Cytotoxicity of new alkylamino- and phenylamino-containing polyfluorinated derivatives of 1,4-naphthoquinone

Article abstract of DOI:10.1016/j.ejmech.2010.02.009

Fluorinated derivatives of 1,4-naphthoquinone are highly potent inhibitors of Cdc25A and Cdc25 phosphatases and growth of tumor cells. Five new N-substituted polyfluorinated derivatives of 2-amino-1,4-naphthoquinone were synthesized and their mutagenic and antioxidant properties in Salmonella cells, as well as cytotoxicity in human myeloma (RPMI 8226), human mammary adenocarcinoma (MCF-7), mouse fibroblasts (LMTK) and primary mouse fibroblast cells (PMF) were studied. 2-tert-Butylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (1) inhibited the growth of normal control and tumor cells at the same concentration. Three compounds: 2-diethylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (2), 2-ethylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (3), 2-phenylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (4) exhibited a 50% decrease in the growth of cancer cells at low and comparable concentrations (2.4-8.6 μM) while being remarkably less cytotoxic toward normal LMTK and PMF cells. Quinones (1)-(4), but not 2-phenylamino-3-methyl-5,6,7,8-tetrafluoro-1,4-naphthoquinone (5), efficiently suppressed spontaneous mutagenesis in Salmonella cells, while all compounds 1-5 decreased the mutagenic effect of H₂O₂ on bacterial cells. Their possible perspectives as anticancer drugs are shortly discussed.

Full text of DOI:10.1016/j.ejmech.2010.02.009

European Journal of Medicinal Chemistry 45 (2010) 2321–2326
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Cytotoxicity of new alkylamino- and phenylamino-containing polyfluorinated
derivatives of 1,4-naphthoquinone
Ol’ga D. Zakharova a, Ludmila P. Ovchinnikova b, Leonid I. Goryunov c, Nadezhda M. Troshkova c,
,b,
Vitalij D. Shteingarts c, Georgy A. Nevinsky a
*
a Institute of Biological Chemistry and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, 8 Lavrentiev Ave., 630090 Novosibirsk, Russia
b Institute of Cytology and Genetics, Siberian Division of Russian Academy of Sciences, 10 Lavrentiev Ave., 630090 Novosibirsk, Russia
c N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Division of Russian Academy of Sciences, 9 Lavrentiev Ave., 630090 Novosibirsk, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
Fluorinated derivatives of 1,4-naphthoquinone are highly potent inhibitors of Cdc25A and Cdc25 phos-
phatases and growth of tumor cells. Five new N-substituted polyfluorinated derivatives of 2-amino-1,4-
naphthoquinone were synthesized and their mutagenic and antioxidant properties in Salmonella cells, as
well as cytotoxicity in human myeloma (RPMI 8226), human mammary adenocarcinoma (MCF-7), mouse
fibroblasts (LMTK) and primary mouse fibroblast cells (PMF) were studied. 2-tert-Butylamino-3,5,6,7,8-
pentafluoro-1,4-naphthoquinone (1) inhibited the growth of normal control and tumor cells at the same
concentration. Three compounds: 2-diethylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (2), 2-
ethylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (3), 2-phenylamino-3,5,6,7,8-pentafluoro-1,4-
naphthoquinone (4) exhibited a 50% decrease in the growth of cancer cells at low and comparable
Received 27 October 2009
Received in revised form
1 February 2010
Accepted 3 February 2010
Available online 10 February 2010
Keywords:
Fluorinated derivatives of
1,4-naphthoquinones
Antitumor
concentrations (2.4–8.6 mM) while being remarkably less cytotoxic toward normal LMTK and PMF cells.
Quinones (1)–(4), but not 2-phenylamino-3-methyl-5,6,7,8-tetrafluoro-1,4-naphthoquinone (5), effi-
ciently suppressed spontaneous mutagenesis in Salmonella cells, while all compounds 1–5 decreased the
mutagenic effect of H2O2 on bacterial cells. Their possible perspectives as anticancer drugs are shortly
discussed.
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
Several of numerous investigated substances were found to
inhibit the enzymes of Cdc25 family [6,7], and NSC 95397 (2,3-
Cyclin-dependent kinases (Cdk) are central regulators of the
eukaryotic cell cycle, which phosphorylate proteins responsible for
the activation of structural and regulatory genes in transitions
between G1, S, G2, and M cell cycle phases. Cdc25A, B and C are
members of the family of dual specificity protein phosphatases
regulating cyclin-dependent kinases by removing phosphate
groups from the cyclin-dependent kinases and thus activating the
cyclin–Cdk complexes, which control the cell cycle progression
[1,2]. Therefore, abnormalities in kinases and phosphatases, which
are the key enzymes of these protein phosphorylation signaling
pathways, are closely linked with many human diseases including
cancer. Cdc25A and Cdc25B have been shown to be overexpressed
in a number of tumors of various origins [3]. The putative
involvement of the Cdc25 phosphatases in tumorigenesis makes
them potential targets for cancer therapy [4,5].
