2,3-Disubstituted-1,4-naphthoquinones, 12H-benzo[b]phenothiazine-6,11-diones and related compounds: Synthesis and Biological evaluation as potential antiproliferative and antifungal agents

Article abstract of DOI:10.1016/j.ejmech.2008.06.025

A series of 2-chloro-3-arylsulfanyl-[1,4]naphthoquinones (2), 2,3-bis-arylsulfanyl-[1,4]naphthoquinones (3) and 12H-benzo[b]phenothiazine-6,11-diones and their analogs 6-8 were synthesized and evaluated for their antiproliferative activity against human c

Full text of DOI:10.1016/j.ejmech.2008.06.025

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European Journal of Medicinal Chemistry 44 (2009) 1086e1092
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Original article
2,3-Disubstituted-1,4-naphthoquinones, 12H-benzo[b]phenothiazine-6,
11-diones and related compounds: Synthesis and Biological
evaluation as potential antiproliferative and antifungal agents
a
a
b
Vishnu K. Tandon a, , Hardesh K. Maurya , Ashutosh Tripathi , G.B. ShivaKeshava ,
*
Praveen K. Shukla b, Pallavi Srivastava c, Dulal Panda
c,**
a Department of Chemistry, University of Lucknow, Lucknow 226007, India
b Division of Fermentation Technology, Central Drug Research Institute, Lucknow 226001, India
c School of Biosciences and Bioengineering, Indian Institute of Technology, Bombay, Powai, Mumbai 400076, India
Received 7 January 2008; received in revised form 14 June 2008; accepted 20 June 2008
Available online 2 July 2008
Abstract
A series of 2-chloro-3-arylsulfanyl-[1,4]naphthoquinones (2), 2,3-bis-arylsulfanyl-[1,4]naphthoquinones (3) and 12H-benzo[b]phenothia-
zine-6,11-diones and their analogs 6e8 were synthesized and evaluated for their antiproliferative activity against human cervical cancer
(HeLa) cells. Compounds 3a and 3b were found to possess most potent antiproliferative and cell killing ability. Compounds 1e8 were also
evaluated for antifungal activities. The structureeactivity relationship of these compounds was studied and the results show that compound
2a (MIC50 ¼ 1.56 mg/mL) exhibited in vitro potent antifungal activity compared to the clinically useful antifungal drug Fluconazole
(MIC50 ¼ 2.0 mg/mL) against Sporothrix. schenckii. Compound 2a (MIC50 ¼ 1.56 mg/mL) also exhibited same antifungal activity compared
to clinically useful drug Amphotericin-B (MIC50 ¼ 1.56 mg/mL) against Trichophyton. mentagraphytes.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Antiproliferative; 1,4-Naphthoquinone derivatives; Cytotoxic, human cervical cancer (HeLa) cells; Antifungal
1. Introduction
The recent pharmacophore modelling approach and three-
dimensional quantitative structureeactivity relationship (3D-
QSAR)/comparative molecular similarity indices analysis
(CoMSIA) methods applied to 2,3-disubstituted-1,4-naphtho-
quinones and heterocyclic 1,4-naphthoquinones I (Fig. 1) in
human promyelocytic leukemia HL-60 cell line have explained
the pronounced cytotoxic activity of these derivatives [1]. The
spirohydantoin derivatives II (Fig. 1) possessing two nitrogen
atoms in the same ring and sulfur atom in the dihydro-
thieno[2,3-b]naphtha-4,9-dione system were found to possess
potential cytoxic activity [10].
The profound biological activity exhibited by naphtho[2,3-
b][1,4]-thiazine-5,10-diones III (Fig. 1) by incorporation of
additional nitrogen or sulfur atoms in six-membered hetero-
cyclic ring retaining the quinone chromophore has led to
development of lead molecule III [6].
