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COMPOSITIONS FOR TRANSFECTING A NUCLEIC ACID MOLECULE INTO A CELL COMPRISING BENZO-FUSED HETEROCYCLIC COMPOUNDS GRAFTED TO A CATIONIC POLYMER, AND THEIR APPLICATIONS

20220325299 · 2022-10-13

    Inventors

    Cpc classification

    International classification

    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 (II) 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, Z.sup.4, Z.sup.5, Z.sup.6, Z.sup.7, X.sub.1, X.sub.2, R.sub.3, P.sup.+, R, T, U 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 (II) 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: ##STR00321## wherein: Y.sup.1, Y.sup.2 and Y.sup.3, which may be identical or different, represent C or N, with the proviso that at least two of Y.sup.1, Y.sup.2 and Y.sup.3 are N, and with the further proviso that at least one, but no more than two, of Y.sup.1, Y.sup.2 and Y.sup.3 are substituted by Z.sup.1, Z.sup.2 and Z.sup.3 respectively; Z.sup.1 represents H, X.sub.1-R.sub.3-X.sub.2-P.sup.+, X.sub.1-R3-P.sup.+, X.sub.1-X.sub.2-P.sup.+, R3-X.sub.2-P.sup.+, X.sub.1-P.sup.+, R3-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, 0H, 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-R3-X.sub.2-P.sup.+, X.sub.1-R3-P.sup.+, X.sub.1-X.sub.2-P.sup.+, R3-X.sub.2-P.sup.+, X.sub.1-P.sup.+, R3-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, 0H, guanidine, halogen, X.sub.1-R3-X.sub.2-P.sup.+, X.sub.1-R3-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, T, U and V, which may be identical or different, represent C or N, with the proviso that the six-membered ring of (II) contains no more than 2 N atoms; Z.sup.4, Z.sup.5, Z.sup.6 and Z.sup.7, which may be identical or different, represent 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, an amine, a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkylamine, a C.sub.1-C.sub.12 alkoxy, C.sub.2-C.sub.18 alkylidene, 0H, guanidine, or halogen; or (i) Z.sup.4 and Z.sup.5 or (ii) Z.sup.5 and Z.sup.6 or (iii) Z.sup.6 and Z.sup.7 together form a fused, optionally substituted 6-membered aryl or heteroaryl; with the proviso that: 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.+.

    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: (i) Y.sup.1 and Y.sup.3 represent N, Y.sup.2 represents C; or (ii) Y.sup.1 and Y.sup.2 represent N, Y.sup.3 represents C; or (iii) Y.sup.2 and Y.sup.3 represent N, Y.sup.1 represents C; or (iv) Y.sup.1, Y.sup.2 and Y.sup.3 represent N.

    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.+, 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 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, even more preferably Z.sup.2 represents CH.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, or a linear or branched, saturated or unsaturated C.sub.6-C.sub.18 aryl-C.sub.1-C.sub.18 alkyl, more preferably fluorobenzyl or 4-hydroxyphenethyl.

    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.1-1)+, 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 in claim 1; 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.12alkoxy, 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.

    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 in claim 1; 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.

    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 in claim 1; 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.12alkoxy, 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.

    9. 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.+, 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 in claim 1; more 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.12alkoxy, 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.

    10. 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.

    11. The composition according to claim 10, wherein the graft cationic polymer is a linear or branched PEI, more preferably a linear PEI.

    12. 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%.

    13. 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, even more preferably the graft cationic polymer has an average Mw of 6, 8, 10, 15, 22 or 30 kDa, preferably of 6, 8, 10, 15 or 30 kDa.

    14. The composition according to claim 1, wherein: (i) R, T, U and V represent C; or (ii) R, T, U and V, which may be identical or different, represent C or N, with the proviso that the six-membered ring contains no 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; preferably (i) R, T, U and V represent C.

    15. The composition according to claim 1, wherein Z.sup.4, Z.sup.5, Z.sup.6 and Z.sup.7, which may be identical or different, represent H, OH, halogen, halogen-substituted C.sub.1-C.sub.12 alkyl, an amine, a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkylamine, 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.4, Z.sup.5, Z.sup.6 and Z.sup.7, which may be identical or different, represent H, CH.sub.3, NH.sub.2, or OCH.sub.3.

    16. The composition according to claim 1, wherein Z.sup.1 represents 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 in claim 1; 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 and P.sup.+ represents a linear or branched PEI, preferably a linear PEI.

    17. The composition according to claim 1, wherein Z.sup.2 represents a linear or branched, saturated or unsaturated C.sub.1-C.sub.6 alkyl, preferably Z.sup.2 represents CH.sub.3.

    18. The composition according to claim 1, wherein Z.sup.4, Z.sup.5, Z.sup.6 and Z.sup.7 represent H.

    19. The composition according to claim 1, wherein one of Z.sup.4, Z.sup.5, Z.sup.6 or Z.sup.7 represents a linear or branched, saturated or unsaturated C.sub.1-C.sub.6 alkyl, preferably one of Z.sup.4, Z.sup.5, Z.sup.6 or Z.sup.7 represents CH.sub.3.

    20. The composition according to claim 1, wherein Z.sup.4 and Z.sup.6 represent a linear or branched, saturated or unsaturated C.sub.1-C.sub.6 alkyl, preferably Z.sup.4 and Z.sup.6 represent CH.sub.3.

    21. The composition according to claim 1, wherein Z.sup.5 and Z.sup.6 represent a linear or branched, saturated or unsaturated C.sub.1-C.sub.6 alkyl, preferably Z.sup.5 and Z.sup.6 represent CH.sub.3.

    22. The composition according to claim 1, wherein: Y.sup.1 and Y.sup.3 represent N, Y.sup.2 represents C; and R, T, U and V represent C; and Z.sup.2, Z.sup.4 and Z.sup.6 represent a linear or branched, saturated or unsaturated C.sub.1-C.sub.6 alkyl, preferably Z.sup.2, Z.sup.4 and Z.sup.6 represent CH.sub.3.

    23. The composition according to claim 22, wherein 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.+, 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.

    24. The composition according to claim 1, wherein: Y.sup.1 and Y.sup.3 represent N, Y.sup.2 represents C; and Z.sup.1 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; and wherein: (i) R, T, U and V represent C; preferably R, T, U and V represent C, and/or Z.sup.2 represents H, CH.sub.3, SCH.sub.3, CF.sub.3, phenyl, isopropyl, propyl, CH.sub.2—NH—CH.sub.3, CH.sub.2-O-CH.sub.3, or CH.sub.2-F, and/or Z.sup.4 represents H, CH.sub.3, and/or Z.sup.5 represents H, F, OCH.sub.3, carboxyphenyl, tert-butyl, Cl, 0H, or CH.sub.3, and/or Z.sup.6 represents H, CH.sub.3, or F, and/or Z.sup.7 represents H; or (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.sup.2 represents H, CH.sub.3, and/or Z.sup.4 represents H, NH.sub.2, N(CH.sub.3).sub.2, and at least one of Z.sup.5, Z.sup.6 or Z.sup.7 represents H; or (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.sup.2 represents H, CH.sub.3, and/or Z.sup.4 represents H, NH.sub.2, N(CH.sub.3).sub.2, and at least one of Z.sup.5, Z.sup.6 or Z.sup.7 represents H; or (iv) one of R, T, U or V represents N; preferably one of R, T, U or V represents N and at least one of Z.sup.2, Z.sup.4, Z.sup.5, Z.sup.6 or Z.sup.7 represents H; or (v) R, T, U and V represent C, and Z.sup.5 and Z.sup.6 together form an optionally substituted naphtalene; preferably R, T, U and V represent C, Z.sup.5 and Z.sup.6 together form a naphtalene, and/or Z.sup.2 represents CH.sub.3, and/or Z.sup.4 represents H, and/or Z.sup.7 represents H.

    25. The composition according to claim 1, wherein: Y.sup.1 and Y.sup.2 represent N, Y.sup.3 represents C; or Y.sup.2 and Y.sup.3 represent N, Y.sup.1 represents C; and Z.sup.1 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; and wherein: (i) R, T, U and V represent C; preferably R, T, U and V represent C, and at least one of Z.sup.4, Z.sup.5, Z.sup.6 or Z.sup.7 represents H; or (ii) R, T, U and V, which may be identical or different, represent C or N, with the proviso that the six-membered ring contains no more than 1 N atom; preferably, one of R, U or V represents N and at least one of Z.sup.4, Z.sup.5, Z.sup.6 or Z.sup.7 represents H.

    26. The composition according to claim 1, wherein: Y.sup.1, Y.sup.2 and Y.sup.3 represent N; and Z.sup.1 or 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.+, wherein X.sub.1, X.sub.2, R.sub.3 and P.sup.+ are as defined in claim 1; and wherein: (i) R, T, U and V represent C; preferably R, T, U and V represent C, and/or Z.sup.4 represents H, and/or Z.sup.5 represents H, CH.sub.3, OCH.sub.3, and/or Z.sup.6 represents H, CH.sub.3, and/or Z.sup.7 represents H; or (ii) R, T, U and V, which may be identical or different, represent C or N, with the proviso that the six-membered ring contains no more than 1 N atom; preferably, one of R, T or U represents N and at least one of Z.sup.4, Z.sup.5, Z.sup.6 or Z.sup.7 represents H.

    27. The composition according to claim 1, wherein the at least one compound of general formula (II) is selected from the group consisting of the following compounds: ##STR00322## ##STR00323## ##STR00324## ##STR00325## ##STR00326## ##STR00327## ##STR00328## ##STR00329## ##STR00330## ##STR00331## ##STR00332## ##STR00333## ##STR00334## ##STR00335## ##STR00336## ##STR00337## ##STR00338##

    28. The composition according to claim 27, wherein the at least one compound of general formula (II) is selected from the group consisting of the following compounds: ##STR00339## ##STR00340## ##STR00341## ##STR00342## ##STR00343##

    29. The composition according to claim 28, wherein the at least one compound of general formula (II) is compound 1.42, 1.57 or 1.65.

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

    31. 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.

    32. The method of claim 31, wherein the method is performed to transfect at least one nucleic acid molecule, preferably at least one DNA, into a stem cell, said composition comprising (i) the compound 1.42, and (ii) an acceptable excipient, buffering agent, cell culture medium, or transfection medium.

    33. The method of claim 31, wherein the method is performed to transfect at least one nucleic acid molecule, preferably at least one DNA, into a neuronal cell, said composition comprising (i) the compound 1.65 or the compound 1.60, and (ii) an acceptable excipient, buffering agent, cell culture medium, or transfection medium.

    34. 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.

    35. 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.

    36. The method according to claim 35, for the production of AAV or LV, said composition comprising (i) at least the compound 1.42, and (ii) an acceptable excipient, buffering agent, cell culture medium, or transfection medium.

    37. The method according to claim 35, for the production of LV, said composition comprising (i) at least the compound 1.57, and (ii) an acceptable excipient, buffering agent, cell culture medium, or transfection medium.

    38. The method according to claim 35, 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.

    39. The method according to claim 38, further comprising a step of performing wherein said recombinant virus is for use in in vivo applications for cell therapy or for gene therapy, wherein the recombinant virus is used in vivo.

    Description

    BRIEF DESCRIPTION OF THE FIGURES

    [0129] FIG. 1. 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.

    [0130] FIG. 2. Percentage of GFP expression after transfection of Caco-2, Hep G2, MDCK and MCF-10A with compounds of Example 4. The ratio 1:3 and 1:4 indicate the ratio of μg of DNA per μL of compound.

    [0131] FIG. 3. Percentage of GFP expression after transfection of HepG2 cells with jetPEI® (0% grafting) and compounds 1.57, 1.64, 1.42, 1.72, and 1.56 comprising 7%, 14%, 22%, 25% and 30% of grafting extent of 2-methyl-benzimadazole to the linear PEI 22 kDa, respectively.

    [0132] FIG. 4. Percentage of GFP expression after transfection of primary dermal fibroblasts (HPDF) and primary endothelial cells (HUVEC) with compounds 1.41, 2.03, 2.05, 1.42, 2.08 and LipoFectamine® 3000 as a commercial reference.

    [0133] FIG. 5. Transfection of Primary rat cortex neurons (RCN) and primary rat hippocampal neurons (RHN).

    [0134] FIG. 6. 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/4 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.

    [0135] FIG. 7. Production of lentivirus particles from suspension HEK-293T cells. Lentivirus expressing the GFP reporter gene was produced in HEK-293Tcells grown in suspension in FreeStyle F17 media. Cells were seeded and cultured for 3 days before being transfected by 4 plasmids with PEIpro® or various compounds at ratio 1:2 μg total DNA/4 reagent. Lentivirus titers (transducing unit, TU/mL) were determined 72 hours post-transfection.

    [0136] FIG. 8. Gel electrophoresis showing genome editing in HEK293 cells after transfection of plasmid CRISPR Cas9 targeting the HRPT-1 gene (plasmid p38285) with compound 1.42. Two days after the transfection, the genomic DNA was extracted and the targeted HPRT-1 focus was amplified by PCR. After digestion by the T7 endonuclease I, the PCR products were run on a 2% agarose gel and stained with ethidium bromide. Cas9-induced cleavage HPTR-1 bands (650 and 430 bp) and the uncleaved HPTR-1 band (1083 bp) were visualized and quantified on the gel, then the genome editing efficiency was determined (INDEL %). The INDEL % was 33.48+/−7.08% for the plasmid p38285, where no INDEL event was detected with the plasmid pCONTROL.

    [0137] FIG. 9. Transfection efficiency of human mesenchymal stem cells (hMSC) with the plasmid pCMV-EGFP and compound 1.42. A) Observation of hMSC by phase contrast and fluorescence microscopy 24 hours after transfection with 400 ng of pCMV-EGFP and 2 μL of compound 1.42. B) GFP expression analysis by flow cytometry 24 hours after transfection of hMSC with 400 ng of pCMV-EGFP and 0.4, 0.6 and 0.8 μL of compound 1.42, with 500 ng of pCMV-EGFP and 0.5, 0.75 and 14 of compound 1.42, and 500 ng of pCMV-EGFP and 0.75 and 1.54 of Lipofectamine 3000 reagent.

    [0138] FIG. 10. Chemical structure of a compound of general formula (II).

    [0139] FIG. 11. Percentage of GFP expression after transfection of Hep G2 cells with compounds of Example 10. The ratio 1:3 and 1:4 indicate the ratio of μg of DNA per μL of compound.

