Anti-tubercular agents. Part 5: Synthesis and biological evaluation of benzothiadiazine 1,1-dioxide based congeners

Article abstract of DOI:10.1016/j.ejmech.2010.07.015

In an effort to discover new and effective chemotherapeutic agents from this laboratory for the treatment of tuberculosis, here in we describe the synthesis and biological evaluation of a series of novel benzothiadiazine 1,1-dioxide (BTD) based congeners by using rifampicin, streptomycin; ciprofloxacin and amphotericin as positive controls. Further, to understand structural requirements for exploring the structure activity relationship of BTDs, cytotoxicity and in vivo study of recently reported potent molecule 4 (MIC = 1 μg/mL) is also discussed.

Full text of DOI:10.1016/j.ejmech.2010.07.015

European Journal of Medicinal Chemistry 45 (2010) 4545e4553
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Anti-tubercular agents. Part 5: Synthesis and biological evaluation
of benzothiadiazine 1,1-dioxide based congeners
,
a
a
a
a
Ahmed Kamal a , Rajesh V.C.R.N.C. Shetti , Shaik Azeeza , S. Kaleem Ahmed , P. Swapna ,
*
A. Malla Reddy a, Inshad Ali Khan b, Sandeep Sharma b, Sheikh Tasduq Abdullah c
a Division of Organic Chemistry-I, Indian Institute of Chemical Technology, Tarnaka, Hyderabad-500 607, India
b Clinical Microbiology Division, Indian Institute of Integrative Medicine, Jammu-180 001, India
c PK-PD Toxicology Division, Indian Institute of Integrative Medicine, Jammu-180 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In an effort to discover new and effective chemotherapeutic agents from this laboratory for the treatment
of tuberculosis, here in we describe the synthesis and biological evaluation of a series of novel benzo-
thiadiazine 1,1-dioxide (BTD) based congeners by using rifampicin, streptomycin; ciprofloxacin and
amphotericin as positive controls. Further, to understand structural requirements for exploring the
structure activity relationship of BTDs, cytotoxicity and in vivo study of recently reported potent molecule
Received 29 April 2010
Received in revised form
5 July 2010
Accepted 6 July 2010
Available online 14 July 2010
4 (MIC ¼ 1
mg/mL) is also discussed.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Anti-tubercular agents
Benzothiadiazine 1,1-dioxides
M. tuberculosis
MDR-and XDR-TB
1. Introduction
In this perspective, from past six years our research efforts have
been focused towards the identification of novel molecules based
on the scaffolds phenazine, thiolactomycin, benzothiadiazine etc.
eventually to develop new drugs for the treatment of tuberculosis
[11e16]. In order to understand the structural requirements at
piperazine/piperidine moiety of the benzothiadiazine 1,1-dioxides
(BTDs) and based on the structure activity relationship (SAR) of the
nitroimidazole-containing current anti-TB leads PA-824 (1) and
OPC-67683 (2) here, we describe the synthesis and biological
evaluation of novel BTD congeners (Fig. 1) [17,18]. Benzothiadiazine
1,1-dioxides constitute important class of cyclic sulfonamides with
broad spectrum activity against bacteria, fungi and Mycobacterium
tuberculosis. In an effort to develop new and effective chemother-
apeutic agents for the treatment of tuberculosis, recently from this
laboratory several series of compounds based on benzothiadiazine
1,1-dioxide scaffold (3,4) with in vitro antimycobacterial activity
have been reported. Amongst all the compounds tested, compound
4 has displayed good inhibition against M. tuberculosis with an MIC
Drugs to treat tuberculosis (TB) are available for over half
a century now, and yet the incidence of the disease globally
continues to rise year by year. According to world health organi-
zation (WHO), it is estimated that more than 9 million new cases
arise each year and nearly 1.8 million deaths are occurring around
the world [1e3]. In addition, the rate of drug-resistant isolates is
approaching half a million per year [4e6]. Furthermore, the co-
infection with human immunodeficiency virus (HIV) has worsened
the situation. The convergence of HIV and TB also posses difficult
problems, not only because viral infection increase mortality from
TB but also optimization of further difficulties such as the rifam-
picin induces CYP 450 enzymes along with inhibition of RNA
polymerization [7,8]. Further, rifampicin is known to have major
pharmacokinetic interactions with certain anti-HIV drugs [9,10].
These problems demand renewed efforts towards the development
of novel chemical entities with different mechanism of action to
control the mortality from TB.
of 1
mg/mL [14]. These results have encouraged us for the synthesis
of another library of new BTD related molecules by systematic
linkage with 5-nitrofuranes and 5-nitrothiophenes. Subsequently,
these newly synthesized molecules have been tested against
Gram-positive, Gram-negative, fungi, and M. tuberculosis. In addi-
tion, to explore the SAR of promising BTDs, the cytotoxicity and
* Corresponding author. Tel.: þ91 40 27193157; fax: þ91 40 27193189.
E-mail address: ahmedkamal@iict.res.in (A. Kamal).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.015

4546
A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 4545e4553
O
N
O
O
N
O
O2N
OCF3
N
O
O2N
OCF3
N
N
PA-824 (1)
OPC-67683 (2)
O
1
S
O
O
O
8
S
7
6
N
N
N 2
H
N
3
NO2
N
O
N
Me
3
N
H
N 4
Ph
N
H
5
O
4
Fig. 1. Potent anti-tubercular compounds.
in vivo efficacy of recently reported molecule 4 against M. tuber-
culosis is also discussed.
3. Pharmacology
3.1. Antimycobacterial assay
2. Chemistry
The antimycobacterial activities of novel BTD congeners 10aef,
11aec and 12aef were evaluated against M. tuberculosis H37Rv. All
the compounds were initially screened against M. tuberculosis H37Rv
To further enhance structural diversity of 4H-1,2,4-
benzothiadizine 1,1-dioxides, we have synthesized other BTD
library with an emphasis on modification piperazine/piperidine by
conjugation with 5-nitrofurfuraldehyde/5-nitrothiophen-2-alde-
hyde. ThesyntheticpathwaysemployedinpreparationofvariousBTD
series are illustrated in Scheme 1. The benzothiadizine 1,1-dioxide
substituted at the 3-positionwith piperazine or isonipicotinic methyl
ester was obtained by the reaction of 3-chloro-4-alkyl/aryl-4H-1,2,4-
benzothiadiazine 1,1-dioxanes (5aec) with N-Boc piperazine/iso-
nipicotinic methyl ester. Later the Boc-protection was removed by
treating with trifluoroacetic acid at 0 ꢀC and subsequently utilized for
the synthesis of the series of compounds 10aef by employing with
5-nitrofurfuraldehyde/5-nitrothiophen-2-aldehyde and sodium tri-
acetoxy borohydride in good yields. The 5-nitrofurane conjugated
BTD series with amido linkage (11aec) was prepared by the coupling
of 5-nitro-2-furoic acid and 3-piperazino-4-alkyl/aryl-4H-1,2,4-ben-
zothiadiazine 1,1-dioxide with EDC and HOBt. Similarly, compounds
12aef was achieved by the condensation reaction between 5-nitro-
at the single concentration of 100 (mg/mL) by Tube Method with
minor modifications. The active compounds from this screening
were further tested for Minimum Inhibitory Concentration (MIC)
determination using the broth microdilution assay [19,20]. The tubes
and microtiter plates were incubated for 3e4 weeks at 37 ꢀC in CO2
incubator and read visually for the absence of growth turbidity.
3.2. Cytotoxicity assay
Compound 4 was evaluated for cytotoxic effect on AML-12
hepatocytes cell lines using MTT assay, in a 96 well plate format.
