JP2005535570 | New pyrimidone derivative |
WO/2000/053607 | CAMPTOTHECIN DERIVATIVES HAVING ANTITUMOR ACTIVITY |
JP2020028302 | PD-1, RECEPTOR FOR B7-4, AND USE THEREFOR |
BIOSCA ROMANILLOS ARNAU (ES)
BOUZÓN ARNÁIZ INÉS (ES)
MUÑOZ-TORRERO LÓPEZ-IBARRA DIEGO (ES)
MARTÍNEZ ARCE ELSA (ES)
REOLID COLL PAU (ES)
FUNDACION PRIVADA INST DE SALUD GLOBAL BARCELONA (ES)
UNIV BARCELONA (ES)
WO2011065980A2 | 2011-06-03 | |||
WO2009109457A2 | 2009-09-11 | |||
WO2011065980A2 | 2011-06-03 |
GB2011457A | 1979-07-11 | |||
EP2053094A2 | 2009-04-29 |
VLACHOU, D.ZIMMERMANN, T.CANTERA, RJANSE, C. JWATERS, A. P.KAFATOS, F. C.: "Real-time, in vivo analysis of malaria ookinete locomotion and mosquito midgut invasion.", CELL. MICROBIOL., vol. 6, no. 7, 2004, pages 671 - 685
STOKES, B.H.DHINGRA, S.K.RUBIANO, K.MOK, S.STRAIMER, J.GNADIG, N.F. ET AL.: "Plasmodium falciparum K13 mutations in Africa and Asia impact artemisinin resistance and parasite fitness", ELIFE, 2021, pages 10
STRAIMER, J.GNADIG, N.FWITKOWSKI, B.AMARATUNGA, C.DURU, V.RAMADANI, A.P.DACHEUX, MKHIM, N.ZHANG, L.LAM, S.: "K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates", SCIENCE, vol. 347, 2015, pages 428 - 431
PORTUGALIZA, H.PLLORA-BATLLE, O.ROSANAS-URGELL, A.CORTES, A.: "Reporter lines based on the gexp02 promoter enable early quantification of sexual conversion rates in the malaria parasite Plasmodium falciparum.", SCI REP, vol. 9, 2019, pages 14595
CLAIMS 1. A compound of formula (I) or a pharmaceutically acceptable salt thereof for use as a medicament, wherein: A is an anion; L is a diradical selected from the group consisting of 1,2-phenylenebis(methylene), 1,3- phenylenebis(methylene), 1,4-phenylenebis(methylene) and a (C2-C12)-oligomethylene chain, wherein one to three methylene groups of the (C2-C12)-oligomethylene chain can be substituted by oxygen atoms; R1, R2, R7, and R8 are radicals independently selected from the group consisting of hydrogen, (C1-C4)-alkyl, hydroxy-(C1-C4)-alkyl, cyano-(C1-C4)-alkyl, (C1-C4)-alkanoyloxy-(C1-C4)-alkyl, phenyl, (C1-C4)-alkanoyl, (C1-C4)-alkanesulfonyl, benzenesulfonyl, naphthalenesulfonyl, and p- toluenesulfonyl, or alternatively, when taken in combination, R1-N-R2 and R7-N-R8 are independent- ly selected from the group consisting of azetidine, pyrrolidine, piperidine, azepine, piperazine, 4- (C1-C2)-alkylpiperazine, and morpholine ring; R3 and R6 are radicals independently selected from the group consisting of hydrogen, (C1-C4)- alkyl, nitro, amino, (C1-C4)-alkylamino, (C1-C4)-alkanamido, (C1-C4)-alkanesulfonamido, benzene- sulfonamido, naphthalenesulfonamido, and p-toluenesulfonamido; and R4 and R5 are radicals independently selected from the group consisting of hydrogen, (C1-C4)- alkyl, halogen, amino, (C1-C4)-alkylamino, (C1-C4)-alkanamido, (C1-C4)-alkanesulfonamido, ben- zenesulfonamido, naphthalenesulfonamido, p-toluenesulfonamido, amino-(C1-C4)-alkyl, (C1-C2)- alkylamino-(C1-C4)-alkyl, (C1-C4)-alkanamido-(C1-C4)-alkyl, (C1-C4)-alkanesulfonamido-(C1-C4)- alkyl, benzenesulfonamido-(C1-C4)-alkyl, naphthalenesulfonamido-(C1-C4)-alkyl, and p- toluenesulfonamido-(C1-C4)-alkyl. 2. The compound for use according to claim 1, wherein the compound is selected from the group consisting of: 1,1'-(decane-1,10-diyl)bis{3-methyl-4-[(E)-4-(pyrrolidin-1-yl)styryl]pyridin-1-ium} dibromide; 1,1'-(decane-1,10-diyl)bis{3-methyl-4-[(E)-4-(piperidin-1-yl)styryl]pyridin-1-ium} dibromide; 1,1'-(decane-1,10-diyl)bis{3-methyl-4-[(E)-4-morpholinostyryl]pyridin-1-ium} dibromide; 1,1'-(decane-1,10-diyl)bis{3-methyl-4-[(E)-4-(piperazin-1-yl)styryl]pyridin-1-ium} dibromide; 1,1'-(decane-1,10-diyl)bis{4-[(E)-4-(diphenylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(decane-1,10-diyl)bis{4-[(E)-4-(dimethylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(decane-1,10-diyl)bis{4-[(E)-4-(dimethylamino)-2-nitrostyryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(decane-1,10-diyl)bis{4-{(E)-4-[(2-cyanoethyl)methylamino]styryl}-3-methylpyridin-1-ium} di- bromide; 1,1'-(decane-1,10-diyl)bis{4-{(E)-4-[bis(2-acetoxyethyl)amino]styryl}-3-methylpyridin-1-ium} dibro- mide; 1,1'-(decane-1,10-diyl)bis{4-{(E)-4-[bis(2-hydroxyethyl)amino]styryl}-3-methylpyridin-1-ium} dibro- mide; 1,1'-(ethane-1,2-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(propane-1,3-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(butane-1,4-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(pentane-1,5-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(hexane-1,6-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(heptane-1,7-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(octane-1,8-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(nonane-1,9-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(decane-1,10-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(undecane-1,11-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(dodecane-1,12-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(3-oxapentane-1,5-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dichloride; 1,1'-(3,6-dioxaoctane-1,8-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} diiodide; 1,1'-(3,6,9-trioxaundecane-1,11-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dichlo- ride; 1,1'-[1,2-phenylenebis(methylene)]bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibro- mide; 1,1'-[1,3-phenylenebis(methylene)]bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibro- mide; 1,1'-[1,4-phenylenebis(methylene)]bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibro- mide; 1,1'-(decane-1,10-diyl)bis{4-[(E)-4-(diethylamino)styryl]pyridin-1-ium} dibromide; 1,1'-(decane-1,10-diyl)bis{3-bromo-4-[(E)-4-(diethylamino)styryl]pyridin-1-ium} dibromide; 1,1'-(decane-1,10-diyl)bis{3-chloro-4-[(E)-4-(diethylamino)styryl]pyridin-1-ium} dibromide; and 1,1'-(decane-1,10-diyl)bis{3-amino-4-[(E)-4-(diethylamino)styryl]pyridin-1-ium} dibromide. 3. The compound for use according to claim 2, wherein the compound is of formula (VI) (VI) or a pharmaceutically acceptable salt thereof. 4. The compound for use according to claim 3, wherein A is bromide. 5. A compound of formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment, prevention or amelioration of malaria, or symptoms, complications and/or sequelae thereof, wherein: A is an anion; L is a diradical selected from the group consisting of 1,2-phenylenebis(methylene), 1,3- phenylenebis(methylene), 1,4-phenylenebis(methylene) and a (C2-C12)-oligomethylene chain, wherein one to three methylene groups of the (C2-C12)-oligomethylene chain can be substituted by oxygen atoms; R1, R2, R7, and R8 are radicals independently selected from the group consisting of hydrogen, (C1-C4)-alkyl, hydroxy-(C1-C4)-alkyl, cyano-(C1-C4)-alkyl, (C1-C4)-alkanoyloxy-(C1-C4)-alkyl, phenyl, (C1-C4)-alkanoyl, (C1-C4)-alkanesulfonyl, benzenesulfonyl, naphthalenesulfonyl, and p- toluenesulfonyl, or alternatively, when taken in combination, R1-N-R2 and R7-N-R8 are independent- ly selected from the group consisting of azetidine, pyrrolidine, piperidine, azepine, piperazine, 4- (C1-C2)-alkylpiperazine, and morpholine ring; R3 and R6 are radicals independently selected from the group consisting of hydrogen, (C1-C4)- alkyl, nitro, amino, (C1-C4)-alkylamino, (C1-C4)-alkanamido, (C1-C4)-alkanesulfonamido, benzene- sulfonamido, naphthalenesulfonamido, and p-toluenesulfonamido; and R4 and R5 are radicals independently selected from the group consisting of hydrogen, (C1-C4)- alkyl, halogen, amino, (C1-C4)-alkylamino, (C1-C4)-alkanamido, (C1-C4)-alkanesulfonamido, ben- zenesulfonamido, naphthalenesulfonamido, p-toluenesulfonamido, amino-(C1-C4)-alkyl, (C1-C2)- alkylamino-(C1-C4)-alkyl, (C1-C4)-alkanamido-(C1-C4)-alkyl, (C1-C4)-alkanesulfonamido-(C1-C4)- alkyl, benzenesulfonamido-(C1-C4)-alkyl, naphthalenesulfonamido-(C1-C4)-alkyl, and p- toluenesulfonamido-(C1-C4)-alkyl. 6. A pharmaceutical composition comprising an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein: A is an anion; L is a diradical selected from the group consisting of 1,2-phenylenebis(methylene), 1,3- phenylenebis(methylene), 1,4-phenylenebis(methylene) and a (C2-C12)-oligomethylene chain, wherein one to three methylene groups of the (C2-C12)-oligomethylene chain can be substituted by oxygen atoms; R1, R2, R7, and R8 are radicals independently selected from the group consisting of hydrogen, (C1-C4)-alkyl, hydroxy-(C1-C4)-alkyl, cyano-(C1-C4)-alkyl, (C1-C4)-alkanoyloxy-(C1-C4)-alkyl, phenyl, (C1-C4)-alkanoyl, (C1-C4)-alkanesulfonyl, benzenesulfonyl, naphthalenesulfonyl, and p- toluenesulfonyl, or alternatively, when taken in combination, R1-N-R2 and R7-N-R8 are independent- ly selected from the group consisting of azetidine, pyrrolidine, piperidine, azepine, piperazine, 4- (C1-C2)-alkylpiperazine, and morpholine ring; R3 and R6 are radicals independently selected from the group consisting of hydrogen, (C1-C4)- alkyl, nitro, amino, (C1-C4)-alkylamino, (C1-C4)-alkanamido, (C1-C4)-alkanesulfonamido, benzene- sulfonamido, naphthalenesulfonamido, and p-toluenesulfonamido; and R4 and R5 are radicals independently selected from the group consisting of hydrogen, (C1-C4)- alkyl, halogen, amino, (C1-C4)-alkylamino, (C1-C4)-alkanamido, (C1-C4)-alkanesulfonamido, ben- zenesulfonamido, naphthalenesulfonamido, p-toluenesulfonamido, amino-(C1-C4)-alkyl, (C1-C2)- alkylamino-(C1-C4)-alkyl, (C1-C4)-alkanamido-(C1-C4)-alkyl, (C1-C4)-alkanesulfonamido-(C1-C4)- alkyl, benzenesulfonamido-(C1-C4)-alkyl, naphthalenesulfonamido-(C1-C4)-alkyl, and p- toluenesulfonamido-(C1-C4)-alkyl, together with appropriate amounts of pharmaceutically acceptable excipients or carriers. 