NEW SYNTHETIC AGONISTS OF TLR4 RECEPTOR
20240299431 ยท 2024-09-12
Inventors
Cpc classification
C07H1/00
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
C07F7/188
CHEMISTRY; METALLURGY
A61K39/39
HUMAN NECESSITIES
A61P37/06
HUMAN NECESSITIES
C07F9/6552
CHEMISTRY; METALLURGY
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
C07H23/00
CHEMISTRY; METALLURGY
International classification
A61K39/39
HUMAN NECESSITIES
C07H23/00
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
Abstract
The present invention relates to new synthetic molecules with agonist activity of human Toll-like Receptor 4 (TLR4), compositions comprising them and uses thereof for the treatment of diseases in which it is useful to induce or increase an immune response. These new syntethic molecules differ from other similar agonists due to the simplicity of the formula, the ease and cheapness of preparation and the possibility of further chemical processing to modify the physicochemical properties and allow conjugation to other molecules (for example protein antigens).
Claims
1. A compound of formula 1 ##STR00065## wherein R.sub.1 is a saturated C.sub.5-C.sub.15 alkyl chain, wherein R.sub.2 is a saturated C.sub.5-C.sub.15 alkyl chain, wherein R.sub.3 is a saturated C.sub.5-C.sub.15 alkyl chain, wherein R.sub.4 is any substituent that can be linked by means of a bond between C.sub.6 and a suitable atom and/or any substituent which possesses an oxygen or a nitrogen atom that can bind to C.sub.6.
2. The compound according to claim 1, wherein R.sub.4 chain is an hydroxyl group (OH), a phosphate group (PO.sub.4.sup.2-), an azide group (N.sub.3), an amine group (NH.sub.2), an acyl group (O(C?O)R) or an alkyl group (OR) or a glycosyl group.
3. The compound according to claim 1, wherein R.sub.1, R.sub.2, R.sub.3, differ from each other.
4. The compound according to claim 1, wherein at least two of R.sub.1, R.sub.2, and R.sub.3, are identical.
5. The compound according to claim 1, wherein at least one of R.sub.1, R.sub.2, and R.sub.3, is free from OH substituents on position 2.
6. The compound according to claim 1, wherein at least one of R.sub.1, R.sub.2 or R.sub.3, is free from any substituent.
7. The compound according to claim 1, wherein R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 and R.sub.4?OH, or R.sub.1=R.sub.3=C.sub.13H.sub.27; R.sub.2=C.sub.11H.sub.23 and R.sub.4?OH, or R.sub.1=R.sub.2=R.sub.3=C.sub.9H.sub.19 and R.sub.4?OH, or R.sub.1=R.sub.2=R.sub.3=C.sub.13H.sub.27 and R.sub.4?OH, or R.sub.1=R.sub.3=C.sub.9H.sub.19; R.sub.2=C.sub.1H.sub.23 and R.sub.4?OH, or R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 and R.sub.4=PO.sub.4.sup.2-, or R.sub.1=R.sub.2=R.sub.3=C.sub.9H.sub.19 and R.sub.4=PO.sub.4.sup.2-, or R.sub.1=R.sub.2=R.sub.3=C.sub.13H.sub.27 and R.sub.4=PO.sub.4.sup.2-, or R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 and R.sub.4=OC.sub.3H.sub.7, or R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 and R.sub.4=O(C?O)C.sub.6H.sub.8(OH).sub.3, or R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 and R.sub.4=NH.sub.2 R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 and R.sub.4=O(C?O)CCH.sub.3(CH.sub.2OH).sub.2 R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 and R.sub.4=OCH(CHOH).sub.3CH(CH.sub.3)O R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 and R.sub.4=OCH(CHOH).sub.3CH(CH.sub.2OH)O R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 and R.sub.4=OCH(CHOH).sub.3(CH.sub.2)O.
8. The compound of claim 1 wherein said compound is an ? anomer of the compound of formula 1.
9. The compound of claim 1 wherein said compound is an ? anomer of the compound of formula 1.
10. A method of treating a disease that requires or benefits from immunostimulation by activating the TLR4 receptor comprising administering to a subject a therapeutically effective amount of the compound according to claim 1.
11. The method according to claim 10 wherein said diseases are cancer, allergies, infectious diseases, cardiovascular diseases, obesity-dependent metabolic diseases, neuronal degeneration, apoptosis, autoimmune disorders, viral infections, bacterial infections, autoimmune diseases.
12. A vaccine adjuvant consisting of the compound as defined in claim 14.
13. A vaccine composition comprising the compound as defined in claim 1, at least one pharmaceutically acceptable carrier and at least one pharmaceutically acceptable immunogenic antigen.
14. The vaccine composition according to claim 13, wherein said compound is the sole adjuvant present in said composition.
15. A pharmaceutical composition comprising the compound as defined in claim 1 and at least one pharmaceutically acceptable excipient and/or carrier.
16. The pharmaceutical composition according to claim 15, further comprising at least one additional active principle.
17. The pharmaceutical composition according to claim 15, in a form for oral, parenteral, nasal, aerosol, sublingual, rectal, vaginal, topical or systemic administration.
18. The pharmaceutical composition according to claim 15 in the form of suspension, emulsion, ointment, cream, spray, granulate, powder, solution, capsule, pill, tablet, lyophilized product, lozenge, aerosol, nebulization, or injection.
19. A method of treatment of diseases that require or benefit from an immunostimulation by activating the TLR4 receptor, comprising administering the pharmaceutical composition of claim 15 to a subject in need thereof.
20. The method according to claim 19, wherein said diseases are cancer, allergies, infectious diseases, cardiovascular diseases, obesity-dependent metabolic diseases, neuronal degeneration, apoptosis, autoimmune disorders, viral infections, bacterial infections, autoimmune diseases.
21. An intermediate of formula 1i ##STR00066## wherein R.sub.1 is a saturated C.sub.5-C.sub.15 alkyl chain.
22. A method for the preparation of an intermediate of formula 1i ##STR00067## comprising the following steps 1) Selective acylation of the amino group in the C.sub.2 position of glucosamine hydrochloride by reaction with acyl chloride in the presence of sodium bicarbonate. 2) Protection by selective silylation of hydroxyl in position C.sub.6 by reaction with tert-butyldimethylsilyl chloride (TBDMSCI) in the presence of imidazole.
23. A method for the preparation of a compound of formula 1, ##STR00068## wherein R.sub.1 is a saturated C.sub.5-C.sub.15 alkyl chain, wherein R.sub.2 is a saturated C.sub.5-C.sub.15 alkyl chain, wherein R.sub.3 is a saturated C.sub.5-C.sub.15 alkyl chain, wherein R.sub.4 is any substituent that can be linked by means of a bond between C.sub.6 and a suitable atom and/or any substituent which possesses an oxygen or a nitrogen atom that can bind to C.sub.6, comprising the following steps: 1) Selective acylation of the amino group in the C.sub.2 position of glucosamine hydrochloride by reaction with acyl chloride in the presence of sodium bicarbonate; 2) Protection by selective silylation of hydroxyl in position C.sub.6 by reaction with tert-butyldimethylsilyl chloride (TBDMSCI) in the presence of imidazole, thereby obtaining the intermediate as defined in claim 22; 3) Selective acylation of hydroxyls in positions C.sub.1 and C.sub.3 by reaction with acyl chloride in the presence of triethylamine and N, N-dimethyl aminopyridine (DMAP); 4) Phosphorylation of hydroxyl in the C.sub.4 position by reaction with dibenzyl N, N-diisopropylphospharamidite in the presence of triflate imidazolium, followed by oxidation of phosphite to phosphate via metachloroperbenzoic acid; 5) Deprotection of hydroxyl from silane in position C.sub.6 through the presence of sulfuric acid in catalytic quantities; and 6) Deprotection of phosphate from benzyls in position C.sub.4 and optionally deprotection of benzyls on any substituent in position C.sub.6 through hydrogenation catalyzed by Palladium on Carbon (Pd/C).
24. The method of claim 23, further comprising a step 5i) after step 5) and before step 6): 5i) Phosphorylation of hydroxyl in position C.sub.6 by reaction with dibenzyl N, N-diisopropylphospharamidite in the presence of triflate imidazolium, followed by oxidation of phosphite to phosphate via metachloroperbenzoic acid wherein said deprotection step 6) is carried out in position C.sub.4 and C.sub.6, and wherein the resulting R.sub.4 is a phosphate group (PO.sub.4.sup.2-).
25. The method of claim 23 wherein the compound of formula 1 is one of R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 and R.sub.4=PO.sub.4.sup.2-, or R.sub.1=R.sub.2=R.sub.3=C.sub.9H.sub.19 and R.sub.4=PO.sub.4.sup.2-, or R.sub.1=R.sub.2=R.sub.3=C.sub.13H.sub.27 and R.sub.4=PO.sub.4.sup.2-.
26. The method of claim 23, further comprising a step 5ii) after step 5) and before step 6): 5ii) Acylation of hydroxy in C.sub.6 position by reaction with carboxylic acid in the presence of a suitable condensing agent and catalyst, or with acyl chloride in the presence of a suitable catalyst, wherein said deprotection step 6) is carried out in position C.sub.4 and wherein the resulting R.sub.4 is an acyl group.
27. The method of claim 26 wherein in said acylation step 5ii) said suitable condensing agent and catalyst are 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N, N-dimethyl aminopyridine (DMAP).
28. The method of claim 23, further comprising a step 5iii) after step 5) and before step 6): 5iii) Glycosylation of hydroxyl in C.sub.6 position by reaction of a glycosyl chloride donor or of a glychoside tioethyl (Set) donor in the presence of a suitable activator, catalyst and a molecular sieve wherein the resulting R.sub.4 is a glycosyl group.
29. The method of claim 23, further comprising a step 5iv) after step 5) and before step 6): 5iv) Alkylation of hydroxyl in C.sub.6 by reaction of a stabilized alkyl chloride in the presence of a suitable activator, catalyst and a molecular sieve wherein the resulting R.sub.4 is n alkyl group.
30. The method of claim 28, wherein in said glycosylation step 5iii) and alkylation step 5iv) said suitable activator is silver (I) oxide or NIS (N-iodosuccinimide), said suitable catalyst is trifilic acid or HOFox (3,3-difluoroxindole), and said suitable molecular sieve is a water scavenger.
31. The method of claim 23, further comprising a step 5v) and a step 5vi) after step 5) and before step 6): 5v) Tosylation of position C.sub.6 by reaction of tosyl chloride in presence of triethylamine as base and of a suitable catalyst 5vi) Azide instertion in position C.sub.6 by reaction with sodium azide in the presence of tetrabutylammonium iodide.
32. The method of claim 31, wherein in said tosylation step 5v) said suitable catalyst is N, N-dimethyl aminopyridine (DMAP).
