COMPOSITION FOR TRANSFECTING CELLS WITH NUCLEIC ACID MOLECULES COMPRISING TRIAZOLE COMPOUND GRAFTED TO A CATIONIC POLYMER AND USE THEREOF

Abstract

The present invention relates to a composition for transfecting a nucleic acid molecule into a cell and its use. The present invention relates to a composition suitable for transfecting a nucleic acid molecule into a cell, preferably a eukaryotic cell, comprising (i) at least one compound of general formula (I), preferably general formula (III), or a tautomer, mesomers, racemates, enantiomers, diastereomers, or mixtures thereof, or an acceptable salt thereof, and (ii) an acceptable excipient, buffer, cell culture medium, or transfection medium, wherein Y 1 , Y 2 , Y 3 , Z 1 , Z 2 , Z 3 , X1, X2, R3, P + , R and V are as defined herein. The present invention also relates to uses of said compositions and to methods for the in vitro or ex vivo transfection of living cells.

Description

[Technical Field]

[0001] The present invention relates to a composition for transfecting a nucleic acid molecule into a cell, comprising a heterocyclic compound, particularly a triazole derivative, grafted onto a cationic polymer, and its use. The present invention relates to a composition suitable for transfecting a nucleic acid molecule into a cell, preferably a eukaryotic cell, comprising (i) at least one compound of general formula (I), preferably at least one compound of general formula (III), or a tautomer, mesomers, racemates, enantiomers, diastereomers, or mixtures thereof, or an acceptable salt thereof, and (ii) an acceptable excipient, buffer, cell culture medium, or transfection medium, wherein Y 1 , Y 2 , Y 3 , Z 1 , Z 2 , Z 3 , X1, X2, R3, P + , R and V are as defined herein. The present invention also relates to uses of said compositions and to methods for the in vitro or ex vivo transfection of living cells. [Background technology]

[0002] Gene transfer is the process of introducing copies of an exogenous gene into a living cell and inducing the synthesis of that gene product. Transfection is the process of deliberately and artificially introducing nucleic acid (DNA or RNA) into a eukaryotic cell using non-viral methods. Transfection has been of fundamental importance to the development of modern biology and medicine and has provided much of our knowledge about gene function and regulation.

[0003] Transfection according to the present invention can be achieved in a variety of cells, including mammalian and insect cells, primary cells, cell lines, stable cells, or tumor cells. Transfection is a powerful tool for in vitro genomic research by offering the possibility to express new foreign proteins in cells or to overexpress or silence naturally occurring proteins.

[0004] Transfection according to the present invention can be applied therapeutically via ex vivo or in vivo protocols. Nucleic acid-based therapy using non-viral vectors can target various diseases, genetic disorders, immune disorders, cancer, or viral infections in various tissues / organs or tumors. Cell targeting is achieved by different mechanisms and depends on the nature and properties of the transfection reagent, the method or protocol, the composition or formulation, and the route of administration (Kaestner et al., 2015).

[0005] In bioproduction, transfection according to the present invention can be used to generate stable cell clones that overproduce recombinant proteins, peptides, or antibodies. More recently, transfection techniques that allow transient gene expression (TGE) have become a valuable method for rapidly producing moderate levels of recombinant proteins or antibodies, useful in research and process development. Transient gene expression processes are conveniently applied to the production of recombinant viruses, such as adeno-associated viruses (AAVs), lentiviruses (LVs), or adenoviruses (Merten et al., 2016; Van Der Loo and Wright, 2015). Such processes involve transfecting cells with multiple expression vectors (plasmids) that express different components required for virus production, including capsid proteins, helper proteins, envelope proteins, viral polymerases or regulators, or viral genomes. High-producing cells, such as HEK293 and derivative cells, HeLa, BHK-21, A549, or insect cells, are used for virus production. Transfection can be achieved in adherent or suspension-adapted cells cultured at high cell density in serum-containing medium or in protein-free, chemically defined or fully synthesized medium.

[0006] Transfection is a method for introducing various components into cells that are necessary to induce genome modification, manipulation, or editing, such as zinc finger nucleases, CRE / LOX proteins, or CRISPR Cas-9 proteins.

[0007] DNA transfection uses plasmid DNA to induce gene expression driven by promoters of proteins or peptides and / or nucleic acids, such as messenger RNA, long RNA, microRNA, small hairpin RNA, small interfering RNA, etc.

[0008] In almost all cases, plasmid DNA has been used for transfection due to its inherent stability and ability to integrate into the host genome, resulting in stable gene expression, or to remain in the nucleus in an episomal form, resulting in transient gene expression. However, a subset of cells, termed "difficult-to-transfect" cells (HTT), are resistant to DNA transfection or exhibit lower levels of transfection and gene expression compared to standard transformed cell lines routinely used in laboratory conditions. These "difficult-to-transfect" cells exhibit transfection efficiencies of less than 50% when transfected with previous-generation commercially available transfection reagents, such as LipoFectAmine® 2000 and 3000 (ThermoFisher), TransIT reagents® (MirusBio), FuGene® (Promega), XtremeGene® (Roche), jetPRIME® (Polyplus-transfection), or ViaFect® (Promega).

[0009] A recent advancement in improving gene expression efficiency in HTT cells is transfection with messenger RNA (mRNA) sequences rather than plasmid DNA constructs, which has been shown to significantly improve transfection and gene expression levels in most cell types, particularly difficult HTT cells. This advantage is explained by the fact that transfected mRNA does not need to reach the nucleus for cellular function, in contrast to DNA transfection, where nuclear access and penetration are major limitations. While the transfer of plasmid DNA is not well understood, efficient DNA transfection is primarily associated with active cell proliferation, where transfected DNA can diffuse into the intranuclear space during nuclear membrane breakdown. DNA transfection is ineffective in most postmitotic or non-dividing cells. The majority of HTT cells, such as neurons or other cell types derived from neural tissue, primary blood cells such as dendritic cells or macrophages, or primary hepatocytes, have been shown to undergo low levels of mitosis or even no mitosis. However, in other HTT cells, the low transfection efficiency may be explained by other factors such as cell fragility, poor binding of the transfection agent to the cell plasma membrane, poor endocytic capacity, or inefficient intracellular transport of the transfected DNA to the nucleus.

[0010] Transfection of plasmid DNA is the most common method for overexpressing proteins in cells grown in culture. Many methods for introducing genetic DNA material into cells involve the use of reagents such as calcium phosphate, cationic liposomes, peptides, or polymers. Transfection failures are generally attributed to the reagent. There is still a need to improve the efficiency of transfection reagents, particularly for HTT cells, through new concepts and next-generation reagents.

[0011] DNA transfection in eukaryotic cells involves combining or mixing polyanionic DNA molecules with a reagent to form transfection complexes or aggregates. Among the most commonly used reagents, cationic lipids, peptides, or polymers are well suited to interacting with negatively charged DNA. When cationic reagents are used in excess, positively charged complexes or aggregates are generated. These complexes can interact with negatively charged glycosaminoglycans, such as heparan sulfate, present on the cell plasma membrane (Labatmoleur et al., 1996; Mislick and Baldeschwieler, 1996). Plasma membrane binding of the complex induces cellular internalization or uptake by the endocytic mechanism. The transfection complex is transported to endosomes, where the transfection reagent exhibits membrane fusion activity and / or membrane destabilization by endosomolysis, releasing the DNA into the cytoplasm. After release from the endosome, the transfected DNA must diffuse into the perinuclear space and penetrate into the nucleus. Due to the large size of plasmid DNA, it cannot diffuse through the nuclear pore complex, making nuclear import the limiting step.

[0012] Cationic liposomes or aggregates are one of the major classes of non-viral vectors for DNA transfection, consisting of cationic lipids combined or formulated with other types of lipids, such as phospholipids or cholesterol, to generate positively charged liposomes, vesicles, or micelles that can bind negatively charged DNA and negatively charged cell membranes, ultimately transfecting cells. In the prior art, the first synthetic cationic lipid was N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) by Felgner et al. When combined with dioleoylphosphatidylethanolamine in a 1:1 ratio, DOTMA formed cationic liposomes capable of transfecting cells in vitro. Based on the positively charged trimethylammonium polar head group, other monocationic lipids, such as 1,2-bis(oleyloxy)-3,3-(trimethylammonium)propane chloride (DOTAP), have been developed. Other prior art compounds are based on polycationic polar head groups, such as the lipids dioctadecylamidoglycylspermine (DOGS) or dipalmitoylphosphatidylethanolamidospermine (DPPES) described by Behr et al. in 1989, in which carboxyspermine is used instead of the ammonium group, or the phospholipid moiety is replaced with a cholesterol derivative such as 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-CHOL) (Gao and Huang, 1991). Since these pioneering studies, many cationic lipids have been designed with the goal of creating new cationic lipid reagents with improved transfection efficiency. Many of these reagents are commercially available, with the recent LipoFectAmine 3000® reagent representing the most advanced commercially available cationic lipid reagent. However, limitations remain, as transfection is not effective in all cell types, and cytotoxicity remains a significant concern with cationic lipid systems.

[0013] Cationic polymers, with the advantage of providing a high density of charged amino groups in their backbones, are the second major class of transfection reagents. Cationic polymers, which possess a positive charge at physiological pH, can complex DNA into particles or aggregates, initiate cell binding, and induce cellular uptake via endocytosis. Polylysine (PLL) was the first polymer used, but demonstrated very limited transfection efficiency (Wu and Wu, 1987; Zenke et al., 1990). Addition of additives such as weak bases like chloroquine (Erbacher et al., 1996) or membrane-fusogenic peptides like influenza peptides (Planck et al., 1994) to buffer the acidic pH of destabilized endosomes and better induce DNA release into the cytoplasm, respectively, can improve efficiency. Behr et al. showed that polyethyleneimine (PEI) is a more effective polymer for transfection than PLL (Boussif et al., 1995). PEI has a high density of amino groups and is not fully protonated at physiological pH. After endocytosis of PEI-complexed DNA, the polymer's buffering capacity induces "proton sponge" activity, resulting in vesicle swelling and endosome lysis, ultimately releasing the DNA into the cytoplasm without the aid of additives (Boussif et al., 1995; Sonawane et al., 2003). While both branched and linear PEI are effective for transfection, the linear form has been shown to be more efficient (Itaka et al., 2004), not inhibited by serum, and less toxic than the branched form. Over the past two decades, numerous strategies have been developed to improve PEI transfection efficiency, reduce toxicity, or propose biodegradable PEI-based polymer alternatives.

[0014] Much research has focused on optimizing the inherent proton-sponging endosomolytic activity of PEI by grafting histidyl or benzyl residues (US8658150, Chandrashekhar et al., 2012) onto the polymer. Other modifications, such as the addition of hydrophilic groups (EP2070970), have been explored to increase the solubility and reduce cytotoxicity of DNA / PEI complexes. Hydrophobic functional groups have been added to PEI using N-acyl groups (EP0262641), to increase the biodegradability of the polymer, or to generate lipopolymers (US20090022746, WO2006 / 041617). Higher gene transfection efficiencies have been observed in various cell lines. However, efficiency in "difficult-to-transfect" cells remains very limited.

[0015] Other cationic polymers, such as chitosan (Erbacher et al., 1998), polyamidoamine (PAMAM) dendrimers (Tomalia et al., 1985; Haensler and Szoka, 2003), degraded or fractured dendrimers (Tang et al., 1996), structurally flexible dendrimers (Liu et al., 2011), polyaminoesters (Little et al., 2004), poly(α[4-aminobutyl]-L-glycolic acid) (Akinc et al., 2003), cationic cyclodextrins, and the like, have been shown to be effective in reducing the urinary acid content of cationic polymers. Amphiphiles ( Cryan et al., 2004 ), poly( N -methylvinylamine) ( Drean et al., 2018 ), poly(2- N -dimethylaminoethyl) methacrylate (PDMAEMA), polyallylamine ( Boussif et al., 1999 ), polyornithine ( Dong et al., 1993 ), polyarginine ( Alhakamy et al., 2013 ), polyhistidine ( Putman et al., 2003 ), and cell-penetrating peptides (CPPs) ( Gupta, 2005 ) have been described for DNA transfection.

[0016] It has been reported that cationic polymers such as PEI can transfect postmitotic cells (Brunner et al.). However, in the absence of mitosis and subsequent nuclear membrane breakdown, it has been shown that plasmid DNA cannot enter the nucleus through the nuclear pore complex due to its large size (>1 kbp) (Lukacs et al., 2000). Upon release from endosomes, DNA remains bound to some cationic polymers, which contributes to protection from degradation by nucleases (Lechardeur et al., 1999). DNA can interact with cytoplasmic proteins, particularly dynein, which allows microtubule-based transport toward the nucleus or binding to transcription factors with NLS signals, which can guide DNA into the nuclear pore complex via the importin pathway (Bai et al., 2017).

[0017] Cationic polymers are a class of delivery reagents suitable for in vivo use in gene therapy approaches, where DNA / cationic polymer complexes are directly injected via various administration routes, such as intravenous, intraperitoneal, intradermal, intratumoral, or intracerebral injection. Cationic polymers formulated with acceptable excipients and / or buffers are suitable for in vivo gene transfer. In particular, PEI has been reported as an effective polymer for in vivo use (Boussif et al., 1995).

[0018] Heterocyclic compounds such as pyrazole, imidazole, or triazole derivatives, especially triazole derivatives, exhibit a wide range of biological activities due to their unique structural characteristics and electron-rich environment. Triazole derivatives may have properties that affect the pH inside the endosome. Triazoles can also contribute to hydrogen bonding with nucleic acids. The addition of cycloalkyl or aryl moieties to triazoles may provide additional hydrophobic interactions, such as π-π stacking with nucleic acid bases. Overall, these properties may offer the opportunity to fine-tune interactions with nucleic acids and develop new DNA carriers. [Prior art documents] [Patent documents]

[0019] [Patent Document 1] US8658150 [Patent Document 2] EP2070970 [Patent Document 3] EP0262641 [Patent Document 4] US20090022746 [Patent Document 5] WO2006 / 041617 Summary of the Invention [Problem to be solved by the invention]

[0020] The present inventors provide a method for improving transfection reagents by using aromatic heterocyclic compounds, particularly triazole derivatives, to fine-tune their affinity and binding to nucleic acid molecules, e.g., DNA, optimize buffering capacity under acidic conditions, and / or increase diffusion, binding, and uptake within the nucleus.

[0021] It is therefore an object of the present invention to provide a more efficient transfection composition or formulation for transfecting nucleic acid molecules into cells.

[0022] Another object of the present invention is to provide a method for transfecting a nucleic acid molecule using said composition or a formulation containing such a composition for administration to a cell.

