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 Scheme 1 and Scheme 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-pyrrolidine-carbohydrazide 3, which crystallized from the reaction mixture after cooling. Condensation of compound 3 with aromatic aldehydes and acetone gave hydrazone-type derivatives – 1-(3-hydroxyphenyl)-5-oxo-N'-(phenylmethylidene)-3-pyrrolidinecarbohydrazides 4a–c and 1-(3-hydroxy- phenyl)-N'-(1-methylethylidene)-5-oxo-3-pyrrolidinecarbohydrazide (5). Then diketones – 2,4-pentanedione 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 ¹H-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 ¹H- and ¹³C-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 ¹³C-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 ¹H-NMR spectrum.

The monophenoxy- and diphenoxy substituted naphthoquinone derivatives 10, 11, containing different substituents in pyrrolidinone moiety, were synthesized by reactions of 1-(3-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic acid and their derivatives 1, 2, 4c, 6–8 with 2,3-dichloro-1,4-naphtho-quinone (9).

3-Substituted 1-[3-(3-chloro-1,4-dioxo-1,4-dihydro-naphthalene-2-yloxy)phenyl]-5-oxopyrrolidine derivatives 10b–f were obtained by stirring the mixture of the respective 1-(3-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic derivatives 2, 4c, 6–8, 2,3-dichloro-1,4-naphthoquinone (9), and sodium carbonate in dimethyl sulfoxide for 24 hours at room temperature (Scheme 2). The reaction was finished by diluting the reaction mixture with water, causing the products to precipitate. It was noticed that, along with the target products, small amounts of 2,3-disubstituted derivatives 11 were formed. Therefore, reactions of 2,3-dichloro-1,4-naphthoquinone (9) with an excess of hydroxyl-substituted compounds 1, 2, 4c, 6–8 were carried out, affording 1-[3-({3-[3-(4-carboxy-2-oxo-1-pyrrolidinyl)-phenoxy]-1,4-dioxo-1,4-dihydro-2-naphthalenyl}oxy)phenyl]-5-oxo-3-pyrrolidinecarboxylic acid and its derivatives 11a–f.

The structures of all synthesized compounds were confirmed by mass spectrometry, IR, ¹H-, and ¹³C-NMR spectroscopy data. A comparison of the ¹H-NMR spectra of 10 and 11 revealed the integral intensities of the proton signals to confirm the formation of monosubstituted naphthoquinone in the first and disubstituted derivative in the second case. These spectra showed identical signals for the functional group protons.

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 monophenoxy-substituted 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 B-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). For S. aureus: For A. niger:

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.

The ¹H- and ¹³C-NMR spectra were recorded on Varian Unity Inova (300 MHz, 75 MHz) spectrometer operating on Fourier transform mode, using DMSO-d₆ and CDCl₃ as solvents and TMS as an internal standart (chemical shifts in δ, ppm). IR spectra (ν, cm⁻¹) were recorded on a Perkin Elmer Spectrum BX FT–IR spectrometer, KBr tablets. Mass spectra were obtained on Waters ZQ 2000 spectrometer using the atmospheric pressure chemical ionization (APCI) mode and operating at 25 V. Elemental analyses were performed on CE-440 elemental analyzer. Melting points were determined on automatic APA1 melting point apparatus and are uncorrected. TLC was performed on Merck, Silica gel 60 F₂₅₄ (Kieselgel 60 F₂₅₄) silica gel plates.

Methyl 1-(3-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylate (2). A mixture of 5-oxopyrrolidine 1 (17.7 g, 0.08 mol), methanol (100 mL) and catalytic amount of conc. H₂SO₄ was refluxed for 5 h. Then the solvent was evaporated. The precipitated product was neutralized with 5% sodium bicarbonate solution and filtered off, washed with 2-propanol. The crude product was recrystallized from 2-propanol. White crystals, (15.9 g, 85%), m.p. 188–189 °C; ν_max/cm⁻¹ 1660 and 1740 (C=O) and 3159 (OH). ¹H-NMR (DMSO-d₆): δ_H 2.65–2.86 (2H, m, CH₂CO), 3.38–3.49 (1H, m, CH), 3.67 (3H, s, CH₃), 3.88–4.06 (2H, m, NCH₂), 6.52–7.28 (4H, m, H_Ar), 9.50 (1H, s, OH). ¹³C-NMR (DMSO-d₆) δ_C 34.8 (CH), 35.1 (CH₂CO), 49.8 (CH₂N), 52.1 (CH₃), 106.7, 109.8, 111.3, 129.4, 140.0, 157.5 (C_Ar), 171.4, 173.1 (C=O). MS, m/z, (%) = 258 ([M+Na]⁺ 100); Anal. Calcd for C₁₂H₁₃NO₄ (235.24): C, 61.27; H, 5.57; N, 5.95. Found: C, 61.34; H, 5.51; N, 5.89.

