Synthesis, structure analysis, and antibacterial activity of some novel 10-substituted 2-(4-piperidyl/phenyl)-5,5-dioxo[1,2,4]triazolo[1,5-b][1,2,4]benzothiadiazine derivatives

Article abstract of DOI:10.1016/j.bmcl.2007.07.043

A new series of 10-substituted 5,5-dioxo-5,10-dihydro[1,2,4]triazolo[1,5-b]-[1,2,4]benzothiadiazine arylsulfonamide derivatives (10a-j and 13a-f) was synthesized. The structures of these compounds were confirmed on the basis of spectral data, elemental an

Full text of DOI:10.1016/j.bmcl.2007.07.043

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5400–5405
Synthesis, structure analysis, and antibacterial activity
of some novel 10-substituted 2-(4-piperidyl/phenyl)-5,5-
dioxo[1,2,4]triazolo[1,5-b][1,2,4]benzothiadiazine derivatives
Ahmed Kamal,a,* M. Naseer A. Khan,a K. Srinivasa Reddy,a K. Rohini,a
G. Narahari Sastry,b B. Sateeshb and B. Sridharc
aBiotransformation Laboratory, Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 007, India
bMolecular Modelling Group, Indian Institute of Chemical Technology, Hyderabad 500 007, India
cLaboratory of X-ray Crystallography, Indian Institute of Chemical Technology, Hyderabad 500 007, India
Received 28 March 2007; revised 11 July 2007; accepted 13 July 2007
Available online 6 August 2007
Abstract—A new series of 10-substituted 5,5-dioxo-5,10-dihydro[1,2,4]triazolo[1,5-b]-[1,2,4]benzothiadiazine arylsulfonamide deriv-
atives (10a–j and 13a–f) was synthesized. The structures of these compounds were confirmed on the basis of spectral data, elemental
analysis, X-ray analysis, and quantum chemical calculations. These compounds were evaluated for their efficacy as antibacterial
agents against various Gram-positive and Gram-negative strains of bacteria. Amongst these compounds 10f and 10i were the most
active compounds against Escherichia coli and 13e against E. coli as well as Bacillus subtilis. Moreover, other compounds also
showed potent inhibitory activity in comparison to the standard drugs.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The demand for novel chemotherapeutic antibacterial
agents remains attractive in the field of medicinal chem-
istry. After many years of extensive studies on the struc-
tural modification of known antibacterial scaffolds, it is
increasingly becoming difficult to deliver new leads. The
focus of such antibacterials’ research has, therefore,
moved to the identification of novel chemical classes
of bacterial targets. In fact, in the past 40 years only
two new chemical classes of antibiotics, oxazolidinone
(linezolid)1 and the lipopeptide (daptomycin),2 have
been introduced to the market and existing antibiotics
are directed at a small number of targets, mainly cell
wall, DNA, and protein biosynthesis.
sulfonamides as antibacterial agents in the early 1930s
was the beginning of one of the most fascinating areas
of chemotherapeutic agents and once used successfully
in the treatment of a variety of bacterial infections.
However, the rapid emergence of sulfonamide resistance
organisms and the development of more potent drugs
have limited their clinical use. Some organisms are resis-
tant to all approved antibiotics and can only be treated
with experimental and potentially toxic drugs. For these
reasons, there is an overwhelming need to develop more
effective antibacterial agents to treat infections caused
by antibiotic resistant bacterial pathogens.
Sulfonamides exert their effect by targeting on dihydro-
pteroate synthase (DHPS) enzyme, which catalyzes folic
acid pathway in bacteria and some eukaryotic cells,9 but
it is not present in human cells.10 This is the basis for the
selective effect of sulfonamides on bacteria and for their
broad spectrum of antibacterial activity. 4H-1,2,4-Ben-
zothiadiazine 1,1-dioxides can be considered as cyclic
sulfonamide class of molecules and have been exten-
sively studied as potassium channel openers, for exam-
ple, diazoxide.11 Moreover, these compounds have
exhibited marked inhibitory activity12 against Gram-po-
sitive bacteria and recently we have investigated this
structural core for its anti-Mycobacterium activity.13
The sulfonamide group is considered as a pharmaco-
phore which is present in a number of biologically active
molecules, particularly in antimicrobial agents.3–5 In
addition, numerous sulfonamide derivatives have been
reported as carbonic anhydrase inhibitors,6 anticancer-
ous,7 and anti-inflammatory agents.8 The discovery of
Keywords: Triazolobenzothiadiazines; Aryl sulfonamides; Antibacte-
rial activity; X-ray crystallography; Quantum chemical calculations.
