ES2368300B1 - LIPOPHYLIC DERIVATIVES OF NUCLEIC ACIDS. - Google Patents

Abstract

Lipophilic derivatives of nucleic acids. # This invention describes new derivatives of pRNA. The modified pRNA according to the invention improves its entry into cells and its stability to degradation by nucleases, increasing the inhibitory capacity of pRNAs. These new compounds contain lipids bound to a bridging molecule present at the terminal end of the pRNA duplex via ether bonds. The synthesis of said compounds is also described.

Description

Lipophilic derivatives of nucleic acids.

The present invention relates to a compound of pRNA and one or more lipids and their synthesis processes. In addition, pharmaceutical compositions comprising the compounds of the invention, as well as their uses in medicine, are also described. Therefore the present invention belongs to the field of biotechnology technique.

Prior art

Interference RNA (RNAi) is an important mechanism of gene regulation that can be used for specific gene silencing. The process is initiated by double-stranded RNAs that are called pRNA, small interfering RNAs (in English small interfering RNAs, siRNAs). The pRNAs, complementary to a particular messenger RNA, bind to a protein complex known as RISC. The complex formed by the union of the antisense or guide chain with RISC catalyzes the efficient degradation of a specific messenger RNA, causing a decrease in the Diana protein. In recent years, numerous studies have been conducted in order to use this phenomenon of natural gene regulation as the basis for a new therapy aimed at the specific reduction of a previously determined protein. Thus, such therapy could be used for the treatment of diseases known to be caused by overexpression of genes such as cancer or inflammatory diseases.

[D. Brumcot et al. RNAi therapeutics: a potential new class of pharmaceutical drugs. Nature Chemical Biology 2 (2006), pages 711-719; A. de Fougerolles et al. RNA interference in vivo: toward synthetic small inhibitory RNA-based therapeutics Methods in Enzymology 392 (2005), pages 278-296; C. Chakraborty Potentiality of small interfering RNAs (siRNA) as recent therapeutic targets for gene-silencing. Current Drug Targets 8 (2007) pages. 469-482]. In order for this regulation mechanism to become an effective treatment, it is necessary that the pRNAs produce a prolonged inhibitory effect, can be effectively distributed in the organism and can be effectively directed towards the damaged organ or tissue or cell group.

Various approaches have been made in the state of the art to improve the entry of pRNA into the cell. For example Wolfrum, et al. (Nature Biotechnology 25 number 10, October 2007) describe describes oligonucleotide conjugates with lipid chains to improve the delivery of siRNAs. The technique used to improve cell entry is the introduction of a cholesterol molecule through an amide bond. Ueno, et al. (Bioorganic & Medicinal Chemistry. 16 (16), 2008, Pp 7698-7704), like Wolfrum, describes oligonucleotide conjugates with lipid chains to improve pRNA delivery. Although the conjugates described by Ueno contain in the glycerol binding nexus, the lipid chain is not linked to it by means of an ether type bond, but a carbamate type bond is used. Some of the problems presented by the derivatives described by Ueno are related to the introduction of the modification in the guide chain, since these changes in the pRNAs significantly reduce the silencing capacity of the pRNAs. In addition, the derivatives described by Ueno were not able to pass through the cell membrane without a transfection agent.

Description of the invention

In this invention new derivatives of pRNA are described. The proposed modifications can be introduced at both the 3 ’and the 5’ end, either of the guide or companion chain of a pRNA duplex. The modified pRNA according to the invention improves its entry into cells and its stability to degradation by nucleases, increasing the inhibitory capacity of pRNAs. Therefore, in the present invention new compounds of pRNA are described that facilitate cell administration and are more stable to nucleases, which makes them more effective in inhibiting gene expression, such as in inhibiting the necrosis gene Mouse alpha tumor involved several inflammatory diseases such as Crown disease, rheumatoid arthritis and cancer. These new compounds contain lipids bound to a bridging molecule present at the terminal end of the pRNA duplex via ether bonds. The synthesis of said compounds is also described.

The presence of the lipid groups joined by ether type bonds at any of the terminal ends, both of the guide chain or the companion chain, improves the thermodynamic stability of the resulting duplexes. These pRNA duplexes covalently linked to aliphatic groups in the 3 'terminal position can be transfected into human cells and the compounds efficiently enter the cells where they trigger the interfering RNA mechanism similar to unmodified pRNA duplexes inducing the specific inhibition of the gene whose sequence is complementary to the sequence of pRNAs. In addition, the modified pRNAs have a much higher stability to the nucleases present in the serum than the unmodified pRNAs, so the pRNAs described in this invention can maintain gene silencing for longer than the unmodified pRNAs.

Therefore, a first aspect of the present invention is a pRNA compound and a lipid covalently bonded together represented by the following formula (I):

where:

And it is a duplex of pRNA linked by a phosphate type bond;

W is an optional group, and is one, or several, substituents that may be present in each methylene unit and selected,

or independently select, from H, C1-C3 alkyl or Z3; Z1, Z2 and Z3 are independently selected from H, C1-C6 alkyl, -NR3R4 and -O-R5; R1 and R2 are independently selected from HyC1-C6-alkyl; R3, R4 and R5 are independently selected from H, a lipid, C1-C6 alkyl; n is an integer that is selected from 1, 2, 3,4 and 5; m is an integer that is selected from 0, 1, 2,3 and 4; with the proviso that at least Z1, Z2 and / or Z3 is -O-lipid.

The pRNA compounds that contain aliphatic and aromatic groups in the 3 'terminal position can be synthesized with good yields. Therefore, a second aspect of the present invention is a process for solid phase synthesis of the compounds as defined by the first aspect or in any of its particular embodiments, characterized in that it comprises the following steps [M. H. Caruthers et al. Chemical synthesis of deoxyoligonucleotides by the phosphoramidite method. Methods in Enzymology 154 (1987) 287-313]:

i) preparing a lipid derivative containing a group that can generate a phosphate group,

ii) synthesize one of the pRNA chains using the solid phase synthesis procedure,

iii) bind the lipid derivative to the pRNA chain via a phosphate bond,

iv) break the bond between the solid support and the product and eliminate the protective groups obtaining one of the chains of the pRNA and

v) mixing equivalent amounts of the guide and companion chains to generate the pRNA.

Another process for solid phase synthesis of the compounds comprises the following steps: i) join the lipid and the bridge, that is the molecule that will finally be interspersed between the pRNA and the lipid,

to form at least one -O-lipid ether bond,

ii) bind the compound obtained in step (i) to a solid support

iii) sequentially assemble one of the pRNA chains and the bridge-O-lipid compound on the support,

iv) break the bond between the solid support and the product and eliminate the protective groups obtaining one of the

chains of the pRNA, v) mixing equivalent amounts of the guide and companion chains to generate the duplex of pRNA.

A third aspect is the pharmaceutical compositions comprising the pRNA compounds of the present invention and at least one pharmaceutically acceptable carrier or excipient.

The last aspect is the use of the pharmaceutical compositions of the present invention for the preparation of a medicament.

Detailed description of the invention

The compounds of the invention have at least one lipid linked by an ether bond. Examples of lipids useful for the present invention, both for those that are linked by ether bonding and for those that are not, are C8-C20 alkyl, with or without unsaturations, fatty acids, acylglyceride, cerido, phospholipids, phosphoglycerides, phospholipid glycolipids, glycolipids , cerebrosides, gangliosides, terpenoids, steroids, eicosanoids, prostaglandins or vitamins A, D, E and K. Among these lipids, those preferred are those selected from C8-C20 alkyl, fatty acids, phospholipids, phosphoglycerides, phospholipolipids, and glycolipids. The lipids of the present invention must have at least one group capable of being converted into an ether bond. This group can be found naturally in the lipid, such as the presence of an alcohol group, or it may have been introduced into the lipid by chemical reactions relevant and known in the art.

By C8-C20 alkyl lipids are meant lipid chains comprising between 8 and 20 carbon atoms. These can be linear or branched chain and can have instalations, either in the form of double bonds or triple bonds and in which there may be hydroxyl, epoxy or carbocyclic ring functions. Examples of these lipids are octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl and bombicol.

By fatty acids is understood as straight chain or branched alkanoic acids that can carry double or triple bonds and in which there may be hydroxyl, epoxy or carbocyclic ring functions. Examples of fatty acids are octanoic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, arachidonic acid, crepeninic acid and aleuritic acid.

By acylglyceride is meant a group of molecules that are formed by glycerin and one or more fatty acids that form ester bonds with the alcohol groups of the glycerin. Examples are monoglycerides, diglycerides and triglycerides.

By cerid is meant a group of molecules that are esters of fatty acids with high molecular weight alcohols. Examples are beeswax, skin wax and etc.

Phospholipids means a group of molecules that are formed by a lipid that contains a phosphate group. The most abundant are phosphoglycerides and phospholipolipids. Phosphoglycerides means a group of molecules that are formed by glycerin esterified by one or two fatty acids and that contains a phosphate group at one of the glycerin positions. Some examples are phosphatidylcholine or lecithin, phosphatidylethanolamine or cephalin, phosphatidylserine, phosphatidylinositol, phosphatidyldiglycerol and etc. Phospholipolipids means a group of molecules that are formed by sphyngosin and that contain a phosphate group. Some examples are fi ngomielin, ceramidaphosphorylethanolamine or fi ngosine-1-phosphate.

By glycolipids is meant a group of molecules that are formed by a lipid that contains at least one carbohydrate. The most abundant are the glycosipolipids that are formed by a ceramide (fatty acid + sphincin) and a short chain carbohydrate. These can be classified into cerebrosides, globosides and gangliosides. By cerebrosides is meant glycospholipids that contain a single carbohydrate linked by a beta-glycosidic junction. Examples are galactocerebrosides that contain galactose such as frenosine, or glucocerebrósidos of nerve tissue.

Gangliosides means a group of sphincolipids that contain complex oligosaccharides. Some examples are N-acetyl neuramic acid or sialic acid or gangliosides present in the gray matter of the brain.

Terpenoids means a group of molecules formed by various isoprene units. The majority are unsaturated hydrocarbons of formula (C5H8) n which may contain alcohol groups and ketones. If they contain two isoprene molecules, they are called monoterpenes, if they contain three isoprene molecules they are called sesquiterpenes, those of C20, diterpenes, C25 sesterterpenes, C30 triterpenes and so on. Some examples are myrcene, limonene, pinene, geraniol, menthol, camphor, farnesol, manool, eudesmol, guayol, abyssic acid or gibberellic acid.

By steroids is meant a family of molecules derived from cyclopentaneperhydrophenanthrene that usually has a branched chain of 8 carbon atoms in one of the positions of the cyclopentane ring. Some examples are cholesterol, ergosterol, stigmasterol, ergocalciferol, precalciferol, ecdysone, colic acid, pregnalone, progesterone, cortisone or tetosterone.

Eicosanoids are understood as a group of molecules caused by the oxygenation of fatty acids of 20 carbon atoms. Some examples are arachidonic acid, prostacyclines, prostagladins, thromboxanes, leukotrienes or certain hydroxy acids.

By prostagladins is meant a group of substances of about 20 carbon atoms present in a wide variety of animal tissues that have a cyclopentane with two linear aliphatic chains that can contain various hydroxyl, ketone, carboxylic and double bond groups. Some examples are prostaglandin E or its derivatives.

In a preferred embodiment Z2 is -O-lipid. While in another embodiment it is Z1 is -O-lipid. In another embodiment both Z1 and Z2 are independently -O-lipid groups.

Relatively short bridges are usually preferred, that is to say that n is 1, 2 or 3, and more preferably that n is

1. It is also preferred that m be 0, 1 or 2, and more preferably that it be 0. Obviously in case m is greater than 1 there may be different groups W. These groups W do not have to be all identical but can be independently selected from the various meanings of W specified above.

In a preferred embodiment R1 and R2 are independently selected from H and C1-C3-alkyl, preferably from H and methyl.

Preferably the derivatizations are introduced at the 3 ′ terminal position of the pRNA duplex.

In a preferred embodiment the bridging molecule that finally in the compound of formula (I) will be introduced between the lipid, or lipids, and Y, come from the introduction, between the phosphate bond and the lipid group, of a threoninol molecule, glycerol , 2-amino-1,3-propanediol, 2-amino-1,4-butanediol, 3-amino-1,4-butanediol, 3, -amino-1,2-propanediol, hydroxypyrolinol, serinol, or any other aminoalkyl diol . Preferably the bridge comes from glycerol.

The length of the pRNA (Y) chain of the compounds of the invention is usually between 15 and 40 nucleotides per chain, more preferably between 15 and 40, and still more preferably between 19 and 25 nucleotides per chain.

Additionally, the pRNA duplex may comprise modifications, such as for example substitutions selected from 2'-O-methyl-ribonucleotide or 2'-deoxyribonucleotide or 2'-methoxyethyl ribonucleotide or 2'-fluoro-deoxyribonucleotide, or 2'-fluoro- arabinonucleotide, restricted conformation nucleosides (such as those known in English with the acronym LNA or HNA), RNA with phosphorothioate bonds, or abbasic residues or ribitoles or nucleosides containing modified bases such as 5-methylcytosine, 5-methyluracil, 4-alkynylcytosine, 5-alkynyluracil, 5halogenocytosine, 5-halogenouracil, or other modified pyrimidines, 7-deazaadenine, 7-deazaguanine, hypoxanthine or other modified purines.

The duplex of pRNA (Y) can inhibit and / or silence, being this inhibition / silencing totally or partially with respect to a control, the expression of various genes of pharmacological interest such as tumor necrosis factor alpha (TNFα), endothelial growth factor and vascular (VEGF), VEGF receptor (VEGF R1 / 2), granulocyte colony suppressor (GM-CSF-1) and macrophage (M-CSF-1), angiopoietin (ANGPT), apolipoprotein (ApoB), vascular neovascularization (CNV), carboxykinase phosphoenol pyruvate (PEPCK), human epidermal growth factor receptor (HER2), macrophage inflammatory protein (MIP2), N-methyl-D aspartate (NMDA) receptor, keratocyte-derived cytokine ( KC), delta opium receptor (DOR), Discoidine domain receptor (DDR1), PIV phosphoprotein gene (PIV-P), heme oxygenase (HMOX1), caveloin, dopamine transporter, green fluorescent protein and / or protein of Swing's sarcoma (EWS-FL / 1). Preferred genes for silencing / inhibiting are tumor necrosis factor alpha (TNFα), endothelial and vascular growth factor (VEGF), VEGF receptor (VEGF R1 / 2), granulocyte colony suppressor factor (GM-CSF-1 ) and macrophages (M-CSF-1). TNF-α is an important mediator of apoptosis both in inflammation and in the immune response. In addition, the overexpression of TNFα is con fi rmed in triggering the pathogenesis of many human diseases. Therefore, the inhibition of this cytokine is relevant from the biomedical point of view. PEPCK is an important protein in the gluconeogenesis process and is overexpressed in diabetes. It is responsible for an increase in hepatic glucose production in diabetic patients and in animal models.

