Synthesis and antibacterial and antifungal properties of novel S-, N-, N,S-, and S,O-substituted 1,4-naphthoquinone derivatives

Article abstract of DOI:10.1080/10426507.2012.727515

A novel series of substituted 1,4-naphthoquinone derivatives were synthesized and evaluated for their antibacterial and antifungal activity. The structures of the novel products were characterized by spectroscopic methods. Among the tested compounds, 2,2′

Full text of DOI:10.1080/10426507.2012.727515

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Synthesis and Antibacterial and
Antifungal Properties of Novel S-,
N-, N,S-, and S,O-Substituted 1,4-
Naphthoquinone Derivatives
Cemil Ibis a , Amac Fatih Tuyun b , Zeliha Ozsoy-Gunes c , Sibel
Sahinler Ayla a , Maryna V. Stasevych d , Rostyslav Ya. Musyanovych d
, Olena Komarovska-Porokhnyavets d & Volodymyr Novikov d
a Department of Chemistry , Engineering Faculty, Istanbul
University , Istanbul , Turkey
b Department of Chemical Engineering , Engineering & Architecture
Faculty, Beykent University , Istanbul , Turkey
c Hasan Ali Yucel Education Faculty , Department of Elementary
Education, Division of Science Education, Istanbul University ,
Istanbul , Turkey
d Department of Technology of Biologically Active Substances ,
Pharmacy and Biotechnology, National University “Lviv Polytechnic”
, Lviv , Ukraine
Accepted author version posted online: 12 Sep 2012.Published
online: 05 Jul 2013.
To cite this article: Cemil Ibis , Amac Fatih Tuyun , Zeliha Ozsoy-Gunes , Sibel Sahinler Ayla , Maryna
V. Stasevych , Rostyslav Ya. Musyanovych , Olena Komarovska-Porokhnyavets & Volodymyr Novikov
(2013) Synthesis and Antibacterial and Antifungal Properties of Novel S-, N-, N,S-, and S,O-Substituted
1,4-Naphthoquinone Derivatives, Phosphorus, Sulfur, and Silicon and the Related Elements, 188:7,
955-966, DOI: 10.1080/10426507.2012.727515
To link to this article: http://dx.doi.org/10.1080/10426507.2012.727515
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Phosphorus, Sulfur, and Silicon, 188:955–966, 2013
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426507.2012.727515
SYNTHESIS AND ANTIBACTERIAL AND ANTIFUNGAL
PROPERTIES OF NOVEL S-, N-, N,S-, AND
S,O-SUBSTITUTED 1,4-NAPHTHOQUINONE
DERIVATIVES
Cemil Ibis,1 Amac Fatih Tuyun,2 Zeliha Ozsoy-Gunes,3
Sibel Sahinler Ayla,1 Maryna V. Stasevych,4
Rostyslav Ya. Musyanovych,4 Olena
Komarovska-Porokhnyavets,4 and Volodymyr Novikov4
1Department of Chemistry, Engineering Faculty, Istanbul University, Istanbul,
Turkey
2Department of Chemical Engineering, Engineering & Architecture Faculty,
Beykent University, Istanbul, Turkey
3Hasan Ali Yucel Education Faculty, Department of Elementary Education,
Division of Science Education, Istanbul University, Istanbul, Turkey
4Department of Technology of Biologically Active Substances, Pharmacy and
Biotechnology, National University “Lviv Polytechnic”, Lviv, Ukraine
GRAPHICAL ABSTRACT
Abstract A novel series of substituted 1,4-naphthoquinone derivatives were synthesized and
evaluated for their antibacterial and antifungal activity. The structures of the novel products
were characterized by spectroscopic methods. Among the tested compounds, 2,2ꢁ,3,3ꢁ-alkoxy
Received 11 August 2012; accepted 27 August 2012.
Financial support from TUBITAK for the Ukraine-Turkey agreement (Project No. 109T617) and from the
State Agency on Science, Innovations and Informatization of the Ukraine (Project No. M/309-2011) is gratefully
acknowledged.
Address correspondence to Cemil Ibis, Department of Chemistry, Engineering Faculty, Istanbul University,
Istanbul, Turkey. E-mail: ibiscml@istanbul.edu.tr
955

956
C. IBIS ET AL.
substituted naphthoquinones, S,O-substituted naphthoquinone, and N,S-substituted naphtho-
quinone derivatives are the most potent antifungals against C. tenuis. 2,3-Thio-2ꢁ,3ꢁ-alkoxy
substituted naphthoquinones are the most effective antifungal compounds against A. niger.
Supplemental materials are available for this article. Go to the publisher’s online edition of
Phosphorus, Sulfur, and Silicon and the Related Elements to view the free supplemental file.
Keywords N,S-substituted 1,4-naphthoquinones; heterocyclic ethers with quinone group;
antibacterial activity; antifungal activity
INTRODUCTION
The 1,4-naphthoquinone moiety represents an important structural motive with
quinonoid structure which exists in many natural or synthetic intermediates from dyes
to pharmaceuticals. 1,4-Naphthoquinone derivatives show important biological properties
such as antibacterial, antifungal, antiviral, anticancer, and anti-inflammatory effects.1–5
Thio or amino derivatives of 1,4-naphthoquinones have been studied widely because of
these important biological properties.6–8 The biological activity of 1,4-naphthoquinone is
due to the presence of two conjugated carbonyl groups which have the ability to accept one
or two electrons to form the corresponding radical anion or dianion species.9 The impor-
tant activity properties of piperazine and homopiperazine analogs are also known from the
literature.10
The synthesis of novel nitrogen and sulfur containing naphthoquinone compounds as
biologically active agents and their spectroscopic characterization is the aim of this study.
