Synthesis and Antioxidative Potency of Novel Amidino Substituted Benzimidazole and Benzothiazole Derivatives

Herein we present the synthesis of versatile amidino substituted benzothiazole 3–10 and benzimidazole 12–19 derivatives substituted with the variable number of hydroxy and methoxy groups. Furthermore, the synthesized compounds were explored for their antioxidative activity in vitro by using three biological assays, namely DPPH, ABTS and FRAP. The obtained results indicated that the variable number of hydroxy groups together with the type of the amidino substituent strongly influenced the antioxidative activity and reducing power of tested compounds. The most promising antioxidative activity showed trihydroxy substituted compounds 6, 10, 15 and 19. In general, it was noticed that unsubstituted amidino group induced the more pronounced activity in comparison to derivatives bearing 2-imidazolinyl group.


INTRODUCTION
ENZIMIDAZOLES and benzothiazoles, amongst all nitrogen heterocycles, are one of the most important building motifs of many essential and physiologically active natural, semisynthetic or synthetic pharmaceuticals. [1]eing fundamental structural parts of a number of biologically active derivatives, there has been an increasing interest in medicinal chemistry in the role of both heterocycles due to their possible pharmacological, chemical or industrial applications. [2,3]Their derivatives display a broad spectrum of different biological features such as anticancer, antiviral, antibacterial, antifungal, antiinflammatory etc. [4][5][6] In our research group we have published several publications regarding antiproliferative and antitumor activity of versatile benzimidazole and benzothiazole derivatives bearing different amidino supstituents. [7,8]mong all amidino substituted benzazole derivatives, the ones with the cyclic amidino substituent, namely 2imidazolinyl group, showed the most significant antiproliferative activity in vitro with IC50 values in submicromolar range of concentrations. [9]Almost all previously synthesized active derivatives have been designed to have cationic amidino substituents which have significantly improve their biological activity.Amidine substituents placed at the termini of the molecule have great importance in the molecule -biological target interactions allowing the formation of the stable complex with biologically important molecules.Also, 2-imidazolinyl substituted tetracyclic benzimidazole derivatives showed pronounced selectivity towards colon carcinoma cells being B strong DNA intercalators and inhibited human topoisomerase II. [10]On the other hand, acyclic benzimidazoles and benzothiazoles have been proven to be a selective DNA minor groove binders which was confirmed with several spectroscopic methods.Regarding the 2-phenylbenzothiazole derivatives, we have shown that their antiproliferative activity strongly depends on the type of amidino group as well as on their position on the targeted molecule. [7,9]Nowadays, the attention of medicinal chemists is focused also on the development of the novel antioxidative agents, especially after it has been evidenced that oxidative damage of important biomacromolecules is directly connected with the pathogenesis of various diseases. [11,12]Thus, the reactive oxygen species (ROS) through the damage of cellular biomacromolecules like proteins, lipids or DNA/RNA may be playing an important role in the development of cancer, atherosclerosis, aging or rheumatoid arthritis. [13,14]One of the chain reactions caused by ROS is lipid peroxidation which could generate mutagenic products with carcinogenic properties. [15]Many natural and synthetic products possess antioxidative capacity and are studied as a promising antioxidants which could reduce the rate of ROS production and the oxidation of biomacromolecules. [16]Several publications described the antioxidative activity and potential of benzimidazole and benzothiazole derivatives as antioxidative agents.B. Zhou and co-authors have been published the antioxidative activity of a series of 2-aryl benzimidazole derivatives (Figure 1). [17]Obtained results revealed that derivative bearing a hydroxy group at the 5-position of benzimidazole nuclei posses a comparable or better antioxidant activity in comparison to standard antioxidant tert-butylhydroquinone (TBHQ).On the other hand a series of 2-aryl substituted benzothiazoles (Figure 1) has also show antioxidative activity and significant radical scavenging potential due to the presence of electron donating substituent. [18]ll the above mentioned facts guided us to design and synthesize novel amidino substituted benzazole derivatives in order to evaluate their antioxidant potential.The obtained results are discussed in terms of SAR to define the influence of the type of amidino group and heterocyclic nuclei as well as the number of hydroxy groups attached on the phenyl ring on the antioxidative activity of prepared derivatives.

GENERAL METHODS
Melting points were determined by means of Original Kofler Mikroheitztisch apparatus (Reichert, Wien).The 1 H NMR and the 13 C NMR spectra were recorded with the Bruker Avance DPX-300 or Bruker AV-600 using TMS as internal standard.Chemical shifts are reported in parts per million (ppm) relative to TMS.LC-MS was performed on the Agilent 6120 Quadrupole coupled to the Agilent 1290 Infinity II UHPLC using electrospray ionization (ESI).

