AntioxidAnt Activity of Alkoxy derivAtives of ( quinolinе-4-ylthio ) carboxylic acids

a selection of potential bioactive molecules including alkoxy derivatives of (quinoline-4-ylthio)carboxylic acids using virtual screening has been carried out and their biological activity was determined. The studied substances proved to be low-toxic, biologically active compounds. it was established that 4-thio derivatives of quinoline exhibit pronounced antiradical, antioxidant effects and can act as preventive antioxidants, radio and cytoprotectors.

a selection of potential bioactive molecules including alkoxy derivatives of (quinoline-4-ylthio)carboxylic acids using virtual screening has been carried out and their biological activity was determined.The studied substances proved to be low-toxic, biologically active compounds.it was established that 4-thio derivatives of quinoline exhibit pronounced antiradical, antioxidant effects and can act as preventive antioxidants, radio and cytoprotectors.k e y w o r d s: 4-thioquinolines, mercaptocarboxylic acids, PaSS-prognosis, toxicity, antiradical and antioxidant activity.
O ne of the most important tasks of the medical and pharmaceutical research is the extension of range of modern medication via the development of new biologically active compounds.The development methods are based on the synthetic modifications of the structures of these molecules to reduce toxicity and enhance selectivity [1].Thus, nitrogen-containing heterocyclic compounds, in particular quinoline derivatives, are a promising base for creating new effective medical substances.Nowadays various quinoline derivatives are used as synthons in organic synthesis and molecu lar design as well as effective bioactive compounds.There is a particular interest in mercaptoderivatives of six-membered nitrogen-containing heterocycles, among which many bioactive molecules affecting the processes of free radical oxidation (FRO) were found [2][3][4].
During FRO a series of products of interactions between free radicals (FR) and between FR and biological macromolecules was formed [5][6][7][8][9].Thus, along with reactive oxygen species (ROS) other reactive radicals (such as peroxides, epoxides, aldehydes, ketones, alkohols, dialdehydes etc.) are formed.These radicals can interact with certain functional groups of proteins that may lead to their polymerizations and can damage amino acid residues, particularly SH-, SCH-groups of cysteine and methionine, NH-group of lysine etc.All these processes cause the modifications of proteins (including enzymes), alterations in their activity, damage of bio antioxidants (such as vitamins, ubiquinone, steroid hormones etc.).Changes in phospholipid compositions can also be caused, when oxidation products form in the hydrophobic part, which induces ion transport and also alters protein conformations and lipids composition.This in turn leads to changes in structural and functional characteristics of membranes.The same processes can be observed in DNA structure of defective cells.Free radicals can react both directly with DNA nitrogenous bases, forming their modified derivatives (including 8-azaguanin) and indirectly through the secondary and end-products of lipid peroxidation (LPO), including malondialdehyde and its derivatives [10].
In most of pharmacological and clinical studies antioxidant effects of medications and physiologically active substances (PAS) are studied using the complex models of experimental pathology (acute cerebrovascular accident, myocardial infarction, emotionally painful stress, toxic liver damage, etc.).It should be noted that in many cases these methods do not allow to sufficiently evaluate the initial antioxidant and antiradical effects of the studied PAS, since these effects could be indirect, that is resultant of complex biochemical and physiological processes , which occur in vitro and do not reflect the initial molecular mechanism of the studied PAS action in biophagy [6].
It is highly necessary to apply methods of primary assessment of antioxidant and antiradical proper ties of PAS in experiments in vitro to explore AOA of potential medication, particularly in the early phases of biological screening.At the same time, it is reasonable to use several models of free-radical reactions initiation in experiments in vitro, which reflect various phases of complex chain reactions of FRO activation [8,10].
The aim of this work is to select potential antioxidants, radio-and cytoprotector among alkoxy derivatives (quinoline-4-ylthio)carboxylic acids (using virtual screening), to study their acute toxicity, antioxidant activity in experiments in vitro and assess the prospects of these compounds as potential bioactive molecules.

