Design, synthesis, biological evaluation, and antioxidant and cytotoxic activity of heteroatom-substituted 1,4-naphtho- and benzoquinones

Article abstract of DOI:10.1248/cpb.c15-00607

In the present paper, we report the synthesis, characterization, and biological evaluation as antifungal, antibacterial, antioxidant, and cytotoxic/anticancer agents of N-, S-, O-substituted-1,4-naphtho- and 2,5-bis(amino-substituted)-1,4-benzoquinone der

Full text of DOI:10.1248/cpb.c15-00607

Vol. 63, No. 12
Chem. Pharm. Bull. 63, 1029–1039 (2015)
1029
Regular Article
Design, Synthesis, Biological Evaluation, and Antioxidant and Cytotoxic
Activity of Heteroatom-Substituted 1,4-Naphtho- and Benzoquinones
,a
Nahide Gülşah Deniz,* Cemil Ibis,a Zeliha Gokmen,a Maryna Stasevych,b Volodymyr Novikov,b
Olena Komarovska-Porokhnyavets,b Mustafa Ozyurek,c Kubilay Guclu,c Didem Karakas,d and
Engin Ulukayae
a Division of Organic Chemistry, Department of Chemistry, Engineering Faculty, Istanbul University; Avcilar,
b
Istanbul 34320, Turkey: Institute of Chemistry and Chemical Technology, Department of Technology of Biologically
Active Substances, Pharmacy and Biotechnology, Lviv Polytechnic National University; Lviv 79013, Ukraine:
c Division of Analytical Chemistry, Department of Chemistry, Engineering Faculty, Istanbul University; Avcilar,
d
Istanbul 34320, Turkey: Department of Biology, Faculty of Arts and Sciences, Uludag University; Bursa 16059,
e
Turkey: and Department of Clinical Biochemistry, Faculty of Medicine, Uludag University; Bursa 16059, Turkey.
Received August 4, 2015; accepted September 19, 2015
In the present paper, we report the synthesis, characterization, and biological evaluation as antifun-
gal, antibacterial, antioxidant, and cytotoxic/anticancer agents of N-, S-, O-substituted-1,4-naphtho- and
2,5-bis(amino-substituted)-1,4-benzoquinone derivatives. In the synthesized compounds, antimicrobial activ-
ity at low concentrations against Escherichia coli B-906, Staphylococcus aureus 209-P, and Mycobacterium
luteum B-917 bacteria and Candida tenuis VKM Y-70 and Aspergillus niger F-1119 fungi in comparison
with controls was identified. 2-(N-Diphenylmethylpiperazin-1-yl)-3-chloro-1,4-naphthoquinone 9a was the
most potent, with a minimum inhibitory concentration value of 3.9µg/mL against test culture M. luteum.
The synthesized compounds were screened for their antioxidant capacity using the cupric-reducing antioxi-
dant capacity (CUPRAC) method. 2,2ꢀ-[1-(2-Aminoethyl)piperazin-1-yl]-3,3ꢀ-dichloro-bis(1,4-naphthoquinone)
10 showed the highest antioxidant capacity, with a 0.455 CUPRAC-trolox equivalent antioxidant capacity
(TEAC) coefficient. Other parameters of antioxidant activity (scavenging effects on OH·, O2·-, and H2O2) of
these compounds were also determined. The cytotoxic activity of the compounds was investigated by employ-
ing the sulforhodamine B cell viability assay against A549 (lung), MCF-7 (breast), DU145 (prostate), and
HT-29 (colon) cancer cell lines. Compound 10 exhibited the most powerful cytotoxic activity at a concentra-
tion of 20µM against all cell lines. In addition to the strongest antioxidant activity of compound 10, it also
had lowest IC50 values (<3µM), warranting further in vivo studies due to its anticancer activity.
Key words quinone; antimicrobial activity; antioxidant activity; cupric-reducing antioxidant capacity method;
reactive oxygen species-scavenging activity; cytotoxicity
Quinonic compounds are of great importance to understand the heterocyclic ring were considerably important factors to
different processes that are related to biology.1) The quinone affect the biological activities.8,9) The presence of amino, thio-
structure is common in numerous natural products that are or chloro-moiety on the quinones was considerably important
associated with antitumor, antibacterial, antimalarial and anti- factor to effect antifungal activity.10) Quinones, in particular
fungal activities.2) Furthermore, several reports have appeared naphthoquinone derivatives, have been repeatedly isolated
in literature about anticancer activities of quinones against from lower as well as higher species of plants, and are found
various cancer cell lines.3–5)
frequently in animals. In addition to quinones possessing a
1,4-Naphthoquinones are widely distributed in nature and biological function in cell metabolism as electron carriers,
there are many clinically important antitumor drugs contain- other compounds of this class have been found active against
ing a quinone nucleus, such as anthracyclines, mitoxantrones bacteria and fungi.11,12)
and saintopin, that show excellent anticancer activity. These
Antioxidants have received increased attention in the re-
anticancer agents are effective inhibitors of DNA topoisome- cent years from medical researchers and nutritionists for their
rase and it is generally accepted that the cytotoxicity of qui- potential activities in the prevention of several degenerative
none analogues results from the inhibition of DNA topoisom- diseases such as cancer, cardiovascular disorder as well as
erase-II.6) Quinone analogues can also induce the formation of aging.13) Because this activity is related with compounds ca-
semiquinone radicals, which can transfer an electron to oxy- pable of protecting a biological system against the potential
gen to produce superoxide. The radical process is catalyzed by harmful effects of oxidative processes. In recent years, several
flavoenzymes such as reduced nicotinamide adenine dinucleo- works have been published on structure–activity analysis on
tide phosphate (NADPH)-cytochrome-P-450 reductase. Both compounds with antioxidant activities. For examples: Kuwa-
the superoxide and semiquinone radical anions of naphtho- hara et al.14) also studied the antioxidant property of polyhy-
quinone analogues can generate the hydroxyl radical, which droxylated naphthoquinone pigment from shells of purple sea
is known to cause DNA strand breaks.7) Structure–activity urchin anthocidarin, crassispina; Talcott et al.15) also evaluated
relationship studies from quinonoid compounds indicated that antioxidant ability of Menadion against microsomal lipid per-
the number and position of nitrogen (N) atoms substituted in oxidation in the presence of physiologic reductase NADPH.
*To whom correspondence should be addressed. e-mail: yurdakul@istanbul.edu.tr
© 2015 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
Vol. 63, No. 12 (2015)
Chart 1
Chart 2
On the other hand, there are several reports available on anti- well known that the reactions of 1 and 16 with nucleophiles
oxidant activity of synthetic compounds.16)
proceed by nucleophilic substitution whereas nucleophilic ad-
Consequently, the synthesis of new active derivatives with dition reactions of 1,4-naphtho- and benzoquinones are aug-
potential applications in this area and prepared by simple mented by oxidative addition pathway.
chemical procedures should be of increasing interest. Here
N-, S-Substituted-1,4-naphthoquinone 4a and bis(thio)-
we described the synthesis, characterization, antimicrobial, substituted-1,4-naphthoqinone 5 were obtained from reaction
antioxidant and cytotoxic/anticancer activities of 1,4-naphtho- of 1 with N-(diphenylmethyl)piperazine 2a and ethanethiol
and benzoquinone derivatives. Their structures of synthesized 3. Compounds 7a and 8 were synthesized from the reaction
compounds were characterizated by using micro analysis, of 1 with 2a and benzylthiol 6. The synthesis and spectral
Fourier transform (FT)-IR, 1H-NMR, 13C-NMR, MS, ultraviolet- characterization of compound 5 were previously reported.17)
visible spectroscopy (UV-Vis).
