Synthesis and Microbiological Evaluation of New 2- and 2,3-Diphenoxysubstituted Naphthalene-1,4-diones with 5-Oxopyrrolidine Moieties

New 3-substituted 1-(3-hydroxyphenyl)-5-oxopyrrolidine derivatives containing hydrazone, azole, diazole, oxadiazole fragments, as well as 2-phenoxy- and 2,3-diphenoxy-1,4-naphthoquinone derivatives were synthesized. The structure of all compounds has been confirmed by NMR, IR, mass spectra, and elemental analysis data. Methyl 1-{3-[(3-chloro-1,4-dioxo-1,4-dihydro-2-naphthalenyl)oxy]phenyl}-5-oxo-3-pyrrolidinecarboxylate demonstrated potential antibacterial and antifungal activities as determined by diffusion and serial dilution methods, while N'-[(4-bromophenyl)methylidene]-1-{3-[(3-chloro-1,4-dioxo-1,4-dihydro-2-naphthalenyl)oxy]phenyl}-5-oxo-3-pyrrolidinecarbohydrazide and 2-{3-[4-(1,2,3-oxadiazol-5-yl)-2-oxo-1-pyrrolidinyl]phenoxy}-3-{3-[4-(1,3,4-oxadiazol-2-yl)-2-oxo-1-pyrrolidinyl]phenoxy}naphthoquinone showed antifungal activity against Candida tenuis and Aspergillus niger at low concentrations, as determined by the serial dilution method. The substitution of the methoxy fragment by N-containing substituents in monophenoxy substituted naphthoquinones was found to decrease their activity against Mycobacterium luteum. Besides, introduction of the second phenoxy substituted fragment increased the antifungal activity against Candida tenuis and Aspergillus niger at lower concentrations.

The incidence of fungal and bacterial infections has increased dramatically in recent years. The widespread use of antifungal and antibacterial drugs and the development of resistance against them of fungal and bacterial infections has led to serious health hazards. The resistance against wide spectrum antifungal and antibacterial agents has prompted the discovery of new antifungal and antibacterial drugs. The amino and thioether derivatives of 1,4-naphthoquinones have extremely rich biological activities because of their redox potentials [15]. The recent pharmacophore modelling approach and three dimensional quantitative structure-activity (3D-QSAR)/comparative molecular similarity indices analysis (CoMSIA) methods applied to 2,3-disubstituted-1,4-naphthoquinones and heterocyclic 1,4-naphthoquinones in human promyelocytic leukemia HL-60 cell line have explained the pronounced cytotoxic activity of these derivatives [11]. The natural naphthoquinone products alkannin and shikonin and their derivatives are active against Gram-positive bacteria such as Staphylococcus aureus, Enterococcus faecium and Bacillus subtilis. 2,3-Diamino-1,4-naphthoquinone itself was found to act as an antibacterial agent against Staphylococcus aureus, with IC 50 values ranging from 30 to 125 µg/mL [16]. The prevalence of strains of Staphylococcus aureus resistant to conventional antibiotics has increased to high levels in some hospitals. The biological activity of several well-known and widely used anthracycline antibiotics such as daunomycin and doxorubicin is thought to be associated to the hydroxyquinone structure. Moreover, equivalent active sites are also present in the tetracycline antibiotics as well as in myxopyronin. The antibacterial effect is also related to naphthoquinones from vegetal origin and isoxazolyl-naphthoquinones. In addition, the fungitoxic effect of 1,4-naphto-quinones and the antiviral activity of some hydroxyquinones have been described [17]. Various 2,3-disubstituted 1,4-naphthoquinone derivatives can be prepared from 2,3-dichloro-1,4naphthoquinone by its reactions with amino-and hydroxy substituted compounds. In our previous study [18], a series 2-and 2,3-disubstituted [2,4-dioxotetrahydropyrimidin-1(2H)-yl)phenoxy]naphthalene-1,4-diones were synthesized, and some of them exhibited antimicrobial activity against Staphylococcus aureus, Mycobacterium luteum, Candida tenuis and Aspergillus niger.