RESULTS AND DISCCUSSION
The novel compounds 3 and 4 were obtained by the reaction of 2,3-dichloro-1,4-
naphthoquinone 1 and diol 2 with potassium carbonate in acetonitrile. Compound 3 is a
novel intramolecular compound with ether structure (Scheme 1). This reaction consists of a
substitution with concomitant Michael-Type addition followed by chloride elimination.8,11
The novel compound 4 is also an interesting heterocyclic compound that is formed by the
same reaction type as 3. Two carbon atom signals of the carbonyl groups were observed at
179.79 and 178.55 ppm in the 13C NMR spectra of 3. However, the carbon atom signals of
the two carbonyl groups of 4 were observed at 177.74 ppm as one peak only.
The novel compounds 6, 7, and 8 were synthesized by the reaction of 2,3-dichloro-1,4-
naphthoquinone 1 and thioalcohol 5 in ethanol solution of sodium carbonate. The IR spectra
of the monothio-substituted naphthoquinone 7 and the dithio-substituted naphthoquinone
8 showed broad bands at 3512 and 3390 cm−1 for the OH stretching, respectively, but
compound 6 did not show any band at this region of the IR spectrum because of the ring
closure.
The N-substituted naphthoquinone 10 was obtained by the reaction of 2,3-dichloro-
1,4-naphthoquinone 1 and compound 9 with sodium carbonate in dichloromethane. The
molecular ion peak of 10 was identified at m/z 334 [M]+ in the positive ion mode for
ESI technique. The dithio-substituted compound 12 and the ethoxy-monothio-substituted
compound 13 were obtained by the reaction of 2,3-dichloro-1,4-naphthoquinone 1 and
compound 11 with sodium carbonate in ethanol. The molecular ion peak of compounds
12 and 13 was identified at m/z 487 [M]+ and 367 [M]+ in the positive ion mode for ESI

ANTIBACTERIAL AND ANTIFUNGAL PROPERTIES
957
Scheme 1
1
technique, respectively. In the H NMR spectrum of 13, the methylene protons (OCH2)
were observed at 4.19 ppm as a multiplet.
The dithio-substituted compounds 16, 17, and 1812 were obtained by the reaction
of 2,3-dichloro-1,4-naphthoquinone 1 and a mixture of 14 and 15 with triethylamine in
chloroform (Scheme 2). No band was observed in OH region of the IR spectra of compound
16. However, compounds 17 and 1812 showed broad bands at 3369 and 3382 cm−1 for the
OH stretching.
Scheme 2
The carbonyl carbon atom signals were observed at 179.1 ppm in the 13C NMR
spectrum of 17. The novel compounds 20, 21, 22, and the known compound 18l2 were
obtained by the reaction of 2,3-dichloro-1,4-naphthoquinone 1 and a mixture of 14 and 19
with triethylamine in chloroform. Compound 18 was synthesized by our group recently.12

958
C. IBIS ET AL.
The IR spectra of compounds 20 and 21 showed broad bands at 3349 and 3357 cm−1
,
respectively, for the OH stretching. The carbonyl carbon atom signals in the 13C NMR
spectrum of 22 were observed at 178.40 and 183.30 ppm.
The N,S-substituted compounds 25, 26, and 28 were obtained from the reactions of
compound 236 (Scheme 3). Compound 25 is a monosubstituted homopiperazine derivative
and was formed by an addition–elimination reaction. The IR spectrum of 25 showed
characteristic amine band (NH) at 3411 cm−1. The 13C NMR spectra of 28 exhibited a
carbonyl carbon signal of the ester groups at 155.82 ppm while the carbonyl carbon signals
of the quinone moiety were observed at 182.04 and 181.85 as two peaks.
Scheme 3
The mass spectra of compounds 25, 26, and 28 in the positive ion mode for ESI
technique confirmed the proposed structure: the molecular ion peaks were identified at m/z
413[M]+, 478 [M+Na]+, and 493 [M+Na]+, respectively.
Biological Study
Herein, we have synthesized different (hetero)cyclic naphthoquinones and evaluated
their antifungal activity against fungi Candida tenuis VKM Y-70 and Aspergillus niger F-
1119 by the diffusion method13 and the serial dilution method.14 The antibacterial activity
of the compounds was elucidated against Escherichia coli B-906, Staphylococcus aureus
209-P, and Mycobacterium luteum B-917 by the diffusion method and the serial dilution
method as shown in Tables S1 and S2 (Supplemental Materials are available online). Their
activities were compared with those of the known antibacterial agent Vancomycine and the
antifungal agent Nystatin.
Afterwards, on the basis of structure–activity relationship of antifungal activity of the
(hetero)cyclic quinone derivatives, we have further synthesized and screened compounds
of 10, 20, and 25 for antibacterial and antifungal activity by the diffusion method as shown
Table S1.

ANTIBACTERIAL AND ANTIFUNGAL PROPERTIES
959
Table 1 Half-wave potentials for the 1st wave and electrochemical data for novel naphthoquinone derivatives
(10−3 m) in DMF/TBAP 0.1 M, ν = 100 mV·s−1
Compound
Ep(Ic) (V)
Ep(IIc) (V)
ꢀEp1a (mV)
Ep1, 1/2b (V)
3
7
−0.468
−0.654
−0.594
−0.430
−0.571
−0.542
−0.546
−0.693
−0.867
−0.923
−1.146
−1.084
−1.266
−1.118
−1.144
−1.324
108
94
84
102
117
114
108
137
−0.4140
−0.6070
−0.5520
−0.3790
−0.5125
−0.4850
−0.3920
−0.6245
10
12
13
17
20
22
aꢀEp1 = Epa1 − Epc1.
bE1,1/2 = (Epa1 + Epc1)/2.