Cells were incubated in Dulbecco’s Modified Eagle’s Medium
(DMEM) containing 10% fetal calf serum (FCS) with the test material
(2e100
m
g/mL) for 24 h at 37 ꢀC in CO2 incubator. After the
completion of incubation 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) was added and cells were further
incubated for 3 h at 37 ꢀC in CO2 incubator. Formation of formazan
salt by mitochondrial dehydrogenases, and was determined by
furfuraldehyde/5-nitrothiophen-2-aldehyde
hydrazide of BTD as depicted in Scheme 1.
and
isonipicotinic
O
O
O
S
(iii)
(iv)
N
O
O
10a R = Me, X = O
10b R = Et, X = O
10c R = Ph, X = O
10d R = Me, X = S
10e R = Et, X = S
10f R = Ph, X = S
O
O
S
(ii)
N
R
N
N
S
N
(i)
N
NO2
NO2
N
N
10a-f
X
N
N
R
N
R
N
O
NH
N
O
O
S
c
7a-
Boc
N
S
6a-c
N
N
R
11a R = Me
11b R = Et
11c R = Ph
N
R
Cl
11a-c
O
5a-c
O
O
O
5a R = Me
O
O
O
O
5b R = Et
5c R = Ph
S
S
S
(v)
(vi)
(vii)
N
N
N
12a R = Me, X = O
12b R = Et, X = O
12c R = Ph, X = O
12d R = Me, X = S
12e R = Et, X = S
12f R = Ph, X = S
N
R
N
N
R
N
N
R
N
H
N
CO2Me
CONHNH2
N
8a-c
12a-f
9a-c
O
X
NO2
Scheme 1. Synthetic route of benzothiadiazine 1,1-dioxide based congeners (10aef, 11aeb and 12aef): Reagents and conditions; (i) N-Boc piperazine, TEA, THF, 0 ꢀC to rt; (ii) TFA,
DCM, 0 ꢀC; (iii) 5-nitro-2-furaldehyde/5-nitro-2-thiophenaldehyde, sodiumtriacetoxyborohydride, THF, molecular sieves 3 Å; (iv) 5-nitro-2-furoic acid, EDCI, HoBt, dry DMF;
(v) isonipicotinic acid methyl ester; TEA, THF, 0 ꢀC; (vi) hydrazine hydrate, ethanol, reflux; (vii) 5-nitro-2-furaldehyde/5-nitro-2-thiophenaldehyde, cat. AcOH, ethanol, reflux.

A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 4545e4553
4547
Elisa reader at 565 nm (Multiskan Spectrum; Thermo Electron
Corporation, USA). The percentage cytotoxicity was calculated with
respect to the untreated cells.
adjusted to reach an optical comparison to that of a 0.5 McFarland
standard; resulting in a suspension containing approximately
1e2 ꢂ 108 CFU/mL. Mueller-Hinton agar plates were inoculated by
streaking the swab over the entire sterile agar surface. This
procedure was repeated by streaking two more times, rotating the
plate approximately 60ꢀ each time to ensure even distribution of
the inoculum. As a final step, the rim of the agar was also swabbed.
After allowing the inoculum to dry at room temperature, 6 mm
diameter wells were prepared in the agar with the help of sterilized
cork borer. The different concentrations of the test compounds
3.3. Drug
The compound 4 was studied (>99.5 potency) in-vivo at
a concentration of 25 mg/kg. Rifampicin from SigmaeAldrich
(St. Louis, MO) at 10 mg/kg was also tested as positive control. For
in vivo studies the compound and rifampicin were dissolved in pure
DMSO. In case of treatment by oral route the compound was dis-
solved in 50% polyetheleneglycol (PEG) in water. The final
concentration of DMSO was not more than 0.1% v/v.
(100e150
mg/mL) were prepared by dissolving in dimethyl sulf-
oxide (DMSO) and introduced into duplicate wells. The plates were
incubated at 37 ꢀC for 18 h. Subsequently, the plates were examined
for bacterial growth inhibition and the inhibition zone diameter
3.4. Animals
(IZD) measured to the nearest millimeter. DMSO (150 mg/mL)
loaded in agar well was used as negative control. The standard
antibiotics streptomycin and ciprofloxacin were used as positive
controls and DMSO was used as negative control.
The institutional animal Ethics committee of the Indian Institute
of Integrative Medicine (IIIM, Jammu) approved all experimental
protocols with animals and the use of animals. The animals were
bred and maintained under standard husbandry conditions; viz.
humidity, temperature (25 ꢁ 2 ꢀC). 6week-old female Balb/c mice,
20e22 gm by weight were used for the efficacy studies. They were
allowed to acclimatize for a week before the commencement of the
experiment. Feed and water were given ad libitum.
3.8. Antifungal assay
The method followed for antifungal bioassay is similar to that
followed for antibacterial assay where the medium is potato
dextrose agar 39 g/L. All the test compounds were studied for their
antifungal activity at concentration 100e150 mg/mL using DMSO as
3.5. Infection with M. tuberculosis H37Rv
a solvent. The solvent did not exhibit any activity at the concen-
trations used. The treated and the controls were kept in an incu-
bator at 28 ꢁ 2 ꢀC for 48 h. Inhibition zones were measured to the
nearest millimeter. Three replicates were maintained for each
The tubercle bacilli was grown by the culturing of bacteria
maintained on Lowenstein-Jensen slant into Middlebrook 7H9
(Difco Laboratories, Detroit, Mich.) supplemented with 0.5% (v/v)
glycerol, 0.25% (v/v) Tween 80 (Himedia, Mumbai India), and 10%
ADC (albumin dextrose catalase, Becton Dickinson, Sparks, MD)
medium on a rotary shaker for 10 days at 37 ꢀC. Infections with
M. tuberculosis were administered by the intranasal route with
treatment. Amphotericin-B (50
4. Results and discussion
4.1. Antimycobacterial activity
mg/mL) was used as positive control.
approximately 1.5 ꢂ 106 CFU in a 25
ml volume per mouse.
The prepared novel congeners of benzothiadiazine 1,1-dioxide
3.6. Assessment of bacterial load
were tested for in vitro antituberculosis activity against M. tuber-
culosis H37Rv at the single concentration of 16 mg/mL. The active
Eight infected mice were sacrificed from the control group one
week post infection (before the start of treatment) to obtain the
initial CFU load of M. tuberculosis H37Rv. In the group treated with
compound 4, the mice were sacrificed after 4 week of treatment. The
lungs and spleen of the sacrificed mice were aseptically removed and
homogenized in 1 mL of sterile normal saline containing 0.05% of
Tween 80. Viable bacteria were quantitated by plating 10-fold serial
dilutions in duplicates on Middlebrook 7H10 agar supplemented
with 10% OADC and incubated at 37 ꢀC in 5% CO2 for 4e6 weeks. CFU
per organ sample was counted and expressed as log10 CFU per organ.
The efficacy of compound 4 was determined as the log10 reduction in
bacterial numbers in treated groups compared to the untreated
control group. CFU during in-vivo studies were determined on
Middlebrook 7H10 plates supplement with 10% OADC containing
compounds from this screening were further tested for Minimum
Inhibitory Concentration (MIC) determination using the broth
microdilution assay [21e25]. The MIC is defined as the lowest
concentration of the compound showing no visible turbidity. Two
drugs in clinical use, rifampicin and isoniazid were included in
these assays as reference drugs. All these compounds have shown
activity between 2 and 16
Table 1.
mg/mL and the results are summarized in
In the first of series conjugates 10aef, the furan/thiophene units
are linked to BTD moiety through methylene bridge and among
them the nitrofuran containing BTD conjugates (10aec) exhibits
good anti-tubercular activity. The most active conjugate (MIC ¼ 2)
of the series 10c contains phenyl group at 4th position of BTD, when
this group is replaced with ethyl (10b) and methyl (10a) groups the
activity gradually decreases. Whereas, the isostructural thiophene
containing conjugates show moderate activity, this indicates the
importance of nitrofuran in effecting the anti-tubercular activity.
Moreover, the replacement of methylene group with carbonyl
group (11aec) leads to dramatic loss of activity. In final series of
conjugates, piperazine moiety has been replaced with iso-
nipicotinic acid hydrazide linker. As expected nitrofuran conjugates
(12aec) have found to be superior to nitrothiophene counterparts
(12def) and among the conjugate 12b have shown promising anti-
tubercular activity. However, these analogs are not as effective as
piperazine containing BTD conjugates, thus emphasizing the
potential of piperazine-BTDs with methylene linkage as an attrac-
tive moiety for anti-tubercular drug designing.
vancomycin 10
mg/mL, amphotericin B 10 mg/mL, nalidaxic acid
25 g/mL. Drugs were added to minimize the chances of contami-
m
nation during the incubation period. This drug concentration had no
effect on M. tuberculosis growth. Rifampicin was also tested at 10 mg/
kg body wt as positive control.
3.7. Antibacterial assay
The antibacterial screening of the synthesized compounds was
determined by the well diffusion method according to Linday.
Three to five identical colonies from each agar plate were lifted
with a sterile wire loop and transferred into a tube containing 5 mL
of Nutrient Agar. The turbidity of each bacterial suspension was

4548
A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 4545e4553
Table 1
Table 3
Antimycobacterial activity of benzothiadiazine 1,1-dioxide based congeners in
Preliminary antibacterial and antifungal activity of benzothiadiazine 1,1-dioxide
congeners against Gram-negative bacteria and C. albicans fungus (zone of inhibition
in mm).
m
g/mL.