[0023] To improve the transfection efficiency of cationic polymers, we conducted a structural screening of substituted heterocyclic compounds, particularly imidazole, triazole, and pyrazole derivatives. These substituted heterocyclic compounds were grafted onto cationic polymers of various molecular weights, particularly polyethyleneimine (PEI) polymers, to fine-tune the conjugates. Many variations were proposed to define the optimal structure for facilitating transfection of nucleic acid molecules such as DNA. Hydrophobic heterocycles were developed, which could serve as binding motifs for cytoplasmic proteins, potentially promoting nuclear import. [Means for solving the problem]

[0024] The present invention provides a composition suitable for transfecting a nucleic acid molecule into a cell, preferably a eukaryotic cell, comprising (i) at least one compound of general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or an acceptable salt thereof, and (ii) an acceptable excipient, buffer, cell culture medium, or transfection medium,

[0025] [ka]

[0026] During the ceremony, - Y 1 , Y 2 and Y 3 may be the same or different and represent C or N, provided that Y 1 , Y 2 and Y 3 At least two of the following are N, and Y 1 , Y 2 and Y 3 At least one and two or less of 1 , Z 2 and Z 3 is replaced by -Z 1 H, X1-R3-X2-P +, X1-R3-P + , X1-X2-P + , R3-X2-P + , X1-P + , R3-P + , or X2-P + or Z 1 does not exist, -Z 2 is H, straight or branched chain saturated or unsaturated C1-C 18 Alkyl, C6-C 18 Aryl, straight or branched chain, saturated or unsaturated, C6-C 18 Aryl-C1~C 18 Alkyl, straight or branched chain, saturated or unsaturated, C2-C 18 Heteroalkyl, C5-C 10 Heteroaryl, halogen, OH, linear or branched saturated or unsaturated C1-C 18 Alkylamines, C1-C 12 Alkoxy, straight or branched chain, saturated or unsaturated, C1-C 18 Alkyl-C1~C 12 Alkoxy, X1-R3-X2-P + , X1-R3-P + , X1-X2-P + , R3-X2-P + , X1-P + , R3-P + , or X2-P + or Z 2 does not exist, -Z 3 is H, straight or branched chain saturated or unsaturated C1-C 18 Alkyl, C6-C 18 Aryl, straight or branched chain, saturated or unsaturated, C6-C 18 Aryl-C1~C 18 Alkyl, C5-C 10 Heteroaryl, straight or branched chain, saturated or unsaturated, C2-C 18 Heteroalkyl, C2-C 18 Alkylidene, OH, guanidine, halogen, X1-R3-X2-P + , X1-R3-P + , X1-X2-P +, R3-X2-P + , X1-P + , R3-P + , or X2-P + or Z 3 does not exist, X1 and X2 may be identical or different and represent CO or CH2; - R3 is (CH2) m , (CH2) m -CHCH3-(CH2) n -, (CH2) m -C(CH3)2-(CH2) n -, (CH2) m -O-(CH2) n -, (CH2) m -S-(CH2) n -, (CH2) m represents —CH2—O—, m represents an integer between 1 and 3, preferably m is equal to 2, and n represents an integer between 1 and 3, -P + represents a grafted cationic polymer, which is a polyamine containing a secondary amine, a tertiary amine, a mixture of primary and secondary amines, a mixture of primary and tertiary amines, a mixture of secondary and tertiary amines, or a mixture of primary, secondary, and tertiary amines; - R or V is H, linear or branched, saturated or unsaturated C1-C 18 Alkyl or cycloalkyl, C6-C 18 Aryl, straight or branched chain, saturated or unsaturated, C6-C 18 Aryl-C1~C 18 Alkyl, straight or branched chain, saturated or unsaturated, C2-C 18 Heteroalkyl, straight or branched chain, saturated or unsaturated, C1-C 24 Esters, C5-C 10 Heteroaryl, C5-C 10 Heterocyclyl, straight or branched chain, saturated or unsaturated, C1-C 18 Alkyl-C5~C 10 Heteroaryl, X1-R3-X2-P + , X1-R3-P + , X1-X2-P +, R3-X2-P + , X1-P + , R3-P + , or X2-P + Represents, however, -Z 1 , Z 2 , Z 3 , only one of R or V is X1-R3-X2-P + , X1-R3-P + , X1-X2-P + , R3-X2-P + , X1-P + , R3-P + , or X2-P + , preferably X1-R3-X2-P + The present invention relates to a composition comprising:

[0027] In a preferred embodiment of the present invention, a composition suitable for transfecting a nucleic acid molecule into a cell, preferably a eukaryotic cell, comprises (i) at least one compound of general formula (III), or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or an acceptable salt thereof, and (ii) an acceptable excipient, buffer, cell culture medium, or transfection medium,

[0028] [ka]

[0029] During the ceremony, -Z 1 H, X1-R3-X2-P + , X1-R3-P + , X1-X2-P + , R3-X2-P + , X1-P + , R3-P + , or X2-P + or Z 1 does not exist, -Z 2 is H, straight or branched chain saturated or unsaturated C1-C 18 Alkyl, C6-C 18Aryl, straight or branched chain, saturated or unsaturated, C6-C 18 Aryl-C1~C 18 Alkyl, straight or branched chain, saturated or unsaturated, C2-C 18 Heteroalkyl, C5-C 10 Heteroaryl, halogen, OH, linear or branched saturated or unsaturated C1-C 18 Alkylamines, C1-C 12 Alkoxy, straight or branched chain, saturated or unsaturated, C1-C 18 Alkyl-C1~C 12 Alkoxy, X1-R3-X2-P + , X1-R3-P + , X1-X2-P + , R3-X2-P + , X1-P + , R3-P + , or X2-P + or Z 2 does not exist, -Z 3 is H, straight or branched chain saturated or unsaturated C1-C 18 Alkyl, C6-C 18 Aryl, straight or branched chain, saturated or unsaturated, C6-C 18 Aryl-C1~C 18 Alkyl, C5-C 10 Heteroaryl, straight or branched chain, saturated or unsaturated, C2-C 18 Heteroalkyl, C2-C 18 Alkylidene, OH, guanidine, halogen, X1-R3-X2-P + , X1-R3-P + , X1-X2-P + , R3-X2-P + , X1-P + , R3-P + , or X2-P + or Z 3 does not exist, X1 and X2 may be identical or different and represent CO or CH2; - R3 is (CH2) m , (CH2) m -CHCH3-(CH2) n-, (CH2) m -C(CH3)2-(CH2) n -, (CH2) m -O-(CH2) n -, (CH2) m -S-(CH2) n -, (CH2) m represents —CH2—O—, m represents an integer between 1 and 3, preferably m is equal to 2, and n represents an integer between 1 and 3, -P + represents a grafted cationic polymer, which is a polyamine containing a secondary amine, a tertiary amine, a mixture of primary and secondary amines, a mixture of primary and tertiary amines, a mixture of secondary and tertiary amines, or a mixture of primary, secondary, and tertiary amines; - R or V is H, linear or branched, saturated or unsaturated C1-C 18 Alkyl or cycloalkyl, C6-C 18 Aryl, straight or branched chain, saturated or unsaturated, C6-C 18 Aryl-C1~C 18 Alkyl, straight or branched chain, saturated or unsaturated, C2-C 18 Heteroalkyl, straight or branched chain, saturated or unsaturated, C1-C 24 Esters, C5-C 10 Heterocyclyl, C5-C 10 Heteroaryl, straight or branched chain, saturated or unsaturated, C1-C 18 Alkyl-C5~C 10 Heteroaryl, X1-R3-X2-P + , X1-R3-P + , X1-X2-P + , R3-X2-P + , X1-P + , R3-P + , or X2-P + represents however, -Z 1 , Z 2 or Z 3 At least one of Z 1 or Z 3 exists, -Z 1, Z 2 , Z 3 , only one of R or V is X1-R3-X2-P + , X1-R3-P + , X1-X2-P + , R3-X2-P + , X1-P + , R3-P + , or X2-P + Represents. DETAILED DESCRIPTION OF THE INVENTION

[0030] In certain embodiments of the above compositions, Z 1 , Z 2 or Z 3 and preferably only one of Z 1 or Z 3 exists.

[0031] The term "tautomer," as defined herein, refers to structural isomers that differ only in the position of hydrogen atoms and electrons. Examples of tautomers include, but are not limited to, ketone-enol, enamine-imine, amide-imidic acid, lactam-lactim, nitroso-oxime, ketene-ynol, amino acid, or phosphite-phosphonic acid.

[0032] The term "mesomer" or "meso compound," as defined herein, refers to an optically inactive stereoisomer that has two or more chiral centers.

[0033] The terms "racemate" or "racemic mixture," as defined herein, refer to a mixture of equal parts of two enantiomers.

[0034] The term "enantiomer," as defined herein, refers to a stereoisomer that is a mirror image, or enantiomer.

[0035] The term "diastereomers," as defined herein, refers to isomers of compounds that have two or more chiral centers but which are not mirror images of one another.

[0036] The term "acceptable excipient" as defined herein refers to a pharmaceutically acceptable vehicle, which is any substance or combination of substances that is physiologically acceptable, i.e., suitable for use in a composition that comes into contact with a host, particularly a human, and is therefore non-toxic. This may refer to any conventional type of solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid. Examples of suitable acceptable excipients include, but are not limited to, glucose, galactose, lactose, dextrose, maltose, mannitol, sucrose, trehalose, polyethylene glycol, or pluronic acid.

[0037] The term "buffering agent," as defined herein, refers to an agent that adjusts, maintains, or controls the pH of a solution. Buffering agents can be either weak acids or weak bases, including buffered solutions. Examples of suitable buffering agents include, but are not limited to, sodium carbonate, sodium bicarbonate, sodium hydroxide, calcium bicarbonate, calcium citrate, sodium citrate, magnesium hydroxide, magnesium bicarbonate, potassium acetate, tris acetate, sodium acetate, monobasic potassium phosphate, potassium carbonate, potassium bicarbonate, potassium citrate, or magnesium oxide.

[0038] The term "cell culture medium" or "transfection medium" as defined herein refers to a serum-containing medium, a synthetic medium, an animal component-free medium, or a chemically defined medium, particularly a medium for keeping cells alive or for growing, differentiating, or expanding cells, or for enhancing transfection.

[0039] As defined in this specification, "C1 to C 18 The term "alkyl" refers to any monovalent radical of a straight or branched hydrocarbon chain containing from 1 to 18 carbon atoms. The term "C1-C6 alkyl" refers to an alkyl group having from 1 to 6 carbon atoms. Suitable C1-C 18Examples of alkyl groups include, but are not limited to, C1-C4 alkyl groups such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, or t-butyl, C6-C8 alkyl groups such as n-hexyl, n-heptyl, or n-octyl, as well as n-pentyl, 2-ethylhexyl, 3,5,5-trimethylhexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, or n-octadecyl.

[0040] As defined in this specification, "C1 to C 12 The term "alkoxy" refers to a group of formula -OR', where R' is a C1-C 12 Alkyl. Suitable C1-C 12 Examples of alkoxy groups include, but are not limited to, C1-C6 alkoxy groups such as methoxy (-OCH3), ethoxy (-OCH2CH3), t-butoxy (-OC(CH3)3), or -O(CH2)5CH3.

[0041] As defined in this specification, "C6 to C 18 The term "aryl" refers to any monovalent radical of an aromatic hydrocarbon containing 6 to 18 carbon atoms. 18 Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, or phenanthrenyl.

[0042] As defined in this specification, "C6 to C 18 Aryl-C1~C 18 The term "alkyl" refers to an aryl group, as defined herein, attached to an alkyl group, as defined herein. 18 Aryl-C1~C 18Examples of alkyl groups include, but are not limited to, benzyl, phenylethyl (or phenethyl), phenylpropyl, phenylbutyl, phenylpentyl, phenylhexyl, naphthylmethyl, naphthylethyl, naphthylpropyl, naphthylbutyl, naphthylpentyl, naphthylhexyl, anthracenylmethyl, anthracenylethyl, anthracenylpropyl, anthracenylbutyl, anthracenylpentyl, anthracenylhexyl, phenanthrenylmethyl, phenanthrenylethyl, phenanthrenylpropyl, phenanthrenylbutyl, phenanthrenylpentyl, or phenanthrenylhexyl.

[0043] As defined in this specification, 18 The term "heteroalkyl" refers to an alkyl group, as defined herein, substituted with one or more heteroatoms such as O, N, or S.

[0044] As defined in this specification, 10 The term "heteroaryl" refers to any monovalent radical of a monocyclic or bicyclic 5- to 10-membered aromatic group containing 1 to 3 heteroatoms independently selected from oxygen, nitrogen, and sulfur. Suitable C5-C 10 Examples of heteroaryl groups include, but are not limited to, furyl, thienyl, pyrrolyl, pyrazoyl, imidazolyl, isoxazolyl, isothiazolyl, thiazolyl, oxazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1-benzofuryl, 1-benzothienyl, indolyl, benzimidazolyl, indazolyl, 1,2-benzisoxazolyl, 2,1-benzisoxazolyl, 1,2-benzisothiazolyl, 2,1-benzisothiazolyl, benzothiazolyl, benzoxazolyl, benzotriazolyl, pyridyl, pyridinium, quinolinyl, quinolinium, isoquinolinyl, isoquinolinium, pyridazinyl, cinnolinyl, phthalazinyl, pyrimidinyl, quinazolinyl, pyrazinyl, or quinoxalinyl.

[0045] As defined in this specification, "C1 to C 18The term "alkylamine" refers to any monovalent group of a straight or branched hydrocarbon chain containing 1 to 18 carbon atoms in which one of the hydrogen atoms bonded to a carbon atom has been replaced by an amino group. 18 Examples of alkylamines include -(CH2) where n is an integer between 1 and 18. n -NH2, -CH2NHCH3, -CH2CH(CH3)-NH2, or -(CH2) where n represents an integer between 1 and 6. n Examples include, but are not limited to, N(CH3)2.

[0046] As defined in this specification, "C1 to C 18 Alkyl-C1~C 12 The term "alkoxy" refers to an alkyl group, as defined herein, attached to an alkoxy group, as defined herein.

[0047] As defined in this specification, 18 The term "alkylidene" refers to a divalent group derived from an alkane by removing two hydrogen atoms from the same carbon atom, the free valence being part of a double bond (=CR). 18 Examples of alkylidene include, but are not limited to, =CH2, =CH(CH2CH3), or =C(CH3)2.

[0048] The term "halogen" as defined herein refers to an atom of F, Cl, Br, or I.

[0049] As defined in this specification, "C1 to C 24 The term "ester" refers to a group of formula -C(O)OR'', where R'' is a C1-C 24 Alkyl, particularly C1-C as defined herein 18 It is alkyl.

[0050] As defined in this specification, 10The term "heterocyclyl" refers to any monovalent monocyclic or bicyclic 5- to 10-membered ring containing one or more heteroatoms such as O, N, or S. Examples of suitable heterocyclyl groups include, but are not limited to, piperidinyl, piperazinyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, or azepanyl, and the like.

[0051] Unless otherwise stated, the groups and radicals defined herein can be unsubstituted or substituted with one or more substituents such as halogen, alkyl, alkoxy, aryl, heteroaryl, haloalkyl, haloalkoxy, alkoxycarbonyl, alkanoyl, aroyl, formyl, nitrile, nitro, amido, alkylthio, alkylsulfinyl, alkylsulfonyl, arylthio, arylsulfinyl, arylsulfonyl, amino, alkylamino, arylamino, dialkylamino, diarylamino, and the like.

[0052] In certain embodiments of the present invention, the composition further comprises at least one nucleic acid molecule to be transfected into the cells. Preferably, the nucleic acid molecule is selected from the group consisting of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), DNA / RNA hybrid, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), CRISPR guide RNA, and expression vectors encoding the nucleic acid molecule, in particular plasmids encoding the nucleic acid molecule or expressing the nucleic acid molecule, such as siRNA, microRNA, shRNA, CRISPR guide RNA, etc. Preferably, the nucleic acid molecule is DNA.

[0053] When separate nucleic acids are provided in the compositions of the invention, they may be all DNA molecules, or all RNA molecules, or a mixture of DNA and RNA molecules, or molecules comprising an association of DNA and RNA strands.

[0054] The nucleic acid molecule may be single-stranded or double-stranded and may contain modified or unmodified bases.

[0055] As used herein, the terms "polynucleotide," "nucleic acid," "oligonucleotide," and "nucleic acid molecule" are used interchangeably to designate these nucleic acid molecules.

[0056] The composition of the present invention may be used in accordance with the disclosure provided herein as a combination of a nucleic acid molecule and at least one compound of general formula (I) (including any specific embodiment disclosed herein), preferably at least one compound of general formula (III), and an acceptable excipient, buffer, cell culture medium, or transfection medium. Alternatively, it may be used as a cell culture or expanded cells, where isolated cells have been treated with the transfection formulation before providing as a culture and / or expanded cells. Otherwise, the composition of the present invention encompasses, in one embodiment, cells or cell cultures or expanded cells into which the formulation has been introduced by transfection according to the present invention. The cells are particularly mammalian cells, preferably human cells. The cells may be dividing or non-dividing cells.

