New synthetic agonists for the TLR4 receptor.

Abstract

The present invention relates to novel synthetic molecules with agonistic activity of human Toll-like receptor 4 (TLR4), compositions containing them, and their use for the treatment of diseases in which it is useful to induce or increase an immune response. These novel synthetic molecules differ from other similar agonists due to their simple formula, their ease and cheapness of preparation, and the possibility of further chemical processing to modify their physicochemical properties and allow them to be conjugated with other molecules (e.g. protein antigens).

Description

[Technical field]

[0001] explanation The present invention relates to novel synthetic molecules having agonistic activity of the human Toll-like receptor 4 (TLR4), compositions containing them, and their use, particularly for the treatment of diseases in which it is useful to induce or enhance an immune response. [Background technology]

[0002] prior art Innate immunity is the first line of defense of higher organisms against pathogens and cellular injury. It is based on the recognition by specific protein receptors of specific molecular structures associated with pathogens or cellular injury (PAMPs and DAMPs, respectively). Such receptors are known as pattern recognition receptors (PRRs) and may be of different types, depending on their intracellular, cytoplasmic or membrane localization, and on their function.

[0003] The best-studied receptors are the Toll-like receptor (TLR) family, whose main role is to recognize various PAMPs, alert the body to the presence of pathogens through inflammation, recruit other immune cells to fight infection, and initiate the process of developing adaptive immunity, the most appropriate and specific defense against such threats.

[0004] Indeed, the innate immune response to a pathogen may be important in determining both the nature and strength of the adaptive immune response.

[0005] For this reason, the development of TLR activators (agonists) is of pharmaceutical relevance when inflammatory stimulation is beneficial from a therapeutic point of view: examples are cancer immunotherapeutics and vaccine adjuvants. In the first case, proinflammatory activity can lead to a reactivation of the immune system in the tumor environment, destroying the tumor (Bhatia S, Miller NJ, Lu H et al. Intratumoral G100, a TLR4 agonist, induces antitumor immune response and tumor regression in patients with Merkel cell carcinoma. Clin Cancer Res. 2019; 25(4): 1185-1195. doi: 10.1158 / 1078-0432. CCR-18-0469).

[0006] In the second case, pro-inflammatory activity is advantageous since modern vaccines no longer use the whole inactivated pathogen, but its subunits, which are not able to stimulate the correct inflammatory response without adjuvation. To date, there are various small molecules capable of binding and activating TLR receptors, some of which are used as adjuvants: for example, imidazoquinoline TLR7 / 8 agonists such as imiquimod and resiquimod, as well as Pam2CS-type TLR2 / TLR6 agonists and TLR4 agonists such as monophosphoryl lipid A (MPL) and aminoalkyl glucosaminide-4-phosphates (AGPs, also called corixa compounds, CRX).

[0007] Among the TLRs, TLR4 is of high pharmacological interest: its activation is the most efficient way to stimulate innate and adaptive immunity. Indeed, TLR4 exhibits two distinct cellular functional mechanisms that lead to a greater and more heterogeneous release of inflammatory cytokines, eliciting a more complete immune response.

[0008] The natural agonist of TLR4 is lipopolysaccharide (LPS), the main component of the outer membrane of gram-negative bacteria, which is divided into three parts: a long polysaccharide chain called the O-antigen, a short oligosaccharide called the core, and finally, lipid A (lpd A), the immunogenic part of the molecule, formed by two glucosamines, generally attached to two phosphates and a variable number of acyl chains.

[0009] The agonistic activity of lipid A is based on its binding affinity (ability to bind) to the TLR4 co-receptor, myeloid differentiation factor 2, or MD-2, and entails the formation of a (TLR4 / MD-2 / LPS)2 complex on the surface of innate immune cells, i.e., macrophages and dendritic cells.

[0010] The activation process of the TLR4 receptor by LPS begins with the interaction of individual LPS molecules or aggregates in solution with lipid-binding proteins (LBPs) that form complexes with LPS molecules, which are then transferred from the LBPs to their co-receptor CD14, which then transfers the LPS molecules from MD-2.

[0011] However, Lpd A is toxic even at picogram amounts and as a result cannot be used pharmacologically (Molinaro A, Holst O, Lorenzo F Di et al. Chemistry of lipid a: At the heart of innate immunity. Chem-A Eur J. 2015;21(2):500-519. doi:10.1002 / chem.201403923).

[0012] Synthetic and natural molecules with structures similar to lipid A but with attenuated endotoxicity are interesting candidates as vaccine adjuvants with a view to maintaining immune stimulating activity while eliminating toxic effects.

[0013] Monophosphoryl lipid A (MPL) is a molecule identical to lipid A except for the lack of the C1 phosphate group. Despite this similarity, its inflammatory activity is only 0.1% of that of the native molecule, and its pharmacological profile is so good that it has been approved by the FDA for use as a vaccine adjuvant. The molecule is currently used in the Cervarix and Fendrix vaccines. However, the MPL adjuvant currently in use is chemically heterogeneous, since it is produced directly from native LPS.

[0014] Moreover, the synthesis of these disaccharide compounds is very long and complicated, resulting in a final price of about 200 eur / mg. For this reason, it is interesting to further develop analogues of lipid A with monosaccharide structures, with the same or even improved advantageous characteristics of the known analogues, preferably obtained through a simpler synthetic route.

[0015] Examples of lipid A analogs of monosaccharide structure known in the art are exemplified by synthetic compounds designated AGPs (also known as CRX adjuvants, Corixa), which contain monosaccharide units linked by glycosidation with units of aminoalkyl aglycone N-acylate.

[0016] AGP is a potent agonist of TLR4, is chemically homogeneous, and is produced by chemical synthesis.

[0017] Additionally, simpler lipid A analogs that are effective in activating TLR4 include monophosphorylated monosaccharide derivatives that mimic either the reduced or non-reduced portions of lipid A (scheme below). [ka]

[0018] Other examples of compounds known in the art are represented by the compounds GLA63 and GLA60 (scheme above) which contain a glucopyranoside skeleton phosphorylated at C4, a linear chain with 14 carbons at C2 and a branched chain at C3 (14+14 carbons in GLA63 or 14+12 carbons in GLA60) (Motohiro Matsuura, Makoto Kiso and Akira Hasegawa Infect. Immun. 1999, 67(12), 6286-6292). These monosaccharides, which partially mimic lipid A and mimic the monosaccharide lipid X, the biosynthetic precursor of lipid A, are active in stimulating the production of TLR4-dependent cytokines TNF-α and IL-6 in both mouse and human cells.

[0019] Also known in the art is the compound SDZ MRL 953, which exhibits a strong activity in stimulating the release of inflammatory cytokines, such as interleukin-6 (IL-6), interleukin-8 (IL-8) and TNF-α factors, in mouse macrophages and neutrophil granulocytes, accompanied by a reduction in the release of proinflammatory cytokines of at least 10% in galactosamine-sensitized mice compared to the parent endotoxin (Salmonella abortus equi). 4 A factor of at least 10 4 ) showed a reduction in toxicity.

[0020] In experimental microbial infection models, the compounds proved highly prophylactically effective when administered prophylactically in either one or three doses to myelosuppressed or immunodeficient mice.

[0021] The SDZ MRL 953 and the effective dose to achieve a 50% response vary depending on the infectious agent and route of administration. However, in all cases, the EC 50 is approximately 10 times that obtained with the endotoxin Salmonella abortus equi. 3 It was more than double.

[0022] However, thanks to its low toxicity, the therapeutic index of this molecule, expressed as LD25 / ED75, is significantly improved compared to endotoxins and ranges from about 5 to >500, depending on the infectious agent and route of administration.

[0023] The compounds also proved to be efficient in inducing tolerance to endotoxin: repeated administration of the compounds induces a transient tolerance (≧1 week) to endotoxin-related lethality risk.

[0024] All these positive results were also confirmed in a model of progressive sepsis caused by Escherichia coli, where antibiotic treatment already proved inefficient: pretreatment with a single dose of SDZ MRL 953, 1 day before microbial inoculation, dramatically increased the curative effect of the administered antibiotic. Thus, increasing the dose of the immune stimulant in the combination therapy significantly increased the long-term survival rate.

[0025] Due to the tolerability demonstrated by SDZ MRL 953 in animals and in vivo, this compound was subsequently tested in human models.

[0026] Based on the known antitumor activity of endotoxin from Salmonella abortus equi and its immune-stimulating properties, Kiani et al. (A. Kiani, A. Tschiersch, E. Gaboriau, F. Otto, A. Seiz, H.-P. Knopf, P. Stutz, L. Farber, U. Haus, C. Galanos, R. Mertelsmann, and R. Engelhardt, Blood, 1997, 1673-1683) carried out a randomized, double-blind, phase I study with control medium, administering SDZ MRL 953 to tumor-affected patients to evaluate, firstly, its biological effect and the safety of its administration in humans and, secondly, its influence on the response to subsequent endotoxin (LPS) challenge.

[0027] Administration of SDZ MRL 953 has proven safe and well tolerated. The same SDZ MRL 953 increases granulocyte counts and serum levels of G-CSF and interleukin-6 (IL-6), but not the serum levels of the proinflammatory cytokines TNF-α, IL-1b, and IL-8.

[0028] Therefore, SDZ MRL 953 has three relevant features: 1) high tolerability and low toxicity, 2) the ability to induce G-CSF production and, consequently, 3) the ability to stimulate nonspecific immune resistance expressed by an increase in the intracellular group of primary defenses.

[0029] Despite these positive results in clinical use of SDZ MRL 953, The mechanism of action of this molecule has yet to be studied in molecular detail.

[0030] The synthesis of SDZ MRL 953 is complicated by the fact that a glucosamine core is attached to the C2, C3, and C4 positions of 3(R)-hydroxymyristic acid chains as enantiomerically pure. 3-Hydroxymyristic acid is commercially available as a racemate, but the enantiomerically pure 3(R)-hydroxymyristic acid needs to be isolated from the racemate before it can be used in the synthesis of SDZ MRL 953.

[0031] WO2019 / 092572 discloses a further class of compounds having a triacylated monophosphorylglucosamine core and, in particular, one phosphate group at the C1 position of FP112. [ka]

[0032] FP112 is a compound similar to the reducing end of lpd A and SDZ MRL 953, but with three fully saturated unsubstituted acyl chains. This compound is significantly easier to synthesize than compounds known in the art, since the generation of optically pure acyl chains is not required for its synthesis.

[0033] FP112 has an impressive pharmacological profile, as it is able to stimulate the release of multiple inflammatory cytokines, the most notable of which are IL-1α, IL-1β, IL-6, TNF-α and IFNβ.

[0034] FP112 has been extensively tested in vitro using Hek-Blue, Raw-Blue and THP-1 cells and consistently demonstrated low toxicity and good pro-inflammatory activity already at concentrations of 10 μM. Summary of the Invention

[0035] The authors of the present invention have identified a group of novel compounds with a triacylated monophosphorylglucosamine core, different from those known in the art, which are effective agonists of the TLR4 receptor. Advantageously, the novel compounds are more versatile than those described in the art, since they can be functionalized with various groups of interest. The authors of the present invention have also developed new methods of synthesis of the novel compounds, which are simpler, faster, and cheaper than those disclosed in the art, as well as methods of synthesis of other compounds known in the art.

[0036] The authors of the present invention provide herein novel compounds of formula 1, [ka] R1 is a saturated C5-C 15 is an alkyl chain, R2 is a saturated C5-C 15 is an alkyl chain, R3 is a saturated C5-C 15 is an alkyl chain, R4 is any substituent known to those skilled in the art that can be linked by means of a bond between C6 and an appropriate atom and / or any substituent having an oxygen or nitrogen atom that can be bonded to C6.

[0037] Said compounds are outwardly similar to those disclosed in WO2019 / 092572, but have several advantages compared to the same compounds. The first advantage is shown by the fact that the molecular structure of the compounds of formula 1 makes them more stable: in fact, although the phosphate group is known to be one of the best leaving groups in organic chemistry, the phosphate group at C4 position of the compounds of formula 1 has proven to be much more stable than the phosphate group at C1 present in the above-mentioned prior art compounds.

[0038] As an example, the compound of formula 1 is more stable in the desilylation reaction (reaction 5 in the synthesis of FP20 and reaction 6 in the synthesis of FP11).

[0039] The instability of the C1 phosphate during the synthesis of FP112 is a major problem, and phosphate decomposition was observed even under mild operating conditions, forming two major dephosphorylated by-products (as shown in the experimental section). Moreover, in the case of FP11, the purification yield id low, averaging close to 50%.

[0040] In FP20, the desilylation was proceeded with good yields, without issues of cleavage of the anomeric phosphate, in 90% yield and with negligible formation of by-products.

[0041] The second advantage is that it provides a simpler method for the chemical synthesis of the compounds of the present invention, requiring only six synthetic steps to obtain the final compound; moreover, in the method of the present invention, purification by chromatography column is sufficient three times, thereby avoiding the waste of a large amount of purification solvent. As a result, the synthesis is cheaper, and therefore it is possible to synthesize larger amounts at the laboratory level, facilitating the industrial scalability of the process.

[0042] Finally, a major advantage of the compounds of the invention is that the base molecule can be modified with various functional groups at the C6 position, thereby making it possible to provide molecules that can combine the TLR4 agonist and additional functions. The different structure of the novel compounds of the invention provides the compounds with higher stability and provides the C6 position for functionalization. In FP11, functionalization of C6 is difficult: most chemical reagents for functionalization cleave the C1 phosphate, similar to what occurs during desilylation. For FP20 compounds and derivatives, the anomeric phosphate is missing and functionalization of C6 is feasible. This allows the skilled person to customize the compound for a particular desired activity by modifying the functional group at C6, for example by increasing its solubility and / or bioavailability, by adding target-specific substituents, by conjugating the molecule with other functional substituents.

[0043] Therefore, the object of the present invention is to: 1. A compound of formula 1, [ka] R1 is a saturated C5-C 15 is an alkyl chain, R2 is a saturated C5-C 15 is an alkyl chain, R3 is a saturated C5-C 15 is an alkyl chain, R4 is any substituent that can be linked by a bond between C6 and a suitable atom and / or any substituent that has an oxygen or nitrogen atom that can be bonded to C6, and; A vaccine adjuvant comprising a compound of formula 1; a vaccine composition comprising a compound of formula 1, at least one pharma- ceutically acceptable carrier, and at least one pharma- ceutically acceptable immunogenic antigen; A pharmaceutical composition comprising a compound of formula 1 and at least one pharma- ceutically acceptable excipient and / or carrier; Use.

[0044] is an intermediate of formula 1i, [ka] R1 is a saturated C5-C 15 It is an alkyl chain.

