Compositions for transfecting nucleic acid molecules into cells comprising heterocyclic compounds grafted onto cationic polymers and their use

Abstract

The present invention relates to a composition for transfecting nucleic acid molecules into cells and its use. The present invention relates to a composition suitable for transfecting nucleic acid molecules into cells, preferably eukaryotic cells, the composition comprising (i) at least one compound of general formula (II) or its tautomer, mesomorph, racemate, enantiomer, diastereomer or mixture, 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 , Z 4 , Z 5 , Z 6 , Z 7 , X1, X2, R3, P + , R, T, U and V are as defined in the present specification. The present invention also relates to the use of the composition, and to a method for in vitro or ex vivo transfection of living cells.

Description

[0001] The present invention relates to a composition for transfecting nucleic acid molecules into cells, comprising a benzo-fused heterocyclic compound grafted onto a cationic polymer, and its use. The present invention relates to a composition suitable for transfecting nucleic acid molecules into cells, preferably eukaryotic cells, comprising (i) at least one compound of general formula (II) or its tautomer, mesomorph, racemate, enantiomer, diastereomer or mixture, 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 in the present specification. The present invention also relates to the use of the composition, and to a method for in vitro or ex vivo transfection of living cells.

[0002] Gene transfer is the process of introducing copies of foreign genes into living cells in order to induce the synthesis of gene products. Transfection is the process of intentionally and artificially introducing nucleic acids (DNA or RNA) into eukaryotic cells by means of non-viral methods. Transfection is crucial to the development of modern biology and medicine and has provided a lot of knowledge about gene function and regulation.

[0003] Transfection according to the present invention can be achieved in various cells including mammalian cells and insect cells, and can be achieved in primary cells, cell lines, stable cells or tumor cells. Transfection is a powerful tool for in vitro genomic studies by providing the possibility of expressing new foreign proteins in cells or overexpressing or silencing naturally occurring proteins.

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

[0005] In biological production, transfection according to the present invention can be used to generate stable cell clones that overproduce recombinant proteins, peptides or antibodies. Recently, transfection allowing transient gene expression (TGE) is becoming a valuable method for rapidly producing medium-level recombinant proteins or antibodies, which can be used in research and process development stages. The transient gene expression process is advantageously used to produce recombinant viruses, such as adeno-associated viruses (AAV), slow viruses (LV) or adenoviruses (Merten et al., 2016; Van Der Loo and Wright, 2015). Such processes consist of transfecting many expression vectors (plasmids), which express different components necessary for producing viruses in cells, including capsid proteins, auxiliary proteins, envelope proteins, viral polymerases or regulators, or viral genomes. High-yield cells are used for virus production, such as HEK293 and derivative cells, HeLa, BHK-21, A549 or insect cells. Transfection can be achieved at high cell densities in adherent or suspension-adapted cells cultured in serum-containing media, or in protein-free, chemically defined, or completely synthetic media.

[0006] Transfection is a method to introduce different components necessary to induce genome modification, engineering or editing, such as zinc finger nucleases, CRE / LOX proteins or CRISPR Cas-9 proteins, into cells.

[0007] DNA transfection uses plasmid DNA that triggers gene expression driven by promoters of proteins or peptides and / or nucleic acids (such as messenger RNA, long RNA, micro RNA, short hairpin RNA, short interfering RNA, etc.).

[0008] In most cases, plasmid DNA has been used for transfection purposes because of its inherent stability and ability to integrate into the host genome to produce stable gene expression or to remain in the cell nucleus in an episomal form to provide transient gene expression. However, some cells, designated as "hard-to-transfect" cells (HTT), are either insensitive to DNA transfection or exhibit low levels of transfection and gene expression when compared to standard transformed cell lines routinely used in laboratories. 2000&3000(ThermoFisher)、TransIT (MirusBio), (Promega), (Roche), (Polyplus-transfection) or (Promega)), these "difficult to transfect" cells showed less than 50% transfection efficiency.

[0009] The latest progress in improving the efficiency of gene expression in HTT cells is the use of messenger RNA (mRNA) sequences instead of plasmid DNA constructs for transfection, which shows significantly increased transfection and gene expression levels in most cell types, especially in challenging HTT cells. The benefit is explained by the fact that the main limitation of DNA transfection is to reach and penetrate the cell nucleus, in contrast, the transfected mRNA does not need to reach the cell nucleus to carry out cellular effects. Plasmid DNA input is not clear, but effective DNA transfection is mainly related to the active proliferation rate of cells, where the transfected DNA may diffuse into the nuclear space during nuclear membrane breakdown. In most post-mitotic cells or non-dividing cells, DNA transfection is ineffective. Most HTT cells exhibit low levels of mitosis or no mitosis, such as neuronal cells or other cell types derived from neural tissue, primary blood cells (such as dendritic cells or macrophages), or primary hepatocytes. However, for other HTT cells, the low transfection efficiency could be explained by other factors, such as cell fragility, poor binding of the transfected material to the cytoplasmic membrane, low endocytic capacity or inefficient intracellular transport to the nucleus of the transfected DNA.

[0010] Transfection of plasmid DNA is the most common method used to overexpress proteins in cultured cells. Most methods for introducing genetic DNA material into cells involve the use of reagents such as calcium phosphate, cationic liposomes, peptides or polymers. When transfection fails, the reagent is often considered the culprit. There is still a need to improve the efficiency of transfection reagents, especially for HTT cells, through new concepts and new generation reagents.

[0011] DNA transfection in eukaryotic cells involves combining or mixing polyanionic DNA molecules with reagents to form transfection complexes or aggregates. Among the most commonly used reagents, cationic lipids, peptides or polymers are suitable for interacting with negatively charged DNA. If an excess of cationic reagents is used, a complex or aggregate with positive characteristics is generated. Such complexes can interact with negatively charged glycosaminoglycans (such as heparan sulfate) present on the plasma membrane (Labatmoleur et al., 1996, Mislick and Baldeschwieler, 1996). The cell membrane binding of the complex induces cell internalization or uptake through an endocytic mechanism. The transfection complex is transported to endosomes, where the transfection reagent exhibits membrane instability by promoting fusion activity and / or endosomal dissolution to release DNA in the cytoplasm. After release from the endosome, the transfected DNA must diffuse to the perinuclear space and infiltrate the nucleus. Nuclear import is a limiting step because plasmid DNA cannot diffuse through the nuclear pore complex due to its large size.

[0012] Among non-viral vectors for DNA transfection, cationic liposomes or aggregates are one of the main categories, which include combining or formulating cationic lipids with other types of lipids (such as phospholipids or cholesterol) to generate positively charged liposomes, vesicles or micelles, which can bind negatively charged DNA and bind to negatively charged cell membranes, ultimately achieving cell transfection. 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 forms cationic liposomes that can transfect cells in vitro. Based on the positively charged trimethylammonium polar head, other monocationic lipids such as 1,2-bis(oleoyloxy)-3,3-(trimethylammonium) propane chloride (DOTAP) have been developed. Other prior art compounds are based on polycationic polar heads, such as the lipids described by B Behr et al., 1989, dioctadecylamidoglycylspermine (DOGS) or dipalmitoyl phosphatidylethanolamidospermine (DPPES), in which carboxyspermine is used instead of the ammonium group, or the phospholipid moiety is replaced by a cholesterol derivative ((Gao & Huang, 1991) such as 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-CHOL)). Since these pioneering works, many cationic lipid designs have been aimed at generating novel cationic lipid reagents with improved transfection efficiency. Many of these reagents are commercially available, and the most recent The reagents represent the most advanced cationic lipid reagents available on the market. However, limitations are still observed, as transfection is not efficient in all cell types and cytotoxicity remains a major concern with cationic lipid systems.

[0013] Cationic polymers represent the second largest class of transfection reagents, with the advantage of providing a high density of charged amino groups in their backbone. Cationic polymers with positive charges at physiological pH can compound DNA into particles or aggregates, initiate cell binding and trigger cell internalization by endocytosis. Polylysine (PLL) is the first polymer used, but shows very limited transfection efficiency (Wu and Wu, 1987, Zenke et al., 1990). When additives such as weak bases (such as chloroquine (Erbacher et al., 1996)) or fusion peptides (such as influenza peptides (Planck et al., 1994)) are added to buffer the acidic pH of unstable endosomes and induce more DNA release in the cytoplasm, its efficiency can be improved. Behr et al. have demonstrated that polyethyleneimine (PEI) is a polymer that is more effective than PLL in transfection (Boussif et al., 1995). PEI has a high density of amino groups and is not fully protonated at physiological pH. After the DNA complexed with PEI is endocytosed, the polymer has a buffering capacity that induces a "proton sponge" activity, leading to vesicle swelling and endosome dissolution, ultimately releasing DNA in the cytoplasm without the help of additives (Boussif et al., 1995; Sonawane et al., 2003). Both branched and linear PEI are effective in transfection, but when compared to branched forms, linear topology has been shown to be more effective (Itaka et al., 2004), less inhibited by the presence of serum, and less toxic. Over the past two decades, many strategies have been developed to improve the transfection efficiency of PEI, reduce its toxicity, or propose alternatives to biodegradable PEI-based polymers.

[0014] Many works focus on optimizing the intrinsic proton sponge endosomal dissolving activity of PEI by grafting histidyl or benzyl residues (US8658150, Chandrashekhar et al., 2012) onto polymers. Other modifications have been explored, such as adding hydrophilic groups (EP2070970) to increase the solubility of DNA / PEI complexes and reduce cytotoxicity. Hydrophobic functional groups are added to PEI to increase the biodegradability of polymers using N-acyl groups (EP0262641), or to generate lipopolymers (US20090022746, WO2006 / 041617). Higher gene transfection efficiency is observed in various cell lines. However, the efficiency in "difficult to transfect" cells remains very limited.

[0015] Other cationic polymers for DNA transfection have been described, such as chitosan ( Erbacher et al., 1998 ), polyamidoamine (PAMAM) dendrimers ( Tomalia et al., 1985 , Haensler and Szoka, 2003 ), degradable or ruptured dendrimers ( Tang et al., 1996 ), structurally flexible dendrimers ( Liu et al., 2011 ), polyamide esters ( Little et al., 2004 ), poly(α[4-aminobutyl]-L-glycolic acid) ( Akinc et al., 2005 ). et al., 2003), cationic cyclodextrin amphiphiles (Cryan et al., 2004), poly(N-methylvinylamine) (Dréan 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 (CPP) (Gupta, 2005).

[0016] Cationic polymers such as PEI are reported to be able to transfect post-mitotic cells (Brunner et al.). However, in the absence of mitosis and subsequent nuclear membrane rupture, it has been demonstrated that plasmid DNA cannot enter the nucleus through the nuclear pore complex due to its large size>1kbp (Lukacs et al., 2000). Once released from the endosome, DNA remains associated with some cationic polymers, which helps protect it from nuclease degradation (Lechardeur et al., 1999). It is known that DNA can interact with proteins present in the cytoplasm, particularly dynein, thereby allowing microtubule-based movement to the nucleus or allowing the binding of transcription factors with NLS signals, which may guide DNA to the nuclear pore complex through the importin pathway (Bai et al., 2017).

[0017] Cationic polymers represent a class of delivery agents suitable for in vivo use in gene therapy protocols, where the DNA / cationic polymer complex is injected directly via different routes of administration, 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 is reported to be an effective polymer for in vivo use (Boussif et al., 1995).

[0018] Due to their special structural features and electron-rich environment, benzimidazole-containing derivatives bind to a variety of targets (such as DNA or proteins) and exhibit a broad spectrum of biological activities (Gaba and Mohan, 2016). The benzimidazole ring is structurally similar to purine bases. Many benzimidazole derivatives were found to be minor groove binders of DNA (Ivanov et al., 2013; Gao et al., 2013). Changing the chemical structure of the benzimidazole ligand may modulate the DNA binding mode and sequence selectivity (Bazhulina et al., 2009; Tari et al., 2017). Benzimidazole derivatives also have the ability to accumulate in the nucleus, such as Hoechst 33258 compounds. Other derivatives were found to inhibit H / K ATPase activity (Fellenius et al., 1981). Minor modifications of the benzimidazole ring such as methyl, ethyl, and amino groups may modulate the pKa value, with a range of 5-6 being of interest for buffering capacity (Brown and Mora-Diez, 2006).

[0019] The present inventors provide a way to improve transfection reagents by using benzo-fused heterocyclic compounds to fine-tune affinity and binding to nucleic acid molecules (e.g., DNA), optimize buffering capacity under acidic conditions and / or increase diffusion, binding and uptake in the cell nucleus.

[0020] Therefore, an object of the present invention is to provide a more effective transfection composition or to provide a formulation for transfecting nucleic acid molecules into cells.

[0021] Another object of the present invention is to provide a method for transfecting nucleic acid molecules using the composition or a preparation comprising such a composition for administration into cells.

[0022] The inventors have performed a structure-based screen for substituted heterocyclic compounds (especially benzimidazoles, benzopyrazoles, benzotriazoles) to improve transfection efficiency by cationic polymers. Such substituted heterocyclic compounds are grafted onto cationic polymers of various molecular weights, especially polyethyleneimine (PEI) polymers, in order to fine-tune the conjugates. Many variations have been proposed in order to define the optimal structure to promote transfection of nucleic acid molecules (such as DNA). Heterocycles that exhibit hydrophobic properties have been developed and may represent binding motifs for cytoplasmic proteins, thereby potentially promoting nuclear import.

[0023] The present invention relates to a composition suitable for transfecting nucleic acid molecules (preferably deoxyribonucleic acid (DNA)) into cells (preferably eukaryotic cells), the composition comprising (i) at least one compound of general formula (II) or its tautomer, mesomer, racemate, enantiomer, diastereomer or mixture, or an acceptable salt thereof, and (ii) an acceptable excipient, buffer, cell culture medium or transfection medium:

[0024]

[0025] in:

[0026] -Y 1 , Y 2 and Y 3 Can be the same or different, representing C or N, provided that Y 1 , Y 2 and Y 3 At least two of them are N, and further condition is Y 1 , Y 2 and Y 3 At least one but no more than two of them are Z 1 , Z 2 and Z 3 replace;

[0027] -Z 1 Represents 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;

[0028] -Z 2 Represents H, straight chain or branched chain saturated or unsaturated C 1- C 18 Alkyl, C 6- C 18 Aryl, straight chain or branched chain, saturated or unsaturated C6-C 18 Aryl-C1-C 18 Alkyl, linear or branched saturated or unsaturated C 2- C 18 Heteroalkyl, C5-C 10 Heteroaryl, halogen, OH, straight or branched saturated or unsaturated C 1- C 18 Alkylamine, C 1- C 12 Alkoxy, linear or branched saturated or unsaturated C1- C 18 Alkyl-C 1- 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;

[0029] -Z 3 Represents H, straight chain or branched chain saturated or unsaturated C 1- C 18 Alkyl, C 6- C 18 Aryl, straight chain or branched chain, saturated or unsaturated C6-C 18 Aryl-C1-C 18 Alkyl, C5-C 10 Heteroaromatic, straight or branched chain saturated or unsaturated C 2- C 18 Heteroalkyl, C 2- C 18 Alkylene, 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;

[0030] -X1 and X2 can be the same or different and represent CO or CH2;

[0031] -R3 represents (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 -CH2-O-, wherein m represents an integer from 1 to 3, and n represents an integer from 1 to 3;

[0032] -P + represents a grafted cationic polymer which is a polyamine comprising a secondary amine, a tertiary amine, a mixture of a primary and secondary amine, a mixture of a primary and tertiary amine, a mixture of a secondary and tertiary amine, or a mixture of a primary, secondary and tertiary amine;

[0033] - R, T, U and V may be identical or different and represent C or N, provided that the six-membered ring of formula (II) contains not more than 2 N atoms;

[0034] -Z 4 , Z 5 , Z 6 and Z 7 Can be the same or different, representing H, straight or branched chain saturated or unsaturated C 1- C 18 Alkyl, C 6- C 18 Aryl, straight chain 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, amine, straight chain or branched saturated or unsaturated C1-C 18 Alkylamine, C1-C 12 Alkoxy, C 2- C 18 alkylene, OH, guanidine or halogen; or (i) Z 4 and Z 5 or (ii) Z 5 and Z 6 or (iii) Z 6 and Z 7 together form a fused optionally substituted 6-membered aryl or heteroaryl group;

[0035] The conditions are:

[0036] -Z 1 , Z 2 or Z 3 Only one of them represents X1-R3-X2-P + 、X1-R3-P + 、X1-X2-P + 、R3-X2-P + 、X1-P + 、R3-P + or X2-P + .

[0037] As defined herein, the term "tautomer" refers to structural isomers that differ only in the position of hydrogen atoms and electrons. Examples of tautomers include, but are not limited to, keto-enols, enamine-imines, amide-imidic acids, lactam-lactim, nitroso-oximes, enone-alkynols, amino acids, or phosphites-phosphonates.

[0038] As defined herein, the term "mesomer" or "meso compound" refers to a stereoisomer having two or more chiral centers but not being optically active.

[0039] As defined herein, the term "racemate" or "racemic mixture" refers to a mixture of two enantiomers in equal proportions.

[0040] As defined herein, the term "enantiomer" refers to a stereoisomer that is a mirror image, ie, a mirror image isomer.

[0041] As defined herein, the term "diastereomer" refers to isomers of a compound with more than one chiral center that are not mirror images of one another.

[0042] As defined herein, the term "acceptable excipient" refers to a pharmaceutically acceptable carrier, which is any physiologically acceptable substance or combination of substances, i.e. suitable for use in compositions contacted with a host (particularly a human being), and is therefore nontoxic. It may refer to any conventional type of solid, semisolid 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.

[0043] As defined herein, the term "buffer" refers to an agent that adjusts, maintains or controls the pH of a solution. A buffer can be a weak acid or a weak base that may comprise a buffered solution. Examples of suitable buffers 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, monopotassium phosphate, potassium carbonate, potassium bicarbonate, potassium citrate or magnesium oxide.

[0044] As defined herein, the term "cell culture medium" or "transfection medium" refers to a serum-containing medium, a synthetic medium, an animal component-free medium or a chemically defined medium, especially a medium used to maintain cell survival, or for growth, for differentiation or for expansion of cells, or for enhancing transfection.

[0045] As defined herein, the term "C1-C 18"Alkyl" represents any monovalent group of a straight or branched hydrocarbon chain containing 1 to 18 carbon atoms. The term "C1-C6 alkyl" represents an alkyl group having 1 to 6 carbon atoms. Suitable C1-C 18 Examples of alkyl groups include, but are not limited to, C1-C4 alkyl groups (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl), C6-C8 alkyl groups (e.g., 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.

[0046] As defined herein, the term "C1-C 12 "Alkoxy" represents a group having the formula -OR', wherein R' is C1-C 12 Suitable C1-C 12 Examples of alkoxy groups include, but are not limited to, C1-C6 alkoxy groups, such as methoxy (-OCH3), ethoxy (-OCH2CH3), tert-butoxy (-OC(CH3)3), or -O(CH2)5CH3.

[0047] As defined herein, the term "C6-C 18 "Aryl" represents any monovalent group of an aromatic hydrocarbon containing 6 to 18 carbon atoms. Suitable C6-C 18 Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, or phenanthrenyl.

[0048] As defined herein, the term "C6-C 18 Aryl-C1-C 18 "Alkyl" represents an aryl group as defined herein combined with an alkyl group as defined herein. Suitable C6-C 18 Aryl-C1-C 18 Examples 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, phenanthracenylmethyl, phenanthracenylethyl, phenanthracenylpropyl, phenanthracenylbutyl, phenan ... or phenanthracenylhexyl.

[0049] As defined herein, the term "C2-C 18 "Heteroalkyl" represents an alkyl group as defined herein substituted with one or more heteroatoms (eg O, N or S).

[0050] As defined herein, the term "C5-C 10"Heteroaryl" represents any monovalent group 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, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, isoxazolyl, isothiazolyl, thiazolyl, oxazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1-benzofuranyl, 1-benzothienyl, indolyl, benzimidazolyl, indazolyl, 1,2-benzisoxazolyl, 2,1-benzisoxazolyl, 1,2-benzisothiazolyl, 2,1-benzisothiazolyl, benzothiazolyl, benzoxazolyl, benzotriazolyl, pyridinyl, pyridinium, quinolyl, quinolinium, isoquinolyl, isoquinolinium, pyridazinyl, cinnolinyl, phthalazinyl, pyrimidinyl, quinazolinyl, pyrazinyl, or quinoxalinyl.

[0051] As defined herein, the term “C 1- C 18 "Alkylamine" represents any monovalent radical 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 is replaced by an amino group. Suitable C1-C 18 Examples of alkylamines include, but are not limited to, -(CH2) n -NH2 (wherein n represents an integer from 1 to 18), -CH2NHCH3, -CH2CH(CH3)-NH2 or -(CH2) n N(CH3)2, wherein n represents an integer from 1 to 6.

[0052] As defined herein, the term "C1-C 18 Alkyl-C1-C 12 "Alkoxy" represents an alkyl group as defined herein bonded to an alkoxy group as defined herein.

[0053] As defined herein, the term “C 2- C 18 "Alkylene" means a divalent radical derived from an alkane by removing two hydrogen atoms from the same carbon atom, the free valences being part of a double bond (=CR2). Suitable C 2- C 18 Examples of alkylene include, but are not limited to, =CH2, =CH(CH2CH3), or =C(CH3)2.

[0054] As defined herein, the term "halogen" represents an F, Cl, Br or I atom.

[0055] As defined herein, the term "C1-C 24"Ester" means a group having the formula -C(O)OR" where R" is C1-C 24 Alkyl, especially C1-C 18 alkyl.

[0056] As defined herein, the term "C5-C 10 "Heterocyclyl" refers to any monovalent group of a 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.

[0057] Unless otherwise mentioned, the groups and radicals defined above may be unsubstituted or substituted by 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 and diarylamino.

[0058] In a specific embodiment of the present invention, the composition further comprises at least one nucleic acid molecule to be transfected into the cell. Preferably, the nucleic acid molecule is selected from: deoxyribonucleic acid (DNA), ribonucleic acid (RNA), DNA / RNA hybrid, short interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), CRISPR guide RNA, and an expression vector encoding the nucleic acid molecule, in particular a plasmid encoding the nucleic acid molecule, or a plasmid expressing the nucleic acid molecule (such as siRNA, microRNA, shRNA, CRISPR guide RNA). Even more preferably, the nucleic acid molecule is deoxyribonucleic acid (DNA).