Materials and Methods
PASS (Prediction of Activity Spectra for substances) software was used for the prediction of biological activity of the studied compounds.The probability of the biological activity was expressed as probability indicator P a (active) and indicator P i (inactive).Prediction was based on the analysis of structure of chemical compounds and on the analysis of structure-activity relationship (from known data) [13].
Wild type male mice with weight 20-24 g were used in experiments for assessment of acute toxicity parameters of the studied compounds.The substances were introduced intraperitonealy.Acute toxicity was determined by express-method [14].
Antioxidant activity of the synthesized compounds in vitro using free radicals initiation processes was assessed accordingly to [15,16].
Methods used in our work for the assessment of the studied compounds AOA, in vitro (n = 5) differed in the mechanism of free radical initiation, in the substrates for oxidation and the marker products [15].
assessment of inhibition of auto oxidation of adrenaline to adrenochrome.Nonenzymatic oxidation of adrenaline to adrenochrome in alkaline me-dium leads to accumulating of superoxide radicals.Rate of the reaction in biological systems depends on superoxide dismutase enzyme activity although this reaction in a chemical system in vitro may be used for quantitative assessment AOA of the studied compounds.
assessment of aOa of the studied compounds by evaluation of nO-radical inhibition is based on sodium nitroprusside photoinduction which results in accumulating of NO • -radicals.Rate of ascorbate oxidation was measured at d = 265 nm.
assessment of aOa of the studied compounds by evaluation of inhibition of proteins oxidative modifications, caused by Fenton's reagent is based on quantitative analysis of proteins oxidized amino acid residues which are formed during FRO initiating by Fenton's reagent in vitro using reaction with 2,4-dinitrophenylhydrazine (2,4-DNPH).
All of the mentioned above methods were reported in the work [15].
assessment of aOa of the studied compounds by differential pulse voltammetry.The peak wave intensity of oxygen reduction on platinum cathode in the presence of the studied compounds characterizes their relative AOA which is associated with the interaction between studied compounds and active products of oxygen reduction [17].The relative antiradical and antioxidant activities of the studied compounds were determined by analyzing the alterations in the wave peaks of reduction of hydroxyl radicals (E = -0.2V) and hydrogen peroxide (E = -1.1 V) on copper cathode under the presence of the studied compounds [18].
The statistical data processing was performed using Student's t-test parametric criteria, Mann-Whitney nonparametric test and Microsoft Excel TM.The confidential interval of 95% (P < 0.05) was used in the statistical analysis.
The probability of having antioxidant, membrane and radioprotective effects in these com-експериментальні рОбОти pounds approaches 47%.Thus, further studies of these compounds are considered to be promising.Structures of the derivatives of (quinoline-4-ylthio) carboxylic acids conform to Lipinski's rule of five which determine criteria for the prediction of any compound bioavailability, based on the observation of simple molecular characteristics (such as molecular weight, molar refractivity, lipophily, numbers of hydrogen bond donors and acceptors) [19].
It should be noted that prediction analysis indicated the presence of membrane protective effects in studied compounds and the absence of negative effects such as high toxicity, terato-, cancer-, mutagens and embryotoxicity.
Studying of quinoline derivatives acute toxicity showed that their DL 50 are 930 mg/kg and higher.It allows to consider these compounds as low-toxic or nontoxic substances (IV and V toxicity classes respectively).Introducing alkoxygroups (methoxy-or ethoxy-) into benzene ring of quinolone structure considerably reduces toxicity that was proved in the works [11,12].Compounds which have two alkoxygroups in their structure (marked as BО) are less toxic than compounds having only one alkoxygroup (marked as O).
Excessive levels of ROS, particularly superoxide radicals, produced by neurocytes at ischemia condition lead to the expression of proapoptotic proteins, anti-inflammatory cytokines and activation of inducible NO synthase.Superoxide radical is the main component of the reaction resulted in formation the most reactive cytotoxins such as hydroxyl R = Alk; R′ = OAlk; R 2 = H, CH3, CH 2 COOH, NH 2 ; n = 0; 1 radical and nitric oxide.So, the positive effect of quinoline thioderivatives on reducing of ROS level is of a particular interest.Thus, all studied compounds showed considerable anti-radical activity in experiments using the model of superoxide production (Table 2).It was interesting to note that the compounds which contain two alkoxy groups in benzene ring of quinoline heterocycle and electron-acceptor substituents (carboxy-, carboxyalkyl-or amino groups) in the mercaptocarboxylic acid residue (compounds BО-3, BО-6 and BО-7) were the most effective.A similar characteristic was observed when using the model of NO-radical initiation.