[0045] A method for preparing an intermediate of formula 1i, wherein R1 is a saturated C5-C 15 is an alkyl chain, [ka] The following process 1) Selective acylation of the amino group at the C2 position of glucosamine hydrochloride by reaction with acyl chloride in the presence of sodium bicarbonate; 2) selective silylation protection of the hydroxyl group at C6 by reaction with tert-butyldimethylsilyl chloride (TBDMSCl) in the presence of imidazole; Includes.

[0046] A process for preparing a compound of formula 1, comprising: [ka] R1 is a saturated C5-C 15 is an alkyl chain, R2 is a saturated C5-C 15 is an alkyl chain, R3 is a saturated C5-C 15 is an alkyl chain, R4 is any substituent that can be linked by a bond between C6 and a suitable atom and / or any substituent having an oxygen or nitrogen atom that can be bonded to C6, and can be prepared by the process described below: 1) Selective acylation of the amino group at C2 position of glucosamine hydrochloride by reaction with acyl chloride in the presence of sodium bicarbonate; 2) selectively protecting the hydroxyl group at C6 position by reaction with tert-butyldimethylsilyl chloride (TBDMSCl) in the presence of imidazole, thereby obtaining the intermediate of claim 20; 3) selective acylation of the hydroxyl groups at the C1 and C3 positions by reaction with acyl chloride in the presence of triethylamine and N,N-dimethylaminopyridine (DMAP); 4) phosphorylation of the hydroxyl group at C4 by reaction with dibenzyl N,N-diisopropylphospharamidite in the presence of triflate imidazolium, followed by oxidation of the phosphite to phosphate via metachloroperbenzoic acid; 5) deprotection of the hydroxyl group from the silane at the C6 position in the presence of a catalytic amount of sulfuric acid; and 6) deprotection of the phosphate from the C4 position and, optionally, the benzyl at the C6 position via hydrogenation catalyzed by palladium on carbon (Pd / C); Includes.

[0047] A process for preparing a compound of formula X, comprising: [ka] R1 is a saturated C5-C 15 is an alkyl chain, R2 is a saturated C5-C 15 is an alkyl chain, R3 is a saturated C5-C 15 It is an alkyl chain. R4 is OH, and each of R1, R2 and R3 does not contain an -OH substituent at the C2 position; 1) Selective acylation of the amino group at C2 position of glucosamine hydrochloride by reaction with acyl chloride in the presence of sodium bicarbonate; 2) selectively protecting the hydroxyl group at C6 position by reaction with tert-butyldimethylsilyl chloride (TBDMSCl) in the presence of imidazole, thereby obtaining the intermediate of formula 1i as described in claim 20; 3) Complete acylation of the hydroxyl groups at C1, C3 and C4 positions by reaction with acyl chloride in the presence of triethylamine and N,N-dimethylaminopyridine (DMAP); 4) Selective diacylation at C1 by reaction with ethylenediamine in the presence of acetic acid; 5) phosphorylation of the hydroxyl group at C1 position by reaction with dibenzyl N,N-diisopropylphospharamidite in the presence of triflate imidazolium, followed by oxidation of the phosphite to phosphate via metachloroperbenzoic acid; 6) deprotection of the hydroxyl group from the silane at C6 position in the presence of catalytic amounts of sulfuric acid; 7) Deprotection of the phosphate from the C1 position and, optionally, the benzyl at the C6 position via hydrogenation catalyzed by palladium on carbon (Pd / C).

[0048] Use of an intermediate compound according to claim 22 for the synthesis of a compound of formula 1, [ka] R1 is a saturated C5-C 15 is an alkyl chain, R2 is a saturated C5-C 15 is an alkyl chain, R3 is a saturated C5-C 15 is an alkyl chain, R4 is any substituent that can be linked by a bond between C6 and a suitable atom and / or any substituent that has an oxygen or nitrogen atom that can be bonded to C6.

[0049] and, Use of an intermediate of formula 1i as defined in any one of the embodiments disclosed herein for the synthesis of a compound of formula X, [ka] R1 is a saturated C5-C 15 is an alkyl chain, R2 is a saturated C5-C 15 is an alkyl chain, R3 is a saturated C5-C 15 is an alkyl chain, R4 is OH, and each of R1, R2 and R3 does not contain an -OH substituent at the C2 position. [Brief description of the drawings]

[0050] [Figure 1] Figure 1. Activity of FP compounds on TLR4 and TLR2. HEK-Blue™ hTLR4 (A) and HEK-Blue™ TLR2 (B) cells were treated with the indicated concentrations of compounds FP20, FP21, FP22, FP23 and FP24, MPLA, LPS (100 ng / mL) and Pam2CSK4 (1 ng / mL) and incubated for 16-18 h. Results were normalized with respect to stimulation with LPS alone (A) or Pam2CSK4 (B) and expressed as percentage of the mean ± SEM of at least three independent experiments (treated vs. untreated: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0051] [Diagram 2] Figure 2. Activity of FP compounds on human and mouse macrophages. THP-1-X Blue™ (A) and RAW-Blue™ (B) cells were treated with the indicated concentrations of compounds FP20, FP21, FP22, FP23 and FP24, MPLA and LPS (100 ng / mL) and incubated for 16-18 h. Results were normalized with respect to stimulation with LPS alone and expressed as percentage of the mean ± SEM of at least three independent experiments (treated vs. untreated: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0052] [Diagram 3]Figure 3. Cell viability. THP-1 cells differentiated into macrophages were treated with increasing concentrations of FP20, FP21, FP22, FP23 and FP24 (0.1-50 μM) and LPS (100 ng / mL). To assess solvent toxicity, vehicle (DMSO) was added at the same concentrations (0.1-50 μM). Data were normalized to the (untreated) control and expressed as percentage of the mean ± SEM of at least three independent experiments (treated vs. untreated: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0053] [Figure 4] Figure 4. Cell viability. RAW-Blue cells were treated with increasing concentrations of FP20, FP21, FP22, FP23, and FP24 (0.1, 1, 10, 25, 50 μM) and LPS (100 ng / mL). To assess vehicle toxicity, vehicle (DMSO) was added at the same concentrations (0.1, 1, 10, 25, 50 μM). Data were normalized to the (untreated) control and expressed as percentage of the mean ± SEM of at least three independent experiments (treated vs. untreated: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0054] [Diagram 5] Figure 5. HEK-Blue hTLR4, HEK-Blue Null and HEK-Blue hTLR2 cells were treated as indicated and incubated for 18 h. Supernatants were harvested and SEAP levels were quantified by the QUANTI-blue method. Data were normalized to stimulation with S-LPS (A, B), IL-1β (C) or PAM2CSK4 (D) and expressed as the mean percentage ± SD of three independent experiments (treated vs. untreated: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0055] [Figure 6] Figure 6. A) Mouse body weight over 7 days after adjuvant administration (n=4 per treatment). B) Antibody response to OVA immunization using MPLA, FP112 and FP11 as adjuvants after prime immunization (day 22 post-vaccination) and booster immunization (day 19) (n=8 per treatment). For statistical comparison, the areas under each curve were examined by Brown-Forsythe test and Welch's one-way ANOVA test with an alpha of 0.05.

[0056] [Figure 7] Figure 7. 1H NMR of compound 25 (impurity 1 of step 6 in the synthesis of FP11), where the cleavage of the phosphate at C-1 can be observed by the multiplicity (d) of the signal at 6.04 ppm. When a phosphate is present at C-1, H-1 has a multiplicity of dd due to HP coupling.

[0057] [Figure 8] Figure 8. 1H NMR of compound 26 (impurity 2 of step 6 in the synthesis of FP11), where the cleavage of the phosphate at C-1 can be observed by the multiplicity (d) of the signal at 6.01 ppm. When a phosphate is present at C-1, H-1 has a multiplicity of dd due to HP coupling. In this case, the silane is also cleaved, observed by the lack of their signals at 0 ppm and 0.85 ppm.

[0058] [Figure 9] FIG. 9. 13C NMR of compound 26 (impurity 2 from reaction 6 in the synthesis of FP11), which confirms the structure of compound 26 observed in FIG.

[0059] [Figure 10]Figure 10. Activity of FP200 diphosphate compound on human macrophages. Differentiated THP-1-X Blue cells were treated with the indicated concentrations of compounds FP11, FP112, FP20, FP200, FP21, MPLA and LPS (100 ng / mL) and incubated for 16-18 hours. Results were normalized with respect to stimulation with LPS alone and expressed as percentage of the mean ± SEM of at least three independent experiments (treated vs. untreated: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0060] [Figure 11] Figure 11 - Activity of FP207 on human macrophages. Differentiated THP1-XBlue (trademark) cells were treated with the indicated concentrations of FP20, FP207, MPLA and LPS (100 ng / mL) and incubated for 16-18 hours. Results were normalized with respect to stimulation with LPS alone and expressed as percentage of the mean ± SEM of at least three independent experiments (treated vs. untreated: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0061] [Figure 12] Figure 12 - Cell viability. Differentiated THP1-XBlue™ cells were treated with increasing concentrations of FP20 and FP207 (0.1-25 μM), MPLA and LPS (100 ng / mL). Data were normalized to the (untreated) control and expressed as percentages of the mean ± SEM of at least three independent experiments.

[0062] Glossary As used herein, the term "TLR4 receptor agonist" refers to a compound that selectively binds to the TLR4 receptor, induces a conformational change in the receptor, and then generates an intracellular stimulus by eliciting a response similar to that induced by the receptor's natural ligand. In the case of TLR4, substances described as agonists bind to the co-receptor MD-2, which then non-covalently binds to TLR4, thereby generating the receptorial complex (TLR4 / MD-2 / agonist)2, initiating a signal cascade that leads to the activation of nuclear transcription factors and the synthesis of inflammatory cytokines (mainly TNF-α and various types of interleukins) from the cell surface.

[0063] As used herein, the compound identified as FP112 refers to a compound having the formula represented below: [ka] R1=R2=R3=C=OC 11 H 23 and R4=H, which is disclosed as FP112 in WO2019 / 092572.

[0064] As used herein, the bond in C1 of the following formula 1 has the meaning generally intended in organic chemistry. [ka] It indicates that the compound may be in either the α or β anomeric conformation.

[0065] As used herein, the bond in C1 of the following formula 1α has the meaning generally intended in organic chemistry: [ka] Indicates that the compound is in the alpha anomeric conformation.

[0066] As used herein, the bond in C1 of the following formula 1α has the meaning generally intended in organic chemistry: [ka] Indicates that the compound is in the β anomeric conformation.

[0067] In this specification, the term "catalytic amount" means the amount or concentration of a substance used in a chemical reaction to obtain a catalytic effect. In particular, in this specification, the term "catalytic amount" may be replaced by "in the range of 0.5% to 1% of the volume / volume concentration." DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] Detailed Description of the Invention The present invention relates to a compound of formula 1, [ka] R1 is a saturated C5-C 15 is an alkyl chain, R2 is a saturated C5-C 15 is an alkyl chain, R3 is a saturated C5-C 15 is an alkyl chain, R4 is any substituent known to those skilled in the art that can be linked by a bond between C6 and an appropriate atom and / or any substituent that has an oxygen or nitrogen atom that can be bonded to C6.

[0069] Thus, according to this specification, each alkyl chain, R1, R2 or R3, can be C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 It may be an alkyl chain.

[0070] According to this specification, each of R1, R2 and R3 can be different or the same alkyl chain, as defined above.

[0071] In one embodiment of the present invention, at least two of R1, R2 and R3 are the same.

[0072] According to one embodiment of the present invention, the R1, R2 and R3 chains do not contain an -OH substituent on the C2 position. The lack of a hydroxyl at the C2 position advantageously allows for a shorter and more efficient synthetic route, thereby eliminating various protection and deprotection steps of the hydroxyl group, reducing the cost of the synthesis and making the synthetic process scalable and industrializable for drug manufacturing.

[0073] According to another embodiment of the invention, the R1, R2 and R3 chains do not contain any substituents.

[0074] As mentioned above, the R4 chain at the C6 position can be optionally functionalized with a substituent of interest, provided that the TLR4 agonist activity is not destroyed.

[0075] By way of non-limiting example, R4 can be a hydroxyl group (OH), a phosphate group (PO4 2- ), an azide group (N3), an amine group (NH2), an acyl group (O(C=O)R), an alkyl group (OR) or a glycosyl group.

[0076] The suitability of R4 at C6 for functionalization is a highly advantageous feature of the compounds of the invention. As mentioned above, depending on the TLR4 agonist function and the R4 substituents selected, it is indeed possible to provide molecules that can combine any other function of interest. For example, it is surprising that the phosphate group (PO4 2-) can be used to obtain increased solubility and bioavailability. Indeed, according to teachings in the art (WO2019 / 092572), the presence of two phosphate groups in the agonists described therein resulted in the loss of TLR4 agonist activity. On the other hand, the novel compounds of the present invention surprisingly retain TRL4 agonist activity even with a second phosphate group, thereby improving the solubility of the compounds themselves. As known to those skilled in the art, improved solubility is an important advantage, as it improves the delivery, bioavailability and stability of the compounds containing them. Hence, the simple presence of an additional phosphate at C6 can significantly improve the efficiency of pharmaceutical or vaccine compositions.

[0077] Another important advantage of the ability to functionalize the compounds of the invention at C6 is that, when used as vaccine adjuvants, they can also be conjugated with antigens or antigenic epitopes or with other adjuvants to improve and extend their activity.

[0078] Additionally, the compounds may be conjugated to target-specific molecules, thereby improving delivery to preferred sites.

[0079] Moreover, when used in antitumor compositions, the compounds of the present invention can be functionalized by linking them with additional, different agents to improve their effectiveness.

[0080] By way of example, it is possible to use substituents that do not contain hydrogen atoms capable of forming hydrogen bonds in order to improve the lipophilic effect, or to use substituents that are characterized by the presence of hydrogen atoms capable of forming hydrogen bonds in order to improve the solubility in water, which is inversely proportional to the lipophilicity.

[0081] A further advantage deriving from the possibility of utilizing a wide variety of substituents as R4 are the steric effects that can result, for example, from using substituents that tend to restrict free rotation around a simple bond and therefore reduce the number of energetically accessible conformations.

[0082] If one of these conformations is biologically active, the "stiffening" effect increases the affinity of the molecule for the receptor.

[0083] Another important example is represented by functionalization with a linker at the C6 position. Non-limiting examples of suitable linkers are represented by disulfides (RSS-R'), hydrazones (R'R''C=N-NH2), peptides or thioethers (R'-S-R'') or the like.