In recent years increasing recognition of biological activi-
ties of natural and synthetic 1,4-naphthoquinone derivatives
has stimulated enormous interest in this class of compounds
[1e7]. The clinical importance of this class of compounds
has led to the development of new agents in which retaining
the core 1,4-naphthoquinone moiety could exhibit variety of
biological effects including cytotoxic [8e11], antiviral [12],
molluscidal [13], anti-inflammatory, antiplatelet, antiallergic
[14,15], antimalarial [16], antileishmanial [17], antibacterial,
antifungal [18e27] and antiproliferative activities [28].
* Corresponding author. Tel.: þ91 9415066847; fax: þ91 522 26848.
** Corresponding author.
E-mail address: vishnutandon@yahoo.co.in (V.K. Tandon).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.06.025

V.K. Tandon et al. / European Journal of Medicinal Chemistry 44 (2009) 1086e1092
1087
R
insertion reaction of nitrene 5 with benzene moiety occurred
to form cyclized products 6e8. The regioselective intramolec-
ular CeH bond insertion reaction of nitrene with aromatic
compounds has earlier been reported by Kim et al. [29]. All
synthesized compounds were characterized by H NMR and
IR spectral data and elemental analytical data.
R
O
O
N
O
O
O
S
NH
CH3
OH
S
O
R
1
N
HN
N
CH3
O
I
II
III
2.2. Antiproliferative activities
Fig. 1. Lead antiproliferative [10] and antifungal agents [6].
The cells undergo many morphological and biochemical
changes during apoptotic cell death. Annexin V is known to
bind to the exposed phosphotidylserine residue in the early
stage of apoptosis [33]. Propidium Iodide (PI) binds to
DNA; however, it enters into the cells only after the disruption
of the plasma membrane [34]. FITC-conjugated Annexin V
and PI were used to stain the early apoptotic cells and the
late apoptotic/necrotic cells, respectively.
In the course of a medicinal chemistry program aimed at
discovering 1,4-naphthoquinone derivatives containing sulfur
and nitrogen atoms endowed with cytotoxic activity, we have
synthesized a series of 2-chloro-3-arylsulfanyl-[1,4]naphtho-
quinones 2, 2,3-bis-arylsulfanyl-[1,4]naphthoquinones 3,
12H-benzo[b]phenothiazine-6,11-diones (6,7) and 7H-14-
thia-7-aza-benzo[a]naphthacene-8,13-dione (8).
The effects of compounds 1e8 on the proliferation of cer-
vical cancer (HeLa) cells were determined by incubating the
HeLa cells with different concentrations (1, 5 and 20 mM) of
compounds 1e8 for 24 h. The inhibitory effects of these
compounds on the proliferation of HeLa cells are provided
in Table 1. Some of the compounds inhibited proliferation
strongly. Compounds 3a and 3b were found to be the most ac-
tive compounds showing 100% inhibition of HeLa cell prolif-
eration at 20 mM concentration, while Noscapine [38] showed
50% inhibition of HeLa cell proliferation at 34 mM concentra-
tion. In the presence of 3a and 3b, HeLa cells were found to be
stained with both of the dyes indicating that these agents
induce apoptotic death in HeLa cell (Fig. 2). Further, the
cells showed characteristic of apoptosis, i.e. cell rounding,
swelling and blebbing in the presence of these compounds.
To find out the mechanism of cell death, we analyzed the ef-
fects of 3a and 3b on the organizations of cellular microtubules
and chromosomes of the HeLa cells by fluorescence micros-
copy. These compounds did not affect the cellular microtubules
and chromosomes of the HeLa cells (data not shown). Further,
3a and 3b did not block cell cycle progression at mitosis. For
example, percentage of cells in mitosis were found to be
3.4 ꢀ 0.8, 3.5 ꢀ 1.1, 3.8 ꢀ 0.9 and 4.2 ꢀ 1.2 in the absence
and presence of 5, 10 and 20 mM of 3b compound.
The study of structureeactivity relationship of 1e8 re-
vealed that 2,3-bis-arylsulfanyl substituted-[1,4]naphthoqui-
nones 3a and 3b showed pronounced antiproliferative
activity against human cervical cancer (HeLa) cells referred
in Table 1, than the cyclic analogs 6e8. The introduction of
methoxy group in aromatic ring enhances antiproliferative
activity at concentration 1 and 5 mM whereas replacement of
phenyl by naphthyl group caused decrease in antiproliferative
activity. The fact that the acyclic analogs are more potent than
the corresponding cyclic analogs has also been reported by
Miguel del Corral et. al. [39].