[0059] When different nucleic acids are provided in the composition of the invention, these may be all DNA molecules or all RNA molecules, or may be a mixture of DNA and RNA molecules or associated molecules comprising DNA and RNA strands.

[0060] The nucleic acid molecules may be single-stranded or double-stranded and may contain modified or unmodified bases.

[0061] The terms "polynucleotide," "nucleic acid," "oligonucleotide," and "nucleic acid molecule" are used interchangeably herein to designate these nucleic acid molecules.

[0062] According to the disclosure provided herein, the composition of the present invention can be used as a preparation of a nucleic acid molecule and at least one compound of formula (II) (including any specific embodiment thereof disclosed herein) and an acceptable excipient, buffer, cell culture medium or transfection medium. Alternatively, it can be used as a cell culture or as an expanded cell (expanded cells), wherein the separated cells have been treated with the preparation for transfection before being provided as a culture and / or expanded cells. In other words, as an embodiment, the composition of the present invention covers cells or cell cultures or expanded cells, wherein the preparation has been introduced by transfection according to the present invention. In particular, the cell is a mammalian cell, preferably a human cell. The cell can be a dividing cell or a non-dividing cell.

[0063] In a specific embodiment of the present invention, the composition of the present invention comprises 1 to 5, preferably at least two different compounds of general formula (II), or their tautomers, mesomers, racemates, enantiomers, diastereomers or mixtures, or acceptable salts thereof.

[0064] In a specific embodiment of the present invention, at least one preferred compound of formula (II) as defined herein is a compound wherein: (i) Y 1 and Y 3 Represents N, Y 2 represents C; or (ii) Y 1 and Y 2 Represents N, Y 3 represents C; or (iii) Y 2 and Y 3 Represents N, Y 1 represents C; or (iv) Y 1 , Y 2 and Y 3 Represents N.

[0065] In a specific embodiment of the present invention, at least one preferred compound of formula (II) as defined herein is a compound wherein: (i) Z 1 represents H; or (ii) Z 1 Represents 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 + , where X1, X2, R3 and P + As defined herein; more preferably Z1 Represents X1-R3-X2-P + , wherein X1 represents CH2, X2 represents CO, and R3 represents (CH2) m , wherein m represents an integer from 1 to 3, preferably m is equal to 2.

[0066] In a specific embodiment of the present invention, at least one preferred compound of formula (II) as defined herein is a compound wherein: (i) Z 2 Represents H, C 1- C 12 Alkoxy, either straight-chain or branched, saturated or unsaturated C1-C 18 Alkyl, preferably 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 Represents 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 + , where X1, X2, R3 and P + As defined herein; more preferably Z 2 Represents X1-R3-X2-P + , wherein X1 represents CH2, X2 represents CO, and R3 represents (CH2) m , wherein m represents an integer from 1 to 3, preferably m is equal to 2.

[0067] In a specific embodiment of the present invention, at least one preferred compound of formula (II) as defined herein is a compound wherein: (i) Z 3 Represents H, straight chain or branched chain, saturated or unsaturated C1-C 18 Alkyl, preferably a linear or branched saturated or unsaturated C1-C6 alkyl, or a linear or branched saturated or unsaturated C6-C 18 Aryl-C1-C 18 alkyl, preferably fluorobenzyl or 4-hydroxyphenethyl; or (ii) Z 3 Represents 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 + , where X1, X2, R3 and P + As defined herein; more preferably Z 3 Represents X1-R3-X2-P + , wherein X1 represents CH2, X2 represents CO, and R3 represents (CH2) m , wherein m represents an integer from 1 to 3, preferably m is equal to 2.

[0068] In a preferred embodiment of the present invention, if (i) Z 1 Represents 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 + , where X1, X2, R3 and P + As defined herein; more preferably Z 1 Represents X1-R3-X2-P + , wherein X1 represents CH2, X2 represents CO, and R3 represents (CH2) m , wherein m represents an integer from 1 to 3, preferably m is equal to 2, then (ii) Z 2 Represents H, C 1- C 12 Alkoxy, either straight-chain or branched, saturated or unsaturated C1-C 18 Alkyl, preferably linear or branched saturated or unsaturated C1-C6 alkyl; more preferably Z 2 represents H, CH3, CF3 or OCH3; and / or (iii) Z 3 Represents H, straight or branched chain saturated or unsaturated C 1- C 18 Alkyl, preferably a linear or branched saturated or unsaturated C1-C6 alkyl, or a linear or branched saturated or unsaturated C6-C 18 Aryl-C1-C 18 The alkyl group is preferably a fluorobenzyl group or a 4-hydroxyphenethyl group.

[0069] In another preferred embodiment of the present invention, if (i) Z 2 Represents 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 + , where X1, X2, R3 and P + As defined herein; more preferably Z 2 Represents X1-R3-X2-P + , wherein X1 represents CH2, X2 represents CO, and R3 represents (CH2) m , wherein m represents an integer from 1 to 3, preferably m is equal to 2, then (ii) Z 1 represents H; and / or (iii) Z 3 Represents H, straight chain or branched chain, saturated or unsaturated C1-C 18 Alkyl, preferably a linear or branched saturated or unsaturated C1-C6 alkyl, or a linear or branched saturated or unsaturated C6-C 18 Aryl-C1-C 18 The alkyl group is preferably a fluorobenzyl group or a 4-hydroxyphenethyl group.

[0070] In another preferred embodiment of the present invention, if (i) Z 3 Represents 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 + , where X1, X2, R3 and P + As defined herein; more preferably Z 3 Represents X1-R3-X2-P + , wherein X1 represents CH2, X2 represents CO, and R3 represents (CH2) m , wherein m represents an integer from 1 to 3, preferably m is equal to 2, then (ii) Z 1 represents H; and / or (iii) Z 2 Represents H, C1-C 12 Alkoxy, either straight-chain or branched, saturated or unsaturated C1-C 18 Alkyl, preferably linear or branched saturated or unsaturated C1-C6 alkyl; more preferably Z 2 represents H, CH3, CF3 or OCH3.

[0071] In a specific embodiment of the present invention, at least one preferred compound of formula (II) as defined herein is a compound wherein: (i) Z 1 , Z 2 or Z 3 Only one of them represents 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 + , where X1, X2, R3 and P + As defined herein; more preferably Z 1 , Z 2 or Z 3 Only one of them represents X1-R3-X2-P + , wherein X1 represents CH2, X2 represents CO, and R3 represents (CH2) m , wherein m represents an integer from 1 to 3, preferably m is equal to 2; and / or (ii) Z 1 represents H; and / or (iii) Z 2 Represents H, C1-C 12 Alkoxy, either straight-chain or branched, saturated or unsaturated C1-C 18 Alkyl, preferably linear or branched saturated or unsaturated C1-C6 alkyl; more preferably Z 2 represents H, CH3, CF3 or OCH3; and / or (iv) Z 3 represents H, or a straight or branched chain saturated or unsaturated C 1- C 18 The alkyl group is preferably a linear or branched saturated or unsaturated C1-C6 alkyl group.

[0072] In a specific embodiment of the present invention, at least one preferred compound of formula (II) as defined herein is a compound wherein: the grafted cationic polymer is selected from: linear or branched polyethyleneimine (PEI), PEI dendrimers, polypropyleneimine (PPI), poly(amidoamine) (PAA) and dendrimers (PAMAM), cationic cyclodextrins, polyalkylamines, polyhydroxyalkylamines, poly(butyleneimine) (PBI), spermine, N-substituted polyallylamines, N-substituted chitosans, N-substituted polyornithines, N-substituted polylysines (PLL), N-substituted polyvinylamines, poly(β-amino esters), hyperbranched poly(amino esters) (h-PAE), reticulated poly(amino esters) (n-PAE), poly(4-hydroxy-1-proline ester) (PHP-ester) and poly-β-amino acids, preferably linear or branched PEI, more preferably linear PEI.

[0073] The grafting rate of the grafted cationic polymer may be 1 to 50%, preferably 5 to 30%, and more preferably 20%.

[0074] As defined herein, the term "grafting rate" refers to the number of monomers grafted onto primary, secondary or tertiary amino groups via side chains divided by the number of total monomers present in the original cationic polymer. The grafting rate will depend 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 rate can be determined by measurement methods well known in the art, such as by NMR.

[0075] The average molecular weight (Mw) of the grafted cationic polymer may be from 1 kDa to 500 kDa, preferably from 1 kDa to 50 kDa, more preferably from 5 kDa to 50 kDa or from 1 kDa to 15 kDa. In particular, the average molecular weight (Mw) of the grafted cationic polymer may be from 6, 8, 10, 15, 22 or 30 kDa, preferably from 6, 8, 10, 15 or 30 kDa.

[0076] The grafted cationic polymer may be associated with a counterion such as chloride, phosphate, citrate, acetate, propionate, carbonate, succinate, sulfonate, sulfate, or carboxylate.

[0077] In one embodiment of the invention, at least one preferred compound of formula (II) as defined herein is a compound wherein: (i) R, T, U and V represent C; or (ii) R, T, U and V, which may be the same or different, represent C or N, provided that the six-membered ring of formula (II) contains not more than 1 N atom; preferably, one of R, T, U or V represents N; or (iii) R and U represent N and T and V represent C; or R and T represent N and U and V represent C; or R and V represent N and T and U represent C; or T and U represent N and R and V represent C; or T and V represent N and R and U represent C. In a preferred embodiment of this first specific embodiment of the invention, at least one preferred compound of formula (II) as defined herein is a compound wherein: (i) R, T, U and V represent C.

[0078] In one embodiment of the present invention, at least one preferred compound of formula (II) as defined herein is a compound wherein: Z 4 , Z 5 , Z 6 and Z 7 may be the same or different and represent H, OH, halogen, or halogen-substituted C1-C 12 Alkyl, amine, straight chain or branched chain saturated or unsaturated C1-C 18 Alkylamine, C 1- C 12 Alkoxy, either straight-chain or branched, saturated or unsaturated C1-C 18 Alkyl, preferably linear or branched saturated or unsaturated C1-C6 alkyl; more preferably Z 4 , Z 5 , Z 6 and Z 7 can be the same or different and represent H, CH3, NH2 or OCH3.

[0079] In a particular embodiment of the present invention, at least one preferred compound of formula (II) as defined herein is a compound wherein: Z 4 , Z 5 , Z 6 and Z 7 Represents H.

[0080] In a particular embodiment of the present invention, at least one preferred compound of formula (II) as defined herein is a compound wherein: Z 1 Represents X1-R3-X2-P + , where X1, X2, R3 and P + As defined in claim 1; more preferably Z 1 Represents X1-R3-X2-P+ , wherein X1 represents CH2, X2 represents CO, and R3 represents (CH2) m , wherein m represents an integer from 1 to 3, preferably m is equal to 2, and P + represents linear or branched PEI, preferably linear PEI.

[0081] In a particular embodiment of the present invention, at least one preferred compound of formula (II) as defined herein is a compound wherein: Z 2 Represents a straight chain or branched chain saturated or unsaturated C 1- C6 alkyl, preferably Z 2 Stands for CH3.

[0082] In a particular embodiment of the present invention, at least one preferred compound of formula (II) as defined herein is a compound wherein: Z 4 , Z 5 , Z 6 or Z 7 One of them represents a straight or branched saturated or unsaturated C 1- C6 alkyl, preferably Z 4 , Z 5 , Z 6 or Z 7 One of them represents CH3.

[0083] In a particular embodiment of the present invention, at least one preferred compound of formula (II) as defined herein is a compound wherein: Z 4 and Z 6 Represents a straight chain or branched chain saturated or unsaturated C 1- C6 alkyl, preferably Z 4 and Z 6 Stands for CH3.

[0084] In a particular embodiment of the present invention, at least one preferred compound of formula (II) as defined herein is a compound wherein: Z 5 and Z 6 Represents a straight chain or branched chain saturated or unsaturated C 1- C6 alkyl, preferably Z 5 and Z 6 Stands for CH3.

[0085] In a particular embodiment of the present invention, at least one preferred compound of formula (II) as defined herein is a compound wherein:

[0086] -Y 1 and Y 3 Represents N, Y 2represents C; and

[0087] - R, T, U and V represent C; and

[0088] -Z 2 , Z 4 and Z 6 Represents a straight chain or branched chain saturated or unsaturated C 1- C6 alkyl, preferably Z 2 , Z 4 and Z 6 Stands for CH3.

[0089] In a particular embodiment of the present invention, at least one preferred compound of formula (II) as defined herein is a compound wherein:

[0090] -Y 1 and Y 3 Represents N, Y 2 represents C; and

[0091] - R, T, U and V represent C; and

[0092] -Z 2 , Z 4 and Z 6 represents a linear or branched saturated or unsaturated C1-C6 alkyl group, preferably Z 2 , Z 4 and Z 6 represents CH3; and

[0093] -Z 1 Represents 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 + , where X1, X2, R3 and P + As defined in claim 1; preferably Z 1 Represents X1-R3-X2-P + , wherein X1 represents CH2, X2 represents CO, and R3 represents (CH2) m , wherein m represents an integer from 1 to 3, preferably m is equal to 2.

[0094] In a preferred embodiment of the present invention, at least one compound of formula (II) is a benzimidazole derivative, wherein:

[0095] -Y 1and Y 3 Represents N, Y 2 represents C; and

[0096] -Z 1 or Z 3 Represents 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 + , where X1, X2, R3 and P + As defined above; more preferably Z 1 or Z 3 Represents X1-R3-X2-P + , wherein X1 represents CH2, X2 represents CO, and R3 represents (CH2) m , wherein m represents an integer from 1 to 3, preferably m is equal to 2; and

[0097] in:

[0098] (i) R, T, U and V represent C; preferably R, T, U and V represent C, and / or Z 2 represents H, CH3, SCH3, CF3, phenyl, isopropyl, propyl, CH2-NH-CH3, CH2-O-CH3 or CH2-F, and / or Z 4 represents H, CH3, and / or Z 5 represents H, F, OCH3, carboxyphenyl, tert-butyl, Cl, OH or CH3, and / or Z 6 represents H, CH3 or F, and / or Z 7 represents H; or

[0099] (ii) T and V represent N, and R and U represent C; preferably T and V represent N, and R and U represent C, and / or Z 2 represents H, CH3, and / or Z 4 represents H, NH2, N(CH3)2, and Z 5 , Z 6 or Z 7 At least one of represents H; or

[0100] (iii) R and U represent N, and T and V represent C; preferably R and U represent N, and T and V represent C, and / or Z 2 represents H, CH3, and / or Z 4 represents H, NH2, N(CH3)2, and Z5 , Z 6 or Z 7 At least one of represents H; or

[0101] (iv) one of R, T, U or V represents N; preferably one of R, T, U or V represents N, and Z 2 , Z 4 , Z 5 , Z 6 or Z 7 At least one of represents H; or

[0102] (v) R, T, U and V represent C, and Z 5 and Z 6 together to form an optionally substituted naphthalene; preferably R, T, U and V represent C, Z 5 and Z 6 Together they form naphthalene, and / or Z 2 represents CH3, and / or Z 4 Represents H, and / or Z 7 Represents H.

[0103] Preferred benzimidazole derivatives of the present invention correspond to compounds 1.01 to 1.42, 1.51 to 1.72, 1.74 to 1.77 and 1.79 as disclosed in Table 1.

[0104] In another preferred embodiment of the present invention, at least one compound of formula (II) is a benzopyrazole derivative, wherein:

[0105] -Y 1 and Y 2 Represents N, Y 3 Representative C; or Y 2 and Y 3 Represents N, Y 1 represents C; and

[0106] -Z 1 or Z 3 Represents 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 + , where X1, X2, R3 and P + As defined above; more preferably Z 1 or Z 3 Represents X1-R3-X2-P +, wherein X1 represents CH2, X2 represents CO, and R3 represents (CH2) m , wherein m represents an integer from 1 to 3, preferably m is equal to 2; and

[0107] in:

[0108] (i) R, T, U and V represent C; preferably R, T, U and V represent C, and Z 4 , Z 5 , Z 6 or Z 7 At least one of represents H; or

[0109] (ii) R, T, U and V may be the same or different and represent C or N, provided that the six-membered ring contains not more than 1 N atom; preferably, one of R, U or V represents N, and Z 4 , Z 5 , Z 6 or Z 7 At least one of them represents H.

[0110] Preferred benzopyrazole derivatives of the present invention correspond to compounds 1.43 to 1.50 as disclosed in Table 1.

[0111] In another preferred embodiment of the present invention, at least one compound of formula (II) is a benzotriazole derivative, wherein:

[0112] -Y 1 , Y 2 and Y 3 represents N; and

[0113] -Z 1 or Z 2 Represents 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 + , where X1, X2, R3 and P + As defined above; more preferably Z 1 or Z 2 Represents X1-R3-X2-P + , wherein X1 represents CH2, X2 represents CO, and R3 represents (CH2) m , wherein m represents an integer from 1 to 3, preferably m is equal to 2; and

[0114] in:

[0115] (i) R, T, U and V represent C; preferably R, T, U and V represent C, and / or Z 4 Represents H, and / or Z 5 represents H, CH3, OCH3, and / or Z 6 represents H, CH3, and / or Z 7 represents H; or

[0116] (ii) R, T, U and V may be the same or different and represent C or N, provided that the six-membered ring contains not more than 1 N atom; preferably, one of R, T or U represents N, and Z 4 , Z 5 , Z 6 or Z 7 At least one of them represents H.

[0117] Preferred benzotriazole derivatives of the present invention correspond to compounds 2.01 to 2.18 as disclosed in Table 1.

[0118] According to a specific embodiment of the present invention, preferred compounds of formula (II) correspond to compounds 1.01 to 1.72, 1.74 to 1.77 and 1.79 as disclosed in Table 1, and compounds 2.01 to 2.18, more preferably compounds 1.42, 1.57 and 1.65.

[0119] Table 1. Structures of preferred compounds of formula (II) according to the invention.

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130] Therefore, in a particular embodiment of the present invention, at least one compound of general formula (II) is selected from:

[0131]

[0132]

[0133]

[0134]

[0135] In a preferred embodiment of the invention, at least one compound of formula (II) is selected from the group consisting of: 1.09, 1.10, 1.11, 1.14, 1.15, 1.17, 1.18, 1.19, 1.20, 1.25, 1.27, 1.28, 1.30, 1.35, 1.36, 1.37, 1.41, 1.42, 1.57, 1.60, 1.65, 2.03, 2.04, 2.05, 2.08, 2.09 and 2.10.

[0136] The at least one compound of general formula (II) can be prepared according to various methods well known in the art.

[0137] The present invention also relates to a composition of the present invention for in vivo use in cell transformation, cell therapy or gene therapy, wherein the cell transformation is carried out by taking up exogenous nucleic acid using the composition of the present invention. The cell may be a eukaryotic cell, in particular a mammalian cell, in particular a human cell, in particular a primary cell, and may be a dividing cell or a non-dividing cell.

[0138] The present invention also relates to a method for in vitro or ex vivo transfection of living cells, which comprises introducing the composition of the present invention into cells. The living cells can be provided or maintained in a serum-containing culture medium, a synthetic culture medium, an animal component-free culture medium or a chemically defined culture medium.

[0139] The present invention also relates to the in vitro or ex vivo use of the composition of the present invention for transfecting at least one nucleic acid molecule into a cell, a cell line or a plurality of cells, preferably a cell, a cell line or a plurality of cells selected from the group consisting of mammalian cells, insect cells, primary cells, adherent cells, suspension cells, dividing cells (such as stem cells), non-dividing cells (such as neuronal cells) and cancer cells, the cell, cell line or a plurality of cells being optionally organized into spheroids, organoids, 2D or 3D cell cultures, or provided as fiber or matrix cultures, and / or within a bioreactor.

[0140] As defined herein, the term "adherent cell" refers to a cell that requires a solid support for growth and is 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, HEK 293 cells, Hep G2 cells, SNN-BE(2) cells, BAE-1 cells, or SH-SY5Y cells.

[0141] As defined herein, the term "suspension cells" 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, Yac 1 cells, Jurkat cells, THP-1 cells, K562 cells, or U266B1 cells.

[0142] As defined herein, the term "spheroid" refers to a spherical heterogeneous cell aggregate that maintains a three-dimensional architecture in culture.

[0143] As defined herein, the term "organoid" refers to a three-dimensional structure formed by a collection of organ-specific cell types that self-organizes in a manner similar to in vivo.

[0144] As defined herein, the term "fibrous or matrix culture" refers to a three-dimensional cell culture support composed of insoluble elastic fibers or extracellular proteins self-organized into a matrix.

[0145] The transfection can be stable or transient, standard or reverse.

[0146] As disclosed herein, the composition of the present invention may comprise a plurality of different nucleic acids for co-transfection, these nucleic acids being particularly selected from: a plurality of plasmid DNAs, plasmid DNA and oligonucleotides, plasmid DNA and mRNA.

[0147] The at least one nucleic acid molecule to be transfected can be a gene encoding a protein, a protein fragment, a peptide or an antibody or its functional antigen binding region (especially its VH and / or VL chain). The protein can be selected from: a reporter protein, a fluorescent protein, an enzyme, a structural protein, a receptor, a transmembrane protein, a therapeutic protein, a cytokine, a toxin, an oncoprotein, an anti-oncogene, a pro-apoptotic protein, an anti-apoptotic protein, a polymerase, a transcription factor and a capsid protein.

[0148] The present invention also relates to the in vitro or ex vivo use of the composition of the present invention for genome engineering, for cell reprogramming (especially for reprogramming differentiated cells into induced pluripotent stem cells (iPC)), for differentiated cells or for gene editing. Such use can be carried out in vitro or ex vivo cell culture for the production of biological preparations, for the preparation of cells for therapeutic purposes, or for the study of cell function or behavior, especially the cell expansion step after transfection, or can be carried out in vivo for therapeutic purposes in a host in need thereof.