[0084] Linkers as described above allow the preparation of antibody-drug conjugates (ADCs), which are conjugated molecules comprising an antibody linked to a biologically active anti-cancer payload or drug (such as a compound of the invention), making it possible to obtain a combinatorial effect between the antibody and the compound of the invention.

[0085] A further interesting example is the possibility of inserting a glycosyl group at C6, such that the resulting compounds would have two advantages: improved water solubility due to the presence of a hydrophilic glycosyl group, and theoretically better affinity for the receptor by mimicking the core part of LPS.

[0086] Suitable substituents depending on the properties desired are known to those of skill in the art.

[0087] According to the present invention, the compound of formula 1 may be an α-anomer having formula 1α, or a β-anomer having formula 1β. [ka] [ka]

[0088] According to some possible non-limiting embodiments, the compound of formula 1 can be selected from the α-anomer or β-anomer form of the compound of formula 1, R1=R2=R3=C 11 H 23 and R4=OH, or R1=R3=C 13 H 27 ;R2=C 11 H 23 and R4=OH, or R1=R2=R3=C9H 19 and R4=OH, or R1=R2=R3=C 13 H 27 and R4=OH, or R1=R3=C9H 19 ;R2=C 11 H 23 and R4=OH, or R1=R2=R3=C 11 H 23 , and R4=PO4 2- or R1=R2=R3=C9H 19 , and R4=PO4 2- or R1=R2=R3=C 13 H 27 , and R4=PO4 2- or R1=R2=R3=C 11 H 23 and R4=OC3H7, or R1=R2=R3=C 11 H 23 and R4=O(C=O)C6H8(OH)3, or R1=R2=R3=C 11 H 23 and R4=NH2, R1=R2=R3=C 11 H 23 , and R4=O(C=O)CCH3(CH2OH)2, R1=R2=R3=C 11 H23 and R4 = OCH(CHOH)3CH(CH3)O, R1=R2=R3=C 11 H 23 , and R4=OCH(CHOH)3CH(CH2OH)O, R1=R2=R3=C 11 H 23 , and R4 = OCH(CHOH)3(CH2)O.

[0089] In a preferred embodiment, the compound is the β-anomer of a compound of formula 1, for example:

[0090] Compound FP20:R1=R2=R3=C 11 H 23 With R4=OH, [ka]

[0091] Compound FP21:R1=R3=C 13 H 27 R2=C 11 H 23 With R4=OH, [ka]

[0092] Compound FP22:R1=R2=R3=C9H 19 With R4=OH, [ka]

[0093] Compound FP23:R1=R2=R3=C 13 H 27 With R4=OH, [ka]

[0094] Compound FP24:R1=R3=C9H 19 R2=C11 H 23 With R4=OH, [ka]

[0095] Compound FP200:R1=R2=R3=C 11 H 23 R4=PO4 2- having [ka]

[0096] Compound FP202:R1=R2=R3=C9H 19 R4=PO4 2- having [ka]

[0097] Compound FP203:R1=R2=R3=C 13 H 27 R4=PO4 2- has. [ka]

[0098] In a further embodiment according to the invention, the compound of formula 1 having an additional substituent at the C6 position may be selected from:

[0099] Compound FP204:R1=R2=R3=C 11 H 23 With R4=OC3H7, [ka]

[0100] Compound FP205:R1=R2=R3=C 11 H 23 With R4=O(C=O)C6H8(OH)3, [ka]

[0101] Compound FP206:R1=R2=R3=C 11 H 23 With R4=NH2, [ka]

[0102] Compound FP207:R1=R2=R3=C 11 H 23 With R4=O(C=O)CCH3(CH2OH)2, [ka]

[0103] Compound FP20Rha:R1=R2=R3=C 11 H 23 With R4 = OCH(CHOH)3CH(CH3)O, [ka]

[0104] Compound FP20Glc:R1=R2=R3=C 11 H 23 With R4 = OCH(CHOH)CH(CHOH)O, [ka]

[0105] Compound FP20Man:R1=R2=R3=C 11 H 23 With R4 = OCH(CHOH)CH(CHOH)O, [ka]

[0106] Compound FP20Gal-α:R1=R2=R3=C 11 H 23 With R4 = OCH(CHOH)CH(CHOH)O, [ka]

[0107] Compound FP20Gal-β:R1=R2=R3=C 11 H 23 With R4 = OCH(CHOH)CH(CHOH)O, [ka]

[0108] Compound FP20Lyx:R1=R2=R3=C 11 H 23 With R4 = OCH(CHOH)3CH2O. [ka]

[0109] The compounds of the invention having formula 14, 15, 16 or 19 are mixtures of anomers (diastereoisomers) of the sugar attached at C6.

[0110] Compounds of the invention having formula 17 or 18 are the pure α and β anomers of glucose attached at C6, respectively.

[0111] In one embodiment, compounds having formula 2 are preferred.

[0112] In the present specification, such a compound is also referred to as compound FP20, where R1, R2 and R3 are -C 11 H 23 and R4 is -OH.

[0113] The data reported in the Examples section demonstrate particular and advantageous features of the aforementioned compounds.

[0114] According to the present specification and based on the experimental data obtained, it is clear that the compounds described and claimed are effective agonists of the TLR4 receptor. "Agonist of a receptor" (receptor agonist) is as generally defined in the literature, i.e., a substance capable of binding a specific receptor at the binding site of the endogenous ligand. Thus, as the name suggests, the former competes with the latter for binding to said site.

[0115] After binding with a natural ligand, the receptor undergoes a conformational change that mediates its biological activity at the cellular level. Agonists are molecules with intrinsic activity that can mimic the effect of the ligand. Binding to the receptor induces a conformational change similar to that caused by binding to the endogenous ligand.

[0116] In the present case, each of the agonists disclosed and claimed is a selective agonist for the TLR4 receptor.

[0117] Considering the technical features observed in the compounds of formula (1) as defined in the present specification and claims, said compounds are useful as active ingredients or adjuvants in the treatment of diseases that benefit from the activation of the TLR4 receptor, i.e. diseases in which activation of the immune system, in particular the innate activity, has a therapeutic or prophylactic effect.

[0118] Therefore, diseases that require or benefit from activation of the TLR4 receptor include all diseases whose treatment or prevention is improved by activation of the TLR4 receptor and the innate immune response elicited by activation of said receptor.

[0119] Non-limiting examples of such diseases are represented by tumors, allergies, infectious diseases such as viral infections, cardiovascular diseases, obesity-dependent metabolic diseases, neuronal degeneration, apoptosis, autoimmune disorders, bacterial infections, autoimmune diseases represented by IBD, Crohn's disease or rheumatoid arthritis.

[0120] The compound of formula 2, also identified herein as compound FP20, can be compared to the compound of formula FP112, which has the formula represented below: [ka] R1=R2=R3=C=OC 11 H 23 R4=H is disclosed in WO2019 / 092572 and is designated FP112 therein due to the fact that it has the same R1, R2, R3 and R4 substituents, but the phosphate group is on the C1 position in FP112 and on the C4 position in FP20.

[0121] Experiments carried out in vitro with FP112 using cells Hek-Blue, Raw-Blue and THP-1, briefly reported in the examples below, demonstrated low toxicity and good pro-inflammatory activity at a concentration of 10 μM. Furthermore, experiments carried out in vivo with FP112 to test its tolerability and efficacy as a vaccine adjuvant demonstrated no collateral damage with the administration of 10 μg of FP112, and its efficacy as a vaccine adjuvant was comparable to that of MPLA. The data reported in the examples section show that, with respect to experiments carried out with Hek-Blue, Raw-Blue and THP-1 cells, the activity of the compounds of the present invention is comparable to that of the agonist compounds disclosed in WO2019 / 092572, which also proved to be cytotoxic in vivo and effective as vaccine adjuvants.

[0122] Moreover, the compounds of the present invention are improved with respect to the compounds disclosed in WO2019 / 092572, since the phosphate group at C1 in the prior art is relocated to C4 in the present invention, thereby allowing the alkyl chain to be located at C1 rather than C4 in the compounds of the present invention, resulting in improved activity of the substituent at C6. Indeed, WO2019 / 092572 discloses in experiment 2 that the compound named FP111 in the specification has two phosphate groups: one at C1 and the other at C6, which proved to be completely inactive as TLR4 agonists in tests carried out on HEK-Blue™ hTLR4 cells. In WO2019 / 092572, it was speculated that the lack of activity was due to the presence of two phosphates in the molecule, and that the number of phosphates in the molecule must be less than or equal to one to maintain their TLR4 agonist activity.

[0123] Surprisingly, the authors of the present invention have discovered that the compounds disclosed and claimed in the present application, such as, for example, compounds of formulae 7, 8 and 9, which have two phosphate groups, one at C4 and the other at C6, surprisingly maintain TLR4 agonist activity in the same test in which compound FP111 disclosed in WO2019 / 092572 is completely inactive. This finding is unexpected and demonstrates a relevant advantage of the compounds of the present invention compared to the prior art, since it demonstrates that the position of the phosphate group in the triacylated monophosphorylglucosamine core can significantly change the activity of such compounds.

[0124] Thus, the present invention also provides a compound of formula 1 in any one of the embodiments disclosed herein or claimed herein as a vaccine adjuvant.

[0125] The relevance of immune response adjuvants in vaccine compositions is known: in fact, vaccine adjuvants substantially increase the efficacy of the vaccine and the development of immunity in the treated subject against the antigens present in the vaccine.

[0126] An object of the present invention is therefore also a vaccine composition comprising a compound of formula 1 or a mixture thereof as defined in any one of the embodiments of the present specification or claims.

[0127] Thus, a vaccine composition according to the invention may, in any one of the above embodiments, comprise a compound of formula 1 as described herein or a mixture thereof, at least one pharma- ceutically acceptable carrier, and at least one antigenic compound capable of inducing a desired immune response, such as an immunogenic antigen.

[0128] Suitable vaccine carriers are known to those skilled in the art.

[0129] The pharmaceutical carrier can be selected to aid in the release of the antigenic component(s) from the composition over an extended period of time. The carrier can include water-soluble or water-insoluble substances.

[0130] The water-soluble substance is a substance that plays a role in controlling the infiltration of water into the pores of the drug dispersion.

[0131] One water-soluble substance or a combination of two or more water-soluble substances may be used.

[0132] Specifically, the water-soluble substance may be selected from one or more of the group consisting of synthetic polymers (e.g., polyethylene glycol, polyethylene polypropylene glycol), sugars (e.g., sucrose, mannitol, glucose, sodium chondroitin sulfate), polysaccharides (e.g., dextran), amino acids (e.g., glycine and alanine), inorganic salts (e.g., sodium chloride), organic salts (e.g., sodium citrate), and proteins (e.g., gelatin and collagen, and mixtures thereof).

[0133] Furthermore, when the water-soluble substance is an amphipathic substance that dissolves in both organic solvents and water, it has the effect of controlling the release of, for example, lipophilic drugs by changing its solubility. The amphipathic substance includes, but is not limited to, one or more selected from the group consisting of polyethylene glycol or its derivatives, polyoxyethylene polyoxypropylene glycol or its derivatives, fatty acid esters of sugars and sodium alkylsulfates, and more specifically, polyethylene glycol, polyoxyl 40 stearate, polyoxyethylene polyoxypropylene glycol, polyoxyethylene-polyoxypropylene glycol, polyoxyethylene- polyoxypropylene glycol, sucrose esters of fatty acids, sodium lauryl sulfate, sodium oleate, sodium chloride, and sodium deoxycholate (or sodium deoxycholate (DCA)) having an average molecular weight of more than 1500.

[0134] In addition, the water-soluble substance may comprise a substance selected from one or more of the group consisting of drugs, peptides, proteins, glycoproteins, polysaccharides or antigenic substances used as vaccines.

[0135] When a water-insoluble carrier is present, it may include a substance that serves to control the infiltration of water into the pores of the drug dispersion. One water-insoluble substance or a combination of two or more water-insoluble substances may be used.

[0136] The water-insoluble material may specifically be selected from one or more of the group of water-insoluble polymers, including water-insoluble acrylates, methacrylates and other carboxy polymers, resins and latexes, waxes, lipids, including phospholipids, and lipoproteins.

[0137] A person skilled in the art knows the amounts of carriers and any further excipients commonly used in pharmaceutical or vaccine compositions.

[0138] In one embodiment, the pharmaceutical carrier may comprise about 1% to 20% by weight, preferably about 10% to 20% by weight, based on the total weight of the vaccine composition.

[0139] The compositions according to the present invention may comprise one or a mixture of compounds as defined and claimed herein.

[0140] The compositions of the invention may be prepared in the form of a single mixture of adjuvant and antigen, or in the form of different mixtures for simultaneous or sequential administration of the components.

[0141] In a particular embodiment, the compound of formula 1 described in the present invention is the only adjuvant present in the vaccine composition.

[0142] The present invention also relates to a pharmaceutical composition comprising a compound of formula 1 as described in any one of the embodiments provided herein or in the claims, or a mixture thereof, and at least one pharma- ceutically acceptable excipient and / or carrier.

[0143] The composition may further comprise one or more additional therapeutically active principles.

[0144] The pharmaceutical composition may also be formulated in the form of an association of two or more active ingredients.

[0145] The pharmaceutical compositions of the present invention may comprise one or more compounds of formula 1 as described in any one of the embodiments provided herein or in the claims as the sole active ingredient, or may also comprise additional active ingredients, such as antitumor active ingredients, kinase inhibitors, cytotoxic compounds, and at least one pharma- ceutically acceptable carrier or excipient.

[0146] Pharmaceutical compositions can be formulated for oral, parenteral, nasal, aerosol, sublingual, rectal, vaginal, topical, intravenous or systemic administration.

[0147] Suitable conventional carriers and / or excipients for suspensions, emulsions, ointments, creams, sprays, granulates, powders, solutions, capsules, pills, tablets, lyophilized formulations, troches, aerosols, nebulization, injections or the like can be selected by those of skill in the art.

[0148] A further object of the present invention is a pharmaceutical composition according to any one of the embodiments disclosed herein for use in the treatment or as an adjuvant in the treatment of a disease requiring or benefiting from immune stimulation by activating the TLR4 receptor.

[0149] Diseases that require or benefit from immune stimulation by activating the TLR4 receptor are known in the art and include cancer, allergies, infectious diseases, cardiovascular diseases, obesity-dependent metabolic diseases, neurodegeneration, apoptotic diseases, autoimmune disorders, viral infections, bacterial infections, autoimmune diseases. Examples of autoimmune diseases are represented by IBD, Crohn's disease or rheumatoid arthritis.

[0150] According to the present invention, the composition may contain 0.01-50 mg of the compound of the present invention or a mixture thereof per daily dose, for example 0.01-50 mg of substance per Kg of body weight (animal tests).