Compound
Mol. Formula
17H19N5O5S
C18H21N5O5S
C22H21N5O5S
C17H19N5O4S2
R
X
C log P
MIC mg/mL
Compd E. coli
K. pneumoiae
100 50
P. aeruginosa
100 50
C. albicans
100 50
10a
10b
10c
10d
10e
10f
11a
11b
11c
12a
12b
12c
C
Me
Et
Ph
Me
Et
Ph
Me
Et
Ph
Me
Et
O
O
O
S
S
S
O
O
O
O
O
O
S
2.30
2.81
2.33
2.86
3.39
4.22
1.34
1.87
2.70
0.75
1.28
2.25
1.34
1.87
2.85
0.46
8
4
2
>16
4
>16
16
>16
>16
>16
4
100
50
m
g/mL
mg/mL
m
g/mL
mg/mL
m
g/mL
m
g/mL
m
g/mL
mg/mL
10a
10b
10c
10d
10e
10f
11a
11b
11c
12a
12b
12c
12d
12e
12f
26
30
20
16
15
16
e
15
16
14
e
22
19
15
12
14
13
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
17
16
15
13
11
12
e
10
10
10
e
18
10
11
17
14
12
12
10
17
14
21
16
17
20
18
11
e
C
18H21N5O4S2
C22H21N5O4S2
C17H17N5O6S
C18H19N5O6S
C22H19N5O6S
C19H20N6O6S
C20H22N6O6S
C24H22N0O6S
e
e
e
e
e
e
e
e
09
e
10
e
e
e
e
e
e
Ph
Me
Et
16
20
16
25
15
14
15
15
10
e
11
15
16
10
13
14
11
16
14
15
e
10
10
12
10
e
10
12d
12e
12f
C
19H20N6O5S2
>16
>16
>16
0.25
C20H22N6O5S2
C24H22N6O5S2
S
S
e
11
e
Ph
e
Rifampicin
e
10
12
11
10
12
10
e
e
e
e
4.2. Antibacterial and antifungal activity
STR
CIP
AT-B
21
>40
e
22
20
e
29
30
e
e
e
Owing to the continual emergence of multi-drug resistance to
clinically available antibiotics against bacterial and fungal patho-
gens [26,27], further these BTD congeners were also tested for
antibacterial activity against a panel of Gram-positive bacteria like
Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis
and Gram-negative bacteria such as Escherichia coli, Klebsiella
pneumoniae, and Pseudomonas aeruginosa. Where as the antifungal
activity was evaluated against Candida albicans (MTCC 227). The
inhibitory zones (in mm) were determined by using agar well
method (cup plate method) [28,29]. The well established antibi-
otics like ciprofloxacin, streptomycin, and amphotericin B were
used as positive controls against bacteria and fungi respectively. In
all determinations, tests were performed in duplicate and results
were reported as mean of at least three determinations. All the
benzothiadiazine congeners have exhibited moderate to good
antibacterial activity at preliminary screening and the results are
assessed in zone of inhibition (mm) in Tables 2 and 3.
16
The test were performed in duplicate and repeated thrice; (e), bacteria are resistant
to the compound at the concentrations; STP, Streptomycin; CIP, Ciprofloxacin
(50 mg/mL); AT-B, Amphotericin (100 units); E. coli (Escherichia coli); K. pneumoniae
(Klebsiella pneumoniae); P. aeruginosa (Pseudomonas aeruginosa); C. albicans
(Candida albicans).
At primary screening, the compounds 10a,10b,11a,11c,12b,12c,
12e, and 12f showed significant inhibition against all the bacteria
tested and were not strain dependent except the compound 11b
exhibited no activity against these bacterial strains. Further, the
active compounds 10a,10b and 12b were evaluated for antibacterial
and antifungal activity at different concentrations 10, 25, 50, 75,
and 100 mg/mL and the results are outlined in Table 4. The prepared
BTD congeners 10aef, 11aec and 12aef have exhibited interesting
trends in structure activity relationship (SAR) studies. The amido
linkage between piperazine of BTD and 5-nitrofurane has shown
remarkable loss in antimicrobial activity (11aec). Where as the
aforementioned moieties linked through methylene spacer (10aef)
Table 2
Preliminary antibacterial activity of benzothiadiazine 1,1-dioxide based congeners
against gram-positive bacteria (zone of inhibition in mm).
Table 4
Antifungal activity of active compounds in different concentrations expressed in
zone of inhibition (mm).
Compd
S. aureus
S. epidermis
B. subtillis
Copmd
Conc
S. a
S. e
B. s
E. c
P. a
K. p
C. a
A. n
A. s
100
50
100
50
100
50
m
g/mL
m
g/mL
m
g/mL
m
g/mL
m
g/mL
m
g/mL
10a
10
25
50
75
100
e
10
13
15
18
25
9
12
15
20
29
34
e
e
NT
e
NT
e
NT
e
10
14
17
19
12
16
20
23
e
10
12
17
19
10a
10b
10c
10d
10e
10f
11a
11b
11c
12a
12b
12c
12d
12e
12f
17
10
15
12
11
12
18
e
15
16
14
10
10
09
17
e
24
25
20
16
18
14
e
16
18
15
14
12
e
23
23
19
18
16
14
e
e
e
e
e
e
e
e
e
e
e
13
e
e
e
e
12
17
19
11
14
18
e
e
e
e
e
e
10b
12b
10
25
50
75
100
13
16
20
21
25
9
e
14
20
25
29
34
e
10
11
15
20
22
NT
e
NT
e
NT
e
12
12
15
24
11
17
19
23
12
16
22
29
10
e
e
e
e
e
e
e
e
e
15
16
18
e
e
17
18
22
10
14
15
15
10
12
13
10
e
19
18
21
11
18
19
15
10
e
e
10
12
10
e
10
25
50
75
100
12
14
20
22
23
e
e
24
31
36
40
<10
10
14
20
24
e
NT
10
15
17
20
NT
e
NT
10
10
12
12
e
12
15
19
22
e
15
14
12
11
13
22
12
16
16
e
e
e
12
14
e
e
>40
STR
CIP
AT-B
22
32
e
18
35
e
17
32
e
Cipro
At-B
Nys
50
20
20
26
NT
NT
34
NT
NT
32
NT
NT
38
NT
NT
40
NT
NT
21
NT
NT
NT
14
e
NT
23
26
NT
14
16
The test were performed in duplicate and repeated thrice; (e), bacteria are resistant
to the compound at the concentrations; STP, Streptomycin; CIP, Ciprofloxacin
Cipro, Ciprofloxacin (50
mg/mL); AT-B, Amphotericin-B (100 units) Nys, Nystatin-1
(20 g/mL); NT, (Not Tested) S. a (Staphylococcus aureus); S. e (Staphylococcus epi-
m
(50
m
g/mL); AT-B, Amphotericin (100 units); S. aureus (Staphylococcus aureus);
dermidis); B. s (Bacillus subtillis); E. c (Escherichia coli); K. p (Klebsiella pneumoniae);
P. a (Pseudomonas aeruginosa); C. a (Candida albicans); A. n (Aspergillus. niger).
S. epidermis (Staphylococcus epidermidis); B. subtillis (Bacillus subtillis).

A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 4545e4553
4549
Table 5
Cytotoxicity of compound 4.
compounds based on this potentially valuable BTD pharmacophore
is underway at our laboratories to improve better in vivo potency in
mouse models.
Sample code
Conc (
m
g/mL)
% Cytotoxicity
4
2
5
2.4
9.0
5. Conclusion
10
25
50
100
9.1
8.5
8.7
14.9
In conclusion, this work demonstrates the synthesis, and in vitro
activity evaluation of new benzothiadiazine 1,1-dioxide based
congeners against M. tuberculosis. Amongst, the compound 10c has
shown significant inhibition against M. tuberculosis having MIC
2 mg/mL. Owing to the spread of drug resistance to many clinically
have led to significant enhance in activity against both Gram-
positive and -negative strains. Interestingly, among isonipicotinic
hydrazones of BTDs having conjugation 5-nitrofuran/5-nitro-
thiophene (12aef) have shown moderate to weak inhibitory
activity except the compound 12b, which has displayed excellent
inhibitory activity against Gram-positive, Gram-negative and fungi.
used antibacterial and antifungal antibiotics, these congeners also
tested against Gram-positive and -negative bacteria and fungi. The
compounds 10a, 10b and 12b have shown good inhibition against
bacterial strains. In addition, the in vivo efficacy of earlier reported
promising compound 4 of this class is also discussed. The promising
bacteriostatic activity of this compound 4 at in vivo reveals that the
synthesis of newer molecules based on benzothiadiazine pharma-
cophore could provide a lead optimization in the development of
novel antimycobacterial agents.