Synthesis and Structural Peculiarities of New Compounds
In this work, we describe the synthesis of new 2-and 2,3-diphenoxy-substituted 1,4-naphthquinone derivatives containing hydrazone, azole, diazole and oxadiazole fragments, and investigation their microbiological activity. The target compounds 2-11 were synthesized as illustrated in Schemes 1 and 2. Methyl 1-(3-hydroxyphenyl)-5-oxo-3-pyrrolidinecarboxylate (2) was synthesized by esterification of 1-(3-hydroxyphenyl)-5-oxo-3-pyrrolidine carboxylic acid (1) with an excess of methanol under reflux in the presence of a catalytic amount of sulphuric acid (Scheme 1). Reaction of ester 2 with hydrazine hydrate in 2-propanol under reflux gave 1-(3-hydroxyphenyl)-5-oxo-3-pyrrolidinecarbohydrazide 3, which crystallized from the reaction mixture after cooling. Condensation of compound 3 with aromatic aldehydes and acetone gave hydrazone-type derivatives -1-(3hydroxyphenyl)-5-oxo-N'-(phenylmethylidene)-3-pyrrolidinecarbohydrazides 4a-c and 1-(3-hydroxyphenyl)-N'-(1-methylethylidene)-5-oxo-3-pyrrolidinecarbohydrazide (5). Then diketones -2,4pentanedione and 2,5-hexanedione -were used in condensation reaction with carbohydrazide 3, dimethylpyrazole, and dimethylpyrrole derivatives 6,7 were thus obtained. The reactions were carried out in 2-propanol in the presence of acetic or hydrochloric acids as catalysts. The formation of heterocyclic systems in compounds 6 and 7 has been confirmed by the characteristic 1 H-NMR peak signals at 6.23 ppm and 5.65 ppm, attributed to the CH group proton in the dimethylpyrazole moiety and two protons of CH groups in the dimethylpyrrole one, respectively.  Compounds 4a-c, containing amide and azomethine groups, theoretically can exist as an inseparable mixture of four isomers. The amide group determines the splitting of resonances in 1 H-and 13 C-NMR spectra due to the restricted rotation around the (E/Z) amide bond. The lone pair of the nitrogen atom in the azomethine group affects the neighbouring atoms and causes formation of geometrical isomers (cis/trans) [19][20][21]. The 13 C-NMR spectra of 4a-c exhibited a double set of resonances of CO, N=CH, pyrrolidinone ring carbons and even some of the benzene ring ones. The data presented above allow us to conclude that the cis/trans geometrical isomers of the azomethine group are not observed. The E/Z ratio of amide conformers can be easily quantified by NMR spectroscopy, and it is about 40/60 for compounds 4a-c. Geometrical isomers are not formed in the case of compound 5, since two identical terminal methyl substituents are positioned at the double bond; therefore, only a mixture of s-cis and s-trans rotamers is observed in the 1 H-NMR spectrum.

Antimicrobial Evaluation of Synthesized Compounds
The synthesized 2,3-disubstituted naphthalene-1,4-diones 10b-f and 11b-f were evaluated for their antibacterial and antifungal activity against Escherichia coli В-906, Staphylococcus aureus 209-Р, Mycobacterium luteum В-917 (as nonpathogenic test bacteria culture representative of the genus Mycobacterium, which are classified as acid resistant Gram-positive bacteria, because they have no outer cell membrane in their cell walls), Candida tenuis VCM Y-70, and Aspergillus niger VCM F-1119 strains by the diffusion [22] and serial dilution techniques [23] (determination of minimal inhibition concentrations, MIC). Their activity was compared with that of the known antibacterial agent vancomycin and the antifungal agent nystatin.
As one can see from the data presented in Table 1, S. aureus was low-sensitive to monophenoxysubstituted compounds 10b and 10f and not sensitive to other mono-and disubstituted naphthoquinones at the investigated concentrations (diffusion method). The bacterial strain Mycobacterium luteum was low-sensitive to naphthoquinones 10b and 11b, for which the diameter of the inhibition zones at 0.5% concentration was 11.4-12.0 mm. Test-culture E. coli В-906 appeared not to be sensitive to the monophenoxy-and diphenoxy-substituted naphthoquinones investigated by the diffusion method. For all compounds 10b-f and 11b-f the growth of bacteria E. coli and S. aureus was observed at the study concentrations, indicating the absence of biocidic activity of these compounds against the studied bacteria.  The evaluation of antifungal activity (diffusion method) has revealed the monophenoxy-substituted naphthoquinones 10b, 10f to show a moderate fungicidal effect against C. tenuis and A. niger at 0.5% concentration (the inhibition zone diameter for strain C. tenuis was 15.0 and 14.4 mm and for the one for A. niger was 15.0 and 17.4 mm, respectively).
Evaluation by the serial dilution method of compounds 10b, 10d and 11f showed a minimal inhibition action against the M. luteum bacterial strain at concentrations of 62.5, 250 and 125 μg/mL, respectively ( Table 2). Compounds 10b-11f didn't show antibacterial activity against E. coli and S. aureus by the serial dilution method. Evaluation by the serial dilution method revealed the MIC for substances 10b,f,c to be 1.9-62.5 μg/mL against C. tenuis, and for the one for 10b,f,c and 11f to be 3.9-62.5 μg/mL against A. niger.
Thus, some promising compounds, especially 10c and 11f, with antifungal activity against Candida tenuis and Aspergillus niger fungi at low concentrations were identified among the synthesized compounds by the serial dilution method. Compounds with antibacterial activity against gram-positive bacteria M. luteum at concentrations 62.5-250 μg/mL were identified. The compound 10b had antibacterial and antifungal activities as determined by diffusion and serial dilution methods.
The substitution of the methoxy fragment by N-containing substituents in monophenoxysubstituted naphthoquinones decreased antibacterial activity against Mycobacterium luteum. Besides, introduction of the second phenoxy substituted fragment increased the antifungal activity against Candida tenuis and Aspergillus niger at lower concentrations. Based on these data, the following correlation between structure and antibacterial and antifungal activities of the investigated naphthoquinones was made (diffusion method). The antimicrobial activity of the tested compounds correlates with their structure. It has been observed that synthesized naphthoquinones containing chlorine atoms in the 2,3-disubstituted naphthoquinone moiety show more significant antibacterial and antifungal activities in comparison with 2,3-diphenoxy-substituted naphthoquinone derivatives. 2,3-Disubstituted derivatives 10b and 11b containing methyl carboxylate groups in the pyrrolidinone ring exhibited the highest biological activity. Transformation of this group to hydrazone, dimethylpyrrole, dimethylpyrazole or oxazole substituents decreased the biological activity.