bis[2-hydroxyethylthio]-1,4-naphthoquinone) from the National
Cancer Institute Library was shown to be the most potent Cdc25
inhibitor [8]. p-Naphthoquinones and 7-aminoquinoline-5,8-
quinones are core structures of potential inhibitors of Cdc25
phosphatases [8,9], including NSC 663284 [10]. Previous studies
suggested that quinoline-5,8-quinones can inactivate the Cdc25
family phosphatases either by Michael addition [11] or oxidation of
the catalytic cysteine [12]. Quinoline-5,8-quinone derivatives
substituted at the C2 and C4 positions were shown to effectively
inhibit Cdc25B and cancer cell growth [13].
Overall, in vivo use of quinones presents a major challenge
because of their toxicity [14,15]. Although most quinones have been
reported to inhibit Cdc25 by sulfhydryl arylation at the quinone
nucleus, the redox properties of quinones can also generate reactive
oxygen species (ROS) [14], which may be toxic to normal tissues
and thus reduce the therapeutic utility of quinones. Quinone radi-
cals can also damage DNA and mitochondria through the formation
of H2O2 and other ROS and reactive nitrogen species (RNS) [16–18].
One strategy for overcoming the intrinsic toxicity of quinones
might be to use derivatives that are more stable in their reduced
* Corresponding author. Institute of Biological Chemistry and Fundamental
Medicine, Siberian Division of Russian Academy of Sciences, 8 Lavrentiev Ave.,
630090 Novosibirsk, Russia. Tel.: þ7 383 3356226; fax: þ7 383 3333677.
E-mail address: nevinsky@niboch.nsc.ru (G.A. Nevinsky).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.009

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O.D. Zakharova et al. / European Journal of Medicinal Chemistry 45 (2010) 2321–2326
state and thus are less likely to initiate formation of radicals and
indiscriminately damage cells. Interestingly, the fluorinated
quinone compound F-Cpd5 (5,6,7,8-tetrafluoro-2-(2-mercaptoe-
thanol)-3-methyl-[1,4]-naphtoquinone) in contrast to its parent
compound 2-(2-mercaptoethanol)-3-methyl-[1,4]-naphtoquinone
(Cpd5) was predicted not to generate ROS [15]. The calculated
reduction potential of F-Cpd5 was suggestive of much higher
possible therapeutic index [13]. This was supported by the obser-
vation that F-Cpd5 generated significantly less ROS than Cpd5; F-
Cpd5 was three times more potent than Cpd5 in inhibiting the
hepatoma Hep3B cell growth [18–20]. Also, F-Cpd5 inhibited
mitogen-induced DNA synthesis in normal rat hepatocytes 12-fold
less than in Hep3B cells [19].
quinone 4 (w90% in the product mixture) alongside with the prod-
ucts of its further transformation (w10%). Quinone 4 was isolated by
TLC in 62% yield. After 48 h, quinone 7 with aniline gave compound 5
(w80% in a product mixture) alongside with the products of its
further transformation (w20%). Quinone 5 was isolated by TLC in 26%
yield.
Compounds 1–5 have been characterized by 1H and 19F NMR
spectra, elemental analysis, or high resolution mass-spectroscopy.
Indicative was the position and appearance of the F3 signals of
quinones 1–4 compared with that of the starting quinone 6 (d, ppm
vs. hexafluorobenzene as an internal reference: F2, F3 22.1; F5, F8
25.5; F6, F7 18.6). In the spectrum of quinone 3, this signal was
significantly high-field shifted (d 5.3 ppm) relative to quinone 6 by
The rationale to study inhibitors of Cdc25 phosphatases is
looking for better inhibitors of tumor cells growth having no toxic,
mutagenic or carcinogenic properties. Since all naphthoquinones
including the polyfluorinated compound F-Cpd5 inhibit Cdc25
phosphatases [8–20], it is reasonable to suggest that some new
polyfluorinated naphthoquinones will also inhibit these enzymes
to some extent. As only the data concerning toxic, mutagenic or
carcinogenic properties could provide a solid judgement regarding
possible applications of new compounds as antitumor drugs,
a measurement of their affinity for Cdc25 phosphatases seems to be
of secondary importance. Taking this into account, we have recently
synthesized four new n-butylamino and two sulfur-containing
derivatives of polyfluoro-1,4-naphthoquinone and analyzed their
mutagenic and antioxidant properties using special type of
Salmonella cells, as well as their cytotoxicity in human and mouse
tumor cells, and primary mouse fibroblast cells [21]. Interestingly,
all six compounds demonstrated different antioxidant and muta-
genic properties in the bacterial system and efficiency to suppress
the growth of cancer cells at significantly lower concentrations
than normal cells.