4.3. Cytotoxicity
In continuation of the efforts towards the development of new
and effective anti-tubercular agents for the treatment of tubercu-
losis, recently we have reported the in vitro antimycobacterial
activity of various analogues based on benzothiadiazine 1,1-dioxide
scaffold. Amongst all compounds evaluated in this study, the
compound 4 has displayed good activity against M. tuberculosis
6. Experimental section
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 per-
formed with Merck (Navi Mumbai, India) 60e120 mesh silica gel.
Spectral patterns are designated as s, singlet; d, doublet; dd, double
doublet; t, triplet; bs, broad singlet; m, multiplet. 1H NMR spectra
were recorded on Gemini (200 MHz) (Varian Inc, Palo Alto, CA,
USA) or [Avance (300 MHz); Bruker, Fallanden, Switzerland]
with an MIC of 1 mg/mL [14]. The cytotoxic effect of compound 4
have been determined on AML-12 hepatocytes cell lines using MTT
assay, in a 96 well plate format. The cells were incubated in pres-
ence of compound 4 at concentrations ranging from 2, 5, 10, 50,
100 mg/mL. After 24 h exposure, viability was assessed on the basis
of cellular conversion of MTT into a formazan product using the
Promega Cell Titer 96 non-radioactive cell proliferation assay and
the results are outlined Table 5. The compound 4 was found to be
instruments. Chemical shifts (d) are reported in ppm, downfield
from internal TMS standard and LC-MS was recorded on instru-
ment LC-MSD-Trap-SL. ESI spectra were recorded on Micro mass,
Quattro LC using ESIþ software with capillary voltage of 3.98 kV
and ESI mode positive ion trap detector. Elemental analyses were
performed on an elemental analyzer (Model: VARIO EL, Elementar,
Hanau, Germany). Melting points were determined with an Elec-
trothermal melting point apparatus and are uncorrected. Starting
materials and reagents were either commercially available or
purchased from Lancaster (Alfa Aesar, Johnson Matthey Co, Ward
Hill, MA, USA), SigmaeAldrich (St Louis, MO, USA) and Spec-
trochem Pvt Ltd (Mumbai, India). Elemental analysis was within
ꢁ0.4% of the theoretical values.
non-toxic upto 100
mg/mL and is advanced for in vivo efficacy
studies in mice infected with M. tuberculosis.
4.4. In vivo study
The compound 4 was evaluated for in vivo efficacy in six week-
old female balb/c mice using rifampicin at 10 mg/kg body weight as
standard drug. The compound 4 at a dose of 25 mg/kg body weight
showed a decreased the bacterial load in lung by 1 log where no
significant decrease log10 CFU in spleen when compares with early
control and is considered to be promising in reducing bacterial
count in lung tissues (Table 6). The good in vivo bacteriostatic
activity of compound 4, suggest that this benzothiadiazine 1,1-
dioxide scaffold might serve as a lead for the development of new
potential anti-tubercular agents. Further synthesis of new
6.1. Preparation of 3-(N-boc-piperazinyl)-4-alkyl/aryl-4H-1,2,4-
benzothiadiazine 1,1-dioxides (6aec)
To a solution of 3-chloro-4-alkyl/aryl-4H-1,2,4-benzothiadiazine
1,1-dioxides (5aec) (1 mmol) and triethylamine (3 mmol) in dry
dichloromethane (25 mL), N-Boc piperazine (1.5 mmol) was added
and the reaction mixture was stirred at room temperature for 12 h.
After the completion of reaction as indicated by TLC, the solvent
was removed under reduced pressure and the product was purified
by column chromatography by using ethyl acetate, hexane (4:6)
system gives the product.
Table 6
In vivo activity of compound 4 against M. tuberculosis.
Treatment groupa
nb
Log10 CFU/organ ꢁSD
Lung
Spleen
Early Control
Late Control
RIF (10 mg/Kg)
Compound 4 (25 mg/Kg)
6
6
5.8 ꢁ 0.21
6.6 ꢁ 0.41
3.8 ꢁ 0.21
4.8 ꢁ 0.40
2.2 ꢁ 0.06
3.5 ꢁ 0.05
NDd
2.0 ꢁ 0.11
6
5c
6.1.1. 3-(N-Boc-piperazinyl)-4-methyl-4H-1,2,4-benzothiadiazine
1,1-dioxide (6a)
In vivo efficacy studies of compound 4.
a
Treatment was started 1 week after mice received y1.5 ꢂ 106 viable myco-
bacteria intranasal. The drugs were evaluated at the following doses: RIF, 10 mg/kg;
compound 4 25 mg/kg.
The compound 6a was prepared according to the above
described method using 5a (230 mg, 1 mmol). Yield 3.266 mg, 70%;
1H NMR (CDCl3, 300 MHz):
d
7.92 (d, 1H, J ¼ 7.5 Hz, BTD-H), 7.59 (t,
b
Number of mice per group.
One mouse found dead during therapy.
ND Below detection limit (<50 CFU).
c
1H, J ¼ 6.8 Hz, BTD-H), 7.37 (t, 1H, BTD-H), 7.26 (d, 1H, BTD-H), 3.63
d
(s, 3H, eNCH3), 3.53e3.46 (m, 8H, 4ꢂ piperazine-CH2), 1.47 (s, 9H,

4550
A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 4545e4553
Boc-3ꢂCH3); LCMS: m/z 381 (M þ 1)þ, 325 (M ꢃ 57)þ, 281
(M ꢃ 100)þ.
piperdine-CH2), 3.69 (s, 3H, eCO2CH3), 3.19 (td, 2H, piperdine-CH2),
2.60e2.51 (m, 1H, J ¼ 3.8 Hz, piperdine-CH), 1.78e2.05 (m, 4H, 2ꢂ
piperdine-CH2), 1.19 (t, 3H, eNCH2CH3); ESIMS: m/z 352 (M þ 1)þ,
353 (Mþ2)þ.
6.1.2. 3-(N-Boc-piperazinyl)-4-ethyl-4H-1,2,4-benzothiadiazine
1,1-dioxide (6b)
The compound 6b was prepared according to the above
described method using 5b (244 mg, 1 mmol). Yield 283 mg, 72%;
6.3.3. 3-(N-Isonipicotinic methyl ester)-4-phenyl-4H-1,2,4-
benzothiadiazine 1,1-dioxide (8c)
1H NMR (CDCl3, 300 MHz):
d
7.91 (d, 1H, J ¼ 7.5 Hz, BTD-H), 7.59 (t,
The compound 8c was prepared according to the method
1H, J ¼ 6.8 Hz, BTD-H), 7.37 (t,1H, BTD-H), 7.26 (td,1H, BTD-H), 4.06
(q, 2H, J ¼ 6.8 Hz, eNCH2CH3), 3.51e3.42 (m, 8H, 4ꢂ piperazine-
CH2), 1.47 (s, 9H, Boc-3ꢂCH3), 1.21 (t, 3H J ¼ 6.8 Hz, eNCH2CH3);
LCMS: m/z 394 (M þ 1)þ, 338 (M ꢃ 57)þ. 294 (M ꢃ 100)þ.
described for 8a using 5c (292 mg, 1 mmol). Yield 279 mg, 70%; 1H
NMR (CDCl3, 300 MHz):
d
7.90 (dd, 1H, J ¼ 7.5 Hz, BTD-H), 7.53e7.49
(m, 4H, J ¼ 4.5 Hz, BTD-H, AreH), 7.37e7.46 (m, 2H, AreH),
7.29e7.35 (m, 2H, BTD-H, AreH), 3.95e3.86 (dt, 2H, piperdine-
CH2), 3.62 (s, 3H, eCO2CH3), 3.07e2.96 (td, 2H, piperdine-CH2),
2.40e2.29 (m, 1H, J ¼ 3.8 Hz, piperdine-CH), 1.68e1.62 (m, 2H,
piperdine-CH2), 1.27e1.20 (m, 2H, piperdine-CH2); NCMS: m/z
400.6 (M þ 1)þ, 422.5 (M þ Na)þ.
6.1.3. 3-(N-Boc-piperazinyl)-4-phenyl-4H-1,2,4-benzothiadiazine
1,1-dioxide (6c)
The compound 6c was prepared according to the above
described method using 5c (292 mg, 1 mmol). Yield 332 mg, 75%;1H
NMR (CDCl3, 300 MHz):
d
7.90 (d, 1H, J ¼ 7.5 Hz, BTD-H), 7.54e7.52
6.4. 3-(N-Isonipicotinic methyl ester)-4-alkyl/aryl-4H-1,2,4-
benzothiadiazine 1,1-dioxides (9aec)
(m, 3H, J ¼ 3.8 Hz, BTD-H, 2ꢂ AreH), 7.42e7.48 (m, 3H J ¼ 9.0 Hz,
BTD-H, 2ꢂ AreH), 7.3e7.35 (m, 2H J ¼ 9.05 Hz, BTD-H, AreH), 3.41
(t, 4H, 2ꢂ piperazine-CH2), 3.08 (t, 4H, 2ꢂ piperazine-CH2), 1.40 (s,
9H, 3ꢂ Boc-CH3); ESIMS: m/z 443 (M þ 1)þ, 387 (M ꢃ 57)þ, 343
(M ꢃ 100)þ.
