Synthesis and anticancer activities of new benzothiadiazinyl hydrazinecarboxamides and anilino[1,2,4]triazolo[1,5-b][1,2,4]thiadiazine 5,5-diones

Article abstract of DOI:10.2174/157340611795564259

Two series of compounds (5-14 and 15-23) based on the scaffolds of 2-(1,1-dioxido-4-phenyl-4Hbenzo[ e][1,2,4]thiadiazin-3-yl)-N-(4-methoxyphenyl) hydrazinecarboxamide (5) and 2-((4-methoxyphenyl)amino)-10- phenyl-10H-benzo[e] [1,2,4]triazolo[1,5-b][1,2,4]

Full text of DOI:10.2174/157340611795564259

Medicinal Chemistry, 2011, 7, 165-172
165
Synthesis and Anticancer Activities of New Benzothiadiazinyl Hydrazine-
carboxamides and Anilino[1,2,4]triazolo[1,5-b][1,2,4]thiadiazine 5,5-diones
Ahmed Kamal, a,* Y. V. V. Srikanth, a Md. Ashraf, a M. Naseer A. Khan, a Thokhir Basha Shaik, a Shasi V.
Kalivendi, a Nitasha Surib and A. K. Saxena b
a Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 607, India
b Pharmacology Division, Indian Institute of Integrative Medicine, Canal Road, Jammu, 180001, India
Abstract: Two series of compounds (5-14 and 15-23) based on the scaffolds of 2-(1,1-dioxido-4-phenyl-4H-
benzo[e][1,2,4]thiadiazin-3-yl)-N-(4-methoxyphenyl)hydrazinecarboxamide (5) and 2-((4-methoxyphenyl)amino)-10-
phenyl-10H-benzo[e][1,2,4]triazolo[1,5-b][1,2,4]thiadiazine 5,5-dioxide (15) respectively, were designed and synthesized.
These compounds were tested for anticancer activity against various cancer cell lines including lung, ovary, prostate,
breast and colon cancers. They exhibited moderate to good inhibitory activity against the above cell lines and compound 9
was found to be the most active one from these two series. Further studies showed that cancer cell growth inhibition by
compounds 22 and 23 could be in part due to the inhibition of tubulin polymerization, with the IC50 values of 4.70 and
5.25 μM, respectively.
Keywords: Hydrazinecarboxamides, triazolo benzothiadiazines, anticancer activity, tubulin polymerization.
1. INTRODUCTION
(3) and 1,2,4-triazolo benzothiadiazines possess anticancer
activity [20-24].
The chemotherapy currently available for cancer treat-
ment is associated with limitations including normal cell
toxicity and tumor cell resistance. To overcome these chal-
lenges, new and effective cytotoxic agents with novel mode
of action are urgently needed. Drug discovery and develop-
ment of potent anticancer agents based on the lead candi-
dates have attracted much attention of many medicinal
chemists [1-2].
In the last decade triazole derivatives have attracted
much interest for the development of potent anticancer
agents [25-27]. 1,2,4-Triazole-3,5-diamine analog is reported
as a novel and potent anticancer CDK inhibitor [28]. Further,
triazole derivatives possess anticancer activity by inhibition
of tubulin polymerization [29]. We have shown that various
heterocyclic compounds including triazolobenzothiadiazine
ring system are potential pharmacophores against various
cancer cell lines [22-24, 30-34]. We were interested in ex-
ploring the alteration in benzodiathiazine ring system with-
out changing the triazoloanilino moiety. In this report, we
present the design, synthesis and in vitro evaluation of tria-
zolobenzothiadiazine and hydrazinecarboxamides as anti-
cancer agents based on our earlier studies [22-24] on this
scaffold (Fig. 2). Further, the effect of substitutents on the
nitrogen of benzothiadiazine ring system and anilino ring has
been investigated.
Benzene sulfonamides represent an important class of
therapeutic agents in the current drug design and discovery
efforts. Moreover, both aryl and hetero arylsulfonamides are
known to possess anticancer activity and their mode of ac-
tion is different from the traditional anticancer agents [3-5].