an electron-donating effect of the ethylamino group. Compared to
this, in the spectra of quinones 1, 2 and 4 the F3 signals were
somewhat shifted to the lower field (1 16.5, 2 18.7, 4 28.1 ppm),
presumably due to the greater or lesser suppression of this effect by
the out-of-plane deviation of bulky substituted amino groups by
their steric interaction with F3 and, in the case of 4, by nitrogen
p-
conjugation with the phenyl substituent. In all these cases, the F3
signal looked like a singlet somewhat widened by weak spin
couplings with F5–F8. The F5–F8 signals in the spectra of 1–5 were
located (d
23–27 for F5, F8 and 14–20 ppm for F6, F7) closely to their
counterparts in the 19F NMR spectra of 6 and 7, their multiplicities
being typical for ortho disubstituted tetrafluorobenzenes (3JF5,F6,
3JF6,F7, 3JF7 8 19–20, 4JF5,F7, 4JF5,F8, 4JF6,F8 10–16 Hz). The NH
,F
resonances in the 1H spectra of quinones 1, 3 and 4 appeared as
slightly widened singlets at
d 5.39, 5.74 and 7.13 ppm respectively;
in case of 5, between 7.3 and 7.4 ppm.
3. Biological studies
At the first step, we have analyzed the inhibition of the growth of
four cell lines by quinones 1–5 using tumor cell lines from human
myeloma RPMI 8226, human mammary adenocarcinoma MCF-7,
mouse fibroblasts LMTK and primary mouse fibroblast cell line
(PMF). Fig.1 shows typical curves of inhibition by compounds 1–4 of
the growth of RPMI cells. The results obtained for compounds 1–5
with all types of cells are summarized in Table 1. It can be seen that
quinone 1 demonstrated nearly the same IC50 for cancer cells RPMI
In this study, five new polyfluorinated derivatives of 1,4-naph-
thoquinone containing tert-butylamino, diethylamino, ethylamino,
and phenylamino groups were synthesized and their cytotoxicity
against cancer and normal cells as well as ability to protect bacterial
cell from mutagenesis were compared.
2. Chemistry
and MCF-7 (2.5–3.5
6.3 M) cells. Compound 4 was characterized by the same IC50
values for two types of cancer cells (2.4 M), which was comparable
m
M) and mouse PMF and LMTK (IC50 ¼ 3.1–
Syntheses of compounds 1–4 were based on amino-
m
defluorination of hexafluoro-1,4-naphthoquinone
6 with tert-
m
butyl-, diethyl-, ethylamine and aniline, respectively. Compound 5
was prepared by phenylaminodefluorination of 2-metylpenta-
fluoro-1,4-naphthoquinone (7) (Scheme 1).
Quinone 6 reacted easily with aliphatic amines at 17–27 ꢀC to
afford corresponding 2-alkylaminopentafluoro-1,4-naphthoquin-
ones 1–3. After 20 h with tert-BuNH2, quinone 6 gave a mixture of 1
(w80%) and the starting compound 6 (w20%). Longer reaction times
did not change the percentage of the major product 1, but, besides 6,
products of the further transformations of quinone 1 were also found
in the reaction mixture. Quinone 1 was isolated by TLC in 62% yield.
Quinones 2 and 3 were obtained for 2 h virtually as individual
compounds in practically quantitative isolated yields. The reaction of
starting compound 6 with aniline at room temperature for 15 h gave
Fig. 1. Effects of four fluorinated compounds on the growth of RPMI cells. The average
Scheme 1. Synthesis of compounds 1–5.
error in three experiments for any compound concentration did not exceed 5–15%.

O.D. Zakharova et al. / European Journal of Medicinal Chemistry 45 (2010) 2321–2326
2323
Table 1
Cytotoxicity (IC50) of polyfluorinated derivatives of 1,4-naphthoquinone towards different mammalian cell lines.
