A new class of lipids for delivering active ingredients to cells

Abstract

The present invention relates to lipids of formula (I), compositions comprising lipids of formula (I), methods for producing lipids of formula (I) and / or compositions, lipids of formula (I) and / or compositions for use as pharmaceuticals, the use of lipids of formula (I) and / or compositions as vectors for delivering active ingredients to targets, organs, cells and / or tissues, methods for transfecting cells with nucleic acids and / or proteins and / or peptides and / or polysaccharides and / or lipids and / or small organic or inorganic molecules and / or any kind of bioactive molecules using lipids of formula (I) and / or compositions, transfected cells obtained by this method, and kits comprising lipids of formula (I) and / or compositions.

Description

[Technical Field]

[0001] The present invention relates to lipids of formula (I), compositions comprising lipids of formula (I), methods for producing lipids of formula (I) and / or compositions, lipids of formula (I) and / or compositions for use as pharmaceuticals, the use of lipids of formula (I) and / or compositions as vectors for delivering active ingredients to targets, organs, cells and / or tissues, methods for transfecting cells with nucleic acids and / or proteins and / or peptides and / or polysaccharides and / or lipids and / or small organic or inorganic molecules and / or any kind of bioactive molecules using lipids of formula (I) and / or compositions, transfected cells obtained by this method, and kits comprising lipids of formula (I) and / or compositions. [Background technology]

[0002] Gene therapy, including nucleic acid-mediated therapy, is today one of the most promising opportunities for curing diseases that are ineffective by conventional means. These innovative protocols involve the safe intracellular delivery of bioactive components using a wide variety of delivery vehicles. Intracellular delivery of bioactive drugs to cells, organs, and / or organisms has applications in a wide range of fields, from biology to medicine. In biology, the transfection of nucleic acids such as DNA or RNA can be used, in particular, to study the regulation of gene expression, the clarification of their function, silencing or editing, and their overexpression. Similarly, intracellular transport of peptides, proteins, polysaccharides, lipids, organic and inorganic small molecules, and any other bioactive molecules, allows for the study of fundamental biological mechanisms and the development of therapeutic drugs. Nucleic acid therapeutics are susceptible to degradation by nucleases and cannot penetrate cells due to their size and charge. This is also true for various proteins, peptides, or small molecules. Therefore, the development of cell-targeted drug delivery systems using clinically translatable systems is crucial to providing opportunities to address a range of life-threatening diseases.

[0003] Among the numerous techniques used to introduce nucleic acids into cells, lipids, particularly cationic lipids (positively charged at physiological pH) in organized or unorganized system forms (e.g., liposomes, monolayer or multilayer vesicles, hexagonal and / or micelles), and / or mixtures of lipids, are perhaps the most widely used. They form complexes with nucleic acids, such as lipoplexes or lipopolyplexes or lipid nanoparticles (LNPs), enabling the delivery of nucleic acids, proteins, and peptides across the cell membrane. Positively charged lipid formulations form complexes with negatively charged nucleic acids. The resulting positively charged complex can then bind to the negatively charged cell membrane. The exact mechanism by which nucleic acid / chemical complexes cross the cell membrane is unknown, but endocytosis is thought to be involved in the process of cellular uptake. The efficiency of chemical methods is highly dependent on factors such as pH, nucleic acid / reagent ratio, salt content, or temperature. Consequently, predicting how chemical reagents will behave in transfection, especially in in vivo applications, is clearly difficult.

[0004] Among all the major known cationic lipid structures, the following can be cited as examples in a non-exclusive manner: monovalent cationic lipids in the form of quaternary ammonium salts such as DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP (1,2-dioleoyl-3-trimethylaluminium propane), or DDAB (dioctadecyldimethylammonium bromide), and pyridinium such as SAINT-2 (N-methyl-4-(dioleyl)methylpyridinium chloride). Monovalent cationic lipids in the form of monoxide salts, polyvalent cationic lipids in the form of lipospermine such as DOGS (5-carboxyspermylglycine-dioctadecylamine) and DOSPA (2,3-dioleyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-propaneammonium trifluoroacetate), polyvalent cationic lipids in the form of lipopolylysine, and cationic cholesterol derivatives such as DC-Chol (3-[N-(N'-N',-dimethylaminomethane)-carbamoyl]cholesterol).

[0005] The use of cationic lipids (formulated with neutral or helper lipids, or unformulated) for transfection offers numerous advantages, as they are non-immunogenic, unlike viral drugs, easy to use, allow for the delivery of nucleic acids without size limitations, possess high affinity for any type of nucleic acid, and can be produced in large quantities. Furthermore, lipofection mediated by formulated cationic lipids (delivery of nucleic acids by lipid-based formulations) is the most widely used laboratory method for transfecting cells in vitro due to its ease of use, its efficacy against a wide variety of cell types, particularly adherent cells, in or without serum, and its versatility for nucleic acid delivery. These advantages justify numerous research programs dealing with the development of cationic lipids as transfection vectors both in vitro and in vivo. However, these synthetic vectors are little to no effective for transfection of certain cell types, particularly cells in suspension, such as lymphocytes, stem cells, and primary cells (non-dividing). The reduced effectiveness of cationic lipids when transfecting specific cells can be explained by the lipoplex's poor ability to adhere to the surface of these cells.

[0006] Thus, various approaches using specific ligands such as transferrin, epidermal growth factor (EGP), monoclonal antibodies, or peptides, coupled or bound to copolymers formulated with cationic polymers or cationic lipids, have been successfully tested. However, quiescent cells are also very difficult to transfect using lipofection because the complex present in the cytosol is prevented from passing through the pores of the nuclear membrane and therefore transcribed due to the absence of means to target the nucleus, such as nuclear localization sequences. To address this problem, several approaches using ternary complexes (lipoplexes: cationic lipids and nucleic acids + nuclear targeting elements) based on histone and peptide nuclear localization sequences have been studied. However, the addition of these targeting elements to cationic lipids makes the formulation and preparation of the complexes difficult, and therefore their use is very limited. Furthermore, most importantly, the in vivo efficacy of lipofection remains too low, limited by the enzymatic degradation of the complexes, their pharmacology, and the presence of transfection inhibitors in body fluids and mucus, such as proteins and polysaccharides that strongly inhibit transfection. The main parameters influencing the efficacy of lipoplex transfection have been extensively studied, suggesting that the best levels of in vivo gene expression are achieved using high lipid / nucleic acid ratios that result in significant toxicity. Therefore, the in vivo cytotoxicity of cationic lipids remains a major drawback of this strategy, mainly due to the low biodegradability of cationic lipids, which are not naturally present in cells, as well as the use of permanently positively charged lipids, which are known to be detrimental to cell survival.

[0007] Various efforts have been made to address these problems, such as the synthesis of lipids with reducing medium-sensitive binding in the lipid chain to promote their metabolism, or the incorporation of vinyl ether groups into the spacer arms of cationic lipids. Since vinyl groups are sensitive to acidic pH, they allow for rapid cleavage of lipids in the acidic medium of endosomes, leading to destabilization of the lipoplex structure embedded in the endosome membrane and thus enabling the early release of DNA into the cytosol. Similarly, families of cationic lipids with spacer arms containing linear or cyclic orthoester groups that are hydrolyzable at acidic pH and stable at physiological pH have been described, although there are numerous examples of cationic lipids containing ester groups, such as DMT-M(Gly) and DOTM(Gly)-tetraesters.

[0008] Similarly, to address the issues of biodegradability and lack of targeting, and to add further biocompatible features, amino acid-based lipids with cleavable linkers have been designed and developed (International Publication 2009 / 106713 A3). These molecules have shown significant progress in transfection efficiency, safety, and the expanded range of transfection applications to various biomolecules due to their innovative design. However, the presence of a permanent positive charge(s) at physiological pH minimizes their in vivo efficiency, despite better biodegradability associated with their structure in intracellular media.

[0009] Recent reports have demonstrated that the combination of a permanently positive charge and a non-degradable backbone is the two main obstacles to safe and efficient in vivo gene delivery. Therefore, a new generation of lipids has been developed to reduce the toxicity of permanently charged cationic lipids without compromising transfection efficacy. Based on the same structural model (polar nitrogen-based head, linker region, and two or more non-polar hydrocarbon tails), they exhibit an optimal pKa value of 6.2–7.0, which is designed to be positively charged at acidic pH, enabling efficient encapsulation of nucleic acids such as DNA or RNA into lipid nanoparticles (LNPs) and effective cytoplasmic release of DNA or RNA upon protonation (endosomal pathway) under acidic conditions. On the other hand, they are neutral under physiological conditions (pH=7.0–7.4), resulting in nonspecific interactions with bodily fluid components and tissues, higher biocompatibility, and significantly lower toxicity compared to permanently charged cationic lipids. Lipids with appropriate pKa values ​​alter their electrostatic charge to ensure proper vector formation, optimal in vivo circulation, and efficient cytosolic release of therapeutic cargo. Specifically, such ionizable lipids should be designed to be positively charged at acidic pH during the production of lipid nanoparticles (LNPs) so that electrostatic interactions with nucleic acids (NA) can be maximized (high NA condensation = high loading efficiency). The lipids should then be converted to near-neutral under physiological conditions to prevent rapid capture by immune cells during systemic circulation. Furthermore, ionizable lipids should become positive again upon exposure to the typical acidic environment of endosomes, thus destabilizing the endosomal membrane and promoting cytosolic delivery of gene cargo. Finally, in the cytosol, a neutral pH is favorable for the release of NA from ionizable lipids, thus allowing their free interaction with cellular mechanisms. Therefore, ionizable lipids are important components of nanoparticle formulations, as they play a role in protecting nucleic acids from degradation and releasing NA into cells.

[0010] Ionizable lipids, when encapsulating biomolecules such as nucleic acids, are associated with specific lipid components to supramolecular structures known as lipid nanoparticles (LNPs), stable nucleic acid lipid particles (SNALPs), lipid polycation-DNA (LPDs), solid lipid nanoparticles (SLNs), or nanostructured lipid carriers (NLCs). These components provide LNPs with further features that enhance the positive effects of being supported by ionizable components such as neutral phospholipids or sterol derivatives. Furthermore, when addressing in vivo injection, polyethylene glycol (PEG)-modified lipids (PEGylated) are included in LNP formulations to increase the circulating stability of LNP formulations, reduce nonspecific adverse interactions, decrease aggregation, and thus improve nucleic acid delivery efficiency. The diverse properties of lipids and their ability to be readily regulated and modified make lipids ideal candidates for the transport of long, fragile nucleic acids such as DNA, mRNA, or saRNA. In recent years, the number of research studies and experiments toward the design and synthesis of such LNPs, and in particular the design and synthesis of ionizable lipids, has increased exponentially. Several recent examples can be found in comprehensive reviews (An ionizable lipid toolbox for RNA delivery, Han et al., Nature Communications 12, 7233 (2021); Lipid nanoparticles for mRNA delivery, Hou et al., Nature reviews materials 6, pages 1078-1094 (2021)). The molecules described in these different studies highlighted the fact that ionizable lipids used in LNPs can have different structures that can positively impact nucleic acid delivery, particularly in vivo. Therefore, several ionizable lipid structures have been described in recent years: - Unsaturated ionizable lipids (e.g., DLin-MC3-DMA, A6, OF-02, A18-Iso5-2DC18), - Multi-tailed ionizable lipids (e.g., SM102, ALC-0315, A9, lipid 2,2(8,8); 98N12-5, C12-200, cKK-E12 and 9A1P9), - Ionizable polymer lipids (e.g., 7C1 and G0-C14), - Acid-sensitive ionizable lipids (e.g., L319, 304O3, OF-Deg-Lin, and 306-O12B) - Ionizable lipids in the branched tail (e.g., 306Oi10 and FTT5).

[0011] To date, formulations using lipid nanoparticles (LNPs) are the most advanced delivery platform for gene therapy and vaccines, and also promising candidates for treating a variety of diseases. LNPs were recently approved by the FDA for the treatment of amyloidosis by siRNA delivery, and more recently, for a widely distributed mRNA-LNP-based SARS-CoV-2 vaccine. Interestingly, ionizable lipids efficient for siRNA (such as DLin-MC3-DMA) did not share the same structural pattern as those used for efficient mRNA delivery (such as SM102 and ALC-0315). This demonstrates the need to develop a reliable platform for the synthesis of a broad range of ionizable lipids that can provide components perfectly tailored to the requirements of each possible application. A single, general-purpose LNP-based synthesis platform would be more convenient, cost-effective, and in some way the safest for nucleic acid therapeutics. Furthermore, despite the current leaps forward in LNPs, there are still some concerns about some inherent toxicity of these nanocarriers, particularly the in vivo distribution and cellular uptake of LNPs, which are affected by their surface composition as well as colloidal stability. As a result, there is a need for a new generation of tunable lipids that possess higher biodegradability and biocompatibility, a better ability to form small and polydisperse nanoparticles, a diverse and / or broader pKa range for targeting different tissues, diseases, or harsh biological environments, and higher compatibility with multiple nucleic acids or other types of molecules, such as proteins, antibodies, and peptides.

[0012] The inventors have, surprisingly, discovered a new class of lipids, in particular a new class of tunable lipids, that can be used to deliver active drugs to cells without having the aforementioned drawbacks.

[0013] In particular, the inventors have discovered a novel, versatile family of lipids, preferably a tunable family of lipids, that is compatible with the presence of serum, non-toxic, and enables effective in vitro, ex vivo, and in vivo delivery of all types of biomolecules, such as nucleic acids, peptides, proteins, polysaccharides, and lipids, to living cells.

[0014] In particular, the inventors have surprisingly discovered a novel class of lipids that can contain a wide variety of amine-based terminal groups that enable reversible fine-tuning of positive charge, thereby conferring a net pKa value to this new class of lipids that matches the formulations of LNPs related to the efficient delivery of biomolecules to cells. The innovative structures of these new lipids, preferably new tunable lipids, combine the following: - The amphiphilic properties of lipids for forming organized structures (such as liposomes, lipid nanoparticles, and micelles) that enable the transport and vectorization of active molecules toward their targets, their properties for forming non-covalent complexes with negatively charged nucleic acids, and their properties for destabilizing cell membranes.

[0015] - Advantageously, the lipid head group, preferably a tunable lipid head group, is composed of a chemical moiety centered on a nitrogen atom, and is itself part of a chemical group selected to be protonated only at an acidic pH, while otherwise remaining neutral. Furthermore, the arrangement of these groups, preferably the arrangement of these tunable groups, interacts with several types of biomolecules, including nucleic acids, peptides, and proteins, and is completely nontoxic.

[0016] - The ability to rapidly degrade in the intracellular environment due to the presence of a spacer arm between the lipophilic moiety and the head, preferably a tunable head, which incorporates functional groups with bonds sensitive to its environment (e.g., pH, oxidation-reduction, and / or enzymes) and / or ester bonds in the hydrophobic chain, which can be cleaved in the cytosolic medium. The compounds resulting from the degradation are naturally occurring molecules (fatty acids, amino acid products) that are readily metabolized by cells. The biodegradability of these molecules makes them non-cytotoxic as a result.

[0017] Accordingly, the present invention provides an innovative solution for generating delivery systems such as lipid nanoparticles or liposomes or lipid-based particles using a completely new set of lipid molecules that, through carefully selected molecular design, can efficiently complex, protect, and deliver a wide range of diverse active ingredients. The present invention carefully describes the association of lipophilic regions that mix several hydrophobic moieties, e.g., alkyl chains, unsaturated groups, and optionally bioreducing groups that provide increased biocompatibility and / or biodegradability to these molecules, in appropriate proportions. This strategic selection is completed by incorporating a linker pattern that plays a dual role in maintaining the correct distance between the hydrophobic and hydrophilic moieties and contributing to the biodegradability of the overall structure. Finally, the polar head consists of a chemical moiety centered on a nitrogen atom, which itself is part of a chemical group selected to be protonated only at acidic pH (i.e., pH = 3.5–6.9) but remain uncharged at higher pH.

[0018] Special attention has been paid to incorporating biodegradable or bioreducible moieties into the lipid chemical structure. In fact, such moieties provide molecules with faster metabolism and removal from the body after the active pharmaceutical ingredient has been delivered to the target region.

[0019] Furthermore, all compounds claimed in this invention are components of pharmaceutical formulations, which means that their efficacy may depend on the selection of other components of the formulation, their assembly (self-assembly) using careful experimental procedures, such as microfluidic processing, high-pressure flow or extrusion systems or any other controlled formulation procedure. [Prior art documents] [Patent Documents]

[0020] [Patent Document 1] International Publication No. 2009 / 106713 A3 [Non-patent literature]

[0021] [Non-Patent Document 1] An ionizable lipid toolbox for RNA delivery,Han et al.,Nature Communications 12,7233(2021) [Non-Patent Document 2] Lipid nanoparticles for mRNA delivery,Hou et al.,Nature reviews materials 6,pages 1078-1094(2021) [Overview of the project]

[0022] This invention relates to a lipid of formula (I), its stereoisomer, or a pharmaceutically acceptable salt thereof: [ka] During the ceremony, -R comprises one or more branched or linear, unsaturated or saturated, optionally fluorinated alkyl chains containing 6 to 48 carbon atoms, preferably 10 to 36 carbon atoms, and possibly containing one or more heteroatoms other than fluorine; or one or more cyclic or polycyclic groups known to be lipophilic and possibly containing one or more heteroatoms, such as steroid groups, polycyclic aromatic groups, or alkaloid derivative groups; or natural or synthetic lipids, which may contain one or more heteroatoms; or a combination thereof; -Sp comprises one or more branched or linear, unsaturated or saturated, possibly fluorinated alkyl chains, comprising 6 to 24 carbon atoms, preferably 10 to 18 carbon atoms, and may contain one or more heteroatoms and / or bioreducible bonds; and -Z s This includes one or more branched or linear, unsaturated or saturated, possibly fluorinated alkyl chains containing 2 to 36 carbon atoms, preferably 2 to 24 carbon atoms, and which may contain one or more heteroatoms other than fluorine, and / or one or more bioreducing bonds covalently bonded to linear or cyclic nitrogen-based chemical moieties; It features the following: Lipids do not contain any positive charge at pH levels between 7.0 and 7.4, but they exhibit one or more positive charges when the pH is below 7.0, preferably between 2.0 and 7.0, more preferably between 4.0 and 7.0, and even more preferably between 4.5 and 7.0; R corresponds to equation (II): [ka] During the ceremony, - Identical or different N1 and N2 represent a linear, branched saturated or unsaturated hydrocarbon group containing 1 to 8 carbon atoms, preferably 1 to 4 carbon atoms, used to terminate the carbon chain, and preferably, identical or different N1 and N2 are any of the methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, or t-butyl groups; - Identical or different R1 and R2 represent linear, branched, and / or cyclic saturated or unsaturated hydrocarbon groups comprising 6 to 24 carbon atoms, preferably 10 to 18 carbon atoms, and may contain one or more heteroatoms; -Identical A and B represent OC(O), C(O)NH-, -NHCO-, OC(O)-O, NH-C(O)-NH, NH-C(O)-O, OC(O)-NH, -SC(O)-S-, -OC(S)-S-, -SC(O)-O, -NH- or -S- group; -a is an integer between 1 and 4, preferably an integer equal to 1, 2, or 3; -b is an integer between 0 and 6, preferably an integer equal to 0 or 1; -E1 and E2, whether identical or different, represent -C(O)O-, -C(O)-NH-, -NH-, -O-, or -S- groups; -c is an integer between 0 and 2; -d is an integer equal to 0 or 1; -d1 is an integer between 0 and 6, preferably an integer equal to 0 or 1; -d2 is an integer between 0 and 6, preferably an integer between 0 and 2; and -e is an integer between 0 and 6, preferably between 0 and 2, more preferably between 0 and 1; • Sp corresponds to general formula (III): [ka] During the ceremony, -Gr represents a linear or branched hydrocarbon group comprising 1 to 15 carbon atoms, preferably 1 to 8 carbon atoms, and may contain one or more heteroatoms; -m is an integer equal to 0 or 1; -RAc represents an amino acid radical; and -n is an integer equal to 0 or 1; and Zs corresponds to equation (IV): [ka] During the ceremony, - Identical or different S1 and S2 represent a linear or branched hydrocarbon group comprising 1 to 15 carbon atoms, preferably 1 to 6 carbon atoms, preferably selected from nitrogen, oxygen, sulfur, bromine, iodine, chlorine, and fluorine, and more preferably one or more heteroatoms selected from nitrogen, sulfur, bromine, iodine, chlorine, and fluorine; -D represents one or more vinyl ether groups, one or more acylhydrazone groups, one or more carbonate bonds, disulfide bonds, ester bonds or carbonate bonds, or one or more photosensitive groups; -p is an integer equal to 0 or 1; -Q is a branched hydrocarbon group comprising 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and one or more heteroatoms, which may be covalently bonded on one side to an S2, RAc, Gr, or R group and on the other side to at least two Q and / or Z groups; -q is an integer between 0 and 8, preferably between 0 and 3; -Z is a nitrogen (N)-centered terminal ionizable moiety, and preferably Z represents a tertiary aliphatic amine such as an N,N-dialkylamine, or a cyclic amine moiety such as an N-substituted pyrrolidine, N-substituted piperidine, N-substituted morpholine, N-substituted piperazine, or N-substituted pyridine; where Z contains no positive charge in the pH range of 7.0 to 7.4, but exhibits one or more positive charges in the pH range of 2.0 to 7.0, preferably 4.0 to 7.0, and more preferably 4.5 to 7.0.

[0023] -r is an integer between 1 and 16, preferably between 1 and 8, preferably, if q is equal to 1, r is at least equal to 2, and if r is greater than 1, the Z groups may be the same or different; and -s is an integer equal to 1 or 2, The following compounds are excluded from general formula (I): [ka] and [ka] Furthermore In equation (III): • Gr is either absent, or Gr acts as a spacer arm, corresponding to a molecular pattern represented as -W4-Y4-W4-; Here, Y4 represents a bridged alkylene group containing 1 to 8 carbon atoms, preferably 1 to 4 carbon atoms, and W4 represents a C(O)-O, C(O)-NH, C(O)-S, S(O), S(O)-O, C(S)-O, -O-, or -NH- group. base; or ·Gr corresponds to formula -W4-Y4-W4-, Here, Y4 has the same meaning as previously defined, being linked on the one hand to the lipophilic region R, on the other hand to the RAc radical, or directly to the S1, Q, or Z group by a carbonate or carbamate bond; and In formula (III), the amino acid radical RAc is selected from aspartic acid, glutamic acid, isoleucine, leucine, lysine, ornithine, glycine, and phenylalanine, and more preferably selected from aspartic acid, glutamic acid, glycine, ornithine, and lysine.

[0024] The present invention also relates to a composition comprising a lipid of the previously defined formula (I).

[0025] The present invention also relates to a method for producing lipids of formula (I) and / or compositions as previously defined.

[0026] The present invention also relates to lipids of the previously defined formula (I) and / or previously defined compositions for use as pharmaceuticals.

[0027] The present invention also relates to the use of lipids of the previously defined formula (I) and / or compositions of the previously defined formula as vectors for delivering active ingredients to subjects, organs, cells, and / or tissues.

[0028] The present invention also relates to a method for transfecting cells with nucleic acids and / or proteins and / or peptides and / or polysaccharides and / or lipids and / or small organic or inorganic molecules and / or any kind of bioactive molecule using lipids of formula (I) and / or compositions of the formula defined above.

[0029] The present invention also relates to transfected cells obtained by the previously defined method.

[0030] The present invention also relates to a kit comprising a lipid of formula (I) as previously defined and / or a previously defined composition, further comprising nucleic acids and / or proteins and / or peptides and / or polysaccharides and / or lipids and / or small organic or inorganic molecules and / or any kind of bioactive molecule, and optionally comprising instructions for use.

[0031] definition "Lipid" is preferably understood as an organic amphiphilic molecule having nonpolar hydrophobic hydrocarbon chain regions and polar hydrophilic regions in its structure, linked together by chemical spacers. "Tunable lipid" is preferably understood as a previously defined lipid having hydrophilic regions that may contain one or more positive charges depending on the pH of its direct environment, allowing the user to adjust its chemical properties by selecting the correct pH. Preferably, it does not exhibit a permanent positive charge at physiological pH (7.0-7.4) but becomes cationic as the pH of its physiological environment decreases. Preferably, due to its amphiphilicity, it is generally insoluble in water and somewhat soluble in organic solvents. Preferably, the term "tunable lipid" also means that such a lipid, when properly treated, can form self-assembled structures in an aqueous environment.

[0032] "Biodegradable lipids" are preferably organic amphiphilic molecules whose structure includes nonpolar hydrophobic hydrocarbon chain regions and polar hydrophilic regions linked to each other by chemical spacers, and which are capable of degradation of their chemical and physical properties and can be completely degraded under biological conditions when exposed to microorganisms, aerobic and anaerobic processes. Furthermore, the compounds or chemical entities resulting from the degradation of these molecules are biocompatible.

[0033] "Biodegradable bond" or "biodegradable atomic group" is preferably understood to mean that the designed chemical bond or atomic group can be degraded, i.e., destroyed, in a biological environment under biological conditions when exposed to microorganisms, aerobic and anaerobic processes. Furthermore, the compounds or chemical entities resulting from the degradation of this bond or atomic group are preferably biocompatible.

[0034] "Biocompatibility" is preferably understood to mean that a molecule, atomic group, or chemical entity can come into contact with a living system without causing any adverse effects.

[0035] "Hydroplosable bond" or "Hydroplosable atomic group" is preferably understood to mean that the designed chemical bond or atomic group can be broken down, or destroyed, by the action of water molecules in a biological environment. This action can be induced or accelerated by specific pH conditions.

[0036] "Bioreducible bond" or "bioreducible atomic group" is preferably understood to mean that the designed chemical bond or atomic group can be reduced, i.e., destroyed, in a biological environment according to an oxide reduction pathway.

[0037] Examples of bioreducible bonds or bioreducible atomic groups include, but are not limited to, disulfide bonds, ester bonds, vinyl ether bonds, and / or acyl-hydrazone bonds.

[0038] "Photosensitive bond" or "photosensitive atomic group" is preferably understood to mean that a designed chemical bond or atomic group can be broken, or destroyed, by irradiation with light of a specific wavelength.

[0039] Examples of photosensitive bonds or photosensitive atomic groups include, but are not limited to, diazo groups, nitrobenzyl groups, and / or phenacyl groups.

[0040] In the present invention, a “lipophilic atomic group” is preferably understood to mean that the atomic group includes a nonpolar hydrocarbon chain region that reduces the atomic group’s ability to dissolve in an aqueous environment. It is widely understood that the term lipophilic can be replaced with the term hydrophobic without changing its meaning. Therefore, in the present invention, the terms “lipophilic” and “hydrophobic” may be used interchangeably.

[0041] In this invention, "hydrophilic group" is understood to mean a group that includes a polar region, typically centered on heteroatoms such as oxygen, nitrogen, sulfur, and / or phosphorus, which enhances the group's ability to dissolve in water. It is widely understood that hydrophilicity can be replaced by the term oleophobicity without changing its meaning. Therefore, in this invention, the terms "hydrophilic" and "oleophobicity" may be used interchangeably.

[0042] The term "terminal ionizable portion" is preferably understood to mean that Z is a chemical group that has the ability to attract or release protons in response to changes in the pH of the medium. In the present invention, ionizable is preferably understood to mean that the overall pKa of Z gives this group the potential to attract one or more protons available in the medium to acquire one or more positive charges when the pH is low. As a result, it is considered that Z can attract one or more protons when the pH of the medium is below the physiological pH value, i.e., pH < 7.4. Preferably, it is considered that Z can attract one or more protons available in the medium to acquire one or more positive charges when the pH is in the range of 2 to 7.4, preferably 4 to 7.4, and more preferably 4.5 to 7.

[0043] Examples of terminally ionizable groups include, but are not limited to, N,N-dialkylaliphatic tertiary amines that incorporate or do not incorporate heteroatoms, such as oxygen, sulfur, nitrogen and / or phosphorus, halogens, or combinations thereof.

[0044] Preferably, in the present invention, the terminal ionizable portion Z has no charge at physiological pH due to its pKa value. Preferably, although the terminal ionizable portion Z has no charge at physiological pH, when the pH of the medium is below the value of physiological pH, i.e., pH < 7.4, Z can attract one or more protons to acquire one or more positive charges. Preferably, although the terminal ionizable portion Z has no charge at physiological pH, when the pH is in the range of 4 to 7.4, preferably 4.5 to 7, Z can attract one or more protons available in the medium to acquire one or more positive charges.

[0045] The terminal position of the ionizable moiety is preferably understood as the pKa of the terminal ionizable moiety directly bearing the overall pKa of the molecule defined by formula (I). As a result, the pKa of the terminal ionizable moiety directly bears the total charge of the molecule defined by formula (I). Therefore, since the terminal ionizable moiety Z preferably has no charge at physiological pH due to its pKa value, the molecule defined by formula (I) preferably has no charge at physiological pH. Preferably, since Z can attract one or more protons available in the medium to acquire one or more positive charges when the pH is in the range of 4 to 7.4, the molecule defined by formula (I) can acquire one or more positive charges when the pH is in the range of 4 to 7.4. More preferably, since Z can attract one or more protons available in the medium to acquire one or more positive charges when the pH is in the range of 4.5 to 7, the molecule defined by formula (I) can acquire one or more positive charges when the pH is in the range of 4.5 to 7.

[0046] "pH of the medium" is preferably understood to mean the pH of the medium containing the compound of the present invention.

[0047] "Physiological pH" is understood to be a pH in the range of preferably 7.0 to 7.4.

[0048] In the present invention, unless otherwise specified, "heteroatom" is understood to be an atom preferably selected from nitrogen, oxygen, sulfur, phosphorus, and halogen.

[0049] In the present invention, unless otherwise specified, "halogen" is preferably understood to mean bromine, iodine, chlorine, and fluorine.

[0050] "Fluorination" is understood to preferably mean the inclusion of one or more fluorine atoms.

[0051] "Containing one or more heteroatoms other than fluorine" is understood to preferably mean containing one or more atoms selected from nitrogen, oxygen, sulfur, phosphorus, and halogens other than fluorine.

[0052] A "hydrocarbon group" is preferably understood as any group containing one or more carbon atoms, which may be bonded to one or more hydrogen atoms.

[0053] "One or more" is preferably understood as one, two, three, four, five, six, seven, eight, nine, or ten, more preferably one, two, three, four, or five, and even more preferably one, two, or three.

[0054] "Transfection" is understood as the process of introducing natural, wild-type, or recombinant genetic material or proteins into target cells, preferably via synthetic or nonviral delivery systems. The nature of the genetic material introduced and the mechanisms involved in this process depend on the type of nucleic acid, protein, or small molecule used. For example, the genetic material introduced can encode a wide variety of genes for specific purposes, such as reporter genes, enzymes, endonucleases, nucleases or recombinases for genome editing, short hairpin RNA, antisense RNA for gene silencing, mRNA, tRNA, receptors, such as manipulated antigen receptors (including, but not limited to, T cell receptors, chimeric antigen receptors, and chimeric cytokine receptors), growth factors, hormones, cell surface proteins, secreted proteins, signaling proteins, or any polypeptides, and / or proteins or nucleic acids for therapeutic purposes.

[0055] "Biologically active molecule," "active molecule," "biological activator," and / or "active ingredient" are preferably understood as any molecule or polymer that has specific activity in cells and is used in a number of fields ranging from cell biology to medicine. Examples of such molecules or polymers include, but are not limited to, nucleic acids, polysaccharides, lipids, polynucleotides, morpholinooligonucleotides, aptamers, proteins, peptides, polypeptides, peptoids, small organic or inorganic molecules, drugs, or any compound of pharmaceutical interest, antigens, and mixtures thereof.

[0056] "Organic or inorganic small molecules (plural)" is preferably understood to be molecules of known structure and molecular weight, which are not polymeric and preferably have a molecular weight of less than 1500 g / mol.

[0057] A drug(s) or any compound of pharmaceutical interest may be any molecule or compound, including its salts or derivatives, that can exert desired effects on cells, tissues, tumors, organs, or subjects, such as biological, physiological, and / or cosmetic effects. Examples of drugs(s) or any compounds of pharmaceutical interest include, but are not limited to, oncological agents (including chemotherapeutic agents, hormonal agents, immunotherapeutic agents, and / or radiotherapeutic agents), lipid-lowering agents, antiviral agents, anti-inflammatory compounds, antidepressants, stimulants, analgesics, antibiotics, contraceptives, antipyretics, vasodilators, anti-angiogenic agents, cellular vasoadhesive agents, signaling inhibitors, cardiovascular agents, such as antiarrhythmic agents, hormones, vasoconstrictors, and / or steroids. More specifically, examples of drugs or any compounds of pharmaceutical interest include, but are not limited to, folate or sodium valproate, antibodies, antigens, lymphokines, interleukins, necrosis factors and apoptosis factors, interferons, growth factors, tissue plasminogen activators, factor VIIIc, erythropoietin, insulin, calcitosine, thymidine kinases, and combinations thereof.

[0058] Any drug(s) or compound of pharmaceutical interest may be therapeutically active on its own, or may be a prodrug that becomes active upon further modification.

[0059] Examples of nucleic acids include, but are not limited to, deoxyribonucleic acid (DNA) such as genes or plasmid DNA (pDNA), linear coding DNA, artificial chromosomes, ribonucleotide acids (RNA) such as antisense oligonucleotides (ASOs) or messenger RNA (mRNA), small interfering RNA (siRNA), self-amplifying RNA (saRNA), circular RNA (cirRNA), microRNA (miRNA), single-stranded guide RNA (sgRNA)-mediated CRISPR-Cas9 systems, double-stranded RNA (dsRNA), short hairpin RNA (shRNA), ribozymes, or modified nucleic acids such as peptide nucleic acids (PNA), locked nucleic acids (LNA), morpholino oligonucleotides and / or aptamers. Nucleic acids may be natural or artificial. Nucleic acids may be of animal, human, plant, bacterial, or viral origin. Nucleic acids can be used to express, modify, downregulate, or silence the translation (i.e., expression) of a gene of interest. Their therapeutic function may be that of a gene or mRNA encoding a target polypeptide and / or protein in a host cell, or that they may have an antisense function that controls gene expression, transcription to RNA, or translation to protein. They can also act as ribozymes or interfering RNAs (such as sRNA, shRNA, or miRNA) involved in gene expression. They can also edit genes by correcting or adding mutations (such as CRISPR-Cas9).

[0060] Non-limiting examples of target genes include, but are not limited to, genes associated with metabolic diseases and disorders (such as liver disease and liver damage), genes associated with cell proliferation, tumorigenesis and / or cell transformation (including cell proliferation disorders such as cancer), angiogenic genes, receptor ligand genes, immunomodulatory genes (such as those associated with inflammation and autoimmune responses), genes associated with viral infection and survival, and / or genes associated with neurodegenerative disorders.

[0061] The nucleic acids present in lipid-nucleic acid particles can be in any form. For example, the nucleic acids may be single-stranded DNA or RNA, double-stranded DNA or RNA, or DNA-RNA hybrids. Non-limiting examples of double-stranded RNA include siRNA and miRNA. Non-limiting examples of single-stranded RNA include mRNA. Non-limiting examples of double-stranded DNA include plasmids and linearized DNA. The nucleic acids may optionally contain one or more modified nucleotides, including but not limited to 2'-O-methylnucleotides, 2'-deoxynucleotides, 2'-O-(2-methoxyethyl)nucleotides, locked nucleic acid (LNA) nucleotides, 5-C-methylnucleotides, 4'-thionucleotides, aminonucleotides, 2'-C-allyl nucleotides, and mixtures thereof.

[0062] For use in the fields of gene therapy, vaccines, and immunotherapy, the nucleic acids may be advantageously DNA and RNA, and preferably comprise an expression cassette consisting of one or more sequences of DNA or RNA encoding a polypeptide of interest under the control of one or more promoters and transcriptional terminators that are active in target cells.

[0063] A "polypeptide" is preferably understood as any amino acid chain, regardless of its size. Therefore, this term can encompass both peptides and proteins.

[0064] "Target cells" are preferably understood as any cells that can be targeted for transfection.

[0065] "Cell" is preferably understood as a single and / or isolated cell, or a cell that is part of a multicellular entity such as a tissue, organism, or cell culture. A cell may be a primary cell or an established cell line, or a stem cell or progenitor cell.

[0066] As used herein, the term “primary cells” may refer to cells that are well known in the art, isolated from tissue, and established for in vitro proliferation.

[0067] Cells may be, but are not limited to, eukaryotic cells, such as endothelial cells, epithelial cells, fibroblasts, hepatocytes, hematopoietic cells (including, but not limited to, lymphocytes and / or monocytes, macrophages, natural killers, and dendritic cells), muscle cells, nerve cells (in particular, all types of neurons including, but not limited to, cortical, hippocampal, sensory neurons, motor neurons, posterior pathway ganglia, oligodendrocytes, cerebellar granule cells, neural stem cells and basket cells, Betz cells, mesospinous neurons, Purkinje cells, pyramidal cells, Renshaw cells, granule cells or anterior horn cells and glial cells and astrocytes), stem cells, embryonic stem cells, hematopoietic stem cells, germ cells, tumor cells, lymphoid cell lineages, and / or somatic cells.

[0068] As used herein, eukaryotic cells may refer to any cells of a multicellular eukaryote, including cells of animal origin such as vertebrates. Preferably, the target cells may be mammalian cells. As used herein, the term “mammalian cells” is well known in the art and may refer to any cells belonging to or originating from animals classified in the class Mammalia.

[0069] Cells can be prokaryotic cells (such as bacteria), plant cells, insect cells, yeast cells, or parasitic cells.

[0070] Cells may be differentiated, and may be totipotent, pluripotent, pluripotent, oligopotent, unipotent, or pluripotent. Cells may be embryonic stem cells or induced pluripotent stem cells (iPS), or may be differentiated from embryonic or iPS cells. Transfected cells may be non-permissible, resistant to infection, or susceptible to infection. Tumor cells and cell lines known in the art may include, but are not limited to, lymphoma cell lines (e.g., Jurkat, CEM, H9, Daudi, SUP-M2, and KARPAS-299), dendritic cell lines (e.g., DC2.4 and Mutu), natural killer cell lines (e.g., NK-92 and KHYG-1), epithelial cell lines (e.g., NIH-3T3, COS, CHO, and HEK293), pancreatic tumor cells (e.g., PANC-1), stem cell lines (KG1a), breast cancer cells (e.g., MCF-7, T74D, and MDA-MB361), neuroblastoma (e.g., SH-SY5Y and N2a), fibrosarcoma (e.g., HT1080), astrocytoma (e.g., 1321N1), and / or endothelial cells (e.g., HUVEC, HMEC, and HCAEC).

[0071] "Signal transduction molecules and molecules that may be involved in signal transduction pathways within target cells" preferably include activators or inhibitors of protein kinase C or phospholipase signaling pathways, for example, activators or inhibitors of cellular metabolic pathways (e.g., AMPK signaling, insulin receptor signaling, and glutamine metabolic pathways), activators or inhibitors of TLR pathways (such as Toll-like receptors), activators or inhibitors of PRR pathways (pattern recognition receptors), activators or inhibitors of PAMP pathways (pathogen-associated molecular patterns), activators or inhibitors of DAMP pathways (injury-associated molecular patterns), AhR ligands (aryl hydrocarbon receptor ligands), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), cytosolic DNA sensors (CDSs), C-type lectin receptors (CLRs), inflammasomes, or activators or inhibitors of inflammation or autophagy pathways, and activators of the JAK / STAT pathway. It is understood that these are inhibitors, activators or inhibitors of ubiquitin and ubiquitin-like / proteasome pathways, activators or inhibitors of the PI3K / AKT pathway, activators or inhibitors of tyrosine kinases, activators or inhibitors of the MAP kinase pathway, activators or inhibitors of GPCRs, activators or inhibitors of the STING pathway, activators or inhibitors of calcium, cAMP signaling pathways, cell cycle, checkpoint control and DNA repair mechanisms, activators or inhibitors of apoptotic pathways, modifiers of reactive oxygen species (such as ROS and NOS), activators or inhibitors of immune checkpoints, modifiers of protein synthesis and RNA degradation processes, modifiers of key elements that play a role at the chromatin or DNA level (e.g., activators or inhibitors of protein acetylation, histone lysine methylation, DNA methylation and nuclear receptor signaling), and modifiers of microtubule and actin dynamics pathways.

[0072] "Lipid of formula (I)" is preferably understood to be the lipid of the present invention.

[0073] Preferably, as used herein, the terms “conjugated” and “coupled” have the same meaning.

[0074] Detailed explanation For the purposes of this application, unless otherwise specified, the range of values ​​shown includes the extreme values.