7. A compound of formula (I) (I) or a pharmaceutically acceptable salt thereof, wherein: A is an anion; L is a diradical selected from the group consisting of ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, nonamethylene, decameth- ylene, undecamethylene, dodecamethylene, 3-oxapentamethylene, 3,6-dioxaoctamethylene, 3,6,9- trioxaundecamethylene, 1,2-phenylenebis(methylene), 1,3-phenylenebis(methylene), and 1,4- phenylenebis(methylene); R1, R2, R7, and R8 are radicals independently selected from the group consisting of hydrogen, (C1-C4)-alkyl, hydroxy-(C1-C4)-alkyl, cyano-(C1-C4)-alkyl, (C1-C4)-alkanoyloxy-(C1-C4)-alkyl, phenyl, (C1-C4)-alkanoyl, (C1-C4)-alkanesulfonyl, benzenesulfonyl, naphthalenesulfonyl, and p- toluenesulfonyl or alternatively, when taken in combination, R1-N-R2 and R7-N-R8 are independent- ly selected from the group consisting of azetidine, pyrrolidine, piperidine, azepine, piperazine, 4- (C1-C2)-alkylpiperazine, and morpholine ring; R3 and R6 are radicals independently selected from the group consisting of hydrogen, (C1-C4)- alkyl, nitro, amino, (C1-C4)-alkylamino, (C1-C4)-alkanamido, (C1-C4)-alkanesulfonamido, benzene- sulfonamido, naphthalenesulfonamido, and p-toluenesulfonamido; and R4 and R5 are radicals independently selected from the group consisting of hydrogen, (C1-C4)- alkyl, halogen, amino, (C1-C4)-alkylamino, (C1-C4)-alkanamido, (C1-C4)-alkanesulfonamido, ben- zenesulfonamido, naphthalenesulfonamido, p-toluenesulfonamido, amino-(C1-C4)-alkyl, (C1-C2)- alkylamino-(C1-C4)-alkyl, (C1-C4)-alkanamido-(C1-C4)-alkyl, (C1-C4)-alkanesulfonamido-(C1-C4)- alkyl, benzenesulfonamido-(C1-C4)-alkyl, naphthalenesulfonamido-(C1-C4)-alkyl, and p- toluenesulfonamido-(C1-C4)-alkyl; with the proviso that when R1, R2, R3, R4, R5, R6, R7 and R8 are hydrogen, L is not 1,4- phenylenebis(methylene); with the proviso that when R1, R2, R7 and R8 are methyl and R3, R4, R5 and R6 are hydrogen, L is not ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, decamethylene, dodecamethylene, 3,6-dioxaoctamethylene, 1,2-phenylenebis(methylene), 1,3- phenylenebis(methylene) or 1,4-phenylenebis(methylene); with the proviso that when R1, R2, R7 and R8 are ethyl and R3, R4, R5 and R6 are hydrogen, L is not trimethylene, tetramethylene, 3-oxapentamethylene, 1,2-phenylenebis(methylene) or 1,4- phenylenebis(methylene); with the proviso that when R1, R2, R7 and R8 are phenyl and R3, R4, R5 and R6 are hydrogen, L is not trimethylene, tetramethylene, pentamethylene, hexamethylene, octamethylene, decameth- ylene, dodecamethylene, 1,2-phenylenebis(methylene), 1,3-phenylenebis(methylene) or 1,4- phenylenebis(methylene); with the proviso that when R1, R2, R7 and R8 are HO-CH2-CH2- and R3, R4, R5 and R6 are hy- drogen, L is not tetramethylene; with the proviso that when R1 and R7 are ethyl, R2 and R8 are HO-CH2-CH2- and R3, R4, R5 and R6 are hydrogen, L is not ethylene, trimethylene, tetramethylene, pentamethylene, hexameth- ylene or heptamethylene; with the proviso that when R1 and R7 are methyl, R2 and R8 are NC-CH2- and R3, R4, R5 and R6 are hydrogen, L is not 1,2-phenylenebis(methylene) or 1,4-phenylenebis(methylene), with the proviso that when R1 and R7 are acetyl, R2, R3, R4, R5 R6 and R8 are hydrogen, L is not 1,4-phenylenebis(methylene); with the proviso that when R1, R2, R3, R6, R7 and R8 are methyl and R4 and R5 are hydrogen, L is not trimethylene or 1,4-phenylenebis(methylene); with the proviso that when R1, R2, R4, R5, R7 and R8 are methyl and R3 and R6 are hydrogen, L is not trimethylene, tetramethylene or decamethylene; with the proviso that when R1, R2, R7 and R8 are ethyl, R3 and R6 are hydrogen and R4 and R5 are methyl, L is not decamethylene; with the proviso that when R1-N-R2 and R7-N-R8 are pyrrolidino and R3, R4, R5 and R6 are hy- drogen, L is not trimethylene or 1,4-phenylenebis(methylene); with the proviso that when R1-N-R2 and R7-N-R8 are piperidino and R3, R4, R5 and R6 are hy- drogen, L is not trimethylene, 1,2-phenylenebis(methylene), 1,3-phenylenebis(methylene) or 1,4- phenylenebis(methylene); and with the proviso that when R1-N-R2 and R7-N-R8 are 4-methylpiperazino and R3, R4, R5 and R6 are hydrogen, L is not trimethylene. 8. The compound according to claim 7, wherein R1 = R7, R2 = R8, R3 = R6, and R4 = R5 or alterna- tively, when used in combination, R1-N-R2 = R7-N-R8, R3 = R6, and R4 = R5. 9. The compound according to claim 8, wherein: R1, R7 and R2, R8 are radicals independently selected from the group consisting of hydrogen, methyl, ethyl, phenyl, 2-hydroxyethyl, 2-cyanoethyl, and 2-(acetyloxy)ethyl, or alternatively, when taken in combination, R1-N-R2 = R7-N-R8 is selected from the group consisting of pyrrolidine, piper- idine, piperazine and morpholine ring; R3, R6 are radicals selected from the group consisting of hydrogen, methyl, nitro, amino, acet- amido, methanesulfonamido, benzenesulfonamido, and p-toluenesulfonamido; and R4, R5 are radicals selected from the group consisting of hydrogen, methyl, chloro, bromo, fluoro, iodo, amino, acetamido, methanesulfonamido, benzenesulfonamido, p-toluenesulfonamido, aminomethyl, acetamidomethyl, methanesulfonamidomethyl, benzenesulfonamidomethyl, and p- toluenesulfonamidomethyl. 10. The compound according to claim 9, wherein the compound is selected from the group consist- ing of: 1,1'-(decane-1,10-diyl)bis{3-methyl-4-[(E)-4-(pyrrolidin-1-yl)styryl]pyridin-1-ium} dibromide; 1,1'-(decane-1,10-diyl)bis{3-methyl-4-[(E)-4-(piperidin-1-yl)styryl]pyridin-1-ium} dibromide; 1,1'-(decane-1,10-diyl)bis{3-methyl-4-[(E)-4-morpholinostyryl]pyridin-1-ium} dibromide; 1,1'-(decane-1,10-diyl)bis{3-methyl-4-[(E)-4-(piperazin-1-yl)styryl]pyridin-1-ium} dibromide; 1,1'-(decane-1,10-diyl)bis{4-[(E)-4-(diphenylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(decane-1,10-diyl)bis{4-[(E)-4-(dimethylamino)-2-nitrostyryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(decane-1,10-diyl)bis{4-{(E)-4-[(2-cyanoethyl)methylamino]styryl}-3-methylpyridin-1-ium} di- bromide; 1,1'-(decane-1,10-diyl)bis{4-{(E)-4-[bis(2-acetoxyethyl)amino]styryl}-3-methylpyridin-1-ium} dibro- mide; 1,1'-(decane-1,10-diyl)bis{4-{(E)-4-[bis(2-hydroxyethyl)amino]styryl}-3-methylpyridin-1-ium} dibro- mide; 1,1'-(ethane-1,2-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(propane-1,3-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(butane-1,4-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(pentane-1,5-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(hexane-1,6-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(heptane-1,7-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(octane-1,8-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(nonane-1,9-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(undecane-1,11-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(dodecane-1,12-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide; 1,1'-(3-oxapentane-1,5-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dichloride; 1,1'-(3,6-dioxaoctane-1,8-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} diiodide; 1,1'-(3,6,9-trioxaundecane-1,11-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dichlo- ride; 1,1'-[1,2-phenylenebis(methylene)]bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibro- mide; 1,1'-[1,3-phenylenebis(methylene)]bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibro- mide; 1,1'-[1,4-phenylenebis(methylene)]bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibro- mide; 1,1'-(decane-1,10-diyl)bis{4-[(E)-4-(diethylamino)styryl]pyridin-1-ium} dibromide; 1,1'-(decane-1,10-diyl)bis{3-bromo-4-[(E)-4-(diethylamino)styryl]pyridin-1-ium} dibromide; 1,1'-(decane-1,10-diyl)bis{3-chloro-4-[(E)-4-(diethylamino)styryl]pyridin-1-ium} dibromide; and 1,1'-(decane-1,10-diyl)bis{3-amino-4-[(E)-4-(diethylamino)styryl]pyridin-1-ium} dibromide. |
Table 3. Chemical structures of compounds tested for in vitro antimalarial activity.
AID-X-2020 belongs to a chemical family where no antimalarial drugs have been described so far. Thus, an in vitro study on the inhibition effect of AID-X-2020 on each stage of the parasite was per- formed.
EXAMPLE 2: Inhibition effect of AID-X-2020 on each stage of the parasite P. falciparum in vitro
The objective of this experiment was the evaluation of the inhibition effect of AID-X-2020 on each stage of the parasite: rings, early trophozoites, mature trophozoites and schizonts. 2.1 Materials and methods For stage of growth inhibition analysis, P. falciparum cultures were synchronized at ring or tropho- zoite stages by repeated treatment with 5% sorbitol or 70% Percoll, respectively. Half of each cul- ture remained untreated and the other half was treated with the IC 80 of AID-X-2020. At different time points, culture samples were stained with Giemsa and the number of rings, early and mature trophozoites and schizonts was counted by microscopic examination of at least 100 pRBCs for each sample. Pictures were taken with a Nikon Eclipse 50i microscope equipped with a DS-Fi1 camera (Nikon). 2.2 Results When added to ring stages at its IC 80 , AID-X-2020 arrested the life cycle of the pathogen at tropho- zoite stage (FIG.1), whereas when the drug was delivered to cultures containing late Plasmodium forms, the parasites were able to complete their intraerythrocytic maturation and egress the pRBC, although their growth inside the new invaded RBCs became arrested at early trophozoite stage. EXAMPLE 3: Effect of AID-X-2020 on the in vitro aggregation of Aβ40 In order to determine the mechanism of action of AID-X-2020 on the inhibition of P. falciparum that was previously proved in vitro, the effect of AID-X-2020 on Aβ40 aggregation in vitro was first stud- ied. Amyloid-β peptide fragment 1-40 (Aβ40) was used as a model of a highly aggregative peptide to evaluate the potential antiaggregatory effect of AID-X-2020. Firstly, an in vitro analysis of Aβ40 aggregation was performed by ThT fluorescence assay. Secondly, to dismiss the possibility that the decrease in ThT fluorescence was due to the presence of AID-X-2020, the Aβ40 samples treated with AID-X-2020 were examined by transmission electron microscopy (TEM). 3.1 Materials and methods 3.1.1 In vitro analysis of Aβ40 aggregation For the in vitro analysis of Aβ peptide fragment 1-40 (Aβ40) aggregation, 1 mg of Aβ40 (GenScript) was dissolved in 500 µL of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Fluka) under vigorous stirring for 1 h and sonicated for 30 min in a sonication bath. Afterwards, the solution was stirred for 1 h and maintained at 4 °C for 30 min. Aliquots were prepared, HFIP was evaporated under a nitrogen stream for a few seconds and the peptide was stored at ‒20 °C. Prior to use, aliquots of Aβ40 were resuspended with DMSO, sonicated for 10 min, further diluted to 25 µM in PBS containing different concentrations of test compounds (0.1 µM AID-X-2020, 1 µM AID-X-2020 and 10 µM AID-X-2020), and incubated for 24 h at 37 °C and 1400 rpm. The final sample always contained less than 5% DMSO to avoid interference of this solvent on Aβ40 amyloid fibril formation. Finally, ThT treatment was performed as described above. 