Result of micro analysis and melting point for compound
8 were given in the releated literature.18) However, there
is no spectroscopic data for compound 8. The piperazine
Chemistry
A series of N-, S-, O-substituted-1,4-naphthoquinones (4a, ring (2-position) and chlorine atom (3-position) substituted
5, 7a, 8, 9a, b, 10, 12) and 2,5-bis(aminosubstituted)-1,4- compounds 9a, b were obtained from reactions of 1 with N-
benzoquinones (17, 18) are synthesized from the reactions of (diphenylmethyl)piperazine 2a or 1-piperonylpiperazine 2b
2,3-dichloro-1,4-naphthoquinone 1 and p-chloranil 16 with (Chart 1). The reaction of 2,3-dichloro-1,4-naphthoquinone
different nucleophilic compounds (2a, c, 3, 6, 11, 13) under 1 with (2-aminoethyl)piperazine 2c resulted in the formation
the aerobic condition as illustrated in Charts 1, 2 and 3. It is of intramolecular cyclization to yield heterocyclic diquinone

Vol. 63, No. 12 (2015)
Chem. Pharm. Bull.
1031
2,2′-[1-(2-aminoethyl)piperazin-1-yl]-3,3′-dichloro-bis(1,4- the UV region around at 204–210, 214–248 and 276–291nm
naphthoquinone) 10 (Chart 2). In the mass spectrum of com- (π–π* electronic transitions). In addition, a third lower en-
pounds 4a, 7a, 8 and 9a, b, the accurate mass measurements ergy transition appeared as a broad band in the visible region
of the molecular ion peaks were noticed at m/z 469 [M]+, 531 between 455 and 512nm for 4a, 7a, 9a, b, 10 and 12. This
[M]+, 403 [M]+, 443 [M]+ and 412 [M]+, respectively.
absorption is typical of N-substituted quinones22,23) and is
The reaction of 2,3-dichloro-1,4-naphthoquinone 1 with assigned to charge-transfer transitions and weak n–π* transi-
(2-aminoethyl)piperazine 2c resulted in the formation of tions of the carbonyl group in the quinone unit.24)
intramolecular cyclization to yield heterocyclic diquinone
Mono(thio)-substituted naphthoquinone compounds contain-
2,2′-[1-(2-aminoethyl)piperazin-1-yl]-3,3′-dichloro-bis(1,4- ing chlorine atom were not observed potentially due to the
naphthoquinone) 10 (Chart 2). In the positive ion mode of decreased thiol amount in the medium of the reaction, while
electrospray ionization (ESI) mass spectrum for compound 10 mono(thio)-substituted naphthoquinone containing ethoxy
the respective molecular ion peak was observed at m/z (%) 510 group (3-position) compounds were obtained successfully. The
(100) [M]+. The cleavage of the chlorine ion from compound synthesis and characterization of compounds 14 and 15 were
10 of the molecular ion gave to rise fragment F1 at m/z (%) previously reported.25) In present study, properties of biologi-
473 (100) [M−37]+ which was the base peak.
Brun et al.19) and Xu et al.20) synthesized the compound 12 have been investigated.
cal activity and antioxidant capacity of compounds 14 and 15
entitled 2-chloro-3-[2-morpholin-4-yl)ethylamino][1,4]-naph-
Brun et al.,19) Xu et al.20) and Hsu et al.21) reported
thoquinone in the presence of triethylammonium acetate that the high biological activities of the compound 12
(0.6mL) in ether (40mL) and the presence of anhydrous which was obtained by the reaction of 2,3-dichloro-1,4-
ethanol, respectively. We synthesized the compound 12 by napthoquinone 1 with 11. We want to investigate the
the different procedure according to general method 2 as biological activity properties of the target compound
given in Experimental. Brun et al. reported the CDC25 phos- [(2-aminoethyl)morpholin-1-yl)]-substituted-1,4-benzoquinone
phatase inhibitory activity of naphthoquinone derivatives in 17. For this reason, 2,5-bis[4-(2-aminoethyl)morpholin-1-yl]-
literature.19) Hsu et al. showed that the compound 12 was the 3,6-dichloro-1,4-benzoquinone 17 and 2,5-bis[1-piperonyl-
most potent to induce cell death in human A549 lung cancer piperazin-1-yl]-3,6-dichloro-1,4-benzoquinone 18 were syn-
cells.21) Xu et al.20) investigated the biological properties of thesized from reactions of p-chloranil 16 with 11 and 2b,
compound 12 as antiproliferative agents and 20S proteasome respectively in Chart 3.
inhibitors20) showed features that biological activity properties
The reactions were found to be exceptionally selective and
of compound 12 encouraged us to synthesize this compound leads to only 2,5-bis(aminosubstituted)-3,6-dichloro-1,4-ben-
again and to investigate antifungal, antibacterial properties zoquinones of the corresponding amine. From these reactions
and antioxidant capacity of compound 12. It is confirmed we could not obtain 2,6-bis(aminosubstituted)-1,4-benzoqui-
that the product formed between the reaction of compounds none derivatives. The steric factors arising from the sub-
1 and 4-(2-aminoethyl)morpholine 11 was compound 12. The stituent effect predominates in these reactions. The result of
proposed mechanism for the reaction of 1 with 11 and the selective formation of 2,5-isomer may be assumed to be due to
synthesis pathway of 12 were illustrated in Chart 2. Alkenes attack of two amines to 1,4-benzoquinone. For such attack to
generally undergo nucleophilic addition reactions. However, give exclusive product of one isomer would require approach
alkenes undergo nucleophilic addition reactions in the pres- of two amines from the furthest possible distance. Thus, ex-
ence of electron withdrawing groups (EWG’s) bound to sp2- clusively 2,5-isomer were formed due to electrostatic reasons
hybrided C atoms and this is known as the Michael reaction. for compounds 17 and 18. The results agree well with the
First, an addition of the attacking reagent to the C, C double corresponding mechanism in the similar compounds.24,26,27)
bond occurs, and in a second step the intermediate product is The 13C-NMR spectra of benzoquinone unit gave one carbonyl
stabilized by elimination of hydrogen chloride as illustrated in signals spectra at 171.29 and 175.00ppm in compounds 17
Chart 2.
and 18, respectively. If 2,6-isomer was obtained from these
UV-Vis electronic absorption spectra of 4a, 7a, 8, 9a, b reactions, we must determine two different carbonyl signals
showed the expected benzene and naphthoquinone bands in in the 13C-NMR spectra for 17 and 18. FT-IR spectrum in
Chart 3

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Chem. Pharm. Bull.
