Synthesis and Characterization of Novel Hydrazone Based Anti-mutagenic and Antioxidative Agents

Article history: Received on: 04/08/2015 Revised on: 07/09/2015 Accepted on: 24/09/2015 Available online: 12/11/2015 The present study was designed to produce novel hydrazine and evaluate their biological properties including antioxidant, antityrosinase and antimutagenic. 4-allyloxybenzoyl hydrazine (1) reacts with 5-acetyl-1,3-dimethyl barbituric acid (2) and 2-Isonitrosoacetophenone (3) in the presence of acetic acid as a catalyst to produce the hydrazone derivatives 4 and 5 in high yields respectively. The new hydrazone derivatives 4 and 5 have been fully characterized by using multinuclear NMR ( 1 H, 13 C) spectroscopy and elemental analysis. The compounds 4 and 5 were studied for their antioxidant and tyrosinase enzyme inhibition activity. In addition the mutagenic and antimutagenic activities were evaluated by Ames Salmonella/ microsome mutagenicity test. The results showed that both of compounds exhibited significant antioxidative and antimutagenic activity and compound 5 has shown moderate tyrosinase inhibition activity. This study suggested that these compounds could be considered as novel bioactive agents in pharmaceutical area.


INTRODUCTION
There are several biologically active organic compounds present significant application in the bioorganic chemistry.Most of the common features of these compounds have several donor atoms such as nitrogen, sulphur or oxygen which interact to biological material in vivo or in vitro environments (Wang et al., 2010;Chugunova et al., 2014;Glinma et al., 2015).Oximes and barbiturates have been known for a long time as highly biologically active compounds (Laxmi et al., 2012;Angelusiu et al., 2010;Savini et al., 2002).Besides on behalf of their biological importance, hydrazone derivatives containing barbiturate or oxime moiety possess diverse pharmacological activities.These types compounds were named by hydrazone-barbiturates and hydrazone-oximes which are typically act as tridentate, bidentate or monodentate compounds reacting through the oxygen or imine nitrogens depending on the reaction conditions (Neumann et al., 2014;Giziroglu et al., 2013;Gup et al., 2006).Antioxidant and antimutagenic effects of organic compounds are important parameters due to their prevention and treatment capabilities on several diseases (Spada et al., 2008;Parvathy et al., 2010;Salleh et al., 2014).Interests in search for new antioxidants and antimutagens have grown dramatically over past years because of their significant biological, industrial and economic impact.In this context, several researchers investigated novel compounds for use as antioxidative and antimutagenic food or drug additives (Siddaiah et al., 2007;Parvathy et al., 2009).
Survey of literature revealed that there are several reports on antimicrobial, antitubercular, anticonvulsant, antitumor and anti-inflammatory activities concerning the structures of such molecules with hydrazone, barbiturate and oxime groups (Abele et al., 2010;Neumann et al., 2014;Zheng et al., 2010;Zaragoza-Dörwald, F. 2012).Interestingly, although over the years, too many compounds including hydrazone-barbiturates and hydrazone-oximes moiety have been synthesized, there are very few examples mentioned about antioxidant activity, tyrosinase inhibition activity and mutagenic or antimutagenic potential (Giziroglu et al., 2013;Khan et al., 2011;Zaragoza-Dörwald, F. 2012).Due to the limited information, we selected antioxidant, antimutagenic and tyrosinase inhibitory properties of new hydrozane derivatives as biological activity parameters.
In this study we aimed to synthesize new hydrazone derivatives containing barbiturate and oxime moiety by bringing together 4-allyloxybenzoyl hydrazine (1) and 5-acetyl-1,3dimethyl barbituric acid (2) or 2-Isonitrosoacetophenone (3).These compounds are fully characterized by multinuclear NMR ( 1 H, 13 C) spectroscopy and elemental analysis.Antioxidant potentials of the compounds 4 and 5 were determined by eight assays including total antioxidant, reducing power, radical scavenging and metal chelating activity.We also investigate their tyrosinase enzyme inhibition activity because of its relation with antioxidant properties including chelating activity in biochemical applications (Woo and Je 2013).The screening of their mutagenic and antimutagenic activities was investigated by Ames Salmonella/microsome mutagenicity test (Maron and Ames 1983).

General
Commercially available reagents were used without further purification.4-allyloxybenzoyl hydrazine was prepared by refluxing ethyl 4-allyloxybenzoate (10 mmol) with hydrazine hydrate (2.5 mL) for 4 h (Shanker et al., 2013;Komurcu et al., 1995).1,3-dimethyl-5-acetyl-barbituric acid (2) was prepared according to the reported procedures (Jursic et al., 2011).Melting points were measured with an Electro thermal 9200 melting point apparatus and the values are uncorrected. 1H, 13 C NMR spectra were recorded on a Bruker Ultrashield 400 Plus NMR spectrometer.Chemical shifts are reported in ppm downfield from Me 4 Si and were referenced to solvent peaks.Elemental analyses were done on a Leco-932.