They act as carbonic anhydrase inhibitors [6], cyclooxy-
genase inhibitors [7] and arrests cell cycle in G1 phase [8],
recently some compounds from this series have reached the
clinical studies [9]. On the other hand, sulfonylurea deriva-
tive such as sulofenur (LY 186641) (1, Fig. 1) has been
clinically evaluated in several solid tumors models [10, 11]
because of apparent lack of toxicity to proliferating normal
tissues [12, 13]. 1,2,4 Benzothiadiazine 1,1-dioxide ring sys-
tem and 10-hydroxy-3,10-dihydro-2H-benzo[e]imidazo[1,2-
b][1,2,4]thidiazine 5,5 dioxide (2) exhibit potent anticancer
and antiviral activities by inhibition of ribonucleotide reduc-
tase [14,15]. These benzothiadiazine analogs have shown to
exhibit antitumor activity by inhibition of cyclin dependent
kinases (CDKs) [16-18] and tubulin polymerization [19].
Various derivatives of 1,2,4-triazolo1,2,4-benzodithiazines
2. RESULTS AND DISCUSSION
2.1. Chemistry
The preparation of the starting materials, 3-hydrazino-4-
methyl/phenyl-4H-1,2,4-benzothiadiazine 1,1-dioxides (4a
and b) was accomplished by the synthetic sequences as pre-
viously reported [34] (Scheme 1). The synthesis of N1-
phenyl-2-(4-phenyl/methyl-1,1-dioxo-1,4-dihydro-1ꢀ6,2,4-
benzothiadiazin-3-yl)-1-hydrazinecarboxamide (5-14) were
obtained by reaction of 4a and b with different aryl isocy-
anates in dry benzene at room temperature. Next, 2-anilino-
10-phenyl/methyl-5,10-dihydro-5ꢀ6-benzo[e][1,2,4]triazolo
[1,5-b][1,2,4]thiadiazine- 5,5-dione derivatives (15-23) were
obtained by refluxing compounds 5-14 in phosphorous oxy-
chloride [32] (Scheme 1).
*Address correspondence to this author at the Division of Organic Chemis-
try, Indian Institute of Chemical Technology, Hyderabad 500 607, India;
Tel: +91-40-27193157; Fax: +91-40-27193189;
E-mail: ahmedkamal@iict.res.in
1573-4064/11 $58.00+.00
© 2011 Bentham Science Publishers Ltd.

166 Medicinal Chemistry, 2011, Vol. 7, No. 3
Kamal et al.
O
O
S
O
O
O
O
NH
S
Cl
O
S
S
N
N
N
N
HN
O
NH
H3CO
N
N
OH
Cl
Cl
(1, LY-186641)
2
3
Fig. (1). Chemical structures of sulfonyl urea derivative (1, LY-186641), imidazo benzothiadiazine derivative (2) and triazolo dithiadiazine
derivative (3).
O
O
O
O
S
Cl
O
S
S
N
N
N
N
N
N
NH
NH
H3CO
N
R1
R2
R
3
15-23
Fig. (2). Design of triazolobenzothiadiazines.
O
O
O
O
O
O
N
S
S
S
N
b
N
N
N
a
H
H
NH R2
N
N
N
N
R2
N
NHNH2
N
H
R1
R1
O
R1
15-23
4a: R1 = CH3
4b: R1 = Ph
5-14
Scheme1. Reagents and conditions: (a) aryl isocyanates, benzene, r.t.; (b) POCl3, 110 o C, 3-4 h.
both triazolo (5 and 15) and carboxamido moieties (11 and
20) were found to be inactive. Interestingly, substitution of
meta or para chloro on the phenyl ring has enhanced the
inhibitory effect in compounds 6, 16, 7 and 17, where R1 =
Ph, however, it showed less effect in compounds 12, 21, 13
and 22 where R1 = Me. Similarly, 3,5-di(trifluoromethyl)
substituents also increased the inhibitory effect in com-
pounds 8 and 18 where R1 = Ph and showed less effect in
compounds 14 and 23, where R1 = Me.
2.1. Anticancer Activity
All the synthesized compounds have been evaluated for
their anticancer activity against 6 different human cancer cell
lines (A-549, IGR-OV-1, PC-3, T-47-D, MCF-7, and CoLo-
205. Overall, 10 hydrazinocarboxamide derivatives and 9
triazolobenzothiadiazine analogues were synthesized; their
structures and corresponding activities are presented in
Scheme 1, Tables 1 and 2. The test compounds were added
at a single concentration (100 ꢀM) and results for each com-
pound were reported as percentage test cell growth inhibition
compared with untreated control cells using sulforhodamine
B (SRB) method [35]. Paclitaxel, mitomycin, adriamycin
and 5-fluorouracil were used as positive controls in this as-
say. Some of the compounds have exhibited potential growth
inhibitory effect in almost all the cell lines. Hydrazinocar-
boxamide analogues (6-10, 14) displayed better growth in-
hibitory potency than their corresponding triazoloben-
zothiadiazine (16-18, 20-23) analogues. However, com-
pounds 5, 11-13, 15 and 19 did not show growth inhibitory
effect (data not shown) at the concentration used in the as-
say. Compound 9 was found to be the most active from these
series, which showed 99% growth inhibition against prostate
cancer cell line and strong inhibition in most of the cell lines.