    [0140] 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 media. Cells were seeded and cultured for 3 days before being transfected by 3 plasmids (pAAV-RC2 vector expressing Rep and Cap from Cell BIOLABS, INC., pALD-X.sub.80, Helper vector expressing Adeno E2A, Adeno E4 and Adeno VA helper factors from ALDEVRON, and pAAV-GFP control vector expressing the GFP under the control of a CMV promoter from Cell BIOLABS, INC.) with PEIpro® or various compounds. 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 their best ratio of μg of DNA per μL of compound (ratio 1:2 for PEIpro®, 1.42, 1.73, 1.74, 1.76, 1.80, 1.75 and ratio 1:5 for BPEI 25K, BPEI 10K, PAA, PVA, PLL, 1.78 and 1.77.

    DESCRIPTION OF THE PREFERRED EMBODIMENTS

    Examples

    Experimental Section

    Material and Methods

    Cell Culture

    [0141] 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.

    [0142] 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.

    [0143] 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.

    [0144] 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.

    [0145] 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.

    [0146] Transfection Assay (96-Well Format)

    [0147] 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 (II) (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.

    [0148] 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).

    [0149] Transfection Assay of Primary Cells

    [0150] HUVEC Human Umbilical Vein Endothelial Cells (Promocell) were seeded at 20 000 cells per well (24-well plate format) in 500 μL of Endothelial Cell Growth Medium with supplementMix (Promocell) and incubated at 37° C. in a 5% CO.sub.2 in air atmosphere. Three days later, the complete medium was removed and replaced by 500 μL of DMEM supplemented with 2% FBS. Then, 500 ng of pCMV-EGFPLuc DNA (Clontech) was added in 50 μL of NaCl buffer, mixed with a vortex and incubated for 5 minutes at rt. Then, 1.5 μL of a compound of general formula (II) (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 (50 μL) was added into the well and the plate was incubated at 37° C. in a 5% CO.sub.2 in air atmosphere. After 2 h of incubation, the medium was removed and replaced by 500 μL of Endothelial Cell Growth Medium with supplementMix (Promocell) and cells were incubated at 37° C. in a 5% CO.sub.2 in air atmosphere. One day post-transfection, the GFP expression was analysed by flow cytometry.

    [0151] Primary human dermal fibroblasts were obtained from Pr. Stéphane Viville (Centre Hospitalier Universitaire, Strasbourg, France). The 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 of penicillin and 100 μg/mL of streptomycin at 37° C. in a 5% CO.sub.2 in air atmosphere. For the transfection, the cells were seeded at 40 000 cells per well (24-well plate format) precoated with 0.1% gelatin in 500 μL of DMEM 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, and incubated for 24 h at 37° C. in a 5% CO.sub.2 in air atmosphere. Then, 500 ng of pCMV-EGFPLuc DNA (Clontech) was added in 50 μL of NaCl buffer, mixed with a vortex and incubated for 5 minutes at rt. Then, 1.5 μL of a compound of general formula (II) (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 (50 μL) was added into the well and the plate was incubated at 37° C. in a 5% CO.sub.2 in air atmosphere. After 4 h of incubation, the medium was removed and replaced by 500 μL of complete DMEM, and cells were incubated at 37° C. in a 5% CO.sub.2 in air atmosphere. One day post-transfection, the GFP expression was analysed by flow cytometry.

    [0152] Primary rat Cortex Neurons (RCN, ThermoFisher) were seeded at 100 000 cells per well (48-well plate format) pre-coated with D-poly-lysine (Sigma) in 0.5 mL of complete neurobasal medium (ThermoFisher) supplemented with B27 supplement (ThermoFisher) and 0.5 mM glutamine, and cells were incubated at 37° C. in a 5% CO.sub.2 in air atmosphere. Every two days, half of the complete medium volume was changed. After 4 days, 150 ng of pCMV-EGFPLuc DNA (Clontech) was added in 25 μL of OPTIMEM, mixed with a vortex and incubated for 5 minutes at rt. Then, 0.15 μL of a compound of general formula (II) (at 7.5 mM nitrogen concentration) were added onto the diluted DNA, mixed with a vortex and incubated for 10 minutes at rt. 250 μL of complete medium was removed and the formulated DNA solution (25 μL) was added into the well and the plate was incubated at 37° C. in a 5% CO.sub.2 in air atmosphere. After 4 h of incubation at 37° C. in a 5% CO.sub.2 in air atmosphere, 250 μL of the complete medium was added per well, and cells were incubated at 37° C. in a 5% CO.sub.2 in air atmosphere. Cells were also transfected with 150 ng of pCMV-EGFPLuc complexed with LipoFectAmine® 3000 (ratio 1 μg: 1.5 μL), LipoFectAmine® 2000 (ratio 1 μg: 4 μL) in OPTIMEM and jetPEI® (ratio 1 μg: 2 μL) in 150 mM NaCl according to the recommended commercial protocols. One day post-transfection, the GFP expression was observed using a ZOE™ Fluorescent Cell Imager (Biorad).

    [0153] Recombinant Virus Production

    [0154] 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.

    [0155] 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.

    [0156] 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.

    [0157] 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).

    [0158] 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.

    [0159] 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).

    [0160] 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.

    [0161] CRISPR Cas9 DNA Transfection

    [0162] HEK293 (ECACC 85120602) human embryonic epithelial kidney cells were grown in Eagle MEM medium with 10% FBS supplemented with 2 mM Glutamine, 0.1 mM non-essential amino acids, 200 U/mL of penicillin and 200 μg/mL of streptomycin. One day before transfection, 12 500 cells were added per well (96-well plate format) in 125 μL of complete medium and the plate was incubated for 24 hours at 37° C. in a 5% CO.sub.2 in air atmosphere.

    [0163] The plasmid pSpCas9 BB-2A-GFP (9.3 kb) from GenScript (Leiden, Netherlands) used for the transfection experiment expressed a version of the Streptococcus pyogenes Cas9 protein (CRISPR Associated Protein 9) with an N and C terminal nuclear localization signal (NLS) under control of the CBh promoter. This plasmid contains a G(N)20 gRNA (guide RNA) and the gRNA scaffold sequences under control of the U6 promoter. The G(N)20 gRNA was designed to target the HPRT-1 (hypoxanthine phosphoribosyltransferase) human gene at the sequence position 38285 (targeted cleavage site by the CRISPR Cas9) generating the plasmid p38285. A second G(N) 20 gRNA was designed to generate the plasmid pCONTROL which is not able to trigger a CRISPR event in human cells.

    [0164] On the day of transfection, 100 ng of plasmid p38285 or pCONTROL was added in 12 μL of OPTIMEM. Then, 0.1 μL of compound 1.42 at 7.5 mM amine concentration was added onto the diluted plasmid, mixed with a vortex and incubated for 10 minutes at rt. The complexed plasmid was added into the well and the plate was incubated 37° C. in a 5% CO.sub.2 in air atmosphere.

    [0165] Two days post-transfection, the medium was removed and cells were washed with PBS. Genomic DNA was isolated with the addition of 50 μL of QuickExtract™ DNA Extraction Solution 1.0 (Epicentre) per well followed by an incubation at 65° C. for 6 minutes, then at 98° C. for 2 minutes and storage at 4° C. The HPRT-1 targeted genomic DNA (250 ng) was amplified by PCR using the Primer HPRT1 mix (IDT) and the Q5® Hot Start High-Fidelity 2× Master Mix (New England Biolabs®). The following PCR conditions were used in a iCycler™ 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) were combined with 1.5 μL of 10× NEBuffer 2 (NEB) and 1.5 μL of nuclease free water (total volume of 18 μL) and denatured then re-annealed with thermocycling at 95° C. for 10 minutes, 95 to 85° C. at −2° C./second; 85 to 25° C. at −0.3° C./second. The re-annealed DNA was incubated with 1 μl of T7 Endonuclease I (10 U/μl, NEB) at 37° C. for 15 minutes. 19 μL of T7 Endonuclease reaction was combined with 2 μL of loading buffer and analyzed on a 2% TAE agarose gel electrophoresed for 45 minutes at 100 V in the presence of Quick Load 100 μb DNA ladder (New England Biolabs®). The gel was stained with ethidium bromide for 30 min. Cas9-induced cleavage bands (827 and 256 bp) and the uncleaved band (1083 bp) were visualized on a G:Box transilluminator (Syngene) and quantified using GeneTools software. The INDEL % was calculated using the following formula: INDEL %=100*[1−(1−((intensities of cleaved bands)/(intensities of cleaved bands and uncleaved band)))].

    [0166] Transfection Assay of Stem Cells

    [0167] 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 of penicillin and 100 μg/mL of streptomycin at 37° C. in a 5% CO.sub.2 in air atmosphere.

    [0168] HMSC were seeded at 12 000 cells per well (24-well plate format) in 500 μL of MSC complete growth medium (Lonza) and incubated at 37° C. in a 5% CO.sub.2 in air atmosphere. Three days after the complete medium was removed and replaced by 500 μL of MSC Basal Medium. Then, pCMV-EGFP DNA (Clontech) was added in 50 μL of NaCl buffer, mixed with a vortex and incubated for 5 minutes at rt. Then, compound 1.42 (at 7.5 mM nitrogen concentration) was added onto the diluted DNA, mixed with a vortex and incubated for 10 minutes at rt. The formulated DNA solution (50 μL) was added into the well and the plate was incubated at 37° C. in a 5% CO.sub.2 in air atmosphere. After 4 h of incubation, the medium was removed and replaced by 500 μL of MSC complete growth medium and cells were incubated at 37° C. in a 5% CO.sub.2 in air atmosphere. One day post-transfection, the GFP expression was analysed by flow cytometry or observed using a ZOE™ Fluorescent Cell Imager (Biorad).

    Example 1. General Procedure for the Preparation of Grafted Polymers

    [0169] ##STR00118##

    Step 1: N-Alkylation of Heterocycles

    [0170] In an oven-dried round-bottom flask under argon was added the corresponding heterocycle (1 equiv.) and DMF (2 ml/mmol of starting material). The solution was cooled to 0° C. and Sodium Hydride (60% dispersion in mineral oil, 1.2 equiv.) was added by portion. The mixture was slowly warmed up to room temperature over 1 h. Then, the corresponding ester was added dropwise and the reaction was stirred at room temperature for 4-12 h. The mixture was quenched by addition of water (10 mL/1 mL of DMF) and the aqueous layer was extracted with EtOAc. (5×2 mL/1 mL of DMF). The combined organic extracts were washed with brine and dried over anhydrous MgSO.sub.4. After filtration, the solvent was removed in vacuo and the resulting oil was purified by column chromatography (EtOAc 20 to 50% in heptane).

    Step 2: Saponification of Acid Moieties

    [0171] To a solution of ester in EtOH (2 mL/mmol of ester) was added dropwise a 5M solution of NaOH (0.2 mL/mmol of ester), and the mixture was stirred at room temperature overnight. Then, the solvent was removed in vacuo and the residue was purified by column chromatography on SiO.sub.2 using MeOH 5% in DCM+AcOH 1% or using Acetonitrile 0 to 100% in H.sub.2O.

    Step 3: Grafting

    [0172] 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 or by UF using Amicon Ultra 15 with HCl 50 mM.

    Step 4: Procedure for Grafting PLL (22K, Sigma-Aldrich) with EDCl and NHS. At room temperature and in microwave vial, poly-L-lysine (1 equiv.) was dissolved in 25-mM MES (pH 6.5) buffer to which was added 4-imidazoleacetic acid (sodium salt) (0.75 to 1.5 equiv.). This solution was used to dissolve EDCl (1.5 to 3 equiv). NHS (1 equiv.) was dissolved in MES buffer and was added immediately to the poly-L-lysine solution. The vial was sealed and stirred for 24 h at room temperature. The product was then purified by dialysis against water or on Amicon ultra 15 with water.

    Example 2. Syntheses of (i) Compounds of Formula (II) of the Invention (benzimidazole, benzopyrazole and benzotriazole Derivatives), i.e. 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

    Synthesis of Product 1.01

    [0173] ##STR00119##

    [0174] Intermediate 1.01a was prepared analogously to the general procedure, step 1 (Example 1). Yield=60%; m=2.30 g; .sup.1H 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).

    ##STR00120##

    [0175] Intermediate 1.01b was prepared analogously to the general procedure, step 2 (Example 1). Yield=45%; m=1.00 g; .sup.1H NMR (400 MHz, Methanol-d.sub.4) 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).

    ##STR00121##

    [0176] Product 1.01 was prepared analogously to the general procedure, step 3 (Example 1). Yield=94%; m=117 mg; .sup.1H NMR (D.sub.2O) δ: .sup.1H 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).

    Synthesis of Product 1.02

    [0177] ##STR00122##

    [0178] Intermediate 1.02a was prepared analogously to the general procedure, step 1 (Example 1). Yield=73%; m=857 mg; .sup.1H 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).

    ##STR00123##

    [0179] Intermediate 1.02b was prepared analogously to the general procedure, step 2 (Example 1). Yield=67%; m=48 mg; .sup.1H 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).

    ##STR00124##

    [0180] Product 1.02 was prepared analogously to the general procedure, step 3 (Example 1). Yield=85%; m=12 mg; .sup.1H 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).

    Synthesis of Product 1.03

    [0181] ##STR00125##

    [0182] Intermediate 1.03a was prepared analogously to the general procedure, step 2 (Example 1). Yield=100%; m=303 mg; .sup.1H 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).

    ##STR00126##

    [0183] Intermediate 1.03b was prepared analogously to the general procedure, step 1 (Example 1). Yield=26%; m=327 mg; .sup.1H 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).

    ##STR00127##

    [0184] Product 1.03 was prepared analogously to the general procedure, step 3 (Example 1). Yield=88%; m=13 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 9.20-8.32 (m, 2H), 4.51-2.93 (m, 21H), 2.88-1.68 (m, 7H).

    Synthesis of Product 1.04

    [0185] ##STR00128##

    [0186] Intermediate 1.04a was prepared analogously to the general procedure, step 1 (Example 1). Yield=51%; m=597 mg; .sup.1H NMR (400 MHz, Chloroform-d) δ 8.41 (dd, J=4.8, 1.5 Hz, 1H), 8.21 (s, 1H), 8.09 (dd, J=8.1, 1.5 Hz, 1H), 7.29-7.22 (m, 1H), 4.40 (t, J=6.8 Hz, 2H), 4.10 (q, J=7.1 Hz, 2H), 2.42-2.17 (m, 4H), 1.22 (t, J=7.1 Hz, 3H).