Specific sequences of pRNAs useful for the present invention are the sequence 5 ’GAGGCUGAGACAUAGG CAC-dT-dT-3’ (SEQ ID NO: 1) or 5’-GUGCCUAUGUCUCAGCCUC-dT-dT-3 ’(SEQ ID NO: 2). The sequence SEQ ID NO: 1 corresponds to the guiding or antisense sequence of a fragment of the MusFusculus TNFα RNA at whose 3 'end 2 thymine have been added. The sequence SEQ ID NO: 2 corresponds to the accompanying sequence or complementary sense to SEQ ID NO: 1 (except for the two thymine nucleotides of the 3 ′ end) in which two thymine are also added at its 3 ′ end.

In a preferred embodiment, in which the bridge comes from glycerol and Z2 is -O-lipid, the compounds of the present invention have the following formula:

Or they can also include two lipids

In addition, the lipids of the present invention may incorporate additional derivatizations, such as the introduction of amino groups. These derivatizations are useful, because they allow the introduction of additional groups or functionalities on the lipid.

The present invention is not only limited to pRNA compounds that comprise pRNA duplexes, but also refers to single chain compounds, that is compounds of formula (II):

where:

And it is a simple chain of pRNA;

W is an optional group, and is one, or several, substituents that may be present in each methylene unit and selected,

or independently select, from H, C1-C3 alkyl or Z3; Z1, Z2 and Z3 are independently selected from H, C1-C6 alkyl, -NR3R4 and -O-R5; R1 and R2 are independently selected from HyC1-C6-alkyl; R3, R4 and R5 are independently selected from H, a lipid, C1-C6 alkyl; n is an integer that is selected from 1, 2, 3,4 and 5; m is an integer that is selected from 0, 1, 2,3 and 4; with the proviso that at least Z1, Z2 and / or Z3 is -O-lipid.

Said single chain can be either a sequence of the RNA guide chain, or a sequence of the RNA accompanying chain.

A first process for the synthesis of pRNA compounds is based on a solid phase synthesis process characterized in that it comprises at least the following stages:

i) joining the lipid and the bridge to form at least one -O-lipid ether bond,

ii) bonding the compound obtained in step (i) to a solid support,

iii) sequentially assemble one of the pRNA chains and the bridge-O-lipid compound on the support,

iv) break the bond between the solid support and the product and eliminate the protective groups obtaining one of the chains of the pRNA and

v) mixing equivalent amounts of the guide and companion chains to generate the pRNA duplex.

The solid support used in the synthesis process can be chosen from glass, silica gel, porous glass, silicon oxide, polystyrene, polyethylene glycol, polystyrene-polyethylene glycol, polyamide, acrylamide, polyvinyl chloride, teflon, teflon derivatives, paper and cellulose.

In a particular embodiment the lipid is attached to a solid support and at least one of the pRNA chains is synthesized using the solid phase synthesis method using the solid support functionalized with the lipid.

In another embodiment, the lipid is bound to a solid support and at least one of the pRNA chains is synthesized by successive addition of nucleoside derivatives that constitute the chain sequence including phosphoramidites, H-phosphonates, diester phosphates and monoester phosphates. In a more preferred embodiment, in the process, the lipid is bound to a solid support and at least one of the pRNA chains is synthesized by successive addition of the phosphoramidites of the nucleosides that constitute the chain sequence.

Example 1 of the present invention describes the introduction of lipids with ether bonding at the 3 'end of the pRNA sequence.

A second process for the synthesis of pRNA compounds is based on a solid phase synthesis process characterized in that it comprises at least the following stages:

i) preparing a lipid derivative containing a group that can generate a phosphate group.

ii) sequentially assemble one of the pRNA chains using the solid phase synthesis procedure.

iii) bind the lipid derivative to the pRNA chain via a phosphate bond.

iv) break the bond between the solid support and the product and eliminate the protective groups obtaining one of the pRNA chains.

v) mixing equivalent amounts of the guide and companion chains to generate the pRNA.

The solid support used in the synthesis process can be chosen from glass, silica gel, porous glass, or any surface of silicon oxide, polystyrene, polyethylene glycol, polystyrene-polyethylene glycol, acrylamide or other polyamides, polyvinyl chloride, teflon and its derivatives, paper and cellulose. Preferred oxide supports are selected from glass, silica gel, porous glass, silicon oxide, polystyrene, polyethylene glycol, polystyrene-polyethylene glycol, polyamide, acrylamide, polyvinyl chloride, teflon, paper and cellulose.

In another particular embodiment at least one of the pRNA chains is synthesized by successive addition of nucleoside derivatives that constitute the chain sequence including phosphoramidites, H-phosphonates, diester phosphates and monoester phosphates and then the lipid using the same derivatives (phosphoramidites, H-phosphonates, diester phosphates and monoester phosphates). In a more preferred embodiment, the process is characterized in that at least one of the pRNA chains is synthesized by successive addition of phosphoramidite derivatives of the nucleosides and then the lipid is added using also a lipid phosphoramidite.

Example 2 of the present invention describes the introduction of modifications at the 5 ′ end of the pRNA sequence.

As mentioned above, the lipids present in the compounds of the present invention are capable of incorporating additional groups, such as amino, to make further derivatizations possible. In example 3 of the present invention possible processes for the incorporation of said amino groups are described in particular.

The pharmaceutical compositions of the third aspect of the invention comprise at least one pharmaceutically acceptable carrier or excipient. Excipients include any inert or non-active material used in the preparation of a pharmaceutical dosage form. For example, tablet excipients include, but are not limited to.

a: calcium phosphate, cellulose, starch or lactose. Liquid dosage forms also include oral liquids for example in the form of liquors or suspensions, as well as injectable solutions. The pharmaceutical composition can be formulated for transdermal administration in patch form. All of the above described compositions may optionally contain one or more of each of the following excipients: vehicles, diluents, colorants, flavoring agents, lubricants, solubilizing agents, disintegrants, binders and preservatives.

A particular type of excipient that any pharmaceutical composition of the invention can comprise are transfection agents. These can be added to the pharmaceutical composition to improve even if the properties thereof, or to vectorize it. Preferred transfection agents are selected from one or mixtures of the following compounds lipofectin, liptopofectamine, oligofectamine, effectene, cellfectin, DOTAP, DOPE, fugene, polyethylene glycol, cholesterol, polyethyleneimide (PEI), Jet-polyethyleneimide, cell penetration peptides, peptides Trojans, TAT peptide, penetratin, oligoarginine, poly-lysine, rabies virus glycoprotein, gold nanoparticles, dendrimers, carbon nanotubes, cationic lipids and liposomes.

The pRNA compounds and the pharmaceutical compositions described herein may have various medical uses, but the preferred ones are for the treatment of cancer, inflammation, colitis, ulcerative colitis, Crohn's disease and / or rheumatoid arthritis. In general, the compounds / compositions of the present invention are useful for the preparation of medicaments for gene silencing.

The preferred pharmaceutical composition for the present invention is presented in a form adapted to oral or parenteral administration, preferably parenteral.

The administration of the compounds of this invention can be carried out by any method that releases the compound, preferably to the desired tissue. These procedures include oral, intravenous, intramuscular, subcutaneous or intramedullary, intraduodenal, etc. Preferably the pharmaceutical composition of the present invention can be administered locally by injection in an area near the region of interest (intramuscularly, subcutaneously, intradermally), injection in an area near the region of interest or intravenous injection.

For the purpose of parenteral administration, they can be used as well as sterile aqueous solutions. If necessary, such aqueous solutions can be adequately buffered, and the liquid diluent first became isotonic with enough saline or glucose. These aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes. In this regard, all sterile aqueous media employed can be easily obtained by standard techniques well known to those skilled in the art.

De fi nitions

Lipids are a set of organic molecules, most of them present in living beings, composed mainly of carbon and hydrogen and to a lesser extent oxygen, although they can also contain phosphorus, sulfur and nitrogen, which have as their main characteristic being hydrophobic or insoluble in water and soluble in organic solvents such as benzene and chloroform. Most lipids cannot be used directly for covalent binding with a small interfering RNA. For this, the lipids of the present invention must have at least one hydroxyl group in order to form the ether bond. This hydroxyl group can be found in the lipid, or it can be introduced by techniques known in the art, such as the reduction of carboxylic groups.

The small interfering RNAs, or pRNA, (in English "siRNA") are small fragments of double-stranded RNA of synthetic or natural origin complementary to the sequence of the genes that are used for the specific inhibition of the genes of which they are complementary . The pRNAs are formed by two RNA chains that are called a guide chain (in English "guide strand" or "antisense strand") and an accompanying chain (in English "passenger strand" or "sense strand"). The pRNA guide chain binds to a protein complex called the induction RNA silencer complex (abbreviated as "RISC") and the resulting complex is responsible for cellular degradation of the complementary messenger RNA to the guide chain. For the binding of the guide chain to the RISC complex it is necessary to administer the double stranded RNA or pRNA since the guide chain alone cannot bind to the RISC complex.

The term "solid support" refers to an organic or inorganic polymer that is used to facilitate the synthesis of oligonucleotides, peptides or other organic compound by the solid phase synthesis process. In this procedure one of the reagents is covalently bound to the solid support and the other reagents are added sequentially. The products of the reaction bind to the reagent fixed in the support facilitating the isolation by filtration of the product. The materials that are normally used as supports for solid phase synthesis are glass, silica gel, polystyrene, polyethylene glycol, polyamides and cellulose.

By the term "gene expression" is meant the cellular production of a particular protein encoded by the corresponding gene. In general, the gene found in the chromosomes present in the nucleus of the cells is transcribed generating a messenger RNA molecule that is released in the cytoplasm. There the messenger RNA sequence directs the synthesis of the protein. The result of this process, which is known as gene expression, is the synthesis of a protein whose sequence is encoded by the corresponding gene.

Description of the fi gures

Figure 1 describes the efficiency of silencing of the modified RNAi with lipids in the accompanying chain using protocol A (with transfection agent). HeLa cells were co-transfected with 250 ng of pCAm TNF-α plasmid using lipofectamine as a transfection vector, an hour later 50 nM of each of the pRNAs were transfected using Oligofectamine. After 48 hours, TNF-α levels were measured by ELISA. The data represent the means ± ES, n = 3 and are compared with a random sequence. *** p <0.001. ANOVA test. s.m is the pRNA constituted by the two unmodified chains (guide, 49 / companion, 50); Chol, is the pRNA constituted by an accompanying chain modified in 3 ’with cholesterol (51) and an unmodified guide chain (49); 106 is the pRNA constituted by an accompanying chain modified in 3 ’with lipid C14 (41) and an unmodified guide chain (49); 107 is the pRNA constituted by an accompanying chain modified in 3 'by a C18 lipid (42) and the unmodified guide chain (49); 110 is the pRNA constituted by a 3'-modified 3 'side chain with a C14 lipid and containing various type 2-O-methyl modi fi cations (43) and the unmodified guide chain (49); 111 is the pRNA constituted by an accompanying chain with a modification of the C14N lipid at the 5 ’end (44) and an unmodified guide chain (49); 114 is the pRNA constituted by an accompanying chain modified with lipid C28 at the 5 ’end (45) and an unmodified guide chain (49); Scr is the pRNA constituted by an unmodified random sequence (guide 52, companion 53).

Figure 2 describes the efficiency of silencing of the modified RNAi with lipids in the accompanying chain using protocol B (without transfection agent). HeLa cells were co-transfected with the plasmid pCAm TNF-α as in the previous case. One hour later, 100 nM of each pRNA was added directly to the cells without using any transfection reagent. After 48 hours protein levels were measured by ELISA. The data represent the means ± ES, n = 3 and are compared with a random sequence. * p <0.05, ANOVA test. s.m is the pRNA constituted by the two unmodified chains (guide, 49 / companion, 50); Chol, is the pRNA constituted by an accompanying chain modified in 3 ’with cholesterol (51) and an unmodified guide chain (49); 106 is the pRNA constituted by an accompanying chain modified in 3 ’with lipid C14 (41) and an unmodified guide chain (49); 107 is the pRNA constituted by an accompanying chain modified in 3 'by a C18 lipid (42) and the unmodified guide chain (49); 110 is the pRNA constituted by a 3'-modified 3 'side chain with a C14 lipid and containing various type 2-O-methyl modi fi cations (43) and the unmodified guide chain (49); 111 is the pRNA constituted by an accompanying chain with a modification of the C14N lipid at the 5 ’end (44) and an unmodified guide chain (49); 114 is the pRNA constituted by an accompanying chain modified with lipid C28 at the 5 ’end (45) and an unmodified guide chain (49); Scr is the pRNA constituted by an unmodified random sequence (guide 52, companion 53).

Figure 3 describes the efficiency of silencing of the modified RNAi with lipids in the guide chain. HeLa cells were co-transfected with 250 ng of pCAm TNF-α plasmid using lipofectamine as a transfection vector, an hour later 50 nM of each of the pRNAs were transfected using Oligofectamine. After 48 hours, TNF-α levels were measured by ELISA. The data represent the means ± ES, n = 3 and are compared with a random sequence. *** p <0.001. ANOVA test. s.m is the pRNA constituted by the two unmodified chains (guide, 49 / companion, 50); 108 is the pRNA constituted by a guide chain modified at end 3 ’by lipid C14

(39) and the unmodified accompanying chain (50); 109 is the pRNA constituted by a modified guide chain at the 3 ’end with lipid C18 (40) and the unmodified accompanying chain (50); Scr is the pRNA constituted by an unmodified random sequence (guide 52, companion 53).