Evaluation of the antibacterial activity of the synthesized compounds showed that the
MIC (minimum inhibitory concentration) of 25 is 15.6 μg/mL for S. aureus, but 20 was
the most potent with MIC = 7.8 μg/mL for M. luteum (Table S2).
Notable antifungal activities against C. tenuis at 3.9 μg/mL were observed for 4, 13,
and 25. Evaluation of antifungal activities against test-culture C. tenuis of 8, 20, and 22
showed MIC = 15.6 μg/mL.
The MIC of 3, 6, and 10 against test-culture C. tenuis were determined at 31.2 μg/mL.
A notable activity of 22 was observed against A. niger fungi at 3.9 μg/mL. Evaluations of
antifungal activity of 10 showed MIC = 7.8 μg/mL against test-culture A. niger. MIC of 6
and 13 were determined at 15.6 μg/mL against test-culture A. niger.
We have synthesized compound 23 with a slight antifungal activity in our previous
study.6 The antifungal activity significantly increased for the derivatives of compound 23
after the substitution with a homopiperazine group, as determined by the diffusion and the
serial dilution method.
Figure 1 Cyclic voltammogram of compound 22 in DMF+0.1 M TBAP on Glassy Carbon Electrode at
0.1 V s−1 (Color figure available online).

960
C. IBIS ET AL.
Electrochemical Study
Some of the novel naphthoquinone derivatives were studied by cyclic voltammetry
in aprotic media (DMF) using tetrabutylammonium perchlorate (TBAP, 0.10 M) as sup-
porting electrolyte at 100 mVs−1 15
.
The electrochemical parameters, including cathodic
1
peak potentials (Epc1 and Epc2), the half-wave peak potentials (E1, ), and the difference
2
between the first oxidation and reduction processes (ꢀEp) are given in Table 1.
The cyclic voltamogram of 22 is shown in Figure 1 for DMF + 0.1 m TBAP on
Glassy Carbon Electrode at 0.1 Vs−1
.
CONCLUSION
A convenient synthetic route for the preparation of 2- and 2,3-substituted naphthalene-
1,4-diones from 2,3-dichloro-1,4-naphthoquinone has been reported. The compounds, es-
pecially 13, 22, and 25, have been shown to exhibit antibacterial and antifungal properties.
Some further compounds (3, 6, 7, 16, 17, and 28) did not show any significant antifungal
or antibacterial activity against fungi and bacteria species. Among the tested compounds 4,
13, and 25 are the most effective antifungal compounds against C. tenuis whereas 22 is the
most potent antifungal compound against A. niger.
EXPERIMENTAL
Melting points were measured using a Bu¨chi B-540 melting point apparatus and are
uncorrected. Elemental analyses were performed on a Thermo Finnigan Flash EA 1112
elemental analyzer. IR spectra (ν/cm−1) were recorded as KBr pellets or Nujol mulls on a
Perkin Elmer Precisely Spectrum One FTIR spectrometer. 1H NMR (499.74 MHz) and 13
C
NMR (125.66 MHz) spectra (δ/ppm) were recorded in CD3OD, CDCl3, or DMSO-d6 on a
Varian Unity INOVA spectrometer. Mass spectra were obtained on a Thermo Finnigan LCQ
Advantage MAX LC/MS/MS spectrometer using the ESI technique. Products were isolated
by column chromatography on silica gel (Fluka silica gel 60, particle size 63–200 μm).
Thin-layer chromatography (TLC) was performed on Merck silica gel plates (60F254), and
detection was carried out with ultraviolet light (254 nm). All chemicals were reagent grade
and used without further purification.
Cyclic voltammetry measurements were performed in a conventional three-electrode
cell using a computer controlled system of a Gamry Reference 600 Model potentio-
stat/galvanostat. A glassy carbon disc was used as a working electrode. The surface of
the working electrode was polished with alumina before each run. A platinum wire served
as the counter electrode. The reference electrode was an Ag/AgCl electrode. Electro-
chemical grade TBAP in extra pure DMF was employed as the supporting electrolyte at
a concentration of 0.10 m. Prior to each run solutions were purged with nitrogen. Mea-
surements were made over a potential range between +1 and −2 V with a step rate of
0.1 Vs−1
.
General Procedure 1: Synthesis of Alkoxy-1,4-Naphthoquinones
Potassium carbonate (1.52 g) was dissolved in acetonitrile (65 mL). 2,3-Dichloro-
1,4-naphthoquinone (1) and alcohol (2) were added to the solution. Without heating, the
mixture was stirred for 6–8 h. The color of the solution changed quickly, and the extent

ANTIBACTERIAL AND ANTIFUNGAL PROPERTIES
961
of the reaction was monitored by TLC. After filtration of the reaction mixture, the filtrate
was extracted in a Soxhlet extractor with CH2Cl2. After recovery of the solvent, the crude
product was purified by column chromatography.
