Synthesis and In Vitro Biological Evaluation of Aminonaphthoquinones and Benzo[b]phenazine-6,11-dione Derivatives as Potential Antibacterial and Antifungal Compounds

A series of 2-arylamino-3-chloro-1,4-naphthoquinone derivatives (3a–h) by the reaction of 2,3-dichloro-1,4-naphthoquinone with aryl amines (2a–h) and benzo[b]phenazine-6,11-dione derivatives (4a–c) by the treatment of 2-arylamino-3-chloro-1,4naphthoquinone derivatives (3a–h) with sodium azide were synthesized and tested for their in vitro antibacterial and antifungal activities. The results suggest that compounds 3d and 3g had potent antifungal activity against Candida albicans (MIC = 78.12 μg/mL). All synthesized compounds (3a–h, 4a–c) possessed activity against E. faecalis with MIC values of between 312.5 and 1250 μg/mL. Benzo[b]phenazine-6,11-dione derivatives (4a–c) were mostly active against Gram-positive bacteria. The structures of the new members of the series were established on the basis of their spectral properties (IR, H NMR, C NMR, and mass spectrometry).


Introduction
Quinones are active compounds used widely as raw materials in pharmaceuticals and agrochemical industries. Particularly, (hetero)cyclic quinone moieties not only exist in many natural products and pharmaceutical compounds, but also are well-known and versatile building blocks for the synthesis of quinones derived from benzoquinone, naphthoquinone, or anthracenequinone condensed with five-membered heterocycles [1][2][3] such as isoxazoles [4], six-membered heterocycles [3,5], and seven-membered heterocycles [6,7] such as 1,4-benzodiazepines [8] in order to evaluate their important bioactivities. Therefore, among the bioactive quinones, 1,4naphthoquinones have been extensively studied since those ones contain two ketone groups as a crucial pharmacophore for their bioactivities because of their ability to accept electrons [9]. A considerable number of natural and synthetic 1,4-naphthoquinones have shown an interesting variety of biological properties, such as antimalarial [10][11][12], antibacterial [13][14][15][16], antifungal [17][18][19][20], antitumor [21,22], antiinflammatory [23,24], and antiallergic [25,26] activities, due to their redox potentials [27]. Some important compounds shown in Figure 1 are good examples for emphasizing their important biological properties [28,29]. All findings showed that the position and the number of nitrogen atoms in the structure could improve the redox potential of the quinone system for biological properties [30]. It has been reported that, generally, increase of the number of the nitrogen atoms and the rings enhances the activities [19].

Results and Discussion
2.1. Chemistry. It is known that the reactions of 2,3-dichloro-1,4-naphthoquinone (1) with aryl and alkyl amines proceed by nucleophilic substitution [15-17, 28, 39-43]. A series of 2-arylamino-1,4-naphthoquinone derivatives (3a-h) were synthesized via the nucleophilic substitution reaction of 2,3dichloro-1,4-naphthoquinone (1) by appropriate aryl amines (2a-h) in refluxing ethanol as shown in Scheme 1. The aminonaphthoquinones (3a-h) were obtained in around 55-60% yields as dark orange, red, dark red, and purple solid. Structures of the aminonaphthoquinone derivatives were confirmed by spectroscopic methods comprising 1 H and 13 C NMR, IR, and MS. In the MS of aminonaphthoquinones, the molecular ion peaks of compounds 3a, 3f, and 3g were observed at 343 [M] + , 362 [M-H] + , and 362 [M-H] + , respectively. Some of the IR spectra of aminonaphthoquinones revealed the absorption bands of the N-H group at around 3300 cm −1 and of >C=O moiety at 1683 cm −1 . The 1 H NMR spectra exhibited aromatic protons at around 6.50-8.00 ppm. The methylene protons of alkoxy groups in 3a appeared at around 3.5-4 ppm as a singlet. The methylene protons of compound 3d (-OCH 2 -) were observed as a triplet at 3.96 ppm. The singlet peak at around 8-9 ppm was assigned to the NH proton in aminonaphthoquinones. In addition, aromatic protons of aminonaphthoquinones are displayed at 6.50-8.00 ppm. In the 13 C NMR spectra, characteristic signals of two carbonyl carbons of aminonaphthoquinones were visible at chemical shift at around 175 and 182 ppm.
at 90-100 ∘ C overnight afforded the expected benzo[b]phenazine-6,11-dione derivatives (4a-c). The reaction is believed to proceed via the formation of the unstable intermediate compound (2-arylamino-3-azido-1,4-naphthoquinones) as mentioned in the literature [29].  Table 1 reveal that compounds 3d and 3g have exhibited moderate activity against both Gram-positive and Gram-negative bacteria, except E. coli for 3d. All of the synthesized compounds (3a-h, 4a-c) possessed activity against E. faecalis with MIC values of between 312.5 and 1250 g/mL. In addition to E. faecalis, all of the compounds, except 4b, possessed activity against P. aeruginosa with MIC values of between 625 and 1250 g/mL. The synthesized compounds, except 3e and 4a, also showed good antibacterial activity against S. epidermidis. The test-culture E. coli appeared not to be susceptible to synthesized compounds except that 3g. Evaluation of the antifungal activity of the synthesized compounds showed that 3d and 3g were the most potent with MIC (minimum inhibition concentration) 78.12 g/mL for C. albicans ( Table 1). The results also reveal that compounds 3d and 3g were the most active among the synthesized compounds; they have both antibacterial and antifungal activities.
On the other hand, benzo[b]phenazine-6,11-dione derivatives (4a-c) were mostly active against Gram-positive bacteria. We found that replacing the sulfonic acid group (-SO 3 H) position in 2-arylamino-3-chloro-1,4-naphthoquinones from the meta position to the para position led to activity loss. Additionally, replacing the sulfonic acid group (-SO 3 H) at the para position by a sulfonamide group (-SO 2 NH 2 ) and trifluoromethyl (-CF 3 ) did not show any progress against C. albicans but showed an increase in activity against some of the Gram-negative bacteria. By contrast, replacing this sulfonic acid group (-SO 3 H) at the para position by a hexyloxy group (-O(CH 2 ) 5 CH 3 ) led to an increase in activity against C. albicans and no significant increase in activity against some of the Gram-positive and Gram-negative bacteria. Changing this hexyloxy group (-O(CH 2 ) 5 CH 3 ) by the methoxy groups at different positions, unfortunately, led to activity loss again. Coupling constants J are given in Hz. IR spectra were recorded as ATR on either Thermo Scientific Nicolet 6700 spectrometer or Alpha T FTIR spectrometer. Mass spectra were obtained on either a Thermo Finnigan LCQ Advantage MAX MS/MS spectrometer equipped with ESI (electrospray ionization) sources or GC-MS Shimadzu QP2010 Plus. Melting points (mp) were determined with an Electrothermal IA9000 series and were uncorrected.