[0149] The present invention also relates to the in vitro or ex vivo use of the composition of the present invention in the following: (i) producing biological preparations, especially biological preparations encoding recombinant proteins, peptides or antibodies; or (ii) producing recombinant viruses, such as adeno-associated viruses (AAV), lentiviruses (LV), adenoviruses, oncolytic viruses or baculoviruses, wherein the composition comprises multiple nucleic acid molecules for co-transfection, such as multiple plasmids; or (iii) producing viral or virus-like particles, wherein the composition comprises multiple nucleic acid molecules for co-transfection, such as multiple plasmids.

[0150] Therefore, the present invention also relates to a method for producing the following: (i) a biological preparation, especially a biological preparation encoding a recombinant protein, peptide or antibody; or (ii) a recombinant virus, such as adeno-associated virus (AAV), lentivirus (LV), adenovirus, oncolytic virus or baculovirus, wherein the composition of the present invention comprises a variety of nucleic acid molecules for co-transfection; or (iii) viral or virus-like particles, wherein the composition of the present invention comprises a variety of nucleic acid molecules for co-transfection. As defined herein, the term "biological preparation" refers to a protein or nucleic acid or a combination thereof, a living body (such as a cell or virus), a cell compartment, an organoid and a tissue.

[0151] In a specific embodiment of the invention, the method is for producing AAV or LV, and the composition comprises (i) at least compound 1.42, and (ii) an acceptable excipient, buffer, cell culture medium or transfection medium.

[0152] In a specific embodiment of the invention, the method is for producing LV, and the composition comprises (i) at least compound 1.57, and (ii) an acceptable excipient, buffer, cell culture medium or transfection medium.

[0153] In a specific embodiment of the present invention, the in vitro or ex vivo use of the composition of the present invention or the method is used to produce recombinant viruses, the composition comprising a variety of expression vectors (such as plasmid vectors) for transfection in adherent or suspended cells (such as HEK293 and derivative cells, HeLa, BHK-21, A549 or insect cells), wherein the vector (especially the plasmid) is a construct expressing viral structural sequences and a transfer vector genome for virus or virus-like production and optionally expressing a molecule of interest encoded by the transfer vector genome.

[0154] In a specific embodiment of the present invention, the recombinant virus is used for in vivo use in cell therapy or gene therapy.

[0155] In a specific embodiment of the present invention, the present invention relates to the in vitro or ex vivo use of a composition of the present invention for transfecting at least one nucleic acid molecule into stem cells, said composition comprising (i) compound 1.42, and (ii) an acceptable excipient, buffer, cell culture medium or transfection medium.

[0156] In a specific embodiment of the present invention, the present invention relates to the in vitro or ex vivo use of a composition of the present invention for transfecting at least one nucleic acid molecule into neuronal cells, the composition comprising (i) compound 1.65 or compound 1.60, and (ii) an acceptable excipient, buffer, cell culture medium or transfection medium.

[0157] In a specific embodiment of the present invention, the present invention relates to the in vitro or ex vivo use of the composition of the present invention in producing recombinant viruses (such as adeno-associated virus (AAV) or lentivirus (LV)), wherein the composition comprises (i) at least one compound selected from compounds 1.42 and 1.57, and (ii) an acceptable excipient, buffer, cell culture medium or transfection medium. Preferably, the composition comprising compound 1.42 is used to produce AAV; the composition comprising a compound selected from compounds 1.42 and 1.57 is used to produce LV.

[0158] Unless stated otherwise, all of the above embodiments may be combined together. Thus, features described in the context of separate embodiments may be combined in a single embodiment.

[0159] Other features and advantages of the present invention will become apparent from the following embodiments and will also be described in the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0160] Figure 1. The 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 DNA / μL compound.

[0161] Figure 2 . The percentage of GFP expression after transfection of Caco-2, Hep G2, MDCK and MCF-10A with the compound of Example 4. The ratios 1:3 and 1:4 indicate the ratio of μg DNA / μL compound.

[0162] Figure 3 .use (0% grafting) and the percentage of GFP expression after transfection of HepG2 cells with compounds 1.57, 1.64, 1.42, 1.72 and 1.56, which contain 7%, 14%, 22%, 25% and 30% grafting degree of 2-methyl-benzimidazole to linear PEI 22kDa, respectively.

[0163] Figure 4 . Using compounds 1.41, 2.03, 2.05, 1.42, 2.08 and as commercial reference The percentage of GFP expression after 3000 transfection of primary dermal fibroblasts (HPDF) and primary endothelial cells (HUVEC).

[0164] Figure 5. Transfection of primary rat cortical neurons (RCN) and primary rat hippocampal neurons (RHN).

[0165] Figure 6 . AAV-2 was produced from suspension HEK-293T cells. AAV-2 vectors expressing the GFP reporter gene were produced in HEK-293T cells grown in suspension in FreeStyle F17 medium. Cells were seeded and cultured for 3 days and then incubated with 1:2 or 1:3 μg DNA / μL reagent. or various compounds were transfected with three plasmids (pAAV-RC2 vector expressing Rep and Cap; pHelper vector expressing Adeno E2A, Adeno E4, and Adeno VA cofactors; and pAAV-GFP control vector expressing GFP controlled by the CMV promoter). 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 transfection.

[0166] Figure 7.Generate lentiviral particles from suspension HEK-293T cells. Lentivirus expressing the GFP reporter gene was produced in HEK-293T cells grown in suspension in FreeStyle F17 medium. Cells were seeded and cultured for 3 days and then incubated with 1:2 μg total DNA / μL reagent. Or various compounds were transfected with four plasmids. Lentivirus titers (transduction units, TU / mL) were measured 72 hours after transfection.

[0167] Figure 8 .Gel electrophoresis of genome editing in HEK293 cells after transfection of plasmid CRISPR Cas9 targeting the HRPT-1 gene (plasmid p38285) with compound 1.42 is shown. Two days after transfection, genomic DNA was extracted and the HPRT-1 focus was targeted by PCR amplification. After digestion by T7 endonuclease I, the PCR product was electrophoresed on a 2% agarose gel and stained with ethidium bromide. The Cas9-induced cut HPTR-1 bands (650 and 430bp) and the uncut HPTR-1 bands (1083bp) were visualized and quantified on the gel, and the genome editing efficiency (INDEL%) was then determined. The INDEL% of plasmid p38285 was 33.48+ / -7.08%, with no INDEL events detected for plasmid pCONTROL.

[0168] Fig. 9 Transfection efficiency of human mesenchymal stem cells (hMSCs) with plasmid pCMV-EGFP and compound 1.42. A) hMSCs were observed by phase contrast and fluorescence microscopy 24 hours after transfection with 400ng pCMV-EGFP and 2μL compound 1.42. B) GFP expression analysis by flow cytometry 24 hours after transfection of hMSCs with 400ng pCMV-EGFP and 0.4, 0.6 and 0.8μL compound 1.42, with 500ng pCMV-EGFP and 0.5, 0.75 and 1μL compound 1.42, and with 500ng pCMV-EGFP and 0.75 and 1.5μL Lipofectamine 3000 reagent.

[0169] Fig.10 .Chemical structure of the compound of general formula (II).

[0170] Fig.11 . The percentage of GFP expression after transfection of Hep G2 cells with the compound of Example 10. The ratios 1:3 and 1:4 indicate the ratio of μg DNA / μL compound.

[0171] Fig.12. Production of AAV-2 from suspension HEK-293T cells. AAV-2 vectors expressing the GFP reporter gene were produced in HEK-293T cells grown in suspension in FreeStyle F17 medium. The cells were seeded and cultured for 3 days and then Or various compounds were transfected by 3 plasmids (pAAV-RC2 vector expressing Rep and Cap from Cell BIOLABS, INC., pALD-X80; helper vector expressing Adeno E2A, Adeno E4 and Adeno VA helper from ALDEVRON; and pAAV-GFP control vector expressing GFP controlled by CMV promoter from Cell BIOLABS, INC.). AAV titer (transducing unit, TU / mL) was determined 72 hours after transfection. The results are expressed as the optimal ratio of μg DNA / μL compound (for 1.42, 1.73, 1.74, 1.76, 1.80, 1.75, the ratio is 1:2, and for BPEI 25K, BPEI 10K, PAA, PVA, PLL, 1.78 and 1.77, the ratio is 1:5) Relative AAV-2 transducing units / mL (TU / mL) compared to transfection. Example

[0172] Experimental Section

[0173] Materials and methods

[0174] Cell culture

[0175] Caco-2 ( HTB-37 TM ) Human colon 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.

[0176] MCF 10A ( CRL-10317 TM ) Human mammary epithelial cells were grown in medium supplemented with SingleQuots TM Supplements and growth factors (Lonza) and 100 ng / ml cholera toxin in MEBM (Lonza).

[0177] Hep G2 ( HB-8065 TM ) 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.

[0178] MDCK ( CCL-34 TM ) Madin-Darby canine kidney epithelial cells were grown in MEM (Ozyme) containing 10% FBS supplemented with 2 mM glutamine and 100 U / mL penicillin and 100 μg / mL streptomycin.

[0179] 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 an atmosphere of 5% CO2 in air.

[0180] Transfection Assay (96-well Format)

[0181] The day before transfection, Caco-2, MCF 10A, Hep G2 and MDCK cells were seeded in 125 μ L complete medium respectively with 10000, 25000, 25000, 10000 cells per well (96 well format), and at 37 ℃, containing 5% CO2 in air atmosphere, incubated. On the transfection day, 200ng pCMV-EGFPLuc DNA (Clontech) was added into 20 μ L OPTIMEM (Thermo Fisher), vortex mixed, and incubated 5 minutes at room temperature (rt). Then, 0.6 or 0.8 μ L compound of general formula (II) (under 7.5mM nitrogen concentration) was added to the diluted DNA, vortex mixed, and incubated 10 minutes at room temperature. Transfection DNA solution (20 μ L) was added in the well, and the plate was incubated 24 hours at 37 ℃, containing 5% CO2 in air atmosphere.

[0182] For GFP expression analysis, one day after transfection, the cell culture medium was removed and 50 μL of trypsin-EDTA (1×, Lonza) was added per well and the plate was incubated for 5 minutes at 37° C. 150 μL of complete culture medium was added to neutralize the trypsin and GFP expression (2000 events) was analyzed by flow cytometry (Exc 488 nm, Em 520 nm) using a Guava easyCyte 6HT cell counter (Millipore).

[0183] Transfection assay of primary cells

[0184] HUVEC human umbilical vein endothelial cells (Promocell) are seeded in the endothelial cell growth medium containing supplementMix (Promocell) in 500 μ L with 20000 cells per well (24-well plate format), and at 37 ℃, containing 5%CO2, incubated in an air atmosphere. After three days, complete culture medium is removed, and 500 μ L of DMEM supplemented with 2% FBS is replaced. Then, 500 ng pCMV-EGFPLuc DNA (Clontech) is added in 50 μ L NaCl buffer, vortex mixed, and incubated at room temperature for 5 minutes. Then, 1.5 μ L of the compound of general formula (II) (under 7.5 mM nitrogen concentration) is added to the diluted DNA, vortex mixed, and incubated at room temperature for 10 minutes. Transfection DNA solution (50 μ L) is added in the hole, and the plate is incubated at 37 ℃, containing 5%CO2, in an air atmosphere. After 2 hours of incubation, the medium was removed and replaced with 500 μL of endothelial cell growth medium containing supplementMix (Promocell), and the cells were incubated at 37° C. in an atmosphere of 5% CO 2 in air. One day after transfection, GFP expression was analyzed by flow cytometry.

[0185] Primary human dermal fibroblasts were obtained from Pr. Stéphane Viville (Centre Hospitalier Universitaire, Strasbourg, France). Cells 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 an atmosphere of 5% CO 2 in air. For transfection, cells were seeded at 40,000 cells per well (24-well plate format, pre-coated with 0.1% gelatin) in 500 μL of DMEM 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 and incubated at 37° C. in an atmosphere of 5% CO 2 in air for 24 hours. Then, 500ng pCMV-EGFPLuc DNA (Clontech) is added in 50 μL NaCl buffer, vortex mixed, and incubated at room temperature for 5 minutes. Then, 1.5 μL compound of general formula (II) (under 7.5mM nitrogen concentration) is added on the diluted DNA, vortex mixed, and incubated at room temperature for 10 minutes. Transfection DNA solution (50 μL) is added in hole, and plate is incubated at 37 ℃, containing 5%CO in air atmosphere. After incubation for 4 hours, culture medium is removed, and replaced with 500 μL complete DMEM, and cells are incubated at 37 ℃, containing 5%CO in air atmosphere. One day after transfection, GFP expression is analyzed by flow cytometry.

[0186] Primary rat cortical neurons (RCN, ThermoFisher) were seeded with 100,000 cells per well (48-well plate format, pre-coated with D-polylysine (Sigma)) in 0.5 mL complete neurobasal medium (ThermoFisher) supplemented with B27 supplement (ThermoFisher) and 0.5 mM glutamine, and the cells were incubated at 37° C. in an air atmosphere containing 5% CO . Every two days, half of the complete medium volume was replaced. After 4 days, 150 ng pCMV-EGFPLuc DNA (Clontech) was added to 25 μL OPTIMEM, vortexed, and incubated at room temperature for 5 minutes. Then, 0.15 μL of the compound of formula (II) (at 7.5 mM nitrogen concentration) was added to the diluted DNA, vortexed, and incubated at room temperature for 10 minutes. 250 μL of complete medium was removed and the prepared DNA solution (25 μL) was added to the wells and the plates were incubated at 37°C in an atmosphere of 5% CO2 in air. After incubation at 37°C in an atmosphere of 5% CO2 in air for 4 hours, 250 μL of complete medium was added to each well and the cells were incubated at 37°C in an atmosphere of 5% CO2 in air. The cells were also incubated with 150 ng of OPTIMEM according to the recommended commercial protocol. 3000 (ratio 1μg:1.5μL), 2000 (ratio 1μg:4μL) and 150mM NaCl One day after transfection, ZOE TM The expression of GFP was observed using a fluorescent cell imager (Biorad).

[0187] Recombinant virus production

[0188] HEK-293T( CRL-3216 TM ): 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.

[0189] For adherent cells, HEK-293T cells were plated at 145 cm 2 5x10 in Petri dish 6 Cells were seeded 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 and incubated at 37° C. in an atmosphere of 5% CO 2 in air.

[0190] AAV-2 was produced in HEK-293T cells by co-transfection of three plasmids (pAAV-RC2 vector expressing Rep and Cap; pHelper vector expressing Adeno E2A, Adeno E4 and Adeno VA auxiliary factors; and pAAV-GFP control vector expressing GFP controlled by CMV promoter). Transfection complexes were prepared with pAAV-RC2, pHelper and pAAV-GFP at a ratio of 2:2:1, respectively (10 μg of total DNA per Petri dish). The plasmids were diluted in OPTIMEM with a total volume of 1.5 mL. Then, 20 or 30 μL of the compound was added to the diluted DNA, vortexed and incubated at room temperature for 10 minutes. The transfection complex was added to the wells, and the plate was incubated for 72 hours at 37°C in an atmosphere of 5% CO2 in air.

[0191] For suspension cells, HEK-293T cells were plated at 1x10 6 10 cells / mL were inoculated 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). The cells were incubated at 37°C in an atmosphere of 8% CO2 in air for 24 hours with agitation (130 rpm). Plasmids (pAAV-GFP–pAAV-RC2–pHelper in a ratio of 2:2:1) were diluted in 3 mL of FreeStyle F17. Compounds were then added to the diluted DNA (at a ratio of 2 or 3 μL per μg DNA), vortexed, and incubated at room temperature for 10 minutes. Transfection complexes were added to the cells (1×10 6 2 μg DNA per cell) and the plates were incubated at 37° C. in an atmosphere of 8% CO 2 in air under agitation (130 rpm) for 72 hours.

[0192] ViraSafe containing pRSV-REV packaging vector, pCgpV packaging vector and pCMV-VSV-G envelope vector TM Lentiviral particles were produced using the Pantropic lentiviral packaging system (Catalog No. VPK-20, CELL BIOLABS INC.). The pLenti6.3 / V5-GW / EmGFP expression control vector was from Thermo Fisher.

[0193] HEK-293T cells were cultured at 1x10 6 125 mL Erlenmeyer flasks (Corning) were inoculated with 27 mL FreeStyle F17 supplemented with 4% glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, and 0.1% Pluronic. The cells were incubated at 37°C in an atmosphere of 8% CO2 in air for 24 hours under agitation (130 rpm). Plasmids (pRSV-REV–pCgpV–pCMV-VSV-G-pLenti6.3 in a ratio of 1:1:1:3) were diluted in 3 mL FreeStyle F17. Compounds were then added to the diluted DNA (at a ratio of 2 μL per μg DNA), vortexed, and incubated at room temperature for 10 minutes. The transfection complexes were added to the cells (1×10 6 2 μg DNA per cell) and the plates were incubated at 37° C. in an atmosphere of 8% CO 2 in air under agitation (130 rpm) for 72 hours.

[0194] After infection of licensed HT 1080 cells for lentiviral vectors and HEK-293T cells for AAV-2 vectors in 96-wells, and in the presence of polybrene (8 μg / mL), the transduction units (TU / mL) were determined by using viral vectors expressing the GFP reporter gene. GFP expression was analyzed by cell counting 72 hours after transduction to determine the transduction units.

[0195] CRISPR Cas9 DNA transfection

[0196] HEK293 (ECACC 85120602) human embryonic epithelial kidney cells were grown in Eagle MEM medium containing 10% FBS supplemented with 2mM glutamine, 0.1mM non-essential amino acids, 200U / mL penicillin and 200μg / mL streptomycin. One day before transfection, 12500 cells were added to each well (96-well plate format) in 125μL complete medium, and the plate was incubated at 37°C, in an air atmosphere containing 5% CO2 for 24 hours.

[0197] The plasmid pSpCas9 BB-2A-GFP (9.3 kb) from GenScript (Leiden, Netherlands) used for transfection experiments expresses a version of the Streptococcus pyogenes Cas9 protein (CRISPR-associated protein 9) with N- and C-terminal nuclear localization signals (NLS) controlled by the CBh promoter. The plasmid contains G(N)20 gRNA (guide RNA) and a gRNA scaffold sequence controlled by the U6 promoter. The G(N)20 gRNA is designed to target the HPRT-1 (hypoxanthine phosphoribosyltransferase) human gene at sequence position 38285 (the targeted cleavage site of CRISPR Cas9), thereby generating plasmid p38285. The second G(N)20 gRNA is designed to generate plasmid pCONTROL, which cannot trigger CRISPR events in human cells.

[0198] On the day of transfection, 100 ng of plasmid p38285 or pCONTROL was added to 12 μL OPTIMEM. Then, 0.1 μL of compound 1.42 was added to the diluted plasmid at 7.5 mM amine concentration, vortexed, and incubated at room temperature for 10 minutes. The composite plasmid was added to the wells, and the plate was incubated at 37°C in an atmosphere of 5% CO2 in air.

[0199] Two days after transfection, the culture medium was removed and the cells were washed with PBS. 50 μL of QuickExtract TM Genomic DNA was isolated using DNA Extraction Solution 1.0 (Epicentre), followed by incubation at 65°C for 6 min, then at 98°C for 2 min and storage at 4°C. Hot Start Hi-Fi 2X Master Mix (New England ) was used to amplify HPRT-1 targeted genomic DNA (250 ng). TMThe following PCR conditions were used in a thermal cycler (Biorad): 1) incubation at 95°C for 5 minutes, 2) 35 cycles (98°C for 20 seconds, 68°C for 15 seconds, 72°C for 30 seconds), 3) incubation at 72°C for 2 minutes, and then stored at 4°C. 15 μL of amplified PCR DNA (250 ng) was combined with 1.5 μL 10X NEBuffer2 (NEB) and 1.5 μL nuclease-free (total volume 18 μL) and denatured, then reannealed at 95°C for 10 minutes by thermal cycling, reaching 95 to 85°C at -2°C / sec; reaching 85 to 25°C at -0.3°C / sec. The reannealed DNA was incubated at 37°C for 15 minutes with 1 μL T7 endonuclease I (10 U / μl, NEB). 19 μL of T7 endonuclease reaction was mixed with 2 μL of loading buffer and analyzed on a 2% TAE agarose gel loaded with a 100 pb DNA ladder (New England Biolabs, NY) in a fast loading column. ) were subjected to electrophoresis at 100 V for 45 minutes in the presence of . The gel was stained with ethidium bromide for 30 minutes. The Cas9-induced cleavage bands (827 and 256 bp) and the uncut band (1083 bp) were visualized on a G:Box transilluminator (Syngene) and quantified using GeneTools software. INDEL% was calculated using the following formula: INDEL% = 100 * [1-(1-((strength of the cut strip) / (strengths of the cut and uncut strips)))].

[0200] Transfection assay of stem cells

[0201] Primary human mesenchymal stem cells (hMSC, Reference PT-2501, Lonza) were grown in MSC basal medium (Lonza) supplemented with GA-1000 (MSC growth medium, Lonza), 2 mM glutamine, and 100 U / mL penicillin and 100 μg streptomycin at 37°C in an atmosphere of 5% CO2 in air.

[0202] HMSC is seeded in 500 μ L MSC complete growth medium (Lonza) with 12000 cells per well (24-well plate format), and is incubated at 37 ° C, containing 5% CO2 in an air atmosphere. After three days, the complete medium is removed and replaced with 500 μ L MSC basal medium. Then, pCMV-EGFP DNA (Clontech) is added in 50 μ L NaCl buffer, vortexed, and incubated at room temperature for 5 minutes. Then, compound 1.42 (under 7.5mM nitrogen concentration) is added to the diluted DNA, vortexed, and incubated at room temperature for 10 minutes. The prepared DNA solution (50 μ L) is added to the hole, and the plate is incubated at 37 ° C, containing 5% CO2 in an air atmosphere. After incubation for 4 hours, the culture medium is removed, and replaced with 500 μ L MSC complete growth medium, and the cells are incubated at 37 ° C, containing 5% CO2 in an air atmosphere. One day after transfection, the cells were analyzed by flow cytometry or by using ZOE TM The expression of GFP was observed using a fluorescent cell imager (Biorad).

