Design and synthesis of 3,4-dichloro-5-methylpyrrolamide DNA gyrase B inhibitors

Po odkritju prvega znanega antibiotika – penicilina – se je začela doba množičnega odkrivanja novih protibakterijskih učinkovin, ki so močno pripomogle k izboljšanju življenjskega standarda. Sčasoma se je odkrivanje novih učinkovin upočasnilo, vedno večja težava pa je postala tudi zelo hitro razvijajoča se odpornost bakterij na obstoječe antibiotike. Danes je razvoj novih protibakterijskih učinkovin osredotočen na odkrivanje novih tarč, novih mehanizmov delovanja na že poznane tarče, oziroma na optimizacijo obstoječih razredov učinkovin. Ena izmed najbolj obetavnih tarč je encim DNA-giraza, ki sodi v skupino topoizomeraz tipa IIa, in sodeluje pri podvajanju in prepisovanju DNA tako, da katalizira uvedbo in odstranitev dodatnega zvitja molekule DNA. DNA-giraza je tetramerni encim, ki je sestavljen iz dveh podenot DNA-giraza A (GyrA) in dveh podenot DNA-giraza B (GyrB). GyrA katalizira cepitev in ponovno združitev dvojnoverižne molekule DNA, energijo za to reakcijo pa pridobi s pomočjo hidrolize molekule ATP, katere vezavno mesto se nahaja na podenoti GyrB. V okviru magistrske naloge smo s pomočjo strukturno podprtega načrtovanja načrtovali in sintetizirali trinajst potencialnih zaviralcev DNA-giraze B s skupnim 3,4-dikloro-5-metilpirolamidnim fragmentom. Vse spojine smo ovrednotili z encimskimi testi na DNA-girazo iz bakterije Escherichia coli, nekatere pa tudi na topoizomerazi IV iz bakterij E. coli in Staphylococcus aureus. Spojinam smo določili tudi protibakterijsko aktivnost na dveh grammpozitivnih bakterijskih sevih Enterococcus faecalis in S. aureus ter na gramnegativnem bakterijskem sevu Escherichia coli. Spojini (4-(3,4-dikloro-5-metil-1H-pirol-2-karboksamido)-3-izopropoksibenzoil)glicin (18c) in 3,4-dikloro-5-metil-N-(4-(((5-okso-4,5-dihidro-1,3,4-oksadiazol-2-il)metil)karbamoil)fenil)-1H-pirol-2-karboksamid (20a) sta pokazali zelo dobri zaviralni aktivnosti na DNA-girazo iz bakterije E. coli, saj sta bili srednji inhibitorni koncentraciji za spojino 18c 62,0 nM, za spojino 20a pa 60,8 nM. Najboljši rezultat zaviranja bakterijske rasti je pokazala spojina 20a, in sicer 64 %, izmerjen na bakterijskem sevu S. aureus, in 51 % na bakterijskem sevu E. coli po 24-urni izpostavitvi 50 μM koncentraciji testirane spojine. Iz pridobljenih rezultatov lahko povzemamo, da so spojine, sintetizirane v okviru te magistrske naloge, pomembna osnova za nadaljnji razvoj zaviralcev DNA-giraze B, vendar bo potrebno njihove lastnosti v prihodnje še optimizirati, predvsem z namenom doseganja boljšega protibakterijskega delovanja.After the discovery of the first known antibiotic – the penicillin – the so-called “antibiotic era” began. This was a time of very fast development of antibiotics, which greatly improved the quality of human life. After the initial period, the rate of development of new antibacterials gradually slowed down, which was accompanied by another problem – the increasing resistance of bacteria to known antibiotics. The development of new antibacterials now focuses on discovering new targets, new mechanisms of action on the existing targets, and on optimizing the properties of marketed drugs. One of the most promising antibacterial targets is DNA gyrase, an enzyme that belongs to the type IIa topoisomerases, and is involved in replication and transcription of DNA by introducing and removing DNA supercoils. It is a tetrameric enzyme that consists of two DNA gyrase A subunits (GyrA) and two DNA gyrase B subunits (GyrB). GyrA is responsible for breaking and reuniting the two-stranded DNA molecule, while GyrB hydrolyses the ATP molecule and provides the energy for the action of GyrA. With the aid of structure based design we designed and synthesized thirteen new potential DNA gyrase B inhibitors with 3,4-dichloro-5-methylpyrrolamide substructures. All of the prepared compounds were tested against DNA gyrase from Escherichia coli, and some of them also against topoisomerases IV from E. coli and Staphylococcus aureus. The compounds were additionally evaluated for their antimicrobial properties against two Gram-positive bacterial strains, Enterococcus faecalis and S. aureus and one Gram-negative bacterial strain, E. coli. The compounds that were the most potent in inhibiting the DNA gyrase of E. coli were (4-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-3-isopropoxybenzoyl)glycine (18c) and 3,4-dichloro-5-methyl-N-(4-(((5-oxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)methyl)carbamoyl)phenyl)-1H-pyrrole-2-carboxamide (20a), with 62,0 nM and 60,8 nM IC50 values, respectively. Compound 20a displayed a promising, 64 %, inhibition of growth of S. aureus bacteria and 51 % inhibition of growth of E. coli bacteria after 24 h exposure to 50 μM concentration of the tested compound. To summarize, the results of our work represent an important contribution to the development of new GyrB inhibitors, however, especially with the aim of improving their antibacterial effects, their structures should still be optimized in the future