[0075] A first object of the present invention relates to lipids, preferably adjustable biodegradable lipids of formula (I), or stereoisomers thereof, or pharmaceutically acceptable salts thereof: [ka] During the ceremony: -R represents a lipophilic region, and preferably R comprises one or more branched or linear, unsaturated or saturated, possibly fluorinated alkyl chains containing 6 to 48 carbon atoms or 6 to 24 carbon atoms, preferably 10 to 36 carbon atoms, more preferably 10 to 18 carbon atoms, and may contain one or more heteroatoms other than fluorine; or one or more cyclic or polycyclic groups known to be lipophilic and which may contain one or more heteroatoms, such as steroid groups which may contain one or more heteroatoms, polycyclic aromatic groups which may contain one or more heteroatoms, or alkaloid derivative groups which may contain one or more heteroatoms; or natural or synthetic lipids which may contain one or more heteroatoms; or a combination thereof.

[0076] -Sp represents a spacer region used to separate a lipophilic region from a hydrophilic polar region, preferably a hydrophilic tunable polar region, and more preferably Sp comprises one or more branched or linear, unsaturated or saturated, possibly fluorinated alkyl chains, comprising 6 to 24 carbon atoms, preferably 10 to 18 carbon atoms, and may contain one or more heteroatoms and / or bioreducible bonds; and -Z s This represents a hydrophilic polar region, preferably a hydrophilic, tunable polar region, used to complex biologically active components and efficiently deliver them into cells. More preferably, Z sIt contains 2 to 36 carbon atoms, preferably 2 to 24 carbon atoms, and may contain one or more heteroatoms other than fluorine, and one or more branched or straight-chain, unsaturated or saturated, optionally fluorinated alkyl chains, and / or preferably has no charge at physiological pH, but has a positive charge when the pH of the medium is below the pKa value of the entity, and contains a bio-reductive bond covalently bonded to one or more straight-chain or cyclic nitrogen-based chemical moieties. This polar region can incorporate a bio-reductive molecular pattern that enables more rapid release of biologically active compounds and improvement of the biocompatibility of the whole molecule.

[0077] Preferably, Z s By the definition of, the lipid of the present invention, preferably the adjustable lipid of the present invention, does not contain any positive charge at physiological pH (7.0 - 7.4), which means that the pKa of the molecule covered by formula (I) is always less than 7.0.

[0078] Preferably, Z s By the definition of, the lipid of the present invention, preferably the adjustable lipid of the present invention, does not contain any positive charge at physiological pH (7.0 - 7.4), but shows one or several positive charges when the pH of the medium is below the pKa of the molecule covered by formula (I), which is always less than 7.0. Preferably, Z s By the definition of, the lipid of the present invention, preferably the adjustable lipid of the present invention, does not contain any positive charge at physiological pH (7.0 - 7.4), but shows one or several positive charges when the pH of the medium is in the range of 2.0 - 7.0, preferably 4.0 - 7.0, more preferably 4.5 - 7.0.

[0079] In other words, preferably, Z s cannot contain a primary aliphatic amine group, a succinic acid group, and / or a carboxylic acid group combined with a tertiary amine group (however, Z s can contain a carboxylic acid group and does not contain a tertiary amine group, and Z s can contain a tertiary amine group and does not contain a carboxylic acid group).

[0080] Examples of nitrogen-based chemical components include, but are not limited to, amines, amides, oximes, hydroxylamines, carbamates, isocyanates, isothiocyanates, or cyanohydrins, aliphatic or aromatic compounds, or combinations thereof.

[0081] Examples of cyclic or polycyclic groups that are known to be lipophilic and may contain one or more heteroatoms include, but are not limited to, cycloalkanes, cycloalkenes, cycloalkynes, and / or steroid groups.

[0082] Examples of steroid groups include, but are not limited to, cholesterol and its derivatives, ergosterol, estradiol, stigmasterol, testosterone, progesterone, and / or androsterone.

[0083] Examples of cycloalkanes include, but are not limited to, cyclopentane, cyclohexane, cyclooctane, cyclodecane, bicyclohexane, and / or tricyclohexane.

[0084] Examples of cycloalkenes include, but are not limited to, cyclopropene, cyclobutene, cyclopentene, cyclohexene, cycloheptene, 1,3-cyclohexadiene, 1,4-cyclohexadiene, 1,5-cyclooctadiene, cis-cyclooctene, and / or trans-cyclooctene.

[0085] Examples of cycloalkynes include, but are not limited to, cyclododecine, cyclodecine, cyclononine, and / or cyclooctin.

[0086] "Cholesterol derivatives" are preferably understood to be cholesterol, bile acids, vitamin D2, progestins, glucocorticoids, estrogens, androgens, phytosterols, and / or oxysterols.

[0087] Examples of polycyclic aromatic groups include, but are not limited to, naphthalene derivatives, dansyl derivatives, and / or anthracene derivatives.

[0088] "Naphthalene derivatives" are preferably understood to be 1-naphthoic acid, 1-naphthol, 1-nitronaphthalene and / or 1-halogenonaphthalene.

[0089] "Dansyl derivative" is preferably understood to be 5-(dimethylamino)naphthalene-1-sulfone.

[0090] "Anthracene derivatives" are preferably understood to be anthracene, monohydroxyanthracene, and / or polyhydroxyanthracene.

[0091] Examples of alkaloid derivatives include, but are not limited to, higrine, retronesin, indicine, N-acetylcholine, atropine, morphine, mescaline, adrenaline, putrescine, spermine, theobromine, cathinone, sedamine, coninine, matrine, swinesonine, pipecolic acid, ioforesin, amrensin, berberine, glaucine, imerbin, muscimol, serotonin, quinine, ergotamine, colchicine, solanidine, cyclopamine, batrachotoxin, and / or isoquinoline.

[0092] Examples of natural or synthetic lipids include, but are not limited to, phosphatidylcholine or its derivatives, phosphatidylethanolamine or its derivatives, phosphatidylinositol or its derivatives, sphingomyelin or its derivatives, phosphatidic acid or its derivatives, lysophosphatidylcholine or its derivatives, lysophosphatidylethanolamine or its derivatives, glycero-3-phosphocholine or its derivatives, and / or glycerol or its derivatives.

[0093] "Bioreducing bond" is preferably understood to be a disulfide bond, ester bond, vinyl ether bond, and / or acyl-hydrazone bond.

[0094] A "bioreducible molecular pattern" is preferably understood as a biocompatible or bio-inspired atomic group that can be degraded or degraded under biological conditions when exposed to microorganisms, aerobic and anaerobic processes, resulting in increased biocompatibility of the entire molecule. Examples of bioreducible molecular patterns include, but are not limited to, difunctional ribose groups, ethylene oxide groups, and / or amino acid groups.

[0095] Preferably, the following compounds are excluded from general formula (I): [ka] and [ka]

[0096] Preferably, in the previously defined equation (I), R corresponds to equation (II): [ka] During the ceremony, - Identical or different N1 and N2 represent a linear, branched saturated or unsaturated hydrocarbon group containing 1 to 8 carbon atoms, preferably 1 to 4 carbon atoms, used to terminate the carbon chain, and preferably identical or different N1 and N2 are any of the methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, or t-butyl groups; - Identical or different R1 and R2 represent linear, branched, and / or cyclic saturated or unsaturated hydrocarbon groups comprising 6 to 24 carbon atoms, preferably 10 to 18 carbon atoms, and may contain one or more heteroatoms; -Identical A and B represent OC(O), C(O)NH-, -NHCO-, OC(O)-O, NH-C(O)-NH, NH-C(O)-O, OC(O)-NH, -SC(O)-S-, -OC(S)-S-, -SC(O)-O, -NH- or -S- groups, or identical or different A and B represent OC(O), C(O)NH-, -NHCO-, -NH- or O- groups, preferably identical A and B represent OC(O), C(O)NH-, -NHCO-, OC(O)-O, NH-C(O)-NH, NH-C(O)-O, OC(O)-NH, -SC(O)-S-, -OC(S)-S-, -SC(O)-O, -NH- or -S- groups; -a is an integer between 1 and 4, preferably an integer equal to 1, 2, or 3; -b is an integer between 0 and 6, preferably an integer equal to 0 or 1; -E1 and E2, whether identical or different, represent -C(O)O-, -C(O)-NH-, -NH-, -O-, or -S- groups; -c is an integer between 0 and 2; -d is an integer equal to 0 or 1; -d1 is an integer between 0 and 6, preferably an integer equal to 0 or 1; -d2 is an integer between 0 and 6, preferably an integer between 0 and 2; -e is an integer between 0 and 6, preferably between 0 and 2, and more preferably between 0 and 1.

[0097] Preferably, in the previously defined formula (II), if a is an integer equal to 4, the following compounds are excluded: - [ka] Here, according to equation I, Sp does not exist, and Zs is CH2-CH2-N(CH3)2, and according to equation II, N1=N2=CH3, R1=R2=C 17 H 30Therefore, identical A and B represent -C(O)NH-, a=4, E1 represents a -C(O)O- group, E2 does not exist, and b=c=e=d1=d2=0; - [ka] Here, according to equation I, Sp does not exist, and Zs is CH2-CH2-CH2-N(CH3)2, and according to equation II, N1=N2=CH3, R1=R2=C 17 H 30 Therefore, identical A and B represent -C(O)NH-, a=4, E1 represents a -C(O)O- group, E2 does not exist, and b=c=e=d1=d2=0; - [ka] Here, according to equation I, Sp does not exist, and Zs is CH2-CH2-N(CH3)2, and according to equation II, N1=N2=CH3, R1=R2=C 17 H 30 Therefore, identical A and B represent -C(O)NH-, a=4, E1 represents a -C(O)NH- group, E2 does not exist, and b=c=e=d1=d2=0; - [ka] Here, according to equation I, Sp does not exist, and Zs is CH2-CH2-CH2-N(CH3)2, and according to equation II, N1=N2=CH3, R1=R2=C 17 H 30 Therefore, identical A and B represent -C(O)NH-, a=4, E1 represents a -C(O)NH- group, E2 does not exist, and b=c=e=d1=d2=0.

[0098] Preferably, in formula (II) above, if either or both R1 and R2 are branched, it will likely be understood that each alkyl chain in the branched molecular pattern is terminated by the N1 and / or N2 moieties as previously defined.

[0099] Preferably, in equation (II) above, if d is equal to 0, then d1 and d2 automatically become equal to 0, and it will probably be understood that E2 does not exist.

[0100] Preferably, in the previously defined formula (I), Sp corresponds to the general formula (III): [ka] During the ceremony, -Gr represents a linear or branched hydrocarbon group comprising 1 to 15 carbon atoms, preferably 1 to 8 carbon atoms, and may contain one or more heteroatoms; -m is an integer equal to 0 or 1; -RAc represents an amino acid radical; and -n is an integer equal to 0 or 1.

[0101] "Amino acid radical" is preferably understood as an atomic group consisting of an amino acid, where the amino acid is covalently bonded to a Gr or R group on one side and to one or more Zs groups on the other side.

[0102] Therefore, in equation (III) defined earlier, if m and / or n are equal to 0, then Gr and RAc may not exist.

[0103] Preferably, in the previously defined equation (I), Z s This corresponds to equation (IV): [ka] During the ceremony, - Identical or different S1 and S2 represent a linear or branched hydrocarbon group comprising 1 to 15 carbon atoms, preferably 1 to 6 carbon atoms, preferably selected from nitrogen, oxygen, sulfur, bromine, iodine, chlorine, and fluorine, and more preferably one or more heteroatoms selected from nitrogen, sulfur, bromine, iodine, chlorine, and fluorine; -D represents a functional group that can incorporate at least one bond sensitive to its environment, the bond being sensitive to stimuli such as a decrease in pH (e.g., a vinyl ether or carbonate bond or acylhydrazone group sensitive to acidic media), changes in redox potential (e.g., a disulfide bond cleaved in a reducing medium), enzymes (e.g., an ester bond cleaved by an endogenous esterase), or light radiation (e.g., having a photosensitive group), so that it is stable in extracellular media and rapidly cleaved in intracellular media; preferably, D represents one or more vinyl ether groups, one or more acylhydrazone groups, one or more carbonate bonds, disulfide bonds, ester bonds, or one or more photosensitive groups; -p is an integer equal to 0 or 1; -Q is a branched hydrocarbon group comprising 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and one or more heteroatoms, which may be covalently bonded on one side to an S2, RAc, Gr, or R group and on the other side to at least two Q and / or Z groups; -q is an integer between 0 and 8, preferably between 0 and 3; -Z is preferably a nitrogen (N)-centered terminal ionizable moiety incorporating a chemical functional group that is uncharged at physiological pH and can be positively charged when the pH of the medium drops below the pKa value of the entity; more preferably, Z is uncharged at physiological pH (7.0-7.4) and has one or more positive charges when the pH of the medium is in the range of 2.0-7.0, preferably 4.0-7.0, more preferably 4.5-7.0.

[0104] More preferably, Z represents a tertiary aliphatic amine such as an N,N-dialkylamine, or a cyclic amine moiety such as an N-substituted pyrrolidine, N-substituted piperidine, N-substituted morpholine, N-substituted piperazine, or N-substituted pyridine. The substituent includes, but is not limited to, one or more alkyl groups such as methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, or t-butyl groups, and / or aromatic rings based on benzene or naphthalene groups; where the substituent may include one or more heteroatoms; -r is an integer between 1 and 16, preferably between 1 and 8, preferably, if q is equal to 1, r is at least equal to 2, and if r is greater than 1, the Z groups may be the same or different; and -s is an integer equal to 1 or 2.

[0105] Examples of photosensitive groups include, but are not limited to, diazo, nitrobenzyl, and / or phenacyl groups.

[0106] "Amino acid radical" is preferably understood as an atomic group consisting of an amino acid, where the amino acid is covalently bonded to a Gr or R group on one side and to one or more S1, Q, or Z groups on the other side.

[0107] Preferably, in the previously defined formula (IV), Z can be written according to formula (V): [ka] During the ceremony, -R3 and R4 are the same or different and selected from C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl groups, which may be substituted with one or more methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl or t-butyl groups, or R3 and R4 may bond to form a heterocycle which may be substituted with one or more methyl, ethyl, n-propyl, i-propyl, s-butyl, i-butyl or t-butyl groups, for example, pyrrole, pyrrolidine, pyridine, piperazine, morpholine, piperidine or indoline which may be substituted with one or more methyl, ethyl, n-propyl, i-propyl, s-butyl, i-butyl or t-butyl groups; -N is nitrogen; -V is O, S, N(R6), C(O), C(O)O, OC(O), C(O)N(R6), N(R6)C(O), O(O)N(R6), N(R6)C(O)O, C(O)S, C(S)O, S(O), S(O)(O), C(S), or a heterocycle which may be substituted, for example, pyrrole, pyrrolidine, pyridine, piperazine, morpholine, piperidine, or indoline which may be substituted with one or more methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, or t-butyl groups, where R6 is hydrogen (H), or a C1-C which may be substituted with one or more methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, or t-butyl groups 10 Alkyl, C2-C 10 Alkenyl or C2-C 10 It is an alkynyl; preferably, R6 is a hydrogen (H), a C1 alkyl (methyl) group, and one or more heteroatoms such as -O, -NH, -S and mixtures thereof, C2, C3, C4, C5, C6, C7, C8, C9 and C 10 Selected from the group consisting of alkyl, alkenyl, and alkynyl groups; -U may be absent or substituted with one or more methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, or t-butyl groups, preferably substituted with one or more heteroatoms or heteroatomic groups selected from nitrogen, oxygen, sulfur, bromine, iodine, chlorine, fluorine, phosphorus, or combinations thereof, C1-C 12 Alkyl, C2-C 12 Alkenyl, or C2-C 12 It is alkinyl.

[0108] Preferably, R3 and R4 may each be independently substituted with one or more methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, or t-butyl groups, such as C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C2-C3 alkyl, C2-C4 alkyl, C2-C5 alkyl, C2-C6 alkyl, C3-C4 alkyl, C3-C5 alkyl, C3-C6 alkyl, C4-C5 alkyl, C4-C6 alkyl, and C5-C6 alkyl groups. These are C2-C3 alkenyl, C2-C4 alkenyl, C2-C5 alkenyl, C2-C6 alkenyl, C3-C4 alkenyl, C3-C5 alkenyl, C3-C6 alkenyl, C4-C5 alkenyl, C4-C6 alkenyl, C5-C6 alkenyl, C2-C3 alkynyl, C2-C4 alkynyl, C2-C5 alkynyl, C2-C6 alkynyl, C3-C4 alkynyl, C3-C5 alkynyl, C3-C6 alkynyl, C4-C5 alkynyl, C4-C6 alkynyl, or C5-C6 alkynyl.

[0109] Preferably, R3 and R4 are both methyl groups, ethyl groups, or a combination of one methyl group and one ethyl group.

[0110] Preferably, R3 and R4 bond to form an optionally substituted heterocyclic ring containing 1, 2, 3, 4, 5, or 6 or more carbon atoms and 1, 2, 3, or 4 or more heteroatoms, such as nitrogen (N), oxygen (O), sulfur (S), and mixtures thereof. The optionally substituted heterocyclic ring may include: -2 to 10 carbon atoms and 1 to 3 heteroatoms such as nitrogen (N), oxygen (O) and / or sulfur (S); or - Imidazoles, triazoles (such as 1,2,3-triazoles, 1,2,4-triazoles), pyrazoles, thiazoles, pyrrole, furans, oxazoles, isoxazoles, oxazolines, oxazolidines, oxazolidines, oxadiazoles, tetrazoles, pyrrole, pyrrolidines, pyridines, piperazines, morpholines, piperidines, or indolines, which may be substituted with an aromatic ring based on a benzene or naphthalene group, which may contain one or more alkyl groups such as methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, or t-butyl groups, and / or one or more heteroatoms; or - 5 carbon atoms and 1 nitrogen atom, Here, the heterocyclic ring may be substituted with substituents such as hydroxyl (-OH) groups at the ortho, meta, and / or para positions; or - An imidazole group which may be substituted with an aromatic ring based on a benzene or naphthalene group which may contain one or more alkyl groups such as methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl or t-butyl groups, and / or one or more heteroatoms.

[0111] In the above formula (II), if R1 and R2 are different, their structures can be described according to formulas (VI a) and (VI b): [ka] During the ceremony, - Same or different Ak f1、 Ak j1 , Ak f2、 Akj2 This represents a linear or branched hydrocarbon group comprising 1 to 22 carbon atoms, preferably 1 to 12 carbon atoms, and may also contain one or more heteroatoms; -f1 and f2 are integers equal to 0 or 1; -j1 and j2 are integers equal to 0 or 1; -The same or different Xg1, Xi1, Xg2 and Xi2 may be O, S, N(R7), C(O), C(O)O, OC(O), C(O)N(R7), N(R7)C(O), O(O)N(R7), N(R7)C(O)O, C(O)S, C(S)O, S(O), S(O)(O), C(S), where R7 may be hydrogen (H) or substituted with one or more methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl or t-butyl groups, C1-C 10 Alkyl, C2-C 10 Alkenyl or C2-C 10 R7 is an alkynyl; more preferably, R7 may contain hydrogen (H), a C1 alkyl(methyl) group, and one or more heteroatoms such as -O, -NH, -S and mixtures thereof, C2, C3, C4, C5, C6, C7, C8, C9 and C 10 The group is selected from the group consisting of alkyl, alkenyl, and alkynyl groups. Preferably, Xg1, Xi1, Xg2, and Xi2 are independently represented by N(R7), C(O)N(R7), N(R7)C(O), OCO(O)N(R7), and N(R7)C(O)O, while if they happen to have different properties, it will probably be understood that R7 can have several different properties within the same molecule among the possibilities shown above; -g1 and g2 are integers equal to 0 or 1; -i1 and i2 are integers equal to 0 or 1; - Identical or different I h1 and I h2 Both represent a straight or branched unsaturated hydrocarbon chain containing 2 to 16 carbon atoms, preferably 2 to 8 carbon atoms and at least one unsaturated atom, more preferably the same or different I h1 and Ih2 The following are selected independently: [ka] and [ka]

[0112] -h1 and h2 are integers equal to 0 or 1; -J1 and J2 are branched hydrocarbon groups comprising 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and / or one or more heteroatoms; J1 is on the one hand Ak f1 , Ak j1 , or Xg1, Xi1, or I h1 J2 may be covalently bonded to either of the bases and to A on the other, and J2 may be covalently bonded to A on one side. f2 , Ak j2 , Xg2 and Xi2, or I h2 It may be covalently bonded to either B or B on the other; -l1 and l2 are integers equal to 0 or 1; preferably, if l1 is equal to 0, Ak f1 , Ak j1 , or Xg1, Xi1, or I h1 It is understood that one of the groups is covalently bonded to A. Similarly, if l2 is equal to 0, then Ak f2 , Ak j2 , Xg2 and Xi2, or I h2 One of the groups is covalently bonded to B; -k1 and k2 are integers between 1 and 3, and preferably, if l1 is equal to 1, then k1 is at least equal to 2. Similarly, if l2 is equal to 1, then k2 is at least equal to 2.

[0113] In equation (IV) above, if R1 and R2 are the same, their structure can be described according to equation (VII): [ka] During the ceremony, - Same or different Ak f and Ak j This represents a linear or branched hydrocarbon group comprising 1 to 22 carbon atoms, preferably 1 to 12 carbon atoms, and may also contain one or more heteroatoms; -f is an integer equal to 0 or 1; -j is an integer equal to 0 or 1; - The same or different Xg and Xi may be O, S, N(R7), C(O), C(O)O, OC(O), C(O)N(R7), N(R7)C(O), O(O)N(R7), N(R7)C(O)O, C(O)S, C(S)O, S(O), S(O)(O), C(S), where R7 may be hydrogen (H) or substituted with one or more methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl or t-butyl groups, C1-C 10 Alkyl, C2-C 10 Alkenyl or C2-C 10 R7 is an alkynyl; more preferably, R7 may contain hydrogen (H), a C1 alkyl(methyl) group, and one or more heteroatoms such as -O, -NH, -S and mixtures thereof, C2, C3, C4, C5, C6, C7, C8, C9 and C 10 Selected from the group consisting of alkyl, alkenyl, and alkynyl groups; -g is an integer equal to 0 or 1; -i is an integer equal to 0 or 1; -I h I represents a straight or branched unsaturated hydrocarbon chain containing 2 to 16 carbon atoms, preferably 2 to 8 carbon atoms and at least one unsaturated atom, more preferably I h teeth, [ka] and [ka] Selected from.

[0114] -h is an integer equal to 0 or 1; -J is a branched hydrocarbon group comprising 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and / or one or more heteroatoms. -J is on the one hand Ak j Or Xi or I h or Xg or Ak f The base may be covalently bonded to A or B on the other side; -l is an integer equal to 0 or 1; preferably, if l is equal to 0, Ak j Or Xi or I h or Xg or Ak f It is understood that one of them is covalently bonded to A, and the other to B; -k is an integer between 1 and 3; preferably, if l is equal to 1, then k is understood to be at least equal to 2.

[0115] Preferably, in the previously defined formula (I), R incorporates an amino acid that acts as a "biocompatible template".

[0116] Examples of amino acids that act as "biocompatible templates" include, but are not limited to, glutamic acid, aspartic acid, and / or ornithine.

[0117] A "biocompatible template" is understood to be, preferably, a novel lipid, preferably a novel modifiable lipid, such as a polyunsaturated amino acid that acts as the basis for the structural skeleton of glutamic acid, aspartic acid, lysine, and ornithine, each presenting three chemical functional groups that can react with a wide variety of components according to the basic principles of peptide coupling chemistry. In the case of glutamic acid and aspartic acid, two carboxylic acid functional groups and one primary amine are freely available for coupling, while in the case of lysine and ornithine, two amine functional groups and one carboxylic acid functional group are available.

[0118] More specifically, a "biocompatible template" is understood to preferably be a polyunsaturated amino acid that acts as the basis for the structural backbone of these new lipids, and it is understood that these amino acids may belong to the L-type or D-type.

[0119] Examples of biocompatible templates may include atomic groups defined by R that do not belong to the amino acid library but constitute precursors to amino acid biosynthesis. Examples of such atomic groups defined by R that do not belong to the amino acid library but constitute precursors to amino acid biosynthesis include, but are not limited to, α-ketoglutaric acid, L-malic acid, D-malic acid, derivatives of oxaloacetate, or combinations thereof.

[0120] Preferably, in the previously defined formula (I), R is selected from the following: [ka] (Formula VIII) In the formula, R 1、 R 2、 N1 and N2 are as previously defined.

[0121] More preferably, in the previously defined formula (I), R is selected from the following: [ka] (Formula VIII) In the formula, R 1、 R 2、 N1 and N2 are as previously defined.

[0122] Preferably, in the previously defined formulas (VI a) and (VI b), the same or different J1 and J2 correspond to the formula -CO-Y1-NH-Y2-N-[Y3-NH]2- or -NH-Y2-N-[Y3-NH]2-, In the formula, the same or different Y1, Y2, and Y3 represent a crosslinked alkylene group containing 1 to 8 carbon atoms, preferably 1 to 4 carbon atoms.

[0123] Preferably, Y1 represents methylene, and Y2 and Y3 represent a bridged alkylene group containing 1 to 4 carbon atoms, more preferably 2 carbon atoms.

[0124] Therefore, preferably, J1 and J2 are such that J1 is Ak f1 , Ak j1 , or Xg1, Xi1, or I h1 J2 has at least two repetitions of one of the groups, and on the other side A, and three functional groups that allow for the formation of a covalent bond, with Ak on one side and J2 on the other. f2 , Ak j2 , Xg2 and Xi2, or I h2 It can optionally be covalently bonded to at least two iterations of one of the bases, and to B on the other.

[0125] Preferably, if R1 and R2 are the same, then J1 and J2 are the same and can be named J as defined in the same way as J1 and J2. Therefore, in this case, J is, J is, on the one hand Ak f , Ak j , or Xg, Xi1, or I h It has at least two repeats of the group, and on the other hand, it has A and B and three functional groups that can optionally form a covalent bond.

[0126] In certain embodiments, in the previously defined formula (III), Gr may be absent, or Gr may function as a spacer arm, thus corresponding to a molecular pattern represented as -W4-Y4-W4-; Here, Y4 represents a bridged alkylene group containing 1 to 8 carbon atoms, preferably 1 to 4 carbon atoms, and W4 represents an -O-, -CO-, or -NH- group.

[0127] In a particular embodiment, in the previously defined formula (III), Gr corresponds to formula CO-Y4-CO, In the formula, Y4 has the same meaning as previously defined, and is linked by an amide bond to the lipophilic region R on the one hand, to the RAc radical on the other hand, or directly to the S1, Q, or Z group.

[0128] Preferably, in the previously defined formula (III), Gr may be absent, or Gr may function as a spacer arm, thus corresponding to a molecular pattern represented as -W4-Y4-W4-; Here, Y4 represents a bridged alkylene group containing 1 to 8 carbon atoms, preferably 1 to 4 carbon atoms, and W4 represents a C(O)-O, C(O)-NH, C(O)-S, S(O), S(O)-O, C(S)-O, -O-, or -NH- group.

[0129] More preferably, in the previously defined formula (III), Gr corresponds to formula -W4-Y4-W4-, In the formula, Y4 has the same meaning as previously defined, and is linked by a carbonate or carbamate bond to the lipophilic region R on the one hand, to the RAc radical on the other hand, or directly to the S1, Q, or Z group.

[0130] Preferably, the amino acid whose radical is represented by RAc in the previously defined formula (III) is selected from aspartic acid, glutamic acid, alanine, arginine, asparagine, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan, and valine.

[0131] Preferably, the amino acid whose radical in the previously defined formula (III) is represented by RAc is selected from aspartic acid, glutamic acid, isoleucine, leucine, lysine, ornithine, glycine, and phenylalanine, more preferably selected from aspartic acid, glutamic acid, glycine, ornithine, and lysine, more preferably selected from aspartic acid, glutamic acid, isoleucine, leucine, lysine, and phenylalanine, more preferably selected from aspartic acid, glutamic acid, and lysine. However, this amino acid may also be selected from rarer amino acids, such as β-alanine, γ-aminobutyric acid, α-aminoadipic acid, hydroxyproline, hydroxylysine, phenylserine, α,β-diaminopimelic acid, ornithine, and any other modified amino acid, any amino acid is suitable because, by definition, it comprises two functional groups, one of which is a carboxylic acid and the other is an amine, allowing for covalent bonding to a spacer arm Gr or R on one side and to at least one S1 or Q or Z group on the other. Preferably, the selection of amino acids depends on the desired value to be given to s in formula (IV), in particular, to the extent that it must contain at least three functional groups to allow s to be equal to 2, while it is sufficient to contain only two functional groups for s to be equal to 1. Preferably, RAc is an amino acid radical belonging to the L type. However, RAc can also be a D type amino acid radical.

[0132] Preferably, in formula (IV), S1 can correspond to a molecular pattern represented as -W5-Y5-, and S2 can correspond to a pattern represented as -Y6-W6-, where the same or different Y5 and Y6 represent a crosslinked alkylene group containing 1 to 8 carbon atoms, preferably 1 to 4 carbon atoms, while the same or different W5 and W6 represent a -CO-, -NH-, or -O- group. In this case, preferably, S1 forms a covalent bond with an RAc, Gr, or R group on the one hand and with a D group on the other, and S2 forms a covalent bond with a D group on the one hand and with a Q or Z group on the other.

[0133] Preferably, in the previously defined formula (IV), D represents an ester (-CO-O-), carbonate (-OC(O)-O-), disulfide (-SS-), vinyl ether (-OC=C-), or acylhydrazone (CO-NR-N=CR'R) group, more preferably an ester (-CO-O-), disulfide (-SS-), vinyl ether (-OC=C-), or acylhydrazone (CO-NR-N=CR'R) group, with ester and disulfide groups being particularly preferred. In this case, preferably, these groups form a covalent bond with an S1 group on one side and with an S2 group on the other.

[0134] Preferably, Q corresponds to a molecular pattern represented as -CO-Y7-NH-Y8-N-[Y9-NH]2- or -NH-Y8-N-[Y9-NH]2-, where the same or different Y7, Y8, and Y9 represent a crosslinked alkylene group containing 1 to 8 carbon atoms, preferably 1 to 4 carbon atoms. Furthermore, Y7 preferably represents methylene, and Y8 and Y9 preferably represent a crosslinked alkylene group containing 1 to 4 carbon atoms, more preferably 2 carbon atoms. Thus, in this case, preferably, Q has three functional groups that enable it to form covalent bonds with S2 or RAc or Gr or R on the one hand, and with at least two Q and / or Z groups on the other hand.

[0135] Preferably, in the previously defined formula (IV), Z represents, on the one hand, an ionizable molecular pattern that can optionally form covalent bonds with Q or S2 or RAc or Gr or R, and on the other hand, one or two basic reactive functional groups (e.g., amines and alcohols) that can optionally form covalent bonds with one or two other Z groups.

[0136] An "ionizable molecular pattern" is preferably understood as a pattern of chemical functional groups that incorporate at least one nitrogen atom, are uncharged at physiological pH (pH = 7.0 to 7.4), and can become positively charged when the pH of the medium falls below the pKa value of the substance.

[0137] Preferably, the “ionizable molecular pattern” is understood to be a pattern of chemical functional groups that preferably incorporate at least one nitrogen atom, are chargeless at physiological pH (pH = 7.0 to 7.4), and exhibit one or more positive charges when the pH of the medium is in the range of 2.0 to 7.0, preferably 4.0 to 7.0, more preferably 4.5 to 7.0.

[0138] More preferably, the compound of formula (I) is selected from the following: [ka] JPEG2026523074000028.jpg208159JPEG2026523074000029.jpg195159JPEG2026523074000030.jpg210159 JPEG2026523074000031.jpg205159JPEG2026523074000032.jpg201159JPEG2026523074000033.jpg135159

[0139] This invention also relates to the following compounds: [ka] JPEG2026523074000035.jpg205159JPEG2026523074000036.jpg210159JPEG2026523074000037.jpg189159 JPEG2026523074000038.jpg201159JPEG2026523074000039.jpg174159JPEG2026523074000040.jpg199159

[0140] Another object of the present invention relates to a mixture comprising one or more lipids of the previously defined formula (I).

[0141] The lipids of formula (I) defined earlier can exist in the form of a racemic mixture.

[0142] A mixture containing the lipid of formula (I) as defined above may be concentrated with one diastereoma of the lipid of formula (I) as defined above. In particular, a mixture of lipids of formula (I) as defined above may have a diastereoma excess of at least 95%, at least 90%, at least 80%, or at least 70%.

[0143] A mixture containing the lipid of formula (I) as defined above may be concentrated with one enantioma of the lipid of formula (I) as defined above. In particular, a mixture of lipids of formula (I) as defined above may have an enantioma excess of at least 95%, at least 90%, at least 80%, or at least 70%.

[0144] A mixture containing the lipid of formula (I) defined earlier may be chiral pure. In particular, the lipid of formula (I) defined earlier may be in the form of a single optical isomer.

[0145] A mixture containing the lipid of formula (I) defined earlier may be concentrated with one optical isomer of the lipid of formula (I) defined earlier.

[0146] When a double bond (such as a carbon-carbon double bond or a carbon-nitrogen double bond) is present, isomerism (such as cis / trans or E / Z isomerism) can exist in the stereochemistry of the double bond. When the stereochemistry of a double bond is shown in a chemical structure, it is preferably understood that corresponding isomers may also exist. The amount of isomers present can vary depending on the relative stability of the isomers and the energy required for conversion between them. Therefore, in practice, some double bonds may exist in only a single stereochemistry, while others may exist as an inseparable equilibrium mixture of stereochemistrys (for example, when the relative stabilities are similar and the energy of conversion is low).

[0147] Lipids of formula (I) as defined above may contain one or more biodegradable groups. These biodegradable groups may contain one or more bonds that are susceptible to bond disruption reactions in a biological environment (e.g., organisms, organs, tissues, cells, or organelles). Examples of functional groups containing biodegradable bonds include, but are not limited to, esters, dithiols, and / or oximes.

[0148] The present invention also relates to a method for preparing a lipid of formula (I) as previously defined, and a method for preparing a mixture containing a lipid of formula (I) as previously defined.

[0149] Accordingly, the present invention also relates to a method for preparing a lipid of the previously defined formula (I) or a mixture comprising a lipid of the previously defined formula (I).

[0150] For example, a method for preparing a lipid of formula (I) as defined above, or a mixture containing a lipid of formula (I) as defined above, may include the following steps: - The step of bonding one or two chemical functional groups of a selected biocompatible template to a hydrophobic (or lipophilic) atomic group defined by formula (I) and / or (II); -Optionally, a step of introducing a spacer region using the remaining chemical functional groups of the biocompatible template, which now also includes a hydrophobic (or lipophilic) region. This spacer region can be defined according to formula (I) and / or (III) and may contain one or more environmentally sensitive chemical bonds or entities that can enhance lipophilicity, preferably tunable lipophilicity. This spacer may also contain chemical functional groups that can bond to the polar region previously defined in formula (I); -Finally, the step of introducing a polar region, preferably a tunable polar region, which acts as the hydrophilic portion of the entire chemical entity. This polar region can be constructed according to formula (IV) defined earlier. In the case of a spacer region pre-bonded to a biocompatible template, this polar region can be chemically bonded to the spacer region. If a spacer region is not present, the polar region can be directly bonded to the biocompatible template using one or two of its remaining chemical functional groups.

[0151] This process can be modified by switching its steps depending on the overall synthesis process selected to obtain the target lipid, preferably a tunable lipid. This means that, in some cases, for example, the polar region may be bonded first to the spacer region, and then this new entity may be bonded to a biocompatible template. Those skilled in the art can select a suitable process to obtain the lipid of formula (I) as defined above or a mixture containing the lipid of formula (I) as defined above.

[0152] Lipids of formula (I) as defined above, or mixtures containing lipids of formula (I) as defined above, may also be prepared by well-known organic synthesis methods, including the methods described in the examples of the present invention.

[0153] Examples of synthetic methods that may be used to synthesize lipids of formula (I) as defined above or mixtures containing lipids of formula (I) as defined above include, but are not limited to, coupling methods involving carbodiimides, such as diisopropylcarbodiimide or uronium salt-based coupling agents, such as HBTU, or nitrogen-based protecting group chemistry (e.g., Greene's Protective Groups in Organic Synthesis, TW. Greene et al., Wiley-Interscience New York City, 1999).

[0154] In particular, the lipids of formula (I) defined earlier may contain at least one protonable or deprotonable group such that the entire lipid may be positively charged at a first pH (3.0 to 6.9, preferably in the range of 4.5 to 6.9) below the physiological pH (pH in the range of 7.0 to 7.4), and neutral at a second pH higher than the first pH, preferably above the physiological pH. It will be understood by those skilled in the art that the addition or removal of protons as a function of pH is an equilibrium process, and that references to charged or neutral lipids refer to the properties of major species, and that not all lipids are required to exist in charged or neutral forms.

[0155] The lipid of formula (I) defined above may have two or more protonable or deprotonable groups, or it may be zwitterionic.

[0156] The pKa value of the lipid of formula (I) defined earlier may be lower than the pH observed in a physiological medium (e.g., a pH in the range of 7.0 to 7.4). Preferably, the lipid of formula (I) defined earlier has an overall pKa value in the range of 3.0 to 6.9, preferably 4.5 to 6.90.

[0157] In the present invention, the pKa value preferably corresponds to a dimensionless number related to the acid dissociation constant of the corresponding acid species, and is therefore a quantitative measure of the acidity of the chemical species in solution.

[0158] Preferably, the pKa values ​​of the lipids of formula (I) defined earlier are finely tuned to remain below the values ​​observed in physiological media (e.g., pH in the range of 7.0 to 7.4). Thus, at this pH, all lipids of formula (I) defined earlier can exist in solution, mostly in their deprotonated form (e.g., basic form). It will be understood by those skilled in the art that the pKa constant values ​​defined above can cover the acidity of the lipids of formula (I) defined earlier. Therefore, the present invention may also encompass lipids of formula (I) that have some protonable moieties (protonable lipids) and exist mainly in their neutral form (e.g., deprotonated form) at physiological pH.

[0159] Preferably, the protonable lipids according to the present invention have a pKa for protonable groups in the range of about 4 to about 7. Thus, such lipids are cationic at lower pH, but the modifiable lipids, preferably formulations containing modifiable lipids, are largely (but not completely) surface-neutralized at the physiological pH around pH 7.4. One advantage of choosing this value for pKa is that at least some nucleic acids associated with the outer surface lose their electrostatic interaction at the physiological pH and are removed by simple dialysis, thus significantly reducing the particle's sensitivity to clearance.

[0160] Advantageously, the lipids of formula (I) defined earlier can incorporate a neutral nitrogen-centered chemical function that allows them to be protonated at acidic pH, preferably strongly acidic pH, while remaining neutral at physiological pH.

[0161] Therefore, the lipid of formula (I) defined earlier may be in its free form, as well as its pharmaceutically acceptable salts and stereoisomers. Accordingly, the lipid of the present invention, preferably the adjustable lipid, may be in its protonated form, i.e., cationic form.

[0162] "Free lipids" are preferably understood as lipids in their non-salted form. Free lipids can be regenerated by treating lipid salts with a suitable dilute aqueous solution of base, such as an aqueous solution of NaOH, an aqueous solution of potassium carbonate, an aqueous solution of ammonia, and / or an aqueous solution of sodium bicarbonate, preferably a dilute aqueous solution of NaOH, a dilute aqueous solution of potassium carbonate, a dilute aqueous solution of ammonia, and / or a dilute aqueous solution of sodium bicarbonate.

[0163] pharmaceutically acceptable salts of lipids of formula (I) as defined above can be synthesized by conventional chemical methods from lipids of formula (I) that contain a basic or acidic moiety. In general, salts of basic lipids can be prepared by ion-exchange chromatography or by reacting a free base with a stoichiometric amount or excess of a desired salt-forming inorganic or organic acid in a suitable solvent or a variety of solvent combinations. Similarly, salts of acidic lipids can be formed by reaction with a suitable inorganic or organic base.

[0164] Examples of pharmaceutically acceptable salts of the lipids of formula (I) defined earlier include, but are not limited to, non-toxic salts of the lipids of formula (I) defined earlier, which are formed by reacting basic lipids with inorganic or organic acids.

[0165] Examples of non-toxic salts include, but are not limited to, those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid, as well as salts prepared from organic acids such as formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmo acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, and / or trifluoroacetic acid (TFA).

[0166] If the lipid of formula (I) defined earlier is an acidic lipid, a suitable “pharmaceutically acceptable salt” refers to a salt prepared from a pharmaceutically acceptable non-toxic base, including inorganic and organic bases. Salts prepared from pharmaceutically acceptable inorganic non-toxic bases include, but are not limited to, salts of aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganese(III), manganese(II), potassium, sodium, zinc, and mixtures thereof. Preferably, the base is selected from ammonium, calcium, magnesium, potassium, and / or sodium. Salts prepared from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, salts of primary amines, secondary amines, tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydravamin, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and mixtures thereof.

