Dizajniranje, sinteza i in vitro ispitivanja derivata propiofenona kao potencijalnih inhibitora enzima HIV-1 proteaze

Abstract

humane imunodeficijencije (HIV), kao i njegovog supstrata, dovelo je do razvoja specifičnih inhibitora. Razvoj i klinička primjena inhibitora HIV-1 proteaze, gotovo odmah nakon identifikacije ovog enzima, kao ciljnog mjesta dejstva, predstavlja jedan od najuspješnijih primjera racionalnog dizajna ljekova. Ispitivanja su pokazala da anti-HIV-1 proteaznu aktivnost, između ostalih, imaju i propiofenonski derivati (halkoni).U postupku ispitivanja supstituisanih derivata halkona koji pokazuju anti-HIV-1 proteaznu aktivnost ovo istraživanje se baziralo na docking studijama. Primjenom kompjuterskog programa ChemOffice v7.0 Ultra optimizovani su komercijalno dostupni inhibitori HIV proteaze, osnovne trans-1,3-diaril-2-propen-1-onske strukture i mono, di i tri supstituisani derivati trans-1,3-diaril-2-propen-1-onske strukture. Ispitivanje potencijalnih interakcija halkona (derivata trans-1,3-diaril-2-propen-1-on) sa aminokiselinama u aktivnom mjestu HIV-1 proteaze kao i uticaja različitih supstituenata na pozicioniranje i formiranje dodatnih interakcija u aktivnom mjestu je izvedeno docking studijama, primjenom programa AutoDock Vina. Validacija izvedene docking studije je izvršena poređenjem konformacije liganda iz kokristala sa ciljnim mjestom (koja je određena primjenom X-ray difrakcije) sa konformacijom iz docking proračuna (predviđena konformacija liganda na vezivnom mjestu enzima) pri čemu je dobijena RMSD od 0,71 Å potvrdila uspješno predviđanje. Za vizuelizaciju interakcija ispitivanih jedinjenja sa ciljnim mjestom korišćen je računarski program Discovery Studio Visualizer 2017. Rezultati docking-a ukazuju da stabilizaciji kompleksa halkon-enzim u najvećoj mjeri doprinosi orto- disupstitucija prstena B fenolnim grupama. Broj vodoničnih veza je veći, što ide u prilog podatku da povećanje broja vodoničnih veza smanjuje mogućnost mutacija. Prsten B je potrebno supstituisati grupama sa jakim –I efektom (fluoro grupe), kao i voluminoznim grupama sa jakim –I efektom (trifluorometil grupa). Na osnovu sprovedenih docking studija izvršen je izbor polaznih orto- i para-, mono- i di- supstituisanih benzaldehida kao i orto- i para-, mono- i di- supstituisanih acetofenona u cilju sinteze derivata halkona koji su pokazali najbolju aktivnost. Reakcijom aldolne kondenzacije (Claisen-Schimdt-ova reakcija) polaznih jedinjenja dobijeno je 17 novih supstituisanih derivata halkona. Za prečišćavanje dobijenih jedinjenja korišćena je preparativna dry flash hromatografija na koloni ili preparativna tankoslojna hromatografija na silikagelu. Jedinjenja su dodatno prečišćena prekristalizacijom. Fizičko-hemijska karakterizacija sintetisanih jedinjenja je izvršena određivanjem tačke topljenja i primjenom instrumentalnih metoda: IC, NMR i MS-MS spektroskopijom.Korišćenjem tri RP-TLC sistema (acetonitril-voda, etanol-voda i aceton-voda) izračunati su parametri, S i C0 na osnovu kojih je procijenjeno retenciono ponašanje sintetisanih jedinjenja. Eksperimentalno dobijene vrijednosti particionih koeficijenata (LogP) su korelisane sa kompjuterski izračunatim LogP vrijednostima, dobijenim primjenom programa ChemDraw 17 i MarvinSketch. Najveći stepen podudarnosti postoji između C0 vrijednosti dobijenih za hromatografski sistem acetonitril-voda i izračunatih LogP vrijednosti (r vrijednost od 0,80 do 0,84) pa se jednačina koja opisuje ovu zavisnost može upotrijebiti za predviđanje retencije novih halkona. Istovremeno i sistem acetonitril-voda je odabran kao najpouzdaniji za detaljnije proučavanje odnosa između strukture i retencije kongenerisanih halkona. Formirana su tri QSRR modela korišćenjem C0, koji je određen u RP-TLC sistemu acetonitril-voda, kao zavisna promjenljiva i odabrani molekulski deskriptori, kao nezavisne promjenljive...

