Sandra Šegan

Second generation steroidal 4-aminoquinolines are potent, dual-target inhibitors of the botulinum neurotoxin serotype A metalloprotease and P. falciparum malaria.

Significantly more potent second generation 4-amino-7-chloroquinoline (4,7-ACQ) based inhibitors of the botulinum neurotoxin serotype A (BoNT/A) light chain were synthesized. Introducing an amino group at the C(3) position of the cholate component markedly increased potency (IC50 values for such derivatives ranged from 0.81 to 2.27 μM). Two additional subclasses were prepared: bis(steroidal)-4,7-ACQ derivatives and bis(4,7-ACQ)cholate derivatives; both classes provided inhibitors with nanomolar-range potencies (e.g., the Ki of compound 67 is 0.10 μM). During BoNT/A challenge using primary neurons, select derivatives protected SNAP-25 by up to 89%. Docking simulations were performed to rationalize the compounds’ in vitro potencies. In addition to specific residue contacts, coordination of the enzyme’s catalytic zinc and expulsion of the enzyme’s catalytic water were a consistent theme. With respect to antimalarial activity, the compounds provided better IC90 activities against chloroquine resistant (CQR) malaria than CQ, and seven compounds were more active than mefloquine against CQR strain W2.

Correlation between structure, retention and activity of cholic acid derived cis–trans isomeric bis‐steroidal tetraoxanes

Both quantitative structure-retention (QSRR) and quantitative structure-activity relationship (QSAR) studies have been performed to correlate the molecular characteristics of seven pairs of cis-trans isomeric bis-steroidal tetraoxanes with their reversed-phase thin-layer chromatography (RPTLC) retention as well as with their antiproliferative activity. 2D and 3D molecular descriptors as whole molecule representations together with retention parameters as well as with biological activity data were subjected to the multivariate statistical analysis (principal component analysis--PCA and hierarchical cluster analysis--HCA) in order to determine the most influential factors governing the retention and activity against human cervix carcinoma (HeLa) and human malignant melanoma (Fem-X) cell lines. Both QSRR and QSAR models were built by means of the partial least-squares (PLS) statistical method. It was found that hydrogen bond donating (HBD), hydrogen bond accepting (HBAcc), hydrophilic surface percentage (%HS) and hydrophilic-lipophilic balance (HLB) exhibit the strongest influence on retention. The most prominent factors affecting antiproliferative activity of the investigated substances are those relating to the size and shape of a molecule such as: connectivity indices, refractivity (Ref), surface area (SA), molecular volume and weight, polarizability (Pol) and those regarding the ability of hydrogen bonding (HB).

Determination of the soil-water partition coefficients (log KOC) of some mono- and poly-substituted phenols by reversed-phase thin-layer chromatography.

In order to determine the soil-water partition coefficient for eleven mono- and poly-substituted phenolic compounds, for which there is still no literature data available, the possibility of using thin-layer chromatography (TLC) as a means for rapid and reliable logK(OC) estimation was examined. A series of chromatographically derived descriptors: R(M)(0), b, C(0) and PC1 (first principal component), calculated from retention data obtained under reversed-phase conditions, were used for the assessment of models as well as for a direct calibration procedure. The final calibration models are discussed with regard to the achieved accuracy and statistical quality, the type of descriptors used and the corresponding chromatographic conditions. The estimated logK(OC) values of the studied phenols were compared with those obtained by other means: (a) the present OECD guideline based on an HPLC technique; (b) the KOCWIN software package, available free of charge from the US Environmental Protection Agency web site and (c) general LSER models established by Nguyen and coworkers, and Poole and coworkers. The proposed method showed the best agreement with the results obtained by the OECD procedure, followed by the LSER models of Poole and Nguyen. Lower quality correlations were achieved with the KOCWIN calculated values, especially those predicted by molecular connectivity indices.

Quantitative analysis of arbutin and hydroquinone in strawberry tree (Arbutus unedo L., Ericaceae) leaves by gas chromatography-mass spectrometry.

