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A series of novel 2-alkythio-4-chloro-N-[imino-(heteroaryl)methyl]benzenesulfonamide derivatives, 8–24, were synthesized in the reaction of the N-(benzenesulfonyl)cyanamide potassium salts 1–7 with the appropriate mercaptoheterocycles. All the synthesized compounds were evaluated for their anticancer activity in HeLa, HCT-116 and MCF-7 cell lines. The most promising compounds, 11–13, molecular hybrids containing benzenesulfonamide and imidazole moieties, selectively showed a high cytotoxic effect in HeLa cancer cells (IC50: 6–7 μM) and exhibited about three times less cytotoxicity against the non-tumor cell line HaCaT cells (IC50: 18–20 μM). It was found that the anti-proliferative effects of 11, 12 and 13 were associated with their ability to induce apoptosis in HeLa cells. The compounds increased the early apoptotic population of cells, elevated the percentage of cells in the sub-G1 phase of the cell cycle and induced apoptosis through caspase activation in HeLa cells. For the most active compounds, susceptibility to undergo first-phase oxidation reactions in human liver microsomes was assessed. The results of the in vitro metabolic stability experiments indicated values of the factor t½ for 11–13 in the range of 9.1–20.3 min and suggested the hypothetical oxidation of these compounds to sulfenic and subsequently sulfinic acids as metabolites. [ABSTRACT FROM AUTHOR]

With metabolism being one of the main routes of drug elimination from the body (accounting for removal of around 75% of known drugs), it is crucial to understand and study metabolic stability of drug candidates. Metabolically unstable compounds are uncomfortable to administer (requiring repetitive dosage during therapy), while overly stable drugs increase risk of adverse drug reactions. Additionally, biotransformation reactions can lead to formation of toxic or pharmacologically active metabolites (either less‐active than parent drug, or even with different action). There were numerous approaches in estimating metabolic stability, including in vitro, in vivo, in silico, and high‐throughput screening to name a few. This review aims at describing separation techniques used in in vitro metabolic stability estimation, as well as chemometric techniques allowing for creation of predictive models which enable high‐throughput screening approach for estimation of metabolic stability. With a very low rate of drug approval, it is important to understand in silico methods that aim at supporting classical in vitro approach. Predictive models that allow assessment of certain biological properties of drug candidates allow for cutting not only cost, but also time required to synthesize compounds predicted to be unstable or inactive by in silico models. [ABSTRACT FROM AUTHOR]

The aim of this study was to identify cytochrome P450 (CYP) isoforms that participate in the metabolism of some novel arylpiperazine derivatives developed by authors as well as their potency to inhibit reactions catalyzed by identified lead metabolizing enzyme. Such studies allow to predicting possible drug-drug interactions that might occur during co-administration of studied compounds with other drugs that are metabolized by an identified enzyme. The compounds were incubated in vitro together with the isolated CYP isoforms. After the incubation, samples were analyzed by liquid chromatography coupled with mass spectrometry. The results showed the main contribution of CYP3A4 isoform in biotransformation of the investigated derivatives. With CYP3A4 being the main CYP isoform responsible for the metabolism of arylpiperazine derivatives and at the same time being the main metabolizing enzyme for almost 50% of all drugs, a high chance of in vivo drug-drug interactions emerged. Therefore, IC50 values were also determined using testosterone hydroxylation as a probe reaction, specific for CYP3A4. The resulting values ranged from 6.13 to 15.85 µM, which places studied derivatives as moderate or weak inhibitors of CYP3A4. Those results, combined with the conclusion that all of the arylpiperazine derivatives are also metabolized to some extent by other CYP isoforms (providing alternative metabolic pathways), result in a conclusion that studied arylpiperazines might be safe for co-administration with other CYP3A4 substrates. [ABSTRACT FROM AUTHOR]

A series of novel 2-alkylthio-4-chloro- N-(5-oxo-4,5-dihydro-1,2,4-triazin-3-yl)benzenesulfonamide derivatives 12-46 have been synthesized by the reaction of aminoguanidines with an appropriate alpha-oxo-acids hydrates in glacial acetic acid. All the synthesized compounds were evaluated for their anticancer activity against HeLa, HCT-116, and MCF-7 human tumor cell lines. Two compounds 33 and 34 displayed outstanding cytotoxic effect selectively toward HeLa cancer cells ( IC50 = 19 μ m) and did not exhibit toxicity to the non-cancerous HaCaT cells. QSAR analysis determined the most important parameters controlling cytotoxic activity of 5-oxo-1,2,4-triazines against HeLa cells. QSAR model showed five significant descriptors: HATS6s ( GETAWAY descriptor), RDF125 m (radial distribution function), SpMax7_Bh(p) (Burden descriptor), SM3_G (3D matrix descriptor), and Hy (hydrophilic factor). The apoptotic potential of the most active compounds was thoroughly analyzed through various assays: cells' morphology, DNA fragmentation, mitochondrial potential disruption, and phosphatidylserine translocation. Selected compounds were tested for metabolic stability in the presence of pooled human liver microsomes and NADPH. Compound 34 was the most resistant for human metabolism ( t1/2 = 38.5 min) and can be pointed as a hit compound for further investigations. [ABSTRACT FROM AUTHOR]

