COMPOSITIONS FOR TRANSFECTING A NUCLEIC ACID MOLECULE INTO A CELL COMPRISING TRIAZOLE COMPOUNDS GRAFTED TO A CATIONIC POLYMER, AND THEIR APPLICATIONS

Abstract

Disclosed are compositions for transfecting a nucleic acid molecule into a cell and their applications. Specifically, this relates to a composition suitable for transfecting a nucleic acid molecule into a cell, preferably a eukaryotic cell, including (i) at least one compound of general formula (I), preferably of general formula (III), or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or an acceptable salt thereof, and (ii) an acceptable excipient, buffering agent, cell culture medium, or transfection medium, wherein Y.sup.1, Y.sup.2, Y.sup.3, Z.sup.1, Z.sup.2, Z.sup.3, X.sub.1, X.sub.2, R.sub.3, P.sup.+, R and V are as defined in the description. Also disclosed are uses of the composition and to a method for in vitro or ex vivo transfection of live cells.

Claims

1. A composition suitable for transfecting a nucleic acid molecule into a cell, preferably a eukaryotic cell, comprising (i) at least one compound of general formula (III) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or an acceptable salt thereof, and (ii) an acceptable excipient, buffering agent, cell culture medium, or transfection medium: ##STR00144## wherein: Z.sup.1 represents H, X.sub.1—R.sub.3—X.sub.2—P.sup.+, X.sub.1—R.sub.3—P.sup.+, X.sub.1—X.sub.2—P.sup.+, R.sub.3—X.sub.2—P.sup.+, X.sub.1—P.sup.+, R.sub.3—P.sup.+, or X.sub.2—P.sup.+; or Z.sup.1 is absent; Z.sup.2 represents H, a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkyl, C.sub.6-C.sub.18 aryl, a linear or branched, saturated or unsaturated C.sub.6-C.sub.18 aryl-C.sub.1-C.sub.18 alkyl, a linear or branched, saturated or unsaturated C.sub.2-C.sub.18 heteroalkyl, C.sub.5-C.sub.10 heteroaryl, halogen, OH, a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkylamine, a C.sub.1-C.sub.12 alkoxy, a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkyl-C.sub.1-C.sub.12 alkoxy, X.sub.1—R.sub.3—X.sub.2—P.sup.+, X.sub.1—R.sub.3—P.sup.+, X.sub.1—X.sub.2—P.sup.+, R.sub.3—X.sub.2—P.sup.+, X.sub.1—P.sup.+, R.sub.3—P.sup.+, or X.sub.2—P.sup.+; or Z.sup.2 is absent; Z.sup.3 represents H, a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkyl, C.sub.6-C.sub.18 aryl, a linear or branched, saturated or unsaturated C.sub.6-C.sub.18 aryl-C.sub.1-C.sub.18 alkyl, C.sub.5-C.sub.10 heteroaryl, a linear or branched, saturated or unsaturated C.sub.2-C.sub.18 heteroalkyl, C.sub.2-C.sub.18 alkylidene, OH, guanidine, halogen, X.sub.1—R.sub.3—X.sub.2—P.sup.+, X.sub.1—R.sub.3—P.sup.+, X.sub.1—X.sub.2—P.sup.+, R.sub.3—X.sub.2—P.sup.+, X.sub.1—P.sup.+, R.sub.3—P.sup.+, or X.sub.2—P.sup.+; or Z.sup.3 is absent; X.sub.1 and X.sub.2, which may be identical or different, represent CO or CH.sub.2; R.sub.3 represents (CH.sub.2).sub.m, (CH.sub.2).sub.m—CHCH.sub.3—(CH.sub.2).sub.n—, (CH.sub.2).sub.m—C(CH.sub.3).sub.2—(CH.sub.2).sub.n—, (CH.sub.2).sub.m—O—(CH.sub.2).sub.n—, (CH.sub.2).sub.m—S—(CH.sub.2).sub.n—, (CH.sub.2).sub.m—CH.sub.2—O—, with m representing an integer between 1 and 3 and n representing an integer between 1 and 3; P.sup.+ represents a graft cationic polymer, which is a polyamine comprising secondary amines, tertiary amines, a mixture of primary and secondary amines, a mixture of primary and tertiary amines, a mixture of secondary and tertiary amines, or a mixture of primary, secondary and tertiary amines; R or V represents H, a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkyl or cycloalkyl, a C.sub.6-C.sub.18 aryl, a linear or branched, saturated or unsaturated C.sub.6-C.sub.18 aryl-C.sub.1-C.sub.18 alkyl, a linear or branched, saturated or unsaturated C.sub.2-C.sub.18 heteroalkyl, a linear or branched, saturated or unsaturated C.sub.1-C.sub.24 ester, a C.sub.5-C.sub.10 heterocyclyl, a C.sub.5-C.sub.10 heteroaryl, a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkyl-C.sub.5-C.sub.10 heteroaryl, X.sub.1—R.sub.3—X.sub.2—P.sup.+, X.sub.1—R.sub.3—P.sup.+, X.sub.1—X.sub.2—P.sup.+, R.sub.3—X.sub.2—P.sup.+, X.sub.1—P.sup.+, R.sub.3—P.sup.+, or X.sub.2—P.sup.+; with the provisos that: at least one of Z.sup.1, Z.sup.2 or Z.sup.3 is present; and only one of Z.sup.1, Z.sup.2, Z.sup.3, R or V represents X.sub.1—R.sub.3—X.sub.2—P.sup.+, X.sub.1—R.sub.3—P.sup.+, X.sub.1—X.sub.2—P.sup.+, R.sub.3—X.sub.2—P.sup.+, X.sub.1—P.sup.+, R.sub.3—P.sup.+, or X.sub.2—P.sup.+.

2. The composition according to claim 1, further comprising at least one nucleic acid molecule to be transfected in a cell, preferably a nucleic acid molecule selected from the group consisting of a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a DNA/RNA hybrid, a short interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a messenger RNA (mRNA), a CRISPR guide RNA, and an expression vector encoding said nucleic acid molecule, in particular a plasmid encoding said nucleic acid molecule or a plasmid expressing said nucleic acid molecule.

3. The composition according to claim 2, wherein the at least one nucleic acid molecule is a DNA.

4. The composition according to claim 1, wherein R or V represents H, methyl, ethyl, propyl, cyclopropyl, isopropyl, sec-butyl, cyclopentyl, phenyl, fluorophenyl, benzyl, pyridine, 2-pyridine, 3-pyridine, fluorobenzyl, substituted morpholinyl, substituted piperazinyl, 4-hydroxybenzyl, or 4-hydroxyphenethyl; more preferably R or V represents methyl, ethyl, propyl, cyclopropyl, isopropyl, sec-butyl, cyclopentyl, phenyl, benzyl, fluorobenzyl, 4-hydroxyphenethyl, 2-pyridine or 3-pyridine.

5. The composition according to claim 1, wherein: (i) only one of Z.sup.1, Z.sup.2 or Z.sup.3 represents X.sub.1—R.sub.3—X.sub.2—P.sup.+, X.sub.1—R.sub.3—P.sup.+, X.sub.1—X.sub.2—P.sup.+, R.sub.3—X.sub.2—P.sup.+, X.sub.1—P.sup.+, R.sub.3—P.sup.+, or X.sub.2—P.sup.+, wherein X.sub.1, X.sub.2, R.sub.3 and P.sup.+ are as defined in claim 1; preferably only one of Z.sup.1, Z.sup.2 or Z.sup.3 represents X.sub.1—R.sub.3—X.sub.2—P.sup.+, wherein X.sub.1 represents CH.sub.2, X.sub.2 represents CO, and R.sub.3 represents (CH.sub.2).sub.m, with m representing an integer between 1 and 3, preferably m is equal to 2; and/or (ii) Z.sup.1 represents H; and/or (iii) Z.sup.2 represents H, a C.sub.1-C.sub.12 alkoxy, or a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkyl, preferably a linear or branched, saturated or unsaturated C.sub.1-C.sub.6 alkyl; more preferably Z.sup.2 represents H, CH.sub.3, CF.sub.3 or OCH.sub.3; and/or (iv) Z.sup.3 represents H, or a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkyl, preferably a linear or branched, saturated or unsaturated C.sub.1-C.sub.6 alkyl.

