Cytotoxic activity of 3-(5-phenyl-3H-[1,2,4]dithiazol-3-yl)chromen-4-ones and 4-oxo-4H-chromene-3-carbothioic acid N-phenylamides

Article abstract of DOI:10.1016/j.ejmech.2009.11.001

6/6,7-Substituted-3-formylchromones (8a-g) were reacted with 2 equivalents thiobenzamide (9) in refluxing toluene to furnish substituted-3-(5-phenyl-3H-[1,2,4]dithiazol-3-yl)chromen-4-ones (10a-g) in high yields. Similarly, when substituted-2-anilino-3-formylchromones (8a-d) were reacted with thiobenzamide (9, 2 equivalents) in refluxing xylene, 4-oxo-4H-chromene-3-carbothioic acid N-phenylamides (11a-d) were obtained in high yields. All the compounds (10a-g) and (11a-d) display significant cytotoxic activity against a number of human cancer cell lines. Among these compounds 10e (IC₅₀?=?10?μM), 10b (IC₅₀?=?14.6?μM) and 10a (IC₅₀?=?10.5?μM) showed maximum cytotoxic activity on neuroblastoma. Also, the compound 10c (IC₅₀?=?10.5?μM) showed maximum cytotoxic activity on ovarian cancer cell line.

Full text of DOI:10.1016/j.ejmech.2009.11.001

European Journal of Medicinal Chemistry 45 (2010) 790–794
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Cytotoxic activity of 3-(5-phenyl-3H-[1,2,4]dithiazol-3-yl)chromen-4-ones
and 4-oxo-4H-chromene-3-carbothioic acid N-phenylamides
,
Tilak Raj a, Richa Kaur Bhatia a, Ashish kapur a, Madhunika Sharma b, A.K. Saxena b, M.P.S. Ishar a
*
a Bio-Organic and Photochemistry Laboratory, Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar 143 005, Punjab, India
b Pharmacology Division, Indian Institute of Integrative Medicine, Jammu 180 001, Jammu & Kashmir, India
a r t i c l e i n f o
a b s t r a c t
Article history:
6/6,7-Substituted-3-formylchromones (8a–g) were reacted with 2 equivalents thiobenzamide (9) in
refluxing toluene to furnish substituted-3-(5-phenyl-3H-[1,2,4]dithiazol-3-yl)chromen-4-ones (10a–g) in
high yields. Similarly, when substituted-2-anilino-3-formylchromones (8a–d) were reacted with thio-
benzamide (9, 2 equivalents) in refluxing xylene, 4-oxo-4H-chromene-3-carbothioic acid N-phenyl-
amides (11a–d) were obtained in high yields. All the compounds (10a–g) and (11a–d) display significant
Received 4 August 2009
Received in revised form
26 October 2009
Accepted 2 November 2009
Available online 6 November 2009
cytotoxic activity against
a
number of human cancer cell lines. Among these compounds 10e
M) showed maximum cytotoxic activity on
(IC50 ¼ 10
m
M), 10b (IC50 ¼ 14.6
m
M) and 10a (IC50 ¼ 10.5
m
Keywords:
Dithiazoles
neuroblastoma. Also, the compound 10c (IC50 ¼ 10.5
m
M) showed maximum cytotoxic activity on ovarian
cancer cell line.
Anticancer activity
Chromanyldithiazoles
N-Phenylthioamides
Anticancer agents
Topoisomerase inhibitors
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
antioxidation and reversal of multidrug resistance [27]. For example,
psorospermin (5) a natural antitumor antibiotic [28], which is appar-
entlyanalkylating agent resembling pluramycin A (6), has beenshown
to intercalate with DNA and its alkylating potential is significantly
increased in the presence of topoisomerase-II [29,30] and recently,
chloro/flurochromones (7) have been designed as potential top-
oisomerase inhibitors, which exhibit high anticancer activity against
Ehrlich ascites cancer cells, in vitro, as well as EAC implanted mice [31].
Taking cognizance of high anticancer activity of both five
membered heterocycles, in particular, dithiazoles, and chromones
derivatives, it was decided to synthesize substituted-3-(5-phenyl-
3H-[1,2,4]dithiazol-3-yl)chromen-4-ones (10a–g), possessing both
dithiazole and chromone moieties, and substituted-4-oxo-4H-
chromene-3-carbothioic acid N-phenylthioamide (11a–d) accord-
ing to the earlier reported procedure [32,33] and evaluate their
cytotoxic activity against number of human cancer cell line.