[0057] In a particular embodiment of the present invention, the composition according to the present invention comprises one to five, preferably at least two, different compounds of general formula (I), preferably general formula (III), or tautomers, mesomers, racemates, enantiomers, diastereomers, or mixtures thereof, or acceptable salts thereof.

[0058] In certain embodiments of the present invention, at least one preferred compound of general formula (I) as defined herein is a compound having the formula: 1 and Y 3 represents N and Y 2 represents C, or (ii) Y 1 and Y 2 represents N and Y 3 represents C, or (iii) Y 2 and Y 3represents N and Y 1 represents C, or (iv) Y 1 , Y 2 and Y 3 represents N. (iv) Y 1 , Y 2 and Y 3 When represents N, at least one preferred compound of general formula (I) as defined herein corresponds to a compound of general formula (III).

[0059] Since the structure of the compound of general formula (III) is symmetrical, R and V may be interchanged, and Z 1 and Z 3 may be interchanged. Therefore, the definition for R also applies to V, and Z 1 The definition for Z 3 also applies to

[0060] In certain embodiments of the present invention, at least one preferred compound of general formula (I), preferably general formula (III), as defined herein, is selected from the group consisting of: (i) Z 1 represents H, or (ii) Z 1 X1-R3-X2-P + , X1-R3-P + , X1-X2-P + , R3-X2-P + , X1-P + , R3-P + , or X2-P + , preferably X1-R3-X2-P + wherein X1, X2, R3 and P + is as defined herein, more preferably Z 1 X1-R3-X2-P + wherein X1 represents CH2, X2 represents CO, and R3 represents (CH2) m and m represents an integer between 1 and 3, preferably m is equal to 2.

[0061] In certain embodiments of the present invention, at least one preferred compound of general formula (I), preferably general formula (III), as defined herein, is selected from the group consisting of: (i) Z2 But H, C1~C 12 Alkoxy or straight or branched chain saturated or unsaturated C1-C 18 alkyl, preferably a linear or branched, saturated or unsaturated C1-C6 alkyl, more preferably Z 2 represents H, CH3, CF3 or OCH3, even more preferably Z 2 represents CH3, or (ii) Z 2 But X1-R3-X2-P + , X1-R3-P + , X1-X2-P + , R3-X2-P + , X1-P + , R3-P + , or X2-P + , preferably X1-R3-X2-P + wherein X1, X2, R3 and P + is as defined herein, more preferably Z 2 But X1-R3-X2-P + wherein X1 represents CH2, X2 represents CO, and R3 represents (CH2) m and m represents an integer between 1 and 3, preferably m is equal to 2.

[0062] In certain embodiments of the present invention, at least one preferred compound of general formula (I), preferably general formula (III), as defined herein, is selected from the group consisting of: (i) Z 3 H, straight or branched chain saturated or unsaturated C1-C 18 Alkyl, preferably linear or branched, saturated or unsaturated C1 to C6 alkyl, or linear or branched, saturated or unsaturated C6 to C 18 Aryl-C1~C 18 alkyl, preferably fluorobenzyl or 4-hydroxyphenethyl; or (ii) Z 3 But X1-R3-X2-P + , X1-R3-P + , X1-X2-P + , R3-X2-P + , X1-P + , R3-P +, or X2-P + , preferably X1-R3-X2-P + wherein X1, X2, R3 and P + is as defined herein, more preferably Z 3 But X1-R3-X2-P + wherein X1 represents CH2, X2 represents CO, and R3 represents (CH2) m and m represents an integer between 1 and 3, preferably m is equal to 2.

[0063] In a preferred embodiment of the present invention, (i) Z 1 But X1-R3-X2-P + , X1-R3-P + , X1-X2-P + , R3-X2-P + , X1-P + , R3-P + or X2-P + , preferably X1-R3-X2-P + wherein X1, X2, R3 and P + is as defined herein, more preferably Z 1 X1-R3-X2-P + wherein X1 represents CH2, X2 represents CO, and R3 represents (CH2) m wherein m represents an integer between 1 and 3, preferably m is equal to 2; (ii) Z 2 H, C1~C 12 Alkoxy or straight or branched chain saturated or unsaturated C1-C 18 alkyl, preferably a linear or branched, saturated or unsaturated C1 to C6 alkyl, more preferably Z 2 represents H, CH3, CF3 or OCH3, and / or (iii) Z 3 is H, straight or branched chain saturated or unsaturated C1-C 18 Alkyl, preferably straight-chain or branched, saturated or unsaturated C1 to C6 alkyl, or straight-chain or branched, saturated or unsaturated C6 to C 18 Aryl-C1~C 18alkyl, preferably fluorobenzyl or 4-hydroxyphenethyl, and / or (iv) R or V is H, a linear or branched, saturated or unsaturated C1-C 18 Alkyl or cycloalkyl, C6-C 18 Aryl, straight or branched chain, saturated or unsaturated, C6-C 18 Aryl-C1~C 18 Alkyl, straight or branched chain, saturated or unsaturated, C2-C 18 Heteroalkyl, straight or branched chain, saturated or unsaturated, C1-C 24 Esters, C5-C 10 Heterocyclyl, C5-C 10 Heteroaryl or straight or branched chain saturated or unsaturated C1-C 18 Alkyl-C5~C 10 represents heteroaryl.

[0064] In another preferred embodiment of the present invention, (i) Z 2 But X1-R3-X2-P + , X1-R3-P + , X1-X2-P + , R3-X2-P + , X1-P + , R3-P + , or X2-P + , preferably X1-R3-X2-P + wherein X1, X2, R3 and P + is as defined herein, more preferably Z 2 But X1-R3-X2-P + wherein X1 represents CH2, X2 represents CO, and R3 represents (CH2) m wherein m represents an integer between 1 and 3, preferably m is equal to 2; (ii) Z 1 represents H, and / or (iii) Z 3 is H, straight or branched chain saturated or unsaturated C1-C 18 Alkyl, preferably straight-chain or branched, saturated or unsaturated C1 to C6 alkyl, or straight-chain or branched, saturated or unsaturated C6 to C 18 Aryl-C1~C18 alkyl, preferably fluorobenzyl or 4-hydroxyphenethyl, and / or (iv) R or V is H, a linear or branched, saturated or unsaturated C1-C 18 Alkyl or cycloalkyl, C6-C 18 Aryl, straight or branched chain, saturated or unsaturated, C6-C 18 Aryl-C1~C 18 Alkyl, straight or branched chain, saturated or unsaturated, C2-C 18 Heteroalkyl, straight or branched chain, saturated or unsaturated, C1-C 24 Esters, C5-C 10 Heterocyclyl, C5-C 10 Heteroaryl or straight or branched chain saturated or unsaturated C1-C 18 Alkyl-C5~C 10 represents a heteroaryl.

[0065] In another preferred embodiment of the present invention, (i) Z 3 But X1-R3-X2-P + , X1-R3-P + , X1-X2-P + , R3-X2-P + , X1-P + , R3-P + or X2-P + , preferably X1-R3-X2-P + wherein X1, X2, R3 and P + is as defined herein, more preferably Z 3 But X1-R3-X2-P + wherein X1 represents CH2, X2 represents CO, and R3 represents (CH2) m wherein m represents an integer between 1 and 3, preferably m is equal to 2; (ii) Z 1 represents H, and / or (iii) Z 2 H, C1~C 12 Alkoxy or straight or branched chain saturated or unsaturated C1-C 18 alkyl, preferably a linear or branched, saturated or unsaturated C1 to C6 alkyl, more preferably Z2 represents H, CH3, CF3 or OCH3, and / or (iv) R or V is H, a linear or branched, saturated or unsaturated C1-C 18 Alkyl or cycloalkyl, C6-C 18 Aryl, straight or branched chain, saturated or unsaturated, C6-C 18 Aryl-C1~C 18 Alkyl, straight or branched chain, saturated or unsaturated, C2-C 18 Heteroalkyl, straight or branched chain, saturated or unsaturated, C1-C 24 Esters, C5-C 10 Heterocyclyl, C5-C 10 Heteroaryl or straight or branched chain saturated or unsaturated C1-C 18 Alkyl-C5~C 10 represents heteroaryl.

[0066] In another preferred embodiment of the present invention, (i) R or V is X1-R3-X2-P + , X1-R3-P + , X1-X2-P + , R3-X2-P + , X1-P + , R3-P + , or X2-P + , preferably X1-R3-X2-P + wherein X1, X2, R3 and P + is as defined herein, more preferably Z 3 But X1-R3-X2-P + wherein X1 represents CH2, X2 represents CO, and R3 represents (CH2) m wherein m represents an integer between 1 and 3, preferably m is equal to 2; (ii) Z 1 represents H, and / or (iii) Z 2 H, C1~C 12 Alkoxy or straight or branched chain saturated or unsaturated C1-C 18 alkyl, preferably a linear or branched, saturated or unsaturated C1-C6 alkyl, and / or (iv) Z 3is H, straight or branched chain saturated or unsaturated C1-C 18 Alkyl, preferably linear or branched, saturated or unsaturated C1-C6 alkyl, or linear or branched, saturated or unsaturated C6-C18 aryl-C1-C 18 It represents alkyl, preferably fluorobenzyl or 4-hydroxyphenethyl.

[0067] In a particular embodiment of the present invention, at least one preferred compound of general formula (I), preferably general formula (III), as defined herein, comprises (i) Z 1 , Z 2 or Z 3 Only one of the X1-R3-X2-P + , X1-R3-P + , X1-X2-P + , R3-X2-P + , X1-P + , R3-P + , or X2-P + , preferably X1-R3-X2-P + wherein X1, X2, R3 and P + is as defined herein, more preferably Z 1 , Z 2 or Z 3 Only one of the X1-R3-X2-P + wherein X1 represents CH2, X2 represents CO, and R3 represents (CH2) m where m is an integer between 1 and 3, preferably m is equal to 2, and / or (ii) Z 1 represents H, and / or (iii) Z 2 But H, C1~C 12 Alkoxy or straight or branched chain saturated or unsaturated C1-C 18 alkyl, preferably a linear or branched, saturated or unsaturated C1-C6 alkyl, more preferably Z 2 represents H, CH3, CF3 or OCH3, and / or (iv) Z 3 is H or a linear or branched, saturated or unsaturated C1-C 18The compound is an alkyl, preferably a linear or branched, saturated or unsaturated C1 to C6 alkyl.

[0068] In a particular embodiment of the present invention, at least one preferred compound of general formula (I), preferably general formula (III), as defined herein, is selected from the group consisting of: (i) R or V is selected from the group consisting of X1-R3-X2-P + , X1-R3-P + , X1-X2-P + , R3-X2-P + , X1-P + , R3-P + , or X2-P + , preferably X1-R3-X2-P + wherein X1, X2, R3 and P + is as defined herein, more preferably Z 3 But X1-R3-X2-P + wherein X1 represents CH2, X2 represents CO, and R3 represents (CH2) m wherein m represents an integer between 1 and 3, preferably m is equal to 2; (ii) Z 3 exists and Z 3 is H, straight or branched chain saturated or unsaturated C1-C 18 Alkyl, preferably straight-chain or branched, saturated or unsaturated C1 to C6 alkyl, or straight-chain or branched, saturated or unsaturated C6 to C 18 Aryl-C1~C 18 alkyl, preferably fluorobenzyl or 4-hydroxyphenethyl.

[0069] In a particular embodiment of the present invention, at least one preferred compound of the defined general formula (I), preferably general formula (III), is a compound in which the grafted cationic polymer is selected from the group consisting of linear or branched polyethyleneimine (PEI), PEI dendrimer, polypropyleneimine (PPI), poly(amidoamine) (PAA) and dendrimer (PAMAM), cationic cyclodextrin, polyalkylamine, polyhydroxyalkylamine, poly(butylenimine) (PBI), spermine, N-substituted polyallylamine, N-substituted chitosan, N-substituted polyornithine, N-substituted polylysine (PLL), N-substituted polyvinylamine, poly(β-amino ester), hyperbranched poly(amino ester) (h-PAE), network poly(amino ester) (n-PAE), poly(4-hydroxy-l-proline ester) (PHP-ester) and poly-β-amino acid. Preferably, the grafted cationic polymer is linear or branched PEI, more preferably linear PEI.

[0070] The grafted cationic polymer may have a graft ratio in the range of 1 to 50%, preferably 5 to 30%, more preferably 20%.

[0071] The term "grafting ratio" as defined herein refers to the number of grafted monomers on primary or secondary amino groups via side chains divided by the total number of monomers present in the original cationic polymer. The grafting ratio depends on the molecular weight of the cationic polymer, the chemical reactivity of the side chains grafted onto the polymer, or the biological effect obtained. The grafting ratio can be determined by methods well known in the art, such as NMR.

[0072] The grafted cationic polymer may have an average molecular weight (Mw) in the range of 1 kDa to 500 kDa, preferably 1 kDa to 50 kDa, more preferably 5 kDa to 50 kDa or 1 kDa to 15 kDa. In particular, the grafted cationic polymer may have an average molecular weight (Mw) of 6, 8, 10, 15, 22 or 30 kDa, preferably 6, 8, 10, 15 or 30 kDa.

[0073] The grafted cationic polymer may be associated with counterions such as chloride, phosphate, citrate, acetate, propionate, carbonate, succinate, sulfonate, sulfate, or carboxylate.

[0074] In a particular embodiment of the present invention, at least one preferred compound of general formula (I), preferably general formula (III), as defined herein, comprises Y 1 , Y 2 , Y 3 , Z 1 , Z 2 , Z 3 , X1, X2, R3 and P + is as defined herein, and R or V is H, a linear or branched, saturated or unsaturated C1-C 18 Alkyl or cycloalkyl, C6-C 18 Aryl, straight or branched chain, saturated or unsaturated, C6-C 18 Aryl-C1~C 18 Alkyl, straight or branched chain, saturated or unsaturated, C2-C 18 Heteroalkyl, straight or branched chain, saturated or unsaturated, C1-C 24 Esters, C5-C 10 Heterocyclyl, C5-C 10 Heteroaryl, straight or branched chain, saturated or unsaturated, C1-C 18 Alkyl-C5~C 10 Heteroaryl, X1-R3-X2-P + , X1-R3-P + , X1-X2-P + , R3-X2-P + , X1-P + , R3-P +, or X2-P + is a compound represented by the formula:

[0075] Preferably, R or V represents H, methyl, ethyl, propyl, cyclopropyl, isopropyl, sec-butyl, cyclopentyl, phenyl, fluorophenyl, benzyl, pyridine, 2-pyridine, 3-pyridine, fluorobenzyl, substituted morpholinyl, substituted piperazinyl, 4-hydroxybenzyl or 4-hydroxyphenethyl, more preferably R or V represents methyl, ethyl, propyl, cyclopropyl, isopropyl, sec-butyl, cyclopentyl, phenyl, benzyl, fluorobenzyl, 4-hydroxyphenethyl, 2-pyridine or 3-pyridine.

[0076] Z 1 , Z 2 , Z 3 The most preferred embodiments of compounds of formula (III) with respect to X1, X2, R3 and P are as defined herein for compounds of formula (I).

[0077] In certain embodiments of the present invention, preferred compounds are 1 , Z 2 or Z 3 Only one of the following, preferably Z 1 But X1-R3-X2-P + , X1-R3-P + , X1-X2-P + , R3-X2-P + , X1-P + , R3-P + , or X2-P + , preferably X1-R 3 -X2-P + wherein X1, X2, R3 and P + is as defined in formula (I), preferably formula (III).