Compound 8 and its corresponding triazolo analogue 18 also
exhibited strong inhibitory effect (>80%) in most cases.
Compounds with 4-methoxy substituent on phenyl rings of
2.2. Inhibition of Tubulin Polymerization
Since these new compounds possess a triazole moiety, it
is interesting to investigate their effects on tubulin polymeri-
zation. One possible explanation of compounds showing
anticancer activity is the inhibition of tubulin polymerization
to form functional microtubules as it is observed with an-
timitotic agents such as nocodazole and colchicines. The
polymerization of purified tubulin was investigated by moni-
toring the fluorescence at 360/420 nm excitation/emission
o
using Varioscan multimode plate reader at 37 C with and
without the compounds at 3 ꢀM (final concentration) which
was diluted in PEM buffer from the stock solution concen-
tration of 1mM in DMSO. Amongst the molecules exam-
ined, 22 and 23 suppressed the tubulin polymerization in
comparison to the positive control compound (podo-
phylltoxin). The compounds 22, 23 also demonstrated a dose

Synthesis and Anticancer Activities of New Benzothiadiazinyl
Medicinal Chemistry, 2011, Vol. 7, No. 3 167
Table 1. Various Hydrazinocarboxamide and Triazolobenzothiadiazine Analogues (5-23)
Compd
5, 15
R1
R2
OCH3
Cl
6, 16
7, 17
Cl
CF3
8, 18
CF3
H3CO
9
tBu
10, 19
OCH3
-CH3
-CH3
-CH3
11, 20
12, 21
13, 22
Cl
Cl
CF3
-CH3
14, 23
CF3
Table 2. Percentage of Growth Inhibition for Selected Cancer Cell Lines by Hydrazinocarboxamide and Triazolobenzothiadiazine
Compounds at 100 ꢀM Concentrations
Lung
Ovary
Prostate
PC-3
Breast
T-47-D
Breast
Colon
Compd
A-549
IGR-OV-1
MCF-7
CoLo-205
6
7
8
9
80
73
80
91
65
56
83
71
72
72
95
99
55
55
53
79
74
64
81
90
74
62
94
87

168 Medicinal Chemistry, 2011, Vol. 7, No. 3
Kamal et al.
Table 2. contd….
Lung
Ovary
Prostate
PC-3
Breast
T-47-D
Breast
Colon
Compd
A-549
IGR-OV-1
MCF-7
CoLo-205
a
10
14
16
17
18
-
-
53
52
50
52
88
67
50
64
62
a
a
a
a
a
-
-
-
-
a
77
69
84
72
59
82
56
62
64
74
66
85
-
a
-
94
a
a
a
a
a
20
21
-
60
69
54
-
-
-
-
-
64
14
47
61
46
29
37
-
16
-
53
31
43
-
10
-
22
23
b
b
b
b
Paclitaxel c
Mitomycin c
Adriamycind
5-Fluorouracilc
63
-
-
-
-
b
b
b
b
b
-
-
67
-
-
-
b
b
b
b
-
-
-
71
63
-
b
b
b
b
b
-
-
-
-
-
79
a Growth inhibition <50%, b not tested, c Inhibition at 10 ꢀM concentration, d Inhibition at 1 ꢀM concentration.
140
120
100
80
60
40
20
0
Compounds
Fig. (3). Effect of compounds at 3 ꢀM concentrations on tubulin polymerization.
3. CONCLUSIONS
dependent tubulin inhibition with IC50 values of 4.70 and
5.25 ꢀM, respectively. Although compounds 8, 9 and 18
exhibited strong growth inhibitory effect against most of the
cancer cell lines, but were found to be inactive in possessing
anti-tubulin activity. In contrast, compounds 22 and 23,
which showed better inhibitory effect on tubulin, were found
to exhibit mild to moderate growth inhibitory effect on dif-
ferent cancer cell lines. Probably, the compounds 8, 9 and 18
mediate cytotoxic effects by targeting pathways other than
tubulin polymerization. Further structural modification of
these lead compounds and studies on other targets are likely
to provide new insights into the mechanism of cancer cell
cytotoxicity by these compounds.