1-(3-Hydroxyphenyl)-5-oxopyrrolidine-3-carbohydrazide (3). A mixture of ester 2 (11.0 g, 0.047 mol), 2-propanol (100 mL) and 98% hydrazine hydrate (8.0 g, 0.16 mol) was refluxed for 5 h. The reaction mixture was cooled, the precipitate was filtered off, washed with 2-propanol and purified by recrystallization from water. White crystals, (10.1 g, 92%), m.p. 208–209 °C; ν_max/cm⁻¹ 1697 and 1660 (C=O), 3131 (OH), 3314 and 3253 (NH, NH₂). ¹H-NMR (DMSO-d₆): δ_H 2.55–2.75 (2H, m, CH₂CO), 3.07–3.19 (1H, m, CH), 3.74–3.98 (2H, m, NCH₂), 4.29 (2H, br. s, NH₂), 6.52–7.26 (4H, m, H_Ar), 9.27 (1H, s, NH), 9.48 (1H, s, OH). ¹³C-NMR (DMSO-d₆) δ_C 33.9 (CH), 35.8 (CH₂CO), 50.7 (CH₂N), 106.6, 109.7, 111.2, 129.4, 140.2, 157.5 (C_Ar), 171.6, 171.9 (C=O). MS, m/z, (%) = 258 ([M+Na]⁺ 100); Anal. Calcd for C₁₁H₁₃N₃O₃ (235.24): C, 56.16; H, 5.57; N, 17.86. Found: C, 56.22; H, 5.32; N, 17.84.

A mixture of hydrazide 3 (3.5 g, 0.015mol), the corresponding aldehyde (0.016 mol) and 2-propanol (30 mL) was refluxed for 3 h. The reaction mixture was cooled, the precipitate was filtered off and washed with 2-propanol.

1-(3-Hydroxyphenyl)-5-oxo-N'-(phenylmethylene)pyrrolidine-3-carbohydrazide (4a). White crystals, (4.36 g, 91%), m.p. 194–195 °C; ν_max/ cm⁻¹ 1590 (C=N), 1664 and 1693 (C=O), 3225 (OH) and 3334 (NH). ¹H-NMR (DMSO-d₆): δ_H (E/Z, 40/60) 2.68–2.89 (2H, m, CH₂CO), 3.27–3.38 (1H, m, CH), 3.89–4.14 (2H, m, NCH₂), 6.52–7.77 (9H, m, H_Ar), 8.04, 8.22 (1H, 2s, NCH), 9.47 (1H, s, OH), 11.57, 11.63 (1H, 2s, NH). ¹³C-NMR (DMSO-d₆) δ_C 32.7, 34.7, 35.0, 32.7 (CH, CH₂CO), 50.1, 50.5 (CH₂N), 106.6, 109.8, 111.1, 126.8, 126.9, 128.8, 129.3, 129.8, 130.0, 134.0, 140.1, 140.2, 143.6, 146.9, 157.5, 168.6, (C_Ar, NCH), 171.7, 171.9, 173.5 (C=O). MS, m/z, (%) = 346 ([M+Na]⁺ 100); Anal. Calcd for C₁₈H₁₇N₃O₃ (323.35): C, 66.86; H, 5.30; N, 13.00. Found: C, 66.75; H, 5.24; N, 13.08.

1-(3-Hydroxyphenyl)-N'-[(4-methoxyphenyl)methylene]-5-oxopyrrolidine-3-carbohydrazide (4b). White crystals, (4.68 g, 89%), m.p. 200–201 °C (from dioxane); ν_max/cm⁻¹ 1600 (C=N), 1609 and 1661, (C=O), 3088 (OH) and 3231 (NH). ¹H-NMR (DMSO-d₆): δ_H (E/Z, 40/60) 2.65–2.85 (2H, m, CH₂CO), 3.24–3.37 (1H, m, CH), 3.85–4.14 (2H, m, NCH₂), 6.51–7.69 (8H, m, H_Ar), 7.98, 8.16 (1H, 2s, NCH), 9.47 (1H, s, OH), 11.44, 11.50 (1H, 2s, NH). ¹³C-NMR (DMSO-d₆) δ_C 32.7, 34.7, 35.0, 35.8 (CH, CH₂CO), 50.1, 50.6 (CH₂N), 55.2 (OCH₃), 106.6, 109.8, 111.1, 114.3, 126.7, 128.4, 128.6, 129.3, 140.2, 143.4, 146.8, 157.5, 160.6, 160.8, 168.4 (C_Ar, N=CH), 171.8, 171.9, 173.2 (C=O). MS, m/z, (%) = 376,6 ([M+Na]⁺ 100); Anal. Calcd for C₁₉H₁₉N₃O₄ (353.38): C, 64.58; H, 5.42; N, 11.89. Found: C, 64.42; H, 5.39; N, 11.79.