[0151] The present invention also provides novel methods for the synthesis of compounds of formula 1 as defined herein and of compounds disclosed in formula X of WO2019 / 092572, [ka] R1 is a saturated C7-C 15 is an alkyl chain, R2 is a saturated C7-C 15 is an alkyl chain, R3 is a saturated C7-C 15 is an alkyl chain, R4 is OH, and each of R1, R2 and R3 does not contain an -OH substituent at the C2 position; and with respect to the synthesis of intermediates of formula 1i: [ka] R1 is saturated C5-C 15 It is an alkyl chain. According to one embodiment of the present invention, said R1 does not include any substituents.

[0152] The compound of formula 1, like the compounds disclosed in WO2019 / 092572, can be synthesized in a simpler, industrially scalable manner compared to the known synthetic methods in the art for the compounds of WO2019 / 092572 and SDZ MRL953. The latter requires the insertion of three (R)-3-hydroxymyristic acid acyl chains. Optically pure compounds (R-enantiomers) are not commercially available, only racemic mixtures are commercially available. In addition, (R)-3-hydroxymyristic acid requires a reaction to protect the hydroxyl group at position 3 before the condensation reaction with the sugar. The method disclosed in WO2019 / 092572 for synthesizing the compound of formula X defined above is already simplified with respect to the synthetic method disclosed for SDZ MRL953, since there is no substituent on the acyl chain, but still includes 10 steps; numerous purifications by chromatography columns and several key steps, such as the formation of low molecular weight azides. Moreover, the method disclosed in WO2019 / 092572 has a very low yield (about 8-9%), which makes the entire process uneconomical.

[0153] The compounds provided in the present invention exhibit biological activity comparable, if not superior, to compounds in the art and can be synthesized by much simpler, industrially scalable methods.

[0154] Therefore, an object of the present invention is a process for the preparation of an intermediate of formula 1i, [ka] R1 is saturated C5-C 15 is an alkyl chain, The method includes the following steps: 1) Selective acylation of the amino group at the C2 position of glucosamine hydrochloride by reaction with acyl chloride in the presence of sodium bicarbonate; 2) Selective silylation protection of the hydroxyl group at C6 by reaction with tert-butyldimethylsilyl chloride (TBDMSCl) in the presence of imidazole.

[0155] The present invention therefore also relates to intermediates of formula 1i, [ka] R1 is saturated C5-C 15 It is an alkyl chain.

[0156] According to one embodiment of the present invention, said R1 does not contain any substituents.

[0157] Furthermore, the present invention relates to a method for preparing a compound of formula 1 as defined in any of the above embodiments and claims, [ka] R1 is a saturated C5-C 15 is an alkyl chain, R2 is a saturated C5-C 15 is an alkyl chain, R3 is a saturated C5-C 15 is an alkyl chain, R4 is any substituent known to those skilled in the art that can be linked by a bond between C6 and a suitable atom and / or any substituent that has an oxygen or nitrogen atom that can be bonded to C6; The process includes the following steps: 1) Selective acylation of the amino group at the C2 position of glucosamine hydrochloride by reaction with acyl chloride in the presence of sodium bicarbonate; 2) selective silylation of the hydroxyl group at C6 by reaction with tert-butyldimethylsilyl chloride (TBDMSCl) in the presence of imidazole to give intermediates of formula 1i as defined in the previous embodiment; 3) selective acylation of the hydroxyl groups at the C1 and C3 positions by reaction with acyl chloride in the presence of triethylamine and N,N-dimethylaminopyridine (DMAP); 4) phosphorylation of the hydroxyl group at C4 position by reaction with dibenzyl N,N-diisopropylphospharamidite in the presence of triflate imidazolium, followed by oxidation of the phosphite to phosphate via metachloroperbenzoic acid; 5) deprotection of the hydroxyl group from the silane at C6 position in the presence of catalytic amounts of sulfuric acid; 6) Deprotection of the phosphate from the benzyl at C4 position via hydrogenation catalyzed by palladium on carbon (Pd / C) and, optionally, deprotection of the benzyl on any substituent at C6 position.

[0158] The process may alternatively comprise steps 3 to 6 starting from an intermediate of formula 1i as defined above and in the claims.

[0159] In one embodiment, the above synthesis method may comprise an additional step 5i) after step 5) and before step 6), 5i) phosphorylation of the hydroxyl group at C6 by reaction with dibenzyl N,N-diisopropylphospharamidite in the presence of triflate imidazolium, followed by oxidation of the phosphite to phosphate via metachloroperbenzoic acid; and The resulting R4 is a phosphate group (PO4 2- ).

[0160] When carried out, this method provides compounds of formula 1 where R4 is a phosphate group, such as compounds of formulas 7, 8 and 9.

[0161] Alternatively, the synthesis method may further comprise, after step 5) and before step 6), step 5ii) instead of step 5i): 5ii) Acylation of the hydroxy group at C6 position by reaction with a carboxylic acid in the presence of a suitable condensing agent and catalyst, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N,N-dimethylaminopyridine (DMAP), or by reaction with an acyl chloride in the presence of a suitable catalyst, such as N,N-dimethylaminopyridine (DMAP). The resulting R4 is an acyl group. When carried out, this method leads to compounds of formula 1 where R4 is an acyl group, such as compounds of formulas 10 and 11.

[0162] In a preferred embodiment, the suitable condensing agent and catalyst are 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N,N-dimethylaminopyridine (DMAP).

[0163] Alternatively, the synthesis method may further comprise, after step 5) and before step 6), step 5iii), instead of steps 5i) and 5ii): 5iii) Glycosylation of the hydroxyl group at C6 by reaction with a glycosyl chloride donor in the presence of silver(I) oxide as an activator, triflic acid as a catalyst and molecular sieves as water scavengers; or Glycosylation of the hydroxyl group at the C6 position by reaction of a glycosyl thioethyl (Set) donor in the presence of NIS (N-iodosuccinimide) as an activator and HOFox (3,3-difluorooxindole) as a catalyst and molecular sieves as a water scavenger; The resulting R4 is a glycosyl group.

[0164] Alternatively, the synthesis method may further comprise, after step 5) and before step 6), step 5iv) instead of steps 5i), 5ii) and 5iii): 5iv) Alkylation of the hydroxyl group at C6 by reaction of stabilized alkyl chlorides in the presence of silver(I) oxide as activator, trifluoromethanesulfonic acid as catalyst and molecular sieves as moisture scavenger. The resulting R4 is an n-alkyl group.

[0165] Alternatively, the synthesis method may further comprise, after step 5) and before step 6), steps 5v) and 5vi) instead of steps 5i), 5ii), 5iii) and 5iv): 5v) Tosylation at C6 by reaction of tosyl chloride in the presence of triethylamine as base and DMAP as catalyst; 5vi) azide insertion at C6 by reaction with sodium azide in the presence of tetrabutylammonium iodide; The resulting R4 is an azido group.

[0166] Alternatively, the synthesis method may further comprise, after step 5) and before step 6), steps 5vii) and 5viii) instead of steps 5i), 5ii), 5iii) and 5iv): 5vii) Glycosylation of the hydroxyl group at the C6 position by reaction of a glycosyl chloride donor bearing a picoloyl group in the presence of Bi(OTf)3 as the sole activating agent. The resulting R4 is a glycosyl group. 5viii) Removal of the picoloyl group by reaction with Cu(OAc)2.

[0167] According to the present specification, the acylation described in reactions 1), 3), and 5ii) can be carried out according to the methods commonly used by chemical engineers. For example, the acylation can be carried out using acyl chloride or carboxylic acid in the presence of other common condensing agents, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or dicyclohexylcarbodiimide (DCC).

[0168] The condensations mentioned in reactions 1) and 3) can be carried out with alkyl chains of different lengths, ranging from 5 to 15 carbon atoms, thereby obtaining different derivatives of the molecule described in formula 1.

[0169] The protection of C6 described in reaction 2) can be carried out according to the most common techniques known to those skilled in the chemical art, one of which is silylation in the presence of various non-nucleophilic bases such as triethylamine, diisopropylethylamine or sodium bicarbonate and catalysts such as N,N-dimethylaminopyridine (DMAP).

[0170] The phosphorylation of C4 described in reaction 4) can be carried out according to the most common techniques known to those skilled in the art of chemistry, such as the insertion of phosphites in the presence of different acidic pH buffers, such as but not limited to tetrazole or 4,5-dicyanoimidazole. The subsequent oxidation can be carried out by reaction with mild oxidizing agents, such as but not limited to dimethyldioxirane (DMDO) or tert-butylperoxide (tBuOOH).

[0171] The deprotection of C6 described in reaction 5) can be carried out by the most common techniques known to those skilled in the art of chemistry, such as the use of tetrabutylammonium fluoride (TBAF), acetic acid (AcOH) or various types of acidic resins, i.e. IRA 120 H + , IRC 120H + or desilylation in the presence of Dowex® 50W or the like.

[0172] The synthetic process according to the invention allows the preparation of compounds of formula 1 in a facile and industrially scalable manner.

[0173] As stated above, the present invention encompasses both the α and β anomers of the above defined compounds of formula 1. The inventors have surprisingly discovered that depending on the temperature and amount of appropriate catalyst in acylation step 3, either the α or β anomer may be obtained.

[0174] Thus, if the β anomer is desired, the acylation step 3) is carried out at temperatures ranging from −78° C. to 0° C. and with amounts of DMAP ranging from 0.05 to 0.2 equivalents.

[0175] In a preferred embodiment, to synthesize the β anomer, acylation step 3 is carried out at −20° C. with 0.1 equivalents of DMAP.

[0176] On the other hand, if the α anomer is desired, the acylation step 3) is carried out at a temperature ranging from 20° C. to 50° C. using a range of 2 to 2.5 equivalents of DMAP.

[0177] In a preferred embodiment, to synthesize the alpha anomer, acylation step 3 is carried out with 2.02 equivalents of DMAP at a temperature of about 30°C.

[0178] The methods defined herein can be used to synthesize one of the embodiments of the compounds of formula 1 disclosed herein (such as compounds of formulas 2-12). Those skilled in the art will know the substituents to use based on their general knowledge in organic chemistry.

[0179] Advantageously, the present invention also provides a method for the synthesis of a compound of formula X, [ka] R1 is a saturated C5-C 15 is an alkyl chain, R2 is a saturated C5-C 15 is an alkyl chain, R3 is a saturated C5-C 15 It is an alkyl chain. R4 is OH, and each of R1, R2 and R3 does not contain an -OH substituent at the C2 position; The process includes the following steps: 1) Selective acylation of the amino group at the C2 position of glucosamine hydrochloride by reaction with acyl chloride in the presence of sodium bicarbonate; 2) selective silylation of the hydroxyl group at C6 by reaction with tert-butyldimethylsilyl chloride (TBDMSCl) in the presence of imidazole to give intermediates of formula 1i as defined in the previous embodiment; 3) Complete acylation of the hydroxyl groups at C1, C3 and C4 positions by reaction with acyl chloride in the presence of triethylamine and N,N-dimethylaminopyridine (DMAP); 4) Selective diacylation at C1 by reaction with ethylenediamine in the presence of acetic acid; 5) phosphorylation of the hydroxyl group at C1 position by reaction with dibenzyl N,N-diisopropylphospharamidite in the presence of triflate imidazolium, followed by oxidation of the phosphite to phosphate via metachloroperbenzoic acid; 6) deprotection of the hydroxyl group from the silane at the C6 position in the presence of a catalytic amount of a 5% solution of sulfuric acid in water; 7) Deprotection of the phosphate from the benzyl at C4 position via hydrogenation catalyzed by palladium on carbon (Pd / C) and, optionally, deprotection of the benzyl on any substituent at C6 position.

[0180] According to one embodiment of the present invention, said R1R2 and R3 do not contain any substituents.

[0181] The present invention also relates to the use of an intermediate compound of formula 1i as defined in any one of the embodiments disclosed herein for the synthesis of a compound of formula 1: [ka] R1 is a saturated C5-C 15 is an alkyl chain, R2 is a saturated C5-C 15 is an alkyl chain, R3 is a saturated C5-C 15 is an alkyl chain, R4 is any substituent that can be linked by a bond between C6 and a suitable atom and / or any substituent that has an oxygen or nitrogen atom that can be bonded to C6.

[0182] According to one embodiment of the present invention, said R1R2 and R3 do not contain any substituents.

[0183] Furthermore, the present invention relates to the use of an intermediate of formula 1i, as defined in any one of the embodiments disclosed herein, for the synthesis of a compound of formula X: [ka] R1 is a saturated C5-C 15 is an alkyl chain, R2 is a saturated C5-C 15 is an alkyl chain, R3 is a saturated C5-C 15 is an alkyl chain, R4 is OH, and each of R1, R2 and R3 does not contain an -OH substituent at the C2 position.

[0184] According to one embodiment of the present invention, said R1R2 and R3 do not contain any substituents.

[0185] An object of the present invention is also a process for the preparation of a pharmaceutical formulation or a vaccine composition comprising the steps of the process described above and at least one step of mixing said product obtained in 6) of pharma-ceutically acceptable grade with at least one pharma-ceutically acceptable carrier and / or excipient.

[0186] In any part of this specification and claims, the term "comprising" may be replaced by the term "consisting of."

[0187] Pursuant to Article 170bis of the Italian Patent Law, the following is hereby declared: All experiments involving cells were performed using commercially available cells; With regard to the mouse models used in the described experiments, the obligations deriving from national or EU regulations and, in particular, the provisions referred to in paragraph 6 of Legislative Decree No. 206 of 12 April 2001 and Legislative Decree No. 224 of 8 July 2003 have been fulfilled. EXAMPLES

[0188] chemistry All reagents and solvents were purchased from commercial sources and used without further purification unless otherwise stated. Reactions were monitored by thin layer chromatography (TLC) performed on Silica Gel 60 F254 plates (Merck®). Flash chromatographic purification was performed using silica gel 60 60-75 μm from commercial sources.

[0189] 1H and 13C NMR spectra were recorded on a Bruker Advance 400 using TopSpin® software or an NMR Varian 400 using Vnmrj software. Chemical shifts are expressed in ppm for Me4Si; coupling constants are expressed in Hz. The multiplicity of the 13C spectra was subtracted by APT experiments.

[0190] Synthesis of 13 [ka] Glucosamine hydrochloride 12 (10 g, 46.5 mmol, 1 eq.) and NaHCO3 (10.54 g, 126 mmol, 2.7 eq.) were dissolved in water (120 ml). Lauroyl chloride (11.20 g, 51.2 mmol, 1.1 eq.) previously dissolved in THF (120 ml) was then added dropwise to the solution at 0° C. The reaction was stirred for 5 h, after which the solution was filtered. A white solid was obtained, which was washed with water and THF at 4° C. The excess water was then coevaporated with toluene under reduced pressure to give the desired product 13 as a white powder in 60% yield (10.10 g). The compound was used without further purification.