33. The method of claim 23, further comprising a step 5vii) and a step 5viii) after step 5) and before step 6): 5vii) Glycosylation of hydroxyl in C.sub.6 position by reaction of a glycosyl chloride donor bearing a picoloyl group in the presence of Bi(OTf).sub.3 as sole activator. wherein the resulting R.sub.4 is a glycosyl group. 5viii) Picoloyl group removal by reaction with Cu(OAc).sub.2.
34. The method of claim 24 wherein said acylation step 3) is carried out at a temperature ranging from ?78? C. of 0? C. and an amount of catalyst ranging from 0.05 to 0.2 equivalents thereby obtaining a ?-anomer of said compound of formula 1, preferably a temperature of ?20? C. and an amount of catalyst of 0.1 equivalents.
35. The method of claim 24, wherein said acylation step 3) is carried out at a temperature ranging from 20? C. to 50? C. and an amount of catalyst ranging from 2 to 2.5 equivalents thereby obtaining an ?-anomer of said compound of formula 1, preferably a temperature of 30? C. and an amount of catalyst of 2.02 equivalents.
36. Use of an intermediate compound as defined in claim 21 for the synthesis of compounds of formula 1, ##STR00069## wherein R.sub.1 is a saturated C.sub.5-C.sub.15 alkyl chain, wherein R.sub.2 is a saturated C.sub.5-C.sub.15 alkyl chain, wherein R.sub.3 is a saturated C.sub.5-C.sub.15 alkyl chain, wherein R.sub.4 is any substituent that can be linked by means of a bond between C.sub.6 and a suitable atom and/or any substituent which possesses an oxygen or a nitrogen atom that can bind to C.sub.6.
37. A method for the preparation of a compound of formula X ##STR00070## wherein R.sub.1 is a saturated C.sub.5-C.sub.15 alkyl chain, wherein R.sub.2 is a saturated C.sub.5-C.sub.15 alkyl chain, wherein R.sub.3 is a saturated C.sub.5-C.sub.15 alkyl chain, wherein R.sub.4 is OH and wherein each of R.sub.1, R.sub.2 and R.sub.3 is free from OH substituents in position C.sub.2 comprising the following steps: 1) Selective acylation of the amino group in the C.sub.2 position of glucosamine hydrochloride by reaction with acyl chloride in the presence of sodium bicarbonate. 2) Protection by selective silylation of hydroxyl in position C.sub.6 by reaction with tert-butyldimethylsilyl chloride (TBDMSCI) in the presence of imidazole obtaining the intermediate of formula 1i as defined in claim 21. 3) Complete acylation of hydroxyls in positions C.sub.1, C.sub.3 and C.sub.4 by reaction with acyl chloride in the presence of triethylamine and N, N-dimethyl aminopyridine (DMAP). 4) selective diacylation of position C.sub.1 by reaction with ethylendiamine in presence of acetic acid 5) Phosphorylation of hydroxyl in the C.sub.1 position by reaction with dibenzyl N, N-diisopropylphospharamidite in the presence of triflate imidazolium, followed by oxidation of phosphite to phosphate via metachloroperbenzoic acid. 6) Deprotection of hydroxyl from silane in position C.sub.6 through the presence of sulfuric acid in catalytic quantities. 7) Deprotection of phosphate from benzyls in position C.sub.4 and optionally deprotection of benzyls on any substituent in position C.sub.6 through hydrogenation catalyzed by Palladium on Carbon (Pd/C).
38. Use of an intermediate compound as defined in claim 21 for the synthesis of compounds of formula X, ##STR00071## wherein R.sub.1 is a saturated C.sub.5-C.sub.15 alkyl chain, wherein R.sub.2 is a saturated C.sub.5-C.sub.15 alkyl chain, wherein R.sub.3 is a saturated C.sub.5-C.sub.15 alkyl chain, wherein R.sub.4 is OH and wherein each of R.sub.1, R.sub.2 and R.sub.3 is free from OH substituents in position C.sub.2.
Description
DETAILED DESCRIPTION OF THE FIGURES
[0093]
[0094]
[0095]
[0096]
[0097]
[0098]
[0099]
[0100]
[0101]
[0102]
[0103]
[0104] Differentiated THP1-XBlue? were treated with the indicated concentrations of FP20, FP207, MPLA and LPS (100 ng/mL) and incubated for 16-18 hours. The results were normalized with respect to stimulation with LPS alone and were expressed as a 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).
[0105]
[0106] Differentiated THP1-XBlue? cells were treated with increasing concentrations of FP20 and FP207 (0.1-25 ?M), MPLA and LPS (100 ng/mL). Data were normalized to (untreated) control and expressed as a percentage of the mean?SEM of at least three independent experiments.
GLOSSARY
[0107] In the present description, the term TLR4 receptor agonist denotes a compound that selectively binds to the TLR4 receptor inducing a conformational change of said receptor, in turn generating an intracellular stimulation by triggering a response similar to that induced by the natural ligand of said receptor. In the case of TLR4, the substances described as agonists bind to co-receptor MD-2, in turn non-covalently bound to TLR4, thereby generating the receptorial complex (TLR4/MD-2/agonist).sub.2, which from the cell surface initiates a signal cascade leading to activation of nuclear transcription factors and synthesis of pro-inflammatory cytokines (mainly TNF-? and various interleukin types).
[0108] In the present description, the compound identified as FP112 refers to a compound having the formula represented below
##STR00012##
[0109] wherein R.sub.1=R.sub.2=R.sub.3=C?OC.sub.11H.sub.23 and R.sub.4=H, disclosed as FP112 in WO2019/092572.
[0110] In the present description, the bond in C.sub.1 as represented in Formula 1 below has the meaning commonly intended in organic chemistry
##STR00013##
[0111] and indicates that the the compound can be either in the a or in the p anomer conformation.
[0112] In the present description, the bond in C.sub.1 as represented in Formula 1a below has the meaning commonly intended in organic chemistry.
##STR00014##
[0113] and indicates that the the compound is in the a anomer conformation.
[0114] In the present description, the bond in C.sub.1 as represented in Formula 1a below has the meaning commonly intended in organic chemistry.
##STR00015##
[0115] and indicates that the the compound is in the p anomer conformation.
[0116] In the present description, the term catalytic quantities means an amount or a concentration of a substance used in a chemical reaction such as to obtain a catalytic effect. In particular, in the description the term catalytic quantities can be substituted by a range from 0.5% to 1% of volume/volume concentration.
DETAILED DESCRIPTION OF THE INVENTION
[0117] The present invention relates to a compound of formula 1
##STR00016##
wherein R.sub.1 is a saturated C.sub.5-C.sub.15 alkyl chain,
[0118] wherein R.sub.2 is a saturated C.sub.5-C.sub.15 alkyl chain,
[0119] wherein R.sub.3 is a saturated C.sub.5-C.sub.15 alkyl chain,
[0120] wherein R.sub.4 is any substituent known by the skilled person that can be linked by means of a bond between C.sub.6 and a suitable atom and/or any substituent which possesses an oxygen or a nitrogen atom that can bind to C.sub.6.
[0121] Therefore, according to the present description, each alkyl chain, R.sub.1, R.sub.2, or R.sub.3, can be a C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11, C.sub.12, C.sub.13, C.sub.14, C.sub.15 alkyl chain.
[0122] According to the description each of R.sub.1, R.sub.2, and R.sub.3 can be a different or identical alkyl chain as defined above.
[0123] In one embodiment of the invention, at least two of R.sub.1, R.sub.2, and R.sub.3 are identical.
[0124] According to an embodiment of the invention said R.sub.1, R.sub.2, and R.sub.3 chains are free from OH substituents on position C.sub.2. The absence of hydroxyls in position C.sub.2 advantageously allows a shorter and more efficient synthetic route, eliminating various protection and de-protection steps of the hydroxyl groups thereby, reducing the costs of synthesis and making this synthetic process scalable and industrializable for drugs production.
[0125] According to another embodiment of the invention, said R.sub.1, R.sub.2, and R.sub.3 chains are free from any substitutent.
[0126] As described above, the R.sub.4 chain in position C can be any functionalization substituent of interest provided that the TLR4 agonist activity is not disrupted.
[0127] According to a non limiting example, the R.sub.4 can be an hydroxyl group (OH), a phosphate group (PO.sub.4.sup.2-), an azide group (N.sub.3), an amine group (NH.sub.2), an acyl group (O(C?O)R), an alkyl group (OR) or a glycosyl group.
[0128] The suitability of R.sub.4 in position C to functionalization is an extremely advantageous feature of the compounds of the invention. As described above, it is indeed possible to provide molecules in which the agonist function of TLR4 and any other function of interest depending on the selected substituent of R.sub.4 may be combined. For example, it is surprisingly possible to obtain an increase in solubility and bioavailability using a phosphate group (PO.sub.4.sup.2-). In fact, according to the teachings in the art (WO2019/092572) the presence of two phosphate groups in the agonists described therein resulted in the absence of TLR4 agonist activity. On the contrary, the new compounds of the invention surprisingly retain the TRL4 agonist activity also with a second phosphate group thereby improving the solubility of the compound itself. As known by the skilled person, improved solubility is an important advantage as it ameliorates the delivery, the bioavailability of the compound and the stability of a composition comprising it. Hence, the simple presence of an additional phosphate in C.sub.6, can significatively improve the efficiency of a pharmaceutical or vaccine composition.
[0129] Another important advantage of the possible functionalization in C of the compounds of the invention is that, when used as a vaccine adjuvant it is also possible to conjugate it to an antigen or to an antigenic epitope, or to conjugate it to a different adjuvant to improve and expand its activity.
[0130] In addition, the compound can be conjugated to a target-specific molecule thereby improving its delivery at a site of preference.
[0131] Furthermore, when used in an anti-tumoral composition, the compound of the invention may be functionalized by linking it to additional, different, drugs in order to improve its effectiveness.
[0132] By way of example, it is possible to use substituents free of hydrogen atoms capable of forming hydrogen bonds in order to improve the lipophilic effect, or substituents characterized by the presence of hydrogen atoms capable of forming hydrogen bonds in order to improve solubility in water, which is inversely related to lipophilicity.
[0133] A further advantage deriving from the possibility of exploiting a large variety of substituents as R.sub.4, is the steric effect that can derive for example by using a substituent which tends to limit the free rotation around simple bonds, and therefore to reduce the number of energetically accessible conformations.
[0134] When the bioactive one is present among these conformations, the stiffening effect can increase the affinity of the molecule for the receptor.
[0135] Another important example may be represented by the functionalization with a linker in position C.sub.6. A non limiting example of suitable linkers si represented by a disulfide (RSSR), an hydrazone (RRC?NNH.sub.2), a peptide or a thioeters (RSR) or the like.
[0136] Linkers such as the ones described above allow the preparation of an Antibody-Drug-Conjugate (ADC) which is a complex molecule comprising an antibody linked to a biologically active anticancer payload or drug (such as the compounds of the invention), allowing to obtain a combination effect between the antibody and the compound of the present invention.
[0137] A further interesting instance is the possibility to insert a glycosyl group in C.sub.6, as the resulting compound would have two advantages: an improved water solubility due to the presence of the hydrophilic glycosyl group and theoretically a better affinity for the receptor due to the mimicking of the core portion of LPS.