General Procedure for the Preparation of Benzo[b]phenazine-6,11-dione Derivatives (4a-c).
Compounds 4a-c were prepared using the following general procedure according to the reported literature [29,36,37]: to a solution of the corresponding 2-arylamino-3-chloro-1,4-naphthoquinone (3ad) in DMF (15 mL), sodium azide (20 mmol), suspended in 2.5 mL water, was added and refluxed. The reaction mixture was diluted with dichloromethane and the organic phase was washed twice with water and then dried over CaCl 2 . After evaporating the solvent, the crude product was purified by column chromatography on silica gel to yield the corresponding benzo[b]phenazine-6,11-dione derivatives (4a-c). -6,11-dione (4a [44,45]. Mueller-Hinton broth for bacteria and RPMI-1640 medium for yeast strain were used as the test medium. Serial twofold dilutions ranging from 5000 mg/L to 4.8 mg/L were prepared in medium. The inoculum was prepared using a 4-6 h broth culture of each bacteria type and 24 h culture of yeast strains adjusted to a turbidity equivalent to 0.5 McFarland standard, diluted in broth media to give a final concentration of 5 × 10 5 cfu/ml for bacteria and 5 × 10 3 cfu/mL for yeast in the test tray. The trays were covered and placed into plastic bags to prevent evaporation. The trays containing Mueller-Hinton broth were incubated at 35 ∘ C for 18-20 h while the trays containing RPMI-1640 medium were incubated at 35 ∘ C for 46-50 h. The MIC was defined as the lowest concentration of compound giving complete inhibition of visible growth. As control, antimicrobial effects of the solvents were investigated against test microorganisms. According to values of the controls, the results were evaluated. The MIC values of the compounds are given in Table 1.

Conclusions
In conclusion, we have synthesized a series of 2-arylamino-3-chloro-1,4-naphthoquinone derivatives (3a-h) and benzo[b]phenazine-6,11-dione derivatives (4a-c) which were given by reacting 2-arylamino-3-chloro-1,4-naphthoquinone derivatives (3a-d) with sodium azide through known chemical routes. The structures of the five new compounds (3a, 3g, and 4a-c) have been confirmed by means of different spectroscopic methods. These new compounds possess high solubility in various organic solvents such as chloroform and dichloromethane while they are insoluble in water and these compounds have shown good stability. On the basis of screening data for the presented compounds, the in vitro antimicrobial activities were evaluated against different Gram-positive and Gram-negative bacterial strains in addition to the antifungal activities. The test-culture E. coli appeared not to be susceptible to most of the synthesized compounds. Results revealed that compounds 3d and 3g have remarkable activity against both Gram-positive and Gram-negative bacteria and against the tested fungi (C. albicans), while all of the synthesized compounds (3a-h, 4a-c) possessed activity against E. faecalis with MIC values of between 312.5 and 1250 g/mL. Benzo[b]phenazine-6,11-dione derivatives (4ac) were mostly active against Gram-positive bacteria.