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

Authors: Semih Kurban; Nahide Gulsah Deniz; Cigdem Sayil; Mustafa Ozyurek; Kubilay Guclu; Maryna Stasevych; Viktor Zvarych; Olena Komarovska-Porokhnyavet; Volodymyr Novikov
Journal: Heteroatom Chemistry
 (2019)

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## Abstract

A series of new 1,4-naphtho- and benzoquinone derivatives possessing N-, S-, O-substituted groups which has not been reported yet has been synthesized from 2,3-dichloro-1,4-naphthoquinone1 and 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione15 involving a Michael addition. In the synthesized compounds, antimicrobial activity at low concentrations againstEscherichia coliB-906,Staphylococcus aureus209-P, andMycobacterium luteumB-917 bacteria andCandida tenuisVKM Y-70 andAspergillus nigerF-1119 fungi in comparison with controls was identified. 2-Chloro-3-((2-(piperidin-1-yl)ethyl)amino)naphthalene-1,4-dione3g and 2,5-dichloro-3-ethoxy-6-((2,4,6-trifluorophenyl)amino)cyclohexa-2,5-diene-1,4-dione17were the most potent, with a minimum inhibitory concentration value of 15.6μg/mL against test-cultureM. luteum and S. aureus,respectively. Furthermore, in this work, a catalase activity of benzo- and naphthoquinone derivatives was examined for the first time. The catalase activity of benzo- and naphthoquinone derivatives was determined, showing that compound3g had significant inhibition activity for catalase enzyme.


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## Body

## 1. 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 1,4-naphthoquinones 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 H2O2 from the cells. For this purpose, the activities of the cells of this enzyme decompose H2O2 generated as a result of the cell activities H2O and O2 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 H2O2 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,1H NMR, 13C NMR, MS, and UV-Vis spectroscopy.


## 2. 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.1H NMR and 13C NMR spectra were recorded on Bruker Avance III 500 MHz, Chemical shifts δ (ppm) were reported relative to tetramethylsilane (TMS) with the solvent resonance employed as the internal standard. 1H NMR and 13C NMR spectra in CDCl3 refer to the solvent signal center at δ = 7.26 and δ = 77.0 ppm, respectively. Moisture was excluded from the glass apparatus using CaCl2 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-1,4-naphthoquinone (Fluka).


### 2.2. Antibacterial and Antifungal Evaluations [13, 14]

Tested microorganisms included the following: bacteriaEscherichia coli B-906, Staphylococcus aureus 209-P, andMycobacterium luteum B-917 and fungiCandida tenuis VKM Y-70andAspergillus 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 109 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 flat-bottomed 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.


### 2.3. Catalase Enzyme Inhibition Activity of Quinone Derivatives

Catalase activity was determined by the rate of H2O2 decomposition, measured spectrophotometrically at 450 nm using the method described by Bekdeser et al. [15]. The reaction mixtures contained 1.0 mM H2O2, 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.


### 2.4. General Procedure for the Synthesis of N-, N,N- N,O- N,S-, and S,S- Substituted Naphtho- and Benzoquinone Compounds3a,3c,3d,3f,3g, 4e,6b,7a,17-25

Sodium carbonate was dissolved in ethanol (60 mL), and equimolar amounts of 2,3-dichloro-1,4-naphthoquinone1 and amines or thiols were added slowly. The mixture was heated between 20-45°C and it was stirred in a single reaction vessel between 2 and 11 h. Similarly, sodium carbonate was dissolved in ethanol (50 mL), and equimolar amounts of 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione15 and amines were added slowly. Without heating, the mixture was stirred in a single reaction vessel between 3-6 h. The color of the solution quickly changed (from yellow to red color), and the extent of the reaction was monitored by TLC. Chloroform (30 mL) was added to the reaction mixture. The organic layer was separated, washed with water (4 × 30 mL), and dried with Na2SO4. After the solvent was evaporated, the residue was purified by column chromatography on silica gel.


### 2.5.2-Phenylamino-3-chloro-naphthalene-1,4-dione (3a) [16, 17]

Compound3a was synthesized from aniline2a (0.4 ml, 4.404 mmol) and 2,3-dichloro-1,4-naphthoquinone1 (1 g, 4.404 mmol) according to the general method. Yield: 94.7%. Red crystal. M.p.: 215-216°C. Rf (1PET:1CHCl3): 0.44. FT-IR (KBr): υ (cm−1) = 3065, 2918 (C-H.), 1673 (C=O), 1588, 1537 (C=C), 3238 (N-H).


### 2.6.2-Chloro-3-((2,5-difluorobenzyl)amino)naphthalene-1,4-dione (3c)

Compound3cwas synthesized from (2,5-difluorophenyl) methanamine2c (0.308 ml, 2.634 mmol) and 2,3-dichloro-1,4-naphthoquinone1 (0.6 g, 2.634 mmol) according to the general method. Yield: 77.9%. Orange crystal. Rf [PET/CHCl3(5:2)]: 0.52. M.p.: 123–125°C. FT-IR (KBr) (cm−1): 3276 (N-H), 3019 (C-Harom.), 2925, 2851 (C-Haliph.), 1676 (C=O), 1576 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 5.00 (d, 2H,J 6.68 Hz, -NH-CH2), 6.21 (bs, 1H, NH), 6.88-7.00 (m, 3H, CHarom), 7.55-7.66 (tt, 2H,J 7.54, 1.46 Hz, CHnapht.), 7.96-8.08 (dd, 2H,J 7.72, 1.46 Hz, CHnapht.). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 155.6, 157.8 (-F-Carom), 115.6, 116.7, 116.9 (CHarom), 129.8 (Carom), 143.7 (-NH-Cnapht.), 115.8 (-Cl-Cnapht.), 126.9,128.8, 130.9, 132.4 (CHnapht.), 132.7, 135.0 (Cnapht.), 177.1, 180.2 (C=O). MS [+ESI] = m/z 334.1 [M+H]+, Anal. Calc. for C17H10ClF2NO2 (333.04): C 61.18, H 3.02, N 4.20. Found: C 61.41, H 3.34, N 4.14%. UV-vis [CHCl3, λmax (nm)(logε)]: 210(2.2), 274(2.4), 336(1.4), 559(1.5).


### 2.7.2-Chloro-3-((2,3-difluorophenyl)amino)naphthalene-1,4-dione (3d)

Compound3d was synthesized from 2,3-difluoroaniline2d (0.287 g, 2.212 mmol) and 2,3-dichloro-1,4-naphthoquinone1 (0.5 g, 2.212 mmol) according to the general method. Yield: 71.1%. Orange crystal. Rf [PET/CHCl3(5:2)]: 0.51. M.p.: 106–109°C. FT-IR (KBr) (cm−1): 3019 (C-Harom.), 2925 (C-Haliph.), 1650 (C=O), 1520 (C=C), 3340 (N-H). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 7.33 (bs, 1H, NH), 6.81-6.03, 7.45-7.48 (m, 3H, CHarom), 7.63-7.75 (tt, 2H,J 7.54, 1.56 Hz, CHnapht.), 8.04-8.14 (dd, 2H,J 7.71, 1.56 Hz, CHnapht.). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 129.8 (-NH-Carom), 146.2, 149.7 (-F-Carom), 114.5, 121.4, 123.0 (CHarom), 135.1 (-NH-Cnapht.), 116.6 (-Cl-Cnapht.), 127.1, 127.2, 130.9, 141.5 (CHnapht.), 133.2, 132.2 (Cnapht.), 177.5, 179.9 (C=O). MS [-ESI] = 318.2 [M-H]−, Anal. Calc. for C16H8ClF2NO2 (319.02): C 60.11, H 2.52, N 4.38. Found: C 60.12, H 2.50, N 4.40%. UV-vis [CHCl3, λmax (nm)(logε)]: 223(3.9), 274(4.0), 348(3.2), 453(3.1).


### 2.8.2-Chloro-3-((4-methylpentan-2-yl)amino)naphthalene-1,4-dione (3f)

Compound3f was synthesized from 4-methylpentan-2-amine2f (0.433 ml, 3.083 mmol) and 2,3-dichloro-1,4-naphthoquinone1 (0.7 g, 3.083 mmol) according to the general method. Yield: 89.6%. Orange crystal. Rf [PET/CHCl3 (2:1)]: 0.48. M.p.: 98–99°C. FT-IR (KBr) (cm−1): 3015 (C-Harom.), 2959, 2928 (C-Haliph.), 1643 (C=O), 1600, 1573 (C=C), 3322 (N-H). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 0.85 (d, 3H,J 7.52 Hz, -CH3), 0.87 (d, 3H,J 6.62 Hz, -CH3), 1.20 (d, 3H,J 6.37 Hz, -CH3), 4.65-4.75, 1.56-1.66 (m, 2H, -CH), 1.44-1.51, 1.26-1.34 (m, 2H, -CH2), 5.81 (bs, 1H, NH), 7.52-7.66 (tt, 2H,J 7.54, 1.56 Hz, CHnapht.), 7.94-8.06 (dd, 2H,J 7.71, 1.56 Hz, CHnapht.). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 22.4, 22.6, 22.9 (-CH3), 25.2, 48.2 (-CH-), 47.1 (-CH2-), 143.5 (-NH-Cnapht.), 126.9 (-Cl-Cnapht.), 128.8, 129.8, 130.9, 132.2 (CHnapht.), 132.5, 135.0 (Cnapht.), 176.4, 182.1 (C=O). MS [+ESI] = m/z 292.1 [M+H]+, Anal. Calc. for C16H18ClNO2 (291.78) C 65.86, H 6.22, N 4.80. Found: C 65.82, H 6.24, N 4.81%. UV-vis [CHCl3, λmax (nm)(logε)]: 238(3.2), 277(3.4), 343(3.4), 469(2.6).


### 2.9.2-Chloro-3-((2-(piperidin-1-yl)ethyl)amino)naphthalene-1,4-dione (3g) [18]

Compound3g was synthesized from 2-(piperidin-1-yl) ethan-1-amine2 g (0.317 ml, 2.202 mmol) and 2,3-dichloro-1,4-naphthoquinone1 (0.5 g, 2.202 mmol) according to the general method. Yield: 80.9%. Red crystal. Rf [PET/CHCl3 (3:1)]: 0.48. M.p.: 108–110°C. FT- IR (KBr) (cm−1): 3016 (C-Harom.), 2938, 2853 (C-Haliph.), 1677 (C=O), 1603, 1573 (C=C), 3355 (N-H). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 1.36-1.58 (m, 6H, -CH2circle-), 2.34-2.42 (m, 4H, -N-CH2circle), 2.53 (t, 2H,J 5.96 Hz, -CH2-N-), 3.80 (t, 2H,J 5.88 Hz, -NH-CH2), 6.98 (bs, 1H, NH), 7.48-7.63 (tt, 2H,J 7.55, 1.46 Hz, CHnapht.), 7.86-8.02 (dd, 2H,J 7.25, 1.46 Hz, CHnapht). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 24.4, 26.1 (CH2piper.), 41.2 (-NH-CH2), 56.8 (-N-CH2), 53.8 (-N-CH2piper.), 144.8 (-NH-Cnapht.), 110.9 (-Cl-Cnapht.), 126.5, 129.8, 132.2 (CHnapht.), 132.7, 134.6 (Cnapht.), 176.2, 180.4 (C=O). MS [+ESI] = m/z 319.2 [M+H]+, Anal. Calc. for C17H19ClN2O2 (318.11): C 64.05, H 6.01, N 8.79. Found: C 63.97, H 6.26, N 8.69%. UV-vis [CHCl3, λmax (nm)(logε)]: 211(2.8), 277(3.4), 339(2.3), 473(2.5).