Abstract The phenolic glycoside arbutin and its metabolite with uroantiseptic activity hydroquinone occur naturally in the leaves of various medicinal plants and spices. In this study, an extraction procedure coupled with gas chromatography-mass spectrometry (GC-MS) was developed to determine arbutin and hydroquinone content in strawberry tree (Arbutus unedo L., Ericaceae) leaves. The method showed good linearity (R2>0.9987) in the tested concentration range (0.5-200 μg mL-1), as well as good precision (RSD<5 %), analytical recovery (96.2-98.0 %), and sensitivity (limit of detection=0.009 and 0.004 μg mL-1 for arbutin and hydroquinone, respectively). The results obtained by the validated GC-MS method corresponded well to those obtained by high performance liquid chromatography (HPLC) method. The proposed method was then applied for determining arbutin and hydroquinone content in methanolic leaf extracts. The amount of arbutin in the leaves collected on the island of Koločep (6.82 mg g-1 dry weight) was found to be higher (tpaired=43.57, tc=2.92) in comparison to the amount of arbutin in the leaves collected on the island of Mali Lošinj (2.75 mg g-1 dry weight). Hydroquinone was not detected in any of the samples. The analytical features of the proposed GC-MS method demonstrated that arbutin and hydroquinone could be determined alternatively by gas chromatography. Due to its wide concentration range, the method could also be suitable for arbutin and hydroquinone analysis in leaves of other plant families (Rosaceae, Lamiaceae, etc.). Fenolni glikozid arbutin i hidrokinon, njegov metabolit s uroantiseptičkim djelovanjem, prirodni su sastojci lišća različitih biljaka koje se koriste u terapeutske svrhe te začinskih biljaka. U ovom radu optimiran je postupak ekstrakcije te je razvijena i validirana plinskokromatografska metoda uz detekciju masenim spektrometrom (GC-MS) za određivanje sadržaja arbutina i hidrokinona u lišću obične planike (Arbutus unedo L., Ericaceae). Metoda je pokazala dobru linearnost (R2>0,9987) u ispitivanom koncentracijskom rasponu (0,5-200 μg mL-1), kao i dobru preciznost (RSD<5 %), analitički povrat (96,2-98,0 %) i osjetljivost (granica detekcije=0,009 μg mL-1 za arbutin i 0,004 μg mL-1 za hidrokinon). Rezultati dobiveni validiranom GC-MS metodom u dobroj su suglasnosti s rezultatima dobivenim metodom tekućinske kromatografije visoke učinkovitosti (HPLC). Predložena metoda je primijenjena za određivanje sadržaja arbutina i hidrokinona u metanolnom ekstraktu lišća. Sadržaj arbutina određen u lišću obične planike prikupljenom na otoku Koločepu (6,82 mg g-1 suhe mase) bio je veći u usporedbi sa sadržajem arbutina u lišću prikupljenom na otoku Malom Lošinju (2,75 mg g-1 suhe mase). Hidrokinon nije detektiran ni u jednom uzorku. Analitičke značajke predložene GC-MS metode pokazale su da se arbutin i hidrokinon mogu alternativno određivati plinskom kromatografijom. S obzirom na široki koncentracijski raspon, metoda je pogodna za analizu arbutina i hidrokinona, ne samo u lišću obične planike, nego također i u lišću biljaka drugih porodica (Rosaceae, Lamiaceae, itd.).

Quantitative structure retention/activity relationships of biologically relevant 4-amino-7-chloroquinoline based compounds.