1-(3-Hydroxyphenyl)-N'-[(4-bromophenyl)methylene]-5-oxopyrrolidine-3-carbohydrazide (4c). White crystals, (5.27 g, 88%), m.p. 255–256 °C (from dioxane); ν_max/cm⁻¹ 1599 (C=N), 1663 and 1698 (C=O), 2973 (OH) and 3172 (NH). ¹H-NMR (DMSO-d₆): δ_H (E/Z, 40/60) 2.64–2.89 (2H, m, CH₂CO), 3.26–3.40 (1H, m, CH), 3.85–4.18 (2H, m, NCH₂), 6.49–7.70 (8H, m, H_Ar), 8.01, 8.19 (1H, 2s, NCH), 9.47 (1H, s, OH), 11.63, 11.71 (1H, 2s, NH). ¹³C-NMR (DMSO-d₆) δ_C 32.7, 34.7, 34.9, 35.7 (CH, CH₂CO), 50.0, 50.5 (CH₂N), 106.6, 109.8, 111.1, 123.0, 123.3, 128.7, 128.9, 129.3, 131.7, 133.4, 140.1, 140.2, 142.4, 145.7, 157.5 (C_Ar), 171.6, 171.9 173.6 (C=O). MS, m/z, (%) = 403 ([M+H]⁺ 95), 405 ([M+H+2]⁺ 100). Anal. Calcd for C₁₈H₁₆BrN₃O₃ (402.25): C, 53.75; H, 4.01; N, 10.45. Found: C, 53.31; H, 4.19; N, 10.71.

1-(3-Hydroxyphenyl)-N'-isopropylidene-5-oxopyrrolidine-3-carbohydrazide (5). A mixture of compound 3 (1.0 g 4.3 mmol) and dry 2-propanone (30 mL) was refluxed for 4 h. The solvent was evaporated under vacuum, and the precipitated product was filtered off, washed with ethyl ether and purified by recrystallization from 2-propanol. White crystals, (0.8 g, 68%), m.p. 185–186 °C; IR ν_max/cm⁻¹ 1597 (C=N), 1675, 1659 (C=O), 3180 (OH) and 3235 (NH). ¹H-NMR (DMSO-d₆): δ_H (E/Z, 55/45)1.87, 1.88 (3H, 2s, CH₃), 1.93 (3H, s, CH₃), 2.59–2.80 (2H, m, CH₂CO), 3.34–3,47 (1H, m, CH), 3.77–4.06 (2H, m, NCH₂), 6.51–7.29 (4H, m, H_Ar), 9.46 (1H, s, OH), 10.21, 10.30 (1H, 2s, NH). ¹³C-NMR (DMSO-d₆) δ_C 17.1, 17.6, 24.9, 25.2 (2CH₃), 32.8, 34.2, 35.1, 35.9 (CH, CH₂CO), 50.2, 50.9 (CH₂N), 106.6, 106.6, 109.7, 109.8, 111.1, 129.3, 140.2, 140.3, 151.2, 156.1, 157.4, 168.6 (C_Ar), 171.9, 172.0, 173.6 (C=O). MS, m/z, (%) = 298.6 ([M+Na]⁺ 100); Anal. Calcd for C₁₄H₁₇N₃O₃ (275.31): C, 61.08; H, 6.22; N, 15.26. Found: C, 60.98; H, 6.31; N, 15.30.