[0167] Under physiological conditions, the deprotonated acidic moieties, such as carboxyl groups, in a compound can be anionic, and their electronic charge may be internally balanced with the cation charge of the protonated or alkylated basic moiety; therefore, the lipids of formula (I) defined earlier can potentially be internal salts or zwitterions.

[0168] Preferably, if the lipid of formula (I) is a zwitterion, the deprotonated acidic moiety should have a pKa in the range of 4.5 to 7.0, and its negative electron charge does not cancel out the cation charge of the protonated or alkylated basic moiety in a medium having a pH value in the range of 4.5 to 7.0.

[0169] The lipids of formula (I) defined earlier can be formulated with one or more active ingredients into active supramolecular structures called "lipid particles."

[0170] Examples of lipid particles include, but are not limited to, liposomes, lipoplexes, micelles, and lipid picoparticles, microparticles, and / or nanoparticles.

[0171] A "liposome" is preferably understood as a structure having a lipid-containing membrane surrounding an aqueous interior.

[0172] A "lipoplex" is preferably understood as a system obtained by complexing a nucleic acid with a lipid composition containing one or more lipids. These complexes are typically obtained by adding a nucleic acid solution to a lipid dispersion, followed by gentle mixing and short incubation.

[0173] Therefore, another object of the present invention relates to lipid particles comprising one or more lipids of the previously defined formula (I) or to compositions comprising the previously defined lipid particles.

[0174] Therefore, the lipid particles and compositions containing lipid particles as defined above may contain one or more lipids of formula (I) as defined above.

[0175] The present invention also relates to a composition comprising a lipid of the previously defined formula (I).

[0176] A composition containing the lipid of formula (I) as defined above may further contain one or more active ingredients, and: - One or more neutral lipids, and / or - One or more components that can reduce aggregation of the composition, and / or - One or more sterols or sterol derivatives, and / or - One or more lipids having a targeted ligand It may also include

[0177] Neutral lipids, when present, can be any lipid species that exist in either an uncharged form or a zwitterionic form at physiological pH. Preferably, the neutral lipids contain saturated fatty acids with potentially mono- or di-unsaturated carbon chains having a carbon chain length in the range of C8 to C 22 and including saturated fatty acids with potentially mono- or di-unsaturated carbon chains having a carbon chain length in the range of C 10 to C 20It contains mono- or diunsaturated fatty acids having carbon chain lengths in the range of . More preferably, examples of neutral lipids include phospholipids, such as diacylphosphatidylethanolamine, diacylphosphatidylcholine, dihydrosphingomyelin, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine, 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), di Examples of these include, but are not limited to, myristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, dieryloylphosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE), lysophosphatidylcholine and dilinoleoylphosphatidylcholine, nonphosphorus-containing lipids such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine lauryl sulfate, alkylaryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, sphingomyelin, cerebrosides, cephalins, and mixtures thereof.

[0178] The neutral lipids that may be present in the composition of the present invention may include, or consist of, one or more phospholipids (such as cholesterol-free lipid compositions).

[0179] The sterols that may be present in the composition of the present invention may include or consist of cholesterol or its derivatives (such as lipid compositions not containing phospholipids).

[0180] Examples of components that can reduce the aggregation of the composition include polyethylene glycol (PEG)-modified lipids (PEG-modified lipids), polyethylene glycol (PEG) that may be substituted, conjugated, and / or coupled, hydrophilic polymers other than PEG, poloxamers, monosialoganglioside (Gml), polyamide oligomers (PAO), and mixtures thereof, but are not limited thereto.

[0181] Examples of PEG-modified lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (such as PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified 1,2-diacyloxypropane-3-amines, PEG-modified diacylglycerols and dialkylglycerols, mPEG (molecular weight = 2000)-diastearoylphosphatidylethanolamine (PEG-DSPE), mPEG (molecular weight = 2000)-dimyristoylphosphatidylethanolamine (PEG-DMPE), mPEG (molecular weight = 2000)-dipalmitoylphosphatidylethanolamine (PEG-DPPE), mPEG (molecular weight = 2000)-dioleoylphosphatidylethanolamine (PEG-DOPE), and 1-(monomethoxypolyethylene glycosides). Examples include, but are not limited to, PEG-2,3-dimyristoylglycerol (PEG-DMG), N-[(methoxypoly(ethylene glycol)2000 carbamyl]-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA), pegylated phosphatidylethanolamine (PEG-PE), 4-O-(2',3'-ditetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl)butanediolate (PEG-S-DMG), pegylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamates, such as ()-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl) carbamate or 2,3-di(tetradecanoxy)propyl-N-(c)-methoxy(polyethoxy)ethyl) carbamate, and mixtures thereof.

[0182] "Polyethylene glycol (PEG)" is preferably understood as a linear, water-soluble polymer of repeating ethylene glycol units having two terminal hydroxyl groups. PEGs are classified by their molecular weight and include, but are not limited to, monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol succinate (MePEG-S), monomethoxypolyethylene glycol succinimidyl succinate (MePEG-SNHS), monomethoxypolyethylene glycolamine (MePEG-NH), monomethoxypolyethylene glycol torecylate (MePEG-TRES), monomethoxypolyethylene glycol imidazolyl carbonyl (MePEG-IM), and such compounds containing terminal hydroxyl groups instead of terminal methoxy groups (e.g., HO-PEG-S, HO-PEG-S NHS, HO-PEG-NH), as well as mixtures thereof.

[0183] The PEG portion of the PEG conjugate lipid of the present invention may have an average molecular weight in the range of 550 daltons to 10,000 daltons, preferably in the range of 750 daltons to 5,000 daltons (for example, 1,000 daltons to 5,000 daltons, 1,500 daltons to 3,000 daltons, 750 daltons to 3,000 daltons, 750 daltons to 2,000 daltons), and more preferably 2,000 daltons or 750 daltons.

[0184] PEG may be substituted with alkyl, alkoxy, acyl, or aryl groups.

[0185] PEG can be directly conjugated to lipids or linked to lipids via a linker moiety. Any linker moiety suitable for coupling PEG to lipids can be used, including but not limited to non-ester-containing and ester-containing linker moieties. Preferably, the linker moiety is a non-ester-containing linker moiety.

[0186] Suitable non-esterified linker moieties include, but are not limited to, amides (-C(O)NH-), aminos (-NR-), carbonyls (-C(O)-), carbamates (-NHC(O)O), ureas (-NHC(O)NH), disulfides (-SS-), ethers (-O-), succinyls (-(O)CCHCHC(O-), succinamidyl (-NHC(O)CHCHC(O)NH-), and combinations thereof (such as linkers containing both carbamate and amide linker moieties). Preferably, a carbamate linker is used to couple PEG to lipids.

[0187] Suitable ester-containing linker moieties include, but are not limited to, carbonates (-OC(O)O-), succinoyl phosphate esters (-O-(O)POHO-), sulfonate esters, and combinations thereof.

[0188] Phosphatidylethanolamines having acyl chain groups with various chain lengths and degrees of saturation can be conjugated to PEG.

[0189] Such phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art. 10 ~C 20 Phosphatidylethanolamine containing saturated or unsaturated fatty acids having carbon chain lengths in the range of C can be used, or phosphatidylethanolamine containing mono- or diunsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can be used. Preferably, C 10 ~C 20Phosphatidylethanolamine containing saturated or unsaturated fatty acids having a carbon chain length within the range is used. More preferably, suitable phosphatidylethanolamines include, but are not limited to, dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), dioleoyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylethanolamine (DSPE), and mixtures thereof.

[0190] PEG can be coupled with the diacylglycerol (DAG) moiety. "Diacylglycerol" or "DAG" is preferably understood to be a compound having two aliphatic acyl chains each independently having 2 to 30 carbons bonded to the 1- and 2-positions of glycerol by ester linkages. The acyl chains may be saturated or may have various degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauroyl (C 12 ), myristoyl (C 14 ), palmitoyl (C 16 ), stearoyl (C 18 ), and / or icosanoyl (C 20 ). Preferably, the two acyl chains are the same, more preferably both acyl chains are myristoyl (such as dimyristoyl) or both are stearoyl (such as distearoyl).

[0191] PEG can be coupled with the dialkyloxypropyl (DAA) moiety. "Dialkyloxypropyl" or "DAA" is preferably understood to be a compound having two alkyl chains each independently having 2 to 30 carbons. The alkyl chains may be saturated or may have various degrees of unsaturation. Preferably, the PEG-DAA conjugate is a PEG-didecyloxypropyl (C 10 ) conjugate, a PEG-dilauryl oxypropyl (C 12 ) conjugate, a PEG-dimyristyloxypropyl (C 14) Conjugate, PEG-Dipalmityloxypropyl (C 16 ) Conjugate, or PEG-distearyloxypropyl (C 18 ) It is a conjugate.

[0192] When PEG is coupled, it preferably has an average molecular weight of 750 or 2,000 daltons.

[0193] Preferably, the terminal hydroxyl group of PEG is substituted with a methyl group, thereby generating a terminal methoxy group.

[0194] Components that can reduce aggregation of the composition may also be selected from other hydrophilic polymers other than PEG. Examples of suitable hydrophilic polymers other than PEG include, but are not limited to, polyglycerol, polysarcosine, polyphosphotriesters, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized cellulose such as hydroxymethylcellulose or hydroxyethylcellulose, and mixtures thereof, preferably polyglycerol, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized cellulose such as hydroxymethylcellulose or hydroxyethylcellulose, and mixtures thereof.

[0195] Components that can reduce aggregation of the composition may also be selected from the poloxamer family. A “poloxamer” is understood to be a well-known class of nonionic triblock copolymers, preferably consisting of a central hydrophobic chain of polyoxypropylene (PPO) [PEO-PPO-PEO] surrounded by two hydrophilic chains belonging to the polyethylene oxide (PEO) family. These poloxamers may be linear or X-shaped, the latter being known as poloxamines. An example of a poloxamer is meloxapole, also known in the literature as an “inverse poloxamer,” which presents a hydrophilic block based on polyethylene oxide polymer surrounded by two hydrophobic blocks based on polypropylene oxide polymer. Poloxamers may be named after manufacturer trade names such as “Pluronic,” “Symperonic,” or “Tetronic.” For example, suitable poloxamers for use as components that can reduce aggregation of a composition may be selected from F127, F108, F68, F87, P123, L61, L64, F88, or F98.

[0196] Poloxamers may be coupled to other components that can reduce aggregation, such as polyethylene glycol (PEG)-modified lipids (PEG-modified lipids) and / or polyethylene glycol (PEG) which may be substituted, conjugated and / or coupled as defined above.

[0197] Examples of monosialogangliosides (Gml) include, but are not limited to, gangliosides GD1b, GQ1b, GT1b, GD1a, GM4, and / or GD3.

[0198] Examples of polyamide oligomers (PAOs) include, but are not limited to, polyamide 6 dimers, polyamide 6 trimers, polyamide 6 tetramers, polyamide 6 cyclic dimers, polyamide 6 cyclic trimers, and / or polyamide 6 cyclic tetramers.

[0199] The composition may also contain one or more sterols or sterol derivatives, such as cholesterol and / or its derivatives. Examples of cholesterol derivatives include polar analogs such as 5C-cholestanol, 5C-coprostanol, cholesteryl-(2-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol, nonpolar analogs such as 5C-cholestane, cholestenone, 5C-cholestanone, 5C-coprostanone, and cholesteryl decanoate, and mixtures thereof, but are not limited thereto. Preferably, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)-butyl ether.

[0200] The composition may further comprise one or more lipids having a targeting ligand.

[0201] Examples of targeting ligands that can be conjugated to lipids include any peptide, protein, antibody, sugar, aptamer, organic and inorganic small molecules, such as RGDF peptide, A54 peptide, cRGD, Lyp-1 peptide, hEGF ligand, anti-CD44 antibody, HIV trans-activating transcription activation peptide, Ega1 nanobody, hyaluronic acid, galactose, mannose, transferrin, folate, and mixtures thereof, but are not limited thereto.

[0202] Examples of lipids that can be conjugated to a targeting ligand include phospholipids, such as dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), dioleoyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylethanolamine (DSPE), and mixtures thereof, or sterols, such as cholesterol, 5C-cholestanol, 5C-coprostanol, cholesteryl-(2-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol, and mixtures thereof, but are not limited thereto.

[0203] The composition may further contain one or more active ingredients, such as nucleic acids, polysaccharides, lipids, polynucleotides, morpholino oligonucleotides, aptamers, proteins, peptides, polypeptides, peptoids, organic or inorganic small molecules, any compound of a drug or pharmaceutical interest, an antigen, or a mixture thereof, in order to form picoparticles, microparticles, nanoparticles, liposomes, lipoplexes, or micelles having lipids of the previously defined formula (I).

[0204] More specifically, examples of drugs or any compounds of pharmaceutical interest include, but are not limited to, folate or sodium valproate, antibodies, antigens, lymphokines, interleukins, necrosis factors and apoptosis factors, interferons, growth factors, tissue plasminogen activator, factor VIIIc, erythropoietin, insulin, calcitosine, thymidine kinases, and combinations thereof.

[0205] The composition may further comprise adjuvants capable of specifically targeting determinants on and / or within cells. These adjuvants may be covalently or noncovalently bonded to the lipid of formula (I) or any other component in the composition comprising the lipid of formula (I).

[0206] Examples of adjuvants that can specifically target determinants on the surface and / or inside of cells include, but are not limited to, ligands of receptors expressed on the surface of target cells, e.g., sugars, folates, transferrin, insulin, hormones, prostaglandins, peptides, antibodies, nanobodies, metabolites, vitamins, or any other molecules capable of recognizing extracellular receptors, or elements of intracellular vectorization for targeting specific compartments, e.g., mitochondria, the nucleus, or the cytoplasm, e.g., nuclear, cytoplasmic, or mitochondrial localization signals, e.g., sugars, ligands or ligand fragments, hormones, peptides or polypeptides, antibodies (e.g., polyclonal antibodies, monoclonal antibodies, antibody fragments, humanized antibodies, recombinant antibodies, and recombinant human antibodies), proteins, cytokines, growth factors, apoptotic factors, metabolites, vitamins, aptamers, differentiation-inducing factors, cell surface receptors and their ligands, hormones, or any other molecules capable of recognizing extracellular receptors, as well as mixtures thereof. Adjuvants can also be fluorophores such as rhodamine, fluorescein, or biotin.

[0207] The composition may further include signaling molecules and molecules that may be involved in signaling pathways within target cells.

[0208] The lipids of formula (I) defined earlier may be combined with other lipid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, saccharides, glycerol, cyclodextrin, magnetic crystals or particles, particles based on organic or inorganic compounds, histones, deoxycholic acid, proteins, or combinations thereof to form the lipid particles defined earlier. Therefore, the compositions defined earlier may also include other lipid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, saccharides, glycerol, cyclodextrin, magnetic crystals or particles, particles based on organic or inorganic compounds, histones, deoxycholic acid, proteins, or combinations thereof.

[0209] The present invention also relates to a method for preparing a previously defined composition.

[0210] For example, a method for preparing the previously defined composition may include the following steps: - A step of preparing an organic or aqueous phase by dissolving one or more lipids of the present invention, preferably the modifiable lipids of the present invention, one or more neutral lipids as previously defined, one or more components that can reduce aggregation of the previously defined composition, and optionally one or more sterol derivatives as previously defined, and optionally one or more lipids having a previously defined targeted ligand, and optionally further components as previously defined. When preparing an organic phase, the selected organic solvent must be miscible with water and may include, but is not limited to, ethanol, tetrahydrofuran, and / or acetonitrile; - A step of preparing an organic or aqueous phase containing one or more previously defined active ingredients. Preferably, the pH of this organic or aqueous phase is controlled to be lower than the pKa of the lipid of formula (I) previously defined, preferably the adjustable lipid of formula (I) previously defined; - A step of mixing both phases to form a composition containing lipid particles that encapsulate one or more active ingredients delivered in a lipid structure.

[0211] Appropriate physical methods for enabling the correct mixed phase include, but are not limited to, microfluidic mixing, microinjection, extrusion, T-type mixed flow focusing, pressure-driven flow focusing, microfluidization, sonication, and combinations thereof.

[0212] Advantageously, active ingredients can be complexed using the nitrogen-containing portion of the lipid of formula (I) defined earlier, preferably the modifiable lipid of the present invention, thereby enhancing their delivery and preventing their degradation.

[0213] In particular, lipids of formula (I) as defined earlier, preferably tunable lipids of formula (I) as defined earlier, are especially attractive for delivering active ingredients for several reasons: they contain nitrogen-based groups for interacting with the active ingredient to be delivered, e.g., DNA, RNA or other polynucleotides, and other negatively charged active ingredients, e.g., folate or sodium valproate, or other negatively charged drugs of interest. They are also extremely useful for buffering pH, causing osmotic lysis of endosomes, and protecting the delivered drug. Furthermore, they can be synthesized from commercially available starting materials, and / or they can be pH-responsive and designed to have the correct broad range of desired pKas (4 to 6.9, preferably in the range of 4.5 to 6.9).

[0214] Therefore, using lipids and / or lipid particles of the previously defined formula (I), the active ingredient can be protected and delivered to the desired target site (such as a cell, tissue, and / or organ).

[0215] The active ingredients delivered by picoparticles, microparticles, nanoparticles, liposomes, lipoplexes, or micelles may be in gaseous, liquid, or solid form.

[0216] Preferably, the lipids of formula (I) defined earlier are biocompatible and biodegradable, and the picoparticles, microparticles, nanoparticles, liposomes, lipoplexes, or micelles formed as defined earlier are also biodegradable and biocompatible and can be used to provide controlled and sustained release of the active ingredient to be delivered.

[0217] The previously defined compositions may be combined with pharmaceutical excipients to form pharmaceutical compositions.

[0218] Therefore, another object of the present invention relates to a pharmaceutical composition comprising the previously defined composition and pharmaceutical excipients.

[0219] The present invention also relates to the first therapeutic use of lipids of the previously defined formula (I), lipid particles, and / or compositions.

[0220] In particular, lipids of formula (I) as defined earlier, lipid particles as defined earlier, and / or compositions as defined earlier can be used to treat and / or prevent and / or diagnose diseases and / or disorders.

[0221] Therefore, the present invention also relates to the use of a previously defined lipid of formula (I), a previously defined lipid particle, and / or a previously defined composition for the manufacture of pharmaceuticals.

[0222] The present invention also relates to lipids of the previously defined formula (I), lipid particles, and / or compositions for use as pharmaceuticals.

[0223] The present invention also relates to a method for treating and / or preventing and / or diagnosing a disease and / or disorder, comprising the use of a lipid of formula (I) as previously defined, a lipid particle as previously defined, and / or a composition as previously defined.

[0224] Examples of diseases and disorders include, but are not limited to, cancer, bacterial or viral infections, inflammatory and genetic disorders, and / or metabolic disorders such as diabetes.

[0225] Lipids of formula (I) as defined above, lipid particles as defined above, and / or compositions as defined above are particularly suitable for delivering active ingredients such as nucleic acids, polysaccharides, lipids, polynucleotides, morpholino oligonucleotides, aptamers, proteins, peptides, polypeptides, peptoids, small organic or inorganic molecules, drugs, or any compounds of pharmaceutical interest, antigens, or mixtures thereof, to subjects, organs, cells, and / or tissues.

[0226] The subject(s) may be living organisms such as humans(s), animals(s), and / or plants(s).

[0227] The tissues may be, but are not limited to, muscle tissue, connective tissue, epithelial tissue, or nerve tissue. Methods for obtaining samples from various tissues and for establishing primary and immortalized cell lines are well known in the art (see, for example, Freshney, RICulture of Animal Cells: A Manual of Basic Technique, Fifth Edition, 2005 Ed: John Wiley & Sons, Inc.).

[0228] Therefore, another object of the present invention relates to the use of lipids of the previously defined formula (I), previously defined lipid particles and / or previously defined compositions as vectors for delivering active ingredients such as nucleic acids (or more), polysaccharides (or more), lipids (or more), polynucleotides (or more), morpholino oligonucleotides (or more), aptamers (or more), proteins (or more), peptides (or more), polypeptides (or more), peptoids (or more), organic or inorganic small molecules (or more), drugs (or more), or any compound of pharmaceutical interest, antigens (or more), or mixtures thereof, to subjects (or more), organs (or more), cells (or more), and / or tissues (or more).

[0229] The present invention relates to lipids of the previously defined formula (I), previously defined lipid particles, and / or previously defined compositions for use as vectors for delivering active ingredients such as nucleic acids (multiple), polysaccharides (multiple), lipids (multiple), polynucleotides (multiple), morpholino oligonucleotides (multiple), aptamers (multiple), proteins (multiple), peptides (multiple), polypeptides (multiple), peptoids (multiple), organic or inorganic small molecules (multiple), drugs (multiple), or any compound of a pharmaceutical interest, antigens (multiple), or mixtures thereof, to subjects (multiple), organs (multiple), cells (multiple), and / or tissues (multiple).

[0230] Lipids of formula (I) defined earlier, lipid particles defined earlier, and / or compositions defined earlier can be used in vitro, ex vivo, and in vivo.

[0231] The present invention also relates to a method for delivering an active ingredient, such as nucleic acids, polysaccharides, lipids, polynucleotides, morpholino oligonucleotides, aptamers, proteins, peptides, polypeptides, peptoids, organic or inorganic small molecules, drugs, or any compound of a pharmaceutical interest, antigens, or mixtures thereof, to a target, organism, organ, cell, and / or tissue, comprising the step of contacting the active ingredient with a lipid of formula (I) as defined above, lipid particles as defined above, and / or a composition as defined above.

[0232] This method can be performed in vivo, ex vivo, or in vitro.

[0233] Lipids of formula (I) and / or lipid particles as defined above may be included in any pharmaceutical, cosmetic, veterinary, diagnostic, and / or research product composition.

[0234] Therefore, the previously defined compositions may be pharmaceutical, cosmetic, veterinary, diagnostic, and / or research product compositions.

[0235] Therefore, the present invention also relates to the use of the previously defined compositions in pharmaceutical, cosmetic, veterinary, diagnostic, and / or research products.

[0236] Lipids of formula (I) and / or lipid particles as defined above may also be used for other purposes, and for example, as materials for coatings, additives, excipients and / or bioengineering purposes.

[0237] Therefore, another object of the present invention relates to the use of lipids of the previously defined formula (I) and / or previously defined lipid particles as materials for coatings, additives, excipients and / or materials for bioengineering purposes.

[0238] Examples of materials for biotechnology purposes include, but are not limited to, filter membranes, synthetic biological tissues, and / or hydrogels.

[0239] The present invention also relates to a method for transfecting cells with nucleic acids and / or proteins and / or peptides and / or polysaccharides and / or lipids and / or small organic or inorganic molecules and / or any kind of bioactive molecule using lipids of formula (I) as previously defined, lipid particles and / or compositions as previously defined.

[0240] This method may include the step of contacting cells with a lipid of formula (I) as defined earlier, a lipid particle and / or a composition as defined earlier, and nucleic acids and / or proteins and / or peptides and / or polysaccharides and / or lipids and / or small organic or inorganic molecules and / or any kind of bioactive molecule.

[0241] The present invention also relates to transfected cells obtained by this method.

[0242] Transfected cells can be used in cell therapy, preferably cell gene therapy or CAR T cell therapy.

[0243] The present invention also relates to the use of lipids of the previously defined formula (I), lipid particles, and / or compositions for transfecting cells with nucleic acids and / or proteins and / or peptides and / or polysaccharides and / or lipids and / or small organic or inorganic molecules and / or any kind of bioactive molecule.

[0244] The present invention also relates to lipids of the previously defined formula (I), lipid particles, and / or compositions for transfecting cells with nucleic acids and / or proteins and / or peptides and / or polysaccharides and / or lipids and / or small organic or inorganic molecules and / or any kind of bioactive molecule.

[0245] According to the present invention, any type of cell culture material or cell culture method can be used on a two-dimensional (2D) or three-dimensional (3D) support to transfect cells called "target cells." Preferably, the 2D apparatus can be used to transfect cells in an adherent manner (cells adhere to the bottom of the wells), a suspension manner (cells remain suspended in the culture medium), or a co-culture (two or more different cell types separated or not separated by an insert), for example, treated or untreated cell culture plates and dishes (1536-6 well cell culture plates, 35 mm, 60 mm, 90 mm, 100 mm Petri dishes) or cell flasks (e.g., 25 cm²). 2 75cm 2 or 150cm 2The present invention is defined as a support that enables cultivation in a cell culture medium. The present invention may also be applied to 3D matrices such as hydrogels or solid 3D scaffolds of various compositions such as collagen, atelocollagen, glycosane, polystyrene, hyaluronic acid, polycaprolactone and / or polyethylene glycol, used as cell culture materials or in cell culture methods.

[0246] Lipids of formula (I) as defined earlier, lipid particles as defined earlier, and / or compositions as defined earlier may also be used in combination with molecules known to affect the transfection procedure. Examples of molecules known to affect the transfection procedure include, but are not limited to, recombinant or non-recombinant polypeptides, liposomes, cationic lipids, cationic polymers such as polyethyleneimine, protamine sulfate, polybrene, poly-L-lysine, DEAE dextran, polylactic acid-coglycolic acid, chitosan, amphiphilic polymers such as chemically modified or unmodified poloxamers, polyethylene glycol and its derivatives, anionic polymers such as starch and hyaluronic acid, and combinations thereof. These molecules known to affect the transfection procedure can be used before, during, and / or after transfection. Therefore, these molecules known to affect the transfection procedure can be added to cells from one week to four weeks before and / or after transfection, more preferably from one day to seven days before and / or after transfection.

[0247] The present invention also relates to the use of magnetic-based particles, preferably magnetic-based nanoparticles, such as iron-based nanoparticles, in the in-cellulose delivery of a previously defined active ingredient, together with a previously defined lipid of formula (I), a previously defined lipid particle, and / or a previously defined composition.

[0248] The present invention also relates to a method for in-cellulose delivery of a previously defined active ingredient, using magnetic-based particles, preferably magnetic-based nanoparticles, such as iron-based nanoparticles, together with a previously defined lipid of formula (I), a previously defined lipid particle, and / or a previously defined composition.

[0249] When the lipid of formula (I) as previously defined, the lipid particles and / or the composition as previously defined are used in combination with magnetic-based particles, preferably magnetic-based nanoparticles, a permanent magnetic field, an oscillating magnetic field, or an electrically mediated magnetic field can be applied to the experiment after the cells, the lipid of formula (I) as previously defined, the lipid particles and / or the composition as previously defined, and the magnetic-based nanoparticles have been brought into contact. This process can be continued for 10 seconds to 96 hours, preferably 1 minute to 48 hours, and more preferably 5 minutes to 4 hours.

[0250] Preferably, the previously defined composition further comprises nucleic acids as active ingredients for forming nucleic acid-lipid particles, preferably nucleic acid-lipid nanoparticles, together with the lipid of formula (I) previously defined.

[0251] Nucleic acid-lipid nanoparticles may also contain other previously defined active ingredients, such as drugs or any compound of the previously defined pharmaceutical interest.

[0252] The active components of nucleic acid-lipid particles can be used as templates for generating one or more target proteins.

[0253] Nucleic acids can effectively encode polypeptides of pharmaceutical interest that, during their expression in host cells, enable the improvement of dysfunction in recipient organisms.

[0254] As a result, the compositions of the present invention can be used in in vitro and in vivo research and development or in vivo and ex vivo gene therapy and / or nucleic acid-based therapy and / or cell therapy.

[0255] Nucleic acids can also effectively encode polypeptides that can generate or induce an immune response against them in humans or animals.

[0256] As a result, the compositions of the present invention find particular applications in gene therapy and / or cell therapy, such as vaccines, and in immunotherapy, particularly immunotherapy for treating or preventing cancer or bacterial or viral infections.

[0257] Accordingly, the present invention also relates to previously defined compositions for use in gene therapy and / or nucleic acid-based therapy and / or cell therapy, for example, in the field of vaccines and immunotherapy.

[0258] Examples of cell therapies include, but are not limited to, cell gene therapy and / or CAR T cell therapy.

[0259] In particular, the present invention relates to previously defined compositions for use in treating and / or preventing cancer, bacterial or viral infections, inflammatory and hereditary disorders, and metabolic disorders such as diabetes.

[0260] The present invention also relates to the use of previously defined compositions for treating or preventing cancer, bacterial or viral infections, inflammatory and genetic disorders, and metabolic disorders such as diabetes.

[0261] The present invention also relates to methods for treating or preventing cancer, bacterial or viral infections, inflammatory and genetic disorders, and metabolic disorders such as diabetes, comprising administering a previously defined composition.

[0262] Lipids of formula (I) as defined earlier, lipid particles as defined earlier, and / or compositions as defined earlier can exhibit an extended circulatory life after intravenous (iv) injection and can accumulate at distal sites (including sites physically separated from the administration site), and therefore can be used for systemic application.

[0263] Lipids of formula (I) as defined earlier, lipid particles as defined earlier, and / or compositions as defined earlier may be used for topical, cutaneous, oral, rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, intracerebral, subcutaneous, intraocular, transdermal, intratracheal, intravitreal and / or intraperitoneal administration, preferably topical, cutaneous, oral, rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, intratracheal and / or intraperitoneal administration.

[0264] The present invention also relates to a kit comprising a lipid of formula (I) as previously defined, a lipid particle as previously defined and / or a composition as previously defined, further comprising nucleic acids and / or proteins and / or peptides and / or polysaccharides and / or lipids and / or small organic or inorganic molecules and / or any kind of bioactive molecule, and optionally comprising instructions for use. [Brief explanation of the drawing]

[0265] [Figure 1] Reaction scheme for synthesizing molecule I: i) oleylamine, DIC, HOBt, DMF / CHCl3; ii) piperidine, DMF; iii) dimethylglycine (DMG), pyBOP, DIPEA, DMF / DCM. [Figure 2] Reaction scheme for synthesizing molecule II: i) N,N-dimethylaminobutyric acid, TCFH, methylimidazole, MeCN / DCM. [Figure 3] Reaction scheme for synthesizing molecule III: i) oleylamine, DIC, HOBt, DMF / CHCl3; ii) piperidine, DMF; iii) dimethylaminobutyric acid (DMABA), DIC, HOBt, DMF / CHCl3. [Figure 4]Reaction scheme for synthesizing molecule IV: i) diethylaminoethanol, disuccinimidyl carbonate, Et3N, MeCN; ii) 1b, Et3N, DCM. [Figure 5] Reaction scheme for synthesizing molecule V: i) Boc2O, NaOH, H2O / t-BuOH; ii) 3-aminopropylimidazole, HBTU, DIPEA, DMF / DCM; iii) TFA, DCM; iv) Linoleic acid, HBTU, DIPEA, DMF / DCM. [Figure 6] Reaction scheme for synthesizing molecule VI: i) 3-aminopropylimidazole, COMU, DIPEA, DMF; ii) TFA, DCM; iii) 2-hexyldecanoic acid, COMU, DIPEA, DMF; iv) piperidine, DMF; v) linoleic acid, HBTU, DIPEA, DMF. [Figure 7] Reaction scheme for synthesizing molecule VII: i) p-TsOH, toluene reflux, Dean Stark; ii) N,N-dimethylglycine, TCFH, methylimidazole, MeCN / DCM. [Figure 8] Reaction scheme for synthesizing molecule VIII: i) N,N-dimethylaminobutyric acid, TCFH, methylimidazole, MeCN / DCM. [Figure 9] Reaction scheme for synthesizing molecule 9a: i) phthalimide, PPh3, DIAD, THF; ii) NH2NH2, 70°C; iii) concentrated HCl, 70°C. [Figure 10] Reaction scheme for synthesizing molecule IX: i) 9a, DIC, HOBt, Et3N, DMF / CHCl3; ii) piperidine; iii) DMABA, pyBOP, DIPEA, DMF / DCM. [Figure 11] Reaction scheme for synthesizing molecule X: i) Oleic acid, HBTU, DIPEA, HOBt, DCM / DMF. [Figure 12] Reaction scheme for synthesizing molecule XI: i) Lauric acid, HBTU, DIPEA, HOBt, DMF. [Figure 13]Reaction schemes for synthesizing molecule XII: i) 9a, DIC, HOBt, Et3N, DMF / CHCl3; ii) piperidine; iii) DMABA, pyBOP, DIPEA, DMF / DCM. [Figure 14] Reaction scheme for synthesizing molecule XIII: i) DMG, HBTU, DIPEA, HOBt, DCM / DMF; ii) TFA, DCM; iii) DIC, HOBt, Et3N, DCM / DMF; iv) TFA, DCM, room temperature; v) Linoleic acid, HBTU, HOBt, DIPEA, DMF / DCM. [Figure 15] Reaction scheme for synthesizing molecule 14d: i) DIC, HOBt, Et3N, DCM / DMF; ii) TFA, DCM; iii) N,N-dimethylaminobutyric acid, TCFH, methylimidazole, MeCN / DCM; iv) TBAF, THF 0°C; v) DIC, HOBt, Et3N, DCM / DMF, vi) TFA, DCM. [Figure 16] Reaction scheme for synthesizing molecule XIV: i) Linoleic acid, HBTU, DIPEA, HOBt, DMF / DCM. [Figure 17] Reaction scheme for synthesizing molecule 15d: d: i) Boc2O, EtOH, 97%; ii) Lauric acid, DIC, HOBt, Et3N, DCM / DMF; iii) Trifluoroacetic acid (TFA), DCM; iv) Fmoc-Glu(OH), DIC, HOBt, Et3N, DCM / DMF; piperidine, room temperature. [Figure 18] Reaction scheme for synthesizing molecule XV: i) N,N-dimethylglycine, HBTU, DIPEA, DMF. [Figure 19] Reaction scheme for synthesizing molecule XVI: i) (Boc)-Orn(Boc)OH, DIC, HOBt, Et3N, DCM / DMF; ii) TFA, DCM; iii) DMABA, HBTU, DIPEA, DMF. [Figure 20] Reaction scheme for synthesizing molecule XVII: i) PyBOP, DIPEA, DCM / DMF; ii) 3-(dimethylamino)propane-1,2-diol, ppTs, toluene reflux. [Figure 21]Reaction scheme for synthesizing molecule XVIII: i) NaBH4 EtOH; ii) N,N-dimethylaminobutyric acid, TCFH, methylimidazole, MeCN / DCM. [Figure 22] Reaction scheme for synthesizing molecule XIX: i) 2-{[dimethyl(2-methyl-2-propanyl)silyl]oxy}-1,3-propanediol, p-TsOH, toluene / THF; ii) TBAF, THF, 0°C; iii) imidazole, MeCN; iv) TCFH, methylimidazole, MeCN / DCM. [Figure 23] Reaction scheme for synthesizing molecule 20b: b) i) Boc2O, NaOH, t-BuOH, H2O; ii) (Z)-2-nonen-1-ol; DIC, HOBt, Et3N, DCM / DMF; iii) TFA, DCM; iv) α-ketoglutaric acid, PyBOP, DIPEA. [Figure 24] Reaction scheme for synthesizing molecule XX: i) 2-{[dimethyl(2-methyl-2-propanyl)silyl]oxy}-1,3-propanediol; ii) TBAF, THF, 0°C; iii) N,N-dimethylaminobutyric acid, TCFH, methylimidazole, MeCN / DCM. [Figure 25] Reaction scheme for synthesizing molecule XXI: i) NaBH4 EtOH; ii) DSC, Et3N, DCM; iii) TBDMSCl, imidazole, DMF; iv) Et3N, DCM; v) TBAF, THF. [Figure 26] Reaction scheme for synthesizing molecule XXII: i) N,N-dimethylethylenediamine, TCFH, methylimidazole, MeCN / DCM; ii) TFA, DCM; iii) 4,6-heptadecadiic acid, HBTU, HOBt, DIPEA, DMF. [Figure 27] Reaction scheme for synthesizing molecule XXIII: i) 20a, COMU, DIPEA, DMF; ii) TFA, DCM; iii) Tetradecylamine, DIC, HOBt, Et3N, DMF; iv) Piperidine, DMF; v) DMG, TCFH, 1-methylimidazole, MeCN / DCM. [Figure 28]Reaction scheme for synthesizing molecule XXIV: i) dodecylamine, HOBt, DIC, DMF / CHCl3; ii) piperidine; iii) 20a, pyBOP, DIPEA, DMF; iv) piperidine; v) DMABA, 1-methylimidazole, TCFH, MeCN. [Figure 29] Reaction scheme for synthesizing molecule XXV: i) Fmoc-Orn(Boc)-OH, HOBt, DIC, Et3N, DMF / DCM; ii) Piperidine; iii) BocOrn(Boc)OH, HBTU, DIPEA, DMF; iv) TFA, DCM; v) Linoleic acid, pyBOP, DIPEA, DMF / DCM. [Figure 30] Reaction scheme for synthesizing molecule XXVI: i) 4-(1-pyrrolidinyl)-1-butanol, DCC, DMAP, DCM; ii) piperidine, DMF; iii) BocOrn(Boc)OH, HBTU, DIPEA, DMF / DCM; iv) TFA, DCM; v) linoleic acid, pyBOP, DIPEA, DMF. [Figure 31] Reaction scheme for synthesizing molecule XXVII: i) ethanolamine, EDC·HCl, HOBt, DMF; ii) TFA, DCM; iii) DMABA, HBTU, DIPEA, DMF; iv) Fmoc-Orn(Boc)-OH, DCC, DMAP, Et3N; v) piperidine, DMF; vi) Boc-Orn(Boc)-OH, HBTU, DIPEA, DMF; vii) TFA, DCM; viiii) linoleic acid, pyBop, DIPEA, DMF / DCM. [Figure 32] Reaction scheme for synthesizing molecule XXVIII: i) DIC, HOBt, Et3N, DCM / DMF; ii) TFA, DCM; iii) Linoleic acid, HBTU, DIPEA, HOBt, DMF / DCM. [Figure 33] Reaction scheme for synthesizing molecule XXIX: i) DIC, HOBt, Et3N, DCM / DMF; ii) TFA, DCM; iii) Linoleic acid, HBTU, DIPEA, HOBt, DMF / DCM. [Figure 34]Reaction scheme for synthesizing molecule XXX: i) DIC, HOBt, Et3N, DCM / DMF; ii) TFA, DCM; iii) BocOrn(Boc)OH, DIC, HOBt, Et3N, DCM / DMF; iv) TFA, DCM; v) Linoleic acid, PyBOP, DIPEA, DMF. [Figure 35] Reaction scheme for synthesizing molecule 31a: i) 3-butyl-2-hepten-1-ol, DCC, DMAP, DCM; ii) TFA, DCM. [Figure 36] Reaction scheme for synthesizing molecule XXXI: i) 31a, DIC, HOBt, DMF / CHCl3; ii) piperidine, DMF; iii) DMABA, HBTU, DIPEA, DMF. [Figure 37] Reaction scheme for synthesizing molecule 32c: i) Boc2O, EtOH; ii) 2-hexyldecanoic acid, DCC, DMAP, DCM; iii) TFA, DCM; iv) FmocGluOH, DIC, HOBt, DMF, CHCl3; v) NaN3, DMF, 50°C. [Figure 38] Reaction schemes for synthesizing molecules XXXII, XXXIII, and XXXIV: i) DMABA, HBTU, DIPEA, DMF / DCM; ii) Propylene oxide, MeOH, 60°C; iii) DMG, HBTU, DIPEA, DMF / DCM. [Figure 39] Reaction scheme for synthesizing molecule XXXV: i) Hexadecylamine, DIC, HOBt, DMF / CHCl3; ii) TFA, Et3Si, DCM; iii) 32b, COMU, DIPEA, DMF; iv) Piperidine, DMF; v) DMG, HBTU, DIPEA, DMF / DCM. [Figure 40] Reaction schemes for synthesizing molecules XXXVI and XXXVII: i) malic acid, EDC, Cl, HOBt, NET3, DMF / DCM; ii) DMABA, EDC, DMAP, DCM; iii) DMG, EDC, DMAP, DCM. [Figure 41]Reaction scheme for synthesizing molecule 38c: i) succinic anhydride, NEt3, DCM / DMF; ii) 2-hexyldecanol, DCC, DMAP, DCM; iii) TFA, DCM; iv) FmcoGluOH, DIC, HOBt, NEt3, DMF / DCM; v) NaN3, DMF, 50°C. [Figure 42] Reaction scheme for synthesizing molecules XXXVIII and XXXIX: i) DMG, HBTU, DIPEA, DMF / DCM; ii) Propylene oxide, MeOH, 60°C. [Figure 43] Reaction scheme for synthesizing molecule XL: i) succinic anhydride, DMAP, DCM / DMF; ii) 32b, COMU, DIPEA, DMF; iii) TFA, DCM; iv) 40a, COMU, DIPEA, DMF; v) piperidine, DMF; vi) DMG, HBTU, DIPEA, DMF. [Figure 44] Reaction scheme for synthesizing molecule XLI: i) 3-(dimethylamino)-1,2-propanediol, I2, PPh3, imidazole, DCM; ii) TFA, DCM; iii) 32b, COMU, DIPEA, DMF; iv) NaN3, DMF; v) Dodecanoic acid, PyBOP, DIPEA, DMF. [Figure 45] Evaluation of LNP mRNA formulations in transfection procedures using mRNA encoding GFP protein: HeLa cell lines were transfected with escalating doses of mRNA (0.1–20 μg) formulated in lipid nanoparticles based on adjustable lipids I and XIV, or with mRNA complexed within the same range using a commercially available transfection reagent (Tr Reag). [Figure 45A] After 24 hours of incubation, the percentage of GFP-positive cells was evaluated by flow cytometry. [Figure 45B] After 24 hours of incubation, the percentage of surviving, unpermeabilized cells was assessed by flow cytometry. [Figure 46]Evaluation of LNP mRNA formulations in transfection procedures using mRNA encoding GFP protein: Jurkat T cell lines were transfected with escalating doses of mRNA (0.1–20 μg) formulated in lipid nanoparticles based on adjustable lipids I and XIV, or with mRNA complexed within the same range using a commercially available transfection reagent (Tr Reag). [Figure 46A] After 24 hours of incubation, the percentage of GFP-positive cells was evaluated by flow cytometry. [Figure 46B] After 24 hours of incubation, the percentage of surviving, unpermeabilized cells was assessed by flow cytometry. [Figure 47] Evaluation of LNP mRNA formulations in transfection procedures using mRNA encoding the GFP protein: PANC-1 cancer cell lines were transfected with escalating doses of mRNA (0.1–2 μg) formulated in lipid nanoparticles based on adjustable lipids I, XXXIV, XXXV, XXXVIII, and XXXVI. After 24 hours of incubation, the percentage of GFP-positive cells was assessed by flow cytometry. [Figure 48] Evaluation of LNP mRNA formulations in transfection procedures using mRNA encoding the GFP protein: RAW264.7 macrophage cell lines were transfected with escalating doses of mRNA (1–20 μg) formulated in lipid nanoparticles based on adjustable lipids I, XXXIV, XXXV, and XXXVIII. After 24 hours of incubation, the percentage of GFP-positive cells was assessed by flow cytometry. [Figure 49] Evaluation of LNP mRNA formulations in transfection procedures using mRNA encoding the GFP protein: THP-1 monocyte cell lines were transfected with escalating doses of mRNA (1–20 μg) formulated in lipid nanoparticles based on adjustable lipids I, XXXIV, XXXVI, and XXXVIII. After 24 hours of incubation, the percentage of GFP-positive cells was evaluated by flow cytometry. [Figure 50A]Bioluminescence signals 3 hours after ip administration of three mRNA-LNP preparations in nude mice: F-Luc mRNA-LNP I, F-Luc mRNA LNP XIV, and F-Luc mRNA LNP DOTAP. [Figure 50B] Dynamics of bioluminescence signals over 25 hours after ip administration of two mRNA-LNP preparations: F-Luc mRNA-LNP I and F-Luc mRNA LNP XIV (a dose equivalent to 10 μg of mRNA) in nude mice. [Figure 51] Relative gene expression from firefly luciferase in different organs using modifiable lipids. [Figure 51A] Relative gene expression in mouse-derived liver tissue using 10 μg of Luc-mRNA for F-Luc-mRNA I, F-Luc-mRNA VI, and F-Luc-mRNA XIV in IP. [Figure 51B] Relative gene expression in mouse-derived lung tissue using 10 μg of Luc-mRNA for F-Luc-mRNA I, F-Luc-mRNA VI, and F-Luc-mRNA XIV in IP. [Figure 51C] Relative gene expression in mouse-derived kidney tissue using 10 μg of Luc-mRNA for F-Luc-mRNA I, F-Luc-mRNA VI, and F-Luc-mRNA XIV in IP. [Figure 51D] Relative gene expression in mouse-derived spleen tissue using 10 μg of Luc-mRNA for F-Luc-mRNA I, F-Luc-mRNA VI, and F-Luc-mRNA XIV in IP. [Figure 52] Relative gene expression from firefly luciferase in different organs 6 hours after injection using adjustable lipids: relative gene expression in mouse-derived lung, liver, and spleen tissues using F-Luc-mRNA I, F-Luc-mRNA XXXIII, and F-Luc-mRNA XXXIV, and 10 μg of Luc-mRNA; after IV administration of three mRNA-LNP preparations. [Figure 53]Humoral responses to OVA subunit vaccine (10 μg) and mRNA(OVA)-LNP (mRNA dose equal to 10 μg based on adjustable lipids I and XIV) in C57BL6J mice, either in the absence or in the presence of vaccine adjuvant (alum 1X) after subcutaneous prime boost injection (D0-14). [Figure 53A] Monitoring of total anti-OVA IgG production over time. [Figure 53B] Monitoring of anti-OVA IgG1 production over time. [Figure 53C] Monitoring of anti-OVA IgG2c production over time. [Figure 54A] Th1 humoral biomarker (IgG2c / IgG1) against OVA subunit vaccine (10 μg) and mRNA(OVA)-LNP (mRNA dose equal to 10 μg based on adjustable lipids I and XIV) in the absence or presence of vaccine adjuvant (alum 1X) during subcutaneous prime boost injection (D0-14) in C57BL6J mice. [Figure 54B] CD8+ T cell response to OVA subunit vaccine (10 μg) and mRNA(OVA)-LNP (mRNA dose equal to 10 μg based on adjustable lipids I and XIV) in the absence or presence of vaccine adjuvant (alum 1X) during subcutaneous prime boost injection (D0-14) in C57BL6J mice. [Figure 55] Evaluation of LNP DNA formulations in transfection procedures using DNA encoding the F-Luc protein: Jurkat T cell lines were transfected with incremental doses of DNA (0.1–10 μg) formulated into lipid nanoparticles based on adjustable lipid XI, or complexed within the same range using a commercially available transfection reagent (MTX reagent, OZ Biosciences). After 48 hours of incubation, the luminescence of lysed transfected cells was evaluated using the Luciferase Assay Kit (OZ Biosciences). [Figure 56]Confocal micrographs of fluorescently labeled LNPs encapsulating Cy5-labeled NTsiRNAs using adjustable lipid XI after 4 hours of contact with HEK-293 cells. Cell nuclei were labeled with Hoechst dye, siRNAs with cyanine 5, and LNPs with DiO. The left image shows LNP internalization after 4 hours of exposure. The right image shows siRNA-Cy5 uptake. [Figure 57] Evaluation of LNP siRNA formulations in transfection procedures using GFP-encoding siRNA: HEK-293 cell lines modified to stably express GFP were transfected with escalating doses of siRNA (5 nM to 100 nM) formulated into lipid nanoparticles based on lipids I and XI, adjustable in two different N / P ratios (4 and 6), or conjugated within the same range using commercially available transfection reagents (Lullaby, OZ Biosciences SAS). After 72 hours of incubation, mean GFP fluorescence was evaluated by flow cytometry. [Figure 58] Evaluation of LNP siRNA formulations in transfection procedures using GFP-encoding siRNA: THP-1 cell lines modified to stably express GFP were transfected with escalating doses of siRNA (5 nM to 100 nM) formulated into lipid nanoparticles based on lipids I and XI, adjustable in two different N / P ratios (4 and 6), or conjugated within the same range using commercially available transfection reagents (Lullaby, OZ Biosciences SAS). After 72 hours of incubation, the mean GFP fluorescence was evaluated by flow cytometry. [Modes for carrying out the invention]