Vol. 63, No. 12 (2015)
KBr showed the following important absorption bands for
The test-cultures E. coli, S. aureus, C. tenuis and A. niger
compounds 17 and 18. In the FT-IR spectra of compounds appeared not to be sensitive to all compounds (by diffusion
17 and 18; two typical strong quinonic carbonyl absorptions method, see Table 1). Compound 15 was sensitive at a concen-
were observed between at 1614 and 1676cm−1 for compounds tration of 0.1%, and the diameter of the inhibition zone was
17 and 18. The (–NH) absorption appeared at 3245cm−1 for 13.4mm (Table 1). The M. luteum bacteria strain was sensitive
compound 17. For compound 18, no bands were observed to compound 15 at a concentration of 0.5% and the diameter
in the region 3200–3450cm−1 attributable to the streching of the inhibition zone was 14.4mm. Compound 10 has moder-
vibration of the bonded (–NH) group. In the mass spectrum ate activity against C. tenuis (12.3mm at 0.5% concentration).
of compounds 17 and 18, the accurate mass measurements of Compounds 4a, 7a, 9a, b, 10, 12 and 14 (at 0.1% concentra-
the molecular ion peaks were noticed at m/z 433 [M]+ and 613 tion) have no antibacterial and antifungal activity against E.
[M]+, respectively.
coli, S. aureus, M. luteum, C. tenuis and A. niger at 0.5 and
0.1% evaluated concentrations by diffusion method (Table 1).
The biological activity results of the synthesized com-
Results and Discussion
Antibacterial and Antifungal Activities The profound pounds were classified as follows: the antimicrobial activities
antifungal and antibacterial activity exhibited by quinone were considered as significant when the minimum inhibition
compounds has prompted us to synthesize new N,S,O- concentration (MIC) was 100µg/mL or less; moderate, when
substituted-1,4-naphtho- and 2,5-bis(aminosubstituted)-1,4- the MIC was 100.0–500.0µg/mL; weak, when the MIC was
benzoquinones. In our new endeavors, we have synthesized 500.0–1.000µg/mL; and inactive when the MIC was above
new 1,4-naphtho- and benzoquinones and evaluated their anti- 1.000µg/mL. According to the literature30) the compounds
bacterial and antifungal activity by diffusion method28) and containing N-phenyl piperazine ring similiar the compound
serial dilution method29) with a view to search new perspec- 9a showed more significant antibacterial activity among the
tive compounds having broad spectrum of biological activity. other similiar compounds. Evaluation of the antibacterial ac-
Antibacterial and antifungal activity of compounds 4a, 7a, 9a, tivity of the synthesized compound showed that 9a was the
b, 10, 12, 14 and 15 were elucidated against Escherichia coli most potent with MIC=3.9 µg/mL for M. luteum (for control
B-906, Staphylococcus aureus 209-P, and Mycobacterium lu- MIC=7.8 µg/mL). Compounds 4a, 7a and 9b showed sig-
teum B-917, Candida tenuis VKM Y-70 and Aspergillus niger nificant anti-Mycobacterium activity with MIC value in the
F-1119 by diffusion method in Table 1 and by serial dilution range of 15.6µg/mL in serial dilution assay against M. luteum.
method as shown in Tables 2 and 3. Activities of quinone Evaluation of antibacterial activity of synthesized compounds
compounds were compared with those of the known anti- showed that 4a and 9b have MIC=62.5µg/mL; 7a and 12 have
bacterial agent Vancomycin and antifungal agent Nystatin MIC=125.0 µg/mL; 14 has MIC=250.0µg/mL for S. aureus.
(control C).
Compounds 10 and 12 have MIC=250.0 and 125.0µg/mL,
Table 1. Antibacterial and Antifungal Activities of Compounds by Diffusion Method
Inhibition diameter of microorganism growth (mm)
Antibacterial activity Antifungal activity
S. aureus
Compound
Concentration (%)
E. coli
M. luteum
C. tenuis
A. niger
4a
7a
0.5
0.1
0.5
0.1
0.5
0.1
0.5
0.1
0.5
0.1
0.5
0.1
0.5
0.1
0.5
0.1
0.5
0.1
0.5
0.1
0.5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
14
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
15
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
19
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
20
0
0
9a
0
0
9b
0
0
10
12.3
0
12
0
0
14
0
0
15
14.4
13.4
0
17
0
18
0
0
Control*
18
* 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 activity of the
synthesized compounds.

Vol. 63, No. 12 (2015)
Chem. Pharm. Bull.
1033
Table 2. Antibacterial Activities of Compounds by Serial Dilution
Table 3. Antifungal Activities of Compounds by Serial Dilution Method
Method
MIC (µg/mL)
Compound
MIC (µg/mL)
C. tenuis
A. niger
Compound
E. coli
S. aureus
M. luteum
4a
7a
9a
9b
250.0
500.0
62.5
500.0
*
4a
7a
9a
9b
*
62.5
125.0
*
15.6
15.6
3.9
*
500.0
125.0
+
*
31.2
*
62.5
+
125.0
250.0
+
15.6
125.0
125.0
+
10
+
125.0
10
+
*
12
14
125.0
12
14
+
+
+
+
+
+
250.0
15
15
*
17
500.0
500.0
7.8 0.2
500.0
500.0
15.6 0.8
17
250.0
125.0
+
18
18
+
+
Control
Control
31.2 0.8
3.9 0.2
7.8 0.2
+: Growth of microorganisms. *: In the investigated concentrations the indexes of
biocidic effect were not determined.
+: Growth of microorganisms. *: In the investigated concentrations the indexes of
biocidic effect were not determined.
Table 4. Calibration Equations of Synthesized Compounds, Linear Ranges and TEAC Coefficients (N=3)
Compound
Linear range (molL−1
)
Calibration equation
r
TEAC*
4a
5
0.012–0.049
0.025–0.098
0.006–0.049
0.070–0.143
0.023–0.095
0.049–0.172
0.051–0.123
0.024–0.097
N.D.
y=5220c+0.021
y=3850c+0.221
y=4900c+3.58×10−3
y=3726c+0.267
y=3822c+2.0×10−3
y=5822c+0.032
y=7605c+0.113
y=2735c+0.031
N.D.
0.9981
0.9978
0.9919
0.9928
0.9951
0.9928
0.9980
0.9929
N.D.
0.313
0.231
0.293
0.223
0.229
0.349
0.455
0.164
N.D.
7a
8
9a
9b
10
12
14
15
17
18
0.046–0.120
0.065–0.132
0.026–0.100
y=5340c+0.134
y=2400c+0.048
y=5665c+0.065
0.9951
0.9961
0.9950
0.320
0.144
0.340
*TEAC=εcompound/εTR (TR: trolox); εTR=1.67×104 Lmol−1 cm−1. N.D.: Not defined.
respectively, for M. luteum in Table 2.
capacity (Table 4). Among the synthesized compounds, 10
Significant antifungal activity for 9b was observed against showed the highest antioxidant capacity, and CUPRAC-TEAC
C. tenuis fungi at 31.2µg/mL. Evaluation of antifungal activ- coefficients (in parentheses) decreased in the following order:
ity of compounds 4a, 7a, 9a, 10, 12, 14 and 15 showed their 10 (0.455)>9b (0.349)>18 (0.340)≥15 (0.320)>4a (0.313)>7a
activity in concentrations 62.5–500.0µg/mL against test- (0.293)>5 (0.231)>9a (0.229). Compound 10, showed the
culture C. tenuis. Compounds 4a, 7a, 9a, b, 10, 12, 14 and 15 highest antioxidant capacity, possibly due to its dimeric struc-
showed moderate antifungal activity with MIC value in the ture, similar to the observation that polymeric polyphenols
range of 125.0–500.0µg/mL against A. niger in Table 3.
had higher antioxidant activity than their monomeric ana-
Summary of obtained data of antibacterial activity in Table logues.