25 μM ThT was added to each sample and fluorescence was measured by exciting samples at 440 nm and recording the emission from 465 nm to 600 nm. A blank measurement of each sample was done before adding ThT. 3.1.2 Transmission electron microscopy (TEM) For in vitro peptide aggregation analysis, 25 µM Aβ40 solution was prepared as explained above and incubated for 48 h at 37 °C and 1400 rpm in the presence of growing concentrations of AID-X- 2020 (0, 0.1, 1 and 10 µM). A carbon-coated copper grid was deposited on top of a 50-µL drop of each solution for 30 min. Then, the excess liquid was removed with filter paper and the grid was placed on top of a water drop for 30 s and finally negatively stained for 2 min with 20 µL of 2% ura- nyl acetate. Samples were observed using a JEM 1010 transmission electron microscope (JEOL Ltd., Japan). Images were acquired using a CCD Orius camera (Gatan, Inc., USA). 3.2 Results According to ThT fluorescence assays (FIG.2(A)), AID-X-2020 is a strong inhibitor of the aggrega- tion of Aβ40. Even at 10 nM, well below its IC 50 in P. falciparum cultures, AID-X-2020 prevents Aβ40 fibrillogenesis to a large extent. Therefore, inhibition of protein aggregation might be the main mechanism responsible for the antimalarial activity of this compound. To discard the possibility that the decrease in ThT fluorescence observed in Aβ40 aggregation as- says resulted from a steric hindrance imposed to ThT binding of amyloid fibrils by the presence of AID-X-2020, TEM was used to examine Aβ40 samples treated with AID-X-2020 (FIG.2(B)). TEM images showed that the amyloid fibril aggregates of AID-X-2020-containing samples were smaller and more fragmented than those present in control untreated Aβ40, supporting the role of this compound as a protein aggregation inhibitor. EXAMPLE 4: Study of the correlation between the in vitro antimalarial activity of AID-X-2020 and the disruption of protein homeostasis in the treated parasites To explore if there was a correlation between the observed in vitro antimalarial activity of AID-X- 2020 and a disruption of protein homeostasis in the treated parasites, dot blots and Western blots of protein extracts of AID-X-2020-treated P. falciparum cultures were performed, where the pres- ence of ubiquitinated proteins and of amyloid fibrils was examined. 4.1 Materials and methods Cultures of the P. falciparum 3D7 strain were sorbitol synchronized in ring stages, and after 24 h were treated for 90 min with AID-X-2020 concentrations ranging from 33 nM to 33 µM. After that time, cultures were spun down and pellets were washed once with ice-cold PBS supplemented with EDTA-free protease inhibitor cocktail (PIC, Roche; 1 PIC tablet/10 mL PBS). For anti-ubiquitin Western blots, PBS was also supplemented with 20 mM N-ethylmaleimide. Once washed, parasite pellets were treated with 0.15% saponin at 4 °C for 15 min and washed by centrifugation (10,000× g, 15 min, 4 °C) with appropriately supplemented PBS until no hemoglobin was observed in the supernatant. Protein extracts were quantified with the bicinchoninic acid assay (ThermoFisher Sci- entific), following the manufacturer’s instructions. For dot blots, 4-μL drops of saponin extract containing 0.5 or 1 mg/mL protein were spotted on a nitrocellulose membrane. Once protein extracts were completely absorbed by the membranes, these were incubated for 3 h in blocking solution: 5% milk powder in TBS-Tween (200 mM tris- base, 1.5 M NaCl, 0.1% Tween-20). The blocked membranes were washed 3 × 5 min with TBS- Tween and incubated overnight at 4 °C with rabbit polyclonal anti-amyloid fibrils OC antibody (Milli- pore) diluted 1:500 in blocking solution or mouse monoclonal anti-spectrin α/β (Sigma) 1:10,000 in TBS-Tween. For Western blots, 15 μg of saponin-extracted proteins were incubated for 5 min at 95 °C diluted in Laemmli solution (0.14 M SDS, 0.125 M tris-HCl, pH 6.8, 20% glycerol, 10% 2-mercaptoethanol, 3 mM bromophenol blue) and resolved by SDS-PAGE in 12% bis-tris acrylamide (Bio-Rad) gels run at 80 V until samples entered the resolving gel and at 120 V afterwards. Proteins were transferred from the gel to polyvinylidene difluoride membranes activated with methanol 100%. After transfer- ence, membranes were blocked with blocking solution for 1 h at room temperature, washed 3 × 5 min with TBS-Tween and probed overnight at 4 °C with rabbit polyclonal anti-ubiquitin antibody (Cell Signaling Technology) diluted 1:1,000 in blocking solution, or mouse monoclonal anti-spectrin α/β (Sigma) 1:10,000 in TBS-Tween. Then, membranes were washed 5 times with TBS-Tween and incubated for 1 h with either goat anti-rabbit (Upstate) or goat anti-mouse (Amersham Life Science, Inc.) IgG-horseradish peroxidase conjugate antibody diluted 1:10,000 in TBS-Tween. After probing with secondary antibodies, for both dot blot and Western blot membranes, four washes with TBS- Tween and one last wash with TBS were done and peroxidase substrate (ECL Prime Western Blot- ting Detection Reagent, Amersham Life Science, Inc.) was poured on the membrane and chemilu- minescent signal was measured in a LAS 4000 reader (ImageQuant) at different exposure times. 4.2 Results At the concentration of 90 nM, close to its IC 50 in vitro, AID-X-2020 treatment of P. falciparum cul- tures led to a reduction in ubiquitinated proteins along time (FIG.3(A)), consistent with an inhibitory effect on protein aggregation. Exposure for only 90 min to >3 µM AID-X-2020 inhibited amyloid fibril formation (FIG. 3(D)) but resulted in a concomitant increase in ubiquitinated proteins (FIG. 3(C)). These results suggested a causal effect between decreasing protein aggregation and a deleterious effect on the parasite ultimately leading to a rapid generalized deregulation of proteostasis. EXAMPLE 5: Assay on the effect of the combination of AID-X-2020 and artemisinin Artemisinin, one of the most potent antimalarials in use, has been described to cause protein dam- age/unfolding and to inhibit folding of newly synthesized proteins, likely inducing protein aggrega- tion. As shown in the previous examples, AID-X-2020 appears to inhibit protein aggregation, the opposite action of artemisinin. Thus, this study was performed to confirm the capacity of AID-X- 2020 to inhibit protein aggregation. 5.1 Materials and methods For assays of AID-X-2020 and artemisinin, serial dilutions of both compounds were prepared at different concentration ratios (1:0, 0:1, 1:1, 1:2, 2:1, 1:5 and 5:1). To assess the synergistic effect of AID-X-2020 and artemisinin, IC 50 values for each individual compound in the mixtures were calculated as described above and plotted in an isobologram (“x” value = AID-X-2020 IC 50 and “y” value = artemisinin IC 50 ). Fractional inhibitory concentration (FIC) values were calculated by dividing the IC 50 of one of the compounds in the mixture by the IC 50 of the same compound in the 1:0 or 0:1 ratio mixture. 5.2 Results When P. falciparum cultures were treated with artemisinin and AID-X-2020 combined in different ratios, the resulting ∑FIC values were always higher than 1.5 (FIG.3(E)), which indicates an an- tagonistic action of both drugs. Thus, parasite viability was significantly higher than expected if the activities of both drugs were additive, thus reinforcing the hypothesis that AID-X-2020 has protein aggregation inhibitory activity that antagonizes the antimalarial effect of artemisinin, and vice versa. EXAMPLE 6: Effect of AID-X-2020 on the pathogen’s insect stage (the gametocyte to ooki- nete transition) Because Plasmodium is known to have protein aggregates in the mosquito stages of Plasmodium, the effect of AID-X-2020 was tested on one of the key steps of the pathogen’s development in the insect, namely the gametocyte to ookinete transition. Gametocytes are the sole stage of malaria parasites present in the blood circulation capable of transmitting the infection to the mosquito vec- tor following their ingestion by a blood-feeding Anopheles female. Transmission-blocking strategies are therefore one of the main approaches being explored to disrupt the life cycle of Plasmodium, but active drugs at this critical step are scarce. 6.1 Materials and methods 6.1.1 Assay of gametocyte to ookinete transition inhibition Eight days before ookinete production, 200 µL of Plasmodium berghei CTRP-GFP (which express- es GFP when reaching ookinete stage) (Vlachou, D. et al., 2004) in cryopreservation solution (RBC pellet:RPMI:30% glycerol in water, 1:1:2) was administered i.p. to a BALB/c mouse. Four days later this mouse was the donor to infect i.p. with 5×10 7 parasitized red blood cells in 200 µL of PBS three mice that one hour before the infection had been pretreated i.p. with phenylhydrazine (120 µL of a 10 mg/mL solution in PBS). For ookinete production, up to 1 mL of blood carrying gametocytes was collected from each animal by intracardiac puncture and diluted in 30 mL of ookinete medium: 10.4 g/L of RPMI supplemented with 2% w/v NaHCO3, 0.05% w/v hypoxanthine, 0.02% w/v xanthurenic acid, 50 U/mL penicillin and 50 µg/mL streptomycin (Invitrogen), 20% heat-inactivated fetal bovine serum (FBS, Invitrogen), 25 mM HEPES, pH 7.4. The effect of AID-X-2020 and DONE3TCl on gametocyte to ookinete conversion was assessed in three independent replicas by plating 120 µL of each culture mixed with growing concentrations of the compounds in 96-well plates and incubated for 24 h at 21 °C with orbital shaking at 50 rpm.24 h later, samples were diluted 1:100 in PBS and analyzed in a LSRFortessa TM flow cytometer (BD Biosciences) set up with the 5 lasers, 20 parameters standard configuration. The GFP positive cell population was selected and counted using 488 nm laser excitation and a 525/40 nm emission col- lection filter. BD FACSDiva software (BD biosciences) was used in data collection, and Flowing Software 2.5.1 (Turku Centre for Biotechnology) was used for analysis. 