[0266] Examples I. Materials and Methods: a) Material Most solvents and reagents were obtained from VWR Prolabo (Briare, France), Sigma-Aldrich SA (St Quentin-Fallavier, France), TCI Europe NV (Zwijndrecht, Belgium), and Bachem Biochimie SARL (Voisin-le-Bretonneux, France). Unless otherwise specified, all reagents were purchased at reagent grade and used without further purification. Unless otherwise specified, all amino acids used in this study were derived from natural amino acids (i.e., L-isomers).

[0267] b) Method Thin-layer chromatography (TLC) is performed on an aluminum plate coated with silica gel 60 Fasa (Merck). Compounds are colored under UV light (254 nm) by immersion in ninhydrin coloring solution (0.2% in butanol) for compounds with nitrogen-based functional groups, followed by heating at 150°C, or by immersion in cerium / concentrated molybdate coloring solution (90 / 10 / 15 / 1) followed by heating at 110°C for sulfur-containing compounds. The synthesized products are purified using a silica chromatography column. Flash chromatography separation is performed using silica gel 60 (230-400 mesh ASTM) (Merck). Particle size and zeta potential measurements: Mean hydrodynamic particle size and charge were measured in Grade 2 water using the Malvern Nano ZS instrument and DTS software (Malvern Instruments, UK) with dynamic light scattering (DLS) and laser Doppler velocimetry (LDV), respectively. Various amounts of cationic poloxamer were diluted in 100 μl of water and mixed with an equal volume of the same water containing DNA. Measurements were performed in automated mode, and results are presented as mean + / - SEM, n=3. Each mean represents the average of 30 measurements.

[0268] Synthesis of molecule I 2-amino-N,N'-bis[(Z)-octadeca-9-enyl]pentanediamide (1b) Dissolve Fmoc-glutamic acid (10 mmol; 3.69 g) in 80 mL of anhydrous DMF under inert atmosphere with stirring. Continuously add DIC (32 mmol; 4.96 mL) and then HOBt (32 mmol; 4.32 g) in 20 mL of anhydrous DMF to the reaction medium. Stir the reaction mixture for 3 hours, then add oleylamine (30 mmol; 11.6 mL) in 3100 mL of anhydrous CHCl dropwise to the reaction medium. After 36 hours, add 30 mL of piperidine and stir the reaction medium for a further 4 hours. Stop the reaction and evaporate the reaction medium to dryness under crude vacuum. Return the residue to 100 mL of  and heat until dissolved. Place the solution in a Falcon tube at -20°C. Wash the formed precipitate several times by centrifugation with cold  and Et2O and dry under high vacuum. Obtain a white solid in 93% yield (9.3 mmol; 6.0 g). TLC:Rf=0.4(DCM:MeOH 95 / 5(v / v)).

[0269] 2-[[2-(dimethylamino)acetyl]amino]-N,N'-bis[(Z)-octadeca-9-enyl]pentanediamide(I) N,N'-dimethylglycine (0.28 mmol; 29 mg) was placed in a 25 mL flask under an inert atmosphere and then dissolved in 2 mL of anhydrous DMF. DIPEA (0.63 mmol; 109 μL) and pyBOP (0.3 mmol; 156 mg) were successively added to the solution. The reaction medium was stirred at ambient temperature for 15 minutes under an inert atmosphere, and then a solution of 1b (0.25 mmol; 162 mg) in 2 mL of anhydrous DMF and 2 mL of anhydrous DCM was added to the reaction medium. After 24 hours, the reaction was stopped and the reaction medium was evaporated to dryness under high vacuum. The coupling product was then purified by flash chromatography on silica gel (elution gradient of 4-10% MeOH in DCM). In this way, the product was isolated in 34% yield (0.171 mmol; 125 mg). TLC: Rf = 0.2 (DCM: MeOH 95 / 5 (v / v)).

[0270] Synthesis of molecule II 2-[4-(dimethylamino)butanoylamino]-N'-[(Z)-heptadeca-9-enyl]-N-[(Z)-octadeca-9-enyl]pentanediamide(II) Under argon, with vigorous stirring, dissolve N,N-dimethylaminobutyric acid (DMABA, 40 mg, 0.23 mmol) in 4 mL of a dry 1 / 2 (v / v) mixed solution of MeCN and DCM already containing methylimidazole (74 μL, 0.92 mmol). After complete dissolution, add 2 mL of a DCM solution containing compound 1b (0.25 mmol, 161 mg) with stirring. After cooling the mixture to 0°C, introduce chloro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate (TCFH) (98 mg, 0.35 mmol) all at once. After addition, warm the reaction mixture at room temperature for 12 hours, then concentrate. The resulting residue is then resuspended in 15 mL of ethyl acetate, washed twice with water and once with brine, dried over sodium sulfate, filtered, and concentrated. The product was recovered by flash chromatography on silica gel using a DCM / methanol mixture as the eluent. A gradient in the range of 98 / 2 to 90 / 10 (DCM / MeOH, v / v) was used. 149 mg (0.2 mmol, 85% yield) of sticky solid was recovered. TLC (DCM / MeOH: 95 / 5) rf: 0.3.

[0271] Synthesis of molecule III 2-amino-N,N'-bis[(Z)-octadeca-9-enyl]butanediamide (3a) Compound 3a was isolated by coupling Fmoc-aspartic acid (2.8 mmol; 1 g) with oleylamine (8.4 mmol; 3.26 mL) using DIC (8.1 mmol; 1.26 mL), HOBt (9.25 mmol; 1.25 g), and then piperidine (8 mL) in DMF (30 mL) and CHCl3 (30 mL), following the procedure described for compound 1b. Reaction time: 96 hours at room temperature; yield: 68% (1.9 mmol; 1.2 g, white solid; TLC: Rf = 0.4 (DCM: MeOH 95 / 5 (v / v)).

[0272] 2-[4-(dimethylamino)butanoylamino]-N,N'-bis[(Z)-octadeca-9-enyl]butanediamide(III) DMABA (0.25 mmol; 162 mg) was placed in a dry 25 mL flask under an inert atmosphere and then dissolved in 2.5 mL of anhydrous DMF with stirring. DIC (0.75 mmol; 116 μL) and HOBt (0.75 mmol; 101 mg) were successively added to the reaction medium. After stirring the reaction medium at ambient temperature under an inert atmosphere for 3 hours, a solution of 3a (0.25 mmol; 158 mg) in 2.5 mL of anhydrous DCM was added to the reaction medium together with triethylamine (0.88 mmol; 122 μL). After 48 hours, the reaction was stopped and the reaction medium was evaporated to dryness under high vacuum. The coupling product was then purified by flash chromatography using silica gel (elution gradient of 4-10% MeOH in DCM). In this way, the product was isolated in 12% yield (0.03 mmol; 22 mg). TLC:Rf=0.3(DCM:MeOH 95 / 5(v / v)).

[0273] Synthesis of Molecular IV 2-(diethylamino)ethyl-N-[4-[[(Z)-heptadeca-9-enyl]amino]-1-[[(Z)-octadeca-9-enyl]carbamoyl]-4-oxo-butyl]carbamate(IV) N,N'-disuccinimidyl carbonate (1.5 mmol; 384 mg) is added with stirring to a solution of diethylaminoethanol (1 mmol; 132 μL) in 5 mL of anhydrous MECN. After 5 minutes, triethylamine (3 mmol; 420 μL) is added, and the reaction medium is stirred under an inert atmosphere and at ambient temperature until all alcohol is consumed. Once the reaction is complete, the solvent is evaporated, and the product is dried under high vacuum. The residue is returned to 5 mL of anhydrous DCM while adding 1b (0.5 mmol; 323 mg) and triethylamine (2 mmol; 140 μL) to the reaction medium. After stirring under an inert atmosphere at ambient temperature for 12 hours, 10 mL of DCM is added, and the organic layer is washed with aqueous solution of NH2Cl and brine. The organic layer is dried over MgSO4 and evaporated to dryness under high vacuum. The residue is purified by flash chromatography on silica gel (elution gradient of 2-8% MeOH in DCM). In this manner, the product was isolated in 15% yield (0.08 mmol; 59 mg). TLC: Rf = 0.3 (DCM: MeOH 95 / 5 (v / v)).

[0274] Synthesis of molecule V 5-amino-2-(tert-butoxycarbonylamino)pentanoic acid / (Boc-Orn(Boc)OH(5a)) Dissolve NaOH (24.7 mmol; 987 mg) in 25 mL of water in a 100 mL flask. Add a solution of ornithine hydrochloride (11 mmol; 1.9 g) in 8 mL of t-BuOH to the reaction medium, and then add a solution of Boc2O (23.7 mmol; 5.2 g) also dissolved in 8 mL of t-BuOH to the reaction medium. Add Boc2O dropwise to the first solution. Stir at ambient temperature for 16 hours, then add 50 mL of petroleum ether and extract the aqueous layer three times with 50 mL of ether. Wash the organic layer twice with NaHCO3. Acidify the pH of the aqueous phase to 2 with concentrated HCl and extract again five times with 100 mL of Et2O. Wash the organic extract with water and brine, dry with MgSO4, and evaporate to dryness under high vacuum. The obtained oil was crystallized with petroleum ether, filtered, the filtrate evaporated, and crystallized again to obtain a white powder in 85% yield (20.1 mmol; 6.7 g).

[0275] 5-((3-(1H-imidazole-1-yl)propyl)amino)-5-oxopentan-1,4-diaminium(5b) 3-aminopropylimidazole (2 mmol; 238 μL) is placed in a dry 25 mL flask under an inert atmosphere and then dissolved in 10 mL of anhydrous DMF with stirring. 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (1.5 mmol; 569 mg) and DIPEA (2 mmol; 340 μL), dissolved in 20 mL of DCM, are continuously added to the reaction medium. The reaction mixture is then maintained at ambient temperature under an inert atmosphere for 15 minutes, after which a solution of compound 8a (1.5 mmol, 500 mg) in 1 mL of anhydrous DCM and 3 mL of DMF is added to the reaction medium. Reaction time: 48 hours at room temperature. The crude product is returned to 50 mL of  and washed three times with water and brine. The organic layer is dried over MgSO4 and evaporated. The product was crystallized in petroleum ether, dried, and then dissolved in 60 mL of DCM. 3 mL of trifluoroacetic acid was added to the reaction medium. The reaction mixture was then maintained overnight at ambient temperature with stirring. The reaction medium was evaporated to dryness under crude vacuum. The final residue was returned to DCM four times consecutively and to Et2O four times, followed by evaporation to dryness. The reaction was quantitative (1.5 mmol, 701 mg).

[0276] (9Z,12Z)-N-[5-(3-imidazole-1-ylpropylamino)-4-[[(9Z,12Z)-octadeca-9,12-dienoyl]amino]-5-oxopentyl]octadeca-9,12-dienamide(V). Molecule V was isolated by coupling linoleic acid (1.5 mmol, 468 μL) with compound 5b (0.6 mmol; 280 mg) using HBTU (1.5 mmol; 569 mg) and DIPEA (3 mmol; 509 μL) dissolved in 6 mL of DCM and 4 mL of DMF, following the procedure described for molecule 5b. Reaction time: 48 hours at room temperature; Purification: Silica gel chromatography gradient 98 / 2~94 / 6 (DCM / MeOH, v / v). Yield: 45% (206 mg, 0.27 mmol, TLC (DCM / MeOH: 90 / 10) Rf: 0.3).

[0277] Synthesis of molecule VI 5-((3-(1H-imidazole-1-yl)propyl)amino)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-oxopentan-1-aminium(6a) Fmoc-ornithine (Boc)-OH (1 mmol, 454 mg) is placed in a dry 50 mL flask under an inert atmosphere, and then dissolved in 10 mL of anhydrous DMF with stirring. 1-Cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU 1.1 mmol; 471 mg) and DIPEA (2 mmol; 346 μL) are continuously added to the reaction medium. The reaction mixture is then maintained under an inert atmosphere and at 0°C for 15 minutes, after which 3-aminopropylimidazole (1.1 mmol; 131 μL) is added to the reaction medium with stirring. The solution is left overnight under an inert atmosphere and then heated to ambient temperature. The reaction medium is evaporated to dryness under crude vacuum. The crude product is returned to 50 mL of ELISA and washed with 1 M HCl, NaHCO3, and brine. The organic layer is dried over MgSO4 and evaporated. The product is used without further purification. TLC: Rf = 0.4 (DCM: MeOH 90 / 10 (v / v)).

[0278] The product is returned to 50 mL of DCM and 2 mL of TFA is added. The reaction mixture is then maintained overnight at ambient temperature with stirring. The reaction medium is evaporated to dryness under crude vacuum. The final residue is returned to DCM four times consecutively and to Et2O four times, then evaporated to dryness. The reaction is quantitative; yield 66% (2 steps, 380 mg, 0.66 mmol).

[0279] N-[4-amino-5-(3-imidazole-1-ylpropylamino)-5-oxopentyl]-2-hexyl-decanamide(6b) 2-hexyldecanoic acid (1.5 mmol, 440 μL) was placed in a dry 50 mL flask under an inert atmosphere and then dissolved in 15 mL of anhydrous DMF with stirring. COMU (1.5 mmol; 642 mg) and DIPEA (3 mmol; 519 μL) were successively added to the reaction medium. The reaction mixture was then maintained at 0°C for 15 minutes under an inert atmosphere, and then 6a (570 mg) dissolved in 5 mL of anhydrous DMF was added to the reaction medium with stirring. The solution was left overnight under an inert atmosphere and then heated to ambient temperature. The reaction medium was evaporated to dryness under crude vacuum. The crude product was returned to 50 mL of  and washed with 1 M HCl, NaHCO3 and brine. The organic layer was dried over MgSO4 and purified by flash chromatography on silica gel (elution gradient of 2-5% MeOH in DCM). The intermediate product was thus isolated in 80% yield (0.8 mmol; 560 mg). TLC:Rf=0.5(DCM:MeOH 90 / 10(v / v)).

[0280] Return the product to 20 mL of DMF and add 5 mL of piperidine. Then, maintain the reaction mixture overnight at ambient temperature with stirring. Evaporate the reaction medium to dryness under coarse vacuum. Polish the residue several times in ether, filter it, and use it as is.

[0281] (9Z,12Z)-N-[4-(2-hexyldecanoylamino)-1-(3-imidazole-1-ylpropylcarbamoyl)butyl]octadeca-9,12-dienamide(VI) Molecule VI was isolated by coupling linoleic acid (4 mmol, 1.1 g) with crude compound 6b (0.8 mmol; 382 mg) using HBTU (4 mmol; 1.5 mg) and DIPEA (6 mmol; 1 mL) dissolved in 20 mL of DMF, following the procedure described for molecule 5b. Reaction time: 48 hours at room temperature; Purification: Silica gel chromatography gradient 96 / 4~90 / 10 (DCM / MeOH, v / v). Yield: 22% (130 mg, 0.18 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.4).

[0282] Synthesis of molecule VII Bis[(9Z,12Z)-octadeca-9,12-dienyl]2-aminopentanedioate (7a) L-glutamic acid (220 mg, 1.5 mmol), linoleyl alcohol (1.16 mL, 3.75 mmol), and p-toluenesulfonic acid hydrate (715 mg, 3.75 mmol) are dissolved in dry toluene (30 mL) with stirring. The flask is equipped with a Dean-Stark trap and a condenser. The reaction mixture is then stirred under reflux for 18 hours, cooled, and concentrated. The residue is then returned to DCM and washed twice with saturated sodium bicarbonate solution, twice with water, and once with brine. The organic extract is dried over magnesium sulfate, filtered, and concentrated under vacuum. The product is recovered by flash chromatography on silica gel using a gradient of petroleum spirit / ethyl acetate (100 / 0 v / v ~ 70 / 30 v / v) mixture as the eluent. 322 mg (0.5 mmol, 30% yield) of colorless oil is recovered. TLC (PS / EA: 80 / 20) rf: 0.4.

[0283] Bis[(9Z,12Z)-octadeca-9,12-dienyl]-2-[[2-(dimethylamino)acetyl]amino]pentanedioate(VII) Molecule VII was isolated by coupling DMG (24 mg, 0.23 mmol) with compound 7a (0.25 mmol, 161 mg) using methylimidazole (74 μL, 0.92 mmol) and TCFH (98 mg, 0.35 mmol) dissolved in 4 mL of a 2 / 1 (v / v) solution of MeCN and DCM, following the procedure described for Molecule II. Reaction time: 12 hours at 0°C to room temperature; Purification: Silica gel chromatography gradient 98 / 2 to 90 / 10 (DCM / MeOH, v / v). Yield: 72% (sticky solid 124 mg, 0.17 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.2).

[0284] Synthesis of molecule VIII Bis[(9Z,12Z)-octadeca-9,12-dienyl]2-[4-(dimethylamino)butanoylamino]pentanedioate(VIII) Molecule VIII was isolated by coupling DMABA (40 mg, 0.23 mmol) with compound 7a (0.25 mmol, 161 mg) using methylimidazole (74 μL, 0.92 mmol) and TCFH (98 mg, 0.35 mmol) dissolved in 4 mL of a 2 / 1 (v / v) solution of MeCN and DCM, following the procedure described for Molecule II. Reaction time: 12 hours at 0°C to room temperature; Purification: Silica gel chromatography gradient 98 / 2 to 90 / 10 (DCM / MeOH, v / v). Yield: 72% (sticky solid 136 mg, 0.180 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.2).

[0285] Synthesis of molecule IX [(9Z,12Z)-Octadeca-9,12-Dienyl]ammonium chloride (9a) A solution of linoleyl alcohol (7.6 mmol; 2.02 g) and diisopropyl azodicarboxylate (12 mmol; 6.4 mL) in 14 mL of THF was added to a solution of PPh3 (9 mmol, 2.36 g) and phthalimide (9 mmol; 1.32 g) in 10 mL of THF over 20 minutes at 0°C. The reaction medium was left overnight to rise to ambient temperature. After the complete conversion of the alcohols, hydrazine hydrate (2 mL, 50 wt%) was added, and the resulting solution was heated under reflux for 6 hours. The solution was cooled to ambient temperature, and 10 mL of concentrated HCl was slowly added. The reaction medium was then refluxed for 2 hours, then cooled to ambient temperature, and stirred overnight. The precipitate was removed by filtration, and the solvent was removed under vacuum. The residual solid was dissolved in water and extracted three times with 40 mL of DCM. The aqueous layer was concentrated to obtain ammonium in 53% yield (4.02 mmol; 1.21 g).

[0286] 2-amino-N,N'-bis[(9Z,12Z)-octadeca-9,12-dienyl]pentanediamide(9b) Compound 9b was obtained in two steps by first reacting Fmoc-glutamic acid (0.6 mmol; 222 mg) with compound 9a (1.5 mmol; 453 mg) in the presence of DIC (1.92 mmol; 297 μL), HOBt (1.92 mmol; 259 mg), and Et3N (3 mmol; 408 μL) dissolved in 6 mL of DMF and 36 mL of CHCl. ​​Then, piperidine (3 mL) was added after 36 hours. Reaction time: 40 hours at room temperature; yield: 84% (white solid, 0.5 mmol; 380 mg), TLC (DCM / MeOH: 95 / 5) Rf: 0.2).

[0287] 2-[4-(dimethylamino)butanoylamino]-N,N'-bis[(9Z,12Z)-octadeca-9,12-dienyl]pentanediamide(IX) Following the procedure described for compound I, molecule IX was obtained by coupling DMABA (0.3 mmol; 50 mg) with compound 9b (0.2 mmol; 129 mg) using DIPEA (0.6 mmol; 100 μL) and pyBOP (0.3 mmol; 156 mg) dissolved in 4 mL of DMF. Reaction time: 24 hours at room temperature; Purification: Silica gel chromatography gradient 98 / 2~92 / 8 (DCM / MeOH, v / v). Yield: 45% (0.09 mmol; 68 mg), TLC (DCM / MeOH: 95 / 5) Rf: 0.3).

[0288] Synthesis of molecule X (Z)-N-[5-(3-imidazole-1-ylpropylamino)-4-[[(Z)-octadeca-9-enoyl]amino]-5-oxopentyl]octadeca-9-enamide(X) Oleic acid (251 μL, 0.8 mmol) is dissolved under argon in 4 mL of DMF solution already containing DIPEA (175 μL, 1 mmol) and HOBt (122 mg, 0.9 mmol). After dissolution, 4 mL of DCM solution containing compound 5b (0.25 mmol, 117 mg) is added with stirring. The mixture is cooled to 0°C, and then HBTU (341 mg, 0.9 mmol) is gradually introduced over 30 minutes. After addition, the reaction mixture is warmed to room temperature for 12 hours, stirred for a further 36 hours, and then concentrated. The resulting residue is then resuspended in 30 mL of ethyl acetate and left at -20°C for 3 hours. The resulting liquid and solid are then separated by centrifugation at 3000 rpm, and this procedure is repeated twice. All liquids are collected, discarding the solid, and concentrated under vacuum. The product is recovered by flash chromatography using silica gel with the DCM / methanol mixture as the eluent. A gradient in the range of 99 / 1 to 90 / 10 (DCM / MeOH, v / v) was used. 102 mg (0.133 mmol, yield 53%) of a white, creamy solid was recovered. TLC (DCM / MeOH: 95 / 5) rf: 0.2.

[0289] Synthesis of Molecular XI N-[4-(dodecanoylamino)-5-(3-imidazole-1-ylpropylamino)-5-oxopentyl]dodecanamide(XVI) Following a similar protocol to that for compound X, but using lauric acid (0.8 mmol, 160 mg), compound XI is obtained as a white powder in 68% yield (0.17 mmol, 103 mg). TLC (DCM / MeOH: 95 / 5) rf: 0.25.

[0290] Synthesis of molecule XII 2-amino-N,N'-bis[(9Z,12Z)-octadeca-9,12-dienyl]butanediamide (12a) Compound 12a was obtained in two steps by first reacting Fmoc-aspartic acid (0.6 mmol; 213 mg) with compound 9a (2 mmol; 604 mg) in the presence of DIC (1.92 mmol; 297 μL), HOBt (1.92 mmol; 259 mg), and Et3N (3 mmol; 408 μL) dissolved in 6 mL of DMF and 36 mL of CHCl. ​​Then, piperidine (3 mL) was added after 36 hours. Reaction time: 40 hours at room temperature; yield: 69% (white solid, 0.41 mmol; 260 mg), TLC (DCM / MeOH: 90 / 10) Rf: 0.4).

[0291] 2-[4-(dimethylamino)butanoylamino]-N,N'-bis[(9Z,12Z)-octadeca-9,12-dienyl]butanediamide(XII) Following the procedure described for compound I, molecule XII was obtained by coupling DMABA (0.6 mmol; 101 mg) with compound 12a (0.4 mmol; 260 mg) using DIPEA (1.2 mmol; 207 μL) and pyBOP (0.6 mmol; 312 mg) dissolved in 8 mL of DMF. Reaction time: 24 hours at room temperature; Purification: Silica gel chromatography gradient 98 / 2~92 / 8 (DCM / MeOH, v / v). Yield: 33% (0.13 mmol; 97 mg), TLC (DCM / MeOH: 95 / 5) Rf: 0.3).

[0292] Synthesis of molecule XIII 2-[2-[[2-(dimethylamino)acetyl]amino]ethyldisulfanyl]ethylammonium (13a) DMG (1 mmol, 103 mg) was placed in a 50 mL flask under argon, and then dissolved in 10 mL of DMF with stirring. HBTU (1 mmol; 379 mg) and DIPEA (2 mmol; 346 μL) were successively added to the reaction medium. The reaction mixture was then maintained at room temperature under an inert atmosphere for 15 minutes, after which 4 mL of DCM and a solution of Boc-cystamine (0.83 mmol; 186 mg) in 2 mL of DMF were added to the reaction medium. After stirring at room temperature under argon for 16 hours, the reaction was stopped, and the reaction medium was evaporated to dryness under crude vacuum. The crude product was returned to 50 mL of  and washed three times with water and brine. The organic layer was dried over MgSO4 and evaporated. The product was used without further purification (TLC: Rf = 0.2 (DCM: MeOH 90 / 10 (v / v))).

[0293] Next, the purified product is dissolved in 40 mL of DCM, and then 2 mL of trifluoroacetic acid is added to the reaction medium. The reaction mixture is then maintained at room temperature with stirring for 12 hours. The reaction medium is evaporated to dryness under vacuum. The final residue is returned to DCM four times and to Et2O four times in succession, and then evaporated to dryness. Yield 83% (2 steps, 0.83 mmol, 291 mg).

[0294] [5-[2-[2-[[2-(dimethylamino)acetyl]amino]ethyldisulfanyl]ethylamino]-5-oxo-4-[(2,2,2-trifluoroacetyl)ammonio]pentyl]-(2,2,2-trifluoroacetyl)ammonium(13b) Boc-L-Orn(Boc)-OH (365 mg, 1.1 mmol) is dissolved in 10 mL of a 9 / 1 v / v mixture of dry DCM / DMF under argon in a three-necked round-bottom flask equipped with a dropping funnel. Subsequently, HOBt (162 mg, 1.2 mmol) and DIC (201 μL, 1.3 mmol) are added, and the reaction mixture is stirred at room temperature for 3 hours to increase the turbidity of the medium. Then, compound 13a (351 mg, 1 mmol) and Et3N (210 μL, 1.5 mmol), dissolved in 10 mL of a 9 / 1 v / v mixture of dry DCM / DMF, are added dropwise through a dropping funnel over 30 minutes. After the addition is complete, the reaction mixture is stirred at room temperature for 36 hours to concentrate to dryness. The crude mixture is then resuspended in 20 mL of ethyl acetate and left at -20°C for 3 hours. Next, the obtained liquid and solid are separated by centrifugation at 3000 rpm, and this procedure is repeated three times. The solid residue is collected, then washed three times with diethyl ether, and dried overnight under vacuum.

[0295] The crude compound is dissolved in 25 mL of dry DCM under argon. Then, 190 μL (2.5 mmol) of trifluoroacetic acid (TFA) is slowly added, and the reaction proceeds until complete conversion is demonstrated by TLC (2 hours). The mixture is then concentrated and co-evaporated three times with 20 mL of DCM, and three times with 20 mL of diethyl ether (Et2O), and dried overnight under high vacuum. The product is then used without further purification (0.63 mmol, 345 mg, 63%).

[0296] (9Z,12Z)-N-[5-[2-[2-[[2-dimethylamino)acetyl]amino]ethyldisulfanyl]ethylamino]-4-[[(9Z,12Z)-octadeca-9,12-dienoyl]amino]-5-oxopentyl]octadeca-9,12-dienamide(XIII) Following the procedure described for compound X, molecule XIII was obtained by coupling linoleic acid (404 μL, 1.3 mmol) with compound 13b (0.63 mmol, 345 mg) using DIPEA (263 μL, 1.5 mmol), HOBt (183 mg, 1.35 mmol), and HBTU (530 mg, 1.4 mmol) dissolved in 6 mL of DMF and 6 mL of DCM. Reaction time: 18 hours at 0°C to room temperature; Purification: Silica gel chromatography gradient 99 / 1 to 90 / 10 (DCM / MeOH, v / v). Yield: 78% (white solid 429 mg, 0.49 mmol, TLC (DCM / MeOH: 90 / 10) Rf: 0.4).

[0297] Synthesis of molecule XIV a) Synthesis of molecule 14d 2,5-Diamino-N-[2-[tert-butyl(dimethyl)silyl]oxyethyl]pentanamide bis-trifluoroacetate (14a) Following the procedure described for compound 13b, molecule 14a was obtained in two steps by first coupling Boc-L-Orn(Boc)-OH (3.65 g, 11 mmol) with 2-[[tert-butyl(dimethyl)silyl]oxy]ethylamine (2.04 mL, 10 mmol) using HOBt (1.62 g, 12 mmol), DIC (2.01 mL, 13 mmol), and Et3N (2.10 mL, 15 mmol) dissolved in 10 mL of DMF and 90 mL of DCM. Deprotection was carried out using 1.9 mL (25 mmol) of TFA in 250 mL of DCM. Reaction time: 72 hours at room temperature, followed by 6 hours at room temperature. Yield: 71% (3.45 g, 7.1 mmol white solid).

[0298] N-[2-[tert-butyl(dimethyl)silyl]oxyethyl]-2,5-bis[4-(dimethylamino)butanoylamino]pentanamide(14b) Molecule 14b was isolated by coupling DMABA (400 mg, 2.4 mmol) with compound 14a (485 mg, 1 mmol) using methylimidazole (650 μL, 8 mmol) and TCFH (1.13 g, 4 mmol) dissolved in 50 mL of a 2 / 1 (v / v) solution of MeCN and DCM, following the procedure described for Molecule II. Reaction time: 18 hours at 0°C to room temperature; Purification: Silica gel chromatography gradient 98 / 2 to 90 / 10 (DCM / MeOH, v / v). Yield: 82% (white solid 423 mg, 0.82 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.5).

[0299] 2,5-Bis[4-(dimethylamino)butanoylamino]-N-(2-hydroxyethyl)pentanamide(14c) Compound 14b (258 mg, 0.5 mmol) was dissolved in 12 mL of dry THF. Then, tetrabutylammonium fluoride (TBAF, 1 M, 2 mmol, 2 mL in THF) was introduced, and the reaction mixture was stirred at 0°C for 2 hours. After concentrating the medium, the resulting residue was resuspended in 15 mL of ethyl acetate, washed twice with water and once with brine, dried over sodium sulfate, filtered, and concentrated to obtain the pure compound in 95% yield (0.48 mmol, 193 mg).

[0300] [1-[2-[2,5-bis[4-(dimethylamino)butanoylamino]pentanoylamino]ethoxycarbonyl]-4-[(2,2,2-trifluoroacetyl)ammonio]butyl]-(2,2,2-trifluoroacetyl)ammonium(14d) Following the procedure described for compound 13b, molecule 14d was obtained in a two-step sequence by first coupling Boc-L-Orn(Boc)-OH (92 mg, 0.275 mmol) with compound 14c (100 mg, 0.25 mmol) using HOBt (41 mg, 0.3 mmol), DIC (50.1 μL, 0.33 mmol), and Et3N (53 μL, 0.375 mmol) dissolved in 3 mL of a 9 / 1 v / v mixture of dry DCM / DMF. Deprotection was performed after intermediate purification using 76 μL (1 mmol) of TFA in 2.5 mL of DCM. Reaction time: 36 hours at room temperature, followed by 2 hours at room temperature. Yield: 79% (140 mg, 0.197 mmol colorless syrup).

[0301] b) Synthesis of molecule XIV 2-[2,5-bis[4-(dimethylamino)butanoylamino]pentanoylamino]ethyl-2,5-bis[[(9Z,12Z)-octadeca-9,12-dienoyl]amino]pentanoate(XIV).

[0302] Following the procedure described for compound X, molecule XIV was obtained by coupling linoleic acid (81 μL, 0.33 mmol) with compound 14d (0.125 mmol, 88.8 mg) using DIPEA (70 μL, 0.3 mmol), HOBt (45 mg, 0.33 mmol), and HBTU (106 mg, 0.28 mmol) dissolved in 2 mL of DMF and 2 mL of DCM. Reaction time: 36 hours at 0°C to room temperature; Purification: Silica gel chromatography gradient 98 / 2 to 90 / 10 (DCM / MeOH, v / v). Yield: 83% (white solid 108 mg, 0.104 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.2).

[0303] Synthesis of molecule XV a) Synthesis of molecule 15d tert-butyl N-(4-hydroxybutyl)carbamate (15a) Dissolve 4-aminobutan-1-ol (10 mmol, 922 μL) in 20 mL of anhydrous ethanol. Cool the mixture to 0°C. Then, while stirring at 0°C, add 2.3 mL (10 mmol) of Boc2O dropwise over 30 minutes. Allow the reaction to proceed for 48 hours, then concentrate the reaction mixture under vacuum. Redissolve the oily residue in 20 mL of a 1 / 1 (v / v) mixture of DCM and water. Slip the phase with a funnel and extract the aqueous layer three times with 5 mL of DCM. Wash the combined organic extract with brine, dry over magnesium sulfate, filter, and concentrate to dryness. Use the oil without further purification (yield 97%, 1.84 g, 9.7 mmol). TLC (DCM / MeOH: 95 / 5) rf: 0.7.

[0304] 4-(tert-butoxycarbonylamino)butyldodecanoate (15b) Compound 15b is obtained by coupling lauric acid (3.3 mmol, 661 mg) with compound 15a (3 mmol, 567 mg) using a solution of DIC 557 μL (3.6 mmol), HOBt 486 mg (3.6 mmol), and triethylamine (546 μL, 3.9 mmol) in 56 mL of DCM and 4 mL of DMF, following the procedure described in Molecular III. The crude mixture is concentrated and then resuspended in 40 mL of ethyl acetate and left at -20°C for 3 hours. The resulting liquid and solid are then separated by centrifugation at 3000 rpm, and this procedure is repeated three times. All liquids are collected and concentrated under vacuum. The product is recovered by flash chromatography with silica gel using a petroleum spirit / ethyl acetate (80 / 20 v / v) mixture as the eluent. 781 mg (2.1 mmol, yield 71%) of colorless oil is recovered. TLC (PS / EA: 80 / 20) rf: 0.5.