N-(2,5-Dimethyl-1H-pyrrol-1-yl)-1-(3-hydroxyphenyl)-5-oxopyrrolidine-3-carboxamide (6). To a solution of hydrazide 3 (3.0 g, 0.013 mol) in 2-propanol (40 mL) 2,5-hexanedione (4.6 g, 0.04 mol) and glacial acetic acid (3 mL) were added. The reaction mixture was stirred and refluxed for 6 h. Then it was cooled to room temperature. The precipitated product was filtered off, washed with ethyl ether and recrystallized from 2-propanol. White crystals, (2.8 g, 70%), m.p. 170–171 °C; ν_max/cm⁻¹ 1603, 1676 (C=O), 3046 (OH) and 3247 (NH). ¹H-NMR (DMSO-d₆): δ_H 1.99 (6H, s, 2CH₃), 2.67–2.94 (2H, m, COCH₂), 3.39–3.51 (1H, m, CH), 3.88–4.15 (2H, m, NCH₂), 5.65 (2H, 2, 2CH), 6.53–7.28 (m, 4H, H_Ar), 9.51 (1H, s, OH), 10.92 (1H, s, NH). ¹³C-NMR (DMSO-d₆) δ_C 10.8 (2CH₃), 33.9 (CH), 35.7 (CH₂CO), 50.4 (CH₂N), 103.0, 106.7, 109.9, 111.3, 126.7, 129.4, 140.1, 157.5 (C_pyrrole, C_Ar), 171.4, 171.8 (2C=O). MS, m/z, (%) = 336 ([M+Na]⁺ 100); Anal. Calcd for C₁₇H₁₉N₃O₃ (313.36): C, 65.16; H, 6.11; N, 13.41. Found: C, 65.22; H, 6.06; N, 13.45.

4-[(3,5-Dimethyl-1H-pyrazol-1-yl)carbonyl]-1-(3-hydroxyphenyl)pyrrolidin-2-one (7). A mixture of hydrazide 3 (3.0 g, 0.013 mol), 2,4-pentanedione (4.0 g, 0.04 mol), 2-propanol (30 mL) and a catalytic amount of hydrochloric acid was refluxed for 6 h. The solvent was evaporated under vacuum to dryness, and the oily product was triturated with ethyl ether. The obtained solid was filtered off and washed with ethyl ether. The crude product was purified by recrystallization from 2-propanol. White crystals, (2.0 g, 53%), m.p. 165–166 °C; ν_max/cm⁻¹ 1601 (C=N), 1663, 1722, (C=O) and 3148 (OH). ¹H-NMR (DMSO-d₆): δ_H 2.21, (3H, s, CH₃), 2.48 (3H, s, CH₃), 2.76–2.94 (2H, m, COCH₂), 3.94–4.20 (2H, m, NCH₂), 4.39–4.51 (1H, m, CH), 6.23(1H, s, CH), 6.52–7.25 (4H, m, H_Ar), 9.49 (1H, s, OH). ¹³C-NMR (DMSO-d₆) δ_C 13.5, 13.9 (2CH₃), 35.2 (CH), 35.2 (CH₂CO), 50.1 (CH₂N), 106.7, 109.9, 111.2, 111.5, 129.3, 139.9, 143.8, 152.0, 157.4 (C_pyrazole, C_Ar), 171.4, 171.5 (C=O). MS, m/z, (%) = 322 ([M+Na]⁺ 100); Anal. Calcd for C₁₆H₁₇N₃O₃ (299.34): C, 64.20; H, 5.72; N, 14.04. Found: C, 64.31; H, 5.75; N, 14.08.

1-(3-Hydroxyphenyl)-4-[1,3,4]oxadiazol-2-yl-pyrrolidin-2-one (8). A mixture of compound 3 (7.0 g, 0.03 mol), triethyl orthoformate (35.6 g, 0.24 mol) and p-toluenesulfonic acid (1.14 g, 6 mmol) was refluxed for 8 h. After cooling, the precipitate was filtered off, washed with ethyl ether and purified by recrystallization from 2-propanol. White crystals, (3.32 g, 46%), m.p. 181–182 °C; ν_max/cm⁻¹ 1600 (C=N), 1679 (C=O) and 3184 (OH). ¹H-NMR (DMSO-d₆): δ_H 2.81–3.12 (2H, m, CH₂CO), 4.02–4.31 (3H, m, NCH2, CH), 6.52–7.27 (4H, m, H_Ar), 9.22 (1H, s, N=CH), 9.48 (1H, s, OH); ¹³C-NMR (DMSO-d₆) δ_C 27.5 (CH), 35.9 (CH₂CO), 50.6 (CH₂N), 106.9, 109.9, 111.4, 129.3, 139.8, 154.7, 157.4, 166.4 (C_oxadiazole, C_Ar), 170.9 (C=O); MS, m/z, (%) = 268 ([M+Na]⁺ 100); Anal. Calcd for C₁₂H₁₁N₃O₃ (245.24): C, 58.77; H, 4.52; N, 17.13. Found: C, 58.59; H, 4.41; N, 17.19.

A mixture of 2,3-dichloro-1,4-naphthoquinone 9 (0.88 g 3.9 mmol), the corresponding 1-(3-hydroxyphenyl)-5-oxopyrrolidine derivative 2, 4c, 6–8 (3.9 mmol), sodium carbonate (1.0 g) in dimethyl sulfoxide (15 mL) was stirred at room temperature for 24 h. The reaction mixture was diluted with water (70 mL), the precipitate was filtered off and washed with water.