The target compound was obtained by condensation reaction
between 3-(N-isonipicotinicacid methyl ester)-4-alkyl/aryl-4H-
1,2,4-benzothiadiazine 1,1-dioxides (8aec) with hydrazine hydrate
by using ethanol as solvent affords title compound, and it is directly
used for further reaction without further purifications.
6.2. Preparation of 3-piperazinyl-4-alkyl/aryl-4H-1,2,4-
benzothiadiazine 1,1-dioxides (7aec)
6.5. Preparation of 3-[4-{(5-nitro-2-furyl/thienyl)methyl}
piperazinyl]-4-alkyl/aryl-1,2,4-benzothiadiazine 1,1-dioxide
(10aef)
To a solution of 3-(N-Boc-piperazinyl)-4-alkyl/aryl-4H-1,2,4-
benzothiadiazine 1,1-dioxides (6aec) (1 mmol) in dry dichloro-
methane (25 mL) triflouro acetic acid (10 mmol) was added and the
mixture was stirred for 12 h, after completion of reaction as indicate
by TLC the reaction mixture was neutralized by aq NaHCO3 solu-
tion, the isolated product was used directly to the next reaction
without further purifications.
The target compounds was obtained by reductive amination of
3-piperazinyl-4-alkyl/aryl-4H-1,2,4-benzothiadiazine 1,1-dioxide
(7aec) (1 mmol) with corresponding heterocyclic nitroaldehyde in
the presence of sodium triacetoxyborohydride (2.5 mmol), molec-
ular sieves 3 Å, in dry THF as a solvent (30 mL). The reaction mixture
stirred at room temperature for 12 h. After completion of reaction
as indicate by TLC, the solvent was removed under reduced pres-
sure. The crude product thus obtained was purified by column
chromatography using ethyl acetate, hexane (1:1) as eluant to
afford pure compound.
6.3. Preparation of 3-(N-isonipicotinicacid methyl ester)-4-alkyl/
aryl-4H-1,2,4-benzothiadiazine 1,1-dioxide (8aec)
To a solution of 3-chloro-4-alkyl/aryl-4H-1,2,4-benzothiadiazine
1,1-dioxide (5aec) (1 mmol) and triethylamine (3 mmol) in dry
dichloromethane (25 mL), isonipicotinic methyl ester (3 mmol) was
added and the reaction mixture was stirred at room temperature
for 12 h. After completion of reaction as indicated by TLC, the
solvent was removed under reduced pressure and the product was
purified by column chromatography by using ethyl acetate, hexane
(1:1) system gives the product.
6.5.1. 3-[4-{(5-Nitro-2-furyl)methyl}piperazinyl]-4-methyl-1,2,4-
benzothiadiazine 1,1-dioxide (10a)
The compound 10a was prepared according to the above
described method by using compound 7a (280 mg, 1 mmol) and
5-nitro-2-furaldehyde (141 mg, 1 mmol). Yield 283 mg, 70%; IR
(KBr) (ymax/cmꢃ1) 3301, 2910, 1635, 1601, 1556, 1528, 1497, 1430,
6.3.1. 3-(N-Isonipicotinic methyl ester)-4-methyl-4H-1,2,4-
benzothiadiazine 1,1-dioxide (8a)
The compound 8a was prepared according to the above
described procedure by using 5a (230 mg, 1 mmol). Yield 235 mg,
1335, 758; 1H NMR (CDCl3, 300 MHz):
d
7.96 (dd, 1H, J ¼ 7.5 Hz,
BTD-H), 7.41 (t, 1H, J ¼ 7.5 Hz, BTD-H), 7.29 (d, 1H, J ¼ 3.7 Hz, furan-
H), 7.23 (d, 2H, J ¼ 8.3 Hz, BTD-H), 6.51 (d, 1H, J ¼ 3.7 Hz, furan-H),
3.71 (s, 2H, eCH2e), 3.60 (s, 3H, eNCH3), 3.54 (t, 4H, 2ꢂ piperazine-
CH2), 2.67 (t, 4H, 2ꢂ piperazine-CH2); 13C NMR (DMSO-d6,
70%; 1H NMR (CDCl3, 300 MHz):
d
7.89 (d, 1H, J ¼ 7.5 Hz, BTD-H),
7.60 (t, 1H, J ¼ 7.5 Hz, BTD-H), 7.39 (t, 1H, J ¼ 7.5 Hz, BTD-H), 7.26
(d,1H, J ¼ 8.3 Hz, BTD-H), 3.79e3.75 (m, 2H, piperdine-CH2), 3.70 (s,
3H, eCO2CH3), 3.58 (s, 3H, eCH3), 3.14 (t, 2H, piperdine-CH2),
2.60e2.52 (m, 1H, J ¼ 3.8 Hz, piperdine-CH), 1.78e2.05 (m, 4H, 2ꢂ
piperdine-CH2); ESIMS: m/z 338 (M þ 1)þ, 339 (M þ 2)þ.
75 MHz): d 157.6, 154.3, 130.1, 137.3, 134.8, 131.9, 125.4, 123.6, 116.1,
112.1, 56.8, 51.6, 50.2, 39.1; ESIMS: m/z 406 (M þ 1)þ, 429
(M þ Na)þ. Anal. calcd. for C17H19N5O5S: C, 50.36; H, 4.72; N, 17.27.
Found: C, 50.25; H, 4.67, N, 17.19.
6.5.2. 3-[4-{(5-Nitro-2-furyl)methyl}piperazinyl]-4-ethyl-1,2,4-
benzothiadiazine 1,1-dioxide (10b)
6.3.2. 3-(N-Isonipicotinic methyl ester)-4-ethyl-4H-1,2,4-
benzothiadiazine 1,1-dioxide (8b)
The compound 10b was prepared according to the method
described for 10a using compound 7b (294 mg, 1 mmol) and
5-nitro-2-furaldehyde (141 mg, 1 mmol). Yield 251 mg, 60%; IR
(KBr) (ymax/cmꢃ1) 3424, 2925, 1641, 1584, 1540, 1427, 1353, 1299,
The compound 8b was prepared according to the method
described for 8a using 5b (244 mg, 1 mmol). Yield 259 mg, 74%; 1H
NMR (CDCl3, 300 MHz):
d
7.90 (dd, 1H, J ¼ 7.5 Hz, BTD-H), 7.57 (t,
1H, J ¼ 7.5 Hz, BTD-H), 7.37 (t, 1H, J ¼ 7.5 Hz, BTD-H), 7.24 (dd, 1H,
1258, 1166, 1019, 759; 1H NMR (CDCl3, 200 MHz):
d
7.90e7.94 (dd,
J ¼ 8.3 Hz, BTD-H), 4.06 (q, 2H, eNCH2CH3), 3.84e3.74 (m, 2H,
1H, J ¼ 7.8 Hz, BTD-H), 7.57e7.50 (t, 1H, J ¼ 7.0 Hz, BTD-H),

A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 4545e4553
4551
7.38e7.34 (m, 2H, J ¼ 7.8 Hz, 2ꢂ BTD-H), 7.23e7.17 (d, 1H, J ¼ 3.1 Hz,
furan-H), 6.45 (d, 1H, J ¼ 3.1 Hz, furan-H), 4.03 (q, 2H, eNCH2CH3),
3.65 (s, 2H, eCH2e), 3.52 (t, 4H, 2ꢂ piperazine-CH2), 2.60 (t, 4H, 2ꢂ
piperazine-CH2), 1.19 (t, 3H, eNCH2CH3); 13C NMR (DMSO-d6,
1294, 1165, 1139, 1001, 849; 1H NMR (CDCl3, 300 MHz):
d
7.94 (dd,
1H, J ¼ 7.7 Hz, BTD-H), 7.74 (d, 1H, J ¼ 4.1 Hz, thiophene-H),
7.53e7.48 (m, 4H, J ¼ 4.5 Hz, BTD-H, AreH), 7.42e7.36 (m, 2H,
J ¼ 7.5 Hz, BTD-H), 7.33e7.28 (m, 2H, J ¼ 8.3 Hz, BTD-H, AreH),
6.78 (d, 1H, J ¼ 4.1 Hz, thiophene-H), 3.57 (s, 2H, eCH2e),
3.49e3.53 (t, 4H, 2ꢂ piperazine-CH2), 2.25e2.27 (t, 4H, 2ꢂ
75 MHz): d 157.5, 151.3, 137.2, 134.5, 131.8, 127.8, 125.6, 123.6, 115.8,
112.7, 56.8, 51.6, 48.4, 45.4, 27.8; ESIMS: m/z 420 (M þ 1)þ. Anal.
calcd. for C18H21N5O5S: C, 51.54; H, 5.05; N, 16.70. Found: C, 51.50;
H, 5.49, N, 16.68.
piperazine-CH2); 13C NMR (DMSO-d6, 75 MHz):
d 157.6, 151.5,
150.7, 141.3, 138.9, 129.8, 129.1, 128.9, 128.5, 128.4, 125.6, 124.4,
123.3, 120.0, 56.7, 51.6, 48.4. ESIMS: m/z 484 (M þ 1)þ, 485
(M þ 2)þ. Anal. calcd. for C22H21N5O4S2: C, 54.46; H, 4.38; N, 14.48.
