Design, synthesis and in vitro testing of propiophenone derivatives as potential inhibitors of the enzyme HIV-1 protease

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..

Halkoni potencijalni inhibitori HIV‐1 proteaze

The discovery of HIV and the study of molecular mechanisms crucial for the virus replication cycle have led to the identification of important protein structures - potential targets of drug action in AIDS therapy. One of the most significant discoveries is HIV-1 protease (PR), an enzyme that plays a key role in the HIV replication cycle (1). This study aimed to test and demonstrate the interactions of newly synthesized chalcones (1,3-diaryl-2- propen-1-one) as well as three commercial drugs (lopinavir, ritonavir and darunavir) in the active site of HIV-1 protease using in sillico methods. and that the results obtained correlate with the results of in vitro tests on the enzyme itself. The twenty structurally similar chalcone derivatives were synthesized and their physico-chemical characterization was performed. Docking calculations were performed using the Autodock Wine program in the 3D structure of the enzime catalytic site (pdb code: 6B36). The inhibition of enzyme activity was monitored by fluorimetric method (2). The obtained results revealed that all compounds showed anti-HIV-1 protease activity. Compound C1 showed the highest inhibitory activity with an IC 50 values of 0.001 μM which is comparable with commercial product Darunavir. The results obtained indicate that the synthesized compounds can be classified as potential anti-HIV-1 protease inhibitors. Further research is focused on testing the ADMET properties of the synthesized compounds as well as the synthesis of their analogues for which in silico tests have shown satisfactory results.Otkrić e HIV-a i istraživanje molekularnih mehanizama ključnih za ciklus replikacije virusa dovelo je do identifikacije važnih proteinskih struktura - potencijalnih ciljnih mesta dejstva lekova u terapiji AIDS-a. Jedno od najznačajnijih otkrića je HIV-1 proteaza (PR), enzim koji ima ključnu ulogu u ciklusu replikacije HIV-a (1). Ova studija imala je za cilj da primenom in sillico metoda ispita i prikaže interakcije novosintetisanih halkona (1,3-diaril-2- propen-1-ona) kao i tri komercijalna leka (lopinavira, ritonavira i darunavira) u aktivnom mestu HIV-1 proteaze i da dobijene rezultate koreliše sa rezultatima in vitro testova na samom enzimu. Sintetisano je dvadeset strukturno sličnih derivata halkona i izvršena njihova fizičko-hemijska karakterizacija. Docking studije izvedene su u programu Autodock Vine u 3D strukturi enzimskog katalitičkog mesta (pdb kod: 6B36). Inhibicija enzimske aktivnosti određena je primenom fluorimetrijske metode (2). Dobijeni rezultati ukazuju da svih 20 jedinjenja ispoljava anti-HIV-1 proteaznu aktivnost. Jedinjenje HNT1 je pokazalo najveć u inhibitornu aktivnost sa vrednostima IC50 od 0,001 μM što je uporedivo sa komercijalnim proizvodom darunavirom. Dobijeni rezultati ukazuju da se sitetisana jedinjenja mogu klasifikovati kao potencijalni anti-HIV-1 proteazni inhibitori. Dalje istraživanje je usmereno na ispitivanju ADMET osobina sintetisanih jedinjenja kao i sintezi njihovih analoga za koje su in silico ispitivanja pokazala zadovoljavajuće rezultate.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra

Application of liquid chromatography in defining the interaction of newly synthesized chalcones and related compounds with human serum albumin