Compound
IC50
, mg/mL (mM)
RPMI 8226
MCF-7
LMTK
FMS
1 (2-tert-butylamino-3,5,6,7,8-pentafluoro-
1,4-naphthoquinone)
2 (2-diethylamino-3,5,6,7,8-pentafluoro-
1,4-naphthoquinone)
3 (2-ethylamino-3,5,6,7,8-pentafluoro-
1,4-naphthoquinone)
4 (2-phenylamino-3,5,6,7,8-pentafluoro-
1,4-naphthoquinone)
5 (2-phenylamino-3-methyl-5,6,7,8-tetrafluoro-
1,4-naphthoquinone)
Control compound F-Cpd5: 5,6,7,8-tetrafluoro-
2-(2-mercaptoethanol)-3-methyl-
[1,4]naphtoquinone [21]
1.1 ꢁ 0.2 (3.5 ꢁ 0.6)a
2.2 ꢁ 0.6 (6.9 ꢁ 1.8)
0.7 ꢁ 0.1 (2.4 ꢁ 0.1)
0.8 ꢁ 0.03 (2.4 ꢁ 0.1)
7.5 ꢁ 0.5 (22.4 ꢁ 1.3)
4.7 ꢁ 0.29 (14.8 ꢁ 0.9)
0.8 ꢁ 0.1 (2.5 ꢁ 0.4)
2.0 ꢁ 0.2 (6.3 ꢁ 0.7)
1.0 ꢁ 0.1 (3.1 ꢁ 0.3)
9.0 ꢁ 1.8 (28.2 ꢁ 5.6)
9.0 ꢁ 4.3 (30.9 ꢁ 14.4)
2.5 ꢁ 0.3 (7.4 ꢁ 0.7)
45%b
1.3 ꢁ 0.1 (4.1 ꢁ 0.4)
2.5 ꢁ 1.1 (8.6 ꢁ 1.4)
0.8 ꢁ 0.07 (2.4 ꢁ 0.2)
4.5 ꢁ 0.4 (13.4 ꢁ 1.1)
54.9 ꢁ 6.7 (173.0 ꢁ 21.0)
20.0 ꢁ 3.0 (62.7 ꢁ 9.4)
3.2 ꢁ 0.5 (11.0 ꢁ 0.7)
4.0 ꢁ 0.6 (11.8 ꢁ 1.8)
36.0 ꢁ 6.0 (104.4 ꢁ 17.9)
No inhibit. at 173
m
M
n.d.c
a
Mean ꢁ S.D. from three independent experiments.
b
When the cytotoxicity was low, the percent of inhibition of cell growth at the highest used concentration (25
n.d., not determined.
mg/mL) of the compound was determined.
c
with those for 1, while 50% inhibition of LMTK (IC50 ¼ 11.8
mM) and
FMS (IC50 ¼ 7.4
m
M) cells was observed at its 3–5-fold higher
concentrations. Cytotoxicities of 1 and 3 toward the tumorcells were
dependent upon the cell line. While compound 2 suppressed the
growth of MCF 1.7-fold better (4.1
quinone 3 was 3.6-fold more active in the case of RPMI (2.4
MCF (8.6 M) cells. While both 2 and 3 exhibited comparable IC50
(28.2 and 30.9 M) in the case of FMS cells, quinone 2 was w5.7-fold
m
M) than of RPMI cells (6.9
mM),
mM) than
m
m
less cytotoxic than 3 towards LMTK cells. Overall, compounds 2, 3,
and 4 suppressed the growth of LMTK and FMS cells at the
concentrations 3.1–15.3-fold higher than was required for
suppression of the tumor RPMI and MCF cells, while this difference
for quinone 1 was lower (1.1–2.6-fold) (Table 1).
Compound 5 inhibited the growth of cancer MCF (13.4
mM),
RPMI (22.4 M) and LMTK (104.4 M) cells at different and signif-
m
m
icantly higher concentrations compared with 1–4 (Table 1), but the
growth of normal FMS cells was depressed only by 45% at a high
concentration (74.4 mM). The data indicate that compounds 2–5 can
better suppress the growth of tumor cells than of normal
mammalian cells in comparison with quinone 1, while the ratio of
relative cytotoxicities of these derivatives towards different types of
normal and tumor cells is individual for the every compound.
Recently we have synthesized F-Cpd5 as reported [15] and
compared its growth-inhibiting properties with those for several
new compounds [21]. Three of them: 2-n-butylamino-3,5,6,7,8-
pentafluoro-1,4-naphthoquinone (I), (2,2’-dithiodi-2)-3,5,6,7,8-pen-
tafluoro-1,4-naphthoquinon-2-ylamino)ethane (II), and 2-(2’-
methylthioethyl)amino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (III)
exhibited IC50 3.7–5.9-fold lower than that for F-Cpd5 (IC50
M for MCF cells) [21].
The structures of 1–5 resemble that of F-Cpd5 and above
mentioned naphtoquinones (I–III), therefore one could expect
similar effects of all these compounds on the Cdc25 phosphatases
and growth of tumor cells. For RPMI cancer cells, F-Cpd5 demon-
¼
14.8 ꢁ 0.9
m
M for RPMI and 173.0 ꢁ 21.0
m
strated IC50 ¼ 14.8 ꢁ 0.9
mM [21] comparable with the IC50 for the
least active compound 5 but it was 2.1–6.2-fold worse inhibitor
than quinones 1–4, while all compounds 1–5 inhibited the growth
of MCF cells 13–72-fold better than F-Cpd5 (Table 1).
