Synthesis, DNA-binding ability and evaluation of antitumour activity of triazolo[1,2,4]benzothiadiazine linked pyrrolo[2,1-c][1,4]benzodiazepine conjugates

Article abstract of DOI:10.1016/j.bmc.2008.06.056

A series of triazolobenzothiadiazine-pyrrolobenzodiazepine conjugates linked through different alkane spacers have been prepared. These compounds have exhibited significant cytotoxicity against most of the cell lines examined. Compound 5a displays GI₅₀ values from 1.83 to 2.38 μM against seven human tumour cell lines, and is identified as a promising lead compound from this series. Their DNA thermal denaturation studies have also been carried out, and one of the compounds 5c elevates the DNA helix melting temperature of the CT-DNA by 2.6 °C after incubation for 36 h.

Full text of DOI:10.1016/j.bmc.2008.06.056

Bioorganic & Medicinal Chemistry 16 (2008) 7804–7810
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, DNA-binding ability and evaluation of antitumour activity
of triazolo[1,2,4]benzothiadiazine linked pyrrolo[2,1-c]
[1,4]benzodiazepine conjugates
a
a
a
b
b
Ahmed Kamal a, , M. Naseer A. Khan , Y. V. V. Srikanth , K. Srinivasa Reddy , Aarti Juvekar , Subrata Sen ,
*
Nisha Kurian b, Surekha Zingde b
a Chemical Biology Laboratory, Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 007, India
b Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai-410 210, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of triazolobenzothiadiazine-pyrrolobenzodiazepine conjugates linked through different alkane
spacers have been prepared. These compounds have exhibited significant cytotoxicity against most of
the cell lines examined. Compound 5a displays GI50 values from 1.83 to 2.38 lM against seven human
tumour cell lines, and is identified as a promising lead compound from this series. Their DNA thermal
denaturation studies have also been carried out, and one of the compounds 5c elevates the DNA helix
melting temperature of the CT-DNA by 2.6 °C after incubation for 36 h.
Received 1 May 2008
Revised 27 June 2008
Accepted 28 June 2008
Available online 3 July 2008
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
Triazolobenzothiadiazines
Pyrrolobenzodiazepine
Antitumour activity
DNA-binding ability
1. Introduction
sulfonylhydroxyguanidine moiety and exhibit their activity against
several tumour cell lines. This is probably due to the combination
Cancer has proven to be one of the most intractable diseases out
of various human health problems due to uncontrolled cell prolif-
eration, diminished apoptosis, invasion and metastasis. In spite of
increasing number of inhibitory compounds against several molec-
ular targets, there is an obvious need for better compounds. The
identification of lead compounds remains an essential early step
in the development of therapeutic agents that interact with the
molecule of interest. Several analogues of the lead compounds
are then designed and synthesized to define the key recognition
elements for potential activity.1,2
Sulfonylurea derivatives are an important class of chemothera-
peutic agents in medicinal chemistry.3 Compound sulofenur
(LY186641, 1) (Fig. 1) has been clinically evaluated in lung, breast,
colon, ovarian, pancreatic and gastric cancers,4–6 and has reached
clinical trials based on its impressive preclinical activity and appar-
ent lack of toxicity to proliferating normal tissues.7,8 These sulfonyl
compounds exert their biological effect by inhibiting DNA, RNA or
protein synthesis. Further, it is found to accumulate in the cell
mitochondria, which may be target site for antitumour activity
of these compounds.9,10 Both the 1,2,4-benzothiadiazine 1,1-diox-
ide ring system and 2,10-dihydro-10-hydroxy-3H-imidazo[1,2-
of their imino group of guanidine along with the hydroxylamino
group of hydroxyurea. The potent antiviral and anticancer activi-
ties of this class of compounds are because of the inhibition of ribo-
nucleotide reductase.11,12 Moreover, triazole derivatives, for
example, letrozole and anastrozole (arimidex) have shown consid-
erable advances in the treatment of hormone-dependent breast
cancer.13 Further, 1,2,4-triazole-3,5-diamine analogues have also
been reported as novel and potent anticancer cyclin-dependent ki-
nase inhibitors.14 A series of novel 1,4-bis-[4-amino-5-mercapto-
1,2,4-triazol-3-yl-methoxy]phenylenes and their triazolothiadiaz-
ole derivatives are reported to exhibit anticancer activity against
a panel of 60 human tumour cell lines.15 Very recently, we have re-
ported N-methyl/phenyl substituted triazolobenzothiadiazine–
benzothiazole conjugates as potential antitumour agents.16
The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a class of
DNA-binding anticancer compounds that include the naturally
occurring anthramycin and DC-81 (3).17–19 The DNA-interactive
ability and consequential biological effects are a result of the cova-
lent bond formed between the N10/C11 carbinolamine/imine moi-
ety in the central B-ring of the PBD moiety and N2 of the guanine
residue in the minor groove of DNA.20–22 In the past few years, sev-
eral hybrid compounds, in which known antitumour agents teth-
ered to PBD moiety, have been designed, synthesized and
evaluated for their biological activity.2,23–27 Recently, we have been
involved in the development of new synthetic strategies28–30 for
b][1,2,4]benzothiadiazine 6,6-dioxide (2) contain
a built-in
* Corresponding author. Tel.: +91 40 27193157; fax: +91 40 27193189.