6.2 Results In vitro inhibition assays of the gametocyte to ookinete transition in the murine malaria parasite Plasmodium berghei indicated that ca. 0.5 µM AID-X-2020 abolished ookinete production in this model (FIG.4(A)). On the other hand, the protein aggregation inhibitor DONE3TCl, which showed a potent antimalarial activity in in vitro P. falciparum cultures, with an IC 50 of ca.80 nM (Table 2), did not have an observable effect on ookinete development up to a concentration of 2 µM (FIG. 4(B)). AID-X-2020 can then be used to arrest the life cycle of malaria parasites in the mosquito host. EXAMPLE 7: In vivo toxicity assays of AID-X-2020 and DONE3TCl 7.1 Materials and methods BALB/c female and male mice (Janvier Laboratories) were maintained with unlimited access to food and water under standard environmental conditions (20-24 °C and 12/12 h light/dark cycle). Three 100 µL doses of an AID-X-2020 solution prepared to administer 0.0959, 0.3069 and 0.9822 mg/kg were tested. First, the lowest dose was intravenously injected to one female and one male mice. An oxygen stream of 4% isoflurane was used to anesthetize the mice, which were then main- tained during the whole procedure (less than 3 minutes) with 2.5% isoflurane. After the administration, mice were observed and different parameters related to their behavior (lethargy, motility alterations, seizures, coma, automutilation, aggressiveness, vocalizations, ste- reotyped movements) and physical conditions (pain, respiratory disturbances, tachycardia or brad- ycardia, dehydration, hair loss, body weight loss, dermatitis, bad hygiene, pruritus, tearing) were followed. If after 48 h no deleterious effects were observed, the following dose was administered to two other male and female mice. All mice were observed for 14 days after treatment in order to de- tect long-term side effects. The animal care and protocol followed here adhered to the specific national and international guidelines stated in the Spanish Royal Decree 53/2013, which is based on the European regulation 2010/63/UE. 7.2 Results In vivo toxicity assays in mice indicated that AID-X-2020 and DONE3TCl started inducing adverse effects in female mice at 10 mg/kg. In male mice toxicity started being observed at 17 and 3 mg/kg for AID-X-2020 and DONE3TCl, respectively.10 mg/kg then is a tentative maximal dose of refer- ence for future preclinical assays. In human umbilical vein endothelial cell cultures, the AID-X-2020 concentration required for the reduction of cell viability by 50% (CC50) was determined to be 3.4 μM, which resulted in a therapeu- tic index (CC 50 /IC 50 ) of 37.8. Despite this relatively high in vitro toxicity, in vivo toxicity assays indi- cated that AID-X-2020 starts inducing adverse effects in female and male mice at 10 and 17 mg/kg respectively. EXAMPLE 8: Inhibition effect of other compounds of formula (I) on P. falciparum in vitro Eighteen compounds were synthesized, which are representative of compounds included in formu- la (I) other than AID-X-2020, and a study of the antimalarial effect of seventeen of these com- pounds was performed. The antimalarial activity of these compounds was then compared to the antimalarial activity of AID-X-2020 (IC 50 = 0.09 µM). 8.1 Materials and Methods 8.1.1 General aspects of synthesis procedures Steps and conditions referring to general aspects of the synthesis procedures were performed as described in section 1.1.1 of EXAMPLE 1. 8.1.2 Synthesis of 1,1'-(decane-1,10-diyl)bis{4-[(E)-4-(dimethylamino)styryl]-3 -methylpyridin-1-ium} dibromide (EMA357) Compound EMA357 was prepared following the first steps and conditions as described for the syn- thesis of compound AID-X-2020 (section 1.1.2 of EXAMPLE 1 – 1 st paragraph) until 1,1'-(decane- 1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide was obtained. A solution of 1,1'-(decane-1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (257 mg, 0.50 mmol) and 4-(dimethylamino)benzaldehyde (149 mg, 1.00 mmol) in n-butanol (2.5 mL) was treated with six drops of piperidine and the reaction mixture was stirred under reflux for 16 h, and then concen- trated under reduced pressure. The resulting black sticky residue was purified by automatic flash column chromatography (CH 2 Cl 2 / 7N ammonia solution in MeOH 9.5:0.5 to 8:2), to provide 1,1'- (decane-1,10-diyl)bis{4-[(E)-4-(dimethylamino)styryl]-3-meth ylpyridin-1-ium} dibromide (127 mg, 33%) as a red oil; IR (ATR) ν: 3390, 2925, 2852, 1640, 1573, 1525, 1478, 1435, 1364, 1310, 1218, 1185, 1170, 1128, 944, 814 cm -1 ; 1 H NMR (400 MHz, CD 3 OD) δ: 1.29-1.43 (m, 12H), 1.97 (m, 4H), 2.55 (s, 6H), 3.06 (s, 12H), 4.40 (t, J = 7.6 Hz, 4H), 6.79 (d, J = 9.2 Hz, 4H), 7.14 (d, J = 16.0 Hz, 2H), 7.64 (d, J = 9.2 Hz, 4H), 7.81 (d, J = 16.0 Hz, 2H), 8.18 (d, J = 6.8 Hz, 2H), 8.47 (br d, J = 6.8 Hz, 2H), 8.56 (br s, 2H); HRMS-ESI+ m/z calculated for [C 42 H 56 N 4 ] 2+ /2: 308.2247, found: 308.2241. 8.1.3 Synthesis of 1,1'-(decane-1,10-diyl)bis{4-{(E)-4-[(2-cyanoethyl)(methyl)a mino]styryl}-3- methylpyridin-1-ium} dibromide (EMA359) Compound EMA359 was prepared following the first steps and conditions as described for the syn- thesis of compound AID-X-2020 (section 1.1.2 of EXAMPLE 1 – 1 st paragraph) until 1,1'-(decane- 1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide was obtained. A solution of 1,1'-(decane-1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (257 mg, 0.50 mmol) and 3-[(4-formylphenyl)(methyl)amino]propanenitrile (188 mg, 1.00 mmol) in n-butanol (2.5 mL) was treated with six drops of piperidine and the reaction mixture was stirred under reflux for 16 h, and then concentrated under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH 2 Cl 2 / 7N ammonia solution in MeOH 9:1), to provide 1,1'-(decane-1,10-diyl)bis{4-{(E)-4-[(2-cyanoethyl)(methyl)a mino]styryl}-3-methylpyridin-1-ium} di- bromide (204 mg, 48%) as a red solid; mp: 130-131 ºC; IR (ATR) ν: 3377, 2925, 2854, 1633, 1578, 1523, 1478, 1383, 1311, 1218, 1184, 1117, 959, 808 cm -1 ; 1 H NMR (400 MHz, CD3OD) δ: 1.31- 1.43 (m, 12H), 1.97 (m, 4H), 2.56 (s, 6H), 2.76 (t, J = 6.8 Hz, 4H), 3.13 (s, 6H), 3.83 (t, J = 6.8 Hz, 4H), 4.41 (t, J = 7.6 Hz, 4H), 6.87 (d, J = 8.8 Hz, 4H), 7.20 (d, J = 16.0 Hz, 2H), 7.68 (d, J = 8.8 Hz, 4H), 7.82 (d, J = 16.0 Hz, 2H), 8.20 (d, J = 6.8 Hz, 2H), 8.50 (br d, J = 6.8 Hz, 2H), 8.59 (br s, 2H); HRMS-ESI+ m/z calculated for [C 46 H 58 N 6 ] 2+ /2: 347.2356, found: 347.2348. 8.1.4 Synthesis of 1,1'-(decane-1,10-diyl)bis{4-[(E)-4-(diphenylamino)styryl]-3 -methylpyridin-1-ium} dibromide (EMA366) Compound EMA366 was prepared following the first steps and conditions as described for the syn- thesis of compound AID-X-2020 (section 1.1.2 of EXAMPLE 1 – 1 st paragraph) until 1,1'-(decane- 1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide was obtained. A solution of 1,1'-(decane-1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (257 mg, 0.50 mmol) and 4-(diphenylamino)benzaldehyde (273 mg, 1.00 mmol) in n-butanol (2.5 mL) was treated with six drops of piperidine and the reaction mixture was stirred under reflux for 16 h, and then concen- trated under reduced pressure. The resulting black oily residue was purified by automatic flash col- umn chromatography (CH 2 Cl 2 / 7N ammonia solution in MeOH 9:1 to 85:15), to provide 1,1'- (decane-1,10-diyl)bis{4-[(E)-4-(diphenylamino)styryl]-3-meth ylpyridin-1-ium} dibromide (236 mg, 46%) as a red solid; mp: 163-165 ºC; IR (ATR) ν: 3352, 3186, 2929, 2854, 1658, 1581, 1510, 1485, 1388, 1318, 1285, 1222, 1192, 1178, 1138, 973, 820, 757, 697, 571 cm -1 ; 1 H NMR (500 MHz, CD3OD) δ: 1.30-1.43 (m, 12H), 1.98 (tt, J = J’ = 7.5 Hz, 4H), 2.57 (s, 6H), 4.45 (t, J = 7.5 Hz, 4H), 7.00 (d, J = 8.5 Hz, 4H), 7.11-7.17 (m, 12H), 7.29 (d, J = 16.0 Hz, 2H), 7.34 (ddm, J = 8.5 Hz, J’ = 7.5 Hz, 8H), 7.63 (d, J = 8.5 Hz, 4H), 7.81 (d, J = 16.0 Hz, 2H), 8.24 (d, J = 6.5 Hz, 2H), 8.57 (dd, J = 6.5 Hz, J’ = 1.5 Hz, 2H), 8.65 (s, 2H); HRMS-ESI+ m/z calculated for [C 62 H 64 N 4 ] 2+ /2: 432.2560, found: 432.2565. 8.1.5 Synthesis of 1,1'-(decane-1,10-diyl)bis{3-methyl-4-[(E)-4-(pyrrolidin-1-y l)styryl]pyridin-1-ium} dibromide (EMA368) Compound EMA368 was prepared following the first steps and conditions as described for the syn- thesis of compound AID-X-2020 (section 1.1.2 of EXAMPLE 1 – 1 st paragraph) until 1,1'-(decane- 1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide was obtained. A solution of 1,1'-(decane-1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (257 mg, 0.50 mmol) and 4-(pyrrolidin-1-yl)benzaldehyde (175 mg, 1.00 mmol) in n-butanol (2.5 mL) was treated with six drops of piperidine and the reaction mixture was stirred under reflux for 22 h, and then concentrat- ed under reduced pressure. The resulting red oily residue was purified by automatic flash column chromatography (CH 2 Cl 2 / MeOH 10:0 to 9.3:0.7), to provide 1,1'-(decane-1,10-diyl)bis{3-methyl-4- [(E)-4-(pyrrolidin-1-yl)styryl]pyridin-1-ium} dibromide (233 mg, 56%) as a red solid; mp: 167-168 ºC; IR (ATR) ν: 3391, 2928, 2850, 1641, 1617, 1571, 1523, 1478, 1387, 1303, 1218, 1177, 1136, 961, 807 cm -1 ; 1 H NMR (400 MHz, DMSO-d 6 ) δ: 1.21-1.31 (m, 12H), 1.86 (m, 4H), 1.98 (t, J = 5.6 Hz, 8H), 2.48 (s, 6H), 3.01 (t J = 5.6 Hz, 8H), 4.36 (t, J = 7.6 Hz, 4H), 6.62 (d, J = 8.8 Hz, 4H), 7.09 (d, J = 16.0 Hz, 2H), 7.66 (d, J = 8.8 Hz, 4H), 7.89 (d, J = 16.0 Hz, 2H), 8.26 (d, J = 6.8 Hz, 2H), 8.65 (br d, J = 6.8 Hz, 2H), 8.72 (br s, 2H); HRMS-ESI+ m/z calculated for [C 46 H 60 N 4 ] 2+ /2: 334.2404, found: 334.2410. 8.1.6 Synthesis of 1,1'-(decane-1,10-diyl)bis{3-methyl-4-[(E)-4-morpholinostyry l]pyridin-1-ium} di- bromide (EMA377) Compound EMA377 was prepared following the first steps and conditions as described for the syn- thesis of compound AID-X-2020 (section 1.1.2 of EXAMPLE 1 – 1 st paragraph) until 1,1'-(decane- 1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide was obtained. A solution of 1,1'-(decane-1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (514 mg, 1.00 mmol) and 4-morpholinobenzaldehyde (383 mg, 2.00 mmol) in n-butanol (5.0 mL) was treated with six drops of piperidine and the reaction mixture was stirred under reflux for 4 h, and then concentrated under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH 2 Cl 2 / MeOH 10:0 to 9:1), to provide 1,1'-(decane-1,10-diyl)bis{3-methyl-4-[(E)- 4-morpholinostyryl]pyridin-1-ium} dibromide (281 mg, 33%) as a red solid; mp: 153-155 ºC; IR (ATR) ν: 3390, 2925, 2851, 1642, 1580, 1519, 1478, 1447, 1428, 1381, 1358, 1307, 1247, 1222, 1187, 1134, 1110, 1049, 925, 810, 635, 604, 572 cm -1 ; 1 H NMR (400 MHz, CD3OD) δ: 1.30-1.42 (m, 12H), 1.98 (tt, J = J’ = 7.6 Hz, 4H), 2.57 (s, 6H), superimposed in part with the solvent signal 3.30 (t, J = 4.8 Hz, 8H), 3.83 (t, J = 4.8 Hz, 8H), 4.43 (t, J = 7.6 Hz, 4H), 7.02 (d, J = 9.2 Hz, 4H), 7.25 (d, J = 16.0 Hz, 2H), 7.69 (d, J = 9.2 Hz, 4H), 7.81 (d, J = 16.0 Hz, 2H), 8.23 (d, J = 6.8 Hz, 2H), 8.54 (dd, J = 6.8 Hz, J’ = 1.6 Hz, 2H), 8.63 (br s, 2H); HRMS-ESI+ m/z calculated for [C 46 H 60 N 4 O 2 ] 2+ /2: 350.2353, found: 350.2361. 