6. The composition according to claim 1, wherein: if (i) Z.sup.1 represents X.sub.1—R.sub.3—X.sub.2—P.sup.+, X.sub.1—R.sub.3—P.sup.+, X.sub.1—X.sub.2—P.sup.+, R.sub.3—X.sub.2—P.sup.+, X.sub.1—P.sup.+, R.sub.3—P.sup.+, or X.sub.2—P.sup.+, preferably X.sub.1—R.sub.3—X.sub.2—P.sup.+, wherein X.sub.1, X.sub.2, R.sub.3 and P.sup.+ are as defined herein; more preferably Z.sup.1 represents X.sub.1—R.sub.3—X.sub.2—P.sup.+, wherein X.sub.1 represents CH.sub.2, X.sub.2 represents CO, and R.sub.3 represents (CH.sub.2).sub.m, with m representing an integer between 1 and 3, preferably m is equal to 2 then (ii) Z.sup.2 represents H, a C.sub.1-C.sub.12 alkoxy, or a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkyl, preferably a linear or branched, saturated or unsaturated C.sub.1-C.sub.6 alkyl; more preferably Z.sup.2 represents H, CH.sub.3, CF.sub.3 or OCH.sub.3; and/or (iii) Z.sup.3 represents H, a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkyl, preferably a linear or branched, saturated or unsaturated C.sub.1-C.sub.6 alkyl, or a linear or branched, saturated or unsaturated C.sub.6-C.sub.18 aryl-C.sub.1-C.sub.18 alkyl, preferably fluorobenzyl or 4-hydroxyphenethyl; and/or (iv) R or V represents H, a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkyl or cycloalkyl, a C.sub.6-C.sub.18 aryl, a linear or branched, saturated or unsaturated C.sub.6-C.sub.18 aryl-C.sub.1-C.sub.18 alkyl, a linear or branched, saturated or unsaturated C.sub.2-C.sub.18 heteroalkyl, a linear or branched, saturated or unsaturated C.sub.1-C.sub.24 ester, a C.sub.5-C.sub.10 heterocyclyl, a C.sub.5-C.sub.10 heteroaryl, or a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkyl-C.sub.5-C.sub.10 heteroaryl.

7. The composition according to claim 1, wherein: if (i) Z.sup.2 represents X.sub.1—R.sub.3—X.sub.2—P.sup.+, X.sub.1—R.sub.3—P.sup.+, X.sub.1—X.sub.2—P.sup.+, R.sub.3—X.sub.2—P.sup.+, X.sub.1—P.sup.+, R.sub.3—P.sup.+, or X.sub.2—P.sup.+, preferably X.sub.1—R.sub.3—X.sub.2—P.sup.+, wherein X.sub.1, X.sub.2, R.sub.3 and P.sup.+ are as defined herein; more preferably Z.sup.2 represents X.sub.1—R.sub.3—X.sub.2—P.sup.+, wherein X.sub.1 represents CH.sub.2, X.sub.2 represents CO, and R.sub.3 represents (CH.sub.2).sub.m, with m representing an integer between 1 and 3, preferably m is equal to 2 then (ii) Z.sup.1 represents H; and/or (iii) Z.sup.3 represents H, a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkyl, preferably a linear or branched, saturated or unsaturated C.sub.1-C.sub.6 alkyl, or a linear or branched, saturated or unsaturated C.sub.6-C.sub.18 aryl-C.sub.1-C.sub.18 alkyl, preferably fluorobenzyl or 4-hydroxyphenethyl; and/or (iv) R or V represents H, a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkyl or cycloalkyl, a C.sub.6-C.sub.18 aryl, a linear or branched, saturated or unsaturated C.sub.6-C.sub.18 aryl-C.sub.1-C.sub.18 alkyl, a linear or branched, saturated or unsaturated C.sub.2-C.sub.18 heteroalkyl, a linear or branched, saturated or unsaturated C.sub.1-C.sub.24 ester, a C.sub.5-C.sub.10 heterocyclyl, a C.sub.5-C.sub.10 heteroaryl, or a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkyl-C.sub.5-C.sub.10 heteroaryl.

8. The composition according to claim 1, wherein: if (i) Z.sup.3 represents X.sub.1—R.sub.3—X.sub.2—P.sup.+, X.sub.1—R.sub.3—P.sup.+, X.sub.1—X.sub.2—P.sup.+, R.sub.3—X.sub.2—P.sup.+, X.sub.1—P.sup.+, R.sub.3—P.sup.+, or X.sub.2—P.sup.+, preferably X.sub.1—R.sub.3—X.sub.2—P.sup.+, wherein X.sub.1, X.sub.2, R.sub.3 and P.sup.+ are as defined herein; more preferably Z.sup.3 represents X.sub.1—R.sub.3—X.sub.2—P.sup.+, wherein X.sub.1 represents CH.sub.2, X.sub.2 represents CO, and R.sub.3 represents (CH.sub.2).sub.m, with m representing an integer between 1 and 3, preferably m is equal to 2 then (ii) Z.sup.1 represents H; and/or (iii) Z.sup.2 represents H, a C.sub.1-C.sub.12 alkoxy, or a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkyl, preferably a linear or branched, saturated or unsaturated C.sub.1-C.sub.6 alkyl; more preferably Z.sup.2 represents H, CH.sub.3, CF.sub.3 or OCH.sub.3; and/or (iv) R or V represents H, a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkyl or cycloalkyl, a C.sub.6-C.sub.18 aryl, a linear or branched, saturated or unsaturated C.sub.6-C.sub.18 aryl-C.sub.1-C.sub.18 alkyl, a linear or branched, saturated or unsaturated C.sub.2-C.sub.18 heteroalkyl, a linear or branched, saturated or unsaturated C.sub.1-C.sub.24 ester, a C.sub.5-C.sub.10 heterocyclyl, a C.sub.5-C.sub.10 heteroaryl, or a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkyl-C.sub.5-C.sub.10 heteroaryl.

9. The composition according to claim 1, wherein: if (i) R or V represents X.sub.1—R.sub.3—X.sub.2—P.sup.+, X.sub.1—R.sub.3—P.sup.+, X.sub.1—X.sub.2—P.sup.+, R.sub.3—X.sub.2—P.sup.+, X.sub.1—P.sup.+, R.sub.3—P.sup.+, or X.sub.2—P.sup.+, preferably X.sub.1—R.sub.3—X.sub.2—P.sup.+, wherein X.sub.1, X.sub.2, R.sub.3 and P.sup.+ are as defined herein; more preferably Z.sup.3 represents X.sub.1—R.sub.3—X.sub.2—P.sup.+, wherein X.sub.1 represents CH.sub.2, X.sub.2 represents CO, and R.sub.3 represents (CH.sub.2).sub.m, with m representing an integer between 1 and 3, preferably m is equal to 2 then (ii) Z.sup.1 represents H; and/or (iii) Z.sup.2 represents H, a C.sub.1-C.sub.12 alkoxy, or a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkyl, preferably a linear or branched, saturated or unsaturated C.sub.1-C.sub.6 alkyl; and/or (iv) Z.sup.3 represents H, a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkyl, preferably a linear or branched, saturated or unsaturated C.sub.1-C.sub.6 alkyl, or a linear or branched, saturated or unsaturated C.sub.6-C.sub.18 aryl-C.sub.1-C.sub.18 alkyl, preferably fluorobenzyl or 4-hydroxyphenethyl.