Code
Oxidative modification of neuron membrane proteins under ischemia leads to disorders in generation and transmission of nerve impulses as well as receptor desensitization, pore formation and resultant initiation of apoptosis and cognitive deficit.One of the key mechanisms of action of many known antioxidant-neuroprotectors is their ability to inhibit the processes of oxidative modification of proteins (OMP) and accumulating of carbonyl and carboxyl products such as aldehyde phenylhydrazone (APH) and carboxy phenylhydrazone (CPH).Therefore investigation of the compounds, which are capable of inhibiting processes of OMP is a promising direction [20].
The derivatives of (quinoline-4-ylthio)carboxylic acid showed AOA also when using the model of inhibition of oxidative modification of proteins.These compounds reduced rate of OMP (APH and CPH) rather efficiently.The compounds BО-3 and BО-7 demonstrated the highest activity in the mentioned above models of FRO initiation (Table 3).
Analysis of the total AOA of the most promising compounds BО-3, BО-5 and BО-6 using method of differential pulse voltammetry allowed to establish the following: the studied compounds affect the wave of oxygen reduction on platinum catho-  de, reducing the height of the wave peak (Fig. 2).The intensities of these reductions are different.We determined the relative alteration of the height of oxygen wave to compare the obtained results.The relative alteration was defined as a quotient of the height of initial peak of oxygen wave divided by the height of oxygen wave peak in the presence of the studied compounds (their concentrations were the same).

T a b l e 2. antiradical activity of the studied compounds (10 -6 М) in vitro assessed by evaluation of inhibition of superoxide (O
The obtained data presented in Table 4 indicated that BО-3 showed the highest total AOA and BО-5 showed the lowest total AOA.Method of simultaneous generating of hydroxyl radicals and hydrogen peroxide during electrochemical reduction of oxygen on copper cathode (special pulse mode) allows a more extensive understanding of molecular mechanism of AOA [18].Addition of the studied compounds into electrochemical cell allows observation of the results of their interactions both with hydroxyl radicals and with soluble oxygen itself.Quinoline derivatives BО-3 and BО-6 decreased wave peaks of hydroxyl radicals and hydrogen peroxide, whereas BО-5 at low concentrations increased wave peaks of hydroxyl radicals causing pro-radical effect.Compound (BО-5) showed antiradical effects at concentrations of 0.3 mM and higher, whereas antioxidative effect occurred at concentrations less than 0.2 mM (Fig. 3).
The relative rate of antiradical and antioxidant activity of the studied compounds were determined in relation to initial height of wave peak of hydroxyl radicals and wave peak of hydrogen peroxide to the height of peaks in the presence of studied compounds.It was established that compound BО-3 showed the highest effect (Table 4).Furthermore, it should be noted that quinoline thioderivatives have effect on oxygen wave itself.The wave height is raised and potential of the peak is shifted to the anode side.It is evident that oxygen forms complexes Thus, high antioxidant, membrane, cyto-, and radioprotector effects of alkoxy derivatives (2-methyl quinoline-4-ylthio)carboxylic acids were revealed using virtual screening.It has been shown that the studied compounds are low or nontoxic.Introduction of two alkoxy groups to quinoline ring leads to lower toxicity.It was observed that alkoxy derivatives of (2-methylquinoline-4-ylthio)carboxylic acids revealed significant AOA, when using the models of initiating of FRO in vitro (such as generating of superoxide-anion and nitric oxide and also by inhibition of oxidative modification of proteins).Introduction of electron acceptor functional groups into structure of mercapto carboxylic acids enhanced their action.It was determined that compound BО-3 showed the highest effect on total wave of electrochemically generated ROS according to the results of pulse voltammetry.The obtained results have proved the appropriateness of study of quinoline

AntioxidAnt Activity of Alkoxy derivAtives of (quinolinе-4-ylthio)carboxylic acids
UDC 547.831.1:577.1 < 0.05 in relation to intact; b P < 0.05 in relation to control; c P < 0.05 in relation to thiotriazoline; d P < 0.05 in relation to emoxypine; e P < 0.05 in relation to ACC.