Synthesis , Antimicrobial Properties , and Inhibition of Catalase Activity of 1 , 4-Naphtho-and Benzoquinone Derivatives Containing N-, S-, O-Substituted

1Division of Organic Chemistry, Department of Chemistry, Engineering Faculty, Istanbul University-Cerrahpasa, Istanbul, Turkey 2Division of Analytical Chemistry, Department of Chemistry, Engineering Faculty, Istanbul University-Cerrahpasa, Istanbul, Turkey 3Faculty of Arts and Sciences, Department of Chemistry, Aydın Adnan Menderes University, Aydın, Turkey 4Department of Technology of Biologically Active Substances, Pharmacy and Biotechnology, Lviv Polytechnic National University, Lviv, Ukraine


Introduction
Natural and synthetic quinonoid compounds are well-known substances which possess a variety of biological properties such as anticancer, antibacterial, or antimalarial drugs as well as fungicides [1].The heterocyclic derivatives of 1,4naphthoquinones have been identified that have potent biological activities towards viral [2], molluscicidal [3], malarial [4], leishmanial [5], cancer [6], and bacterial and fungal diseases [7] due to their redox potentials [8].Some of these pharmacological effects have been attributed to the formation of DNA-damaging anion-radical intermediates formed by bioreduction of the quinone nucleus.Quinones are known to inhibit electron transport involved in photosynthesis and mitochondrial respiration.Quinone-based fungicides are classified as "organic fungicides" and are known multisite inhibitors.This may be advantageous in the prevention of resistance development in fungal pathogens.Similarly, quinone-based natural herbicides were also described with multisite inhibitors.
As a part of a program directed towards the design and synthesis of N-, S-, O-substituted quinones as potential antibacterial, antifungal, and anticancer agents, we have reported the synthesis and antimicrobial as well as anticancer activities of N-, S-, O-substituted quinones [6,9,10].This paper describes the synthesis, characterization, and discovering promising pharmacologically active compounds.In this work, a catalase activity of benzo-and naphthoquinone derivatives was examined for the first time.The catalase enzyme plays an important role in removing toxic H 2 O 2 from the cells.For this purpose, the activities of the cells of this enzyme decompose H 2 O 2 generated as a result of the cell activities H 2 O and O 2 before dispersion into the body tissues.The catalase enzyme also exhibits peroxidic activity on compounds (i.e., formaldehyde, phenols, formic acid, and alcohols).In this reaction, low molecular weight alcohols serve as an electron donor.In addition to having peroxidase activity, this enzyme can use one molecule of H 2 O 2 as an electron donor and the other as an oxidant [11,12].
Consequently, the synthesis of new active derivatives with potential applications in this area and prepared by simple chemical procedures should be of increasing interest.
Here we described the synthesis, characterization, antimicrobial activity, and inhibition of catalase of 1,4-naphtho-and benzoquinone derivatives.Their structures of synthesized compounds were characterized by using elemental analysis, FT-IR, 1 H NMR, 13 C NMR, MS, and UV-Vis spectroscopy.