10. The composition according to claim 1, wherein: if (i) R or V represents X.sub.1—R.sub.3—X.sub.2—P.sup.+, X.sub.1—R.sub.3—P.sup.+, X.sub.1—X.sub.2—P.sup.+, R.sub.3—X.sub.2—P.sup.+, X.sub.1—P.sup.+, R.sub.3—P.sup.+, or X.sub.2—P.sup.+, preferably X.sub.1—R.sub.3—X.sub.2—P.sup.+, wherein X.sub.1, X.sub.2, R.sub.3 and P.sup.+ are as defined herein; more preferably Z.sup.3 represents X.sub.1—R.sub.3—X.sub.2—P.sup.+, wherein X.sub.1 represents CH.sub.2, X.sub.2 represents CO, and R.sub.3 represents (CH.sub.2).sub.m, with m representing an integer between 1 and 3, preferably m is equal to 2 then (ii) Z.sup.3 is present and Z.sup.3 represents H, a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkyl, preferably a linear or branched, saturated or unsaturated C.sub.1-C.sub.6 alkyl, or a linear or branched, saturated or unsaturated C.sub.6-C.sub.18 aryl-C.sub.1-C.sub.18 alkyl, preferably fluorobenzyl or 4-hydroxyphenethyl.

11. The composition according to claim 1, wherein the graft cationic polymer is selected from the group consisting of a linear or branched polyethyleneimine (PEI), PEI dendrimers, a polypropyleneimine (PPI), Poly(amidoamine) (PAA) and dendrimers (PAMAM), cationic cyclodextrin, polyalkylamine, a polyhydroxyalkylamine, poly(butyleneimine) (PBI), spermine, a N-substituted polyallylamine, N-substituted chitosan, a N-substituted polyornithine, a N-substituted polylysine (PLL), a N-substituted polyvinylamine, poly(β-amino ester), hyperbranched poly(amino ester) (h-PAE), networked poly(amino ester) (n-PAE), poly(4-hydroxy-1-proline ester) (PHP-ester) and a poly-β-aminoacid.

12. The composition according to claim 11, wherein the graft cationic polymer is a linear or branched PEI, more preferably a linear PEI.

13. The composition according to claim 1, wherein the graft cationic polymer has a grafting ratio ranging from 1 to 50%, preferably from 5 to 30%, more preferably is 20%.

14. The composition according to claim 1, wherein the graft cationic polymer has an average molecular weight (Mw) ranging 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.

15. The composition according to claim 14, wherein the graft cationic polymer has an average molecular weight (Mw) of 6, 8, 10, 15, 22 or 30 kDa, preferably of 6, 8, 10, 15 or 30 kDa.

16. The composition according to claim 1, wherein the at least one compound of general formula (III) is selected from the group consisting of the following compounds: ##STR00145## ##STR00146## ##STR00147## ##STR00148## ##STR00149## ##STR00150## ##STR00151## ##STR00152## ##STR00153##

17. The composition according to claim 16, wherein the at least one compound of general formula (III) is selected from the group consisting of the following compounds: ##STR00154## ##STR00155## ##STR00156## ##STR00157##

18. The composition according to claim 17, wherein the at least one compound of general formula (III) is compound 2.22.

19. A method for in vitro or ex vivo transfection of live cells comprising introducing in the cells the composition according to claim 2.

20. A method for in vitro or ex vivo transfection of at least one nucleic acid molecule into a cell, cell line or cells, preferably a cell, cell line or cells selected from the group consisting of a mammalian cell, an insect cell, a primary cell, an adherent cell, a suspension cell, a dividing cell such as a stem cell, a non-dividing cell such as a neuronal cell, and a cancer cell, said cell, cell line or cells being optionally organized into spheroids, organoids, 2D or 3D cell culture, or provided as fibre or matrix culture, and/or within a bioreactor, the method comprising introducing the composition of claim 2 into the cell, the cell line, or the cells.

21. A method for genome engineering, for cell reprogramming, for differentiating cells, or for gene-editing, comprising applying to the genome, cells, or gene the composition according to claim 2.

22. A method for the production of: (i) biologics, in particular biologics encoding a recombinant protein, peptide or antibody, the method comprising applying the composition of claim 2; or (ii) recombinant virus, such as adeno-associated virus (AAV), lentivirus (LV), adenovirus, oncolytic virus, or baculovirus, the method comprising applying the composition of claim 2, said composition comprising multiple nucleic acid molecules for co-transfection; or (iii) viral or virus-like particles, the method comprising applying the composition according to claim 2, said composition comprising multiple nucleic acid molecules for co-transfection.

23. The method according to claim 22, for the production of AAV, said composition comprising (i) at least one compound selected from the group consisting of compounds 2.22, 2.23, 2.43, 2.44, 2.47, 2.54, 2.57, 2.60 and 2.61 and (ii) an acceptable excipient, buffering agent, cell culture medium, or transfection medium.

24. The method according to claim 22, for the production of LV, said composition comprising (i) at least the compound 2.22, and (ii) an acceptable excipient, buffering agent, cell culture medium, or transfection medium.

25. The method according to claim 22, for the production of recombinant virus, said composition comprising a plurality of expression vectors such as plasmid vectors to transfect in an adherent or suspension cell, such as HEK293, HeLa, BHK-21, A549 or insect cells, wherein said vectors, in particular plasmids, are construct expressing viral structural sequences and transfer vector genome for virus or virus-like production and optionally expressing molecules of interest encoded by the transfer vector genome.

26. The method according to claim 25, further comprising a step of performing cell therapy or gene therapy, wherein the recombinant virus is used in vivo.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0126] FIG. 1. Chemical structure of a compound of general formula (I).

[0127] FIG. 2. Percentage of GFP expression after transfection of Caco-2, Hep G2, MDCK and MCF-10A with compounds of Example 3. The ratio 1:3 and 1:4 indicate the ratio of μg of DNA per μL of compound.

[0128] FIG. 3. 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 media. Cells were seeded and cultured for 3 days before being transfected by 3 plasmids (pAAV-RC2 vector expressing Rep and Cap, pHelper vector expressing Adeno E2A, Adeno E4 and Adeno VA helper factors, and pAAV-GFP control vector expressing the GFP under the control of a CMV promoter) with PEIpro® or various compounds at ratio 1:2 or 1:3 μg DNA/μL reagent. AAV titers (transducing unit, TU/mL) were determined 72 hours post-transfection. The results are expressed as relative AAV-2 transducing Units/mL (TU/mL) in comparison to PEIpro® transfection at ratio 1:2 and 1:3.

[0129] FIG. 4. Production of lentivirus particles from suspension HEK-293T cells. Lentivirus expressing the GFP reporter gene was produced in HEK-293T cells grown in suspension in FreeStyle F17 media. Cells were seeded and cultured for 3 days before being transfected by 4 plasmids with PEIpro® or compound 2.22 at ratio 1:2 μg total DNA/μL reagent. Lentivirus titers (transducing unit, TU/mL) were determined 72 hours post-transfection.

[0130] FIG. 5. Chemical structure of a compound of general formula (III).

[0131] FIG. 6. Percentage of GFP expression after transfection of Hep G2 cells with compounds 2.22 and 2.53 to 2.61. The ratio 1:3 and 1:4 indicate the ratio of μg of DNA per μL of compound.