[0203] Example 1. General procedure for preparing grafted polymers

[0204]

[0205] Step 1: N-alkylation of heterocycle

[0206] Under argon, the corresponding heterocycle (1 equivalent) and DMF (2 ml / mmol raw material) are added into a drying round-bottom flask. The solution is cooled to 0 ° C, and sodium hydride (60% dispersed in mineral oil, 1.2 equivalents) is added by portion. The mixture is slowly warmed to room temperature over 1 hour. Then, the corresponding ester is added dropwise, and the reaction is stirred at room temperature for 4-12 hours. The mixture is quenched by adding water (10 mL / 1 mL DMF), and the aqueous layer is extracted with EtOAc. (5x2 mL / 1 mL DMF). The combined organic extracts are washed with brine and dried over anhydrous MgSO4. After filtration, the solvent is removed in vacuo, and the resulting oil is purified by column chromatography (heptane containing 20 to 50% EtOAc).

[0207] Step 2: Saponification of the acid moiety

[0208] To a solution of the ester in EtOH (2 mL / mmol of ester) was added dropwise a 5M NaOH solution (0.2 mL / mmol of ester), and the mixture was stirred at room temperature overnight. The solvent was then removed in vacuo, and the residue was purified by column chromatography on SiO2 using DCM + 1% AcOH containing 5% MeOH or using H2O containing 0 to 100% acetonitrile.

[0209] Step 3: Grafting

[0210] Water (4mL / mmol raw material) containing cationic polymer (1 equivalent) is added in a round-bottom flask, followed by N-methylmorpholine or NMM (2 equivalents). Carboxylate (0.3-1 equivalent) is added, followed by MeOH (polymer of 16mL / mmol). After stirring for 10 minutes, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride or DMTMM (0.6-2 equivalent) is added, and the mixture is stirred at room temperature for 12-24 hours. Then, MeOH is removed in vacuo, water (4mL / mmol raw material) is added, followed by 3M HCl solution (1mL / mmol raw material). Residue is purified using dialysis cassettes in a 50mM HCl bath, or residue is purified by UF using Amicon Ultra15 with HCl 50mM.

[0211] Step 4: Procedure for grafting PLL (22K, Sigma-Aldrich) with EDCI and NHS.

[0212] At room temperature and in a microwave vial, poly-L-lysine (1 equivalent) is dissolved in 25-mM MES (pH 6.5) buffer, to which 4-imidazoleacetic acid (sodium salt) (0.75 to 1.5 equivalent) is added. This solution is used to dissolve EDCI (1.5 to 3 equivalents). NHS (1 equivalent) is dissolved in MES buffer and added immediately to the poly-L-lysine solution. The bottle is sealed and stirred at room temperature for 24 hours. The product is then purified by dialysis with water, or the product is purified with water on Amicon ultra 15.

[0213] Example 2. Synthesis of: (i) compounds of formula (II) of the present invention (benzimidazole, benzopyrazole and benzotriazole derivatives), namely compounds 1.01 to 1.72, 1.74 to 1.77, 1.79 and 2.01 to 2.18; and (ii) imidazole derivatives, such as compounds 1.73, 1.78 and 1.80

[0214] -Synthesis of Product 1.01

[0215] Intermediate 1.01a was prepared analogously to general procedure 1 (Example 1). Yield = 60%; m = 2.30 g; 1 H NMR (400 MHz, CHLOROFORM-d) δ 7.94 (s, 1H), 7.81–7.72 (m, 1H), 7.43–7.36 (m, 1H), 7.26 (ddd, J = 13.1, 7.5, 4.9 Hz, 2H), 4.24 (t, J = 7.0 Hz, 2H), 4.09 (q, J = 7.4 Hz, 2H), 2.28 (t, J = 7.0 Hz, 2H), 2.16 (p, J = 7.0 Hz, 2H), 1.20 (t, J = 7.1 Hz, 3H).

[0216] Intermediate 1.01b was prepared analogously to general procedure 2 (Example 1). Yield = 45%; m = 1.00 g; 1 H NMR (400 MHz, methanol-d4) δ 8.21 (d, J = 2.4 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.33 (dt, J = 17.3, 7.5 Hz, 2H), 4.37 (t, J = 7.4 Hz, 2H), 2.35 (t, J = 7.4 Hz, 2H), 2.18 (p, J = 7.4 Hz, 2H).

[0217] The product 1.01 was prepared similarly to general procedure 3 (Example 1). Yield = 94%; m = 117 mg; 1 H NMR (D2O)δ: 1 H NMR (400 MHz, deuterium oxide) δ 9.29–8.97 (m, 1H), 7.96–7.17 (m, 4H), 4.46 (d, J = 42.4 Hz, 2H), 3.45 (s, 39H), 2.35 (dd, J = 135.3, 58.1 Hz, 4H).

[0218] -Synthesis of Product 1.02

[0219] Intermediate 1.02a was prepared analogously to general procedure 1 (Example 1). Yield = 73%; m = 857 mg; 1 H NMR (400 MHz, CHLOROFORM-d) δ 9.05 (s, 1H), 8.97 (s, 1H), 8.23 ​​(s, 1H), 4.36 (t, J = 7.1 Hz, 2H), 4.05 (q, J = 7.2 Hz, 2H), 2.31 (m, 2H), 2.19 (m, 2H), 1.17 (t, J = 7.2 Hz, 3H).

[0220] Intermediate 1.02b was prepared analogously to general procedure 2 (Example 1). Yield = 67%; m = 48 mg; 1 H NMR (400 MHz, deuterium oxide) δ 9.16 (s, 1H), 8.95 (s, 1H), 8.61 (s, 1H), 4.46–4.37 (m, 2H), 2.26–2.12 (m, 4H).

[0221] The product 1.02 was prepared similarly to general procedure 3 (Example 1). Yield = 85%; m = 12 mg; 1 H NMR (400 MHz, deuterium oxide) δ 9.57–7.98 (m, 3H), 4.60–4.16 (m, 2H), 4.15–3.05 (m, 24H), 2.92–1.71 (m, 4H).

[0222] -Synthesis of Product 1.03

[0223] Intermediate 1.03a was prepared analogously to general procedure 2 (Example 1). Yield = 100%; m = 303 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.86 (s, 1H), 8.77 (s, 1H), 4.25 (t, J = 7.3 Hz, 2H), 2.67 (s, 3H), 2.21 (m, 2H), 2.10–2.01 (m, 2H).

[0224] Intermediate 1.03b was prepared analogously to general procedure 1 (Example 1). Yield = 26%; m = 327 mg; 1 H NMR (400 MHz, CHLOROFORM-d) δ 8.96 (s, 1H), 8.89 (s, 1H), 4.30 (dd, J = 7.8, 6.8 Hz, 2H), 4.08 (q, J = 7.2 Hz, 2H), 2.69 (s, 3H), 2.38 (t, J = 6.9 Hz, 2H), 2.21–2.09 (m, 2H), 1.20 (t, J = 7.1 Hz, 3H).

[0225] The product 1.03 was prepared similarly to the general procedure 3 (Example 1). Yield = 88%; m = 13 mg; 1 H NMR (400 MHz, deuterium oxide) δ 9.20–8.32 (m, 2H), 4.51–2.93 (m, 21H), 2.88–1.68 (m, 7H).

[0226] -Synthesis of Product 1.04

[0227] Intermediate 1.04a was prepared analogously to general procedure 1 (Example 1). Yield = 51%; m = 597 mg; 1 H NMR (400MHz, chloroform-d) δ8.41(dd,J=4.8,1.5Hz,1H),8.21(s,1H),8.09(dd,J=8.1,1.5Hz,1H),7.29–7 .22(m,1H),4.40(t,J=6.8Hz,2H),4.10(q,J=7.1Hz,2H),2.42–2.17(m,4H),1.22(t,J=7.1Hz,3H).

[0228] Intermediate 1.04b was prepared analogously to general procedure 2 (Example 1). Yield = 76%; m = 441 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.21 (s, 1H), 8.18 (dd, J = 4.9, 1.4 Hz, 1H), 7.94 (dd, J = 8.1, 1.4 Hz, 1H), 7.22 (dd, J = 8.1, 4.9 Hz, 1H), 4.17 (t, J = 7.0 Hz, 2H), 2.14 (m, 2H), 2.05 (m, 2H).

[0229] The product 1.04 was prepared similarly to the general procedure 3 (Example 1). Yield = 78%; m = 10 mg; 1 H NMR (400 MHz, deuterium oxide) δ 9.46–7.11 (m, 4H), 4.64–4.15 (m, 1H), 4.14–1.88 (m, 41H).

[0230] -Synthesis of Product 1.05

[0231] Intermediate 1.05a was prepared analogously to general procedure 1 (Example 1). Yield = 77%; m = 783 mg; 1 H NMR (400 MHz, CHLOROFORM-d) δ 8.33 (d, J = 3.7 Hz, 1H), 7.71 (d, J = 3.0 Hz, 1H), 4.24 (m, 2H), 4.11 (m, 2H), 3.53 (s, 6H), 2.31 (m, 2H), 2.19 (m, 2H), 1.23 (m, 3H).

[0232] Intermediate 1.05b was prepared analogously to general procedure 2 (Example 1). Yield = 68%; m = 311 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.88 (s, 1H), 7.87 (s, 1H), 4.05 (t, J = 7.2 Hz, 2H), 3.19 (s, 6H), 2.19–2.11 (m, 2H), 2.01 (dt, J = 8.4, 6.6 Hz, 2H).

[0233] The product 1.05 was prepared similarly to the general procedure 3 (Example 1). Yield = 74%; m = 13 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.62–7.38 (m, 2H), 4.42–1.55 (m, 17H).

[0234] -Synthesis of Product 1.06

[0235] Intermediate 1.06a was prepared analogously to general procedure 1 (Example 1). Yield = 44%; m = 810 mg; 1 H NMR (400 MHz, chloroform-d) δ8.33 (s, 1H), 7.80 (s, 1H), 6.47–6.06 (m, 2H), 4.26 (t, J = 7.0 Hz, 2H), 4.10 (q, J = 7.1 Hz, 2H), 2.32 (td, J = 6.9, 1.0 Hz, 2H), 2.25–2.11 (m, 2H), 1.21 (t, J = 7.1 Hz, 4H)

[0236] Intermediate 1.06b was prepared analogously to general procedure 2 (Example 1). Yield = 99%; m = 710 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.94 (s, 1H), 7.91 (s, 1H), 4.03 (t, J = 7.1 Hz, 2H), 2.07 (ddd, J = 7.9, 7.0, 1.2 Hz, 2H), 2.00–1.89 (m, 2H).

[0237] The product 1.06 was prepared similarly to the general procedure 3 (Example 1). Yield = 99%; m = 13 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.78–7.72 (m, 2H), 4.33–1.07 (m, 38H).

[0238] -Synthesis of Product 1.07

[0239] Intermediate 1.07a was prepared analogously to general procedure 1 (Example 1). Yield = 61%; m = 2.7 g; 1 H NMR (400 MHz, CHLOROFORM-d) δ 7.70 (dq, J = 7.0, 3.9, 3.3 Hz, 1H), 7.33 (dt, J = 5.8, 2.5 Hz, 1H), 7.30–7.20 (m, 2H), 4.49–4.40 (m, 2H), 4.18–4.07 (m, 2H), 2.87–2.78 (m, 2H), 2.67 (d, J = 2.2 Hz, 3H), 1.21 (td, J = 7.2, 2.2 Hz, 3H).

[0240] Intermediate 1.07b was prepared analogously to general procedure 2 (Example 1). Yield = 17%; m = 400 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.71 (d, J = 7.4 Hz, 1H), 7.62 (d, J = 7.3 Hz, 1H), 7.49 (dd, J = 7.3, 3.8 Hz, 2H), 4.60 (q, J = 5.8 Hz, 2H), 2.93 (q, J = 5.8 Hz, 2H), 2.81 (d, J = 2.8 Hz, 3H).

[0241] The product 1.07 was prepared similarly to general procedure 3 (Example 1). Yield = 96%; m = 134 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.03–7.09 (m, 4H), 4.34–2.28 (m, 32H).

[0242] -Synthesis of Product 1.08

[0243] Intermediate 1.08a was prepared analogously to general procedure 1 (Example 1). Yield = 73%; m = 3.0 g; 1 H NMR (400 MHz, CHLOROFORM-d) δ 7.76–7.70 (m, 1H), 7.26 (tdd, J = 9.4, 6.6, 3.4 Hz, 3H), 4.82 (s, 2H), 4.25 (q, J = 7.2 Hz, 2H), 2.61 (d, J = 1.6 Hz, 3H), 1.33–1.24 (m, 3H).

[0244] Intermediate 1.08b was prepared analogously to general procedure 2 (Example 1). Yield = 27%; m = 700 mg; 1 H NMR (D2O)δ:1 H NMR (400 MHz, deuterium oxide) δ 7.63 (ddt, J = 18.8, 6.4, 2.9 Hz, 2H), 7.50 (dq, J = 6.5, 3.5 Hz, 2H), 5.15 (d, J = 2.7 Hz, 2H), 2.74 (d, J = 2.7 Hz, 3H).

[0245] The product 1.08 was prepared similarly to general procedure 3 (Example 1). Yield = 100%; m = 130 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.88–7.28 (m, 4H), 5.71–5.17 (m, 2H), 4.12–3.12 (m, 45H), 2.89–2.41 (m, 3H).

[0246] -Synthesis of Product 1.09

[0247] The product 1.09 was prepared similarly to the general procedure 3 (Example 1). Yield = 100%; m = 150 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.87–7.21 (m, 4H), 4.15–2.49 (m, 29H).

[0248] -Synthesis of Product 1.10

[0249] Intermediate 1.10a was prepared analogously to general procedure 1 (Example 1). Yield = 28%; m = 610 mg; 1 H NMR (400 MHz, CHLOROFORM-d) δ 7.74–7.66 (m, 1H), 7.34–7.27 (m, 1H), 7.31–7.21 (m, 2H), 4.20–4.08 (m, 4H), 3.43 (td, J = 6.6, 1.5 Hz, 0H), 2.64 (d, J = 1.5 Hz, 3H), 2.35 (t, J = 7.2 Hz, 2H), 1.98–1.67 (m, 5H), 1.32–1.19 (m, 3H).

[0250] Intermediate 1.10b was prepared analogously to general procedure 2 (Example 1). Yield = 71%; m = 386 mg; 1H NMR (400 MHz, methanol-d4) δ 7.82 (d, J = 7.4 Hz, 1H), 7.76–7.69 (m, 1H), 7.59–7.49 (m, 2H), 4.45 (t, J = 7.5 Hz, 2H), 2.85 (d, J = 2.2 Hz, 3H), 2.40 (t, J = 7.2 Hz, 2H), 1.96 (p, J = 8.0, 7.3 Hz, 2H), 1.74 (q, J = 7.7 Hz, 2H).

[0251] The product 1.10 was prepared analogously to general procedure 3 (Example 1). Yield = 98%; m = 170 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.90–7.00 (m, 4H), 4.42–3.02 (m, 14H), 2.84–2.56 (m, 2H), 2.55–2.11 (m, 2H), 1.98–1.25 (m, 6H).

[0252] -Synthesis of product 1.11

[0253] Intermediate 1.11a was prepared analogously to general procedure 1 (Example 1). Yield = 30%; m = 1.44 g; 1 H NMR (400 MHz, CHLOROFORM-d) δ 7.76–7.67 (m, 1H), 7.30 (dd, J = 6.3, 3.0 Hz, 1H), 7.25 (dd, J = 6.2, 3.1 Hz, 2H), 4.13 (p, J = 7.3 Hz, 4H), 2.62 (s, 3H), 2.31 (t, J = 7.4 Hz, 2H), 1.84 (p, J = 7.5 Hz, 2H), 1.69 (p, J = 7.5 Hz, 2H), 1.48–1.36 (m, 2H), 1.26 (t, J = 7.1 Hz, 3H).

[0254] Intermediate 1.11b was prepared analogously to general procedure 2 (Example 1). Yield = 75%; m = 970 mg; 1 H NMR (400MHz, methanol-d4) δ7.57(d,J=7.6Hz,1H),7.50(d,J=7.7Hz,1H),7.27(p,J=7.4Hz,2H),4.24(t,J=7.4Hz,2H), 2.63(d,J=2.2Hz,3H), 2.30(t,J=7.4Hz,2H), 1.85(p,J=7.8Hz,2H), 1.67(p,J=7.5Hz,2H), 1.43(p,J=7.8Hz,2H).

[0255] The product 1.11 was prepared analogously to general procedure 3 (Example 1). Yield = 100%; m = 206 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.80–7.24 (m, 4H), 4.43–3.03 (m, 13H), 2.83–2.50 (m, 3H), 2.45–1.97 (m, 2H), 1.91–1.05 (m, 6H).

[0256] -Synthesis of Product 1.12

[0257] Intermediate 1.12a was prepared analogously to general procedure 1 (Example 1). Yield = 85%; m = 3.63 g; 1 H NMR (400 MHz, CHLOROFORM-d) δ 7.78–7.69 (m, 1H), 7.36–7.28 (m, 1H), 7.26 (s, 1H), 7.24 (d, J = 4.1 Hz, 1H), 4.24–4.13 (m, 4H), 2.86 (d, J = 1.3 Hz, 3H), 2.40 (t, J = 7.0 Hz, 2H), 2.19 (dp, J = 14.1, 6.9 Hz, 2H), 1.34–1.25 (m, 3H).

[0258] Intermediate 1.12b was prepared analogously to general procedure 2 (Example 1). Yield = 36%; m = 1.19 g; 1 H NMR (400 MHz, methanol-d4) δ 7.59 (d, J = 7.2 Hz, 1H), 7.48 (d, J = 7.2 Hz, 1H), 7.30–7.19 (m, 2H), 4.26 (t, J = 7.3 Hz, 2H), 2.78 (d, J = 2.2 Hz, 3H), 2.38 (t, J = 7.1 Hz, 2H), 2.10 (p, J = 7.3 Hz, 2H).

[0259] The product 1.12 was prepared analogously to general procedure 3 (Example 1). Yield = 100%; m = 217 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.80–6.90 (m, 4H), 4.52–3.09 (m, 12H), 3.05–1.63 (m, 7H).

[0260] -Synthesis of product 1.13

[0261] Intermediate 1.13a was prepared analogously to general procedure 1 (Example 1). Yield = 33%; m = 750 mg; 1 H NMR (400MHz, methanol-d4) δ7.82–7.66(m,2H),7.53–7.36(m,2H),4.46(t,J=7.8Hz,1H),4.0 9(q,J=7.4Hz,1H), 2.46(t,J=6.9Hz,2H), 2.15(t,J=7.5Hz,2H), 1.23(t,J=7.3Hz,3H).

[0262] Intermediate 1.13b was prepared analogously to general procedure 2 (Example 1). Yield = 53%; m = 360 mg; 1 H NMR (400MHz, methanol-d4) δ7.80(t,J=6.7Hz,2H),7.52(t,J=7.9Hz,1H),7.44(t,J=7 .8Hz, 1H), 4.51 (t, J = 7.9Hz, 2H), 2.47 (t, J = 7.1Hz, 2H), 2.17 (p, J = 7.4Hz, 2H).

[0263] The product 1.13 was prepared analogously to general procedure 3 (Example 1). Yield = 100%; m = 150 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.39–5.98 (m, 4H), 4.49–0.19 (m, 17H).

[0264] -Synthesis of product 1.14

[0265] The product 1.14 was prepared analogously to general procedure 3 (Example 1). Yield = 93%; m = 237 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.94–6.59 (m, 4H), 4.62–1.17 (m, 28H).

[0266] -Synthesis of Product 1.15

[0267] Intermediate 1.15a was prepared analogously to general procedure 2 (Example 1). Yield = 91%; m = 1.19 g; 1H NMR (400 MHz, CHLOROFORM-d) δ 7.73–7.66 (m, 1H), 7.34 (dd, J = 6.7, 2.7 Hz, 1H), 7.24 (dd, J = 4.6, 2.0 Hz, 2H), 4.24–4.09 (m, 4H), 2.64 (s, 3H), 2.37 (t, J = 6.8 Hz, 2H), 2.12 (p, J = 7.1 Hz, 2H), 1.25 (td, J = 7.1, 1.5 Hz, 3H).

[0268] Intermediate 1.15b was prepared analogously to general procedure 2 (Example 1). Yield = 31%; m = 1.46 g; 1 H NMR (CDCl3) δ: 1 H NMR (400 MHz, deuterium oxide) δ 7.71–7.64 (m, 1H), 7.60 (d, J = 6.9 Hz, 1H), 7.52–7.41 (m, 2H), 4.35–4.26 (m, 2H), 2.75 (d, J = 2.6 Hz, 3H), 2.24 (td, J = 7.1, 2.6 Hz, 2H), 2.06 (q, J = 7.5 Hz, 2H).

[0269] The product 1.15 was prepared analogously to general procedure 3 (Example 1). Yield = 100%; m = 143 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.84–7.10 (m, 4H), 4.51–3.09 (m, 22H), 3.07–1.61 (m, 6H).

[0270] -Synthesis of product 1.16

[0271] Intermediate 1.16a was prepared analogously to general procedure 1 (Example 1). Yield = 75%; m = 2.4 g; 1 H NMR (400 MHz, CHLOROFORM-d) δ 7.85–7.79 (m, 1H), 7.75–7.68 (m, 2H), 7.51 (dq, J = 6.1, 3.3, 2.3 Hz, 3H), 7.44–7.22 (m, 3H), 4.33 (t, J = 7.4 Hz, 2H), 4.05 (q, J = 7.1 Hz, 2H), 2.25 (t, J = 6.8 Hz, 2H), 2.13 (p, J = 7.0 Hz, 2H), 1.27–1.17 (m, 3H).

[0272] Intermediate 1.16b was prepared analogously to general procedure 2 (Example 1). Yield = 42%; m = 920 mg; 1 H NMR (400MHz, methanol-d4) δ8.11(d,J=7.1Hz,1H),7.94(d,J=7.2Hz,2H),7.86(dd,J=16.2,7.3Hz,2H) ,7.79(d,J=7.2Hz,2H),7.71(d,J=6.5Hz,2H),4.62(t,J=7.4Hz,2H),2.44(s,2H),2.22(s,2H).

[0273] The product 1.16 was prepared analogously to general procedure 3 (Example 1). Yield = 96%; m = 162 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.36–6.92 (m, 9H), 4.57–2.85 (m, 21H), 2.76–1.33 (m, 4H).

