Sinteza in vrednotenje 3,4-dikloro-5-metil-N-fenilpirolamidnih zaviralcev DNA-giraze B

Križaj, Nika (author), Zidar, Nace (mentor)

Odpornost bakterij proti antibiotikom predstavlja enega večjih svetovnih problemov, saj se rezistenca pojavi kmalu zatem, ko je antibiotik uveden v terapijo. Odkritje novega antibiotika bi predstavljalo velik napredek v boju proti bakterijski odpornosti. Ena izmed tarč z dobrim potencialom je DNA-giraza, ki spada v skupino encimov imenovanih topoizomeraze. DNA-giraza je zgrajena iz dveh podenot A in dveh podenot B, ki predstavljata tarči delovanja protibakterijskih učinkovin. V okviru magistrske naloge smo sintetizirali tri zaviralce DNA-giraze B s 3,4-dikloro-5-metil-N-fenilpirolamidnim skeletom. Z namenom tvorbe dodatnih in močnejših interakcij v vezavnem mestu encima, smo na osnovni skelet vezali različne substituente. Z določanjem srednje zaviralne koncentracije (IC50) na DNA-girazi iz Escherichia coli, smo ovrednotili aktivnost sintetiziranih spojin in vpliv različnih substituentov na njihovo vezavo. Sinteza spojin je potekala po treh ločenih sinteznih poteh. Tekom sinteze smo spojinam določili identiteto, fizikalno-kemijske lastnosti in čistost s kromatografskimi (tankoplastna in kolonska kromatografija, tekočinska kromatografija visoke ločljivosti) in spektroskopskimi metodami (jedrska magnetna resonanca, infrardeča spektroskopija, masna spektrometrija) ter merjenjem temperature tališča. Kot najbolj aktivna spojina se je izkazala spojina 20 (IC50 = 243 nM), ostali spojini (12 in 7) sta bili slabše aktivni (IC50 > 1µM). Vzrok za slabšo aktivnost je mogoče pripisati vezavi benzilnega (7) in metoksi etilnega (12) substituenta na dušikov atom amidne skupine, saj ta dva substituenta ne tvorita ugodnih interakcij v t.i. lipofilnih tleh encima. Močnejše delovanje so imele spojine, ki so imele alifatske oz. aromatske substituente vezane neposredno na orto mesto (glede na pirolamidni del) osrednjega fenilnega obroča. Spojina 20, s sulfonamidno skupino na desnem delu molekule, je ohranila del aktivnosti, vendar je bila ta slabša v primerjavi s spojinami, ki imajo na tem mestu vezane kisle heterocikle, npr. 1,3,4-oksadiazol-2-onski obroč ali tetrazol. Čeprav v okviru magistrske naloge nismo uspeli pripraviti spojin z močno zaviralno aktivnostjo na encimu DNA-giraza, smo pridobili informacije, ki bodo uporabne pri načrtovanju novih zaviralcev DNA-giraze B.

• Language: Slovenian
• Keywords: DNA-giraza, zaviralec, protibakterijska učinkovina, N-fenilpirolamid, načrtovanje učinkovin
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2022
• PID: 20.500.12556/RUL-137358
• Publication date in RUL: 14.06.2022

Secondary language

• Language: English
• Title: The synthesis and evaluation of 3,4-dichloro-5-methyl-N-phenylpyrrolamide inhibitors of DNA gyrase B

Abstract

Antibiotic resistance is one of the world's biggest problems. Resistance develops shortly after antibiotics are introduced into therapy. The discovery of a new antibiotic would be an important step in the fight against antibiotic resistance. One of the potential targets is DNA gyrase, which belongs to a group of enzymes called topoisomerases. DNA gyrase consists of two subunits A and two subunits B, which are targets for antibacterial agents. In this master's thesis, we synthesized three DNA gyrase B inhibitors with 3,4-dichloro-5-methyl-N-phenylpyrrolamide core structure. To form additional and stronger interactions at the enzyme binding site, we introduced different substituents to the core structure. By determining the half-maximal inhibitory concentration (IC50) on Escherichia coli DNA gyrase, we evaluated the activity of the synthesized compounds and the effect of the different substituents on binding affinity. The synthesis was carried out by three synthetic routes. During the synthesis, the identity, physicochemical properties, and purity of the compounds were determined by chromatographic (thin-layer and column chromatography, high-performance liquid chromatography) and spectroscopic methods (nuclear magnetic resonance, infrared spectroscopy, mass spectrometry), and by melting point measurements. Compound 20 proved to be the most active (IC50 = 243 nM), while the other compounds (7 and 12) were less active (IC50 > 1µM). The reason for the lower activity can be attributed to the benzyl (7) and methoxyethyl (12) substituents attached to the nitrogen atom of the amide group, as they do not form favorable interactions in the so-called lipophilic floor of the enzyme compared to the compounds with aliphatic or aromatic substituents attached directly to the ortho site (with respect to the pyrrolamide moiety) of the central phenyl ring. Compound 20, with the sulfonamide group attached to the right side of the molecule, retained some activity, but it was lower compared to compounds with acidic heterocycles, e.g., the 1,3,4-oxadiazol-2-one ring or tetrazole. Although we did not prepare compounds with improved inhibitory activity against DNA gyrase, we obtained information that will be useful in the design of new DNA gyrase B inhibitors.

• Keywords: DNA gyrase, inhibitor, antibacterial agent, N-phenylpyrrolamide, drug design