Suitable substituents depending on the desired property are known to the skilled person.
[0138] According to the present invention, the compound of formula 1 may be an ?-anomer having formula 1a or an ?-anomer having formula 1?.
##STR00017##
##STR00018##
[0139] According to some possible non-limiting embodiments, the compound of formula 1 may be selected from compounds in the form of ?-anomer or ?-anomer of compounds of Formula 1, wherein [0140] R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 and R.sub.4=OH, or [0141] R.sub.1=R.sub.3=C.sub.13H.sub.27; R.sub.2=C.sub.11H.sub.23 and R.sub.4=OH, or [0142] R.sub.1=R.sub.2=R.sub.3=C.sub.9H.sub.19 and R.sub.4=OH, or [0143] R.sub.1=R.sub.2=R.sub.3=C.sub.13H.sub.27 and R.sub.4=OH, or [0144] R.sub.1=R.sub.3=C.sub.9H.sub.19; R.sub.2=C.sub.11H.sub.23 and R.sub.4=OH, or [0145] R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 and R.sub.4=PO.sub.4.sup.2-, or [0146] R.sub.1=R.sub.2=R.sub.3=C.sub.9H.sub.19 and R.sub.4=PO.sub.4.sup.2- or [0147] R.sub.1=R.sub.2=R.sub.3=C.sub.13H.sub.27 and R.sub.4=PO.sub.4.sup.2-, or [0148] R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 and R.sub.4=OC.sub.3H.sub.7, or [0149] R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 and R.sub.4=O(C?O)C.sub.6H.sub.8(OH).sub.3, or [0150] R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 and R.sub.4=NH.sub.2 [0151] R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 and R.sub.4=O(C?O)CCH.sub.3(CH.sub.2OH).sub.2 [0152] R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23and R.sub.4=OCH(CHOH).sub.3CH(CH.sub.3)O [0153] R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23and R.sub.4=OCH(CHOH).sub.3CH(CH.sub.2OH)O [0154] R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 and R.sub.4=OCH(CHOH).sub.3(CH.sub.2)O.
In a preferred embodiment the compounds are ?-anomer of compounds of Formula 1 such as:
[0155] Compound FP20: with R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 R.sub.4=OH
##STR00019## [0156] compound FP21: with R.sub.1=R.sub.3=C.sub.13H.sub.27 R.sub.2=C.sub.11H.sub.23 R.sub.4=OH
##STR00020## [0157] compound FP22: with R.sub.1=R.sub.2=R.sub.3=C.sub.9H.sub.19 R.sub.4=OH
##STR00021## [0158] compound FP23: with R.sub.1=R.sub.2=R.sub.3=C.sub.13H.sub.27 R.sub.4=OH
##STR00022## [0159] compound FP24: with R.sub.1=R.sub.3=CH.sub.19 R.sub.2=C.sub.11H.sub.23 R.sub.4=OH
##STR00023## [0160] compound FP200: with R.sub.1=R.sub.2=R.sub.3=C?H.sub.23 R.sub.4=PO.sub.4.sup.2-
##STR00024## [0161] compound FP202: with R.sub.1=R.sub.2=R.sub.3=C.sub.9H.sub.19 R.sub.4=PO.sub.4.sup.2-
##STR00025## [0162] compound FP203: with R.sub.1=R.sub.2=R.sub.3=C.sub.13H.sub.27 R.sub.4=PO.sub.4.sup.2-
##STR00026##
[0163] Further embodiments according to the present invention, wherein the compound of formula 1 having additional substituent on position C may be selected from the following ones: [0164] compound FP204: with R.sub.1=R=R.sub.3=C.sub.11H.sub.23 R.sub.4=OC.sub.3H.sub.7
##STR00027## [0165] compound FP205: with R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 R.sub.4=O(C?O)C.sub.6H.sub.8(OH).sub.3
##STR00028## [0166] compound FP206: with R.sub.1=R.sub.2=R.sub.3=C.sub.11H-23 R.sub.4=NH.sub.2
##STR00029## [0167] compound FP207: with R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 R.sub.4=O(C?O)CCH.sub.3(CH.sub.2OH).sub.2
##STR00030## [0168] compound FP20Rha: with R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 R.sub.4=OCH(CHOH).sub.3CH(CH.sub.3)O
##STR00031## [0169] compound FP20GIc: with R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 R.sub.4=OCH(CHOH).sub.3CH(CH.sub.2OH)O
##STR00032## [0170] Compound FP20Man: with R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 R.sub.4=OCH(CHOH).sub.3CH(CH.sub.2OH)O
##STR00033## [0171] Compound FP20Gal-?: with R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 R.sub.4=OCH(CHOH).sub.3CH(CH.sub.2OH)O
##STR00034## [0172] Compound FP20Gal-?: with R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 R.sub.4=OCH(CHOH).sub.3CH(CH.sub.2OH)O
##STR00035## [0173] Compound FP20Lyx: with R.sub.1=R.sub.2=R.sub.3=C.sub.11H.sub.23 R.sub.4=OCH(CHOH).sub.3CH.sub.2O
##STR00036##
[0174] Compounds of the present invention having formula 14, 15, 16 or 19 are mixtures of anomers (diastereoisomers) of the sugars bound to C.sub.6.
[0175] Compounds of the present invention having formula 17 or 18 are respectively the pure alpha and beta anomers of glucose bound in C.sub.6.
[0176] In one embodiment, the compound having formula 2 is preferred.
[0177] In the present description, such a compound is also referred as compound FP20, wherein R.sub.1, R.sub.2 and R.sub.3 are C.sub.11H.sub.23 and R.sub.4 is OH.
[0178] Data reported in the Examples section show the peculiar advantageous features of the above-indicated compounds.
[0179] According to the present description, and on the basis of experimental data obtained, it is evident that the compounds as described and claimed are effective agonists of TLR4 receptor. By agonist of a receptor (receptor agonist) it is meant as is commonly defined in the literature, i.e., a substance able to bind a specific receptor in the binding site for the endogenous ligand. Therefore, as the name suggests, the former competes with the latter for the binding with said site.
[0180] Following binding with the natural ligand, the receptor encounters conformational changes that mediate its biological activity at cell level. Agonists are molecules having inherent activity able to mimic ligand effects. When binding to the receptor, they cause conformational changes of an extent similar to those caused by binding with the endogenous ligand.
[0181] In the case of the present description, each agonist disclosed and claimed is an agonist selective for the TLR4 receptor.
[0182] Given the technical features observed for compounds of formula (1) as defined in the present description and in the claims, said compounds are useful as active principles or as adjuvants in the treatment of diseases benefiting from a TLR4 receptor activation, i.e., in diseases in which an activation of the immune system, particularly of the innate activity, has a therapeutic or prophylactic effect.
[0183] Therefore, among diseases requiring or benefiting from a TLR4 receptor activation, are included all the diseases whose treatment or whose prevention are improved by TLR4 receptor activation and by the innate immune response triggered by the activation of said receptor.
[0184] A non-limiting example of such diseases is represented by tumours, allergies, infectious diseases such as viral infectious, cardiovascular diseases, obesity-dependent metabolic diseases, neuronal degeneration, apoptosis, autoimmune disorders, bacterial infections autoimmune diseases. An example of autoimmune diseases is represented by IBD, Chron's disease or rheumatoid arthritis.
[0185] The compound of formula 2, herein also identified as compound FP20, can be compared with the compound of formula FP112 having the formula represented below
##STR00037##
[0186] wherein R.sub.1=R.sub.2=R.sub.3=C?OC.sub.11H.sub.23 R.sub.4=H, disclosed in WO2019/092572, named therein as FP112, due to the fact that they have the same R.sub.1, R.sub.2, R.sub.3 and R.sub.4 substituents, but the phosphate group is on position C.sub.1 for FP112 and on position C.sub.4 for FP20.
[0187] Experiments in vitro conducted for FP112 on cells Hek-Blue, Raw-Blue and THP-1 briefly reported in the examples below, demonstrated a low toxicity and a good pro-inflammatory activy at a concentration of 10 ?M. Furthermore, experiments in vivo conducted with FP112 to test its tolerability and effectiveness as vaccine adjuvant, demonstrated no collateral damage from the administration of 10 ?g of FP112, and an effectiveness as vaccine adjuvant comparable to that of MPLA.Data reported in the Examples section show that for experiments conducted on Hek-Blue, Raw-Blue and THP-1 cells, show that the activity of the compounds of the invention is comparable with the activity of the agonists compounds disclosed in WO2019/092572 which proved to be also non cytotoxic and effective as vaccine adjuvants in vivo.
[0188] Additionally, the compounds of the present invention are improved with respect to the compounds disclosed in WO2019/092572 due to the re-location of the phosphate group in C.sub.1 in the prior art, in position C.sub.4 in the present invention, thereby allowing the positioning of the alkyl chains in C.sub.1 in the compounds of the present invention rather than in C.sub.4, which resulted in a enchanced activity of the substituents in C.sub.6. In fact, WO2019/092572 discloses in Experiment 2 that a compound named therein as FP111, having two phosphate groups: one in position C.sub.1 and the other one in position C proved completely inactive as TLR4 agonist in a test conducted on HEK-Blue? hTLR4 cells. In WO2019/092572 it was speculated that the absence of activity could be due to the presence of two phosphates in the molecule and that the number of phosphates in the molecule had to be no more than 1 in order to maintain their TLR4 agonist activity.
[0189] Surprisingly, the Authors of the present invention discovered that the compounds disclosed and claimed in the present application, such as, by way of example compounds of formula 7, 8 and 9, bearing two phosphate group, one in position C.sub.4 and another one in position C.sub.6, surprisingly maintained the TLR4 agonist activity in the same tests in which compound FP111 disclosed in WO2019/092572 resulted completely inactive. This finding was unexpected and proves a relevant advantage of the compounds of the invention over the prior art as it demonstrates that the position of phosphate groups in a triacylated monophosphoryl glucosamine core, can significantly vary the activity of such compound.
[0190] The invention therefore also provides the compound of formula 1 in any one of the embodiments disclosed in the description or in the claims as vaccine adjuvant.
[0191] The relevance of immune response adjuvants in vaccine composition is known. Vaccine adjuvants, in fact, substantially increase vaccine effectiveness and development of immunity, in the treated subject, toward antigens present in the vaccine.
[0192] Therefore, object of the present invention is also a vaccine composition comprising the compound of formula 1 as defined in any one of the embodiments in the description or in the claims or a mixture thereof.
[0193] The vaccine composition according to the invention can therefore comprise the compound of formula 1 as described herein or a mixture thereof, in any one of the above-listed embodiments, at least one pharmaceutically acceptable carrier and at least one antigenic compound able to induce a desired immune response, such as an immunogenic antigen.
[0194] Suitable vaccine carriers are known to the skilled person.