### 2.10.2-Ethoxy-3-((2-methyl-4-oxo-4H-chromen-7-yl)amino)naphthalene-1,4-dione (4e)

Compound4e was synthesized from 7-amino-2-methyl-4H-chromen-4-one2e (0.385 g, 2.202 mmol) and 2,3-dichloro-1,4-naphthoquinone1 (0.5 g, 2.202 mmol) according to the general method. Yield: 54.9%. Red crystal. Rf [PET/CHCl3(5:2)]: 0.58. M.p.: 140–142°C. FT-IR (KBr) (cm−1): 2971 (C-Harom.), 2926, 2850 (C-Haliph.), 1682 (C=O), 1599, 1520 (C=C), 3306 (N-H). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 0.83 (t, 3H,J= 7.52 Hz, CH3ethoxy), 1.17 (bs, 3H, CH3), 4.01-4.04 (q, 2H,J 7.06 Hz, O-CH2ethoxy), 5.65 (bs, 1H, CH2), 6.08 (bs, 1H, O-CH), 6.28-6.29 (d, 1H,J 7.06 Hz, CHphenyl), 7.44-7.46 (d, 1H,J 7.06 Hz, CHphenyl), 7.54-7.66 (tt, 2H,J 7.53, 1.46 Hz, CHnapht.), 5.96 (bs, 1H, NH), 7.94-8.09 (dd, 2H,J 7.7, 1.46 Hz, CHnapht.). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 11.0 (-CH3ethoxy), 14.1 (-CH3subst.), 68.2 (-O-CH2ethoxy), 109.9, 112.2, 119.4, 127.0 (CHsubst.), 120.5 (Csubst.), 167.3 (CH3-Csubst.), 162.0 (C-Osubst.), 160.8 (NH-Csubst.), 173.8 (C=Osubst.), 134.9 (O-Cnapht.), 115.5 (-NH-Cnapht.), 128.0, 128.4, 130.8, 130.9 (CHnapht.), 132.5 132.6 (Cnapht.), 176.9, 180.5 (C=O). MS [-ESI] = m/z 372.9 [M-2H]−, Anal. Calc. for C22H17NO5 (375.11): C 70.39, H 4.56, N 3.73. Found: C 70.24, H 4.45, N 3.80%. UV-vis [CHCl3, λmax (nm)(logε)]: 243(3.0), 276(3.1), 344(2.3), 468(2.1).


### 2.11.2,3-Bis((4-fluorophenyl)thio)naphthalene-1,4-dione (6b) [19]

Compound6bwas synthesized from 4-fluorobenzenethiol5b (0.375 ml, 3.523 mmol) and 2,3-dichloro-1,4-naphthoquinone1 (0.4 g, 1.761 mmol) according to the general method. Yield: 79.1%. Orange crystal. Rf [PET/CHCl3 (2:1)]: 0.55. M.p.: 175–177°C. FT-IR (KBr) (cm−1): 3008 (C-Harom.), 2923, 2853 (C-H), 1665 (C=O), 1586 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 7.41-7.46 ppm (m, 4H, CHarom), 7.01-7.06 (m, 4H, CHarom), 7.68-7.72 (m, 2H, CHnapht), 7.96-8.00 (m, 2H, CHnapht). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 128.4 (-S-Carom), 161.6-163.6 (-F-Carom), 116.3-116.5, 133.7-133.9 (CHarom), 132.6 (-S-Cnapht), 127.2, 133.6 (CHnapht), 148.9 (Cnapht), 178.7, 178.7 (C=O). MS [+ESI] = m/z 411 [M+H]+, Anal. Calc. for C22H12F2O2S2 (410.02): C 64.38, H 2.95, S 15.62. Found: C 64.38, H 2.94, S 15.62%. UV-vis [CHCl3, λmax (nm)(logε)]: 211(2.6), 253(2.5), 342(1.9), 458(1.7).


### 2.12.2-(Ethylthio)-3-(phenylamino)naphthalene-1,4-dione (7a) [20]

Compound7awas synthesized from ethanethiol5a (0.110 ml, 1.762 mmol) and 2-chloro-3-(phenylamino) naphthalene-1,4-dione3a(0.5 g, 1.762 mmol) according to the general method. Yield: 91.5%. Red crystal. Rf [PET/CHCl3 (5:2)]: 0.57 M.p.: 92–94°C. FT-IR (KBr) (cm−1): 3008 (C-Harom.), 2923, 2853 (C-H), 1665 (C=O), 1586 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 0.96 (t, 3H,J 7.38 Hz, CH3), 2.56 (q, 2H,J 7.39 Hz, S-CH2), 6.97 (d, 2H,J 7.74 Hz, CHarom), 7.08 (t, 1H,J 7.44 Hz, CHarom), 7.27 (t, 2H,J 7.56 Hz, CHarom), 7.58-7.68 (tt, 2H,J 7.53, 1.46 Hz, CHnapht.), 7.76 (bs, 1H, NH), 8.00-8.09 (dd, 2H,J 7.7, 1.46 Hz, CHnapht). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 14.5 (-CH3), 28.0 (SCH2-), 122.5, 124.7, 126.6 (CHarom.), 124.5 (S-Cnapht.), 126.8 (-NH-Cnapht.), 129.4, 130.8, 132.8 (CHnapht.), 134.6, 138.5 (Cnapht.), 145.0 (NH-C), 180.5, 181.1 (C=O). MS [-ESI] = m/z 308.01 [M-H]-, Anal. Calc. for C18H15NO2S (309.08): C 69.88, H 4.69, N 4.53. Found: C 70.04, H 4.88, N 4.57%. UV-vis [CHCl3, λmax (nm)(logε)]: 210(2.4), 283(2.6), 382(1.9), 511(1.7).


### 2.13.2-Chloro-3-(o-tolylamino)naphthalene-1,4-dione (9) [16, 21]

Compound9was synthesized from o-toluidine8 (0.235 g, 2.202 mmol) and 2,3-dichloro-1,4-naphthoquinone1 (0.5 g, 2.202 mmol) according to the general method. Yield: 72.7%. Red crystal. Rf [PET/CHCl3 (1:1)]: 0.41. M.p.: 162–163°C. FT-IR (KBr) (cm−1): 3060, 2946 (C-H.), 1672 (C=O), 1595, 1573 (C=C), 3244 (N-H).


### 2.14. General Procedure for the Synthesis of N,N-Substituted Naphthoquinone Compounds (1-Methylbenzo[b]phenazine-6,11-dione11and 2-Methylbenzo[b]phenazine-6,11-dione14 [22])

Mono substituted naphthoquinone derivatives9 [21] and13 (1 mol) were dissolved in DMF (100 mL) and sodium azide (NaN3) (2 mol) dissolved in 10 ml of water was slowly added. The reaction was heated to reflux with stirring. The color of the solution quickly changed (from yellow to red color), and the extent of the reaction was monitored by TLC. Chloroform (40 mL) was added to the reaction mixture. The organic layer was separated, washed with water (4 × 50 mL), and dried with Na2SO4. After the solvent was evaporated, the residue was purified by column chromatography on silica gel.


### 2.15.1-Methylbenzo[b]phenazine-6,11-dione (11)

Compound11 was synthesized from sodium azide10(0.131 g, 2.015 mmol) and 2-chloro-3-(o-tolylamino) naphthalene-1,4-dione9 [21] (0.3 g, 1.007 mmol) according to the general method. Yield: 75.7%. Dark blue crystal. Rf [PET/CHCl3(1:1)]: 0.53. M.p.: 139–141°C. FT-IR (KBr)(cm−1): 3019 (C-Harom.), 2926, 2860 (C-H), 1624 (C=O), 1524 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 2.30 (bs, 3H, -CH3arom), 7.13 (d, 1H,J 7.81 Hz, -CHarom-), 7.08 (t, 1H,J 7.83 Hz, -CHarom-), 6.37 (d, 1H,J 7.88 Hz, -CHarom-), 7.53-7.61 (m, 2H, CHnapht.), 7.95-7.99 (m, 2H, CHnapht.). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 18.1 (CH3arom) 114.1, 120.7, 128.0 (-CHarom), 132.6 (CH3-Carom), 137.7, 139.0 (-N=Carom), 131.7, 133.7 (-N-Cnapht.), 121.8, 126.0, 126.2 (CHnapht.), 130.6, 130.7 (Cnapht.), 180.7, 180.8 C=O). MS [+ESI] = 276.1 [M+2H]+. Anal. Calc. for C17H10N2O2 (274.07): C 74.44, H 3.68, N 10.21. Found: C 74.63, H 3.45, N 10.18%. UV-vis [CHCl3, λmax (nm)(logε)]: 241(2.5), 298(2.6), 429(1.2), 544(1.6).


### 2.16.2-Chloro-3-(m-tolylamino)naphthalene-1,4-dione (13) [16, 23]

Compound13was synthesized from m-toluidine12(0.188 g, 1.761 mmol) and 2,3-dichloro-1,4-naphthoquinone1 (0.4 g, 1.761 mmol) according to the general method. Yield: 75.3%. Red crystal. Rf [PET/CHCl3 (1:1)]: 0.41. M.p.: 177–179°C. FT-IR (KBr) (cm−1): 3045, 2915 (C-H.), 1675 (C=O), 1593, 1560 (C=C), 3237 (N-H).


### 2.17.2-Methylbenzo[b]phenazine-6,11-dione (14) [22]

Compound14 was synthesized from sodium azide10 (0.087 g, 1.344 mmol) and 2-chloro-3-(m-tolylamino) naphthalene-1,4-dione13 (0.2 g, 0.672 mmol) according to the general method. Yield: 73.0%. Dark navy blue crystal. Rf [PET/CHCl3)(2:1)]: 0.50. M.p.: 193–195°C.IR (KBr) (cm−1): 3019 (C-Harom.), 2926, 2850 (C-H), 1616 (C=O), 1577, 1522 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 2.35 (bs, 3H, -CH3arom), 7.18-7.22 (t, 1H,J 7.81 Hz, -CHarom-), 6.78-6.82 (d, 1H,J 7.49 Hz, -CHarom-), 6.57-6.60 (d, 1H,J 7.52 Hz, -CHarom-), 7.63-7.69 (m, 2H, CHnapht.), 8.04-8.08 (m, 2H, CHnapht.). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 21.5 (CH3arom) 119.0, 120.3, 122.4 (-CHarom.), 132.8 (CH3-Carom), 139.1, 139.7 (-N=Carom), 131.8, 133.7 (-N-Cnapht.), 115.5, 126.0, 126.2 (CHnapht.), 128.9, 129.5 (Cnapht.), 180.7, 180.8 (C=O). MS [+ESI] = m/z 276.0 [M+2H]+, Anal. Calc. for C17H10N2O2 (274.07): C 74.44, H 3.68, N 10.21. Found: C 74.49, H 3.34, N 10.19%. UV-vis [CHCl3, λmax (nm)(logε)]: 212(2.2), 241(2.1), 298(2.2), 539(1.2).


### 2.18.2,5-Dichloro-3-ethoxy-6-((2,4,6-trifluorophenyl)amino)cyclohexa-2,5-diene-1,4-dione (17)

Compound17 was synthesized from 2,4,6-trifluoroaniline16 (0.597 g, 2.440 mmol) and 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione15 (1.0 g, 4.067 mmol) according to the general method. Yield: 28.3%. Red crystal. Rf [PET/CHCl3(3:1)]: 0.51. M.p.: 131–132°C. FT-IR (KBr) (cm−1): 3341 (N-H), 3018, 2956 (C-Harom), 2923, 2851 (C-H), 1712, 1640 (C=O), 1588-1522 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 0.80 (t, 3H,J 7.08 Hz, -CH3ethoxy), 4.33 (q, 2H,J 7.05 Hz, -O-CH2ethoxy), 6.52 (bs, 1H, -NH-), 6.63-6.67 (m, 6H, -CHarom). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 100.5, 100.5 (-CHarom), 150.3-152.1, 152.1-154.0 (C-F-), 124.3 (NH-Carom), 14.2 (-CH3ethoxy), 68.1 (-CH2ethoxy), 146.9 (-NH-Cbenzo), 114.2, 118.9 (C-Cl-), 158.0 (C-O-), 164.5, 166.1 (C=O). MS [-ESI] = m/z 364.0 [M]−, Anal. Calc. for C14H8Cl2F3NO3 (364.98): C 45.93, H 2.20, N 3.83. Found: C 45.98, H 2.19, N 3.94%. UV-vis [CHCl3, λmax (nm)(logε)]: 225(2.2), 301(2.7), 377(1.4), 462(1.2).


### 2.19.2,5-Dichloro-3,6-bis((2,4,6-trifluorophenyl)amino)cyclohexa-2,5-diene-1,4-dione (18)

Compound18 was synthesized from 2,4,6-trifluoroaniline16 (0.597 g, 2.440 mmol) and 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione15 (1.0 g, 4.067 mmol) according to the general method. Yield: 39.2%. Light yellow crystal. Rf [PET/CHCl3(3:1)]: 0.55. M.p.: 154–156°C. FT-IR (KBr) (cm−1): 3380 (N-H), 3019, 2961 (C-Harom), 2927, 2858 (C-H), 1716 (C=O), 1519 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 4.26 (bs, 2H, -NH-), 7.93-7.98 (m, 4H, -CHarom). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 101.2 (-CHarom), 153.9-155.8, 155.8-157.3 (C-F-), 124.3 (NH- Carom), 141.0 (-NH-Cbenzo), 111.3 (C-Cl-), 166.0 (C=O). MS [-ESI] = m/z 464.9 [M-H]−, Anal. Calc. for C18H6Cl2F6N2O2 (465.97 g/mol): C 46.28, H 1.29, N 6.00. Found: C 46.40, H 1.25, N 6.09%. UV-vis [CHCl3, λmax (nm)(logε)]: 247(3.1), 292(3.0), 377(2.7), 462(2.4).