*
Corresponding author. Tel.: +91 40 27193157; fax: +91 40
27193189; e-mail: ahmedkamal@iict.res.in
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.043

A. Kamal et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5400–5405
5401
On the basis of these findings and in continuation of our
search on 1,2,4-benzothiadiazine derivatives with poten-
tial biological activity, we have synthesized14 new mole-
cules based on 1,2,4-benzothiadiazine 1,1-dioxide ring
system by incorporating arylsulfonamide functionality.
The arylsulfonamide structural motif is well known for
exhibiting antibacterial activity.15–20 This article de-
scribes the synthesis and antibacterial activity of some
novel triazolobenzothiadiazine linked arylsulfonamides
(10a–j and 13a–f). The structure of these molecules has
been confirmed by X-crystallography and the rearrange-
ment of the ring is explained on the basis of relative
energies and proton affinities. Almost all compounds
showed promising antibacterial activity against Gram-
positive and Gram-negative bacterial strains.
1,2,4-benzothiadiazine 1,1-dioxides (6a and b) and
isonipecotic acid (7) in phosphorus oxychloride.22 The
target sulfonamide linked triazolobenzothiadiazines
10a–j were obtained by the condensation of 8a and b
with substituted arylsulfonyl chlorides (9a–e) in pyridine23
(Scheme 1).
A probable mechanism22 of the reaction course is shown
in Scheme 2. Treatment of 3-hydrazino-4-methyl-4H-
1,2,4-benzothiadiazine 1,1-dioxide and isonipecotic acid
with POCl3 gives 3-(N0-piperidinoyl)benzothiadiazine
hydrazide of type A, which undergoes chlorination to
give chloro derivative of type B. This undergoes intra-
molecular ring closure with evolution of HCl to give
triazolo[4,3-b][1,2,4]benzothiadiazine (C). Protonation
of triazole ring D in acidic condition to form a sulfonyl
chloride E by SO2–N bond cleavage takes place which in
turn cyclizes to more stable triazolo[1,5-b][1,2,4]benzo-
thiadiazines (8a and b) with minimization of potential
energies. Structures of the compounds 8a and b, and
final products 10a–j (Scheme 1) were confirmed by
the elemental analysis, IR, 1H, 13C NMR, and mass
The preparation of the starting materials, 3-hydrazino-
4-methyl/phenyl-4H-1,2,4-benzothiadiazine 1,1-dioxides
(6a and b), was accomplished by the synthetic sequences
as previously reported21 (Scheme 1). The synthesis of
triazolo fused benzothiadiazines (8a and b) was carried
out by refluxing 3-hydrazino-4-methyl/phenyl-4H-
R
N
NHR
O
O
S
O
a
Cl
N C O
NH
+
O
S
Cl
3a-b
O
2
1a-b
b
COOH
O
N
O
O
O
O
O
S
S
c
S
d
N
NH
N
R
Cl
N
R
NHNH2
N
R
4a-b
O
N
H
5a-b
6a-b
7
NH
e
O
O
x
S
N
N
e
N
N
R
C
O
S
O
O
O
O
O
S
N
N
Y
S
O
S
N
N
Cl
+
N
f
N
O
NH
N
Y
X
N
R
N
R
X
10a-j
9a-e
8a-b
9a: X = Y = H
9b: X = Y = Cl
8a: R = CH3
8b: R = C6H5
10a : R = Ph, X = Y = H
10b : R = Ph, X = Y = Cl
9c: X = NHAc, Y = H
9d: X = F, Y = H
10c : R = Ph, X = NHAc, Y = H
10d : R = Ph, X = F, Y = H
10e : R = Ph, X = CH3, Y = H
10f : R = CH3, X = Y = H
10g : R = CH3, X = Y = Cl
10h : R = CH3, X =F, Y = H
10i : R = CH3, X = NHAc, Y = H
10j : R = CH3, X = CH3, Y = H
9e: X = CH3, Y = H
Scheme 1. Reagents and conditions: (a) CH3NO2, 0 ꢁC, rt, 30 min; (b) AlCl3, 120 ꢁC, 30 min; (c) PCl5, 190 ꢁC, 30 min; (d) N2H4ÆH2O, CHCl3, 10 ꢁC;
(e) POCl3, 110 ꢁC, 3–4 h; (f) pyridine, rt, 4 h.