[0132] FIG. 7. Production of AAV-2 from suspension HEK-293T cells with compounds 2.22 and 2.53 to 2.61. AAV-2 vectors expressing the GFP reporter gene were produced in HEK-293T cells grown in suspension in FreeStyle F17 media. Cells were seeded and cultured for 3 days before being transfected by 3 plasmids (pAAV-RC2 vector expressing Rep and Cap, pHelper vector expressing Adeno E2A, Adeno E4 and Adeno VA helper factors, and pAAV-GFP control vector expressing the GFP under the control of a CMV promoter) with PEIpro® or various compounds at ratio 1:2 μg DNA/μL reagent. AAV titers (transducing unit, TU/mL) were determined 72 hours post-transfection. The results are expressed as relative AAV-2 transducing Units/mL (TU/mL).

[0133] FIG. 8. Influence of the amount of DNA transfected and the ratio of compound 2.22 per μg DNA on the 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 media. Cells were seeded and cultured for 3 days before being transfected by 3 plasmids (pAAV-RC2 vector expressing Rep and Cap, pHelper vector expressing Adeno E2A, Adeno E4 and Adeno VA helper factors, and pAAV-GFP control vector expressing the GFP under the control of a CMV promoter) with compound 2.22 (formulated at 15 mM nitrogen concentration) at different ratio of μg DNA/μL reagent (ratio 1:1.5 to 1:3). AAV titers (transducing unit, TU/mL) were determined 72 hours post-transfection. The results are expressed as relative AAV-2 transducing Units/mL (TU/mL). The cell viability was determined 72 hours post-transfection with a Trypan blue assay.

[0134] FIG. 9. Influence of time of DNA complexation with compound 2.22 on the 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 media. Cells were seeded and cultured for 3 days before being transfected by 3 plasmids (pAAV-RC2 vector expressing Rep and Cap, pHelper vector expressing Adeno E2A, Adeno E4 and Adeno VA helper factors, and pAAV-GFP control vector expressing the GFP under the control of a CMV promoter) with compound 2.22 (formulated at 15 mM nitrogen concentration) at a ratio 1:2 of DNA/μL reagent and with 1 μg DNA/million cells. AAV titers (transducing unit, TU/mL) were determined 72 hours post-transfection. The results are expressed as relative AAV-2 transducing Units/mL (TU/mL).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

EXAMPLES

Experimental Section

Material and Methods

[0135] Cell Culture

[0136] Caco-2 (ATCC® HTB-37™) human colon epithelial cells were grown in DMEM 4.5 g/L glucose with 20% FBS supplemented with 1% non-essential amino acids, 1 mM sodium pyruvate, 2 mM glutamine and 100 U/mL of penicillin and 100 μg/mL of streptomycin at 37° C. in a 5% CO.sub.2 in air atmosphere.

[0137] MCF 10A (ATCC® CRL-10317™) human mammary epithelial cells were grown in MEBM (Lonza) supplemented with SingleQuots™ Supplements and Growth Factors (Lonza) and 100 ng/ml cholera toxin at 37° C. in a 5% CO.sub.2 in air atmosphere.

[0138] Hep G2 (ATCC® HB-8065™) human hepatocarcinoma cells were grown in MEM (Ozyme) with 10% FBS supplemented with 1% non-essential amino acids, 1 mM sodium pyruvate, 2 mM glutamine and 100 U/mL of penicillin and 100 μg/mL of streptomycin at 37° C. in a 5% CO.sub.2 in air atmosphere.

[0139] MDCK (ATCC® CCL-34™) Madin-Darby canine kidney epithelial cells were grown in MEM (Ozyme) with 10% FBS supplemented with 2 mM glutamine and 100 U/mL of penicillin and 100 μg/mL of streptomycin at 37° C. in a 5% CO.sub.2 in air atmosphere.

[0140] 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 of penicillin and 100 μg/mL of streptomycin at 37° C. in a 5% CO.sub.2 in air atmosphere.

[0141] Transfection Assay (96-Well Format)

[0142] One day before transfection, Caco-2, MCF 10A, Hep G2 and MDCK Cells were seeded at 10 000, 25 000, 25 000, 10 000 cells per well (96-well plate format), respectively, in 125 μL of their respective complete medium and incubated at 37° C. in a 5% CO.sub.2 in air atmosphere. On the day of transfection 200 ng of pCMV-EGFPLuc DNA (Clontech) was added in 20 μL of OPTIMEM (Thermo Fisher), mixed with a vortex and incubated for 5 minutes at room temperature (rt). Then, 0.6 or 0.8 μL of a compound of general formula (I), preferably of general formula (III) (at 7.5 mM nitrogen concentration) were added onto the diluted DNA, mixed with a vortex and incubated for 10 minutes at rt. The transfection DNA solution (20 μL) was added into the well and the plate was incubated for 24 hours at 37° C. in a 5% CO.sub.2 in air atmosphere.

[0143] For the GFP expression analysis, one day post-transfection, the cell culture medium was removed and 50 μL of trypsin-EDTA (1×, Lonza) were added per well and the plate was incubated for 5 minutes at 37° C. 150 μL of complete medium were added to neutralize the trypsin, and the GFP expression was analysed (2000 events) by flow cytometry (Exc 488 nm, Em 520 nm) using a Guava easyCyte 6HT cytometer (Millipore).

[0144] Recombinant Virus Production

[0145] HEK-293T (ATCC® CRL-3216™): Human embryonic kidney cell is a highly transfectable derivative of human embryonic kidney 293 cells, and contains the SV40 T-antigen. HEK-293T cells are widely used for recombinant virus production, gene expression and protein production.

[0146] For adherent cells, HEK-293T cells were seeded at 5×10.sup.6 cells in 145 cm.sup.2 petri dishes in 15 mL of DMEM 4.5 g/L glucose supplemented with 10% FBS, 2 mM glutamine and 100 U/mL of penicillin and 100 μg/mL of streptomycin, and incubated at 37° C. in a 5% CO.sub.2 in air atmosphere.

[0147] AAV-2 was produced in HEK-293 T cells using the AAV-2 Helper Free Packaging System (catalog number VPK-402, Cell BIOLABS, INC.) by co-transfection of 3 plasmids, pAAV-RC2 vector expressing Rep and Cap, pHelper vector expressing Adeno E2A, Adeno E4 and Adeno VA helper factors, and pAAV-GFP control vector expressing the GFP under the control of a CMV promoter. Transfection complexes (10 μg total DNA per petri dish) were prepared with a ratio of 2:2:1 with pAAV-RC2, pHelper and pAAV-GFP, respectively. Plasmids were diluted in a total volume of 1.5 mL of OPTIMEM. Then, 20 or 30 μL of compounds were added onto the diluted DNA, mixed with a vortex and incubated for 10 minutes at rt. Transfection complexes were added onto the cells and the plate was incubated for 72 h at 37° C. in a 5% CO.sub.2 in air atmosphere.

[0148] For suspension cells, HEK-293T cells were seeded at 1×10.sup.6 cells/mL in 27 mL of FreeStyle F17 supplemented with 4% Glutamine, 100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.1% Pluronic in 125 mL flask Erlenmeyer (Corning). Cells were incubated for 24 h at 37° C. in an 8% CO.sub.2 in air atmosphere under agitation (130 rpm). Plasmids (pAAV-GFP-pAAV-RC2—pHelper at ratio 2:2:1) were diluted in 3 mL of FreeStyle F17. Then, compounds were added onto the diluted DNA (ratio 2 or 3 μL per μg of DNA), mixed with a vortex and incubated for 10 minutes at rt. Transfection complexes were added onto the cells (2 μg DNA per 1×10.sup.6 cells) and the plate was incubated for 72 h at 37° C. in a 8% CO.sub.2 in air atmosphere under agitation (130 rpm).