    ##STR00129##

    [0187] Intermediate 1.04b was prepared analogously to the general procedure, step 2 (Example 1). Yield=76%; m=441 mg; .sup.1H 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).

    ##STR00130##

    [0188] Product 1.04 was prepared analogously to the general procedure, step 3 (Example 1). Yield=78%; m=10 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 9.46-7.11 (m, 4H), 4.64-4.15 (m, 1H), 4.14-1.88 (m, 41H).

    Synthesis of Product 1.05

    [0189] ##STR00131##

    [0190] Intermediate 1.05a was prepared analogously to the general procedure, step 1 (Example 1). Yield=77%; m=783 mg; .sup.1H 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).

    ##STR00132##

    [0191] Intermediate 1.05b was prepared analogously to the general procedure, step 2 (Example 1). Yield=68%; m=311 mg; .sup.1H 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).

    ##STR00133##

    [0192] Product 1.05 was prepared analogously to the general procedure, step 3 (Example 1). Yield=74%; m=13 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.62-7.38 (m, 2H), 4.42-1.55 (m, 17H).

    Synthesis of Product 1.06

    [0193] ##STR00134##

    [0194] Intermediate 1.06a was prepared analogously to the general procedure, step 1 (Example 1). Yield=44%; m=810 mg; .sup.1H NMR (400 MHz, Chloroform-d) δ 8.33 (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)

    ##STR00135##

    [0195] Intermediate (1.06b) was prepared analogously to the general procedure, step 2 (Example 1). Yield=99%; m=710 mg; .sup.1H 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).

    ##STR00136##

    [0196] Product 1.06 was prepared analogously to the general procedure, step 3 (Example 1). Yield=99%; m=13 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.78-7.72 (m, 2H), 4.33-1.07 (m, 38H).

    Synthesis of Product 1.07

    [0197] ##STR00137##

    [0198] Intermediate 1.07a was prepared analogously to the general procedure, step 1 (Example 1). Yield=61%; m=2.7 g; .sup.1H 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).

    ##STR00138##

    [0199] Intermediate 1.07b was prepared analogously to the general procedure, step 2 (Example 1). Yield=17%; m=400 mg; .sup.1H 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).

    ##STR00139##

    [0200] Product 1.07 was prepared analogously to the general procedure, step 3 (Example 1). Yield=96%; m=134 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.03-7.09 (m, 4H), 4.34-2.28 (m, 32H).

    Synthesis of Product 1.08

    [0201] ##STR00140##

    [0202] Intermediate 1.08a was prepared analogously to the general procedure, step 1 (Example 1). Yield=73%; m=3.0 g; .sup.1H 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).

    ##STR00141##

    [0203] Intermediate 1.08b was prepared analogously to the general procedure, step 2 (Example 1). Yield=27%; m=700 mg; .sup.1H NMR (D.sub.2O) δ: .sup.1H 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).

    ##STR00142##

    [0204] Product 1.08 was prepared analogously to the general procedure, step 3 (Example 1). Yield=100%; m=130 mg; .sup.1H 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).

    Synthesis of Product 1.09

    [0205] ##STR00143##

    [0206] Product 1.09 was prepared analogously to the general procedure, step 3 (Example 1). Yield=100%; m=150 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.87-7.21 (m, 4H), 4.15-2.49 (m, 29H).

    Synthesis of Product 1.10

    [0207] ##STR00144##

    [0208] Intermediate 1.10a was prepared analogously to the general procedure, step 1 (Example 1). Yield=28%; m=610 mg; .sup.1H 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).

    ##STR00145##

    [0209] Intermediate 1.10b was prepared analogously to the general procedure, step 2 (Example 1). Yield=71%; m=386 mg; .sup.1H NMR (400 MHz, Methanol-d.sub.4) 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).

    ##STR00146##

    [0210] Product 1.10 was prepared analogously to the general procedure, step 3 (Example 1). Yield=98%; m=170 mg; .sup.1H 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).

    Synthesis of Product 1.11

    [0211] ##STR00147##

    [0212] Intermediate 1.11a was prepared analogously to the general procedure, step 1 (Example 1). Yield=30%; m=1.44 g; .sup.1H 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).

    ##STR00148##

    [0213] Intermediate 1.11b was prepared analogously to the general procedure, step 2 (Example 1). Yield=75%; m=970 mg; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.57 (d, J=7.6 Hz, 1H), 7.50 (d, J=7.7 Hz, 1H), 7.27 (p, J=7.4 Hz, 2H), 4.24 (t, J=7.4 Hz, 2H), 2.63 (d, J=2.2 Hz, 3H), 2.30 (t, J=7.4 Hz, 2H), 1.85 (p, J=7.8 Hz, 2H), 1.67 (p, J=7.5 Hz, 2H), 1.43 (p, J=7.8 Hz, 2H).

    ##STR00149##

    [0214] Product 1.11 was prepared analogously to the general procedure, step 3 (Example 1). Yield=100%; m=206 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) 6 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).

    Synthesis of Product 1.12

    [0215] ##STR00150##

    [0216] Intermediate 1.12a was prepared analogously to the general procedure, step 1 (Example 1). Yield=85%; m=3.63 g; .sup.1H 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).

    ##STR00151##

    [0217] Intermediate 1.12b was prepared analogously to the general procedure, step 2 (Example 1). Yield=36%; m=1.19 g; .sup.1H NMR (400 MHz, Methanol-d.sub.4) 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).

    ##STR00152##

    [0218] Product 1.12 was prepared analogously to the general procedure, step 3 (Example 1). Yield=100%; m=217 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.80-6.90 (m, 4H), 4.52-3.09 (m, 12H), 3.05-1.63 (m, 7H).

    Synthesis of Product 1.13

    [0219] ##STR00153##

    [0220] Intermediate 1.13a was prepared analogously to the general procedure, step 1 (Example 1). Yield=33%; m=750 mg; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.82-7.66 (m, 2H), 7.53-7.36 (m, 2H), 4.46 (t, J=7.8 Hz, 1H), 4.09 (q, J=7.4 Hz, 1H), 2.46 (t, J=6.9 Hz, 2H), 2.15 (t, J=7.5 Hz, 2H), 1.23 (t, J=7.3 Hz, 3H).

    ##STR00154##

    [0221] Intermediate 1.13b was prepared analogously to the general procedure, step 2 (Example 1). Yield=53%; m=360 mg; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.80 (t, J=6.7 Hz, 2H), 7.52 (t, J=7.9 Hz, 1H), 7.44 (t, J=7.8 Hz, 1H), 4.51 (t, J=7.9 Hz, 2H), 2.47 (t, J=7.1 Hz, 2H), 2.17 (p, J=7.4 Hz, 2H).

    ##STR00155##

    [0222] Product 1.13 was prepared analogously to the general procedure, step 3 (Example 1). Yield=100%; m=150 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.39-5.98 (m, 4H), 4.49-0.19 (m, 17H).

    Synthesis of Product 1.14

    [0223] ##STR00156##

    [0224] Product 1.14 was prepared analogously to the general procedure, step 3 (Example 1). Yield=93%; m=237 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.94-6.59 (m, 4H), 4.62-1.17 (m, 28H).

    Synthesis of Product 1.15

    [0225] ##STR00157##

    [0226] Intermediate 1.15a was prepared analogously to the general procedure, step 2 (Example 1). Yield=91%; m=1.19 g; .sup.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).

    ##STR00158##

    [0227] Intermediate 1.15b was prepared analogously to the general procedure, step 2 (Example 1). Yield=31%; m=1.46 g; .sup.1H NMR (CDCl.sub.3) δ: .sup.1H 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).

    ##STR00159##

    [0228] Product 1.15 was prepared analogously to the general procedure, step 3 (Example 1). Yield=100%; m=143 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.84-7.10 (m, 4H), 4.51-3.09 (m, 22H), 3.07-1.61 (m, 6H).

    Synthesis of Product 1.16

    [0229] ##STR00160##

    [0230] Intermediate 1.16a was prepared analogously to the general procedure, step 1 (Example 1). Yield=75%; m=2.4 g; .sup.1H 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).

    ##STR00161##

    [0231] Intermediate 1.16b was prepared analogously to the general procedure, step 2 (Example 1). Yield=42%; m=920 mg; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.11 (d, J=7.1 Hz, 1H), 7.94 (d, J=7.2 Hz, 2H), 7.86 (dd, J=16.2, 7.3 Hz, 2H), 7.79 (d, J=7.2 Hz, 2H), 7.71 (d, J=6.5 Hz, 2H), 4.62 (t, J=7.4 Hz, 2H), 2.44 (s, 2H), 2.22 (s, 2H).

    ##STR00162##

    [0232] Product 1.16 was prepared analogously to the general procedure, step 3 (Example 1). Yield=96%; m=162 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.36-6.92 (m, 9H), 4.57-2.85 (m, 21H), 2.76-1.33 (m, 4H).

    Synthesis of Product 1.17

    [0233] ##STR00163##

    [0234] Product 1.17 was prepared analogously to the general procedure, step 3 (Example 1). Yield=99%; m=170 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.82-7.03 (m, 4H), 4.40-2.94 (m, 19H), 2.94-1.41 (m, 7H).

    Synthesis of Product 1.18

    [0235] ##STR00164##

    [0236] Intermediate 1.18a was prepared analogously to the general procedure, step 1 (Example 1). Yield=44%; m=2.29 g; .sup.1H NMR (400 MHz, Chloroform-d) δ 7.81-7.73 (m, 1H), 7.37 (dd, J=6.6, 2.8 Hz, 1H), 7.32-7.20 (m, 2H), 4.19 (dq, J=14.2, 7.3 Hz, 4H), 3.22 (hept, J=6.9 Hz, 1H), 2.40 (t, J=6.8 Hz, 2H), 2.14 (p, J=7.1 Hz, 2H), 1.48 (dd, J=6.8, 1.5 Hz, 6H), 1.29 (tt, J=7.1, 1.1 Hz, 3H).

    ##STR00165##

    [0237] Intermediate 1.18b was prepared analogously to the general procedure, step 2 (Example 1). Yield=61%; m=1.25 g; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.60 (dd, J=17.9, 7.6 Hz, 2H), 7.28 (t, J=7.0 Hz, 2H), 4.34 (t, J=7.8 Hz, 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).

    ##STR00166##

    [0238] Product 1.18 was prepared analogously to the general procedure, step 3 (Example 1). Yield=94%; m=167 mg; .sup.1H 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).

    Synthesis of Product 1.19

    [0239] ##STR00167##

    [0240] Intermediate 1.19a was prepared analogously to the general procedure, step 1 (Example 1). Yield=41%; m=700 mg; .sup.1H NMR (400 MHz, Chloroform-d) δ 7.78-7.70 (m, 1H), 7.39-7.32 (m, 1H), 7.31-7.20 (m, 2H), 4.24-4.12 (m, 4H), 2.87 (t, J=7.7 Hz, 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).

    ##STR00168##

    [0241] Intermediate 1.19b was prepared analogously to the general procedure, step 2 (Example 1). Yield=97%; m=610 mg; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.64-7.55 (m, 2H), 7.29 (t, J=7.1 Hz, 2H), 4.32 (t, J=7.7 Hz, 2H), 3.33 (s, 2H), 2.97 (t, J=7.4 Hz, 2H), 2.42 (t, J=6.9 Hz, 2H), 2.10 (p, J=7.0 Hz, 2H), 1.92 (h, J=7.6 Hz, 2H), 1.09 (td, J=7.4, 2.2 Hz, 3H).

    ##STR00169##

    [0242] Product 1.19 was prepared analogously to the general procedure, step 3 (Example 1). Yield=95%; m=166 mg; .sup.1H 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).

    Synthesis of Product 1.20

    [0243] ##STR00170##

    [0244] Intermediate 1.20a was prepared analogously to the general procedure, step 1 (Example 1). Yield=100%; m=1.54 g; .sup.1H 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).

    ##STR00171##

    [0245] Intermediate 1.20b was prepared analogously to the general procedure, step 2 (Example 1). Yield=69%; m=1.05 g; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.64 (dd, J=17.8, 7.8 Hz, 2H), 7.31 (dt, J=17.0, 7.4 Hz, 2H), 4.79 (s, 2H), 4.34 (t, J=7.9 Hz, 2H), 2.95 (s, 3H), 2.41 (t, J=7.2 Hz, 2H), 2.07 (t, J=8.2 Hz, 2H), 1.48 (s, 9H).

    ##STR00172##

    [0246] Product 1.20 was prepared analogously to the general procedure, step 3 (Example 1). Yield=98%; m=183 mg; .sup.1H 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).

    Synthesis of Product 1.21

    [0247] ##STR00173##

    [0248] Intermediate 1.21a was prepared analogously to the general procedure, step 1 (Example 1). Yield=63%; m=2.63 g; .sup.1H NMR (400 MHz, Chloroform-d) δ 7.77-7.70 (m, 1H), 7.44-7.37 (m, 1H), 7.32-7.20 (m, 2H), 4.74 (d, J=2.1 Hz, 2H), 4.33-4.24 (m, 2H), 4.10 (qd, J=7.2, 1.9 Hz, 2H), 3.37 (d, J=2.0 Hz, 3H), 2.35 (td, J=6.9, 2.0 Hz, 2H), 2.13 (p, J=7.1 Hz, 2H), 1.22 (td, J=7.1, 2.0 Hz, 3H).

    ##STR00174##

    [0249] Intermediate 1.21b was prepared analogously to the general procedure, step 2 (Example 1). Yield=63%; m=1.63 g; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.65 (ddd, J=18.3, 8.2, 2.8 Hz, 2H), 7.39-7.25 (m, 2H), 4.76 (t, J=2.3 Hz, 2H), 4.43-4.34 (m, 2H), 3.44 (s, 3H), 2.41 (td, J=7.2, 6.5, 3.7 Hz, 2H), 2.17 (q, J=7.8 Hz, 2H).

    ##STR00175##

    [0250] Product 1.21 was prepared analogously to the general procedure, step 3 (Example 1). Yield=91%; m=172 mg; .sup.1H 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).

    Synthesis of Product 1.22

    [0251] ##STR00176##

    [0252] Product 1.22 was prepared analogously to the general procedure, step 3 (Example 1). Yield=42%; m=129 mg; .sup.1H 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).

