having a =O on C<NUM>, said chain being free from -OH substituents on C<NUM>,
wherein R<NUM> is a saturated C<NUM>-C<NUM> aliphatic chain having a =O on C<NUM>, said chain being free from -OH substituents on C<NUM>;
wherein R<NUM> is a hydrogen atom (H) or O-R<NUM> is a phosphate group (PO<NUM><NUM>-), and
wherein R1, R2 or R3 have no other substituents besides =O in C1 position.