2. Results and discussion
2.1. Chemistry
The precursors, 2-chloro-3-phenylsulfanyl-[1,4]naphtho-
quinone (2) required for the synthesis of 6e8, have been syn-
thesized according to Scheme 1. The reaction of 2,3-dichloro-
1,4-naphthoquinone (1) with aryl thiols afforded a mixture of
2-chloro-3-arylsulfanyl-[1,4]naphthoquinones (63e80%) (2)
and 2,3-bis-arylsulfanyl-[1,4]naphthoquinones (20e37%) (3).
This reaction involves nucleophilic displacement of Cl atom
in 2,3-dichloro-1,4-naphthoquinone (1) with sulfur nucleo-
phile resulting in the formation of 2. Nucleophilic displace-
ment reaction of 2 with arylthiols (1:2) resulted exclusively
in the formation of 2,3-bis-arylsulfanyl-[1,4]naphthoquinones
(3) as exhibited in Scheme 1.
The synthesis of 12H-benzo[b]phenothiazine-6,11-diones
(6,7) and their analog 7H-14-thia-7-aza-benzo[a]naphtha-
cene-8,13-dione (8) was carried out according to Scheme 2.
However, the nucleophilic displacement reaction of 2 with
NaN3 in DMFeH2O (10:1) afforded 12H-benzo[b]phenothia-
zine-6,11-diones and their analogs 6e8 as the only isolated
products as exhibited in Scheme 2. The mechanism of forma-
tion of 6e8 from 2 involves nucleophilic displacement of Cl
by N3 followed by elimination of N2 and formation of nitrene
5. Intramolecular cyclization further leads to formation of 6e
8. In these reactions regioselective intramolecular CeH bond
O
O
O
Cl
SAr
SAr
SAr
(i)
+
ArSH
+
Cl
Cl
O
O
(a) Ar=C6H5
(b) Ar=3-OMe-C6H5
(c) Ar=1-C10H7
O
3
1
2
2.3. Antifungal activity
2 ArSH
(i)
Comparison of antifungal activity of compounds 1e8 re-
ferred in Table 2 with that of antifungal drug Miconazole
Scheme 1. Reagents and conditions: (i) EtOH, room temp.

1088
V.K. Tandon et al. / European Journal of Medicinal Chemistry 44 (2009) 1086e1092
O
O
O
O
S
R
S
S
R
R
(i)
N
N
N
Cl
N
N
N
O
O
2
4
(a) Ar=C6H5
(b) Ar=3-OMe-C6H5
(c) Ar=1-C10H7
-N2
O
O
O
O
5
S
7
4
6a
4a
5a
3
R
S
S
8
9
6
R
12
5c
11
2
12a
11a
6-7
10a
N
N
1
10
N
H
H
O
O
5
8
(6) R=H,
(7) R=OCH3,
Scheme 2. Reagents and conditions: (i) NaN3, DMF, H2O, D.
showed that compound 2a had better activity against Candida.
albicans and Cryptococcus neoformans and had same antifungal
profile against Aspergillus fumigatus while compound 2b had
same activity against C. albicans. On comparison of antifungal
activity with that of antifungal drug Nystatin, compound 2a had
better activity against Sporothrix schenckii and significant activ-
ity against C. albicans. Compound 2c also exhibited better ac-
tivity against S. schenckii when compared with Nystatin.
Compound 2a exhibited better activity than clinically prevalent
antifungal drug Fluconazole against S. schenckii. Compound 2a
also exhibited similar antifungal activity when compared with
Amphotericin-B against Trichophyton mentagraphytes.