    Synthesis of Product 1.23

    [0253] ##STR00177##

    [0254] Intermediate 1.23a was prepared analogously to the general procedure, step 1 (Example 1). Yield=29%; m=523 mg; .sup.1H NMR (400 MHz, Chloroform-d) δ 7.83 (d, J=7.8 Hz, 1H), 7.52 (d, J=7.9 Hz, 1H), 7.37 (dt, J=17.6, 7.3 Hz, 2H), 5.75 (d, J=47.8 Hz, 2H), 4.38 (t, J=7.6 Hz, 2H), 4.15 (q, J=7.3 Hz, 2H), 2.41 (t, J=6.8 Hz, 2H), 2.21 (p, J=7.0 Hz, 2H), 0.98-0.79 (m, 3H).

    ##STR00178##

    [0255] Intermediate 1.23b was prepared analogously to the general procedure, step 2 (Example 1). Yield=67%; m=343 mg; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.75-7.63 (m, 2H), 7.44-7.27 (m, 2H), 5.70 (dd, J=47.9, 3.0 Hz, 2H), 4.47-4.36 (m, 2H), 2.41 (q, J=5.5, 4.5 Hz, 2H), 2.17 (q, J=7.8 Hz, 2H).

    ##STR00179##

    [0256] Product 1.23 was prepared analogously to the general procedure, step 3 (Example 1). Yield=99%; m=166 mg; .sup.1H 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).

    Synthesis of Product 1.24

    [0257] ##STR00180##

    [0258] Product 1.24 was prepared analogously to the general procedure, step 3 (Example 1). Yield=91%; m=117 mg; .sup.1H NMR 400 MHz, Deuterium Oxide) δ 7.95-7.17 (m, 4H), 4.54-2.39 (m, 24H), 2.07-1.56 (m, 2H).

    Synthesis of Product 1.25

    [0259] ##STR00181##

    [0260] Product 1.25 was prepared analogously to the general procedure, step 3 (Example 1). Yield=81%; m=137 mg; .sup.1H NMR 400 MHz, Deuterium Oxide) δ 7.82-7.05 (m, 4H), 4.62-2.27 (m, 22H), 2.12-1.74 (m, 2H).

    Synthesis of Product 1.26

    [0261] ##STR00182##

    [0262] Product 1.26 was prepared analogously to the general procedure, step 3 (Example 1). Yield=81%; m=101 mg; .sup.1H NMR 400 MHz, Deuterium Oxide) δ 7.94-7.18 (m, 4H), 4.71-2.18 (m, 25H), 2.05-1.82 (m, 2H).

    Synthesis of Product 1.27

    [0263] ##STR00183##

    [0264] Product 1.27 was prepared analogously to the general procedure, step 3 (Example 1). Yield=91%; m=173 mg; .sup.1H NMR 400 MHz, Deuterium Oxide) δ 7.87-7.02 (m, 4H), 4.51-2.23 (m, 17H), 2.10-1.67 (m, 2H).

    Synthesis of Product 1.28

    [0265] ##STR00184##

    [0266] Product 1.28 was prepared analogously to the general procedure, step 3 (Example 1). Yield=100%; m=152 mg; .sup.1H 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).

    Synthesis of Product 1.29

    [0267] ##STR00185##

    [0268] Intermediate 1.29a was prepared analogously to the general procedure, step 1 (Example 1). Yield=86%; m=788 mg; .sup.1H NMR (400 MHz, Chloroform-d) δ 7.57-7.46 (m, 0.5H), 7.27 (dd, J=9.4, 2.4 Hz, 0.5H), 7.16 (dd, J=9.3, 4.4 Hz, 0.5H), 6.99-6.81 (m, 1.5H), 4.06 (ddt, J=9.6, 7.1, 4.4 Hz, 4H), 2.52 (s, 2H), 2.28 (t, J=6.7 Hz, 2H), 2.02 (p, J=6.9 Hz, 2H), 1.18 (t, J=7.1 Hz, 3H).

    ##STR00186##

    [0269] Intermediate 1.29b was prepared analogously to the general procedure, step 2 (Example 1). Yield=100%; m=786 mg, .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.60 (ddd, J=14.5, 8.8, 4.5 Hz, 1H), 7.38 (dd, J=49.0, 9.0 Hz, 1H), 7.12 (dt, J=16.4, 9.6 Hz, 1H), 4.32 (dt, J=12.6, 7.7 Hz, 2H), 2.69 (s, 3H), 2.43 (d, J=6.9 Hz, 2H), 2.11 (h, J=6.7 Hz, 2H).

    ##STR00187##

    [0270] Product 1.29 was prepared analogously to the general procedure, step 3 (Example 1). Yield=100%; m=187 mg; .sup.1H 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).

    Synthesis of Product 1.30

    [0271] ##STR00188##

    [0272] Intermediate 1.30a was prepared analogously to the general procedure, step 1 (Example 1). Yield=63%; m=1.7 g; .sup.1H NMR (400 MHz, Chloroform-d) δ 7.63 (d, J=8.7 Hz, 0H), 7.27 (d, J=8.8 Hz, 1H), 6.99-6.86 (m, 2H), 4.27-4.15 (m, 4H), 3.93 (dd, J=7.3, 1.3 Hz, 3H), 2.65 (dd, J=3.7, 1.3 Hz, 3H), 2.43 (q, J=6.8 Hz, 2H), 2.18 (p, J=7.1 Hz, 2H), 1.33 (td, J=7.2, 1.3 Hz, 3H).

    ##STR00189##

    [0273] Intermediate 1.30b was prepared analogously to the general procedure, step 2 (Example 1). Yield=100%; m=676 mg; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.54 (dd, J=32.8, 8.9 Hz, 1H), 7.18 (dd, J=33.3, 2.4 Hz, 1H), 7.01 (ddd, J=21.3, 8.9, 2.3 Hz, 1H), 4.33 (q, J=6.8, 6.3 Hz, 2H), 3.92-3.84 (m, 3H), 2.70 (d, J=8.0 Hz, 3H), 2.43 (q, J=6.7 Hz, 2H), 2.12 (p, J=7.0 Hz, 2H).

    ##STR00190##

    [0274] Product 1.30 was prepared analogously to the general procedure, step 3 (Example 1). Yield=100%; m=175 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) 6 7.80-6.57 (m, 3H), 4.44-2.96 (m, 22H), 2.80-1.38 (m, 7H).

    Synthesis of Product 1.31

    [0275] ##STR00191##

    [0276] Intermediate 1.31a was prepared analogously to the general procedure, step 1 (Example 1). Yield=50%; m=820 mg; .sup.1H NMR (400 MHz, Chloroform-d) δ 7.94-7.21 (m, 8H), 7.05 (d, J=1.3 Hz, 1H), 5.08 (d, J=1.3 Hz, 2H), 4.05 (td, J=8.1, 7.5, 2.9 Hz, 2H), 3.92 (dtd, J=16.2, 7.8, 6.5 Hz, 2H), 2.50 (dd, J=12.5, 1.4 Hz, 3H), 2.19 (q, J=6.3 Hz, 2H), 1.94 (p, J=7.0 Hz, 2H), 1.04 (ddd, J=14.3, 7.9, 6.5 Hz, 3H).

    ##STR00192##

    [0277] Intermediate 1.31b was prepared analogously to the general procedure, step 2 (Example 1). Yield=73%; m=580 mg; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.01 (d, J=8.9 Hz, 1H), 7.83-7.48 (m, 7H), 4.34 (tt, J=7.3, 3.0 Hz, 2H), 2.71-2.65 (m, 2H), 2.41 (ddt, J=10.2, 7.4, 4.2 Hz, 2H), 2.12 (h, J=7.2 Hz, 2H).

    ##STR00193##

    [0278] Product 1.31 was prepared analogously to the general procedure, step 3 (Example 1). Yield=84%; m=171 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.92-6.49 (m, 8H), 4.50-3.10 (m, 17H), 3.03-1.78 (m, 7H).

    Synthesis of Product 1.32

    [0279] ##STR00194##

    [0280] Intermediate 1.32a was prepared analogously to the general procedure, step 1 (Example 1). Yield=67%; m=1.29 mg; .sup.1H NMR (400 MHz, Chloroform-0 6 7.81-7.51 (m, 1H), 7.37-7.23 (m, 2H), 4.23-4.08 (m, 4H), 2.62 (d, J=4.3 Hz, 3H), 2.41-2.32 (m, 2H), 2.12 (pd, J=6.9, 2.4 Hz, 2H), 1.38 (d, J=6.9 Hz, 9H), 1.25 (td, J=7.2, 2.0 Hz, 3H).

    ##STR00195##

    [0281] Intermediate 1.32b was prepared analogously to the general procedure, step 2 (Example 1). Yield=81%; m=1.02 mg; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.48 (s, 1H), 7.41 (dd, J=8.5, 5.2 Hz, 1H), 7.23 (ddd, J=17.6, 8.5, 1.7 Hz, 1H), 4.16 (q, J=7.8 Hz, 2H), 2.50 (s, 3H), 2.26 (dt, J=13.7, 7.0 Hz, 2H), 1.90 (p, J=7.2 Hz, 2H), 1.33 (d, J=7.3 Hz, 9H).

    ##STR00196##

    [0282] Product 1.32 was prepared analogously to the general procedure, step 3 (Example 1). Yield=97%; m=176 mg; .sup.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).

    Synthesis of Product 1.33

    [0283] ##STR00197##

    [0284] Intermediate 1.33a was prepared analogously to the general procedure, step 2 (Example 1). Yield=49%; m=1.72 g; .sup.1H 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).

    ##STR00198##

    [0285] Intermediate 1.33b was prepared analogously to the general procedure, step 2 (Example 1). Yield=54%; m=920 mg; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.38 (d, J=8.2 Hz, 1H), 7.19 (t, J=7.4 Hz, 1H), 7.07 (d, J=7.4 Hz, 1H), 4.28 (t, J=7.3 Hz, 2H), 3.33 (d, J=3.3 Hz, 2H), 2.66 (d, J=2.7 Hz, 3H), 2.58 (s, 3H), 2.37 (t, J=6.3 Hz, 2H), 2.08 (h, J=7.7 Hz, 2H).

    ##STR00199##

    [0286] Product 1.33 was prepared analogously to the general procedure, step 3 (Example 1). Yield=100%; m=188 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.62-6.76 (m, 3H), 4.46-3.03 (m, 15H), 2.87-1.36 (m, 10H).

    Synthesis of Product 1.34

    [0287] ##STR00200##

    [0288] Intermediate 1.34a was prepared analogously to the general procedure, step 1 (Example 1). Yield=91%; m=1.55 g; .sup.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).

    ##STR00201##

    [0289] Intermediate 1.34b was prepared analogously to the general procedure, step 2 (Example 1). Yield=60%; m=840 mg; .sup.1H NMR (400 MHz, Methanol-d.sub.4) 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).

    ##STR00202##

    [0290] Product 1.34 was prepared analogously to the general procedure, step 3 (Example 1). Yield=100%; m=193 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) 6 7.83-6.65 (m, 3H), 4.52-3.09 (m, 15H), 3.04-2.32 (m, 5H), 2.30-1.72 (m, 2H).

    Synthesis of Product 1.35

    [0291] ##STR00203##

    [0292] Intermediate 1.35a was prepared analogously to the general procedure, step 1 (Example 1). Yield=63%; m=1.16 g; .sup.1H NMR (400 MHz, Chloroform-d) δ 7.59-7.45 (m, 1H), 7.24-7.02 (m, 2H), 4.15 (td, J=8.5, 6.9, 5.0 Hz, 4H), 2.61 (d, J=3.7 Hz, 3H), 2.47 (d, J=8.6 Hz, 3H), 2.35 (td, J=6.8, 4.7 Hz, 2H), 2.17-2.05 (m, 2H), 1.25 (td, J=7.2, 3.0 Hz, 3H).

    ##STR00204##

    [0293] Intermediate 1.35b was prepared analogously to the general procedure, step 2 (Example 1). Yield=100%; m=1.07 g; .sup.1H NMR (400 MHz, Methanol-d.sub.4) 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).

    ##STR00205##

    [0294] Product 1.35 was prepared analogously to the general procedure, step 3 (Example 1). Yield=100%; m=163 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.61-6.56 (m, 3H), 4.43-3.06 (m, 20H), 2.88-1.52 (m, 10H).

    Synthesis of Product 1.36

    [0295] ##STR00206##

    [0296] Intermediate 1.36a was prepared analogously to the general procedure, step 1 (Example 1). Yield=100%; m=1.35 g; .sup.1H NMR (400 MHz, Chloroform-d) δ 7.74-7.11 (m, 2H), 7.04 (dtd, J=18.1, 9.2, 2.4 Hz, 1H), 4.76 (s, 2H), 4.35-4.23 (m, 2H), 4.14 (qd, J=7.2, 2.9 Hz, 2H), 2.89 (d, J=3.5 Hz, 3H), 2.38 (t, J=7.1 Hz, 2H), 2.08-2.00 (m, 2H), 1.48 (s, 9H), 1.25 (s, 3H).

    ##STR00207##

    [0297] Intermediate 1.36b was prepared analogously to the general procedure, step 2 (Example 1). Yield=61%; m=810 mg; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.61 (dd, J=8.2, 4.0 Hz, 1H), 7.37 (dd, J=30.5, 9.2 Hz, 1H), 7.08 (dt, J=22.3, 9.4 Hz, 1H), 4.77 (s, 2H), 4.32 (dt, J=14.4, 7.3 Hz, 2H), 2.95 (s, 3H), 2.40 (t, J=7.2 Hz, 2H), 2.05 (q, J=7.4 Hz, 2H), 1.47 (s, 9H).

    ##STR00208##

    [0298] Product 1.36 was prepared analogously to the general procedure, step 3 (Example 1). Yield=98%; m=183 mg; .sup.1H 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).

    Synthesis of Product 1.37

    [0299] ##STR00209##

    [0300] Product 1.37 was prepared analogously to the general procedure, step 3 (Example 1). Yield=93%; m=155 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.54-6.54 (m, 3H), 4.39-2.94 (m, 15H), 2.93-1.52 (m, 10H).