[0191] 1H NMR(400MHz,DMSO)δ7.68(d,J=8.1Hz,1H),7.52(d,J=7.7Hz,3H),6.46(d,J=6.3Hz,1H),6.37(d,J= 4.0Hz,3H),4.97-4.86(m,7H),4.81(d,J=4.7Hz,1H),4.62(d,J=5.0Hz,3H),4.53(t,J=5.7Hz,1H), 4.43(dd,J=9.5,4.3Hz,4H),3.73-3.42(m,18H),3.34-3.22(m,2H),3.16-3.09(m,3H),3.04(d,J=1 4.1Hz, 2H), 2.13-2.03 (m, 8H), 1.56-1.37 (m, 9H), 1.26 (d, J=14.5Hz, 67H), 0.86 (t, J=6.8Hz, 13H).

[0192] 13C NMR(101MHz,DMSO)δ173.31,172.82,96.11,91.05,77.21,74.74,72.49,71.59,71.32,70.83,61.58,57.57,54.73,40.59,40.38 ,40.17,39.96,39.75,39.54,39.33,36.18,35.74,31.77,29.53,29.49,29.43,29.37,29.23,29.18,29.14,25.78,22.56,14.42.

[0193] Synthesis of 14 [ka] To a solution of 13 (3 g, 8.3 mmol, 1 eq.) and imidazole (850 mg, 12.4 mmol, 1.5 Eq.) in dimethylsulfoxide (166 ml, 0.05 M), a solution of TBDMSCl (1.4 g, 9.1 mmol, 1.1 eq.) in DCM (15 ml) was added dropwise under inert atmosphere in an ice bath. The solution was then allowed to warm to room temperature and stirred overnight. The reaction, monitored by TLC (DCM / MeOH 9:1), was then stopped and the solution was concentrated under reduced pressure. It was then diluted with AcOEt and washed three times with NH4Cl. The organic phase thus obtained was dried over Na2SO4 and the solvent was removed on a rotavapor. The raw product thus obtained (3.65 g) was resuspended in EtPet at 0 °C for 30 min. The suspension was then filtered under vacuum and the desired compound was recovered as a white solid. After purification, 3.5 g of compound 14 was obtained as a whiteish solid in 85% yield.

[0194] 1H NMR(400MHz,DMSO)δ7.62(d,J=7.9Hz,1H),6.43(d,J=6.4Hz,1H),4.90(t,J=6.5 Hz,1H),4.77(t,J=9.1Hz,1H),4.42(t,J=7.0Hz,1H),3.86(d,J=10.8Hz,1H),3. 66(dd,J=11.0,4.6Hz,1H),3.30(d,J=7.9Hz,1H),3.14-2.98(m,1H),2.06(t,J= 7.4Hz, 1H), 1.48 (s, 1H), 1.24 (s, 3H), 0.94-0.74 (m, 2H), 0.05 (d, J=3.0Hz, 1H).

[0195] 13C NMR(101MHz,DMSO)δ173.21,95.93,77.09,74.83,70.82,63.61,57.54,40.61,40.40,40.20,39.99,39.78,39.57, 39.36,36.20,31.78,29.54,29.51,29.45,29.39,29.20,29.14,26.41,25.76,22.57,18.64,14.41,-4.66,-4.67.

[0196] Synthesis of 15 [ka] Compound 14 (2.0 g, 4.2 mmol, 1 eq.) and 4-dimethylaminopyridine (26 mg, 0.2 mmol, 0.05 Eq.) were dissolved in anhydrous THF (84 ml, 0.05 M) under Ar atmosphere. Triethylamine (2.4 ml, 17.2 mmol, 4.1 Eq.) and lauroyl chloride (2.10 ml, 8.5 mmol, 2.0 eq.) were added dropwise to the solution at -20°C. The reaction was stirred at -20°C for 2 h and then controlled by TLC (EtPet / AcOEt 6:4). The solution was then diluted with AcOEt and washed with 1 M HCl. The organic phase thus obtained was dried over Na2SO4 and the solvent was removed on a rotavapor. The crude product thus obtained (4 g) was purified using flash column chromatography (Tol / AcOEt 9:1). After purification, 2.1 g of compound 15 was obtained in 50% yield.

[0197] 1H NMR (400 MHz, DMSO) δ 7.80 (d, J = 9.5 Hz, 1H), 5.56 (d, J = 8.9 Hz, 1H), 5.38 (d, J = 5.9 Hz, 1H), 4.92 (dd, J = 10.6, 8.6 Hz, 1H), 3.83 (dd, J = 10.4, 5.8 Hz, 2H), 3.76 - 3.70 (m, 1H), 3.38 (dd, J = 14.3, 8.5 Hz, 2H), 2.30 - 2.14 (m, 7H), 1.94 (t, J = 7.3 Hz, 2H), 1.44 (dd, J = 25.9, 6.4 Hz, 10H), 1.24 (d, J = 2.4 Hz, 75H), 0.90 - 0.81 (m, 24H), 0.07 - 0.01 (m, 6H).

[0198] 13C NMR (101 MHz, DMSO) δ 174.93, 172.72, 172.34, 171.74, 92.49, 77.48, 75.66, 67.73, 62.54, 52.26, 40.65, 40.44, 40.23, 40.02, 39.82, 39.61, 39.40, 36.08, 34.13, 33.94, 31.78, 31.74, 29.59, 29.52, 29.50, 29.44, 29.39, 29.35, 29.30, 29.19, 29.00, 28.93, 28.75, 26.26, 25.70, 24.95, 24.77, 22.55, 18.54, 14.39, 14.36, -4.71, -4.78.

[0199] Synthesis of 16

Chemical formula

[0200] After TLC analysis, the reaction was quenched with 15 ml of saturated NaHCO3 solution and concentrated on a rotavapor. The mixture was then diluted with AcOEt and washed three times with saturated NaHCO3 solution and three times with 1M HCl solution. The organic phase was collected, dried over Na2SO4 and the solvent was removed on a rotavapor.

[0201] The crude product thus obtained was purified by flash column chromatography (EtPet / acetone 9:1) to give 2.41 g of pure compound 16 as a yellow oil in 91% yield.

[0202] 1H NMR (400 MHz, CDCl3) δ 7.34 - 7.25 (m, 10H), 5.61 (d, J = 8.7 Hz, 1H), 5.44 (d, J = 9.6 Hz, 1H), 5.16 (dd, J = 10.8, 9.1 Hz, 1H), 5.00 (dd, J = 8.1, 2.8 Hz, 2H), 4.96 - 4.91 (m, 2H), 4.53 (q, J = 9.2 Hz, 1H), 4.23 (dt, J = 10.8, 9.5 Hz, 1H), 3.91 (dd, J = 11.9, 1.8 Hz, 1H), 3.78 (dd, J = 11.9, 4.6 Hz, 1H), 3.56 (ddd, J = 9.6, 4.4, 1.7 Hz, 1H), 2.31 (td, J = 7.5, 3.5 Hz, 2H), 2.19 (t, J = 7.7 Hz, 2H), 2.07 - 2.01 (m, 2H), 1.61 - 1.37 (m, 6H), 1.33 - 1.10 (m, 50H), 0.92 - 0.83 (m, 19H), 0.03 - -0.03 (m, 6H).

[0203] 13C NMR (101 MHz, CDCl3) δ 174.43, 172.75, 172.40, 135.52, 128.60, 128.56, 127.88, 127.83, 92.59, 77.31, 77.00, 76.68, 76.23, 76.16, 72.94, 72.89, 69.56, 69.51, 69.46, 61.63, 52.79, 36.76, 34.08, 33.94, 31.89, 29.65, 29.60, 29.49, 29.47, 29.43, 29.37, 29.33, 29.25, 29.11, 29.01, 25.82, 25.58, 24.63, 24.58, 22.66, 18.32, 14.07, -5.19, -5.32.

[0204] Synthesis of 17

Chem.

[0205] 1H NMR(400MHz,CDCl3)δ7.40-7.27(m,1H),5.63(d,J=8.8Hz,1H),5.45(d,J=9.6Hz,1H), 5.18(dd,J=10.7,9.3Hz,1H),5.08-4.91(m,1H),4.54(q,J=9.5Hz,1H),4.26(dd,J=19 .9,9.3Hz,1H),3.87-3.74(m,1H),3.47(d,J=9.7Hz,1H),2.40-2.24(m,1H),2.10-1.9 1(m,1H),1.61-1.46(m,1H),1.46-1.33(m,1H),1.33-1.01(m,5H),0.92-0.83(m,1H).

[0206] 13C NMR(101MHz,CDCl3)δ174.11,172.77,172.49,128.94,128.84,128.72,128.66,128.2 6,127.95,92.61,77.33,77.01,76.69,75.90,75.87,72.46,72.42,72.15,72.10,70.2 3, 70.17, 70.10, 60.23, 52.78, 36.71, 34.03, 33.71, 31.90, 29.67, 29.62, 29.49, 29.44, 29.38, 29.34, 29.32, 29.26, 29.23, 29.04, 29.01, 25.56, 24.59, 24.48, 22.66, 14.09.

[0207] Synthesis of 1 [ka] Compound 17 (50 mg, 0.05 mmol, 1 Eq) was dissolved in a mixture of DCM (2.5 mL) and MeOH (2.5 mL) and placed under an Ar atmosphere. Pd / C catalyst (10 mg, 20% m / m) was then added to the solution. The reaction environment was then subsequently degassed under an H2 atmosphere. The solution was stirred for 2 h, after which the H2 was removed and the reaction was monitored by TLC (EtPet / Acetone 8:2).

[0208] Triethylamine (100 μL) was then added to the reaction and stirred for 15 min. Afterwards, the solution was filtered through a PALL 4549T Acrodisc 25 mm syringe filter with GF / 0.45 μm Nylon to remove the Pd / C catalyst, and the solvent was evaporated on a rotavapor. The crude product was resuspended in a DCM / MeOH solution and purified by IRA 120H. + After stirring for 30 minutes, IRA 120H was added. + The mixture was filtered, the solvent removed on a rotavapor, and the crude was resuspended in DCM / MeOH and treated with IRA 120 Na + After stirring for 30 minutes, IRA 120 Na + It was filtered and the solvent was removed on a rotavapor.

[0209] (45 mg) of 1 was obtained as a white powder in quantitative yield.

[0210] 1H NMR(400MHz,cd3od)δ5.75(d,J=8.9Hz,1H),5.28(t,J=9.8Hz,1H),4.28(q,J=9.7Hz,1H),4.06(t,J=9.6Hz,1H),3.89-3.74(m,2 H),3.62(t,J=9.2Hz,1H),2.42-2.25(m,5H),2.09(t,J=7.6Hz,2H),1.56(d,J=6.4Hz,7H),1.29(s,53H),0.90(t,J=6.6Hz,9H).

[0211] 13C NMR(101MHz,MeOD)δ174.69,173.32,172.00,92.16,76.22,76.17,72.81,72.7 8,72.20,72.14,60.30,52.82,48.23,48.02,47.81,47.59,47.38,47.17,46.96 ,36.05,33.64,33.55,31.67,31.66,29.45,29.39,29.38,29.35,29.26,29.20,29.19,29.14,29.07,29.05,29.02,28.92,28.74,25.58,24.38,22.31,13.00.

[0212] Synthesis of 18 [ka] Compound 17 (2.36 g, 2.4 mmol, 1 eq.) and imidazole triflate (1.4 g, 5.4 mmol, 2.25 Eq.) were dissolved in DCM (121 mL, 0.02 M) under inert atmosphere. Dibenzyl N,N-diisopropyl phosphoramidite (1.83 g, 5.3 mmol, 2.2 eq.) was added to the solution at 0° C. The reaction was monitored by TLC (EtPet / acetone 9:1); after 30 min, depletion of the substrate was detected. The solution was then cooled at −20° C. and metachloroperbenzoic acid (1.66 g, 9.7 mmol, 4 Eq) dissolved in 17 ml of DCM was added dropwise. After 30 min, the reaction was allowed to warm to room temperature and left stirring overnight.

[0213] After TLC analysis, the reaction was quenched with 15 ml of saturated NaHCO3 solution and concentrated on a rotavapor. The mixture was then diluted with AcOEt and washed three times with saturated NaHCO3 solution and three times with 1M HCl solution. The organic phase was collected, dried over Na2SO4 and the solvent was removed on a rotavapor.

[0214] The crude material thus obtained was purified by flash column chromatography (EtPet / acetone 9:1) to give 2.41 g of pure compound 18 as a yellow oil in 91% yield.

[0215] 1H NMR(400MHz,CDCl3)δ7.33-7.18(m,21H),5.66(d,J=8.8Hz,1H),5.51(d,J=9.5Hz,1H),5.18(dd,J=10.6,9.2Hz,1H ),5.02(dd,J=10.8,3.3Hz,4H),5.00-4.95(m,2H),4.94-4.88(m,2H),4.49-4.43(m,1H),4.42-4.36(m,1H),4.25( dd,J=19.8,9.3Hz,1H),4.16(ddd,J=11.8,7.1,5.0Hz,1H),3.74(dd,J=9.5,4.2Hz,1H),2.19(dt,J=15.9,7.0Hz,5 H),2.07-2.01(m,2H),1.49(dt,J=14.0,7.1Hz,4H),1.45-1.36(m,2H),1.34-1.11(m,54H),0.88(t,J=6.8Hz,10H).

[0216] 13C NMR(101MHz,CDCl3)δ174.22,172.82,172.18,135.79,135.72,135.33,128.62,128.5 8,128.52,128.05,128.00,127.96,92.44,74.11,72.59,72.39,69.38,65.25,52.68.

[0217] Synthesis of 6 [ka] Compound 18 (57 mg, 0.05 mmol, 1 Eq) was dissolved in a mixture of DCM (2.5 mL) and MeOH (2.5 mL) and placed under Ar atmosphere. Pd / C catalyst (10 mg, 20% m / m) was then added to the solution. The reaction environment was then subsequently degassed under H2 atmosphere. The solution was stirred for 2 h, after which the H2 was removed and the reaction was monitored by TLC (EtPet / Acetone 8:2).