5402
A. Kamal et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5400–5405
COOH
O
O
O
O
S
S
N
NH
POCl3
N
H
N
+
N
R
N
H
N
R
NHNH2
N
H
O
A
NH
Cl
NH
O
O
O
O
O
O
S
S
S
N
HCl
NH
NH
N
N
N
N
N
N
H
N
R
-HCl
N
R
N
N
R
Cl
D
C
B
O
S
O
O
O
Cl
HN
S
N
N
N
N
NH
NH
-HCl
N
N
R
N
R
E
8a-b
Scheme 2. Proposed mechanism of the formation of 10-substituted 2-piperidyl[1,2,4]triazolo[1,5-b][1,2,4]benzothiadiazines 8a and b.
spectrometry. However, the spectroscopic data did not
allow straightforward discrimination between expected
benzo[e][1,2,4]triazolo[4,3-b][1,2,4]thiadiazines’ C struc-
ture and the alternative benzo[e][1,2,4]triazolo[1,5-
b][1,2,4]thiadiazines 8a and b (Scheme 1). Therefore,
X-ray crystallography24 was undertaken on representa-
tive compound 10b to investigate further discrete struc-
tural aspects of this compound. The compound 10b was
subjected to single-crystal X-ray analysis to prove the
alternative benzo[e][1,2,4]triazolo[1,5-b][1,2,4]thiadiazines
8a and b (Fig. 1 and Scheme 1). Crystal of compound
10b was grown by slow evaporation from a mixture of
solvents, that is, methanol, chloroform, and diisopropy-
lether (2:3:1).
the compounds 6a and b and p-amino benzoic acid
(11) by refluxing in phosphorus oxychloride followed
by the condensation of 12a and b with arylsulfonyl chlo-
rides (9a–e) in pyridine (Scheme 3).
Quantum chemical calculations were employed to cor-
roborate the observed experimental results. A two-mod-
el system was studied to explain the preferential
formation of the structure 8a over C (Fig. 2). All the cal-
culations were carried out by using the Gaussian 03 pro-
gram package.25 Geometry optimization and frequency
calculations were performed on all the structures consid-
ered at B3LYP/6-31G* level. Further, the proton affini-
ties were also calculated at the same level of theory.
Similarly, 3-(N0-arylsulfonyl)triazolobenzothiadiazine
derivatives (13a–f) were obtained by the reaction of
Both these structures (8a and C) were found to be
minima on the potential energy surface. The calculated
relative energies are given in parentheses. Based on the
calculated relative energies it was observed that 8a is
more stable than C and these energy values are shown
in Figure 2. Highest occupied molecular orbital and
lowest unoccupied molecular orbital gap for 8a is
4.44 eV and C is 4.35 eV. This is evident as the
HOMO–LUMO difference is higher, stability of the
molecule is considered as higher. Furthermore, in order
to explain the reactivity of these molecules a gas-phase
proton affinity (PA) has been calculated for the follow-
ing reaction:
B + Hþ ꢀ BHþ
where B is the base form and BH+ is the protonated
form. The PAs were calculated by using the equation
given below.
Figure 1. ORTEP view showing the atom-labeling scheme with
thermal ellipsoids drawn at 30% probability for compound 10b.

A. Kamal et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5400–5405
5403
O
Cl
COOH
O
O
O
O
O S
S
S
a
N
N
Y
N
N
+
+
NH2
N
R
NHNH2
N
R
NH2
X
12a-b
9a-e
6a-b
11
12a: R = CH3
12b: R = C6H5
b
O
O
S
O
N S
N
N
H
N
O
13a: R = Ph, X = Y = H
Y
13b: R = Ph, X = F, Y = H
13c: R = Ph, X = Y = Cl
N
R
13d: R = Ph, X = CH3, Y = H
13e: R = Ph, X = NHAc, Y = H
13f : R = CH3, X = NHAc, Y = H
13a-f
X
Scheme 3. Reagents and conditions: (a) POCl3, 110 ꢁC, 3–4 h; (b) pyridine, rt, 4 h.
1.462
1.468
1.456
1.779
1.408
1.461
1.729
1.547
1.460
1.772
1.397
1.727
1.390
1.384
2
1.390
1.539
1.319
1.543
1.554
1
1.397
1.391
1.498
1.415
1.399
1.472
1.409
1.488
3
1.373
5
2
1.398
1
1.414
1.398
1.303
1.385
1.552
1.380
1.407
1.315
3
5
4
1.464
1.391
1.409
1.393
1.383
1.464
1.553
4
1.306
1.465
8a (0.0 kcal/mol)
C (6.8 kcal/mol)
˚
Figure 2. Optimized geometries (A) and relative energies of model systems 8a and C at B3LYP/6-31G* level.
tive bacterial strains of Escherichia coli MTCC
448, Pseudomonas aeruginosa MTCC 424, Klebsiella
pneumoniae MTCC 618, Staphylococcus epidermidis
MTCC 435, Bacillus subtilis MTCC 441, and Vibrio species.