    Synthesis of Product 1.38

    [0301] ##STR00210##

    [0302] Intermediate 1.38a was prepared analogously to the general procedure, step 1 (Example 1). Yield=26%; m=300 mg; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.30 (d, J=8.6 Hz, 1H), 6.96 (s, 1H), 6.80 (dd, J=8.8, 2.3 Hz, 1H), 4.21 (t, J=7.6 Hz, 2H), 4.07 (q, J=7.4 Hz, 1H), 3.62 (d, J=2.5 Hz, 1H), 2.56 (s, 3H), 2.41 (q, J=6.6 Hz, 2H), 2.09 (p, J=7.3 Hz, 2H), 1.21 (t, J=7.3 Hz, 2H).

    ##STR00211##

    [0303] Intermediate 1.38b was prepared analogously to the general procedure, step 2 (Example 1). Yield=54%; m=68 mg; .sup.1H NMR (D.sub.2O) δ: .sup.1H NMR (400 MHz, Methanol-d.sub.4) 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).

    ##STR00212##

    [0304] Product 1.38 was prepared analogously to the general procedure, step 3 (Example 1). Yield=100%; m=54 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.80-6.40 (m, 4H), 4.35-1.30 (m, 22H).

    Synthesis of Product 1.39

    [0305] ##STR00213##

    [0306] Intermediate 1.39a was prepared analogously to the general procedure, step 2 (Example 1). Yield=70%; m=1.6 g; .sup.1H 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).

    ##STR00214##

    [0307] Intermediate 1.39b was prepared analogously to the general procedure, step 2 (Example 1). Yield=58%; m=840 mg; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 12.27 (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=7.5 Hz, 2H), 2.66 (s, 3H), 2.37 (t, J=7.2 Hz, 2H), 2.03 (p, J=7.3 Hz, 2H).

    ##STR00215##

    [0308] Product 1.39 was prepared analogously to the general procedure, step 3 (Example 1). Yield=95%; m=174 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.21-6.03 (m, 6H), 4.47-0.63 (m, 22H).

    Synthesis of Product 1.40

    [0309] ##STR00216##

    [0310] Product 1.40 was prepared analogously to the general procedure, step 3 (Example 1). Yield=97%; m=198 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.19-6.14 (m, 6H), 4.51-0.73 (m, 24H).

    Synthesis of Product 1.41

    [0311] ##STR00217##

    [0312] Intermediate 1.41a was prepared analogously to the general procedure, step 2 (Example 1). Yield=45%; m=1.3 g; .sup.1H 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).

    ##STR00218##

    [0313] Intermediate 1.41b was prepared analogously to the general procedure, step 2 (Example 1). Yield=79%; m=1.0 g; .sup.1H NMR (400 MHz, Methanol-d.sub.4) 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).

    ##STR00219##

    [0314] Product 1.41 was prepared analogously to the general procedure, step 3 (Example 1). Yield=99%; m=161 mg; .sup.1H 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).

    Synthesis of Product 1.42

    [0315] ##STR00220##

    [0316] Intermediate 1.42a was prepared analogously to the general procedure, step 2 (Example 1). Yield=53%; m=418 mg; .sup.1H 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).

    ##STR00221##

    [0317] Product 1.42 was prepared analogously to the general procedure, step 3 (Example 1). Yield=84%; m=25 mg; .sup.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).

    Synthesis of Product 1.43

    [0318] ##STR00222##

    [0319] Intermediate 1.43a was prepared analogously to the general procedure, step 1 (Example 1). Yield=75%; m=1.05 g; .sup.1H NMR (400 MHz, Chloroform-d) δ 8.51 (dd, J=4.5, 1.6 Hz, 1H), 8.05 (dd, J=8.0, 1.6 Hz, 1H), 8.00 (s, 1H), 7.10 (dd, J=8.0, 4.5 Hz, 1H), 4.64-4.56 (m, 2H), 4.07 (q, J=7.1 Hz, 2H), 2.36-2.22 (m, 4H), 1.19 (t, J=7.2 Hz, 3H).

    ##STR00223##

    [0320] Intermediate 1.43b was prepared analogously to the general procedure, step 2 (Example 1). Yield=76%; m=778 mg; .sup.1H 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).

    ##STR00224##

    [0321] Product 1.43 was prepared analogously to the general procedure, step 3 (Example 1). Yield=81%; m=94 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.35-6.54 (m, 4H), 4.52-2.63 (m, 9H), 2.59-1.42 (m, 4H).

    Synthesis of Product 1.44

    [0322] ##STR00225##

    [0323] Intermediate 1.44a was prepared analogously to the general procedure, step 1 (Example 1). Yield=15%; m=211 mg; .sup.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).

    ##STR00226##

    [0324] Intermediate 1.44b was prepared analogously to the general procedure, step 2 (Example 1). Yield=100%; m=887 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) 6 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).

    ##STR00227##

    [0325] Product 1.44 was prepared analogously to the general procedure, step 3 (Example 1). Yield=97%; m=103 mg; .sup.1H 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).

    Synthesis of Product 1.45

    [0326] ##STR00228##

    [0327] Intermediate 1.45a was prepared analogously to the general procedure, step 1 (Example 1). Yield=43%; m=602 mg; .sup.1H 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).

    ##STR00229##

    [0328] Intermediate 1.45b was prepared analogously to the general procedure, step 2 (Example 1). Yield=100%; m=592 mg; .sup.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).

    ##STR00230##

    [0329] Product 1.45 was prepared analogously to the general procedure, step 3 (Example 1). Yield=100%; m=84 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 9.67-8.10 (m, 4H), 4.23-3.06 (m, 16H), 2.86-1.90 (m, 4H).

    Synthesis of Product 1.46

    [0330] ##STR00231##

    [0331] Product 1.46 was prepared analogously to the general procedure, step 3 (Example 1). Yield=89%; m=68 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.63-6.72 (m, 4H), 4.57-2.78 (m, 20H), 2.75-1.47 (m, 4H).

    Synthesis of Product 1.47

    [0332] ##STR00232##

    [0333] Product 1.47 was prepared analogously to the general procedure, step 3 (Example 1). Yield=97%; m=73 mg; .sup.1H 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).

    Synthesis of Product 1.48

    [0334] ##STR00233##

    [0335] Intermediate 1.48a was prepared analogously to the general procedure, step 1 (Example 1). Yield=59%; m=820 mg; .sup.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).

    ##STR00234##

    [0336] Intermediate 1.48b was prepared analogously to the general procedure, step 2 (Example 1). Yield=100%; m=175 mg; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.01 (s, 1H), 7.74 (dt, J=8.2, 1.0 Hz, 1H), 7.59 (dq, J=8.5, 0.9 Hz, 1H), 7.40 (ddd, J=8.6, 6.9, 1.1 Hz, 1H), 7.15 (ddd, J=7.9, 6.8, 0.8 Hz, 1H), 4.52-4.44 (m, 2H), 2.28-2.12 (m, 4H).

    ##STR00235##

    [0337] Product 1.48 was prepared analogously to the general procedure, step 3 (Example 1). Yield=83%; m=105 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.18-6.91 (m, 5H), 4.14-1.72 (m, 42H).

    Synthesis of Product 1.49

    [0338] ##STR00236##

    [0339] Product 1.49 was prepared analogously to the general procedure, step 3 (Example 1). Yield=52%; m=39 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.21-6.38 (m, 5H), 4.47-1.37 (m, 25H).

    Synthesis of Product 1.50

    [0340] ##STR00237##

    [0341] Product 1.50 was prepared analogously to the general procedure, step 3 (Example 1). Yield=16%; m=13 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.19-6.27 (m, 5H), 4.42-1.05 (m, 23H).

    Synthesis of Product 1.51

    [0342] ##STR00238##

    [0343] Product 1.51 was prepared analogously to the general procedure, step 3 (Example 1). Yield=91%; m=12 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.65-7.82 (m, 2H), 4.39-2.88 (m, 36H), 2.68-1.91 (m, 3H).

    Synthesis of Product 1.52

    [0344] ##STR00239##

    [0345] Intermediate 1.52a was prepared analogously to the general procedure, step 1 (Example 1). Yield=76%; m=440 mg; .sup.1H NMR (400 MHz, Chloroform-d) δ 8.40 (dd, J=4.8, 1.6 Hz, 1H), 8.18 (s, 1H), 8.07 (dd, J=8.1, 1.6 Hz, 1H), 7.30-7.20 (m, 1H), 4.40 (td, J=6.9, 1.7 Hz, 2H), 4.10 (q, J=7.1 Hz, 2H), 2.43-2.20 (m, 4H), 1.22 (td, J=7.2, 0.6 Hz, 3H).

    ##STR00240##

    [0346] Intermediate 1.52b was prepared analogously to the general procedure, step 2 (Example 1). Yield=50%; m=597 mg; .sup.1H 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).

    ##STR00241##

    [0347] Product 1.52 was prepared analogously to the general procedure, step 3 (Example 1). Yield=71%; m=12 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 9.59-6.98 (m, 4H), 4.13-2.68 (m, 17H), 2.68-0.79 (m, 3H).

    Synthesis of Product 1.53

    [0348] ##STR00242##

    [0349] Intermediate 1.53a was prepared analogously to the general procedure, step 1 (Example 1). Yield=65%; m=153 mg; .sup.1H 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).

    ##STR00243##

    [0350] Intermediate 1.53b was prepared analogously to the general procedure, step 2 (Example 1). Yield=100%; m=105 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) 6 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).

    ##STR00244##

    [0351] Product 1.53 was prepared analogously to the general procedure, step 3 (Example 1). Yield=82%; m=12 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 9.47-6.92 (m, 4H), 4.19-2.88 (m, 20H), 2.92-1.60 (m, 4H).

    Synthesis of Product 1.54

    [0352] ##STR00245##

    [0353] Product 1.54 was prepared analogously to the general procedure, step 3 (Example 1). Yield=83%; m=12 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 9.20-8.32 (m, 2H), 4.51-2.93 (m, 16H), 2.88-1.68 (m, 7H).

    Synthesis of Product 1.55

    [0354] ##STR00246##

    [0355] Product 1.55 was prepared analogously to the general procedure, step 3 (Example 1). Yield=97%; m=169 mg; .sup.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).

    Synthesis of Product 1.56

    [0356] ##STR00247##

    [0357] Product 1.56 was prepared analogously to the general procedure, step 3 (Example 1). Yield=100%; m=187 mg; .sup.1H NMR (D.sub.2O) δ: 7.41-6.35 (m, 2H), 4.44-2.96 (m, 23H), 2.85-1.34 (m, 20H).

    Synthesis of Product 1.57

    [0358] ##STR00248##

    [0359] Product 1.57 was prepared analogously to the general procedure, step 3 (Example 1). Yield=100%; m=26 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.64-6.35 (m, 2H), 4.50-3.01 (m, 62H), 3.05-1.41 (m, 13H).

    Synthesis of Product 1.58

    [0360] ##STR00249##

    [0361] Product 1.58 was prepared analogously to the general procedure, step 3 (Example 1). Yield=72%; m=19 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.55-6.59 (m, 2H), 4.41-3.11 (m, 39H), 3.05-1.37 (m, 13H).

    Synthesis of Product 1.59

    [0362] ##STR00250##

    [0363] Product 1.59 was prepared analogously to the general procedure, step 3 (Example 1). Yield=70%; m=22 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.80-6.15 (m, 2H), 4.50-3.21 (m, 22H), 3.17-0.97 (m, 13H).

    Synthesis of Product 1.60

    [0364] ##STR00251##

    [0365] Product 1.60 was prepared analogously to the general procedure, step 3 (Example 1). Yield=73%; m=23 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.98-6.15 (m, 2H), 4.62-3.06 (m, 22H), 2.99-1.39 (m, 13H).

    Synthesis of Product 1.61

    [0366] ##STR00252##

    [0367] Product 1.61 was prepared analogously to the general procedure, step 3 (Example 1). Yield=84%; m=25 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.64-6.35 (m, 2H), 4.57-3.13 (m, 25H), 3.13-1.50 (m, 13H).

    Synthesis of Product 1.62

    [0368] ##STR00253##

    [0369] Product 1.62 was prepared analogously to the general procedure, step 3 (Example 1). Yield=68%; m=21 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.74-6.35 (m, 2H), 4.50-3.01 (m, 24H), 2.97-1.41 (m, 13H).

    Synthesis of Product 1.63

    [0370] ##STR00254##

    [0371] Product 1.63 was prepared analogously to the general procedure, step 3 (Example 1). Yield=35%; m=13 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.62-6.39 (m, 2H), 4.57-3.01 (m, 16H), 2.90-1.21 (m, 13H).

    Synthesis of Product 1.64

    [0372] ##STR00255##

    [0373] Product 1.64 was prepared analogously to the general procedure, step 3 (Example 1). Yield=85%; m=18 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.90-6.11 (m, 2H), 4.52-3.09 (m, 30H), 3.11-1.39 (m, 13H).

    Synthesis of Product 1.65

    [0374] ##STR00256##

    [0375] Product 1.65 was prepared analogously to the general procedure, step 3 (Example 1). Yield=84%; m=21 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.80-6.15 (m, 2H), 4.60-3.17 (m, 19H), 3.09-1.25 (m, 13H).

    Synthesis of Product 1.66

    [0376] ##STR00257##

    [0377] Product 1.66 was prepared analogously to the general procedure, step 3 (Example 1). Yield=72%; m=17 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.49-6.19 (m, 2H), 4.57-3.05 (m, 13H), 3.03-1.37 (m, 4H).

    Synthesis of Product 1.67

    [0378] ##STR00258##

    [0379] Intermediate 1.67a was prepared analogously to the general procedure, step 1 (Example 1). Yield=85%; m=395 mg; .sup.1H 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).

    ##STR00259##

    [0380] Product 1.67 was prepared analogously to the general procedure, step 3 (Example 1). Yield=69%; m=22 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.64-6.35 (m, 2H), 4.52-3.01 (m, 25H), 2.94-1.02 (m, 13H).

    Synthesis of Product 1.68

    [0381] ##STR00260##

    [0382] Intermediate 1.68a was prepared analogously to the general procedure, step 2 (Example 1). Yield=66%; m=247 mg; .sup.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).

    ##STR00261##

    [0383] Product 1.68 was prepared analogously to the general procedure, step 3 (Example 1). Yield=74%; m=24 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.80-6.25 (m, 2H), 4.52-1.32 (m, 33H).

    Synthesis of Product 1.69

    [0384] ##STR00262##

    [0385] Intermediate 1.69a was prepared analogously to the general procedure, step 1 (Example 1). Yield=76%; m=332 mg; .sup.1H 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).

