EffEct of Pd ( II ) and ni ( II ) coordInatIon comPounds wIth 4-amIno-3-mErcaPto-5-mEthyl-1 , 2 , 4-trIazolE on thE mItochondrIal dEhydrogEnasEs actIvIty

pd(II) and Ni(II) complex compounds: [pd(AmmT)2]cl2 (1), [pd(AmmT)4]cl2 (2) and [Ni(AmmT)2(h2O)2](NO3)2 (3) with 4-amino-3-mercapto-5-methyl-1,2,4-triazole (AmmT) have been synthesized. The spectral characteristics of 1, 2 were studied by 1h (13c) Nmr and UV-Vis spectroscopy. X-ray diffraction studies established that all complexes contain the AmmT molecule, which are coordinated to the central metal ion in the thione tautomeric form. At the ratio m : l = 1 : 2 ligand is coordinated in bidentate chelate manner by the nitrogen of aminoand sulfur of mercapto group (compounds 1, 3). But the molar ratio m : l = 1 : 4 leads to monodentate coordination of AmmT molecules only by sulfur of mercaptogroup (complex 2). Vacant coordination sites of the metal ion are occupied by water molecules (complex 3). The screening of complexes 1−3 and starting compounds [АММТ, k2pdcl4 (4), Ni(NO3)2∙6h2O (5)]by their mitochondrial dehydrogenase activity have been performed by us for the first time, resulting in established that the pd(II) complexes (1, 2), pd(II) salt (4) and AmmT normalize the activity of mitochondrial dehydrogenases of cancer HeLa cells, identified by MTT-test. In contrast, the Ni(II) complex (3) and Ni(II) salt (5) do not stimulate the activity of mitochondrial dehydrogenases. It has been found, that all investigated compounds do not affect on the cell cycle and the level of apoptotic cells as well as do not show a toxic effect. Thus, these results indicate that AmmT and pd(II) complexes may be used as modifiers of mitochondrial respiration, which dysfunction is particularly evident in the tumor cells.

pd(II) and Ni(II) complex compounds: [pd(AmmT) 2 ]cl 2 (1), [pd(AmmT) 4 ]cl 2 (2) and [Ni(AmmT) 2 (h 2 O) 2 ](NO 3 ) 2 (3) with 4-amino-3-mercapto-5-methyl-1,2,4-triazole (AmmT) have been synthesized.The spectral characteristics of 1, 2 were studied by 1 h ( 13 c) Nmr and UV-Vis spectroscopy.X-ray diffraction studies established that all complexes contain the AmmT molecule, which are coordinated to the central metal ion in the thione tautomeric form.At the ratio m : l = 1 : 2 ligand is coordinated in bidentate chelate manner by the nitrogen of amino-and sulfur of mercapto group (compounds 1, 3).But the molar ratio m : l = 1 : 4 leads to monodentate coordination of AmmT molecules only by sulfur of mercaptogroup (complex 2).Vacant coordination sites of the metal ion are occupied by water molecules (complex 3).The screening of complexes 1−3 and starting compounds [АММТ, k 2 pdcl 4 (4), Ni(NO 3 ) 2 •6h 2 O (5)]by their mitochondrial dehydrogenase activity have been performed by us for the first time, resulting in established that the pd(II) complexes (1,2), pd(II) salt (4) and AmmT normalize the activity of mitochondrial dehydrogenases of cancer HeLa cells, identified by MTT-test.In contrast, the Ni(II) complex (3) and Ni(II) salt (5) do not stimulate the activity of mitochondrial dehydrogenases.It has been found, that all investigated compounds do not affect on the cell cycle and the level of apoptotic cells as well as do not show a toxic effect.Thus, these results indicate that AmmT and pd(II) complexes may be used as modifiers of mitochondrial respiration, which dysfunction is particularly evident in the tumor cells.k e y w o r d s: 1,2,4-triazole, pd(II) and Ni(II) complexes, 1 Н ( 13 С) NMR spectroscopy, the mitochondrial dehydrogenases activity, level of apoptotic cells.
I n recent decades a considerable attention has been paid to the development of methods for the synthesis of transition metal complexes, such as platinum with bioactive organic ligands, due to their high activity and stability in physiological media and their potential as drugs [1,2].
