Synthesis and microbiological evaluation of new 2- and 2,3- diphenoxysubstituted naphthalene-1,4-diones with 5-oxopyrrolidine moieties

Article abstract of DOI:10.3390/molecules171214434

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 bee

Full text of DOI:10.3390/molecules171214434

Molecules 2012, 17, 14434-14448; doi:10.3390/molecules171214434
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Microbiological Evaluation of New 2- and
2,3-Diphenoxysubstituted Naphthalene-1,4-diones with
5-Oxopyrrolidine Moieties
Aušra Voskienė 1, Birutė Sapijanskaitė 1, Vytautas Mickevičius 1, Ilona Jonuškienė 1,*,
Maryna Stasevych 2, Olena Komarovska-Porokhnyavets 2, Rostyslav Musyanovych 2 and
Volodymyr Novikov 2
1
Department of Organic Chemistry, Kaunas University of Technology, Radvilėnų pl. 19,
LT-50254 Kaunas, Lithuania; E-Mails: ausra.voskiene@ktu.lt (A.V.);
birute.sapijanskaite@ktu.lt (B.S.); vytautas.mickevicius@ktu.lt (V.M.)
2
Department of Technology of Biologically Active Substances, Pharmacy Biotechnology,
National University “Lviv Politechnic” Bandera str. 12, 79013, Lviv-13, Ukraine;
E-Mails: maryna_s@inbox.ru (M.S.); olkomarovska@gmail.com (O.K.-P.);
rostykm71@ukr.net (R.M.); vnovikov@polynet.lviv.ua (V.N.)
* Author to whom correspondence should be addressed; E-Mail: ilona.jonuskiene@ktu.lt;
Tel.: +37-061-643-642; Fax: +37-037-300-152.
Received: 9 October 2012; in revised form: 26 November 2012 / Accepted: 3 December 2012 /
Published: 5 December 2012
Abstract: 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

Molecules 2012, 17
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luteum. Besides, introduction of the second phenoxy substituted fragment increased the
antifungal activity against Candida tenuis and Aspergillus niger at lower concentrations.
Keywords: 3-substituted 1-(3-hydroxyphenyl)-5-oxopyrrolidine derivatives; 1,4-naphtho-
quinone; antibacterial activity; antifungal activity
1. Introduction
Quinones are important compounds widely spread in Nature. Compounds containing
1,4-naphthoquinone moiety exhibit a broad spectrum of biological activities such as cytotoxic [1,2]
antiviral [3,4] anti-inflammatory [5,6] antimalarial [7], antibacterial [8,9], antifungal [10] and
antiproliferative properties [11]. Naphthoquinones are particularly important in dye chemistry, and
recently they have been used for new infrared dyes in optical recording media [12–14]. The aim of this
research was to synthesize some new 2,3-disubstituted 1,4-naphthoquinone derivatives and to screen
their antibacterial and antifungal activity.
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 IC50 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-1,4-
naphthoquinone 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.

Molecules 2012, 17
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2. Results and Discussion
2.1. 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-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
1
heterocyclic systems in compounds 6 and 7 has been confirmed by the characteristic 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.
Scheme 1. Synthesis of 3-substituted 1-(3-hydroxyphenyl)-5-oxopyrrolidines.
HO
O
N
CH3
CH3
H
HO
O
N
N
N
CH3
H
N
5
O
N
6
O
H3C
CH3COCH2CH2COCH3
CH3COCH3
HO
OH
O
OH
O
HO
CH3
CH3
O
N
CH3OH, H+
NH2NH2
CH3COCH2COCH3
N
N
N
N
O
H
N
N
NH2
3
O
O
O
O
7
O
CH3
OH
HC(OC2H5)3
1
2
RCHO
HO
O
N
HO
O
N
O
R = a) C6H5; b) 4-OCH3-C6H4;
c) 4-Br-C6H4.
H
N
8
N
N
N
R
O
4a-c
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

Molecules 2012, 17
14437
nitrogen atom in the azomethine group affects the neighbouring atoms and causes formation of
geometrical isomers (cis/trans) [19–21]. 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.
Scheme 2. Synthesis of phenoxy-substituted 1,4-naphthoquinone derivatives.
O
O
O
N
Cl
A
O
R1
10b-f
O
HO
O
R1
Cl
Cl
O
+
N
N
R1
B
O
O
O
O
9
1, 2, 4c, 6-8
O
HO
HO
O
O
N
A. 1 equiv.
B. 2 equiv.
, DMSO, Na2CO3
11a-f
N
R1
O
R1
N
, DMSO, Na2CO3
R1
a
f
c
e
b
d
CH3
H3C
O
Br
O
O
N
O
O
N
O
1
N
R
N
N
N
OH
O
CH3
N
H
N
H
H3C
CH3