Figure 4 describes the efficiency of silencing of the modified RNAi with lipids in the guide chain. HeLa cells were co-transfected with 250 ng of pCAm TNF-α plasmid using lipofectamine as a transfection vector, one hour later 100 nM of each of the pRNAs were transfected directly onto the cells. After 48 hours, TNF-α levels were measured by ELISA. The data represent the means ± ES, n = 3 and are compared with a random sequence. *** p <0.001, ** p <0.01. ANOVA test. Ye. it is the pRNA constituted by the two unmodified chains (guide, 49 / companion, 50); 108 is the pRNA constituted by a guide chain modified at the 3 ’end by the lipid C14 (39) and the unmodified accompanying chain (50); 109 is the pRNA constituted by a modified guide chain at the 3 ’end with lipid C18 (40) and the unmodified accompanying chain (50); Scr is the pRNA constituted by an unmodified random sequence (guide 52, companion 53).

Figure 5 describes the efficiency of silencing lipid-modified pRNAs with amino groups in the accompanying chain using protocol A (with transfection agent). HeLa cells were co-transfected with 250 ng of pCAm TNF-α plasmid using lipofectamine as a transfection vector, an hour later 50 nM of each of the pRNAs were transfected using Oligofectamine. After 48 hours, TNF-α levels were measured by ELISA. The data represent the means ± ES, n = 3 and are compared with a random sequence. *** p <0.001. ANOVA test. s.m is the pRNA constituted by the two unmodified chains (guide, 49 / companion, 50); 126 is the pRNA constituted by an accompanying chain modified at the 3 ’end with the lipid NH2 C12 (46) and the unmodified guide chain (49); 127 is the pRNA constituted by an accompanying chain modified at the 3 ’end with the lipid NH2 triazole C12 (47) and the unmodified guide chain (49); 128 is the pRNA constituted by an accompanying chain modified at the 3 ’end with the lipid NH2 C4 triazole C12 (48) and the unmodified guide chain (49); Scr is the pRNA constituted by an unmodified random sequence (guide 52, companion 53).

Figure 6 describes the efficiency of silencing lipid-modified pRNAs with amino groups in the accompanying chain using protocol B (without transfection agent). HeLa cells were co-transfected with 250 ng of pCAm TNF-α plasmid using lipofectamine as a transfection vector, one hour later 100 nM of each of the pRNAs were transfected directly onto the cells. After 48 hours, TNF-α levels were measured by ELISA. The data represent the means ± ES, n = 3 and are compared with a random sequence. *** p <0.001, ** p <0.01. ANOVA test. s.m is the pRNA constituted by the two unmodified chains (guide, 49 / companion, 50); 126 is the pRNA constituted by an accompanying chain modified at the 3 ’end with the lipid NH2 C12 (46) and the unmodified guide chain (49); 127 is the pRNA constituted by an accompanying chain modified at the 3 ’end with the lipid NH2 triazole C12 (47) and the unmodified guide chain (49); 128 is the pRNA constituted by an accompanying chain modified at the 3 ’end with the lipid NH2 C4 triazole C12 (48) and the unmodified guide chain (49); Scr is the pRNA constituted by an unmodified random sequence (guide 52, companion 53).

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

Examples

All reactions have been carried out under a positive atmosphere of argon and in anhydrous solvents. Reagents obtained from commercial sources and anhydrous solvents were used directly without further purification. Some solvents were distilled before use and dried using conventional methods. The homogeneity of the products obtained was confirmed by TLC and the expected spectroscopic data were obtained for each of the synthesized compounds. Chemical shifts are described in parts per million (ppm) in relation to the singlet of CHCl3 at δ = 7.24 ppm for proton NMR spectra and in the central triplet signal of CDCl3 at δ = 77.0 ppm for NMR-13C spectra. IR spectra were measured fi lm in a BOMEM MB-120 spectrometer. The thin layer chromatography (TLC) was performed on silica gel chromatopholios (Alugram Sil G / UV). The MALDI spectra were obtained on a Fisons VG Tofspec mass spectrometer and a Fisons VG Plattform II. The oligonucleotides were prepared in an automatic Applied Biosystems model 3400 synthesizer.

Example 1

Introduction of lipids with ether bond at the 3 ’end of the pRNA sequence

Scheme 1 details the process. The alkylation reaction between the initial product 1 and the alkyl bromide 2a or the mesylate 2b, prepared following the protocol described in the literature (Marcel YL Holman RT (1968) Synthesis of 14C-labeled polyunsaturated fatty acids Chemical Physics of Lipids Vol. 2, pp. 173-182; Heyes, J., Palmer, L., Bremner, K., Maclachlan, I. (2005) Cationic lipid saturation in fl uences intracellular delivery of encapsulated nucleic acids. Journal of Controlled Release Vol. 107, pp 276-287) it was performed at toluene reflux and yielded the corresponding alkylation products 3a and 3b in good yields (78%). Hydrolysis of acetonide with pTsOH in methanol gave the corresponding diols 4a and 4b in good yield. Then, the selective protection of the primary alcohols was carried out by reaction with monomethoxytrityl chloride (MMTrCl) and N, N-dimethylaminopyridine (DMAP) in pyridine. The lipid analog protected with the dimethoxytrityl group was synthesized in a similar manner but using dimethoxytrityl chloride (DMTrCl). The alcohols protected with the MMTr group 5a and 5b were purified by column chromatography with good yields (80% for 5a and 67% for 5b). The functionalization of the solid support of the controlled pore glass type (CPG) was carried out following the usual protocols obtaining the solid supports functionalized with the corresponding lipids 7a (C14) and 7b (C18).

Scheme 1

Synthesis of controlled pore glass supports (CPG)

Example 2

Introduction of modi fi cations at the 5 ’end of the pRNA sequence

The synthetic route used in the synthesis of lipophosphoramidites 11 and 18 is described in Schemes 2-4. The alkylation reaction between alcohol 8 and the corresponding alkyl bromide was carried out following the procedure described in the literature (Marcel YL Holman RT (1968) Synthesis of 14C-labeled polyunsaturated fatty acids Chemical Physics of Lipids Vol. 2, pp 173- 182). Deprotection of the allyl group was carried out using tetrakistriphenylphosphine and N, N-dimethylbarbituric acid as catalysts. The corresponding alcohol 10 was characterized by 1 H-NMR. Phosphoramidite 11 (C28) was then synthesized using the usual protocols. The resulting product was used directly in the synthesis of oligonucleotides.

Scheme 2

Synthesis of phosphoramidite 11 (C28) used in the synthesis of oligonucleotides containing a lipid at the 5 ’end

The starting materials for the synthesis of the compounds of the invention are either commercial or can be obtained by methods known in the art. For example, phosphoramidite 13 (C18) was synthesized starting from alcohol 12 which was obtained from commercial sources.

Scheme 3

Synthesis of phosphoramidite 13 (C18) used in the synthesis of oligonucleotides containing a lipid at the 5 ’end

Compound 16 was synthesized in good yields following the protocol described in the literature (Moss R.A, Jiang W. (1995) Cis / trans isomerization in azobenzene-chain liposomes. Langmuir vol.11, pp 4217-4221). Deprotection of the trityl group was performed with a 20% tri-fluoroacetic acid solution, obtaining the desired product 17 (76%). Phosphoramidite 18a (C14N) was synthesized using standard protocols. Similarly, phosphoramidite 18b (C18N) was prepared.

Scheme 4

Synthesis of phosphoramidites 18a (C14N) and 18b (C18N) used in the synthesis of oligonucleotides containing a lipid at the 5 ’end

Example 3

Procedures for incorporating amino groups into the pRNA sequence

The synthesis of lipid derivatives and triazole derivatives is shown in Schemes 5 and 6, respectively. The alkylation reaction between solquetal 19 and the corresponding 1,2-dibromododecane 20 in DMF using sodium hydride (60%) as the base yielded the alkylation product 21. On the other hand, the alkylation reaction of 19 with stannate of di-n-butyl in the presence of 2.0 equivalents of cesium fluoride in DMF, obtaining 21 after purification by silica gel column chromatography (Gonçalves AG, Noseda MD, Duarte MER, Grindley TB (2007) Semisynthesis of long- chain alykl ether derivatives of sulfonated oligosaccharides via dibutylstannylene acetal intermediates. J. Org. Chem. Vol. 72, pp9896-9904).

Nucleophilic displacement of the bromide group with sodium azide in DMF yielded the corresponding azide 22 with excellent yields. Then the Staudinger reduction reaction followed by the introduction of the corresponding protecting group into the amino group gave the N-protected compound 23 in good yields which was purified by silica gel chromatography.

The acetonide hydrolysis and removal of the Boc group was carried out simultaneously using a treatment with a solution of tri-fluoroacetic acid in dichloromethane to obtain the desired product as a tri-fluoroacetate. This compound was treated with ethyl tri-fluoroacetate, obtaining the desired compound 24. The reaction of this compound with dimethoxytrityl chloride (DMTr) was carried out in pyridine using DMAP as a catalyst to obtain product 25 in 58% yield.

Scheme 5

Synthesis of derivative protected with DMT group 25

Since one of the intermediates of this synthesis is azide 22, the possibility of exploring the use of the 1,3-dipolar cycloaddition reaction to generate new polar head lipids was considered. Recently, the synthesis of a series of lipid-containing oligonucleotides prepared through a 1,3dipolar cycloaddition reaction that improve cell entry and facilitate intracellular addressing has been described (Godeau G, Staedel C, Barthélémy P (2008) Lipid- conjugated oligonucleotides via “click chemistry” efficiently inhibit hepatitis C virus translation.

J. Med. Chem. Vol. 51 pp 4374-4376)). Azide 22 was reacted with commercial alkynes 26-28 using the usual conditions of "click" chemistry (Rostovtsev VV, Green GL, Fokin VV, Sharpless KB (2002) A stepwise Huisgen cycloaddition process: copper (I) -catalyzed regioselective "ligation" of azides and terminal alkynes. Angew. Chem. Int. Ed. Vol. 41, pp. 2596-2599). Expected products 29a-b and 32 were obtained which were isolated as a single regioisomer by silica gel chromatography (Table 1).

Scheme 6

Synthesis of derivatives with triazole groups

TABLE 1

Cycloaddition reaction between compound 22 and alkynes 26-28

The use of a mild acidic medium for the deprotection of acetonide yielded the corresponding diol 30b with good yield. Protection of the hydroxyl group with the monomethoxytrityl group (MMTr) was carried out with compound 30b following the procedure described in the literature obtaining the corresponding protected alcohol 31.

Scheme 7

Synthesis of the lipid derivative protected with the MMTr group 31

Finally, the synthesis of the lipid derivative 35 is detailed in Scheme 8. After the 1,3dipolar cycloaddition between azide 22 and alkyne 28, hydrolysis of acetonide with p-toluenesulfonic acid (pTsOH) was carried out, generating product 33 with excellent yields that was purified by silica gel chromatography. Basic hydrolysis yielded the corresponding amine which was reacted with ethyl tri-fluoroacetate. The product obtained was used directly without purification in the next reaction in which the primary alcohol group was protected with the DMTr group to obtain compound 35.

Scheme 8

Synthesis of the lipid derivative protected with the DMTr 35 group

Compounds 25, 31 and 35 were used for the functionalization of solid supports of controlled pore glass (CPG) using succinyl type joints as described in the literature (Gupta KC, Kumar P, Bhatia D, Sharma AK (1995 ) A rapid method for the functionalization of polymer supports for solid phase oligonucleotide synthesis. Nucleosides, Nucleotides vol. 14, pp 829-832). To achieve this objective, the DMTr and MMTr derivatives described above were reacted with succinic anhydride, followed by coupling of the corresponding hemisuccinates with functionalized CPG with amino groups obtaining the glass supports properly functionalized with lipids 36-38 (Scheme 9).

Scheme 9

Synthesis of controlled pore glass (CPG) supports functionalized with lipids 36-38

The synthesis of lipid functionalized oligoribonucleotides for use in interfering RNA experiments is described below. Tumor necrosis factor alpha (TNF-α) has been chosen as the target gene. This protein is one of the most important mediators of the apoptosis process as well as is involved in the processes of inflammation and immunity. In addition, the implication of the overexpression of TNF-α in several human diseases has been demonstrated and therefore the inhibition of this protein is medically relevant. The following sequences were chosen: 1) anti-TNFα antisense: SEQ ID NO: 1 and anti-TNFα sense: SEQ ID NO:

2. The sequence of the unmodified anti-TNFα duplex RNA-pRNA has been described in the literature and has inhibitory activity of the mouse TNFα mRNA [D.R. Sorensen et al. Gene silencing by systemic delivery of synthetic siRNA in adult mice. Journal of Molecular Biology 327 (2003) pages 761-766].

Seven oligoribonucleotides with C14 oC18 chain-derived lipids have been prepared: two guide chains with C14 (39) and C18 (40) derivatives at the 3 'end and five accompanying chains with the same lipids (Table 2). Oligonucleotides with the sequence of the accompanying chain 41 and 42 with C14 and C18 derivatives at the 3 'end. Oligonucleotides 44 and 45 contain the C14NyC28 derivatives at the 5 'end (Table 2) and were synthesized using phosphoramidites 18a-b. The oligonucleotide with the sequence of the accompanying chain 43 has the C14 derivative at the 3 ′ end in addition to several 2’-O-methyl-RNA units throughout its sequence without the two final thymidines. Type 2’-O-methyl-RNA units have been used in the literature to protect RNA chains from nuclease degradation. The position of the 2'-O-methyl-RNA units (mC = 2'-O-methyl-C and mU = 2'-O-methyl-U) in oligonucleotide 43 is 5'-GUGC (mC) UAUG ( mU) (mC) U (mC) AG (mC) C (mU) C-3 '(SEQ ID NO: 5) -C14 lipid.

The assembly of the RNA sequences was performed in a DNA synthesizer. The RNA monomers had the t-butyldimethylsilyl (TBDMS) group for the protection of the 2 ′ hydroxyl. The coupling yields of the monomers were about 97-98% per stage. The last DMTr was removed at the end of the sequence assembly since partial elimination was detected during fluoride treatment. The final deprotection of the oligonucleotides was carried out by two consecutive treatments: I) concentrated aqueous ammonia-ethanol 3: 1 at 55 ° C, and II) triethylamine tris (hydro fl uoride) of triethylamine in N-methylpyrrolidone at 65 ° C. HPLC puri fi cation yielded a major product with the expected molecular weight (Table 2).