[0218] Triethylamine (100 μL) was added to the reaction and stirred for 15 min. The solution was filtered through a PALL 4549T Acrodisc 25 mm syringe filter with GF / 0.45 μm Nylon to remove the Pd / C catalyst, and the solvent was evaporated on a rotavapor. The crude product was resuspended in a DCM / MeOH solution and purified by IRA 120 H + After stirring for 30 minutes, IRA 120 H + The mixture was filtered, the solvent removed on a rotavapor, and the crude was resuspended in DCM / MeOH and treated with IRA 120 Na + After stirring for 30 minutes, IRA 120 Na + It was filtered and the solvent was removed on a rotavapor.

[0219] (45 mg) of 6 was obtained as a white powder in quantitative yield.

[0220] 1H NMR(400MHz,cd3od)δ5.77(d,J=8.8Hz,1H),5.32-5.23(m,1H),4.39(dd,J=18.9,9.5Hz,1H),4.21(d,J=9.7Hz,3H),4.10-4.00(m,1H), 3.80(d,J=9.2Hz,1H),2.44-2.24(m,6H),2.09(t,J=7.6Hz,2H),1.55(dd,J=13.5,6.9Hz,10H),1.39-1.24(m,79H),0.96-0.82(m,33H).

[0221] 13C NMR(101MHz,MeOD)δ174.87,173.81,91.22,72.86,72.80,71.25,68.94,68.86,68.80,64 .65,64.61,52.10,48.24,48.03,47.82,47.61,47.39,47.18,46.97,36.10,35.63,33.76 ,33.64,33.55,33.40,31.69,31.63,29.26,29.22,29.18,29.15,29.11,29.06,29.03,28.98,28.84,28.78,25.68,25.62,24.69,24.63,24.38,22.35,22.32,13.06,13.03,7.82.

[0222] Synthesis of 19 [ka]

[0223] Compound 17 (100 mg, 0.1 mmol, 1 eq.) and silver(I) oxide (140 mg, 0.6 mmol, 6 eq.) were dissolved in toluene (1 mL, 0.1 M) under an inert atmosphere. Allyl bromide (51 μL, 0.6 mmol, 6 eq.) was added to the solution at RT. The reaction was left stirring overnight.

[0224] After TLC analysis (EtPet / Acetone 8:2), the reaction was stopped and the solution was filtered over a celite pad. The organic liquid phase was collected and the solvent was removed on a rotavapor.

[0225] The crude material thus obtained was purified by flash column chromatography (EtPet / Acetone 8:2) to give 50 mg of pure compound 19 as a yellow oil in 50% yield.

[0226] 1H NMR (400 MHz, CDCl3) δ 7.38 - 7.22 (m, 10H), 5.91 - 5.77 (m, 1H), 5.64 (d, J = 8.8 Hz, 1H), 5.25 - 5.07 (m, 3H), 5.04 - 4.91 (m, 4H), 4.56 (q, J = 9.3 Hz, 1H), 4.26 (dd, J = 19.8, 9.3 Hz, 1H), 3.99 - 3.90 (m, 2H), 3.73 (dd, J = 11.0, 1.5 Hz, 1H), 3.69 (dd, J = 9.6, 4.4 Hz, 1H), 3.60 (dd, J = 11.0, 4.4 Hz, 1H), 2.40 - 2.24 (m, 2H), 2.18 (dd, J = 16.1, 8.4 Hz, 2H), 2.03 (dd, J = 15.1, 7.1 Hz, 2H), 1.62 - 1.53 (m, 2H), 1.49 (dd, J = 14.2, 7.2 Hz, 2H), 1.41 (dt, J = 13.2, 6.8 Hz, 2H), 1.34 - 1.10 (m, 51H), 0.88 (t, J = 6.8 Hz, 9H).

[0227] 13C NMR (101 MHz, CDCl3) δ 174.33, 172.77, 172.41, 135.48, 134.43, 128.64, 128.59, 127.92, 117.20, 92.70, 77.32, 77.00, 76.68, 75.29, 75.23, 73.21, 73.15, 72.83, 72.47, 69.67, 69.63, 67.81, 52.83, 36.74, 34.05, 33.93, 31.89, 29.65, 29.62, 29.60, 29.49, 29.44, 29.36, 29.33, 29.31, 29.27, 29.23, 29.10, 29.00, 25.55, 24.57, 24.50, 22.65, 14.07.

[0228] Synthesis of 21

Chem.

[0229] After completion of the reaction, the solution was diluted with AcOEt and washed with saturated NaHCO3 solution three times.The organic phase thus obtained was dried over Na2SO4 and the solvent was removed on a rotavapor.

[0230] The crude material thus obtained was purified by flash column chromatography (EtPet / acetone 85:15) to give 100 mg of pure compound 21 as a white powder in 65% yield.

[0231] Synthesis of 23 [ka] Compound 17 (100 mg, 0.1 mmol, 1 eq.), compound 22 (57 mg, 0.13 mmol, 1.25 eq.) and powdered 3a molecular sieves (50 mg) were dissolved in toluene (1 mL, 0.1 M) under inert atmosphere and stirred for 1 h. Silver(I) oxide (46 mg, 0.2 mmol, 2 Eq.) was then added to the solution at RT, which was then cooled to 0° C. and trifluoromethanesulfonic acid (4.4 μL, 0.05 mmol, 0.5 eq.) was added. The reaction was then left stirring overnight at room temperature.

[0232] After TLC analysis (EtPet / acetone 8:2), the reaction was stopped and the solution was filtered on a celite pad. The organic phase was collected, diluted with AcOEt and washed three times with NaHCO3. The organic phase was collected, dried over Na2SO4 and evaporated.

[0233] The crude material thus obtained was purified by flash column chromatography (toluene / acetone 85:15) to give 50 mg of a mixture of diastereoisomers of compound 23 as a yellow oil in 40% yield.

[0234] 1H NMR(400MHz,CDCl3)δ7.40-7.20(m,38H),5.65(d,J=8.8Hz,1H),5.61(s,1H),5.40(s,1H) ),5.24-5.18(m,1H),5.14(s,1H),4.91(d,J=11.6Hz,9H),4.69(s,2H),4.62(d,J=2.2Hz ,4H),4.57-4.37(m,2H),4.23(s,1H),2.40-2.26(m,3H),2.13(ddd,J=13.7,7.6,4.4Hz, 3H), 2.05 (s, 3H), 1.57 (s, 6H), 1.47-1.36 (m, 3H), 1.29 (s, 79H), 0.88 (t, J=6.8Hz, 14H).

[0235] 13C NMR(101MHz,CDCl3)δ174.23,172.81,172.43,138.66,138.63,135.24,128.75,128.69,128.67,128.62,128.50,128.40,1 28.31,128.27,128.20,128.06,127.92,127.88,127.71,127.64,127.56,127.41,127.39,98.55,92.54,80.36,79.72,77. 32, 77.00, 76.68, 75.36, 74.99, 72.76, 72.51, 71.93, 69.72, 69.66, 69.63, 69.58, 68.09, 64.99, 52.74, 36.77, 34.03, 33.89, 31.89, 30.90, 29.66, 29.60, 29.49, 29.46, 29.36, 29.34, 29.22, 29.09, 29.02, 25.59, 24.58, 24.46, 22.66, 17.97, 14.09.

[0236] Synthesis of 24 [ka] Compound 23 (70 mg, 0.05 mmol, 1 Eq) was dissolved in a mixture of DCM (2.5 mL) and MeOH (2.5 mL) and placed under an Ar atmosphere. Pd / C catalyst (10 mg, 20% m / m) was then added to the solution. The reaction environment was then subsequently degassed under an H2 atmosphere. The solution was stirred for 2 h, after which the H2 was removed and the reaction was monitored by TLC (EtPet / Acetone 8:2).

[0237] Triethylamine (100 μL) was then added to the reaction and stirred for 15 min. Afterwards, the solution was filtered through a PALL 4549T Acrodisc 25 mm syringe filter with GF / 0.45 μm Nylon to remove the Pd / C catalyst, and the solvent was evaporated on a rotavapor. The crude product was resuspended in a DCM / MeOH solution and purified by IRA 120 H + After stirring for 30 minutes, IRA 120 H + The mixture was filtered, the solvent removed on a rotavapor, and the crude was resuspended in DCM / MeOH and treated with IRA 120 Na + After stirring for 30 minutes, IRA 120 Na + It was filtered and the solvent was removed on a rotavapor.

[0238] (50 mg) of 24 was obtained as a white powder in quantitative yield as a mixture of diastereoisomers.

[0239] 1H NMR(400MHz,MeOD)δ5.76(dd,J=8.8,4.7Hz,1H),5.29(dd,J=10.5,9.1Hz,1H),4.76(d, J=1.2Hz,1H),4.36(dd,J=18.8,9.4Hz,1H),4.08(ddt,J=19.6,14.2,5.9Hz,4H),3.91(d d,J=3.4,1.6Hz,2H),3.83-3.75(m,2H),3.75-3.62(m,5H),3.44-3.34(m,3H),2.48-2. 27(m,7H),2.14-2.08(m,2H),1.60(s,11H),1.40-1.21(m,96H),0.92(t,J=6.8Hz,17H).

[0240] 13C NMR(101MHz,MeOD)δ174.68,173.39,171.99,101.03,92.10,75.14,72.96,72.66,72.32,70.90,70.57,68.47,65.71,52.71,48.23,48.02,4 7.81,47.59,47.38,47.17,46.96,36.06,33.66,33.57,31.69,29.39, 29.30,29.09,28.95,28.76,28.44,25.59,24.42,22.33,16.62,13.03.

[0241] Synthesis of 29 [ka] Compound 17 (100 mg, 0.11 mmol, 1 eq.) and compound 28 (24 mg, 0.11 mmol, 1.1 eq.) were dissolved in dry DCM (1 ml, 0.1 M) under Ar atmosphere. EDC (23 mg, 0.12 mmol, 1.2 eq.) and DMAP (0.112 mg, 0.01 mmol, 0.1 eq.) were then added to the solution at 0° C. Afterwards, the solution was allowed to warm to room temperature and stirred overnight. The reaction, monitored by TLC (EtPet / acetone 8:2), was then stopped and the solution was concentrated under reduced pressure. It was then diluted with AcOEt and washed three times with HCl. The organic phase thus obtained was dried over Na2SO4 and the solvent was removed on a rotavapor. The crude product thus obtained (550 mg) was purified using flash column chromatography (EtPet / acetone 85:15). After purification, 17 mg of compound 29 was obtained in 40% yield.

[0242] 1H NMR (400 MHz, CDCl3) δ 7.43 (dd, J = 6.8, 2.9 Hz, 1H), 7.36 - 7.19 (m, 10H), 5.60 (d, J = 8.7 Hz, 1H), 5.45 (s, 1H), 5.36 (d, J = 9.7 Hz, 1H), 5.15 (dd, J = 10.7, 9.1 Hz, 1H), 4.97 (t, J = 9.9 Hz, 1H), 4.93 - 4.80 (m, 1H), 4.67 (ddd, J = 18.4, 12.5, 2.3 Hz, 2H), 4.51 (q, J = 9.3 Hz, 1H), 4.22 (td, J = 12.5, 7.2 Hz, 1H), 3.79 (dd, J = 9.5, 3.1 Hz, 1H), 3.64 (d, J = 10.2 Hz, 1H), 2.36 - 2.23 (m, 1H), 2.21 - 2.09 (m, 1H), 2.09 - 1.99 (m, 1H), 1.04 (s, 2H), 0.98 - 0.75 (m, 5H).

[0243] 13C NMR (101 MHz, CDCl3) δ 172.86, 134.44, 129.73, 128.98, 128.68, 128.27, 128.08, 127.97, 127.89, 126.22, 126.09, 101.61, 92.55, 77.31, 76.99, 76.67, 73.09, 72.47, 72.27, 69.82, 67.34, 61.47, 52.67, 50.48, 42.54, 36.74, 34.02, 31.89, 29.60, 29.44, 29.33, 28.99, 25.57, 24.50, 22.66, 19.14, 17.39, 14.08.

[0244] Synthesis of 30

Chem.

[0245] 1 H NMR(400MHz,MeOD)δ5.76(d,J=8.9Hz,1H),5.33-5.24(m,1H),4.54-4.38( m,2H),4.37-4.29(m,1H),4.14-4.05(m,1H),3.89(d,J=7.3Hz,1H),3.78(q ,J=9.0Hz,1H),3.67(dd,J=16.4,11.6Hz,4H),3.56(t,J=6.6Hz,1H),2.49 -2.27(m,5H),2.23-2.08(m,2H),1.59(d,J=6.4Hz,7H),1.22-1.15(m,3H).

[0246] Synthesis of 32 [ka] Compound 17 (100 mg, 0.1 mmol, 1 eq.), compound 31 (110 mg, 0.2 mmol, 2 eq.) and powdered 3a molecular sieves (330 mg) were dissolved in DCM (2 mL, 0.2 M) under inert atmosphere and stirred for 1 h. NIS (45 mg, 0.2 mmol, 2 Eq.) and HOFox (8.5 mg, 0.5 mmol, 0.5 eq.) were then added to the solution at RT. The reaction was then stirred at room temperature for 1.5 h.

[0247] After TLC analysis (EtPet / acetone 8:2), the reaction was stopped and the solution was filtered on a cotton pad. The organic liquid phase was collected, diluted with AcOEt, and washed with Na2S2O3 three times. The organic phase was collected, dried over Na2SO4, and evaporated.

[0248] The crude material thus obtained was purified by flash column chromatography (EtPet / acetone 80:20) to give 120 mg of a mixture of diastereoisomers of compound 32 as a yellow oil in 84% yield.

[0249] 1H NMR(400MHz,CDCl3)δ7.40-7.20(m,32H),7.11(ddd,J=13.3,6.8,2.7Hz,2H),5.62(dd,J=8.8,3.8Hz,1H),5.34(d,J=9.5Hz,1H),5.27(d,J=9. 6Hz,0H),5.13(td,J=10.9,8.9Hz,1H),5.00-4.85(m,6H),4.79(dd,J=10.9,2.5Hz,1H),4.73(d,J=11.0Hz,1H),4.70-4.64(m,2H),4.63-4.54( m,2H),4.53-4.45(m,1H),4.44-4.21(m,4H),3.93(t,J=9.3Hz,1H),3. 87-3.75(m,3H),3.70-3.51(m,5H),3.46-3.33(m,1H),2.22(t,J=7.6Hz ,1H),2.13(dt,J=17.2,7.7Hz,3H),2.03(q,J=7.3Hz,3H),1.50(s,3H),1.40(p,J=7.1Hz,3H),1.22(d,J=16.1Hz,52H),0.88(t,J=6.7Hz,10H).