Molecules 2012, 17
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1
The structures of all synthesized compounds were confirmed by mass spectrometry, IR, H-, and
1
13C-NMR spectroscopy data. A comparison of the H-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.
2.2. 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 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 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.
Table 1. Bactericidal and fungicidal activities of 10b–f and 11b–f determined by
diffusion method.
Inhibition diameter of microorganism growth, mm
Concentration,
Compound
%
S. aureus
M. luteum
C. tenuis
A. niger
15.0
14.0
7.0
0.5
0.1
0.5
0.5
0.1
0.5
0.1
0.5
0.5
0.5
0.5
0.1
8.0
7.0
0
11.4
8.7
0
15.0
13.0
0
10b
10c
10f
8.0
6.0
0
0
14.4
10.0
0
17.4
14.4
6.0
0
12.0
7.0
0
11b
0
0
0
11c
11d
11e
11f
C *
0
0
7.0
0
0
0
7.0
0
0
0
7.0
0
0
0
9.0
15.0
18.0
19.0
20.0
* Vancomycin was used as a control in the tests of antibacterial activity of the synthesized compounds and
Nystatin was used in the tests of antifungal action.

Molecules 2012, 17
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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.
Table 2. Bactericidal and fungicidal activity of 10b–f and 11b–f determined by the serial
dilution method.
MIC, μg/mL
Compound
M. luteum
C. tenuis
62.5
1.9
A. niger
10b
10c
10d
10e
10f
11b
11c
11d
11e
11f
62.5
7.8
3.9
+
+
250.0
+
+
+
+
+
31.2
31.2
250
62.5
15.6
3.9
62.5
+
+
+
+
+
+
+
+
125.0
7.8
+ – growth of microorganisms.
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:
10b>10f>10c≈10d≈10e≈11b≈11c≈11d≈11e≈11f
Increase of antibacterial activity

Molecules 2012, 17
For A. niger:
14440
10f≈10b>11f>10c≈11c≈11d≈11e>11b≈10d≈10e
Increase of antifungal activity
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.
3. Experimental
3.1. General
1
The H- 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 , ppm). IR spectra (, 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; νmax/cm−1 1660 and 1740 (C=O) and 3159
(OH). 1H-NMR (DMSO-d6): δH 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, HAr), 9.50 (1H, s, OH). 13C-NMR (DMSO-d6) δC 34.8 (CH),
35.1 (CH2CO), 49.8 (CH2N), 52.1 (CH3), 106.7, 109.8, 111.3, 129.4, 140.0, 157.5 (CAr), 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; νmax/cm−1 1697 and 1660
(C=O), 3131 (OH), 3314 and 3253 (NH, NH2). 1H-NMR (DMSO-d6): δH 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, HAr), 9.27
(1H, s, NH), 9.48 (1H, s, OH). 13C-NMR (DMSO-d6) δC 33.9 (CH), 35.8 (CH2CO), 50.7 (CH2N),

Molecules 2012, 17
14441
106.6, 109.7, 111.2, 129.4, 140.2, 157.5 (CAr), 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.
3.2. General Synthetic Procedure for the Synthesis of Hydrazones 4a–c
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; νmax/ cm−1 1590 (C=N), 1664 and 1693 (C=O), 3225 (OH) and 3334
1
(NH). H-NMR (DMSO-d6): δH (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, HAr), 8.04, 8.22 (1H, 2s, NCH), 9.47 (1H, s, OH), 11.57,
11.63 (1H, 2s, NH). 13C-NMR (DMSO-d6) δC 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, (CAr, 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); νmax/cm−1 1600 (C=N), 1609 and
1
1661, (C=O), 3088 (OH) and 3231 (NH). H-NMR (DMSO-d6): δH (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, HAr), 7.98, 8.16 (1H,
13
2s, NCH), 9.47 (1H, s, OH), 11.44, 11.50 (1H, 2s, NH). C-NMR (DMSO-d6) δC 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 (CAr, 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); νmax/cm−1 1599 (C=N), 1663 and 1698
(C=O), 2973 (OH) and 3172 (NH). 1H-NMR (DMSO-d6): δH (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, HAr), 8.01, 8.19 (1H, 2s, NCH),
9.47 (1H, s, OH), 11.63, 11.71 (1H, 2s, NH). 13C-NMR (DMSO-d6) δC 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 (CAr), 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

Molecules 2012, 17
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ν
max/cm−1 1597 (C=N), 1675, 1659 (C=O), 3180 (OH) and 3235 (NH). 1H-NMR (DMSO-d6): δH (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, HAr), 9.46 (1H, s, OH), 10.21, 10.30 (1H, 2s, NH).
13C-NMR (DMSO-d6) δC 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 (CAr),
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, .4 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; νmax/cm−1 1603,
1676 (C=O), 3046 (OH) and 3247 (NH). 1H-NMR (DMSO-d6): δH 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,
13
HAr), 9.51 (1H, s, OH), 10.92 (1H, s, NH). C-NMR (DMSO-d6) δC 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 (Cpyrrole, CAr), 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, .4 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; νmax/cm−1 1601 (C=N), 1663, 1722, (C=O) and 3148 (OH).
1H-NMR (DMSO-d6): δH 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, HAr), 9.49 (1H, s, OH).
13C-NMR (DMSO-d6) δC 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 (Cpyrazole, CAr), 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; νmax/cm−1 1600
1
(C=N), 1679 (C=O) and 3184 (OH). H-NMR (DMSO-d6): δH 2.81–3.12 (2H, m, CH2CO), 4.02–4.31
(3H, m, NCH2, CH), 6.52–7.27 (4H, m, HAr), 9.22 (1H, s, N=CH), 9.48 (1H, s, OH); 13C-NMR
(DMSO-d6) δC 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 (Coxadiazole, CAr), 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.