Design, synthesis and evaluation of anticancer agents continues to
be a major area of activity, because, despite the progress made in
chemotherapy of cancer, complete control of malignancies is still
adistinctdream[1–5]. Majoreffortsaredirectedtowardsevaluationof
small molecules with minimal toxicity to normal cells [6–9] and
heterocycliccompoundshave emerged as important candidates in the
treatment of cancers [10–13]. The known anticancer five membered
heterocycles (Fig. 1) include triazole derivative, 3-arylamine-5-
(hetro)aryl-1,2,4-triazole (1), which acts as tubulin polymerization
inhibitorbybindingtothecolchicinesbindingsiteontubulin[14], 3-S-
alkylated-5-(hetero)aryl-1,2,4-triazole (2), a somatostatin, sst2/sst5
binding agonist [15,16], 2-amino-1,3,4-thiadiazole derivative, 2-(4-
flurophenylamino)-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole
(3),
involved with apoptotic mechanisms and angiogenesis [17–24], and
dithiazole derivatives, 4-chloro-5-heteroimmino-1,2,3-dithiazoles
(4a–e) [25].
On the otherhand, chromone and xanthone derivatives (Fig. 2)also
display high anticancer activity [26] with novel mechanisms, such
as carcinogens inactivation, antiproliferation, cell cycle arrest,
induction of apoptosis and differentiation, inhibition of angiogenesis,
2. Results and Discussion
2.1. Chemistry
2.1.1. Synthesis of chromanyl-1,2,4-dithiazoles and N-
phenylthioamides
The substituted-3-(5-phenyl-3H-[1,2,4]dithiazol-3-yl)chromen-
4-ones (10a–g) were obtained in high yield when substituted-3-
* Corresponding author. Tel.: þ91 183 2258802x3321; fax: þ91 183 2258820.
E-mail address: mpsishar@yahoo.com (M.P.S. Ishar).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.11.001

T. Raj et al. / European Journal of Medicinal Chemistry 45 (2010) 790–794
791
H3C
N
N
NH
N
N
H
N
N
N
HN
N
NH
S
H3C
CH3
O
O
O
H2N
O CH3
2
1
NC
N
S
H
a) R=Me, b) R=Ph
c) R=Bn, d) R=Ph(CH2)2
e) R=Ph(CH2)3
N
Cl
HO
N
F
N
R
N N
S
N
OH
S
3
4
Fig. 1. Some five membered heterocycles as anticancer agents.
O
O
O
R1
R2
CH3
F/Cl
O
O
CH3
OH
R
O
O
OCH3
CH3
n
O
X
N
N
/ H
n = 1,2
O
N(CH3)2
n
X
O
O
X = O, N-CH3, NH, CH2
CH3
CO2CH3
OH
O
H
R1, R2 = O, R = H
(H3C)2N
H3C
CH3
R1, R2 = O, R = OH
R1= OH, R2, R = H, Alkyl
O
5
6
7
Fig. 2. Some chromone based anticancer agents.
formylchromones (8a–g, Z ¼ H) were reacted with two equivalents
of thiobenzamide (9) in refluxing toluene. The-oxo-4H-chromene-
3-carbothioic acid N-phenylamides (11a–d) were synthesized by
the reaction of substituted-2-anilino-3-formylchromones (8a–d,
Z ¼ NHPh) with 2 equivalents of thiobenzamide (9) in refluxing
xylene. All the compounds were purified by column chromatog-
raphy (Scheme 1, Table 1) using neutral (pH w 7) silica 60–120
mesh (Loba Cheme, 30 g, packed in hexane) and eluted with 1–5%
ethylacetate in hexane. All the compounds have been characterized
by detailed spectroscopic (IR, 1H and 13C NMR, mass) and elemental
analysis. Structures of compounds 10a and 11c were further
confirmed by X-ray crystallography (CCDC-286229 for 10a and
702090 for 11c) [32,33].