### 2.20.2-Chloro-3,6-diethoxy-5-((4-fluorobenzyl)amino)cyclohexa-2,5-diene-1,4-dione (19)

Compound19 was synthesized from (4-fluorophenyl) methanamine2h (0.275 ml, 2.440 mmol) and 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione15 (0.6 g, 2.440 mmol) according to the general method. Yield: 21.1%. Dark red crystal. Rf [(PET/CHCl3(5:2)]: 0.58. M.p.: 60–62°C. FT-IR (KBr) (cm−1): 3345 (N-H), 3001 (C-Harom), 2982, 2929 (C-H), 1682 (C=O), 1570 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 1.37 (t, 3H,J 7.06 Hz, -CH3ethoxy), 1.36 (t, 3H,J 7.06 Hz, -CH3ethoxy), 4.12-4.52 (m, 4H, -O-CH2ethoxy), 1.71 (d, 2H,J 4.97 Hz, -NH-CH2-), 5.66 (bs, 1H, -NH-), 6.13-6.35 (m, 4H, -CHarom-).13C NMR (125.66 MHz, CDCl3) δ (ppm) = 171.5-173.3 (-C-Farom), 140.5, 138.6, 110.8, 111.4 (-CHarom), 144.3 (-Carom), 29.5 (-N-CH2-) 70.3, 71.5, 15.9, 20.7 (-CH2ethoxy), (-CH3ethoxy), 125.8 (-Cbenzo-NH-) 102.0 (C-Cl-), 143.9, 154.4 (C-O-), 171.7, 172.8 (C=O). MS [-ESI] = m/z 352.3 [M-H]−, Anal. Calc. for C17H17ClFNO4 (353.08): C 57.72, H 4.84, N 3.96. Found: C 57.89, H 4.58, N 3.98%. UV-vis [CHCl3, λmax (nm)(logε)]: 217(1.7), 241(1.8), 298(2.1), 430(0.3).


### 2.21.2-Chloro-5-ethoxy-3,6-bis((4-fluorobenzyl)amino)cyclohexa-2,5-diene-1,4-dione (20)

Compound20 was synthesized from (4-fluorophenyl) methanamine2h (0.275 ml, 2.440 mmol) and 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione15 (0.6 g, 2.440 mmol) according to the general method. Yield: 37.3%. Orange crystal. Rf [PET/CHCl3(5:2)]: 0.55. M.p.: 82–84°C. FT-IR (KBr) (cm−1): 3300 (N-H), 3009, 2961 (C-Harom), 2930, 2874 (C-H), 1682 (C=O), 1624, 1579 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 1.35 (t, 3H,J 7.05 Hz, -CH3ethoxy), 4.40 (q, 2H,J 7.05 Hz, -O-CH2ethoxy), 4.50 (d, 4H,,J 8.09 Hz, -NH-CH2-), 5.81, 6.78 (bs, 2H, -NH-), 7.08-7.65 (m, 8H, -CHarom-).13C NMR (125.66 MHz, CDCl3) δ (ppm) = 174.2-176.3 (-C-Farom), 123.4, 125.9, 128.9, 131.0 (-CHarom), 132.4 (-Carom), 29.0, 30.3 (-N-CH2-), 71.3, 15.9 (-CH2ethoxy) ve (-CH3ethoxy), 154.1, 153.1 (-Cbenzo-NH-), 105.9 (C-Cl-), 129.6 (C-O-), 171.5, 175.3 (C=O). MS [+ESI] = m/z 433.3 [M+H]+, Anal. Calc. for C22H19ClF2N2O3 (432.11): C 61.05, H 4.42, N 6.47. Found: C 61.14, H 4.76, N 6.51%. UV-vis [CHCl3, λmax (nm)(logε)]: 205(1.9), 240(1.7), 303(2.1), 430(0.5).


### 2.22.2-Chloro-5,6-diethoxy-3-((4-fluorobenzyl)amino)cyclohexa-2,5-diene-1,4-dione (21)

Compound21 was synthesized from (4-fluorophenyl) methanamine2h (0.275 ml, 2.440 mmol) and 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione15 (0.6 g, 2.440 mmol) according to the general method. Yield: 18.2%. Dark red crystal. Rf [PET/CHCl3(5:2)]: 0.51. M.p.: 104–106°C. FT-IR (KBr) (cm−1): 3242 (N-H), 3003-2957 (C-Harom), 2926, 2855 (CH), 1690 (C=O), 1579-1519 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 1.35 (t, 3H,J 7.05 Hz, -CH3ethoxy), 1.37 (t, 3H,J 7.05 Hz, -CH3ethoxy), 4.19-4.53 (q, 4H,J 7.05 Hz, -O-CH2ethoxy), 1.68 (d, 2H,J 4.97 Hz, -NH-CH2-), 5.49 (bs, 1H, -NH-), 6.15-6.30 (m, 4H, -CHarom-). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 163.6-161.7 (-C-Farom), 129.4, 129.6, 116.0, 116.2 (-CHarom), 132.9 (-Carom), 29.8 (-N-CH2-), 68.2, 71.6, 11.0, 14.1 (-CH2ethoxy) ve (-CH3ethoxy), 143.5 (-Cbenzo-NH-), 128.7 (C-Cl-), 135.9, 142.0 (C-O-), 169.9, 174.2 (C=O). MS [-ESI] = m/z 352.33 [M-H]−, Anal. Calc. for C17H17ClFNO4 (353.08): C 57.72, H 4.84, N 3.96. Found: C 57.92, H 4.68, N 3.98%. UV-vis [CHCl3, λmax (nm)(logε)]: 206(2.0), 241(2.1), 298(2.4), 430(0.5).


### 2.23.2,5-Diethoxy-3,6-bis((4-fluorobenzyl)amino)cyclohexa-2,5-diene-1,4-dione (22)

Compound22 was synthesized from (4-fluorophenyl)methanamine2h (0.275 ml, 2.440 mmol) and 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione15 (0.6 g, 2.440 mmol) according to the general method. Yield: 16,4%. Pale pink crystal. Rf [PET/CHCl3 (5:2)]: 0.48. M.p.: 226–228°C. FT-IR (KBr) (cm−1): 3244 (N-H), 3019 (C-Harom), 2929, 2850 (CH), 1663 (C=O), 1586 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 0.84 (t, 6H,J 7.46 Hz, -CH3ethoxy), 4.10-4.18 (m, 4H, -O-CH2ethoxy), 4.88 (d, 2H,J 6.18 Hz, -NH-CH2-), 6.00 (bs, 2H, -NH-), 6.98-7.64 (m, 8H, -CHarom-).13C NMR (125.66 MHz, CDCl3) δ (ppm) = 161.2, 163.0 (-C-Farom), 130.8, 131.0, 112.9,113.1 (-CHarom), 140.2 (-Carom), 32.0 (-N-CH2-), 71.4, 15.9 (-CH2ethoxy) ve (-CH3ethoxy), 122.4 (-Cbenzo-NH-), 132.7 (C-O-), 175.1 (C=O). MS [+ESI] = m/z 445.0 [M+2H]+, Anal. Calc. For C24H24F2N2O4 (443.17): C 65.15, H 5.47, N 6.33. Found: C 65.08, H 5.80, N 6.27%. UV-vis [CHCl3, λmax (nm)(logε)]: 219(3.7), 245(3.8), 462(2.4), 557(2.6).


### 2.24.2,5-Dichloro-3-((2,5-difluorobenzyl)amino)-6-ethoxycyclohexa-2,5-diene-1,4-dione (23)

Compound23 was synthesized from (2,5-difluorophenyl)methanamine2c (0.290 g, 2.033 mmol) and 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione15 (0.5 g, 2.033 mmol) according to the general method. Yield: 52.4%. Dark purple crystal. Rf [PET/CHCl3(3:1)]: 0.47. M.p.: 73–74°C. FT-IR (KBr) (cm−1): 3346 (N-H), 3020 (C-Harom), 2927, 2856 (CH), 1667 (C=O), 1522 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 1.36 (t, 3H,J 7.05 Hz, -CH3ethoxy), 4.62 (q, 2H,J 7.03 Hz, -O-CH2ethoxy), 4.95 (d, 2H,J 6.72 Hz, -NH-CH2), 6.27 (bs, 1H, -NH-), 6.85-7.15 (m, 3H, -CHcircle).13C NMR (125.66 MHz, CDCl3) δ (ppm) = 116.4, 117.5, 120.3 (-CHarom), 126.5 (-Carom), 155.5-157.4, 157.8-159.7 (C-F-), 42.4 (-CH2-NH-), 16.1 (-CH3ethoxy), 68.0 (-CH2ethoxy), 156.4 (-NH-Cbenzo), 115.8, 119.4 (C-Cl-), 162.2 (C-O-), 175.9, 173.8 (C=O). MS [-ESI] = m/z 360.0 [M-H]−, Anal. Calc. for C15H11Cl2F2NO3 (361.01): C 49.75, H 3.06, N 3.87. Found: C 49.96, H 3.00, N 3.91. UV-vis [CHCl3, λmax (nm)(logε)]: 219(2.2), 241(2.4), 311(2.3), 492(1.2).


### 2.25.2,3,5-Trichloro-6-((2-(piperidin-1-yl)ethyl)amino)cyclohexa-2,5-diene-1,4-dione (24)

Compound24 was synthesized from 2-(piperidin-1-yl) ethan-1-amine2 g (0.208 g, 1.626 mmol) and 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione15 (0.4 g, 1.626 mmol) according to the general method. Yield: 33.3%. Dark red crystal. Rf [PET/CHCl3 (3:1)]: 0.51. M.p.: 60–62°C. FT-IR (KBr) (cm−1): 3332 (N-H), 3019, 2932 (CH), 1639 (C=O), 1522 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 1.22-1.32, 2.44-2.76 (m, 10H, -Ncircle-CH2), 3.15 (t, 2H,J 5.67 Hz, -CH2circle-N-), 4.17-4.20 (m, 2H, -NH-CH2), 8.03 (bs, 1H, -NH). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 20.0, 23.8, 23.9, 54.1, 54.2 (-CH2circle), 44.0, 56.1 (-N-CH2-CH2-N-), 148.4 (-C-NH-), 120.0, 140.1, 142.7 (C-Cl-), 162.3, 174.9 (C=O). MS [+ESI] = m/z 339.4 [M+2H]+, Anal. Calc. for C13H15Cl3N2O2 (337.63): C 46.25, H 4.48, N 8.30. Found: C 46.22, H 4.72, N 8.25%. UV-vis [CHCl3, λmax (nm)(logε)]: 217(2.8), 293(1.9), 354(2.0), 587(1.9).


### 2.26.2-Chloro-5-ethoxy-3,6-bis((2-(piperidin-1-yl)ethyl)amino)cyclohexa-2,5-diene-1,4-dione (25)

Compound25 was synthesized from 2-(piperidin-1-yl)ethan-1-amine2 g (0.208 g, 1.626 mmol) and 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione15 (0.4 g, 1.626 mmol) according to the general method. Yield: 36.3%. Dark red crystal. Rf [PET/CHCl3 (3:1)]: 0.54. M.p.: 241–243°C. FT-IR (KBr) (cm−1): 3295 (N-H), 3019, 2923 (CH), 1678 (C=O), 1570 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 0.81 (t, 3H,J 7.03 Hz,-CH3ethoxy), 1.15-1.55, 2.10-2.24 (m, 20H, -CH2circle-N-), 2.26-2.28, 2.38-2.41 (m, 4H, -Ncircle-CH2), 4.13-4.23 (m, 4H, -NH-CH2), 4.50 (q, 2H,J= 5.35 Hz, O-CH2ethoxy-), 5.67, 5.90 (bs, 2H, -NH). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 23.0, 23.7, 55.7 (-CH2circle), 44.4, 58.6 (-N-CH2-CH2-N-), 14.8 (-CH3ethoxy), 66.1 (-CH2ethoxy) 136.3, 140.5 (-C-NH-), 100.2 (C-Cl-), 127.8 (C-O-), 169.2, 175.0 (C=O). MS [+ESI] = 441.4 [M+2H]+, Anal. Calc. for C22H35ClN4O3 (439.0): C 60.19, H 8.04, N 12.76. Found: C 60.14, H 8.21, N 12.68%. UV-vis [CHCl3, λmax (nm)(logε)]: 223(2.8), 292(3.0), 333(1.7), 483(1.5).