Molecules 2012, 17
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3.3. General Synthetic Procedure for the Synthesis of Compounds 10b–f
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), Rf 0.56. Yellow crystals, (1.0 g, 61%), m.p. 144–145 C; νmax/cm−1 1663, 1679, 1692 and
1736 (C=O). 1H-NMR (DMSO-d6): δH 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, HAr). 13C-NMR (DMSO-d6) δC 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 (CAr), 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), Rf 0.63. Yellow crystals, (2.1 g, 90%), m.p. 185–186 C;
νmax/cm−1 1594 (C=N), 1630, 1676, 1690, 1725 (C=O) and 3215 (NH). 1H-NMR (DMSO-d6): δH (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, HAr), 11.65, 11.71 (1H, 2s, NH). 13C-NMR (DMSO-d6) δC 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
(CAr), 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): δH 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,
13
6.22 (1H, 2s, C=CH), 6.94–8.15 (8H, m, HAr). C-NMR (DMSO-d6) δC 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 (Cpyrazole, CAr), 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; νmax/cm−1 1578 (C=N), 1650, 1676, 1696, 1710 (C=O) and
1
3313 (NH). H-NMR (DMSO-d6): δH 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,

Molecules 2012, 17
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HAr), 10.88 (1H, s, NH); 13C-NMR (DMSO-d6) δC 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 (Cpyrrole, CAr), 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), Rf 0.29. Yellow crystals, (1.4 g, 82%), m.p. 195–196 C; νmax/cm−1 1576, 1597 (C=N), 1678,
1
1698 and 1743 (C=O). H-NMR (DMSO-d6): δH 2.84–3.12 (2H, m, COCH2), 4.06–4.34 (3H, m, CH,
NCH2), 6.96–8.15 (8H, m, HAr), 9.23 (1H, s, N=CH). 13C-NMR (DMSO-d6) δC 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 (CAr, Coxadiazole), 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.
3.4. General Synthetic Procedure for the Synthesis of Compounds 11a–f
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; νmax/cm−1 1671, 1700, 1730, (C=O) and 3072
1
(OH). H-NMR (DMSO-d6): δH 2.58–2.76 (4H, m, 2COCH2), 3.10–3.23 (2H, m, 2CH), 3.80–3.97
13
(4H, m, 2NCH2), 6.84–8.07 (12H, m, HAr). C-NMR (DMSO-d6) δC 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
(CAr), 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), Rf 0.29. Orange crystals, (0.9 g,
45%), m.p. 90–91 C; νmax/cm−1 1676, 1703 and 1737 (C=O). 1H-NMR (DMSO-d6): δH 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
13
(12H, m, HAr). C-NMR (DMSO-d6) δC 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 (CAr), 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.

Molecules 2012, 17
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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), Rf 0.58. Yellow crystals, (1.2 g, 38%), m.p. 189–190 C; νmax/cm−1 1591
(C=N), 1678, 1693, 1731 (CO) and 3209 (NH). 1H-NMR (DMSO-d6): δH (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, HAr),
13
11.63, 11.69 (2H, 2s, 2NH). C-NMR (DMSO-d6) δC 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, CAr), 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),
Rf 0.72. Yellow crystals, (1.1 g, 43%), m.p. 136–137 C; νmax/cm−1 1587 (C=N), 1677, 1706 and 1723
1
(C=O). H-NMR (DMSO-d6): δH 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
13
(12H, m, HAr). C-NMR (DMSO-d6) δC 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, (Cpyrrazole
,
CAr), 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), Rf 0.33. Orange crystals, (0.6 g, 23%), m.p. 193–194 C; νmax/cm−1 1649,
1
1676, 1693, 1715 (C=O) and 3252 (NH). H-NMR (DMSO-d6): δH 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, HAr), 10.90 (2H, s, 2NH). 13C-NMR (DMSO-d6) δC 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 (Cpyrrole, CAr), 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), Rf 0.23. Orange crystals, (0.52 g, 49%), m.p. 150–151 C;
1
νmax/cm−1 1578, 1604 (C=N), 1677 and 1705 (C=O). H-NMR (DMSO-d6): δH 2.82–3.08 (4H, m,
13
2COCH2), 3.99–4.26 (6H, m, CH, NCH2), 6.90–8.07 (12H, m, HAr), 9.21 (2H, s, 2N=CH). C-NMR
(DMSO-d6) δC 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 (CAr, Coxadiazole), 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.

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3.5. 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 − 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 °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 106 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.
4. 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.
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 the diffusion and
serial dilution methods. 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

Molecules 2012, 17
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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.
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© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).