Found: C, 54.41; H, 4.35, N, 14.40.
6.5.3. 3-[4-{(5-Nitro-2-furyl)methyl}piperazinyl]-4-phenyl-1,2,4-
benzothiadiazine 1,1-dioxide (10c)
The compound 10c was prepared according to the method
described for 10a using compound 7b (342 mg, 1 mmol) and 5-
nitro-2-furaldehyde (141 mg, 1 mmol). Yield 280 mg, 60%; IR (KBr)
6.6. Preparation of 3-[4-{(5-nitro-2-furyl)carbonyl}piperainyl]-
4alkyl/aryl-1,2,4-benzothiadiazine 1,1-dioxides (11aec)
(
ymax/cmꢃ1) 3452, 2854, 1639, 1595, 1568, 1534, 1498, 1265, 1135,
784; 1H NMR (CDCl3, 300 MHz):
d
7.90 (d, 1H, J ¼ 7.5 Hz, BTD-H),
The target compounds were prepared by amide bond formation
between 3-piperazinyl-4-alkyl/aryl-4H-1,2,4-benzothiadiazine 1,1-
dioxides (7aec)(1 mmol) and 4-nirofuroic acid (1.5 mmol) in dry
DMF, coupling agent EDC (2 mmol) and hydroxy benzotriazole
(2 mmol), were added and the reaction mixture was stirred at
room temperature for 12 h, After completion of reaction as indi-
cated by TLC, the reaction was pored into crushed ice (10 g) and
extract with ether and the crude product purified by column
chromatography by using ethyl acetate, hexane (8:2) system gives
the product.
7.52e7.48 (m, 4H, J ¼ 4.5 Hz, BTD-H, AreH), 7.42e7.39 (m, 2H,
J ¼ 7.5 Hz, AreH), 7.33e7.28 (m, 2H, J ¼ 8.3 Hz, BTD-H, AreH), 7.19
(d, 1H, J ¼ 3.8 Hz, furan-H), 6.35 (d, 1H, J ¼ 3.8 Hz, furan-H),
3.6e3.49 (m, 6H, 2ꢂ piperazine-CH2, CH2e), 2.64 (t, 4H, 2ꢂ piper-
azine-CH2); 13C NMR (DMSO-d6, 75 MHz):
d 157.6, 151.5, 150.7,
141.3, 138.9, 129.8, 129.1, 128.9, 128.5, 128.4, 125.6, 124.4, 116.2,
112.4, 56.7, 51.6, 48.4; ESIMS: m/z 468 (M þ 1)þ, 469 (M þ 2)þ. Anal.
calcd. for C22H21N5O5S: C, 56.52; H, 4.53; N, 14.98. Found: C, 56.49;
H, 4.51, N, 14.92.
6.5.4. 3-[4-{(5-Nitro-2-thienyl)methyl}piperazinyl]-4-methyl-
1,2,4-benzothiadiazine 1,1-dioxide (10d)
6.6.1. 3-[4-{(5-Nitro-2-furyl)carbonyl}piperainyl]-4-methyl-1,2,4-
benzothiadiazine 1,1-dioxide (11a)
The compound 10d was prepared according to the method
described for 10a using compound 7a (280 mg, 1 mmol) and 5-
nitrothiophene-2-aldehyde (157 mg, 1 mmol). Yield 255 mg, 60%;
IR (KBr) (ymax/cmꢃ1) 3301, 2910, 1631, 1580, 1563, 1430, 1335, 1256,
The compound 11a was prepared according to the above
described method using compound 7a (280 mg,1 mmol) and 5-nitro-
2-furoic acid (157 mg, 1 mmol). Yield 293 mg, 70%; IR (KBr) (ymax
cmꢃ1) 3301, 2910,1694,1640,1601,1556,1528,1497,1420,1380,1260,
1138, 894; 1H NMR (DMSO-d6, 200 MHz):
/
1158, 815; 1H NMR (CDCl3, 300 MHz):
d
7.89 (d, 1H, J ¼ 7.5 Hz, BTD-
d
7.75 (d, 1H, J ¼ 7.4 Hz,
H), 7.74 (d, 1H, J ¼ 4.1 Hz, thiophene-H), 7.54 (t, 1H, J ¼ 3.7 Hz, BTD-
H), 7.36 (t, 1H, J ¼ 7.5 Hz, BTD-H), 7.23 (d, 1H, J ¼ 8.3 Hz, BTD-H),
6.85 (d, 1H, J ¼ 4.1 Hz, thiophene-H), 3.71 (s, 2H, eCH2e), 3.60 (s,
3H, eNCH3), 3.56 (t, 4H, 2ꢂ piperazine-CH2), 2.62 (t, 4H, 2ꢂ
BTD-H), 7.68 (td, 1H, J ¼ 8.1 Hz, BTD-H), 7.64 (d, 1H, J ¼ 3.4 Hz, furan-
H), 7.54 (d,1H, J ¼ 7.5 Hz, BTD-H), 7.41 (t,1H, J ¼ 7.4 Hz, BTD-H) 7.26 (d,
1H, J ¼ 3.4 Hz, furan-H), 3.87e3.9 (m, 4H, 2ꢂ piperazine-CH2), 3.78 (s,
3H, eNCH3), 3.63e3.66 (m, 4H, 2ꢂ piperazine-CH2); 13C NMR
piperazine-CH2); 13C NMR (DMSO-d6, 75 MHz):
d
157.6, 151.5, 150.7,
(DMSO-d6, 75 MHz): d 157.2, 155.5, 153.4, 147.3, 137.4, 136.2, 135.8,
141.3, 137.3, 134.8, 131.9, 129.8, 125.4, 123.5, 56.8, 51.6, 48.4, 39.1.
ESIMS: m/z 421 (M)þ, 422 (M þ 1)þ. Anal. calcd. for C17H19N5O4S2:
C, 48.44; H, 4.54; N, 16.62. Found: C, 48.35; H, 4.50, N, 16.58.
125.5, 124.6, 124.0, 116.5, 112.1, 56.8, 47.4, 43.2, ESIMS: m/z 420
(M þ 1)þ, 421 (M þ 2)þ, 442 (M þ Na)þ. Anal. calcd. for C17H17N5O6S:
C, 48.68; H, 4.09; N, 16.70. Found: C, 48.64, H, 4.01, N, 16.67.