The chromatographic behaviour of series of 4-amino-7-chloroquinoline (4,7-ACQ) based compounds was studied by reversed-phase thin-layer chromatography (RPTLC) with binary mobile phases containing water and the organic modifiers, DMSO or acetone. The lipophilicity of the studied compounds was determined by extrapolation of retention parameters RM to pure water content in mobile phase. In order to obtain some basic insight into the chromatographic behaviour and structural features of investigated compounds, PCA was performed on both chromatographic data (RM values) and calculated 2D and 3D structural descriptors. Both QSRR and QSAR models were built by means of the partial least squares (PLS) statistical method. It was found that descriptors which encode hydrophobic (dispersive) interactions have positive influence on retention, while influence of descriptors encoding polar interactions was negative. According to the obtained PLS model for inhibition of botulinum neurotoxin serotype A light chain, hydrophobic interactions influence positively on the mechanism of action of the investigated 4,7-ACQ, while polar interactions are less favoured. Contrary, the results of PLS modelling of activity against Plasmodium falciparum strains (W2, D6 and TM91C235) indicate that higher polarity of 4,7-ACQ contribute to their higher antimalarial activity.

Correlation study of retention data and antimalarial activity of 1,2,4,5-mixed tetraoxanes with their molecular structure descriptors and LSER parameters

The chromatographic behavior of mixed 1,2,4,5-tetraoxanes, cholic and deoxycholic acid derivatives with distinct biological activity, was examined by high-performance thin-layer chromatography in order to correlate their structure and retention. Chromatographic systems were consisted of RP-18 or CN-silica as stationary phase, and binary mixtures of water with methanol, dioxane or acetone as mobile phase. Based on the respective retentions, the lipophilicity of the investigated compounds was determined. Multiple linear regression and partial least squares have been used to select variables that best describe the behavior of the investigated compounds in chromatographic systems and to quantify influences of most important parameters. The validation and cross-validation of the QSRR model suggest its applicability for prediction and understanding of retention of congeners. The models indicate the importance of nonpolar properties of the solutes and their ability for hydrophobic interactions, as well as the importance of proton donating abilities, hydrophilic and π interactions pointing out on that way the possible separation mechanism in the studied chromatographic systems. Observed correlations between structure and biological activity of mixed 1,2,4,5-tetraoxanes, indicate that the antimalarial activity against W2 and D6 Plasmodium falciparum strains, is governed by hydrophobic feature (measured with lipophilicity parameter), hydrophilic feature (measured with HLB, %HS, HB and HBA descriptors), and electronic feature (HOMO).

Sugar Profile of Kernels as a Marker of Origin and Ripening Time of Peach (Prunus persicae L.)

Large amounts of fruit seeds, especially peach, are discarded annually in juice or conserve producing industries which is a potential waste of valuable resource and serious disposal problem. Regarding the fact that peach seeds can be obtained as a byproduct from processing companies their exploitation should be greater and, consequently more information of cultivars’ kernels and their composition is required. A total of 25 samples of kernels from various peach germplasm (including commercial cultivars, perspective hybrids and vineyard peach accessions) differing in origin and ripening time were characterized by evaluation of their sugar composition. Twenty characteristic carbohydrates and sugar alcohols were determined and quantified using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC/PAD). Sucrose, glucose and fructose are the most important sugars in peach kernels similar to other representatives of the Rosaceae family. Also, high amounts of sugars in seeds of promising hybrids implies that through conventional breeding programs peach kernels with high sugar content can be obtained. In addition, by the means of several pattern recognition methods the variables that discriminate peach kernels arising from diverse germplasm and different stage of maturity were identified and successful models for further prediction were developed. Sugars such as ribose, trehalose, arabinose, galactitol, fructose, maltose, sorbitol, sucrose, iso-maltotriose were marked as most important for such discrimination.

Estimation of Lipophilicity of Some Polyoxygenated Steroids by the Means of Normal-Phase Thin-Layer Chromatography

Unmodified silica gel in combination with two mobile phases, acetone–n-hexane and acetonitrile–dichloromethane, was used in order to evaluate the capability of normal-phase (NP) chromatography on bare silica gel in estimation of lipophilicity of some polyoxygenated steroids. Soczewinski equation coefficients were employed as a measure of lipophilicity. The RM0 values obtained in NP-systems were correlated with those derived by extrapolation from reversed-phase (RP) systems. In addition, retention data, i.e., lipophilicity parameters determined in NP systems were compared with logP values calculated by use of several commercial computer programs. The results showed that chromatographic parameters RM0, and m obtained in NP system consisted of silica gel as stationary phase and acetone–n-hexane as mobile phase, are acceptable as the measures of lipophilicity of polyoxygenated steroids. The mechanism of retention was discussed.