A series of 1-[(imidazolidin-2-yl)imino]-1 H-indole analogues of hypotensive α2-AR agonists, 1-[(imidazolidin-2-yl)imino]-1 H-indazoles, was synthesized and tested in vitro for their activities at α1- and α2-adrenoceptors as well as imidazoline I1 and I2 receptors. The most active 1-[(imidazolidin-2-yl)imino]-1 H-indoles displayed high or moderate affinities for α1- and α2-adrenoceptors and substantial selectivity for α2-adrenoceptors over imidazoline-I1 binding sites. The in vivo cardiovascular properties of indole derivatives 3 revealed that substitution at C-7 position of the indole ring may result in compounds with high cardiovascular activity. Among them, 7-fluoro congener 3g showed the most pronounced hypotensive and bradycardic activities in this experiment at a dose as low as 10 μg/kg i.v. Metabolic stability of the selected compounds of type 3 was determined using both in vitro and in silico approaches. The results indicated that these compounds are not vulnerable to rapid first-phase oxidative metabolism. [ABSTRACT FROM AUTHOR]

A series of novel 5-substituted 2-(arylmethylthio)-4-chloro-N-(5-aryl-1,2,4-triazin-3-yl) benzenesulfonamide derivatives 27–60 have been synthesized by the reaction of aminoguanidines with an appropriate phenylglyoxal hydrate in glacial acetic acid. A majority of the compounds showed cytotoxic activity toward the human cancer cell lines HCT-116, HeLa and MCF-7, with IC50 values below 100 μM. It was found that for the analogues 36–38 the naphthyl moiety contributed significantly to the anticancer activity. Cytometric analysis of translocation of phosphatidylserine as well as mitochondrial membrane potential and cell cycle revealed that the most active compounds 37 (HCT-116 and HeLa) and 46 (MCF-7) inhibited the proliferation of cells by increasing the number of apoptotic cells. Apoptotic-like, dose dependent changes in morphology of cell lines were also noticed after treatment with 37 and 46. Moreover, triazines 37 and 46 induced caspase activity in the HCT-116, HeLa and MCF-7 cell lines. Selected compounds were tested for metabolic stability in the presence of pooled human liver microsomes and NADPH, both R2 and Ar = 4-CF3-C6H4 moiety in 2-(R2-methylthio)-N-(5-aryl-1,2,4-triazin-3-yl)benzenesulfonamides simultaneously increased metabolic stability. The results pointed to 37 as a hit compound with a good cytotoxicity against HCT-116 (IC50= 36 μM), HeLa (IC50 = 34 μM) cell lines, apoptosis-inducing activity and moderate metabolic stability. [ABSTRACT FROM AUTHOR]

In the drug development pipeline, metabolic stability is not only one of the most limiting factors when it comes to drug candidate approval but is also one of the most resource-consuming and time-consuming ones regarding in vitro studies. With the popularization of the in silico approach in the drug discovery process and the application of many excellent chemometric techniques in predicting the properties of compounds, the authors focused on developing a model able to estimate the in vitro half-life of arylpiperazine derivatives. The aim of the presented study was to develop a regression model and compare the performance of three potent methods (multiple linear regression (MLR), orthogonal partial least squares (OPLS), and support vector machine (SVM)) when predicting in vitro half-life values. Of the three studied techniques, SVM proved to be the most accurate ( RMSESVM = 0.054 for both sets, RMSEMLR = 0.072 for both sets, and RMSEOPLS = 0.073 for both sets) and provided the best predictability ( Q2 = 0.8410; for MLR, Q2 = 0.7263; and for OPLS, Q2 = 0.7490). Hence, SVM regression provides valuable predictions and seems to be a promising technique not only for developing regression models but also for studying quantitative structure-metabolic stability relationships. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