    ##STR00263##

    [0386] Product 1.69 was prepared analogously to the general procedure, step 3 (Example 1). Yield=74%; m=30 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.48-6.39 (m, 2H), 4.46-3.01 (m, 18H), 2.88-0.77 (m, 13H).

    Synthesis of Product 1.70

    [0387] ##STR00264##

    [0388] Product 1. 70 was prepared analogously to the general procedure, step 3 (Example 1). Yield=63%; m=20 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.72-6.29 (m, 2H), 4.48-3.21 (m, 24H), 3.09-1.19 (m, 13H).

    Synthesis of Product 1.71

    [0389] ##STR00265##

    [0390] Product 1. 71 was prepared analogously to the general procedure, step 3 (Example 1). Yield=80%; m=28 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.62-6.31 (m, 2H), 4.43-3.09 (m, 18H), 3.01-0.82 (m, 15H).

    Synthesis of Product 1.72

    [0391] ##STR00266##

    [0392] Product 1.72 was prepared analogously to the general procedure, step 3 (Example 1). Yield=86%; m=30 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.55-6.25 (m, 2H), 4.55-3.06 (m, 17H), 2.94-1.23 (m, 13H).

    Synthesis of Product 1.73

    [0393] ##STR00267##

    [0394] 1.73a: 4-DMAP and Et.sub.3N were added to a solution of 4-imidazoleacetic acid in THF/H.sub.2O. Boc.sub.2O in THF was then added at 0° C. and the mixture was stirred at rt overnight. EtOAc was added and HCl 3M was added to have the aqueous phase at pH 4. Aqueous phase was removed and organic phase was washed with brine. Dried over Na.sub.2SO.sub.4 and evaporated under vacuum to give the product as a white solid. Yield=54%; m=300 mg; .sup.1H NMR (400 MHz, 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).

    ##STR00268##

    [0395] Product 1.73: In a microwave sealed tube was added PEI22k.HCl (1 eq) in water followed by NMM (2 eq). The acid was dissolved in MeOH and added to the PEI. After stirring 10 min, DMTMM was added and the mixture was stirred overnight at rt. Solvent were evaporated and co evaporation with ethanol was done. TFA was added at 0° C. and stirred for 3 h. TFA was evaporated and the product was purified on Amicon Ultra 15 (3 kD) with 6*10 mL HCl 50 mM.

    [0396] Product 1.73. Yield=84%; m=58 mg; .sup.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).

    Synthesis of Product 1.74

    [0397] ##STR00269##

    [0398] Product 1.74 was prepared analogously to the general procedure, step 3 using branched polyethyleneimine (bPEI, 25K, Sigma-Aldrich). Yield=94%; m=282 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.42-6.52 (m, 2H), 4.45-1.51 (m, 33H).

    Synthesis of Product 1.75

    [0399] ##STR00270##

    [0400] Product 1.75 was prepared analogously to the general procedure, step 3 using branched polyethyleneimine (bPEI, 10K, Alfa Aesar). Yield=99%; m=351 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.37-6.50 (m, 2H), 4.57-1.44 (m, 29H).

    Synthesis of Product 1.76

    [0401] ##STR00271##

    [0402] Product 1.76 was prepared analogously to the general procedure, step 3 using poly(allyamine) (PAA, 15K, Sigma-Aldrich). Yield=99%; m=146 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.43-6.38 (m, 2H), 4.46-0.74 (m, 41H).

    Synthesis of Product 1.77

    [0403] ##STR00272##

    [0404] Product 1.77 was prepared analogously to the general procedure, step 3. Yield=34%; m=41 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.44-6.93 (m, 2H), 4.53-4.06 (m, 5H), 3.15-1.08 (m, 40H).

    Synthesis of Product 1.78

    [0405] ##STR00273##

    [0406] Product 1.78 was prepared analogously to the general procedure, step 4 using poly(vinylamine) (PLL, 22K, Sigma-Aldrich). Yield=99%; m=37 mg; .sup.1H 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).

    Synthesis of Product 1.79

    [0407] ##STR00274##

    [0408] Product 1.79 was prepared analogously to the general procedure, step 3 using poly(vinylamine) (PVA, 25K, Polysciences). Yield=78%; m=139 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.49-6.52 (m, 2H), 4.60-0.96 (m, 26H).

    Synthesis of Product 1.80

    [0409] ##STR00275##

    [0410] Product 1.80 was prepared analogously to the general procedure, step 4. Yield=98%; m=68 mg; .sup.1H 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).

    Synthesis of Product 2.01

    [0411] ##STR00276##

    [0412] Intermediate 2.01a was prepared analogously to the general procedure, step 1 (Example 1). Yield=17%; m=1.00 g; .sup.1H NMR (400 MHz, Chloroform-d) δ 7.95 (d, J=8.4 Hz, 1H), 7.45 (d, J=8.3 Hz, 1H), 7.38 (t, J=7.6 Hz, 1H), 7.30-7.22 (m, 1H), 4.68-4.57 (m, 2H), 4.01 (qd, J=7.1, 1.6 Hz, 2H), 2.30-2.16 (m, 4H), 1.13 (td, J=7.1, 1.6 Hz, 3H).

    ##STR00277##

    [0413] Intermediate 2.01b was prepared analogously to the general procedure, step 2 (Example 1). Yield=85%; m=830 mg; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 8.03-7.96 (m, 1H), 7.82 (d, J=8.5 Hz, 1H), 7.58 (t, J=7.3 Hz, 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).

    ##STR00278##

    [0414] Product 2.01 was prepared analogously to the general procedure, step 3 (Example 1). Yield=100%; m=189 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 7.41-6.55 (m, 4H), 4.58-3.02 (m, 14H), 2.90-1.31 (m, 3H).

    Synthesis of Product 2.02

    [0415] ##STR00279##

    [0416] Intermediate 2.02a was prepared analogously to the general procedure, step 1 (Example 1). Yield=34%; m=2.00 g; .sup.1H 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).

    ##STR00280##

    [0417] Intermediate 2.02b was prepared analogously to the general procedure, step 2 (Example 1). Yield=53%; m=1.00 g; .sup.1H NMR (400 MHz, Methanol-d.sub.4) 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).

    ##STR00281##

    [0418] Product 2.02 was prepared analogously to the general procedure, step 3 (Example 1). Yield=100%; m=166 mg; .sup.1H 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).

    Synthesis of Product 2.03

    [0419] ##STR00282##

    [0420] Product 2.03 was prepared analogously to the general procedure, step 3 (Example 1). Yield=93%; m=143 mg; .sup.1H 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).

    Synthesis of Product 2.04

    [0421] ##STR00283##

    [0422] Product 2.04 was prepared analogously to the general procedure, step 3 (Example 1). Yield=92%; m=133 mg; .sup.1H 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).

    Synthesis of Product 2.05

    [0423] ##STR00284##

    [0424] Intermediate 2.05a was prepared analogously to the general procedure, step 1 (Example 1). Yield=35%; m=1.94 g; .sup.1H NMR (400 MHz, Chloroform-d) δ 7.87 (d, J=8.5 Hz, 0H), 7.77 (s, 0H), 7.40 (d, J=8.4 Hz, 0H), 7.26 (s, 1H), 7.15 (d, J=8.5 Hz, 1H), 4.64 (q, J=6.7 Hz, 2H), 4.08 (qd, J=7.1, 2.4 Hz, 2H), 2.48 (dd, J=8.6, 1.9 Hz, 3H), 2.29 (pd, J=6.8, 2.2 Hz, 4H), 1.19 (td, J=7.2, 2.0 Hz, 3H).

    ##STR00285##

    [0425] Intermediate 2.05b was prepared analogously to the general procedure, step 2 (Example 1). Yield=70%; m=1.53 g; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.90-7.83 (m, 1H), 7.76 (s, 0H), 7.70 (dd, J=8.8, 2.4 Hz, 0H), 7.59 (s, 1H), 7.44 (d, J=8.7 Hz, 0H), 7.31 (d, J=8.6 Hz, 1H), 4.77 (q, J=7.0 Hz, 2H), 3.33 (d, J=3.3 Hz, 2H), 2.56 (dd, J=13.3, 2.5 Hz, 3H), 2.36 (t, J=7.4 Hz, 2H), 2.30-2.22 (m, 2H).

    ##STR00286##

    [0426] Product 2.05 was prepared analogously to the general procedure, step 3 (Example 1). Yield=86%; m=136 mg; .sup.1H 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).

    Synthesis of Product 2.06

    [0427] ##STR00287##

    [0428] Intermediate 2.06a was prepared analogously to the general procedure, step 1 (Example 1). Yield=40%; m=2.23 g; .sup.1H NMR (400 MHz, Chloroform-d) δ 7.77 (d, J=8.7 Hz, 1H), 7.63 (s, 1H), 7.25 (d, J=8.7 Hz, 1H), 4.81 (td, J=6.5, 1.9 Hz, 2H), 4.15 (qd, J=7.2, 2.0 Hz, 2H), 2.53 (s, 3H), 2.48-2.35 (m, 4H), 1.27 (td, J=7.1, 1.9 Hz, 3H).

    ##STR00288##

    [0429] Intermediate 2.06b was prepared analogously to the general procedure, step 2 (Example 1). Yield=41%; m=793 mg; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.74 (dd, J=8.9, 2.4 Hz, 1H), 7.61 (s, 1H), 7.28 (d, J=8.8 Hz, 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).

    ##STR00289##

    [0430] Product 2.06 was prepared analogously to the general procedure, step 3 (Example 1). Yield=85%; m=128 mg; .sup.1H 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).

    Synthesis of Product 2.07

    [0431] ##STR00290##

    [0432] Intermediate 2.07a was prepared analogously to the general procedure, step 1 (Example 1). Yield=14%; m=538 mg; .sup.1H 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).

    ##STR00291##

    [0433] Intermediate 2.07b was prepared analogously to the general procedure, step 2 (Example 1). Yield=79%; m=416 mg; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.59 (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).

    ##STR00292##

    [0434] Product 2.07 was prepared analogously to the general procedure, step 3 (Example 1). Yield=49%; m=73 mg; .sup.1H 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).

    Synthesis of Product 2.08

    [0435] ##STR00293##

    [0436] Intermediate 2.08a was prepared analogously to the general procedure, step 1 (Example 1). Yield=12%; m=483 m; .sup.1H 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).

    ##STR00294##

    [0437] Intermediate 2.08b was prepared analogously to the general procedure, step 2 (Example 1). Yield=94%; m=444 mg; .sup.1H NMR (400 MHz, Methanol-d.sub.4) 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).

    ##STR00295##

    [0438] Product 2.08 was prepared analogously to the general procedure, step 3 (Example 1). Yield=81%; m=129 mg; .sup.1H 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).

    Synthesis of Product 2.09

    [0439] ##STR00296##

    [0440] Intermediate 2.09a was prepared analogously to the general procedure, step 1 (Example 1). Yield=33%; m=2.22 g; .sup.1H NMR (400 MHz, Chloroform-d) δ 7.94-7.87 (m, 1H), 7.30-7.19 (m, 2H), 4.93 (t, J=6.1 Hz, 2H), 4.31 (qd, J=7.2, 2.0 Hz, 2H), 4.07 (d, J=1.9 Hz, 3H), 2.59 (dt, J=16.1, 5.2 Hz, 4H), 1.43 (td, J=7.2, 1.9 Hz, 3H).

    ##STR00297##

    [0441] Intermediate 2.09b was prepared analogously to the general procedure, step 2 (Example 1). Yield=70%; m=1.49 g; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.72 (dd, J=9.1, 2.5 Hz, 1H), 7.14 (s, 1H), 7.08 (d, J=9.3 Hz, 1H), 4.75 (q, J=3.8 Hz, 2H), 3.89 (d, J=2.6 Hz, 3H), 2.35 (d, J=2.9 Hz, 4H).

    ##STR00298##

    [0442] Product 2.09 was prepared analogously to the general procedure, step 3 (Example 1). Yield=90%; m=146 mg; .sup.1H 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).

    Synthesis of Product 2.10

    [0443] ##STR00299##

    [0444] Intermediate 2.10a was prepared analogously to the general procedure, step 1 (Example 1). Yield=23%; m=1.69 g; .sup.1H NMR (400 MHz, Chloroform-d) δ 7.78 (dd, J=9.1, 1.9 Hz, 1H), 7.34 (dd, J=9.1, 1.9 Hz, 0H), 7.26 (d, J=2.3 Hz, 0H), 7.05 (dd, J=9.0, 2.2 Hz, 0H), 6.89 (dd, J=9.1, 2.2 Hz, 1H), 6.73 (d, J=2.2 Hz, 1H), 4.55 (dtd, J=13.6, 6.7, 1.9 Hz, 2H), 4.01 (q, J=7.1 Hz, 2H), 3.78 (dd, J=6.6, 1.8 Hz, 3H), 3.04 (s, 0H), 2.22 (ddd, J=19.8, 7.7, 4.2 Hz, 4H), 1.13 (td, J=7.1, 1.8 Hz, 3H).

    ##STR00300##

    [0445] Intermediate 2.10b was prepared analogously to the general procedure, step 2 (Example 1). Yield=70%; m=1.16 g; .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.81 (dd, J=9.1, 2.7 Hz, 1H), 7.67 (dd, J=8.9, 2.7 Hz, 0H), 7.33 (s, 0H), 7.24-7.15 (m, 1H), 7.05 (dd, J=9.1, 2.7 Hz, 1H), 4.73 (qd, J=7.1, 2.3 Hz, 2H), 3.91 (dd, J=13.2, 2.8 Hz, 3H), 3.33 (d, J=3.2 Hz, 0H), 2.35 (t, J=6.7 Hz, 2H), 2.26 (t, J=7.3 Hz, 2H).

    ##STR00301##

    [0446] Product 2.10 was prepared analogously to the general procedure, step 3 (Example 1). Yield=96%; m=153 mg; .sup.1H 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).

    Synthesis of Product 2.11

    [0447] ##STR00302##

    [0448] Intermediate 2.11a was prepared analogously to the general procedure, step 1 (Example 1). Yield=77%; m=1.09 g; .sup.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).

    ##STR00303##

    [0449] Intermediate 2.11b was prepared analogously to the general procedure, step 2 (Example 1). Yield=100%; m=1.16 g; .sup.1H 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).

    ##STR00304##

    [0450] Product 2.11 was prepared analogously to the general procedure, step 3 (Example 1). Yield=56%; m=43 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.95-6.92 (m, 3H), 4.11-2.95 (m, 18H), 2.90-1.23 (m, 5H).