Structureeactivity relationship in 1e8 revealed that pres-
ence of chlorine atom in 1,4-naphthoquinone nucleus seemed
essential for potent antifungal activity as revealed in Table 2 in
compounds 2(aec). Replacement of chlorine atom by SAR in
3(aec) as well as their cyclic analogs 6e8 did not result in an-
tifungal activity. Replacement of one chlorine atom by phenyl
group in 1 resulted in profound antifungal activity leading to
formation of 2a compared to replacement by 3-methoxy phe-
nyl and 1-naphthyl substituent leading to formation of 2b and
2c respectively.
Table 1
Inhibition of cell proliferation by compounds 1e8
Compound
Conc. (mM)
Inhibition (%)
SD (ꢀ)
2a
2a
1
5
15
29
73
39
48
74
0
2.6
3.4
5.8
2.7
3.5
7.6
0
2a
2b
20
1
2b
2b
5
20
1
2c
2c
5
22
69
51
60
100
64
76
100
22
30
46
5.5
6.5
42
8.5
20
77
12
19
42
28
43
49
50
5.5
4.4
4.6
3.7
0
2c
3a
20
1
3a
3a
5
20
1
3b
3b
3.8
4.2
0
5
3. Conclusion
3b
3c
20
1
3.8
2.4
3.5
1.2
2.2
4.5
2.5
3.7
5.6
2.7
4.8
6.4
4.2
3.6
4.8
3.7
In conclusion, we have synthesized a series of 2-chloro-3-aryl-
sulfanyl-[1,4]naphthoquinones (2), 2,3-bis-arylsulfanyl-[1,4]naph-
thoquinones (3) and 12H-benzo[b]phenothiazine-6,11-diones and
their analogs (6e8). The promising compounds 3a and 3b
have exhibited potent antiproliferative activity compared to
the other derivatives of the series against cervical cancer
(HeLa) cells. Compound 7 also exhibited significant antipro-
liferative activity but lesser compared to 3a and 3b. Our
results suggest that both 3a and 3b have potent antiprolifera-
tive and cell killing ability. Amongst the most promising
antifungal compounds 2a showed better antifungal activity
than clinically prevalent antifungal drug Fluconazole against
S. schenckii and same antifungal activity when compared
with amphotericin-B against T. mentagraphytes. Further
work is in progress for drug development of compounds 2a,
3a and 3b.
3c
3c
5
20
1
6
6
5
6
20
1
7
7
5
7
20
1
8
8
5
8
20
1
1
1
5
1
Noscapine [38]
20
34
Compounds 1e8 inhibit HeLa cell proliferation in a concentration dependent
manner. Inhibition of proliferation was determined after one cell cycle using
Sulforhodamine B.

V.K. Tandon et al. / European Journal of Medicinal Chemistry 44 (2009) 1086e1092
1089
Fig. 2. Compounds 3a and 3b induce apoptosis in HeLa cells: HeLa cells were incubated with 5 mM 3a or 3b for 24 h and stained with Annexin V and Propidium
Iodide (PI) using Annexin V apoptosis detection kit. Panel 1 shows cell morphology using differential interference contrast (DIC) microscopy. Panel 2 shows An-
nexin V staining, panel 3 shows PI staining and panel 4 is a merged image of panel 2 and panel 3. Bar 20 mm.
4. Experimental
(NMR) spectra were recorded on PerkineElmer model R.32
spectrometer using TMS as an internal reference. All com-
pounds showed satisfactory elemental analysis for C, H and
N. Progress of reactions and purity of compounds were mon-
itored by thin layer chromatography (TLC), which was per-
formed on silica gel G and compounds were detected with
iodine vapors, where required. Spectral analyses and elemental
microanalyses were carried out by SAIF division of Central
Drug Research Institute, Lucknow, India. Most reagents
were purchased from Lancaster, SigmaeAldrich and Merck.
4.1. Materials and methods
The reagents and the solvents used in this study were of an-
alytical grade and were used without further purification. The
melting points were determined on an electrically heated
Townson Mercer melting point apparatus and are uncorrected.