In summary, the synthesis and screening of anticancer ac-
tivity for novel series of a N1-phenyl-2-(4-phenyl/methyl-
1,1-dioxo-1,4-dihydro-1ꢀ6,2,4-benzothiadiazin-3-yl)-1-hyd-
razinecarboxamide (5-14) and 2-anilino-10-phenyl/methyl-
5,10-dihydro-5 ꢀ6-benzo[e][1,2,4]triazolo[1,5-b][1,2,4] thia-
diazine-5,5-dione derivatives (15-23) were investigated.
Compounds 6, 7, 8, 9, 18 showed higher potency against all
the cell lines tested, and compound 9 was found to be the
most active from this series. Compounds 22 and 23 have
shown significant tubulin inhibitory activity. These prelimi-
nary results demonstrate that the potent compounds from
these series may further be optimized for better anticancer
leads.

Synthesis and Anticancer Activities of New Benzothiadiazinyl
Medicinal Chemistry, 2011, Vol. 7, No. 3 169
drazinecarboxamide (8) The title compound was obtained
from (24, 290 mg, 1 mmol) and 3,5-di(trifluoromethyl)
phenyl isocyanate (306 mg, 1.2 mmol) as described for 5.
4. EXPERIMENTAL SECTION
4.1. General
Chemistry: All chemicals and reagents were obtained
from Aldrich (Sigma–Aldrich, St. Louis, MO, USA),
Lanchester (Alfa Aesar, Johnson Matthey Company, Ward
Hill, MA, USA) or Spectrochem Pvt. Ltd (Mumbai, India)
and were used without further purification. Reactions were
monitored by TLC, performed on silica gel glass plates con-
taining 60 F-254, and visualization on TLC was achieved by
UV light or iodine indicator. Column chromatography was
Yield: 441 mg 81%; mp 169-171 oC; 1H NMR (300
MHz, CDCl3) ꢀ 8.49 (bs, 1H);8.14 (bs, 1H); 7.95 (dd, J =
7.5, 1.5 Hz, 1H); 7.81-7.70 (m, 4H); 7.61-7.54 (m, 2H);
7.48-7.31 (m, 3H); 6.49-6.43 (m, 2H); LRMS (ESI) m/z 544
[M+].
4.2.5. N1-[4-(tert-Butyl)-2-methoxyphenyl]-2-(1,1-dio-
xo-4-phenyl-1,4-dihydro-1ꢀ6,2,4-benzo thiadiazin-3-yl)-1-
hydrazinecarboxamide (9) The title compound was obtai-
ned from (24, 290 mg, 1 mmol) and 4-(tert-butyl)-2-
methoxyphenyl isocyanate (205 mg, 1.2 mmol) as described
for 5.
1
performed with Merck 60–120 mesh silica gel. H and 13C
NMR spectra were recorded on Gemini Varian-VXR-unity
(200, 400, 500 MHz) or Bruker UXNMR/XWIN-NMR (300
MHz) instruments. Chemical shifts (ꢀ) are reported in ppm
downfield from internal TMS standard. ESI spectra were
recorded on Micro mass, Quattro LC using ESI+ software
with capillary voltage 3.98 kV and ESI mode positive ion
trap detector. Melting points were determined with an Elec-
trothermal melting point apparatus, and are uncorrected.
o
1
Yield: 410 mg, 83%; mp 220-222 C; H NMR (200
MHz, CDCl3+DMSO-d6) ꢀ 8.19 (bs, 1H); 8.02 (bs, 1H); 7.90
(bs, 1H); 7.79-7.53 (m, 6H); 7.39-7.10 (m, 2H); 6.96 (dd, J =
8.1, 2.2 Hz, 1H); 6.77-6.62 (m, 2H); 6.37 (d, J = 8.8 Hz,
1H); 3.74 (s, 3H); 1.26 (s, 9H); LRMS (ESI) m/z 517
[M+Na]+.