[0078] In certain embodiments of the invention, preferred compounds are those in which only one of R or V is X1-R3-X2-P + , X1-R3-P + , X1-X2-P+ , R3-X2-P + , X1-P + , R3-P + , or X2-P + , preferably X1-R3-X2-P + wherein X1, X2, R3 and P + is as defined in formula (I), preferably formula (III).

[0079] In certain embodiments of the present invention, preferred compounds are 1 , Y 2 and Y 3 represents N. These compounds correspond to the compounds of general formula (III).

[0080] In certain embodiments of the present invention, preferred compounds are 3 Straight or branched chain saturated or unsaturated C6-C 18 Aryl-C1~C 18 The compounds are those which represent alkyl, preferably fluorobenzyl or 4-hydroxyphenethyl.

[0081] In certain embodiments of the invention, preferred compounds are those in which R represents H, methyl, propyl, isopropyl, cyclopropyl, benzyl, fluorobenzyl, pyridine, 2-pyridine, 3-pyridine, phenyl, fluorophenyl, substituted morpholinyl, or substituted piperazinyl.

[0082] In certain embodiments of the present invention, preferred compounds are those in which V is H, X1-R3-X2-P + , X1-R3-P + , X1-X2-P + , R3-X2-P + , X1-P + , R3-P + , or X2-P + , preferably X1-R3-X2-P + wherein X1, X2, R3 and P + is as defined in formula (I), preferably formula (III).

[0083] In a preferred embodiment of the present invention, the preferred compounds are (i) Y 1 , Y 2 and Y 3 represents N, and / or (ii) V is X1-R3-X2-P + , X1-R3-P + , X1-X2-P + , R3-X2-P + , X1-P + , R3-P + , or X2-P + , preferably X1-R3-X2-P + wherein X1, X2, R3 and P + is as defined in formula (I), and / or (iii) R represents H, and / or (iv) Z 3 is a compound in which fluorobenzyl or 4-hydroxyphenethyl is represented.

[0084] In a preferred embodiment of the present invention, the preferred compounds are (i) Y 1 , Y 2 and Y 3 represents N, and / or (ii) Z 1 X1-R3-X2-P + , X1-R3-P + , X1-X2-P + , R3-X2-P + , X1-P + , R3-P + or X2-P + , preferably X1-R3-X2-P + and / or (iii) V represents H; and / or (iv) R represents benzyl, fluorobenzyl, pyridine, 2-pyridine, 3-pyridine, methyl, propyl, isopropyl, cyclopropyl, phenyl, fluorophenyl, substituted morpholinyl, or substituted piperazinyl.

[0085] According to a particular embodiment of the present invention, preferred compounds correspond to compounds 2.19 to 2.61, preferably compounds 2.19, 2.22, 2.23, 2.42, 2.43, 2.44, 2.46, 2.47, 2.54, 2.55, 2.56, 2.57, 2.58, 2.59, 2.60 and 2.61, as disclosed in Table 1.

[0086] [Table 1A]

[0087] [Table 1B]

[0088] [Table 1C]

[0089] [Table 1D]

[0090] [Table 1E]

[0091] In certain embodiments of the present invention, the at least one compound of general formula (III) is selected from the group consisting of the following compounds:

[0092] [Table 2A]

[0093] [Table 2B]

[0094] [Table 2C]

[0095] In a preferred embodiment of the present invention, at least one compound of general formula (III) is selected from the group consisting of the following compounds: 2.19, 2.22, 2.42, 2.43, 2.44, 2.46, 2.47, 2.54, 2.55, 2.56, 2.57, 2.58, 2.59, 2.60 and 2.61, and even more preferably is compound 2.22.

[0096] At least one compound of general formula (I), preferably general formula (III), can be prepared according to various methods well known in the art.

[0097] The present invention also relates to a composition according to the invention for use in in vivo applications for cell transformation, cell therapy or gene therapy by uptake of exogenous nucleic acid using the composition of the invention. The cells may be eukaryotic cells, in particular mammalian cells, in particular human cells, in particular primary cells, and may be either dividing or non-dividing cells.

[0098] The present invention also relates to a method for the in vitro or ex vivo transfection of living cells, which may be provided or maintained in serum-containing, synthetic, animal-component-free or chemically defined medium, comprising introducing into the cells a composition according to the invention.

[0099] The present invention also relates to the in vitro or ex vivo use of a composition according to the invention for transfecting at least one nucleic acid molecule into cells, cell lines or cells, preferably selected from the group consisting of mammalian cells, insect cells, primary cells, adherent cells, suspension cells, dividing cells, such as hepatocytes, non-dividing cells, such as neuronal cells, and cancer cells, said cells, cell lines or cells optionally organized into spheroids, organoids, 2D or 3D cell cultures or provided as fiber or matrix cultures and / or in a bioreactor.

[0100] The term "adherent cells" as defined herein refers to cells that require a solid support for growth and are therefore anchorage-dependent. Examples of adherent cells include, but are not limited to, MRC-5 cells, HeLa cells, Vero cells, NIH-3T3 cells, L293 cells, CHO cells, BHK-21 cells, MCF-7 cells, A549 cells, COS cells, HEK293 cells, Hep G2 cells, SNN-BE(2) cells, BAE-1 cells, or SH-SY5Y cells.

[0101] The term "suspension cells" as defined herein refers to cells that do not require a solid support for growth and are therefore anchorage-independent. Examples of suspension cells include, but are not limited to, NSO cells, U937 cells, Namalawa cells, HL60 cells, WEHI231 cells, Yacl cells, Jurkat cells, THP-1 cells, K562 cells, or U266B1 cells.

[0102] The term "spheroid," as defined herein, refers to a spherical heterogeneous aggregate of cells in culture that retains a three-dimensional structure.

[0103] The term "organoid," as defined herein, refers to a three-dimensional structure made up of a collection of organ-specific cell types that self-organize in a manner similar to that in vivo.

[0104] The term "fiber or matrix culture" as defined herein refers to a three-dimensional cell culture support composed of insoluble elastic fibers or extracellular proteins self-assembled into a matrix.

[0105] The transfection may be stable or transient, standard or reverse.

[0106] As disclosed herein, the composition according to the present invention may comprise a plurality of different nucleic acids for co-transfection, in particular those selected from the group consisting of a plurality of plasmid DNAs, a plasmid DNA and an oligonucleotide, a plasmid DNA and an mRNA.

[0107] The at least one nucleic acid molecule to be transfected may be a protein, a protein fragment, a peptide, or a gene encoding an antibody or a functional antigen-binding region thereof, in particular the VH and / or VL chain thereof. The protein may be selected from the group consisting of reporter proteins, fluorescent proteins, enzymes, structural proteins, receptors, transmembrane proteins, therapeutic proteins, cytokines, toxins, oncogenic proteins, anti-oncogenes, pro-apoptotic proteins, anti-apoptotic proteins, polymerases, transcription factors, and capsid proteins.

[0108] The present invention also relates to the in vitro or ex vivo use of the compositions according to the invention for genome engineering, cell reprogramming, in particular the reprogramming of differentiated cells into induced pluripotent stem cells (iPCs), cell differentiation or gene editing. Such use can be carried out in in vitro or ex vivo cell culture for the production of biologics, the preparation of cells for therapeutic purposes or the study of cell function or behavior, in particular for the step of cell expansion after transfection, or can be carried out in vivo for therapeutic purposes in a host in need thereof.

[0109] The present invention also relates to the in vitro or ex vivo use of a composition according to the invention: (i) in the production of a biological product, in particular a biological product encoding a recombinant protein, peptide or antibody; or (ii) in the production of a recombinant virus, such as an adeno-associated virus (AAV), a lentivirus (LV), an adenovirus, an oncolytic virus or a baculovirus, wherein the composition comprises multiple nucleic acid molecules, such as multiple plasmids for co-transfection; or (iii) in the production of a virus or virus-like particle, wherein the composition comprises multiple nucleic acid molecules, such as multiple plasmids for co-transfection.

[0110] Thus, the present invention also relates to methods for (i) the production of biologics, in particular biologics encoding recombinant proteins, peptides or antibodies, or (ii) the production of recombinant viruses, such as adeno-associated viruses (AAV), lentiviruses (LV), adenoviruses, oncolytic viruses or baculoviruses, in which the composition according to the invention comprises multiple nucleic acid molecules for co-transfection, or (iii) the production of viruses or virus-like particles, in which the composition according to the invention comprises multiple nucleic acid molecules for co-transfection.

[0111] In a preferred embodiment of the method for producing AAV, the composition comprises (i) at least one compound selected from the group consisting of compounds 2.22, 2.23, 2.43, 2.44, 2.47, 2.54, 2.57, 2.60, and 2.61, and (ii) an acceptable excipient, buffer, cell culture medium, or transfection medium.

[0112] In a preferred embodiment of the method for producing LV, the composition comprises (i) at least Compound 2.22, and (ii) an acceptable excipient, buffer, cell culture medium, or transfection medium.

[0113] The term "biologic" as defined herein refers to a biological entity such as a protein, or nucleic acid, or a combination thereof, a cell or virus, a cell compartment, an organoid, and a tissue.

[0114] In a particular embodiment of the invention, said in vitro or ex vivo use of a composition or said method according to the invention is for the production of recombinant viruses, wherein said composition comprises a plurality of expression vectors, such as plasmid vectors, for transfecting adherent or suspension cells, such as HEK293 and derivative cells, HeLa, BHK-21, A549 or insect cells, wherein said vectors, in particular plasmids, are constructs expressing viral structural sequences and transfer vector genomes for virus or virus-like production, and optionally expressing a molecule of interest encoded by the transfer vector genome.

[0115] In certain embodiments of the invention, the recombinant virus is for use in in vivo applications for cell therapy or gene therapy.

[0116] In a specific embodiment of the invention, the invention relates to the in vitro or ex vivo use of a composition according to the invention in the production of a recombinant virus, such as an adeno-associated virus (AAV) or a lentivirus (LV), said composition comprising (i) at least one compound selected from the group consisting of compounds 2.22, 2.23, 2.42, 2.43, 2.44, 2.46, 2.47, 2.54, 2.57, 2.60, and 2.61, and (ii) an acceptable excipient, buffer, cell culture medium, or transfection medium. Preferably, a composition comprising compound 2.22 is used in the production of LV, and a composition comprising at least one compound selected from the group consisting of compounds 2.22, 2.23, 2.43, 2.44, 2.47, 2.54, 2.57, 2.60, and 2.61 is used in the production of AAV.

[0117] Unless otherwise stated, all of the above-described embodiments may be combined with each other. Thus, features that are described in the context of separate embodiments may also be combined in a single embodiment.

[0118] Other features and advantages of the invention will become apparent from the examples which follow and are illustrated in the drawings. [Brief explanation of the drawings]

[0119] [Figure 1] Chemical structure of the compound of general formula (I). [Figure 2] Percentage of GFP expression after transfection of Caco-2, Hep G2, MDCK and MCF-10A with the compound of Example 3. The ratios 1:3 and 1:4 indicate the ratio of μg of DNA per μL of compound. [Figure 3] AAV-2 production from suspension HEK-293T cells. AAV-2 vectors expressing a GFP reporter gene were produced in HEK-293T cells grown in suspension in FreeStyle F17 medium. After seeding and culturing for 3 days, the cells were transfected with three plasmids (pAAV-RC2 vector expressing Rep and Cap; pHelper vector expressing Adenoviral E2A, Adenoviral E4, and Adenoviral VA helper factors; and pAAV-GFP control vector expressing GFP under the control of a CMV promoter) together with PEIpro® or various compounds at a ratio of 1:2 or 1:3 μg DNA / μL reagent. AAV titers (transducing units, TU / mL) were determined 72 hours after transfection. Results are expressed as relative AAV-2 transducing units / mL (TU / mL) compared to PEIpro® transfections at ratios of 1:2 and 1:3. [Figure 4] Production of lentiviral particles from suspension HEK-293T cells. Lentivirus expressing a GFP reporter gene was produced in HEK-293T cells grown in suspension in FreeStyle F17 medium. After seeding and culturing for 3 days, the cells were transfected with the four plasmids together with PEIpro® or compound 2.22 at a ratio of 1:2 μg total DNA / μL reagent. Lentiviral titers (transducing units, TU / mL) were determined 72 hours after transfection. [Figure 5] Chemical structure of the compound of general formula (III). [Figure 6] Percentage of GFP expression after transfection of Hep G2 cells with compounds 2.22 and 2.53–2.61. The ratios 1:3 and 1:4 indicate the ratio of μg of DNA per μL of compound. [Figure 7] AAV-2 production from suspension HEK-293T cells using compounds 2.22 and 2.53–2.61. AAV-2 vectors expressing a GFP reporter gene were produced in HEK-293T cells grown in suspension in FreeStyle F17 medium. Cells were seeded and cultured for 3 days, after which they were transfected with three plasmids (pAAV-RC2 vector expressing Rep and Cap; pHelper vector expressing Adenoviral E2A, Adenoviral E4, and Adenoviral VA helper factors; and pAAV-GFP control vector expressing GFP under the control of a CMV promoter) together with PEIpro® or various compounds at a 1:2 μg DNA / μL reagent ratio. AAV titers (transducing units, TU / mL) were determined 72 hours after transfection. Results are expressed as relative AAV-2 transducing units / mL (TU / mL). [Figure 8]The effect of the amount of transfected DNA and the ratio of compound 2.22 per μg of DNA on AAV-2 production from suspension HEK-293T cells was investigated. AAV-2 vectors expressing a GFP reporter gene were produced in HEK-293T cells grown in suspension in FreeStyle F17 medium. After seeding and culturing for 3 days, the cells were transfected with three plasmids (pAAV-RC2 vector expressing Rep and Cap; pHelper vector expressing Adenoviral E2A, Adenoviral E4, and Adenoviral VA helper factors; and pAAV-GFP control vector expressing GFP under the control of a CMV promoter) along with compound 2.22 (formulated at a 15 mM nitrogen concentration) at different ratios of DNA μg / μL (1:1.5 to 1:3). AAV titers (transducing units, TU / mL) were determined 72 hours after transfection. Results are expressed as relative AAV-2 transducing units / mL (TU / mL). Cell viability was determined 72 hours after transfection using a trypan blue assay. [Figure 9] Effect of DNA complexation time with compound 2.22 on AAV-2 production from suspension HEK-293T cells. AAV-2 vectors expressing a GFP reporter gene were produced in HEK-293T cells grown in suspension in FreeStyle F17 medium. After seeding and culturing for 3 days, the cells were transfected with three plasmids (pAAV-RC2 vector expressing Rep and Cap, pHelper vector expressing Adenoviral E2A, Adenoviral E4, and Adenoviral VA helper factors, and pAAV-GFP control vector expressing GFP under the control of a CMV promoter) together with compound 2.22 (formulated at a nitrogen concentration of 15 mM) at a DNA / reagent μL ratio of 1:2, at 1 μg DNA / 10 cells. AAV titers (transducing units, TU / mL) were determined 72 hours after transfection. Results are expressed as relative AAV-2 transducing units / mL (TU / mL). [Example]

[0120] Experimental Section Materials and Methods cell culture Caco-2 (ATCC® HTB-37™) human colonic epithelial cells were grown in DMEM 4.5 g / L glucose containing 20% ​​FBS supplemented with 1% non-essential amino acids, 1 mM sodium pyruvate, 2 mM glutamine, and 100 U / mL penicillin and 100 μg / mL streptomycin at 37°C in 5% CO2 atmosphere.

[0121] MCF 10A (ATCC® CRL-10317™) human mammary epithelial cells were grown in MEBM (Lonza) supplemented with SingleQuots™ Supplements and Growth Factors (Lonza) and 100 ng / ml cholera toxin at 37°C in 5% CO2 in air.