The antibacterial activity was compared with those of
some standard antibacterial agents like sulfanilamide
and sulfadiazine. From the results in Table 1, most of
the compounds showed broad spectrum of inhibitory
activity against bacterial strains. Compounds 10f, 10i,
and 13e were found to be the most active against
E. coli (MIC 8 lg/disk); however, these compounds
showed comparable activity with sulfadiazine against
other organisms. Similarly, compounds 13a, 13d, and
13e were very effective against B. subtilis. All these com-
pounds displayed more potent inhibitory activity than
the standard drugs against K. Pneumoniae. Overall,
good to improved antibacterial activity was observed
for most of the compounds against all the bacterial
strains used in the study and their MIC ranged from
16 to 64 lg/disk in comparison to the controls.
PA = ꢀDH = DEtot + DZPE + 5/2 RT
where DH is the enthalpy change of the protonation
reaction, Etot is the electron energy at 0 K, ZPE is the
zero point correction energy, R is the molar gas con-
stant, and T is the absolute temperature in Kelvin.
In the case of 8a, proton additions at the N2 and N4
positions gave proton affinities 240.6 and 238.0 kcal/
mol, respectively, whereas in case of C, proton addition
at the N4 and N3 positions gave proton affinities 248.1
and 252.1 kcal/mol, respectively. These proton affinity
results depict that the proton addition takes place pref-
erably in C. These results also indicate that the ring rear-
rangement takes place in structure C. Moreover, overall
results of quantum chemical calculations are in accor-
dance with the experimental observations and confirm
that the combined relative energies and proton affinities
provide rationale for the observed rearrangement that
takes place in this reaction.
The antibacterial activity of the synthesized compounds
(10a–j and 13a–f) was tested by disk diffusion meth-
od26,27 against various Gram-positive and Gram-nega-
In summary, the synthesis and screening of antibacte-
rial activity for a novel series of 10-substituted
5,5-dioxo[1,24,]triazolo[1,5-b][1,2,4]benzothiadiazine

5404
A. Kamal et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5400–5405
Table 1. Antibacterial activity of compounds of [1,2,4]triazolo[1,5-b]-benzothiadiazine arylsulfonamide derivatives (10a–j and 13a–f)
Compound
MIC (lg/disk)
K. pneumoniaea S. epidermidisb
E. colia
P. aeruginosaa
B. subtilisb
Vibrio spsa
10a
10b
10c
10d
10e
10f
10g
10h
10i
16
16
16
16
16
8
16
32
32
32
32
64
64
32
32
64
32
32
16
32
32
32
>512
32
32
32
32
32
16
32
16
16
32
64
32
64
32
32
16
32
>64
>64
32
32
32
32
32
32
16
32
32
32
32
16
32
32
32
32
>512
32
16
16
16
16
16
16
16
32
32
16
8
32
16
32
16
32
32
32
32
16
32
32
32
32
32
32
32
>512
16
16
16
8
10j
16
16
16
16
16
8
13a
13b
13c
13d
13e
13f
SA
SZ
16
16
8
8
16
>128
16
16
>128
16
a Gram-negative.
b Gram-positive; SA, sulfanilamide; SZ, sulfadiazine.
Ed.; John Wiley & Sons: New York, 1996; pp 527–
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6. Supuran, C. T.; Scozzafava, A.; Casini, A. Med. Res. Rev.
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arylsulfonamide derivatives was investigated. Moreover,
rearrangement of the fused triazolobenzothiadiazine
was studied by using X-ray crystallography and quantum
chemical calculations. Compounds 10f, 10i, and 13e were
found to be the most active against E. coli and 13a, 13d,
and 13e against B. subtilis. Almost all the compounds
have shown significant antibacterial activity in compari-
son to the controls. Therefore, such compounds would
represent a fruitful matrix for the development of a new
class of antibacterial agents using triazolobenzothiadi-
azine arylsulfonamides as a promising scaffold for further
investigations.
10. Huovinen, P.; Sundstro¨m, L.; Swedberg, G.; Skold, O.
Antimicrob. Agents Chemother. 1995, 39, 279.
11. de Tullio, P.; Dupont, L.; Francotte, P.; Counerotte, S.;
Lebrun, P.; Pirotte, B. J. Med. Chem. 2006, 49, 6779.
