Novel 2,3-disubstituted 1,4-naphthoquinone derivatives and their metal complexes - Synthesis and in vitro cytotoxic effect against mouse fibrosarcoma L929 cells

Article abstract of DOI:10.1016/j.jorganchem.2011.10.036

Quinones have various pharmacological properties including antibacterial, antifungal, antiviral, anti-inflammatory, antipyretic and anticancer activity. Two novel 2,3-disubstituted 1,4-naphthoquinones and their metal complexes were synthesised, characterized and tested. The cytotoxic potential of the novel 2,3-disubstituted 1,4-naphthoquinones and their metal complexes was studied against L929 murine fibroblasts cells grown in vitro. The treatment resulted in a concentration-dependent cytotoxicity as indicated by MTT assay.

Full text of DOI:10.1016/j.jorganchem.2011.10.036

Journal of Organometallic Chemistry 700 (2012) 13e19
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Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Novel 2,3-disubstituted 1,4-naphthoquinone derivatives and their metal
complexes e Synthesis and in vitro cytotoxic effect against mouse fibrosarcoma
L929 cells
a
,
b
c
c
*
Violeta-Carolina Niculescu , Nicolae Muresan , Aurora Salageanu , Catalin Tucureanu ,
d
e
d
Gabriela Marinescu , Liviu Chirigiu , Costinel Lepadatu
National Research and Development Institute for Cryogenics and Isotopes Technologies e ICIT, 4th Uzinei Street, 240050 Ramnicu Valcea, Romania
Faculty of Chemistry, University of Craiova, 165 Calea Bucuresti, 200144 Craiova, Romania
National Research and Development Institute for Microbiology and Immunology, 103 Spl. Independentei, 050096 Bucharest, Romania
Institute of Physical Chemistry, 202 Spl. Independentei, 060021 Bucharest, Romania
Faculty of Pharmacy, University of Medicine and Pharmacy, 2-4 Petru Rares Street, 200349 Craiova, Romania
a
b
c
d
e
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 June 2011
Received in revised form
Quinones have various pharmacological properties including antibacterial, antifungal, antiviral, anti-
inflammatory, antipyretic and anticancer activity. Two novel 2,3-disubstituted 1,4-naphthoquinones and
their metal complexes were synthesised, characterized and tested. The cytotoxic potential of the novel
11 August 2011
2,3-disubstituted 1,4-naphthoquinones and their metal complexes was studied against L929 murine
Accepted 19 October 2011
fibroblasts cells grown in vitro. The treatment resulted in a concentration-dependent cytotoxicity as
indicated by MTT assay.
Keywords:
Cytotoxic
Ó 2011 Elsevier B.V. All rights reserved.
Complex
Fibroblast
In vitro
Naphthoquinone
1. Introduction
1,4-naphthoquinone pharmacophore is known to impart
pronounced biological effects in 1,4-naphthoquinone derivatives,
Research in the field of cancer therapy has led to the develop-
ment of many modern medicines and therapeutics with promising
anticancer activity. Among the many natural and synthetic
compounds explored for anticancer potential, compounds with
quinone containing moieties form a major part. Quinones are widely
distributed compounds in nature and they are reported to exhibit
diverse pharmacological properties including anticancer activity
leading to antitumour [5,6], antiproliferative [7], antimycobacterial
[8], antiplatelet, anti-inflammatory, antiallergic [9], antimalarial
[10] and antileishmanial activities [11]. The incorporation of
sulphur atom in 1,4-naphthoquinone derivatives has led to anti-
fungal, antibacterial, antiviral, and anticancer activities [12e16].
It has been reported that menadione (an analogue of 1,4-
naphthoquinone) could induce reactive oxygen species (ROS),
which mediate DNA damage in many human cultured cells, sug-
gesting that this activity might be important for its therapeutic
effects of naphthoquinone derivatives [17,18].