[0274] -Synthesis of product 1.17

[0275] The product 1.17 was prepared analogously to general procedure 3 (Example 1). Yield = 99%; m = 170 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.82–7.03 (m, 4H), 4.40–2.94 (m, 19H), 2.94–1.41 (m, 7H).

[0276] -Synthesis of product 1.18

[0277] Intermediate 1.18a was prepared analogously to general procedure 1 (Example 1). Yield = 44%; m = 2.29 g; 1 H NMR (400MHz, chloroform-d) δ7.81–7.73(m,1H),7.37(dd,J=6.6,2.8Hz,1H),7.32–7.20(m,2H),4.19(dq,J=14.2,7.3Hz,4H),3.22( hept,J=6.9Hz,1H),2.40(t,J=6.8Hz,2H),2.14(p,J=7.1Hz,2H),1.48(dd,J=6.8,1.5Hz,6H),1.29(tt,J=7.1,1.1Hz,3H).

[0278] Intermediate 1.18b was prepared analogously to general procedure 2 (Example 1). Yield = 61%; m = 1.25 g;1 H NMR (400MHz, methanol-d4) δ7.60(dd,J=17.9,7.6Hz,2H),7.28(t,J=7.0Hz,2H),4.34(t,J=7.8Hz,2H), 3.41 (h, J = 7.0 Hz, 1H), 2.42 (t, J = 6.9 Hz, 2H), 2.10 (p, J = 7.1 Hz, 2H), 1.45 (dd, J = 6.8, 2.1 Hz, 6H).

[0279] The product 1.18 was prepared analogously to general procedure 3 (Example 1). Yield = 94%; m = 167 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.79–7.22 (m, 4H), 4.42–3.06 (m, 17H), 2.82–2.50 (m, 3H), 2.46–2.01 (m, 2H), 1.93–1.00 (m, 6H).

[0280] -Synthesis of product 1.19

[0281] Intermediate 1.19a was prepared analogously to general procedure 1 (Example 1). Yield = 41%; m = 700 mg; 1 H NMR (400MHz, chloroform-d) δ7.78–7.70(m,1H),7.39–7.32(m,1H),7.31–7.20(m,2H),4.24–4.12(m,4H),2.87(t,J=7.7Hz,2H),2. 39 (t, J = 6.8 Hz, 2H), 2.13 (p, J = 7.1 Hz, 2H), 1.96 (h, J = 7.5 Hz, 2H), 1.28 (td, J = 7.2, 1.6 Hz, 3H), 1.09 (td, J = 7.3, 1.6 Hz, 3H).

[0282] Intermediate 1.19b was prepared analogously to general procedure 2 (Example 1). Yield = 97%; m = 610 mg; 1 H NMR (400MHz, methanol-d4) δ7.64–7.55(m,2H),7.29(t,J=7.1Hz,2H),4.32(t,J=7.7Hz,2H),3.33(s,2H),2.97(t,J =7.4Hz, 2H), 2.42 (t, J = 6.9Hz, 2H), 2.10 (p, J = 7.0Hz, 2H), 1.92 (h, J = 7.6Hz, 2H), 1.09 (td, J = 7.4, 2.2Hz, 3H).

[0283] The product 1.19 was prepared analogously to general procedure 3 (Example 1). Yield = 95%; m = 166 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.85–6.97 (m, 4H), 4.53–2.81 (m, 19H), 2.77–2.25 (m, 2H), 2.21–1.48 (m, 4H), 1.06–0.73 (m, 3H).

[0284] -Synthesis of Product 1.20

[0285] Intermediate 1.20a was prepared analogously to general procedure 1 (Example 1). Yield = 100%; m = 1.54 g; 1 H NMR (400 MHz, CHLOROFORM-d) δ 7.72 (d, J = 7.3 Hz, 1H), 7.44 (d, J = 7.6 Hz, 1H), 7.32–7.19 (m, 2H), 4.77 (s, 2H), 4.29 (t, J = 7.8 Hz, 2H), 4.09 (q, J = 7.1 Hz, 2H), 2.85 (s, 3H), 2.39–2.31 (m, 2H), 2.07–1.96 (m, 2H), 1.44 (s, 9H), 1.21 (t, J = 7.1 Hz, 3H).

[0286] Intermediate 1.20b was prepared analogously to general procedure 2 (Example 1). Yield = 69%; m = 1.05 g; 1 H NMR (400MHz, methanol-d4) δ7.64 (dd, J=17.8, 7.8Hz, 2H), 7.31 (dt, J=17.0, 7.4Hz, 2H), 4.79 (s, 2H), 4.34(t,J=7.9Hz,2H),2.95(s,3H),2.41(t,J=7.2Hz,2H),2.07(t,J=8.2Hz,2H),1.48(s,9H).

[0287] The product 1.20 was prepared analogously to general procedure 3 (Example 1). Yield = 98%; m = 183 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.92–6.90 (m, 4H), 4.53–3.08 (m, 18H), 3.00–2.77 (m, 3H), 2.72–1.73 (m, 4H).

[0288] -Synthesis of product 1.21

[0289] Intermediate 1.21a was prepared analogously to general procedure 1 (Example 1). Yield = 63%; m = 2.63 g; 1 H NMR (400MHz, chloroform-d) δ7.77–7.70(m,1H),7.44–7.37(m,1H),7.32–7.20(m,2H),4.74(d,J=2.1Hz,2H),4.33–4.24(m,2H),4.10( qd, J=7.2, 1.9Hz, 2H), 3.37 (d, J=2.0Hz, 3H), 2.35 (td, J=6.9, 2.0Hz, 2H), 2.13 (p, J=7.1Hz, 2H), 1.22 (td, J=7.1, 2.0Hz, 3H).

[0290] Intermediate 1.21b was prepared analogously to general procedure 2 (Example 1). Yield = 63%; m = 1.63 g; 1 H NMR (400MHz, methanol-d4) δ7.65 (ddd, J=18.3, 8.2, 2.8Hz, 2H), 7.39–7.25 (m, 2H), 4.76 (t, J=2.3Hz ,2H),4.43–4.34(m,2H),3.44(s,3H),2.41(td,J=7.2,6.5,3.7Hz,2H),2.17(q,J=7.8Hz,2H).

[0291] The product 1.21 was prepared analogously to general procedure 3 (Example 1). Yield = 91%; m = 172 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.92–7.09 (m, 4H), 5.08–4.82 (m, 2H), 4.51–4.06 (m, 2H), 4.00–2.98 (m, 16H), 2.77–2.27 (m, 2H), 2.22–1.65 (m, 2H).

[0292] -Synthesis of product 1.22

[0293] The product 1.22 was prepared analogously to general procedure 3 (Example 1). Yield = 42%; m = 129 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.95–7.13 (m, 4H), 5.17–4.86 (m, 2H), 4.54–4.14 (m, 2H), 4.10–3.01 (m, 22H), 2.98–2.32 (m, 2H), 2.30–1.70 (m, 2H).

[0294] -Synthesis of product 1.23

[0295] Intermediate 1.23a was prepared analogously to general procedure 1 (Example 1). Yield = 29%; m = 523 mg; 1 H NMR (400MHz, chloroform-d) δ7.83(d,J=7.8Hz,1H),7.52(d,J=7.9Hz,1H),7.37(dt,J=17.6,7.3Hz,2H),5.75(d,J=47.8Hz, 2H), 4.38(t,J=7.6Hz,2H), 4.15(q,J=7.3Hz,2H), 2.41(t,J=6.8Hz,2H), 2.21(p,J=7.0Hz,2H), 0.98–0.79(m,3H).

[0296] Intermediate 1.23b was prepared analogously to general procedure 2 (Example 1). Yield = 67%; m = 343 mg; 1 H NMR (400MHz, methanol-d4) δ7.75–7.63(m,2H),7.44–7.27(m,2H),5.70(dd,J=47.9,3 .0Hz,2H),4.47–4.36(m,2H),2.41(q,J=5.5,4.5Hz,2H),2.17(q,J=7.8Hz,2H).

[0297] The product 1.23 was prepared similarly to general procedure 3 (Example 1). Yield = 99%; m = 166 mg; 1 H NMR 400 MHz, deuterium oxide) δ 7.96–7.21 (m, 4H), 6.02–5.95 (m, 2H), 4.59–4.14 (m, 2H), 3.48–3.24 (m, 17H), 2.91–1.66 (m, 4H).

[0298] -Synthesis of product 1.24

[0299] The product 1.24 was prepared analogously to general procedure 3 (Example 1). Yield = 91%; m = 117 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.95–7.17 (m, 4H), 4.54–-2.39 (m, 24H), 2.07–-1.56 (m, 2H).

[0300] -Synthesis of product 1.25

[0301] The product 1.25 was prepared analogously to general procedure 3 (Example 1). Yield = 81%; m = 137 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.82–7.05 (m, 4H), 4.62–-2.27 (m, 22H), 2.12–-1.74 (m, 2H).

[0302] -Synthesis of product 1.26

[0303] The product 1.26 was prepared analogously to general procedure 3 (Example 1). Yield = 81%; m = 101 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.94–7.18 (m, 4H), 4.71–-2.18 (m, 25H), 2.05–-1.82 (m, 2H).

[0304] -Synthesis of product 1.27

[0305] The product 1.27 was prepared analogously to general procedure 3 (Example 1). Yield = 91%; m = 173 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.87–7.02 (m, 4H), 4.51–-2.23 (m, 17H), 2.10–-1.67 (m, 2H).

[0306] -Synthesis of product 1.28

[0307] The product 1.28 was prepared similarly to general procedure 3 (Example 1). Yield = 100%; m = 152 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.95–7.20 (m, 4H), 4.56–3.11 (m, 24H), 2.99–2.34 (m, 6H), 2.28–1.68 (m, 2H).

[0308] -Synthesis of product 1.29

[0309] Intermediate 1.29a was prepared analogously to general procedure 1 (Example 1). Yield = 86%; m = 788 mg; 1H NMR (400MHz, chloroform-d) δ7.57–7.46(m,0.5H),7.27(dd,J=9.4,2.4Hz,0.5H),7.16(dd,J=9.3,4.4Hz,0.5H),6.99–6.81(m,1. 5H), 4.06 (ddt, J = 9.6, 7.1, 4.4Hz, 4H), 2.52 (s, 2H), 2.28 (t, J = 6.7Hz, 2H), 2.02 (p, J = 6.9Hz, 2H), 1.18 (t, J = 7.1Hz, 3H).

[0310] Intermediate 1.29b was prepared analogously to general procedure 2 (Example 1). Yield = 100%; m = 786 mg, 1 H NMR (400MHz, methanol-d4) δ7.60 (ddd, J=14.5, 8.8, 4.5Hz, 1H), 7.38 (dd, J=49.0, 9.0Hz, 1H), 7.12 (dt, J=16. 4, 9.6Hz, 1H), 4.32 (dt, J = 12.6, 7.7Hz, 2H), 2.69 (s, 3H), 2.43 (d, J = 6.9Hz, 2H), 2.11 (h, J = 6.7Hz, 2H).

[0311] The product 1.29 was prepared similarly to the general procedure 3 (Example 1). Yield = 100%; m = 187 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.12–6.68 (m, 3H), 4.44–3.03 (m, 16H), 2.90–2.27 (m, 5H), 2.18–1.71 (m, 2H).

[0312] -Synthesis of Product 1.30

[0313] Intermediate 1.30a was prepared analogously to general procedure 1 (Example 1). Yield = 63%; m = 1.7 g; 1 H NMR (400MHz, chloroform-d) δ7.63(d,J=8.7Hz,0H),7.27(d,J=8.8Hz,1H),6.99–6.86(m,2H),4.27–4.15(m,4H),3.93(dd,J=7 .3, 1.3Hz, 3H), 2.65 (dd, J = 3.7, 1.3Hz, 3H), 2.43 (q, J = 6.8Hz, 2H), 2.18 (p, J = 7.1Hz, 2H), 1.33 (td, J = 7.2, 1.3Hz, 3H).

[0314] Intermediate 1.30b was prepared analogously to general procedure 2 (Example 1). Yield = 100%; m = 676 mg; 1 H NMR (400MHz, methanol-d4) δ7.54(dd,J=32.8,8.9Hz,1H),7.18(dd,J=33.3,2.4Hz,1H),7.01(ddd,J=21.3,8.9,2.3Hz,1H) ,4.33(q,J=6.8,6.3Hz,2H),3.92–3.84(m,3H),2.70(d,J=8.0Hz,3H),2.43(q,J=6.7Hz,2H),2.12(p,J=7.0Hz,2H).

[0315] The product 1.30 was prepared analogously to general procedure 3 (Example 1). Yield = 100%; m = 175 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.80–6.57 (m, 3H), 4.44–2.96 (m, 22H), 2.80–1.38 (m, 7H).

[0316] -Synthesis of product 1.31

[0317] Intermediate 1.31a was prepared analogously to general procedure 1 (Example 1). Yield = 50%; m = 820 mg; 1 H NMR (400MHz, chloroform-d) δ7.94–7.21(m,8H),7.05(d,J=1.3Hz,1H),5.08(d,J=1.3Hz,2H),4.05(td,J=8.1,7.5,2.9Hz,2H),3.92(dtd,J=1 6.2,7.8,6.5Hz,2H),2.50(dd,J=12.5,1.4Hz,3H),2.19(q,J=6.3Hz,2H),1.94(p,J=7.0Hz,2H),1.04(ddd,J=14.3,7.9,6.5Hz,3H).

[0318] Intermediate 1.31b was prepared analogously to general procedure 2 (Example 1). Yield = 73%; m = 580 mg; 1H NMR(400MHz, methanol-d4)δ8.01(d,J=8.9Hz,1H),7.83–7.48(m,7H),4.34(tt,J=7.3,3.0H z,2H),2.71–2.65(m,2H),2.41(ddt,J=10.2,7.4,4.2Hz,2H),2.12(h,J=7.2Hz,2H).

[0319] The product 1.31 was prepared analogously to general procedure 3 (Example 1). Yield = 84%; m = 171 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.92–6.49 (m, 8H), 4.50–3.10 (m, 17H), 3.03–1.78 (m, 7H).

[0320] -Synthesis of product 1.32

[0321] Intermediate 1.32a was prepared analogously to general procedure 1 (Example 1). Yield = 67%; m = 1.29 mg; 1 H NMR (400MHz, chloroform-d) δ7.81–7.51(m,1H),7.37–7.23(m,2H),4.23–4.08(m,4H),2.62(d,J=4.3Hz,3H) ,2.41–2.32(m,2H),2.12(pd,J=6.9,2.4Hz,2H),1.38(d,J=6.9Hz,9H),1.25(td,J=7.2,2.0Hz,3H).

[0322] Intermediate 1.32b was prepared analogously to general procedure 2 (Example 1). Yield = 81%; m = 1.02 mg; 1 H NMR (400MHz, DMSO-d6) δ7.48(s,1H),7.41(dd,J=8.5,5.2Hz,1H),7.23(ddd,J=17.6,8.5,1.7Hz,1H),4.16 (q, J=7.8Hz, 2H), 2.50 (s, 3H), 2.26 (dt, J=13.7, 7.0Hz, 2H), 1.90 (p, J=7.2Hz, 2H), 1.33 (d, J=7.3Hz, 9H).

[0323] The product 1.32 was prepared analogously to general procedure 3 (Example 1). Yield = 97%; m = 176 mg; 1H NMR (400 MHz, deuterium oxide) δ 7.80–7.04 (m, 3H), 4.49–4.04 (m, 2H), 3.94–2.93 (m, 15H), 2.81–2.27 (m, 5H), 2.17–1.70 (m, 2H), 1.46–0.82 (m, 9H).

[0324] -Synthesis of product 1.33

[0325] Intermediate 1.33a was prepared analogously to general procedure 2 (Example 1). Yield = 49%; m = 1.72 g; 1 H NMR (400 MHz, CHLOROFORM-d) δ 7.30–7.18 (m, 2H), 7.14 (d, J = 6.5 Hz, 1H), 4.31–4.22 (m, 3H), 4.25–4.17 (m, 1H), 2.75 (s, 6H), 2.57–2.41 (m, 2H), 2.21 (p, J = 7.2 Hz, 2H), 1.35 (td, J = 7.1, 1.8 Hz, 3H).

[0326] Intermediate 1.33b was prepared analogously to general procedure 2 (Example 1). Yield = 54%; m = 920 mg; 1 H NMR (400MHz, methanol-d4) δ7.38(d,J=8.2Hz,1H),7.19(t,J=7.4Hz,1H),7.07(d,J=7.4Hz,1H),4.28(t,J=7.3Hz, 2H), 3.33 (d, J = 3.3Hz, 2H), 2.66 (d, J = 2.7Hz, 3H), 2.58 (s, 3H), 2.37 (t, J = 6.3Hz, 2H), 2.08 (h, J = 7.7Hz, 2H).

[0327] The product 1.33 was prepared analogously to general procedure 3 (Example 1). Yield = 100%; m = 188 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.62–6.76 (m, 3H), 4.46–3.03 (m, 15H), 2.87–1.36 (m, 10H).

[0328] -Synthesis of product 1.34

[0329] Intermediate 1.34a was prepared analogously to general procedure 1 (Example 1). Yield = 91%; m = 1.55 g; 1H NMR (400 MHz, CHLOROFORM-d) δ 7.68–7.53 (m, 1H), 7.33–7.16 (m, 2H), 4.14 (pd, J = 7.4, 1.6 Hz, 4H), 2.61 (d, J = 1.5 Hz, 3H), 2.35 (td, J = 6.9, 3.5 Hz, 2H), 2.09 (h, J = 5.8, 4.7 Hz, 2H), 1.26 (tdd, J = 6.9, 4.5, 1.5 Hz, 3H).

[0330] Intermediate 1.34b was prepared analogously to general procedure 2 (Example 1). Yield = 60%; m = 840 mg; 1 H NMR (400 MHz, methanol-d4) δ 7.85 (s, 1H), 7.75–7.60 (m, 1H), 7.45–7.38 (m, 1H), 4.39 (q, J = 7.6, 6.5 Hz, 2H), 2.77 (t, J = 2.9 Hz, 3H), 2.49 (t, J = 6.6 Hz, 2H), 2.13 (p, J = 7.3 Hz, 2H).

[0331] The product 1.34 was prepared analogously to general procedure 3 (Example 1). Yield = 100%; m = 193 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.83–6.65 (m, 3H), 4.52–3.09 (m, 15H), 3.04–2.32 (m, 5H), 2.30–1.72 (m, 2H).

[0332] -Synthesis of product 1.35

[0333] Intermediate 1.35a was prepared analogously to general procedure 1 (Example 1). Yield = 63%; m = 1.16 g; 1 H NMR (400MHz, chloroform-d) δ7.59–7.45(m,1H),7.24–7.02(m,2H),4.15(td,J=8.5,6.9,5.0Hz,4H),2.61(d,J=3.7 Hz,3H),2.47(d,J=8.6Hz,3H),2.35(td,J=6.8,4.7Hz,2H),2.17–2.05(m,2H),1.25(td,J=7.2,3.0Hz,3H).

[0334] Intermediate 1.35b was prepared analogously to general procedure 2 (Example 1). Yield = 100%; m = 1.07 g; 1H NMR (400 MHz, methanol-d4) δ 7.82–7.64 (m, 1H), 7.61–7.49 (m, 1H), 7.39 (t, J = 10.2 Hz, 1H), 4.45 (tt, J = 7.6, 3.2 Hz, 2H), 2.85 (d, J = 3.2 Hz, 3H), 2.66–2.44 (m, 5H), 2.17 (p, J = 7.7 Hz, 2H).

[0335] The product 1.35 was prepared analogously to general procedure 3 (Example 1). Yield = 100%; m = 163 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.61–6.56 (m, 3H), 4.43–3.06 (m, 20H), 2.88–1.52 (m, 10H).

[0336] -Synthesis of product 1.36

[0337] Intermediate 1.36a was prepared analogously to general procedure 1 (Example 1). Yield = 100%; m = 1.35 g; 1 H NMR (400MHz, chloroform-d) δ7.74–7.11(m,2H),7.04(dtd,J=18.1,9.2,2.4Hz,1H),4.76(s,2H),4.35–4.23(m,2H),4.14( qd,J=7.2,2.9Hz,2H),2.89(d,J=3.5Hz,3H),2.38(t,J=7.1Hz,2H),2.08–2.00(m,2H),1.48(s,9H),1.25(s,3H).

[0338] Intermediate 1.36b was prepared analogously to general procedure 2 (Example 1). Yield = 61%; m = 810 mg; 1 H NMR (400MHz, methanol-d4) δ7.61(dd,J=8.2,4.0Hz,1H),7.37(dd,J=30.5,9.2Hz,1H),7.08(dt,J=22.3,9.4Hz,1H),4. 77(s,2H),4.32(dt,J=14.4,7.3Hz,2H),2.95(s,3H),2.40(t,J=7.2Hz,2H),2.05(q,J=7.4Hz,2H),1.47(s,9H).

[0339] The product 1.36 was prepared analogously to general procedure 3 (Example 1). Yield = 98%; m = 183 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.77–6.66 (m, 3H), 4.73–4.51 (m, 2H), 4.47–3.01 (m, 17H), 2.99–2.78 (m, 3H), 2.73–1.70 (m, 4H).

[0340] -Synthesis of product 1.37

[0341] The product 1.37 was prepared analogously to general procedure 3 (Example 1). Yield = 93%; m = 155 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.54–6.54 (m, 3H), 4.39–2.94 (m, 15H), 2.93–1.52 (m, 10H).

[0342] -Synthesis of product 1.38

[0343] Intermediate 1.38a was prepared analogously to general procedure 1 (Example 1). Yield = 26%; m = 300 mg; 1 H NMR (400MHz, methanol-d4) δ7.30(d,J=8.6Hz,1H),6.96(s,1H),6.80(dd,J=8.8,2.3Hz,1H),4.21(t,J=7.6Hz,2H),4.07(q, J=7.4Hz,1H),3.62(d,J=2.5Hz,1H),2.56(s,3H),2.41(q,J=6.6Hz,2H),2.09(p,J=7.3Hz,2H),1.21(t,J=7.3Hz,2H).

