Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/06—Pyrimidine radicals
  • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/06—Pyrimidine radicals
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

• the present invention relates to new amphiphilic compounds, their preparation process and their applications in particular to transfection.
• One method of choice is the introduction of specific genetic material into a cell or transfection.
• the compounds used for this purpose such as the liposome formulation of a mono cationic lipid (N- [1- (2,3-Dioleoyloxy)] - N, N, N- trimethylammonium propane methylsulfate in sterile water ( concentration 1 mg / ml) called DOTAP), polyethyleneimine (PEI) or lipofectamine, have a low rate of effectiveness.
• X represents an oxygen or sulfur atom, or a group -CH 2 -
• B represents a purine or pyrimidine base, such as uracil, adenine, guanine, cytosine, thymine or hypoxanthine, optionally substituted, or a base non-natural mono-or bi-cyclic heterocylic, each cycle comprising 4 to 7 links, optionally substituted, Li and L 2 , identical or different, represent an oxycarbonyl group -O-CO-, thiocarbamate -O-CS-NH-, carbonate -O-CO-O-, carbamate -O-CO-NH-, an oxygen atom, or alternatively, Li and L 2, together, form a ketal group of formula
• Ri and R 2 represent a linear or branched alkyl chain containing from 2 to 30 carbon atoms, preferably from 6 to 25 carbon atoms, in particular from 8 to 25 carbon atoms, saturated or partially unsaturated, possibly totally or partially fluorinated, optionally substituted on the chain end carbon by a fluorine atom or by a benzyl or naphthyl ester or ether and R 3 represents a hydroxy, amino group, a phosphate, phosphonate, phosphatidylcholine, O-alkylphosphatidylcholine group, phosphatidylethanolamine, O-alkyl-phosphatidylethanolamine, O- alkylphosphate, thiophosphate, phosphonium, a group NH 2 -R 4 , NHR R 5 or NR R 5 R 6 in which R 4l R 5 and R 6 , identical or different, represent an atom d hydrogen or a linear or branched alkyl or hydroxy
• CO-V- in which V and W identical or different represent a bond -O -, - S-, or -NH-, Z can take the meanings of R 3 , Ri and R 2 , and n can take the values of 1 at 500 and A T and A 2 which are identical or different, represent a linear or branched alkyl radical containing from 1 to 5 carbon atoms or linear or branched acyl radical containing from 2 to 5 carbon atoms, with the exception of the compounds in which: - X represents an oxygen atom,
• R 3 represents a hydroxy group and either L1 and L2, identical represent an oxycarbonyl group, Ri and R 2 identical represent a C 2 to C ⁇ 7 alkyl, and B represents cytosine, adenosine or a 6-methoxypurine, or Li and L 2 together form a ketal group, identical Ri and R 2 represent methyl and B represents adenosine.
• X represents an oxygen atom.
• Preferred compounds of preferred formula (I) are those in which: X represents an oxygen or sulfur atom, - B represents a purine or pyrimidine base such as uracil, adenine, guanine, cytosine, thymine or hypoxanthine, Li and L 2 , identical or different, represent an oxycarbonyl group -O-CO-, thiocarbamate -O-CS-NH-, carbonate -O-CO-O-, carbamate -O-CO-NH-, an oxygen atom, - Ri and R 2 , identical or different, represent a linear or branched alkyl chain containing from 2 to 30 carbon atoms, preferably from 6 to 25 carbon atoms, in particular from 8 to 25 carbon atoms, saturated or partially unsaturated, possibly totally or partially fluorinated, optionally substituted on the chain end carbon by a fluorine atom or by a benzyl or naphthyl ester or ether and R 3 represents a
• - B represents cytosine, adenosine or 6-methoxypuhne
• - Ri and R 2 identical represent a C 2 to C 17 alkyl and - R 3 represents a hydroxy group.
• Other preferred compounds of formula (I) are those in which R 3 represents an amino group, a phosphate, phosphonate, phosphatidylcholine, O-alkylphosphatidylcholine, phosphatidylethanolamine, O-alkyl-phosphatidylethanolamine, O-alkylphosphate, thiophosphate, phosphonium group.