[0250] 13C NMR(101MHz,CDCl3)δ174.35,172.75,172.22,138.97,138.22,138.03,128.74,128.69,128.67,128.59,128.45,128.35,128.29,128.2 2,128.12,128.02,127.91,127.89,127.83,127.80,127.73,127.69,127.63,127.48,127.43,103.88,97.27,92.65,92.54,84.49,81.90 ,79.78,77.61,77.50,77.34,77.02,76.70,75.58,74.95,74.64,73.43,73.33,73.27,72.80,72.71,70.19,69.73,68.41,52.92,36.83,34.03,33.94,31.93,29.69,29.64,29.51,29.47,29.39,29.37,29.33,29.26,29.14,29.04,25.64,24.60,24.53,24.36,22.69,14.12.

[0251] Synthesis of 34 [ka] Compound 17 (100 mg, 0.1 mmol, 1 eq.), compound 33 (110 mg, 0.2 mmol, 2 eq.) and powdered 3a molecular sieves (330 mg) were dissolved in DCM (2 mL, 0.2 M) under inert atmosphere and stirred for 1 h. The reaction was cooled at 0 °C and Bi(OTf)3 (50 mg, 0.075 mmol, 0.75 Eq) was added. The reaction was then left stirring at RT overnight.

[0252] After TLC analysis (EtPet / acetone 7:3), the reaction was stopped and the solution was filtered on a celite pad. The organic phase was collected, diluted with AcOEt and washed three times with NaHCO3. The organic phase was collected, dried over Na2SO4 and evaporated.

[0253] The crude material thus obtained was purified by flash column chromatography (EtPet / acetone 70:30) to separate the two diastereoisomers, affording 125 mg of total compound 34 (α+β) as a yellow oil in 94% yield.

[0254] 34α 1H NMR(400MHz,CDCl3)δ8.75(dt,J=4.6,1.4Hz,1H),8.06(d,J=7.8Hz,1H),7.89(td,J=7.7 ,1.8Hz,1H),7.47(ddd,J=7.6,4.7,1.2Hz,1H),7.40-7.13(m,30H),5.53(d,J=8.8Hz,1H ),5.32(d,J=9.7Hz,1H),5.10(dd,J=10.8,8.9Hz,1H),4.99-4.90(m,5H),4.89(d,J=3.5 Hz,1H),4.83(d,J=11.7Hz,1H),4.75(d,J=11.8Hz,1H),4.68(dd,J=11.7,6.1Hz,2H),4. 61(d,J=11.4Hz,1H),4.40-4.30(m,2H),4.30-4.25(m,1H),4.16-4.09(m,1H),4.05(dd, J=10.2,2.9Hz,2H),3.98(dd,J=10.1,2.7Hz,1H),3.90(d,J=2.5Hz,1H),3.78(td,J=9.5 ,5.4Hz,3H),2.26(q,J=7.4Hz,2H),2.15(d,J=7.8Hz,2H),2.06-1.99(m,3H),1.49(q,J= 7.4Hz, 4H), 1.41 (p, J = 7.3Hz, 1H), 1.23 (d, J = 7.7Hz, 61H), 0.88 (qt, J = 3.8, 1.8Hz, 12H).

[0255] 13C NMR(101MHz,CDCl3)δ174.24,172.55,172.30,164.62,149.93,147.89,138.46,138.29,137.14,128.66,128.39,128.30,128.07,127.87,127.69,127.63,127.46,126.92,125.40,97.29,92.61,79.00,76.34,75.16,74.57,73.59,73.19,72.81,69.70,68.80,65.18,65.03,52.82,36.82,33.97,31.92,29.64,29.52,29.36,29.30,29.14,29.06,25.63,24.60,22.69,14.11。

[0256] 34β 1H NMR(400MHz,CDCl3)δ8.73(dd,J=4.9,1.7Hz,1H),7.95(d,J=7.8Hz,1H),7.78(td,J=7.8,1.8Hz,1H),7.46-7.41(m,1H),7.29(ddddd,J=21.3,16.3,13.5,8.4,4.4Hz,27H),5.63(d,J=8.8Hz,1H),5.36(d,J=9.6Hz,1H),5.12(dd,J=10.8,8.9Hz,1H),4.97(d,J=11.7Hz,1H),4.95-4.86(m,5H),4.81(d,J=11.8Hz,1H),4.71(d,J=11.9Hz,1H),4.65(dd,J=11.2,5.6Hz,2H),4.39(d,J=8.0Hz,1H),4.48-4.34(m,2H),4.34-4.22(m,2H),3.90-3.82(m,3H),3.68(t,J=4.6Hz,1H),3.64(dd,J=9.2,5.7Hz,1H),3.51(dd,J=9.8,2.9Hz,1H),2.19-2.06(m,3H),2.02(t,J=7.7Hz,2H),1.49(t,J=7.3Hz,2H),1.40(p,J=7.2Hz,1H),1.35-1.04(m,55H),0.88(td,J=6.8,2.1Hz,10H)。

[0257] 13C NMR(101MHz, CDCl3)δ174.24,172.75,172.16,164.53,149.91,147.67,138.78,138.53,138.20,137. 00,128.65,128.62,128.51,128.36,128.28,128.25,128.04,127.98,127.62,127.57,127.51,126.90 ,125.35,104.08,92.54,81.88,79.16,74.41,73.25,72.85,72.11,69.77,68.46,64.19,52.87,36.77,33.89,31.92,29.69,29.63,29.36,29.34,29.22,29.12,28.99,25.59,24.60,24.34,22.69,14.12.

[0258] Synthesis of 35 [ka] Under an inert atmosphere, to a solution of 34 (75 mg, 0.5 mmol, 0.5 eq.) in a 3:1 mixture of DCM / MeOH (5 mL, 0.1 M) was added Cu(OAc)2 (15 mg, 0.75 mmol, 1.5 eq.) at RT.

[0259] The solution was left stirring for ca. 2 h and then monitored by TLC (EtPet / AcOEt 6:4).

[0260] The solvent is evaporated on a rotavapor and the solution is purified by flash chromatography (EtPet / AcOEt 6:4) without further purification.

[0261] 55 mg of compound 35 was recovered in 80% yield.

[0262] 1H NMR(400MHz,CDCl3)δ7.40-7.22(m,29H),5.62(d,J=8.7Hz,1H),5.38(d,J=9.6Hz,1H),5.12(dd,J=10.8,8.9Hz,1H),4.97-4.88(m,6H),4.80(d,J=11.9Hz,1H),4.71(d,J=11.9Hz,1H),4.65(dd,J=13.1,11.3Hz,2H),4.39-4.36(m,1H),4.35(d,J=5.5Hz,1H),4.33(d,J=7.4Hz,1H),4.27(dt,J=10.8,7.4Hz,1H),3.86-3.80(m,2H),3.72(d,J=3.3Hz,2H),3.72-3.63(m,2H),3.47(dd,J=9.7,2.9Hz,1H),3.39(dd,J=11.5,4.8Hz,1H),3.31(dd,J=7.2,4.9Hz,1H),2.16(dd,J=8.8,7.1Hz,2H),2.09(ddd,J=8.7,7.2,5.1Hz,2H),2.03(t,J=7.7Hz,2H),1.51(q,J=7.3Hz,2H),1.41(dq,J=14.9,7.0Hz,4H),1.33-1.09(m,55H),0.88(td,J=6.9,2.0Hz,10H)。

[0263] 13C NMR(101MHz,CDCl3)δ174.21,172.80,172.24,138.79,138.52,138.27,135.34,128.72,128.66,128.64,128.53,128.40,128.26,128.10,128.06,127.89,127.64,127.59,127.51,103.82,92.52,82.12,79.15,75.18,75.09,74.94,74.20,73.75,73.69,73.44,73.24,72.79,69.87,69.81,67.94,62.16,52.74,36.77,33.91,33.86,31.93,29.69,29.64,29.51,29.47,29.36,29.33,29.29,29.23,29.11,29.00,25.59,24.59,24.39,22.70,14.12。

[0264] Biology The ability of compounds FP20, FP21, FP22, FP23 and FP24 to selectively activate TLR4 was first investigated in specific HEK reporter cell lines. HEK-Blue™ hTLR4 and HEK-Blue™ hTLR2 (InvivoGen) are cell lines designed to study the activation of human TLR4 and TLR2 receptors, respectively, by monitoring the activation of the transcription factors NF-κB and AP-1. Stimulation with TLR4 ligands (in the case of HEK-Blue hTLR4) or TLR2 ligands (in the case of HEK-Blue hTLR2) activates NF-κB and AP-1, inducing the production and release of the SEAP reporter gene (phosphatase secreted embryonic alkaline) into the extracellular environment. Analysis of the reporter gene was performed using the QUANTI-Blue™ colorimetric assay (InvivoGen), a substrate for SEAP, which generates a colored product whose absorbance is read at 630 nm. The agonist activity of the molecules was tested by treating HEK-Blue hTLR4 cells for 18 hours with increasing concentrations of the compounds (0.1-1-10-25 μM), using MPLA (0.1-1-10 μM) and S-LPS (100 ng / mL) as a reference and positive receptor activation control, respectively. The results obtained show that the molecules FP20, FP21, FP22, FP23 and FP24 are able to induce the activation of TLR4 in a dose-dependent manner (FIG. 1a). The molecules were subsequently tested using the HEK-Blue hTLR2 cell line, with the aim of excluding the activation of this receptor. In this regard, HEK-Blue hTLR2 cells were treated with the same compounds and at the same concentrations as in the tests previously performed, and the compound PAM2CSK4 was used as a positive control for TLR2 activation. As expected, stimulation with PAM2CSK4 induced strong activation of TLR2, whereas treatment with compounds FP20, FP21, FP22, FP23 and FP24 did not result in any activation (FIG. 1b).

[0265] Following the results obtained from the screening test with HEK cells, the biological activity of compounds FP20, FP21, FP22, FP23 and FP24 was investigated in human and mouse macrophage cell lines. The THP-1-X Blue™ cell line, monocytes differentiated into macrophages after treatment with 100 ng / mL PMA, and RAW-Blue™ were used. Similar to HEK-Blue cells, THP-1 X-Blue and RAW-Ox stably express the SEAP reporter gene under the control of the transcription factors NF-κB and AP-1. The cells were treated as described above. The results show that all compounds induce the activation of NF-κB in both human (Figure 2a) and mouse macrophages (Figure 2b); the FP23 compound was the only one that was not statistically significant in human macrophages. Furthermore, in the RAW-blue strain, the compound is active at the lowest concentration tested (0.1 μM), whereas in THP-1-X-Blue it is significantly active from a 100-fold higher concentration (10 μM).

[0266] To evaluate the cytotoxicity of compounds FP20, FP21, FP22, FP23 and FP24, macrophage-differentiated THP-1-X-Blue cells (Figure 3) and RAW-Blue cells (Figure 4) were treated with increasing concentrations of the test compounds (0.1, 1, 10, 25, 50 μM). The toxicity of the compounds was evaluated by MTT viability assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The results obtained show that the compounds are non-toxic to the THP-1-X-Blue cell line, but at the highest concentration tested (50 μM), cell viability decreases after treatment with FP20 and FP23 (Figure 3). The results for the mouse macrophage line show an increased toxicity of the compounds at a concentration of 50 μM, whereas only compound FP23 showed toxicity at a concentration of 25 μM.

[0267] Next, FP200 activity was evaluated in the human monocytic cell line THP-1-X-Blues described above. Cells were treated with compounds FP11, FP112, FP20, FP200, FP21 in increasing concentrations (0.1, 1, 10, 20 μM), with MPLA (0.1-1-10 μM) and S-LPS (100 ng / mL) used as reference and positive receptor activation controls, respectively. Results show that all compounds induce NF-κB activation in a dose-dependent manner.

[0268] The MPA-functionalized compound FP207 was tested in vitro using human THP-1-XBlue cells to assess NF-κB activation. The molecule was tested at the same concentrations as those used for FP22 and FP23.

[0269] Again, the results reveal that compound FP207 activates TLR4 and induces the production of transcription factors. Its agonist activity has a concentration-dependence as in the previous case. Surprisingly, it is more active than FP20 and is comparable to LPS at 25 μM. This is a great result, since it allows to use a lower amount of product to obtain the same inflammatory effect compared to FP20, which is advantageous in two respects: pharmacologically, it reduces any possible side effects; on the other hand, economically it means that less expenses are needed to achieve better results and a larger public.

[0270] Preliminary studies to determine the toxicity of this functionalized compound were performed in triplicate. However preliminary, these data suggest that the molecule is non-toxic in the concentration range of 1-25 mM.

[0271] In vitro and in vivo data for the compounds disclosed in WO2019 / 092572 are provided below.

[0272] TLR4 activation by synthetic agonists The ability of FP molecules to activate human TLR4 was assessed using HEK-Blue hTLR4 cells, a HEK293-derived cell line stably transfected with the LPS receptors CD14, TLR4, and MD-2, and the reporter gene secreted embryonic alkaline phosphatase (SEAP) under the control of two TLR4-dependent transcription factors (NF-κB and AP-1). HEK-Blue hTLR4 cells were treated with increasing concentrations (0.1-25 μM) of FP11, FP112, and FP111 for 18 h. Stimulation with smooth chemotype LPS (S-LPS) served as a positive control for activation of the TLR4-mediated pathway.

[0273] The molecules FP11 and FP112 induced the release of SEAP reporter protein in the medium in a concentration-dependent manner, indicating that both compounds activate NF-κB and AP-1, whereas FP111 was inactive (Fig. 5A). The three compounds did not inhibit LPS-induced SEAP production, suggesting a lack of TLR4 antagonism (Fig. 5B). The lack of activity in HEK-Blue Null cells, which harbor the same SEAP reporter gene but lack the LPS receptor, confirmed that FP11 and FP112 both act through TLR4 (Fig. 5C). To confirm selectivity for TLR4 compared to TLR2, the molecules were also tested in HEK-Blue cells expressing human Toll-like receptor 2 (hTLR2), which resulted in no agonist activity (Fig. 5D). These data are consistent with the in vitro binding results and suggest that FP11 and FP112 are specific TLR4 agonists that interact directly with the co-receptor MD-2.