2.2. Pharmacology
In vitro cytotoxic studies of dithiazoles (10a–g) and (11a–d) were
carried out on different cancer cell lines according to the protocol of
Skehan et al. [34–36]. The cytotoxic effects of chromanyldithiazoles
and N-phenylthioamides were observed on colon (COLO-205),
prostrate (PC-3), ovary (OVCAR-5), lungs (A-549), liver (HEP-2) and
neuroblastoma (IMR-32) cancer cell lines. The cytotoxic effects are
reported in terms of % age inhibitory concentration (Table 2) and
IC50 Values (mM), which is the concentration required to inhibit
cancer cell proliferation by 50% after exposure of cells to test
compounds, have also been determined (Table 3).
In the case of colon cell line (COLO-205) the maximum inhibi-
tion 71% (100
m
M) was observed for 11a with IC50 ¼ 72.6 followed
by 57% (IC50 ¼ 76.7) for 10c at same concentration. In the case of
O
S S
N
x1
x2
Z = H
Ph
S
O
Table 1
O
O
2
10
Reaction yield (%) of the products 10 and 11.
x1
x2
Ph
NH2
H
9
Entry
Chromones
Reaction time (h)
Yield of 10 (%)
Z ¼ H
Yield of 11 (%)
Z ¼ NHPh
Xylene reflux
H
N
O
8
z
O
O
x1
1
2
3
4
5
6
7
8a
8b
8c
8d
8e
8f
5
6
6
6
7
7
7
70
70
73
70
74
72
73
70
75
60
78
–
a X1 = H,
c X1 = H,
X2 = H, b X1 = F, X2 = H,
X2 = Cl, d X1 = Cl, X2 = Cl
e X1 = Cl, X2 = H, f X1 = F, X2 = Cl
Z = NHPh
S
x2
11
g
X1 = CH3, X2 = H
–
–
8g
Scheme 1. Synthesis of chromanyl-1,2,4-dithiazoles and N-phenylthioamides..

792
T. Raj et al. / European Journal of Medicinal Chemistry 45 (2010) 790–794
Table 2
In vitro cytotoxicity of compounds (10a–g) and (11a–d) against different human cancer cell lines.
Compounds/
standard
drugs
Conc.
M)
% Growth inhibition against human cancer cell linesa
(
m
COLO-205
Colon
PC-3
Prostate
OVCAR-5
Ovary
A-549
Lung
HEP-2
Liver
IMR-32
Neuroblastoma
10a
10b
10c
10d
10e
10f
10g
11a
11b
11c
11d
5-Fu
Mito-C
Paclitaxel
Adriamycin
100
100
100
100
100
100
100
100
100
100
100
20
18
37
57
50
12
45
32
71
26
38
16
12
44
–
23
32
81
70
62
76
40
63
45
55
16
20
41
–
11
27
91
68
71
75
54
58
49
47
2
42
55
76
52
73
57
67
79
70
66
3
48
51
31
51
69
52
43
32
32
41
4
82
75
53
77
92
80
53
53
59
56
18
–
26
65
–
–
–
67
93
–
–
55
57
10
10
01
–
45
55
–
–
–
a
%age growth inhibition, %age inhibition caused by the compounds and standard drugs at various concentrations.
prostrate cancer cell line (PC-3) the maximum inhibition 81% at
100
M was shown by 10c with IC50 ¼ 38.4, followed by 76%
(IC50 ¼ 56.9) for 10f. For ovarian cancer cell line (OVCAR-5) the
maximum inhibition 91% (100 M) was observed for 10c
design, synthesis and evaluation of chromone based molecules as
potential topoisomerase inhibitor anticancer agents [31]; plausibly
presently investigated molecules may be having similar mode of
action.
m
m
(IC50 ¼ 10.5), followed by 75% at the same concentration for 10f
(IC50 ¼ 61.7). In the case of the lung cancer cell line (A-549) the
2.3. Structure activity relationship
maximum inhibition 79% at 100
mM was observed for 11a with
IC50 ¼ 52.8, followed by 76% for 10c with IC50 ¼ 57.5 at same
concentration. In the case of liver cell line (HEP-2) the maximum
inhibition observed was 69% (IC50 ¼ 74.9) for 10e.The inhibitory
effect on CNS was also evaluated using (IMR-32) cell lines in the
latter case maximum inhibition observed was 92% (IC50 ¼ 10) for
Though systematic establishment of SAR has not been taken up,
however, apparently compounds (10b,e Fig. 3) bearing electron
withdrawing groups at the positions C6 are found to be more active,
whereas, the compounds 10a,c bearing electron withdrawing
groups at position C7 or unsubstituted at C6 and C7 position are
relatively less active. The compound bearing electron releasing
groups at C6 position have low activity. In the case of N-phenyl-
thioamides 11a, derivatives unsubstituted at C6 or C7 are found to
be highly active, whereas compound 11b bearing electron with-
drawing group at C6 showed moderate activity.