Determination of the reducing activity of the stable radical 1,1-diphenyl-picrylhydrazyl (DPPH)
The determination of reducing activity of the stable DPPH radical was measured according to previously reported procedure with modification. [21]Briefly, to solution of DPPH (final concentration 100µM) in methanol was added equal volume of the tested compounds various concentrations dissolved in DMSO.The assay was carried out in a 96 well microtiter plate.Methanol and DMSO was used as control solution.Sample blank were also performed.After 30 min in dark at room temperature the absorbance was recorded at 517 nm on microplate reader µQuant (Biotec Inc.).All measures were done in triplicate and averaged.The results were presented as IC50 in Table 1.

Free radical scavenging ability by the use of a stable ABTS radical cation (2,2'-azinobis-(3-ethylbenzthiazoline-6sulphonic acid)
The total antioxidant activity assay was adjusted for microplate reader. [22]For the standard ABTS assay, ABTS •+ was prepared by mixing an ABTS stock solution (7 mM in water) with 2.45 mM potassium persulfate and left in dark at room temperature for 12-16 h until reaching a stable oxidative state.Before analysis, the ABTS •+ solution was diluted 100 times with methanol to obtain an absorbance of 0.700 (0.01 at 734 nm).The radical was stable in this form for more than two days when stored in the dark at room temperature.Standards and solutions of tested compounds (20 µL) were mixed with working ABTS•+ radical cation solution (280 µL) to each well the microplate, shake and incubate at room temperature for 5 min.The decrease of absorbance at 734 nm was recorded by microplate reader µQuant (Biotec Inc.).Methanol (without ABTS •+ solution) was used as a control and BHT as reference compound.All compounds were tested in triplicate and averaged.Results were expressed as IC50 and presented in Table 1.

Determination of Ferric Reducing/Antioxidant Power (FRAP assay)
The FRAP method was adjusted for a 96-well microplate according to previously reported procedure. [23]The antioxidant capacity of the tested compounds was estimated as their power to reduce the TPTZ-Fe(III) to TPTZ-Fe(II) complex, which is fast, simple and reproducible.A solution of 10 mM TPTZ and 20 mM ferric chloride was diluted in 300 mM sodium acetate buffer (pH 3.6) at a ratio of 1:1:10.The tested compound solution (20 µl) was added to the 96-well microplate followed by working FRAP solution (280 µl).The mixture was shaken and incubated 30 min at 37 °C in the dark.Final concentration of tested compounds was 0.01 mM for all except for compounds 6, 10, 15 and 19 (250 µM).The absorbance at 593 nm was recorder using microplate reader µQuant (Biotec Inc.).For the standard curve construction ferrous sulphate (FeSO4 x 7H2O) was used and range of standard curve was 20-2000 µmol/L.All results were then expressed as Fe 2+ equivalents (Fe 2+ µmol).All compounds were tested in triplicate and the results were averaged and presented in Table 1.

Statistical analysis
Calculation of IC50 value is performed by using GraphPadPrism software (GraphPad Software, Inc. La Jolla, CA USA 2012).Briefly, individual concentration effect curves are generated by plotting the logarithm of the concentration of tested compounds (X) against corresponding percent inhibition values (Y) using least squares fit.All experiments were tested at least three times.Results were expressed as mean ± standard deviation (SD) and analyzed using the Student's t-test to assess the statistical significance using the Statistica software (STATISTICA 2010 program, Tulsa, OK, USA).P values < 0.05 were regarded as significant.
The structures of all newly prepared benzothiazole and benzimidazole derivatives were determined by using 1 H and 13 C NMR spectroscopy and mass spectrometry.NMR analysis based on the values of H-H coupling constants and chemical shifts in the 1 H and 13 C NMR spectra confirmed the structures of compounds.Furthermore, IR spectroscopy was used for the monitoring of Pinner reaction due to the synthesis of main precursors 2a, 2b, 11a and 11b.