It is known that some compounds interacting with many cell
targets at the same time may be polyfunctional and possess anti-
oxidant properties, or, on the contrary, may be mutagenic or
carcinogenic [22–24]. Some quinone derivatives can act as top-
oisomerase inhibitors via DNA intercalation and reduction of
quinone moiety by oxidoreductases [25–27] and form ROS/RNS
Fig. 2. Analysis of the mutagenic and antioxidant activity of compounds 1–5 by
a standard Ames test [28] using the S. typhimurium strain TA102 in the absence (A) and
in the presence of 3 mM H2O2 (B). The average error in three experiments for any
compound concentration did not exceed 5–15%. The number of revertants in the
absence of H2O2 was taken for 100%.

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O.D. Zakharova et al. / European Journal of Medicinal Chemistry 45 (2010) 2321–2326
Table 2
IC50 values characterizing suppression of spontaneous and H2O2-induced mutagenesis by polyfluorinated derivatives of 1,4-naphthoquinone.
Compound
IC50, mg/mL (m
M)a
Suppression of spontaneous
mutagenesis (from 100 to 50%)
Suppression of H2O2-induced and spontaneous
mutagenesis
From 150 to 100%
From 100 to 50%
1 (2-tert-butylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone)
2 (2-diethylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone)
3 (2-ethylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone)
4 (2-phenylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone)
5 (2-phenylamino-3-methyl-5,6,7,8-tetrafluoro-1,4-naphthoquinone)
Control compound F-Cpd5: 5,6,7,8-tetrafluoro-2-(2-mercaptoethanol)
-3-methyl-[1,4]naphtoquinone [21]
3.9 ꢁ 0.4 (12.2 ꢁ 1.2)
5.4 ꢁ 0.5 (18.5 ꢁ 1.9)
0.2 ꢁ 0.03 (0.69 ꢁ 0.06)
0.2 ꢁ 0.03 (0.59 ꢁ 0.06)
0.45 ꢁ 0.4 (1.4 ꢁ 0.14)
0.25 ꢁ 0.03 (0.86 ꢁ 0.09)
0.14 ꢁ 0.015 (0.094 ꢁ 0.009)
0.03 ꢁ 0.003 (0.089 ꢁ 0.009)
0.25 ꢁ 0.02 (0.75 ꢁ 0.08)
n.d.c
2.2 ꢁ 0.2 (6.9 ꢁ 0.7)
0.78 ꢁ 0.08 (2.7 ꢁ 0.27)
0.3 ꢁ 0.03 (1.0 ꢁ 0.1)
0.05 ꢁ 0.005 (0.15 ꢁ 0.015)
7.3 ꢁ 0.7 (21.8 ꢁ 2.2)
(0.44)
24% at 50
(0.81)
mg/ml (149 m
M)b
a
Mean ꢁ S.D. from three independent experiments.
b
When the suppression of revertant formation was low, the percent of suppression at the highest used concentration (50
n.d., not determined.
m
g/mL) of the compound was determined.
c
that damage DNA [16,17]. Thus, drug may be more successful when
decreased the frequency of mutants, while at higher concentrations
it was a more effective suppressor of reversions (Fig. 2B). While
the reference F-Cpd5 possessed low activity toward tumor
they are not mutagenic at least at the therapeutic concentrations.
The Salmonella typhimurium strain TA102 is often used both for
evaluation of mutagenicity of different compounds and for detec-
tion of their antioxidant properties as judged from suppression of
spontaneous mutagenesis in this strain and from a decrease in the
mutagenisity of known oxidants, usually H2O2 [28].
The mutagenic and antioxidant activities of compounds 1–5
were first estimated in the Ames test [28] using the S. typhimurium
strain TA102 similar to [29]. The test for mutation induction in the
Ames assay is performed by calculating the reversion frequencies
from histidine auxotrophy to prototrophy in a response to different
mutagenic substances. The compound concentration that causes
50% bacterial revertants growth inhibition (IC50) was determined.
Quinone 5 did not markedly affect the reversion frequencies (24% of
cells (IC50 ¼ 14.8–173
mM), it suppressed efficiently spontaneous
M) and, especially, H2O2-indiced mutagenesis
M). Similar behavior was previously observed for
(IC50 ¼ 0.81
(IC50 ¼ 0.44
m
m
2-(20-methylthioethyl)amino-3,5,6,7,8-pentafluoro-1,4-naph-
thoquinone (VI), which became w20-fold more effective supp-
ressor of H2O2-dependent mutagenesis [21]. One cannot exclude
that compounds 2, 4 and previously described F-Cpd5 and VI
possessing higher activity in the presence than in the absence of
H2O2 can be metabolized or react with components of mammalian
cells or cell peroxides to yield products with a higher protective
function or general cytotoxicity.
