Sinteza konjugatov benzotiazolpirolamidnih zaviralcev DNA-giraze B s siderofori

Kocmur, Petra (author), Tomašič, Tihomir (mentor)

Zaradi množične uporabe protibakterijskih učinkovin se je pojavila odpornost bakterij proti protibakterijskim učinkovinam, ki z leti dobiva vse večje razsežnosti. Bakterijska DNA-giraza in topoizomeraza IV sta encima, ki uravnavata topologijo DNA pri njenem podvajanju. Oba encima imata podobno strukturo, zato lahko z isto učinkovino zaviramo oba encima hkrati in na ta način dosežemo protibakterijsko delovanje. V primeru takšnih učinkovin se razvoj odpornosti bakterij zaradi mutacij tarče upočasni. Siderofori so molekule, ki vežejo železo v okolju in ga prenesejo v bakterijsko celico. Ta sistem privzema lahko zlorabimo s strategijo trojanskega konja, in sicer tako, da bakterija privzame siderofor, na katerega je vezana protibakterijska učinkovina. Na ta način lahko povečamo privzem učinkovine v bakterijo. Naš cilj je bil sinteza novih potencialnih zaviralcev DNA-giraze B, in sicer konjugatov zaviralcev encima z mimetiki sideroforov. Slednje smo v več stopnjah sintetizirali iz kojične kisline, kot zaviralce DNA-giraze B pa smo uporabili derivate benzotiazolpirolamida. Za spremljanje poteka reakcij in identifikacijo spojin smo uporabljali kromatografske in spektroskopske metode. Poleg znanega zaviralca DNA-giraze smo želeli pripraviti tudi novega, vendar smo imeli težave pri uvedbi hidroksilne skupine na benzotiazolni obroč. Konjugacija zaviralca s sideroforom je bila uspešna in tako smo pridobili končni spojini 21 in 22. Določili so jima zaviralno aktivnost na rekombinantni DNA-girazi ter aktivnost proti po Gramu pozitivnim in negativnim bakterijam. Nobena od pripravljenih spojin ni dosegla želenega protibakterijskega delovanja. Na rekombinanti DNA-girazi sta dosegli zaviralno aktivnost v mikromolarnem območju, želeli pa smo doseči nanomolarno območje. Vzrok za nizko aktivnost je najverjetneje v tem, da siderofora končnih spojin v primerjavi s podobnimi predhodno sintetiziranimi spojinami, ki imajo večjo aktivnost, tvorita šibkejše vezi s tarčo. Izsledki magistrske naloge pomembno prispevajo k nadaljnjemu razvoju potencialnih protibakterijskih učinkovin z uporabo strategije trojanskega konja, s katero želimo povečati privzem zaviralca v bakterijsko celico z izkoriščanjem sistema za transport železa.

• Language: Slovenian
• Keywords: DNA-giraza, siderofor, zaviralec, strategija trojanskega konja, konjugat
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-107973
• Publication date in RUL: 11.06.2019

Secondary language

• Language: English
• Title: Synthesis of conjugates of benzothiazolepyrrolamide-based DNA gyrase B inhibitors with siderophores

Abstract

Due to the extensive use of antibacterial agents bacterial resistance to antibiotics has emerged, which has over the years become a serious health threat. Bacterial DNA gyrase and topoisomerase IV are enzymes that regulate the topology of DNA during its replication. Both enzymes have a similar structure, which offers the possiblity of dual-targeting with one antibacterial drug. In this way, the development of bacterial resistance due to target mutations against such an agent slows down. Siderophores are molecules that bind iron in the environment and transfer it to a bacterial cell. This system of iron uptake can be abused by the Trojan horse strategy in such a way that the bacteria uptake the siderofor-antibacterial agent conjugate. In this way, we can increase the uptake of the active substance into the bacterium. Our goal was to synthesise new DNA-gyrase inhibitors and their conjugates with siderophore mimetics. The latter were prepared from the kojic acid in several reaction steps, and the benzotiazolpyrrolamide derivatives were used as inhibitors of DNA gyrase B. Chromatographic and spectroscopic methods were used to monitor the course of the reactions and identify the compounds. In addition to the known DNA-gyrase inhibitor, we also wanted to prepare a novel inhibitor, but the synthesis of 5-hydroxy substituted benzothiazole ring failed. The conjugation of the siderophore mimetic to the known DNA gyrase inhibitor was successful and final compounds 21 and 22 were obtained. The inhibitory activity on recombinant DNA-gyrase and activity against Gram-positive and Gram-negative bacteria were determined, but unfortunately, none of the prepared compounds achieved the desired antibacterial activity. On DNA recombinants, the inhibitory activity in the micromolar range was achieved, but we wanted to achieve a nanomolar range. The cause of low activity is most likely that the siderophores of the final compounds, in comparison with similar previously synthesized compounds having a higher activity, create weaker bonds to the target. The results of the master's thesis make a significant contribution to the further development of potential antibacterial agents using the Trojan horse strategy, which aims to increase the entry of the inhibitor into the bacterial cell by exploiting the iron transport system.

• Keywords: DNA gyrase, siderophore, inhibitor, Trojan horse strategy, conjugate