8.1.7 Synthesis of 1,1'-(butane-1,4-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-m ethylpyridin-1-ium} dibromide (PRC-5) A mixture of 1,4-dibromobutane (0.23 mL, 416 mg, 1.93 mmol) and 3,4-dimethylpyridine (0.49 mL, 467 mg, 4.36 mmol) was heated to 120 °C for 3 h. Then, isopropanol (2 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room temperature the resulting brown residue was washed with ice-cold Et 2 O (15 mL) and the remaining beige solid was dried in vacuo, taken up in MeOH (5 mL) and treated with cold Et 2 O (2 × 15 mL), drawing off the liquids. After drying the residue in vacuo, 1,1'-(butane-1,4-diyl)bis(3,4- dimethylpyridin-1-ium) dibromide (757 mg, 88%) was obtained as a beige solid; mp: 109-110 ºC; IR (ATR) ν: 3431, 3360, 3041, 1641, 1621, 1512, 1472, 1460, 1234, 1149, 1027, 847, 706 cm -1 ; 1 H NMR (400 MHz, CD3OD) δ: 2.10 (tm, J = 7.2 Hz, 4H), 2.48 (s, 6H), 2.59 (s, 6H), 4.62 (br t, J = 7.2 Hz, 4H), 7.87 (d, J = 6.4 Hz, 2H), 8.71 (dd, J = 6.4 Hz, J’ = 1.6 Hz, 2H), 8.81 (br s, 2H); HRMS- ESI+ m/z calculated for [C 18 H 26 N 2 ] 2+ /2: 135.1043, found: 135.1051. A solution of 1,1'-(butane-1,4-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (669 mg, 1.55 mmol) and 4-(diethylamino)benzaldehyde (604 mg, 3.41 mmol) in n-butanol (8 mL) was treated with twelve drops of piperidine and the reaction mixture was stirred under reflux for 4 h, and then con- centrated under reduced pressure. The resulting oily residue was purified twice by automatic flash column chromatography (CH 2 Cl 2 / 7N ammonia solution in MeOH 95:5 to 85:15), to provide 1,1'- (butane-1,4-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methyl pyridin-1-ium} dibromide (511 mg, 44%) as a red solid; mp: 275-276 ºC; IR (ATR) ν: 3406, 2964, 1641, 1577, 1522, 1484, 1467, 1434, 1402, 1372, 1350, 1307, 1262, 1220, 1191, 1132, 1067, 975, 810 cm -1 ; 1 H NMR (400 MHz, CD3OD) δ: 1.21 (t, J = 7.2 Hz, 12H), 2.07 (m, 4H), 2.54 (s, 6H), 3.49 (q, J = 7.2 Hz, 8H), 4.48 (m, 4H), 6.76 (d, J = 9.2 Hz, 4H), 7.10 (d, J = 16.0 Hz, 2H), 7.61 (d, J = 9.2 Hz, 4H), 7.81 (d, J = 16.0 Hz, 2H), 8.17 (d, J = 6.8 Hz, 2H), 8.47 (dd, J = 6.8 Hz, J’ = 1.6 Hz, 2H), 8.56 (br s, 2H); HRMS- ESI+ m/z calculated for [C 40 H 52 N 4 ] 2+ /2: 294.2091, found: 294.2098. 8.1.8 Synthesis of 1,1'-(octane-1,8-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-m ethylpyridin-1-ium} dibromide (PRC-8) A mixture of 1,8-dibromooctane (0.37 mL, 546 mg, 2.00 mmol) and 3,4-dimethylpyridine (0.49 mL, 467 mg, 4.36 mmol) was heated to 120 °C for 4 h. Then, isopropanol (2 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room temperature the resulting brown residue was washed with ice-cold Et 2 O (15 mL) and the remaining brown sticky oil was dried in vacuo, taken up in MeOH (5 mL) and treated with cold Et 2 O (2 × 15 mL), drawing off the liquids. After drying the residue in vacuo, 1,1'-(octane-1,8-diyl)bis(3,4- dimethylpyridin-1-ium) dibromide (510 mg, 52%) was obtained as a beige solid; mp: 183-184 ºC; IR (ATR) ν: 3459, 3026, 2925, 2853, 1639, 1511, 1484, 1460, 1444, 1393, 1337, 1230, 1141, 1020, 951, 839, 711 cm -1 ; 1 H NMR (400 MHz, CD3OD) δ: 1.41 (m, 8H), 2.00 (tt, J = 7.6 Hz, J’ = 7.2 Hz, 4H), 2.48 (s, 6H), 2.59 (s, 6H), 4.53 (t, J = 7.6 Hz, 4H), 7.86 (d, J = 6.4 Hz, 2H), 8.68 (dd, J = 6.4 Hz, J’ = 1.6 Hz, 2H), 8.77 (br s, 2H); HRMS-ESI+ m/z calculated for [C 22 H 34 N 2 ] 2+ /2: 163.1356, found: 163.1358. A solution of 1,1'-(octane-1,8-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (410 mg, 0.84 mmol) and 4-(diethylamino)benzaldehyde (328 mg, 1.85 mmol) in n-butanol (4.5 mL) was treated with six drops of piperidine and the reaction mixture was stirred under reflux for 17 h, and then concentrat- ed under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH 2 Cl 2 / MeOH 95:5 to 85:15), to provide 1,1'-(octane-1,8-diyl)bis{4-[(E)-4- (diethylamino)styryl]-3-methylpyridin-1-ium} dibromide (230 mg, 34%) as a red solid; mp: 235-236 ºC; IR (ATR) ν: 3393, 2970, 2928, 1641, 1570, 1521, 1478, 1404, 1350, 1310, 1259, 1219, 1185, 1128, 1076, 960, 807, 702 cm -1 ; 1 H NMR (400 MHz, CD3OD) δ: 1.21 (t, J = 7.2 Hz, 12H), 1.41 (m, 8H), 1.87 (tt, J = J’ = 7.2 Hz, 4H), 2.54 (s, 6H), 3.48 (q, J = 7.2 Hz, 8H), 4.40 (t, J = 7.2 Hz, 4H), 6.76 (d, J = 8.8 Hz, 4H), 7.10 (d, J = 16.0 Hz, 2H), 7.61 (d, J = 8.8 Hz, 4H), 7.79 (d, J = 16.0 Hz, 2H), 8.16 (d, J = 6.8 Hz, 2H), 8.46 (dd, J = 6.8 Hz, J’ = 1.6 Hz, 2H), 8.55 (br s, 2H); HRMS-ESI+ m/z calculated for [C 44 H 60 N 4 ] 2+ /2: 322.2404, found: 322.2417. 8.1.9 Synthesis of 1,1'-(nonane-1,9-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-m ethylpyridin-1-ium} dibromide (PRC-25) A mixture of 1,9-dibromononane (0.41 mL, 577 mg , 2.02 mmol) and 3,4-dimethylpyridine (0.49 mL, 467 mg, 4.36 mmol) was heated to 120 °C for 3 h. Then, isopropanol (2 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room temperature, the resulting brown residue was washed with ice-cold Et 2 O (2 × 20 mL) and the re- maining brown sticky oil was dried in vacuo, taken up in MeOH (1 mL) and treated with cold Et 2 O (2 × 20 mL), drawing off the liquids. After drying the residue in vacuum, 1,1'-(nonane-1,9- diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (665 mg, 66%) was obtained as a brown oil that so- lidified on standing; mp: 88-90 ºC; IR (ATR) ν: 3435, 2933, 2852, 1638, 1505, 1469, 1335, 1228, 1152, 1138, 1023, 834, 754, 729, 707 cm -1 ; 1 H NMR (400 MHz, DMSO-d 6 ) δ: 1.17-1.31 (m, 10H), 1.88 (tt, J = J’ = 7.2 Hz, 4H), 2.39 (s, 6H), 2.51 (s, 6H), 4.49 (t, J = 7.2 Hz, 4H), 7.94 (d, J = 6.4 Hz, 2H), 8.84 (br d, J = 6.4 Hz, 2H), 8.94 (br s, 2H); HRMS-ESI+ m/z calculated for [C 23 H 36 N 2 ] 2+ /2: 170.1434, found 170.1435. A solution of 1,1'-(nonane-1,9-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (250 mg, 0.50 mmol) and 4-(diethylamino)benzaldehyde (195 mg, 1.10 mmol) in n-butanol (2.5 mL) was treated with four drops of piperidine and the reaction mixture was stirred under reflux for 4 h, and then concentrated under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH 2 Cl 2 / MeOH 95:5 to 85:15). Then fractions containing the desired product were evaporated and the residue was dissolved in hot isopropanol (15 mL) and after addition of Et 2 O (30 mL) and drawing off the liquids, 1,1'-(nonane-1,9-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3- methylpyridin-1-ium} dibromide (193 mg, 47%) was obtained as a red solid; mp: 138-142 ºC; IR (ATR) ν: 3381, 2920, 2852, 1642, 1567, 1519, 1482, 1402, 1347, 1308, 1258, 1217, 1185, 1126, 1070, 954, 807 cm -1 ; 1 H NMR (400 MHz, DMSO-d 6 ) δ: 1.13 (t, J = 7.2 Hz, 12H), 1.22-1.31 (m, 10H), 1.87 (tt, J = J’ = 7.2 Hz, 4H), 2.48 (s, 6H), 3.43 (q, J = 7.2 Hz, 8H), 4.36 (t, J = 7.2 Hz, 4H), 6.74 (d, J = 9.2 Hz, 4H), 7.08 (d, J = 16.0 Hz, 2H), 7.64 (d, J = 9.2 Hz, 4H), 7.87 (d, J = 16.0 Hz, 2H), 8.26 (d, J = 6.8 Hz, 2H), 8.66 (dd, J = 6.8 Hz, J’ = 1.6 Hz, 2H), 8.73 (br s, 2H); HRMS-ESI+ m/z calculated for [C 45 H 62 N 4 ] 2+ /2: 329.2482, found: 329.2485. 8.1.10 Synthesis of 1,1'-(undecane-1,11-diyl)bis{4-[(E)-4-(diethylamino)styryl]- 3-methylpyridin-1- ium} dibromide (PRC-14) A mixture of 1,11-dibromoundecane (0.47 mL, 627 mg, 2.00 mmol) and 3,4-dimethylpyridine (0.49 mL, 467 mg, 4.36 mmol) was heated to 120 °C for 3 h. Then, isopropanol (2 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room temperature, the resulting brown residue was washed with ice-cold Et 2 O (2 × 20 mL) and the re- maining brown sticky oil was dried in vacuum, taken up in CH 2 Cl 2 (1 mL) and treated with cold Et 2 O (2 × 20 mL), drawing off the liquids. After drying the residue in vacuum, 1,1'-(undecane-1,11- diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (0.98 g, 93%) was obtained as a brown oil; IR (ATR) ν: 3403, 3020, 2924, 2853, 1638, 1510, 1481, 1466, 1389, 1229, 1146, 1026, 843, 707 cm -1 ; 1 H NMR (400 MHz, CD3OD) δ: 1.29-1.42 (m, 14H), 1.99 (tt, J = J’ = 7.6 Hz, 4H), 2.48 (s, 6H), 2.59 (s, 6H), 4.52 (t, J = 7.6 Hz, 4H), 7.86 (d, J = 6.4 Hz, 2H), 8.67 (dd, J = 6.4 Hz, J’ = 1.6 Hz, 2H), 8.76 (br s, 2H); HRMS-ESI+ m/z calculated for [C 25 H 40 N 2 ] 2+ /2: 184.1590, found: 184.1598. A solution of 1,1'-(undecane-1,11-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (396 mg, 0.75 mmol) and 4-(diethylamino)benzaldehyde (293 mg, 1.65 mmol) in n-butanol (3.75 mL) was treated with six drops of piperidine and the reaction mixture was stirred under reflux for 4 h, and then con- centrated under reduced pressure, to afford 1,1'-(decane-1,10-diyl)bis{4-[(E)-4- (diethylamino)styryl]-3-methylpyridin-1-ium} dibromide (180 mg, 28%) as a red solid, which was used in the following step without further purification; mp: 81-82 ºC; IR (ATR) ν: 3389, 2924, 2847, 1641, 1572, 1522, 1478, 1405, 1351, 1310, 1259, 1217, 1186, 1157, 1133, 1075, 1008, 959, 808, 783, 701 cm -1 ; 1 H NMR (400 MHz, DMSO-d 6 ) δ: 1.12 (t, J = 6.8 Hz, 12H), 1.20-1.31 (m, 14H), 1.86 (tt, J = J’ = 7.2 Hz, 4H), 2.48 (s, 6H), 3.43 (q, J = 6.8 Hz, 8H), 4.36 (t, J = 7.2 Hz, 4H), 6.74 (d, J = 9.2 Hz, 4H), 7.08 (d, J = 16.0 Hz, 2H), 7.64 (d, J = 9.2 Hz, 4H), 7.88 (d, J = 16.0 Hz, 2H), 8.26 (d, J = 6.8 Hz, 2H), 8.65 (br d, J = 6.8 Hz, 2H), 8.73 (br s, 2H); HRMS-ESI+ m/z calculated for [C 47 H 66 N 4 ] 2+ /2: 343.2638, found: 343.2640. 8.1.11 Synthesis of 1,1'-(dodecane-1,12-diyl)bis{4-[(E)-4-(diethylamino)styryl]- 3-methylpyridin-1- ium} dibromide (PRC-15) A mixture of 1,12-dibromododecane (656 mg, 2.00 mmol) and 3,4-dimethylpyridine (0.49 mL, 467 mg, 4.36 mmol) was heated to 120 °C for 3 h. Then, isopropanol (2 mL) was added and the reac- tion mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room tem- perature, the resulting brown residue was washed with ice-cold Et 2 O (2 × 20 mL) and the remaining brown sticky oil was dried in vacuum, taken up in CH 2 Cl 2 (1 mL) and treated with cold Et 2 O (2 × 20 mL), drawing off the liquids. After drying the residue in vacuum, 1,1'-(dodecane-1,12-diyl)bis(3,4- dimethylpyridin-1-ium) dibromide (850 mg, 78%) was obtained as a brown oil that solidified on standing; mp: 82-83 ºC; IR (ATR) ν: 3438, 3373, 2994, 2923, 2853, 1636, 1516, 1466, 1387, 1332, 1228, 1147, 1027, 856, 837, 721, 709, 619, 592 cm -1 ; 1 H NMR (400 MHz, CD3OD) δ: 1.28-1.41 (m, 16H), 1.99 (tt, J = J’ = 7.6 Hz, 4H), 2.48 (s, 6H), 2.59 (s, 6H), 4.52 (t, J = 7.6 Hz, 4H), 7.86 (d, J = 6.4 Hz, 2H), 8.68 (dd, J = 6.4 Hz, J’ = 1.6 Hz, 2H), 8.77 (br s, 2H); HRMS-ESI+ m/z calculated for [C 26 H 42 N 2 ] 2+ /2: 191.1669, found: 191.1675. A solution of 1,1'-(dodecane-1,12-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (379 mg, 0.70 mmol) and 4-(diethylamino)benzaldehyde (273 mg, 1.54 mmol) in n-butanol (3.5 mL) was treated with six drops of piperidine and the reaction mixture was stirred under reflux for 4.5 h, and then concentrated under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH 2 Cl 2 / MeOH 95:5), to provide 1,1'-(dodecane-1,12-diyl)bis{4-[(E)- 4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide (116 mg, 19%) as a red solid; mp: 112- 114 ºC; IR (ATR) ν: 3400, 2971, 2927, 2852, 1641, 1574, 1519, 1477, 1435, 1403, 1348, 1312, 1259, 1219, 1188, 1157, 1126, 1077, 961, 824, 789, 571 cm -1 ; 1 H NMR (400 MHz, DMSO-d 6 ) δ: 1.13 (t, J = 7.2 Hz, 12H), 1.19-1.32 (m, 16H), 1.88 (tt, J = J’ = 7.2 Hz, 4H), 2.48 (s, 6H), 3.43 (q, J = 7.2 Hz, 8H), 4.36 (t, J = 7.2 Hz, 4H), 6.74 (d, J = 8.8 Hz, 4H), 7.08 (d, J = 16.0 Hz, 2H), 7.64 (d, J = 8.8 Hz, 4H), 7.88 (d, J = 16.0 Hz, 2H), 8.26 (d, J = 6.8 Hz, 2H), 8.66 (br d, J = 6.8 Hz, 2H), 8.73 (br s, 2H); HRMS-ESI+ m/z calculated for [C 48 H 68 N 4 ] 2+ /2: 350.2717, found: 350.2721. 