12. (a) Di Bella, M.; Monzani, A.; Andrisano, M. G.; Fabio,
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Acknowledgments
13. Kamal, A.; Reddy, K. S.; Ahmed, S. K.; Khan, M. N. A.;
Sinha, R. K.; Yadav, J. S.; Arora, S. K. Bioorg. Med.
Chem. 2006, 14, 650.
The authors M.N.A.K., K.S.R., and K.R. thank CSIR,
New Delhi, for the award of research fellowship.
14. General procedures for the synthesis of compounds 10a–j.
3-Hydrazino-4-methyl/phenyl-4H-1,2,4-benzothiadiazine
1,1-dioxides (6a and b, 6.63 mmol) and piperidine 4-
carboxylic acid (7, 7.79 mmol) were taken in phosphorus
oxychloride (5 mL) and refluxed for 4–5 h in oil bath
under nitrogen atmosphere. The reaction mixture was then
cooled to room temperature, poured onto crushed ice, and
neutralized with sodium bicarbonate. The aqueous layer
was extracted with ethyl acetate (4· 50 mL) dried over
anhydrous Na2SO4, and concentrated under vacuum to
get 8a and b. Compounds 8a and b (1 mmol) and
substituted benzenesulfonyl chlorides (9a–e, 1.2 mmol)
were taken in dry pyridine (5 mL) and stirred at room
temperature until starting materials were consumed as
monitored by TLC. The reaction mixture was diluted with
1 M HCl, then the obtained precipitate was filtered, dried,
and further purified by column chromatography (hexane/
ethyl acetate) to afford the final products (10a–j). Spectral
data for compound 10b: 1H NMR (300 MHz, CDCl3):
8.12 (d, J = 8.30, 1H), 7.98 (d, J = 8.30 Hz, 1H), 7.60–7.70
(m, 3H), 7.52 (m, 2H), 7.32–7.44 (m, 4H), 6.74 (d,
J = 8.30 Hz, 1H), 3.79 (td, J = 12,84, 3.77 Hz, 2H), 2.91
(dt, J = 10.57, 3.77 Hz, 2H), 2.74–2.86 (m, 1H), 2.05 (dd,
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.07.043.
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24. Crystal data of 10b. C25H21Cl2N5O4S2; Data were
collected on a Bruker SMART Apex CCD detector
with graphite monochromated Mo Ka radiation
27. Antibacterial assay: All the test compounds were dissolved
in DMF of 2 mg/mL. Empty sterilized disks of 6 mm were
impregnated with compounds in the range from 8 to
64 lg/disk and placed in triplicate in the medium inocu-
lated with fresh bacteria (1–5 · 104 cfu mLꢀ1) on the agar
surface of the culture plates. The plates were incubated at
35 ꢁC for 24 h for zone of inhibition, if any, around the
disks. Lowest concentration of the test substance exhib-
iting no visible growth of bacteria on the plate was
considered to be minimum inhibitory concentrations.
Sulfanilamide and sulfadiazine were used as positive
controls whereas, the equivalent amount of solvent
(DMF) did not exhibit any activity in the assay.
28. SMART & SAINT. Software Reference manuals. Ver-
sions 5.625 &, 6.28a Bruker Analytical X-ray Systems Inc.:
Madison, Wisconsin, U.S.A., 2001.
29. Sheldrick, G. M. SHELXS97 and SHELXL97, Programs
for Crystal Structure Solution and Refinement; University
of Gottingen: Germany, 1997.
˚
(k = 0.71073 A). Data collection was performed and
unit cell was initially refined using SMART. The
ꢀ
˚
crystals were triclinic, P1, with a = 10.5944(5) A, b =
˚
˚
11.0090(5) A, c = 11.5280(5) A, a = 88.034(1)ꢁ, b =
3
˚
84.082(1)ꢁ, c = 80.500(1)ꢁ, V = 1318.84(10) A , Z = 2,
q
calc = 1.487 Mg/m3, l = 0.447 mmꢀ1, and F(000) = 608.
Data reduction was performed using SAINT.28 The
structure was solved by direct methods using SHELXS97
and refinement was carried out by full-matrix least-squares
technique using SHELXL97.29 Anisotropic displacement
parameters were included for all non-hydrogen atoms. All
H atoms were included using a riding model. The final R
value (R1) was 0.0326 for 4231 observed [I > 2r(I)]
reflections and 0.0357 for all 4639 reflections using 343
parameters, and goodness-of-fit was 1.041. Full crystallo-
graphic details of 10b have been deposited at the
Cambridge Crystallographic Data Centre and allocated
the deposition number CCDC 641916.