6.5.5. 3-[4-{(5-Nitro-2-thienyl)methyl}piperazinyl]-4-ethyl-1,2,4-
benzothiadiazine 1,1-dioxide (10e)
6.6.2. 3-[4-{(5-Nitro-2-furyl)carbonyl}piperainyl]-4-ethyl-1,2,4-
benzothiadiazine 1,1-dioxide (11b)
The compound 10e was prepared according to the method
described for 10a using compound 7b (294 mg, 1 mmol) and 5-
nitrothiophene-2-aldehyde (157 mg, 1 mmol). Yield 295 g, 68%; IR
(KBr) (ymax/cmꢃ1) 3324, 2905, 1631, 1574, 1533, 1427, 1333, 1264,
The compound 11b was prepared according to the above
described method using compound 7b (294 mg, 1 mmol) and
5-nitro-2-furoic acid (157 mg, 1 mmol). Yield 294 mg, 68%; IR (KBr)
(
ymax/cmꢃ1) 2925, 1694, 1641, 1580, 1540, 1427, 1335, 1299, 1258,
1258, 1156, 1009, 859; 1H NMR (CDCl3, 300 MHz):
d
7.89 (d, 1H,
1166, 818; 1H NMR (DMSO-d6, 200 MHz):
d
7.75 (d, 1H, J ¼ 7.4 Hz,
J ¼ 7.5 Hz, BTD-H), 7.74 (d, 1H, J ¼ 4.1 Hz, thiophene-H), 7.54 (td,
1H, J ¼ 3.7 Hz, BTD-H), 7.36 (t, 1H, J ¼ 7.5 Hz, BTD-H), 7.23 (d, 1H,
J ¼ 8.3 Hz, BTD-H), 6.85 (d, 1H, J ¼ 4.1 Hz, thiophene-H), 4.02 (q, 2H,
eNCH2CH3), 3.71 (s, 2H, eCH2e), 3.56 (t, 4H, 2ꢂ piperazine-CH2),
2.62 (t, 4H, 2ꢂ piperazine-CH2), 1.12 (t, 3H, eNCH2CH3); 13C NMR
BTD-H), 7.67 (td, 1H, J ¼ 8.1 Hz, BTD-H), 7.64 (d, 1H, J ¼ 3.4 Hz,
furan-H), 7.57 (d, 1H, J ¼ 7.5 Hz, BTD-H), 7.41 (t, 1H, J ¼ 7.4 Hz, BTD-
H) 7.27 (d, 1H, J ¼ 3.4 Hz, furan-H), 4.15 (q, 2H, J ¼ 6.6 Hz,
eNCH2CH3), 3.88e3.91 (m, 4H, 2ꢂ piperazine-CH2), 3.64e3.68 (m,
4H, 2ꢂ piperazine-CH2), 1.12 (t, 3H J ¼ 6.6 Hz, eNCH2CH3); 13C NMR
(DMSO-d6, 75 MHz):
d
157.6, 151.5, 150.7, 141.3, 138.9, 137.3, 134.8,
(DMSO-d6, 75 MHz): d 157.1, 155.2, 153.4, 146.2, 137.3, 136.4, 135.8,
131.9, 129.8, 125.4, 123.5, 56.8, 51.6, 48.4, 45.4, 27.8. ESIMS: m/z 436
(M þ 1)þ, 437 (M þ 2)þ. Anal. calcd. for C18H21N5O4S2: C, 49.64; H,
4.86; N, 16.08. Found: C, 49.59; H, 4.82, N, 15.98.
132.6, 125.3, 124.6, 123.8, 116.5, 112.1, 57.2, 43.5, 45.2, 27.8. ESIMS:
m/z 434 (M þ 1)þ. Anal. calcd. for C18H19N5O6S: C, 49.85; H, 4.42; N,
16.16. Found: C, 49.80; H, 4.39, N, 16.12.
6.5.6. 3-[4-{(5-Nitro-2-thienyl)methyl}piperazinyl]-4-phenyl-1,2,4-
benzothiadiazine 1,1-dioxide (10f)
6.6.3. 3-[4-{(5-Nitro-2-furyl)carbonyl}piperainyl]-4-phenyl-1,2,4-
benzothiadiazine 1,1-dioxide (11c)
The compound 10f was prepared according to the method
described for 10a using compound 7c (342 mg, 1 mmol) and
5-nitrothiophene-2-aldehyde (157 mg, 1 mmol). Yield 338 mg,
69%; IR (KBr) (ymax/cmꢃ1) 3411, 2928, 1640, 1587, 1530, 1493, 1355,
The compound 11c wasprepared according tothe above described
method using compound 7c (342 mg, 1 mmol) and 5-nitro-2-furoic
acid (157 mg, 1 mmol). Yield 342 g, 71%; IR (KBr) (ymax/cmꢃ1) 3244,
2910,1637,1598,1528,1477,1323,1271,1148, 756; 1H NMR (DMSO-d6,

4552
A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 4545e4553
200 MHz):
d
7.90 (d, 1H, J ¼ 7.8 Hz, BTD-H), 7.58e7.45 (m, 5H,
2-furaldehyde (141 mg, 1 mmol). Yield 365 mg, 70%. IR (KBr) (ymax
cmꢃ1) 3160, 3025,1664,1636,1596,1565,1530,1477,1320,1265,1138,
758; 1H NMR (DMSO-d6 200 MHz):
10.3 (bs, 1H, eNH), 8.04 (s, 1H,
eCH]Ne), 7.90 (d,1H, J ¼ 7.5Hz, BTD-H), 7.64(m, 4H, J ¼ 3.2 Hz, BTD-
H, AreH, furan-H), 7.50e7.32 (m, 5H, J ¼ 7.5Hz, BTD-H, AreH), 7.05 (d,
1H, J ¼ 3.8 Hz, furan-H), 3.78e3.49 (m, 4H, piperdine-CH2e), 2.29 (m,
1H, piperdine-CHe), 2.01e1.78 (m, 4H, piperdine-CH2e). NCMS: m/z
523 (Mþ 1)þ, 545 (Mþ Na)þ. Anal. calcd. forC24H22N6O6S:C, 55.17;H,
4.24; N, 16.08. Found: C, 55.10; H, 4.19, N, 16.00.
/
J ¼ 3.1 Hz, BTD-H, AreH, furan-H), 7.26e7.52 (m, 4H, J ¼ 7.8, 3.9 Hz,
BTD-H, AreH), 7.16 (d, 1H, J ¼ 3.1 Hz, furan-H), 3.87e3.91 (m, 4H, 2ꢂ
piperazine-CH2), 3.59e3.71 (m, 4H, 2ꢂ piperazine-CH2); 13C NMR
d
(DMSO-d6, 75 MHz):
d 157.4, 156.1, 146.7, 138.1, 136.2, 135.4, 132.5,
130.4, 129.2, 129.0, 125.3, 124.4, 124.2, 115.4, 112.3, 58.5, 43.2. ESIMS:
m/z 482 (M þ 1)þ, 504 (M þ Na)þ; Anal. calcd. for C22H19N5O6S: C,
54.88; H, 3.98; N, 14.95. Found: C, 54.82; H, 3.84, N, 14.87.
6.7. Preparation of N0-4-[(E/Z)-1-(5nitro-2-furyl/thinyl)
methylidene]-1-(4-alkyl/aryl 1,1-dioxo-1,4-dihydro1,2,4-
benzothiadazin-3-yl)-4-piperdin carbohyrdazide (12aef)
6.7.4. N0-4-[(E/Z)-1-(5-nitro-2-thinyl)methylidene]-1-(4-methyl-
1,1-dioxo-1,4-dihydro1,2,4-benzothiadazin-3-yl)-4-piperdin
carbohyrdazide (12d)
The compound 12d was prepared according to the method
described for 12a using compound 9a (338 mg, 1 mmol) and
5-nitrothiophene-2-aldehyde (157 mg,1 mmol). Yield 323 mg, 68%;
IR (KBr) (ymax/cmꢃ1) 3245, 3152, 1672, 1637, 1597, 1528, 1528, 1273,
To a solution of 3-(N-isonipicotinicmethylester)-4-alkyl/aryl-4H-
1,2,4-benzothiadiazine 1,1-dioxanes (9aec) (1 mmol) and catalytic
amount of acetic acid in ethanol (25 mL), 5-nitro-2-furyl/thinly
carboxaldehyde (1.5 mmol) was added and the reaction mixture was
stirred at room temperature for 12 h. After completion of reaction as
indicated by TLC, the solvent was filtered and the product was
washed with methanol thrice (3 ꢂ 10 mL) and finally the precipitate
was recrystallized from hot methanol to afford desire compound.
1105, 690; 1H NMR (DMSO-d6, 200 MHz):
d 11.2 (bs, 1H, eNH), 8.06
(s, 1H, eCH]Ne), 7.94 (d, 1H, J ¼ 7.4 Hz, BTD-H), 7.75 (d, 1H,
J ¼ 4.1 Hz, thiophene-H), 7.64 (d,1H, J ¼ 7.4 Hz, BTD-H), 7.54 (d,
1H, J ¼ 7.5 Hz, BTD-H), 7.41 (t, 1H, J ¼ 7.4 Hz, BTD-H) 7.26 (d, 1H,
J ¼ 4.4 Hz, thiophene-H), 3.87e3.72 (m, 4H, piperdine-CH2e), 3.78
(s, 3H, eNCH3), 2.20 (m, 1H, piperdine-CHe), 2.13e1.78 (m, 4H,
6.7.1. N0-4-[(E/Z)-1-(5-nitro-2-furyl)methylidene]-1-(4-methyl-1,1-
dioxo-1,4-dihydro1,2,4-benzothiadazin-3-yl)-4-piperdin
carbohydrazide (12a)
The compound 12a was prepared according to the above
described method using compound 9a (338 mg,1 mmol) and 5-nitro-
piperdine-CH2e); 13C NMR (DMSO-d6, 75 MHz):
d 157.4, 154.0,
150.3, 146.5, 137.5, 137.2, 136.2, 132.7, 130.5, 128.8, 125.3, 124.6,
123.2, 40.5, 36.7, 32.1, 29.8. ESIMS: m/z 477 (M þ 1)þ, 479 (M þ 3)þ.