E-mail address: ahmedkamal@iict.res.in (A. Kamal).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.06.056

A. Kamal et al. / Bioorg. Med. Chem. 16 (2008) 7804–7810
7805
O
O
S
O
O
NH
N
HO
H
S
N
N
HN
O
N
H3CO
N
O
OH
Cl
(3, DC-81)
2
(1, LY-186641)
O
O
S
N
N
(CH2)n
O
O
S
O
H
N
R
N
S
N
N
N
O
N
H
( )n
N
H3CO
N
N
R
O
N
H3CO
O
5a-f
4
Figure 1. Chemical structures of sulfonylurea derivative (1, LY-186641), imidazobenzothiadiazine derivative (2), DC-81 (3), benzothiadiazine–PBD conjugates (4) and
triazolobenzothiadiazine–PBD conjugates (5a–f).
the preparation of PBD ring system as well as in the design of struc-
turally modified PBDs and their hybrids, in continuation of our ef-
forts towards the search for more potent anticancer agents.31–34
In the previous studies, we had reported35 new type of benzo-
thiadiazine–pyrrolobenzodiazepine conjugates (4) linked through
different 3-piperazinyl alkane spacers as significant cytotoxic and
DNA-binding agents. Based on these preliminary investigations,
we herein report the synthesis, DNA-binding ability and cytotoxic-
ity of triazolo fused benzothiadiazine linked through various al-
kane spacers to the C8-position of the A ring of PBD ring system
(5) to explore its potential as a new class of anticancer agents
(Fig. 1 and Scheme 1).
methods reported in our earlier studies31–35 and mercapto precur-
sors (7a and b) using K2CO3 in acetone to provide the nitrothioac-
etal intermediates 9a–f. Finally, these upon reduction with
SnCl2ꢀ2H2O in methanol followed by deprotection using HgCl2/
CaCO3 afford the desired PBD conjugates 5a–f (Scheme 2).
2.2. Biological activity
2.2.1. Cytotoxicity
Compounds 5a–f have been evaluated for their in vitro cytotox-
icity in selected human cancer cell lines of lung, breast, oral, colon,
prostate and ovary by using sulforhodamine B (SRB) method.37,38
The compounds exhibiting GI50 6 10ꢁ5 M (10
lM) are considered
to be active on the respective cell lines. The results expressed as
2. Results and discussion
2.1. Chemistry
GI50 are reported in Table 1. All the compounds 5a–f have exhib-
ited cytotoxicity with GI50 values ranging from 0.22 to 30.30
in comparison to adriamycin (GI50 0.16–7.25 M).
lM
l
Compounds 5a and 5c, substituted by a methyl group at the 10
position of benzothiadiazine ring with odd number of alkyl spacers
(n = 3 and 5) exhibit more activity than their counterpart with an
even number of alkyl spacer (5b; n = 4). Replacement of 10-methyl
group with a phenyl group in benzothiadiazine ring system (5d–f)
has decreased the activity, and on increasing the number of car-
bons in the alkyl spacer results in some increase in activity. It is as-
sumed that the alkyl spacer length and substituents at position 10
of the triazolobenzothiadiazine ring could play an important role
in the increase or decrease of the binding ability based on the
favourability of interaction with DNA. Compound 5c has exhibited
strong effect against DWD (AW13516),39 Gurav (AW8507),39
The preparation of mercaptotriazolobenzothiadiazine interme-
diates (7a and b) has been accomplished by the reaction of 3-
hydrazino-4-methyl/phenyl-4H-1,2,4-benzothiadiazine 1,1-diox-
ides (6a and b), which have been obtained by previously reported
method16 and carbon disulfide (CS2) in ethanolic KOH under reflux
conditions followed by the acidification with 1 M HCl to afford the
mercapto substituted derivatives36 (7a and b) as illustrated in
Scheme 1.
The synthesis of C8-linked triazolobenzothiadiazine–PBD
conjugates (5a–f) has been carried out from the (2S)-N-{4-[3-bro-
moalkoxy-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxalde-
hydediethylthioacetal (8), which has been prepared by the
O
O
SH
N
O
O
O
S-K+
N
O
S
S
S
N
N
(i)
(ii)
N
NHNH2
N
N
R
N
R
N
N
R
6a,b
7a,b
7a: R = CH3
7b: R = C6H5
Scheme 1. Reagents and conditions: (i) CS2, KOH, EtOH, reflux, 12 h; (ii) dil HCl.

7806
A. Kamal et al. / Bioorg. Med. Chem. 16 (2008) 7804–7810
studies with duplex form of calf thymus (CT)-DNA by using re-
O
SH
N
O
ported procedures.40,41 Thermal denaturation studies showed that
S
O
NO2
CH(SEt)2
N
Br (H2C)n
these compounds stabilized the thermal helix ? coil or melting
N
stabilization (DTm) for the CT-DNA duplex at pH 7.0, where PBD/
N
R
N
H3CO
DNA molar ratio is 1:5. Interestingly, all the benzothiadiazine–
PBD conjugates (5a–f) elevate the helix melting temperature of
CT-DNA in the range of 0.5–2.6 °C at 0 h and also examined after
18 and 36 h incubation at 37 °C (Table 2). Compound 5c showed
O
7a,b
R = CH3, C6H5
8a-c
(i)
n = 3-5
the highest
DTm of 2.1 °C at 0 h which increased up to 2.6 °C after
36 h incubation. The spacers in compounds 5a, 5c, 5d and 5f ap-
pear to impart the molecules a better fit in the minor groove of
DNA, leading to enhanced binding affinity. In the same experiment,
NO2
(CH2)n
H3CO
9a-f
O
O
S
O
CH(SEt)2
S
N
N
N
the naturally occurring DC-81 exhibits a
DTm of 0.7 °C upon incu-
N
N
R
bation under similar conditions. This result shows the effect on
DNA-binding affinity by linking the benzothiadiazine moiety to
PBD through different alkyl spacers at C8-postion of the DC-81. It
is expected that the anticancer activity of these compounds could
be due to a similar mechanism to that of PBD, that is by DNA-bind-
ing and by the action of sulfonyl urea derivatives on the inhibition
of DNA, RNA or protein synthesis.