2 showed that the compounds 4a, 7a, 9a, b and 12 were sensi-
Reactive Oxygen Species (ROS) Scavenging Activity
tive to bacteria S. aureus and especially 9a to M. luteum, 9a, Hydroxyl radical (OH·) is the most reactive free radical that
b were sensitive to fungies C. tenuis and A. niger in Table 3.
can be formed from superoxide anion (O2·−) and hydrogen
Antioxidant Capacity The newly synthesized compounds peroxide (H2O2), in the presence of metal ions.33) In the pres-
4a, 5, 7a, 8, 9a, b, 10, 12, 14, 15, 17, 18 were screened for ent study, the hydroxyl radical scavenging (HRS)-CUPRAC
their antioxidant capacity by using the cupric-reducing anti- method was used for determining the HRS activity of sub-
oxidant capacity (CUPRAC) methods31,32) against trolox as the stituted quinonic compounds. The results are shown in Table
standard reference compound. The linear calibration equations 5. The % inhibition ratio values of compounds in this assay
of these compounds (as absorbance in a 1cm cell vs. molar were in the range of 60.6 to 76.7. The compounds 10 and 18
concentration) gave the molar absorption coefficient (ε) as the were found to show better inhibition compared to other com-
slope. The CUPRAC molar absorption coefficient of the tested pounds with % inhibition values 75.10 and 76.70, respectively.
compound divided by that of trolox under the same condi- The compounds 12, 17 and 9b showed poor (OH·) scavenging
tions gave the trolox equivalent antioxidant capacity (TEAC), activity.
or TEAC coefficient of that compound tested for antioxidant
Hydrogen peroxide is thought to be the major precursor

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Chem. Pharm. Bull.
Vol. 63, No. 12 (2015)
of highly reactive free radicals, and it has been reported to drogen peroxide scavenging (HPS) compounds like 10 and 4a
induce apoptosis in cells of the central nervous system.34) The are also the ones with high HPS activity, as measured in this
ability of the compounds to scavenge H2O2 was determined work.
according to the method of literatures.35,36) A concentration-
Superoxide anion radical (O2·−) have gained great attention
dependent assay was carried out with newly synthesized due to their important role in the progression of a number
substituted quinonic compounds. Thus, the free radical scav- of human diseases and carcinogenesis. So it is important to
enging activities of newly synthesized compounds decreased eliminate excessive (O2·−) in vivo to prevent important dis-
in the order of 10>4a>15>18>5, showing a parallelism with eases.37) In this study, superoxide anion radical scavenging
that of CUPRAC-TEAC. It can be seen that the leading hy- (SARS) activity of newly synthesized compounds was evalu-
ated according to the method of Yu et al.38) Superoxide anion
(O2·−) can be formed from dissolved oxygen by PMS-reduced
nicotinamide adenine dinucleotide (NADH) coupling reac-
Table 5. ROS Scavenging Activity of Synthesized Compounds (N=3)
tion, and (O2·−) reduces the yellow dye (NBT2+) to produce a
blue formazan, of which the absorbance value is measured at
560nm. Antioxidants are able to inhibit formazan formation.
The decrease of absorbance with antioxidants indicates the
consumption of (O2·−) in the reaction mixture.
HRS-CUPRAC
HPS-CUPRAC
SARS activity
(% Inh.)
Compound
(% Inh.)
(% Inh.)
4a
5
74.10
62.15
70.60
65.75
68.20
61.25
75.10
60.70
65.50
62.70
60.60
76.70
35.15
30.21
8.86
76.24
N.D.
N.D.
23.45
42.10
14.15
78.45
N.D.
14.92
19.45
N.D.
N.D.
7a
8
15.60
11.48
15.73
39.85
8.07
The order of SARS activity of the synthesized compounds
in terms of % inhibition ratio was: 10>4a>9a>8>15.
9a
9b
10
12
14
15
17
18
Table 6. IC50 Values after 48h of Treatment with Compounds 9b and 10
in MCF-7 and DU145 Cell Lines Were Determined by ATP Assay
17.44
35.65
8.58
Cell lines
Compound 9b*
Compound 10*
MCF-7
9.42µM
2.16µM
2.52µM
30.51
DU145
15.96µM
N.D.: Not defined.
* IC50 is defined as the dose inhibiting 50% of viability.
Fig. 1. Viability (%) of A549, MCF-7, DU145 and HT-29 Cells Treated with 20µM Concentration of 11 Different Compounds (4a, 7a, 9a, b, 10, 12,
14, 15, 17) for 48h
After treatment period, the SRB assay was performed to measure cell viability. ANOVA was used to demonstrate statistical significance between different doses with
a Tukey’s multiple comparison post test. * denotes statistically significant differences in comparison with negative control (NC). * p≤0.05; **p≤0.01; and ***p≤0.001.

Vol. 63, No. 12 (2015)
Chem. Pharm. Bull.
1035
Fig. 2. Viability of MCF-7 and DU145 Cells after the Treatment with Varying Concentrations of 9b and 10 for 48h Was Measured by ATP Viability Assay
#
* (for 9b) and (for 10) denotes statistically significant differences in comparison with negative control (NC) * p≤0.05; **p≤0.01; and ***p≤0.001.
Cytotoxic Activity A549, MCF-7, DU145 and HT-29 cells ing the charge transfer complexes that have enormous applica-
were treated with different compounds (4a, 7a, 9a, b, 10, 12, tions ranging from sensors, magnetic materials to chemistry
14, 15, 17) at 20µM concentration (the arbitrary dose of our of drugs. The aim of this study was synthesis, characteriza-
laboratory for cytotoxicity screening studies) for 48h. After tion, evaluation of biological properties, antioxidant capacity
the treatment period, cytotoxic effects of compounds were and anticancer activity of some naphtho- and benzoquinone
investigated by employing sulforhodamine B (SRB) cell vi- compounds. All new compounds were characterized on the
ability assay (Table 6).
basis of nuclear magnetic resonance spectroscopy (1H- and
Based on SRB viability results, it was found that compound 13C-NMR), MS, FT-IR and UV-Vis. Among the synthesized
10 exhibited the most powerful cytotoxic activity against all compounds with antimicrobial activity at low concentrations
cell lines. In addition, the SRB results showed that compound against S. aureus, M. luteum bacteria and C. tenuis and A.
9b had strong cytotoxic effect on MCF-7 and DU145 cell lines niger fungi in comparison with control were identified. The M.
(Fig. 1).
luteum bacteria strain was sensitive to compound 15 at a con-
Based on the SRB results, two cell lines (MCF-7 and centration of 0.5% and the diameter of the inhibition zone was
DU145) and two compounds (9b, 10) to were selected to 14.4mm. Antibacterial activity showed that compounds 4a,
perform ATP viability assay. Although SRB and ATP assays 7a, 9a, b and 12 are sensitive to bacteria, S. aureus and espe-
measure cell viability, ATP assay is considered to be more cially to M. luteum, 9a and b are sensitive to fungies C. tenuis
sensitive than SRB assay due to its being luminescence-based and A. niger. 2-(N-Diphenylmethylpiperazin-1-yl)-3-chloro-1,4-
assay.39) For this purpose, MCF-7 and DU145 cells were treat- naphthoquinone 9a was the most potent with MICs (minimum
ed with different concentrations (0.39–50µM) of compounds inhibition concentrations)=3.9 µg/mL against test culture M.