A series of novel N-acyl-4-chloro-5-methyl-2-(R¹-methylthio)benzenesulfonamides 18-47 have been synthesized by the reaction of N-[4-chloro-5-methyl-2-(R¹-methylthio) benzenesulfonyl]cyanamide potassium salts with appropriate carboxylic acids. Some of them showed anticancer activity toward the human cancer cell lines MCF-7, HCT-116 and HeLa, with the growth percentages (GPs) in the range from 7% to 46%. Quantitative structure-activity relationship (QSAR) studies on the cytotoxic activity of N-acylsulfonamides toward MCF-7, HCT-116 and HeLa were performed by using topological, ring and charge descriptors based on the stepwise multiple linear regression technique (MLR). The QSAR studies revealed three predictive and statistically significant models for the investigated compounds. The results obtained with these models indicated that the anticancer activity of N-acylsulfonamides depends on topological distances, number of ring system, maximum positive charge and number of atom-centered fragments. The metabolic stability of the selected compounds had been evaluated on pooled human liver microsomes and NADPH, both R¹ and R² substituents of the N-acylsulfonamides simultaneously affected them. [ABSTRACT FROM AUTHOR]

A bstract A series of new N' -(2-alkylthio-4-chloro-5-methylbenzenesulfonyl)-1-(5-phenyl-1 H -pyrazol-1-yl)amidine derivatives have been synthesized and evaluated in vitro by MTT assays for their antiproliferative activity against cell lines of colon cancer HCT-116, cervical cancer HeLa and breast cancer MCF-7. The studied compounds display selective activity mainly against HCT-116 and HeLa cells. Thus, five compounds show selective cytotoxic effect against HCT-116 (IC 50  = 3–10 μM) and HeLa (IC 50  = 7 μM). Importantly, the noticed values of IC 50 for four compounds are almost 4-fold lower for HeLa than non-malignant HaCaT cells. More-in-depth biological research revealed that the treatment of HCT-116 and HeLa with active compound resulted in increased numbers of cells in sub-G1 phase in a time dependent manner, while non-active derivative does not influence cell cycle. Metabolic stability assays using liver microsomes and NADPH provide important information on compounds susceptibility to phase 1 biotransformation reactions. [ABSTRACT FROM AUTHOR]

A series of new 2-(2-alkylthiobenzenesulfonyl)-3-(phenylprop-2-ynylideneamino)guanidine derivatives have been synthesized and evaluated in vitro by MTT assays for their antiproliferative activity against cell lines of colon cancer HCT-116, cervical cancer HeLa and breast cancer MCF-7. The obtained results indicated that these compounds display prominent cytotoxic effect. The best anticancer properties have been observed for derivatives 44 (IC 50 = 6–18 μM) and 45 (IC 50 = 8–14 μM). Very good results of antiproliferative assays have been also shown for compounds 26 , 36 , and 46 and noticeable anticancer profile has been found for set of derivatives 34 – 39 . Based on results of MTT assays the structure-activity relationships have been drawn. More in-depth biological research revealed that compounds 26 , 33 , 37 , 39 , 41 and 43 display cytotoxic effect only against cancer cells and do not inhibit the growth of non-malignant HaCaT cells. Furthermore, the novel series of derivatives have shown good metabolic stability, especially among the pharmacologically active compounds. To obtain a deeper insight into the molecular description of compounds activity the QSAR studies have been applied. Support vector machines (SVM) have been used to developed QSAR models for predicting the anti-proliferative activity of novel derivatives. The obtained SVM models have shown prognostic ability for HCT-116 and HeLa cell lines and as a result these models may be useful for further development of structurally similar derivatives with better biological properties. [ABSTRACT FROM AUTHOR]

A series of novel 2-benzylthio-4-chloro-5-(5-substituted 1,3,4-oxadiazol-2-yl)benzenesulfonamides ( 4 – 27 ) have been synthesized as potential anticancer agents. MTT assay was carried out to determine the cytotoxic activity against three human cancer cell lines: colon cancer HCT-116, breast cancer MCF-7 and cervical cancer HeLa as well as to determine the influence on human keratinocyte cell line HaCaT. Relatively high (IC 50 : 7–17 μM) cytostatic activity and selectivity against HeLa cell line was found for compounds 6, 7, 9–11 and 16. While compounds 23 – 27 bearing styryl moieties attached to a 1,3,4-oxadiazole ring at position 5, exhibited significant activity against two and/or three cancer cell lines with IC 50 : 11–29 μM. Further quantitative structure-activity relationships based on molecular descriptors calculated by DRAGON software, were investigated by Orthogonal Projections to Latent Structures (OPLS) technique and Variable Influence on Projection (VIP) analysis. Considering molecular descriptors with the highest influence on projection (highest VIP values) lipophilicity of tested compounds was pointed as main factor affecting activity towards HCT-116 cell line, while structural parameters associated with presence of styryl substituent in position 5 of 1,3,4-oxadiazole ring were identified as essential for activity towards MCF-7 breast cancer. In vitro tests for metabolic stability in the presences of pooled human liver microsomes and NADPH showed that some of the most active compounds 26 and 27 presented favorable metabolic stability with t 1/2 in the range of 28.1–36.0 min. [ABSTRACT FROM AUTHOR]