4.2. General Procedures
4.2.1. N1-(4-Methoxyphenyl)-2-(1,1-dioxo-4-phenyl-
1,4-dihydro-1ꢀ6,2,4-benzothiadiazin-3-yl)-1-
4.2.6. N1-(2-naphthyl)-2-(1,1-dioxo-4-phenyl-1,4-dihy-
dro-1ꢀ6,2,4-benzothiadiazin-3-yl)-1-hydrazinecarboxam-
ide (10) The title compound was obtained from (24, 290 mg,
1 mmol) and 2-naphthyl isocyanate (203 mg, 1.2 mmol) as
described for 5.
hydrazinecarboxamide (5) The title compound was ob-
tained from 3-hydrazino-4-phenyl-4H-1,2,4-benzothiadia-
zine 1,1-dioxide (24, 290 mg, 1 mmol) was taken in dry ben-
zene to this 4-methoxy phenyl isocyanate (179 mg, 1.2
mmol) was slowly added and kept for overnight solid sub-
stance was formed filtered and washed with ether dried to
form pure substances.
0
1
Yield: 407 mg, 89%; mp 247-249 C; H NMR (300
MHz DMSO-d6 ) ꢀ 8.81 (bs, 1H); 8.68 (bs, 1H); 8.49 (s, 1H);
8.17 (d, J = 7.9 Hz, 1H); 7.91 (dd, J = 9.1, 2.1 Hz, 1H); 7.86
(d, J = 7.2 Hz ,1H); 7.78-7.66 (m, 4H); 7.65-7.43 (m, 6H);
7.41-7.32 (m, 1H); 6.33 (d, J = 8.3 Hz, 1H).; LRMS(ESI)
m/z 481 [M+Na]+.
o
1
Yield: 363 mg, 83%; mp 181-184 C; H NMR (300
MHz, DMSO-d6) ꢀ 8.53 (bs, 2H); 8.17 (bs, 1H); 7.83 (d, J =
7.4 Hz,1H); 7.77-7.64 (m, 3H); 7.63-7.43 (m, 3H); 7.41-7.26
(m, 3H); 6.84 (d, J = 8.3 Hz, 2H); 6.32 (d, J = 8.3 Hz, 1H);
3.70 (s, 3H); LRMS (ESI) m/z 461 [M+Na]+.
4.2.7. N1-(4-Methoxyphenyl)-2-(4-methyl-1,1-dioxo-
1,4-dihydro-1ꢀ6,2,4-benzothiadiazin-3-yl)-1-
hydrazinecarboxamide (11) The title compound was obtai-
ned from (25, 226 mg, 1 mmol) and 4-methoxy phenyl isoc-
yanate (179 mg, 1.2 mmol) as described for 5.
4.2.2. N1-(3-chlorophenyl)-2-(1,1-dioxo-4-phenyl-1,4-
dihydro-1ꢀ6,2,4-benzothiadiazin-3-yl)-1-
hydrazinecarboxamide (6) The title compound was obtai-
ned from 3-hydrazino-4-phenyl-4H-1,2,4-benzothiadiazine
1,1-dioxide (24, 290 mg, 1 mmol) and 3-chloro phenyl isoc-
yanate (184 mg, 1.2 mmol) as described for 5.
o
1
Yield: 342 mg, 91%; mp 226-228 C; H NMR (300
MHz, CDCl3+DMSO-d6 ) ꢀ 9.73 (bs, 1H); 8.80 (s, 1H); 8.39
(s, 1H); 7.91-7.57 (m, 3H); 7.56-7.30 (m, 3H); 7.00-6.88 (m,
2H); 3.79 (s, 3H); 3.65 (s, 3H); LRMS (ESI) m/z 399
[M+Na]+.
o
1
Yield: 380 mg, 86%; mp 152-154 C; H NMR (400
MHz, DMSO-d6) ꢀ 8.57 (bs, 1H); 8.45 (bs, 1H); 7.83 (d, J =
7.2 Hz,1H); 7.77-7.64 (m, 5H); 7.60-7.45 (m, 3H); 7.37 (t, J
= 7.2 Hz, 1H); 7.28 (t, J = 9.0 Hz, 1H); 7.06-6.97 (m, 1H);
6.33 (d, J = 9.0 Hz ,1H); LRMS(ESI) m/z 442 [M+].
4.2.8. N1-(3-Chlorophenyl)-2-(4-methyl-1,1-dioxo-1,4-
dihydro-1ꢀ6,2,4-benzothiadiazin-3-yl) -1-hydrazinecarbo-
xamide (12) The title compound was obtained from (25, 226
mg, 1 mmol) and 3-chloro phenyl isocyanate (184 mg, 1.2
mmol) as described for 5.