9b and 10 for 48h and then ATP cell viability assay was per- luteum bacteria. It has been observed that naphthoquinones
formed (Fig. 2). It was found that both compounds exhibited 9 containing chlorine atom in the position 3 of 1,4-naphtho-
statistically significant anti-growth effects in a dose-dependent quinone moiety show more significant antibacterial activ-
manner compared to negative control (NC). IC50 values were ity against M. luteum in comparison with thiosubstituted-1,4-
calculated on the basis of the results of the ATP assay and naphthoqinones 4 and 7 and control. The antioxidant proper-
were shown in Table 6. IC50 values of compound 9b were 9.42 ties of the synthesized compounds have also been tested using
and 15.96µM for MCF-7 and DU145 cell lines, respectively. CUPRAC method in which 2,2′-[1-(2-aminoethyl)piperazin-1-
It was found that compound 10 had lower IC50 values when yl]-3,3′-dichloro-bis(1,4-naphthoquinone) 10 exhibited better
compared to compound 9b (2.16 µM for MCF-7 and 2.52µM for antioxidant capacity (CUPRAC-TEAC: 0.455) than the other
DU145 cells). These results demonstrated that compound 10 compounds. Moreover, the related compound exhibited high
had more strong cytotoxic activity, compared to compound 9b. ROS scavenging properties (depending on the type of ROS)
In the literature, IC50 values of quinones have a broad compared to the other compounds. Cytotoxicity assay (SRB
range. Dolan et al., reported that IC50 values of different assay) showed that compound 10 exhibited the most power-
naphthoquinones was ranging from 1.1 to 10.8µM for MCF-7 ful cytotoxic activity against four different cancer cell lines
cells.3) In the other study, IC50 values were determined be- (A549, MCF-7, DU145, HT-29). Interestingly, it has also the
tween 0.88 and 43.5µM for MCF-7 cells.40) Copeland et al. most powerful antioxidant capacity. IC50 values of compounds
determined IC50 values of different quinones as 1, 3, 1.5, 3 9b and 10 found 9.42 and 15.96µM for MCF-7 cells and
and 10µM for LNCaP, CWR-22, PC-3, DU-145 and HS-5 cells, DU145 cell lines, respectively. In addition, compound 10 had
respectively.41)
lower IC50 values when compared to compound 9b (2.16 µM
for MCF-7 and 2.52µM for DU145 cells).
Consequently, the synthesis of new active derivatives with
Conclusion
The quinone compounds deserve the great prominence by potential applications in this area and prepared by simple
exhibiting a broad spectrum of biological activities and form- chemical procedures should be of increasing interest. In

1036
Chem. Pharm. Bull.
Vol. 63, No. 12 (2015)
conclusion, we have synthesized a series of 1,4-naphtho- and brs, Hpiper), 4.3 (1H, s, –CH<), 7.0–8.0ppm (14H, m, Harom).
benzoquinone derivatives that are the promising candidates 13C-NMR (125.66Hz, CDCl3) δ: 13.76 (CH3), 33.87 (S-CH2),
with respect to biological activity as potential antibacterial, 44.62, 51.61 (N-CH2), 73.83 (–CH<), 125.55, 126.53, 126.76,
antifungal, antioxidant and anticancer agents.
126.91, 127.26, 127.47, 127.57, 127.81, 127.85, 128.00, 128.69,
129.03, 131.08, 131.38, 131.87, 132.65, 133.17, 133.61, 136.64
(CHarom
, Carom), 146.09 (=C-S), 162.60 (=C-N), 180.68,
Experimental
Melting points were measured on a Buchi B-540 melting 181.10 ppm (C=O). MS [+ESI]: m/z 469 [M]+. Anal. Calcd for
point apparatus. TLC plates silica 60F254 (Merck, Darmstadt), C29H23N2S1O2 (M=468.62g/mol) C, 74.32; H, 6.02; N, 5.97; S,
detection with ultraviolet light (254nm). Elemental analyses 6.48. Found C, 74.30; H, 6.06; N, 5.98; S, 6.43%.
were performed on a Thermo Finnigan Flash EA 1112 El-
2-(N-Diphenylmethylpiperazin-1-yl)-3-benzylsulfa-
emental analyser. IR spectra were recorded in KBr pellets in nyl-1,4-naphthoquinone (7a) Compound 7a was synthe-
Nujol mulls on a PerkinElmer, Inc. Precisely Spectrum One sized from the reaction of 1 (1.0g, 4.4mmol) with 2a (1.1g,
FT-IR spectrometry. UV spectra in CHCl3 were recorded on 4.4mmol) and 6 (0.54g, 4.4mmol) and according to gen-
1
PerkinElmer, Inc. Lambda 35 UV/VIS Spectrometer. H- and eral method 1. Red solid, Yield: 1.8g, 81%, mp: 126–127°C,
13C-NMR spectra were recorded on VarianUNITYINOVA op- Rf=0.42 (CHCl3), FT-IR (in KBR pellet, cm−1): 3026 (Ar-H),
erating at 500MHz. Mass spectra were obtained on a Thermo 2894, 2816 (C-H), 1666 (C=O), 1593, 1521 (C=C), 1281
Finnigan LCQ Advantage MAX LC-MS/MS spectrometer (C-N). UV-Vis [CHCl3, λ (log ε)]: 206 (4.1), 220 (4.1), 241
1
according to ESI probe. Products were isolated by column (4.2), 291 (4.1), 512 (3.5). H-NMR (499.74MHz, CDCl3): 2.4
chromatography on Silica gel (Fluka Silica gel 60, particle (4H, brs, Hpiper), 3.4 (t, J=4.88Hz, 4H, Hpiper), 3.9 (s, 2H,
size 63–200µm). All chemicals were reagent grade and used S-CH2), 4.2 (1H, s, –CH<), 7.0–8.1ppm (m, 24H, Harom).
without further purification. Moisture was excluded from the 13C-NMR (125.66Hz, CDCl3) δ: 38.15 (S-CH2), 51.53, 51.98
glass apparatus using CaCl2 drying tubes. Solvents, unless (N-CH2), 75.14 (–CH<), 120.95, 125.21, 125.47, 125.53, 125.95,
otherwise specified, were of reagent grade and distilled once 126.07, 126.51, 126.95, 127.08, 127.20, 127.24, 127.34, 127.38,
prior to use.
127.45, 127.54, 127.74, 127.97, 128.16, 131.05, 131.52, 131.99,
General Methods for the Synthesis of 1,4-Naphtho- and 132.62, 137.06 (CHarom, Carom), 141.32 (=C-S), 154.87 (=C-N),
Benzoquinones
General Method 1
180.68, 181.01ppm (C=O). MS [+ESI]: m/z 531 [M]+, MS/
MS [+ESI]: m/z 363 [M−168]+. Anal. Calcd for C34H30N2O2S1
Sodium carbonate (1.52g) was dissolved (60mL) in ethanol. (M=530.69g/mol) C, 76.95; H, 5.69; N, 5.27. Found C, 76.97;
2,3-Dichloro-1,4-naphthoquinone 1 and nuchleophilies (2a, 3, H, 5.64; N, 5.65%.