[0195] The pharmaceutical carrier may be selected to assist release of the antigen component(s) over an extended period of time from the composition. The carrier may include a water-soluble or water-insoluble substance.
[0196] A water-soluble substance is a substance which plays a role in controlling infiltration of water into the interstices of the drug dispersion.
[0197] One water-soluble substance, or a combination of two or more water-soluble substances may be used.
[0198] The water-soluble substance specifically may be selected from one or more of the groups consisting of synthetic polymers (eg. polyethylene glycol, polyethylene polypropylene glycol), sugars (eg. sucrose, mannitol, glucose, sodium chondroitin sulfate), polysaccharides (e.g. dextran), amino acids (eg. glycine and alanine), mineral salts (eg. sodium chloride), organic salts (eg. sodium citrate) and proteins (eg. gelatin and collagen and mixtures thereof).
[0199] In addition, when the water-soluble substance is an amphiphilic substance, which dissolves in both an organic solvent and water, it has an effect of controlling the release of, for example, a lipophilic drug by altering the solubility thereof. An amphiphilic substance includes, but not limited to, one or more selected from the group consisting of polyethylene glycol or a derivative thereof, polyoxyethylene polyoxypropylene glycol or a derivative thereof, fatty acid ester and sodium alkylsulfate of sugars, and more specifically, polyethylene glycol, polyoxy stearate 40, polyoxyethylenepolyoxypropylene-glycol, polyoxyethylene-polyoxypropylene-glycol, polyoxyethylene-polyoxypropylene-glycol, sucrose esters of fatty acids, sodium lauryl sulfate, sodium oleate, sodium chloride, sodium desoxycholic acid (or sodium deoxycholic acid (DCA)) of which mean molecular weights are more than 1500.
[0200] In addition, the water-soluble substance may include a substance selected from one or more of the groups consisting of drugs, peptides, proteins, glycoproteins, polysaccharides, or an antigenic substance used as vaccines.
[0201] A water-insoluble carrier, when present, may include a substance which plays a role in controlling infiltration of water into the interstices of the drug dispersion. One water-insoluble substance, or a combination of two or more water-insoluble substances may be used.
[0202] The water-insoluble substance specifically may be selected from one or more of the groups of water insoluble polymers, resins and latexes including water-insoluble acrylates, methacrylates and other carboxy polymers, waxes, lipids including phospholipids and lipoproteins.
[0203] The skilled person knows the amount of carrier and optional further eccipients commonly used in a pharmaceutical or in a vaccine composition.
[0204] In an embodiment, the pharmaceutical carrier may constitute from approximately 1% to 20% by weight, preferably approximately 10% to 20% by weight, based on the total weight of the vaccine composition.
[0205] The composition according to the invention may comprise one of the compounds as defined and claimed herein, or a mixture thereof.
[0206] The composition 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 a concomitant or sequential administration of the components.
[0207] In a particular embodiment, the compound of formula 1 described in the present invention is the sole adjuvant present in the vaccine composition.
[0208] The present invention also relates to a pharmaceutical composition, comprising a compound of formula 1 described in any one of the embodiments provided in the description or in the claims or a mixture thereof and at least one pharmaceutically acceptable excipient and/or carrier.
[0209] The composition may further comprise one or more additional therapeutically active principle.
[0210] Said pharmaceutical composition can also be formulated in the form of an association of a plurality of active principles.
[0211] The pharmaceutical composition of the invention may comprise as sole active principle one or more compounds of formula 1 according to any one of the embodiments provided in the description or in the claims, or could also comprise additional active principles, such as anti-tumour active principles, kinase inhibitors, cytotoxic compounds and at least one pharmaceutically acceptable carrier or excipient.
[0212] The pharmaceutical composition may be formulated for oral, parenteral, nasal, aerosol, sublingual, rectal, vaginal, topical, endovenous or systemic administration. suitable conventional carriers and/or excipients for suspension, emulsion, ointment, cream, spray, granulate, powder, solution, capsule, pill, tablet, lyophilized product, lozenge, aerosol, nebulization, injection, or others can be selected by the person skilled in the art.
[0213] Further object of the present invention is the pharmaceutical composition according to any one of the embodiments herein disclosed for use in the treatment, or as an adjuvant in the treatment, of diseases that require or benefit from an immunostimulation by activating the TLR4 receptor.
[0214] Diseases that require or benefit from an immunostimulation by activating the TLR4 recepto, are known in the art and comprise cancer, allergies, infectious diseases, cardiovascular diseases, obesity-dependent metabolic diseases, neuronal degeneration, apoptotic diseases, autoimmune disorders, viral infections, bacterial infections, autoimmune diseases. An example of autoimmune diseases is represented by IBD, Chron's disease or rheumatoid arthritis.
[0215] According to the invention, the composition may comprise 0.01 to 50 mg of compound of the invention or of a mixture thereof per daily dosage, by way of example 0.01 to 50 mg of substance per Kg of body weight (test on animals).
[0216] The invention also provides a new method for the synthesis of compounds of formula 1 as herein defined as well as for the synthesis of the compounds disclosed in WO2019/092572 of formula X
##STR00038##
[0217] wherein R.sub.1 is a saturated C.sub.7-C.sub.15 alkyl chain,
[0218] wherein R.sub.2 is a saturated C.sub.7-C.sub.15 alkyl chain,
[0219] wherein R.sub.3 is a saturated C.sub.7-C.sub.15 alkyl chain,
[0220] wherein R.sub.4 is OH and wherein each of R.sub.1, R.sub.2 and R.sub.3 is free from OH substituents in position C.sub.2, and for the synthesis of an intermediate of of formula 1i
##STR00039## [0221] wherein R.sub.1 is a saturated C.sub.4-C.sub.15 alkyl chain. According ot an embodiment of the invention, said R.sub.1 is free from any substituent
[0222] The compounds of formula 1, as well as the compounds disclosed in WO2019/092572, can be synthesized in a simpler and industrially scalable way compared to the synthesis methods knonw in the art for the compounds of WO2019/092572 as well as for SDZ MRL953. The latter requires the insertion of three acyl chains of (R)-3-hydroxymyristic acid. The optically pure compound (R-enantiomer) is not commercially available, as only the racemic mixture is marketed. Moreover, (R)-3-hydroxymyristic acid requires a reaction of protection of the hydroxyl group in 3 position prior to the condensation reaction with the sugar. The method disclosed in WO2019/092572 for the synthesis of compounds of formula X as defined above, although already simplified with respect to the synthesis method disclosed for SDZ MRL953, due to the absence of substituents on the acyl chains, still comprises 10 steps; a number of purifications by chromatographic column and some critical steps, such as the formation of a low molecular weight azide. Additionally, the method disclosed in WO2019/092572 has an extremely low yield (about 8-9%), which makes the whole process uneconomic.
[0223] The compounds provided in the present invention exhibit biological activities comparable to, if not even better than, the compounds of the art and can be synthesized in a much simpler and industrially scalable way.
[0224] Hence, an object of the present invention is a method for the prepation of an intermediate of formula 1i
##STR00040##
[0225] wherein R.sub.1 is a saturated C.sub.5-C.sub.15 alkyl chain, comprising the following steps [0226] 1) Selective acylation of the amino group in the C.sub.2 position of glucosamine hydrochloride by reaction with acyl chloride in the presence of sodium bicarbonate. 2) Protection by selective silylation of hydroxyl in position C by reaction with tert-butyldimethylsilyl chloride (TBDMSCI) in the presence of imidazole.
[0227] Therefore, present invention also relates to an intermediate of formula 1i
##STR00041##
[0228] wherein R.sub.1 is a saturated C.sub.5-C.sub.15 alkyl chain.
[0229] According to an embodiment of the invention, said R.sub.1 is free from any substituent.
[0230] Further, the present invention relates to a method for the preparation of compounds of formula 1 as defined in any of the embodiments above and in the claims
##STR00042##
[0231] wherein R.sub.1 is a saturated C.sub.5-C.sub.15 alkyl chain,
[0232] wherein R.sub.2 is a saturated C.sub.5-C.sub.15 alkyl chain,
[0233] wherein R.sub.3 is a saturated C.sub.5-C.sub.15 alkyl chain,
[0234] wherein R.sub.4 is any substituent known by the skilled person that can be linked by means of a bond between C.sub.6 and a suitable atom and/or any substituent which possesses an oxygen or a nitrogen atom that can bind to C comprising the following steps: [0235] 1) Selective acylation of the amino group in the C.sub.2 position of glucosamine hydrochloride by reaction with acyl chloride in the presence of sodium bicarbonate. [0236] 2) Protection by selective silylation of hydroxyl in position C by reaction with tert-butyldimethylsilyl chloride (TBDMSCI) in the presence of imidazole obtaining the intermediate of formula 1i as defined in the previous described embodiments. [0237] 3) Selective acylation of hydroxyls in positions C.sub.1 and C.sub.3 by reaction with acyl chloride in the presence of triethylamine and N, N-dimethyl aminopyridine (DMAP). [0238] 4) Phosphorylation of hydroxyl in the C.sub.4 position by reaction with dibenzyl N, N-diisopropylphospharamidite in the presence of triflate imidazolium, followed by oxidation of phosphite to phosphate via metachloroperbenzoic acid. [0239] 5) Deprotection of hydroxyl from silane in position C through the presence of sulfuric acid in catalytic quantities. [0240] 6) Deprotection of phosphate from benzyls in position C.sub.4 and optionally deprotection of benzyls on any substituent in position C through hydrogenation catalyzed by Palladium on Carbon (Pd/C).
[0241] The method can alternatively start from the intermediate of formula 1i as defined above and in the claims and may comprise steps 3-6.
[0242] In one embodiment, the method of synthesis describved above may comprise an additional step 5i) after step 5) and before step 6)
[0243] 5i) Phosphorylation of hydroxyl in position C by reaction with dibenzyl N, N-diisopropylphospharamidite in the presence of triflate imidazolium, followed by oxidation of phosphite to phosphate via metachloroperbenzoic acid, and wherein the resulting R.sub.4 is a phosphate group (PO.sub.4.sup.2-).
[0244] When carried out, this method leads to compounds of formula 1 wherein R.sub.4 is a phosphate group, such as compounds of formulas 7, 8 and 9.
[0245] Alternatively, the method of synthesis may further comprise a step 5ii) instead of step 5i) after step 5) and before step 6):
[0246] 5ii) Acylation of hydroxyl in C position either by reaction with carboxylic acid in the presence of a suitable condensing agent and catalyst, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N, N-dimethyl aminopyridine (DMAP), or by reaction with acyl chloride in the presence of a suitable catalyst, such as N, N-dimethyl aminopyridine (DMAP).
[0247] wherein the resulting R.sub.4 is an acyl group. When carried out, this method leads to compounds of formula 1 wherein R.sub.4 is an acyl group, such as compounds of formulas 10 and 11.
[0248] In a preferred embodiment, said suitable condensing agent and catalyst are 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N, N-dimethyl aminopyridine (DMAP).