The complexation enhances the bioavaila bility of organic substances as well as metal ions improving their transport through the cell mem branes.Furthermore, the coordination of organic compounds to the transition metal ions extends the range of their biological targets.The nitrogencon taining heterocycle derivatives such as 1,2,4triazole are the imidazole structural analogs (which are the components of nucleosides) and widely used as bio active ligands [3,4].
The triazole ring contains three Ndonor reac ting sites, which are capable to coordinate transi tion metal ions.The insertion of additional donor functional groups (amino, mercapto, hydroxyl, carboxy, etc.) into the triazole structure allows obtaining the polydentate chelating ligand systems forming stable chelate metalocycles with transition metal ions which show antiproliferative [5] and cy tostatic [6] effects.Further studies of complexation reactions with substituted 1,2,4triazoles and the de експериментальні роботи velopment of methods for the synthesis of complexes on their base are the promising direction in design of new pharmaceutical drugs, including anticancer ones.
It is known that the characteristic features of tumor cells are high level of glycolysis, low level of the mitochondrial respiration and weakened Pasteur effect (the suppression of the glycolysis by the res piration) [7].This fact is also confirmed by the sup pression of the activity in transformed cells of such crucial components of the mitochondrial respiration as cytochrome C, cytochrome oxidase and succinate dehydrogenase [8][9][10].Tumor cells are characterized by the development of the lactic acidosis, which is associated with alterations in the activity and isoen zyme spectrum of lactate dehydrogenase [11].The decrease in the activity of the pyruvate dehydroge nase complex induces the accumulation of pyruvic acid.These alterations cause the tissue hypoxia, re sulting redox system abnormalities [12].Therefore, the development of methods for the synthesis of low toxicity compounds, which could be used for the me tabolism restoration in a case of malignant growth of cells, is essential nowadays.As shown below, Pd(II) and Ni(II) complexes (1-3) with 4amino3mercap to5methyl1,2,4 triazole (AMMT) can be attributed to such type of compounds.
It is known from the open literature, that the reaction AMMT with Pd(II) ions, depending on the reagents ratio, forms three complexes, which con tain 1, 2 or 4 molecules of Smonodentate coordi nated ligand [13].The Ni(II) complex synthesized by Sen A. K. and coworkers [14] contains two mole cules of ligand with the S,Nbidentate coor dination of AMMT.In contrast to results obtained by Grap S. R. and coworkers [13], complex 1 was obtained by the interaction between AMMT and Pd(II) ions.This complex contains two molecules of bidentate chelate ligands coordinated to metal ion by the nitrogen of amino group and sulfur of thione group.Complexes 1 and 2 [13], 3 [14] have been used for the study of their effects on the mitochondrial dehydrogenase activity in HeLa tumor cell line in comparison with the analogous effects of inorganic salts such as K 2 PdCl 4 (4), Ni(NO 3 ) 2 •6H 2 O (5) and un coordinated AMMT.
It has been established that complex com pounds Pd(II) 1, 2 and AMMT are able to enter freely into cells and serve as a substrate for mito chondrial dehydrogenase, in particular for succi nate dehydrogenase.Moreover these complexes are also able to enhance the functional activity of the mitochondrial dehydrogenase and improve the mi tochondrial respiration in general.It allows one to consider the complex compounds based on AMMT and palladium inorganic salts as potential agents for the restoration of the metabolism under condition of the malignant growth.materials and methods 1 H ( 13 C) NMR spectra were recorded on Bruker Avance DRX500 (500 (124.75)MHz) spectrometers with dimethylsulfoxided 6 as a solvent in the presen ce of tetramethylsilane.
The crystal structure of complex 1 was deter mined by a singlecrystal Xray diffraction method.Measurements were performed on XCalibur 3 dif fractometer (MoKα radiation, graphite monochro mator, λ = 0.71073 Å) at room temperature.The structure was solved by the direct method and re fined by full matrix least-square method against F 2 with the anisotropic approximation for nonhy drogen atoms using SHELXTL program package.The PdCl 2 , K 2 PdCl 4 and Ni(NO 3 ) 2 •6H 2 O (Merck, Cermany) were used for the synthesis of complex compounds.