Methyl 1-{3-[(3-chloro-1,4-dioxo-1,4-dihydro-2-naphthalenyl)oxy]phenyl}-5-oxo-3-pyrrolidinecarboxylate (10b). The crude product was purified by chromatography on a silica gel 60 column (2-propanone-hexane, 1:1), R_f 0.56. Yellow crystals, (1.0 g, 61%), m.p. 144–145 °C; ν_max/cm⁻¹ 1663, 1679, 1692 and 1736 (C=O). ¹H-NMR (DMSO-d₆): δ_H 2.66–2.86 (2H, m, COCH₂), 3.38–3.50 (1H, m, CH), 3.66 (3H, s, CH₃), 3.91–4.11 (2H, m, NCH₂), 6.92–8.15 (8H, m, H_Ar). ¹³C-NMR (DMSO-d₆) δ_C 34.8 (CH), 35.1 (CH₂CO), 49.7 (CH₂N), 52.1 (OCH₃), 107.3, 111.6, 114.3, 126.5, 126.6, 129.7, 130.5, 131.3, 133.5, 134.4, 134.6, 140.3, 152.3, 156.3 (C_Ar), 171.8, 172.9, 177.7, 178.1 (C=O). MS, m/z, (%) = 449 ([M+Na]⁺ 100), 451 ([M+Na+2]⁺ 35); Anal. Calcd for C₂₂H₁₆ClNO₆ (425.83): C, 62.05; H, 3.79; N, 3.29. Found: C, 62.10; H, 3.88; N, 3.18.

N'-[(4-Bromophenyl)methylidene]-1-{3-[(3-chloro-1,4-dioxo-1,4-dihydro-2-naphthalenyl)oxy]phenyl}-5-oxo-3-pyrrolidinecarbohydrazide (10c). The crude product was purified by chromatography on a silica gel 60 column (ethyl acetate–hexane, 1:1), R_f 0.63. Yellow crystals, (2.1 g, 90%), m.p. 185–186 °C; ν_max/cm⁻¹ 1594 (C=N), 1630, 1676, 1690, 1725 (C=O) and 3215 (NH). ¹H-NMR (DMSO-d₆): δ_H (E/Z, 40/60) 2.68–2.91 (2H, m, COCH₂), 3.30–3.39 (1H, m, CH), 3.93–4.17 (2H, m, NCH₂), 6.93–8.20 (13H, m, N=CH, H_Ar), 11.65, 11.71 (1H, 2s, NH). ¹³C-NMR (DMSO-d₆) δ_C 32.7, 34.7, 35.0, 35.7 (CH, CH₂CO), 49.9, 50.4 (CH₂N), 107.2, 107.4, 111.5, 114.2, 114.3, 123.0, 123.3, 126.4, 126,.6, 128.7, 128.8, 129.7, 130.5, 131.2, 131.7, 133.3, 133.4, 134.4, 134.5, 140.4, 142.4, 145.8, 152.3, 156.3, 168.6 (C_Ar), 172.1, 172.3, 173.4, 177.7, 178.0 (C=O). Anal. Calcd for C₂₈H₁₉BrClN₃O₅ (592.82): C, 56.73; H, 3.23; N, 7.09. Found: C, 56.85; H, 3.36; N, 6.98.

2-Chloro-3-{3-[4-(3,5-dimethylpyrazole-1-carbonyl)-2-oxopyrrolidin-1-yl]phenoxy}-[1,4]naphthoquinone (10d). The crude product was recrystallized from ethanol. Yellow crystals, (1.4 g, 72%), m.p. 130–131 °C; ν_max/cm⁻¹ 1572 (C=N), 1667, 1673, 1698 and 1735 (C=O). ¹H-NMR (DMSO-d₆): δ_H 2.19 (3H, s, CH₃), 2.47 (3H, s, CH₃), 2.77–2.96 (2H, m, COCH₂), 3.97–4.24 (2H, m, NCH₂), 4.40–4.52 (1H, m, CH), 6.21, 6.22 (1H, 2s, C=CH), 6.94–8.15 (8H, m, H_Ar). ¹³C-NMR (DMSO-d₆) δ_C 13.5, 14.0 (2CH₃), 35.2 (CH), 35.5 (CH₂CO), 50.2 (CH₂N), 107.4, 111.6, 114.4, 126.5, 126.6, 129.7, 130.5, 131.3, 133.4, 134.4, 134.6, 140.3, 143.8, 152.1, 152.3, 156.3 (C_pyrazole, C_Ar), 171.8, 172.5, 177.7, 178.1 (C=O). MS, m/z, (%) = 513 ([M+Na]⁺ 100); 515 ([M+Na+2]⁺ 35) Anal. Calcd for C₂₆H₂₀ClN₃O₅ (489.92): C, 63.74; H, 4.11; N, 8.58. Found: C, 63.65; H, 3.98; N, 8.49.