4.2.3. N1-(4-Chlorophenyl)-2-(1,1-dioxo-4-phenyl-1,4-
dihydro-1ꢀ6,2,4-benzothiadiazin-3-yl)-1-
hydrazinecarboxamide (7) The title compound was ob-
tained from (24, 290 mg, 1 mmol) and 4-chloro phenyl iso-
cyanate (184 mg, 1.2 mmol) as described for 5.
o
1
Yield: 331 mg, 87%; mp 252-253 C; H NMR (400
MHz, DMSO-d6 ) ꢀ 9.71 (bs, 1H); 9.11 (bs, 1H); 8.58 (bs,
1H); 7.80-7.66 (m, 4H); 7.55 (d, J = 8.9 Hz, 1H); 7.42 (t, J =
8.9, 7.2 Hz, 1H); 7.30 (t, J = 7.2, 8.9 Hz, 1H); 7.02 (d, J =
7.2 Hz, 1H); 3.57 (s, 3H); LRMS (ESI) m/z 403 [M+Na]+.
o
1
Yield: 393 mg, 89%; mp 208-210 C; H NMR (300
MHz, DMSO-d6) ꢀ 8.54 (bs, 1H); 8.36 (bs, 1H); 7.82 (d, J =
7.4 Hz, 1H); 7.77-7.63 (m, 2H); 7.63-7.42 (m, 5H); 7.41-
7.21 (m, 4H); 6.32 (d, J = 8.5 Hz, 1H); LRMS (ESI) m/z 442
[M+].
4.2.9. N1-(4-Chlorophenyl)-2-(4-methyl-1,1-dioxo-1,4-
dihydro-1ꢀ6,2,4-benzothiadiazin-3-yl) -1-hydrazinecarbo-
xamide (13) the title compound was obtained from (25, 226
mg, 1 mmol) and 4-chloro phenyl isocyanate (184 mg, 1.2
mmol) as described for 5.
4.2.4. N1-[3,5-Di(trifluoromethyl)phenyl]-2-(1,1-dioxo-
4-phenyl-1,4-dihydro-1ꢀ6,2,4-benzo thiadiazin-3-yl)-1-hy-

170 Medicinal Chemistry, 2011, Vol. 7, No. 3
Kamal et al.
o
1
Yield: 343 mg, 90%; mp 248-249 C; H NMR (200
MHz, DMSO-d6 ) ꢀ 9.77 (bs, 1H); 9.09 (s, 1H); 8.55 (s, 1H);
7.86-7.70 (m, 2H); 7.69-7.50 (m, 3H); 7.49-7.29 (m, 3H);
3.62 (s, 3H); LRMS (ESI) m/z 403 [M+Na]+.
4.3.5. 2-(2-Naphthylamino)-10-phenyl-5,10-dihydro-
5ꢀ6-benzo[e][1,2,4]triazolo[1,5-b][1,2,4]thiadiazine-5,5-
dione (19) The title compound was obtained from 10 (458
mg, 1 mmol) and POCl3 (10 mL) as described for 24.
o
1
4.2.10. N1-[3,5-Di(trifluoromethyl)phenyl]-2-(4-methyl-
1,1-dioxo-1,4-dihydro-1ꢀ6,2,4-benzothiadiazin-3-yl)-1-hy-
drazinecarboxamide (14) The title compound was obtained
from (25 226 mg, 1 mmol) and 3,5-di(trifluoromethyl) phen-
yl isocyanate (306 mg, 1.2 mmol) as described for 5.
Yield: 383 mg, 87%; mp 269-272 C; H NMR (300
MHz, DMSO-d6) ꢀ 9.30(s, 1H); 8.34-8.04 (m, 2H), 7.92-7.35
(m, 13H), 6.77(d, J = 8.8 Hz, 1H); LRMS (ESI) m/z 441
[M+1]+.
4.3.6. 2-(4-Methoxyanilino)-10-methyl-5,10-dihydro-
5ꢀ6-benzo[e][1,2,4]triazolo[1,5-b][1,2,4]thiadiazine-5,5-
dione (20) The title compound was obtained from 11 (376
mg, 1 mmol) and POCl3 (10 mL) as described for 15.
o
1
Yield: 415 mg, 86%; mp 274-276 C; H NMR (300
MHz, DMSO-d6) ꢀ 9.77 (bs, 1H), 8.96 (s, 1H); 8.24 (s, 2H),
7.80-7.68 (m, 2H), 7.65 (s, 1H); 7.56 (d, J = 8.5 Hz 1H);
7.43 (t, J = 7.6 Hz 1H), 3.62 (s, 3H); LRMS (ESI) m/z 505
[M+Na]+.
o
1
Yield: 293 mg, 82%; mp 256-258 C; H NMR (300
MHz, CDCl3) ꢀ 8.12 (dd, J = 8.1, 1.5 Hz, 1H), 7.84-7.67 (m,
1H), 7.54-7.23 (m, 3H), 6.90 (d, J = 8.8 Hz, 2H), 6.58 (d, J =
1.5 Hz, 1H), 3.81 (s, 3H), 3.78(s, 3H); LRMS (ESI) m/z 358
[M+1]+.