O
(ii), (iii)
N
(CH2)n
O
O
S
O
H
S
3. Conclusion
N
N
N
H3CO
N
The synthesis and evaluation of anticancer activity of a new ser-
ies of benzothiadiazine–PBD conjugates has been investigated.
Triazolobenzothiadiazine pharmacophore linked through an alkyl
spacer at C8-position of the PBD ring system has emerged as po-
tential antitumour agent. Compound 5c has exhibited promising
N
R
O
5a-f
5a: R = CH3, n = 3
5b: R = CH3, n = 4
5c: R = CH3, n = 5
5d: R = C6H5, n = 3
5e: R = C6H5, n = 4
5f: R = C6H5, n = 5
activity (GI50 0.22 lM) against AW8507 cell line (oral cancer). Most
Table 2
Scheme 2. Reagents and conditions: (i) K2CO3, acetone, reflux, 24 h; (ii)
SnCl2ꢀ2H2O, CH3OH, 4 h, reflux; (iii) HgCl2, CaCO3, CH3CN/H2O, 12 h, rt.
Thermal denaturation data for triazolobenzothiadiazine–PBD conjugates (5a–f) with
calf thymus (CT)-DNA
Compound
[PBD]:[DNA] molar ratiob
D
Tm (°C)a after incubation at 37 °C
A2780 (GI50 1.95, 0.22 and 1.92
2.45–28.50 M against other cell lines, whereas compound 5a
exhibited significant activity in GI50 range of 1.83–2.29 M against
all the cell lines used in this study. Compound 5f has shown selec-
tivity against all the cell lines of oral cancer with GI50 value up to
lM, respectively) and a GI50 of
0 h
18 h
36 h
l
5a
5b
5c
5d
5e
5f
DC-81
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1.2
0.5
2.1
1.0
0.5
1.2
0.3
1.5
0.5
2.4
1.2
0.6
1.3
0.7
1.7
0.7
2.6
1.5
0.7
1.5
0.7
l
1.78 lM. Therefore, from these data as illustrated in Table 1, it ap-
pears that there is potential in this new type of PBD conjugates for
further exploitation with additional structural modifications.
a
For CT-DNA alone at pH 7.00 0.01, Tm = 69.2 °C 0.01 (mean value from 10
Tm values are 0.1–0.2 °C.
For a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA concentration = 100
and ligand concentration = 20 M in aqueous sodium phosphate buffer [10 mM
sodium phosphate + 1 mM EDTA, pH 7.00 0.01].
separate determinations), all
D
2.2.2. DNA-binding affinity: thermal denaturation studies
The DNA-binding ability for these triazolobenzothiadiazine–
PBD conjugates (5a–f) has been examined by thermal denaturation
b
l
M
l
Table 1
GI50 valuesa (in
lM) for compounds 5a–f in selected human cancer cell lines
Compound
A549b
HOP62b
Zr-75-1c
MCF7c
AW13516d
AW8507d
KBd
2.38
26.40
25.60
—
—
2.46
1.72
Colo205e
SiHaf
PC3g
A2780h
5a
5b
5c
5d
5e
2.29
2.28
2.45
—
29.71
30.30
7.25
2.13
—
—
—
—
2.19
25.90
28.50
26.01
—
2.06
2.29
—
—
—
2.24
26.60
1.95
—
1.98
1.78
0.10
2.24
2.29
0.22
—
25.80
2.48
0.17
1.93
2.11
2.19
—
—
25.60
0.14
2.12
27.00
27.90
—
—
2.30
0.18
2.15
25.50
25.51
—
28.80
2.40
1.81
1.83
2.17
1.92
30.02
2.27
2.01
0.16
5f
ADR
2.35
0.14
24.00
1.79
—
0.17
—, means GI50 values not attained at the concentrations used; ADR, adriamycin.
a
50% growth inhibition and the values are mean of three determinations.
Lung cancer.
Breast cancer.
Oral cancer.
Colon cancer.
Cervix cancer.
Prostate cancer.
Ovarian cancer.
b
c
d
e
f
g
h

A. Kamal et al. / Bioorg. Med. Chem. 16 (2008) 7804–7810
7807
of the compounds have demonstrated appreciable cytotoxicity
against selected cell lines. Moreover, these conjugates also exhib-
ited DNA-binding ability and this investigation provides some use-
ful lead towards the discovery of potential anticancer agents.
indicated by TLC, potassium carbonate was removed by suction
filtration and the solvent was removed under vacuum. The crude
product thus obtained was purified by column chromatography
using ethyl acetate/hexane (1:1) as eluant to afford the pure com-
pound 9a.