1-{3-[(3-Chloro-1,4-dioxo-1,4-dihydro-2-naphthalenyl)oxy]phenyl}-N-(2,5-dimethyl-1H-pyrrol-1-yl)-5-oxo-3-pyrrolidinecarboxamide (10e). The crude product was recrystallized from ethanol. Yellow crystals, (1.8 g, 90%), m.p. 181–182 °C; ν_max/cm⁻¹ 1578 (C=N), 1650, 1676, 1696, 1710 (C=O) and 3313 (NH). ¹H-NMR (DMSO-d₆): δ_H 1.97 (3H, s, CH₃), 1.99 (3H, s, CH₃), 2.68–2.96 (2H, m, COCH₂), 3.40–3.52 (1H, m, CH), 3.94–4.17 (2H, m, NCH₂), 5.64 (2H, s, 2CH), 6.94–8.16 (8H, m, H_Ar), 10.88 (1H, s, NH); ¹³C-NMR (DMSO-d₆) δ_C 10.8 (2CH₃), 33.8 (CH), 35.7 (CH₂CO), 50.2 (CH₂N), 102.9, 107.4, 111.6, 114.2, 126.4, 126.6, 129.7, 130.5, 131.2, 133.3, 134.4, 134.5, 140.3, 152.3, 156.2 (C_pyrrole, C_Ar), 171.6, 171.7, 177.6, 177.9 (C=O). MS, m/z, (%) = 527 ([M+Na]⁺ 100); 529 ([M+Na+2]⁺ 35)Anal. Calcd for C₂₇H₂₂ClN₃O₅ (503.95): C, 64.35; H, 4.40; N, 8.34. Found: C, 64.22; H, 4.39; N, 8.40.

2-Chloro-3-[3-(4-[1,3,4]oxadiazol-2-yl-2-oxopyrrolidin-1-yl)phenoxy]-[1,4]naphthoquinone (10f). The crude product was purified by chromatography on a silica gel 60 column (2-propanone-hexane, 1:1), R_f 0.29. Yellow crystals, (1.4 g, 82%), m.p. 195–196 °C; ν_max/cm⁻¹ 1576, 1597 (C=N), 1678, 1698 and 1743 (C=O). ¹H-NMR (DMSO-d₆): δ_H 2.84–3.12 (2H, m, COCH₂), 4.06–4.34 (3H, m, CH, NCH₂), 6.96–8.15 (8H, m, H_Ar), 9.23 (1H, s, N=CH). ¹³C-NMR (DMSO-d₆) δ_C 27.5 (CH), 36.0 (CH₂CO), 50.6 (CH₂N), 107.4, 111.7, 114.5, 126.5, 126.6, 129.8, 130.5, 131.3, 133.5, 134.4, 134.6, 140.2, 152.3, 154.9, 156.3, 166.3 (C_Ar, C_oxadiazole), 171.4, 177.7, 178.1 (C=O). MS, m/z, (%) = 459 ([M+Na]⁺ 100); 461 ([M+Na+2]⁺ 35) Anal. Calcd for C₂₂H₁₄ClN₃O₅ (435.83): C, 60.63; H, 3.24; N, 9.64. Found: C, 60.51; H, 3.31; N, 9.69.

A mixture of 2,3-dichloro-1,4-naphthoquinone 9 (3.3 mmol, 0.75 g), the corresponding 1-(3-hydroxyphenyl)-5-oxopyrrolidine derivative 1, 2, 5–7, 8c (6.6 mmol), sodium carbonate (2.0 g) and dimethyl sulfoxide (15 mL) was stirred at room temperature for 24 h. The reaction mixture was diluted with water (70 mL), the precipitate was filtered off and washed with water.