Antimicrobial Activity
The synthesized compounds were tested for their in vitro antimicrobial and antifungal activity against bacteria Escherichia coli B-906, Staphylococcus aureus 209-P, Mycobacterium luteum B-917 and fungi Candida tenuis VCM Y-70, Aspergillus niger VCM F-1119 by the diffusion method in agar (method A) and by the serial dilution method (method B).
Method А. Determination of antimicrobial and antifungal activity by diffusion method in agar. Antimicrobial and antifungal activity has been tested by diffusion in agar on solid nutrient medium (beef-extract agar for bacteria, wort agar for fungi). Petri plates containing 20 mL of nutrient medium were used for all tested microorganisms. The inoculums (the microbial loading − 10 9 cells (spores)/1 mL) was spread on the surface of the solidified media and Whatman no.1 filter paper discs (6 mm in diameter) impregnated with the test compound solution (0.1% and 0.5%) were placed on the plates. The duration of bacteria incubation was 24 h at 35 °С and the one of fungi incubation was 48-72 h at 28-30 С. The antimicrobial effect and degree of activity of the tested compounds were evaluated by measuring the zone diameters. The results were compared with well known drugs (Table 1). Every experiment was repeated three times.
Method B. Determination of minimal inhibitory (MIC), minimal bactericidal (MBC) and minimal fungicidal (MFC) concentrations using serial dilution method. The tested compounds were added to the nutrient medium (beef-extract broth for bacteria and wort for fungi) as solutions in dimethyl sulfoxide (DMSO) by ensuring needed concentration (0.9-500.0 μg/mL). Bacteria and fungi inoculum was inoculated into nutrient medium (the microbial loading was 10 6 cells (spores)/1 mL). The duration of bacteria incubation was 24 h at 35 С and the one of fungi incubation was 48-72 h at 28-30 С. The results were estimated by the microorganism growth measured by degree of microbial turbidity in nutrient medium. Minimal inhibitory concentration (MIC) of any compound is defined as the lowest concentration which completely inhibits visible growth (turbidity on liquid nutrient medium).
Determination of MBC and MFC. Visually transparent nutrient medium solutions were sowed on the sterile agar medium (beef-extract agar for bacteria, wort agar for fungi). The duration of bacteria incubation was 24 h at 35 С and the one of fungi incubation was 48-72 h at 28-30 С. In the absence of microorganism colony growth on the incubated Petri plate, minimal bactericidal (MBC) and minimal fungicidal (MFC) concentrations of the investigated compounds were identified. The test was repeated three times.

Conclusions
New 3-substituted 1-(3-hydroxyphenyl)-5-oxopyrrolidine derivatives contained hydrazone, azole, diazole, oxadiazole fragments, 2-phenoxy-and 2,3-diphenoxy-1,4-naphthoquinones were synthesized. showed antifungal activity against Candida tenuis and Aspergillus niger at low concentrations as determined by the serial dilution method. The substitution of the methoxy fragment by N-containing substituents in monophenoxy substituted naphthoquinones was found to decrease their activity against Mycobacterium luteum. Besides, introduction of the second phenoxy substituted fragment increased the antifungal activity against Candida tenuis and Aspergillus niger at lower concentrations.