The literature offers data related to the complex compounds
formed by transition metals and ligands having conjugated double
bonds [19e21]. The special interest about these complexes is due,
among others, to the fact that the complex compounds readily
participate in reversible electron-transfer reactions. Literature
mentions the important biologic-active, antimalaric, antiviral and
antitumoural properties of this type of ligands; the same properties are
shown by the complexes that these ligands form with metal ions,
which act in the biological structures as essential microelements
[19e21]. The importance of these compounds can be exemplified by
[1,2]. One of the mechanisms by which quinones could induce
cytotoxicity was induction of oxidative stress [3]. According to this
proposed mechanism, quinones are first reduced to hydroquinones
or semiquinone radicals by cellular reductases at the expense of
NADPH. Then, both hydroquinones and semiquinone radicals
undergo rapid autoxidation with the regeneration of the parent
quinones and the concomitant formation of superoxide [3]. These
quinones with the ability to induce oxidative stress are responsible
for initiation of tissue damage selectively in tumour cells and this
seems to be a promising approach for targeting cancer cells [4].
*
Corresponding author. Tel.: þ40 25 073 2744; fax: þ40 25 073 2746.
E-mail addresses: violeta@icsi.ro, caroletyy@yahoo.com (V.-C. Niculescu).
0
022-328X/$ e see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2011.10.036

14
V.-C. Niculescu et al. / Journal of Organometallic Chemistry 700 (2012) 13e19
naphthoquinone compounds in terms of cytotoxicity by MTT
assay [36], using L929 mouse fibroblasts.
2. Methods
2.1. Chemicals
The reagents and the solvents for the synthesis were used without
further purification. 2-amino-3-chloro-1,4-naphthoquinone and 2,3-
dichloro-1,4-naphthoquinone were purchased from Fluka (Stein-
heim, Germany), chloroacetic acid from Merck (Darmstadt, Germany),
ethanol, methanol, dilituric acid, anhydrous nickel chloride and hexa-
hydrate chloroplatinicacid fromSigmaeAldrich(Steinheim, Germany),
metallic sodium from Aldrich (Steinheim, Germany), thiourea, NaOH
and glacial acetic acid from UTCHIM (Ramnicu Valcea, ROMANIA) and
Water Chromasolv from Riedel-de Haën (Seelze, Germany).
Fig. 1. 3-alkyl- or3-alkylamino-substitutedderivativesof2-hydroxy-1,4-naphthoquinone
or 2-chloro-1,4-naphthoquinones.
the interesting biological activities associated with many 3-alkyl- or 3-
alkylamino-substituted derivatives of 2-hydroxy-1,4-naphthoquinone
or 2-chloro-1,4-naphthoquinones (Fig. 1) [22].
Further need for a study of compounds of this type is illustrated
by the observation that the ortho-amino quinoid unit is present in
many antitumoural antibiotics such as actinomycins, mitomycin C,
porfiromycin and streptonigrin [23].
2
.2. Instruments
Melting points (m.p.) of the ligands and metal complexes were
determined by open capillary method using Sunsim electric
melting point apparatus and were uncorrected. Elemental analysis
of C, H, N, S and O was performed with a Thermo Finnigan Flash
In this study, in order to continue the existing investigations on
2
,3-disubstituted 1,4-naphthoquinones [24], we introduced
substituents groups containing N, S or O atoms, knowing the fact
that in many natural structures, such as proteins or aminoacids,
these groups are responsible for various biological activities.
Moreover, the potential biological activity of the new synthesized
compounds can be cumulated due to the 1,4-naphthoquinone
properties. Also, because there are few literatures [25e27]
regarding the complexes of the general [M-N2S2] type, especially
metal complexes with naphthoquinonic ligands, we hope that the
data provided by this paper will contribute to enrich the knowledge
in this insufficiently studied field.
Cell lines are useful models for doing research since they
provide large amounts of consistent cells for prolonged use and
because most cellular characteristics are maintained, reliable
experimental data can be compared among research reports, in
which the same cell lines are used. L929 is a cell line popular in
many experiment aspects such as material biocompatibility testing
2
000 Automatic Elemental Analyzer. The metal content was
determined using an Analytic Jena NOVAA 300 Absorption Atomic
Spectrophotometer. The IR spectra were recorded in the region
ꢀ1
4
000e400 cm on a FT-IR MAGNA 750 NICOLET in anhydrous KBr
pellets. A Jasco V-670 UVeVis spectrophotometer was used to
perform the electronic spectra.