Yield 600 mg, 85%; mp 119–121 °C; 1H NMR (300 MHz, CDCl3):
d 8.02 (dd, J = 8.0, 1.4 Hz, 1H), 7.76 (dt, J = 7.3, 1.4 Hz, 1H), 7.66 (s,
1H), 7.40–7.25 (m, 2H), 6.80 (s, 1H), 4.86 (d, J = 3.6 Hz, 1H), 4.75–
4.62 (m, 1H), 4.27 (t, J = 5.8 Hz, 2H), 3.94 (s, 3H), 3.80 (s, 3H),
3.54 (dt, J = 6.5, 1.4 Hz, 2H), 3.32–3.18 (m, 2H), 2.90–2.65 (m,
4H), 2.40 (t, J = 6.5 Hz, 1H), 2.30–1.80 (m, 2H), 1.42–1.24 (m, 7H),
4. Experimental
4.1. General
All chemicals and reagents were obtained from Aldrich
(Sigma–Aldrich, St. Louis, MO, USA), Lancaster (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 containing 60 F-254, and visualization on TLC was achieved
by UV light or iodine indicator. Column chromatography was
performed with Merck 60–120 mesh silica gel. 1H and 13C NMR
spectra were recorded on Gemini Varian-VXR-unity (200 MHz) or
Bruker UXNMR/XWIN-NMR (300 M Hz) instruments. Chemical
shifts (d) are reported in ppm downfield from internal TMS stan-
dard. LC–MS was recorded on instrument LC-MSD-Trap-SL. ESI
spectra were recorded on Micro mass, Quattro LC using ESI+ soft-
ware with capillary voltage 3.98 kV and ESI mode positive ion trap
detector. High-resolution mass spectra (HRMS) were recorded on
QSTAR XL Hybrid MS/MS mass spectrometer. Elemental analysis
was within 0.4% of the theoretical values. Elemental analyses
were performed on a elemental analyzer, Model: VARIO EL,
Elementar, Germany. Melting points were determined with an
Electrothermal melting point apparatus, and are uncorrected.
0.99–0.85 (m, 2H); HRMS (ESI) m/z calcd for
C29H37N6O7S4
[M+H]+, 709.1606; found, 709.1578; IR (KBr) ( max/cmꢁ1): 2921,
m
2872, 1626, 1596, 1526, 1422, 1339, 1278, 1216, 1184, 1032.
4.2.4. (2S)-N-{4-[4-[3-(10-Methyl-3-sulfanyl-5,10-dihydro-5-
benzo[e][1,2,4]triazolo[4,3-b][1,2,4]thiadiazine-5,5-dione]butyl]-
oxy]-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehydedie-
thylthioacetal (9b)
This compound was prepared according to the method de-
scribed for compound 9a, employing compound 8b (535 mg,
1 mmol) and compound 7a (2268 mg, 1 mmol).
Yield 560 mg, 78%; mp 80–82 °C; 1H NMR (300 MHz, CDCl3): d
8.01 (dd, J = 8.1, 1.5 Hz, 1H), 7.80–7.72 (m, 1H), 7.64 (s, 1H),
7.36–7.27 (m, 2H), 6.80 (s, 1H), 4.86 (d, J = 3.7 Hz, 1H), 4.74–4.65
(m, 1H), 4.18–4.04 (m, 2H), 3.94 (s, 3H), 3.79 (s, 3H), 3.30–3.20
(m, 2H), 2.85–2.65 (m, 4H), 2.35–2.20 (m, 1H), 2.10–2.02 (m,
6H), 1.90–1.58 (m, 1H), 1.35 (q, J = 7.3 Hz, 4H), 1.28–1.24 (m,
2H), 0.95–0.90 (m, 2H); HRMS (ESI) m/z calcd for C30H39N6O7S4
[M+H]+, 723.1763; found, 723.1752, [M+H]+; IR (KBr)
(mmax/
cmꢁ1): 2925, 2870, 1672, 1637, 1582, 1520, 1423, 1335, 1275,
1220, 1177, 1017.
4.2. General procedures
4.2.1. Synthesis ofcompound 10-methyl-3-sulfanyl-5,10-dihydro-
5l6-benzo[e][1,2,4] triazolo[4,3-b][1,2,4]thiadiazine-5,5-dione (7a)
A mixture of 3-hydrazino-4-methyl-4H-1,2,4-benzothiadiazine
1,1-dioxide (6a, 1.5 g, 6.63 mmol) was treated with CS2 (5 mL)
and KOH (715 mg, 21 mmol) and refluxed in ethanol for 8 h to give
potassium salt of compound 6a. On further acidification of this
with 1 M HCl, precipitate was obtained, which was filtered and
washed with water and recrystallized from ethanol to give the
pure compound 7a.
4.2.5. (2S)-N-{4-[5-[3-(10-Methyl-3-sulfanyl-5,10-dihydro-5-
benzo[e][1,2,4]triazolo[4,3-b][1,2,4]thiadiazine-5,5-dione]pentyl]-
oxy]-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde-
diethylthioacetal (9c)
This compound was prepared according to the method de-
scribed for compound 9a, employing compound 8c (549 mg,
1 mmol) and compound 7a (268 mg, 1 mmol).
Yield 670 mg, 91%; mp 51–53 °C; 1H NMR (200 MHz, CDCl3): d
8.10 (d, J = 8.0 Hz, 1H), 7.77 (t, J = 8.0 Hz, 1H), 7.65 (s, 1H), 7.45–
7.25 (m, 2H), 6.80 (s, 1H), 4.86 (d, J = 3.6 Hz, 1H), 4.75–4.64 (m,
1H), 4.18–4.05 (m, 2H), 3.95 (s, 3H), 3.18 (s, 3H), 3.25 (t,
J = 7.3 Hz, 4H), 2.87–2.65 (m, 4H), 2.40–2.19 (m, 1H), 2.15–1.80
(m, 4H), 1.78–1.60 (m, 1H), 1.35 (p, J = 3.6 Hz, 4H), 1.30–1.20 (m,
4H), 0.98–0.80 (m, 2H); HRMS (ESI) m/z calcd for C31H40N6O7S4Na
Yield 1.33 g, 75%; mp 156–158 °C; 1H NMR (200 MHz, CDCl3): d
8.00 (d, J = 7.4 Hz, 1H), 7.76 (t, J = 7.4 Hz, 1H), 7.34 (t, J = 8.1 Hz,
2H), 3.64 (s, 3H); MS (LC) m/z 269 [M+H]+.