[0344] Intermediate 1.38b was prepared analogously to general procedure 2 (Example 1). Yield = 54%; m = 68 mg; 1 H NMR (D2O)δ: 1 H NMR (400 MHz, methanol-d4) δ 7.54 (dd, J = 8.4, 3.1 Hz, 1H), 7.41–7.29 (m, 1H), 7.09–6.98 (m, 1H), 4.33–4.22 (m, 2H), 2.63 (s, 3H), 2.37 (t, J = 6.9 Hz, 2H), 2.09 (p, J = 7.1 Hz, 2H), 1.56 (s, 9H).

[0345] The product 1.38 was prepared similarly to general procedure 3 (Example 1). Yield = 100%; m = 54 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.80–6.40 (m, 4H), 4.35–1.30 (m, 22H).

[0346] -Synthesis of product 1.39

[0347] Intermediate 1.39a was prepared analogously to general procedure 2 (Example 1). Yield = 70%; m = 1.6 g; 1 H NMR (400 MHz, CHLOROFORM-d) δ 8.00 (s, 1H), 7.88–7.81 (m, 1H), 7.81–7.75 (m, 1H), 7.52 (s, 1H), 7.26 (ddt, J = 8.4, 6.7, 4.2 Hz, 2H), 4.11–3.96 (m, 4H), 2.52 (s, 3H), 2.24 (t, J = 6.8 Hz, 2H), 2.02 (p, J = 7.1 Hz, 2H), 1.11 (td, J = 7.1, 1.6 Hz, 3H).

[0348] Intermediate 1.39b was prepared analogously to general procedure 2 (Example 1). Yield = 58%; m = 840 mg; 1 H NMR (400MHz, DMSO-d6) δ12.27(s,1H),8.10(s,1H),8.04(s,1H),8.05–7.95(m,2H),7.47–7. 34(m,2H),4.31(t,J=7.5Hz,2H),2.66(s,3H),2.37(t,J=7.2Hz,2H),2.03(p,J=7.3Hz,2H). The product 1.39 was prepared analogously to general procedure 3 (Example 1). Yield = 95%; m = 174 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.21–6.03 (m, 6H), 4.47–0.63 (m, 22H).

[0349] -Synthesis of product 1.40

[0350] The product 1.40 was prepared analogously to general procedure 3 (Example 1). Yield = 97%; m = 198 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.19–6.14 (m, 6H), 4.51–0.73 (m, 24H).

[0351] -Synthesis of product 1.41

[0352] Intermediate 1.41a was prepared analogously to general procedure 2 (Example 1). Yield = 45%; m = 1.3 g; 1 H NMR (400 MHz, CHLOROFORM-d) δ 7.63 (s, 1H), 7.26 (s, 1H), 4.33 (q, J = 7.2 Hz, 4H), 2.77 (s, 3H), 2.58–2.49 (m, 8H), 2.29 (p, J = 7.2 Hz, 2H), 1.44 (td, J = 7.2, 1.5 Hz, 3H).

[0353] Intermediate 1.41b was prepared analogously to general procedure 2 (Example 1). Yield = 79%; m = 1.0 g; 1 H NMR (400 MHz, methanol-d4) δ 7.34 (s, 2H), 4.28–4.19 (m, 2H), 2.61 (s, 3H), 2.43–2.30 (m, 8H), 2.08 (p, J = 7.9 Hz, 2H).

[0354] The product 1.41 was prepared analogously to general procedure 3 (Example 1). Yield = 99%; m = 161 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.55–6.76 (m, 2H), 4.53–2.95 (m, 20H), 2.89–2.39 (m, 5H), 2.36–1.49 (m, 8H).

[0355] -Synthesis of product 1.42

[0356] Intermediate 1.42a was prepared analogously to general procedure 2 (Example 1). Yield = 53%; m = 418 mg; 1 H NMR (400 MHz, deuterium oxide) δ 6.98 (s, 1H), 6.82 (s, 1H), 3.90 (t, J = 7.3 Hz, 2H), 2.41 (s, 3H), 2.37 (s, 3H), 2.30 (s, 3H), 2.08–2.01 (m, 2H), 1.94–1.77 (m, 2H).

[0357] The product 1.42 was prepared analogously to general procedure 3 (Example 1). Yield = 84%; m = 25 mg; 1H NMR (400 MHz, deuterium oxide) δ 7.48–6.58 (m, 2H), 4.48–3.14 (m, 25H), 2.95–1.67 (m, 12H).

[0358] -Synthesis of product 1.43

[0359] Intermediate 1.43a was prepared analogously to general procedure 1 (Example 1). Yield = 75%; m = 1.05 g; 1 H NMR (400MHz, chloroform-d) δ8.51(dd,J=4.5,1.6Hz,1H),8.05(dd,J=8.0,1.6Hz,1H),8.00(s,1H),7.10(dd,J =8.0, 4.5Hz, 1H), 4.64–4.56 (m, 2H), 4.07 (q, J = 7.1Hz, 2H), 2.36–2.22 (m, 4H), 1.19 (t, J = 7.2Hz, 3H).

[0360] Intermediate 1.43b was prepared analogously to general procedure 2 (Example 1). Yield = 76%; m = 778 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.30–8.22 (m, 1H), 8.06–7.96 (m, 1H), 7.90–7.81 (m, 1H), 7.09–7.01 (m, 1H), 4.23 (t, J = 6.4 Hz, 2H), 2.04–1.89 (m, 4H).

[0361] The product 1.43 was prepared analogously to general procedure 3 (Example 1). Yield = 81%; m = 94 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.35–6.54 (m, 4H), 4.52–2.63 (m, 9H), 2.59–1.42 (m, 4H).

[0362] -Synthesis of product 1.44

[0363] Intermediate 1.44a was prepared analogously to general procedure 1 (Example 1). Yield = 15%; m = 211 mg; 1H NMR (400 MHz, CHLOROFORM-d) δ 8.52 (dd, J = 4.4, 1.3 Hz, 1H), 8.19 (d, J = 1.0 Hz, 1H), 7.77 (dt, J = 8.5, 1.2 Hz, 1H), 7.24 (dd, J = 8.6, 4.4 Hz, 1H), 4.46–4.38 (m, 2H), 4.04 (q, J = 7.2 Hz, 2H), 2.28–2.13 (m, 4H), 1.17 (t, J = 7.1 Hz, 3H).

[0364] Intermediate 1.44b was prepared analogously to general procedure 2 (Example 1). Yield = 100%; m = 887 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.24 (dd, J = 4.5, 1.3 Hz, 1H), 7.92 (d, J = 1.0 Hz, 1H), 7.76 (dt, J = 8.7, 1.2 Hz, 1H), 7.20 (dd, J = 8.7, 4.4 Hz, 1H), 4.21–4.13 (m, 2H), 2.02–1.87 (m, 4H).

[0365] The product 1.44 was prepared analogously to general procedure 3 (Example 1). Yield = 97%; m = 103 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.92–7.42 (m, 4H), 4.61–4.25 (m, 2H), 4.04–3.09 (m, 10H), 2.67–1.88 (m, 5H).

[0366] -Synthesis of product 1.45

[0367] Intermediate 1.45a was prepared analogously to general procedure 1 (Example 1). Yield = 43%; m = 602 mg; 1 H NMR (400 MHz, CHLOROFORM-d) δ 9.00 (s, 1H), 8.29 (d, J = 5.6 Hz, 1H), 8.04 (d, J = 0.8 Hz, 1H), 7.61 (dd, J = 5.6, 1.3 Hz, 1H), 4.61–4.52 (m, 2H), 4.09 (q, J = 7.1 Hz, 2H), 2.34–2.21 (m, 4H), 1.20 (t, J = 7.1 Hz, 3H).

[0368] Intermediate 1.45b was prepared analogously to general procedure 2 (Example 1). Yield = 100%; m = 592 mg; 1H NMR (400 MHz, deuterium oxide) δ 8.77 (s, 1H), 8.04–7.93 (m, 2H), 7.57 (dd, J=5.8, 1.3 Hz, 1H), 4.37–4.29 (m, 2H), 2.07–1.93 (m, 4H).

[0369] The product 1.45 was prepared analogously to general procedure 3 (Example 1). Yield = 100%; m = 84 mg; 1 H NMR (400 MHz, deuterium oxide) δ 9.67–8.10 (m, 4H), 4.23–3.06 (m, 16H), 2.86–1.90 (m, 4H).

[0370] -Synthesis of product 1.46

[0371] The product 1.46 was prepared similarly to general procedure 3 (Example 1). Yield = 89%; m = 68 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.63–6.72 (m, 4H), 4.57–2.78 (m, 20H), 2.75–1.47 (m, 4H).

[0372] -Synthesis of product 1.47

[0373] The product 1.47 was prepared similarly to the general procedure 3 (Example 1). Yield = 97%; m = 73 mg; 1 H NMR (400 MHz, deuterium oxide) δ 9.12–7.56 (m, 4H), 4.58–4.36 (m, 2H), 4.09–3.01 (m, 19H), 2.90–1.77 (m, 4H).

[0374] -Synthesis of product 1.48

[0375] Intermediate 1.48a was prepared analogously to general procedure 1 (Example 1). Yield = 59%; m = 820 mg; 1H NMR (400 MHz, CHLOROFORM-d) δ 7.84 (s, 1H), 7.57 (dt, J = 8.2, 1.1 Hz, 1H), 7.27 (dd, J = 8.5, 1.1 Hz, 1H), 7.28–7.18 (m, 1H), 6.98 (ddd, J = 8.0, 6.6, 1.1 Hz, 1H), 4.31 (t, J = 6.5 Hz, 2H), 3.94 (q, J = 7.1 Hz, 2H), 2.18–2.03 (m, 4H), 1.06 (t, J = 7.1 Hz, 3H).

[0376] Intermediate 1.48b was prepared analogously to general procedure 2 (Example 1). Yield = 100%; m = 175 mg; 1 H NMR (400MHz, methanol-d4) δ8.01(s,1H),7.74(dt,J=8.2,1.0Hz,1H),7.59(dq,J=8.5,0.9Hz,1H),7.40(d dd,J=8.6,6.9,1.1Hz,1H),7.15(ddd,J=7.9,6.8,0.8Hz,1H),4.52–4.44(m,2H),2.28–2.12(m,4H).

[0377] The product 1.48 was prepared similarly to general procedure 3 (Example 1). Yield = 83%; m = 105 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.18–6.91 (m, 5H), 4.14–1.72 (m, 42H).

[0378] -Synthesis of product 1.49

[0379] The product 1.49 was prepared similarly to general procedure 3 (Example 1). Yield = 52%; m = 39 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.21–6.38 (m, 5H), 4.47–1.37 (m, 25H).

[0380] -Synthesis of Product 1.50

[0381] The product 1.50 was prepared analogously to general procedure 3 (Example 1). Yield = 16%; m = 13 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.19–6.27 (m, 5H), 4.42–1.05 (m, 23H).

[0382] -Synthesis of product 1.51

[0383] The product 1.51 was prepared analogously to general procedure 3 (Example 1). Yield = 91%; m = 12 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.65–7.82 (m, 2H), 4.39–2.88 (m, 36H), 2.68–1.91 (m, 3H).

[0384] -Synthesis of product 1.52

[0385] Intermediate 1.52a was prepared analogously to general procedure 1 (Example 1). Yield = 76%; m = 440 mg; 1 H NMR (400MHz, chloroform-d) δ8.40(dd,J=4.8,1.6Hz,1H),8.18(s,1H),8.07(dd,J=8.1,1.6Hz,1H),7.30–7.20(m ,1H),4.40(td,J=6.9,1.7Hz,2H),4.10(q,J=7.1Hz,2H),2.43–2.20(m,4H),1.22(td,J=7.2,0.6Hz,3H).

[0386] Intermediate 1.52b was prepared analogously to general procedure 2 (Example 1). Yield = 50%; m = 597 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.12 (s, 1H), 8.09 (dd, J = 4.9, 1.4 Hz, 1H), 7.85 (dd, J = 8.1, 1.4 Hz, 1H), 7.14 (dd, J = 8.1, 4.9 Hz, 1H), 4.08 (t, J = 7.0 Hz, 2H), 2.27–1.73 (m, 4H).

[0387] The product 1.52 was prepared similarly to the general procedure 3 (Example 1). Yield = 71%; m = 12 mg; 1 H NMR (400 MHz, deuterium oxide) δ 9.59–6.98 (m, 4H), 4.13–2.68 (m, 17H), 2.68–0.79 (m, 3H).

[0388] -Synthesis of product 1.53

[0389] Intermediate 1.53a was prepared analogously to general procedure 1 (Example 1). Yield = 65%; m = 153 mg;1 H NMR (400 MHz, CHLOROFORM-d) δ 8.57 (dd, J = 4.8, 1.5 Hz, 1H), 8.25 (s, 1H), 7.84 (dd, J = 8.1, 1.5 Hz, 1H), 7.28–7.22 (m, 1H), 4.32 (t, J = 7.2 Hz, 2H), 4.12 (q, J = 7.1 Hz, 2H), 2.37–2.28 (m, 2H), 2.25–2.12 (m, 2H), 1.23 (t, J = 7.1 Hz, 3H).

[0390] Intermediate 1.53b was prepared analogously to general procedure 2 (Example 1). Yield = 100%; m = 105 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.41 (dd, J = 4.9, 1.5 Hz, 1H), 8.38 (s, 1H), 8.08 (dd, J = 8.2, 1.5 Hz, 1H), 7.38 (dd, J = 8.2, 4.9 Hz, 1H), 4.31 (t, J = 6.9 Hz, 2H), 2.25–2.06 (m, 4H).

[0391] The product 1.53 was prepared similarly to general procedure 3 (Example 1). Yield = 82%; m = 12 mg; 1 H NMR (400 MHz, deuterium oxide) δ 9.47–6.92 (m, 4H), 4.19–2.88 (m, 20H), 2.92–1.60 (m, 4H).

[0392] -Synthesis of product 1.54

[0393] The product 1.54 was prepared similarly to general procedure 3 (Example 1). Yield = 83%; m = 12 mg; 1 H NMR (400 MHz, deuterium oxide) δ 9.20–8.32 (m, 2H), 4.51–2.93 (m, 16H), 2.88–1.68 (m, 7H).

[0394] -Synthesis of product 1.55

[0395] The product 1.55 was prepared analogously to general procedure 3 (Example 1). Yield = 97%; m = 169 mg; 1H NMR (400 MHz, deuterium oxide) δ 7.74–7.19 (m, 4H), 4.41–3.00 (m, 13H), 2.83–2.52 (m, 3H), 2.48–1.99 (m, 2H), 1.95–1.00 (m, 6H).

[0396] -Synthesis of product 1.56

[0397] The product 1.56 was prepared similarly to general procedure 3 (Example 1). Yield = 100%; m = 187 mg; 1 H NMR(D2O)δ:7.41–6.35(m,2H),4.44–2.96(m,23H),2.85–1.34(m,20H).

[0398] -Synthesis of product 1.57

[0399] The product 1.57 was prepared similarly to general procedure 3 (Example 1). Yield = 100%; m = 26 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.64–6.35 (m, 2H), 4.50–3.01 (m, 62H), 3.05–1.41 (m, 13H).

[0400] -Synthesis of product 1.58

[0401] The product 1.58 was prepared similarly to general procedure 3 (Example 1). Yield = 72%; m = 19 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.55–6.59 (m, 2H), 4.41–3.11 (m, 39H), 3.05–1.37 (m, 13H).

[0402] -Synthesis of product 1.59

[0403] The product 1.59 was prepared similarly to the general procedure 3 (Example 1). Yield = 70%; m = 22 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.80–6.15 (m, 2H), 4.50–3.21 (m, 22H), 3.17–0.97 (m, 13H).

[0404] -Synthesis of Product 1.60

[0405] The product 1.60 was prepared analogously to general procedure 3 (Example 1). Yield = 73%; m = 23 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.98–6.15 (m, 2H), 4.62–3.06 (m, 22H), 2.99–1.39 (m, 13H).

[0406] -Synthesis of product 1.61

[0407] The product 1.61 was prepared similarly to general procedure 3 (Example 1). Yield = 84%; m = 25 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.64–6.35 (m, 2H), 4.57–3.13 (m, 25H), 3.13–1.50 (m, 13H).

[0408] -Synthesis of product 1.62

[0409] The product 1.62 was prepared similarly to general procedure 3 (Example 1). Yield = 68%; m = 21 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.74–6.35 (m, 2H), 4.50–3.01 (m, 24H), 2.97–1.41 (m, 13H).

[0410] -Synthesis of product 1.63

[0411] The product 1.63 was prepared similarly to general procedure 3 (Example 1). Yield = 35%; m = 13 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.62–6.39 (m, 2H), 4.57–3.01 (m, 16H), 2.90–1.21 (m, 13H).

[0412] -Synthesis of product 1.64

[0413] The product 1.64 was prepared similarly to general procedure 3 (Example 1). Yield = 85%; m = 18 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.90–6.11 (m, 2H), 4.52–3.09 (m, 30H), 3.11–1.39 (m, 13H).

[0414] -Synthesis of product 1.65

[0415] The product 1.65 was prepared similarly to general procedure 3 (Example 1). Yield = 84%; m = 21 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.80–6.15 (m, 2H), 4.60–3.17 (m, 19H), 3.09–1.25 (m, 13H).

[0416] -Synthesis of product 1.66

[0417] The product 1.66 was prepared similarly to general procedure 3 (Example 1). Yield = 72%; m = 17 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.49–6.19 (m, 2H), 4.57–3.05 (m, 13H), 3.03–1.37 (m, 4H).

[0418] -Synthesis of product 1.67

[0419] Intermediate 1.67a was prepared analogously to general procedure 1 (Example 1). Yield = 85%; m = 395 mg; 1 H NMR (400 MHz, deuterium oxide) δ 6.93 (d, J = 1.5 Hz, 1H), 6.80 (s, 1H), 3.84 (t, J = 7.3 Hz, 2H), 2.40 (s, 3H), 2.37 (s, 3H), 2.30 (s, 3H), 2.07 (t, J = 7.4 Hz, 2H), 1.64–1.53 (m, 2H), 1.51–1.35 (m, 2H).

[0420] The product 1.67 was prepared similarly to general procedure 3 (Example 1). Yield = 69%; m = 22 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.64–6.35 (m, 2H), 4.52–3.01 (m, 25H), 2.94–1.02 (m, 13H).

[0421] -Synthesis of product 1.68

[0422] Intermediate 1.68a was prepared analogously to general procedure 2 (Example 1). Yield = 66%; m = 247 mg; 1H NMR (400 MHz, deuterium oxide) δ 7.03 (s, 1H), 6.83 (s, 1H), 4.19 (t, 2H), 2.48 (t, J = 7.7, 6.8 Hz, 2H), 2.43 (s, 3H), 2.35 (s, 3H), 2.29 (s, 3H).

[0423] The product 1.68 was prepared similarly to general procedure 3 (Example 1). Yield = 74%; m = 24 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.80–6.25 (m, 2H), 4.52–1.32 (m, 33H).

[0424] -Synthesis of product 1.69

[0425] Intermediate 1.69a was prepared analogously to general procedure 1 (Example 1). Yield = 76%; m = 332 mg; 1 H NMR (400 MHz, deuterium oxide) δ 6.82 (s, 1H), 6.71 (s, 1H), 3.69 (t, J = 7.3 Hz, 2H), 2.31 (d, J = 2.5 Hz, 6H), 2.22 (s, 3H), 1.99 (q, J = 7.7 Hz, 2H), 1.54–1.41 (m, 2H), 1.40–1.30 (m, 2H), 1.14–0.98 (m, 2H).

[0426] The product 1.69 was prepared similarly to general procedure 3 (Example 1). Yield = 74%; m = 30 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.48–6.39 (m, 2H), 4.46–3.01 (m, 18H), 2.88–0.77 (m, 13H).

[0427] -Synthesis of Product 1.70

[0428] The product 1.70 was prepared similarly to general procedure 3 (Example 1). Yield = 63%; m = 20 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.72–6.29 (m, 2H), 4.48–3.21 (m, 24H), 3.09–1.19 (m, 13H).

[0429] -Synthesis of product 1.71

[0430] The product 1.71 was prepared analogously to general procedure 3 (Example 1). Yield = 80%; m = 28 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.62–6.31 (m, 2H), 4.43–3.09 (m, 18H), 3.01–0.82 (m, 15H).

[0431] -Synthesis of product 1.72

[0432] The product 1.72 was prepared similarly to general procedure 3 (Example 1). Yield = 86%; m = 30 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.55–6.25 (m, 2H), 4.55–3.06 (m, 17H), 2.94–1.23 (m, 13H).

[0433] -Synthesis of product 1.73

[0434]

[0435] 1.73a: 4-DMAP and Et3N were added to a solution of 4-imidazoleacetic acid in THF / H2O. Boc2O in THF was then added at 0°C and the mixture was stirred at room temperature overnight. EtOAc was added and 3M HCl was added to bring the pH of the aqueous phase to 4. The aqueous phase was removed and the organic phase was washed with brine. Drying over Na2SO4 and evaporation in vacuo gave the product as a white solid. Yield = 54%; m = 300 mg; 1 H NMR (400MHz, MeOD) δ 8.14 (d, J = 1.4 Hz, 1H), 7.42 (d, J = 1.4 Hz, 1H), 3.59 (s, 2H), 1.63 (s, 9H).

[0436]

[0437] Product 1.73: Water containing PEI22k.HCl (1 eq.) was added into a microwave sealed tube followed by NMM (2 eq.). The acid was dissolved in MeOH and added to the PEI. After stirring for 10 minutes, DMTMM was added and the mixture was stirred overnight at room temperature. The solvent was evaporated and co-evaporated with ethanol. TFA was added at 0°C and stirred for 3 hours. TFA was evaporated and the product was purified on an Amicon Ultra 15 (3kD) with 6*10 mL HCl 50 mM.

[0438] Product 1.73. Yield = 84%; m = 58 mg; 1H NMR (400 MHz, deuterium oxide) δ 8.74–8.64 (m, 1H), 7.46–7.31 (m, 1H), 4.50–3.26 (m, 16H).