3,3ꢁ-{[(Ethane-1,2-diylbis(sulfanediyl)]bis(ethane-2,1-diyl)bis(oxy)bis(2-chloro-
1,4-naphthoquinone}(3) and7,8,10,11,13,14,23,24,26,27,29,30-dodecahydrodinaphtho
[2,3-b:2ꢁ,3ꢁ-n][1,4,7,10,13,16,19,22]tetraoxatetrathiacyclotetracosine-5,16,21,32-
tetraone (4) were synthesized from 1 (1 g, 4.38 mmol) and 2 (0.80 mL, 4.38 mmol) by
use of the general procedure 1.
3: Yellow solid. Yield: 1.21 g (49%). M.p. 93.5–95 ◦C. Rf: 0.61 (CHCl3). IR: 3309,
3061 (CHarom), 2975, 2937, 2919 (CHaliph), 1596, 1570 (C C), 1678, 1659 (C O).
1H NMR (CDCl3): 2.80 (t, 4H, SCH2CH2S), 2.91 (t, 4H, SCH2), 4.62 (t, 4H, OCH2),
7.61–7.65 (m, 4H, CHarom), 7.93–7.94 (m, 2H, CHarom), 7.97–7.99 (m, 2H, CHarom).
13C NMR (CDCl3): 32.38 (SCH2), 32.93 (SCH2CH2S), 73.68 (OCH2), 134.61, 134.18,
131.20, 130.92, 129.20, 127.19, 127.08, 110.00 (Carom), 127.75 (=CCl), 156.38 ( C O),
179.79, 178.55 (C O). MS [+ESI]: m/z = 585 [M+Na]+. Anal. Calcd. for C26H20Cl2O6S2
(563.469): C, 55.42; H, 3.58; S, 11.38. Found C, 53.98; H, 3.52; S, 10.27.
4: Red oil. Yield: 1.12 g (45%). Rf: 0.1 (CH2Cl2). IR: 3391 (CHarom), 2924 (CHaliph),
1589, 1539 (C C), 1679 (C O). 1H NMR (CDCl3): 3.16 (t, 4H, SCH2CH2S), 3.20 (t, 4H,
SCH2), 4.65 (t, 4H, OCH2), 7.46–7.60 (m, 4H, CHarom), 7.83–7.92 (m, 2H, CHarom). 13C
NMR (CDCl3): 34.24 (SCH2), 32.25 (SCH2CH2S), 61.27 (OCH2), 125.75, 131.31, 134.09
(Carom), 167.68 ( C O), 177.74 (C O). MS [ESI]: m/z = 674 [M+H]+. Anal. Calcd. for
C32H32O8S4 (672.852): C, 57.12; H, 4.79; S, 19.06. Found C, 57.96; H, 4.78; S, 18.54.
General Procedure 2: Synthesis of Monosulfanyl-, Disulfanyl-, and
Ethoxy-Sulfanyl-1,4-Naphthoquinones
Sodium carbonate (1.52 g) was dissolved in ethanol (65 mL). 2,3-Dichloro-1,4-
naphthoquinone (1) and thiol (5 and 11) were added to the solution. Without heating, the
mixture was stirred for 6–8 h. The color of the solution changed quickly, and the extent
of the reaction was monitored by TLC. The reaction mixture was extracted in a Soxhlet
extractor with CH2Cl2. After recovery of the solvent, the crude product was purified by
column chromatography.
4-Propyl-3,4-dihydro-2H-naphtho[2,3-b][1,4]oxathiepine-6,11-dione (6),
2-chloro-3-[(1-hydroxyhexan-3-yl)thio]-1,4-naphthoquinone (7), and 2,3-bis[(1-hydro-
xyhexan-3-yl)-thio]-1,4-naphthoquinone (8) were synthesized from 1 (1 g, 4.38 mmol)
and 5 (0.61 mL, 4.38 mmol) by use of the general procedure 2. ◦
6: Orange solid. Yield: 0.51 g (40%). M.p. 146.5–148 C. Rf: 0.71 (CHCl3). IR:
1
3307, 3068 (CHarom), 1587, 1551 (C C), 1663 (C O). H NMR (CDCl3): 0.86 (t, 3H,
CH3), 1.37–1.74 [m, 4H, (CH2)2], 2.43–2.51 (m, 1H, SCH), 4.51–4.55 (m, 2H, OCH2CH2),
4.90–4.95 (m, 2H, OCH2), 7.58–7.63 (m, 2H, CHarom), 7.96–8.01 (m, 2H, CHarom). 13C
NMR (CDCl3): 13.94 (CH3), 20.29, 34.61 [(CH2)2], 37.53 (SCH2), 71.47 (OCH2), 45.03
(OCH2CH2), 133.89, 131.95, 131.17, 126.86, 126.66 (Carom), 129.74 (=C–S), 156.50
( C O), 183.34, 178.50 (C O). MS [+ESI]: m/z = 311 [M+Na]+. Anal. Calcd. for
C16H16O3S (288.361): C, 66.64; H, 5.59; S, 11.12. Found C, 66.92; H, 5.70; S, 12.17.
7: Red oil. Yield: 0.29 g (20%). Rf: 0.22 (CHCl3). IR: 3512 (OH), 3071 (CHarom),
2957, 2931, 2871 (CHaliph), 1591, 1549 (C C), 1664, 1655 (C O). 1H NMR (CDCl3): 0.85
(t, 3H, CH3), 1.37–1.95 (m, 4H, (CH2)2), 4.50–4.54 (m, 2H, OCH2CH2), 2.42–2.49 (m, 1H,
SCH), 4.88–4.94 (m, 2H, OCH2), 7.57–7.61 (m, 2H, CHarom), 7.93–7.98 (m, 2H, CHarom).