4.2.2. 10-Phenyl-3-sulfanyl-5,10-dihydro-5l6-benzo[e][1,2,4]-
triazolo[4,3-b][1,2,4]thiadiazine-5,5-dione (7b)
The compound 7b was prepared according to the method de-
scribed for compound 7a, employing compound 6b (1.5 g,
5.20 mmol).
[M+Na]+, 759.1739; found, 759.1752; IR (KBr) ( max/cmꢁ1): 2924,
m
2853, 1643, 1587, 1563, 1519, 1488, 1446, 1470, 1352, 1274,
1218, 1175, 723.
Yield 1.40 g, 82%; mp 133–135 °C; 1H NMR (200 MHz, CDCl3):
8.03 (dd, J = 7.3, 1.4 Hz, 1H), 7.70–7.54 (m, 4H), 7.53–7.40 (m,
2H), 7.31 (t, J = 7.3 Hz, 1H), 6.62 (d, J = 8.1 Hz, 1H); MS (LC) m/z
331 [M+H]+.
4.2.6. (2S)-N-{4-[3-[3-(10-Phenyl-3-sulfanyl-5,10-dihydro-5-
benzo[e][1,2,4]triazolo[4,3-b][1,2,4]thiadiazine-5,5-dione]-
propyl]oxy]-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxal-
dehydediethylthioacetal (9d)
This compound was prepared according to the method de-
scribed for compound 9a by employing compound 8a (521 mg,
1 mmol) and compound 7b (330 mg, 1 mmol).
4.2.3. Synthesis of (2S)-N-{4-[3-[3-(10-Methyl-3-sulfanyl-5,10-
dihydro-5-benzo[e][1,2,4]triazolo[4,3-b][1,2,4]-thiadiazine-5,5-
dione]propyl]oxy]-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-
carboxaldehydediethylthioacetal (9a)
To a solution of (2S)-N-[4-(3-bromobutyl)oxy-5-methoxy-2-
nitrobenzoyl)pyrrolidine-2-carboxarbaldehydediethylthioacetal
8a (521 mg, 1 mmol) in acetone (10 mL) anhydrous K2CO3
(552 mg, 4 mmol) and the 3-mercaptotriazolobenzothiadiazine
(7a, 268 mg, 1 mmol) were added. The reaction mixture was
heated to reflux for 4–6 h. After completion of the reaction as
Yield 600 mg, 84%; mp 60–62 °C; 1H NMR (300 MHz, CDCl3): d
8.13 (dd, J = 8.1, 1.5 Hz, 1H), 7.70–7.62 (m, 4H), 7.60–7.52 (m, 1H),
7.48–7.35 (m, 3H), 6.82 (s, 1H), 6.77 (d, J = 8.1 Hz, 1H), 4.88 (d,
J = 3.7 Hz, 1H), 4.76–4.64 (m, 1H), 4.24 (t, J = 6.0 Hz, 2H), 3.97 (s,
3H), 3.40 (t, J = 6.6, 1.5 Hz, 2H), 3.32–3.22 (m, 1H), 2.90–2.70 (m,
4H), 2.43–2.36 (m, 1H), 2.18–1.90 (m, 2H), 1.88–1.50 (m, 2H), 1.37
(q, J = 7.3 Hz, 4H), 1.30–1.24 (m, 2H), 0.98–0.92 (m, 2H); MS (LC)

7808
A. Kamal et al. / Bioorg. Med. Chem. 16 (2008) 7804–7810
m/z 793.2 [M+Na]+; IR (KBr) (
1553, 1519, 1485, 1420, 1351, 1218, 1185, 1040, 720.
m
max/cmꢁ1): 2923, 2866, 1639, 1579,
2H), 0.87 (p, J = 5.1 Hz, 1H); MS (LC) m/z 555.1 [M+H]+; IR (KBr)
(m
max/cmꢁ1): 2924, 2854, 1586, 1560, 1514, 1489, 1446, 1351,
1260, 1219, 1175, 1024. Anal. Calcd for C25H26N6O5S2: C, 54.14; H,
4.2.7. (2S)-N-{4-[4-[3-(10-Phenyl-3-sulfanyl-5,10-dihydro-5-benzo-
[e][1,2,4]triazolo[4,3-b][1,2,4]thiadiazine-5,5-dione]butyl]oxy]-
5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehydedi-
ethylthioacetal (9e)
This compound was prepared according to the method described
for compound 9a, employing compound 8b (535 mg, 1 mmol) and
compound 7b (330 mg, 1 mmol).
4.72; N, 15.15. Found: C, 54.35; H, 4.81; N, 15.42.
4.2.10. 7-Methoxy-8-{4-(10-methyl-3-sulfanyl-5,10-dihydro-5-
benzo[e][1,2,4]triazolo[4,3-b][1,2,4]thiadiazine-5,5-dione)]
butyl}oxy-(11aS)-1,2,3,11a-5H-pyrrolo[2,1-c][1,4]benzodiaze-
pin-5-one (5b)
The compound 5b was prepared according to the method de-
scribed for the compound 5a, employing the compound 9b
(720 mg, 1 mmol) to afford the compound 5b.