    Synthesis of Product 2.12

    [0451] ##STR00305##

    [0452] Intermediate 2.12a was prepared analogously to the general procedure, step 1 (Example 1). Yield=18%; m=254 mg; .sup.1H NMR (400 MHz, Chloroform-d) δ 8.53 (dd, J=4.4, 1.5 Hz, 1H), 7.82 (dd, J=8.4, 1.5 Hz, 1H), 7.24 (dd, J=8.4, 4.4 Hz, 1H), 4.54 (t, J=6.8 Hz, 2H), 3.89 (q, J=7.1 Hz, 2H), 2.20-2.05 (m, 4H), 1.01 (t, J=7.1 Hz, 3H).

    ##STR00306##

    [0453] Intermediate 2.12b was prepared analogously to the general procedure, step 2 (Example 1). Yield=98%; m=242 mg; .sup.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).

    ##STR00307##

    [0454] Product 2.12 was prepared analogously to the general procedure, step 3 (Example 1). Yield=75%; m=54 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 9.06-6.76 (m, 3H), 4.16-2.96 (m, 20H), 2.93-1.52 (m, 4H).

    Synthesis of Product 2.13

    [0455] ##STR00308##

    [0456] Intermediate 2.13a was prepared analogously to the general procedure, step 1 (Example 1). Yield=43%; m=736 mg; .sup.1H 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).

    ##STR00309##

    [0457] Intermediate 2.13b was prepared analogously to the general procedure, step 2 (Example 1). Yield=100%; m=707 mg; .sup.1H 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).

    ##STR00310##

    [0458] Product 2.13 was prepared analogously to the general procedure, step 3 (Example 1). Yield=95%; m=65 mg; .sup.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).

    Synthesis of Product 2.14

    [0459] ##STR00311##

    [0460] Intermediate 2.14a was prepared analogously to the general procedure, step 1 (Example 1). Yield=52%; m=600 mg; .sup.1H 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).

    ##STR00312##

    [0461] Intermediate 2.14b was prepared analogously to the general procedure, step 2 (Example 1). Yield=97%; m=550 mg; .sup.1H 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).

    ##STR00313##

    [0462] Product 2.14 was prepared analogously to the general procedure, step 3 (Example 1). Yield=95%; m=68 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 10.08-8.17 (m, 3H), 4.21-2.84 (m, 25H), 2.83-1.64 (m, 4H).

    Synthesis of Product 2.15

    [0463] ##STR00314##

    [0464] Product 2.15 was prepared analogously to the general procedure, step 3. Yield=88%; m=74 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.98-7.01 (m, 3H), 4.07-3.06 (m, 14H), 2.75-1.45 (m, 4H).

    Synthesis of Product 2.16

    [0465] ##STR00315##

    [0466] Product 2.16 was prepared analogously to the general procedure, step 3 (Example 1). Yield=46%; m=42 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 8.97-6.90 (m, 3H), 4.07-2.81 (m, 12H), 2.60-1.43 (m, 4H).

    Synthesis of Product 2.17

    [0467] ##STR00316##

    [0468] Product 2.17 was prepared analogously to the general procedure, step 3 (Example 1). Yield=97%; m=82 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 10.03-8.05 (m, 3H), 4.20-3.04 (m, 14H), 2.81-1.60 (m, 4H).

    Synthesis of Product 2.18

    [0469] ##STR00317##

    [0470] Product 2.18 was prepared analogously to the general procedure, step 3 (Example 1). Yield=97%; m=82 mg; .sup.1H NMR (400 MHz, Deuterium Oxide) δ 10.02-8.30 (m, 3H), 3.54 (s, 15H), 2.93-1.88 (m, 4H).

    Example 3. Compounds 1.01 to 1.50

    [0471] Screening of transfection activity Compounds 1.01 to 1.50 were evaluated for their ability to transfect DNA in four different cell lines. Many cell lines were first transfected with commercially available transfection reagents (see Table 2). The plasmid pCMV-EGFPLuc encoding the Green Fluorescent Protein (GFP) was used and the transfection efficiency in 96-well plate format was determined by analyzing the percentage of cells expressing the GFP (% GFP) by cytometry assay one day post-transfection. Table 2 presents the results of commercial reagents used in their optimal conditions for the four 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 results indicated that these cells were relatively difficult to transfect as the highest transfection efficiencies were inferior to 40%, 28%, 40% and 22% in Caco-2, Hep G2, MDCK and MCF-10A, respectively. These cell lines were selected to screen the transfection activity of compounds 1.01 to 1.50.

    TABLE-US-00002 TABLE 2 Transfection of Caco-2, Hep G2, MDCK and MCF-10A with commercially available reagents (jetPEI ®, jetPRIME ® form Polyplus-transfection, ViaFect ® from Promega, TransIT-XT2 ® from MirusBio, X-tremeGENE 9 ® from Roche Life Science, LipoFectamine ® 3000 from Thermo Fisher). Ratio μg DNA _μL Amount of transfection Cell line Commercial reagent DNA/well reagent % GFP CaCo-2 Viafect 150 ng 1_4  5.92 X-tremeGENE9 150 ng 1_6  7.06 TransFectin 150 ng 1_4 22.15 jetPRIME 150 ng 1_2 31.19 jetPEI 200 ng 1_3 15.9  LipoFectamine 3000 150 ng 1_3 39.92 HepG2 Viafect 150 ng 1_4  3.21 X-tremeGENE9 150 ng 1_6 11.34 TransIT-X2 150 ng 1_4  8.06 TransFectin 150 ng 1_4  9.77 jetPRIME 150 ng 1_3 10.77 jetPEI 200 ng 1_3 16.16 LipoFectamine 3000 200 ng 1_3 27.77 MDCK Viafect 150 ng 1_4 11.64 TransIT-X2 150 ng 1_4 14.63 jetPRIME 150 ng 1_3 13.5  jetPEI 200 ng 1_2 15.27 LipoFectamine 3000 150 ng 1_3 39.5  MCF-10A Viafect 150 ng 1_4  6.80 X-tremeGENE9 150 ng 1_6  3.82 TransIT-X2 150 ng 1_4  8.58 jetPRIME 150 ng 1_3 21.40 jetPEI 200 ng 1_2 8.0 LipoFectamine 3000 150 ng 1_3 17.75

    [0472] The screening of compounds 1.01 to 1.50 (FIG. 1) 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 1.01 to 1.50 (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 compounds 1.01 to 1.50.

    [0473] The activity results of the compounds comprising a benzimidazole ring, wherein Y.sup.1═Y.sup.3═N, Y.sup.2═C, and R, T, U, V form a benzene ring, showed an efficient transfection with the 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 with a similar profile of efficiency in the four cell lines tested. Among these compounds comprising a benzimidazole ring, when Z.sup.2 is different of H, many compounds showed an improved efficiency when compared to jetPEI®, 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 wherein Z.sup.2═CH.sub.3, or 1.14 wherein Z.sup.2═CF.sub.3, or 1.17 wherein Z.sup.2═S—CH.sub.3, 1.18 wherein Z.sup.2═isopropyl, or 1.19 wherein Z.sup.2═propyl, 1.20 wherein Z.sup.2═CH.sub.2—NH—CH.sub.3, 1.21, 1.22 wherein Z.sup.2═CH.sub.2—O—CH.sub.3. Other substitutions on the benzene ring on position Z.sup.4, Z.sup.5, or Z.sup.6 provided very efficient compounds such as 1.30 wherein Z.sup.5═O—CH.sub.3, 1.32 wherein Z.sup.6═isopropyl, 1.35, 1.36 wherein Z.sup.6═CH.sub.3, 1.37 wherein Z.sup.4═CH.sub.3, 1.41, 1.42 wherein Z.sup.5═Z.sup.6═CH.sub.3. Taken together, the data indicated that a chemical diversity could be introduced on compounds comprising a benzimidazole ring, which might favour the transfection efficiency.

    [0474] The activity results of compounds comprising a benzopyrazole ring (1.44, 1.45, 1.48, 1.49, 1.50), wherein Y.sup.1═Y.sup.2═N, Y.sup.3═C, and R, T, U, V form a benzene ring, showed a moderate transfection efficiency when compared to jetPEI®. Therefore, the compound 1.49 showed high level of transfection, particularly in MDCK cells. Introduction of amino groups in Z.sup.6 (1.43, 1.46) or Z.sup.4 (1.47) was also tolerated in transfection.

    Example 4. Compounds 2.01 to 2.18

    [0475] Screening of Transfection Activity

    [0476] Compounds 2.01 to 2.18 were screened in transfection (FIG. 2) 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 0.84 of one compound of the invention (one compound of 2.01 to 2.18) (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.

    [0477] Benzotriazole ring derivatives (2.01 to 2.10) were found to be the most interesting compounds of Example 4 according to their transfection activity in transfection of Caco-2, Hep G2, MCF-10A, and MDCK. The grafting position to the polymer Y.sup.1 or Y.sup.2 (Z.sup.1 or Z.sup.2) seemed to have low impact on the transfection but the grafting extent on the polymer of benzotriazole ring influenced more the results. The addition of methyl or methoxy groups on position Z.sup.5 and/or Z.sup.6 might improve the transfection activity as exemplified by compounds 2.05, 2.08, 2.09 or 2.10.

    Example 5. Heterocycle Grafting Extent and Cationic Polymer Length

    [0478] The grafting extent with heterocycle ring to the cationic polymer is a key factor to modulate the transfection activity. FIG. 3 exemplified the grafting impact with 2-methyl benzimidazole to the linear PEI of 22 kDa where a grafting extent from 14% to 25% provided very efficient compounds in transfection, with an optimal closed to 20%, when compared to jetPEI®. This is also exemplified with the compounds 1.28, 1.25, and 1.27 grafted to the linear PEI 10 kDa showing also that a grafting extent around 20% was of interest in transfection (FIG. 1).

    [0479] Additionally, the cationic polymer length might influence the transfection activity as exemplified by the compounds 1.24, 1.25, and 1.26 where the same grafting extent with 2-methyl benzimidazole ring was performed on polymer having a mean molecular of 22, 10 and 6 kDa, respectively (FIG. 1).

    Example 6. Transfection of Primary Cells

    [0480] A selection among the best compounds of Examples 3 and 4 was tried to transfect primary cells, known to be difficult to transfect (FIG. 4), such as primary dermal fibroblasts (HPDF) and primary endothelial cells (HUVEC). Primary cells were cultured with their specific media conditions in 24-well format plate (see Material and Methods). The cells were transfected with 500 ng of pCMV-EGFPLuc complexed with 1.5 μL of compound 1.41, 2.03, 2.05, 1.42 or 2.08 in 50 μL of BUFFER (ratio 1 μg DNA: 3 μL of compounds) or with LipoFectAmine® 3000 (ratio 1 μg DNA:1.5 μL reagent) according to the recommended protocol. After 2 or 4 h, the transfection medium was removed and replaced by complete medium. The GFP expression analysis was performed 24 h after transfection by cytometry analysis.

    [0481] The GFP expression results showed that all the compounds selected were able to transfect HPDF and HUVEC cells more efficiently than the commercial reference LipoFectamine® 3000, reaching about 50% of transfection efficiency of HPDF with compound 2.03 and more than 70% transfection efficiency with compounds 1.41 and 1.42. These results indicated the great potential of the compounds of the invention with a possible diversity of the heterocycle grafted to the cationic polymer.

    [0482] Transfection of primary neurons is known to be very difficult as these non-dividing cells have a very limited access for the transfected DNA to the nucleus. In addition, these cells are very fragile. The inventors have tested compounds closed to the structure 1.42 such as compounds 1.56 to 1.72, and found that compound 1.65 or 1.60 shoved very impressive transfection results of primary neurons. FIG. 5 exemplified the results obtained with compound 1.65 when compared to commercially available transfection reagents.

    [0483] Primary rat cortex neurons (RCN) and primary rat hippocampal neurons (RHN) were cultivated for 4 days in complete medium, and were then transfected with 150 ng of pCMV-EGFP.sub.Luc plasmid complexed with either 0.15 μL of compound 1.65 in 25 μL of OPTIMEM or 0.6 μl of LipoFectAmine® 2000 (ratio 1 μg: 4 μL) and jetPEI® (ratio 1 μg: 2 μL) according to the recommended commercial protocols. The cells were observed 24 h post-transfection by using a fluorescent cell imager.

    [0484] jetPEI® was found not to be effective to transfect both RCN and RHN where LipoFectAmine® 2000 provided significant level of transfection efficiency. Therefore, compound 1.65 was shown to nicely transfect both RCN and RHN with a higher efficiency without affecting the cell morphology (the cell dendrites were clearly observables). In contrast, the morphology of cells transfected with LipoFectAmine® 2000 was clearly affected with few remaining dendrites indicating toxicity effect.

    Example 7. Bioproduction of Recombinant Virus

    [0485] DNA transfection is one of the frequently 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).

    [0486] 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.

    [0487] Many compounds of Examples 3 and 4 were tested for the production of AAV-2 and FIG. 6 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.

    [0488] 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. 7).

    Example 8. CRISPR Cas9 DNA Transfection

    [0489] The CRISPR-Cas9 technology was used to introduce a deletion in the targeted human HPRT-1 gene. A plasmid encoding both the spCas9 protein and the guide RNA was introduced by the transfection into HEK293 cells with compound 1.42.

    [0490] Two days post-transfection, the genomic DNA was extracted and submitted to PCR using HPRT-1 specific primers. The genome editing event was analysed by the T7 Endonuclease assay and visualized on agarose gel and quantified using Ethidium Bromide staining to determine the % INDEL (percentage of insertion/deletion CRISPR event). The transfected plasmid p38285 expressing the spCas9 and the specific HPRT-1 guide RNA showed the presence of the two expected bands on the gel at 650 bp and 430 bp (FIG. 8). The % INDEL (Insertion—deletion mutation event) was 33.48+/−7.08%. The specificity of the CRISPR Cas9 transfection was shown as specific signals of cleaved band was observed after transfection of the plasmid targeting the HPRT-1 gene and not with the plasmid control. The experiment demonstrated that compound 1.42 used for the transfection was efficient to induce a CRISRP Cas9 genome modification without Generating Off-Targets Events.