Detailed knowledge of the structure of HIV-1 protease, an enzyme which plays a key role in the life cycle of the virus, as well as its substrate, has led to the development of specific inhibitors. The development and clinical application of HIV-1 protease inhibitors, almost immediately after the identification of the enzyme, as a drug target, is one of the most successful examples of rational drug design. Studies have shown that, among others, propiophenone derivatives (chalcones) have anti-HIV-1 protease activity. Study of substituted chalcone derivatives showing anti-HIV-1 protease activity was based on docking. Using the computer program ChemOffice v7.0 Ultra, commercially available HIV protease inhibitors, basic trans-1,3-diaryl-2-propen-1-one structures and mono, di and three substituted trans-1,3-diaryl-2-propen-1-one derivatives were optimized. Investigation of potential interactions of chalcone (trans-1,3-diaryl-2-propen-1-one derivatives) with amino acids in the active site of HIV-1 protease as well as the influence of different substituents on the positioning and formation of additional interactions in the active site was performed by docking using the AutoDock Vina program. Validation of the performed docking study was carried out by comparing the conformation of the ligand from the co-crystal with the target site (determined by X-ray diffraction) with the conformation from the docking calculation (docked pose) where the obtained RMSD of 0.71 Å confirmed the successful prediction. The computer program Discovery Studio Visualizer 2017 was used to visualize the interactions of the tested compounds with the target site. The docking results indicate that the ortho- disubstitution of ring B by phenolic groups contributes the most to the stabilization of the chalcone enzyme complex. The number of hydrogen bonds is higher, which supports the fact that increasing the number of hydrogen bonds reduces the possibility of mutations. Ring B should be substituted with groups with strong -I effect (fluoro groups) as well as voluminous groups with strong -I effect (trifluoromethyl group). Based on the conducted docking studies, the initial ortho- and para-, mono- and di-substituted benzaldehydes as well as ortho- and para-, mono- and di-substituted acetophenones were selected in order to synthesize the chalcone derivatives that showed the best activity. The aldol condensation reaction (Claisen-Schimdt reaction) of the starting compounds gave 17 new substituted chalcone derivatives. For the purification of the obtained compounds, preparative column flash chromatography or preparative thin layer chromatography on silica gel was used. The compounds were further purified by recrystallization. Physico-chemical characterization of the synthesized compounds was performed by determining the melting point and applying instrumental methods: IC, NMR and MS-MS spectroscopy. Using three RP-TLC systems (acetonitrile-water, ethanol-water and acetone-water), the parameters, S and C0, were calculated on the basis of which the retention behavior of the synthesized compounds was estimated. The experimentally obtained values of partition coefficients (LogP) were correlated with computer-calculated LogP values, obtained using ChemDraw 17 and MarvinSketch. The highest degree of agreement exists between the C0 values obtained for the acetonitrile-water chromatographic system and the calculated LogP values (r value from 0.80 to 0.84), so the equation describing this dependence can be used to predict the retention of new chalcones. At the same time, the acetonitrile-water system was selected as the most reliable for a more detailed study of the relationship between the structure and retention of chalcones. Three QSRR models were formed using C0, which was determined in the RP-TLC system acetonitrile-water, as a dependent variable, and selected molecular descriptors, as independent variables. The obtained statistical parameters, r = 0.83, R2 pred = 0.94 as well as the result of cross-validation Q2 = 0.68 indicate a high prognostic ability of the SVM (C0) model...