[0439] -Synthesis of product 1.74

[0440]

[0441] Product 1.74 was prepared similarly to general procedure 3 using branched polyethyleneimine (bPEI, 25K, Sigma-Aldrich). Yield = 94%; m = 282 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.42–6.52 (m, 2H), 4.45–1.51 (m, 33H).

[0442] -Synthesis of product 1.75

[0443]

[0444] Product 1.75 was prepared similarly to general procedure 3 using branched polyethyleneimine (bPEI, 10K, Alfa Aesar). Yield = 99%; m = 351 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.37–6.50 (m, 2H), 4.57–1.44 (m, 29H).

[0445] -Synthesis of product 1.76

[0446]

[0447] Product 1.76 was prepared similarly to general procedure 3 using poly(allylamine) (PAA, 15K, Sigma-Aldrich). Yield = 99%; m = 146 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.43–6.38 (m, 2H), 4.46–0.74 (m, 41H).

[0448] -Synthesis of product 1.77

[0449]

[0450] The product 1.77 was prepared similarly to general procedure 3. Yield = 34%; m = 41 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.44–6.93 (m, 2H), 4.53–4.06 (m, 5H), 3.15–1.08 (m, 40H).

[0451] -Synthesis of product 1.78

[0452]

[0453] Product 1.78 was prepared similarly to general procedure 4 using poly(vinylamine) (PLL, 22K, Sigma-Aldrich). Yield = 99%; m = 37 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.74 (s, 1H), 7.03 (s, 1H), 4.43–4.09 (m, 2H), 3.75–2.85 (m, 11H), 2.66–2.41 (m, 4H), 2.03–0.88 (m, 14H).

[0454] -Synthesis of product 1.79

[0455]

[0456] Product 1.79 was prepared similarly to general procedure 3 using poly(vinylamine) (PVA, 25K, Polysciences). Yield = 78%; m = 139 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.49–6.52 (m, 2H), 4.60–0.96 (m, 26H).

[0457] -Synthesis of Product 1.80

[0458]

[0459] The product 1.80 was prepared similarly to general procedure 4. Yield = 98%; m = 68 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.63–8.50 (m, 1H), 7.36–7.27 (m, 1H), 4.34–4.17 (m, 1H), 3.79–3.66 (m, 2H), 3.63–2.84 (m, 7H), 2.59–2.15 (m, 3H), 1.90–1.15 (m, 9H).

[0460] -Synthesis of Product 2.01

[0461] Intermediate 2.01a was prepared similarly to general procedure 1 (Example 1). Yield = 17%; m = 1.00 g; 1H NMR (400MHz, chloroform-d) δ7.95(d,J=8.4Hz,1H),7.45(d,J=8.3Hz,1H),7.38(t,J=7.6Hz,1H),7.30–7.22(m ,1H),4.68–4.57(m,2H),4.01(qd,J=7.1,1.6Hz,2H),2.30–2.16(m,4H),1.13(td,J=7.1,1.6Hz,3H).

[0462] Intermediate 2.01b was prepared analogously to general procedure 2 (Example 1). Yield = 85%; m = 830 mg; 1 H NMR (400MHz, methanol-d4) δ8.03–7.96(m,1H),7.82(d,J=8.5Hz,1H),7.58(t,J=7.3Hz,1H),7.45 (t, J = 8.0 Hz, 1H), 4.80 (dt, J = 7.0, 4.3 Hz, 2H), 2.37 (t, J = 7.0 Hz, 2H), 2.30 (q, J = 7.0 Hz, 2H).

[0463] The product 2.01 was prepared similarly to the general procedure 3 (Example 1). Yield = 100%; m = 189 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.41-6.55 (m, 4H), 4.58-3.02 (m, 14H), 2.90-1.31 (m, 3H).

[0464] -Synthesis of Product 2.02

[0465] Intermediate 2.02a was prepared analogously to general procedure 1 (Example 1). Yield = 34%; m = 2.00 g; 1 H NMR (400 MHz, CHLOROFORM-d) δ 7.73 (ddt, J = 7.4, 4.1, 2.2 Hz, 2H), 7.25 (ddt, J = 9.4, 4.0, 2.2 Hz, 2H), 4.68 (dd, J = 7.3, 5.5 Hz, 2H), 3.99 (ddd, J = 9.1, 7.2, 6.0 Hz, 2H), 2.40–2.12 (m, 4H), 1.11 (tt, J = 7.3, 1.3 Hz, 3H).

[0466] Intermediate 2.02b was prepared analogously to general procedure 2 (Example 1). Yield = 53%; m = 1.00 g; 1H NMR (400 MHz, methanol-d4) δ 7.86 (dd, J = 6.9, 3.4 Hz, 2H), 7.42 (dd, J = 6.9, 3.4 Hz, 2H), 4.83 (d, J = 13.0 Hz, 2H), 2.37 (d, J = 4.3 Hz, 4H).

[0467] The product 2.02 was prepared similarly to general procedure 3 (Example 1). Yield = 100%; m = 166 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.83-6.50 (m, 4H), 4.61-3.90 (m, 2H), 3.88-2.51 (m, 11H), 2.49-1.35 (m, 4H).

[0468] -Synthesis of Product 2.03

[0469] The product 2.03 was prepared similarly to the general procedure 3 (Example 1). Yield = 93%; m = 143 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.95-6.76 (m, 4H), 4.57-4.09 (m, 2H), 3.96-2.52 (s, 17H), 2.45-1.61 (m, 4H).

[0470] -Synthesis of product 2.04

[0471] The product 2.04 was prepared similarly to general procedure 3 (Example 1). Yield = 92%; m = 133 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.98-6.60 (m, 4H), 4.67-4.17 (m, 2H), 4.15-2.69 (m, 18H), 2.52-1.45 (m, 4H).

[0472] -Synthesis of product 2.05

[0473] Intermediate 2.05a was prepared analogously to general procedure 1 (Example 1). Yield = 35%; m = 1.94 g; 1H NMR (400MHz, chloroform-d) δ7.87(d,J=8.5Hz,0H),7.77(s,0H),7.40(d,J=8.4Hz,0H),7.26(s,1H),7.15(d,J=8.5Hz,1H),4.64(q,J= 6.7Hz, 2H), 4.08 (qd, J=7.1, 2.4Hz, 2H), 2.48 (dd, J=8.6, 1.9Hz, 3H), 2.29 (pd, J=6.8, 2.2Hz, 4H), 1.19 (td, J=7.2, 2.0Hz, 3H).

[0474] Intermediate 2.05b was prepared analogously to general procedure 2 (Example 1). Yield = 70%; m = 1.53 g; 1 H NMR (400MHz, methanol-d4) δ7.90–7.83(m,1H),7.76(s,0H),7.70(dd,J=8.8,2.4Hz,0H),7.59(s,1H),7.44(d,J=8.7Hz,0H),7.31(d,J= 8.6Hz, 1H), 4.77 (q, J = 7.0Hz, 2H), 3.33 (d, J = 3.3Hz, 2H), 2.56 (dd, J = 13.3, 2.5Hz, 3H), 2.36 (t, J = 7.4Hz, 2H), 2.30–2.22 (m, 2H).

[0475] The product 2.05 was prepared similarly to the general procedure 3 (Example 1). Yield = 86%; m = 136 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.02-6.48 (m, 3H), 4.61-4.10 (m, 2H), 4.05-2.81 (m, 17H), 2.47-1.49 (m, 7H).

[0476] -Synthesis of Product 2.06

[0477] Intermediate 2.06a was prepared similarly to general procedure 1 (Example 1). Yield = 40%; m = 2.23 g; 1H NMR (400MHz, chloroform-d) δ7.77(d,J=8.7Hz,1H),7.63(s,1H),7.25(d,J=8.7Hz,1H),4.81(td,J=6.5,1. 9Hz, 2H), 4.15 (qd, J=7.2, 2.0Hz, 2H), 2.53 (s, 3H), 2.48–2.35 (m, 4H), 1.27 (td, J=7.1, 1.9Hz, 3H).

[0478] Intermediate 2.06b was prepared analogously to general procedure 2 (Example 1). Yield = 41%; m = 793 mg; 1 H NMR (400MHz, methanol-d4) δ7.74(dd,J=8.9,2.4Hz,1H),7.61(s,1H),7.28(d,J=8.8Hz,1H),4. 78 (q, J = 4.0 Hz, 2H), 3.33 (d, J = 3.2 Hz, 1H), 2.50 (d, J = 2.5 Hz, 3H), 2.36 (d, J = 3.0 Hz, 4H).

[0479] The product 2.06 was prepared similarly to general procedure 3 (Example 1). Yield = 85%; m = 128 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.92-6.35 (m, 3H), 4.66-4.20 (m, 2H), 4.11-2.83 (m, 17H), 2.67-1.45 (m, 7H).

[0480] -Synthesis of product 2.07

[0481] Intermediate 2.07a was prepared analogously to general procedure 1 (Example 1). Yield = 14%; m = 538 mg; 1 H NMR (400 MHz, CHLOROFORM-d) δ 7.58 (s, 2H), 4.75 (t, J = 6.4 Hz, 2H), 4.11 (q, J = 7.0 Hz, 2H), 2.39 (s, 8H), 2.35 (d, J = 6.4 Hz, 2H), 1.27–1.19 (m, 3H).

[0482] Intermediate 2.07b was prepared analogously to general procedure 2 (Example 1). Yield = 79%; m = 416 mg; 1H NMR (400MHz, methanol-d4) δ7.59 (d, J = 2.8 Hz, 2H), 4.75 (q, J = 4.6 Hz, 2H), 3.33 (d, J = 3.3 Hz, 1H), 2.41 (d, J = 2.7 Hz, 6H), 2.33 (d, J = 4.1 Hz, 4H).

[0483] The product 2.07 was prepared similarly to general procedure 3 (Example 1). Yield = 49%; m = 73 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.34-6.60 (m, 2H), 4.68-4.19 (m, 2H), 4.07-2.84 (m, 18H), 2.69-1.33 (m, 10H).

[0484] -Synthesis of product 2.08

[0485] Intermediate 2.08a was prepared analogously to general procedure 1 (Example 1). Yield = 12%; m = 483m; 1 H NMR (400 MHz, CHLOROFORM-d) δ 7.75 (s, 1H), 7.24 (d, J = 13.6 Hz, 1H), 4.63 (t, J = 6.4 Hz, 2H), 4.14–4.04 (m, 2H), 2.38 (d, J = 10.2 Hz, 6H), 2.33–2.21 (m, 4H), 1.21 (td, J = 7.2, 1.8 Hz, 3H).

[0486] Intermediate 2.08b was prepared analogously to general procedure 2 (Example 1). Yield = 94%; m = 444 mg; 1 H NMR (400 MHz, methanol-d4) δ 7.72 (s, 1H), 7.56 (s, 1H), 4.73 (td, J = 7.0, 2.2 Hz, 2H), 2.45 (dd, J = 14.7, 2.7 Hz, 6H), 2.35 (t, J = 7.2 Hz, 2H), 2.30–2.20 (m, 2H).

[0487] The product 2.08 was prepared similarly to general procedure 3 (Example 1). Yield = 81%; m = 129 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.79-6.33 (m, 2H), 4.55-3.99 (m, 2H), 3.93-2.78 (m, 18H), 2.67-1.22 (m, 10H).

[0488] -Synthesis of product 2.09

[0489] Intermediate 2.09a was prepared analogously to general procedure 1 (Example 1). Yield = 33%; m = 2.22 g; 1 H NMR (400MHz, chloroform-d) δ7.94–7.87(m,1H),7.30–7.19(m,2H),4.93(t,J=6.1Hz,2H),4.31(qd,J=7. 2, 2.0Hz, 2H), 4.07 (d, J = 1.9Hz, 3H), 2.59 (dt, J = 16.1, 5.2Hz, 4H), 1.43 (td, J = 7.2, 1.9Hz, 3H).

[0490] Intermediate 2.09b was prepared similarly to general procedure 2 (Example 1). Yield = 70%; m = 1.49 g; 1 H NMR (400MHz, methanol-d4) δ7.72 (dd, J=9.1, 2.5Hz, 1H), 7.14 (s, 1H), 7.08 (d, J=9. 3Hz, 1H), 4.75 (q, J = 3.8Hz, 2H), 3.89 (d, J = 2.6Hz, 3H), 2.35 (d, J = 2.9Hz, 4H).

[0491] The product 2.09 was prepared similarly to general procedure 3 (Example 1). Yield = 90%; m = 146 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.87-6.34 (m, 3H), 4.57-4.12 (m, 2H), 4.03-2.76 (m, 20H), 2.58-1.29 (m, 4H).

[0492] -Synthesis of Product 2.10

[0493] Intermediate 2.10a was prepared analogously to general procedure 1 (Example 1). Yield = 23%; m = 1.69 g; 1H NMR (400MHz, chloroform-d) δ7.78 (dd, J=9.1, 1.9Hz, 1H), 7.34 (dd, J=9.1, 1.9Hz, 0H), 7.26 (d, J=2.3Hz,0H),7.05(dd,J=9.0,2.2Hz,0H),6.89(dd,J=9.1,2.2Hz,1H),6.73(d,J=2.2 Hz, 1H), 4.55 (dtd, J=13.6, 6.7, 1.9Hz, 2H), 4.01 (q, J=7.1Hz, 2H), 3.78 (dd, J=6.6, 1. 8Hz, 3H), 3.04 (s, 0H), 2.22 (ddd, J=19.8, 7.7, 4.2Hz, 4H), 1.13 (td, J=7.1, 1.8Hz, 3H).

[0494] Intermediate 2.10b was prepared analogously to general procedure 2 (Example 1). Yield = 70%; m = 1.16 g; 1 H NMR (400MHz, methanol-d4) δ7.81(dd,J=9.1,2.7Hz,1H),7.67(dd,J=8.9,2.7Hz,0H),7.33(s,0H),7.24–7.15(m,1H),7.05(dd,J=9.1,2.7H z, 1H), 4.73 (qd, J = 7.1, 2.3Hz, 2H), 3.91 (dd, J = 13.2, 2.8Hz, 3H), 3.33 (d, J = 3.2Hz, 0H), 2.35 (t, J = 6.7Hz, 2H), 2.26 (t, J = 7.3Hz, 2H).

[0495] The product 2.10 was prepared similarly to the general procedure 3 (Example 1). Yield = 96%; m = 153 mg; 1 H NMR (400 MHz, deuterium oxide) δ 7.99-6.22 (m, 3H), 4.49-4.03 (m, 2H), 3.96-2.80 (m, 21H), 2.63-1.55 (m, 4H).

[0496] -Synthesis of product 2.11

[0497] Intermediate 2.11a was prepared analogously to general procedure 1 (Example 1). Yield = 77%; m = 1.09 g; 1H NMR (400 MHz, CHLOROFORM-d) δ 8.83–8.57 (m, 1H), 8.40–8.19 (m, 1H), 7.42–7.30 (m, 1H), 4.89–4.78 (m, 2H), 4.17–4.04 (m, 2H), 2.52–2.28 (m, 5H), 1.22 (tdd, J = 7.1, 4.1, 0.9 Hz, 3H).

[0498] Intermediate 2.11b was prepared analogously to general procedure 2 (Example 1). Yield = 100%; m = 1.16 g; 1 H NMR (400 MHz, deuterium oxide) δ 8.59–8.49 (m, 2H), 8.31–8.08 (m, 2H), 7.42–7.32 (m, 2H), 4.70–4.63 (m, 2H), 4.63–4.54 (m, 2H), 2.29–2.16 (m, 2H), 2.20–2.05 (m, 7H).

[0499] The product 2.11 was prepared similarly to the general procedure 3 (Example 1). Yield = 56%; m = 43 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.95–6.92 (m, 3H), 4.11–2.95 (m, 18H), 2.90–1.23 (m, 5H).

[0500] -Synthesis of product 2.12

[0501] Intermediate 2.12a was prepared analogously to general procedure 1 (Example 1). Yield = 18%; m = 254 mg; 1 H NMR (400MHz, chloroform-d) δ8.53 (dd, J=4.4, 1.5Hz, 1H), 7.82 (dd, J=8.4, 1.5Hz, 1H), 7.24 (dd, J=8.4, 4. 4Hz, 1H), 4.54 (t, J = 6.8Hz, 2H), 3.89 (q, J = 7.1Hz, 2H), 2.20–2.05 (m, 4H), 1.01 (t, J = 7.1Hz, 3H).

[0502] Intermediate 2.12b was prepared analogously to general procedure 2 (Example 1). Yield = 98%; m = 242 mg; 1H NMR (400 MHz, deuterium oxide) δ 8.59 (dq, J = 4.5, 1.5 Hz, 1H), 8.21 (dt, J = 8.5, 1.6 Hz, 1H), 7.52 (ddt, J = 8.5, 4.5, 1.4 Hz, 1H), 4.70–4.62 (m, 2H), 2.21–2.04 (m, 4H).

[0503] The product 2.12 was prepared similarly to the general procedure 3 (Example 1). Yield = 75%; m = 54 mg; 1 H NMR (400 MHz, deuterium oxide) δ 9.06–6.76 (m, 3H), 4.16–2.96 (m, 20H), 2.93–1.52 (m, 4H).

[0504] -Synthesis of product 2.13

[0505] Intermediate 2.13a was prepared analogously to general procedure 1 (Example 1). Yield = 43%; m = 736 mg; 1 H NMR (400 MHz, CHLOROFORM-d) δ 9.41 (d, J = 1.4 Hz, 1H), 8.44 (d, J = 6.1 Hz, 1H), 7.74 (dd, J = 6.1, 1.4 Hz, 1H), 4.87 (t, J = 6.5 Hz, 2H), 4.11 (q, J = 7.1 Hz, 2H), 2.50–2.39 (m, 2H), 2.41–2.31 (m, 2H), 1.22 (t, J = 7.1 Hz, 3H).

[0506] Intermediate 2.13b was prepared analogously to general procedure 2 (Example 1). Yield = 100%; m = 707 mg; 1 H NMR (400 MHz, deuterium oxide) δ 9.25 (d, J = 1.4 Hz, 1H), 8.27 (dd, J = 6.3, 1.2 Hz, 1H), 7.77 (dt, J = 6.4, 1.6 Hz, 1H), 4.79 (t, J = 6.9 Hz, 2H), 2.26 (dqd, J = 7.8, 6.9, 0.9 Hz, 2H), 2.11 (ddd, J = 8.0, 7.1, 1.0 Hz, 2H).

[0507] The product 2.13 was prepared similarly to the general procedure 3 (Example 1). Yield = 95%; m = 65 mg; 1H NMR (400 MHz, deuterium oxide) δ 10.06–7.98 (m, 3H), 4.07–2.89 (m, 27H), 2.83–2.11 (m, 4H).

[0508] -Synthesis of product 2.14

[0509] Intermediate 2.14a was prepared analogously to general procedure 1 (Example 1). Yield = 52%; m = 600 mg; 1 H NMR (400 MHz, CHLOROFORM-d) δ 9.51–9.16 (m, 1H), 8.60–8.50 (m, 1H), 7.96–7.52 (m, 1H), 4.90–4.71 (m, 2H), 4.10 (dq, J = 8.6, 7.1 Hz, 2H), 2.43–2.30 (m, 4H), 1.23 (t, J = 7.2 Hz, 3H).

[0510] Intermediate 2.14b was prepared analogously to general procedure 2 (Example 1). Yield = 97%; m = 550 mg; 1 H NMR (400 MHz, deuterium oxide) δ 9.27–9.11 (m, 1H), 8.43–8.27 (m, 1H), 7.95–7.69 (m, 1H), 4.84–4.63 (m, 2H), 2.28–2.05 (m, 4H).

[0511] The product 2.14 was prepared similarly to the general procedure 3 (Example 1). Yield = 95%; m = 68 mg; 1 H NMR (400 MHz, deuterium oxide) δ 10.08–8.17 (m, 3H), 4.21–2.84 (m, 25H), 2.83–1.64 (m, 4H).

[0512] -Synthesis of product 2.15

[0513] The product 2.15 was prepared similarly to general procedure 3. Yield = 88%; m = 74 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.98–7.01 (m, 3H), 4.07–3.06 (m, 14H), 2.75–1.45 (m, 4H).

[0514] -Synthesis of product 2.16

[0515] The product 2.16 was prepared similarly to the general procedure 3 (Example 1). Yield = 46%; m = 42 mg; 1 H NMR (400 MHz, deuterium oxide) δ 8.97–6.90 (m, 3H), 4.07–2.81 (m, 12H), 2.60–1.43 (m, 4H).

[0516] -Synthesis of product 2.17

[0517] The product 2.17 was prepared similarly to the general procedure 3 (Example 1). Yield = 97%; m = 82 mg; 1 H NMR (400 MHz, deuterium oxide) δ 10.03–8.05 (m, 3H), 4.20–3.04 (m, 14H), 2.81–1.60 (m, 4H).

[0518] -Synthesis of product 2.18

[0519] The product 2.18 was prepared similarly to the general procedure 3 (Example 1). Yield = 97%; m = 82 mg; 1 H NMR (400 MHz, deuterium oxide) δ 10.02–8.30 (m, 3H), 3.54 (s, 15H), 2.93–1.88 (m, 4H).

[0520] Example 3. Compounds 1.01 to 1.50

[0521] Screening for transfection activity

[0522] Compounds 1.01 to 1.50 were evaluated for their ability to transfect DNA into four different cell lines. Many cell lines were first transfected with commercially available transfection reagents (see Table 2). Plasmid pCMV-EGFPLuc encoding green fluorescent protein (GFP) was used, and one day after transfection, the percentage of cells expressing GFP (% GFP) was analyzed to determine the transfection efficiency of the 96-well plate format by using a cell counting assay. Table 2 presents the results of commercial reagents used under their optimal conditions for the following four 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). The results show that these cells are relatively difficult to transfect because the highest transfection efficiencies in Caco-2, Hep G2, MDCK and MCF-10A are less than 40%, 28%, 40% and 22%, respectively. These cell lines were selected to screen the transfection activity of compounds 1.01 to 1.50.

[0523] Table 2. Commercially available reagents ( From Polyplus-transfection From Promega From MirusBio X-tremeGENE from Roche Life Science From Thermo Fisher 3000) were transfected into Caco-2, Hep G2, MDCK and MCF-10A.