Antioxidative Activity
In this research we have synthesized benzimidazole and benzothiazole derivatives which we envisioned to show protection against free radicals attack as an index of pharmacological usefulness.To determine antioxidant activity of different benzazoles, the reducing activity of the stable radical 1,1-diphenyl-picrylhydrazyl (DPPH), free radical scavenging ability by the use of radical cation 2,2azinobis-(3-ethylbenzthiazoline-6-sulphonic acid (ABTS) and ferric reducing/antioxidant power (FRAP) were evaluated.The evaluation was conducted by in vitro methods which require a spectrometric approach. [25]he radical scavenging assay using stable radical DPPH indicates the ability of tested compounds to donate proton/electron.The DPPH final concentration was 100 µM and results were read after 30 min and presented in Table 1.The results were expressed as IC50 values with the exception of 5, 7, 9, 12 and 17 which did not exhibit activity under assay condition.Based on experimental results presented, among all synthesized compounds, benzo- Moreover, it was noticed that the type of the amidino group attached on the heteroaromatic scaffold also influenced the antioxidative activity.Thus, derivatives bearing the unsubstituted amidino group 6 and 15 showed higher activity in comparison to the derivatives with 2imidazolinyl group 10 and 19.In addition, there were no any differences in the antioxidative activity between benzothiazole or benzimidazole derivatives substituted with amidino group 6 and 15.Furthermore, 2-imidazolinyl substituted benzothiazole derivative 10 compared to the same benzimidazole analogue 19 showed significantly higher antioxidative activity but lower than control BHT.
Following assay employed in the testing of antioxidant ability of benzazole derivatives was ABTS radical cation assay at pH 4 as well. [26]The ability of pure compounds to decrease the color reacting directly with ABTS •+ radical is a measure of their antioxidant capacity.In addition, ABTS •+ radicals are more stable then DPPH radicals and could be used to evaluate hydrophilic and lipophilic compounds. [23]The results are presented as IC50 in Table 1.
Obtained results revealed a higher number of active compounds in comparison to the DPPH assay.The different number of hydroxy groups attached on the phenyl ring strongly influenced the antioxidative activity with the trihydroxy substituted derivatives being the most active ones.Trihydroxy substituted benzothiazole 6 and 10 and benzimidazole The reducing power of investigated compounds was determined by FRAP assay.This method is based on the ability of compounds to reduce ferric tripyridyl triazine complex (TPTZ) to ferrous (Fe 2+ ) form producing an intense blue colour as result.
The reaction progress is monitored by change in the absorbance at 593 nm.Higher increase in the absorbance signifies higher reduction potential of investigated compounds.The FRAP assay results of tested compounds are presented in Figure 2.
Presented results revealed that only four derivatives 6, 10, 15 and 19 showed meaningful ferric reducing antioxidative activity.Structure-activity relationship revealed also the presence of similar activity pattern as in the DPPH assay.The presence of three hydroxy substituents on the phenyl ring might play the crucial role for ferric reducing antioxidant.

CONCLUSION
Within this manuscript, we present the design and synthesis of amidino substituted benzothiazole and benzimidazole derivatives directly attached to the phenyl ring with the variable number of hydroxy and methoxy groups.The prepared compounds were synthesized to explore their antioxidative activity in vitro.
Furthermore, our attention was to study SAR and the influence of the number of methoxy and hydroxy groups, the type of the heteroaromatic nuclei attached to the phenyl ring as well as the type of the amidino group placed either on the benzothiazole or benzimidazole moiety on the antioxidative activity.
The results obtained from the used antioxidative assays indicated that the variable number of hydroxy groups strongly influenced the antioxidative activity and reducing power of tested compounds, among which compounds 6, 10, 15 and 19 showed the most pronounced activity.Concerning the type of the amidino substituent placed at the heteroaromatic scaffold, it could be noticed, that in general unsubstituted amidino group had a greater impact on the increase of antioxidative activity.Furthermore, there was no significant difference in the antioxidative activity among benzothiazole and benzimidazole derivatives.All obtained results pointed out that the new series of synthesized derivatives demonstrated a high potential for expansion and optimization of their antioxidative activity.
15 and 19 derivatives showed similar activity pattern as in the DPPH assay with the 2-imidazolinyl substituted benzimidazole 19 showing the weakest activity.Moreover, derivatives bearing two hydroxyl groups 4, 8, 13 and 17 also showed good antioxidative activity.Thus, benzimidazole derivatives 13 and 17 exerted improved activity in comparison to the same benzothiazole analogues 4 and 8 with the compounds bearing 2-imidazolinyl group being more active.Due to the replacement of one hydroxy group with the methoxy group attached on the phenyl ring, antioxidative activity was decreased with the benzimidazole derivatives 14 and 18 showing more pronounced activity.The most active compounds 4, 6, 8, 10, 13, 15 and 17 which exhibited antioxidant activity higher then BHT ranging from 6.2 to 13.3 µM (P<0.05).The compounds 18 and 19 showed almost similar results as control compound BHT (35.7±1.3 µM, 32.0±3.7 µM; respectively).

Figure 2 .
Figure 2. Reducing power of tested compounds measured as FRAP (P < 0.01 for all compounds vs BHT).

Table 1 .
IC50 values of 2-arylbezimidazoles and benzothiazoles for DPPH and ABTS free radical scavenging activity* ,b Values with different superscripts in the same column are significantly different (P < 0.05) by the t-test (compounds vs control BHT).
* Values are presented as means ± standard deviation.a