6) were added slowly to this solution. Without heating, the
2,3-Bis(Benzylsulfanyl)-1,4-naphthoquinone (8) Com-
mixture was stirred for 6h. The colour of the solution quickly pound 8 was synthesized from the reaction of 1 (1.0g,
changed and the extent of the reaction was monitored by TLC. 4.4mmol) with 2a (1.1g, 4.4mmol) and 6 (0.54g, 4.4mmol)
Chloroform (30mL) was added to the reaction mixture. The according to general method 1. Red crystal, Yield: 0.3g, 17%,
organic layer was washed with water (4×30mL), and dried mp: 183–184°C (183°C14)), Rf=0.52 (CHCl3), FT-IR (in KBR
with Na2SO4. After the solvent was evaporated the residue pellet, cm−1): 3020 (Ar-H), 2892, 2805 (C-H), 1651 (C=O),
was purified by column chromatography on silica gel.
General Method 2
1591, 1463 (C=C). UV-Vis [CHCl3, λ (log ε)]: 208 (4.8), 214
1
(4.8), 241 (4.8), 284 (4.5), 468 (3.9). H-NMR (499.74MHz,
2,3-Dichloro-1,4-naphthoquinone 1 and nuchleophilies (2b, CDCl3): 4.2 (s, 4H, S-CH2), 7.1–8.1ppm (m, 14H, Harom).
c, 11) were stirred in chloroform (25mL) for 8h. Chloroform 13C-NMR (125.66Hz, CDCl3) δ: 38.23 (S-CH2), 125.76, 126.39,
(30mL) was added to the reaction mixture. The organic layer 127.46, 127.53, 128.17, 128.39, 131.88, 132.40, 136.18 (CHarom
,
was washed with water (4×30mL), and dried with Na2SO4. Carom), 146.69 (=C-S), 178.09ppm (C=O). MS [+ESI]: m/z 403
After the solvent was evaporated the residue was purified by [M]+, 425 [M+Na]+, MS/MS [+ESI]: m/z 303 [(M+Na)−123]+,
column chromatography on silica gel.
General Method 3
281 [M−123]+. Anal. Calcd for C24H18S2O2 (M=402.538 g/
mol). C, 71.61; H, 4.51; S, 15.93. Found C, 71.55; H, 4.52; S,
p-Chloranil 16 and nuchleophilies (2b, 11) were stirred in 15.95% (C, 71.30; H, 4.50%14)).
triethylamine (1mL) in chloroform (25mL) for 6h. The extent
2-(N-Diphenylmethylpiperazin-1-yl)-3-chloro-1,4-naph-
of the reaction was monitored by TLC. Chloroform (30mL) thoquinone (9a) Compound 9a was synthesized from the re-
was added to the reaction mixture. The organic layer was action of 1 (1.0g, 4.4mmol) with 2a (2.2g, 8.8mmol) accord-
washed with water (4×30mL), and dried with Na2SO4. After ing to general method 1. Brown oil, Yield: 2.8g, 72%. Rf=0.41
solvent recovery, the crude product was purified by chroma- (CHCl3), FT-IR (in KBR pellet, cm−1): 3025 (Ar-H), 2959,
tography.
2813 (C-H), 1648 (C=O), 1592, 1556 (C=C), 1282 (C-N). UV-
2-(1-Ethylsulfanyl)-3-(1-N-diphenylmethylpiperazin- Vis [CHCl3, λ (log ε)]: 204 (4.6), 225 (4.0), 237 (4.0), 276 (4.0),
1-yl)-1,4-naphthoquinone (4a) Compound 4a was synthe- 480 (3.4). H-NMR (499.74MHz, CDCl3): 2.5 (4H, brs, Hpiper),
1
sized from the reaction of 1 (1.0g, 4.4mmol) with 2a (1.1g, 3.6 (4H, brs, Hpiper), 4.2 (1H, s, –CH<), 7.0–8.0ppm (14H, m,
4.4mmol) and 3 (0.27g, 4.4mmol) according to general meth- Harom). 13C-NMR (125.66Hz, CDCl3) δ: 50.66, 51.74 (N-CH2),
od 1. Red solid, Yield: 1.2g, 58%, mp: 124–125°C, Rf=0.45 75.21 (–CH<), 121.59, 125.48, 125.83, 126.14, 126.49, 126.93,
(CHCl3), FT-IR (in KBR pellet, cm−1): 3050 (Ar-H), 2957, 127.19, 127.24, 127.62, 127.83, 129.01, 130.45, 130.63, 131.35,
2923 (C-H), 1664 (C=O), 1592, 1537 (C=C), 1279 (C-N). UV- 131.95, 132.98 (CHarom, Carom), 141.28 (=C-N), 148.9 (=C-Cl),
Vis [CHCl31, λ (log ε)]: 206 (3.5), 226 (3.4), 248 (3.5), 286 (3.4), 176.94, 180.81ppm (C=O). MS [+ESI]: m/z 443 [M]+. Anal.
498 (2.5). H-NMR (499.74MHz, CDCl3): 1.2 (t, J=7.32Hz, Calcd for C27H23N2O2Cl1 (M=442.94g/mol) C, 73.21; H, 5.23;
3H, CH3), 3.00 (q, 2H, S-CH2), 2.5 (4H, brs, Hpiper), 3.6 (4H, N, 6.32. Found C, 73.20; H, 5.26; N, 6.31%.

Vol. 63, No. 12 (2015)
Chem. Pharm. Bull.
1037
2-[1-Piperonylpiperazin-1-yl]-3-chloro-1,4-naphthoqui- Anal. Calcd for C18H26N4O4Cl2 (M=433.33g/mol): C, 49.89; H,
none (9b) Compound 9b was synthesized from the reaction 6.04; N, 12.92%. Found: C, 49.90; H, 6.06; N, 12.83%.
of 1 (1.0g, 4.4mmol) with 2b (2.9g, 13.2mmol) accord-
2,5-Bis[1-piperonylpiperazin-1-yl]-3,6-dichloro-1,4-ben-
ing to general method 2. Red solid, Yield: 1.2g, 66%. mp: zoquinone (18) Compound 18 was synthesized from the
169–170°C, Rf=0.41 (CH2Cl2), FT-IR (in KBR pellet, cm−1): reaction of 16 (1.0g, 4.06mmol) with 2b (3.58g, 13.2mmol)
3030 (Ar-H), 2908, 2887, 2809, 2768 (C-H), 1676, 1637 (C=O), according to general method 3. Brown solid, mp: 197–198°C.
1592, 1558 (C=C). UV-Vis [CHCl3, λ (log ε)]: 210 (4.0), 221 Yield: 2.1g, 84%. Rf=0.32 [CHCl3 :EtAc (3:1)], FT-IR (in
1
(4.6), 246 (4.4), 282 (4.6), 491 (3.8). H-NMR (499.74MHz, KBR pellet, cm−1): 3013 (Ar-H), 2946, 2910, 2895, 2853, 2814,
CDCl3): 2.6 (brs, 4H, Hpiper), 3.4 (s, 2H, N-CH2), 3.6 (4H, brs, 2765 (C-H), 1656 (C=O), 1577, 1499 (C=C). UV-Vis [CHCl3,
Hpiper), 5.84 (s, 2H, O-CH2-O), 6.6–8.1ppm (7H, m, Harom). λ (log ε)]: 204 (4.0), 220 (4.0), 228 (4.6), 233 (4.4), 244 (4.4),
1
13C-NMR (125.66Hz, CDCl3) δ: 51.33, 61.05 (N-CH2), 53.68 283 (4.1), 384 (3.8), 438 (4.1). H-NMR (499.74MHz, CDCl3):
(N-CH2), 101.19 (O-CH2-O), 108.18, 109.82, 122.77, 123.16, 2.5 (s, 8H, Hpiper), 3.4 (s, 4H, N-CH2), 3.5 (t, J=4.8Hz, 8H,
126.71, 127.05, 130.95, 131.63, 131.80, 133.25, 134.25, 147.16 Hpiper), 5.87 (s, 4H, O-CH2-O), 6.6–6.8ppm (m, 6H, Harom).