8.1.12 Synthesis of 1,1'-(3,6-dioxaoctane-1,8-diyl)bis{4-[(E)-4-(diethylamino)st yryl]-3-methylpyridin- 1-ium} diiodide (PRC-20) A mixture of 1,2-bis(2-iodoethoxy)ethane (0.27 mL, 548 mg, 1.48 mmol) and 3,4-dimethylpyridine (0.37 mL, 353 mg, 3.29 mmol) was heated to 120 °C for 3 h. Then, isopropanol (2 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room temperature, the resulting brown residue was washed with ice-cold Et 2 O (2 × 20 mL) and the remaining brown sticky oil was dried in vacuum, taken up in MeOH (1 mL) and treated with cold Et 2 O (2 × 20 mL), drawing off the liquids. After drying the residue in vacuum, 1,1'-(3,6-dioxaoctane- 1,8-diyl)bis(3,4-dimethylpyridin-1-ium) diiodide (620 mg, 72%) was obtained as a brown oil that so- lidified on standing; mp: 135-136 ºC; IR (ATR) ν: 3426, 3029, 2983, 2937, 2878, 1636, 1510, 1476, 1450, 1369, 1353, 1327, 1297, 1247, 1230, 1217, 1139, 1102, 1091, 1026, 992, 977, 912, 886, 813, 713, 704, 609, 558 cm -1 ; 1 H NMR (400 MHz, DMSO-d 6 ) δ: 2.39 (s, 6H), 2.53 (s, 6H), 3.48 (s, 4H), 3.84 (t, J = 5.2 Hz, 4H), 4.64 (t, J = 5.2 Hz, 4H), 7.95 (d, J = 6.0 Hz, 2H), 8.71 (dd, J = 6.0 Hz, J’ = 1.6 Hz, 2H), 8.81 (br s, 2H); HRMS-ESI+ m/z calculated for [C 20 H 30 N 2 O 2 ] 2+ /2: 165.1148, found 165.1147. A solution of 1,1'-(3,6-dioxaoctane-1,8-diyl)bis(3,4-dimethylpyridin-1-ium ) diiodide (409 mg, 0.70 mmol) and 4-(diethylamino)benzaldehyde (273 mg, 1.54 mmol) in n-butanol (3.5 mL) was treated with six drops of piperidine and the reaction mixture was stirred under reflux for 4.5 h, and then concentrated under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH 2 Cl 2 / MeOH 95:5), to provide 1,1'-(3,6-dioxaoctane-1,8- diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-iu m} diiodide (200 mg, 32%) as a red solid; mp: 71-72 ºC; IR (ATR) ν: 3417, 2967, 2920, 2859, 1640, 1567, 1519, 1477, 1435, 1404, 1350, 1307, 1258, 1218, 1184, 1154, 1128, 1072, 1009, 812, 700, 603 cm -1 ; 1 H NMR (400 MHz, DMSO- d 6 ) δ: 1.12 (t, J = 7.2 Hz, 12H), 2.44 (s, 6H), 3.43 (q, J = 7.2 Hz, 8H), 3.54 (s, 4H), 3.85 (t, J = 5.2 Hz, 4H), 4.53 (t, J = 5.2 Hz, 4H), 6.72 (d, J = 8.8 Hz, 4H), 7.04 (d, J = 16.0 Hz, 2H), 7.61 (d, J = 8.8 Hz, 4H), 7.83 (d, J = 16.0 Hz, 2H), 8.22 (d, J = 6.8 Hz, 2H), 8.54 (br d, J = 6.8 Hz, 2H), 8.61 (br s, 2H); HRMS-ESI+ m/z calculated for [C 42 H 56 N 4 O 2 ] 2+ /2: 324.2196, found: 324.2197. 8.1.13 Synthesis of 1,1'-(3,6,9-trioxaundecane-1,11-diyl)bis{4-[(E)-4-(diethylam ino)styryl]-3- methylpyridin-1-ium} dichloride (PRC-28) A mixture of bis[2-(2-chloroethoxy)ethyl] ether (0.26 mL, 307 mg, 1.33 mmol), 3,4-dimethylpyridine (0.49 mL, 467 mg, 4.36 mmol), and NaI (15 mg, 0.10 mmol) was heated to 120 °C for 4 h. Then, isopropanol (1.5 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mix- ture was allowed to cool down to room temperature, the resulting brown residue was washed with ice-cold Et 2 O (2 × 20 mL) and the remaining brown sticky oil was dried in vacuum, taken up in MeOH (1 mL) and treated with cold Et 2 O (2 × 20 mL), drawing off the liquids. After drying the resi- due in vacuum, 1,1'-(3,6,9-trioxaundecane-1,11-diyl)bis(3,4-dimethylpyridin -1-ium) dichloride (467 mg, 79%) was obtained as a brown oil; IR (ATR) ν: 3371, 3043, 2923, 2871, 1639, 1510, 1480, 1449, 1233, 1086, 1026, 931, 836, 706 cm -1 ; 1 H NMR (400 MHz, DMSO-d 6 ) δ: 2.38 (s, 6H), 2.52 (s, 6H), 3.38 (tm, J = 4.8 Hz, 4H), 3.49 (tm, J = 4.8 Hz, 4H), 3.87 (t, J = 4.8 Hz, 4H), 4.67 (t, J = 4.8 Hz, 4H), 7.93 (d, J = 6.4 Hz, 2H), 8.75 (dd, J = 6.4 Hz, J’ = 1.6 Hz, 2H), 8.85 (br s, 2H); HRMS- ESI+ m/z calculated for [C 22 H 34 N 2 O 3 ] 2+ /2: 187.1279, found 187.1292. A solution of 1,1'-(3,6,9-trioxaundecane-1,11-diyl)bis(3,4-dimethylpyridin -1-ium) dichloride (190 mg, 0.43 mmol) and 4-(diethylamino)benzaldehyde (164 mg, 0.93 mmol) in n-butanol (2.2 mL) was treated with four drops of piperidine and the reaction mixture was stirred under reflux overnight, and then concentrated under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH 2 Cl 2 / MeOH 95:5 to 85:15), to provide 1,1'-(3,6,9- trioxaundecane-1,11-diyl)bis{4-[(E)-4-(diethylamino)styryl]- 3-methylpyridin-1-ium} dichloride (55 mg, 17%) as a red oil; IR (ATR) ν: 3364, 2970, 2925, 2859, 1640, 1573, 1522, 1479, 1436, 1406, 1352, 1311, 1260, 1223, 1187, 1156, 1132, 1075, 1011, 814, 700, 607 cm -1 ; 1 H NMR (400 MHz, DMSO-d 6 ) δ: 1.12 (t, J = 7.2 Hz, 12H), 2.44 (s, 6H), superimposed in part 3.42 (tm, J = 4.8 Hz, 4H), 3.43 (q, J = 7.2 Hz, 8H), 3.52 (tm, J = 4.8 Hz, 4H), 3.86 (t, J = 4.8 Hz, 4H), 4.54 (t, J = 4.8 Hz, 4H), 6.73 (d, J = 8.8 Hz, 4H), 7.05 (d, J = 16.0 Hz, 2H), 7.62 (d, J = 8.8 Hz, 4H), 7.85 (d, J = 16.0 Hz, 2H), 8.22 (d, J = 6.8 Hz, 2H), 8.57 (br d, J = 6.8 Hz, 2H), 8.63 (br s, 2H); HRMS-ESI+ m/z calculat- ed for [C 44 H 60 N 4 O 3 ] 2+ /2: 346.2327, found: 346.2328. 8.1.14 Synthesis of 1,1'-[1,4-phenylenebis(methylene)]bis{4-[(E)-4-(diethylamino )styryl]-3- methylpyridin-1-ium} dibromide (PRC-29) A mixture of 1,4-bis(bromomethyl)benzene (527 mg, 2.00 mmol) and 3,4-dimethylpyridine (0.49 mL, 467 mg, 4.36 mmol) was heated to 120 °C for 2 h. Then, isopropanol (4 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room temperature, the resulting brown residue was washed with ice-cold Et 2 O (2 × 20 mL) and the re- maining brown sticky oil was dried in vacuum, taken up in MeOH (1 mL) and treated with cold Et 2 O (2 × 20 mL), drawing off the liquids. After drying the residue in vacuum, 1,1'-[1,4- phenylenebis(methylene)]bis(3,4-dimethylpyridin-1-ium) dibromide (813 mg, 85%) was obtained as a white solid; mp: >300 ºC; IR (ATR) ν: 3008, 2984, 2920, 1634, 1507, 1484, 1448, 1364, 1339, 1297, 1248, 1223, 1142, 1039, 1027, 961, 931, 887, 866, 770, 744, 608, 557 cm -1 ; 1 H NMR (400 MHz, DMSO-d 6 ) δ: 2.37 (s, 6H), 2.50 (s, 6H), 5.74 (s, 4H), 7.59 (s, 4H), 7.96 (d, J = 6.4 Hz, 2H), 8.94 (dd, J = 6.4 Hz, J’ = 1.6 Hz, 2H), 9.03 (br s, 2H); HRMS-ESI+ m/z calculated for [C 22 H 26 N 2 ] 2+ /2: 159.1043, found 159.1032. A solution of 1,1'-[1,4-phenylenebis(methylene)]bis(3,4-dimethylpyridin-1- ium) dibromide (231 mg, 0.48 mmol) and 4-(diethylamino)benzaldehyde (156 mg, 0.88 mmol) in n-butanol (2 mL) was treat- ed with four drops of piperidine and the reaction mixture was stirred under reflux overnight, and then concentrated under reduced pressure. The resulting black oily residue was purified by auto- matic flash column chromatography (CH 2 Cl 2 / MeOH 95:5). The fractions containing the desired product were evaporated and the resulting oily residue was dissolved in hot isopropanol (15 mL) and treated with Et 2 O (40 mL), drawing off the liquid. After drying the residue in vacuum, 1,1'-[1,4- phenylenebis(methylene)]bis{4-[(E)-4-(diethylamino)styryl]-3 -methylpyridin-1-ium} dibromide (320 mg, 84%) was obtained as a red solid; mp: 246-248 ºC; IR (ATR) ν: 3380, 2969, 2910, 1635, 1567, 1520, 1478, 1446, 1356, 1312, 1260, 1211, 1185, 1151, 1128, 1074, 1013, 970, 813, 701, 645 cm- 1 ; 1 H NMR (500 MHz, DMSO-d 6 ) δ: 1.12 (t, J = 7.0 Hz, 12H), 2.46 (s, 6H), 3.43 (q, J = 7.0 Hz, 8H), 5.62 (s, 4H), 6.73 (d, J = 9.0 Hz, 4H), 7.06 (d, J = 16.0 Hz, 2H), 7.58 (s, 4H), 7.63 (d, J = 9.0 Hz, 4H), 7.88 (d, J = 16.0 Hz, 2H), 8.27 (d, J = 6.5 Hz, 2H), 8.76 (dd, J = 6.5 Hz, J’ = 1.5 Hz, 2H), 8.83 (br s, 2H); HRMS-ESI+ m/z calculated for [C 44 H 52 N 4 ] 2+ /2: 318.2091, found: 318.2090. 8.1.15 Synthesis of 1,1'-[1,3-phenylenebis(methylene)]bis{4-[(E)-4-(diethylamino )styryl]-3- methylpyridin-1-ium} dibromide (PRC-31) A mixture of 1,3-bis(bromomethyl)benzene (527 mg, 2.00 mmol) and 3,4-dimethylpyridine (0.49 mL, 467 mg, 4.36 mmol) was heated to 120 °C for 2 h. Then, isopropanol (4 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room temperature, the resulting brown residue was washed with ice-cold Et 2 O (2 × 20 mL) and the re- maining brown sticky oil was dried in vacuum, taken up in MeOH (1 mL) and treated with cold Et 2 O (2 × 20 mL), drawing off the liquids. After drying the residue in vacuum, 1,1'-[1,3- phenylenebis(methylene)]bis(3,4-dimethylpyridin-1-ium) dibromide (785 mg, 82%) was obtained as a brown solid; mp: 152-153 ºC; IR (ATR) ν: 3460, 3414, 3006, 2920, 1636, 1509, 1480, 1450, 1387, 1362, 1340, 1225, 1163, 1139, 1020, 856, 842, 740, 705, 692, 652, 613 cm -1 ; 1 H NMR (500 MHz, DMSO-d 6 ) δ: 2.40 (s, 6H), 2.52 (s, 6H), 5.77 (s, 4H), 7.47-7.55 (m, 3H), 7.80 (t, J = 2.0 Hz, 1H), 7.98 (d, J = 6.0 Hz, 2H), 8.94 (dd, J = 6.0 Hz, J’ = 1.5 Hz, 2H), 9.10 (br s, 2H); HRMS-ESI+ m/z calculated for [C 22 H 26 N 2 ] 2+ /2: 159.1043, found 159.1042. A solution of 1,1'-[1,3-phenylenebis(methylene)]bis(3,4-dimethylpyridin-1- ium) dibromide (231 mg, 0.48 mmol) and 4-(diethylamino)benzaldehyde (156 mg, 0.88 mmol) in n-butanol (2 mL) was treat- ed with four drops of piperidine and the reaction mixture was stirred under reflux overnight, and then concentrated under reduced pressure. The resulting black oily residue was purified by auto- matic flash column chromatography (CH 2 Cl 2 / MeOH 100:0 to 95:5). The fractions containing the desired product were evaporated and the resulting oily residue was dissolved in hot isopropanol (15 mL) and treated with Et 2 O (40 mL), drawing off the liquid. After drying the residue in vacuum, 1,1'-[1,3-phenylenebis(methylene)]bis{4-[(E)-4-(diethylamino )styryl]-3-methylpyridin-1-ium} dibro- mide (20 mg, 5%) was obtained as a red solid; mp: 66-67 ºC; IR (ATR) ν: 3347, 2969, 2920, 1639, 1572, 1523, 1479, 1438, 1406, 1350, 1310, 1261, 1213, 1187, 1151, 1128, 1076, 1013, 971, 808, 785, 740, 700 cm -1 ; 1 H NMR (500 MHz, DMSO-d 6 ) δ: 1.13 (t, J = 7.0 Hz, 12H), 2.48 (s, 6H), 3.43 (q, J = 7.0 Hz, 8H), 5.64 (s, 4H), 6.74 (d, J = 9.0 Hz, 4H), 7.08 (d, J = 16.0 Hz, 2H), 7.49-7.51 (m, 3H), 7.60 (br s, 1H), 7.64 (d, J = 9.0 Hz, 4H), 7.90 (d, J = 16.0 Hz, 2H), 8.29 (d, J = 7.0 Hz, 2H), 8.74 (dd, J = 7.0 Hz, J’ = 1.5 Hz, 2H), 8.82 (br s, 2H); HRMS-ESI+ m/z calculated for [C 44 H 52 N 4 ] 2+ /2: 318.2091, found: 318.2090. 