## 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.1H NMR and 13C NMR spectra were recorded on Bruker Avance III 500 MHz, Chemical shifts δ (ppm) were reported relative to tetramethylsilane (TMS) with the solvent resonance employed as the internal standard. 1H NMR and 13C NMR spectra in CDCl3 refer to the solvent signal center at δ = 7.26 and δ = 77.0 ppm, respectively. Moisture was excluded from the glass apparatus using CaCl2 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-1,4-naphthoquinone (Fluka).


## 2.2. Antibacterial and Antifungal Evaluations [13, 14]

Tested microorganisms included the following: bacteriaEscherichia coli B-906, Staphylococcus aureus 209-P, andMycobacterium luteum B-917 and fungiCandida tenuis VKM Y-70andAspergillus 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 109 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 flat-bottomed 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.


## 2.3. Catalase Enzyme Inhibition Activity of Quinone Derivatives

Catalase activity was determined by the rate of H2O2 decomposition, measured spectrophotometrically at 450 nm using the method described by Bekdeser et al. [15]. The reaction mixtures contained 1.0 mM H2O2, 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.


## 2.4. General Procedure for the Synthesis of N-, N,N- N,O- N,S-, and S,S- Substituted Naphtho- and Benzoquinone Compounds3a,3c,3d,3f,3g, 4e,6b,7a,17-25

Sodium carbonate was dissolved in ethanol (60 mL), and equimolar amounts of 2,3-dichloro-1,4-naphthoquinone1 and amines or thiols were added slowly. The mixture was heated between 20-45°C and it was stirred in a single reaction vessel between 2 and 11 h. Similarly, sodium carbonate was dissolved in ethanol (50 mL), and equimolar amounts of 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione15 and amines were added slowly. Without heating, the mixture was stirred in a single reaction vessel between 3-6 h. The color of the solution quickly changed (from yellow to red color), and the extent of the reaction was monitored by TLC. Chloroform (30 mL) was added to the reaction mixture. The organic layer was separated, washed with water (4 × 30 mL), and dried with Na2SO4. After the solvent was evaporated, the residue was purified by column chromatography on silica gel.


## 2.5.2-Phenylamino-3-chloro-naphthalene-1,4-dione (3a) [16, 17]

Compound3a was synthesized from aniline2a (0.4 ml, 4.404 mmol) and 2,3-dichloro-1,4-naphthoquinone1 (1 g, 4.404 mmol) according to the general method. Yield: 94.7%. Red crystal. M.p.: 215-216°C. Rf (1PET:1CHCl3): 0.44. FT-IR (KBr): υ (cm−1) = 3065, 2918 (C-H.), 1673 (C=O), 1588, 1537 (C=C), 3238 (N-H).


## 2.6.2-Chloro-3-((2,5-difluorobenzyl)amino)naphthalene-1,4-dione (3c)

Compound3cwas synthesized from (2,5-difluorophenyl) methanamine2c (0.308 ml, 2.634 mmol) and 2,3-dichloro-1,4-naphthoquinone1 (0.6 g, 2.634 mmol) according to the general method. Yield: 77.9%. Orange crystal. Rf [PET/CHCl3(5:2)]: 0.52. M.p.: 123–125°C. FT-IR (KBr) (cm−1): 3276 (N-H), 3019 (C-Harom.), 2925, 2851 (C-Haliph.), 1676 (C=O), 1576 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 5.00 (d, 2H,J 6.68 Hz, -NH-CH2), 6.21 (bs, 1H, NH), 6.88-7.00 (m, 3H, CHarom), 7.55-7.66 (tt, 2H,J 7.54, 1.46 Hz, CHnapht.), 7.96-8.08 (dd, 2H,J 7.72, 1.46 Hz, CHnapht.). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 155.6, 157.8 (-F-Carom), 115.6, 116.7, 116.9 (CHarom), 129.8 (Carom), 143.7 (-NH-Cnapht.), 115.8 (-Cl-Cnapht.), 126.9,128.8, 130.9, 132.4 (CHnapht.), 132.7, 135.0 (Cnapht.), 177.1, 180.2 (C=O). MS [+ESI] = m/z 334.1 [M+H]+, Anal. Calc. for C17H10ClF2NO2 (333.04): C 61.18, H 3.02, N 4.20. Found: C 61.41, H 3.34, N 4.14%. UV-vis [CHCl3, λmax (nm)(logε)]: 210(2.2), 274(2.4), 336(1.4), 559(1.5).


## 2.7.2-Chloro-3-((2,3-difluorophenyl)amino)naphthalene-1,4-dione (3d)

Compound3d was synthesized from 2,3-difluoroaniline2d (0.287 g, 2.212 mmol) and 2,3-dichloro-1,4-naphthoquinone1 (0.5 g, 2.212 mmol) according to the general method. Yield: 71.1%. Orange crystal. Rf [PET/CHCl3(5:2)]: 0.51. M.p.: 106–109°C. FT-IR (KBr) (cm−1): 3019 (C-Harom.), 2925 (C-Haliph.), 1650 (C=O), 1520 (C=C), 3340 (N-H). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 7.33 (bs, 1H, NH), 6.81-6.03, 7.45-7.48 (m, 3H, CHarom), 7.63-7.75 (tt, 2H,J 7.54, 1.56 Hz, CHnapht.), 8.04-8.14 (dd, 2H,J 7.71, 1.56 Hz, CHnapht.). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 129.8 (-NH-Carom), 146.2, 149.7 (-F-Carom), 114.5, 121.4, 123.0 (CHarom), 135.1 (-NH-Cnapht.), 116.6 (-Cl-Cnapht.), 127.1, 127.2, 130.9, 141.5 (CHnapht.), 133.2, 132.2 (Cnapht.), 177.5, 179.9 (C=O). MS [-ESI] = 318.2 [M-H]−, Anal. Calc. for C16H8ClF2NO2 (319.02): C 60.11, H 2.52, N 4.38. Found: C 60.12, H 2.50, N 4.40%. UV-vis [CHCl3, λmax (nm)(logε)]: 223(3.9), 274(4.0), 348(3.2), 453(3.1).


## 2.8.2-Chloro-3-((4-methylpentan-2-yl)amino)naphthalene-1,4-dione (3f)

Compound3f was synthesized from 4-methylpentan-2-amine2f (0.433 ml, 3.083 mmol) and 2,3-dichloro-1,4-naphthoquinone1 (0.7 g, 3.083 mmol) according to the general method. Yield: 89.6%. Orange crystal. Rf [PET/CHCl3 (2:1)]: 0.48. M.p.: 98–99°C. FT-IR (KBr) (cm−1): 3015 (C-Harom.), 2959, 2928 (C-Haliph.), 1643 (C=O), 1600, 1573 (C=C), 3322 (N-H). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 0.85 (d, 3H,J 7.52 Hz, -CH3), 0.87 (d, 3H,J 6.62 Hz, -CH3), 1.20 (d, 3H,J 6.37 Hz, -CH3), 4.65-4.75, 1.56-1.66 (m, 2H, -CH), 1.44-1.51, 1.26-1.34 (m, 2H, -CH2), 5.81 (bs, 1H, NH), 7.52-7.66 (tt, 2H,J 7.54, 1.56 Hz, CHnapht.), 7.94-8.06 (dd, 2H,J 7.71, 1.56 Hz, CHnapht.). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 22.4, 22.6, 22.9 (-CH3), 25.2, 48.2 (-CH-), 47.1 (-CH2-), 143.5 (-NH-Cnapht.), 126.9 (-Cl-Cnapht.), 128.8, 129.8, 130.9, 132.2 (CHnapht.), 132.5, 135.0 (Cnapht.), 176.4, 182.1 (C=O). MS [+ESI] = m/z 292.1 [M+H]+, Anal. Calc. for C16H18ClNO2 (291.78) C 65.86, H 6.22, N 4.80. Found: C 65.82, H 6.24, N 4.81%. UV-vis [CHCl3, λmax (nm)(logε)]: 238(3.2), 277(3.4), 343(3.4), 469(2.6).


## 2.9.2-Chloro-3-((2-(piperidin-1-yl)ethyl)amino)naphthalene-1,4-dione (3g) [18]

Compound3g was synthesized from 2-(piperidin-1-yl) ethan-1-amine2 g (0.317 ml, 2.202 mmol) and 2,3-dichloro-1,4-naphthoquinone1 (0.5 g, 2.202 mmol) according to the general method. Yield: 80.9%. Red crystal. Rf [PET/CHCl3 (3:1)]: 0.48. M.p.: 108–110°C. FT- IR (KBr) (cm−1): 3016 (C-Harom.), 2938, 2853 (C-Haliph.), 1677 (C=O), 1603, 1573 (C=C), 3355 (N-H). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 1.36-1.58 (m, 6H, -CH2circle-), 2.34-2.42 (m, 4H, -N-CH2circle), 2.53 (t, 2H,J 5.96 Hz, -CH2-N-), 3.80 (t, 2H,J 5.88 Hz, -NH-CH2), 6.98 (bs, 1H, NH), 7.48-7.63 (tt, 2H,J 7.55, 1.46 Hz, CHnapht.), 7.86-8.02 (dd, 2H,J 7.25, 1.46 Hz, CHnapht). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 24.4, 26.1 (CH2piper.), 41.2 (-NH-CH2), 56.8 (-N-CH2), 53.8 (-N-CH2piper.), 144.8 (-NH-Cnapht.), 110.9 (-Cl-Cnapht.), 126.5, 129.8, 132.2 (CHnapht.), 132.7, 134.6 (Cnapht.), 176.2, 180.4 (C=O). MS [+ESI] = m/z 319.2 [M+H]+, Anal. Calc. for C17H19ClN2O2 (318.11): C 64.05, H 6.01, N 8.79. Found: C 63.97, H 6.26, N 8.69%. UV-vis [CHCl3, λmax (nm)(logε)]: 211(2.8), 277(3.4), 339(2.3), 473(2.5).


## 2.10.2-Ethoxy-3-((2-methyl-4-oxo-4H-chromen-7-yl)amino)naphthalene-1,4-dione (4e)

Compound4e was synthesized from 7-amino-2-methyl-4H-chromen-4-one2e (0.385 g, 2.202 mmol) and 2,3-dichloro-1,4-naphthoquinone1 (0.5 g, 2.202 mmol) according to the general method. Yield: 54.9%. Red crystal. Rf [PET/CHCl3(5:2)]: 0.58. M.p.: 140–142°C. FT-IR (KBr) (cm−1): 2971 (C-Harom.), 2926, 2850 (C-Haliph.), 1682 (C=O), 1599, 1520 (C=C), 3306 (N-H). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 0.83 (t, 3H,J= 7.52 Hz, CH3ethoxy), 1.17 (bs, 3H, CH3), 4.01-4.04 (q, 2H,J 7.06 Hz, O-CH2ethoxy), 5.65 (bs, 1H, CH2), 6.08 (bs, 1H, O-CH), 6.28-6.29 (d, 1H,J 7.06 Hz, CHphenyl), 7.44-7.46 (d, 1H,J 7.06 Hz, CHphenyl), 7.54-7.66 (tt, 2H,J 7.53, 1.46 Hz, CHnapht.), 5.96 (bs, 1H, NH), 7.94-8.09 (dd, 2H,J 7.7, 1.46 Hz, CHnapht.). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 11.0 (-CH3ethoxy), 14.1 (-CH3subst.), 68.2 (-O-CH2ethoxy), 109.9, 112.2, 119.4, 127.0 (CHsubst.), 120.5 (Csubst.), 167.3 (CH3-Csubst.), 162.0 (C-Osubst.), 160.8 (NH-Csubst.), 173.8 (C=Osubst.), 134.9 (O-Cnapht.), 115.5 (-NH-Cnapht.), 128.0, 128.4, 130.8, 130.9 (CHnapht.), 132.5 132.6 (Cnapht.), 176.9, 180.5 (C=O). MS [-ESI] = m/z 372.9 [M-2H]−, Anal. Calc. for C22H17NO5 (375.11): C 70.39, H 4.56, N 3.73. Found: C 70.24, H 4.45, N 3.80%. UV-vis [CHCl3, λmax (nm)(logε)]: 243(3.0), 276(3.1), 344(2.3), 468(2.1).