962
C. IBIS ET AL.
13C NMR (CDCl3): 13.92 (CH3), 20.26, 34.58 [(CH2)2], 37.5 (SCH2), 45.02 (OCH2CH2),
71.46 (OCH2), 133.89, 131.90, 131.11, 129.73, 126.82, 126.62 (Carom), 127.51 (=C–Cl),
156.46 (=C–S), 183.31, 178.47 (C O). MS [+ESI]: m/z = 305 [M-OH]+. Anal. Calcd.
for C16H17ClO3S (324.822): C, 59.16; H, 5.28; S, 9.87. Found C, 60.49; H, 5.95; S, 9.79.
8: Red oil. Yield: 0.28 g (15%). Rf: 0.12 (CHCl3). IR: 3390 (OH), 3070 (CHarom),
1
2957, 2931, 2872 (CHaliph), 1591 (C C), 1660, 1652 (C O). H NMR (CD3OD): 0.93
(t, 3H, CH3), 1.49–1.87 (m, 4H, [CH2)2], 3.3 (s, 2H, OH), 3.71 (m, 2H, OCH2CH2), 1.91
(m, 1H, SCH), 4.15 (m, 2H, HOCH2), 7.35–7.76 (m, 2H, CHarom), 8.00–8.03 (m, 2H,
CHarom). 13C NMR (CD3OD): 13.11 (CH3), 19.83, 38.67 [(CH2)2], 38.51 (SCH2), 46.03
(HOCH2CH2), 59.39 (HOC), 133.59, 133.40, 126.66 (Carom), 149.21 ( C O), 179.12
(C O). MS [+ESI]: m/z = 445 [M+Na]+. Anal. Calcd. for C22H30O4S2 (422.601): C,
62.53; H, 7.16; S, 15.18. Found C, 60.50; H, 7.09; S, 14.75.
2,3-Bis[(4-tert-butylphenyl)thio]-1,4-naphthoquinone (12) and 2-[(4-tert-butyl
phenyl)-thio]-3-ethoxy-1,4-naphthoquinone (13) were synthesized from 1 (1 g,
4.38 mmol) and 11 (0.73 g, 4.38 mmol) by use of the general procedure 2.
12: Red solid. Yield: 0.61 g (28%). M.p. 78.1–79.7 ◦C. Rf: 0.50 (Petroleum
ether/CH2Cl2 1:1). IR: 3070 (CHarom), 2961, 2903, 2867 (CHaliph), 1665 (C O), 1590
(C C). 1H NMR (CDCl3): 1.21 (s, 18H, CH3), 7.16–7.25 (m, 8H, CHarom), 7.54–7.56 (m,
2H, CHarom), 7.87–7.89 (m, 2H, CHarom). 13C NMR (CDCl3): 30.2, 33.6 (C, CH3), 125.1,
126.1, 129.2, 130.1, 131.8, 132.6, 147.4, 149.9 (CHarom, Carom), 177.8 (C=O). MS [+ESI]:
m/z = 487 [M]+. Anal. Calcd. for C30H30O2S2 (486.70): C, 74.04; H, 6.21; S, 13.18. Found
C, 72.98; H, 6.34; S, 12.60.
13: Red solid. Yield: 0.19 g (12%). M.p. 64.9–65.7 ◦C. Rf: 0.40 (Petroleum
ether/CH2Cl2 1:1). IR: 3065 (CHarom), 2955, 2903, 2867 (CHaliph), 1661 (C O), 1591,
1
1544, 1560 (C C). H NMR (CDCl3): 0.99 (t, 3H, CH3), 1.19 (s, 9H, CH3), 4.19 (q,
2H, OCH2), 7.19–7.22 (m, 2H, CHarom), 7.26–7.28 (m, 2H, CHarom), 7.56–7.58 (m, 2H,
CHarom), 7.92–7.95 ppm (m, 2H, CHarom). 13C NMR (CDCl3): 14.3, 30.2, 33.5 (C, CH3),
68.8 (OCH2), 124.9, 125.4, 125.7, 128.9, 130.2, 130.4, 131.1, 131.3, 132.5, 132.8, 149.7
(CHarom, Carom), 157.5 (O–C–C O), 178.6, 181.1 (C=O). MS [+ESI] m/z = 367 [M+H]+.
Anal. Calcd. for C22H22O3S (366.48): C, 72.10; H, 6.05; S, 8.75. Found C, 71.79; H, 6.05;
S, 8.57.
General Procedure 3. Reactions of Mixtures of Two Thiols with
1,4-Naphthoquinones
2,3-Dichloro-1,4-naphthoquinone (1.0 g, 4.38 mmol) was dissolved in CHCl3
(50 mL). Subsequently, the mixture of two thiols (8) (4.38 mmol) and triethylamine (cat-
alytic amount) was added to the solution. Without heating, the mixture was stirred for 4–6 h.
The color of the solution changed quickly, and the extent of the reaction was monitored
by TLC. The reaction mixture was extracted in a Soxhlet extractor with CH2Cl2. After
recovery of the solvent, the crude product was purified by column chromatography.
2,3-Bis(nonylthio)-1,4-naphthoquinone (16), 2-[(6-hydroxyhexyl)thio]-3-(nonyl-
thio)-1,4-naphthoquinone(17), and 2,3-bis[(6-hydroxyhexyl)thio]-1,4-naphthoquinone
(18)12 were synthesized from 1 (0.5 g, 2.19 mmol), 14 (0.3 mL, 2.19 mmol), and 15
(0.35 g, 2.19 mmol) by use of the general procedure 3.