Defining the interaction of newly synthesized compounds with plasma proteins is an important step in the drug development process. Chromatographic techniques can be successfully used in predicting the biopharmaceutical and pharmacokinetic properties of newly synthesized compounds. The aim of this study is to investigate and isolate the most important molecular properties that affect the interaction of 20 newly synthesized chalcones and commercial compounds (lopinavir, ritonavir, darunavir and ivermectin) with human serum albumin (HSA). The retention behaviour of the selected compounds was tested on a CHIRALPAK®HSA column. A mixture of phosphate buffer (pH 7.0) and isopropanol (80:20 volume ratio) was used as the mobile phase, and the support vector method was used to form the quantitative structure retention relationship (QSRR) model. Based on the obtained values of retention parameters, it was observed that halogenated derivatives show the strongest, and methylated chalcone derivatives the weakest interaction with HSA. By correlating the retention and physicochemical properties of the tested compounds, it was shown that the structural (SDSCH) and electronic properties (MAXQ, EEM_F1) groups have the greatest influence on the retention behaviour and the interaction of the tested compounds with HSA. The obtained QSRR model can be applied in the prediction of the retention characteristics of new, structurally related chalcone derivatives on HSA stationary phase

Применатечнехроматографијеудефинисањуинтеракцијановосинтетисаниххалконаисроднихсупстанцисахуманимсерумскималбуминима

Defining the interaction of newly synthesized compounds with plasma proteins is an important step in the drug development process. Chroma-tographic techniques can be successfully used in predicting the biopharmaceut-ical and pharmacokinetic properties of newly synthesized compounds. The aim of this study is to investigate and isolate the most important molecular pro-perties that affect the interaction of 20 newly synthesized chalcones and com-mercial compounds (lopinavir, ritonavir, darunavir and ivermectin) with human serum albumin (HSA). The retention behaviour of the selected compounds was tested on a CHIRALPAK®HSA column. A mixture of phosphate buffer (pH 7.0) and isopropanol (80:20 volume ratio) was used as the mobile phase, and the support vector method was used to form the quantitative structure retention relationship (QSRR) model. Based on the obtained values of retention para-meters, it was observed that halogenated derivatives show the strongest, and methylated chalcone derivatives the weakest interaction with HSA. By correl-ating the retention and physicochemical properties of the tested compounds, it was shown that the structural (SDSCH) and electronic properties (MAXQ, EEM_F1) groups have the greatest influence on the retention behaviour and the interaction of the tested compounds with HSA. The obtained QSRR model can be applied in the prediction of the retention characteristics of new, structurally related chalcone derivatives on HSA stationary phase.Дефинисањеинтеракцијеновосинтетисанихједињењасапротеинимаплазмејеважанкоракупроцесуразвојалекова. Хроматографскетехникесемогууспешнo корис-титиупредикцијибиофармацеутскихифармакокинетичкихособинановосинтетисанихједињења. Циљовоградајеиспитивањеииздвајањенајзначајнијихмолекулскихособинакојеутичунаинтеракцију 24 новосинтетисаниххалконаињимасроднихједињењасахуманимсерумскималбумином (HSA). Ретенционопонашањеодабранихједињењајеиспитанона CHIRALPAK®HSA колони. Каомобилнафазакоришћенајесмешафос-фатногпуфера (pH 7,0) иизопропанола (запр. однос 80:20). Уформирању QSRR моделакоришћенајеметодавектораподршке. Наосновудобијенихвредностиретенционихпараметарауоченоједахалогенованидериватипоказујунајјачу, аметилованидериватихалконанајслабијуинтеракцијуса HSA. Доводећиукорелацијуретенцијуифизичко-хе-мијскасвојстваиспитиванихједињењапоказалоседаструктурне (SDSCH) иелек-тронскеособине (MAXQ,EEM_F1) групанајвишеутичунаретенционопонашањеиинтеракцијуиспитиванихједињењаса HSA. Добијени QSRR моделсеможеприменитиупредикцијиретенционихкарактеристиканових, структурно-сроднихдериватахалконана HSA стационарнојфази