(CHarom, Carom), 147.99 (=C-N), 150.09 (=C-Cl), 178.14, 13C-NMR (125.66Hz, CDCl3) δ: 50.80, 61.66 (N-CH2),
181.98ppm (C=O). MS [+ESI]: m/z 412 [M+H]+. Anal. Calcd 52.58 (N-CH2), 99.91 (O-CH2-O), 106.90, 108.40, 114.81,
for C22H19N2O4Cl1 (M=410.86g/mol) C, 64.31; H, 4.66; N, 121.21, 130.66, 145.75 (CHarom, Carom), 146.73 (=C-N), 147.45
6.81. Found C, 64.10; H, 4.68; N, 6.78%.
(=C-Cl), 175.00ppm (C=O). MS [+ESI]: m/z 613 [M]+, MS/
2,2′-[1-(2-Aminoethyl)piperazin-1-yl]-3,3′-dichloro- MS [+ESI]: m/z 577 [M−37]+. Anal. Calcd for C30H30N4O6Cl2
bis(1,4-naphthoquinone) (10) Compound 10 was synthe- (M=613.502g/mol). C, 58.73; H, 4.92; N, 9.13%. Found C,
sized from the reaction of 1 (1.0g, 4.4mmol) with 2c (0.57g, 58.74; H, 4.98; N, 9.28%.
4.4mmol) according to general method 2. Red solid. mp:
167–168°C, Yield: 1.9g, 86%. Rf=0.42 (CHCl3), FT-IR (in
Antibacterial and Antifungal Evaluation
KBR pellet, cm−1): 3330 (N-H), 3067 (Ar-H), 2947, 2815
Diffusion Method25) Antibacterial activity of compounds
(C-H), 1670 (C=O), 1574, 1524 (C=C). UV-Vis [CHCl3, λ (log was evaluated by diffusion in peptone on nutrient medium
ε)]: 207 (4.5), 219 (4.5), 237 (4.5), 257 (4.4), 268 (4.5), 472 (meat-extract agar for bacteria; wort agar for fungi). The
1
(3.8). H-NMR (499.74MHz, CDCl3): 2.9 (s, Hpiper, 4H), 3.4 (t, microbial loading was 109 cells (spores)/1mL. The required
J=6.84Hz, 2H, NH-CH2-CH2-), 3.6 (q, NH-CH2-, 2H), 3.7 (s, incubation periods were as: 24h at 35°C for bacteria and
Hpiper, 4H), 4.2 (t, J=5.37Hz, NH), 7.4–8.4ppm (m, 4H, Harom). 48–72h at 28–30°C for fungi. The results were recorded by
13C-NMR (125.66Hz, CDCl3) δ: 47.87 (NH-CH2-CH2-), 49.35 measuring the zones surrounding the disk. Control disk con-
(NH-CH2-), 52.43, 56.27 (CH2piper), 121.64, 123.71, 125.70, tained Vancomicine (for bacteria), Oxacilinum (for bacteria),
125.76, 125.96, 128.97, 130.36, 130.53, 131.50, 132.30, 133.22, or Nistatine (for fungi) as a standard.
133.84 (CHarom, Carom), 143.51 (=C-N), 148.52 (=C-Cl), 179.49,
Serial Dilution Method26) Testing was performed in a
180.75ppm (C=O). MS [+ESI]: m/z 510 [M]+, MS/MS [+ESI]: flat-bottomed 96-well tissue culture plate. The tested com-
m/z 473 [M−37]. Anal. Calcd for C26H21N3O4Cl2 (M=510.38 g/ pounds were dissolved in dimethyl sulfoxide (DMSO) to the
mol). C, 61.18; H, 4.14; N, 8.23. Found C, 61.19; H, 4.15; N, necessary concentration. The exact volume of solution of
8.28%.
compounds is brought in nutrient medium. The inoculum of
2-[4-(2-Aminoethyl)morpholin-1-yl]-3-chloro-1,4-naph- bacteria and fungi was in nutrient medium (meat-extract agar
thoquinone (12) Compound 12 was synthesized from the for bacteria; wort agar for fungi). The duration of incubation
reaction of 1 (1.0g, 4.4mmol) with 11 (1.72g, 13.2mmol) was at 37°C for bacteria and 30°C for fungi over 24–72h. The
according to general method 2. Dark red solid, Yield: 0.9g, results were estimated according to the presence or absence of
64%, mp: 140–141°C, Rf=0.50 (EtAc), FT-IR (in KBR pel- microorganism growth.
let, cm−1): 3342 (N-H), 3210 (Ar-H), 2966, 2849 (C-H), 1672
The microorganisms that were tested included the follow-
(C=O), 1571, 1519 (C=C). UV-Vis [CHCl3, λ (log ε)]: 205 ing: bacteria E. coli B-906, S. aureus 209-P, and M. luteum
(4.5), 212 (4.5), 223 (4.5), 238 (4.5), 272 (4.3), 455 (3.5). Anal. B-917 and fungi C. tenuis VKM Y-70, and A. niger F-1119.
Calcd for C16H17N2O3Cl1 (M=320.77g/mol) C, 59.91; H, 5.34;
N, 8.73. Found C, 59.89; H, 5.35; N, 8.75%).
Antioxidant Capacity The CUPRAC reagent solutions
were prepared as follows: CuCl2 solution (10mM) was pre-
2,5-Bis[4-(2-aminoethyl)morpholin-1-yl]-3,6-dichlo- pared in distilled water, ammonium acetate solution (1.0M,
ro-1,4-benzoquinone (17) Compound 17 was synthesized pH=7) and neocuproine solution (7.5mM) were prepared in
from the reaction of 16 (1.0g, 4.06mmol) with 11 (2.1g, pure ethanol for the CUPRAC assay, as a difference from the
16.13mmol) according to general method 3. Yellow solid. original CUPRAC method31) where ammonium acetate buffer
mp: 205–206°C. Yield: 1.5g (85%). Rf=0.52 (EtAc). FT-IR is prepared in distilled water. The solutions of all other com-
(in KBR pellet, cm−1): 3245 (N-H), 3013 (Ar-H), 2980, 2956, pounds were freshly prepared in DMSO.
2929, 2894, 2855, 2814 (C-H), 1614 (C=O), 1560, 1492 (C=C).