16: Red oil. Yield: 0.13 g (26%). Rf: 0.78 (CHCl3). IR: 2954, 2924, 2854 (CHaliph),
1660, 1652 (C O), 1592 (C C). 1H NMR (CDCl3): 0.79 (t, 6H, CH3), 1.17–1.20 (m, 20H,

ANTIBACTERIAL AND ANTIFUNGAL PROPERTIES
963
CH2), 1.31–1.36 (m, 4H, CH2), 1.52–1.57 (m, 4H, CH2), 3.19 (t, J = 7.32 Hz, 4H, SCH2),
7.58–7.60 (m, 2H, CHarom), 7.95–7.97 (m, 2H, CHarom). 13C NMR (CDCl3): 13.1, 21.6,
27.7, 28.1, 28.2, 28.4, 29.5, 30.8 (CH2, CH3), 33.9 (SCH2), 125.8, 132.1, 132.3 (CHarom
,
Carom), 146.8 (S C C O), 177.9 ppm (C=O). MS [-ESI] m/z = 474 [M]−. Anal. Calcd.
for C28H42O2S2 (474.76): C, 70.84; H, 8.92; S, 13.51. Found C, 70.90; H, 8.27; S, 12.76.
17: Red oil. Yield: 0.16 g (32%). Rf: 0.43 (CHCl3). IR 3369 (OH), 3069 (CHarom),
2923, 2853 (CHaliph), 1658 (C O), 1591 (C C). 1H NMR (CDCl3): 0.77 (t, J = 6.8 Hz,
3H, CH3), 1.15–1.19 (m, 10H, CH2), 1.28–1.37 (m, 6H, CH2), 1.38–1.49 (m, 2H, CH2),
1.50–1.59 (m, 4H, CH2), 1.95 (s, 1H, OH), 3.18 (t, 4H, SCH2), 3.53 (t, 2H, OCH2),
7.58–7.59 (m, 2H, CHarom), 7.93–7.94 (m, 2H, CHarom). 13C NMR (CDCl3): 14.3, 22.8,
25.5, 28.7, 28.9, 29.3, 29.4, 29.6, 30.5, 30.6, 32.0, 32.7 (CH2, CH3), 35.0, 35.2 (SCH2), 62.9
(OCH2), 126.9, 133.2, 133.6 (CHarom, Carom), 147.7, 148.2 (S C C O), 179.1 (C=O).
MS [+ESI] m/z = 471 [M+Na]+. Anal. Calcd. for C25H36O3S2 (448.69): C, 66.92; H,
8.09; S, 14.29. Found C, 66.35; H, 7.90; S, 14.20.
◦
1812: Red solid. Yield: 0.11 g (22%). M.p. 70.3–70.6 C. Rf: 0.3 (CH2Cl2/EtAc
3:1). IR: 3382, 3304 (OH), 2922, 2854 (CHaliph), 1654 (C O), 1589 (C C). Anal.
Calcd. for C22H30O4S2 (422.61): C, 62.53; H, 7.16; S, 15.17. Found C, 62.79; H, 7.05;
S, 15.57.
2,3-Bis[(6-hydroxyhexyl)thio]-1,4-naphthoquinone (18)12, 2-[(6-hydroxyhexyl)
thio]-3-[(3-hydroxypropyl)thio]-1,4-naphthoquinone (20), 2,3-bis[(3-hydroxypropyl)
thio]-1,4-naphthoquinone (21), and 8,9,19,20-tetrahydrodinaphtho[2,3-b:2ꢁ,3ꢁ-i]
[1,4,8,11]dioxadithia-cyclotetradecine-5,11,16,22(7H,18H)-tetraone (22) were synthe-
sized from 1 (0.5 g, 2.19 mmol), 14 (0.3 mL, 2.19 mmol), and 19 (0.38 mL, 2.19 mmol)
by use of the general procedure 3.
1812: Red solid. Yield: 0.11 g (22%). M.p. 70.3–70.6 ◦C. Rf: 0.3 (CH2Cl2/EtAc 3:1).
IR: 3382, 3304 (OH), 2922, 2854 (CHaliph), 1654 (C O), 1589 (C C). Anal. Calcd. for
C22H30O4S2 (422.61): C, 62.53; H, 7.16; S, 15.17. Found C, 62.79; H, 7.05; S, 15.57.
20: Red oil. Yield: 0.13 g (16%). Rf: 0.17 (CHCl3/EtAc 2:1). IR: 3349 (OH), 3065
1
(CHarom), 2930, 2856 (C–Haliph), 1657 (C O), 1591 (C C). H NMR (CDCl3): 1.19 (s,
1H, OH), 1.29–1.33 (m, 2H, CH2), 1.37–1.43 (m, 2H, CH2), 1.45–1.51 (m, 2H, CH2),
1.54–1.61 (m, 2H, CH2), 1.79–1.84 (m, 2H, CH2), 1.93 (s, 1H, OH), 3.22 (t, J = 7.3 Hz,
2H, SCH2), 3.29 (t, 2H, SCH2), 3.53–3.56 (m, 2H, OCH2), 3.71 (t, 2H, OCH2), 7.59–7.63
(m, 2H, CHarom), 7.94–7.97 (m, 2H, CHarom). 13C NMR (CDCl3): 24.3, 27.3, 29.3, 30.5,
31.5 (CH2), 31.9, 33.9 (SCH2), 59.9, 61.6 (OCH2), 125.9, 131.9, 132.5 (CHarom, Carom),
146.1, 147.8 (S C C O), 177.9, 178.0 (C=O). MS [+ESI] m/z = 404 [M+Na]+. Anal.