[0122] Hep G2 (ATCC® HB-8065™) human hepatoma cells were grown in MEM (Ozyme) containing 10% FBS supplemented with 1% non-essential amino acids, 1 mM sodium pyruvate, 2 mM glutamine, and 100 U / mL penicillin and 100 μg / mL streptomycin at 37°C in 5% CO2 atmosphere.

[0123] MDCK (ATCC® CCL-34™) Madin-Darby canine kidney epithelial cells were grown in MEM (Ozyme) containing 10% FBS supplemented with 2 mM glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in 5% CO2 atmosphere.

[0124] Primary human dermal fibroblasts were grown in DMEM (Ozyme) supplemented with 10% FBS, 1% non-essential amino acids, 1 mM sodium pyruvate, 2 mM glutamine, and 100 U / mL penicillin and 100 μg / mL streptomycin at 37°C in 5% CO atmosphere.

[0125] Transfection assay (96-well format) One day before transfection, Caco-2, MCF 10A, Hep G2, and MDCK cells were seeded in 125 μL of complete medium at 10,000, 25,000, and 10,000 cells per well (96-well plate format) and incubated at 37°C in 5% CO2 atmosphere. On the day of transfection, 200 ng of pCMV-EGFPLuc DNA (Clontech) was added to 20 μL of OPTIMEM (Thermo Fisher Scientific), vortexed, and incubated at room temperature (rt) for 5 minutes. Next, 0.6 or 0.8 μL of a compound of formula (I), preferably formula (III), (7.5 mM nitrogen concentration) was added to the diluted DNA, vortexed, and incubated at room temperature for 10 minutes. The transfection DNA solution (20 μL) was added to the wells, and the plate was incubated at 37° C. in 5% CO 2 atmosphere for 24 hours.

[0126] For GFP expression analysis, one day after transfection, cell culture medium was removed, 50 μL of trypsin-EDTA (1x, Lonza) was added per well, and the plates were incubated at 37°C for 5 min. Trypsin was neutralized by adding 150 μL of complete medium, and GFP expression was analyzed (2000 events) by flow cytometry (excitation 488 nm, emission 520 nm) using a Guava easyCyte 6HT cytometer (Millipore).

[0127] Recombinant virus production HEK-293T (ATCC® CRL-3216™): Human embryonic kidney cells are highly transfectable derivatives of human embryonic kidney 293 cells and contain the SV40 T-antigen. HEK-293T cells are widely used for recombinant virus production, gene expression, and protein production.

[0128] For adherent cells, HEK-293T cells were cultured at 145 cm in 15 mL of DMEM 4.5 g / L glucose supplemented with 10% FBS, 2 mM glutamine, and 100 U / mL penicillin and 100 μg / mL streptomycin. 2 5 x 10 Petri dishes 6 The cells were seeded and incubated at 37°C in 5% CO2 in air.

[0129] AAV-2 was produced in HEK-293T cells using the AAV-2 Helper-Free Packaging System (Cat. No. VPK-402, Cell BioLabs, Inc.) by cotransfecting three plasmids: the pAAV-RC2 vector expressing Rep and Cap; the pHelper vector expressing Adenoviral E2A, Adenoviral E4, and Adenoviral VA helper factors; and the pAAV-GFP control vector expressing GFP under the control of a CMV promoter. Transfection complexes (10 μg total DNA per Petri dish) were prepared using pAAV-RC2, pHelper, and pAAV-GFP at a 2:2:1 ratio. The plasmids were diluted in OPTIMEM in a total volume of 1.5 mL. Then, 20 or 30 μL of compound was added to the diluted DNA, vortexed, and incubated at room temperature for 10 minutes. The transfection complexes were added to the cells, and the plates were incubated at 37°C in a 5% CO2 atmosphere for 72 hours.

[0130] For suspension cells, HEK-293T cells were cultured at 1 × 10 in 27 mL of FreeStyle F17 supplemented with 4% glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, and 0.1% pluronic acid in a 125 mL Erlenmeyer flask (Corning). 6The cells were seeded at 1 × 10 cells / mL. The cells were incubated at 37°C with 8% CO2 in an ambient atmosphere with agitation (130 rpm) for 24 hours. The plasmid (pAAV-GFP-pAAV-RC2-pHelper, ratio 2:2:1) was diluted with 3 mL of FreeStyle F17. Compounds were then added to the diluted DNA (at a ratio of 2 or 3 μL per μg of DNA), vortexed, and incubated at room temperature for 10 minutes. The transfection complex was added to the cells (1 × 10 cells / mL). 6 2 μg of DNA per cell) and the plates were incubated at 37°C in an 8% CO atmosphere with agitation (130 rpm) for 72 hours.

[0131] Lentiviral particles were produced using the ViraSafe® Lentiviral Packaging System, Pantropic (Cat. No. VPK-20, CELL BIOLABS) containing the pRSV-REV packaging vector, pCgpV packaging vector, and pCMV-VSV-G envelope vector. The pLenti6.3 / V5-GW / EmGFP expression control vector was from Thermo Fisher.

[0132] HEK-293T cells were cultured at 1 × 10 in 27 mL of FreeStyle F17 supplemented with 4% glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, and 0.1% Pluronic in a 125 mL Erlenmeyer flask (Corning). 6 The cells were seeded at 1 × 10 cells / mL. The cells were incubated at 37°C with 8% CO2 in an ambient atmosphere with agitation (130 rpm) for 24 hours. The plasmid (pRSV-REV-pCgpV-pCMV-VSV-G-pLenti6.3, ratio 1:1:1:3) was diluted with 3 mL of FreeStyle F17. Compounds were then added to the diluted DNA (at a ratio of 2 μL per μg of DNA), vortexed, and incubated at room temperature for 10 minutes. The transfection complex was added to the cells (1 × 10 cells / mL). 62 μg of DNA per cell), and the plates were incubated at 37°C with agitation (130 rpm) in an 8% CO atmosphere for 72 hours.

[0133] Transducing units (TU / mL) were determined by using viral vectors expressing a GFP reporter gene after infection of permissive HT1080 cells for lentiviral vectors and HEK-293T cells for AAV-2 vectors in 96-well plates in the presence of polybrene (8 μg / mL). GFP expression was analyzed by cytometry 72 hours after transduction to determine transducing units.

[0134] Example 1 General procedure for the preparation of graft polymers Step 1: Grafting To a round-bottom flask, cationic polymer (1 equiv.) in water (4 mL / mmol starting material) was added, followed by N-methylmorpholine or NMM (2 equiv.). Carboxylate (0.3–1 equiv.) was added, followed by MeOH (16 mL / mmol polymer). After stirring for 10 min, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride or DMTMM (0.6–2 equiv.) was added, and the mixture was stirred at room temperature for 12–24 h. The MeOH was then removed in vacuo, and water (4 mL / mmol starting material) was added, followed by 3 M HCl (1 mL / mmol starting material). The residue was purified using a dialysis cassette in a 50 mM HCl bath.

[0135] Step 2: Synthesis of triazoles via "click" chemistry starting from acids

[0136] [ka]

[0137] To a 2:1 (v / v) solution of nBuOH and water was added alkyne (1 equiv.), azide (1 equiv.), CuSO (0.01 equiv.), and sodium ascorbate (0.03 equiv.). The reaction was stirred at room temperature for 24 h. NaOH (5 M, 2 equiv.) was then added, and the organic solvent was removed in vacuo. The residue was purified by reverse-phase flash chromatography using 0–100% CHCN in water as the eluent.

[0138] Step 3: Synthesis of triazoles via "click" chemistry starting from esters

[0139] [ka]

[0140] To a 2:1 (v / v) solution of nBuOH and water was added alkyne (1 equiv.), azide (1 equiv.), CuSO (0.01 equiv.), and sodium ascorbate (0.03 equiv.). The reaction was stirred at room temperature for 24 h. NaOH (5 M, 2 equiv.) was then added, and the organic solvent was removed in vacuo. The residue was purified by reverse-phase flash chromatography using 0–100% CHCN in water as the eluent.

[0141] Step 4: Saponification of the ester moiety To a solution of the ester in EtOH, a 3 M solution of LiOH was added dropwise, and the mixture was stirred at room temperature over the weekend. The solvent was then removed in vacuo, and the residue was purified by reverse-phase FC on SiO2 using a Biotage Flash purification system with HO / MeCN as the eluent. The resulting acid was lyophilized to give a solid.

[0142] Step 5: Synthesis of triazoles via ruthenium-catalyzed “click” chemistry starting from esters. Cp*RuCl(cod) was added to a microwave vial. The vial was then evacuated and backfilled with argon (3x). Alkyne (1.1 equiv.), arsine (1 equiv.), and toluene were added to the vial under Ar, and the mixture was stirred at room temperature overnight. The toluene was evaporated, and the product was purified by reverse-phase chromatography using HO and MeCN.

[0143] The ester was retaken in EtOH and NaOH 1M (1.1 eq) and stirred until completion (followed by HPLC). EtOH was evaporated and the product was purified by reverse phase chromatography using HO and MeCN. The product was lyophilized.

[0144] Step 6: Synthesis of 1,2,3-triazoles. Triazole and K2CO3 in MeCN at 80 °C. R-Br was added dropwise and stirred at 80 °C overnight. Filtered and the solid was washed with MeCN. The filtrate was evaporated and purified by reverse phase chromatography (H2O:MeCN). Two fractions were collected.

[0145] The ester was retaken in EtOH and NaOH 1M (1.1 eq) and stirred until complete. The EtOH was evaporated and the product purified by reverse phase chromatography H2O:MeCN.

[0146] Example 2 Synthesis of Compounds of the Invention - Synthesis of product 2.19

[0147] [ka]

[0148] Intermediate 2.19a was prepared analogously to the general procedure, step 2 (example 1). Yield=87%; m=520 mg; 1H NMR (400 MHz, heavy water) δ 7.75 (s, 1H), 7.34 - 7.25 (m, 2H), 7.18 - 7.04 (m, 2H), 5.50 (s, 2H), 2.65 (t, J = 7.2 Hz, 2H), 2.17 (t, J = 7.1 Hz, 2H), 1.55 (dq, J = 23.6, 7.8 Hz, 3H).

[0149] [ka]

[0150] Product 2.19 was prepared analogously to general procedure, step 1 (example 1). Yield=36%; m=21 mg; 1 H NMR (400 MHz, heavy water) δ 8.12 - 6.41 (m, 5H), 5.68 - 4.93 (m, 2H), 4.05 - 2.88 (m, 17H), 2.79 - 0.87 (m, 8H).

[0151] - Synthesis of product 2.20

[0152] [ka]

[0153] Intermediate 2.20a was prepared analogously to general procedure, step 2 (example 1). Yield=51%; m=261 mg; 1 H NMR (400 MHz, heavy water) δ 7.75 (s, 1H), 7.34 - 7.25 (m, 2H), 7.18 - 7.04 (m, 2H), 5.50 (s, 2H), 2.65 (t, J = 7.2 Hz, 2H), 2.17 (t, J = 7.1 Hz, 2H), 1.64 - 1.45 (m, 3H).

[0154] [ka]

[0155] Product 2.20 was prepared analogously to general procedure, step 1 (example 1). Yield=71%; m=31 mg; 1 H NMR (400 MHz, heavy water) δ 8.17 - 6.68 (m, 5H), 5.60 - 5.28 (m, 2H), 4.10 - 2.93 (m, 27H).

[0156] - Synthesis of product 2.21

[0157] [ka]

[0158] Intermediate 2.21a was prepared analogously to general procedure, step 2 (example 1). Yield=27%; m=148 mg; 1 H NMR (400 MHz, heavy water) δ 7.71 (s, 1H), 7.32 - 7.24 (m, 2H), 7.09 (td, J = 8.8, 2.0 Hz, 2H), 5.55 - 5.46 (m, 2H), 2.86 (t, J = 7.5 Hz, 2H), 2.45 (t, J = 7.5 Hz, 2H).

[0159] [ka]

[0160] Product 2.21 was prepared analogously to general procedure, step 1 (example 1). Yield=29%; m=12 mg; 1 H NMR (400 MHz, heavy water) δ 8.90 - 6.37 (m, 5H), 5.58 - 5.25 (m, 2H), 4.20 - 2.91 (m, 36H).

[0161] - Synthesis of product 2.22

[0162] [ka]

[0163] Intermediate 2.22a was prepared analogously to general procedure, step 2 (example 1). Yield=28%; m=78 mg; 1 H NMR (400 MHz, methanol-d4) δ 7.57 (s, 1H), 6.93 - 6.84 (m, 2H), 6.68 - 6.60 (m, 2H), 4.54 - 4.45 (m, 2H), 3.04 (t, J = 7.4 Hz, 2H), 2.99 - 2.91 (m, 2H), 2.53 - 2.44 (m, 2H).

[0164] [ka]

[0165] Product 2.22 was prepared analogously to general procedure, step 1 (example 1). Yield=87%; m=44 mg; 1 H NMR (400 MHz, heavy water) δ 7.82 - 6.34 (m, 5H), 4.60 - 4.06 (m, 2H), 4.00 - 3.07 (m, 22H), 3.06 - 2.24 (m, 7H).

[0166] - Synthesis of product 2.23

[0167] [ka]

[0168] Intermediate 2.23a was prepared analogously to general procedure, step 2 (example 1). Yield=87%; m=258 mg; 1H NMR (400 MHz, methanol-d4) δ 7.53 (s, 1H), 6.98 - 6.87 (m, 2H), 6.75 - 6.63 (m, 2H), 4.53 (t, J = 7.1 Hz, 2H), 3.08 (t, J = 7.1 Hz, 2H), 2.69 (t, J = 7.6 Hz, 2H), 2.26 - 2.16 (m, 2H), 1.91 (tt, J = 8.3, 6.9 Hz, 2H).

[0169] [ka]

[0170] Product 2.23 was prepared analogously to general procedure, step 1 (example 1). Yield=100%; m=48 mg; 1 H NMR (400 MHz, heavy water) δ 8.14 - 6.01 (m, 5H), 4.62 - 4.11 (m, 2H), 3.99 - 2.76 (m, 26H), 2.73 - 0.92 (m, 8H).

[0171] - Synthesis of product 2.24

[0172] [ka]

[0173] Intermediate 2.24a was prepared analogously to general procedure, step 2 (example 1). Yield=67%; m=379 mg; 1 H NMR (400 MHz, heavy water) δ 7.62 (s, 1H), 7.24 - 7.14 (m, 2H), 7.05 - 6.92 (m, 2H), 5.35 (s, 2H), 2.54 (t, J = 7.6 Hz, 2H), 2.08 (t, J = 7.5 Hz, 2H), 1.73 (tt, J = 8.2, 7.0 Hz, 2H).

[0174] [ka]

[0175] Product 2.24 was prepared analogously to general procedure, step 1 (example 1). Yield=97%; m=42 mg; 1 H NMR (400 MHz, heavy water) δ 7.90 - 6.74 (m, 5H), 5.57 - 5.15 (m, 2H), 4.19 - 3.11 (m, 35H), 2.91 - 1.47 (m, 6H).

[0176] - Synthesis of product 2.25

[0177] [ka]

[0178] Product 2.25 was prepared analogously to general procedure, step 1 (example 1). Yield=85%; m=41 mg; 1 H NMR (400 MHz, heavy water) δ 7.84 - 6.46 (m, 5H), 5.54 - 4.94 (m, 2H), 4.15 - 3.11 (m, 26H), 2.97 - 1.11 (m, 8H).

[0179] - Synthesis of product 2.26

[0180] [ka]

[0181] Product 2.26 was prepared analogously to general procedure, step 1 (example 1). Yield=80%; m=44 mg; 1 H NMR (400 MHz, heavy water) δ 8.00 - 6.36 (m, 5H), 5.60 - 4.93 (m, 2H), 4.12 - 3.01 (m, 19H), 2.79 - 0.93 (m, 8H).