Yield 675 mg, 85%; mp 64–66 °C; 1H NMR (300 MHz, CDCl3): d
8.13 (dd, J = 8.1, 1.5 Hz, 1H), 7.68–7.60 (m, 4H), 7.58–7.50 (m, 1H),
7.45–7.34 (m, 3H), 6.81 (s 1H), 6.74 (d, J = 8.1 Hz, 1H), 4.87 (d,
J = 3.7 Hz, 1H), 4.75–4.66 (m, 1H), 4.17–4.06 (m, 2H), 3.94 (s, 3H),
3.32–3.20 (m, 4H), 2.88–2.66 (m, 4H), 2.36–2.18 (m, 1H), 2.15–
1.90 (m, 2H), 1.88–1.46 (m, 1H), 1.35 (q, J = 7.3 Hz, 4H), 1.30–1.20
(m, 2H), 0.96–0.84 (m, 4H); HRMS (ESI) m/z calcd for C35H40N6O7S4-
Yield 330 mg, 58%; mp 132–134 °C; ½a D25
ꢃ
+174.64 (c 0.5, CHCl3);
1H NMR (200 MHz, CDCl3): d 1H NMR (200 MHz, CDCl3): d 8.11 (d,
J = 7.5 Hz, 1H), 7.75 (t, J = 7.5 Hz, 1H), 7.66–7.50 (m, 2H), 7.53 (s,
1H), 7.42–7.20 (m, 2H), 6.81 (s, 1H), 4.22–3.96 (m, 4H), 3.82 (s,
3H), 3.74–3.40 (m, 4H), 3.21 (t, J = 6.5 Hz, 2H), 2.38–2.23 (m, 1H),
2.15–1.85 (m, 1H), 1.75–1.56 (m, 2H), 1.24 (s, 2H), 0.95–0.78 (m,
Na [M+Na]+, 807.1739; found, 807.1727; IR (KBr) ( max/cmꢁ1): 2924,
m
2855, 1640, 1579, 1553, 1519, 1485, 1420, 1351, 1255, 1218, 1184,
1H); MS (LC) m/z 569 [M+H]+; IR (KBr) ( max/cmꢁ1): 2928, 2858,
m
1058, 744.
1576, 1552, 1510, 1491, 1449, 1355, 1265, 1172. Anal. Calcd for
C26H28N6O5S2: C, 54.92; H, 4.96; N, 14.78. Found: C, 54.61; H, 4.86;
4.2.8. (2S)-N-{4-[5-[3-(10-Phenyl-3-sulfanyl-5,10-dihydro-5-
benzo[e][1,2,4]triazolo[4,3-b][1,2,4]thiadiazine-5,5-dione]pentyl]-
oxy]-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde-
diethylthioacetal (9f)
This compound was prepared according to the method described
for compound 9a, employing compound 8c (549 mg, 1 mmol) and
compound 7b (330 mg, 1 mmol).
N, 14.97.
4.2.11. 7-Methoxy-8-{5-(10-methyl-3-sulfanyl-5,10-dihydro-5-
benzo[e][1,2,4]triazolo[4,3-b][1,2,4]thiadiazine-5,5-dione)]-
pentyl}oxy-(11aS)-1,2,3,11a-5H-pyrrolo[2,1-c][1,4] benzodiaze-
pin-5-one (5c)
The compound 5c was prepared according to the method de-
scribed for the compound 5a, employing the compound 9c
(736 mg, 1 mmol) to afford the compound 5c.
Yield 640 mg, 86%; mp 74–76 °C; 1H NMR (300 MHz, CDCl3): d
8.13 (dd, J = 9.0, 1.5 Hz, 1H), 7.72–7.50 (m, 5H), 7.48–7.34 (m, 3H),
6.80 (s, 1H), 6.74 (d, J = 9.0 Hz, 1H), 4.87 (d, J = 3.7 Hz, 1H), 4.76–
4.75 (m, 1H), 4.10 (dt, J = 6.0, 1.5 Hz, 1H), 3.95 (s, 3H), 3.30–3.15
(m, 3H), 2.88–2.65 (m, 4H), 2.35–2.20 (m, 1H), 2.15–2.05 (m, 1H),
1.90 (p, J = 7.5 Hz, 4H), 1.72–1.60 (m, 1H), 1.42–1.20 (m, 7H), 0.96–
0.80 (m, 4H); HRMS (ESI) m/z calcd for C36H43N6O7S4 [M+H]+,
Yield 360 mg, 62%; mp 90–92 °C; ½a D25
ꢃ
+181.00 (c 0.5, CHCl3); 1H
NMR (200 MHz, CDCl3): d 8.10 (d, J = 7.3 Hz, 1H), 7.78 (t, J = 7.3 Hz,
1H), 7.65 (d, J = 4.4 Hz, 1H), 7.50 (s, 1H), 7.48–7.21 (m, 3H), 6.80 (s,
1H), 4.09 (d, J = 6.6 Hz, 1H), 3.95 (s, 3H), 3.80 (s, 3H), 3.76–3.48 (m,
4H), 3.26 (t, J = 6.6 Hz, 2H), 2.40–2.20 (m, 1H), 2.16–1.80 (m, 2H),
1.76–1.54 (m, 3H), 1.26 (s, 2H), 0.96–0.80 (m, 1H); HRMS (ESI) m/z
calcd for C27H31N6O5S2 [M+H]+, 583.1763; found, 583.1781; IR
799.2076; found, 799.2039; IR (KBr) (m
max/cmꢁ1): 2928, 2850,
1642, 1582, 1555, 1518, 1482, 1421, 1358, 1249, 1211, 1164, 1054,
741.