Calcd. for C19H24O4S2 (380.53): C, 59.97; H, 6.36; S, 16.85. Found C, 60.77; H, 7.19; S,
15.92.
21: Orange solid. Yield: 0.11 g (15%). Rf: 0.10 (CHCl3/EtAc 2:1). IR: 3357 (OH),
2967, 2924, 2853 (CHaliph), 1658 (C O), 1590 (C C). 1H NMR (CDCl3): 1.81–1.90 (m,
4H, CH2), 2.75 (t, 2H, SCH2), 3.32 (t, J = 6.8 Hz, 2H, SCH2), 3.70 (t, 2H, OCH2),
3.74 (t, J = 5.9 Hz, 2H, OCH2), 7.62–7.64 (m, 2H, CHarom), 7.97–7.99 (m, 2H, CHarom).
13C NMR (CDCl3): 31.9 (CH2), 35.4 (SCH2), 61.3 (OCH2), 127.2, 133.2, 133.9 (CHarom
,
Carom), 148.3 (S C C O), 179.2 (C=O). MS [–ESI] m/z = 338 [M]−. Anal. Calcd. for
C16H18O4S2 (338.45): C, 56.78; H, 5.36; S, 18.95. Found C, 56.83; H, 6.16; S, 17.42.
◦
22: Orange solid. Yield: 0.6 g (47%). M.p. 165.2–165.6 C. Rf: 0.71 (CHCl3/EtAc
2:1). IR: 3076 (CHarom), 2966, 2942, 2925, 2845 (CHaliph), 1666, 1643 (C O), 1589, 1546
(C C). 1H NMR (CDCl3): 2.17–2.22 (m, 4H, CH2), 3.24 (t, 4H, SCH2), 4.77 (t, J = 5.9 Hz,
4H, OCH2), 7.58–7.61 (m, 2H, CHarom), 7.93–7.98 (m, 2H, CHarom). 13C NMR (CDCl3):

964
C. IBIS ET AL.
27.7 (CH2), 30.0 (SCH2), 71.9 (OCH2), 126.6, 126.9, 130.6, 131.1, 131.8 (CHarom, Carom),
133.9 (S C C O), 156.8 (O–C–C O), 178.4, 183.3 (C=O). MS [+ESI] m/z = 517
[M+Na]+. Anal. Calcd. for C26H20O6S2 (492.57): C, 63.40; H, 4.09; S, 13.02. Found C,
63.76; H, 4.25; S, 13.98.
General Procedure 4. Amination of 1,4-Naphthoquinones
Na2CO3 (1.52 g) was dissolved in CH2Cl2 (50 mL). 2,3-Dichloro-1,4-
naphthoquinone (1) or 2-chloro-3-[(4-chlorobenzyl)thio]-1,4-naphthoquinone (23)
and a secondary cyclic amine were added to the solution. Without heating, the mixture
was stirred for 4–6 h. The color of the solution changed quickly, and the extent of the
reaction was monitored by TLC. The reaction mixture was extracted in a Soxhlet extractor
with CH2Cl2. After recovery of the solvent, the crude product was purified by column
chromatography.
2-Chloro-3-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)-1,4-naphthoquinone (10) was
synthesized from 1 (0.4 g, 1.15 mmol) and 9 (0.163 g, 2.30 mmol) by use of the general
procedure 4.
10: Red solid. Yield: 1.28 g (87%). M.p. 148–149.5 ◦C. Rf: 0.59 (CHCl3). IR: 3072
1
(CHarom), 2954, 2900, 2860 (CHaliph), 1589, 1548 (C C), 1677, 1640 (C O). H NMR
(CDCl3): 1.83 (t, 4H, CH2piperidine), 3.57 (t, 4H, CH2piperidine), 3.93 (s, 4H, OCH2CH2O),
7.54–7.61 (m, 2H, CHarom), 7.89–7.91 (d, 1H, CHarom), 7.98–8.00 (d, 1H, CHarom). 13C
NMR (CDCl3): 36.23, 49.94 (CH2piperidine), 64.65 (OCH2CH2O), 134.20, 133.24, 131.83,
131.68, 126.71, 123.51 (Carom), 106.90 (=CCl), 150.68 ( C N), 181.98, 178.19 (C O).
MS [+ESI]: m/z = 334 [M]+. Anal. Calcd. for C17H16ClNO4 (333.766): C, 61.18; H, 4.83;
N, 4.20. Found C, 61.31; H, 5.08, N, 4.23.
2-[(4-Chlorobenzyl)thio]-3-(1,4-diazepan-1-yl)-1,4-naphthoquinone (25) was
synthesized from 236 (0.6 g, 1.72 mmol) and 24 (0.18 g, 1.72 mmol) by use of the general
procedure 4.
25: Red oil. Yield: 0.60 g (84%). IR: 3411 (NH), 2962, 2822 (CHaliph), 1592,
1
1556 (C C), 1632 (C O). H NMR (CD3OD): 1.97 (s, 1H, NH), 2.16–2.30 (m, 2H,
3
CH2homopiper.), 3.40–3.50 (m, 4H, CH2homopiper.), 3.60 (t, J = 8.30 Hz, 2H, CH2homopiper.),
3.77 (t, 3J = 4.88 Hz, 2H, CH2homopiper.), 4.02 (s, 2H, SCH2), 7.14–7.21 (m, 4H, CHarom),
7.67–7.73 (m, 2H, CHarom), 7.90–7.92 (d, 3J = 7.32 Hz, 1H, CHarom), 7.94–7.96 (d, 3J =
7.32 Hz, 1H, CHarom). 13C NMR (CD3OD): 52.98, 51.92, 45.52, 25.83, 16.64 (CH2homopiper.),
37.64 (SCH2), 136.80, 132.88, 132.60, 132.11, 130.71, 130.54, 128.52, 128.43, 128,35,
126.54 (Carom), 133.54 (=C–S), 155.33 ( C N), 182.71, 182.31 (C O). MS [+ESI]: m/z
= 413 [M]+. Anal. Calcd. for C22H21ClN2O2S (412.932): C, 63.99; H, 5.13; N, 6.78; S,