Antioxidant and Tyrosinase inhibition activity Tyrosinase inhibition activity
Tyrosinase inhibitory activity was measured using a modified dopachrome method with L-3,4-dihydroxyphenylalanine (L-DOPA) as substrate, as previously reported (Zengin et al., 2014a).Sample solution (25 µL) was mixed with tyrosinase solution (40 µL) and phosphate buffer (100 µL, pH 6.8) in a 96well microplate and incubated for 15 min at 25 C.The reaction was then initiated with the addition of L-DOPA (40 µL).Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (tyrosinase) solution.The sample and blank absorbance were read at 492 nm after 10 min incubation at 25 C.Absorbance of the blank was subtracted from that of the sample.

Total antioxidant activity by phosphomolybdenum method
Total antioxidant activity of the samples was evaluated by phosphomolybdenum method (Giziroglu et al., 2013).Sample solution (0.2 mL) was combined with 2 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate).The sample absorbance was read at 695 nm after 90 min incubation at 95 C.
Effect of the samples on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical were estimated according to a method reported by Sarikurkcu (2011) and Giziroglu et al. (2013).Sample solution (1 mL) was added to a 4 mL of 0.004% methanol solution of DPPH.Sample absorbance was read at 517 nm after 30 min incubation at room temperature in dark.
Scavenging activities of the samples on ABTS cation radical was measured according to the method of Zengin et al. (2014a).Briefly, ABTS .+radical cation was produced directly by reacting 7 mM ABTS solution with 2.45 mM potassium persulfate and allowing the mixture to stand for 12-16 h in dark at the room temperature.Prior to beginning the assay, ABTS solution was diluted with methanol to obtain an absorbance of 0.700 ± 0.02 at 734 nm.Sample solution (1 mL) was added to ABTS solution (2 mL) and mixed.Sample absorbance was read at 734 nm after 30 min incubation at room temperature.
Superoxide anion radical scavenging activity was carried out in riboflavin-light-nitroblue tetrazolium (NBT) system (Sarikurkcu et al., 2014).Sample solution (0.25 mL) was added to reaction mixture containing riboflavin (0.1 mL, 0.1 mg/mL), ethylenediaminetetraacetic acid (EDTA) (0.1 mL, 12 mM), NBT (0.05 mL, 1 mg/mL), phosphate buffer (1 mL, 50 mM, pH 7.8) and 1-butanol (0.5 mL).The reaction mixture was illuminated for 10 min at room temperature and the sample absorbance was read at 560 nm.Un-illuminated reaction mixture was used as a blank.Absorbance of the blank was subtracted from that of the sample Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which can be measured by Griess reaction (Sarikurkcu et al., 2014).Sample solution (0.5 mL) was mixed with sodium nitroprusside (0.5 mL, 5 mM) in phosphate buffer (0.2 M, pH 7.4) and incubated for 150 min at room temperature.Similarly, a blank was prepared by adding sample solution (0.5 mL) to phosphate buffer (0.5 mL).Diluted Griess reagent (1 mL, 1:1) was added to the incubated sample and allowed to stand for 30 min.The sample and blank absorbance were read at 548 nm.Absorbance of the blank was subtracted from that of the sample.

Reducing power
The reducing power was investigated using cupric ion reducing (CUPRAC) and ferric reducing antioxidant power (FRAP) methods, as previously described in the literature.
The cupric ion reducing activity (CUPRAC) was determined according to the method of Zengin et al. (2014b).Sample solution (0.5 mL) was added to a premixed reaction mixture containing CuCl 2 (1 mL, 10 mM), neocuproine (1 mL, 7.5 mM) and NH 4 Ac buffer (1 mL, 1 M, pH 7.0).Similarly, a blank was prepared by adding sample solution (0.5 mL) to a premixed reaction mixture (3 mL) without CuCl 2 .Then, the sample and blank absorbance were read at 450 nm after 30 min incubation at room temperature.Absorbance of the blank was subtracted from that of the sample.