The relative cytotoxicities of the compounds under study in the
suppression of all types of mammalian cell growth (Table 1) overall
did not correlate with their ability to decrease the bacterial rever-
tant formation, since quinones 1, 2 and 5 restrained tumor cell
growth better than the formation of bacterial revertants while 3
and 4 showed an opposite tendency (Tables 1 and 2). Taken
together, one may suggest these polyfluorinated naphthoquinones
to play a dual role, being both antioxidants suppressing sponta-
neous and H2O2-induced formation of revertants and potential
inhibitors of cell phosphatases. However, one cannot exclude that
these compounds may be to some extent cytotoxic toward
mammalian and bacterial cells, without being associated with the
inhibition of Cdc25 phosphatases.
According to [18–20], functionalized polyfluoro-1,4-naphtho-
quinones may be more promising inhibitors of Cdc25 as compared
to 1,4-naphthoquinone. It is known that some quinone derivatives
can form ROS/RNS and damage DNA [16,17]. A complete absence of
a compound-dependent increase in the mutant formation in the
presence and in the absence of H2O2 indicates that quinones 1–5 are
not mutagenic themselves and counteract both spontaneous muta-
genesis and the toxic and mutagenic effects of H2O2. It should be
mentioned that compounds 1–4 can be considered effective antiox-
idants, while 5 possesses reduced antioxidant activity.
suppression at 149
icant decrease (50% at at 3.9 ꢁ 0.4 and 5.4 ꢁ 0.5
and 18.5 ꢁ1.9 M, respectively) in the spontaneous appearance of
mutants, while complete suppression of cell growth was observed
at concentrations ꢂ10–50 g/mL (Fig. 2A, Table 2). At the same
time, compounds 3 and 4 displayed the significantly lower and
nearly the same IC50 values (0.2 ꢁ 0.03 g/mL or 0.6–0.7 M) and
completely suppressed the appearance of mutants at approximately
0.5 and 2.5 g/mL (1.6 and 7.4 M), respectively (Fig. 2A, Table 2).
mM), while compounds 1 and 2 caused a signif-
mg/mL or 12.2 ꢁ 1.2
m
m
m
m
m
m
It is known that some compounds reacting as antioxidants
efficiently decrease the toxic and mutagenic effect of H2O2 [28,29].
In the Ames test, H2O2 was added to TA102 cells at the optimal
concentration, 3 mM [28,29], and the test compound concentra-
tions varied (Fig. 2B). At low concentrations (ꢃ0.05
mg/mL or 0.09–
1.4 M), all five 1,4-naphthoquinone derivatives under study
m
suppressed efficiently the H2O2-dependent formation of mutants
from 144 to 100% of revertants that was observed for the control
containing no H2O2. The dependencies observed at higher
concentrations of 1 and 3 (Fig. 2B) were comparable with those
found for 3 and 4 in the absence of H2O2 (Fig. 2A, Table 2). At the
same time, quinones 2 and 4 decreased the formation of revertant
cells from 100 to 50% in the presence of H2O2 at 4–7-fold lower
concentrations (0.78 ꢁ 0.08 and 0.05 ꢁ 0.005,
m
g/mL or 2.7 ꢁ 0.27
Since quinone 1 possesses practically the same cytotoxicity
towards cancer and control normal mammalian cells, it cannot be
considered promising as an inhibitor of Cdc25 and a specific
suppressor of cancer cell growth.
and 0.15 ꢁ 0.055 M, respectively) than in the absence of hydrogen
m
peroxide. Interestingly, while without H2O2 compound 1 was
a better suppressor of spontaneous revertant formation than 2, in
the presence of H2O2 a reverse situation was observed (Fig. 2, Table
2). Compound 5, demonstrating no detectable effect on the
frequency of the spontaneous cell reversion up to high concentra-
4. Conclusion
tion, 50
m
g/mL (149
m
M) (Fig. 2A), was a very good suppressor of
Polyfluorinated derivatives of 1,4-naphthoquinone are less
active in generation of ROS and may be promising inhibitors of
Cdc25 as compared to 1,4-naphthoquinone itself [15,18–20]. Our
H2O2-dependent mutant formation and effectively decreased it
from 144 to 100% at 0.25
mg/mL (0.75
mM) (Fig. 2B). At conce-
ntrations 0.5–5.0 g/mL, compound
m
5
only slightly (w10%)
data indicate that compounds
3
(2-ethylamino-3,5,6,7,8-

O.D. Zakharova et al. / European Journal of Medicinal Chemistry 45 (2010) 2321–2326
2325
pentafluoro-1,4-naphthoquinone) and 4 (2-phenylamino-3,5,6,7,8-
pentafluoro-1,4-naphthoquinone) show the best potential, exerting
a stronger cytotoxic effect against cancer cells in comparison with
normal mammalian cells (Table 1) and efficiently protecting
bacterial cells from mutagenesis both in the presence and the
absence of H2O2, all investigated properties of these two
compounds being better than for F-Cpd5, but comparable with the
analogous properties of previously described naphthoquinones (I)
and (II). Compound 2 is of lower efficiency in both cases (Table 2).