Anal. calcd. for C19H20N6O5S2: C, 47.89; H, 4.23; N, 17.64. Found: C,
47.84; H, 4.20, N, 17.60.
2-furaldehyde (141 mg, 1 mmol). Yield 468 mg, 85%; IR (KBr) (ymax
cmꢃ1) 3301, 3102, 2910,1694,1601,1556,1528,1497,1430,1335,1287,
1210,1170,1138, 1108, 887; 1H NMR (DMSO-d6, 200 MHz):
10.2 (bs,
/
6.7.5. N0-4-[(E/Z)-1-(5-nitro-2-furyl)methylidene]-1-(4-ethyl-1,1-
dioxo-1,4-dihydro1,2,4-benzothiadazin-3-yl)-4-piperdin
carbohyrdazide (12e)
d
1H, eNH), 7.75 (d,1H, J ¼ 7.4 Hz, BTD-H), 7.68 (m,1H, J ¼ 8.1 Hz, BTD-
H), 7.64 (d,1H, J ¼ 3.1 Hz, furan-H), 7.54 (d,1H, J ¼ 7.5 Hz, BTD-H), 7.50
(s,1H, eCH]Ne), 7.41 (t,1H, J ¼ 7.4 Hz, BTD-H) 7.26 (d,1H, J ¼ 3.1 Hz,
furan-H), 3.85e3.72 (m, 4H, piperdine-CH2e), 3.78 (s, 3H, eNCH3),
2.20e2.25 (m, 1H, piperdine-CHe), 2.13e1.8 (m, 4H, piperdine-
The compound 12e was prepared according to the method
described for 12a using compound (9b) (352 mg, 1 mmol) and 5-
nitrothiophene-2-aldehyde (157 mg, 1 mmol). Yield 294 mg, 60%;
IR (KBr) (ymax/cmꢃ1) 3270, 3107, 2928, 1695, 1601, 1556, 1491, 1427,
CH2e); 13C NMR (DMSO-d6, 75 MHz):
d
157.4, 154.6, 152.0, 151.3,
1335, 1285, 1148, 673; 1H NMR (DMSO-d6, 200 MHz):
d 10.5 (bs, 1H,
133.8,132.7,127.8,125.3,124.6,124.0,115.1,112.7, 40.5, 38.2, 36.7, 24.8.
ESIMS: m/z 461 (M þ 1)þ. Anal. calcd. for C19H20N6O6S: C, 49.56; H,
4.38; N, 18.25. Found: C, 49.52; H, 4.34, N, 18.20.
eNH), 8.01 (s, 1H, eCH]Ne), 7.78e7.86 (m, 2H, J ¼ 7.5, 4.77 Hz,
BTD-H, thiophene-H), 7.68 (m, 1H, J ¼ 4.5 Hz, BTD-H), 7.64 (m, 1H,
J ¼ 7.5 Hz, BTD-H), 7.43 (m, 1H, J ¼ 8.3 Hz, BTD-H), 7.27 (d, 1H,
J ¼ 4.8 Hz, thiophene-H), 4.03 (q, 2H, eNCH2CH3), 3.72e3.65 (m,
4H, piperdine-CH2e), 2.21 (m, 1H, piperdine-CHe), 2.13 (m, 4H,
piperdine-CH2e), 1.15 (t, 3H, eNCH2CH3); 13C NMR (DMSO-d6,
6.7.2. N0-4-[(E/Z)-1-(5-nitro-2-furyl)methylidene]-1-(4-ethyl-1,1-
dioxo-1,4-dihydro1,2,4-benzothiadazin-3-yl)-4-piperdin
carbohyrdazide (12b)
75 MHz):
d 157.1, 154.9, 151.2, 147.1, 137.5, 137.3, 136.4, 132.6, 131.7,
The compound 12b was prepared according to the method
described for 12a using compound 9b (352 mg, 1 mmol) and 5-
nitro-2-furaldehyde (141 mg, 1 mmol). Yield 355 mg, 70%; IR (KBr)
128.9, 125.3, 124.6, 123.8, 45.4, 40.7, 30.2, 29.2, 27.8. ESIMS: m/z 491
(M þ 1)þ, 493 (M þ 3)þ. Anal. calcd. for C20H22N6O5S2: C, 48.97; H,
4.57; N, 17.13. Found: C, 48.90; H, 4.57, N, 17.19.
(
ymax/cmꢃ1) 3455, 2950, 1645, 1287, 1528, 1495, 1465, 1427, 1538,
1353, 1295, 1167, 1103, 885; 1H NMR (DMSO-d6, 200 MHz):
d
10.2
6.7.6. N0-4-[(E/Z)-1-(5-nitro-2-furyl)methylidene]-1-(4-phenyl-1,1-
dioxo-1,4-dihydro1,2,4-benzothiadazin-3-yl)-4-piperdin
carbohyrdazide (12f)
(bs,1H, eNH), 8.02 (s,1H, eCH]Ne), 7.90 (d,1H, J ¼ 7.5 Hz, BTD-H),
7.68 (d, 1H, J ¼ 3.2 Hz, furan-H), 7.64 (m, 1H, J ¼ 7.5 Hz, BTD-H), 7.43
(m, 1H, J ¼ 8.3 Hz, BTD-H), 7.29 (d, 1H, J ¼ 4.4 Hz, BTD-H), 6.95 (d,
1H, J ¼ 3.2 Hz, furan-H), 4.03 (q, 2H, eNCH2CH3), 3.79e3.70 (m, 4H,
piperdine-CH2e), 2.25 (m, 1H, piperdine-CHe), 2.15e1.8 (m, 4H,
piperdine-CH2e), 1.19 (t, 3H, -NCH2CH3); 13C NMR (DMSO-d6,
The compound 12f was prepared according to the method
described for 12a using compound (9c) (400 mg, 1 mmol) and
5-nitrothiophene-2-aldehyde (157 mg, 1 mmol). Yield 323 g, 60%;
IR (KBr) (ymax/cmꢃ1) 3250, 3090, 1708, 1601, 1597, 1570, 1523, 1556,
75 MHz):
d
157.1, 154.8, 152.2, 137.3, 136.2, 132.6, 127.6, 125.3, 124.6,
1333, 1285, 1169, 643; 1H NMR (DMSO-d6, 200 MHz):
d 11.0 (bs, 1H,
123.6, 115.1, 112.7, 44.2, 40.7, 30.2, 27.8. ESIMS: m/z 473.1 (M ꢃ 1)þ,
475 (M þ 1)þ. Anal. calcd. for C20H22N6O6S: C, 50.63; H, 4.67; N,
17.71. Found: C, 50.60; H, 4.67, N, 17.65.
eNH), 8.05 (s, 1H, eCH]Ne), 7.92 (d, 1H, J ¼ 7.5 Hz, BTD-H),
7.64e7.78 (m, 4H, J ¼ 4.5 Hz, BTD-H, AreH, thiophene-H),
7.50e7.32 (m, 5H, J ¼ 7.5 Hz, BTD-H, AreH), 7.25 (d, 1H, J ¼ 4.8 Hz,
thiophene-H), 3.75e3.45 (m, 4H, piperdine-CH2e), 2.29 (m, 1H,
piperdine-CHe), 1.92 (m, 4H, piperdine-CH2e); 13C NMR (DMSO-
6.7.3. N0-4-[(E/Z)-1-(5-nitro-2-furyl)methylidene]-1-(4-phenyl-1,1-
dioxo-1,4-dihydro1,2,4-benzothiadazin-3-yl)-4-piperdin
carbohyrdazide (12c)
The compound 12c was prepared according to the method
described for 12a using compound 9c (400 mg, 1 mmol) and 5-nitro-
d6, 75 MHz):
d 157.4, 155.2, 151.0, 141.2, 138.1, 136.2, 134.4, 132.5,
130.4, 129.2, 129.1, 126.8, 125.5, 124.7, 123.9, 40.2, 31.2, 28.2. ESIMS:
m/z 539 (M þ 1)þ, 561 (M þ Na)þ. Anal. calcd. for C24H22N6O5S2: C,
53.52; H, 4.12; N, 15.60. Found: C, 53.49; H, 4.08, N, 15.58.

A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 4545e4553
4553
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535e568.
Acknowledgement
We thank to Department of Science Technology (DST: SR/S1/OC-
42/2008), New Delhi for financial assistance. The authors RVCRNCS,
SA, PS, AMR, SS, are thankful to CSIR, New Delhi for the award of
their research fellowship.
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