Metal chelating activity on ferrous ions
Metal chelating activity on ferrous ions was determined by the method described by (Zengin et al. (2014b).Briefly, sample solution (2 mL) was added to FeCl 2 solution (0.05 mL, 2 mM).The reaction was initiated by the addition of 5 mM ferrozine (0.2 mL).Similarly, a blank was prepared by adding sample solution (2 mL) to FeCl 2 solution (0.05 mL, 2 mM) and water (0.2 mL) without ferrozine.Then, the sample and blank absorbance were read at 562 nm after 10 min incubation at room temperature.Absorbance of the blank was subtracted from that of the sample

Bacterial strains
S. typhimurium TA98 and S. typhimurium TA100 were used for the mutagenity and antimutagenity tests.The strains were analyzed according to the procedure in literature.(Mortelmans and Zieger 2000;Oh et al., 2008;Basgedik et al., 2011)

Mutagenic and antimutagenic activity Viability assays and determination of test concentrations
Cytotoxic dose of the compounds 4 and 5 were determined by the method of Mortelmans and Zeiger (2000).The toxicity of the compounds 4 and 5 toward S. typhimurium TA98 and TA100 were estimated according to the procedure in literature (Santana-Rios et al., 2001;Yu et al., 2001;Gulluce et al., 2001).

Mutagenicity and antimutagenicity tests
In this study, the plate incorporation method was used to assess the results of mutagenicity and antimutagenicity assays as previously described in the literature (Maron and Ames, 1983).The known mutagens 4-nitro-o-phenylenediamine (4-NPD) 3 µg/plate) for S. typhimurium TA98 and sodium azide (NaN 3 ) (8 µg/plate) for S. typhimurium TA100 were used as positive controls and Dimethyl sulfoxide was used as a negative control in mutagenicity and antimutagenicity tests.

Statistical analysis
All the assays were carried out in triplicate.The results were expressed as mean values and standard deviation (mean ± SD).Differences between the values were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's honestly significant difference post hoc test (α=0.05).All the analyses were carried out by using SPSS v22.0 software.

Synthesis and spectroscopic studies
The new hydrazone derivatives 4 and 5 were prepared by reaction of 4-allyloxybenzoyl hydrazine 1 with 5-acetyl-1,3dimethyl barbituric acid 2 and 2-isonitrosoacetophenone 3 in the presence of EtOH along with catalytic amount of acetic acid (Fig. 1).
The compound 4 and 5 are soluble in DMF, DMSO, CHCl 3 , Ethanol however, it is insoluble in water and other common organic solvents such as toluene, hexane, pentane, diethyl ether etc.
The new barbiturate derivative 4 and 5 are nonhydroscopic and characterized by 1 H, and 13 C NMR spectroscopy and elemental analysis.According to literature reports, unlike compound 5, compound 4 may exist in three tautomeric forms as shown in Fig. 2 (DaSilva et al., 2003;Giziroglu et al., 2013).
To decide which tautomeric form of the compound 4 in the solution, we checked the 13 C NMR spectra.The 13 C NMR spectra of 4 in CDCl 3 display two peaks belonging to -NH-C=C carbons at 87.8 and 118.0 ppm reveals that this compound exist in tautomeric form(II) in the solution (DaSilva et al., 2003;Giziroglu et al., 2013).Another distinctive signal in 13 C NMR varies from 115.9 to 154.0 ppm indicates aromatic and allylic CH carbons.The 1 H NMR spectra of compound 4 show signals at 2.71, 3.26 and 3.28 ppm can be attributed to -CH 3 protons.The characteristic allyl protons are observed at 5.28, 5.36 and 5.98 ppm.The NH proton bound to carbonyl of the compound 4 was observed at 13.72 whereas the NH proton bound alkenyl group appeared at the higher field at 7.80 ppm.
Examination of 1 H NMR spectra of compound 5 in DMSO-d6 solution revealed that the chemical shift of the oxime and amidic protons have characteristic values at 13.18 and 12.63 ppm as broad signals, respectively.The allylic protons are observed at 5.28, 5.44 and 6.07 ppm.The 13 C NMR spectra of 5 in DMSO-d6 solution exhibit that the chemical shift of the amidic carbonyl carbons have characteristic values at 161.3.The peaks at 68.4 attributed to carbon of -C-O-Ar.Another 13 C NMR signals varies from 114.8 to 145.2 ppm indicates aromatic and allylic -CH carbons. 1 H NMR spectras for 4 and 5 are shown in Fig. 3.
All of spectral results were confirmed by elemental analysis, and the molecular formula was determined as C 18 H 20 N 4 O 5 for 4 and C 18 H 17 N 3 O 3 for 5. ) radicals, cupric ion reducing (CUPRAC), ferric reducing antioxidant power (FRAP) and total antioxidant activity phosphomolybdenum methods.Four different radicals (DPPH, ABTS, NO radical, O 2 radical) were used for define of radical-scavenging activity of compounds 4 and 5.The basis of test methods is that the antioxidants react with the free radical and changes their absorbencies.The degree of the changing in absorbance indicates the scavenging potential of the samples.Usually the radical scavenging activity was dependent on concentration and was increased with amount of the sample.The results were presented in Table 1 and it is important to note that the lower IC 50 value of sample indicate greater radical scavenging.We would like to note that the compound 4 and 5 with IC 50 values 4.99 and 6.81 mM respectively greater than those of trolox (7.29 mM) for superoxide anion radical scavenging activity.The Tyrosinase enzyme inhibition analysis showed us the compound 5 display good inhibition activity against mushroom tyrosinase.The tyrosinase inhibition of the synthesized compounds were spectrophotometrically measured and the results are expressed as kojic acid equivalents (Table 1) The compound 5 which has IC 50 value of 3.90 for tyrosinase inhibition has also more effective metal chelating activity among the both of compounds.The results showed us the oxime and imine groups cause increase in inhibitory activity.Therefore we can conclude that the inhibition mechanism could be explained that copper ions in the tyrosinase enzyme were complexed by oxime and imine groups.In the other words oxime group could be oxidized by tyrosinase enzyme.Total antioxidant activities reflect the capacity of a nonenzymatic, antioxidant defense system.The phosphomolybdenum test system is based on the reduction of molybdenum by the antioxidative compounds following the forming of a green molybdenum (V) complex, which show strong absorbance at 695 nm.Total antioxidant capacity of sample is presented as equivalents of ascorbic acid (Table 2).According to test results compound 4 has better activity than compound 5.When we look at the reducing power assay, we used two different test system namely cupric ion reducing (CUPRAC) and ferric reducing antioxidant power (FRAP).In these methods reduction of metal ions can be monitored by calculating the absorption changes.Therefore, the absorption changes are entirely based on the total reducing power of the electron donating antioxidants present in the reaction mixture.As it can be seen in Table 2, CUPRAC result followed the same tendency as those obtained using the phosphomolybdenum total antioxidant capacity.Surprisingly there is no correlation of FRAP test with phosphomolybdenum total antioxidant capacity.