2.3. In vitro cytotoxicity assay
In order to determine the cytotoxicity of the compounds, the
in vitro cytotoxicity MTT assay was performed using L929 mouse
fibroblasts (kindly provided by Dr. Ronald Doyle, University of
Louisville School of Medicine, Louisville, KY, USA).
The cells were cultivated in Dulbecco’s Modified Eagle Medium
(
1
1
DMEM) (SigmaeAldrich, Inc. St. Louis, MO) supplemented with
0% fetal bovine serum (FBS) (BIOCHROM AG, Berlin, Germany) and
00 U/mL penicillin-streptomycin (Lonza, Verviers, Belgium) and
[
[
28e30], drug cytotoxicity testing [31,32] and cell biology studies
33e35].
ꢁ
incubated at 37 C in a humidified atmosphere with 5% CO
bottom 96-well tissue culture plates (starting concentration:
ꢂ 105 cells/mL, 100 L/well).
Test compounds were dissolved in dimethylsulphoxide (DMSO)
Merck, Darmstadt, Germany) at a final concentration of 1% in
culture medium.
2
, in flat-
In order to develop new antitumoural drugs with less secondary
effects, the final purpose of this study was to evaluate the potential
biological activity of the novel synthesized 2,3-disubstituted 1,4-
6
m
(
Fig. 2. 2-acetamino-3-mercapto-1,4-naphthoquinone.
Fig. 3. 2-mercapto-3(5-nitrobarbituro)-1,4-naphthoquinone.

V.-C. Niculescu et al. / Journal of Organometallic Chemistry 700 (2012) 13e19
15
Scheme 1. Synthesis of AMNQ.
Ten binary dilutions were made by serial dilution in culture
media from a starting concentration of 0.200 mg/mL. After incu-
(Tecan Group Ltd., Mannedorf, Switzerland) at 540 nm. Each dilution
was tested in two replicates. The 50% inhibition concentration (IC50)
was determined by curve fitting using “ic50 package” in R [37,38].
bation, cells were exposed to serial dilutions test samples for 24 h at
ꢁ
3
7 C in a humidified atmosphere with 5% CO
2
. Viability of the cells
was measured by MTT assay (MTT: (3-[4,5-dimethylthiazol-2-yl]-
,5-diphenyl tetrazolium bromide) purchased from SigmaeAldrich
3. Results and discussion
2
Corp., St. Louis, MO, USA) [36]. MTT solution was added to cell
cultures in final concentration of 0.5 mg/mL and cells were incu-
bated for 3 h.
3.1. Synthesis
The new naphthoquinonic ligands containing X ¼ S, Y ¼ N as
donor atoms were: 2-acetamino-3-mercapto-1,4-naphthoquinone
(AMNQ) (Fig. 2) and 2-mercapto-3(5-nitrobarbituro)-1,4-
naphthoquinone (MNBNQ) (Fig. 3). These ligands were synthe-
sized according the Schemes 1 And 2.
Thereafter,100 mL of lysis solution (20% sodium dodecyl sulphate
in 50% N,N-dimethylformamide, 0.4% acetic acid, and 0.04 N HCl)
was added and the conversion of MTT to formazan by metabolically
viable cells was measured by a Tecan Sunrise microplate reader
Scheme 2. Synthesis of MNBNQ.

16
V.-C. Niculescu et al. / Journal of Organometallic Chemistry 700 (2012) 13e19
Table 1
2
Preparation details and elemental analyses for AMNQ and [Pt(AMNQ) ].
ꢁ
tPSAa
Compound
AMNQ
Mol wt calcd
mp ( C)
Appearance
Brown
Yield %
93
Element
Calc./found%
clogPb
83.47
263.27
140
C
54.75/54.74
3.42/3.42
5.32/5.31
24.34/24.35
12.17/12.18
40.06/40.05
2.24/2.26
3.89/3.87
17.79/17.77
8.91/8.89
H
N
O
S
1.3462
[Pt(AMNQ)
2
]
719.60
170
149.36
Red-brown microcrystalline
95
C
H
N
O
S
5.9268
Pt
27.11/27.16
a
tPSA e molecular polar surface area.
clogP e octanol/water partition coefficient.
b
Table 2
2
Preparation details and elemental analyses for MNBNQ and [Ni(MNBNQ) ].