IR spectra were recorded on FTIR 8201 PC, Schimadzu Spec-
trophotometers on KBr discs. Nuclear magnetic resonance
Table 2
Structures and in vitro antifungal activity for compounds 1e8 (MIC: mg/mL)
Compounds
MIC (mg/mL)
C. albicans
C. neoformans
S. schenckii
T. mentagraphytes
A. fumigatus
Candida parapsilosis
2a
2b
6.25
25
50
6.25
25
50
1.56
3.12
6.25
50
1.56
3.12
6.25
>50
50
12.5
25
6.25
25
50
2c
3a
50
>50
>50
>50
>50
>50
>50
0.78
25
>50
>50
>50
>50
>50
>50
3.12
12.5
3.5
>50
>50
>50
>50
>50
>50
1.56
>50
>50
>50
>50
>50
>50
3b
3c
>50
>50
50
>50
>50
50
6
7
>50
50
8
>50
1.56
<0.78
1
0.78
1.56
a
a
Miconazole
Nystatin
12.5
a
a
a
7.8
1.0
13.2
2.0
Fluconazole
Amphotericin-B
1.0
0.78
0.5
1.56
2.0
2.0
a
a
a
0.39
a
Activity not reported.

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V.K. Tandon et al. / European Journal of Medicinal Chemistry 44 (2009) 1086e1092
4.2. General procedure for the synthesis of 2-chloro-3-
arylsulfanyl-[1,4]naphthoquinones (2) and 2,3-bis-
arylsulfanyl-[1,4]naphthoquinones (3)
(s, 6H, 2 ꢃ OCH3), 6.79e7.26 (m, 8H, Ar-H), 7.68 (m, 2H,
C6eH and C7eH), 8.00 (m, 2H, C5eH and C8eH). Anal.
Calcd. for C24H18O4S2 (434.53): C, 66.34; H, 4.18; S, 14.76.
Found: C, 66.56; H, 4.32; S, 14.92.
A
mixture of 2,3-dichloro-1,4-naphthoquinone (1)
(10 mmol) and aryl thiols (12 mmol) in abs. EtOH (50 mL)
was stirred vigorously for 3e8 h at 40 ꢁC. The resulting solu-
tion was concentrated in vacuo and the residue comprising of
2 and 3 was subjected to column chromatography on silica
gel using EtOAc/hexane (1:25) and the product was crystallized
with suitable solvent to give 2 in 63e80% yield. Analogous re-
action of 2,3-dichloro-1,4-naphthoquinone (1) (10 mmol) and
aryl thiols (20 mmol) in abs. EtOH (50 mL) gave the product
3 in 90e95% yield after crystallization with suitable solvent.
4.2.6. 2,3-Bis(naphthalen-1-ylthio)naphthalene-1,4-
dione (3c)
Rf ¼ 0.3; dark orange crystals after crystallization with
EtOAc/hexane; 90% yield; mp 190 ꢁC; IR (KBr): 1591and
1
1665 (pC]O of quinone) cmꢂ1; H NMR (CDCl3): d 7.44
(m, 6H, Ar-H), 7.69 (m, 4H), 7.80 (m, 2H, C6eH and C7e
H), 7.98 (m, 2H, C5eH and C8eH), 8.09 (m, 4H, Ar-H).
Anal. Calcd. for C30H18O2S2 (474.59): C, 75.92; H, 3.82; S,
13.51. Found: C, 75.89; H, 3.78; S, 13.48.
4.2.1. 2-Chloro-3-phenylsulfanyl-[1,4]naphthoquinones (2a)
Rf ¼ 0.4; orange needles after crystallization with EtOAc/
hexane; 80% yield; mp 124 ꢁC; IR (KBr): 1590 and 1666
4.3. General procedure for the synthesis of 12H-
benzo[b]phenothiazine-6,11-diones and 7H-14-thia-7-
aza-benzo[a]naphthacene-8,13-dione (6e8)
1
(pC]O of quinone) cmꢂ1; H NMR (CDCl3): d 7.27 (s, 3H,
Ar-H), 7.40 (m, 2H, Ar-H), 7.65 (m, 2H, C6eH and C7eH),
8.08 (m, 2H, C5eH and C8eH). Anal. Calcd. for C16H9ClO2S
(300.76): C, 63.90; H, 3.02; S, 10.66. Found: C, 63.68; H,
2.98; S, 10.58; Beilstein test: [35] Cl positive.