• R 4 , R 5 and R 6 identical or different, represent a hydrogen atom or a linear or branched alkyl or hydroxyalkyl radical containing from 1 to 5 carbon atoms, or R 3 represents a cyclodextrin residue or a ZW-CO-CH 2 -CH 2 - (- N-CO-CH 2 -CH 2 -) n - N - A1 CH 2 -V- A2 or - (- CH 2 -CH-) n - I CO-V- in which V and W identical or different represent a bond -0 -, - S-, or -NH-, Z can take the meanings of R 3 , Ri and R 2l and n can take the values from 1 to 500 and Ai and A 2 identical or different represent a linear or branched alkyl radical containing from 1 to 5 atoms of ca linear or branched carbon or acyl containing
• - B represents a purine or pyrimidine base chosen from uracil, adenine, guanine, cytosine, thymine
• - X represents an oxygen or sulfur atom
• - L 1 and L 2 represent an oxycarbonyl, thiocarbamate, carbonate or carbamate group, and are preferably identical
• - Ri and R 2 represent an undecyl, tridecyl, pentadecyl, heptadecyl radical, nonadecyl, cis, cis, cis, cis-4,7,10,13-nonadec-tetraenyl, arachidonyl, c s-8-heptadecenyl
• - R 3 represents an amino radical, methylamine, dimethylamine, trimethylamine, O-alkylphosphatidylcholine and - the counterion, if
• the invention relates to compounds of formula (I) above, in which Li and L 2 , together, form a ketal or thiocetal group, corresponding to the formula (la) below:
• R 3 represents an amino, phosphate, phosphonate, phosphatidylcholine group, O-alkylphosphatidylcholine, phosphatidylethanolamine, O-alkylphosphatidylethanolamine, O-alkylphosphate, thiophosphate, phosphonium, an NH 2 -R 4 group , NHR R 5 or NR 4 R 5 R 6 in which R, R 5 and R 6 , identical or different represent a hydrogen atom or a linear or branched alkyl or hydroxyalkyl radical containing 1 to 5 carbon atoms, or R 3 represents a cyclodextrin residue or a ZW-CO-CH 2 -CH 2 - (- N-CO -CH 2 -CH 2 -) n - N
• n is advantageously between 1 and 500, preferably between 1 and 100, especially between 1 and 50, very particularly between 1 and 10.
• linear or branched alkyl containing 1 to 5 carbon atoms is meant for example a methyl, ethyl, propyl radical , i-propyl, n-butyl, i-butyl, tert-butyl, preferably methyl or ethyl.
• the purine or pyrimidine base, or the non-natural heterocyclic base can be substituted with at least one substituent chosen, for example, from a halogen, an amino group, a carboxy group, carbonyl group, carbonylamino group, hydroxy group, azido, cyano, alkyl, cycloalkyl, perfluoroalkyl, alkyloxy (e.g. methoxy), oxy carbonyl, vinyl, ethynyl, propynyl, acyl etc.
• substituent chosen, for example, from a halogen, an amino group, a carboxy group, carbonyl group, carbonylamino group, hydroxy group, azido, cyano, alkyl, cycloalkyl, perfluoroalkyl, alkyloxy (e.g. methoxy), oxy carbonyl, vinyl, ethynyl, propynyl, acyl etc.
• non-natural heterocyclic base means a base other than uracil, adenine, guanine, cytosine, thymine or hypoxanthine, which does not exist in nature.
• R 3 represents a cationic group, for example a phosphonium or NR RsR ⁇ group as defined above
• the counterion can be chosen, for example, from tosylate, halide, nitrate, sulfate, sulfonate, and thiosulfate anions.
• the present invention also relates to a process for the preparation of the new compounds of formulas (I) as defined above as of their salts in which Li and L 2 , which are identical, represent an oxycarbonyl, thiocarbonyl, carbamate or carbonate group and R 3 represents an NH-R 4 , NR R 5 or NR 4 R 5 R 6 group in which R 4 , R 5 and R ⁇ , identical or different, represent a hydrogen atom or a linear or branched alkyl radical containing from 1 to 5 atoms carbon, or R 3 represents a cyclodextrin residue or a ZW-CO-CH 2 -CH 2 - (- N-CO-CH 2 -CH 2 -) n - N - ⁇ II CH 2 -V- A 2 or - (- CH 2 -CH-) n -
• CO-V- in which V and W identical or different-represent a bond -O -, - S-, or -NH-, Z can take the meanings of R 3 , Ri and R 2 , n can take the values of 1 at 500 and Ai and A 2 identical or different represent a linear or branched alkyl radical containing from 1 to 5 carbon atoms or linear or branched acyl radical containing from 2 to 5 carbon atoms, characterized in that a derivative is reacted of formula (II)
• Ts represents a residue of an activating agent of a hydroxyl such as a tosyl or mesyl residue, which is reacted in an acid medium and then with a compound of formula R ⁇ - LR 7 (V) or R 2 -L 2 -R 7 (V) in which Ri, R 2 , - ⁇ and L 2 have the meaning already indicated and R 7 represents a residue of an activating agent such as carbonyldiimidazole, dicyclohexylcarbodiimide, hydroxybenzotriazole, or thiocarbonyldiimidazole, or a halogen atom, to obtain a compound of formula (VI)
• R 4 , R 5 and R 6 identical or different, represent a hydrogen atom or a linear or branched alkyl or hydoxyalkyl radical containing from 1 to 5 carbon atoms, to obtain the expected compound of formula (I), that isolate if desired.