Experimental
2.1.Material and Methods.Infrared (FT-IR) spectra were recorded for liquids as film and for solids as KBr discs on a Perkin Elmer Precisely Spectrum One FTIR spectrometry.Microanalyses were carried out with a Thermo Finnigan Flash EA 1112 Elemental analyser.Mass spectra were obtained on a Thermo Finnigan LCQ Advantage MAX LC/MS/MS spectrometer according to either APCI or ESI techniques. 1 H NMR and 13 C NMR spectra were recorded on Bruker Avance III 500 MHz, Chemical shifts  (ppm) were reported relative to tetramethylsilane (TMS) with the solvent resonance employed as the internal standard. 1H NMR and 13 C NMR spectra in CDCl 3 refer to the solvent signal center at  = 7.26 and  = 77.0ppm, respectively.Moisture was excluded from the glass apparatus using CaCl 2 drying tubes.Spectrophotometric catalase enzyme activity measurements of synthesized compounds were performed by using a Perkin Elmer Lambda 35 UV-Vis spectrophotometer using a pair of matched quartz cuvettes of 1 cm thickness.
The following chemicals were supplied from the corresponding sources: sodium carbonate, sodium sulfate, aniline, ethanethiol, 2,3-diaminopyridine, 4-fluorobenzylamine, 2-(piperidin-1-yl)ethan-1-amine, 2,4,6-trifluoroaniline, 4-fluorothiophenol, 2,3-difluoroaniline, and 1,3-dimethylbutylamine from Merck Chemicals (Darmstadt, Germany); acetone, absolute ethanol, and neocuproine (Nc) from Sigma-Aldrich Chemicals (Steinheim, Germany); 2,3-dichloro-1,4naphthoquinone (Fluka).[13,14].Tested microorganisms included the following: bacteria Escherichia coli B-906, Staphylococcus aureus 209-P, and Mycobacterium luteum B-917 and fungi Candida tenuis VKM Y-70 and Aspergillus niger F-1119.The antimicrobial activity of compounds was evaluated by diffusion in peptone on a nutrient medium (meat-extract agar for bacteria and wort agar for fungi).The microbial loading was 10 9 cells (spores)/1 mL.The required incubation periods were 24 h at 35 ∘ C for bacteria and 48-72 h at 28-30 ∘ C for fungi.The results were recorded by measuring the zones surrounding the disk.The control disk contained vancomycin (for bacteria) or nystatin (for fungi) as a standard.Testing was performed in a flatbottomed 96-well tissue culture plate.The tested compounds were dissolved in DMSO applying the necessary concentration.The exact volume of the solution of compounds is brought into a nutrient medium.The bacteria and fungi were inoculated in a nutrient medium (meat-extract agar for bacteria and wort agar for fungi).The duration of incubation was 24-72 h at 37 ∘ C for bacteria and 30 ∘ C for fungi.The results were estimated according to the degree of the growth inhibition.

Catalase Enzyme Inhibition Activity of Quinone Derivatives.
Catalase activity was determined by the rate of H 2 O 2 decomposition, measured spectrophotometrically at 450 nm using the method described by Bekdeser et al. [15].The reaction mixtures contained 1.0 mM H 2 O 2 , 3.691 U mL −1 catalase solution, and 1.0 mM synthesized compound.This mixture (total volume 2.6 mL) was then incubated at 25 ∘ C.After 30 min incubation period, the optical CUPRAC sensor was taken out and immersed in a test tube consisted of 2.0 mL of the incubation reaction mixture + 6.2 mL of EtOH.After 30 min agitation, the colored membrane was taken out and its absorbance was recorded at 450 nm and activities were expressed in U mL −1 .
In the second step of this study, different molar amount of N-substituted naphthoquinone compounds 9 [16,21], 13 [16,23] was reacted with sodium azide in DMF.The phenazine compounds 11 and 14 [22] were synthesized and compound 11 has not yet been described in the literature (Scheme 2).
In 1 H NMR spectrum of compounds 17-23, the hydrogen signals were observed at between  = 6.1-8.0 ppm as multiplet peak, assigned to the (-CH arom ).In the 13