[0249] Alternatively, the method of synthesis may further comprise a step 5iii) instead of step 5i) and 5ii) after step 5) and before step 6):
[0250] 5iii) Glycosylation of hydroxyl in C position by reaction of a glycosyl chloride donor in the presence of silver (I) oxide as activator, of triflic acid as catalyst and molecular sieves as water scavenger
[0251] Or
[0252] Glycosylation of hydroxyl in C position by reaction of a glycosyl thioethyl (Set) donor in the presence of NIS (N-iodosuccinimide) as activator and HOFox (3,3-difluoroxindole) as catalyst and molecular sieves as water scavenger, wherein the resulting R.sub.4 is a glycosyl group.
[0253] Alternatively, the method of synthesis may further comprise a step 5iv) instead of step 5i), 5ii) and 5iii) after step 5) and before step 6):
[0254] 5iv) Alkylation of hydroxyl in C by reaction of a stabilized alkyl chloride in the presence of silver (I) oxide as activator, of triflic acid as catalyst and molecular sieves as water scavenger wherein the resulting R.sub.4 is n alkyl group.
[0255] Alternatively, the method of synthesis may further comprise a step 5v) and a step 5vi) instead of step 5i), 5ii), 5iii) and 5iv) after step 5) and before step 6):
[0256] 5v) Tosylation of position C by reaction of tosyl chloride in presence of triethylamine as base and of DMAP as catalyst
[0257] 5vi) azide instertion in position C by reaction with sodium azide in the presence of tetrabutylammonium iodide
[0258] wherein the resulting R.sub.4 is an azide group.
[0259] Alternatively, the method of synthesis may further comprise a step 5vii) and a step 5viii) instead of step 5i), 5ii), 5iii) and 5iv) after step 5) and before step 6):
[0260] 5vii) Glycosylation of hydroxyl in C position by reaction of a glycosyl chloride donor bearing a picoloyl group in the presence of Bi(OTf).sub.3 as sole activator. wherein the resulting R.sub.4 is a glycosyl group.
[0261] 5viii) Picoloyl group removal by reaction with Cu(OAc).sub.2.
[0262] According to the present description, the acylations described in reactions 1), 3), 5ii) can be carried out according to methods commonly used by technicians in the chemical field. By way of example, acylations can be carried out using acyl chloride or carboxylic acid in presence other common condensing agents, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or dicyclohexylcarbodiimide (DCC).
[0263] The condensations referred to in reactions 1) and 3) can be carried out using alkyl chains of different lengths, between 5 and 15 carbon atoms, thereby obtaining different derivatives of the molecule described by formula 1.
[0264] The protection of C described by reaction 2) can be carried out in accordance with the most common techniques known to those skilled in the chemical field. One of said common techniques is silylation in the presence of various non nucleophilic bases, such as triethylamine, diisopropylethylamine or sodium bicarbonate and catalyst such as N, N-dimethyl aminopyridine (DMAP).
[0265] The phosphorylation of C.sub.4 described by reaction 4) can be carried out according to the most common techniques known to those skilled in the chemical field, such as phosphite insertion in 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 different mild oxidants, such as but not limited to dimethyldioxirane (DMDO) or tert-Butyl peroxide (tBuOOH).
[0266] The deprotection of C described by reaction 5) can be carried out according to the most common techniques known to those skilled in the chemical field. such as desilylation in the presence of tetrabutylammonium fluoride (TBAF), acetic acid (AcOH) or various types of acidic resins, i.e. IRA 120 H.sup.+, IRC 120 H.sup.+ or Dowex? 50W.
[0267] The process of synthesis according to the invention enables to make in an easy and industrially scalable way the compounds of formula 1.
[0268] As stated above, the invention encompasses both a as well as p anomers of the compound of formula 1 as defined above. The inventors have surprisingly found that, depending on the temperature and amount of a suitable catalyst of the acylation step 3, ? or ? anomers can be obtained.
[0269] Therefore, when a ? anomer is desired, the acylation step 3) is carried out at a temperature ranging from ?78? C. to 0? C. and with an amount of DMAP ranging from 0.05 to 0.2 equivalents.
[0270] In a preferred embodiment, in order to synthesise a ? anomer acylation step 3 is carried out at ?20? C. with 0.1 equivalents of DMAP.
[0271] On the other hand, when an ? anomer is desired, the acylation step 3) is carried out at a temperature ranging from 20? C. to 50? C. with an amount of DMAP ranging from 2 to 2.5 equivalents.
[0272] In a preferred embodiment, in order to synnthesise an ? anomer acylation step 3 is carried out at a temperature of about 30? C. with 2.02 equivalents of DMAP.
[0273] The methods as defined herein allow the synthesis of each one of the embodiments of the compounds of formula 1 (such as compounds of fomulas 2-12) as disclosed in the present specification. The skilled person will know the substituents to use based on the common knowledge in organic chemistry.
[0274] Advantageously, the invention also provides a method for the synthesis of a compound of formula X
##STR00043##
[0275] wherein R.sub.1 is a saturated C.sub.5-C.sub.15 alkyl chain,
[0276] wherein R.sub.2 is a saturated C.sub.5-C.sub.15 alkyl chain,
[0277] wherein R.sub.3 is a saturated C.sub.5-C.sub.15 alkyl chain,
[0278] wherein R.sub.4 is OH and wherein each of R.sub.1, R.sub.2 and R.sub.3 is free from OH substituents in position C.sub.2
[0279] comprising the following steps: [0280] 1) Selective acylation of the amino group in the C.sub.2 position of glucosamine hydrochloride by reaction with acyl chloride in the presence of sodium bicarbonate. [0281] 2) Protection by selective silylation of hydroxyl in position C by reaction with tert-butyldimethylsilyl chloride (TBDMSCI) in the presence of imidazole obtaining the intermediate of formula 1i as defined in the previous described embodiments. [0282] 3) Complete acylation of hydroxyls in positions C.sub.1, C.sub.3 and C.sub.4 by reaction with acyl chloride in the presence of triethylamine and N, N-dimethyl aminopyridine (DMAP). [0283] 4) selective diacylation of position C.sub.1 by reaction with ethylendiamine in presence of acetic acid [0284] 5) Phosphorylation of hydroxyl in the C.sub.1 position by reaction with dibenzyl N, N-diisopropylphospharamidite in the presence of triflate imidazolium, followed by oxidation of phosphite to phosphate via metachloroperbenzoic acid. [0285] 6) Deprotection of hydroxyl from silane in position C through the presence of a 5% solution of sulfuric acid in water in catalytic quantities. [0286] 7) Deprotection of phosphate from benzyls in position C.sub.4 and optionally deprotection of benzyls on any substituent in position C through hydrogenation catalyzed by Palladium on Carbon (Pd/C).
[0287] According to an embodiment of the invention, said R.sub.1 R.sub.2 and R.sub.3 are free from any substituent.
[0288] The present invention also relates to the use of an intermediate compound of formula 1i, as defined in any one of the embodiments herein disclosed, for the synthesis of compounds of formula 1
##STR00044##
[0289] wherein R.sub.1 is a saturated C.sub.5-C.sub.15 alkyl chain,
[0290] wherein R.sub.2 is a saturated C.sub.5-C.sub.15 alkyl chain,
[0291] wherein R.sub.3 is a saturated C.sub.5-C.sub.15 alkyl chain,
[0292] wherein R.sub.4 is any substituent that can be linked by means of a bond between C.sub.r and a suitable atom and/or any substituent which possesses an oxygen or a nitrogen atom that can bind to C.sub.6.
[0293] According to an embodiment of the invention, said R.sub.1 R.sub.2 and R.sub.3 are free from any substituent.
[0294] Furthermore, the present invention relates to the use of an intermediate of formula 1i as defined in any one of the embodiments herein disclosed, for the synthesis of compounds of formula X
##STR00045##
[0295] wherein R.sub.1 is a saturated C.sub.5-C.sub.15 alkyl chain,
[0296] wherein R.sub.2 is a saturated C.sub.5-C.sub.15 alkyl chain,
[0297] wherein R.sub.3 is a saturated C.sub.5-C.sub.15 alkyl chain,
[0298] wherein R.sub.4 is OH and wherein each of R.sub.1, R.sub.2 and R.sub.3 is free from OH substituents in position C.sub.2.
[0299] According to an embodiment of the invention, said R.sub.1 R.sub.2 and R.sub.3 are free from any substituent.
[0300] Object of the invention is also a process for the preparation of pharmaceutical formulations or of vaccine compositions comprising the steps of the above process, and at least one step wherein the product obtained at 6) in a pharmaceutically acceptable grade is mixed with at least one pharmaceutically acceptable carrier and/or excipient.
[0301] In any part of the present description and claims the term comprising can be substituted by the term consisting of.
[0302] In compliance with Art. 170bis of the Italian patent law it is herein declared that: all experiments involving cells were carried out on commercially available cells with reference to model mice used in the described experiments, the obligations deriving from the national or EU regulations, and in particular, from the provisions referred to in paragraph 6 of Legislative Decree No. 206 of 12 Apr. 2001 and 8 Jul. 2003 no. 224, have been fulfilled.
EXAMPLES
Chemistry
[0303] All reagents and solvents were purchased from commercial source and used without further purifications, unless stated otherwise. Reactions were monitored by thin-layer chromatography (TLC) performed over Silica Gel 60 F254 plates (Merck?). Flash chromatography purifications were performed on silica gel 60 60-75 ?m from commercial source.
[0304] 1H and 13C NMR spectrum were recorded with Bruker Advance 400 with TopSpin? software, or with NMR Varian 400 with Vnmrj software. Chemical shifts are expressed in ppm respect Me4Si; coupling constants are expressed in Hz. The multiplicity in the 13C spectra was deducted by APT experiments.
Synthesis of 13
[0305] ##STR00046##
[0306] Glucosamine hydrochloride 12 (10 g, 46.5 mmol, 1 eq.) and NaHCO.sub.3 (10.54 g, 126 mmol, 2.7 eq.) were dissolved in water (120 ml). Then, previously dissolved lauroyl chloride (11.20 g, 51.2 mmol, 1,1 eq.) in THF (120 ml) was added dropwise to the solution at 0? C. Reaction was stirred for 5 h, then solution was filtered. A white solid was obtained, which was washed with 4? C. water and THF. Excess water was then coevaporated with toluene under reduced pressure, to obtain the desired product 13 as a white powder in 60% yield (10.10 g). Compound was used without further purification.
[0307] 1H NMR (400 MHz, DMSO) ? 7.68 (d, J=8.1 Hz, 1H), 7.52 (d, J=7.7 Hz, 3H), 6.46 (d, J=6.3 Hz, 1H), 6.37 (d, J=4.0 Hz, 3H), 4.97-4.86 (m, 7H), 4.81 (d, J=4.7 Hz, 1H), 4.62 (d, J=5.0 Hz, 3H), 4.53 (t, J=5.7 Hz, 1H), 4.43 (dd, J=9.5, 4.3 Hz, 4H), 3.73-3.42 (m, 18H), 3.34-3.22 (m, 2H), 3.16-3.09 (m, 3H), 3.04 (d, J=14.1 Hz, 2H), 2.13-2.03 (m, 8H), 1.56-1.37 (m, 9H), 1.26 (d, J=14.5 Hz, 67H), 0.86 (t, J=6.8 Hz, 13H).