(Table goes to next page) TABLE 2

Mass spectrometry data and melting temperatures (Tm, ° C) of lipid-functionalized RNA oligonucleotides

Next, the effect of lipids on the stability of the pRNA duplex was determined (Table 2). The thermal stability of the modified and unmodified duplexes was measured in a solution of 100 mM potassium acetate, 2 mM magnesium acetate, and 30 mM HEPES-KOH at pH 7.4. The melting temperatures (Tm) of the lipid functionalized duplexes were similar to those of the unmodified duplex (Tm) modified duplex 83.6-84.3 ° C, unmodified duplex 83.5) except for the duplex containing the oligonucleotide 43 which recorded a Tm of 86.9. The increase in Tm is due to the duplex stabilizing properties of the 2’-O-methyl-RNA units.

The accompanying anti-TNFα sequence SEQ ID NO: 2 was assembled on CPG 36-38 supports obtaining oligoribonucleotides with the lipid amino molecules at the 3 'end (46-48, Table 3).

TABLE 3

Mass spectrometry characterization of RNA derivatives containing polar lipid molecules

The inhibitory power of the tumor necrosis factor gene of the pRNAs described in this invention was then determined. The accompanying and guide chains designed for the inhibition of TNF-α expression previously described [D.R. Sorensen et al. Gene silencing by systemic delivery of synthetic siRNA in adult mice. Journal of Molecular Biology 327 (2003) pages 761-766] were hybridized and the inhibitory capacity of the resulting duplexes was evaluated.

TABLE 4

RNA oligonucleotides used as controls

TABLE 5

RNA duplexes in which the inhibitory effect of TNF-α gene expression has been evaluated

First, the inhibitory properties of pRNAs in HeLa cells were evaluated. These cells do not express mouse TNF-α so the cells were transfected with a plasmid containing the TNF-α gene. Two different protocols were studied:

A) First, HeLa cells were transfected with 250 ng of the plasmid expressing mouse TNF-α (pCAm TNF-α) using lipofectin and 1 h later transfected with the duplex pRNA (50 nM) using oligofectamine. After 48 h, the amount of TNF-α produced by the cells was determined by an ELISA assay.

B) HeLa cells were transfected with 250 ng of the plasmid expressing mouse TNF-α (pCAm TNF-α) using lipofectin as described in protocol A. After 1 h the cells were treated with the pRNA duplex (100 nM) without using any transfection reagent. After 48 h, the amount of TNF-α produced by the cell was analyzed by an ELISA assay.

First, the conjugated pRNAs with C14, C18 and C28 lipids in the sense chain were analyzed. Figure 1 shows the quantity TNF-α produced after 48 h of the addition of the pRNA duplexes using protocol A (using lipofectin / oligofectamine). Figure 2 shows the result using protocol B (without transfection agent). Both modified and unmodified duplex pRNAs designed against TNF-α produced an inhibition of TNF-α production of 80-90% compared to a random sequence control pRNA duplex (protocol A). This result indicates that the introduction of lipid molecules at the 3 'and 5' end of the sense or antisense chains of a duplex does not affect the inhibitory properties of the resulting pRNA duplex in HeLa cells. The removal of the transfection agent (protocol B) decreased the inhibition of TNF-α probably due to the difficulty of crossing the cellular membrane of the pRNA without a transfection agent. However, four modified pRNAs (107, 110, 111 and 114) are capable of inducing gene silencing in the absence of a transfection agent, probably due to the hydrophobic characteristics of the modifications incorporated in the pRNAs.

The modifications made in the antisense chain of the anti-TNF-α pRNA duplex were then evaluated and it was observed that when they were transfected in the presence of a transfection agent such as oligofectamine, they were as efficient in silencing the protein (Figure 3), as they are carried out in the sense chain, indicating that the modification in the antisense chain does not prevent it from being taken by the RISC to perform its function of binding to the corresponding mRNA to inhibit the production of the protein.

When these modified antisense pRNAs are transfected directly on the cells without using any type of transfection agent, the inhibition disappears (Figure 4).

Finally, lipids were evaluated with polar groups, aminos and triazolaminos in the 12-carbon alkyl chain, at the 3 ’end of the sense chain of anti TNF-α pRNA. HeLa cells were transfected with oligofectamine and without oligofectamine and it was found that in the first case, the pRNAs inhibit approximately 65% of the TNF-α protein production compared to a control pRNA (scr) (Figure 5). While when the pRNA molecules were added directly to the cells without any transfection reagent, an almost total loss of the silencing capacity of the pRNAs was observed (Figure 6).

In conclusion, it has been shown that RNAs containing lipid groups at the 3 'or 5' ends can be efficiently synthesized using the reagents developed in this invention. The presence of lipids does not inhibit the ability to bind to its complementary chain, but quite the opposite in most cases has a duplex stabilizing effect. Duplex of pRNA with covalently linked lipids can be efficiently administered to human cells using transfection agents and are compatible with the RNA interference mechanism causing specific inhibition of gene expression. In addition, some of the pRNA duplexes described in this invention are capable of entering the cell interior without the need for a transfection agent.

Example 4

Procedures used in different reactions of the present invention

4.1. General procedure used in alkylation reactions

Solquetal 1 (100 mg, 0.757 mmol) and 60% sodium hydride (NaH, 60.4 mg, 1.51 mmol, 2.0 eq) were stirred in toluene (3.0 mL) and under argon for 20 minutes The corresponding alkyl bromide 2a (420 mg, 1.51 mmol, 2.0 eq) or, where appropriate, the corresponding mesylate 2b (520 mg, 1.51 mmol) were dissolved in toluene (2.0 mL) and the solution was added dropwise. The reaction mixture was heated to reflux under an argon atmosphere. The mixture was diluted with more toluene (10 mL) and the resulting mixture was cooled in an ice bath while the hydride was destroyed by careful addition of cold water. The mixture was washed with water (5.0 mL) and saturated NaCl solution (5.0 mL), using ethanol to help phase separation if necessary. The organic phase was dried with sodium sulfate and evaporated. The crude obtained was purified by column chromatography on silica gel (cyclohexane (Ch): ethyl acetate (AcOEt) 0% to 3%).

2,2-dimethyl-4- (tetradecyloxymethyl) -1,3-dioxolane, 3a

Yield: 78%; IR (fi lm) 2925, 2854, 1462, 1379, 1370, 1117.4 cm -1; 1H-NMR (400 MHz, CDCl3) δ 4.26 (q, J = 6.0 Hz, 1H), 4.05 (dd, J = 8.2 Hz, 6.4 Hz; 1H), 3.73 (dd, J = 8.2 Hz, 6.4 Hz; 1H), 3.51 (m, 2H), 3.43 (m, 2H), 1.60 (m, 4H), 1.42 (s , 3H), 1.36 (s, 3H), 1.25 (m, 20H), 0.88 (t, J = 6.6 Hz, 3H); 13C-NMR (400 MHz, CDCl3) δ 109.6, 74.9, 72.1, 72.0, 67.1, 32.1, 30.0, 29.9, 29.8, 29.8, 29.8, 29.8, 29.7, 29.6, 29.5, 26.9, 26.2, 25.6, 22.9, 14.3; HR ESI MS: m / z calculated for C20H40 Na O3 (M + + 23), 351.2870, experimental m / z 351.2862; calculated for C40H80 Na O6 (2M + + 23), 679.5847, experimental m / z 679.5835.

2,2-dimethyl-4 - ((((10Z, 13Z) -octadeca-10,13-dienyloxy) methyl) -1,3-dioxolane, 3b

Yield: 77%; IR (fi lm) 3008, 2986, 2926, 2856, 1457, 1379, 1369, 1214, 1120, 847 cm -1; 1H-NMR (400 MHz, CDCl3) δ 5.34 (m, 4H), 4.25 (q, J = 5.8 Hz, 1H), 4.04 (dd, J = 14.6 Hz, 8, 2 Hz, 1H), 3.71 (dd, J = 14.6 Hz, 8.2 Hz, 1H), 3.45 (m, 4H), 2.76 (t, J = 5.8 Hz, 2H ), 2.00 (m, 4H), 1.56 (m, 2H), 1.41 (s, 3H), 1.35 (s, 3H), 1.29 (m, 20H), 0.88 (t, J = 6.8 Hz, 3H); 13C-NMR (400 MHz, CDCl3) δ 132.1, 128.9, 110.0, 72.7, 72.1, 70.5, 64.5, 32.1, 30.0, 29.9, 29.8, 29.8, 29.8, 29.6, 29.5, 26.3, 22.9, 14.3; HR ESI MS: m / z calculated for C24H44 Na O3 (M + + 23), 403.3183, experimental m / z 403.3176.

General procedure used in deprotection reactions

A solution of dioxolane 3a (1.0 eq) or 3b (1.0 eq) and p-toluenesulfonic acid monohydrate (0.5 eq) in methanol (5.0 mL) was stirred at room temperature for 16 hours. The solvent was evaporated and the residue was purified by column chromatography on silica gel eluted with dichloromethane (DCM): 0% to 2% methanol (MeOH).

3- (tetradecyloxy) propan-1,2-diol, 4a

Yield: 72%; IR (fi lm) 2980, 2850, 2390, 2387 cm -1; 1H-NMR (400 MHz, CDCl3) δ 3.87 (m, 1H), 3.74 (dd, J = 11.4 Hz, 3.9 Hz, 1H), 3.67 (dd, J = 11, 4 Hz, 5.0 Hz, 1H), 3.54 (m, 2H), 3.48 (m, 2H), 1.60 (m, 4H), 1.27 (m, 20H), 0.89 (t, J = 6.8 Hz, 3H); 13C-NMR (400 MHz, CDCl3) δ 72.8, 72.0, 70.5, 64.5, 32.1, 30.0, 29.9, 29.8, 29.8, 29.8, 29.6, 29.5, 26.3, 22.9, 14.3; HR ESI MS: m / z calculated for C17H36 Na O3 (M + + 23), 311,2557, experimental m / z 311,2555.

3 - ((10Z, 13Z) -octadeca-10,13-dienyloxy) propan-1,2-diol, 4b

Yield: 70%; IR (fi lm) 3105, 2990, 2365, 1530, 1329 cm -1; 1H-NMR (400 MHz, CDCl3) δ 5.33 (m, 4H), 3.83 (m, 1H), 3.64 (m, 2H), 3.47 (m, 4H), 2.74 ( m, 2H), 2.01 (m, 4H), 1.55 (m, 2H), 1.27 (m, 18H), 0.86 (t, J = 6.8 Hz, 3H); 13C-NMR (400 MHz, CDCl3) δ 130.4, 130.3, 130.2, 130.2, 128.2, 128.1, 128.1, 128.0, 72.6, 72.5, 72.0, 72.0, 70.8, 70.7, 64.4, 64.3, 31.8, 31.7, 29.9, 29.8, 29.7, 29.7, 29, 7, 29.6, 29.6, 29.5, 29.5, 29.4, 29.4, 29.4, 27.4, 26.3, 26.2, 25.8, 25.8, 22.8, 22.7, 14.3; HR ESI MS: m / z calculated for C21H40 Na O3 (M + + 23), 363.2698, found m / z 363.2697.

4.2. General procedure used for protection with the MMTr group

Diols 4a or 4b (1.0 eq) were co-evaporated with pyridine and dissolved in anhydrous pyridine (2 mL). N, N-Dimethylpyridine (DMAP) (0.1 eq) and monomethoxytrityl chloride ((MeO) TrCl) (1.5 eq) were added at room temperature and the mixture was stirred at 40 ° C for 16 hours. The reaction was stopped by the addition of MeOH (5 mL), the mixture is concentrated in vacuo and the residue is purified by column chromatography on silica gel (DCM: MeOH: TEA 98: 1: 1).

1 - ((4-methoxyphenyl) diphenylmethoxy) -3- (tetradecyloxy) propan-2-ol, 5a

Yield: 80%; IR (fi lm) 3200, 1630, 1521, 1232, 990 cm -1; 1H-NMR (400 MHz, CDCl3) δ 7.37 (m, 2H), 7.23 (m, 5H), 7.19 (m, 5H), 6.77 (m, 1H), 6.74 ( m, 1H), 3.86 (m, 1H), 3.73 (s, 3H), 3.46 (dd, J = 9.7 Hz, 4.2 Hz, 1H), 3.39 (dd, J = 9.7 Hz, 6.4 Hz, 1H), 3.35 (m, 2H), 3.11 (m, 2H), 2.34 (width d, 1H), 1.47 (m, 2H ), 1.19 (m, 22H), 0.81 (t, J = 6.6 Hz, 3H); 13C-NMR (400 MHz, CDCl3) δ 158.9, 158.7, 150.0, 147.3, 144.6, 135.7, 130.5, 129.4, 128.7, 128.6, 128.1, 128.0, 128.0, 127.3, 127.1, 123.9, 113.4, 113.3, 86.5, 81.9, 72.2, 71.8, 70, 1, 64.7, 55.4, 55.4, 32.1, 30.0, 29.9, 29.8, 29.8, 29.8, 29.7, 29.5, 26.3, 22.9, 14.3; HR ESI MS: m / z calculated for C38H54 Na O5 (M + + 23), 613.7623, experimental m / z 613.7622.

1 - ((4-methoxyphenyl) diphenylmethoxy) -3 - ((10Z, 13Z) -octadeca-10,13-ienyloxy) propan-2-ol, 5b

Yield: 67%; IR (fi lm) 3300, 2980, 2732, 1600, 1475, 1200, 995 cm -1; 1H-NMR (400 MHz, CDCl3) δ 7.57 (m, 1H), 7.36 (m, 2H), 7.20 (m, 7H), 7.10 (d, J = 8.8 Hz, 2H), 6.75 (d, J = 8.8 Hz, 2H), 5.29 (m, 4H), 3.87 (m, 1H), 3.70 (s, 3H), 3.45 ( dd, J = 9.7 Hz, 4.2 Hz, 1H), 3.37 (m, 1H), 3.10 (m, 2H), 3.01 (m, 2H), 2.69 (t, J = 6.5 Hz, 2H), 2.42 (width d, 1H), 1.96 (m, 2H), 1.46 (m, 2H), 1.20 (m, 18H), 0.80 (t, J = 6.7 Hz, 3H); 13C-NMR (400 MHz, CDCl3) δ 158.8, 149.9, 147.4, 144.6, 139.5, 136.1, 130.5, 129.4, 128.6, 128.1, 128.0, 128.0, 127.3, 127.1, 123.9, 113.4, 86.5, 81.9, 72.3, 71.8, 70.1, 64.7, 55, 4, 31.7, 29.9, 29.9, 29.8, 29.7, 29.6, 29.5, 27.5, 27.4, 26.3, 25.8, 22.8, 14.3; HR ESI MS: m / z calculated for C42H58 Na O5 (M + + 23), 665.8249, experimental m / z 665.8250.