• reaction of the derivative of formula (II) with acetone is carried out in the presence of a catalytic amount of an acid such as sulfuric acid
• reaction of the derivative of formula (III) with an activating agent of a hydroxyl is carried out in pyridine in particular using a sulfonyl halide which is preferably a tosyl halide, mesyl or trifluoromethanesulfonyl such as their chloride or bromide.
• a sulfonyl halide which is preferably a tosyl halide, mesyl or trifluoromethanesulfonyl such as their chloride or bromide.
• reaction of the derivative of formula (IV) with the compound of formula (V) or (V) is preceded by acidification with trifluoroacetic acid and is carried out in water.
• the present invention also relates to a process for the preparation of the new compounds of formula (I) as defined above, as well as their salts, in which Li and L 2 , identical represent an oxycarbonyl, thiocarbonyl, carbamate, or carbonate group and R 3 represents a phosphate, phosphonate, phosphatidylcholine, O- alkylphosphatidylcholine, O-alkylphosphatidylethanolamine, O-alkylphosphatidylethanolamine, O-alkylphosphate, thiophosphate or phosphonium group what is reacted with a compound of formula (III) above with a compound of formula (VIT)
• X represents an oxygen or sulfur atom
• Rs and R 9 identical or different represent a radical, linear or cyclic or branched alkyl or O-alkyl containing from 1 to 20 carbon atoms and Hal represents a halogen atom
• B has the meaning already indicated
• R 3 represents a phosphate, O-alkylphosphate, phosphonate, thiophosphate or phosphonium group, which is reacted if necessary with a trialkylamine of formula (VII) as defined above, for obtaining a compound of formula (VIII) in which R 3 represents a phosphatidylcholine or phosphatidylethanolamine group, which is then reacted in an acid medium and then with a compound of formula R1-L 1 -R7 (V) or R 2 -L 2 -R 7 (V) in which Ri, R 2 , L1 and L 2 have the meaning already indicated and R represents an activating agent such as carbonyl
• the reaction of the compound of formula (III) above with the compound of formula (VIT) is carried out in the presence of triethylamine (approximately 0.5 M) in tetrahydrofuran under an inert atmosphere at 0 ° C for one hour and then at room temperature for about 18 h.
• concentrations preferably used are approximately 0.3 M for the compound of formula (III) and approximately 0.4 M for the compound VII '.
• the reaction of the compound of formula (VIII) with the trialkylamine (VII) is carried out with a large excess of trialkylamine (approximately 200 equivalents) in the presence of a binary mixture of acetonitrile / tetrahydrofuran approximately 1/1 at approximately 70 ° C for approximately 48 h.
• concentration preferably used is approximately 0.2 M for the compound of formula (VII).
• the present invention also relates to a process for the preparation of the new compounds of formula (I) or (la), as defined above, as well as their salts, in which Li and L 2 , together, form a ketal group or thioketal, in which R 3 represents an NH-R 4 , NR 4 R 5 or NR 4 R 5 R6 group in which R 4 , R5 and R 6 , identical or different, represent a hydrogen atom or a linear alkyl radical or branched containing from 1 to 5 carbon atoms, or R 3 represents a cyclodextrin residue or a residue ZW-CO-CH 2 -CH 2 - (- N-CO-CH 2 -CH 2 -) n - N - A ⁇ II CH 2 -V- A 2 or - (- CH 2 -CH-) n - I CO-V- in which V and W, identical or different, represent a bond -0 -, - S-, or -NH-, Z can take
• Ts represents a residue of an activating agent of a hydroxyl such as a tosyl or mesyl residue, which is reacted with a compound of formula (VII) as defined previously R 4
• the reaction of the derivative of formula (II) with hentriacontan-16-one is carried out in the presence of a catalytic amount of an acid, such as paratoluenesulfonic acid (APTS),
• APTS paratoluenesulfonic acid
• the reaction of the derivative of formula (III ') with an activating agent of a hydroxyl is carried out in pyridine in particular using a sulfonyl halide which is preferably a halide of tosyle, mesyle or trifluorométhanesulfonyle, like their chloride or bromide.
• a binary solvent such as a tetrahydrofuran / acetonitrile mixture.