[0274] Adjuvant activity and in vivo toxicity of FP11 and FP112: OVA immunization experiments The ability of FP11 and FP112 to induce immune responses in vivo was compared with MPLA by evaluating antibody production in C57Bl / 6 mice immunized with chicken ovalbumin (OVA) as a model antigen. First, the toxicity of FPs was evaluated in a pilot experiment in which mice were subcutaneously injected with 10 μg of FP11 and FP112. The results showed that the two test adjuvants had no obvious adverse effects on mice, as assessed by measuring local reactions at the injection site as well as the weight and alertness state of the animals over a 7-day period (Figure 6A). Next, the tested adjuvants were mixed with ovalbumin (OVA) and immunized into mice. Antibody induction was evaluated 21 days after immunization. The results showed that mice immunized with the test adjuvants showed slightly higher levels of anti-OVA total IgG after prime immunization compared to OVA-immunized controls and significantly lower levels compared to MPLA-OVA-immunized animals (Figure 6B, prime immunization). In contrast, after receiving a boost immunization on day 22 and testing ova-specific antibody titers 14 days later, IgG levels in FP112-immunized mice were higher than those in the FP11-immunized group (Figure 6B, booster immunization). These data indicate that, consistent with the in vitro and in cells results, FP112 is a more effective adjuvant in vivo than FP11, with potency comparable to or even higher than that of MPLA.

[0275] Impurity 1 in step 6 of FP11 synthesis, reference figure 7 [ka] 11H NMR (400 MHz, CDCl3) δ 6.04 (d, J = 9.7 Hz, 1H), 5.63 (dd, J = 9.7, 7.3 Hz, 1H), 3.87 (dt, J = 7.5, 3.9 Hz, 1H), 3.77 - 3.70 (m, 3H), 3.68 (dd, J = 10.5, 4.3 Hz, 1H), 2.36 (dd, J = 14.7, 6.9 Hz, 2H), 2.32 - 2.26 (m, 3H), 1.67 (dt, J = 15.3, 7.7 Hz, 2H), 1.59 (dt, J = 20.5, 7.1 Hz, 4H), 1.27 (d, J = 16.5 Hz, 62H), 0.91 - 0.84 (m, 20H), 0.05 (d, J = 8.4 Hz, 7H).

[0276] Impurity 2 in Step 6 in the synthesis of FP11, see Reference Figure 8 ( 1 1H NMR) and 9 ( 13 13C NMR)

Chemical Structure

[0277] 13C NMR(101MHz,CDCl3)δ174.14,173.65,173.12,91.40,77.32,77.01,76.69,70.37,69.67,68.54,61.19,52.49,36.68,34.19,34.13,31.90,29.66,29.63,29.60,29.54,29.52,29.46,29.39,29.34,29.33,29.29,29.27,29.19,29.15,25.60,24.92,23.82,22.66,14.08。

Claims

1. A compound of formula 1, 【Chemical 1】 R １ is a saturated C ５ -C １５ alkyl chain, R ２ is a saturated C ５ -C １５ alkyl chain, R ３ is a saturated C ５ -C １５ alkyl chain, and R ４ is a compound which is C ６ and any substituent which can be linked by a bond between appropriate atoms, and / or any substituent having an oxygen or nitrogen atom which can be bonded to C ６ .

2. R ４ The lock is a hydroxyl group (OH), a phosphate group (PO ４ ２－ ), an azide group (N ３ ), an amine group (NH ２ ), an acyl group (O(C=O)R) or an alkyl group (OR) or a glycosyl group, the compound according to claim 1.

3. R １ = R ２ = R ３ = C １１ H ２３ 、 and R ４ = OH, or R １ = R ３ = C １３ H ２７ ; R ２ = C １１ H ２３ 、and R ４ = OH, or R １ = R ２ = R ３ = C ９ H １９ 、 and R ４ = OH, or R １ = R ２ = R ３ = C １３ H ２７ 、 and R ４ = OH, or R １ = R ３ = C ９ H １９ ; R ２ = C １１ H ２３ 、and R ４ = OH, or R １ = R ２ = R ３ = C １１ H ２３ 、and R ４ = PO ４ ２－ 、or R １ = R ２ = R ３ = C ９ H １９ 、and R ４ = PO ４ ２－ 、or R １ = R ２ = R ３ = C １３ H ２７ 、and R ４ = PO ４ ２－ 、or R １ = R ２ = R ３ = C １１ H ２３ 、and R ４ = OC ３ H ７ 、or R １ = R ２ = R ３ = C １１ H ２３ 、and R ４ = O(C = O)C ６ H ８ (OH) ３ 、or R １ = R ２ = R ３ = C １１ H ２３ 、and R ４ = NH ２ 、 R １ = R ２ = R ３ = C １１ H ２３ 、and R ４ = O(C = O)CCH ３ (CH ２ OH) ２ , R １ = R ２ = R ３ = C １１ H ２３ 、and R ４ = OCH(CHOH) ３ CH(CH ３ )O, R １ = R ２ = R ３ = C １１ H ２３ 、and R ４ = OCH(CHOH) ３ CH(CH ２ OH)O, R １ = R ２ = R ３ = C １１ H ２３ 、and R ４ = OCH(CHOH) ３ (CH ２ )O, The compound according to claim 1, wherein

4. The compound according to claim 1, wherein the compound is the α-anomer or β-anomer of the compound of formula 1.

5. A vaccine adjuvant comprising the compound according to any one of claims 1 to 4.

6. A vaccine composition comprising the compound according to any one of claims 1 to 4, at least one pharmaceutically acceptable carrier and at least one pharmaceutically acceptable immunogenic antigen.

7. The vaccine composition according to claim 6, wherein the compound is the only adjuvant present in the composition.

8. A pharmaceutical composition comprising the compound according to any one of claims 1 to 4 and at least one pharmaceutically acceptable excipient and / or carrier.

9. The pharmaceutical composition according to claim 8, further comprising at least one additional active ingredient.

10. The pharmaceutical composition according to claim 8, which is in a form for oral, parenteral, nasal, aerosol, sublingual, rectal, intravaginal, topical or systemic administration.

11. The pharmaceutical composition according to any one of claims 8, which is in the form of a suspension, emulsion, ointment, cream, spray, granule, powder, solution, capsule, pill, tablet, lyophilized preparation, troche, aerosol, spray or injection.

12. The pharmaceutical composition according to any one of claims 8, for use as an active ingredient or adjuvant in the treatment of a disease that requires immune stimulation by activating the TLR4 receptor or that benefits from immune stimulation.

13. The pharmaceutical composition for use according to claim 12, wherein the disease is cancer, allergy, infectious disease, cardiovascular disease, obesity-dependent metabolic disease, neurodegeneration, apoptosis, autoimmune disorder, viral infection, bacterial infection, autoimmune disease.

14. An intermediate of formula 1i, [Chemical 2] R １ is an intermediate having a saturated C ５ -C １５ alkyl chain.

15. A method for preparing the intermediate of formula 1i, [Chemical Formula 3] Step 1) Selective acylation of the amino group at the C ２ position of glucosamine hydrochloride by reaction with an acyl chloride in the presence of sodium hydrogen carbonate, 2) Protection by selective silylation of the hydroxyl group at the C ６ position by reaction with tert-butyldimethylsilyl chloride (TBDMSCl) in the presence of imidazole, comprising the method.

16. A method for preparing the compound of formula 1, 【Chemical 4】 R １ is a saturated C ５ -C １５ alkyl chain, R ２ is a saturated C ５ -C １５ alkyl chain, R ３ is a saturated C ５ -C １５ alkyl chain, R ４ is a C ６ and any substituent that can be linked by a bond between appropriate atoms and / or a C ６ and any substituent having an oxygen or nitrogen atom that can be bonded to it, Step: 1) Selectively acylating the amino group at the C ２ position of glucosamine hydrochloride by reaction with an acyl chloride in the presence of sodium hydrogen carbonate; 2) By reacting with tert-butyldimethylsilyl chloride (TBDMSCl) in the presence of imidazole, protecting the hydroxyl group at the C ６ position by selectively silylating it, thereby obtaining the intermediate according to claim 14; 3) By reaction with an acyl chloride in the presence of triethylamine and N,N-dimethylaminopyridine (DMAP), selectively acylate the hydroxyl groups at positions C １ and C ３ ; 4) By reaction with dibenzyl N,N - diisopropylphosphoramidite in the presence of triflate imidazolium, phosphorylation of the hydroxyl group at the C ４ position, followed by oxidation of the phosphite to phosphate via meta - chloroperbenzoic acid; 5) Deprotecting the hydroxyl group from the silane at the C ６ position through the presence of a catalytic amount of sulfuric acid; and 6) Deprotecting the phosphate from the benzyl at the C position via hydrogenation catalyzed by palladium on carbon (Pd / C), and optionally deprotecting the benzyl on any substituent at the C position ４ position, and optionally deprotecting the benzyl on any substituent at the C ６ position comprising the method.

17. After step 5) and before step 6), step 5i): 5i) By reaction with dibenzyl N,N - diisopropylphosphoramidite in the presence of triflate imidazolium, the hydroxyl group at the C ６ position is phosphorylated, followed by oxidation of the phosphite to phosphate via meta - chloroperbenzoic acid, and the deprotection step 6) is carried out at the C ４ and C ６ positions, The resulting R4 is a phosphate group (PO ４ ２－ ). The method according to claim 16, further comprising

18. The compound of formula 1 is R １ = R ２ = R ３ = C １１ H ２３ and R ４ = PO ４ ２－ or R １ = R ２ = R ３ = C ９ H １９ and R ４ = PO ４ ２－ or R １ = R ２ = R ３ = C １３ H ２７ and R ４ = PO ４ ２－ , one of the

19. After step 5) and before step 6), step 5ii): 5ii) By reacting with a carboxylic acid in the presence of a suitable condensing agent and catalyst or reacting with an acyl chloride in the presence of a suitable catalyst, acylating the hydroxy group at the C ６ position, and the deprotection step 6) is carried out at the C ４ position. The resulting R ４ is an acyl group, The method according to claim 16, further comprising

20. In the acylation step 5ii), the suitable condensing agent and catalyst are 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N,N-dimethylaminopyridine (DMAP), the method according to claim 19.

21. After step 5) and before step 6), step 5iii): 5 iii) Glycosylation of the hydroxyl group at the C ６ position by reaction of a glycosyl chloride donor or a glycoside thioethyl (Set) donor in the presence of a suitable activator, catalyst and molecular sieve, The method according to claim 19, further comprising.

22. After step 5) and before step 6), step 5iv): 5iv) By the reaction of the stabilized alkyl chloride in the presence of a suitable activator, catalyst and molecular sieve, alkylate the hydroxyl group of C ６ ; The resulting R ４ is n alkyl groups, The method according to claim 19, further comprising.

23. In the glycosylation step 5iii) and alkylation step 5iv), the suitable activator is silver(I) oxide or NIS (N-iodosuccinimide), the suitable catalyst is trifluoromethanesulfonic acid or HOFox (3,3-difluorooxyindole), and the suitable molecular sieve is a water scavenger, the method according to claim 21.

24. After step 5) and before step 6), steps 5v) and 5vi): 5 v) Tosylate the C position by the reaction of tosyl chloride in the presence of triethylamine as a base and a suitable catalyst ６ position, 5 vi) Inserting an azide at the C position by reaction with sodium azide in the presence of tetrabutylammonium iodide ６ position, The method according to claim 16, further comprising.

25. In the tosylation step 5v), the suitable catalyst is N,N-dimethylaminopyridine (DMAP), the method according to claim 24.

26. After step 5) and before step 6), steps 5vii) and 5viii): 5 vii) Glycosylation of the hydroxyl group at the C-position by the reaction of a glycosyl chloride donor having a picoloyl group in the presence of Bi(OTf) as the sole activator ３ and ６ the hydroxyl group at the C-position The resulting R ４ is a glycosyl group, 5 viii) Remove the picoloyl group by reaction with Cu(OAc) ２ and The method according to claim 16, further comprising.

27. The acylation step 3) is carried out at a temperature in the range of -78 °C to 0 °C and an amount of catalyst in the range of 0.05 to 0.2 equivalents, thereby obtaining the β-anomer of the compound of formula 1, preferably carried out at a temperature of -20 °C and an amount of catalyst of 0.1 equivalent, the method according to any one of claims 16 to 26.

28. The acylation step 3) is carried out at a temperature in the range of 20 °C to 50 °C and an amount of catalyst in the range of 2 to 2.5 equivalents, thereby obtaining the α-anomer of the compound of formula 1, preferably carried out at a temperature of 30 °C and an amount of catalyst of 2.02 equivalents, the method according to any one of claims 16 to 26.

29. Use of the intermediate compound according to claim 14 for the synthesis of the compound of formula 1, 【Chemical Formula 5】 R １ is a saturated C ５ -C １５ alkyl chain, R ２ is a saturated C ５ -C １５ alkyl chain, R ３ is a saturated C ５ -C １５ alkyl chain, R ４ is a use of an intermediate compound which is any substituent which may be linked by a bond between C ６ and any appropriate atom and / or any substituent having an oxygen or nitrogen atom which may be bonded to C ６ bonded thereto.

30. A method for preparing a compound of formula X, [Chemical Formula 6] R １ is a saturated C ５ -C １５ alkyl chain, R ２ is a saturated C ５ -C １５ alkyl chain, and R ３ is a saturated C ５ -C １５ alkyl chain R ４ is OH, and R １ , R ２ and R ３ each do not contain an -OH substituent at the ２ C position, Steps: 1) Selectively acylating the amino group at the C2 position of glucosamine hydrochloride by reaction with an acyl chloride in the presence of sodium bicarbonate, 2) By reacting with tert-butyldimethylsilyl chloride (TBDMSCl) in the presence of imidazole, the hydroxyl group at the C ６ position is selectively silylated for protection to obtain the intermediate of formula 1i according to claim 14, 3) By reaction with an acyl chloride in the presence of triethylamine and N,N-dimethylaminopyridine (DMAP), the hydroxyl groups at the C １ , C ３ and C ４ positions are completely acylated, 4) By reacting with ethylenediamine in the presence of acetic acid, selectively diacylate the C １ position, 5) By reaction with dibenzyl N,N - diisopropylphosphoramidite in the presence of triflate imidazolium, the hydroxyl group at the C １ position is phosphorylated, followed by oxidation of the phosphite to phosphate via meta - chloroperbenzoic acid, 6) C in the presence of a catalytic amount of sulfuric acid ６ deprotecting the hydroxyl group from the silane at the position; 7) Deprotecting the phosphate from the benzyl at the C position via hydrogenation catalyzed by palladium on carbon (Pd / C), and optionally deprotecting the benzyl on any substituent at the C position ４ position, and optionally deprotecting the benzyl on any substituent at the C ６ position. The method comprising.

31. 15. Use of an intermediate compound according to claim 14 for the synthesis of a compound of formula X, comprising: 【Chemical Formula 7】 R １ is a saturated C ５ -C １５ alkyl chain, and R ２ is saturated C ５ -C １５ is an alkyl chain, R ３ is saturated C ５ -C １５ is an alkyl chain, R ４ is OH and R １ , R ２ and R ３ Each of the ２ Use of intermediate compounds that do not contain -OH substituents at positions.

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