[0305] 4-Dodecanoyloxybutylammonium trifluoroacetate (15c) Compound 15b (781 mg, 2.1 mmol) is dissolved in 40 mL of dry DCM under argon. Then, 2 equivalents (4.2 mmol, 321 μL) of trifluoroacetic acid (TFA) are slowly added, and the reaction is allowed to proceed until complete conversion is demonstrated by TLC (2 hours). The mixture is then concentrated and co-evaporated three times with 20 mL of DCM, and three times with 20 mL of diethyl ether (Et2O), and dried overnight under high vacuum. The product is then used without further purification (2.08 mmol, 768 mg, 99%).

[0306] 4-[[4-amino-5-(4-dodecanoyloxybutylamino)-5-oxo-pentanoyl]amino]butyl dodecanoate (15d) Compound 15d is obtained by first coupling N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-glutamic acid (Fmoc-Glu-OH, 259 mg, 0.7 mmol) with compound 15c (768 mg, 2.08 mmol) using HOBt (331 mg, 2.45 mmol), DIC (325 μL, 2.1 mmol), and Et3N (394 μL, 2.8 mmol) dissolved in chloroform (7 mL) and DMF (7 mL), and then removing the Fmoc group using piperidine (2 mL), following the two-step procedure described for molecule 1b. The crude mixture is then concentrated and resuspended in 40 mL of ethyl acetate and left at -20°C for 3 hours. The resulting liquid and solid are then separated by centrifugation at 3000 rpm, and this procedure is repeated three times. All liquids are collected, discarding the solid, and concentrated under vacuum. The product was recovered by flash chromatography using silica gel with a DCM / methanol mixture as the eluent. A gradient in the range of 98 / 2 to 94 / 6 (DCM / MeOH, v / v) was used. 163 mg (0.25 mmol, 36% yield) of a white viscous solid was recovered. TLC (DCM / MeOH: 95 / 5) rf: 0.2.

[0307] b) Synthesis of molecule XV 4-[[4-[[2-(dimethylamino)acetyl]amino]-5-(4-dodecanoyloxybutylamino)-5-oxo-pentanoyl]amino]butyl dodecanoate (XV) Following the procedure described for compound X, molecule XV was obtained by coupling DMG (34 mg, 0.325 mmol) with compound 15d (0.25 mmol, 163 mg) using DIPEA (90 μL, 0.5 mmol), HOBt (50 mg, 0.375 mmol), and HBTU (142 mg, 0.375 mmol) dissolved in 2 mL of DMF and 2 mL of DCM. Reaction time: 36 hours at 0°C to room temperature; Purification: Silica gel chromatography gradient 99 / 1 to 92 / 8 (DCM / MeOH, v / v). Yield: 48% (white solid 89 mg, 0.12 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.4).

[0308] Synthesis of molecule XVI 4-[[4-[2,5-bis(tert-butoxycarbonylamino)pentanoylamino]-5-(4-dodecanoyloxybutylamino)-5-oxo-pentanoyl]amino]butyldodecanoate (16a) Boc-L-Orn(Boc)-OH (365 mg, 1.1 mmol) is dissolved in 10 mL of a 9 / 1 v / v mixture of dry DCM / DMF under argon in a three-necked round-bottom flask equipped with a dropping funnel. Subsequently, HOBt (162 mg, 1.2 mmol) and DIC (201 μL, 1.3 mmol) are added, and the reaction mixture is stirred at room temperature for 3 hours. Then, compound 15d (654 mg, 1 mmol) and Et3N (210 μL, 1.5 mmol), dissolved in 10 mL of a 9 / 1 v / v mixture of dry DCM / DMF, are added dropwise through a dropping funnel over 30 minutes. After the addition is complete, the reaction mixture is stirred at room temperature for 36 hours and concentrated to dryness. The crude mixture is then resuspended in 20 mL of ethyl acetate and left at -20°C for 3 hours. Next, the obtained liquid and solid are separated by centrifugation at 3000 rpm, and this procedure is repeated three times. The solid residue is collected and subsequently washed three times with diethyl ether, and then dried overnight under vacuum to obtain the pure desired compound as a white solid in 85% yield (823 mg, 0.85 mmol). TLC (DCM / MeOH: 95 / 5) rf: 0.4.

[0309] 4-[[5-(4-dodecanoyloxybutylamino)-4-(2-methylhexanoylamino)-5-oxo-pentanoyl]amino]butyl dodecanoate bis-trifluoroacetate (16b) The Boc protecting group was removed from compound 16a (823 mg, 0.85 mmol) dissolved in 18 mL of DCM and 2 equivalents (1.7 mmol, 130 μL) of TFA under argon at room temperature for 3 hours, following the procedure described for compound 15c. The product was isolated in 98% yield (0.833 mmol, 803 mg).

[0310] 4-[[4-[2,5-bis[4-(dimethylamino)butanoylamino]pentanoylamino]-5-(4-dodecanoyloxybutylamino)-5-oxo-pentanoyl]amino]butyldodecanoate (XVI) Molecule XVI was isolated by coupling DMABA (109 mg, 0.65 mmol) with compound 22b (0.5 mmol, 482 mg) using DIPEA (305 μL, 1.75 mmol) and HBTU (663 mg, 1.75 mmol) in 8 mL of DMF, following the procedure described for molecule 5b. Reaction time: 48 hours at 0°C to room temperature; Purification: Silica gel chromatography gradient 99 / 1 to 90 / 10 (DCM / MeOH, v / v). Yield: 56% (white solid 278 mg, 0.28 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.25).

[0311] Synthesis of molecule XVII N,N'-bis[(Z)-octadeca-9-enyl]-2-oxopentanediamide(17a) α-Ketoglutaric acid (146 mg, 1 mmol) was dissolved in a 4 / 1 (v / v) mixture of DCM / DMF under argon. Then, DIPEA (558 μL, 3.2 mmol) was added, and the reaction mixture was stirred for 5 minutes. PyBOP (1.56 g, 3 mmol) was introduced, and the mixture was stirred for a further 5 minutes until it turned orange. Finally, oleylamine (1.16 mL, 3 mmol) was added dropwise over 15 minutes, and the reaction mixture was maintained at room temperature for 48 hours before being concentrated. The oily residue was returned to 30 mL of toluene and left at -20°C for 3 hours, and the resulting solid was removed by centrifugation at 3000 rpm. The remaining liquid was concentrated, and the product was purified by flash chromatography using silica gel with a DCM / methanol mixture as the eluent. A gradient in the range of 99 / 1 to 95 / 5 (DCM / MeOH, v / v) was used. A bright yellow solid of 310 mg (0.48 mmol, yield 48%) was recovered. TLC (DCM / MeOH: 95 / 5) rf: 0.45.

[0312] 4-[(dimethylamino)methyl]-N-[(Z)-octadeca-9-enyl]-2-[3-[[(Z)-octadeca-9-enyl]amino]-3-oxopropyl]-1,3-dioxolane-2-carboxamide(XVII) Compound 17a (161 mg, 0.25 mmol), 3-(dimethylamino)propane-1,2-diol (59 μL, 0.5 mmol), and pyridinium-p-toluenesulfonate (PPTS, 11 mg, 0.05 mmol) were dissolved in dry toluene (20 mL) with stirring. A round-bottom flask equipped with a Dean-Stark trap and condenser was used. The reaction mixture was then stirred under reflux for 18 hours, cooled, and concentrated. The residue was then returned to DCM and washed twice with saturated sodium bicarbonate solution, twice with water, and once with brine. The organic extract was dried over magnesium sulfate, filtered, and concentrated under vacuum. The product was recovered by flash chromatography with silica gel using a gradient of DCM / methanol (100 / 0 v / v ~ 95 / 5 v / v) mixture as the eluent. 63 mg (0.085 mmol, 34% yield) of a colorless solid was recovered. TLC (DCM / MeOH:97 / 3) rf:0.3.

[0313] Synthesis of molecule XVIII 2-Hydroxy-N,N'-bis[(Z)-octadeca-9-enyl]pentanediamide (18a) Compound 17a (0.25 mmol, 161 mg) is dissolved in 2.5 mL of anhydrous ethanol. The reaction mixture is then cooled to 0°C, and sodium borohydride (19 mg, 0.5 mmol) is added in two equal portions. The reaction is then carried out at 0°C for 3 hours, after which the mixture is quenched at 0°C with 5 mL of saturated ammonium chloride aqueous solution. The aqueous phase is then extracted with ethyl acetate (3 × 10 mL). The combined organic extracts are washed with water and brine, dried over magnesium sulfate, filtered, and concentrated. The pure product (149 mg, 0.23 mmol) is recovered as a colorless oil and used directly in the next step (yield 91%).

[0314] [4-[[(Z)-octadeca-9-enyl]amino]-1-[[(Z)-octadeca-9-enyl]carbamoyl]-4-oxo-butyl]4-(dimethylamino)butanoate(XVIII) DMABA (40 mg, 0.24 mmol) was linked to compound 18a (129 mg, 0.2 mmol) using methylimidazole (65 μL, 0.8 mmol) and TCFH (113 mg, 0.4 mmol dissolved in 3.5 mL of a dry 2 / 1 (v / v) mixed solution of MeCN and DCM), and the esterification yielding molecule XVIII was achieved by adapting the procedure described for compound II. Reaction time: 18 hours at 0°C to room temperature; Purification: Silica gel chromatography gradient 98 / 2 to 90 / 10 (DCM / MeOH, v / v). Yield: 61% (white solid 93 mg, 0.122 mmol, TLC (DCM / MeOH: 90 / 10) Rf: 0.4).

[0315] Synthesis of molecule XIX 5-Hydroxy-N-[(Z)-Octadeca-9-Enyl]-2-[3-[[(Z)-Octadeca-9-Enyl]amino]-3-Oxopropyl]-1,3-Dioxane-2-Carboxamide (19a) Ketalization of molecule 19a (161 mg, 0.25 mmol) using 2-{[dimethyl(2-methyl-2-propanyl)silyl]oxy}-1,3-propanediol (103 mg, 0.5 mmol) and PPTS (11 mg, 0.05 mmol) dissolved in toluene (20 mL) was carried out for 18 hours according to the procedure described for compound XVII.

[0316] Next, hydroxyl functional groups were released using TBAF (1 M, 1 mmol, 1 mL in THF) in 5 mL of dry THF, following the procedure described in 14c. After concentrating the medium, the target product was recovered by flash chromatography with silica gel using a DCM / methanol mixture as the eluent. A gradient in the range of 98 / 2 to 90 / 10 (DCM / MeOH, v / v) was used. 49 mg of white solid (0.068 mmol, yield 27%, 2 steps) was recovered. TLC (DCM / MeOH: 90 / 10) rf: 0.5.

[0317] 4-Imidazole-1-ylbutanoic acid (19b) Dissolve 4-bromobutyric acid (10 mmol, 1.67 g) in dry MeCN (100 mL). Then, introduce imidazole (10.5 mmol, 714 mg), stir the reaction mixture overnight at 60°C, and then concentrate it. Next, precipitate the resulting solid in diethyl ether, filter it, and wash it three times with cold diethyl ether. Use the product (9.6 mmol, 1.48 g, 96%) without further purification.

[0318] [2-[3-[[(Z)-octadeca-9-enyl]amino]-3-oxopropyl]-2-[[(Z)-octadeca-9-enyl]carbamoyl]-1,3-dioxan-5-yl]4-imidazole-1-ylbutanoate(XIX) Compound XIX was isolated by ligating compound 19b (15.4 mg, 0.1 mmol) with compound 19a (72 mg, 0.1 mmol) using methylimidazole (33 μL, 0.4 mmol) dissolved in 2 mL of a 2 / 1 (v / v) mixed solution of MeCN and DCM, and TCFH (113 mg, 0.4 mmol), and adapting the procedure described for compound II. Reaction time: 18 hours at 0°C to room temperature; Purification: Silica gel chromatography gradient 98 / 2 to 90 / 10 (DCM / MeOH, v / v). Yield: 52% (white solid 44 mg, 0.122 mmol, TLC (DCM / MeOH: 90 / 10) Rf: 0.3).

[0319] Synthesis of molecule XX a) Synthesis of molecule 20b [(Z)-non-2-enyl]4-aminobutanoate trifluoroacetate (20a) The Boc protecting group was first introduced using 4-aminobutyric acid (1.03 g, 10 mmol), NaOH (440 mg, 11 mmol), and Boc2O (10.5 mmol, 2.41 mL) in a 1 / 1 mixture of water and tert-BuOH, following the procedure described for compound 5a. The reaction time was 12 hours.

[0320] Following the procedure described for compound 13b, the vacuum-dried crystalline compound (2.03 g) was then coupled with (Z)-2-nonen-1-ol (1.77 mL, 10.5 mmol) for 36 hours using DIC (1.7 mL, 11 mmol), HOBt (1.49 g, 11 mmol), and Et3N (2.1 mL, 15 mmol) dissolved in 100 mL of a 9 / 1 v / v mixture of dry DCM / DMF. Thus, after the initial workup, the amine functional group was released for 6 hours using 1.3 mL of TFA in 60 mL of DCM. After further workup, the product was used in its as-purity state without further purification (8.8 mmol, 4.97 g, 88% in 3 steps).

[0321] [(Z)-non-2-enyl]4-[[5-[[4-[(Z)-non-2-enoxy]-4-oxo-butyl]amino]-4,5-dioxo-pentanoyl]amino]butanoate(20b) Compound 17a was isolated by ligating compound 20a (813 mg, 2.5 mmol) with α-ketoglutaric acid (146 mg, 1 mmol) using DIPEA (558 μL, 3.2 mmol) dissolved in 20 mL of a 4 / 1 (v / v) mixture of DCM / DMF under argon, and PyBOP (1.56 g, 3 mmol), following the procedure described above. Reaction time: 48 hours at room temperature; Purification: Silica gel chromatography gradient 98 / 2~92 / 8 (DCM / MeOH, v / v). Yield: 42% (bright yellow solid 237 mg, 0.42 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.25).

[0322] b) Synthesis of molecule XX [(Z)-non-2-enyl]4-[3-[5-hydroxy-2-[[4-[(Z)-non-2-enoxy]-4-oxobutyl]carbamoyl]-1,3-dioxan-2-yl]propanoylamino]butanoate (20c) Ketalization of molecule 20b (565 mg, 1 mmol) using 2-{[dimethyl(2-methyl-2-propanyl)silyl]oxy}-1,3-propanediol (412 mg, 2 mmol) and PPTS (44 mg, 0.2 mmol) dissolved in toluene (100 mL) was carried out for 18 hours according to the procedure described for compound XVII.

[0323] Next, hydroxyl functional groups were released using TBAF (1M, 4 mmol, 4 mL in THF) in 20 mL of dry THF, following the procedure described for 2 hours at 0°C. After concentrating the medium, the target product was recovered by flash chromatography with silica gel using a DCM / methanol mixture as the eluent. A gradient in the range of 98 / 2 to 90 / 10 (DCM / MeOH, v / v) was used. 83 mg (0.13 mmol, 13%, 2 steps) of white solid was recovered. TLC (DCM / MeOH: 95 / 5) rf: 0.35.

[0324] [(Z)-non-2-enyl]4-[3-[5-[4-(dimethylamino)butanoyloxy]-2-[[4-[(Z)-non-2-enoxy]-4-oxobutyl]carbamoyl]-1,3-dioxan-2-yl]propanoylamino]butanoate(XX) Compound XX was isolated by adapting the procedure described for compound II, using methylimidazole (65 μL, 0.8 mmol) and TCFH (113 mg, 0.4 mmol) dissolved in 5 mL of a 2 / 1 (v / v) mixed solution of MeCN and DCM, and by linking compound DMABA (40 mg, 0.24 mmol) with compound 20c (83 mg, 0.13 mmol). Reaction time: 18 hours at 0°C to room temperature; Purification: Silica gel chromatography gradient 98 / 2 to 90 / 10 (DCM / MeOH, v / v). Yield: 64% (white solid 63 mg, 0.083 mmol, TLC (DCM / MeOH: 90 / 10) Rf: 0.25).

[0325] Synthesis of molecule XXI 3-[tert-butyl(dimethyl)silyl]oxy-N-[3-[tert-butyl(dimethyl)silyl]oxypropyl]propan-1-amine(21a) Dissolve 3-(3-hydroxypropylamino)-propan-1-ol (10 mmol, 1.33 g) in dry DMF (100 mL). After cooling the solution to 0°C, introduce imidazole (21 mmol, 1.43 g) and tert-butyldimethylsilyl chloride (25 mmol, 3.75 g) in four equal portions over 1 hour. Stir the reaction mixture overnight at room temperature and then concentrate. The resulting residue is then redissolved in 100 mL of DCM, washed twice with saturated sodium carbonate solution, twice with water, and once with brine, dried over sodium sulfate, filtered, and concentrated. The product is recovered by flash chromatography using silica gel with a DCM / Et2O (50 / 50 v / v) mixture as the eluent. 3.4 g of oily solid (9.4 mmol, 94% yield) is obtained. TLC(DCM / Et2O(50 / 50 v / v))rf:0.5.

[0326] [(Z)-non-2-enyl]4-[[4-(2,5-dioxopyrrolidine-1-yl)oxycarbonyloxy-5-[[4-[(Z)-non-2-enoxy]-4-oxo-butyl]amino]-5-oxo-pentanoyl]amino]butanoate(21b) Compound 20b (565 mg, 1 mmol) is first reduced as described for molecule 18a by using sodium borohydride (76 mg, 2 mmol) that has been reacted in 10 mL of anhydrous ethanol at 0°C for 3 hours. The crude compound is then dissolved in 10 mL of dry DCM. Triethylamine (1.3 mmol, 182 μL) is added, followed 5 minutes later by the addition of N,N-disuccinimidyl carbonate (307 mg, 1.2 mmol). After stirring the solution at room temperature for 12 hours, the organic matter is washed with saturated ammonium chloride solution, water, and brine. The organic phase is then dried over magnesium sulfate, filtered, and concentrated. The product is recovered by flash chromatography with silica gel using a 95 / 5 v / v DCM / methanol mixture as the eluent. 580 mg of white solid (0.82 mmol, 82% yield in 2 steps) is recovered. TLC (DCM / MeOH:95 / 5) rf:0.4.

[0327] [(Z)-non-2-enyl]4-[[4-[bis(3-hydroxypropyl)carbamoyloxy]-5-[[4-[(Z)-non-2-enoxy]-4-oxo-butyl]amino]-5-oxo-pentanoyl]amino]butanoate(XXI) Compound 21a (181 mg, 0.5 mmol) is dissolved in 5 mL of a 1 / 1 (v / v) mixture of dry DCM and DMF under argon. Then, 2 equivalents (1 mmol, 140 μL) of t Et3N are slowly added, and the reaction mixture is stirred for 5 minutes. Next, compound 21b (0.5 mmol, 353 mg) is added, and the mixture is stirred at room temperature for 24 hours. The mixture is then concentrated and redissolved in 80 mL of ethyl acetate. The organic phase is then washed with saturated aqueous ammonium chloride solution, washed twice with Milli-Q water and once with brine, dried over MgSO4, filtered, and concentrated. The resulting crude product is then redissolved in 25 mL of dry THF. Next, TBAF (1 M, 4 mmol, 4 mL in THF) is introduced, and the reaction mixture is stirred at 0°C for 4 hours. After the medium is concentrated, the target product is recovered by flash chromatography using silica gel with the DCM / methanol mixture as the eluent. A gradient in the range of 98 / 2 to 94 / 6 (DCM / MeOH, v / v) was used. 400 mg of colorless oil (0.55 mmol, 55% yield, 2 steps) was recovered. TLC (DCM / MeOH: 95 / 5) rf: 0.4.

[0328] Synthesis of molecule XXII [5-[2-(dimethylamino)ethylamino]-5-oxo-4-[(2,2,2-trifluoroacetyl)ammonio]pentyl]-(2,2,2-trifluoroacetyl)ammonium(22a) Compound 22a was isolated in two steps by first using methylimidazole (299 μL, 3.7 mmol) and TCFH (520 mg, 1.85 mmol) dissolved in 28 mL of a 1 / 2 (v / v) mixed solution of MeCN and DCM, and then by linking compound Boc-L-Orn(Boc)-OH (365 mg, 1.1 mmol) with N,N-dimethylethylenediamine (66 μL, 0.6 mmol), adapting the procedure described for compound II. Reaction time: 18 hours at 0°C to room temperature; Purification: Silica gel chromatography gradient 98 / 2 to 95 / 5 (DCM / MeOH, v / v). Yield: 77% (185 mg, 0.46 mmol, molecular weight = 402.5 g / mol, 77%). Next, using 76 μL (1 mmol) of TFA in 10 mL of DCM, the procedure described for compound 15c is adapted to release the hydroxyl functional group until complete (2 hours). Normal work-up is performed to obtain the compound as a colorless solid (0.245 mmol, 97 mg, 98%).

[0329] N-[5-[2-(dimethylamino)ethylamino]-4-(heptadeca-4,6-diinoylamino)-5-oxopentyl]heptadeca-4,6-diinamide(XXII) Molecule XXII was isolated by coupling heptadeca-4,6-diic acid (193 mg, 0.74 mmol) with compound 22b (0.245 mmol, 97 mg) using DIPEA (175 μL, 1 mmol) and HBTU (341 mg, 0.9 mmol) in 4 mL of DMF and 4 mL of DCM, following the procedure described for molecule X. Reaction time: 36 hours at 0°C to room temperature; Purification: Silica gel chromatography gradient 95 / 5 to 90 / 10 (DCM / MeOH, v / v). Yield: 49% (white solid 83 mg, 0.120 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.15).

[0330] Synthesis of molecule XXIII 4-(9H-fluoren-9-ylmethoxycarbonylamino)-5-[[4-[(Z)-non-2-enoxy]-4-oxo-butyl]amino]-5-oxo-pentanoic acid (23a) Fmoc-Glu(OtBu)-OH (2.5 mmol; 1.06 g) was placed in a 100 mL flask under argon, and then dissolved in 40 mL of anhydrous DMF with stirring. COMU (2.5 mmol; 1.07 g) and DIPEA (5 mmol; 865 μL) were successively added to the reaction medium. The reaction mixture was then maintained under argon at 0°C for 15 minutes, after which 20a (4.9 mmol; 1.67 g) dissolved in 10 mL of anhydrous DMF was added to the reaction medium with stirring. The solution was warmed to room temperature overnight, then stopped and evaporated to dryness under vacuum. The crude product was returned to 50 mL of siRNA and washed with 1 M HCl, NaHCO3, and brine. The organic layer was dried over MgSO4 and purified by flash chromatography on silica gel (elution gradient of 30-60% siRNA in petroleum ether). In this manner, the intermediate is isolated in 74% yield (1.85 mmol; 1.2 g). TLC: Rf = 0.3 (siRNA: petroleum ether 40 / 60 (v / v)), then dissolved in 90 mL of DCM containing 10 mL of trifluoroacetic acid. The reaction mixture is then stirred overnight at room temperature. The reaction medium is evaporated to dryness. The mixture is then concentrated and co-evaporated three times with 50 mL of DCM, then three times with 50 mL of diethyl ether (Et2O), and dried overnight under high vacuum (74%, 1.85 mmol, 1.07 g in two steps).

[0331] [(Z)-non-2-enyl]4-[[2-amino-5-oxo-5-(tetradecylamino)pentanoyl]amino]butanoate(23b) Molecule 23b was isolated in two consecutive steps following the procedure described for molecule 1b by coupling tetradecylamine (1.5 mmol; 320 mg) with compound 23a (1 mmol; 579 mg) using DIC (2 mmol; 313 μL), HOBt (2 mmol; 270 mg), and Et3N (2 mmol; 272 μL) in 18 mL of DMF and 330 mL of CHCl. ​​Piperidine (10 mL) was added after 30 hours, and the mixture was stirred for a further 6 hours. Reaction time: 36 hours at room temperature; yield: 61% (0.61 mmol; 337 mg) TLC: Rf = 0.4 (DCM: MeOH 90 / 10 (v / v)).

[0332] [(Z)-non-2-enyl]4-[[2-[[2-(dimethylamino)acetyl]amino]-5-oxo-5-(tetradecylamino)pentanoyl]amino]butanoate(XXIII) Compound XXIII was isolated by adapting the procedure described for compound II, by ligating compound DMG (0.23 mmol; 24 mg) with compound 23b (0.3 mmol; 170 mg) using methylimidazole (0.81 mmol; 65 μL) dissolved in 5 mL of a 2 / 1 (v / v) mixed solution of MeCN and DCM, and TCFH (0.28 mmol; 79 mg). Reaction time: 12 hours at 0°C to room temperature; Purification: Silica gel chromatography gradient 98 / 2 to 95 / 5 (DCM / MeOH, v / v). Yield: 83% (white solid 121 mg, 0.19 mmol, TLC (DCM / MeOH: 90 / 10) Rf: 0.4).

[0333] Synthesis of molecule XXIV 2-amino-N,N'-didodecyl-butanediamide (24a) Following the procedure described for compound 1b, molecule 24a was obtained in two steps by coupling Fmoc-aspartic acid (3 mmol; 1.07 g) with dodecylamine (9 mmol; 2.07 mL) using DIC (10.5 mmol; 1.66 mL) and HOBt (10.5 mmol; 1.42 g) dissolved in 40 mL of DMF and 340 mL of CHCl. ​​After stirring at room temperature for 24 hours, Fmoc deprotection was performed by introducing 9 mL of piperidine and stirring at room temperature for 4 hours. Reaction time: 24 hours + 4 hours at room temperature; Yield: 82% (2.46 mmol; 1.12 g); TLC: Rf = 0.4 (DCM: MeOH 95 / 5 (v / v)).

[0334] [(Z)-non-2-enyl]-4-[[2-amino-5-[[3-(dodecylamino)-1-(dodecylcarbamoyl)-3-oxopropyl]amino]-5-oxopentanoyl]amino]butanoate(24b) 20a (1.89 mmol; 1.09 g) was placed in a dry 100 mL flask under argon, and then dissolved in 20 mL of anhydrous DMF with stirring. DIPEA (5.7 mmol; 987 μL) and pyBOP (2.46 mmol; 1.28 g) were successively added to the solution. After stirring the reaction medium under argon at room temperature for 15 minutes, a solution of 24a (2.46 mmol; 1.12 g) in 10 mL of anhydrous DMF and 5 mL of anhydrous DCM was added to the reaction medium. After stirring at room temperature for 36 hours, 7 mL of piperidine was added, and the reaction medium was stirred for a further 4 hours. The reaction was stopped, and the reaction medium was evaporated to dryness. The coupling product was then purified by flash chromatography on silica gel (elution gradient of 0-5% MeOH in DCM). The product was thus isolated in 42% yield (0.79 mmol; 636 mg). TLC:Rf=0.5(DCM:MeOH 90 / 10(v / v)).

[0335] [(Z)-non-2-enyl]-4-[[2-[4-(dimethylamino)butanoylamino]-5-[[3-(dodecylamino)-1-(dodecylcarbamoyl)-3-oxo-propyl]amino]-5-oxo-pentanoyl]amino]butanoate(XXIV) Compound XXIV was isolated by adapting the procedure described for compound II, by linking compound 24b (0.26 mmol; 210 mg) with DMABA (0.2 mmol; 34 mg) using methylimidazole (0.7 mmol; 120 μL) dissolved in 5 mL of a 2 / 1 (v / v) mixed solution of MeCN and DCM, and TCFH (0.6 mmol; 170 mg). Reaction time: 12 hours at 0°C to room temperature; Purification: Silica gel chromatography gradient 98 / 2 to 90 / 10 (DCM / MeOH, v / v). Yield: 61% (white solid 147 mg, 0.16 mmol, TLC (DCM / MeOH: 90 / 10) Rf: 0.3).

[0336] Synthesis of molecule XXV tert-butyl-N-[4-amino-5-[2-[2-[[2-(dimethylamino)acetyl]amino]ethyldisulfanyl]ethylamino]-5-oxopentyl]carbamate (25a) Place Fmoc-Orn(Boc)-OH (1 mmol; 1.07 g) in a dry 50 mL flask under an inert atmosphere, then dissolve it in 10 mL of anhydrous DMF while stirring. Continuously add a solution of DIC (1.2 mmol; 188 μL) in 1 mL of anhydrous DMF, followed by a solution of HOBt (1.2 mmol; 162 mg), to the reaction medium. Maintain the reaction mixture at ambient temperature under an inert atmosphere for 2.5 hours. Add a solution of 13a (0.83 mmol; 292 mg) and triethylamine (2 mmol; 281 μL) in 6 mL of anhydrous DCM dropwise to the reaction medium. Stir under an inert atmosphere at ambient temperature for 24 hours, then add 3 mL of piperidine and stir the reaction medium for a further 4 hours. Stop the reaction and evaporate the reaction medium to dryness under crude vacuum. Return the residue to 20 mL of  and wash three times with water and brine. The organic layer was dried over MgSO4 and purified by flash chromatography on silica gel (elution gradient of 2-6% MeOH in DCM). The product was isolated in this manner with an 87% yield (0.72 mmol; 485 mg). TLC: Rf = 0.3 (DCM: MeOH 90 / 10 (v / v)).

[0337] Return the product to 20 mL of DMF, add 2 mL of piperidine, and stir the reaction medium for 4 hours. Stop the reaction and evaporate the reaction medium to dryness under coarse vacuum. Polish the residue several times in petroleum ether, filter it, and use it as is.

[0338] [4-Azaniumyl-5-[[4-Azaniumyl-1-[2-[2-[[2-(dimethylamino)acetyl]amino]ethyldisulfanyl]ethylcarbamoyl]butyl]amino]-5-oxopentyl]ammonium(25b) First, Boc-L-Orn(Boc)-OH (1.4 mmol; 465 mg) is placed in a pre-dried 25 mL flask under an inert atmosphere, and then dissolved in 10 mL of anhydrous DMF with stirring. HBTU (1.4 mmol; 531 mg) and DIPEA (2.8 mmol; 485 μL) are continuously added to the solution. After stirring the reaction medium at ambient temperature for 15 minutes under an inert atmosphere, a solution of 25a (0.72 mmol; 325 mg) in 1 mL of anhydrous DMF and 1 mL of anhydrous DCM is added to the reaction medium. After 12 hours, the reaction is stopped, and the reaction medium is evaporated to dryness under high vacuum. The crude product is returned to 100 mL of siRNA and washed with 1 M HCl, NaHCO3, and brine. The organic layer is dried over MgSO4 and purified by flash chromatography on silica gel (elution gradient of 2-5% MeOH in DCM). In this manner, the product is isolated in 63% yield (2 steps, 392 mg, 0.51 mmol). The obtained compound (0.1 mmol; 79 mg) is dissolved in 5 mL of DCM, and then 250 μL of trifluoroacetic acid is added to the reaction medium. The reaction mixture is then maintained at ambient temperature with stirring for 4 hours. The reaction medium is evaporated to dryness under crude vacuum. The final residue is returned to DCM four times and to Et2O four times in succession, and then evaporated to dryness. The reaction is quantitative.

[0339] (9Z,12Z)-N-[5-[[1-[2-[2-[[2-(dimethylamino)acetyl]amino]ethyldisulfanyl]ethylcarbamoyl]-4-[[(9Z,12Z)-octadeca-9,12-dienoyl]amino]-butyl]amino]-4-[[(9Z,12Z)-octadeca-9,12-dienoyl]amino]-5-oxopentyl]octadeca-9,12-dienamide(XXV)

[0340] Compound XXV was isolated by adapting the procedure described for compound 1b, using DIPEA (2 mmol; 353 μL) dissolved in 15 mL of DMF and PyBOP (1.8 mmol; 936 mg) to concatenate compound linoleic acid (1.8 mmol; 505 mg) with compound 25b (0.51 mmol; 411 mg). Reaction time: 24 hours at room temperature; Purification: Silica gel chromatography gradient 98 / 2~94 / 6 (DCM / MeOH, v / v). Yield: 26% (white solid 166 mg, 0.13 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.4).

[0341] Synthesis of molecule XXVI 4-Pyrrolidine-1-ylbutyl2-amino-5-(tert-butoxycarbonylamino)pentanoate (26a) Fmoc-Orn(Boc)-OH (0.5 mmol; 227 mg) is placed in a 50 mL flask under argon and then dissolved in 20 mL of DCM. DCC (0.6 mmol; 124 mg) and DMAP (0.1 mmol; 11 mg) are added to the reaction medium, followed by the addition of 4-(1-pyrrolidinyl)-1-butanol (0.7 mmol; 100 mg). The mixture is stirred under argon at room temperature for 15 hours, after which the reaction is stopped and the reaction medium is evaporated to dryness. The crude product is returned to 20 mL of DCM and washed with 1 M HCl, NaHCO3 and brine. The organic layer is dried over MgSO4 and purified by flash chromatography on silica gel (elution gradient of 4-10% MeOH in DCM). The intermediate product is thus isolated and then returned to 20 mL of DMF, 2 mL of piperidine is added, and the reaction medium is stirred for 4 hours. The reaction is stopped, and the reaction medium is evaporated to dryness under a rough vacuum. The residue is polished several times in petroleum ether, filtered, and used as is (yield 82%, 2 steps, 0.41 mmol, 147 mg).

[0342] [4-Azaniumyl-5-[[4-Azaniumyl-1-(4-Pyrrolidine-1-ylbutoxycarbonyl)butyl]amino]-5-oxopentyl]ammonium(26b) Compound 26b was obtained following the two-step procedure used for the synthesis of compound 25b. First, Boc-L-Orn(Boc)-OH (0.7 mmol; 233 mg) and compound 26a (0.41 mmol; 147 mg) were coupled using HBTU (0.7 mmol; 265 mg) and DIPEA (1.4 mmol; 243 μL) dissolved in 9 mL of DMF, and the reaction mixture was then stirred at room temperature for 16 hours. After isolation and purification of the intermediate (flash chromatography on silica gel, elution gradient of 2-5% MeOH in DCM), the amine functional group was released by reacting with TFA (1 mL) in 10 mL of DCM for 12 hours. Yield: 51% (2 steps, 149 mg, 0.21 mmol).

[0343] 4-Pyrrolidine-1-ylbutyl-2-[2,5-bis[[(9Z,12Z)-octadeca-9,12-dienoyl]amino]pentanoylamino]-5-[[(9Z,12Z)-octadeca-9,12-dienoyl]amino]pentanoate(XXVI) Compound XXVI was isolated by adapting the procedure described for compound 1b, using DIPEA (1.4 mmol; 247 μL) dissolved in 8 mL of DMF and PyBOP (0.74 mmol; 385 mg) to linoleic acid (0.74 mmol; 206 mg) and compound 26b (0.21 mmol; 149 mg). Reaction time: 24 hours at room temperature; Purification: Silica gel chromatography gradient 98 / 2~94 / 6 (DCM / MeOH, v / v). Yield: 32% (white solid 78 mg, 0.07 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.3).

[0344] Synthesis of molecule XXVII [4-Azaniumyl-5-(2-hydroxyethylamino)-5-oxopentyl]ammonium (27a) A solution of Boc-L-Orn(Boc)-OH (2 mmol; 665 mg) in DMF was stirred at room temperature, and EDC·HCl (2.4 mmol; 460 mg) and HOBt (2.4 mmol; 324 mg) were successively added to the reaction medium. The reaction mixture was stirred under argon at room temperature for 1 hour, after which ethanolamine (10 mmol; 604 μL) was added. After stirring for a further 24 hours at room temperature, the reaction was stopped and the reaction medium was evaporated to dryness. The residue was returned to 50 mL of water, the amide was extracted three times with CHCl3, the organic layer was dried over MgSO4, and purified by flash chromatography on silica gel (elution gradient of 2-6% MeOH in DCM). The isolated intermediate product (1.82 mmol; 683 mg) was then dissolved in 90 mL of DCM, and then 5 mL of trifluoroacetic acid was added to the reaction medium. The reaction mixture was then maintained at ambient temperature with stirring for 16 hours. Next, the mixture is concentrated, co-evaporated three times with 50 mL of DCM, and co-evaporated three times with 50 mL of diethyl ether (Et2O), and dried overnight under high vacuum. Yield: 91% (2 steps, 734 mg, 1.82 mmol).

[0345] 2,5-Bis[4-(dimethylamino)butanoylamino]-N-(2-hydroxyethyl)pentanamide(27b) Compound 27b was isolated by coupling DMABA (4.55 mmol, 763 mg) with compound 27a (1.82 mmol; 734 mg) using DIPEA (9 mmol; 1.55 mL) and HBTU (4.55 mmol; 1.72 g) dissolved in 30 mL of DCM and 5 mL of DMF, adapting the procedure described for compound 5b. Reaction time: 48 hours at room temperature; Purification: Silica gel chromatography gradient 98 / 2~95 / 5 (DCM / MeOH, v / v). Yield: 51% (white solid 373 mg, 0.93 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.4).

[0346] 2-[2,5-bis[4-(dimethylamino)butanoylamino]pentanoylamino]ethyl 2-amino-5-(tert-butoxycarbonylamino)pentanoate(27c) Compound 27c was obtained following the two-step procedure used for the synthesis of compound 26a. First, Fmoc-L-Orn(Boc)-OH (1 mmol; 454 mg) and compound 27b (0.93 mmol; 373 mg) were coupled using DCC (1.2 mmol; 248 mg) and DMAP (0.2 mmol; 22 mg) dissolved in 40 mL of DCM, and the reaction mixture was then stirred at room temperature for 16 hours. After isolation and purification of the intermediate (flash chromatography on silica gel, elution gradient of 2-5% MeOH in DCM), the Fmoc group was released by reacting with piperidine (2 mL) in 20 mL of DMF for 4 hours. Yield: 76% (2 steps, 437 mg, 0.71 mmol).

[0347] [4-Azaniumyl-5-[[4-Azaniumyl-1-[2-[2,5-Bis[4-(dimethylamino)butanoylamino]pentanoylamino]ethoxycarbonyl]butyl]amino]-5-oxopentyl]ammonium(27d) Compound 27d was obtained by following the two-step procedure experienced for the synthesis of compound 25b. First, Boc-L-Orn(Boc)-OH (1.4 mmol; 465 mg) and compound 27c (0.71 mmol; 437 mg) were coupled using HBTU (1.4 mmol; 531 mg) and DIPEA (2.8 mmol; 485 μL) dissolved in 15 mL of DMF, and the reaction mixture was then stirred at room temperature for 16 hours. After isolation and purification of the intermediate (flash chromatography on silica gel, elution gradient of 2-5% MeOH in DCM), the amine functional group was released by reacting with TFA (2 mL) in 30 mL of DCM for 12 hours. Yield: 59% (2 steps, 408 mg, 0.42 mmol).

[0348] 2-[2,5-bis[4-(dimethylamino)butanoylamino]pentanoylamino]ethyl 2-[2,5-bis[[(9Z,12Z)-octadeca-9,12-dienoyl]amino]pentanoylamino]-5-[[(9Z,12Z)-octadeca-9,12-dienoyl]amino]pentanoate(XXVII) Compound XXVII was isolated by adapting the procedure described for compound 1b, using DIPEA (2 mmol; 353 μL) dissolved in 16 mL of DMF and 2 mL of DCM, and PyBOP (1.5 mmol; 780 mg), by ligating linoleic acid (1.5 mmol; 421 mg) with compound 27d (0.42 mmol; 408 mg). Reaction time: 24 hours at room temperature; Purification: Silica gel chromatography gradient 98 / 2~94 / 6 (DCM / MeOH, v / v). Yield: 26% (white solid 156 mg, 0.11 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.3).

[0349] Synthesis of molecule XXVIII [4-methyl-5-[[4-[[2-methyl-5-[(2,2,2-trifluoroacetyl)ammonio]pentanoyl]amino]-5-oxo-5-(4-pyrrolidine-1-ylbutoxy)pentyl]amino]-5-oxo-pentyl]-(2,2,2-trifluoroacetyl)ammonium-bis-trifluoroacetate (28a) Compound 28a was obtained following the two-step procedure experienced for the synthesis of compound 13b. First, Boc-L-Orn(Boc)-OH (365 mg, 1.1 mmol) and compound 13a (274 mg, 0.5 mmol) were ligated using HOBt (162 mg, 1.2 mmol) and DIC (201 μL, 1.3 mmol) dissolved in 20 mL of a 9 / 1 v / v mixture of dry DCM / DMF also containing Et3N (210 μL, 1.5 mmol), and the reaction mixture was then stirred at room temperature for 72 hours. After isolation and purification of the intermediate compound, the amine functional group was released by reacting it with TFA (190 μL) in 25 mL of DCM for 2 hours. Yield: 48% (2 steps, 0.24 mmol, 234 mg).