[0182] - Synthesis of product 2.27

[0183] [ka]

[0184] Intermediate 2.27a was prepared analogously to the general procedure, step 3 (example 1). Yield=65%; m=305 mg; 1 H NMR (400 MHz, chloroform-d) δ 8.03 (s, 1H), 8.00–7.91 (m, 2H), 7.31–7.21 (m, 2H), 5.36 (s, 2H), 3.96 (s, 3H).

[0185] [ka]

[0186] Intermediate 2.27b was prepared analogously to the general procedure, step 4 (example 1). Yield=35%; m=97 mg; 1 H NMR (400 MHz, heavy water) δ 8.07 (s, 0H), 7.71 - 7.59 (m, 1H), 7.19 - 7.04 (m, 1H), 4.96 (s, 1H).

[0187] [ka]

[0188] Product 2.27 was prepared analogously to general procedure, step 1 (example 1). Yield=67%; m=28 mg; 1 H NMR (400 MHz, heavy water) δ 9.33 - 7.35 (m, 5H), 6.13 - 5.19 (m, 2H), 4.17 - 3.22 (m, 42H).

[0189] - Synthesis of product 2.28

[0190] [ka]

[0191] Intermediate 2.28a was prepared analogously to the general procedure, step 3 (example 1). Yield=62%; m=272 mg; 1 H NMR (400 MHz, methanol-d4) δ 8.59 (d, J = 5.0 Hz, 1H), 8.50 (s, 1H), 8.10 (dt, J = 7.9, 1.1 Hz, 1H), 7.93 (td, J = 7.8, 1.8 Hz, 1H), 7.38 (ddd, J = 7.6, 4.9, 1.2 Hz, 1H), 5.45 (s, 2H), 3.83 (s, 3H).

[0192] [ka]

[0193] Intermediate 2.28b was prepared analogously to general procedure, step 4 (example 1). Yield=94%; m=236 mg; 1 H NMR (400 MHz, heavy water) δ 8.44 - 8.38 (m, 1H), 8.23 ​​(s, 1H), 7.89 - 7.74 (m, 2H), 7.31 (ddd, J = 6.0, 5.0, 2.8 Hz, 1H), 5.02 (s, 2H).

[0194] [ka]

[0195] Product 2.28 was prepared analogously to general procedure, step 1 (example 1). Yield=47%; m=23 mg; 1 H NMR (400 MHz, heavy water) δ 8.06 - 6.11 (m, 5H), 5.55 - 4.96 (m, 2H), 4.26 - 2.20 (m, 20H).

[0196] - Synthesis of product 2.29

[0197] [ka]

[0198] Intermediate 2.29a was prepared analogously to the general procedure, step 3 (example 1). Yield=81%; m=355 mg; 1 H NMR (400 MHz, methanol-d4) δ 9.08 (s, 1H), 8.64–8.46 (m, 2H), 8.28 (tt, J = 6.3, 1.6 Hz, 1H), 7.55 (dd, J = 8.0, 4.7 Hz, 1H), 5.44 (s, 2H), 3.84 (s, 2H).

[0199] [ka]

[0200] Intermediate 2.29b was prepared analogously to general procedure, step 4 (example 1). Yield=88%; m=287 mg; 1 H NMR (400 MHz, heavy water) δ 8.76 (dd, J = 2.3, 0.9 Hz, 1H), 8.40 (dd, J = 5.0, 1.6 Hz, 1H), 8.23 ​​(s, 1H), 8.07 (ddd, J = 8.0, 2.3, 1.6 Hz, 1H), 7.42 (ddd, J = 8.0, 5.0, 0.9 Hz, 1H), 5.01 (s, 2H), 1.09 (t, J = 7.1 Hz, 2H).

[0201] [ka]

[0202] Product 2.29 was prepared analogously to general procedure, step 1 (example 1). Yield=76%; m=29 mg; 1H NMR (400 MHz, heavy water) δ 9.28 - 7.21 (m, 5H), 5.94 - 5.16 (m, 2H), 4.19 - 2.35 (m, 19H).

[0203] - Synthesis of product 2.30

[0204] [ka]

[0205] Product 2.30 was prepared analogously to general procedure, step 1 (example 1). Yield=66%; m=32 mg; 1 H NMR (400 MHz, heavy water) δ 9.47 - 7.87 (m, 5H), 6.08 - 5.50 (m, 2H), 4.32 - 2.94 (m, 50H).

[0206] - Synthesis of product 2.31

[0207] [ka]

[0208] Intermediate 2.31a was prepared analogously to the general procedure, step 3 (example 1). Yield=82%; m=354 mg; 1 H NMR (400 MHz, heavy water) δ 9.46 - 7.68 (m, 5H), 6.03 - 5.32 (m, 2H), 4.28 - 2.83 (m, 50H). 1 H NMR (400 MHz, methanol-d4) δ 8.34 (s, 1H), 7.88–7.80 (m, 2H), 7.51–7.41 (m, 2H), 7.41–7.32 (m, 1H), 5.39 (s, 2H), 3.83 (s, 3H).

[0209] [ka]

[0210] Intermediate 2.31b was prepared analogously to the general procedure, step 4 (example 1). Yield=99%; m=325 mg; 1 H NMR (400 MHz, heavy water) δ 8.08 (s, 1H), 7.71 - 7.62 (m, 2H), 7.46 - 7.37 (m, 2H), 7.41 - 7.30 (m, 1H), 4.94 (s, 2H).

[0211] [ka]

[0212] Product 2.31 was prepared analogously to general procedure, step 1 (example 1). Yield=58%; m=24 mg; 1 H NMR (400 MHz, heavy water) δ 8.60 - 6.52 (m, 6H), 5.90 - 5.15 (m, 2H), 4.23 - 2.90 (m, 32H).

[0213] - Synthesis of product 2.32

[0214] [ka]

[0215] Intermediate 2.32a was prepared analogously to the general procedure, step 3 (example 1). Yield=87%; m=380 mg; 1 H NMR (400 MHz, methanol-d4) δ 8.82–8.45 (m, 3H), 7.92 (s, 2H), 5.45 (s, 2H), 3.84 (s, 3H).

[0216] [ka]

[0217] Intermediate 2.32b was prepared analogously to general procedure, step 4 (example 1). Yield=100%; m=351 mg; 1 H NMR (400 MHz, heavy water) δ 8.49 - 8.43 (m, 2H), 8.34 (s, 1H), 7.70 - 7.64 (m, 2H), 5.02 (s, 2H).

[0218] [ka]

[0219] Product 2.32 was prepared analogously to general procedure, step 1 (example 1). Yield=91%; m=32 mg; 1 H NMR (400 MHz, heavy water) δ 9.17 - 8.00 (m, 5H), 6.13 - 5.23 (m, 2H), 4.21 - 3.01 (m, 74H).

[0220] - Synthesis of product 2.33

[0221] [ka]

[0222] Intermediate 2.33a was prepared analogously to general procedure, step 2 (example 1). Yield=59%; m=49 mg; 1 H NMR (400 MHz, heavy water) δ 7.90 (s, 1H), 4.40 - 4.32 (m, 2H), 3.69 - 3.62 (m, 6H), 2.52 - 2.45 (m, 4H), 2.14 - 2.00 (m, 4H).

[0223] [ka]

[0224] Product 2.33 was prepared analogously to general procedure, step 1 (example 1). Yield=89%; m=15 mg;1 H NMR (400 MHz, heavy water) δ 8.63 - 7.84 (m, 1H), 4.52 - 4.23 (m, 3H), 4.13 - 2.86 (m, 27H), 2.74 - 1.54 (m, 4H).

[0225] - Synthesis of product 2.34

[0226] [ka]

[0227] Intermediate 2.34a was prepared analogously to the general procedure, step 2 (example 1). Yield=48%; m=51 mg; 1 H NMR (400 MHz, heavy water) δ 7.92 (s, 1H), 7.35 - 7.26 (m, 2H), 7.09 - 7.01 (m, 2H), 6.98 (tt, J = 7.4, 1.1 Hz, 1H), 4.41 - 4.33 (m, 2H), 3.71 (s, 2H), 3.14 - 3.07 (m, 4H), 2.69 - 2.61 (m, 4H), 2.14 - 2.00 (m, 4H).

[0228] [ka]

[0229] Product 2.34 was prepared analogously to general procedure, step 1 (example 1). Yield=98%; m=17 mg; 1 H NMR (400 MHz, heavy water) δ 8.67 - 7.86 (m, 1H), 7.64 - 6.61 (m, 5H), 4.66 - 4.19 (m, 3H), 4.11 - 3.09 (m, 31H), 2.80 - 1.74 (m, 4H).

[0230] - Synthesis of product 2.35

[0231] [ka]

[0232] Intermediate 2.35a was prepared analogously to the general procedure, step 2 (example 1). Yield=19%; m=20 mg; 1 H NMR (400 MHz, heavy water) δ 8.24 (d, J = 4.9 Hz, 2H), 7.95 (s, 1H), 6.64 (t, J = 4.9 Hz, 1H), 4.40 - 4.33 (m, 2H), 3.81 - 3.77 (m, 2H), 3.67 - 3.60 (m, 4H), 2.66 - 2.58 (m, 4H), 2.12 - 2.00 (m, 4H).

[0233] [ka]

[0234] Product 2.35 was prepared analogously to general procedure, step 1 (example 1). Yield=44%; m=7 mg; 1 H NMR (400 MHz, heavy water) δ 8.58 - 7.90 (m, 3H), 7.07 - 6.43 (m, 1H), 4.57 - 4.19 (m, 3H), 4.23 - 2.99 (m, 32H), 2.83 - 1.68 (m, 4H).

[0235] - Synthesis of product 2.36

[0236] [ka]

[0237] Intermediate 2.36a was prepared analogously to general procedure, step 2 (example 1). Yield=51%; m=78 mg; 11H NMR (400 MHz, deuterium oxide) δ 8.39 (ddd, J = 5.0, 1.7, 1.0 Hz, 1H), 8.21 (s, 1H), 7.85 - 7.71 (m, 1H), 7.30 (ddd, J = 7.3, 5.0, 1.5 Hz, 1H), 4.43 - 4.35 (m, 2H), 2.20 - 2.03 (m, 4H).

[0238]

Chem.

[0239] Product 2.36 was prepared in the same manner as in the general procedure, Step 1 (Example 1). Yield = 77%; m = 14 mg; 1 1H NMR (400 MHz, deuterium oxide) δ 9.15 - 7.11 (m, 5H), 4.57 - 4.15 (m, 1H), 4.07 - 2.86 (m, 13H), 2.74 - 1.68 (m, 4H).

[0240] - Synthesis of Product 2.37

[0241]

Chem.

[0242] Intermediate 2.37a was prepared in the same manner as in the general procedure, Step 2 (Example 1). Yield = 14%; m = 38 mg; 1 1H NMR (400 MHz, deuterium oxide) δ 8.00 (s, 1H), 7.57 - 7.47 (m, 2H), 7.09 - 6.95 (m, 2H), 4.26 (t, J = 7.0 Hz, 2H), 2.08 (t, J = 7.5 Hz, 2H), 1.76 (p, J = 7.2 Hz, 2H), 1.49 - 1.35 (m, 2H).

[0243]

Chem.

[0244] Product 2.37 was prepared analogously to general procedure, step 1 (example 1). Yield=24%; m=9 mg; 1 H NMR (400 MHz, heavy water) δ 8.23 ​​- 6.04 (m, 5H), 4.39 - 2.72 (m, 18H), 2.70 - 0.56 (m, 6H).

[0245] - Synthesis of product 2.38

[0246] [ka]

[0247] Intermediate 2.38a was prepared analogously to the general procedure, step 2 (example 1). Yield=11%; m=27 mg; 1 H NMR (400 MHz, heavy water) δ 7.97 (s, 1H), 7.51 (dd, J = 8.7, 5.3 Hz, 2H), 7.02 (t, J = 8.9 Hz, 2H), 4.23 (t, J = 7.1 Hz, 2H), 2.04 (t, J = 7.5 Hz, 2H), 1.76 (p, J = 7.2 Hz, 2H), 1.45 (p, J = 7.6 Hz, 2H), 1.21 - 1.09 (m, 2H).

[0248] [ka]

[0249] Product 2.38 was prepared analogously to general procedure, step 1 (example 1). Yield=18%; m=6 mg; 1 H NMR (400 MHz, heavy water) δ 8.31 - 6.11 (m, 5H), 4.33 - 2.72 (m, 21H), 2.68 - 0.15 (m, 6H).

[0250] - Synthesis of product 2.39

[0251] [ka]

[0252] Intermediate 2.39a was prepared analogously to the general procedure, step 2 (example 1). Yield=35%; m=11 mg; 1 H NMR (400 MHz, heavy water) δ 8.01 (s, 1H), 7.60 - 7.53 (m, 2H), 7.39 - 7.25 (m, 3H), 4.24 (t, J = 7.1 Hz, 2H), 2.09 (t, J = 7.5 Hz, 2H), 1.76 (p, J = 7.2 Hz, 2H), 1.48 - 1.35 (m, 2H).

[0253] [ka]

[0254] Product 2.39 was prepared analogously to general procedure, step 1 (example 1). Yield=53%; m=133 mg; 1 H NMR (400 MHz, heavy water) δ 8.49 - 6.15 (m, 6H), 4.52 - 2.83 (m, 21H), 2.66 - 0.54 (m, 6H).

[0255] - Synthesis of product 2.40

[0256] [ka]

[0257] Intermediate 2.40a was prepared analogously to general procedure, step 2 (example 1). Yield=76%; m=184 mg; 1H NMR (400 MHz, heavy water) δ 7.88 (s, 1H), 7.54 - 7.47 (m, 2H), 7.35 - 7.20 (m, 3H), 4.14 (t, J = 7.1 Hz, 2H), 2.04 (t, J = 7.5 Hz, 2H), 1.76 - 1.64 (m, 2H), 1.43 (p, J = 7.6 Hz, 2H), 1.21 - 1.06 (m, 2H).

[0258] [ka]

[0259] Product 2.40 was prepared analogously to general procedure, step 1 (example 1). Yield=12%; m=4 mg; 1 H NMR (400 MHz, heavy water) δ 7.94 - 6.37 (m, 6H), 4.43 - 2.84 (m, 19H), 2.68 - 0.23 (m, 8H).

[0260] - Synthesis of product 2.41

[0261] [ka]

[0262] Intermediate 2.41a was prepared analogously to the general procedure, step 2 (example 1). Yield=26%; m=46 mg; 1 H NMR (400 MHz, heavy water) δ 7.41 (s, 1H), 7.20 - 7.01 (m, 5H), 4.08 (t, J = 7.0 Hz, 2H), 3.78 (s, 2H), 2.00 (t, J = 7.5 Hz, 2H), 1.72 - 1.50 (m, 2H), 1.32 (tt, J = 15.0, 9.9 Hz, 2H).

[0263] [ka]

[0264] Product 2.41 was prepared analogously to general procedure, step 1 (example 1). Yield=75%; m=26 mg; 1 H NMR (400 MHz, heavy water) δ 7.99 - 6.38 (m, 6H), 4.32 - 1.58 (m, 30H).

[0265] - Synthesis of product 2.42

[0266] [ka]

[0267] Intermediate 2.42a was prepared analogously to general procedure, step 2 (example 1). Yield=57%; m=96 mg; 1 H NMR (400 MHz, heavy water) δ 7.15 (s, 1H), 7.05 - 6.86 (m, 5H), 3.92 (t, J = 7.2 Hz, 2H), 3.67 (s, 2H), 1.97 (t, J = 7.6 Hz, 2H), 1.54 - 1.42 (m, 2H), 1.39 - 1.27 (m, 2H), 1.05 - 0.92 (m, 2H).