[0149] Lentivirus particles were produced using the ViraSafe™ Lentiviral Packaging System, Pantropic (Catalog Number VPK-20, CELL BIOLABS INC.) containing pRSV-REV packaging vector, pCgpV Packaging Vector and pCMV-VSV-G Envelop Vector. pLenti6.3/V5-GW/EmGFP Expression Control Vector was from Thermo Fisher.

[0150] HEK-293T cells were seeded at 1×10.sup.6 cells/mL in 27 mL of FreeStyle F17 supplemented with 4% Glutamine, 100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.1% Pluronic in 125 mL flask Erlenmeyer (Corning). Cells were incubated for 24 h at 37° C. in an 8% CO.sub.2 in air atmosphere under agitation (130 rpm). Plasmids (pRSV-REV-pCgpV-pCMV-VSV-G-pLenti6.3 at ratio 1:1:1:3) were diluted in 3 mL of FreeStyle F17. Then, compounds were added onto the diluted DNA (ratio 2 μL per μg of DNA), mixed with a vortex and incubated for 10 minutes at rt. Transfection complexes were added onto the cells (2 μg DNA per 1×10.sup.6 cells) and the plate was incubated for 72 h at 37° C. in an 8% CO.sub.2 in air atmosphere under agitation (130 rpm).

[0151] The transducing unit (TU/mL) was determined by using virus vectors expressing the GFP reporter gene after infection of permissive HT 1080 cells for lentivirus vectors and HEK-293T cells for AAV-2 vectors in 96-well and in presence of polybrene (8 μg/mL). The GFP expression was analysed by cytometry 72 h after transduction to determine the transducing units.

Example 1. General Procedure for the Preparation of Grafted Polymers

Step 1: Grafting

[0152] In a round-bottom flask was added the cationic polymer (1 equiv.) in water (4 mL/mmol of starting material) followed by N-methyl morpholine or NMM (2 equiv.). The carboxylate (0.3-1 equiv.) was added followed by MeOH (16 mL/mmol of polymer). After stirring 10 min, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride was added or DMTMM (0.6-2 equiv.) and the mixture was stirred 12-24 h at room temperature. Then, MeOH was removed in vacuo, water (4 mL/mmol of starting material) followed by a solution of 3M HCl (1 mL/mmol of starting material) were added. The residue was purified using a dialysis cassette in a 50 mM HCl bath.

Step 2: Synthesis of Triazole by «Click» Chemistry Starting from an Acid

[0153] ##STR00055##

[0154] Alkyne (1 equiv.), azide (1 equiv.), CuSO.sub.4 (0.01 equiv) and sodium ascorbate (0.03 equiv) were added to a 2:1 (v/v) solution of nBuOH and water. The reaction was stirred at room temperature for 24 h. Then, NaOH (5M, 2 equiv.) was added and the organic solvent was removed in vacuo. The residue was purified by reversed phase flash chromatography using 0 to 100% CH.sub.3CN in water as eluant.

Step 3: Synthesis of Triazole by «Click» Chemistry Starting from an Ester

[0155] ##STR00056##

[0156] Alkyne (1 equiv.), azide (1 equiv.), CuSO.sub.4 (0.01 equiv) and sodium ascorbate (0.03 equiv) were added to a 2:1 (v/v) solution of nBuOH and water. The reaction was stirred at room temperature for 24 h. Then, NaOH (5M, 2 equiv.) was added and the organic solvent was removed in vacuo. The residue was purified by reversed phase flash chromatography using 0 to 100% CH.sub.3CN in water as eluant.

Step 4: Saponification of the Ester Moiety

[0157] To a solution of ester in EtOH was added dropwise a 3M solution of LiOH, and the mixture was stirred at rt for the week-end. Then, the solvent was removed in vacuo and the residue was purified by reverse phase FC on SiO.sub.2 using H.sub.2O/MeCN as eluant using a Biotage Flash purification system. The acid obtained was lyophilized to yield a solid.

Step 5: Synthesis of Triazole by Ruthenium Catalyzed «Click» Chemistry Starting from an Ester

[0158] Cp*RuCl (cod) was added to a microwave vial. The vial was then evacuated and backfilled with Argon (3×). Alkyne (1.1 eq.); alcyne (1 eq.) and toluene were added to the vial under Ar and the mixture was stirred at rt overnight. Toluene was evaporated and the product was purified on reverse phase chromatography using H2O and MeCN.

[0159] The ester was retaken in EtOH and NaOH 1M (1.1 eq.) and stirred until completion (followed by HPLC). EtOH was evaporated and the product was purified by reverse phase chromatography using H2O and MeCN. The product was lyophilized.

Step 6: Synthesis of 1,2,3-triazole

[0160] Triazole and K.sub.2CO.sub.3 in MeCN at 80° C. Add R—Br dropwise and stirred at 80° C. overnight. Filtrate and washed the solid with MeCN. The filtrate was evaporated and purified by reverse phase chromatography (H.sub.2O:MeCN). Two fractions were collected.

[0161] The esters were retaken in EtOH and NaOH 1M (1.1 eq) and stirred until completion. EtOH was evaporated and the product was purified by reverse phase chromatography H.sub.2O:MeCN.

Example 2. Syntheses of Compounds of the Invention

Synthesis of Product 2.19

[0162] ##STR00057##

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

##STR00058##

Product 2.19 was prepared analogously to the general procedure, step 1 (Example 1). Yield=36%; m=21 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.12-6.41 (m, 5H), 5.68-4.93 (m, 2H), 4.05-2.88 (m, 17H), 2.79-0.87 (m, 8H).

Synthesis of Product 2.20

[0163] ##STR00059##

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

##STR00060##

Product 2.20 was prepared analogously to the general procedure, step 1 (Example 1). Yield=71%; m=31 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.17-6.68 (m, 5H), 5.60-5.28 (m, 2H), 4.10-2.93 (m, 27H).

Synthesis of Product 2.21

[0164] ##STR00061##

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

##STR00062##

Product 2.21 was prepared analogously to the general procedure, step 1 (Example 1). Yield=29%; m=12 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.90-6.37 (m, 5H), 5.58-5.25 (m, 2H), 4.20-2.91 (m, 36H).

Synthesis of Product 2.22

[0165] ##STR00063##

Intermediate 2.22a was prepared analogously to the general procedure, step 2 (Example 1). Yield=28%; m=78 mg; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.57 (s, 1H), 6.93-6.84 (m, 2H), 6.68-6.60 (m, 2H), 4.54-4.45 (m, 2H), 3.04 (t, J=7.4 Hz, 2H), 2.99-2.91 (m, 2H), 2.53-2.44 (m, 2H).

##STR00064##

Product 2.22 was prepared analogously to the general procedure, step 1 (Example 1). Yield=87%; m=44 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.82-6.34 (m, 5H), 4.60-4.06 (m, 2H), 4.00-3.07 (m, 22H), 3.06-2.24 (m, 7H).

Synthesis of Product 2.23

[0166] ##STR00065##

Intermediate 2.23a was prepared analogously to the general procedure, step 2 (Example 1). Yield=87%; m=258 mg; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.53 (s, 1H), 6.98-6.87 (m, 2H), 6.75-6.63 (m, 2H), 4.53 (t, J=7.1 Hz, 2H), 3.08 (t, J=7.1 Hz, 2H), 2.69 (t, J=7.6 Hz, 2H), 2.26-2.16 (m, 2H), 1.91 (tt, J=8.3, 6.9 Hz, 2H).

##STR00066##

Product 2.23 was prepared analogously to the general procedure, step 1 (Example 1). Yield=100%; m=48 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.14-6.01 (m, 5H), 4.62-4.11 (m, 2H), 3.99-2.76 (m, 26H), 2.73-0.92 (m, 8H).