synthesis of ammt and the complexes 1-3 AMMT was synthesized according to [14] (22.2 mg, 0.125 mM) was dissolved in the mixture of 20 ml ethanol and 2 ml 2N HCl un der stirring and heating.The solution of AMMT (32.5 mg, 0.25 mM) in 15 ml of ethanol was slowly added to the hot reaction mixture under constant stirring.The reaction mixture was heated at 60 °C for 1 h.The resulting orange solution was left in a dark place for crystallization.After 10 days, the orange needlelike crystals were formed.The crys tals were filtered, washed with ethanol and ether and then dried at room temperature.Yield 49.7 mg (84%).T dec.= 143-144 °C.NMR 1 H, δ ppm: 2.24 s (3H, CH 3 ); 6.18 w.s.(2H, NH 2 ); 9.58 s ( 1 H, NH thiazole ).NMR 13  The complex [pd(AmmT) 4 ]cl 2 (2).The synthe sis was carried out in accordance with [13].15 ml of the hot aqueous solution of K 2 PdCl 4 (32.6 mg, 0.1 mM) was slowly added to 15 ml of the hot aque ous solution of AMMT (52.1 mg, 0.4 mM) with stirring.The resulting redorange solution was left to crystallize.After 5 days, the red crystals were formed.The crystals were filtered, washed with etha nol and ether and then dried at room tempera ture.The yield: 57.2 mg (82%).T dec.= 199-200 °C.NMR 1 H, δ ppm: 2.29 s (3H, CH 3 ); 6.14 w. s (2H, NH 2 ); 9.49 s (1H, NH thiazole ).NMR 13  The identity of structure of synthesized complex and the com pound previously obtained in accordance with [13] was confirmed by X-ray diffraction method.
The complex [Ni(AmmT) 2 (h 2 O) 2 ](NO 3 ) 2 (3).The synthesis was carried out in accordance with [14].The hot solution of AMMT (65.1 mg, 0.5 mM) in ethanol (15 ml) was slowly added to the hot solu tion of Ni(NO 3 ) 2 •6H 2 O (72.7 mg, 0.25 mM in ethanol (15 ml) under constant stirring.The resulting light green solution was heated for 2 h at 60 °C and then left to crystallize in a dark place.After 56 days the light green crystals were formed which then were filtered, washed with ethanol ether and then dried at room temperature.The yield: 94.6 mg (79%  (4), Ni(NO 3 ) 2 •6h 2 O ( 5)) biological activity screening.HeLa cervical carcinoma cell line was used for the assessment of mitochondrial respiration level, cell cycle characteristic and apo ptosis level in the presence of the compounds 15 and AMMT.In order to assess mitochondrial res piration level the cells were incubated with studied compounds for 24 hours in 96well plates under standard culture conditions at 37 °C, 5% CO 2 and 100% humidity.Cells were grown in the RPMI1640 medium (Sigma, USA) with addition of 10% the fetal calf serum (Sigma, USA), 2 mM Lglutamine and 40 µg/ml gentamicin.The initial cells concentration was 2.5•10 4 cells/ml.Cells were seeded in a volume of 100 µl.Compounds 1-5 and AMMT were added to cells after 4hours of adaptation in the concen trations range 4.0•10 6 -1.3•10 4 M in the volume of 100 µl.Cells were cultured under standard condi tions for 24 h.Cell via bility was assessed by routine counting method in the Goryaev chamber by the trypan blue dye inclusion into dead cells.
The flow cytometric method was used to deter mine the cell cycle phase distribution and the apop tosis level [19].For this purpose cells were seeded in 6-well plates at a concentration of 2.5•10 4 cells/ ml in 3 ml of the total culture medium volume and incubated with compounds 15 and AMMT (at con centration 2.5•10 5 M each) for 48 h at standard con ditions.At least 5•10 5 cells were used for the sample preparation.The cells were precipitated by the cen trifugation at 1000 g for 5 min.The supernatant was removed and cells were washed with the saline or 1.0 M sodium phosphate buffer (PBS, pH 7.2).Cells were resuspended in 200 µl PBS, then the 300 µl of 1.0 M citrate buffer (pH 6.8) containing 0.1% Triton X100 was added.After 1 min, 10 µl of ribonuclea se and 10 µl of propidium iodide PI (Sigma, USA) for DNA staining were added [19].The mixture was incubated for 10 min at 37 °C in the dark and ad ditionally for 30 min at room temperature.Then it was centrifuged at 1000 g for 10 min and the su pernatant was removed.Then cells were fixed by adding 400 µl PBS with 0.4% formalin and the DNA contents were analyzed in samples.The flow-cyto metric analyses were performed with FACS Calibur (Becton Dickinson, USA), which has two lasers with wavelengths of 488 and 625 nm.The narrowband filter 585/42 nm was used for the measurement of PI fluorescence.The data analysis was performed using CellQuest and ModFit LT 2.0 (BDIS, USA) software.