• the present invention also relates to a process for the preparation of the new compounds of formula (I) in which and L 2 , together, form a ketal or thioketal group or of formula (la), as defined above, as well as of their salts, in which R 3 represents a phosphate, phosphonate, phosphatidylcholine, O- alkylphosphatidylcholine, phosphatidylethanolamine, O- alkylphosphatidylethanolamine, O-alkylphosphate, thiophosphate or phosphonium group, characterized in that a compound of formula (III) is reacted ') above with a compound of formula (VIT) in which X represents an oxygen or sulfur atom, R 8 and R 9 , identical or different, represent a radical, linear or cyclic or branched alkyl or O-alkyl containing from 1 to 20 carbon atoms and Hal represents an atom halogen, to obtain a compound of formula (I) in which Li and L 2
• R 3 represents a phosphate, O-alkylphosphate, phosphonate, thiophosphate or phosphonium group, which is reacted if necessary with a trialkylamine of formula (VII) as defined above, for obtaining a phosphate alkylamine compound of formula (Ia) in which R3 represents a phosphatidylcholine or phosphatidylethaloamine group, which is isolated if desired; then, if we want to obtain a compound of formula (la) in which B has the meaning already indicated and R 3 represents an O-alkylphosphatidylcholine or O-alkylphosphatidylethanolamine group, a compound of formula R 10 -J is reacted with the meaning already indicated, with a compound (la) in which B has the meaning already indicated and R 3 represents a phosphatidylcholine or phosphatidylethanolamine group.
• the preferentially used concentrations are approximately 0.3 M for the compound of formula (III ') and approximately 0.4 M for the compound (VIT), the reaction of the compound of formula (la) with trialkylamine (VII) is carried out with a large excess of trialkylamine (approximately 200 equivalents) in the presence of a binary mixture of acetonitrile / tetrahydrofuran approximately 1/1 at approximately 70 ° C for approximately 48 h.
• the concentration preferably used is approximately 0.2 M for the compound of formula (la).
• the invention also relates to the use of the compounds of formula (I)
• - X represents an oxygen or sulfur atom, or a group -CH 2 -
• B represents a purine or pyrimidine base, such as uracil, adenine, guanine, cytosine, thymine or hypoxanthine, optionally substituted, or a non-natural mono-or bi-cyclic heterocylic base each cycle of which has 4 to 7 links, optionally substituted, - Li and L 2 , identical or different, represent an oxycarbonyl group -O-CO-, thiocarbamate -O-CS-NH- , carbonate -O-CO-O-, carbamate -O-CO-NH-, an oxygen atom, or else, Li and L 2 ⁇ together, form a ketal group of formula or a thiocetal group of formula
• Ri and R 2 represent a linear or branched alkyl hydrophobic chain containing from 2 to 30 carbon atoms, preferably from 6 to 25 carbon atoms, in particular from 8 to 25 carbon atoms, saturated or partially unsaturated, optionally totally or partially fluorinated, optionally substituted on the chain end carbon by a fluorine atom or by a benzyl or naphthyl ester or ether and R 3 represents a hydroxy, amino group, a phosphate, phosphonate, phosphatidylcholine, O-alkylphosphatidylcholine group , phosphatidylethanolamine, O-alkylphosphatidylethanolamine, thiophosphate, phosphonium, an NH 2 -R 4 group , NHR 4 R 5 or NR 4 R 5 R 6 in which R 4 , R 5 and R ⁇ , which are identical or different, represent a hydrogen atom or a linear or branched alkyl or hydroxyalky
• said biologically active molecules or macromolecules can be chosen from active principles, nucleic acids such as DNA or RNA molecules, antisense DNA, PNA, messenger RNA, RNA interference (siRNA's), plasmids, duplex oligonucleotides, single-stranded oligonucleotides, triplex oligonucleotides, peptides, polysaccharides, etc.
• nucleic acids such as DNA or RNA molecules, antisense DNA, PNA, messenger RNA, RNA interference (siRNA's), plasmids, duplex oligonucleotides, single-stranded oligonucleotides, triplex oligonucleotides, peptides, polysaccharides, etc.
• - anticancer agents such as taxol derivatives, AraC (cytarabine), troxacitabine, navelbine, vinflunine, acronycin and its derivatives , analogs of rebeccamycin, anti-angiogenic molecules, retinoic acid, arsenic, cis platinum and its derivatives, intercalating agents, 5-FU, Idoxuridine (IdU) or DMDC;
• - antiviral agents such as, for example, cyclic nucleoside derivatives such as BVDU (anti-herpes), TFT (anti-herpes), ribavirin and analogues (anti-HCV,) AZT, ddC, d4T, 3TC, CBV, ddl, abacavir (anti-HIV, anti-HBV), lavidarabine or AraA (anti-HBV, anti-HZV, anti-HSV); acyclic taxol derivatives, AraC (cytar
• the compounds of formula (I) can also forming covalent complexes, with different stoichiometries, with certain active principles, such as cis-platinum and its derivatives.