Sodium azide (30 mmol) was added to a stirred solution of
2-chloro-3-arylsulfanyl-[1,4]naphthoquinones (2) in DMF
(15 mL) and H2O (1.5 mL). The reaction mixture was stirred
for 4 h at 100 ꢁC. The resulting solution was poured onto
crushed ice (50 g) and the solid precipitate was filtered, dried
and subjected to column chromatography on silica gel using
EtOAc/hexane (1:15) and the product was crystallized with
suitable solvent to give 6e9 in 67e85% yield.
4.2.2. 2-Chloro-3-(3-methoxy)phenyl sulfanyl-[1,4]napht-
hoquinones (2b)
Rf ¼ 0.5; orange needles after crystallization with EtOAc/
hexane; 63% yield; mp 108 ꢁC; IR (KBr): 1589 and 1665
1
(pC]O of quinone) cmꢂ1; H NMR (CDCl3): d 3.79 (s, 3H,
4.3.1. 6H-Benzo[b]phenothiazine-6,11(12H )-dione (6)
Red crystals after crystallization with EtOAc/hexane; 85%
yield; mp 110 ꢁC; IR (KBr): 1592 and 1665 (pC]O of qui-
;
none), 3305 (NeH) cmꢂ1 1H NMR (CDCl3): d 6.00 (bh,
1H, NH), 7.32 (m, 2H, Ar-H), 7.49 (m, 2H, Ar-H), 7.71 (m,
2H, C6eH and C7eH), 8.15 (m, 2H, C5eH and C8eH).
Anal. Calcd. for C16H9NO2S (279.31): C, 68.80; H, 3.25; N,
5.01; S, 11.48. Found: C, 68.78; H, 3.22; N, 4.97; S, 11.51.
OCH3), 6.85e7.20 (m, 4H, Ar-H), 7.73 (m, 2H, C6eH and
C7eH), 8.15 (m, 2H, C5eH and C8eH). Anal. Calcd. for
C17H11ClO3S (330.79): C, 61.73; H, 3.35; S, 9.69. Found:
C, 61.92; H, 3.52; S, 9.80; Beilstein test: [35] Cl positive.
4.2.3.
dione (2c)
Rf ¼ 0.5; orange crystals after crystallization with EtOAc/
hexane; 65% yield; mp 180 ꢁC; IR (KBr): 1586 and 1662
(pC]O of quinone) cmꢂ1 1H NMR (CDCl3): d 7.50 (m,
2-Chloro-3-(naphthalen-1-ylthio)naphthalene-1,4-
4.3.2.
dione (7)
3-Methoxy-6H-benzo[b]phenothiazine-6,11(12H )-
;
3H, Ar-H), 7.77 (m, 4H, Ar-H), 7.98 (m, 2H, C6eH and
C7eH), 8.17 (m, 2H, C5eH and C8eH). Anal. Calcd. for
C20H11ClO2S (350.82): C, 68.47; H, 3.16; S, 9.14. Found:
C, 68.50; H, 3.14; S, 9.10; Beilstein test: [35] Cl positive.
Red crystals after crystallization with EtOAc/hexane; 79%
yield; mp 145e147 ꢁC; IR (KBr): 1594 and 1662 (pC]O of
quinone), 3310 (NeH) cmꢂ1; 1H NMR (CDCl3): d 3.79 (s, 3H,
OCH3), 6.00 (bh, 1H, NH), 6.77 (d, 1H, J ¼ 6.5 Hz, C1eH),
7.08 (m, 1H, C3eH), 7.38 (m, 1H, C2eH), 7.63 (m, 2H,
C6eH and C7eH), 8.09 (m, 2H, C5eH and C8eH). Anal.
Calcd. for C17H11NO3S (309.34): C, 66.01; H, 3.58; N, 4.53;
S, 10.37. Found: C, 59.98; H, 3.60; N, 4.50; S, 10.40.
