Sinteza in vrednotenje N-fenilpirolamidnih zaviralcev DNA-giraze B s piperidin-3-aminskim in pirolidin-3-aminskim substituentom

Rašić, Una (author), Zidar, Nace (mentor)

Mikroorganizmi postajajo vedno bolj odporni na protimikrobne snovi. Kljub prizadevanju številnih podjetij za razvoj novih protimikrobnih učinkovin, se je število novih zdravil v zadnjih letih izjemno zmanjšalo, pri čemer je prišlo na tržišče le nekaj protimikrobnih učinkovin iz novih strukturnih razredov. Zaradi pomembne vloge pri preživetju celic, so topoizomeraze pomembna tarča raziskav za odkrivanje protibakterijskih in protitumornih učinkovin. Bakterije izražajo dve različni topoizomerazi tipa IIA, znani kot DNA-giraza in topoizomeraza IV. Ta dva encima sta v evkariontih odsotna in sta hkrati bistvena za preživetje bakterij. Učinkovine, ki se vežejo na DNA-girazo, delujejo preko dveh glavnih mehanizmov, nas pa je zanimal mehanizem zaviranja ATP-vezavnega mesta na DNA-girazi B. V zadnjih letih so odkrili veliko strukturno raznolikih razredov zaviralcev DNA-giraze B, ki imajo dobre aktivnosti na tarčo in protibakterijske lastnosti, vendar imajo nekatere pomanjkljivosti, kot so neugodne fizikalno-kemijske lastnosti ali toksičnost. Na podlagi znanih struktur smo zasnovali dve seriji ATP-kompetitivnih zaviralcev DNA-giraze B. Prva serija je na benzenovem obroču imela vezan piperidin-3-aminski substituent, druga serija pa je imela pripet pirolidin-3-aminski substituent. Obema serijama je bil skupen N-fenilpirolamidni del molekule. Učinkovitost pripravljenih spojin smo ovrednotili z izvedbo bioloških testov na encimih DNA-giraza in topoizomeraza IV iz bakterij E. coli in S. aureus, protibakterijsko aktivnost pa smo določili na osmih različnih po Gramu pozitivnih in po Gramu negativnih bakterijskih sevih. Najboljšo zaviralno aktivnost na encim DNA-giraza je dosegla spojina 14 s srednjo zaviralno koncentracijo (IC50) 2,59 nM. Pri spojini 8 vrednost IC50 prav tako sega v nanomolarno območje in znaša 27,2 nM. Spojini sta dosegli odlične rezultate tudi pri protibakterijskem testiranju. Minimalna zaviralna koncentracija (MIK90) je bila pri spojinah 8 in 14 najnižja pri po Gramu negativni bakteriji K. pneumoniae in je znašala 0,125 µM oz. 0,03125 µM. Spojina 14 je dosegla boljše zaviranje vseh bakterijskih sevov od spojine 8, njena vrednost MIK90 pri bakteriji E. coli pa je znašala 4 µM. N-Fenilpirolamidne spojine, ki smo jih pripravili v okviru diplomske naloge, so se izkazale kot učinkoviti zaviralci DNA-giraze B z zelo dobim protibakterijskim delovanjem. Pripravljene spojine tako predstavljajo zelo dobro osnovo za načrtovanju spojin, ki bi tvorile še močnejše interakcije v ATP-vezavnem mestu encima in bi imele ustrezne fizikalno-kemijske lastnosti.

• Language: Slovenian
• Keywords: DNA-giraza B, protibakterijska učinkovina, N-fenilpirolamid, piperidin, pirolidin
• Work type: Bachelor thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-121343
• Publication date in RUL: 05.10.2020

Secondary language

• Language: English
• Title: Synthesis and evaluation of N-phenylpyrrolamides with piperidin-3-amine and pyrrolidin-3-amine substitutents as inhibitors of DNA gyrase B

Abstract

Microorganisms are becoming increasingly resistant to antimicrobials. Despite the efforts of many companies to develop new antimicrobials, the number of new drugs has declined dramatically in recent years, with only a few antimicrobials from the new structural classes entering the market. Due to their important role in cell survival, topoisomerases are important research targets for the discovery of antibacterial and antitumor agents. Bacteria express two different type IIA topoisomerases, DNA gyrase and topoisomerase IV. These two enzymes are absent in eukaryotes and are essential for bacterial survival. Drugs that bind to DNA gyrase act through two main mechanisms, among which we focused on the inhibition of the ATP-binding site on DNA gyrase B. In recent years, many structurally diverse classes of DNA gyrase B inhibitors have been discovered that have good on-target activities and antibacterial properties, but have some drawbacks such as nonoptimal physicochemical properties or toxicity. Based on the known structures of DNA gyrase B inhibitors, two series of ATP-competitive DNA gyrase B inhibitors were designed. The first series had piperidine-3-amine substituent bound on the benzene ring, and the second series had a pyrrolidine-3-amine substituent attached. The N-phenylpyrrolamide portion of the molecule was common to both series. The efficacy of the prepared compounds was evaluated by performing biological tests on DNA gyrase and topoisomerase IV enzymes from E. coli and S. aureus, and antibacterial activity was determined on eight different Gram-positive and Gram-negative bacterial strains. The strongest inhibitory activity on the DNA gyrase enzyme was achieved by compound 14 with a mean inhibitory concentration (IC50) of 2.59 nM. For compound 8, the IC50 value was also in the nanomolar range and was 27.2 nM. The compounds also achieved excellent results in antibacterial testing. The minimum inhibitory concentrations (MIC90) of compounds 8 and 14 were the lowest in Gram-negative K. pneumoniae, 0.125 μM and 0.03125 µM respectively. Compound 14 showed stronger inhibition of all bacterial strains compared to compound 8, which had a MIC90 value of 4 µM in E. coli. The N-phenylpyrrolamide compounds prepared in the framework of this diploma thesis proved to be effective inhibitors of DNA gyrase B with very good antibacterial activities. The prepared compounds thus represent a very good basis for the design of compounds that would form even stronger interactions in the ATP-binding site of the enzyme and would have appropriate physicochemical properties.

• Keywords: DNA gyrase B, antibacterial agent, N-phenylpyrrolamide, piperidine, pyrrolidine