Long-Chain 4-Aminoquinolines as Quorum Sensing Inhibitors in Serratia marcescens and Pseudomonas aeruginosa

Antibiotic resistance has become a serious global threat to public health; therefore, improved strategies and structurally novel antimicrobials are urgently needed to combat infectious diseases. Here we report a new type of highly potent 4-aminoquinoline derivatives as quorum sensing inhibitors in Serratia marcescens and Pseudomonas aeruginosa, exhibiting weak bactericidal activities (minimum inhibitory concentration (MIC) > 400 μM). Through detailed structure-activity study, we have identified 7-Cl and 7-CF3 substituted N-dodecylamino-4-aminoquinolines (5 and 10) as biofilm formation inhibitors with 50% biofilm inhibition at 69 μM and 63 μM in S. marcescens and P. aeruginosa, respectively. These two compounds, 5 and 10, are the first quinoline derivatives with anti-biofilm formation activity reported in S. marcescens. Quantitative structure-activity relationship (QSAR) analysis identified structural descriptors such as Wiener indices, hyper-distance-path index (HDPI), mean topological charge (MTC), topological charge index (TCI), and log D(o/w)exp as the most influential in biofilm inhibition in this bacterial species. Derivative 10 is one of the most potent quinoline type inhibitors of pyocyanin production described so far (IC50 = 2.5 μM). While we have demonstrated that 5 and 10 act as Pseudomonas quinolone system (PQS) antagonists, the mechanism of inhibition of S. marcescens biofilm formation with these compounds remains open since signaling similar to P. aeruginosa PQS system has not yet been described in Serratia and activity of these compounds on acylhomoserine lactone (AHL) signaling has not been detected. Our data show that 7-Cl and 7-CF3 substituted N-dodecylamino-4-aminoquinolines present the promising scaffolds for developing antivirulence and anti-biofilm formation agents against multidrug-resistant bacterial species.

Linear modeling of the soil-water partition coefficient normalized to organic carbon content by reversed-phase thin-layer chromatography

Soil-water partition coefficient normalized to the organic carbon content (KOC) is one of the crucial properties influencing the fate of organic compounds in the environment. Chromatographic methods are well established alternative for direct sorption techniques used for KOC determination. The present work proposes reversed-phase thin-layer chromatography (RP-TLC) as a simpler, yet equally accurate method as officially recommended HPLC technique. Several TLC systems were studied including octadecyl-(RP18) and cyano-(CN) modified silica layers in combination with methanol-water and acetonitrile-water mixtures as mobile phases. In total 50 compounds of different molecular shape, size, and various ability to establish specific interactions were selected (phenols, beznodiazepines, triazine herbicides, and polyaromatic hydrocarbons). Calibration set of 29 compounds with known logKOC values determined by sorption experiments was used to build simple univariate calibrations, Principal Component Regression (PCR) and Partial Least Squares (PLS) models between logKOC and TLC retention parameters. Models exhibit good statistical performance, indicating that CN-layers contribute better to logKOC modeling than RP18-silica. The most promising TLC methods, officially recommended HPLC method, and four in silico estimation approaches have been compared by non-parametric Sum of Ranking Differences approach (SRD). The best estimations of logKOC values were achieved by simple univariate calibration of TLC retention data involving CN-silica layers and moderate content of methanol (40-50%v/v). They were ranked far well compared to the officially recommended HPLC method which was ranked in the middle. The worst estimates have been obtained from in silico computations based on octanol-water partition coefficient. Linear Solvation Energy Relationship study revealed that increased polarity of CN-layers over RP18 in combination with methanol-water mixtures is the key to better modeling of logKOC through significant diminishing of dipolar and proton accepting influence of the mobile phase as well as enhancing molar refractivity in excess of the chromatographic systems.