## 2.11.2,3-Bis((4-fluorophenyl)thio)naphthalene-1,4-dione (6b) [19]

Compound6bwas synthesized from 4-fluorobenzenethiol5b (0.375 ml, 3.523 mmol) and 2,3-dichloro-1,4-naphthoquinone1 (0.4 g, 1.761 mmol) according to the general method. Yield: 79.1%. Orange crystal. Rf [PET/CHCl3 (2:1)]: 0.55. M.p.: 175–177°C. FT-IR (KBr) (cm−1): 3008 (C-Harom.), 2923, 2853 (C-H), 1665 (C=O), 1586 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 7.41-7.46 ppm (m, 4H, CHarom), 7.01-7.06 (m, 4H, CHarom), 7.68-7.72 (m, 2H, CHnapht), 7.96-8.00 (m, 2H, CHnapht). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 128.4 (-S-Carom), 161.6-163.6 (-F-Carom), 116.3-116.5, 133.7-133.9 (CHarom), 132.6 (-S-Cnapht), 127.2, 133.6 (CHnapht), 148.9 (Cnapht), 178.7, 178.7 (C=O). MS [+ESI] = m/z 411 [M+H]+, Anal. Calc. for C22H12F2O2S2 (410.02): C 64.38, H 2.95, S 15.62. Found: C 64.38, H 2.94, S 15.62%. UV-vis [CHCl3, λmax (nm)(logε)]: 211(2.6), 253(2.5), 342(1.9), 458(1.7).


## 2.12.2-(Ethylthio)-3-(phenylamino)naphthalene-1,4-dione (7a) [20]

Compound7awas synthesized from ethanethiol5a (0.110 ml, 1.762 mmol) and 2-chloro-3-(phenylamino) naphthalene-1,4-dione3a(0.5 g, 1.762 mmol) according to the general method. Yield: 91.5%. Red crystal. Rf [PET/CHCl3 (5:2)]: 0.57 M.p.: 92–94°C. FT-IR (KBr) (cm−1): 3008 (C-Harom.), 2923, 2853 (C-H), 1665 (C=O), 1586 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 0.96 (t, 3H,J 7.38 Hz, CH3), 2.56 (q, 2H,J 7.39 Hz, S-CH2), 6.97 (d, 2H,J 7.74 Hz, CHarom), 7.08 (t, 1H,J 7.44 Hz, CHarom), 7.27 (t, 2H,J 7.56 Hz, CHarom), 7.58-7.68 (tt, 2H,J 7.53, 1.46 Hz, CHnapht.), 7.76 (bs, 1H, NH), 8.00-8.09 (dd, 2H,J 7.7, 1.46 Hz, CHnapht). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 14.5 (-CH3), 28.0 (SCH2-), 122.5, 124.7, 126.6 (CHarom.), 124.5 (S-Cnapht.), 126.8 (-NH-Cnapht.), 129.4, 130.8, 132.8 (CHnapht.), 134.6, 138.5 (Cnapht.), 145.0 (NH-C), 180.5, 181.1 (C=O). MS [-ESI] = m/z 308.01 [M-H]-, Anal. Calc. for C18H15NO2S (309.08): C 69.88, H 4.69, N 4.53. Found: C 70.04, H 4.88, N 4.57%. UV-vis [CHCl3, λmax (nm)(logε)]: 210(2.4), 283(2.6), 382(1.9), 511(1.7).


## 2.13.2-Chloro-3-(o-tolylamino)naphthalene-1,4-dione (9) [16, 21]

Compound9was synthesized from o-toluidine8 (0.235 g, 2.202 mmol) and 2,3-dichloro-1,4-naphthoquinone1 (0.5 g, 2.202 mmol) according to the general method. Yield: 72.7%. Red crystal. Rf [PET/CHCl3 (1:1)]: 0.41. M.p.: 162–163°C. FT-IR (KBr) (cm−1): 3060, 2946 (C-H.), 1672 (C=O), 1595, 1573 (C=C), 3244 (N-H).


## 2.14. General Procedure for the Synthesis of N,N-Substituted Naphthoquinone Compounds (1-Methylbenzo[b]phenazine-6,11-dione11and 2-Methylbenzo[b]phenazine-6,11-dione14 [22])

Mono substituted naphthoquinone derivatives9 [21] and13 (1 mol) were dissolved in DMF (100 mL) and sodium azide (NaN3) (2 mol) dissolved in 10 ml of water was slowly added. The reaction was heated to reflux with stirring. The color of the solution quickly changed (from yellow to red color), and the extent of the reaction was monitored by TLC. Chloroform (40 mL) was added to the reaction mixture. The organic layer was separated, washed with water (4 × 50 mL), and dried with Na2SO4. After the solvent was evaporated, the residue was purified by column chromatography on silica gel.


## 2.15.1-Methylbenzo[b]phenazine-6,11-dione (11)

Compound11 was synthesized from sodium azide10(0.131 g, 2.015 mmol) and 2-chloro-3-(o-tolylamino) naphthalene-1,4-dione9 [21] (0.3 g, 1.007 mmol) according to the general method. Yield: 75.7%. Dark blue crystal. Rf [PET/CHCl3(1:1)]: 0.53. M.p.: 139–141°C. FT-IR (KBr)(cm−1): 3019 (C-Harom.), 2926, 2860 (C-H), 1624 (C=O), 1524 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 2.30 (bs, 3H, -CH3arom), 7.13 (d, 1H,J 7.81 Hz, -CHarom-), 7.08 (t, 1H,J 7.83 Hz, -CHarom-), 6.37 (d, 1H,J 7.88 Hz, -CHarom-), 7.53-7.61 (m, 2H, CHnapht.), 7.95-7.99 (m, 2H, CHnapht.). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 18.1 (CH3arom) 114.1, 120.7, 128.0 (-CHarom), 132.6 (CH3-Carom), 137.7, 139.0 (-N=Carom), 131.7, 133.7 (-N-Cnapht.), 121.8, 126.0, 126.2 (CHnapht.), 130.6, 130.7 (Cnapht.), 180.7, 180.8 C=O). MS [+ESI] = 276.1 [M+2H]+. Anal. Calc. for C17H10N2O2 (274.07): C 74.44, H 3.68, N 10.21. Found: C 74.63, H 3.45, N 10.18%. UV-vis [CHCl3, λmax (nm)(logε)]: 241(2.5), 298(2.6), 429(1.2), 544(1.6).


## 2.16.2-Chloro-3-(m-tolylamino)naphthalene-1,4-dione (13) [16, 23]

Compound13was synthesized from m-toluidine12(0.188 g, 1.761 mmol) and 2,3-dichloro-1,4-naphthoquinone1 (0.4 g, 1.761 mmol) according to the general method. Yield: 75.3%. Red crystal. Rf [PET/CHCl3 (1:1)]: 0.41. M.p.: 177–179°C. FT-IR (KBr) (cm−1): 3045, 2915 (C-H.), 1675 (C=O), 1593, 1560 (C=C), 3237 (N-H).


## 2.17.2-Methylbenzo[b]phenazine-6,11-dione (14) [22]

Compound14 was synthesized from sodium azide10 (0.087 g, 1.344 mmol) and 2-chloro-3-(m-tolylamino) naphthalene-1,4-dione13 (0.2 g, 0.672 mmol) according to the general method. Yield: 73.0%. Dark navy blue crystal. Rf [PET/CHCl3)(2:1)]: 0.50. M.p.: 193–195°C.IR (KBr) (cm−1): 3019 (C-Harom.), 2926, 2850 (C-H), 1616 (C=O), 1577, 1522 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 2.35 (bs, 3H, -CH3arom), 7.18-7.22 (t, 1H,J 7.81 Hz, -CHarom-), 6.78-6.82 (d, 1H,J 7.49 Hz, -CHarom-), 6.57-6.60 (d, 1H,J 7.52 Hz, -CHarom-), 7.63-7.69 (m, 2H, CHnapht.), 8.04-8.08 (m, 2H, CHnapht.). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 21.5 (CH3arom) 119.0, 120.3, 122.4 (-CHarom.), 132.8 (CH3-Carom), 139.1, 139.7 (-N=Carom), 131.8, 133.7 (-N-Cnapht.), 115.5, 126.0, 126.2 (CHnapht.), 128.9, 129.5 (Cnapht.), 180.7, 180.8 (C=O). MS [+ESI] = m/z 276.0 [M+2H]+, Anal. Calc. for C17H10N2O2 (274.07): C 74.44, H 3.68, N 10.21. Found: C 74.49, H 3.34, N 10.19%. UV-vis [CHCl3, λmax (nm)(logε)]: 212(2.2), 241(2.1), 298(2.2), 539(1.2).


## 2.18.2,5-Dichloro-3-ethoxy-6-((2,4,6-trifluorophenyl)amino)cyclohexa-2,5-diene-1,4-dione (17)

Compound17 was synthesized from 2,4,6-trifluoroaniline16 (0.597 g, 2.440 mmol) and 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione15 (1.0 g, 4.067 mmol) according to the general method. Yield: 28.3%. Red crystal. Rf [PET/CHCl3(3:1)]: 0.51. M.p.: 131–132°C. FT-IR (KBr) (cm−1): 3341 (N-H), 3018, 2956 (C-Harom), 2923, 2851 (C-H), 1712, 1640 (C=O), 1588-1522 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 0.80 (t, 3H,J 7.08 Hz, -CH3ethoxy), 4.33 (q, 2H,J 7.05 Hz, -O-CH2ethoxy), 6.52 (bs, 1H, -NH-), 6.63-6.67 (m, 6H, -CHarom). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 100.5, 100.5 (-CHarom), 150.3-152.1, 152.1-154.0 (C-F-), 124.3 (NH-Carom), 14.2 (-CH3ethoxy), 68.1 (-CH2ethoxy), 146.9 (-NH-Cbenzo), 114.2, 118.9 (C-Cl-), 158.0 (C-O-), 164.5, 166.1 (C=O). MS [-ESI] = m/z 364.0 [M]−, Anal. Calc. for C14H8Cl2F3NO3 (364.98): C 45.93, H 2.20, N 3.83. Found: C 45.98, H 2.19, N 3.94%. UV-vis [CHCl3, λmax (nm)(logε)]: 225(2.2), 301(2.7), 377(1.4), 462(1.2).


## 2.19.2,5-Dichloro-3,6-bis((2,4,6-trifluorophenyl)amino)cyclohexa-2,5-diene-1,4-dione (18)

Compound18 was synthesized from 2,4,6-trifluoroaniline16 (0.597 g, 2.440 mmol) and 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione15 (1.0 g, 4.067 mmol) according to the general method. Yield: 39.2%. Light yellow crystal. Rf [PET/CHCl3(3:1)]: 0.55. M.p.: 154–156°C. FT-IR (KBr) (cm−1): 3380 (N-H), 3019, 2961 (C-Harom), 2927, 2858 (C-H), 1716 (C=O), 1519 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 4.26 (bs, 2H, -NH-), 7.93-7.98 (m, 4H, -CHarom). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 101.2 (-CHarom), 153.9-155.8, 155.8-157.3 (C-F-), 124.3 (NH- Carom), 141.0 (-NH-Cbenzo), 111.3 (C-Cl-), 166.0 (C=O). MS [-ESI] = m/z 464.9 [M-H]−, Anal. Calc. for C18H6Cl2F6N2O2 (465.97 g/mol): C 46.28, H 1.29, N 6.00. Found: C 46.40, H 1.25, N 6.09%. UV-vis [CHCl3, λmax (nm)(logε)]: 247(3.1), 292(3.0), 377(2.7), 462(2.4).


## 2.20.2-Chloro-3,6-diethoxy-5-((4-fluorobenzyl)amino)cyclohexa-2,5-diene-1,4-dione (19)

Compound19 was synthesized from (4-fluorophenyl) methanamine2h (0.275 ml, 2.440 mmol) and 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione15 (0.6 g, 2.440 mmol) according to the general method. Yield: 21.1%. Dark red crystal. Rf [(PET/CHCl3(5:2)]: 0.58. M.p.: 60–62°C. FT-IR (KBr) (cm−1): 3345 (N-H), 3001 (C-Harom), 2982, 2929 (C-H), 1682 (C=O), 1570 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 1.37 (t, 3H,J 7.06 Hz, -CH3ethoxy), 1.36 (t, 3H,J 7.06 Hz, -CH3ethoxy), 4.12-4.52 (m, 4H, -O-CH2ethoxy), 1.71 (d, 2H,J 4.97 Hz, -NH-CH2-), 5.66 (bs, 1H, -NH-), 6.13-6.35 (m, 4H, -CHarom-).13C NMR (125.66 MHz, CDCl3) δ (ppm) = 171.5-173.3 (-C-Farom), 140.5, 138.6, 110.8, 111.4 (-CHarom), 144.3 (-Carom), 29.5 (-N-CH2-) 70.3, 71.5, 15.9, 20.7 (-CH2ethoxy), (-CH3ethoxy), 125.8 (-Cbenzo-NH-) 102.0 (C-Cl-), 143.9, 154.4 (C-O-), 171.7, 172.8 (C=O). MS [-ESI] = m/z 352.3 [M-H]−, Anal. Calc. for C17H17ClFNO4 (353.08): C 57.72, H 4.84, N 3.96. Found: C 57.89, H 4.58, N 3.98%. UV-vis [CHCl3, λmax (nm)(logε)]: 217(1.7), 241(1.8), 298(2.1), 430(0.3).