• the compounds of formula (I) above also have very interesting properties. They are capable of forming supramolecular assemblies such as micelles, inverted micelles, liposomes (uni lamellar, multi lamellar, small or giant), hexagonal phases, gel phases, toroids, emulsions, micro emulsions, helices, tubes, fibers or disks, alone or in combination with an active principle or a genetic material, said supramolecular assemblies also being an object of the invention.
• the use of said assemblies for the transport and / or in-vitro, ex-vivo and in-vivo delivery of biologically active molecules or macromolecules, in particular biologically active molecules or macromolecules as defined above, represents a subsequent subject of the invention.
• the compounds of formula (I) or said supramolecular assemblies can enter into the formulation of pharmaceutical compositions. These can in particular be in solid form, solutions, emulsions or gels. In these pharmaceutical compositions, they can be present at concentrations by weight generally ranging from 0.01 to almost 100%, in particular from 0.05 to 5%, and from 0.01 to almost 100% for the solutions and emulsions. and from 1 to 50% for gels.
• the compounds of formula (I) above as well as the supramolecular assemblies as defined above) can be used alone or included in a formulation in which they can represent from 1 to 99%, comprising known amphiphilic compounds, nonionic , for example polyethylene glycol, cationic, anionic, zwitterionic derivatives (DMPC, DLPC, DPPC, DSPC, DOPC, DMPE, DOPE, DPPE, etc.) including cationic lipids (such as DOTAP), polycationic compounds (like the PEI).
• DOTAP cationic lipids
• the present application also relates to a composition for the transfer of genes comprising a gene and a compound of formula (I) as defined above.
• said compound of formula (I) is in the form of a supramolecular assembly as defined above.
• the above formulations can be used according to different protocols for gene transfer as for transfection as electroporation, particle bombardment, delivery by liposomes, delivery by gel, delivery by vector. no viral and receptor-mediated genes.
• they can be used in solution or gel, for example as a culture medium, or deposited on a support surface such as on mica or silica in particular for growing cells.
• the genes can in particular be used in the form of plasmids.
• the gel form is particularly advantageous for the purposes of the invention.
• the present application also relates to a method for the transfer of genes in which a gene is transferred into a host organism using a compound of formula (I) as defined above.
• the compounds of formula (I) and the above supramolecular formulations or assemblies, in particular the gel form can be used for the transfection of nonadherent cells.
• the invention also relates, according to a subsequent aspect, to the use of compounds of formula (I) above and supramolecular assemblies of said compounds as described above for the delivery and / or transport in vitro, ex vivo and in vivo of marker molecules and / or having biological activity, in particular lanthanide or actinide salts.
• the supramolecular assemblies of compounds according to the invention mentioned above can be used to complex salts of actinides or lanthanides, in particular salts of cerium, thorium or uranium, in particular in the form of microspheres.
• said microspheres are formed in an aqueous phase.
• - B represents cytosine, adenosine or a 6-methoxypurine
• R 3 represents a hydroxy group
• active principle also called pro-drug
• the substituent B may represent a base corresponding to a base present in the chemical structure of said active principle, for example a purine or pyrimidine base, optionally substituted, or an unnatural heterocyclic base, mono- or bicyclic, optionally substituted, for example , triazole, thiazole benzimidazole or pyridine-2-one.
• 5-fluorouridine 5-FU
• 5-trifluorouridine TFT
• 5-lodouridine 5 -IU
• the compounds of formula (I) can also be used as a carrier structure for a luminescent probe, for example when the substituent B represents a fluorescent molecule, for example a purine derivative such as 2-aminopurine.
• Preferred compounds for the purposes of the uses mentioned above are compounds of formula (I) in which: X represents an oxygen or sulfur atom, B represents a purine or pyrimidine base such as uracil, adenine, guanine, cytosine, thymine or hypoxanthine, Li and L 2 , identical or different, represent an oxycarbonyl group -O-CO-, thiocarbamate -O-CS-NH-, carbonate -O-CO-O-, carbamate -O-CO-NH-, an oxygen atom, and Ri, R 2 , and R 3 are as defined above.
• Stage A 5'-dimethoxytrityluridine 2 g of chlorodimethoxytrityl, 1.1 g of uridine and a catalytic amount of N, N-dimethyl aminopyridine (DMAP) are dissolved in 25 ml of pyridine. Mix for 24 h at room temperature. The pyridine is removed under reduced pressure and the resulting crude product is purified on silica gel (eluent DCM / methanol 95/5) to obtain 2.14 g of the expected product.