4.3.1. 2-(4-Methoxyanilino)-10-phenyl-5,10-dihydro-
5ꢀ6-benzo[e][1,2,4]triazolo[1,5-b][1,2,4]thiadiazine-5,5-
dione (15) The title compound (15) was obtained from by
reaction of corresponding urea compound 5 (438 mg, 1
mmol) with POCl3 (10 mL) and then reflux the reaction mix-
4.3.7. 2-(3-Chloroanilino)-10-methyl-5,10-dihydro-
5ꢀ6-benzo[e][1,2,4]triazolo[1,5-b] [1,2,4] thiadiazine-5,5-
dione (21) The title compound was obtained from 12 (483
mg, 1 mmol) and POCl3 (10 mL) as described for 15.
Yield: 313 mg, 86%; mp 250-252 oC;1H NMR (300
MHz, DMSO-d6 ) ꢀ 8.12 (d, J = 8.0 Hz, 1H), 8.02-7.80 (m,
1H), 7.76-7.71 (m, 1H), 7.66-7.42 (m, 3H), 7.25 (t, J = 8.0
Hz, 1H), 6.90 (d, J = 8.0 Hz, 1H), 3.85 (s, 3H); LRMS (ESI)
m/z 362 [M+].
o
ture at 110 C for 3 h. Then the reaction mixture was poured
in crushed ice and quenched with NaHCO3 and extracted in
ethyl acetate (4x25 mL) from the ice cold aqueous layer and
dried over anhydrous Na2SO4. The resulting products (15)
were purified by recrystallization from methanol.
o
1
Yield: 340 mg, 81%; mp 196-198 C; H NMR (200
MHz, CDCl3) ꢀ 8.15 (dd, J = 7.7, 1.4 Hz, 1H), 7.73-7.56 (m,
3H), 7.55-7.31 (m, 5H), 6.87 (d, J = 8.4 Hz, 2H), 6.73 (d, J =
8.4 Hz, 1H), 6.54 (s, 1H), 3.79 (s, 3H); LRMS (ESI) m/z 421
[M+1]+.
4.3.8.
2-(4-Chloroanilino)-10-methyl-5,10-dihydro-
5ꢀ6-benzo[e][1,2,4]triazolo[1,5-b] [1,2,4] thiadiazine-5,5-
dione (22) The title compound was obtained from 13 (483
mg, 1 mmol) and POCl3 (10 mL) as described for 15.
4.3.2.
2-(3-Chloroanilino)-10-phenyl-5,10-dihydro-
5ꢀ6-benzo[e][1,2,4]triazolo[1,5-b][1,2,4]thiadiazine-5,5-
dione (16) The title compound was obtained from 6 (442
mg, 1 mmol) and POCl3 (10 mL) as described for 15.
o
1
Yield: 326 mg, 90%; mp 304-306 C; H NMR (300
MHz, DMSO-d6 ) ꢀ 8.16-8.06 (m, 1H), 8.00-7.79 (m, 1H),
7.70-7.41 (m, 4H), 7.25 (d, J = 8.6 Hz, 2H), 3.85 (s, 3H);
LRMS (ESI) m/z 362 [M+].
o
1
Yield: 360 mg, 85%; mp 171-173 C; H NMR (400
MHz, DMSO-d6 ) ꢀ 8.58 (bs, 1H), 7.83 (d, J = 7.2 Hz, 1H);
7.78-7.64 (m, 4H), 7.62-7.46 (m, 3H); 7.44-7.24 (m, 3H),
7.02 (t, J = 7.2 Hz, 1H), 6.33 (d, J = 8.9 Hz, 1H), LRMS
(ESI) m/z 447 [M+Na]+.
4.3.9.
2-[3,5-Di(trifluoromethyl)anilino]-10-methyl-
5,10-dihydro-5ꢀ6-benzo[e][1,2,4]triazolo [1,5-b][1,2,4]thi-
adiazine-5,5-dione (23) The title compound was obtained
from 14 (482 mg, 1 mmol) and POCl3 (10 mL) as described
for 15.