Mutagenic and antimutagenic properties
The hydrazone derivatives 4 and 5 which were tested at three different concentrations, including 5, 0.5, 0.05 mg/plate for 4 and 2, 0.2, 0.02 mg/plate for 5, did not exhibit any mutagenic effect in the mutagenicity assays performed with S. typhimurium TA98 and TA100.The possible antimutagenic potential of the compounds 4 and 5 were examined against 4-NPD and NaN 3 in S. typhimurium TA 98 and TA 100, respectively.The results were evaluated by using standard plate incorporation method and summarized in Table 3.In the antimutagenicity assays performed with TA98 and TA100 strains, the compound 5 exhibited strong antimutagenic effects at 2, 0.2 and 0.02 mg/plate concentrations.The compound 5 also exhibited moderate antimutagenic effects at 2 and 0.2 mg/plate concentrations on TA100 strain.The strongest antimutagenic activity was observed at 2 mg/ plate concentration against S. typhimurium TA98 strain but the 0.02 mg/plate concentration of 5 didn't exhibit any antimutagenic effect against S. typhimurium TA100.The compound 4 showed antimutagenic effects at 5 and 0.5 mg/plate concentrations.The strongest antimutagenic activity was observed at 5 mg/ plate concentration against S. typhimurium TA100 strain.concentration (IC50: mM) at which 50% of the radicals were scavenged.** Inhibition concentration (IC50: mM) at which 50% of the Fe 2+ferrozine complex were inhibited.*** Inhibition concentration (IC50: mM) at which 50% of the tyrosinase enzyme were inhibited.

CONCLUSION
In this report we synthesized and characterized of new hydrazone based antimutagenic and antioxidative compounds.For the structural characterization various spectroscopic techniques were used.These compounds are new examples of aroylhydrazone derivatives which are known as biologically active compounds.For the first time we explored mutagenic and antimutagenic activity of a member of these kinds of compounds and it has shown significant antimutagenic activity.In addition we studied their tyrosinase inhibition activity and antioxidative properties in vitro by several assay systems.The modifications of the aroyl hydrazone moiety to increase of water solubility as well as their several biological activities are under active investigation.

a
Values expressed are means ± S.D. of three parallel measurements.The regression analysis was carried out in Microsoft Excel between percent inhibition of mutagenicity and log values of concentrations of the samples.

Table 1 :
Inhibition concentration of the samples and standards for radical scavenging, metal chelating and tyrosinase enzyme inhibitory activities a .Data marked with different letters within the same column indicate significant difference statistically (p < 0.05).b EDTA, disodium edentate.c na, not active.
a * Inhibition

Table 2 :
Effective concentration of the samples and standards for reducing power and total antioxidant activity phosphomolybdenum method a Data marked with different letters within the same column indicate significant difference statistically (p < 0.05).* Effective concentration (EC50: mM) at which the absorbance was 0.5 for reducing power and total antioxidant activity. a

Table 3 :
The antimutagenicity assay results of the comppounds 4 and 5 for S. typhimurium TA98 and TA100 bacterial strains.NPD and NaN3 were used as positive controls for S. typhimurium TA98 and TA100 strains, respectively.