ꢁ
tPSAa
Compound
MNBNQ
Mol wt calcd
mp ( C)
Appearance
Yield %
94
Element
Calc./found%
clogPb
361.29
142
153.43
e
Purple-brown
C
46.54/46.52
1.95/1.97
11.63/11.60
31.00/31.02
8.88/8.89
H
N
O
S
1.2946
[
Ni(MNBNQ)
2
]
779.25
154
286.40
.9334
Dark brown microcrystalline
96
C
43.16/43.14
1.55/1.57
10.78/10.77
28.74/28.72
8.23/8.24
H
N
O
S
1
Ni
7.54/7.56
Values calculated with ChemBioDraw, v. 11.0.01, Cambridge Soft.
a
tPSA e molecular polar surface area.
clogP e octanol/water partition coefficient.
b
Table 3
Infrared absorption frequencies (cmꢀ1) for AMNQ and [Pt(AMNQ)
].
2
Compound
AMNQ
n
NeH
n
NePt
n
SH
n
C¼O
d
NH
n
CeN
n
CeH
n
CeS
3346.09
m
e
2231.18
s
1673.15
m
1590.83
s
1280.23
m
841.95
w
594.56
s
[
Pt(AMNQ)
2
]
3423.11
w
3867.15
s
2360.71
s
1604.51
s
e
1275.12
m
841.30
w
e
w e weak; m e medium; s e strong.
Synthesis of AMNQ: 2-amino-3-chloro-1,4-naphthoquinone
0.053 mol) and chloroacetic acid (0.074 mol) in absolute ethanol
100 mL) were stirred and refluxed at 120 C for 3 h. The reaction
mixture was then cooled and after filtration the intermediary, 2-
amino-3-chloro-1,4-naphthoquinone was formed. This compound
obtain the final product, AMNQ, 1 N acetic acid solution was added
to the reaction mixture. The product was then filtered, washed with
pure water and dried. The purification of AMNQ was achieved by
consecutive dissolution in 1 N NaOH solution and precipitation in
1 N acetic acid solution.
(
(
ꢁ
(
(
0.022 mol) was then stirred and refluxed in absolute ethanol
80 mL) with thiourea (0.422 mol) at 120 C for 2 h. The reaction
Synthesis
of
MNBNQ:
2,3-dichloro-1,4-naphthoquinone
ꢁ
(0.048 mol) and dilituric acid (0.049 mol) in absolute ethanol
(100 mL) were stirred and refluxed at 120 C for 3.5 h. The reaction
mixture was then cooled and after filtration the red intermediary, 2-
dilituro-3-chloro-1,4-naphthoquinone was formed. This compound
ꢁ
mixture volume was then half reduced by distillation under
vacuum, 100 mL pure water and 10 g (0.4 mol) NaOH were added to
the solution and the mixture was refluxed another 2 h. In order to
Table 4
nfrared absorption frequencies (cmꢀ1) for MNBNQ and [Ni(MNBNQ)
2
].
a
a
a
a
Compound
MNBNQ
n
NeH
n
NeNi
n
SeH
n
C¼O
n
C¼O
n
NO2
n
C¼C
n
CeN
n
CeN
n
CeS
3417.24
m
e
2854.48
w
1836.77
w
1673.37
s
1569.57
m
1524.5
w
1386.05
w
1285.63
s
697.43
w
[Ni(MNBNQ)
2
]
3415.33
3742.10
m
e
1836.02
1687.20
1549.85
1524.64
1390.52
e
693.13
1642.39
i
s
w
m
w
w
w
w e weak; m e medium; s e strong.
a
N cycle from the ligand and the complex.

V.-C. Niculescu et al. / Journal of Organometallic Chemistry 700 (2012) 13e19
17
2
Fig. 4. Electronic spectra of AMNQ and [Pt(AMNQ) ].