2,3-bis (tetradecyloxy) propan-1-ol, 10

A mixture of alcohol 9 (100 mg, 0.191 mmol), N, N-dimethylbarbituric acid (80 mg, 0.514 mmol, 2.7 eq) and catalyst (12.3 mg, 0.011 mmol, 0.05 eq) were dissolved in anhydrous tetrahydrofuran (THF) (2.5 mL) and the mixture was heated 90 ° C in a sealed tube for 16 hours. The solvent was evaporated and the residue was purified by silica gel column chromatography (DCM: MeOH 0% to 1%) to obtain a white solid. Yield: 97%; 1H-NMR (400 MHz, CDCl3) δ 3.72 (m, 1H), 3.61 (m, 2H), 3.50 (m, 2H), 3.30 (m, 2H), 2.91 ( m, 2H), 2.15 (m, 2H), 1.54 (m, 8H), 1.26 (m, 12H), 0.88 (t, J = 6.88Hz, 6H).

N, N-dimethyl-2- (tetradecyloxy) -3- (trityloxy) propan-1-amine, 16

The alcohol 15 (300 mg, 0.830 mmol) and 60% NaH (133 mg, 3.32, 4.0 eq) were suspended in anhydrous THF (4.5 mL) and the mixture was stirred for 15 min at room temperature. Alkyl bromide (575 mg, 2.1 mmol, 2.0 eq) was added dropwise. The reaction mixture was heated at reflux for 16 hours. The reaction mixture was cooled to 0 ° C and water (1.5 mL) was added slowly and carefully. The solvent was evaporated and the residue was dissolved in dichloromethane (10 mL). The solution was washed with water (2 x 20 mL) and NaCl solution (2 x 20 mL). The organic phase was dried with sodium sulfate and the solvent was evaporated. The crude is purified by column chromatography on silica gel (Ch: TEA

99: 1 → Ch: AcOEt: TEA 98: 1: 1). Yield: 77%; IR (fi lm) 3059, 2924, 2853, 2818, 2766, 1490, 1449, 1074, 909 cm -1; 1H-NMR (400 MHz, CDCl3) δ 7.39 (m, 4H), 7.19 (m, 11H), 3.45 (m, 1H), 3.38 (m, 2H), 3.08 ( m, 2H), 2.31 (s, 6H), 1.49 (m, 2H), 1.18 (m, 22H), 0.81 (t, J = 6.6 Hz, 3H); 13C-NMR (400 MHz, CDCl3) δ 144.4, 128.9, 127.9, 127.1, 86.7, 77.9, 70.7, 70.6, 64.9, 61.7, 60.2, 46.5, 45.7, 32.1, 30.4, 30.0, 29.9, 29.8, 29.6, 26.4, 22.9, 14.4; HR ESI MS: m / z calculated for C38H56 NO2 (M + + H), 558.4306, found m / z 558.4298.

3- (dimethylamino) -2- (tetradecyloxy) propan-1-ol, 17

Alcohol 16 (358 mg, 0.642 mmol) was dissolved in a 20% mixture of tri-fluoroacetic acid (TFA) in DCM (2: 8). The mixture was stirred for 2 hours at room temperature. The organic layer was brought to basic pH by the addition of a 1M NaOH solution and extracted twice with DCM (20 mL). The organic layer was dried with sodium sulfate and the solvent was evaporated. The residue was purified by silica gel column chromatography (DCM: MeOH: TEA 90: 9: 1). Yield: 76%; IR (fi lm) 3110, 2960, 2874, 1530, 1470, 1065, 864 cm -1; 1H-NMR (400 MHz, CDCl3) δ 3.79 (dd, J = 10.9 Hz, 4.1 Hz, 1H), 3.68 (m, 1H), 3.48 (m, 5H), 2 , 60 (m, 2H), 2.32 (s, 6H), 1.54 (m, 2H), 1.25 (m, 20H), 0.88 (t, J = 6.6 Hz, 3H) ; 13C-NMR (400 MHz, CDCl3) δ 75.3, 70.1, 65.6, 62.9, 46.5, 32.1, 30.3, 29.9, 29.8, 29.8, 29.8, 29.8, 29.6, 29.5, 26.3, 22.9, 14.3; HR ESI MS: m / z calculated for C19H41NO2 (M + + H), 316.2143, experimental m / z 316.2142.

4.3. General procedure used in the synthesis of lipo-phosphoramidites 11, 13 and 18

To a solution formed by alcohol 10, in DCM (2.0 mL), N, N-diisopropylethylamine (2.0 eq) was added dropwise and at room temperature. The solution was then cooled to 0 ° C and β-cyanoethyl-N, N-diisopropylaminochlorophosphine (1.0 eq) was added. The resulting mixture was taken out of the ice bath and allowed to reach room temperature to be stirred for 2 hours. The reaction mixture was diluted with more DCM (10 mL) and washed with 10 mL of 0.5M cooled sodium bicarbonate. The organic layer was dried with anhydrous sodium sulfate and evaporated to obtain a colorless oil. A vacuum pump was used to remove the last traces of solvent and N, Ndiisopropylethylamine. The crude was used directly without further purification.

2,3-bis (tetradecyloxy) propyl 2-cyanoethyl diisopropylphosphoramidite, 11

Yield: 80%; 1H-NMR (300 MHz, CDCl3) δ 4.12 (m, 2H), 3.79 (m, 1H), 3.47 (m, 8H), 3.26 (m, 1H), 2.69 ( m, 2H), 2.56 (m, 2H), 1.48 (m, 4H), 1.18 (m, 44H), 1.12 (d, J = 6.7 Hz, 6H), 1, 10 (d, J = 6.7 Hz, 6H), 0.81 (t, J = 6.6 Hz, 6H); 31P-NMR (300 MHz, CDCl3) δ 150.1.

2-cyanoethyl (10Z, 13Z) -octadeca-10,13-dienyl diisopropylphosphoramidite, 13

Yield: 100%, 1H-NMR (300 MHz, CDCl3) δ 5.38 (m, 4H), 3.83 (m, 2H), 3.60 (m, 4H), 2.80 (t, J = 6.8 Hz, 2H), 2.63 (t, J = 6.7 Hz, 2H), 2.08 (m, 4H), 1.62 (m, 2H), 1.36 (m, 18H) , 0.84 (t, J = 6.8 Hz, 3H); 31P-NMR (300 MHz, CDCl3) δ 149.8.

2-Cyanoethyl 3- (dimethylamino) -2- (tetradecyloxy) propyl diisopropylphosphoramidite, 18

Yield: 69%; 1H-NMR (400 MHz, CDCl3) δ 4.18 (m, 2H), 3.88 (m, 1H), 3.56 (m, 6H), 2.76 (m, 2H), 2.63 ( m, 2H), 2.26 (s, 6H), 1.56 (m, 2H), 1.25 (m, 22H), 1.19 (m, 12H), 0.88 (t, J = 6 , 5 Hz, 3H); 31P-NMR (300 MHz, CDCl3) δ 150.1.

4 - ((12-bromododecyloxy) methyl) -2,2-dimethyl-1,3-dioxolane, 21

Alcohol 19 (200 mg; 1.51 mmol) was reacted with dibutyltin oxide (751 mg, 3.02 mmol; 2.0 eq) in dry methanol (10 mL) at reflux for 3h. The methanol was then removed and traces of water were removed by azeotropic distillation with toluene. The reaction mixture was concentrated to dryness and 1.12 dibromododecane (991 mg, 3.02 mmol; 2.0 eq), cesium fluoride (458 mg, 3.02 mmol; 2.0 eq) and anhydrous DMF ( 10 mL) The reaction mixture was maintained 16 h at room temperature. The solvent was evaporated and the residue was purified by silica gel column chromatography (Ch: AcOEt 1% to 4%). Yield: 47%; IR (fi lm) 2989, 2931, 2858, 1455, 1367, 1255, 1212, 1115, 1054, 845 cm -1; 1H-NMR (400 MHz, CDCl3) δ 4.24 (q, J = 6.0 Hz, 1H), 4.03 (dd, J = 14.6 Hz, 8.2 Hz; 1H), 3.71 (dd, J = 8.2 Hz, 14.6 Hz; 1H), 3.49 (m, 2H), 3.40 (m, 2H), 3.38 (t, J = 6.8 Hz, 2H ), 1.83 (q, J = 7.1 Hz, 2H), 1.55 (m, 2H), 1.40 (s, 3H), 1.34 (s, 3H), 1.25 (m , 16H); 13C-NMR (125 MHz, CDCl3) δ 109.9, 75.4, 72.5, 72.4, 67.5, 34.6, 33.4, 30.2, 30.1, 30.1, 30.0, 30.0, 29.4, 28.8, 27.4, 26.6, 26.0; HR ESI MS: m / z calculated for C18H35BrO3 Na (M + Na), 401.1661; experimental, m / z 401,1662; calculated for C36H70O6NaBr2 (2M + Na), 779.3427; Experimental, 779.3431.

4 - ((12-azidododecyloxy) methyl) -2,2-dimethyl-1,3-dioxolane, 22

Compound 21 (100 mg, 0.264 mmol) was dissolved in 5 mL of anhydrous DMF. To the solution was added NaN3 (103 mg, 1.58 mmol; 6.0 eq) and the mixture was stirred and heated at 70 ° C for 48 h. The reaction mixture was cooled to 0 ° C and water was added carefully. The solvent was evaporated and the residue was purified by silica gel column chromatography (Ch: AcOEt 1% to 3%). Yield: 93%; IR (fi lm) 2935, 2858, 2363, 2344, 2093, 1251, 1077 cm -1; 1H-NMR (400 MHz, CDCl3) δ 4.26 (q, J = 6.1 Hz, 1H), 4.06 (dd, J = 14.6 Hz, 8.23 Hz; 1H), 3.72 (dd, J = 8.22 Hz, 14.6 Hz; 1H), 3.51 (m, 2H), 3.43 (m, 2H), 3.25 (t, J = 6.97 Hz, 2H ), 1.59 (m, 6H), 1.42 (s, 3H), 1.36 (s, 3H), 1.27 (m, 14H); 13C-NMR (125 MHz, CDCl3) δ 109, 3, 74.7, 71.8, 71.7, 66.9, 51.4, 29.5, 29.4, 29.4, 29.4, 29.4, 29.1, 28.7, 26.7, 26.6, 26.0, 25.3; HR ESI MS: m / z calculated for C18H36N3O3 (M +), 342.2755; experimental, m / z 342.2751; calculated for C18H35N3O3K (M + K), 380.2312; Experimental, 380.2310.

tert-butyl 12 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) dodecylcarbamate, 23

Azide 22 (85 mg, 0.249 mmol) was mixed together with PPh3 (131 mg, 0.498 mmol; 2.0 eq) in 3 mL of anhydrous tetrahydrofuran (THF). The reaction was stirred for 2 h at room temperature. Water (500 μL) was then added dropwise. The mixture was stirred for 16 h at room temperature. The solvent was evaporated and the crude was dried, yielding the corresponding amine. This amine was dissolved in 2.0 mL of DCM; and triethylamine (TEA) (69 μL, 0.498 mmol; 2.0 eq) was added dropwise together with Boc2O (82 mg, 0.373 mmol; 1.5 eq). The reaction mixture was stirred 5 h at room temperature. The organic layer was extracted with more DCM (5 mL) and washed with water (3 x 10 mL). The organic layer was dried with anhydrous sodium sulfate. The solvent was evaporated and the residue was purified by silica gel column chromatography (Ch: AcOEt 4% to 9%). Yield: 65%; IR (fi lm) 3020, 2935, 2850, 1699, 1506, 1371, 1216, 1166, 1046 cm -1; 1H-NMR (400 MHz, CDCl3) δ 4.49 (m, 1H), 4.26 (m, 1H), 4.04 (dd, J = 14.6 Hz, 8.2 Hz; 1H), 3 , 72 (dd, J = 14.6 Hz, 8.2 Hz; 1H), 3.50 (m, 2H), 3.42 (m, 2H), 3.08 (m, 2H), 1.56 (m, 2H), 1.43 (s, 9H), 1.41 (s, 3H), 1.35 (s, 3H), 1.24 (m, 18H); 13C-NMR (125 MHz, CDCl3) δ 110.0, 75.4, 72.5, 72.4, 67.6, 30.7, 30.2, 30.1, 29.9, 29.1, 27.4, 27.4, 26.7, 26.1; HR ESI MS: m / z calculated for C23H45NO5 (M ++ 1), 416.3370; Experimental, m / z 416,3370.

N- (12- (2,3-dihydroxypropoxy) dodecyl) -2,2,2-tri-fluoroacetamide, 24

Compound 23 (60.0 mg, 0.161 mmol) was dissolved in a mixture of DCM / 10% TFA (5 mL) at room temperature for 20 minutes. The solvent was evaporated to obtain the corresponding salt tri-fluoroacetate. This product was dissolved in anhydrous DCM (5.0 mL) and TEA was added dropwise (45 µL, 0.322 mmol). The reaction mixture was cooled to 0 ° C and ethyl tri-fluoroacetate (22.0 µL, 0.177 mmol) was added. The reaction mixture was stirred for 30 minutes, extracting the organic layer with more DCM (3 x 10 mL), which was subsequently washed with water (3 x 10 mL). The organic layer was dried with anhydrous sodium sulfate. The solvent was evaporated and the residue was purified by silica gel column chromatography (DCM: 2% MeOH). Yield: 87%; IR (fi lm) 3333, 2924, 2854, 1694, 1552, 1471, 1185 cm -1; 1H-NMR (400 MHz, CDCl3) δ 6.42 (broad s, 1H), 3.05 (m, 1H), 3.72 (dd, J = 11.4 Hz, 3.8 Hz; 1H), 3.65 (dd, J = 11.4 Hz, 5.2 Hz; 1H), 3.51 (m, 2H), 3.49 (m, 2H), 3.35 (m, 2H), 1, 57 (m, 2H), 1.26 (m, 20H); 13C-NMR (125 MHz, CDCl3) δ 73.2, 72.5, 71.0, 64.9, 40.6, 30.2, 30.1, 30.1, 30.1, 30.0, 29.7, 29.6, 27.3, 26.7; 19F-NMR δ -76.3 (reference CFCl3); HR ESI MS: m / z calculated for C17H32F3NO4 (M ++ 23), 394.2175; experimental, m / z 342.2176.