## 2.21.2-Chloro-5-ethoxy-3,6-bis((4-fluorobenzyl)amino)cyclohexa-2,5-diene-1,4-dione (20)

Compound20 was synthesized from (4-fluorophenyl) methanamine2h (0.275 ml, 2.440 mmol) and 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione15 (0.6 g, 2.440 mmol) according to the general method. Yield: 37.3%. Orange crystal. Rf [PET/CHCl3(5:2)]: 0.55. M.p.: 82–84°C. FT-IR (KBr) (cm−1): 3300 (N-H), 3009, 2961 (C-Harom), 2930, 2874 (C-H), 1682 (C=O), 1624, 1579 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 1.35 (t, 3H,J 7.05 Hz, -CH3ethoxy), 4.40 (q, 2H,J 7.05 Hz, -O-CH2ethoxy), 4.50 (d, 4H,,J 8.09 Hz, -NH-CH2-), 5.81, 6.78 (bs, 2H, -NH-), 7.08-7.65 (m, 8H, -CHarom-).13C NMR (125.66 MHz, CDCl3) δ (ppm) = 174.2-176.3 (-C-Farom), 123.4, 125.9, 128.9, 131.0 (-CHarom), 132.4 (-Carom), 29.0, 30.3 (-N-CH2-), 71.3, 15.9 (-CH2ethoxy) ve (-CH3ethoxy), 154.1, 153.1 (-Cbenzo-NH-), 105.9 (C-Cl-), 129.6 (C-O-), 171.5, 175.3 (C=O). MS [+ESI] = m/z 433.3 [M+H]+, Anal. Calc. for C22H19ClF2N2O3 (432.11): C 61.05, H 4.42, N 6.47. Found: C 61.14, H 4.76, N 6.51%. UV-vis [CHCl3, λmax (nm)(logε)]: 205(1.9), 240(1.7), 303(2.1), 430(0.5).


## 2.22.2-Chloro-5,6-diethoxy-3-((4-fluorobenzyl)amino)cyclohexa-2,5-diene-1,4-dione (21)

Compound21 was synthesized from (4-fluorophenyl) methanamine2h (0.275 ml, 2.440 mmol) and 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione15 (0.6 g, 2.440 mmol) according to the general method. Yield: 18.2%. Dark red crystal. Rf [PET/CHCl3(5:2)]: 0.51. M.p.: 104–106°C. FT-IR (KBr) (cm−1): 3242 (N-H), 3003-2957 (C-Harom), 2926, 2855 (CH), 1690 (C=O), 1579-1519 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 1.35 (t, 3H,J 7.05 Hz, -CH3ethoxy), 1.37 (t, 3H,J 7.05 Hz, -CH3ethoxy), 4.19-4.53 (q, 4H,J 7.05 Hz, -O-CH2ethoxy), 1.68 (d, 2H,J 4.97 Hz, -NH-CH2-), 5.49 (bs, 1H, -NH-), 6.15-6.30 (m, 4H, -CHarom-). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 163.6-161.7 (-C-Farom), 129.4, 129.6, 116.0, 116.2 (-CHarom), 132.9 (-Carom), 29.8 (-N-CH2-), 68.2, 71.6, 11.0, 14.1 (-CH2ethoxy) ve (-CH3ethoxy), 143.5 (-Cbenzo-NH-), 128.7 (C-Cl-), 135.9, 142.0 (C-O-), 169.9, 174.2 (C=O). MS [-ESI] = m/z 352.33 [M-H]−, Anal. Calc. for C17H17ClFNO4 (353.08): C 57.72, H 4.84, N 3.96. Found: C 57.92, H 4.68, N 3.98%. UV-vis [CHCl3, λmax (nm)(logε)]: 206(2.0), 241(2.1), 298(2.4), 430(0.5).


## 2.23.2,5-Diethoxy-3,6-bis((4-fluorobenzyl)amino)cyclohexa-2,5-diene-1,4-dione (22)

Compound22 was synthesized from (4-fluorophenyl)methanamine2h (0.275 ml, 2.440 mmol) and 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione15 (0.6 g, 2.440 mmol) according to the general method. Yield: 16,4%. Pale pink crystal. Rf [PET/CHCl3 (5:2)]: 0.48. M.p.: 226–228°C. FT-IR (KBr) (cm−1): 3244 (N-H), 3019 (C-Harom), 2929, 2850 (CH), 1663 (C=O), 1586 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 0.84 (t, 6H,J 7.46 Hz, -CH3ethoxy), 4.10-4.18 (m, 4H, -O-CH2ethoxy), 4.88 (d, 2H,J 6.18 Hz, -NH-CH2-), 6.00 (bs, 2H, -NH-), 6.98-7.64 (m, 8H, -CHarom-).13C NMR (125.66 MHz, CDCl3) δ (ppm) = 161.2, 163.0 (-C-Farom), 130.8, 131.0, 112.9,113.1 (-CHarom), 140.2 (-Carom), 32.0 (-N-CH2-), 71.4, 15.9 (-CH2ethoxy) ve (-CH3ethoxy), 122.4 (-Cbenzo-NH-), 132.7 (C-O-), 175.1 (C=O). MS [+ESI] = m/z 445.0 [M+2H]+, Anal. Calc. For C24H24F2N2O4 (443.17): C 65.15, H 5.47, N 6.33. Found: C 65.08, H 5.80, N 6.27%. UV-vis [CHCl3, λmax (nm)(logε)]: 219(3.7), 245(3.8), 462(2.4), 557(2.6).


## 2.24.2,5-Dichloro-3-((2,5-difluorobenzyl)amino)-6-ethoxycyclohexa-2,5-diene-1,4-dione (23)

Compound23 was synthesized from (2,5-difluorophenyl)methanamine2c (0.290 g, 2.033 mmol) and 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione15 (0.5 g, 2.033 mmol) according to the general method. Yield: 52.4%. Dark purple crystal. Rf [PET/CHCl3(3:1)]: 0.47. M.p.: 73–74°C. FT-IR (KBr) (cm−1): 3346 (N-H), 3020 (C-Harom), 2927, 2856 (CH), 1667 (C=O), 1522 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 1.36 (t, 3H,J 7.05 Hz, -CH3ethoxy), 4.62 (q, 2H,J 7.03 Hz, -O-CH2ethoxy), 4.95 (d, 2H,J 6.72 Hz, -NH-CH2), 6.27 (bs, 1H, -NH-), 6.85-7.15 (m, 3H, -CHcircle).13C NMR (125.66 MHz, CDCl3) δ (ppm) = 116.4, 117.5, 120.3 (-CHarom), 126.5 (-Carom), 155.5-157.4, 157.8-159.7 (C-F-), 42.4 (-CH2-NH-), 16.1 (-CH3ethoxy), 68.0 (-CH2ethoxy), 156.4 (-NH-Cbenzo), 115.8, 119.4 (C-Cl-), 162.2 (C-O-), 175.9, 173.8 (C=O). MS [-ESI] = m/z 360.0 [M-H]−, Anal. Calc. for C15H11Cl2F2NO3 (361.01): C 49.75, H 3.06, N 3.87. Found: C 49.96, H 3.00, N 3.91. UV-vis [CHCl3, λmax (nm)(logε)]: 219(2.2), 241(2.4), 311(2.3), 492(1.2).


## 2.25.2,3,5-Trichloro-6-((2-(piperidin-1-yl)ethyl)amino)cyclohexa-2,5-diene-1,4-dione (24)

Compound24 was synthesized from 2-(piperidin-1-yl) ethan-1-amine2 g (0.208 g, 1.626 mmol) and 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione15 (0.4 g, 1.626 mmol) according to the general method. Yield: 33.3%. Dark red crystal. Rf [PET/CHCl3 (3:1)]: 0.51. M.p.: 60–62°C. FT-IR (KBr) (cm−1): 3332 (N-H), 3019, 2932 (CH), 1639 (C=O), 1522 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 1.22-1.32, 2.44-2.76 (m, 10H, -Ncircle-CH2), 3.15 (t, 2H,J 5.67 Hz, -CH2circle-N-), 4.17-4.20 (m, 2H, -NH-CH2), 8.03 (bs, 1H, -NH). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 20.0, 23.8, 23.9, 54.1, 54.2 (-CH2circle), 44.0, 56.1 (-N-CH2-CH2-N-), 148.4 (-C-NH-), 120.0, 140.1, 142.7 (C-Cl-), 162.3, 174.9 (C=O). MS [+ESI] = m/z 339.4 [M+2H]+, Anal. Calc. for C13H15Cl3N2O2 (337.63): C 46.25, H 4.48, N 8.30. Found: C 46.22, H 4.72, N 8.25%. UV-vis [CHCl3, λmax (nm)(logε)]: 217(2.8), 293(1.9), 354(2.0), 587(1.9).


## 2.26.2-Chloro-5-ethoxy-3,6-bis((2-(piperidin-1-yl)ethyl)amino)cyclohexa-2,5-diene-1,4-dione (25)

Compound25 was synthesized from 2-(piperidin-1-yl)ethan-1-amine2 g (0.208 g, 1.626 mmol) and 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione15 (0.4 g, 1.626 mmol) according to the general method. Yield: 36.3%. Dark red crystal. Rf [PET/CHCl3 (3:1)]: 0.54. M.p.: 241–243°C. FT-IR (KBr) (cm−1): 3295 (N-H), 3019, 2923 (CH), 1678 (C=O), 1570 (C=C). 1H NMR (499.74 MHz, CDCl3) δ (ppm) = 0.81 (t, 3H,J 7.03 Hz,-CH3ethoxy), 1.15-1.55, 2.10-2.24 (m, 20H, -CH2circle-N-), 2.26-2.28, 2.38-2.41 (m, 4H, -Ncircle-CH2), 4.13-4.23 (m, 4H, -NH-CH2), 4.50 (q, 2H,J= 5.35 Hz, O-CH2ethoxy-), 5.67, 5.90 (bs, 2H, -NH). 13C NMR (125.66 MHz, CDCl3) δ (ppm) = 23.0, 23.7, 55.7 (-CH2circle), 44.4, 58.6 (-N-CH2-CH2-N-), 14.8 (-CH3ethoxy), 66.1 (-CH2ethoxy) 136.3, 140.5 (-C-NH-), 100.2 (C-Cl-), 127.8 (C-O-), 169.2, 175.0 (C=O). MS [+ESI] = 441.4 [M+2H]+, Anal. Calc. for C22H35ClN4O3 (439.0): C 60.19, H 8.04, N 12.76. Found: C 60.14, H 8.21, N 12.68%. UV-vis [CHCl3, λmax (nm)(logε)]: 223(2.8), 292(3.0), 333(1.7), 483(1.5).