Synthesis of Product 2.24

[0167] ##STR00067##

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

##STR00068##

Product 2.24 was prepared analogously to the general procedure, step 1 (Example 1). Yield=97%; m=42 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.90-6.74 (m, 5H), 5.57-5.15 (m, 2H), 4.19-3.11 (m, 35H), 2.91-1.47 (m, 6H).

Synthesis of Product 2.25

[0168] ##STR00069##

Product 2.25 was prepared analogously to the general procedure, step 1 (Example 1). Yield=85%; m=41 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.84-6.46 (m, 5H), 5.54-4.94 (m, 2H), 4.15-3.11 (m, 26H), 2.97-1.11 (m, 8H).

Synthesis of Product 2.26

[0169] ##STR00070##

Product 2.26 was prepared analogously to the general procedure, step 1 (Example 1). Yield=80%; m=44 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.00-6.36 (m, 5H), 5.60-4.93 (m, 2H), 4.12-3.01 (m, 19H), 2.79-0.93 (m, 8H).

Synthesis of Product 2.27

[0170] ##STR00071##

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

##STR00072##

Intermediate 2.27b was prepared analogously to the general procedure, step 4 (Example 1). Yield=35%; m=97 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.07 (s, OH), 7.71-7.59 (m, 1H), 7.19-7.04 (m, 1H), 4.96 (s, 1H).

##STR00073##

Product 2.27 was prepared analogously to the general procedure, step 1 (Example 1). Yield=67%; m=28 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 9.33-7.35 (m, 5H), 6.13-5.19 (m, 2H), 4.17-3.22 (m, 42H).

Synthesis of Product 2.28

[0171] ##STR00074##

Intermediate 2.28a was prepared analogously to the general procedure, step 3 (Example 1). Yield=62%; m=272 mg; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.59 (d, J=5.0 Hz, 1H), 8.50 (s, 1H), 8.10 (dt, J=7.9, 1.1 Hz, 1H), 7.93 (td, J=7.8, 1.8 Hz, 1H), 7.38 (ddd, J=7.6, 4.9, 1.2 Hz, 1H), 5.45 (s, 2H), 3.83 (s, 3H).

##STR00075##

Intermediate 2.28b was prepared analogously to the general procedure, step 4 (Example 1). Yield=94%; m=236 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.44-8.38 (m, 1H), 8.23 (s, 1H), 7.89-7.74 (m, 2H), 7.31 (ddd, J=6.0, 5.0, 2.8 Hz, 1H), 5.02 (s, 2H).

##STR00076##

Product 2.28 was prepared analogously to the general procedure, step 1 (Example 1). Yield=47%; m=23 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.06-6.11 (m, 5H), 5.55-4.96 (m, 2H), 4.26-2.20 (m, 20H).

Synthesis of Product 2.29

[0172] ##STR00077##

Intermediate 2.29a was prepared analogously to the general procedure, step 3 (Example 1). Yield=81%; m=355 mg; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 9.08 (s, 1H), 8.64-8.46 (m, 2H), 8.28 (tt, J=6.3, 1.6 Hz, 1H), 7.55 (dd, J=8.0, 4.7 Hz, 1H), 5.44 (s, 2H), 3.84 (s, 2H).

##STR00078##

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

##STR00079##

Product 2.29 was prepared analogously to the general procedure, step 1 (Example 1). Yield=76%; m=29 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 9.28-7.21 (m, 5H), 5.94-5.16 (m, 2H), 4.19-2.35 (m, 19H).

Synthesis of Product 2.30

[0173] ##STR00080##

Product 2.30 was prepared analogously to the general procedure, step 1 (Example 1). Yield=66%; m=32 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 9.47-7.87 (m, 5H), 6.08-5.50 (m, 2H), 4.32-2.94 (m, 50H).

Synthesis of Product 2.31

[0174] ##STR00081##

Intermediate 2.31a was prepared analogously to the general procedure, step 3 (Example 1). Yield=82%; m=354 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 9.46-7.68 (m, 5H), 6.03-5.32 (m, 2H), 4.28-2.83 (m, 50H). .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.34 (s, 1H), 7.88-7.80 (m, 2H), 7.51-7.41 (m, 2H), 7.41-7.32 (m, 1H), 5.39 (s, 2H), 3.83 (s, 3H).

##STR00082##

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

##STR00083##

Product 2.31 was prepared analogously to the general procedure, step 1 (Example 1). Yield=58%; m=24 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.60-6.52 (m, 6H), 5.90-5.15 (m, 2H), 4.23-2.90 (m, 32H).

Synthesis of Product 2.32

[0175] ##STR00084##

Intermediate 2.32a was prepared analogously to the general procedure, step 3 (Example 1). Yield=87%; m=380 mg; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.82-8.45 (m, 3H), 7.92 (s, 2H), 5.45 (s, 2H), 3.84 (s, 3H).

##STR00085##

Intermediate 2.32b was prepared analogously to the general procedure, step 4 (Example 1). Yield=100%; m=351 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.49-8.43 (m, 2H), 8.34 (s, 1H), 7.70-7.64 (m, 2H), 5.02 (s, 2H).

##STR00086##

Product 2.32 was prepared analogously to the general procedure, step 1 (Example 1). Yield=91%; m=32 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 9.17-8.00 (m, 5H), 6.13-5.23 (m, 2H), 4.21-3.01 (m, 74H).

Synthesis of Product 2.33

[0176] ##STR00087##

Intermediate 2.33a was prepared analogously to the general procedure, step 2 (Example 1). Yield=59%; m=49 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.90 (s, 1H), 4.40-4.32 (m, 2H), 3.69-3.62 (m, 6H), 2.52-2.45 (m, 4H), 2.14-2.00 (m, 4H).

##STR00088##

Product 2.33 was prepared analogously to the general procedure, step 1 (Example 1). Yield=89%; m=15 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.63-7.84 (m, 1H), 4.52-4.23 (m, 3H), 4.13-2.86 (m, 27H), 2.74-1.54 (m, 4H).

Synthesis of Product 2.34

[0177] ##STR00089##

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

##STR00090##

Product 2.34 was prepared analogously to the general procedure, step 1 (Example 1). Yield=98%; m=17 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.67-7.86 (m, 1H), 7.64-6.61 (m, 5H), 4.66-4.19 (m, 3H), 4.11-3.09 (m, 31H), 2.80-1.74 (m, 4H).

Synthesis of Product 2.35

[0178] ##STR00091##

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

##STR00092##

Product 2.35 was prepared analogously to the general procedure, step 1 (Example 1). Yield=44%; m=7 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.58-7.90 (m, 3H), 7.07-6.43 (m, 1H), 4.57-4.19 (m, 3H), 4.23-2.99 (m, 32H), 2.83-1.68 (m, 4H).

Synthesis of Product 2.36

[0179] ##STR00093##

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

##STR00094##

Product 2.36 was prepared analogously to the general procedure, step 1 (Example 1). Yield=77%; m=14 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 9.15-7.11 (m, 5H), 4.57-4.15 (m, 1H), 4.07-2.86 (m, 13H), 2.74-1.68 (m, 4H).

Synthesis of Product 2.37

[0180] ##STR00095##

Intermediate 2.37a was prepared analogously to the general procedure, step 2 (Example 1). Yield=14%; m=38 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.00 (s, 1H), 7.57-7.47 (m, 2H), 7.09-6.95 (m, 2H), 4.26 (t, J=7.0 Hz, 2H), 2.08 (t, J=7.5 Hz, 2H), 1.76 (p, J=7.2 Hz, 2H), 1.49-1.35 (m, 2H).

##STR00096##

Product 2.37 was prepared analogously to the general procedure, step 1 (Example 1). Yield=24%; m=9 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.23-6.04 (m, 5H), 4.39-2.72 (m, 18H), 2.70-0.56 (m, 6H).