N- (12- (3- (bis (4-methoxyphenyl) (phenyl) methoxy) -2-hydroxypropoxy) dodecyl) -2,2,2-tri-fluoroacetamide, 25

Compound 24 (52 mg, 0.134 mmol) was reacted with dimethoxytrityl chloride (DMTrCl) (48 mg, 0.141 mmol; 1.2 eq) and DMAP (8 mg, 0.07 mmol; 0.5 eq) in 1 , 5 mL of anhydrous pyridine. The reaction was heated to 40 ° C and kept stirring overnight. Then, methanol (0.5 mL) was added and the solvent was evaporated. The residue was purified by silica gel column chromatography (Hex / AcOEt 15%). Yield: 58%; IR (fi lm) 3276, 3072, 2927, 2851, 1716, 1673, 1607, 1502, 1462, 1390, 1248, 1176, 1038 cm -1; 1H-NMR (400 MHz, CDCl3) δ 7.67 (m, 4H), 7.40 (m, 2H), 7.28 (m, 3H), 7.01 (broad s, 1H), 6.81 (d, J = 8.9 Hz, 4H), 3.92 (m, 1H), 3.76 (s, 6H), 3.74 (m, 1H), 3.51 (m, 2H), 3 , 43 (m, 2H), 3.35 (m, 1H), 3.17 (m, 2H), 2.41 (broad d, 1H), 1.85 (m, 2H), 1.57 (m , 9H), 1.25 (m, 9H); 13C-NMR (125 MHz, CDCl3) δ 162.7, 158.6, 149.9, 145.0. 136.3, 136.2, 130.2, 128.3, 127.9, 126.9, 123.9, 113.2, 86.2, 72.3, 71.8, 70.1, 64, 7, 55.4, 55.3, 55.3, 50.8, 40.2, 36.6, 31.6, 29.9, 29.8, 29.7, 29.7, 29.6, 29.6, 29.6, 29.3, 29.1, 26.8, 26.2; 19F-NMR δ -74.3 (reference CFCl3); HR ESI MS: m / z calculated for C38H50F3NO6 (M ++ 23), 696.3482; Experimental, m / z 696,3490.

4.4. General procedure used in click-type reactions (29a-b and 32)

In a mixture (1: 1) of water and tert-butyl alcohol (1.0 mL), azide 22 (1.0 eq) and alkynes (26-28) (1.0 eq) were added. To the mixture was added dropwise sodium ascorbate (10%) and copper (II) sulfate pentahydrate (1%). The heterogeneous mixture was vigorously stirred overnight. The reaction mixture was diluted with DCM and the organic layer was dried with anhydrous Na2SO4 and the solvent was evaporated to dryness. The residue was purified by column chromatography on silica gel.

tert-butyl (1- (12 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) dodecyl) -1H-1,2,3-triazol-4-yl) methylcarbamate, 29a

Yield: 69%; IR (fi lm) 2929, 2861, 1713, 1500, 1458, 1369, 1250, 1169, 1050, 752 cm -1; 1H-NMR (400 MHz, CDCl3) δ 7.43 (s, 1H), 5.09 (s wide, 1H), 4.32 (d, J = 5.9 Hz; 2H), 4.24 (t , J = 7.2 Hz; 2H), 4.19 (m, 1H), 3.98 (dd, J = 14.6 Hz, 8.2 Hz; 1H), 3.65 (dd, J = 14 , 6 Hz, 8.2 Hz; 1H), 3.44 (m, 2H), 3.38 (m, 2H), 1.81 (m, 2H), 1.49 (m, 2H), 1, 37 (s, 9H), 1.35 (s, 3H), 1.29 (s, 3H), 1.21 (m, 16H); 13C-NMR (125 MHz, CDCl3) δ 156.0, 122.4, 109.5, 79.5, 74.9, 72.1, 72.0, 67.1, 50.6, 36.4, 36.3, 30.4, 29.7, 29.7, 29.7, 29.6, 29.6, 29.5, 29.1, 28.5, 26.9, 26.6, 26, 2, 25.6; ); HR ESI MS: m / z calculated for C26H48N4O5 (M ++ 1), 497.3697; experimental, m / z 497.3698; calculated for C26H48N4O5 Na (M ++ 23), 519.3516; Experimental, m / z 519,3517.

(9H- fl uoren-9-yl) methyl (1- (12 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) dodecyl) -1H-1,2,3-triazol-4 -il) methylcarbamate, 29b

Yield: 58%; IR (fi lm) 2930, 2858, 1721, 1523, 1448, 1370, 1250, 1120, 1049, 758, 738 cm -1; 1H-NMR (400 MHz, CDCl3) δ 7.74 (d, J = 7.5 Hz; 2H), 7.57 (d, J = 7.5 Hz; 2H), 7.47 (s, 1H) , 7.38 (t, J = 7.3Hz; 2H), 7.29 (t, J = 7.4 Hz; 2H), 5.48 (broad d, 1H), 4.46 (d, J = 6.0 Hz; 2H), 4.40 (d, J = 7.0 Hz; 2H), 4.31 (t, J = 7.2 Hz; 1H), 4.25 (t, J = 6, 0 Hz; 2H), 4.22 (m, 2H), 4.05 (dd, J = 14.6 Hz, 8.2 Hz; 1H), 3.72 (dd, J = 14.6 Hz, 8 , 2 Hz; 1H), 3.50 (m, 2H), 3.42 (m, 2H), 1.87 (m, 2H), 1.55 (m, 2H), 1.41 (s, 3H ), 1.35 (s, 3H), 1.27 (m, 16H); 13C-NMR (125 MHz, CDCl3) δ 156.3, 143.8, 141.2, 127.6, 127.0, 125.0, 119.9, 109.3, 74.7, 71.9, 71.8, 66.9, 50.3, 47.2, 36.5, 30.2, 29.5, 29.5, 29.4, 29.4, 29.3, 28.9, 26, 7, 26.4, 26.0, 25.4; HR ESI MS: m / z calculated for C36H50N4O5 (M ++ 1), 619.3853; experimental, m / z 619.3858; calculated for C36H50N4O5 Na (M ++ 23), 641.3673; Experimental, m / z 641.3675.

2- (4- (1- (12 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) dodecyl) -1H-1,2,3-triazol-4-yl) butyl) isoindolin-1,3-dione, 32

Yield: 89%; IR (fi lm) 2919, 2854, 2363, 2342, 1710, 1053 cm -1; 1H-NMR (400 MHz, CDCl3) δ 7.81 (m, 2H), 7.69 (m, 2H), 7.27 (s, 1H), 4.27 (t, J = 7.24 Hz; 2H), 4.22 (m, 1H), 4.04 (dd, J = 14.6 Hz, 8.2 Hz; 1H), 3.69 (m, 3H), 3.49 (m, 2H) , 3.41 (m, 2H), 2.74 (t, J = 7.0 Hz, 2H), 1.85 (m, 2H), 1.72 (m, 4H), 1.54 (m, 2H), 1.40 (s, 3H), 1.34 (s, 3H), 1.26 (m, 16H); 13C-NMR (125 MHz, CDCl3) δ 168.6, 134.1, 132.3, 123.3, 109.5, 74.9, 72.1, 72.0, 67.1, 50.4, 37.8, 30.5, 29.7, 29.7, 29.7, 29.6, 29.5, 29.2, 28.2, 26.9, 26.8, 26.7, 26, 2, 25.6, 25.3; HR ESI MS: m / z calculated for C32H48N4O5 (M ++ 1), 569.3697; experimental, m / z 569.3697 calculated for C32H48N4O5 Na (M ++ 23), 591.3516; experimental, m / z 591.3516.

(9H-Fluoren-9-yl) methyl (1- (12- (2,3-dihydroxypropoxy) dodecyl) -1H-1,2,3-triazol-4-yl) methylcarbamate, 30b

Compound 29b (48 mg; 0.078 mmol) was dissolved in a mixture of CH2Cl2 / 3% TFA (2.5 mL). The reaction mixture was stirred at room temperature for 30 minutes. The solvent was evaporated to dryness, washing three times with more CH2Cl2. The residue was purified by silica gel column chromatography (CH2Cl2 / 2% MeOH). Yield: 72%; IR (fi lm) 3329, 2926, 2858, 1715, 1522, 1441, 1256, 1127, 1043, cm -1; 1H-NMR (400 MHz, CDCl3) δ 7.76 (d, J = 7.5 Hz; 2H), 7.58 (d, J = 7.3 Hz; 2H), 7.38 (t, J = 7.3 Hz; 2H), 7.28 (m, 3H), 5.83 (wide s, 1H), 5.31 (s, 2H), 4.64-4.13 (m, 5H), 3 , 76 (m, 4H), 3.47 (m, 4H), 1.89 (m, 2H), 1.56 (m, 2H), 1.26 (m, 16H); 13C-NMR (400 MHz, CDCl3) δ 156.4, 143.6, 141.1, 127.5, 126.8, 124.9, 119.8, 72.3, 71.6, 70.3, 70.2, 66.7, 64.0, 53.2, 50.6, 47.0, 29.9, 29.5, 29.3, 29.2, 29.2, 29.2, 29, 2, 29.1, 28.7, 26.3, 25.8; HR ESI MS: m / z calculated for C33H47N4O5 (M ++ 1), 579.3540; experimental, m / z 579.3539 calculated for C33H46N4O5 Na (M ++ 23), 601.3360; experimental, m / z 601.3358.

(9H-Fluoren-9-yl) methyl (1- (12- (2-hydroxy-3 - ((4-methoxyphenyl) diphenylmethoxy) propoxy) dodecyl) -1H-1,2,3-triazol-4-yl) methylcarbamate, 31

Compound 30b (32 mg; 0.055 mmol) was dissolved with DMAP (3 mg; 0.0275 mmol; 0.5 eq) and monomethoxytrityl chloride (MMTrCl) (26 mg, 0.083 mmol; 1.5 eq) in pyridine ( 1.5 mL) The reaction was heated to 40 ° C and stirred overnight. Methanol (1.0 mL) was added and the solvent was evaporated. The residue was purified by column chromatography on silica gel (CH2Cl2: 1% MeOH). Yield: 45%; IR (fi lm) 3064, 2930, 2855, 1726, 1607, 1505, 1449, 1252, 1074, 755 cm -1; 1H-NMR (400 MHz, CDCl3) δ 7.69 (d, J = 7.5 Hz; 1H), 7.50 (d, J = 7.5 Hz; 1H), 7.35 (m, 4H) , 7.21 (m, 4H), 7.13 (m, 1H), 6.73 (d, J = 8.9 Hz; 1H), 5.37 (m, 1H), 4.38 (dd, J = 22.9 Hz, 5.8 Hz; 4H), 4.18 (m, 4H), 3.87 (m, 1H), 3.71 (s, 3H), 3.45 (m, 2H) , 3.38 (m, 2H), 3.10 (m, 2H), 2.48 (c, J = 7.2 Hz; 2H), 1.80 (m, 2H), 1.46 (m, 2H), 1.18 (m, 16H); 13C-NMR (400 MHz, CDCl3) δ 158.8, 156.6, 144.6, 144.0, 141.5, 135.7, 130.5, 128.7, 128.6, 128.0, 127.9, 127.2, 1271, 125.2, 122.2, 120.0, 113.3, 86.5, 72.3, 71.8, 70.0, 67.0, 64.7, 55.4, 50.6, 47.4, 46.4, 36.7, 30.4, 29.8, 29.7, 29.7, 29.6, 29.5, 29.2, 26, 7, 26.7, 26.3; HR ESI MS: m / z calculated for C53H62N4O6 (M ++ 23), 873.4558; experimental, m / z 873.4555 calculated for C106H124N8O12 Na (2M ++ 23), 1723.9230; Experimental, m / z 1723.9234.

2- (4- (1- (12- (2,3-dihydroxypropoxy) dodecyl) -1H-1,2,3-triazol-4-yl) butyl) isoindolin-1,3-dione, 33

Compound 32 (30 mg, 0.052 mmol; 1.0 eq) was reacted with pTsOH (5 mg, 0.026 mmol; 0.5 eq) in methanol (1.0 mL). The reaction mixture was stirred at room temperature for 5 h. The methanol was evaporated and the residue was purified by silica gel column chromatography (CH2Cl2: 2% MeOH). Yield: 100%; IR (fi lm) 3349, 2924, 2850, 1684, 1517, 1251, 1170, 1123, 1046 cm -1; 1H-NMR (400 MHz, CDCl3) δ 7.82 (m, 2H), 7.69 (m, 2H), 7.28 (s, 1H), 4.27 (t, J = 7.2 Hz; 2H), 3.85 (m, 1H), 3.69 (m, 3H), 3.63 (m, 1H), 3.49 (m, 2H), 3.44 (m, 2H), 2, 74 (m, 2H), 2.53 (broad d, 2H), 1.85 (m, 2H), 1.72 (m, 4H), 1.55 (m, 2H), 1.23 (m, 18H); 13C-NMR (400 MHz, CDCl3) δ 168.6, 134.1, 132.3, 123.4, 72.7, 72.0, 70.6, 64.4, 50.4, 37.8, 30.5, 29.7, 29.6, 29.6, 29.6, 29.5, 29.5, 29.1, 28.2, 26.8, 26.6, 26.2, 25, 3; HR ESI MS: m / z calculated for C29H44N4O5 (M ++ 1), 529.3384; experimental, m / z 529.3384; Calculated for C29H44N4O5 Na (M ++ 23), 551.3190; experimental, m / z 551,3200.