One milliliter CuCl2, 1mL Nc solution, and 1mL NH4Ac
UV-Vis [CHCl3, λ (log ε)]: 202 (3.7), 213 (3.8), 226 (3.8), 234 solution were added to xmL of the tested compound, followed
1
(3.9), 243 (4.2), 361 (4.4), 536 (2.5). H-NMR (499.74MHz, by (1.1−x)mL DMSO. The absorbance of the final solution
CDCl3): δ=2.4 (t, J=8.3Hz, 8H, Hmorph), 3.6 (t, J=7.32Hz, 8H, (of 4.1mL total volume) at 450nm was read against a reagent
Hmorph), 2.58 (t, J=6.35Hz, 4H, NH-CH2-CH2), 3.9 (q, 4H, blank after 30min standing at room temperature.31) The absor-
NH-CH2), 7.73 (s, 2H, NH) 13C-NMR (125.66MHz, CDCl3): bance of the emerging cuprous neocuproine chromophore was
δ=39.76 (NH-CH2-CH2-), 55.47 (NH-CH2-), 52.02, 65.93 correlated to tested compound concentration.
(CH2morph), 139.49 (=C-N), 144.41 (=C-Cl), 171.29ppm (C=O).
MS [+ESI]: m/z 433 [M]+, MS/MS [+ESI]: m/z 396 [M−37]+,

1038
Chem. Pharm. Bull.
Vol. 63, No. 12 (2015)
ROS Scavenging Activity
HRS Activity
mixture in the absence and presence of scavenger, respec-
tively.
Cytotoxic Activity
Chemicals and Cell Culture
All compounds were dissolved in DMSO at a concentration
OH· in aqueous media were generated through the Fenton
system and spectrophotometrically determined—via hydrox-
ylation of a probe—by the modified CUPRAC method.35)
To a test tube were added 1.5mL of phosphate buffer (pH of 25mM as a stock solution. Further dilutions were made in
7.0), 0.5mL of 10mM sodium salicylate, 0.25mL of 20mM culture medium. DMSO final concentration was 0.1%. All
ethylenediaminetetraacetic acid disodium salt (EDTA), tested cells, A549 (lung), MCF-7 (breast), DU145 (prostate)
0.25mL of 20mM FeCl2 solution, 18mL H2O, (x) mL sample and HT-29 (colon), were cultured in RPMI 1640 medium sup-
solution (x varying between 0.1 and 0.5mL) at a concentra- plemented with penicillin G (100U/mL), streptomycin (100µg/
tion of 3.0×10−5 M, and 0.5mL of 10mM H2O2 rapidly in this mL), L-glutamine, and 10% fetal bovine serum at 37°C in a
order. The mixture in a total volume of 5mL was incubated humidified atmosphere containing 5% CO2.
for 10min in a water bath kept at 37°C. After incubation, the
The SRB Viability Assay
To detect the cytotoxicity of compounds against tested cell
reaction was stopped with adding 0.5mL of 268U mL−1 cata-
lase solution, and mixed for 30s. Final mixtures (0.5mL of lines, firstly all compounds were used at 20µM concentrations.
the incubation solution) were subjected to the HRS-CUPRAC Cells were seeded at a density of 5×103 cells per well of 96-
method. The HRS activity (%) of samples was calculated well culture plate in 100µL medium in triplicate. After 24h,
using the equation:
compounds were added at 20µM concentrations and cells were
incubated with these different compounds for 48h.
At the end of treatment, 50µL of 50% trichloroacetic acid
HRS (%)=[(A0 − A)/A0]×100
where A0 and A are the CUPRAC absorbances of the system (TCA) was added and fixation was allowed to proceed for 1h
in the absence and presence of sample, respectively.
HPS Activity
at 4°C. After fixation, the supernatant was discarded and the
plate was washed with deionized water five times. TCA-fixed
The ability of compounds to scavenge H2O2 was determined cells were stained with 50µL of SRB solution (0.4% in 1%
according to the method of Özyürek et al.35) To a test tube acetic acid) for 30min at room temperature. After staining,
were added 0.7mL of phosphate buffer (pH 7.4), 0.4mL of the unbound SRB was washed out with 1% acetic acid and
1mM H2O2, 0.4mL of 0.1mM CuCl2·2H2O in this order (H2O2 air-dried. Bound SRB was solubilized with 150µL of Tris
incubation solution, used as reference). To the other two test base (10mM, pH 10.0) and then plate was shaken for 10min at
tubes were added 0.5mL of phosphate buffer (pH 7.4), 0.4mL 150rpm. The 96-well plate was read by a spectrophotometer
of 1.0mM H2O2, 0.2mL sample (3.0×10−5 M), and 0.4mL of at 570nm.
0.1 mM CuCl2. 2H2O solution rapidly in this order (named as
The ATP Viability Assay
scavenger solutions-I, -II). The mixtures in a total volume of
MCF-7 and DU145 were seeded as exactly the same as done
1.5mL were incubated for 30min in a water bath kept at 37°C. in the SRB assay. After 24h, compounds (9b and 10) were
At the end of this period, to both reference and scavenger added at different concentrations (0.39–50µM) and cells were
solution-I was added 0.4mL H2O, and to scavenger solution-II incubated with these compounds for 48h.
was added 0.4mL of 268U mL–1 catalase solution, and mixed
In order to carry out the ATP assay, 150µL of medium
for 30s. Final mixtures (1.0mL of the incubation solution) was removed from each well and 50µL of somatic cell ATP
were subjected to the HPS-CUPRAC method. The HPS activ- releasing agent (ATP Bioluminescent Somatic Cell Assay Kit,
ity (%) of sample was calculated using the equation:
Sigma-Aldrich, St. Louis, MO, U.S.A.) was added. After mix-
ing thoroughly, the microplate was allowed to stand on the
bench for 20–30min at room temperature. At the end of the
HPS (%)=[(A0 − (A − A2)) /A0]×100
1
where A0 is the CUPRAC absorbance of reference H2O2 in- incubation 50µL of mixture from each well was transferred to
cubation solution, A1 and A2 are the CUPRAC absorbances of a white non-translucent plate. To each well of the plate, 50µL
scavenger solutions-I and -II, respectively.
SARS Activity
luciferin–luciferase reagent (ATP Bioluminescent Somatic
Cell Assay Kit, Sigma-Aldrich) was added and the 96-well
The superoxide anion radicals (O2·−) were generated in vitro plate was measured using a count integration time of 1s at
in a non-enzymatic system (PMS-NADH) and determined luminometer (Bio-Tek, VT, U.S.A.).
spectrophotometrically by nitroblue tetrazolium (NBT) re-
Statistical Analyses All of the statistical analyses of
duction method described by Yu et al.38) To a test tube were cell viability assays were performed by using the Graphpad
added 2.3mL DMSO, 0.2mL of sample (3.0×10−5 M), 2mL of Prism 6.0 statistical software for Windows. All results were
468µM NADH, 1mL of 300µM NBT, in this order. The reac- expressed as mean standard deviation (S.D.).
tion was started by adding 1mL of 60µM PMS solution to the
incubation mixture. The mixture in a total volume of 6.5mL
Acknowledgment The authors would like to express their
was incubated for 5min in a water bath kept at 25°C, and the gratitude to Scientific Research Projects Coordination Unit
absorbance was read at 560nm against DMSO. Decreased of Istanbul University for financial support (Project Number:
absorbance of the incubation reaction mixture indicated in- NP-45621).
creased superoxide anion radical scavenging activity. The
SARS (%) of sample was calculated using the equation:
Conflict of Interest The authors declare no conflict of
interest.
SARS (%)=[(A0 − A)/A0]×100
where A0 and A are the absorbances of the incubation reaction

Vol. 63, No. 12 (2015)
Chem. Pharm. Bull.
(2005).
1039
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