8.1.16 Synthesis of 1,1'-(decane-1,10-diyl)bis{4-[(E)-4-(diethylamino)styryl]pyr idin-1-ium} dibromide (PRC-39) A mixture of 1,10-dibromodecane (0.44 mL, 587 mg, 1.96 mmol) and 4-methylpyridine (0.43 mL, 412 mg , 4.42 mmol) was heated to 120 °C for 3 h. Then, isopropanol (2 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room temperature, the resulting brown residue was washed with ice-cold Et 2 O (2 × 20 mL) and the re- maining brown sticky oil was dried in vacuo, taken up in MeOH (1 mL) and treated with cold Et 2 O (2 × 20 mL), drawing off the liquids. After drying the residue in vacuum, 1,1'-(decane-1,10- diyl)bis(4-methylpyridin-1-ium) dibromide (751 mg, 79%) was obtained as a orange oil that solidi- fied on standing; mp: 56-57 ºC; IR (ATR) ν: 3350, 3013, 2920, 2852, 1638, 1516, 1469, 1379, 1219, 1171, 1030, 828, 730, 710 cm -1 ; 1 H NMR (400 MHz, DMSO-d 6 ) δ: 1.17-1.31 (m, 12H), 1.87 (tt, J = J’ = 7.2 Hz, 4H), 2.61 (s, 6H), 4.53 (t, J = 7.2 Hz, 4H), 7.99 (d, J = 6.4 Hz, 4H), 8.96 (d, J = 6.4 Hz, 4H); HRMS-ESI+ m/z calculated for [C 22 H 34 N 2 ] 2+ /2: 163.1356, found 163.1360. A solution of 1,1'-(decane-1,10-diyl)bis(4-methylpyridin-1-ium) dibromide (243 mg, 0.50 mmol) and 4-(diethylamino)benzaldehyde (156 mg, 0.88 mmol) in n-butanol (2. mL) was treated with four drops of piperidine and the reaction mixture was stirred under reflux overnight, and then concen- trated under reduced pressure. The resulting black oily residue was purified by automatic flash col- umn chromatography (CH 2 Cl 2 / MeOH 100:0 to 95:5). The fractions containing the desired product were evaporated and the resulting oily residue was dissolved in hot isopropanol (15 mL) and treat- ed with Et 2 O (2 × 40 mL), drawing off the liquids. After drying the residue in vacuum, 1,1'-(decane- 1,10-diyl)bis{4-[(E)-4-(diethylamino)styryl]pyridin-1-ium} dibromide (132 mg, 33%) was obtained as a red solid; mp: 157-159 ºC; IR (ATR) ν: 3404, 2969, 2920, 1646, 1572, 1521, 1471, 1434, 1403, 1348, 1325, 1272, 1192, 1169, 1150, 1011, 827, 704, 572, 561 cm -1 ; 1 H NMR (400 MHz, CD3OD) δ: 1.21 (t, J = 7.2 Hz, 12H), 1.31-1.42 (m, 12H), 1.96 (tt, J = J’ = 7.2 Hz, 4H), 3.48 (q, J = 7.2 Hz, 8H), 4.40 (t, J = 7.2 Hz, 4H), 6.76 (d, J = 8.8 Hz, 4H), 7.05 (d, J = 16.0 Hz, 2H), 7.59 (d, J = 8.8 Hz, 4H), 7.83 (d, J = 16.0 Hz, 2H), 7.95 (d, J = 6.8 Hz, 4H), 8.55 (d, J = 6.8 Hz, 4H); HRMS-ESI+ m/z calculated for [C 44 H 60 N 4 ] 2+ /2: 322.2404, found: 322.2395. 8.1.17 Synthesis of 1,1'-(decane-1,10-diyl)bis{3-bromo-4-[(E)-4-(diethylamino)st yryl]pyridin-1-ium} dibromide (PRC-42) A mixture of 1,10-dibromodecane (0.44 mL, 587 mg, 1.96 mmol) and 3-bromo-4-methylpyridine (0.49 mL, 759 mg, 4.41 mmol) was heated to 120 °C for 3 h. Then, isopropanol (2 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room temperature, the resulting brown residue was washed with ice-cold Et 2 O (2 × 20 mL) and the remaining bluish sticky oil was dried in vacuo, taken up in MeOH (1 mL) and treated with cold Et 2 O (2 × 20 mL), drawing off the liquids. After drying the residue in vacuum, 1,1'-(decane-1,10- diyl)bis(3-bromo-4-methylpyridin-1-ium) dibromide (605 mg, 48%) was obtained as a brown oil that solidified on standing; mp: 63-64 ºC; IR (ATR) ν: 3454, 3387, 2988, 2921, 2851, 1632, 1497, 1465, 1375, 1317, 1186, 1174, 1084, 1031, 864, 719, 697, 579 cm -1 ; 1 H NMR (400 MHz, DMSO-d 6 ) δ: 1.19-1.32 (m, 12H), 1.90 (tt, J = J’ = 7.2 Hz, 4H), 2.61 (s, 6H), 4.54 (t, J = 7.2 Hz, 4H), 8.16 (d, J = 6.4 Hz, 2H), 9.04 (dd, J = 6.4 Hz, J’ = 1.6 Hz, 2H), 9.49 (d, J = 1.6 Hz, 2H); HRMS-ESI+ m/z calcu- lated for [C 22 H 32 Br 2 N 2 ] 2+ /2: 241.0461, found 241.0453. A solution of 1,1'-(decane-1,10-diyl)bis(3-bromo-4-methylpyridin-1-ium) dibromide (322 mg, 0.50 mmol) and 4-(diethylamino)benzaldehyde (195 mg, 1.10 mmol) in n-butanol (2 mL) was treated with four drops of piperidine and the reaction mixture was stirred under reflux overnight, and then concentrated under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH 2 Cl 2 / MeOH 100:0 to 90:10), to provide 1,1'-(decane-1,10- diyl)bis{3-bromo-4-[(E)-4-(diethylamino)styryl]pyridin-1-ium } dibromide (45 mg, 9%) as a purple sol- id; mp: 136-138 ºC; IR (ATR) ν: 3396, 2969, 2924, 2852, 1634, 1567, 1520, 1454, 1406, 1350, 1293, 1269, 1171, 1149, 1075, 1011, 806, 689, 567 cm -1 ; 1 H NMR (400 MHz, CD3OD) δ: 1.22 (t, J = 7.2 Hz, 12H), 1.31-1.43 (m, 12H), 1.97 (tt, J = J’ = 7.6 Hz, 4H), 3.51 (q, J = 7.2 Hz, 8H), 4.39 (t, J = 7.6 Hz, 4H), 6.79 (d, J = 9.2 Hz, 4H), 7.24 (d, J = 16.0 Hz, 2H), 7.64 (d, J = 9.2 Hz, 4H), 7.94 (d, J = 16.0 Hz, 2H), 8.23 (d, J = 6.8 Hz, 2H), 8.52 (br d, J = 6.8 Hz, 2H), 9.02 (d, J = 1.2 Hz, 2H); HRMS-ESI+ m/z calculated for [C 44 H 58 Br 2 N 4 ] 2+ /2: 400.1509, found: 400.1512. 8.1.18 Synthesis of 1,1'-(decane-1,10-diyl)bis{3-chloro-4-[(E)-4-(diethylamino)s tyryl]pyridin-1-ium} dibromide (PRC-50) A mixture of 1,10-dibromodecane (0.44 mL, 587 mg, 1.96 mmol) and 3-chloro-4-methylpyridine (484 mg, 3.79 mmol) was heated to 120 °C for 3 h. Then, isopropanol (2 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room temperature, the resulting brown residue was washed with ice-cold Et 2 O (2 × 20 mL) and the re- maining bluish sticky oil was dried in vacuo, taken up in MeOH (1 mL) and treated with cold Et 2 O (2 × 20 mL), drawing off the liquids. After drying the residue in vacuum, 1,1'-(decane-1,10-diyl)bis(3- chloro-4-methylpyridin-1-ium) dibromide (614 mg, 58%) was obtained as a bluish oil that solidified on standing to a light pink solid; mp: 167-168 ºC; IR (ATR) ν: 3411, 2945, 2924, 2896, 2859, 1638, 1504, 1468, 1449, 1366, 1311, 1250, 1219, 1200, 1169, 1099, 1025, 1007, 955, 865, 769, 726, 703, 596 cm -1 ; 1 H NMR (400 MHz, DMSO-d 6 ) δ: 1.21-1.31 (m, 12H), 1.90 (tt, J = J’ = 7.6 Hz, 4H), 2.61 (s, 6H), 4.54 (t, J = 7.6 Hz, 4H), 8.18 (d, J = 6.4 Hz, 2H), 9.01 (dd, J = 6.4 Hz, J’ = 1.6 Hz, 2H), 9.43 (d, J = 1.6 Hz, 2H); HRMS-ESI+ m/z calculated for [C 22 H 32 Cl 2 N 2 ] 2+ /2: 197.0966, found 197.0967. A solution of 1,1'-(decane-1,10-diyl)bis(3-chloro-4-methylpyridin-1-ium) dibromide (222 mg, 0.40 mmol) and 4-(diethylamino)benzaldehyde (156 mg, 0.88 mmol) in n-butanol (1,6. mL) was treated with four drops of piperidine and the reaction mixture was stirred under reflux overnight, and then concentrated under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH 2 Cl 2 / 7N ammonia solution in MeOH 100:0 to 90:10), to provide 1,1'-(decane-1,10-diyl)bis{3-chloro-4-[(E)-4-(diethylamino)s tyryl]pyridin-1-ium} dibromide (54 mg, 15%) as a purple solid; mp: 124-126 ºC; IR (ATR) ν: 3396, 2967, 2924, 2852, 1633, 1572, 1520, 1455, 1405, 1349, 1294, 1269, 1176, 1150, 1074, 1037, 1012, 972, 917, 808, 699, 612, 570 cm -1 ; 1 H NMR (500 MHz, CD3OD) δ: 1.22 (t, J = 7.0 Hz, 12H), 1.32-1.42 (m, 12H), 1.97 (tt, J = J’ = 7.5 Hz, 4H), 3.51 (q, J = 7.0 Hz, 8H), 4.39 (t, J = 7.5 Hz, 4H), 6.79 (d, J = 9.0 Hz, 4H), 7.25 (d, J = 16.0 Hz, 2H), 7.64 (d, J = 9.0 Hz, 4H), 7.96 (d, J = 16.0 Hz, 2H), 8.26 (d, J = 6.5 Hz, 2H), 8.49 (br d, J = 6.5 Hz, 2H), 8.91 (d, J = 1.5 Hz, 2H); HRMS-ESI+ m/z calculated for [C 44 H 58 Cl 2 N 4 ] 2+ /2: 356.2014, found: 356.2020. 8.1.19 Synthesis of 1,1'-(decane-1,10-diyl)bis{4-[(E)-4-(diethylamino)-2-hydroxy styryl]-3- methylpyridin-1-ium} dibromide (PRC-69) Compound PRC-69 was prepared following the first steps and conditions as described for the syn- thesis of compound AID-X-2020 (section 1.1.2 of EXAMPLE 1 – 1 st paragraph) until 1,1'-(decane- 1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide was obtained. A solution of 1,1'-(decane-1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (307 mg, 0.60 mmol) and 5-(diethylamino)-2-formylphenyl acetate (309 mg, 1.31 mmol) in n-butanol (3 mL) was treated with nine drops of DBU and the reaction mixture was stirred under microwave irradiation at 140 ºC, 1 bar, 80 W for 15 min. Then, it was concentrated under reduced pressure and the resulting black oily residue was purified by automatic flash column chromatography (CH 2 Cl 2 / isopropanol 90:10 to 75:25). The fractions containing the desired product were treated with Cs 2 CO 3 in MeOH, until the colour changed from red to blue. The solution was evaporated under reduced pressure and the solid was suspended in CH 2 Cl 2 . The suspension was filtered, the filtrate was evaporated, and the resulting solid was washed with EtOAc (5 × 10 mL), drawing off the liquids. After drying the residue in vacuum, 1,1'-(decane-1,10-diyl)bis{4-[(E)-4-(diethylamino)-2-hydroxy styryl]-3-methylpyridin-1- ium} dibromide (12 mg, 2%) was obtained as a blue solid; mp: 169-171 ºC; IR (ATR) ν: 3277, 2924, 2853, 1556, 1512, 1395, 1373, 1350, 1243, 1186, 1104, 1075, 1011, 962, 771, 682 cm -1 ; 1 H NMR (400 MHz, CD3OD) δ: 1.18 (t, J = 7.2 Hz, 12H), 1.23-1.40 (m, 12H), 1.89 (m, 4H), 2.37 (s, 6H), 3.41 (q, J = 7.2 Hz, 8H), 4.18 (t, J = 7.2 Hz, 4H), 6.01 (d, J = 2.0 Hz, 2H), 6.16 (dd, J = 9.2 Hz, J’ = 2.0 Hz, 2H), 6.89 (d, J = 15.2 Hz, 2H), 7.45 (dm, J = 9.2 Hz, 2H), 7.89 (d, J = 7.2 Hz, 2H), 8.05 (d, J = 7.2 Hz, 2H), 8.11 (br s, 2H), 8.30 (d, J = 15.2 Hz, 2H); HRMS-ESI+ m/z calculated for [C 46 H 64 N 4 O 2 ] 2+ /2: 352.2509, found: 352.2514. 8.1.20 P. falciparum growth inhibition assay The assay on P. falciparum growth inhibition for compounds EMA357, EMA359, EMA366, EMA368, EMA377, PRC-5, PRC-8, PRC-14, PRC-15, PRC-20, PRC-25, PRC-28, PRC-29, PRC- 31, PRC-39, PRC-42, and PRC-50was performed following the same steps and conditions used in section 1.1.2 of EXAMPLE 1. The results obtained were then compared to the values previously obtained for AID-X-2020 (IC 50 = 0.09 µM). 8.2 Results The results obtained indicated that all the compounds exhibit good antimalarial activity, especially EMA357, EMA359, EMA377, PRC-8, PRC-14, PRC-15, PRC-25, PRC-39, PRC-42, and PRC-50, which have a potent antimalarial activity, similar to AID-X-2020. Particularly, PRC-25 shows the highest antimalarial activity among the tested compounds, with an IC 50 of 47 nM (Table 4). Table 4. In vitro antimalarial activity of EMA357, EMA359, EMA366, EMA368, EMA377, PRC-5, PRC-8, PRC-14, PRC-15, PRC-20, PRC-25, PRC-28, PRC-29, PRC-31, PRC-39, PRC-42, and PRC-50.