[0524]

[0525] Compounds 1.01 to 1.50 ( 1.01 to 1.50) were prepared in 96-well plates by transfecting 200 ng of pCMV-EGFPLuc DNA (Clontech) complexed with 0.6 μL or 0.8 μL of one compound of the invention (i.e., one selected from compounds 1.01 to 1.50) (under 7.5 mM nitrogen concentration). Figure 1 ) were screened to define the ratio of 1 μg DNA / 3 μL compound (ratio 1:3) or 1 μg DNA / 4 μL compound (ratio 1:4). The percentage of cells expressing GFP (% GFP) was determined by cell counting one day after transfection. As a control, a 22 KDa linear polyethyleneimine and representing the parent cationic polymer backbone of compounds 1.01 to 1.50 was transfected.

[0526] Compounds containing a benzimidazole ring (wherein Y 1 =Y 3 =N,Y 2 =C, and R, T, U, V form a benzene ring) showed that transfection using products 1.07 to 1.08, 1.14 to 1.22, 1.25, 1.27 to 1.30, 1.32, 1.35 to 1.37, 1.41, 1.42 was effective in the four cell lines tested and had similar efficiency attributes. Among these compounds containing a benzimidazole ring, when Z 2 When H is not present, many compounds are Showing improved efficiency, such as 1.09, 1.10, 1.15, 1.27, 1.28, 1.30, 1.32, 1.35, 1.36, 1.37, 1.41, 1.42, where Z 2 =CH3; or 1.14, where Z 2 =CF3; or 1.17, where Z 2 =S-CH3; 1.18, where Z2 = isopropyl; or 1.19, wherein Z 2 = propyl; 1.20, where Z 2 =CH2-NH-CH3; 1.21, 1.22, where Z 2 =CH2-O-CH3. At position Z 4 , Z 5 or Z 6 Other substitutions on the phenyl ring at 1.30 provide very effective compounds such as 1.30, in which Z 5 =O-CH3; 1.32, where Z 6 = isopropyl; 1.35, 1.36, where Z 6 =CH3; 1.37, where Z 4 =CH3; 1.41, 1.42, where Z 5 =Z 6 =CH3. Taken together, the data suggest that chemical diversity can be introduced into compounds containing a benzimidazole ring, which may benefit transfection efficiency.

[0527] When with When compared, compounds containing a benzopyrazole ring (1.44, 1.45, 1.48, 1.49, 1.50, wherein Y 1 =Y 2 =N,Y 3 =C, and R, T, U, V form a benzene ring) showed moderate transfection efficiency. Therefore, compound 1.49 showed high levels of transfection, especially in MDCK cells. 6 (1.43, 1.46) or Z 4 The introduction of an amino group into (1.47) is also acceptable.

[0528] Example 4. Compounds 2.01 to 2.18

[0529] Screening for transfection activity

[0530] Compounds 2.01 to 2.18 ( 2.01 to 2.18 ) were screened in a transfection similar to that previously described for the compounds of Example 3 in 96-well plates by transfecting 200 ng of pCMV-EGFPLuc DNA (Clontech) complexed with 0.6 μL or 0.8 μL of one compound of the invention ( 2.01 to 2.18 ) (at 7.5 mM nitrogen concentration). Figure 2 ), thereby defining a ratio of 1 μg DNA / 3 μL compound or a ratio of 1 μg DNA / 4 μL compound, respectively.

[0531] The benzotriazole ring derivatives (2.01 to 2.10) were found to be the most interesting compounds in Example 4 based on their transfection activity in transfecting Caco-2, Hep G2, MCF-10A and MDCK. 1 or Y 2 (Z 1 or Z 2 ) seems to have less influence on transfection, but the degree of grafting on the benzotriazole ring polymer has a greater influence on the results. 5 and / or Z 6 Addition of methyl or methoxy groups may enhance transfection activity as exemplified by compounds 2.05, 2.08, 2.09 or 2.10.

[0532] Example 5. Heterocyclic Grafting Degree and Cationic Polymer Length

[0533] The degree of grafting of heterocycles to cationic polymers is a key factor in regulating transfection activity. Figure 3 The grafting effect of 2-methylbenzimidazole on 22 kDa linear PEI is illustrated, where In comparison, a grafting degree of 14% to 25% provides very effective compounds in transfection, with the optimum being close to 20%. This is also exemplified by compounds 1.28, 1.25 and 1.27 grafted to linear PEI 10 kDa, which also demonstrates that a grafting degree of about 20% is of interest in transfection ( Figure 1 ).

[0534] Additionally, cationic polymer length may affect transfection activity, as exemplified by compounds 1.24, 1.25, and 1.26, in which polymers with average molecular weights of 22 kDa, 10 kDa, and 6 kDa, respectively, were grafted to the same extent with 2-methylbenzimidazole rings ( Figure 1 ).

[0535] Example 6. Transfection of primary cells

[0536] Attempts were made to select the best compounds from Examples 3 and 4 to transfect primary cells known to be difficult to transfect ( Figure 4 ), such as primary dermal fibroblasts (HPDF) and primary endothelial cells (HUVEC). Primary cells were cultured in 24-well format using their specific media conditions (see Materials and Methods). Cells were incubated with 500 ng of pCMV-EGFPLuc complexed with 1.5 μL of 50 μL of buffer containing compound 1.41, 2.03, 2.05, 1.42, or 2.08 (ratio 1 μg DNA: 3 μL compound) or with 3000 (ratio 1 μg DNA: 1.5 μL reagent) was used for transfection. After 2 or 4 hours, the transfection medium was removed and replaced with complete medium. 24 hours after transfection, GFP expression analysis was performed by cell counting analysis.

[0537] GFP expression results showed that all selected compounds were able to compare well with the commercial reference 3000 more effectively transfected HPDF and HUVEC cells, among which compound 2.03 achieved a transfection efficiency of about 50% for HPDF, and compounds 1.41 and 1.42 had transfection efficiencies exceeding 70%. These results indicate that the compounds of the present invention have great potential, in which a variety of heterocycles may be grafted onto cationic polymers.

[0538] It is known that transfection of primary neurons is very difficult because these non-dividing cells have very limited access to the nucleus for the transfected DNA. In addition, these cells are very fragile. The inventors have tested compounds close to structure 1.42 (such as compounds 1.56 to 1.72) and found that compounds 1.65 or 1.60 showed very impressive primary neuron transfection results. Figure 5 illustrates the results obtained when compound 1.65 is compared with commercially available transfection reagents.

[0539] Primary rat cortical neurons (RCN) and primary rat hippocampal neurons (RHN) were cultured in complete medium for 4 days according to the recommended commercial protocol and then transfected with 150 ng of pCMV-EGFPLuc plasmid complexed with 0.15 μL of 25 μL of OPTIMEM containing compound 1.65 or with 0.6 μL of 2000 (ratio 1μg:4μL) and (ratio 1 μg:2 μL) complex. 24 hours after transfection, cells were observed by using a fluorescent cell imager.

[0540] Discover Both RCN and RHN could not be efficiently transfected, while 2000 provided a significant level of transfection efficiency. Therefore, it was demonstrated that compound 1.65 transfected both RCN and RHN well with higher efficiency and did not affect cell morphology (cell dendrites were clearly visible). The morphology of cells transfected with 2000 was clearly affected, with few remaining dendrites, indicating a toxic effect.

[0541] Example 7. Biological production of recombinant viruses

[0542] DNA transfection is one of the common techniques for biological production of recombinant proteins and viruses through transient gene expression (TGE) processes. Regarding the production of AAV and lentivirus, the most commonly used method is transfection for delivering viral and therapeutic genes into producer cell lines (HEK293 adherent or suspension cells). In most systems, co-transfection of many plasmids is performed by chemical methods, such as coprecipitation with calcium phosphate or by cationic polymers such as polyethyleneimine (PEI) (such as (Polyplus-transfection))-mediated transfection.

[0543] AAV and lentiviral particles were produced from HEK-293T cells by transient co-transfection of several plasmids containing the gene of interest and the necessary viral components to produce complete recombinant viral particles. AAV-2 and lentiviral vectors expressing the GFP reporter gene were produced with various compounds, and viral productivity was determined by assessing transducing units (TU / mL) 3 days after transfection. Productivity levels were compared with the use of a widely used adherent and suspension virus production system. The productivity levels obtained with the transfection reagents were compared.

[0544] Many of the compounds of Examples 3 and 4 were tested for AAV-2 production, and Figure 6 Some of the results obtained are presented. Some compounds performed similarly in terms of viral production when used in a 1:2 ratio for transfection (1 μg total DNA per μL compound). Similar, but most of these compounds significantly increased viral titers by 3- to 8-fold.

[0545] Similarly, lentivirus was produced in suspension HEK-293T cells after co-transfection of four plasmids (pRSV-REV packaging vector, pCgpV packaging vector, pCMV-VSV-G envelope vector, and pLenti6.3 / V5-GW / EmGFP expression control vector). Lentivirus titers (TU / mL) were determined 72 hours after transfection ( Figure 7 ).

[0546] Example 8. CRISPR Cas9 DNA transfection

[0547] CRISPR-Cas9 technology was used to introduce deletions into the targeted human HPRT-1 gene. Plasmids encoding both the spCas9 protein and guide RNA were introduced into HEK293 cells by transfection with compound 1.42.

[0548] Two days after transfection, genomic DNA was extracted and PCR was performed using HPRT-1 specific primers. Genome editing events were analyzed by T7 endonuclease assay and visualized on agarose gels and quantified using ethidium bromide staining to determine % INDEL (percentage of insertion / deletion CRISPR events). Transfected plasmid p38285 expressing spCas9 and specific HPRT-1 guide RNA showed the presence of two expected bands of 650 bp and 430 bp on the gel ( Figure 8 ). %INDEL (Insertion-deletion mutation event) was 33.48+ / -7.08%. The specificity of CRISPR Cas9 transfection was shown as the specific signal of the cleavage band observed after transfection of the plasmid targeting the HPRT-1 gene, which was not observed using the plasmid control. The experiment demonstrated that compound 1.42 used for transfection effectively induced CRISRP Cas9 genome modification without generating off-target events.

[0549] Example 9. Transfection assay of stem cells

[0550] Primary human mesenchymal stem cells (hMSCs) were transfected with compound 1.42 and different amounts of pCMV-EGFP DNA, 400 and 500 ng per well, in a 24-well plate format ( Fig. 9 ). Different volumes of compound 1.42 were used per DNA amount. One day after transfection, GFP expression was analyzed by flow cytometry or by using ZOE TM Cells were observed using a fluorescent cell imager. For the 400ng DNA condition, the stepwise increase in transfection efficiency was quantified when the volume of compound 1.42 was increased to achieve optimal transfection of up to 60% GFP-positive cells. For the 500ng DNA condition, the optimal condition of more than 60% GFP-positive cells was obtained with 0.75 μL of compound 1.42. As a control, transfection with Lipofectamine 3000 reagent provided a transfection efficiency of less than 10%. These results indicate that optimization of transfection conditions can be achieved by changing both the amount of DNA transfected and the volume of the compound.

[0551] Example 10. Compounds 1.73 to 1.80

[0552] The inventors have performed some comparative data using imidazole derivatives such as compounds 1.73, 1.78 and 1.80 (see Table 3). The synthesis of the compounds is reported in Example 2.

[0553] Table 3. Imidazole derivatives

[0554]

[0555]

[0556] Screening for transfection activity

[0557] Compounds 1.73 to 1.80 were evaluated for their ability to transfect DNA (pCMV-EGFPLuc) into Hep G2 cells and transfection efficiency in a 96-well plate format was determined by analyzing the percentage of cells expressing GFP (% GFP) by flow cytometry one day after transfection ( Fig.11 Compounds 1.73 to 1.80 were mixed with compound 1.42 (where Z 2 , Z 4 and Z 6 =CH3). This benzene ring derivative was grafted onto many cationic polymers, including branched PEI (25K or 10K), poly(allylamine) (PAA, 15K), polylysine (PLL, 22K) or poly(vinylamine) (PVA, 25K). The presence of the benzimidazole ring showed higher transfection efficiency when compared to the unmodified parent polymer. Compounds 1.42 and 1.74, in which the parent polymer is PEI, particularly show this effect. In addition, when compared to compound 1.73 comprising an imidazole ring grafted onto linear PEI 22K, compound 1.42 with a benzimidazole ring showed higher transfection efficiency.

[0558] Biological production of recombinant viruses

[0559] Compounds 1.73 to 1.80 were tested for AAV-2 production and Fig.12 Results obtained at the optimal ratio μg DNA / μL compound are presented. The presence of the benzimidazole ring showed higher AAV-2 production rates when compared to the unmodified parent polymer, especially for compounds 1.42 and 1.74 (linear 22K or branched 25K, respectively) where the parent polymer was PEI. Compound 1.42 showed significantly higher virus production when compared to compound 1.73 comprising an imidazole ring grafted onto linear PEI 22K.

[0560] in conclusion

[0561] Many compounds based on the grafting of polyamines with heterocycles of formula (II) show improved properties to induce gene expression in "difficult to transfect" cells (such as cancer cells, primary cells, non-dividing cells), or to increase the production rate of biologics (such as viruses, AAV or LV).

[0562] When compared with the unmodified parent linear PEI When compared to the linear modified PEI containing an imidazole ring, or compared to the cationic polymers that exhibited benzimidazole or imidazole grafts in the four cancer cell lines tested, many compounds containing benzimidazole rings of Examples 3 and 10 (where Y 1 =Y 3 =N,Y 2 =C, and R, T, U, V form a benzene ring) showed higher transfection efficiency. Among the various structures tested, when Z 2 Not H, especially Z 2 =CH3, and Z 4 , Z 5 or Z 6 Not H, especially Z 4 , Z 5 or Z 6 =CH3, these compounds provided the highest transfection efficiency.

[0563] The compounds of Example 3 containing a benzopyrazole ring (wherein Y 1 =Y 2 =N,Y 3 =C, and R, T, U, V form a benzene ring) showed promising activity in transfection.

[0564] These results were also confirmed after transfection of primary cells (particularly non-dividing cells such as primary neurons, as well as fragile cells and "difficult to transfect cells" such as primary fibroblasts, endothelial cells or stem cells).

[0565] Many compounds of Example 4 (especially the polyamine grafted with a benzotriazole derivative) showed high transfection efficiency, similar to the best compound of Example 3.

[0566] The selected compounds of Examples 3 and 4 also showed an increased production rate of biologics such as AAV or LV, indicating that the combined effect of high transfection efficiency and gene expression in the cells resulted in high viral titers expressed as transducing units. The increased viral production rate was observed regardless of the type of transfected cells (e.g. adherent or suspended). The results obtained suggest that such compounds may also be of interest for the production of other biologics such as recombinant proteins, peptides or antibodies.

[0567] In summary, the compounds of formula (II) of the present invention represent new agents for transfection and bioproduction purposes, where a careful optimization of the chemical structure can be adapted to each application, cell type or transfection condition.

[0568] Those skilled in the art can adapt the transfection method using the compound of formula (II) of the present invention to the specific cell type, cell culture conditions or cell culture device used. Specifically, the amount of DNA, the amount of transfection reagent, the volume of the transfection complex, the conditions of DNA mixing with the transfection reagent, the culture medium for the preparation of the transfection complex can be changed. In addition, the transfection method using the compound of formula (II) of the present invention can be suitable for industrial use, particularly for large-scale applications in bioreactors for both adherent cells and suspended cells.

[0569] Those skilled in the art can use acceptable excipients or buffers to make the transfection method using the compound of formula (II) of the present invention suitable for in vivo application. The compound of formula (II) can be mixed with DNA to generate a DNA complex suitable for direct injection into animals or humans. It is known that particularly low-salt buffers (such as TRIS, phosphate, or citrate buffer) or excipients (such as glucose, dextrose or maltose) can provide acceptable formulations for direct injection into animals and humans. Many mixing methods of DNA with compounds of formula (II) are suitable because they can generate formulations containing small-sized particles (non-aggregated DNA complexes) that can be injected by various routes of administration.

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Claims

1. A composition for transfecting a nucleic acid molecule into a cell, the composition comprising (i) at least one compound of general formula (II) or an acceptable salt thereof, and (ii) an acceptable excipient or cell culture medium: in: -(i)Y 1 and Y 3 Represents N, Y 2 Represents C; Z 1 Represents X1-R3-X2-P + ; Z 2 Represents H, CH3, SCH3, CF3, phenyl, isopropyl, propyl, CH2-NH-CH3, CH2-O-CH3; Z 3 Does not exist; Z 4 Represents H or CH3; Z 5 represents H, F, OCH3, tert-butyl, or CH3; Z 6 represents H or CH3; and Z 7 represents H; or -(ii)Y 1 , Y 2 and Y 3 Represents N; Z 1 Represents X1-R3-X2-P + , or Z 1 Does not exist; Z 2 Represents X1-R3-X2-P + , or Z 2 does not exist; the condition is Z 1 or Z 2 Only one of them represents X1-R3-X2-P + ; Z 3 Does not exist; Z 4 Represents H; Z 5 represents H, CH3, or OCH3; Z 6 represents H, CH3, or OCH3; Z 7 Represents H; -X1 represents CH2; -X2 represents CO; -R3 represents (CH2) m , wherein m represents an integer from 1 to 3; -P + represents a grafted cationic polymer, which is a linear polyethyleneimine (PEI), whose grafting rate is 7 to 30%, and whose average Mw is 8, 10, 15, 22 or 30 kDa; -R, T, U and V stand for C.

2. The composition according to claim 1, wherein the cell culture medium is a transfection medium.

3. The composition of claim 1, wherein the cell is a eukaryotic cell. The composition according to claim 1 , further comprising at least one nucleic acid molecule to be transfected into cells.

5. The composition of claim 4, 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, and an expression vector encoding the nucleic acid molecule.

6. The composition of claim 5, wherein the at least one nucleic acid molecule is selected from the group consisting of short interfering RNA (siRNA), micro RNA (miRNA), short hairpin RNA (shRNA), messenger RNA (mRNA) and CRISPR guide RNA.

7. The composition according to claim 5, wherein the expression vector encoding the nucleic acid molecule is a plasmid encoding the nucleic acid molecule or a plasmid expressing the nucleic acid molecule.

8. The composition of claim 5, wherein the at least one nucleic acid molecule is DNA.

9. The composition according to claim 1, wherein the grafting rate of the grafted cationic polymer is 14 to 25%.

10. The composition according to claim 9, wherein the grafting rate of the grafted cationic polymer is 20%, 22% or 23%.

11. The composition of claim 1, wherein the grafted cationic polymer has an average Mw of 10, 15 or 22 kDa.

12. The composition of claim 11, wherein the grafted cationic polymer has an average Mw of 22 kDa.

13. A composition for transfecting a nucleic acid molecule into a cell, the composition comprising (i) at least one compound or an acceptable salt thereof, and (ii) an acceptable excipient or cell culture medium, wherein the at least one compound is selected from the following compounds:

14. The composition according to claim 13, wherein the at least one compound is selected from the following compounds:

15. The composition of claim 14, wherein the at least one compound is compound 1.42, 1.57 or 1.

65.

16. A method for in vitro or ex vivo transfection of living cells, comprising introducing the composition according to claim 4 into said cells.

17. Use of the composition according to claim 4 for transfecting at least one nucleic acid molecule into a cell in vitro or ex vivo.

18. In vitro or ex vivo use of a composition according to claim 17, wherein the cells are organized into spheroids, organoids, 2D or 3D cell cultures, or provided as fiber or matrix cultures, and / or within a bioreactor.

19. The in vitro or ex vivo use of the composition according to claim 17, wherein the cells are selected from the group consisting of mammalian cells, insect cells, primary cells, adherent cells, suspension cells, dividing cells, non-dividing cells and cancer cells.

20. The in vitro or ex vivo use of the composition according to claim 19, wherein the dividing cells are stem cells.

21. An in vitro or ex vivo use of the composition according to claim 19, wherein the non-dividing cells are neuronal cells.

22. The in vitro or ex vivo use according to claim 17, for transfecting at least one nucleic acid molecule into stem cells, the composition comprising (i) compound 1.42, and (ii) an acceptable excipient or cell culture medium.

23. The in vitro or ex vivo use according to claim 22, wherein the at least one nucleic acid molecule is at least one DNA.

24. The in vitro or ex vivo use according to claim 17, for transfecting at least one nucleic acid molecule into neuronal cells, the composition comprising (i) compound 1.65 or compound 1.60, and (ii) an acceptable excipient or cell culture medium.

25. The in vitro or ex vivo use according to claim 24, wherein the at least one nucleic acid molecule is at least one DNA.

26. The composition according to claim 4 is used for in vitro or ex vivo use of genome engineering, for cell reprogramming, for differentiating cells or for gene editing.

27. A method for producing: (i) a biologic, wherein a composition according to claim 4 is used; or (ii) a recombinant virus, wherein a composition according to claim 4 is used, said composition comprising multiple nucleic acid molecules for co-transfection; or (iii) a viral or virus-like particle, wherein a composition according to claim 4 is used, said composition comprising multiple nucleic acid molecules for co-transfection.

28. The method of claim 27, wherein (i) the biologic is a biologic encoding a recombinant protein, peptide, or antibody; or (ii) the recombinant virus is an adeno-associated virus (AAV), a lentivirus (LV), an adenovirus, an oncolytic virus, or a baculovirus.

29. The method of claim 27 for producing AAV or LV, the composition comprising (i) at least compound 1.42, and (ii) an acceptable excipient or cell culture medium.

30. The method of claim 27 for producing LV, wherein the composition comprises (i) at least compound 1.57, and (ii) an acceptable excipient or cell culture medium.

31. The method of claim 29 or 30, wherein the cell culture medium is a transfection medium.

32. The method according to claim 27, for producing recombinant viruses, said composition comprising a plurality of expression vectors for transfection in adherent cells or suspension cells or insect cells, wherein said vectors are constructs expressing viral structural sequences for virus or virus-like production and transfer vector genomes.

33. The method of claim 32, wherein the expression vector is a plasmid vector.

34. The method of claim 32, wherein the adherent or suspension cells are HEK293, HeLa, BHK-21 or A549.

35. The method of claim 32, wherein the vector is a construct that further expresses a molecule of interest encoded by the transfer vector genome.

36. The method of claim 32, wherein the recombinant virus is used for in vivo application of cell therapy or gene therapy.

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