7.77. Found C, 64.07; H, 4.99, N, 6.56; S, 7.59.
2-[(4-Chlorobenzyl)thio]-3-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)-1,4-
naphthoquinone (26) was synthesized from 236 (0.48 g, 1.38 mmol) and 9 (0.39 g,
2.76 mmol) by use of the general procedure 4.
26: Purple oil. Yield: 0.53 g (84%). Rf: 0.47 (CHCl3). IR: 3054 (CHarom), 2958,
1
2928, 2881 (CHaliph), 1591, 1536 (C C), 1668, 1636 (C O). H NMR (CDCl3): 1.76
3
3
(t, J = 5.86 Hz, 4H, CH2piperidine), 3.38 (t, J = 5.37 Hz, 4H, CH2piperidine), 3.93 (s, 4H,
OCH2CH2O), 3.97 (s, 2H, SCH2), 7.01–7.04 (d, 3J = 8.30 Hz, 2H, CHarom), 7.06–7.08 (d,
3J = 8.30 Hz, 2H, CHarom), 7.54–7.63 (m, 2H, CHarom), 7.87–7.89 (d, 3J = 7.32 Hz, 1H,
CHarom), 8.00–8.02 (d, 3J = 7.32 Hz, 1H, CHarom). 13C NMR (CDCl3): 35.89, 51.05, 106.71
(Cpiperidine), 64.40 (OCH2CH2O), 38.27 (SCH2), 136.60, 132.86, 132.77, 131.99, 130.24,

ANTIBACTERIAL AND ANTIFUNGAL PROPERTIES
965
128.39, 126.65, 126.24, 126.20 (Carom), 122.33 (=C–S), 156.24 ( C N), 181.79, 181.73
(C O). MS [+ESI]: m/z = 478 [M+Na]+. Anal. Calcd. for C24H22ClNO4S (455.954): C,
63.22; H, 4.86; N, 3.07; S, 7.03. Found C, 60.91; H, 4.70, N, 3.28; S, 6.49.
Ethyl 4-{3-[(4-Chlorobenzyl)thio]-1,4-dioxo-1,4-dihydronaphthalen-2-yl}pipe
razine-1-carboxylate (28) was synthesized from 236 (0.4 g, 1.15 mmol) and 27 (0.36 g,
2.30 mmol) by use of the general procedure 4.
28: Purple oil. Yield: 0.47 g (87%). Rf: 0.33 (CHCl3). IR: 3056 (CHarom), 2979, 2925
(CHaliph), 1592, 1536 (C C), 1699, 1669, 1639 (C O). 1H NMR (CDCl3): 1.22 (t, 3J =
3
3
7.32 Hz, 3H, CH3), 3.26 (t, J = 4.88 Hz, 4H, CH2piperazine), 3.50 (t, J = 5.37 Hz, 4H,
3
CH2piperazine), 4.02 (s, 2H, SCH2), 4.11 (q, 2H, OCH2), 7.02–7.04 (d, J = 8.79 Hz, 2H,
CHarom), 7.08–7.11 (d, 3J = 8.78 Hz, 2H, CHarom), 7.57–7.65 (m, 2H, CHarom), 7.88–7.90
(d, 3J = 7.81 Hz, 1H, CHarom), 8.01–8.03 (d, 3J = 7.32 Hz, 1H, CHarom). 13C NMR (CDCl3):
14.89 (CH3), 52.63, 29.93 (CH2piperazine), 61.87 (OCH2), 136.74, 133.21, 133.03, 132.13,
130.48, 128.74, 127.48, 126.95, 126.60 (Carom), 124.48 (=C–S), 155.72 ( C N), 182.04,
181.85, 155.82 (C O). MS [+ESI]: m/z = 493 [M+Na]+. Anal. Calcd. for C24H23ClN2O4S
(470.968): C, 61.21; H, 4.92; N, 5.95; S, 6.81. Found C, 58.42; H, 4.92, N, 5.34; S, 5.41.
Antifungal and Antibacterial Evaluation
Diffusion Technique. The antibacterial activities were evaluated by diffusion in
peptone on nutrient medium (meat-extract agar for bacteria; wort agar for fungi). The
microbial l◦oading was 109 cells (spores)/1 mL. ◦The required incubation periods were
24 h at 35 C for bacteria and 48–72 h at 28–30 C for fungi. The results were recorded
by measuring the zones surrounding the disk. Control disk contained Vancomycine (for
bacteria) or Nystatine (for fungi) as a standard.
Serial Dilution Technique. Testing was performed in a flat-bottomed 96-well
tissue culture plate. The tested compounds were dissolved in DMSO, and arriving the
necessary concentration. The exact volume of solution of compounds is brought in nutrient
medium. The inoculum of bacteria and fungi was inoculated in nutrient medium (meat-
extract agar for bacteria; wort agar for fungi). The duration of incubation was at 37 ◦C for
bacteria and 30 ◦C for fungi during 24–72 h. The results were estimated according to the
presence or absence of microorganism growth.
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