1-[3-({3-[3-(4-Carboxy-2-oxo-1-pyrrolidinyl)phenoxy]-1,4-dioxo-1,4-dihydro-2-naphthalenyl}oxy)phenyl]-5-oxo-3-pyrrolidinecarboxylic acid (11a). The crude product was purified by dissolving them in sodium hydroxide solution (5%), filtering the solution, and acidifying the filtrate with acetic acid up to pH 6. Orange crystals, (1.4 g, 72%), m.p. 170–171 °C; ν_max/cm⁻¹ 1671, 1700, 1730, (C=O) and 3072 (OH). ¹H-NMR (DMSO-d₆): δ_H 2.58–2.76 (4H, m, 2COCH₂), 3.10–3.23 (2H, m, 2CH), 3.80–3.97 (4H, m, 2NCH₂), 6.84–8.07 (12H, m, H_Ar). ¹³C-NMR (DMSO-d₆) δ_C 35.7 (CH), 35.9 (CH₂CO), 50.7 (CH₂N), 107.1, 107.3, 111.7, 113.9, 126.0, 129.3, 130.8, 134.3, 140.2, 145.3, 145.4, 156.4, 156.4 (C_Ar), 172.6, 174.8, 179.9 (C=O). MS, m/z, (%) = 619 ([M+Na]⁺ 100); Anal. Calcd for C₃₂H₂₄N₂O₁₀ (596.56): C, 64.43; H, 4.06; N, 4.70. Found: C, 64.39; H, 3.98; N, 4.66.

Methyl 1-{3-[(3-{3-[4-(methoxycarbonyl)-2-oxo-1-pyrrolidinyl]phenoxy}-1,4-dioxo-1,4-dihydro-2-naphthalenyl)oxy]phenyl}-5-oxo-3-pyrrolidinecarboxylate (11b). The crude product was chromatographed over a silica gel 60 column (2-propanone-hexane, 1:1), R_f 0.29. Orange crystals, (0.9 g, 45%), m.p. 90–91 °C; ν_max/cm⁻¹ 1676, 1703 and 1737 (C=O). ¹H-NMR (DMSO-d₆): δ_H 2.64–2.83 (4H, m, 2COCH₂), 3.35–3.48 (2H, m, 2CH), 3.66 (6H, s, 2CH₃), 3.84–4.04 (4H, m, 2NCH₂), 6.88–8.07 (12H, m, H_Ar). ¹³C-NMR (DMSO-d₆) δ_C 34.7 (2CH), 34.9 (2CH₂CO), 49.7 (2CH₂N), 52.1 (2OCH₃), 107.3, 111.8, 113.9, 126.0, 129.4, 130.8, 134.3, 139.9, 145.6, 156.6 (C_Ar), 171.6, 172.9, 179.9 (C=O). MS, m/z, (%) = 647 ([M+Na]⁺ 100); Anal. Calcd for C₃₄H₂₈N₂O₁₀ (624.61): C, 65.38; H, 4.52; N, 4.48. Found: C, 65.44; H, 4.49; N, 4.52.

N'-[(4-Bromophenyl)methylidene]-1-{3-[(3-{3-[4-({2-[(4-bromophenyl)methylidene]hydrazino}carbonyl)-2-oxo-1-pyrrolidinyl]phenoxy}-1,4-dioxo-1,4-dihydro-2-naphthalenyl)oxy]phenyl}-5-oxo-3-pyrrolid-inecarbohydrazide (11c). The crude product was chromatographed over a silica gel 60 column (ethyl acetate–methanol, 11:1), R_f 0.58. Yellow crystals, (1.2 g, 38%), m.p. 189–190 °C; ν_max/cm⁻¹ 1591 (C=N), 1678, 1693, 1731 (CO) and 3209 (NH). ¹H-NMR (DMSO-d₆): δ_H (E/Z, 40/60) 2.69–2.83 (4H, m, 2COCH₂), 3.23–3.39 (2H, m, 2CH), 3.84–4.11 (4H, m, 2NCH₂), 6.86–8.19 (22H, m, 2N=CH, H_Ar), 11.63, 11.69 (2H, 2s, 2NH). ¹³C-NMR (DMSO-d₆) δ_C 32.6, 34.6, 35.0, 35.7, (2CH, 2CH₂CO), 49.9, 50.4 (2CH₂N), 107.3, 111.6, 114.0, 123.0, 123.2, 126.0, 128.7, 128.8, 129.3, 130.8, 131.7, 133.3, 134.3, 140.1, 142.4, 145.7, 156.6 (2N=CH, C_Ar), 172.1, 173.4, 179.9 (C=O). Anal. Calcd for C₄₆H₃₄Br₂N₆O₈ (958.63): C, 57.64; H, 3.58; N, 8.77. Found: C, 57.51; H, 3.39; N, 8.69.