[0308] 13C NMR (101 MHz, DMSO) ? 173.31, 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.
Synthesis of 14
[0309] ##STR00047##
[0310] 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 TBDMSCI (1.4 g, 9.1 mmol, 1.1 eq.) in DCM (15 ml) was added dropwise under inert atmosphere in ice bath. Subsequently, the solution was allowed to return at room temperature and stirred overnight. Reaction, monitored by TLC (DCM/MeOH 9:1), was then stopped and the solution concentrated under reduced pressure. Then it was diluted with AcOEt and washed three times with NH.sub.4Cl. Organic phase thus obtained was dried with Na.sub.2SO.sub.4 and solvent was removed by rotavapor. Raw product thus obtained (3.65 g) was resuspended in EtPet at 0? C. for 30 min. Then, suspension was filtered under vacuum and desired compound was recovered as a white solid. After purification, 3.5 g of compound 14 as a whiteish solid were obtained, in 85% yield.
[0311] 1H NMR (400 MHz, DMSO) ? 7.62 (d, J=7.9 Hz, 1H), 6.43 (d, J=6.4 Hz, 1H), 4.90 (t, J=6.5 Hz, 1H), 4.77 (t, J=9.1 Hz, 1H), 4.42 (t, J=7.0 Hz, 1H), 3.86 (d, J=10.8 Hz, 1H), 3.66 (dd, J=11.0, 4.6 Hz, 1H), 3.30 (d, J=7.9 Hz, 1H), 3.14-2.98 (m, 1H), 2.06 (t, J=7.4 Hz, 1H), 1.48 (s, 1H), 1.24 (s, 3H), 0.94-0.74 (m, 2H), 0.05 (d, J=3.0 Hz, 1H).
[0312] 13C NMR (101 MHz, DMSO) ? 173.21, 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.
Synthesis of 15
[0313] ##STR00048##
[0314] 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. Reaction was stirred for two hours at ?20? C., then controlled by TLC (EtPet/AcOEt 6:4). Subsequently, solution was diluted in AcOEt and washed with 1M HCl. Organic phase thus obtained was dried with Na.sub.2SO.sub.4 and solvent was removed by rotavapor. Raw product thus obtained (4 g) was purified using flash column chromatography (Tol/AcOEt 9:1). After purification, 2.1 g of compound 15 were obtained, in 50% yield.
[0315] 1H NMR (400 MHz, DMSO) ? 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).
[0316] 13C NMR (101 MHz, DMSO) ? 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.
Synthesis of 16
[0317] ##STR00049##
[0318] Compound 15 (2.12 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-diisopropylphosphoramidite (1.83 g, 5.3 mmol, 2.2 eq) was added to the solution at 0? C. Reaction was monitored by TLC (EtPet/acetone 9:1); after 30 min, substrate depletion was detected. Solution was then cooled at ?20? C. and meta-chloroperbenzoic 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 return to RT and left stirring overnight.
[0319] After TLC analysis, reaction was quenched with 15 ml of a saturated NaHCO.sub.3 solution and concentrated by rotavapor. The mixture was then diluted in AcOEt and washed 3 times with a saturated NaHCO.sub.3 solution and three times with a 1 M HCl solution. The organic phase was recovered, dried with Na.sub.2SO.sub.4 and solvent was removed by rotavapor. Crude thus obtained was purified by flash column chromatography (EtPet/acetone 9:1). 2.41 g of pure compound 16 were obtained as a yellow oil in a 91% yield.
[0320] 1H NMR (400 MHz, CDC3) ? 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).
[0321] 13C NMR (101 MHz, CDCl3) ? 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.
Synthesis of 17
[0322] ##STR00050##
[0323] Compound 16 (2.41 g, 2.4 mmol, 1 Eq) was dissolved in acetone (48 mL) and a 5% v/v solution of H.sub.2SO.sub.4 in H.sub.2O was added at RT (480 ?L, 1% v/v). Solution was left stirring for 8 h and monitored by TLC (EtPet/Acetone 8:2). After reaction completion, solution was diluted in AcOEt and washed three times with a saturated NaHCO.sub.3 solution. Organic phase thus obtained was dried with Na.sub.2SO.sub.4 and solvent was removed by rotavapor. Raw product thus obtained was purified by flash column chromatography (EtPet/Acetone 85:15). After purification (2.1 g) of compound 17 was obtained as a white solid in a 90% yield.
[0324] 1H NMR (400 MHz, CDCl3) ? 7.40-7.27 (m, 1H), 5.63 (d, J=8.8 Hz, 1H), 5.45 (d, J=9.6 Hz, 1H), 5.18 (dd, J=10.7, 9.3 Hz, 1H), 5.08-4.91 (m, 1H), 4.54 (q, J=9.5 Hz, 1H), 4.26 (dd, J=19.9, 9.3 Hz, 1H), 3.87-3.74 (m, 1H), 3.47 (d, J=9.7 Hz, 1H), 2.40-2.24 (m, 1H), 2.10-1.91 (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).
[0325] 13C NMR (101 MHz, CDCl3) ? 174.11, 172.77, 172.49, 128.94, 128.84, 128.72, 128.66, 128.26, 127.95, 92.61, 77.33, 77.01, 76.69, 75.90, 75.87, 72.46, 72.42, 72.15, 72.10, 70.23, 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.
Synthesis of 1
[0326] ##STR00051##
[0327] 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 put under Ar atmosphere. Pd/C catalyser (10 mg, 20% m/m) was then added to the solution. Gases were then removed in reaction environment, which was subsequently put under H.sub.2 atmosphere. The solution was allowed to stir for 2 h, then H.sub.2 was removed and reaction monitored by TLC (EtPet/acetone 8:2). Triethylamine (100 ?L) was then added to reaction, which was stirred for 15 min. Solution was subsequently filtered on syringe filters PALL 4549T Acrodisc 25 mm with GF/0.45 ?m Nylon to remove Pd/C catalyser and solvents were evaporated by rotavapor. Crude product was resuspended in a DCM/MeOH solution and IRA 120 H.sup.+ was added. After 30 min stirring, IRA 120 H.sup.+ was filtered, solvents were removed by rotavapor, the crude resuspended in DCM/MeOH and IRA 120 Na.sup.+ was added. After 30 min stirring, IRA 120 Na.sup.+ was filtered and solvents were removed by rotavapor. (45 mg) of 1 were obtained as a white powder in a quantitative yield.
[0328] 1H NMR (400 MHz, cd3od) ? 5.75 (d, J=8.9 Hz, 1H), 5.28 (t, J=9.8 Hz, 1H), 4.28 (q, J=9.7 Hz, 1H), 4.06 (t, J=9.6 Hz, 1H), 3.89-3.74 (m, 2H), 3.62 (t, J=9.2 Hz, 1H), 2.42-2.25 (m, 5H), 2.09 (t, J=7.6 Hz, 2H), 1.56 (d, J=6.4 Hz, 7H), 1.29 (s, 53H), 0.90 (t, J=6.6 Hz, 9H).
[0329] 13C NMR (101 MHz, MeOD) ? 174.69, 173.32, 172.00, 92.16, 76.22, 76.17, 72.81, 72.78, 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.
Synthesis of 18
[0330] ##STR00052##
[0331] 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-diisopropylphosphoramidite (1.83 g, 5.3 mmol, 2.2 eq) was added to the solution at 0? C. Reaction was monitored by TLC (EtPet/acetone 9:1); after 30 min, substrate depletion was detected. Solution was then cooled at ?20? C. and meta-chloroperbenzoic 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 return to RT and left stirring overnight.
[0332] After TLC analysis, reaction was quenched with 15 ml of a saturated NaHCO.sub.3 solution and concentrated by rotavapor. The mixture was then diluted in AcOEt and washed 3 times with a saturated NaHCO.sub.3 solution and three times with a 1 M HCl solution. The organic phase was recovered, dried with Na.sub.2SO.sub.4 and solvent was removed by rotavapor. Crude thus obtained was purified by flash column chromatography (EtPet/acetone 9:1). 2.41 g of pure compound 18 were obtained as a yellow oil in a 91% yield.
[0333] 1H NMR (400 MHz, CDCl3) ? 7.33-7.18 (m, 21H), 5.66 (d, J=8.8 Hz, 1H), 5.51 (d, J=9.5 Hz, 1H), 5.18 (dd, J=10.6, 9.2 Hz, 1H), 5.02 (dd, J=10.8, 3.3 Hz, 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, 9.3 Hz, 1H), 4.16 (ddd, J=11.8, 7.1, 5.0 Hz, 1H), 3.74 (dd, J=9.5, 4.2 Hz, 1H), 2.19 (dt, J=15.9, 7.0 Hz, 5H), 2.07-2.01 (m, 2H), 1.49 (dt, J=14.0, 7.1 Hz, 4H), 1.45-1.36 (m, 2H), 1.34-1.11 (m, 54H), 0.88 (t, J=6.8 Hz, 10H).
[0334] 13C NMR (101 MHz, CDCl3) ? 174.22, 172.82, 172.18, 135.79, 135.72, 135.33, 128.62, 128.58, 128.52, 128.05, 128.00, 127.96, 92.44, 74.11, 72.59, 72.39, 69.38, 65.25, 52.68.
Synthesis of 6
[0335] ##STR00053##
[0336] 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 put under Ar atmosphere. Pd/C catalyser (10 mg, 20% m/m) was then added to the solution. Gases were then removed in reaction environment, which was subsequently put under H.sub.2 atmosphere. The solution was allowed to stir for 2 h; then H.sub.2 was removed and reaction monitored by TLC (EtPet/acetone 8:2).
[0337] Triethylamine (100 ?L) was then added to reaction, which was stirred for 15 min. Solution was subsequently filtered on syringe filters PALL 4549T Acrodisc 25 mm with GF/0.45 ?m Nylon to remove Pd/C catalyser and solvents were evaporated by rotavapor. Crude product was resuspended in a DCM/MeOH solution and IRA 120 H.sup.+ was added. After 30 min stirring, IRA 120 H.sup.+ was filtered, solvents were removed by rotavapor, the crude resuspended in DCM/MeOH and IRA 120 Na.sup.+ was added. After 30 min stirring, IRA 120 Na.sup.+ was filtered and solvents were removed by rotavapor. (45 mg) of 6 were obtained as a white powder in a quantitative yield.
[0338] 1H NMR (400 MHz, cd3od) ? 5.77 (d, J=8.8 Hz, 1H), 5.32-5.23 (m, 1H), 4.39 (dd, J=18.9, 9.5 Hz, 1H), 4.21 (d, J=9.7 Hz, 3H), 4.10-4.00 (m, 1H), 3.80 (d, J=9.2 Hz, 1H), 2.44-2.24 (m, 6H), 2.09 (t, J=7.6 Hz, 2H), 1.55 (dd, J=13.5, 6.9 Hz, 10H), 1.39-1.24 (m, 79H), 0.96-0.82 (m, 33H).