10e, followed by 82% (IC50 ¼ 16.5) and 75% (IC50 ¼ 14.6) at 100
mM
concentration for 10a and 10b, respectively. The results indicate
that some of the dithiazoles such as (10b,e) are active on neuro-
blastoma, while the compound (10c) is active against ovarian
cancer cells. It is pertinent to mention here that compounds 10a,b,e
have IC50 value of 16.5, 14.6 and 10.0, respectively, against neuro-
blastoma (IMR-32 cells). Adriamycin is a DNA alkylating agent and
topoisomerase-II inhibitor, and is known to be active on the
neuroblastoma (IC50 ¼ 1.7). Also, recently, Ishar et al. reported the
3. Conclusions
Chromanyl-1,2,4-dithiazole(10a–g) and N-Phenylthioamides
(11a–d) were synthesized and evaluated for cytotoxic activity
against human cancer cell lines. The results of investigations indi-
cate that in most of the experimental observations, maximum
activity was observed when chromone ring either bears electron
withdrawing groups at C6 and C7 position (10e,c,f) or unsub-
stituted (10a). The compounds 10e,b,a are active on neuroblastoma,
10c on ovary, 10c,d,e,f on prostrate and 10c and 11a on lung cancer
cell lines; no significant activity was observed on liver and colon
cancer cell lines. These molecules shall serve as useful ‘Lead’ for
further development.
Table 3
IC50 value for compounds (10a–g) and (11a–d) against different human cancer cell
lines.
Compounds IC50
(m
M)a
COLO-205 PC-3
OVCAR-5 A-549 HEP-2 IMR-32
Prostate Ovary Lung Liver Neuroblastoma
>100 >100 16.5
Colon
10a
10b
10c
10d
10e
10f
10g
11a
11b
11c
11d
>100
>100
76.7
>100
>100
>100
>100
72.6
>100
>100
38.4
60.0
52.7
56.9
>100
60.6
>100
90.8
95.4
–
>100
>100
10.5
65.2
66.5
61.7
93.7
86.4
>100
>100
>100
–
91.1
57.5
98.6
>100 95.1
14.6
95.0
71.3
93.5
66.5
52.8
71.9
63.6
71.2
–
98.0
74.9
97.6
>100 70.7
>100 95.8
>100 89.8
>100 92.7
>100 89.9
58.0
10
56.8
4. Experimental protocols
Starting materials, reagents and solvents were purchased from
commercial suppliers and purified/distilled/crystallized before use.
JEOL AL-300FT (300 MHz) NMR spectrometer was used to record
>100
>100
>100
5-Flurouracil 21
–
–
1H and 13C NMR (75 MHz) spectra. Chemical shifts (
d) are reported
Mito-C
Paclitaxel
Adriamycin
–
–
–
1.5
–
–
–
2.7
–
–
2.7
–
1.5
–
–
–
–
1.7
as downfield displacements from TMS used as internal standard
and coupling constants (J) are reported in Hz. IR spectra were
recorded with Shimadzu FT-IR-8400S spectrophotometer on KBr
pellets. Mass spectra, ESI-method, were recorded on Bruker Dal-
tonics Esquire 300 mass spectrometer. Elemental Analyses were
a
IC50, 50% inhibitory concentration represents the mean from dose response
curves of number of experiments.

T. Raj et al. / European Journal of Medicinal Chemistry 45 (2010) 790–794
793
Chromanyl dithiazoles
N-Phenylthioamides
Electron withdrawing
group at C6 position
enhance activity
H
S
S
O
O
Electron withdrawing
group at C6 position
enhance activity
O
O
N
N
S
Electron withdrawing
group at C7 inhances
the activity
Compounds with
electron withdrawing
group at C7 show high
activity
Fig. 3. Structure activity relationship.
carried out on a Thermoelectron EA-112 elemental analyzer and are
reported in percent atomic abundance. All melting points are
uncorrected and measured in open glass-capillaries on a Veego
(make) MP-D digital melting point apparatus. X-ray analysis was
recorded at Bruker SMART APEX diffractometer equipped with low-
temperature device and the structure was solved by direct methods
using SHELXS 97 software (Sheldrick, 1997).