[Display omitted] • Drug properties significant for further development were estimated. • Chosen properties were metabolic stability, lipophilicity, membrane permeability and drug-protein binding. • 3D models of studied compounds were optimized using DFT to provide dataset for analysis. • Combining both datasets allowed a creation of predictive models. • Created models allow accurate prediction of estimated properties. In recent years mental disorders, such as depression, have become an alarmingly frequent problem in modern society. Therefore, a need for novel anti-depressant agents is ever-growing. With low drug acceptance rate, industry is struggling to introduce new anti-depressant agents, and every single tool helping to change this state of things is of immense value. In this study, a set of potential novel anti-depressant derivatives with confirmed pharmacological activity was subjected to a number of assays. A group of four drug properties, assessed in early stages of drug development were chosen for those assays. Lipophilicity, membrane permeability and drug-protein binding were estimated using biomimetic chromatography methods, while metabolic stability was estimated using in-vitro enzymatic assay followed by Liquid chromatography coupled with mass spectrometry (LC-MS) analysis. Afterwards, 3D models of studied compounds were geometrically optimized using Density Functional Theory (DFT) molecular mechanics protocol in order to resemble real-state energies and structure of those derivatives. After calculating molecular descriptors from optimized 3D models, a matrix combining data from assays and molecular descriptors was created. Using Genetic Algorithm Multiple Linear Regression (GA-MLR) on this matrix allowed to create four mathematical models, one for each of the studied properties, which accurately predicts properties crucial in early drug-development stages. Such models can severely help in reducing costs of drug-development, by allowing exclusion of extremely unfavorable derivatives – even before the stage of synthesis. [ABSTRACT FROM AUTHOR]

• The most popular way to assess in-vitro metabolic stability is based on phase I metabolism. • Adding phase II co-factors to the assay should provide stability value based on both phases. • Calculated half-lives do not differ significantly regardless of the presence of phase II co-factors in the assay. • It is possible, that phase II metabolism has no significant impact on in-vitro half-life values. Metabolic stability plays a crucial role in assessing the safety of potential drug candidates. Assays to designate in vitro half-life are well established and are focused on phase I metabolism using human liver microsomes. However, such an assay can easily be modified to include phase II metabolism. Yet, to authors best knowledge, no attempts at designating in vitro half-life values that includes phase II enzymes were performed. That means, that all current metabolic stability estimations reflect only partial information – as only phase I of metabolism takes place in currently used assays. The following study uses originally modified metabolic stability assay, coupled with LC–MS analysis to provide in vitro half-life values for four pirydo[1,2-c]pirymidine derivatives. Testosterone was used as a positive control. The assay was modified using alamethicin (a pore-forming agent which enhances membrane transport, allowing for phase II reactions) and UDPGA (Uridine 5′-diphosphoglucuronic acid) – co-factor for most common phase II biotransformation reaction – glucuronidation. In vitro half-life values estimated using two assays (a standard one comprising sole phase I enzymes and modified one – providing phase II reactions) have been compared using U Mann-Whitney's test for statistical significance. Values for phases I + II were slightly higher, however, the difference was not statistically significant. Such results lead to the conclusion, that in vitro half-life values are not strongly influenced by phase II biotransformation in the case of the considered compounds. [ABSTRACT FROM AUTHOR]

• The 3D-printed sorbent is coupled to the metabolic stability test of drug candidates. • Customizable sorbent enhances the performance of LC-MS analysis at low cost of sample clean-up. • The additional extraction step improves the precision of drug candidates' t 1/2 determination. Metabolic stability tests are one of the fundamental steps at the preclinical stages of new drug development. Microsomes, used as a typical enzymatic model of liver biotransformation, can be a challenging matrix for analytical scientists due to a high concentration of cellular proteins and membrane lipids. In the work, we propose a new procedure integrating biotransformation reaction with SPME-like protocol for sample clean-up. It is beneficial to increase the overall quality of results in contrary to the typical protein precipitation approach. A set of ten arylpiperazine analogs, six of which are considered promising drug candidates (and four are accepted drugs) were used as a probe to assess the goodness of the newly proposed approach. In order to promote an efficient extraction protocol, a new, miniaturized shape of a sorbent, suitable to perform the extraction in 100 µL of the sample has been designed. Termination of the biotransformation process by protein denaturation with hot water was additionally evaluated. A quantitative structure-property relationship (QSPR) study using Orthogonal Partial Least Squares (OPLS) technique to reveal insights to the sorption mechanism was also performed. The obtained results showed the new 3D-printed sorbent can be an attractive basis for the new sample preparation approach for metabolic stability studies and an alternative for commercially available protocols based on solid-phase microextraction (SPME) or solid-phase extraction (SPE) principles. [ABSTRACT FROM AUTHOR]