2,3-Bis(3-{4-[(3,5-dimethyl-1H-pyrazol-1-yl)carbonyl]-2-oxo-1-pyrrolidinyl}phenoxy)naphthoquinone (11d). The crude product was chromatographed over a silica gel 60 column (2-propanone-hexane, 1:1), R_f 0.72. Yellow crystals, (1.1 g, 43%), m.p. 136–137 °C; ν_max/cm⁻¹ 1587 (C=N), 1677, 1706 and 1723 (C=O). ¹H-NMR (DMSO-d₆): δ_H 2.19, (6H, s, 2CH₃), 2.46 (6H, s, 2CH₃), 2.75–2.92 (4H, m, 2COCH₂), 3.89–4.16 (4H, m, 2NCH₂), 4.37–4.49 (2H, m, 2CH), 6.22 (2H, s, 2C=CH), 6.88–8.05 (12H, m, H_Ar). ¹³C-NMR (DMSO-d₆) δ_C 13.2, 13.7 (CH₃), 34.8 (CH), 34.9 (CH₂CO), 49.8 (CH₂N), 107.2, 111.3, 111.5, 113.9, 125.8, 129.1, 130.5, 134.1, 139.7, 143.6, 145.3, 151.8, 156.3, (C_pyrrazole, C_Ar), 171.4, 172.2, 179.7 (C=O). MS, m/z, (%) = 775 ([M+Na]⁺ 100); Anal. Calcd for C₄₂H₃₆N₆O₈ (752.79): C, 67.01; H, 4.82; N, 11.16. Found: C, 67.19; H, 4.91; N, 11.20.

N-(2,5-Dimethyl-1H-pyrrol-1-yl)-1-[3-({3-[3-(4-{[(2,5-dimethyl-1H-pyrrol-1-yl)amino]carbonyl}-2-oxo-1-pyrrolidinyl)phenoxy]-1,4-dioxo-1,4-dihydro-2-naphthalenyl}oxy)phenyl]-5-oxo-3-pyrrolid-inecarboxamide (11e). The crude product was chromatographed over a silica gel 60 column (2-propanone-hexane, 1:1), R_f 0.33. Orange crystals, (0.6 g, 23%), m.p. 193–194 °C; ν_max/cm⁻¹ 1649, 1676, 1693, 1715 (C=O) and 3252 (NH). ¹H-NMR (DMSO-d₆): δ_H 1.97 (6H, s, 2CH₃), 1.98 (6H, s, 2CH₃), 2.65–2.93 (4H, m, 2COCH₂), 3.37–3.49 (2H, m, 2CH), 3.87–4.11 (4H, m, 2NCH₂), 5.64 (4H, s, 4CH), 6.90–8.07 (12H, m, H_Ar), 10.90 (2H, s, 2NH). ¹³C-NMR (DMSO-d₆) δ_C 10.6 (4CH₃), 33.6 (2CH), 35.5 (2CH₂CO), 49.9 (2CH₂N), 102.7, 107.1, 111.4, 113.7, 125.8, 126.4, 129.2, 130.5, 134.1, 139.8, 145.4, 156.4 (C_pyrrole, C_Ar), 171.4, 179.6 (C=O). MS, m/z, (%) = 803 ([M+Na]⁺ 100); Anal. Calcd for C₄₄H₄₀N₆O₈ (780.84): C, 67.68; H, 5.16; N, 10.76. Found: C, 67.59; H, 5.21; N, 10.79.

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 (11f). The crude product was chromatographed over a silica gel 60 column (2-propanone-hexane, 5:1), R_f 0.23. Orange crystals, (0.52 g, 49%), m.p. 150–151 °C; ν_max/cm⁻¹ 1578, 1604 (C=N), 1677 and 1705 (C=O). ¹H-NMR (DMSO-d₆): δ_H 2.82–3.08 (4H, m, 2COCH₂), 3.99–4.26 (6H, m, CH, NCH₂), 6.90–8.07 (12H, m, H_Ar), 9.21 (2H, s, 2N=CH). ¹³C-NMR (DMSO-d₆) δ_C 27.4 (2CH), 35.9 (2CH₂CO), 50.5 (2CH₂N), 107.4, 111.9, 114.2, 126.0, 129.4, 130.8, 134.3, 139.9, 145.6, 154.8, 156.6, 166.2 (C_Ar, C_oxadiazole), 171.2, 179.9 (C=O). MS, m/z, (%) = 667 ([M+Na]⁺ 100); Anal. Calcd for C₃₄H₂₄N₆O₈ (644.41): C, 63.35; H, 3.75; N, 13.04. Found: C, 63.22; H, 3.80; N, 12.97.

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 − 10⁹ 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 °C and the one of fungi incubation was 48–72 h at 28–30 °C. 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 10⁶ cells (spores)/1 mL). The duration of bacteria incubation was 24 h at 35 °C and the one of fungi incubation was 48–72 h at 28–30 °C. 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 °C and the one of fungi incubation was 48–72 h at 28–30 °C. 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.