N- (4- (1- (12- (3- (bis (4-methoxyphenyl) (phenyl) methoxy) -2-hydroxypropoxy) dodecyl) -1H-1,2,3-triazol-4-yl) butyl) -2,2,2tri fl uoroacetamide, 35

Compound 33 (30 mg, 0.060 mmol) was dissolved in a mixture of 3% DCM / TFA and stirred at room temperature for 1 h. The solvent was evaporated and the residue was dissolved in anhydrous DCM (1.5 mL). TEA (38 μL) was added dropwise at room temperature. The reaction mixture was cooled 0 ° C and EFTA (8.0 µL) was added dropwise. The reaction mixture was stirred for 30 minutes at 0 ° C. The organic layer was washed with water and aqueous NaCl solution (3 x 10 mL) and dried with anhydrous Na2SO4 and concentrated to dryness. The product obtained was used in the next step without further purification. The product obtained was reacted with MMTCl (27.8 mg, 0.09 mmol; 1.5 eq) in the presence of DMAP (4.0 mg, 0.03 mmol; 0.5 eq) in 1.0 mL of pyridine The reaction mixture was heated to 40 ° C and stirred overnight. The reaction was stopped by the addition of methanol (1.0 mL) and the solvent was concentrated to dryness. The residue was purified by silica gel column chromatography (DCM / MeOH / TEA 98: 1: 1). Yield: 39%; IR (fi lm) 3065, 2935, 2860, 1715, 1650, 1593, 1454, 1230, cm − 1; 1H-NMR (400 MHz, CDCl3) δ 7.41 (m, 2H), 7.31 (m, 4H), 7.26 (m, 4H), 6.82 (d, J = 8.8 Hz, 4H), 4.29 (t, J = 7.2 Hz; 2H), 3.94 (m, 1H), 3.78 (s, 6H), 3.51 (m, 2H), 3.42 ( m, 4H), 3.17 (m, 2H), 2.99 (m, 3H), 2.74 (t, J = 7.0 Hz, 2H), 1.87 (m, 2H), 1, 75 (m, 2H), 1.67 (m, 2H), 1.53 (m, 2H), 1.28 (m, 16H); 13C-NMR (400 MHz, CDCl3) δ 158.6, 150.0, 147.4, 145.0, 136.2, 130.2, 128.3, 128.0, 126.9, 124.0, 120.9, 113.2, 86.2, 72.3, 71.8, 70.1, 64.6, 55.4, 50.4, 39.8, 30.5, 29.8, 29, 7, 29.6, 29.5, 29.2, 28.1, 26.7, 26.4, 26.3, 24.9; 19F-NMR δ -73.5 (reference CFCl3); HR ESI MS: m / z calculated for C44H59N4O6 (M ++ 1), 796.4434; experimental, m / z 796.44353 calculated for C44N59N4O6 (M ++ 23), 819.3745; Experimental, m / z 819,3742.

General procedure used for the functionalization of polymeric supports, 7a and 7b, 36, 37, 38

The alcohols 5a-b, 26, 31 and 35 were reacted with succinic anhydride (1.5 eq) in the presence of DMAP (1.5 eq) in anhydrous DCM (1 mL). The reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with DCM (5 mL) and the organic layer was washed with a 0.1M Na2HPO4 aqueous solution. The organic layer was dried with sodium sulfate and the solvent was evaporated to dryness. The products obtained (hemisuccinates) were used directly without further purification.

2,2-Dithio-bis- (5-nitropyridine) (DTB, 0.1 mmol) was dissolved in 400 µL of acetonitrile and the solution was mixed with a solution containing hemisuccinate (0.1 mmol) and DMAP (0 , 1 mmol) in acetonitrile (500 μL). Finally, a solution of triphenylphosphine (TPP) (0.1) dissolved in acetonitrile (200 μL) was added, all at room temperature.

The mixture was stirred with the vortex for a few seconds and added to a syringe containing the polymeric controlled pore glass support (LCAA-CPG) (500 mg, 0.05 mmol of amino groups) and allowed to react for 2 h room temperature. The reaction was stopped by the addition of methanol (500 μL). The polymeric support was washed with methanol (3 x 10 mL), acetonitrile (3 x 10 mL) and diethyl ether (3 x 10 mL). The support was dried under vacuum and the residual amino groups were blocked by acetylation with a mixture of acetic anhydride / pyridine / tetrahydrofuran (500 µL) and 1-methylimidazole in tetrahydrofuran (500 µL). After 30 minutes, the polymeric support was washed with methanol (3 x 10 mL), acetonitrile (3 x 10 mL) and diethyl ether (3 x 10 mL). The resulting support was dried first in the air and then applying vacuum. The polymeric support was stored at 4 ° C. The degree of functionalization of the supports was determined by elimination of the DMTr or MMTr groups in a small aliquot and reading the absorbance of the DMTr cation at 500 nm or that of the MMTr cation at 475 nm detached from the supports during removal. The degree of functionalization of the prepared supports was between 15-35 nmol / g.

4.5. Oligonucleotide synthesis

The oligoribonucleotides were prepared using a synthesizer from the Applied Biosystems Model 3400 commercial house using 2-cyanoethyl phosphoramidites and tert-butyldimethylsilyl (TBDMS) type protectors for the protection of the hydroxyl group in the 2 ′ position. The following solutions were used: 0.4 M 1H-tetrazole in ACN (catalyst); 3% trichloroacetic acid in DCM (destritilation), acetic anhydride / pyridine / tetrahydrofuran (1: 1: 8) (capping A), 10% N-methylimidazole in tetrahydrofuran (capping B), 0.01 M iodine in tetrahydrofuran / pyridine / Water

(7: 2: 1) (oxidation). In the RNA sequences, the last DMT was removed since the DMT group is not completely stable to fluoride treatment. The average yield per stage of addition of a nucleotide was about 97-98% for RNA monomers. The polymeric supports obtained were treated with a concentrated solution of ammonia-ethanol (3: 1) for 1 h at 55 ° C. The supports were washed with ethanol and the resulting solutions were combined and evaporated to dryness. The resulting products were treated with 0.15 mL of triethylaminetris (hydro fl uoride) / triethylamine / N-methylpyrrolidone (4: 3: 6) for 2.5 h at 65 ° C in order to eliminate TBDMS groups. The reactions were stopped by the addition of 0.3 mL of isopropoxytrimethylsilane and 0.75 mL of ether. The resulting mixtures were stirred and cooled to 4 ° C. A precipitate formed which was centrifuged at 7000 rpm for 5 min at 4 ° C. The precipitates were washed with ether and centrifuged again. The residues were dissolved in water and the conjugates were purified by HPLC. Column: Nucleosil 120-10 C18 (250x4 mm); a linear gradient of 20 min from 0% to 50% B was used; with a flow of 3 mL / min. The composition of the solutions used in the HPLC were: solution A: 5% acetonitrile (ACN) in 100 mM triethylammonium acetate (pH 6.5) and solution B: 70% ACN in 100 mM triethylammonium acetate pH 6.5. Puri fi ed products were analyzed by MALDI-TOF mass spectrometry. The homogeneity of the purified conjugates is determined by analytical HPLC and electrophoresis in polyacrylamide gels and all presented a purity greater than 90%.

Analytical HPLC purity analysis is performed using an XBridgeTM OST C18 column (Waters), 2.5 μm, 4.6 x 50 mm using a 10-minute linear gradient from 0% to 35% B, fl ow with a fl ow 1 mL / min The AyB solutions are the same as those described above.

Electrophoresis in polyacrylamide gels under denaturing conditions were carried out in a Hoefer Scienti fi c SE-600 apparatus. The gels (8 M urea, 20%, with a 19: 1 acrylamide / bisacrylamide ratio), had dimensions of 14 x 16 x 0.1 cm and a voltage of 400 V was applied for 5 h. Three micrograms of RNA were loaded per pocket. The gel was stained with Stains-all (Sigma).

MALDI-TOF spectra were performed on a Perseptive Voyager DETMRP mass spectrometer, equipped with a 337 nm nitrogen laser using a 3ns pulse. The matrix used contained 2,4,6-trihydroxyacetophenone (THAP, 10 mg / mL in ACN / water 1: 1) and ammonium citrate (50 mg / mL in water).

Oligonucleotides

The following RNA sequences were obtained from commercial sources (Sigma-Proligo, Dharmacon): sense or companion chain negative control (scr) 5'-CAGUCGCGUUUGCGACUGG-dT-dT-3 '(SEQ ID NO: 3), antisense or guide chain negative control (scr) 5'-CCAGUCGCAAACGCGACUG-dT-dT-3 '(SEQ ID NO: 4), antisense chain or anti-TNF-α guide: SEQ ID NO: 1 and anti-TNF-α sense or companion chain: SEQ ID NO: 2. The monomers of the RNA type are detailed in capital letters, the abbreviation dT corresponds thymidine. The accompanying anti-TNF-α chain with 3 'end cholesterol (SEQ ID NO: 2-cholesterol) was prepared from the commercial solid support named cholesterol-tetraethylene glycol (TEG) -3'-CPG (Glen Research). The anti-TNF-α siRNA sequences had been described pRNA in the literature to inhibit mouse TNF-α mRNA [D.R. Sorensen et al. Gene silencing by systemic delivery of synthetic siRNA in adult mice. Journal of Molecular Biology 327 (2003) pages 761-766].

4.6. Cell cultures, transfection and cell assays

HeLa cells were cultured under usual conditions: 37 ° C, 5% CO2, modified Dulbecco Eagle medium, 10% fetal bovine serum, 2 mM L-glutamine, supplemented with penicillin (100 U / ml) and streptomycin (100 mg / ml). All experiments were performed at a fl uence of 40-60%. HeLa cells were transfected with 250 ng of the plasmid expressing the mouse TNF-α gene using lipofectin (Invitrogen) and following the manufacturer's instructions.

Protocol A: After one hour after transfection, HeLa cells expressing m-TNF-α were transfected with 50nM pRNA (SEQ ID NO: 2 / SEQ ID NO: 1, [DR Sorensen et al. Gene silencing by systemic delivery of synthetic siRNA in adult mice Journal of Molecular Biology 327 (2003) pages 761-766] designed to inhibit TNF-α, using oligofectamine (Invitrogen) The concentration of TNF-α in cell culture supernatants was determined using the enzyme-linked immunoabsorption assay (ELISA) (Bender MedSystems) following the manufacturer's instructions.

Protocol B: After one hour of transfection with the plasmid, the cells were treated with duplex pRNA (100 nM) without the use of any transfection agent. After 48 h the amount of TNF-α produced by the cells was analyzed by the enzyme-linked immunoabsorbent assay (ELISA).

Claims (36)

1. A compound of pRNA and a lipid covalently bonded together represented by the following formula (I): where: And it is a duplex of pRNA linked by a phosphate type bond; W is an optional group, and is one, or several, substituents that may be present in each methylene unit and selected, or independently select, from H, C1-C3 alkyl or Z3; Z1, Z2 and Z3 are independently selected from H, C1-C6 alkyl, -NR3R4 and -O-R5; R1 and R2 are independently selected from HyC1-C6-alkyl; R3, R4 and R5 are independently selected from H, a lipid, C1-C6 alkyl; n is an integer that is selected from 1, 2, 3,4 and 5; m is an integer that is selected from 0, 1, 2,3 and 4; with the proviso that at least Z1, Z2 and / or Z3 is -O-lipid.

2.

The pRNA compound according to the preceding claim, characterized in that the lipid chain is a C8-C20 alkyl, fatty acids, acylglyceride, cerido, phospholipids, phosphoglycerides, phosphospholipids, glycolipids, cerebrosides, gangliosides, terpenoids, steroids, eicosanoids or vitamins A, D , EyK.

3.

The pRNA compound according to any of the preceding claims, characterized in that the lipid is selected from C8-C20 alkyl, fatty acids, phospholipids, phosphoglycerides, phospholipolipids and glycolipids.

Four.

The pRNA compound according to any of the preceding claims, characterized in that Z2 is -O-lipid.

5.

The pRNA compound according to any of the preceding claims, characterized in that Z1 is -O-lipid.

6.

The pRNA compound according to any of the preceding claims, characterized in that Z1 and Z2 are independently -O-lipid.

7.

The pRNA compound according to any of the preceding claims, characterized in that n is 1, 2 or 3, preferably 1.

8.

The pRNA compound according to any of the preceding claims, characterized in that m is an integer that is selected from 0.1 to 2, preferably m is 0.

9.

The pRNA compound according to any of the preceding claims, characterized in that R1 and R2 are independently selected from H and C1-C3 alkyl, preferably R1 and R2 are H.

10.

The pRNA compound according to any of the preceding claims, characterized in that the derivatization is introduced at the 3 ′ position of the pRNA duplex.

eleven.

The pRNA compound according to any of the preceding claims, characterized in that the bridge present in the compound of formula (I) between the lipid, or lipids, and Y comes from a molecule of threoninol, glycerol, 2-amino-1,3- Propandiol, 2-amino-1,4-butanediol, 3-amino-1,4-butanediol, 3, -amino-1,2-propanediol, hydroxyprolinol or serinol, preferably comes from glycerol.

12.

The pRNA compound according to any of the preceding claims, characterized in that the siRNA duplex (Y) has between 15 and 40 nucleotides per chain.

13.

The pRNA compound according to any of the preceding claims, characterized in that the pRNA duplex (Y) has between 19 and 25 nucleotides per chain.

14.

The pRNA compound according to any of the preceding claims, characterized in that the pRNA chain comprises at least one substitution selected from 2'-O-methyl-ribonucleotide, 2'-deoxyribonucleotide, 2'-methoxyethyl-ribonucleotide, 2'-fluoro- deoxyribonucleotide, 2'-fl uoro-arabinonucleotide, RNA with phosphorothioate bonds, abbasic residues, ribitoles, 5-methylcytosine, 5-methyluracil, 4-alkynylcytosine, 5-alkynyluracil, 5halogenocytosine, 5-halogenouracil, 7-deazaaanthine, 7-deazaaanthin .

fifteen.

The pRNA compound according to any of the preceding claims, characterized in that said pAR-Ni (Y) inhibits / totally or partially inhibits the expression of at least one of the following genes: tumor necrosis factor alpha (TNFα), factor of Endothelial and vascular growth (VEGF), VEGF receptor (VEGF R1 / 2), granulocyte colony suppressor (GM-CSF-1) and macrophage (M-CSF-1), angiopoietin (ANGPT), apolipoprotein (ApoB) ), vascular neovascularization (CNV), carboxykinase phosphoenol pyruvate (PEPCK), human epidermal growth factor receptor (HER2), macrophage inflammatory protein (MIP2), N-methyl-D aspartate (NMDA) receptor, cytokine derived from keratocytes (KC), delta opium receptor (DOR), discoidin domain receptor (DDR1), PIV phosphoprotein gene (PIV-P), heme oxygenase (HMOX1), caveloin, dopamine transporter, green fluorescent protein and protein of Swing's sarcoma (EWS-FL / 1).

16.

The pRNA compound according to the preceding claim, characterized in that said pRNA (Y) totally / partially inhibits / silences the expression of at least one of the following genes tumor alpha necrosis factor (TNFα), endothelial and vascular growth factor ( VEGF), VEGF receptor (VEGF R1 / 2), granulocyte colony suppressor factor (GM-CSF-1) and macrophage (M-CSF-1).

17.

The pRNA compound according to any of the preceding claims, characterized in that the pRNA sequence is SEQ ID NO: 1SEQ ID NO: 2.

18.