[0268] [ka]

[0269] Product 2.42 was prepared analogously to general procedure, step 1 (example 1). Yield=87%; m=27 mg; 1 H NMR (400 MHz, heavy water) δ 7.58 - 6.61 (m, 6H), 4.55 - 0.72 (m, 33H).

[0270] - Synthesis of product 2.43

[0271] [ka]

[0272] Intermediate 2.43a was prepared analogously to the general procedure, step 2 (example 1). Yield=24%; m=36 mg; 1 H NMR (400 MHz, heavy water) δ 8.78 (s, 1H), 8.43 - 8.37 (m, 1H), 8.29 (s, 1H), 8.09 (dt, J = 8.1, 1.9 Hz, 1H), 7.43 (ddd, J = 8.0, 5.0, 0.9 Hz, 1H), 4.38 (t, J = 7.0 Hz, 2H), 2.06 (t, J = 7.5 Hz, 2H), 1.91 - 1.79 (m, 2H), 1.54 - 1.42 (m, 2H), 1.26 - 1.14 (m, 2H).

[0273] [ka]

[0274] Product 2.43 was prepared analogously to general procedure, step 1 (example 1). Yield=38%; m=17 mg; 1 H NMR (400 MHz, heavy water) δ 9.38 - 7.80 (m, 5H), 4.58 - 0.92 (m, 31H).

[0275] - Synthesis of product 2.44

[0276] [ka]

[0277] Intermediate 2.44a was prepared analogously to the general procedure, step 2 (example 1). Yield=25%; m=38 mg; 1H NMR (400 MHz, heavy water) δ 8.45 - 8.40 (m, 1H), 8.24 (s, 1H), 7.85 - 7.79 (m, 2H), 7.34 - 7.30 (m, 1H), 4.37 (t, J = 7.0 Hz, 2H), 2.05 (t, J = 7.4 Hz, 2H), 1.91 - 1.79 (m, 2H), 1.54 - 1.42 (m, 2H), 1.27 - 1.14 (m, 2H).

[0278] [ka]

[0279] Product 2.44 was prepared analogously to general procedure, step 1 (example 1). Yield=64%; m=29 mg; 1 H NMR (400 MHz, heavy water) δ 8.79 - 7.46 (m, 5H), 4.56 - 2.83 (m, 23H), 2.72 - 0.78 (m, 8H).

[0280] - Synthesis of product 2.45

[0281] [ka]

[0282] Product 2.45 was prepared analogously to general procedure, step 1 (example 1). Yield=49%; m=18 mg; 1 H NMR (400 MHz, heavy water) δ 8.01 - 6.82 (m, 5H), 5.62 - 5.23 (m, 2H), 4.04 - 3.18 (m, 66H), 2.95 - 1.63 (m, 6H).

[0283] - Synthesis of product 2.46

[0284] [ka]

[0285] Product 2.46 was prepared analogously to general procedure, step 1 (example 1). Yield=93%; m=156 mg; 1 H NMR (400 MHz, heavy water) δ 8.98 - 8.37 (m, 3H), 8.27 (s, 1H), 7.90 (s, 1H), 4.61 - 4.32 (m, 2H), 4.05 - 3.13 (m, 18.5H), 2.56 - 2.18 (m, 2H), 2.12 - 1.79 (m, 2H), 1.73 - 1.42 (m, 2H), 1.42 - 1.14 (m, 2H).

[0286] - Synthesis of product 2.47

[0287] [ka]

[0288] Product 2.47 was prepared analogously to general procedure, step 1 (example 1). Yield=99%; m=44 mg; 1 H NMR (400 MHz, heavy water) δ 8.00 - 6.02 (m, 5H), 4.66 - 4.05 (m, 1H), 3.97 - 2.01 (m, 32H).

[0289] - Synthesis of product 2.48

[0290] [ka]

[0291] Product 2.48 was prepared analogously to general procedure, step 1. Yield=28%; m=83 mg; 1 H NMR (400 MHz, heavy water) δ 7.75 - 7.23 (s, 1H), 7.15 - 6.38 (m, 4H), 4.64 - 4.17 (m, 2H), 4.14 - 2.00 (m, 25H).

[0292] - Synthesis of product 2.49

[0293] [ka]

[0294] Product 2.49 was prepared analogously to general procedure, step 1. Yield=5%; m=13 mg; 1 H NMR (400 MHz, heavy water) δ 7.78 - 7.32 (s, 1H), 7.20 - 6.31 (m, 4H), 4.66 - 4.31 (m, 2H), 4.22 - 2.20 (m, 27H).

[0295] - Synthesis of product 2.50

[0296] [ka]

[0297] Product 2.50 was prepared similarly to general procedure, step 1. Yield=18%; m=29 mg; 1 H NMR (400 MHz, heavy water) δ 7.71 - 7.19 (s, 1H), 7.15 - 6.34 (m, 4H), 4.65 - 4.09 (m, 2H), 4.06 - 0.57 (m, 26H).

[0298] - Synthesis of product 2.51

[0299] [ka]

[0300] Product 2.51 was prepared analogously to general procedure, step 1. Yield=36%; m=13 mg; 1H NMR (400 MHz, heavy water) δ 7.68 - 7.45 (m, 1H), 7.14 - 6.62 (m, 4H), 4.67 - 4.42 (m, 2H), 4.39 - 4.17 (m, 5H), 3.38 - 2.73 (m, 16H), 2.58 - 2.33 (m, 2H), 1.99 - 1.01 (m, 35H).

[0301] - Synthesis of product 2.52

[0302] [ka]

[0303] Product 2.52 was prepared analogously to general procedure, step 1. Yield=43%; m=63 mg; 1 H NMR (400 MHz, heavy water) δ 7.84 - 7.31 (s, 1H), 7.28 - 6.48 (m, 4H), 4.69 - 4.33 (m, 2H), 4.30 - 1.04 (m, 25H).

[0304] - Synthesis of product 2.53

[0305] [ka]

[0306] Intermediate 2.53a was prepared analogously to the general procedure, steps 3 and 4. Yield=34%; m=151 mg; 1 H NMR (400 MHz, MeOD) δ 6.69 (s, 1H), 3.39 - 3.21 (m, 2H), 1.20 - 0.94 (m, 4H), 0.26 (s, 9H).

[0307] [ka]

[0308] Product 2.53 was prepared analogously to general procedure, step 1. Yield=37%; m=25 mg; 1 H NMR (400MHz, heavy water) δ 8.07 - 7.88 (m, 1H), 4.60 - 3.35 (m, 2H), 4.08 - 3.05 (m, 24H), 2.90 - 1.97 (m, 4H), 1.55 - 1.05 (m, 9H)

[0309] - Synthesis of product 2.54

[0310] [ka]

[0311] Intermediate 2.54a was prepared analogously to the general procedure, steps 3 and 4. Yield=69%; m=287 mg; 1 H NMR (400 MHz, MeOD) δ 6.64 (s, 1H), 3.32 (d, J = 6.9 Hz, 2H), 1.17 - 1.00 (m, 4H), 0.90 (tt, J = 8.4, 5.0 Hz, 1H), -0.02 - -0.18 (m, 2H), -0.22 - -0.35 (m, 2H).

[0312] [ka]

[0313] Product 2.54 was prepared analogously to general procedure, step 1. Yield=34%; m=24 mg; 1 H NMR (400MHz, heavy water) δ 7.99 - 7.78 (m, 1H), 4.61 - 4.38 (m, 2H), 4.06 - 3.26 (m, 21H), 2.66 - 1.95 (m, 5H), 1.15 - 1.01 (m, 2H), 0.88 - 0.70 (m, 2H).

[0314] - Synthesis of product 2.55

[0315] [ka]

[0316] Intermediate 2.55a was prepared analogously to the general procedure, step 3. Yield = 52%; m = 251 mg; 1H NMR (500 MHz, CDCl3) δ 7.28 (s, 1H), 4.39 (t, J = 6.9 Hz, 2H), 4.14 (q, J = 7.1 Hz, 2H), 3.28 - 3.09 (m, 1H), 2.34 (t, J = 6.9 Hz, 2H), 2.22 (p, J = 7.0 Hz, 2H), 2.11 (s, 2H), 1.88 - 1.56 (m, 6H), 1.26 (t, J = 7.1 Hz, 3H).

[0317] [ka]

[0318] Intermediate 2.55b was prepared analogously to the general procedure, step 4. Yield = quantitative; m = 223 mg; 1 H NMR (400 MHz, MeOD) δ 7.65 (s, 1H), 4.35 - 4.21 (m, 2H), 3.12 - 2.96 (m, 1H), 2.15 - 1.91 (m, 6H), 1.77 - 1.49 (m, 6H).

[0319] [ka]

[0320] Product 2.55 was prepared analogously to general procedure, step 1. Yield=40%; m=22 mg; 1¹H NMR (400MHz, heavy water) δ 8.04 - 7.83 (m, 1H), 4.59 - 4.34 (m, 2H), 4.07 - 3.04 (m, 25H), 2.60 - 1.97 (m, 6H), 1.80 - 1.47 (m, 6H)

[0321] - Synthesis of product 2.56

[0322]

change

[0323] Intermediate 2.56aを, general Nashou, Suppress 3 and the same modulated formula. Yield=80%; m=350mg; 1 ¹H NMR (400 MHz, CDCl₃) δ 7.26 (s, 1H), 4.38 (t, J = 6.9 Hz, 2H), 4.14 (q, J = 7.1 Hz, 2H), 3.09 (septue, J = 6.9 Hz, 1H), 2.34 (dd, J = 7.4, 6.4 Hz, 2H), 2.29 - 2.14 (m, 2H), 1.31 (s, 3H), 1.29 (s, 3H), 1.26 (td, J = 7.1, 0.6 Hz, 3H).

[0324]

change

[0325] Intermediate 2.56bを, general Nashu, Suppress 4 and the same modulated formula. Yield=quantitative; m=340mg; 1 H NMR (400 MHz, MeOD) δ 7.75 (d, J = 0.8 Hz, 1H), 4.39 (td, J = 6.4, 5.8, 2.8 Hz, 2H), 3.03 (pd, J = 6.9, 0.7 Hz, 1H), 2.25 - 2.05 (m, 4H), 1.30 (s, 3H), 1.29 (s, 3H).

[0326] [Chemical]

[0327] Product 2.56 was prepared in the same general procedure as Step 1. Yield = 45%; m = 32 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.09 - 7.90 (m, 1H), 4.61 - 4.36 (m, 2H), 4.09 - 3.20 (m, 21H), 3.17 - 2.97 (m, 1H), 2.62 - 2.03 (m, 4H), 1.45 - 1.16 (m, 6H).

[0328] - Synthesis of Product 2.57

[0329] [Chemical]

[0330] Intermediate 2.57a was prepared in the same general procedure as Step 5. Yield = 55%; m = 287 mg; 1H NMR (400 MHz, CDCl3) δ 7.73 - 7.66 (m, 2H), 7.56 - 7.40 (m, 4H), 7.38 - 7.27 (m, 2H), 4.39 (t, J = 7.1 Hz, 2H), 4.31 (t, J = 6.9 Hz, 1H), 4.14 (q, J = 7.1 Hz, 2H), 4.04 (q, J = 7.1 Hz, 1H), 2.48 (s, 3H), 2.43 (t, J = 7.0 Hz, 2H), 2.30 (s, 2H), 2.31 - 2.23 (m, 1H), 2.27 - 2.19 (m, 2H), 2.15 - 2.03 (m, 1H), 1.26 (t, J = 7.1 Hz, 3H), 1.19 (t, J = 7.1 Hz, 2H).

[0331] [Chemical]

[0332] Intermediate 2.57b was prepared analogously to the general procedure, step 4. Yield = quantitative; m = 280 mg; 1 H NMR (400 MHz, MeOD) δ 7.79 - 7.10 (m, 5H), 4.56 - 4.17 (m, 2H), 2.57 - 1.89 (m, 7H).

[0333] [ka]

[0334] Product 2.57 was prepared analogously to general procedure, step 1. Yield=55%; m=44 mg; 1 H NMR (400MHz, heavy water) δ 7.68 - 6.86 (m, 5H), 4.53 - 3.03 (m, 20H), 2.62 - 1.58 (m, 7H).

[0335] - Synthesis of product 2.58

[0336] [ka]

[0337] Intermediate 2.58a was prepared analogously to the general procedure, step 5. Yield=49%; m=224 mg; 1 H NMR (400 MHz, CDCl3) δ 4.35 - 4.25 (m, 2H), 4.19 - 4.08 (m, 2H), 3.23 - 2.96 (m, 1H), 2.42 - 2.34 (m, 4H), 2.22 - 2.09 (m, 2H), 1.36 - 1.30 (m, 6H), 1.29 - 1.18 (m, 3H).

[0338] [ka]

[0339] Intermediate 2.58b was prepared analogously to the general procedure, step 4. Yield = quantitative; m = 219 mg; 1 H NMR (400 MHz, MeOD) δ 4.37 - 4.27 (m, 2H), 3.31 - 3.18 (m, 1H), 2.33 (s, 3H), 2.27 - 2.13 (m, 2H), 2.15 - 2.03 (m, 2H), 1.34 (s, 3H), 1.32 (s, 3H).

[0340] [ka]

[0341] Product 2.58 was prepared analogously to general procedure, step 1. Yield=64%; m=48 mg; 1 H NMR (400MHz, heavy water) δ 4.49 - 4.22 (m, 2H), 3.99 - 3.03 (m, 20H), 2.76 - 1.87 (m, 7H), 1.34 - 1.04 (m, 6H).

[0342] - Synthesis of product 2.59

[0343] [ka]

[0344] Intermediate 2.59a was prepared analogously to the general procedure, step 3. Yield=41%; m=199 mg; 1H NMR (400 MHz, CDCl3) δ 7.25 (s, 1H), 4.39 (t, J = 6.9 Hz, 2H), 4.14 (q, J = 7.1 Hz, 2H), 2.96 (h, J = 7.0 Hz, 1H), 2.34 (td, J = 7.1, 1.0 Hz, 2H), 2.21 (p, J = 7.0 Hz, 2H), 1.75 - 1.62 (m, 1H), 1.60 - 1.46 (m, 1H), 1.41 - 1.16 (m, 8H), 0.90 (t, J = 7.3 Hz, 3H).

[0345] [ka]

[0346] Intermediate 2.59b was prepared analogously to the general procedure, step 4. Yield = quantitative; m = 194 mg; 1 H NMR (400 MHz, MeOD) δ 7.75 (s, 1H), 4.44 - 4.35 (m, 2H), 2.92 (h, J = 7.0 Hz, 1H), 2.19 - 2.08 (m, 4H), 1.74 - 1.49 (m, 2H), 1.47 - 1.10 (m, 6H), 0.91 (t, J = 7.4 Hz, 3H).

[0347] [ka]

[0348] Product 2.59 was prepared analogously to general procedure, step 1. Yield=58%; m=44 mg; 1H NMR (400MHz, heavy water) δ 7.98 - 7.77 (m, 1H), 4.58 - 4.31 (m, 2H), 4.07 - 3.16 (m, 20H), 3.05 - 2.77 (m, 1H), 2.60 - 1.98 (m, 4H), 1.66 - 1.42 (m, 2H), 1.34 - 1.04 (m, 5H), 0.92 - 0.64 (m, 3H).

[0349] - Synthesis of product 2.60

[0350] [ka]

[0351] Intermediate 2.60a was prepared analogously to the general procedure, step 6. Yield=44%; m=586 mg; 1 H NMR (400 MHz, CDCl3) δ 7.75 - 7.68 (m, 1H), 7.58 (s, 1H), 4.48 (td, J = 6.8, 1.9 Hz, 2H), 4.21 - 4.04 (m, 2H), 2.43 - 2.29 (m, 2H), 2.29 - 2.19 (m, 2H), 1.33 - 1.18 (m, 3H).