Quinone 5 is unlikely a promising candidate for being an anticancer
drug owing to its low activity in restraining the growth of cancer
cells and suppressing reversions in bacterial cells.
From quinone 6 and aniline (0.067 g, 0.719 mmol), 2-phenyl-
amino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone 4 (0.136 g, 62%)
was obtained after purification by TLC (Silufol, CHCl3–CCl4, 1:1) as
wine-red crystals, mp 211–212 ꢀC. 19F NMR: /ppm 28.1 (br s, F3),
d
26.6 (dt, F8), 24.9 (dt, F5), 20.2 (ddt, F6, J6,3 ¼ 4.5 Hz),16.3 (dt, F7), J5,6
,
J6,7, J7,8 19.2–20.1 Hz, J5,7, J5,8, J6,8 10.8–12.7 Hz; 1H NMR:
d/ppm
7.34–7.42 (m, 2H, 2CH), 7.20–7.27 (m, 1H, CH), 7.09–7.17 (m, 2H,
2CH), 7.13 (br s, 1H, NH). HRMS (Mþ$
339.03186, found 339.03150.
, C14H10F5NO2) calcd
A mixture of quinone 7 (0.200 g, 0.763 mmol), aniline (0.142 g,
1.526 mmol) and dioxan (2 mL) was stirred for 48 h and worked up
as mentioned above. 2-Phenylamino-3-methyl-5,6,7,8-tetrafluoro-
1,4-naphthoquinone 5 (0.067 g, 26%) was obtained after purifica-
tion by TLC (Silufol, CHCl3–CCl4, 1:2) as red crystals, mp 155–156 ꢀC.
5. Experimental protocols
19F NMR:
d
/ppm 25.2 (dt, F5), 23.5 (ddd, F8),18.8 (dt, F7),14.6 (dt, F6),
J5,6, J6,7, J7,8 19.5–19.9 Hz, J5,7, J6,8 15.5–12.8 Hz, J5,8, 10.0 Hz; 1H NMR:
5.1. Chemistry
d/ppm 7.32–7.42 (m, 3H, 2CH, NH), 7.13–7.23 (m, 1H, CH), 6.95–7.05
(m, 2H, 2CH), 1.72 (s, 3H, CH3). HRMS (Mþ$, C17H9F4NO2) calcd
335.0569, found 335.0569.
Commercially supplied diethylamine, tert-butylamine, and
aniline were purified by vacuum distillation (0.03 mm Hg); ethyl-
amine hydrobromide was used without purification. Quinone 6 was
prepared according to [30], and compound 7 according to [31].
Dioxan was vacuum distilled (0.1 mm Hg) and dried by molecular
sieves. 19F and 1H spectra were recorded on a Bruker AV-300 NMR
spectrometer at 282 MHz (19F) or 300.13 MHz (1H) and calibrated
according to the chemical shifts of hexafluorobenzene and acetone
or chloroform, respectively. Molecular masses were determined on
a Finnigan MAT-8200 HRMS instrument.
5.2. Biological experiments
5.2.1. Determination of mutagenisity of compounds
In the Ames test, the histidine-dependent strain of S. typhimurium
TA102 was used, which carries a mutation at the histidine operon
[28]. The mutagenic activity of the samples was analyzed by the
standard method without metabolic activation [28]. A liquid
culture of TA102 was obtained by 16-h growth of cells from a frozen
stock at 37 ꢀC in LB medium with penicillin. Then cells were plated
on minimal glucose agar, antibiotics and histidine at the density
sufficient to obtain isolated colonies. A separate bacterial colony
was inoculated into LB medium (5 mL) containing ampicillin
5.1.1. Typical procedure for the synthesis of quinones 1–5
A mixture of ethylamine hydrobromide (0.070 g, 0.556 mmol),
KOH (0.094 g, 1.675 mmol) and dioxan (2 mL) was stirred for
30 min at room temperature. The precipitate was centrifuged,
quinone 6 (0.101 g, 0.379 mmol) was added, and the mixture was
stirred at room temperature for 2 h. The red precipitate was
collected by centrifugation, washed with water, dried and purified
by sublimation (150 ꢀC, 0.03 mm Hg) to give 2-ethylamino-
(50
(130 rpm) for 15 h at 37 ꢀC.