(KBr) (m
max/cmꢁ1): 2926, 2860, 1589, 1561, 1489, 1446, 1351,
1260, 1216, 1177, 1100, 1021, 755. Anal. Calcd for C27H30N6O5S2:
4.2.9. Synthesis of 7-methoxy-8-{3-(10-methyl-3-sulfanyl-5,10-
dihydro-5-benzo[e]-[1,2,4]triazolo[4,3-b][1,2,4]thiadiazine-5,5-
dione)]propyl}oxy-(11aS)-1,2,3,11a-5H-pyrrolo[2,1-c ][1,4]-
benzodiazepin-5-one (5a)
To a solution of compound 9a (706 mg, 1 mmol) in methanol
(20 mL), SnCl2ꢀ2H2O (5 mmol) was added and refluxed for 1–2 h.
The methanol was evaporated in vacuum and the aqueous layer
was then carefully adjusted to pH 8 with 10% NaHCO3 solution and
then extracted with ethyl acetate (20ꢂ 30 mL). The combined organ-
ic phase was dried over anhydrous Na2SO4 and evaporated under
vacuum to afford the amino diethyl thioacetal, which due to poten-
tial stability problems42 proceeded for the next step.
C, 55.66; H, 5.19; N, 14.42. Found: C, 55.93; H, 5.34; N, 14.70.
4.2.12. 7-Methoxy-8-{3-(10-phenyl-3-sulfanyl-5,10-dihydro-5-
benzo[e][1,2,4]triazolo[4,3-b][1,2,4]thiadiazine-5,5-dione)]
propyl}oxy-(11aS)-1,2,3,11a-5H-pyrrolo[2,1-c][1,4] benzodiaze-
pin-5-one (5d)
The compound 5d was prepared according to the method de-
scribed for the compound 5a, employing the compound 9d
(770 mg, 1 mmol) to afford the compound 5d.
Yield 400 mg, 65%; mp 124–126 °C; ½a D25
ꢃ
+144.62 (c 0.5, CHCl3);
1H NMR (200 MHz, CDCl3): d 8.20–8.08 (m, 1H), 7.74–7.50 (m, 6H),
7.48–7.35 (m, 4H), 6.80 (d, J = 5.4 Hz, 1H), 6.74 (s, 1H), 4.30–4.10
(m, 2H), 3.95 (s, 3H), 3.90–3.50 (m, 2H), 3.36 (t, J = 7.0 Hz, 2H),
2.45–4.25 (m, 1H), 2.16–1.96 (m, 1H), 1.33–1.23 (m, 2H), 0.98–
0.82 (m, 2H); HRMS (ESI) m/z calcd for C30H29N6O5S2 [M+H]+,
A
solution of amino diethyl thioacetal (1 mmol), HgCl2
(2.26 mmol) and CaCO3 (2.46 mmol) in CH3CN/water (4:1) was stir-
red slowly at room temperature until TLC indicated complete loss of
starting material (12 h). The reaction mixture was diluted with ethyl
acetate(30 mL)andfilteredthroughaCelitebed. Theclearyellowor-
ganic supernatant was extracted with saturated 5% NaHCO3 (20 mL)
and brine (20 mL), and the combined organic phase was dried over
anhydrous Na2SO4. The organic layer was evaporated in vacuum
and purified by column chromatography (MeOH/EtOAc, 2:98) to
give the final product 5a.
617.1640; found, 617.1640; IR (KBr) (m
max/cmꢁ1): 2929, 2852,
1597, 1552, 1484, 1437, 1353, 1256, 1221, 1186, 1024, 746. Anal.
Calcd for C30H28N6O5S2: C, 58.43; H, 4.58; N, 13.63. Found: C,
58.70; H, 4.42; N, 13.91.
4.2.13. 7-Methoxy-8-{4-(10-phenyl-3-sulfanyl-5,10-dihydro-5-
benzo[e][1,2,4]triazolo[4,3-b][1,2,4]thiadiazine-5,5-dione)]
butyl}oxy-(11aS)-1,2,3,11a-5H-pyrrolo[2,1-c][1,4] benzodiaze-
pin-5-one (5e)
The compound 5e was prepared according to the method de-
scribed for the compound 5a, employing the compound 9e
(784 mg, 1 mmol) to afford the compound 5e.
Yield 280 mg, 51%; mp 86–88 °C; ½a D25
ꢃ
+152.80 (c 0.5, CHCl3); 1H
NMR (200 MHz, CDCl3): d 8.08 (dd, J = 7.9, 1.4 Hz, 1H), 7.80–7.64 (m,
2H), 7.52 (s, 1H), 7.46–7.24 (m, 2H), 6.84 (d, J = 2.2 Hz, 1H), 4.25 (t,
J = 6.6 Hz, 1H), 4.00–3.88 (m, 4H), 3.80 (s, 3H), 3.72–3.35 (m, 2H),
2.48–2.24 (m, 1H), 2.16–1.95 (m, 2H), 1.78–1.52 (m, 2H), 1.25 (s,

A. Kamal et al. / Bioorg. Med. Chem. 16 (2008) 7804–7810
7809
Yield 410 mg, 66%; mp 76–78 °C; ½a D25
ꢃ
+135.32 (c 0.5, CHCl3); 1H
Percentage growth was expressed as the (ratio of average absor-
bance of the test well to the average absorbance of the control wells)
multiplied by 100. Growth inhibition of 50% (GI50) was calculated
from [(Ti ꢁ Tz)/(C ꢁ Tz)] ꢂ 100 = 50, which is the drug concentration
resulting in a 50% reduction in the net protein increase (as measured
by SRB staining) in control cells during the drug incubation.
Here, Tz is the optical density at time zero OD of control; and Ti,
the OD of test growth in the presence of drug.