Antimicrobial Studies.
The profound antifungal and antibacterial activity exhibited by quinone compounds has prompted us to synthesize new heteroatom substituted 1,4naphtho-and benzoquinones.In our new endeavors, we have synthesized new 1,4-naphtho-and benzoquinones and evaluated their antibacterial and antifungal activity by diffusion [13] and serial dilution [14] methods with a view to search new perspective compounds having broad spectrum of biological activity.Antibacterial and antifungal activity of compounds 3c, 3d, 3f, 3g, 6b, 11, 17, 21, and 25 was elucidated against Escherichia coli B-906, Staphylococcus aureus 209-P, Mycobacterium luteum B-917, Candida tenuis VKM Y-70, and Aspergillus niger F-1119 by diffusion method (Tables 1 and 2) and by serial dilution method as shown in Tables 3 and 4.      Activities of quinone compounds were compared with those of the known antibacterial agent vancomycin and antifungal agent nystatin (control C).The test-culture E. coli appeared not to be sensitive to any compounds except that 3g.Compound 3g has moderate activity against E. coli at a concentration of 0.5% and the diameter of the inhibition zone was 11 mm by diffusion method.Compounds 3d and 3g have strong activity against S. aureus (16 and 20 mm at 0.5% concentration) and have moderate activity at a concentration of 0.1% (the diameter of the inhibition zones were 12 and 14 mm).The M. luteum strain was sensitive to compounds 3g, 6b, and 17 at a concentration of 0.5% and the diameter of the inhibition zone was 20 and 11 mm, respectively (Table 1).Compound 3d has good antibacterial activity against M. luteum at concentration of 0.5% and the diameter of the inhibition zone was 24 mm by diffusion method (for vancomycin was 18 mm).Compounds 3d and 3g were found to exhibit strong antibacterial activity against S. aureus and M. luteum (at concentration of 0.5%) on Antifungal activity against C. tenuis was observed for 6b, 21, and 25 at concentration of 0.5% (d = 15, 7 and 15 mm, respectively).Compound 17 showed antifungal activity against A. niger at 0.5% concentration (d = 15 mm) by the diffusion method (Table 2).Compounds 3c, 3f, and 11 have no antibacterial and antifungal activity against E. coli, S. aureus, M. luteum, C. tenuis, and A. niger at 0.5 and 0.1% evaluated concentrations by diffusion method (Tables 1 and 2).
The biological activity results of the synthesized compounds were classified as follows: the antimicrobial activities were considered as significant when the minimum inhibition concentration (MIC) was 100 g/mL or less; moderate, when the MIC was 100.0-500.0g/mL; weak, when the MIC was 500.0-1.000g/mL; and inactive when the MIC was above 1.000 g/mL.Evaluation of the antibacterial activity of the synthesized compounds showed that 3g and 17 was the most potent with MIC=15.6 g/mL for M. luteum and S. aureus, respectively (Table 3).Evaluation of antibacterial activity of synthesized compounds showed that 3d and 3g have MIC=31.2g/mL for M. luteum and S. aureus, respectively (Table 3).
Significant antifungal activity for 17 and 25 was observed against C. tenuis fungi at 15.6 and 31.2 g/mL, respectively.Evaluation of antifungal activity of compounds 3c, 3g, and 21 showed their activity in concentrations 62.5-500.0g/mL against test-culture C. tenuis (Table 4).Compounds 3g, 17, 21, and 25 showed moderate antifungal activity with MIC value in the range of 125.0-500.0g/mL against A. niger in Table 4.

Catalase Enzyme Inhibition Activity of Quinone Derivatives.
Catalase is a common heme containing enzyme found in nearly all living organisms that are exposed to O 2 , where it functions to catalyze the decomposition of H 2 O 2 to H 2 O and O 2 .Compounds 3c, 3d, 3f, 3g, 4e, 6b, 7a, 14, 17, 19, 22, and 23 were tested in vitro for their catalase activities and the results are shown in Table 5 and Figure 1.As shown in Figure 1, compound 3g caused significant elevation of catalase activity.

Conclusion
In this study we have done, the aim is to synthesize known and unknown quinone derivatives by reacting quinone compounds with some nucleophiles such as containing sulfur, nitrogen, and oxygen atoms in various conditions.In the synthesized compounds, antimicrobial activity at low concentrations against E. coli, S. aureus, and M. luteum bacteria and C. tenuis and A. niger fungi in comparison with controls was identified.Furthermore, a catalase activity of benzo-and naphthoquinone derivatives was examined for the first time in this work.Their structures of new synthesized compounds were determined by microanalysis, FT-IR, 1 H NMR, 13 C NMR, MS, and UV-Vis.
Compound 3d has good antibacterial activity against test-culture M. luteum at concentration of 0.5% and the diameter of the inhibition zone was 24 mm by diffusion method (for vancomycin was 18 mm).Compounds 3d and 3g were found to exhibit high antibacterial activity against S. aureus and M. luteum (at concentration of 0.5%) on comparison with antibacterial drug vancomycin evaluated by diffusion method.Then, inhibitory activities of the benzoand naphthoquinone derivatives against catalase enzyme were measured and especially 3g exhibited better catalase enzyme inhibition activity than the other quinone derivatives.

18 *
Vancomycin was used as a control in the tests of antibacterial activity of the synthesized compounds.

20 *
Nystatin was used in the tests of antifungal activity of the synthesized compounds.

Table 1 :
Antibacterial activity of the compounds determined by diffusion method.

Table 2 :
Antifungal activity of the compounds determined by diffusion method.

Table 3 :
Antibacterial activity of the compound determined by serial dilution method.

Table 4 :
Antifungal activity of the compounds determined by serial dilution method.

Table 5 :
Catalase enzyme activities of the compounds.