2
Fig. 5. Electronic spectra of MNBNQ and [Ni(MNBNQ) ].
(
0.022mol)wasthenstirredandrefluxedinabsoluteethanol(80mL)
ꢁ
with thiourea (0.44 mol) at 120 C for 1.5 h. The reaction mixture
volume was then half reduced by distillation under vacuum, 100 mL
pure water and 10 (0.4 mol) g NaOH were added to the solution and
the mixture was refluxed another 2 h. In order to obtain the final
product, MNBNQ, 1 N acetic acid solution was added to the reaction
mixture. The product was then filtered, washed with pure water and
dried. The purification of AMNQ was achieved by consecutive
dissolution in 1 N NaOH solution and precipitation in 1 N acetic acid
solution.
The transitional metals complexes of Pt (II) and Ni (II) with
naphthoquinonic ligands were prepared by the procedure
described by Jensen and Nielsen [39]. The complexes are micro-
crystalline powders, with melting points higher than that of the
pure ligand (Tables 1 and 2). They are air-stable, insoluble in ordi-
nary organic solvents (such as chloroform, acetone, ether, ethanol,
or carbon tetrachloride), sparingly soluble in dichlorethane and
dioxane and soluble in dimethylformamide (DMF) or dime-
thylsulphoxide (DMSO).
The absorption bands due to vibrations groups not involved in
the coordination appear in the infrared spectra of the free ligand
and of the complex in the same spectral regions, with unchanged or
at most slightly modified intensities, because of the electromeric
effects due to the coordination. The peaks assignment was made
according to Pretsch et al. [40]. The characteristic bands of the NeH,
CeN, CeS and SeH groups, which appear in the IR spectra of the
ligand as intense or medium intense bands in the complex spectra
the bands are modified in intensity and appear at modified
frequencies or they cannot be detected, proving the involvement of
these groups in the coordination. [Pt(AMNQ)
2 2
] and [Ni(MNBNQ) ]
are also characterized by the appearance of new bands at
ꢀ
1
ꢀ1
3
867.15 cm
and 3742.10 cm , respectively, which can be
assigned to
n(MeN) frequencies. So, IR spectra indicate the partic-
ipation of both sulphur and nitrogen atoms in coordination to the
metal.
In order to establish the coordination geometry of the new
complexes, a spectral analyses in the visible and UV range was
performed. The bands observed in the electronic absorption spectra
of the studied complexes were assigned according to Gray and
Ballhausen [41] and Vanquickenborne [42]. The absorption bands
that appear in the UV range are considered to be the band for the
ligands.
3
.2. Spectra interpretation
In order to explain the structures of the metal complexes, we
must admit that AMNQ and MNBNQ act as bidentate ligands. The
assumption has been confirmed by the infrared spectra for the free
ligands and complex compounds. These results are showed in
Tables 3 and 4.
Fig.
4
presents the electronic spectra for AMNQ and
]. The absorption spectrum of AMNQ presents a band
[Pt(AMNQ)
2
2
Fig. 6. Configuration of [Pt(AMNQ) ].

18
V.-C. Niculescu et al. / Journal of Organometallic Chemistry 700 (2012) 13e19
Fig.
Ni(MNBNQ)
centred at w395 nm that is attributed to a
5
presents the electronic spectra for MNBNQ and
]. The electronic spectrum of MNBNQ presents a band
* transition. The
] complex presents a band at 375 nm. Most of the Ni
II) complexes present strong absorption in the
[
2
p
/ p
[Ni(MNBNQ)
2
(
1
2
a
ꢀ
ꢀ
1
5 000e25 000 cm (666e400 nm) region and another one in the
3 000e30 000 cm (435e333 nm) interval. The square-planar Ni
1
(
II) complexes differ from the octahedral or tetrahedral complexes
ꢀ1
because no electronic transitions occur below 10 000 cm . This is
a consequence of the large field splitting in a square complex; the
energy separation between the dx2ey2 orbital and the next lowest
ꢀ
1
orbital is invariably greater than 10 000 cm
.