The cells distribution in different cell cycle phases were assessed with flow cytometry.Cells were seeded in 6well plates at a concentration of 2.5•10 4 cells/ml in 3 ml of the total culture medium volume.Cells were incubated with compounds 15 and AMMT (at concentration 2.5•10 5 M each) for 48 h under standard conditions.Cells pro portions in different phases of the cell cycle was measured by flow cytometry with the argon laser (λ excitation = 488 µm, λ emission = 585/40 µm, Becton Dickinson, USA) in accordance with standards for dyeing [19].The apoptotic index was assessed by staining cells with PI, which is selectively bound with DNA intercalating sites.
The assessment of the mitochondrial dehydro genase activity in the presence of studied compounds was performed by using the MTT assay [15].MTT (3(4,5dimethylthiazol2yl)2,5diphenyltetrazo lium bromide) is the yellow monotetrazolium salt (Fig. 1).Its reduction is the most often used method for measurements of the cell proliferation and cyto toxicity [16].
The biochemical essence of this method is based on the fact that mitochondrial dehydrogena ses of living cells are capable to cleave tetrazolium rings with formation of insoluble purple crystals (formazan).The dehydrogenase reduction activity considerably depends on the intracellular concen tration of NADH and NADPH, which is driven by the presence of extracellular glucose [17].Mitochon drial succinate dehydrogenase and cytochrome C are mainly involved in the MTT reduction [18].Thus, compounds affecting the MTT reduction rate may serve as modifiers of the mitochondrial respiration.
MTT (20 µl) was added to the culture medium 4 h before the termination of the cells incubation in order to achieve the final concentration of 0.6 mM.Formazan crystals formed after the incubation with MTT were dissolved in 100 µl of dimethylsulfoxide.The plate was analyzed on the spectrophotometer at 540 nm.
The graphical and statistical data processing (calculation of means and standard deviation, statis tical analysis of the reliability, differences in values of all described experiments according to Student's ttest standard criteria) were performed using the software Microsoft Excel™ 2010.All experimental results are expressed as a value ± standard deviation (m ± m).Values with p ≤ 0.05 were considered as reliable.

results and discussion
The tendency of AMMT to the thiolthione tau tomerism and the presence of various nucleophilic reactive sites in the molecule, which cause its poly dentate nature, determine the possibility to form transition metal complexes with various composi tions and structures as well as the way of the ligand coordination dependence on synthesis conditions and the metal nature.The synthesis of complexes 13 were performed in accordance with the scheme shown in Fig. 2.
The composition and structure of all synthe sized compounds were determined by the elemental analysis, Xray diffraction study, UVVis spectra and nuclear magnetic resonance (NMR) on proton ( 1 H) and carbon ( 13 C) nucleus.The complex 1 is novel and the general form of the molecule 1 according to the Xray diffraction data is represented on Fig. 3.The Complex 1 crystallizes in the triclinic crystal system, space group P1, with following unit cell pa rameters: a = 8.076 (19) 7)N( 8) in com parison with the original AMMT [20] are slightly elongated to 0.029/0.023and 0.019/0.033Ǻ due to the coordination of sulfur and nitrogen atoms with metal ions.
The analyses of UVVis spectra of AMMT and complexes 13 were carried out after their de composition into Gaussian components (Table 1).The wide absorption band of the AMMT solution at 40383 cm 1 consists of three components that corre spond to π→π* electron transitions (C=N) of the tri azole ring [23].It should be noted that AMMT thiolic tautomer dominates in solutions [20], as a result the π→π* electron transitions of the (C=S) group are not

Fig. 1. Scheme of reduction of 3-(4,5-dimethyltiazole-2-yl)-2,5-diphenyltetrazole bromide to crystallized formazan Fig. 2. Scheme of the synthesis of palladium(II) and nickel(II) complex compounds with AmmT
visualized.In contrast to AMMT, one of Gaussian components in spectra of complexes 13 corresponds to π→π* electron transitions of the (C=S) group of the thione tautomeric form of coordinated ligand molecules in the region of 28221-28775 cm 1 (Tab le 1).Furthermore, in 1 and 2 complexes spectra the Gaussian component at 24153 and 23835 cm 1 cor responds to dd electron transitions in the Pd 2+ ion.Thus, UVVis data prove the similarity in structures of complexes in solid states and in solutions.