stock solution of 2 ꢂ 10ꢁ3
mM was prepared. 5-Flurouracil and
Mitomycin-C were dissolved in double distilled water and stock
solution of 2 ꢂ 103
m
M was prepared. Stock solutions were further
diluted with complete growth medium supplemented with 50
mg/
ml gentamycin to obtain desired concentration. All the cells were
maintained in RPMI-1640 medium, supplemented with fetal
bovine serum (10%), 100 units/ml penicillin and 100 mg/ml strep-
tomycin (complete medium). The cells were seeded into 96 well
cell culture plates (1 ꢂ 104 cells/100
ml/well) and incubated in CO2
4.1. Synthesis
incubator (37 ꢀC, 5% CO2, 95% relative humidity) for 24 h. After 24 h,
compounds 10a–g, 11a–d and positive controls (100 ml/well) were
added in quadruplets and the plates were further incubated in CO2
incubator for 48 h. Suitable controls were also included in each
4.1.1. Synthesis of 1,2,4-dithiazoles (10c,d)
Synthesis of 3-(5-phenyl-3H-[1,2,4]dithiazol-3-yl)chromen-
4-ones and 4-oxo-4H-chromene-3-carbothioic acid N-phenyl-
amides had been earlier [31,32]. Two new compounds 10c,d
bearing electron withdrawing groups (Cl) at C6 and C7 position
were synthesized by the same procedure and characterized
detailed spectroscopic analysis.
experiment. After 48 h chilled trichloro acetic acid (50% w/v, 50 ml)
was laid gently on top of the medium in all the wells. The plates
were incubated at 4 ꢀC for one hour to fix the cells. All the contents
of the wells were gently pipetted out and discarded. The plates
were washed five times with distilled water to remove trichloro
acetic acid, growth medium, low molecular weight metabolites and
serum proteins etc. The plates were air-dried. Sulphorhodamine-B
(0.4% SRB in 1% acetic acid, 100 ml/well) was added to each well of
the 96 well plates for 30 min. Excess of the dye was washed off
using 1% acetic acid and the plates were air-dried. Tris buffer
4.1.1.1. 7-Chloro-3-(5-phenyl-3H-[1,2,4]dithiazol-3-yl)-chromen-4-
one (10c). Yield: 89%; Light orange crystalline solid, mp
140–143 ꢀC (chloroform: hexane, 1:1); UV (MeOH): 307, 247 nm;
IR (KBr): nmax 1645, 1517, 1220 cmꢁ1 1H NMR (200 MHz, CDCl3):
;
d
¼ 8.15 (d, 1H, J ¼ 1.8 Hz ArH), 7.95 (dd, 2H, J ¼ 7.5 and 1.6 Hz,
ArH), 7.73 (s, 1H, C2H), 7.58–7.08(m, 6H, 5-Ar H and C5 H); 13C
(10 mM, pH 10.5, 100 ml/well) was added to each well and plates
0
were shaken on a mechanical stirrer for 10 min and O. D. was
recorded on ELISA reader at 540 nm. Viability of cells was evaluated
by trypan blue exclusion method immediately before setting up the
experiment for cytotoxicity determination. Cells with >98%
viability were used in the assay [37].
0
NMR (50 MHz, CDCl3):
d
¼ 175.2 (C4), 170.7 (C3 ), 156.4 (q), 152.7
(C2), 132.4 (q), 131.4 (C7), 129.2 (CH), 128.9 (C5), 127.2 (CH), 126.4
0
(CH), 126.3 (q), 124.6 (C6), 122.4 (C8), 118.2 (C3), 82.7 (C5 ); MS
(ESI): m/z 359 (M þ Naþ); Anal. calcd. For C17H10ClNO2S2: C, 56.74;
H, 2.80; N, 3.89; Found C, 56.62; H, 2.67 and N, 3.76%.
4.1.1.2. 6,7-Dichloro-3-(5-phenyl-3H-[1,2,4] dithiazol-3-yl)-chromen-
4-one (10d). Yield: 73%; solid, Light orange crystalline solid, mp
183–186 ꢀC (chloroform: hexane, 1:1); UV (MeOH): 306, 253,
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DMSO and stock solution of 2 ꢂ 104
mM was prepared. Stock solu-
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mg/ml gentamycin to obtain test concentration of
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m

794
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