## 3. Results and Discussion

### 3.1. Chemistry

In this study that we have done, reactions of thiol and amine compounds with 2,3-dichloro-1,4-naphthoquinone and 2,3,5,6-tetrachloro-1,4-benzoquinone as a starting compounds were investigated. Firstly, the multicomponent reactions of 2,3-dichloro-1,4-naphthoquinone1 with various thiol and amine nucleophiles were investigated. Similarly, 2,3,5,6-tetrachloro-1,4-benzoquinone15 with various amine nucleophiles was investigated. As shown in Scheme 1, the reaction of1 with different amines2a,2c,2d,2e,2f,2 gin ethanol in the presence of Na2CO3 gave known and unknown compounds3a [16, 17],3c,3d,3f,3g [18],4e. Compound6b[19] obtained the reaction of1 with5b.The reaction of3awith5a gave compound7a [20]. When1 reacted with an equimolar amount of various amines and thiols in ethanol in the presence of sodium carbonate solution at room temperature but under different conditions, the corresponding products (3c,3d,3f,3g,4e,6b,7a) were obtained in different yields. All synthesized compounds were confirmed by spectroscopic methods comprising 1H NMR and 13C NMR, FT-IR, elemental analysis, and MS.Scheme 1
The synthesis of N-, S-, O-substituted naphthoquinone derivatives (3a, 3c-d,3f-g,4e,6b,7a).In the second step of this study, different molar amount of N-substituted naphthoquinone compounds9 [16, 21],13 [16, 23] was reacted with sodium azide in DMF. The phenazine compounds11and14 [22] were synthesized and compound11 has not yet been described in the literature (Scheme 2).Scheme 2
The synthesis of phenazine compounds11 and14 via the condensation reaction of naphthoquinones9 and13.In the last step of this study, 2,3,5,6-tetrachloro-1,4-benzoquinone15 compound was reacted with compounds containing N-nucleophiles(2b,2c,2 g,16)that novel benzoquinones (17-25) not yet described in the literature were synthesized in Scheme 3. The synthesis, spectroscopic data (1H NMR, 13C NMR, MS, UV, FT-IR), elemental analysis, and melting points of compounds were reported in studies. The 1H NMR signal of the hydrogen atoms of the naphthoquinone unit of compounds3c,3d,3f,3g,4e,7a was observed at (CH) δ= 7.9-8.1 and 7.5-7.7 ppm like as doublet of doublets and triplet of triplets, respectively. Similarly,6b,11,14 were observed at (CH) δ= 7.9-8.1 and 7.5-7.7 ppm like as multiplets, respectively. Substituted aromatic ring hydrogens showed peaks around 6.8-7.4 ppm. Aliphatic groups in compounds3f,7a were shifted to a higher field and displayed peaks at 0.8-1.2 ppm. The 13C NMR spectra of compound3d gave two carbonyl signals at 177.5 and 179.9 ppm (C=O). Unlike other studies, the carbon atoms attached to the fluorine atoms in the3c compound give cleavage peaks 155.6, 157.8 ppm (F-Carom) in aromatic unit. Compound3d gave one carbonyl signal at 116.6 ppm (-Cl-Cnapht.) similarly giving a single peak at 126.9 ppm in the compound3f. The FT-IR spectra of compounds3c,3d,3f,3g,4e,7a showed bands around at 3300 cm−1 for the (–NH) stretching. Also, (C-Harom) bond was observed ν = 3000 cm−1. With the aid of the positive ion mode of electron spray ionization (ESI) mass spectrum of the compounds3c,6b, and3f, the respective molecular ion peaks were observed atm/z(%) 334 (100) [M+H]+, 411 (100) [M+H]+, 292 (100) [M+H]+, respectively.Scheme 3
The synthesis of N,O-substituted benzoquinone derivatives.2-Chloro-3-(o-tolylamino)naphthalene-1,4-dione9 and sodium azide10 required for the synthesis of11, similarly, 2-chloro-3-(m-tolylamino)naphthalene-1,4-dione13 and sodium azide (NaN3)10 required for the synthesis of compound14 have been synthesized according to Scheme 2. The nucleophilic displacement reaction of compound9 with sodium azide (NaN3) in DMF-H2O (10:1) afforded 1-methylbenzo[b]phenazine-6,11-dione11 and this analog 2-methylbenzo[b]phenazine-6,11-dione14 as the only isolated products as exhibited in Scheme 2. The proposed mechanism of condensation reaction of naphthoquinones agrees well with the related literatures [24, 25]. Both synthesized compounds were characterized by using the 1H NMR, 13C NMR, FT-IR spectral data, and elemental analysis. The first compound11was obtained by an interesting ring closure and is a phenazine derivative. The 13C NMR spectra of compound11 gave two carbonyl signals at 180.7 and 180.8 ppm (C=O). The FT-IR spectra of compounds11 and14 showed bands at 3019 cm−1 for the (C-Harom) stretching and (–NH) bonds were not observed in the FT-IR. 1H NMR peak of the hydrogen atoms of the naphthoquinone group gave on (CHarom) δ= 7.53-7.61 ppm and 7.95-7.99 ppm as multiplets for compound11. Molecular ion peaks were observed atm/z(%) 276.1 (100) [M+2H]+. The UV-Vis spectroscopy values for compound14 were also observed at 212(2.2), 241(2.1), 298(2.2), 539(1.2).It is known that the reactions of 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4 dione15 with amines proceed by Michael addition reaction. A series of 2-arylamino-1,4-benzoquinone derivatives17-25 were synthesized via the nucleophilic substitution reaction of 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione15 by appropriate aryl amines2c,2 g,2h,16 in ethanol as shown in Scheme 3. The reactions were found to be exceptionally selective and lead to mainly 2- and/or 2,5-bis(amino substituted)-3,6-dichloro-1,4-benzoquinones of the corresponding amine. From these reactions we could not obtain 2,6-bis(amino substituted)-1,4-benzoquinone derivatives. The steric factors arising from the substituent effect predominates in these reactions. The result of selective formation of 2,5-isomer may be assumed to be due to attack of two amines to 1,4-benzoquinone. For such attack to give exclusive product of one isomer (2,5-) would require approach of two amines from the furthest possible distance. Thus, exclusively 2- and/or 2,5-isomer were formed due to electrostatic reasons for compounds17-25. The results agree well with the corresponding mechanism in the similar compounds [6, 26].In1H NMR spectrum of compounds17-23, the hydrogen signals were observed at between δ = 6.1-8.0 ppm as multiplet peak, assigned to the (-CHarom). In the 13C NMR, characteristic signals of two carbonyl carbons of benzoquinones were visible at around 175.9 and 173.8 ppm. For compound23, substitute ethoxy group carbons (-CH2etoxy) and (-CH3etoxy) at 68.0 and 16.1 ppm, respectively. Like the naphthoquinone derivatives, carbon atoms attached to the fluorine atoms in the23 compound give cleavage peaks 155.5 and 157.8 ppm (F-Carom). The FT-IR spectra of compounds17-25showed the absorption bands of the N–H group at around 3240-3380 cm−1. The characteristic stretching band of carbonyl groups (C = O) was observed at between ν = 1650-1700 cm−1. In the MS of quinone derivatives, the molecular ion peaks of compounds17,22,and23were observed at 364 (100) [M]−, 445 (100) [M+2H]+, 360 (100) [M-H]−.


### 3.2. Antimicrobial Studies

The profound antifungal and antibacterial activity exhibited by quinone compounds has prompted us to synthesize new heteroatom substituted 1,4-naphtho- 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 compounds3c,3d,3f,3g,6b,11,17,21, and25 was elucidated againstEscherichia coli B-906,Staphylococcus aureus 209-P,Mycobacterium luteum B-917, Candida tenuis VKM Y-70, andAspergillus 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).Table 1
Antibacterial activity of the compounds determined by diffusion method.
Compound Concentration% Diameter of inhibition of growth of microorganisms, mm E. coli S. aureus M. luteum 3c 0.5 0 0 0 0.1 0 0 0 3d 0.5 0 16 24 0.1 0 12 12 3f 0.5 0 0 0 0.1 0 0 0 3g 0.5 11 20 20 0.1 0 14 16 6b 0.5 0 0 11 0.1 0 0 8 11 0.5 0 0 0 0.1 0 0 0 17 0.5 0 10 11 0.1 0 6 9 21 0.5 0 0 0 0.1 0 0 0 25 0.5 0 0 0 0.1 0 0 0 Control ∗ 0.5 14 15 18 ∗Vancomycin was used as a control in the tests of antibacterial activity of the synthesized compounds.Table 2
Antifungal activity of the compounds determined by diffusion method.
Compound Concentration% Diameter of inhibition of growth of microorganisms, mm C. tenuis A. niger 3c 0.5 0 0 0.1 0 0 3d 0.5 0 0 0.1 0 0 3f 0.5 0 0 0.1 0 0 3g 0.5 0 0 0.1 0 0 6b 0.5 15 0 0.1 13 0 11 0.5 0 0 0.1 0 0 17 0.5 0 15 0.1 0 10 21 0.5 7 12 0.1 0 6 25 0.5 15 10 0.1 0 0 Control ∗ 0.5 19 20 ∗Nystatin was used in the tests of antifungal activity of the synthesized compounds.Table 3
Antibacterial activity of the compound determined by serial dilution method.
Compound Microorganism E. coli S. aureus M. luteum MIC (μg/mL) 3c + + + 3d + 62.5 31.2 3f + + + 3g 250.0 31.2 15.6 6b + + + 11 + + + 17 + 15.6 62.5 21 + + + 25 + 500.0 250.0 Control 31.2 ± 0.8 3.9 ± 0.2 7.8 ± 0.2 +: growth of microorganisms.Table 4
Antifungal activity of the compounds determined by serial dilution method.
Compound Microorganism C. tenuis A. niger MIC (μg/mL) 3c 125.0 + 3d + + 3f + + 3g 500.0 500.0 6b + + 11 + + 17 15.6 250.0 21 62.5 125.0 25 31.2 250.0 Control 7.8 ± 0.2 15.6 ± 0.8 +: growth of microorganisms.The test-cultureE. coli appeared not to be sensitive to any compounds except that3g. Compound3g has moderate activity againstE. coli at a concentration of 0.5% and the diameter of the inhibition zone was 11 mm by diffusion method. Compounds3dand3g have strong activity againstS. 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). TheM. luteum strain was sensitive to compounds3g,6b, and17at a concentration of 0.5% and the diameter of the inhibition zone was 20 and 11 mm, respectively (Table 1). Compound3d has good antibacterial activity againstM. luteum at concentration of 0.5% and the diameter of the inhibition zone was 24 mm by diffusion method (for vancomycin was 18 mm). Compounds3dand3gwere found to exhibit strong antibacterial activity againstS. aureus andM. luteum (at concentration of 0.5%) on comparison with antibacterial drug vancomycin evaluated by diffusion method.Antifungal activity againstC. tenuis was observed for6b,21, and25at concentration of 0.5% (d = 15, 7 and 15 mm, respectively). Compound17 showed antifungal activity againstA. niger at 0.5% concentration (d = 15 mm) by the diffusion method (Table 2). Compounds3c,3f, and11have no antibacterial and antifungal activity againstE. coli, S. aureus, M. luteum, C. tenuis, andA. 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 100μg/mL or less; moderate, when the MIC was 100.0–500.0 μg/mL; weak, when the MIC was 500.0–1.000 μg/mL; and inactive when the MIC was above 1.000 μg/mL. Evaluation of the antibacterial activity of the synthesized compounds showed that3g and17 was the most potent with MIC=15.6 μg/mL forM. luteum andS. aureus, respectively (Table 3).Evaluation of antibacterial activity of synthesized compounds showed that3d and3ghave MIC=31.2 μg/mL forM. luteum andS. aureus, respectively (Table 3).Significant antifungal activity for17 and25was observed againstC. tenuisfungi at 15.6 and 31.2 μg/mL, respectively. Evaluation of antifungal activity of compounds3c, 3g,and21showed their activity in concentrations 62.5–500.0μg/mL against test-cultureC. tenuis(Table 4). Compounds3g,17,21, and25 showed moderate antifungal activity with MIC value in the range of 125.0–500.0μg/mL againstA. nigerin Table 4.


### 3.3. Catalase Enzyme Inhibition Activity of Quinone Derivatives

Catalase is a common heme containing enzyme found in nearly all living organisms that are exposed toO2, where it functions to catalyze the decomposition of H2O2 to H2O and O2. Compounds3c,3d,3f,3g,4e,6b,7a,14,17,19,22, and23 were testedin vitro for their catalase activities and the results are shown in Table 5 and Figure 1. As shown in Figure 1, compound3g caused significant elevation of catalase activity.Table 5
Catalase enzyme activities of the compounds.
Compound Catalase activities (U/mL) 3c 0.599 3d 0.705 3f 0.715 3g 0.722 4e 0.689 6b 0.606 7a 0.608 14 0.470 17 0.581 19 0.550 22 0.709 23 0.585Figure 1
Catalase enzyme activities of the compounds, UmL-1.