Synthesis of Product 2.38

[0181] ##STR00097##

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

##STR00098##

Product 2.38 was prepared analogously to the general procedure, step 1 (Example 1). Yield=18%; m=6 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.31-6.11 (m, 5H), 4.33-2.72 (m, 21H), 2.68-0.15 (m, 6H).

Synthesis of Product 2.39

[0182] ##STR00099##

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

##STR00100##

Product 2.39 was prepared analogously to the general procedure, step 1 (Example 1). Yield=53%; m=133 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.49-6.15 (m, 6H), 4.52-2.83 (m, 21H), 2.66-0.54 (m, 6H).

Synthesis of Product 2.40

[0183] ##STR00101##

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

##STR00102##

[0184] Product 2.40 was prepared analogously to the general procedure, step 1 (Example 1). Yield=12%; m=4 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.94-6.37 (m, 6H), 4.43-2.84 (m, 19H), 2.68-0.23 (m, 8H).

Synthesis of Product 2.41

[0185] ##STR00103##

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

##STR00104##

Product 2.41 was prepared analogously to the general procedure, step 1 (Example 1). Yield=75%; m=26 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.99-6.38 (m, 6H), 4.32-1.58 (m, 30H).

Synthesis of Product 2.42

[0186] ##STR00105##

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

##STR00106##

Product 2.42 was prepared analogously to the general procedure, step 1 (Example 1). Yield=87%; m=27 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.58-6.61 (m, 6H), 4.55-0.72 (m, 33H).

Synthesis of Product 2.43

[0187] ##STR00107##

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

##STR00108##

Product 2.43 was prepared analogously to the general procedure, step 1 (Example 1). Yield=38%; m=17 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 9.38-7.80 (m, 5H), 4.58-0.92 (m, 31H).

Synthesis of Product 2.44

[0188] ##STR00109##

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

##STR00110##

Product 2.44 was prepared analogously to the general procedure, step 1 (Example 1). Yield=64%; m=29 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.79-7.46 (m, 5H), 4.56-2.83 (m, 23H), 2.72-0.78 (m, 8H).

Synthesis of Product 2.45

[0189] ##STR00111##

Product 2.45 was prepared analogously to the general procedure, step 1 (Example 1). Yield=49%; m=18 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.01-6.82 (m, 5H), 5.62-5.23 (m, 2H), 4.04-3.18 (m, 66H), 2.95-1.63 (m, 6H).

Synthesis of Product 2.46

[0190] ##STR00112##

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

Synthesis of Product 2.47

[0191] ##STR00113##

Product 2.47 was prepared analogously to the general procedure, step 1 (Example 1). Yield=99%; m=44 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.00-6.02 (m, 5H), 4.66-4.05 (m, 1H), 3.97-2.01 (m, 32H).

Synthesis of Product 2.48

[0192] ##STR00114##

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

Synthesis of Product 2.49

[0193] ##STR00115##

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

Synthesis of Product 2.50

[0194] ##STR00116##

Product 2.50 was prepared analogously to the general procedure, step 1. Yield=18%; m=29 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.71-7.19 (s, 1H), 7.15-6.34 (m, 4H), 4.65-4.09 (m, 2H), 4.06-0.57 (m, 26H).

Synthesis of Product 2.51

[0195] ##STR00117##

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

Synthesis of Product 2.52

[0196] ##STR00118##

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

Synthesis of Product 2.53

[0197] ##STR00119##

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

##STR00120##

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

Synthesis of Product 2.54

[0198] ##STR00121##

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

##STR00122##

product 2.54 was prepared analogously to the general procedure, step 1. Yield=34%; m=24 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.99-7.78 (m, 1H), 4.61-4.38 (m, 2H), 4.06-3.26 (m, 21H), 2.66-1.95 (m, 5H), 1.15-1.01 (m, 2H), 0.88-0.70 (m, 2H).

Synthesis of Product 2.55

[0199] ##STR00123##

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

##STR00124##

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

##STR00125##

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

Synthesis of Product 2.56

[0200] ##STR00126##

Intermediate 2.56a was prepared analogously to the general procedure, step 3. Yield=80%; m=350 mg; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.26 (s, 1H), 4.38 (t, J=6.9 Hz, 2H), 4.14 (q, J=7.1 Hz, 2H), 3.09 (hept, J=6.9 Hz, 1H), 2.34 (dd, J=7.4, 6.4 Hz, 2H), 2.29-2.14 (m, 2H), 1.31 (s, 3H), 1.29 (s, 3H), 1.26 (td, J=7.1, 0.6 Hz, 3H).

##STR00127##

Intermediate 2.56b was prepared analogously to the general procedure, step 4. Yield=quantitative; m=340 mg; .sup.1H NMR (400 MHz, MeOD) δ 7.75 (d, J=0.8 Hz, 1H), 4.39 (td, J=6.4, 5.8, 2.8 Hz, 2H), 3.03 (pd, J=6.9, 0.7 Hz, 1H), 2.25-2.05 (m, 4H), 1.30 (s, 3H), 1.29 (s, 3H).

##STR00128##

Product 2.56 was prepared analogously to the general procedure, step 1. Yield=45%; m=32 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.09-7.90 (m, 1H), 4.61-4.36 (m, 2H), 4.09-3.20 (m, 21H), 3.17-2.97 (m, 1H), 2.62-2.03 (m, 4H), 1.45-1.16 (m, 6H).

Synthesis of Product 2.57

[0201] ##STR00129##

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

##STR00130##

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

##STR00131##

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

Synthesis of Product 2.58

[0202] ##STR00132##

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

##STR00133##

Intermediate 2.58b was prepared analogously to the general procedure, step 4. Yield=quantitative; m=219 mg; .sup.1H NMR (400 MHz, MeOD) δ 4.37-4.27 (m, 2H), 3.31-3.18 m 1H) 2.33 (s, 31H), 2.27-2.13 (m, 2H), 2.15-2.03 (m, 2H), 1.34 (s, 3H), 1.32 (s, 3H).

##STR00134##

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

Synthesis of Product 2.59

[0203] ##STR00135##

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

##STR00136##

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

##STR00137##

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

Synthesis of Product 2.60

[0204] ##STR00138##

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

##STR00139##

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

##STR00140##

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

Synthesis of Product 2.61

[0205] ##STR00141##

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

##STR00142##

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

##STR00143##

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

Example 3. Compounds 2.19 to 2.26

[0206] Screening of Transfection Activity

[0207] Compounds 2.19 to 2.26 were evaluated for their ability to transfect DNA in four different cell lines, Caco-2 (human colon epithelial cells), Hep G2 (human hepatocarcinoma cells), MDCK (Madin-Darby canine kidney epithelial cells) and MCF-10A (human mammary epithelial cells). The screening of compounds (FIG. 2) was performed in 96-well plate by transfecting 200 ng of pCMV-EGFPLuc DNA (Clontech) complexed with 0.6 or 0.8 μL of one compound of the invention, i.e. one compound selected from the group consisting of compounds 2.19 to 2.26 (at 7.5 mM nitrogen concentration), defining a ratio of 1 μg DNA/3 μL of compound (ratio 1:3) or a ratio of 1 μg DNA/4 μL of compound (ratio 1:4), respectively. The percentage of cells expressing the GFP (% GFP) was determined by cytometry assay one day post-transfection. A transfection was performed with jetPEI® as a control which is a linear polyethylenimine of 22 KDa and represents the parental cationic polymer backbone of the tested compounds.

[0208] The compounds 2.19 to 2.26 represent polymers wherein the triazole ring was used to graft fluorobenzyl or hydroxyphenol (or 4-hydroxyphenethyl) moiety and wherein the cationic polymer is grafted to R or V of the formula (III). All the compounds showed significant transfection activity whereas the best compound was dependent to the cell line used.