Coordination compounds are also stable in di methyl sulfoxide solutions, which are indicated by 1 H ( 13 C) NMR spectra of complexes 1 and 2. The analy sis of 1 H NMR of the starting AMMT proved that in solution AMMT exists as the thiolic tautomer since the spectrum contains the singlet at 13.39 ppm which corresponds to the proton signal of the SH functional group.This singlet is absent in the spectra of com plexes 1 and 2, but a wide singlet at 9.58/9.49ppm is observed.This singlet corresponds to the proto nated nitrogen atom of the triazole moie ty.The wide singlet of the NH 2 group in spectra of complexes is shifted downfield by ∆δ = +0.67/+0.63ppm.There are three carbon signals in 13 C NMR spectra of com plexes 1 and 2 in positions 1, 2, 3 at 166.49/165.42,149.19/149.18,10.48/10.50ppm, respectively.The signal C(1) of the complex 1 has the largest down field shift (∆δ = +1.02ppm) in comparsion with free

Fig. 3. molecular structure of the complex 1 according to X-ray diffraction analysis
AMMT that may be related to its bidentate coordina tion to the central metal ion.Compounds 13 are soluble in water, which al lowed us to compare their action on metabolism of HeLa cells with the action of original compounds of AMMT and inorganic metal salts such as K 2 PdCl 4 (4), Ni (NO 3 ) 2 •6H 2 O (5) in vitro.
The screening of complex compounds 1-3, AmmT and metal salts 4, 5 with regard to hela cells in the mTT assay.Obtained data allow to sug gest that the incubation of HeLa cells with com pounds 15 and AMMT in vitro in a certain way affect their metabolism.In particular, MTT assay re sults showed (Fig. 4), that the compound 1 increases mitochondrial dehydrogenases activities in the con centration range from 0.032 to 0.062 mM.A similar effect for the compound 2 at concentrations 0.008, 0.032, 0.062, 0.125 mM was observed.However, the relationship between the increase/decrease of the dehydrogenase activity and the concentration of compounds was not found.AMMT activated mito chondrial dehydrogenase in the concentration range of 0.008-0.016mM.The concentration range for palladium inorganic salt 4 was 0.0160.032mM.It should be noted that cells which were incubated with compounds 3 and 5 and control cells did not show statistically significant discrepancies.Thus, the com plex compound 3 and nickel inorganic salt 5 (which was used for the synthesis of complex compound 3) do not exhibit the biological activity, as well as the toxic effect.
Thus, studied complexes 1, 2, as well as AMMT are able to enter into cells like MTT, moreo ver, they can compete with MTT, acting as substrates for mitochondrial dehydrogenases.Taking into con sideration, that the MTT concentration was 0.6 mM (time of incubation in the presence of MTT was 4 hours), and studied compounds have been screened in much smaller ranges of concentrations for 24 hours, it is obvious that the compounds 1, 2, as well as AMMT and palladium inorganic salt 5 penetrate into the cell without damage to the cell membrane.They are also accelerating the energy transfer, spe cifically affecting the mitochondrial respiration rate.The compound 3 and nickel inorganic salt 5 do not show such an activity in the presence of MTT but cytotoxic effect also was not observed.It should be noted that unlike MTT the studied compounds did not stain cells.
