Synthesis and evaluation of DNA gyrase A inhibitor ciprofloxacin conjugates with benzothiazolepyrrolamide-based DNA gyrase B inhibitors

V boju proti bakterijski odpornosti obetavno strategijo predstavlja razvoj večtarčnih zaviralcev bakterijskih encimov. Z omenjeno strategijo lahko zmanjšamo sposobnost bakterij za razvoj tarčne odpornosti proti protibakterijskim spojinam. Ena od privlačnih tarč za razvoj protibakterijskih spojin je encim DNA-giraza, ki ga sestavljata dve podenoti imenovani DNA-giraza A in dve podenoti imenovani DNA-giraza B. Ta encim uvrščamo med topoizomeraze tipa IIA, ki med podvajanjem uravnavajo topologijo DNA molekule. Tekom magistrske naloge smo načrtovali in sintetizirali novo serijo petih konjugatov med zaviralcem DNA-giraze A ciprofloksacinom in benzotiazolpirolamidnima zaviralcema DNA-giraze B: 2-(3,4-dikloro-5-metil-lH-pirol-2-karboksamido)benzo[d]tiazol-6-karboksilno kislino ali zaviralcem z dodanim benziloksi fragmentom, 4-(benziloksi)-2-(3,4-dikloro-5-metil-1H-pirol-2-karboksamido)benzo[d]tiazol-6-karboksilno kislino. Zaviralca DNA-giraze A in DNA-giraze B smo povezali na mestu C7 piperazina ciprofloksacina in pri tem, z izjemo direktnega konjugata, uporabili različne distančnike (glicinski, β-alaninski, etrski). Z encimskim testom smo ovrednotili zaviralno aktivnost končnih spojin na DNA-girazi iz bakterije Escherichia coli, pri najaktivnejši spojini tudi iz bakterije Staphylococcus aureus. Spojinam smo določili tudi protibakterijsko delovanje proti trem po Gramu pozitivnim, petim po Gramu negativnim bakterijam in trem dodatnim sevom E. coli: divjem sevu ter dvema mutiranima bakterijskima sevoma. Najmočnejši zaviralec sintetizirane serije je bila spojina 25 z vrednostjo srednje zaviralne koncentracije (IC50) 14 ± 9 nM na DNA-girazi iz E. coli in 1,09 µM na DNA-girazi iz bakterije S. aureus. Ta spojina vsebuje zaviralec DNA-giraze B z dodanim benziloksi fragmentom, ki je s ciprofloksacinom povezan z etrskim distančnikom. Najmočnejše protibakterijsko delovanje je izkazala spojina 12 z vrednostmi minimalne zaviralne koncentracije (MIC) proti bakterijam Klebsiella pneumoniae, E. coli in Enterobacter cloacae spp. cloacae v območju 0,5-4 µg/mL ter vrednostjo MIC proti bakteriji Enterococcus faecalis 16 µg/mL. S sintetiziranimi spojinami smo dosegli delovanje na obe podenoti DNA-giraze, saj so spojine aktivne proti odpornim sevom z mutacijo posamezne podenote (DNA-giraze A ali DNA-giraze B) tega encima. Rezultati magistrske naloge dajejo pomembne informacije za odkrivanje novih dvojnih zaviralcev z delovanjem na obe podenoti DNA-giraze ter s tem zmanjšano verjetnostjo za razvoj odpornosti.A promising strategy in the fight against bacterial resistance is the development of multi-target inhibitors of bacterial enzymes. With the mentioned strategy the ability of bacteria to develop target-based resistance against antibacterial agents can be reduced. One of the attractive targets for the development of antibacterial agents is bacterial enzyme DNA gyrase, which consists of two DNA gyrase A subunits and two DNA gyrase B subunits. The enzyme belongs to type IIA topoisomerases which are enzymes that regulate the topology of DNA molecule during replication. We designed and synthesized a new series of five conjugates between DNA gyrase A inhibitor ciprofloxacin and benzothiazolepyrrolamide-based DNA gyrase B inhibitor: 2-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)benzo[d]thiazole-6-carboxylic acid or a DNA gyrase B inhibitor with added benzyloxy fragment, 4-(benzyloxy)-2-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)benzo[d]thiazole-6-carboxylic acid. With the exception of the direct conjugate, DNA gyrase A and DNA gyrase B inhibitors were linked via various linkers (glycine, β-alanine, ether) at the C7 piperazine of ciprofloxacin. In an enzyme assay we evaluated the inhibitory activity of the final compounds against DNA gyrase from Escherichia coli, for the most active compound also from Staphylococcus aureus. We also determined their antibacterial activity against three Gram-positive, five Gram-negative bacterial strains and three additional E. coli strains: wild-type strain and two mutated bacterial strains. The most active compound of the synthesized series was compound 25 with a half-maximal inhibitory concentration (IC50) of 14 ± 9 nM against DNA gyrase from E. coli and an IC50 value of 1.09 µM against DNA gyrase from S. aureus. It contains a DNA gyrase B inhibitor with added benzyloxy fragment and it is connected to ciprofloxacin through an ether linker. The most potent antibacterial activity was displayed by compound 12 with minimum inhibitory concentration (MIC) values between 0.5-4 µg/mL against Klebsiella pneumoniae, E. coli and Enterobacter cloacae spp. cloacae and with the MIC value of 16 µg/mL against Enterococcus faecalis. With the synthesized compounds we reached activity against both subunits of DNA gyrase since the compounds are active against resistant strains that carry mutations either at subunit DNA gyrase A or DNA gyrase B. The results of the master\u27s thesis provide important information for the discovery of new dual-targeting inhibitors that inhibit both subunits of DNA gyrase and thereby reducing the probability for the development of bacterial resistance