• DMAP N, N-dimethyl aminopyridine
• Stage B 5'-dimethoxytrityl-2 ', 3'-dimyristoyl uridine.
• 0.50 g of the product obtained in Stage A is dissolved in 100 ml of freshly distilled dichloromethane (DCM), 0.46 g of myristic acid, 0.41 g of dicyclohexylecarbodiimide (DCC), and 0.24 g of N, N-dimethylaminopyridine (DMAP).
• DCM dichloromethane
• DCC dicyclohexylecarbodiimide
• DMAP N, N-dimethylaminopyridine
• Stage C 2 ', 3'-dimyristoyl uridine An excess of a 3% solution of trichloroacetic acid is dissolved in 50 ml of freshly distilled DCM 0.78 g of the product obtained in Stage B of Example 1 from DCM. The mixture is brought under stirring for 30 min at room temperature under an inert atmosphere. 3 ml of methanol are then added to the reaction mixture. The organic phase is washed 3 times with 20 ml of water, and dried over sodium sulfate. The residual methylene chloride is removed under reduced pressure. It is further dried under very reduced pressure for 1 h. The resulting white powder is then recrystallized from 20 ml of freshly distilled DCM. 0.464 g of the expected product is obtained.
• Example 2 2 ′, 3′-distearoyl uridine 1.30 g of the product obtained in Stage A of Example 1 are dissolved in 100 ml of freshly distilled DCM, 2.37 g of stearic acid, 1.71 g of DCC, and 1.00 g of DMAP. The mixture is brought under stirring for 48 h at room temperature under an inert atmosphere. After filtration, the organic phase is washed 3 times with 20 ml of water, and dried over sodium sulfate. The residual methylene chloride is removed under reduced pressure. It is further dried under high vacuum for 1 h.
• the resulting white powder was dissolved in 50 ml of freshly distilled dichloromethane and a 3% excess of trichloroethylacetic acid in DCM is added to the solution under an inert atmosphere. After 15 min, 5 ml of methanol are added to the solution. The organic phase is washed 3 times with 20 ml of water, and dried over sodium sulfate. The solvent is removed under reduced pressure and 1.1 g of the expected product are obtained after crystallization from methylene chloride.
• Example 3 bis (2 ', 3'-myristoyl) -5' - (phosphocholine) -uridine
• Stage A 1- (6-hvdroxymethyl-2.2-dimethyltetrahvdro-furo f3.4-d ⁇ 1, 31dioxol-4-yl) -1 H-pyrimidine-2,4-dione
• 2.33 g of paratoluene sulfonic acid are added to 1g of uridine in 180 ml of anhydrous acetone. After 4 h at room temperature, the acetone is evaporated and the residual crude product is dissolved in 200 ml of ethyl acetate.
• Stage B Uridine-oxo-dioxaphospholane 30 ml of freshly distilled tetrahydrofuran (THF) and 803 ⁇ l of triethanolamine (TEA) are added to 0.8 g of the compound obtained in Stage A, under an inert atmosphere. The mixture is cooled to 0 ° C. and 414 ⁇ l of 2-chloro-2-oxo-1, 3,2-dioxaphospholane are added dropwise. The reaction mixture is stirred at room temperature for 15 h. Then, the triethanolamine salts are removed by filtration under suction at 0 ° C. Most of the solvent is evaporated under reduced pressure at 0 ° C and
• Stage C Uridine acetonide phosphocholine 4 ml of anhydrous trimethylamine are condensed at -50 ° C. under an inert atmosphere. 10 ml of dry acetonitrile at -40 ° C and the crude product obtained in stage B are added to the cold trimethylamine. The reaction mixture is stirred at 60 ° C for 48 h. After evaporation of the residual trimethylamine at room temperature and filtration, a solid is isolated. White. The raw material was successively triturated with ethyl acetate and dichloromethane. 1.35 g of the expected product are thus obtained in the form of a hygroscopic solid after drying under very reduced pressure.
• Stage D Uridine phosphocholine 0.20 g of the compound obtained in Stage C are mixed for 24 h at room temperature in 1.5 ml of 98% formic acid. The excess formic acid is evaporated together with ethanol. Crystallization from a methanol-ethanol binary mixture produces 0.127 g of the expected product in the form of a white hygroscopic solid.
• RF 0.72 (reverse phase, MeOH / H 2 O: 8/2).