[0350] (9Z,12Z)-N-[5-[[4-[2,5-bis[[(9Z,12Z)-octadeca-9,12-dienoyl]amino]pentanoylamino]-5-[2-[2-[[2-(dimethylamino)acetyl]amino]ethyldisulfanyl]ethylamino]-5-oxopentyl]amino]-4-[[(9Z,12Z)-octadeca-9,12-dienoyl]amino]-5-oxopentyl]octadeca-9,12-dienamide(XXXX)

[0351] Compound XXVIII was isolated by adapting the procedure described for compound X, using DIPEA (263 μL, 1.5 mmol), HBTU (530 mg, 1.4 mmol), and HOBt (183 mg, 1.35 mmol) dissolved in 6 mL of DMF and 6 mL of DCM, and by linking linoleic acid (404 μL, 1.3 mmol) with compound 28a (0.21 mmol, 204 mg). Reaction time: 36 hours at 0°C to room temperature; Purification: Silica gel chromatography gradient 95 / 5 to 90 / 10 (DCM / MeOH, v / v). Yield: 54% (white solid 179 mg, 0.11 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.3).

[0352] Synthesis of molecule XXIX [5-[[1-[2-[2,5-bis[4-(dimethylamino)butanoylamino]pentanoylamino]ethoxycarbonyl]-4-[[2-methyl-5-[(2,2,2-trifluoroacetyl)ammonio]pentanoyl]amino]butyl]amino]-4-methyl-5-oxopentyl]-(2,2,2-trifluoroacetyl)ammonium(29a)

[0353] Compound 29a is obtained by following the two-step procedure experienced for the synthesis of compound 13b. First, Boc-L-Orn(Boc)-OH (183 mg, 0.55 mmol) and compound 14d (178 mg, 0.25 mmol) are ligated using HOBt (81 mg, 0.6 mmol) and DIC (100 μL, 0.65 mmol) dissolved in 10 mL of a 9 / 1 v / v mixture of dry DCM / DMF also containing 105 μL, 0.75 mmol of Et3N, and the reaction mixture is then stirred at room temperature for 72 hours. After isolation and purification of the intermediate compound, the amine functional group is released by reacting TFA (95 μL) in 15 mL of DCM for 2 hours. Yield: 51% (2 steps; 0.127 mmol, 145 mg).

[0354] 2-[2,5-bis[4-(dimethylamino)butanoylamino]pentanoylamino]ethyl 2,5-bis[2,5-bis[[(9Z,12Z)-octadeca-9,12-dienoyl]amino]pentanoylamino]pentanoate (XXXIX) Compound XXXIX was isolated by adapting the procedure described for compound X, using DIPEA (132 μL, 0.75 mmol), HBTU (265 mg, 0.7 mmol), and HOBt (92 mg, 0.68 mmol) dissolved in 6 mL of DMF and 6 mL of DCM, and by ligating linoleic acid (202 μL, 0.65 mmol) with compound 29a (0.1 mmol, 113 mg). Reaction time: 36 hours at 0°C to room temperature; Purification: Silica gel chromatography gradient 93 / 7 to 90 / 10 (DCM / MeOH, v / v). Yield: 54% (white solid 179 mg, 0.11 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.2).

[0355] Synthesis of molecule XXX [5-Oxo-5-(4-pyrrolidine-1-ylbutoxy)-4-[(2,2,2-trifluoroacetyl)ammonio]pentyl]-(2,2,2-trifluoroacetyl)ammonium(30a) Compound 30a is obtained by following the two-step procedure experienced for the synthesis of compound 13b. First, Boc-L-Orn(Boc)-OH (10 mmol, 3.32 g) and 4-(1-pyrrolidinyl)-1-butanol (1.43 g, 10 mmol) are ligated using HOBt (1.49 g, 11 mmol) and DIC (1.7 mL, 11 mmol) dissolved in 100 mL of a 9 / 1 v / v mixture of dry DCM / DMF also containing Et3N (2.1 mL, 15 mmol), and the reaction mixture is then stirred at room temperature for 36 hours. After isolation and purification of the intermediate compound, the amine functional group is released by reacting TFA (1.3 mL) in 60 mL of DCM for 2 hours. Yield: 83% (2 steps; 8.3 mmol, 3.76 g).

[0356] [4-methyl-5-[[4-[[2-methyl-5-[(2,2,2-trifluoroacetyl)ammonio]pentanoyl]amino]-5-oxo-5-(4-pyrrolidine-1-ylbutoxy)pentyl]amino]-5-oxo-pentyl]-(2,2,2-trifluoroacetyl)ammonium(30b) Compound 30b is obtained by following the two-step procedure experienced for the synthesis of compound 13b. First, Boc-L-Orn(Boc)-OH (1.46 g, 4.4 mmol) and 30a (906 mg, 2 mmol) are ligated using HOBt (648 mg, 4.8 mmol) and DIC (800 μL, 5.2 mmol) dissolved in 40 mL of a 9 / 1 v / v mixture of dry DCM / DMF also containing Et3N (2.1 mL, 15 mmol), and the reaction mixture is then stirred at room temperature for 72 hours. After isolation and purification of the intermediate compound, the amine functional group is released by reacting it with TFA (760 μL) in 30 mL of DCM for 6 hours. Yield: 44% (2 steps; 8.3 mmol, 772 mg).

[0357] 4-Pyrrolidine-1-ylbutyl-2,5-bis[2,5-bis[[(9Z,12Z)-octadeca-9,12-dienoyl]amino]pentanoylamino]pentanoate(XXX) Compound XXX was isolated by adapting the procedure described for compound X, using DIPEA (263 μL, 1.5 mmol), HBTU (530 mg, 1.4 mmol), and HOBt (183 mg, 1.35 mmol) dissolved in 6 mL of DMF and 6 mL of DCM, and by ligating linoleic acid (404 μL, 1.3 mmol) with compound 30b (0.25 mmol, 219 mg). Reaction time: 36 hours at 0°C to room temperature; Purification: Silica gel chromatography gradient 95 / 5 to 90 / 10 (DCM / MeOH, v / v). Yield: 59% (white solid 227 mg, 0.148 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.25).

[0358] Synthesis of molecule XXXI [4-(3-butylhept-2-enoxy)-4-oxo-butyl]ammonium (31a) 3-Butyl-2-hepten-1-ol (5.5 mmol; 1.11 mL) was added with stirring to a solution of N-BOC-γ-aminobutyric acid (5 mmol, 1.02 g), DCC (5.5 mmol; 1.13 g), and DMAP (1 mmol; 122 mg) dissolved in 50 mL of anhydrous DCM. The reaction mixture was then maintained at ambient temperature under an inert atmosphere for 16 hours. After the complete consumption of the acid, the reaction medium was evaporated to dryness under crude vacuum. The crude product was returned to 50 mL of DCM and washed with 1 M HCl, NaHCO3, and brine. The organic layer was dried over MgSO4 and then purified by flash chromatography on silica gel (elution gradient of 0-50% Â in petroleum ether). The product was thus isolated in 79% yield (3.95 mmol; 1.4 g). TLC: Rf = 0.4 (ethyl: petroleum ether 30 / 70 (v / v)).

[0359] Next, the residue (3.95 mmol; 1.4 g) is dissolved in 100 mL of DCM, and then 5 mL of trifluoroacetic acid is added to the reaction medium. The reaction mixture is then maintained overnight at ambient temperature with stirring. The reaction medium is evaporated to dryness under crude vacuum. The final residue is returned to DCM four times and to Et2O four times in succession, and then evaporated to dryness. The reaction is quantitative.

[0360] 3-Butylhept-2-enyl 4-[[4-amino-5-[[4-(3-butylhept-2-enoxy)-4-oxo-butyl]amino]-5-oxo-pentanoyl]amino]butanoate (31b) Following the procedure described for compound 15d, molecule 31b was obtained in two steps by coupling (Fmoc-Glu-OH, 259 mg, 0.7 mmol) with compound 31a (1.82 mmol; 672 mg) using HOBt (331 mg, 2.45 mmol), DIC (325 μL, 2.1 mmol), and Et3N (394 μL, 2.8 mmol) dissolved in chloroform (7 mL) and DMF (7 mL), and stirring for 48 hours. The Fmoc group was then removed by stirring with piperidine (2 mL) at room temperature for 4 hours. Reaction time: 48 hours + 4 hours at room temperature; Purification: Silica gel chromatography gradient 95 / 5~90 / 10 (DCM / MeOH, v / v). Yield: 41% (0.287 mmol; 179 mg); TLC: Rf = 0.4 (DCM: MeOH 90 / 10 (v / v)).

[0361] 3-Butylhept-2-enyl-4-[[5-[[4-(3-butylhept-2-enoxy)-4-oxo-butyl]amino]-4-[4-(dimethylamino)butanoylamino]-5-oxo-pentanoyl]amino]butanoate(XXXI) Compound XXXI was isolated by coupling DMABA (0.72 mmol, 121 mg) with compound 31b (0.287 mmol; 179 mg) using DIPEA (1.42 mmol; 245 μL) and HBTU (0.72 mmol; 271 mg) dissolved in 2.5 mL of DCM and 2.5 mL of DMF, adapting the procedure described for compound 5b. Reaction time: 24 hours at room temperature; Purification: Silica gel chromatography gradient 98 / 2~90 / 10 (DCM / MeOH, v / v). Yield: 38% (white solid 81 mg, 0.11 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.2).

[0362] Synthesis of molecule XXXII a) Synthesis of molecule 32c tert-butyl N-(4-hydroxybutyl)carbamate (32a) Dissolve 4-aminobutan-1-ol (10 mmol, 922 μL) in 20 mL of anhydrous ethanol. Cool the mixture to 0°C. Then, while stirring at 0°C, add 2.3 mL (10 mmol) of Boc2O dropwise over 30 minutes. Allow the reaction to proceed for 48 hours, then concentrate the reaction mixture under vacuum. Redissolve the oily residue in 20 mL of a 1 / 1 (v / v) mixture of DCM and water. Slip the phase with a funnel and extract the aqueous layer three times with 5 mL of DCM. Wash the combined organic extract with brine, dry over magnesium sulfate, filter, and concentrate to dryness. Use the oil without further purification (yield 97%, 1.84 g, 9.7 mmol). TLC (DCM / MeOH: 95 / 5) rf: 0.7.

[0363] 4-((2-hexyldecanoyl)oxy)butane-1-aminium(32b) 2-hexyldecanoic acid (8.8 mmol; 2.3 g) was placed in a dry 100 mL flask under an inert atmosphere and then dissolved in 45 mL of DCM while stirring. DCC (10.6 mmol; 1.34 g) and DMAP (2 mmol; 244 mg) were added to the reaction medium, followed by the addition of 4-boc-amino-1-butanol (10.6 mmol; 2 g). After stirring at ambient temperature under an inert atmosphere for 15 hours, the medium was filtered to remove DCU and washed with NH4Cl, NaHCO3, and brine. The organic layer was dried over MgSO4 and purified by flash chromatography on silica gel (elution gradient of 5-15% EtOAC in petroleum ether). In this way, the product was isolated in 88% yield (7.8 mmol; 3.34 g). TLC: Rf = 0.5 (EtOAC: EP 10 / 90 (v / v))

[0364] The oily compound is dissolved in 100 mL of dry DCM under argon. Then, 2 mL of trifluoroacetic acid (TFA) is added, and the reaction is allowed to proceed until complete conversion is demonstrated by TLC (5 hours). The mixture is then concentrated and co-evaporated three times with 100 mL of DCM, then three times with 100 mL of diethyl ether (Et2O), and dried overnight under high vacuum. The product is then used without further purification (7.9 mmol, 3.4 g, quantitative yield).

[0365] 4-[[4-amino-5-[4-(2-hexyldecanoyloxy)butylamino]-5-oxo-pentanoyl]amino]butyl-2-hexyldecanoate(32c) Dissolve Fmoc-glutamic acid (2.6 mmol; 959 mg) in 20 mL of anhydrous DMF under an inert atmosphere with stirring. Continuously add DIC (7.9 mmol; 1.22 mL) and then HOBt (7.9 mmol; 1.07 g) to the reaction medium. Stir the reaction mixture for 3 hours, then add a solution of 32b (7.9 mmol; 3.4 g) in 330 mL of anhydrous CHCl dropwise to the reaction medium. After 48 hours, stop the reaction and evaporate the reaction medium to dryness under crude vacuum. Return the residue to 50 mL of siRNA and filter to remove DIU. Wash the organic layer with water and brine, dry over MgSO4, and evaporate. Dissolve the crude product in 50 mL of DMF and add NaN3 (5.2 mmol; 338 mg). Heat the reaction medium at 50°C for 12 hours. After complete deprotection of Fmoc (TLC Rf=0.5 DCM:MeOH 95 / 5(v / v)), the medium was evaporated and the crude product was purified by flash chromatography on silica gel (elution gradient of 2-5% MeOH in DCM). In this way, the product was isolated in 62% yield (1.61 mmol; 1.23 g).

[0366] Synthesis of molecules XXXII;XXXIII;XXXIV 4-[[4-[4-(dimethylamino)butanoylamino]-5-[4-(2-hexyldecanoyloxy)butylamino]-5-oxo-pentanoyl]amino]butyl 2-hexyldecanoate (XXXII) DMABA (0.26 mmol, 44 mg) was placed in a dry 25 mL flask under an inert atmosphere and then dissolved in 2 mL of anhydrous DMF while stirring. HBTU (0.26 mmol; 99 mg) and DIPEA (0.3 mmol; 51 μL) were successively added to the reaction medium. The reaction mixture was then maintained at ambient temperature under an inert atmosphere for 15 minutes, after which a solution of 32c (0.09 mmol; 69 mg) in 1 mL of anhydrous DCM was added to the reaction medium. After stirring at ambient temperature under an inert atmosphere for 48 hours, the reaction was stopped, and the reaction medium was evaporated to dryness under crude vacuum. The product was purified by flash chromatography using silica gel (elution gradient of 4-10% MeOH in DCM). The product was thus isolated in 65% yield (0.05 mmol; 52 mg). TLC: Rf = 0.3 (DCM: MeOH 90 / 10 (v / v))

[0367] 4-[[4-[bis(2-hydroxypropyl)amino]-5-[4-(2-hexyldecanoyloxy)butylamino]-5-oxo-pentanoyl]amino]butyl 2-hexyldecanoate (XXXIII) 32c (0.25 mmol; 190 mg) is dissolved in 5 mL of MeOH in a dry 50 mL flask under an inert atmosphere. A large excess of propylene oxide (1 mL) is added dropwise, and the reaction medium is heated to 60°C for 16 hours. Then, methanol is evaporated, and the crude product is purified by flash chromatography on silica gel (elution gradient of 2-5% MeOH in DCM). In this way, the product is isolated in 56% yield (0.14 mmol; 123 mg). TLC: Rf = 0.6 (DCM: MeOH 90 / 10 (v / v))

[0368] 4-[[4-[[2-(dimethylamino)acetyl]amino]-5-[4-(2-hexyldecanoyloxy)butylamino]-5-oxo-pentanoyl]amino]butyl 2-hexyldecanoate (XXXIV) Following the procedure described for compound XXXII, molecule XXXIV was obtained by coupling DMG (0.25 mmol, 26 mg) with 32c (0.25 mmol, 190 mg) using DIPEA (1 mmol; 170 μL) and HBTU (0.5 mmol; 190 mg) dissolved in 4 mL of DMF and 1 mL of DCM. Reaction time: 36 hours at room temperature; Purification: Silica gel chromatography gradient 96 / 4~90 / 10 (DCM / MeOH, v / v). Yield: 60% (125 mg, 0.15 mmol, TLC (DCM / MeOH: 90 / 10) Rf: 0.3).

[0369] Synthesis of molecule XXXV 4-(9H-fluoren-9-ylmethoxycarbonylamino)-5-(hexadecylamino)-5-oxopentanoic acid (35a) Dissolve Fmoc-Glu(OtBu)-OH (1 mmol; 426 mg) in 10 mL of anhydrous DMF under an inert atmosphere with stirring. Add DIC (1.6 mmol; 247 μL) and then HOBt (1.6 mmol; 216 mg) successively to the reaction medium. Stir the reaction mixture for 3 hours, then add a solution of hexadecylamine (1.6 mmol; 387 mg) in 3100 mL of anhydrous CHCl dropwise to the reaction medium. After 48 hours, stop the reaction and evaporate the reaction medium to dryness under a rough vacuum.

[0370] The residue is returned to 50 mL of siRNA and filtered to remove DIU. The organic layer is washed with water and brine, dried over MgSO4, and evaporated. The white product is washed several times with ether and then dissolved in 15 mL of DCM. After stirring the medium under argon, 4 mL of TFA and 500 μL of triethylsilane are added. After 15 hours, the tert-butyl group is completely deprotected (TLC Rf = 0.4 siRNA / EP 40 / 60 (v / v)). The mixture is then concentrated and co-evaporated three times with 50 mL of DCM, and three times with 50 mL of diethyl ether (Et2O), and dried overnight under high vacuum. The product is then used without further purification (0.71 mmol, 421 mg). 71% yield in two steps.

[0371] 4-[[4-amino-5-(hexadecylamino)-5-oxo-pentanoyl]amino]butyl 2-hexyldecanoate (35b) 35a (0.48 mmol, 289 mg) is placed in a dry 50 mL flask under an inert atmosphere, and then dissolved in 10 mL of anhydrous DMF while stirring. COMU (0.5 mmol; 214 mg) and DIPEA (1 mmol; 180 μL) are continuously added to the reaction medium. The reaction mixture is then maintained at 0°C for 15 minutes under an inert atmosphere, and then 32b (0.5 mmol; 214 mg) is added to the reaction medium while stirring. The solution is left overnight under an inert atmosphere and then heated to ambient temperature. The mixture is evaporated to dryness under a rough vacuum. The residue is returned to 50 mL of  and heated until dissolved. The solution is placed in a Falcon tube at -20°C. The formed precipitate is washed several times by centrifugation using cold  and dried under high vacuum. A white solid is obtained. TLC: Rf = 0.4 (DCM: MeOH 95 / 5 (v / v)).

[0372] The product is returned to 30 mL of DMF, and 2 mL of piperidine is added. The reaction mixture is then maintained overnight at ambient temperature with stirring. The reaction medium is evaporated to dryness under crude vacuum. The product is then purified by flash chromatography using silica gel (elution gradient of 2-5% MeOH in DCM). In this manner, the pure compound is isolated in 50% yield (0.24 mmol; 160 mg). TLC: Rf = 0.6 (DCM: MeOH 90 / 10 (v / v))

[0373] 4-[[4-[[2-(dimethylamino)acetyl]amino]-5-(hexadecylamino)-5-oxo-pentanoyl]amino]butyl 2-hexyldecanoate (XXXV) Following the procedure described for compound XXXIV, molecule XXXV was obtained by coupling DMG (0.2 mmol, 21 mg) with 35a (0.1 mmol, 68 mg) using DIPEA (0.4 mmol; 68 μL) and HBTU (0.2 mmol; 76 mg) dissolved in 2 mL of DMF and 1 mL of DCM. Reaction time: 36 hours at room temperature; Purification: Silica gel chromatography gradient 96 / 4~90 / 10 (DCM / MeOH, v / v). Yield: 67% (51 mg, 0.067 mmol, TLC (DCM / MeOH: 90 / 10) Rf: 0.3).

[0374] Synthesis of molecules XXXVI and XXXVII 4-[[4-[4-(2-hexyldecanoyloxy)butylamino]-3-hydroxy-4-oxo-butanoyl]amino]butyl 2-hexyldecanoate (36a) Malic acid (1.57 mmol; 209 mg) was placed in a dry 50 mL flask under an inert atmosphere and then dissolved in 25 mL of anhydrous DCM with stirring. HOBt (4 mmol; 540 mg) and 32b (4.67 mmol; 2 g) in 2 mL of DMF were successively added to the reaction medium, followed by the addition of NEt3 (0.5 mmol; 51 μL). The reaction mixture was then maintained with stirring, and EDC,Cl (4.7 mmol; 902 mg) was added to the reaction medium. After stirring at ambient temperature for 20 hours under an inert atmosphere, the reaction was stopped, and the reaction medium was evaporated to dryness under crude vacuum. The product was purified by flash chromatography using silica gel (elution gradient of 40-80% EtOAC in EP). In this way, the product was isolated in 55% yield (0.86 mmol; 650 mg). TLC:Rf=0.5(EtOAc:EP 50 / 50(v / v))

[0375] 4-[[4-[4-(2-hexyldecanoyloxy)butylamino]-3-hydroxy-4-oxo-butanoyl]amino]butyl 2-hexyldecanoate (XXXVI) DMABA (0.2 mmol; 34 mg) and DMAP (0.05 mmol; 6.1 mg) were dissolved in 2 mL of anhydrous DCM using molecular sieves under an inert atmosphere with stirring. 36a (0.13 mmol; 100 mg) and then EDC (0.2 mmol; 38 mg) were continuously added to the reaction medium. After 48 hours, the reaction was stopped and the reaction medium was evaporated to dryness under crude vacuum. The residue was returned to 20 mL of DCM and washed with water and brine. The organic layer was dried over MgSO4 and evaporated. The crude product was purified by flash chromatography on silica gel (elution gradient of 4-10% MeOH in DCM). The product was thus isolated in 66% yield (0.09 mmol; 74 mg).

[0376] Synthesized 4-[[3-[2-(dimethylamino)acetyl]oxy-4-[4-(2-hexyldecanoyloxy)butylamino]-4-oxo-butanoyl]amino]butyl 2-hexyldecanoate (XXXVII) Following the procedure described for compound XXXVI, molecule XXXVII was obtained by coupling DMG (0.2 mmol, 21 mg) with 36a (0.1 mmol, 75 mg) using DMAP (0.04 mmol; 5 mg) and EDC (0.3 mmol; 58 mg) dissolved in 2 mL of DCM. Reaction time: 36 hours at room temperature; Purification: Silica gel chromatography gradient 100 / 0~96 / 4 (DCM / MeOH, v / v). Yield: 65% (55 mg, 0.065 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.7).

[0377] Synthesis of molecule 38c 4-[2-[2-(tert-butoxycarbonylamino)ethyldisulfanyl]ethylamino]-4-oxo-butanoic acid (38a) Boc-cystamine (5.3 mmol; 1.34 g) is dissolved in 20 mL of a 1 / 1 (v / v) mixture of dry DCM and DMF under argon. Triethylamine (7.9 mmol; 1.1 mL) is then slowly added, and the reaction mixture is stirred for 5 minutes. Succinic anhydride (10.6 mmol, 1.06 g) is then added, and the mixture is stirred at room temperature for 12 hours. The reaction mixture is then concentrated, and the crude product is purified by flash chromatography on silica gel (elution gradient of 2-8% MeOH in DCM). The product is thus isolated in 93% yield (4.9 mmol; 1.6 g).

[0378] 2-[2-[[4-(2-hexyldecoxy)-4-oxo-butanoyl]amino]ethyldisulfanyl]ethylammonium (38b) 38a (0.6 mmol, 194 mg) is placed in a dry 25 mL flask under argon, and then dissolved in 6 mL of DCM with stirring. DCC (1 mmol; 206 mg) and DMAP (0.1 mmol; 12 mg) are added to the reaction medium. The reaction mixture is then stirred for 10 minutes, after which 2-hexyldecanol (0.7 mmol; 170 mg) is introduced. The mixture is stirred under argon at room temperature for 16 hours, then the reaction is stopped and the medium is evaporated under vacuum. The crude product is returned to 20 mL of DCM and washed with 1 M HCl, NaHCO3 and brine. The organic extract was dried over MgSO4 and purified by flash chromatography on silica gel (elution gradient of 20-50% siRNA in EP, TLC: Rf=0.5(siRNA / EP 50 / 50(v / v))). It was then dissolved in 20 mL of DCM with the addition of 1 mL of trifluoroacetic acid. The reaction mixture was then maintained overnight at room temperature with stirring. The reaction medium was evaporated, co-evaporated four times in DCM and four times in Et2O, and then dried. Yield 84% (2 steps, 0.51 mmol, 299 mg).

[0379] 2-Hexyldecyl 4-[2-[2-[[4-amino-5-[2-[2-[[4-(2-hexyldecoxy)-4-oxo-butanoyl]amino]ethyldisulfanyl]ethylamino]-5-oxo-pentanoyl]amino]ethyldisulfanyl]ethylamino]-4-oxo-butanoate (38c) Dissolve Fmoc-glutamic acid (0.2 mmol; 74 mg) in 2 mL of anhydrous DMF under an inert atmosphere with stirring. Continuously add DIC (0.5 mmol; 77 μL) and then HOBt (0.5 mmol; 68 mg) to the reaction medium. Stir the reaction mixture for 3 hours, then add a solution of 38a (0.5 mmol; 300 mg) and NEt3 (0.6 mmol; 84 μL) in 32 mL of anhydrous CHCl dropwise to the reaction medium. After 48 hours, stop the reaction and evaporate the reaction medium to dryness under crude vacuum. Return the residue to 50 mL of  and filter to remove DIU. Wash the organic layer with water and brine, dry over MgSO4, and evaporate. Dissolve the crude product in 10 mL of DMF and add NaN3 (20 mg). Heat the reaction medium at 50°C for 16 hours. After complete deprotection of Fmoc (TLC Rf=0.4 DCM:MeOH 95 / 5(v / v)), the medium is evaporated and the crude product is purified by flash chromatography on silica gel (elution gradient of 2-6% MeOH in DCM). In this way, the product is isolated in 46% yield (0.09 mmol; 100 mg).

[0380] Synthesis of molecules XXXVIII and XXXIX 2-Hexyldecyl 4-[2-[2-[[4-[[2-(dimethylamino)acetyl]amino]-5-[2-[2-[[4-(2-hexyldecoxy)-4-oxo-butanoyl]amino]ethyldisulfanyl]ethylamino]-5-oxo-pentanoyl]amino]ethyldisulfanyl]ethylamino]-4-oxo-butanoate (XXXVIII) Following the procedure described for compound XXXIV, molecule XXXVIII was obtained by coupling DMG (0.2 mmol, 21 mg) with 38c (0.09 mmol, 100 mg) using DIPEA (0.4 mmol; 68 μL) and HBTU (0.2 mmol; 76 mg) dissolved in 2 mL of DMF. Reaction time: 36 hours at room temperature; Purification: Silica gel chromatography gradient 98 / 2~92 / 8 (DCM / MeOH, v / v). Yield: 42% (51 mg, 0.04 mmol, TLC (DCM / MeOH: 90 / 10) Rf: 0.3).

[0381] 2-Hexyldecyl 4-[2-[2-[[4-[bis(2-hydroxypropyl)amino]-5-[2-[2-[[4-(2-hexyldecoxy)-4-oxo-butanoyl]amino]ethyldisulfanyl]ethylamino]-5-oxo-pentanoyl]amino]ethyldisulfanyl]ethylamino]-4-oxo-butanoate(XXXIX) Following the procedure described for compound XXXIII, molecule XXXIX was obtained by alkylating 38c (0.07 mmol, 75 mg) with a large excess of propylene oxide (500 μL) in 2 mL of MeOH. Reaction time: 16 hours at room temperature; Purification: Silica gel chromatography gradient 98 / 2~90 / 10 (DCM / MeOH, v / v). Yield: 13% (10 mg, 0.009 mmol, TLC (DCM / MeOH: 90 / 10) Rf: 0.3).

[0382] Synthesis of molecule XL 4-[(Z)-non-2-enoxy]-4-oxo-butanoic acid (40a) Dissolve cis-2-noneol (35 mmol; 5.92 mL) in 70 mL of a 1 / 2 (v / v) mixture of dry DCM and DMF under argon. Slowly add DMAP (35 mmol; 4 g) and stir the reaction mixture for 5 minutes. Then add succinic anhydride (53 mmol, 5.3 g) and stir the mixture at room temperature for 48 hours. Then concentrate the reaction mixture, return the crude product to 50 mL of toluene, and wash with 1 M HCl, water, and brine. Dry the organic layer over MgSO4 and evaporate. A clear oil is obtained in 97% yield (34 mmol; 8.3 g).

[0383] [4-(9H-fluorene-9-ylmethoxycarbonylamino)-5-[4-(2-hexyldecanoyloxy)butylamino]-5-oxopentyl]ammonium(40b) FmocOrn(Boc)OH (1 mmol, 454 mg) was placed in a dry 50 mL flask under an inert atmosphere, and then dissolved in 10 mL of anhydrous DMF with stirring. COMU (1.2 mmol; 514 mg) and DIPEA (3 mmol; 509 μL) were successively added to the reaction medium. The reaction mixture was then maintained at 0°C for 15 minutes under an inert atmosphere, and then 32b (1.3 mmol; 560 mg) was added to the reaction medium with stirring. The solution was left overnight under an inert atmosphere and then heated to ambient temperature. The mixture was evaporated to dryness under a rough vacuum. The residue was returned to 50 mL of  and washed with 1 M HCl, water, and brine. The organic layer was dried over MgSO4 and evaporated. The product was then dissolved in 50 mL of DCM with 2.5 mL of TFA. After 15 hours, the mixture is concentrated and co-evaporated three times with 50 mL of DCM, then three times with 50 mL of diethyl ether (Et2O), and dried overnight under high vacuum. The product is then used as is without further purification (850 mg).

[0384] 4-[[2-amino-5-[[4-[(Z)-non-2-enoxy]-4-oxo-butanoyl]amino]pentanoyl]amino]butyl 2-hexyldecanoate (40c) 40a (0.8 mmol; 194 mg) is placed in a dry 50 mL flask under an inert atmosphere, and then dissolved in 10 mL of anhydrous DMF with stirring. COMU (0.9 mmol; 385 mg) and DIPEA (2 mmol; 340 μL) are continuously added to the reaction medium. The reaction mixture is then maintained at 0°C for 15 minutes under an inert atmosphere, and then 40b (1 mmol, 850 mg) dissolved in 5 mL of DMF is added to the reaction medium with stirring. The solution is left overnight under an inert atmosphere and then heated to room temperature. The mixture is evaporated to dryness under crude vacuum. The residue is returned to 50 mL of  and washed with 1 M HCl, water, and brine. The organic layer is dried over MgSO4 and evaporated. The product is then dissolved in 20 mL of DMF containing 4 mL of piperidine. After 15 hours, the mixture is concentrated, and the crude product is purified by flash chromatography on silica gel (elution gradient of 2–8% MeOH in DCM). In this way, the product is isolated in 42% yield (0.42 mmol; 280 mg).

[0385] 4-[[2-[[2-(dimethylamino)acetyl]amino]-5-[[4-[(Z)-non-2-enoxy]-4-oxo-butanoyl]amino]pentanoyl]amino]butyl 2-hexyldecanoate (XL) Following the procedure described for compound XXXIV, molecule XL was obtained by coupling DMG (0.2 mmol, 21 mg) with 40c (0.15 mmol, 100 mg) using DIPEA (0.4 mmol; 68 μL) and HBTU (0.3 mmol; 114 mg) dissolved in 5 mL of DMF. Reaction time: 36 hours at room temperature; Purification: Silica gel chromatography gradient 98 / 2~92 / 8 (DCM / MeOH, v / v). Yield: 42% (47 mg, 0.06 mmol, TLC (DCM / MeOH: 90 / 10) Rf: 0.3).

[0386] Synthesis of molecule XLI 5-[3-(dimethylamino)-2-hydroxypropoxy]-4-(9H-fluoren-9-ylmethoxycarbonylamino)-5-oxopentanoic acid (41a) Iodine (1.5 mmol; 171 mg) is dissolved in 20 mL of anhydrous DCM using a molecular sieve under an inert atmosphere with stirring. Triphenylphosphine (1.5 mmol; 393 mg), imidazole (3.3 mmol; 224 mg), and then Fmoc-Glu(OtBu)-OH (1 mmol; 426 mg) are successively added to the reaction medium. The reaction mixture is then stirred for 10 minutes, after which 3-(dimethylamino)-1,2-propanediol (1.5 mmol; 178 μL) is added dropwise to the reaction medium. After 20 hours, 30 mL of DCM is added, the organic layer is washed twice with 1 M HCl and brine, dried over MgSO4, and evaporated. The crude product is purified by flash chromatography on silica gel (elution gradient of 40-80% EtOAC in EP). In this way, the product is isolated in 90% yield (0.9 mmol; 500 mg). TLC: Rf = 0.5 (ا:EP 50 / 50 (v / v))

[0387] Next, the product is dissolved in 50 mL of DCM with 2.5 mL of TFA. After 15 hours, the mixture is concentrated and co-evaporated three times with 50 mL of DCM, then three times with 50 mL of diethyl ether (Et2O), and dried overnight under high vacuum. The product is then used as is without further purification.

[0388] 4-[[4-amino-5-[3-(dimethylamino)-2-hydroxy-propoxy]-5-oxo-pentanoyl]amino]butyl 2-hexyldecanoate (41b) 41a (0.5 mmol, 242 mg) is placed in a dry 50 mL flask under an inert atmosphere, and then dissolved in 5 mL of anhydrous DMF with stirring. COMU (0.6 mmol; 257 mg) and DIPEA (1.5 mmol; 255 μL) are added successively to the reaction medium. The reaction mixture is then maintained at 0°C for 15 minutes under an inert atmosphere, and then 32b (0.6 mmol; 257 mg) dissolved in 4 mL of DCM / DMF is added to the reaction medium with stirring. The solution is left overnight under an inert atmosphere and then heated to room temperature. The mixture is evaporated to dryness under a rough vacuum. The residue is returned to 50 mL of  and washed with 1 M HCl, water, and brine. The organic layer is dried over MgSO4 and evaporated. The product is then dissolved in 10 mL of DMF containing 350 mg of NaN, and the reaction medium is heated at 50°C. After 15 hours, the mixture was concentrated, and the crude product was purified by flash chromatography on silica gel (elution gradient of 2-5% MeOH in DCM). The product was thus isolated in 34% yield (0.17 mmol; 96 mg). TLC: Rf = 0.5 (DCM / MeOH 95 / 5 (v / v))

[0389] 4-[[5-[3-(dimethylamino)-2-hydroxypropoxy]-4-(dodecanoylamino)-5-oxopentanoyl]amino]butyl 2-hexyldecanoate (XLI) 41b (0.17 mmol, 96 mg) was placed in a dry 25 mL flask under an inert atmosphere and then dissolved in 5 mL of anhydrous DMF with stirring. PyBOP (0.22 mmol; 116 mg) and DIPEA (0.4 mmol; 70 μL) were successively added to the reaction medium. The reaction mixture was then maintained under an inert atmosphere for 5 minutes, after which dodecanoic acid (0.2 mmol; 54 mg) was added to the reaction medium with stirring. After 20 hours, the mixture was evaporated to dryness under crude vacuum. The crude product was purified by flash chromatography using silica gel (elution gradient of 1-5% MeOH in DCM). In this way, the product was isolated in 45% yield (0.076 mmol; 56 mg). TLC: Rf = 0.6 (DCM / MeOH 95 / 5 (v / v)).

[0390] Other synthesis methods: Synthesis of the molecule N,N'-bis[(Z)-octadeca-9-enyl]-2-[[4-oxo-4-(2-pyrrolidine-1-ylethylamino)butanoyl]amino]pentanediamide 4-Oxo-4-(2-pyrrolidine-1-ylethylamino)butanoic acid 2-Pyrrolidine-1-ylethaneamine (251 μL, 2 mmol) was dissolved in 20 mL of a 1 / 1 (v / v) mixture of dry DCM and DMF under argon. Then, 2 equivalents (4 mmol, 560 μL) of triethylamine (Et3N) were slowly added, and the reaction mixture was stirred for 5 minutes. Then, succinic anhydride (4 mmol, 400 mg) was added, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was then concentrated and redissolved in 80 mL of ethyl acetate. The organic phase was then washed with saturated aqueous ammonium chloride solution, washed twice with Milli-Q water and once with brine, dried over MgSO4, filtered, and concentrated. This post-treatment, after high vacuum drying TLC (DCM / MeOH: 95 / 5) rf: 0.7, yielded a clean expected product in 79% yield (339 mg, 1.58 mmol).

[0391] N,N'-Bis[(Z)-Octadeca-9-enyl]-2-[[4-Oxo-4-(2-pyrrolidine-1-ylethylamino)butanoyl]amino]pentanediamide The molecule N,N'-bis[(Z)-octadeca-9-enyl]-2-[[4-oxo-4-(2-pyrrolidine-1-ylethylamino)butanoyl]amino]pentanediamide was converted to MeCN and DCM 2 / 1(v / ) according to the procedure described for compound 2-[4-(dimethylamino)butanoylamino]-N'-[(Z)-heptadeca-9-enyl]-N-[(Z)-octadeca-9-enyl]pentanediamide. v) Compound oxo-4-(2-pyrrolidine-1-ylethylamino)butanoic acid (54 mg, 0.25 mmol) was isolated from the coupling of compound 2-amino-N,N'-bis[(Z)-octadeca-9-enyl]pentanediamide (0.25 mmol, 161 mg) using methylimidazole (74 μL, 0.92 mmol) and TCFH (98 mg, 0.35 mmol) in 4 mL of mixed solution. Reaction time: 12 hours at room temperature; Purification: Silica gel chromatography gradient 98 / 2~90 / 10 (DCM / MeOH, v / v). Yield: 65% (135 mg, 0.16 mmol, white solid TLC (DCM / MeOH: 90 / 10) rf: 0.4).

[0392] Synthesis of the molecule 2-[[4-[2-[2-[[2-(dimethylamino)acetyl]amino]ethyldisulfanyl]ethylamino]-4-oxobutanoyl]amino]-N,N'-bis[(Z)-octadeca-9-enyl]pentanediamide 4-[2-[2-(tert-butoxycarbonylamino)ethyldisulfanyl]ethylamino]-4-oxo-butanoic acid Compound 4-[2-[2-(tert-butoxycarbonylamino)ethyldisulfanyl]ethylamino]-4-oxo-butanoic acid was obtained by reacting Boc-cystamine (5.3 mmol; 1.34 g), succinic anhydride (10.6 mmol; 1.06 g), and triethylamine (7.95 mmol; 1.1 mL) in 20 mL of DCM and 5 mL of DMF, according to the method described for compound 4-oxo-4-(2-pyrrolidine-1-ylethylamino)butanoic acid. Reaction time: 16 hours at room temperature; Purification: Silica gel chromatography gradient 98 / 2~95 / 5 (DCM / MeOH, v / v). Yield: 93% (1.6 g, 4.92 mmol, TLC (DCM / MeOH: 90 / 10) Rf: 0.6).

[0393] 2-[2-[[4-[[4-[[(Z)-octadeca-9-enyl]amino]-1-[[(Z)-octadeca-9-enyl]carbamoyl]-4-oxo-butyl]amino]-4-oxo-butanoyl]amino]ethyldisulfanyl]ethylammonium trifluoroacetate 4-[2-[2-(tert-butoxycarbonylamino)ethyldisulfanyl]ethylamino]-4-oxo-butanoic acid (1.6 mmol; 500 mg) is placed in a dry 100 mL flask under an inert atmosphere, and then dissolved in 40 mL of DCM while stirring. DIC (2.3 mmol; 360 μL) and HOBt (2.3 mmol; 314 mg) dissolved in 5 mL of anhydrous DMF are continuously added to the reaction medium. The reaction mixture is then maintained at ambient temperature under an inert atmosphere for 2.5 hours. A solution of 2-amino-N,N'-bis[(Z)-octadeca-9-enyl]pentanediamide (2.2 mmol; 1.4 g) in 10 mL of anhydrous DCM containing triethylamine (2.9 mmol; 400 μL) and 5 mL of anhydrous DMF is added to the reaction medium while stirring. After 48 hours, the reaction is stopped, and the reaction medium is evaporated to dryness under crude vacuum. The intermediate product was first purified by flash chromatography on silica gel (elution gradient of 2-8% MeOH in DCM), then dissolved in 33 mL of DCM in a 50 mL flask, and 2.5 mL of trifluoroacetic acid was added to the reaction medium. The reaction mixture was then maintained overnight at ambient temperature with stirring. The reaction medium was evaporated to dryness under crude vacuum. To remove excess trifluoroacetic acid, the final residue was returned to DCM four times consecutively and then to Et2O four times, followed by evaporation to dryness. In this way, the pure product was isolated in 42% yield (0.68 mmol; 657 mg, 2 steps).