4.2.4. 2,3-Bis(phenylthio)naphthalene-1,4-dione(3a)
Rf ¼ 0.3; dark orange crystals after crystallization with
EtOAc/hexane; 95% yield; mp 150 ꢁC; IR (KBr): 1591and
1
1660 (pC]O of quinone) cmꢂ1; H NMR (CDCl3): d 7.34
(m, 10H, Ar-H), 7.68 (m, 2H, C6eH and C7eH), 7.99 (m, 2H,
C5eH and C8eH). Anal. Calcd. for C22H14O2S2 (374.48): C,
70.56; H, 3.77; S, 17.13. Found: C, 70.52; H, 3.74; S, 17.09.
4.3.3. 7H-14-Thia-7-aza-benzo[a]naphthacene-8,13-
dione (8)
Red crystals after crystallization with EtOAc/hexane; 78%
yield; mp 151 ꢁC; IR (KBr): 1592 and 1665 (pC]O of
4.2.5. 2,3-Bis(3-methoxyphenylthio)naphthalene-1,4-
dione (3b)
1
quinone), 3304 (NeH) cmꢂ1; H NMR (CDCl3): d 6.12 (br
Rf ¼ 0.3; dark orange crystals after crystallization with
s, 1H, NH), 7.40 (m, 4H, naphthyl), 7.70 (m, 2H, naphthyl-
H), 7.78 (m, 2H, C6eH and C7eH), 8.19 (m, 2H, C5eH and
C8eH). Anal. Calcd. for C20H11NO2S (329.37): C, 72.93; H,
EtOAc/hexane; 93% yield; mp 80 ꢁC; IR (KBr): 1589 and
1
1662 (pC]O of quinone) cmꢂ1; H NMR (CDCl3): d 3.77

V.K. Tandon et al. / European Journal of Medicinal Chemistry 44 (2009) 1086e1092
1091
3.37; N, 4.25; S, 9.74. Found: C, 73.18; H, 3.54; N, 4.34; S,
9.92.
chem. Co. MO, USA) for fungal strains. The concentration
range of tested compounds was 50e0.36 mg/mL for standard
compounds. Initial inoculums of fungal strain were maintained
at 1e5 ꢃ 103 cells/mL. These plates were incubated in a moist
chamber at 35 ꢁC and absorbance at 492 nm was recorded on
Versa Max micro plate reader (Molecular devices, Sunnyvale,
USA) after 48 h for C. albicans and C. parapsilosis, 72 h for
A. fumigatus, S. schenckii and C. neoformans and 96 h for T.
mentagraphytes while bacterial strains were incubated for
24 h. MIC was determined as 90% inhibition of growth with re-
spect to control was observed by using SOFTmax Pro 4.3 Soft-
ware (Molecular Devices, Sunnyvale, USA).
4.4. In vitro antiproliferative assay
The evaluation of antiproliferative activities of compounds
1e8 against human cervical cancer (HeLa) cells was accom-
plished by cell culture, immuno-fluorescence microscopy
and Annexin/Propidium Iodide staining as shown below.
4.4.1. Cell culture
Human cervical cancer (HeLa) cells were grown in Eagle’s
minimal essential medium (Himedia) with 10% (v/v) fetal bo-
vine serum at 37 ꢁC in a humidified atmosphere of 5% CO2.
All compounds were dissolved in DMSO and the final concen-
tration of DMSO was less than 0.1%. Cells were seeded at
1 ꢃ 105 cells/mL in 96-well tissue culture plates. After 24 h,
medium was replaced by fresh medium containing DMSO as
vesicle (control) or compounds 1e8 and was incubated for
one cell cycle (24 h). The cells were stained with 0.4% Sulfo-
rhodamine B, and the concentration of the protein-bound dye
was detected by measuring the absorbance at 560 nm [30].
Acknowledgements
We thank Director, SAIF of Central Drug Research Insti-
tute, Lucknow, India for spectral, microanalyses data reported
in the manuscript. H.K.M. thanks U.G.C., New Delhi, India
for award of J.R.F/S.R.F and D.P. thanks Swarnajayanti
Fellowships.
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