Evaluation of the lipophilicity of chalcones by RP-TLC and computational methods

Retention behaviour of twenty-one chalcones synthesized in our laboratory was tested in three thin-layer chromatography (RP-TLC) systems (acetonitrile–water, ethanol–water and acetone–water) and chromatography parameters R0 M, S and C0 were calculated. The most suitable RP-TLC system (acetonitrile–water) and chromatography parameter (C0) for lipophilicity prediction of tested compounds were selected on the basis of the highest correlations with calculated logP values. In selected system, compound 12 had the highest, whereas 47 had the lowest C0 value. QSRR analysis was performed and three models representing relationships between C0 and selected molecular descriptors were created—MLR(C0), PLS(C0) and SVM(C0). Interpretation of molecular descriptors which form statistically the most reliable SVM(C0) model identified the most important structural and physico-chemical properties that influence retention behaviour of tested compounds. In addition, descriptors with the highest influence on R0 M as well as on C0 calculated in the remaining two RP-TLC systems were dentified and interpreted

Differences in antioxidant potential of chalcones in human serum: In vitro study

An imbalance between oxidants and antioxidants in favour of oxidants, potentially leading to damage, is termed oxidative stress. Antioxidants (AO), either enzymatic or non-enzymatic, are the ones that can reduce diverse effects of pro-oxidants such as DNA, proteins and lipids damage. Chalcones (1,3-diaryl-2-propen- 1-ones) are open chain flavonoids that are widely biosynthesized in plants. Aim of this study was to test antioxidative potency of 15 chalcones (Chs) in in vitro model in serum (native conditions), so as with exogenously induced oxidative stress. Material and methods: Oxidative stress was induced in serum samples of healthy individuals with 0.25 mmol/L terc-buthyl-hydroperoxide (TBH), and then we monitored the effects of various concentrations of chalcones on oxidative stress parameters: total antioxidative status (TAS), total oxidative status (TOS), total concentration of sulfhydryl group (SHG) and prooxidative-antioxidative balance (PAB), and calculated prooxidative score, antioxidative score, and oxy score (OS). Nonparametric repeated measures ANOVA (Friedman's test) was used for comparison of antioxidative potency of samples with 15 different chalcones, in a native state and upon TBH influence. Spearman's nonparametric correlation analysis was used for estimation of relation between different parameters. Results: Negative Oxy Score (OS) values for Chs11-15 showed significantly stronger antioxidative potency compared to other investigated chalcones (p < 0.05). Ch11, Ch13 and Ch14 remained with negative OS even after TBH addition, whereas OS of Ch12 and Ch15 became positive, with small nominal values. Samples with Ch11 and Ch13 showed significant negative correlation between TAS and TOS (p < 0.05 for both), but in Ch14 sample the negative correlation existed between TAS and PAB (p < 0.05). Conclusion: Lower value of OS (i.e. better antioxidative potency) was noticed in samples with Ch11-Ch15. Electron-donor effects of substituent groups as a structural part of these chalcones could explain its antioxidative capability. Phenolic and methyl groups are responsible for antioxidative ability enhancement of five chalcones with the best activity

3D-QSAR, molecular docking and in silico ADMET studies of propiophenone derivatives with anti-HIV-1 protease activity

HIV protease inhibitors are one of the most important agents for the treatment of HIV infection. In this work, molecular modeling studies combining 3D-QSAR, molecular docking, MESP, HOMO, and LUMO energy calculations were performed on propiophenone derivatives to explore structure activity relationships and structural requirements for the inhibitory activity. The aim of this study was to create a field point–based 3D-QSAR (3D-Quantitative structure-activity relationship) model by using chalcone structures with anti-HIV-1 protease activity from our previous study and to design new potentially more potent and safer inhibitors. The developed model showed acceptable predictive and descriptive capability as represented by standard statistical parameters R2 (0.94) and Q2 (0.59). High correlation between experimental and predicted activities of training set is noticed. All compounds fit into the defined applicability domain. The derived pharmacophoric features were further supported by MESP and Mulliken charge analysis using density functional theory. Statistically significant variables from 3D-QSAR were used to define key structural characteristics which enhance anti-HIV-1 protease activity. This information has been used to design new structures with anti-HIV-1 protease activity. Docking studies were conducted to understand the interactions in predicted compounds. All the compounds were subjected to in silico ADMET profiling in order to select the best potential drug candidates

Pharmacovigilance as an imperative of modern medicine - experience from Montenegro

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