• Stage E Bis- (2 ', 3'-myristoyl) -5' - (phosphocholine) -uridine 185 mg of myristic acid, 148 mg of DCC and 88 mg of DMAP in 20 ml of anhydrous dimethylformamide. After 70 h at room temperature, the dicyclohexylurea (DCU) is removed by filtration. The solvent is evaporated and the residual solid is triturated twice in 25 ml of ether. 123 mg of expected product, after chromatography (LH 20 DCM / MeOH 5/5). RF: 0.26 (reverse phase, DCM / MeOH 5/5).
• Example 4 bis- (2 ', 3'-stearoyl) -5' - (phosphocholine) uridine
• uridine phosphocholine 555 mg of stearic acid, 402 mg of DCC, 238 mg of DMAP in 20 ml are added. anhydrous dimethylformamide. After 72 h at room temperature, the solvent is evaporated and the residual solid is dissolved in 20 ml of methylene chloride. The DCU is removed by filtration and the solvent is evaporated.
• Example 5 bis- (2 ', 3'-arachidoyl) -5' - (phosphocholine) uridine
• Example 6 bis- (2 ', 3'-oleoyl) -5' - (phosphocholine) uridine
• Example 7 O-Ethyl-bis- (2 ', 3'-oleoyl) -5' - (phosphocholine) -uridine (O-Ethyl DOUPC) 90 mg of bis- (2 ', 3'-oleoyl) -5' - (phosphocholine) uridine of Example 6 are dissolved in 2 ml of anhydrous DCM under an inert atmosphere and protected from light. 25 ⁇ l of ethyl triflate are added to the mixture and the reaction medium is stirred, protected from light for 12 h. The solvent is then evaporated and the residual ethyl triflate is removed under reduced pressure.
• Example 8 Bis- (2 ', 3'-oleoyl) -5' - (phosphocholine) -adenosine (DQAPC) Stage A: 5 '- (phosphocholine) -adenosine 150 mg of 2', 3 '- (isopropylidene) - 5 '- (phosphocholine) -adenosine and 181 mg of paratoluenesulfonic acid monohydrate are dissolved in 25 ml of methanol. The mixture is heated to reflux. After 4 h, the solvent is evaporated under reduced pressure, and the crude product is dissolved in 4 ml of anhydrous ethanol and crystallized after addition of ethyl acetate.
• Stage B Bis- (2 ', 3'-oleoyl) -5' - (phosphocholine) -adenosine (DOAPC) 172 mg of carbonyl diimidazole are added to 337 ⁇ l of oleic acid dissolved in 5 ml of anhydrous dimethylformamide (DMF) under argon. After 5 h of stirring at room temperature, the reaction medium is transferred to a flask containing 320 mg of 5 'tosylate (phosphocholine) -adenosine and 274 mg of paratoluenesulfonic acid in 5 ml of anhydrous DMF. The mixture is stirred for 24 hours at room temperature under argon (the pH is kept below 7).
• DMF dimethylformamide
• the DMF is distilled under reduced pressure and the crude product is purified on an exclusion column, Sephadex LH20, DCM: MeOH 5/5. 123 mg of a highly hygroscopic product are isolated. (Yield: 23%).
• Example 9 Bis- (2 ', 3'-C ⁇ H5Fr7 ⁇ ) -5' - (phosphocholine) -uridine 720 mg of acid 4,4,5,5,6,6,7,7,8,8,8,9 , 9,10,10,11, 11, 11-heptadecafluoro-undecanoic, 303 mg of DCC and 180 mg of DMAP are added to 200 mg of uridine phosphocholine in 12 ml of anhydrous DMF. After 24 h at room temperature, the DMF is evaporated and the residual solid is dissolved in 20 ml of a dichloromethane /: methanol mixture 1: 1. The DCU is removed by filtration and the solvent is evaporated.
• Uridine Palmitonide In a 50 ml two-necked flask, 1 g of hentriacontan-16-one (2.22 mmol), 2.7 g of uridine (11, 11 mmol) and 0.42 g of are weighed. APTS (2.22 mmol). The reactor is placed under an atmosphere of anhydrous nitrogen, then 20 ml of anhydrous THF and 1.85 ml of triethyl orthoformate are added. The reaction medium is stirred and brought to reflux. After 18 h, the reaction is stopped by the addition of 0.6 ml of TEA. The reaction medium is poured into 50 ml of water containing 50 g of ice and 4 g of sodium hydrogencarbonate.
• Example 11 Uridine-oxo-dioxaphospholane palmitonide To 0.5 g of uridine palmitonide obtained in Example 9, 7 ml of freshly distilled THF and 187 ⁇ l of triethylamine are added under an inert atmosphere. The mixture is cooled to 0 ° C. and 109 ⁇ l of 2-chloro-2-oxo-1, 3,2-dioxaphospholane are added dropwise. The reaction mixture is stirred at room temperature for 15 h. Then, the triethylamine salts are removed by filtration under suction at 0 ° C. Most of the solvent is evaporated under reduced pressure at 0 ° C and 3 ml of the residual solution are used directly without purification additional to the next stage.