A compound of pRNA and a lipid covalently bonded together represented by the following formula (II):

where: And it is a simple chain of pRNA; W is an optional group, and is one, or several, substituents that may be present in each methylene unit and selected, or independently select, from H, C1-C3 alkyl or Z3; Z1, Z2 and Z3 are independently selected from H, C1-C6 alkyl, -NR3R4 and -O-R5; R1 and R2 are independently selected from HyC1-C6-alkyl; R3, R4 and R5 are independently selected from H, a lipid, C1-C6 alkyl; n is an integer that is selected from 1, 2, 3,4 and 5; m is an integer that is selected from 0, 1, 2,3 and 4; with the proviso that at least Z1, Z2 and / or Z3 is -O-lipid. 19. A process for the synthesis of the pRNA compounds as defined in any one of claims 1 to 17, based on the solid phase synthesis process, characterized in that it comprises at least the following steps: i) joining the lipid and the bridge to form at least one -O-lipid ether bond, ii) bonding the compound obtained in step (i) to a solid support, iii) sequentially assemble one of the pRNA chains and the bridge-O-lipid compound on the support, iv) break the bond between the solid support and the product and eliminate the protective groups obtaining one of the chains of the pRNA and v) mixing equivalent amounts of the guide and companion chains to generate the pRNA duplex.

twenty.

The process as defined in the preceding claim, characterized in that the solid support is selected from glass, silica gel, porous glass, silicon oxide, polystyrene, polyethylene glycol, polystyrene-polyethylene glycol, polyamide, acrylamide, polyvinyl chloride, teflon, paper and cellulose.

twenty-one.

The process as defined in any of the two preceding claims, characterized in that the lipid is attached to a solid support and at least one of the pRNA chains is synthesized using the solid phase synthesis method using the solid support functionalized with the lipid. .

22

The process as defined in any of the three preceding claims, characterized in that the lipid is attached to a solid support and at least one of the pRNA chains is synthesized by successive addition of nucleoside derivatives that constitute the chain sequence including phosphoramidites, H-phosphonates, diester phosphates and monoester phosphates.

2. 3.

The process as defined in any of the four preceding claims, characterized in that the lipid is bound to a solid support and at least one of the pRNA chains is synthesized by successive addition of the phosphoramidites of the nucleosides that constitute the chain sequence. .

24.

A process for the synthesis of the pRNA compounds as defined in any one of claims 1 to 17, based on the solid phase synthesis process, characterized in that it comprises at least the following steps:

i) preparing a lipid derivative containing a phosphorylating group that serves to generate a phosphate group between the lipid and the pRNA, ii) synthesize one of the pRNA chains using the solid phase synthesis procedure. iii) bind the lipid derivative to the pRNA chain via a phosphate bond, iv) break the bond between the solid support and the product and eliminate the protective groups obtaining one of the chains of the pRNA and v) mixing equivalent amounts of the guide and companion chains to generate the pRNA.

25.

A process as defined in any one of claims 19 to 24, characterized in that the solid support is selected from glass, silica gel, porous glass, or any surface of silicon oxide, polystyrene, polyethylene glycol, polystyrene-polyethylene glycol, acrylamide or others. polyamides, polyvinyl chloride, teflon and its derivatives, paper and cellulose.

26.

A process as defined in any one of claims 19 to 25, characterized in that the solid support is selected from glass, silica gel, porous glass, silicon oxide, polystyrene, polyethylene glycol, polystyrene polyethylene glycol, polyamide, acrylamide, polyvinyl chloride, teflon , paper and cellulose.

27.

The process as defined in any of claims 19 to 26, characterized in that at least one of the pRNA chains is synthesized by successive addition of nucleoside derivatives that constitute the chain sequence including phosphoramidites, H-phosphonates, diester phosphates and monoester phosphates

and then the lipid is added using the same derivatives (phosphoramidites, H-phosphonates, diester phosphates and monoester phosphates).

28.

The process as defined in claim 27, characterized in that at least one of the pRNA chains is synthesized by successive addition of phosphoramidite derivatives of the nucleosides and then the lipid is added using also a lipid phosphoramidite.

29.

A pharmaceutical composition comprising any of the pRNA compounds as described in claims 1-18 and minus a pharmaceutically acceptable excipient or carrier.

30

The pharmaceutical composition according to the preceding claim, characterized in that it further comprises a transfection agent.

31.

The pharmaceutical composition according to the preceding claim, characterized in that the transfection agent is selected from the list comprising the following compounds: lipofectin, liptopofectamine, oligofectamine, effectene, cellfectin, DOTAP, DOPE, fugene, polyethylene glycol, cholesterol, polyethyleneimide (PEI), Jet-polyethyleneimide, cell penetration peptides, Trojan peptides, TAT peptide, penetratin, oligoarginine, poly-lysine, rabies virus glycoprotein, gold nanoparticles, dendrimers, carbon nanotubes, cationic lipids, liposomes and any combination thereof.

32

The pharmaceutical composition according to any of claims 29 to 31, wherein it is presented in a form adapted to oral or parenteral administration, preferably parenteral.

33.

The use of the pRNA compounds according to any one of claims 1 to 18 or of the compositions according to any of the four preceding claims for the preparation of a medicament.

34. The use according to the preceding claim for the preparation of a medicament for gene silencing. 35. The use according to any of the two preceding claims for the preparation of a medicament for the treatment of any of the following conditions: cancer, inflammation, ulcerative colitis, Crohn's disease or rheumatoid arthritis. SEQUENCE LIST <110> Higher Council for Scientific Research University of Barcelona CIBER-BBN <120> Lipophilic derivatives of nucleic acids <130> ES1641.437 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Fragment of the guiding or antisense sequence of the MusFusculus TNF alpha RNA to which two thymine have been added at the 3 'end. <400> 1 <210> 2 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Fragment of the accompanying sequence or sense of the TNF alpha RNA of Mus musculus to which two thymine have been added at the 3 ’end. <400> 2 <210> 3 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Fragment of a guide or antisense sequence of RNA from Euglena gracilis to which two thymine have been added at the 3 ’end. <400> 3 <210> 4 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Fragment of an accompanying sequence or sense of RNA from Euglena gracilis to which two thymine have been added at the 3 ’end. <400> 4 <210> 5 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Fragment of the accompanying sequence or sense of the Mus musculus TNF alpha RNA to which two thymine have been added at the 3 'end and some nucleotides have been methylated. <220> <221> misc_RNA <222> (5) .. (5) <223> The nucleotide defined as "n" corresponds to a nucleotide "cm" in accordance with the list of modified nucleotides in Table 2 of Appendix 2 of Standard ST.25 <220> <221> misc_feature <222> (5) .. (5) <223> nesa, c, g, tou <220> <221> misc_RNA <222> (10) .. (10) <223> The nucleotide defined as "n" corresponds to a nucleotide "um" in accordance with the modified nucleotide list in Table 2 of Appendix 2 of Standard ST.25 <220> <221> misc_feature <222> (10) .. (11) <223> nesa, c, g, tou <220> <221> misc_RNA <222> (11) .. (11) <223> The nucleotide defined as "n" corresponds to a nucleotide "cm" in accordance with the list of modified nucleotides in Table 2 of Appendix 2 of Standard ST.25 <220> <221> misc_RNA <222> (13) .. (13) <223> The nucleotide defined as "n" corresponds to a nucleotide "cm" in accordance with the list of modified nucleotides in Table 2 of Appendix 2 of Standard ST.25 <220> <221> misc_feature <222> (13) .. (13) <223> nesa, c, g, tou <220> <221> misc_RNA <222> (16) .. (16) <223> The nucleotide defined as "n" corresponds to a nucleotide "cm" in accordance with the list of modified nucleotides in Table 2 of Appendix 2 of Standard ST.25 <220> <221> misc_feature <222> (16) .. (16) <223> nesa, c, g, tou <220> <221> misc_RNA <222> (18) .. (18) <223> The nucleotide defined as "n" corresponds to a nucleotide "cm" in accordance with the list of modified nucleotides in Table 2 of Appendix 2 of Standard ST.25 <220> <221> misc_feature <222> (18) .. (18) <223> nesa, c, g, tou <400> 5 SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 201030611 SPAIN Date of submission of the application: 04.27.2010 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: See Additional Sheet RELEVANT DOCUMENTS

Category

Documents cited Claims Affected

TO

YOSHIHITO UENO et al. “Synthesis and silencing properties of siRNAs possessing lipophilic groups at their 3’-termini” BIOORGANIC & MEDICINAL CHEMISTRY, vol 16, 2008, pages 7698-7704. Page 7698, left column; page 7699, right column, section 2 and scheme 1; and table 1. 1-35

TO

CHRISTIAN WOLFRUM et al. "Mechanisms and optimization of in vivo delivery of lipophilic siRNAs" NATURE BIOTECHNOLOGY, vol 25, no. 10, 2007, pages 1149-1157. Summary; page 1149, left column, second paragraph; page 1150, left column; and figure 1. 1-35

TO

US 20070207966 A1 (KIM et al.) 06.09.2007, paragraphs [0009], [0010]. 1-35

TO

JÜRGEN SOUTSCHEK et al. "Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs" NATURE, vol 432, 2004, pages 173-178. Summary; page 177, left column, second and third paragraphs. 1-35

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 24.10.2011

Examiner S. González Peñalba Page 1/5

REPORT OF THE STATE OF THE TECHNIQUE Application number: 201030611 CLASSIFICATION OBJECT OF THE APPLICATION A61K48 / 00 (2006.01) A61K47 / 48 (2006.01) C12N15 / 87 (2006.01) Minimum documentation sought (classification system followed by classification symbols) A61K, C12N Electronic databases consulted during the search (name of the database and, if possible, terms of search used) INVENES, EPODOC, WPI, BIOSIS, EMBASE, NPL, MEDLINE, XPESP, EBI State of the Art Report Page 2/5  WRITTEN OPINION Application number: 201030611 Date of Written Opinion: 24.10.2011 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims Claims 1-35 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims Claims 1-35 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/5  WRITTEN OPINION Application number: 201030611 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

YOSHIHITO UENO et al. “Synthesis and silencing properties of siRNAs possessing lipophilic groups at their 3’-termini” BIOORGANIC & MEDICINAL CHEMISTRY, vol 16, 2008, pages 7698-7704. Page 7698, left column; page 7699, right column, section 2 and scheme 1; and table 1.

D02

CHRISTIAN WOLFRUM et al. "Mechanisms and optimization of in vivo delivery of lipophilic siRNAs" NATUREBIOTECHNOLOGY, vol 25, no. 10, 2007, pages 1149-1157. Summary; page 1149, left column, second paragraph; page 1150, left column; and figure 1.

D03

US 20070207966 A1 (KIM et al.) 06.09.2007

D04

JÜRGEN SOUTSCHEK et al. "Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs" NATURE, vol 432, 2004, pages 173-178. Summary; page 177, left column, second and third paragraphs.

2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The present patent application as drafted refers to a compound of pRNA and a lipid covalently linked to each other by phosphate bond (ether type) (claims 1, 4-9, 14). The type of lipid chain that binds is also claimed (claims 2,3), as well as that derivatization is introduced at the 3 ′ position of the pRNA duplex (claim 10). The binding bridge present between the lipid and the pRNA molecule comes from, among other compounds, glycerol (claim 11); the pRNA sequence is also claimed (claims 12, 13 and 17). Said pRNA inhibits or partially or totally silences the expression of at least one gene (claims 15, 16). The pRNA chain that binds to the lipid compound can be a single chain (claim 18). And finally, the synthesis procedure of the pRNA compounds covalently bound to lipids (claims 19-28), the pharmaceutical composition containing said compounds (claims 29-32) and the use thereof (claims 3335) are claimed. NEW AND INVENTIVE ACTIVITY. LP ARTS 6 AND 8. Document D01 refers to siRNA (short interfering RNAs) conjugated to lipophilic groups at the 3 'end, the lipophilic group that is bound to the siRNA is, among others, cholesterol (see page 7698, column left). These modified siRNAs can more easily cross the plasma membranes of cells in vivo and be used for gene specific silencing (see page 7699, right column). In addition, the bridge present in the modified siRNAs between the siRNA and the lipid comes from a glycerol molecule (see page 7699, section 2). In the present patent application it is specified that the lipid groups are linked by ether bonds and in said document D01 the link used is carbamate type (see page 7699, scheme 1), and on the other hand, the specific siRNA sequences are other than those of said document D01 (see page 7700, table 1). Document D02 refers to that conjugates of siRNAs with lipids can silence the expression of certain genes in vivo. Conjugated siRNAs were synthesized with bile acids, long chain fatty acids, cholesterol, among others and short hip. The absorption of these conjugated siRNAs depends on the interactions with lipoprotein particles (see summary). The effect of these siRNAs on the expression of apolipoprotein B (apo B) was studied and it was observed that conjugates of short-chain fatty acids, such as lauroyl (C12), miristoyl (C14) and palmitoyl (C16) did not reduce Apo B transcription levels (see page 1150, left column), while long chain, including cholesterol, did. On the other hand, the binding of these lipid compounds to the SiRNAs is of the covalent type (see page 1149, left column, second paragraph), but they are not of the ether type, nor is the sequence of siRNA the same as in the present application. patent (see page 1150, figure 1). Document D03 describes a method for the introduction of small interfering RNAs into the cell. The method comprises contacting the cell with a complex formed by a mixture of a small interfering RNA and a conjugate comprising a cholesterol residue covalently linked to an oligoarginine. The interfering RNA is directed to the endothelial and vascular growth factor (see paragraphs [0009] and [0010]). State of the Art Report Page 4/5  WRITTEN OPINION Application number: 201030611 Document D04 deals with conjugates of siRNAs and cholesterol for the silencing of genes involved in certain diseases (see summary). In this study it has been shown how the conjugated compounds of siRNAs with cholesterol and bound to apoB have biological activity, while the conjugated compounds of apoB-siRNAs do not show such activity, which demonstrates the importance of the cholesterol molecule in these conjugates of siRNAs (see page 177, left column, second paragraph). The siRNAs used in said document are different from those of the present invention (see page 177, left column, second paragraph). In none of the documents cited above does a compound of pRNA and a covalently bound lipid of formula (I) appear. Nor is there any evidence in said documents, alone or in combination, that directs the person skilled in the art towards the invention defined in claims 1-35. Therefore, claims 1-35 of the present patent application are considered to meet the requirements of novelty and inventive activity according to articles 6 and 8 of the LP. State of the Art Report Page 5/5