[0352] [ka]

[0353] Intermediate 2.60b was prepared analogously to the general procedure, step 4. Yield = quantitative; m = 567 mg; 1 H NMR (400 MHz, MeOD) δ 8.01 (d, J = 1.3 Hz, 1H), 7.71 (d, J = 1.2 Hz, 1H), 4.52 - 4.43 (m, 2H), 2.23 - 2.10 (m, 4H).

[0354] [ka]

[0355] Product 2.60 was prepared analogously to general procedure, step 1. Yield=46%; m=33 mg; 1 H NMR (400MHz, heavy water) δ 8.09 - 7.95 (m, 1H), 7.89 - 7.76 (m, 1H) 4.59 - 4.41 (m, 2H), 4.04 - 3.12 (m, 16H), 2.55 - 1.99 (m, 4H).

[0356] - Synthesis of product 2.61

[0357] [ka]

[0358] Intermediate 2.61a was prepared analogously to the general procedure, step 6. Yield=27%; m=362 mg; 1 H NMR (400 MHz, CDCl3) δ 7.59 (s, 2H), 4.51 (td, J = 6.6, 1.1 Hz, 2H), 4.13 (qd, J = 7.1, 1.2 Hz, 2H), 2.36 - 2.26 (m, 4H), 1.24 (td, J = 7.1, 1.1 Hz, 3H).

[0359] [ka]

[0360] Intermediate 2.61b was prepared analogously to the general procedure, step 4. Yield = quantitative; m = 350 mg; 1 H NMR (400 MHz, MeOD) δ 7.54 (s, 2H), 4.42 - 4.34 (m, 2H), 2.16 - 2.00 (m, 4H).

[0361] [ka]

[0362] Product 2.61 was prepared analogously to general procedure, step 1. Yield=41%; m=29 mg; 1 H NMR (400MHz, heavy water) δ 7.84 - 7.67 (m, 2H), 4.60 - 4.39 (m, 2H), 4.08 - 3.12 (m, 16H), 2.57 - 1.99 (m, 4H).

[0363] Example 3 Compounds 2.19~2.26 Screening for transfection activity Compounds 2.19–2.26 were evaluated for DNA transfection ability in four different cell lines: Caco-2 (human colon epithelial cells), Hep G2 (human hepatoma cells), MDCK (Madin-Darby canine kidney epithelial cells), and MCF-10A (human mammary epithelial cells). Compound screening (Figure 2) was performed in 96-well plates by transfecting 200 ng of pCMV-EGFPLuc DNA (Clontech) complexed with 0.6 or 0.8 μL of one compound of the present invention, i.e., one compound selected from the group consisting of compounds 2.19–2.26 (7.5 mM nitrogen concentration), at a ratio of 1 μg DNA / 3 μL compound (1:3 ratio) or 1 μg DNA / 4 μL compound (1:4 ratio), respectively. The percentage of cells expressing GFP (% GFP) was determined by cytometry assay one day after transfection. As a control, transfection was performed with jetPEI®, a 22 KDa linear polyethyleneimine, which represents the parent cationic polymer backbone of the test compounds.

[0364] Compounds 2.19-2.16 represent polymers in which a triazole ring is used to graft a fluorobenzyl or hydroxyphenol (or 4-hydroxyphenethyl) moiety, and a cationic polymer is grafted to R or V of formula (III). All compounds showed significant transfection activity, but the optimal compound depended on the cell line used.

[0365] Example 4 Bioproduction of recombinant viruses using compounds 2.22, 2.23, 2.41, 2.42, 2.43, 2.46, and 2.47 DNA transfection is one of the most commonly used techniques for the bioproduction of recombinant proteins and viruses through the transient gene expression (TGE) process. For AAV and lentivirus production, the most commonly used method is transfection to deliver viruses and therapeutic genes to producer cell lines, such as HEK293 adherent or suspension cells. In many systems, co-transfection of multiple plasmids is achieved by chemical methods, such as co-precipitation with calcium phosphate or transfection via cationic polymer polyethyleneimine (PEI), such as PEIpro® (Polyplus-transfection), which is commercially recommended for this type of bioproduction of recombinant viruses.

[0366] AAV and lentiviral particles were produced in HEK-293T cells by transient cotransfection of several plasmids containing the gene of interest and necessary viral components to generate complete recombinant viral particles. AAV-2 and lentiviral vectors expressing a GFP reporter gene were produced using various compounds, and viral productivity was determined by assessing transducing units (TU / mL) 3 days after transfection. Productivity levels were compared to those obtained with PEIpro® transfection reagent, a widely used reagent for adherent and suspension viral production systems.

[0367] Many of the compounds of Example 3 were prepared by grafting the triazole ring with a benzyl (2.41 or 2.42) or pyridinyl (2.43-2.46) moiety, and the cationic polymer was grafted at position Z of formula (III). 1 The compounds were tested for AAV-2 production, as were other compounds linked to the triazole ring in Figure 3. Figure 3 shows some of the results. At a 1:2 ratio (1 μg total DNA per μL of compound) used for transfection, several compounds exhibited virus productivity similar to PEIpro®, but most compounds significantly increased virus titers by 3-8 fold. This improvement was observed for most compounds and was enhanced when used at a 1:3 ratio, with compound 2.43 showing the highest increase in virus titer, exceeding 10 fold.

[0368] Similarly, lentivirus was produced in suspension HEK-293T cells after cotransfection with four plasmids (pRSV-REV packaging vector, pCgpV packaging vector, pCMV-VSV-G envelope vector, and pLenti6.3 / V5-GW / EmGFP expression control vector). Lentiviral titers (TU / mL) were determined 72 hours after transfection (Figure 4). The use of compound 2.22 at a 1:3 ratio resulted in an approximately 10-fold improvement in LV production yield compared to productivity with PEIpro®.

[0369] Example 5 Compound 2.53~2.61 Screening for transfection activity Compounds 2.53-2.61 were screened in transfection wells in 96-well plates by transfecting 200 ng of pCMV-EGFPLuc DNA (Clontech) complexed with 3, 0.6, or 0.8 μL of one compound of the invention (7.5 mM nitrogen concentration) as described above for the compound of Example 3, and determining the ratio of 1 μg DNA / 3 μL compound or 1 μg DNA / 4 μL compound, respectively ( FIG. 6 ).

[0370] Compounds 2.53 to 2.61 are compounds in which the cationic polymer is Z of formula (III) 1 These compounds represent compounds having a triazole ring linked to the R or V position of formula (III), with various alkyl or cycloalkyl moieties added at the R or V position of formula (III). Figure 6 shows that grafting alkyl or cycloalkyl moieties at the R or V position of the triazole ring results in compounds that are efficient in transfection, as exemplified by compounds 2.54, 2.56, 2.58, and 2.57. Surprisingly, compounds 2.60 and 2.61, which have unsubstituted triazole rings at the R and V positions of formula (III), were unable to efficiently transfect Hep G2 cells.

[0371] Bioproduction of recombinant viruses Compounds 2.53-2.61 were tested for AAV-2 production, and Figure 7 shows the results obtained for compounds at a 1:2 ratio of DNA μg / μL of reagent. AAV titers (transducing units, TU / mL) were determined 72 h after transfection. Results are expressed as relative AAV-2 transducing units / mL (TU / mL).

[0372] Compound 2.22 was used as a positive control. Compounds 2.54 and 2.57 showed promising results, which correlated with the transfection activity shown in Figure 6. In contrast to the transfection studies in Hep G2 cells, compounds 2.60 and 2.61, with R and V = H, showed high levels of AAV-2 production in HEK-293T cells.

[0373] Example 6 Key parameters for the production of AAV-2 from suspension HEK-293T cells Recombinant virus production was achieved by co-transfecting multiple plasmids into HEK293 cells. Virus productivity was significantly affected by the total amount of plasmid and the volume of transfection reagent. Figure 8 shows AAV-2 production using compound 2.22 (formulated at a nitrogen concentration of 15 mM). Different amounts of plasmid were used to transfect suspension HEK293-T cells. Multiple ratios of transfection reagents were also tested, and the viral yield was measured at 10% on the day of transfection. z The results were expressed as μg DNA per μL of reagent per cell. The results indicate that viral productivity depends on the amount of transfected plasmid. Furthermore, for each amount of transfected DNA, the optimal productivity depends on the ratio of μg DNA per μL of reagent. This example demonstrates that transfection conditions using compounds of formula (III) can be easily adapted to achieve optimal viral productivity. Figure 9 shows the effect of DNA complex formation time with compound 2.22 on AAV-2 production from suspension HEK-293T cells. To achieve high viral production yields, a minimum DNA complex formation time of 15 minutes is required before adding the transfection complex to the cell culture. Longer DNA complex formation times beyond 15 minutes can be used without affecting viral yields, indicating good stability of the transfection complex in terms of viral production activity. This property indicates that compound 2.22 is particularly suitable for large-scale applications in bioreactors, where the time window during transfer of the transfection complex mixture needs to be adapted depending on the cell culture volume.

[0374] conclusion Many compounds based on grafting polyamines with heterocycles of formula (I), preferably formula (III), have shown improved performance in inducing gene expression in "difficult to transfect" cells, such as cancer cells, or in enhancing the productivity of biologics, such as viruses, AAV, or LV.

[0375] Many of the compounds of Examples 3, 4 or 5, especially polyamines grafted with benzyl, fluorobenzyl, hydroxyphenyl, 4-hydroxyphenethyl, pyridine or phenyltriazole derivatives, showed high transfection efficiency.

[0376] Selected compounds of Examples 3, 4, and 5 also showed improved productivity of biologics such as AAV or LV, indicating that the combined effect of high transfection efficiency and intracellular gene expression leads to high virus titers, expressed as transducing units. Furthermore, improved virus productivity was observed regardless of the type of cells transfected, e.g., adherent or suspension cells. The results indicated that such compounds may also be interesting for the production of other biologics, such as recombinant proteins, peptides, or antibodies.

[0377] In summary, the compounds of formula (I), preferably formula (III), of the present invention represent novel reagents for transfection and bioproduction purposes that can be tailored for each application, cell type or transfection conditions through careful optimization of the chemical structure.

[0378] Those skilled in the art can adapt the transfection method using the compounds of general formula (I), preferably general formula (III), of the present invention for in vivo use using acceptable excipients or buffers. The compounds of general formula (I), preferably general formula (III), can be mixed with DNA to produce DNA complexes suitable for direct injection into animals or humans. In particular, low-salt buffers such as Tris, phosphate, or citrate buffers, or excipients such as glucose, dextrose, or maltose, are known to provide formulations acceptable for direct injection into animals and humans. Many of the methods for mixing DNA with compounds of general formula (I), preferably general formula (III), are suitable because they can produce formulations containing small particles (non-aggregated DNA complexes) that can be injected via various routes of administration.

[0379] References TIFF0007772689000100.tif215170TIFF0007772689000101.tif222170TIFF0007772689000102.tif132170

Claims

1. A composition for transfecting a cell with a nucleic acid molecule, comprising: (i) at least one compound of general formula (III), or an acceptable salt thereof; and (ii) an acceptable excipient, buffer, cell culture medium, or transfection medium, 【Chemistry 1】 During the ceremony, （１） -Z 1 does not exist, -Z 2 does not exist, -Z 3 represents p-fluorobenzyl or 4-hydroxyphenethyl; - R represents H, -V,X 1 -R 3 -X 2 -P+, X 1 represents CH 2 , X 2 represents CO, R 3 represents (CH 2 ) m , m is an integer between 1 and 3, and P + represents a grafted cationic polymer which is linear or branched polyethyleneimine (PEI), or （２） - Z 1 represents X 1 -R 3 -X 2 -P + , where X 1 represents CH 2 , X 2 represents CO, R 3 represents (CH 2 ) m , m represents an integer between 1 and 3, and P + represents a graft cationic polymer which is linear or branched PEI; - Z 2 does not exist, - Z 3 does not exist, V represents H or methyl; R represents cyclopropyl, isopropyl, phenyl, benzyl, 2-pyridine or 3-pyridine; composition.

2. The composition of claim 1, further comprising at least one nucleic acid molecule to be transfected into the cell.

3. 3. The composition of claim 2, wherein the at least one nucleic acid molecule is selected from the group consisting of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), a DNA / RNA hybrid, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a messenger RNA (mRNA), a CRISPR guide RNA, and an expression vector encoding the nucleic acid molecule.

4. The composition of claim 3 , wherein the at least one nucleic acid molecule is DNA.

5. 5. The composition according to claim 1, wherein the grafted cationic polymer is linear PEI.

6. 6. The composition according to claim 1, wherein the grafted cationic polymer has a grafting ratio of 1 to 50%.

7. 7. The composition according to any one of claims 1 to 6, wherein the grafted cationic polymer has an average molecular weight (Mw) in the range of 1 kDa to 500 kDa.

8. 8. The composition of claim 7, wherein the grafted cationic polymer has an average molecular weight (Mw) of 10 or 22 kDa.

9. The at least one compound of general formula (III) is the following compound: Table 1A Table 1B 9. The composition of any one of claims 1 to 8, selected from the group consisting of:

10. The at least one compound of general formula (III) is the following compound: Table 2 10. The composition of claim 9, selected from the group consisting of:

11. 11. The composition of claim 10, wherein the at least one compound of general formula (III) is compound 2.

22.

12. 12. A method for in vitro or ex vivo transfection of living cells, comprising introducing into the cells a composition according to any one of claims 2 to 11.

13. In vitro or ex vivo use of a composition described in any one of claims 2 to 11 for transfecting at least one nucleic acid molecule into a cell, a cell line or a plurality of cells, wherein the cell, cell line or a plurality of cells are organized or not organized into spheroids, organoids, 2D or 3D cell cultures, or are provided as fiber or matrix cultures and / or in a bioreactor.

14. In vitro or ex vivo use of the composition described in claim 13, wherein the cell, cell line, or plurality of cells is selected from the group consisting of mammalian cells, insect cells, primary cells, adherent cells, suspension cells, dividing cells, non-dividing cells, and cancer cells.

15. 12. In vitro or ex vivo use of a composition according to any one of claims 2 to 11 for genome engineering, cell reprogramming, cell differentiation or gene editing.

16. 12. A method comprising the in vitro or ex vivo use of a composition according to any one of claims 2 to 11, A method for producing (i) a biologic encoding a recombinant protein, peptide, or antibody, or (ii) a recombinant virus, wherein the composition comprises multiple nucleic acid molecules for co-transfection, or (iii) a virus or virus-like particle, wherein the composition comprises multiple nucleic acid molecules for co-transfection.

17. 17. The method of claim 16, wherein the recombinant virus is selected from the group consisting of an adeno-associated virus (AAV), a lentivirus (LV), an adenovirus, an oncolytic virus, or a baculovirus.

18. 18. The method of claim 17 for producing AAV, wherein the composition comprises (i) at least one compound selected from the group consisting of compounds 2.22, 2.23, 2.43, 2.44, 2.47, 2.54, and 2.57, and (ii) an acceptable excipient, buffer, cell culture medium, or transfection medium.

19. 18. The method of claim 17 for producing LV, wherein the composition comprises (i) at least compound 2.22, and (ii) an acceptable excipient, buffer, cell culture medium, or transfection medium.

20. 18. The method of claim 16 or 17 for producing a recombinant virus, wherein the composition comprises a plurality of expression vectors for transfecting adherent cells, suspension cells or insect cells, the vectors being constructs that express viral structural sequences and a transfer vector genome for virus or virus-like production, and that may or may not express a molecule of interest encoded by the transfer vector genome.

21. 21. The method of claim 20, wherein the recombinant virus is for use in in vivo applications for cell therapy or gene therapy.

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