## 3.1. Chemistry

In this study that we have done, reactions of thiol and amine compounds with 2,3-dichloro-1,4-naphthoquinone and 2,3,5,6-tetrachloro-1,4-benzoquinone as a starting compounds were investigated. Firstly, the multicomponent reactions of 2,3-dichloro-1,4-naphthoquinone1 with various thiol and amine nucleophiles were investigated. Similarly, 2,3,5,6-tetrachloro-1,4-benzoquinone15 with various amine nucleophiles was investigated. As shown in Scheme 1, the reaction of1 with different amines2a,2c,2d,2e,2f,2 gin ethanol in the presence of Na2CO3 gave known and unknown compounds3a [16, 17],3c,3d,3f,3g [18],4e. Compound6b[19] obtained the reaction of1 with5b.The reaction of3awith5a gave compound7a [20]. When1 reacted with an equimolar amount of various amines and thiols in ethanol in the presence of sodium carbonate solution at room temperature but under different conditions, the corresponding products (3c,3d,3f,3g,4e,6b,7a) were obtained in different yields. All synthesized compounds were confirmed by spectroscopic methods comprising 1H NMR and 13C NMR, FT-IR, elemental analysis, and MS.Scheme 1
The synthesis of N-, S-, O-substituted naphthoquinone derivatives (3a, 3c-d,3f-g,4e,6b,7a).In the second step of this study, different molar amount of N-substituted naphthoquinone compounds9 [16, 21],13 [16, 23] was reacted with sodium azide in DMF. The phenazine compounds11and14 [22] were synthesized and compound11 has not yet been described in the literature (Scheme 2).Scheme 2
The synthesis of phenazine compounds11 and14 via the condensation reaction of naphthoquinones9 and13.In the last step of this study, 2,3,5,6-tetrachloro-1,4-benzoquinone15 compound was reacted with compounds containing N-nucleophiles(2b,2c,2 g,16)that novel benzoquinones (17-25) not yet described in the literature were synthesized in Scheme 3. The synthesis, spectroscopic data (1H NMR, 13C NMR, MS, UV, FT-IR), elemental analysis, and melting points of compounds were reported in studies. The 1H NMR signal of the hydrogen atoms of the naphthoquinone unit of compounds3c,3d,3f,3g,4e,7a was observed at (CH) δ= 7.9-8.1 and 7.5-7.7 ppm like as doublet of doublets and triplet of triplets, respectively. Similarly,6b,11,14 were observed at (CH) δ= 7.9-8.1 and 7.5-7.7 ppm like as multiplets, respectively. Substituted aromatic ring hydrogens showed peaks around 6.8-7.4 ppm. Aliphatic groups in compounds3f,7a were shifted to a higher field and displayed peaks at 0.8-1.2 ppm. The 13C NMR spectra of compound3d gave two carbonyl signals at 177.5 and 179.9 ppm (C=O). Unlike other studies, the carbon atoms attached to the fluorine atoms in the3c compound give cleavage peaks 155.6, 157.8 ppm (F-Carom) in aromatic unit. Compound3d gave one carbonyl signal at 116.6 ppm (-Cl-Cnapht.) similarly giving a single peak at 126.9 ppm in the compound3f. The FT-IR spectra of compounds3c,3d,3f,3g,4e,7a showed bands around at 3300 cm−1 for the (–NH) stretching. Also, (C-Harom) bond was observed ν = 3000 cm−1. With the aid of the positive ion mode of electron spray ionization (ESI) mass spectrum of the compounds3c,6b, and3f, the respective molecular ion peaks were observed atm/z(%) 334 (100) [M+H]+, 411 (100) [M+H]+, 292 (100) [M+H]+, respectively.Scheme 3
The synthesis of N,O-substituted benzoquinone derivatives.2-Chloro-3-(o-tolylamino)naphthalene-1,4-dione9 and sodium azide10 required for the synthesis of11, similarly, 2-chloro-3-(m-tolylamino)naphthalene-1,4-dione13 and sodium azide (NaN3)10 required for the synthesis of compound14 have been synthesized according to Scheme 2. The nucleophilic displacement reaction of compound9 with sodium azide (NaN3) in DMF-H2O (10:1) afforded 1-methylbenzo[b]phenazine-6,11-dione11 and this analog 2-methylbenzo[b]phenazine-6,11-dione14 as the only isolated products as exhibited in Scheme 2. The proposed mechanism of condensation reaction of naphthoquinones agrees well with the related literatures [24, 25]. Both synthesized compounds were characterized by using the 1H NMR, 13C NMR, FT-IR spectral data, and elemental analysis. The first compound11was obtained by an interesting ring closure and is a phenazine derivative. The 13C NMR spectra of compound11 gave two carbonyl signals at 180.7 and 180.8 ppm (C=O). The FT-IR spectra of compounds11 and14 showed bands at 3019 cm−1 for the (C-Harom) stretching and (–NH) bonds were not observed in the FT-IR. 1H NMR peak of the hydrogen atoms of the naphthoquinone group gave on (CHarom) δ= 7.53-7.61 ppm and 7.95-7.99 ppm as multiplets for compound11. Molecular ion peaks were observed atm/z(%) 276.1 (100) [M+2H]+. The UV-Vis spectroscopy values for compound14 were also observed at 212(2.2), 241(2.1), 298(2.2), 539(1.2).It is known that the reactions of 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4 dione15 with amines proceed by Michael addition reaction. A series of 2-arylamino-1,4-benzoquinone derivatives17-25 were synthesized via the nucleophilic substitution reaction of 2,3,5,6-tetrachlorocyclohexa-2,5-diene-1,4-dione15 by appropriate aryl amines2c,2 g,2h,16 in ethanol as shown in Scheme 3. The reactions were found to be exceptionally selective and lead to mainly 2- and/or 2,5-bis(amino substituted)-3,6-dichloro-1,4-benzoquinones of the corresponding amine. From these reactions we could not obtain 2,6-bis(amino substituted)-1,4-benzoquinone derivatives. The steric factors arising from the substituent effect predominates in these reactions. The result of selective formation of 2,5-isomer may be assumed to be due to attack of two amines to 1,4-benzoquinone. For such attack to give exclusive product of one isomer (2,5-) would require approach of two amines from the furthest possible distance. Thus, exclusively 2- and/or 2,5-isomer were formed due to electrostatic reasons for compounds17-25. The results agree well with the corresponding mechanism in the similar compounds [6, 26].In1H NMR spectrum of compounds17-23, the hydrogen signals were observed at between δ = 6.1-8.0 ppm as multiplet peak, assigned to the (-CHarom). In the 13C NMR, characteristic signals of two carbonyl carbons of benzoquinones were visible at around 175.9 and 173.8 ppm. For compound23, substitute ethoxy group carbons (-CH2etoxy) and (-CH3etoxy) at 68.0 and 16.1 ppm, respectively. Like the naphthoquinone derivatives, carbon atoms attached to the fluorine atoms in the23 compound give cleavage peaks 155.5 and 157.8 ppm (F-Carom). The FT-IR spectra of compounds17-25showed the absorption bands of the N–H group at around 3240-3380 cm−1. The characteristic stretching band of carbonyl groups (C = O) was observed at between ν = 1650-1700 cm−1. In the MS of quinone derivatives, the molecular ion peaks of compounds17,22,and23were observed at 364 (100) [M]−, 445 (100) [M+2H]+, 360 (100) [M-H]−.


## 3.2. Antimicrobial Studies

The profound antifungal and antibacterial activity exhibited by quinone compounds has prompted us to synthesize new heteroatom substituted 1,4-naphtho- 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 compounds3c,3d,3f,3g,6b,11,17,21, and25 was elucidated againstEscherichia coli B-906,Staphylococcus aureus 209-P,Mycobacterium luteum B-917, Candida tenuis VKM Y-70, andAspergillus 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).Table 1
Antibacterial activity of the compounds determined by diffusion method.
Compound Concentration% Diameter of inhibition of growth of microorganisms, mm E. coli S. aureus M. luteum 3c 0.5 0 0 0 0.1 0 0 0 3d 0.5 0 16 24 0.1 0 12 12 3f 0.5 0 0 0 0.1 0 0 0 3g 0.5 11 20 20 0.1 0 14 16 6b 0.5 0 0 11 0.1 0 0 8 11 0.5 0 0 0 0.1 0 0 0 17 0.5 0 10 11 0.1 0 6 9 21 0.5 0 0 0 0.1 0 0 0 25 0.5 0 0 0 0.1 0 0 0 Control ∗ 0.5 14 15 18 ∗Vancomycin was used as a control in the tests of antibacterial activity of the synthesized compounds.Table 2
Antifungal activity of the compounds determined by diffusion method.
Compound Concentration% Diameter of inhibition of growth of microorganisms, mm C. tenuis A. niger 3c 0.5 0 0 0.1 0 0 3d 0.5 0 0 0.1 0 0 3f 0.5 0 0 0.1 0 0 3g 0.5 0 0 0.1 0 0 6b 0.5 15 0 0.1 13 0 11 0.5 0 0 0.1 0 0 17 0.5 0 15 0.1 0 10 21 0.5 7 12 0.1 0 6 25 0.5 15 10 0.1 0 0 Control ∗ 0.5 19 20 ∗Nystatin was used in the tests of antifungal activity of the synthesized compounds.Table 3
Antibacterial activity of the compound determined by serial dilution method.
Compound Microorganism E. coli S. aureus M. luteum MIC (μg/mL) 3c + + + 3d + 62.5 31.2 3f + + + 3g 250.0 31.2 15.6 6b + + + 11 + + + 17 + 15.6 62.5 21 + + + 25 + 500.0 250.0 Control 31.2 ± 0.8 3.9 ± 0.2 7.8 ± 0.2 +: growth of microorganisms.Table 4
Antifungal activity of the compounds determined by serial dilution method.
Compound Microorganism C. tenuis A. niger MIC (μg/mL) 3c 125.0 + 3d + + 3f + + 3g 500.0 500.0 6b + + 11 + + 17 15.6 250.0 21 62.5 125.0 25 31.2 250.0 Control 7.8 ± 0.2 15.6 ± 0.8 +: growth of microorganisms.The test-cultureE. coli appeared not to be sensitive to any compounds except that3g. Compound3g has moderate activity againstE. coli at a concentration of 0.5% and the diameter of the inhibition zone was 11 mm by diffusion method. Compounds3dand3g have strong activity againstS. 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). TheM. luteum strain was sensitive to compounds3g,6b, and17at a concentration of 0.5% and the diameter of the inhibition zone was 20 and 11 mm, respectively (Table 1). Compound3d has good antibacterial activity againstM. luteum at concentration of 0.5% and the diameter of the inhibition zone was 24 mm by diffusion method (for vancomycin was 18 mm). Compounds3dand3gwere found to exhibit strong antibacterial activity againstS. aureus andM. luteum (at concentration of 0.5%) on comparison with antibacterial drug vancomycin evaluated by diffusion method.Antifungal activity againstC. tenuis was observed for6b,21, and25at concentration of 0.5% (d = 15, 7 and 15 mm, respectively). Compound17 showed antifungal activity againstA. niger at 0.5% concentration (d = 15 mm) by the diffusion method (Table 2). Compounds3c,3f, and11have no antibacterial and antifungal activity againstE. coli, S. aureus, M. luteum, C. tenuis, andA. 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 100μg/mL or less; moderate, when the MIC was 100.0–500.0 μg/mL; weak, when the MIC was 500.0–1.000 μg/mL; and inactive when the MIC was above 1.000 μg/mL. Evaluation of the antibacterial activity of the synthesized compounds showed that3g and17 was the most potent with MIC=15.6 μg/mL forM. luteum andS. aureus, respectively (Table 3).Evaluation of antibacterial activity of synthesized compounds showed that3d and3ghave MIC=31.2 μg/mL forM. luteum andS. aureus, respectively (Table 3).Significant antifungal activity for17 and25was observed againstC. tenuisfungi at 15.6 and 31.2 μg/mL, respectively. Evaluation of antifungal activity of compounds3c, 3g,and21showed their activity in concentrations 62.5–500.0μg/mL against test-cultureC. tenuis(Table 4). Compounds3g,17,21, and25 showed moderate antifungal activity with MIC value in the range of 125.0–500.0μg/mL againstA. nigerin Table 4.


## 3.3. Catalase Enzyme Inhibition Activity of Quinone Derivatives

Catalase is a common heme containing enzyme found in nearly all living organisms that are exposed toO2, where it functions to catalyze the decomposition of H2O2 to H2O and O2. Compounds3c,3d,3f,3g,4e,6b,7a,14,17,19,22, and23 were testedin vitro for their catalase activities and the results are shown in Table 5 and Figure 1. As shown in Figure 1, compound3g caused significant elevation of catalase activity.Table 5
Catalase enzyme activities of the compounds.
Compound Catalase activities (U/mL) 3c 0.599 3d 0.705 3f 0.715 3g 0.722 4e 0.689 6b 0.606 7a 0.608 14 0.470 17 0.581 19 0.550 22 0.709 23 0.585Figure 1
Catalase enzyme activities of the compounds, UmL-1.


## 4. 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 againstE. coli,S. aureus, andM. luteumbacteria andC. tenuisandA. nigerfungi 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, 1H NMR, 13C NMR, MS, and UV-Vis.Compound3d has good antibacterial activity against test-cultureM. luteum at concentration of 0.5% and the diameter of the inhibition zone was 24 mm by diffusion method (for vancomycin was 18 mm). Compounds3d and3g were found to exhibit high antibacterial activity againstS. aureus andM. luteum (at concentration of 0.5%) on comparison with antibacterial drug vancomycin evaluated by diffusion method. Then, inhibitory activities of the benzo- and naphthoquinone derivatives against catalase enzyme were measured and especially3g exhibited better catalase enzyme inhibition activity than the other quinone derivatives.



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