[0339] 13C NMR (101 MHz, MeOD) ? 174.87, 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.
Synthesis of 19
[0340] ##STR00054##
[0341] Compound 17 (100 mg, 0.1 mmol, 1 eq.) and silver (1) oxide (140 mg, 0.6 mmol, 6 Eq) were dissolved in toluene (1 mL, 0.1 M) under inert atmosphere. Allyl bromide (51 ?L, 0.6 mmol, 6 eq) was added to the solution at RT. Reaction was left stirring overnight. After TLC analysis (EtPet/acetone 8:2), reaction was halted and solution filtered on a celite pad. The organic liquid phase was recovered and solvent was removed by rotavapor.
[0342] Crude thus obtained was purified by flash column chromatography (EtPet/acetone 8:2). 50 mg of pure compound 19 were obtained as a yellow oil in a 50% yield.
[0343] 1 H NMR (400 MHz, CDCl3) ? 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).
[0344] 13C NMR (101 MHz, CDCl3) ? 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.
Synthesis of 21
[0345] ##STR00055##
[0346] Compound 17 (100 mg, 0.1 mmol, 1 eq.) and compound 20 (48 mg, 0.11 mmol, 1.1 Eq) were dissolved in DCM (1 mL, 0.1 M) under inert atmosphere. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (192 mg, 0.12 mmol, 1.2 eq) and dimethyl N,N aminopiridine (DMAP) was added to the solution at 0? C. Reaction was allowed to return to RT and left stirring overnight.
[0347] After reaction completion, solution was diluted in AcOEt and washed three times with a saturated NaHCO.sub.3 solution. Organic phase thus obtained was dried with Na.sub.2SO.sub.4 and solvent was removed by rotavapor.
[0348] Crude thus obtained was purified by flash column chromatography (EtPet/acetone 85:15). 100 mg of pure compound 21 were obtained as a white powder in a 65% yield.
Synthesis of 23
[0349] ##STR00056##
[0350] Compound 17 (100 mg, 0.1 mmol, 1 eq.), compound 22 (57 mg, 0.13 mmol, 1.25 eq.) and powdered 3 a molecular sieves (50 mg) were dissolved in toluene (1 mL, 0.1 M) under inert atmosphere and allowed to stir for 1 h. Silver (1) oxide (46 mg, 0.2 mmol, 2 Eq) was then added to the solution at RT, which was then cooled to 0? C. and triflic acid (4,4 ?L, 0.05 mmol, 0.5 eq.) was added. Reaction was then left stirring overnight at RT. After TLC analysis (EtPet/acetone 8:2), reaction was halted and solution filtered on a celite pad. The organic liquid phase was recovered, diluted in AcOEt and washed three times with NaHCO.sub.3. Organic phase was recovered, dried over Na.sub.2SO.sub.4 and evaporated. Crude thus obtained was purified by flash column chromatography (toluene/acetone 85:15). 50 mg of a mixture of diastereoisomers of compound 23 were obtained as a yellow oil in a 40% yield.
[0351] 1H NMR (400 MHz, CDCl3) ? 7.40-7.20 (m, 38H), 5.65 (d, J=8.8 Hz, 1H), 5.61 (s, 1H), 5.40 (s, 1H), 5.24-5.18 (m, 1H), 5.14 (s, 1H), 4.91 (d, J=11.6 Hz, 9H), 4.69 (s, 2H), 4.62 (d, J=2.2 Hz, 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, 4.4 Hz, 3H), 2.05 (s, 3H), 1.57 (s, 6H), 1.47-1.36 (m, 3H), 1.29 (s, 79H), 0.88 (t, J=6.8 Hz, 14H).
[0352] 13C NMR (101 MHz, CDCl3) ? 174.23, 172.81, 172.43, 138.66, 138.63, 135.24, 128.75, 128.69, 128.67, 128.62, 128.50, 128.40, 128.31, 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.
Synthesis of 24
[0353] ##STR00057##
[0354] 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 put under Ar atmosphere. Pd/C catalyser (10 mg, 20% m/m) was then added to the solution. Gases were then removed in reaction environment, which was subsequently put under H.sub.2 atmosphere. The solution was allowed to stir for 2 h, then H.sub.2 was removed and reaction monitored by TLC (EtPet/acetone 8:2). Triethylamine (100 ?L) was then added to reaction, which was stirred for 15 min. Solution was subsequently filtered on syringe filters PALL 4549T Acrodisc 25 mm with GF/0.45 ?m Nylon to remove Pd/C catalyser and solvents were evaporated by rotavapor. Crude product was resuspended in a DCM/MeOH solution and IRA 120 H.sup.+ was added. After 30 min stirring, IRA 120 H.sup.+ was filtered, solvents were removed by rotavapor, the crude resuspended in DCM/MeOH and IRA 120 Na.sup.+ was added. After 30 min stirring, IRA 120 Na.sup.+ was filtered and solvents were removed by rotavapor. (50 mg) of 24 were obtained as a white powder in a quantitative yield, as a mixture of diastereoisomers.
[0355] 1H NMR (400 MHz, MeOD) ? 5.76 (dd, J=8.8, 4.7 Hz, 1H), 5.29 (dd, J=10.5, 9.1 Hz, 1H), 4.76 (d, J=1.2 Hz, 1H), 4.36 (dd, J=18.8, 9.4 Hz, 1H), 4.08 (ddt, J=19.6, 14.2, 5.9 Hz, 4H), 3.91 (dd, J=3.4, 1.6 Hz, 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=6.8 Hz, 17H).
[0356] 13C NMR (101 MHz, MeOD) ? 174.68, 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, 47.81, 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.
Synthesis of 29
[0357] ##STR00058##
[0358] 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. Then, EDC (23 mg, 0.12 mmol, 1.2 eq.) and DMAP (0.112 mg, 0.01 mmol, 0.1 eq.) were added to the solution at 0? C. Subsequently, the solution was allowed to return at room temperature and was stirred overnight. Reaction, monitored by TLC (EtPet/Acetone 8:2), was then stopped and the solution concentrated under reduced pressure. Then it was diluted with AcOEt and washed three times with HCl. Organic phase thus obtained was dried with Na2SO4 and solvent was removed by rotavapor. Raw product thus obtained (550 mg) was purified using flash column chromatography (EtPet/Acetone 85:15). After purification, 17 mg of compound 29 were obtained, in 40% yield.
[0359] 1H NMR (400 MHz, CDCl3) ? 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).
[0360] 13C NMR (101 MHz, CDCl3) ? 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.
Synthesis of 30
[0361] ##STR00059##
[0362] Compound 29 (30 mg, 0.03 mmol, 1 eq.) was dissolved in a 1:1 mixture of dry 0CM and dry MeOH (3 ml, 0.01 M) under Ar atmosphere. Then, Pd/C catalyst (6 mg), 20% i/i) was added always under inert atmosphere. The reaction is left under vacuum for some minutes. Then, the hydrogen was added, and the reaction stirred overnight at room temperature. Reaction, monitored by TLC (Toluene/Acetone 85:15), was then stopped. The hydrogen was completely removed and the Ar atmosphere restored. Triethylamine (60 ?l) was added to the solution that stirred for 30 minutes. Then, the catalyst was removed removed by filtration on syringe filters PALL 4549T Acrodisc 25 mm with GF/0.45 ?m Nylon to remove Pd/C catalyser and solvents were evaporated by rotavapor. Raw compound was resuspende in DCM/MeoH 1:1 and IRA 120 H+ was added to the solution. After 30 minutes, the acid resin was removed, and sodic resin was added instead. The solution was stirred again for 30 minutes; then Solvent was removed by rotavapor, obtaining 52.9 mg of compound 30 were obtained, in >99% yield.
[0363] .sup.1H NMR (400 MHz, MeOD) ? 5.76 (d, J=8.9 Hz, 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=7.3 Hz, 1H), 3.78 (q, J=9.0 Hz, 1H), 3.67 (dd, J=16.4, 11.6 Hz, 4H), 3.56 (t, J=6.6 Hz, 1H), 2.49-2.27 (m, 5H), 2.23-2.08 (m, 2H), 1.59 (d, J=6.4 Hz, 7H), 1.22-1.15 (m, 3H).
Synthesis of 32
[0364] ##STR00060##
[0365] 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 allowed to stir 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. Reaction was then left stirring c.a 1.5 h at RT.
[0366] After TLC analysis (EtPet/acetone 8:2), reaction was stopped and solution filtered on a cotton pad. The organic liquid phase was recovered, diluted in AcOEt and washed three times with Na.sub.2S2O.sub.3. Organic phase was recovered, dried over Na.sub.2SO.sub.4 and evaporated. Crude thus obtained was purified by flash column chromatography (EtPet/acetone 80:20). 120 mg of a mixture of diastereoisomers of compound 32 were obtained as a yellow oil in a 84% yield.
[0367] 1H NMR (400 MHz, CDCl3) ? 7.40-7.20 (m, 32H), 7.11 (ddd, J=13.3, 6.8, 2.7 Hz, 2H), 5.62 (dd, J=8.8, 3.8 Hz, 1H), 5.34 (d, J=9.5 Hz, 1H), 5.27 (d, J=9.6 Hz, OH), 5.13 (td, J=10.9, 8.9 Hz, 1H), 5.00-4.85 (m, 6H), 4.79 (dd, J=10.9, 2.5 Hz, 1H), 4.73 (d, J=11.0 Hz, 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=9.3 Hz, 1H), 3.87-3.75 (m, 3H), 3.70-3.51 (m, 5H), 3.46-3.33 (m, 1H), 2.22 (t, J=7.6 Hz, 1H), 2.13 (dt, J=17.2, 7.7 Hz, 3H), 2.03 (q, J=7.3 Hz, 3H), 1.50 (s, 3H), 1.40 (p, J=7.1 Hz, 3H), 1.22 (d, J=16.1 Hz, 52H), 0.88 (t, J=6.7 Hz, 10H).
[0368] 13C NMR (101 MHz, CDCl3) ? 174.35, 172.75, 172.22, 138.97, 138.22, 138.03, 128.74, 128.69, 128.67, 128.59, 128.45, 128.35, 128.29, 128.22, 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.
Synthesys of 34
[0369] ##STR00061##
[0370] 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 allowed to stir for 1 h. Reaction was cooled at 0? C. and Bi(OTf).sub.3 (50 mg, 0.075 mmol, 0.75 Eq) and was then added to the solution. Reaction was then left stirring overnight at RT.
[0371] After TLC analysis (EtPet/acetone 7:3), reaction was stopped and solution filtered on a celite pad. The organic liquid phase was recovered, diluted in AcOEt and washed three times with NaHCO.sub.3. Organic phase was recovered, dried over Na.sub.2SO.sub.4 and evaporated. Crude thus obtained was purified by flash column chromatography (EtPet/acetone 70:30), which allowed to separate the two diastereoisomers. 125 mg of of total compound 34 (?+?) were obtained as a yellow oil in a 94% yield.