4.3.3.
2-(4-Chloroanilino)-10-phenyl-5,10-dihydro-
5ꢀ6-benzo[e][1,2,4]triazolo[1,5-b][1,2,4]thiadiazine-5,5-
dione (17) The title compound was obtained from 7 (442
mg, 1 mmol) and POCl3 (10 mL) as described for 15.
o
1
Yield: 375 mg, 86%; mp 300-302 C; H NMR (300
MHz, DMSO-d6 ) ꢀ 10.74 (bs, 1H), 8.28-8.14 (m, 1H); 7.94
(t, J = 7.5 Hz, 1H), 7.79-7.64 (m, 1H), 7.61 (s, 3H), 7.52 (t, J
= 7.5 Hz , 1H), 3.85 (s, 3H); LRMS(ESI) m/z 464 [M+1]+.
o
1
Yield: 348 mg, 82%; mp 210-212 C; H NMR (300
MHz, DMSO-d6) ꢀ 10.14 (bs, 1H); 8.25 (d, J = 7.6 Hz, 1H);
7.80-7.61 (m, 6H), 7.56 (d, J = 8.9 Hz, 2H), 7.49 (t, J = 7.6
Hz, 1H), 7.37 (d, J = 8.9 Hz, 2H), 6.71 (d, J = 8.5 Hz, 1H);
LRMS (ESI) m/z 447 [M+Na]+.
In vitro Cytotoxicity Evaluation
The synthesized compounds (5-23) have been evaluated
for their in vitro cytotoxicity in selected human cancer cell
lines of lung (A-549), ovary (IGR-OV-1), prostate (PC-3),
breast (T-47-D and MCF-7) and colon (CoLo-205) origin. A
protocol of 48 h continuous drug exposure has been used and
a sulforhodamine B (SRB) protein assay [35] has been used
to estimate cell growth.
4.3.4. 2-[3,5-di(trifluoromethyl)anilino]-10-phenyl-5,
10-dihydro-5ꢀ6-benzo[e][1,2,4]triazolo[1,5-b][1,2,4]
thiadiazine-5,5-dione (18) The title compound was ob-
tained from 8 (544 mg, 1 mmol) and POCl3 (10 mL) as de-
scribed for 15.
o
1
Yield: 426 mg, 81%; mp 260-263 C; H NMR (300
MHz, DMSO-d6) ꢀ 10.79 (bs, 1H); 8.27 (d, J = 7.7 Hz, 1H);
8.13 (s, 2H), 7.81-7.61 (m, 6H), 7.58 (s, 1H), 7.51 (t, J = 7.7
Hz, 1H), 6.75 (d, J = 8.5 Hz, 1H); LRMS (ESI) m/z 526
[M+].
Tubulin Polymerization Assay
The assay was performed employing the fluorescence
based tubulin polymerization assay kit (BK011) obtained
from Cytoskeleton Inc. The reaction mixture contained PEM

Synthesis and Anticancer Activities of New Benzothiadiazinyl
Medicinal Chemistry, 2011, Vol. 7, No. 3 171
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buffer [[80mM PIPES (pH-6.9), 1mM MgCl2, 1mM EGTA,
10% glycerol], Tubulin (2mg/ml), and GTP (1mM) in the
presence or absence of test compound (3ꢀM) at 37 oC. Tubu-
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(excitation and emission wavelengths of 360 nm and 420 nm
respectively) using Varioscan multimode plate reader at.
Flouresence units were recorded at every 60 sec intervals for
up to 1 h. Podophyllotoxin was used as a positive control in
each assay.
The IC50 value was defined as the drug concentration re-
quired to inhibit 50% of tubulin assembly compared to con-
trols. The reaction mixture for these experiments include:
tubulin (2 mg/mL) in PEM buffer, GTP (1mM), in the
presence or absence of test compounds at 1.5, 3, 4.5 and 6
[14]
[15]
ꢀM
concentrations,
different
concentrations
of
podophyllotoxin was used as positive control in this study.
Polymerization was monitored by increase in the flouresence
at 360/420 nm of exitation/emission using Varioscan
[16]
o
multimode plate reader at 37 C. Flouresence was monitored
at every 1 min for 1 h.
ACKNOWLEDGMENTS
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The authors Y.V.V.S and M.A thank UGC, New Delhi,
for the award of research fellowship.
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Received: November 03, 2011
Revised: March 02, 2011
Accepted: March 09, 2011