    Example 9. Transfection assay of Stem Cells

    [0491] Primary hyman mesenchymal stem cells (hMSC) were transfected with the compound 1.42 and different amount of pCMV-EGFP DNA, 400 and 500 ng per well in 24-well plate format (FIG. 9). Various volumes of compound 1.42 were used per amount of DNA. One day post-transfection, the GFP expression was analysed by flow cytometry or the cells were observed using a ZOE™ Fluorescent Cell Imager. For the conditions with 400 ng of DNA, a progressive increase of the transfection efficiency was quantified when the volume of compound 1.42 was increased to reach an optimal transfection up to 60% of GFP positive cells. For the conditions at 500 ng DNA, the best condition was obtained with 0.75 μL of compound 1.42 with more than 60% of GFP positive cells. As a control, the transfection performed with the Lipofectamine 3000 reagent provided a transfection efficiency inferior to 10%. These results show that an optimization of the transfection conditions can be realized by varying both the DNA amount transfected and the volume of compound.

    Example 10. Compounds 1.73 to 1.80

    [0492] The inventors carried out some comparative data using imidazole derivatives such as compounds 1.73, 1.78 and 1.80 (see Table 3). Synthesis of said compounds is reported in Example 2.

    TABLE-US-00003 TABLE 3 Imidazole derivatives Polymer Com- Molecular Heterocycle pound Structure weight grafting 1.73 [00318]embedded image 22k 29% 1.78 [00319]embedded image 22k 30% 1.80 [00320]embedded image 22k 45%

    [0493] Screening of Transfection Activity

    [0494] Compounds 1.73 to 1.80 were evaluated for their ability to transfect DNA (pCMV-EGFP.sub.Luc) in Hep G2 cells and the transfection efficiency in 96-well plate format was determined by analyzing the percentage of cells expressing the GFP (% GFP) by cytometry assay one day post-transfection (FIG. 11). Compounds 1.73 to 1.80 were compared to the compound 1.42 comprising a benzimidazole ring grafted to the linear PEI 22K wherein Z.sup.2, Z.sup.4 and Z.sup.6═CH.sub.3. 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 efficiencies when compared to the unmodified parental polymers. This effect was particularly shown with the compounds 1.42 and 1.74 wherein the parental polymer is PEI. In addition, the compound 1.42 with a benzimidazole ring showed a higher transfection efficiency when compared to the compound 1.73 comprising an imidazole ring grafted to the linear PEI 22K.

    [0495] Bioproduction of Recombinant Virus

    [0496] Compounds 1.73 to 1.80 were tested for the production of AAV-2 and FIG. 12 presents the results obtained at the best ratio μg DNA/4 compound. The presence of the benzimidazole ring showed higher AAV-2 productivities when compared to the unmodified parental polymers, particularly shown with the compounds 1.42 and 1.74 wherein the parental polymer is PEI (linear 22K or branched 25K, respectively). Compound 1.42 showed a significant higher virus production when compared to the compound 1.73 comprising an imidazole ring grafted to the linear PEI 22K.

    CONCLUSION

    [0497] Many compounds based on grafting of polyamine with heterocycles of formula (II) showed improved performances to induce gene expression in “hard to transfect” cells such as cancer cells, primary cells, non-dividing cells or to increase the productivity of biologics such as viruses, AAV or LV.

    [0498] Many compounds of Examples 3 and 10 comprising a benzimidazole ring wherein Y.sup.1═Y.sup.3═N, Y.sup.2═C, and R, T, U, V form a benzene ring showed higher transfection efficiency when compared to the unmodified parental linear PEI (jetPEI®) or compared to linear modified PEI comprising an imidazole ring or compared to cationic polymers exhibiting benzimidazole or imidazole grafts in the four cancer cell lines tested. Among the various structures tested, when Z.sup.2 is different of H, and particularly with Z.sup.2═CH.sub.3, and Z.sup.4, Z.sup.5 or Z.sup.6 are different of H, particularly with Z.sup.4, Z.sup.5 or Z.sup.6═CH.sub.3, the compounds provided the highest transfection efficiencies.

    [0499] Many compounds of Example 3 comprising a benzopyrazole ring wherein Y.sup.1═Y.sup.2═N, Y.sup.3═C, and R, T, U, V form a benzene ring showed promising activity in transfection.

    [0500] These results were also confirmed after transfection of primary cells, particularly non-dividing cells, such as primary neurons, but also fragile cells and “hard to transfect cells” such as primary fibroblasts, endothelial cells or stem cells.

    [0501] Many compounds of Example 4, particularly polyamine grafted with benzotriazole derivatives showed high transfection efficiencies, similarly to the best compounds of Example 3.

    [0502] Selected compounds of Examples 3 and 4 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.

    [0503] Taken together, the compounds of formula (II) 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.

    [0504] The person skilled in the art can adapt the transfection method with the compounds of formula (II) of the invention to a particular cell type, cell culture conditions or cell culture devices used. In particular, the amount of DNA, amount of transfection reagent, volume of transfection complexes, conditions of mixing of DNA and transfection reagent, medium of transfection complex preparation can vary. In addition, the transfection method with the compounds of formula (II) of the invention can be adapted for industrial uses, particularly at large scale applications in bioreactors for both adherent and suspension cells.

    [0505] The person skilled in the art can adapt the transfection method with the compounds of formula (II) of the invention for in vivo applications with an acceptable excipient or buffering agent. The compounds of formula (II) 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 formula (II) 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

    [0506] Kaestner L, Schol A, Lipp P. Conceptual and technical aspects of transfection and gene delivery. Bioorganic & Medicinal Chemistry Letters, 25(6), 1171-1176 (2015). [0507] Merten O W, Hebben M, Bovolenta C. Production of lentiviral vectors. Mol Ther Methods Clin Dev. 3:16017 (2016). [0508] Van der Loo J C, Wright J F. Progress and challenges in viral vector manufacturing. Hum Mol Genet. 25(R1):R42-52 (2015). [0509] Labat-Moleur F, Steffan A M, Brisson C, Perron H, Feugeas O, Furstenberger P, Oberling F, Brambilla E, Behr J P. An electron microscopy study into the mechanism of gene transfer with lipopolyamines. Gene Ther., 3(11):1010-7 (1996). [0510] Mislick K A, Baldeschwieler J D. Evidence for the role of proteoglycans in cation-mediated gene transfer. Proc Natl Acad Sci USA, 93(22):12349-54 (1996). [0511] Feigner P L, Gadek T R, Holm M, Roman R, Chan H W, Wenz M, Northrop J P, Ringold G M, Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci USA, 84(21):7413-7 (1987). [0512] Behr J P, Demeneix B, Loeffler J P, Perez-Mutul J. Efficient gene transfer into mammalian primary endocrine cells with lipopolyamine-coated DNA. Proc Natl Acad Sci USA, 86: 6982-6986 (1989). [0513] Gao X, Huang L. A novel cationic liposome reagent for efficient transfection of mammalian cells. Biochem Biophys Res Commun., 179(1):280-5 (1991). [0514] Wu G Y, Wu C H. Receptor-mediated in vitro gene transformation by a soluble DNA carrier system. The Journal of Biological Chemistry, 262, 4429-4432 (1987). [0515] Zenke M, Steinlein P, Wagner E, Cotton M, Beug H, Birnstiel M L. Receptor-mediated endocytosis of transferrin-polycation conjugates: an efficient way to introduce DNA into hematopoietic cells. Proc Natl Acad Sci USA, 87(10) 3655-3659 (1990). [0516] Erbacher P, Roche A C, Monsigny M, Midoux P. Putative role of chloroquine in gene transfer into a human hepatoma cell line by DNA/lactosylated polylysine complexes. Exp Cell Res., 225(1):186-94 (1996). [0517] Plank C, Oberhauser B, Mechtler K, Koch C, Wagner E. The influence of endosome-disruptive peptides on gene transfer using synthetic virus-like gene transfer systems. J Biol Chem., 269(17):12918-24 (1994). [0518] Boussif O, Lezoualc′h F, Zanta M A, Mergny M D, Scherman D, Demeneix B, Behr J P. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci USA., 92(16): 7297-7301 (1995). [0519] Sonawane N D, Szoka F C Jr, Verkman A S. Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes. J Biol Chem., 278(45):44826-31 (2003). [0520] Itaka K, Harada A, Yamasaki Y, et al. In situ single cell observation by fluorescence resonance energy transfer reveals fast intracytoplasmic delivery and easy release of plasmid DNA complexed with linear polyethylenimine. J Gene Med, 6: 76-84 (2004). [0521] Chandrashekhar C, Pons B, Muller C D, Tounsi N, Mulherkar R, Zuber G. Oligobenzylethylenimine enriches linear polyethylenimine with a pH-sensitive membrane-disruptive property and leads to enhanced gene delivery activity. Acta Biomaterialia, 9(2):4985-4993 (2012). [0522] Erbacher P, Zou S, Bettinger T, Steffan A M, Remy J S. Chitosan-based vector/DNA complexes for gene delivery: biophysical characteristics and transfection ability. Pharm Res., 15(9):1332-9 (1998). [0523] Tomalia D A, Baker D, Dewald J, Hall M, Kallos G, Martin S, Roeck J, Ryder, Smith J. A New Class of Polymers: Starburst-Dendritic Macromolecules Polymer Journal 17, 117-132 (1985). [0524] Haensler J, Szoka F C. Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconjugate Chem., 45, 372-379 (1993). [0525] Tang M X, Redemann C T, Szoka F C Jr. In vitro gene delivery by degraded polyamidoamine dendrimers. Bioconjug Chem., 7(6):703-14 (1996). [0526] Liu X, Wu J, Yammine M, Zhou J, Posocco P, Viel S, Liu C, Ziarelli F, Fermeglia M, Pricl S, Victorero G, Nguyen C, Erbacher P, Behr J P, Peng L. Structurally flexible triethanolamine core PAMAM dendrimers are effective nanovectors for DNA transfection in vitro and in vivo to the mouse thymus. Bioconjug Chem., 22(12):2461-73 (2011). [0527] Little S R, Lynn D, Ge Q, Anderson D G, Puram S V, Chen J, Eisen H, Langer R. From The Cover: Poly-amino ester-containing microparticles enhance the activity of non-viral genetic vaccines. Proc Natl Acad Sci USA, 101(26), 9534-9539 (2004). [0528] Akinc A, Anderson D G, Lynn D M, Langer R. Synthesis of poly(beta-amino ester) s optimized for highly effective gene delivery. Bioconjug Chem., 14:979-988 (2003). [0529] Cryan S, Donohue R, Ravoo B J, Darcy R, O'Driscoll C M. Cationic cyclodextrin amphiphiles as gene delivery vectors. Journal of Drug Delivery Science and Technology, 14(1), Pages 57-62 (2004). [0530] Dréan M, Debuigne A, Jérôme C, Goncalves C, Midoux P, Rieger J, Guegan P. Poly(N-methylvinylamine)-Based Copolymers for Improved Gene Transfection. Macromol Biosci., 18(4): e1700353 (2018). [0531] Boussif O, Delair T, Brua C, Veron L, Pavirani A, Kolbe H V. Synthesis of polyallylamine derivatives and their use as gene transfer vectors in vitro. Bioconjug Chem. 10(5):877-83 (1999). [0532] Dong Y, Skoultchi Al, Pollard J W. Efficient DNA transfection of quiescent mammalian cells using poly-L-ornithine. Nucleic Acids Res., 21(3): 771-772 (1993). [0533] Alhakamy N A, Berkland C J. Polyarginine molecular weight determines transfection efficiency of calcium condensed complexes. Mol Pharm., 10(5):1940-8 (2013). [0534] Putnam D, Zelikinb A N, lzumrudovc V A, Langer R. Polyhistidine-PEG:DNA nanocomposites for gene delivery Biomaterials 24:4425-4433 (2003). [0535] Gupta B, Levchenko T S, Torchilin V P. Intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides. Advanced Drug Delivery Reviews, 57(4), 637-651 (2005). [0536] Brunner S, FOrtbauer E, Sauer T, Kursa M, Wagner E. Overcoming the Nuclear Barrier: Cell Cycle Independent Nonviral Gene Transfer with Linear Polyethylenimine or Electroporation. Mol. Ther., 5(1), 80-86 (2002). [0537] Lukacs G L, Haggie P, Seksek O, Lechardeur D, Freedman N, Verkman A S. Size-dependent DNA mobility in cytoplasm and nucleus. J Biol Chem. 275(3):1625-9 (2000). [0538] Lechardeur D, Sohn K J, Haardt M, Joshi P B, Monck M, Graham R W, Beatty B, Squire J, O'Brodovich H, Lukacs G L. Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer. Gene Ther. 6(4):482-97 (1999). [0539] Bai H, Schiralli Lester G M, Petishnok L C, Dean D A. Cytoplasmic transport and nuclear import of plasmid DNA. Bioscience Reports 37, BSR20160616 (2017). [0540] Gaba M, Mohan C. Development of drugs based on imidazole and benzimidazole bioactive heterocycles: recent advances and future directions. Medicinal Chemistry Research, 25(2), 173-210 (2016). [0541] Ivanov A A, Susova O Y, Salyanov V I, Kirsanov K I, Zhuze A L. A new series of biologically active DNA minor groove binders based on bisbenzimidazole and benzimidazole-pyrrole motives. Journal of Biomolecular Structure and Dynamics, 31: 1, 52-53 (2013). [0542] Gao C, Li B, Zhang B, Sun Q, Li L, Li X, Chen C, Tan C, Liu H, Jiang Y. Synthesis and biological evaluation of benzimidazole acridine derivatives as potential DNA-binding and apoptosis-inducing agents. Bioorganic & Medicinal Chemistry, 23, 1800-1807 (2015). [0543] Bazhulina N P, Nikitin A M, Rodin S A, Surovaya A N, Kravatsky Y V, Pismensky V F, Archipova V S, Martin R, Gursky G V. Binding of Hoechst 33258 and its derivatives to DNA. J Biomol Struct Dyn. 26(6):701-18, (2009). [0544] Tari Ö, Gümüş F, Açik L, Aydin B. Synthesis, characterization and DNA binding studies of platinum(II) complexes with benzimidazole derivative ligands. Bioorg Chem. 74:272-283 (2017). [0545] Fellenius E, Berglindh T, Sachs G, Olbe L, Elander B, Sjostrand S E, Wallmark B. Substituted benzimidazoles inhibit gastric acid secretion by blocking (H++K+) ATPase. Nature. 290(5802):159-61 (1981). [0546] Brown T N, Mora-Diez N. Computational determination of aqueous pKa values of protonated benzimidazoles (Part 2). J Phys Chem B. 110(41):20546-54 (2006).