The Ames test was carried out using the double-layer method as
described in [28]. The overnight culture of bacteria (100 l) con-
mg/mL) and tetracycline (2 mg/mL), and grown with shaking
m
taining one of the tested compounds in different concentrations
and, if required, 3 mM H2O2, was mixed at 42 ꢀC with 2 mL of liquid
0.6% top agar. The mixture was poured onto plates with a minimal
medium containing 0.2% glucose and 3% agar, taking care to
distribute the mixture uniformly on the surface of the solid agar.
The plates were incubated for 48 h at 37 ꢀC, and the revertants were
counted. The cells incubated with H2O2 in the absence of
compounds analyzed were used as positive controls, and the cells
grown in the absence of H2O2 and antioxidants served as negative
controls for mutation induction. The results are expressed as
mean ꢁ standard deviation of at least 3 independent experiments.
3,5,6,7,8-pentafluoro-1,4-naphthoquinone
3
(0.095 g, 88%) as
d
wine-red crystals, mp 184–185 ꢀC. 19F NMR:
/ppm 25.8 (dt, F8),
24.2 (ddd, F5), 19.8 (ddt, F6, J6,3 ¼ 4.1), 15.1 (dt, F7), 5.3 (br s, F3), J5,6
,
J6,7, J7,8 19.7–19.8 Hz, J5,7, J5,8, J6,8 10.1–12.1 Hz; 1H NMR:
d/ppm 5.39
(br s, 1H, NH), 3.58 (m, 2H, CH2), 1.29 (dt, 3H, J ¼ 7.2, 0.9 Hz, CH3).
Elemental anal.: calcd for C10H6F5NO2: C, 49.50; H, 2.08; N, 4.81.
Found: C, 49.23; H, 1.85; N, 4.86.
A mixture of quinone 6 (0.174 g, 0.653 mmol), corresponding
amine and dioxan (1.5 mL) was stirred for 20 h (for 1) or 2 h (for 2)
or 15 h (for 4) and poured into water (6 mL). The precipitate was
collected by centrifugation, dried in vacuum (0.03 mm Hg) at room
temperature for 2 h, and purified as described below.
5.2.2. Cytotoxicity assays
From quinone 6 and tert-butylamine (0.086 g, 1.170 mmol), 2-
tert-butylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone 1 (0.128 g,
62%) was obtained after purification by TLC (Silufol, CHCl3–hexane,
Tumor cell lines from human myeloma RPMI 8226, human
mammary adenocarcinoma MCF-7, mouse fibroblasts LMTK and
primary mouse fibroblast cell line (PMF) (w2000 cells per well)
were incubated for 24 h at 37 ꢀC in IMDM or RPMI 1640 medium
(5% CO2) and then were treated with compounds 1–5. After 72 h of
cell incubation, the relative amount of live cells was determined
3:1) as orange crystals, mp 186–190 ꢀC.19F NMR: /ppm 26.0 (dt, F8),
d
24.2 (ddt, F5, J5,3 ¼ 2.0),19.8 (ddt, F6, J6,3 ¼ 4.5),16.5 (br s, F3),15.1 (dt,
F7), J5,6, J6,7, J7,8 19.4–19.8 Hz, J5,7, J5,8, J6,8 10.1–12.3 Hz; 1H NMR:
d/
ꢄ
ppm 5.74 (br s,1H, NH),1.43 (s, 9H, 3CH3). HRMS (Mþ , C14H10F5NO2)
calcd 319.06316, found 319.06247.
using
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl
tetrazolium
bromide (a standard colorimetric MTT-test [32]) and the drug
concentration that caused 50% cell growth inhibition (IC50) was
determined. The results are expressed as mean ꢁ standard devia-
tion of at least 3 independent experiments.
From quinone 6 and diethylamine (0.071 g, 0.971 mmol), 2-
diethylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone 2 (0.200 g,
96%) was obtained as dark-red crystals, mp 90–92 ꢀC. 19F NMR:
d/
ppm 22.9 (m, F8), 22.7 (m, F5), 18.7 (br s, F3), 17.4 (ddt, F6, J6,3 ¼ 3.8),
15.1 (dt, F7), J5,6, J6,7, J7,8 18.7–19.4 Hz, J5,7, J6,8 8.8–9.1 Hz; 1H NMR:
d/
Acknowledgments
ppm 3.45 (dq, 4H, J ¼ 7.0, 1.9 Hz, 2CH2), 1.26 (dt, 6H, J ¼ 0.6,
ꢄ
7.0 Hz, 2CH3). HRMS (Mþ , C14H10F5NO2) calcd 319.06316, found
This research was made possible in part by grants from the
Presidium of the Russian Academy of Sciences (‘‘Molecular and
319.06220.

2326
O.D. Zakharova et al. / European Journal of Medicinal Chemistry 45 (2010) 2321–2326
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