Example 4. Bioproduction of Recombinant Virus with Compounds 2.22, 2.23, 2.41, 2.42, 2.43, 2.46 and 2.47

[0209] DNA transfection is one of the mainly used technologies in the bioproduction of recombinant proteins and viruses by a process of transient gene expression (TGE). Concerning the production of AAV and lentivirus the most commonly used method is the transfection to deliver the viral and therapeutic genes in the producer cell lines, HEK293 adherent of suspension cells. In most systems, the co-transfection of many plasmids is performed by a chemical method, such as the co-precipitation with the calcium phosphate or the transfection mediated with the cationic polymer polyethylenimine (PEI), such as PEIpro® (Polyplus-transfection) commercially recommended for such a bioproduction of recombinant virus.

[0210] AAV and lentivirus particles were produced from HEK-293T cells through transient co-transfection of several plasmids containing the gene of interest and necessary viral components to produce full recombinant virions. AAV-2 and lentivirus vectors expressing the GFP reporter gene were produced with various compounds and the virus productivity was determined by assessing the transducing unit (TU/mL) 3 days post-transfection. The levels of productivity were compared to those obtained with the PEIpro® transfection reagent extensively used in adherent and suspension virus production systems.

[0211] Many compounds of Example 3 were tested for the production of AAV-2 as well as other compounds wherein the triazole ring was grafted by benzyl (2.41 or 2.42) or pyridinyl (2.43 to 2.46) moiety and wherein the cationic polymer was linked to the triazole ring in position Z.sup.1 of the formula (III). FIG. 3 presents some of the results obtained. At a ratio of 1:2 (1 μg total DNA per μL of compound) used for the transfection, some compounds performed similarly in virus productivity than PEIpro® but most of them increased significantly by 3- to 8-fold the viral titer. This improvement was confirmed for most of the compounds and enhanced by using a ratio of 1:3 with the highest increase of viral titer superior to 10-fold for compound 2.43.

[0212] Similarly, lentiviruses were produced in suspension HEK-293T cells after co-transfection of 4 plasmids (pRSV-REV packaging vector, pCgpV Packaging Vector, pCMV-VSV-G Envelop Vector and pLenti6.3/V5-GW/EmGFP Expression Control Vector). Lentivirus titers (TU/mL) were determined 72 hours post-transfection (FIG. 4). An improvement of the LV production yield of about 10-fold was obtained when compared to the productivity with PEIpro® by using the compound 2.22 at a ratio of 1:3.

Example 5. Compounds 2.53 to 2.61

[0213] Screening of Transfection Activity

[0214] Compounds 2.53 to 2.61 were screened in transfection (FIG. 6) similarly as previously described for compounds of Example 3, in 96-well plate by transfecting 200 ng of pCMV-EGFPLuc DNA (Clontech) complexed with 0.6 or 0.8 μL of one compound of the invention (at 7.5 mM nitrogen concentration), defining a ratio of 1 μg DNA/3 μL of compound or ratio of 1 μg DNA/4 μL of compound, respectively.

[0215] Compounds 2.53 to 2.61 represent compounds having a triazole ring wherein the cationic polymer is linked at Z.sup.1 of the formula (III) and wherein various alkyl or cycle moiety where added on position R or V of the formula (III). FIG. 6 shows that grafting of alkyl or cycloalkyl moiety at the position R or V on the triazole ring provides efficient compounds in transfection as exemplified by the compounds 2.54, 2.56, 2.58 or 2.57. Surprisingly, compounds 2.60 and 2.61 with unsubstituted triazole ring on position R and V of the formula (III) were not able to transfect efficiently the Hep G2 cells.

[0216] Bioproduction of Recombinant Virus

[0217] Compounds 2.53 to 2.61 were tested for the production of AAV-2 and FIG. 7 presents the results obtained of compounds at ratio 1:2 μg DNA/μL reagent. AAV titers (transducing unit, TU/mL) were determined 72 hours post-transfection. The results are expressed as relative AAV-2 transducing Units/mL (TU/mL).

[0218] The compound 2.22 was used as a positive control. Compounds 2.54 and 2.57 showed promising results and in correlation with the transfection activity presented in FIG. 6. Contrary to the experiments of transfection in Hep G2 cells, compounds 2.60 and 2.61 wherein R and V═H showed high levels of AAV-2 productivity in HEK-293T cells

Example 6. Key Parameters for the Production of AAV-2 from Suspension HEK-293T Cells

[0219] The production of recombinant virus is achieved by co-transfection of many plasmids in HEK293 cells. The virus productivity is greatly influenced by the total amount of plasmids and the volume of transfection reagent. FIG. 8 illustrates the AAV-2 production using the compound 2.22 (formulated at 15 mM nitrogen concentration). Different amounts of plasmids were used to transfect the HEK293-T cells in suspension. Many ratios of transfection reagent were also tested and expressed as μg DNA/μL reagent per millions of cells the day of transfection. The results show that the virus productivity depends on the amount of plasmids transfected. In addition, for each amount of DNA transfected, the optimal productivity depends on the ratio of μg DNA/μL reagent. This example illustrates the transfection conditions with the compounds of formula (III) can be adapted easily to obtain an optimal virus productivity. FIG. 9 presents the influence of time of DNA complexation with the compound 2.22 on the production of AAV-2 from suspension HEK-293T cells. A minimal time of DNA complexation of 15 minutes before adding the transfection complexes into the cell culture is required to obtained high yield of virus production. A longer time of DNA complexation above 15 minutes can be used without affecting the virus yield, indicating a good stability of the transfection complexes in virus production activity. This property indicates that the compound 2.22 is particularly suitable for large scale applications in bioreactors where the time window during the transfer of the transfection complexes mixture needs to be adapted according to the cell culture volume.

CONCLUSION

[0220] Many compounds based on grafting of polyamine with heterocycles of formula (I), preferably of formula (III) showed improved performances to induce gene expression in “hard to transfect” cells such as cancer cells, or to increase the productivity of biologics such as viruses, AAV or LV.

[0221] Many compounds of Example 3, 4 or 5, particularly polyamine grafted with benzyl, fluorobenzyl, hydroxyphenyl, 4-hydroxyphenethyl, pyridine or phenyl triazole derivative showed high transfection efficiencies.

[0222] Selected compounds of Example 3, 4 or 5 also showed improved productivity of biologics such as AAV or LV, indicating a combined effect of high transfection efficiency and gene expression in cells resulting in high virus titers expressed as transducing units. Improved virus productivity was observed whatever the type of transfected cells, e.g. adherent or in suspension. The results obtained indicated that such compounds might be also of interest to produce other biologics such as recombinant proteins, peptides or antibodies.

[0223] Taken together, the compounds of formula (I), preferably of formula (III) of the invention represent novel reagents for transfection and bioproduction purposes wherein a fine optimisation of the chemical structure may be adapted for each application, cell types or transfection conditions.

[0224] The person skilled in the art can adapt the transfection method with the compounds of general formula (I), preferably of general formula (III) of the invention for in vivo applications with an acceptable excipient or buffering agent. The compounds of general formula (I), preferably of general formula (III) can be mixed with DNA to generate DNA complexes suitable for direct injection into animals or humans. Particularly low salt buffering agents such as TRIS, phosphate, or citrate buffer or excipient such as glucose, dextrose, or maltose are known to provide acceptable formulation for direct injection into animals and humans. Many mixture methods between the DNA and the compounds of general formula (I), preferably of general formula (III) are suitable as they are able to generate formulation containing small size particles (non aggregated DNA complexes) that can be injected through various routes of administration.

REFERENCES

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