[0394] Synthesis of molecules 2-[[4-[2-[2-[[2-(dimethylamino)acetyl]amino]ethyldisulfanyl]ethylamino]-4-oxo-butanoyl]amino]-N,N'-bis[(Z)-octadeca-9-enyl]pentanediamide and 2-[[4-[2-[2-[4-(dimethylamino)butanoylamino]ethyldisulfanyl]ethylamino]-4-oxo-butanoyl]amino]-N,N'-bis[(Z)-octadeca-9-enyl]pentanediamide

[0395] 2-[[4-[2-[2-[[2-(dimethylamino)acetyl]amino]ethyldisulfanyl]ethylamino]-4-oxo-butanoyl]amino]-N,N'-bis[(Z)-octadeca-9-enyl]pentanediamide DMG (0.4 mmol, 41 mg) is placed in a dry 25 mL flask under an inert atmosphere, and then dissolved in 2 mL of anhydrous DMF while stirring. 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, 0.4 mmol; 152 mg) and DIPEA (0.8 mmol; 136 μL) are continuously added to the reaction medium. Next, the reaction mixture was maintained at ambient temperature under an inert atmosphere for 15 minutes, after which a solution of 2-[2-[[4-[[4-[[(Z)-octadeca-9-enyl]amino]-1-[[(Z)-octadeca-9-enyl]carbamoyl]-4-oxo-butyl]amino]-4-oxo-butanoyl]amino]ethyldisulfanyl]ethylammonium trifluoroacetate (0.2 mmol; 198 mg) in 1 mL of anhydrous DCM and 3 mL of DMF was added to the reaction medium. After stirring at ambient temperature under an inert atmosphere for 48 hours, the reaction was stopped and the reaction medium was evaporated to dryness under crude vacuum. The product was purified by flash chromatography with silica gel (elution gradient of 2-8% MeOH in DCM). In this way, the product was isolated in 32% yield (0.064 mmol; 60 mg). TLC:Rf=0.4(DCM:MeOH 95 / 5(v / v)).

[0396] 2-[[4-[2-[2-[4-(dimethylamino)butanoylamino]ethyldisulfanyl]ethylamino]-4-oxo-butanoyl]amino]-N,N'-bis[(Z)-octadeca-9-enyl]pentanediamide The molecule 2-[[4-[2-[2-[4-(dimethylamino)butanoylamino]ethyldisulfanyl]ethylamino]-4-oxo-butanoyl]amino]-N,N'-bis[(Z)-octadeca-9-enyl]pentanediamide is prepared in 6 mL of DMF and DCM according to the procedure described for the molecule 2-[[4-[2-[[2-(dimethylamino)acetyl]amino]ethyldisulfanyl]ethylamino]-4-oxo-butanoyl]amino]-N,N'-bis[(Z)-octadeca-9-enyl]pentanediamide. The compound was isolated by coupling DMABA (0.6 mmol, 101 mg) with 2-[2-[[4-[[(Z)-octadeca-9-enyl]amino]-1-[[(Z)-octadeca-9-enyl]carbamoyl]-4-oxo-butyl]amino]-4-oxo-butanoyl]amino]ethyldisulfanyl]ethylammonium trifluoroacetate (0.3 mmol, 298 mg) using HBTU (0.6 mmol, 227 mg) and DIPEA (1.2 mmol, 204 μL) in 2 mL. Reaction time: 48 hours at room temperature; Purification: Silica gel chromatography gradient 98 / 2~92 / 8 (DCM / MeOH, v / v). Yield: 35% (110 mg, 0.10 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.4).

[0397] Synthesis of the molecular bis[(9Z,12Z)-octadeca-9,12-dienyl]2-[[4-oxo-4-(4-pyrrolidine-1-ylbutoxy)butanoyl]amino]pentanediate 4-[[4-[(9Z,12Z)-octadeca-9,12-dienoxy]-1-[(9Z,12Z)-octadeca-9,12-dienoxy]carbonyl-4-oxobutyl]amino]-4-oxobutanoic acid The compound bis[(9Z,12Z)-octadeca-9,12-dienyl]2-aminopentanedioate (1 mmol; 644 mg) was placed in a dry 50 mL flask under an inert atmosphere, and then dissolved in 15 mL of DCM and 5 mL of anhydrous DMF with stirring. Succinic anhydride (1.5 mmol; 150 mg) and triethylamine (2 mmol; 280 μL) were added to the reaction medium. After stirring at ambient temperature for 16 hours under an inert atmosphere, the reaction was stopped, and the reaction medium was evaporated to dryness under high vacuum. The crude product was purified by flash chromatography using silica gel (elution gradient of 0-6% MeOH in DCM). The product was obtained in 91% yield (0.91 mmol; 677 mg). TLC: Rf = 0.5 (DCM: MeOH 95 / 5 (v / v)).

[0398] Bis[(9Z,12Z)-octadeca-9,12-dienyl]2-[[4-oxo-4-(4-pyrrolidine-1-ylbutoxy)butanoyl]amino]pentanedioate 4-(1-pyrrolidinyl)-1-butanol (0.7 mmol; 100 mg) is added with stirring to a solution of 4-[[4-[(9Z,12Z)-octadeca-9,12-dienoxy]-1-[(9Z,12Z)-octadeca-9,12-dienoxy]carbonyl-4-oxo-butyl]amino]-4-oxo-butanoic acid (0.47 mmol; 350 mg), N,N'-dicyclohexylcarbodiimide (DCC, 0.7 mmol; 145 mg), and 4-(dimethylamino)pyridine (DMAP, 0.094 mmol; 11 mg) in 18 mL of DCM. After stirring at ambient temperature for 15 hours under an inert atmosphere, the reaction is stopped and the reaction medium is evaporated to dryness under high vacuum. The crude product was returned to 20 mL of DCM and washed with 1 M HCl, NaHCO3, and brine. The organic layer was dried over MgSO4 and purified by flash chromatography on silica gel (elution gradient of 2–10% MeOH in DCM). The product was thus isolated in 73% yield (0.34 mmol; 298 mg). TLC: Rf = 0.3 (DCM: MeOH 95 / 5 (v / v)).

[0399] Synthesis of the molecular bis[(9Z,12Z)-octadeca-9,12-dienyl]2-[[4-[3-[bis(2-hydroxyethyl)amino]propylamino]-4-oxo-butanoyl]amino]pentanediate Bis[(9Z,12Z)-octadeca-9,12-dienyl]2-[[4-[3-[bis(2-hydroxyethyl)amino]propylamino]-4-oxo-butanoyl]amino]pentanedioate The molecular bis[(9Z,12Z)-octadeca-9,12-dienyl]2-[[4-[3-[bis(2-hydroxyethyl)amino]propylamino]-4-oxo-butanoyl]amino]pentanediate is converted to DMF according to the procedure described for the molecular 2-[[4-[2-[2-[[2-(dimethylamino)acetyl]amino]ethyldisulfanyl]ethylamino]-4-oxo-butanoyl]amino]-N,N'-bis[(Z)-octadeca-9-enyl]pentanediamide Compound 4-[[4-[(9Z,12Z)-octadeca-9,12-dienoxy]-1-[(9Z,12Z)-octadeca-9,12-dienoxy]carbonyl-4-oxo-butyl]amino]-4-oxo-butanoic acid (0.4 mmol, 298 mg) was isolated by coupling with N-(3-aminopropyl)diethanolamine (0.5 mmol, 76 μL) using HBTU (0.4 mmol, 152 mg) and DIPEA (0.8 mmol, 136 μL) dissolved in 8 mL. Reaction time: 16 hours at room temperature; Purification: Silica gel chromatography gradient 98 / 2~90 / 10 (DCM / MeOH, v / v). Yield: 66% (0.26 mmol, 234 mg), TLC (DCM / MeOH: 95 / 5) Rf: 0.2).

[0400] Synthesis of the molecule 2-[[2-[2,5-bis[4-(dimethylamino)butanoylamino]pentanoylamino]acetyl]amino]ethyl 4-[[4-[[(9Z,12Z)-octadeca-9,12-dienyl]amino]-1-[[(9Z,12Z)-octadeca-9,12-dienyl]carbamoyl]-4-oxo-butyl]amino]-4-oxo-butanoate tert-butyl-N-[4-(tert-butoxycarbonylamino)-5-[[2-(2-hydroxyethylamino)-2-oxo-ethyl]amino]-5-oxo-pentyl]carbamate The molecule tert-butyl-N-[4-(tert-butoxycarbonylamino)-5-[[2-(2-hydroxyethylamino)-2-oxo-ethyl]amino]-5-oxo-pentyl]carbamate was isolated by coupling Boc-L-Orn(Boc)-OH (1.5 mmol, 500 mg) with 2-amino-N-(2-hydroxyethyl)acetamide (2 mmol; 236 mg) using HBTU (1.5 mmol; 569 mg) and DIPEA (2 mmol; 340 μL) dissolved in 20 mL of DMF, following the procedure described for the molecule 2-[[4-[2-[2-[[2-(dimethylamino)acetyl]amino]ethyldisulfanyl]ethylamino]4-oxo-butanoyl]amino]-N,N'-bis[(Z)-octadeca-9-enyl]pentanediamide. Reaction time: 48 hours at room temperature; Purification: Silica gel chromatography gradient 96 / 4~90 / 10 (DCM / MeOH, v / v). Yield: 62% (402 mg, 0.93 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.3).

[0401] 4-[[4-[[(9Z,12Z)-octadeca-9,12-dienyl]amino]-1-[[(9Z,12Z)-octadeca-9,12-dienyl]carbamoyl]-4-oxo-butyl]amino]-4-oxo-butanoic acid Molecular 4-[[4-[[(9Z,12Z)-octadeca-9,12-dienyl]amino]-1-[[(9Z,12Z)-octadeca-9,12-dienyl]carbamoyl]-4-oxo-butyl]amino]-4-oxo-butanoic acid was obtained using 20 mL of a 1 / 1 (v / v) mixture of dry DCM and DMF containing 2-amino-N,N'-bis[(9Z,12Z)-octadeca-9,12-dienyl]pentanediamide (1.28 g, 2 mmol), Et3N (4 mmol, 560 μL), and succinic anhydride (4 mmol, 400 mg), dissolved under argon, following the procedure described for the synthesis of molecular 4-oxo-4-(2-pyrrolidine-1-ylethylamino)butanoic acid. Reaction time: 18 hours, yield 78% (1.16 g, 1.56 mmol), TLC (DCM / MeOH: 95 / 5) rf: 0.5.

[0402] [4-Azaniumyl-5-[[2-[2-[4-[[4-[[(9Z,12Z)-Octadeca-9,12-Dienyl]amino]-1-[[(9Z,12Z)-Octadeca-9,12-Dienyl]Carbamoyl]-4-Oxo-Butyl]amino]-4-Oxo-Butanoyl]Oxyethylamino]-2-Oxo-Ethyl]amino]-5-Oxo-Pentyl]ammonium Compound 4-[[4-[[(9Z,12Z)-octadeca-9,12-dienyl]amino]-1-[[(9Z,12Z)-octadeca-9,12-dienyl]carbamoyl]-4-oxo-butyl]amino]-4-oxo-butanoic acid (0.3 mmol, 224 mg) was placed in a dry 25 mL flask under argon, and then dissolved in 5 mL of DCM with stirring. DCC (0.6 mmol; 124 mg) and DMAP (0.1 mmol; 12 mg) were added to the reaction medium. Next, the reaction mixture is stirred for 10 minutes, after which 4-[[4-[[(9Z,12Z)-octadeca-9,12-dienyl]amino]-1-[[(9Z,12Z)-octadeca-9,12-dienyl]carbamoyl]-4-oxo-butyl]amino]-4-oxo-butanoic acid (0.4 mmol; 173 mg) is introduced. After stirring under argon at room temperature for 16 hours, the reaction is stopped and the medium is evaporated under vacuum. The crude product is returned to 20 mL of DCM and washed with 1 M HCl, NaHCO3 and brine. The organic extract was dried over MgSO4 and purified by flash chromatography on silica gel (elution gradient of 2-5% MeOH in DCM, TLC: Rf = 0.5 (DCM: MeOH 90 / 10 (v / v))). It was then dissolved in 20 mL of DCM with the addition of 1 mL of trifluoroacetic acid. The reaction mixture was then maintained overnight at room temperature with stirring. The reaction medium was evaporated, co-evaporated four times in DCM and four times in Et2O, and then dried. Yield 84% (2 steps, 0.252 mmol, 299 mg).

[0403] 2-[[2-[2,5-bis[4-(dimethylamino)butanoylamino]pentanoylamino]acetyl]amino]ethyl 4-[[4-[[(9Z,12Z)-octadeca-9,12-dienyl]amino]-1-[[(9Z,12Z)-octadeca-9,12-dienyl]carbamoyl]-4-oxo-butyl]amino]-4-oxo-butanoate The molecule 2-[[2-[2,5-bis[4-(dimethylamino)butanoylamino]pentanoylamino]acetyl]amino]ethyl 4-[[4-[[(9Z,12Z)-octadeca-9,12-dienyl]amino]-1-[[(9Z,12Z)-octadeca-9,12-dienyl]carbamoyl]-4-oxo-butyl]amino]-4-oxo-butanoate was prepared using the procedure described for the molecule 2-[[4-[[2-[[2-(dimethylamino)acetyl]amino]ethyldisulfanyl]ethylamino]-4-oxo-butanoyl]amino]-N,N'-bis[(Z)-octadeca-9-enyl]pentanediamide, in 6 mL of DMF and DCM. The compound [4-azaniumyl-5-[[2-[2-[4-[[4-[[(9Z,12Z)-octadeca-9,12-dienyl]amino]-1-[[(9Z,12Z)-octadeca-9,12-dienyl]carbamoyl]-4-oxo-butyl]amino]-4-oxo-butanoyl]oxyethylamino]-2-oxo-ethyl]amino]-5-oxo-pentyl]ammonium (0.25 mmol; 290 mg) was isolated by coupling with 2 equivalents of DMABA (0.6 mmol; 168 mg) using HBTU (0.6 mmol; 227 mg) and DIPEA (1.2 mmol; 204 μL) dissolved in 2 mL. Reaction time: 12 hours at room temperature; Purification: Silica gel chromatography gradient 98 / 2~90 / 10 (DCM / MeOH, v / v). Yield: 58% (0.15 mmol; 117 mg), TLC (DCM / MeOH: 95 / 5) Rf: 0.2).

[0404] Synthesis of the molecule 4-[[4-[[4-[4-[4-[(dimethylamino)methyl]triazol-1-yl]butylamino]-4-oxo-butanoyl]amino]-5-(4-dodecanoyloxybutylamino)-5-oxo-pentanoyl]amino]butyldodecanoate 4-[[4-(4-dodecanoyloxybutylamino)-1-(4-dodecanoyloxybutylcarbamoyl)-4-oxo-butyl]amino]-4-oxo-butanoic acid The molecule 4-[[4-(4-dodecanoyloxybutylamino)-1-(4-dodecanoyloxybutylcarbamoyl)-4-oxo-butyl]amino]-4-oxo-butanoic acid was isolated using the procedure described for compound 4-oxo-4-(2-pyrrolidine-1-ylethylamino)butanoic acid, by dissolving compound 4-[[4-amino-5-(4-dodecanoyloxybutylamino)-5-oxo-pentanoyl]amino]butyl dodecanoate (491 mg, 0.75 mmol) in 4 mL of a 1 / 1 (v / v) mixture of DCM and DMF under argon, along with Et3N (1.5 mmol, 210 μL) and succinic anhydride (1.5 mmol, 150 mg). Reaction time: 12 hours at room temperature. Yield 82% (463 mg, 0.62 mmol, TLC (DCM / MeOH: 95 / 5) rf: 0.6).

[0405] 4-[[4-[[4-(4-azidobutylamino)-4-oxo-butanoyl]amino]-5-(4-dodecanoyloxybutylamino)-5-oxo-pentanoyl]amino]butyldodecanoate Compound 4-[[4-(4-dodecanoyloxybutylamino)-1-(4-dodecanoyloxybutylcarbamoyl)-4-oxo-butyl]amino]-4-oxo-butanoic acid (231 mg, 0.31 mmol) is dissolved in 3 mL of a 9 / 1 v / v mixture of dry DCM / DMF in a three-necked round-bottom flask equipped with a dropping funnel under argon. Subsequently, HOBt (55 mg, 0.4 mmol) and DIC (62 μL, 0.4 mmol) are added, and the reaction mixture is stirred at room temperature for 2 hours and 30 minutes to increase the turbidity of the medium. Then, 4-azidobutylamine (1 M in DCM, 470 μL, 0.47 mmol) and Et3N (87 μL, 0.62 mmol), dissolved in 3 mL of a 9 / 1 v / v mixture of dry DCM / DMF, are added dropwise through a dropping funnel over 30 minutes. After the addition is complete, the reaction mixture is stirred at room temperature for 33 hours and concentrated to dryness. The crude mixture is then resuspended in 20 mL of ethyl acetate and left at -20°C for 3 hours. The resulting liquid and solid are then separated by centrifugation at 3000 rpm, and this procedure is repeated three times. All liquids are collected, discarding the solid, and concentrated under vacuum. The remaining oil is then dried overnight under vacuum to obtain the desired compound (182 mg, 0.21 mmol). TLC (DCM / MeOH:98 / 2) rf:0.5.

[0406] 4-[[4-[[4-[4-[4-[(dimethylamino)methyl]triazol-1-yl]butylamino]-4-oxo-butanoyl]amino]-5-(4-dodecanoyloxybutylamino)-5-oxo-pentanoyl]amino]butyldodecanoate Copper sulfate heptahydrate (0.012 mmol, 3 mg), ascorbic acid (Sigma, 0.06 mmol, 12 mg), and triphenylphosphine (0.012 mmol, 3 mg) are mixed together in 1 mL of dry DMSO with stirring. While stirring this solution (for 15 minutes), the compounds 4-[[4-[[4-(4-azidobutylamino)-4-oxo-butanoyl]amino]-5-(4-dodecanoyloxybutylamino)-5-oxo-pentanoyl]amino]butyldodecanoate (87 mg, 0.1 mmol) and N,N-dimethylpropargylamine (108 μL, 1 mmol) are dissolved in 4 mL of THF. After the starting materials are completely dissolved, the copper-based yellowish solution is added to the reaction flask and stirred with the starting materials at room temperature for 12 hours. The reaction mixture is then diluted with 10 mL of water and extracted with 4 × 20 mL of DCM. Next, the organic extracts were combined, dried over magnesium sulfate, filtered, and concentrated under vacuum. The product was recovered by flash chromatography with silica gel using a DCM / methanol mixture as the eluent. A gradient ranging from 99 / 1 to 90 / 10 (DCM / MeOH, v / v) was used. 135 mg (0.145 mmol, 49% yield) of white solid was recovered. TLC (DCM / MeOH: 95 / 5) rf: 0.3. 4-[[1-[[4-[(Z)-non-2-enoxy]-4-oxo-butyl]carbamoyl]-4-oxo-4-(tetradecylamino)butyl]amino]-4-oxo-butanoic acid (31a) Synthesis of the molecule [(Z)-non-2-enyl]-4-[[2-[[4-[3-[bis(2-hydroxyethyl)amino]propylamino]-4-oxo-butanoyl]amino]-5-oxo-5-(tetradecylamino)pentanoyl]amino]butanoate

[0407] Molecular 4-[[1-[[4-[(Z)-non-2-enoxy]-4-oxo-butyl]carbamoyl]-4-oxo-4-(tetradecylamino)butyl]amino]-4-oxo-butanoic acid was isolated using compound [(Z)-non-2-enyl]4-[[2-amino-5-oxo-5-(tetradecylamino)pentanoyl]amino]butanoate (83 mg, 0.15 mmol) dissolved in 3 mL of DCM under argon, Et3N (0.4 mmol, 56 μL), and succinic anhydride (0.3 mmol, 30 mg), following the procedure described for compound 4-oxo-4-(2-pyrrolidine-1-ylethylamino)butanoic acid. Reaction time: 16 hours at room temperature. Purification: Silica gel chromatography gradient 100 / 0~95 / 5 (DCM / MeOH, v / v). Yield: 91% (white solid 89 mg, 0.14 mmol, TLC (DCM / MeOH: 90 / 10) Rf: 0.4).

[0408] [(Z)-non-2-enyl]-4-[[2-[[4-[3-[bis(2-hydroxyethyl)amino]propylamino]-4-oxo-butanoyl]amino]-5-oxo-5-(tetradecylamino)pentanoyl]amino]butanoate(XXXI) Compound [(Z)-non-2-enyl]-4-[[2-[[4-[3-[bis(2-hydroxyethyl)amino]propylamino]-4-oxo-butanoyl]amino]-5-oxo-5-(tetradecylamino)pentanoyl]amino]butanoate was dissolved in 4 mL of a 2 / 1 (v / v) mixed solution of MeCN and DCM, and methylimidazole (0.4 mmol; 32 μL) and TCFH (0.14 mmol; 40 mg) were used to prepare compound 4-[[1-[[4-[(Z)-non-2-enyl] Compound 2-[4-(dimethylamino)butanoylamino]-N'-[(Z)-heptadeca-9-enyl]-N-[(Z)-octadeca-9-enyl]pentanediamide was isolated by linking 0.14 mmol; 89 mg of [xy]-4-oxo-butyl]carbamoyl]-4-oxo-4-(tetradecylamino)butyl]amino]-4-oxo-butanoic acid with N-(3-aminopropyl)diethanolamine (0.12 mmol; 18 μL), adapting the procedure described for compound 2-[4-(dimethylamino)butanoylamino]-N'-[(Z)-heptadeca-9-enyl]pentanediamide. Reaction time: 12 hours at 0°C to room temperature; Purification: Silica gel chromatography gradient 98 / 2 to 90 / 10 (DCM / MeOH, v / v). Yield: 64% (white solid 61 mg, 0.08 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.3).

[0409] Synthesis of the molecule [(Z)-non-2-enyl]4-[[2-[[4-[2-[2-[2,5-bis[[2-(dimethylamino)acetyl]amino]pentanoylamino]ethyldisulfanyl]ethylamino]-4-oxo-butanoyl]amino]-5-oxo-5-(tetradecylamino)pentanoyl]amino]butanoate 2-[2-[[4-[[1-[[4-[(Z)-non-2-enoxy]-4-oxo-butyl]carbamoyl]-4-oxo-4-(tetradecylamino)butyl]amino]-4-oxo-butanoyl]amino]ethyldisulfanyl]ethylammonium Boc-cystamine (0.28 mmol; 91 mg) is placed in a 25 mL flask under argon and then dissolved in 2 mL of anhydrous DMF while stirring. HBTU (0.28 mmol; 106 mg) and DIPEA (0.56 mmol; 97 μL) are continuously added to the solution. After stirring the reaction medium under argon at room temperature for 15 minutes, a solution of compound [(Z)-non-2-enyl]4-[[2-amino-5-oxo-5-(tetradecylamino)pentanoyl]amino]butanoate (0.14 mmol; 89 mg) in 1 mL of anhydrous DMF and 1 mL of anhydrous DCM is added to the reaction medium. After 12 hours, the reaction is stopped and the reaction medium is evaporated to dryness under high vacuum. The crude product is returned to 10 mL of siRNA and washed with 1 M HCl, NaHCO3 and brine. The organic layer was dried over MgSO4 and purified by flash chromatography on silica gel (elution gradient of 2-5% MeOH in DCM). The isolated intermediate (0.1 mmol; 79 mg) was dissolved in 5 mL of DCM, and then 250 μL of trifluoroacetic acid was added to the reaction medium. The reaction mixture was then maintained at room temperature with stirring for 4 hours. The mixture was then concentrated and co-evaporated three times with 50 mL of DCM, and three times with 50 mL of diethyl ether (Et2O), and dried overnight under high vacuum. Yield: 67% (2 steps, 84 mg, 0.094 mmol).

[0410] [4-Azaniumyl-5-[2-[2-[[4-[[1-[[4-[(Z)-Non-2-Enoxy]-4-Oxo-Butyl]Carbamoyl]-4-Oxo-4-(Tetradecylamino)Butyl]Amino]-4-Oxo-Butanoyl]Amino]Ethyldisulfanyl]Ethylamino]-5-Oxo-Pentyl]Ammonium Compound [4-azaniumyl-5-[2-[2-[[4-[[1-[[4-[(Z)-non-2-enoxy]-4-oxo-butyl]carbamoyl]-4-oxo-4-(tetradecylamino)butyl]amino]-4-oxo-butanoyl]amino]ethyldisulfanyl]ethylamino]-5-oxo-pentyl]ammonium is obtained by following the two-step procedure experienced for the synthesis of compound 2-[2-[[4-[[1-[[4-[(Z)-non-2-enoxy]-4-oxo-butyl]carbamoyl]-4-oxo-4-(tetradecylamino)butyl]amino]-4-oxo-butanoyl]amino]ethyldisulfanyl]ethylammonium. First, Boc-L-Orn(Boc)-OH (0.2 mmol; 66 mg) and compound 32a (0.1 mmol; 90 mg) were ligated together using HBTU (0.2 mmol; 76 mg) and DIPEA (0.4 mmol; 69 μL) dissolved in 3 mL of DMF at room temperature for 16 hours. After isolation and purification of the intermediate, the amine functional group was released by reacting it with TFA (250 μL) in 5 mL of DCM for 1 hour. Yield: 61% (2 steps, 69 mg, 0.061 mmol).

[0411] [(Z)-non-2-enyl]4-[[2-[[4-[2-[2-[2,5-bis[[2-(dimethylamino)acetyl]amino]pentanoylamino]ethyldisulfanyl]ethylamino]-4-oxo-butanoyl]amino]-5-oxo-5-(tetradecylamino)pentanoyl]amino]butanoate Following the procedure described for compound 2-[[2-(dimethylamino)acetyl]amino]-N,N'-bis[(Z)-octadeca-9-enyl]pentanediamide, the molecule [(Z)-non-2-enyl]4-[[2-[[4-[2-[2-[2,5-bis[[2-(dimethylamino)acetyl]amino]pentanoylamino]ethyldisulfanyl]ethylamino]-4-oxo-butanoyl]amino]-5-oxo-5-(tetradecylamino)pentanoyl]amino]butanoate is converted to DMF The compound [4-azaniumyl-5-[2-[2-[[4-[[1-[[4-[(Z)-non-2-enoxy]-4-oxo-butyl]carbamoyl]-4-oxo-4-(tetradecylamino)butyl]amino]-4-oxo-butanoyl]amino]ethyldisulfanyl]ethylamino]-5-oxo-pentyl]ammonium (0.06 mmol; 68 mg) is obtained by coupling DMG (0.18 mmol; 19 mg) with the compound [4-azaniumyl-5-[2-[2-[[4-[[1-[[4-[(Z)-non-2-enoxy]-4-oxo-butyl]carbamoyl]-4-oxo-4-(tetradecylamino)butyl]amino]-4-oxo-butanoyl]amino]ethyldisulfanyl]ethylamino]-5-oxo-pentyl]ammonium (0.06 mmol; 68 mg) using DIPEA (0.4 mmol; 69 μL) and pyBOP (0.2 mmol; 104 mg) dissolved in 3 mL. Reaction time: 48 hours at room temperature; Purification: Silica gel chromatography gradient 98 / 2~90 / 10 (DCM / MeOH, v / v). Yield: 32% (0.02 mmol; 21 mg), TLC (DCM / MeOH: 95 / 5) Rf: 0.2).

[0412] Synthesis of the molecule 3-butylhepto-2-enyl-4-[[2-[[4-[3-[bis(2-hydroxyethyl)amino]propylamino]-4-oxo-butanoyl]amino]-5-oxo-5-(tetradecylamino)pentanoyl]amino]butanoate a) Synthesis of the molecule [4-(3-butylhept-2-enoxy)-4-oxobutyl]ammonium [4-(3-butylhept-2-enoxy)-4-oxo-butyl]ammonium 3-Butyl-2-hepten-1-ol (5.5 mmol; 1.11 mL) is added, with stirring, to a solution of N-Boc-γ-aminobutyric acid (5 mmol, 1.02 g), DCC (5.5 mmol; 1.13 g), and DMAP (1 mmol; 122 mg) dissolved in 50 mL of anhydrous DCM. The reaction mixture is then maintained under argon at room temperature for 16 hours. After the complete consumption of the acid, the reaction medium is evaporated to dryness under vacuum. The crude product is returned to 50 mL of DCM and washed with 1 M HCl, NaHCO3, and brine. The organic layer is dried over MgSO4 and then purified by flash chromatography on silica gel (elution gradient of 0-50% Â in petroleum ether) to obtain an intermediate (3.95 mmol; 1.4 g), which is dissolved in 100 mL of DCM in the presence of 5 mL of TFA. The reaction mixture is then maintained overnight at ambient temperature with stirring. Next, the mixture is concentrated, co-evaporated three times with 50 mL of DCM, and co-evaporated three times with 50 mL of diethyl ether (Et2O), and dried overnight under high vacuum. Yield: 79% (2 steps, 1.46 g, 3.95 mmol).

[0413] b) Synthesis of the molecules 3-butylhept-2-enyl-4-[[2-[[4-[3-[bis(2-hydroxyethyl)amino]propylamino]-4-oxo-butanoyl]amino]-5-oxo-5-(tetradecylamino)pentanoyl]amino]butanoate and 4-pyrrolidine-1-ylbutyl-4-[[1-[[4-(3-butylhept-2-enoxy)-4-oxo-butyl]carbamoyl]-4-oxo-4-(tetradecylamino)butyl]amino]-4-oxo-butanoate 5-[[4-(3-butylhept-2-enoxy)-4-oxo-butyl]amino]-4-(9H-fluoren-9-ylmethoxycarbonylamino)-5-oxo-pentanoic acid The molecule 5-[[4-(3-butylhept-2-enoxy)-4-oxo-butyl]amino]-4-(9H-fluoren-9-ylmethoxycarbonylamino)-5-oxopentanoic acid is used in DMF The compound 4-(9H-fluoren-9-ylmethoxycarbonylamino)-5-[[4-[(Z)-non-2-enoxy]-4-oxobutyl]amino]-5-oxopentanoic acid is obtained by adapting the two-step method described for the synthesis of 4-(9H-fluoren-9-ylmethoxycarbonylamino)-5-[[4-[(Z)-non-2-enoxy]-4-oxobutyl]amino]-5-oxopentanoic acid by coupling Fmoc-Glu(OtBu)-OH (2.5 mmol; 1.06 g) with the compound [4-(3-butylhept-2-enoxy)-4-oxobutyl]ammonium 4.9 mmol; 1.67 g while stirring in the presence of COMU (2.5 mmol; 1.07 g) and DIPEA (5 mmol; 865 μL) dissolved in 40 mL. Reaction time: 18 hours at 0°C to room temperature; Purification: Silica gel chromatography gradient 70 / 30~40 / 60 (EP / EA, v / v). Next, the ester functional groups were successfully cleaved using TFA (20 mL) in 20 mL of DCM while stirring at room temperature for 24 hours. The mixture was then concentrated and co-evaporated three times with 50 mL of DCM, and three times with 50 mL of diethyl ether (Et2O), and dried overnight under high vacuum. Yield: 74% (2 steps, 1.12 g, 1.85 mmol).

[0414] 3-Butylhept-2-enyl-4-[[2-amino-5-oxo-5-(tetradecylamino)pentanoyl]amino]butanoate Following the procedure described for compound 2-amino-N,N'-bis[(Z)-octadeca-9-enyl]pentanediamide, molecular 3-butylhept-2-enyl-4-[[2-amino-5-oxo-5-(tetradecylamino)pentanoyl]amino]butanoate is obtained in two steps by coupling tetradecylamine (2.8 mmol; 592 mg) with compound 5-[[4-(3-butylhept-2-enoxy)-4-oxo-butyl]amino]-4-(9H-fluoren-9-ylmethoxycarbonylamino)-5-oxopentanoic acid (0.06 mmol; 68 mg) using DIC (3.7 mmol; 579 μL) and HOBt (3.7 mmol; 500 mg) dissolved in 330 mL of CHCl containing 30 mL of DMF and Et3N (3 mmol; 408 μL). After stirring at room temperature for 36 hours, Fmoc deprotection was performed by introducing 10 mL of piperidine and stirring at room temperature for 4 hours. Reaction time: 36 hours + 4 hours at room temperature; Yield: 77% (1.42 mmol; 826 mg); TLC: Rf = 0.4 (DCM: MeOH 90 / 10 (v / v)).

[0415] 4-[[1-[[4-(3-butylhept-2-enoxy)-4-oxo-butyl]carbamoyl]-4-oxo-4-(tetradecylamino)butyl]amino]-4-oxo-butanoic acid Molecular 4-[[1-[[4-(3-butylhept-2-enoxy)-4-oxo-butyl]carbamoyl]-4-oxo-4-(tetradecylamino)butyl]amino]-4-oxo-butanoic acid was isolated using the procedure described for compound 4-oxo-4-(2-pyrrolidine-1-ylethylamino)butanoic acid, with compound 3-butylhept-2-enyl-4-[[2-amino-5-oxo-5-(tetradecylamino)pentanoyl]amino]butanoate (1.42 mmol; 826 mg), Et3N (2.1 mmol; 295 μL), and succinic anhydride (2.1 mmol; 210 mg) dissolved in 20 mL of DCM and 10 mL of DMF under argon. Reaction time: 16 hours at room temperature. Purification: Silica gel chromatography gradient 98 / 2~92 / 8 (DCM / MeOH, v / v). Yield: 88% (white solid 850 mg, 1.25 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.4).

[0416] 3-Butylhepto-2-enyl-4-[[2-[[4-[3-[bis(2-hydroxyethyl)amino]propylamino]-4-oxo-butanoyl]amino]-5-oxo-5-(tetradecylamino)pentanoyl]amino]butanoate Compound 3-butylhept-2-enyl-4-[[2-[[4-[3-[bis(2-hydroxyethyl)amino]propylamino]-4-oxo-butanoyl]amino]-5-oxo-5-(tetradecylamino)pentanoyl]amino]butanoate was dissolved in 15 mL of a 2 / 1 (v / v) mixed solution of MeCN and DCM, and methylimidazole (1.5 mmol; 120 μL) and TCFH (0.6 mmol; 170 mg) were used to prepare compound 4-[[1-[[4-(3-butylhept-2- The compound 2-[4-(dimethylamino)butanoylamino]-N'-[(Z)-heptadeca-9-enyl]-N-[(Z)-octadeca-9-enyl]pentanediamide was isolated by linking enoxy)-4-oxo-butyl]carbamoyl]-4-oxo-4-(tetradecylamino)butyl]amino]-4-oxo-butanoic acid (0.5 mmol; 340 mg) with N-(3-aminopropyl)diethanolamine (0.6 mmol; 91 μL), adapting the procedure described for compound 2-[4-(dimethylamino)butanoylamino]-N'-[(Z)-heptadeca-9-enyl]pentanediamide. Reaction time: 12 hours at 0°C to room temperature; Purification: Silica gel chromatography gradient 98 / 2 to 94 / 6 (DCM / MeOH, v / v). Yield: 61% (white solid 256 mg, 0.31 mmol, TLC (DCM / MeOH: 90 / 10) Rf: 0.3).

[0417] 4-Pyrrolidine-1-ylbutyl-4-[[1-[[4-(3-butylhept-2-enoxy)-4-oxo-butyl]carbamoyl]-4-oxo-4-(tetradecylamino)butyl]amino]-4-oxo-butanoate Compound 4-pyrrolidinyl-1-ylbutyl-4-[[1-[[4-(3-butylhept-2-enoxy)-4-oxo-butyl]carbamoyl]-4-oxo-4-(tetradecylamino)butyl]amino]-4-oxo-butanoate was isolated by coupling compound 4-[[1-[[4-(3-butylhept-2-enoxy)-4-oxo-butyl]carbamoyl]-4-oxo-4-(tetradecylamino)butyl]amino]-4-oxo-butanoic acid (0.5 mmol; 340 mg) with 4-(1-pyrrolidinyl)-1-butanol (0.7 mmol; 100 mg) using DCC (0.7 mmol; 145 mg) and DMAP (0.1 mmol; 12 mg) dissolved in 20 mL of DCM. Reaction time: 15 hours at room temperature; Purification: Silica gel chromatography gradient 98 / 2~90 / 10 (DCM / MeOH, v / v). Yield: 58% (white solid 234 mg, 0.29 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.3).

[0418] Synthesis of the molecule [(Z)-non-2-enyl]-4-[[2-[[4-[2-[2-[2,5-bis[[2-(dimethylamino)acetyl]amino]pentanoylamino]ethyldisulfanyl]ethylamino]-4-oxo-butanoyl]amino]-5-[[3-(dodecylamino)-1-(dodecylcarbamoyl)-3-oxo-propyl]amino]-5-oxo-pentanoyl]amino]butanoate 2-[2-[[4-[[4-[[3-(dodecylamino)-1-(dodecylcarbamoyl)-3-oxopropyl]amino]-1-[[4-[(Z)-non-2-enoxy]-4-oxobutyl]carbamoyl]-4-oxobutyl]amino]-4-oxobutanoyl]amino]ethyldisulfanyl]ethylammonium Compound 2-[2-[[4-[[4-[[3-(dodecylamino)-1-(dodecylcarbamoyl)-3-oxopropyl]amino]-1-[[4-[(Z)-non-2-enoxy]-4-oxobutyl]carbamoyl]-4-oxobutyl]amino]-4-oxobutanoyl]amino]ethyldisulfanyl]ethylammonium is obtained by following the two-step procedure experienced for the synthesis of compound 2-[2-[[4-[[4-[[(Z)-octadeca-9-enyl]amino]-1-[[(Z)-octadeca-9-enyl]carbamoyl]-4-oxobutyl]amino]-4-oxobutanoyl]amino]ethyldisulfanyl]ethylammonium trifluoroacetate. First, compound 4-[2-[2-(tert-butoxycarbonylamino)ethyldisulfanyl]ethylamino]-4-oxo-butanoic acid (0.9 mmol; 292 mg) and compound [(Z)-non-2-enyl]-4-[[2-amino-5-[[3-(dodecylamino)-1-(dodecylcarbamoyl)-3-oxo-propyl]amino]-5-oxo-pentanoyl]amino]butanoate (0.6 mmol; 484 mg) are linked together using 12 mL of DMF and 4 mL of DCM containing Et3N (0.8 mmol; 112 μL) and HOBt (0.9 mmol; 122 mg); then the reaction mixture is stirred at room temperature for 48 hours. After isolation and purification of the intermediate (flash chromatography on silica gel, elution gradient of 0-5% MeOH in DCM), the amine functional group was released by reacting TFA (550 μL) in 11 mL of DCM for 12 hours. Yield: 38% (2 steps, 265 mg, 0.23 mmol).

[0419] [4-Azaniumyl-5-[2-[2-[[4-[[4-[[3-(Dodecylamino)-1-(Dodecylcarbamoyl)-3-Oxopropyl]amino]-1-[[4-[(Z)-Non-2-Enoxy]-4-Oxobutyl]carbamoyl]-4-Oxobutyl]amino]-4-Oxobutanoyl]amino]Ethyldisulfanyl]Ethylamino]-5-Oxopentyl]ammonium Compound 2-[2-[[4-[[4-[[3-(dodecylamino)-1-(dodecylcarbamoyl)-3-oxopropyl]amino]-1-[[4-[(Z)-non-2-enoxy]-4-oxobutyl]carbamoyl]-4-oxobutyl]amino]-4-oxobutanoyl]amino]ethyldisulfanyl]ethylammonium is obtained by following the two-step procedure experienced for the synthesis of compound 2-[2-[[4-[[1-[[4-[(Z)-non-2-enoxy]-4-oxobutyl]carbamoyl]-4-oxo-4-(tetradecylamino)butyl]amino]-4-oxobutanoyl]amino]ethyldisulfanyl]ethylammonium. First, Boc-L-Orn(Boc)-OH (0.4 mmol; 132 mg) and compound 2-[2-[[4-[[4-[[3-(dodecylamino)-1-(dodecylcarbamoyl)-3-oxopropyl]amino]-1-[[4-[(Z)-non-2-enoxy]-4-oxobutyl]carbamoyl]-4-oxobutyl]amino]-4-oxobutanoyl]amino]ethyldisulfanyl]ethylammonium (0.22 mmol; 254) are linked together using HBTU (0.4 mmol; 152 mg) and DIPEA (0.8 mmol; 138 μL) dissolved in 6 mL of DMF; then, the reaction mixture is stirred at room temperature for 16 hours. After isolation and purification of the intermediate (flash chromatography on silica gel, elution gradient of 2-10% MeOH in DCM), the amine functional group was released by reacting TFA (350 μL) in 5 mL of DCM for 12 hours. Yield: 63% (2 steps, 176 mg, 0.14 mmol).