As it is indicated earlier, MTT is a substrate for succinate dehydrogenase (SDH), which is involved in the electron transport chain to molecular oxygen.Using of the SDH inhibitors has showed that the in hibition of the activity of this enzyme leads to the decrease of the MTT reduction rate [16].The SDH, which participates both in the tricarboxylic acid cy cle and respiratory chain, plays an important role in the mitochondria function as well as in cell func tions in general.The SDH catalyzes the reversib le succinic acid (succinate) oxidation to fumaric acid in the citric acid cycle.The oxidation of 1 succi nate equivalent leads to the formation of 2 adeno sine triphosphate (ATP) equivalents.Thus, electrons are transferred from SDH into respiratory chain to coenzyme Q. Mitochondrial SDH of mammals is composed of four subunits: two hydrophilic and two hydrophobic.The first two subunits: A (flavoprotein) and B (ironsulfur protein) are hydrophilic.The sub unit A contains the covalently bonded flavin adenine dinucleotide (FAD), which acts as a cofactor of the succinate dehydrogenase reaction.The subunit B contains three ironsulfur clusters [2Fe2S], [4Fe4S] and [3Fe4S].The other two subunits: C and D, are hydrophobic and membrane anchored [21].It should be noted that, although genes which are responsible for the SDH subunits expression are "housekeeping genes", mutations in these genes cause the malignant tumor.Thus, mutations in subunits B, C and D may cause paragliomas or pheochromocytoma.Mutations in the subunit A may cause leiomyoma, leiomyosar coma or renalcell carcinoma [22].
It is known that the respiratory chain enzymes and the Krebs cycle dehydrogenases determine the rate of the aerobic oxidation, which is the main path way of the energy generation.The complex com pounds synthesized in this work, such as 1, 2 and AMMT, can be involved in these metabolic conver sions.
effect of synthesized compounds on the mitotic cycle and apoptosis level in the hela cell line.The effect of these compounds on the mitotic cycle and apoptosis level of HeLa cell line was the next step of our study.The obtained results have showed that the synthesized complex compounds 13 as well as AMMT and inorganic salts of palladium 4 and nickel 5 do not affect the mitotic cycle since the number of cells in phases G 0 /G 1 , G 2 /M, S, G 2 /M+S of the cell cycle in the presence of studied compounds (Table 2) do not differ from control cells.
The studied compounds did not affect the ap optotic cells level (Fig. 3) and did not cause cyto toxic/cytostatic effects, so their activity apparently is aimed at the regulation of the cellular metabolism.The studied compounds in their effects are similar to MTT which was used in work for their screening .Some of them, in particular 1, 2 and 4, exhibit syn ergy with AMMT.They enhance the mitochondrial SDH activity, however do not stain cells, in contrast to MTT.Compounds 3 and 5 were inactive against a background of the MTT.
It was interesting that palladium salt 4 showed an activating and inducing effect on mitochondrial dehydrogenases.It may indicate its role as an inor ganic catalyst.The synthesized palladium complex compounds 1 and 2 demonstrated a greater activity.The compound 2 showed the effect within a wide range of concentrations.AMMT, which was used as a starting substance for their synthesis, demonstrated higher activity in comparison with the synthesized complexes, but within a little smaller range of con centrations.It should also be noted that nickel salt 5 did not lead to changes in the mitochondrial respira tion activity.The similar effects observed with the complex 3 confirm that the compound 5 may block the AMMT biological activity.
Thus, AMMT and palladium inorganic salt 4 can be used for the synthesis of biologically active complex compounds, including those, which may be involved in the regulation of cell metabolism.
It is known that MTT can be reduced by other nonmitochondrial dehydrogenases, such as Nethyl maleimid (NEM) sensitive flavin oxidase [16].It is obvious that studied compounds act by similar mechanisms.The decrease in activities of these en zymes leads to the decrease of the tissue respiration rate and to the decrease of the energy supply to an organism.
Three AMMT complex compounds with Pd 2+ (1, 2) and Ni 2+ (3) ions have been synthesized in this study.The singlecrystal Xray diffraction method showed that in presented complexes AMMT exists in thione tautomeric form as monodentate or biden tate ligand with the S or S,Ndonor coordination to the central metal ion.The synthesized watersoluble complex compounds were proposed as potential bio logically active agents.
The biological activity of the complexes 13 in comparison with the activity of starting AMMT, as well as inorganic salts K 2 PdCl 4 , Ni(NO 3 ) 2 •6H 2 O were studied.It was shown that the biologically ac tive AMMT and its complexes with Pd 2+ ions can be used as agents that induce the mitochondrial respi ration.Nickel nitrate blocks the AMMT biological activity, however the synthesized on its base com plex 3 does not affect cell metabolism, cell cycle and apoptosis.Palladium complexes 1, 2 induce the mi tochondrial dehydrogenases activity and do not af fect the cell cycle and apoptosis level, moreover the compound 2 exhibits the biological effect in a much wider range of concentrations, in comparison with the compound 1.