• Example 13 Adenosine palmitonide In a 25 ml two-necked flask, 1.17 g of palmitone diethoxycetal (2.22 mmol), 0.975 g of adenosine paratoluenesulfonate (2.22 mmol) and 0.042 g of APTS are placed. , 22 mmol). The reactor is placed under an atmosphere of anhydrous nitrogen, then 8 ml of anhydrous THF and 2 ml of anhydrous dimethylformamide (DMF) are added. The reaction medium is stirred and brought to reflux. After 15 hours, the reaction is stopped by the addition of 5 ml of anhydrous TEA.
• DMF dimethylformamide
• the reaction medium is poured into 50 ml of a saturated sodium hydrogen carbonate solution, the aqueous phase is extracted with twice 60 ml of dichloromethane. The organic phase is washed with 30 ml of water, dried over sodium sulfate and the solvent is evaporated under reduced pressure.
• EXAMPLE 14 Adenosine-oxo-dioxaphospholane palmitonide 8 ml of adenosine palmitonide is added to 0.5 g (0.71 mmol) of adenosine palmitonide.
• Example 15 Adenosine phosphocholine palmitonide (PAPC)
• PAPC Adenosine phosphocholine palmitonide
• 4 ml of anhydrous trimethylamine is condensed at -50 ° C. under an inert atmosphere.
• 2 ml of dry acetonitrile and 4 ml of crude adenosine-oxo-dioxaphospholane palmitonide obtained in Example 14 are added to the cold trimethylamine.
• the tube is sealed and the reaction mixture is stirred at 65 ° C. for 48 hours. h.
• Example 16 Use of 2 ', 3'-dioleoyl-5' - (N, N, N-trimethylammonium) uridine tosylate for transfection
• Preliminary transfection studies were carried out taking into account the published work [M. C. Pedroso de Lima et al., Advance Drug Delivery Reviews 47 (2001) 277-294]. These tests carried out on cell lines of HCT 116, Calu 6, Hela, HEK 293 types with the plasmid p-TRACER have shown for the 2 ', 3'-dioleoyl-5' - (N, N, N-trimethylammonium) tosylate ) uridine with similar efficacy to that of DOTAP. In addition, cytotoxicity was found to be less or almost nonexistent in the case of 2 ', 3'-dioleoyl-5' - (N, N, N-trimethylammonium) uridine tosylate.
• Example 17 Formation of a gel usable for transfection 1- A mixture in aqueous phase of the compound (la) Uridine phosphocholine stearonide (SUPC) also called 2 ', 3'-pentatriacontan-18-onide-5' - ( phosphocholine), prepared analogously to the palmitonide PUPC compound of Example 12 (at concentrations between 0.001 and 10%) and of a cationic compound (PEI, lipofectamine, DOTAP, DOTAU etc). The suspension obtained is then heated then cooled over 5 cycles of rise and fall over temperature ranges from -20 ° C to + 60 ° C.
• SUPC Uridine phosphocholine stearonide
• DOTAP DOTAU
• Tm phase transition temperatures
• the suspension obtained is heated and then cooled over 5 temperature cycles from -20 ° C. to 60 ° C.
• the transfection can then be carried out as described in D. Luo, K. Haverstick, N. Belcheva, E. Han and WM Saltzman, Macromolecules, 2002, 35, 3456.
• the plasmid can be added to the mixture obtained at the end of the first step at a temperature below Tm.
• a gel can be produced from the DSUPC compound of Example 4 or the PUPC compound of Example 12.
• Example 18 Formation of a gel of a compound of formula (which can be used for transfection The procedure is as in Example 17, except that the plasmid / cationic compound complex is produced independently of the gel. complex is then added at a temperature higher than Tm to the gel previously formed. In this case the gel was produced by suspending SUPC (at concentrations between 0.001 and 10%) in the aqueous phase. heated then cooled over 5 cycles of rise and fall over temperature ranges from -20 ° C. to 60 ° C. According to the same protocol, a gel can be produced from the DSUPC compound of Example 4 or from the PUPC compound from example 12.
• SUPC at concentrations between 0.001 and 10%
• Example 19 Preparation of microspheres comprising actinides or lanthanides 25 mg of bis- (2 ', 3'-stearoyl) -5' - (phosphocholine) uridine prepared in Example 4 (DSUPC) lyophilized are hydrated for 2 min at 75 ° C by
• microspheres can be prepared with cerium III nitrate or uranium nitrate.

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