NMR (200 MHz, CDCl3): d 8.12 (d, J = 8.5 Hz, 1H), 7.70–7.48 (m,
7H), 7.46–7.30 (m, 3H), 6.77 (d, J = 4.6 Hz, 1H), 6.71 (s, 1H), 4.08
(d, J = 6.2 Hz, 1H), 3.92 (s, 3H), 3.86–3.42 (m, 2H), 3.23 (t,
J = 6.2 Hz, 2H), 2.38–2.21 (m, 1H), 2.14–1.80 (m, 3H), 1.35–1.18
(m, 3H), 0.98–0.80 (m, 2H); HRMS (ESI) m/z calcd for C31H31N6O5S2
[M+H]+, 631.1797; found, 631.1785; IR (KBr) ( max/cmꢁ1): 2918,
m
1591, 1552, 1481, 1357, 1251, 1236, 1159, 1021, 754. Anal. Calcd
for C31H30N6O5S2: C, 59.03; H, 4.79; N, 13.32. Found: C, 59.35; H,
4.66; N, 13.58.
4.4. Thermal denaturation studies
4.2.14. 7-Methoxy-8-{5-(10-phenyl-3-sulfanyl-5,10-dihydro-5-
benzo[e][1,2,4]triazolo[4,3-b][1,2,4]thiadiazine-5,5-dione)]
pentyl}oxy-(11aS)-1,2,3,11a-5H-pyrrolo[2,1-c][1,4]benzodiaze-
pin-5-one (5f)
Compoundswere subjectedto thermaldenaturationstudieswith
duplex-form CT-DNA using reported method.40 Working solutions
in aqueous buffer (10 mM NaH2PO4/Na2HPO4, 1 mM Na2EDTA, pH
7.00 + 0.01) containing CT-DNA (100
lM in phosphate) and the
The compound 5f was prepared according to the method de-
scribed for the compound 5a, employing the compound 9f
(798 mg, 1 mmol) to afford the compound 5f.
PBD (20 M) were prepared by addition of concentrated PBD solu-
l
tions in DMSO to obtain a fixed [PBD]/[DNA] molar ratio of 1:5.
The DNA–PBD solutions were incubated at 37 °C for 0, 18 and 36 h
prior to analysis. Samples were monitored at 260 nm using a
Beckman–Coulter DU 800 spectrophotometer fitted with high
performance temperature controller, and heating was applied at
1 °C minꢁ1 in the 40–90 °C range. DNA helix ? coil transition tem-
peratures (Tm) were obtained from the maxima in the d(A260)/dT
derivative plots. Results are given as means standard deviation
from three determinations and are corrected for the effects of DMSO
co-solvent using a linear correction term.41 Drug-induced altera-
Yield 640 mg, 68%; mp 84–86 °C; ½a D25
ꢃ
+179.00 (c 0.5, CHCl3); 1H
NMR (300 MHz, CDCl3): d 8.13 (dd, J = 8.0, 1.46 Hz, 1H), 7.66 (d,
J = 5.1 Hz, 1H), 7.65–7.48 (m, 6H), 7.46–7.32 (m, 3H), 6.78 (d,
J = 5.1 Hz, 1H), 6.72 (s, 1H), 4.22 (dd, J = 5.8, 2.1 Hz, 1H), 4.07 (t,
J = 6.5 Hz, 1H), 3.94 (s, 3H), 3.90–3.50 (m, 2H), 3.19 (t, J = 6.5 Hz,
2H), 2.40–2.25 (m, 1H), 2.06 (d, J = 7.3 Hz, 1H), 1.97–1.75 (m, 2H),
1.73–1.50 (m, 2H), 1.41–1.18 (m, 2H), 0.98–0.80 (m, 2H); 13C NMR
(75 MHz, CDCl3): 165.2, 164.6, 162.3, 153.8, 150.8, 147.8, 140.8,
137.5, 136.0, 134.4, 130.8, 130.2, 128.9, 124.4, 123.8, 121.8, 120.0,
117.3, 111.5, 110.4, 68.7, 56.1, 53.7, 46.6, 31.0, 29.5, 28.7, 28.3,
tions in DNA melting behavior are given by
DTm = Tm(DNA +
PBD) ꢁ Tm(DNA alone), where the Tm value for the PBD-free
CT-DNA is 69.2 0.01 °C. The fixed [PBD]/[DNA] ratio used did not
result in binding saturation of the host DNA duplex for any
compound examined.
25.0, 24.1; HRMS (ESI) m/z calcd for
645.1953; found, 645.1947; IR (KBr) (
C
32H31N6O5S2 [M+H]+,
m
max/cmꢁ1): 2922, 2860,
1602, 1556, 1475, 1438, 1361, 1248, 1230, 1162, 1026, 755. Anal.
Calcd for C32H30N6O5S2: C, 59.61; H, 5.00; N, 13.03. Found: C,
59.92; H, 5.17; N, 12.88.
Acknowledgments
The author and M.N.A.K. thank CSIR, New Delhi; Y.V.V.S. thanks
UGC, New Delhi; and N.K. thanks DBT for the award of research fel-
lowships. We are also thankful to the Department of Biotechnology
(BT/PR/7037/Med/14/933/2006), New Delhi; for financial
assistance.
4.3. In vitro evaluation of cytotoxic activity
The synthesized compounds (5a–f) have been evaluated for their
in vitro cytotoxicity in selected human cancer cell lines of breast (Zr-
75-1), lung (A-549), colon (Colo205), oral (DWD, Gurav), prostate
(PC-3) and ovarian (A2780) origin. A protocol of 48 h continuous
drug exposure has been used and a sulforhodamine B (SRB) protein
assay has been used to estimate cell viability or growth.
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