[0420] [(Z)-non-2-enyl]-4-[[2-[[4-[2-[2-[2,5-bis[[2-(dimethylamino)acetyl]amino]pentanoylamino]ethyldisulfanyl]ethylamino]-4-oxo-butanoyl]amino]-5-[[3-(dodecylamino)-1-(dodecylcarbamoyl)-3-oxo-propyl]amino]-5-oxo-pentanoyl]amino]butanoate The compound [(Z)-non-2-enyl]-4-[[2-[[4-[2-[2-[2,5-bis[[2-(dimethylamino)acetyl]amino]pentanoylamino]ethyldisulfanyl]ethylamino]-4-oxo-butanoyl]amino]-5-[[3-(dodecylamino)-1-(dodecylcarbamoyl)-3-oxo-propyl]amino]-5-oxo-pentanoyl]amino]butanoate was dissolved in 15 mL of a 2 / 1 (v / v) mixed solution of MeCN and DCM, and then in methylimidazole (1.05 mmol; 84 μL) and TCFH. Compound II was isolated by coagulating compound [4-azaniumyl-5-[2-[2-[[4-[[4-[[3-(dodecylamino)-1-(dodecylcarbamoyl)-3-oxopropyl]amino]-1-[[4-[(Z)-non-2-enoxy]-4-oxobutyl]carbamoyl]-4-oxobutyl]amino]-4-oxobutanoyl]amino]ethyldisulfanyl]ethylamino]-5-oxopentyl]ammonium (0.14 mmol; 178 mg) with DMG (0.35 mmol; 36 mg) using the procedure described for compound II. Reaction time: 12 hours at 0°C to room temperature; Purification: silica gel chromatography gradient 98 / 2 to 90 / 10 (DCM / MeOH, v / v). Yield: 44% (white solid 82 mg, 0.06 mmol, TLC (DCM / MeOH: 95 / 5) Rf: 0.2).

[0421] II-Results Encapsulation of mRNA into lipid nanoparticles using microfluidic formulations Next, mRNA was encapsulated via a microfluidic formulation of lipid nanoparticles using the lipids described in the present invention. First, all lipid components (i.e., adjustable lipids, helper neutral lipids, sterol derivatives, and PEG lipids) were separately dissolved in glass vials at 25 mg / mL in a 99 / 1 (v / v) solution of chloroform in methanol at different molar ratios (Table 1). Then, the required amount of each component was introduced into a flask and evaporated at 45°C for 2 hours under gentle vacuum. This process yielded a dry lipid film, which was then dried overnight under high vacuum. The lipid film was then dissolved in sterile filtered EtOH and sonicated for 10 minutes. The volume of ethanol was selected according to the desired final formulation volume required. Meanwhile, mRNA was dissolved in 1 mM citrate buffer at pH=4 to a final concentration of 0.125 mg / mL of mRNA solution.

[0422] Formulation into lipid nanoparticles is carried out on a MicroMixer chip using Dolomite Microfluidics' μ-encapsulation system. Briefly, the ethanol lipid solution and mRNA solution are injected into the system, and the formed LNPs are collected at the outlet. Pump parameters are optimized for each formulation depending on the lipids used, the target final concentration of the required lipids, and the size of the selected nucleic acid. As a baseline, a flow rate ratio (FRR) equal to 3 was used, and a total flow rate of 1000 μL / min could be set. The collected LNPs are then dialyzed overnight against PBS + 5% w / v sucrose, filtered through a sterile 0.45 μm filter, and stored at -80°C. [Table 1] Table 1: Molar composition used for LNP formation based on adjustable lipids

[0423] Characterization of mRNA LNP The size and charge of the recovered LNPs are determined using DLS (Malvern Instruments NanoZS) by dissolving 50 μL of the recovered solution in 950 μL of MilliQ water. The formed LNPs are then destabilized by adding 1% v / v Triton X-100 and incubating at 30°C for 1 hour by vortexing. After disruption of the formulation, a 1 / 100 aqueous solution of SyBr® Gold is prepared, and 0.5 μL of it is added to 49.5 μL of the resulting mixture containing Triton X-100. SyBr® Gold is similarly added to 49.5 μL of the solution containing undisrupted LNPs and 49.5 μL of the mRNA mother solution. The fluorescence of each sample is then read using a Victor (Perkin Elmer) fluorometer (excitation: 495 nm; emission: 537 nm) after 30 minutes of incubation at room temperature. The encapsulation efficiency (ee(%)) can be easily obtained from these fluorescence measurements using the following formula.

number

[0424] This method has been applied to several modifiable lipids as described in the present invention (Table 2). For this first step of screening, GFP-encoding 5-methoxyuridine-modified mRNA (OZ Biosciences, catalog number: MRNA15-1000) was encapsulated. [Table 2] Table 2: Main physical properties of mRNA (GFP) LNPs based on adjustable lipids I, II, V, VI, XIV, and XV

[0425] All lipids tested were shown to be able to form nanoparticles when associated with neutral phospholipids, sterol derivatives and PEG lipids under classical microfluidic handling, and high encapsulation efficiencies were achieved. Regarding the physicochemical properties, most of the lipids enabled the obtaining of small LNPs, and DLS measurements showed values of about 100 nm. In particular, for lipids I, V and XV, LNPs with sizes in the range below 100 nm could be obtained with a polydispersity index of about 0.1. Interestingly, the zeta potential values were different for each lipid even when the N / P ratio was maintained constant. Indeed, the surface charge of the LNPs could vary such that it was quite negative when lipid V was used and positive when lipid XIV was used. This means that the adjustable lipid geometries used have a clear impact on the future physical properties and possibly the biological behavior of the corresponding LNPs. As a result, one skilled in the art will have to test several lipids corresponding to the formulas shown in the present invention and select the most appropriate lipid according to the properties desired for the LNPs.

[0426] Cells Human cervical cancer (HeLa) and human pancreatic cancer (PANC-1) and mouse macrophage (RAW 264-7) cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM, Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS, St. Louis, MO, USA), 2 mM final L-glutamine, 100 units / ml penicillin and 100 μg / ml streptomycin (Lonza, Walkersville, MD, USA). Human immortalized Jurkat T cells and human leukemic monocytes (THP-1) were cultured in Roswell Park Memorial Institute medium (RPMI, Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS, St. Louis, MO, USA), 2 mM final L-glutamine, 100 units / ml penicillin and 100 μg / ml streptomycin (Lonza, Walkersville, MD, USA). All cell lines were grown in 75 cm 2Cells were cultured in flasks (Sarstedt AG&Co., Germany) at 37°C in a 5% CO2 atmosphere in a humidified incubator (Sanyo, Tokyo, Japan). Before the transduction assay, they were treated with trypsin at a concentration of 80% to maintain an exponential mitotic rate. Transfection experiments were performed in 24-well plates (Sarstedt AG&Co., Germany).

[0427] In vitro transfection of GFP mRNA using LNP Next, two LNP formulations based on lipids I and XIV, respectively, were tested in in vitro transfection experiments to demonstrate their efficacy in efficiently delivering mRNA while enabling mRNA gene expression. Transfection efficiency was monitored by flow cytometry by evaluating the percentage of transfected cells and the percentage of viable cells using flow cytometry. mRNA amounts ranging from 0.1 μg to 20 μg were tested, and RmesFect, a commercially available transfection reagent (a cationic lipid with a permanent positive charge) specifically for mRNA delivery, was used as a positive control.

[0428] These results (Figure 45) demonstrate the efficacy of LNPs based on adjustable lipids for transfecting cell lines using a wide range of mRNA amounts, up to 90% of which are positive GFP cells. The values ​​achieved proved to be better than those observed with more classical transfection reagents while maintaining cell viability. In fact, in terms of the percentage of viable cells, the adjustable lipids formulated as LNPs remained harmless compared to transfection reagents that resulted in a rapid decline in cell viability, with only 30% of cells still surviving when using 1 μg of mRNA (Figure 45).

[0429] These results clearly demonstrate that the efficacy of adjustable lipid-based LNPs is efficient in in vitro transfection settings, at a level comparable to standard transfection reagents, particularly at high nucleic acid doses, without impairing cell viability.

[0430] These results confirmed in another known cell model that transfection is difficult in laboratory settings using non-viral transfection reagents (Figure 46). Figure 46 shows the results of transfecting the suspension cell line Jurkat T cells as described above and using LNPs based on adjustable lipids I and XIV. The observed results are consistent with those observed in HeLa cells. Transfection efficiency could reach 40%, but at the same time, commercially available reagents only yield 10% positive cells at the maximum mRNA amount used. Furthermore, with respect to the percentage of viable cells after transfection, the adjustable lipids formulated as LNPs remained the safest across the entire range of mRNA usage, whereas the transfection reagents used as a control resulted in decreased cell viability with increasing mRNA levels, with only 40% of cells still surviving when using 5 μg of mRNA. In vitro transfection of GFP mRNA in cancer cell lines using LNPs

[0431] Next, several LNP formulations based on lipids I, XXXIV, XXXV, XXXVIII, XXXVI, and XXXIII were tested in in vitro transfection experiments to demonstrate their efficacy in efficiently delivering mRNA while enabling mRNA gene expression. Since LNPs show promising prospects due to their ability to efficiently deliver genetic material to cancer cells, the inventors decided to evaluate the efficacy of the compounds of the present invention in cancer cell lines. The inventors selected PANC-1 cells, a human pancreatic cancer cell line isolated from pancreatic cancer of pancreatic ductal cell origin, as a model and monitored them by flow cytometry by evaluating the percentage of transfected cells and the percentage of viable cells using flow cytometry. [Table 3] Table 3: Main physical properties of mRNA (GFP) LNPs based on adjustable lipids I, XXXIV, XXXV, XXXVIII, XXXVI, and XXXIII

[0432] Here again, all the lipids tested were proven to be able to form nanoparticles when associated with neutral phospholipids, sterol derivatives, and PEG lipids under classical microfluidic conditions, achieving high encapsulation efficiency. Regarding physicochemical properties, most of the lipids allowed for the acquisition of small LNPs, with DLS measurements showing values ​​of approximately 100 nm. In particular, for lipids I and XXXV, LNPs with sizes in the range of less than or close to 100 nm could be obtained with a polydispersity index of approximately 0.1. Interestingly, the zeta potential values ​​differed for each lipid, even when the N / P ratio was kept constant. In fact, the surface charge of the LNPs could vary, taking significantly negative values ​​when using lipid XXXVIII and positive values ​​when using lipid XXXV. This means that the geometry of the tunable lipids used has a clear influence on the future physical properties and possibly their biological behavior of the corresponding LNPs.

[0433] Next, five LNP formulations based on lipids I, XXXIV, XXXV, XXXVIII, XXXVI, and XXXIII were tested in in vitro transfection experiments in cancer cell lines to demonstrate their efficacy in efficiently delivering mRNA while enabling mRNA gene expression. Transfection efficiency was monitored by flow cytometry by evaluating the percentage of transfected cells. mRNA doses ranging from 0.1 μg to 20 μg were tested (Figure 47).

[0434] These results (Figure 47) demonstrate the efficacy of LNPs based on adjustable lipids for transfecting cancer cell lines using a wide range of mRNA levels, with the best lipid candidates achieving transfection efficiencies exceeding 90% of GFP-positive cells. The values ​​achieved proved to be better than those observed with more classical transfection reagents while maintaining cell viability.

[0435] These results clearly demonstrate that the efficacy of adjustable lipid-based LNPs is efficient in in vitro transfection settings at levels comparable to standard transfection reagents, particularly at high nucleic acid doses, without impairing cell viability.

[0436] In vitro transfection of GFP mRNA in macrophage and monocyte cell lines using LNPs Macrophages, multifunctional phagocytic cells, play a crucial role in many physiological activities, including immunomodulation, scavenging of exogenous antigens, removal of endogenous cellular debris, tissue repair, regeneration, and fibrosis. Furthermore, macrophages are long-lived cells distributed throughout the human body, with lifespans ranging from several months to several years. Thus, these cells play a vital role in innate immunity and the pathogenesis of several chronic and inflammatory diseases through the induction or dissipation of inflammation and tissue repair. Monocytes, on the other hand, are important components of the innate immune system. They are a source of many other important elements of the immune system, such as macrophages and dendritic cells. Monocytes play a role in both inflammatory and anti-inflammatory processes that occur during the immune response. Knowing this, the inventors evaluated the efficacy of the lipids of the present invention to efficiently transfect one macrophage cell line and one monocyte cell line using eGFP-encoding mRNA. The inventors chose to evaluate several lipid formulations shown in Table 3: RAW 264-7, a mouse macrophage cell line, and THP-1, monocytes isolated from the peripheral blood of patients with acute monocytic leukemia.

[0437] Lipid nanoparticles based on lipids I, XXXIII, XXXIV, and XXXVIII were first evaluated in the adherent RAW 264-7 cell line, with mRNA test doses ranging from 0.1 μg to 20 μg (Figure 48). After 24 hours, the inventors observed that different LNPs exhibited different transfection behaviors, which may be related to their different geometries. Thus, LNPs based on lipid XXXIII yielded only about 40% GFP-positive cells at their optimal RNA dose, while lipids XXXIV and XXXVIII consistently resulted in higher transfection rates across a wider range of experimental conditions. Overall, the four formulations provided consistent transfection rates in this RAW 264-7 cell line, expanding the application area of ​​the molecules and methods presented in this invention.

[0438] The results of transfection of the THP-1 cell line (Figure 49) proved to be more difficult, as even under optimal conditions, using lipid XXXVIII resulted in approximately 50% transfected cells overall. On the other hand, lipid XXXIV, which has proven efficient in other cell lines, did not reach the same level of transfected THP-1 cells. Lipids I and XXXVI showed similar dose-dependent transfection profiles, peaking at approximately 20% transfected cells at the maximum mRNA usage.

[0439] This new series of experiments provides clear evidence that the lipids of the present invention can be efficiently used to transfect cell lines derived from immune cells such as macrophages or monocytes. Interestingly, the inventors were also able to notice that LNPs based on the same lipid exhibited different transfection patterns depending on the cell line used. This is fully visible in the case of lipid XXXIV, which proved to be a very beneficial LNP component for transfecting macrophage cell lines, but was not as efficient for macrophages. This observation is further evidence that those who wish to use LNP-driven transfection should rely on a broad library of LNP components, particularly with respect to ionizable modifiable lipids. This diversity ensures a reliable and efficient solution for mRNA transfection in a wide variety of cell lines, even those that are difficult to transfect.

[0440] In vivo transfection of F-Luc mRNA using LNPs; efficiency and in vivo distribution LNPs have been described as highly potent gene delivery vehicles for in vivo applications due to their harmless nature and efficacy in efficiently protecting and delivering a wide range of nucleic acids. The modifiable lipids described in this invention have further advanced their safe properties, as their overall design and synthesis meet these important criteria and have been developed to further broaden their use in vivo.

[0441] Lipids I, VI, and XIV were formulated into LNPs according to the molar compositions shown previously (Table 1). 5-methoxyuridine-modified mRNA encoding firefly luciferase (OZ Biosciences, catalog number: MRNA16-1000) was encapsulated in the LNPs. To demonstrate the innovative properties of moduloable lipids, further LNPs containing well-known cationic lipids were selected for comparison. DOTAP was chosen due to its widely documented in vivo behavior and its permanent positive charge at physiological pH. The main physical properties of the resulting LNPs are shown in Table 4. [Table 4] Table 4: Main physical properties of mRNA(F-Luc)LNPs based on adjustable lipids I, VI, XIV, and cationic lipid DOTAP

[0442] Overall, all LNP formulations exhibited consistent physicochemical properties, controlled and reproducible size, charge, polydispersity, and encapsulation rates suitable for use in small animals. Three different LNPs were used to administer doses equivalent to 10 μg of mRNA via ip (intraperitoneal) injection into nude mice. Adjustable lipid I and XIV, as well as DOTAP-based LNPs, were selected. Bioluminescence was monitored over time using the IVIS imaging system. Bioluminescence evaluation demonstrated that all three LNPs successfully delivered F-Luc mRNA to mice. Indeed, luminescence could be observed in all animals at different rates three hours after injection (Figures 50A and B). Specifically, F-Luc mRNA expression was higher in mice administered with the adjustable lipid-based LNPs, but much lower when using DOTAP. Interestingly, Figure 50B, which monitors the dynamics of the bioluminescence signal over time, shows a significant difference between the two LNP formulations. In fact, LNPs based on adjustable lipid I produced the highest overall luminescence signal, which was maximal at 6 hours post-injection, while LNPs based on lipid XIV produced the maximum bioluminescence signal earlier than the other formulations, at just 3 hours. These results demonstrate not only the in vivo efficiency of the adjustable lipids claimed in this invention, but also the complementary behaviors that may arise from their structural diversity, thereby providing a formulation capable of efficiently delivering nucleic acids with different kinetics.

[0443] In vivo distribution studies involving adjustable lipid-based LNPs (Lipids I, VI, and XIV) were also performed in nude mice. To evaluate nucleic acid delivery to various organs, luciferase mRNA formulated into three different LNP formulations was administered via ip injection at a 10 μg RNA dose. Liver, lung, kidney, and spleen were harvested and analyzed by RT-PCR 6 hours after injection. As previously observed, adjustable lipid-based LNPs are appropriately efficient in enabling the observation and quantification of luciferase protein at 6 hours after ip injection. Lipid-based LNPs provided high protein expression rates observed in the lungs and spleen, and to a lower degree in the kidneys. Lipid-based LNPs showed high delivery in the spleen after 6 hours. Interestingly, this formulation could also deliver mRNA to the liver at a selected N / P ratio, a rate comparable to that observed in the lungs. Lipid-based LNPs, on the other hand, provided the highest gene expression rate in the lungs, as high as that observed in the spleen. Furthermore, it was the most efficient among the three formulations tested in expressing luciferase in the kidney. This in vivo distribution study demonstrated the efficiency of LNPs based on moduloable lipids for transporting nucleic acids in different organs in vivo. Moreover, the complementary nature of the results, which allows for different expression rates in organs depending on the selected moduloable lipid, is a clear demonstration of the importance of the structural diversity provided by the present invention, which enables organ targeting through the selection of moduloable lipid structures.

[0444] In vivo intravenous (iv) injection of F-Luc mRNA using LNPs; efficiency and biodistribution To further investigate the efficacy of the molecules described in this invention, the inventors decided to test them in vivo under different experimental settings. The inventors again incorporated mRNA into LNPs and injected them intravenously into wild-type mice. Lipids I, XXXIII, and XXXIV were formulated into LNPs according to the molar compositions shown previously (Table 5). 5-methoxyuridine-modified mRNA encoding firefly luciferase (OZ Biosciences, catalog number: mRNA16-1000) was encapsulated in the LNPs. The main physical properties of the resulting LNPs are shown in Table 5. [Table 5] Table 5: Main physical properties of mRNA(F-Luc)LNPs based on adjustable lipids I, XXXIII, and XXXIV

[0445] All LNP formulations, again here, exhibit consistent physicochemical properties, controlled and reproducible size, charge, polydispersity, and encapsulation rates suitable for use in small animals. Three different LNPs were used to deliver doses equivalent to 10 μg of mRNA to nude mice via IV injection. In vivo distribution studies (shown in Figure 52) were performed to evaluate nucleic acid delivery to various organs. Liver, lung, kidney, and spleen were collected and analyzed by RT-PCR 24 hours after injection. As previously observed, LNPs based on adjustable lipids are appropriately efficient in allowing observation and quantification of luciferase protein at levels 6 hours after IV injection. LNPs based on lipids XXXIII and XXXIV provide high protein expression rates, particularly in the liver and spleen, and to a lower degree in the lung. LNPs based on lipid XXXIV showed high delivery in the spleen at 6 hours. Interestingly, this formulation can also deliver mRNA to the liver at a selected N / P ratio. On the other hand, the lipid XXXIII-based LNP provided the highest gene expression rate in the spleen, which was almost as high as the rate observed in the liver. It was also the most efficient among the three formulations tested in expressing luciferase in the lungs. On the other hand, the lipid I-based LNP was very efficient when administered intravenously, but when used intravenously, it provided more modest results, with only an average luciferase expression rate that was spread almost equally among the lungs, liver, and spleen. This in vivo distribution study demonstrated the efficiency of modifiable lipid-based LNPs for transporting nucleic acids in different organs in vivo. Again, the different expression rates in organs depending on the selected modifiable lipid are a clear demonstration of the importance of the structural diversity provided by the present invention, which enables organ targeting through the selection of modifiable lipid structures.

[0446] In vivo immunization experiment; delivery of antigen OVA mRNA using LNPs. Recent trials have clearly demonstrated the community's need to develop a new generation of vaccines that are easy to handle and safe for patients, while remaining efficient in immunization. In this context, the emergence of vaccine-based LNPs presents a clear opportunity to address pathologies that are ineffective with more classical vaccine approaches. The modifiable lipids presented in this document could be envisioned as strong pillars of future nucleic acid-based vaccines due to their groundbreaking characteristics in terms of efficiency and safety. Therefore, LNPs based on modifiable lipids I and XIV were formulated to encapsulate unmodified mRNA encoding the ovalbumin (OVA) antigen (OZ Biosciences, catalog number: MRNA42-1000), and their physicochemical properties were determined (Table 6). [Table 6] Table 6: Main physical properties of mRNA(OVA)LNPs based on adjustable lipids I and XIV.

[0447] The two LNP formulations exhibited consistent and reproducible size, polydispersity, charge, and encapsulation efficiency. The two LNPs were used in immunization studies to demonstrate their efficacy and potential compared to classical recombinant subunit vaccines. C57BL6J mice underwent prime-boost (D0-14) immunization with OVA antigen delivered by mRNA-based LNP or recombinant protein (alone or enhanced with an aluminum-based adjuvant). To characterize the immunophenotyping of the mice, serum and splenocytes were collected at different time points (D0, D14, D21, D28) and analyzed by ELISA and Elispot. Mice were immunized via the sc (subcutaneous) route at 100 μL / injection with 10 μg OVA subunit and 10 μg mRNA doses. Results are shown in Figures 53 and 54, demonstrating that the mRNA-OVA LNP formulations are efficient at priming a strong humoral response during subcutaneous prime-boost injection (D0-14). Indeed, a strong antibody response was measured after prime-boost immunization. Furthermore, no significant signal was observed when a dose of 10 μg of OVA antigen was administered directly alone. Of note is the importance of boosting (D14) for obtaining a long-lasting immune response. Both lipid I and lipid XIV-based mRNA-OVA LNPs are efficient at generating an immune response after sc administration of mRNA, particularly with respect to the initial response of IgG2c. mRNA-OVA LNP formulations are efficient at priming a strong TH1 humoral response comparable to that of the best TH1 adjuvants at subcutaneous prime-boost injection (D0-14). mRNA-OVA LNP formulations are also efficient at priming a strong anti-OVA CD8 T cell response comparable to one of the most efficient adjuvants at subcutaneous prime-boost injection (D0-14), as indicated by IFNγ Elispot results and IL-18 production (not shown).

[0448] Embedding of DNA into lipid nanoparticles using microfluidic formulations; the efficacy of modifiable lipids for DNA delivery was further evaluated, and several lipids belonging to the present invention were selected for embossing plasmid DNA into LNPs. After initial screening, lipids I and XI were selected as the basic modifiable compounds for formulation. The same microfluidic procedure used for mRNA embossing was converted to DNA application. The ratios between all lipid components used with mRNA (i.e., modifiable lipids, helper neutral lipids, sterol derivatives, and PEG lipids) were maintained with respect to DNA (see Table 1), and the microfluidic formulation procedure remained unchanged. As a baseline, the flow rate ratio (FRR) was equal to 3, and the total flow rate was set to 1000 μL / min. The recovered LNPs were then dialyzed overnight against PBS + 5% w / v sucrose, filtered through a sterile 0.45 μm filter, and stored at -20°C. Plasmid DNA (pVectOZ) was designed and generated by OZ Biosciences. Three plasmids encoding Ppt, eGFP, and F-Luc were tested. Therefore, LNPs based on adjustable lipids I and XI were formulated with physicochemical properties determined accordingly (Table 7). [Table 7] Table 7: Main physical properties of LNPs based on pVectOZ plasmids with adjustable lipids I and XI.

[0449] The results shown in Table 7 clearly highlight the trend of adjustable lipids I and XI for the efficient encapsulation of various DNA plasmids in LNPs. Due to the larger size of DNA compared to mRNA, the size of the resulting nanostructures is maintained below 200 nm, and the N / P ratio is adjusted so that the polydispersity index is less than 0.1, which makes these structures suitable for further testing and provides good insight into their medium-term stability.

[0450] In vitro transfection of F-Luc DNA using LNPs Next, LNP formulations based on lipid XI were tested in in vitro transfection experiments to demonstrate their efficacy in efficiently delivering DNA while enabling DNA gene expression. Jurkat T cells were used. Transfection efficiency was assessed after 48 hours of incubation using the Luciferase Assay Kit (OZ Biosciences) by evaluating the luminescence intensity (Perkin Elmer Victor NOVO luminometer) obtained after lysing of transfected cells in the presence of luciferin, magnesium, and ATP. DNA amounts ranging from 0.1 μg to 10 μg were tested, and MTX reagent (OZ Biosciences SAS), a commercially available transfection reagent (a cationic lipid with a permanent positive charge) specifically for DNA delivery, was used as a positive control.

[0451] These results (Figure 55) again demonstrate the efficacy of adjustable lipid-based LNPs in transfecting suspension cell lines that are difficult to transfect using a wide range of DNA amounts, with the measured increase in luminescence directly correlated with the amount of introduced DNA. The luminescence values ​​achieved were proven to be similar to those observed with more classical transfection reagents while maintaining cell viability. These results clearly demonstrate the efficacy of adjustable lipid-based LNPs in in vitro DNA transfection at levels comparable to standard transfection reagents, particularly at high nucleic acid doses, without impairing cell viability.

[0452] Enc...

Claims

1. A lipid of formula (I), its stereoisomer, or a pharmaceutically acceptable salt thereof, 【Chemistry 1】 During the ceremony, -R comprises one or more branched or linear, unsaturated or saturated, optionally fluorinated alkyl chains containing 6 to 48 carbon atoms, preferably 10 to 36 carbon atoms, and may contain one or more heteroatoms other than fluorine; or one or more cyclic or polycyclic groups known to be lipophilic and which may contain one or more heteroatoms, such as steroid groups which may contain one or more heteroatoms, polycyclic groups which may contain one or more heteroatoms, or alkaloid derivative groups which may contain one or more heteroatoms; or natural or synthetic lipids which may contain one or more heteroatoms; or a combination thereof; -Sp comprises one or more branched or linear, unsaturated or saturated, possibly fluorinated alkyl chains, comprising 6 to 24 carbon atoms, preferably 10 to 18 carbon atoms, and may contain one or more heteroatoms and / or bioreducible bonds; and -Z ｓ It comprises one or more branched or linear, unsaturated or saturated, possibly fluorinated alkyl chains containing 2 to 36 carbon atoms, preferably 2 to 24 carbon atoms, and which may contain one or more heteroatoms other than fluorine, and / or bioreducing bonds covalently bonded to one or more linear or cyclic nitrogen-based chemical moieties; It features the following: Lipids do not contain any positive charges at pH levels between 7.0 and 7.4, but they exhibit one or more positive charges when the pH is below 7.0, preferably between 2.0 and 7.0, more preferably between 4.0 and 7.0, and even more preferably between 4.5 and 7.0; R corresponds to equation (II): 【Chemistry 2】 During the ceremony, - Same or different N １ and N ２ This represents a linear, branched saturated or unsaturated hydrocarbon group containing 1 to 8 carbon atoms, preferably 1 to 4 carbon atoms, used to terminate the carbon chain, preferably the same or different N １ and N ２ is one of the following groups: methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, or t-butyl group; - Same or different R １ and R ２ This represents a linear, branched, and / or cyclic saturated or unsaturated hydrocarbon group comprising 6 to 24 carbon atoms, preferably 10 to 18 carbon atoms, and may contain one or more heteroatoms; - The same A and B represent O-C(O), C(O)NH-, -NHCO-, O-C(O)-O, NH-C(O)-NH, NH-C(O)-O, O-C(O)-NH, -S-C(O)-S-, -O-C(S)-S-, -S-C(O)-O, -NH- or -S- group; -a is an integer between 1 and 4, preferably an integer equal to 1, 2, or 3; -b is an integer between 0 and 6, preferably an integer equal to 0 or 1; - the same or different E １ and E ２ represents a -C(O)O-, -C(O)-NH-, -NH-, -O- or -S- group; -c is an integer between 0 and 2; -d is an integer equal to 0 or 1; -d １ is an integer between 0 and 6, preferably d １ is an integer equal to 0 or 1; -d ２ is an integer between 0 and 6, preferably d ２ is an integer between 0 and 2; and -e is an integer between 0 and 6, preferably between 0 and 2, more preferably between 0 and 1; Sp corresponds to general formula (III): 【Transformation 3】 During the ceremony, -Gr represents a linear or branched hydrocarbon group comprising 1 to 15 carbon atoms, preferably 1 to 8 carbon atoms, and may contain one or more heteroatoms; -m is an integer equal to 0 or 1; -RAc represents an amino acid radical; and -n is an integer equal to 0 or 1; and Zs corresponds to equation (IV): 【Chemistry 4】 During the ceremony, - The same or different S １ and S ２ This represents a linear or branched hydrocarbon group comprising 1 to 15 carbon atoms, preferably 1 to 6 carbon atoms, preferably selected from nitrogen, oxygen, sulfur, bromine, iodine, chlorine, and fluorine, and more preferably one or more heteroatoms selected from nitrogen, sulfur, bromine, iodine, chlorine, and fluorine; -D represents one or more vinyl ether groups, one or more acylhydrazone groups, one or more carbonate bonds, disulfide bonds, ester bonds or carbonate bonds, or one or more photosensitive groups; -p is an integer equal to 0 or 1; -Q comprises 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and one or more heteroatoms, on the one hand S ２ Alternatively, it is a branched hydrocarbon group that may be covalently bonded to an RAc, Gr, or R group on the other side, and to at least two Q and / or Z groups on the other side; -q is an integer between 0 and 8, preferably between 0 and 3; -Z is a nitrogen (N)-centered terminal ionizable moiety, and preferably Z represents a tertiary aliphatic amine such as an N,N-dialkylamine, or a cyclic amine moiety such as an N-substituted pyrrolidine, N-substituted piperidine, N-substituted morpholine, N-substituted piperazine, or N-substituted pyridine; where Z contains no positive charge at pH in the range of 7.0 to 7.4, but exhibits one or more positive charges at pH in the range of 2.0 to 7.0, preferably 4.0 to 7.0, and more preferably 4.5 to 7.0; -r is an integer between 1 and 16, preferably between 1 and 8, preferably, if q is equal to 1, r is at least equal to 2, and if r is greater than 1, the Z groups may be the same or different; and -s is an integer equal to 1 or 2, The following compounds are excluded from general formula (I): 【Transformation 5】 and 【Transformation 6】 Furthermore In equation (III): - Gr does not exist, or Gr acts as a spacer arm, -W ４ -Y ４ -W ４ Corresponds to the molecular pattern shown as -; Here, Y ４ represents a crosslinked alkylene group containing 1 to 8 carbon atoms, preferably 1 to 4 carbon atoms, W ４ This represents a C(O)-O, C(O)-NH, C(O)-S, S(O), S(O)-O, C(S)-O, -O-, or -NH- group. base; or Gr is given by formula -W ４ -Y ４ -W ４ - corresponds to, Here, Y ４ This has the same meaning as previously defined, on the one hand to the lipophilic region R, on the other hand to the RAc radical, or directly to S １ Alternatively, the Q or Z group is linked by a carbonate or carbamate bond; and In formula (III), the amino acid radical RAc is selected from aspartic acid, glutamic acid, isoleucine, leucine, lysine, ornithine, glycine, and phenylalanine, and more preferably selected from aspartic acid, glutamic acid, glycine, ornithine, and lysine. A lipid of formula (I), its stereoisomer, or a pharmaceutically acceptable salt thereof.

2. Z corresponds to equation (V), 【Transformation 7】 During the ceremony, -R ３ and R ４ C may be the same or different and may be substituted with one or more methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl or t-butyl groups. １ -C ６ Alkyl, C ２ -C ６ Alkenil or C ２ -C ６ Selected from Alkinyl, or R ３ and R ４ However, they may bond to form a heterocycle which may be substituted with one or more methyl, ethyl, n-propyl, i-propyl, s-butyl, i-butyl or t-butyl groups, for example, pyrrole, pyrrolidine, pyridine, piperazine, morpholine, piperidine or indoline which may be substituted with one or more methyl, ethyl, n-propyl, i-propyl, s-butyl, i-butyl or t-butyl groups; -N is nitrogen; -V is O, S, N(R ６ ), C(O), C(O)O, OC(O), C(O)N(R ６ ), N (R ６ )C(O), OC(O)N(R ６ ), N (R ６ ) C(O)O, C(O)S, C(S)O, S(O), S(O)(O), C(S), or a heterocycle which may be substituted, for example, pyrrole, pyrrolidine, pyridine, piperazine, morpholine, piperidine, or indoline which may be substituted with one or more methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, or t-butyl groups, where R ６ However, C may be substituted with hydrogen (H) or one or more methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl or t-butyl groups. １ -C １０ Alkyl, C ２ -C １０ Alkenil or C ２ -C １０ It is an alkynyl; preferably, R ６ However, hydrogen (H), C １ C, which may or may not incorporate an alkyl (methyl) group, and one or more heteroatoms such as -O, -NH, -S, and mixtures thereof. ２ , C ３ , C ４ , C ５ , C ６ , C ７ , C ８ , C ９ and C １０ Selected from the group consisting of alkyl, alkenyl, and alkynyl groups; and -U may be absent or substituted with one or more methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, or t-butyl groups, preferably substituted with one or more heteroatoms or heteroatomic groups selected from nitrogen, oxygen, sulfur, bromine, iodine, chlorine, fluorine, or phosphorus. １ -C １２ Alkyl, C ２ -C １２ Alkenil, or C ２ -C １２ Alkinyl is The lipid of formula (I) as described in claim 1.

3. A lipid of formula (I) according to claim 1 or 2, wherein, ・R １ and R ２ They differ, and their structures are represented by equations (VI a) and (VI b), respectively: 【Transformation 8】 Here, - Same or different Ak ｆ１、 Ak ｊ１ , Ak ｆ２、 Ak ｊ２ However, it represents a linear or branched hydrocarbon group comprising 1 to 22 carbon atoms, preferably 1 to 12 carbon atoms, and may contain one or more heteroatoms; -f1 and f2 are integers equal to 0 or 1; -j1 and j2 are integers equal to 0 or 1; - the same or different Xg １ , Xi １ , Xg ２ and Xi ２ are selected from O, S, N(R ７ ), C(O), C(O)O, OC(O), C(O)N(R ７ ), N(R ７ ), N(R ７ ), OC(O)N(R ７ ), N(R ７ ), N(R １ ), C(S)O, S(O), S(O)(O) and C(S), where R １０ is hydrogen (H), or C[[ID=2,5]] ２ alkyl, C[[ID=##]] １０ alkenyl or C ２ alkynyl which may be substituted with one or more methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl or t-butyl groups; more preferably, R ７ is hydrogen (H), C １ [[ID=##]]alkyl (methyl) group, and C ２、 C ３ , C ４ , C ５ , C ６ , C ７ , C ８ , C ９ and C １０ alkyl, alkenyl and alkynyl groups selected from the group consisting of; preferably, Xg １、 Xi １ , Xg ２ and Xi ２ are independently represented by N(R ７ ), C(O)N(R ７ ), N(R ７ ), OC(O)N(R ７ ), N(R ７ ), N(R ７ ), and it will probably be understood that R ７ can have several different properties among those shown above within the same molecule; -g1 and g2 are integers equal to 0 or 1; -i1 and i2 are integers equal to 0 or 1; - Identical or different I ｈ１ and I ｈ２ However, both represent a straight or branched unsaturated hydrocarbon chain containing 2 to 16 carbon atoms, preferably 2 to 8 carbon atoms and at least one unsaturated atom, and more preferably the same or different I ｈ１ and I ｈ２ but, 【Chemistry 9】 and 【Chemistry 10】 Selected independently of; -h1 and h2 are integers equal to 0 or 1; -J １ and J ２ However, it is a branched hydrocarbon group containing 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and / or one or more heteroatoms; J １ However, on the other hand, Ak ｆ１ , Ak ｊ１ , or Xg １ , Xi １ , or I ｈ１ One of the groups may be covalently bonded to A, and J ２ However, on the other hand, Ak ｆ２ , Ak ｊ２ Xg ２ and Xi ２ , or I ｈ２ It may be covalently bonded to either B or B on the other; - l1 and l2 are integers equal to 0 or 1; preferably, if l1 is equal to 0, Ak ｆ１ , Ak ｊ１ , or Xg １ , Xi １ , or I ｈ１ It is understood that one of the groups is covalently bonded to A; similarly, if l2 is equal to 0, then Ak ｆ２ , Ak ｊ２ Xg ２ and Xi ２ , or I ｈ２ One of the groups is covalently bonded to B; and -k1 and k2 are integers between 1 and 3, preferably, if l1 is equal to 1, then k1 is equal to at least 2; similarly, if l2 is equal to 1, then k2 is equal to at least 2; or ・R １ and R ２ If they are identical, their structures can be represented by formula (VII): 【Chemistry 11】 During the ceremony, - Same or different Ak ｆ and Ak ｊ However, it represents a linear or branched hydrocarbon group comprising 1 to 22 carbon atoms, preferably 1 to 12 carbon atoms, and may contain one or more heteroatoms; -f is an integer equal to 0 or 1; -j is an integer equal to 0 or 1; - If Xg and Xi are the same or different, O, S, N(R) ７ ), C(O), C(O)O, OC(O), C(O)N(R ７ ), N (R ７ )C(O), OC(O)N(R ７ ), N (R ７ ) Selected from C(O)O, C(O)S, C(S)O, S(O), S(O)(O), C(S), where R ７ However, C may be substituted with hydrogen (H) or one or more methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl or t-butyl groups. １ -C １０ Alkyl, C ２ -C １０ Alkenil or C ２ -C １０ It is an alkynyl; more preferably, R ７ However, hydrogen (H), C １ C may contain an alkyl (methyl) group, and one or more heteroatoms such as -O, -NH, -S, and mixtures thereof. ２ , C ３ , C ４ , C ５ , C ６ , C ７ , C ８ , C ９ and C １０ Selected from the group consisting of alkyl, alkenyl, and alkynyl groups; -g is an integer equal to 0 or 1; -i is an integer equal to 0 or 1; -I ｈ However, it represents a straight or branched unsaturated hydrocarbon chain containing 2 to 16 carbon atoms, preferably 2 to 8 carbon atoms and at least one unsaturated atom, more preferably I ｈ but, 【Chemistry 12】 and 【Chemistry 13】 Selected from; -h is an integer equal to 0 or 1; -J is a branched hydrocarbon group comprising 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and / or one or more heteroatoms; J is Ak on one side. ｊ Or Xi or I ｈ or Xg or Ak ｆ The base may be covalently bonded to A or B on the other side; - l is an integer equal to 0 or 1; preferably, if l is equal to 0, Ak ｊ Or Xi or I ｈ or Xg or Ak ｆ It is understood that one of them is covalently bonded to A on the one hand and to B on the other; and -k is an integer between 1 and 3; preferably, if l is equal to 1, it is understood that k is at least equal to 2. Lipids of formula (I).

4. R is, 【Chemistry 14】 (Formula VIII) Selected from, in the formula, R １、 R ２、 N １ and N ２ as defined in a prior claim, A lipid of formula (I) as described in any one of claims 1 to 3.

5. The compound of formula (I) above, 【Chemistry 15】 【change】 【change】 【change】 【change】 【change】 【change】 A lipid of formula (I) according to any one of claims 1 to 4, selected from the above.

6. A composition comprising a lipid of formula (I) as described in any one of claims 1 to 5.

7. It further contains one or more active ingredients, and - One or more neutral lipids, and / or - One or more components that can reduce aggregation of the composition, and / or - One or more sterols or sterol derivatives, and / or - One or more lipids having a targeted ligand The composition according to claim 6, which may further contain the following.

8. A method for producing a lipid of formula (I) according to any one of claims 1 to 5 and / or a composition according to claim 6 or 7.

9. A lipid of formula (I) according to any one of claims 1 to 5 and / or a composition according to claim 6 or 7, for use as a pharmaceutical.

10. Use of a lipid of formula (I) according to any one of claims 1 to 5 and / or the composition according to claim 6 or 7 as a vector for delivering an active ingredient to a target(s), organ(s), cell(s), and / or tissue(s).

11. A method for transfecting cells with nucleic acids and / or proteins and / or peptides and / or polysaccharides and / or lipids and / or small organic or inorganic molecules and / or any kind of bioactive molecule, using a lipid of formula (I) according to any one of claims 1 to 5 and / or the composition according to claim 6 or 7.

12. Transfected cells obtained by the method of claim 11.

13. A kit comprising a lipid of formula (I) according to any one of claims 1 to 5 and / or the composition according to claim 6 or 7, further comprising nucleic acids and / or proteins and / or peptides and / or polysaccharides and / or lipids and / or small organic or inorganic molecules and / or any type of bioactive molecule, and optionally comprising instructions for use.

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