The transitions assignments prove the involvement of the CeS
group to coordination. This conclusion is sustained also by the
absorption maxima shift in the complexes, for the
tion, according to a hypsochromic effect.
p / p* transi-
The structures of the complexes were explained using the
molecular orbital approach. The results of the spectral interpreta-
tion confirmed the proposed structures for the new synthesized
ligands and also the square-planar configuration for the transitional
2
metal complexes. These complexes are the [ML ] type, where
2
Fig. 7. Configuration of [Ni(MNBNQ) ].
M ¼ Pt (II) or Ni(II) and L ¼ AMNQ or MNBNQ (Figs. 6 and 7). In
order to explain this composition, we must admit that AMNQ and
MNBNQ act as bidentate ligands.
with a maximum at 410 nm, which is characteristic to a
transition of the aromatic moiety. The electronic spectrum of the
complex consists in a broad absorption band in the region
p / p*
2
00e800 nm (most probably this broad band is an overlap of
3.3. Evaluation of cytotoxicity
bands: the band characteristic for the free ligand, the charge
8
transfer band, and ded transitions characteristic for Pt (II) (d ) ion).
To explore the in vitro antitumor activity of novel naph-
thoquinones derivatives, cultures of mouse fibrosarcoma L929 cells
were treated with the two ligands (AMNQ and MNBNQ) and metal
The majority of compounds of Pt (II) are four-coordinated with
a square-planar stereochemistry. According to the literature data
[
41], electronic spectrum of a compound containing Pt (II) shows
complex [Pt(AMNQ) ] for 24 h, and cell viability was determined by
2
8
1
three ded spin allowed transitions (d ion, ground state
A1g).
MTT assay (Fig. 8). Each determination represents the average
2þ
These ones allow ded transitions corresponding to the transitions
from the three lower d levels to the empty dx2ꢀy2 orbital. The
ground state is A1g and the excited states, corresponding to these
means of two replicates. The metal complex of MNBNQ with Ni
could not be tested due to low solubility. Both novel synthesized
naphthoquinonic ligands were cytotoxic for L929 fibrosarcoma cell
line. Samples at high concentration interfere with detection,
resulting in an artificial increase in signal (due to absorbance
overlap), although no sign of reduced MTT crystals were visible at
microscopic examination. The IC50 differed between these two
1
1
1
1
g
transitions are A2g, B1g and E . These three bands are observed in
the region 470e525 nm, 405e448 nm and 335e394 nm and are
attributed to 1
transitions.
A
1g
/
1
A
2g
,
1
A
/
1
B
1g and to
1
A
1g
/
1
E
g
1g
Fig. 8. Cells viability determined by MTT assay.

V.-C. Niculescu et al. / Journal of Organometallic Chemistry 700 (2012) 13e19
19
ligands: 0.0088 mg/mL (95% CI: 0.0074e0.0104) for AMNQ and
[4] M.T. Smith, C.G. Evans, H. Thor, O. Sten, Quinones-induced Oxidative Injury to
Cells and Tissues, in: S. Helmet (Ed.), Oxidative Stress, Academic Press Inc,
London, 1985, pp. 91e110.
0
.0022 mg/mL (95% CI: 0.0018e0.0027) for MNBNQ, respectively.
Similar results were reported by Srinivas et al. [18] with
a structurally related naphthoquinone, plumbagin, in human
cervical cancer cell line, ME-180.
[
[
[
[
[
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furanorpyranringare importantgroupsforcytotoxicitytocancercell
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induced cell death was attributed to the induction of apoptosis. Our
data do not allow for a conclusion regarding the mechanisms by
which the new synthesized compounds AMNQ and MNBNQ exer-
ted their cytotoxic effect on L929 and further studies are needed to
elucidate if cell apoptosis or necrosis occurred. Results of a QSAR
study have shown that the cytotoxic activities of 1,4-naph-
thoquinones depend largely on their hydrophobicity [44].
Also, it has been showed the steric interactions of the substit-
uents at either position-2 or position-6 might be also important for
cytotoxic effect [45].
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[
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[
[
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[
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