34?
[0372] 1 H NMR (400 MHz, CDCl3) ? 8.75 (dt, J=4.6, 1.4 Hz, 1H), 8.06 (d, J=7.8 Hz, 1H), 7.89 (td, J=7.7, 1.8 Hz, 1H), 7.47 (ddd, J=7.6, 4.7, 1.2 Hz, 1H), 7.40-7.13 (m, 30H), 5.53 (d, J=8.8 Hz, 1H), 5.32 (d, J=9.7 Hz, 1H), 5.10 (dd, J=10.8, 8.9 Hz, 1H), 4.99-4.90 (m, 5H), 4.89 (d, J=3.5 Hz, 1H), 4.83 (d, J=11.7 Hz, 1H), 4.75 (d, J=11.8 Hz, 1H), 4.68 (dd, J=11.7, 6.1 Hz, 2H), 4.61 (d, J=11.4 Hz, 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, 2.9 Hz, 2H), 3.98 (dd, J=10.1, 2.7 Hz, 1H), 3.90 (d, J=2.5 Hz, 1H), 3.78 (td, J=9.5, 5.4 Hz, 3H), 2.26 (q, J=7.4 Hz, 2H), 2.15 (d, J=7.8 Hz, 2H), 2.06-1.99 (m, 3H), 1.49 (q, J=7.4 Hz, 4H), 1.41 (p, J=7.3 Hz, 1H), 1.23 (d, J=7.7 Hz, 61H), 0.88 (qt, J=3.8, 1.8 Hz, 12H).
[0373] 13C NMR (101 MHz, CDCl3) ? 174.24, 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.
34?
[0374] 1H NMR (400 MHz, CDCl3) ? 8.73 (dd, J=4.9, 1.7 Hz, 1H), 7.95 (d, J=7.8 Hz, 1H), 7.78 (td, J=7.8, 1.8 Hz, 1H), 7.46-7.41 (m, 1H), 7.29 (ddddd, J=21.3, 16.3, 13.5, 8.4, 4.4 Hz, 27H), 5.63 (d, J=8.8 Hz, 1H), 5.36 (d, J=9.6 Hz, 1H), 5.12 (dd, J=10.8, 8.9 Hz, 1H), 4.97 (d, J=11.7 Hz, 1H), 4.95-4.86 (m, 5H), 4.81 (d, J=11.8 Hz, 1H), 4.71 (d, J=11.9 Hz, 1H), 4.65 (dd, J=11.2, 5.6 Hz, 2H), 4.39 (d, J=8.0 Hz, 1H), 4.48-4.34 (m, 2H), 4.34-4.22 (m, 2H), 3.90-3.82 (m, 3H), 3.68 (t, J=4.6 Hz, 1H), 3.64 (dd, J=9.2, 5.7 Hz, 1H), 3.51 (dd, J=9.8, 2.9 Hz, 1H), 2.19-2.06 (m, 3H), 2.02 (t, J=7.7 Hz, 2H), 1.49 (t, J=7.3 Hz, 2H), 1.40 (p, J=7.2 Hz, 1H), 1.35-1.04 (m, 55H), 0.88 (td, J=6.8, 2.1 Hz, 10H).
[0375] 13C NMR (101 MHz, CDCl3) ? 174.24, 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.
Synthesis of 35
[0376] ##STR00062##
[0377] 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) under inert atmosphere, Cu(OAc).sub.2 (15 mg, 0.75 mmol, 1.5 eq.) was added at RT. Solution is left stirring for c.a. 2 h and then monitored by TLC (EtPet/AcOEt 6:4).
[0378] Solvent is evaporated by rotavapor and solution is purified by flash chromatography (EtPet/AcOEt 6:4) without further purification.
[0379] 55 mg of compound 35 were recovere in a 80% yield
[0380] 1H NMR (400 MHz, CDCl3) ? 7.40-7.22 (m, 29H), 5.62 (d, J=8.7 Hz, 1H), 5.38 (d, J=9.6 Hz, 1H), 5.12 (dd, J=10.8, 8.9 Hz, 1H), 4.97-4.88 (m, 6H), 4.80 (d, J=11.9 Hz, 1H), 4.71 (d, J=11.9 Hz, 1H), 4.65 (dd, J=13.1, 11.3 Hz, 2H), 4.39-4.36 (m, 1H), 4.35 (d, J=5.5 Hz, 1H), 4.33 (d, J=7.4 Hz, 1H), 4.27 (dt, J=10.8, 7.4 Hz, 1H), 3.86-3.80 (m, 2H), 3.72 (d, J=3.3 Hz, 2H), 3.72-3.63 (m, 2H), 3.47 (dd, J=9.7, 2.9 Hz, 1H), 3.39 (dd, J=11.5, 4.8 Hz, 1H), 3.31 (dd, J=7.2, 4.9 Hz, 1H), 2.16 (dd, J=8.8, 7.1 Hz, 2H), 2.09 (ddd, J=8.7, 7.2, 5.1 Hz, 2H), 2.03 (t, J=7.7 Hz, 2H), 1.51 (q, J=7.3 Hz, 2H), 1.41 (dq, J=14.9, 7.0 Hz, 4H), 1.33-1.09 (m, 55H), 0.88 (td, J=6.9, 2.0 Hz, 10H).
[0381] 13C NMR (101 MHz, CDCl3) ? 174.21, 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.
Biology
[0382] The ability of compounds FP20, FP21, FP22, FP23 and FP24 to selectively activate TLR4 was initially investigated on specific HEK reporter cell lines. HEK-BIue? hTLR4 and HEK-BIue? hTLR2 (InvivoGen) are cell lines designed to study the activation of human TLR4 and TLR2 receptors, respectively, by monitoring the activation of transcription factors NF-kB and AP-1. Stimulation with TLR4 ligands (in the case of HEK-Blue hTLR4) or with TLR2 ligands (in the case of HEK-Blue hTLR2) activates NF-kB and AP-1, inducing the production and release of the SEAP reporter gene (phosphatase secreted embryonic alkaline) in the extracellular environment. The analysis of the reporter gene was performed using the QUANTI-Blue? colorimetric assay (InvivoGen), a substrate of SEAP, which generates a chromogenic 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 ?M) and using MPLA (0.1-1-10 ?M) 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 (
[0383] Following the results obtained from screening tests on HEK cells, the biological activity of compounds FP20, FP21, FP22, FP23 and FP24 was investigated in human and mouse macrophage cell lines. THP-1-X BIue? cell lines, monocytes differentiated into macrophages following treatment with PMA 100 ng/mL, and RAW-BIue? were used. Similarly to HEK-Blue cells, THP-1 X-Blue and RAW-Ox stably express the SEAP reporter gene, under the control of transcription factors NF-kB and AP-1. The cells were treated as previously described. The results show that all compounds induce the activation of NF-kB, both on human (
[0384] To evaluate the cytotoxicity of compounds FP20, FP21, FP22, FP23 and FP24, THP-1-X-Blue cells differentiated into macrophages (
[0385] FP200 activity was then assessed on human monocytes cell line THP-1-X-Blues, described before. Cells were treated with compounds FP11, FP112, FP20, FP200 and FP21 in increasing concentrations (0.1, 1, 10, 20 ?M) and using MPLA (0.1-1-10 ?M) and S-LPS (100 ng/mL) as a reference and positive receptor activation control, respectively. The result shows that all compounds induce the activation of NF-kB in a dose dependent manner.
[0386] Compound FP207, functionalized with with MPA, was tested in vitro on human THP-1-XBlue cells to evaluate the NF-kB activation. The molecule was tested at the same concentrations used for FP22 and FP23.
[0387] Also in this case, the results revealed that compound FP207 is active on TLR4 and induces the production of the transcription factor. Its agonist activity has a concentration-dependent manner as the previous case. Astonishingly, it is more active than FP20 and also comparable to LPS at 25 ?M. This is a great result because it will be possible to use less quantity of product to obtain the same inflammatory effect compared to FP20, which will be advantageous in two ways: pharmacologically any possible collateral effect is reduced; while economically this means that less expenses are required to achieve greater results and larger public.
[0388] A preliminary test to investigate the toxicity of this functionalized compound was performed, even though without a triplicate. However preliminary, those data suggest that the molecule is non-toxic in the concentration range from 1 to 25 mM.
[0389] In vitro and vivo data regarding the compounds disclosed in WO2019/092572 are provided below.
TLR4 Activation by Synthetic Agonists
[0390] The ability of FP molecules to activate human TLR4 was assessed using HEK-Blue hTLR4 cells. These are a HEK293-derived cell line stably transfected with the LPS receptors CD14, TLR4 and MD-2 and a reporter gene, secreted embryonic alkaline phosphatase (SEAP) placed under the control of two TLR4-dependent transcription factors (NF-kB and AP-1). The HEK-Blue hTLR4 cells were treated with increasing concentrations (0.1-25 ?M) of FP11, FP112 and FP111 over 18 hours. Stimulation with smooth chemotype of LPS (S-LPS) served as a positive control for the activation of the TLR4-mediated pathway.
[0391] Molecules FP11 and FP112 induced the release of the SEAP reporter protein in the medium in a concentration-dependent manner, indicating that both compounds activate NF-kB andAP-1, while FP111 was inactive (
Adjuvant Activity of FP11 and FP112 and In Vivo Toxicity: OVA Immunization Experiments
[0392] The ability of FP11 and FP112 to induce immune responses in vivo was compared to MPLA by evaluating antibody production in C.sub.57Bl/6 mice immunized with chicken ovalbumin (OVA) as a model antigen. Has been first evaluated the toxicity of FPs in a pilot experiment in which mice were injected subcutaneously with 10 ?g of the FP11 and FP112. The results showed that the two test adjuvants had no obvious adverse effect on mice, as assessed by the local response at the injection site and by determining the animal weight and state of alertness over 7 days (
Impurity 1 of Step 6 in FP11 Synthesis, Reference FIG. 7
[0393] ##STR00063##
[0394] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 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).
Impurity 2 of step 6 in FP11 Synthesis, reference
##STR00064##
[0395] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 6.01 (d, J=9.1 Hz, 1H), 5.40-5.30 (m, 1H), 5.21 (t, J=6.7 Hz, 1H), 5.10-4.99 (m, 2H), 4.22 (td, J=10.7, 3.4 Hz, 1H), 4.04 (ddd, J=10.2, 4.3, 2.0 Hz, 1H), 3.68 (d, J=11.7 Hz, 1H), 3.61-3.53 (m, 1H), 2.32-2.19 (m, 5H), 2.18-2.06 (m, 3H), 1.62-1.46 (m, 8H), 1.34-1.18 (m, 62H), 0.87 (t, J=6.8 Hz, 11H).
[0396] .sup.13C NMR (101 MHz, CDCl.sub.3) ? 174.14, 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.