EXAMPLE 9: AID-X-2020 is also active against chloroquine- and artemisinin-resistant P. fal- ciparum strains The aim of this experiment was to assess whether AID-X-2020 is active against chloroquine- and artemisinin-resistant P. falciparum strains, and thus to evaluate the potential of a new antimalarial resistant drug compared to existing antimalarials. 9.1 Materials and Methods 9.1.1 P. falciparum growth inhibition assays P. falciparum parasites of chloroquine-sensitive (3D7 strain) and chloroquine- and artemisinin- resistant strains were processed as described in section 1.1.3 of EXAMPLE 1. In particular, the P. falciparum resistant strains were the following: (1) strain W2 MRA-157 (BEI Re- sources, managed by ATCC) which is chloroquine-resistant; (2) strains 3D7 harboring the K13 mu- tations associated to artemisinin resistance, either M579I or R561H (both provided by Dr. David A. Fidock and referenced in Stokes, B.H. et al., 2021); (3) Cam 3.II strain which is chloroquine and sulfadoxine/pyrimethamine-resistant; and (4) Cam 3.II strain modified to carry K13 mutations asso- ciated to artemisinin-resistant strains, either R561H or R539T, therefore being multiresistant strains. All Cam 3.II strains (3) and (4) were provided by Dr. David A. Fidock and are referenced in Straimer, J. et al., 2015 and Stokes, B.H. et al., 2021. Then, the required amount of AID-X-2020 was added to each well at different concentrations (0.0001, 0.001, 0.01, 1 and 10 µM) and triplicates. The effect of AID-X-2020 of chloroquine- and artemisinin-resistant strains was compared with the activity of AID-X-2020 of P. falciparum parasites of chloroquine-sensitive 3D7 strain. 9.2 Results Results confirm that AID-X-2020 was also strongly active against the chloroquine-resistant W2 strain (IC 50 of 90 ± 1 nM), and several artemisinin-resistant strains with IC 50 values ranging from 90 to 160 nM, in comparison to parental 3D7 and Cam3.II strains (FIG.5). The results do not show a significant IC 50 increase relative to the corresponding parental strains except for M579I (p value of 0.02; all other p values > 0.15), in agreement with a mode of action for AID-X-2020 different from that of artemisinin. This result is also consistent with the calculated IC 50 of AID-X-2020 in the chlo- roquine-resistant P. falciparum W2 strain (FIG.5(A)), undistinguishable from its activity in the chlo- roquine-sensitive 3D7 strain. Results suggest that resistance is slowed down significantly for AID-X-2020, a molecule belonging to a chemical family where no antimalarials have been described so far. Thus, the sensitivity to AID-X-2020 of the chloroquine-resistant W2 strain indicates that the antimalarial mode of action of AID-X-2020 is not related to that of chloroquine. Similarly, data showing a sensitivity of artemisinin- resistant strains to AID-X-2020 similar to that of the corresponding parental non-resistant lines, strongly suggest that the antimalarial modes of action of both drugs are not related. Indeed, an an- tagonistic action of artemisinin and AID-X-2020 was observed in P. falciparum cultures, indicating that their main effects on the pathogen are opposed (EXAMPLE 5). Therefore, in this scenario, it can be concluded that AID-X-2020 is a potential new antimalarial resistant drug compared to the existing antimalarials. EXAMPLE 10: AID-X-2020 is also able to disassemble preformed Aß40 fibrils EXAMPLE 3 confirmed that AID-X-2020 is a strong inhibitor of the aggregation of Aß40 in for- mation, suggesting that inhibition of protein aggregation was the responsible mechanism for the antimalarial activity of this compound. In the present example, inventors further tested the possibil- ity of disaggregation of already formed aggregative peptides. 10.1 Materials and Methods 10.1.1 In vitro assay of aggregation and disaggregation of aggregative peptides To test the effect of AID-X-2020 on already formed amyloid fibrils, Aβ40 DMSO solutions were di- luted to 25 µM in PBS and incubated as above in order to allow fibril formation. Then, AID-X-2020 was added at different concentrations (0.1 µM AID-X-2020, 1 µM AID-X-2020 and 10 µM AID-X- 2020) and the mixture was incubated in the same conditions for another 24 h. The final samples always contained less than 5% DMSO to avoid interference of this solvent on Aβ40 amyloid fibril formation. Finally, ThT treatment and TEM analysis was performed as described in section 3.1 of EXAMPLE 3. The analysis of aggregation inhibition and disaggregation performed with aggregative peptides present in P. falciparum proteins were conducted in the same way. Aggregation inhibition and disaggregation activities were also tested with six aggregative peptides present in P. falciparum proteins with SEQ ID NO: 1-6 (NVNIYN, LYWIYY, NFNNIYH, NNFYYNN, LISFIL, LQSNIG) in order to determine the effects on AID-X-2020 directly on P. falciparum pep- tides. AID-X-2020 was added in this case at 0.1 µM and 1 µM. 10.2 Results AID-X-2020 at concentrations > 90 nM was also found to disassemble preformed Aβ40 fibrils (FIG. 6). Both in vitro aggregation inhibition and disaggregation assays show the presence of character- istic amorphous aggregates at 0.1 µM AID-X-2020, which might represent an intermediate species between mature Aβ40 fibrils and the disassembled protofibrillar structures found at higher drug concentrations. These aggregation inhibition and disaggregation activities were also observed with the six aggregative peptides present in P. falciparum proteins (FIG.7). Results confirm the in vitro activity of AID-X-2020 as inhibitor of the aggregation of a model amyloi- dogenic peptide like Aβ40 and of aggregative peptides present in P. falciparum proteins, as already demonstrated in EXAMPLES 3, 4 and 5. The present example further demonstrates the disaggre- gating activity of AID-X-2020, beyond its capacity to inhibit protein aggregation. EXAMPLE 11: Subcellular localization of AID-X-2020 in pRBCs 11.1 Materials and Methods 11.1.1 Fluorescence microscopy For AID-X-2020 staining, a P. falciparum 3D7 culture was incubated in RPMIc for 30 min at 37 °C with 4.5 µM of the compound and 4 μg/ml of Hoechst 33342. For colocalization studies, 0.5 µM of ER Tracker TM Green (BODIPY™ FL Glibenclamide, Thermo Fisher Scientific) was included in the solution. Cells were placed in an 8-well LabTek TM II chamber slide system (Thermo Fisher Scientific), rinsed with warm PBS and diluted 1:20 for their observa- tion in a Leica TCS SP5 confocal microscope (Leica Camera, Mannheim, Germany) equipped with a 63× objective of 1.4 NA. Hoechst 33342 was excited with a diode laser at 405 nm, ER Tracker Green with the 488 nm line of an argon laser, and AID-X-2020 with a diode-pumped solid-state la- ser at 561 nm. The corresponding fluorescence emissions were collected in the ranges of, respec- tively, 415-460, 490-590, and 600-700 nm. To avoid crosstalk between the different fluorescence signals, sequential line scanning was per- formed. To quantify Manders’ overlap coefficient, images were analyzed using the Just Another Colocalization Plugin (JACoP) in the Fiji software. To avoid fixation artifacts, all the fluorescence microscopy data presented in this work were obtained with live cells. 11.1.2 Correlative light and electron microscopy (CLEM) A 0.5% parasitemia RBC culture was prepared for CLEM by allowing its binding to concanavalin. Briefly, a µ-Dish 35 mm, High, Grid-500 (ibidi GmbH, Gräfelfing, Germany) was coated for 20 min at 37 °C with a 50 mg/ml concanavalin A solution in ddH 2 O and wells were rinsed with pre-warmed PBS before parasite seeding. P. falciparum-infected RBCs washed twice with PBS were deposited into the dish and incubated for 10 min at 37 °C; afterwards, unbound RBCs were washed away with three PBS rinses. Seeded RBCs were then incubated with 3 µM AID-X-2020, and nuclei were counterstained with 2 µg/ml Hoechst 33342. The preparation was observed with a Zeiss LSM880 confocal microscope (Carl Zeiss, Jena, Germany), with respective λex/em for AID-X-2020 and Hoechst 33342 of 405/415-520 nm and 561/565-600 nm. Images were obtained from areas corre- sponding to a specific coordinate of the dish-grid by tile scans that were stitched into larger mosa- ics. A bright field image facilitated the recognition of the grid coordinates from the plate where the cells selected for CLEM were located. After confocal image acquisition, cells were washed three times with TEM fixation buffer (2% para- formaldehyde and 2.5% glutaraldehyde in PBS) for 5 min each. Then, the fixation buffer was changed to 1% osmium tetroxide and 0.8% potassium ferricyanide in fixation buffer and incubated at 4 °C for 45 min, followed by three 5-min washes with ddH 2 O. Then, a dehydration procedure was performed by gradually increasing ethanol concentration: 50% (10 min), 70% (10 min), 80% (10 min), 90% (5 min, 3×), 96% (5 min, 3×), and 100% (5 min, 3×). At this point, the plastic part of the dish was carefully separated from the crystal part containing the samples, which was embed- ded in Spurr resin by successive incubations with different proportions of resin/ethanol, starting with 1/3 for 1 h, 1/1 for 1 h, 3/1 for 1 h and 1/0 overnight. After the embedding procedure, a BEEM® capsule containing polymerized Spurr resin was filled with a small volume of liquid resin in order to obtain an interphase in which the dish was placed. The BEEM® capsule was incubated at 70 °C for 72 h, and the crystal part of the dish was removed by alternatively immersing samples in liquid nitrogen and boiling water. When the crystal was broken, cells remained attached to the res- in, which was further cut in a microtome with a diamond blazer in order to obtain 100 nm-thick resin slides, which were mounted on a carbon-coated copper grid and negatively stained with 2% uranyl acetate for 2 min and washed with ddH 2 O for 1 min. Samples were observed in a JEM 1010 trans- mission electron microscope. Images were processed for CLEM analysis using the CORRELIA plug-in in the Fiji software (version 2.0.0-pre-8). 11.2 Results Confocal fluorescence microscopy colocalization analysis (FIG.8(A)) and correlative light and elec- tron microscopy data (FIG. 8(B)) confirmed the presence of AID-X-2020 mainly in the parasite’s cytosol, particularly in association with endoplasmic reticulum regions. The cytosolic localization of the protein aggregation inhibitor AID-X-2020 in Plasmodium rough ER regions, where proteins are being synthesized, is consistent with the likely role of this drug in dis- rupting a yet to be described parasite’s aggresome. EXAMPLE 12: Quantitative analysis of protein aggregation in live Plasmodium cells after treatment with AID-X-2020 The aim of this experiment was to directly prove the level of protein aggregation in live Plasmodium cells following AID-X-2020 treatment. 12.1 Materials and Methods 12.1.1 Quantitative analysis of protein aggregation in live P. falciparum cultures To directly probe the level of protein aggregation in live Plasmodium cells following AID-X-2020 treatment, a ThT-based method to measure protein aggregation in parasite cultures was devel- oped. P. falciparum cultures enriched in early stages were treated with the IC 10 (27 nM) and IC 50 (90 nM) of AID-X-2020 or left untreated. After 90 min, 4 h and 30 h, a Percoll purification was done in order to isolate parasitized cells from uninfected RBCs. After Percoll purification, the pellets of late stage parasites and a control non-infected RBC suspension containing the same proportion of cells than the purified cultures were resuspended in 50 µl of lysis buffer (4.5 mg/ml NaCl in water supple- mented with EDTA-free protease inhibitor cocktail, PIC, Hoffman-La Roche, Basel, Switzerland; 1 PIC tablet/10 ml water) and incubated overnight, at 4 °C under stirring, with the objective of releas- ing their inner content. After this time, lysed samples were spun down and the protein content in the supernatant was quantified with the bicinchoninic acid assay (Thermo Fisher Scientific), follow- ing the manufacturer’s instructions.30 µg of protein from each supernatant were further diluted with PBS to a final volume of 70 µl and plated on a 96-well black plate in triplicates. ThT fluorescence was measured as described above. 12.2 Results ThT fluorescence of culture extracts, normalized to have equal protein content, exhibited a reduced emission spectrum in samples that had been treated for only 90 min with 90 nM AID-X-2020, the compound’s in vitro IC 50 (FIG. 9). After 4 h of treatment, the decrease in ThT fluorescence was more evident, even for cultures treated with the IC 10 of AID-X-2020 (27 nM). This reduced signal could still be clearly detected after 30 h of 90 nM treatment (FIG.10). These results indicating a relevant decrease in aggregated protein load in live parasites following AID-X-2020 treatment at physiologically relevant concentrations are supportive of a mode of action of this drug consisting in the inhibition of protein aggregation in the pathogen. EXAMPLE 13: Activity of AID-X-2020 on Plasmodium gametocytes EXAMPLE 6 studied the inhibition of the gametocyte to ookinete transition in order to determine whether AID-X-2020 was able to arrest the life cycle of malaria parasites in the mosquito host. Contrarily, the objective of the present study was to determine if AID-X-2020 is capable of targeting gametocytes, the sexual phase of the malaria parasites’ life cycle and the sole stage of malaria parasites present in the blood circulation capable of transmitting the infection to the mosquito vec- tor, and where drugs active at this critical step are scarce. 13.1 Materials and Methods Cultures of the P. falciparum NF54-gexp02-Tom strain (developed and authenticated by Portugali- za, H.P. et al., 2019 and kindly provided by Prof. Alfred Cortés), were maintained in standard con- ditions in RPMI medium supplemented with 0.5% Albumax II and 2 mM choline, synchronized in ring stages with sorbitol lysis, and diluted to 2% parasitemia. To trigger sexual conversion, choline was removed from the medium and cultures were maintained in the same conditions for 48 h after synchronization (cycle 0). In the next cycle (cycle 1), parasites were treated with 50 mM N- acetylglucosamine (GlcNac) in order to kill asexual parasites, and maintained in RPMI supple- mented with 10% human serum. Medium was refreshed daily and GlcNac was kept during 4 days. To determine the effect of AID-X-2020 and DONE3TCl in early gametocytes, the culture was dis- tributed in triplicates (200 µl/well, 96-well plates) and drugs were added in cycle 1 and maintained for 48 h in the culture. Controls of untreated parasites as well as of parasites treated with a lethal dose of chloroquine were prepared. Gametocytemia was monitored daily by light microscopy until the majority of parasites (~ 90%) could be identified as stage V gametocytes. At that point, Giemsa smears of each well were prepared and mature gametocytes were manually counted (10,000 cells were counted for each replica by two investigators blinded to group assignment). To test the effect of AID-X-2020 and DONE3TCl on mature gametocytes, cultures were grown for 14 days, when the majority of the parasites could be identified as stage V gametocytes. Afterwards, the culture was treated for 48 h with the drugs and the gametocytemia determined as above. 13.2 Results AID-X-2020 efficiently blocked the development of P. falciparum early and mature stage V gameto- cytes in vitro with respective IC 50 of 95 ± 3 nM and 103 ± 3 nM (FIG.11), close to that obtained for the asexual blood stages (EXAMPLE 6). For the amyloid pan-inhibitor aminoquinoline DONE3TCl the IC 50 values on early and late gametocytes were 285 ± 56 nM and 78 ± 12 nM, respectively. Therefore, besides its activity against Plasmodium asexual blood stages, AID-X-2020 displays marked activity against the sexual phase of the malaria parasites’ life cycle, paving the way for the exploration of this drug as a potential multi-stage antiplasmodial therapy. EXAMPLE 14: Microscopic observation of clinical samples 14.1 Materials and Methods Clinical malaria samples were provided by the Centre de Salut Internacional i Malalties Transmis- sibles Drassanes-Vall d’Hebron. Five µL of blood from a malaria-infected person were diluted in 100 µL of RPMIc. The solution was stained with 2 µg/mL Hoechst 33342 and 18 µM AID-X-2020 and incubated for 30 min before centrifuging at 5000× g for 30 s and discarding the supernatant. The pellet was then resuspended in 100 µL of PBS and 5 µL of the suspension were placed on a 8 well slide (Ibidi) with 200 µL of PBS. The sample was observed in a IX-51 Olympus fluorescence microscope with a 100× objective and a SPRED filter (λex: 586/20 nm, λem: 628/32 nm). 14.2 Results Microscopic observation of AID-X-2020-stained clinical blood samples of a P. falciparum infection allowed the identification of the circulating ring forms of the parasite that are detected in malaria diagnosis.
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